key: cord-008881-579ronfq authors: nicholson, karlg; prestage, howard; cole, peterj; turner, georges; bauer, sallyp title: multisite intradermal antirabies vaccination: immune responses in man and protection of rabbits against death from street virus by postexposure administration of human diploid-cell-strain rabies vaccine date: 1981-10-24 journal: lancet doi: 10.1016/s0140-6736(81)91402-1 sha: doc_id: 8881 cord_uid: 579ronfq lymphocyte transformation, production of neutralising antibody, and the development of antirabies igg antibody were studied in ten healthy volunteers in response to 0·8 ml of human diploid-cell strain (hdcs) rabies vaccine administered on one occasion in divided doses in 8 intradermal (i.d.) sites. all ten volunteers rapidly developed substantial titres of rabies antibody, and eight of the ten had t lymphocytes that were immunologically stimulated by hdcs rabies-virus antigen. postexposure treatment with 0·8 ml of hdcs vaccine given at 4 i.d. sites completely protected fourteen rabbits from death by street virus. the results suggest that in developing countries patients could be protected with small volumes of potent tissue-culture vaccine administered intradermally shortly after exposure. lymphocyte transformation, production of neutralising antibody, and the development of antirabies igg antibody were studied in ten healthy volunteers in response to 0&middot;8 ml of human diploid-cell strain (hdcs) rabies vaccine administered on one occasion in divided doses in 8 intradermal (i.d.) sites. all ten volunteers rapidly developed substantial titres of rabies antibody, and eight of the ten had t lymphocytes that were immunologically stimulated by hdcs rabies-virus antigen. postexposure treatment with 0&middot;8 ml of hdcs vaccine given at 4 i.d. sites completely protected fourteen rabbits from death by street virus. the results suggest that in developing countries patients could be protected with small volumes of potent tissue-culture vaccine administered intradermally shortly after exposure. introduction in 1975, forty-five people severely bitten by rabid dogs and wolves in iran were treated after exposure with a new rabies vaccine produced in cultures of human diploid cells. all except one also received one injection of rabies immune serum. this treatment, in contrast to past experience with other vaccines, resulted in protection of all individuals against rabies. this resounding success has been repeated in trials in germany and the u.s.a. using 5 or 6 doses of human diploid-cell strain (hdcs) rabies vaccine and human rabies immune globulin.', thus, almost a century after the post exposure treatment of man began, effective antirabies prophylaxis appears to have been achieved. with few exceptions, rabies is a problem of impoverished areas of the world, where the annual per-caput expenditure on health care is often far less than the cost of a single 1 ml dose of hdcs vaccine. as a consequence, potent tissueculture vaccine is seldom used in the third world. the need for an effective but less expensive method of treatment prompted us to investigate the possibility of administering potent vaccine more economically and efficiently than at present. our previous studies have shown that substantial titres of antibody can be achieved with small quantities of hdcs vaccine administered by the intradermal (i.d.) route and that the cost of vaccination can be reduced considerably.4-7 in this paper we report the antibody and cellmediated immune response of man to multisite i.d. vaccination and application of the method to postexposure protection of rabbits. it appears possible that the i.d. route could be applied successfully to the post exposure treatment of man. approval for the volunteer study was given by northwick park hospital ethical committee. hdcs vaccine (0 -8 ml) was given i.d., on single occasion, to ten volunteers at 8 sites on the medial and lateral aspects of the upper arms and thighs. blood samples for tests of lymphocyte transformation and antibody determination were taken before vaccination and 10, 14, 21, and 42 days later. a further blood sample for antibody titration was taken on day 100. four subjects were vaccinated with a dermojet injector;* the six remaining volunteers were given vaccine with a 25-gauge needle and tuberculin syringe. no volunteer had received antirabies vaccine previously. the nuchal muscles of forty-two new zealand white rabbits were inoculated with 50 rabbit ld50 ofa first mouse-brain-passage arcticfox rabies virus isolate in two separate sites (0 -5 ml each side). 8 h later fourteen rabbits were given 1-0 0 ml of hdcs vaccine intramuscularly (i.m.) into the left forelimb, and fourteen others received 4 i.d. injections of 0-2 2 ml of vaccine into each limb. the remaining fourteen were used as controls and received no prophylaxis. each animal was then observed for 12 months for signs of rabies. none of the rabbits had been exposed to rabies previously or had been immunised against the disease. in both studies whole-virion hdcs rabies vaccine (i'institut merieux; lot r 0220; antigenic value 10-8) inactivated with 9-propiolactone was used. all blood samples were titrated for rabies neutralising antibody with the mouse neutralisation test;9 titres in iu/ml were calculated by reference to the international standard antiserum to rabies virus (statens seruminstitut, copenhagen, denmark). in addition, the sera were assayed for igg rabies antibody by enzyme immunoassay (elisa) using a modification of the method used for the detection of coronavirus antibodiesl&deg; (k. g. nicholson, h. prestage, unpublished). absorbance values were read at 405 nm on a flow laboratories titertek multiskan spectrophotometer 20, 30, 45, and 60 min after addition of the substrate. rabies antibody was considered present when the optical-density reading of the test sample was greater than the mean + 2 standard deviations of comparable dilutions of 8 negative control sera. an enriched t-lymphocyte population was obtained by passing a thrice-washed mononuclear-cell suspension taken from the top of a ficoll-triosil gradient (pharmacia fine chemicals, uppsala, sweden) twice through a nylon-fibre column." the cell suspension obtained contained less than 2% b lymphocytes as judged by staining with polyvalent fluorescein-labelled antihuman immunoglobulin reagent. an enriched b-lymphocyte population was obtained by sedimenting rosettes formed between t lymphocytes and sheep red blood cells." lymphocytes from human cord blood and a non-vaccinated subject were used as controls. hdcs rabies-virus vaccine (l'institut merieux; lot r 0220) was exhaustively dialysed against phosphate-buffered saline (pbs) and adjusted to the original volume with pbs for use as antigen. phytohaemagglutinin (pha; purified grade, wellcome laboratories, beckenham) was used as control at a concentration of 0-2 2 mitogenic units/ml. cultures containing 10 1 of antigen or mitogen and 200 1 of cell suspension containing 2 x 105 lymphocytes were established in microtitre plates then pulsed with tritiated thymidine and harvested as described previously.' because of the ph indicator in the vaccine, needle inoculation immediately resulted in magenta-coloured skin blebs at each injection site. vaccination with the dermojet injector was generally quicker, but bleb formation was less satisfactory and in some areas where the skin was especially soft it appeared that all the vaccine had entered the subcutaneous tissue. this method of inoculation was also associated with lower titres of antibody than occurred after needle inoculation (figs. 1 six subjects who were inoculated with a needle and syringe. by 10 days, neutralising antibody ranged between 1 3 and 8 7 iu/ml (geometric mean titre [gmt] 3 0 iu/ml) and antirabies igg ranged between 1/345 and 1/1250 (gmt 1/693). peak titres of virus neutralising antibody were found on day 14 (gmt 14' 3 iu/ml), but substantial titres were still present 100 days after vaccination. the increments (cpm) in lymphocyte transformation are expressed as a ratio to the cpm values of non-stimulated control cultures (table). the results show that t lymphocytes from eight of ten vaccinees were significantly stimulated in vitro 14 to 42 days after vaccination. this blast transformation occurred with cells from three of four people given vaccine by the dermojet injector and from five ofthe six people inoculated with a needle and syringe. there was no significant difference between the transformation increments of the two groups, and no correlation was found between the transformation increment and the titre of antirabies igg or nine of fourteen rabbits developed paralysis and died after infection with 50 rabbit ldso of street-rabies virus. in these animals, forelimb paralysis developed within 13 to 38 days of infection and progressed to complete paralysis and death 2 to 7 days later. postexposure treatment with a single dose of hdcs vaccine reduced the mortality significantly; the administration of i -0 ml of vaccine i.m. gave significant (p=0'018, fisher's exact test) but incomplete protection, two of fourteen animals developing paralysis and dying after incubation periods of 13 and 20 days. none of fourteen rabbits died after receiving 4 i.d. inoculations of 0 . 2 ml of the vaccine in each limb (p = 0' 0006). britain, [4] [5] [6] [7] 12, 13 france, 14 and germanyls,16 that the administration of hdcs vaccine by the intradermal route is followed by substantial titres of virus-neutralising antibody with occasional mild local and systemic reactions. it has also been shown that 4 or 8 doses of 0 1 ml given in separate sites on a single occasion rapidly induce high titres of antibody. i ,7 the present report confirms these observations and shows in addition that the early production of virus-neutralising antibody is accompanied by high titres of antirabies igg. this early igg response may be most important, for it is now well established that neutralising antibody of the igg class, unlike igm neutralising antibody, confers protection on animals challenged with rabies17 and may be the key to successful postexposure treatment of man. the possible role of cell-mediated immunity in rabies infection is poorly understood, but it too may be an important component of the host's immune response. we have reported that transformation of lymphocytes occurred with cells taken from eight of ten vaccinees after the first 4 x 1 ml doses of an established postexposure regimen for hdcs vaccine.' in the present study we separated the lymphocyte subpopulations and have shown that the blast transformation is a t-cell response. furthermore, it occurred in the same proportion of vaccinees (eight often) as was found in the previous study, but with only a quarter of the volume of vaccine. clearly, if high titres of neutralising antibody and a cell-mediated response are both important for protection, the present study suggests that they can be obtained equally well with much smaller quantities of vaccine than are used at present. we further showed that rabbits could be completely protected by injecting 4 x 0 -2 ml doses of hdcs vaccine intradermally 8 h after intranuchal infection with street-rabies virus. by contrast, nine of the fourteen controls (64%) died from rabies with incubation periods of 13 to 38 days (mean 22 days). opponents to the administration of rabies vaccine by the i.d. route claim that it is technically difficult, especially in the elderly and the very young. nevertheless, many vaccines are routinely administered by the i.d. route with apparent success and considerable financial savings. there seems to be ample experimental evidence to justify a postexposure study of hdcs vaccine administered by the i.d. route; we believe that this would be both ethical and potentially of great importance for developing countries. k. g. n. was partly supported by an mrc-eli lilly travelling fellowship at the u.s. national center for disease control, atlanta, ga. g. s. t. was partly supported by mrc grant no. 9 976/661. we thank dr c. charbonnier and l'lnstitut merieux for the gift of the vaccine requests for reprints should be addressed to k. g. n. successful protection of humans exposed to rabies infection postexposure treatment with the new human diploid cell rabies vaccine and antirabies serum post-exposure use of human diploid cell culture rabies vaccine deitch mw postexposure trial of a human diploid cell strain rabies vaccine immunization with a human diploid cell strain of rabies virus vaccine: two year results studies with human diploid cell strain rabies vaccine and human rabies immunoglobulin in man human diploid cell strain rabies vaccine rapid prophylactic immunisation of volunteers with small doses immune responses of humans to a human diploid cell strain of rabies virus vaccine: lymphocyte transformation, production of virus-neutralizing antibody, and induction of interferon l'injecteur sans aiguille "dermo-jet" presse m&eacute quantitative assay and potency test of antirabies serum and immunoglobulin macnaughton mr enzyme linked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus 229 e group viruses purification of human t and b lymphocytes immunogenicity and acceptability of a human diploid cell culture rabies vaccine in volunteers rabies prophylaxis simplified resultats de la vaccination antirabique preventive par le vaccin inactive concentre souche rabies pm/w138-1503-3m cultivee sur cellules diploides humaines developments in biological standardisation prophylactic immunization of humans against rabies by intradermal inoculation of human diploid cell culture vaccine a large scale antirabies immunisation study in humans using hdcs vaccine. who consultation on cell culture rabies vaccines and their protective effect in man immunoglobulin igg and igm antibody responses to rabies vaccine transformed the management of diabetes and it is now administered to about 4 million diabetics throughout the world. although the frequency of side-effects is relatively low, efforts have been made over the years to improve the quality and extend the range of insulin preparations. the pace of these changes has accelerated during the past decade. introduction of high-purity insulin and of preparations of insulin from a single species of animal have been followed by developments of two kinds: the availability for therapy of insulin containing the aminoacid sequence of the natural human hormone and the construction of continuous-delivery systems to administer insulin under conditions that more closely mimic its natural secretion in the body. the changes in quality and type of insulin available have posed problems of nomenclature.recognition that the molecular structure of insulin can influence tolerance to, and the side-effects of, therapy led to the introduction in the british pharmacopoeia (bp) in 1975' of a requirement that all formulations be labelled with the species of origin. at that time, the bp contained no monograph for bulk insulin, but the marketing of a variety of purified insulins made such a monograph desirable, and it appeared in the bp 1980.2 the monograph covers insulin of either porcine or bovine origin that has been purified beyond the stage of conventional crystalline insulin. this has had several consequences. first, the monograph required a title and "insulin" was taken. had the title been "purified insulin" or similar, to draw a distinction with crystalline insulin, the problem might arise of a name for material of yet higher quality (already available as "monocomponent" or "pro-insulin free" preparations). this is one reason why a *professor turner is vice-chairman and dr calam a member of the nomenclature committee of the british pharmacopoeia commission. monograph for a drug substance rarely contains any qualifying adjective indicating degree of purity. however, the title "insulin" had been for many years a synonym for insulin injection, and it was necessary to delete it as an alternative name for that preparation. second, a unique situation arose in that the monographs for formulations continued to specify a minimum potency of 23 iu/mg for the insulin used, against the higher figure in the bulk monograph, thus allowing insulin not of bp quality to be incorporated into bp formulations. this was the result of decisions not to delete conventional formulations, satisfactory for many patients, from the pharmacopoeia and not to add a set of separate monographs for formulations containing the bp grade of insulin, which would require additional titles. another factor was involved in that control of six of the nine insulin formulations is exerted by monographs in the european pharmacopoeia. for these, no unilateral change in the requirements can be made by a national authority, but revision must await agreement by the european pharmacopoeia commission, with a further lapse of time before adoption. this factor illustrates the point that pharmacopoeial decisions in the united kingdom, including those involving nomenclature, cannot be taken without considering the international constraints that apply and the international implications that may result. during the past eighteen months another aspect of insulin nomenclature has been discussed by the british pharmacopoeia commission and its committees concerned with nomenclature and with hormones. this arose from availability, for testing and clinical trial, of insulin possessing the structure of the natural human hormone, the prospect of its wider use, and the eventual need for a monograph for it. that one method of production of such insulin uses geneticengineering techniques suggests that the wider implications must be considered. thus, in the following discussion, it should be remembered that the final outcome should, so far as possible, set a pattern that can be applied to other hormones and therapeutic substances (such as growth hormone and interferons) obtained by novel techniques.the structure of human insulin was established in 19603 and was found to differ from that of the porcine hormone only in the presence of threonine in place of alanine at position 30 of the b chain. the total synthesis of insulin having the human sequence was achieved in an elegant manner by workers at ciba geigy in 1974,4 but the process is not economically viable under present conditions. this material key: cord-000366-u4649rtx authors: shan, tongling; lan, daoliang; li, linlin; wang, chunmei; cui, li; zhang, wen; hua, xiuguo; zhu, caixia; zhao, wei; delwart, eric title: genomic characterization and high prevalence of bocaviruses in swine date: 2011-04-15 journal: plos one doi: 10.1371/journal.pone.0017292 sha: doc_id: 366 cord_uid: u4649rtx using random pcr amplification followed by plasmid subcloning and dna sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate h18 (pbov1-h18) and porcine bocavirus 2 isolate a6 (pbov2-a6) which differed by 51.8% in their ns1 protein. phylogenetic analysis indicated that pbov1-h18 was very closely related to a ∼2 kb central region of a porcine bocavirus-like virus (pbo-likev) from sweden described in 2009. pbov2-a6 was very closely related to the porcine bocavirus genomes pbov-1 and pbov2 from china described in 2010. among 340 fecal samples collected from different age, asymptomatic swine in five chinese provinces, the prevalence of pbov1-h18 and pbov2-a6 related viruses were 45–75% and 55–70% respectively, with 30–47% of pigs co-infected. pbov1-a6 related strains were highly conserved, while pbov2-h18 related strains were more diverse, grouping into two genotypes corresponding to the previously described pbov1 and pbov2. together with the recently described partial bocavirus genomes labeled v6 and v7, a total of three major porcine bocavirus clades have therefore been described to date. further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses. members of the parvoviridae family are small non-lipid enveloped viruses with a diameter of 18-26 nm, icosahedral symmetry (t = 1), encoded by a single-stranded linear dna genome of approximately 4,000 to 6,000 nucleotides (nt) [1] . the family includes two subfamilies: densovirinae, parvovirinae. the subfamily of densovirinae contains four genera: densovirus, iteravirus, brevidensovirus and pefudensovirus, which infect only invertebrates [1] . the parvovirinae subfamily is currently subdivided into five genera: parvovirus, erythrovirus, dependovirus (adeno-associated virus), amdovirus, and bocavirus, infecting vertebrates [1] . the bocavirus genus was recently assigned by the international committee on taxonomy of viruses (ictv) [1] to parvovirus genomes containing a third orf (labeled np1) between the ns1 and vp1/vp2 genes [2] . bocaviruses were first identified in bovine and canine [3, 4] , samples from which it derives its genus name [1, 5] . presently, the bocavirus genus contains eight members: bovine parvovirus, canine minute virus (cnmv), human bocavirus 1-4 (hbov1-4), a gorilla bocavirus and a partially sequenced chimpanzee bocavirus [1, 6, 7] . the first human bocavirus (hbov) was found in the nasopharyngeal secretion of a child with respiratory problems using a methodology closely related to that used here [8] . hbov has been associated with lower respiratory tract symptoms and possibly diarrhea [5, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] , and shows a very low degree of genetic variability worldwide [6, 23, 24] . hbov2 was first reported in the stool of pakistani children with non-polio acute flaccid paralysis (afp) [10] , and then in australian children and chinese children with diarrhea [9, 25] . hbov3 was first reported in the stool of australian children with diarrhea [9] and then in stool from nigerian, tunisian, nepalese and us children [5] . hbov4 was reported in the stool of children with afp from nigeria and tunisia [5] . hbov 1/2/3 were also detected in untreated sewage water from throughout the us [26] . recently, a novel bocavirus was identified in the feces of captive gorillas with diarrhea [6] and from wild gorillas and chimpanzees [7] . in 2009, a porcine bocalike virus (pbo-likev) was reported in swine feces with postweaning multisystemic wasting syndrome in sweden and 1854 bp of its partial genome sequenced [27] . in 2010, the nearly complete genomes of distinct porcine bocaviruses provisionally named pbov1 and pbov2 were characterized from feces of swine in china [28] . finally, partial genome sequences of 2.4 kb from another clade of porcine bocaviruses labeled 6v and 7v where also identified yielding three major bocavirus groups in swine (pbo-likev, pbov1/pbov2, and 6v/7v). random amplification and sequencing has been used to discover novel virus in human and animal [8, 10, . in this study, we found in swine feces highly distinct bocavirus whose genome we tentatively named porcine bocavirus 1 (pbov1-h18), and porcine bocavirus 2 (pbov 2-a6). the nearly complete genomes of both viruses were acquired and are described here. pbov1-h18 and pbov2-a6 were also screened for in 340 stool samples of asymptomatic swine from five provinces of china. a total of 340 porcine stool samples from different aged swine were collected from 17 middle or large-scale porcine farms (200-2,000 sows each) in five provinces of china from april 2008 to october 2009, of which 80 were collected from four farms located in shanghai, 60 from three farms located in jiangsu province, 120 from six farms located in anhui province, 20 from a farm located in shandong province, and 60 from three farms located in guizhou province and stored at 280uc (table 1 ). one stool sample was randomly selected from each of the 17 farms. the samples were suspended in pbs (0.01 m phosphate, ph7.2-7.4, 0.15nacl), vortexed, centrifuged at 15000 g, and filtered through a 0.22-mm filter to remove eukaryotic-and bacterial-cell-sized particles [45, 46, 54, 55] . the filtrates were then treated with benzonase, dnase and rnase to digest non-particleprotected nucleic acid as reported [54, 55] . viral nucleic acids were then extracted using the tianamp virus dna/rna kit (tiangen biotech, beijing, co., ltd.). viral cdna synthesis was performed by incubation of the extracted viral rna/dna with 100 pmol of primer k-8n [56] with a degenerate 39 end and the use of superscript reverse transcriptase, and the opposite strand of the cdna was generated after melting and reannealing and primer extension using klenow dna polymerase [45, 46, [54] [55] [56] . pcr of extension products was performed as reported previously using the k-8 primer (k-8n without the degenerate 39 end) [54] . this protocol amplifies both viral rna and dna genomes [54, 55] . random rt-pcr dna products ran as smears on agarose gel and were gel purified (axygen, ca, usa), then subcloned into pmd-18t plasmid vector (takara, japan) for sequencing. the sequences were then screened for sequence similarities using tblastx and blastn against the nr database in genbank. sequences of each pcr product were assembled using seqman ii program (dnastar, inc). the identification of open reading the near-full genomes of pbov1-h18, pbov2-a6 and the partial ns1 and vp1 sequences from the diagnostic npcr have been deposited in genbank under accession numbers hq291308-hq291309 and hq291310-hq291343. seventeen porcine samples stool supernatants from 17 farms were analyzed using a generic viral particle-protected nucleic acid enrichment procedure followed by random amplification of extracted rna and dna (see materials and methods) [46, [54] [55] [56] . amplified dna was then subcloned and 1190 plasmid inserts were sequenced (70 for each of 17 samples). nine samples (totaling 82 subclones) showed the presence of fragments whose virtual translation products were related to canine and human bocaviruses using blastx. twenty-four clones from pig sample h18 and 26 clones from pig sample a6 showed significant similarity with bocaviruses. h18 derived sequences showed high (.99%) identity with the recently described porcine bocavirus-like virus (pbo-likev), the only porcine bocavirus reported at the time of these experiments (genbank gu902971) [27] . sequences from porcine sample a6 showed low identity with those of h18 and pbo-likev. the h18 and a6 samples were selected for targeted viral genome amplification and sequencing. the 24 sequences from h18 were assembled to form a continuous sequence of approximately 2700 nucleotides that appeared to lack .1300 and 1000 nucleotides from the 59and 39 ends of its genome. pcr primers based on the available h18 sequences and regions highly conserved between hbov2 (fj170278) and canine bocavirus (fj214110) were used to amplify the nearly complete bocavirus genome we provisionally called pbov1-h18 (5267 nt). to confirm this genome sequence, this sequence was re-amplified using 6 sets of pcr primers generating overlapping fragments of the genome which were directly sequenced. using the same method, the nearly complete bocavirus genome (5117 nt) from sample a6 was also sequenced and was provisionally labeled pbov2-a6. using an open reading frame (orf) finder (http://www.ncbi. nlm.nih.gov/gorf/gorf.html), three orf were found in both genomes (figure 1 ). the orfs of pbov1-h18 were 636 aa for ns1, 219 aa for np1 and 621 aa for vp1/vp2. the orfs of pbov2-a6 were 703 aa for ns1, 221 aa for np1 and 704 aa for vp1/vp2. the possible splicing of bocavirus ns1 transcripts recently shown to extend the length of ns1 proteins was not investigated here [6, 57] . nucleic acids were extracted from 340 porcine stool samples. pbov1-h18 related sequences were screened for using nested pcr with primers amplifying a 530-bp fragment of the np1/vp1 region. the electrophoretic bands of the expected size were subcloned and sequenced. the results showed that the prevalence of pbov1-h18 related viruses was high in china with 215 out of 340 (63.2%) porcine samples positive ( to determine the genetic relationship of pbov1-h18 and pbov2-a6 with recently described porcine bocaciruses and bocaviruses from other host species, both nucleotide and amino acid alignments were generated and used for phylogenetic analyses. when the whole genomes were considered porcine bocaviruses as a group (except for the v6/v7 variants with only np1 sequences available), were most closely related to the canine bocavirus cnmv (figure 2a ). phylogenetic analyses of the 3 orfs -ns1, np1 and vp1/vp2 -were also performed ( figure 2b-g) . in all three regions, pbov1-h18 was most closely related to the chinese pbov1 and pbov2 recently reported by cheng et al [28] . pbov2-a6 was closely related in np1 to the first reported partial porcine bocavirus sequence pbo-likev from sweden, the only region available for comparison (fig. 2c, f ) [27] . table 2 numerically shows the protein similarities between pbov1-h18 and pbov2-a6 and other porcine and non-porcine bocaviruses. the partial vp1 sequence of pbov2-a6 related variants from different farms was also phylogentically analyzed and fell into two major clades we named pbov2 genotype 1 and 2 (pbov2-g1 and pbov2-g2). the two previously described chinese pbov1 and pbov2 ''species'' grouped within these two genotypes. pbo-likev was originally found in swine with postweaning multisystemic wasting syndrome (pmws) in 2009 when approximately 35% of its genome sequence was reported [27] . the nearly full genomes of two distantly related porcine bocaviruses labeled pbov1 and pbov2 as well as two partial genomes labeled v6 and v7 were then reported in 2010 [28] . these bocaviruses grouped into 3 phylogenetic clades containing pbo-likev, pbov1/pbov2, and v6/v7. in the present study, we characterize two nearly complete bocavirus genomes, one of which (pbov1-h18) grouped with the pbo-likev clade while the second (pbov2-a6) fell with the pbov1/pbov2 clade. we provisionally named the genome from the h18 sample pbov1-h18 since its closest homologue, pbo-likev, was the first reported porcine bocavirus [27] . the virus from sample a6 was provisionally named pbov2-a6 since it phylogenetically groups with the second reported set of porcine bocaviruses containing both pbov1 and pbov2 [28] . no close homologues of the v6/v7 clade were identified in this study. under this proposed classification scheme, the viruses labeled pbov1 and pbov2 by cheng et al, therefore both belong to the pbov2 clade, the second reported clade of porcine bocaviruses [28] . under this proposed taxonomic classification, the v6 and v7 bocaviruses [28] belong to the still only partially characterized pbov3 clade. sequence analysis of the pbov2 clade showed that their vp1/ vp2 genes were highly diverse and could be classified into two genotypes ( figure 3 ). the partial np1 and vp1 genes of pbov1-h18 related viruses detected in this study were more highly conserved (99-100% identity), consistent with a recent report by zhai et al reporting pbov1 in chinese pigs using partial vp1/ vp2 nested pcr and sequencing [58] . zhai et al also found a high prevalence of pbov1 (69% in weanling piglets) with a higher frequency of pbov1 in animals also infected with pcv2, prrsv, pttv or csfv and in pigs with respiratory symptoms versus healthy pigs [58] . members of the bocavirus genus contain an 3 rd orf of unknown function labeled np1 gene. recently another parvovirus (ppv4) was identified in porcine feces that also contained a central 3 rd orf, although unrelated in sequence to the bocaviruses np1 [59] . none of the orfs of ppv4 clustered phylogenetically with the bocaviruses (data not shown) but instead clustered with members of the parvovirus genus [59] . ppv4 is therefore unrelated to the bocaviruses reported here. hbov1 has been associated with respiratory symptoms while other hbov may be associated with diarrhea and acute flaccid paralysis [5, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] 25] . a gorilla bocavirus was detected in captive animals in the us experiencing severe diarrhea [6] . pbov1 was found in pigs with pmws in sweden [27] and in pigs with respiratory tract symptoms in china [58] . in the present study, both pbov1 and pbov2 were highly prevalent in both asymptomatic swine from five provinces of china. further studies are needed to examine possible associations between infections with these different porcine bocaviruses, the viral loads excreted, the presence of co-infections and various porcine diseases. virus taxonomy: the eighth report of the international committee on taxonomy of viruses animal bocaviruses: a brief review complete nucleotide sequence and genome organization of bovine parvovirus recovery and characterization of a minute virus of canines human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections identification and characterization of a new bocavirus species in gorillas widespread infection of chimpanzees and gorillas with homologues of human parvoviruses b19, parv4 and human bocavirus in the wild cloning of a human parvovirus by molecular screening of respiratory tract samples a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool human bocavirus human bocavirus and acute wheezing in children detection of human bocavirus in canadian children in a 1-year study human bocavirus infection in hospitalized children during winter bocavirus infection in hospitalized children human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus respiratory infections in children clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong human bocavirus infection in children with gastroenteritis role of human bocavirus infections in outbreaks of gastroenteritis human bocavirus-a novel parvovirus to infect humans no gastroenteric bocavirus in high risk patients stool samples human bocavirus infection in children with acute gastroenteritis and healthy controls human bocavirus in an immunocompromised child presenting with severe diarrhea the first detection of human bocavirus 2 infections in china frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the united states detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome identification and nearly full-length genome characterization of novel porcine bocaviruses identification of a third human polyomavirus a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species cardioviruses are genetically diverse and cause common enteric infections in south asian children genetic detection and characterization of lujo virus, a new hemorrhagic feverassociated arenavirus from southern africa identification of cardioviruses related to theiler's murine encephalomyelitis virus in human infections a metagenomic survey of microbes in honey bee colony collapse disorder viral metagenomics clonal integration of a polyomavirus in human merkel cell carcinoma complete genome sequence of a highly divergent astrovirus isolated from a child with acute diarrhea identification of a novel polyomavirus from patients with acute respiratory tract infections the complete genome of klassevirus-a novel picornavirus in pediatric stool klassevirus 1, a previously undescribed member of the family picornaviridae, is globally widespread novel borna virus in psittacine birds with proventricular dilatation disease new adenovirus species found in a patient presenting with gastroenteritis new dna viruses identified in patients with acute viral infection syndrome discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin a highly divergent picornavirus in a marine mammal a highly prevalent and genetically diversified picornaviridae genus in south asian children recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent genomic characterization of novel human parechovirus type a new arenavirus in a cluster of fatal transplant-associated diseases characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r462q rnasel variant a newly discovered human pneumovirus isolated from young children with respiratory tract disease rapid identification of known and new rna viruses from animal tissues metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis characterization of virus isolates by particle-associated nucleic acid pcr characterization of the gene expression profile of human bocavirus high prevalence of a novel porcine bocavirus in weanling piglets with respiratory tract symptoms in china identification and molecular cloning of a novel porcine parvovirus key: cord-005789-jngjusk2 authors: selden, richard f; skośkiewicz, marek j.; howie, kathleen burke; russell, paul s.; goodman, howard m. title: regulation of human insulin gene expression in transgenic mice date: 1986 journal: nature doi: 10.1038/321525a0 sha: doc_id: 5789 cord_uid: jngjusk2 insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1, 2). it is synthesized by the (β)-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. insulin consists of two polypeptide chains (a and b, linked by disulphide bonds) that are derived from the proteolytic cleavage of proinsulin, generating equimolar amounts of the mature insulin and a connecting peptide (c-peptide). humans, like most vertebrates, contain one proinsulin gene(3,4), although several species, including mice(5) and rats(6,7), have two highly homologous insulin genes. we have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. we report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent. comparison of huv np and mv np iges in binding inhibition assays. various concentrations of huvnp-ige (e) and mvnp-ige (0) were used to compete the binding of radiolabelled mvnp-ige to polyvinyl microtitre plates that had been coated with a, sheep anti-human e antiserum (seward laboratory); b, (nip-caph4-bsa; c, ac38 antiidiotypic antibody; d, ac146 anti-idiotypic antibody; e, rabbit anti-mv np antiserum. binding was also carried out in the presence of mvnp-igm antibody jw1/2/2 (ref. 32) (_) as well as in the presence of jw5/1/2 (0), which is an igm antibody that differs from jw1/2/2 at 13 residues mainly located in v h cdr2 (m.s.n., unpublished results). values of binding are relative to the binding in the absence of inhibitor. between .a-strands depends on loop size and specific interactions of the loop back to the .a-sheet. however, in the same class of variable domains (v h, v k or va) these interactions are usually conserved (ref. 5 and a. m. lesk and c. chothia, personal communication). while human monoclonal antibodies have therapeutic potential in human disease, they can be difficult to prepare 17 and treatment of patients with mouse monoclonal antibodies often increases the titre of circulating antibody against the mouse immunoglobulinl8. as chimaeric antibodies containing human constant domains l2 ,19.20 and variable domains made by grafting mouse cdrs into human frs, could have therapeutic potential, we wondered whether the huvnp-ige antibody loses antigenic determinants associated with the mv np variable region (idiotopes). the binding of huvnp-ige and mvnp-ige to both monoclonal and polyclonal anti-idiotypic antibodies directed against the mv np domain was examined by using inhibition assays. as shown in fig. 3d , the huvnp-ige antibody has lost the mv np idiotypic determinant recognized by antibody ac146 (ref. 21) , furthermore, huvnp-ige also binds the antibody ac38 (ref. 21 ) less well (fig.3c) , therefore it is not surprising that huvnp-ige has lost many of the determinants recognized by a polyclonal rabbit anti-idiotypic antiserum (fig. 3e) . while the loss of idiotypic determinants that accompanies 'humanizing' of the v h region is reassuring in view of potential therapeutic applications, it does suggest that the recognition of the hapten and of anti-idiotypic antibodies is not equivalent. thus the huvnp-ige antibody retains hapten binding but has lost idiotypic determinants, indicating that the immunoglobulin uses different sites to bind hapten and anti-idiotypic antibodies. it appears, therefore, that both fr and cdr side chains form the binding site for these anti-idiotopes, but mainly cdr side chains interact with hapten. we thank c. milstein for suggesting this project, k. rajewsky and m. reth for the anti-idiotypic antibodies ac38 and ac146, and a. m. lesk, c. chothia, r. j. leatherbarrow and c. milstein for helpful discussions. j.f. is a fellow of the jane coffin childs memorial fund for medical research. received insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1, 2). it is synthesized by the il-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. insulin consists of two polypeptide chains (a and b, linked by disulphide bonds) that are derived from the proteolytic cleavage of pro insulin, generating equimolar amounts of the mature insulin and a connecting peptide (c-peptide). humans, like most vertebrates, contain one proinsulin gene 3 ,4, although several species, including mice! and rats 6 ,7, have two highly homologous insulin genes. we have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. we report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent. the human insulin gene that we have used to generate transgenic mice is contained within a 12.5-kilobase (kb) ecori fragment that was isolated from a genomic library4. of 46 mice born after one series of single-cell embryo microinjections, three contained human insulin gene sequences as detected by southern hybridization analysis (fig. 1a) . a human c-peptide radioimmunoassay (behringwerke) was used to monitor expression of the human insulin gene in the transgenic mice and their offspring. several hundred transgenic and control mice have been analysed under a variety of physiological circumstances (see below), and the transgenic mice show variable levels of human c-peptide in their sera, whereas control mice show no such expression. the tissue specificity of human insulin gene expression in these transgenic mice was examined by both rna analyses and pancreatic islet function studies. northern hybridizations using a human insulin complementary dna probe demonstrated that total pancreatic rna from both transgenic (fig. 1b , lane 11) and control (lane 1) mice hybridized to the human insulin cdna probe, whereas rnas from transgenic spleen, kidney, brain, lung, liver, salivary gland, intestine, heart and muscle (lanes 2-10, respectively) showed no hybridization. because no transgenic tissues other than pancreas were found to express detectable levels of insulin rna, insulin expression in both transgenic and control pancreas was studied to determine whether the transgenic pancreas is a site of human insulin expression. pancreatic islets from six transgenic and six control mice were isolated by collagenase digestion as described previously8,9 and cultured in groups of 80-100 islets per tissue culture well. the following day, aliquots of media were taken and human cpeptide levels measured. the samples from the transgenic islet wells contained 250-650 ng ml-1 of human c-peptide, but the control islet wells contained no detectable human c-peptide. the cultured transgenic islets continued to express human cpeptide for several days. from these experiments, we conclude that the major site of human insulin expression in these transgenic mice is the endocrine pancreas. transgenic and control pancreas were stained with immunoperoxidase using a guinea pig anti-porcine insulin antibody and a goat anti-human c-peptide antibody (fig. 2) . the anti-porcine insulin antibody cross-reacted with both human and mouse insulin, and islets from both transgenic (fig. 2a) and control (fig. 2b) mice were stained. the size, distribution and number of islets were essentially the same in transgenic and control mice. the anti-human c-peptide antibody showed little or no cross-reactivity with mouse c-peptide, however, and the transgenic islets (fig. 2c) were stained using this antibody whereas the control islets (fig. 2d) were not. these immunohistochemistry data are consistent with the northern analysis and islet function studies presented above, and demonstrate that the transgenic islets were specifically expressing human insulin. glucose and human c-peptide levels in the transgenic mice were studied under a variety of physiological conditions to determine whether normal glucose homeostasis was being preserved and whether expression of the human insulin gene was being regulated appropriately in these mice. blood glucose regulation was studied by glucose tolerance tests. transgenic offspring of mouse 16 and non-transgenic siblings were fasted overnight, given an intraperitoneal (i.p.) injection of glucose, and bled at various times after injection to determine serum glucose levels. the glucose tolerance curve from the transgenic mice was similar to that from the control mice (fig. 3a) . of particular importance is the finding that the fasting and maximally stimulated glucose levels as well as the kinetics of neither the mouse insulin cdna nor gene sequences have been determined, but since the coding re~ions of the human and rat insulin mrnas share 81 % sequence homology 5, it was expected that both transgenic and control pancreatic rnas would hybridize to the human insulin cdna probe. methods. a 12.5-kb ecori fragment containing the human insulin gene' was isolated from pbr322 sequences by preparative gel electrophoresis and electroelution l6 . fertilized mouse eggs for microinjection were recovered in cumulus from the oviducts of (c57 x c3h)f i females that had mated with fi males several hours earlier. approximately 1,000 copies of the human insulin gene fragment were microinjected into the male pronucleus of each fertilized egg. microinjected eggs were implanted into the oviducts of i-day pseudopregnant icr foster mothers and carried to term 17 • several weeks after the birth of animals that developed from microinjected eggs, total genomic dna was prepared l8 from mouse tails. for the southern blot analysis in a, 8 flg of total genomic dna for each mouse were digested with pvuii, subjected to electrophoresis on a 0.9% agarose gel and transferred to nitrocellulosel 9 • the filter was then prehybridized overnight, hybridized to a 32p_iabelled genomic human insulin probe, washed and exposed to x-ray film. the genomic human insulin probe is a fragment that extends from a bglii site at -169 with respect to the transcriptional start site of the human insulin gene to an avai site at +644 (ref. 4 ). in addition to promoter sequences, this fragment contains the first intervening sequence, the first exon (including sequences encoding the signal peptide, b-peptide and a portion of c-peptide), and a portion of the second intervening sequence. approximately 75% of the genomic human insulin probe consists of non coding sequences that are not highly conserved between species, which explains its limited cross-hybridization with the endogenous mouse insulin genes (which can be detected only after long exposures). total cellular rna was isolated from tissue samples by the guanidinium isothiocyanate/ caesium chloride technique 20 . for northern blot analysis, 4 flg of total rna from each sample was subjected to electrophoresis on a 1.2% agarose-formaldehyde denaturing gel and transferred to nitrocellulose filters l6 . the filter was then prehybridized, hybridized to a 32p_iabelled human insulin cdna is probe, washed and exposed to x-ray film. fig. 2 immunoperoxidase staining of transgenic and control pancreas. pancreas samples were immersed in liquid nitrogen and 4-llm tissue sections were prepared using a cryostat. immunoperoxidase staining of serial sections was performed as described previousll 1 , using either guinea pig anti-porcine insulin antibody (a and b) or goat anti-human c-peptide antibody (c and d). a, transgenic pancreas stained with anti-insulin antibody (arnel products). b, control pancreas stained with antiinsulin antibody. c, transgenic pancreas stained with anti-human cpeptide antibody (behringwerke). d, control pancreas stained with antihuman c-peptide antibody (photographically enhanced to allow visualization of islets). other transgenic tissues, including liver, adrenal and thyroid, are not stained using the guinea pig anti-porcine insulin antibody (not shown). data are similar to glucose tolerance results previously reported for both mice 22 and humans 2 . preliminary experiments suggest that mouse 20 also expresses human c-peptide and responds to a glucose tolerance test, and more extensive studies will be performed when mouse 20 and mouse 38 have generated large colonies. an assay specific for mouse c-peptide is not available, and we were unable to compare levels of endogenous mouse c-peptide with levels of human c-peptide. the return to basal glucose levels were similar for the transgenic and control animals. in addition, intravenous (i.v.) administration of glucagon increased serum glucose levels by -50% within 15 min in both transgenic and control mice. taken together, these results strongly suggest that serum glucose levels were appropriately modulated in the transgenic mice. the weights of the transgenic mice, growth rates, feeding behaviour, reproductive capability and longevity appeared normal. the role of human insulin in the regulation of blood glucose levels in transgenic mice was investigated by performing a glucose tolerance test on transgenic and control mice (fig. 3b) . no human c-peptide was detected in the sera of fasting transgenic mice, but within 10 min of i.p. administration of glucose, human c-peptide appeared in the serum, and peak levels were attained within -20 min. by 45 min post-glucose, human cpeptide levels fell to values approaching the pre-stimulation or basal level. this pattern of human c-peptide expression correlates closely with the glucose tolerance curves presented above, and suggests that serum human insulin levels were being appropriately regulated by glucose. the control mice did not express any detectable human c-peptide, indicating that the human gene must have been the source of the human c-peptide in the transgenic animals. insulin is regulated by several other factors, including amino acids and certain pharmacological agents. an i.v. amino-acid infusion test was performed on fasting transgenic and control mice and human c-peptide levels in the serum were determined. peak human c-peptide levels were seen within 5 min of aminoacid infusion and declined gradually over the next 40 min (fig. 3c) . similarly, serum human c-peptide levels responded to tolbutamide, a sulphonylurea derivative known to promote insulin release lo (fig. 3d) . within 20 min of i.v. tolbutamide administration, serum human c-peptide levels peaked, then decreased rapidly over the next 10 min. tolbutamide has been used clinically to diagnose insulinomas ll because in normal subjects serum insulin (or c-peptide) levels rapidly return to normal from their tolbutamide-induced peak, but in insulinoma patients elevated insulin levels persist. that the transgenic mice quickly regained basal serum human c-peptide levels supports the conclusion that their insulin expression was tightly regulated. we have demonstrated that the human insulin gene is expressed in the pancreas of transgenic mice. cell-type-and tissue-specific expression of h uman 12 and rae z -14 insulin genes has been documented in two other laboratories. a 230-base-pair (bp) region (from -103 bp to -333 bp with respect to the transcriptional start site) of the rat insulin i promoter was reported to be sufficient to allow tissue-specific expression of insulin/ chloramphenicol acetyltransferase fusion genes in a hamster pancreatic cellline lz .i3. similarly, a rat insulin ii/ simian virus 40 large-t antigen fusion gene has been reported to cause the development of islet cell tumours in transgenic mice l4 • as both of these studies used fusion genes, the regulation of circulating human insulin could not be studied. serum insulin levels are regulated by glucose, amino acids, proteins and drugs such as the sulphonylurea derivatives. the human insulin gene in these transgenic mice is regulated appropriately by all of these agents, and serum glucose homeostasis is normal. these transgenic animals can therefore now be used to study several critical aspects of the physiological regulation of insulin gene expression, including the mechanisms controlling serum insulin and total f3-cell insulin levels. because at least one additional insulin gene is being expressed in the transgenic mice and total insulin rna and protein levels are approximately the same as in control mice, the question of dosage compensation can be investigated. moreover, our tolbutamide results indicate that drugs thought to affect human insulin metabolism can now be tested in an in vivo animal system. in a more general sense, the in vivo effects of various pharmacological agents on human gene expression and protein function can therefore be evaluated in a non-human setting. finally, it is noteworthy that a 12.5-kb dna fragment contains sufficient information for the appropriate physiological regulation of insulin levels in these transgenic mice. the organism's ability to modulate foreign dna sequences and proteins on a minute to minute basis clearly has important implications for both molecular biology and gene therapy. we thank dr tom wagner for instruction in microinjection, victoria roman for technical assistance and patrick mattoon for animal care. the human c-peptide assay was the gift of dr h. h. schoene and behringwerke, ag. this work was supported by grants from hoechst ag and the nih (am-07055). genetic recombination of dna is one of the fundamental mechanisms underlying the evolution of dna-based organisms and results in their diversity and adaptability. the importance of the role of recombination is far less evident for the rna-based genomes that occur in most plant viruses and in many animal viruses. rna recombination has been shown to promote the evolutionary variation of picornaviruses l -4, it is involved in the creation of defective interfering (di) rnas of positive-and negative-strand virusess--9 and is implicated in the synthesis of the messenger rnas of influenza virus lo and coronavirus ll • however, rna recombination has not been found to date in viruses that infect plants. in fact, the lack of di rnas and the inability to demonstrate recombination in mixedly infected plants has been regarded as evidence that plants do not support recombination of viral rnas. here we provide the first molecular evidence for recombination of plant viral rna. for brome mosaic virus (bmv), a plus-stranded, tripartite-genome virus of monocots, we show that a deletion in the 3' end region of a single bmv rna genomic component can be repaired during the development of infection by recombination with the homologous region of either of the two remaining wild-type bmv rna components. this result clearly shows that plant viruses have available powerful recombinatory mechanisms that previously were thought to exist only in animal hosts, thus they are able to adapt and diversify in a manner comparable to animal viruses. moreover, our observation suggests an increased versatility of viruses for use as vectors in introducing new genes into plants. molecular cloning: a laboratory manual proc. natn. acad. sc' us.a. 78 key: cord-016657-w30hed7w authors: blatt, amy j. title: geographic medicine date: 2014-09-29 journal: health, science, and place doi: 10.1007/978-3-319-12003-4_8 sha: doc_id: 16657 cord_uid: w30hed7w this chapter uses a sub-discipline of medicine, known as geographic medicine, to describe how human movements contribute to the transmission of parasites on spatial scales that exceed the limits of its natural habitat. traditionally, public health programs have focused on the health of populations, whereas the practice of medicine has focused on the health of individuals. it should be noted, however, that the population health management owes much to the effective delivery of clinical care. this chapter demonstrates how public health is intimately linked to patient care through human movement. nearly a century ago, people typically did not develop a disease where it is contracted or even close to that place. today, daily travel is a common way of life in modern metropolitan areas. large, localized mosquito populations in areas that people visit regularly may be both reservoirs and hubs of infection, even if people only pass through those locations briefly. by examining of the role of human movement across different scales, this chapter examines how public health communities can use information on pathogen transmission to increase the effectiveness of disease prevention programs and clinical care. in 2008, the united states' institute of medicine convened an expert committee on the u.s. commitment to global health and reaffi rmed the notion that local health and local health care are linked to sources of disease and disability occurring elsewhere in the world [ 1 ] : the last three chapters of this book will illustrate the importance of geography in the practice of medicine, both on a local and global scale. the current chapter will highlight the role of geographic forces in the fi eld of travel medicine (also known as geographic medicine) and emphasize the role of geographic knowledge and thought in a study of disease that views humans has vectors and hosts. chapter 9 will introduce a relatively new discipline, called geospatial medicine, that utilizes the advances in geospatial data and technology to uncover the genetic, social, and environmental effects of disease as it occurs throughout human development. this is an exciting new fi eld in which there is a tremendous potential for geographers to take center stage in answering some of the hard research questions that scientists have been grappling with for a long time. lastly, chap. 10 puts the fi nishing touches on the new portrait of medical geography that we have been painting, and frames it in a more centralized role on the national stage of the u.s. health care reform. a main objective of this book is to present a new model of patient care that emphasizes the patient's geographic and medical history, against the backdrop of contemporary globalization, while taking advantage of the current advances in geospatial data and technologies. the current chapter will describe the main principles of travel medicine, and emphasize how human movements contribute to the transmission of parasites on spatial scales that exceed the limits of its natural habitat. studies have shown that the ability to identify the sources (origins) and sinks (destinations) of imported infections due to human travel and locating the high-risk sites of parasite importation could greatly improve the control and prevention programs [ 3 , 4 ] . for instance, a group of researchers have combined mobile phone data with a high-resolution malaria prevalence map to analyze the regional travel patterns of nearly 15 million individuals over the course of a year in kenya, in the context of parasitic dispersals. in this way, they were able to identify and map the sources and sinks of human and parasite travel in this country ( fig. 8.1 ) [ 5 ] . to describe the challenges of travel medicine on human terms -in 2013, an estimated 1.1 billion travelers crossed international borders, including an estimated 28.5 million u.s. travelers [ 6 , 7 ] . an increase in the number of international travelers and local commuters has caused a surge of global and regional epidemics in the past several decades [ 8 ] . travel-related risks include infections from food, vectors, and bodily fl uids. most travelers (20-70 %) report health problems while traveling, but many do not seek pre-travel advice -such as vaccinations, prophylactics, and therapeutic medications [ 9 , 10 ] . moreover, the risk of travel-related diseases is 2.3 times higher in those with an underlying medical conditions than in health individuals [ 10 ] . given the unique health situations of travelers, there is a need for better characterization of the geographical and environmental risk factors underlying travel-related illnesses. to address these concerns, this chapter describes how human travelers who are infected can serve as an important route for the transmission of the virus from their place of origin to their place of destination. by understanding the effects of human movement on disease transmission, researchers can appropriately identify high-risk areas for effective intervention and control. historically, epidemiologists have viewed human movement from two main perspective. the fi rst perspective is from that of the populations of susceptible hosts moving into high-risk areas. the second perspective is from that of populations of infected hosts moving into susceptible populations. movements of infected hosts across different spatial scales affect pathogen transmission in a variety of ways. noted historian and geographer r. mansell prothero published one of the fi rst studies describing the role of human movements in epidemiology based on his experience in africa, in 1977. drawing on the geographic literature concerning diffusion and migration processes, he discussed the relevance of these movement patterns to public health in his seminal paper published in the international journal of epidemiology . he carefully outlined the differences between circulatory and migratory movements and categorized these movements by their spatial scale (i.e., a rural-urban gradient) and temporal scale (i.e., the time and timing of displacements). circulatory movements are those in which the individuals return home after some period, and migratory movements are those which usually result in permanent changes of residence. prothero's argument was that knowledge of the nature of these movements would help inform the public's understanding of the incidence and prevalence of disease on a population level and provide informed options for control [ 11 ] . for instance, seasonal migratory movements from one rural area to another for agriculture could potentially expose individuals to different areas where the risk of african trypanosomiasis, or malaria, is high [ 12 ] . at broad spatial scales (e.g., national, international), individual movements can drive pathogen introduction and reintroduction. for example, the global spread of dengue virus via shipping routes was characterized by periodic, large, spatial displacements. globalization and mass air transportation have changed the transmission of pathogens by dramatically shortening the time required to travel around the earth. at fi ner scales (e.g., regional, urban-rural, intra-urban), movement associated with work, recreation, and transient migration into high-risk areas not only lead to individual infection, but also contribute to local transmission when infected hosts return home and infect other individuals ( fig. 8. 2 ) [ 13 ] . indeed, vector-borne diseases place an enormous burden on public health and require effi cient control strategies that are developed through an understanding of the origin (or sources) of infections and the relative importance of human movement at different scales. a number of social and environmental factors -such as human population density, regional settlement patterns, population movements, precipitation, and other weather-related factors -contribute to local and regional transmission dynamics [ 14 ] . human movement -which determines exposure to vectors -is a key aspect of vector ecology that is poorly understood. this is due to the variations in exposure based on individual host movement that can strongly infl uence the pathogen's transmission dynamics [ 15 ] . the types of movement most relevant for exposure will depend on site-specifi c differences, the ecology of the arthropod vector, human behavior, and the relative scale of host and vector movement. for instance -although fi ne-scale host movements are not important to pathogens transmitted by vectors that are able to move long distances in search of a host -these fi ne-scale host movements are very important for pathogens transmitted by sessile vectors. aedes aegypti is the principal vector of dengue virus. it bites during the day, disperses only short distances, and is heterogeneously distributed within urban areas. humans, on the other hand, move frequently and allocate different amounts of time to multiple locations on a regular basis. not only does this infl uence the individual risk of infection with dengue virus but it also infl uences overall patterns of transmission [ 16 ] . in addition, commuting and non-commuting patients have different diffusion patterns and determinants in a dengue epidemic. non-commuters (e.g., elderly adults and housewives) may initiate a local epidemic, whereas commuters carrying the virus to geographically distant areas can cause a large-scale epidemic [ 17 ] . dengue is a global threat and is endemic or epidemic in almost every country located in the tropics (fig. 8.3 ). while new tools (such as vaccines, antiviral drugs and improved diagnostics) are being developed, better use should be made of the interventions that are currently available. along with comprehensive tracking of commuting cases, the concomitant rapid notifi cation and diagnosis of non-commuting cases can enable the appropriate interventions and faster response times, thus preventing subsequent large-scale epidemics. as illustrated in the previous sections of this chapter, human movement is a largely ignored variable in the study and diagnosis of pathogen exposures. there are few, if any, well-established clinical guidelines that utilize a person's place history in the diagnosis of infected individuals, despite the fact that disease management protocols can be made more effective and effi cient by targeting the sources or agents of transmission. the study of human movement is critical to identifying high-risk factors (e.g., hostpreferences) because these factors are always conditioned by exposure rates which are, in turn, closely related to variations in human movement and behavior. although geographical movement is becoming increasing easy to measure through advanced geospatial technologies, a patient's movements and place history have been largely ignored by the medical community. quantifying and describing human movements provides valuable information necessary to predict disease outbreaks and to evaluate control alternatives to halt epidemics [ 18 -20 ] . in addition, the ability to apply this knowledge to a variety of diseases creates an opportunity to identify common areas where infection occurs across multiple diseases and to leverage public health programs to target the most important locations that serve as sources for more than one disease. by examining of the role of human movement across different scales, public health communities can use this valuable information on pathogen transmission to increase the effectiveness of disease prevention programs. as transmission rates are reduced through intervention efforts, scientists can expect the importance of heterogeneity in exposure to increase and play an even more important role in pathogen persistence. therefore, characterization of human and population movements can facilitate not only the elimination of disease, but also help to prevent its return [ 15 ] . key considerations in the analysis of human movement patterns include, but are not limited to: spatial scale, the type and periodicity of movement, and the time period of observation. spatial scale refers to the geographical extent of the pathogen and the spatial interpretation of the data. the geographical extent of the pathogen and its transmission can be determined by the disease dynamic -i.e., the spread of a pathogen to new geographical areas versus a sustained transmission at a given location. if the transmission is local, then the relevant movements will be those placing the susceptible hosts in high risk locations at times when infection risk is high. assumptions regarding the importance of movements should be made with care because heterogeneity in exposure can have a dramatic effect on infection risk. the type of movement refers to what the researcher is aiming to measure. for instance, is the study interested in the sites where individuals spend their time on a 8.3 geographic considerations in human movement regular basis (high spatial and temporal resolution) or when they are traveling outside of their home city? what is the value of travel information (outside of an urban area) that specifi es exactly where people go? are specifi c routes important, or should only destinations be considered? these specifi c details will depend on the nature of the questions, systems, and resources and methods involved for measuring movements. finally, the time period of observation is concerned with how long to observe individual movements for. the correct answer will depend on the questions being asked and available resources. in the case of dengue, infection can occur up to 2 weeks prior to the manifestation of symptoms. for a retrospective study, 14-15 days would be an appropriate observation period. conversely, in a prospective study, the length of the observation period will depend on the relative importance of rare movements. studies of human movements in developed societies reveal markedly regular patterns, especially during the work-week [ 19 , 21 , 22 ] . additionally, there may be significant instability in movements on weekends or at other times (e.g., vacations). for regular movements during the work week, at least 2 weeks of observation are desired. for more variable movements/times, longer observation periods will be necessary. over the last century, human civilizations and urbanization have witnessed a huge increase in mobility and population growth. the combined effect of these factors means that, despite great improvements in hygiene, sanitation and vector control, the containment of disease remains one of the biggest challenges of our modern contemporary society. the importance of increased human mobility in disease transmission cannot be under-stated. on national and global scales, the airline transport network has played a key role in the global dissemination of infl uenza and sars [ 23 ] . in addition, migrants, tourists, and commercial travelers also have a signifi cant infl uence on the spread of hiv [ 24 ] . on a local scale, human movements in metropolitan areas are frequent and extensive, but are often composed of highly structured commuting patterns between the home and places of employment, education, or business. the ability to quantify human movement and its effects are vital in the ongoing development of strategies to eradicate vector-borne diseases (such as dengue) from urban centers [ 25 ] . using a meta-population analysis where mobile humans connect with static mosquito subpopulations in a very structured pattern, a group of researchers found that, due to frequency dependent biting, infection incidence in the human and mosquito populations is independent of the duration of contact [ 26 ] . the researchers hypothesize that, since the biting rate is frequency dependent (and independent of the density of the human population), a mosquito will bite the same number of people per day. in addition, their modeled results indicate that people who travel regularly to areas with large mosquito populations form a high-risk group, and have a relatively high level of infection compared to people that travel regularly to patches with small mosquito populations. furthermore, extensive variation in human movement patterns causes the number of interactions between human and mosquito populations to increase. more variable human movements increases the likelihood that people will carry the infection from these highly infested areas to mosquito subpopulations where the pathogen has died out. therefore, a large mosquito population in a frequently visited area may be suffi cient to ensure infection is endemic, even if there are relatively few mosquitoes elsewhere. when people do not vary their travel patterns very much and there is no direct connectivity between mosquito populations, the transit corridor can signifi cantly enhance disease persistence by acting as a reservoir and hub. if people vary the areas they visit even occasionally, the effect of the transit corridor is overridden [ 26 , 27 ] . mosquito and human movements become even more important as remote rural villages are connected to each other by mass transportation networks. nearly a century ago, it was observed that people do not develop a disease where it is contracted or even close to that place [ 28 ] . today, widespread mass transportation makes that observation even more relevant. the incidence and persistence of vector-borne diseases on relatively small spatial scales may be strongly infl uenced by infectious humans who remain mobile because the infection is mild or silent. increased human movement on a local scale may be a key factor behind increased incidence of vector-borne diseases. in modern metropolitan areas, daily travel is a common way of life. distant subpopulations of mosquitoes may be connected by this daily movement. large, localized mosquito populations in areas that people visit regularly may be both reservoirs and hubs of infection, even if people only pass through those locations briefl y. this implies that surveillance with the goal of controlling vector-borne disease may be a much greater challenge than originally anticipated. ultimately, successful public health intervention must focus on both hosts and vectors. large mosquito populations that are also visited by a large fraction of the human population need to be identifi ed. it is vital to employ surveillance strategies that reveal the variability in the distribution of mosquitoes and target areas where the mosquito population is signifi cant and human movement is extensive. further study of networks formed by human movement in urban areas are need, and cell phone records are one potential source of such detailed information [ 19 ] . committee on the u.s. commitment to global health. the u.s. commitment to global health: recommendations for the public and private sectors a global view of health -an unfolding series stable and unstable malaria hotspots in longitudinal cohort studies in kenya international population movements and regional plasmodium falciparum malaria elimination strategies quantifying the impact of human mobility on malaria international tourism results and prospects for international trade administration (2013) u.s. travel to international destinations increased three percent in 2012. department of commerce, international trade administration airports, localities and disease: representations of global travel during the h1n1 pandemic advising the traveler health risks of travelers with medical conditions -a retrospective analysis disease and mobility: a neglected factor in epidemiology population mobility and trypanosomiasis in africa dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem climate change and risk projection: dynamic spatial models of tsetse and african trypanosomiasis in kenya the role of human movement in the transmission of vector-borne pathogens using geographic information systems to analyze the distribution and abundance of aedes aegypti in africa: the potential role of human travel in determining the intensity of mosquito infestation population movement and vector-borne disease transmission: differentiating spatial-temporal diffusion patterns of commuting and non-commuting dengue cases modelling disease outbreaks in realistic urban social networks understanding individual human mobility patterns large-scale spatial-transmission models of infectious disease what about people in spatial science habitual travel behaviour: evidence from a six-week travel diary the role of the airline transportation network in the prediction and predictability of global epidemics travel and the spread of hiv-1 genetic variants dengue and dengue hemorrhagic fever man bites mosquito: understanding the contribution of human movement to vector-borne disease dynamics household and community transmission parameters from fi nal distributions of infections in households stegomyia indices and their value in yellow fever control key: cord-017537-ztdz4a2s authors: bologna, mauro title: biological agents and bioterrorism date: 2014-09-18 journal: detection of chemical, biological, radiological and nuclear agents for the prevention of terrorism doi: 10.1007/978-94-017-9238-7_1 sha: doc_id: 17537 cord_uid: ztdz4a2s for this very stimulating course, i want to share with you some of my studies and even some of my scientific and phylosophical considerations on biological agents living in the environment and their relations with humans, in the very wide concepts of ecological relationships, parasitism, immunolgical defenses and infectious disease mechanisms. all these concepts must be studied and considered in the event of criminal use of biological agents (bioterrorism) aimed at harming human populations in time and in geographical space. terrorism is the use of violence to condition societies or governments in their political choices. bioterrorism is the use (or menace of use) of biological agents to enact terrorism events and induce generalized fear concerning negative consequences in target populations. (a) use of poison darts/arrows (primitive populations): mostly for hunting, but also for battles against enemies. from here derived many of the advances of toxicology, the science of toxic substances (the word "toxicology" derives from the greek words "toxon" = arch and "farmakon" = poison; toxicon farmakon = poison for arch hunting): (b) from such practice derives the knowledge we have of stricnin, curare, ouabain, aconite, other plant/animal poisons primarily devised for hunting and fi ghting. (c) impingement of darts in decomposing cadavers or putrefaction soil (or manure >> tetanus) before throwing at enemies (new guinea, tribal combats; sciites 400 b.c.) (d) last but not least, the use of fear that humans have of beasts. here comes the example of hannibal (from carthago), leading the ships of prusia i, king of bithynia (west turkey) in a battle of year 184 b.c. against eumene ii (attalides, pergamon); he won that naval battle because he managed to throw canisters full of reptiles at the enemy ships, causing terror and uncoordinated reactions leading to his victory. he therefore used fear as a weapon: snakes were not even harmful (not poisonous), but big was the surprise and reactions were out of control! (e) in recent history, we can see that first world war (also named the war of chemistry) contributed to the development and use of nervine gases , chemical weapons banned everywhere but still existing in some countries. second world war (also named the war of physics) led to the development and use of the atomic bomb. thepeace treaties: geneva protocol ruled against chemical weapons (1925) and was followed later by additions concerning bacteriologic war. not all the states however subscribed it. most states anyway have banned, in time, chemical and bacteriological weapons by 1975. today, how scared should we be of biological and chemical terrorism? well, since these are lethal and cheap weapons, they are of considerable concern, because they may be seen as the atomic bomb of poors and represent remarkable threats to peace in local confl icts and in terroristic attacks worldwide. we should all know more on the subject and do extensive prevention. albert einstein once said "i do not know by what weapons the third world war will be fought, but for sure the fourth will be fought with stones". well, we do not want any more world wars, for sure, period. life began on earth with unicellular beings: primitive bacteria and algae, ∼3.5 billion years ago (archean age). biological evolution of species produced today's forms of life, as we observe them. millions of species co-exist and share the stage (biosphere). humans (we) pretend to have absolute priority, but … share the stage in such a crowded environment on earth means to learn, respect, understand and prevent. on this planet we have millions of different species of live beings, with variable proportions in the biosphere and in different ecosystems: they are all co-existing, interacting and competing for food and survival. this encompasses the very universal phenomena of competition and of parasitism. one of the most recent and precise evaluations of the number of species existing on planet earth (2012), but still very approximate and provisional, (well illustrated and summarized in national geographic, 2013) fi nds evidence of more than 5,000 species of mammals, 10,000 species of birds, 12,000 species of reptiles, 15,000 species of amphibians, 45,000 species of fi sh, 150,000 species of crustaceans, 200,000 species of mollusks, 600,000 species of aracnids, fi ve million species of insects and many, many millions (unestimable indeed) species of bacteria, viruses and other microrganisms. are we many on earth? is there enough work for the immune system of each living multicellular organism to distinguish "self" from possibly harmful "not self"? species of microrganisms ascertained as pathogenic for humans are indeed a very small fraction of the existing species: we come to know them better because we study the diseases connected with them, but we ignore a lot about the great number of other, presumably innocuous species. in general, different species interacting may set a parasitic relationship , in which the larger animal ( the host ) may receive harm (food loss or disease) and the smaller one ( the parasite ) may get advantages (more food, protection). both must preserve their identity and prevent contamination by foreign genetic material (immunologic surveillance, bilaterally). some parasites can even live within the hosts (endoparasitism), like some bacteria and all viruses. three types of interactions may occur between a microrganism and a human host: (a) symbiotic relationship, in which the microrganism and the host both benefi t; (b) commensal relationship, in which the microrganism gains but the host suffers no harm; and (c) a true parasytic relationship, in which the microrganism gains and the host is harmed. symbiosis offers frequently mutual advantages and remains very stable in time. pathogens are a minimal part of existing microrganisms. we humans host some advantageous bacterial populations (intestine, surface germs on the skin, commensal germs on the mucosae): we indeed are also made of the germs living in/on our body. indeed, only one cell out of ten in our body is a human cell: the rest are bacteria (the so-called micro-biome). among these, we count billions of bacteria in the intestines, useful for many functions (vitamin production, competition with pathogens, contribution to metabolism, etc.). the balance between host and parasites depends on two basic forces, an aggressive force by the parasite depending on survival/proliferation/invasion capacity of the parasite itself and a defensive force by the host depending on the immune mechanisms (phagocytosis, cellular and humoral immune reactions). in this balancing of opposite forces the parasitic relationship is played by the contendents. if we have prevalence of parasite, we may have disease (and eventually death) of the host, but if we have prevalence of host defence we may have control (and eventually elimination) of parasites. in the light of recent concern and interest about the potential for biological terrorism (biofarware) there are several diseases and bacterial toxins that must be considered in particular, like anthrax [ 1 , 2 ] , smallpox [ 3 , 4 ] , plague [ 5 ] , botulinum toxin [ 6 ] , and tularemia [ 7 ] . a very detailed discussion of such diseases and other infectious diseases with similar risks in terms of bioterrorism goes beyond the scopes of this concise chapter, but some features of these and other infectious diseases representing important threats in the biofarware fi eld will be mentioned. in this respect, we may distinguish in time diseases which are: smallpox is a highly contagious disease (incubation 10-12 days) caused by the smallpox virus, an orthopoxvirus. it causes death in up to 30 % of infected subjects. indigenous infection has been eradicated (last case, ethyopia, 1990 -who). the main concern for outbreaks of smallpox is today from bioterrorism. smallpox is characterized by severe constitutional symptoms (fever, headache, extreme malaise) and a characteristic pustular rash. treatment is supportive; prevention involves vaccination, which, because of its risks (eczema, encephalitis, etc.), is done selectively. pathogenesis of smallpox demonstrates that the virus is transmitted from person to person by direct contact or inhalation of droplet nuclei. clothing and bed linens can also transmit infection. most contagions are in the fi rst 7-10 days after the skin rash appears. once crusts form, infectivity declines. the virus invades the oropharyngeal and respiratory mucosa, multiplies in regional lymphnodes, causing viremia and localization in small blood vessels of the skin (rash) and rarely in cns (encephalitis). offi cially, smallpox is dead on earth. there are no longer cases detected in the world population since 1990, but can we destroy the samples of smallpox virus existing in some virology laboratories around the world? certainly not [ 3 ] , because we could no longer prepare vaccine doses without live virus samples to start from. and without vaccine, a small amount of wild virus could ignite a wide epidemic killing a large proportion of the human population, since the vaccination is no longer mandatory in any country and a large percentage of young populations have no longer been vaccinated after the early 1990s. poliomyelitis is an acute infection caused by a poliovirus. manifestations include a nonspecifi c minor illness (abortive poliomyelitis), sometimes aseptic meningitis without paralysis (nonparalytic poliomyelitis) and, less often, fl accid weakness of various muscle groups (paralytic poliomyelitis). diagnosis is clinical, although laboratory diagnosis is possible. treatment is supportive. vaccination is available, still mandatory in many countries, although soon legislations may change. childhood vaccination produces immunity in 95 % of recipients. declared cases worldwide have diminished remarkably, but some areas with particularly poor sanitary services or with confl icts preventing health services to operate are recording increased numbers of cases recently (syria, 2013; china 2013). polioviruses have three serotypes. the virus enters the mouth via the fecal-oral route, then enters the lymphoid tissues of the gi tract. if not contained, infection may enter the cns with signifi cant damage in spinal cord and brain, specifi cally to nerves controlling motor and autonomic function (breathing). spreading is through the enteric route. vaccine is live, attenuated virus, able to immunize many contacts respect to the vaccinated subjects (community vaccination strategies; problems in nomad populations). anthrax is caused by bacillus anthracis , toxin producing, encapsulated, aerobic or facultative anaerobic organisms. anthrax, an often fatal disease of animals, is transmitted to humans by contact with infected animals or their products (woolsorter's disease). in humans, infection tipically occurs through the skin. inhalation infection is less common; oropharingeal, meningeal and gi infections are rare. for inhalation and gi infections, nonspecifi c local symptoms are typically followed in several days by severe systemic illness, shock and often death. empyric treatment is with cyprofl oxacin or doxycycline. a vaccine is available (antitoxin). pathogenesis of anthrax takes place since bacillus anthracis readily forms spores when germs encounter dry environment -a condition unfavorable for growth . spores resist destruction and can remain viable in soil, wool, and animal hair for decades. spores germinate and multiply in favourable conditions (wet skin, tissue, blood) and can give human disease by contact (papules, black eschars, contagious also via fomites) ingestion (raw meat > fever, nausea, vomiting, diarrhea), and inhalation (fl u-like illness, respiratory distress, cyanosis, shock, coma). of note is the anthrax bioterrorist attack through mailings (using spores in powder form) that took place in the usa in 2001 (us postal service, washington dc), event that highly sensitized the public to the global theme of bioterroristic attacks. plague is caused by yersinia pestis (formerly named pasteurella pestis ). short bacillus with hairpin shape, infects wild rodents and can infect humans via tick bites. symptoms are either severe pneumonia or massive lymphadenopathy with high fever, often progressing to septicemia. diagnosis is epidemiologic and clinical, confi rmed by culture and serologic testing. treatment is with streptomycin or doxycycline. unfortunately, a vaccine is not available for plague. tularemia is a febrile disease caused by francisella tularensis ; it may resemble typhoid fever: symptoms are a primary local ulcerative lesion, regional lymphadenopathy, profound systemic symptoms, and, occasionally, atypical pneumonia. diagnosis is primarily epidemiologic and clinical and supported by serologic tests. treatment is with streptomycin, gentamycin and other antibiotics. tetanus is an acute poisoning from a neurotoxin produced by clostridium tetani. symptoms are intermittent tonic spasms of voluntary muscles. spasm of the masseters accounts for the name "lockjaw" (trismus). incubation requires 2-10 days. diagnosis is clinical. treatment with immune globulin and intensive support. only unbound toxin can be neutralized. a vaccine is available, with a good extent of preventive protection. botulism is a neuromuscular poisoning due to clostridium botulinum toxin. botulism may occur without infection if toxin is ingested. symptoms are symmetric cranial nerve palsies accompanied by a symmetric descending weakness and fl accid paralysis without sensory defi cits. diagnosis is clinical and by laboratory idenifi cation of toxin. treatment is with antitoxin and support therapies. tbc is a chronic, progressive infection by mycobacterium tuberculosis , often with a long period of latency following initial infection. it occurs most commonly in the lungs, with productive cough, chest pain and dyspnea. diagnosis is most often by sputum culture and smear. tbc can involve any tissue (organ disease). treatment is with multiple antimicrobial drugs. forms of multiresistant tb bacteria are becoming more and more frequent. coronavirus infections in humans most frequently cause common cold symptoms; however in 2002, a relatively new coronavirus caused an outbreak of severe acute respiratory syndrome (sars), which was much more severe than other coronavirus infections. sars is an infl uenza-like disease leading to progressive respiratory insuffi ciency with signifi cant mortality rate. first detected in china (guandong, 2002), the sars epidemic spread to more than 30 countries. in mid-july 2003, there were >8,000 cases with >800 deaths (10 % mortality). then the outbreak subsided and no new cases have been identifi ed from 2004 to 2012. in 2012 a new similar epidemic (sustained by the virus ncov, novel coronavirus) started in middle east (arabia), with an estimated mortality above 40 %. later the ncov epidemic has been named mers (middle east respiratory syndrome) and is being studied as a new zoonosis trasmitted to humans from dromedary camels. studies are currently in progress, with great attention by the international sanitary authorities [ 9 ] . who in 2013 indeed alarmed many countries against the new sars-like coronavirus responsible of mers, that infected at the moment of this writing (december 2013; www.who.int/en ) more than 160 persons (arabia, great britain, france, germany, tunisia, italy, abu dhabi, united arab emirates, etc.) with reduced infective capacity as compared to sars, but still highly lethal and communicable via close contacts (family members). the latest available numbers call for 163 ascertained diagnoses in humans, with 71 deaths (mortality, 43.5 %). updates can be found at the following web sites: www.who.int/en ; www.cdc.gov and (recommendations for clinicians) emergency.cdc.gov , emphasizing the need to consider the novel (ncov) coronavirus when treating patients with a severe respiratory illness who have recently traveled to the arabian peninsula (or close contacts of the travelers). marburg and ebola are fi loviruses that cause hemorrhage, multiple organ failure and high mortality rates. diagnosis is with enzyme-linked immunosorbent assay, pcr or electron microscopy. treatment is supportive. strict isolation and quarantine measures are necessary to contain outbreaks. incubation 5-10 days. marburg virus has been identifi ed in bats and in primates. human to human transmission occurs via skin and mucous membranes contact (humans/primates). filoviruses can affect intestines (nausea, vomiting, diarrhea), respiratory tract (cough, pharingitis), liver (jaudice), cns (delirium, stupor, coma), and cause hemorrhagic phenomena (petechiae, frank bleeding) with high mortality rates (up to 90 % with ebola virus). survivors recover very slowly and may develop long lasting complications (hepatitis, uveitis, orchitis) with only supportive care available: no specifi c antivirals nor vaccines are available fo fi lovirus infections. bunyaviridae contain the genus hantavirus (four serogroups, nine viruses) causing hemorrhagic fevers with renal and pulmonary consequences, starting with fl u-like symptoms and evolving with severe renal and pulmonary consequences. lethal in 10-15 % of cases. lassa fever is an often fatal arenavirus infection occurring mostly in africa. it may involve multiple organs, except cns. treated with ribavirin. no vaccinations are available so far for hantavirus infections. outbreaks of such infections have been recorded in nigeria, liberia, central africa, with some rare imported cases in the usa and in the united kingdom. the animal reservoir of such viruses is in wild african rats ( mastomys natalensis ), frequently found in african houses. direct human to human transmission is documented via urine, feces, saliva or blood. mortality (up to 45 %) can be reduced by prompt ribavirin treatment. universal hygiene precautions, airborne isolation and surveillance of contacts are essential. last but not least … we must mention now infl uenza! flu viruses are in nature among the most rapidly changing (mutating) organisms through their ability to infect a variety of hosts: birds (migrating waterfowl -ducks-, stantial poultry -chickens-), mammals (pigs, felines) and humans. in south east asia (mostly in china, but also in viet-nam, laos, thailand, etc.) it is very common to have mixed farms of pigs, poultry and ducks, attended by humans. every year, new strains appear in se-asia, favoured by the recyprocal passage between migrating birds (mostly fowl), pigs and chickens, with exposure of many humans in farms, markets, rooster fi ghting sports, and food preparation places. a common say in china tells that "anything with four legs (except chairs) and anything that fl ies (except airplanes), can be eaten". with this phylosophy, there is generally a lot to be desired in food safety and in general hygienic prevention in such geographical areas. after the avian fl u h5n1 of 2005-2006, highly lethal but unable to give human to human contagion, new combinations of fl u strains are expected and feared, with high lethality and high human to human transmissibility. on this widely interesting theme for the world diffusion of new virus strains with pandemic potential, i wrote in 2010 together with the colleague virologist aldo lepidi a book entitled "pandemics -virology, pathology and prevention of infl uenza" (bollati boringhieri publisher, turin, italy , 2010) [ 10 ] . in summary, we can see that a continuous surveillance is being devoted worldwide to the appearance of new strains of infl uenza viruses, in order to isolate as soon as possible potentially pandemic new strains and to prepare biological stocks suitable for massive vaccine preparations in due time to prevent the global spreading of potentially lethal new variants of the infl uenza viruses. examples in time recall the cases of the highly lethal pandemics known as "spanish fl u" in 1917-1918 (in excess of 40 million deaths worldwide), "asian fl u" in 1956 (in excess of 100,000 deaths worldwide) and "hong kong fl u" in 1978 (in excess of 700,000 deaths worldwide). the basic question is: when the new pandemic will strike ? sometimes soon, as international experts say. the so called "avian fl u" came close to that, but sometimes in the future new mutations may emerge with the potential of being much worse. in conclusion of this wide although rapid overview of the most frequent or alarming causes of microrganism-related human diseses with potential interest for bioterrorism, i hope to have provided suffi cient matter for discussion and for further future diffusion of medical and microbiological culture that may be useful for prevention and the betterment of human social relationships and for peace promotion. anthrax as a biological weapon: medical and public health management anthrax as a biological weapon, 2002: updated recommendations for management smallpox as a biological weapon: medical and public health management addressing the unthinkable: preparing to face smallpox plague as a biological weapon: medical and public health management. working group on civilian biodefense botulinum toxin as a biological weapon: medical and public health management tularemia as a biological weapon: medical and public health management the merck manual of diagnosis and therapy, 19th edn deadly mers coronavirus not yet a global concern pandemie¸ virologia, patologia e prevenzione dell'infl uenza (pandemics -virology, pathology and prevention of infl uenza) key: cord-254265-8i86c8kt authors: camps, marta; ricart, sílvia; dimova, veselka; rovira, núria; muñoz‐almagro, carmen; garcia, juan jose; pons‐odena, martí; marcos, mª angeles; pumarola, tomàs title: prevalence of human metapneumovirus among hospitalized children younger than 1 year in catalonia, spain date: 2008-06-12 journal: j med virol doi: 10.1002/jmv.21209 sha: doc_id: 254265 cord_uid: 8i86c8kt human metapneumovirus was discovered recently respiratory virus implicated in both upper and lower respiratory tract infection. in children, the clinical symptoms of human metapneumovirus are similar to those produced by respiratory syncytial virus, ranging from mild to severe diseases such as bronchiolitis and pneumonia. the aim of the present study was to describe the prevalence of human metapneumovirus and other common respiratory viruses among admitted to hospital infants. from january 2006 to june 2006, 99 nasopharyngeal aspirates were collected from hospitalized children younger than 12 months in order to study respiratory viruses. human metapneumovirus detection was performed by cell culture and two rt‐pcr targeting on polymerase and fusion genes. the latter gene was used for phylogenetic analysis. in 67/99 children (67%) at least one viral pathogen was identified, the viruses detected most frequently were respiratory syncytial virus (35%), human metapneumovirus (25%) and rhinovirus (19%). the results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among children less than 12 months; (2) children infected with human metapneumovirus were significantly older than those infected by respiratory syncytial virus; (3) human metapneumovirus was associated more frequently with pneumonia whereas respiratory syncytial virus was only detected in patients with bronchiolitis; (4) there was a clear epidemiological succession pattern with only a small overlap among the viruses detected most frequently; (5) all human metapneumovirus samples were clustered within sublineage a2. j. med. virol. 80:1452–1460, 2008. © 2008 wiley‐liss, inc. human metapneumovirus was discovered recently as a respiratory virus implicated in both upper and lower respiratory tract infection ranging from mild to severe disease in all age groups [van den hoogen et al., 2001] . since then, it has been reported in europe, asia, australia, south africa and america [peret et al., 2002; bastien et al., 2003; jpma et al., 2004; rao et al., 2004; sloots et al., 2006; brooks et al., 2007] , suggesting worldwide distribution. seroprevalence studies have shown that this virus has been present among humans for over five decades [van den hoogen et al., 2001; hamelin et al., 2004] . human metapneumovirus was not discovered previously due to its difficulty to grow in traditional cell cultures, although it has slow growth in the rhesus monkey kidney (llc-mk2) cell line [deffrasnes et al., 2005] . although monoclonal antibodies (mabs) are being developed [landry et al., 2005; percivalle et al., 2005] , these are not available commercially for direct antigen detection in nasopharyngeal aspirate (npa), and molecular assays remain the main approach available currently for human metapneumovirus identification. human metapneumovirus has a negative-strand rna genome of approximately 13 kb encapsidated by a helicoidal nucleocapsid and covered with a lipid bilayer [van den hoogen et al., 2002; biacchesi et al., 2003] . it has been classified in the pneumovirus subfamily of the paramyxovirus family, which also contains the respiratory syncytial virus and metapneumovirus genus. currently, two main genotypes a and b with the subtypes a1, a2, b1, and b2 are identified by molecular methods [biacchesi et al., 2003] . the clinical symptoms of human metapneumovirus are similar to those caused by respiratory syncytial virus, ranging from wheeze, cough, fever, bronchiolitis, and even pneumonia [wolf et al., 2006] . although some studies have detected human metapneumovirus in all age groups, human metapneumovirus infections could be more severe in children, elderly, and immunocompromised patients [pelletier et al., 2002; bastien et al., 2003; cane et al., 2003; falsey et al., 2003] . however, several epidemiological and clinical features have yet to be established firmly. the aim of the present study was to describe the role of human metapneumovirus and other common respiratory viruses including: influenza virus a, b, and c, parainfluenza 1-4 viruses, adenoviruses, respiratory syncytial virus a and b, rhinovirus, coronavirus 229e and oc43 and enterovirus as bronchiolitis, and bronchopneumonia pathogens among hospitalized children younger than 1 year, taking into account that in this age group respiratory viruses are the main etiological agents of lower respiratory tract infections [shay et al., 1999; smyth and openshaw, 2006; bush and thomson, 2007] . additionally, in human metapneumovirus positive samples, a comparative phylogenetic analysis of fusion (f) gene sequences was carried out to study the potential circulation of distinct human metapneumovirus genotypes among patients included. the present study falls within the framework of the ''study of prevalence, clinical and epidemiological features associated to human metapneumovirus infection among infants and adult population'' awarded by the fondo de investigaciones sanitarias (fis), spain. from january 2006 to june 2006, children younger than 12 months hospitalized in a 345-bed children's hospital (hospital sant joan de déu, barcelona, spain) with bronchiolitis or bronchopneumonia, were studied prospectively. within the first 24 hr of admission, a nasopharyngeal aspirate was collected and delivered to the laboratory of microbiology at the hospital clínic of barcelona, where it was processed to study respiratory viruses. a questionnaire about clinical and epidemiological features was also completed during the initial consultation. routine bacteriological testing (of blood and urine) was only performed in all febrile infants who have toxic manifestations, defined as a clinical appearance consistent with the sepsis syndrome [baraff et al., 1993] . other respiratory samples were obtained according to clinical indication or the decision of the attending physician. bronchiolitis was defined as an acute infection of the lower airway, characterized by increased respiratory effort (tachypnea) and expiratory wheezing and/or crackles associated with common viral infection symp-toms (rhinorrhea, cough) in infants younger than 1 year. the presence of lower respiratory infection (wheezing and/or crackles) with a focal infiltrate in the chest x-ray was considered as a bronchopneumonia. exclusion criteria were underlying medical conditions (cystic fibrosis, metabolic diseases, neurological diseases) and patients with two or more previous episodes of wheeze without the presence of radiological infiltrates. criteria for pediatric intensive care unit (picu) admission were: acute respiratory failure with hemoglobin saturation <90% and >40% fraction of inspired oxygen (fio 2 ) supplementation, common episodes of apnea and sepsis according to the international consensus conference on pediatric sepsis [goldstein et al., 2005] . informed consent was obtained from the patients' parents. the ethical committees of the hospital clínic and hospital sant joan de déu approved the study protocol. specimens for immunofluorescence assay (ifa) were suspended in pbs (phosphate buffered saline), when necessary, and spotted onto a slide that was air-dried and fixed with cold acetone and then stained with a pool of fluorescein-conjugated antibody to influenza virus a, influenza virus b, human parainfluenza virus 1-3, adenovirus, and respiratory syncytial virus (respiratory panel 1, viral screening and identification kit; light diagnostics, chemicon international temecula, usa). simultaneously, specimens were inoculated into mdck (madin darby canine kidney), hep-2 (human caucasian larynx carcinoma squamous cell) and llc-mk2 (rhesus monkey kidney epithelial cell) cell lines (vircell, granada, spain) for isolation of the viruses mentioned above and human metapneumovirus. mdck and llc-mk2 tube cultures were incubated at 33 and 378c, respectively, and maintained with growth essential medium [emem (ebss) þ 2 mm glutamine þ 1% non-essential amino acids (neaa)] adding 12 ml (500 mg/ml) of trypsin/edta for optimal growth of influenza viruses and human metapneumovirus. hep-2 cell line was maintained with the same medium plus 2% fetal bovine serum and incubated at 378c. cell cultures were examined twice weekly for the development of a cytopathic effect; positive cultures were harvested and stained for conventional virus identification (ifa). in ifa negative cases, a rt-pcr (reverse transcription-polymerase chain reaction) for common respiratory viruses and human metapneumovirus was undertaken. upon sample collection, an aliquot of each fresh specimen was collected to be used for rt-pcr analysis. nucleic acids from either dna/rna viruses present in the nasopharyngeal secretion or in infected cell cultures were extracted from 200 ml of specimen using nucli-sense easymag (biomérieux, nl-5281 rm boxtel, the netherlands) according to the manufacturer's instructions. the lysis buffer included 500 molecules of the cloned-amplified product used as an internal control in each reaction tube in order to exclude false negative results due to non-specific inhibitors or extraction failure. two independent multiplex nested rt-pcr assays able to detect from 1 to 10 copies of viral genomes were carried out using techniques described previously [coiras et al., 2003; coiras et al., 2004] . one rt-pcr assay detected influenza virus a, influenza virus b, influenza virus c, respiratory syncytial virus a, respiratory syncytial virus b, and adenovirus. another rt-pcr assay examined human parainfluenza virus 1-4a and 4b, human coronavirus 229e, human coronavirus oc43, and the generic detection of enterovirus and rhinovirus. in order to detect human metapneumovirus, extracted rna was used as a template for cdna synthesis by random primers according to the manufacturer's instructions (first strand cdna synthesis kit, roche diagnostics, mannheim, germany). amplification reaction was carried out using specific primers amplifying a conserved fragment of 170 bp in the polymerase gene (l): lf, 5 0 cat gcc cac tat aaa agg tca g 3 0 ; lr, 5 0 cac ccc agt ctt tct tga aa 3 0 , as described elsewhere . the pcr conditions comprised 45 cycles at 948c for 1 min (denaturalizaton), 508c for 50 sec (annealing), 728c for 1 min (extension) and a final extension at 728c for 10 min. to detect human metapneumovirus types a and b, in addition to the widely used primers of l gene that do not hybridize to type b [sarasini et al., 2006 ], a second nested pcr test using fusion gene (f) primers was performed. first round pcr used the following primers: ff1, 5 0 ttc gtt cta gga gca a 3 0 and fr, 5 0 gtc ttc ctg tcg taa ctt tg 3 0 . the second round used an inner forward primer ff 5 0 atg cca aca tct gca gga c 3 0 and the same reverse primer as in the first step, obtaining a final product of 450 pb. amplified products were analyzed by electrophoresis on 2% agarose gel, stained with ethidium bromide. fusion gene primers were either selected from published protocols or designed originally based on the sequence data of a canadian strain available from genbank, national center for biotechnology information (genbank) (accession no. ay297749) [biacchesi et al., 2003] . as an human metapneumovirus positive control, rna of human metapneumovirus (subtype a2, according to strain canada 97/83) was used in the pcr test. each set of rt-pcr reaction included a positive control for extraction and amplification handling obtained from our viral lysates, when available, and a negative control (viral transport medium containing no nucleic acid). all positive results were confirmed by two sequential assays. in addition, a second nested pcr targeting a fragment of the f gene was also used for further phylogenetic analysis and to confirm the previous l gene positive results and identify probable human metapneumovirus type b [bouscambert-duchamp et al., 2005; banerjee et al., 2007] . cdna synthesized previously was used as a template for the first f gene pcr amplification. the reaction mix contained ff1/fr primers and termocycler conditions were 35 cycles at 948c for 1 min (denaturation), 458c for 2 min (annealing), 728c for 2 min (extension) and a final extension step of 728c for 10 min. the second round pcr used an inner primer, ff, and the same reverse primer used before. in this pcr, annealing reaction was performed at 508c instead of 488c, and other conditions and reaction volumes were identical to those applied in the first round. pcr reactions that produced proper bands were purified on qiaquick gel extraction kit (qiagen, izasa, spain) and sequenced with bigdye terminator v3.1 (applied biosystems, foster city, usa) in an abi prism 3700 dna analyzer (applied biosystems, foster city, usa). sequences were aligned with different human metapneumovirus subtype strains from canada, netherlands, and japan (accession nos. ay145295, ay145298, ay145289, ay145292, ay145287, ay145294, ay145299, ay145297, ay145301, ay145296, dq362940.1, and ay312232) [bastien et al., 2003; galiano et al., 2006; huck et al., 2006] and avian metapneumovirus c sequence (accession no. ay590688) was used as an outgroup [govindarajan et al., 2004] . nucleotide sequence alignments were generated using the clustalw algorithm of the mega software [kumar et al., 2004] and the total length of each sequence was 302 nucleotides. phylogenetic and molecular analyses were conducted using mega version 3.0. phylogenetic trees were constructed by the phylip program package, version 3.66 (felsenstein, department of genetics, university of washington, seattle, wa), using the neighbor-joining method supplied by the treeview 32 program. prevalence of human metapneumovirus was calculated as the number of children positive by pcr for human metapneumovirus divided by the number of children in the same age group admitted during the same period and hospitalized with lower respiratory tract infection. quantitative variables were described with means ae standard deviation and significant differences between respiratory syncytial virus and human metapneumovirus groups were analyzed by using mann-whitney u-test. qualitative variables were reported as frequencies and percentages. comparison of proportions was determined by w 2 or fisher's exact test. probability values of p < 0.05 were considered significant. analysis was performed using the graph-pad prism3 statistical program (graphpad software, inc., san diego, ca). from january to june 2006, of the 120 children younger than 12 months who were hospitalized for lower respiratory tract infection, 99 with nasopharyngeal aspirate for respiratory viruses detection were included prospectively. the median age of the patients was 3.9 ae 4.1 months (range: 14 days to 12 months) and included 59 males (59%) and 40 females (41%). clinical diagnoses at the time of admission were bronchiolitis (74%) and (26%) bronchopneumonia, of which 67% and 72%, respectively, had an etiological diagnosis. in 67/99 children (67%), at least one viral pathogen was identified; 55/99 (55%) had an infection with one of the viruses investigated, whereas 12/99 (12%) had a coinfection of at least two viruses. a total of 80 viruses were identified, while 32/99 (33%) patients remained without an etiological diagnosis. the most frequent virus detected was respiratory syncytial virus (35%) followed by human metapneumovirus (25%) and rhino-virus (19%) (fig. 1) . viral co-infections were observed in 12 (12%) cases, with adenovirus and rhinovirus the most common viruses identified. in one patient with bronchopneumonia, a triple infection was detected (respiratory syncytial virus þ adenovirus þ rhinovirus). blood and urine cultures were obtained from 32 patients with criteria of toxic manifestations. none of the specimens of blood or urine grew a pathogen. three bronchial aspirates and one bronchoalveolar lavage were collected. an haemophilus influenzae and a streptococcus pneumoniae were recovered from bronchial aspirate and bronchoalveolar lavage, respectively. diagnosis of bronchiolitis was significantly more frequent in respiratory syncytial virus single-infected children (p < 0.001), whereas human metapneumovirus infection was significantly more common among infants with pneumonia (p < 0.001) ( table i) . human metapneumovirus infected children were significantly older (mean age: 6.2 ae 5.14 months) than those infected by respiratory syncytial virus (mean age: 2.5 ae 3.37 months) (p ¼ 0.009) (tables ii, iii) . thirteen patients required hospitalization to the pediatric intensive care unit (picu); 11 were first diagnosed as bronchiolitis and two as bronchopneumonia. eleven out of 13 patients admitted to the picu had an etiological diagnosis: 2 respiratory syncytial virus, 3 rhinovirus, 1 human metapneumovirus, 1 human metapneumovirus þ rhinovirus, 2 respiratory syncytial virus þ adenovirus, 1 h. influenzae and 1 s. pneumoniae þ respiratory syncytial virus. the duration of hospitalization was 2-14 days (mean 6.2 ae 3.8). although the differences were not statistically significant, patients with a dual viral infection required more days to recover (8.3 ae 4.9 days) than those with a single infection (5.28 ae 3.25 days). overall, 20 human metapneumovirus cases were detected by rt-pcr, of which 5 could also be recovered from the llc-mk2 cell line. all samples that were j. med. virol. doi 10.1002/jmv human metapneumovirus positive by rt-pcr targeting l gene were also confirmed by rt-pcr based on the f gene. sequencing the f amplicons of human metapneumovirus strains identified from npa and their subsequent phylogenetic analysis showed that only type a2 of human metapneumovirus circulated among patients included during the study period (fig. 2) . all human metapneumovirus samples tested were found in the same cluster sharing a nucleotide identity of 88.4% with a slightly higher amino acid similarity of 89%. notably, when the same sequences were examined leaving out p15 and p19 patients, the similarity increased to 91%. in the phylogenetic tree, both sequences seemed to form a different cluster within the subtype a2 (fig. 2) . the present study is based on the first half of the year, thus it is difficult to establish a distribution of viruses detected during the follow-up. nonetheless, during these 6 months, there were different distribution patterns among the viruses detected most frequently (fig. 3) . acute respiratory tract infections are an important cause of morbidity and mortality in children, and it is well known that a high percentage of these infections are caused by respiratory viruses [williams et al., 2002; jennings et al., 2004; meissner, 2005; pierangeli et al., 2007] . to date, respiratory syncytial virus has been an important trigger of acute respiratory tract infection, although the role of other respiratory viruses such as rhinovirus or human metapneumovirus [van den hoogen et al., 2001] , human coronavirus-nl63 [van der hoek et al., 2005] , human coronavirus-hku1 [woo et al., 2005] and human bocavirus [allander et al., 2007] are gaining attention increasingly. in the present report, respiratory viruses were involved in 67% of either bronchiolitis or bronchopneumonia patients admitted to hospital. despite the fact that both human metapneumovirus and respiratory syncytial virus are common pathogens in children, patients infected with human metapneumovirus were significantly older than respiratory syncytial virusinfected patients, a finding that has been reported elsewhere [williams et al., 2006; wolf et al., 2006; chung et al., 2007] . despite the fact that a limited range of age population has been selected, similar results to previous studies were found [wright et al., 1989; chung et al., 2007; weigl et al., 2007] , suggesting that during the first years of life respiratory viruses are the most important pathogens causing severe respiratory tract infections resulting in hospital admission. several studies have demonstrated that human metapneumovirus is more likely to be associated with bronchiolitis in early childhood [xepapadaki et al., 2004; garcia-garcia et al., 2007; do carmo debur et al., 2007] . however, other investigations including the present study found that human metapneumovirus was associated more frequently with bronchopneumonia compared to human metapneumovirus-bronchiolitis cases [choi et al., 2006; brooks et al., 2007] . these variable results may reflect that human metapneumovirus is not exclusively a bronchiolitis pathogen and can, also cause bronchopneumonia among infants. it probably indicates that human metapneumovirus causes a spectrum of lower respiratory tract illness, with a tendency toward the more severe end, namely pneumonia. in the study sample, human metapneumovirus was present in 25% of patients causing either bronchiolitis or broncopneumonia, a percentage second only to respiratory syncytial virus (35%) and surpassing rhinovirus (19%) . compared to other studies, a higher rate of human metapneumovirus infection was obtained, considering that it is responsible normally for 5-10% of hospitalizations of children suffering from acute respiratory tract infections [jpma et al., 2004; deffrasnes et al., 2007] . however, these rates reflect data from only the first half of the year, corresponding to the peak infection period and thus, increasing the proportion of human metapneumovirus cases. probably, it has led to an overestimate of the real prevalence. dual viral infections are frequent in childhood, mainly in infants, but to date they were not associated with an increased severity of illness [williams et al., 2004; wolf et al., 2006; van woensel et al., 2006 ]. in contrast, other studies have found an increase of 5-to 10-fold in the severity of disease in human metapneumovirus and respiratory syncytial virus dual infections [greensill et al., 2003; semple et al., 2005] . the present study showed 12% of viral coinfections, and although no human metapneumovirus plus respiratory syncytial virus cases were detected, coinfected patients required longer recovery. thirteen patients required hospitalization to the pediatric intensive care unit, and respiratory syncytial virus and rhinovirus were the main respiratory viruses involved. either as a single pathogen or in co-infection, they required more days in the pediatric intensive care unit. interestingly, one rhinovirus-infected patient spent 11 days at the pediatric intensive care unit, although further studies are needed to evaluate its role in the severity of illness. respiratory syncytial virus has been studied widely and in most cases involved in severe acute respiratory infections, however, it has been shown that rhinovirus can also be a life-threatening virus among children with lower respiratory infection [guittet et al., 2003; calvo et al., 2007] . currently, four distinct major human metapneumovirus phylogenetic lineages, a1, a2, b1, and b2 have been described . during the study period, 20 human metapneumovirus cases were detected, all belonging to type a2. maertzdorf et al. [2004] highlighted that some strains, particularly b1 and b2 sublineages, could not be detected by the widely used l6-l7 pair primers published by van den hoogen et al. [2001] . nonetheless, all samples were tested by either l and f genes rt-pcr, so it was considered that during the follow-up only human metapneumovirus subtype a2 circulated among patients included in this study. interestingly, in a study undertaken during the same year based on 171 children with upper respiratory infection attended in different primary care centers of catalonia, it was observed a cocirculation of both lineages among 10 human metapneumovirus identified (unpublished data). recently, huck et al. [2006] described a novel human metapneumovirus sublineage within a2 group, which they divided into a2a and a2b. in the present report, the sequences within the group a2 shared a nucleotide identity of 88.4%. when patients p15 and p19 were excluded, the identity of sequences reached 91%, suggesting that these patients may belong to another cluster. one limitation of this study is that follow-up was done during a short period of time, even though respiratory syncytial virus, human metapneumovirus and rhinovirus cases occurred in a defined succession with only small overlap between the viruses, as has been reported in other studies [choi et al., 2006; pierangeli et al., 2007] . further studies would be valuable to investigate how these seasonal patterns change, taking into account either the entire year and a wider age group, thus establishing a true pattern of respiratory viruses. overall, the results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among hospitalized children less than 12 months, and therefore an important cause of severe lower respiratory tract infection in this age group; (2) children infected with human metapneumovirus were significantly older (mean 6.2 months) than those infected by respiratory syncytial virus (mean 2.5 months); (3) human metapneumovirus was associated more frequently to pneumonia whereas respiratory syncytial virus was only detected in bronchiolitis cases; (4) human metapneumovirus is associated with a spectrum of lower respiratory tract illness, with a greater predominace, in this population, with pneumonia; (5) although respiratory syncytial virus and human metapneumovirus circulate mainly during the winter season, there was a clear succession with a small overlap between viruses; (6) all human metapneumovirus sequences were clustered within sublineage a2. human bocavirus and acute wheezing in children human metapneumovirus infections among children with acute respiratory 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respiratory syncytial virus infection ten years' experience with year-round active surveillance of up to 19 respiratory pathogens in children estimates of world-wide distribution of child deaths from acute respiratory infections human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia the tucson children's respiratory study. ii. lower respiratory tract illness in the first year of life human metapneumovirus as a causative agent of acute bronchiolitis in infants key: cord-005080-r01ii1bu authors: butler, colin d.; corvalan, carlos f.; koren, hillel s. title: human health, well-being, and global ecological scenarios date: 2005-02-22 journal: ecosystems doi: 10.1007/s10021-004-0076-0 sha: doc_id: 5080 cord_uid: r01ii1bu this article categorizes four kinds of adverse effects to human health caused by ecosystem change: direct, mediated, modulated, and systems failure. the effects are categorized on their scale, complexity, and lag-time. some but not all of these can be classified as resulting from reduced ecosystem services. the articles also explores the impacts that different socioeconomic–ecologic scenarios are likely to have on human health and how changes to human health may, in turn, influence the unfolding of four different plausible future scenarios. we provide examples to show that our categorization is a useful taxonomy for understanding the complex relationships between ecosystems and human well-being and for predicting how future ecosystem changes may affect human health. the interconnection between ecosystems and human activity is complex, important, and poorly understood. ecosystems support human health and well-being through their provisioning, regulating, cultural, and supporting services (butler and others 2003) . shortages of food, fiber, and other ecosystem products adversely affect human health via many direct and indirect pathways. the regulating functions of ecosystems that affect health include the purification of air and fresh water, the reduction of flooding and drought, and range limitation of certain vector-borne diseases. ecosystems also impact mental well-being through provision of cultural services, for example by providing totemic species and sacred groves, landscapes, and water bodies. these influence the aesthetic, recreational, educational, cultural, and spiritual aspects of the human experience. ecosystem changes have also altered the epidemiology of communicable and noncommunicable diseases, including through some pathways that would not be considered as arising from a reduced ecosystem regulating service. this article explores the impact that different plausible future scenarios (cumming and others 2005; raskin 2005 ) may have on health, and it also suggests how changes to health may feedback on and hence modify the course of the future. in doing so, we have categorized the effects of ecosystem change on human health into a useful taxonomy which can help predict which future ecosystem changes will impact human health and how. although positive scenarios are conceivable, in this article we are mainly concerned with how adverse ecosystem changes and reduced ecosystem services may harm future human health. until the very recent past, most human induced ecological changes have had favorable effects on human society and health. human health, judged by average life expectancy, has increased substantially, as has population size (riley, 2001; tuljapurkar and others 2000) . the reasons for these increases are well-known and include the mutually reinforcing and interacting elements of improved knowledge, technology, and social organization, including of public health (horiuchi 2000; szreter 1999) . a fundamental contributory factor has been the increased human capacity to modify ecosystems, for example, by increasing food supplies, by restricting populations of large carnivores, and by providing more fiber for fuel and shelter. this success is not unqualified. some adverse effects of our increased capacity to modify ecosystems on human health can already be seen. the transformation of ecosystems to provide certain benefits has reduced the scale and integrity of many ecosystems (pimentel and others 2000) . reduced ecosystem integrity decreases their ability to provide some ecosystem services, which can, in turn, have negative impacts on human health. this relationship is unlikely to be linear and may contain thresholds beyond which incremental loss of ecosystem services has a disproportionately negative effect on human-health and well-being. examples of the negative impacts of ecosystem change on human health abound. national life expectancy has fallen in several countries, including many parts of sub-saharan africa, haiti, russia, north korea, and ethiopia (farmer and others 2003; shkolnikov and others 2001; united nations population divisional 1999) . reduced ecosystem services may explain a part of these declines in national life expectancy and thus may be an underrecognized factor in the slowed rate of increase in global life expectancy. the problems of decreased provision of ecosystem services are often unequally distributed, with the majority of the burden falling on the poor. additionally, poor populations frequently lack the income and other means to substitute or partly compensate for reduced ecosystem services (for example, by boiling microbiologically contaminated water). in addition to the effect of ecosystem services on human health, human health itself influences access to critical ecosystem services and can modify the environmental impacts of human populations. for example, the aids epidemic in sub-saharan africa has reduced the provisioning ecosystem service of food supply (de waal and whiteside 2003) . the high prevalence of yellow fever and malaria delayed the construction of the panama canal, and sleeping sickness still limits human settlement and thereby affects human access to ecosystem services in parts of central africa (bhalla 2002) . a major challenge in this field is to apply real world data to conceptual models (miranda and others 2002) , to validate the models, and to develop approaches that can serve as a vehicle for generating hypothesis-driven research. although realization of these goals remains distant, the conceptual frameworks which will stimulate data acquisition and analysis in this field are developing (butler and others, 2003) . figure 1 shows one such framework, linking natural and social systems with human wellbeing, of which health is an important component. here we introduce four categories of adverse effects on human health due to ecosystem change as a means to help understand the impacts of the different ecological scenarios on human health and well-being. in ascending order of scale, complexity, and lag-time, we call these adverse effects direct, mediated, modulated, and systems failure (see table 1). we emphasize that, at their margins, these categories overlap because drivers may differ on temporal and spatial scales. figure 2 graphically presents these concepts and provides a preliminary attempt to approximate the quantitative impact of the different categories. direct (adverse) health effects are manifested through the immediate impacts of the loss of a useful ecosystem service, such as the provision of sufficient food, clean water, fertile soil or the restriction of erosion and flooding. direct effects occur as the result of physical factors but do not include pathogens per se. miranda and others, 2002) climate change has recently been recognized as causing a substantial change in the lake tanganyika ecosystem. the fish catch has decreased due to a climate-related reduction in the nutrient supply (o'reilly and others 2003) . this reduction in ecosystem services places additional economic and nutritional stresses on an already poor and vulnerable human population. although data to measure the health effect of this reduced catch are unlikely to be available, the effect is probably adverse because it causes reduced income and reduced nutrition (verschuren 2003) . another example is the collapse of cod fishing in the north atlantic, which caused widespread unemployment, mental distress, and social dislocation, but little if any true under nutrition because the social mechanisms operating in canada were able to partially substitute for the lost provisioning services once supplied by the fishery. a third example of a direct health effect from a reduced ecosystem service is the disruption and physical injury caused by flooding. there is increasing recognition that floods are caused by the interaction of climatic and landuse changes (hellin and others, 1999; zhang and others, 2000) . there is also increasing evidence that mental and physical health is enhanced by contact with nature (friedman and thomas 1995) . reduction of the cultural services that ecosystems provide is likely to contribute to the already enormous burden of disease caused by impaired mental health. compared to direct effects, mediated effects have increased causal complexity and, in some cases, involve pathogens. some mediated effects have the potential for high rates of illness and death. there is also often a longer lag between the ecosystem change and the health outcome than for direct effects. however, by definition, mediated effects are insufficient in scale to cause the larger-scaled social collapse that we define as a modulated effect. many infectious and some chronic diseases fall in this category. the epidemiology of many communicable diseases is related to ecological factors. some major nonvector-borne diseases, including tuberculosis, measles, and influenza, are thought to have crossed into human populations because of close contact with domesticated animals (mcneil 1976; daszak and others, 2000; oxford and others, 2002) . changes to biodiversity may be associated with increased numbers of disease-transmitting insects. although contested, there are suggestions that malaria may also have become a significant human disease following the development of agriculture (pennisi 2002; joy and others 2003) . more recently, variant creutzfeld-jacob disease, nipah virus, and hendra virus illustrate novel infectious diseases that have entered human populations because of changed and more intensive animal feeding and farming practices (waltner-toews and lang 2000). the emergence of nipah virus may also have been related to bats fleeing from the intense drought and el niñ o-related fires in indonesia (epstein and others, 2003) . the long list of other infectious diseases related to ecosystem change (patz and others, 2000) includes schistosomiasis (li and others, 2000) , cholera (pascual and others, 2000) , and lyme disease (jones and others, 1998; blockstein 2000) . in many of these cases, the disease has emerged as a result of increased food-producing capacity of ecosystems-a provisioning ecosystem service-for example, by animal domestication, irrigation, dams, and other intensive farming practices. a tradeoff has been the unforeseen increase in the incidence and prevalence of many of these communicable diseases. some mediated health effects have also led to migration, while others have prevented the colonization of certain areas. for example, malaria has long restricted human settlement in lowland areas, including the terai in nepal, and many parts of equatorial africa. some chronic, noninfectious diseases can also be classified as mediated effects of ecosystem change, including allergies, asthma, and some forms of cancer and chronic lung disease. for example, lung cancer and pulmonary fibrosis have become particularly common in the region around the shrunken aral sea, as pesticide-contaminated dust from this human-made desert is inhaled (o'hara and others 2000). both long-distance dust transport and more localized air pollution are also related to ecosystem service change and have been linked with a number of diseases, including asthma and atopy (monteil 2002) . there is also increasing evidence that air pollution, often exacerbated by ecosystem change such as land clearing and fires, may aggravate heart disease (pyne 2002) . future ecosystem change, such as desertification, leading to a decrease in the ecosystem provisioning service of clean air, could thus alter the epidemiology of these diseases. these diseases are classified as mediated rather than direct because their connection to changed ecosystems is more complex than are direct effects. direct and mediated health effects are analogous to the direct and indirect health effects of climate change. in that classification, direct (adverse) health effects include phenomena such as heat stroke, while indirect effects include changes to certain vector-borne diseases because of altered patterns of temperature, humidity, and rainfall, and other effects secondary to extreme weather and adverse economic effects. it is possible that a novel emerging disease could escape from a remote ecosystem to enter the wider human population, as the plague and hiv probably did. however, at least in the case of hiv, its really major (modulated) impact depended on powerful social cofactors, including severe poverty, social practices and taboos, and poor governance (butler 2000a (butler , 2000b . the ecological factors that underlie the recent sars outbreak remain unclear (enserink and normile 2003) , but its origin and amplification in a region of china, characterized by extremely dense populations of humans and domesticated animals and by poor public health services (anonymous 2003) , is consistent with the view that human-dominated ecosystems today harbor more danger to population health than does the ''wild'' (oxford and others 2002) . a plausible example of this principle could be the widespread transmission of multi-or even omni drug-resistant tuberculosis emerging from a prison (tanne 1999; dye and others, 2002) . this scenario would have severe economic implications, especially for aviation and other industries perceived as increasing the probability of disease spread. we also identify a larger-scale, more lagged, and more causally complex adverse consequence of adverse ecosystem change, than either direct or mediated effects, which we call a modulated or tertiary effect (figure 2 ). these effects include episodes of state failure, or of nascent or realized large-scale social and economic collapse. the role of environmental factors in the causation of large-scale conflicts, state failure, and social collapse is controversial (deudney 1991; gleick 1991; homer-dixon 1994; uvin 1996; cramer 2002) . we agree that causation for the phenomena is complex, but we assert that reduced ecosystem services and other adverse ecosystem changes are frequently a component of the causal webs that lead to these phenomena (butler and others 2003) . this may be of increasing significance in the near future as evidence accrues that ecosystems are being changed more frequently and at larger scales. there is compelling evidence that reduced ecosystem services were a causal factor for several large-scale social collapses and catastrophes, from both archeological sources (weiss and bradley 2001) and more recent history. two ancient cases are the collapse of the ancient mesopotamian and the mayan civilizations, contributed to, respectively, by increased salinity (jacobsen and adams 1958) and drought (haug and others 2003) . two more recent examples are the irish famine of the 1840s and the rwandan genocide in 1994. the irish famine was caused by the spread of a potato fungus (wilson 1995) interacting with a refusal by the british government to supply an effective substitute, such as famine relief (sen 2000, pp 170-5) . the rwandan genocide also occurred as a result of the interaction of multiple factors, including poor governance, long-standing ethnic hatred, and rapid population growth. the violence was inflicted mainly by a large number of unemployed young men (mesquida and weiner 1996; potts 1999) , displaced from a livelihood in farming because of the shortage of fertile arable land, thus losing a key ecosystem service (andré and platteau 1998; butler-2000a) . in these examples adverse health effects are likely to be larger than those from mediated effects, although in some cases state failure may be limited to small populations, such as for the people of easter island or the norse in medieval greenland. inevitably, ecosystem service changes that contribute to modulated effects will be embedded in a mosaic of social, economic, and political cofactors. in turn, many of these cofactors are likely to have at least partial ecosystem change-dependent causation. depending on the knowledge, bias, and experience of the observer, the causal role of ecological factors in state failure may sometimes be underestimated, or even totally denied. for example, rotberg (2002) identifies the roots of state failure as based in ethnic, religious, linguistic, or other intercommunal enmity. he argues that state failure is ''man-made, not merely accidental nor-fundamentally-caused geographically, environmentally, or externally.'' we do not claim that reduced ecosystem services or other ecosystem changes that lead to adverse health effects are always a ''fundamental'' factor in state failure, but they are often important and usually identifiable. the enmity that rotberg refers to often arises over the distribution of diminishing per capita ecosystem services. there may be increasing recognition of this. for example, o'reilly and others (2003) concludes, in discussing the potential for further reduction in the ecosystem provisioning service of lake tanganyika, that ''the human implications of such subtle, but progressive, environmental changes are potentially dire in this densely populated region of the world, where large lakes are essential natural resources for regional economies.'' ecosystem services as a significant element in state failure may be underrecognized due to our tendency to discount the future possibility of thresholds or emergence. thresholds refer to sudden, nonlinear changes that result from a small increment and that are not intuitively predictable without prior experience (alley and others 2003; waldrop 1992; may 1999) . emergence refers to the new property that becomes apparent beyond the threshold. modulated and systems failure effects (described below) are emergent phenomena that become apparent when linked socioecological systems pass a threshold, caused by the interaction of numerous social, political, and ecological elements. we also describe an even larger scale phenomenon than state failure, as a result of coalescing, interacting modulated effects. we call this phenomenon ''systems failure.'' the increasing connections that insulate diverse human communities from scarcity also create large-scale vulnerabilities, magnifiable by feedbacks such as collapsed global trade, terrorism, technological breakdown, and radiating failure of institutions and governance. collapse could occur on a regional, continental, or even global scale. it is also possible, however, that a reverse state, ''systems success,'' could occur. large-scale epidemics exacerbated by chronic food insecurity, poor governance, and wide-scale conflict are plausible elements of this pathway. drug-resistant bacteria, in part driven by the excessive use of antibiotics in animal husbandry, could contribute to this, as could the emergence of new viruses. however, we stress that novel infectious agents are unlikely to lead to modulated or systems failure effects without significant cofactors. only modulated and systems failure effects are likely to be of sufficient scale to alter the unfolding of the various ecological and socioeconomic scenarios that are described elsewhere in this issue of ecosystems. cumming and others (2005) and raskin (2005, this issue) review several socioecological scenarios that may unfold over this century. although all plausible futures are influenced by the same driving forces, these forces evolve in different ways in the different scenarios. demographic, economic, political, cultural, and social factors -including health -are codependent so that each factor will continually influence other factors. each scenario will influence and be influenced by ecological factors as well. just as in the past, the future world will contain a mixture of familiar and novel situations. scenarios seek to coalesce these myriad possibilities into a limited number of theme futures that have strongly plausible elements. although there are dozens of names for the existing environmental scenarios, most can be categorized into a surprisingly small group of core pathways, as described by cumming and others (this issue) . the names of the four scenarios considered in this article are market forces, reformed market, value change, and higher fences (see table 2 ). it is difficult to succinctly describe these scenarios, but a number of axes can be identified along which they vary. for example, there is a spectrum identifiable between comparative economic deregulation (market forces) to a neo-keynesian model, here called reformed market. another spectrum can be identified between a concerted attempt to protect existing ecosystem services (the ''value change'' scenario) and a laissez faire approach to ecosystems and the nonliving environment, such as climate change (the ''market forces'' scenario). a third spectrum is identifiable between trade deregulation (''reformed market'') and continuing or even increased protectionism (''higher fences''). as well, the trend of global income distribution can be predicted from these scenarios, from a continuing increase (''higher fences'') to a marked decrease (''reformed market'' and ''value change''). the state of health for high-and low-income populations can be predicted, largely consequent to the anticipated change in income for each group. three of these scenarios, as very briefly described, are essentially optimistic, because they all assume an increase in income for high-and low-income populations. however, in the higher-fences world, it is conceivable that incomes will decline for populations that currently have a low income, and the increased global inequality in this scenario could exacerbate tensions between low and income populations. cumming and others (2005) examine the assumptions made about ecosystem resilience in each of four scenarios and find that the outcome of different scenarios is influenced by this resilience. table 2 lists some key terms concerning health for both high-and low-income populations in these four scenarios. it also shows our estimation of the probability of changes in the ''health gap,'' that is, the gap between high-and low-income populations with respect to health. at present, for example, the burden of disease from diarrhea (of which nearly 90% can be attributed to unsafe water, poor sanitation, and hygiene) is over 100 times higher in the least developed countries than in developed ones. the health gaps between high-and low-income populations have been systematically estimated using a measure called ''disability adjusted life years'' which accounts for total years of life lost because of disease and also for partial years of life lost because of disability (world health report 2002) . it is likely that in three of the four scenarios the health gap will decrease. this is because three of these scenarios assume gradual socioeconomic convergence between populations that are currently rich and poor. these scenarios postulate continued advances in science, technology, and the dissemination of information and expertise. the response to the sars outbreak illustrates the potential for a coordinated response to events that are perceived as sufficiently threatening. if convergence between rich and poor populations occurs, then coordinated responses to numerous health problems that continue to affect poor populations are likely. advances could improve new vaccines, attention to ''orphan'' diseases, and dis-tribution of the improved seeds and agricultural techniques needed to enhance food security. in these scenarios the health of high-income populations is also likely to improve, though not as rapidly as for low-income populations. in only one scenario -called ''barbarization'' (raskin, this issue) or ''higher fences'' (cumming and others, this issue) -is the health gap likely to increase. in this scenario, poor populations are increasingly ignored by the remaining population. food security of the poor is likely to further diminish, perhaps leading to positive feedbacks as malnutrition impairs cognitive development and further hinders education and the chance of skillful social and political responses. in this scenario the health of high-income populations is unlikely to be ideal: we identify obesity, diabetes, and anxiety as likely to increase, with their negative effects only partially countered by improved medical technology. modulated effects could sabotage even optimistic scenarios. table 3 provides an estimation of the chance of systems failure, depending on assumptions concerning the resilience of ecosystems and the linked socioeconomic system. cumming and others (2005) have defined ecosystem resilience as the capacity to absorb anthropogenic impacts without the loss of essential structure or functions. we suggest that it is meaningful to assume that human populations are characterized by social resilience, which modifies their capacity to effectively deal with stress (carpenter and others 2001) . from a public health perspective, resilience may be defined as the capacity of society to respond to problems and challenges, over the short and long term, in ways that protect and advance public health, over the short and long term. affluence, education, social cooperation, technological capability, and flexibility are important determinants of the size of social buffers and human resilience. we suggest that systems failure effects are more likely to occur in the market-forces and higher-fences scenarios, because inequality between high-and low-income populations is likely to be the greatest in these scenarios. the most optimistic future for human health is likely to be if both ecological and social systems prove highly resilient. in this case both major ecosystem and social services are likely to be preserved even if ecosystem changes (currently perceived by many as adverse) continue to occur at a high rate. on the other hand, the chance of further modulated or even systems failure effects occurring is increased if ecosystem and social systems prove to be brittle. because the size of ecosystem service buffers is falling and the extent of ecosystem service resilience is uncertain, it is prudent to reduce further ecosystem damage as rapidly as possible. however, it is also prudent to conduct this in a way that minimizes harm to human health. continuing tradeoffs are likely to be required. for example, improving the health of populations in indonesia may require further clearing of forests to generate more income. but this process cannot be continued indefinitely. on the other hand, excessively strict protection of the surviving ecosystems could reduce the size of the socioeconomic buffer, thus perhaps increasing the longterm risk of a modulated effect. in reality, economic and social forces make an extremely rapid transition to full protection of surviving ecosystems unlikely, but it may be just as risky to delay protection in response to only short-term socioeconomic concerns. the causal relationship between ecosystem service change and impact upon human health remains incompletely understood. we have explored how ecosystem services impact human health and have proposed that adverse ecological changes can interact and feedback with dysfunctional social responses, leading to the development of states that we have termed mediated and systems failure. we have grouped the myriad possible interactions and cascading responses between ecosystem services and public health into four categories, a previously undescribed taxonomy which might help us better comprehend how ecosystem change and human health interact to affect the way the future unfolds. we believe that this is an important conceptual advance that will be useful for understanding the relationship between human health and changes in ecosystem services. continued analysis of cases studies, using both quantitative and qualitative methods, will advance our understanding of these relationships. several regions of the world, characterized by substantial ecological and social stresses, may be useful ''natural laboratories'' for this purpose, including sub-saharan africa, indonesia, and northern india. direct and mediated effects, although they may lead to some important changes to the path of the future, are unlikely to seriously compromise the development of regional or global civilization. despite occasional and localized setbacks, human health is likely to generally improve if future ecosystem changes result only in events such as occasional flooding, periodic disease outbreaks, and episodes of air pollution. on the other hand, modulated and systems failure effects, if they occur, have the power to alter the course of society in significant ways. if negative this will cause substantial harm to human health and well-being, and, by exacerbating poor governance, could also further erode ecosystem services. such events could derail even the most optimistic scenario. however, we do not deny the chance that positive systems effects could emerge, especially if ecosystem and social resilience remain high. current trends toward an increasingly large environmental footprint, further climate change, depletion of fossil fuels, and the erosion of existing ecological and social buffers are disturbing and unlikely to be sustainable (mcmichael 2001) . on the other hand, the increasing capacity to conceptualize, diagnose, and modify the global environment gives hope that humanity will self-organize in ways that can sustain both its social and ecological functions (crutzen 2002) . abrupt climate change land relations under unbearable stress: rwanda caught in the malthusian trap emerging stronger from the china crisis pan african group takes lead against the tsetse fly lyme disease and the passenger pigeon? entrapment: global ecological 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survival after acute myocardial infarction in the cardiac arrhythmia suppression trial (cast) environment and security: the clear connections climate and the collapse of maya civilization rainfall characteristics of hurricane mitch environmental scarcities and violent conflict: evidence from cases greater lifetime expectations salt and silt in ancient mesopotamian agriculture chain reactions linking acorns to gypsy moth outbreaks and lyme disease risk early origin and recent expansion of plasmodium falciparum epidemiology of schistosoma japonicum in china: morbidity and strategies for control in the dongting lake region how the biosphere is organized human frontiers, environments and disease: past patterns plagues and peoples human collective aggression: a behavioral ecology perspective policy concepts and applications dust clouds and spread of infection alternative projections of mortality and disability by cause 1990-2020: global burden of disease study exposure to airborne dust contaminated with pesticide in the aral sea region climate change decreases aquatic ecosystem productivity of lake tanganyika world war i may have allowed the emergence of ''spanish'' influenza cholera dynamics and el niñ o-southern oscillation effects of environmental change on emerging parasitic diseases malaria's beginnings: on the heels of hoes? ecological integrity: integrating environment, conservation, and health. washington dc the population policy pendulum. needs to settle near the middle and acknowledge the importance of numbers small particles add up to big disease risk global scenarios: background review for the millennium ecosystem assessment rising life expectancy: a global history the new nature of nation-state failure development as freedom changes in life expectancy in russia in the 1990s rapid economic growth and the four ds of disruption, deprivation, disease and death: public health lessons from 19th-century britain for 21st-century china? drug resistant tb is spreading worldwide a universal pattern of mortality decline in the g7 countries world population prospects: the 1998 revision. the demographic impact of hiv/aids tragedy in rwanda: the political ecology of conflict global change: the heat on lake tanganyika complexity. the emerging science at the edge of order and chaos a new conceptual base for food and agricultural policy: the emerging model of links between agriculture, food, health, environment and society what drives societal collapse? infectious disease: an ecological perspective world health organization china's forest policy for the 21st century we thank the millennium ecosystem assessment for providing many opportunities to discuss the main ideas expressed in this article with colleagues. we also thank professor d. crawford-brown and two anonymous reviewers for their comments and suggestions. key: cord-023647-dlqs8ay9 authors: nan title: sequences and topology date: 2003-03-21 journal: curr opin struct biol doi: 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family members a novel neutrolphfl chemmtttactant generated duan8 an ln~ammmtory reaction in the l~mt peritoneal cal~lt~ tt~ t~t~o -l~tl'~t~tloil~ ~ amino acid seque~tce and structural relmtmmhip to interkukin-& b~ffx~m j the multlfimctinna 6-methylmllcyllc acid syn~ ge~e of ~~ ~ its ge~e structmm ieimive to tl~t of other po~lyketide symhase~. f.urj b/odaem 1990 mammalkm ublquitin carrier prmmtmh but not i~:i~k, ame ltdated to the 20-kda yeast 182, rad6. bk~chem b/qohys res commun chambers gk: sequence. structure and evolution of the c.ene codin b for ~t-gi~erol-3-phe~plmte ~rdrotfm~ in om,qt~ the cotaplete sequence of bogu/ktmm nenrotoxin type-#, and com~ with other clostrldhtl neugoto~hm if: a pamlly of cxam~fltutive c/bbp-llkc dna blndln~ proteins attenuate the il-l~t induced, ni~b mediated trans-activation of the ansiotemflnogen gene acute-phase response element different fort~ of ultmhithomx proteim generated by alternative spttcim~ are functionally equivalent evolution of collagen-iv genes from a 54-batm pair faton --a role for lntrmm ht gem~ evolution evolution of the insulin superfamlly tcetins are structoraily related sertoli cell proteim who~ ~on is tightly coupled to the iprtsence of germ cells ivarie r~ a bovine homolo s to the human myolletti c determination factor myf~ sequence conservation and 3' proce~ing of transcripts proteiu sertne threonine phoephatmes -an expanding family coppes zl divergence of duplicate genes in three sciaenid species (perciformes) from the south co~t of uruguay coasfaneda m: rrs~j~o~a (mu-~--a~) repetitive dna seqmmce l~vointion in 3 ~hically mstinct isolates. cor~0 bnz~n physiol repetitive seq~ce involvement in the duplication and divergence of mouse lysozyme genes the structure of a subtermlnal nut/e/6 a6/ds res 1990 schoofs i~ h~ between amino acid sequenc~ of ~ v~'lt~tm'stte peptide hormones and peptides ~mlated fi-on~ invertebrate sources. corn# bm&.n mg~ol bun'nng s, ~us r& lqatelet gtycoprotetn nb-ma protein antssonim from snake venoms ---evidence for s fumlly of p~telet-~sgqpttlon lnhll~tol~ hikher plant orilgins and the whylogeny of gt~en allpte simihtrity between the t~ ~ sindln s proteins abf1 how big is the univet~ of e~otm worklwide diffegences in the ~ncideace of type ! diabetes are ammciated with amino acid variation at pos/tion 57 of the hi~-dq ~ chain yeast general trtnscelptimt l~ctor gf! --sequence requirements for binding to dna mad evointhmky commrvttion. nudeg m/ds res concerted ]rv~ution of primate mplm smelllte dna. e'~kmce foe tm an~mt~ sequence sbm'ed by goal~ md human x ~e alpha ~ttdllte the nuchl~m~ sequence of etve ribommaal protein genea from the o/anene. of ~~ impacattom concem~ the mtytosene~ relationship bet~-en cyanelles and chloropluts wmslanoer l~ a new member of a secretory protein gene family in the dipteran c~t~onomot~ tentaus ~ a variant repeat stracture the ~r sequence ~ --die.inn on the x-chromosome and y-chromosome of a large set of closely related sequence~, most of wmda are i~eudogene~ ba~ttmo~e l~ cloning of the pso dna binding subutdt of nf-kapi~-b -homolo~" to gel and dortml l-~te two-monooxr~muse from m~ --clon~ nucleotide sequence, and primary structu~ homology within an enzyme family genetic hot~o~n~ty ~ acute and chronic acute forms of spinal muscular atrophy genetic variants of bovine ~-lactogiobulin --a novel wild.type ~-lacto#obulin w and ~ts primary sequence. b/or (~rn h0tt0e sey/er l~ltogh~ dna evolution in the olmcm species subgroup of drooophll~ f mot evot lovell-badge l~ a gene mapldng to the sex-determining gegion of the mouse y chromommae ~ a member of a novel ~ of zmbryonk~ly genes ~titmte 1,2-dioxy~mm~ from p~.udomotm~ pustfi~mtion, characterization, ~md compm'tson of the f.mtymes from psemffmmm~m ta~o~k-ron/and aaammms~ spec~clties of the peptidyl prolyl cis-tratm isomeric activities of cydophmn and fk-506 bindh~ protein --evidence for the existence of a family of distinct enzymes. b~x/aem/ary mltochondrl~ dna evolution in primates -tt-atmltion gate has been extremely low in the lemug homeobox containing genes in the nematode ~enorbabd/f~ elk.gamin nucleic ac shdic add fateesses of ~ • voluttomu.y origins have serine active sites f~entlal arginlne residues dewact-rrer l~ the 188 ltilm0omal rna ~-quence of the s~t anemone anemom~s ssdcmta and its evolutionary intuition amomqg other eukaryotes inferred b'om s~l,.m.~ comlmrttmas of a heat shock g~ae in two nematorl~ the l~'/o multtgene family of ok~hag of cdna ~ for the ~ omin of human complement component ca~bi~una protein, seqaenoe homolo~ with thc a c~t~:~a~h proc natl acad s¢t usa1990 highly conserved core domain and unique n terminus with presumptive regulatory moti~ in a hmman tata factor (l'lql~) [letter] identification cimractertzaflon of a novel member of the nerve growth fmctor/besln.dertved neurotrophic factor family ~ bind8 to s~dlfmme [eal(~-so4)l~l-lcer ] and has a sequence homology with other pt'otelns that bind sulfated glycoconjut~tes anllllo acid seqmmce of clnnamomin, a new member of the elicitin family, and its comparison to cryptogein and capsicetn soluble and mtmo[~tle~ioc~ta~l h~ low-ml~n|ty adenomne binding protein (adenotin) --properties and homology with mtmmall~la and avian stress protelus. b~-/~om/stry edolatlon of complementary dna$ f~lcoding a cerebellum-enriched nuclear factor-i family that activates tt'anscription from the mouse m~.lin basic protein promoter ye~mt mltochondrlal dna polymet'ase is related to the family a dna polymerases nudeotide and deduced amino add sequence of a human cdna (nqo2) corresponding to a second membeg of the nad(p)h --quinone oxldoreductase gene family --extensive polymorphism at the nqo 2 gene locus on chgomo~ome-6. b/oc.heraistry ult~ sltnlltt'leles a~llolltll enzyme pterin binding sites as demonstrated by a monoeinnal amiidiotypic antibody blundell tl molecular anatomy: phylogenetic relationship* derived from three~limenslonal structure~ of proteins subfamily structure and evolution of the hnmtn 1.1 family of repetitive scquence~. f mot evo 3 selmt~te mltochondrlal dna sequences are contiguous in htlmsa~ genol~ic dna l~t~lit~ within mmmm~lla~ sogl~tol deh~ --the prlmm'y structure of the human liver enzyme heterogeneous modifications of the l14/alo ltrote~a of ibtegleuldn-~t cells are concentrated in a/,ti~hly r~qg~.titlv ~ amino-t~ vaults.ell rebofmcleoprotein structures are msl~ conserved among higher and lower e~tes rnas le~d support to the monophyletic nature of the ~erla lmmunoloslcal ~lmllmtties ~etween cytosolic and partictdate tissue trans#utamilsc. febs lat mans~ti x#tope m~w~zed by a protective m~aodonm antibody is identical to the sta~e-specific embryonic antlgen-l. proc naa acad sa o~ 1990 the murg3 gene of t-brucei contains multiple dom.l.m of extensive editinil and is hofaoin~m~ to a subultit of nadh dehy~ neparm-bindl~ nenrotrophtc x~tor (hbnf) and mk, member's of z new i~mily of homolosous~ developmentally l~ted proteitm pugmattion and strucrmml ~on of pttcentel nad + .mtked 15-hydroxyproma#andm dehydtoffmase ~ the primary structure reveals the enzyme to belon 8 to the short-alcohol l)ehydrogena~ l~mlly. b/ochemistry structores and homologies of carbohydrate ~pho~ system ep~l~[ln, a ~o~a-gmjoclated mudn, is generated by a polymorphlc gene encodin8 splice variants with alternative amino termini a new member of the leucine zipper class of proteins that binds to the hia drct promoter. sc/ence attalysi~ of cdna for human ~ ajudgyrin i~dicltes a repeated structure with homology to tissue-differentiation a~td cell-cycle control protein the b subunlt of a rat hetefomeric ocaat-binding transcription factor shoes a striking sequence identity with the yeast hap2 transcription factor homology to mouse s-if and sequence similarity to yeast pt~2 stgucttu'e and evolution of the 02 small nuclear rna multigene family in primates: gene amplification under nat-¢wal selectinn? ident~catinn of an additional member of the proteln.tyrushle-phosp~ family --l*vidence f~ alternative spliclog in the tyrmine phosphzmme domain a 8~le am~o acid difference dis~ishes the human and the rat sequences of statlmaln, a ubiquitous intracehular pho~phoproteln ~ with cell item comp~ison of the seve~le~ gene* of drosop~ffa t~'ff~ end ma4~ muty, an adenine ~ active on g-a mislmirs, has homology to ~t evolution of largesubunit iutna structuge --the ~cation of imvetbe~t d3 dommin amon8 mmjor phyiolpmetic groups discrepancy in diveqlenoe of the mltodtondrlal and nuclear genomes of m sensor/and y~ j mot evot 1~90 adenylate deamll~t~. a mt~flige~e fam~ in p..m~,n, and rats isolmion and structure of ceerol#m, itna,~le hat~ peptmes, from the smm~m, ~ mo~ comp a~a rmm~ i~ vmotocin ge~ of the teleom f.,xott intro~ botany. ~ hot~ ot'l~mization. b~hemioy the adb gene areal share features of sequence structure and nudeast~protected sites. m0/cell bto/1990 the amino-acid sequence of multip/e lectins of the #.corn barnacle m~us-lgo~ and its homology with .animal ]~'tllls. bioclx'm btqobys acta amino add ~.-quence of mtmkey erythrocyte glycophorba mk. its amino acid ~'qu~'~icc ]f][~ a stri~tl~ homology with that of human glycophorin a flsp~r p& drtmophila proliferating cell nuclear antigen. structural and functional homology with its mammalian coonterpart phylogeny of n|trogen*me s~queac~ in ][~mnkla and other nlteogen-fixing ml~m$ vertebrate prot~mlne c~ne evolution.1. sequence alignments and gene structure florin l~ a major styl~ matrix polypeptid~ (sp41) is a member of the f~thogenesia-reiated proteins superciass complete amino acid sequence of rat kidney ornithine aminoteat~fet-~e --identity with ijver omithine aminotransferme. l bnxl;em (tokyo) rlbonuclease p --function and variation. j b/o/~bem the primary strum of glycoprotein-m from bovine adrenal medullary granules --sequence similarity with bnmmn serum protein-40,40 and rat sertoli cell giycoprotein-2 compm'ative ~quence/umlysis of m~mmantan f'a~or ix protaotegs the amino acid sequence of the b nman l~ia polymet'a~-h 33-kda subunit hrpb 33 is highly cotmerved among eukaryotes phylogenetic conservation of atylsulfatases --cdna cloturing and expre~ion of hnman aryisul~t~e-b. j b/o/cbem c.oll/l~'vlltion and diversity in fatnllies of coated vemcle adaptlns cllaracterizaflon of petel porcine bone sialoproteins, soca'~ted phosphopgotein ! (sppi, osteopontin), bone siaioprotein, and a 23.kda glycoprotetn ~ demonstration that the 23-kda glycoprotein is derived from the carboxyl terminus of sppi characterization of matteuccin, the 2.2s storag~ prote~ of the ostcich fern -evolutionary iteiatinnshlp to angiosperm seed storage ~ a new mmber of the glutamine-rlch protein gene family is characterized by the absence of internal lgepe~ts and the androgen control of its expression in the subm*ndlbuiar gland of pad 2 novel insect n~ with homology to peptides of the vea'te~ tachykinin family identircation of a novel platelet-derived neutrophli-chcmaotgctic po~ with structural homology to piatelet-factor-4 a novel repeated dna sequoncc located in the intergenic regions of ba~tceial chromosomes. nuc2eic.,k:ids res the proianlin storage protellx¢ of cere~ seeds ~ structure and evolution functional analysis of the 3'-terminal part of the balbiani ring gene by hlterspecies sequence comparison dr= mammaban ~yl phosphate symhetase (cp*) --cdna sequence and evolution of the cl m domain of the syrian hamster multifunctional protein cad mammalian dihydroorotase --nudeotide sequence, peptide sequences, and evolution of the imhydroorotsse domain of the multifunctinnal protein cad a receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins the control of flower morphogenesis in a~..ffd~um majusthe protein shows homoinff~ to transcription factors an element of symmetry in ytmst tata-box binding protein transcription factor-lid --consequence of an ancestra/ duplication? c-type natciuretic peptide (cnp): a new member of nateinretic peptide family identified in porcine brain evolution of antioxidant m~: ediol-dependent petoxidm~.s and thiol~ ~umong ptocaryotes towards the evolution of ribozymes alkyl hydroperoxide reductase from sa/mone/ta ~ur/um --sequence and homology to thinredoxin reductase and other fiavoprotein disuliide oxidoreducmses fc: nonuniform evolution of duplicated, developmentally controlled c~azrion genes in a sillumoth the fission yeast cutl + gene regulates spindle pole body duplication and has homolosy to the buddin structural homology b~ween the hnmmn fur gene product mad the sub---like protea~ encoded by ye~t/~x2. nuc~ a¢/ds res 1990 nudeotide sequences and novel steuctut~ features of hnm=. and cimm~ lighter ~# primary stt~t~ and expression of a nuclear-coded subunit of complex-n n~ to protetm specified by the chtoropiast genome. b/0chera bnfhys r~ commun a novel gene member of the human giycophorin-a and glycophorin-b genc fatuily -molecular cloning and expression the x-chromosome of monotremes shares a highly conserved region with the eutherlan and marsupial x-o~romosomes despite the absence of x-chromosome ittactt~tion c~lract~tion and or~= nl~tion of dna sequences adjacent to the evidence for a new fmily of evolutionarily conserved homeobox genes elellatltlll and albolabrin purified peptides from viper venoms --homologies with the rgds domain of flbrinogen and yon willebrand pactor measurement of $~tiv~-site homology between potato and l~bbit muscle alpha-glum phosphoryiases through use of a iane~r free energy relationship white 1~ weiss 1~ the neuroflbromatosis typed gene encodes a protein related to gap the dna damage-inducible gcne-dinl of saocbarom3q~ewcet~#.s/ae encodes a regulatory subunit of elbonucleotide reductase and is identical to gnr3 fhlgegprinting of ne~lr-homogeneous dna hgase-i and ligase-h from eh,m~n cells --similarity of their amp-binding domains control of m11na st~mlity in • chnoc~qg.~um, by 3'inverted ltepeats: effects of stem and loop mutations on degradation ofxtmba mlna/n vt~ nuc/e~ ac alternative messenger rna structures of the ciil-gene of bacteriophage ~. determine the rate of its tt'ansbttion initiation alternative mrna structures of the cm genc of bacta~ophage ~ detc:'mine the rate of its translation initiation. j mo/b~0/1989 a model fog iina editing in klnetopiastid mltochondrla --guide rna molecules transcribed from max/circle dna provide the edited information elements and coding sequences. j mol bio11989, 210:417-427 . chang c-y, ~ d-a, mohandas til chung b-c: stt~ctut~e, ~-quence, chromo~maal location, and evolution of the human fercedoxin gene family. dna cell b/o/1990, 9:205-212 key: cord-005159-6agnsbyd authors: turner, bryan stanley; dumas, alex title: vulnerability, diversity and scarcity: on universal rights date: 2013-07-12 journal: med health care philos doi: 10.1007/s11019-013-9500-6 sha: doc_id: 5159 cord_uid: 6agnsbyd this article makes a contribution to the on-going debates about universalism and cultural relativism from the perspective of sociology. we argue that bioethics has a universal range because it relates to three shared human characteristics,—human vulnerability, institutional precariousness and scarcity of resources. these three components of our argument provide support for a related notion of ‘weak foundationalism’ that emphasizes the universality and interrelatedness of human experience, rather than their cultural differences. after presenting a theoretical position on vulnerability and human rights, we draw on recent criticism of this approach in order to paint a more nuanced picture. we conclude that the dichotomy between universalism and cultural relativism has some conceptual merit, but it also has obvious limitations when we consider the political economy of health and its impact on social inequality. the generic concepts of 'ethics of rights' and 'ethics of duties' (patrão neves 2009)-found implicitly in most official bioethics documents-can be viewed as two relevant ideas for a sociological study of human rights and global health policy. they identify basic human needs and socio-cultural conditions that should be safeguarded by political institutions. the fact that health is now considered a basic good within international conventions is an important point of departure for universal rights to health (unesco 2011) . the duties that are associated with these rights are also expressed by the moral obligation to develop a social contract that would achieve a modicum of social justice by for example reducing social inequalities. both dimensions of the ethics debate (rights and duties) converge on the notion of 'institution'. in sociology, the problems of developing universal institutions to achieve a civilized level of social protection, while respecting personal autonomy, lie at its core. in an effort to promote 'multidisciplinary and pluralistic dialogue' (unesco 2005) in bioethics, this article makes a contribution to ongoing debates about universalism and cultural relativism from the perspective of sociology. we argue that bioethics has a universal range because it relates to three shared human characteristics,-human vulnerability, institutional precariousness and scarcity of resources. these three components of our argument provide support for a related notion of 'weak foundationalism' that emphasizes the universality and interrelatedness of human experience, rather than their cultural differences. after presenting a theoretical position on vulnerability and human rights, we draw on recent criticism of this approach in order to paint a more nuanced picture. we conclude that the dichotomy between universalism and cultural relativism has some conceptual merit, but it also has obvious limitations when we consider the political economy of health and its impact on social inequality. the idea that different cultures produce not only different ethics and values but also vastly different ways of experiencing the world has become the dominant assumption of both anthropology and sociology. in terms of philosophical anthropology, our social being-in-the-world is deeply rooted in distinctive and separate sets of cultural practices, often referred to simply as 'habitus' (bourdieu 2000) . the implication is that we cannot assume that the experiences of sickness and disease, and experiences of the body are universal and it follows that some assumptions of western bioethics cannot be generalized. in sociology the problem of relativism occurs under the general discussion of 'social constructionism', namely that the phenomena of the social world have no consistent or permanent essence; they are always and already produced by social conditions. perhaps the classic illustration of the argument was the work of margaret lock (1993) on the cross-cultural experience of menopause in american and japanese women. she found that, while the discomforts of menopause in the united states were widely prevalent, japanese women did not experience negative symptoms to the same extent. medical sociologists therefore concluded that the social construction of menopause was at the source of its medicalization in some areas of the word. while social constructionism is a basic premise of modern anthropology and sociology, it has certain limitations in the context of rights. we defend the idea some conditions such as human vulnerability, precariousness institutions and scarcity of resources, are common to human societies and can serve as a grounding for future research in bioethics. in short we defend a position that we call 'weak foundationalism'. without rejecting cultural relativism, we argue that humans share a physical embodiment, which has significant consequences regardless of cultural variations. for example, the prospect of post-humanism is threatening to alter what it is to be human and is generating many ethical questions that appear to go beyond cultures or religious denomination; it is in this perspective that the study of embodiment in social sciences is central to ethical life (frank 2012: 395) . we also elaborate the notion of institutional precariousness that occurs in context of scarcity. the result is that over many issues we have to co-operate through mutual recognition just in order to survive. we start with the observation that cultural relativism runs up against at least two obvious counter arguments. the first is that the notion of cultural specificity is contradicted by the widespread assumption in the social sciences that globalization is the dominant form of social change in the modern world. globalism is especially evident in the fact that the world is shaped by a common technology and production system. for example, access to medical technology, international vaccination co-ordination efforts, and sharing of information through the world health organisation can be viewed as proof that most countries are to some degree part of globalized networks. while the interaction between global and local cultures often results in hybrid cultures that sociologists describe as a process of 'glocalization', there are important common processes that result in shared problems and experiences. medical anthropologists, by grasping the relativist implications of her work, can too easily ignore one of the conclusions of margaret lock's research, which was that japanese women would come to acquire menopausal difficulties as a result of globalization. this first point is supported primarily by the nature of human ageing, demographic data and considerations on the specificity of the social classification of disease. let us take two examples of the emergence of a common 'health world' with respect to globalization and health. perhaps the most important demographic revolution of the late twentieth century was the decline in female total fertility rates and the greying of human populations. this demographic change is more or less uniform regardless of cultural differences and especially religious differences. by the beginning of this century, only four countries in the world have a fertility rate above five, and half the world's population now live in societies that have fertility rates that are near or below the replacement level (macinnes and pérez diaz 2009: 150) . obviously there are important differences. china's one-child policy is very different from the demographic situation of the united states, but there are common global processes: the improvement in female education, the availability of contraceptives, rising prosperity of the middle classes and changing attitudes towards children. in association with changing fertility, there is the longer life expectancy and lower death rates that translate into a strong trend of ageing of the world's population. for most societies demography is central to various health, labour and economic policies. it would also be possible to construct a list of such shared health circumstances related to ageing-cancer, alzheimer's disease, strokes, and so forth. with globalization, there is the rapid transmission of conditions such as hiv/aids, sars, and the annual influenza outbreak. there are also more 'exotic' problems such as the arrival and spread of west nile virus to texas where 118 people died and 3,000 were infected in the summer of 2012. we can therefore legitimately argue that in the past humans lived in communities that were more or less isolated and hence diseases with geographically and culturally specific. this communal autonomy and isolation was relative. in the medieval world, the bubonic plague devastated human communities across much of europe. the modern world is very different. an outbreak of sars in east asia can reach ottawa in a matter of days if not hours. another example would be diabetes. there is a worldwide epidemic of diabetes. it is clearly widespread among urban, sedentarized and developed societies from australia to the united states, where lack of exercise, fast food and urbanization contribute to its rising incidence among young people. obviously more efficient detection and monitoring contribute to the growth of the disease, but it is also widespread among indigenous peoples from australian aboriginals to native americans. the second counter argument is the widespread, if not universal, acceptance of human rights. sociologists have suggested that the cultural contexts of moral debate are not as radically incommensurable as many philosophers suggest, and thus the process of globalization has provided a counter-balance to national and cultural diversity (mouzelis 2011). the contemporary almost universal acceptance of human rights suggests that the globalization of the principles of the declaration of 1948 can mitigate if not overcome the fragmentation and diversity of human cultures. there are of course many well-known problems with human rights, such as the difference between the acceptance and enforcement of rights (woodiwiss 2009 ). human rights began to emerge on the global political agenda in the 1970 s when growing dissatisfaction with the historic the role of states in the international order and widespread recognition of the failures of communism opened up opportunities for rethinking the role of rights in international affairs. human rights emerged as a serviceable ideology for a variety of social movements such as women's internationalism, political dissidents in poland and hungary, and as the basis of global ngo activity. the presidency of jimmy carter, who in his inauguration in 1977 declared an absolute commitment to human rights as the basis of american foreign policy, was also an important development. however, the critical turning-point occurred when academic lawyers came to embrace human rights as the normative framework of international law. these lawyers, who began to question the prevailing realist doctrines of international relations theory, embraced human rights as part of their core business (moyn 2010) . one standard argument against human rights has been that they are western and individualistic. but even this argument has lost a lot of traction. the so-called 'asian values debate' has more or less disappeared. at one stage both mahatir in malaysia and lee kwan yew in singapore sought to ground a view of human rights in confucianism with its emphasis on the family, order and respect, but for critics of these societies such values were thought to be a screen to hide the authoritarianism of their respective regimes (kamaludeen and turner 2012) . although the spread of human rights is far from complete, there is a growing network of international law that is binding on nations. the united nations convention on the law of the sea (1982) is a significant illustration of this development (charney and smith 2002) . the growth of legally binding relations within the european community has also been seen by legal scholars as an important example of legal internationalism. for example in 1951 the treaty establishing the european coal and steel community made provision for an independent court, the court of justice, to interpret and enforce of the treaty's provisions. another example is the creation of the european court of human rights in 1959. these international legal relations have multiplied with juridical globalization in clear recognition of the need to develop a set of universal norms to address global concerns relating to major issues, especially the environment (charney 1993) . in addition, important normative instruments developed in bioethics and human rights over the last decades (e.g., declaration of helsinki, belmont report, european convention on bioethics, universal declaration of bioethics and human rights) have identified a number of shared human conditions that should be preserved through political means. the notion of shared vulnerability-that is commonly used in bioethics as an answer to relativistic claims in health policy-is a good example in this regard. generally speaking, the notion of vulnerability holds two meanings. first, the word refers to a universal and persistent character of human beings (e.g., kottow 2004; luna 2009; patrão neves 2009; ruof 2004) . in some respect, it holds an ontological priority over other bioethical principles (solbakk 2011) . second, it holds a more variable status, which is dependent on a sociocultural context. socioeconomic inequalities increase vulnerability, and humans thus become vulnerated and, as a consequence, more susceptible to disease and shorter lives (kottow 2004) . essentially, global rights institutions and conventions protect humans because they are vulnerable. the arguments invoking a 'bioethics of protection' or a 'duty to aid' often put forward the significance of international solidarity as an answer to health inequalities (e.g., schramm and braz 2008; london 2005) . as stated in a recent report of the international bioethics committee: ''vulnerability might provide a bridge between the moral 'strangers' of a pluralistic society, thereby enhancing the value of solidarity rather than mere individual interest'' (unesco 2013: 2). economic development does not automatically reduce the vulnerability of every sector of society, and hence there is a continuing need for basic forms of protection. vulnerability, diversity and scarcity 665 with respect to recent biotechnological developments, various treaties and conventions on the integrity of the human species testify to the existence of a global risk society. in 'protecting the endangered human ' annas, andrews and isasi (2002) suggest an international treaty prohibiting cloning and inheritable alterations in response to species altering technology: 'prevention … must be based on the recognition that all human are the same, rather than on an emphasis on our difference ' (2002: 136) . we believe that sociological arguments about globalization and human rights can contribute to philosophical debates in bioethics since the empirical findings of sociological research have an obvious bearing on bioethics and health policy. however we do not want to present a counter argument in terms of various empirical examples. we need to develop our position at a much more fundamental and conceptual level. these examples from our discussion so far indicate that what human beings share in common, even when they are profoundly divided by culture and religion, is their ontological vulnerability. this point has been emphasized in vulnerability and human rights, in which turner (2006) argued from a sociological perspective that the concept of vulnerability, which is derived from the latin vulnus or 'wound', recognises the corporeal dimension of human existence, namely our embodiment; it describes the condition of sentient, embodied creatures, who are exposed to the dangers of their natural environment, and who are conscious of their precarious circumstances. our vulnerability signifies our capacity to be open to wounding, and therefore to be open to the world. this theme of human vulnerability clearly has strong religious connotations. it can be easily related to the christian tradition the symbol of which is the cross of jesus. but it can also be recognized in the teachings of the buddha. in a discussion of the buddhist idea of dukkha or suffering, robert bellah (2011:532) notes that it can also be translated as meaning that life is 'unsatisfactory'. one reason life is less than satisfactory is because we experience it as transient and tragic. he concludes that 'fundamentally it is the recognition of the vulnerability and fragility of life' (bellah 2011: 532) . one might also relate this concept of human vulnerability to the shi'ite tradition of islam with its profound sense of martyrdom and suffering. these comparisons suggest that vulnerability is not cultural specific but speaks to the human condition as a shared ontology. human beings are ontologically vulnerable and insecure, and their natural environment, uncertain. in order to protect themselves from the uncertainties and challenges of the everyday world, they must build social institutions (especially political, familial and cultural institutions) that come to constitute 'society'. we need a certain level of trust in order to build companionship and friendship to provide us with mutual support in times of uncertainty. we need the creative force of ritual and the emotional ties of common festivals to renew social life and to build effective institutions, and we need the comforts of social institutions as means of fortifying our individual precarious existence. because we are vulnerable, it is necessary to build political institutions to provide for our collective security. these institutions are, however, themselves precarious and they cannot begin to function without effective leadership, political wisdom and good fortune to provide an enduring and reliable social environment. however rituals typically go wrong; social norms offer no firm or enduring blue-print for action in the face of rapid social change; and the guardians of social values-priests, academics, lawyers and politicians-turn out to be all too easily open to corruption, mendacity and self interest. nevertheless the uncertainties and contingencies of everyday life also generate inter-societal patterns of dependency and connectedness, and in psychological terms this shared world of risk and uncertainty results in sympathy, empathy and trust without which society would not be possible. all social life is characterised by this contradictory, unstable and delicate balance between scarcity, solidarity and security. in its report on the principle of respect for human vulnerability and personal integrity, the international bioethics committee notably indicates that the 'most significant worldwide barrier to improving the levels of attainment of health through health care interventions is the scarcity of resources' (unesco 2011: 29) . drawing on sociology, in recent publications we have placed greater emphasis on this problem of scarcity (especially on the political economy of scarcity), because we believe that debates about human rights have often neglected some of the basic economic problems associated with rights claims. the idea of scarcity has been a basic assumption of economics in which, considering its most generic meaning, it signifies a shortage of means to achieve desirable ends of action. a shortage of income means that i cannot purchase basic commodities to satisfy needs such as food and shelter. adam smith in the wealth of nations recognized the often negative consequences of swings between years of plenty and years of scarcity, and in the latter case for example in 1,740 workers could often be hired for less than subsistence. our arguments relating to vulnerability and precariousness also have an economic dimension by grasping the relationship between vulnerability and economic analysis of environment. in the entropy law and the economic process, nicholas georgescu-roegen (1971) argued that waste is an unavoidable aspect of the development process of modernization, and that human beings inevitably deplete natural resources and create environmental pollution. economic progress merely speeds up the inevitable exhaustion of the earth's natural resources. georgescu-roegen's theory showed that classical economics had neglected the problem of natural scarcity, thinking that technology and entrepreneurship could eventually solve the problem described by thomas malthus of population growth in relation to fixed resources. his economic theory of waste applied the ideas of alfred lotka (1925) on biology to the accumulation of capital. human beings have to rely on what lotka called 'exosomatic instruments' to develop the environment, unlike animals which depend on 'endosomatic instruments'. in some respects this distinction is an old anthropological argument. reptiles evolve wings to fly; human beings create aeroplanes. however, wings involve low entropy solutions and do not deplete natural resources; technological solutions, such as jet-propelled aeroplanes, are high entropic solutions that use up finite energy. because humans are ontologically vulnerable, they develop high entropy strategies that have the unfortunate consequence of creating a precarious environment. more importantly, the entropy law implies a pessimistic conclusion that social conflict is inevitable. because resources are scarce, humans degrade their environment, and they must consequently compete within limited space. these malthusian conditions of social conflict in modern times have been further exacerbated by the mechanization of violence and by the de-stabilising impact of new wars. we can as a result interpret social citizenship as an institutional attempt to reduce conflict through, typically modest, income redistribution in the framework of the nation state, and human rights as conflict-reducing instruments between and within states. as argued by etzioni (1993) , increased social divisions and power of lobby groups can be linked to moral relativism. although this assertion has been criticized, it shows that systems that privileges the virtues of the market and individual freedom, fail to nurture the roots of the community (turner and rojek 2001) . while recognizing the common vulnerability of human beings, as sociologists we cannot ignore the precariousness of human institutions and the basic condition of scarcity. in order to engage with other human beings as moral agents worthy of our respect, there has to be mutual recognition. this basic starting point of ethics is referred to as 'recognition ethics' (williams 1997) . in a human community, this basic act of recognition requires some degree of equality. for example, hegel's master-slave analysis takes account of the fact that neither slave nor master can arrive at mutual recognition, because the master perceives the slave as his property, while the slave is too lowly to recognise the master. hence, without some degree of social equality, there can be no ethical community, and hence a system of rights and obligations cannot function. material scarcity undercuts the roots of social community without which conscious, rational agency is always compromised. taking their cue from the critique of liberal theories of rights by karl marx (1818-1883) , sociologists have remained sceptical about human rights traditions that have no corresponding social policies to secure some minimum level of equality through strategies of redistribution such as progressive taxation (waldron 1987) . rights to individual freedoms without democratic egalitarianism are thought to be merely symbolic not real claims for recognition. without some degree of equality, however basic, bioethics can have no real purchase on the social world. recognition requires some basic redistribution. the vulnerability thesis has received some criticism because it is very relevant to some human rights but not to others. it is limited by its inability to explain the individual rights of liberalism. in fact, it is often is used to prevent excess freedom that may increase inequalities. it can also be criticised on the grounds that we do not automatically feel responsible for the suffering of others. relativism 'opens the door' to moral queuing principles in function of interest groups and political agendas. in luc boltanski's distant suffering (1999) , there has been some discussion about whether we can sympathize with those with whom we are not connected. our argument that embodiment is a valid basis for the defence the universalism of human rights is partly grounded in the notion of the ubiquity of human misery and suffering. in 1850 arthur schopenhauer opened his essay 'on the suffering of the world' (2004) with the observation that every 'individual misfortune, to be sure, seems an exceptional occurrence; but misfortune in general is the rule'. while the study of misery and misfortune has been the stuff of philosophy and theology, there is little systematic study of these phenomena by sociologists. one exception is barrington moore (1970:11) who argues in reflections on the causes of human misery that 'suffering is not a value in its own right. in this sense any form of suffering becomes a cost, and unnecessary suffering an odious cost'. in general political opposition to human misery becomes a stand-point that can transcend and unite different cultures and values. a critic might object that suffering is too variable in its cultural manifestations and too indefinite in its meanings and local significance to provide such a common, indeed universal, standpoint. what actually constitutes human vulnerability, diversity and scarcity 667 suffering might well turn out to be culturally and historically specific. those who take note of the cultural variability of suffering have made similar arguments against a common standard of disability. although one could well accept this anthropological argument on the grounds that suffering involves essentially the devaluation of a person as a consequence of accident, affliction or torture, pain is less variable. whereas bankruptcy for example could involve some degree of variable psychological suffering through a loss of face, a toothache is a toothache. if we claim that disability is a social condition (basically the loss of social rights) and thus relative, we might argue that impairment is the underlying condition about which there is less political dispute or philosophical uncertainty. in short, some conditions or states of affairs are less socially constructed than others. suffering is often, perhaps always, a threat to our dignity, which is obviously culturally variable. pain by signalling a deeper somatic malfunction is a threat to our existence. yet another criticism is the medical technology paradox. the more medical science improves our global health condition, the less vulnerable we are. therefore technological progress could make this vulnerability thesis historically specific. in principle if we live longer, because we have become less vulnerable with advances in medical technology, then the relevance of human rights might well diminish. this paradox however helps us to sharpen our argument, which is that we are human, because we are vulnerable. the irony of medical advances is that we could only finally escape our vulnerability by ultimately escaping from our own humanity. technological change threatens to create a post-human world in which, with medical progress, we could in principle live forever. this criticism presents an interesting argument, but there are two potentially important counter-arguments. the first is that, if we could significantly increase our life expectancy, then we would live longer but in all probability with higher rates of discomfort and disability. the quantity of life might increase in terms of years, but there would be a corresponding decline in its quality. a post-human world is a medical utopia that has all the negative features of a brave new world. secondly, medical improvements in the advanced societies are likely to increase the inequality between societies, creating a more unequal and insecure international order. in such a risk society, where human precariousness increases and human vulnerability decreases, the need for human rights protection would continue to be important. the prospect of living forever might require us to inhabit, in max weber's pessimistic metaphor, an 'iron cage' in which our existence is by courtesy of lifesupport machines. a post-human world would in principle require a different ethical system namely a post-human ethics (fukuyama 2004) . scarcity is nonetheless at the centre of bioethics. for many scholars, scarcity is regarded as socially constructed in the sense that it is produced by a consumer culture in which expectations are elastic and diverse. the theory of positional goods suggests that demand for status goods can be controlled only with great difficulty (hirsch 1977) . our notion of inescapable vulnerability may be questioned by the optimism often generated by medical technologies that promise to provide replacement organs, brain implants, and a wealth of interventions aims to extend life 'indefinitely'. the task of bioethics is to address the problems of scarcity in societies of abundance and to consider the consequences of medical technology that will increase social inequality. with the scarcity of resources, there is always social competition and conflict-even in the richest societies of the developed world (turner and rojek 2001) . the occupy wall street slogan-we are the 99%-may become a relatively permanent feature of social movements in this century. there are few discussions on the nature of scarcity in terms of bioethics. if scarcity itself is not a product of modernity, globalization, or ageing populations, new technologies are important factors involved in the politics of life. bioethics will need to consider its relations to humans suffering and protective institutions. geriatric technologies are bringing new standards of longevity and quality of life, and are generating new social and ethical questions. characteristics of patients such as age, capacity to pay, degree of success of medical intervention, and social value of the individual, are all deciding factors that are used to different degrees that determine access to health care in the face of scarcity (moody 2002) . the opportunity costs of massive investments of health care for older populations are also being evaluated in terms medical ethics and social justice. ageing societies are faced with the difficult questions of 'choosing who's to live', and under what conditions, by limiting resources for the very old (walters 1996) . researchers in biogerontology have revived the medical utopia of wanting to significantly extending life well beyond the current human life span, situated approximately at 125 years. whether this life extension is achievable or not is somewhat irrelevant for our discussion. however, the justifications for funding such a project have been interpreted as 'cutting through ethics' turner 2007, 2013) . our criticism of cultural relativism does not endorse a pure foundationalist approach; we recognize that societies are different and have different value systems. however, we cannot minimise the import of universalist claims because there are shared similarities between humans and potent social forces such as globalization that shape and reshape human experiences. perhaps bioethics is deemed to follow a version of the 'glocalization' model, where, on the one hand, it would acknowledge and act upon the fact that globalized forces are being opposed to the legitimate resistance of local cultures, and on the other hand, it would strongly promote universal thresholds when in comes to health and human rights. our contribution to the understanding of conventional bioethics is also based in the strong assumption that there is always a struggle over scarce resources and that scarcity will continue to dominate the lives of large sections of the population, even within the wealthiest countries (bury 2000) . bioethics needs political economy. if we do not hold any firm foundationalist arguments in contexts of scarcity, we must recognize the inflation of demand for health technologies, increased competition for scarce resources and increased health inequalities. we note that our argument is somewhat similar to the position taken by hervé juvin (2010) in the coming of the body. for juvin, globalized societies are market-driven and characterized by individualism, indeterminacy, increased concerns over health and body appearance. without a strong and forceful legal framework that overrides individual investments in biomedicine, social inequalities will increase further eroding social and intergenerational relations. opposition to austerity measures in many european societies in 2012 may become a regular feature of street politics with growing unemployment and increasing inequality. indignation against visible inequality may evolve into political rage (reich 2012). furthermore, a strict opposition between universalism and cultural relativism is problematic because related forms of ethics are characterized by mutual recognition and empathy between people of different cultures. these forms of ethics also recognize cultural identity as a key component of agency, and without sufficient agency it is difficult to mobilize individuals to preserve their institutions. political anthropology has been dealing with these tensions for some time; however they are mainly framed in efforts to safeguard cultural diversity, which is quite different from the problem of sustaining human rights and bioethics. sociology has brought more attention towards increasing social inequalities. amongst other things, income inequality underlines new power struggles over life and health between the rich and the poor areas of the world. assuming there is a connection between health and wealth, relativism can nourish liberalism in biomedicine to the expense of vulnerable groups. post-humanists, for example, are transforming the discursive space in which bioethical debates are taking pace, and are proposing a detraditionalization of biomedical practices,-a process described as a moving away from nature and tradition that is essentially market-driven (giddens 1995) . this opposition to 'tradition' is radically changing the foundations of a politics of life. contemporary health care systems and research policies are faced with ethical questions that are derived from the relationship between the 'infinite demand' for health care services and the 'finite systems' of institutions (foucault 1988) . scarcity is thus creating an 'ethic of limits' in which universal claims for global health are being challenged by various forms of relativism. in this regard, a sharper focus on social inequalities in bioethics within the on-going discussion on cultural diversity will certainly clarify universal thresholds regarding health status and reinforce key objectives of social justice that are central to all major conventions in human right and bioethics. protecting the endangered human: toward an international treaty prohibiting cloning and inheritable alterations religion in human evolution distant suffering: morality, media and politics pascalian meditations. cambridge: polity press. bury, m. 2000. health, ageing and the lifecourse universal international law international maritime boundaries the life extension project: a sociological critique statecraft and soulcraft: foucault on prolonging life the spirit of community social security the varieties of my body: pain, ethics and illusion the entropy law and the economic process affluence, poverty, and the idea of a post-scarcity society. geneva: united nations research institute for social development the social limits to growth the coming of the body governing as gardening: reflections on soft authoritarianism in singapore. citizenship studies vulnerability: what kind of principle is it? medicine encounters with aging. mythologies of menopause in japan and north america justice and the human development approach to international element of physical biology elucidating the concept of vulnerability. layers not labels transformations of the world's population: the demographic revolution aging and controversies reflections on the causes of human misery and upon certain proposals to eliminate them encyclopedia, genealogy and tradition: a sociocultural critique of macintyre's three moral discourses the last utopia. human rights in history beyond outrage. what has gone wrong with our economy and our democracy, and to fix it vulnerability, vulnerable populations, and policy on the suffering of the world bioethics of protection: a proposal for the moral problems of developing countries the principle of respect for human vulnerability and global bioethics vulnerability and human rights universal declaration of bioethics and human rights report of the international bioethics committee of unesco on social responsibility and health report of the international bioethics committee of unesco on the principle of respect for human vulnerability and personal integrity nonsense on stilts. bentham, burke and marx on the rights of man introduction. in choosing who's to live: ethics and aging hegel's ethics of recognition taking the sociology of rights seriously key: cord-022381-x15ki4xv authors: goldblum, randall m.; goldman, armond s. title: immunological components of milk: formation and function date: 2012-12-02 journal: handbook of mucosal immunology doi: 10.1016/b978-0-12-524730-6.50056-7 sha: doc_id: 22381 cord_uid: x15ki4xv nan tion of milk fat. the substrate for milk fat synthesis derives from two sources. fatty acids are synthesized from glucose by mammary alveolar cells that contain fatty acid synthetase. these fatty acids have somewhat shorter chain lengths (10-16 carbons) than those synthesized in adipose tissue because of the presence of a mammary gland-specific enzyme called thioesterase ii. in addition, plasma triacylglycerols are cleaved within the mammary gland by the enzyme lipoprotein lipase. the resulting fatty acids are transported into the mammary alveolar cells, where they are reesterified with glycol to make the neutral fat molecules that coalesce to form milk fat globules. alveolar cells have a columnar shape with copious endoplasmic reticulum surrounding the nucleus in the basal region. during milk secretion, the golgi apparatus and secretory vesicles become more numerous toward the apical pole. abundant cytoplasmic lipid droplets enlarge as they move toward the luminal end of the cell. as they press into the apical plasma membrane, the droplets are released into the milk as membrane-bound milk fat globules (moyer-mileur and chan, 1989) . other globules that are formed during this process contain fewer lipids but larger amounts of other cytoplasmic constituents (patton and huston, 1985) . the third pathway transports certain small molecules including sodium, potassium, chloride, and glucose across the apical membrane of the cell. the secretion of the monovalent ions is effected by the electrical gradient across this membrane. the fourth pathway transfers proteins and possible other substances from the interstitial space to the lumen using receptors and intracellular vesicles. this mechanism will be considered in the description of the formation of secretory ig a (siga) in milk via polymeric immunoglobulin receptors. finally, in addition to soluble components, certain cells and cellular membranes enter human colostrum and milk. epithelial cells or their membranes are shed directly into milk. most b cells that home to the mammary gland remain sessile, whereas many t cells, macrophages, and neutrophils that have entered the lamina propria pass through the intercellular junctions of alveolar cells into the milk (brandtzaeg, 1983) . the mechanisms that attract the leukocytes to the mammary gland and trigger the migration of those cells into the milk are considered in a subsequent section. milk secretions are stored in the alveoli and small ducts until they are ejected during nursing. epithelial cells of the alveoli and ductules are surrounded by contractile basket-like epithelial cells. the ejection of milk from the breast is mediated by neuroendocrine events that culminate in the contraction of those myoepithelial cells. as a result of stimulation of sensory nerves at the nipple and areola during nursing, oxytocin is released from the hypothalamus into the posterior lobe of the pituitary gland and then into the peripheral circulation. oxytocin triggers myoepithelial cells to contract, forcing milk into the larger ducts and finally through the orifice of the nipple. human milk from early in lactation differs from most other external secretions because it contains viable leukocytes. the concentration of leukocytes is highest in colostrum and declines rapidly during the first months of lactation. table i displays estimates of the numbers of neutrophils, macrophages, and lymphocytes in human milk during the first months of lactation. these numbers are based on morphological characterisitics. however, distinguishing neutrophils from macrophages in human milk is difficult because the morphology of both cells is dominated by the large amount of lipid-containing vesicles in the cytoplasm (smith and goldman, 1968 ). the mechanism by which maternal leukocytes enter the milk is poorly understood. however, one potentially important clue to this process is the finding that essentially all these cells have surface markers or physiological features of activated cells. since most of the surface markers of activation on milk leukocytes are also present on leukocytes found in sites of inflammation, and are known to be important in homing and egress of leukocytes from the vascular compartment, the mechanism of migration of leukocytes into the milk may be similar to that involved in inflammation. although the array and mechanisms of production of inflammatory mediators in the lactating mammary gland are not well understood, several cytokines that may be involved in leukocyte migration have been detected in human milk, as described in later sections of this chapter. the following sections discuss our current knowledge of the morphology and in vitro function of the milk leukocytes. macrophages may have been first photographed in human milk by the french microscopist alfred donne (1844). however, little attention was paid to the cells in milk until smith and goldman (1968) described milk cells with the morphology and phagocytic activity consistent with activated macrophages. the concentration of macrophages in early milk is usually greater than that of their counterparts in peripheral blood, the monocytes. more recent studies have demonstrated that the milk macrophages are more motile than their counterparts in blood (özkaragöz et al, 1988; mushtaha et al, 1989) and display a pattern of surface markers associated with activation (keeney et al, 1993) . these cells also actively produce toxic oxygen radicals (tsuda et ah, 1984) . attributing other activities to the milk macrophages is difficult since most studies of milk leukocytes have used unseparated cells. the role of the milk macrophage in vivo has not been established. early in lactation, the concentration of neutrophils in milk approaches that in peripheral blood (table i) . neutrophils in human milk have been demonstrated to be phagocytic (smith and goldman, 1968) . however, the adherence, response to chemotactic factors, and motility of these cells are less than those of neutrophils from peripheral blood özkaragöz et al., 1988) . studies of surface markers suggest that some of these functional features may be the result of prior activation of the neutrophils (keeney et al., 1993) . activation of neutrophils may occur during the process of egression from the vascular space, may relate to the process by which large numbers of milk globules are engulfed by neutrophils in milk, or may be the result of exposure to cytokines demonstrated to exist in milk (see subsequent text). although the concentration of lymphocytes in human milk is small relative to that in peripheral blood, these cells are consistently present in milk obtained during the first few months of lactation. approximately 80% of milk lymphocytes are t cells (wirt et al, 1992) . the precise distribution of t-cell subpopulations in milk lymphocytes is controversial, since different investigators have reported numbers of cd4 + and cd8 + cells similar to those in peripheral blood (keller et al, 1986 ), a cd8 + -cell predominance (richie et al, 1982) , or moderate increase in the proportion of cd8 + cells relative to peripheral blood (wirt et al, 1992) . these differences may be the result of selection of certain subsets during the fractionation of milk or of the analytic limitations of direct fluorescent microscopy. these problems were avoided in the last study by using flow cytometry of unfractionated milk (wirt et al, 1992) . as in the case for other milk leukocytes, the increased display of certain surface phenotypic markers, including cd45ro, cd25 (il-2r), and hla-dr, suggests that the t lymphocytes are activated memory cells (wirt et al, 1992) . milk lymphocytes can be activated to proliferate using mitogens, but their responses are weaker than those of peripheral blood t cells (parmely et al, 1976; goldblum et al, 1981) . the spectrum of antigen-specific responses, as measured by proliferation, is thought to differ from that of peripheral blood lymphocytes from the same donor (parmely et al, 1976) , but the t-cell receptor repertoire of milk t lymphocytes has not been investigated. although evidence exists for the production of interferon (emodi and just, 1974; keller et al, 1981; bertotto et al, 1990 ) and monocyte chemotactic factor (keller et al, 1981) , the full array of cytokines produced by milk cells has not been determined. in addition to viable cells, several different types of particles are suspended in human milk, including casein micelles, globules packed with lipid, and globular structures containing less lipid but more cytoplasmic structures (patton and huston, 1985) . with low speed centrifugation, some of these particles sediment with the cells whereas many of the membrane-bound lipid-filled particles rise to the surface and coalesce. understanding this particulate structure of milk may be important, since several studies suggest that some host defense factors are compartmentalized within these structures. for instance, centrifugation of human milk causes the concentration of lysozyme to increase approximately fivefold over the value obtained by sampling milk prior to centrifugation (goldblum et al, 1975) . crago et al (1979) demonstrated that some of the siga antibodies in human milk are contained in lipid-filled particles. little is known about the direct effects on host defenses of the particles suspended in human milk, although some of them may be neutrophil activators (keeney et al, 1992) or may interfere with the attachment of enterobacteria to epithelial cells (schroten et al, 1992) . a. immunoglobulins and ig transport fragments. the major class of immunoglobulins in human milk is ig a. in contrast, mature cow's milk contains predominantly igg. the structure and function of siga, which makes up at least 80% of the milk iga, is discussed in chapters 7 and 11, respectively. chapter 21 considers the distribution and characteristics of the cells that produce immunoglobulins within the mammary gland. the demonstration of a very high density of igaland iga2-producing cells in the lactating mammary gland (brandtzaeg, 1983) helps explain why human colostrum and milk contain the highest concentrations of siga of any secretions. the high proportion of siga in human milk of the iga2 isotype (-40%) relative to plasma (10%) also must be related to the isotype distribution of these cells in the mammary gland. these findings also provide evidence that most of the iga secreted into the milk is produced locally within the breast, rather than transported from the plasma. the mechanism of antigenic sensitization and migration of iga-committed b cells into the mammary gland is considered in several earlier chapters. briefly, current evidence indicates that many of the iga antibody responses detected in human milk originate from antigenic stimulation at specialized mucosal sites in the intestinal and respiratory tracts (goldblum et al, 1975; roux et al., 1977) . iga-committed b cells emerging from mucosal sites, such as peyer's patches in the small intestine, migrate preferentially to other mucosal sites, including the lactating mammary gland. migration to the mammary gland is under hormonal regulation (weisz-carrington et al., 1978) . the wide array of specific antibodies found in human milk suggests that some of these cells are derived from memory b cells rather than from recent antigenic exposure. in the breast, b cells mature into plasma cells, the predominant product of which is polymeric ig a. as in other secretory mucosae, transport of immunoglobulins into colostrum and milk is accomplished predominantly by the polymeric immunoglobulin receptor (pigr). chapter 10 describes in detail the mechanism by which pigr mediates specific binding, endocytosis, and transcellular transport of immunoglobulins. proteolytic cleavage of pigr at the apical membrane of the mammary alveolar cell releases the polymeric ig a molecule, covalently complexed to a fragment of pigr termed secretory component (sc). this complex is called siga. some of the pigr molecules are transported and cleaved by the epithelial cells without any attached immunoglobulin. the resulting proteolytic fragment, free sc, is also present in high concentrations in human milk. the function of free secretory component has not been established clearly. one study (wilson and christi, 1991) suggests that these molecules may be able to inhibit the enzyme phospholipase a 2 , a function that could reduce inflammatory reactions along mucosal surfaces as well as, perhaps, the fluid accumulation produced by some intestinal pathogens (peterson and ochoa, 1989) . from this brief outline of the immunogenesis of siga, we can deduce that this system is well adapted for the production, and secretion into milk, of specific antibodies against pathogenic microorganisms to which the mother's mucosal immune system has been exposed. since the mother-infant pair normally shares many environmental exposures, this system may be ideal for protecting the infant from potential pathogens entering the environment. as indicated in chapter 52, several epidemiological studies have shown that the presence in milk of specific siga antibodies against enteric pathogens diminishes the incidence or severity of diarrhea caused by bacterial and viral organisms. immunoglobulins of the other major isotypes also are found in human milk, although at lower concentrations than in plasma. igm in milk is in its typical pentameric form, although some of the molecules have noncovalently attached sc, suggesting that igm is transported into the milk via the pigr. however, the binding of free sc in milk could generate the same complexes. the antibody specificity of igm in milk has not been tested extensively, but seems to parallel that of iga when examined. the presence of igm antibodies with attached sc should be considered when designing studies of siga in human milk, since detection systems based on anti-sc antibodies also may detect secretory igm (meilander et al., 1985) . in cow milk, igg is the major isotype. however, only small amounts of each of the subclasses of igg have been detected in human milk. the relative proportion of igg4 is greater in milk than in serum, suggesting that more of this isotype may be produced locally or selectively transported by the interstitium (keller et al, 1983) . this hypothesis has not been borne out in a more recent investigation (mehta et al., 1991) . nonetheless, the total amount of igg4 is so low that attributing biological functions to this or the other igg isotypes will be difficult. the concentration of igd in human milk is very low, even relative to serum concentrations (keller et al., 1984) . ige is essentially absent from human milk (underdown et al., 1976) . antibodies against a large number of microbes and specific antigens have been detected in human milk. table ii provides a summary of some of these specificities. this list should not be considered complete or thought to imply functional significance of the presence of antibodies of particular specificity, since it respresents a summary of studies from groups with particular interest in the antigens tested. the stability of the immunoglobulins in human milk has been the subject of a number of studies, most of which have concentrated on siga. the majority of the iga molecules in postnatal milk seems to be intact, based on western blots using anti-á chain antibodies as developing reagents (cleveland et al., 1991) . thus, during active lactation, little degradation must occur within the mammary gland. following the fate of the siga within the infant is difficult. fecal excretion of siga has been examined in low birth weight and full-term infants (schanler et al, 1986; prentice et al, 1987; davidson and lönnerdal, 1987) . the finding that siga excretion in the feces was 30 times higher in low birth weight infants receiving human milk than in similar infants fed cow milk formulas strongly suggested that a portion of the ingested milk survived the whole gastrointestinal tract. however, when expressed as a proportion of the siga fed, approximately 9% of the siga was recovered as siga (schanler et al., 1986) . the function of mucosal immunoglobulins is discussed in detail in chapter 11. of potential importance for milk immunoglobulins is the amplification of the effect of other milk defense factors by ig a. for instance, some of the lactoferrin in milk is found complexed with ig a (arnold et al., 1977) . these complexes may have enhanced activity since they can be targeted to surfaces of pathogenic microorganisms where lactoferrin could function to chelate selectively the iron needed by the microorganism for growth. microorganisms that are resistant to the lytic action of lysozyme may become more susceptible in the presence of siga and complement (adinolfi et al., 1966) . galactosyltransferase enzyme also complexes tightly with ig a, although the functional significance of these complexes is not known (mcguire et al., 1989) . b. lysozyme. lysozyme, a 12-kda single polypeptide, catalyzes the hydrolysis of ßl-4 linkages between nacetylmuramic acid and 2-acetylamino-2-deoxy-d-glucose groups in bacterial cell walls, leading to direct lysis of susceptible bacteria, predominantly those without an extensive cell wall. as indicated earlier, interaction with iga and complement may expand the antimicrobial range of activity of this secretory enzyme. the quantity of lysozyme supplied to the infant each day are presented in table iii . although the concentration varies during lactation the amount delivered appears to remain relatively constant for at least the first 4 months (butte et al., 1984) . these concentrations are among the highest of any secretion (jolles and jolles, 1967) . the relative stability of lysozyme against acid denaturation and tryptic digestion makes it well suited to function in the gastrointestinal tract of the recipient infant. however, the fate of the ingested lysozyme is not clear. low birth weight infants who are fed human milk excrete about eight times more lysozyme in their stool than do cow milk-fed infants (schanler et al., 1986) . lactoferrin is the whey protein with the highest concentration in human milk. the daily amount of lactoferrin ingested by the infant at various stages of lactation are shown in table iii . a gradual decline is seen in the amount transferred to the infant, beginning soon after initiation of lactation. a single chain glycoprotein of 79 kda, lactoferrin consists of two globular lobes, each with two domains surrounding a binding cleft for an atom of ferric iron and a bicarbonate ion. the major function of lactoferrin is the chelation of iron. in that respect, apolactoferrin robs the siderophilins of microorganisms of iron that is essential for their growth (spik et al., 1978) . the lactoferrin in human milk is well suited for this role, since 90% of the molecules are devoid of iron (fransson and lönnerdal, 1980) . several other functions have been suggested for lactoferrin. the finding of a specific receptor for lactoferrin on the mucosa of the upper bowel suggested that lactoferrin might enhance the uptake of milk iron by the infant (davidson and lönnerdal, 1988) . the low degree of iron saturation in milk lactoferrin makes this function seem unlikely, unless iron was transferred from other compartments during the digestion process. some evidence also suggests that lactoferrin has trophic effects on enterocytes (nichols et al., 1987) and may inhibit complement activity (kijlstra and jeurissen, 1982) . few studies have been done on the disposition of milk lactoferrin in the infant. one study was carried out on infants with enterostomies, which allowed the gut contents to be recovered before entering the colon (hambreus et al., 1989) . the results indicated that 9-32% of the ingested lactoferrin could be recovered from this site. low birth weight infants fed a human milk preparation excreted almost 200 times more lactoferrin in their stool than similar infants fed a cow milkbased formula (schanler et al., 1986) . however, only 3% of the ingested lactoferrin was recovered in the fecal sample. in addition, a major portion of the excreted lactoferrin was partially digested, resulting in molecules of unknown activity (goldman et al., 1990) . infants fed human milk also had a larger amount of lactoferrin and lactoferrin fragments in their urine (goldblum et al., 1989) ; the molecular sizes of those fragments were similar to those in the stools (goldman et al., 1990) . another study using stable isotope methods also suggested that some lactoferrin derived from the milk may a data modified from butte et al. (1984) . be absorbed intact from the intestinal tract and then excreted into the urinary tract (hutchens et al, 1991) . if this occurs, the absorbed lactoferrin must be cleared rapidly since human milk ingestion does not increase the serum concentration of lactoferrin (schanler et al., 1986) . many of the components of the classical and alternative complement pathway have been detected in human milk (ballow et al., 1974; nakajima et al., 1977) . however, with the exception of c3, the concentrations of these factors are very low. the activity of the complement system in human milk is not likely to be great, although interactions with other milk constituents may allow some function (adinolfi et al., 1966) . e. bioactive peptides. several cytokines have been quantified by immunoassays in human milk, including interleukin-lß (il-lß; munoz et al., 1990) , tumor necrosis factor a (tnf-a; mushtaha et al, 1989; rudioff et al., 1992a) and il-6 (saito et al, 1991; rudioff et al, 1992b) . the functions of these factors in the infant remain to be elucidated. however, studies of the leukocytes in the milk suggest that some of these cytokines may be active (söder, 1987; mushtaha ei a/., 1989) . of special interest is the finding that incubation of peripheral blood leukocytes with human milk causes monocytes and neutrophils to become activated. further, addition of neutralizing antibodies against human tnfa abrogated the activating effects of milk on monocytes (mushtaha et al, 1989) . some fragments of casein, the ß-casomorphines, which are created during proteolysis of casein in the gastrointestinal tract, are biologically active (teschenmacher, 1987) . ß-casomorphines not only have endorphin effects but may be immunoregulatory as well (parker et al, 1984) . several different isoforms of prolactin have been demonstrated in human milk that apparently are produced by posttranslational modifications in the mammary gland. although the function of each of these isoforms is not delineated, the basic protein molecule has been found to influence the development of t cells in animal model systems (chikanza and panay, 1991; gala, 1991; rovensky et al,\99\) and to enhance the formation of specific antibodies in serum and milk (ijaz et al, 1990) . the array of growth factors in human milk includes epidermal growth factor (egf), insulin, transforming growth factor ß (tgf-js; hooton et al, 1991) , and mammary gland derived growth factor (kidwell et al, 1987) . some of these factors have been postulated to aid in the postnatal development of the mucosal barriers of the intestinal and respiratory tracts. however, the in vivo effects of these factors are not well characterized. a. lactobacillus growth factors. human milk contains high levels of a growth-promoting activity for lactobacillus bifidus var. pennsylvania (györgy et al, 1974) . this activity, which is essentially absent from cow milk, is generated by oligosaccharides (györgy et al, 1974) , glycopeptides, and proteins (nichols et al, 1975; bezkorovainy et al, 1979) . similar activity associated with caseins also may be the result of oligosaccharide moieties on that protein (bezkorovainy and topouzian, 1981) . the role of these factors in host defense may be related to the predominance of lactobacillus in the bacterial flora in the colon of infants fed human milk. the large amount of acetic acid produced by these organisms suppresses the growth of enteropathogens. b. oligosaccharides and glycoconjugates. human milk is rich in oligosaccharides that appear to be formed in the mammary epithelium by the same galactosyltransferases that glycosylate proteins and peptides, using lactose as the acceptor molecule. various biological activities have been attributed to the whole group of oligosaccharides (holmgren et al, 1981) and, more recently, to individually characterized moieties, including fucosylated oligosaccharides that inhibit the hemagglutinin activity of the classical strain of vibrio cholerae and protect against the heat-stable toxin of escherichia coli (newburg et al, 1990) . mannose-containing glycoproteins and glycolipids interfere with the fimbria-mediated binding of e. coli (holmgren et al, 1987; wold et al, 1990) . the attachment of haemophilus influenzae and streptococcus pneumoniae to epithelial cells is inhibited by saccharides containing the disaccharide subunit af-acetylglucosamine (l-3)-ß-galactose (andersson et al, 1986) . these units may exist as free oligosaccharide or in glycoproteins or peptides. in any case, molecules with these structures may act as false receptors for the lectinlike adherence structures on the microorganism and thereby protect the infant from colonization or infection with these pathogens. although an in vivo role for these oligosaccharides is suggested by animal models (otnaess and svennerholm, 1982; cleary et al, 1983; ashkanazi et al, 1992) few human studies have been done that pertain to this question. a. unsaturated fatty acids and monoglycerides. free fatty acids and monoglycerides are produced by the digestion of milk triglycerides by bile salt-stimulated lipases or lipoprotein upases in human milk. in addition, the lingual and gastric lipase activities of the recipient infant are active on the milk triglycerides (hosmosh, 1990) . the lipid products have several host defense activities including disruption of enveloped viruses (stock and frances, 1940; welch et al, 1979; issacs et al, 1986; thromar et al, 1987) , which may prevent coronavirus infection in the intestinal tract (resta et al, 1985) . the fatty acids and monoglycerides also may provide some defense against intestinal parasites such as giardia lamblia (gillin et al, 1983 (gillin et al, ,1985 herneil et al, 1987) . b. á-tocopherol and /3-carotene. two vitamins found in human milk (chapell et al, 1985) also may have host defense activity. high levels of á-tocopherol in milk may serve as an antioxidant, but additionally this vitamin is known to stimulate the development of immunity (tengerdy et al, 1981; bendich et al, 1986) . ß-carotene, another potent antioxidant, is present in high concentrations in the mammary gland at parturition. this agent is released from the tissue into milk during the first few days of lactation (chapell et al., 1985) . as a result of the ingestion of á-tocopherol and ß-carotene in human milk, the blood levels of these two agents rise substantially in the recipient infant (chapell et al., 1985; ostrea et al., 1986) . these and other agents in human milk may regulate inflammatory responses and immune functions of the infant. despite the identification and quantification in human milk of many factors that have the potential to protect the lactating breast and the recipient infant, little currently is known about how these factors function in vivo. progress in this area has been limited by the types of studies that can be carried out in human infants and by the large species differences in milk composition and function that make experimental animal studies difficult to apply to humans. however, certain patterns of factors may provide clues to unique in vivo function of human milk. in contrast to many mucosal glands, which function on a continuous basis, the mammary gland secretion of immune factors is restricted largely to periods of lactation. the factors that regulate the onset, quality, and quantity of the human milk are only partially understood. prolactin and other lactogenic hormones are essential for the onset and maintenance of lactation. an array of growth factors including egf, insulin, and mammary gland derived growth factors that are concentrated in human milk (kidwell et al., 1987) also may play a role in these processes. the same proximity of the mucosal surfaces to the external environment that leads to extensive exposure to microorganisms and other antigens allows the mucosal immune system to defend against infections without the need for the extensive inflammatory and phagocytic responses that are typical of the systemic defenses. thus, if factors in milk can reduce the adherence, colonization, or growth of microorganisms in the infant's respiratory or intestinal tract, the incidence or severity of infection would decrease correspondingly without producing much physiological abnormality in the infant. we have hypothesized previously that a characteristic of the immune system in human milk is the absence of phlogistic factors and the presence of agents with anti-inflammatory activity . demonstrations that infants who receive mother's milk that contains specific antibodies against an enteric pathogen still have culture-proven infections but less diarrhea than those receiving milk without those antibodies (glass et al., 1983 ; also see chapter 52) are in keeping with this hypothesis. in that respect, the lower morbidity of breast-fed infants infected with rotavirus was not found to be related to the levels of specific antibodies in the milk (duffy et al., 1986) . this result suggests that other agents, including anti-inflammatory factors, may be responsible for some of the protection against certain pathogens. several studies suggest that breast feeding may have effects that last much longer than the breast-feeding period. for instance, breast-fed infants have a lower incidence of juvenile diabetes mellitus (mayer et al., 1988; ) and crohn's disease (koletzko et al, 1989 ) than those fed formulas. retrospective analysis also suggests a diminished risk of lymphomas after breast-feeding (davis et al., 1988) . whether these long-term effects are the result of mucosal immune factors in human milk is unclear. however, speculating that breast-feeding alters the development of the infant's immune system or protects against certain infections during a critical developmental period, thereby preventing illnesses that become manifest later in life, is interesting. serological properties of tja antibodies to escherichia coli present in human colostrum inhibition of attachment of streptococcus pneumoniae and haemophilus influenzae by human milk and receptor oligosaccharides a bactericidal effect for human milk lactoferrin the effect of human milk on the adherence of enterohemorrhagic e. coli to rabbit intestinal cells developmental aspects of complement components in the newborn. the presence of complement components and c3 proactivator (properdin factor b) in human colostrum dietary vitamin e requirement for optimum immune responses in the rat human breast milk t cells display the phenotype and functional characteristics of memory t cells bifidobacterium bifidus var. pennsylvanicus growth promoting activity of human milk casein and its derivatives isolation of a glycopeptide fraction with lactobacillus bifidus subspecies pennsylvanicus growth-promoting activity from whole human milk casein the secretory immune system of lactating human mammary glands compared with other exocrine organs daily ingestion of immunologic components in human milk during the first four months of life vitamin a and e content of human milk at early stages of lactation hypothalamic-pituitary mediated modulation of immune function: prolactin as a neuroimmune peptide protection of suckling mice from the heat-stable enterotoxin of escherichae coli by human milk characterization of secretory component in amniotic fluid: identification of new forms of secretory ig a human colostral cells. i. separation and characterization the persistence of human milk proteins in the breast-fed infant specific binding of lactoferrin to brush-border membrane: ontogeny and effect of glycan chain infant feeding in childhood cancer cours de microscopie modulation of rotavirus enteritis during breast-feeding interferon production by lymphocytes in human milk iron in human milk prolactin and growth hormone in the regulation of the immune system human milk kills parasitic protozoa cholatedependent killing of giardia lamblia by human milk. infect. immun protection against cholera in breast-fed children by antibodies in breast milk antibody forming cells in human colostrum after oral immunization human milk banking i. effects of container upon immunologic factors in mature milk human milk feeding enhances the urinary excretion of immunologic factors in low birth weight infants immunologic factors in human milk during the first year of lactation anti-inflammatory properties of human milk molecular forms of lactoferrin in stool and urine from infants fed human milk undialyzable growth factors for lactobacillus bifidus var. pennsylvanicus lactoferrin content in feces in illeostomy-operated children fed human milk reduced risk of iddm among breast fed children. the colorado iddm registry lingual and gastric upases killing of giardia lamblia by human milk lipases: an effect mediated by lipolysis of milk lipids nonimmunoglobulin fraction of human milk inhibits bacterial adhesion (hemagglutination) and enterotoxin binding of escherichia coli and vibrio cholerae receptor-like glycocompounds in human milk that inhibit classical and el tor vibrio cholerae cell adherence (hemagglutination) inhibition of bacterial adhesion and toxin binding by glycoconjugate and oligosaccharide receptor analogues in human milk human colostrum contains an activity that inhibits the production of il-2 origin of intact lactoferrin and its dn a-binding fragments found in the urine of human milkfed preterm infants. evaluation of stable isotopic enrichment neuroimmunomodulation of the in vivo anti-rotavirus humoral immune response membranedisruptive effect of human milk: inactivation of enveloped viruses human tear and human milk lysozymes activated neutrophils and nutraphil activators in human milk: increased expression of cd11b and decreased expression of l-selectin lymphokine production by human milk lymphocytes local production of igg4 in human colostrum igd-a mucosal immunoglobulin? t cell subsets in human milk production of growth factors by normal human mammary cells in culture modulation of classical c3 convertase of complement by tear lactoferrin role of infant feeding practices in development of crohn's disease in childhood a human milk galactosyltransferase is specific for secreted, but not plasma reduced risk of iddm among breast fed children immunoglobulin g subclasses in human colostrum and milk secretory iga antibody response against pyexcheria coli antiens in finfants in relation to exposure composition and properties of submicellar casein complexes in colloidal phosphate-free skim milk milk membranesorigin, content, changes during lactation and nutritional importance interleukin-1/3 in human colostrum chemokinetic agents for monocytes in human milk: possible role of tumor necrosis factor-á complement system in human colostrum fucosylated oligosaccharides of human milk protect suckling mice from heat-stable enterotoxin of escherichia coli human lactoferrin stimulates thymidine incorporation into dna of rat crypt cells isolation and characterization of several glycoproteins from human colostral whey influence of breast-feeding on the restoration of the low serum concentration of vitamin e and 0-carotene in the newborn infant non-immunoglobulin fraction of human milk protects against enterotoxin-induced intestinal fluid secretion the motility of human milk macrophages in collagen gels immunostimulating hexapeptide from human casein: amino acid sequence, synthesis, and biological properties in vitro studies on the t-lymphocyte population of human milk human lactation: milk components and methodologies role of prostaglandins and camp in the secretory effects of cholera toxin the nutritional role of breast milk iga and lactoferrin isolation and propagation of a human enteric coronavirus distribution of t lymphocyte subsets in human colostrum origin of iga-secreting plasma cells in the mammary gland evidence for immunomodulation properties of prolactin in in vitro and in vivo situations tumor necrosis factor-á in human milk interleukin-6 (il-6) in human milk detection of il-6 in human milk and its involvement in iga production enhanced fecal excretion of selected immune factors in very low birth weight infants fed fortified human milk inhibition of adhesion of s-fimbriated escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of protective function of mucins in inoimmunoglobulin fraction the cells of human colostrum. i. in vitro studies of morphology and functions isolation of interleukin-1 from human milk bacteriostasis of a milk-sensitive strain of escherichia coli by immunoglobulins and iron-binding proteins in association the inactivation of the virus of epidemic influenza by soaps vitamin e, immunity and disease resistance human lactation 3: the effects of human milk on the recipient infant decreased response of human milk leukocytes to chemoattractant peptides inactivation of enveloped viruses and killing of cells by fatty acids and monoglycerides oxygen metabolism of human colostral macrophages the relative paucity of ige in human milk hormonal induction of the secretory immune system in the mammary gland effect of antiviral lipids, heat, and freezing on the activity of viruses in human milk activated-memory t lymphocytes in human milk gravidin, an endogenous inhibitor of phospholipase a2 activity, is a secretory component of ig a secretory iga carries oligosaccharide receptors for escherichia coli type 1 fimbrial lectin key: cord-006257-rnskg79a authors: majer, m.; behrens, f.; weinmann, e.; mauler, r.; maass, g.; baumeister, h. g.; luthardt, t. title: diarrhea in newborn cynomolgus monkeys infected with human rotavirus date: 1978 journal: infection doi: 10.1007/bf01642161 sha: doc_id: 6257 cord_uid: rnskg79a of six newborn cynomolgus monkeys (macaca fascicularis) naturally delivered and normally nursed five developed diarrhea after oral administration of human rotavirus. virus excretion was observed in the stool of four animals. this virus was transmitted to four out of six other monkeys causing diarrhea in only one animal. using electron microscopy rotaviruses were detected in stools of infants and young children in various parts of the world, and their etiologic role in infantile gastroenteritis seems to be established (1) . further progress, however, is hampered by the lack of a productive 'in vitro' system for the propagation of the human rotavirus and of an established animal model for the disease. successful infection of piglets (2) , gnotobiotic calves (3), lambs (4) , and rhesus monkeys (5) with the human rotavirus was recently described. only colostrum-deprived monkeys delivered by caesarean section developed diarrhea after infection. in search of a more feasible animal model using non-human primates, we inoculated juvenile and newborn cynomolgus monkeys (macaea faseicularis) with the human rotavirus. the virus was isolated by ultracentrifugation of a clarified 10 ~ stool suspension from fecal specimens of four children hospitalized with acute gastroenteritis. the pellet was resuspended in eagle's minimum essential medium supplemented with 2% bovine serum albumin and hepes buffer ph 7.9. the virus suspension was distributed in 5.0 ml samples and stored at --80 °c. this corresponds to a 0.3 % stool suspension. the virus was identified by typical morphology in electron microscopy and by counterimmunoelectrophoresis using a hyperimmune calf diarrhea virus serum. the calf diarrhea virus for immunization purposes was obtained by courtesy of dr. g.n. wood, compton, berks., great britain. the virus was shown to be serologically related to human rotavirus (6, 7). four juvenile cynomolgus monkeys approximately four to six months old were inoculated with 5.0 ml of human rotavirus suspension by stomach tube. four similar animals constituted the control group. stool samples were collected before inoculation and during the ten following days, and were examined by electron microscopy for the presence of rotavirus. the method has been described in detail (8); serum samples were collected before and 14 days after inoculation and they were assayed for complement-fixing antibodies using the calf diarrhea virus as described previously (9) . three out of four inoculated animals had diarrhea on days 1, 3, and 7 respectively. no virus excretion, however, was detected. most animals had low initial antibody titers which remained essentially unchanged during the observation period. thus the etiology of the diarrhea remains uncertain. in the next experiment six newborn naturally delivered and normally nursed animals of the same species were inoculated within 24 hrs of delivery as described previously. one additional animal served as an uninoculated control. five infected animals developed diarrhea lasting two days on the average (table 1) . four out of six animals excreted the virus which was identified by typical morphology and by counterimmunoelectrophoresis. virus excretion lasted 1.8 days on the average. serum samples were collected from the mothers at the time of inoculation and six weeks after, and from the babies six weeks after inoculation only. seroconversion occurred in three mothers during the observation period indicating a possible natural infection from their infected babies (table 1) . only low antibody titers were observed in the babies. virus-containing stool of the animal no. 3257 (see tablel) was used to inoculate six other newborn monkeys. table 2 shows that virus excretion was observed over a similar (5, 10) . however, newborn cynomolgus monkeys which were naturally delivered and normally nursed, seem to be a promising animal model for the study of human rotavirus infection. at present we are using this model to examine the protective value of orally administered specific immunoglobu!ins. acute enteritis associated with reovirus-like agent propagation of infantile gastroenteritis virus (orbi-group) in conventional and germfree piglets diarrhea in gnotobiotic calves caused by the reovirus-like agent of human infantile gastroenteritis human rotavirus in lambs: infection and passive protection induction of diarrhea in colostrum-deprived newborn rhesus monkeys with the human reovirus-like agent of infantile gastroenteritis neonatal calf diarrhea: identification of reoviruslike (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy iv.: new complement fixation test for the human reovirus-like agent of infantile gastroenteritis viren als ursache der akuten gastroenteritis im s/iuglings-und kleinkinderatter seroepidemiological investigations on the epidemiology of human rotavirus infections 0rbiviruses and gastroenteritis key: cord-254559-3kgfwjzd authors: neo, jacqueline pei shan; tan, boon huan title: the use of animals as a surveillance tool for monitoring environmental health hazards, human health hazards and bioterrorism date: 2017-05-31 journal: veterinary microbiology doi: 10.1016/j.vetmic.2017.02.007 sha: doc_id: 254559 cord_uid: 3kgfwjzd abstract this review discusses the utilization of wild or domestic animals as surveillance tools for monitoring naturally occurring environmental and human health hazards. besides providing early warning to natural hazards, animals can also provide early warning to societal hazards like bioterrorism. animals are ideal surveillance tools to humans because they share the same environment as humans and spend more time outdoors than humans, increasing their exposure risk. furthermore, the biologically compressed lifespans of some animals may allow them to develop clinical signs more rapidly after exposure to specific pathogens. animals are an excellent channel for monitoring novel and known pathogens with outbreak potential given that more than 60 % of emerging infectious diseases in humans originate as zoonoses. this review attempts to highlight animal illnesses, deaths, biomarkers or sentinel events, to remind human and veterinary public health programs that animal health can be used to discover, monitor or predict environmental health hazards, human health hazards, or bioterrorism. lastly, we hope that this review will encourage the implementation of animals as a surveillance tool by clinicians, veterinarians, ecosystem health professionals, researchers and governments. zoonosis is derived from the greek words "zoon" (animals) and "nosos" (disease), referring to any infectious diseases transmitted from animals to humans, either directly or indirectly (world health organization, 2016) . as the global human population increases, so will anthropogenic pressures on wildlife and the environment, augmenting the likelihood of zoonotic pathogen spillover from animal to human populations. the world health organization (who) identifies zoonoses as emerging threats and describe them as previously occurring phenomena that have an increasing trend and expansion in geographical, host or vector range. more than 60 % of all emerging infectious diseases are from zoonoses (mackenzie and jeggo, 2013) . despite acting as the main reservoir for only 3 % of the known zoonoses, humans are the main source of identification for disease outbreaks (frank, 2008) . as such, epidemiological relationships agents in an analogous manner to humans and manifest similar disease symptoms. some animals have biologically compressed lifespans, consequently developing clinical signs more rapidly after exposure to specific pathogens. furthermore, they may be more susceptible to contaminants than humans and they do not share some human behaviors that may confound investigation results (e.g. smoking). table 1 provides a list of websites containing information related to the use of animals for surveillance. proper utilization of animals for surveillance may allow the early identification of epidemics, which facilitates mitigation of its magnitude, or prevention of its occurrence (chomel, 2003; kahn, 2006) . this is due to the ability of animals to: 1) exhibit changes in the occurrence or prevalence of a pathogen or disease with time, 2) serve as markers for on-going exposure risk, 3) allow examination of hypotheses on the ecology of pathogens, and 4) provide information on the efficiency of disease control measures (mccluskey, 2003) . this review attempts to highlight animal illnesses, deaths, biomarkers or sentinel events, to remind human and veterinary public health programs that animal health can be used to discover, monitor or predict environmental and human health hazards, or bioterrorism. animals useful for surveillance mostly exist in the environment as hosts of naturally cycling pathogens. they can be utilized in passive, active or sentinel surveillance programs. passive (reactive) surveillance involves the spontaneous reporting of disease data from the animal sector to veterinary authorities (hoinville et al., 2013) . data reported can include illnesses or deaths in animals, or notifiable diseases that must be reported by law. the data is then analyzed to observe disease trends and identify potential outbreaks. active (proactive) surveillance on the other hand involves calling on animal facilities to interview workers and to review animal health records to identify diseases under surveillance. it also involves actively monitoring domestic or wild animals for biomarkers. the choice of surveillance type depends on the characteristics of a pathogen and the objective of the program. passive surveillance is best employed when the objective of the program is targeted towards early detection of outbreaks or monitoring the extent of disease for making decisions on control strategies; whereas, active surveillance is best employed when a disease is targeted for elimination. passive surveillance is the least time consuming, labor intensive and expensive of the three forms of surveillance, and covers an extensive range. however, because it relies heavily on reports from veterinarians who receive little incentive for reporting, the data reported is frequently incomplete and delayed. underreporting of disease suspicions is also known to be a major cause of disease control failure (fao, 2011) and multiple studies have been conducted to better comprehend the decisionmaking processes behind underreporting so as to develop recommendations for improved passive surveillance (bronner et al., 2014; delabouglise et al., 2016; paul et al., 2013; sawford et al., 2012; thompson et al., 2016) . in contrast, active surveillance demands more time and resources and is thus less commonly employed. however, it provides more complete and accurate data than passive surveillance. a study comparing active and passive animal surveillance in chad concluded that for monitoring of existing diseases, the less expensive passive surveillance is better (marr and calisher, 2003) . historian plutarch mentioned that flock of ravens displayed unusual behavior and died subsequently as alexander entered babylon. 1878 several hundred livestock deaths death of several hundred livestock in lake alexandrina, australia allowed the identification of cyanobacteria nodularia spumigena in water. despite warnings issued, there was undescribed illness was reported in one individual after consuming contaminated water (codd et al., 1994) . 20th century canaries in coal mines coal miners in the u.k. 9 and the u.s. 10 brought canaries into coalmines as an early-warning signal for toxic gases including methane and carbon monoxide. the birds, being more sensitive, would become sick before the miners, who would then have a chance to escape or put on protective respirators (burrell and seibert, 1914). 1956 minamata disease cats from a fishing village, minamata developed a neurological disease. people of minamata later displayed similar symptoms. investigations later found that effluent from a factory had polluted surrounding waters resulting in accumulation of mercury in fish (takeuchi et al., 1977 (takeuchi et al., ). 1962 chicken sentinels chickens as sentinels for surveillance of arboviruses like wnv, wee 11 and sle 12 viruses (rainey et al., 1962) . rabbits warn of nerve gases during transportation rabbits were placed in small cages in railcars during transportation of nerve gases and sudden animal mortality would warn of gas release (brankowitz, 1987) . april 1979 sverdlovsky anthrax release anthrax was accidentally released from a soviet military microbiology facility. livestock died at a greater distance of 60 km from the plant, compared to human cases which occurred within a narrow 4 km zone downwind of the facility (meselson et al., 1994) . west nile virus wnv was reintroduced into the u.s., where it caused the ongoing epizootic in birds with a spillover of infections to humans and equines (chancey et al., 2015) . chickens on alert in kuwait u.s. marine corps employed chickens for the detection of nerve and blister agents. they were meant to act as a backup to false alarms the automated detectors were notorious for (ember, 2003 (ember, ). 2004 dog, livestock, wildlife deaths death of dogs, livestock and wildlife in the buccaneer bay lake, eastern nebraska, u.s. allowed identification of cyanobacteria anabaena, microcystis, oscillatoria and aphanizo-menon in water. >50 incidences of skin lesions, rash, gastroenteritis and/or headache were reported in humans. warnings were issued (walker et al., 2008 (walker et al., ). 2005 plague cases in yulong county of the yunnan province, china serologic survey found antibodies against the f1 antigen from domestic dogs around the affected county, demonstrating that domestic dogs could serve as animals for plague surveillance (li et al., 2008) . windblown lead carbonate in esperance, western australia windblown lead carbonate causing huge number of bird deaths in esperance, western australia, prevents lead exposure to esperance community (gulson et al., 2009). suited for chad's conditions. whereas, for monitoring of rare diseases, active surveillance of animal herds is required to complete passive surveillance (ouagal et al., 2010) . occasionally, when high-quality data about a specific disease is required, animal sentinels are intentionally deployed for surveillance. these animals receive greater attention than would be possible with active or passive surveillance. sentinel surveillance is less extensive in terms of range and personnel involved compared to passive surveillance, but often yields more detailed data. it is best employed when thorough investigation of each animal or site is necessary, however it may not be as effective for detecting diseases outside the demarcated limits of the sentinel sites. examples of events involving the various forms of animal surveillance spanning 323 bce and the 21st century can be found in table 2 . the review concludes with the one health approach. a sentinel is a naïve animal which is intentionally placed in an environment of potential infection that is monitored at short time intervals to detect infection. if the sentinel is deployed close to human populations, the sentinel should react to the infectious agent (but not become infectious), thereby providing early warning of human health hazards in the environment (van der schalie et al., 1999) . a classic example of an animal sentinel system is the well-known canary in the coalmine (burrell and seibert, 1914) . canaries are sensitive to the effects of poisonous gases, particularly carbon monoxide, and were routinely taken into the mines to warn of dangers. its inclination to sing much of the time, coupled with its brightly colored plumage offered both "audio and visual" cues to the miners. if the canary stopped singing and/or fell from its perch, this was the signal for the miners to don their respirators or evacuate. many miners owe their existence and livelihood to this historic animal sentinel. besides canaries, other animal species have also been used as sentinels of toxic chemical exposure. for example, birds, horses, cats, guinea pigs, rats, mice and rabbits were employed as sentinels for chemical agent exposure during world war i (wwi) and wwii. until 1969, rabbits were placed in small cages in railcars during the transportation of nerve gases and sudden animal mortality would warn of gas release (brankowitz, 1987) . although technological advancements have since resulted in the more widespread use of electronic detectors for detecting toxic chemicals, animal sentinels are still superior because of the complexity and likeness of the animal and human physiology. this is evidenced by more recent uses of animals, in particular avian species, as sentinels of toxic chemical exposure. for example, on march 20, 1995, the tokyo underground trains were hit by synchronized chemical terrorist attacks (national research council of the national academies, 2005). the aum shinrikyo religious sect dispensed a concoction of military nerve agent, sarin, killing twelve and injuring thousands. as a precaution, the japanese policemen carried canaries à the very primitive animal sentinel à in cages with them to warn of the presence of toxic gases during raids. another recent example of the use of avian species as sentinels of toxic chemical exposures was in 2003, when u.s. marine corps employed chicken sentinels at the kuwaiti staging area despite the deployment of automated detectors (ember, 2003) . the chickens were employed to complement the m22 ionmobility spectrometer, which was used to tag nerve and blister agents. they were meant to act as a backup to false alarms the automated detectors were notorious for. other than as sentinels of toxic chemical exposure, the avian species has also proven itself to be a valuable sentinel of disease outbreaks. for example, chickens have been used as sentinels for the surveillance of arboviruses like west nile virus (wnv), western equine encephalomyelitis (wee) and st. louis encephalitis (sle) viruses (moore et al., 1993) . they are amenable to and can tolerate arboviral infections with little or no symptoms, developing antibodies within a week of being bitten by an infected mosquito. they produce low tittered viremia, are cheap to purchase, robust and easily bled (biweekly or monthly during the peak season from june to october), making them excellent sentinel animals of arboviruses. despite providing accurate spatiotemporal information on virus transmission, the relationship between mosquito transmission and percentage of bird and mosquito infections in a particular region still needs to be determined in order to precisely evaluate human risk. in order to improve their use as sentinels, chicken interferon-a can be administered perorally in drinking water, where it acts as an adjuvant, inducing rapid seroconversion in chickens after infection by low pathogenic avian influenza (lpai). these chickens are called 'super-sentinels' since they are able to detect clinically inapparent lpai (marcus et al., 2007) . lpai strains are the most widespread, and can mutate into highly pathogenic avian influenza (hpai) strains, which can lead to human transmission and potential fatalities. thus, by placing super-sentinel chickens in locations prone to bird flu outbreaks, for example live-bird markets, this would allow early detection of lpai, thereby buying time for its control. in spite of the value of animal sentinels in monitoring the presence of pathogens or chemicals in the surroundings, there are ethical concerns regarding the deliberate exposure of animals to danger by placing them at sites of suspected contamination. consequently, the surveillance of extant animals in their natural habitats could act as an alternative means to warn of human, veterinary or environmental health hazards. animals in many habitats can be studied to monitor health hazards in the environment (reif, 2011) . environmental health hazards refer to both natural and unnatural contaminants in air, water, soil or food, which can potentially lead to acute or chronic health issues in humans (national research council (u.s.) and committee on animals as monitors of environmental hazards, 1991) . a variety of marine species are excellent surveillance tools of environmental stress and potential health threats for humans. for example, the mussel watch program actively analyzes sediment and bivalve tissue chemistry for a suite of organic contaminants and trace metals to identify trends at over 300 selected u.s. coastal sites from 1986 to today. it is designed to identify deleterious changes in the marine habitat and indicate potential human health concerns (kim et al., 2008) . anomalocardia brasiliana is also a good surveillance tool for actively monitoring contamination levels of coliforms in shellfish harvesting regions in brazil's northeast coast (lima-filho et al., 2015) . mussels, clams and oysters are particularly suitable for us as surveillance tools because they are able to bioaccumulate many chemicals (o'connor and lauenstein, 2006) , as well as concentrate microbial organisms and pathogens (kueh and chan, 1985) to concentrations in excess of 1000 fold (grodzki et al., 2014) . thus, the high concentrations of chemical and pathogens make it easier to detect environmental and health threats in these organisms. moreover, improved sequencing technologies have led to the monitoring of bivalves via genomics, epigenomics, transcriptomics, proteomics and metabolomics (suarez-ulloa et al., 2013) . such integrative omic studies will make powerful tools in the biomonitoring of marine pollution. besides marine species, cats can be potentially used as a passive surveillance tool for monitoring toxic chemicals in the aquatic ecosystem. in 1956, japanese veterinarians discovered a neurological disease in cats in the minamata fishing village (takeuchi et al., 1977) . it was called "dancing cat fever" because the cats displayed convulsions and involuntary jumping movements. however, this disease was not investigated rigorously until similar symptoms also manifested in the people of minamata. as a result of subsequent epidemiologic studies in minamata, researchers realised that effluent from a factory had polluted surrounding waters resulting in the accumulation of mercury in fish. subsequent consumption of contaminated fish by fishermen and their families resulted in high mercury concentrations in their brains, kidneys and livers. had the authorities paid more attention to the cats' disease symptoms, this could have been prevented. nevertheless, this episode raised the awareness of cats as a surveillance tool for monitoring mercury poisoning, food safety and public health throughout the world. besides mercury poisoning, there have been increasing numbers of reports of human or animal illnesses or deaths associated with harmful cyanobacteria blooms in freshwater systems. hence, in a similar way, the surveillance of fish, dogs or livestock can provide important early warnings of cyanobacteria-associated environmental hazards (hilborn and beasley, 2015) . more recently in 2006, the "birds dropping from the sky" phenomenon demonstrated how passive monitoring of bird dieoffs alerted the community of esperance, western australia to a case of lead poisoning in the environment (gulson et al., 2009) . during that period, the community was alarmed by the sudden death of more than 9000 birds. this sparked an urgent investigation which eventually revealed that the birds had died of lead poisoning. the lead ore concentrate had originated from the handling of lead carbonate concentrate at the megallan mine 600 km north of esperance. the western australia department of health and local shire council measured lead concentrations in rainwater from household tanks (the main source of drinking water) and discovered that 10% of households had lead concentrations exceeding who guidelines of 10 mg/l. although the death of numerous native bird species was tragic, it triggered the investigation, which ultimately prevented the catastrophic exposure of lead to the community. the clear and present dangers of emerging infectious diseases have propelled world governments to enhance animal surveillance activities (gubernot et al., 2008) . due to the zoonotic origin of most human health hazards, it is thought that animals may have a greater susceptibility to zoonotic pathogens, thereby justifying their use as surveillance tools for monitoring human health hazards. for example, in 1999, death and illness in multiple avian species aided investigators in identifying wnv as the root of the encephalitis outbreak in humans in new york. during those periods, the unusually high numbers of encephalitis cases in humans was concurrent with a surge in dying crows with neurological symptoms similar to encephalitis patients (eidson et al., 2001) . this prompted investigations, which identified wnv as the cause of the outbreak, and demarcated its geographical limits since the crows were amplification hosts for viral transmission. the cdc, u.s. geological survey national wildlife health centre and u.s. department of agriculture have since been involved in the battle against wnv. strategies are currently in place to consolidate data on human, mosquito, bird, chicken and veterinary cases of west nile infection in the arbonet system (u.s. geological survey and cdc, 2016). other than birds, dogs could also act as surveillance tools for monitoring wnv circulation. seroconversion was detected in juvenile dogs 6 weeks before wnv appeared in humans in houston, texas (resnick et al., 2008) . hence, active surveillance of wnv seroprevalence in birds and dogs can be used for monitoring wnv activity. additionally, the active surveillance of swine and live bird markets or supply abattoirs at the human-animal interface could be used to monitor the risk of hpai to human and animal populations. in 2009 and 2010, the h1n1 swine flu pandemic claimed more than 18,138 lives. this new strain resulted when a triple reassortment of northern american swine, bird and human flu viruses further combined with a eurasian pig flu virus (trifonov et al., 2009) . also in 2009 was the h5n1 avian influenza outbreak which led to the intense surveillance of wild ducks for avian flu viruses in europe (globig et al., 2009) . in eastern asia, wild swans are an ideal surveillance species as there is vast geographical overlap between whooper swan distributions and h5n1 outbreak areas (newman et al., 2009) . other hosts of hpai include cats and dogs (cleaveland et al., 2006; kuiken et al., 2005) . these studies show that active surveillance of suitable animal species through serosurveys could provide early warnings of hpai foci, accelerating public health investigation and action. bats are also important animals from a surveillance perspective. they have long life spans, are highly mobile and are increasingly well adapted to human environments due to habitat loss as a result of land use changes. they live in close proximity to humans, and interact with livestock and domestic animals that are potential intermediate hosts for pathogens. bats are the natural reservoir of severe acute respiratory syndrome coronavirus (sars-cov) (lau et al., 2005; li et al., 2005) , which was responsible for the sars-cov outbreak in 2003, with 8422 known infected cases and 916 confirmed human deaths worldwide (who, 2003) . besides sars-cov, bats are also reservoir hosts to filoviruses like ebola (leroy et al., 2009) and marburg (towner et al., 2007) viruses; paramyxoviruses like hendra (halpin et al., 2000) and nipah (yob et al., 2001) viruses; rubulaviruses like tioman (chua et al., 2001) and menangle (philbey et al., 1998) viruses; and the australian fruit bat lyssavirus (fraser et al., 1996) . there are however various challenges associated with the active surveillance of bats. firstly, the collection of blood and fluid samples from bats is dangerous given their highly infectious nature. secondly, the collection of bat specimens is difficult in remote areas. thirdly, bats are sensitive to disturbances and may migrate as a consequence of investigations, making it difficult to locate bat colonies. hotspots with high human and bat population density have thus been identified to focus bat surveillance efforts on areas with the highest probability of the emergence of zoonoses (jones et al., 2008) . dromedaries may also be potentially useful as surveillance tools. in particular, active surveillance of dromedaries in herds and large abattoirs could potentially reveal the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection. in 2012, mers-cov was first detected in humans in saudi arabia. sera from dromedary camels across and beyond the arabian peninsula were found to harbor high levels of antibodies against mers-cov (reusken et al., 2013) . indeed, viral sequencing revealed nucleotide polymorphism signatures, indicative of cross-species transmission (chu et al., 2014; memish et al., 2014) . this suggests that human mers-cov infections could have been zoonotically acquired from camels and that their surveillance could reveal mers-cov foci. mosquitoes, aedes aegypti and potentially aedes albopictus, transmit brazilian zika virus (zikv) among humans. in 2015, the first zikv infection was confirmed, and within a few months it was declared by the who to be a public health emergency of international concern (aziz et al., 2016) . health authorities have found zikv disease to be associated with auto-immune and neurological complications, and microcephaly in babies. transmission has been rampant in various regions and zikv is expected to spread to new territories. hence, the active surveillance of mosquitoes could enable the evaluation of vector control measures to determine the efficacy of zikv outbreak interventions. bioterrorism is the intentional release of microorganisms or biological agents to cause disease or death in humans, animals or plants to influence government conduct or threaten civilian population (cdc, 2007) . since more than 80 % of bioweapons are zoonoses, animals are likely to be at high risk (ryan, 2008) and thus the surveillance of animals may provide early warning of a bioterrorist attack (rabinowitz et al., 2006) . farm animals like sheep and cows are potentially valuable surveillance tools for passively monitoring the production or release of bioterrorism weapons in rural areas. bacillus anthracis, the causative agent of anthrax, which has fatality rates of near 100 % in both humans and animals, has been identified by cdc as one of the most likely biological agents to be used (cdc, 2016) . moreover, anthrax can form resilient spores that persist for decades in soil. during wwii, the british government was experimenting the use of anthrax on gruinard island. despite efforts to decontaminate the island, the long-lasting contamination of the soil by anthrax spores put the island under quarantine for 48 years before it was considered safe for human use ("britain's anthrax island," 2001) . this highlights the importance of passively monitoring random cases in animals to identify anthrax hot spots. in 1979, b. anthracis spores were inadvertently released from a soviet military microbiology facility in sverdlovsky (meselson et al., 1994) . livestock 60 km away from the plant died, whereas human cases occurred within 4 km downwind of the facility. analysis showed that the dosage of b. anthracis at which sheep and cows became ill was more than an order of magnitude lower than the dosage required to affect humans. this analysis therefore suggests that livestock are much more susceptible than humans to b. anthracis and would be ideal for use as surveillance tools for b. anthracis since lower dosages at greater distances from the source were sufficient to affect the animals. furthermore, animals like domestic dogs and rodents spend more time outdoors and have greater exposure to the environment than humans, making them great surveillance tools for monitoring plague. yersinia pestis, the etiological agent of plague, has also been identified by cdc as a bioterrorism agent (cdc, 2016) . it appeared in humans in the u.s. in the 1900s (link, 1955; lipson, 1972) , and became established enzootically in wild rodents by the mid-1940s. it is hypothesized that plague is maintained by reservoir species like rodents, and that carnivores become ill following the ingestion of plague-infected rodents. in 2005, 5 plague cases were confirmed in the yulong county of the yunnan province, china (li et al., 2008) . a survey of serum samples of domestic dogs in and around the affected county confirmed that they could be used for active plague surveillance. the dynamics of infectious diseases are highly variable. they are determined by infecting dose, pathogen characteristics, host susceptibility and transmission routes. it is therefore important to have a framework for the proper evaluation of animals to determine if they are suitable for use as sentinels for surveillance. to this end, the halliday et al. (2007) conceptual framework effectively evaluates animals as sentinel populations for various surveillance aims and ecological settings (halliday et al., 2007) . there are three fundamental components of the sentinel framework: the pathogen under surveillance, the target population, and the sentinel population. the sentinel framework produces a sentinel response which could take the form of seroconversion, current infection, morbidity, mortality and changes in morphology or behavior. in addition to the sentinel response, other sentinel practical factors and host factors also influence the detectability of the sentinel response, which eventually determines the utility of the sentinel. as alluded to earlier, although there are numerous benefits in using animal sentinels, its use is associated with ethical concerns which might be alleviated by the surveillance of existing animals in their natural habitats. in spite of the obvious potential of animals as a surveillance tool for monitoring environmental damage, risks to human health and bioterrorism, animals currently appear to be underutilized as surveillance tools. a likely reason is that human and animal health surveillance efforts mostly stem from disparate initiatives, resulting in data being kept in entirely separate databases (scotch et al., 2009) . there is thus an increasing need for interdisciplinary integration between human and veterinary medicine, and communication before we can completely exploit the benefits of animals for surveillance (bisdorff et al., 2016; wendt et al., 2015) . to this end, committees have been established to increase awareness of the mutual reliance between human, animal, plant, microbial, and ecosystem health (rabinowitz et al., 2013) . this includes the 'one medicine' or 'one health' initiatives like the one health commission, the one health initiative and the comparative clinical science foundation (zinsstag et al., 2011) . the term 'one medicine' was coined in 1976 by calvin w. schwabe in recognition of the mutualism of human and animal health, nutrition and livelihood (schwabe, 1984) . this mutualism is further supported by the close genomic relationship of humans and animals (peters et al., 2007) . today, the appreciation of the interdependence of the well-being and health of humans, animals and the ecosystems, evolved the term 'one medicine' towards 'one health', to include public health, ecology and broader societal dimensions (zinsstag et al., 2011) . in support of one health, predict was launched in 2009 (usaid, 2016a). the predict project is part of united states agency for international development's (usaid's) emerging pandemic threats (ept) program, designed to identify zoonotic viral threats with pandemic potential at wildlife-human viral transmission interfaces (kelly et al., 2016) . it has successfully improved surveillance and laboratory capabilities for monitoring humans (that have had animal contact) and wildlife for new and known pathogens with outbreak potential; defined ecological and human causes of zoonosis; and reinforced and perfected models for predicting outbreaks. it became the largest zoonotic virus surveillance project worldwide, successfully identifying and predicting the emergence of pathogens from wildlife. it also established infrastructure and expertise required for the operation of pandemic threat surveillance and diagnostics to support the one health workforce (ohw). the huge success of ept led to the launch of ept 2 which aims to discover diseases of known and unknown origin; minimize the possibility of disease outbreaks by reducing human activities that promote disease spillover; boost national readiness; and ultimately to reduce the repercussions of novel zoonotic pathogen emergence (usaid, 2016b). in southeast asia, there is the one health network south east asia platform supported by the european union to promote collaboration, networking and sharing between southeast asian one health programs (massey university new zealand, 2014). it presently hosts two programs, lacanet and comacross. lacanet is a cambodia and laos effort aimed at improving detection of zoonotic diseases, developing capabilities for surveillance, promoting regional and national collaborations, and researching into land-use change and wildlife trade à the two main causes of disease emergence. on the other hand, comacross is a thailand, cambodia and laos effort aimed at assembling a multidisciplinary framework to address complex one health problems and to improve integration between public health, animal health, agriculture and livestock, ecology, environmental science, social science and engineering. besides the one health network south east asia platform, in southeast asia, there is also biodivhealthsea supported by the french anr cp&es. it investigates the impact of global changes and global governance on zoonotic diseases, biodiversity and health (morand et al., 2014) . together with comacross, biodivhealthsea and a few other bodies have proposed in a southeast asian interdisciplinary conference that ecosystems could reveal potentially harmful developments for human health (walther et al., 2016) . they made recommendations for the implementation of the one health approach; future research direction; education, training, and capacity building; potential science-policy interactions; and ethical and legal considerations. these recommendations should influence legislation and enforcement, thereby strengthening the health and resilience of southeast asian societies. as typical with large programs, ept, ept 2, lacanet, comacross and biodivhealthsea will need to apportion sufficient funds and resources for conducting surveillance, laboratory diagnosis, consumables, equipment and infrastructure. it is therefore critical that they continue to collaborate with partners like the cdc, who, ohw, food and agriculture of the united nations, institut pasteur du cambodge and lao-oxford-mahosot hospital-wellcome trust research unit, to strengthen their capacity in surveillance and laboratory capabilities, and to ensure that efforts are not replicated. it is also important that one health programs continue to receive the support and funds that they need to sustain their work. the extent of support for animal disease surveillance in communities is largely built upon its understanding of the dangers of zoonoses to human health, trade and the economy, rather than out of interest in wildlife health. accordingly, it is crucial to boost public awareness of the importance of wildlife health to societies. lastly, governments are occasionally unwilling to announce potential disease outbreaks for various reasons, including preventing the disruption of trade. hence, it is important that reporting of wildlife diseases be standardized and made necessary, and for a reporting global clearinghouse to be established. altogether, 'one health' is a unifying paradigm encouraging integration and leverage of existing capabilities among clinicians, veterinarians, ecosystem health professionals, researchers and governments. ultimately, this will hopefully lead to the development and application of sustainable and effective community health interventions, thereby reducing zoonotic disease emergence, outbreaks and repercussions, and addressing some of the biggest multidisciplinary challenges of the 21st century. none. zika virus: global health challenge, threat and current situation active animal health surveillance in european union member states: gaps and opportunities chemical weapons movement history compilation. office of the program manager for chemical munitions (demilitarization and binary britain's anthrax island why do farmers and veterinarians not report all bovine abortions, as requested by the clinical brucellosis surveillance system in france? experiments with small animals and carbon monoxide bioterrorism overview [www document 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utility and strategies for design and implementation of a national companion animal infectious disease surveillance system bivalve omics: state of the art and potential applications for the biomonitoring of harmful marine compounds the outbreak of minamata disease (methyl mercury poisoning) in cats on northwestern ontario reserves improving animal disease detection through an enhanced passive surveillance platform marburg virus infection detected in a common african bat geographic dependence, surveillance, and origins of the 2009 influenza a (h1n1) virus west nile virus human provisional emerging pandemic threats [www document emerging pandemic threats 2 program [www document nebraska experience summary of probable sars cases with onset of illness from 1 biodiversity and health: lessons and recommendations from an interdisciplinary conference to advise southeast asian research, society and policy zoonotic disease surveillance?inventory of systems integrating human and animal disease information nipah virus infection in bats (order chiroptera) in peninsular malaysia from one medicine to one health and systemic approaches to health and well-being the writing of this review was supported by dso national laboratories. key: cord-007101-m0fs2f2a authors: wang, mei; donovan, sharon m. title: human microbiota-associated swine: current progress and future opportunities date: 2015-05-19 journal: ilar j doi: 10.1093/ilar/ilv006 sha: doc_id: 7101 cord_uid: m0fs2f2a gnotobiotic (gn) rodent models have provided insight into the contributions of the gut microbiota to host health and preventing disease. however, rodent models are limited by several important physiological and metabolic differences from humans, and many rodent models do not dependably replicate the clinical manifestations of human diseases. due to the high degree of similarity in anatomy, physiology, immunology and brain growth, the domestic pig (sus scrofa) is considered a clinically relevant model to study factors influencing human gastrointestinal, immune, and brain development. gnotobiotic piglet models have been developed and shown to recapitulate key aspects of gn rodent models. human microbiota-associated (hma) piglets have been established using inocula from infants, children, and adults. the gut microbiota of recipient hma piglets was more similar to that of the human donor than that of conventionally reared piglets harboring a pig microbiota. moreover, bifidobacterium and bacteroides, two predominant bacterial groups of infant gut, were successfully established in the hma piglets. thus, the hma pig model has the potential to be a valuable model for investigating how the gut microbiota composition changes in response to environmental factors, such as age, diet, vaccination, antibiotic use and infection. the hma also represents a robust model for screening the efficacy of preand probiotic interventions. lastly, hma piglets can be an ideal model with which to elucidate microbe–host interactions in human health and disease due to the similarities to humans in anatomy, physiology, developmental maturity at birth, and the pathophysiology of many human diseases. the human gut is colonized by a complex microbial community with a population approximately 3 to 10 times greater than the total number of host cells of which the body consists (björkstén et al. 2001) . germ-free (gf) animal studies have shown that gut microbiota and their hosts do not simply coexist, but rather form a mutualistic relationship . it is now clear that the structure and functions of the gut microbiota play a crucial role in human health through its contributions in fermentation of undigested carbohydrates, vitamin biosynthesis, regulation of energy storage, maturation of the immune system, pathogen colonization resistance, and brain development (douglas-escobar et al. 2013; li et al. 2014) . alteration in the composition of the gut microbiota has been associated with digestive tract diseases, including necrotizing enterocolitis (nec) (mai et al. 2011; wang et al. 2009 ) and inflammatory bowel diseases (ibd) (aomatsu et al. 2012; michail et al. 2012; schwiertz, jacobi et al. 2010; walker et al. 2011) . additionally, strong evidence from human studies and animal models links intestinal microbiota dysbiosis with a broad-range of immune, metabolic, and neurodevelopmental disorders , including asthma (vael et al. 2011) , eczema (gore et al. 2008; wang et al. 2008) , obesity (karlsson et al. 2012; ley et al. 2006; schwiertz, taras et al. 2010; turnbaugh et al. 2009 ), and autism (kang et al. 2013; parracho et al. 2005; wang et al. 2013) . defining the mechanistic underpinnings whereby the intestinal microbiota influences human health and disease has been hampered by individual variation in host genetics and microbiota and ethical concerns of using invasive procedures in human subjects, particularly infants and children. animal models, especially gn rodents, have been extensively employed for exploring the cross talk between the host and commensal bacteria (chow et al. 2010; gootenberg and turnbaugh 2011; leser and mølbak 2009; smith et al. 2007) . comparative studies of gf and conventional (cv) mice have demonstrated that the gut microbiota profoundly impacts host biology, ranging from intestinal morphology and motility, mucosal and systemic immunity, to absorptive and metabolic functions (smith et al. 2007 ). the term cv refers to an animal or human colonized by the microorganisms normally associated with its particular species. for example, gf mice have shorter ileal villi and crypt, and slower rate of small intestinal cell turnover than cv mice (smith et al. 2007; yi and li 2012) . furthermore, gf animals have fewer and smaller peyer's patches and mesenteric lymph nodes and greatly reduced fecal iga than animals raised under specific pathogenfree (spf) conditions (honda and takeda 2009; macpherson and harris 2004; round and mazmanian 2009) . moreover, gene expression profile of mouse ileal epithelium is altered in the absence of commensal bacteria . while gn rodent models have provided insight into host-microbe interactions, rodent models are limited by several important physiological and metabolic differences from humans (graham and aman 1987; heinritz et al. 2013 ). more importantly, many rodent models do not dependably replicate clinical manifestation observed in human diseases (lunney 2007) . therefore, more clinically relevant animal models are needed. nonhuman primates are good models for humans because they share significant physiological, metabolic, biochemical, and genetic similarity with humans; however, expensive housing, long lifespan, and ethical concerns limit their use (puiman and stoll 2008; shen 2010) . the domestic pig (sus scrofa) is closely related to the human in terms of anatomy, physiology, and genetics, and is considered the preferred nonprimate model for humans (dawson 2011; guilloteau et al. 2010; meurens et al. 2012; odle et al. 2014 ). in addition, the piglet is an excellent model for infectious diseases (meurens et al. 2012) . the goal of this review is to highlight the usefulness and limitations of the cv pig as a model for human gastrointestinal physiology, immunology, and neurodevelopment. in addition, findings of recent studies using gn and human microbiota-associated (hma) pigs, and future directions with the model will be discussed. pigs have served as biomedical models for decades. advantages of the swine model are highlighted in table 1 . swine have high genome and protein sequence homology with humans, which facilitates understanding of gene-microbiome interactions and the availability of molecular probes and antibodies. for example, when porcine reagents are not available, antibodies and probes directed against human proteins and gene sequences often cross-react with porcine samples (lunney 2007) . from a nutritional perspective, pigs and humans are omnivorous, whereas rodents are granivorous. in terms of the gastrointestinal anatomy and physiology, pigs are also more similar to humans than are rodents (guilloteau et al. 2010; odle et al. 2014) . also, both pigs and human are colon fermenters, whereas fermentation take place in the cecum of rodents (heinritz et al. 2013) . pigs are also immunologically similar to humans. for example, porcine immune responses more closely resemble human responses than mouse responses with >80% of parameters studied, whereas the immune response in mice was more similar to the human in <10% of comparisons (dawson 2011) . humans and pigs also share similar brain growth and development patterns. the major brain growth spurt of the pig extends from late prenatal to the early postnatal period, resembling that of the human, which is different from other animals including rats (dobbing and sands 1979) . additionally, gross anatomical features such as gyral pattern and gray and white matter distribution of the piglet brain are comparable to those of human infants . furthermore, the possiblity of using pigs from the same litter and similar disease progression make the pig an excellent model for human gastrointestinal physiology, immunology, and neural development. due to similarities in immune function, pigs are also an outstanding model for infectious diseases and vaccine development (meurens et al. 2012) and have been used extensively to study infectious diseases relevant to human health, including respiratory (bordetella pertussis [elahi et al. 2007] , cornona virus [saif 1996 ], influenza viruses [khatri et al. 2010] , mycobacterium tuberculosis [gil et al. 2010] , pseudomonas aeruginosa, and staphylococcus aureus [nielsen et al. 2009 ]) and gastrointestinal pathogens (cryptosporidium parvum [vítovec and koudela 1992] , helicobacter pylori [nedrud 1999] , hepatitis e virus [krawczynski et al. 2011] , norovirus [cheetham et al. 2006 ], and rotavirus ). conventional piglets are extensively used for studies of early nutrition on gastrointestinal, immune and neural development (guilloteau et al. 2010; odle et al. 2014; rytych et al. 2012 ); however, a major limitation of the cv piglet model is that the gut microbiota of piglets differs from that of human infants. phyla-level gut bacterial composition of mother-fed or formula-fed (ff) 3month-old infants ) and 21-day-old piglets (unpublished observations) are compared in figure 1 . while differences between mother-fed or ff neonates of both species can be appreciated, marked differences in the gut microbiota table 1 advantages of the swine model • omnivorousnutritional requirement and physiology similar to human • high genome and protein sequence similarities with human • immune system more closely resembles human • brain growth and development patterns similar to human ○ the major brain growth spurt similar to human ○ gross anatomical features of the brain are comparable to that of human infants • body sizeallowing various surgical manipulation and collection of adequate quantity of samples. • large litter size (10-12 piglets/litter) • similar disease progression ○ metabolic diseases, such as obesity and heart disease ○ infectious diseases (e.g., influenza viruses, rotavirus, helicobacter pylori, and neisseria meningitides infection) conrad et al. (2012) ; dobbing and sands (1979) ; lunney (2007) ; meurens et al. (2012) between neonates of the two species exist. for example, actinobacteria (mainly bifidobacterium) predominates (>50% of 16s rrna sequences) in both breastfed (bf) and ff infants, whereas little actinobacteria (<0.2% of 16s rrna sequences) is detectable in piglets. the predominant phyla in both sow-reared (sr) and fffed piglets are bacteroidetes and firmicutes, which is more similar to the adult human . additionally, both sr and ff piglets have greater microbial diversity than human infants. establishment of the intestinal microbiota after birth plays a vital role in development of the neonatal gastrointestinal and immune systems (adlerberth and wold 2009; sjögren et al. 2009 ). recent data have also shed light on the ability of microbiota to influence brain development and behavior (collins et al. 2012; desbonnet et al. 2014; diaz heijtz et al. 2011 ). however, differences in the native gut microbiota between the infant and the piglet complicate direct translation of results from piglets to humans. a solution to this problem is to develop piglets harboring a human gut microbiota. gnotobiotic animals are animals colonized with known strains of bacteria or microbiota. they are delivered by cesarean section (or sterile hatching of eggs) under aseptic conditions and are raised within sterile isolators and fed sterile water and food in order to control their exposure to microorganisms (butler 2009; gustafsson et al. 1957 ). germ-free animals are gnotobiotic animals that have been maintained free from microorganisms, including bacteria, fungi, viruses, and parasites throughout their life. gnotobiotic experiments take advantages of highly controlled, repeatable experimental design, which reduces interindividual variation. as of the writing of this review, over 500 publications have used gn piglets. gnotobiotic pigs have been used to study the impact of bacterial colonization on the host, including organ growth, intestinal morphology, physiology, and immune development (table 2) . relative to cv pigs, gf pigs have smaller thyroid and liver size, but larger spleen, lung, heart, and gall bladder mass at 7 weeks of age (shurson et al. 1990 ). shirkey and colleagues (2006) investigated the effects of colonization of different bacterial species on small intestinal morphology and observed that the relative length of the small intestine (si) was smaller in gf and mono-associated (ma) piglets than in cv piglets at postnatal day 13. they also showed that gf and ma piglets had lower relative weights of proximal si regions than that of cv piglets. this is consistent with previous findings reporting that the si thickness of gf pigs was lower than cv pigs (shurson et al. 1990 ). in addition, gf and ma piglets had shorter crypt depths, longer villi height, reduced lamina propria cellularity, and smaller peyer's patches in their si compared to their cv counterparts (shirkey et al. 2006; willing and van kessel 2007) . the intestinal microbiota also affects brush border enzyme activities. aminopeptidase n and lactase phlorizin hydrolase activities were lower in si enterocytes of cv piglets in comparison with piglets maintained gf or monoabbreviations: bf, breast-fed; ff, formula-fed; sr, sow-reared. associated with nonpathogenic escherichia coli or lactobacillus fermentum at 14 days of age (willing and van kessel 2009) . shorter villus height was observed in cv pig enterocytes , thus the lower enzyme activity in cv pigs may be partly explained by reduced cell maturity or mature cell number. in addition, reduced enzyme activity in cv pigs could be due to microbial brush border enzyme deactivation (willing and van kessel 2009) . several studies have investigated the role of bacterial colonization on the host immune development. haverson and colleagues (2007) compared the immunological structure of the lamina propria in the jejunum of gf piglets with piglets associated with two strains of commensal e.coli between 1 and 4 days of age. by two days after transfaunation, they found that mono-association of gf piglets with e. coli increased the numbers of dendritic and t cells in diffuse lymphoid tissue of the jejunum. additionally, si expression of proinflammatory cytokines interleukin-1β (il-1β) and il-6 were higher in gf and ma piglets compared with cv piglets at postnatal day 13 (shirkey et al. 2006) . other studies have shown effects on systemic immunity as well; relative to gf piglets, serum immunoglobulin level of piglets colonized with a mixture of defined bacteria was significantly greater on the first 6 weeks of life (butler et al. 2000) . gene microarray profiling of the si epithelium in gf and cv piglets confirmed the essential role of a commensal microbiota for normal development of the host intestinal transcriptome. genes involved in transcription, cell proliferation and differentiation, nutrient transport and metabolism, xenobiotic metabolism and immune responsiveness were upregulated in gn piglets bearing a microbiota from cv piglets versus gf piglets (chowdhury et al. 2007) . despite the fact that gn piglets differ in aspects of gastrointestinal and immune development relative to cv piglets, gn pigs still provide a unique and powerful model for study of enteric diseases that affect both humans and pigs. for example, gn piglets have been used to investigate disease pathogenesis and/or immunity to rotavirus , enterohemorragic escherichia coli (brady et al. 2011) , clostridium difficile (steele et al. 2010) , and shigella dysenteriae type i (jeong et al. 2010) infections, among others (meurens et al. 2012) . furthermore, beneficial effects of probiotics (azevedo et al. 2012; liu et al. 2013 ) and the application of vaccination (jeong et al. 2013 ) have been tested with gn pig infection models. in most gn pig studies, pigs were colonized with single or multiple strains of bacteria. these studies are useful for delineating the physiological functions of specific microbes, but the effects of single or multiple bacteria on the host are not representative of a complex microbiota. recently, hma animal models have been developed, both in rodents and pigs. the hma rodent model has been used to investigate how the gut microbiome is influenced by dietary components and, in turn, influences host health and disease (gootenberg and turnbaugh 2011) . for example, production of equol from dietary soy isoflavone, the microbial reduction of cholesterol, the effects of a defined diet changes on the gut microbial community structure and functions, and the biogeography and assembly of the gut microbiota have all been studied in hma rodent models (gootenberg and turnbaugh 2011) . additionally, hma mice have been important for understanding the role of the microbiota in a variety of human diseases (gootenberg and turnbaugh 2011) , including the biological effects of microbiota obtained from obese adults or children with kwashiorkor (smith et al. 2013 ). these studies have definitively proven that the clinical signs and symptoms commonly associated with many human diseases could be recapitulated by transferring the microbiome and, in the case of organ growth gf pigs had a smaller thyroid and liver, but larger spleen, lung, heart, and gall bladder than cv at 7 weeks of age. shurson et al. (1990) relative si length & weight in gf and ma pigs, the relative length of si was reduced compared with cv at postpartum day 13. shirkey et al. (2006) compared to gf and ma, relative weight of proximal si regions was higher for cv; while higher relative weight in the distal regions was reported in gf. shirkey et al. (2006) the si thickness of gf pigs was reduced compared with cv. shurson et al. (1990) gf pigs had fewer leukocytes and lower proportion of mature neutrophils in blood at 7 weeks of age. shurson et al. (1990) mono-associated gf pigs with escherichia coli strains increased numbers of dendritic and t cells in diffuse lymphoid tissue of the jejunum 2 days post-association. haverson et al. (2007) serum immunoglobulin level in piglets colonized with a mixture of defined bacteria was significantly higher than in gf piglets in the first 6 weeks of life. butler et al. (2000) si expression of proinflammatory cytokines il-1β and il-6 were higher in gf and ma pigs relative to cv at postnatal day 13. shirkey et al. (2006) abbreviations: cv, conventional; gf, germ-free; ma, mono-associated; si, small intestine. kwashiorkor, providing a similar diet (smith et al. 2013) . however, due to the differences in anatomy and physiology between rodents and humans, some important members of human gut microbiota, such as bifidobacterium do not readily colonize the rodent gut (raibaud et al. 1980 ). thus, results obtained from the use of rodent models may be difficult to extrapolate to humans, especially human infants, who are extensively colonized with bifidobacterial species. several studies have investigated the possibility of transfaunation of gut microbiota from humans to piglets (table 3 ). in the study of pang and colleagues (2007) , piglets delivered by cesarean section were housed in an spf barrier system and were inoculated orally with a fecal suspension collected from a healthy 10-year-old boy. the culture-independent analysis of the gut microbiota of recipient piglets and the human donor revealed that the microbiota of hma piglet was more similar to that of human donor than to that of cv piglets. moreover, bifidobacterium and bacteroides, two predominant bacterial groups of the infant gut, were successfully established in the gastrointestinal tract of piglets. furthermore, introduction of solid food during the weaning period significantly altered the gut microbiota in hma piglets; this change in the gut microbiota is similar to that observed in human infants (table 3) . in another study, piglets derived by cesarean section were inoculated with human infant or adult microbiota (table 3 ). the piglets were housed in sterile isolators and maintained on infant formula or solid diet for swine. high throughput sequencing of the 16s rrna v6 region was used to monitor to what extent the transplanted human microbiota changed in piglets over time. when infant stool was transferred, the microbiota composition of the hma piglets converged toward that of the human donor. in contrast, the microbiota of hma piglets harboring the adult human microbiota did not converge toward the composition of the donor even 20 days postinoculation. in a more recent study (zhang et al. 2014) , piglets derived by hysterectomy were inoculated with a suspension of fecal samples obtained from a bf infant between 17 and 23 days postpartum. the piglets were maintained in germ-free isolators and fed sterilized infant formula. sequencing the v4 region of 16s rrna genes showed that hma pigs harbored a microbiota similar to that of the infant donor. collectively, these studies demonstrate the feasibility of transplantation of a complex human gut microbiota to piglets. additionally, in comparison with the cv counterparts, the intestinal immunity of hma piglets is well developed (che et al. 2009 ), whereas that of hma rodents is not (imaoka et al. 2004 ). therefore, the hma piglet model provides a significantly improved system for research on gut ecology and host-microbe interactions, particularly when the human infant is the population of interest (pang et al. 2007; zhang et al. 2013 zhang et al. , 2014 . the hma pig model has been used in several recent publications to study dietary prebiotics and probiotics and for infection models. the first use of hma piglets was described by shen and colleagues (2010) , who studied the prebiotic activity of shortchain fructo-oligosaccharides (scfos). the piglets were inoculated with fecal suspension from a 27-year-old man and fed basal diets alone (control) or supplemented with scfos at 0.5 g/kg body weight daily for 37 days after birth. the composition of the fecal microbiota was monitored by denaturing gradient gel electrophoresis and quantitative polymerase chain reaction (pcr). as demonstrated previously in human trials (bouhnik et al. 1999; , supplementation of scgos increased the abundance of bifidobacterium. the bifidogenic effect of gos (3 g/l of formula) has also been examined in newborn cv piglets; however, no significant increase in fecal bifidobacteria abundance was detected after 15 days of supplementation. differences in bifidogenic effects of fos observed in cv and hma piglets may partly due to differences in bifidobacterium species composition between hma and cv piglets. for example, hma piglets harbor bifidobacterium of human origin, such as b. longum, b. breve, b. catenulatum, and b. adolescentis, while bifidobacterium found in the gut of piglets are b. suis, b. globosum, and b. pseudolongum (harrman and knol 2005; heinritz et al. 2013) . previous studies have shown prebiotics stimulate bifidobacteria species differently. for example, a mixture of scgos and polydextrose in infant formula increased b. longum but not b. catenulatum counts (scalabrin et al. 2012) . because of the important role of bifidobacterium in preventing intestinal infection, promoting gut integrity, and modulating the host immune homeostasis (gibson and roberfroid 1995) , stimulating the growth of gut bifidobacterium is considered as a marker of prebiotic effect (roberfroid et al. 2010) . therefore, hma piglets provide a more attractive model than cv piglets for evaluation of potential prebiotics. another application of the hma piglet model is for testing therapeutic interventions, such as probiotics and vaccination on host immune response and gut microbiota. wen and colleagues (2014) tested dose-dependent effects of lactobacillus rhamnosus gg (lgg) on the immune response to human rotavirus (hrv) vaccination in the hma pig model. they observed that the human gut microbiota stimulated neonatal immune development, as evidenced by a significant increase in the frequencies of interferon (ifn)-γ producing t cells and a decrease in the frequencies of cd4+cd25-foxp3+ regulatory t cells (tregs), and il-10-or tgf-β-producing tregs in hrv-vaccinated pigs. furthermore, the higher dose of lgg (14 doses, up to a 10 9 colony-forming-unit [cfu]/dose), but not the lower dose (9 doses, up to 10 6 cfu/dose), increased the lgg counts in the intestinal contents of hma pigs and significantly enhanced hrv-specific ifn-γ-producing t cell responses. moreover, oral supplementation of lgg prevented the changes in gut microbial composition caused by hrv infection (zhang et al. 2014 ). the hma pig model for studies of human gut microbiome because of the important role of human microbiota in the maintenance of health and causation of disease, several international efforts have designed to the study of human microbiota in recent years, including the human microbiome project (hmp) (http:// commonfund.nih.gov/hmp/index [human microbiome consortium 2012]) and the metagenomics of the human intestinal tract (metahit) (www.metahit.eu ]) initiatives. while much progress has been made by describing the composition of the gut microbiome in the human population and linking it to age-and health-related outcomes, much of the data at this point are associative. furthermore, confounding factors that influence the composition of the gut microbiota are difficult or impossible to control at the present time in human studies. these factors include individual variation in the host genetics and microbiota, current and past environmental exposures, and dietary nutrient composition and caloric load (gootenberg and turnbaugh 2011) . the hma pig model provides the ability to minimize many of the confounding variables mentioned above and, as such, will be valuable for studying microbiota composition change due to external factors, such as age, diet, viral infections, vaccination, and antibiotic use on the development of gut microbiota. human donors 10-y-old boy (n = 1) adults (n = 10; 50-70 y) 3-mo-old bf baby (n = 1) adults (n = 10; 50-70 y) 17-23 d-old bf infant (n = 1) fecal inoculation 1 ml of 5% fecal suspension 3 ml of 10% fecal suspension 3 ml of 10% fecal suspension 3 ml of 10% fecal suspension 1 ml of 5% fecal suspension • gn pigs carried a microbiota similar to the human donor's microbiota transplantation of gut microbiota from human to piglets is feasible. the pig intestine can be colonized with human fecal microbiota to generate a realistic model of human gi tract. human gut microbiota could be transplanted to and colonize gn pigs. abbreviations: bf, breast-fed; cv, conventional; d, day; eric-pcr, enterobacterial repetitive intergenic consensus sequence-pcr; gi, gastrointestinal; gn, gnotobiotic; hma, human microbiota-associated, qpcr, quantitative pcr; spf, specific pathogen free; ttge, temperature gradient gel electrophoresis; y, year. the hma pig model for investigating the role of microbiota on normal development establishment of the gut microbiota after birth plays an important role in stimulating the development of the neonatal gastrointestinal, immune and neural systems. studies in gf animals have shown that colonization of the commensal microbiota is required for normal intestinal epithelial cell proliferation and migration, and maintenance of villus morphology (shirkey et al. 2006; van kessel 2007, 2009 ). gf animals do not develop normal lymph node architecture and have a reduced antibody production (macpherson and harris 2004; round and mazmanian 2009) . evidence for the role of gut microbiota in neural development is intriguing, and mechanistic data is rapidly emerging. diaz heijtz and colleagues (2011) investigated the impact of colonization of gut microbiota on the mammalian brain development and behavior and reported that gf mice displayed increased motor activity and reduced anxiety compared to spf mice with a normal gut microbiota. additionally, gf mice exposed to gut microbiota early in life showed characteristics similar to spf mice, including reduced expression of synaptophysin and psd-95, two proteins that are specifically involved in synaptogenesis pathways (diaz heijtz et al. 2011) . studies of gastrointestinal, immune, and neural development often require tissue collection from the gastrointestinal tract, immune organs, and brain; however, due to ethical concerns and the limitation of invasive procedure, collecting tissue samples from human subjects is extremely difficult or impossible. therefore, clinically relevant animal models are needed. because of the high degree of similarity in anatomy, physiology, immunology, and brain growth and development patterns between pigs and humans, piglets are considered an ideal model for research on gastrointestinal, immune, and brain development. previous studies have shown environmental factors, such as diet and the use of antibiotics, pre-and probiotics, modify the composition of gut microbiota . germ-free piglets colonized with human intestinal communities provide a tool for examining the environmental factors on the establishment of gut microbiota and how the resultant microbiota impacts the development of gastrointestinal, immune, and neural systems. as previously discussed, symbiotic host-microbiota interactions play a key role in maintaining homeostasis. shifts in the bacterial composition of the human gut microbiota have been associated with several human disorders. table 4 summarizes association between gut microbiota change and microbiota-associated diseases. much of the information regarding the role of gut microbiota in human diseases comes from cross-sectional studies in which microbial community structures are altered in subjects with disease compared to healthy controls. however, it remains unclear whether changes in gut microbiota composition are the cause or the consequence of the diseases. studies designed to access a causative role for the gut microbiota are critically needed. understanding dysbiosis in human subjects is challenging because of the extraordinary complexity of the gut ecosystem and the tremendous variability in microbiota between healthy individuals (gill et al. 2006) . hma pigs provide an excellent model for isolating microbiota as an environmental factor in disease models. for example, fecal samples could be collected from lean and obese humans or individuals suffering from microbiota-associated diseases, such as ibd and nec, and healthy controls and then used to colonize gf pigs. using hma pigs, together with metabolomics, metaproteomics, host gene expression profiling, and metatrascriptomics, we may be able to delineate the role of gut microbiota in diseases at the cellular and molecular level. using hma piglets to identify potential biomarkers of microbiota-associated diseases through the use of metabolomics and metaproteomics has implications for development of diagnostic and therapeutic strategies for both infectious and noninfectious conditions. however, a current limitation is the completeness of bioinformatics repositories for metabolomics and proteomics. pre-and probiotics have been studied in recent decades as a way to modulate gut microbial composition and functions (ducatelle et al. 2014 ). prebiotics are defined as "a selectively fermented ingredient that results in specific changes, in the composition and/ or activity of the gastrointestinal microbiota, thus, conferring benefit(s) upon host health" (roberfroid et al. 2010, p. s2) . prebiotics have the potential to stimulate the growth of beneficial bacteria, such as bifidobacterium and lactobacillus. probiotics are "live microorganisms which when consumed in adequate amounts, confer a health benefit on the host" (fao/who 2001, p. 2). bifidobacterium and lactobacillus are the most commonly used probiotics (walsh et al. 2014) . other bacterial genera such as akkermansia and faecalibacterium have also been reported as potential probiotics (thomas et al. 2014) . pre-and/or probiotic intervention has been used successfully for promoting health and prevention or treatment of some microbiota-associated disorders, such as eczema, ibd, nec, and obesity in human studies (kadooka et al. 2010; li et al. 2014) ; however, the mechanisms underlying the beneficial effects of pre-or probiotics remain incompletely understood. understanding the impact of pre-or probiotics on the gut microbiota and host requires carefully controlled studies in which potential confounding variables such as host genotype, diet, and environmental exposure can be controlled. gnotobiotic animals can be reared under well-controlled conditions, representing one way to constrain some of these variables. recently, gn mice harboring a mixture of 15 species of human gut microbiota were studied prior to and after gavage with five fermented milk strains (mcnulty et al. 2011 ). the results revealed only a minimal change in the composition of the microbiota, whereas significant changes in the expression of microbiome-encoded enzymes in numerous metabolic pathways, especially the pathways related to carbohydrate metabolism, were observed (mcnulty et al. 2011 ). compared to rodents, pigs colonized with human microbiota are more similar to humans in anatomy, physiology, microbiota, and genetics, providing a more attractive model for 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the transplanted human gut microbiota in gnotobiotic pigs a pig model of the human gastrointestinal tract this work was supported in part by hatch funds (project illu-971-346) distributed by the division of nutritional sciences at the university of illinois through its vision 20/20 program. key: cord-016364-80l5mua2 authors: menotti-raymond, marilyn; o’brien, stephen j. title: the domestic cat, felis catus, as a model of hereditary and infectious disease date: 2008 journal: sourcebook of models for biomedical research doi: 10.1007/978-1-59745-285-4_25 sha: doc_id: 16364 cord_uid: 80l5mua2 the domestic cat, currently the most frequent of companion animals, has enjoyed a medical surveillance, as a nonprimate species, second only to the dog. with over 200 hereditary disease pathologies reported in the cat, the clinical and physiological study of these feline hereditary diseases provides a strong comparative medicine opportunity for prevention, diagnostics, and treatment studies in a laboratory setting. causal mutations have been characterized in 19 felid genes, with the largest representation from lysosomal storage enzyme disorders. corrective therapeutic strategies for several disorders have been proposed and examined in the cat, including enzyme replacement, heterologous bone marrow transplantation, and substrate reduction therapy. genomics tools developed in the cat, including the recent completion of the 2-fold whole genome sequence of the cat and genome browser, radiation hybrid map of 1793 integrated coding and microsatellite loci, a 5-cm genetic linkage map, arrayed bac libraries, and flow sorted chromosomes, are providing resources that are being utilized in mapping and characterization of genes of interest. a recent report of the mapping and characterization of a novel causative gene for feline spinal muscular atrophy marked the first identification of a disease gene purely from positional reasoning. with the development of genomic resources in the cat and the application of complementary comparative tools developed in other species, the domestic cat is emerging as a promising resource of phenotypically defined genetic variation of biomedical significance. additionally, the cat has provided several useful models for infectious disease. these include feline leukemia and feline sarcoma virus, feline coronavirus, and type c retroviruses that interact with cellular oncogenes to induce leukemia, lymphoma, and sarcoma. mankind has held a centuries-long fascination with the cat. the earliest arch eological records that have been linked to the domestication of felis catus date to approximately 9500 years ago from cyprus, 1 with recent molecular genetic analyses in our laboratory suggesting a middle eastern origin for domestication (c. driscoll et al., unpublished observations) . currently the most numerous of companion animals, numbering close to 90 million in households across the united states (http://www.appma.org/ press_industrytrends.asp), the cat enjoys a medical surveillance second only to the dog and humankind. in this chapter we review the promise of the cat as an important model for the advancement of human hereditary and infectious disease and the genomic tools that have been developed for the identification, and characterization of genes of interest. for many years we have sought to characterize genetic organization in the domestic cat and to develop genomic resources that establish f. catus as a useful animal model for human hereditary disease analogues, neoplasia, genetic factors associated with host response to infectious disease, and mammalian genome evolution. 2, 3 to identify genes associated with inherited pathologies that mirror inherited human conditions and interesting pheno-types in the domestic cat, we have produced genetic maps of sufficient density to allow linkage or association-based mapping exercises. [4] [5] [6] [7] [8] [9] [10] [11] the first genetic map of the cat, a physical map generated from a somatic-cell hybrid panel, demonstrated the cat's high level of conserved synteny with the human genome, which offered much promise for the future application of comparative genomic inference in felid mapping and association exercises. 12 several radiation hybrid (rh) and genetic linkage (gl) maps have since been published. [4] [5] [6] [7] [8] [9] 11, 13, 14 although previous versions of the cat gene map, based on somatic cell hybrid and zoo fish analysis, 15 ,16 revealed considerable conservation of synteny with the human genome, these maps provided no knowledge of gene order or intrachromosomal genome rearrangement between the two species, information that is critical to applying comparative map inference to gene dis covery in gene-poor model systems. radiation hybrid (rh) mapping has emerged as a powerful tool for constructing moderate-to high-density gene maps in vertebrates by obviating the need to identify interspecific polymorphisms critical for the generation of genetic linkage maps. 7 the most recent rh map of the cat 8 includes 1793 markers: 662 coding loci, 335 selected markers derived from the cat 2x whole genome sequence targeted at breakpoints in conserved synteny between human and cat, and 797 short tandem repeat (str) loci. the strategy used in developing the current rh map was to target gaps in the feline-human comparative map, and to provide more definition in breakpoints in regions of conserved synteny between cat and human. the 1793 markers cover the length of the 18 feline autosomes and the x chromosome at an average spacing of one marker every 1.5 mb (megabase), with fairly uniform marker density. 8 an enhanced comparative map demonstrates that the current map provides 86% and 85% comparative coverage of the human and canine genomes, respectively. 8 ninety-six percent of the 1793 cat markers have identifi able orthologues in the canine and human genome sequences, providing a rich comparative tool, which is critical in linkage mapping exercises for the identification of genes controlling feline phenotypes. figure 25 -1 presents a graphic display of each cat chromosome and blocks of conserved syntenic order with the human and canine genomes. 8 one hundred and fifty-two cat-human and 134 cat-dog homologous synteny blocks were identified. alignment of cat, dog, and human chromosomes demonstrated different patterns of chromosomal rearrangement with a marked increase in interchromosomal rearrangements relative to human in the canid lineage (89% of all rearrangements), as opposed to the more frequent intrachromosomal rearrangements in the felid lineage (95% of all rearrangements) since divergence from a common carnivore ancestor 55 my ago. with an average spacing of 1 marker every 1.5 mb in the feline euchromatic sequence, the map provided a solid framework for the chromosomal assignment of feline contigs and scaffolds during assembly of the cat genome assembly, 17 and served as a comparative tool to aid in the identification of genes controlling feline phenotypes. as a complement to the rh map of the cat, a third generation linkage map of 625 strs is currently nearing completion. the map has been generated in a large multigeneration domestic cat pedigree (n = 483 informative meioses). 18 previous first-and second-generation linkage maps of the cat were generated in a multigeneration interspecies pedigree generated between the domestic cat and the asian leopard cat, prionailurus bengalensis, 7 to facilitate the mapping and integration of type i (coding) and type ii (polymorphic str) loci. 7 the current map, which spans all 18 autosomes with single linkage groups, has twice the str density of previous maps, providing a 5-cm resolution. there is also greatly expanded coverage of the x chromosome, with some 75 str loci. marker order between the current generation rh and gl maps is highly concordant. 8 approximately 85% of the strs are mapped in the most current rh map of the cat, 8 which provides reference and integration with type i loci. whereas the third-generation linkage map is composed entirely of str loci, the sequence homology of extended genomic regions adjacent to the str loci in the cat 2x whole genome sequence, 17 to the dog's homologous region, 19 has enabled us to obtain identifiable orthologues in the canine and human genome sequences for over 95% of the strs. thus, practically every str acts as a "virtual" type 1 locus, with both comparative anchoring and linkage map utility. combined with the cat rh map, these genomic tools provide us with the comparative reference to other mammalian genomes critical for linkage and association mapping. the domestic cat is one of 26 mammalian species endorsed by the national human genome research institute (nhgri) human genome annotation committee for a "light" 2-fold whole genome sequence, largely to capture the pattern of genome variation and divergence that characterizes the mammalian radiations (http:// www.hgsc.bcm.tmc.edu/projects/bovine/, http://www.broad.mit. edu/mammals/). although light genome coverage provides limited sequence representation, (∼80%), 20 one of the rationales for these light genome sequences included "enhancing opportunities for research on species providing human medical models." the 2-fold assembly of the domestic cat genome has recently been completed for a female abyssinian cat, "cinnamon," 17 and a 7x whole genome sequencing effort is planned in the near future. a total of 9,161,674 reads were assembled to 817,956 contigs, covering 1.642 gb with an n50 (i.e., half of the sequenced base pairs are in contigs <n50) contig length of 2378 bp. assembled supercontigs (n = 217,790) had an n50 length of 117 kb 17 (http:// hosted. abcc.ncifcrf.gov/cgi-gin/gbrowse/cat/). the estimated size of the genome was 2.7 gb and the genome coverage was approximately 2-fold, predicting an average inclusion of 80-85% of the eukaryotic genome sequence. 21 feline coding genes were identified using a comparative approach based upon sequence homology and syntenic orthology of neighboring gene homologues in the genomes of six index mammal species (human, chimp, mouse, rat, cow, and dog). the results revealed nearly 21,080 feline genes plus 132,493 conserved sequence blocks (csbs) used to build the gene map, 17 depending upon the framework rh map of 1794 ordered type 1 markers. 8 the 2x feline genome sequence detects 83% of human genes, 89% of chimp or cattle genes, and 92% of dog genes based upon sequence identity to approximately 1000 bp of reciprocal base match 17 between the cat sequence and the genome sequence of the six index mammals. a genome browser has been developed from the cat assembly, named genome annotation region field (garfield), which provides a physical map of the 18 autosomes and the x chromosome, which can be inspected for sequence representation, including genes and the proportion of that gene available in the 2x cover, single nucleotide polymorphisms (snps), and strs, which can be used in linkage and association mapping, and other genome features (http://ccr.cancer.gov/ labs/lab). figure a total of 421,000 snp variants were identified in cinnamon's sequence, representing an incidence of 1/600 bp. 17 approximately 43% of cinnamon's genome was heterozygous and 57% homozygous, which was not unexpected in a breed cat that is also the member of a highly inbred pedigree for retinal atrophy. 17, 22 long stretches of alternating homozygous and heterozygous segments were observed ( figure 25 -3), which represent the consequences of close inbreeding during the domestication process, and the more recent generation of fancy breeds and inbred disease pedigree. 22 similar patches of homozygous/heterozygous segments were observed in the recently released whole genome sequence of the dog. 19 the length of the segments is influenced by breedspecifi c history including effective population sizes, use of popular sires, and population bottlenecks. 23, 24 linkage disequilibrium (ld) mapping has recently emerged as a powerful approach in humans for association mapping. 25 long stretches of linkage disequilibrium in the target population greatly facilitate the success of the strategy and decrease the number of markers required for analysis. 25 the extended lds observed in dog breeds, up to one hundred times the length observed in human populations, 26 is proving to be a powerful mapping strategy for identification of genes associated with breedspecifi c phenotypes, 27 including hereditary pathologies. 28, 29 the potential of this mapping approach in cat breeds was evaluated by examining breed-specific patterns of common segment homozygosity in 24 cat fancier association (cfa) (http://www.cfainc.org) breeds. 17 the level of homozygosity refl ected in a group of 665 snps was roughly half that seen in dogs, 26 likely reflecting a more extensive recent inbreeding within heterozygosity across cinnamon's chromosomes is represented in nonoverlapping windows of 100 kb. black represents regions with more than two snps per 100 kb while gray represents the homozygous region (less than two snp, 0/100 kb). white represents gaps in the chromosome assembly. dog versus cat breeds. 30 this level of homozygosity was used to estimate 19 that some 45,000 equivalently spaced snp variants would be required for a linkage disequilibrium/haplotype-based association genome search of a complex heritable disease within cat breeds. recently, tyrosinase-related protein 1 (tyrp1), one of the key enzymes in the melanogenic pathway, was linked to two coat color variants in the cat by association mapping in 38 cat breeds due to extensive ld. 31 two dna polymorphisms in tyrp1, an a3g substitution in the signal peptide and an in-frame insertion tyrp1-421ins17/18, were associated with the chocolate (b) allele. a premature uag stop codon at position 100 of tyrp1 was associated with a second allele of the b locus, cinnamon (bl). 32 snp discovery is planned in the 7x whole genome sequencing of the cat through a resequencing strategy of selected genomic regions in several cat breeds as was recently performed in the 7x whole genome sequencing of the dog. 19 the majority of hereditary pathologies in the domestic cat for which the gene defect has been elucidated have resulted from the analysis of candidate genes (table 25-1) . however, with the availability of a detailed comparative map, and integration with developing gl and rh maps, and the cat 2x whole genome sequence, linkage and association-based mapping techniques have recently identified causative mutations for hereditary disease genes, 33, 34 as well as several feline phenotypes (table 25-1) . 18, 32, once a genomic region is implicated from association-based or linkage mapping exercises, fine mapping has been accomplished by development of new strs or snps in the targeted region using the cat 2x whole genome sequence data accessed sequence tagged sites (sts), utilizing sequence information from the cat 2x whole genome sequencing effort, which ultimately identifi ed an ∼140 kb deletion and a novel gene candidate, lix1 34 ( figure 25-4) . though the function of lix1 is unknown, the predicted secondary structure is compatible with a role in rna metabolism. an exon sequence screen of 25 human sma cases, not otherwise explicable by mutations at the smn1 locus, failed to identify comparable lix1 mutations. 34 the smn1 gene product, smn, is a ubiquitously expressed protein member of multiple ribonucleoprotein complexes with diverse roles in rna metabolism, splicing, and transport in all cells. 62, 63 a central focus of sma research remains to discern the disease mechanism(s) and to understand why the primary disease pathology is localized to spinal lower motor neurons when all cells require smn function. lix1 expression is largely restricted to the central nervous system (cns), primarily in spinal motor neurons, thus offering an explanation of the tissue restriction of pathology in feline sma. determination of lix1 function may well provide fresh insight into the mechanisms of human sma pathology, impetus for more targeted therapeutics, and answers to fundamental questions of motor neuron development, maintenance, and/or function. through the cat genome browser, garfield. 17 a recent report of the mapping and characterization of a novel gene causative of feline spinal muscular atrophy 34 marked the first identification of a disease gene purely from positional reasoning. human spinal muscular atrophies (smas) are a genetically heterogeneous group of neuropathies that varies in clinical severity, from lethal in infancy to onset of mild weakness in adulthood, but all are characterized by neurogenic muscle atrophy due to degeneration of lower motor neurons of the spinal cord. 58 for approximately 97% of people affected with sma, disease pathology is attributable to a mutation in the smn1 gene, on human chromosome 5q13, which is subject to a high frequency of deletions and gene conversion events with the divergent and only partially functional centromeric copy/copies of the duplicated smn2 locus. 59, 60 a domestic cat model of sma has been described that is a model of autosomal recessive juvenile-onset sma. 61 with the feline smn gene excluded as the disease locus, 61 a full genome linkage scan was conducted in a pedigree segregating for sma. 61 the disease phenotype was linked to chromosome a1q, 34 in a region of conserved synteny to human chromosome 5q15. fine mapping was accomplished with development of new strs and the world's veterinary schools produce thousands of practitioners each year, most of whom carefully document genetic and chronic diseases of our pets. the result is a comprehensive veterinary literature that has described over 200 feline hereditary pathologies 17 (http://www.angis.org.au/). the clinical and physiological study of these feline hereditary diseases provides a strong comparative medicine opportunity for prevention, diagnostics, and treatment studies in a laboratory setting. additionally, large animal homologues are similar to humans in natural genetic diversity and offer the possibility of evaluating long-term effects of treatment. 64 to date, causal mutations have been characterized in 19 felid genes that cause hereditary disease (table 25 -1). the largest representation comes from lysosomal storage enzyme disorders that arise from defects in genes playing a role in degradation of macromolecules targeted to the lysosomes. many of the genes that cause these pathologies have been mapped in the cat. 65 corrective therapeutic strategies have been proposed and examined in the cat, including enzyme replacement, heterologous bone marrow transplantation, and substrate reduction therapy. 64, 66 limitations to these treatment strategies include high morbidity and mortality, limited positive outcomes, incomplete response to therapy, cost, and in some cases requirements for continuous lifelong therapy. 66 gene therapy poses the most recent of intervention strategies. feline models have been important in elucidating molecular pathogenesis and are now playing a critical role in evaluating and optimizing the range of therapeutic strategies prior to clinical trials in humans. the mucopolysaccharidoses are disorders that result from the defi ciency of lysosomal enzymes involved in the degradation of mucopolysaccharides. the cat offers homologous models for mucopolysaccharidosis types i, vi, and vii. mucopolysaccharidosis type i (mps i), which results from a genetic deficient activity of the enzyme α-l-iduronidase (idua), can lead to mental retardation, growth abnormalities, and shortened life span in humans. 67 naturally occurring models have been characterized in the cat 68,69 and dog. 70 immune responses can nullify the effect of gene corrective therapy. it has been demonstrated that cats, but not dogs, mount a potent ctl response to canine idua after neonatal gene therapy, which can be prevented with transient ctla4-ig. 71 the efficacy of neonatal retroviral therapy has also been explored in the cat. 66 the cat model, additionally, provides an ideal system to study mechanisms of brain neurodegeneration and neural-directed strategies, especially given a large body of preexisting literature on cat neurology. mps vi or marteaux-lamy disease, deficient activity of arylsulfatase b (arsb), is characterized in humans and cats with growth retardation, coarse facial features, corneal opacity, and skeletal deformities. [72] [73] [74] [75] the feline model also exhibits abnormal lysosomal storage in occasional neurons and glia distributed throughout the cerebral cortex. 76 fibroblast-mediated in vitro gene therapy has been examined in this cat model. 77 recently, an adeno-associated vector containing feline arsb has demonstrated gene therapy-based correction of corneal clouding in the mps vi cat. 78 mps vii results from deficiency of β-glucuronidase (gusb), which in humans manifests as cartilaginous and bony malformations, growth and mental retardation, abdominal organ enlargement, and corneal clouding. 79 naturally occurring animal models have been described in mice, 80 dogs, 81 and the cat. 82 enzymatic activity has been restored in fibroblasts and restored by retroviral gene transfer of rat gusb cdna. as gusb is an essential housekeeping enzyme, this feline model is important for examination of exogenous genes and gene product delivery to a variety of tissue types, and could prove especially valuable due to extensive research conducted on the anatomy and physiology of the cat central nervous and visual systems. three different serotypes of adeno-associated viral constructs of gusb demonstrated the efficacy of this vector to achieve gene transfer in the normal cat brain, as a model for the efficacy of this construct in a large mammalian brain. 83 defi ciency of lysosomal α-mannosidase leads to an accumulation of mannose-rich oligosaccharides, 84 which leads to mental retardation, recurrent infections, skeletal changes and hearing impairment. 85 this feline model was initially important in achieving bone marrow transplantation as corrective strategy for neuronal storage diseases of the cns. 86, 87 recently, adeno-associated viral (aav) constructs of feline α-mannosidase were used to demonstrate the efficacy of cns gene therapy. treated cats exhibited widespread improvement of neuropathology, showing the effi cacy of this treatment in a large mammalian brain for cns correction of human lysosomal enzyme defi ciencies. 88 lipoprotein lipase (lpl) is a crucial enzyme involved in the regulation of lipoprotein and lipid metabolism ability to thrive. 89 cats with lpl deficiency display a remarkably similar phenotype to humans, including severe pancreatitis, chylomicronemia, and failure to thrive. 46 there is currently no adequate treatment for this pathology in humans. cats could prove to be the most valuable animal model of lpl deficiency, as of numerous animal model systems examined including the mouse, the cat most closely resembles the lipoprotein pattern and lipid transport system of humans. recently, aav-mediated transfer of a human lpl s447x variant into feline muscle cells demonstrated correction of the hypertriglyceridemia associated with feline pathology; this offers much promise for treatment of human lpl defi ciency. 90 a separate class of lysosomal storage disorder characterized in the cat is the gangliosidoses, g m1 and g m2 , which are heritable neurodegenerative diseases. a deficiency of lysosomal β-galactosidase results in the neuronal accumulation of the g m1 ganglioside, while the degradation of the g m2 ganglioside is initiated by coordinated action of at least three gene products, the α and β submits of β-n-acetylhexosaminidase and the g m2 activator (gm2a) protein. 42, 91 mutations in any of these enzymes result in an accumulation of gangliosides g m1 and g m2 in the lysosomes of affected neurons, resulting in progressive deterioration of the cns. feline models have been especially important in characterizing the pathobiology and molecular biology of these diseases. g m2 -gangliosidosis has been characterized in cat models defi cient in the g m2 activator protein and hexb, 40, 42, 92, 93 exhibiting remarkably similar pathology to human sandhoff's disease. 91 limited reduction in g m2 neuronal storage has been reported following bone marrow therapy. 86 feline models will be important in the development of therapeutic strategies for these disorders. mucolipidosis ii (i-cell disease) is caused by deficient activity of the enzyme n-acetylglucosamine-1-phosphotransferase, which leads to a failure to internalize enzymes into lysosomes. the cat is the only known animal model for this pathology. 94 congenital diseases of feline muscle and neuromuscular junction have been reviewed by gashen et al. 95 some pathologies have been observed in isolated breeds, including hypokalemic myopathy of burmese cats, 96 glycogen storage disease type iv in norwegian forest cats, 44 and myopathy observed in the devon rex. 97 the cat is the only reported animal model for type iv glycogen storage disease. myotonia congenital, 95,98 muscular dystrophy (dystrophin defi cient), 52 and laminin α 2 defi ciency 52, 99 have also been reported in the cat. x-linked muscular dystrophy in humans is characterized by progressive degeneration of skeletal and cardiac muscle. mutations in humans lead to either an absence of or abnormality in the protein product dystrophin. 100, 101 a deletion in the dystrophin muscle promoter characterized in the cat eliminates expression of muscle and purkinje neuronal dystrophin isoforms. 52 marked clinical heterogeneity is observed in these models, from severe disability exhibited in human and dog, to minor muscle fi brosis and an actual regenerative process leading to muscle hypertrophy in mouse and cat. [102] [103] [104] these different sequellae could be important in characterizing immediate and secondary consequences of the lack of dystrophin 105 and points out the importance of multiple animal models. hypertrophic cardiomyopathy is a clinically heterogeneous myocardial disease contributing to one of the most common causes of sudden cardiac death in young adults. 106 the cat represent the first spontaneous large animal model for this familial disease 47 and will prove to be valuable for examining pathophysiological processes and therapeutic interventions. there is a continued demand for alternative mammalian models for studying human diseases. compared to traditional murine models, the cat's more similar physiology, increased size, and longevity have made it ideal for testing the safety and efficacy of some therapeutic modalities in naturally occurring feline disease models. [107] [108] [109] [110] additionally, cats have a genetically heterogeneous background, similar to humans. a good example of a human disease in need of a better mammalian model is cystic fibrosis, as mice targeted with the most common human mutation (∆f508) in the cystic fibrosis transmembrane receptor (cftr) fail to spontaneously develop the same opportunistic lung infections that plague human patients with cystic fi brosis. 111, 112 one group has been working to produce a ferret cystic fibrosis model through gene targeting of somatic cells for nuclear transfer. 113, 114 while reproductive cloning has a consistently low success rate (1-4%) across mammalian species, 115 targeting genetic loci through homologous recombination in somatic cells is currently the only viable method for producing knockout models in all mammalian species other than the mouse, for which targeted embryonic stem cells are routinely used. nuclear transfer of targeted fibroblasts has been successfully used to produce viable α-1,3-galactosyltransferase knockout pig and cow models for xenotransplantation studies. 116, 117 with the successful reproductive cloning of cats by several groups, 118-120 the development of gene-targeted cat models through nuclear transfer is now feasible. 121 the imminent release of the annotated feline genome project, 17 integration of recombination and radiation hybrid maps, [5] [6] [7] [8] [9] [10] 122 and availability of multiple pac, bac, 123 flow-sorted autosome, and y-chromosome libraries 124, 125 (j. pecon-slattery et al., unpublished observations) at the laboratory of genomic diversity will facilitate efforts by researchers to develop new cat models of specific human genetic diseases. the coat color loci influence the development, maturation, and migration of melanocytes as well as the synthesis of melanin and the formation, transport, and transfer of melanosomes. genes involved in these processes often have pleiotropic effects, which impact other important biochemical pathways. coat color loci in the mouse have been known to be part of diverse cellular, developmental, and physiological processes and in some cases to be implicated in pathologies such as anemia, sterility, and neurological disorders. [126] [127] [128] the cat is an excellent model system with which to study coat color phenotypes. at least nine different coat color loci have been identified in the cat, 129 and several are now characterized on a molecular genetic level including, a (nonagouti) responsible for melanism, 18 b (brown), which changes black pigmentation to brown or variants of brown, 32 c (color), causing the darker pigmentation at extremities (i.e., ears, tail), observed in siamese and burmese cats 32,37 and albinism, 36 and d (dilute), which causes dilution of expected color (i.e., black pigmentation appears gray). 35 the cat is also unique in mammalian species in exhibiting a variation in coat pattern, demonstrating agouti (a) (nonpatterned coat) and variants of the t (tabby) locus, which affect striping and spotting patterns. the cat has provided several useful models for infectious disease. these include feline leukemia and feline sarcoma virus, feline coronavirus, and type c retroviruses that interact with cellular oncogenes to induce leukemia, lymphoma, and sarcoma. [130] [131] [132] historically, many of the human oncogenes that defi ne signal transduction pathways were originally discovered in the context of feline leukemia virus interaction in cat models. the cat provides the only naturally occurring model for human aids pathogenesis, in its endemic fatal transmissible feline immunodefi ciency virus (fiv). 133, 134 similar to its close phylogenetic relative hiv, fiv induces cd4-t lymphocyte depletion in affected cats, an immune system collapse, and susceptibility to adventitious microbial agents as a prelude to wasting disease and death. 133, 135 interestingly, over 10 wildcat species (including lions, leopards, cheetahs, ocelots, pumas and other big cats) are endemic with their own species-specific strain of fiv [136] [137] [138] [139] [140] [141] ; however, unlike strains in domestic cats, the wildcat fiv strains do not appear to cause acute immunodeficiency in the wildcat species, perhaps a consequence of historic natural selection of host genetic resistance to the fatal virus. 139, 142 lion and puma-specific fiv strains have recently been demonstrated to utilize novel, more promiscuous mechanisms for cell entry than fiv, suggesting a divergent tropism and biological properties of these viruses. 143 the world health organization reported a new human respiratory illness outbreak (severe acute respiratory syndrome, sars) that emerged in guangdong province, china in 2003. 144, 145 sequence analysis demonstrated that the infectious agent was a previously unrecognized coronavirus. 146, 147 an animal model demonstrating clinical symptoms and pathology of sars-infected patients has not been reported. 148 of interest, the recent report of a highly virulent feline coronavirus epidemic in captive african cheetahs, a disease model for human sars, illustrates the critical role of ancestral population genetic variation. 132 in addition, cats injected with the sars virus developed clinical symptoms, an important insight in implicating the virus in the sars epidemic. [149] [150] [151] [152] the feline panleukopenia (feline distemper) virus has revealed a natural history parable in its abrupt transformation of the cat virus to an epidemic, fatal canine parvovirus, that emerged in the world's puppy population in 1978. 153 in contrast, the canine distemper virus, which is normally restricted to canid species, precipitously adapted to and decimated east african lions in 1994, killing one-third of the lions in the serengeti ecosystem within a 9-month outbreak. 154 a clear involvement of host defense mechanisms in these and other infectious disease episodes renders the cats and their pathogens an excellent candidate species for characterizing the interaction of microbial adaptation and host disease gene defenses. given the critical importance of infectious disease in scores of chronic and acute human disease, there are powerful research opportunities in the cat family. 142, 155 finally, recent concern over the emergence of avian flu h5n1 has shown a strong susceptibility of cats, both domestic and large cats, again raising possibilities for pathogenesis and therapy development. 156, 157 with the development of genomic resources in the cat (table 25 -2) and the application of complementary comparative tools developed in other species, the domestic cat is emerging as a promising resource of phenotypically defined genetic variation of biomedical significance. exploration of similar resources in other species, particularly the dog and mouse, has provided important insight into otherwise unexplained biomedical disorders. early taming of the cat in cyprus the feline genome project integration of the feline radiation hybrid and linkage maps radiation hybrid mapping of 304 novel microsatellites in the domestic cat genome a genetic linkage map of microsatellites in the domestic cat (felis catus) a 1.5 megabase resolution radiation hybrid map of the cat genome and comparative analysis with the canine and human genomes second-generation 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cardiomyopathy a missense mutation in nacetylglucosamine-1-phosphotransferase causes mucolipidosis ii in domestic shorthair cats two mutations within a feline mucopolysaccharidosis type vi colony cause three different clinical phenotypes molecular basis of feline beta-glucuronidase deficiency: an animal model of mucopolysaccharidosis vii deletion of the dystrophin muscle promoter in feline muscular dystrophy mutation analysis of feline niemann-pick c1 disease a tyrosinase gene missense mutation in temperature-sensitive type i oculocutaneous albinism. a human homologue to the siamese cat and the himalayan mouse molecular basis of erythrocyte pyruvate kinase (rpk) deficiency in cats cats lack a sweet taste receptor nomenclature for the description of human sequence variations spinal muscular atrophy identifi cation and characterization of a spinal muscular atrophy-determining gene quantitative analysis of survival motor neuron copies: identification of subtle smn1 mutations in patients 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arylsulfatase b defi ciency: a model of maroteaux-lamy syndrome mucopolysaccharide storage disease in three families of cats with arylsulfatase b defi ciency: leukocyte studies and carrier identifi cation animal model of human disease: mucopolysaccharidosis vi maroteaux-lamy syndrome, arylsulfatase b-deficient mucopolysaccharidosis in the siamese cat abnormal neuronal metabolism and storage in mucopolysaccharidosis type vi (maroteaux-lamy) disease evaluation of fi broblast-mediated gene therapy in a feline model of mucopolysaccharidosis type vi phenotypic rescue after adeno-associated virus-mediated delivery of 4-sulfatase to the retinal pigment epithelium of feline mucopolysaccharidosis vi the metabolic and molecular basis of inherited disease beta-glucuronidase deficiency in a dog: a model of human mucopolysaccharidosis vii feline mucopolysaccharidosis vii due to beta-glucuronidase defi ciency adeno-associated virus vector-mediated transduction in the cat brain disorders of glycoprotein degradation: α-mannosidosis, β-mannosidosis, sialidosis, aspartylglucosaminuria, and carbohydrate-deficient glycoprotein syndrome biochemical studies on a case of feline mannosidosis bone marrow transplantation corrects the enzyme defect in neurons of the central nervous system in a lysosomal storage disease bone marrow transplantation in animal models of lysosomal storage diseases effective gene therapy for an inherited cns disease in a large animal model familial lipoprotein lipase deficiency and other causes of the chylomicronemia syndrome correction of feline lipoprotein lipase deficiency with adeno-associated virus serotype 1-mediated gene transfer of the lipoprotein lipase s447x beneficial mutation characterization of a new model of gm2 gangliosidosis (sandhoff's disease) in korat cats the metabolic and molecular bases of inherited diseases gm2-gangliosidosis variant 0 (sandhoff-like disease) in a family of japanese domestic cats inheritance, biochemical abnormalities, and clinical features of feline mucolipidosis ii: the fi rst animal model of human i-cell disease congenital diseases of feline muscle and neuromuscular junction hereditary myopathy of devon rex cats congenital myotonia in related kittens congenital myotonia in 2 domestic cats laminin alpha 2 (merosin)-defi cient muscular dystrophy and demyelinating neuropathy in two cats dystrophin: the protein product of the duchenne muscular dystrophy locus dystrophin defi ciency causes lethal muscle hypertrophy in cats the complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein functional regeneration in the hindlimb skeletal muscle of the mdx mouse feline muscular dystrophy with dystrophin defi ciency muscular dystrophies: expanding our knowledge in companion animals the molecular genetic basis for hypertrophic cardiomyopathy large animal models and gene therapy autologous transplantation of retrovirally transduced bone marrow or neonatal blood cells into cats can lead to long-term engraftment in the absence of myeloablation visual resolution with retinal implants estimated from recordings in cat visual cortex restoration of auditory nerve synapses in cats by cochlear implants generation and characterization of a delta f508 cystic fibrosis mouse model effi cient killing of inhaled bacteria in deltaf508 mice: role of airway surface liquid composition progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer cloned ferrets produced by somatic cell nuclear transfer somatic cell nuclear transfer production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning 3-galactosyltransferase-gene knockout in cattle using a single targeting vector with loxp sequences and cre-expressing adenovirus a cat cloned by nuclear transplantation birth of african wildcat cloned kittens born from domestic cats cats cloned from fetal and adult somatic cells by nuclear transfer nuclear transfer in cats and its application development of a feline whole-genome radiation hybrid panel and comparative mapping of human chromosome 12 and 22 loci the pattern of phylogenomic evolution of the canidae conservation of human vs. feline genome organization revealed by reciprocal chromosome painting the coal colors of mice: a model for mammalian gene action and interaction from white spots to stem cells: the role of the kit receptor in mammalian development molecular and developmental genetics of mouse coat color robinson's genetics for cat breeders and veterinarians feline leukemia virus feline oncoretroviruses plagues and adaptation: lessons from the felidae models for sars and aids the feline immunodeficiency virus fiv infection of the domestic cat: an animal model for aids toward a detailed characterization of feline immunodeficiency virus-specific t cell immune responses and mediated immune disorders worldwide prevalence of lentivirus infection in wild feline species: epidemiologic and phylogenetic aspects a lion lentivirus related to feline immunodeficiency virus: epidemiologic and phylogenetic aspects coadaptation and immunodeficiency virus: lessons from the felidae patterns of feline immunodeficiency virus multiple infection and genome divergence in a free-ranging population of african lions seroprevalence and genomic divergence of circulating strains of feline immunodeficiency virus among felidae and hyaenidae species genomic prospecting feline lentiviruses demonstrate differences in receptor repertoire and envelope structural elements sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus animal-to-human sars-associated coronavirus transmission? civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates pet theory comes to the fore in fight against sars virology: sars virus infection of cats and ferrets the emergence and evolution of canine parvovirus-an example of recent host range mutation a canine distemper virus epidemic in serengeti lions (panthera leo) plague time: how stealth infections cause cancer, heart disease, and other deadly ailments germany says people in areas with bird flu should keep cats indoors h5n1 virus attachment to lower respiratory tract infl uenza a virus (h5n1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts avian h5n1 infl uenza in cats feline friend or potential foe? comparative gene mapping in the domestic cat (felis catus) zoo-fish analysis: cat and human karyotypes closely resemble the putative ancestral mammalian karyotype patterns of y and x chromosome dna sequence divergence during the felidae radiation phylogenetic reconstruction of the felidae using 16s rrna and nadh-5 mitochondrial genes complete nucleotide sequence of the domestic cat (felis catus) mitochondrial genome and a transposed mtdna tandem repeat (numt) in the nuclear genome comparative genome organization of human, murine, and feline mhc class ii region key: cord-018354-o6pmuhd8 authors: mine, yoichi; gómez, oscar a.; muto, ako title: human security in east asia: assembling a puzzle date: 2018-12-07 journal: human security norms in east asia doi: 10.1007/978-3-319-97247-3_1 sha: doc_id: 18354 cord_uid: o6pmuhd8 this chapter describes the motivation of the research project, provides the theoretical framework of the entire book, and gives a summary of the findings of the case study chapters. in the process of diffusion of human security norms in east asia, several features have emerged. first, east asians have accepted a comprehensive definition of human security regarding the perception of threats. second, east asians tend to think that human security and state security are complementary. third, the constituent elements of the human security norms such as freedom from fear and from want, freedom to live in dignity, protection, and empowerment are already accepted by east asian nations. we need an extra effort to elevate human security to a full-fledged norm in the region. human security is an international norm concerned with global public interest, or a concept that aims to be an international norm such as human rights, the sustainable development goals (sdgs), and corporate social responsibility (csr). in general terms, norms denote codes of desirable (or undesirable) behaviors shared in a specific community. one of the strongest norms common in human society is that "homicide is evil." even when the death penalty and war are allowed, they are considered exceptions to this norm. written norms become statutes and formal regulations, while social consciousness supporting specific codes of conduct can also be called norms. 2 normative sciences not only describe facts but also inquire into "how the object ought to be," covering logic, ethics, and aesthetics. the study of norms can be part of such an intellectual exercise: we describe and evaluate what people consider to be appropriate behaviors, nationally and internationally. let us try to answer the above question. why does human security not fade out? it is because the international community needs this concept. though not explicitly using this term, the un can be thought of as being originally organized to realize human security beyond international security. the originality of human security as an international norm lies in its attempt to shift the referent object of security from "states" to "individuals" and to urge various actors to conduct themselves accordingly. the two world wars in the twentieth century claimed large numbers of human lives and stripped as many of their dignity and property in the all-out wars between nation states. in order not to repeat such calamities, the un conferred on its security council the authority to limit the sovereignty of states threatening international peace and security and to impose military sanctions under international law. in the un, state sovereignty is not necessarily an inviolable sanctuary, even though "non-interference" remains a major norm in international society. it is often assumed that hobbes' "realism" and kant's "idealism" are poles apart. however, if the nation-state is invented to overcome the havoc caused by the war of every man against every man (hobbes 1996 (hobbes [originally 1651 , and a world federation is shaped to avoid the devastation caused by the war of every state against every other state (kant 1977 (kant [originally 1795 ), these two world views are conterminous in a single spectrum. in this light, the ultimate objective of both nation states and international organizations is to realize the security of individuals by ensuring freedom for all people. therefore, it is of pressing importance to evaluate government functions on the extent to which they serve this objective. although we cannot deny the crucial roles of nation states and national governments, the strong nation states are those that effectively serve the security of individuals living in their territories, not those that demand citizens' sacrifice for state security too easily. in a nutshell, the normative message of human security boils down to a powerful proposition that the ultimate objective of governance at all levels is to provide security (or ensure freedoms) for every individual. the core message of human security is thus very simple, but many other intentions and meanings have been subsumed in this concept along the way. if the objective is the security of individual persons, we must be able to characterize the core constitutive elements of such a secure state, as well as the principal means to achieve that goal, which can be described as norms themselves. human security is being formed as a "norm-complex" in which different existing norms are combined and nested under the umbrella of human security (kurusu 2005) . 3 this hybrid nature of human security is observable in the consensus-based resolution on the definition of human security adopted by the united nations general assembly (unga) in september 2012. 4 that resolution stipulates that human security is "an approach to assist member states in identifying and addressing widespread and cross-cutting challenges to survival, livelihood and dignity of their people." according to the resolution, a common understanding on the notion of human security includes: "(a) the right of people to live in freedom and dignity, free from poverty and despair. all individuals, especially vulnerable people, are entitled to freedom from fear and freedom from want, with an equal opportunity to enjoy all their rights and fully develop their human potential." the resolution then enumerates certain qualifications of the concept: "(b) human security calls for people-centred, comprehensive, context-specific and preventionoriented responses that strengthen the protection and empowerment of all people and all communities," "(c) human security recognizes the interlinkages between peace, development and human rights, and equally considers civil, political, economic, social and cultural rights," "(d) the notion of human security is distinct from the responsibility to protect and its implementation," and "(e) human security does not entail the threat or the use of force or coercive measures" and "does not replace state security." human security thus makes much of "national ownership," local contexts and bottom-up initiatives, and pays respect to all generations of human rights. based on the characterization of human security in past documents, including this unga resolution as well as the commission on human security (2003) and undp (1993 undp ( , 1994 , we defined the practice of human security for the present research as follows: to ensure three freedoms (freedom from fear, freedom from want, and freedom to live in dignity) for individuals and communities vulnerable to large-scale and cross-border threats, by combining protection from above and empowerment from below. although this definition may still feel too complicated, with careful attention, one finds that the concept has been made dynamic by incorporating new elements into a set of established norms. let us discuss three points. first, while taking the concept of "freedoms from fear and want" as a given, human security brought in the third element, "dignity." realizing a world free from "fear and want" is the ideal of the universal declaration of human rights, and these two freedoms can be represented by civil liberties and socio-economic rights. they are embedded in the national constitutions of many nations as well as in international human rights law. on the other hand, dignity corresponds to a moral attitude when aiming at the realization of these freedoms: to express respect for humanity, recognizing that every human being has intrinsic worth (rosen 2012) . it is impossible to think of the human rights of the dead, even though we do think of the dignity of the dead. this is because dignity is a relational concept, and practical methods to respect the irreplaceability of others depend on local cultural contexts. 5 second, while human security does not deny the importance of protection, it incorporates the element of "empowering" people from below as a complement to protecting them from above. empowerment is a process that enables people to become the masters of their own lives and may require the redistribution of power and resources between the powerful and the powerless. in the context of social development, friedman (1992) developed a theory of empowerment focusing on community development and livelihood support. women's empowerment has been incorporated into both the millennium development goals (mdgs) and, more recently, the sdgs. self-evaluation tools for empowerment processes have also been developed (fetterman et al. 2015) . if practitioners of human security want to translate empowerment into practice, it is important for them to unambiguously respect the agency of local people while avoiding their protracted dependence on assistance wherever possible. by reinforcing the power not only of individuals but also of communities and local governments, the excessive power of national governments can also be effectively checked. in human security, it is important to lower the level of the focus of empowerment from the national to the subnational and down to the community level. thus, in the human security discourse, by adding the concepts of dignity and empowerment, the elements of culture and agency have been grafted onto existing norms of human rights and humanitarian intervention. this deserves more attention as a new value that has been added to the human security idea. in east asia, where social hierarchy is relatively strongly rooted, the concept of dignity based on the premise that individuals are embedded in society can be accepted more easily than the concept of empowerment that might "disturb" public order. however, as a counterbalance to public authorities' sometimes excessively paternalistic protection, the emphasis on empowerment is undeniably of great significance in this region. 6 the third source of power that can dynamize human security is the awareness that human society is in danger. mahbub ul haq, a pakistani economist and the first advocate of human security in the un, wrote: "a powerful, revolutionary idea, the emerging concept of human security forces a new morality on all of us through a perception of common threats to our very survival (…) while great religions often move the human spirit through the sublimeness of their messages, they also carry in their messages the fear of eventual punishment. much human change comes from a fear for human survival (haq 1995, 116) ." we cannot fully control the forces of nature or the fate of humanity. in envisioning a sustainable future for human beings and nature, the human security idea is expected to contribute to the realization of the sdgs through its emphasis on serious and pervasive threats (downside risks) and people's vulnerability to these. human security as defined in the unga resolution makes much of national ownership in organizing human security action. the implication of this approach will be discussed further in the rest of this volume. modern international norms involving many and diverse stakeholders tend to be complex, which relates to the ways a norm is established. there is a normative process of norm-making: in other words, a desirable process that is the standard way of setting a new norm. wise people may gather to put bonum commune of humankind into statutory forms and diffuse this downward. however, the actual processes of norm creation and diffusion are a little different. for an idea to be established as a norm, it must be internalized in the minds of the members of society irrespective of whether it is legally enforced or not. for this purpose, it is desirable for as many parties as possible at the center and at the periphery to actively participate in the process of norm-making instead of passively waiting for the advent of a new norm. in this process, both universal and local values tend to slot into a new norm, thereby making it hybrid, composite, or complex. international norms are said to have life cycles. at the beginning, "norm entrepreneurs" 7 propose a new norm, which is accepted by several states (the norm emergence stage). then, after a certain "tipping point," the norm diffuses quickly and prevails throughout international society (the norm cascade stage). finally, the norm is internalized in every country and becomes "taken for granted" (the internalization stage) (finnemore and sikkink 1998) . however, as clarified by amitav acharya in the case of the security regime in southeast asia, foreign norms may be opposed, modified, or displaced by existing local norms in local space. norms are not simply accepted or rejected but are also localized (acharya 2004; . conversely, new norms that are (re)created by local actors in the periphery may eventually reach the core nations and/or challenge global powers (acharya 2011; towns 2012) . as indicated by the concept of bricolage in cultural anthropology (lévi-strauss 1962) , people living in communities bring together various indigenous and foreign materials to ingeniously create a new modality of life. proposed norms are to diffuse or fade out while being transformed vertically from the un headquarters to a small village, and horizontally across diverse world regions and nations. the process of initiation, diffusion, and regeneration of a norm is called "norm dynamics." as described above, the concept of human security was first advocated by a group of norm entrepreneurs at the undp, consisting of mahbub ul haq and others. after that, several countries including canada reinterpreted the human security concept, and this gave rise to an offshoot norm called responsibility to protect (r2p), which defined the conditions for international society to intervene into a sovereign state with military and/or non-military measures to directly protect citizens from the horror of "genocide, war crimes, ethnic cleansing and crimes against humanity." 8 on the other hand, countries including japan, thailand, and the philippines understood the nature of threats in broader and more comprehensive ways and tried to redefine human security to avoid confrontation between state sovereignty and humanitarian imperatives by emphasizing prevention and sensitivity to local contexts. it should be noted that the comprehensive human security initiative of the latter group, maintaining the universality of un-based messages, has passed through the process of localization in asia. a radical change of international norms is often triggered by a dispute or a grave event (sandholtz and stiles 2009) . the prime minister of japan, keizo obuchi, officially advocated human security for the first time in singapore in 1998 after the asian financial crisis (he was foreign minister at the time) (kurusu 2011) . the commission on human security, which released the final report on the comprehensive human security approach in 2003, was co-chaired by the former united nations high commissioner for refugees (unhcr) sadako ogata and the nobel prize-winning economist amartya sen, a combination of east asian and south asian universal figures (chs 2003). pitsuwan and caballero-anthony (2014) relate the effects of the financial crises, as well as the multiple humanitarian crises, that have made evident the significance of human security as a "compelling normative framework." still, they argue "that as far as institutionalizing human security in its security practices, … asean still has a long way to go," particularly because of gaps in economic security, protection from disasters and of minorities and migrants, among others. in the rest of this introductory chapter, we discuss how the concept of human security has been received in east asia in terms of the perspective of norm dynamics. what do asian countries accept, reject, or remodel of the idea of human security born in the un? in this book, the so-called asean plus three countries (the member states of the association of southeast asian nations (asean) plus china, japan, and south korea) is defined as east asia. in this region that has experienced "miraculous" growth (world bank 1993), the nexus between economic development and human insecurities is prominent. 9 japan is not the only country that has accepted the human security norm in asia. the late surin pitsuwan, a member of the commission on human security and distinguished fellow of the jica research institute, persevered in his effort to diffuse the concept of human security in southeast asia, serving as the minister of foreign affairs of thailand and then as secretary general of asean. as discussed in chap. 11, in 2002 the gov-ernment of thailand set up the first government ministry in the world bearing the name of human security: the ministry of social development and human security. in thailand, knowledge on human security had been widely diffused among academic researchers, but the practice of human security canalized by the establishment of this ministry came to focus on the social welfare of the vulnerable: persons with disabilities, the elderly, children, women, and ethnic minorities. the philippines also paid attention to human security as soon as the 1994 undp report was released, and multiple efforts of localization can be enumerated, including the design of a "human security index." there have been attempts of co-option as well. an antiterrorism law called the "human security act" was enacted in 2007, inviting criticism from filipino civil society (chap. 8). application of the concept of human security in thailand and the philippines headed in the opposite directions of benign welfare and hardline public order. the chinese government does not often mention human security, but chap. 3 argues that china articulates a vision similar to this concept and practices it without saying so. that is partly because china, a permanent member of the un security council, is expected to promote international norms embraced by the un system. the acceptance of human security by way of participation in multilateral stages is applicable to south korea as well. in 2010, south korea became a member of the organisation for economic co-operation and development-development assistance committee (oecd-dac). in addition, ban ki-moon promoted human security in his capacity as un secretary-general. also, the government of south korea has occasionally referred to the importance of human security in addresses by its president and foreign minister (chap. 10). thus, in east asia, several countries have accepted the concept of human security to varying degrees under government initiatives. in the meantime, local scholars have also accumulated academic inquiries. in addition to two major single-authored books (howe 2013; nishikawa 2010) , a train of edited volumes on human security in the east asian contexts has been published (kassim 2011; peou 2009; teh 2012; tow et al. 2000 tow et al. , 2013 umegaki et al. 2009 ). moreover, with relatively limited circulation, the proceedings, commentaries, and policy recommendations based on international conferences held in bangkok, seoul, jakarta, and so on, have been published one after another (banpasirichote et al. 2012; hernandez and kraft 2012; thabchumpon 2012; unesco 2004 unesco , 2007 wun'gaeo 2004) . these publications have shared a certain feature: authors based in east asia transmit messages mainly to readers within the region. these earlier studies, especially most of the edited volumes, discuss how concrete issues can be interpreted using the concept of human security and how those issues can be addressed on the ground. however, there is little research that digs into the processes by which individual countries in the east asian region have accepted the human security norm in their own ways. the country-by-country analyses in this book are expected to fill this gap. in 2003, when the final report of the commission on human security was published, sadako ogata returned to japan to take the helm of the japan international cooperation agency (jica). under her presidency, the human security idea became embedded in the spirit of the agency. when a part of the japan bank for international cooperation (jbic) and jica were integrated to set up the new jica in 2008, the jica research institute was established and launched several international research projects related to human security. then, in 2013, a research project to directly investigate the norm dynamics of human security in east asia was set up. based on a common questionnaire, researchers from 11 east asian countries were to work on interview surveys and document research to elucidate the present status of human security in each country (see fig. 1.1) . the researchers participating in the project-the authors of the chapters in this book-are a combination of senior and young scholars specializing in international relations, political science, development studies and other disciplines and working for universities and think tanks in various parts of the region. the researchers agreed to ask questions about the following three topics in the interviews: first, local perceptions of threats (the ranking of human security issues that are considered important in each country and in the east asian region); second, the ways of (selective) acceptance of the concept of human security (the understanding of freedoms from fear, from want and to live in dignity, the strategy for combining protection and empowerment, and the understanding of preparedness for calamities, and so on); and third, the question of national sovereignty (whether to allow foreign actors to operate within the country in case of natural disasters and violent conflict, as well as whether to take action in territories of other countries in such a case). at the same time, respondents were allowed to change the combination of interview questions to adapt to their countries' unique circumstances. in addition, it was agreed that the researchers would welcome responses criticizing human security. the interviewees included government officials, lawmakers, researchers at universities and think tanks, nongovernmental organization (ngo) activists, religious leaders, journalists, business persons, and international organization staff. though they were not necessarily statistically representative, in-depth interviews were conducted (some of the survey activities included anthropological interviews with villagers in the countryside and focus group discussions). the interviews reached more than a hundred, and two workshops for chapter authors were organized in tokyo and manila. in the next section, we put together the research outcomes in the light of norm dynamics, including the localization processes. first, let us think about what threats to human security we face. classifying the sources of threats to human security into those derived from the physical system (the earth), from the living system (animals and plants), and from the social system (human beings), akihiko tanaka called for a clearer understanding of the mechanism in which these threats bring about human insecurities. to that end, close collaboration between different academic disciplines including the natural sciences and engineering, the biological and ecological sciences, and the social sciences and the humanities is required (tanaka 2015) . in our surveys of the 11 east asian countries, local experts were asked to enumerate the threats to human security. though priority ranking varies from country to country, an integrated list of threats arranged according to the above three systems can be as follows: climate change, typhoons/ cyclones, floods, volcano eruptions, earthquakes, tsunami, infectious diseases such as severe acute respiratory syndrome (sars), avian influenza and hiv/aids, food crises, lack of basic health and education, environmental pollution, urbanization, extreme poverty, unemployment, migration, human trafficking, violent conflicts, interstate military conflicts, religious intolerance, organized crime, oppression from the government, and so forth. meanwhile, the undp's human development report 1994 listed seven main categories of human security: economic, food, health, environmental, personal, community, and political security (undp 1994, chap. 2) . in the case studies of cambodia (chap. 2), thailand (chap. 11), the philippines (chap. 8), and vietnam (chap. 12), human security challenges are classified in line with these seven categories. these areas correspond not only to the divisions of the un specialized agencies but also to government ministries, so that the classification could be accepted as familiar and practical. such a diversity of threats largely overlaps with the so-called nontraditional security (nts) issues. while military threats from foreign states are considered "traditional," many threats that simultaneously affect multiple countries are of a non-military nature and fall into the category of "non-traditional" threats. as pointed out in the cases of china (chap. 3), indonesia (chap. 4), malaysia (chap. 6), the philippines (chap. 8), south korea (chap. 10), and vietnam (chap. 12), there is growing interest in nts among policymakers and researchers in china, south korea and in the asean countries, which seems to have contributed to the acceptance of human security in the region (caballero-anthony et al. 2006; caballero-anthony and cook 2013; li 2010) . however, there is substantial difference between the nts and the human security approach: while the actors that address such diverse threats still concentrate on the national governments in the former, more emphasis is placed on peer collaboration between states and other actors in the latter. the role of national armies in coping with human security challenges should be limited. chapter 4 presents the opinion of an indonesian military officer who argued that the term "security" should not be used until the poverty level or the impact of a disaster exceeded a certain threshold and becomes a real threat to the survival of all citizens. if every threat was considered a security challenge, the military would be overwhelmed by the resulting deluge of duties. human security is regarded as a principle of official development assistance (oda) policies in japan, and to a lesser extent, in south korea. as described in chap. 5, in japan, the idea to combine efforts toward development and peacebuilding has gradually taken root under this framework. as an added value of human security, japanese interviewees emphasized the importance of a "comprehensive approach" in which diverse actors (including ngos and private firms) cooperate, as well as the significance of working among grassroots people and paying more attention to real needs in the field. meanwhile, most experts pointed out that human security challenges lie on the domestic front, too. people who were familiar with the concept of human security interpreted the great east japan earthquake and the resultant fukushima disaster as a typical human security issue. in addition, the aging population and a possible collapse of social security in the future can also be serious domestic human insecurity issues. in terms of domestic human security challenges, the case of singapore as presented in chap. 9 is also revealing. while singapore has achieved a high degree of human security as a developed country in southeast asia, this small city-state is also going through acute human insecurities such as growing inequalities, increasing psychological stresses on citizens, the survival race between small enterprises, and discrimination against migrants and minorities. here, social media cuts two ways by spreading messages virally: it can mobilize good will but may also deeply wound people. in singapore, with strong administrative control from above, empowerment is supposed to be of great significance. besides, singapore assists neighboring countries in the form of philanthropy, even though this is not officially classified as oda. it is pointed out that the philippines has also provided humanitarian assistance while receiving assistance itself (chap. 8) . in the great east japan earthquake, japan, a major provider of oda, received goodwill support from many countries including recipients of japan's assistance (chap. 5) . it is noteworthy that the line separating providers from recipients of oda is blurred in the case of humanitarian crises. a country that has faced a series of exceptionally acute threats to human security is cambodia (chap. 2). this country is considered "a showcase of human insecurities" that started with the genocide under the pol pot regime (it is said that around 2 million people were killed in a country with a population of 8 million). the interviewees enumerated contemporary sources of threats in cambodia such as the government, natural disasters, diseases, political insecurity, and land issues. some respondents pinpointed the problem of the "government approach, relying on the heavy presence of security forces and legal means to threaten and detain people." it is widely perceived that cambodian society has been destabilized and that human security has been threatened despite (or due to) recent economic growth. one of the topical concerns in east asia that has wider political implications is the north korean issue (chap. 10). an emergency on the korean peninsula could bring about an exodus of refugees and other situations, which may potentially give rise to grave human insecurities both regionally and globally. the risk of military conflict over maritime interests could also be a threat to human security, as voiced by several countries. the necessity to address cross-border issues such as human trafficking, air pollution, infectious diseases, food security, and cybersecurity was also pointed out by many interviewees. how far has the concept of human security permeated east asian countries so that stakeholders can jointly address the multiple threats described thus far? as pointed out in those chapters that discuss the experiences in the philippines (chap. 8), malaysia (chap. 6), and thailand (chap. 11), east asian experts did not fully understand the difference between human security and human rights or human development, while activists in civil society tended to use the discourse of human rights more often than that of human security. however, even though the human security norm has not prevailed in east asia, the concept has been accepted at least partially, as argued in several of the case study chapters. the survey carried out in vietnam (chap. 12) broke down human security into the seven security categories of the undp and found that all these elements were inscribed into the vietnamese constitution and other laws. in addition, even when interviewees were not familiar with the concept of human security, they "were able to quickly connect the abstract concepts of 'freedom from fear', 'freedom from want', and 'freedom to live in dignity' to specific examples in their lives." human security in vietnam "can be said to be a jigsaw puzzle, in which the pieces are identified, but have not been put together." the surveys in indonesia (chap. 4) and south korea (chap. 10) also found, by examining official documents, that the elements of human security defined in this chapter, such as the three freedoms, protection, and empowerment, were all written into these documents to varying degrees (the former in domestic policies and the latter in oda policies). in addition, people who were interviewed in cambodia pointed out that the three freedoms were closely linked to each other in substance (chap. 2). what is the most important element among the components that make up human security? the study of japan (chap. 5) presented the expert opinion that the third "freedom to live in dignity" could be a real added value of the human security approach, indicating that "dignity is an idea of waiting and caring." the survey in the philippines also mentioned that the concept of dignity had potential to lead human security to a higher dimension and emphasized the importance of local contexts. moreover, people in cambodia said that having dignity is associated with "having a moral character; with notions of respect, pride, and having value and independence; and of helping others and having an honest character." a rural resident made a candid remark: "dignity is most important because it is about no discrimination, having rights to do what we want, not being looked down upon by wealthy people." while the expectation of state protection was found in many interview results, empowerment was mostly referred to in general terms. however, protection and empowerment make an effective pair in reality. empowerment leads to a series of concepts that value people's agency, such as ownership, self-help support, resilience, and capacity development in the practice of development cooperation, while the same concept is expected to promote collaboration between governments and civil society in domestic policies. given that the asian approach to human security tends to give relative weight to the role of states as discussed below, the counterbalance of empowerment is needed all the more in this region. in many countries, we also asked the interviewees whether foreign support should be accepted in case their own country suffers an uncontrollable crisis due to a natural disaster or violent conflict (and whether their country should support neighboring countries in case the latter suffers the same situations). the common pattern of responses to these hypothetical situations was that foreign support was undesirable during political unrest but welcome when a natural disaster occurs. it was also preferred that the support should be provided in multilateral rather than unilateral frameworks, as mentioned in the studies on malaysia (chap. 6), the philippines (chap. 8), and vietnam (chap. 12). these reactions illustrate that east asians tend to think that state security could be compromised in favor of humanitarian concerns in certain emergency situations, especially in case of natural disasters. it should be remembered that sadako ogata stressed that human security and state security complement one another (ogata 2003) . 10 as to the role of states in realizing human security, both a loose consensus and a subtle disagreement could be found among east asian countries. the case study of china argues as follows (chap. 3). on the one hand, we can establish the causal connection that state security contributes to human security. the idea that people should not be easily sacrificed for national objectives is absolutely correct because human beings are not means but ends in themselves. on the other hand, national security and personal security can be compatible. the perception that states are a "necessary evil" is not a chinese but a western idea. east asians naturally expect a great deal from their governments: people expect the governments to protect them just like parents protect their children. this represents a view of states as benevolent and "paternalistic." the relationship in which a stable state guarantees people's security is also expressed in the case study of vietnam (chap. 12). on the "right" of this view of states, there is another understanding that human security is part of state security, that is, state security subordinates human security, not vice versa. in this research project, such a view was expressed by government officers from indonesia (chap. 4) and malaysia (chap. 6) . from the government side, however, some added that the role of the military in human security should be strictly limited. indonesian interviewees opined that military operations should be firmly placed under civilian control, even though the military effectively responded to the earthquake and tsunami in 2004. this is because they consider that the military is essentially not trained to respond to nonmilitary threats, and it is often better to entrust the duty of maintaining public order to police forces in disaster situations. on the "left" side, there are countries with impressive traditions of civil society activism such as the philippines (chap. 8) and thailand (chap. 11), which have strongly influenced the trajectories of acceptance of human security. in the case studies of malaysia (chap. 6) and singapore (chap. 9), dynamic and strained relationships between the government and civil society are vividly depicted. the chapter on malaysia places expectations in consolidating human security through empowerment of local governments, more active dialogues between the government and civil society, and regional cooperation through the networks of asean and ngos, against the backdrop of the government repression of free speech, religious intolerance, and the surge of rohingya refugees. when severe threats to human security are actualized, the relationship between state sovereignty and human security may become extremely tense. as described in chap. 7, when cyclone nargis hit myanmar in 2008, the military government refused to accept foreign aid, even while lowland residents were caught in the flooding. it is said that the dead and missing persons numbered nearly 140,000. though western countries such as france threatened to make a r2p-type humanitarian intervention, the government of myanmar rejected such operations and instead decided to accept coordinated assistance from organizations such as asean and the un. this multilateral collaboration has become a model for humanitarian operations in east asia. the case study of japan (chap. 5) warns that the concept of human security could be "politicized" in the contexts of domestic debates on security and securitization. in contrast, the study of thailand (chap. 11) voices concern that human security is now too "depoliticized," arguing that the concept has been reduced to the practice of social welfare and is now rarely discussed in thai diplomatic contexts. however, behind the activities of the ministry of social development and human security seeking to improve the well-being of the socially vulnerable seems to lie the buddhist concept of mercy as well as an attempt to integrate human security with the concept of sufficiency economy advocated by king rama ix. these dynamics of politicization, depoliticization, and local reinterpretation are interesting in terms of the "norm localization" discussed in this chapter. keeping in mind the urgent problems including land grabbing and king sihanouk's political legacy in cambodia, chap. 2 emphasizes the importance of "cooperative leadership" based on the spirit of tolerance and compromise. the key to ensuring human security in cambodia is to realize voluntary collaboration among opposing political parties, between the government and civil society, and between the central and local governments, and to make the government listen to the voice of the people. different countries have different perceptions as to which state and non-state actors should be valued as against others. however, we can safely say that there is a shared understanding in the region that diverse actors, including national governments, should coordinate each other's activities to secure freedoms and development for individual persons in the face of serious and pervasive threats. just as a world where autonomous villages cease to make decisions on their own affairs is hard to imagine, it is unlikely that the governments of nation states will cease to make their own decisions. national governments are important because most of them have strong powers and the authority to ensure security for individuals by utilizing well-developed institutions, resources, and national cohesion. however, overly powerful state security mechanisms require an antidote, which can be the human security norm. as history illustrates, when pluralist thinking that endorses critical roles played by non-state actors is denied, the world as well as national politics go awry. 11 the purpose of the association of world peoples is not only to promote the security of nations but also to ultimately promote the security of all human beings. in this sense, the security council of the un could be renamed a human security council. in the practice of human security, neither "western individualism" nor "oriental despotism" is required in their pure forms; it seems that asian versions of human security have begun walking along the middle road between the two. in east asian nations, perceptions of diverse threats as sources of insecurities largely overlap, and therefore the conditions for collective action to address common threats also seem to be maturing. even though the term human security is not officially used very often, in this research it was found that the constituent elements of human security, namely, the three freedoms as well as protection and empowerment, have been accepted more or less in all parts of east asia. if regional spaces for dialogues are provided, the human security idea may diffuse in the short term like a cascade. an international network of experts sharing the value of a specific norm and assuming key roles in its diffusion as well as policy coordination is called an epistemic community (haas 1992) . in the process of this research, we witnessed the emergence of a bridged community with a shared interest in human security in east asia. the process of this research endeavor itself might be part of the formation of such a community. lastly, we would like to pay notice to the fact that the outcomes of this research reflect not only east asia's potential unity but also its actual diversity. once we zoom in to the regional space of east asia, we can see a kaleidoscopic diversity of human security stakeholders and their values. this is the reason why this book is entitled human security norms rather than the human security norm. the latter is only in the making: there remain forces that resist the idea of human security, while east asian nations are developing their own human security norms with different interpretations and preferences. the country-by-country analyses in subsequent chapters are based on independent research, which seems to have succeeded in shedding light upon the diversity of the history, society, and political economy of the region. the chapters are arranged in the alphabetical order of countries, so readers can start with any chapter while referring to the comparative analysis in chap. 13. we return to the issue of east asia's diversity in chap. 14, in which we will suggest a direction to proceed with the practice of human security in this region. notes 1. martin and owen (2014) present a stock-taking collection of reflections on the concept and its application. bourbeau (2015) captures the multidisciplinary nature of the study of security, including human security. 2. in international relations, norms are defined as "shared expectations about appropriate behavior held by a community of actors" (finnemore 1996, 22) or "collective expectations for the proper behavior of actors with a criticizing the proposition that human rights make sense only when they are legally guaranteed, amartya sen argues that strong moral imperatives of what to do and not to do make up human rights. these imperatives may call for legislation, but legal provision is not a prerequisite for human rights see also the discussions of "composite norms united nations general assembly, follow-up to paragraph 143 on human security of the 2005 world summit outcome the concept of dignity was explicitly introduced to the human security discourse in the commission on human security (2003) as one of the triad of "survival, livelihood and dignity annan (2005) has introduced "freedom to live in dignity" into the agenda of the un reform, and tadjbakhsh and chenoy (2007) have attempted to incorporate dignity fully into the human security perspective. on the other hand, while the concept of empowerment is widely diffused in social movements in the americas and south asia after a norm is established by idealistic "norm entrepreneurs," a different group of pragmatic actors called "message entrepreneurs human development and human security correspond to the upside and the downside of economic growth, and to the dual policy challenges of "growth with equity" and "downturn with security for the role of states to promote human security, see bae and maruyama (2015), a collaborative work by american and japanese scholars of human security how ideas spread: whose norms matter? norm localization and institutional change in asian regionalism larger freedom: towards development, security and human rights for all: report of the secretary-general human security, changing states and global responses: institutions and practices mainstreaming human security: asian perspectives. bangkok: chula global network and international development studies program implementation and world politics: how international norms change practice security: dialogue across disciplines non-traditional security in asia: issues, challenges and framework for action non-traditional security in asia: dilemmas in securitization chs (commission on human security). 2003. human security now. new york: commission on human security dignity: a history the cambridge handbook of human dignity: interdisciplinary perspectives empowerment evaluation: knowledge and tools for self-assessment, evaluation capacity building, and accountability national interests in international society international norm dynamics and political change empowerment: the politics of alternative development international norm dynamics and the 'end of poverty': understanding the millennium development goals introduction: epistemic communities and international policy coordination reflections on human development mainstreaming human security in asean integration (three volumes). quezon city: institute for strategic and development studies leviathan the protection and promotion of human security in east asia perpetual peace: a philosophical sketch strategic currents: issues in human security in asia introduction: alternative perspectives on national security japan as an active agent for global norms: the political dynamism behind the acceptance and promotion of 'human security la pensée sauvage china and non-traditional security in asia routledge handbook of human security human security in southeast asia human security and state security human security in east asia: challenges for collaborative action human security in southeast asia: 20 years in review dignity: its history and meaning international norms and cycles of change the concept of the political development, rights, and human security human security: concepts and implications toward a theory of human security human security: securing east asia's future critical connections: human rights, human development and human security asia's emerging regional order: reconciling traditional and human security new approaches to human security in the asia-pacific: china, japan and australia. harnham: ashgate norms and social hierarchies: understanding international policy diffusion 'from below human insecurity in east asia united nations development programme) proceedings of the international conference on human security in east asia. seoul: korean national commission for unesco the east asian miracle: economic growth and public policy human security now: strengthening policy networks in southeast asia key: cord-029209-v2w0i2ex authors: gilder, alexander title: international law and human security in a kaleidoscopic world date: 2020-07-15 journal: indian journal of international law doi: 10.1007/s40901-020-00109-w sha: doc_id: 29209 cord_uid: v2w0i2ex international law is being challenged by a multitude of new actors and networks that do not fit within the traditional westphalian system. similarly, security is increasingly undermined by, for example, economic, health, and environmental threats that can affect individuals’ daily lives and know no state boundaries. this is the kaleidoscopic world as outlined by edith brown weiss. the concept of ‘human security’ has been advanced to inform decision-making on threats to security in the interest of individuals in a bottom-up manner. this article looks forward to methods that can counter what could be perceived as a legitimacy crisis in international law. first, some of the current challenges which international law faces are explained ranging from globalisation, the declining state-based order, and decentralised security threats. second, the concept of human security is defined, and its contents expounded. lastly, the thesis is advanced that a conceptual framework of human security can reorientate international law to be responsive to the kaleidoscopic world by using un peace operations as an example of where human security could have a profound impact. global issues are increasingly kaleidoscopic to the extent that international law is undermined by a multitude of new actors and networks that do not fit within the traditional westphalian system. similarly, security is increasingly threatened by, for example, cross-border threats of violence, health, environmental crises, and more that become intertwined, can affect individuals' daily lives, and are not halted by traditional state boundaries. for instance, terrorism in the sahel, famine in nigeria, the spread of viruses such as ebola and coronavirus, and the displacement of the rohingya are a few examples from recent years where a broad notion of security has been undermined. international law fails to adequately respond to these challenges and implement new, bottom-up approaches where the international community is responsive to the needs identified by individuals. this article understands the world to be ''kaleidoscopic'', as argued by edith brown weiss, where the actors and coalitions engaging in the international system are constantly changing. 1 developments are swift where crises can appear and quickly cross borders amidst an everconnected world linked through information technology. brown weiss stresses that international law more than ever needs to be viewed as legitimate by both those who create it, importantly states, and those affected by it. 2 this is because in recent years there has been unprecedented backlash against the international system, and more broadly globalisation, by states. well known examples of this trend include the sceptical voice of president donald trump, the uk's referendum to leave the european union, and the global rise in nationalist politics. in addition, globalisation and access to information technology has resulted in the spread of fake news, unprecedented monitoring of individuals by states, more visible economic inequalities, and the contraction of space for civil society. 3 the concept of 'human security' can be used as a conceptual framework to allow international law to better focus its attention on the individual and be responsive to the needs of persons affected by insecurity. the term ''human security'' is used throughout the article to denote the non-legal concept as espoused by both the un development programme (undp) and commission on human security which will be expounded in sect. 3. 4 it is argued that international actors, particularly the un, would be able to utilise human security to advance a human-centred approach to international law and specifically, international peace and security. human security would provide for the participation of a diverse range of non-state actors who previously have been unable or find it incredibly difficult to permeate the international system. by doing so the international system will remain relevant in this time of rapid change and pushback against the prevailing international order. the example of un peace operations is used to demonstrate how a human security approach could positively influence the practice of the un and their state partners toward the inclusion of individuals as participants. the approach argued for in this article does not purport to be a panacea that will enable a reformulation of international law in an iconoclastic manner. 5 nor does this article address the full range of discussions that can be had such as, how such a conceptual framework would be implemented, how a human security approach fits within scholarship from third world approaches to international law (twail) and also how human security relates to liberal peace, but there is not the space in one article to address these questions. instead, the article aims to generate debate and future critical examination of how international law can accommodate for a rapidly interconnected and globalised world. 2 the kaleidoscopic world, security and the legitimacy of international law individuals are not typically included in the making of international law or the decision-making of states and the normative international system has little scope for accommodating non-state actors in general. 6 instead the international system is viewed through the prism of geography, due to the principles of sovereignty and equality of (territorial) states, and the question is whether such a state-based, geographic approach is relevant in the twenty first century. 7 on the one hand it is claimed that state sovereignty will remain relevant over the coming decades. 8 however, daniel bethlehem has argued that, [w] hile the geography of statehood is likely to remain at the root of the international system, it is becoming increasingly less important as people, goods, services, and funds flow across borders; as individuals and corporations engage directly with one another without the intermediation of states or of their paraphernalia; as virtual space takes on dimensions and an importance that rivals physical space in the world of transactions, communications, and other engagements; as regional and multilateral integration arrangements between states reduce the importance of boundaries; as international and non-governmental organizations proliferate and operate transnationally on the basis of technical mandates that transcend, or endeavour to transcend, narrow sovereign interests. 9 the international system has, in practice, become progressively about attempting to regulate a diverse range of both public and private actors interacting across the globe regardless of state borders. what is true is that international law has undergone a process of ''humanization''. 10 for instance, ruti teitel's concept of ''humanity's law'' provides a framework for the international community to recognise the interests of a multitude of actors, including individuals. 11 in a similar vein, anne peters has identified that 'law ultimately should be guided and justified by the concerns of the persons affected by them.' 12 peters argues in her expansive study that 'an orientation towards the individual in communities justifies international law as a whole.' 13 when examining the subjects of international law, rosalyn higgins rejects the notion that only states can be subjects as a legal fiction. 14 higgins advocates a view that individuals are participants in the international legal system but notes how individuals are 'extremely handicapped' with little access to the international arena. 15 in the subsequent sections, this article will propose a framework which encourages both access to the international system by individuals and for their interests to be at the forefront of decision-making. the author agrees with peters that international law requires an orientation towards the individual to remain legitimate and this article sets out a possible method of achieving such an orientation for international peace and security. consequently, the focus of this paper is on the implications of a globalised and interconnected world on individual security as opposed to the general 'humanization' of international law. law in general exists to both protect and regulate values we deem worthy, one of which is security. security is an 'elastic and dynamic concept' and can be understood in a variety of different ways. 16 one very broad definition of security is that it means the absence of threats. 17 conventionally, the referent object of security has been the state. that is to say, if the state is able to maintain safety and order internally and is sufficiently capable of repelling external threats then those living within the state are also perceived to be secure. since the second world war the notion of security has been liberalised to become denationalised and both globalised and individualised. 18 there has been increased attention given to a human-centric concept of security where the absence of threats for individuals, not only the state as a whole, has been given increased value. the rise in interventions by the un security council, the creation of the international human rights law system, the codification of and enforcement of humanitarian law, un agencies focused on global security and development, amongst other advancements have brought a postmodern concept of security to the forefront where the individual is the referent object of security, not the state. nevertheless, a state-centric view of security remains present and states pursue their own global security goals with little regulation which may detract from the security of individuals. traditionally, the state was able to respond effectively to crises within its own borders and the state was the primary provider of security. the 15 reality today is that in an increasingly globalised world an action or event in one state can either rapidly have ramifications elsewhere in the globe or a crisis that would once have been isolated can cut to the very core of a person's ability to survive. 19 the global value of security can thus no longer be purely regulated by states. advocacy groups and coalitions can form on the internet nearly instantly and dissolve just as fast. issues can trend on twitter in an instant bringing the concerns of a previously isolated community to global attention and disappear just hours later. however, the discourse on social media can also be seized upon by persons, either locally or from overseas, seeking to undermine the credibility of a crisis. those persons can easily and rapidly spread pernicious misinformation that threatens local communities. 20 one potent example is the use of whatsapp in india to spread misinformation, which has led to a number of deaths and violent attacks since 2015, but notably hitting a peak in 2018. 21 it is increasingly difficult for states to regulate online activities and rapidly shared misinformation can quickly create a security crisis. climate change, for example, could be at the root of a food scarcity crisis in sub-saharan africa that then results in violence threatening the physical integrity of communities and massive internal displacement of persons. such a crisis could cross-borders, create a storm of both accurate and false reports on social media, and ultimately at the centre of the situation are individuals seeking security, likely in the form of protection and assistance, from either their government or the wider international community. in a kaleidoscopic world a sub-national or national crisis can rapidly become a global security issue. for instance, where refugees flee a situation in their home country irrespective of state borders the security problem trickles over into other states and is no longer contained. the situation then becomes a topic of worldwide debate in news media and social media platforms where a variety of stakeholders are able to reach out globally to have their voices heard. brown weiss argues the international legal system will need to adapt to 19 bethlehem has noted that there are considerable cross-border challenges related to public health, the environment and food security in light of how transboundary the world has become. see bethlehem, supra note 7, 16-7. 20 22 not only is the world increasingly kaleidoscopic but the international legal system is suffering from both international norm fatigue and backlash. an example given by james crawford is the uk's dispute over the finding of the un working group on arbitrary detention in relation to julian assange in 2016. 23 a few of the plethora of examples of norm fatigue and backlash include: the unclear status of transatlantic trade and investment partnership (ttip) negotiations; us withdrawal from the trans-pacific partnership (tpp); russian unsigning of the rome statute of the international criminal court; the campaign of the african union against the international criminal court which has led to high profile cases of withdrawal, and later rescinding of the withdrawals, from the rome statute including burundi, the gambia and south africa; the uk's withdrawal from the eu; and the us withdrawal from the paris agreement. the russian seizure of crimea is a potent example of how the tides have shifted since post-cold war liberalism in international affairs during the 1990s towards rejection of the norms that underpin the international legal system. 24 coupled with backlash is dissatisfaction with international standard-setting such as scepticism surrounding the economic liberalisation agendas of the world trade organization (wto) and international financial institutions (ifis), and apprehension over increased security council activism. 25 there has been gradual disengagement of some nations with the international system which can possibly affect the so-called sedimentary formations of international law. crawford explains that states can remove some layers of international law's sedimentary formation, but some are more difficult to erode. 26 for example, top layers of multilateral treaties can be easily affected by states, for instance by choosing to withdraw, but the solid base of the sediment, the principles, norms and institutions, are more difficult to erode even amid the current political discourse. current disengagement and backlash though does not only extend to treaties. instead it also includes the un, part of 22 brown weiss 2019, supra note 1. 23 similar to the demands of the kaleidoscopic world, crawford argues that international law will only survive if it manages to transform and develop over time. 30 the changing attitude of states towards international law is a concurrent trend that will ultimately affect the functioning of the un in combatting increasingly linked threats that affect the core of individual's lives. a return ''back to westpahlia'' to address the legitimacy crisis of international law has been described by richard collins as deeply unpopular. 31 instead, what is needed is 'to ensure that international law reflects shared values that bind people together and that it provides processes that all regard as fair and as ensuring accountability by states, non-state actors, and the myriad of other actors, especially individuals.' 32 collins explains that the direct participation of individuals in international law-making is a chimera and that 'the only plausible prospect appears to be to enhance the participatory role and normative influence of ngos and other nonstate actors as a nascent form of ''global civil society''.' 33 nevertheless, participation of the kaleidoscopic world's growing range of non-state actors, whether that be individuals or civil society groups who are more visible than ever, is crucial and this article argues the international system requires a new framework which can assist with 27 accommodating those actors and refocus the attention of international actors toward inclusive decision-making processes. the following section will outline the content of human security which can serve as a conceptual framework for international law to be responsive to the kaleidoscopic world. 3 what is human security? the concept of human security seeks to respond to new interlinkages in peoples' lives and can allow international law to react to the changing demands of a world where movements and the concerns of local people can be communicated across the globe in an instant. human security has two key elements: (1) it aims to shift the referent object of security from the state to the individual giving the individual intrinsic value and placing the interests of the individual ahead of the state; (2) it gives rise to a broader view on what can cause insecurity and that many threats are interconnected and reinforcing. 34 past initiatives had attempted to draw attention to the linkages between peace and development and called on the international community to consider more than only traditional threats to peace. for instance, the brandt commission in 1980 stated 'the basis for any world or national order must be people and respect for their essential rights' deeming a state-centric, military focused notion of security as unfit for the world. 35 the palme commission in 1982 introduced the idea of ''common security'' and recognised that states need to consider both economic progress to ensure the freedom from want and more traditional, military-based notions of security to ensure the freedom from fear. 36 food security challenges, different energy sources, and the environment as a cause of conflict. 38 the undp built on the work of the earlier commissions and advanced the notion of ''sustainable human development''. 39 under a new administrator, gus speth, the undp sought to be increasingly involved in times of crisis aspiring to coordinate the un's peace operations, political and humanitarian efforts, and development efforts. 40 security became a concern for the undp because the needs of people during different forms of emergencies, be it a natural disaster, war, or humanitarian crisis, are inseparable from sustainable development. 41 the result was the 1994 iteration of the undp's human development report which coined human security. the report attempted to create a vision of human security that could be adopted by both states and the un in furthering social development. 42 the team behind the report sought to create an approach that 'focuses on building human capabilities to confront and overcome poverty, illiteracy, diseases, discrimination, restrictions on political freedom, and the threat to violent conflict.' 43 the report has been argued to represent a 'broader normative shift leading to the strengthening of the position of individual human beings at the international scene.' 44 following the 1994 report various middle powers including canada, norway, and japan adopted human security approaches in their foreign policies. 45 in 2001, the commission on human security was created by secretary-general kofi annan and published its human security now report two years later. 46 the commission defined human security as: to protect the vital core of all human lives in ways that enhance human freedoms and human fulfilment. human security means protecting fundamental freedoms-freedoms that are the essence of life. it means protecting people from critical (severe) and pervasive (widespread) threats and situations. it means using processes that build on people's strengths and aspirations. it means creating political, social, environmental, economic, military and cultural systems that together give people the building blocks of survival, livelihood and dignity. 47 the definition places a focus on pervasive and widespread threats in an attempt to narrow human security to the core threats to survival and make the concept more practical for operationalisation. 48 following the report, a number of human security-based initiatives were created within the un. namely, a human security unit (hsu) and an advisory board for human security. the hsu in particular has helped promote and institutionalise the human security concept within the un. 49 it is suggested here that human security is based upon five principles, namely, (1) existing rights and norms, (2) a focus on the vital core identified in a bottom-up manner, (3) a concern for vulnerability and building resilience, (4) preventative protection, and (5) the empowerment of people to act on their own behalf and implement solutions to security threats. 50 others have argued the human security discourse requires a change in values for the international community but instead the operationalisation of human security provides a new avenue by which we can advance existing values and goals of the international system and ensure that they serve the needs of individuals. 51 respect for international legal norms such as human rights law and humanitarian law, the rule of law, accountability through legal mechanisms, and good governance are inherent to human security. 52 the commission on human security argues a rights-based human security approach 'reorients humanitarian strategies towards enhancing 46 commission on human security, supra note 4. 47 people's capabilities, choices and security.' 53 the commission articulates the concept of human security as one which allows for informed decision-making where human rights are examined in relation to a range of actors involved, not through a state-centric perspective. in that sense, human security can constructively refocus human rights responses to reflect the public interest, and not solely in relation to state obligations and state methods for realising rights. 54 in general, international law 'must be at the heart of human security' and the latter encompasses disciplines of human rights law, humanitarian law, international criminal law and refugee law. 55 those disciplines are often seen in isolation and human security has the capacity to be a lens by which international actors examine a situation and ensure a range of legal regimes are used to protect individuals. by grounding human security in existing legal regimes, it can be utilised to give a universal, coherent framework that focuses the attention on protecting individuals from immediate and pervasive threats identified by those affected. human security has been criticised in the past as ''nothing more than a shopping list'' of wants and desires due to its wide-ranging definitions where any number of issues are securitised. 56 the co-chair of the commission on human security recognised that it would be wholly impracticable to address all possible security issues identified by a multitude of actors. 57 to rectify this the commission focused the attention of human security on the ''vital core'' which is what people hold to be the ''essence of life'' and ''crucially important''. 58 consequently, the vital core is able to shift the focus on a needs basis for different individuals and groups. the commission's definition of human security, quoted above, speaks of 'critical (severe) and pervasive (widespread) threats and situations' to denote a focus on security threats which cut to the core a person's activities and functions, and large-scale, recurrent dangers. 59 individuals and groups affected deem to be essence of their lives. in this way security is personalised and not rigid based on what the international community, and consequently states, determine to be the most critical and pervasive threats. if a human security approach was adopted, international actors such as the un would need to seek views of individuals and groups to ascertain the core factors undermining security in a given crisis. the international actor concerned can then provide a response which will lead to meaningful improvements in the eyes of those affected. the commission further identified that particular groups can be disproportionately affected by security threats. namely, people on the move, women, children, the elderly, the disabled, the indigenous, and the missing. 60 when, in 2012, the un general assembly defined human security vulnerable people were singled out as being of particular concern. 61 however, human security demands more than merely identifying where a group could be at exceptional risk. martha fineman has argued that the partial antidote to vulnerability is resilience. resilience is defined as 'what provides an individual with the means and ability to recover from harm or setbacks.' 62 to embody a human security approach international actors would need to first identify the peculiarities of vulnerability in a given situation and second contribute to building resilience which would ultimately address the root cause of the vulnerability. for instance, the un regularly empowers women to participate in politics which within a society where women have traditionally been subjugated could alleviate vulnerability in the longterm. the final two principles, preventative protection and empowerment, are advanced to ensure people are shielded from severe and widespread threats on the one hand and on the other that people's opportunities are enhanced and built upon to provide a future situation where individuals and communities are more resilient to threats. protection may need to be offered to combat a range of threats, not only physical harm, and protection responses should be preventative where possible. international law and human security in a kaleidoscopic world security has a concern for more than threats of violence and as such protection must go further than ensuring only physical integrity. instead an intervention could address foreseen health or environmental crises and use institution building programmes to build the capacity of the state in question to be better prepared for future risks. the empowerment aspect of the commission's framework is thought to have more potential and shows how human security can respond to crises in a legitimate and focused way compared to existing security practices. 64 where people are adequately protected, they can be empowered to 'make better choices, and actively prevent and mitigate the impact of insecurities.' 65 the commission argues that where people are empowered they can address issues locally and can mobilise others by gaining international attention for food shortages or protest human rights violations. today over half of the world have access to the internet which gives an unprecedented opportunity for individuals to be empowered and use the internet to gain global attention for an ongoing security issue. a notable, and inspiring example is that of alaa salah, a student who led protests against former president omar al-bashir's regime in early-2019. photographs and videos of salah gained worldwide attention first on social media and later in mainstream news media outlets. salah helped mobilise a large number of people in opposition to al-bashir's government and the image was dubbed ''the image of the revolution''. 66 powerful examples such as this demonstrate the power of social media in communicating concerns of populations to a wider audience across the globe. 4 can human security make international law more responsive to human need? it has previously been argued that human security is a concept which is not useful for policymaking because its competing policy goals are too difficult to reconcile. 67 unworkable in practice.' 68 however, it has conversely been claimed that the un, with regards to peace operations, have been working on a human security-based agenda since 1999 regardless of the fact the term is absent from major policy documents. 69 in addition, the un has been said to have responsibility for an integrated approach to human security and ultimately 'promote a positive concept [of peace] by addressing the underlying conditions and prerequisites for a stable and durable peace.' 70 in a kaleidoscopic world, the legitimacy of actors such as the un and states are increasingly challenged. the question becomes, are the current international institutions and legal system fit for purpose? as noted above, international law has developed into a system more focused on humanity and the position of individuals. 71 emmanuelle jouannet, for example, argues that international law has become more than a means of social regulation, and is 'becoming used to transform international society in order to make up for economic, social or equitable imbalances.' 72 however, it has been predicted that over the coming decades international law will find it difficult to formally incorporate the diverse and innumerable non-state actors involved in this transformation into international institutions and legal processes. 73 human security would give guidance and orientation to international actors allowing them to engage with non-state actors and ultimately provide better preventative protection and the alleviation of vulnerabilities. 74 a human security approach would seek to respond to global problems by ensuring bottom-up engagement with local actors and decentralising decision-making to open up discussions with a multitude of parties in the interests of increased legitimacy and accountability. how human security is utilised, 'all depends on what human security is understood to be: a political agenda for governments, a rallying cry that forges ad hoc or sustained coalitions of states on single issues, a common concern that brings together single-issue civil society groups under a uniting umbrella, an academic problem, or a new research category.' 75 human security is described as a concept by most literature. therefore, human security is a collection of interrelated ideas and can be a guide to interpretation. specifically, as an agenda-setting concept, it determines what are the most relevant issues and brings to the forefront neglected problems that have previously not been included in national and international security debates or have been on the periphery. barbara von tigerstrom has said that human security is 'a concept that is designed to be used in a variety of ways, including in the interpretation and development of legal norms.' 76 furthermore, gerd oberleitner claims that 'a human security approach to international law can reinforce and strengthen attempts to bring international law into line with the requirements of today's world.' 77 by using human security as an agenda-setting concept international actors will be able to react to the needs of individuals and provide effective responses that solidify the continued relevance of international law. following on from the arguments of von tigerstrom and oberleitner, shireen daft argues that human security can be a 'synthesised overarching framework'. 78 daft says that human security can have legal character by serving as a framework for the expression of existing norms with human security providing a principled future direction for how international law tackles security threats. 79 daft believes this is possible if clear principles of human security are articulated with roots and relevance in existing international law. 80 daft's argument lends well to the current position of states since most believe human security should be pursued under existing international legal frameworks and not through new legal obligations. 81 australia has already advanced the view that human security can provide a normative framework which 75 oberleitner, supra note 54, 592-3. 76 can ensure collective actions are providing preventative protection, empowerment to build resilience, and direct benefits to populations. 82 other states agreed with, for example, qatar arguing that by using a framework for human security states will be compelled to be proactive. 83 likewise, india stated that human security can be implemented and used as a framework to respond to current challenges, not only as a policy goal. 84 if human security is not to be a legal concept in its own right what it can do is harness existing international law to pursue human-centric operational goals. 85 a human-centred approach to international law is already evident in a number of developments over the last couple of decades. for instance, the 1997 ottawa treaty, the 1998 rome statute of the international criminal court, and the 2000 optional protocol to the convention on the rights of the child have been argued to embody a shift toward giving greater importance to human security as opposed to national security. 86 however, hitoshi nasu notes that the adoption of humancentred treaties does not necessarily mean the concept of human security is being implemented because states may simply address issues they consider a threat, in a manner they consider appropriate, while not taking into consideration the views of individuals facing insecurity. 87 to counter this deficiency, it is proposed in this paper that international law can be focused toward the inclusion of individual views on insecurity by the un implementing a conceptual framework of human security. due to shared goals there is direct relevance of human security for the un and the organisation represents an ideal vehicle for implementing a human security framework. the un has long advanced the needs of individuals and a sophisticated international human rights framework, development programmes, and the protection of civilians by un peacekeeping forces are evidence of this. it could be criticised that the implementation of human security is an unattainable utopian goal but involving individuals as participants, using preventative methods of protection, and empowering people to 82 be involved in security processes are, on some level, achievable facets of the human security concept. a human security framework would allow the un to adapt to future security developments in the face of a new breed of crises brought by an interconnected and globalised world. in addition, the framework would provide consistency of approach by using a core set of individual-focused principles as the backbone of the organisation's activities. a conceptual framework of human security, in a similar manner to that argued by daft, should be applied to the activities of the un and importantly the resolutions of the general assembly and security council. the principles of human security (i.e. its basis as a rights-based concept, the vital core, recognition of vulnerability, and preventative protection and empowerment) can have transformative potential as a conceptual framework. human security is transformative because '[i]t is an idea that seeks to reopen analysis of the world's priorities, to produce new integrated methodologies in analysing the most complex and pressing problems, and to give greater voice to individuals and communities in searching for solutions.' 88 human security does not entail additional legal obligations on the part of states but does represent an alternate expression of existing legal principles to create an approach where individuals are the referent object of security. individuals may then determine which threats to security, and consequently related legal rights, require the immediate attention of the international community. under such a framework, the un and its member states can open an equal dialogue with national, regional, and local actors to determine if efforts in a broad range of security-related areas are 'having unintended negative effects on people's security ''on the ground'' in spite of good intentions, or whether they are achieving the goals articulated in terms of benefit to affected populations.' 89 also, decision making, for instance by the security council, may not explicitly threaten human security but choices may be made that do not reflect the immediate needs of individuals and not respect human security. resources may not be used most efficiently to the benefit of empowering individuals and reducing their vulnerability. it has previously been claimed that the 'ability to link diverse issues and encourage a coordinated approach constitutes the most important contribution of a human security approach.' 90 drawing on benedek's belief that human security and human rights should not be used interchangeably but can ''redefine and refine'' our approaches to peace, security and human rights, a conceptual framework of human security has a role to play in the international community. the implementation of a human security approach must be undertaken across the full spectrum of actors. at the un-level, the un general assembly has adopted a definition of the concept and has also held a thematic debates on the subject. 91 however, it is the un security council which has the primary responsibility to maintain international peace and security under article 24 of the un charter and can take action under chapters vi and vii of the charter to either settle a dispute through peaceful means or take enforcement measures to maintain or restore international peace and security. 92 therefore, the security council is unique in its position to articulate threats to international peace and security, advance what it deems to be an important agenda, and coordinate a human security approach. 93 trina ng suggests that if the security council adopted a framework of human security then its interpretation of international peace and security would be radically altered resulting in a limitless expanded application of chapter vii. 94 while it is true human security would affect what the security council recognises as a serious threat or acute danger it would be doing so on the basis of bottom-up information from individuals and civil society. in a time where non-state actors are calling for action on various global issues it is suggested here that the security council should engage with a broader notion of security that is responsive to the needs of individuals even if the application of chapter vii is altered. 1950) ) states that in a situation where the security council is unable to exercise its primary responsibility for the maintenance of international peace and security then the general assembly may immediately consider the matter at hand and make recommendations to maintain or restore international peace and security. this was used in 1956 to create unef i but missions are, under most circumstances, created by the un security council states are a necessary adjunct to any security action taken by the security council as states are the ultimate decision makers in the procedure of the council and troops must be contributed by states for collective security action. as such, a human security approach at the un would necessarily need to be acceptable to states. a potential threat to this support is the above mentioned scepticism that human security should not entail additional legal obligations or erode state sovereignty. however, this is not the case as barry buzan reiterates that, states are a 'necessary condition for individual security because without the state it is not clear what other agency is to act on behalf of individuals.' 95 human security is, in the first instance, provided by states. states must consider that they are sovereign only so long as they are effective in developing conditions and preventing conflict to provide for the wellbeing of individuals under their control. 96 human security does not disregard the importance of state infrastructure and provisions for protecting its own population. instead, 'strengthening the function of states is the major course of human security.' 97 where a state is failing and its institutions no longer functioning there can be profound damage to human security. 98 the second level of human security is the responsibility of actors such as the un to act in defence of human security where a state fails to do so. this second level is where the use of a human security framework by the un security council fits in. the third tier of human security is where empowered individuals, communities, and civil society provide for their own security. this is where people themselves become involved in the participatory process of security. 99 that is to say, individuals are active subjects in identifying and implementing solutions to security issues. 100 this can be undertaken by individuals being given platforms to voice security concerns and the proposal of solutions, especially in post-conflict situations where a society is being rebuilt. 101 the next section will provide an example of un practice which can be informed by the concept of human security to include bottom-up engagement and an inclusive decision-making process which recognises individuals as valuable participants. a conceptual framework of human security could have a profound effect on the mandating of peace operations by involving meaningful engagement with individuals. this is an ideal example since un missions in the field are necessarily engaged in activities with local communities and national reconciliation efforts that would benefit from a bottom-up, human security approach to decision-making and mandate implementation. it has previously been argued by howe, kondock and spijkers that while peace operation mandates do not use the language of human security they are impacted by the un's wider human security agenda which includes the protection of civilians, r2p and pursuit of the millennium development goals. 102 both minusma and minusca have carried out a number of activities aimed at promoting human rights and establishing the rule of law. minusma has been directly involved in the training of malian defence and security forces and police since 2013 and understands 'long-term reconciliation will not be possible without the promotion and defence of the human rights of all communities in the north.' 103 the focus on human rights, both as a task to assist the malian authorities with and as a monitoring and reporting task for the un, has since been carried over into each renewal of minusma's mandate. 104 as for minusca, human rights and extending the rule of law forms a crucial part of its mandate with the mission working to revitalise the justice system in the wake of widespread human rights abuses and sexual violence. a hybrid special criminal court has been established to try 'serious crimes, including serious violations of human rights and international humanitarian law, including conflict-related sexual violence as well as grave violations of the rights of the child, that constitute a threat to peace, stability or security'. 105 both missions have also sought to re-establish the rule of law by training judges and magistrates and reopening courts with the ultimate goal of ending impunity. 106 implementation of the vital core would allow the un to determine what is crucially important for local people to be able to respond in a bottom-up manner. missions can demonstrate a concern for the vital core by opening space for dialogue with individuals. prior to minusma's deployment the un's preliminary assessment mission held talks with 'a broad cross section of malian society' to make recommendations for how the un can provide assistance. 107 a key method of engaging with communities in both minusma and minusca has been through the use of community liaison assistants (clas). clas aim to improve communication with local communities and to gain 'a better understanding of the population's concerns and expectations of the mission' and are 'critical links' between the mission and communities in remote areas. 108 clas speak the local language, can find out the risks to communities, act as mediators, and communicate outcomes to both civilian and military contingents of an operation. clas are able to work with communities to formulate protection strategies and alert mechanisms for community security. 109 through to 2017 minusma remained committed to improving 'community outreach' in the north to respond to the needs of the local population. 110 however, it is worth noting that while the missions contain examples of community outreach it is unclear to what extent local views on security in actuality inform the security council's decisions on mandate priorities or the un's decisions on what initiatives it will pursue within the country as part of the mandate. the concept of human security recognises vulnerability as a crucial factor in decision-making. the mandates of minusma and minusca typically recognise the vulnerability of women and children and call for specific protection and the deployment of child protection advisors and women protection advisors. 111 other groups have been identified as vulnerable in mali and the car such as displaced persons, 112 the elderly, 113 disabled, 114 and muslim communities. 115 to what extent resilience is built is another matter. resilience can be read into some un activities such as the creation of employment opportunities for young people, the promotion of gender equality, and the involvement of women in the peace process. 116 however, the focus of the un remains on protection rather than resilience building initiatives. both minusma and minusca have protection of civilian (poc) mandates. for a human security-based analysis the question is whether the poc mandates take preventative action and look beyond only physical harm. the poc mandates themselves specify the protection of civilians 'under imminent threat of physical violence'. 117 the missions have utilised so-called 'robust' force to establish control of territory and deter armed groups from harming civilians. 118 in one sense this can be seen as preventative as the un is taking steps to mitigate harm to civilians. another key area of protection for the missions is the prevention of sexual violence and abuse against women and children by providing training to a multitude of actors and assisting the host governments with introducing preventative measures. 119 the missions clearly focus on physical protection but wider human security concerns can be seen where vulnerabilities identified above are linked to the type of protection offered, as with preventing sexual violence against women and children. empowerment requires giving agency to local people to be able to make choices for their future and demand improvements which enhances their security. the un has made a concerted effort to empower women in mali and the car to have active roles in the peace process and national politics. 120 the security council acknowledges 'the significant contribution that women can have in conflict prevention, peace building and mediation efforts.' 121 the missions have also undertaken and supported actions that provide space for people to be empowered to act on their own behalf in identifying and implementing solutions to the crisis. for instance, local peace and reconciliation committees, initially established by minusca in 2015, contain elected persons from within a community and 'are expected to solve local conflicts and promote peace through mediation and dialogue.' 122 this is remarkably similar to the human security aim of giving people the ability to act on their own behalf and on the behalf of others. 123 local peace committee activities have run community awareness campaigns, reopened markets, and facilitated dialogue between farmers and herders to resolve differences peacefully. 124 human security is undeniably a difficult concept to quantify. though the examples above outline some areas of current un practice which do operationalise aspects of human security. however, there is much more which could be achieved with the application of a conceptual framework for human security. for instance, there is an overwhelming focus of un peace operations on protecting individuals from physical harm. human security goes beyond physical protection and necessitates a concern for harms rooted in, for example, environmental, health, or economic causes. the un mission in liberia (unmil) did provide support during an ebola outbreak but more could have been done in this regard. 125 the security council declared the ebola outbreak in 2014 a threat to international peace and security and took a major leadership role during the crisis. 126 that being said, un peace operations operating in affected areas did not have their mandates modified to include the new concern for health security. under a conceptual framework for human security, the protection of health security would have become a key concern for the un missions in response to local needs in areas hit by the ebola virus. going further, if un peace operations adopted the conceptual framework there would not need to be a global pandemic to result in un attention on health threats. instead, un missions in the field would actively work on improving access to health services with other partners if that need is identified by a local population. value can be added to un peace operations by orientating practice toward initiatives like informing decision-making with local views on security needs, the building of resilience to a range of vulnerabilities, and allowing for dialogue within communities as well as the empowerment of local people to engage on national and international levels. under the conceptual framework, un missions would need to explicitly identify and plan initiatives that build resilience and prioritise 123 commission on human security, supra note 4, 11. 124 empowerment at the local level to ensure communities are able to resolve conflict peacefully and engage with national reconciliation. while the un implements some wider empowerment related strategies, either on its own or in partnership with host governments, they do not feature as focal points of mission examples given above. empowerment is fundamental for the international actor to gain a local-level understanding of the conflict and interlinkages between security threats and a conceptual framework for human security would assist the un in achieving meaningful empowerment. this case study has served as a starting point for international lawyers to begin to consider and visualise how the concept of human security could be further implemented in other un activities and the application of international law with individuals as participants. the concept of human security can be a valuable tool to reorientate international law, and importantly the work of the un, toward a human-centric notion of security. by doing this, international law would remain legitimate in the face of declining state-centric attitudes and would be able to respond to new security challenges in the interlinked kaleidoscopic world. human security would allow new actors to penetrate the international system and ensure that the existing structures remain legitimate in a time where coalitions and decentralised global movements are ever present. particularly in situations of conflict, it is crucial that the voices of local communities, national groups and other non-state actors are able to access the global discourse in a formal manner as opposed to relying on social media traction to garner attention for their concerns. the earlier mentioned example of sudan is one success story of how social media influenced the international community's discussion of a crisis. however, there are countless other conflicts which to do not attract the same level of attention. a conceptual framework of human security would provide space for the un to engage with non-state actors in a meaningful way. the example of peace operations shows the beginnings of an approach that implicitly takes into account the principles of human security. the implementation of these principles could be expanded to ensure international responses and decision-making are locally-informed and prepare communities for future security threats. a conceptual framework of human security would provide an approach in which the un, and states, could achieve human-centric, locally-informed peace in a globalised and interconnected world where individuals are able to permeate international discourse more than ever before. there are of course many unanswered questions and other scholars may wish to continue this debate by linking the conceptual framework to twail scholarship, the women peace and security agenda, critiques of liberal peacebuilding, and much more. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. publisher's note springer 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of the secretary-general on the situation in the central african republic report of the secretary-general on the situation in the central african republic key: cord-005872-w1x1i0im authors: volk, t.; kox, w.j. title: endothelium function in sepsis date: 2000 journal: inflamm res doi: 10.1007/s000110050579 sha: doc_id: 5872 cord_uid: w1x1i0im endothelial cells can be the prime target for an infection and infected endothelial cells may serve as an initiating system for a systemic response as these cells are able to secrete many mediators known to be of paramount importance. endothelial cell functions in turn are regulated by these circulating mediators. cellular interactions with leukocytes revealed protective and destructive functions. single cell and animal studies indicate that endothelial permeability is increased and apart from clinical obvious edema formation in septic patients, the endothelial component remains unknown. endothelial coagulation activation has been shown in vitro, however human data supporting an endothelial procoagulatory state are lacking. defects in endothelium dependent vasoregulation in animal models are well known and again human studies are largely missing.¶an imbalanced production of reactive oxygen species including nitric oxide has been found to be involved in all endothelial functions and may provide a common link which at present can be supported only in animal studies. sepsis is considered the leading cause of death in noncoronary intensive care units. it has been defined as a systemic inflammatory reaction to an infection. among many cellular disturbances endothelial cells play a major role in the pathogenesis of this disease as these cells are critically involved in maintaining a delicate balance between vasoconstriction and vasodilation, blood cell adherence and nonadherence, anticoagulation and procoagulation, permeability and thightness. all these functions are believed to be imbalanced and impairment is believed to precede clinically recognizable alterations (e.g. bleeding, edema, organ dysfunction and shock) but it is by no means clear whether these changes are the cause or consequence of endothelial dysfunction in sepsis. endothelial functions have largely been studied in vitro from the first successful attempts of culturing isolated human endothelial cells dating back to the early 70ties. endothelial cells from and within different organs or different species behave differently and tend to change properties the longer they are kept in culture. to overcome isolation variabilities several endothelial cell lines are currently available which have retained at least some features. however, experiments from single cell cultures are flawed in many ways and results from these may never be of any relevance to medical practice. it is inherent to any cell culturing that data obtained from these experiments often are restricted to cell type, time of culture and general working conditions. in vitro stressed endothelial cells almost uniquely tend to activate a functional program leading to a proinflammatory, procoagulatory and hyperpermeable phenotype. in this review we want to summarize investigations of disturbed endothelial functions including infection, mediator and cellular interactions, permeability, coagulation and vasoactivc properties from molecular findings to patient care. staphylococcus aureus is the most prevalent bacterial pathogen isolated from patients with blood stream infection in north america [1] . s. aureus has been reported to directly infect human umbilical vein endothelial cells (huvec) thereby inducing secretion of cytokines and functional upregulation of adhesion molecules [2] . internalized s. aureus may lead to apoptosis in huvec [3] or persistence as small colony variants [4] . group a streptococci can enter huvec [5] , a process which may render these cells particularly sen-sitive to otherwise subtoxic concentrations of hydrogen peroxide [6] . group b streptococci (gbs) are the most common cause of neonatal sepsis and pneumonia. gbs-induced endothelial cell injury can be confirmed by histological findings at autopsy, in animal studies and in vitro [7] . gbs invasion and subsequent damage of endothelial cells may be inhibited by cytochalasin d in huvec implicating that cytoskeletal interactions are important for toxicity. however, invasion of brain microvascular endothelial cells by gbs may be dose dependently cytotoxic due to beta-hemolysin production [8] . streptococcus pneumoniae may enter activated endothelial cells using paf-receptors [9] . this receptor engagement may also serve as a sorting signal for endothelial transcytosis [10] . infection and activation of endothelial cells by listeria monocytogenes is believed to be a critical component of the pathogenesis of this disease and includes ceramide generation, transcription factor activation and increases in adhesion molecule expression on huvec [11] . listeria have been described to enter huvec either directly via internalin b or by a cell-to-cell spread from infected monocytes [12] . gram negative bacteria have lipopolysaccarides (lps) within their cell wall. cellular binding of lps usually is accomplished by cd14. endothelial cells lack cd14 receptors and lps effects on endothelial cells generally require the presence of cd14 in the serum. lps effects from gram negative live bacteria (b. fragilis, e. cloacae, h. influenzae, k pneumoniae) on endothelial cells have been demonstrated by transcription factor activation and subsequent surface expression of e-selectin and tissue factor which was not seen from viable or heat-killed gram-positive bacteria (s. aureus, e. faecalis, s. pneumoniae) [13] . neisseria meningitidis adherence on endothelial cells has been reported to be influenced by pilus protein c expression and cd66 on endothelial surfaces [14, 15] and may cause tissue factor expression due to the presence of lps within the cell wall. haemophilus influenzae generally is not toxic to endothelial cells except three clones of biogroup aegyptius causing brazilian purpuric fever [16] . toxicity has been reported to be independent of endotoxin, phagocytosis and replication since irradiation, cycloheximide, cytochalasin d and methylamine have no effect on the ability of the bacterium to invade and cause a cytotoxic response. piliated pseudomonas aeruginosa adheres to and enters human endothelial cells leading to progressive damage [17] or may persist by lysis of endosomal membranes [18] . escherichia coli may invade human brain microvascular endothelial cells involving specialized proteins [19, 20] . endothelial infection by chlamydia pneumoniae also activates endothelial cells to produce cytokines and adhesion molecules and become procoagulant [21] [22] [23] . bartonella quintana, the cause of trench fever transmitted by the body louse, has recently been implicated in culture-negative endocarditis and bacteraemia amongst homeless people [24] . infection and damage of endothelial cells by b. quintana has been demonstrated in vitro and in vivo [25] . interaction of bartonella henselae with endothelial cells may result in bacterial aggregation on the cell surface and the subsequent internalisation of the bacterial aggregate by a unique struc-ture, the invasome [26] . rickettsia rickettsii, an obligate intracellular gram-negative bacterium which causes rocky mountain spotted fever, inhibits endothelial apoptosis allowing to remain inside the host endothelium [27] but is also known to cause a proinflammatory endothelial phenotype. rickettsia conorii causes mediterranean spotted fever which causes endothelial infection with secretion of cytokines, increases in adhesion molecules and induction of surface tissue factor [28, 29] . attachment of borrelia burgdorferi, the agent inducing lyme disease, to endothelial cells may be accomplished by cd14, alpha(v)beta3 and alpha5beta1 integrins or different classes of proteoglycans [30] [31] [32] . b. burgdorferi activates the transcription of chemokine and adhesion in molecule gene expression in endothelial cells [33] . plosmodium infected erythrocytes may bind to endothelial cells via p-selectin, cd36, intercellular adhesion molecule 1 (icam-1) or platelet endothelial cell adhesion molecule 1 (pecam-1) on the endothelial surface [34] [35] [36] . phagocytosed live candida albicans stimulates cytokine secretion and inducible cyclooxygenase expression in endothelial cells [37, 38] . some viral diseases are known to primarily infect endothelial cells and alter their function. dengue virus infection of huvec leads to production of chemoattractant proteins rantes and il-8 [39] , herpes and measles virus infection of brain microvascular endothelial cells increases lymphocyte adhesion by increasing icam-1 [40] and measles virus and cytomegalovirus increases tissue factor expression on huvec [41, 42] . hemorrhagic fever caused by hantaviruses may be accomplished by integrin mediated endothelial infection [43] . on the other hand, ebola virus infects endothelial cells with a transmembrane glycoprotein and inhibits inflammatory responses [44] . exotoxins are known to directly activate endothelial cells. platelet activating factor (paf), no˙and pgi 2 secretion from huvec has been demonstrated by e. coli hemolysin via inositolphosphate/diacylglycerol formation and by s. aureus alpha-toxin via transmembrane ca 2+ entry [45] . there is increasing evidence that hemolytic uremic syndrome results from the systemic action of verocytotoxin producing e. coli on vascular endothelial cells [46] . alpha toxin from clostridium perfringens is a phospholipase c and has been reported to induce adhesion molecule expression and secretion of chemokines from endothelial cells [47] . brain capillary endothelial cells have been reported to express mbec1, a protein that may serve as the c. perfringens enterotoxin receptor [48, 49] . the activity of small gtpase rho has been shown to be altered by c. difficile toxin b [50] and pasteurella mulrocida toxin [51] , which leads to alterations of endothelial permeability. p. aeruginosa exotoxin a may directly injure endothelial cells by a motif shared by many toxins [52] . listeriolysin and phosphatidylinositol-specific phospholipase c secreted from l. monocytogenes has been shown to induce phosphatidyinositol metabolism and diacylglycerol formation in the absence of bacterial uptake by the endothelial cells [53] . some bacterial exotoxins have been used for years as pharmacological tools like pertussis toxin for studying g-protein dependent endothelial cell functions. lipoteichoic acid and peptidoglycan from cell wall components of gram positive bacteria have been shown to induce sepsis in animal models. while lipoteichoic acid has been reported to directly activate endothelial cells [54] , peptidoglycan seems monocyte dependent [55] . most in vitro experiments, however, were performed with different sources of lps as a surrogate activator modelling gram negative bacterial infection. endothelial stimulation using lps in relevant concentrations are usually performed in the presence of serum containing soluble cd14-receptor because endothelial cells generally lack cd14. alternatively, proinflammatory cytokines including tumor necrosis factor alpha (tnf-a), interleukins (il-1, il-4) and interferon gamma (ifng) acting on endothelial cells have extensively been investigated and shown to alter endothelial in vitro functions [56, 57] . the inflammatory response in endothelial cells has been linked to an alteration in reactive oxygen production including superoxide (o 2 -˙) , hydrogen peroxide (h 2 o 2 ), nitric oxide (no˙), hydroxyl radicals (oh˙) and secondary reaction products thereof. o 2 -˙i s believed to be present in unstressed conditions in less than nanomolar quantities within cells. mitochondrial and cytoplasmatic superoxide dismutase readily reacts with o 2 -˙f orming h 2 o 2 which has been measured in micromolar concentrations in human blood. sources of endothelial o 2 -˙p roduction apart from mitochondrial leakage may include metabolism of cytp450 or other metabolic byproducts and production by a nadh oxidase system or by nitric oxide synthase in the absence of l-arginine. some bacterial pathogens are known to be able to produce h 2 o 2 by themselves, however interaction with the endothelium may greatly enhance cellular alterations. streptococcal hemolysin, streptolysin s, is capable of interacting with h 2 o 2 to injure vascular endothelial cells [6] . iron bound to the p. aeruginosa siderophore, pyochelin, augments oxidantmediated endothelial cell injury by modification of transferrin to form iron complexes capable of catalyzing the formation of oh˙from o 2 -˙a nd h 2 o 2 [58] . r. rickettsii infection of the endothelial cell line ea.hy 926 and huvec has been demonstrated to cause glutathione depletion, a major intracellular antioxidant, and reduced glutathione peroxidase activity leading to increased amounts of intracellular peroxide [59] . an increase in reactive oxygen species production has been shown in endothelial cells after incubation with lps, il-1, tnf-a and ifn-g [60] [61] [62] [63] [64] [65] . tnf-a has many times been reported to increase, e.g., adhesion molecule expression inhibitable by various antioxidants [62, [66] [67] [68] . nitric oxide, like o 2 -˙, is chemically not very reactive at all. endothelial production has been established by either constitutive no-synthase (nos) iii in a ca 2+ and phosphorylation dependent manner or by inducible nos ii. direct biochemical actions of no˙include metalcomplex containing proteins, other radical species, oxygen and oxygen derivatives leading to oxidation, nitrosation and nitration. relevant concentrations in vivo have been reported to range from nm to 100 mm. no˙directly reacts with oxyhemoglobin (fe 2 -o 2 ) leading to methemoglobin (fe 3+ ) and nitrate (no 3 -). no˙reacts with o 2 forming nitrite (no 2 -) via intermediate no 2 and n 2 o 3 . mainly n 2 o 3 is participating in n-or s-nitrosylations leading to nitrosamines or nitrosothioles, the latter may serve as a circulating source or as a transporter across cell membranes. reduced glutathione has a high affinity to n 2 o 3 and may also be important in toxicity related to no˙autoxidation. toxicity related cellular targets of no˙may include inhibition of cytochrome c oxidase, inhibition of catalase or dna damage. on the other hand, no˙has been reported to inhibit iron catalysed fenton reaction and to inhibit lipid peroxidation. iron nitrosyl formation in heme containing proteins are the best characterized reactions for no˙in biology. this type of interaction includes very sensitive stimulation of guanylate cyclase and inhibition of cytochrome c oxidase. e. coli hemolysin and s. aureus alpha toxin induce no˙formation in cultured porcine pulmonary endothelial cells [69] . transformed mouse endothelial cells stimulated by the combination of ifn-g and tnf-a killed intracellular r.conorii by a mechanism that required the synthesis of no˙ [70] . ifn-g, tnf-a and il-1 stimulated murine endothelial cells have been shown to kill schistosoma mansoni through the production of nitric oxide [71] . both no˙and o 2 -˙p roduction may have a limited influence on endothelial viability under resting conditions. cultured bovine and porcine aortic endothelial cells showed decreased no˙production after 1 h of lps incubation which was related to decreases in capacitative ca 2+ signals [72] . posttranscriptional destabilization of nos iii mrna may have accounted for this early decrease in no˙production [73] . nos ii, which is believed to produce larger amounts of no˙is also known to be regulated by many proinflammatory stimuli with significant cell type variablilities. at sites of endothelial involvement in an inflammatory process both vascular non-endothelial and non-resident cells are known to produce no˙. as the amount and physiological consequences of no˙produced from noss at the microcirculatory level is not known, it may well serve also to reduce inflammatory processes as recently implicated from a coculture experiment [74] . that an increase in reactive oxygen species is present in human sepsis has been documented by almost any study trying to quantify secondary reaction products by several methods, however the cellular sources remain speculative ( table 2 ). in 1990 beckman et al. showed that the presence of both no˙and o 2 -˙p roduced peroxynitrite (onoo -) which may decompose to produce ho˙like molecules and thereby kill endothelial cells [75] . at ph 7 its lifetime is in the order of a second. half of the onoo formed is rapidly equilibrated to peroxynitrous acid (onooh) and breaks down to no 3 11 sepsis immunohistochemical endothelial nitrotyrosine≠ [176] 3 septic shock no 2 -/no 3 -, plasmatic nitrotyrosine≠ [177] 3 septic lung injury immunohistochemical endothelial nitrotyrosine≠ antioxidant capacity was defined as the ability of plasma to inhibit * ferryl myoglobin production by hydrogen peroxide addition to metmyoglobin or ** oxo-iron induced damage to deoxyribose, phospholipids and dna. tbars, thiobarbituric acid reactive substances; xod, xanthine oxidase. models and therefore question the view that this molecule solely is detrimental. toxicity induced by an interaction between hydrogen peroxide and nitric oxide has led to conflicting results. rat lung microvascular endothelial cells and porcine pulmonary artery endothelial cells exposed to h 2 o 2 have been reported to be protected by no˙donors [76, 77] , whereas toxicity in bovine aortic endothelial cells [78] and in rat liver microvascular endothelial cells [79] was increased in the presence of no˙donors. no˙in the presence of h 2 o 2 may also produce oh˙like molecules independent of the presence of iron [80] . neutrophils may add to the complexity of toxic reactions. myeloperoxidase from activated neutrophils, which produces hocl and oh˙molecules in the presence of o 2 -˙, has recently been demonstrated to convert no 2 into no 2 , thereby damaging endothelial cells [81] . high doses of reactive oxygen species (including no˙) have been shown to cause apoptosis of endothelial cells, whereas low doses were protective [82] . in mice disseminated endothelial apoptosis has been suggested to be responsible for organ failure and shock induced by endotoxin or tnf-a [83] . both lps or tnf-a usually do not cause endothelial cell death unless protein synthesis is blocked probably due to simultaneous increases in antiapoptotic protein synthesis [84] . however, postmortal investigation in humans deceased from or with sepsis did not confirm these results [85] . most of the genes activated in vitro during the endothelial stress response are controlled by at least two transcription factor families: activator protein 1 (ap-1) and nuclear factor kappa b (nfkb). nfkb has gained wide interest as a target in inflammatory diseases as it seems to be invariably upregulated [86] . a variety of agents including cytokines and reactive oxygen stress cause ikb to dissociate from the complex after phosphorylation by ikb-kinase complex. h 2 o 2 has been reported to activate transcription factor nfkb in porcine aortic endothelial cells [87] whereas huvec were unresponsive [88] . no˙has in most cases been shown to inhibit transcription factor nfkb and its influence on transcription factor ap-1 is unclear. inhibition of nfk-b as a therapeutic means has been suggested. however, as this transcription factor is also involved in protective gene regulation, its inhibition can make cells sensitive to e.g. tnf-a [89] . data on activated intracellular signalling pathways in sepsis patients are scarce but include nfkb within mononuclear cells [90] . usually any blood cell is kept off endothelial surfaces. this is believed to be accomplished by net electrical charge, biomechanical characteristics of flowing blood and the secretion of no˙. if blood cells touch the endothelium a ca 2+ -signal is induced [91] , but it is unclear at present whether this has any functional consequence. it is tempting to speculate that ca 2+signals may then in a context sensitive manner augment proadhesive processes or antiadhesive endothelial properties. activated endothelial cells are potent producers of cytokines like il-1, a major proinflammatory cytokine, and chemotactic peptides including il-8 for neutrophils, macrophage inflammatory protein-1 alpha (mip-1a), monocyte chemoattractant proteins 1-4 and rantes (regulated on activa-tion, normal t cell expressed and secreted) for monocytes, t-lymphocytes and dendritic cells, growth related protein (gro) and gamma-interferon-inducible protein (ip-10) for activated t-lymphocytes, epithelial neutrophil activating peptide 78 (ena-78), vascular monocyte adhesion-associated protein (vmap-1) and endothelial monocyteactivating polypeptide ii (emap-ii) for monocytes [92] . many adhesion molecules are expressed on the surface of endothelial cells in a highly complex yet regulated manner. p-selectin, e-selectin, icam-1, vascular cell adhesion molecule 1 (vcam-1), pecam-1 are well known for their stimulus-, cell-, time-and organ specific dependence of expression and their importance in regulation of leukocyteendothelial interactions. moreover, apart from being passive adhesion molecules, all of the above mentioned molecules have been shown to signal inside endothelial cells upon receptor engagement. shed receptors from endothelial surfaces may also serve as endogeneous antiadhesive molecules demonstrating even more the dynamic and complex nature of these processes. soluble forms of adhesion molecules have been shown to be present in high amounts in human sepsis (table 3) , however whether this is beneficial or detrimental is not known and the assumption that these parameters may serve as practical indexes remains to be established. particularly ifn-g has been repeatedly shown to increase endothelial hla-dr expression rendering them capable of mhc class ii restricted interactions with cd4 + t-cells. costimulatory cd80 (b7-1) and cd86 (b7-2) are usually not present on endothelial surfaces leading to the conventional view that endothelial cells are semiprofessional antigen presenting cells. however, under certain conditions both costimulatory molecules and cd40 can be upregulated on endothelial surfaces indicating excessive antigen presentation [93] . migration of lymphocytes into inflamed mouse tis-vol. 49, 2000 endothelial function in sepsis 189 sues has been reported to depend on psgl-1 and esl-1 binding endothelial p-selectin and e-selectin, respectively [94] . this phenotype was found to be restricted to th1 lymphocytes, but human sepsis seems to be predominated by a th2 type [95] . both tnf-a and ifn-g are able to promote transmigration of leukocytes, whereas coapplication of both cytokines inhibit this process [96] . t-cells migrating through the endothelial barrier in a pecam-1-dependent manner are subject to inhibitory signals which may limit their activation in tissues [97] . tnf bound to monocytes has been shown to inhibit endothelial apoptosis, whereas this process is promoted in the presence of lymphocytes [98] . unperturbed endothelial cells express fas-ligand which has been implicated as a means of signalling apoptosis to constitutively fas receptor (cd95) bearing cells, whereas tnf-a treated endothelial cells decrease fas ligand expression thereby allowing leukocyte survival during extravasation [99] . how the endothelium might interact specifically with lymphocytes or monocytes in septic patients can only be speculated on. neutrophils interacting with endothelial cells have repeatedly been shown to cause toxicity due to the release of enzymes and reactive oxygen species. if uncontrolled, these cells are believed to mediate significant tissue damage. however whether uncontrolled activation or rather deactivation is present in human sepsis is a matter of debate. endothelial cells have been reported to inhibit some neutrophil functions [100] . transmigrated neutrophils may actively participate in the endothelial resealing process by the secretion of adenosin precursors [101] . however, massive leukocyte extravasation as one would expect from most animal studies has never been shown in septic patients [102] . endothelium is known to regulate transvascular fluid flux, flux of nutrients, mediators and cells by either paracellular or transcellular vacuolar channel related pathways. lateral junction proteins including the vascular endothelial cadherin-complex, platelet-endothelial cell adhesion molecule-1, occludin, zona occludens-1, recently described junctional adhesion protein, cd151/platelet endothelial tetraspan antigen and cd81/target of antiproliferative antigen are known to participate in this process. paracellular permeability is achieved by either an active contraction or the controlled release of an intrinsic tone mediated in most cases by the action of myosin light chain kinase (mlck) acting on non muscle myosin. generally an increase in the concentration of camp keeps cultured endothelial monolayers tight and cgmp has been reported to assist this function in human aortic and foreskin vessels [103] . endothelial retraction may be initiated by increases in intracellular ca 2+ concentration, but elevation of ca 2+ in the presence of maintained camp-kinase dependent phosphorylation is not edemagenic. these antagonistic effects of ca 2+ and camp in endothelial permeability regulation have recently been reviewed by moore et al. [104] . many reports documented increases in endothelial permeability involving exotoxins like s. aureus alpha-toxin and p. aeruginosa cytotoxin [105, 106] or endotoxins [107] . for example, p. multocida toxin has been shown to activate rho/rho kinase, which inactivates mlc phosphatase. the resulting increase in mlc phosphorylation caused endothelial cell retraction and a rise in endothelial permeability [51] . lps induced increases in paracellular permeability by caspase activated cleavage of adherens junction proteins [108] . counteracting lipid peroxidation during lps activation may inhibit increases in permeability [109] . tnf-a stimulated endothelial cadherin complex is disrupted in a proteasome dependent manner [110] . the resulting increase in permeability has been reported to lower camp and activate phosphediesterase ii and iv [111] . h 2 o 2 induced hyperpermeability of porcine pulmonary endothelial cells has been reported to be effectively reduced by cgmp elevating drugs including phosphodiesterase ii inhibition or no˙donators [112] . the electroneutral na-k-cl cotransport system is thought to function in the maintenance of a selective permeability. il-1, tnf-a and lps upregulate the expression of a bumetanide-sensitive na-k-cl cotransporter subtype in huvec and in murine lung and kidney endothelial cells [113] . septic rats show different increases in albumin flux accross several endothelial beds [114] . increases in venular permeability have been shown to be preventable by the antioxidants n-acetyl-cystein or tirilazad mesylate in e. coli infused rats [115, 116] . il-10, an antiinflammatory cytokine not produced by endothelial cells, was shown to participate as an inhibitor of endothelial permeability induced by lps in mice [117] . clinically, increases in endothelial permeability may be obvious in many septic patients, but only recently venous congestion plethysmography showed a selectively elevated filtration capacity as a measure of endothelial dysfunction in septic patients [118] . which of the many pathways of increased permeability might be turned on and whether it persists remains unknown. in principle endothelial cells are believed to be anticoagulatory by virtue of their surface expression of glycosaminoglycan-antithrombin iii complex, thrombomodulin, heparin releasable tissue factor pathway inhibitor and production of adenosine by ecto-adpases, their secretion of protein s, prostacyclin and no˙. no˙production was shown to participate in heparan sulfate preservation in porcine aortic endothelial cells [119] and may be responsible for prostacyclin secretion by activating cyclooxygenase-1 under resting conditions [120] . endothelial release of plasminogen activator (t-pa) and plasminogen activator inhibitor 1 (pai-1) may determine the fibrinolytic potential of plasma. endothelial cells specifically bind coagulation factors xii, iia, ix, viia and xa. xa was shown to bind to endothelial effector cell protease receptor-1 and thereby cause release of no˙, il-6, il-8, mcp-1 and functional upregulation of icam-1, e-selectin and vcam-1 [121] . when endothelium is perturbed by physical or chemical factors transformation to a prothrombotic surface is invariably seen in in vitro models. thrombomodulin surface expression on huvec can easily be downregulated by lps, il-1 and tnf-a and upregulated by increasing camp [122] . a tnf-a induced decrease in surface thrombomodulin has been suggested via activation of phosphodiesterase ii and iv thereby decreasing camp in baec [111] . vascular endothelial growth factor may counteract il-1, tgf-b and lps induced suppression of both thrombomodulin surface antigen and mrna [123] . adenosin nucleotides are released from damaged as well as lps stimulated and shear stressed huvec [124] . endothelial cells have atp diphosphohydrolase (cd 39) on their surface to degrade atp via adp and amp to adenosine. adenosine is known to have antiaggregatory properties due to stimulation of prostacyelin and no˙production. however, activating endothelial cells with tnf-a has been reported to cause loss of atp diphosphohydrolase activity, which was preventable in the presence of antioxidants [125] . tissue factor (tf) expression on endothelial cells by bacteria, lps, il-1 and tnf-a is well known. tissue factor pathway inhibitor (tfpi), a serine protease inhibitor of xa and xa/viia/tf complex on endothelial surfaces, which immediately blocks tissue factor activation, has been shown to be decreased under proinflammatory conditions. another counterbalancing mechanism includes shear stress in tnf-a stimulated huvec [126] . however, an anticipated increase in endothelial tissue factor expression has not convincingly been demonstrated in animal or human sepsis [127, 128] . fibrinolytic systems on endothelial surfaces are also believed to be altered in sepsis. increases in pai-1 has been reported after stimulation with il-1 or lps [129, 130] . however soluble pai-1 in septic patients was not found to be different from nonseptic patients [131] . once thrombin formation has occured, its cleavage of endothelial proteinase activated receptor (par) in turn may lead to secretion of il-8 and il-6 [132] , upregulation of icam-1 and vcam-1 [133] , relaxation via no˙production or to vasoconstriction by an as yet unidentified factor [134] . these data may demonstrate an interconnection between coagulation activation, inflammation and vasoregulation mediated by the endothelium. coagulation activation is clearly present in septic patients, however endothelial participation in this process is unclear. potent procoagulatorv sources may well include bacterial surfaces per se [135] or monocytes [128] . up to the late 60ies the endothelium was viewed as a passive organ, which at best was able to remove vasoactive hormones in the lung. in 1976-1978 sir john vane's group (noble laureate 1982) reported that endothelial cells can synthesize i series prostaglandins (like prostacyclin) and thereby relax arteries and inhibit platelet aggregation. however, whether pgi 2 regulates basal vascular tone is unclear. after a new technician used an unintended vessel preparation robert furchgott realized after a series of contradicting results that acetylcholine (ach) was no longer able to relax precontracted arteries when endothelium was removed and termed the nonprostanoid mediator an endothelium derived relaxing factor (edrf). in 1986 superoxide was shown to participate in vasoregulation. the presence of superoxide dismutase prolonged the action of edrf whereas addition of o 2 -˙i nactivated edrf. even direct effects of several reactive oxygen species have been suggested to be relevant in cerebral or coronary circulation. collectively robert furchgott, louis ignarro and ferrid murad received the noble laureate in 1998 for demonstrating that no˙is a major edrf. endothelial cells produce more relaxing factors which pharmacologically can be separated from nitric oxide action and these were termed endothelium derived hyperpolarizing factors (edhfs). these factors may particularly be important in coronary and gastrointestinal vessels. epoxyeicosatrienoic acids, anandamide, the endogenous ligand of cannabinoid receptors or simply the release of k + may constitute edhfs. conceptually shear may in larger vessels primarily determine production of no˙, whereas cyclic strain determines physiological edhf release. soon after the discovery of edrf endothelin-1 (et-1), a vasoconstricting peptide produced from endothelial cells was isolated. low doses of et-1 can induce no˙release and subsequent relaxation via et breceptors on endothelial cells. endothelin secretion is thought to occur abluminally leading to et a -receptor activation on smooth muscle cells and subsequent vasoconstriction. many other factors clearly contribute to endothelial control of vasoregulation by e.g. transcellular production of vasoconstricting prostanoids like thromboxane a 2 or prostaglandin h 2 or the enzymatic conversion of angiotensin i to angiotensin ii by angiotensin converting enzyme. a hallmark of sepsis is the heterogeneous pattern of vasoconstriction and vasodilatation in different organs, culminating in a fall in total peripheral vascular resistance concomitant with regional maldistribution of blood flow. vasoactive substances produced by the endothelium under experimental septic conditions are known to be altered by factors such as no˙, pgi 2 , angiotensin converting enzyme (ace) activity, endothelin and adrenomedullin. endothelium dependent vasoregulation has largely been studied in animal models (table 4 ). in 1985 endothelium dependent vasoregulatory failure was seen as a defect in reactive hyperemia related vasodilator release [136] and decreased dilatation of arterioles induced by ach [137] . in a rat model of cecal ligation and puncture decreased vasoconstriction was found after administration of norepinephrine in septic animals which was largely reversible by removal of the endothelium [138] . parker et al. [139] showed in explanted coronary arteries and aortas of guinea pigs treated with lps intraperitoneally for 16 h that endothelium dependent relaxation induced by acetylcholine and adp was depressed, whereas relaxation induced by substance p or receptor independent relaxations by ca 2+ -ionophore a23187 was unaffected. edrf release and bioactivity from explanted aortas of these animals was decreased after adp or ach stimulation, whereas a23187 induced edrf release was unaltered [140] . this group also demonstrated reduced adp and ach responses after 4 h of lps endotoxemia in guinea pigs and that adp may produce constricting thromboxane in septic animals [141] . in contrast, coronary arteries of rabbits treated for 5 weeks with low doses of lps stimulation with ach but not adp showed increased relaxation of explanted vessels [142] . wang et al. [143] isolated subepicardial arterioles from rats treated 48 h intraperitoneally with feces containing life e. coli and showed in a pressurized no-flow chamber that relaxation by alpha 2 agonist clonidine and adp was reduced in an endothelium dependent manner, which could be inhibited by the nos inhibitor lnma. also, in this model mesenteric arter-vol. 49, 2000 endothelial function in sepsis iolar relaxation after adp and clonidine was decreased, whereas in skeletal muscle these agonists caused vasoconstriction [144] . pulmonary arteries of rats treated with lps showed depressed endothelin-1 induced contractions which were even augmented in endothelium denuded vessels. the authors [145] concluded that a vasoconstrictor eicosanoid is produced in lps treated animals by pulmonary endothelium upon et-1 stimulation. swine infused with live p. aeruginosa showed no alteration in endothelium dependent bradykinin and endothelium independent nitroprusside relaxation, whereas ach induced relaxation was found to be reduced in explanted peripheral arteries [146] . chaudry's group investigated endothelium dependent relaxation in rats treated with cecal ligation and puncture. a time dependent alteration was demonstrated with increased ach induced vasorelaxation early after challenge whereas depressed vasodilatation after 5-20 h was found in explanted aortas with no alteration in nitroglycerine induced relaxation [147] . the decreased endothelium dependent response to ach was also found in superior mesenteric arteries and small intestinal arteries [148] . in the same model this group demonstrated a reduction of immunodetectable nos iii in explanted aortas [149] . porcine coronary arterioles incubated for 20 h with e. coli lps (100 mg/ml) decreased bradykinin induced edhf secretion [150] . carotid and coronary arteries from rabbits also showed decreases in edhf-release in an ex-vivo assay after treatment with lps, tnf-a and il-1 [151] . collectively these data indicate that the majority of sepsis models consistently show a disruption of receptor coupled relaxation mechanism leading to an intraendothelial signalling deficit. to quantify endothelial function in humans several methods are available. endothelium dependent relaxation after pharmacological stimulation or flow dependent relaxation after vessel obstruction are frequently quantified by high resolution ultrasound techniques [152] , alternatively flow and size can be determined angiographically. however, definite functional measurements in human sepsis are scarce. endothelium dependent relaxation has been investigated in isolated superficial hand veins of healthy volunteers after lps exposure. reduced vasorelaxation by bradykinin and arachidonic acid in noradrenalin precontracted vessels were noted which persisted for more than 2 days [153] . reactive hyperemia is believed to mainly test endothelial no˙production upon shear stress if vessel diameters and flow is monitored. implications from indirect measurements support an endothelial dysfunction in septic patients [154] [155] [156] . however, data on pharmacological stimulation of endothelium dependent relaxation in humans are currently not available. 192 t. volk +/-ec: presence or absence of endothelium; ne: norepinephrine; ach: acetylcholine; adp, adenosine diphosphate; sp, substance p; a23187, ca 2 + -ionophore; cox, cycloogygenase; snp, sodium nitroprusside; ntg, nitroglycerine; et, endothelin; bk, bradykinin; edhf, endothelium derived hyperpolarizing factor; pres., preserved. table 4 . endothelium dependent relaxation is impaired in animal sepsis models. a normal response to infection or other insults is a self limiting process that through temporal expression of regulators and effector molecules causes resolution. the failure to resolve the causative infection may lead to sepsis. cellular and animal models of sepsis using bacteria, endotoxins, exotoxins, cytokines or some peptides all consistently produce endothelial impairment which is usually regarded as dysfunctional. blocking the majority of pathways used by these inducing agents has often lead to the inhibition of such endothelial alterations. many aspects of these induced alterations can be expected to reveal exciting new pathways and complex interactions at various molecular and cellular level. the past has taught us that inhibitors of presumably activated pathways consistently failed to improve survival in septic patients. this has stimulated many researchers to reconcile the results of experimental and clinical models in sepsis. compared to activating pathways, considerably less is known about how an inflammatory response is endogenously counterregulated. cytokines induce a whole host of signal inhibiting proteins and endogenous counterregulating systems are just beginning to be elucidated. activation of endogenous counterregulatory systems may become the predominant feature of the so-called compensatory antiinflammatory response syndrome. endothelial responses to endogenously present antiinflammatory mediators have hardly been investigated in sepsis models. almost all endothelial cell studies in sepsis indicated that an imbalance in reactive oxygen species production is associated with the above described dysfunctions. animal studies in which endothelial function could be improved pharmacologically also consistently indicate that reversal of imbalanced reactive oxygen production may be a common link (table 4 ). it seems that at times an adequate production of nitric oxide is lacking whereas superoxide and/or derivatives are overproduced. however, as endothelial functional measurements in septic humans become available, we will hopefully get a clearer picture of what might happen in our patients. [199] clp, cecal ligation and puncture; lpo, lipid peroxidation; ros, reactive oxygen species. table 5 . treatment of endothelial vasoregulatory dysfunction in animal sepsis models. bacterial pathogens isolated from patients wit bloodstream infection: frequencies of occurrence and antimicrobial susceptibility patterns from the sentry antimicrobial surveillance program (united states and canada, 1997) staphylococcus aureus infections internalization of staphylococcus aureus by endothelial cells induces apoptosis staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu genetic inactivation of the extracellular cysteine protease enhances in vitro internalization of group a streptococci by human epithelial and endothelial cells interaction of viable group a streptococci and hydrogen peroxide in killing of vascular endothelial cells group b streptococci invade endothelial cells: type iii capsular polysaccharide attenuates invasion invasion of brain microvascular endothelial cells by group b streptococci streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor pneumococcal trafficking across the blood-brain barrier. molecular analysis of a novel bidirectional pathway two distinct phospholipases c of listeria monocytogenes induce ceramide generation, nuclear factor-kappa acivation, and e-selectin expression in human endothelial cells internalin b is essential for adhesion and mediates the invasion of listeria monocytogenes into human endothelial cells activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble cd14 interaction of neisseria maningitidis with the components of the blood-brain barrier correlates with an increased expression of pilc the ndomain of the human cd66a adhesion molecule is a target for opa proteins of neisseria meningitidis and neisseria gonorrhoeae human microvascular endothelial tissue culture cell model for studying pathogenesis of brazilian purpuric fever pseudomonas aeruginosa selective adherence to and entry into human endothelial cells endothelial function in sepsis 193 cincomitant endosome-phagosome fusion and lysis of endosomal membranes account for pseudomonas aeruginosa survival in human endothelial cells endothelial cell glcnac beta 1-4glcnac epitopes for outer membrane protein a enhance traversal of escherichia coli across the blood-brain barrier escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10 characterization of a strain of chlamydia pneumoniae isolated from a coronary atheroma by analysis of the omp1 gene and biological activity in human endothelial cells chlamydia species infect human vascular endothelial cells and induce procoagulant activity signal transduction pathways activated in endothelial cells following infection with chlamydia pneumoniae bartonella (rochalimaea) quintana endocarditis in three homeless men bartonella quintana invades and multiplies within endothelial cells in vitro and in vivo and forms intracellular blebs interaction of bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome nf-kappa b-dependent inhibition of apoptosis is essential for host cellsurvival during rickettsia rickettsii infection rickettsia conorii infection enhances vascular cell adhesion molecule-1-and intercellular adhesion molecule-1-dependent mononuclear cell adherence to endothelial cells il-6 and il-8 production from cultured human endothelial cells stimulated by infect on with rickettsia conorii via a cell-associated il-1 alpha-dependent pathway the role of cd14 in signaling mediated by outer membrane lipoproteins of borrelia burgdorferi integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells different classes of proteoglycans contribute to the attachment of borrelia burgdorferi to cultured endothelial and brain cells borrelia burgdorferi upregulates the adhesion molecules e-selectin, p-selectin, icam-1 and vcam-1 on mouse endothelioma cells in vitro characterization of plasmodium falciparum-infected erythrocyte and p-selectin interaction under flow conditions intercellular adhesion molecule-1 and cd36 synergize to mediate adherence of plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells pecam-1/cd31, an endothelial receptor for binding plasmodium falciparum-infected erythrocytes candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells secreted aspartyl proteinases and interactions of candida albicans with human endothelial cells dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis adhesion molecule expression and lymphocyte adhesion to cerebral endothelium: effects of measles virus and herpes simplex 1 virus measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro effects of viral activation of the vessel wall on inflammation and thrombosis cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins ebola virus inhibits induction of genes by double-stranded rna in endothelial cells human endothelial cell activation and mediator release in response to the bacterial exotoxins escherichia coli hemolysin and staphylococcal alpha-toxin infection by verocytotoxin-producing escherichia coli alpha toxin from clostridium perfringens induces proinflammatory changes in endothelial cells brain capillary endothelial cells express mbec1, a protein that is related to the clostridium perfringens enterotoxin receptors phospholipase c and perfringolysin o from clostridium perfringens upregulate endothelial cellleukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells glucosylation of small gtp-binding rho proteins disrupts endothelial barrier function pasteurella multocida toxin increases endothelial permeability via rho kinase and myosin light chain phosphatase evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome the listerial exotoxins listeriolysin and phosphatidylinositol-specific phospholipase c synergize to elicit endothelial cell phosphoinositide metabolism lipoteichoic acid-induced neutrophil adhesion via e-selectin to human umbilical vein endothelial cells (huvecs) endothelial and epithelial cells do not respond to complexes of peptidoglycan with soluble cd14 but are activated indirectly by peptidoglycan-induced tumor necrosis factor-alpha and interleukin-1 from monocytes cytokines and endothelial cell biology cytokine regulation of endothelial cell function: from molecular level to he bedside pseudomonas siderophore pyochelin enhances neutrophil-mediated endothelial cell injury superoxide dismutase-dependent, catalase-sensitive peroxides in human endothelial cells infected by rickettsia rickettsii superoxide release from interleukin-1b-stimulated human vascular cells: in situ electrochemical measuremeut lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells e-selectin expression in human endothelial cells by tnf-alpha-induced oxidant generation and nf-kappab activation superoxide responses of endothelial cells to c5a and tnf-alpha: divergent signal transduction pathways lactosylceramide mediates tumor necrosis factor-alpha induced intercellular adhesion molecule-1 (icam-1) expression and the adhesion of neutrophil in human umbilical vein endothelial cells effect of antioxidants on lipopolysaccharide-stimulated induction of mangano superoxide dismutase mrna in bovine pulmonary artery endothelial cells ambient but not incremental oxidant generation effects intercellular adhesion molecule 1 induction by tumour necrosis factor alpha in endothelium icam-1 and vcam-1 expression induced by tnf-alpha are inhibited by a glutathione peroxidase mimic glutathione peroxidase mimics prevent tnfalpha-and neutrophilinduced endothelial alterations pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells endothelial cells are activated by cytokine treatment to kill an intravascular parasite, schistosoma mansoni, through the production of nitric oxide escherichia coli endotoxin inhibits agonistmediated cytosolic ca2 + mobilization and nitric oxide biosynthesis in cultured endothelial cells expressional control of the "constitutive" isoforms of nitric oxide synthase (nos i and nos iii) inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide nitric oxide donor prevents hydrogen peroxide-mediated endothelial cell injury nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells protective effects of tetrahydrobiopterin against nitric oxide-induced endothelial cell death endothelial damage induced by nitric oxide: synergism with reactive oxygen species hydroxyl radical formation resulting from the interaction of nitric oxide and hydrogen peroxide halliwell b van d v. formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation lipopolysaccharide induces the antiapoptotic molecules, a1 and a20, in microvascular endothelial cells apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction nuclear factor-kappab: a pivotal transcription factor in chronic inflammatory diseases oxidant-sensitive and phosphorylation-dependent activation of nf-kappa b and ap-1 in endothelial cells endothelial activation by hydrogen peroxide. selective increases of intercellular adhesion molecule-1 and major histocompatibility complex class i adenovirus-mediated expression of a dominant negative mutant of p65/rela inhibits proinflammatory gene expression in endothelial cells without sensitzing to apoptosis role of nfkappab in the mortality of sepsis endothelial function in sepsis initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store chemokines and leukocyte traffic cd40 ligation induced phenotypic and functional expression of cd80 by human cardiac microvascular endothelial cells cd4+ t cells migrate into inflamed skin only if they express ligands for e-and p-selectin t helper cell subset ratios in patients with severe sepsis cutting edge: combined treatment of tnf-alpha and ifngamma causes redistribution of junctional adhesion molecule in human endothelial cells a new role for platelet-endothelial cell adhesion molecule-1 (cd31): inhibition of tcrmediated signal transduction monocytes stimulate expression of the bcl-2 family member, a1, in endothelial cells and confer protection against apoptosis negative regulation of inflammation by fas ligand expression on the vascular endothelium regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury neutrophil-derived 5¢-adenosine monophosphat promotes endothelial barrier function via cd73-mediated conversion to adenosine and endothelial a2b receptor activation neutrophil migration during endotoxemia expression of cgmp-dependent protein kinase i and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin signal transduction and regulation of lung endothelial cell permeability interaction between calcium and camp bacterial exotoxins and endothelial permeability for water and albumin in vitro effects of escherichia coli hemolysin on endothelial cell function endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction bacterial lipopolysaccharide disrupts endothelial monolayer integrity and survival signaling events through caspase cleavage of adherens junction proteins endotoxin-induced changes of endothelial cell viability and permeability: protective effect of a 21-aminosteroid endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-tocell junctions tnf modulates endothelial properties by decreasing camp role of nitric oxide and phosphodiesterase isoenzyme ii for reduction of endothelial hyperpermeability expression of the bumetanide-sensitive na-k-cl cotransporter bsc2 is differentially regulated by fluid mechanical and inflammatory cytokine stimuli in vascular endothelium endothelial barrier resistance in multiple organs after septic and nonseptic challenges in the rat n-acetylcysteine attenuates endotoxin-induced leukocyte-endothelial cell adhesion and macromolecular leakage in vivo effect of the 21-aminosteroid tirilazad mesylate on leukocyte adhesion and macromolecular leakage during endotoxemia endogenous interleukin-10 regulates hemodynamic parameters, leukocyte-endothelial cell interactions, and microvascular permeability during endotoxemia increased microvascular water permeability in patients with septic shock, assessed with venous congestion plethysmography (vcp) endothelial-derived nitric oxide preserves anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells does elevated nitric oxide production enhance the release of prostacyclin from shear stressed aortic endothelial cells? hypotension and inflammatory cytokine gene expression triggered by factor xa-nitric oxide signaling up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor increased release of atp from endothelial cells during acute inflammation loss of atp diphosphohydrolase activity with endothelial cell activation fluid shear stress attenuates tumor necrosis factoralpha-induced tissue factor expression in cultured human endothelial cells cell biology of tissue factor, the principal initiator of blood coagulation on behalf of the subcommittee on tissue factor pathway inhibitor (tfpi) of the scientific and standardization committee of the isth effects of lipopolysaccharide on the expression of fibrinolytic factors in an established cell line from human endothelial cells induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygencentered free radicals plasminogen: an important hemostatic parameter in septic patients potential mechanisms for a proinflammatory vascular cytokine response to coagulation activation thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression intercellular adhesion molecule-1 cd54) and vascular cell adhesion molecule-1 (vcam-1; cd106) dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity activation of the contact-phase system on bacterial surfaces -a clue to serious complications in infectious diseases reactive hyperemic responses of single arterioles are attenuated markedly after intestinal ischemia, endotoxemia and traumatic shock: possible role of endothelial cells failure of microscopic metarterioles to elicit vasodilator responses to acetylcholine, bradykinin, histamine and substance p after ischemic shock, endotoxemia and trauma: possible role of endothelial cells vascular endothelium contributes to decreased aortic contractility in experimental sepsis selective inhibition of endotheliumdependent vasodilator capacity by escherichia coli endotoxemia release of edrf and no in ex vivo perfused aorta: inhibition by in vivo e. coli endotoxemia inhibition of endothelium-dependent vasodilation by escherichia coli endotoxemia chronic endotoxemia and endothelium-dependent vasodilation in coronary arteries chronic septicemia alters alpha-adrenergic mechanisms in the coronary circulation mesenteric and skeletal muscle microvascular responsiveness in subacute sepsis contraction to endothelin-1 in pulmonary arteries from endotoxin-treated rats is modulated by endothelium pulmonary artery endothelial cell function in swine pseudomonas sepsis nitric oxide. to block or enhance its production during sepsis? endothelium-dependent relaxation is depressed at the macro-and microcirculatory levels during sepsis endothelial nitric oxide synthase is downregulated during hyperdynamic sepsis attenuation of endothelium-dependent hyperpolarizing factor by bacterial lipopolysaccharides proinflammatory mediators chronically downregulate the formation of the endothelium-derived hyperpolarizing factor in arteries via a nitric oxide/cyclic gmp-dependent mechanism technical aspects of evaluating brachial artery vasodilatation using high-frequency ultrasound local venous responses to endotoxin in humans reactive hyperemia in patients with septic conditions microvascular function and rheologic changes in hyperdynamic sepsis peripheral vascular tone in sepsis decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction plasma antioxidant potential in severe sepsis: a comparison of survivors and nonsurvivors complement activation and polymorphonuclear neutrophil leukocyte elastase in sepsis correlation with severity of disease xanthine oxidase activity and free radical generation in patients with sepsis syndrome ascorbyl radical formation in patients with sepsis: effect of ascorbate loading the effects of intravenous antioxidants in patients with septic shock nitrogen oxide levels in patients after trauma and during sepsis evidence of increased nitric oxide production in patients with the sepsis syndrome nitrite/nitrate oxide (nox) and cytokine levels in patients with septic shock l-arginine: nitric oxide pathway in endotoxemia and human septic shock relationship between circulating levels of calcitonin gene-related peptide, nitric oxide metabolites and hemodynamic changes in human septic shock assessment of inflammatory cytokines, nitrate/nitrite, type ii phospholipase a2, and soluble adhesion molecules in systemic inflammatory response syndrome plasma nitrite and nitrate concentrations and multiple organ failure in pediatric sepsis endothelial function in sepsis measurements of total plasma nitrite and nitrate in pediatric patients with the systemic inflammatory response syndrome activation of the l-arginine nitric oxide pathway in severe sepsis increased serum nitrite and nitrate concentrations in children with the sepsis syndrome circulating methemoglobin and nitrite/nitrate concentrations as indicators of nitric oxide overproduction in critically ill children with septic shock effect of l-name, an inhibitor of nitric oxide synthesis, on plasma levels of il-6, il-8, tnf alpha and nitrite/nitrate in human septic shock extensive tyrosine nitration in human myocardial inflammation: evidence for the presence of peroxynitrite clinical evidence of peroxynitrite formation in chronic renal failure patients with septic shock evidence for in vivo peroxynitrite production in human acute lung injury elevated von willebrand factor antigen is an early plasma predictor of acute lung injury in nonpulmonary sepsis syndrome increased plasma levels of soluble thrombomodulin in patients with sepsis and organ failure endothelial cell activity varies in patients at risk for the adult respiratory distress syndrome soluble e-selectin levels in sepsis and critical illness. correlation with infection and hemodynamic dysfunction die zirkulierenden adhäsionsmoleküle sicam-1 und sb-selectin bei patienten mit sepsis elevated circulating e-selectin, intercellular adhesion moleculc 1, and von willebrand factor in patients with severe infection increased circulating thrombomodulin in children with septic shock systemic endothelial activation occurs in both mild and severe malaria. correlating dermal microvascular endothelial cell phenotype and soluble cell adhesion molecules with disease severity increased plasma von willebrand factor in the systemic inflammatory response syndrome is derived from generalized endothelial cell activation blood levels of endothelin-1 and thrombomodulin in patients with disseminated intravascular coagulation and sepsis plasma levels of endothelial cell protein c receptor are elevated in patients with sepsis and systemic lupus or erythematosus: lack of correlation with thrombomodulin suggests involvement of different pathological processes demonstration of rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von willebrand factor in patients with mediterranean spotted fever influence of angiotensin-converting enzyme inhibitor enalaprilat on endothelial-derived substances in the critically ill mesenteric and skeletal muscle microvascular responsiveness in subacute sepsis influence of group b streptococci on piglet pulmonary artery response to bradykinin effects of antisense oligonucleotide to inos on hemodynamic and vascular changes induced by lps pentoxifylline maintains vascular endothelial cell function during hyperdynamic and hypodynamic sepsis a novel nonanticoagulant heparin prevents vascular endothelial cell dysfunction during hyperdynamic sepsis effect of endotoxin-enhanced hepatic reperfusion injury on endonthelium-dependent relaxation in rat aorta splanchnic vascular endothelial dysfunction in rat endotoxemia: role of superoxide radicals endothelial dysfunction in a rat model of endotoxic shock. importance of the activation of poly (adp-ribose) synthetase by peroxynitrite endothelial cell dysfunction in septic shock key: cord-023488-jf2xl3vl authors: le duc, james w.; nathanson, neal title: emerging viral diseases: why we need to worry about bats, camels, and airplanes date: 2016-02-12 journal: viral pathogenesis doi: 10.1016/b978-0-12-800964-2.00016-1 sha: doc_id: 23488 cord_uid: jf2xl3vl the emergence of a new viral disease is one of the most dramatic aspects of virology, which often receives widespread attention from the scientific community and the lay public. considering that the discipline of animal virology was established over 100 years ago, it may seem surprising that new virus diseases are still being discovered. how this happens is the subject of this chapter. one of the most dramatic aspects of virology is the emergence of new virus diseases, which often receives widespread attention from the scientific community and the lay public. considering that the discipline of animal virology was established over 100 years ago, it may seem surprising that new virus diseases are still being discovered. how this happens is the subject of this chapter. there are many recent books and reviews (see further reading) that list the plethora of determinants that can lead to the emergence of infectious diseases (table 1) . in this chapter, we concentrate on those determinants that relate to viral pathogenesis and deal only briefly with the many societal and environmental factors that can be instrumental in disease emergence. in some instances, the "emergence" of a viral disease represents the first identification of the cause of a well-recognized disease. an example is la crosse virus, a mosquitotransmitted bunyavirus that was first isolated from a fatal case of encephalitis in 1964. the isolation of the causal agent and the development of serological tests made it possible to distinguish la crosse encephalitis from the rubric of "arbovirus encephalitis, etiology unknown." since that time, about 100 cases have been reported annually, without any significant increase since the 1970s. it appears that the emergence of this "new" disease reflected only the newfound ability to identify this etiologic entity, rather than any true change in its occurrence. hantavirus pulmonary syndrome is another example of the "emergence" of an existing but previously unrecognized disease. in 1993, in the four corners area of the southwestern united states, there occurred a small outbreak of cases of acute pulmonary illness with a high mortality. epidemiologic and laboratory investigation rapidly identified the causal agent, a previously unknown hantavirus, now named sin nombre virus (snv). snv is an indigenous virus of deer mice (peromyscus maniculatus) that are persistently infected and excrete the virus. apparently deer mice produce virus-infected excreta and, when they infest human dwellings, aerosolized fomites can result in occasional human infections. the 1993 outbreak is thought to reflect a transient rise in deer mouse populations associated with an unusual crop of pine nuts, a major food source for these rodents. the recognition of snv soon led to the discovery of other heretofore unrecognized hantaviruses in north, central and south america, many of which also cause serious human disease. on occasion, a virus that is already widespread in a population can emerge as a cause of epidemic or endemic disease, due to an increase in the ratio of cases to infections. such an increase can be caused by either an increase in host susceptibility or enhancement of the virulence of the virus. although counterintuitive, there are some dramatic instances of such phenomena. increase in host susceptibility. poliomyelitis first appeared as a cause of summer outbreaks of acute infantile paralysis in sweden and the united states late in the nineteenth century (figure 1 ). isolated cases of infantile paralysis had been recorded in prior centuries, and an eygptian tomb painting indicates that poliomyelitis probably occurred in early recorded history. why then did poliomyelitis emerge abruptly as an epidemic disease? when personal hygiene and public health were primitive, poliovirus circulated as a readily transmitted enterovirus, and most infants were infected while they still carried maternal antibodies (up to 9-12 months of age). under these circumstances, the virus produced immunizing infections of the enteric tract, but passively acquired circulating antibodies prevented invasion of the spinal cord. with the improvement of personal hygiene in the late-nineteenth century, infections were delayed until 1-3 years of age, after the waning of maternal antibodies. infections now occurred in susceptible children, resulting in outbreaks of infantile paralysis. this reconstruction is supported by seroepidemiological studies conducted in north africa in the 1950s, when epidemic poliomyelitis first emerged in this region. increase in viral virulence. viruses may undergo sudden increases in virulence resulting in emergence of dramatic outbreaks. an outbreak of lethal avian influenza in pennsylvania in 1983 is one documented example. in eastern pennsylvania, avian influenza appeared in chicken farms early in 1983, but the virus was relatively innocuous and most infections were mild. however, in the fall of that year a fatal influenza pandemic spread rapidly through the same farms. when viruses from the spring and fall were compared, it appeared that both isolates had almost identical genomes. the fall virus had acquired a single point mutation in the viral hemagglutinin that facilitated the cleavage of the hemagglutinin. the virus could now replicate outside the respiratory tract, markedly increasing its virulence (discussed in chapter 7, patterns of infection). this point mutation led to the emergence of an overwhelming epizootic, which was only controlled by a widespread slaughter program involving millions of birds. similar outbreaks of avian influenza have occurred subsequently in other countries. a virus that is endemic in a population may "fade out" and disappear, because the number of susceptibles has fallen below the critical level required for perpetuation in that population. if the population is somewhat isolated, the virus may remain absent for many years. during this interval, there will be an accumulation of birth cohorts of children who are susceptible. if the virus is then reintroduced, it can "reemerge" as an acute outbreak. in the years 1900-1950, iceland had a population of about 200,000, which was too small to maintain measles virus, and measles periodically disappeared. when travelers to iceland reintroduced the virus, measles reemerged in epidemic proportions. on occasion, a virus can enter and spread in a region where it had never previously circulated, leading to the emergence of a disease new to that locale. a dramatic example is afforded by the emergence of west nile virus (wnv) in the united states, beginning in 1999 ( figure 2 ). wnv, like most arboviruses, is usually confined to a finite geographic area, based on the range of its vertebrate reservoir hosts and permissive vectors. in an unusual event, wnv was imported into new york city, probably by the introduction of infected vector mosquitoes that were inadvertent passengers on a flight from the middle east, where the virus is enzootic. this hypothesis was supported by the finding that the genomic sequence of the new york isolates was closely related to the sequence of contemporary isolates from israel. some american mosquito species were competent vectors for wnv, and certain avian species such as american crows were highly susceptible. as a result, west nile encephalitis emerged as a significant disease new to the united states. over a period of several years wnv spread across the continent, finally reaching the west coast and many areas in latin america. chikungunya virus (chikv), another mosquito-borne arbovirus, has been endemic for many years in regions of africa and asia where it periodically caused outbreaks of a febrile illness associated with severe arthritis and arthralgia. in 2005-2006, chikv caused a large epidemic on the island of reunion in the indian ocean, apparently associated with a mutation that allowed the virus to be more efficiently transmitted by vector mosquitoes. chikungunya subsequently spread to india and elsewhere in asia, followed by the americas. as of november 2014, transmission of chikv had been documented in 40 countries or territories in the caribbean, central, south and north america, resulting in nearly one million suspected cases each year. it appears that chikv may become established in the new world, where virtually the entire human population currently lacks immunity. zoonotic infections of animals that can be transmitted to humans are a major cause of emerging virus diseases of humans. these viruses are transmitted by direct contact, by virus-laden droplets or aerosols, or by insect vectors. all zoonotic viruses have one or more animal reservoir hosts, which play an important role in the epidemiological dynamics of human infections. although many zoonotic viruses can be transmitted to humans on occasion, their relative ability to spread from human to human determines whether or not they emerge as significant new virus diseases of mankind (table 2 ). most zoonotic viruses that are transmitted to humans cannot be spread directly from person to person, so humans are considered to be "dead-end hosts." one familiar example is rabies, which is enzootic in several animal hosts, such as dogs, skunks, foxes, raccoons, and bats. humans are infected by bite of a rabid animal or by aerosol exposure (in caves with roosting bats). several zoonotic arenaviruses, such as lassa, machupo (bolivian hemorrhagic fever), and junin (argentine hemorrhagic fever) viruses, are likely transmitted from the reservoir host (wild rodents) by inhalation of contaminated aerosols. there are more than 500 viruses-belonging to several virus families-that are also classified as arboviruses (arthropod-borne viruses), based on a vertebrate-arthropod maintenance cycle in nature. arboviruses replicate in both the vertebrate host and the arthropod vector (mosquitoes, ticks, sandflies, and others), and transmission occurs when the vector takes a blood meal. typically, arboviruses have only a few vertebrate hosts and are transmitted by a narrow range of arthropods. humans are not the reservoir vertebrate hosts of most arboviruses, but can be infected by many of these viruses, if they happen to be bitten by an infected vector. in most instances, arbovirus-infected humans are dead-end hosts for several reasons. many arthropod vectors competent to transmit a zoonotic arbovirus prefer nonhuman hosts as a blood source, reducing the likelihood of transmission from human to vector. also, infected humans are usually not sufficiently permissive to experience a high titer viremia, so they cannot serve as effective links in the transmission cycle. there are only a few exceptions: in urban settings, dengue, urban yellow fever, oropouche, and chikungunya viruses can be maintained by an arthropod vector-human cycle. as table 2 shows, a few zoonotic viruses can be transmitted directly from human to human, at least for a few passages, and can emerge as the cause of outbreaks involving a few to several hundred cases. since many viruses in this group cause a high mortality in humans, even a small outbreak constitutes a public health emergency. these viruses belong to many different virus families, and there is no obvious biological clue why they should be able to spread from human to human, in contrast to other closely related viruses. typically, infections are mainly limited to caregivers or family members who have intimate contact with patients, often in a hospital setting. however, transmission is marginal, so that most outbreaks end after fewer than 5-10 serial transmissions, either spontaneously or due to infection-control practices. in the history of modern virology (the last 50 years) there are very few documented instances where zoonotic viruses have established themselves in the human population and emerged as new viral diseases of mankind (table 2 ). most viruses have evolved to optimize their ability to be perpetuated within one or a few host species, and this creates what is sometimes called the "species barrier." in most instances, a virus must undergo some adaptive mutations to become established in a new species. sars coronavirus. in november, 2002, an outbreak of severe acute respiratory disease (sars) began in guangdong province, in southeast china near the hong kong border. in retrospect, the first cases were concentrated in food handlers, who then spread the virus to the general population in that region. although not recognized immediately as a new disease, the outbreak continued to spread both locally and in other parts of china. in february, 2003, a physician who had been treating likely sars patients, traveled to hong kong, where he transmitted sars to a large number of contacts in a hotel. these persons, in turn, spread the infection to singapore, taiwan, vietnam, and canada, initiating a global pandemic that eventually involved almost 30 countries. from patient samples, several research groups isolated a novel coronavirus, which has been named the sars coronavirus (sars cov). clearly, this virus is new to the human population, and there is circumstantial evidence that it was contracted from exotic food animals that are raised for sale in restaurants in guangdong province. recent studies suggest that horseshoe bats (genus rhinolophus) may be the reservoir hosts and palm civets, consumed as food in china, may be the intermediary hosts for sars cov. sars cov went through a large number of human passages (perhaps 25) before being contained by primary control measures, such as respiratory precautions, isolation, and quarantine. the virus has been eliminated from the human population, but the 2003 outbreak showed that it could be maintained by human-to-human transmission. from that perspective, it is potentially capable of becoming an indigenous virus of humans. since many coronaviruses infect the respiratory system and are transmitted by the respiratory route, the sars virus did not have to undergo any change in its pathogenesis. however, the virus did have to replicate efficiently in cells of the human respiratory tract and it is unknown whether this required some adaptive mutations from the virus that is enzootic in its reservoir hosts. middle eastern respiratory syndrome. mers is an acute respiratory disease of humans that was first recognized in 2012, when a novel coronavirus (mers-cov) was isolated from two fatal human cases in saudi arabia and qatar. since that time, more than 1400 clinical cases of mers have been recognized, the great majority in saudi arabia but also in most of the other countries in the arabian peninsula and beyond ( figure 3 ). hospitalized cases of mers are characterized by an acute respiratory disease syndrome (ards) with a fatality rate over 25%. however, evolving evidence suggests that there may be many additional human infections with little or no associated illness. what is the origin of this new disease? based on fragmentary data now available, it appears that mers-cov is likely to be an enzootic virus of one or more species of bats. camels are probably an intermediary host, and humans in close contact with camels can acquire infection. also, mers-cov can spread from human to human, particularly to caretakers or others in close contact with acutely ill patients. however, there are many unknowns in this speculative reconstruction: how do camels become infected? are camels the main source of human infections? what has caused this disease to emerge in 2012, or had it preexisted but was just recognized at that time? is this an evolving outbreak, and if so, what is driving it? several relevant facts have been ascertained from basic studies. mers-cov replicates well in cell cultures obtained from many species, including bats and humans. the pantropic nature of this virus is explained in part by its cellular receptor, dipeptidyl peptidase 4 (dppt4), a widely distributed cell surface molecule. this would facilitate the transmission of this zoonotic infection from bats to camels to humans. of interest, mers-cov infects type ii alveolar pneumocytes while sars cov uses a different cell receptor (angiotensin-converting enzyme 2, ace2) and infects type i alveolar pneumocytes. however, both viruses appear to cause a similar ards. type a influenza virus. genetic evidence strongly implicates avian and porcine type a influenza viruses as the source of some past pandemics of human influenza. it appears that new epidemic strains are often derived as reassortants between the hemagglutinin (and the neuraminidase in some cases) of avian influenza viruses with other genes of existing human influenza viruses. the new surface proteins provide a novel antigenic signature to which many humans are immunologically naã¯ve. the human influenza virus genes contribute to the ability of the reassortant virus to replicate efficiently in human cells. it is thought that reassortment may take place in pigs that are dually infected with avian and human viruses. currently, there is concern that a new pandemic strain of type a influenza could emerge as a derivative of highly virulent avian h5n1 or h7n9 influenza viruses now causing epidemics in domestic chickens in southeast asia. there have been several hundred documented human infections with each of these avian viruses in recent years-mainly among poultry workers-with significant mortality. however, few if any of these infections have spread from human to human, perhaps because the infecting avian virus has not undergone reassortment with a human influenza virus. a critical determinant is that avian influenza viruses preferentially attach to î±2-3 sialic acid receptors while human viruses attach to î±2-6 sialic acid receptors. the 1918 pandemic of influenza is presumed to be an example of a zoonotic influenza virus that crossed the species barrier and became established in humans where it caused an excess mortality estimated at 20-40 million persons. recent viral molecular archaeology has recovered the sequences of the 1918 h1n1 influenza virus from the tissues of patients who died during the epidemic. all of the genes of the reconstructed virus are avian in origin, but it is unknown whether the virus underwent mutations that enhanced its ability to be transmitted within the human population. the reconstructed hemagglutinin of the 1918 virus has been inserted into recombinant influenza viruses, and-in mice-markedly increases the virulence of primary human isolates of influenza virus (table 3) , but the full virulence phenotype appears to require many of the avian influenza genes. several physiological factors play a role in disease enhancement, including increased replication in pulmonary tissues and an enhanced ability to stimulate macrophages to secrete pro-inflammatory cytokines which, in turn, cause severe pneumonitis. human immunodeficiency virus. hiv has emerged as the greatest pandemic in the recent history of medical science. modern methods have made it possible to reconstruct its history in great detail (see chapter 9, hiv/aids). a provisional reconstruction of the sequence of events is summarized in figure 4 . the emergence of hiv may be divided into two phases: what was the zoonotic source of hiv and when did it cross into humans? and when did hiv spread from the first human cases to become a global plague? there have been several transmissions of simian immunodeficiency virus (siv) to humans (sharp and hahn, 2011) , and this account will focus on hiv-1 which appears to have been transmitted to humans in at least four separate instances, identified by individual hiv-1 lineages called groups (m, n, o, p). of these, the most important was the m group of hiv-1, which has been responsible for the vast majority of human infections. furthermore, hiv-1 is most closely related to sivcpz, the siv strain infecting two subpopulations of chimpanzees. different segments of the sivcpz genome, in turn, are closely related to genome segments of two sivs of african monkeys, red-capped monkeys and cercopithecus monkeys. it is hypothesized that chimpanzees, which regularly kill and eat monkeys, were infected during consumption of their prey; and that a recombination event produced sivcpz, which was derived from parts of the genomes of the two acquired monkey viruses. it is speculated that a further transmission from chimpanzees may have occurred during the butchering of nonhuman primates, which occurs in rural africa ( figure 5) . amazingly, genomic similarities tentatively map the substrain of sivcpz that is the ancestor of the m group of hiv-1, to chimpanzees in the southeastern corner of cameroon, a small country in west africa. using a sequence analysis to compare diversity within current isolates, the common parent of the m group can be reconstructed. a molecular clock, derived from dated isolates, indicates that this virus was transmitted to humans during the period 1910-1930. the period from 1930 to 1980 is a mystery (pepin, 2011) , but there are fragmentary data suggesting that the virus persisted as a rare and unrecognized infection in residents of jungle villages in west africa during this time. it has also been proposed that the reuse of unsterilized needles-a frequent practice during the period of colonial rule-could have inadvertently helped to spread the virus. starting about 1980, the virus began to spread more rapidly. it appears that accelerated spread began in the region centered on kinshasa (previously leopoldville) in the democratic republic of the congo (previously the belgian congo, then zaire) and brazzaville, just across the congo river in congo. transmission was exacerbated by the chaos in postcolonial zaire. during the period 1985-2004, hiv infection spread widely in africa as shown in figure 6 . in the countries worst affected, the prevalence of infection among adults aged 15-49 years reached levels higher than 30%. the rapid spread was driven by many factors among which were: (1) a high frequency of concurrent sexual contacts in some segments of the population and the hidden nature of sexual networks; (2) the long asymptomatic incubation period during which infected individuals able to transmit the virus were sexually active; (3) the spread along commercial routes of travel within africa; (4) the failure of health systems to concomitantly with the spread of hiv in africa, the m group of hiv-1 evolved into nine different subtypes (a-d, f-h, j, k), based on sequence diversity. during the spread within africa, there were population bottlenecks that resulted in the predominance of different m group subtypes in different regions. subtype c is most frequent in southern africa, and subtypes a and d are most frequent in eastern africa. during the 1980s, hiv also spread globally, although prevalence rates did not reach the levels seen in some african countries. subtype b is dominant in the western hemisphere and europe, while subtype c is most frequent in india and some other asian countries. this implies that each of these regional epidemics was initiated speculative reconstruction of events following the transmission of sivcpz to humans. this reconstruction is based on data in hahn (2011), pepin (2011) . bushmeat is part of the diet in rural africa. photograph courtesy of billy karesh (2015). by a small number of founder strains of hiv-1, improbable though that may seem. thirty years after its appearance as a global disease, almost 40 million persons have died, and there are more than 30 million people living with hiv/aids. although the global incidence of hiv has fallen slightly since 2010, there are still more than two million new infections each year (2014). ebola hemorrhagic fever. the ebola pandemic of 2014-15 is the most recent emerging virus disease that has riveted the attention of the world. where did it come from? why did it cause a pandemic? why did the international health community fail to control it? where will it end? these questions are discussed below. ebola is a filovirus indigenous to africa that is maintained in one or more reservoir species of wild animals, among which bats are a likely host. the transmission cycle is not well documented but it is thought that humans get infected, either by direct contact with bats, or while slaughtering infected wild animals who may act as intermediate hosts. human-to-human spread can then occur. the pathogenesis of ebola virus infection may be briefly summarized. presumably ebola enters the human host via the mucous membranes or cuts in the skin. the virus infects mononuclear cells including macrophages and dendritic cells and traffics to lymph nodes, whence it spreads to target organs including the liver, spleen, and adrenal glands. it causes a high titer viremia and a dysregulation of the innate immune system. clinically, ebola patients undergo severe vomiting and diarrhea with massive fluid losses and become very dehydrated, with a mortality that varies from 25% to 75%. in fatal cases, the infection of the liver leads to disseminated intravascular coagulopathy, a shock syndrome, and multiorgan failure, although the sequential details are not well understood. infection is transmitted between humans by contact with bodily fluids of patients, but not by aerosols. therefore, the virus is spread most frequently to caregivers or others who are in intimate contact with patients and their fomites. rituals associated with funerals and burial practices often serve to transmit the virus. ebola virus was first isolated in 1976 in an outbreak near the ebola river in the democratic republic of the congo (then called zaire) and almost concurrently in a second outbreak in southern sudan. viruses recovered from these two outbreaks were subsequently shown to be different and are now known as ebola-zaire and ebola-sudan. since that time there have been more than 25 individual outbreaks of ebola disease, mainly in central africa. past outbreaks have been controlled by use of protective garments by caregivers and quarantine of infected or potentially infected contacts. these controlled outbreaks have been limited to no more than â�¼5 serial human-to-human transmissions, and mainly ranged from about 25 to 300 cases. in december, 2013, an ebola-zaire outbreak began in guinea, west africa. it appears that the initial case was in an infant who may have been infected by contact with bats. the outbreak then spread to two contiguous countries, liberia and sierra leone. by the fall, 2014, the epidemic had become a catastrophe, and was raging out of control in several parts of these three countries. although the data are incomplete, it is estimated that there have been at least 20,000 cases (with at least 50% mortality) through february, 2015 (figure 7) . the infection has spread to nigeria, senegal, mali, the united states, and a few european countries, but these invasions have so far been controlled, with limited secondary cases. a global effort to control this pandemic was initiated, with participation from doctors without borders, the red cross, other nongovernment organizations, the world health organization, and the u.s. centers for disease control and prevention. as of march, 2015, it appeared that the epidemic was coming under control. aggressive border screening, both on exit from the affected countries and on arrival at destinations, has limited spread by air travelers, but the porous land borders remain areas of concern. from an epidemiological viewpoint, why did this outbreak of ebola explode into a massive pandemic, in contrast to the many prior outbreaks that were limited to no more than a few hundred cases at most? the outbreak began in guinea and was mainly confined to that country for about 6 months before it spread to neighboring liberia and sierra leone (figure 8 ). during this first 6 months, incidence in guinea varied from a few to about 50 cases a week, and cases were concentrated in rural areas. from a public health viewpoint, this represented a missed opportunity to contain the outbreak. contributing to this omission were a combination of factors: a weak health system fragmented by social disruption, a failure of local health authorities to recognize or respond to the outbreak, the failure of international health organizations such as who to take aggressive action, and local societal norms that brought family and friends into close contact with ebola victims, during their illness and at their funerals (washington post, october 5, 2014; cohen, 2014) . once ebola infections spread from rural villages to urban centers, the outbreak exploded. beginning in the fall of 2014, it was recognized that the pandemic was a global threat. in response, a number of countries and international organizations provided resources to africa, including building facilities, sending equipment, and recruiting personnel to work in the pandemic areas. a major effort was made to get the local population to temporarily change some of their normal social responses to illness and death, to reduce the spread within the population. combined with the efforts of the affected countries, these initiatives started to take hold about january, 2015. however, as of march, 2015, the epidemic was not yet terminated. in retrospect, the international community has acknowledged that it lacks a contingency plan to respond to global outbreaks wherever they may occur. futhermore, because ebola was a "neglected" disease, the tools to combat it had not been developed. the ebola pandemic has spurred crash programs to develop a rapid diagnostic test, drugs and antibodies for treatment, and a vaccine for prevention (product, 2015; mire et al., 2015) . canine parvovirus. cpv is another example of a disease that emerged due to the appearance of a virus new to its host species. in the late 1970s, a highly lethal pandemic disease appeared in the dog populations of the world. the etiologic agent was a parvovirus previously unknown in canines. the sequence of cpv is almost identical to that of feline panleukopenia virus (fpv), an established parvovirus of cats, which causes acutely fatal disease in kittens. cpv has a few point mutations that distinguish it from fpv, and these mutations permit cpv to bind to and infect canine cells, a property not possessed by fpv. it is presumed that these mutations permitted the emergence of a new virus disease of dogs. many mammalian viruses have evolved with their hosts so that different members of a virus family are associated with each host species. furthermore, under natural circumstances, each member of the virus family usually "respects" the species barrier and does not cross into other species, although it spreads readily between individual animals within its host species. diverse mechanisms contribute to the species barrier, including host defenses and viral genes. for a zoonotic virus to establish itself in humans or another new species, it is probable that mutations are needed for full adaptation. this requirement is likely part of the explanation for the rarity of such events. one of the best-studied examples is the transmission of sivcpz, a virus of chimpanzees, to hiv, a human virus. several recent studies have shown that apobec3 and tetherin are two very important gate keepers for transmission of lentiviruses between different primate species (reviewed in sharp and hahn, 2011) . apobec3 proteins represent a powerful first line of defense, believed to be responsible for preventing the transmission of sivs from monkeys to chimpanzees and from monkeys to humans (etienne et al., 2013) . however, apobec3 proteins can be counteracted by the hiv/siv viral infectivity factor (vif), albeit generally in a species-specific fashion. thus, mutations in vif were required for monkey sivs to be able to infect chimpanzees and then humans (letko et al., 2013) . tetherin is a second line of defense, and only hivs that have evolved an effective tetherin antagonism have spread widely in humans (kluge et al., 2014) . different lentiviruses use different viral proteins to antagonize tetherin: hiv-1 group m uses the viral protein u (vpu); hiv-2 group a uses the envelope glycoprotein (env), and hiv-1 group o uses the negative regulatory factor (nef). in contrast, hiv-1 groups n and p, whose spread in the human population has remained very limited, are unable to counteract tetherin efficiently. turning to another virus, recent studies using a mouseadapted strain of ebola virus to infect a panel of inbred mice provided by the collaborative cross (see chapter 10, animal models) found striking variation in the response to ebola virus infection, from complete resistance to severe hemorrhagic fever with 100% mortality. this underlines the role of host genetic background as a determinant of susceptibility. consistent with this are comments of clinicians that there are unpredictable differences in the outcome of individual ebola cases. these studies suggest a dynamic relationship between host and pathogen that may determine when a virus can cross the species barrier and create a new virus disease of humankind. although difficult to document in a rigorous manner, it does appear that new virus diseases of humans (and perhaps of other species) are emerging at an increased tempo. there are a number of reasons for this trend (table 1) . the human population is growing inexorably, and is becoming urbanized even faster. as a result, there are an increasing number of large-crowded cities, which provide an optimal setting for the rapid spread of any newly emergent infectious agent. in the nineteenth century, it was noteworthy that someone could circumnavigate the globe in 80 days, but now it can be done in less than 80 h. however, the incubation period of viral infections (several days to several months) has stayed constant. someone can be infected in one location and-within a single incubation period-arrive at any other site on earth. this enhances the opportunity for a new human virus to spread as a global infection before it has even been recognized, markedly increasing the opportunity for the emergence of a new disease. the same dynamics also apply to viral diseases of animals and plants, which has important economic and social consequences for humankind. the sars pandemic of 2002-2003, described above, is an example of how rapidly and widely a new virus disease can emerge and spread globally. in this instance, it is extraordinary that the disease was brought under control-and eradicated from the human population-by the simple methods of isolation, quarantine, and respiratory precautions. although conceptually simple, a heroic effort was required for success. another example is the 2009 emergence of a novel h1n1 strain of influenza a virus that spread rapidly around the world before a vaccine could be produced. remote areas of the world are now being colonized at a high frequency, driven by population pressures and economic motives, such as the reclamation of land for agriculture or other uses, and the harvest of valuable trees and exotic animals. the construction of new dams, roads, and other alterations of the natural environment create new ecological niches. it is possible that this is the origin of urban yellow fever. yellow fever virus is an arbovirus maintained in a monkey-mosquito cycle in the jungles of south america and africa. humans who entered jungle areas became infected. when they returned to villages or urban centers, an urban cycle was initiated, where aedes aegypti mosquitoes-that are well adapted to the urban environment and preferentially feed on humansmaintained the virus. in the last 25 years, agriculture has undergone a dramatic evolution with the development of "agribusiness." food and food animals are now raised on an unprecedented scale and under very artificial conditions, where the proximity of many members of a single plant or animal species permits an infection to spread like wildfire. furthermore, increasing numbers of plants, animals, and food products are rapidly transported over large distances; we enjoy fresh fruits and vegetables at any time, regardless of the season. international shipment of plants and animals can import viruses into new settings where they may lead to the emergence of unforeseen diseases. one example is the 2003 outbreak of monkeypox that caused about 80 human cases in the united states. this was traced to the importation from africa of gambian giant rats as exotic pets; several rodent species in west africa appear to be reservoir hosts of this poxvirus. monkeypox spread from these animals to pet prairie dogs and from prairie dogs to their owners. because monkeypox causes infections in humans that resemble mild cases of smallpox, this outbreak was a major cause of public health concern. on occasion, a virus has been deliberately introduced into a susceptible population where it caused the emergence of a disease epidemic. for sport, rabbits were imported from europe into australia in the mid-nineteenth century. because of the absence of any natural predator the rabbits multiplied to biblical numbers, and threatened natural grasslands and agricultural crops over extensive areas of southern australia. to control this problem, myxomatosis virus was deliberately introduced in 1950 (fenner, 1983) . this poxvirus is transmitted mechanically by the bite of insects and is indigenous to wild rabbits in south america, in which it causes nonlethal skin tumors. however, myxomatosis virus causes an acutely lethal infection in european rabbits, and its introduction in australia resulted in a pandemic in the rabbit population. following the introduction of myxomatosis virus in 1950, co-evolution of both virus and host were observed. the introduced strain was highly virulent and caused epizootics with very high mortality. however, with the passage of time field isolates exhibited reduced virulence, and there was a selection for rabbits that were genetically somewhat resistant to the virus. strains of moderate virulence probably became dominant because strains of lowest virulence were less transmissible and strains of maximum virulence killed rabbits very quickly. concern has also been raised about disease emergence due to the deliberate introduction of viruses into either human or animal populations, as acts of bioterrorism. because of the shortage of human organs for transplantation recipients, there is considerable research on the use of other species-particularly pigs-as organ donors. this has raised the question whether known or unknown latent or persistent viruses in donor organs might be transmitted to transplant recipients. since transplant recipients are immunosuppressed to reduce graft rejection, they could be particularly susceptible to infection with viruses from the donor species. in the worst scenario, this could enable a foreign virus to cross the species barrier and become established as a new human virus that might spread from the graft recipient to other persons. the impetus to identify a new pathogenic virus usually arises under one of two circumstances. first, a disease outbreak that cannot be attributed to a known pathogen may set off a race to identify a potentially new infectious agent. identification of the causal agent will aid in the control of the disease and in prevention or preparedness for potential future epidemics. sars coronavirus, west nile virus, and sin nombre virus are examples of emergent viruses that were identified in the wake of outbreaks, using both classical and modern methods. alternatively, an important new virus may be discovered as a serendipitous by-product of research directed to a different goal, as was the case with hepatitis b virus (hbv). in this instance, a search for alloantigens uncovered a new serum protein that turned out to be the surface antigen (hbsag) of hbv, leading to the discovery of the virus. when a disease outbreak cannot be attributed to a known pathogen, and where classical virus isolation, propagation, and identification fail, molecular virology is required. hepatitis c virus (hcv), sin nombre and other hantaviruses, certain rotaviruses, and kaposi's sarcoma herpesvirus (hhv8) , are examples of emergent viruses that were first discovered as a result of molecular technologies. below, we briefly describe some methods of viral detection and identification. more detailed information and technical specifics can be found in several current texts. the first question that confronts the investigator faced with a disease of unknown etiology is whether or not it has an infectious etiology? evidence that suggests an infectious etiology is an acute onset and short duration, clinical similarity to known infectious diseases, a grouping of similar illnesses in time and place, and a history of transmission between individuals presenting with the same clinical picture. for chronic illnesses, the infectious etiology may be much less apparent and a subject for debate. faced with a disease that appears to be infectious, the next question is whether it is caused by a virus. a classical example that predates modern virology is the etiology of yellow fever. in a set of experiments that would now be prohibited as unethical, the yellow fever commission, working with the us soldiers and other volunteers in cuba in 1900, found that the blood of a patient with acute disease could transmit the infection to another person by intravenous injection. furthermore, it was shown that the infectious agent could pass through a bacteria-retaining filter and therefore could be considered a "filterable virus." virus isolation in cell culture and animals. the first step in identification of a putative virus is to establish a system in which the agent can be propagated. before the days of cell culture, experimental animals were used for this purpose. many viruses could be isolated by intracerebral injection of suckling mice, and some viruses that did not infect mice could be transmitted to other experimental animals. human polioviruses-because of their cellular receptor requirements-were restricted to old world monkeys and great apes; the virus was first isolated in 1908 by intracerebral injection of monkeys and was maintained by monkey-to-monkey passage until 1949 when it was shown to replicate in primary cultures of human fibroblasts. the modern era of virology (beginning about 1950) can be dated to the introduction of cultured cells as the standard method for the isolation, propagation, and quantification of viruses. there are now a vast range of cell culture lines that can be used for the isolation of viruses, and currently this is the first recourse in attempting to isolate a suspected novel virus. some viruses will replicate in a wide variety of cells but others are more fastidious and it can be hard to predict which cells will support their replication. it is also important to recognize that some viruses will replicate in cell culture without exhibiting a cytopathic effect. an important example is the identification of simian virus 40 (sv40). poliovirus was usually grown in primary cell cultures obtained from the kidneys of rhesus monkeys, but sv40 had escaped detection because it replicated without causing a cytopathic effect. when poliovirus harvests were tested in similar cultures prepared from african green monkeys, a cytopathic effect (vacuolation) was observed, leading to the discovery of sv40 virus in 1960. because inactivated poliovirus vaccine produced from 1955 to 1960 had been prepared from virus grown in rhesus monkey cultures, many lots were contaminated with this previously unknown virus, which inadvertently had been administered to humans. since that time, viral stocks and cell cultures have been screened to exclude sv40 and other potential virus contaminants. a number of methods are available to detect a noncytopathic virus that is growing in cell culture. these include visualization of the virus by electron microscopy, detection of viral antigens by immunological methods such as immunofluorescence or immunocytochemistry, the agglutination of erythrocytes of various animal species by virus bound on the cell surface (hemagglutination), the production of interferon or viral interference, and the detection of viral nucleic acids. detection of nonreplicating viruses. during the period from 1950 to 1980, there was a concerted effort to identify the causes of acute infections of infants and children. in seeking the etiology of diarrheal diseases of infants, it was hypothesized that-in addition to bacteria, which accounted for less than half of the cases-one or more viruses might be responsible for some cases of infantile diarrhea. numerous unsuccessful attempts were made to grow viruses from stools of patients with acute diarrhea. it was conjectured that it might be possible to visualize a putative fastidious virus by electron microscopy of concentrated fecal specimens. when patients' convalescent serum was added to filtered and concentrated stool specimens, aggregates of 70 nm virions were observed in stools from some infants with acute gastroenteritis. the ability of convalescent but not acute illness serum to mediate virion aggregation provided a temporal association of the immune response with an acute diarrheal illness. within 5 years, rotavirus was recognized as the most common cause of diarrhea in infants and young children worldwide, accounting for approximately onethird of cases of severe diarrhea requiring hospitalization. once an emergent virus has been identified, it is necessary to classify it, in order to determine whether it is a known virus, a new member of a recognized virus group, or represents a novel virus taxon. this information provides clues relevant to diagnosis, prognosis, therapy, and prevention. in 1967, an outbreak of acute hemorrhagic fever occurred in laboratory workers in marburg, germany, who were harvesting kidneys from african green monkeys (chlorocebus aethiops, formerly cercopithecus aethiops). in addition, the disease spread to hospital contacts of the index cases, with a total of over 30 cases and 25% mortality. clinical and epidemiological observations immediately suggested a transmissible agent, but attempts to culture bacteria were unsuccessful. however, the agent was readily passed to guinea pigs which died with an acute illness that resembled hemorrhagic fever. after considerable effort, the agent was adapted to tissue culture and shown to be an rna virus. when concentrated tissue culture harvests were examined by electron microscopy, it was immediately recognized that this agent differed from known families of rna viruses, since the virions consisted of very long cylindrical filaments about 70 nm in diameter. this was the discovery of marburg virus, the first recognized member of the filoviruses, which now include marburg and ebola viruses. isolation of a virus from patients suffering from an emergent disease provides an association, but not proof of a causal relationship. formal demonstration that an isolated virus is the causal agent involves several criteria formulated over the past 100 years. these are often called the henle-koch postulates, after two nineteenth-century scientists who first attempted to enunciate the rules of evidence. sidebar 1 summarizes these postulates, which have been modernized in view of current knowledge and experimental methods. the classic version of the henle-koch postulates required that the causal agent be grown in culture. however, as discussed below, a number of viruses that cannot be grown in culture have been convincingly associated with a specific disease. usually, this requires that many of the following criteria can be met: (1) viral sequences can be found in the diseased tissue in many patients, and are absent in appropriate control subjects; (2) comparison of acute and convalescent sera document induction of an immune response specific for the putative causal virus; (3) the disease occurs in persons who lack a preexisting immune response to the putative virus, but not in those who are immune; (4) the implicated virus or a homologous virus causes a similar disease in experimental animals; and (5) epidemiological patterns of disease and infection are consistent with a causal relationship. some very important human diseases-such as hepatitis b and hepatitis c-are caused by viruses that cannot readily be grown in cell culture. experiences with these viruses have given credibility to the view that an infectious etiology can be inferred by clinical and epidemiological observations in the absence of a method for growing the causal agent. also, they have stimulated researchers to devise novel techniques that bypass the requirement for replication in cell culture. furthermore, the application of molecular biology, beginning about 1970, has led to an array of new methods-such as the polymerase chain reaction (pcr), deep sequencing, and genomic databases-that can be applied to the search for unknown viruses. several case histories illustrate the inferences that lead to the hypothesis of a viral etiology, the strategy used to identify the putative causal agent, and the methods exploited by ingenious and tenacious researchers. sin nombre virus. hantavirus pulmonary syndrome was described above, as an example of an emerging virus disease. the disease was first reported in mid-may, 1993, the henle-koch postulates were formulated in 1840 by henle, revised by koch in 1890, and have undergone periodic updates to incorporate technical advances and the identification of fastidious agents with insidious disease pathogenesis. many of the following criteria should be met to establish a causal relationship between an infectious agent and a disease syndrome. 1. the putative causal agent should be isolated from patients with the disease; or the genome or other evidence of the causal agent should be found in patients' tissues or excreta; and less frequently from appropriate comparison subjects. temporally, the disease should follow exposure to the putative agent; if incubation periods can be documented they may exhibit a log-normal distribution. if an immune response to the putative agent can be measured, this response should correlate in time with the occurrence of the disease. subjects with evidence of immunity may be less susceptible than naã¯ve individuals. 3. experimental reproduction of the disease should occur in higher incidence in animals or humans appropriately exposed to the putative cause than in those not so exposed. alternatively, a similar infectious agent may cause an analogous disease in experimental animals. 4. elimination or modification of the putative cause should decrease the incidence of the disease. if immunization or therapy is available, they should decrease or eliminate the disease. 5. the data should fit an internally consistent pattern that supports a causal association. and tissues and blood samples from these cases were tested extensively, but no virus was initially isolated in cell culture. however, when sera from recovered cases were tested, they were found to cross-react with a battery of antigens from known hantaviruses, providing the first lead (in june, 1993) . dna primers were then designed, based on conserved hantavirus sequences, and these were used in a pcr applied to dna transcribed from rna isolated from tissues of fatal cases. sequence of the resulting amplicon suggested that it was a fragment of a putative new hantavirus (july, 1993) , yielding a presumptive identification of the emerging virus within 2 months after the report of the outbreak. an intense effort by three research teams led to the successful isolation of several strains of snv by november, 1993. snv is a fastidious virus that replicates in vero e6 cells but not in many other cell lines. kaposi's sarcoma herpesvirus (hhv8). ks was described over 100 years ago as a relatively uncommon sarcoma of the skin in older men in eastern europe and the mediterranean region. in the 1980s, ks emerged at much higher frequency, as one of the diseases associated with aids. furthermore, ks exhibited an enigmatic epidemiological pattern, since its incidence in gay men was more than 10-fold greater than in other aids patients, such as injecting drug users and blood recipients. these observations led to the hypothesis that ks was caused by a previously undetected infectious agent that was more prevalent among gay men than among other hiv risk groups. however, researchers were unable to isolate a virus from ks tissues. searching for footprints of such a putative agent, chang and colleagues used the method of representational difference analysis to identify dna sequences specific for ks tumor tissue. several dna fragments were identified, and found to be homologous with sequences in known human and primate herpesviruses. in turn, these sequences were used to design primers to obtain the complete genome of a previously undescribed herpesvirus, since named hhv8, human herpesvirus 8. to this date, hhv8 defies cultivation in tissue culture. is computer modeling a useful adjunct to the analysis or control of emerging viral diseases? the 2014-15 ebola pandemic in west africa offers an interesting case study. as the epidemic unfolded, several groups attempted to project how it would evolve (meltzer et al., 2014; butler, 2014) . these projections had very wide confidence limits, and several of them had upper limits in the range of 100,000 or more cases. what the modelers could not foretell was that the infection did not spread across sub-saharan africa, and that several introductions (into countries such as nigeria and mali) were controlled by case isolation, contact tracing, and quarantine. however, modeling did contribute useful insights. dobson (2014) suggested that rapid quarantine (within 5-7 days) of contacts of ebola patients could be critical in epidemic control. one of the most exciting current issues in virology is the emergence of new viral diseases of humans, animals, and plants. even though the era of modern virology has been well established for more than 65 years, virus diseases continue to appear or reemerge. the ebola pandemic of 2014-15 highlights the associated dangers and obstacles to control. there are several explanations for emergence: (1) discovery of the cause of a recognized disease; (2) increase in disease due to changes in host susceptibility or in virus virulence; (3) reintroduction of a virus that has disappeared from a specific population; (4) crossing the barrier into a new species previously uninfected. many zoonotic viruses that are maintained in a nonhuman species can infect humans, but most cause dead-end infections that are not transmitted between humans. a few zoonotic viruses can be transmitted between humans but most fade out after a few person-to-person transmissions. rarely, as in the case of hiv, sars coronavirus, and ebola filovirus, a zoonotic virus becomes established in humans, causing a disease that is truly new to the human species. there are many reasons for the apparent increase in the frequency of emergence of new virus diseases, most of which can be traced to human intervention in global ecosystems. emergent viruses are identified using both classical methods of virology and newer genome-based technologies. once a candidate virus has been identified, a causal relationship to a disease requires several lines of evidence that have been encoded in the henle-koch postulates, guidelines that are periodically updated as the science of virology evolves. identifying, analyzing, and controlling emerging viruses involve many aspects of virological science. virus-host interactions play a key role, to explain persistence in zoonotic reservoirs, transmission across the species barrier, and establishment in human hosts. thus, the issues discussed in many other chapters contribute to our understanding of emerging viral diseases. hendra and nipah viruses: different and dangerous biological control as exemplified by smallpox eradication and myxomatosis sequence-based identification of microbial pathogens: a reconsideration of koch's postulates emerging virus diseases: can we ever expect the unexpected? emerging microbes and infection epidemiology and transmission dynamics of west nile virus disease emerging infections the emergence and evolution of canine parvovirus -an example of recent host range mutation social and environmental risk factors in the emergence of infectious diseases diseases of the central nervous system caused by lymphocytic choriomeningitis virus and other arenaviruses middle east respiratory syndrome coronavirus (mers-cov) evidence and speculation human monkeypox: an emerging zoonosis isolation of the causative agent of hantavirus pulmonary syndrome cultivation of the lansing strain of poliomyelitis virus in cultures of various human embryonic tissue. science 1949 mission to mers origin of hiv-1 in the chimpanzee pan troglodytes genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak a decade after sars: strategies for controlling emerging coronaviruses chikungunya disease outbreak, reunion island enhanced virulence of influenza a viruses with the haemagglutinin of the 1918 pandemic virus mers surges again, but pandemic jitters ease origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia bats are natural reservoirs of sars-like coronavirus the many projected futures of dengue ks-associated herpesvirus, and the criteria for causality in the age of molecular biology genetic identification of a hantavirus associated with an outbreak of acute respiratory illness host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance recent researches concerning etiology, propagation and prevention of yellow fever, by the united states army commission recognition determinants of broadly neutralizing human antibodies against dengue viruses a single mutation in chikungunya virus affects vector specificity and epidemic potential characterization of the reconstructed 1918 spanish influenza pandemic virus molecular changes in a/chicken/ pennsylvania/83(h5n2) influenza virus associated with acquisition of virulence ebola references models overestimate ebola cases breakdown of the year: ebola mathematical models for emerging disease mobilizing ebola survivors to curb the epidemic ebola hemorrhagic fever genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak basic clinical and laboratory features of filoviral hemorrhagic fever ebola virus vaccines: an overview of current approaches. expert review of vaccines estimating the future number of cases in the ebola epidemic -liberia and sierra leone single-dose attenuated vesiculovax vaccines protect primates against ebola makona virus pathogenesis of the viral hemorrhagic fevers product: reebovâ�¢ antigen rapid test kit host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance who ebola response team. ebola virus disease in west africa-the first 9 months of the epidemic and forward projections gene loss and adaptation to hominids underlie the ancient origin of hiv-1 sharp pm aids as a zoonosis: scientific and public health implications nef proteins of epidemic hiv-1 group o strains antagonize human tetherin vif proteins from diverse primate lentiviral lineages use the same binding site in apobec3g the origins of aids origins of hiv and the aids pandemic key: cord-018151-5su98uan authors: lynteris, christos title: introduction: infectious animals and epidemic blame date: 2019-10-12 journal: framing animals as epidemic villains doi: 10.1007/978-3-030-26795-7_1 sha: doc_id: 18151 cord_uid: 5su98uan the introduction to the edited volume summarises the chapters of the volume and discusses their contribution in the context of current historical and anthropological studies of zoonotic and vector-borne disease, with a particular focus on how epidemic blame is articulated in different historical, social and political contexts. of 'emerging infectious diseases' (eid), which configures the rise of new diseases as carrying with it a potential for human extinction. 2 this volume examines the history of the emergence and transformation of epidemiological and public health framings of non-human disease vectors and hosts across the globe. providing original studies of rats, mosquitoes, marmots, dogs and 'bushmeat', which at different points in the history of modern medicine and public health have come to embody social and scientific concerns about infection, this volume aims to elucidate the impact of framing non-human animals as epidemic villains. underlining the ethical, aesthetic, epistemological and political entanglement of non-human animals with shifting medical perspectives and agendas, ranging from tropical medicine to global health, the chapters in this volume come to remind us that, in spite of the rhetoric of one health and academic evocations of multispecies intimacies, the image and social life of non-human animals as epidemic villains is a constitutive part of modern epidemiology and public health as apparatuses of state and capitalist management. 3 whereas the above approaches (including microbiome studies, and 'entanglement' frameworks in medical anthropology) do contribute to a much-needed shift in the intellectual landscape as regards the impact of animals on human health, their practical and political limitations are revealed each time there is an actual epidemic crisis. then, all talk of one health, multispecies relationships and partnerships melts into thin air, and what is swiftly put in place, to protect humanity from zoonotic or vector-borne diseases, is an apparatus of culling, stamping out, disinfection, disinfestation, separation and eradication; what we may call the sovereign heart of public health in relation to animal-borne diseases. 4 for the maintenance and operation of this militarised apparatus, the framing of specific animals as epidemic villains is ideologically and biopolitically indispensable, even when blame of the 'villain' in question lacks conclusive scientific evidence (see thys, this volume). going against the grain of scholarship that in recent years has sought to portray the vilification of animals as hosts and spreaders of disease as a thing of the past, histories of non-human disease hosts and vectors aims to illuminate the continuous importance of this ideological and biopolitical cornerstone of modern epidemiology and public health. representations of animals as enemies, antagonists or sources of danger have, in different forms, shapes and degrees, been part and parcel of human interactions with the non-human world across history. it is, however, only at the turn of the nineteenth century that, as a result of bacteriological breakthroughs, non-human animals began to be systematically identified and framed as reservoirs and spreaders of diseases affecting humans. to take one famous example, before the end of the nineteenth century, rats were not believed to be carriers or spreaders of plague or any other infectious disease. 5 whereas rats had long been considered to be damaging to human livelihood, due to consuming and spoiling food resources, their only redeeming characteristic was, erroneously, widely believed to be their supposed disease-free nature. 6 hence while mid-seventeenth-century plague treatises noted the rat's destructive impact on fabrics and food, no mention of its connection with the disease was made. 7 equally, two centuries later, when in 1849-1850 british colonial officers in india observed that, at the first sight of rat epizootics, garhwali villagers fled to the himalayan foothills in fear of the 'mahamari' disease, they dismissed this behaviour as merely superstitious. 8 however, the bacteriological identification of rats as carriers of plague or mosquitoes as carriers and spreaders of yellow fever and malaria, at the end of the nineteenth century, was itself enabled and indeed complicated by an already-existing stratum of signification which, by the mid-seventeenth century, had led to the introduction of new symbolic, ontological and legal frameworks of thinking about animals as 'vermin'. vermin, in mary fissell's definition, 'are animals whom it is largely acceptable to kill', not because of some inherent characteristic they possess, but because, in specific historical contexts, 'they called into question some of the social relations which humans had built around themselves and animals'. 9 paraphrasing fissell, we may say that, arising in early modern europe, the category 'vermin' problematised animals which devoured or destroyed the products of human labour and the means of human subsistence in terms of an agency or intentionality that confounded human efforts to control them. departing from the structuralist influences of mary douglas, which dominated animal studies in the 1980s (see, for example, robert danton's work on the great cat massacre in 1730 france), and from keith thomas' 'modernisation' reading of vermin as simply animals that were of no use in an increasingly utilitarian world, fissell's discourse analysis of popular texts on vermin from seventeenth-century england was the first to dwell in the social historical reality of the emergence of this notion. 10 however, more recent studies have opposed fissell's idea that what made vermin a threat to 'human civility' was their perceived 'greed and cunning', or their overall 'trickster' character. 11 lucinda cole's recent monograph imperfect creatures argues that, 'what made vermin dangerous was less their breedspecific cleverness or greed than their prodigious powers of reproduction through which individual appetites took on new, collective power, especially in relation to uncertain food supplies'. 12 the two approaches are not mutually exclusive. indeed, if approached anthropologically, they point to an entanglement between symbolic and economic aspects of vermin as threats to 'social integrity', something that is further supported by the association of vermin at the time with vagrancy and the poor. 13 medical historians have in turn noted the association of vermin with miasma in disease aetiologies and public health practices of early modern europe, especially in times of epidemics when extensive legislation against them and prescriptions for their destruction are recorded. 14 this was particularly the case in the context of plague outbreaks that had long been associated with 'putrid' and 'corrupt' vapours, which certain animals, like dogs, pigs, cats and poultry (and their excrements and carcasses), were believed to emanate. 15 as in the late middle ages, the fear of pestilential miasmata emanating from offal and other meat products had led to the spatial regulation of butchery in england and other parts of europe (cf concerns with 'bushmeat' in relation to ebola; thys, this volume), william riguelle has shown that, in the course of the seventeenth century, concerns with 'noxious' animals played an important role in instituting limits of where these could be kept and where they could be allowed to roam in urban environments. 16 the idea of miasma would continue to impact medical thinking into the nineteenth century. as a part of ontologies that escape both the straightjacket of recent anthropological classifications and classical medicalhistorical dichotomies of contagionism/anti-contagionism, the idea of miasma was malleable, adaptable and ambiguous enough to be compatible with, rather than antagonistic to, that of infection and contagion. 17 however, as new medical and biopolitical challenges arose in the context of colonial conquest, the problematisation of animal-derived miasma or 'febrile poison' gave way to concerns about the climate as the driving force of epidemic disease. 18 thus while the dawn of bacteriology, by the 1870s, did not introduce understandings of animals as sources of disease ex nihilo, it did mark a drastic return to this idea, and, at the same time, led to a significant conceptual shift as regards the ontology of the diseases transmitted, and the mechanism involved in this transmission. 19 this transformation was catalysed by an intense medical, economic and political interest and concern over cattle epizootics, which, as historians have shown, catalysed both the emergence of veterinary medicine and the medicalisation of animals across the globe in the second half of the century. 20 as regards infectious diseases affecting humans, the medicalisation of non-human animals and their transformation into 'epidemic villains' involved an interlinked, two-part framing of their epidemiological significance: on the one hand, as spreaders and, on the other hand, as reservoirs of diseases. the historiography of the identification and study of non-human animals as spreaders of infectious diseases has for some time now stopped being the foray of heroic biographies of men like ronald ross, paul-louis simond or carlos chagas. focused on the social, political and epistemological histories of scientific studies of zoonosis and vector-borne diseases, historians, anthropologists and sts scholars have underlined the ways in which, within epidemiology, bacteriology and parasitology, non-human animals constituted active agents in complex networks of power and knowledge, and how they assumed different epistemic value in diverse colonial and metropolitan contexts. 21 framed as spreaders of infectious diseases, animals also came to play an important role in what charles rosenberg has famously described as the dramaturgy of epidemics. 22 assuming a protagonistic role in a series of epidemic and public health dramas, animals came to be seen as the ultimate source of disease outbreaks. no longer simply a nuisance or 'pests', the transformed image of a series of animals (mosquitos, rats, ticks, lice and flies in particular) as enemies of humanity was invested with militaristic tropes and colonial moralities. these animals formed as it were a global repertoire of disease spreaders, while at the same time assuming importantly diverse local forms, often in interaction with concerns and social imaginaries about other, regionally specific, disease hosts and vectors (beetles, bats, sandflies, etc.). while it is not in the scope of this introduction to map these 'glocal' interactions, deborah nadal's chapter in this volume provides a detailed picture of the longue durée of dogs as spreaders of rabies in india. nadal's chapter underlines the complex and important semiotic and ontological workings and re-workings on dogs as spreaders of rabies from colonial india to our times. with dog-borne rabies being recognised as an important public health problem across the globe since the 1870s, in india, where rabies is endemic, human understandings of the particular zoonosis were linked to practices of classifying dogs. for british colonials, distinguishing between rabies-prone and rabies-impervious dogs was key to the imperial project of mastery over both indian society and 'nature'. within the confines of tropical medicine and its biopolitical imperatives, the management of rabies made crucial the definition of dog-human relations in terms of ownership. believed to be able to spontaneously develop rabies, for the british, 'ownerless' dogs presented a distinct danger for the colony. seen as the source of infection amongst owned dogs (which were considered unable to develop spontaneous rabies), these animals, nadal argues, challenged victorian morality and were associated with two key notions: on the one hand the notion of 'stray', with its overtones of vagrancy, and, on the other hand, the notion of the 'pariah'-an anglicised caste term used by british colonials to refer to outcaste or untouchable communities. at the heart of these classifications lied ideas about domesticity and wildness, as well as a pervasive social hierarchical mentality. perceiving street life in general as a threat to colonial rule grouped dogs of distinct social status and social life under one, infectious category. transforming 'strays' from 'vermin' and 'nuisance' into epidemic villains that should be sacrificed in the name of human health was not, however, a frictionless process but, as nadal shows us, one that embroiled indian society in debates about the value of life and compassion (led by both anti-vivisectionists and mahatma gandhi). after 1947, 'catch-and-kill' of dogs for the control of rabies continued unabated but also involved indian society in renewed debate involving civil society activists, animal welfarists and political parties. in nadal's reading, these dog-related conflicts underlined a lingering problem pertaining to the classification of dogs vis-à-vis rabies: the persistence of the term 'stray' (inclusive of its 'pariah' associations). the solution since 2001, nadal argues, has been the emergence of a discourse around 'street dogs', which has marked a shift towards an accommodation between different attitudes towards the particular animals, allowing for the concept that they can be both masterless and hygienic. nadal's chapter thus points out that, at the same time as what we may call high-epidemiology redefined experiences of non-human animals as spreaders of disease, it also instituted regimes of hygienic hope. envisioning and putting in place programs of increasing separation between humans and non-human disease vectors became the hallmark of public health from 1900 onwards. whether this involved rat-proofing, ddt spraying, mosquito nets, the cleaning of streets from stray dogs or the drying of swamps, this sanitary-utopian aspiration to liberate humanity of zoonotic and vectorborne diseases was based on a vision of universal breaking of the 'chains of infection'; a separation and, at the same time, unshackling of humans from disease vectors that was aimed at confining pathogens in the animal realm. 23 in this way, whereas separation from animals was seen as a sufficient means of protection of humans from zoonotic and vector-borne diseases, animals themselves were defined as ultimately hygienically unredeemable-they were, in other words, rendered indistinct from disease. hence, the naturalist ontology of the enlightenment, which in philippe descola's anthropological model defines humans and animals as unified under the rubric of nature, was unsettled by a radical divide that saw disease as a mode of being which was only inherently proper to non-human animals, and only tentatively, or, as sanitary utopians would have it, temporarily, part of the human species. 24 sayer's chapter in this volume focuses on the 1910-1911 plague outbreak in freston (suffolk, uk)-the last outbreak of plague in the history of england-and excavates the epistemological, political, class and colonial history of such a regime of prevention and hope. analysing what she calls 'the vermin landscape' of the outbreak, sayer focuses on non-human animal actors so as to show that, in spite of the widespread epidemiological acceptance of the rat flea (xenopsylla cheopis ) as the true spreader of plague, ideas about locality and class created a medico-juridical matrix where it was the rat that constituted the main object of scientific investigation and public health intervention. situating the suffolk outbreak both within the third plague pandemic and within british imperial science politics, sayer stresses the ways in which suffolk was connected to india, as the prime locus of the pandemic and of plague science in the empire. as the outbreak in suffolk was experienced as an echo of the ongoing devastating epidemic in india, the rat became an object of epidemiological concern and fear. what if infected rats moved from the rural hotspots of the epidemic into urban areas, transforming them into the equivalents of plague-ravaged bombay on english soil? such fears were fostered not just by the perceived natural traits of rats (as invasive of migratory animals), but also through their association with the rural poor. tapping into complex imaginary registers involving victorian systems of class-related disgust, the english rural idyll, and the image of 'the labourer's country cottage […] as literal and figurative representation of the state of the nation', sayer argues that, 'because this rested in turn on the state of the rural labouring class, and that class were said here to be unsanitary and their cottages invaded by rat and plague, the indian racial other therefore ghosted a new category of (dead) undeserving poor'. as epidemic villains, in the eyes of epidemiologists and public health authorities, rats indianised the dwellings of rural labourers in suffolk. as 'plague was equated with "rat plague"', plague also became indian plague, and in turn necessitated control measures and legislation aimed at 'codif [ying] the rat in law and normalis[ing] its destruction'. formulated around an entanglement of class and interspecies relations, the suffolk plague crisis led, on the one hand, to an increasing medico-juridical investment of the rat in england, while, on the other hand, to a systematic neglect of 'the hares, cats, dogs that featured in gamekeepers' and labourers' narratives of the disease'. identifying and investing on a non-human epidemic protagonist (the rat) led to, and indeed required, a disinvestment and neglect of other species involved in the spread of the disease, and-perhaps most crucially-to overlooking the ecological complexity of disease persistence and transmission between different species in any given ecosystem. the rats and mice (destruction) act 1919, 'which tasked every british citizen with a legal obligation to remove rats from their property', was the pinnacle of the configuration of the rat as an epidemic villain in england and of the institutionalisation of sanitary regimes of hope as regards the prevention of animal-borne infection. having conquered the globe by the mid-1920s, this regime of prevention and hope came to an end with the dawn of the emerging infectious diseases framework in the early 1990s, when scientists began to focus on processes leading to new diseases, hitherto of non-human animals, infecting humans and to the 'specie-jump' processes (so-called spillover) leading to this phenomenon: 'rather than revolving around already-existing pathogens and how they circulate in specific ecological contexts, the focus on emergence required a shift of attention to what we may call "viral ontogenesis"'. 25 over the past 30 years, the rise of 'emergence' as the central framework of studying and understanding infectious diseases has led to a radical shift of scales and a reinvestment on zoonotic diseases that has been tied to a shift away from prevention towards preparedness. 26 this is a regime of biosecurity that, as anthropologists like andrew lakoff, frédéric keck and carlo caduff have shown, is based on the anticipation of an unavoidable pandemic catastrophe, and which sets in place technologies of biosecurity that have come to increasingly dominate the realm of global health. 27 envisioned as inevitable and catastrophic, 'emergence' has thus radically transformed the status of animals as epidemic villains. on the one hand, whereas in the sanitary-utopian framework of highepidemiology, animals were considered to be isolatable carriers of disease, in the eid framework infection is rendered inevitable. and, on the other hand, whereas for the sanitary-utopian framework, animal-human infection posed a limited threat to humanity, for eid it poses an unlimited one, or to be precise one associated with existential risk. it is telling that the mytho-historical event defining the conceptual horizon of the sanitaryutopian framework was the black death. believed by 1900 to have been rat-borne bubonic plague, the fourteenth-century pandemic was used by moderns as a key cautionary tale, and at the same time as a potent medical metaphor: black death was something that could 'return' (as hundreds of reports and news items made clear during the third plague pandemic) but whose impact would be effectively limited by grace of modern medicine and sanitation. on the other hand, as caduff has shown, the mytho-historical event defining the conceptual horizon of eid is the 1918 flu pandemic. 28 the political ontology of this event for our contemporary pandemic imaginary is distinctly different from that of the black death for the early-tomid-twentieth-century public. for, as every contemporary epidemiological report and news broadcast makes clear, were an event like 'the spanish flu' to occur again today, globalisation and modern transport would transform it to an event of human extinction proportions; something not only nonpreventable, but whose control, once it has begun, is not guaranteed. both of these mytho-historical events have non-human animals at the heart of their causation narrative: the black death (at least so scientists believed at the time) rats, while the 1918 flu birds, probably chicken. however, while the sanitary myth of origin of the black death portrayed the rat as an ancient enemy of humanity whose days were numbered due to the advancement of science, the eid myth of origin frames chicken as just one example of a host of unknown species from which the 'killer virus' may emerge and against which the only action we can take is being prepared. séverine thys' chapter in this volume explores the consequences of the eid approach to non-human animals, as it applied to 'bushmeat' in the context of the recent ebola epidemic in west africa (2014-2016), with a focus on the impact of epidemiological and public health framings of 'bushmeat' hunting, butchering and consumption. especially affecting 'forest people' in macenta, guinea-conakry, the framing of a fluid host of animals as the source of epidemiologically illicit meat relies on persistent colonial tropes that imagine the 'tropical jungle' as an originally natural realm whose disturbance by human activity leads to the emergence of killer viruses. 29 rehearsed time and again in films like outbreak (1995), this mortal link between nature and culture, thys reminds us, is currently being mediated by the figure of the bat-the in-between figure of a 'rogue' animal, which, james fairhead has shown, is being increasingly deployed as an epidemiological bridge in several zoonotic scenarios (ebola, mers, sars). 30 thys follows other anthropologists in pointing out that this insistence on 'bushmeat' and contact with fruit-bats frames local cultures as pathogenic, in line with paul ewald's notion of 'culture vectors', and thus 'obscure[s] the actual, political, economic, and political-economic drivers of infectious disease patterns'. 31 framed in terms of a 'transgression of species boundaries', ebola spillover events are thus pictured as resulting from a life led according to 'traditional' (and the implication is irrational) classificatory systems that fail to maintain 'us vs. them' boundaries. replete with visual and affective structures of disgust, this view, thys argues, is not challenged by the one health framework, which 'should provide a more nuanced and expanded account of the fluidity of bodies, categories and boundaries' so as to 'generate novel ways of addressing zoonotic diseases, which have closer integration with people's own cultural norms and understandings of human-animal dynamics'. 32 key to this, according to thys, is to recognise and examine the historically dynamic nature of these classificatory and more broadly ontological systems (a view shared by nadal, this volume), and the explanatory models with which they are entangled. thys outlines the complex matrix of uses of non-farmed meat in the region (for nourishment, medicaments, trophies, etc.) and their transformation under the weight of regional and global commodity market networks. one may add that what is often neglected is the fact that 'bushmeat' was used by colonial authorities as a reward to local communities; in angola, for example, the portuguese rewarded local communities with 'bushmeat' for rat-catching in the colonial power's effort to contain plague during the 1930s. 33 the political investments of non-human animals as disease spreaders are further explored in gabriel lopes' and luísa reis-castro's chapter in this volume on the history of the aedes aegypti mosquito in modern brazil. following the social life of the particular mosquito species from the 1950s until today, lopes and reis-castro stress that, while recognising that it has always constituted an 'epidemic villain', we need to pay closer attention to the particular diseases to which this villainous character has been linked to, and to the corresponding political system under which this identification has been undertaken, over the course of modern brazilian history. at the beginning of the twentieth century, aedes aegypti was associated with 'underdevelopment' as a key overarching ailment of brazil, with 'the image of a plagued country swarming with mosquitoes' filled with yellow fever playing an important role in bringing health under the rubric of the state and its modernising agenda. lopes and reis-castro follow gilberto hochman's classic work on the linkage between sanitation and nationbuilding in brazil in stressing that what began as a project of 'civilizing the tropics' by eliminating yellow fever across the country transformed by the early 1930s into a more modest programme of preventing outbreaks in urban centres. 34 by contrast to the liberal nation-building sanitary-utopian visions of oswaldo cruz and his collaborators in the first decades of the twentieth century, in the second half of the 1980s a renewed focus on aedes aegypti was underscored by the politics of democratisation, following the end of the 21-year-long military dictatorship in 1985. as by april 1986 it had become identified with dengue fever, as a new disease to plague urban 'areas marked by racialised histories of state abandonment and violence', the aedes aegypti became associated with a disease that was not as lethal as yellow fever, and which bore with it the sign of social, political and economic restitution. as public health had been the pejorative of left-wing and other democratic forces during the last decade of the dictatorship, calls to control dengue-carrying aedes aegypti as an embodiment of state violence and neglect contributed to the success of the 'sanitary reform movement' and the establishment, in 1988, of brazil's sistema único de saúde. lopes and reis-castro then turn their attention to the latest incarnation of aedes aegypti as a spreader of the zika virus. unfolding during the years of the impeachment (or judicial coup, depending on one's point of view) against dilma rousseff, the appearance of zika in brazil involved aedes aegypti in an international emergency. lopes and reis-castro examine the political struggles around zika-related mosquito control and argue that, focused on social inequality and the 'uneven effects of climate change', this new framing of the aedes aegypti on the one hand continues a longestablished practice of problematising it as a disease vector with specific political and political-economic parameters, while, on the other hand, introducing important gender-related critiques of public health. hence, while the authors claim that, 'the specific kind of virus in mosquitoes' bodies shaped what kind of epidemic villain the mosquito became', they also stress that, 'the mosquito as a vector carried not only three epidemiologically distinct viruses but very different political desires, struggles, and debates'. focusing on the recent zika crisis, in their chapter to this volume gustavo corrêa matta, lenir nascimento da silva, elaine teixeira rabello and carolina de oliveira nogueira in turn argue that the focus on mosquitoes' guilt and on the technological strategies developed to control these vectors unfolded within a context of profound political instability, and at the same time of epistemic uncertainty regarding key epidemiological traits of the disease. framing aedes aegypti as epidemic villains in this context, diverted attention from issues of social, economic and environmental injustice and inequality that were driving determinants of the outbreak, and legitimised the absence of governmental measures regarding the latter in response to the epidemic. the 'enactment of a global enemy, aedes aegypti, as the villain of the epidemic' thus allowed the brazilian government to paint an all-too-familiar and deceptive picture of a promethean struggle of the country as a unified whole (notwithstanding its enormous and often violent class, race, gender and ideological discrepancies and antagonisms) against a vile creature, which was solely held responsible for the disease. drawing on critical medical anthropological perspectives, matta et al. thus underline the structural violence inherent in both the discourse of epidemic villains and in the policies built and legitimated by this discourse. brazil's mosquitocentred policy in the face of zika, financially, politically and morally boosted by the declaration of public health emergency of international concern (pheic) by the who, relied on a securitisation framework that rhymed well with the broader neoliberal turn of the country and mobilised the image of the mosquito as a public enemy to create a spectacle of national unity that obscured 'iniquities, poverty, the skin colour of those bitten by mosquitoes, the house and streets where these fly, and the environment where they lay their eggs'. as mark honigsbaum has shown, disease ecology frameworks, arising in the usa in the 1930s, framed non-human animals not simply as spreaders of infectious diseases but also as their 'reservoirs'. 35 the 'great parrot fever epidemic' of 1929-1930 involved pet parrots in an epidemic panic across the globe, with a particular focus in the usa. as readers of the colonialist bande dessiné exemplar, tintin in the congo (published in the shadow of the epidemic in 1931), may remember, psittacosis (caused by chlamydia psittaci) is a zoonotic disease carried by parrots and parakeets that can infect humans. 36 however, for karl f. meyer, a key contributor to the development of disease ecology, the ability of parrots and parakeets (popular pets at the time in the usa) to be asymptomatic carriers of the disease posed a more important problem that the immediate epidemic crisis; especially, honigsbaum explains, as '[t]hese latent infections were a particular problem in california where during the depression many people supplemented their incomes by breeding parakeets in backyard aviaries'. 37 the discovery that psittacosis was not simply an 'exotic' disease imported to the usa by parrot traders, but one that had established itself endemically in american aviaries transformed the structure of epidemic blame from one focused on an outbreak to one focused on an endemic and, at the same time, from one revolving around an exotic invasion to one regarding unhygienic infrastructures at home. more profoundly, it also contributed to a shift towards a reframing of animal-borne disease in terms of disease ecology, a process which involved several decades of studies and interdisciplinary exchanges, but was ultimately triggered by an integration of charles elton's pathbreaking understanding of animal zoology in the realm of epidemiology. 38 what is less well recognised historically is that the notion of the reservoir had a long history in epidemiological reasoning predating disease ecology. rats in particular were suspected, from as early as 1900, as not only spreading plague (via their flea, xenopsylla cheopis ) but as also contributing to the maintenance of persistence of the disease in given urban settings. 39 indeed, elton's interest in the role of disease in the regulation of animal populations was itself stimulated by earlier russian and chinese studies of the siberian marmot as a host of plague in the inner asian steppes. 40 in chapter 2 of this volume, christos lynteris returns to these studies to examine how the so-called tarbagan became the subject of investigations regarding plague's ability to survive the harsh winters of the region. the question was related to ideas about 'chronic plague', which in the case of the siberian marmot were linked to its hibernation between october and april. using an abundance of visual material, lynteris argues that, on the one hand, tarbagan burrows, which had been epistemic objects ever since the discovery of the species in 1856, and, on the other hand, marmot hibernation, which had been the focus of scientific investigation in relation to host immunity already by 1902, were tied together into an epidemiological duet as a result of the emergency of the manchurian plague epidemic of 1910-1911. there is indeed a crucial metonymic work involved in this tying together the 'mystery of the survival of plague' over winter to marmot hibernation, and marmot underground dwellings. 41 for the three actants in this network of what following genese sodikoff, we may call 'zoonotic semiotics'-latent plague, hibernating marmots, underground burrows-shared and maintained between them an image of 'mystery' and occultation which has been key both to epidemiological reasoning regarding infectious diseases and to the 'pandemic imaginary' underlying understandings of zoonosis. 42 this image of plague taking advantage of unseen biological processes, materialities or infrastructures so it can assume an imperceptible form that would allow it to persevere over either human action against it or environmentally adverse conditions is of course reliant on pasteurian notions virulence, latency and attenuation. yet, more than simply illuminating a reiteration of bacteriological doctrine, what the tarbagan example points out to is a pervasive aspect of epidemiological reasoning; for the assumption that, when plague (or indeed any other disease) is not seen, this is because it is 'hiding', is part of what we may call a cynegetic complex in epidemiology. as john berger once noted, admittedly in a very different context, a key principle (and, one may add, a mythic structure) of cynegetic worlds is that, 'what has vanished has gone into hiding'. 43 in the case of epidemiology, as with other cynegetic cosmologies, this implies an ambivalent relation. 44 on the one hand, microbes are seen as predators of humanity, who lurk and hide so as to better ambush their prey. and on the other hand, as the enduring metaphor of 'virus hunters' amply illustrates, microbes are also seen as humanity's pray-which thus 'hide' to escape being caught and vanquished by us. 45 as frédéric keck has stressed (following chamayou), '[w]hereas pastoral techniques are asymmetrical, relying on the pastor's superior gaze over the flock manifested by sacrifice, cynegetic techniques are symmetrical, as hunters and prey constantly change perspectives when displayed in rituals'. 46 maurits meerwijk's chapter in the present volume shows that this is indeed a historically pervasive framework, which in the case of mosquitoes is carried over from tropical medicine into global health. comparing the discourses of ronald ross and bill gates, meerwijk shows how the cynegetic metaphor comes to encompass not only the pathogens in question but also their vectors. this points out at a transformative ontology underlying epidemiological reasoning, and its obsession with the 'invisibility' of disease, insofar as pathogens are seen, on the one hand, as able to persist by transforming themselves inside non-human animal hosts (by means of attenuation or mutation) and, on the other hand, as able to spread by transforming their hosts into bestial man-hunters. 47 more than simply blaming non-human animals, in epidemiological reasoning, this double transformative ability configures the former into the loci par excellence of pathogenesis and, at the same time, necessitates techniques of rendering host-pathogen relations visible. visual images of non-human animals have played a historically important role in their configuration as epidemic villains. since the dawn of bacteriology, the scientific identification and examination of non-human hosts and vectors of infectious diseases have heavily relied on photographic technologies (including microphotography), diagrams and epidemic cartography. 48 following sayer (this volume), animals have been 'fed into a data-focused visual regime', combining photography, mapping, diagrams and statistical graphs, that seeks to establish points of contact, habitats, interspecies boundaries and other forms of what hannah brown and ann h. kelly have called human/non-human 'material proximities'. 49 in the context of high-modern epidemiology as well as in today's eid framework, these visualisations are part of a project of mastery aimed not so much at the subjugation of nature, as to the control of humanity's relations with nature. 50 diagrammatic images of dissected mosquitoes played a key role in ronald ross' examination of the insects as malaria vectors, as, in later years, the microphotography of anopheles gambiae dissected ovaries would prove an indispensable, soviet-led method for identifying the capacity of a given mosquito to transmit the malaria plasmodium to humans. 51 similarly, nicholas evans has shown, in the course of the third plague pandemic, comparative images between healthy and plague-infected rats became standard visual objects in epidemiological investigations and their published reports. 52 but the visualisation of 'epidemic villains' did not always necessitate their direct representation. in her chapter for this volume, sayer draws an insightful comparison between two sets of visualising rat control, the first in the english port of liverpool and the second in british india. in both cases, the actual rats are imperceptible, with the photographic focus being on humans undertaking carefully orchestrated epidemiological work (rat dissection, flea collection); a fact which, in the case of liverpool, is underlined by the staged poses of the sanitary officers in questions, and, in the case of india, was permeated by colonial racial hierarchies in the representation of lab work. as representations of the relation between pandemic plague, medical science and empire, these images provide reassuring portraits of control in direct dialogue with the image of objectified rats, described by evans, thus 'making rats an integral part of plague'. 53 similarly, with a focus on this relational aspect of human/non-human mastery and its visual regimes, in the second chapter of this volume lynteris illustrates how the epidemic framing of siberian marmots as reservoirs of plague in inner asia relied on photography and the diagrammatic cartography of their burrows. comprising in survey photographs of excavated marmot burrows and diagrammatic depictions of burrow systems, the visual regime constructed around this suspected host of plague following the manchurian plague outbreak of 1910-1911 comes to show, on the one hand, that intrusive practices of epidemiological visualisation were not limited to human dwellings, but also included those of non-human animals (photographing the marmot burrows required their prior excavation), and, on the other hand, that the visual framing of 'epidemic villains' is not limited to the representation of their role as spreaders of diseases. 54 at the same time, the popularisation of the identity of specific mammals, birds and insects as disease spreaders has and continues to be mediated by their visual representation through photography, film and illustration. photographs of 'wet markets' in south china during and in the aftermath of the 2003 sars pandemic have been shown to incorporate a key principle of 'epidemic photography': the depiction of animal-related spaces as potential ground zeros of the 'next pandemic'. 55 the practice of the public vilification of non-human animals and the framing of contact spaces between them and humans as infection hotspots was established for the first time in the course of anti-malarial and anti-yellow fever campaigns in the first decades of the twentieth century, but also during complex public health operations against plague in the context of the third plague pandemic (1894-1959) when the dreaded disease was often visually personified as the rat. 56 indeed, quite often, the image of animals as enemies of humanity assumed anthropomorphic aspects, which under a colonialist gaze, involved racist inflections. in australian newspaper illustrations, for example, plague-carrying rats were depicted having chinese faces, thus both making an aetiological connection between plague and china (plague as an 'oriental disease' arriving from china, by chinese migrants) and fostering broader sinophobic bigotry at the time. 57 in his examination of the framing of 'tiger mosquitoes' (aedes aegypti and aedes albopictus ) in this volume, meerwijk explores the rich visual culture supporting progressive framings of the specific mosquito species as infectious enemies of humanity. in a striking example, meerwijk shows how the diagrammatic juxtaposition of a mosquito and a tiger was used in a public health poster, meant to underline the predatory, man-eating qualities of aedes mosquitos. pointing at a pervasive tendency to talk about and visualise mosquitoes in terms of great predators (tigers, sharks) or 'enemies of humanity' (terrorists, vampires, prostitutes), meerwijk elucidates the work of the fusion between military, cynegetic and sexual metaphors and visual tropes employed in the depiction of mosquitoes across epidemiological paradigms. this is all the more important as the visualisation of animals as 'epidemic villains' was a trope that found application and success beyond epidemiology and public health. non-human animals were charismatic protagonists of political caricatures since the turn of the eighteenth century. in particular, lukas englemann notes, 'the "political bestiary", as gombrich calls the long tradition of depicting political issues through animal characters, acquired widespread popularity in the nineteenth century. the meaning many animals inhabited could be easily exploited to convey strong messages and almost always suggested degradation'. 58 what changed at the turn of the nineteenth century was the introduction of a new aspect in the use of animals in caricature: their infectious nature. with political discourse utilising more and more medical terms at the time, the use of the visual form of the infectious animal to portray one's political enemies became an exemplary field of vilification. to mention only one example, in the course of the moscow trials, soon after the soviet state prosecutor, andrey vyshinsky, publicly pledged 'to stamp out the accursed vermin' who 'should be shot down like rabid dogs', the prolific cartoonist of the pravda, boris efimov (who was present at the trial), produced a striking caricature of leon trotsky and nikolai bukharin as a two-headed rabid dog held on the leash by the hand of the gestapo. 59 however, as engelmann has shown in his examination of caricatures in the course of the 1900 plague outbreak in san francisco, the aim of depicting animals in the context of epidemic crises has not been limited to practices of blaming the former as spreaders or reservoirs of disease. in fact, animals were also used to critique and ridicule bacteriology itself. for example, in the case of san francisco, newspaper caricatures used animals to portray bacteriology 'as a science that formulated its judgments through experiments with animals, not in the treatment of people'. 60 by visualising laboratory animals as 'vermin and pest', englemann argues, bacteriology was portrayed as 'a wasteful expenditure of public funds' and 'the medical laboratory was stripped of its progressive potential and instead appeared as an infliction of damage on the public good'. 61 at the same time, as dawn day biehler has shown in her monograph on pests in twentieth-century us history, images of disease hosts, like rats, have also been used for subaltern purposes, such as the campaigns by the black panther party in the 1960s-1970s against slumlords and the living conditions in african american neighbourhoods. 62 for example, biehler argues, the well-known illustration by emory douglas, 'black misery! ain't we got right to the tree of life?', 'constrast[ed] with images of women afraid of rats; the woman's grip on the rat suggests determination, courage and fury'. 63 here, the rat represented the unhygienic, exploitative and pestilential conditions imposed by white capital on working-class african americans, and the latter's determination to face up to this social injustice. the prolific use of images of non-human animals as 'epidemic villains' in diverse fields of social practice as public health campaigns, political propaganda, the critique of bacteriology and subaltern critiques of power and domination, points at the importance placed on the infectious nature or potential of animals both as a reality and as a metaphor in the modern world. however, whether it is to convey a threat to the national body, or to mock science, the use of these images also points at the fascination and discomfort of moderns towards non-human agency. underlining how epidemiology and public health emerged in relation to, and continue to be informed by framings of non-human animals as epidemic villains, the chapters in this volume explore the layered political, symbolic and epistemic investments of non-human animals, as these have become rhetorically and visually enabled in distinct ways over the past 150 years. whether it is stray dogs as spreaders of rabies in colonial and contemporary india, bushmeat as the source of ebola in west africa, mosquitoes as vectors of malaria, dengue, zika and yellow fever in the global south, or rats and marmots as hosts of plague during the third pandemic, this volume shows framings of non-human animals to be entangled in local webs of signification and, at the same time, to be global agents of modern epidemic imaginaries. civet cats, fried grasshoppers, and david beckham's pajamas: unruly bodies after sars' the pandemic perhaps: dramatic events in a public culture of danger the scale politics of emerging diseases more than one world more than one health: reconfiguring inter-species health i am using animal-borne diseases here as a term inclusive of zoonotic and vector-borne diseases the rat-catcher's prank: interspecies cunningness and scavenging in henry mayhew's for an influential example of the rat being described as disease-free, see cristofano and the plague: a study in the history of public health in the age of galileo the rat would become suspect of carrying plague for the first time during the inaugural outbreak of the third plague pandemic, in 1894 hong kong, with another decade elapsing before the universal acceptance of the link between the animal and human plague. the first scientific study showing the role of the rat and its flea in the propagation of plague was: p. l. simond, 'la propagation de la peste imagining vermin in early modern england the great cat massacre and other episodes in french cultural history religion and the decline of magic: studies in popular beliefs in sixteenth and seventeenth century england imagining vermin in early modern england imperfect creatures: vermin, literature, and the sciences of life on vermin and the poor, see p. camporesi, bread of dreams: food and fantasy in early modern europe animal bodies, renaissance culture filth is the mother of corruption". plague, the poor and the environment in early modern florence que la peste soit de l'animal! la législation à l'encontre des animaux en période d'épidémies dans les villes des pays-bas méridionaux et de la principauté de liège (1600-1670) que la peste soit de l'animal!'; on ideas of miasma emanating from butchered meat see d. r. carr, 'controlling the butchers in late medieval english towns great stenches, horrible sights and deadly abominations": butchery and the battle against plague in late medieval english towns infection," and the logic of quarantine in the nineteenth century fractured states: smallpox, public health and vaccination policy in british india toxic histories: poison and pollution in modern india as kathleen kete has shown, the modern transformation of this connection, before the dawn of bacteriology, was fostered by a sexualisation of the disease, which rendered it comparable to uncontrollable impulses or lust. commenting on kete's work, linda kalof writes: 'since nymphomania and uncontrollable sexual desire in men were considered the result of prolonged sexual abstinence the beast in the boudoir: petkeeping in nineteenth-century paris looking at animals in human history healing the herds: disease, livestock economies, and the globalization of veterinary medicine veterinary research and the african rinderpest epizootic: the cape colony the great epizootic of 1872-73: networks of animal disease in north american urban environments' beastly encounters of the raj: livelihoods, livestock and veterinary health in india animals and disease: an introduction to the history of comparative medicine from coordinated campaigns to watertight compartments: diseased sheep and their investigation in britain, c.1880-1920 unpacking the politics of zoonosis research and policy catching the rat: understanding multiple and contradictory human-rat relations as situated practices blaming the rat? accounting for plague in colonial indian medicine the bacteriological city and its discontents' malarial subjects: empire, medicine and nonhumans in british india what is an epidemic? aids in historical perspective wartime rat control, rodent ecology, and the rise and fall of chemical rodenticides urban mosquitoes, situational publics, and the pursuit of interspecies separation in dar es salaam the colonial disease: a social history of sleeping sickness in northern zaire the mobile workshop: the tsetse fly and african knowledge production cat and mouse: animal technologies, trans-imperial networks and public health from below building out the rat: animal intimacies and prophylactic settlement in 1920s south africa'. american anthropological association (engagement modern" management of rats: british agricultural science in farm and field during the twentieth century of rats, rice, and race: the great hanoi rat massacre, an episode in french colonial history for a more detailed discussion of this process, see c. lynteris, 'zoonotic diagrams: mastering and unsettling human-animal relations' curing their ills: colonial power and african illness rethinking human-nonhuman primate contact and pathogenic disease spillover the scale politics of emerging diseases the pandemic perhaps avian preparedness: simulations of bird diseases and reverse scenarios of extinction in hong kong unprepared: global health in a time of emergency the pandemic perhaps great anticipations human extinction and the pandemic imaginary for discussion, see k. ostherr, cinematic prophylaxis: globalization and contagion in the discourse of world health inclusivity and the rogue bats and the war against "the invisible enemy as fairhead argues, this entanglement of 'native culture' with 'rogue animals' has the effect of transferring the status of the 'rogue' to the 'culture' in question; fairhead, 'technology, inclusivity and the rogue bats and the war against "the invisible enemy"'. see also m. leach and i. scoones, 'the social and political lives of zoonotic disease models: narratives for a discussion of disgust and animal disease, see a. l. olmstead, arresting contagion. science, policy and conflicts over animal disease control it needs to be noted here that, following fissell, the emergence of the early modern notion of 'vermin' was not associated with disgust-something that points to the introduction of this affective and sensory structure in the nineteenth century imagining vermin in early modern england serviço permanente de prevenção e combate à peste bubónica no sul de angola: relatório 1933 (lisboa: agência geral das colónias the sanitation of brazil: nation, state, and public health latent infections, and the birth of modern ideas of disease ecology' tipping the balance blaming the rat? plague and the regulation of numbers in wild mammals évolution de la peste chez la marmotte pendant l'hibernation'. comptes rendus hebdomadaires des séances de l'académie des sciences for a more detailed examination of zoosemiotics in the case of marmots, see c. lynteris, 'speaking marmots, deaf hunters: animal-human semiotic breakdown as the cause of the manchurian pneumonic plague of 1910-11 on the ambivalence as applies to hunters and gatherers, see r. willerslev lessons in medical nihilism. virus hunters, neoliberalism and the aids pandemic in cameroon on chamayou's theory, see grégoire chamayou, manhunts: a philosophical history for a discussion of the mythic ability of pathogens to transform their hosts into man-hunters, see c. lynteris, 'the epidemiologist as culture hero: visualizing humanity in the age of "the next pandemic on diagrams and the configuration of zoonosis, see lynteris the evolution of ebola zoonotic cycles'. contagion material proximities and hotspots: toward an anthropology of viral hemorrhagic fevers' human extinction and the pandemic imaginary seeing cellular debris, remembering a soviet method' blaming the rat? on the practice of intrusive epidemic photography as regards human dwellings, see r. peckham, 'plague views. epidemic, photography and the ruined city the prophetic faculty of epidemic photography: chinese wet markets and the imagination of the next pandemic this 'global visual economy' was so pervasive in fact so as to lead to a retrospective diagnosis of the presence of rats in paintings such as nicholas poussin's 1665 the plague of ashdod as evidence of a pre-bacteriological knowledge of this zoonotic connection; for a critique, see s. barker yellow peril epidemics: the political ontology of degeneration and emergence a plague of kinyounism: the caricatures of bacteriology in 1900 san francisco the sharp weapon of soviet laughter: boris efimov and visual humor a plague of kinyounism', p. 18. 61. ibid pests in the city: flies, bedbugs, cockroaches, and rats (washington key: cord-005262-pi8nkuc3 authors: nan title: program schedule date: 1983 journal: in vitro doi: 10.1007/bf02618062 sha: doc_id: 5262 cord_uid: pi8nkuc3 nan lysis and phagocytosis of antibody-sensitized chicken red texas in search of better media for invertebrate cell culture. convener: f. hink, ohio state univ. standardization of cells, media, and substrates agrigenetics corp factors convener: t. maciag, harvard medical school introduction: t. maciag platelet derived growth factor: purification and characterization. t. deuel. chemistry and function of somatomedin/insulin-like growth factors and their receptors coffee break purification of growth factors from retina and brain. comparative study of edgf {eye derived growth factor) and bdgf (brain derived growth factor) cape/volusia session-in-depth mutants and variants from plant cultures convener: r. henke, u. tennessee genetic characterization of mutants from cell culture: cosegregation of altered enzymatic phenotype with selected culture traits in progeny of regenerated plants heritable somaclonal variation in wheat chromosomal and mendelian variability in cultures and r regenerated plants genetic analysis of alfalfa variants from somatic cell selection rat kidney epithelial cell culture to study metal toxicity. g. cherian. coffee break altered regulation of proliferation and differentiated function in cultured human epidermal cells by chlorinated aromatic hydrocarbons genetic toxicity evaluation of chemicals: implications for animal assays palm beaeh/broward contributed papers differentiation in cultured cells convener vascular smooth muscle cell modulation in vivo and growth potential in vitro. j. grunwald and c. c. haudenschud.* 8:45 154 stimulation of growth and expression of differentiation antigens of human melanocytes by tumor promoters biologic modification of antigenic expression in dibutyryl adenosine 3'-5' cyclic monophosphate in cultured human glioma lines hypermethylation of albumin and alphafetoprotein genes in nonexpressing mouse-rat hybrid cell lines. w. church and j. papaconstantinou.* coffee break initiation of muscle-type phosphorylase {pplj and phosphoglycerate mutase {pgam} synthesis is not nerve-dependent * reversible inhibition of the accumulation of muscle-specific proteins by the differentiation * regulation of differentiation of adult human and rat hepatocytes cultured in a serum-free medium by interaction with another liver cell numbers preceding titles refer to abstracts. capitalization identifies speaker; asterisk (*) identifies member coffee break intrinsic and acquired resistance to bcnu correlates with the near-diploid cells in 4 freshly resected human gliomas differential phenotypic response of normal and neoplastic mouse mammary epithelium in collagen matrix culture poster session odd numbered posters should be manned from 9:00 to 10:30 a.m. even numbered posters should be manned from 10:30 a.m. to noon * 178 polyclonal and monoclonal antibodies that identify breast myoepithelial and breast fibroblast cells trypanosoma cruzi infection of human muscle cells is inhibited by antibodies to parasite surface antigens production of the c3 component of the complement by cultures of rat liver epithelial cells. stimulating effect of supernatants of human preparative density-gradient electrophoresis of cultured human embryonic kidney cells collagen as an attachment factor for cell monolayers on gas-permeable teflon membranes large scale culture of human breast carcinoma ceils to isolate estrogen receptors * 185 evaluation of a new and flexible method for culturing macrophages: attachment to and removal from cytodex microcarriers. c. ostlung, j. clark, and m. kruse.* mutagenesis 186 isolation of epithelial subcomponents of the mouse mammary gland for the tissue level culture studies cytotoxicity and absence of mutagenic activity of vomitoxin (4-deoxynivalenol) in an hepatocyte-mediated mutation assay with v79 chinese pluronic polyols as vehicles for petroleum derivatives in in vitro assays the influence of metabolic substrates upon hepatocyte proliferation in short term primary cultures enzymology 192 induction of transglutaminase activity in transformed human cells by sodium butyrate substrates of cellular transglutaminase using wi38 cells generate isopeptide bonds separation of exponential cultures of v-79 ceils by counterflow centrifugal elutriation: correlation of protein and superoxide coffee 10:20 w-3 propagation of magnolia in tissue culture. i. beiderman cytogerontology convener: l. hayflick, u. florida perspectives in cytogerontology. l. hayflick. dominance of finite vs. infinite in vitro lifespan in somatic cell hybrids * the role of protein and rna synthesis in the inhibition of nuclear dna synthesis in heterokaryons resulting from the fusion of senescent and low passage, actively dividing human diploid fibroblast-like cells * the presence of non-cycling cells at all levels of population doubling of imr-90 fibroblasts. l. n. castor.* reorganization of the genome during aging of human fibroblasts numbers preceding titles refer to abstracts. capitalization identifies speaker; asterisk (*) identifies member plant genetics, inc. r. mans, univ. of florida introduction: k. redenbaugh trauma as a means of initiating change in genome organization and expression. b. mcclintock. characterization of putative transposons in cytoplasmic male sterility of maize coffee break in vitro plant transformation by bacterial co-cultivation and expression of foreign genes in plant cells new developments in the transformation of plant tissues medical college of pennsylvania introduction: j. leighton insights into human cancer using organ cultures. j.c. petricciani* and i. levenbook.* migration of a rat bladder carcinoma cell line on solid substrata and in three-dimensional tissue. r. tchao, w. schroyens, and j. leighton.* anchorage independent survival as an indicator of malignant transformation: morphological and biochemical comparison * coffee break the analysis of some interactive processes in mammary disease experimental surgical pathology using histophysiologie gradient culture amborski fish cell line: persistent infection with a coronavirus. b. lidgerding.* differential interferon sensitivities of lethal and non-lethal strains of rift valley fever virus (rvfv) in vitro numbers preceding titles refer to abstracts. capitalization identifies speaker; asterisk (*) identifies member. j'submitted for wilton r. earle award. #submitted for the honor b * cardiotoxieity of tricyclic antidepressants in cultured rat myocytes. d. acosta* and k. ramos.* good correlation between the cytotoxicity of drugs and their allergic side effects growth: promoters and control conveners: m. absher and u. k. ehmann conditioned medium promotes clonal growth of amniotie fluid cells the effect of retinoic acid on normal and transformed human embryonic lung fibroblasts. b. stanulis-praeger.* response of fibroblasts to bleomyeln suggest subpopulations with variable sensitivity laminin and fibronectin adsorption to a new surface for cell culture: effect on cell attachment and spreading cinemicrographic analysis of mitosis in primary cultures of adult rat hepatocytes salt lake city virus elimination in caladium and gerbera through tissue culture techniquest --effects on marketing numbers preceding titles refer to abstracts. capitalization identifies speaker; asterisk (*} identifies member key: cord-021113-e4ya7llm authors: elliott, david; soifer, eldon title: divine omniscience, privacy, and the state date: 2017-02-02 journal: nan doi: 10.1007/s11153-017-9612-7 sha: doc_id: 21113 cord_uid: e4ya7llm traditional theism teaches that god engages in a relentless form of observation for every human being. if, as is widely supposed, humans have a right to privacy, then it seems that god constantly violates this right. in this paper we argue that there is both a defensible philosophical excuse and justification for this infringement. we also argue that this defense is extensible to human social and political contexts; it provides the vital elements of a theory of just privacy infringement. this theory is broadly compatible both with major forms of political theory (except anarchistic ones) and with the main conceptions of privacy defended in recent philosophical and jurisprudential literature. orthodox or conservative christian, jewish, and islamic traditions teach that god 1 engages in what we will call ''total observation'' 2 for every human being. 3 total observation occurs when a person, or institution of persons, observes every action and mental event of some other person (or persons). this observation could be done in an unobtrusive manner, such that the person being observed is not immediately aware of being watched. it is widely held, however, that, in some form or other, human persons have a right to privacy. if so, it seems that these traditional theologies face a moral problem: god's total observation violates human privacy in a way that seems wrong in most human contexts. in what follows, we will contend that the abrahamic conception of god does indeed imply the infringement of human privacy, but this, nevertheless, is not a serious moral problem facing traditional christian, jewish or islamic theologies. 4 a defensible excuse and justification can be given for this privacy infringement. defending the conceptual integrity of the traditional view is not, however, our only main goal in this paper. we also intend to show that consideration of this alleged problem of divine-human relations yields a significant theory of privacy infringement justification that is usefully applicable to ordinary human social and political contexts. 1 no central claim in this paper is intended to assume or defend the truth or falsity regarding god's existence, nor the truth or falsity of any conservative/traditional theological claims. rather, our main purpose here is to show that, from a purely conceptual understanding of these ideas, important moral claims about privacy violation can be derived. 2 many conservative theologians rely on scriptural sources for their theological beliefs. in this regard, the scriptural basis for the belief that god engages in total observation seems unassailable. luke 12:2-3, for example, records jesus saying: ''nothing is covered up that will not be uncovered, and nothing secret that will not become known. therefore...what you have whispered behind closed doors will be proclaimed from the housetops.'' see also matthew 10:26-27. (all quotations from the bible are from the new revised standard version [nrsv] .) the author of hebrews (at 4:13) also emphasizes our transparency before god: ''before him no creature is hidden, but all are naked and laid bare to the eyes of the one to whom we must render an account.'' in the hebrew bible, the psalmist writes (69:5): ''o lord, you know my folly; the wrongs i have done are not hidden from you.'' (see also psalm 38:9.) and moses maimonides, historically regarded as a venerable defender of orthodox judaism, claims that god's total observation of all persons is one of the most basic beliefs of judaism. in his commentary on the mishnah (''introduction to helek'') maimonides states that the ''tenth principle of faith'' is ' '[t] hat he, the exalted one, knows the works of men and is not unmindful of them.... [his] eyes are open upon all the ways of the sons of men'' (cohen 1968, p. 189 .) maimonides cites jeremiah 22:19 as a source for this principle. and finally, the koran repeatedly states that god (allah) is watching everyone at all times. see 2. 110; 2.233; 2.237; 2.265; 3.156; 8.72; 11.112; 41:40; 49.18; 57.4; 60.3; 64.2. 3 in a fascinating and provocative article, margaret falls-corbitt and f. michael mclain (1992) have argued that this claim is false. they contend that god's capacity for what we call total observation is capable of self-limitation, and hence god can choose at will to observe or not to observe every human thought or action. they openly admit, however, that their thesis ''does not fit well with theistic tradition and its devotional practice' ' (pp. 383-384) . falls-corbitt and mclain, however, insist that their conception of a god who respects human privacy makes this same traditional understanding more complete and consistent (especially on the matter of god's moral perfection). in the course of this paper, we intend to challenge directly the general thrust of their main thesis. (for a different analysis and a critique of the details of their argument see davison 1997.) 4 although all three abramic traditions raise questions regarding privacy, we will hereinafter limit our focus to christianity. we shall begin our discussion by developing further the problem of human privacy posed by traditional jewish, islamic, and christian conceptions of god. as we have just mentioned, theologians in these traditions hold that god engages in a seemingly relentless form of total observation. they also hold that god documents every one of these observations. this appears to be the implication of ''the books, where, at the final judgment, god judges every person according to what has been recorded in ''the books'' and whether a person's name is found in the ''book of life.'' presumably, this documentation is not done as an aid for god's memory, but as a way, through the documentation itself, of making private acts part of a public record, something that other persons can become aware of. the jewish and christian scriptures, however, clearly do not regard human privacy as something of little or no value. at least two biblical narratives offer support for this claim: the exposure of adam and eve after the fall, and the observation of noah's nakedness after the flood by his son ham. 5 in both stories, the narrative suggests either that god values human privacy or that it is something which humans should respect in their relations with each other. in the first case, after realizing they had sinned and were naked, adam and eve ''sewed fig leaves together and made loincloths for themselves'' (genesis 3:7). after cursing ''the serpent'' and the ground ''because of you'', god then ''made garments of skins for the man and for his wife, and clothed them'' (genesis 3:21). this seems to entail that god directly approved of the desire humans typically have to keep certain aspects of themselves hidden from public view. 6 killing an animal for its skin may even be read as a further indication of the importance of privacy. the attempt to clothe themselves with leaves appears insufficient-both as a means of covering that which should be private and as an expression of the seriousness that should be put on the matter. this may be implied by the fact that killing a sentient being instead of vegetation foreshadows the importance given to divine-human relations through animal and blood sacrifice. in the case of noah (genesis 9:20-27), the significance of respecting certain forms of privacy is also emphasized. 7 when noah drank too much wine and passed out naked on the floor of his tent, his son ham did two things which violated noah's privacy: he observed the nakedness of his father, and then he told his two other brothers about it (i.e., he took something that should have remained private and made it public). the severe curse that noah placed on ham (cursing him before god and enslaving him and his descendants [i.e., canaan] to his two other brothers), the blessing given to shem and japheth, and the apparent endorsement of noah's 5 see konvitz (1966, p. 272) . 6 for further discussion of this in connection with the concept of shame, see velleman (2001) . 7 it might be suggested here that what is primarily at stake is the violation of the obligation to honor one's parents, and not the violation of noah's privacy. however, even if honoring one's parent really is the primary issue here, it is difficult to see how the dishonoring that occurs in the story has no connection at all to noah's privacy. on the contrary, it seems central to the dishonour itself. int j philos relig (2017) 82:251-271 253 assessment by god (the canaanites do not fare well from here on in the biblical narrative), all seem to suggest that due respect for a person's privacy is a value that has a fairly strong basis in biblical theology. so at least in human relations, there is a fairly clear requirement that privacy is a value that ought to regulate human affairs. but, of course, in the very same theological tradition this value or constraint appears to be no part of the relation between god and humans. what then would morally allow for god's total observation? a first response to this question might point to a distinction, implicit in what has just been argued, between god-human relations and human-human relations. it could be argued that the moral relation in each case is vastly different. putting the matter this way, however, raises a familiar dilemma. it could mean that god has moral standards which are completely unrecognizable or unknowable to human beings, and hence god may have ''moral'' reasons for total observation which are simply beyond our ordinary moral understanding. on the other hand, it could also mean that, while morality for both god and humans is within the realm of human understanding, it still might be the case that god's moral obligations or privileges are quite different than human obligations and entitlements in virtue of the sort of being that god is. determining which of these two interpretations is correct would involve entering into a long-standing debate that goes back at least to plato's (and/or socrates') arguments in the euthyphro dialog. this is the debate about the proper relationship between god's nature and the ground of morality itself. this paper is not the place to settle this long-standing discussion. we will only state that, along with a number of other philosophers, 8 we would defend the view that the ground of morality is independent of god's will, and hence deny that god could have moral standards that are completely apart from ones that hold for all other moral agents. this position still leaves the possibility, however, that there might be different obligations and entitlements for god, given his position as god, than those which might hold between humans. this is an important idea, one which seems basically correct and one which we shall return to below. another relevant consideration that might make total observation acceptable has to do with its possible consequences. if total observation has no serious consequences for the persons observed, then, from a general consequentialist point of view, it might seem that there would be no serious moral problem with it. even in human affairs, it might be suggested, if the observed is never aware of her observation, and nothing else ever turns up as a possible negative consequence for her, nothing of any moral importance has occurred. however, this response is too philosophically easy. 9 it also distorts a fairly traditional christian understanding of god. one of the observed's expectations or goals in life could be to not be observed about certain matters. thus even if a person is unaware of the observation, her goal has been thwarted. so if the observation is to be morally defensible, a sound argument is needed to show that having an important personal goal thwarted without consent is not a moral affront. in this regard, it is first worth noting that this situation does not describe the situation that we have focused on above with the traditional conception of god's total observation. god's observation is not morally neutral, nor is it purposeless. one purpose of this observation, traditional christian theology tells us, is to keep track of each person's sinful actions or thoughts. and the standard conservative belief is that each person will, at some future time, be held accountable with eternal damnation facing the unredeemed or unrepentant. so god's observation is far from being of no consequence for the observed. but, again, this same tradition usually insists that god is completely justified in practicing total observation. another attempt to address this problem might suggest that god is justified in engaging in total observation simply in virtue of god's omnipotence. god is justified simply because god can undertake total observation. this, however, seems misguided. to state a moral truism: merely being able to do something (e.g., being able to kill another human being) provides no moral warrant for doing that act. the contemporary state equipped with all available technology might well be able to do something fairly close to total observation for some of its citizens. however, this still begs the obvious moral question of whether this state would ever be morally justified in doing such a thing. more plausibly, it might be argued that god's omniscience implies that god cannot avoid engaging in total observation. omniscience seems to imply necessarily that god will just know what everyone is doing/thinking, has done/thought, and so forth. margaret falls-corbitt and michael mclain, however, have suggested that this understanding of omniscience is false. the best way to think of god's omniscience, they claim, is in a self-limiting way, one which allows for the possibility that god is able to respect human privacy by intentionally limiting knowledge of human thoughts and actions. as they put it: if god...values freedom, then a very strong case can be made that god also chooses to limit divine knowledge in a way that respects privacy. that is, though god's knowledge of what is true could include every innermost thought and feeling of each of us, god chooses instead to grant humans the choice of self-disclosure. 10 scott davison, however, has argued that there are sound metaphysical reasons for believing that this claim is false. god's creative and sustaining activities with respect to the universe entail that it is ''extremely difficult to see how god could not know everything about every human person. after all, god is present in every place in which god acts, and [on the traditional view] god acts everywhere, at every time'' [(1997), p. 144 ]. this response is, in our view, sound. but we would press the case against falls-corbitt and mclain to an even more basic level. the straightforward meaning of omniscience logically forbids the idea that god's knowledge could ever be self-limiting. the traditional judeo-christian conception of divine omniscience identifies this quality as a completed state where god knows everything at all times. one way that god's omniscience could be self-limiting is if it is understood as a capacity rather than (more traditionally) as a state of god. consider the logical difference between the standard concept of omnipotence and that of omniscience. omnipotence is, basically, the claim god is an all-powerful being. no difficulty seems to be involved in holding that god's omnipotence can be self-limited. within the realm of the logically possible, 11 god can freely choose to do or not do something. even if god chooses to do nothing, the traditional understanding is that it would still be true that god is omnipotent. omniscience, however, cannot be selflimiting in the way that omnipotence is. while there is no logical contradiction in holding that god knows (at least) everything that everyone is thinking and doing, and has thought and has done, it is contradictory to hold that god's omniscience can be self-limited. for if god were to choose to not know something, then it would simply be false that god knows everything-i.e., that god is omniscient. someone might argue here that god's omnipotence can be exercised in some particular way in order for god to self-limit his own omniscience. but if this were proposed, the claim still amounts to the contradiction that god both knows everything and that god could will that he could not know something. this, again, is an obvious contradiction. most traditional theologians and philosophers of religion who consider the abramic conception of theism, hold that god cannot do what is logically impossible, 12 they typically argue that this is not an imperfection of omnipotence, but the rational exercise and limitation of it. it follows, then, that god's omniscience cannot be self-limiting. and this necessarily follows even if it might be true that it is wrong for god to violate human privacy. falls-corbitt and mclain, then, must either give up any claim to the traditional attribution of omniscience as part of god's essential nature or reject their own conclusion that god has an obligation to respect human privacy. even if it is true, however, that in some sense god cannot help knowing everything about everyone, this at best amounts to a preliminary excuse for god's total observation; it provides no moral justification. 13 and there needs to be such a justification, otherwise god's moral perfection must be denied or significantly compromised. for if it were the case that god innocently engages in total observation, and this observation were itself morally unjustified, then we have a situation where an innocent party would be bringing about something that is morally wrong or evil. and, to say the least, this does not seem to fit well with the traditional idea of god as a morally perfect being, particularly given god's providential nature and capacities. we do not mean to suggest here that it is inconsistent to hold both that a morally perfect being might do something, which was either completely morally innocent or morally justified, and that something evil could happen as a result of the original act. the traditional view of the creation, particularly of free human agents, challenges this idea. for in creating free human beings, where the real possibility existed that such beings could do evil, god, according to the traditional christian account, both did something that was morally permissible (the creation of completely free beings) and which resulted in the presence of evil in the world (e.g., human sin). but the problem here is that god's total observation involves no shift in responsibility from god to humans. humans are not responsible for god's total observation, as they may be in the case of their own sin exercised by free will. whatever responsibility there might be here seems to follow directly from god's omniscient nature. so while omniscience may excuse god's total observation, it simply does not follow without any further argument that human privacy has not been violated, that something morally bad has not occurred. to illustrate this point, consider a rather different but analogous case: a completely insane person, one who is utterly unaware of what he is doing and that it is morally wrong, kills an innocent child. this person would typically be excused on the grounds of insanity, but it remains true that a great moral harm or evil has occurred; an innocent life has been tragically lost. so, again, even if the cause of god's total observation is morally innocent, it is still not obvious that human privacy has not been violated. to show that this is not the case one needs to argue that god's total observation either (a) does not fundamentally disrespect human beings and/or (b) is completely morally justified; it is a great moral good or perhaps even an obligation that god has in virtue of god's moral perfection. in what follows, we will argue that both of these two options are correct. let us begin by considering and responding to arguments which try to show that (a) is false. this continues our discussion of an excuse for god's total observation. in the next section of this paper, we will offer a justification (i.e., [b] ). there are two lines of argument which could attempt to show that god's total observation fundamentally disrespects humans as persons. it could be argued (1) that essential to what it means to be a person is the entitlement of being the sole footnote 13 continued unnecessary. providing an excuse addresses primarily the issue of personal culpability, whereas justification focuses largely on the moral worth of an agent's reasons for doing some particular action. in many cases, providing both forms of argument can be essential to offering a complete moral case for some particular action or concern. so, for example, when an insane person kills another person, an excuse for the insane person (i.e., insanity) is usually acknowledged. but it might also be relevant that, although completely unknown or misunderstood by the insane person, the individual that he killed was in fact posing a lethal threat, and hence the insane person was also justified by self-defense in doing what he did. person who has access to one's own mental life. 14 and (2), it is standardly argued by kantians and other deontologists that autonomy is essential to personhood, and that this is violated in some way by any instance of non-consensual, total observation. these two suggestions share the basic idea that some feature essential to what it means to be a person is violated by god's total observation. both (1) and (2), of course, set out only a prima facie case for privacy violation. so if they are true, they at best still allow that total observation, even if wrong, might still be morally justifiable for some greater moral reason or purpose. 15 a defender of the traditional christian perspective now seems to have two main options. he or she could (i) accept that one or both of (1) and (2) are correct, and hence hold that total observation is always-even for god-prima facie morally wrong or bad, but then argue that some set of higher principles justifies the infringement of human privacy. or he or she could (ii) argue that neither (1) nor (2) is true, and then provide a reason for thinking that god's total observation is morally good or permissible. it is the latter, stronger line (ii) that we want to defend in what follows. let us turn to the first suggestion why god's total observation might disrespect humans as persons, namely, that being a person means that i alone should have direct access to my ''inner'' thoughts and experiences. this conception of a person involves the idea of an individuation of these inner thoughts and experiences as ones which i rightly should have sole and sovereign access to. something is terribly wrong, it is widely believed, if someone else is non-consensually observing or manipulating these inner processes. the idea is that when this happens, i would not be treated in the way that a person should be treated-namely, as a being who has a mental and emotional life which is, in some sense, separate from other persons. we will call this claim the ''separateness of persons thesis.'' it is important to note that for the separateness of persons thesis to bear any moral weight it must do so on its own accord, and not revert to the significance of moral autonomy. that is, the defender of the separateness of persons thesis must show that even if an infringement of the separateness of persons were accepted or welcomed autonomously by any of the parties involved, it would nevertheless be morally wrong. if this cannot be shown, the moral significance of the separateness of persons is reducible to issues of autonomy, and so autonomy rather than the separateness of persons should become our focus for cases of disrespect. it is very difficult, however, to argue that the separateness of persons thesis bears any serious moral weight on its own. consider the following hypothetical situation. through some technological miracle, a can ''connect'' to b in a way that gives 14 falls-corbitt and mclain (1992, p. 375) note something like this objection. they also acknowledge that reiman (1976) is another source of this kind of argument. 15 the prima facie quality of (1) and (2) could be denied, of course, by moral absolutists about privacy. general moral absolutism, however, is a fairly difficult position to defend, even though some christian moral philosophers and theologians may think that it is defensible. but whatever the philosophical defensibility of moral absolutism in general, it is a much more difficult matter, we would suggest, to hold that privacy is an absolute value or principle. no instance of true moral evil or sin would ever be excusable or unassailable simply because it was done ''in private.'' hence privacy should be considered, at best, only a prima facie value or entitlement, even if one's wider ethical position is absolutist. a and b mutual access to their mental experiences, and they can tell which experiences belong to whom. 16 now everything that a experiences, b will experience also-or at least be fully aware that a is having such experiences (and vice versa). if the separateness of persons thesis is alone sufficient, it would have to follow that a and b are fundamentally disrespecting each other's essential natures as persons. and this would be true, even if they had both equally and completely agreed, with full informed consent, to the connection. it would also be true even if the arrangement in no way seriously affected their own individual autonomies or the autonomy of anyone else. the problem here is that while we can think of many practical reasons why people might not, and perhaps should not, want to be connected like this, it is not at all clear that they have violated or devalued their own personhood in so being connected. this problem is further aggravated by the more ordinary fact that the close personal relationships that many people have developed seem to approximate the connection postulated in this hypothetical example. but, provided that all else is equal (e.g., the relationship is not marred by obsessive jealousy, distrust, etc.), it seems just false that such a relationship involves a fundamental disrespect for each member as a person to the extent that it approximates the converse of the separateness of persons thesis. in fact, in ordinary human relations, many people (lovers, life-time companions, family members, etc.) seek and value a similar closeness. no one usually takes anything to be seriously wrong when a close friend or loved one always seems to just know what the other is thinking, or what he or she is up to. in many contexts of love and trust such closeness is highly valued. furthermore, exactly this sort of relation of complete openness seems to be the one that conservative theologians standardly defend as the appropriate way for human beings to relate to god. so it is not at all clear that the separateness of persons thesis alone is sufficient to show that persons in any such relation are in a situation involving moral disrespect. the autonomy of the individual seems to be the crucial issue relating to disrespect. so let us turn to autonomy as a possible way of defending the claim that total observation is prima facie wrong. and, of course, the crucial feature of god's total observation of humans is that it does not depend on our consent. does this mean that god's observation of us would imply that our basic autonomy-and hence our essential nature as persons-is constantly and unjustly violated? it is important to notice first that simply observing someone does not obviously cause a person to lose her autonomy. observation interferes with autonomy only in those cases where the observation is explicitly unwanted. this general point is true both in situations where the observed is completely unaware that she is being observed and also in cases where she is completely aware of the observation. an example may help to clarify and stress these different claims. assume (for the sake of argument) that dealing in certain kinds of very harmful drugs is illegal and immoral. consider an important dealer of these drugs who is carefully and persistently watched (unknown to him) over a long period of time by police, and then is arrested for his crimes. in spite of the unknown observation, and given that there was no other interference with his autonomy, the dealer's activities remained autonomous: it was he who chose what to do, and it was he, without interference from the observing police, who chose to break the law. furthermore, he had no right-i.e., had no moral warrant because of his basic autonomy-to any privacy that would cover his illegal and immoral actions. he could not reasonably argue that his goal of committing an immoral act (say, the murder of a rival dealer) without observation had been violated, and thus his observers had disrespected him as an autonomous being. the point is that he had no moral right to that sort of autonomous action-namely, action which directly violates the autonomy of other people by harming them. so, in general, we could say that this person's autonomy was not violated or interfered with when the observation was unknown. the observation, of course, was unwanted. and in this case, we can reasonably assume that if the dealer had been aware of it, he would have (autonomously) changed his decisions and actions. thus observation seems to interfere with autonomy only when it is explicitly unwanted. the above argument focuses on immoral actions. but what about moral or nonmoral actions that are observed? it is important to note that even in cases where someone is engaged in a morally permissible action and is observed, it does not immediately follow that that person's autonomy has been violated. 17 to support a claim of privacy violation one would have to make the argument that the observation seriously affected the autonomy in this case of the observed person in order get any support for the claim of privacy violation. the point here is that mere observation does not entail that a person's autonomy has been violated. for one thing, people regularly make autonomous choices even though they know that other people either know or are observing what they do. this can be true even if they know that the observers disapprove of what they are doing. put more schematically: it does not follow that if a knew that b wanted to do x, and a disapproves of x, and b knows of a's disapproval of x, and where this disapproval might be coercive on b, then this alone would entail that b would be acting nonautonomously in doing x. practical examples of this principle seem innumerable. almost any adolescent that one can think of is, in at least some respect, acting under similar conditions. that is, she is often doing things where she knows that her parents both know that she is doing such actions and that they strongly disapprove of her doing those same actions. these features alone do not entail that the teenager is acting non-autonomously when, say, she gets some outrageous tattoo inscribed on her forehead. so it equally seems false that a person's autonomy can be compromised simply when someone (a) knows what one is planning to do and (b) disapproves of this plan (and one is aware of this disapproval). some other coercive element would seem to be necessary-e.g., a threat of serious negative outcome, a significant threat to deprive someone of something else that he or she might want, or some other effort to manipulate that person's choices. so merely being observed is not sufficient to entail that a person's autonomy has been violated. but, again, what about those cases where a person wants to do something she has every moral right to do and she does not want to be observed? we think that a satisfactory answer to this question comes from a consideration of the following two points. first, as argued above, god's omniscience entails that god's essential nature requires that all human action is known to god. given the unavoidability of this, god does not disrespect human autonomy because the total observation is something which cannot be avoided. no one is obligated to do something that one simply cannot do. and following the idea that morality is universally applicable to both god and all other moral agents, we must allow this in the case of god as well. this fact can help somewhat to explain the difference between human and divine obligations with respect to privacy. since humans clearly can avoid knowing things about other humans, we have a prima facie obligation-which god cannot have-to respect a person's autonomous choice to not be observed doing something that is morally permissible. 18 the second point is that it is no obvious threat to a person's autonomy that he or she must act under certain conditions over which he or she has had no choiceunless those conditions are such that they completely dictate the choice that a person makes. all human choice and action is performed under certain conditions. we take this, again, to be a widely accepted aspect of human action. so while we have no choice about the terms and conditions of our births, we do have autonomy with respect to many other choices-e.g., whether one will continue to live in the country of one's birth, whether one will go to university or not, and so on. in connection with privacy, one's choice of what things one can expect to be private meets analogous conditions about which one's consent is not relevant. if, for example, i appear in public dressed in a certain manner, it is simply unreasonable for me to expect other people not to notice what i am wearing. this is simply a condition of what it means to exist in a society where i live and make all of my other choices. i ought to, of course, have some control over how much publicity i should be exposed to. so while i cannot avoid the observation of people that i can reasonably expect to meet during the course of my day, it may be wrong for someone to publish what i am wearing in every newspaper in the worldespecially if i am not a ''public'' person. 19 but i clearly should have no moral expectation that i should not be seen if i appear in public and other people are there and happen to observe me. this basic point, generally accepted in human affairs, applies equally to the case of relations between god, humans, and other moral agents. living in the presence of god-which traditional christian, jewish, and muslim believers maintain that all of us do-just entails that i cannot expect god not to know what i do. i can, however, expect that whatever god knows about me will not be broadcast to the universe (at least not normally, and not without considerable moral warrant on god's part for such a broadcast). i can also expect that whatever god knows about me will not be used in some way to treat me unjustly or to defame me maliciously. and if traditional christianity is correct in its claim that god is morally perfect, all of these features would seem to be the case. furthermore, god's knowing what i do usually gives no one else-at least no other human-any knowledge of what i am doing. that remains, for the time being, private. i may some day have to account for what i do now in private before god and everyone else, but in the traditional christian account this ''judgment day'' is something which god, as the rightful moral judge of creation, is completely morally justified in doing. and furthermore, traditional christian theists maintain that in so judging, god will act in such a way that no one will have any legitimate moral complaint. thus the claim that god violates people's privacy with total observation because god does so without their consent is a misguided argument. when i appear every day in public, it is irrelevant that i had no say over whether there is a society out there which is such that its members can know things that are placed before them. my consent about such a condition is not possible. it is simply a fact about social life that i must accept, and then find whatever autonomy i can while living under it. 20 the same point, it seems to us, holds in the case of the abramic conception of god. god's being god is not something that has any possible relation to a matter that my consent could affect. and so it would be quite irrelevant to point out that god's full awareness of what humans are doing is something done without human consent. we have also shown that it need not have any bearing on our autonomy of a sort that would put god in the position of disregarding and disrespecting human beings as persons. thus far, our argument has only shown that god's total observation can be excused. this is sufficient for the negative claim that at least god's complete knowledge of human actions and mental states does not disrespect humans as persons. but we think that there is an argument, consistent with a fairly traditional version of christian theism, which goes much further and would justify god's total observation. if so, this argument will be useful, not merely as a way of understanding the conservative understanding of god-human relations, but as a defensible account of the conditions under which privacy may be infringed. that is, such an argument will provide us with the conditions-to the extent that they are applicable to human affairs-under which a person's privacy ought to be respected, and when it may rightfully be disregarded. traditional christian theism, of course, has specific teachings about many aspects of god's nature, but only three main features are of concern here. first, god is understood to be the sole creator and sustainer of the universe, and thus equally of all life within it. 21 this position, it could be argued, gives god the moral authority to observe all of creation. from the fact that someone has created or generated something, it does not strictly follow that one is thereby in a moral relation to that thing. and it certainly does not follow, without further argument, that one would have ultimate authority over it. but, in spite of this lack of strict entailment, in ordinary morality we do seem to accept a version of this principle. if i have children, and all else is morally equal (e.g., i am a responsible person, am able to raise children properly, etc.), then it is generally accepted that i have particular responsibilities and obligations toward them. i have authority (tempered by morality, of course) over my children that other people do not have largely because i have taken it upon myself to bring them into the world (or to adopt them as my own). it seems reasonable, then, to argue by analogy that god has authority over persons because god is the ultimate source or cause of our appearance in the world and of our continued existence. 22 secondly, traditional christian theology teaches that god is a morally perfect being. as such, god is concerned about establishing perfect justice-cosmic justice, it could be called-in creation. this sort of justice may require that every immoral act done without the knowledge of other human beings will be subjected to judgment and possible punishment by a justified moral authority. the world, it might be said, would be unjust if (say) murderers could get away with killing people simply because they can elude human authorities. since god has both the capabilities and moral perfection necessary to carry out such a task, it can reasonably be argued that god is morally justified in taking up the cause of cosmic justice, and hence engaging in total observation and documentation as the necessary means to achieve this goal. finally, god's perfect goodness may justify total observation. that is, it could be argued that god's purpose in total observation is not merely one of preserving justice; god's goodness means that in total observation, the (objective) needs and interests of human beings are not being violated, rather they are being promoted. this suggests that god's observation may be justified in terms of a maternal and paternal care for all humans (and all creation). there is surely nothing wrongindeed, we standardly believe there is everything right-with a parent who constantly observes her infant child to provide needed care and love. moreover, this reason is quite consistent with the previous reason mentioned, the pursuit of perfect justice. as children grow from helpless infants to active individuals, parents still watch and observe them closely to correct-i.e., to judge and perhaps punishinappropriate behavior. and this correction is, or should be, completely in line with 21 the bible, indeed, begins with ''in the beginning when god created the heavens and the earth'' (genesis 1:1). ephesians 4:4-6 also states: ''for there is one body and one spirit...one god and father of all, who is above all and through all and in all.'' (some translations here use the phrase ''father of us all'', which may leave it open as to whether st. paul, with the word ''us'', is referring only to believers. but the nrsv clearly seems to imply that paul is referring to god as the parent of everyone, believer or not. 22 note that the standard acceptance of this point in human affairs usually involves the idea that (all else being equal, of course) no child under legal age has the right to refuse to accept his or her parents' guardianship. so if the parent-child relation is analogous to the god-human relation, the same point would also hold. but this analogy, as noted further on in this paper, is controversial and requires further defense. their role as loving parents who have their children's well-being at heart. parents also closely watch a child's activities to provide positive encouragement and the capacity for self-development, and to re-enforce the vital fact that the child is loved and cared for. so if, as some conservative theologians standardly maintain, the relation of god to human beings is primarily a maternal/paternal loving one, it would be entirely appropriate for god to be aware of everything that we do. it may be objected that this argument may well apply to children and other nonautonomous persons, but it is inappropriate when applied to autonomous adults. human parents would simply step over an immoral line if they were to engage in the total observation of their autonomous, adult children. a traditional response to this kind of argument would maintain that the original analogy is correct: humans are in an analogous relation to god as infants are to their parents, and hence god's observation is analogously justified. 23 while this response might well be accepted by many conservative christians, we do not believe that it is the most defensible reply to the objection. the response is helpful, however, to the extent that it points to the differences that exist between the relations of god to us, and human parents to their adult children. one of the main reasons why the total observation of autonomous, adult children by parents is morally questionable is that parents cannot support the autonomous actions of their children in the same way that god can through observing our actions. in at least most western cultures, my autonomy is defined in part by my ability to make my own choices irrespective of the wishes of my parents. their will for me is something that i might (rightly and autonomously) follow, but equally it could be something that i could (again, rightly and autonomously) decide to go against. in fact, i could be promoting my well-being and following morality by exercising my autonomy in a way contrary to my parent's wishes. but this would be an improper description of my appropriate relation to god considered as my divine parent. god's will for me and everyone else (on the traditional view) really is what is best for me and everyone else as autonomous beings. similarly, from this same view, god never wills anything for me that is ultimately wrong or evil. and so to the extent that i exercise my autonomy in such a way as to follow god's will for me, i achieve what i really should be and become. in contrast, in the case of human parents, privacy assures to some extent that they will not interfere with my autonomy. and they could easily do this given that when i am an adult, my parents are in no better position than i am to determine what course my life should take. they are, generally speaking, as epistemically and morally fallible as i am. hence, even if they are well-intentioned toward me, their choices or intentions could still seriously and negatively affect my life. in the case of god, however, this sort of privacy is not necessary because none of these conditions hold. moreover, not having privacy, for traditional believers at any rate, is standardly seen as a great good, something which god does that actively promotes one's own well-being. no matter how dark, lost, or unknown to others my situation might be, it is traditionally taught, god is right there with me, caring when no one else can or does, empowering me in every way as i continue to put my trust in god. and since this great good would not be possible without god's knowing what is happening to me or what i am doing at all times, god is morally justified in total observation. and, again, we can say this while affirming the traditional conception of god as a loving parent, without conceding that humans are in a situation that is analogous to their non-autonomous infants or children. 24 a final, but important, point must be stressed about the three criteria we have just presented and defended. the criteria must be understood as jointly necessary conditions for privacy infringement. the reasons for this requirement should be obvious, particularly when we attempt to apply them to human political contexts. a state's having the requisite moral legitimacy alone gives it no moral warrant to observe its citizens voyeuristically. similarly, there must also be some issue of justice and well-being that is actually and immanently at stake. a state's interest in preserving justice alone does not provide sufficient warrant if that state itself lacks moral legitimacy. it could be suggested that there is much more detail that may need to be provided about the conceptual relationship of these criteria to each other. however, it should at least be apparent that these relationships serve to support an adequate theory of privacy infringement justification when they are applied together. so any person, and perhaps any institution of persons, who together has the requisite moral authority (due to being the generative and sustaining cause), has the intention of preserving justice, and is appropriately well-intentioned toward the persons observed, may, according to the above line of argument, engage in total observation. we must now ask whether these generally adequate criteria for god's total observation of humans have any chance of applying to ordinary human political situations. 24 lackey (1985, p. 390) has again raised what might seem to be a telling point against the argument we have just put forward here. he acknowledges that in personal relations people do often waive privacy for the sake of intimacy. but, of course, they do this voluntarily. people who desire closeness to god may waive their privacy, but this should not hold for people who, for whatever reason, do not want such a relationship. ''even if,'' he argues, ''it is a good thing for me to develop an intimate relationship with the deity, it cannot be said that i am morally bound to enter into this relationship. it is permissible for me to refuse, in which case the right to privacy remains in effect''. and so, he concludes, god violates the privacy of those who are unwilling to become close to god. lackey is surely right, here, in claiming that we have no moral duty to become close to god, even though it might be a good thing (objectively) to do so. furthermore, we also agree that humans-whether believers or not-do indeed have a right to privacy that is infringed by god's total observation. however, lackey's argument presupposes that there is no excuse or justification for this interference, whereas we have argued above, in ways not directly addressed by lackey, that there is a defensible excuse and justification. in the case of human families, as we have already argued, there is at least a surface reasonableness to the criteria. parents are standardly the generative source of their children, and this entails that they have certain prima facie rights, privileges, and obligations with respect to the privacy of their children. 25 furthermore, since parents are also responsible for sustaining their children in various ways, this role too seems to give parents the moral authority to observe their children in ways that it would be wrong for other people or institutions to do. part of parental responsibility and privilege is that of raising children according to certain moral standards. and this is widely accepted as justifying, for some period of a child's development, a reasonably close watch over much of what the child says, does, possesses, and so on. finally, as already mentioned, the care and love that parents have for their children morally justifies, up to some limit in development, the attention and observation that they give to their children. they have (or certainly ought to have) the child's best interests at stake. these intentions often require closely watching the child's behavior, communication, habits, and so on. we take the argument which focuses on family life just presented to be fairly obvious and widely accepted. however, when one tries to apply the main criteria for total observation applicable (traditionally) to god and parents to the state, difficult and intractable issues appear to arise. political philosophers profoundly disagree over the generative role of the state in relation to its individual citizens. many communitarians and, at the other end of the spectrum, socialists and marxists, tend to argue that the state plays a vital role in the formation of individuality, and hence the state possesses an authority in areas that might be seen as invasive by liberal political philosophers. on these non-liberal views, furthermore, the role of the state in ensuring the well-being and good of its citizens is not unlike that of a parent. liberals, on the other hand, from libertarians to left-leaning rawlsians, generally hold that the idea of the state as a sustaining parent is objectionably paternalistic. for these theorists, the claim that the state plays a crucial generative role in the formation of individuality is simply false and/or normatively objectionable. it is held that individuals bear primary responsibility for their own choices and thus for their own well-being. hence the suggestion that the state could engage in anything approaching total observation would be prima facie wrong. these preliminary observations seem to imply that, in the application of the three criteria defended above, we should expect fundamental disagreement particularly on the first and third criteria. this disagreement appears to be based largely on foundational issues in political theory; namely, the questions of the legitimacy of the state and the role that paternalism should play in governance. this leaves us with the second criterion that total observation would be justified in the pursuit of perfect justice. it seems here that most political theoretical perspectives should accept some privacy invasions in the pursuit of justice, simply because there does not seem to be any way to pursue many important forms of justice without such invasions. hence the disagreement we should expect regarding the second criterion would only be about the precise content and scope of such invasions. when we examine this matter more closely, however, we think there is in fact a much wider agreement about the three criteria, even among diverse political theoretical perspectives. take, for example, a fairly strong libertarianism which affirms the basic liberty and rights of individuals. such a libertarian would probably deny the generative relation of the state to individuals. in fact, she would argue, the truth is precisely the reverse: the state is generated by the interests and needs of its individual citizens, not the other way around. but even if this is true, any libertarian who defends the idea of state legitimacy will still not completely deny the sustaining qualities of the state. the properly functioning minimal state must acquire normative authority because, in part, it can provide adequate protection for its citizens, and hence allow them to be sustained as a community of individuals. this authority alone justifies the state in undertaking some-perhaps very minimalobservation of its citizens. libertarians also seem committed to some form of the idea that the observation of individuals, and hence some infringement of their privacy, is warranted on the grounds that their well-being as individuals is preserved. if the proper state is the one which offers even minimal protection for its citizens, and even if the legitimacy is based on individual self-interest, it will still be the case that, in some contexts, the state would be justified in violating privacy. unlike in the case of god, of course, we would not have any strong assurances that the information gathered by the state will only be used for morally defensible purposes, and so these contexts would be comparatively few and minimal in extent. so if the minimal protection state is acting correctly, it will sometimes be in an individual's rational self-interest to permit the state to engage in some level of observation, as collectively agreed on by citizens, on the basis of the well-being of all citizens. again, in such cases the observation would have to be fairly minimal and restricted in extent since, unlike in the case of god, we have much less-if any-assurance that the information will always be gathered for morally acceptable purposes. now if we accept that libertarianism is just an extreme or ''strong'' form of liberalism, other ''weaker'' versions of liberalism will yield to very similar considerations. it follows, then, that differing political theoretical commitments and perspectives do not provide any prima facie basis for rejecting the three criteria for total observation, provided these criteria are understood as general criteria. disagreement on political theoretical commitment, then, will appear only as a difference of opinion regarding the precise scope and application of the criteria. philosophers who are so committed should not reject their fundamental justificatory role in determining the value and extent of privacy. we have shown that what might seem to be a serious moral difficulty in christian philosophical theology can be effectively avoided, and that a potentially useful basis for a theory of just privacy infringement can be developed out of a response to this same difficulty. this, we believe, should provide moral and political theorists with a theory of justification regarding privacy infringement in ordinary human affairs. a theory of justification for privacy infringement, however, is only one aspect of what a moral theorist working on privacy needs to provide. some account of what privacy is, its nature or definition, needs to be given as well-even if this account only amounts to the claim that privacy is best understood as a very broad, heterogeneous concept. on the question of the definition of privacy, however, the philosophical literature is considerably unsettled. nevertheless, despite these difficulties, it seems reasonable to hold that any theory of the just infringement of privacy at least owes us some account of what privacy is. 26 in closing, then, we shall restrict our attention to a brief consideration of whether the justificatory argument provided here is relevant to some of the main definitional accounts given to the concept of privacy. three definitional accounts of privacy have held considerable attention. first, privacy is frequently understood to involve the right to control information about oneself and one's associates. 27 issues arise here, of course, as to what the scope and content of this ''information'' should be-i.e., whether it can be any trivial piece of information about myself that i do not want to become public, or whether it should be limited to something like ''personal'' or ''intimate'' information. secondly, privacy has also been conceived as having autonomy over specific (or not so specific) kinds of access to oneself. since one way of gaining access to a person is by getting specific or personal information about that person, access accounts generally allow that control over information may be necessary for privacy. but this concession must be seen as only one instance of all of the various other ways in which people can gain unwanted access to a person. an ex-husband who stealthily watches his former wife bathing is probably not gaining anything like information from his observation-at least not any new information, one might think. however, access-based theorists argue, it seems that the former spouse's privacy has been violated simply by the unauthorized access to her that her ex-husband has acquired. 28 finally, privacy has been given a fairly distinct legal definition by the united states supreme court in a series of well-known cases beginning with griswold v. connecticut (1965) , and including eisenstadt v. baird (1972) and roe v. wade (1973) . the general idea here is that privacy consists in the right to make certain decisions of a ''personal'' nature. constitutional privacy thus is understood as a particular form of autonomy that we should have in being free from state 26 it was not the purpose of this paper to provide any such definition. for our own efforts in this regard see [soifer and elliott (2014, p. 195f) ] where we argue that privacy is best understood in connection with the judgment of others or the concern about one's reputation. this view, or something similar to it, can also be found in prosser (1984 , p. 112), feldman (1994 , johnson (1989) , velleman (2001) , and marmor (2015) . 27 warren and brandeis (1890) can be seen as an example of this way of understanding privacy. even though they conceive of privacy as a specific tort that falls under the wider right that individuals have to be left alone, it is clear that it is certain kinds of information about an individual which needs to be ''left alone.'' a more recent and straightforward example of the informational account can be found in parent (1983) . 28 a widely discussed defense of an access-based account can be found in gavison (1980, p. 428f) . see also solove (2008, pp. 18-21). interference in making certain intimate and personal choices (e.g., family planning and contraceptive use [griswold] or abortion [roe] ). the question now is whether the account of invasion justification defended in this paper is relevant to any of these three main ways of understanding privacy. we would suggest that the account given here is directly relevant for either the information-or access-based conceptions. this is because the paradigm case of privacy violation is total observation. in total observation, whatever else might be at stake, it seems clear that at least information about and/or access to a person is of direct moral concern. total observation acquires complete, non-consensual information about a person. and it equally seems to provide a tremendous extent of non-consensual access. hence our account, which bases its justification from the context of total observation, is directly relevant to these two understandings of privacy. in contrast, constitutional privacy defines privacy largely, if not exclusively, in terms of the exercise of certain kinds of choices, or establishes certain zones of autonomous choice surrounding specific issues. it is therefore not obvious that the privacy invasion theory presented here fits very well with constitutional conceptions of privacy. setting aside cases where people do not want such decisions to be known or observed, as we have argued, people can still make the decisions covered by constitutional privacy, even if they are subject to total observation-although, of course, they would not be able to prevent others from knowing what decisions they had made. so how then can the account of privacy invasion justification we have defended be relevant to constitutional accounts of privacy? an obvious response at this point would be to argue that constitutional ''privacy'' is simply not a true account of privacy at all. many philosophers and jurists have defended just such a claim. 29 for the purposes this discussion, however, we do not want to adopt this line of argument. rather, the concern about constitutional privacy can be addressed by the following considerations. first, our account does apply to those cases of constitutional privacy where people do not want the state either to document or observe their protected choices. it seems that this condition is true for most understandings of constitutional privacy. most people who would insist that citizens in a liberal democracy (should) have the legal right to make (e.g.) family planning decisions also understand-rightly or wrongly-that this same right includes the (prima facie) right not to have such decisions documented or observed by the state. since it is possible, as argued above, for the autonomous actions in question to be carried out in spite of observation, this consideration alone may not seem to address the concern about the relevance of our account for constitutional privacy. furthermore, the state, properly governed by considerations of public good, 29 h.j. mccloskey, in both (1971) and , has argued extensively that it is a mistake to conflate privacy with autonomy (which, of course, is what most constitutional accounts effectively do). parent (1983, p. 284 ) also writes that: ''confusing liberty with privacy only serves to impugn the intellectual integrity of the judiciary.'' the reason for this, parent argues, is that the conflation of privacy as a part of liberty is a conceptual confusion. the defining idea behind ''liberty'' is the absence of external restraints or coercion. but this is clearly distinguishable from the right to privacy which, as he puts it, should only be understood as that which ''condemns the unwarranted acquisition of undocumented personal knowledge'' (p. 274). may have an interest in monitoring such decisions. if so, constitutional privacy understood strictly as a specific kind of autonomy may be protected even when something approaching total observation is in place. that is, it would make sense to think that there are private zones where certain kinds of autonomy can be exercised without direct interference by the state. this last point, however, indicates, not the irrelevance of the account of privacy infringement defended here, but its significant relevance. considerations of justice and well-being (and by implication, the basis of state authority over such matters) do provide clear limits on the exercise of constitutional privacy. personal and/or physical intimacy with another person, which should clearly fall under the scope of constitutional privacy, may have to be documented if justice to all persons is in question because of (say) concerns about communicable disease. in the case of an outbreak of a serious communicable disease (e.g., the ebola virus or a nasty influenza pandemic), people can have their actions severely restricted. people can be forbidden to visit their loved one in a quarantined hospital, even if he or she is seriously ill or dying. 30 such patients are thus often required to suffer and even die alone, physically separated from any society which it is otherwise their right to have. it is difficult to think of a more important, personal, and private matter than that of providing care or holding the hand of a dying loved one. and yet even this invasion into the private lives of people can perhaps be defended in terms of justice and care for the public good. whether measures like these really are justified invasions or not is not, however, the focus of this paper. the point is that the considerations noted are clearly relevant, and this shows that the account defended here is indeed relevant to constitutional understandings of privacy. summa theologica the teachings of maimonides privacy and control privacy and perfect voyeurism god and privacy secrecy, dignity or autonomy? views of privacy as a civil liberty privacy and the limits of law privacy and the judgment of others privacy and the law: a philosophical prelude divine omniscience and human privacy guide for the perplexed what is the right to privacy the political ideal of privacy measures identical or similar to these were put in place some years ago in the sars epidemic in toronto, canada in 2003. during this epidemic, when a patient in a particular hospital was discovered to have sars, the entire hospital would be put under quarantine. the effectiveness of such an extensive quarantine, however, has since been strongly questioned privacy and the right to privacy just looking: voyeurism and the grounds of privacy privacy, morality, and the law philosophical dimensions of privacy: an anthology privacy, intimacy, and personhood severe acute respiratory syndrome: did quarantine help? nonstandard observers and the nature of privacy understanding privacy the coherence of theism is there a god? privacy and autonomy: a reappraisal the genesis of shame. philosophy and public affairs the right to privacy key: cord-016743-k5plq0ja authors: mohammed, yousuf h.; moghimi, hamid r.; yousef, shereen a.; chandrasekaran, navin c.; bibi, césa r.; sukumar, sinduja c.; grice, jeffrey e.; sakran, wedad; roberts, michael s. title: efficacy, safety and targets in topical and transdermal active and excipient delivery date: 2017-01-25 journal: percutaneous penetration enhancers drug penetration into/through the skin doi: 10.1007/978-3-662-53270-6_23 sha: doc_id: 16743 cord_uid: k5plq0ja a key requirement for topical and transdermal active delivery is the effective delivery of an active to a desired target site, to achieve both safe and efficacious outcomes. this chapter seeks to explore the importance of the pharmacological, toxicological and therapeutic properties of actives and excipients, as well as the site of action as complementary components in percutaneous absorption. this is crucial for optimized topical and transdermal product design. properties of the skin to be completely destroyed, allowing most actives to be delivered in high concentrations. this, in turn, may allow the ingress of previously innocuous excipients and external agents which can then lead to skin irritation and allergic responses. accordingly, the key question that should be asked is: "is the transdermal route suitable and required for the proposed therapeutic agent?" obviously, an active such as acetaminophen, with an oral dose in the hundreds of milligrams, can never be delivered at a sufficient percutaneous flux to achieve efficacy. alternatively, the skin provides for a regulated, constant delivery rate and much lower first pass metabolism than oral delivery and so may be the best route of delivery for certain actives. however, many actives for which transdermal delivery could be the only viable option are yet to be formulated effectively. this is largely because their physicochemical properties make them unsuitable candidates for skin permeation (barry 2001) . having an appropriate efficacy and potency for a therapeutic agent is a key pre-formulation consideration and will govern the selection of a transdermal delivery technology strategy. such a strategy must also take into account the various factors affecting skin penetration, such as the condition of the skin to be treated (for example, whether it is healthy or diseased), the physicochemical properties of the penetrating molecule, the formulation in which the penetrating molecule is applied and the required dosing conditions (wiechers 1989 ). an overriding consideration is also the aesthetic or sensorial properties of the product, as perceived by the user -its appearance, feel, odour, residue, stability, etc. accordingly, we support johann wiechers' proposed percutaneous product goal: "both dermal and transdermal delivery aim to achieve one goal summarized in the four r's of delivery: to deliver the right chemical at the right concentration to the right site in (dermal delivery) or beyond (transdermal delivery) the skin for the correct period of time" (wiechers et al. 2004 ). however, we also suggest the inclusion of a fifth r: "…with the right sensorial feel to the consumer". the formulation of a topical product should start with the choice of an appropriate active. the two considerations here are safety and efficacy. hayden et al. (2005) assessed the safety of five sunscreens by determining their in vitro toxicity to human keratinocytes in culture then estimated the equivalent concentration to inhibit 50 % of cells in viable epidermis, after adjustment for the differences in protein binding in the two media ( fig. 23.1 ). it is evident that the observed viable epidermal concentrations after topical application are somewhat less than either the observed ic 50 in cultured keratinocytes or the equivalent estimated ic 50 for viable epidermis, based on adjustments for differences in sunscreen binding in culture and in the viable epidermis. although oxybenzone has an estimated unbound viable epidermal concentration of >50 times or more than those of the other sunscreens, this is still only 20 % of the ic 50 determined in keratinocyte culture. these findings can be summarized by defining a cutaneous margin of safety after topical application as the ratio of concentration of active and excipient associated with toxicity (e.g. ic 50 ) to the viable epidermal concentration of the active or excipient. a desirable margin of safety based on human models should be at least 10 to account for inter-subject variability. toxicities, such as irritancy, photosensitization and corrosivity, are likely to be better defined by the probability of an event occurring in a given patient after a particular exposure and are dependent on patient susceptibility and the nature and conditions of the product used. the second safety measure of interest is the systemic margin of safety which arises when an active applied to the skin for local cutaneous effect enters the systemic circulation and causes systemic side effects. this concept is most highly developed for topically applied corticosteroids and, in this case, is quantified by the extent of suppression of the adrenal gland in the hypothalamic-pituitary-adrenal axis. a systematic literature review of the risk of adrenal axis suppression and skin atrophy after application of topical corticosteroids in plaque psoriasis for the period 1980 to january 2011 found morning cortisol was reduced in 0-48 % of patients in 11 short-term studies (castela et al. 2012) . thong et al. (2007) have listed a range of topical drugs and chemicals known to cause systemic effects. the agents include anti-infectives, antihistamines, minoxidil, insecticides, solvents and steroids. the corollary measure to safety is cutaneous efficacy and, in general, the more potent the topical product used, the less skin flux required to achieve a desired local cutaneous effect after topical application. a commonly used approach, analogous to that described earlier for toxicity, is to estimate the free drug concentration at the local target site (c * ), which, for antivirals, is assumed to be the epidermal basal cell layer (patel et al. 1996) . afouna et al. (1998) analysed the receptor solutions for full thickness hairless mouse skin mounted in franz diffusion cells. assuming that receptor sink conditions applied, c * was estimated as the ratio of the steady state penetration flux (j s ) to in vivo dermis permeability coefficient (k p, d ): these c* values derived for various formulations of bromovinyldeoxyuridine (bvdu) and acyclovir (acv) were then compared with the in vivo efficacy of the formulations against cutaneous her-pes simplex virus type 1 infections in hairless mice. figure 23 .2 shows a composite graph of their findings. it is evident that a similar concentration response relationship exists for the 95 % dimethyl sulfoxide (dmso) + 5 % azone for both bdvu and acv and for the 95 % dmso only formulation for acv, whereas much higher bvdu c* values are needed for the bvdu in 95 % dmso only formulation. they surmise that azone could have two effects. firstly, it may either enhance penetration of the active. this is evident from the 6.5 times higher c* for 5 % acv in 95 % dmso +5 % azone than in the 95 % dmso only formulation. the effect is less for bvdu, where the c* for 5 % bvdu in 95 % dmso +5 % azone is 1.65 times that of 95 % dmso only formulation. secondly, azone may have a synergistic effect with the antiviral agents on viral kill, evident in the shift towards higher potencies of c * for bvdu but not for acv ( fig. 23 .2). a key assumption made in this analysis is that the dermal permeability coefficient (which is normally expressed in the pharmacokinetic literature as a dermal clearance (cl d ) (siddiqui et al. 1989 hayden et al. (2005)) when applied topically, showing the enhanced response that can be achieved by targeted topical application. davis has recast the free drug concentration approach described above in eq. (23.2), which describes the efficacy of the active in terms of both potency of the active and how much penetrates to a target site (davis 2008; jepps et al. 2013) : a major issue not fully recognized in the work of cordero and davis (cordero et al. 2001; davis 2008) is that the fraction of active bound in the in vitro system used to determine potency may differ from that present in skin penetration studies and in vivo. in order to simplify the present description and be consistent with the work reported by cordero and davis, this important correction will not be included in the following discussion. hence, if a cutaneous concentration for a desired effect is defined as c eff and that observed after topical application as c ve , the efficacy is given by the ratio c ve /c eff . c ve is defined by the penetration flux divided by the clearance from the viable epidermis. cordero et al. assumed that this clearance, in turn, was related to the in vitro dermis diffusion and thickness. in reality, as discussed earlier in referring to the work of afouna et al (1998) , this should be in vivo clearance. our own work supports this, suggesting that, firstly, uptake by the blood in the dermal capillaries located just below the viable epidermis is likely to be the rate limiting determinant of clearance in vivo and secondly, carriage of topical non-steroidal anti-inflammatory drugs (nsaids) to deeper tissues below that application site is also dependent on blood flow for highly plasma protein-bound drugs (dancik et al. 2013a, b) . accordingly, we have, for illustrative purposes, assumed both constant binding and clearance and expressed efficacy simply as a ratio of flux divided by the in vitro minimum inhibitory concentration (mic), as originally proposed by mertin and lippold (mertin and lippold 1997) . thus, a greater efficacy is provided by an active with either a higher flux or a greater potency (a open symbols: formulations in dmso without azone; closed symbols: formulations in dmso with 5 % azone (adapted from afouna et al. (1998)) lower dose for the same effect). these concepts are illustrated in fig. 23 .3, where data from cordero et al. and others for saturated skin flux ( fig. 23.3a) , anti-inflammatory activity ( fig. 23 .3b) and efficacy ( fig. 23 .3c) for a range of non-steroidal antiinflammatory drugs are shown (mertin and lippold 1997; wenkers and lippold 2000; cordero et al. 2001; jepps et al. 2013) . in fig. 23 .3a, the shaded area indicates the region of optimal skin penetration occurring at a log p of around 3 (yano et al. 1986; zhang et al. 2009 ). ketorolac and ketoprofen, in this range, consequently have the greatest saturated flux. the more lipophilic actives also have a greater in vitro performance index of anti-inflammatory activity (i.e. lower mic), and so these two compounds also have high efficacy coefficients. diclofenac, although having a less favourable log p of >4, still has the highest predicted efficacy coefficient as a consequence of having the lowest mic. on the other hand, piroxicam has the least saturated flux of all these actives, as well as a relatively high mic, and consequently has the lowest efficacy coefficient of these non-steroidal anti-inflammatory drugs (nsaids). apart from a comparatively higher molecular weight and melting point, hydrogen bonding seems to be playing a pivotal role in the lower flux of piroxicam (anrade and costa 1999) . adrian davis (davis 2007 ) also presented efficacy and systemic toxicity indices for topical immunosuppressive agents used in treating atopic dermatitis, extracted from trottet's phd thesis (trottet 2004 ) at the sci meeting in 2007 (see fig. 23 .4). the corresponding margins of safety for cyclosporin a, tacrolimus and pimecrolimus, calculated as the ratio of the systemic safety indices and the efficacy indices, were 2400, 47 and 457, respectively. the systemic efficacy of many actives, such as anticonvulsants, anti-infective and cardiac drugs, can be defined by plasma concentrations after dosing by a particular route of administration. as shown in fig. 23 .5, in transdermal delivery occurring from a constant rate transdermal passive delivery patch, there is a lag prior to reaching maximal levels and in returning to baseline on patch removal (roberts and walters 2007) . further, after reaching a maximum, the serum levels slowly decline with time, consistent with a reduction in flux due to a gradual depletion in the amount of active in the patch. topical products, especially transdermal patches, are often required to provide constant therapeutically effective plasma concentrations, c ss . the penetration flux j s ideally needed to reach such concentrations is given by eq. a key goal in formulating an active in a transdermal product is to obtain a desired steady state skin penetration flux (amount penetrated over a period of time) of the active. j s is defined by eq. (23.4) (reeve et al. 2013) , where j sat is the flux from a saturated solution (often referred as maximum flux (magnusson et al. 2004 )), c v is concentration of the active in a given formulation and s v is the solubility of the active in the formulation. f v represents the factional solubility of the active in that formulation, as is evident from eq. (23.4), one way to maximize skin penetration is to use a fractional solubility approaching unity. poulsen led a range of studies that showed maximal skin penetration will occur when an active is at saturation in a formulation (poulsen et al. 1978) . as discussed in depth by davis (2008) , if an active is used at a concentration below its solubility in the vehicle, the product will normally have a lower efficacy than if the product was used at saturation. consequently, products may be formulated to contain more active than will actually be needed for an adequate effect, as is the case with transdermal fentanyl patches, which have led to numerous deaths. the danger inherent to fentanyl is its potency (greater than 50-100 times that of morphine) and rapidity of action (jumbelic 2010 ). an optimal dosing strategy demands that the rate of active penetration into the skin will achieve and sustain biologically active free levels at the target site within the specified limits. also, ideally, these conditions should be independent of skin site and skin condition. this is a considerable challenge, given that there is wide variability in skin permeation, depending on the anatomical site (rougier et al. 1988 ) and the condition of the skin, such as in particular disease states (elias and feingold 1992) or levels of hydration (roberts and walker 1993) . as discussed in other chapters in this book, another strategy to increase efficacy is to use a saturated active solution in the vehicle and to further increase either the stratum corneum solubility s sc and/or its diffusivity d sc using co-solvents and (2008)) enhancers, in accordance with eq. (23.5), where h sc is the stratum corneum thickness: however, care has to be applied in representing such saturated fluxes as maximum fluxes because increasingly, formulations are being designed to include a volatile component that may result in enhanced skin penetration due to the generation of a transient supersaturated state. many researchers have now promoted the use of supersaturated solutions (coldman et al. 1969) , with several showing that these can lead to even higher fluxes than those achieved with saturated solutions (lippold 1992; morgan et al. 1998; iervolino et al. 2001; timothy et al. 2002; santos et al. 2012 ). more recently, there is an increasing tendency to either stabilize supersaturated products by using polymers (raghavan et al. 2003) or to generate supersaturation on application to the skin as a consequence of the evaporation of the volatile components in the product . supersaturated systems may be obtained either by design or via solvent evaporation or by mixing of co-solvents. the stability of supersaturated solutions is an important issue, and it would be useful to develop supersaturated systems that are stable throughout the product shelf life. santos et al. (2011) investigated the permeation of fentanyl from supersaturated for-mulations across silicone membranes. supersaturated formulations containing either propylene glycol (pg)/water or pg/ethanol were prepared with varying degrees of saturation (ds) of fentanyl. both pg/water and pg/ethanol formulations showed good correlation between the flux and ds. the enhancement observed for pg/ ethanol formulations confirmed that enhanced active thermodynamic activity was induced due to ethanol evaporation. in further studies, tapestripping was used to show that supersaturation of the active is maintained in the outer layers. however, if the active lacks potency, a penetrating active will still prove to be inefficacious. obviously, an improved evaluation of product efficacy and safety will contribute to better therapeutic outcomes. whereas the determination of bioequivalence for two solid oral dosage forms is based on a comparison of active and/or its metabolite concentrations in blood or urine after dosing from a generic product or that of an innovator in healthy volunteers, such considerations are really only appropriate for transdermal products meant for systemic effect. for most topically acting actives, such an approach is not appropriate as the site of action is local, not systemic. hence, generic dermatological products are only required to demonstrate bioequivalence in clinical trials, including the vasoconstrictor test used to assess the efficacy of topical corticosteroids (shah et al. franz et al. (2009) suggested that a substitute for clinical bioequivalence testing is to define the penetration fluxes of an active through excised human skin. their evaluation, based on glucocorticosteroids and generic tretinoin gels, indicated that the in vitro model had greater sensitivity than the clinical method in detecting small differences between two products (franz et al. 2009 ). it is unclear, however, how this approach can overcome the well-known wide variability in excised human skin permeability. more recently, lehman and franz have assessed the bioequivalence of topical retinoid products using pharmacodynamic assays (lehman and franz 2012) . there is a range of skin and systemic sites often targeted by topical and transdermal delivery, summarized in fig. 23 .6. in this section, we illustrate some of the actives and their formulations used to achieve appropriate targeted delivery by topical or transdermal delivery. the skin is the site of application of numerous cosmetics and cosmeceutical preparations. in this section, we will discuss the importance of targets on active delivery choices. testing for safety of cosmetic preparations will be discussed in the following sections. a chemist's role in overcoming an active's physicochemical properties to make it suitable for topical and transdermal delivery is a complicated task. formulators have made use of physicochemical characteristics like molecular weight to stop an active from penetrating through the skin. one such example is a high molecular weight (>500 da) uva and uvb absorber tinosorb ® m (bisoctrizole, inci: methylene bis-benzotriazolyl tetramethylbutylphenol (and) aqua (and) decyl glucoside (and) propylene glycol (and) xanthan gum) developed by ciba chemicals (now part of basf, ludwigshafen, germany). similarly l'oréal's sunscreen with uvinul ® n539 (2-ethylhexyl 2-cyano-3,3-diphenyl-2-propenoate, inci: octocrylene), parsol ® 1789 (avobenzone; inci: butyl methoxydibenzoylmethane) and mexoryl ® sx (mw-563; ecamsule, inci: terephthalylidene dicamphor sulfon) was formulated to reduce solar-uv-induced skin damage (seité et al. 2000) . sunscreens containing ecamsule (mexoryl ® sx) are exclusive to l'oréal and its brands. chlorhexidine is another example of a molecule which binds extensively to the skin surface due to its cationic nature (judd et al. 2013 ). this ensures that it does not penetrate into the skin and rather provides for a sustained antimicrobial action when used topically (karpanen et al. 2008) . insect repellents are another good example of actives that are not intended to penetrate. penetration retardants and modifiers have been used for this purpose (kaushik et al. 2010 ). on the other hand, terbinafine, a highly lipophilic but equally potent antifungal active, with a log p of 5.5, needs a different approach. when formulated as a microemulsion to enhance its stratum corneum solubility using a mixture of surfactant, co-surfactant and oil, terbinafine exhibited enhanced permeation parameters. microbiological studies of the microemulsion showed better antifungal activity against candida albicans and aspergillus flavus compared to marketed products (baboota et al. 2007 ). retinol, with a high molecular weight and lipophilicity, is another active that is used for topical treatment of acne. these unfavourable properties were overcome by delivering retinol using solid lipid nanoparticles (sln). the suitability of the slns was compared to a nanoemulsion of the same size, with respect to the ability to influence active penetration and distribution within the skin. in both formulations, 500 μg retinol was applied to porcine skin. following the sln dispersion for 6 h, a high retinol concentration was found in the stratum corneum and upper epidermis (approximately 3400 ng or 0.68 % in the top layer). the nanoemulsion, however, only transported 2500 ng (0.5 %) into the upper skin strata (jenning et al. 2000) . for specific disease, conditions where the surface of the skin itself is the target of therapy, formulation and delivery strategy are of greatest importance. creams, lotions, emulsions and other topical delivery vehicles have been used effectively for this. the challenge here is to modify the formulation according to the active characteristics and skin condition. being the largest organ, skin is exposed to the damaging effects of uva and uvb radiation, often leading to erythema. antioxidants such as vitamin a, vitamin e and vitamin c have shown beneficial effects in reducing oxidative damage to critical cellular components (trevithick et al. 1993; stamford 2012) . while the photoprotective effect of topical antioxidants applied before uv exposure is well recognized, the beneficial effect of these compounds when administered after irradiation is less obvious (wu et al. 2012) . only vitamin a has been proven to be benefi-cial in treating long-term photoaged and photodamaged skin (cho et al. 2005) . the stratum corneum is a major barrier to the penetration of lipophilic vitamin e (cassano et al. 2009 ), and hydrophilic vitamin c is prone to excessive oxidation. oxidation of vitamin c is triggered by its ionization in aqueous solution (stamford 2012) . another example where erythema and papular oedema lesions are seen is herpes labialis. in a recent study, iontophoretic application of 5 % acyclovir cream was tested in a placebocontrolled trial for treatment of herpes, among 200 patients with an incipient cold sore outbreak at the erythema or papular oedema lesion stage. a 20 min iontophoretic treatment cycle shortened the median classic lesion healing time by 35 h in the active group when compared to the control (morrel et al. 2006) . steroids have also been popularly used in the treatment of erythema and other inflammatory skin conditions (perry and trafeli 2009; paller 2012) . numerous studies have examined various factors controlling percutaneous absorption rates and dermal clearances of steroids (siddiqui et al. 1989 ). the lipophilicity of steroids has been associated with their tissue accumulation and toxic effects. however, magnusson et al. showed that the tissue accumulation of steroids was not linearly related to their lipophilicity, but the binding of steroids to tissue components led to their accumulation (magnusson et al. 2006 ). long-term corticosteroid usage has been associated with skin deterioration, abnormal skin aging and cutaneous mast cell depletion (lavker and schechter 1985; gonzalez and sethi 2012) . one of the most widely explored aims of cellular targeting has been in the viable epidermis itself. inflammatory conditions broadly known as dermatitis or eczema are largely reflected in epidermal involvement (elias et al. 1999) , including spongiosis, acantosis and parakeratosis, as well as some dermal involvement (freeman-anderson et al. 2008) . the classical treatment for such conditions is the use of topical corticosteroids, which can be classified on a four-point scale of potency (i-iv, mild, moderate, potent, very potent), based on their vasoconstrictor effects in humans. the body site to be treated determines the potency of the steroid to be used. for example, the face should be treated with mild corticosteroids only, such as hydrocortisone and hydrocortisone acetate. care needs to be taken with the more potent steroids and in the treatment of chronic conditions. intermittent dosing can be effective in these circumstances. salicylic acid has long been used for conditions such as psoriasis and ichthyosis that require keratolytic treatment. at lower concentrations, salicylic acid is known to have keratoplastic effects; however at higher concentrations, it is found to be keratolytic (gloor and beier 1984; schwarb et al. 1999) . coal tar, retinoids and anthralin, as well as corticosteroids are some other commonly used treatments for psoriasis (mitra and wu 2010) . psoriatic skin is one such example where the "rigidization" of skin, ascribed to an increase in the levels of cholesterol and fall in the levels of ceramides and the lack of hydration, makes the topical route a big challenge (wertz et al. 1989 ). the intricacies of topical delivery into psoriatic skin have lately been addressed by lipoidal carrier systems, such as liposomes. recent work by ward et al. describes the delivery of novel immunotherapy using a liposomal platform. liposomes synthesized to incorporate clodronate were injected intradermally into kc-tie2 mice. an elimination of the cd11c+, f4 ⁄80+ and cd11b + cells was seen in the skin, and the cd8+ t-cell numbers returned to levels seen in control mice. further, there was also a marked reduction in the levels of interleukin (il)-1a, il-6, il-23 and tumour necrosis factor (tnf α ) (ward et al. 2011) . peptides and proteins such as elafin and psoriasin are known to be highly upregulated in psoriatic skin (madsen et al. 1991; nonomura et al. 1994; namjoshi et al. 2008) . peptide t analogue, dapta, was shown in clinical trials to clear psoriasis lesions and significantly inhibit the monocyte and lymphocyte chemotactic properties of rantes (a beta chemokine found in increased amounts in psoriatic lesions). a better understanding of the role of these peptides may provide new strategies for the treatment of diseases such as psoriasis (harder and schröder 2002) . the synthesis and distribution of melanin contribute to skin and hair colour. however, melanin and pigmentation-associated skin cancers are a major problem, while there is also interest in cosmetic skin whitening treatments where increased epidermal melanin synthesis causes unwanted darkening of the skin. accurate determination of cutaneous melanin in normal skin and in disease conditions requires the collection of skin biopsies. however, our recent work has used multiphoton tomography and fluorescence lifetime imaging to non-invasively measure levels of epidermal melanin, which can be related to the visible skin colour (dancik et al. 2013a, b) . the use of these techniques has implications for lesion diagnosis and assessment of therapeutic progress. proteins and peptides play an important role in skin health as structural components of the skin as well as activators, regulators or inhibitors of certain biochemical processes occurring in the skin (heidl 2009 ). peptides as cosmeceuticals are a fast-growing segment of the personal care industry with an increasing trend towards the use of these agents in skin care regimens for protection from radiation and oxidant damage (brandt et al. 2011) . peptides also play an important role in melanogenesis. melanocyte-inhibiting factor (also known as pro-leu-gly-nh 2 , melanostatin, msh release-inhibiting hormone or mif-1) is an endogenous peptide fragment derived from oxytocin (taleisnik 1971) . melanostatin is shown to inhibit melanin formation in streptomyces bikiniensis nrrlb-1049 and b16 melanoma cells. a patent filed in 2010 describes a "one-step" process for production of esters and acetylated forms of peptides, describing melanostatin (mif-1). this chemical modification approach improved metabolic properties leading to increased efficiency for therapeutic and cosmetic purposes including transdermal and topical administration (skinner 2007) . melanocyte-stimulating hormone analogues that function as melanocortin 1 receptor (mc1-r) agonists have been investigated as potential topical agents to prevent skin photocarcinogenesis. in cultured human melanocytes, tetrapeptide α-msh analogues, ac-his-d-phe-arg-trp-nh 2 , n-pentadecanoyl and 4-phenylbutyryl-his-d-phe-arg-trp-nh 2 were more potent than α-msh in stimulating the activity of melanogenesis, reducing apoptosis and release of hydrogen peroxide and enhancing repair of deoxyribonucleic acid (dna) photoproducts in melanocytes exposed to uv radiation (abdel malek 2006) . melanostatin-5 (aqua-dextran-nonapeptide-1) is a new skin lightening biomimetic peptide. this peptide acts as a α-msh antagonist thus preventing and lightening hyperpigmentation (dhatt 2006) . melanostatin-5 produces noticeable skin lightening by at least 33 % when formulated into a lightening product, and showed continued improvement over time. skin has evolved a highly competent immunological function, in langerhans cells (lcs -estimated population 500-1000 cells mm −2 ) (berman et al. 1983; chen et al. 1985) , which often serves as the first line of defence against many pathogens (babiuk et al. 2000) . the viable epidermis lacks the blood vessels and sensory nerve endings found in the dermis, which makes it an ideal site for pain-free delivery with minimal damage. there are many approaches for non-invasive targeting of viable epidermal cells and dna vaccination against major diseases. there have been attempts at displaying foreign peptides and proteins on virus particles and insertion of mammalian cell-active expression cassettes in baculoviruses to express genes efficiently into many different mammalian cell types (paul et al. 2013 ). pearton et al. showed morphological changes in human lcs stimulated with influenza virus-like particulate vaccines delivered via intradermal injection. the lcs were more dispersed throughout the epidermis, often in close proximity to the basement membrane, and appeared vertically elongated, which provides essential evidence of host response (pearton et al. 2010 ). there are many physical approaches, both old and recent for targeting lcs; we will try to mention them in brief. the most well-known technique was: (a) needle and syringe: this is an inefficient and indirect way of targeting the dendritic cells with dna. it has resulted in modest immune responses (mumper and ledebur 2001) . other disadvantages include risks due to needle stick injuries (gill et al. 2009; sullivan et al. 2010 ). (b) liquid jet injector: this is a needlefree approach using high-speed liquid jet injectors (furth et al. 1995) (mark 2008) . this technique has seen a recent resurgence, with liquid delivered around the lcs in gene transfer and dna vaccination experiments (furth et al. 1995) . current liquid jet injectors typically disrupt the skin in the epidermal and dermal layers to target lcs. liquid jet injectors however have been reported to cause pain to the patients. (c) microneedle arrays: unlike current liquid jet injectors, microneedles can accurately target the viable epidermis, are simple to use when built up as patches and are relatively pain free. mcallister et al. (2003) have shown that they can be made from a range of materials, including silicon, metal and other biodegradable polymers. this makes microneedles a cost-effective method for delivering oligonucleotides to epidermal cells for dna vaccination (kendall 2010) . (d) biolistic microparticle delivery: in this needle-free technique, pharmaceutical or immunomodulatory agents, formulated as particles, are accelerated in a supersonic gas jet to a sufficient momentum to penetrate the skin (or mucosal) layer and achieve a pharmacological effect. sanford and klein (sanford et al. 1987 ) pioneered this innovation with systems designed to deliver dna-coated metal particles (1 μm diameter) into plant cells for genetic modification. follicular targeting has been approached as another way of targeting the lcs (patzelt and lademann 2013) . vogt et al. (2006) found a high density of lcs around hair follicles that were very susceptible to targeting with the use of nanoparticles. nanoparticles of 40 nm diameter, but not larger particles in the micron size range, were delivered via the follicles and were able to diffuse through the follicular infundibulum to be taken up by the adjacent langerhans cells. triggered synovial fibroblasts, as part of a multipart cellular complex, play an important role in the pathogenesis of rheumatoid arthritis (pap et al. 2000; seemayer et al. 2001) . diclofenac is a commonly used non-steroidal anti-inflammatory active for symptom control in osteoarthritis of the knee and soft tissue injuries (bajaj et al. 2011) . although analgesics applied topically can provide a local enhanced active delivery for the superficial joint tissues by direct diffusion, they are unlikely to be effective for the deeper tissue synovial fluid (xi et al. 2013) . recently, fibroblasts have been targeted using diclofenac in vesicles containing various penetration enhancers (manca et al. 2013) . fibroblasts also play an important role in development of hypertrophic scars, which result from an overgrowth of fibrous tissue at the site of an injury. the development of a hypertrophic scar is a complex interplay between fibroblasts and cytokines (tredget et al. 1997 ). zhao et al. recently delivered small interfering ribonucleic acid (sirna) -transforming growth factor beta 1 (tgfβ1)-337 from hydrogel pressure-sensitive adhesive patches in mice and demonstrated a down-regulation in collagen and reduction in scar size. the scar fibroblasts were shown to have significantly reduced by undergoing apoptosis (zhao et al. 2013 ). the skin is an ideal route for targeted delivery of steroidal and non-steroidal anti-inflammatory agents and other analgesics for pain and inflammation. this has been achieved with many techniques. topical nsaids and related solutes are often applied to the skin to target tissues directly below the application site. in a 1999 study, roberts et al. used biopsy and microdialysis techniques to show that most solutes penetrate below dermal capillaries into the subcutaneous and deeper tissues of both rats and human subjects. the selectivity of local penetration is time related, and the concentrations of the nsaids in the underlying tissues at longer times are often defined by recirculation from the systemic blood supply (roberts and cross 1999) . increased depths of penetration may be achieved by the use of vasoactive agents (singh and roberts 1994) . on the other hand, steroids have been shown to accumulate in the skin to a greater extent. magnusson et al. showed a slower penetration and a higher rate of accumulation of progesterone in the viable epidermis of full-thickness skin. these findings were consistent with dermal penetration limitation effects associated with high lipophilicity of the steroids (magnusson et al. 2006 ). previously, pelchrzim et al. also showed a similar reservoir effect of the stratum corneum on corticosteroid penetration, although they considered this to be formulation dependent (pelchrzim et al. 2004 ). ketorolac has been successfully delivered by iontophoresis to human volunteers using silver electrodes with a current of 2 ma for five treatment sessions of 20 min every day in order to avoid the well-known gastrointestinal side effects of its oral delivery (saggini et al. 1996) . transdermal delivery has been successfully used to achieve systemic targeting, by exploiting the rich blood perfusion of the dermis. fentanyl, a widely used opioid, has potency approximately 100-500-fold greater than morphine. its physicochemical and pharmacological properties, together with its pharmacokinetics (short halflife; high first pass effect), make it an ideal candidate to be delivered systemically through the skin. fentanyl has been delivered transdermally using two different techniques. firstly, it is formulated in transdermal patches to be passively delivered for the treatment of chronic pain. however, this technique cannot provide a rapid bolus active input for the relief of acute pain (chelly et al. 2004 ). iontophoresis has also been investigated for systemic fentanyl delivery; for example, as a patient controlled system that could be activated for symptomatic relief (ashburn et al. 1995) . the skin also provides a potential route for the non-invasive delivery of peptides and proteins which are in danger of inactivation when delivered orally, due to chemical and enzymatic degradation, although there are significant challenges involved in achieving this . cosmetics and pharmaceuticals must be formulated not only for efficacy, but also for patient or consumer acceptance. this is influenced by a perception of safety or lack of toxicity. regarding safety and toxicity, it must be understood that for a topically applied active to have either therapeutic or toxic effects, it must first be absorbed through the skin. the toxic effects caused if the chemicals are absorbed can include acute irritation, corrosion and skin sensitization . chemicals applied to the skin can also have direct effects on the stratum corneum, resulting in an alteration in its permeability. this concept has been effectively applied in the development of penetration enhancers. however, the harmful effects of excipients on the skin have been evaluated under increasing scrutiny by regulatory authorities. regulatory considerations will be discussed later in this chapter. topically applied actives causing adverse effects can be classified as either allergens or irritants (zweiman 1990 ). these allergens and irritants not only affect the skin but can also affect other body sites. cutaneous active reactions can be classified into three types of contact dermatitis: (a) allergic contact dermatitis, (b) irritant contact dermatitis and (c) photo contact dermatitis. the role and mechanism of irritation caused by various chemical penetration enhancers has been studied extensively. allergic contact dermatitis (acd) results from an adaptive immune response to the penetration of allergens into the skin. acd progresses in two phases: (a) in the initial sensitization phase, the host is immunized to the allergen, and (b) in the elicitation phase, a rapid secondary immune response is mounted following re-exposure to the allergen which manifests as acd (alenius et al. 2008) . cutaneous photosensitivity can be evoked by both systemic and topical exogenous photosensitizers leading to active photosensitivity and photo contact dermatitis, respectively. photoallergenicity is a well-organized immunological reaction. the current understanding of ordinary contact dermatitis and active hypersensitivity is based on the hapten hypothesis: molecules bind covalently to proteins, and the resulting conjugates can be recognized as immunogenic determinants. likewise, photosensitive chemicals have a haptenic moiety. although the terms structure-activity relationship (sar) and quantitative structure-activity relationship (qsar) are widely used, for skin sensitization, it is more meaningful to describe the relationship as being between chemistry and activity. skin sensitization induction is a multistage process (jowsey et al. 2006) : stage 1, translocation of the sensitizer from the skin surface to the epidermal site of action. this depends on the dose given and duration of exposure; stage 2, covalent reaction of the sensitizer with skin protein. this is strongly dependent on the chemical nature of the sensitizer, in particular electrophilic reactivity and hydrophobicity. the nature of the skin proteins involved in this process is not established. possibilities range from any protein encountered in the skin to highly nucleophilic proteins, "purpose-designed" by evolution, associated with epidermal langerhans cell membranes; stage 3, response of epidermal langerhans cells to the modified protein, resulting in migration to the lymph node and presentation of antigen, leading to antigen recognition by naïve t-lymphocytes resulting in clonal expansion. regulators classify chemicals as being hazardous after an acute dermal contact if they lead to mortality (owing to absorption), cause acute irritation or corrosion and cause skin sensitization on multiple contacts. in the eu, evaluation of skin irritation/corrosion is mandatory for all products likely to be associated with skin exposure. the section below discusses testing protocols and requirements in the eu. the european commission scientific committee on consumer safety (sccp) adopted its most recent guidelines "the sccs's notes of guidance for the testing of cosmetic substances and their safety evaluation, 8th revision" in december 2012 (sccp 2012). these set out procedures for the safety evaluation of cosmetic substances (single ingredients or defined mixtures) and finished cosmetic products. in the guidelines, a cosmetic ingredient is defined as "any chemical substance or mixture of synthetic or natural origin, used in the formulation of cosmetic products." a cosmetic ingredient may be: 1. a chemically well-defined single substance with a molecular and structural formula 2. a complex mixture, requiring a clear definition and often corresponding to a mixture of substances of unknown or variable composition and biological nature 3. a mixture of 1 and 2, used in the formulation of a finished cosmetic product a finished cosmetic product is defined as "any substance or mixture intended to be placed in contact with the external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition or correcting body odours". table 23 .2 illustrates the sccp requirements for testing of chemical and physical properties and the relevant toxicity studies for cosmetic substances. other requirements for sccp evaluation of cosmetic substances are included under the general headings listed in table 23 .3. the sccp guidelines cover the evaluation of safety and microbiological quality of finished cosmetic products under the headings listed in table 23 .4. sccp recognizes the increasing use of synthetic and semisynthetic ingredients, in addition to the ingredients derived from natural products that have been traditionally used in cosmetics. in addition, their requirements for evaluating the safety requirements for cosmetic products are increasingly being brought into line with those for therapeutic products. while dermal toxicity to exogenous materials is seldom associated with mortality, it may involve significant morbidity. ethical concerns have brought about a paradigm shift in the way the skin irritancy of a cosmetic ingredient is evaluated. prior to its phasing out and eventual ban by the european commission, the scientific community has traditionally used in vivo animal testing methods to evaluate the safety of a topically human data functions and uses applied product. there are many in vitro testing models that have been developed and commercialized to provide an alternative to animal testing. models such as episkin ® (episkin snc, lyon, france), skinethic ® (skinethic laboratories (nice, france) and epiderm ® (mat-tek inc., ashland, usa) have reasonable similarities to human tissue in terms of morphology, lipid composition and biochemical markers (netzlaff et al. 2005) . numerous reviews and research articles have been written comparing the different skin models to each other and to human skin (lotte et al. 2002; netzlaff et al. 2005; schafer-korting et al. 2006 neis et al. 2010; gotz et al. 2012; brinkmann et al. 2013) . lotte et al. (2002) studied the permeation of lauric acid, caffeine and mannitol in episkin ® , skinethic ® and epiderm ® and observed that although there was huge variability in permeation data among the skin models, the order of permeation was in line with that observed in ex vivo skin. enzymatic equivalence and gene expression were also compared (hu et al. 2010; neis et al. 2010) . in microarray analysis, hu et al. (2010) found that the expression of a large percentage of the genes was consistent between epiderm ® and human skin indicating the presence of similar metabolic pathways. in a 2005 review, netzlaff et al. evaluated the morphology of commercially available skin models and compared their suitability for testing phototoxicity, irritancy, corrosivity and active transport (netzlaff et al. 2005) . they tabulated comparative morphological features and test results for these parameters. they commented that currently the models come close to reproducing human skin, but only in certain aspects. these skin models are useful in toxicity testing, including phototoxicity testing, and to an extent for drug transport studies. the variability in transport can be attributed to the incompletely developed barrier. before the enforcement of the eu chemicals policy reach (registration, evaluation and authorization of chemicals) in 2009, basketter et al. (2007) published report and recommendations of european centre for the validation of alternative methods (ecvam's) workshop 59 a . this workshop was part of a concerted effort in recognizing the role of percutaneous/dermal absorption process in sensitization. they defined epidermal disposition as the specific location and stressed the quantification of chemicals in the epidermal compartment, which are relevant to local toxicity endpoints/skin sensitization. oecd test guideline (tg) 428 (oecd 2004), for in vitro skin absorption, provides general rules on how to measure systemic availability and concentrations of chemicals that permeate through all layers of the skin and the overall kinetics of skin absorption but does not allow for separate evaluation of epidermal disposition to be derived for local skin toxicity endpoints, such as skin sensitization. hence, this report emphasizes the need to relate the in vivo dosimetry of sensitizers that penetrate into the viable epidermis via the stratum corneum of human skin to the concentrations used in in vitro applications. this requires the need to toxicological profile of the substances stability and physical and chemical characteristics of the finished cosmetic product evaluation of the safety of the finished product microbiological quality: quantitative and qualitative limits challenge testing good manufacturing practice estimate both the applied concentration of chemical onto the skin surface for given finite dose exposure conditions and to relate it to what is applied to cells in vitro as being representative of the target dose of chemical in human skin. the models proposed by the sccp for skin safety tests have been validated against in vivo draize tests (sccp 2012). however, these standalone methods rely heavily on quantification of optical density and colorimetry, which lead to a one-dimensional analysis. moreover, since results obtained from previously conducted human tests studies on model compounds are necessary as reference values for in vitro assays, attention should also be paid to the generation of in vivo data and standardization of in vivo test protocols (ponec 2002) . in the last decade, numerous committees and taskforces have been set up to constantly review the performance of in vitro irritancy and genotoxicity assays (fentem et al. 1998; eskes et al. 2012; pfuhler et al. 2014 ). in vitro assays have shown a very high rate of positive genotoxicity results that do not correlate with in vivo genotoxicity or carcinogenicity (kirkland et al. 2007 ). due to the inability to follow up the in vitro tests with in vivo assays, many potential new products have been de-selected. the cosmetics europe (formerly colipa) genotoxicity task force has driven and funded projects to help address the high rate of misleading positives in in vitro genotoxicity tests (pfuhler et al. 2014 ). the ongoing "3d skin model" project is now validating the use of human reconstructed skin (rs) models in combination with the micronucleus (mn) and comet assays. non-testing methods like [quantitative] structureactivity relationships ([q]sars) or in silico methods are also a valuable way of gaining more toxicological information. formation of chemical categories facilitates the application of read across between similar chemicals based on the assumption that their behaviour will be consistent for their class (adler et al. 2011) . even from before the complete ban on animal testing, alternative techniques to test dermal irritancy and immunogenicity caused by pharmaceutical and cosmetic products were being developed. responses to irritants have been stud-ied in cell culture for a significant period (deleo et al. 1996) . there are other approaches that can be used to complement the validated in vitro methods and satisfy the need for triangulation in safety evaluation. human ex vivo skin is often used as a realistic model for in vivo human skin in transdermal active delivery. the study of detailed cellular and sub-cellular processes in the skin was previously not possible using conventional light microscopic methods, such as widefield and confocal microscopy. although these tools work reliably in cell cultures and thin tissue sections, their application in thick biological samples such as skin is greatly limited due to light-scattering and absorbing properties of biological tissues. however, this is being addressed with the increasing use of multiphoton or two photon microscopy (mpm/tpm). understanding of the process of epidermal and transdermal transports of xenobiotics is important for rational design of topical active delivery and to avoid exposure to toxic and allergenic compounds. mpm allows 3d visualization of penetration and distribution with minimal sample preparation. sanchez et al. have used cellular autofluorescence to show that ex vivo skin viability, including metabolic activity, can be preserved up to 7 days at 4 °c (sanchez et al. 2010 ). mpm has also been used to demonstrate the importance of stratum corneum, in the sensitization phase of contact allergy (samuelsson et al. 2009 ). one of the present drawbacks of mpm is that it tends to be qualitative rather than quantitative; however, the use of fluorescence lifetime imaging (flim) holds promise for quantitative analysis in tissue samples (benati et al. 2011; koenig et al. 2011) . excised skin obtained from elective abdominoplasties is a convenient resource, which when used within its viability provides an excellent model. however, not all research facilities have access to freshly excised skin. skin from elective surgery is donated in goodwill to assist scientific research, and there are ethical constraints on its use for commercial testing. the eu prohibits financial gain through the use of human tissue (netzlaff et al. 2005) . progress is ongoing in the development of advanced skin equivalents with immune and inflammatory equivalency. better culture media to maintain the viability of ex vivo skin and skin biopsies are also under development. chau et al. (2013) recently described an advanced static 3d skin equivalent which combines human keratinocytes, human dendritic cell and fibroblasts combined into a threelayer cellgrown™ tissue culture insert comprising keratinocyte and fibroblast layers at a thickness of 100 μm and 30 μm, respectively. dinitrochlorobenzene (dncb), an established skin sensitizer, was used topically to stimulate this skin model and showed extensive cell response with up-regulation of the cell surface antigens cd86 (cluster of differentiation 86) and hla-dr (an mhc class ii antigen expressed mainly on antigen-presenting cells) on the dendritic cells. another approach to emulate the human skin composition of both somatic and immune cell populations is the use of skin biopsies for in vitro cultures. skin biopsies contain all the immune cell types relevant for the donor at the place of biopsy extraction (giese and marx 2014) . a biopsy from the skin sample can then be used to test for the presence of inflammationrelated human genes using real-time quantitative polymerase chain reaction (rq-pcr). the de novo construction of full skin equivalents and the use of skin biopsies are two complementary and scientifically grounded approaches to model human skin immunity in vitro (giese and marx 2014) . in this chapter, we have discussed some of the major issues facing the formulator: efficacy, safety, toxicity and skin targets. efficacy, defined as the product of potency of the active and the amount delivered to its site of action, and formulation dictate the dosage of a given active. the formulator must be guided by the physicochemical properties of the particular active and knowledge of skin physiology to create a delivery system to target particular skin regions or cell populations. for all topically applied actives and cosmetics, safety and irritancy testing is required. suitable predictive methods of in vitro testing must be estab-lished and validated to replace in vivo animal testing 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authors: arkow, phil title: human–animal relationships and social work: opportunities beyond the veterinary environment date: 2020-09-05 journal: child adolesc social work j doi: 10.1007/s10560-020-00697-x sha: doc_id: 31482 cord_uid: atltc10d a species-spanning approach that incorporates clients’ relationships with their companion animals into family genograms, schools of social work curricula, continuing education, interviews, assessments, and interventions offers increased career opportunities, professional and personal growth and development, and a more comprehensive resolution of clients’ issues, social justice concerns, and the prevention of family violence. this article identifies six reasons why social workers should be cognizant of human–animal relationships and introduces nine ways, with action steps, in which social workers can include these relationships into training and practice outside the more developed field of veterinary social work. these venues include: agencies working in child protection and child sexual abuse; children’s advocacy centers and courthouse facility dogs; animal shelters; domestic violence shelters; public policy advocacy; clinical practice; agencies working with older and disabled populations; veterinary sentinels for intimate partner violence; and pet support services for homeless populations. such attention to the human–animal bond can utilize social workers’ problem-solving skills to improve delivery of services, identify clients’ risk and resiliency factors, enhance social and environmental justice, expand academic inquiry, and increase attention to all of the vulnerable members of families and communities. system arousal, and improving physical fitness by providing an impetus to exercise (friedmann, 2019) . there is, conversely, substantial evidence of animal abuse as a potential precursor and indicator of interpersonal violence often linked to child maltreatment, intimate partner violence and elder abuse (arkow, 2015a) . pets occupy central roles in many interpersonal and intrafamilial relationships (turner, 2006) . they may serve as significant others, confidants, attachment figures, and sources of companionship (mcnicholas & collis, 2006) . they can be vital members of an individual's support system and facilitators to foster social capital, trust, civic participation and a sense of safety and community (wood, martin, christian, houghton, & kawachi, 2017) . the relationships between humans and animals in a household may mirror the status of the health and safety of the people in that family (hoffer, hargreaves-cormany, muirhead & meloy, 2018) . inquiring about children's and adolescents' experiences with animals can help social workers address risk and resilience factors. pets' physical condition and behaviors can provide clues to human experiences and family functioning. human-animal relationships are contextual for learning and resilience in the wake of family violence. strengthening these relationships, and helping people better understand their animals' behaviors, can promote well-being in both species and enable people to leverage inclusion and a sense of belonging in community relationships. animal rights arguments about inherent "speciesism" in humans' relationships with other animals should resonate well with social work's commitment to social justice and fighting oppression (national link coalition, 2019a) . this article explores six reasons why human-animal relationships can be significant to social workers' responsiveness to their clients and nine areas outside the veterinary social work environment where an understanding of clients' interactions with animals can be directed into enhanced professional and personal growth, innovative species-spanning solutions, and potential career opportunities. the american veterinary medical association (2018) estimated that 57% of u.s. households owned a pet, including 76.8 million dogs, 58.3 million cats, plus birds, horses and other companion animals. pets are predominantly found in households self-defined as "family" rather than "non-family" and the highest rates of dog and cat ownership continue to be among households with children. primary responsibility for pets' care continues to rest overwhelmingly with the female members of the household. pet ownership rates are higher in rural areas and lower in densely urbanized cities. there are unexplained racial and ethnic disparities in the rates of pet ownership; pets are found in 64.7% of white households, compared with latino/hispanic (61.4%), asian (46.6%), and black/african american (36.9%). some pet owners describe veterinarians as "the other family doctor" (national link coalition, 2019b) . two studies reported that 87% (risley-curtiss et al. 2006a; risley-curtiss, holley, & wolf, b) and 97% (risley-curtiss et al., 2006a, b) of multi-ethnic pet owners agreed that their pets are members of the family. in short, social workers exploring a client's home life and family dynamics may be missing a significant piece of the puzzle if they neglect to inquire about the client's animals and the attachments, relationships, and problems with them. in addition to appreciating a client's individual and familial attachments or antipathy toward pets, social workers can achieve a fuller understanding of a client's connectivity or isolation from the community by seeing human-animal relationships in a social context. social capital (as contrasted with human capital, economic capital, cultural capital, technological capital, or other community resources) is the connectivity among people which enhances cooperation for mutual benefit. the concept was popularized by putnam (2000) describing the networks and other forces that build social cohesion, personal investment, reciprocity, civic engagement, and interpersonal trust among community residents. notably absent, however, from the work of putnam and other social capital researchers was inclusion of the influence of pets in a community (arkow, 2013) . this knowledge gap was addressed by wood et al. in studies in australia (wood, giles-corti, & bulsara, 2005; wood, giles-corti, bulsara, & bosch, 2007 and the u.s. (2017) which reported companion animal ownership to be positively associated with social capital, civic engagement, perceptions of neighborhood friendliness, and a sense of community. the ability of pets to generate interpersonal communications was greater than minor conversational exchanges among dog-walkers: the visible presence of people walking dogs and the impetus dogs provide for people to be outdoors and use park areas ameliorated negative mental health conditions and gave residents a feeling of greater collective safety and sense of community. companion animal owners were found to be more likely to participate in volunteer, school and sports activities, professional associations and environmental campaigns. they were reported to be more likely to vote and to exchange favors with neighbors. arkow (2019a, 2013) proposed a converse, that the absence of companion animals in communities where rates of pet ownership are lower and incidence of animal problems is greater might contribute to less social connectivity and cohesion. levinthal (2010) used geospatial mapping techniques to correlate the distribution and prevalence of animal neglect, abuse and dog fighting in philadelphia with domestic violence and child maltreatment. she reported a highcrime neighborhood seemed to predict animal abuse, and that animal neglect correlated with demographic, cultural, and structural aspects of block groups, suggesting social disorganization may lead to animal neglect. campbell (2019) used geospatial mapping techniques to report direct correlations between animal control complaints and domestic violence incidents in neighborhoods in indianapolis. bruni (2019) suggested a way to push back on "the degradation of our country's civil culture" can be found in walking one's dog in public spaces. dog walks encourage "honest-to-goodness conversations with actual strangers" that leave their owners "feeling a little less isolated, a little less disconnected" and discourage americans retreating into "increasingly homogeneous enclaves." dog-walking encourages mutual courtesy and reciprocal generosity. "when you're about to bend down and scoop up your beloved's odoriferous bequest, there's no snobbery and no timidity, only solidarity," he wrote. there is something immensely powerful about animals that attracts and motivates humans, a force that is especially compelling with youths. whether discussing pet ownership, fascination with wild animals, or imaginary animals, children are particularly engaged, and asking about animalrelated experiences can provide important information and establish a caring persona and a trusting relationship (melson & fine, 2015; boat, 2010) . a pet is a communication waiting to happen. a recurring theme in the literature is that companion animals are what messent (1983, p. 37) first called "social lubricants," icebreakers who facilitate social support and interpersonal communications (garrity & stallones, 1998) . the nonjudgmental nature and unconditional positive regard of human-animal interactions can be a useful bridge for establishing rapport between therapists and clients (arkow, 2015b). because animals slip under the radar of human defense mechanisms, clients who are fearful, traumatized or under stress may be more willing to talk about their concerns for their animals before opening up and describing their own vulnerabilities (melson & fine, 2015) . fawcett and gullone (2001) reported that even the mere observation of animals can result in reduced physiological responses to stressors and increased positive mood. lange, cox, benert, & jenkins (2006) reported animals can introduce a calming effect, stress-reducing humor, increased feelings of safety, experienced empathy, and motivation among adolescents attending anger management sessions. chandler (2005) wrote that discussing pets can easily segue into a discussion about the client's family support system and how well he or she is utilizing personal resources. an interaction with a therapy animal can enhance social workers' listening responses, convey empathy and help the client access feelings. she observed that it is not the mere presence of an animal, but rather the orchestrated child-dog and child-therapist interactions, that can facilitate client motivation and participation, enhance the relationship with the client, stimulate client focus and attention to task, and reinforce positive client change. margaret mead's oft-quoted adage (1964) that "one of the most dangerous things that can happen to a child is to kill or torture an animal and get away with it" has been substantiated with research. a growing body of evidence suggests that bonds formed or broken with companion animals in childhood reverberate and resonate across the lifespan (jalongo, 2004) . childhood acts of committing or witnessing animal cruelty: may be sentinel warnings that a child is living in a dysfunctional environment and may be exhibiting other antisocial behaviors (gullone, 2012) ; are a prime risk factor for perpetrating animal cruelty, bullying behaviors and violence against humans; and may lead to desensitization, decreased empathy, learned maladaptive coping mechanisms, and unresolved feelings of anger, fear and resentment, particularly if the child is also experiencing co-occurring family violence (ladny & meyer, 2019) . correlations between childhood exposure to animal abuse and bullying behaviors have been reported by several authors (for example, see baldry, 2005; gullone & robertson, 2008; henry & sanders, 2007; parkes & signal, 2017; sanders & henry, 2015; vaughn et al., 2011; walters, 2019) . currie (2006) reported that children exposed to domestic violence were three times more likely to have been cruel to animals than children not exposed to intimate partner violence (ipv). there is increasing academic and programmatic recognition of animal abuse and neglect as sentinel indicators of concurrent or future family violence, particularly child maltreatment and child sexual abuse, ipv and elder abuse. social workers may find that when animals are abused people are at risk, and when people are abused animals may be at risk (arkow, 2019b) . emotional attachments to companion animals are often exploited by abusers in violence-prone households to control and coerce victims in domestic violence, sexual assault, child abuse, and elder abuse situations (ascione & arkow, 1999) and fear of leaving a pet behind is a significant barrier that keeps women and children from extricating themselves from abusive situations (roguski, 2012; ascione, 2007) . households marked by ipv have higher-than-average rates of pet ownership and are extreme high-risk environments when animal abuse is also present: nearly 80% of ipv survivors where suspects also had histories of animal abuse feared they would be killed, 76% had been strangled and 26% had been forced into non-consensual sex. multidisciplinary collaborations were seen as critical in prevention, detection and intervention to address substantial risk of harm for all children, adults and animals residing in the home (campbell, hicks, thompson, & wiehe, 2017) . grief over the loss of a pet is a broad, complex construct, which can be complicated by pet owners' perceptions that they have minimal social support sources and negative veterinary interactions (rémillard, meehan, kelton, & coe, 2017) . the loss of a pet through death or disappearance, and the decisions inherent in determining whether euthanasia of a beloved animal companion is necessary and appropriate, can generate significant emotional trauma for human members of families as well as staffs of veterinary facilities and animal shelters (see, e.g., barnard-nguyen, breit, anderson, & nielsen, 2016; carmack, 2003; dunn, mehler, & greenberg, 2005; laing & maylea, 2018; miller, prout, rourke, lefkowitz, & boyer, 2014; ross, 2005) . social workers who have had training in grief and loss theory are well positioned to be resources for individuals experiencing these emotions and can aid them in making difficult decisions and navigating the options available. because pets generally have shorter lifespans than humans, families are likely to witness significant life-cycle events such as birth, serious illness and death of their animal companions, who are often seen by children as peers and family members. the inevitable death of a pet can bring a profound sense of loss, with patterns of bereavement similar to those experienced with the death of a human family member or friend, and thus an opportunity for social work intervention. several studies have reported that by adolescence the majority of children have experienced pet loss through death or disappearance (melson & fine, 2015) . social workers can play important roles in offering grieving clients opportunities for validation of their feelings, memorialization of the animal, resolution of potential feelings of guilt, and closure. social work's interest in human-animal relationships has its origins in the practitioner-client-patient dynamics of the veterinary hospital or clinic. from modest beginnings at the veterinary hospital of the university of pennsylvania in 1978 (quackenbush, 1981; quackenbush & glickman, 1983) , the field of veterinary social work has grown dramatically in recent years. the term veterinary social work is believed to have been coined in 2002 by elizabeth strand, founding director of the veterinary social work certificate program at the university of tennessee-knoxville. today, dozens of students have been trained in the four areas of veterinary social work: the link between human and animal violence; grief and loss; animal-assisted interactions; and compassion fatigue management. this work may include: • supportive grief support and counseling with end-of-life decisions and follow-up. • advocacy and brokering of resources. • circulating reading materials and educational packets. • crisis intervention. • assessment of suicidal tendencies, mental health issues and domestic violence issues. • facilitation of a pet loss support group for hospital clientele and the community. • staff debriefing sessions. • client consultations and follow-up. • presentations to staff. • referral of mental health services for staff. • recommendations to administrators. • making improvements to client comfort on-site. (larkin, 2016) as the field of veterinary social work gains additional recognition both within and outside the world of social work, additional opportunities will continue to emerge whereby an understanding of human-animal relationships becomes a valuable asset in many aspects of social work practice. this process can begin with something as simple as routinely including pets in family genograms and adding relevant coursework in schools of social work and continuing education as well as field placement opportunities. by demonstrating additional opportunities for social workers to include human-animal relationships in interventions and assessments and to be aware of community resources that can resolve clients' animal-related concerns, social workers can be more holistic and effective in resolving clients' needs and challenges and preventing further abuse of vulnerable members of families and communities. social work's legacy of facilitating collaborative community change can open up many new career opportunities by incorporating human-animal relationships into social work practice. inquiring about the presence (or absence), stability (or turbulence), attachments, dangerousness, history, and status of animals within clients' lives can help social workers to obtain more comprehensive family assessments, validate important intra-familial relationships, build stronger support networks, support resiliency, gain earlier recognition of abusive behaviors, and address clients' animal care concerns with practical, appropriate and affordable solutions. these emerging opportunities include: despite the social reformer origins of child protective services established by humane societies and spcas in the latter third of the 19th century (hoy-gerlach, delgado, sloane, & arkow, 2019), there is a peculiar and unfortunate irony in that child and animal welfare agencies today operate wholly independently with little to no trans-species cross-fertilization of ideas, information or collaboration (arkow, 2010; walker, 1980; zilney & zilney, 2005) . this inter-agency communications gap can have tragic consequences, as with a case in brooklyn, n.y. in which a child whose family was under investigation by child protective services was killed by a dangerous dog in the home (baker & stelloh, 2011) . the author has heard anecdotally of three children under the aegis of child protection agencies in oxford, fla., and st. john, n.b., canada, who were killed by pet snakes. the potential impact of animals in the lives of children cannot be overstated and warrants expanding the ecological lens of child welfare work to include animals (risley-curtiss, 2013). companion animals are found in 67.7% of households with children under age 6 and 74.6% of households with children over age 6 (avma, 2007). melson (2001) reported that pets are more likely to be a part of children's growing up than are siblings or fathers, and that animals are pervasive in children's media, stories, imagination, and play. she reported that words for different animals-dog, cat, duck, horse, bear, bird and cow-are among the first 50 words that toddlers learn; children include dog and cat in their initial productive vocabularies more than any other words except mama and daddy. an estimated 80 to 90% of children first confront the loss of a loved one when a pet dies, disappears, or is abandoned (melson & fine, 2015) . melson (2013) reported that children, from an early age, view animals as living actors who have autonomy, intentionality and feeling. children are often animals' caregivers; since opportunities for and encouragement of nurturing others are rare in childhood, nurturing animals makes up a large proportion of childhood caregiving experiences. because caring for pets is gender-neutral, companion animals may develop innate nurturing skills in boys and feelings of mastery and self-efficacy among children who feel dependent and powerless. many children turn to their pets for reassurance and emotional support during times of stress. companion animals may assist children in feeling security and unconditional love and contribute to a child's cognitive and language development (risley-curtiss, 2013) . companion animals may be sentinels of unsafe environmental conditions, mirroring family tensions and serving as cues in assessments that explore family issues. as one of the sub-systems within a complex family system, the inclusion of questions and observations about the current and past presence of animals in a child's environment, the meaning those animals have for each family member, their care, and whether any of them have been killed or hurt can be important to effective family-centered practice (risley-curtiss, 2013) . children may feel safer talking about their pets' experiences before they disclose their own, thereby opening a friendly channel where children can provide important information (melson & fine, 2006; boat, 2010) . introducing therapy animals into the interview process can further advance this process, easing the stresses of such sessions, establishing rapport, providing the child with a sense of comfort, and creating a less threatening environment (menzies, 2003) , particularly in working with sexuallyabused children (reichert, 1998) . of particular concern is the nexus of animal sexual abuse or bestiality with child sexual abuse. edwards (2019) reported 31.6% of animal sex offenders also had histories of sexually offending children or adults and 10.8% had prior convictions for child pornography. her survey of 456 offenders arrested for bestiality between 1975 and 2015 found at least 213 children and 28 adults had been directly sexually victimized by the offenders, and that in 25 arrests (5.5%) animal pornography had been used to groom a child for sexual behavior. the canadian centre for child protection (2018) reported significant overlaps of animal and human sexual abuse in a study of 38 cases of bestiality. in 82% of cases, sexual abuse of children occurred as frequently as, if not more frequently, than coerced sexual abuse of an animal. in 68% of cases involving both child and animal sexual abuse, the offender was in a position of trust over the child, usually a close family member. child welfare workers can obtain more accurate and useful assessments of child safety and well-being by taking several steps: • in conducting child welfare checks and ongoing case management, look for: potentially abused, neglected or dangerous animals (e.g., aggressive dogs, poisonous reptiles, exotic species, dog-and cock-fighting paraphernalia); animals needing veterinary care; excess numbers of animals; and inadequate food, water or shelter. animal health issues such as fleas or other parasites could have a direct impact on the health of the humans in the home. include these findings in evaluations of the child's living environment, lifestyle and risk factors as potential threats to the child's well-being. such conditions may also be illegal under a jurisdiction's laws. • consider a turbulent history of frequent turnover of animals as potential indicators of a family's inability to make strong, lasting emotional attachments. • include the child's emotional attachment to pets as a key support which may help build resiliency and a protective factor that mitigates. treat the death or disappearance of animals as potentially as emotionally charged as the death of a human family member. • identify whether the child has been traumatized by witnessing or causing the abuse or death of animals. • consider animal maltreatment as a factor that supports a finding of child abuse or neglect. • report suspected animal maltreatment to the animal control/services, humane society/spca, or law enforcement agency in that jurisdiction. 1 the reporter need not prove that animal abuse occurred, but rather introduces the case into those agencies' investigative systems to vet the information and follow through as appropriate. confidentiality restrictions may be waived in reporting to another such law enforcement agency, or when the welfare of the client and others in the household is threatened. establishing channels of communication with animal welfare agencies in advance can simplify the cross-reporting process when a case of suspected abuse occurs. • a history of animal cruelty, and a child's emotional attachments to animals, may have evidentiary importance in court trials, dispositions and hearings involving child maltreatment, custody, visitation, removal, and protection orders. by asking three simple open-ended questions, social workers can learn much about a child's experiences with the animals and humans who share his or her environment: • are there animals at home? • how are they cared for? • are you worried about their welfare? follow-up questions about their names, breeds, play activities, deaths or disappearances, recent health problems or injuries, and secrets the child shares with them may fill in details of the family dynamics, patterns of power and control, and risk and resiliency factors in the child's life. facility animals in children's advocacy centers, casa (court appointed special advocates), guardians ad litem programs, and courtrooms provide emotional support to sexual abuse survivors as they undergo forensic examinations, re-live their experiences, and confront their abusers (labahn, 2015) . as of may, 2020, an estimated 238 dogs are working in courtrooms and children's advocacy centers in 40 states, plus others in canada, australia, chile, and europe (courthouse dogs foundation, 2020a , 2020b , 2020c . elaborate precautions prevent handlers from violating client confidentiality and keep the dog's presence from adversely eliciting sympathy from a jury. judges must balance the accommodation for a vulnerable witness with the potential for prejudice which could impact the defendant's right to a fair trial. extensive guidelines on best practices protect the interests of the animal, the victim, the defendant, and the criminal justice system (courthouse dogs foundation, 2015) . social workers in victim services can be trained to be facility animal handlers or secondary dog handlers to allow the children to spend more time with the dogs after concluding their testimony. they can facilitate interactions between the dogs and distraught family members and stressed facility staff and be resources who connect individuals and institutions with facility animals in their community. nonprofit animal welfare and governmental animal control agencies have historically operated in isolation outside the purview of human services agencies, leading to a "silo" effect in which cross-disciplinary and trans-species collaborations rarely occur (becker & french, 2004) due to increased specialization, avoidance of "mission creep" and fear of violating confidentialities. consequently, interagency cooperation and cross-training is minimal, resulting in a significant barrier to change. meanwhile, animal shelter workers experience significant stressors including animal suffering and euthanasia, responsibility for life, abusive clients, negative public perceptions, and attachments to animals under their care (schneider & roberts, 2016) without recognizing that their counterparts in human services often experience similar stressors. this lack of knowledge and coordination among community systems constricts the potential for creative and effective collaborations and can increase the risk of harm to people and animals in situations where both human and animal abuse co-occur. social workers can facilitate bridging these segregated service delivery systems through the profession's longstanding commitment to community-level action, intervention and change. social workers can work with animal shelters to organize species-spanning community coalitions, link organizational champions, and connect consumers and professionals for the well-being of underserved and at-risk individuals and family members (long & kulkarni, 2013) . social workers can help animal shelters build capacity by coordinating inter-disciplinary interaction and communication, gathering data, conducting research, and building resources. increasing cross-systems knowledge and promoting individual and institutional relationships across systems, particularly vis-à-vis cross-reporting animal, child and elder abuse, can protect vulnerable 1 unlike coordinated state-run child protection systems, animal protection is handled exclusively on the local level by a fragmented patchwork of independent government animal control/services agencies, municipal/county law enforcement, and nonprofit humane societies/spcas. a national directory of animal abuse investigation agencies in over 6,500 jurisdictions is available at https ://natio nalli nkcoa litio n.org/how-do-i-repor t-suspe cted-abuse . populations and develop stronger community services (long & kulkarni, 2013) . numerous animal shelters, often working with juvenile and adult detention centers, have implemented animal-assisted therapy interventions where individuals who have offended, or who are at risk, train dogs with behavior problems who are at risk of being euthanized. using positive reinforcement techniques, these programs teach teamwork, non-violent conflict resolution and collaboration skills to save animals' lives and modify the behaviors of abusive and traumatized individuals (arkow, 2019b). animal shelters appear poised for such systemic change. the service philosophy in the animal shelter community is evolving to recognize that treating the symptoms of animal welfare problems, such as animal homelessness, abuse and neglect, is only a stopgap solution: to be truly effective, underlying community and family dysfunction and violence must be addressed (petlynx, 2011). hoy-gerlach et al. (2019) described promising opportunities for social work field placements in community animal shelters, including: reducing staff and volunteers' compassion fatigue in an exceedingly difficult and emotionally draining work environment; placement of shelter pets as emotional support animals; strengthening community responsiveness to violence through assessing overlaps and differences between child, elder and animal abuse investigations; creating and implementing educational programming across child and animal protection systems; and increasing community awareness of the link between violence to animals and violence to humans. animal control and humane officers frequently have access to pet owners' homes in the course of their investigations, and in the process may observe conditions detrimental to the welfare of children, youth and others. in addition, cruelty investigations which result in the removal of animals from a home could be an additional stressor on the family system and could lead to increased risk for vulnerable family members. social workers can train shelter personnel on the intersectionality of animal abuse and human violence and the procedures for making referrals to social services agencies. other untapped social work opportunities in animal shelters might include: strengthening collaborations with domestic violence shelters and mobile meals programs; directing and expanding pet visitation programs for long-term care facilities and animal-assisted interventions for at-risk populations; developing pet loss grief support groups; developing safety net supportive programming for individuals who experience a medical, economic or housing crisis that temporarily makes it difficult to keep an animal; defusing contentious confrontations with shelter clients; resolving customers' complaints and needs for services; and connecting pet owners with community resources, such as low-cost pet and veterinary services, animal behavioral counselors, pet food banks, and social services agencies. social workers provide essential services to survivors of intimate partner violence and their children in numerous ways, including advocacy, practice and public policy. they serve as advocates in women's shelters, criminal courts, protective order offices, hospital ers, police victim services units, government and nonprofit agencies, military family advocacy centers, fatality review teams, and elsewhere. their work encompasses crisis intervention, investigations, counseling, case management, legal services, public policy, and referrals to and liaison with community resources. all of these aspects of social work practice can take on an additional dimension by incorporating human-animal relationships into the perspective. research findings suggest that in working with pet-owning domestic violence victims, social workers must consider the welfare of the women's pets in order to effectively help the women achieve safety for themselves and their families (strand & faver, 2005) . domestic violence shelters in the 1990s began raising awareness that significant numbers of survivors (usually, but not always, women) and their children are either turned away from safehouses that will not accept their pets or are refusing to leave abusive situations for fear of what would happen to their pets if they left. these fears range from the mundane-that no one remaining at home would provide adequate care-to the tragedies of seeing animals tortured and killed in an emotional extortion that warns partners that they themselves could be the abuser's next victims. the issue reached national awareness in 2006 when susan walsh, 50, told legislators in maine that her husband had retaliated against her and her children and prevented her from leaving a frightening relationship by killing her pets and farm animals: "it wasn't just the cats and the dogs i had, it was the sheep and the chickens -i was terrified for their welfare. i knew if i were to leave, he wouldn't hesitate to kill them. he had done it before." (belluck, 2006) . to address the problem, then-gov. john baldacci signed into law the first of what are now laws in 35 states plus puerto rico and the district of columbia specifically allowing courts to include pets and, in some cases, livestock in domestic violence protection-from-abuse orders. these allow courts to grant petitioners exclusive care, custody and control of animals, and to forbid respondents from harming, taking or disposing of animals or even coming near them (national link coalition, 2019c). abusers' obsessive jealousy and control can become a manipulative tool for power over partners and children by exploiting the vulnerability their emotional attachment to pets. 71% of battered women reported that their abusers had harmed, killed or threatened animals as coercive control (ascione, weber & wood, 1997) . 25% to 40% of abused women delay seeking safety in fear for the welfare of their animals (mcintosh, 2002) . 41% of ipv offenders had histories of animal cruelty (febres et al., 2014) , which is one of the four strongest risk factors for becoming a batterer (walton-moss, manganello, frye, & campbell, 2005) . animals are chosen as soft targets because abusers believe that they can get away with it because police generally don't care about animal abuse (roguski, 2012) . when perpetrators of ipv also have a history of animal abuse, victims experience 20 to 50 violent incidents before contacting police and the risk of lethality to first responders doubles (campbell, thompson, harris, & wiehe, 2018) . the risks encapsulated in these and similar findings are further escalated vis-à-vis the impact on children in these households: 87% of coercive ipv animal abuse incidents occurred in the presence of the woman; 75% occurred in the presence of the children (quinlisk, 1999) . 32% of domestic violence survivors in shelters reported their children had also harmed animals, repeating the intergenerational cycle of violence (ascione, 1998) . batterers have been reported to sexually abuse animals, threaten pets to get children to do something, or force the child to kill the pet (jury, thorburn, & burry, 2018) . the national link coalition (2017) modified the "power and control wheel" frequently used to graphically depict the dimensions of domestic violence to demonstrate how animal abuse is incorporated in abusers' coercive control tactics, as shown in fig. 1 . in response to these situations, domestic violence shelters are developing collaborative foster care programs with local animal welfare agencies to provide off-site "safe havens" for the animal survivors, thereby removing one barrier that prevents families from escaping abuse (ascione, 2000) . more recently, a program called saf-t-sheltering animals and families together-is helping more than 200 domestic violence shelters in the u.s. and other countries build co-sheltering facilities for pets to keep all family members together and safe (phillips, 2019) . grant funding is available to help shelters with capital costs and survivors with veterinary and boarding expenses (national link coalition, 2019d). these concerns dictate bringing social workers into the planning process for innovative and collaborative intakes, assessments, responses, and referrals that incorporate human-animal relationships. serious gaps often separate domestic violence and animal shelters: although concern for the safety of pets and livestock is a barrier to individuals leaving situations of ipv in urban and rural areas, one study reported that 76.92% of animal welfare representatives and 53.33% of human service representatives, respectively, reported no collaboration between their agencies (saskatchewan spca & stops, 2016). acts of animal cruelty in mental health assessments and rehabilitation of abusers and in the specialized domestic violence assessment of risk to children. • including relocation of pets in domestic violence agencies' safety plans (national link coalition, 2014). • obtaining information from local animal welfare and control agencies about prior investigations at the household. • inviting animal-assisted therapy teams into shelters to help comfort survivors. • counseling children regarding incidents of animal maltreatment, death or disappearance of pets that they may have witnessed or committed. • developing community education campaigns to alert the public and cross-train professionals about how animal abuse is linked with ipv. veterinarians, in particular, whose staffs and clients are predominantly female, should begin to recognize a responsibility to serve as resources for survivors of ipv (larkin, 2018; newland, boller, & boller, 2019) . the well-established role of social workers as advocates for social justice provides additional opportunities to advance legislation that recognizes human-animal relationships, the beneficial aspects of pet ownership on individual and community health and well-being, and the adverse effects of animal abuse on human welfare and safety. as an underserved population, animals are classified as property and have long been ignored by the legal system; legislators frequently trivialize campaigns to protect their interests for the simple reasons that animals don't vote and human concerns are widely viewed as being more pressing. however, recognition of the foundation that animal abuse is linked to human violence and therefore improving animal welfare improves human society is generating a new respect for animal welfare legislation. current relevant public policy issues include legislation that would: • allow courts to include pets and/or livestock in protection-from-abuse orders (currently enacted in 35 states, puerto rico and the district of columbia) (national link coalition, 2019c). • allow courts to award custody of pets in divorce and marriage dissolutions based upon the animals' best interests, similar to long-standing similar provisions affecting child custody (four states). • redefine animal abuse when committed as coercive control as also being an act of domestic or dating violence (11 states). • allow acts of violence against animals to be included in criteria for extreme risk protection orders that bar domestic violence abusers from obtaining firearms. • allow courts to appoint pro bono advocates to represent animals' interests in criminal cruelty cases, similar to established court appointed special advocates (casa) provisions for children (three states). • mandate or permit child welfare, adult protection and animal services agencies to cross-report incidents of suspected animal, elder and child abuse to each other, and veterinarians to report suspected animal, child and elder abuse to appropriate agencies, with immunity from civil and criminal liability and professional disciplinary sanctions. • increase penalties for bestiality (now often considered animal sexual abuse) based upon increased evidence of its co-occurrence with child sexual abuse and child pornography. as of august, 2020, having sex with animals is still legal in hawai'i, new mexico, west virginia, and wyoming. • increase penalties for acts of animal cruelty when committed in the presence of a child or adolescent. clinical social workers may become aware of clients' human-animal interactions through recognizing a client's attachments and issues vis-à-vis the animals in his or her environment and by introducing animals for therapeutic purposes to enhance the client-practitioner relationship. in one of the earliest writings on the nexus of social work and human-animal interactions, netting, wilson, & new (1987) outlined seven ways in which social workers can contribute to human-animal bonding: • being sensitive and supportive in counseling clients who have pet-related problems. • being aware of clients' relationships with their pets and assisting in locating support services that include pet care. • being aware of policies that affect pet ownership, such as restrictive housing conditions, and advocating for clients' pet-related interests. • assessing clients to determine their readiness to accept pet-related interventions. • being critical of how pet-related programs are developed and collaborating with animal professionals. • acknowledging potential benefits and problems which may accompany pet-related programs. • linking veterinarians into the human services referral network. more recently, silverman (2018) published a brief guide to ways in which social workers can utilize animals as a bridge between a therapist and patient in private practice. animals can expedite rapport building with patients who have issues with attachment disorders and enhance the motivation to attend the session which improves retention and treatment outcomes. animals may function as a surrogate of the therapist and allow for more ethical therapeutic touch, which could be a corrective experience for those with histories of trauma. fostering the human-animal connection may help patients identify sustainable, long-term support to manage symptoms and maintain functioning after the therapeutic relationship with a clinical social worker has ended. she identified four categories of animals utilized in a helping capacity: • service animals, which are individually trained to do specific tasks for a person with a physical or sensory disability. clinical social workers may recommend that a patient consider having a service animal and identify resources to obtain one. • emotional support animals, a newer and vaguer category, that provide emotional benefits to a person diag-nosed with a mental health disorder that impairs or limits functioning in one or more life domains. • comfort dogs, introduced in disaster responses to offer a calming presence to survivors and first responders. • animal-assisted therapy animals, professionally evaluated to be introduced in treatment plans with intentional, goal-directed activities to complement traditional interventions. social workers should note that spouses and partners may be jealous of a disabled individual's dependence upon and emotional attachment to a service animal. also, the emotional support animal system has a potential for egregious abuses by individuals getting their animals so designated solely to accompany them on airplane flights; online services will provide such documentation from mental health professionals who have never examined the client. human-animal bonds may be particularly robust with older clients and present unique challenges. for individuals who are socially isolated, pets may be a significantly vital source of companionship and emotional support. caring for a pet may be an especially strong motivator for a client to get out of bed, have a daily routine, nurture another being, or go for a walk. the animal may be a last link to a deceased spouse (arkow, 2015a). human-animal social work issues relevant to older adults include: • animal neglect: more than 92% of adult protective services respondents to a national survey reported animal neglect coexisting with a client's inability to care for himself/herself, indicating that reports of animal neglect may be an important warning sign for vulnerable adults' self-neglect (lockwood, 2002) . animals may be neglected by frail elders who lack financial resources, transportation, or physical or mental capacity to care for them adequately (peak, ascione & doney, 2012 ). • self-neglect: frail elders may neglect their own needs by spending limited financial resources on their animals' food and medications. some may refuse to go into hospitals, assisted living or long-term healthcare facilities unless provisions are made for their pets (boat & knight, 2000) . • coercive control: in more than two-thirds of domestic/ elder abuse cases, the perpetrators were family members who may neglect or abuse the elder's pet as a form of control or retaliation, out of frustration over their caretaking responsibilities, or as a way to extract financial assets from the victim (humane society of the u.s., 2005). • bereavement: isolated seniors may experience profound grief and depression upon the death of a beloved pet. some seniors are reluctant to replace departed pets in fear that the animals will outlive them (boat & knight, 2000) . many older adults, particularly the widowed and elderly, are at risk of emotional trauma and experience significant disruptions in eating, sleeping, job-related responsibilities and other daily routines and decreased socialization behaviors following the death of a pet (quackenbush, 1984) . • denied services: home health aides, social workers and other caregivers may be reluctant to enter seniors' dwellings if they fear the presence of aggressive animals or deteriorated environmental conditions linked with animal hoarding or neglect (boat & knight, 2000) . • animal hoarding: animal hoarders may come from any cohort but they are statistically over-represented by older women (patronek & nathanson, 2009 ). animal hoarders (and their children) often live in unhealthy environments surrounded by dozens and even hundreds of living and deceased animals in states of neglect, starvation and suffering. stereotypical hoarders, often labeled as "cat ladies," have been reported as living in a self-fulfilling cycle of social isolation: they gravitate towards animals because they are uncomfortable around people, and other people choose not to associate with them because of their excess number of animals. many are experiencing mental health issues and a collaborative, multi-agency response is invariably required (patronek, loar, & nathanson, 2006) . nathanson (2009) , in identifying four core barriers that limit adult protective services workers' involvement in these cases, called animal hoarding one of the most perplexing and problematic human-animal relationship and a deviant behavior associated with extremely deleterious conditions of comorbid animal and selfneglect. she identified training programs that can better prepare human services professionals to respond to these clients and engage in trans-species and interdisciplinary efforts essential for the safety, health and well-being of the hoarder, human and animal dependents, property, and community. social workers, whether in private practice, nonprofit organizations or governmental adult protective services agencies, can recognize the import of these human-animal relationships, locate support services for the animals and make appropriate referrals including temporary foster care and other pet services for owners who are in need of hospitalization, long-term care or other social services. social work input on multidisciplinary teams can help to resolve the particularly challenging psychosocial aspects of animal hoarding. and social workers should be attuned to the potential that a client's requesting a veterinarian to have all of his or her pets euthanized is a potential sentinel warning sign for suicidal behavior. social workers can help train veterinarians in recognizing this warning sign and responding with appropriate referrals. an emerging frontier is exploring veterinary medicine's response to suspected domestic violence. an incident in deland, fla. in 2018, when a woman being held captive at gunpoint by her abusive boyfriend was able to alert veterinary staff who in turn called the police (robbins, 2018) , brought to national attention what was just beginning to be discussed in professional journals: how should veterinarians and their staffs, the majority of whom now are women (kelly, 2017) , respond to suspected domestic violence in their clientele? (newland et al., 2019; larkin, 2018; allison, satterwhite, ramaswamy, hynek, & agnew-svoboda, 2017) . veterinarians in the united kingdom and new zealand have taken the most proactive responses in addressing this concern. medics against violence, a scottish collaborative of human and veterinary healthcare professionals, created a domestic abuse veterinary initiative to train veterinarians to help pet owners needing to escape domestic violence; the initiative was featured in a british veterinary association guidance for responses to suspected domestic abuse (animal welfare federation and the links group, 2016). in 2015, the scottish government put £115,000 into a national campaign to train 100,000 front-line professionals in the three fields identified as most likely to encounter domestic violence survivors: dentists, veterinarians and hairdressers (paterson, 2015) . the u.k.'s code of professional conduct for veterinary surgeons states, "given the links between animal, child and domestic abuse, a veterinary surgeon or veterinary nurse reporting suspected or actual animal abuse should consider whether a child or adult within that home might also be at risk" (royal college of veterinary surgeons, 2016). the new zealand veterinary association supported a national legislative response to family violence by describing veterinary medicine as a "three-dimensional profession" with a unique voice in issues that transcend animal life, human life and the environment. nzva called for domestic violence protection-from-abuse orders to specifically include animals, and for changing the definition of domestic violence to include "coercive control" which would cover emotional and psychological abuse to family members through threat or harm to pets or farm animals (national link coalition, 2015) . the veterinary council of new zealand (2013), whose code of professional conduct includes a recommendation that veterinarians confronted with situations of animal abuse should consider whether people within that home might also be at risk, published a guidance that included suggestions on preparing the practice and responding to domestic violence. social workers can help to introduce a response to intimate partner violence as a public health matter to a profession which has been reluctant to get involved, due to a lack of training and fears for personal safety, and help veterinary clinics develop protocols for response and dissemination of literature about community domestic violence resources to their clients. they can also coordinate programs linking students at colleges of veterinary medicine with local domestic violence shelters, such as has been done at texas a & m, mississippi state university, and the university of georgia. social workers can respond to the needs of pet owners who are homeless, whose attachments to their animal companions are often stronger compared with the general population (labrecque & walsh, 2011 ). an estimated 5% to 10% of the 3.5 million americans who experience homelessness every year have dogs and cats, with rates as high as 25% in some areas. because the vast majority of homeless shelters do not allow pets, these restrictions deter pet owners from seeking essential shelter (o'reilly-jones, 2019). many individuals who live on the street keep pets, primarily dogs, for emotional support, safety, a sense of responsibility, to combat loneliness (labrecque & walsh, 2011; williams & hogg, 2016; arnott, 2004) , and as social catalysts to attract passers-by who may offer them money (irvine, kahl, & smith, 2012; anderson, snow, & cress, 1994) . social workers can coordinate veterinary and foster care for the animals and advocate for pet-friendly co-shelters for the homeless much as has been done in domestic violence shelters (phillips, 2019) . social workers can participate in such programs as the street dog coalition, operating in 30 states, in which social work, veterinary and medical school students host clinics and provide resources to help the pets of homeless pet owners. an accurate representation of the roles of animals in families, and of social work's responsiveness to these dynamics, is compromised by several factors. despite the above-cited market research data indicating a widespread population of companion animals and their over-representation among families with children, relatively little is known about the racial, ethnic, socioeconomic, age-related, or geographic demographics of pet-owning families, as such information has never been included in the u.s. census (arkow, 2019a). meanwhile, until recently, social workers have historically ignored the central role that companion animals may play in the lives of their clients, adopting an anthropocentric view underpinned by human rights and social justice (laing & maylea, 2018) . this is reflected in reports (national link coalition, 2020) that identified only 24 schools of social work in the u.s. and seven in canada that are believed to include the topic of human-animal relationships in either undergraduate or graduate level study, or in the absence of such courses have a faculty member known to have a specialization in the human-animal bond. it is hoped that publications such as this will begin to address these shortcomings and increase awareness of human-animal relationships in the lives of social workers' clients. the inclusion of human-animal relationships should be considered more widely in training and practice as part of social work's commitment to social and environmental justice and fighting oppression and seen as an expanding opportunity for research, practice, advocacy, and advancing public policy. in the process, additional career opportunities may open up with this species-spanning approach to resolving individual, family and community challenges. such inclusion can begin with something as simple as routinely including companion animals in genograms, ecomapping, and definitions of family support systems (risley-curtiss, 2010; hodgson & darling, 2011) . as assessing clients' needs is an important step in developing the best plan to solve clients' problems, including pet protective factors in clients' ecologies should be considered a relevant environmental factor in social work practice theory (sato, 2011) . collecting information about all the pets and humans in a family communicates interest and concern for the whole family and demonstrates an integrated approach to care that can help in planning appropriate interventions and preventive care. human-animal bond awareness can be further expanded by adding relevant coursework and field placements in schools of social work and training programs in continuing education. given the established links between animal cruelty and other forms of violence within the family system (arkow, 2019b), questions about human-animal interactions and relationships and clients' committing and/or witnessing acts of animal abuse should be systemically, not just optionally, introduced in intakes and assessments. incorporating the significance of human-animal interactions can help modernize what has been an intrinsic anthropocentrism of social work's theoretical foundations. growing opportunities both within and beyond the veterinary environment will help convince educators, researchers and practitioners that this species-spanning approach is worthwhile and offers opportunities for career development, personal fulfillment and improved service delivery. veterinary social work blends the human side of veterinary medicine with the animal side of social work. as awareness of and interest in veterinary social work continues to grow, additional opportunities will emerge whereby social workers with an abiding interest in animals as well as people can help their clients, society, and the non-human members of families and communities. battered women's reports of their partners' and their children's cruelty to animals safe havens 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for children adulthood animal abuse among men arrested for domestic violence the animal-human bond: health and wellness effects of pet contact on human well-being animal cruelty, antisocial behaviour, and aggression: more than a link the relationship between bullying and animal abuse in adolescents: the importance of witnessing animal abuse bullying and animal abuse: is there a connection? pets in the family: practical approaches violence in animal cruelty offenders rediscovering connections between animal welfare and human welfare: creating social work internships at a humane society helping vulnerable adults and their pets confrontations and donations: encounters between homeless pet owners and the public the world's children and their companion animals: developmental and educational significance of the child/ pet bond. washington: association for childhood education international pet abuse as part of intimate partner violence. wellington, nz: national collective of independent women's refuges veterinary medicine is a woman's world. veterinarian's money digest providing comfort, companionship, and relief: the use of courtroom dogs homeless women's voices on incorporating companion animals into shelter services traumatized witnesses: review of childhood exposure to animal cruelty they burn brightly, but only for a short time": the role of social workers in companion animal grief and loss is counseling going to the dogs? an exploratory study related to the inclusion of an animal in group counseling with adolescents for human needs, some veterinary clinics are turning to a professional: social workers see a place for themselves in veterinary practice when domestic violence arrives at the clinic door: how veterinary staff can respond to abused clients and patients the community context of animal and human maltreatment: is there a relationship between animal maltreatment and human maltreatment: does neighborhood context matter? (doctoral dissertation making the connection between animal cruelty and abuse and neglect of vulnerable adults cross-reporting of interpersonal violence and animal cruelty: the charlotte project animals as social supports: insights for understanding animal-assisted therapy the links between animal abuse and family violence, as reported by women entering shelters in calgary communities cultural factors in the cause and prevention of pathological homicide why the wild things are: animals in the lives of children children's ideas about the moral standing and social welfare of non-human species animals in the lives of children animals in the lives of children animal assisted therapy and young people: a review of selected literature social facilitation of contact with other people by pet dogs a therapist's guide to treating grief after the loss of a pet: a three-tier model animal hoarding: slipping into the darkness of comorbid animal and self-neglect three-dimensional" new zealand veterinarians respond to domestic violence the connection between animal cruelty and societal violence and vulnerability schools of social work with humananimal interactions in curriculum the human-animal bond: implications for practice considering the relationship between domestic violence and pet abuse and its significance in the veterinary clinical and educational contexts when fido is family: how landlordimposed pet bans restrict access to housing revisiting a link: animal abuse, bullying, and empathy in australian youth vets enlisted in bid to stop domestic abuse. the national animal hoarding: structuring interdisciplinary responses to help people, animals and communities at risk a theoretical perspective to inform assessment and treatment strategies for animal hoarders adult protective services and animal welfare: should animal abuse and neglect be assessed during adult protective services screening 2010 national urban animal report start-up manual bowling alone: the collapse and revival of american community the pet connection: its influence on our health and quality of life pets, owners, problems, and the veterinarian: applied social work in a veterinary teaching hospital. the compendium on continuing education for the small animal practitioner social work services for bereaved pet owners: a retrospective case study in a veterinary teaching hospital child abuse, domestic violence, and animal abuse: linking the circles of compassion for prevention and intervention individual counseling for sexually abused children: a role for animals and storytelling exploring the grief experience among callers to a pet loss support hotline social work practitioners and the human companion animal bond: a national study expanding the ecological lens in child welfare practice to include other animals she was family:" women of color and their animal human connections the animalhuman bond and ethnic diversity florida woman held captive by boyfriend slips note to vet staff pets as pawns: the co-existence of animal cruelty and family violence pet loss and children: establishing a healthy foundation code of professional conduct for veterinary surgeons, supporting guidance no. 14 (client confidentiality). london: royal college of veterinary surgeons nonhuman animal cruelty, bullying, and behavioral difficulties among women saskatchewan society for the prevention of cruelty to animals and saskatchewan towards offering partnership solutions (stops) to violence social workers' attachments to their pets, organizational structures, and their impact on professional assessment regarding the roles pets play in clients' lives (doctoral dissertation) shelter-specific occupational stress among employees in animal shelters the role of animals as therapeutic aids in private practice battered women's concern for their pets: a closer look the role of companion animals throughout the family life cycle effects of childhood adversity on bullying and cruelty to animals in the united states: findings from a national sample guidance for veterinarians dealing with cases of suspected or actual animal abuse and family violence the link between interpersonal violence and animal abuse a study on the relationship of child abuse and pet abuse animal cruelty and bullying: behavioral markers of delinquency risk or causal antecedents of delinquent behavior? risk factors for interpersonal violence and associated injury among urban women the health and welfare of dogs belonging to homeless people wa: petcare information & advisory service and the centre for the built environment and health (school of population health) the pet connection: pets as a conduit for social capital? more than a furry companion: the ripple effect of companion animals on neighborhood interactions and sense of community social capital and pet ownership-a tale of four cities reunification of child and animal welfare agencies: cross-reporting of abuse in wellington county key: cord-007843-yqdqm4rh authors: shader, richard i. title: zoonotic viruses: the mysterious leap from animals to man date: 2018-07-26 journal: clin ther doi: 10.1016/j.clinthera.2018.06.016 sha: doc_id: 7843 cord_uid: yqdqm4rh nan valley, chikungunya, and ross river fevers and western equine, eastern equine, japanese, la crosse, and st louis encephalitis. mosquito-borne viruses are often labeled as arboviruses, a category that also includes tick-borne viruses. another virus carrier is the fruit bat. i remember walking through a beautiful park in melbourne australia on the way to captain cook's cottage. i was startled by the sight of huge black creatures hanging from trees that arched over the walkway. the racket they made when they all flew away was memorable. they were australian fruit bats. from a park guide, i learned that there are 13 varieties of australian fruit bats, popularly known as flying foxes because their ears and faces are foxlike. fruit bats belong to the family pteropodidae. they are hosts for both nipah and hendra viruses, which can cause disease in various animals and humans. nipah and hendra viruses are both members of the paramyxoviridae family; neither virus is known to cause disease in their host bats. nipah also infects pigs. fruit bats inhabit many other regions and countries, including africa, china, india, and malaysia. coronaviruses infect humans, other mammals, and birds. 6 certain human coronaviruses are involved in the human to human transmission of some cases of the common cold. some coronaviruses are zoonotic, infecting both their hosts and humans and other mammals. examples include severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). the latter infections have been recognized only for the last several decades; it is possible that they have been around much longer but not identified as such. sars is now definitively linked to bats. 7 certain influenza viruses (h5n1, h7n9) can infect birds, chickens, ducks, and other wild and domesticated birds. called bird flu, these viruses can be transmitted to humans who come in contact with the droppings and saliva of infected birds. in rare instances, human disease has resulted from eating undercooked eggs or poultry. similarly, although infrequent, the h1n1 influenza virus can be transmitted from infected pigs to humans, hence its common name of swine flu. questions remain about these cases because some infected persons with sars and mers never came into contact with pigs. 8 even more uncommon is for a virus to be transmitted from an infected human to an animal. two reports i read suggest that humans infected their pets (cats, dogs, ferrets) with the h1n1 virus. 9, 10 from what i could learn, however, the possibility that the animals and humans were independently infected from common sources has not been ruled out. in theory, infected humans could transmit the rabies virus to animals; no cases have been documented. 11 a new antiviral agent sheahan and colleagues 12 have reported that a novel antiviral, previously shown to be active against the acellular ebola virus (zaire ebolavirus), is also active against the coronaviruses sars and mers. developed and named remdesivir (gs-5734) by gilead sciences, it is a nucleotide analog prodrug. gilead also studied it for marburg virus. ebola and marburg are filoviruses. results from other investigations suggest it may be effective against bat viruses and other viruses, such as respiratory syncytial virus and lassa fever virus. [12] [13] [14] remdesivir is not currently approved by the us food and drug administration; however, some countries have authorized premarketing compassionate use. for this issue of clinical therapeutics, our infectious diseases topic editor dr ravi jhaveri has assembled a collection of articles entitled "hot topics in viral diseases." the collection highlights recent controversies in vaccine licensure and recommendation, as well as advances in antiviral therapies for herpesvirus, hepatitis b and c, respiratory syncytial virus, and influenza, with an emphasis on pediatric patients. [15] [16] [17] [18] [19] [20] [21] [22] in closing, i wish to congratulate ravi on his promotion and change of venue. he is moving from the university of north carolina to northwestern university's feinberg school of medicine, where he will become professor of pediatrics and associate chief of infectious diseases at lurie children's hospital. richard i. shader, md polio has not returned to venezuela, who says centers for disease control and prevention. enterovirus and parechovirus surveillance -united states host range of poliovirus is restricted to simians because of a rapid sequence change of the poliovirus receptor gene during evolution origins of hiv and the aids pandemic. cold spring harb perspect med emerging zoonotic viral diseases bats are natural reservoirs for sars-like coronaviruses centers for disease control and prevention. h1n1 flu. the 2009 h1n1 pandemic: summary highlights the health implications influenza virus infection has for pets petmed can you really give your dog or cat the flu exposure to the virus broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses hot topics in viral infections a review of therapeutics in clinical development for respiratory syncytial virus and influenza in children elimination of vertical transmission of hepatitis b in africa: a review of available tools and new opportunities antiviral therapies for herpesviruses: current agents and new directions unique challenges of hepatitis c in infants, children, and adolescents hepatitis c management simplification from test to cure: a framework for primary care providers live attenuated influenza vaccine: is past performance a guarantee of future results? discrepancies between us food and drug administration vaccine licensure indications and advisory committee on immunization practices recommendations: provider knowledge and attitudes number 8 of clinical therapeutics. to view the previous update, see the articles below: jhaveri r. vaccines collins mh, metz sw. progress and works in progress: update on flavivirus vaccine development cortes key: cord-020764-5tq9cr7o authors: vertrees, roger a.; goodwin, thomas; jordan, jeffrey m.; zwischenberger, joseph b. title: tissue culture models date: 2010-05-21 journal: molecular pathology of lung diseases doi: 10.1007/978-0-387-72430-0_15 sha: doc_id: 20764 cord_uid: 5tq9cr7o the use of tissue cultures as a research tool to investigate the pathophysiologic bases of diseases has become essential in the current age of molecular biomedical research. although it will always be necessary to translate and validate the observations seen in vitro to the patient or animal, the ability to investigate the role(s) of individual variables free from confounders is paramount toward increasing our understanding of the physiology of the lung and the role of its cellular components in disease. additionally, it is not feasible to conduct certain research in humans because of ethical constraints, yet investigators may still be interested in the physiologic response in human tissues; in vitro characterization of human tissue is an acceptable choice. the use of tissue cultures as a research tool to investigate the pathophysiologic bases of diseases has become essential in the current age of molecular biomedical research. although it will always be necessary to translate and validate the observations seen in vitro to the patient or animal, the ability to investigate the role(s) of individual variables free from confounders is paramount toward increasing our understanding of the physiology of the lung and the role of its cellular components in disease. additionally, it is not feasible to conduct certain research in humans because of ethical constraints, yet investigators may still be interested in the physiologic response in human tissues; in vitro characterization of human tissue is an acceptable choice. tissue culture techniques have been utilized extensively to investigate questions pertaining to lung physiology and disease. the isolation and propagation of human bronchial epithelial cells has allowed investigators to begin to characterize the interactions and reactions that occur in response to various stimuli. moreover, the culture of human airway smooth muscle has allowed researchers to investigate a pathologic cascade that occurs in asthma as well as other physiologic responses in the smooth muscle of the lung. numerous lung cancer cell lines have been established to investigate their responses to chemotherapy and determine their biologic properties. overall, the use of cultured human lung tissue has provided a windfall of information on the pathogenesis of diseases that affect the lung and on the basic physiology and development of the lung in general. despite this wealth of information in the literature, this chapter is the fi rst to discuss the use of tissue culture models to examine the physiology and pathologic basis of lung diseases. in light of this, we briefl y discuss the history and principles behind the utilization of tissue culture. we then discuss the current use of tissue culture to examine many of the from henrietta lacks were cultivated into the fi rst immortal cell line-"hela." 5 hela cells are still one of the most widely used cell lines today. since the 1950s, tissue culture has become fi rmly established as a mechanism to answer many questions in biomedical research. today, tissue culture is widely used to investigate diseases that affect the lung, and through this work we have been able to increase our understanding of the pathologic cascades that occur in lung diseases, as well as the normal physiologies of the lung. tissue culture is a commonly used generic term for the in vitro cultivation of cells, attributed to the early cultures that generally consisted of heterogeneous cultures of crudely disaggregated tissues. currently, many terms are used that can be encompassed by the term: organ culture, cell culture, primary explants, and ex vivo propagation all deal with the in vitro cultivation of cells or tissues. cell culture in general can be applied either to primary cells (e.g., those with a fi nite life span) or to cell lines (e.g., hela cells). additionally, these cultures can be either a homogenous or a heterogenous group of cells. primary cell culture involves the isolation of cells from a tissue by disaggregation. single cell suspensions from tissues can be completed through either enzymatic digestion of extracellular matrix surrounding the cells-such as with ethylenediaminetetraacetic acid, trypsin, or collagenase-or mechanical disaggregation. these disaggregation procedures have the disadvantage of possibly injuring cells. if the cells of interest are adherent viable cells, they will be separated from nonviable cells when the medium is changed. alternatively, viable cells can be separated from nonviable cells prior to culture by subjecting the single cell suspension to density gradient centrifugation (e.g., hypaque). primary cells have an advantage of possessing many of the biologic properties that they possessed in vivo because they are not transformed. primary cells, unlike cell lines, are not immortal and have only a fi nite survival time in culture before becoming senescent. variant cells, however, as well as those obtained from neoplastic tissue, may proliferate infi nitely, thus becoming immortal in vitro. this will eventually allow the immortal cell to take over the culture and can be thought of as a cell line. in general, primary human cultures will survive for 30-80 passages in vitro, although this number is dependent on cell type, conditions, and possibly other unknown factors. primary cells are widely used to examine the effects of toxins, infectious agents, or other cellular interactions that would not be feasible in vivo. primary cells have a disadvantage of being a heterogeneous mixture of cells upon primary isolation, with the type of cell obtained generally a component of the disag-gregation method used. the most common contaminant seen following isolation of primary cells is cells of mesenchymal origin (e.g., fi broblasts). however, advances have been made that allow the culture of homogenous populations of cells. for instance, cell surface molecules specifi c for the cells of interest may be tagged with monoclonal antibodies. techniques such as fl uorescenceactivated cell sorting or the use of magnetic beads can be utilized to enrich the single cell suspension for the cell type of interest. additionally, some investigators have recently exploited unique characteristics of certain cells, such as the presence of p-glycoprotein or multidrug resistance-associated proteins expressed on endothelial cells, to poison other contaminating cells in culture. 6 another type of primary cell culture is "primary explants." this type of culture is not subjected to a disaggregation procedure like the primary cell technique described earlier. therefore, single cell suspensions do not occur. briefl y, tissue samples are dissected and fi nely minced. these tissue pieces are then placed onto the surface of a tissue culture plate. following plating of tissue pieces, cells have been shown to migrate out of the tissue and onto the tissue culture surface. 7 this technique is useful when cells of interest may become damaged or lost in the disaggregation technique described earlier and is often used to culture human bronchial epithelial cells. 8 cell lines are another useful source of cells to investigate questions in biomedical research. these cells have the advantage of being immortal as opposed to the fi nite life spans that primary cells possess. additionally, they are generally well studied and characterized, leaving few experimental variables to worry about. these cells however, are prone to dedifferentiation-a process by which they lose the phenotypic characteristics of the cell from which they began. many of the early cell lines were established from tumor tissue and as such possess abnormal growth characteristics. newer cell lines have been established by molecular techniques such as inserting a telomerase gene into a cell to allow it to replicate infinitely. 9 because of the phenotypic changes that allow cell lines to replicate infi nitely in culture, they are often a fi rst choice for experiments; however, they are also highly criticized in light of their nonnatural phenotype. organ culture, as the name implies, involves ex vivo culture of the whole or signifi cant portion of the organ. the main advantage to this type of culture is the retention and preservation of the original cell-cell interaction and extracellular architecture. this type of culture may be particularly important when experimental design necessitates the use of an ex vivo system, but researchers still need to retain the original organ architecture to answer questions posed. these types of cultures do not grow rapidly, however, and are therefore not suitable for experiments needing large numbers of a particular cell type. 10 advantages and limitations of tissue culture tissue culture has become the penultimate tool of the reductionist biologist. the utilization of tissue culture as a research methodology has allowed investigators to study isolated interactions in its near-normal environment. these experiments by their very nature introduce artifacts; however, they do minimize the number of confounding variables that may affect a particular experiment. for instance, tissue culture allows investigators to determine the effects of one particular treatment on a particular cell type, which would not be feasible in vivo. additionally, tissue culture models of disease allow investigators to obtain samples and make observations more readily than those done in vivo. however, it is the relative simplicity of experiments done in vitro that allows models of disease or physiology to come under frequent and warranted criticism. these models do not take into consideration the complexity of biologic systems. diminishing possible confounding variables by culturing cells in vitro brings up the constant criticism of how applicable results are because of alterations of the normal cellular environment in vivo. for example, cell-cell interactions in vitro are reduced and unnatural. moreover, the culture does not contain the normal heterogeneity and threedimensional architecture that is seen in vivo. this said, however, tissue culture biology has proved to be successful in many ways. we have briefl y discussed the advantages that experimental systems using tissue culture affords researchers studying physiology and pathogenesis. because of its ability to isolate individual variables and determine their role(s) in physiology, cell culture has become an integral tool in deciphering the pathologic cascades that occur in human disease. diseases that affect lung are no exception. many diseases that affect the lung, and humans in general, are multifactorial. this begs the question how can cell culture, because of its reductionist nature only dealing with a minimal number of variables, help to solve the unknown questions and decipher the components involved in disease? often, clinical observations, and the questions arising therein, have been the launching pad for investigation. for instance, observations of massive infl ammation in the bronchoalveolar lavage samples of patients with acute respiratory disease syndrome (ards), consistent with damage seen in histologic samples, prompted investiga-tors to determine the role(s) of infl ammation in the etiology of ards. through the use of cell culture, investigators were able to determine individual interactions that occurred in the disease process. investigators have utilized culture models employing microcapillary endothelial cells under fl ow conditions to understand the role of proinfl ammatory cytokines in the cytokinesis and emigration of neutrophils in disease. using a model of pulmonary endothelium under fl ow conditions allowed investigators to demonstrate the importance of certain proinfl ammatory cytokines in ards. 11 the role of inhaled toxicants in lung injury, and the mechanism(s) by which they cause disease, is another area of investigation that has utilized cell culture. scientists have developed diverse and unique tissue culture systems that contain air-liquid barriers of lung epithelium and subjected these cells to various gaseous toxicants to determine what occurs following inhalation of various chemicals. utilizing these types of systems, investigators are able to control the exposure time and other variables that may be diffi cult when determining inhaled toxicant effects in vivo. moreover, the use of tissue culture, as opposed to an animal model, allows investigators to observe effects kinetically, without undue changes (e.g., sacrifi ce) and expense in the experimental model. 11 a tissue culture model also permits an investigator to observe multiple changes in real time, such as cellular integrity, cell signaling and intracellular traffi cking, protein expression changes, oxidant-induced cellular damage, and more. deciphering each of these changes in an animal model would be extremely diffi cult; through employing a tissue culture model, researchers are able to tightly control the experimental system while isolating the events of interest. further examples of how tissue culture models are currently being used to elucidate questions in lung physiology and disease are discussed later in the section on lung tissue cell lines. maintaining cells in vitro was initially a very diffi cult task. many characteristics need to be fulfi lled before a successful cell culture occurs. some of these characteristics are dependent on the type of tissue being studied; others may depend on specifi c requirements of the individual cells. various chemically defi ned media are now available commercially to support the growth and differentiation of numerous cell types. the creation of defi ned media has allowed investigators to culture a multitude of cell types while controlling the local environment to answer pertinent questions. for example, glucose can be removed from a culture medium in order to study its effects on cellular metabolism, relative position in the cell cycle, and many other effects. each chemical component is known in these media. additionally, investigators can add growth factors to nourish their cell cultures. the medium chosen when culturing cells in tissue culture must fi t two main requirements: (1) it must allow cells to continue to proliferate in vitro, and (2) it must allow the preservation of the certain specialized functions of interest. 7 the most common medium formulations used currently in lung research are dulbecco's modifi ed eagle's medium, minimum essential medium, rpmi 1640, and ham's f-12. occasionally, investigators develop new medium types to attain a formulation that optimizes their own experimental conditions. fetal bovine serum is a common additive to most tissue culture media, although some investigators choose to forgo this additive for more defi ned supplementation. additionally, others may choose sera from other sources such as human serum when culturing cells of human origin. inactivation of complement by heat treating serum for 1 hr at 56°c was initially very popular in tissue culture. however, it has become clear that this treatment may in fact damage some of the proteinaceous growth factors present in the medium, rendering it less effective. currently, many experts recommend heat inactivation only if the cell type of interest is particularly sensitive to complement. 12 more specifi c examples of medium utilized in lung tissue culture models are given later in the section on lung tissue cell lines. when deciphering if the current culture conditions are suffi cient for the experimental design, the investigator must determine which cellular characteristics are important. not only are the general characteristics, such as adhesion, multiplication, and immortalization of cell types important, but so are tissue-specifi c characteristics. of importance to pulmonary research, the lung is a unique environment to simulate in vitro because of the air-liquid interface. recently, investigators have made use of culture insert wells (e.g., transwells, corning) in order to study this interaction. 6 cell adhesion nearly all normal or neoplastic human epithelial cells will attach with relative ease to tissue culture surfaces. most tissue culture models utilizing tissue of lung origin fi t this description, with the notable exception of small cell lung carcinoma cell lines. however, for culture cells that may loosely adhere, or may not adhere at all, scientists coat tissue culture surfaces with extracellular matrix proteins. incubating tissue culture surfaces with serum, as well as laminin, fi bronectin, or collagen, prior to culture has been shown to improve attachment of fi nicky cells. 8 these treatments also help in replicating the normal attachment of cells to extracellular matrix proteins in vivo. the development of continuous cell lines may be serendipitous, as was the development of early cell lines. in brief, many investigators would continue splitting primary cell cultures until one or more cell clones became immortal. unfortunately, the changes that generally occurred in culture led to cells with abnormal phenotypes that had undergone dedifferentiation. today, many investigators choose to use molecular biology techniques, exploiting our current knowledge of oncogenic viruses and enzymatic processes of cellular aging to transform primary cells in vitro to an immortal phenotype. it is known that the large t antigen present in the sv (simian virus) 40 virus is capable of transforming cells to an abnormal phenotype. 11, 13, 14 moreover, transfection of primary cells with a transposase enzyme has also been shown to induce an immortal phenotypic change while preserving most normal cellular functions and phenotypes. 11 dedifferentiation a commonly encountered problem in tissue culture is dedifferentiation. this loss of phenotype may be insignificant to the research at hand or it may be critical, and it must be dealt with on a case by case basis. when a cell culture undergoes dedifferentiation it is often unclear whether undifferentiated cells took over the culture of terminally differentiated cells or whether a primary cell of interest became immortal under the culture conditions. the functional environment in which cells are cultured is critical when correlating experimental results to those seen in vivo. we previously alluded to the importance of the environment in which cells are cultured when discussing the advantages and limitations of tissue culture. investigators have frequently strived to replicate integral in vivo environments in vitro in order to increase the signifi cance of their experimental results. the development of cell culture insert wells (e.g., transwells, corning) has allowed investigators to culture bronchial or alveolar epithelial cells at an air-liquid interface. this ability allows investigators to begin to replicate a signifi cant aspect of these cells' functional environment in vitro, thereby increasing their understanding of the effects of gaseous particles on pulmonary epithelial cells. alternatively, scientists have also cultured epithelial cells on a roller bottle apparatus. this method allows investigators to determine the amount of time the apical epithelial cell surface is in contact with the air. capillary cell cultures have also come under frequent criticism when cultured in a monolayer in a tissue culture plate. investigators have been able to utilize gel matrices in which capillary cells form tubule-like structures, more closely replicating the architecture these cells maintain in vivo. additionally, endothelial cells are constantly under fl ow conditions in vivo. addressing this condition in vitro has allowed investigators to look at the role of endothelial cells during infl ammation-helping to increase the understanding of the role endothelium plays in acute lung injury. at times, researchers may also choose to determine the effects of soluble factors (e.g., cytokines, hormones, neurotransmitters) from acute patients or animal models in a cell culture model. the milieu of soluble factors present in the serum that may play a role in a disease state is considerable. moreover, these factors may have actions alone that are different when combined with other soluble factors. reconstituting every factor presents a diffi culty in vitro and leaves the possibility that an unknown factor may be missing. to address this, investigators have harvested sera from patients or animal models and used these samples as additives in their media formulations. for instance, through the use of serum samples from an animal model of smoke/burn injury-induced acute lung injury, investigators have demonstrated that use of arteriovenous co 2 removal in acute lung injury signifi cantly reduces apoptotic cell death in epithelial cells. 15 lung tissue cell lines: establishment and signifi cance the diversity of research fi elds utilizing tissue culture models of lung diseases is extensive. in this section, we will give a brief overview of the main lung cell types that are being utilized in research today to answer pressing questions about lung physiology and the pathophysiology of pulmonary disease. included in this discussion is also an overview of cell isolation and culture. the use of normal human bronchial epithelial (hbe) cells is extensively reported in the literature. based on a method pioneered by lechner et al., 16 bronchial fragments obtained from surgery, autopsy, or biopsy specimens may be used as explants. the outgrowth of bronchial epithelial cells occurs readily from these explants when grown in medium supplemented with bovine pituitary extract and epidermal growth factor. alternatively, these cells have also been demonstrated to grow in basal keratinocyte serum-free medium without supplementation; however, they demonstrate a slower growth rate and earlier senescence. 8 cultures of hbe cells are valuable for determining the responses to toxic inhaled pollutants. in vitro exposure systems based on these methods have several advantages. first, in vitro exposure systems can be stringently controlled and reproduced much better than in animal systems; second, individual determination of the cell types' responses to pollutants allows for a better characterization of the individual involvement of the cell type to a biologic response. finally, in vitro determination of the responses to toxic agents allows investigators to observe the reactions of human cells when testing in humans is not feasible because of ethical restraints. in vitro study of the responses of bronchial cells to gaseous pollutants is not without its diffi culties. wallaert et al. 17 have described these constraints well. briefl y, because of the gaseous nature of the pollutants, culture systems should be designed that allow signifi cant exposure times to pollutants while also taking care to inhibit cells from drying out when exposed to air. to facilitate these experiments, roller bottle cultures have been developed that allow cells direct contact with the ambient air. alternatively, cells have been grown on a membrane fi lter and cultured at an air-liquid interface, which allows constant exposure to the experimental treatment. the same type of experiments that are used to determine the responses of cells to inhaled toxicants have also been used to characterize responses to inhaled pharmaceuticals. in addition to the characterization of responses to inhaled agents, epithelial cell cultures, notably alveolar epithelium obtained from fetal lung tissue, have allowed investigators to characterize the liquid transport phenotype that occurs in the developing lung. characterization of the cl − ion secretion system, which occurs in the distal lung epithelium throughout gestation, has been shown to be integral in the stimulation of growth of the developing lung by regulating liquid secretion. likewise, a phenotypic switch of na + absorptive capacity has been described toward the end of gestation, which is important for preparation of the lung to function postpartum and beyond. these culture systems have elucidated important physiologic changes that occur in the developing lung. similar experiments have demonstrated that while ion transport plays a crucial role in this process other hormones and neurotransmitters are also important. pulmonary endothelial cells represent a unique type of endothelium because of their paradoxical responses to hypoxia. this uniqueness highlights the need to utilize cell culture models of pulmonary endothelium as opposed to other endothelia when interested in investigating their role(s) in pulmonary physiology. several investigators have described the isolation and culture of pulmonary endothelial cells. persistent pulmonary hypertension of the newborn, also known as neonatal pulmonary hyper-tension, is caused by a disorder of the pulmonary vasculature from fetal to neonatal circulation, culminating in hypoxemic respiratory failure and death. the inciting events that culminate in neonatal pulmonary hypertension are multifactorial. despite this, decreased production of vasodilator molecules such as nitric oxide and prostaglandin i 2 in the pulmonary endothelium has been shown to be a critical component of disease progression. 18 primary cell cultures of human airway smooth muscle tissue can be obtained utilizing a method described by halayko et al. 19 in which they isolated and characterized airway smooth muscle cells obtained from canine tracheal tissue. briefl y, airway smooth muscle cells were obtained by fi nely mincing tissue and subjecting it to an enzymatic disaggregation solution containing collagenase, type iv elastase, and type xxvii nagarse protease. following generation of a single cell suspension, cells may be grown in dulbecco's modifi ed eagle's medium supplemented with 10% fetal bovine serum. halayko et al. 20 obtained approximately 1.3 × 10 6 smooth muscle cells per gram of tissue using this method. although halayko et al. 21 pioneered this technique using trachealis tissue, many other investigators have obtained airway smooth muscle cells from a variety of biopsy specimens. airway smooth muscle hyperreactivity and hypertrophy has been known for nearly 100 years 2 to be an important end response of asthma. the use of airway smooth muscle in vitro has been vital toward delineating the pathologic steps that occur in asthma, as well as testing of potential therapeutics that may help to decrease the morbidity and mortality of asthma. additionally, the relative paucity of in vivo models of asthma further illustrates the value of isolation and characterization of smooth muscle cells from asthmatic patients in vitro. using airway smooth muscle cell culture, investigators have characterized both the hypertrophic and hyperplastic growth of smooth muscle in individuals. investigation of the potential stimuli that lead to airway smooth muscle proliferation and hypertrophy have led researchers to implicate the mitogen-activated protein kinase family members, extracellular signal-regulated kinase-1 and -2, and the phosphoinositol-3 kinase pathways in pathogenesis. 22 additionally, mediators directing smooth muscle migration have also been observed in vitro and may play a role in the progression of asthma. platelet-derived growth factor, fi broblast growth factor-2, and transforming growth factor-β (tgf-β) have all been shown to play a role in the migratory response of smooth muscle cells seen in asthma. 22 additionally, contractile agonists such as leukotriene e 4 have been shown to potentiate the migratory responses seen with platelet-derived growth factor treatment. 22 human airway smooth muscle cell culture has also been utilized to investigate possible pharmacologic interventions for the treatment of asthma. β 2 -agonists have been shown to decrease the rate of dna synthesis and likewise decrease the hyperplasia seen in airway smooth muscle cells in response to mitogenic stimuli through an increase in cyclic adenosine monophosphate. like β 2agonists, glucocorticoids have similar antiproliferative activities. lung cancer tissue and the development of novel therapeutics culture of neoplastic cells from human tumors has allowed investigators to harvest a wealth of knowledge into the biology of lung cancers; moreover, these cultures have provided potential models to test potential therapeutics. the propagation of lung cancer cells in vitro has been covered in great depth previously. 8 in contrast to primary cell cultures, cultures of neoplastic cells are immortal, allowing their easy growth in culture with less chance of being overgrown by mesenchymal cells such as fi broblasts. the relative ease of growth in culture has led to many cell lines of lung cancer tissue. the national cancer institute, recognizing the need for a variety of lung cancer cell lines (both small cell and non-small cell), helped establish over 300 cell lines. 23 these lines are a wonderful resource for investigators given that they are extensively characterized, and many have full clinical data available. moreover, many of these cell lines are now easily available through the american type culture collection for a modest handling fee. additionally, if investigators do not wish to use currently established lung cancer cell lines, obtaining clinical samples for use in tissue culture models is relatively easy. the same methods used to obtain biopsy specimens for clinical staging can also be used to begin cell cultures. following culture and initial characterization of lung cancer cell lines, many investigators have demonstrated that lung cancer cell lines maintain a similar phenotype after establishment. specifi cally, it has been verifi ed that injection of lung cancer cell lines into nude mice exhibit similar histopathology to the original tumor, indicating minimal change occurred following establishment of the cell lines. small cell lung carcinoma (sclc) cell lines have been established from a multitude of biopsy specimens, including bone marrow, lymph nodes, and pleural effusions. 8, 24 once viable cells have been obtained from clinical samples, cells are easily maintained in a basal cell culture medium such as rpmi 1640 in a humidifi ed incubator at 37°c and 5% co 2 , although the initial isolations of sclc lines utilized hites and acl-4 media. 25 most established sclc cell lines maintain a neuroendocrine phenotype in culture; however, baillie-johnson et al. 24 noticed considerable heterogeneity in the cell lines they established, highlighting the signifi cance that establishing a cell line from the clinical sample of interest may provide investigators with a line that possesses the exact phenotypic properties of interest. small cell carcinoma poses many diffi culties to surgical treatment, owing to its early and widespread metastasis. therefore, combination chemotherapy is generally utilized in treatment. unfortunately, despite initial sensitivities, sclc tumors become resistant to further treatment. utilizing in vitro cultures of sclc cell lines, sethi et al. 26 began to describe how extracellular matrix proteins can protect sclc against apoptosis-inducing chemotherapeutics through β 1 -integrin-mediated survival signals. these data indicate that extracellular matrix proteins surrounding sclc may play a role in the local recurrence seen in patients following chemotherapy in vivo and suggest novel therapeutics aimed at blocking these survival signals. non-small cell lung carcinoma (nsclc) cell lines including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma have all been established. despite the fact that nsclc cells comprise three distinct histologic cell types, all cell types can be established relatively easily. the primary treatment protocol for patients affl icted by nsclc is generally surgical resection of the tumor; therefore, tumor cells for culture are readily available. these cell types can be grown under conditions similar to those described for sclc. infectious diseases play a unique role in lung pathology in light of their roles as either important contributors or consequences of many lung diseases. for instance, certain lung diseases may predispose patients to infection: patients affl icted with obstructive lung diseases, as well as cystic fi brosis patients, commonly suffer from severe and recurrent bacterial infections. additionally, patients may become superinfected following a viral respiratory infection. systemic infections, such as gram-negative bacterial sepsis, may lead to lung diseases such as ards. human type ii alveolar pneumocytes and acute lung injury/acute respiratory distress syndrome pulmonary alveolar type ii cells are a unique cell subset that carries out highly specialized functions that include synthesis and secretion of surfactant, a unique composition of lipoproteins that act to reduce surface tension at the alveolar air-liquid interface. 27 defi ning the molecular mechanisms leading to production of surfactant by type ii pneumocytes is important in many disease processes. the pathogenic sequence that results in ards, the most severe manifestation of alveolar lung injury, is generally thought to be initiated by a systemic infl ammatory response. 28 despite this knowledge, there still exist many questions about the initial triggers and pathologic steps that occur in ards. greater understanding of these steps may help to develop new treatment regimes. currently, treatment of ards consists of mechanical ventilation, which helps to stabilize blood gases. however, mechanical ventilation itself may provoke further infl ammation in the alveoli, thereby decreasing compliance and gas exchange in the alveoli. 29 the cell type of particular interest in ards and diffuse alveolar damage is the type ii pneumocytes. [30] [31] [32] [33] [34] until recently, studies trying to decipher the pathologic sequence in acute lung injury have had to rely on standard lung epithelial cell lines. recently, however, human type ii alveolar epithelial cells (pneumocytes) have been successfully isolated from fetal human lung tissue by collagenase digestion. 35 briefl y, fetal lung tissues were minced and incubated in a serum-free medium containing dibutyryl cyclic adenosine monophosphate for 5 days. the tissue explants were then treated with collagenase and incubated with deae-dextran to eliminate contaminating fi broblasts. cells were then plated onto tissue culture dishes treated with extracellular matrix derived from mdck cells and cultured overnight in waymouth's medium containing 10% serum. these steps resulted in relatively pure populations of human type ii pneumocytes that were then cultured at an air-liquid interface. using these methods, alcorn et al. 35 were able to maintain a primary culture that retained the morphologic and biochemical characteristics of type ii pneumocytes for up to 2 weeks. conventional bioreactors and three-dimensionality: the origins of three-dimensional culture carrel postulated that tissue development was linked to access to nutrient supply, noting that peripheral cells grew readily, and internal cells became necrotic presumably based on their distance from the nutrient source. to circumvent this issue, carrel implemented cultures on silk veils, preventing the plasma clots of the growth media from deforming or becoming spherical, thus facilitating the internal cell's ability to obtain nutrient replenishment. many attempts were made in standard culture systems (bioreactors) and other culture apparatuses to escape the constraints of two-dimensional cell culture, with the intent of yielding high-fi delity human and mammalian tissues, and thus emphasizing the need for development of three-dimensional biology. another famous researcher, leighton, improved on carrel's techniques in the 1950s and 1960s. leighton's major contribution to three-dimensional culture technology was the introduction of the idea of a sponge matrix as a substrate on which to culture tissues. 36 , 37 leighton fi rst experimented on cellulose sponges surrounded by plasma clots resident within glass tubes. he devised a system to grow 1-to 5-mm 3 tissue explants on sponges, using small amounts of chick plasma and embryo extract. after the mixture solidifi ed on the sponge leighton added the nutrient media and inserted the "histoculture" in a roller apparatus to facilitate nutrient mass transfer. he experimented with many sponge combinations, discovering that collagen-impregnated cellulose sponges were optimal for sustaining the growth of native tissue architecture. 3, 38 leighton was successful in growing many different tissue types on the sponge-matrix cultures. 3, 38 leighton also found that c3hba mouse mammary adenocarcinoma cells, when grown on sponge-matrix histoculture, aggregated "much like the original tumor, forming distinct structures within the tumors such as lumina and stromal elements, and glandular structures." an extremely important difference of this threedimensional histoculture from the standard two-dimensional culture is the apparent quiescence of the stromal component and the balanced growth of these cells with regard to the overall culture. leighton further advanced the concept of three-dimensional histoculture to histophysiologic gradient cultures. 39 these cultures are conducted in chambers that allow metabolic exchange between "the pool of medium and the culture chamber by diffusion across a membrane." histophysiologic gradient cultures mimic, to some degree, diffusion in tissues. 38 from the pioneering work of carrel and leighton, other methods of emulating three-dimensional cultures have been developed, such as embedding cells and tissues in collagenous gels of rat tail as per the techniques of nandi and colleagues. many of the advantages of threedimensional cultures seen by leighton, nandi, and others may be attributed to permitting the cells to retain their normal shape and special associations. 3 this global concept will be important as we begin to understand and recall the physical and environmental characteristics of the rotating-wall vessel systems. other methods of three-dimensional culture encompass a technique known as organ culture or culture on a fi lter, a strategy developed by strangeways 40 and fell and robinson. 41 tissue explants were grown on lens paper in a watch glass containing liquid culture medium. browning and trier 42 found "that for some tissues, it is critical to keep the cultures at the air-liquid interface," thus allowing the tissues to experience conditions similar to the in vivo environment. another strategy is the use of three-dimensional cultures known as proto-tissues, or aggregates of cells, used to form spheroids. this technique was popularized by sutherland and colleagues more than 20 years ago when they manipulated aggregates of cells into a spherical confi guration by spinning agitation of the cells in spinner fl asks. 43 this technique produced pseudo-tissue-like organoids useful for research evaluations. each of these methodologies will be of benefi t as we continue to examine strategies for achieving three-dimensional lung tissue constructs. 3, 38 finally, membrane bioreactors are capable of retaining enzymes, organelles, and microbial, animal, and plant cells behind a membrane barrier, trapped in a matrix or adherent to the membrane surface. in 1963, gallup and gerhardt 44 fi rst used the membrane bioreactor for dialysis culture of serratia marcescens. immobilized enzyme microencapsulation was pioneered by chang, 45 but butterworth et al. 46 fi rst developed the enzyme membrane reactor to successfully accomplish starch hydrolysis with α-amylase. likewise, for animal cell culturing, knazek et al. 47 fi rst cultured human choriocarcinoma cells on compacted bundles of amicon fi bers. many reviews on the particular applications of hollow fi ber and immobilized bioreactant bioreactors for enzyme catalysts, microbial cells, and animal cell culture are available. [48] [49] [50] [51] [52] [53] as presented previously, tissue-engineering applications of three-dimensional function and structure are well known in medical science research. 54 in microgravity three-dimensional aggregates form, facilitating the expression of differentiated organotypic assemblies. investigations to determine the effect of composite matrices, spiked with esterifi ed hyaluronic acid and gelatin, to augment osteochondral differentiation of cultured, bone marrow-derived mesenchymal progenitor cells and the effects of the matrix on cellular differentiation have been examined in vitro and in vivo. 54 briefl y, empty and populated matrices cultured for 28 days, with and without tgf-β 1 demonstrated the following results. cells implanted in the matrix produced a robust type ii collagen extracellular matrix in vitro. matrices placed in immunodefi cient mice yielded no differentiation in empty constructs, osteochondral differentiation in loaded implants, and an enhanced level of differentiation in preimplantation in vitro-cultured matrices containing tgf-β 1 . these results demonstrate the utility of three-dimensional matrix for presentation of bone mesenchymal progenitor cells in vivo for repair of cartilage and bone defects as well as indicate the efficacy for in vitro tissue engineering regimes. 54 these techniques lend themselves to microgravity and ground-based research tissue cultures alike. many earth-based laboratories are researching and developing hemopoietic bone marrow cultures of stem cell origin, and three-dimensional confi gurations are providing promising results as illustrated by schoeters and coworkers. 55 they report that murine bone marrow cells, cultured under long-term hemopoietic conditions, produce mineralized tissue and bone matrix proteins in vitro but only when precipitated by the presence of adherent bone stroma cells in three-dimensional collagen matrices. at a concentration of 8 × l0 6 stromal cells, mineralization occurs in 6 days. in contrast, twodimensionally oriented marrow fragments at 1 × 10 7 cells require requires more than 10 days before mineralization can similarly be detected. 55 two-dimensional long-term marrow culture facilitates and enhances expansion of the stromal component and rudimentary differentiation of osteogenic-like cells in the adherent stromal layer as verifi ed by type i collagen or cells positive for alkaline phosphatase. production of osteonectin and osteocalcin, a bone-specifi c protein, combined with calcifi cation is observed only in threedimensional cultures. these studies demonstrate the need for and benefi t of three-dimensionality and the application to the microgravity environment. 55 as we can see, this further reinforces the quest for threedimensionality and the potential of modeling the microgravity environment. investigations clearly show the need for the application of three-dimensional study techniques in lung pathophysiologic studies. interestingly, three-dimensional biology has facilitated full-scale investigations into most areas of tissue engineering, cell biology and physiology, immunology, and cancer research. anchorage-dependent cells are widely cultured on microcarriers. 56 studies show that for the purposes of improved surface-to-volume ratio and scale up, the microcarrier suspension culture provides excellent potential for high-density cell growth. 57 in addition, microcarriers serve well as structural supports for three-dimensional assembly, the composite of which is the basis for threedimensional tissue growth. 58 conventional culture systems for microcarrier cultures (i.e., bioreactors) use mechanical agitation to suspend microcarriers and thus induce impeller strikes as well as fl uid shear and turbulence at the boundary layer between the wall and the fl uid. investigators have attempted to make a complete study of the most effi cient bioreactor designs and agitation regimens. 59 they concluded that virtually all stirred-tank bioreactors operate in the turbulent regimen. it has been demonstrated that bead-to-bead bridging of cells is enhanced signifi cantly at lower agitation rates in a stirred reactor. 60 excessive agitation from either stirring or gas bubble sparging has been documented as a cause of cell damage in microcarrier cell cultures. 61, 62 to overcome the problems induced by these mechanisms, investigators developed alternative culture techniques such as porous microcarriers to entrap cells, 63 increased viscosity of culture medium, 64 bubble-free oxygenation, 65 and improved methods for quiescent inoculation. 66, 67 these steps decreased the damage attributed to turbulence and shear forces but failed to signifi cantly rectify the problems. reactor systems of substantially increased volume exhibit less agitation-related cell damage. this is presumably because of the decreased frequency of cell-microcarrier contact with the agitation devices in the systems. research-scale investigations do not afford the luxury of experimenting with large-scale production systems. therefore, if a large-volume system is indeed more quiescent, an improved bioreactor system should emulate the fl uid dynamics present in the upper regions of large-scale reactors in which cells and microcarriers reside with minimal agitation. the problem, then, is to suspend microcarriers and cells without inducing turbulence or shear while providing adequate oxygenation and nutritional replenishment. the term rotating-wall vessel comprises a family of vessels, batch fed and perfused, that embody the same fl uid dynamic operating principles. these principles are (1) solid body rotation about a horizontal axis that is characterized by (a) colocation of particles of different sedimentation rates, (b) extremely low fl uid shear stress and turbulence, and (c) three dimensional spatial freedom; and (2) oxygenation by active or passive diffusion to the exclusion of all but dissolved gasses from the reactor chamber, yielding a vessel devoid of gas bubbles and gas-fl uid interface (zero head space). 68, 69 three-dimensional models of lung disease current cell culture models have shortcomings resulting in unreliable tumor growth, uncharacteristic tumor development, nonhuman tumors, and inadequate methods of detection. cells propagated under traditional culture conditions differ widely in their expression of differentiated markers, adhesion receptors, and growth factor receptors compared with cells in situ or those grown as tissue-like structures. 70, 71 this is of concern because the phenotypic changes leading to malignant transformation often stem from alterations in the balanced and multifaceted roles of growth factors, receptors, and cytokines (reviewed by herlyn et al. 71 ). with increasing evidence of the importance of adhesive contacts, paracrine cross-talk between different cell types, and signaling cascades that link the cell with a complex substratum, there is now recognition that models must be developed that better simulate these complexities. there is still much to learn about the dynamic relationships among the different phenotypes found in the normal lung and in lung cancers. until a cell culture system is developed that allows differentiation to occur, 72 it is diffi cult to make any fi rm statement about relating effects in cell culture to clinical practice. tissue engineering is very embryonic in development and currently nearly universally focused on building replacement tissues. a new technology developed at the nasa johnson space center used to study colon cancer has been adapted to three-dimensional in vitro lung tissue culture models but has not been reported on to date. rotating-wall vessels are horizontally rotating cylindrical tissue culture vessels that provide controlled supplies of oxygen and nutrients with minimal turbulence and extremely low shear. 69 these vessels suspend cells and microcarriers homogeneously in a nutrient-rich environment, which allows the three-dimensional assembly of cells to tissue. prior to seeding rotating-wall vessels (synthecon, inc, houston, tx), cells were cultured in standard t fl asks (corning, corning, ny) in gtsf-2 medium (1993 psebm) in a humidifi ed 37°c, 5% co 2 incubator. the rotating-wall vessels were seeded with 1-2 mg/ml cultispher-gl microcarriers (hyclone laboratories, inc., logan, ut) followed by beas2-b or bzr-t33 cells (atcc, baltimore, md) at a density of 2 × 10 5 cells/ml. cultures were grown in the rotating-wall vessels for 14-21 days for formation of 3-to 5-mm diameter tumor masses. rotating-wall vessel rotation was initiated at 25 rpm and increased as aggregate size became larger. stationary control cultures were initiated under the same conditions using fep tefl on bags (american fluoroseal, columbia, md). at 24-hour intervals ph, dissolved co 2 , and dissolved o 2 were determined using a corning 238 model clinical blood gas analyzer. glucose concentration was determined using a beckman 2 model clinical glucose analyzer (beckman, fullerton, ca). cell samples were harvested every 48 hr and fi xed with omnifi x (xenetics, tustin, ca) for immunohistochemistry or fi xed with 3% glutaraldehyde/2% paraformaldehyde in 0.1 m cacodylic buffer (electron microscopy sciences, fort washington, pa) for scanning electron microscopy. cancer models already developed by nasa investigators include growth and differentiation of an ovarian tumor cell line, 72-74 growth of colon carcinoma lines, 72 and three-dimensional aggregate and microvillus formation in a human bladder carcinoma cell line. 74 in support as an appropriate model for cancer, even the most rudimentary three-dimensional cellular structures exhibit different phenotypes than cell lines cultured under twodimensional conditions. properties such as responses to tgf-β, drug resistance to cisplatin or cyclophosphamide, and resistance to apoptosis are all altered in various types of cell aggregates. 75 many investigations sustain consistent evidence that cells growing in three-dimensional arrays appear more resistant to cytotoxic chemoagents than cells in monolayer culture. 38 li et al. found that spheroids were more resistant to cytosine arabinoside by 11-fold and methotrexate by 125-fold when compared with single cell suspensions. 76 further monolayer cultures of colon carcinoma cells were sensitive to piericidin c in contrast to responses within in vivo colon tumors or three-dimensional slices of tumors grown in vitro. 77 numerous other investigations have revealed increased levels of drug resistance of spheroids compared with single cell monolayers. 3, 38 questions of poor diffusion and insuffi cient drug absorption within spheroids and a relatively frequent high proportion of resting cells have clouded differences in drug resistance, which could be the result of nutrient deprivation and hypoxia. heppner and colleagues executed precise experiments that confi rmed threedimensional structure and function as the causative agent and was responsible for drug resistance rather than simple inaccessibility to nutrients or the drug concentration. heppner embedded tumor specimens or cell aggregates in collagen gels, exposed the culture to various cytotoxic drugs, and compared the drug responses of the same cells in monolayers. these experiments revealed an increased resistance in the three-dimensional tumor arrays of a remarkable 1,000-fold greater than in monolayer cultures, and a similar result was seen in three-dimensional histocultures in collagen. the tumor cells grew in the presence of drug concentrations that rendered monolayers to a viability less than 0.1% of control cultures. amazingly, heppner observed that the cells became sensitive again when replated as monolayers and fi nally showed that even when exposed to melphalan and 5-fl uorouracil in monolayer cells transferred to collagen gels were again resistant based on three-dimensional architecture. thus, the cells were exposed to the drugs as monolayers, facilitating access to the drugs, and, once the cells were transferred after drug exposure to a threedimensional structure, high resistance to the drugs was sustained. 38, [78] [79] [80] [81] based on the caliber of data referenced above, teicher et al. 82 serially passaged through multiple (10) transfers emt-6 tumors in mice that were treated with thiotepa, cisplatin, and cyclophosphamide over a prolonged 6month period, thus producing extremely drug-resistant tumors in vivo. when these tumors were grown as monolayer cultures, they were as drug sensitive as the parental cells. kobayashi and colleagues 83 grew the same in vivo drug-resistant tumor cell lines as spheroids in threedimensional arrays, and resistance was almost 5,000 times that of the parent line with selected drugs, an example being the active form of cyclophosphamide used in vitro. similarly extreme resistance was also observed to cisplatin and thiotepa. this resistance was not seen in monolayer cultures, even when the monolayers were cultured on traditional extracellular matrix substrates. these experiments reconfi rmed that cells in a three-dimensional array are more drug resistant than monolayer cells in vitro and demonstrated that three-dimensional cellular confi gurations can and do become resistant to super pharmacologic doses of drugs by forming compact structures. 38 rotating-wall vessel tumor models several important human tumor models have been created in rotating-wall vessel cultures, specifi cally, lung, prostate, colon, and ovarian. 14, 58, 73, 84 many of these models involve cancers that are leading killers in our society. we present two such examples in this section, colon and prostate carcinoma. as previously reviewed, the literature indicates the remarkable difference between chemotherapeutic cytotoxicity in two-dimensional and three-dimensional cellular constructs, which may be predicated on a number of criteria. therefore, a threedimensional tumor model that emulates differentiated in vivo-like characteristics would provide unique insights into tumor biology. goodwin et al. 58 detail the fi rst construction of a complex three-dimensional ex vivo tumor in rotatingwall vessel culture composed of a normal mesenchymal base layer (as would be seen in vivo) and either of two established human colon adenocarcinoma cell lines, ht-29, an undifferentiated line, and ht-29km a stable, moderately differentiated subline of ht-29. each of these engineered tumor tissues produced tissue-like aggregates (tlas) with glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. necrosis was minimal throughout the tissue masses up to 60 days of culture while achieving >1.0 cm in diameter. other notable results included enhanced growth of neoplastic colonic epithelium in the presence of mixed normal human colonic mesenchyme. these results mimic the cellular differentiation seen in vivo and are similar to results obtained with other tumor types. prostate carcinoma has also been modeled in the rotating-wall vessel system by several investigators. [85] [86] [87] one of the most comprehensive descriptions of these engineered tissues is detailed by wang et al. 88 in that review, the authors describe the ability of the rotatingwall vessel system to recapitulate human prostate carcinoma (lncap) and bone stroma (mg63) to illuminate the evolution of prostate tumorigenesis to the metastatic condition. in particular, the lncap and arcap models represented in the review are known to be lethal in the human, being androgen independent and metastatic. rotating-wall vessel tla engineering also allowed indepth study of epithelial and stromal interactions, which are the facilitating elements of the continuance of lncap prostate-specifi c antigen production in vitro. when lncap was cultured in three dimensions without stroma, production of prostate-specifi c antigen ceased and metastatic markers were not observed. the authors outline the process of malignant transformation, demonstrating that these metastatic models are only possible in threedimensional tlas and are achieved by specifi c geometric relationships in three-dimensional confi guration. furthermore, they show through direct comparison with other culture systems the advantages of the rotating-wall vessel system to allow synergistic relationships to study this disease state. 88 unlike two-dimensional models, these rotating-wall vessel tumor tissues were devoid of metabolic and nutrient defi ciencies and demonstrated in vivo-like architecture. these data suggest that the rotating-wall vessel affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissues. in this section, we explore the utility of rotating-wall vessel tlas as targets for microbial infection and disease. several studies have been conducted recently that indicate that three-dimensional tissues respond to infective agents with greater fi delity and with a more in vivo-like response than traditional two-dimensional cultures. nickerson et al. 89 describe the development of a threedimensional tla engineered from int-407 cells of the human small intestine, which were used as targets for the study of salmonella typhimurium. in this study, threedimensional tlas were used to study the attachment, invasion, and infectivity of salmonella into human intestinal epithelium. immunocytochemical characterization and scanning and transmission electron microscopic analyses of the three-dimensional tlas revealed that the tlas more accurately modeled human in vivo differentiated tissues than did two-dimensional cultures. the level of differentiation in the int-407 tlas was analogous to that found in previously discussed small intestine tlas 72 and from other organ tissues reconstructed in rotatingwall vessels. analysis of the infectivity studies revealed salmonella attached and infected in a manner significantly different from that in control two-dimensional cultures. during an identical exposure period of infection with salmonella, the three-dimensional tlas displayed a minor loss of structural integrity when compared with the two-dimensional int-407 cultures. furthermore, salmonella demonstrated a greatly reduced ability to adhere, invade, and induce the apoptotic event in these int-407 three-dimensional tlas than in twodimensional cultures. this result is not unlike the in vivo human response. two-dimensional cultures were significantly damaged within several hours of contact with the bacteria; conversely, although "pot marks" could be seen on the surfaces of the three-dimensional tlas, they remained structurally sound. cytokine analysis and expression postinfection of three-dimensional tlas and two-dimensional cultures with salmonella exhibited remarkable differences in expressed levels of interleukin (il)-1α, il-1β, il-6, il-1ra, and tumor necrosis factor-α mrnas. additionally, noninfected three-dimensional tlas constitutively demonstrated elevated levels of tgf-β 1 mrna and prostaglandin e 2 compared with noninfected two-dimensional cultures of int-407. 89 as previously stated, traditional two-dimensional cell monolayers lack adequate fi delity to emulate the infection dynamics of in vivo microbial adhesion and invasion. the respiratory epithelium is of critical importance in protecting humans from disease. exposed to the environment, the respiratory epithelium acts as a barrier to invading microbes present in the air, defending the host through a multilayered complex system. 90 the three major layers of the human respiratory epithelium are pseudostratifi ed epithelial cells, a basement membrane, and underlying mesenchymal cells. ciliated, secretory, and basal epithelial cells are connected by intercellular junctions and anchored to the basement membrane through desmosomal interactions. together with tight junctions and the mucociliary layer, the basement membrane maintains the polarity of the epithelium and provides a physical barrier between the mesenchymal layer and the airway. 91, 92 infi ltrating infl ammatory and immune cells move freely between the epithelial and subepithelial compartments. airway epithelial cells play a vital role in host defense 90 by blocking paracellular permeability and modulating airway function through cellular interactions. ciliated epithelial cells block invasion of countless inhaled microorganisms by transporting them away from the airways. 93 as regulators of the innate immune response, epithelial cells induce potent immunomodulatory and infl ammatory mediators such as cytokines and chemokines that recruit phagocytic and infl ammatory cells that remove microbes and enhance protection. 90, 91, 94, 95 ideally, cell-based models should reproduce the structural organization, multicellular complexity, differentiation state, and function of the human respiratory epithelium. immortalized human epithelial cell lines, such as beas-2b, 96 primary normal human bronchial epithelial cells, 97 and air-liquid interface cultures, 98 are used to study respiratory virus infections in vitro. traditional monolayer cultures (two-dimensional) of immortalized human bronchoepithelial cells represent homogenous lineages. although growing cells in monolayers is convenient and proliferation rates are high, such models lack the morphology and cell-cell and cell-matrix interactions characteristic of human respiratory epithelia. thus, their state of differentiation and intracellular signaling pathways most likely differ from those of epithelial cells in vivo. primary cell lines of human bronchoepithelial cells provide a differentiated model similar to the structure and function of epithelial cells in vivo; however, this state is short lived in vitro. 97, 99 air-liquid interface cultures of primary human bronchoepithelial cells (or submerged cultures of human adenoid epithelial cells) are grown on collagen-coated fi lters in wells on top of a permeable fi lter. these cells receive nutrients basolaterally, and their apical side is exposed to humidifi ed air. the result is a culture of well-differentiated heterogeneous (ciliated, secretory, basal) epithelial cells essentially identical to airway epithelium in situ. 98, 100 although this model shows fi delity to the human respiratory epithelium in structure and function, maintenance of consistent cultures is not only diffi cult and time consuming but also limited to small-scale production and thus limits industrial research capability. true cellular differentiation involves sustained complex cellular interactions [101] [102] [103] in which cell membrane junctions, extracellular matrices (e.g., basement membrane and ground substances), and soluble signals (endocrine, autocrine, and paracrine) play important roles. [104] [105] [106] [107] this process is also infl uenced by the spatial relationships of cells to each other. each epithelial cell has three membrane surfaces: a free apical surface, a lateral surface that connects neighboring cells, and a basal surface that interacts with mesenchymal cells. 108 recently viral studies by goodwin et al. 109 and suderman et al. 110 were conducted with rotating-well vessel-engineered tla models of normal human lung. this model is composed of a coculture of in vitro threedimensional human bronchoepithelial tlas engineered using a rotating-wall vessel to mimic the characteristics of in vivo tissue and to provide a tool to study human respiratory viruses and host-pathogen cell interactions. the tlas were bioengineered onto collagen-coated cyclodextran beads using primary human mesenchymal bronchial-tracheal cells as the foundation matrix and an adult human bronchial epithelial immortalized cell line (beas-2b) as the overlying component. the resulting tlas share signifi cant characteristics with in vivo human respiratory epithelium, including polarization, tight junctions, desmosomes, and microvilli. the presence of tissuelike differentiation markers, including villin, keratins, and specifi c lung epithelium markers, as well as the production of tissue mucin, further confi rm these tlas differentiated into tissues functionally similar to in vivo tissues. increasing virus titers for human respiratory syncytial virus (wtrsva2) and parainfl uenza virus type 3 (wtpiv3 js) and the detection of membrane-bound glycoproteins (f and g) over time confi rm productive infections with both viruses. viral growth kinetics up to day 21 pi with wtrsva2 and wtpiv3 js were as follows: wtpiv3 js replicated more effi ciently than wtrsva2 in tlas. peak replication was on day 7 for wtpiv3 js (approximately 7 log 10 particle forming units [pfu] per milliliter) and on day 10 for wtrsva2 (approximately 6 log 10 pfu/ml). viral proliferation remained high through day 21 when the experiments were terminated. viral titers for severe acute respiratory syndrome-coronavirus were approximately 2 log 10 pfu/ml at 2 day pi. human lung tlas mimic aspects of the human respiratory epithelium well and provide a unique opportunity to study the host-pathogen interaction of respiratory viruses and their primary human target tissue independent of the host's immune system, as there can be no secondary response without the necessary immune cells. these rotating-wall vessel-engineered tissues represent a valuable tool in the quest to develop models that allow analysis and investigation of cancers and infectious disease in models engineered with human cells alone. we have explored the creation of three-dimensional tlas for normal and neoplastic studies and fi nally as targets for microbial infections. perhaps carrel and leighton would be fascinated to know that from their early experiments in three-dimensional modeling and the contributions they made has sprung 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form and function robbins infectious diseases, 6th ed. philadelphia: wb saunders epithelial regulation of innate immunity to respiratory syncytial virus epithelia cells as regulators of airway infl ammation human bronchial epithelial cells with integrated sv40 virus t antigen genes retain the ability to undergo squamous differentiation identifi cation and culture of human bronchial epithelial cells developing differentiated epithelial cell cultures: airway epithelial cells evaluation of anchorageindependent proliferation in tumorigenic cells using the redox dye alamar blue airway epithelium and mucus: intracellular signaling pathways for gene expression and secretion disorganization of stroma alters epithelial differentiation of the glandular stomach in adult mice expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction cell replication of mesenchymal elements in adult tissues. i. the replication and migration of mesenchymal cells in the adult rabbit dermis defi ning conditions to promote the attachment of adult human colonic epithelial cells laminin expression in colorectal carcinomas varying in degree of differentiation infl uence of mammary cell differentiation on the expression of proteins encoded by endogenous balb/c mouse mammary tumor virus genes opinion: building epithelial architecture: insights from three-dimensional culture models threedimensional engineered high fi delity normal human lung tissue-like assemblies (tla) as targets for human respiratory virus infection severe acute respiratory syndrome (sars)-cov infection in a threedimensional human bronchial-tracheal (hbte) tissue-like assembly key: cord-029480-3md13om6 authors: meix-cereceda, pablo title: educational values in human rights treaties: un, european, and african international law date: 2020-07-21 journal: hum rights rev doi: 10.1007/s12142-020-00599-6 sha: doc_id: 29480 cord_uid: 3md13om6 while human rights treaties provide a formidable set of principles on education and values, domestic courts often tend to adjudicate claims in terms of local arguments for or against each particular educational practice. this article explores how international human rights law could inspire the interpretation of domestic law and educational practice, without neglecting specific cultural aspects. firstly, the article reviews the sociological debate on values in education and shows its importance for the legal discussion. secondly, some critical contestations of international cultural human rights are outlined, as well as certain arguments to justify the importance of this model. the study of international law follows: the un, the european court of human rights, and three relevant african charters, as well as every reference to education made by the african commission on human and peoples’ rights and by the african court is examined. lastly, a comparative section reveals a certain cultural commonality inspired by the un treaties, but also reflects some cultural and institutional differences between the european and the african regional systems. educational action in schools. botha (2019, 185) has advocated the importance of international law as a reference and inspiration for interpreting domestic law and practice on educational matters involving human rights. with regard to national legal systems, du plessis (2019, 49 and 2020) has pointed out the exemplarity of the preamble of the south african national education policy act 27 of 1996 when it proclaims that "the education system must be transformed into one which serves the needs and interests and upholds the fundamental rights of all the people of south africa." nevertheless, the concrete result in each learner's education will vary according to a complex interaction of all the aforementioned factors. using a sculptural metaphor, constitutions and international human rights treaties would provide a sediment of values to the educational clay. however, this clay will be modeled, to a greater or a lesser extent, by the hands of all the aforementioned actors. the starting point of this piece is that education always implies the transmission of certain values which, nevertheless, are sometimes in a tension with each other or even contradict each other. therefore, the absorption of values in each learner's case is not easily controlled by one single force but is the result of many influences. the child's own freedom will in all probability play a major role in how these values intertwine and weigh on their moral decisions. the notion of human rights culture has been contested as reflecting a eurocentric philosophy of the individual. indeed, as stoppioni (2020, 2) has recently reminded, "the fundamental problem of the european conception of human rights would be to focus exclusively on the individual and to forget the group". a harsher critique considers that "'human rights' are, as such, a false ideological universality, which masks and legitimizes a concrete politics of western imperialism, military interventions and neocolonialism" (žižek 2005) . these arguments criticize what bonfiglio (2018) has called "human rights colonialism," which the same author has sought to overcome by proposing an intercultural theory of human rights. from an african perspective, justice mokgoro (1998, 8) , of the constitutional court of south africa, has claimed the proximity between human rights and the traditional values of ubuntu, while acknowledging that the communal dimension of ubuntu could enrich the interpretation of western law. in this regard, she mentions in particular the following ideas: -the original conception of law perceived not as a tool for personal defense, but as an opportunity given to all to survive under the protection of the order of the communal entity -communalism which emphasizes group solidarity and interests generally, and all rules which sustain it, as opposed to individual interests, with its likely utility in building a sense of national unity among south africans -the conciliatory character of the adjudication process which aims to restore peace and harmony between members rather than the adversarial approach which emphasizes retribution and seems repressive. the lawsuit is viewed as a quarrel between community members and not as a conflict. the importance of group solidarity requires restoration of peace between them -the importance of public ritual and ceremony in the communication of information within the group -the idea that law, experienced by an individual within the group, is bound to individual duty as opposed to individual rights or entitlement. closely related is the notion of sacrifice for group interests and group solidarity so central to ubuntu(ism) -the importance of sacrifice for every advantage or benefit, which has significant implicants for reciprocity and caring within the communal entity even if they were not written apropos of education, these reflections seem especially relevant to the debate on educational values, in particular with respect to which values the school system ought to promote and the related problem of conflict and restorative justice in schools. the abovementioned critical views, however, cannot undermine the importance of international protection. as will be discussed especially in "african union: commonality and specificities", regional international bodies have both the necessary independence from states and the ability to deliver a relevant view in terms of idiosyncratic proximity. in addition, the lack of adequate solutions in certain domestic cases would prove the necessity of international protection for at least certain categories of subjects. this seems of particular relevance in the case of children, arguably the most vulnerable subjects of human rights. some examples from southern african domestic courts may be helpful to illustrate the latter claim. in some cases, national courts have selected international rulings that may be isolated in a court's body of case law or not applied as a meaningful precedent by the international court itself. for instance, in the arundel school case 2 the constitutional court of zimbabwe ruled against the application of certain pupils who had been obliged to attend school prayer by the new headmistress. the pupils' parents, who were jehova's witnesses, had expressly declared their faith upon the application for admission of their children, and the children had been attending the school before the arrival of the new headmistress. despite the fact that the application had been filed by the parents, the constitutional court expressly declared the right to expel the pupils, which the school had not yet carried out. even more surprising, however, was the fact that the constitutional court based on the european court's reasoning in the case of valsamis of 1996, which has been changed in more recent rulings on similar claims, such as the cases of folgerø (2007), zengin (2007), or yalçın (2014) (see below, "european court of human rights" for details). in other cases, national courts simply refrain from using international law as a source of inspiration, even when it could reinforce the authority of their own rulings. in this regard, the generally protective constitutional court of south africa rarely integrates arguments from international law when ruling on matters concerning children and education. in the case of the juma musjid primary school, 3 the principle of the child's best interest was only discussed in terms of national constitutional law in spite of the importance that international law has in this field (see below "full development of the human personality and best interest of the child"). in this case, an eviction order was sought by the owner of the land where a school was based. the constitutional court mentioned certain international treaties and soft law on the right to education, 4 and even a us supreme court ruling, 5 but no international decisions by african or un bodies were considered. the constitutional court held that the kwazulu-natal high court had failed to take account of the interests of the learners and, instead, had simply "privileged the right to property over the learners' right to a basic education" (para 71). nevertheless, the court considered that the children had been successfully accommodated by other schools and therefore ruled for the owners and granted the order of eviction. another important case from south africa concerned the pregnancy policies adopted by several schools. in the welkom and harmony high schools case, 6 the constitutional court declared these policies unconstitutional for being contrary to a number of rights including non-discrimination, the right to education, and the respect of the child's best interest. 7 as will be discussed below, the african commission on human and peoples' rights has often sought to convince states and schools of the need to ensure school attendance of girls in general, and very especially in the case of pregnancy. nevertheless, and despite a specific allegation by one of the parties, the constitutional court did not mention any rule of international law in its judgment. other situations and cultural practices may deeply affect the full development of children and their right to education, as demonstrated by the achpr's appeals against genital mutilation, child labor, child marriage, and other forms of denial of the right to schooling, as will be discussed in the following sections of this article. while it is difficult to conceive international remedies as a valid instance for reviewing every domestic ruling, they are certainly useful for both denouncing and inspiring educational and legal practice. state authorities-but also individualsshould take notice of the problems detected by international bodies and, with the help of available or new domestic remedies, gradually work toward the improvement of such problems in daily practice. in this regard, the article seeks to provide a comprehensive view of the educational values advanced in three international human rights jurisdictions: the un, the european court of human rights, and the african instruments. part of the elements protected by these jurisdictions is shared by the other systems, but each of them has developed certain specificities. concerning the latter, an effort has been made to examine every allusion to education made by the african court on human and peoples' rights (hereinafter african court) and the african commission on human and peoples' rights (hereinafter achpr). lastly, some comparative considerations on the three systems will be outlined. the universal declaration of human rights (hereinafter udhr) 8 includes a remarkable set of educational values. they have been further developed by some of the major human rights treaties sponsored by the un. four kinds of values seem relevant in this regard. the "full development of the human personality" principle is expressly mentioned in art 26 para 2 of the udhr and has been adopted as the main goal of education by many treaties and even constitutions. in order to grasp the meaning of this principle, it may be worth exploring some interesting variations across different international treaties and declarations. in 1959, 11 years after the udhr, the declaration of the rights of the child 9 would state that education "will promote (the child's) general culture, and enable him, on a basis of equal opportunity, to develop his abilities, his individual judgement" (principle 7). this declaration acknowledged the relationship between a person's awareness of the world that surrounds them ("general culture") and the development of their personality both in terms of their talents ("abilities") and their moral sense ("individual judgment"). thirty years later, the convention on the rights of the child (crc) would rephrase and widen these ideas stating the following: (…) the education of the child shall be directed to: (a) the development of the child's personality, talents and mental and physical abilities to their fullest potential (…). 10 the main purpose thus became the development of talents and abilities, both mental and physical, as part of the more complex concept of the "child's personality." the 1959 declaration, on the other hand, included what is nowadays considered the main goal that should guide not only education but generally any measure concerning children. indeed, after the abovementioned statement, principle 7 went on as follows: the best interests of the child shall be the guiding principle of those responsible for his education and guidance; that responsibility lies in the first place with his parents. full development of the personality and a child's best interest thus appear clearly connected if we consider the udhr and the declaration of the rights of the child jointly. this connection may be useful in order to construe the principle of best interest in some difficult cases. indeed, when the 1959 declaration referred to the best interests 8 adopted 10 december 1948 unga res 217 a(iii) (udhr). 9 declaration of the rights of the child (proclaimed by the general assembly, resolution 1386 (xiv), a/res/ 14/1386, 20 november 1959) (drc). 10 convention on the rights of the child (adopted on 20 november 1989, entered into force 2 september 1990) 1577 unts 3 (crc) art 29 para 1. of the child as "the guiding principle of those responsible for his education", it also specified that this responsibility lies "in the first place with his parents". this phrase certainly means that parents are, prima facie, considered the fittest to decide what that interest may be. nevertheless, there may be some cases where parents may not be able or willing to distinguish between their own interest or belief, on one hand, and the child's best interest on the other: child labor, 11 underage marriage, 12 female genital mutilation (girls' circumcision), 13 or recruitment into militias 14 are only some of the most dramatic examples. in these cases, it may be difficult to justify an intervention of public authorities based solely on the principle of "the child's best interest," as parents might claim that international law enables them "in the first place" to determine that interest in each case. nevertheless, a joint interpretation of this principle and the full development of the child's personality may prove useful in this regard. an individual's personality may keep developing through all stages of life, which means that full development is almost an ideal, or, from a legal perspective, a principle permanently subject to the possibility of progression. this seems even clearer in the case of a child. therefore, if a decision from the parents may pose a clear threat to the child's future development, intervention from public authorities would then be justified insofar as necessary (and not any further) to safeguard the child's future possibilities of personal development. another interesting variation can be found in the international covenant on economic social and cultural rights 15 : (…) education shall be directed to the full development of the human personality and the sense of its dignity (…) human dignity has been considered as a single human right 16 and as a theoretical foundation of the entire legal system (solozábal echavarría 1994 , 2489 and garcía guerrero 2014 and even of the state (häberle 2004, 319) . from a practical perspective, however, if a person's human rights are violated or even threatened, that person may in all probability not lead a decent life. this paragraph from the icescr hence reminds that the right to education is almost literally a "key right," which raises in children and adults awareness of their own dignity, and eventually allows them to fight for their rights by any means acceptable in their society. from an economic 11 the african commission for human and peoples' rights (achpr) sent on 23 march 2018 a letter of urgent appeal to the authorities of uganda "concerning alleged child labour in extractive industries, particularly gold mining." 12 association pour le progrès et la défense des droits des femmes maliennes (apdf) and the institute for human rights and development in africa (ihrda) v republic of mali african court of human and peoples' rights 11 may 2018, especially paras 71-78. in addition, the achpr's 40th activity report (of 2016) acknowledged that "in january 2016, child marriage was declared illegal in zimbabwe by the constitutional court" (para 27.a.ix). 13 the achpr's 34th activity report (2013) found positive developments for human rights in this regard in senegal and the achpr's 36th activity report (of 2014) in four states of the sudan. 14 the achpr's 34th activity report (2013), para 21, denounced the situation in the republic of congo. 15 perspective, education would allow individuals from poorer origins to improve their situation-which is often described with the "social lift" metaphor. 17 these ideas can be summarized in the words of the committee on economic, social and cultural rights (cescr), which conceives education as "an empowerment right". 18 one last important aspect that un-promoted treaties often link to full individual development is the "strengthening of respect for human rights and fundamental freedoms". this phrase is used to describe one of the two basic aims of education according to art 26 para 2 of the udhr (the other one being the full development of the human personality). the phrase between quotes has thus come to represent, in international legal terms, the effort of the society to create a conscience in the child. in this regard, an early but powerful wording can be found in the 1924 geneva declaration of the rights of the child: the child must be brought up in the consciousness that its talents must be devoted to the service of fellow men. it is however the convention on the rights of the child that enshrines the richest catalog of educational and developmental aims. from the perspective of this paper, there are at least two interesting references in its text. firstly, the convention's preamble reminds that the "child should be fully prepared to live an individual life in society". secondly, and elaborating on this idea, art 29 para 1 even claims that "the education of the child shall be directed to: (…) (d) the preparation of the child for responsible life in a free society (…)". two different aspects should be underlined here. on the one hand, this sentence reminds that the child is to be educated in order to become a healthy adult, which prevents exploitation and generally any abusive treatment, considering that a child is not yet an adult. but, on the other hand, the fact that the child will eventually develop into an adult also stresses the need to gradually promote a certain responsibility and maturity. therefore, education should prepare a child to live autonomously in a free society. preparing a child for freedom also means preparing it for responsibility, for making its own rules and taking its own decisions as a useful member of the society, to quote principle 7 of the 1959 un declaration of the rights of the child. this link between individual development and respect for human rights paves the way for the second of the aims mentioned in the general international law. the second aim of education would be clearly political. with different variations, most of the texts include understanding, tolerance, and peace among their political aims for education. a good example can be found in art 26 para 2 of the udhr, according to which, education "shall promote understanding, tolerance and friendship among all nations, racial or religious groups, and shall further the activities of the united nations for the maintenance of peace". the promotion and maintenance of peace has been the most important principle of international law at least since the establishment of the united nations in 1945 (white 1997, 3) . it was enshrined in the charter of the united nations 19 as the maintenance of "international peace and security". the reference to "understanding, tolerance and friendship among all nations, racial or religious groups" would enable the primary goal of maintaining peace. nevertheless, it can be seen as a more ambitious purpose, too. in this respect, it seems that the udhr expects, or even promotes, the emergence of a global society, which in turn would be expected to achieve much more than preventing its peoples from going to war against each other. over 40 years later, the convention on the rights of the child would repeat and widen the ideals of the universal declaration in this respect. the reference to nations and racial and religious groups was then completed with the more flexible "friendship among all peoples, ethnic, national and religious groups and persons of indigenous origin." moreover, the equality of sexes also joined the previous understanding, peace and tolerance. it is probably needless to remind that between the 1948 udhr and the 1989 crc, the international convention on the elimination of all forms of racial discrimination 20 and the convention on the elimination of all forms of discrimination against women 21 had been adopted, putting these problems on the international agenda. international law thus acknowledges the importance that children are made aware of the society's complexity as part of their growth and education process, and of the need to respect and equally treat every individual regardless of their social circumstances. indeed, the relationship between human rights and education can be conceived in dialectic terms. in this regard, promoting human rights in education by celebrating difference 22 could in turn make education a force for social cohesion and understanding. the school system could enhance the child's awareness of different cultural approaches to sensitive or controversial matters, bearing in mind the age and maturity of the learners. for instance, discussion could be encouraged on the role of religion in society, the importance of art, the relationship between freedom of speech and political correctness, environmental conscience, sex or drugs, to name but a few of these controversial matters. a number of authors have underlined these ideas by naming human rights education a human right itself (alfredsson 2001, 273; benedek 2007, 1) . despite the claim for universality of human rights and the importance of promoting mutual understanding, the social circumstances surrounding a child are also important for education, as the third aim from the un treaties conveys. 19 charter of the united nations (adopted 26 june 1945, entered into force 24 october 1945) (un charter) art 1 para 1. 20 international convention on the elimination of all forms of racial discrimination (adopted 7 march 1966, entered into force 4 january 1969) 660 unts 1. 21 convention on the elimination of all forms of discrimination against women (adopted 18 december 1979, entered into force 3 september 1981) 1249 unts 1. 22 mittler (2000, 10) has developed a model of inclusive education. in his words, "inclusion implies a radical reform of the school in terms of curriculum, assessment, pedagogy and grouping of pupils. it is based on a value system that welcomes and celebrates diversity arising from gender, nationality, race, language of origin, social background, level of educational achievement or disability" (emphasis added.) the third kind of values that are found in most declarations and treaties have to do with what sociologists would call the "meso" or middle level of societies. international law thus reveals an awareness of the importance that intermediate bodies (groups) have in a person's life. the family-despite its diminishing size in many societies-is considered the main channel for the flow of values from the groups that make a society to the child. language, cultural values, and moral and religious references, as well as many other traditions, are gradually absorbed by the child in an environment of such high affectivity as the family. all of these ideas could be summarized as the protection of cultural pluralism in education. indeed, even a treaty as sparing in educational matters as the international covenant on civil and political rights includes a reference to religious and moral education, if only to proclaim the right of parents to ensure that such education is delivered "in conformity with their own convictions". 23 nevertheless, it is very difficult to construe this principle as a right to ensure that any education involving moral or even religious contents can only be attempted in conformity with the convictions of families. the reasons would be twofold. on the one hand, the scope of contents potentially affected by moral or religious views is so wide that it would be practically impossible to carry out an institutionalized education. thus, equal access to formal education 24 and other principles that are part of the right to education would be made void. on the other hand, the aforementioned principles of the full development of the human personality and the best interest of the child justify that certain contents of public interest be addressed in a non-indoctrinatory manner, even against parental wishes. sex education has frequently been a controversial matter, 25 but teaching in certain languages in some parts of the world can be just as disputed. 26 however, it is the crc that best captures the essence behind the need to ensure pluralism in education. in its preamble, the states parties acknowledge "taking due account of the importance of the traditions and cultural values of each people for the protection and harmonious development of the child". also, when discussing placement of the child outside its original family, art 20 para 3 mandates that "(…) due regard shall be paid to the desirability of continuity in a child's upbringing and to the child's ethnic, religious, cultural and linguistic background." moreover, art 29 para 3.c places the following values among the aims of education: the development of respect for the child's parents, his or her own cultural identity, language and values, for the national values of the country in which the child is living, the country from which he or she may originate, and for civilizations different from his or her own (emphasis added). however, the importance of all these cultural values does not prevent art 14 para 1 from proclaiming "(…) the right of the child to freedom of thought, conscience and religion". we may inquire about the purpose of stressing cultural specificity in international human rights treaties. this does not seem an appropriate occasion for developing all the complexity of the tension between the universality of human rights and their implementation in different cultural environments. but it may be useful to point out a few considerations on the matter, in particular concerning the topic of this article. cultural traditions may have a great value in collective terms, for example as a way of preserving different valid approaches to the world and to mankind. according to the african court, culture should be construed in its widest sense encompassing the total way of life of a particular group, including the group's languages, symbols such as dressing codes and the manner the group constructs shelters; engages in certain economic activities, produces items for survival; rituals such as the group's particular way of dealing with problems and practicing spiritual ceremonies; identification and veneration of its own heroes or models and shared values of its members which reflect its distinctive character and personality. 27 however, this does not appear as the main argument to ponder here. when discussing child education and values, other considerations seem to take precedence. the rights of parents to instill certain values and, generally, the protection of specific groups and their traditions find a strong justification in the need of a certain psychological stability of the child, a feeling of safety which is crucial for a healthy development of the young who will, over time, become adults. childhood, once again, is not only protected on account of its intrinsic value-which it clearly has, as the right to the child's freedom of conscience underlines-but also because it is the seed of tomorrow's adults that will shape society. a fourth kind of values, still not very common in international law on education, refers to a growing concern in other domains of international law. art 29 para 1 of crc shows this concern in the following terms: [states parties agree that the education of the child shall be directed to (…)] e. the development of respect for the natural environment. given the overwhelming scientific analyses that place human activity at the origin of global warming, and of air, water, and land pollution among many other environmental hazards, the fact that this fundamental value is mentioned as a goal of education in only one of the major conventions on human rights can only be received with surprise. nevertheless, it is a perfectly valid obligation under international law, enshrined in a treaty that is binding for 196 states parties, three more than the member states of the united nations. the usa seems to be the only state not to have ratified or adhered to this convention (of which, however, it is a signatory). the european court of human rights (hereinafter ecthr) has produced approximately 40 judgments and decisions on educational matters. concerning values in education, the court's rulings stem from the second sentence of article 2 of the first additional protocol 28 to the convention. 29 this sentence reads as follows: in the exercise of any functions which it assumes in relation to education and to teaching, the state shall respect the right of parents to ensure such education and teaching in conformity with their own religious and philosophical convictions. in spite of this rather open wording, the ecthr has nevertheless managed to develop a rich case law. it is not possible to examine all of these rulings in detail or to discuss some of the ecthr's excesses. i will therefore limit the analysis to a broad summary of three principles before focusing on how they apply to human rights education, given its relevance for the discussion. firstly, the ecthr has displayed a rather extensive conception of the states' "margin of appreciation". this is especially true in matters of religious symbols, either personal (swiss teachers, 30 turkish students, 31 or french learners 32 ) or institutional. among the latter, the italian "crucifix case" 33 is widely known, but earlier rulings dealt with religious freedom and national celebrations in greek schools. 34 secondly, the ecthr has frequently applied the "best interest of the child" doctrine whenever this interest could be endangered by parental religious or philosophical 28 convictions: the abovementioned cases of kjeldsen 35 and jiménez alonso, 36 or the decision in the konrad v germany case, 37 are some of the most prominent examples on sex education (the first two) and compulsory schooling (the third one). the best interest of the child principle, however, has occasionally been subordinated to a state's margin of appreciation whenever there has been a conflict between the two. for instance, a conflict between the best interest of the child and the french principle of secularism, or the italian defense of the crucifix, or the greek celebration of the national day. most of the cases mentioned in the previous paragraph exemplify this statement. a third group of cases has developed from 2007. these rulings deal with indoctrination in the classroom. they address the instilment of certain religious values through specific ritual practices in compulsory classes for underage learners 38 or a disproportionate attention to certain religious doctrines and a practical neglect of others. 39 the ecthr has upheld the applicants in all such cases. human rights education (hre) was mentioned above as one of the key instruments for education to perform as an empowerment right (see above "promoting understanding, tolerance, and peace"). it can be provided as an autonomous subject or transversally through different subjects, or even as non-formal education. 40 in some cases, however, a vivid controversy has been prompted by the introduction of a specific subject in the school system. this was the case of spain after 2006, when the new subject on "education for citizenship and human rights" was denounced and legally challenged by the political opposition and the catholic church. the subject was deemed constitutional in a number of rulings by both the supreme court 41 and the constitutional court, 42 but the central claim of the opponents to the bill deserves some consideration nevertheless. according to this claim, the curriculum of the subject would have been indoctrinating because it reflected an intent of the state to instill certain moral values. in the opinion of the applicants, such an objective would be the prevalent responsibility of the families and therefore the state should refrain from interfering with it. while the highest domestic courts held that certain values could be legitimately taught by the school system, the case never reached the ecthr and hence the question may remain whether the spanish subject would have been sanctioned by the european court or, on the contrary, whether it would have been declared indoctrinating. however, the ecthr has delivered a number of rulings on the legitimacy of teaching values within the education system. in this respect, the abovementioned cases from norway and turkey are useful precedents. 43 as explained, parts of the curricula in these cases were considered indoctrinating on account of the prevalence granted to certain religious beliefs and rites. interestingly, however, the ecthr made it clear that the teaching of values in school entails a valuable opportunity for young people to appreciate and respect difference and even to experience the commonality of many moral principles that underlie various systems of beliefs. 44 concerning the spanish case, and given that no specific aspects of the curriculum had been challenged before the national courts, it seems arguable that the ecthr would have upheld the decision to introduce the education for citizenship and human rights subject. the misgivings of the applicants concerning the spanish subject stem perhaps from the understanding that state values essentially equal a particular government's values. nonetheless, this is precisely the boundary between acceptable hre and unlawful and indoctrinating ideological reeducation. openness and debate should be part of hre, and teachers should refrain from conveying their own views where the discussion goes beyond the common principles underlying society. this can certainly be a thin line and may not necessarily avoid the risk of complaints by specific parents, but a teacher or educator who adopts a meticulous and open approach would in all probability be in accordance with the view of the ecthr. human rights treaties fostered by the organization of african unity (oau) and its successor the african union (au) include certain interesting obligations from the perspective of values in education. in addition, the african commission on human and peoples' rights (hereinafter the achpr) has provided, and continues to do so, valuable resolutions and other soft law instruments as well as case-based decisions through its communications procedure. the achpr's activity reports are also an invaluable source of information to keep track of both "positive developments" and "areas of concern" for human rights in the continent. 43 see n. 38 and n. 39. 44 case of folgerø, cit. (see n. 38), para 88: "the intention was that the school should not be an arena for preaching or missionary activities but a meeting place for different religious and philosophical convictions where pupils could gain knowledge about their respective thoughts and traditions (…). in the view of the court, these intentions were clearly consonant with the principles of pluralism and objectivity embodied in article 2 of protocol no. 1". and para 89: "(…) from the drafting history it emerges that the idea was that the aim of avoiding sectarianism and fostering intercultural dialogue and understanding could be better achieved with an arrangement, such as here, bringing pupils together within the framework of one joint subject (...) moreover (…) the second sentence of article 2 of protocol no. 1 does not embody any right for parents that their child be kept ignorant about religion and philosophy in their education." a similar reasoning in paras 58 & 59 of the zengin case (see n. 39). the key document of african instruments on human rights is the african charter on human and peoples' rights (hereinafter, african charter). provisions on education, however, appear scarce in the african charter, and there is only one specific reference on art 17 para 1: "every individual shall have the right to education". apparently, and when compared to un provisions on education's goals and values, there is no reference to such values in the basic african instrument. nevertheless, it should be noted that article 17 also refers to free participation in the community's cultural life as a right of every individual (para 2) and to the duty of the state to promote and protect "morals and traditional values recognized by the community" (para 3). this systematic proximity induces the consideration that education, participation in the community's cultural life, and the protection of moral and traditional (non-colonial) values are deeply connected in the african charter's spirit. this interpretation will be further discussed in "african morals, traditional values and cultures, and other specific political principles" below. in contrast to the african charter on human and peoples' rights, the african charter on the rights and welfare of the child 45 displays a rich wording when dealing with educational values. this treaty has been so far ratified by 49 states and signed by another five. only one state (morocco) has done neither. 46 in addition, some provisions of the african charter on democracy, elections, and governance will be discussed. 47 up to the present, 34 states have become parties to it and fifteen others have become signatories. six states, however, have accomplished neither yet (botswana, egypt, eritrea, libya, morocco, and tanzania). 48 let us now focus on the values that should guide education according to these three charters. although this fundamental aim does not differ in substance from the one set in the udhr and the core international human rights instruments, african instruments have included certain elaborations that deserve specific consideration. art 11 para 2 of the african child charter includes a phrase that is almost identical to another in the convention on the rights of the child, which seems a reasonable outcome given the proximity of their respective adoptions (1989 for the crc and 1990 for the acc). the first of the goals mentioned reads as follows: the promotion and development of the child's personality, talents and mental and physical abilities to their fullest potential. 45 african charter on the rights and welfare of the child (adopted 11 july 1990, entered into force 29 november 1999) (african child charter or acc). 46 however, and beyond this initial commonality with un-fostered treaties, the african conception seems particularly concerned about the importance of education-and leisure (udombana 2006 49 )-for individual self-development in general and for promoting awareness of the own rights in particular. not by chance, in 2013, the achpr identified among its "areas of concern" the fact that "many children are not in school despite the provision of free and compulsory education in some state parties, due to socio-cultural and political considerations, among others 50 ; moreover, the achpr has included as a "positive development," among other measures taken to protect the rights of children, "the introduction of school feeding programs in south africa to encourage parents to send their children to school". 51 as well as the fact that certain states "(…) have put in place educational systems that are specifically tailored to suit the mobile lifestyles of their indigenous populations/ communities (namibia) (…)". 52 despite these achievements, certain "socio-cultural" patterns seem to hinder some specific groups, in particular women and girls. many examples can be found in the achpr's activity reports. among these, in 2012, the special rapporteur on the rights of women in africa informed the commission that "the situation of women in the rural areas remains dire, and in the area of education, the problem of girls' access to education despite the progress made by some countries" (sic). 53 more recently, the achpr intensified its appeal in order to denounce the "continuing discriminations and practices against women and girls, including the exclusion of pregnant girls from the education system and refusing them to take public examinations, which violates their right to education and serves to perpetuate other discriminations against them". 54 in this regard, the achpr has also addressed letters of urgent appeal to certain governments that seemed to embrace these convictions, as revealed by "the statement made by the tanzanian authorities on 22 june 2017 to the effect that pregnant girls and teen mothers would no longer be allowed to attend school and continue their education". 55 fully aware of these hindrances, one of the most recent legal instruments in african human rights, the african charter on democracy, elections, and governance, directed its state parties "to provide free and compulsory basic education to all, especially girls, rural inhabitants, minorities, people with disabilities and other marginalized social groups", 56 as well as to ensure "the literacy of citizens above compulsory school age, particularly women, rural inhabitants, minorities, people with disabilities, and other marginalized social groups". 57 49 art 12 of the acc refers to the right "to engage in play and recreational activities appropriate to the age of the child." see also n.j. udombana (2006, 190) . 50 achpr, 34th activity report (2013), para 21.vii. emphasis added. 51 achpr, 36th activity report (2014), p. 11, para v. emphasis added. 52 achpr, 30th activity report (2011), para 251. 53 achpr, combined 32nd and 33rd activity reports (2012), para 251. 54 achpr, 38th activity report (2015), para 41.b.vi. emphasis added. 55 achpr, 43rd activity report (2017). 56 acdeg art 43 para 1. 57 acdeg art 43 para 2. thus, an instrument apparently intended for political rights mandates the establishment of a free and compulsory education system and grants special attention to disadvantaged groups. nevertheless, it must not surprise that a charter on democracy, elections, and governance refers to education in such detail. to quote john dewey's classic book democracy and education, "the realization of a form of social life in which interests are mutually interpenetrating, and where progress, or readjustment, is an important consideration, makes a democratic community more interested than other communities have cause to be in deliberate and systematic education" (dewey 1916, 100-101). more recently, and from the perspective another regional system for the protection of human rights, the inter-american court of human rights considered education as the "epitome of indivisibility and interdependence of all human rights". 58 despite the political importance of education, however, complaints brought before the achpr through the communications procedure have often addressed insufficiency of funding or facilities, as well as a lack of dedicated teaching staff. 59 so far, it may be concluded that african instruments on human rights consider the very accessibility to school education as a key element for the first value that should guide education: the full development of the child's personality. therefore, great interest is placed in countering social and cultural convictions that limit such accessibility. despite its clear connection with the previous aim, the importance that different african instruments attach to this matter recommends devoting a specific subsection to its discussion. after the development of the child's personality, the african child charter considers that education should be directed to: fostering respect for human rights and fundamental freedoms with particular reference to those set out in the provisions of various african instruments on human and peoples' rights and international human rights declarations and conventions 60 the concept of education as an "empowerment right," already mentioned above, has been received in the african instruments on human rights. indeed, this idea was already present in the african charter despite the scarcity of its provisions on education. 61 as such empowerment right, education helps the child-and future adult-to take informed decisions (thus increasing individual freedom) and live in better conditions (improving a certain equality and fostering solidarity). one specific example of better 58 gonzales lluy and others v ecuador inter-american court of human rights series c no 298 (1 september 2015) para 234. see also hevia rivas (2008, 143 and 2010, 29) . 59 socio economic rights and accountability project v. nig., comm. 300/2005, 25th achpr aar annex (may-nov 2008) . the complaint was declared inadmissible due to lack of proof concerning the exhaustion of domestic remedies. 60 acc, art 11 para 2.b. 61 african charter, art 25: "state parties (…) shall have the duty to promote and ensure through teaching, education and publication, the respect of the rights and freedoms contained in the present charter (…)." living conditions would be "understanding of primary health care" (art 11 para 2.e), which is of great importance given the threat of epidemics and viral infections in the continent (yellow fever, malaria, ebola, hiv, or the covid-19 pandemic). the wording of the acc, however, does not aim so much at raising awareness of the own human rights as it does at fostering respect for human rights. both aims (raising awareness and fostering respect) are not completely diverse, but the second might seem more ambitious. moreover, from a political perspective, democracy is enhanced if citizens are more and better educated, and in particular with regard to human rights. education on human rights and democracy (more commonly "human rights education") thus appears as a highly valuable resource. to be sure, the achpr has long been aware of its importance, as it showed with its resolution on human and peoples' rights education 62 of 1993 and other statements. 63 the member states of the african union have, for their part, displayed a clear interest in promoting this kind of education, as the african charter on democracy, elections, and governance demonstrates. according to this instrument, state parties undertake to implement programs and carry out activities designed to promote democratic principles and practices as well as consolidate a culture of democracy and peace. to this end, state parties shall: (…) 4. integrate civic education in their educational curricula and develop appropriate programs and activities. 64 the importance of this obligation is further underlined by another provision. indeed, civic education shall be "systematic and comprehensive" and, more importantly, it shall aim "to encourage full participation of social groups with special needs in democracy and development processes". 65 these provisions of the acdeg allow for three considerations. firstly, education is considered essential for the political participation of social groups (in this case, specifically those with special needs). once again, the idea of education as empowerment, now applied to groups with special needs. thus, the educational aim of fostering respect for human rights appears linked with the value of solidarity within society. secondly, the reference to "(social) groups" reminds of the abovementioned "intermediate bodies." among these, political parties are of particular importance in a parliamentary democracy. political parties, however, would be hardly democratic if their militants were unable to grasp the significance-and debate on the substance-of complex decisions advanced by their leaders. in a presidential democracy, the political 62 resolution on human and peoples' rights education, adopted by the achpr at its 14th session, december 1993. the text is available at https://www.ohchr.org/en/issues/education/training/compilation/pages/5 resolutiononhumanrightseducation (1993) .aspx. 63 in its 35th activity report, of 2013, the achpr highlighted "lesotho's (…) new curriculum for schools which includes components of human rights issues affecting children." 64 acdeg, art 12. emphasis added. 65 acdeg, art 31 para 2. party does not necessarily enjoy the same significance, but the education of those called to vote (especially a pluralistic and non-indoctrinating human rights education) remains of paramount importance. according to experts, "political representation means choosing, electing representatives, selecting the political class" (bonfiglio 2013, 90; similarly, randall 2007, 85-86) , which confirms the importance of education as a crucial element for democracy. however, historically, in african politics, this has not excluded the possibility that the power of granting educational opportunities (e.g., through scholarships) was used, conversely, as an "enormously effective instrument of oneparty consolidation" (coleman and rosberg 1964, 666) or, more brutally, that teachers and health workers "were forced to attend political education meetings" under mugabe's rule in zimbabwe (laakso 2007, 243) . this confirms how strongly education can affect the political system. thirdly, the african conception of civic education appears in close connection with that of "development." if the notion of development may come as a novelty when discussing educational values, other principles deeply connected with it have been present in african human rights instruments since its early days. the ideas of independence, decolonization, and the highly interesting principle of african solidarity have more recently led to an emphasis on economic and social development. this, however, should not be deemed incompatible with the preservation of traditional values, territorial integrity, and african unity, as will be discussed below ("african morals, traditional values and cultures, and other specific political principles"). despite the strong connection of this principle with the full development of the personality, the authors of the african child charter have chosen to present it as a different goal. this principle is closely linked with the "universal" political aims of the core international human rights instruments as well. 66 nevertheless, the preparation for responsible life in freedom does not only apply to children, but also to adults, and, importantly, even to those incarcerated. 67 let us turn to the wording of the acc. its art 11 para 2 provides the following: the education of the child shall be directed to: (…) (d) the preparation of the child for responsible life in a free society, in the spirit of understanding, tolerance, dialogue, mutual respect and friendship among all peoples, ethnic, tribal and religious groups. while the general drafting and many of the elements appear very close, or even identical, to the relevant paragraph of the crc's, 68 some differences should be underlined. the african wording thus features some new elements but, strangely enough, others have been erased. among the latter, "in the spirit of" the crc appeared 66 see above "promoting understanding, tolerance and peace." 67 achpr, 31st activity report (of 2011), para 30. 68 crc, art 29 para 1.d. see above "full development of the human personality and best interest of the child". the references to "peace," "equality of sexes," "friendship among all (…) national groups," and "persons of indigenous origin". the removal of the reference to national groups may be explained by the introduction, instead, of two new and more elaborate paragraphs on the matter (namely, e and f). however, the reasons behind the other three suppressions appear obscure, and the result, difficult to approve. the additions, nevertheless, enrich the text of the provision with some aspects of african legal and political thinking. perhaps the most evident would be the reference to "tribal" groups as a basic organizational form of many african societies, a notion criticized for the "lack of conceptual groundwork and empirical testing" on whether and how ethnicity affects voting preferences (elischer 2013, 25) . moreover, the ideas of "dialog" and "mutual respect" are also an important part of african culture, as the traditional notion of ubuntu expresses well (venter 2004, 149 and le roux 2000, 43) . as vervliet (2009, 64) has written, "in (…) ubuntu, the human person does not stand on his own, but becomes more human in relation with other people". lastly, the reference to the "peoples" is not an innovation of the african instrument, but it certainly acquires a new significance given the importance of this notion in the continent's legal tradition of human rights. it should be noted that the african charter, the achpr, and the african court on human and peoples' rights all bear the reference to the peoples in their very names, thus showing their importance as a subject of rights. human and peoples' rights thus appear closely connected in the african conception. unfortunately, the study of collective rights would require an in-depth approach that clearly exceeds the scope of this article. the un convention on the rights of the child referred to the development of respect for the child's "own cultural identity, language and values, for the national values of the country in which the child is living, the country from which he or she may originate, and for civilizations different from his or her own". receptive to the influence of its immediate un precedent, the african child charter picked up the baton and included four different provisions that expand these ideas. the relevant parts of art 11 para 2 mandate the following: the education of the child shall be directed to: (…) (c) the preservation and strengthening of positive african morals, traditional values and cultures; (…) (e) the preservation of national independence and territorial integrity; (f) the promotion and achievement of african unity and solidarity; article 12, in turn, further elaborates on the right to culture: state parties shall respect and promote the right of the child to fully participate in cultural and artistic life and shall encourage the provision of appropriate and equal opportunities for cultural, artistic, recreational and leisure activity. firstly, from the perspective of moral education, the acc is unorthodox but honest when it accepts that not all african morals, traditional values, and cultures are necessarily "positive" and hence deserve to be promoted. among the positive aspects, the abovementioned notion of ubuntu requires a socially valuable orientation of individual rights. in their definition of the african child, nthontho and ogina (2020) highlight the collective effort behind the education of the young. they illustrate this cultural pattern with the traditional saying that "it takes a village to raise a child". this communalist approach to human rights and education is genuinely african and is not to be found-at least not with the same intensity-in other conceptions. 69 another example of traditional values inspiring human rights can be found in the south african constitutional court's ruling on the khosa case. 70 according to kamga (2018, 641) , when the constitutional court held that "everyone's right to access social security encompasses permanent residents in the country" (including children), it was inspired by the values of ubuntu. on the other hand, a more negative aspect would be the use of corporal punishment. while it was banned by the south african schools act in 1996, this ban was subsequently challenged by 196 independent christian schools. the constitutional court, however, upheld the act in a memorable ruling by justice albi sachs. 71 by limiting its advocacy to the positive inheritance, the acc separates itself from the wording of the older african charter, which did not include any differentiation but rather mandated the protection of any "morals and traditional values recognized by the community." still, despite the more advanced contents of the acc in this regard, basing on the african charter may include some advantages, too. an important one concerns the possibility of using the rather open communications procedure of article 55, thus attracting the attention of the achpr and, perhaps, driving it to use its "good offices" in search of a friendly settlement, or a recommendation, or even to submit the case to the african court. 72 in this regard, certain complaints have been addressed to the achpr despite the strict wording of its article 25 ("every individual shall have the right to education"). among these, for example, a case of 2003 concerning cameroon reflected an attempt by the complainants to demonstrate a linguistic, cultural, and educational undermining of the anglophone parts of the country by the government, thus trying to push the achpr to ponder on the argument of respect for cultural specificity and the protection of linguistic minorities. the achpr, nevertheless, rejected the complaint on article 17 due to lack of sufficient proof 73 (but accepted several others, including certain forms of discrimination such as that of the english language in business transactions 74 ). the notion of culture, in the second place, is not only destined to the stationary status of preservation. the use of the term strengthening in art 11 para 2.c and especially that of participation (linked in article 12 with artistic and with recreational and leisure activities) bring the educational treatment of culture closer to its dynamic essence. education should help the children to understand and preserve their cultural heritage but also to enjoy and develop it. the african charter confirms this interpretation when it includes the right to "freely" "take part in the cultural life of his community" next to the very recognition of the right to education. 75 thirdly, the references to "national independence," "territorial integrity," "african unity," and "african solidarity" are of a more political nature and reflect the historical circumstances to which the rise of the human rights movement in africa was bound. indeed, all of these ideas seem in line with the basic orientations of the organization of african unity since its inception. it should be interesting to remind that in 1963, the african heads of states and governments declared themselves "determined to safeguard and consolidate the hard-won independence as well as the sovereignty and territorial integrity of our states, and to fight against neo-colonialism in all its forms". 76 a purpose later reinforced in the preamble of the very african charter on human and people's rights, where the member states of the oau again showed themselves conscious of their duty to achieve the total liberation of africa, the peoples of which are still struggling for their dignity and genuine independence, and undertaking to eliminate colonialism, neo-colonialism, apartheid, zionism and to dismantle aggressive foreign military bases and all forms of discrimination, particularly those based on race, ethnic group, color, sex, language, religion or political opinions. summarizing, the protection of african cultural values in human rights' instruments reveals the struggle of the continent to become politically independent and economically developed while (dynamically) preserving the best of its traditional identity. one last aim of education according to the african child charter concerns the development of respect for the environment and natural resources. 77 once again, the acc takes up the baton of the un convention on the rights of the child, until then the only un instrument on human rights to have mentioned environmental awareness as an educational value. given the rich resources of the african continent, also from an economic perspective, the african instrument expands the scope in order to instill respect for natural resources as well. indeed, natural resources have historically been over-exploited, either by colonial powers 78 or under self-rule. the latter, nevertheless, were often responsible for some of the first conservation efforts as well (van eeden 2014, 640). 75 african charter art 17 para 2. 76 organization of african unity charter, 25 may 1963, (oau charter) preamble. 77 acc art 11 para 2.g. 78 c.w. de kiewiet (1941, 188) , in his history of south africa, wrote that "in all the great colonial regions of the world the history of the ruthless exploitation of natural resources is a full one." this provision, therefore, points to the aim of delivering the continent from economic exploitation. in doing so, it is perfectly coherent with the african charter's proud declarations that "all peoples shall freely dispose of their wealth and natural resources. this right shall be exercised in the exclusive interest of the people. in no case shall a people be deprived of it." 79 some of the most infamous cases of recent history are closely linked with this defense of the environment against large transnational companies backed by some governments. the most notorious is probably the niger delta dispute, 80 but the aforementioned achpr/kenya case concerning the rights of the ogiek community of the mau forest is a more recent example. both un and regional systems reflect certain humanistic values that, despite different wordings and circumstances, may be considered essentially common to all three conceptions. perhaps the best example is the principle of the full development of the personality (often linked with the child's best interest doctrine), but clearly not the only one. the promotion of understanding, tolerance, and of specific cultural values is also present in every human rights system. on the darker side, the risk of cultural indoctrination has made its appearance in both the european and the african contexts. despite these common aspects, regional systems also reveal specific conceptions that are due to each continent's own history and current challenges. in the african system, the different charters reflect a rather open understanding of cultural values and traditions by granting every individual the right to "freely participate" in cultural life. the historical struggle for political independence, economic development, and environmental protection has also left its mark on educational values. concerning the enforcement of the charters, however, the communications procedure before the achpr may only lead to the adoption of decisions that are technically not binding for the states parties involved. this lack of binding force when discussing individual communications may explain why certain statements throughout the achpr's periodical activity reports have adopted a strongly critical style, for example with regard to access to education for pregnant girls. a more frequent intervention of the african court might be desirable, but both the institutional framework and the system's budget entail limitations. the ecthr, on the other hand, may seem less demanding than the achpr in its requirements to states parties. examples of this are the frequent recourse to the margin of appreciation doctrine and the ecthr's acquiescence to france's broad interpretation of secularism or italy's defense of the crucifix as a symbol of universal values. however, this acquiescence in matters that are highly controversial within certain societies could paradoxically reflect a stronger institutional system and hence a smaller need to react to all challenges. this institutional strength (at least when compared to other human rights systems) arises from the solid position of the ecthr as the only international protector of the echr and from the mandatory nature of its rulings for the 79 african charter art 21 para 1. 80 soc. and econ. rights action ctr. v. nig, comm. 155/96, 15th achpr aar annex v (2000 -2001 . states parties involved. nevertheless, and most importantly, the ecthr receives and adjudicates a far larger number of cases than any other human rights system. this grants the european court the opportunity to rule on each case considering the social, political, and legal specificity of the state concerned, rather than motivated by the need to develop a notion that is valid for the whole continent. it therefore seems to have more occasions to develop a nuanced body of principles. such a system, however, requires greater funding by states parties. the importance of international human rights law: contestations and reasons" and stoppioni protocol to the african charter on human and peoples' rights on the establishment of an african court on human and peoples' rights comm. 266/2003, 26th achpr aar annex the right to human rights education human rights education intercultural constitutionalism: from human rights colonialism to a new 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nations and the maintenance of international peace and security acknowledgments the author wishes to kindly thank prof johan beckmann, prof everard weber and dr. andré du plessis, from the university of pretoria's department of education management & policy studies. this research was made possible by the university of pretoria's visiting professors program. conflict of interest the author declares that he has no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-002728-6oyw5sqv authors: carding, s. r.; davis, n.; hoyles, l. title: review article: the human intestinal virome in health and disease date: 2017-09-04 journal: aliment pharmacol ther doi: 10.1111/apt.14280 sha: doc_id: 2728 cord_uid: 6oyw5sqv background: the human virome consists of animal‐cell viruses causing transient infections, bacteriophage (phage) predators of bacteria and archaea, endogenous retroviruses and viruses causing persistent and latent infections. high‐throughput, inexpensive, sensitive sequencing methods and metagenomics now make it possible to study the contribution dsdna, ssdna and rna virus‐like particles make to the human virome, and in particular the intestinal virome. aim: to review and evaluate the pioneering studies that have attempted to characterise the human virome and generated an increased interest in understanding how the intestinal virome might contribute to maintaining health, and the pathogenesis of chronic diseases. methods: relevant virome‐related articles were selected for review following extensive language‐ and date‐unrestricted, electronic searches of the literature. results: the human intestinal virome is personalised and stable, and dominated by phages. it develops soon after birth in parallel with prokaryotic communities of the microbiota, becoming established during the first few years of life. by infecting specific populations of bacteria, phages can alter microbiota structure by killing host cells or altering their phenotype, enabling phages to contribute to maintaining intestinal homeostasis or microbial imbalance (dysbiosis), and the development of chronic infectious and autoimmune diseases including hiv infection and crohn's disease, respectively. conclusions: our understanding of the intestinal virome is fragmented and requires standardised methods for virus isolation and sequencing to provide a more complete picture of the virome, which is key to explaining the basis of virome‐disease associations, and how enteric viruses can contribute to disease aetiologies and be rationalised as targets for interventions. viruses are thought to be the most abundant and diverse entities on earth, numbering as many as 10 31 virus-like particles (vlps), 1 though this paradigm is likely to change in light of information from large-scale sequencing surveys of marine environments and improved analytical tools. 2 with the advent of new, sequence-based technologies that do not rely on the ability to isolate viruses for their identification, it is now possible to define and characterise viruses in different environmental samples in greater detail than ever before, which has resulted in an increased interest in the role the viral assemblage of the human gut microbiota plays in health and disease. the following reviews our current knowledge on the human intestinal virome. a virome comprises all the nucleic acids (ssdna, dsdna, ssrna and dsrna) belonging to the vlps associated with a particular ecosystem. the human virome is a genetically complex component of the microbiome, with the blood, nose, skin, conjunctiva, mouth, vagina, lungs and gastrointestinal (gi) tract harbouring their own distinct virus assemblages (table s1 ). the genetic content of vlps comprising bacteriophages (phages) that infect bacteria and archaea and, to a much lesser extent, human-, plant-, amoebae-and animal-infecting viruses found along the gi tract constitute the human intestinal virome (figure 1 ). phages can be lytic or lysogenic. lytic (virulent) phages bind to specific host-cell receptors, then penetrate and infect their host cell to hijack its replication and translation machinery to produce virions; once sufficient virions have accumulated (tens to thousands, depending on the phage), the lytic enzymes they produce cause the host cell to lyse, releasing the virions into the surrounding environment where they can infect new host cells. lytic phages can have narrow or broad host ranges, infecting only one strain of a prokaryote or multiple species of closely related prokaryotes. these entities have been used in the past to treat infections, and have recently been revisited as alternatives to antibiotic therapies for a range of infections and as means of controlling food-contaminating bacteria such as listeria spp. 3 their gene products are also of interest in biotechnological and medical applications. 4 the mammalian intestinal virome comprises viruses that infect eukaryotic and prokaryotic cells. it is established soon after birth and is dominated by viruses that infect bacteria (ie, phages). the virome establishes a mutualistic relationship with eukaryotes/prokaryotes, contributing to intestinal homeostasis by influencing microbial ecology and host immunity. composition of the virome is influenced by numerous factors that affect viruses directly (infection) or change host-cell populations (eg, antibiotics, diet). members of the virome may contribute to the pathogenesis of certain diseases via microbial host lysis leading to dysbiosis, infection of epithelial cells, and/or translocation of the compromised or damaged mucosal barrier to gain access to underlying tissues and immune cells, leading to immune activation. dysbiosis can be defined as a microbial imbalance or any changes to the composition of resident microbial communities relative to the community found in healthy individuals. [101] [102] [103] virome association with certain disease states is characterised by changes in diversity, and predominance of specific virotypes (eg, members of the order caudovirales in ibd) (temperate) phages do not kill their host but instead integrate into their host's genome without interfering with its replication, incorporating the phage into its genome as a prophage that is transmitted to its progeny at each cell division. lysogenic phages can be converted to a lytic cycle in response to environmental stressors (eg, antibiotics). phage-host interactions influence host and viral evolution. the ability of phages to transfer genes from one prokaryotic host to another can lead to increased diversification of viral species, and increased antibiotic resistance and/or induction of toxins or virulence factors in prokaryotes. 5 some phages alter the antigenicity of their hosts by producing enzymes that modify the o-antigen component of lipopolysaccharides. 6 modification of surface structures of prokaryotes has the potential to affect microbial interactions with the human host, and influence niche specialisation within the gi tract. 7 presence of clustered regularly interspaced short palindromic repeats (crisprs) confers upon prokaryotes resistance to phage infection and contributes to prokaryotic adaptive immunity. analyses of metagenomic sequence data provide detailed information on phage-host and phage-phage competition within the human faecal microbiome, implying crispr spacers are actively and continuously acquired by prokaryotes in response to the presence of phages in the gi tract. 8 the potential effects of such phage-host interactions on microbiota composition/function or host health are unknown. viruses do not encode universally conserved genes such as the 16s or 18s rrna genes of prokaryotes and eukaryotes, respectively, and are genetically highly diverse. consequently, it is not possible to use metataxonomic approaches such as 16s rrna gene sequencing to characterise vlps within ecosystems. traditionally, classical approaches-mainly microscopy and cultivation-have been relied upon to characterise vlps in the human gut. based on transmission electron microscopy (tem), mucosal samples contain~1.2 9 10 9 vlps/biopsy. 9 vlps have been detected in caecal contents at 10 6 /ml using tem 10 with faeces harbouring 10 8 -10 9 vlps/g wet weight. 10, 11 in all gi contents examined to date by microscopy, the overwhelming majority of vlps have been phages of the order caudovirales, with the human gi tract estimated to harbour 10 15 phages in total. 3, 9, 10, 12 the order caudovirales encompasses most known phages (figure 2 ), comprising the families the only reliable molecular method currently available for routine surveys of the human virome is metagenomics. metagenomics is a culture-independent, molecular-based approach that allows functional and sequence-based analyses of the collective microbial genomes contained in an environmental sample, providing a powerful approach for exploring the ecology of complex microbial communities. 14 it has been used to examine prokaryotic communities associated with the human gut microbiome in health and disease, [15] [16] [17] [18] and to examine viromes associated with different regions of the human body (table s1) . a protocol involving homogenisation of faeces in buffer, centrifugation (to remove cell debris), tangential flow filtration (tff) (to concentrate large-volume samples and isolate vlps), ultracentrifugation and metagenomic reconstruction was used to characterise the first human faecal virome. 19 however, recent improvements in recovery methods give the potential to characterise human-associated vlp assemblages at the molecular level in greater detail than ever before ( sequences associated with this phage were not found in american gut-associated datasets (metagenome or virome), suggesting geographical variation in distribution of gut-associated phages. 7 using a similar metagenomic mining approach but with major capsid protein vp1 specific for the ssdna phage family microviridae, members of the subfamilies gokushovirinae, alpnavirinae and pichovirinae were detected in faecal metagenomes. 24 by using phage genome signature-based recovery with metagenomes, several (predominantly temperate) potential gut-specific phages infecting members of the order bacteroidales were identified. 21 this alignment-free approach was able to resolve phage sequences not readily detected by conventional alignment-driven approaches. the main limitation of this method is the lack of available genome sequences from isolated phages, as "driver sequences" are required to provide baseline information upon which inferences can be made regarding sequence data and host phylogeny (only four such "driver sequences" were required in the bacteroidales study). 21 using co-occurrence profiling, crassphage was identified in publicly available metagenomes, and by crispr analysis and co-occurrence profiling predicted to infect bacteroides or prevotella spp. 22 assembly of sequence data from four deep-sequenced dna viromes (8.1 gb sequence data in total) from two healthy individuals led to recovery of 4301 contigs, representing 72 complete phage genomes plus potentially complete and partial phage genomes. 28 mapped to 160 contigs. a consortium of 155 phages contributed to the "healthy gut phageome" (hgp), though only 23 phages were present in >50% of samples ("core" phages, including crassphage and nine other complete genomes), and 132 were present in 20%-50% of samples ("common" phages); 1679 were present in 2%-19% ("low overlap" phages). the hgp represented only 4% of the total phage community, with the expectation that the hgp will increase in size by analysing additional healthy individuals using deep sequencing. 28 although it has been proposed the hgp may play a role in maintaining and possibly restoring a dysbiotic microbiota, 30 (table s1 ). later work on oral and faecal viromes of 20 individuals over a 6-month period confirmed this, demonstrating phages were persistent in these viromes and readily shared among related and unrelated members of the same household. 38 sharing of phages encoding virulence or antibiotic-resistance genes has implications for shaping of microbiomes of those in close contact with one another, and warrants further study. with respect to antibiotic-resistance genes, their presence may have been over-estimated in human viromes 11, 20, 27, 31, 35, 37 as these entities are thought to be rarely encoded in phages. 39 consequently, care should be taken when analysing data: proper in silico checks with conservative cut-offs are required to refine functional assignments and avoid over-interpretation of data. 39 and geminiviruses were detected in at least one twin of each pair between months 3 and 24 of the study. 46 anelloviruses, associated with host immune status, were most prevalent from 3 months of age and highly divergent from known anelloviruses, with their abundance peaking at 6-12 months of age. the same anelloviruses were detected in faecal samples of the same infant collected 12 months apart, suggesting a persistent or stable source of recurring infection. 46 it was speculated the increase in anelloviruses between 6 46 the initially high phage population is unsustainable because of low numbers of bacteria colonising the gi tract. consequently, the phage assemblage shrinks in size and diversity, relieving pressure on the bacterial community and allowing it to establish and colonise the gut. differences in birth mode (caesarean vs vaginal delivery) and diet (breast-vs formula-feeding; age at which weaning began) were confounders in the study of lim et al. 46 51, 54 circulating phages have also been detected in blood samples after oral administration of antibiotics to patients with bacterial infections, whereas no phages could be detected in their blood prior to antibiotic therapy. 51 it is not known if these phages were of gi origin. the presence of phages in the peripheral blood has been termed "phagemia". 51 however, whether the blood contains its own "baseline" vlp population to which the gi virome can contribute in disease is unknown. in colonic biopsies than healthy individuals (n = 10) (2.9 9 10 9 vs 1.2 9 10 8 vlps/biopsy), and for the cd patients ulcerated mucosa had significantly fewer vlps than nonulcerated mucosa (2.1 9 10 9 vs 4.1 9 10 9 vlps/biopsy). 9 more recent, small-scale studies have been undertaken to characterise the gut virome of ibd. 29,62-64 a pilot metagenomic study of ileal and colonic contents of six cd patients and ileal samples from six non-ibd controls showed cd dna-based viromes had higher phage abundance than control samples. 62 however, the ways in which virome data from healthy controls (n = 8) and patients with ileocolic cd (n = 11) were analysed were found to contribute to interpretation of results. 63 prophages represented the most hits in the cd samples when unassembled reads were compared, but were higher in the control group when the assembled data were compared. analysis based on assembled data showed fewer differences between the cd and control group. nonrarefied data were used to generate estimators of species richness: the viromes of cd patients were less diverse than the healthy patients, but showed greater heterogeneity across samples. the cd and control samples could not be fully separated into two groups based on vlp composition and abundance. a later dna-based study showed differences in cd patients related to disease status (newly diagnosed, active onset, active presurgery) and therapy. 65 similar to their previous study, individual variability and sample origin had a greater effect on the virome than cd, although more over-represented viruses were found in the cd viromes than the healthy, and newly diagnosed patients had higher diversity in faecal and biopsy viromes than those with active disease. patients on steroids and/or immunosuppressors had lower diversity than untreated patients, while those on immunosuppressors only had lower diversity than those on combination therapy or steroids only. the study benefitted from recruiting cd and ulcerative colitis (uc) patients and their family members in the uk (cambridge) and usa (los angeles), thereby controlling for household factors that may influence the microbiome. 29 an increase in phage-associated richness (predominantly caudovirales and microviridae) was seen in ibd faecal viromes compared with those of controls. initial findings were confirmed using two independent and geographically distinct us (boston, chicago) patient cohorts with matched controls. 29 it was suggested decreases in bacterial richness concomitant with increases in phage richness may be due to predator-prey dynamics, but it was unclear how these contribute to the pathogenesis of ibd. also proposed was the virome as a target for therapeutic modulation. 29 few studies of the human virome have considered the effects of contaminants in reagents and nucleic acid extraction kits on viral metagenomes (table s1 ), though these are being increasingly recognised in prokaryote-focused studies. 26, 69, 70 nucleases, proteases and polymerases used in studies are produced in protein expression systems, and sequences associated with these expression vectors have been detected in virome studies. 70 columns used to purify dna may introduce parvovirus-like, circoviruses/densoviruses and iridoviruses sequences into samples, and it has been suggested their use should be avoided in virome studies. 26, 70 efforts should be made to prevent cross-contamination of samples when processing samples for nucleic acid extraction and sequencing. 26, 70 low-biomass samples obtained from non-gi sites will be reliant on pcr-based approaches for the foreseeable future, and these are most likely to be affected by contaminants in reagents. therefore, appropriate negative controls should be included in sequencing studies, to allow the identification of sample-associated sequences rather than those from contaminants. those studies targeting only the vlps of the microbiota will not capture prophage diversity within samples, and will contain little information to allow studies of phage-host interactions or host ranges, limiting in-depth analyses of the overall ecology of the gut microbiome. 21 studying total community metagenomes in conjunction with vlp-derived metagenomes will provide a more-complete picture of virus-host interactions in the human gut. 21 the greatest challenge to integrated studies of the whole gut microbiome is 71 it has been stated that isolating "just a few phage genomes from novel environments will greatly increase our understanding of viral diversity in these environments". 72 (2) there are a number of medical conditions speculated to be linked with viral infections (eg, type i diabetes, chronic fatigue syndrome, obesity) but for which no infectious agent has been found. it has been estimated over half of humaninfecting viruses remain to be discovered. 76 interactions with pets, insects and wild animals will also influence the composition of the human virome. from a pathogen perspective, bush-workers, abattoir workers and individuals exposed to insect bites have been highlighted as likely sources of new humaninfecting viruses. 75 with the advent of high-throughput, inexpensive, sensitive sequencing methods, it has become possible to study the contribution of dsdna, ssdna and rna vlps to the human virome. gene transfer agents, which appear to represent defective phages, have not been examined in the context of the human microbiome or virome, but may also make a contribution to its genetic content. 77, 78 similarly, exosomes-extracellular vesicles of 40-100 nm in diameter consisting of proteins, lipids, mirna, mrna and dna-derived from cells lining the gi epithelium are likely to contribute to nucleic acids found in viromes. 79, 80 outer-membrane vesicles, the equivalent (in size and composition) of eukaryotic exosomes for gram-negative bacteria, may also contribute to what we currently call the virome, as these vesicles have recently been reported to contain dna as well as rna. 81 whereas 16s rrna gene sequence data are readily processed and analysed using packages such as qiime and mothur, no such tools exist for the analyses of sequence data derived from host-associated viromes. 35 tools exist for functional annotation of viromes and estimating viral diversity (table 3) . no easy-to-use pipeline that takes raw reads, strips out host dna, looks for bacterial contaminants then assigns taxonomy and functionality to prokaryote and eukaryote viruses within samples exists, though efforts are being made to generate such tools. these will need to take into account the presence of human endogenous retroviruses, which comprise~8% of the human genome and still possess the ability to encode retrovirus polymerase and envelope protein and can be reactivated by exogenous retroviruses such as hiv. 53 the authors would like to thank sam carding for creating figure 1 . declaration of personal interest: none. here a virus, there a virus, everywhere the same virus? viral ecology comes of age. environ microbiol rep exploiting gut bacteriophages for human health characterization of a bacteriophage-derived murein peptidase for elimination of antibiotic-resistant staphylococcus aureus ecology of prokaryotic viruses bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell comparative (meta)genomic analysis and ecological profiling of human gut-specific bacteriophage φb124-14 crispr targeting reveals a reservoir of common phages associated with the human gut microbiome dysbiosis in inflammatory bowel disease: a role for bacteriophages? characterization of viruslike particles associated with the human faecal and caecal microbiota diversity and abundance of single-stranded dna viruses in human feces optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis metagenomics: genomic analysis of microbial communities richness of human gut microbiome correlates with metabolic markers dietary intervention impact on gut microbial gene richness alterations of the human gut microbiome in liver cirrhosis shotgun metagenomics of 250 adult twins reveals genetic and environmental impacts on the gut microbiome metagenomic analyses of an uncultured viral community from human feces the human gut virome: inter-individual variation and dynamic response to diet genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences a highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes the application of a recently isolated strain of bacteroides (gb-124) to identify human sources of faecal pollution in a temperate river catchment evolution and diversity of the microviridae viral family through a collection of 81 new complete genomes assembled from virome reads new viruses in idiopathic human diarrhea cases, the netherlands viral metagenomics applied to blood donors and recipients at high risk for blood-borne infections viruses in the faecal microbiota of monozygotic twins and their mothers healthy human gut phageome disease-specific alterations in the enteric virome in inflammatory bowel disease the human gut phage community and its implications for health and disease hypervariable loci in the human gut virome viral diversity and dynamics in an infant gut rapid evolution of the human gut virome bacteriophage adhering to mucus provide a non-host-derived immunity effects of long term antibiotic therapy on human oral and fecal viromes quinolone antibiotics induce shiga toxin-encoding bacteriophages, toxin production, and death in mice metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals transmission of viruses via our microbiomes phages rarely encode antibiotic resistance genes: a cautionary tale for virome analyses single-stranded dna library preparation for the sequencing of ancient or damaged dna structure and diversity of ssdna microviridae viruses in two peri-alpine lakes (annecy and bourget, france) towards quantitative viromics for both double-stranded and single-stranded dna viruses distribution of ribonucleic acid coliphages in animals rna viral community in human feces: prevalence of plant pathogenic viruses direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach early life dynamics of the human gut virome and bacterial microbiome in infants longitudinal investigation of the faecal microbiota of healthy full-term infants using fluorescence in situ hybridization and denaturing gradient gel electrophoresis. microbiology improved detection of bifidobacteria with optimised 16s rrna-gene based pyrosequencing 16s rrna gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and pcr primer choice gut dna viromes of malawian twins discordant for severe acute malnutrition bacteriophage translocation altered virome and bacterial microbiome in human immunodeficiency virus-associated acquired immunodeficiency syndrome detection and identification of plasma bacterial and viral elements in hiv/aids patients in comparison to healthy adults method for discovering novel dna viruses in blood using viral particle selection and shotgun sequencing the antibody response to bacteriophage phi-x 174 in newborn premature infants tumor-specific bacteriophages induce tumor destruction through activation of tumor-associated macrophages an enteric virus can replace the beneficial function of commensal bacteria efficacy of sterile fecal filtrate transfer for treating patients with clostridium difficile infection bacteriophage transfer during faecal microbiota transplantation in clostridium difficile infection is associated with treatment outcome virus-plus-susceptibility gene interaction determines crohn's disease gene atg16l1 phenotypes in intestine enteric viruses ameliorate gut inflammation via toll-like receptor 3 and toll-like receptor 7-mediated interferon-b production bacteriophages in gut samples from pediatric crohn's disease patients: metagenomic analysis using 454 pyrosequencing study of the viral and microbial communities associated with crohn's disease: a metagenomic approach metagenomic analysis of microbiome in colon tissue from subjects with inflammatory bowel diseases reveals interplay of viruses and bacteria metagenomic analysis of crohn's disease patients identifies changes in the virome and microbiome related to disease status and therapy, and detects potential interactions and biomarkers modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis evaluation of methods to purify virus-like particles for metagenomic sequencing of intestinal viromes the perioperative lung transplant virome: torque teno viruses are elevated in donor lungs and show divergent dynamics in primary graft dysfunction reagent and laboratory contamination can critically impact sequence-based microbiome analyses direct multiplexed whole genome sequencing of respiratory tract samples reveals full viral genomic information analysis of the virus population present in equine faeces indicates the presence of hundreds of uncharacterized virus genomes the marine viromes of four oceanic regions virfinder: a novel kmer based tool for identifying viral sequences from assembled metagenomic data alignment-free d 2 * oligonucleotide frequency dissimilarity measure improves prediction of hosts from metagenomically-derived viral sequences a roadmap to the human virome human viruses: discovery and emergence prophage-like gene transfer agents-novel mechanisms of gene exchange for methanococcus, desulfovibrio, brachyspira, and rhodobacter species transfer of antibiotic-resistance genes via phage-related mobile elements exosome can prevent rnase from degrading microrna in feces q&a: what are exosomes, exactly? membrane vesiclemediated release of bacterial klebsiella pneumoniae subsp. pneumoniae-bacteriophage combination from the caecal effluent of a healthy woman phage tail-like particles kill clostridium difficile and represent an alternative to conventional antibiotics three new escherichia coli phages from the human gut show promising potential for phage therapy lytic bacteriophage pm16 specific for proteus mirabilis: a novel member of the genus phikmvvirus full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm direct sequencing of human gut virome fractions obtained by flow cytometry comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent phaccs, an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information aclame: a classification of mobile genetic elements, update 2010 metavir: a web server dedicated to virome analysis metavir 2: new tools for viral metagenome comparison and assembled virome analysis virome: a standard operating procedure for analysis of viral metagenome sequences molecular methods of virus detection in lymphoma virsorter: mining viral signal from microbial genomic data viromescan: a new tool for metagenomic viral community profiling vip: an integrated pipeline for metagenomics of virus identification and discovery virusdetect: an automated pipeline for efficient virus discovery using deep sequencing of small rnas phaster: a better, faster version of the phast phage search tool virusseeker, a computational pipeline for virus discovery and virome composition analysis defining dysbiosis and its influence on host immunity and disease dysbiosis in inflammatory bowel disease microbiota and neurological disorders: a gut feeling key: cord-009614-lbjesv8y authors: durmuş tekir, saliha d.; ülgen, kutlu ö. title: systems biology of pathogen‐host interaction: networks of protein‐protein interaction within pathogens and pathogen‐human interactions in the post‐genomic era date: 2012-11-29 journal: biotechnol j doi: 10.1002/biot.201200110 sha: doc_id: 9614 cord_uid: lbjesv8y infectious diseases comprise some of the leading causes of death and disability worldwide. interactions between pathogen and host proteins underlie the process of infection. improved understanding of pathogen‐host molecular interactions will increase our knowledge of the mechanisms involved in infection, and allow novel therapeutic solutions to be devised. complete genome sequences for a number of pathogenic microorganisms, as well as the human host, has led to the revelation of their protein‐protein interaction (ppi) networks. in this post‐genomic era, pathogen‐host interactions (phis) operating during infection can also be mapped. detailed systematic analyses of ppi and phi data together are required for a complete understanding of pathogenesis of infections. here we review the striking results recently obtained during the construction and investigation of these networks. emphasis is placed on studies producing large‐scale interaction data by high‐throughput experimental techniques. despite immense technological advances in medicine, pathogenic organisms remain the source of much human morbidity and mortality. hiv/aids, acute lower respiratory tract infections, hemorrhagic fever, diarrheal diseases, tuberculosis and malaria are particularly notorious for high mortality rates [1] [2] [3] . the continuous emergence of new diseases and drug-resistant pathogens has heightened the global burden of infectious diseases in the 21 st century [1, 4] . to tackle such biological threats, an improved understanding of pathogenic microorganisms and their interactions with host organisms is needed since pathogen-host molecular interactions have crucial roles in initiating, sustaining, or preventing infection. pathogenic microorganisms communicate with human cells through interactions with human proteins both on the surface of the cell and within the interior of the cell. these interactions allow the microorganisms to enter the host cell and manipulate cellular mechanisms in order to use the host cell's capabilities to their own advantage, resulting in infection in the host organism. detailed knowledge of pathogen-host protein interactions may enable us to comprehend the mechanisms of infection and to identify better strategies to prevent or cure infection [5, 6] . however, the identification of new drug and vaccine targets for infectious diseases is only possible when the molecular machinery within individual pathogenic and host organisms is understood. for instance, anti-infection therapeutics should target essential genes in the pathogens which have no homology with human genes [7] . the very first genome sequencing was published in 1977 with the dna sequence for the genome of a virus, bacteriophage phix174 [8] . following the sequencing of the bacterial pathogen haemophilus influenzae in 1995 [9] and the human genome in 2000 [10] , sequence data for prokaryotic and eukaryotic genomes have appeared at an accelerated rate. today, genomic data for most of the pathogen and host organisms are available [11] . these data are used to study individual genes and corresponding proteins as well as to identify intra-and interspecies connections between proteins. in the light of these advances, the initial steps towards complete understanding of infection mechanisms through protein interactions have been recently published. in this review, the efforts to systematic determination and analysis of protein interaction networks underlying infection pathogenesis are summarized (mainly in a chronological order) to present the current picture of the research on infectious diseases. from a classical perspective, a protein is a functional unit that specifies a small, but discrete, part of the cellular physiology of an organism. in the post-genomic era, a protein is seen to function as an element within network of its interaction, and its role should be evaluated within this network together with its interacting partners [12] . advances in genomics and proteomics have been followed by the first large-scale efforts to identify functional networks of interacting proteins using the two-hybrid method [13] [14] [15] , pull-down assays [16, 17] , and protein chips [18] . to increase our understanding of the mechanisms of infection, protein-protein interaction (ppi) networks of pathogenic organisms should be determined in order to capture their functional and structural organizations. pathogenic ppi maps reveal biological pathways and processes, allowing prediction of protein functions and discovery of new drug and vaccine targets. the first genome-wide protein interaction networks were determined for viruses [19] [20] [21] . the first large-scale bacterial networks [22] [23] [24] followed successes in eukaryote mapping [15, [25] [26] [27] . today, the genome-wide ppi maps for a number of pathogens and hosts are available in public databases: bind [28] , biogrid [29] , dip [30] , hprd [31] , intact [32] , mint [33] , mips [34] , reactome [35] and string [36] . primarily due to their small genome size, whole genome ppi maps were first constructed for viruses. the first interaction map of whole proteome was determined for escherichia coli bacteriophage t7, mapping 25 interactions among viral proteins [19] . subsequently, genomewide analyses of important human pathogens, hepatitis c virus [20, 37] , vaccinia virus [21] , herpesviruses [38, 39] , and sars coronavirus [40, 41] were performed through intraviral ppi maps. hepatitis c virus (hcv), a flaviviridae family member causing severe liver disease, is a positive-sense singlestranded rna virus. it encodes only a single polyprotein which is co-or post-translationally processed into at least 10 viral proteins [42] . a controlled two-hybrid strategy based on a random genomic hcv library screen was used by flajolet et al. [20] , resulting in the identification of known and novel ppis. interactions among structural and non-structural proteins were revealed in the study, leading to the conclusion that almost all of the viral proteins encoded by the genome function in the hcv life-cycle, as in the cases of other members of the flaviviridae [43] . the roles of these functional interactions were discussed within the framework of the constructed genome-wide interaction map. interacting domains of the viral polyprotein were also identified to shed light on the development of anti-viral agents [20] . another genome-wide ppi map of hcv was then generated for the viral non-structural proteins [37] . vaccinia virus, well-known as a smallpox vaccine and also the source of potential recombinant vaccines against cancer and infectious diseases, is a member of poxviridae family. it is a large, double-stranded dna virus. poxviruses replicate themselves in the cytoplasm of the host cells without depending on the host's transcriptional machinery. the large genome of vaccinia virus can potentially express more than 200 proteins [44, 45] . mccraith et al. [21] performed a comprehensive two-hybrid analysis of full-length vaccinia virus proteins and detected 37 ppis (including 28 novel interactions) among both characterized and uncharacterized proteins. many of the ppis mapped involved one partner which was known to function in a specific process, coupled with another of unknown function, allowing functions to be assigned to previously unannotated proteins within dna replication, transcription, virion structure, or host evasion. another double-stranded dna virus family is herpesviridae whose members encode 70-170 proteins. herpesviruses cause human diseases such as kaposi sarcoma, b-cell lymphomas, chickenpox, shingles, and nasopharyngeal carcinoma [46] [47] [48] . the genome-wide intraviral protein interaction maps for three members of this family, kaposi sarcoma-associated herpesvirus (kshv), varicella-zoster virus (vzv), and epstein-barr virus (ebv) were generated by two-hybrid and analyzed comprehensively to reveal viral network properties [38, 39] . in the work of uetz et al. [38] , 123 ppis for kshv and 173 ppis for vzv were identified, the largest dataset published to date, allowing the construction of the first viral networks. topological network analyses of these interactome maps indicated that the viral networks appear as a single, highly coupled module ( fig. 1 ) with relatively many hubs and few peripheral nodes [38] in contrast to scale-free cellular networks with well-separated functional modules [49, 50] . just after this study was published, calderwood et al. [39] reported the detection of 43 ppis among ebv proteins. the construction of a ppi map for ebv by merging these interactions with already published ones resulted in a network of 52 proteins with 60 interactions. this large-scale network allowed the prediction of functions of uncharacterized proteins, further defining viral mechanisms. in these consecutive studies [38, 39] , core proteins common to all herpesviruses and noncore ones specific to each strain were investigated thoroughly. the severe acute respiratory syndrome coronavirus (sars-cov) is a positive-sense single-stranded rna virus belonging to the family of the largest rna viruses known, coronaviridae. its genome encodes 14 open reading frames expressing up to 30 structural and non-structural proteins that have roles in viral replication, assembly, and other functions for viral amplification in host cells [40, 41, 51] . for a genome-wide analysis of ppis of sars-cov, interactions between all sars-cov proteins were determined [40, 41] by two-hybrid producing 65 and 40 interactions, respectively. intraviral ppis were analyzed to elucidate the functions of the proteins as well as to identify the essential proteins in viral replication. von brunn et al. [40] compared the intraviral network topology of sars-cov with a previously defined viral network [38] and cellular networks [52] [53] [54] , concluding sars-cov network contained similarities to the kshv network [38] . insights gained into molecular mechanisms and topological network properties provided by the genome-wide analyses of intraviral ppi maps (table 1 ) may be used as a basis for further characterization of the functions and mechanisms of viral proteins, especially for other members of the same virus families. having successfully built genome-wide ppi maps for viruses, similar two-hybrid methodology was applied to construct ppi networks for the larger, more complex genomes of pathogenic bacteria. the first prokaryotic ppi map was built for helicobacter pylori [22] . other large-scale prokaryotic networks eventually emerged for campylobacter jejuni [55] , treponema pallidum [56] mycobacterium tuberculosis [57] , and bacillus subtilis [58] . genome-scale analysis of interacting proteins that assemble into protein complexes were performed for e. coli [23, 24] and mycoplasma pneumoniae [59] . the first large-scale intrabacterial ppi map was constructed for the human gastric pathogen, and gram-negative bacterium h. pylori, identifying 1280 interactions between 46.6% of all 261 bacterial proteins using the twohybrid method [22] . the comparison of these h. pylori ppis with previously described interactions between orthologous e. coli proteins resulted in prediction of protein functions within biological pathways such as chemotaxis and urease activity, essential for h. pylori pathogenicity. in this study, the interacting domains of h. pylori proteins were also identified and used in protein function predictions. interacting domains may serve in mapping new functional domains, providing crucial information for antibacterial drug design studies. gram-negative bacterium e. coli, the main cause of urinary tract infections and a model bacterial system, is one of the best characterized and early studied organisms [60] [61] [62] . however, any large-scale analysis of protein complexes in e. coli was not performed until the studies of butland et al. [23] and arifuzzaman et al. [24] . first, 716 binary interactions involving 83 essential and 152 nonessential proteins, were identified by pull-down assay using tandem affinity purification-mass spectrometry, targeting 1000 orfs (about one-quarter of the e. coli genome) [23] . a small number (15%) of these interactions were already available in dip, bind, and string. ten newly described e. coli ppis were found as orthologous to the interactions reported for h. pylori [22] . the novel interactions were analyzed for functional annotations of uncharacterized proteins, allocating them within ribosome function, rna processing, rna binding, and so on. the graph theoretical analysis of the ppi map of e. coli revealed scale-free behavior and a high correlation between connectivity and the degree of conservation. the genome-wide ppi map of e. coli k-12 strain with 11 511 interactions among 2667 proteins was then constructed by a similar method [24] . the comprehensive analysis of this large-scale network also validated the scale-free nature and the connectivity-conservation correlation found previously [23] . arifuzzaman et al. [24] identified 107 functional units which have roles in metabolic pathways, transcriptional and translational machinery, recombination and flagella assembly. analysis of ppis based on this functional unit categorization provided further functional annotations. the gram-negative, food-borne pathogen c. jejuni is the major cause of gastroenteritis. the proteome-level analysis revealed 11687 interactions involving 80% of 1654 c. jejuni proteins [55] , the most comprehensive bacterial ppi map determined by two-hybrid. a scale-free network was obtained, removing low confidence-scored interactions. this ppi map of c. jejuni was used to identify evolutionarily conserved subnetworks through comparison with protein networks of h. pylori [22] , e. coli [23] and saccharomyces cerevisiae in dip. further analyses of the identified conserved sub-networks allowed the prediction of new c. jejuni interactions using orthologous interactions. this comparative analysis also enabled the identification of essential c jejuni genes based on their orthology to essential genes in other organisms. this comprehensive interactome data were next used to predict protein roles and to map functional pathways such as chemotaxis. the causative agent of syphilis, t. pallidum, has one of the smallest genomes known in extracellular bacteria, encoding 1039 proteins [63] . the global ppi network of t. pallidum, involving 3649 interactions connecting 726 bacterial proteins, was identified by two-hybrid [56] . the high-confidence subset connects 576 proteins by 991 interactions. in that study, an integrated network of dna-metabolism related processes was constructed and 18 proteins were functionally annotated within this network. additionally, various orthologous interactions were predicted for completely sequenced genomes, allowing the description of phylogenetically conserved interaction patterns. atypical pneumonia causing human pathogen, m. pneumoniae also has one of the smallest genomes in self-replicating organisms with 689 protein-encoding genes, making it a good model organism to study proteome organization in prokaryotes [64] . a proteome-wide analysis was performed by tandem affinity purificationmass spectrometry, identifying 62 homo-multimeric and 116 hetero-multimeric protein complexes [59] . about a third of the found hetero-multimeric complexes were observed to interact with proteins forming 35 larger, multiprotein complexes implying higher level of proteome organization and protein multifunctionality, allowing functional annotations of assemblies as well as prediction of biological roles of individual proteins within the complexes. m. tuberculosis causes millions of deaths each year with tuberculosis infection [65] . after computational efforts to construct large-scale ppi maps of m. tuberculosis [66, 67] , its genome-wide network was identified experimentally by two-hybrid [57] . this global network is composed of 8 042 interactions among 2907 proteins which represent 74.1% of the whole proteome. the topological properties of the undirected network of these interactions were calculated and compared with those of the previously defined prokaryotic ppi networks [22-24, 55, 56] . similar scale-free behavior following a power-law distribution was observed. in fact, the networks obtained by pull-down assay [23, 24] differ in values of clustering coefficient from the networks obtained by two-hybrid analysis [22, 55, 56] . moreover, wang et al. [57] performed a cross-species network comparison analysis of m. tuberculosis interactions with the available large-scale ppi data [22-24, 55,56] and identified conserved sub-networks. additionally, the highly connected critical proteins and mechanisms of the protein secretion pathways which have roles in its pathogenesis were revealed. a large-scale ppi network was recently constructed for the gram-positive bacterium b. subtilis (which is rarely pathogenic) by two-hybrid [58] . this network of 793 interactions involves 287 bacterial proteins. due to its role as a model organism, many studies were performed to characterize the biological functions of its ppis in cellular processes [68] [69] [70] . however, many processes remained uncharacterized. hence marchadier et al. [58] performed a comprehensive analysis with the integration of transcriptomic data focusing on cell division, cell responses to stresses, the bacterial cytoskeleton, dna replication and chromosome maintenance. these sequential efforts on construction of large-scale ppi networks for prokaryotes (table 1) constitute the first comprehensive description of the intraspecies mechanisms of the bacterial pathogens. the protozoan pathogen plasmodium falciparum causes malaria which results in deaths of nearly a million of people each year [71] . a comprehensive protein interaction map of this pathogen was generated by two-hybrid, identifying a highly interconnected, scale-free network of 2823 interactions within 1267 proteins (~25% of the predicted p. falciparum proteins) [72] . in this network, 33% of the interactions are between two uncharacterized proteins whereas 49% of the interactions include one such protein. bioinformatic analysis of this network yielded functional annotations of the proteins within the processes; chromatin modification, transcription, messenger rna stability, ubiquitination, and invasion of host cells. more detailed studies of ppis within p. falciparum are required in order to unravel its pathogenesis mechanisms thoroughly. despite the increasing rate in the identification of genome-wide ppi networks, they remain unconstructed for most pathogens. in the light of accelerating advances in genomics, proteomics, and interactomics, large-scale maps for many more organisms are expected to be built in the near future. increasing numbers of ppi networks will allow the comparison of networks across diverse organisms, resulting in generalized conclusions about pathogenic molecular mechanisms. the first examples of such comparative studies have been highlighted in the sections 2.1 and 2.2 above. integration of several highthroughput interaction datasets to generate more detailed networks is also possible, as indicated by recent examples for the e. coli system [73, 74] . the frequency of such integrated networks is expected to increase, owing to the large number of diverse data sets. these will be invaluable in defining whole proteomic maps of the pathogens. one of the most striking results of bioinformatic analyses on the constructed ppi maps is the identification of essential proteins functioning within pathogens. these proteins should be examined thoroughly to test their potential as novel therapeutic targets. the exploration of genome-wide ppi maps of the pathogens permits the assignment of unannotated proteins to biological pathways with function prediction. the proteins annotated to the host invasion processes may provide a launching point for pathogen-host interaction studies. biochemical interactions of pathogens with their hosts are necessary to invade the host organism. these connections between pathogens and hosts include interactions between proteins, nucleotide sequences, and small ligands [75, 76] . however, the protein interactions of pathogen-host systems have been identified as the most important, and therefore the most studied, type of pathogen-host interactions (phis) [76, 77] . since these interspecies crosstalks determine the pathogenesis, focusing on the whole phi system, instead of investigating a pathogen or host individually, may allow us to capture critical mechanisms (i.e. strategies used by pathogens and host immune responses) during infection that cannot be provided by traditional methods. due to a lack of sufficient experimental phi data until recent years, many computational phi prediction methods have been developed [78] [79] [80] [81] [82] [83] [84] . these studies focused mainly on interactions of p. falciparum and human immunodeficiency virus (hiv), as these are some of the most threatening pathogens to humans. very recently experiments have been carried out to determine the first large-scale molecular interactions between human and viruses [39, 85, 86] and bacteria [87, 88] . as a result of an increase in data available for pathogen-host systems, phispecific databases have been introduced such as phibase [89] , virusmint [90] , virhostnet [91] , patric [92] , and phisto [93] . although these advances in data archiving are promising, most data relevant to phi are still buried in the biomedical literature. some rare efforts have been performed to obtain hidden phis from the literature by text mining [94] [95] [96] . as in the case of intraspecies pathogen ppis, large-scale phi data were generated for viral systems before bacterial systems ( table 2 ). the first examples are for commonly observed human pathogens, ebv [39] , hcv [85] and influenza a virus (h1n1 and h3n2) [86] and then recently for hiv [97] . in calderwood et al. [39] , protein interactions between herpesvirus, ebv and human were mapped by twohybrid in conjunction with ebv intraviral ppi mapping, providing 173 phis between 40 ebv proteins and 112 human proteins. a systematic analysis of these interactome maps of ppis and phis enabled hypotheses of the roles of ebv proteins in pathogenesis to be generated. furthermore, intraspecies protein interaction data for human were integrated from databases (bind, dip, hprd, mips) and from the literature [52, 53] to analyze the organization of the human proteins targeted by ebv within human molecular machinery. it was found that ebv proteins tend to target human proteins which are highly connected (hubs) and central to many paths (bottlenecks) in the human ppi network. on the other hand, the degree distribution of the ebv-human protein interaction network could not be fitted to any model because of its incompleteness (fig. 2) . attempts to analyze incomplete maps of ppis and phis are still able to supply a partial understanding of mechanisms underlying infection. a similar thorough analysis was earlier performed with herpesviral protein networks of kshv and vzv and their interaction with the human proteome [38] . in that study, protein interactions between herpesviruses and human were predicted using the interacting orthologs of both proteins in other organisms [54] . combined virus-human networks were constructed by starting with the viral networks, adding their human protein targets, and then adding the cellular interactions among the targeted human proteins. the topological analyses of the combined herpesviruses-human networks revealed distinct properties from both viral and human interactomes providing insights into the impact of the two organisms on each other [38] . a proteome-wide phi map for the flavivirus hcv was mapped by two-hybrid and then by literature mining of previously found interactions between hcv and human [85] . a map of 481 interactions between 11 hcv proteins and 421 human proteins was generated (314 phis by twohybrid). 65% of this phi network included novel interactions. the integrated human network of 44 223 ppis among 9520 proteins [98] was used to evaluate the interplay between hcv and human. very similar behavior to ebv [39] was observed for hcv in terms of attacking hub and bottleneck proteins in the human network. to assess the human pathways targeted by hcv, kegg functional annotation pathways [99] were used. four pathways were detected to be enriched in hcv-targeted human proteins. three of them were associated already with hcv clinical www.biotechnology-journal.com syndromes as insulin, tgf-β and jak/stat pathways. the last enriched pathway, focal adhesion, is a novel observation as a human pathway affected during hcv infection [85] . influenza a is a member of negative-sense singlestranded viruses of orthomyxoviridae family. it is the sources of all flu pandemics infecting multiple species. for h1n1 a/pr/8/34 strain of influenza virus, 31 intraviral ppis among 10 viral proteins and 135 phis between 10 viral and 87 human proteins, most of which are expressed in primary human bronchial cells, were detected by twohybrid [86] . some of the phis constructed had been published previously [100] . the topology of the constructed intraviral network revealed a highly interconnected nature, as observed previously for other viral networks [38, 101] . in the case of the influenza a-human interaction network, important properties about connectivity of proteins were observed. first, viral proteins interact with significant number of human proteins, reflecting the multifunctionality of the small number proteins encoded in rna viruses. second, each of 24 human proteins connects with two or more viral proteins forming virus-human multiprotein complexes. additionally, it was observed that viral proteins generally target human proteins which are highly connected within their own network, as it was the case in herpesviruses-human system [39] . in shapira et al. [86] another phi network was identified for strain of influenza virus, h3n2 a/udorn/72 by the same experimental approach. this phi network consists of 81 interactions between 10 viral and 66 human proteins, reflecting a similar nature to the network for h1n1 strain-human system. this confirms the conserved functions of influenza virus proteins through strains. besides direct physical interactions between viral and human proteins, host responses in bronchial cells to influenza infection was identified by expression profiling, generating a regulatory map of interactions between influenza proteins and their human targets. comprehensive analysis of the physical and regulatory maps of the phi system elucidated human mechanisms involved in infection. for example, nf-κb, mitogen-activated protein kinase, apoptosis, and wnt signaling pathways are regulated through transcriptional and/or physical interactions during influenza a infection. one of the most dangerous human pathogens, hiv, belongs to positive-sense single-stranded rna virus family retroviridae. acquired immunodeficiency syndromecausing hiv has been extensively studied since its first observation near the end of the 20 th century [102] [103] [104] [105] . similar to other rna viruses, hiv has a small genome and depends largely on human cellular machinery to be replicated. identifying the physical contacts between hiv and human proteins during hiv replication is critically important for a full understanding of hiv infection. being one of the most studied pathogens, there are many phi data for hiv-1 in virusmint and phisto. the current phi data have been produced mainly by small-scale experiments [106] [107] [108] . very recently, a global phi network was generated for hiv-human protein complexes by affinity tagging and purification mass spectrometry, producing 497 phis between 16 hiv-1 proteins and 435 human proteins [97] . it was observed that hiv-targeted human proteins are highly conserved across primates. the novel interactions identified in that study requires further work to detail their biological significance in terms of hiv infection. besides whole proteins, domains of the interacting proteins were investigated and the enriched domain types in targeted human proteins were indicated for facilitating future structural modeling studies regarding hivhuman system. the first large-scale interaction networks between viruses and humans [39, 85, 86, 97] provide crucial clues about the viral infections, verifying the critical importance of phi analyses in infection researches. until very recent years, the phi data were scarce for bacterial systems because of lack of any large-scale experiments. the first extensive bacterial phi networks were identified for important human pathogens, bacillus anthracis, francisella tularensis, and yersinia pestis [87] , then another high-throughput experimental study generating phi data of y. pestis was reported [88] . gram-positive bacteria b. anthracis and y. pestis and gram-negative bacterium f. tularensis are respiratory pathogens causing anthrax, bubonic plague, and acute pneumonic disease, respectively. using a two-hybrid assay, large-scale interaction data were generated between these bacteria and human producing 3073 phis between 943 b. anthracis proteins and 1748 human proteins, 4059 phis between 1218 y. pestis proteins and 2108 human proteins, and 1383 phis between 349 f. tularensis proteins and 999 human proteins [87] . the first conclusion of computational analyses of these comprehensive bacteria-human networks, in combination with the integrated human ppi network from databases bind, dip, hprd, intact, mint, mips, and reactome, was that bacterial proteins tend to target hubs and bottlenecks in the human network. secondly, the roles of human proteins targeted by these bacteria were investigated using their gene ontology annotations [109] . the tendency of all three pathogens to target human proteins involved in immune responses was observed as previously reported [110] [111] [112] . besides being effectors of immune signaling, the bacteria-targeted human proteins also have crucial roles in apoptosis [87] . thirdly, the conserved protein interaction modules of the three phi networks were computed [113, 114] for a more systematic comparative analysis. conserved modules revealed common attacks by the bacterial pathogens to same human pathways. subsequently, another phi map was generated for plague causing y. pestis by a different two-hybrid strategy by choosing only potential virulence factors as bait pro-teins [88] . 204 phis were yielded between 66 y. pestis proteins and 109 human proteins and then 23 previously reported phis were integrated to construct a comprehensive network between y. pestis and human. a graph theoretic analysis confirms that y. pestis preferentially targets hub and bottleneck proteins in the human intranetwork as concluded previously for viruses [39, 86] and bacteria [87] . signaling pathways, crucial for human immune system, were found to be enriched in human proteins targeted by y. pestis. these pathways include mitogen-activated protein kinase signaling and toll-like receptor signaling and also pathways functioning in focal adhesion, regulation of cytoskeleton, and leukocyte transendoepithelial migration. finally, y. pestis-targeted human proteins were compared with those targeted by viruses whose phi networks were identified previously. 16 of 109 y. pestis-targeted human proteins are included in phi networks of ebv [39] and hcv [85] indicating the common infection strategies of both viruses and bacteria. the recent detected first large-scale phi networks of bacteria-human systems [87, 88] contribute largely to the understanding of bacterial infection mechanisms with immune evasion. as phi data available for various pathogens increase, a need to analyze comprehensive phi data for all pathogen types together arises in order to draw a generalized picture. although infection mechanisms of individual pathogens have been studied through intraspecies pathogenic ppi maps and interspecies phi maps, a general overview of infection mechanisms was missing until analyses of phi data from different infection agents were attempted [6, 93] . in the absence of large-scale phi networks for bacterial, protozoan and fungal systems, dyer et al. [6] performed the first global analysis of 10 477 protein interactions between 190 pathogen strains of viruses, bacteria, protozoa, and human through properties of targeted 1233 human proteins. diversity of the available phi data was not rich, 98.3% of 10 477 phis belonged to the virushuman systems with 77.9% of the interaction data drawn from hiv -human interaction systems. the importance of the pathogen-targeted proteins was evaluated within the intraspecies human ppi network of 75 457 interactions. these phi and ppi data were integrated from public databases; mint, intact, dip, hprd, reactome, bind and mips. firstly, targeting hub and bottleneck proteins was concluded to be global behavior for all pathogens, as reported for individual pathogen strains previously [39, [86] [87] [88] . gene ontology [109] functions enriched in the targeted human proteins by different pathogens revealed common infection mechanisms. attack of human transcription factors and key proteins that control the cell cycle and regulate apoptosis and transport of genetic material across the nuclear membrane were found to be among the common viral strategies. despite its scarcity (174 interactions in the datset), bacterial phi data allowed identification of specific human proteins that function in the host immune response (via toll-like receptors and i-κb kinase/nf-κb signaling cascade) as a target of bacterial infection strategy [6] . recently we performed another study with comprehensive phi data to explore common and special infection strategies for viruses and bacteria [93] . a significant amount of bacterial phis, constituting 36.5% of all data, was avaiable thanks to dyer et al. [87] . we analyzed 23 435 interactions between 3419 proteins of viral, bacterial, protozoan and fungal pathogens (totally 257 strains) and 5210 proteins of human obtained from phisto (www.phisto.org). to generate the intra species human protein network, 194006 ppis were integrated from biogrid, dip, intact, mint and reactome. the significant amount of bacterial and viral phi data allowed us to focus on comparisons between their specific infection mechanisms. firstly, attacking hub and bottleneck proteins in the human ppi network was verified as a common infection strategy of both bacteria and viruses. furthermore, viruses were observed to target human proteins of much higher connectivity and centrality values in comparison to bacteria. secondly, gene ontology enrichment analysis of the targeted human proteins verified the special mechanisms of bacteria and viruses use to manipulate of human immune defense mechanisms and cellular processes, respectively (as reported in dyer et al. [6] but relying on lower amounts of phi data). a first attempt at the investigation of the human proteins targeted by both bacteria and viruses revealed that attacking human metabolic processes is a common strategy used by both pathogens during infections [93] . global analysis of phi data provides insights into the strategies adapted by bacteria and viruses to subvert human cellular processes and immune system for the infection. however, large-scale phi networks for pathogens other than bacteria and viruses are still undetermined, leaving their pathogenesis mechanisms to be relatively uncharacterized. research on infectious diseases through phis has accelerated within the post-genomic era (fig. 3) . however, large-scale phi networks have been infrequently studied. efforts to identify and analyze large-scale phis for diverse pathogen types would be expected to parallel the acceleration of biotechnology and bioinformatics research. increasing amounts of data available will allow more complete data sets to be compiled, resulting in characterization of topological properties of phi networks. the first attempt to fit the degree distribution of ebv-human interaction network failed due to scarcity of data [39] . on the other hand, bioinformatic analyses of the pathogen-tar-geted human proteins succeeded in unraveling some infection strategies such as targeting human hubs and bottlenecks, subverting cellular processes for the usage of pathogens' own advantages and evasion of immune defenses [6, 39, 85, 87, 88, 93] . the huge amount of data expected to be generated for phi systems will enable us to capture all details of infection processes. potentially leading to the development of new and more efficient therapeutics. conventional treatments for infectious diseases often aim to kill pathogens by targeting their essential proteins. this approach unfortunately forces the pathogens to evolve for survival and consequently selects resistant strains (especially in the case of rna viruses with a high mutation rate). to fight drug-resistant patho gens, novel alternative therapeutics are emerging which target host proteins required by pathogens to replicate and persist within the host organism. if these host factors are indispensable for pathogens, but not essential for host cells, their silencing may inactivate pathogenic activity, allowing them to serve as therapeutic targets [4, 115] . in the light of phi studies, some human factors required by viral and bacterial pathogens have been determined for hiv [115] [116] [117] [118] [119] , hcv [120] , west nile virus [121] , influenza virus [122, 123] , and m. tuberculosis [124] in recent years. despite the efforts reviewed here, the use of systems biology approaches to investigate phi is still considered relatively undeveloped. the availability of new phi network data, together with further topological and functional analyses of pathogen-host systems, are expected to shed more light on infection mechanisms and novel therapeutic targets for infectious diseases in the near future. we particularly thank dr. tunahan çakır for critical reading of the manuscript and for his contributions to figure 3 . the financial support was provided by the research funds of bogaziçi university, through project 5554d. the doctoral scholarship for saliha durmuş tekir is sponsored by tübitak, is gratefully acknowledged. the authors declare no conflict of interest. figure 3 . the number of scientific publications including phi-related terms in pubmed in the post genomic era. the searched phi-related terms: "pathogen host interactions", "host pathogen interactions", "pathogen host interaction", "host pathogen interaction", "pathogen-host interactions", "pathogen-host interaction", "host-pathogen interactions", "host-pathogen interaction". infectious diseases: for considerations the 21st century host-pathogen systems biology filoviruses are ancient and integrated into mammalian genomes new strategies to fight infectious diseases -arms race on a microscale signaling during pathogen infection the landscape of human proteins interacting with viruses and other pathogens antibacterial drug discovery and structure-based design nucleotide sequence of bacteriophage ϕx174 dna whole-genome random sequencing and assembly of haemophilus influenzae rd the sequence of the human genome protein function in the post-genomic era a novel genetic system to detect protein-protein interactions interaction mating reveals binary and ternary connections between drosophila cell cycle regulators toward a functional analysis of the yeast genome through exhaustive two-hybrid screens functional organization of the yeast proteome by systematic analysis of protein complexes systematic identification of protein complexes in saccharomyces cerevisiae by mass spectrometry global analysis of protein activities using proteome chips a protein linkage map of escherichia coli bacteriophage t7 a genomic approach of the hepatitis c virus generates a protein interaction map genome-wide analysis of vaccinia virus protein-protein interactions the protein-protein interaction map of helicobacter pylori interaction network containing conserved and essential protein complexes in escherichia coli largescale identification of protein-protein interaction of escherichia coli k-12 toward a protein-protein interaction map of the budding yeast: a comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins a comprehensive analysis of protein-protein interactions in saccharomyces cerevisiae protein interaction mapping in c. elegans using proteins involved in vulval development the biomolecular interaction network database and related tools: 2005 update the biogrid interaction database: 2011 update the database of interacting proteins: 2004 update human protein reference database the intact molecular interaction database in 2012 mint, the molecular interaction database: 2012 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of the succinyl-coa synthetase of escherichia coli one-step inactivation of chromosomal genes in escherichia coli k-12 using pcr products new partners of acyl carrier protein detected in escherichia coli by tandem affinity purification complete genome eequence of treponema pallidum, the syphilis spirochete complete sequence analysis of the genome of the bacterium mycoplasma pneumoniae epidemiology, strategy, financing mycobacterium tuberculosis interactome analysis unravels potential pathways to drug resistance uncovering new signaling proteins and potential drug targets through the interactome analysis of mycobacterium tuberculosis an expanded view of bacterial dna replication dna polymerase i acts in translesion synthesis mediated by the y-polymerases in bacillus subtilis cell-cycle-dependent spatial sequestration of the dnaa replication initiator protein in bacillus subtilis chemical genetics of plasmodium falciparum a protein interaction network of the malaria parasite plasmodium 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sequence motifs structural similarity-based predictions of protein interactions between hiv-1 and homo sapiens hepatitis c virus infection protein network a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection the human-bacterial pathogen protein interaction networks of bacillus anthracis, francisella tularensis, and yersinia pestis insight into bacterial virulence mechanisms against host immune response via the yersinia pestis-human protein-protein interaction network phi-base update: additions to the pathogen host interaction database virus-mint: a viral protein interaction database virhost-net: a knowledge base for the management and the analysis of proteome-wide virus-host interaction networks patric: the comprehensive bacterial bioinformatics resource with a focus on human pathogenic species infection strategies of bacterial and viral pathogens through pathogen-human protein-protein interactions document classification for mining host pathogen protein-protein interactions text mining for discovery of host-pathogen-interactions literature mining of hostpathogen interactions: comparing feature-based supervised learning and language-based approaches global landscape of hiv-human protein complexes analysis of the human protein interactome and comparison with yeast, worm and fly interaction datasets gene annotation and pathway mapping in kegg the multifunctional ns1 protein of influenza a viruses connecting viral with cellular interactomes gay compromise syndrome how does hiv cause aids? science hiv-1 nef impairs mhc class ii antigen presentation and surface expression hiv-1 tar mir-na protects against apoptosis by altering cellular gene expression anchorage of hiv on permissive cells leads to coaggregation of viral particles with surface nucleolin at membrane raft microdomains human immunodeficiency virus type 1 vpr interacts with antiapoptotic mitochondrial protein hax-1 hiv-1 envelope triggers polyclonal ig class switch recombination through a cd40-independent mechanism involving baff and c-type lectin receptors gene ontology: tool for the unification of biology francisella tularensis induces cytopathogenicity and apoptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication macrophage apoptosis by anthrax lethal factor through p38 map kinase inhibition inhibition of mapk and nf-kb pathways is necessary for rapid apoptosis in macrophages infected with yersinia graemlin: general and robust alignment of multiple large interaction networks conserved patterns of protein interaction in multiple species network-based prediction and analysis of hiv dependency factors identification of host proteins required for hiv infection through a functional genomic screen global analysis of hostpathogen interactions that regulate early-stage hiv-1 replication genome-scale rnai screen for host factors required for hiv replication host cell factors in hiv replication: meta-analysis of genome-wide studies a genome-wide genetic screen for host factors required for hepatitis c virus propagation rna interference screen for human genes associated with west nile virus infection genome-wide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication genome-wide analysis of the host intracellular network that regulates survival of mycobacterium tuberculosis key: cord-007735-ejvv2lxv authors: bowdish, d. m. e.; davidson, d. j.; hancock, r. e. w. title: immunomodulatory properties of defensins and cathelicidins date: 2006 journal: antimicrobial peptides and human disease doi: 10.1007/3-540-29916-5_2 sha: doc_id: 7735 cord_uid: ejvv2lxv host defence peptides are a conserved component of the innate immune response in all complex life forms. in humans, the major classes of host defence peptides include the αand β-defensins and the cathelicidin, hcap-18/ll-37. these peptides are expressed in the granules of neutrophils and by a wide variety of tissue types. they have many roles in the immune response including both indirect and direct antimicrobial activity, the ability to act as chemokines as well as induce chemokine production leading to recruitment of leukocytes to the site of infection, the promotion of wound healing and an ability to modulate adaptive immunity. it appears that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, t cells and epithelial cells. the importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides showgreater susceptibility to bacterial infections. in the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents. that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, t cells and epithelial cells. the importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides show greater susceptibility to bacterial infections. in the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents. human neutrophil peptide hbd human beta defensin tlr toll-like receptor lps lipopolysaccharide lta lipoteichoic acid tnf-α tumour necrosis factor alpha mhc1 major histocompatibility complex class 1 bal bronchoalveolar lavage 1 overview host defence peptides are small (generally less than 50 amino acids), positively charged peptides that are an evolutionarily conserved component of the innate immune response. originally characterised as natural antimicrobial agents, it is becoming increasingly apparent that these peptides have a wide range of immunomodulatory properties that are either complementary to, or independent of, antimicrobial activity. interest in the immunomodulatory functions of these peptides is increasing, and indeed many peptides and proteins with similar characteristics to host defence peptides have been found to have either antimicrobial or immunomodulatory properties in addition to their primary functions. in humans the best-characterised host defence peptides are the defensins and the sole cathelicidin, hcap-18/ll-37. the amino acid sequences of these peptides are summarised in table 1 . in general, the defensins are between 29 and 30 amino acids long (approximately 3.5 kda) and contain three conserved disulphide bridges (white et al. 1995) . the genes for all human defensins are clustered on chromosome 8 (sparkes et al. 1989; maxwell et al. 2003) . they are further subdivided to include the αand β-defensins, a distinction based on the organisation of the three characteristic cystine disulphide bonds. the canonical sequence of the α-defensins is x 1-2 cxcrx 2-3 cx 3 ex 3 gxcx 3 gx 5 ccx 1-4 , where x represents any amino acid residue. these peptides are rich in cysteine, arginine, and aromatic residues . the cysteines are linked 1-6, 2-4, and 3-5. initially these peptides were isolated from the neutrophils and are thus called human neutrophil peptides (hnp)-1 to -3 . the hnps are expressed at the transcriptional level in the bone marrow, spleen and thymus, where they co-localise with peripheral blood leukocytes (zhao et al. 1996) . the three hnps are highly homologous, differing by only one amino acid at the nh 2 terminus. because of the high sequence similarity and difficulties in purifying the individual peptides as well as the high degree in functional similarity, the hnp1-3 are often studied as a group, although certain studies have demonstrated differences in their antimicrobial ) and immunomodulatory activities (chertov et al. 1996) . hnp4 was identified as a hnp due to its structural homology to the hnp1-3 (wilde et al. 1989 ). this gene differs from the other genes of this family by an extra 83-base pair segment that is apparently the result of a recent duplication within the coding region (palfree et al. 1993) . as with the other hnps, hnp4 is found in the neutrophils, but is also called corticostatin because it exhibits corticostatic activity and inhibits corticotrophin-stimulated corticosterone production (singh et al. 1988) . despite its variation from the conserved sequences of hnp1-3, it appears to have much more potent antimicrobial activity (wilde et al. 1989) . two other α-defensins, hd5 and hd6, are found solely in the intestinal tract. hd5 and hd6 were found to be expressed at the transcriptional level solely in the small intestine and in situ hybridisation demonstrated that this expression occurs in the paneth cells bevins 1992, 1993) . southern blot analysis using a nucleotide probe for the conserved signal sequence of the defensins indicated that a number of genes with high homology to hnps exist within the human genome. the β-defensins are expressed in a variety of tissue types, including epithelial cells from the trachea and lung, in the salivary and mammary glands, in a variety of organs such as in the plasma and skin (bensch et al. 1995; zhao et al. 1996; harder et al. 1997; reviewed in lehrer and ganz 2002) . the expression of certain β-defensins is inducible upon stimulation with bacterial components or pro-inflammatory cytokines and thus these peptides are presumed to be an important component of host defence to infection or inflammation. the canonical sequence for the beta defensins is x 2-10 cx 5-6 (g/a) x cx 3-4 cx 9-13 c x4-7 ccx n . the best characterised members of the β-defensin family are hbd1-3; however, the antimicrobial properties of hbd4 have been recently published (garcia et al. 2001 ) and over 20 potential β-defensin homologues have been identified in the human genome based on sequence similarity to hbd1-4 (schutte et al. 2002) . the cathelicidins are an evolutionarily conserved family of host defence peptides which are found in cows, sheep, guinea pigs, rabbits, mice, and primates (reviewed in zanetti 2004) and are characterised by having an evolutionarily conserved n-terminal domain called the cathelin domain. in addition, these peptides have a signal sequence, which is believed to target their delivery to the secondary granules of neutrophils. the c terminal domain, which is released by cleavage of proteases, has both antimicrobial and immunomodulatory properties. despite the conserved nature of the cathelin domain, its function remains unclear, although it has been proposed to block the antimicrobial activity of the cleaved product, presumably as a mechanism which allows storage of the peptide in its inactive form , and there is some evidence that it has anti-protease activity . the sole human cathelicidin ll-37/hcap-18 is found at high concentrations in its unprocessed form in the granules of neutrophils and is processed upon degranulation and release (sorensen et al. 2001) . consequently, it is found at sites of neutrophil degranulation. it is also produced by epithelial cells and is found in a number of tissues and bodily fluids, including gastric juices, saliva, semen, sweat, plasma, airway surface liquid and breast milk (bals et al. 1998c; murakami et al. 2002b; hase et al. 2003; murakami et al. 2005) . generally epithelial cells have been shown to produce the hcap-18 form. although the hcap-18 has been shown to be cleaved by the neutrophil protease, protease 3, when released from neutrophils, it is not entirely clear how or when hcap-18 is cleaved when it is produced by epithelial cells. there are a variety of processed forms of hcap-18 that result from as-yet uncharacterised cleavage processes. for example, a 6-kda form is found in gastric juice (hase et al. 2003) , while numerous cleavage products are found in the sweat (murakami et al. 2002b) . as well, hcap-18 from semen is cleaved to a 38-amino acid antimicrobial peptide all-38 in the vagina, thus potentially providing some antimicrobial protection after sexual intercourse ). there appears to be some overlapping, complementary and possibly even enhanced antimicrobial activity of these isoforms (murakami et al. 2004) ; however, to date there is no information about their immunomodulatory properties. due to the high homology of hnp1-3, they are often classed as a group. the hnps are found exclusively in leukocytes and at their highest concentrations in neutrophils where they are localised to azurophilic granules. the recently described hnp4 is also found in azurophilic granules but at much lower concentrations (wilde et al. 1989) . to date there has been no indication that their expression levels can vary substantially. neutrophils stimulated with il-8, fmlp or phorbol 12-myristate 13-acetate causes degranulation and release of hnp1-3 (chertov et al. 1996) , thus it is believed that relatively high concentrations of these peptides are present at sites of infection or inflammation. interestingly, in the course of infection hnp levels increase systemically. although the mechanism is not entirely clear, the plasma levels of defensins increase significantly in patients experiencing septicaemia or meningitis (from approximately 40 ng/ml to as much as 170,000 ng/ml). the concentration of neutrophil defensins generally correlates with that of il-8, a potent chemoattractant for neutrophils (ashitani et al. 2002) , as well as the presence of other neutrophil components such as elastase (zhang et al. 2002) . presumably this occurs due to neutrophil degranulation in response to bacterial or pro-inflammatory stimuli as concentrations decrease after antibiotic treatment (panyutich et al. 1993) . hd5 and hd6 are expressed throughout the gastrointestinal tract, with the highest levels of expression occurring in the jejunum and ileum (dhaliwal et al. 2003) . specifically, immunohistochemical studies have shown that hd5 is expressed by paneth cells, some villous epithelial cells and in the terminal ileal mucosa (cunliffe et al. 2001) . although there is great variability between individuals, levels of these defensins are increased in certain disease states such as acute coeliac sprue (frye et al. 2000b) and are decreased in others such as hiv-related cryptosporidiosis (kelly et al. 2004 ). in patients with crohn's disease or ulcerative colitis, but not in healthy individuals, hd5 immunoreactive cells are present in the crypt region of a large proportion of colonic samples, indicating that expression in these disease states might be dysregulated (cunliffe et al. 2001) . limited studies have demonstrated that hd5 and hd6 may also be present at low levels in airway epithelial cells (frye et al. 2000a) or the female reproductive tract (quayle et al. 1998 ). the patterns of expression of the β-defensins are markedly different. in general hbd1 is not up-regulated during the course of infection or inflammation or by stimulation with pro-inflammatory cytokines or bacterial components and in many cases the presence of hbd1 is detectable at the transcriptional level but is undetectable at the protein level (o'neil et al. 1999; frye et al. 2000b) . however its constitutive presence in airway surface liquid, in intestinal and colon cell lines and in other tissues and bodily fluids implies that it may be involved in maintenance and homeostasis of these areas (salzman et al. 2003) . the expression levels of hbd1 appear to be quite low, ranging from the pg/ml to ng/ml levels in most bodily fluids. hbd2 is an inducible host defence peptide whose expression is altered under both infectious and inflammatory conditions. it has been found to be up-regulated by pro-inflammatory stimuli in oral epithelial cells and keratinocytes (krisanaprakornkit et al. 2000) , in intestinal and colonic epithelial cell lines (o'neil et al. 1999; ogushi et al. 2001; vora et al. 2004 ) and in various lung epithelial cell lines (singh et al. 1998; becker et al. 2000) . hbd2 is believed to be an important component of host defences, since hbd2 expression is depressed in patients with atopic dermatitis who often present with cases of acute and chronic colonisation by staphylococcus aureus (ong et al. 2002) but is increased in psoriatic skin, a disease in which patients are fairly resistant to bacterial infection. it has been demonstrated to be upregulated by both commensal and pathogenic bacteria in the oral mucosa and keratinocytes, although by different mechanisms (krisanaprakornkit et al. 2002; chung and dale 2004) . hbd2 is inducible during the course of inflammation and infection in the gastrointestinal system, as observed at both the mrna and protein levels. hbd2 expression in intestinal and colonic epithelial cell lines is increased upon stimulation with il-1α, flagellin or bacteria, in an nf-κb-dependent manner (o'neil et al. 1999; ogushi et al. 2001) , and by either lipopolysaccharide (lps) or lipoteichoic acid (lta) in a tlr4 and tlr2-dependent manner (vora et al. 2004) . interestingly, other inflammatory mediators such as tnf-α and lps do not induce hbd2 up-regulation (o'neil et al. 1999) . this may reflect a predominantly intracellular expression pattern of tlr4 in these cells, which has been suggested to be an evolutionary adaptation to the high bacterial load in the intestine (naik et al. 2001; hornef et al. 2002) . increases in hbd2 expression have been detected in inflamed intestinal and colonic tissues by rt-pcr and immunohistochemistry, in crohn's disease and ulcerative colitis (fahlgren et al. 2003) , and in the stomach of patients suffering from helicobacter pylori-induced gastritis (wehkamp et al. 2003) . in the lung, hbd2 has been found to be down-regulated in a variety of infectious and inflammatory diseases including cystic fibrosis and infections (chen et al. 2004) . in lung transplant patients, it has been found to be present at greater than ten times the concentration in patients suffering from bronchiolitis obliterans syndrome, a consequence of rejection of the transplant, compared to transplant patients without signs of rejection (ross et al. 2004) . less is known about the newly characterised hbd3. hbd3 expression and activity is not well characterised; however, it has been demonstrated to be inducibly expressed at the transcriptional level in bronchial epithelial cell lines stimulated with tnf-α, bacteria, or live rhinovirus (harder et al. 2001; duits et al. 2003 ). in addition, this peptide can be induced in amnion cells in response to bacterial components and is found at high concentrations in human fetal membranes, by immunohistology, indicating that it may be involved in maintaining the sterility of the intra-amniotic environment (buhimschi et al. 2004) . the pro-cathelicidin hcap18 is found at high concentrations in the granules of neutrophils and is produced by epithelial cells. the camp-gene promoter that directs the expression of this peptide contains many transcription factor binding sites (larrick et al. 1996) , including a vitamin d response element (wang et al. 2004) . consistent with this, hcap18, and/or its processed product ll-37, has been shown to be up-regulated in sinus epithelial cells, at the transcriptional level, by the bacterial products lps and lta (nell et al. 2004) . it is also increased in bronchial airway cells by il-α (erdag and morgan 2002) . in other cell types, pro-inflammatory cytokines do not increase the expression of this peptide, implying that other signalling pathways may be involved (hase et al. 2003 ). its expression is increased in gastric epithelial cells upon stimulation with a wild-type strain of h. pylori, but not a type iv secretion mutant (hase et al. 2003) , and is found at increased levels in other forms of bacterial infection. it is not entirely clear under what circumstances neutrophils release ll-37. the antimicrobial activity of all host defence peptides is highest in media of low ionic strength, and the activity of most peptides is sensitive to the presence of physiological concentrations of ions such as na + , mg 2+ and ca 2+ . the antimicrobial properties of the β-defensins, for example demonstrate profound salt sensitivity, and in some cases their antimicrobial activity is completely lost at concentrations of 100 mm nacl (bals et al. 1998a (bals et al. , 1998b garcia et al. 2001) . for example, hbd1 is antimicrobial towards gram-negative bacteria at concentrations of 1-10 µg/ml (singh et al. 1998 ), but its antimicrobial activity is almost completely abrogated by the presence of 100 mm sodium ions. although hbd2 is slightly less sensitive to the presence of sodium ions, its antimicrobial activity is reduced from 0.001-0.1 µg/ml in conditions of low ionic strength to 0.5 µg/ml or more in the presence of 100 mm sodium (singh et al. 1998) . of the human β-defensins, hbd3 is the most potent antimicrobial. this peptide is more basic, has a broader spectrum and stronger bactericidal activity against gram-positive and gram-negative bacteria, as well as yeast, and is salt-insensitive at concentrations less than 200 mm na + ions (harder et al. 2001) . most biological fluids, including sputum (halmerbauer et al. 2000) , airway surface liquid (baconnais et al. 1999 ) and serum/plasma (hoshino et al. 2003) contain mg 2+ and ca 2+ at free concentrations between 1 and 2 mm, and the presence of these ions is generally more detrimental to antimicrobial activity than na + alone. the α-defensins are susceptible to concentrations of ca 2+ and mg 2+ as low as 0.5 mm ). in the presence of 100 mm na + ions, the antimicrobial activity of ll-37 is decreased two-to eightfold (turner et al. 1998) , and in the presence of standard tissue culture media, which contains 150 mm nacl and 1-2 mm of mg 2+ and ca 2+ , ll-37 has no killing activity against s. aureus or salmonella typhimurium even at concentrations as high as 100 µg/ml . in some cases, for example in the granules of neutrophils, the concentrations of host defence peptides are estimated to be as great as 10 mg/ml, and there is no doubt that upon ingestion of bacteria these concentrations are sufficient to cause direct antimicrobial activity, despite the presence of divalent cations or other inhibitory substances. however, it is questionable whether these concentrations are reached at mucosal surfaces. for example, in patients suffering from inflammatory lung disease or infection, the concentration of hnps in the bronchoalveolar lavage (bal) has been estimated to be 0.7-1.2 and 10 µg/ml in two different studies (cole et al. 2001; spencer et al. 2003 ). this contrasts with antimicrobial activity in low salt medium in vitro at greater than 10 µg/ml for s. aureus and escherichia coli (nagaoka et al. 2000) , and reduction of the infectivity of adenoviruses in an airway epithelial model at concentrations of between 8-50 µg/ml (spencer et al. 2003) . interestingly, the antimicrobial activity of the host defence peptides may also be inhibited by components of serum. for example, hnp1 has also been demonstrated to possess antiviral activity towards enveloped viruses, but this activity is abrogated by the presence of serum or albumin (daher et al. 1986 ). it is believed but has not been conclusively shown that the high concentrations of α-defensins in neutrophils would overcome any localised serum effects (daher et al. 1986 ). moreover, the antibacterial activity of ll-37 has been demonstrated to be abrogated by the presence of apolipoprotein . in contrast to the antimicrobial activity of these peptides, their immunomodulatory properties are generally studied in the presence of standard tissue culture media, which contains physiologically relevant concentrations of ions and serum proteins. under these conditions, host defence peptides have been demonstrated to induce chemokine production, reduce pro-inflammatory cytokine production, alter transcription, induce proliferation and angiogenesis, induce chemotaxis and alter dendritic cell differentiation. thus the immunomodulatory properties of host defence peptides are unaffected by physiological ion concentrations and it seems possible that they may be the predominant function of these peptides in vivo. this perspective is quite controversial, and will remain so since it is extremely difficult to discriminate between direct and indirect (i.e. through stimulation of innate immunity) mechanisms of killing. one often used argument for the likely antimicrobial function of these peptides is their substantial variation over evolution (e.g. the mouse cramp and human ll-37 peptides share only 67% homology), which could have arisen from the evolutionary pressure of dealing with different pathogens. however, not all antimicrobial proteins are as divergent, and we note that a number of proteins involved in immunity and reproduction show similar "rapid evolution" to the antimicrobial peptides (emes et al. 2003 ). there has been some debate about whether host defence peptides might be directly antimicrobial in vivo. certainly, neutrophil granules and the crypts of the lumen contain sufficiently high concentrations of peptides to ensure substantial antimicrobial activity; however, it is less clear that antimicrobial activity occurs at the lower concentrations in such sites as mucosal surfaces, and it is worth noting that such sites are often heavily colonised by a rich and diverse collection of commensal bacteria. the evidence for antimicrobial activity in certain body sites is in our opinion inconclusive. on the one hand, certain bodily fluids such as sinus fluid (cole et al. 1999 ) and gastric fluids (hase et al. 2003) can directly kill certain micro-organisms, and this antimicrobial activity is ablated or reduced by removal of proteins or immunodepletion with a peptide-specific antibody. however, in certain animal models in which peptides and bacteria are instilled simultaneously, bacterial counts are often not significantly different from mice treated with bacteria alone, despite improved outcome or reduced pro-inflammatory responses (sawa et al. 1998; giacometti et al. 2004) . the difficulties in assessing the role of host defence peptides in vivo are profound, as it is almost impossible to account for synergistic interactions between peptides and other factors, to assess the actual concentrations at the sites of infection and to discriminate the direct antimicrobial activity of peptides from other less direct effects such as enhancement of inflammatory mechanisms (chemotaxis and recruitment of effector cells, enhancement of nonopsonic phagocytosis, etc.). nonetheless, creative experiments and animal models have begun to elucidate the roles of these peptides in vivo. in transgenic mouse model studies in which the expression of certain host defence peptides is ablated, these mice are somewhat more susceptible to infection and carry increased bacterial loads when challenged nizet et al. 2001) . although this was interpreted as being due to direct antimicrobial activity, other components of host defences must be considered. for example, in a mouse model of peritoneal klebsiella pneumoniae infection, small doses of hnp1 (4 ng-4 µg) caused an increase in leukocyte accumulation. in this model, it was the leukocyte accumulation which was linked to hnp1 induced antimicrobial activity, as the reduction in bacterial counts was significantly diminished in leukocytopenic mice. similar results were observed in s. aureus thigh infections (welling et al. 1998) . gain of function studies have found that introducing or increasing the expression of a host defence peptide can reduce bacterial loads in certain animal models of infection. for example, adenovirus transfer of ll-37/hcap-18 into the lungs of mice that were subsequently challenged with pseudomonas aeruginosa led to a reduction in both the bacterial load and in production of the pro-inflammatory cytokine, tnf-α (bals et al. 1999) , and intriguingly, similar gene therapy decreased susceptibility to sepsis induced by lps in the complete absence of bacterial infection. in other models, the simultaneous instillation into the mouse lung of p. aeruginosa and either of hbd2 or a ll-37 derivative led to reduced lung damage and pro-inflammatory cytokine production, but did not affect bacterial counts (sawa et al. 1998 ). there are very few human diseases that are characterised by defects in host defence peptide production, perhaps emphasizing their importance. however, the neutrophils of individuals with specific granule deficiency, a disease characterised by frequent and severe infections, have a reduction in the size of the peroxidase positive, defensins-containing granules (parmley et al. 1989) and are deficient in defensins ). however, it is difficult to assess the extent to which these infections result from the lack of defensins, as these patients are also deficient in other neutrophil components. it is believed that the constitutive production and deposition of neutrophils is of crucial importance to maintaining the immunological balance of the mouth. patients who suffer from morbus kostman, a severe congenital neutropenia, and are treated with g-csf to restore neutrophil level, do not express ll-37 in these cells. one of the manifestations of this disease is frequent and severe infections and periodontal disease (putsep et al. 2002) . it has been proposed that the absence of ll-37 may give a selective advantage to bacteria that at low levels are commensals but at higher levels are responsible for periodontal disease. it is unclear, however, whether ll-37 is directly microbicidal towards common pathogens of the mouth or marshals other defences. although a number of oral bacteria are susceptible to ll-37 (<10 µg/ml) at 10 mm nacl in vitro, far fewer bacteria are susceptible in physiologically more relevant isotonic environments (tanaka et al. 2000) . although ll-37 has been detected in saliva, the actual concentration was not determined (murakami et al. 2002a) . other indirect evidence for the in vivo antimicrobial activity of host defence peptides is that a decreased level of expression often correlates with frequency or severity of disease. for example, hbd2 and ll-37 expression is depressed in patients with atopic dermatitis who often present with cases of acute or chronic colonisation by s. aureus (ong et al. 2002) . in contrast to atopic dermatitis, hbd2 expression is increased in psoriatic skin, a disease in which patients are fairly resistant to bacterial infections (harder et al. 2001; nomura et al. 2003) . whether host defence peptides are directly or indirectly antimicrobial, it is apparent that it is of advantage for bacterial pathogens to subvert their expression or activity. for example, streptococcus pyogenes binds to α 2 -microglobin and secretes a small proteinase which inhibits ll-37 from interacting with the bacteria and thus prevents ll-37 mediated killing (nyberg et al. 2004 ). ll-37 expression has been shown to be decreased in shigella infection, consistent with a proposed mechanism of evasion by this bacterium (islam et al. 2001) . however, it is not clear whether this is a direct down-regulation of expression, or a consequence of denuding the epithelium, with reduced expression in the replacement cells. early experiments with host defence peptides demonstrated that many of these peptides have mitogenic effects on a variety of cells and cell lines. since modest to high concentrations of host defence peptides are found at sites of infection and inflammation, it has been hypothesised that this proliferative effect might be involved in wound healing and re-epithelisation. consistent with this hypothesis, both the human and mouse cathelicidins are up-regulated at sites of incision or wounding, even if the wound is sterile. the appearance of cathelicidins in the skin has been ascribed to both synthesis within epidermal keratinocytes, and deposition from granulocytes that migrate to the site of injury . upon incision, hcap-18 (the precursor to ll-37) has been shown to be up-regulated in the epidermis bordering the wound. this increase in expression at both the rna and protein levels was clearly evident at the migrating front of the wound during re-epithelialisation. levels of hcap-18 decreased following wound closure and eventually returned to baseline levels when the wound was intact and reepithelisation was complete. hcap-18 was found to be an active component in the process of re-epithelisation since antibodies specific for the peptide decreased the rate of re-epithelisation in a concentration-dependent manner (heilborn et al. 2003) . consistent with this observation, low levels of ll-37 (as low as 50 ng/ml) have been demonstrated to increase proliferation in an endothelial cell line (koczulla et al. 2003) . the importance of this peptide in re-epithelisation has been further inferred from its presence in wounds which are healing normally, but its absence in chronic ulcers (heilborn et al. 2003) . hnps are potent mitogens for epithelial cells, squamous cell carcinoma cell lines and fibroblasts in vitro at low concentrations (murphy et al. 1993; . interestingly, in one of the earliest studies of these effects it was demonstrated that the hnps acted synergistically with insulin to induce proliferation (murphy et al. 1993) . in general it has been hypothesised that the mitogenic properties of the neutrophil defensins on non-myeloid cells is an important component of the healing process. however, certain tumours and tumour cell lines have been demonstrated to inappropriately express neutrophil defensins, and in such cases it is believed that this expression might lead to inappropriate proliferation. for example, in a renal carcinoma cell line, the α-defensins hnp1-3 are expressed at both the transcriptional and protein levels. at moderate levels (i.e. ≤12 µg/ml), the defensins had mitogenic activity on a subset of these cell lines. by influencing tumour cell proliferation, α-defensins could potentially modulate tumour progression of renal carcinoma cells (muller et al. 2002) . moderate concentrations (e.g. ≤10 µg/ml ) of neutrophil defensins (hnp1-3) induce proliferation of a lung epithelial cell line in vitro (aarbiou et al. 2002) . consistent with these observations, a combination of hnp1-3 caused a dose-and time-dependent increase in cell migration and wound closure of an airway epithelial cell line, possibly due to an ability to induce the expression of genes involved in proliferation (aarbiou et al. 2004 ). the mitogenic activity of the hnps and cathelicidins does not appear to be shared by the β-defensins. although hbd2 has been demonstrated to be up-regulated in chronic ulcers (butmarc et al. 2004) , it has not been demonstrated to be involved in re-epithelisation. in addition, the β-defensins investigated did not increase the proliferation of epithelial cells, squamous cell carcinoma cell lines or fibroblasts (m. ). an interesting phenomenon which has been observed to occur in response to two cathelicidins, human ll-37 and its mouse homologue cramp, is the induction of angiogenesis, which is the process of blood vessel formation and/or growth. the formation of new blood vessels results in restoration of tissues increasing the oxygen supply and the provision of blood substances and cells to these tissues. as such it is a requirement for tissue repair and wound healing as well as for the marshalling of innate immunity. thus this function is consistent with a role for host defence peptides in the maintenance and repair of tissues. in a chorioallantoic membrane assay, 5 µg of ll-37 induced an increase in blood vessel growth, while in a rabbit hind limb model of angiogenesis, collateral vessel growth and blood flow were increased (koczulla et al. 2003) . interestingly however, despite the known chemotactic properties of this peptide, no inflammatory infiltrate was detected. the angiogenic properties of ll-37 appear to stem from its direct interaction with endothelial cells rather than induction of growth factors. these data are consistent with the observation that cramp knockout mice have reduced vascular structures at the wound edge at the site of injury (koczulla et al. 2003) . a mouse β-defensin, defbd29, has been shown to be involved in vasculogenesis, which is the differentiation of endothelial cells from progenitor cells during blood vessel development, leading to the de novo formation of blood vessels and tubes. tumours expressing defbd29 recruit dendritic cell (dc) precursors via ccr6 and result in enhanced vascularisation and growth in the presence of the cytokine vegf-a (conejo). interestingly, these dcs differentiate to express both dc and endothelial cell markers in response to vegf, indicating that these cells undergo endothelial cell-like specialisation after or during migration to newly formed vessels. this implies that host defence peptides may play important roles in vascular development. it has been observed that there are similarities between chemokines and host defence peptides. indeed, many chemokines have modest antimicrobial activity (hieshima et al. 2003; yang et al. 2003) , while a derivative of the highly active antimicrobial peptide, horseshoe crab polyphemusin is a potent antagonist of cxcr4 (tamamura et al. 1998) . indeed it has been proposed that certain host defence peptides have evolved from duplication of chemokine genes, although this connection is controversial (durr and peschel 2002; yang et al. 2002) ; consistent with this, certain peptides have chemotactic activity. interestingly, unlike the chemokines characterised to date, many host defence peptides appear to have chemotactic activity over a wide range of species, and generally speaking these activities are often observed at concentrations 100fold or more higher than observed with the classical chemokines. hnp1 and -2 have been demonstrated to induce chemotaxis of t cells in vitro at concentrations of between 0.1-100 ng/ml, with maximal activity occurring at less than 10 ng/ml (chertov et al. 1996) . hnp1 is a more potent chemoattractant of monocytes that hnp2, with optimal activity at concentrations of 10 -8 -10 -9 m, while hnp3 failed to induce significant chemotaxis (territo et al. 1989) . conversely, these peptides were not chemotactic for neutrophils (territo et al. 1989) , and indeed a subsequent study demonstrated that hnp1 actually suppressed polymorphonuclear (pmn) migration to formyl-methionyl-leucyl-phenylalanine but not to interleukin 8 (grutkoski et al. 2003) . in balb/c mice, 4 h after subcutaneous injection, a mixture of hnp1-3 was demonstrated to induce infiltration of pmns and mononuclear cells, while in hupbl-scid mice the defensins-induced infiltrate consisted of modest numbers of cd3 + cells (chertov et al. 1996) . interestingly, in contrast to in vitro results, in this animal model study, the infiltration of pmns was observed. however, it is unclear whether pmn infiltration was caused by direct chemotaxis or indirect effects of the peptide treatment. further studies demonstrated that these peptides specifically lead to chemotaxis of immature dendritic cells and naïve, but not memory, t cells (yang et al. 2000a ). collectively, these data indicate that neutrophil granules contain important chemotactic factors which promote the infiltration of cells of both the innate and adaptive immune responses. the β-defensins hbd1 and hbd2 are chemoattractants for immature dendritic cells and memory t, cells with peak activities occurring at 1 µg/ml (yang et al. 1999) . these activities are mediated through the chemokine receptor ccr6, which also binds the chemokine larc. hbd2, but not hbd1, has also been demonstrated to be a chemotactic agent for tnf-α treated human neutrophils (niyonsaba et al. 2004 ), a response that is also mediated through ccr6. ll-37 has been demonstrated to be chemotactic for rat mast cells (niyonsaba et al. 2002b) , mouse mononuclear cells and pmns (chertov et al. 1996) , as well as human neutrophils, monocytes and t cells (de et al. 2000) . as ll-37 has been demonstrated to induce a number of chemokines, there has been some debate as to whether it induces chemotaxis directly or indirectly by induction of classical chemokines. in the rat mast cell model, it appears as though this chemotaxis is a direct effect: as when mast cells are cultured with ll-37 and the supernatants are used for the chemotaxis assay, chemotaxis can be blocked by anti-ll-37 antiserum (niyonsaba et al. 2002b) . host defence peptides may also indirectly enhance chemotaxis by inducing the production of chemokines from a variety of different cell types, including epithelial cells and monocytes. the hnps, for example, have been demonstrated to induce il-8 from lung epithelial cells and cell lines (van wetering et al. 1997; sakamoto et al. 2004 ) and to induce the production of il-1β and il-8 mrna production (sakamoto et al. 2004 ) from a lung epithelial cell line. it is unclear whether the β-defensins have similar chemokine-inducing activities. hbd2, for example, does not induce il-8 expression in bronchial epithelial cells (sakamoto et al. 2004 ). however, in bal from patients with diffuse panbronchiolitis, the hbd2 concentration correlated significantly with the numbers of cells recovered from the bal fluid (total cells, neutrophils, and lymphocytes) (hiratsuka et al. 2003) , implying that there might be a link between this peptide and cellular infiltration to the site of infection. ll-37 has been demonstrated to induce mcp-1 and il-8 release in a mouse macrophage and a human bronchial epithelial cell line, respectively, and both chemokines were increased upon stimulation with ll-37 in whole human blood (scott et al. 2002) . ll-37 has also been demonstrated to induce chemokine transcription (il-8, mcp-1, mcp-3) and release (il-8) in a mitogen-activated protein kinase (mapk)-dependent manner in human peripheral blood derived monocytes . both ll-37 and the hnps are neutrophil-derived peptides which are released upon neutrophil degranulation. these peptides induce the transcription and release of chemokines, specifically il-8, which preferentially attract neutrophils. consequently the presence of these peptides correlates well with that of il-8, a potent chemoattractant for neutrophils (ashitani et al. 2002) , as well as the presence of other neutrophil components such as elastase (zhang et al. 2002 ). it appears that host defence peptides induce chemotaxis in two ways: first through direct chemotactic activity of pmns and mononuclear cells mediated through ccr6 and other as yet to be identified receptors and second through inducing chemokine production which would hypothetically increase the numbers of neutrophils and monocytes at sites of infection. this then would have the net effect of promoting or marshalling cells important in innate immunity to the sites of excessive production (through induction) or deposition (through neutrophil degranulation) of these host defence peptides. this then begs the question as to whether host defence peptides are overtly pro-inflammatory. early experiments determined that a number of host defence peptides from various sources bound to lps from diverse gram-negative bacteria and reduced lps-induced release of pro-inflammatory cytokines (e.g. tnf-α, il-1, il-6) and nitric oxide from monocyte or macrophages and protected mice from lps lethality (larrick et al. 1994 (larrick et al. , 1995 vandermeer et al. 1995; kirikae et al. 1998) . initial studies focussed on the unprocessed form of cathelicidin, hcap-18 (kirikae et al. 1998 ); however, it was later found that the lps-binding properties of the peptide were contained within the processed 37-amino acid c-terminal domain, ll-37 (turner et al. 1998 ). it has been proposed that the anti-endotoxic properties of these peptides are the result of the inhibition of binding of lps to cd14 ) and lipopolysaccharidebinding protein (lbp) (scott et al. 2000) , and/or indirect effects on cells (scott et al. 2002) . ll-37 has been shown to block a number of lps-induced inflammatory responses, including contractility and (nitric oxide) no release in aortic rings (ciornei et al. 2003) , pro-inflammatory cytokine production in a macrophage cell line and in animal models (scott et al. 2002) (ohgami et al. 2003) , suppression of leukocyte infiltration in a model of endotoxin-induced uveitis (ohgami et al. 2003 ) and lethality in animal models of sepsis (scott et al. 2002) . these effects occur at concentrations in the physiological range for ll-37 (1-5 µg/ml) and may reflect a natural role for ll-37 in the body (e.g. balancing of the potential stimulus by endotoxin from commensals). this anti-endotoxin activity appears to correlate with an ability to dampen the pro-inflammatory effects of the gram-positive surface molecule lipoteichoic acid (scott et al. 2002) . it appears that there may be marked differences in the ability of ll-37 and the defensins to inhibit the pro-inflammatory effects of endotoxin. for example, hnp1 and hbd2 are not potent inhibitors of lps-lbp binding (scott et al. 2000) . in ex vivo whole blood experiments, hnp1 was approximately 1,000-fold less potent than bpi at reducing tnf-α in response to gramnegative bacteria and is much less potent in blocking endotoxin activity, as assessed by a surrogate assay, the limulus amoebocyte lysate assay, or in priming pmn for arachidonate release or stimulating leukocyte oxidase activity (levy et al. 1995) . thus, the ability to bind to and neutralise endotoxininduced activity in humans may be more evident for ll-37 and other proteins such as bacterial permeability-inducing protein (bpi) and lbp (weiss 2003) . natural killer cells are cd56 -cd3lymphocytes that are an important component of the innate immune response. they kill transformed and infected cells, but unlike t cells they are active against cells that have decreased or ablated expression of major histocompatibility complex class 1 (mhc1) molecules. the cytolytic properties of nk cells are increased in the presence of cytokines produced by cells of the innate immune response. nk cells themselves produce cytokines, such as ifn-γ, which are involved in the enhancement of both the innate and adaptive immune responses. nk cells contain a wide variety of cytotoxic peptides of which granulysin (nk-lysin) is considered to be the most important (kumar et al. 2001) . recently nk cells have also been demonstrated to express the transcripts for ll-37 and hnp1-3, and these peptides were found in the supernatants of il-2-treated cells consistent with an involvement in the cytotoxic properties of these cells (agerberth et al. 2000) , or alternatively an immunomodulatory role. consistent with these observations, it has been shown that both tlr2 and tlr5 agonists induce the release of hnp1-3 from nk cells into the supernatant and that this release is increased synergistically in the presence of other cytokines found at the site of inflammation (chalifour et al. 2004 ). it is not entirely clear what the role of defensins may be in modulating nk-induced cytotoxicity. in one study it was found that nk mediated cytolysis of the transformed cell line kn62 is decreased in the presence of hnp1-3 in a dose-dependent manner. as well, nk cells treated with hnps had a decreased expression of both cd16 and cd56 ). this study also demonstrated that there are high concentrations of hnps due to the infiltration of neutrophils in colorectal tumours, but not in surrounding healthy tissue. thus the authors hypothesised that the presence of hnps might actually protect cancerous cells from nk cytolysis. a conflicting study demonstrated that pbmcs, treated with opsonin-coated zymosan particles, induced the release of substances that enhanced nk-mediated cytotoxicity. these substances were identified as neutral serine proteases and hnps. of the peptides tested, hnp1 was the most potent, increasing nk-mediated cytolysis optimally at a concentration of 1.25 µg/ml (lala et al. 1992) . clearly, further studies are required to fully elucidate the role that host defence peptides have on nk mediated cytoxicity. monocytes and macrophages do not express high levels of defensins or cathelicidins unless stimulated by lps or pro-inflammatory mediators (agerberth et al. 2000; duits et al. 2002) . however, when thus stimulated, they secrete as yet unidentified factors that stimulate epithelial cells and keratinocytes to produce host defence peptides . monocytes and macrophages are, however, quite responsive to stimulation with these peptides and both ll-37 and the defensins have been demonstrated to induce chemotaxis (territo et al. 1989; de et al. 2000) . it has been noted that host defence peptides are strong inducers of chemokine activity in monocytes (chaly et al. 2000; . interestingly it has been demonstrated that the hnps are able to prevent hiv replication in monocytes and monocyte-derived macrophages and that this property may be due to their ability to induce chemokine production and/or receptor antagonism (guo et al. 2004 ). hnp1 and hnp2 were both demonstrated to induce production of mip-α and mip-1β, the ligands for ccr5 in monocyte-derived macrophages and to prevent replication of a ccr5 tropic strain of the virus, presumably by blocking virus binding to ccr5 (guo et al. 2004 ). there has been some evidence that host defence peptides might work as opsonins (fleischmann et al. 1985; sawyer et al. 1988) . although this property would be predicted to generally enhance that antimicrobial activity associated with these peptides, one study demonstrated that an ll-37 derivative actually promoted infectivity of coxiella burnetii, an intracellular pathogen of macrophages (aragon et al. 1995) . generally, host defence peptides are thought to possess anti-inflammatory properties, as described above. however, in some cases, they may actually enhance some aspects of a pro-inflammatory response. ll-37, for example, has been demonstrated to enhance il-1β processing and release in lps-primed primary human monocytes (elssner et al. 2004) . this property appears to be conserved across a range of host defence peptides from a number of different species (perregaux et al. 2002) . mast cells are distributed throughout the body and are also found in low amounts in the blood. these cells rapidly accumulate at sites of infection, and upon encountering certain bacterial components or pro-inflammatory stimuli they promote the inflammatory response by releasing histamine, which causes vasodilation and thus assists in the recruitment of cells and substances from the blood. two host defence peptides, ll-37 and hbd2 have been demonstrated to be chemotactic for rat mast cells, although they may work by different mechanisms (niyonsaba et al. 2002b (niyonsaba et al. , 2003 . thus mast cells may accumulate at sites of high concentrations of host defence peptides such as at sites of neutrophil degranulation or at epithelial surfaces in vivo. hbd2 and ll-37 as well as the hnp1-3 and hnp homologues from rabbits and guinea pigs have also been demonstrated to induce histamine release (befus et al. 1999; niyonsaba et al. 2001) . this property may be especially important in the development of host defence peptides as drugs, as mast cell degranulation is a potentially detrimental side effect. host defence peptides have been demonstrated to interact with epithelial cells. neutrophil peptides have been demonstrated to induce proliferation (m. , induce chemokine production (van wetering et al. 1997 ) and stimulate cell signalling pathways . ll-37 has been demonstrated to bind to a lung epithelial cell line in a manner which suggests that it may have more than one receptor . it has also been demonstrated that binding and subsequent internalisation is required in order to induce il-8 production . host defence peptides in the adaptive immune response in addition to apparently having multiple roles in innate immunity, it is becoming clear that host defence peptides can modulate the adaptive immune response, and several studies have now demonstrated adjuvant activities of host defence peptides in vivo. the mechanisms involved remain unclear, although these activities could reflect the innate immunity modulating activity of host defence peptides and the fact that there appears to be a strong interconnection between innate and adaptive immunity. the relatively non-immunogenic model antigen ovalbumin (ova) is widely used to study adaptive immune responses. intranasal co-administration of human α-defensins hnp1-3 with ova was shown to enhance the production of ova-specific igg antibodies and ova-specific cd4 + t cells, which produced significantly more ifnγ, il-5, il-6, and il-10 (lillard et al. 1999 ). this indicated the capacity of α-defensins to alter the host response to ova, acting as adjuvants to promote a mixed t helper (th) cell response. in two other recent studies, hnp1, the human β-defensins hbd1 and hbd2 (brogden et al. 2003) , and a simple synthetic peptide klkl 5 klk (fritz et al. 2004) were also demonstrated to be effective adjuvants. the observation that such effects are observed with the model peptide klkl 5 klk suggests the possibility of a relatively non-specific mechanism, and that such activities may therefore be seen with a broad range of host defence peptides. however, the nature of the enhanced responses may depend both on the antigen and the peptide used. in contrast to the mixed th-1/th-2 response enhanced in the hnp1-3-treated animals (lillard et al. 1999) , ova-stimulated splenic lymphoid cell cultures were found to produce significantly decreased levels of ifn-γ, when taken from hbd2-treated mice (brogden et al. 2003 ). on the other hand, although klkl 5 klk induced a strong th-2 type response when co-administered with ova, it enhanced a mixed response when the trivalent influenza split-vaccine fluvirin was used as antigen, with the production of both igg 1 and igg 2 antibodies (fritz et al. 2004 ). interestingly, this report also demonstrated that a peptide could markedly enhance antigen association with a monocytic cell line in vitro, and that co-administration in vivo could result in the formation of a transient depot of antigen at the site of injection. these observations indicate that antigen uptake by antigen-presenting cells (apcs) might be enhanced in the presence of the peptide, and thus influence responses in the presence of klkl 5 klk in vivo. although these studies clearly showed altered humoral and th responses to antigens, the functional consequences of these alterations were not clearly demonstrated. in another study, mice were given an intraperitoneal vaccination combining a b-cell lymphoma idiotype antigen and daily 1 µg injections of human α-defensins. this study also demonstrated adjuvant activity, whereby the defensins led to increased levels of antigen-specific igg antibodies and enhanced ifn-γ production by splenic cells (tani et al. 2000) . moreover, defensins showed mitogenic properties (with a significant increase in the number of splenic b cells) and led to an increase in resistance to tumour challenge. the latter observation raised the possibility that an antigen-specific cytotoxic t cell response was being generated in addition to a humoral response. these studies collectively demonstrate that co-administration of host defence peptides with antigens can enhance and perhaps alter the nature of the host's specific adaptive immune responses in vivo. this raises the question of whether host defence peptides might naturally act as endogenous adjuvants to enhance normal immunological responses, since many peptides can be up-regulated or secreted at sites of infection and inflammation. it is unclear whether the doses used in such studies to assess in vivo immunological processes are within relevant physiological ranges. the physiological significance should be addressed by examining transgenic mice with defective production of host defence peptides, although the issue of possible functional redundancy amongst the many murine defensins must be considered when examining single gene knockouts. the published characterisations of such mice have concentrated on innate responses and have generally not described defects in adaptive immune responses morrison et al. 2002; moser et al. 2002) . however, one mbd-1 knockout model was found to display a defect in generating antibodies to the carbohydrate capsule of pneumococci (c. moser, personal communication) . while this is consistent with an in vivo role for this constitutively expressed defensin in generating an effective humoral response, it is clearly an area requiring further study. regardless of possible physiological significance, the adjuvant effects of host defence peptides are clearly of interest from an immunotherapeutic and vaccinology perspective. in contrast to the studies that have co-administered host defence peptides and antigens, other groups have taken an alternative dna-vaccine approach. this methodology involved immunizing mice with dna plasmids encoding non-immunogenic lymphoma antigens fused to murine β-defensins (biragyn et al. 2001) . successfully transfected cells of an undefined nature should then express the peptide/lymphoma antigen fusion proteins. this strategy represents an attempt to target antigen to immature dendritic cells (idcs), by exploiting the affinity of the β-defensin portion of the fusion proteins for the chemokine receptor ccr6, expressed on idcs. this approach also demonstrated an adjuvant capacity for host defence peptides; however, igg responses were only observed when the plasmid encoded a fusion of the antigen and peptide, and not observed after simple co-administration of peptide and antigen. interestingly, anti-tumour activity was also generated in these mice (most effectively with murine β-defensin 2), but did not correlate with the amplitude of the humoral response (superior with murine β-defensin 3). furthermore, this anti-tumour activity could be transferred to other mice with the delivery of splenocytes, but not serum, from vaccinated animals, indicating the generation of cytotoxic t cells in response to non-immunogenic antigens when fused to peptides. in another recent study, using a similar approach, immunisation of mice with a plasmid fusing the human cathelicidin ll-37 to m-csfr (acting as a tumour antigen in this model) also generated enhanced antigen-specific humoral and cytotoxic responses, and prolonged survival in a tumour model ). ll-37 fusion plasmids were found to be significantly more effective than the m-csfr plasmid alone, or co-administration of separately encoded m-csfr and ll-37 plasmids. these animal studies all demonstrate the adjuvant capacity of host defence peptides in vivo, but the mechanisms underlying these observations have not been fully elucidated. a variety of hypotheses can be proposed, including direct modulation of lymphocyte responses, mitogenic effects, chemotactic capacity, increased apc antigen uptake and consequently enhanced presentation, activity as endogenous danger signals, alterations to the apc cytokine environment, or direct modulation of apc function (fig. 1) . the most obvious mechanisms might include altered antigen uptake (fritz et al. 2004 ) and direct modulation of lymphocyte activity and proliferation (tani et al. 2000) , boosting apc presentation, cellular and humoral responses. this could be further enhanced by direct chemotactic effects of host defence peptides, resulting in the chemotaxis of monocytes, neutrophils, macrophages, idcs, mast cells and t lymphocytes (territo et al. 1989; chertov et al. 1996; yang et al. 1999 yang et al. , 2000a yang et al. , 2000b niyonsaba et al. 2002a niyonsaba et al. , 2002b , and the enhancement of chemokine receptor expression on these cells (scott et al. 2002) . in addition, host defence peptides could act indirectly to stimulate the release of potent traditional chemokines (such as il-8) from epithelial cells (van wetering et al. 1997) , and/or cause mast cell degranulation , enhancing vascular permeability. these direct and indirect chemotactic effects could amplify the inflammatory response and bring key cells of the adaptive immune response to the location of the antigen. while recruiting memory t cells to an infection site may induce a more rapid cellular response to previously encountered antigens, the recruitment of monocytes and idc is likely to be critical to generating the initial response. dendritic cells are sentinel leukocytes that capture antigen in the peripheral tissues and then initiate and orchestrate t cell helper (th-1) responses, the nature of which determines the character of the adaptive immune response (moser and murphy 2000) . this process is critical to generating a successful defence against harmful microbial non-self antigens while maintaining tolerance to self. it is dependent upon the antigen-capturing capabilities of idcs, and antigen-presenting capabilities of mature dendritic cells (mdcs). idcs are derived from circulating haematopoietic precursor cells and predc populations (monocytes and plasmacytoid cells) under the influence of specific cytokines and growth factors (liu 2001; pulendran et al. 2001 ). in the tissues, these cells encounter and take up antigen. stimulation of idcs by conserved structures on certain microbial antigens, acting via the toll-like receptors (tlrs) of the innate immune system (medzhitov and janeway 2000) or by signals from host cytokines, results in dc activation. these activated cells mature to become effective antigen-processing and presenting mdcs, migrate to the secondary lymphoid organs and interact with naïve t-lymphocytes (banchereau et al. 2000) . the characteristics of the mdcs determine the nature and consequences of this interaction, resulting in proliferation and differentiation, or deletion of t cells, and determine the polarisation of the th response (lanzavecchia and sallusto 2001) . whereas steady-state trafficking of non-activated idcs carrying self-antigen is thought to help maintain tolerance, it has been proposed that sustained trafficking of large numbers of highly stimulatory mdcs to the t cell areas is necessary for the generation of an effective t cell proliferative response (lanzavecchia and sallusto 2001) . this would require extensive, repeated recruitment of circulating predcs to the site of infection, with rapid differentiation to replace the "first-line" resident idcs. thus, at the simplest level, it is conceivable that the in vivo effects of host defence peptides on the adaptive immune response are the result of direct and indirect chemotaxis of idcs and monocytes to the site of inflammation. thus, chemotaxis, altered antigen uptake, and mitogenic effects on lymphocytes offer potential mechanisms by which host defence peptides may enhance responses to immunogenic antigens. however, these explanations do not account for the generation of humoral and cytotoxic t lymphocyte responses to non-immunogenic antigens observed in vivo. in these examples, an increase in the number of dcs encountered and the amount of antigen taken up should make no difference to the response in the absence of an activating signal. indeed, theoretically, this might serve to increase host tolerance to these antigens. despite this, host defence peptides clearly enhance an adaptive immune response to non-immunogenic antigens in vivo. on the basis of current literature, we propose three hypotheses that might explain these observations. the first two theories propose that these peptides directly or indirectly provide an activating signal to differentiated idcs concurrent with these cells encountering antigen, while the third proposes peptide modulation of dc differentiation from precursor cells. in one intriguing report, it was demonstrated that murine β-defensin 2 fusion proteins were capable of activating idcs directly in a tlr-4 dependent manner, to produce t helper (th-1) polarised responses . in the context of these dna plasmid vaccines, stimulation of the innate pattern recognition pathways through tlr would occur in close spatial and temporal conjunction to an otherwise non-immunogenic antigen. this suggests that host defence peptides might be capable of functioning as endogenous ligands of innate pattern recognition receptors. however, activation of idcs was not observed with murine β-defensin 2 in the absence of fusion to lymphoma antigen. although this appears to make it improbable that this mechanism is responsible for most of the above-described in vivo observations, it is possible that peptide and antigen concentrations are much higher in co-administration studies, than when relying on dna plasmid expression. thus, if the temporal coordination of tlr4 stimulation and antigen presentation are critical, this may be achieved by high concentration co-administration and depot formation at the site of delivery but, when utilizing a dna vaccine approach, require peptide fusion. however, despite proving effective as an adjuvant for humoral responses (biragyn et al. 2001) , murine β-defensin 3 fusion proteins did not have tlr-4 dependent idc-activating capabilities . furthermore we have seen no evidence of an ability to directly mature human monocyte-derived dc in vitro when studying a range of peptides at or above the putative physiological concentrations, (dj davidson, aj currie,, rew hancock, dp speert, unpublished data). these data indicate that direct activation of idcs may not be an inherent property of host defence peptides. thus, although direct activation of idcs is unlikely to be the basic mechanism underlying host defence peptide adjuvant activities, we cannot rule out a peptide-specific effect in which temporal coordination of tlr4 ligation and chemokine receptor-directed antigen uptake by the same cell are critical. an alternative mechanism to explain altered idc activation is to suggest that the effects of host defence peptides might be indirect, acting to alter the milieu in which these cells encounter antigen. defensins have been shown to increase expression of various cytokines, including il-8, il-6, mcp-1 and gm-csf , in different airway epithelial cells, while ll-37 can induce il-8 and mcp-1 expression in epithelial and monocytic cells (scott et al. 2002) . changes to the cytokine environment may induce a myriad of effects, from the chemotactic activities of mcp-1 and il-8, and cellular differentiation effects of gm-csf, to the enhancement of b cell proliferation and blockade of the suppressive effects of regulatory t cells by il-6 (pasare and medzhitov 2003) . possibly other factors are induced that might activate idcs even in the presence of non-immunogenic antigens. following activation of monocytes with s. aureus or phorbol myristate, human α-defensins at con-centrations as low as 1 nm, can increase the expression of tnf-α and il-1β (chaly et al. 2000) . these cytokines have the potential to directly induce dc maturation, sharing components of activating pathways with tlr, and thus potentially enhancing the generation of highly stimulatory mdcs. however, while such mechanisms might therefore be proposed at sites of inflammation, similar activities in the presence of non-immunogenic antigens are only speculative. the third hypothesis relates to our recent discovery that the human cathelicidin ll-37 can modulate the differentiation of idcs from precursor cells, with consequent impact on th cell polarisation ). the stimulatory nature of dcs is subject to dynamic temporal regulation (langenkamp et al. 2000) and can be modified by precursor cell lineage, the specific antigen captured, the receptors engaged, and the microenvironment for both differentiation and maturation (liu 2001; pulendran et al. 2001; de jong et al. 2002; boonstra et al. 2003) . we demonstrated that ll-37 has the potential to act as an endogenous environmental modifier of dc differentiation ). ll-37-primed dcs displayed significantly upregulated endocytic capacity, modified phagocytic receptor expression and function, up-regulated co-stimulatory molecule expression, enhanced secretion of th-1-inducing cytokines, and promoted th-1 responses in vitro. these results suggest the potential for host defence peptides to exert effects on the adaptive immune system by priming newly differentiating dcs to enhance their antigen uptake and presentation capabilities and influence the nature of the response they will subsequently generate. according to this hypothesis, host defence peptides would not simply affect "first-line" resident idcs, but act upon the differentiating "second-line" dcs that can sustain highly stimulatory presentation of antigen to generate an effective t cell proliferative response. in the context of a physiological role, ll-37-primed idcs might be generated at sites where ll-37 is up-regulated in response to infection or inflammation, be matured by immunogenic antigens, and promote a more robust adaptive immune response. however, ll-37-primed idcs also have increased expression of the co-stimulatory molecule cd86 in vitro in the absence of activating stimuli and any other signs of maturation. if such cells were generated in vivo in the presence of a high concentration depot of host defence peptides at the site of vaccination, or in an area of peptide overexpression by host cells transfected with a dna vaccine, they might be capable of presenting non-immunogenic antigen in a stimulatory context. although enhanced humoral and cytotoxic t-lymphocyte (ctl) responses in dna vaccinated mice are dependent on the fusion of ll-37 and the antigen , this might again relate to issues of local concentration and co-presentation to the same cells. interestingly however, enhanced splenocyte ifn-γ responses were observed not only in mice vaccinated with the ll-37 fusion plasmid, but also in those given separately encoded ll-37 and antigen plasmids. this might reflect the enhanced ifn-γ responses of t cells stimulated with ll-37-primed dcs in vitro. however, further research is required to establish the in vivo significance of the ll-37-priming of dcs observed in vitro. furthermore, the effects of other host defence peptides on dc differentiation have not been described, raising uncertainty about this hypothesis in the context of the in vivo studies using defensins, or synthetic peptides as adjuvants. in conclusion, the potential for host defence peptides to modulate the adaptive immune response is evident, but remains largely undescribed. in addition to further exploration of the effects in vitro, innovative in vivo modelling is a priority to dissect the mechanisms underlying these observations. a clear understanding of the extent and mechanisms of the immunomodulatory effects of host defence peptides will be fundamental to their future development as novel therapeutic agents. however, these early in vivo studies demonstrate great potential for targeting tumours, recalcitrant, antibioticresistant pathogens, infections for which effective vaccines do not exist, and vaccines, which generate suboptimal responses of an inappropriate nature. mammalian host defence peptides were originally discovered as components of the non-oxidative killing mechanisms of neutrophils. in the granules of neutrophils, these peptides are found at sufficiently high concentrations to be antimicrobial. however, it is less clear that this is the case at mucosal surfaces or in other body fluids, especially at sites that already support a rich and diverse normal flora. certain body fluids, including sinus fluid and gastric juices, have innate antimicrobial activity against certain bacteria, and the components that appear to contribute to this include a variety of antimicrobial proteins (e.g. lysozyme, secretory phospholipase a 2 ), as well as peptides (e.g. defensins) (cole et al. 2002) . however, the specific contributions of each of these components to overall antimicrobial activity has not been determined, and given the moderate levels of peptides often found in these fluids, synergy between individual agents working in combination may be important, as been demonstrated for some peptides, and combinations of lysozyme and peptides in vitro (singh et al. 2000; yan and hancock 2001) .this is still further complicated at sites such as the mucosa when considering the abilities of host defence peptides to modulate innate immunity, as discussed extensively in this review. one approach to trying to resolve these mechanisms is to use genetic strategies using either knockout models in animals or specific genetic deficiencies in humans nizet et al. 2001; putsep et al. 2002; salzman et al. 2003) . such studies have clearly demonstrated that defensins and cathelicidins are integral components of host defence in mammals, and that these peptides are required to reduce bacterial load and inhibit infection. however, they do not always permit the discrimination between the various potential mechanisms of host defence peptides, namely direct antimicrobial activity, synergistic activity with other antimicrobial components and/or the broad range of abilities to modulate immunity. indeed, these distinctions may be unimportant, as they all have the same net result, namely the control of potentially dangerous microbes. we hypothesise that all of these mechanisms operate in the body of mammals, and that any given peptide may have different roles in anti-infective immunity according to the body site it is found, its local concentration, the prevailing physiological conditions, and the other antimicrobial and cellular components of immunity at that site. a clear illustration of this complexity is provided by a study of the use of human defensin 1 (hnp-1) to treat experimental peritoneal k. pneumoniae infections (welling et al. 1998 ). in this model, hnp1 injection was shown to markedly reduce bacterial numbers, but the antibacterial effect was associated with an increased influx of leukocytes into the peritoneal cavity, and this was strongly related to the antibacterial effect, as no such activity was observed in leukocytopenic mice. a further challenge to our thinking, and possibly the most profound question in innate immunity, is how mammals manage to support a complex normal flora while retaining the ability to respond to potentially dangerous pathogens. the toll-like receptors (tlrs), which represent one of the major "triggers" of innate immunity, do not really distinguish between the conserved surface molecules from pathogens and commensal organisms. thus it is of interest as to whether host defence peptides may play a role in this delicate dance between symbiosis and pathogenesis. as shown by e. nishimura et al., many commensal bacteria from the oral cavity are quite susceptible to hbd2, while chung and dale indicated that both commensals and pathogenic bacteria can induce this defensin (chung and dale 2004; e. nishimura et al. 2004 ). conversely, putsep et al. compared germ-free and normal mice to conclude that an intestinal microflora does not have a major influence on the production or processing of defensins (putsep et al. 2000) . however, we consider at least one activity of these peptides might play a role for host defence peptides in homeostasis, namely anti-endotoxic activity, which in our experience is expressed at lower concentrations of ll-37 than other immunomodulatory activities. it seems possible that this would provide a mechanism for balancing the po-tential stimulation of tlrs by surface molecules from commensal organisms. innate immunity would then be triggered by local perturbations of peptide concentrations though mechanisms such as degranulation of phagocytes, or specific up-regulation by certain cytokines. in addition, the efficiency of these peptides might the increased by other local factors, for example phagocytes that enter the local site (welling et al. 1998) or local cytokines such as gm-csf that has been shown to enhance mapk signalling by ll-37 . the full spectrum of the immunomodulatory properties of these peptides is not yet known and each new report demonstrates that the range and importance of these immunomodulatory effects is greater than initially suspected. most likely both antimicrobial and immunomodulatory activities are to some degree involved, as this is consistent with the redundant and efficient nature of evolution, and with the concept of innate immunity as a network of overlapping mechanisms. understanding the interplay between host defence peptides and innate and adaptive immunity will expand our knowledge of immunity in general and allow us to develop anti-infective therapies adapted from nature's design that will enhance the efficiency of immune defences. human neutrophil defensins induce lung epithelial cell proliferation in vitro neutrophil defensins enhance lung epithelial wound closure and mucin gene expression in vitro the human antimicrobial and chemotactic peptides ll-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations ll-37 enhances adaptive immune response in murine model challenged with tumour cells a cationic antimicrobial peptide enhances the infectivity of coxiella burnetii elevated levels of alpha-defensins in plasma and bal fluid of patients with active pulmonary tuberculosis ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts mouse beta-defensin 1 is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung the peptide antibiotic ll-37/hcap-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide immunobiology of dendritic cells cd14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium neutrophil defensins induce histamine secretion from mast cells: mechanisms of action hbd-1: a novel beta-defensin from human plasma mediators of innate immunity that target immature, but not mature, dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens toll-like receptor 4-dependent activation of dendritic cells by beta -defensin 2 flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing t helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation the human cationic peptide ll-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes impact of ll-37 on anti-infective immunity defensin-induced adaptive immunity in mice and its potential in preventing periodontal disease the novel antimicrobial peptide beta3-defensin is produced by the amnion: a possible role of the fetal membranes in innate immunity of the amniotic cavity human beta-defensin-2 expression is increased in chronic wounds direct bacterial protein pamp recognition by human nk cells involves tlrs and triggers alpha-defensin production neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells beta-defensins and ll-37 in bronchoalveolar lavage fluid of patients with cystic fibrosis identification of defensin-1, defensin-2, and cap37/azurocidin as t-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils innate immune response of oral and foreskin keratinocytes: utilization of different signaling pathways by various bacterial species effects of human cathelicidin antimicrobial peptide ll-37 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro innate antimicrobial activity of nasal secretions determinants of staphylococcus aureus nasal carriage cationic polypeptides are required for antibacterial activity of human airway fluid tumor-infiltrating dendritic cell precursors recruited by a betadefensin contribute to vasculogenesis under the influence of vegf-a human defensin 5 is stored in precursor form in normal paneth cells and is expressed by some villous epithelial cells and by metaplastic paneth cells in the colon in inflammatory bowel disease direct inactivation of viruses by human granulocyte defensins the cationic antimicrobial peptide ll-37 modulates dendritic cell differentiation and dendritic cell-induced t cell polarization microbial compounds selectively induce th1 cell-promoting or th2 cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals ll-37, the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (fprl1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells intestinal defensin gene expression in human populations cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group a streptococcus rhinovirus increases human beta-defensin-2 and -3 mrna expression in cultured bronchial epithelial cells expression of beta-defensin 1 and 2 mrna by human monocytes, macrophages and dendritic cells chemokines meet defensins: the merging concepts of chemoattractants and antimicrobial peptides in host defense a novel p2x7 receptor activator, the human cathelicidin-derived peptide ll37, induces il-1 beta processing and release comparison of the genomes of human and mouse lays the foundation of genome zoology interleukin-1alpha and interleukin-6 enhance the antibacterial properties of cultured composite keratinocyte grafts increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis opsonic activity of mcp-1 and mcp-2, cationic peptides from rabbit alveolar macrophages the artificial antimicrobial peptide klklllllklk induces predominantly a th2-type immune response to co-injected antigens expression of human alpha-defensin 5 (hd5) mrna in nasal and bronchial epithelial cells differential expression of human alpha-and beta-defensins mrna in gastrointestinal epithelia defensins. natural peptide antibiotics of human neutrophils microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: chediak-higashi syndrome and "specific" granule deficiency human betadefensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity cathelicidin peptide sheep myeloid antimicrobial peptide-29 prevents endotoxin-induced mortality in rat models of septic shock alpha-defensin 1 (human neutrophil protein 1) as an antichemotactic agent for human polymorphonuclear leukocytes alpha-defensins inhibit hiv infection of macrophages through upregulation of cc-chemokines the relationship of eosinophil granule proteins to ions in the sputum of patients with cystic fibrosis a peptide antibiotic from human skin isolation and characterization of human beta-defensin-3, a novel human inducible peptide antibiotic expression of ll-37 by human gastric epithelial cells as a potential host defense mechanism against helicobacter pylori the cathelicidin anti-microbial peptide ll-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium ccl28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity increased concentrations of human betadefensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis tolllike receptor 4 resides in the golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells influence of heart surgery on magnesium concentrations in pediatric patients downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial dna as a potential regulator paneth cells of the human small intestine express an antimicrobial peptide gene defensin-6 mrna in human paneth cells: implications for antimicrobial peptides in host defense of the human bowel paneth cell granule depletion in the human small intestine under infective and nutritional stress protective effects of a human 18-kilodalton cationic antimicrobial protein (cap18)-derived peptide against murine endotoxemia an angiogenic role for the human peptide antibiotic ll-37/hcap-18 inducible expression of human beta-defensin 2 by fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier regulation of human beta-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the nf-kappab transcription factor family granulysin: a novel antimicrobial the differential effects of polymorphonuclear leukocyte secretion on human natural killer cell activity kinetics of dendritic cell activation: impact on priming of th1, th2 and nonpolarized t cells regulation of t cell immunity by dendritic cells human cap18: a novel antimicrobial lipopolysaccharide-binding protein a novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity structural, functional analysis and localization of the human cap18 gene interaction and cellular localization of the human host defense peptide ll-37 with lung epithelial cells defensins of vertebrate animals modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations antibacterial proteins of granulocytes differ in interaction with endotoxin. comparison of bactericidal/permeability-increasing protein, p15s, and defensins mechanisms for induction of acquired host immunity by neutrophil peptide defensins by il-1 signaling, monocyte-derived cells dramatically enhance the epidermal antimicrobial response to lipopolysaccharide dendritic cell subsets and lineages, and their functions in innate and adaptive immunity rapid sequence divergence in mammalian beta-defensins by adaptive evolution innate immunity characterization of the mouse beta defensin 1, defb1, mutant mouse model beta-defensin 1 contributes to pulmonary innate immunity in mice dendritic cell regulation of th1-th2 development human alpha-defensins hnps-1, -2, and -3 in renal cell carcinoma: influences on tumor cell proliferation cathelicidin antimicrobial peptides are expressed in salivary glands and saliva cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense expression and secretion of cathelicidin antimicrobial peptides in murine mammary glands and human milk defensins are mitogenic for epithelial cells and fibroblasts cathelicidin family of antibacterial peptides cap18 and cap11 inhibit the expression of tnf-alpha by blocking the binding of lps to cd14(+) cells synergistic actions of antibacterial neutrophil defensins and cathelicidins absence of toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium bacterial products increase expression of the human cathelicidin hcap-18/ll-37 in cultured human sinus epithelial cells oral streptococci exhibit diverse susceptibility to human beta-defensin-2: antimicrobial effects of hbd-2 on oral streptococci effect of defensin peptides on eukaryotic cells: primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and ll-37 on histamine release and prostaglandin d(2) production from mast cells epithelial cellderived human beta-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase c-dependent pathway a cathelicidin family of human antibacterial peptide ll-37 induces mast cell chemotaxis epithelial cell-derived antibacterial peptides human beta-defensins and cathelicidin: multifunctional activities on mast cells human beta-defensin-2 functions as a chemotactic agent for tumour necrosis factor-alpha-treated human neutrophils innate antimicrobial peptide protects the skin from invasive bacterial infection cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes alpha 2-macroglobulin-proteinase complexes protect streptococcus pyogenes from killing by the antimicrobial peptide ll-37 salmonella enteritidis flic (flagella filament protein) induces human beta-defensin-2 mrna production by caco-2 cells effect of human cationic antimicrobial protein 18 peptide on endotoxin-induced uveitis in rats expression and regulation of the human beta-defensins hbd-1 and hbd-2 in intestinal epithelium endogenous antimicrobial peptides and skin infections in atopic dermatitis the gene encoding the human corticostatin hp-4 precursor contains a recent 86-base duplication and is located on chromosome 8 plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis abnormal peroxidase-positive granules in "specific granule" deficiency toll pathway-dependent blockade of cd4+cd25+ t cell-mediated suppression by dendritic cells antimicrobial peptides initiate il-1 beta posttranslational processing: a novel role beyond innate immunity modulating the immune response with dendritic cells and their growth factors germ-free and colonized mice generate the same products from enteric prodefensins deficiency of antibacterial peptides in patients with morbus kostmann: an observation study gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract increased bronchoalveolar lavage human beta-defensin type 2 in bronchiolitis obliterans syndrome after lung transplantation differential effects of alpha-and betadefensin on cytokine production by cultured human bronchial epithelial cells protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin evaluation of antimicrobial and lipopolysaccharide-neutralizing effects of a synthetic cap18 fragment against pseudomonas aeruginosa in a mouse model interaction of macrophage cationic proteins with the outer membrane of pseudomonas aeruginosa discovery of five conserved beta -defensin gene clusters using a computational search strategy the human antimicrobial peptide ll-37 is a multifunctional modulator of innate immune responses cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (lps) to lps binding protein primary structures of three human neutrophil defensins structure of a novel human granulocyte peptide with anti-acth activity production of beta-defensins by human airway epithelia synergistic and additive killing by antimicrobial factors found in human airway surface liquid human cathelicidin, hcap-18, is processed to the antimicrobial peptide ll-37 by extracellular cleavage with proteinase 3 processing of seminal plasma hcap-18 to all-38 by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina assignment of defensin gene(s) to human chromosome 8p23 the role of human neutrophil peptides in lung inflammation associated with α1-antitrypsin deficiency pharmacophore identification of a chemokine receptor (cxcr4) antagonist, t22 ([tyr(5,12),lys7]-polyphemusin ii), which specifically blocks t cell-line-tropic hiv-1 infection sensitivity of actinobacillus actinomycetemcomitans and capnocytophaga spp. to the bactericidal action of ll-37: a cathelicidin found in human leukocytes and epithelium defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens monocyte-chemotactic activity of defensins from human neutrophils activities of ll-37, a cathelin-associated antimicrobial peptide of human neutrophils effect of defensins on interleukin-8 synthesis in airway epithelial cells neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone protective effects of a novel 32-amino acid c-terminal fragment of cap18 in endotoxemic pigs ) β-defensin-2 expression is regulated by tlr signaling in intestinal epithelial cells cutting edge: 1,25-dihydroxyvitamin d3 is a direct inducer of antimicrobial peptide gene expression apolipoprotein a-i binds and inhibits the human antibacterial/cytotoxic peptide ll-37 defensin pattern in chronic gastritis: hbd-2 is differentially expressed with respect to helicobacter pylori status bactericidal/permeability-increasing protein (bpi) and lipopolysaccharide-binding protein (lbp): structure, function and regulation in host defence against gram-negative bacteria antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation structure, function, and membrane integration of defensins purification and characterization of human neutrophil peptide 4, a novel member of the defensin family regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense synergistic interactions between mammalian antimicrobial defense peptides beta-defensins: linking innate and adaptive immunity through dendritic and t cell ccr6 human neutrophil defensins selectively chemoattract naive t and immature dendritic cells ll-37, the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (fprl1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells mammalian defensins in immunity: more than just microbicidal many chemokines including ccl20/mip-3alpha display antimicrobial activity antimicrobial and protease inhibitory functions of the human cathelicidin (hcap18/ll-37) prosequence cathelicidins, multifunctional peptides of the innate immunity conventional mechanical ventilation is associated with bronchoalveolar lavage-induced activation of polymorphonuclear leukocytes: a possible mechanism to explain the systemic consequences of ventilator-induced lung injury in patients with ards regulation of activities of nk cells and cd4 expression in t cells by human hnp-1, -2, and -3 widespread expression of beta-defensin hbd-1 in human secretory glands and epithelial cells we would like to acknowledge support for the authors' research from the applied food and materials network, the canadian bacterial diseases network and the functional pathogenomics of mucosal immunity program funded by genome prairie and genome bc, with additional funding from inimex pharmaceuticals. rewh holds a canada research chair, dmeb is supported by a cihr studentship, and djd was funded by a wellcome trust uk, international prize travelling research fellowship (060168). key: cord-253844-y6xdcf20 authors: yesudhas, dhanusha; srivastava, ambuj; gromiha, m. michael title: covid-19 outbreak: history, mechanism, transmission, structural studies and therapeutics date: 2020-09-04 journal: infection doi: 10.1007/s15010-020-01516-2 sha: doc_id: 253844 cord_uid: y6xdcf20 purpose: the coronavirus outbreak emerged as a severe pandemic, claiming more than 0.8 million lives across the world and raised a major global health concern. we survey the history and mechanism of coronaviruses, and the structural characteristics of the spike protein and its key residues responsible for human transmissions. methods: we have carried out a systematic review to summarize the origin, transmission and etiology of covid-19. the structural analysis of the spike protein and its disordered residues explains the mechanism of the viral transmission. a meta-data analysis of the therapeutic compounds targeting the sars-cov-2 is also included. results: coronaviruses can cross the species barrier and infect humans with unexpected consequences for public health. the transmission rate of sars-cov-2 infection is higher compared to that of the closely related sars-cov infections. in sars-cov-2 infection, intrinsically disordered regions are observed at the interface of the spike protein and ace2 receptor, providing a shape complementarity to the complex. the key residues of the spike protein have stronger binding affinity with ace2. these can be probable reasons for the higher transmission rate of sars-cov-2. in addition, we have also discussed the therapeutic compounds and the vaccines to target sars-cov-2, which can help researchers to develop effective drugs/vaccines for covid-19. summary: the overall history and mechanism of entry of sars-cov-2 along with structural study of spike-ace2 complex provide insights to understand disease pathogenesis and development of vaccines and drugs. angiotensin-converting enzyme 2 ccl2 chemokine (c-c motif) ligand the sars-cov-2 is a single, positive-strand rna virus that causes severe respiratory syndrome in humans [1] . the coronavirus disease 2019 (covid-19) has emerged as a severe pandemic, claiming more than 0.8 million lives worldwide between december 2019 and august 2020 [2, 3] . compared to sars-cov, sars-cov-2 human-to-human infection is more readily transmitted and spread to almost all continents leading to the who's declaration of a public health emergency of international concern (pheic) on january 30, 2020 [3] [4] [5] . generally, coronaviruses can cause respiratory, gastrointestinal, and central nervous system diseases in humans and animals, threatening humans life and causing economic loss [6, 7] . these viruses also have the capacity to adapt to a new environment through mutations and are programmed to modify host tropism; thus, the threats are constant and long-term [6, 8, 9] . including sars-cov-2, other coronaviruses cross the species barrier into humans, which lead to outbreaks of severe and fatal respiratory diseases. the sars-cov was first identified in bats, and spread to other animals in different geographic regions. the sars-cov outbreak was first emerged in humans in 2003, through transmissions from animals in open-air markets in china [10, 11] . thereafter, a higher number of genetically related viruses were also identified in chinese horseshoes bats (rhinolophus sinicus) [11] [12] [13] . coronaviruses belong to the family coronaviridae and are divided into alpha (α-cov), beta (β-cov), gamma (γ-cov), and delta (δ-cov) coronaviruses. the alpha and betacoronaviruses can infect mammals, and the viruses found in humans are genetically similar to β-cov genus. the β-covs are further divided into different lineages (a, b, c, and d lineages): sars-cov and sars-cov-2 are grouped in lineage b, which has approximately 200 published virus sequences, whereas mers-cov belongs to lineage c, which has ~ 500 viral sequences [11] . the hcov-229e and hcov-nl63 belong to the alphacoronavirus family, whereas hcov-oc43, hcov-hku1 and sars-cov are betacoronaviruses [14] [15] [16] . the phylogenetic analysis shows that sars-cov-2 protein is firmly rooted in the β-genus lineage of bat coronaviruses [14] . the whole genome of sars-cov-2 shares 80% identity with that of sars-cov and is 96% identical to the bat coronavirus batcov-ratg13 [17] . the spike protein sequence similarity between sars-cov-2 and sars-cov is around 76-78%. the rbd alone shares a similarity of 73-76%, and rbm shares 50-53%. in contrast, the human mers-cov is related to tylonycteris bat coronavirus hku4, shares less sequence similarity (~ 54%) and recognizes dpp4 as their receptor. the sequence similarity between sars-cov-2 and sars-cov spike proteins explains the possibility of binding to the same receptor angiotensin converting enzyme 2 (ace2) in the host cell [14] . coronavirus is one of the largest genomes among all rna viruses ranging from 27 to 32 kb. receptor-mediated endocytosis is the main process of virus entry to the host cells. sars-cov-2 uses ace2, a cell-surface receptor that is present in the kidney, blood vessels, heart, and importantly, in the lung at2 alveolar respiratory tract epithelial cells for viral infection [18] . the spike protein, which is responsible for the viral entry, has n-terminal and c-terminal domains, and two major subunits s1 and s2 are present in almost all coronaviruses [6] . one of these s1 or s2 subunits binds with the host receptors and acts as a receptor-binding domain (rbd) (fig. 1a) . next-generation sequencing technology (ngs) revolutionized the biological sciences, including virus discovery. the ngs made it easy to recognize thousands of novel virus sequences from wild animal and human populations around the world. despite these vast coronavirus sequences are published, very little work has been performed for further studies. further, the lack of tools to test these novel viruses and their ability to infect humans hindered the efforts to predict the subsequent zoonotic viral outbreaks [11] . understanding the virology of coronaviruses and the methods to control their spread is currently a necessary task to maintain the global health and economic stability. in this review, we discuss the history of coronaviruses in both humans and animals, their transmissions, mechanism of host cell entry and the structural studies, explaining active and inactive receptor binding of spike protein and the key residues playing an important role in the receptor binding. the drug repurposing and the therapeutic targets for sars-cov-2 are also discussed. until the first identification of human coronaviruses 229e and oc43, in the late 1960s, coronavirus infections were witnessed as harmless for humans [14, 15] . the outbreak of sars-cov in southern china in the winter of 2002, took a fatality rate of 10% of the infected patients [18] [19] [20] . the virus had been rapidly spreading throughout the world, especially in asia, and controlled after july 2003 [21] . viral analysis of the outbreak of sars showed that bats are natural reservoirs for sars-covs, and civet cats and raccoon dogs are the intermediate hosts. in the year 2012, a novel highly pathogenic middle east respiratory syndrome coronavirus (mers-cov) was identified in humans, demonstrating that the coronaviruses are transmitted from animals to humans at any time and with unexpected consequences for the public health [22] . mers-cov, the slow-spreading virus, has affected ~ 1700 people with a fatality rate of ~ 36% [6, 22] . the animal sources of sars-cov-2 infections are bats and sars-cov-2 can be transmitted to cats, pangolins, and dogs [23] . other than humans, the coronaviruses have a big impact on the animal kingdom. animal coronaviruses can cause severe threat to their hosts; since 1984, an unrecognized infection massively spread among the swine population in europe, and later it was identified as porcine epidemic diarrhea coronavirus (pedv), which is derived from the porcine enteric coronavirus tgev [24] . in 2013, the same pedv in less than a year had caused a 100% fatality rate in piglets and rubbing out more than 10% of america's pig population [5, 25, 26] therefore, the total number of human coronaviruses identified has been increasing throughout the years. hcov-229e and hcov-oc43 are the first two discovered human coronaviruses in the 1960s, hcov-nl63 and hcov-hku1 human coronaviruses identified after the sars-cov outbreak in [22] . however, the story continues with the new identification of sars-cov-2 in december 2019 at the seafood wholesale market in wuhan, china. sars-cov-2 is the seventh member of the family of coronaviruses that infects humans and it is different from both mers-cov and sars-cov. although some infections caused by human coronaviruses are mild and associated with common colds, certain animal and human coronaviruses can make a severe impact on the human population. especially in young children, elderly people, and immune-deficient patients, the infections can be lethal [9, [27] [28] [29] . therefore, it is important to understand the mechanism of invading viruses to theirs hosts, transmission and prevention of these processes. the respiratory droplets are the main routes of transmissions; sars-cov-2 can be transmitted to a healthy person if he happens to have contact with the infected person or any of his belongings, including clothes, doorknobs, etc. studies have been reported that aerosol transmission (airborne transmission) is also possible for sars-cov-2, but there is no clear study on neonatal infections (mother to child) [30] [31] [32] [33] [34] [35] [36] . however, the transmission can be avoided by keeping a distance of 2 m between two people, wearing masks while going out, and the isolation of infected people. during the initial phase of the covid-19 outbreak, a dataset was obtained from 1099 patients with laboratoryconfirmed covid-19 from 552 hospitals in 30 provinces of china on january 29, 2020. only 2% of the patients had a history of contact with animals; more than three quarters have either visited the wuhan city or are residents. hence, the outbreak patterns or the source of infection could not be predicted from their study. the incubation period of the sars-cov-2 was from 1 to 12 days; however, the median incubation period was 4 days [32] . the most common symptoms are fever (43% on admission, and 88.7% during hospitalization), cough (67.8%), diarrhea (3.8%), and fatigue [32, 37] . the sars-cov-2 was detected in saliva, blood, sputum, and urine before the development of viral pneumonia, and some patients do not develop pneumonia at all. asymptomatic persons are potential sources of sars-cov-2 infection, which control the transmission dynamics of the current outbreak [32, 38] . the sars-cov-2 first identified in wuhan, china has spread all over the globe. as of august 20, 2020, more than 22 million confirmed infection cases and 0.8 million deaths had been reported across the world, including almost all the countries. the rate of infection or the average number of people getting infected by an individual (r0) was 2.75 in the case of sars pandemic in 2003. the r0 value of ebola 2014 was in the range of 1.51-2.53, and h1n1 influenza 2009 was from 1.46 to 1.48, and for mers, it was around 1. the sars-cov-2 r0 value was estimated to be in the range of 1.5-3.5. the comparison of r0 values of various coronaviruses shows that the difference is minimal. however, the difficulties arising for sars-cov-2 infection are due to the following: (1) basic properties of the viral infection and the infection periods are uncertain, (2) most of the infected individuals do not show symptoms, but are capable of spreading the infection, (3) changing susceptibility of the population in affecting the spread of infection remains unanswered. in addition, there are no control measures for this spread [39] . coronaviruses consist of four structural proteins: the nucleocapsid protein (n) forms the helical capsid to accommodate its genome. the whole structure is further surrounded by a lipid envelope, which is made of s (spike), e (envelope) and m (membrane) proteins. the membrane and the envelope proteins are needed for the virus assembly and the spike protein is for virus entry and host cell recognition [6] . the spike protein forms large protrusions (peplomers) on the virus surface (looks like the virus has crowns), and hence it is named as "corona" (corona is a latin word which means crown). it comprises three segments: (1) large ectodomain, (2) transmembrane domain and (3) intracellular tail. the receptor-binding subunits s1 and s2 are placed in the ectodomain region. during the infection, the s1 binds with the host receptor, and s2 fuses the host and viral membranes, thereby releasing the viral genome into the cell (fig. 1 ). the spike protein is a clove-shaped trimer with three s1 heads and a trimeric s2 stalk [6] . during viral infection, spike protein (~ 1300 amino acid residues) is cleaved by host proteases into receptor binding subunit s1 and membrane fusion subunit s2. during cell entry, the s1 subunit binds directly to the sugar receptors [40] and ace2 of the host cell surface, and the s2 subunit undergoes conformational changes and obtains post-fusion state [41] . during this state, the three pairs of heptad repeat region hr-n and hr-c in trimeric s2 form a six-helix bundle structure [42] . the buried hydrophobic fusion peptides become exposed and insert into the target host membrane. these fusion peptides and the transmembrane anchors are positioned at the end of a six-helix bundle structure, bringing the viral and host membranes to fuse [42, 43] . during this process, a large amount of energy is released, which accelerates the membrane fusion forward. along with this, receptor binding and low ph can also trigger this membrane fusion [6] . since the spike protein has a good binding affinity for sugar receptors of human cells, it uses them as a mechanism of cell entry [6] . notably, the sars-cov-2 has a higher affinity to human ace2 than the sars-cov virus strain. the ectodomain of the sars-cov-2 spike protein binds to the peptidase domain (pd) of ace2 with a k d (equilibrium dissociation constant) of ~ 15 nm [4] . spike protein priming is done by transmembrane protease serine 2 (tmprss2), which is also essential for the entry of sars-cov-2 [44] . generally, sars-cov enters host cells through endocytosis, where its spike protein is processed by cathepsin l and cathepsin b lysosomal proteases. the extracellular proteases, including elastase in the respiratory tract and tmprss2 on the surface of flung cells are also known to activate spike membrane fusion [6] . the viral entry to the host cells can be via: (1) the endocytic pathway and (2) non-endosomal pathway [45] (fig. 1b) . the endocytic pathway, especially clathrindependent endocytosis is extensively studied for sars-cov and mers-cov viral entry. since the sars-cov-2 also uses the same receptor as sars-cov, it is reported that sars-cov-2 also uses the same viral entry mechanism. wang et al. [46] reported clathrin-and caveolaeindependent endocytic pathways for sars-cov entry. despite the common use of endocytic pathway as a viral entry mechanism, the discrepancies about the same are unavoidable. thus, the exact nature of the viral entry is context-dependent, including the type of the virus and the host cells [47] . in addition, the macrophages can also act as a viral reservoir and support minimally for the sars-cov-2 entry and its replication. although the dendritic cells and other immune cells are not infected by sars-cov-2, they may serve as a transporter for the viruses, which is also responsible for the pathogenesis [48, 49] . the s1 subunit of sars-cov spike glycoprotein is predominantly composed of β-strand structures, composed of an n-terminal domain (ntd, residues 14-294) and three c-terminal domains (ctd1, residues 320-516; ctd2, residues 517-578 and ctd3, residues 579-663). the ntd is linked with ctd1 through the linker residues 295-319, where ctd1 acts as a potential rbd for sars-cov and binds explicitly with the ace2 receptor. gui et al. [43] reported that the sars-cov spike glycoprotein trimer could obtain different conformations, which are necessary for effective binding with ace2. all these conformations are termed based on the position of ctd1 of the spike glycoprotein (fig. 2) . the three spike glycoprotein monomers intertwine with each other and form densely packed homotrimer. the head portion of this trimer is taken place by ntds and ctd1s of s1 subunits, where the ctd1s are located at the center and the ntds are located outside of this triangular head. the s2 subunits represented as a stem for this trimer, which is further surrounded by ctd2s and ctd3s of the s1 subunits (fig. 2) . in the inactive state (fig. 2a, b) , s2 subunits (stem portion) are completely covered by the "down" position of ctd1s (head portion), which causes steric clashes for the binding between spike protein and ace2. in the active state (fig. 2c) , two ctd1s adapt the "down" conformation and one ctd1 rotates outward and obtains "up" conformation, which does not cover the s2 subunit and allows the interactions between spike protein and ace2. the "up" position also paves the way for the s2 subunit to expose and insert its fusion peptides into the host cell membrane [43] (fig. 2) . the sequence similarity between sars-cov-2 and sars-cov spike protein explains the possibility of having the same receptor ace2 in the host cell [14] . the sequence and the structural studies revealed the key residues, which are involved in spike-ace2 interactions. the key residue at position 493 in rbd of sars-cov-2 is gln, wherein sars-cov (479 is the corresponding residue in sar-cov) of civets and humans, it is lys and asn, respectively. since the residue 479 of rbd is near to the virus-binding hotspot residue lys31 of human ace2, the lys residue present in civet causes steric clashes and not favoring human ace2 receptor binding. however, the lys479asn mutation revealed that the asn present in 479 of human sars-cov enhances the viral binding with human ace2 receptors. the gln493 in sars-cov-2 rbd is compatible with the human ace2 receptor hotspot residue lys31, which explains its target cell identification [14] . similarly, the residue 501 in sars-cov-2 rbd is asn, wherein civet rbd, it is ser (487 is the corresponding residue in sars-cov), and for humans, it is thr. this residue also plays an important role in making interaction with the hot spot residue lys353 of receptor ace2. ser487thr mutation analysis shows encouraging results upon human ace2 receptor binding and plays a role in human-to-human transmission. thus, the interactions with lys353 will be favorable for threonine (human sars-cov) and asn (sars-cov-2) than serine (civet) [14] . the residues lue455, phe486, and ser494 in sars-cov-2 rbd (tyr442, leu472, and asp480 in human and civet sars-cov) are considered as important for the human ace2 receptor binding. tyr442 of human and civet sars-cov rbds shows unfavorable interactions with hotspot residue lys31 of human ace2; however, lue455 of sars-cov-2 provides favorable interactions. compared with leu472 of human civet sars-cov rbd, phe486 of sars-cov-2 provides better interaction with hotspot residue lys31 of human ace2. although ser494 provides positive support for the human ace2 receptor hotspot residue lys353, the sars-cov asp480 also makes favorable interaction with hotspot residue lys353. throughout this viral entry process, the lys31 and lys 353 of human ace2 receptors are termed as "hot spot" residues, which consist of a salt bridge buried in a hydrophobic environment and contribute critically for the virus and host cell receptor binding. thus, this key residue comparison of sars-cov-2 with the civet and human sars-cov explains how actively sars-cov-2 is choosing and binding with the human ace2 receptors, which is likely to cause the human-to-human transmission [14] . the heterogeneity of amino acids in ace2 receptors is also responsible for the wavering binding affinities between the host and the virus, which is associated with the viral transmission. however, the variants in the host cell receptors (ace2) can confer resistance against the invading pathogens. hussain et al. reported that the mutations s19p and e329g in ace2 disrupt the intermolecular interactions and have low binding affinity with viral spike protein. in addition to the variations in the viral spike protein, ace2 allelic variants can also drive the potential resistance against sars-cov-2 infection [50] . intrinsically disordered proteins (idps) and intrinsically disordered regions (idrs) also play major roles in a number of biological functions, including dna/rna binding, protein binding, and facilitating access to the binding sites between the binding partners [51, 52] . the rna-protein recognition often needs conformational changes in both rna and protein, which is facilitated by the structural flexibility of disordered residues [51] . also, the functional importance of intrinsically disordered regions in proteins includes transcription, translation, post-translational modifications, and cell signaling [51, 53] . the categorization of coronaviruses, based on the intrinsic disorder propensities, can represent useful identification for the viral life cycle and its pathogenicity. the idrs in sars-cov nucleocapsid protein comprise three segments, such as 1-44, 182-247 and 366-422 [54, 55] . the highly flexible intrinsic disordered linker region, which connects the ntd and ctd is rich in serine and arginine residues. an intrinsically disordered domain that flanks the ctd (c-terminal tail peptide) plays a significant role in dimer-dimer association in human coronavirus 229e (hcov-229e). likewise, the coronavirus hku1, has a partially disordered conserved linker loop (amino acids 428-587) structure [56] . hku1 s2 subunit also shows the presence of disordered residues at its protease cleavage site [57] . in sars-cov-2, the attachment of spike protein with the host cell is activated by the host cell enzymes trypsin, cathepsin l, furin and tmprss2 (fig. 1b) . the sequence comparison of sars-cov-2 against other lineage b betacoronaviruses shows that the unique amino acid pattern "rrar" is present at the s1/s2 junction of the spike protein, which is cleaved by the furin enzyme. however, the structure reported for sars-cov-2 spike protein (pdb code: 6vsb), shows that s1/s2 junction is in a disordered, solvent-exposed loop [4] . hence, it has been hypothesized that the unique amino acid sequence "rrar" present in sars-cov-2 is responsible for their effective transmission [58] [59] [60] . the binding with ace2 is governed by the intrinsically flexible receptor binding motif, and the binding interfaces along with the key residues are reported in the literature [61, 62] . in addition, based on our analysis (fig. 3) , the monomeric structure of the spike protein in the free form (pdb id: 6vsb; green color) [4] shows a number of missing residues in the structure. these missing residues of the spike protein attain a stable conformation upon binding to the ace2 receptor and hence, they are termed as disorderto-order transition (dot) residues (pdb id: 6lzg; light golden color). specifically, the regions, leu455 to pro491 and asn501 to val503 show disorder-to-order transitions [61] . these disordered-to-ordered residues are facilitating a better shape complementary and affinity between ace2 and spike protein. interestingly, the key residues responsible for transmission, and interaction with ace2 receptors (already discussed in section "key residues of sars-cov-2 and sars-cov") are overlapping with these mentioned disordered-to-ordered residues. based on these observations, the disorder-to-order conformational change is necessary to facilitate the spike protein binding with its receptor. thus, an in-depth analysis of these disordered residues will shed additional insights on the viral recognition and transmission mechanism. the studies by goh et al. [63] on 1918 h1n1 and h5n1 explain that it is very likely that disordered regions are important for host specificity and recognition, e.g., across species of birds. they also explained the changes created by disorder in crucial regions, increasing the virulence of both the h5n1 and the 1918 h1n1 viruses. therefore, the increasing reports for the intrinsically disordered regions in coronaviruses need to be pointed out. the importance and functional role of intrinsically disordered regions need to be thoroughly studied. this could identify additional targets for drugs to combat coronavirus through the disruption of their packing and assembly process. the receptor binding, along with its membrane fusion, is the initial and important step in the coronavirus infection and serves as primary targets for inhibiting the viral entry. the genome sequencing of sars-cov-2 and the comparison of the genomes of related virus proteins suggested the anti-hiv lopinavir plus ritonavir combination can be likely effective [15, 64, 65] . sars-cov-2 uses the ace2 receptor of at2 cells in the lung as its primary targets. since the viral entry is governed by receptor-mediated endocytosis, ap2-associated protein kinase 1 (aak1), a known regulator of endocytosis, can be a good target to interrupt the virus entry. the janus kinase inhibitor baricitinib, and its binding with the cyclin g-associated kinase (endocytosis regulator) is sufficient to inhibit aak1 [66, 67] . similarly, sunitinib and erlotinib, the oncology drugs, have been shown to inhibit viral infection of cells through the inhibition of aak1 [68] . however, these compounds bring serious side effects and cannot be considered for a safe therapy. fig. 3 structure of the monomeric spike protein (green)-ace2 receptor (blue) complex. the interface residues are shown in light golden. the disordered-to-ordered transition residues (leu455 to pro491 and asn501 to val503) have been marked in the figure based on the pathogenicity studies of sars-cov and mers-cov, an increased amount of inflammatory cytokines in serum is associated with the inflammation and extensive lung damage [69] . similarly, sars-cov-2 also has a high amount of il1b, ifnγ, ip10, and mcp1/ccl2, which lead to t-helper-1 (th1) activation. however, sars-cov-2 is shown to increase the t-helper-2 (th2) cytokines (il4 and il10) that are known to suppress inflammation, which differs from sars and mers-cov infection. in view of cytokines induced by sars-cov-2 and other sars-cov, mers-cov, corticosteroids were frequently used to reduce the inflammation-induced lung injury [2] . arabi et al. [70] examined the combination of interferon beta-1b, lopinavir, and ritonavir combination for the mers infection in saudi arabia. the antiviral nucleotide prodrug remdesivir showed a potent efficacy on mers-cov and sars-cov infections. since sars-cov-2 is an emerging virus, no effective treatment has been developed so far, yet the already available combination of lopinavir and ritonavir is being used [2] . since the sars-cov enters the cell through endocytosis and the lysosomal protease is priming the spike protein, targeting/inhibiting the endosomal acidification or lysosomal cysteine protease can block the sars-cov entry [6] . the view of the activation of sars-cov and sars-cov-2 by tmprss2 might also have therapeutic suggestions. a protease inhibitor, camostat mesylate, which is studied as a tmprss2 inhibitor and has been used to treat humans, is available and could be employed as a defense against the respiratory viruses [71, 72] . autophagy has been implicated in viral replications, which is also responsible for the formation of doublemembrane vesicles (dmv) in the host cells [73] . since the autophagosomes are degraded by lysosomes, inhibitors like lysosomotropic agents have been proposed for sars-cov-2 [74] . the antimalarial drugs hydroxychloroquine (hcq) and chloroquine (cq) have been demonstrated for sars-cov-2 antiviral activity. however, data to support the use of hcq and cq for covid-19 are incomplete [75] . these lysosomotropic agents are helpful for neutralizing the endosome-lysosomal acidic ph, thereby blocking protease activity and subsequently blocking the viral entry. however, how autophagy is implicated in the infection of covs is still under debate [47, 74, 76] . table 1 describes the different inhibitors used for blocking the viral entry. the most studied and the common pathway proposed for all coronaviruses is the endocytic pathway, so blocking that pathway is a big hallmark for treating the disease. identification of potential drugs for sars-cov-2 is a necessary task, so drug repurposing can also work for this scenario. the small molecules, which are already in clinical trials or used for some other diseases and the molecules from the expert's opinion are also listed in table 2 . the table is categorized into three groups: (1) compounds involving drug repurposing, (2) compounds in clinical and pre-clinical study, (3) compounds targeting the mechanism pathway. researchers in government and private sectors are making huge efforts to develop effective vaccines for sars-cov-2. the vaccine development approaches are mainly based on inactivated and attenuated viral protein particles, viral vectors and viral dna/rnas. a novel rna-based vaccine uses a part of the genetic code of spike protein mrna-1273 (clinicaltrials.gov: nct04283461) [139] . several other mrna-based vaccines by curevac (tübingen, germany), bnt162 by biontech (mainz, germany), pfizer (new york, ny, usa) and biontech mrna vaccine (mainz, germany) are in different stages of development [139, 140] . cansino biologics (tianjin, china), the company that developed the vaccine for ebola is also developing a vaccine named ad5-ncov for sars-cov-2. it is a spike protein-based vaccine that is undergoing phase i clinical trials in healthy individuals in wuhan china (clinicaltrials. gov: nct04313127) [141, 142] . the current status of the vaccines under development is available at the milken institute treatment and vaccine tracker: https ://milke ninst itute .org/sites /defau lt/files /2020-03/ covid 19%20tra cker%20032 020v3 -posti ng.pdf [141] . sars-cov-2 is a continuously growing life-threatening disease. this coronavirus has rapidly evolved and spread all over the world, having a mortality of more than 0.8 million lives so far. the exact origin and mechanism of attack and spatial distribution are still not completely explored. the viral and the host cell proteins assisting in invasion process can be a target for treating the infections. the available crystal structures and the binding mechanism proposed by other coronaviruses can help to study the sars-cov-2. the critical structural studies of the viral particles are also helpful to identify the drug targets. the positional changes (up and down conformation) of spike protein trimer determine the active to inactive state. thus, targeting these conformations and developing small molecules or peptides can also stop the viral entry. in addition, phylogenetic analysis and structural studies revealed that the hot spot residues, the role of gln493 in human ace2 receptor binding and the critical residue asn501 of rbd explain the human-tohuman transmission. the intrinsic disorder region and a precise furin-like cleavage site can be responsible for viral cycle and pathogenicity. however, in-depth studies are needed to address this issue, which may represent a potential antiviral strategy. the small molecules, which are in clinical trials, the drug compounds, which 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782iew86 a new species of parvovirus tentatively named human bocavirus 4 (hbov4) was genetically characterized. among 641 feces samples from children and adults the most commonly detected bocaviruses species were hbov2>hbov3>hbov4>hbov1 with hbov2 prevalence of 21% and 26% in nigerian and tunisian children. hbov3 and hbov4 species combined were found in 12/192 cases of non-polio acute flaccid paralysis (afp) from tunisia and nigeria and 0/96 healthy tunisian contacts (p=0.01). evidence of extensive recombination at the np1 and vp1 gene boundary between and within species was found. the multiple species and high degree of genetic diversity seen among the human bocaviruses found in feces relative to the highly homogeneous hbov1 suggest that this world-wide distributed respiratory pathogen may have recently evolved from an enteric bocavirus, perhaps after acquiring an expanded tropism favoring the respiratory track. elucidating the possible role of the newly identified enteric bocaviruses in human diseases including afp and diarrhea will require further epidemiological studies. b19 in 1976, several other human parvoviruses have recently been genetically characterized. parv4 was found in the blood of a febrile adult intravenous drug user [5] , hbov1 in the nasopharyngeal secretion of a child with respiratory problems [6] , hbov2 in the stool of children with non-polio acute flaccid paralysis (afp) [7] , and hbov3 in the stool of australian children with diarrhea [8] . in contrast to parv4 or b19, bocaviruses contain a third open reading frame of unknown function [9, 10] . the first bocavirus identified was in cows [10] , and the name of the genus is derived from its first known hosts (bovine-canine). animal bocaviruses can cause both respiratory and gastrointestinal diseases, as well as embryonic and fetal death [11] . hbov1 infection has been linked with mild-to-severe, primarily lower respiratory tract infections in children, frequently in association with other viral infections [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . hbov1 has also been detected in stool samples, although association with diarrhea appears weaker than with respiratory symptoms [26] [27] [28] [29] [30] [31] . hbov1 strains show a very low degree of genetic variability worldwide [32, 33] . in this study, pan-bocavirus polymerase chain reaction (pcr) primers were designed and used on human stool samples from several countries. all 3 recently identified bocaviruses plus a fourth species, hbov4, were identified. a high degree of genetic diversity, relative to that seen for hbov1, was seen among human bocaviruses in feces. stool samples were collected as part of previous clinical studies and were anonymized. this study was approved by the university of california san francisco committee on human research. samples from nigeria and tunisia were collected as part of the world health organization's poliovirus eradication program from children with non-polio afp between the ages of 4 months and 15 years. stool samples from healthy contacts of case patients from tunisia were matched for age. stool samples from nepal were obtained from adult travelers and resident expatriates with diarrhea with no known pathogens detected by standard microbiologic testing for enteric bacteria; enzyme immunoassay for rotavirus, adenovirus, astrovirus, giardia and cryptosporidium; and reverse transcription pcr for norovirus. stool samples from healthy, asymptomatic control persons were collected from the same population. stool samples from the minnesota department of health are from individuals with diarrhea and healthy individuals, matched for age and residential area code. pcr amplification of bocaviruses. we used nested pcr targeting the vp1/2 region of both hbov1 and hbov2 (nucleotide positions 3233-3808, numbered here and subsequently with use of the hbov2 prototype sequence; genbank accession number fj170278). nucleic acids (both dna and rna) were extracted (qiaamp viral rna mini kit) from 140 ml of clarified stool supernatant and were eluted into 60 ml of water. first-round pcr primers were ak-vp-f1 (5 -cgccgtggct-cctgctct-3 ) and ak-vp-r1 (5 -tgttcgccatcacaaa-agatgtg-3 ) and second-round primers were ak-vp-f2 (5 -ggctcctgctctaggaaataaagag-3 ) and ak-vp-r2 (5 -cctgctgttaggtcgttgttgtatgt-3 ). pcr reactions contained 2.5 u of taq dna polymerase (neb) in 1.1ϫ thermopol reaction buffer with mgcl (2.0 mmol/l), 20 pmol/ l (each) of forward and reverse primers, and 2.5 ml of nucleic acids (first round) or 1 ml of the first-round pcr product (second round) as template in a 50 ml total volume. firstround conditions were 10 cycles at 95њc for 35 s, 58њc for 1 min, and 72њc for 1 min, with a decrease of 0.5њc in annealing temperature each cycle; 30 cycles at 95њc for 30 s, 54њc for 45 s, and 72њc for 45 s; and a final extension at 72њc for 10 min. similar conditions were used for the second pcr round, except that the initial annealing temperatures were 60њc and 58њc in the first and second group of pcr cycles, respectively. amplicons of the appropriate size, as detected by agarose gel electrophoresis, were directly sequenced. the pcr products whose sequences are reported here produced unambiguous dideoxysequencing electrophoregram peaks indicating the predominance (190%) of a single bocavirus variant. the sensitivity of the pan-bocavirus nested pcr was determined using dilution of plasmids containing the target sequences from each of the 4 bocavirus species and was estimated at 10-100 plasmid copies for each species. complete genome sequencing. nearly complete genomes were amplified using pcr primers designed from alignments of hbov1 and hbov2 genomes and were then directly sequenced by primer walking [7] . the terminal sequences were acquired by a modified protocol for rapid amplification of complementary dna ends [5] . the terminal sequences are incomplete because of extensive hairpin structures that prevented extensions to the viral 5 and 3 extremities. distance measurements and phylogenetic analyses. phylogenetic relationships were evaluated with use of mega 4.1 (http:// www.megasoftware.net/mega41.html) [34] . neighbor-joining trees were inferred using a matrix of pair-wise maximum-likelihood distances computed from a nucleotide alignment including the genomes obtained in this study and in genbank, plus a matrix of pam distances computed from the inferred amino acid alignment. recombination analyses. similarity values based on jukes-cantor corrected nucleotide distances between full-length sequences were calculated using the program sequencedist in the simmonic2005 v1.6 sequence editor package [35] . to assess similarity across the genomes, sequence scans were performed using a fragment length of 300 bases and an increment of 9 bases between fragments. for sequence comparisons with hbov1 and hbov2, a mean pair-wise distance was computed using a set of 14 hbov1 and 7 hbov2a sequences. nucleotide sequence accession numbers. the near-full genomes and partial vp1 gene sequences have been deposited in genbank under accession numbers fj973558-fj973563 and gq506558-gq506661. pan-bocavirus pcr primers were designed that could anneal to both hbov1 and hbov2, amplifying a ∼576-nucleotide fragment of the vp1 capsid gene. nucleic acids from human stools collected from nigeria, tunisia, nepal, and the united states were then analyzed using this nested pcr (table 1) . of the 641 samples tested, 101 (16%) had confirmed positive results by pcr sequencing, with the highest prevalence in tunisian children with afp (33%). to determine the phylogenetic relationship of these strains, the sequences were aligned with available sequences of hbov1, hbov2, and hbov3. only 4 of 101 strains (from nigeria and tunisia) grouped with hbov1. the remaining strains shared a more recent common ancestry with hbov2, although the sequences clustered into 4 distinct genetic lineages, labeled hbov2a, hbov2b, hbov3, hbov4 ( figure 1 ). the hbov2a clade included all published hbov2 genotypes [7] , plus 4 new strains from nigeria and tunisia and the recently reported w153 strain from australia [8] . because of the high degree of genetic diversity observed and to reduce splitting of hbov2 into a multitude of genotypes, all strains in that cluster were reclassified into a new, now more diverse genotype a (hbov2a). the nearly full genome of a new hbov2a variant was sequenced (tu-c-114-06) and showed very high protein identity (198%) to the pakistani hbov2 prototype pk5510 (fj170278). the hbov2b cluster included 76 of the 101 bocavirus strains reported here ( figure 1 ). two nearly full hbov2b genome sequences demonstrated a pair-wise amino acid divergence in vp1 of 0.45% (table 2 ) (ni-213 and ni-327; genbank accession numbers fj973560 and fj973559). greater divergence was observed when these strains were compared with the vp1 of hbov2a strains (average divergence, 3.9% [range, 3.3%-4.5%] ( table 2 ). the nucleotide and amino acid distances at the other loci are shown in table 2 . the hbov3 cluster included 11 strains, and the genomes of 2 representative strains, ni-374 and tu-a-210-07, were sequenced (genbank accession numbers fj973563 and fj973562). when compared with hbov2, the ns1 region of both strains showed an average amino acid divergence of 26% in the ns1 region and 9% in the vp1 region. compared with hbov1, the hbov3 strains showed an average amino acid divergence of 12% in the ns1 region and 20% in the vp1 region ( table 2 ). all 11 strains were classified as members of the hbov3 species, recently described by arthur et al [8] . a distantly related variant of hbov3 was also recently identified in a sewage sample in the united states (hbov3b-ca-1-c1; figure 1 ) [36] . to confirm whether this variant represents a second genotype of hbov3 will require full genome sequencing. the hbov4 cluster included 6 strains. the complete genome of 1 representative strain (ni-385) was obtained (genbank accession number fj973561). the ns1 protein of hbov4 (ni-385) showed an average of 11% divergence with hbov2 and 25%-27% divergence with hbov1 or hbov3 (table 2) . for the vp1 protein, the relative divergences were reversed, with an average divergence of 8.5% with hbov3, 9.5% with hbov2, and 19% with hbov1. ni-385, therefore, also appears to be a recombinant, with the 5 genes ns1 and np1 closely related to hbov2 (particularly genotype a) and the 3 vp1 gene slightly more similar to hbov3 than hbov2 (table 2 ). according to the international committee on taxonomy of viruses species demarcation criteria in the genus bocavirus, members of different species must show 15% divergence in their nonstructural gene nucleotide sequences [37] . the genetic distance of ni-385 to its closest relatives in the ns1 gene (hbov2a) was 10.8% (range, 6.8%-12.6%), indicating that ni-385 qualifies, pending international committee on taxonomy of viruses review, as the prototype of a fourth hbov species (hbov4). a distant vp1 (partial) variant of hbov4 was also detected in the united states (us-mn-964-05; figure 1 ). to determine whether the latter variant represents a second hbov4 genotype will require full genome sequencing. nearly complete genomes and phylogenetic analysis of new bocavirus species. in a manner similar to hbov1 and 2, all the new genomes of hbov2, 3, and 4 encoded 3 large open reading frames (orf). the left orf encodes the nonstructural protein (ns), the middle orf encodes np1, and the right orf encodes overlapping vp1/vp2 capsid proteins. conserved motifs associated with rolling circle replication, helicase, and atpase were identified within the ns protein. np1 is a highly phosphorylated protein of currently undetermined function [38] ; np1 differed in length between species, ranging from 214 to 219 amino acids. situated within the vp1-unique (vp1u) region, the phospholipase a2 motifs required for parvovirus infectivity were found in all 6 genomes, together with the calcium-binding loop and catalytic residues. further evidence of recombination in human bocaviruses. to further determine the relationship between members of the bocavirus genus, phylogenetic analyses of ns1, np1, and vp1/ vp2 were performed, with use of both nucleotide sequences and deduced protein sequences (figure 2 ). the ns1 and np1 genes of hbov3 clustered with hbov1, whereas their vp1/2 gene clustered with hbov2. the incongruence in phylogenetic association between loci provided further evidence that hbov3 originated from a recombination event bringing together the ns1/np1 gene of hbov1 and the vp1/2 gene of hbov2 [8] . the likely recombinant origin of hbov4, clustering with hbov2 in the ns1/np1 but with hbov3 in the vp1, is also shown (figure 2) . a scan of sequence divergence between complete genome sequences further supported the hypothesis of past recombi-nation between hbov1 and 2 in the generation of hbov3 and recombination of hbov2 and hbov3 in the generation of hbov4, with both recombination points near the np1 and vp1 junction (figure 3 ). when different hbov2 variants were similarly analyzed for recombination, intraspecies hbov2 recombinants were also detected (data not shown) [7] . diversity among respiratory hbov1 and enteric hbov2-4. we compared the intraspecies diversity of hbov1 with that of hbov2 by use of the partial vp1 sequence data generated with the pan-bocavirus pcr primers available for the greatest number of hbov2, 3, 4 variants ( figure 4a and 4b) . a very low average pair-wise difference was seen for hbov1 collected worldwide ( figure 4a ). hbov2, including both genotypes, was more diversified than hbov1, although hbov2b alone showed a low level of diversity comparable to that of hbov1 (hbov2b generated the large low divergence peak in figure 4a ). the homogeneity of hbov1 and hbov2b can also be visualized in the small branch lengths in figure 1 . when the pair-wise distances of all the enteric (non-hbov1) sequences were plotted ( figure 4c ), the distribution was much larger than that for hbov1. the low level of intraspecies diversity of hbov1 relative to the other species is also reflected in table 2 . the genomic organization of hbov species are remarkably similar to those of animal bocaviruses, except that all hbov ns orfs encode a shorter ns2 protein (630-650 amino acids), compared with the longer ns1 of animal bocaviruses (716-726 amino acids) ( figure 5a ). we noticed in all 4 hbov species the presence of a stretch of encoded amino acids similar to the c-terminus of the longer ns1 of animal bocaviruses overlapping the np orf, but in a different frame ( figure 5a) . genomes of all hbov species were aligned to determine the presence of conserved potential rna splicing signals near the end of the smaller ns2 orf and the putative second exon encoding the c-terminal region of ns1 (figure 5b). the putative ns1 resulting from such a spliced transcript encoded a 750-780 amino acid protein with a carboxy terminus that showed significant similarity to that of the canine and bovine bocaviruses ns1 ( figure 5c ) [39, 40] . a study using rt-pcr for the detection of hbov1 viral transcript in human lung epithelial cells did not detect the ns splicing proposed here [41] . on the other hand, the proposed ns1 rna splicing and ns1 protein expression itself were detected using northern blots and ns1 c-termini specific sera, respectively, in 293 and human epithelial cells transfected with plasmids expressing hbov1 transcripts (jianming qiu, personal communication). most pcr-positive stool samples contained hbov2b (76 of 101), making this genotype the most commonly detected enteric human bocavirus (table 1) . hbov3 was identified in 11 stool samples and hbov4 in 6, making them the second and third most common enteric human bocaviruses, respectively, in the regions analyzed here. hbov1 was detected in only 4 of the 101 positive samples. stool samples from patients with afp in nigeria and tunisia both showed a high prevalence of hbov2a+b (21%-26%). because both patients with afp and healthy contacts from tunisia showed a comparable prevalence of hbov2, no association was observed between afp and hbov2 excretion. the prevalence of hbov2 in adults with diarrhea from the united states and nepal were also compared with those in healthy matched subjects. no associations were observed between hbov2 shedding and diarrhea. although the numbers of hbov3 and hbov4 detected were relatively small, it was noticed that the 8 hbov3-and 4 hbov4-positive samples were found among 192 samples from patients with afp, whereas these 2 species were not detected among 96 healthy tunisian matched contacts. comparing the combined nigerian and tunisian patients with afp infected with hbov3 and hbov4 to tunisian controls yielded a 2-tailed fisher's exact p value of .01, whereas comparing only the tunisian patients with afp with tunisian controls gave a p value of .059. hbov3 was also found in 1 patient with diarrhea from the united states and from 1 healthy person each from the united states and nepal. hbov4 was also found in a patient with diarrhea and 1 healthy adult from the united states. further testing will be needed to confirm this trend of an association of hbov3/4 with afp in children or of the association of hbov2 with diarrhea [8] . we report on a previously uncharacterized species of human bocavirus which we tentatively named hbov4. hbov1, hbov2, and hbov3 were also detected using a pan-pcr approach. hbov2 has been detected in the stool of pakistani children [7] and hbov3 in stool samples of australian children [8] , and both were recognized as recombinant viruses. the newly reported australian hbov3 (eu918736) is closely related to a tunisian strain (ta-210-07) (figure 2) . a highly prevalent genotype of hbov2 (hbov2b), together with partial genomic support for second genotypes of hbov3 and hbov4, were also identified. the availability of novel bocavirus genomes will allow the design of species-specific pcr or microarray oligonucleotides for their detection and for the disease association studies that are now required for the 3 recently characterized enteric human bocavirus species (hbov2, hbov3, and hbov4). based on the phylogenetic clustering observed for a large number of partial vp1 sequences ( figure 1 ) and the distances among full genomes (table 2) , we propose for future classification that hbov strains showing 18% protein and 110% nucleotide difference in the complete vp1 gene should be considered different species, whereas those showing 11.5% protein and 15% nucleotide difference should be considered different genotypes. this vp1-based classification retains the 4 proposed human bocavirus species. the vp1 locus was selected because it is likely to strongly influence tissue tropism and potentially pathogenesis [42] . hbov1 is primarily, although not exclusively [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] , a respiratory virus. we show here a higher prevalence of hbov2 (particularly hbov2b) than hbov1 in stool samples from children and adults of different countries. a recent study testing for hbov1 and hbov2 dna with use of species-specific nested pcrs failed to detect any hbov2 in 16500 respiratory secretion samples from edinburgh and bangkok, whereas hbov1 was found in 3% and 14% of these respiratory samples, respectively [43] . another study found 5 (2%) of 212 nasopharyngeal samples from korean children with acute lower respiratory track infections to be positive for hbov2 dna, but unexpectedly, no hbov1 dna was found [44] . analyzing for hbov1 and hbov2 in both respiratory secretion and stool samples collected from the same individuals will be required to confirm whether the tropism of hbov2 favors the digestive track and is distinct from that of the largely respiratory hbov1. extensive evidence for recombination was observed through full genome analyses, including the likely recombinant origin of hbov3 and hbov4 and the high level of intraspecies recombination between hbov2 variants [7, 8] . the high prev-alence of bocavirus infection does provide the opportunity for coinfections, the first step in generating recombinant viruses. indeed, a hbov3 and hbov4 coinfection was detected based on the pattern of mixed bases in 1 directly sequenced pcr product (data not shown). the frequent detection of hbovs in stool samples from both healthy children and adults supports the likelihood of long periods of viral shedding and/or frequent reinfections. determination of whether symptoms such as diarrhea are associated with acute infection in subsequently healthy viral shedders will require quantitative viral load measurements, analysis of longitudinally collected samples, and serological assays. whether prior infection provides any protection against reinfection with the same or different genotype or species is also unknown. in both diarrhea sample sets analyzed here, consisting of samples from adults, no association with hbov2 shedding was detected. if hbov2 causes diarrhea, it may do so in only a small subset of infected children, possibly those without passively transferred maternal antibodies or without protective immune responses from prior infections. coinfections with other enteric viruses may also exacerbate symptoms. given the very large number of childhood infections (viral shedding prevalence of 120% in some developing countries), even low virulence would translate into a large disease burden. a trend of an association of hbov3/hbov4 detection with afp was detected. the small numbers of cases will require independent confirmation. the neurological damages caused by bovine and canine bocaviruses in their animal hosts provide a precedent for infant nervous system pathogenicity [11] . the totality of hbov1 sequences collected worldwide by multiple groups show very low protein and nucleotide sequence diversity (table 2 and figure 4a ) [35, 36] . in contrast, this single study found substantial diversity among hbov2 and hbov3, a fourth species (hbov4), and extensive viral recombination (figures 3, 4b, and 4c ). assuming comparable rates of evolution, the genetically homogeneous and largely respiratory hbov1, therefore, appears to be the more recently evolved species relative to the more diverse hbov2, 3, and 4 found predominantly in feces. we propose that hbov1 evolved from an enteric bocavirus ancestor that acquired, through mutation and/or recombination, enhanced respiratory tract tropism. single-stranded dna parvoviruses have been shown to have a mutation rate approaching that of rna viruses, and recombination among animal parvoviruses has also been reported [45] [46] [47] . parvoviruses have also demonstrated the capacity to rapidly expand their host species tropism, resulting in a recent pandemic in dogs [42, 48, 49] . a recent study showed that hbov1 could replicate in differentiated human airway epithelial cells [41] . whether hbov2, 3, and 4 show an in vitro tropism and in vivo distribution that is more biased towards digestive track cells will require further study. duration of immunity for canine and feline vaccines: a review review of companion animal viral diseases and immunoprophylaxis forty years of canine vaccination new dna viruses identified in patients with acute viral infection syndrome cloning of a human parvovirus by molecular screening of respiratory tract samples a newly identified bocavirus species in human stool a novel bocavirus associated with acute gastroenteritis in australian children human bocavirus complete nucleotide sequence and genome organization of bovine parvovirus animal bocaviruses: a brief review the association of newly identified respiratory viruses with lower respiratory tract infections in korean children human bocavirus infection human bocavirus in children severe pneumonia and human bocavirus in adult 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study clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong detection of human bocavirus in children hospitalized because of acute gastroenteritis human bocavirus, a respiratory and enteric virus human bocavirus infection in children hospitalized with acute gastroenteritis in china complete coding sequences and phylogenetic analysis of human bocavirus (hbov) genotyping of human bocavirus using a restriction length polymorphism mega: a biologist-centric software for evolutionary analysis of dna and protein sequences structural constraints on rna virus evolution frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the united states virus taxonomy. eighth report of the international committee on taxonomy of viruses virally coded noncapsid protein associated with bovine parvovirus infection molecular characterization of infectious clones of the minute virus of canines reveals unique features of bocaviruses the transcription profile of the bocavirus bovine parvovirus is unlike those of previously characterized parvoviruses human bocavirus can be cultured in differentiated human airway epithelial cells the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses absence of detectable replication of the human bocavirus species 2 in the respiratory tract despite frequent detection in faecal samples human bocavirus 2 in children rates of evolutionary change in viruses: patterns and determinants rapid sequence change and geographical spread of human parvovirus b19; comparison of b19 evolution in acute and persistent infections comparative analysis reveals frequent recombination in the parvoviruses phylogenetic analysis reveals the emergence, evolution and dispersal of carnivore parvoviruses high rate of viral evolution associated with the emergence of carnivore parvovirus we thank dr michael p. busch and blood systems research institute for sustained support and hope biswas for statistical assistance. key: cord-021375-lca26xum authors: voelkner, nadine title: riding the shi: from infection barriers to the microbial city date: 2019-08-23 journal: nan doi: 10.1093/ips/olz016 sha: doc_id: 21375 cord_uid: lca26xum how can a microbial approach to global health security protect life? contemporary infection control mechanisms set the human and the pathogenic microbe against each other, as the victim versus the menace. this biomedical polarization persistently runs through the contemporary dominant mode of thinking about public health and infectious disease governance. taking its cue from the currently accepted germ theory of disease, such mechanisms render a global city like hong kong not only pervasively “on alert” and under threat of unpredictable and pathogenic viruses and other microbes, it also gives rise to a hygiene and antimicrobial politics that is never entirely able to control pathogenic circulation. the article draws on recent advances in medical microbiology, which depart from germ theory, to invoke an ecological understanding of the human-microbe relation. here, while a small number of viruses are pathogenic, the majority are benign; some are even essential to human life. disease is not just the outcome of a pathogenic microbe infecting a human host but emerges from socioeconomic relations, which exacerbate human-animal-microbial interactions. in a final step, the article draws on daoist thought to reflect on the ways that such a microbial understanding translates into life and city dwelling. any visitor to hong kong will realize that disinfection is an urgent and pervasive imperative in contemporary everyday life in this global city. in late 2016, hong kong was on the way to fashioning itself an antimicrobial global city. public signs on multiple surfaces including elevator buttons, escalator handrails, and floor mats duly inform the passer-by of hourly or daily sanitation. at various busy urban spaces, including mtr (metro) stations, walkways, libraries, and office and housing complex receptions, free hand sanitizer dispensers compel the passer-by to engage regularly in the act of public cleansing of the body. similarly, handbag-sized instant hand sanitizer bottles, readily available at every corner store or supermarket, with promises of killing "99.99 percent of germs"; notices of hand-washing rituals; and the donning of masks by individual city dwellers, remind residents of the private acts of virus and bacteria control. all these practices of cleansing nourish an insidious sanitizing imperative in the defense against epidemic infections deeply enmeshed with the pulsating energies of an ambitious global city. it is the experience and fear of infection and death that accompanied the recent experience of the sars (severe acute respiratory syndrome) outbreak in 2003, which precipitated this aggressive stance to potential contagions emerging in hong kong. 1 li (2014) , a scholar of asian architecture, suggests that infection control in the form of public hygiene in contemporary postcolonial hong kong manifests itself in what he has termed "infection barriers." these antimicrobial-like-that is, antibiotic-and antiviral-like-physical and mental barriers aim to prevent the virus and other microbes from settling in the city. basing his interpretation on the traditional conception of the chinese city, "infection barriers" help him to analyze the historico-aesthetics of the city's contemporary urban defense against infectious diseases. li succinctly demonstrates how public hygiene is not only governed through overt public health programs but is affectively knit into both the fabric of the urban architecture and the tissue of the city population. infection barriers take the form of structures and widespread cleansing practices, which in li's eyes render hong kong a hospital disguised as a city. like other modern infection control mechanisms, the story of infection barriers in hong kong is one of setting the human and the virus against each other, as the victim versus the menace. this biomedical polarization, in which an external pathogen threatens the healthy human body, persistently runs through the contemporary dominant mode of thinking about public health and infectious disease governance (macphail 2002 (macphail , 2014 fishel 2015; white 2015; du plessis 2017) , which is rooted in the currently accepted germ theory of disease. it renders a city like hong kong pervasively "on alert"-albeit with chinese characteristics, as will be shown below. indeed, the polarization between the human body and the virus in global health constitutes "a world on alert" (weir and mykhalovskiy 2010; lee and mcinnes 2012) . "global public health vigilance," bolstered by an extensive transnational surveillance apparatus led by the world health organization (who), caters to a world under threat of unpredictable and pathogenic microorganisms and diseases. over the past two decades, a range of global health issues have reached the highest levels of political concern, prompting states and international organizations to respond to such threats in the language, and with the arsenal, of security (rushton and youde 2015) . worryingly, it has been suggested that these security practices render citizens as patients and states as megahospitals (elbe 2010) , as is also the argument in li's description of hong kong. in her excellent "pathography" of global public health's experience with the 2009 h1n1 pandemic, macphail (2014) examines our collective fear of viruses by tracing the h1n1 influenza virus through history and sites of public health activity, particularly in hong kong. the picture she paints is also of anxiety fueling an influenza pandemic narrative in which not only is the virus misunderstood but archaic truths of influenza research dominate (cf. webster 1993) and infection control stifles the development of necessary, novel ideas of infection control (schiffman 2014; lee 2015) . the fallacy of the influenza pandemic narrative lies in it largely misunderstanding the virus and bacteria and their relation to the human. recent advances in medical microbiology have found that the human-virus relationship is not one of opposition per se but of profound entanglement. one cannot be thought without the other, leading some to theorize the figure of homo microbis (helmreich 2014) . in this understanding, few microbes are pathogenic; most are benign, some even essential to human life. indeed, viruses have been and are a vital source of new genetic information, horizontal gene transfer, and genetic diversity in the evolution of life. zoonotic viruses' ability to continuously mutate as they go about infecting hosts and exchanging genetic information renders global health strategies largely obsolete. moreover, there is an increasing realization that antimicrobial interventions such as antivirals and antibiotics are accelerating antimicrobial resistance, making infection control strategies not just obsolete but also counterproductive. thus, a rethinking of the human-virus relationship in (urban) politics and security in hong kong and global health more generally is timely. the article begins by tracing the way the global city of hong kong has been constituted as an antimicrobial city since the sars outbreak in 2003. the antimicrobial stance takes its cue from the scientifically accepted germ theory of disease. in a second step, the article explores the recent claims of medical microbiology, which profoundly depart from the germ theory to invoke an ecological or configurational understanding of the human-microbe relation. here, disease is not just the outcome of a pathogenic microbe infecting a human host but emerges from socioeconomic relations, which intensify human-animal-microbial interactions, thereby leading to pathogenesis-that is, the diseased state (lorimer 2017) . in a final step, the article draws on daoist thought and traditional chinese medicine to reflect on the ways that such an ecological understanding translates into life and city dwelling. sporadic outbreaks of h5n1 in the live bird markets; the spectre of sars whenever someone with an acute, unexplained upper respiratory tract infection is hospitalized; the occasional case of japanese encephalitis; thick, humid air that feels pregnant with microbes; the soundless, nearly invisible mosquitos ubiquitous in the lush, dense patches of forest that cover the territory. contagions seem to originate both from within and from without. (macphail 2014, 79) in early 2003, after having treated patients with atypical pneumonia in a guangzhou hospital, a medical professor arrived in hong kong from guangzhou carrying sars coronaviruses. the arrival of the coronaviruses led to the volatile outbreak of sars in hong kong. hitching a ride on the chinese professor, the pathogenic microbe family began infecting and reproducing among the large pool of hosts-that is, the hospital staff and medical students whom the chinese professor was visiting. during the professor's short stay at the metropole hotel in kowloon, before he himself fatally succumbed to sars, the viruses continued infecting seven other hotel guests, who traveled elsewhere in hong kong, singapore, vietnam, and canada. the viruses moved into the prince of wales hospital while journeying on a local hotel guest who was admitted in early march. there, they moved through the hospital, infecting over one hundred medical and nursing personnel. by early april, coronaviruses entered the housing estate "amoy gardens," where they infected and reproduced among the large pool of hosts living in and beyond this estate (lee 2003) . only a month later, coronavirus activities finally began to slow down and decrease-however, not before having also spread to a number of global cities including beijing, guangzhou, singapore, and hanoi in east asia as well as toronto. as hardy has aptly noted, "the history of the infectious diseases in modern times remains inextricably intertwined with the history of the cities that spawned them" (hardy 1993, 293; see also mcneill 1976) . this can be said too of hong kong. with the social and environmental conditions of a global city, the densely populated city is argued to have provided the necessary breeding and circulation ground for a fast-emerging disease such as sars to spread rapidly in the network of connecting global cities. 2 cities afford microbes a large pool of closely connected human hosts by which a "sustainable chain of transmission" is ensured (harris ali and keil 2008, 4) . "the city is a playground for parasites," notes guardian writer kira cochrane and adds, quoting historian of science barnett (cochrane 2014 ), there are a lot more exciting human beings they can jump on to, . . . lots more opportunities for vectoring and transmission. it's all about movement. parasites love movement. so in that sense the city is an absolutely fantastic place for them. indeed, in hong kong, according to li shiqao, the sars outbreak brought into painful realization the problem with the city's characteristically chinese urban architecture of abundance or "maximum quantities," expressed through the functional building of ever-higher skyscrapers to house more and more people within ever smaller spaces (li 2014, 28-30; also roloff 2007) . this specific urbanity of hong kong emerged with the unique political and economic circumstances that came with the arrival of large waves of mainland chinese refugees after the creation of the people's republic of china in 1949. until the 1960s, an estimated one million people were homeless in hong kong. before they were resettled in tiny spaces in public housing estates by the colonial authority, mainland chinese refugees lived in squalid squatter-settlements, which gave rise to the circulation of diseases and criminality (roloff 2007, 112) . throughout history, cities have responded in different ways to infectious diseases-from the traditional quarantine, to the development of urban hygiene infrastructure through sanitation and waste management systems (loos 1987, 45-49; melosi 1999) , to the more recent establishment of public health systems. "the whole history of urban life," argues medical historian richard barnett, "is of living with parasites and trying to get rid of them" (as quoted in cochrane 2014) . indeed, in the modernization of cities in the late nineteenth century, gandy emphasized the way technopolitical discourses became entangled with advances in the medical sciences such as disease epidemiology to influence developments in civil engineering and planning, as well as public health (gandy 2006, 15) . 3 writing about the inclusion of public hygiene in urban design and architecture, li shiqiao has spoken of "an architecture of bacteria and virus control" emerging in europe during this time (li 2014, 117-18) . this found its expression especially in urban architectural designs of whiteness (which expresses the visual act of bleaching and antisepsis) and homogenous surfaces (which express the medical practice of disinfection) (li 2014, 117-18) . 4 thus, the concern for public hygiene and microbial control has shaped (particularly western) urban architecture since the turn of the twentieth century. chinese cities, on the other hand, first encountered the late nineteenth century european discourse of urban hygiene 5 through "treaty-port cities" such as hong kong, shanghai, and tianjin, which were established to ensure the trading interests of western powers in china (rogaski 2004, 141) . 6 cities like hong kong were to participate in late nineteenth century global trade, at a time when a bubonic plague was also spreading globally. hong kong's crowded architecture rendered it a 3 gandy (2006) referred to this as the emergence of the "bacteriological city," to highlight the complex interactions between disease, water, and urban infrastructure. similar to concepts such as the "sanitary city" and the "hydraulic city" in analyzing the relationship between water and cities in the modernization of cities in late nineteenth century europe, gandy's "bacteriological city" nevertheless goes further in emphasizing the role of scientific advances such as disease epidemiology. 4 according to li (2014, 188) , whiteness symbolizes "the visual act of bleaching, the physical and metaphorical removal of dirt as an antiseptic practice." it is reflective of ventilation and light in hospital design. "shine" is produced of homogenous surfaces that find their cue from the medical practice of disinfection. 5 health strategies in china have traditionally focused on preserving the body. urban hygiene as a rationale was officially incorporated into the qing imperial administration in the early twentieth century and subsequently adopted by the communist government in 1949. urban hygiene was linked to the nationalist project of the mao regime and launched in mass campaigns to eliminate disease and pest (li 2014, 119) . 6 hong kong was created in the mid-nineteenth century as a result of the opium war between the british empire and the qing empire in china. having lost the war, the chinese emperor ceded hong kong in perpetuity to britain in 1842. under british rule, the city was a free trade center with low taxation, a character it still holds today. its british heritage is an intensely neoliberal mentality reflected in a minimum government, few controls on imports and exports, and the lack of a military arm, which laid the ground for its eventual integration into the global economy and evolution into a global city. hygienically challenged city that was particularly vulnerable to infectious diseases, leading to urban hygiene becoming a fundamental feature in governing the colonial, and eventually postcolonial, city (li 2014, 118-19) . historically, hong kong is considered a "naturally diseased space," where new influenzas emerge. with its "year-round humidity and swampy, tropical marshland," from the early times of british colonization hong kong was "seen as a reservoir-pit-of disease" (macphail 2014, 80) . a series of deadly outbreaks in the newly acquired colony led the british to set up hospitals and bring in western medical authorities to ensure there was little disruption to economic flow. the port of hong kong had become indispensable to the british economy and thus necessitated a strategic eye to controlling disease circulation (macphail 2014, 82) . in 1894, hong kong experienced a plague outbreak which led to the establishment in 1906 of what would eventually become the bacteriological institute, its first modern microbiological research center (macphail 2014, 83) . while until the 1890s hong kong's medical officials followed the widely accepted miasma theory in explaining infectious disease circulation, by the early nineteenth century, and with the establishment of the institute, the shift to germ theory in european medical circles had resolutely arrived in hong kong. using the new technology of the microscope, germ theory established that infectious diseases were not caused by atmosphericmiasma or "bad air" but by pathogenic microorganisms such as bacteria, protozoa, fungi, and viruses in the body. microbiologists proved that specific microbes, which spread from person to person, caused infectious diseases. germ theory radically changed the practice of medicine. it influenced how hospital space in hong kong and elsewhere had to be rethought toward including microbiological laboratories and isolation wards (sihn 2017). more importantly, as the accepted scientific theory of disease, which still underlies contemporary biomedicine, germ theory to this day influences and dominates modern sanitary practices and public health. the battle against sars, occurring only five years after hong kong's handover to china in 1997, revealed considerable problems both with the sino-british political arrangement of "one country, two systems" and with the city's public health governance system. sars "can be understood epidemiologically as a virus that tested hong kong's healthcare system and governance to the maximum" (baehr 2008, 147) . while a number of stringent but important measures to control the spread of infections-including isolation and home confinement, regular health checks at the border, and public information sharing-were introduced during the outbreak, the hong kong government's response was widely considered to have been severely delayed and inadequate (ng 2008, 71-73) . 7 in fact, the change in political rule and the insufficient governmental response provoked a distinct cultural reaction to sars by the hong kong people who mistrusted mainland chinese and associated hong kong officials. culturally, baehr found the "mask culture," which arose during this time, a sign of an emerging social solidarity among hong kong people in which they paid tribute to a common good by meeting one's duty not to endanger the wider hong kong community. the plague, or any other pandemic in chinese culture, is traditionally seen as a sign of evil: it "summons up the possibility of a collective death: the extirpation of the social itself" (baehr 2008, 147) . mask-wearing thus "became the quickly improvised, if obligatory, social ritual: failing to don one was met with righteous indignation, a clear sign of ritual violation" (baehr 2008, 150) . in the immediate sars aftermath, partly to be seen to regain control of the battle against infectious diseases and partly to defend its beleaguered image as "asia's world city," the hong kong government aggressively promoted a new culture of urban hygiene (roloff 2007, 74) . building on the "positive hygiene spirit" and mask-culture during the sars outbreak, the city government began working to normalize a mentality of urban hygiene in the hong kong community. shortly after the sars outbreak, it established the governmental organization team clean. its role was to regain hong kong's status as a world-class city by instituting high hygiene standards throughout the city. former chief secretary for administration, donald tsang, declared on may 28, 2003: there are problems emanating from personal hygienic habits, household hygiene unsatisfactory conditions and some environmental unhygienic problems relating to building maintenance and so on. . . . it is a monumental task but it is an important step that hong kong must go through if we aspire to be a city of the first rank and not only as a very successful international financial centre, but in fact a "clean room" in asia. (tsang 2003) the work of team clean began by identifying, defining, and clearing "hygiene blackspots" in nearly one hundred public housing estates. but the city's hygiene efforts extended not only to cleaning up buildings and surrounding infrastructure, it crucially involved changing the behavior of the people of hong kong: we believe that all efforts must begin with the self, extend to the family and the immediate neighbourhood, and then radiate throughout the entire community of hong kong before we can claim a place as a world-class city. (team clean report as quoated in roloff 2007, 97) by orchestrating mass campaigns of hygiene habits through posters, audio announcements, television broadcasts on public transportation, and the display of disinfection stations, public notices of disinfection routines, etc. and by incorporating regular and mandatory hand washing in schools, hong kong actively worked to induce an antimicrobial (sanitizing) imperative in the hong kong people. its stringent urban hygiene regime is creating hygienic world-class citizens who are to carry hong kong back into the top ranks of global cities (roloff 2007, 91) . in li's reading of the post-sars climate in hong kong, an architecture of bacteria and virus control was manifesting in distinctly chinese-style "infection barriers" in the city. historically, the chinese city is "conceived as a set of concentric corporeal defenses of the body, the family, the village, the work unit, and the state family" (li 2014, 133) . the preferred method of defense in traditional cities in china was walls. infection barriers in chinese cities like hong kong, li claims, take on some of the characteristics of the traditional walls in their concentric forms. unlike city walls or gated communities, however, infection barriers manifest according to the principles of antimicrobials. as such, in reproducing the chinese imperative of prudence in preserving the body, they defend the body by "fighting bacteria and viruses from within the tissues of architecture" (li 2014, 130) . in this way, the "monumental task" of transforming hong kong into an antimicrobial global city was underway. li concludes, "in hong kong, the hospital is poised to take over the entire city, spreading its standard practices of hand-washing, maskwearing, and temperature-taking" (li 2014, 130-31) . building on germ theorywhich posits that pathogenic microorganisms such as bacteria, protozoa, fungi, and viruses residing in human bodies spread from person to person to cause infectious diseases-an extreme urban hygiene culture emerged in this global city to prevent germs from spreading. this is not only fueling and normalizing anxieties of infection and disease as i experienced when visiting hong kong in late 2016, it may also not be achieving what it set out to do, namely, securing human life. considering recent advances in gene sequencing in microbiology, through which a "vast diversity of microbial life in, on and around the human body" (lorimer 2017, 544) has been identified as residing in complex relationality with one another, how befitting is it to fight infectious diseases by indiscriminately eliminating microbes through the use of antimicrobials and practicing urban hygiene as in the case of hong kong? what happens when the enemy is not the virus or other microbes but us (methot and alizon 2014, 778) ? like many others caught up in the emerging-disease narrative, which is based on the tenets of germ theory, the story of infection barriers is one of setting the human and the nonhuman virus against each other, as the victim versus the menace. viruses are commonly understood to be "'bad matter' to be prepared for, brought under control, and ultimately eradicated or rendered impotent" (white 2015, 145-46) . unlike the germ theory of disease, which makes a specific microbe responsible for a specific disease (e.g., the coronavirus is responsible for sars), an ecological perspective holds that microbes are not essentially pathogenic (methot and alizon 2014) . rather, as hinchliffe et al. (2016) have argued, disease emerges from the complex entanglement between the immune system of a host and the microbial milieu in and outside of the host. various scholars have noted how, much like hong kong in the face of sars, global public health programs adopt an antimicrobial stance to the control and/or elimination of infectious diseases, however, which might prove to be counterproductive in securing human life (macphail 2014; methot and alizon 2014; fishel 2015 fishel , 2017 white 2015; hinchliffe et al. 2016; du plessis 2017; lorimer 2017, 545) . at the microbial level, ecological microbiologists understand most viruses and bacteria are not pathological; they are benign and even indispensable to human life. scientists are understanding better the causes of infectious diseases, but they also "increasingly hear about beneficial microbes and the consequences of their decline or absence" (lorimer 2017, 544) . 8 causal links remain unclear and contested, however; as lorimer finds, there is "a widespread reappraisal underway in modern medicine of the salutary potential of the microbiome and the therapeutic use of microbes" (lorimer 2017, 544) . humans and microbes seem deeply and irrevocably entangled. this has led anthropologist stefan helmreich to consider the figure of homo microbis, made up of bacteria, viruses, fungi, and protozoa (helmreich 2014 (helmreich , 2015 . what are the implications of this understanding of the human body as mostly microbial to the way we fight infectious diseases in global cities and elsewhere? microbes are everywhere. apart from the ocean floors (helmreich 2009 ), soil, and deep forests, they inhabit nearly all of living matter, including mammals. humans are colonized by many viruses. this collection of viruses found in or on humans is known as the human virome, which is the viral component of the human microbiome-the assemblage of microorganisms including bacteria, fungi, protists, archaea, and viruses residing in/on the human body. only a minority of viruses infect human cells and can cause "acute, persistent, or latent infection"; some viruses are even "integrated into the human genome such as endogenous retroviruses" (wylie, weinstock, and storch 2012) , which are essential to human reproduction. 9 8 "missing microbes" have arguably been linked to several metabolic, immunological, and mental health conditions, such as allergies, obesity, inflammatory bowel disease, and depression. see velasquez-manoff 2012; blaser 2014; and lorimer 2017. 9 endogenous retroviruses are fossil viruses which "began to be integrated into the human genome some 30-40 million years ago and now make up 8% of the genome" (tugnet et al. 2013) . they may be associated with autoimmune diseases such as rheumatoid arthritis, though evidence is still limited. yet, while many mammalian viruses, such as the endogenous retrovirus, picked up mammalian genes during their evolution by subverting these "to provide a selective advantage to the virus," an opposite story can be told too. an endogenous defective retrovirus "has been sequestered to serve an important function in the physiology of a mammalian host" by encoding a protein call syncytin. syncytin has been found to be vital in placenta production, a necessary prerequisite to human reproduction (mi et al. 2000; zimmer 2012 ). there is no scientific consensus on whether viruses are living matter. 10 this is because viruses cannot live without a host cell: "they must invade and 'hijack' a cell's mechanism" so that they can produce the proteins needed for their own reproduction (macphail 2014, 8) . it is in this sense that van regenmortel and mahy (2008) have noted, viruses lead "a kind of borrowed life." their capacity to produce an effect-that is, their efficacy-, like other nonhuman matter, is "not only to impede or block the will and designs of humans but also to act as quasi agents or forces with trajectories, propensities, or tendencies of their own" (bennett 2010, viii) . by not taking seriously their efficacy to alter the course of events, however, their animating role in evolution has until very recently been overlooked. viruses have been and are a vital source of new genetic information, horizontal gene transfer, and genetic diversity in the evolution of life, including increasing and decreasing immunity to certain viruses or bacteria. 11 they have effected change on the direction of human evolution and were involved in the making of human history, most visibly in the form of plagues and diseases (mcneill 1976) . they are essential to sustaining the environment, including sea and freshwater regulation, as well as human reproduction; humans are deeply involved with viruses, in the positive and negative sense (fishel 2015, 158) . the vital role of viruses in human evolution necessitates an epistemological rethinking of evolution, as melissa a. white has noted, "from darwinian models of the 'survival of the fittest' to the phenomenon of emergent life" (white 2015, 146-47) . viruses are able to evolve very quickly, reinventing themselves by mutation and adaptation, thereby "evading immune systems and other means of eradicating them" and "surviving under conditions that would cripple or kill other organisms" (macphail 2014, 9) . essentially, viruses are "packets of pure information" in that they are protein encasements of genetic material in the form of either dna or rna. rna viruses such as influenza a (responsible, for example, for avian flu in 2003, the 1918 flu, and swine flu in 2010) evolve by engaging in "antigenetic drift" when mutating while replicating. zoonotic viruses that are able to jump species also evolve by engaging in "antigenetic shift" during which they exchange "entire genetic segments with other viruses inside a host" (macphail 2014, 9) . it is in this capacity that viruses ought to be recognized as "bioinformatic transport machines." indeed, white argues, "they ought to be considered active participants in creating the potentiality of new conditions of life through their capacity to assemble novel coalitions of genes" (white 2015, 147) . antigenetic shifts can change the surface proteins of the virus (the antigens), which are then no longer recognizable by the host's immune system, thus provoking a slower or no response to fend off the virus. virologists studying influenza a tend to focus on any incremental changes or dramatic shifts in genetic makeup to the viral antigens hemagglutinin (ha) and neuraminidase (na). it is after these specific antigens that influenza a viruses are named by their h and n numbers 10 generally, scientists apply a list of a priori criteria to decide on life. following this, according to virologists van regenmortel and mahy (2008) , viruses today are considered somewhere in-between living and nonliving. similarly, theresa macphail (2104) argues, while viruses ought to be considered to have a "certain type of nontrivial agency," they are usually seen as "liminal objects" "with some of the properties of life, yet they cannot be considered fully 'alive' while outside of a permissive host." they are "organisms at the edge of life" (rybicki 1990) . 11 in fact, historical viral traces in human dna were, until recently, controversially referred to as "junk dna." this noncoding dna makes up a greater portion of the human genome than "segments of dna that actively code for genes" (macphail 2014) . the human genome effectively consists of only two to three percent of coding dna, while the remaining ninety seven to ninety eight percent was thought to be just a "sea of genetic gibberish" with no biological function, hence "junk dna" (hall 2012) . this idea was debunked in 2012 by the encode group, which revealed that "junk dna" was in fact brimming with important genetic information. the encode group produced "a stunning inventory of previously hidden switches, signals and sign posts embedded like runes throughout the entire length of human dna." this essential noncoding dna, as macphail (2014) has noted, is evidence of past infections or "of a long-standing symbiotic partnership" with viruses. viral "junk" may even be responsible for creating new genes and for enabling the immune system to adapt to emerging infections (macphail 2002). (macphail 2014, 10). the pathogenicity of influenza a viruses is established both by their molecular makeup and their ability to cause serious illness in their host species, birds or other. a highly pathogenic virus causes severe or lethal illness in bird or other species populations. it is possible for a virus to "jump" from its host species into a human host, resulting in an usually severe and lethal infection. yet, viruses do not have "motives, or thoughts, or diabolical plans to wreak havoc to our cities"; "their function and purpose (if we can even say that they have one) is to replicate, to evolve, to survive" (macphail 2014, 11) . 12 most viruses, however, infect microorganisms including bacteria in the human microbiome (edwards and rohwer 2005) . these prokaryotic viruses "affect human health by impacting bacterial community structure and function" (relman 2015; also wylie et al. 2012) . because viruses evolve very quickly, the human virome (and thus also the microbiome) is changing all the time. each human virome is different and unique as it evolves and is formed both by preexisting immunity and viral and human genetics as well as by human lifestyle, age, geographic location, and susceptibility to disease, all of which affect individual exposure to viruses (delwart 2013) . from this perspective, diseases are not the outcome of a virus or other microbe infecting a human, as germ theory holds, but emerge from the unique constellation of political and ecological relations that affect the biological interactions of the human, the animal, and the microbe and lead to the potential development of a diseased state-that is, pathogenesis. hinchliffe et al. usefully speak of disease as "multispecies conditions configured by specific socio-ecological 'situations'" (hinchliffe et al. 2016; lorimer 2017, 545) . some scientists already seek a shift toward this ecological or configurational thinking. the emerging diseases and global health security narrative runs on the notion that the bacteria or virus is the enemy against which the global surveillance / health security apparatus must operate (weir and mykhalovskiy 2010) . this narrative rests on the germ theory of disease currently dominating microbiology/bacteriology. in her pathography of the h1n1 influenza pandemic in hong kong in 2009, macphail finds that a few "heretic" microbiologists are challenging the dominant emerging infectious diseases narrative, calling for a new epistemology in which microbes are understood not as enemies but as coinhabitants of the world (macphail 2014, 17) . hong kong biologist frederick c. leung suggests that the problem with the alarmist influenza pandemic discourse lies partly in the way viral genetic data revealed through "signature sequences" has been interpreted by leading influenza scientists such as the virologist robert webster (webster 1993) . speaking against the central focus on the h and n proteins as the key to what makes an influenza virus deadly or not in the dominant influenza surveillance and research discourse, leung and others believe that "the public health orthodoxy has become too ready to see what it has already been prepared to look for and to fear" (macphail 2014, 190) . evolutionary biologist paul w. ewald suggests that although the h and n proteins are most visible to our immune system, it is not certain that they are the reason for a strain's pathogenicity or severity (macphail 2014, 190) . ewald believes that scientists tend to confuse "sources of variation-the mutation and recombination of genes-with the process of evolution by natural selection" (ewald 2000, 22-23) . 12 hong kong biologist frederick c. leung suggests that the problem with the alarmist influenza pandemic discourse lies partly in the way viral genetic data, revealed through "signature sequences," has been interpreted by leading influenza scientists such as the virologist robert webster (1993) . speaking against the central focus on the h and n proteins as the key to what makes an influenza virus deadly or not in the dominant influenza surveillance and research discourse, leung and others believe that "the public health orthodoxy has become too ready to see what it has already been prepared to look for and to fear" (macphail 2014) . evolutionary biologist paul w. ewald (2000) suggests that although the h and n proteins are most visible to our immune system, it is not certain that they are the reason for a strain's pathogenicity or severity. ewald believes that scientists tend to confuse "sources of variation-the mutation and recombination of genes-with the process of evolution by natural selection." to date, our understanding of the human virome, benign viruses, and the virushuman relation remains limited. yet, viruses may not simply be bits of "bad matter" that slow down, disassemble, and debilitate complex systems. rather, viruses might well be approached from another vantage point altogether, as vitalizing forces in complex ecological systems in which humans are not the center. (white 2015, 149) the relation between the human and microbial communities is not antagonistic but symbiotic. while the rapid emergence of infectious diseases is creating a "world on alert," as weir and mykhalovskiy (2010) have observed, it demonstrates the frail character of the ecological balance of this vital symbiosis between humanity and their vital environment (methot and alizon 2014, 782) . the study of the biology and ecology of viruses forces us to appreciate our mostly symbiotic relationship with viruses and other microbes. it compels us to rethink how (human) life has come to be. it is not the darwinian paradigm of "the survival of the fittest" but rather an emerging paradigm revolving around a microbiological understanding of "emergent life" that explains our evolution. it also necessitates that we understand and respond to disease not as the invasion of enemy microbes but as the pathogenesis, that biological mechanism, of a unique constellation of politico-ecological relations and human-animal-microbial interactions, which gives rise to a malady. how can this emerging ontological and epistemological understanding of human life inform the governance of disease? the best defence we have against microbes is our brains, which can surely work out how to live in harmony with the microbes we know, and find non-disruptive ways of combating those that emerge in the future. (crawford 2007, 213) how do we live in harmony with the microbial world but still prevent infectious diseases from developing? how do we move from a potentially destructive antimicrobial perspective such as is embodied in hong kong to a microbial perspective in which we take responsibility for our vital relation with the microbial world? new materialist thinking (cf. hinchliffe 2007; coole and frost 2010; dolphijn and tuin 2012), which takes seriously the efficacy and ecology of human bodies and nonhuman bodies such as the virus and other microbes, invites us to examine how the human-microbe relationship can be rethought in politics. while a number of theorists have begun to conceptualize a new materialist politics and ethics within modern political theory (bennett 2010; connolly 2013; mitchell 2014) , others have looked to indigenous cosmologies, which take into account the world we share with other kinds of beings, to formulate a postanthropocentric politics (kohn 2013; tsing 2015; du plessis 2017) . the daoist cosmology underlying chinese medicine and chinese strategic thought and practices provides a first step to think in distinctly chinese ethical terms about how public health strategies in hong kong and beyond can begin to direct a human-microbial ecology to the advantage of protecting all life. daoist thought takes reality as dynamic, and the regulation of this changing reality is immanent to the interaction of the heterogeneous factors involved, thus emerging spontaneously (jullien 2004 ). sunzi's philosophy strategizes how best to direct a heterogeneous ensemble of human and nonhuman (virus, bacteria, and other nonhuman) bodies, by shifting the inherent potential, the shi, of this ecology to our advantage. in the art of war (515-512 bce), success in war was achieved not through the courage, activity, and talent of any individual fighter but through careful planning instead of actual combat. in fact, combat in chinese thought is not violence; violence is to be avoided. in medicine too, defending the body from danger demanded care for the body manifested in preservation regimes instead of reactive "western drug-and surgery-based medical practice" (li 2014, 83) . chinese medicine is about preserving rather than curing. this can be seen in its guise as a conception of food: it demands specific diet regimes developed from "observations of the characteristics of the vegetation and animals in the season." over time, it came to incorporate a puzzling spectrum of diet-based caring regimes that are still widely practiced today (li 2014, 83) . reflecting on this, medical anthropologist judith farquhar has noted of chinese medicine that it "heals in a world of unceasing transformation." this a priori dynamism in chinese medicine contrasts sharply with the modern western "world of discrete entities characterized by fixed essences, which seem to be exhaustively describable in structural terms." since motion and change are a given, they rarely require explanation with reference to their causes. according to farquhar, "one consequence of this dynamic bias in chinese medicine is that the body and its organs (i.e., anatomical structure) appear as merely contingent effects or by-products of physiological processes" (farquhar 1994 as quoted in needham 2000) . in a comparable vein, methot and alizon (2014) and others (hinchliffe and bingham 2008; lorimer 2017) have argued that pathogenesis results not from any single pathogenic microbe but from the configuration of socio-ecological relations and human-animal-microbe interactions. chinese thought, francois jullien explains, takes reality as an immanently "regulated and continuous process that stems purely from the interaction of the factors in play (which are at once opposed and complementary: the yin and yang)" (jullien 2004, 15) . reality is dynamic, and the regulation of this transformative reality-that is, the order of reality-emerges spontaneously. it is not achieved through external intervention but is "entirely contained within the course of reality, which it directs in an (inherent) fashion, ensuring its viability" (jullien 2004, 15) . two notions are central to this ancient chinese strategy: (a) the notion of a situation or configuration (xing)-that is, as a relation of forces such as are immanent to an ecology-and (b) the notion of the potential (shi) of a situation/configuration. this is commonly illustrated by "a mountain stream that, as it rushes along, is strong enough to carry boulders with it" (jullien 2004, 17) . the configuration (xing) of the mountain consists of a downward-sloping course and narrow channel, while this configuration itself gives rise to the potential (shi) for the rushing stream to carry boulders with it. thus, it is not "what we ourselves personally invest in the situation" that counts so much but rather "the objective conditioning that results from the situation" (jullien 2004, 17) . 13 jullien has argued that shi helps to "illuminate something that is usually difficult to capture in discourse: namely, the kind of potential that originates not in human initiative but instead results from the very disposition of things" (jullien 1995, 13) . bennett (2005, 461) usefully elaborates on this by explaining that "shi is the style, energy, propensity, trajectory, or élan inherent to a specific arrangement of things." for bennett, "shi names the dynamic force emanating from a spatiotemporal configuration rather than from any particular element within it." as "both the membership (of a configuration or assemblage) changes over time and the members themselves undergo internal alteration," as bennett points out, the shi or mood of a configuration also changes (bennett 2005, 461) . in margaret archer's words, everyone in the configuration "possesses autonomous emergent properties which are thus capable of independent variation and therefore of being out of phase with one another in time" (archer 1995, 66) . it is possible that an individual element such 13 biophilosophers deleuze and guattari (2004) describe something comparable when they discuss the milieu as a force field composed of nonhuman and human things (xing). not only do these heterogeneous elements constituting a milieu each entail some form of efficacy, but the milieu as a whole gives rise to a potential to effect (shi). jane bennett (2005) likewise argues that there is an agency that attaches to assemblages of human and nonhuman entities. she contends that this agency of an assemblage is comparable to the chinese strategic notion, shi. as a virus, in an antigenic shift, "becomes out of sync with its (previous) self" and forms new relations within an assemblage, "leaning towards a different set of allies" (bennett 2005, 462) . as such, the members of an assemblage "maintain an energy potentially at odds with the shi" (bennett 2005, 462) . it is for this vibrancy, according to bennett, that the agency of assemblages cannot be understood in terms of passive social structures. crucially, in chinese strategic thought, it is the shi that "can be made to play in one's favour" (jullien 2004, 17) . it is because of their variability that circumstances or ecologies can little by little be turned advantageously by the propensity or efficacy immanent to a situation. a chinese sage, according to jullien, "is inclined to concentrate his attention on the course of things in which he finds himself involved in order to deter their coherence and profit from the way that they evolve" (jullien 2004, 16) . a good general, in turn, "must be able to read and then ride the shi of a configuration of moods, winds, historical trends, and armaments" (bennett 2005, 461) . this "logic of regulated evolution" allows the potential of a situation "to develop of its own accord and to 'carry' us with it" (jullien 2004, 17) . jullien illustrates this by drawing on an old chinese proverb that captures the core idea of this thinking (mencius as quoted in jullien 2004, 16) : "even with a mattock and a hoe to hand, it is better to wait for the moment of ripening." unlike the western tradition of establishing a model that is projected onto a variable reality, according to jullien, chinese thought will concentrate on understanding how things unfold so as to discover their configuration (relationality) in order to come up with alternative ways to effect a more advantageous outcome. thus, instead of constructing an ideal form that we then project on to things, we could try to detect the factors whose configuration is favourable to the task at hand; instead of setting up a goal, we could allow ourselves to be carried by the propensity of things. (jullien 2004, 16) to ride the shi-in other words, to live harmoniously alongside and with the microbial word-we need to identity those factors whose configuration is favorable to human life. daoist ethics and chinese strategic thought lead us to embed homo microbis in an environment that is favorable to all life. as hong kong biologist frederik leung points out, the problem not only lies in the prevailing dominance of germ theory in science, it also lies in the way we relate to the animal and nonhuman world at large with which humans are so deeply entangled. he speaks against the culling of birds and animals as a response to an emerging influenza pandemic (macphail 2014, 192) . in fact, the flu is argued to be unpredictable only because influenza experts "don't understand the basic science" (leung, as quoted in macphail 2014, 193) . viruses naturally undergo constant mutation. in the case of cross-species transmission, such as the sars coronavirus which crossed over from civet cats to humans in 2003, it became clear that "highly pathogenic influenza viruses naturally burn themselves out, . . . they pose no greater risk to humanity than 'normal' influenza viruses" (macphail 2014, 193) . in fact, "the more virulent the virus, the faster the virus dies out. by evolutionary principle" (leung, as quoted in macphail 2014, 194) . leung laments the current focus on the development of influenza vaccines, since intervention through the vaccine encourages further mutation, whereas allowing the virus to take its natural course leads to its natural burn out (macphail 2014, 194) . leung's call for rethinking how we relate to the animal world echoes the recent effort to do just that in the one world one health initiative of scientists, physicians, and veterinarians worldwide to collaborate across disciplines in addressing emerging diseases. in part, this effort came about due to concerns for shared risks across human, animal, and environmental health. the focus, however, has tended to lie on the contamination and transmission of pathogens rather than on the socioeconomic relations underlying disease and health. it has been criticized for reducing diversity and for underappreciating the local, contingent, and practical engagements that first make health possible (hinchliffe 2015) . yet, it is not just how we relate to the animal world that matters. it is also about how we think about life in cities, especially as sars in hong kong in 2003 revealed, a set of absent actors-animals, microbes, airplanes, sewage systems, respiratorsthat had been banished to the margins of our conceptions of urban life, even as they actively contributed to how urban lives were composed and lived. (braun 2008, 251) the sars encounter and the post-sars politics force us to think about the microbes, animals, and many other organisms living amongst us and influencing the "social" collectives of humans. that social collective, the city, needs to be rethought on microbial terms. at the turn of the twentieth century, cities thrived on "bacteriological" (gandy 2006) and "epidemiological" conceptions of urban spaces in europe and north america. they aimed to transform urban spaces and the behavior of city people accordingly (braun 2008, 3) . in his exploration of the chinese city, li shiqiao highlights the key imperative of prudence, understood in terms of "the principle of endurance and an unknown future reward," which firmly runs through chinese conceptual thought (li 2014, 81) . he interpreted this in the post-sars situation of hong kong to be the donning of masks and ultimately striving to become hygienic subjects. yet, it seems prudent to build a microbial instead of an antimicrobial city, to rethink space and the behavior of the city people in hong kong and other (global) cities in ecological and sustainable terms, and to take into account the social-economic relations that intensify human-animal-microbial interactions. this article set out to show how a better understanding of the microbe and its deeply entangled existence with humans is crucial to conceptualizing a better approach to health and life. specifically, it argued for an inclusive approach to the virus and other microbes with which we share the world we live in over an exclusively oppositional approach to eradicating pathogenic circulation, which also eliminates or mutates benign viruses essential to human life. the article began by looking at the antimicrobial politics in chinese postcolonial hong kong since the turn of the century. it particularly focused on the notion of "infection barriers" advanced by li (2014) . li bases his analysis on the traditional conception of the chinese city and the practice of defense in walling the city off against danger. by arguing that, in the struggle against infectious diseases, contemporary practices of urban defense have taken on another guise, namely infection barriers, li is able to demonstrate how the politics of public hygiene operates not only through overt public programs but through infection barriers that are affectively knit into the fabric of the urban architecture and tissue of city dwellers. although his lens on chinese urban defenses takes seriously ancient chinese thought in the making of urban spaces, nonetheless, the politics of infectious disease remains caught up in the prevalent and dominant oppositional narrative of the human against the microbe. infection barriers set up the human and the nonhuman virus against each other, as the victim versus the menace. like antivirals and antibiotics, urban hygiene practices are never entirely able to control pathogenic circulation. this is because urban hygiene proceeds on the basis of a very narrow conception of disease, namely the widely accepted germ theory of disease. by studying the biology of microbes, it becomes possible to grasp more what the virus and other microbes are and do but particularly how they relate to the human. first, the activities of the virus escape the political designs, regimes, and practices because the virus and other microbes are forever in transformation. how then can human efforts be conceived to control pathogenic, while fostering benign, viral circulation? second, viruses play a large and essential part in making up the human. while a small number of viruses are pathogenic, the majority are benign, and some are even essential to human life. strategies to eliminate them may kill pathogenic viruses but essential viruses might also be killed off in the process. as the human cannot be thought without the virus, and attempts to alter or eliminate it (in)directly also affect the human, how might we think differently about urban spaces and public health if we consider the human and the virus not as opposed but as fundamentally interrelated? finally, the article considered daoist thought, to begin to reflect on the governance of microbes, which takes seriously their efficacy, dynamism, and deeply historical relationality with humans. from the biological vantage point of ecologies in which the virus, the human, and many other organisms are fundamentally interconnected forms that vitally depend on, but can also harm, each other, all interventions to control viruses will affect all involved, including the virus as well as the human. thus, it is in the interest of human beings to proceed with interventions to control viruses that cause the least direct or indirect harm to humans. daoist thought offers some ideas for how to strategize public health schemes, which take due consideration of this deep involvement of viruses and humans. public health schemes that develop defenses against pathogenic viral circulations, especially in a densely populated global city such as hong kong, remain important. however, there is also a need to consider and adapt the human practices that both create(d) the easy passages for viral circulation as well as forced viral mutations through the overuse of antiviral (and antibiotic) agents or vaccine development which is thought to have led to antiviral resistance. most of all, rather than fostering an oppositional relation between the human and the virus, there is a need for an appreciation of the vital role of the virus in human lives as well as of how deeply we are entangled with the virus. instead of only arming against pathogenic viral circulation by sanitizing both the urban environment and the minds of city dwellers, as in the case of hong kong, such circulation might be more usefully countered by championing an approach that proceeds on the understanding that the human irrevocably inhabits an ecosystem in which the multiplicity of life forms are deeply interrelated and dependent on each other. much like the chinese general evaluating the course of things immanent to a situation, since humans thrive on a range of other beings doing well, "an interest in human security becomes an interest in biodiversity: thriving is not a zero-sum game between different species, in fact, quite the opposite" (du plessis 2017, 18). global health projects ignore this and are struggling to deliver what they aim to do. modernity at large: cultural dimensions in globalization. public worlds realist social theory: the morphogenetic approach city under siege: authoritarian toleration, mask culture, and the sars crisis in hong kong the agency of assemblage and the north american blackout vibrant matter: a political ecology of things missing microbes: how killing bacteria creates modern plagues thinking the city through sars: bodies, topologies, politics the rise of the network society sick cities: why urban life is breeding new illness fears the fragility of things: self-organizing processes, neoliberal fantasies, and democratic activism new materialisms: ontology, agency, and politics deadly companions: how microbes shaped our history a thousand plateaus: capitalism and schizophrenia a roadmap to the human virome new materialisms: interviews and cartographies when pathogens determine the territory: toward a concept of non-human borders viral metagenomics security and global health: towards the medicalization of security plague time: how stealth infections cause cancers, heart disease, and other deadly ailments microbes the bacteriological city and its discontents journey to the genetic interior the epidemic streets: infectious diseases and the rise of preventive medicine 1865-1900 networked disease: emerging infections in the global city homo microbis: the human microbiome, figural, literal, political more than one world, more than one health: re-configuring interspecies health mapping the multiplicities of biosecurity pathological lives: disease, space and biopolitics the propensity of things: towards a history of efficacy in china how forests think: toward an anthropology beyond the human revealing power in truth global health and international relations. cambridge: polity. lee, shi hung understanding the chinese city. theory, culture and society. london: sage. loos, adolf. 1987. "plumbers parasites, ghosts and mutualists: a relational geography of microbes for global health the viral gene: an undead metaphor recoding life the viral network: a pathography of the h1n1 influenza pandemic. expertise: cultures and technologies of knowledge plagues and people the sanitary city: urban infrastructure in america from colonial times to the present. creating the north american landscape what is a pathogen? toward a process view of host-parasite interactions syncytin is a captive retroviral envelope protein involved in human placental morphogenesis only human? a worldly approach to security part vi: medicine. science and civilisation in china: biology and biological technology globalization of sars and health governance in hong kong under "one country, two systems are viruses alive? the human microbiome and the future practice of medicine hygienic modernity: meanings of health and disease in treaty-port china die sars-krise in hong kong: zur regierung von sicherheit in der global city routledge handbook of global health security the classification of organisms at the edge of life, or problems with virus systematics the global city reorganizing hospital space: the 1894 plague epidemic in hong kong and the germ theory transcript of the press conference with the chief secretary for administration in the conference hall of the central government offices new annexe on may the mushroom at the end of the world: on the possibility of life in capitalist ruins human endogenous retroviruses (hervs) and autoimmune rheumatic disease: is there a link? an epidemic of absence: a new way of understanding allergies and autoimmune diseases influenza global public health vigilance: creating a world on alert. routledge studies in science virus emerging view of the human virome mammals made by viruses i thank the anonymous reviewers and the editors of ips for their generous comments. i am also grateful for insightful comments on earlier versions of the article presented at the eisa convention 2017, ewis 2018, and before the irss research group, university of groningen. i am especially thankful to gitte du plessis, luis lobo-guerrero, and christopher long for thought-provoking nudges. this paper was produced under the aegis of the groningen centre for health and humanities and the aletta jacobs school of public health, university of groningen. key: cord-017225-6ofi6mg5 authors: wei, yuwa title: human rights issues date: 2018-12-10 journal: issues decisive for china&#x02019;s rise or fall doi: 10.1007/978-981-13-3699-7_8 sha: doc_id: 17225 cord_uid: 6ofi6mg5 contemporary china is plagued by a wide range of human rights related issues and problems. in addition to those arising in the areas of religious toleration, judicial practice, treatment of labor and forced abortion, which were extensively reported by the media in the past, some newly emerged problems concerning human rights violation are much more alarming, due to the size of population affected and the degree of challenge caused to the public’s psychological endurance and confidence in the social ethnics and administration of the nation. most of all, these problems concern nearly every chinese citizen’s well-being and impact on their personal prosperity, as well as the prosperity of the nation as a whole. these problems are mainly associated with failures in environmental protection, food safety, and medical security. first white paper on human rights being released in 1991, the white papers of the following years were arranged as a series sequentially informing the progress made and the actions taken in human rights promotion in each year. 7 nevertheless, it is an undeniable fact that contemporary china is plagued by a wide range of human rights related issues and problems. in addition to those arising in the areas of religious toleration, judicial practice, treatment of labor and forced abortion, which were extensively reported by the media in the past, some newly emerged problems concerning human rights violation are much more alarming, due to the size of population affected and the degree of challenge caused to the public's psychological endurance and confidence in the social ethnics and administration of the nation. most of all, these problems concern nearly every chinese citizen's wellbeing and impact on their personal prosperity, as well as the prosperity of the nation as a whole. these problems are mainly associated with failures in environmental protection, food safety, and medical security. achieving modernization has been the core economic objective and a solemn promise of the chinese government. 9 urbanization has been a major means of reaching this goal. this course has been accelerated since the 1990s with an average urban population growth rate of more than 3.4%. 10 nowadays, the number of urban population has reached 7711.6 million, account for 56.1% of the total population of the nation. 11 in the meantime, china has scored an extraordinary gdp growth rate ranging from the lowest 6.8% to the highest 13% between 1991 and 2014. 12 if these are to be seen as modernization or development, the modernization or development droughts and storms, deforestation, desertification, etc., affected more than two billion people and the economic toll multiplied compared with the previous decades. 17 this trend has an even acuter presentation in china. in the past three decades, china experienced extraordinary economic growth, accompanied by rapid industrialization and urbanization. the emergence of many city clusters has resulted in centralized and condensed population in urban areas. the economic development, together with urbanization, has generated substantial environmental problems. nowadays, china is facing nearly all of the world's ecological challenges including climate change, desertification, deforestation, declining water resources, acid rain, soil erosion, air and water pollution and biodiversity loss. 18 apart from increasingly frequent natural disasters caused by climate change, air and water pollution has become a paramount concern to the general public, which is acutely perceived in their daily life. as mentioned in the beginning of this section, the cost of environmental degradation in china is massive and profound, from people's lifespan to economic toll. the link between environment and human rights can be established on basis that physical environment is a precondition for living a life of dignity and worth. good environment is a pre-requisite for the enjoyment of fundamental human rights. 19 in china, for a long time, human rights violations and environmental degradation have not been treated by central and local governments as related issues and addressed in a coherent and integrated way. it has become evident that environmental protection and human rights are prima facie interrelated, interconnected, and mutually responsive, since they both serve the purpose of improving and sustaining the well-being of humanity. 20 human well-being has a basic requirement for safe food, clean air and drinking water, safe shelter and protection from diseases. 21 human activities such as rapid industrialization and urbanization result in environmental degradation 17 including rising of sea levels, air and water pollution, drought, flooding, which in turn, affects people at a large number. at the international law level, the vital role that environment plays in guaranteeing individuals' fundamental human rights has been recognized at a much earlier time. the stockholm declaration of 1972, nairobi declaration of 1982, world charter for nature of 1980, earth summit of 1992, johannesburg conference on sustainable development of 2002, un conference on sustainable development of 2012 have all acknowledged the interdependence between environment and human rights by conveying the message that environment may lead to long term impact on humanity and violation of human rights. 22 the human rights council in its resolution 7/23 of march 2008 and resolution 10/4 of march 2009 focused specifically on human rights and climate change, pointing out that environment change impairs and undermines directly and indirectly the effective enjoyment of human rights. 23 traditional international environmental laws commonly address environmental issues through creating and determining rights and obligations between states. human rights laws, on the other hand, place individuals' rights at the center of the focus. the establishment of the linkage between human rights and environment enables individuals victimized by environmental degradation to seek remedy on human rights violation ground, which traditional environmental laws gives little focus. it is unfortunate that in china's priority agenda, both human rights protection and environment protection have given way to economic development for quite a while. the link between environmental damage and human rights violation has not been articulately addressed either. indeed, it is a high time for china to adopt a comprehensive, human rights based approach to formulate environmental policies for clarifying human rights obligation in environment protection and for identifying and promoting good practice in environment management. there are signs that current central government shows the interest in considering human rights law enforcement as a means of achieving the purpose of environment protection. "environment protection" have become hot words in china's political life since the 18th national congress of the communist party of china (nov. 8-14, 2012) . in his keynote report in the opening ceremony, hu jin tao, the president of china by then, emphasized the term "ecological civilization" for fifteen times. his successor, the current president xi jin ping, in his speeches at the national people's congress 2015 and at the chinese people's political consultative conference 2015, called for environment protection and pointed out that protecting environment is as important as protecting productive forces. 24 with the initiative from top leaders, the governments of all levels have become more and more mobilized to change their focus and give particular importance to environment protection. although the areas like beijing, hebei and shanxi are still frequently blanketed by hazardous smog and problems caused by environmental degradation and climate change are still acute, people are more willingly to adopt an optimistic attitude when speaking likely environmental improvement in the coming decades. nowadays, with the growth of economic prosperity, common chinese people enjoy an ever-abundant supply of food products and are keen to broaden their dining experiences. in today's china, eating occupies nearly all occasions and businesses. visitors to china are likely to find that eating is arranged as a highlight for them by the hosts. some research suggests that the current food consumption pattern in china even poses as an exception to engel's law, that is: as the income of an average chinese family rises, the proportion of income spent on food increases, rather than decreases. 25 some other research finds that in fact, the share of chinese households' income spent on food fell from over half in the 1970s to about one-third nowadays. 26 nevertheless, one-third of household income being spent on food is still spectacularly excessive, compared with 6.7% in the u.s., 9.3% in the u.k., and 6.7% in singapore. 27 while all levels of the population take any possible opportunities to indulge with feasts, disturbing facts concerning food safety emerge. this article concerns itself with some of the most severe problems associated with food safety in china, which mainly refer to the cases involving deliberate contamination of food for profits. these include problems such as manufacturing and trading imitation food (e.g., selling pork as beef), processing and selling contaminated food, adding chemical additives into food products for improving visual appeal, taste, or simply for falsely 24 increasing nutritional value (e.g., adding melamine into baby formula to inflate the protein levels), and producing and trading tainted food (e.g., profiting from recycled oil mainly dredged up from restaurant gutters). 28 worst of all, the above problems are not sporadic but are widespread and rampant, and in some cases, systematic. it needs to be borne in mind that the above issues are the most severe ones and only account for a small part of the avalanche of food scandals. here, incidents relating to sanitation or foodborne illness outbreaks caused by negligence in manufacture and service processes are not taken into account, though they are also serious problems. the right to safe food is a basic human right for all individuals. 29 in addition, safe food should be regarded as an issue of national security for the reason that toxic food poses a serious threat to public health, which in turn leads to destructive social and economic consequences, and eventually has an adverse impact on the national interests of a country. it is high time for the chinese to review their policies and strategies in dealing with social issues, including social justice, equal distribution, anticorruption and clean governments, social ethics and public morality, and public psychology. regulation is surely a necessary instrument to control illegal conduct. however, given the fact that policies and laws are always watered down or compromised in implementation, it is thus particularly important for policy makers to take all the above issues into consideration when shaping the nation's regulatory infrastructure for food safety. when serious food problems have a devastating impact on a large proportion of the population, it is necessary to assess the situation from a human rights perspective while searching for solutions. this section examines china's food safety issues in connection with human rights awareness in the country. it investigates the social, political and cultural causations of the food scandals, and explores possible solutions. before the 1980s, under the planned economy, food problems that arose from manufacturing were extremely rare and the regulatory focus was on sanitation occurring in the service and consumption processes, where the food safety administration was part of the duties of the sanitation and anti-epidemic departments. 31 although it appeared that a legal framework was largely put into place, the food industry was, in fact, less effectively regulated, due to the loose context of the laws and the gaps between the laws and their practice. 32 the situation has deteriorated since the beginning of the new millennium with the introduction of the practice of quality inspection exemption or the regime of self-inspection by enterprises themselves, an initiative launched to reduce the operational costs of food enterprises. 33 the self-inspection regime was a result of the administrative authorities exercising their supervisory powers in an abusive manner while in the discharge of their quality control duties concluding with the imposition of unnecessary and excessive burdens on food producers. 34 however, instead of disciplining administrative conduct, the policy makers opted for a compromise by abolishing food quality inspection. research has shown that the managerial costs in chinese firms are chiefly caused by defects inherent in the governance of these firms. 35 without fundamental improvements in the corporate governance system, 30 34 xu, supra note 32. 35 see, for detailed discussions on managerial costs and corporate governance in chinese firms, yuwa wei, "directors' duties under chinese law -a comparative review", 3(1) university of the costs are likely to continue to inflate. later instances of food safety crises have proven that those who maintain the argument that relaxation in quality control can reduce food companies' operational costs are simply barking up the wrong tree, where the most likely result is noncompliance. it took the government 8 years to realize that giving enterprises free rein in regards to food quality control was an unwise decision. it was at least partly responsible for the current culpable culture in the food industry. in the aftermath of a series of food scandals, policy and regulatory mechanisms have been introduced as the main crisis control efforts. on the current law imposes full responsibility on food producers and manufacturers to ensure that their products meet safety standards throughout each process, including purchase of raw materials, production, storage, package, transport and delivery. 37 in addition, they need to faithfully record the safety management throughout the whole course. however, china's experiences reveal that without fierce scrutiny, the industry is unlikely to comply with such requirements. under the current regime, the supervision and administration are given to local governments at or above the county level, who formulate annual plans for food safety and undertake food sampling inspection. 38 specific branches of government, including the departments of agriculture administration, quality supervision, industrial and commerce administration, and the food and drug administration carry out the routine work of food sampling inspection. 39 (2006) china, § 3 (2009) , an english version is available at <www.fdi.gov.cn/1800000121_39_4037_0_7.html>. the section states: "the food producers and traders shall, in accordance with laws, regulations and food safety standards, carry out productive and operational activities, establish and improve the food safety management system and adopt efficient management measures to ensure food safety. the food producers and traders shall be responsible for the safety of produced and trade food themselves as well as society and the public, and also bear social responsibility." 38 ibid, at § 47. 39 ibid, at § 48. persons with direct responsibility in the above mentioned governments are subject to disciplinary punishment comprising of the recording of serious demerits, demotion, dismissal or expulsion, in the event of a failure to perform their supervisory and administrative duties, particularly where the failure causes a serious social effect. 40 the same penalties are imposed directly on persons responsible for the above mentioned government branches, in the case where negligence is committed, daily supervision and inspection of food samples are omitted, abuse of power occurs, or favouritism is shown in the supervisory and administrative processes. 41 from the above overview of the new regulatory regime, a few inadequacies can be identified. first, the legal (regulatory) framework is far from comprehensive. the supervisory model of food safety mapped out in the new laws can be summarized as: relying mainly on the segment-based approach, supplemented by a categorybased approach. 42 in other words, the administration of food quality is undertaken by multiple inspection and administrative agencies, exercising their authorities in different stages of food production and services and targeting different types of foods. 43 such an arrangement poses a potential risk in regards to conflict of powers, though the new laws allude to the benefits of efficient coordination among these agencies by identifying their authorities and duties in different stages of production and services. 44 given the gaps in the existing laws, it is very likely that china will encounter further challenges in food supervision caused by its inefficient administrative system. speaking from a broader perspective, china is not the only country that has applied a multi-agency approach to food supervision and administration. the united states and japan have also adopted a scheme of assigning duties of food administration to multiple governmental agencies and departments. 45 however, their comprehensive and detailed stipulations on division of responsibilities, quality standards, transparency and public scrutiny have ensured effectiveness of the system and smooth coordination among the agencies. moreover, it is noteworthy that even in countries such as the united states, there are increasing calls for combining their multiple agencies into a single agency to further the operational efficiency. 46 it is therefore necessary for china to improve its food safety administration system to further enhance its regulatory regime on the one hand, and to rationalize the administrative chains and procedures in food supervision on the other. second, the current legal regime does not provide sufficient remedies for victims of food safety, nor does it effectively encourage consumer and community participation in food safety control. the laws have introduced monetary remedies for consumers consisting of damages and a fine of ten times that of the commodity's value. however, the accused are liable for the fine only where evidence shows that they have produced defective products and are at fault. 47 what is more outrageous is that the onus of proof is on the consumers in such cases. 48 the amount of ten times the value of the goods has already been criticized as being too insignificant to be a deterrent. 49 the requirement that consumers need to prove the culpability of producers in making defective foods renders it nearly impossible for the consumers to benefit from the fine, not to mention the conceivable negligible impact that the provision has on food producers. third, the regulatory regime has loopholes and demonstrates too much leniency towards liable producers and traders, as well as those administrative personnel who fail to discharge their duties or show favoritism. among government staff of relevant departments, only those directly responsible for administrative negligence or abuse of powers that result in serious social impact may face disciplinary action. 50 criminal penalties imposed on food producers for breaches of the law are also weak. moreover, no single criminal penalty is available to penalize delinquent public servants. the criminal law of china only punishes the conduct of producing 45 for instance, in the u.s., the powers of food administration are divided amongst the u.s. department of agriculture, the food and drug administration and the environmental protection agency. 46 see chamus burnside-savazzini, "training in an integrated food safety system: focus on food protection officials", food safety magazine (april/may 2011), <www.foodsafetymagazine.com/ article.asp?id=4018&sub=sub1>. 47 the people's congress of the people's republic of china, supra note 36, § 96. 48 the food safety law does not address the issue of burden of proof. hence, the rules of general law apply, rendering that the burden of proof lies upon he who brings the claim. 49 see tonghai liu, "on the shortcomings of the foot safety law and some suggestions on improvement", blog of tonghai liu on legal study net (october 10, 2011), <http://wq.zfwlxt.com/ newlawyersite/blogshow.aspx?itemtypeid=fdf950ea-312b-4519-91ea-9bfb01037315&itemid= de914f78-54bd-4f50-9bdc-9f80015e3473&user=9947>. 50 the people's congress of the people's republic of china, supra note 36, § 61. and selling foods causing serious food poisoning and diseases, and the conduct of producing and selling foods mixed with toxic or harmful non-food substances. 51 it can thus be observed that the scope of application of the criminal law provisions is limited to offences occurring in food production and sale. this leaves the conduct of processing, packaging, storing and transporting defective or harmful foods unpunished. 52 furthermore, the amounts of the penal fines provided in the criminal law are even less than those stipulated in the food safety law, and are subject to a higher threshold in application. 53 finally, the most essential issue to be addressed by the chinese is the enforcement of the laws. according to a survey carried out by the national survey research center at renmin university of china in 2011, 68% of people interviewed attributed the cause of food scandals to administrative inefficiency and supervisory failure. 54 in the meantime, judicial failure to rigorously implement food laws has cast a further shadow over effective law enforcement in the future. it is reported that courts are reluctant to support consumers' claims for damages up to 2 or 10 times the amount of the commodity price provided for in the law of the people's republic of china on the protection of consumer rights and interests 1993 and the food safety law of the people's republic of china 2009. 55 many manufacturers also promise 51 the criminal code of china states: "whoever produces or sells food that does not confirm to hygiene standards in a manner that is sufficient to cause a serious food-poisoning accident or any serious disease caused by foodborne bacteria shall be sentenced to fixed-term imprisonment of not more than 3 years or criminal detention, and concurrently or independently be sentenced to a fine of not less than half of the sum obtained through sale and not more than twice of that. if the offence causes serious harm to human health, the offender shall be sentenced to fixed-term imprisonment of not less than 3 years and not more than 7 years, and concurrently be sentenced to a fine of not less half of the sum obtained through sale and not more than twice of that. if the consequences are especially serious, the offender shall be sentenced to fixed-term imprisonment of not less 7 years or life imprisonment, and concurrently be sentenced to a fine of not less than half of the sum obtained through sale and not more than twice of that or confiscation of property." a person who knowingly mixes the food to be produced or sold with toxic and harmful non-food stuffs is subject to the same penalties. : "when any manufacturer produces any food not conforming to the food safety standards or sells any food knowing its nonconformity with the food safety standards, the customer can demand the manufacturer or the seller to pay a penalty 10 times of the paid amount, in addition to the compensation for the loss thereof." the amount of the compensation provided for in article 49 of the law of the people's republic of china on the protection of consumer rights and interests 1993 is up to two times upon occurrence of fraud. junhai liu, the deputy president of china consumer association pointed out that due to judges' misunderstanding of legal reasoning, consumers' that an amount of compensation of up to 10 times the amount of the commodity price will be paid to consumers upon violation of food safety. however, such claims are likely to be rejected by courts. some judges insist that such an amount appears to be punitive and thus does not have legal basis in contractual relations. the stance of courts in such cases renewed the public's suspicion of judicial corruption. the worst scenario concerns events involving arrest and conviction of consumer advocates. the zhao lianhai case is a typical example. zhao lianhai was a man who became an activist after his son experienced kidney problems linked to contaminated baby formula. he was convicted of inciting public disorder by setting up a web site to help other parents with sick children share information, seek compensation, and organize protests. 56 it is clear that despite legislative efforts, tackling the food safety problem is an overwhelming challenge. compared to assessing legislative improvements, evaluating law enforcement in china presents a far more demanding task and is entangled with a number of social, political, economic and cultural issues. more discussions on enforcing food safety law in china will be carried out in section iii of this article. in summary, much work needs to be done to develop an efficient and effective food safety regulatory regime. this is not a mere legal task, but a social project involving reform of the administrative culture, a change in the business environment and transformation of the perception of the right to safe food for the public. human rights are rights inherent to all human beings, and they are inalienable and fundamental. 57 the international community has reached a common understanding on the universality of human rights. for promoting and protecting human rights and fundamental freedoms of individuals, governments are obliged to act in certain ways or to refrain from specific acts. 58 the most common universal human rights claims for compensation provided in the above provisions are rarely supported by courts in the past. see social and cultural rights 1966 , the right to food is recognized as part of the right to an adequate standard of living. 63 the rome declaration on world food security and world food summit plan of action 1996 reaffirms everyone's right to "have access to safe and nutritious food, consistent with the right to adequate food and the fundamental right of everyone to be free from hunger" and describes food security as: "[w]hen all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life." 64 it is therefore clear that as a part of human rights, the right to food is inalienable and indivisible from other fundamental rights including the right to life, the right to health, the right to know, 65 (13-17 nov. 1996, rome) . 65 here, "right to know" is a term used in environmental law, referring to the legal principle that the individual has the right to know the chemicals to which they may be exposed in their daily living. it comprises two aspects -community right to know (environmental hazard information) and workplace right to know (workplace hazard information). nowadays, the term is used in the field of human rights in reference to the right of access to information concerning citizens' human rights, or their rights under freedom of information laws. for instance see sandra coliver, the justifiable to say that the right to food incorporates at least the following dimensions: availability, accessibility and adequacy. 66 in other words, food must not only be available as a resource and affordable in order for people to be able to satisfy their dietary needs, but must also be safe for human consumption and free from contaminants. everyone must have access to food that is not only nutritionally adequate, but also free from harmful substances, so that it does not endanger people's lives or health. the states parties to the international covenant on economic, social and cultural rights 1966 have agreed to assume the obligation to progressively realize the right to adequate food. the responsibility is interpreted by the committee on economic, social and cultural rights as comprising three types of obligations: respect, protect and fulfil/facilitate. 67 a state government must not interfere with individuals' efforts to access food. 68 it must also protect its people from infringement of their right to adequate food by others. the government should help those who do not already enjoy the right to food by creating opportunities for them to provide for themselves. in the case where people are unable to enjoy the right to food due to events out of the individuals' control (natural disaster for example), the government ought to provide food to them directly. 69 china is a signatory of the universal declaration of human rights and a contracting party of the international covenant on economic, social and cultural rights 1966, the convention on the elimination of all forms of discrimination against women 1979 and the united nations convention on the rights of the child 1989. china also signed the international covenant on civil and political rights 1966 but has yet to ratify it. 70 the government of china is under international obligation to facilitate a pre-warning system regarding threats to the right to adequate food, and to improve coordination between different government and food safety agencies, as well as improve their accountability. violation of the right to food refers right to know: human rights and access to reproductive health information (article 19 -university of pennsylvania press, london-philadelphia, 1995) ; and jacqueline klosek, the right to know (praeger, westport, 2009) . article 19 of the universal declaration of human rights states: "everyone has the right to freedom of opinion and expression; this right includes freedom to hold opinions without interference and to seek, receive and impart information and ideas through any media and regardless of frontiers." 66 jean ziegler, "what is the right to food?", right to food (august 6, 2012) <www.webcitation. org/68cmsst1c>. 67 not only to shortage of food, lack of infrastructure, mal-distribution and inadequate access to food, but also to protection from contaminated and harmful food. most of the recent food scandals in china involve rampant violations of the right to food in the dimension of adequacy -provision of food for safe consumption. while immoral and illegal conduct of individuals and enterprises is ultimately responsible for the outbreak of food safety crises, the government is accountable for inefficiencies in monitoring the enterprises and disciplining personnel in charge of administrating the food industry. as the most populated country on the earth, the chinese government has always paid great attention to food issues and regarded provision of enough food to the people as the first priority. after decades of effort, china has mostly solved the problem of feeding its people. 71 however, new problems associated with agriculture and food have arisen since the commencement of market oriented reforms. it is reported that due to accelerated industrialization and urbanization, arable land has been shrinking and farmland capacity has declined. 72 this constitutes a threat to china's future food security. the government has introduced the national land consolidation plan (2011) (2012) (2013) (2014) (2015) that aims to increase the effective area of arable land by 1.6 million hectares before 2015, though the enforcement of the plan faces challenges such as lack of mechanisms for protection of cultivated land and inefficiencies in the management of land tenure rights. it appears that china has made a significant effort to achieve food sufficiency in the country. food availability and accessibility have been a top agenda item for the government in the past and will continue to remain a central issue in the future. the adequacy aspect received far less attention. the recent outbreak of food scandals is chiefly a result of a lack of oversight within the food industry. this can be partly attributed to the understaffing of supervisory authorities, partly to the misfeasance, malfeasance, nonfeasance and unprofessionalism of administrative officers. it is noteworthy that administrative personnel's unsupportive attitude and behavior towards businesses has been the cause of changes in inspective practice, resulting in a switch of models from official inspection to enterprise self-inspection. 73 under the enterprise self-inspection regime, the working manner of administration is even more unprofessional. for instance, in the recycled buns case, when doing their sample inspection, the governmental inspectors, once arriving at a food factory or workshop, would only come into the administrative office and wait for the factory managerial staff to provide the samples to be examined. it is no surprise that they did not get any clues over the years. 74 in one of the clenbuterol contaminated pork 71 s z gu and y j zhang, "food security in china", in sun honglie and shidong zhao (eds.), area studies -china: regional sustainable development review (vol. 1, 2009) incidents, the problematic pigs were sold from henan pig farms to jiangsu province and had to go through several checkpoints conducted by the trade bureau, health inspection station, veterinary station, etc. however, some inspectors acted as middlemen between pig sellers and slaughterhouses for a commission of rmb10 yuan per pig, which caused wide dissemination of the contaminated pork and pork products. 75 the recycled buns case showed workers in a filthy workshop "recycling" buns by throwing the stale buns into a vat, adding water and flour, and repackaging them to be sold anew. the clenbuterol contaminated pork case involves pig farmers feeding pigs with pig feed mixed with clenbuterol used to make the pigs have leaner bodies, which sell for a higher price. other food scandals involve melamine-contaminated baby milk; pork sold as beef after it was soaked in borax; rice contaminated with cadmium, arsenic-laced soy sauce; popcorn and mushrooms treated with fluorescent bleach; bean sprouts tainted with an animal antibiotic; and wine diluted with sugared water and chemicals. 76 the alarming fact is that most of the food related offences were first discovered and disclosed by journalists, and not the personnel in charge of the surveillance authorities, or even the police or national security authority. it is inexcusable for administrative agencies to claim that ill-training, poor quality equipment and understaffing are the main causes for such an appalling incidence of food scandals in the country, since journalists are by no means in a position to be better trained and better equipped than food inspection agencies or police. as a matter of fact, detection and disclosure of food safety problems is not the expertise of journalists, nor their major business. respect for human rights is becoming a universal principle of good government. 77 this is because many human rights require activity on behalf of government. these activities relate to the duties to respect, protect and fulfil human rights in order to provide the conditions necessary for prosperity and well being of individuals. 78 a good government is committed to protection of the rule of law. 79 hence, it is the state government's responsibility to make good laws and to establish able, professional and corruption-free administrative institutions, with a public service team to guarantee the implementation of the laws. although china's government is quick in making legislative efforts to curtail misconduct in the food industry, it needs to demonstrate its determination and capability of ensuring that the laws are fully abided by and it is accountable for building a law enforcement agency comprised of highly motivated, dedicated, well-trained law enforcers. a typical national food control system comprises the following components: food law and regulation, food control management, inspection services, laboratory services and information, education, communication and training. 80 the capacity of food safety control in a nation is demonstrated by effectiveness, efficiency and sustainability. in china's case, apart from legislative efforts, much needs to be done in the following aspects: law enforcement involves certain members of society acting in an organized manner to promote adherence to the law by investigating and punishing persons who violate the rules and norms governing that society. the enforcement of the law consists of a system comprised of the courts and procuratorates, the judicial and public security agencies, and the administrative agencies, with their descending hierarchy of departments, bureaus, sub-bureaus, and stations. with regard to enforcement of food laws and regulations, administrative mechanisms play a more substantial role, compared with other enforcement instruments. the key is to enhance food control management. this involves development of food control strategies, implementation of risk analysis, operation of a food control program, securing funds and allocating resources, and development of emergency response procedures. 81 development and implementation of food control strategy is concerned with defining food control objectives, identifying priorities of competing interests relating to public investment, state economic interest, food import and export, farmers and producers. 82 an integral, effective food control strategy is particularly important to china, since it will improve the coherence of multiple existing administrative agencies in the country and reduce confusion, duplications of effort, inefficiencies in performance and waste of resources. for the time being, a crucial task china 80 food and agriculture organization of the united nations and world health organization, assuring food safety and quality: guidelines for strengthening national food control systems (joint fao/who publication) 6-9 (rome 2003), <ftp://ftp.fao.org/docrep/fao/006/y8705e/ y8705e00.pdf>. 81 see food and agriculture organization of the united nations and world health organization, supra note 62, at 7. 82 ibid. faces is to consolidate its food control policy at the macro-level by standardizing food quality certification, management, and rating, and in the meantime, to improve the market price system. a market entry certification system should also be put in place. another important task to deal with is to establish a uniform food safety risk analysis scheme to ensure compliance with food regulations and standards. 83 unlike developed countries, the food safety problems in china occur in every stage from food production to service, typically consisting of contamination of raw food materials, cross contamination in manufacture, improper storage and preservation, and unhygienic practices in services. 84 this requires a wide-ranging risk analysis system capable of identifying risk factors at every stage and of providing solutions. in doing so, there is the need to coordinate the risk assessment activities currently exercised by different administrative agencies and to develop a comprehensive scheme for food safety risk assessment, in which a professional team comprising food researchers, toxicological analyzers, pathological analyzers and inspectors play an essential role. attention should also be paid to food safety risk communication improvement. insufficient food risk communication in the past was chiefly responsible for a crisis in customer confidence with china's food industry. 85 until this point, no substantial improvement has been made in this regard. in summary, the flaws of this situation can be attributed to a lack of government guidance, silence on the part of scientific academia, and unreliability of entrepreneurs. 86 addressing this issue, the minister of health recently announced that a plan for food safety risk communication was in development and the press was expected to continue to play a leading role in this aspect. 87 however, without access to channels for obtaining scientific information, the press's capacity for publishing fair and accurate reports is considerably undermined. it is therefore necessary to base risk communication on a system of interactive exchange of information and opinions among all stakeholders, including risk assessors, risk managers, consumers, industries, the academic community and other interested parties. this requires the government to allocate sufficient funds to finance and staff risk communication institutions of various specialities. china has more than 200 million farmers and more than 500,000 food production companies. 88 hence, to some, the food production system seems too vast to 83 see world health organization/food and agriculture organization of the united nations, "about risk analysis in food", <www.who.int/foodsafety/micro/riskanalysis/en/>. 84 allow for meaningful inspection at all stages of the food production process. 89 however, the establishment of a national database for food supervision and inspection and a platform for food safety risk assessment enabling all interested parties to follow the development of food safety will absolutely make a difference to china's food industry. by the end of 2009, the ministry of health formed the expert committee for national food safety risk assessment, which was a significant step toward improvement of china's food safety control. 90 it is also reported that the state council of china has revealed its intention to establish a database of food companies' safety records. 91 according to the plan, companies with poor safety records will be blacklisted and publicized, and will be subject to punishment. 92 better regulation mechanisms, legal and standard systems, as well as technical support systems will also be introduced to improve the overall food safety management level. 93 now, the world is waiting to see how well the plan will be realized. improving inspection services for food safety control is another key element for enhancing the food safety control system in china. food inspection service is a means of ensuring that the food supply is safe for consumers and meets legislative requirements. it is also an instrument for decreasing hazardous intoxicants in foods, including pathogenic bacteria, viruses and parasites, chemical contaminants and mycotoxins. the primary duty of the food safety inspection service is to inspect food manufacturing, processing and handling facilities, importing/exporting of foods and the monitoring of a company's facilities for compliance with the national legal and regulatory requirements. 94 in some countries, the food inspectorate also conducts the functions of investigating food poisoning and injury cases and fraudulent marketing practices, as well as handling consumer complaints. 95 of individual inspectors and the quality of their work. it is thus important to make sure that inspectors have detailed guidance and rules to follow when discharging their responsibilities. their powers and the procedures to be followed should be well defined. inspectors should receive up-to-date training in the new technologies used in processing and manufacturing, including what is required for the control of these technologies in order for them to function at maximum effectiveness and to assure proper performance. they must also be able to evaluate the performance of equipment and instruments used in production to guarantee that they are appropriately controlled and monitored. 96 the impotence of china's food safety inspectorate has been widely reported and acutely recognized by consumers. it is officially perceived that the food safety inspection administration suffers from insufficient resources, a lack of qualified personnel, and deficient coordination among layers of governments and among various institutions, particularly among those administrative agencies at the provincial level and below. 97 apart from the above-mentioned vices, three more factors must be taken into account: corruption, shirking and laziness. china's public services have long suffered from a number of bureaucratic problems such as authoritarianism, routinism, elitism, corruption, shirking of responsibility, deceit, laziness, formalism, red tape, nepotism, seeking special privilege, overstaffing, duplication, ineffectiveness, and over-centralization. 98 upon close study, one can discern that corruption, shirking and laziness are the most prominent problems that induce or exacerbate the other problems. these deep-rooted problems penetrate the food safety inspection system and eventually take their toll on public confidence in the food industry. the government of china has pledged to enhance the ability of the inspectorate. in its food safety decision june 23, 2012, the state council announced that it planned to take 5 years to improve the nation's food safety and food control management. 99 according to the state council's statement, food safety will become a measure of local governments' performance in their annual assessments, in the hope that incorporating food safety as one of the evaluation criteria will make local governments and officials aware of their responsibilities. 100 however, given the institutional decline, erosion of authority of the central government, and loss of control over local agencies and agents in the era of the market economy, effectively rooting out corruption in the food safety administration will certainly demand much 96 greater efforts based on resolute determination. to a certain extent, it represents a social problem and needs to be tackled on a broader scale. to curtail food safety problems on a broader scale, it is necessary to promote general respect for universal human rights in the society. from this point of view, improvement of food safety can be seen as a social project. in the past three decades, in making the transition from a planned economy to a market economy, china placed much emphasis on economic gains and profitability. 101 along with gdp growth, extreme utilitarianism, fickleness and mammonism have become more and more evident and have turned into a driving force in chinese people's daily life. 102 the food safety outbreaks are merely typical consequences of the erosion of social morality and business ethics. food safety outrages spark a new round of calls to promote public morality and human rights. 103 many chinese have come to realize that without a morally healthy social environment, mere economic prosperity does not guarantee a successful society and true advancement. in contrast, it may only carry with it the seeds of corruption and decay. to bring changes to the broader system, the general public needs to step out from their family-centred tradition and self-interested sphere, and endeavor to shape a society where individual equality is upheld, respect for others' human rights is generally aware of, and public interests are greatly valued. the construction of such a society requires the combined efforts of the government, private sectors and the community. while the government is accountable for formulating policies and initiatives, facilitating state agencies and mechanisms, and allocating resources, the business sector and the community are expected to participate in shaping a good society by promoting sustainable business practices, interacting with the government and influencing the policies of governance. it is worth noting that the constitution of china only implicitly addresses citizens' rights to safe food. article 21 provides that "the state . . . promotes health and sanitation activities of a mass character, all for the protection of the people's health"; and article 45 states: "citizens of the people's republic of china have the right to material assistance from the state and society when they are old, ill or disabled." 104 compared with some nations that incorporate the right to healthy food into their constitutions, china is much behind in providing constitutional safeguards for food security. 105 given the challenge of tackling food problems, it may be necessary for china to introduce provisions to protect food safety in the constitution. in the case of combating food safety problems, the food industry shares responsibility with governmental agencies in achieving the objectives of a national food control strategy. involving the industry in national food control activities might be instrumental in overcoming potential problems. the food industry is responsible for the implementation of rules regulating agriculture, food manufacturing and processing practices, as well as a food quality and safety system. 106 the industry should also be obliged to educate and train all employees in food handling and make sure that they understand general food quality and safety system. it can also provide information to consumers through food labeling and advertising. 107 in china's case, the food industry may need to go through a long journey to correct its practices which may also involve a change in the business culture. as members of the community, consumers have a right to quality and safe food and also have responsibilities to prevent food related health hazards. consumers need to understand there is no such thing as an absolutely safe food supply. 108 the right to food for consumers also requires a responsibility to play a role in food control. consumers can actively contribute to food safety control by providing valuable information to the authorities, usually by complaining about product deception and poor quality and by reporting injury and illness caused by food. 109 the authority then needs to provide them with a channel to let their dissatisfaction be known. consumer organizations are expected to play an important role in representing the consumer in the development of a national food control strategy and also in bringing the concerns of consumers to the attention of policy makers and the industry. 110 the chinese government needs to take proactive action in order to ensure that consumers are heard and taken into account. in summary, combating food safety problems depends on a combination of mechanisms including training, business ethics education, administration and supervision, public scrutiny, civil litigation and penal punishment. in other words, combating the food safety problem require a collective effort from the entire nation. the government is certainly accountable for all mandatory activities necessary to ensure the quality and safety of food, including enacting food regulations, operating food inspectorates, organizing analytical services, and enforcing rules. the participation of other stakeholders and interested parties including the industry, consumers, communities and even the society as a whole, is equally crucial. the battle will happen across a gamut of fields involving governmental conduct, social ethics, public relations, business practice, and consumer culture. the medical insurance system of a state directly affects its people's key human rights, namely the right to life and the right to health. the notion of modern medical insurance can be traced back to the introduction of the sickness insurance program in 1883 in germany. the antecedents of similar kinds existed in the form of charity or welfare in some civilizations, such as friendly societies organized by european trade guilds, fraternal organizations, the english poor law 1601, the prussian common law 1974, and the civil war pension program 1862. the sickness insurance program in germany came as a result of the medical reform movement 1848 and a series of political debates and compromises afterwards. 111 the sickness insurance act was enacted in 1883 to facilitate the program. the law allowed the largest segment of workers to benefit from access to public health care through insurance funds. in the event of accidental injury, illness or old age, workers were entitled for minimum indemnity for medical treatment and sick pay for up to 13 weeks. this initial system was financed by workers and employers, with the employers contributed one third, while the workers contributed two thirds to insurance funds. in 1884 germany passed the accident insurance act covering serious and fatal accidents occurring on the job in certain industries. the 1884 act went a step further by placing the entire cost of the liability on the employers. the significance of the german health insurance program introduced in the 1880s lay in that it was for the first time in history that a national health insurance system was institutionalized. 112 following the german practice, industrialized countries commonly introduced social security programs in the following decades, i.g., the uk 111 114 the medical insurance program as an indispensable part of the modern social security system has carried human societies into a new era where the government of a country provides economic assistance to persons faced with unemployment, disability, or agedness for the purpose of advancing the benefits of the members of the community as a whole. 115 the key feature of the scheme is: it is imposed and controlled by the government. before the introduction of social security schemes, the dominant mode of support for people unable to provide for themselves was charitable relief proffered by benevolent societies, sometimes with financial help from the authorities. social security comprises two arms: social assistance and social insurance. 116 the rationale behind modern social security programs is the necessity to maintain social and economic justice. with industrialization, social security has gone through a change from charity to a state responsibility. guaranteeing a certain amount of social well-being and economic security to every individual member in the community has become a duty of a modern state. 117 nowadays, social security has emerged as a human right enshrined in the universal declaration of human rights, which states: after the establishment of the socialist state in 1949, as part of the social welfare or security package china introduced a state funded, central planned health care system, which provided a universal coverage of health care to the chinese population. an innovative feature of the system was the operation of the so call barefoot doctors at health clinics at the village and township level, which served the mass rural citizens accounting for three quarters of the total population. 119 this system was held up as a model for providing universal health care in the developing world in the 1970s by the world health organization (who) and inspired who to launch the "health for all by 2000 a.d." program. 120 under this scheme, public health in china improved dramatically, with the citizens' life expectancy increasing from 35 to 68 years, infant mortality decreasing from 200 to 34 per 1000 live births. 121 the system operated in china for nearly 40 years until the market oriented medical reform took place in the 1980s. as a result, china's health care program has experienced a change from a scheme performing universal social welfare function to a model that is a mixture of free market and medical insurance. however, the reform turns to be a failure which makes china's health care scheme one of the least equitable systems in the world. 122 care reforms ciency and abuse. its sustainability posed a challenge to the leadership in the post-mao era, which had an economic reform scheme at the top of the agenda. by the 1980s, the administrative and financial support for the health care was removed from the national agenda through decentralization before a nationwide health insurance system was provided. 125 with local governments having little incentive to promote local healthcare, individuals of rural areas and urban non-salaried residents primarily relied on out-of-pocket spending for medical and health services. in the meantime, prices for provision of medicines, diagnostic tests, surgical implants, and specialized care were deregulated. as a direct impact, many chinese were in short of medical services and supplies since they simply resolved to not seeking medical help. it was reported in 2003, about 50% of the chinese people refused to see a doctor when ill and 30% of those refused to go to hospital, mainly out of financial concerns. 126 there was still worse to come. after the collapse of the previous model, no strategy was adopted to mitigate the lack of integration and coordination in the later medical system. due to a lack of reliable local clinics, people had to wait in long queues for basic health care, with no scheduled appointments and little privacy. after all the inconvenience, what they could receive was usual a very brief face to face consultation of a few sentences or a couple of minutes. 127 during this period, health care was seen "as a consumption activity rather than a fundamental right of the people or a responsibility of the state". 128 indeed, this unsustainable, reactionary approach had to be subject to review and amendment. 129 as a result, in 2003, the new cooperative medical scheme (ncms) for rural population, mainly funded by the government, was launched. 130 since 2007, a health insurance scheme for urban non-salaried residents, especially for children and old people has been implemented. 131 although china's health care system has appeared to get back on the track of government subsidy since the mid-2000, it is far from offering accessible, affordable, equitable and quality health care services with a universal coverage. some believe that the current health care reform is unlikely to solve the fundamental problems of accessibility, affordability, equity and quality. as long as the buck-passing polity does not fully change and the health policy process is still influenced by interested groups, the problem of dis-125 see jason shafrin, "an olympic post: the history of the chinese healthcare system", healthcare economist (august, 2008), available at <http://healthcare-economist.com/2008/08/ page/3/>. 126 parity inherent in china's health care system will continue to affect the country's social environment. 132 the worst part of china's health care system is the rampant medical corruption stemming from the medical reform over the past decades, particularly in the period between the mid-1980s and the mid-2000s. the usual scenario is that doctors and other staff expect to be paid extra fees (usually in the form of "red envelops" 133 ) to perform operations and take kickbacks from pharmaceutical firms and medicalequipment suppliers. the practice is so prevalent that an unsullied doctor nearly does not exist. 134 almost all practitioners and medical staff tell the same story as the defence of their unclean hands -low salary or underpayment. whistle blowers also tell that doctors have economic income targets allocated by hospitals and hospital administrators for them to meet and exceed. only upon completion of their quotes, they are paid benefit bonuses which can be much higher than their basic salaries. 135 in other words, they are under pressure to charge patients extra fees and to recommend patients to go through unnecessary but expensive tests and procedures as well as purchase expensive medicines. nonetheless, such excuses are not sound enough to defeat professionalism and patients' human rights. besides, on many occasions, they are free to make a choice. for instance, a surgeon or a nurse can always hold up professional dignity by declining a red envelop because it is commonly given privately. moreover, nothing can justify their procurement of briberies and kickbacks from pharmaceutical companies or health care companies. facing a choice between campaigning for improving income or work environment and becoming corrupt, chinese doctors have had less difficulty in choosing to compromise their professional morality in the first place. moreover, nothing can defend the corruptive and negligent conduct of the personnel in governments and the health care system, which have caused further spread of epidemics such as aids. 136 a nation has every reason to become alarmed when seeing medical profession to be infested by the epidemic of corruption. the governments and citizens all have a responsibility to trace the deep root of the phenomenon in their society, culture, policy and ethics in order to find the cures. access to quality health care services is a basic right in a civilized society. it is essential to one of the core human rights -human right to health. 137 the human right to health care means that hospitals, clinics, medicines, and doctors' services must be accessible, available, acceptable, and of good quality for everyone, on an equitable basis, where and when needed. hence, universal access, availability, acceptability, dignity, non-discrimination, accountability, and transparency should be the fundamental principles upon which a health care system is built. many countries have upheld health care as a basic human right in their laws. 138 unfortunately, the failure of the health care scheme has deprived many chinese citizens this right for more than two decades after the medical reform. this renders china's medical reform a truly disgraceful story. when people are deprived their access to hospital and health care services, their human rights, including their rights to health, life and information are potentially compromised and threatened. china is a contracting party to the international covenant on economic, social and cultural rights (icescr) and the united nations convention against corruption (uncac), a signatory of the international covenant on civil and political rights (iccpr). it is thus under treaty obligation to protect individuals against corrupt acts of third parties. though corruption and abuse of the health care scheme committed by practitioners and professionals in hospitals, insurance companies and suppliers of medical and health care goods and services impact on patients' human rights, the guilty individuals are not in direct breach of the uncac. instead, the uncac is directed primarily at member states rather than non-state actors. hence, it is the obligation of the chinese government to prevent corruption involving governments, industries, companies and individuals in fight against corruption and to raise public awareness of the matter. 139 due to the fact that corruption has invaded into every corner of the chinese society, citizens have faced an unfavorable physical and social environment. fraud in project approvals and failure to apply emission control measures are responsible for 137 the human right to health means that everyone has the right to the highest attainable standard of physical and mental health, which includes access to all medical services, sanitation, adequate food, decent housing, healthy working conditions, and a clean environment. 138 xiangming zhou, a study of the health care right (phd thesis, jilin university, 2006) 1. 139 see part ii -articles 12 and 13 uncac. rising pollution in the country. some professions including teachers, doctors, lawyers and those safeguarding consumptive goods are not only important to people's lifestyle and well-being and society's progress, but also play a social role in which trustworthiness is an expectation in every tradition. 140 in china, these professions, because of the rampage of the unethical performance conducted by a majority of the people in the trades, have definitely failed to honor their part of social compact with society, which in turn exacerbates human rights related problems. it is a high time for china to review the ethical conduct of these key professions. the chinese government has an obligation to protect citizens' human rights to health, access to healthy food, safe drinking water, clean air, and a healthy environment. in discharging this obligation, effective mechanisms combating corruption in various forms need to be established in order to sustain the country's social stability and economic prosperity. china -pig farmers told to drug livestock china, fear of fake eggs and 'recycled' buns history of universal human rights -up to ww2 sourcebook: human rights & good governance (asialink project on education in good governance and human rights office of the united 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reform: not just a policy failure, carengie endonwment for international peace an envelope with cash inside if the government is really serious, all the chinese doctors and drug companies will be found guilty because no drug manufacturer or doctor is innocent. if doctors are to be laid off for receiving hongbao, then every chinese doctor will be laid off. but then who will take care of the patients? if the benefit bonus is fully deducted, the doctor only receives the basic salary part. people usually refer the basic salaries when speaking their salaries consequences of a stalled response: epidemic among blood donors in central china sourcebook: human rights & good governance. asialink project on education in good governance and human rights china's environmental crisis social insurance and allied services public health and human rights: evidence-based approaches privatization and its discontents-the evolving chinese health care system training in an integrated food safety system: focus 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by governments at the world summit on sustainable development a study of china's legal system for food safety a study of the health care right chinese vs. western perspectives: understanding contemporary china food safety becomes national priority, china daily what is the right to food? right to food key: cord-196608-k4f79dr4 authors: saha, sovan; halder, anup kumar; bandyopadhyay, soumyendu sekhar; chatterjee, piyali; nasipuri, mita; basu, subhadip title: computational modeling of human-ncov protein-protein interaction network date: 2020-05-05 journal: nan doi: nan sha: doc_id: 196608 cord_uid: k4f79dr4 covid-19 has created a global pandemic with high morbidity and mortality in 2020. novel coronavirus (ncov), also known as severe acute respiratory syndrome coronavirus 2 (sars-cov2), is responsible for this deadly disease. international committee on taxonomy of viruses (ictv) has declared that ncov is highly genetically similar to sars-cov epidemic in 2003 (89% similarity). limited number of clinically validated human-ncov protein interaction data is available in the literature. with this hypothesis, the present work focuses on developing a computational model for ncov-human protein interaction network, using the experimentally validated sars-cov-human protein interactions. initially, level-1 and level-2 human spreader proteins are identified in sars-cov-human interaction network, using susceptible-infected-susceptible (sis) model. these proteins are considered as potential human targets for ncov bait proteins. a gene-ontology based fuzzy affinity function has been used to construct the ncov-human protein interaction network at 99.98% specificity threshold. this also identifies the level-1 human spreaders for covid-19 in human protein-interaction network. level-2 human spreaders are subsequently identified using the sis model. the derived host-pathogen interaction network is finally validated using 7 potential fda listed drugs for covid-19 with significant overlap between the known drug target proteins and the identified spreader proteins. covid-19 evolved in the chinese city of wuhan (hubei province) 1 . the first case of human species getting affected by ncov was observed on 31 december 2019 2 . soon it expands its adverse effect on almost all nations within a very short span of time 3 . world health organization (who) observes that the massive disastrous outbreak of ncov is mainly due to mass community spreading and declares a global health emergency on 30 january 2020 4 . after proper assessment, who presumes its fatality rate to be 4% 5 which urges the global researchers to work together to discover a proper treatment for this pandemic 6, 7 . coronaviridae is the family to which a corona virus belongs. it also infects birds and mammals besides affecting human beings. though the common symptoms of corona virus are common cold, cough etc., but it is accompanied by severe acute chronic respiratory disease along with multiple organ failure leading to human death. severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) are the two major outbreaks in 2003 and 2012 respectively before sars-cov2. the source of origin of sars was located in southern china. its fatality rate was within 14%-15% 8 due to which 774 people lost their lives among 8804 affected cases. saudi arabia was marked as the base for the commencement of mers. 858 persons among 2494 infected cases were defeated in their battle against mers virus. thus it generated a much higher fatality rate of 34.4% 9 when compared to that of sars. all of the three epidemic creators sars, mers and sars-cov2 are biologically included in the genus betacoronavirus which is under the family coronaviridae. both structural and nonstructural proteins are involved in the formation of sars-cov2. out of the two, structural proteins like the envelope (e) protein, membrane (m) protein, nucleocapsid (n) protein and the spike (s) protein play a major role in transmitting the disease by binding with the receptors after entering into human body 10 . no clinically proven and approved vaccine for ncov is available till date though researchers have been trying hard to develop it. so there is an urgent need to understand and analyse the mechanism of disease transmission of this new virus. host-pathogen protein-protein interaction networks (ppin) are very significant for understanding the mechanism of transmission of infection, which is essential for the development of new and more effective therapeutics, including rational drug design. progression of infection and disease results in due to the interaction of proteins in between pathogen and host. pathogen plays an active role in spreading infection. it has been proved to be an acute threat to human lives as it has the mutative capability to adapt itself toward drugs. pathogen and host ppin permits pathogenic microorganisms to utilize host capabilities by manipulating the host mechanisms in order to abscond from the immune responses of the host [11] [12] [13] . detection of target proteins through the analysis of pathogen and host ppin is the central point of research study 14, 15 . topologically significant proteins having higher degree of interactions are generally found to be essential drug targets. however, proteins having less number of interactions or topologically not significant may be involved in the mechanism of infection because of some biological pathway relevance. however, clinically validated human-ncov protein interaction data is limited in the current literature. this has motivated us to develop a new computational model for ncov-human ppi network. we have subsequently validated the proteins involved in the host-pathogen interactions with respect to potential food and drug administration (fda) drugs for covid-19 treatment. key aspects of this research work are highlighted below: • it has been reported that sars-cov has ∼ 89% 16, 17 genetic similarities with ncov. • sars-cov-human protein-protein interaction network has also been studied widely and available in literature [18] [19] [20] . • recently we have developed a computational model to identify potential spreader proteins in a human-sars cov interaction network using sis model 21 . • sequence information of some of the ncov proteins have been released 22 . • gene ontological (go) information (biological process (bp), molecular function (mf), cellular component (cc)) of some of the ncov proteins are also available 22, 23 . • recently we have also developed a method to predict interaction affinity between proteins from the available go graph. 24 . • assessment of interaction affinity between ncov proteins with potential human target/bait proteins, which are susceptible to sars-cov infection, has been done. • fuzzy affinity thresholding is done to detect high quality ncov-human ppin. the selected human proteins are considered as level-1 human spreader nodes of ncov. • level 2 spreader node in ncov-human ppin are detected using spreadability index and validated by sis 21, 25 model. • validation of our developed model is done with respect to the target proteins of the potential fda drugs for covid-19 treatment. 26 our developed computational model of ncov-human ppin contains high quality interactions (hqi) and proteins identified by fuzzy affinity thresholding and spreadability index validated by sis model respectively. sources of input and the generated results always play a crucial role in any computational model which is also true for our proposed model. sars-cov-human ppin serves as a baseline for our model. the potential level-1 and level-2 human spreaders of sars-cov becomes the possible candidate set for selecting level-1 human spreaders of sars-cov2. various datasets have been curated for this purpose which has been outlined below: the dataset 27, 28 consists of all possible interactions between human proteins that are experimentally documented in humans. human proteins are represented as nodes while the physical interactions between proteins are represented by edges. it is a collection of 21557 nodes and includes 342353 edges/interactions. the dataset 18 consists of interactions between sars-cov proteins. it contains 7 unique proteins along with the involvement of 17 interacting edges out of which only the densely connected proteins are considered rather than the isolated ones since the former play a more active role in transmission of infection than the later. the dataset 18 comprises of 118 interactions between sars-cov and human. it is used to fetch the level-1 human interactions of sars-cov. this data is collected from pre-released dataset of available sars-cov2 protein from uniprotkb 22 (https://covid-19.uniprot.org/) which include 14 reviewed sars-cov2 proteins. three types (cc, mf and bp) of go graph are collected from go consortium 23, 29 (http://geneontology.org/). protein to go-annotation map is retrieved from uniprotkb database. seven potential fda drugs : lopinavir 30 , ritonavir 31 , hydroxychloroquine 32, 33 , azithromycin 33 , remdesivir 34-36 , favipi-ravir 37, 38 and darunavir 39 have been identified from the drugbank 40 published white paper 26 which have been used for validation in our proposed model. sars-cov-human ppin (up to level-2) is formed by the combination of sars-cov-human and human-human ppin datasets. sars-cov-human dataset generates the direct level-1 human interactions of sars-cov while human-human ppin dataset is used to fetch the corresponding level-2 human interactions. potential spreader nodes are identified using spreadability index which has been validated by sis model (see figure 1 ) 21 . the selected spreader nodes in sars-cov-human ppin are highlighted in table s1, table s2 and table s3 in the supplementary document. the network view of sars-cov-human ppin at each level and various selected thresholds are also available online (sars-cov level-1 human spreaders, level-1 & level-2:high human spreaders at high threshold of spreadability index, and level-1 & level-2:low human spreaders at low threshold of spreadability index). the go information can be useful to infer the binding affinity of any pair of interacting proteins using three different types of go hierarchical relationship graph (cc, mf and bp) 23 . the fuzzy ppi model has been proposed to find the interaction affinity between the sars-cov2 and human proteins using go based information (please see figure 2 and methods for details). to identify the interactors of sars-cov2 on human using the fuzzy ppi model, a set of candidate proteins are selected as the l1 and l2 spreader nodes of sars-cov using sis model (as depicted in figure 1 ). fuzzy ppi model is constructed from the ontological relationship graphs by evaluating the affinity between all possible go pairs annotated from any target protein pair and finally the fuzzy score of interaction affinity of protein pair is computed from these go pair-wise interaction affinity in to a range of [0,1] (details are discussed in the methods). the heatmap representation of fuzzy interaction affinities (with score ≥ 0.2 for very high specificity ~ 99%) is shown in supplementary figure s1 and table s4 . the high quality interaction (hqi) is retrieved at threshold 0.4, (almost ~ 99.98% specificity) which results total 78 interactions between sars-cov2 and human (37 proteins). the interaction networks predicted from fuzzy-ppi model are shown in figure 3 . human proteins present in the high quality interactions of ncov-human ppin fetched by applying fuzzy affinity threshold are considered as level-1 spreaders. from these level-1 spreaders, corresponding level-2 human interactions are obtained using human-human ppin dataset. spreadability index is thus computed for these level-2 human proteins for the identification of level-2 human spreader nodes. the selection is also verified by sis model. the selected spreader nodes in sarscov-human ppin are highlighted in table s4, table s5 and table s6 in the supplementary document. a sample computational model after proper assessment of all potential drugs as mentioned in the drugbank 40 white paper 26 40, 42 . these targets when searched in our proposed model of ncov-human ppin are found to play an active role of spreader nodes. this reveals the fact that the selected spreader nodes are of biological importance in transmitting infection in a network which actually make them the protein drug targets of the potential fda drugs for covid-19. the target protein hits in our ncov-human ppin for each of the 7 potential fda drugs are highlighted in figure 5 . it can be observed that 4 target protein hits are obtained for hydroxychloroquine, 3 target proteins for ritonavir, 2 target protein hits for each of lopinavir, darunavir and azithromycin and 1 target protein hit for remdesivir and favipiravir. out of these protein targets, ace2 is the most important one since it is considered to be the one of the crucial receptors of human for ncov to transmit infection deep inside the human cell [43] [44] [45] . in any host-pathogen interaction network, identification of spreader nodes is crucial for disease prognosis. not every protein in an interaction network has intense disease spreading capability. in this work, we have used the sars-cov-human ppin network and the spreader nodes at both level-1 and level-2 using the sis model. these spreader nodes are considered for computing the protein interaction affinity score to unmask the level-1 human spreaders of ncov. go annotations have been also taken into consideration along with ppin properties to make this model more effective and significant. with the gradual progress of the work, it has been observed that the selected human spreader nodes, identified by our proposed model, emerge as the potential protein targets of the fda approved drugs for covid-19. the basic hypotheses of the work may be listed as follows: • there is a genetic overlap of ~ 89% (as suggested by ictv) between sars-cov and sars-cov2, which also leads to a significant overlap in spreader proteins between human-sars-cov and human-sars-cov2 protein-interaction network. • fuzzy ppi approach can assess protein interaction affinities at very high specificity with respect to benchmark datasets as shown in figure 6 . high specificity signifies very low false positive rate at a given threshold. thus, at 0.4 threshold (~ 99.9% specificity), the proposed model evaluates high quality positive interactions in human-ncov ppin. finally we propose, that the developed computational model effectively identifies human-ncov ppis with high specificity. ncov-human interactions are inferred from another pandemic initiator sars-cov which is believed to be highly genetically similar to ncov. we also identify the human spreader proteins (up to level-2) using spreadability index, validated through sis model. due to high network density in human interaction network, number of proteins increase with the transition from one level to another. so, our proposed model is also capable of identifying human spreader proteins in level-2 by using spreadability index which is validated by sis model. target proteins of the potential fda drugs for covid-19 are found to overlap with the spreader nodes of the proposed computational ncov-human protein interaction model. target proteins of seven potential fda drugs: lopinavir 30 , ritonavir 31 , hydroxychloroquine 32, 33 , azithromycin 33 , remdesivir 34-36 , favipiravir 37, 38 and darunavir 39 for covid-19 as mentioned in the drugbank white paper 26 overlap with the spreader nodes of the proposed in silico ncov-human protein interaction model (see figure 5 ). though clinical trials for covid-19 vaccine in 2020 are on their way till date but three out of the seven i.e. remdesivir 46 hydroxychloroquine 47 and favipiravir 47 are found to be the most promising as well as effective ones and their protein targets r1ab_sars2, tlr9, ace2, cyp3a4 and abcb1 are also successfully identified as spreader nodes by our proposed model. this assessment reveals the fact that these spreader nodes are indeed have biological relevance relative to disease propagation. our developed computational model for ncov-human ppin consists of two important methodologies 1) identification of spreader nodes by spreadability index along with the validation of sis model and 2) fuzzy ppi model. in ncov-human ppin, the former acts as a pathogen while the host acts as bait. the transmission of infection starts when a pathogen enters a host body and starts infecting its protein which in turn affects its directly or indirectly connected neighborhood proteins. considering this method of transmission, ppin of human and sars-cov are considered to detect spreader nodes. spreader nodes are those nodes proteins which actually transmits the disease fast among its neighbors. but not all the nodes in a ppin are spreaders. so, proper detection of spreader nodes is crucial. thus, spreader nodes are identified by spreadability index which actually measure the transmission capability of a node protein. compactness of ppin and its transferal capability is evaluated using centrality analysis. nodes having high centrality value are usually considered as spreader nodes or the most critical node in a network. spreadability index 21 is one of the centrality based measure which is a combination of three major topological neighborhood based features of a network. they are: 1) node weight 48 2) edge ratio 49 and 3) neighborhood density 49 . nodes having high spreadability index are considered as spreader nodes. the spreader nodes thus identified are also validated by sis model 25 . sis model is implemented with a motive of designing the sars-cov and sars-cov2 outbreak into a disease model consisting of proteins based on their present infection status. a protein can be in either of one of the three states: 1) s: susceptible, which means that every protein is initially susceptible though it is not yet infected but is at risk of getting infected by disease. 2) i: infected, which means that the protein is already infected by the disease and 3) s: susceptible, which means proteins again become susceptible after getting recovered from infected state. this model is implemented in such a way that it generates the overall infection capability of a node after a certain range of iterations. thus the sum of the infection capability of the top selected spreader nodes are computed by this model which is compared against the sum obtained for the selected top critical nodes by other existing centrality measures like betweenness centrality (bc) 50 , closeness centrality (cc) 51 , degree centrality (dc) 52 and local average centrality (lac) 53 . our proposed method of selecting spreader nodes 21 has performed better in comparison to the other existing state-of-the-art. a synthetic ppin is considered in figure 7 to demonstrate the entire methodology of spreadability index. computational analysis of spreadability index of our proposed model with one of the other methodology bc has been highlighted from table 1 to supplementary table s7 . is the total number of edges which are outgoing from the ego network whereas is denoted as the total number of interconnections in the neighborhood of node 49 . of node 3 is 6 while of node 3 is 3 which highlights the fact that node 3 has the highest transmission ability from its ego network to outside when compared to other nodes. node 3 has also the highest spreadability index. but bc failed to rank node 3 in first position. same scenario can be observed for some other nodes in the synthetic network too. besides sis validation result shows that the selected top ranked spreader nodes in this proposed model have the highest infection capability in comparison to the other ranked nodes. the binding affinity between any two interacting proteins can be estimated by combining the semantic similarity scores of the go terms associated with the proteins 15, 24, 54-56 . greater number of semantically similar go annotations between any protein pair indicates higher interaction affinity. fuzzy ppi model is a hybrid approach 24 that utilizes both the topological 57 features of the go graph and information contents 56, 58, 59 of the go terms. go is organized in three independent directed acyclic graphs (dags): molecular function (mf), biological process(bp), and cellular component(cc) 23 . the nodes in each go graph represent go terms and the edges represent different hierarchical relationships. in this work, two most important relations 'is a' and 'part of' has been used for go relations 29 . the semantic similarity between any two proteins is estimated by considering the similarities between their all pairs of annotating gene ontology (go) terms belonging to a particular ontological graph. the similarity of a go term pair is determined by considering certain topological properties (shortest path length) of the go graph and the average information content (ic) 60 of the disjunctive common ancestors (dcas) 54, 55 of the go terms as proposed in 24 . fuzzy ppi first rely on a fuzzy clustering of the go graph where the selection of go terms as cluster center is based on the level of association of that go term in the go graph. the cluster centers are selected based on the proportion measure of go terms. the proportion measure for any go term t is computed as where ( ) , ( ) represents the ascendant and descendant of term and ( ) is the total number of go terms in ontology o. higher value of proportion measure signifies higher coverage of ascendants and descendants associated with the specific node. the go terms for which this proportion measure is above a predefined threshold are selected as cluster centers. in this work, the cluster centers are selected based on the threshold values as suggested in 15, 24 . after selecting the cluster centers, the degree of membership of a go term to each of the selected cluster centers is calculated using its respective shortest path lengths to the corresponding cluster centers. the membership of the go term to a cluster decreases with increase in its shortest path length to the cluster center. the membership function is defined as ( ) where is center and is the width of membership function and is the shortest path length from to . the difference in membership values between the go pair and with respect to each cluster center, are computed to find the weight parameter. the weight parameter is defined as ( ) ( ). this weight value determines how dissimilar two go terms can be with respect to the cluster centers. next, the shared information content (sic) is computed using average ic 60 , of the dcas of go term pair ( ) is determined from three go graphs. the sic is defined as ) represents the disjunctive common ancestors of go-term and .the semantic similarity of protein pair ( ) for each go-type (cc, mf and bp), is estimated by utilizing the maximum similarity of all possible go pairs from the annotations of proteins and for each type of go. the binding affinity of protein pair ( ) is defined as the average of cc, mf and bp based semantic similarity. the fuzzy score of binding affinity is computed by normalizing the binding affinity using max-min normalization. in this work, the fuzzy binding affinity score is computed between the protein pairs of sars-cov2 and human proteins using the available ontological information. finally, with high specificity threshold (please see figure 6 ), high quality interactions are extracted for human-sars-cov2. a novel coronavirus outbreak of global health concern world-health-organization coronavirus disease (covid-19) outbreak statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus statement on the meeting of 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on new coronavirus, now deadly | cidrap genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan the sars-coronavirus-host interactome: identification of cyclophilins as target for pancoronavirus inhibitors analysis of intraviral protein-protein interactions of the sars coronavirus orfeome human coronavirus: host-pathogen interaction detection of spreader nodes and ranking of interacting edges in human-sars-cov protein interaction network uniprot: the universal protein knowledgebase the gene ontology (go) database and informatics resource assessment of semantic similarity between proteins using information content and topological properties of the gene ontology graph the mathematical theory of infectious diseases and its applications (charles griffin & company ltd, 5a crendon street, high wycombe, bucks hp13 6le covid-19 : finding the right fit identifying potential treatments using a data-driven approach large-scale analysis of disease pathways in the human interactome biosnap: network datasets: human protein-protein interaction network gene ontology: tool for the unification of biology coronavirus puts drug repurposing on the fast track a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection emergency access to remdesivir outside of clinical trials china approves antiviral favilavir to treat coronavirus -upi.com taiwan synthesizes anti-viral drug favilavir for covid-19 patients -focus taiwan efficacy and safety of darunavir and cobicistat for treatment of covid-19 -full text view -clinicaltrials drugbank: a knowledgebase for drugs, drug actions and drug targets advanced search -drugbank interaction between raas inhibitors and ace2 in the context of covid-19 ace-2 is shown to be the entry receptor for sars-cov-2: r&d systems covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: what is the evidence? trial shows covid-19 patients recover with gilead's remdesivir clinical trial of favipiravir tablets combine with chloroquine phosphate in the treatment of novel coronavirus pneumonia -full text view -clinicaltrials detecting overlapping protein complexes in ppi networks based on robustness identifying influential spreaders based on edge ratio and neighborhood diversity measures in complex networks the rush in a directed graph, stichting mathematisch centrum the centrality index of a graph lethality and centrality in protein networks a local average connectivity-based method for identifying essential proteins from the network level semantic similarity over the gene ontology: family correlation and selecting disjunctive ancestors measuring semantic similarity between gene ontology terms using information content to evaluate semantic similarity in a taxonomy an improved method for scoring protein-protein interactions using semantic similarity within the gene ontology an information-theoretic definition of similarity semantic similarity based on corpus statistics and lexical taxonomy a mathematical theory of communication. acm sigmobile mob this work is partially supported by the cmater research laboratory of the computer science and engineering department, jadavpur university, india, purse-ii and upe-ii grants. competing interests: the authors declare no competing interests.all supplementary information are freely available for academic and research purpose only.all queries should be send to the corresponding author's email: subhadip.basu@jadavpuruniversity.in key: cord-007726-bqlf72fe authors: rydell-törmänen, kristina; johnson, jill r. title: the applicability of mouse models to the study of human disease date: 2018-11-09 journal: mouse cell culture doi: 10.1007/978-1-4939-9086-3_1 sha: doc_id: 7726 cord_uid: bqlf72fe the laboratory mouse mus musculus has long been used as a model organism to test hypotheses and treatments related to understanding the mechanisms of disease in humans; however, for these experiments to be relevant, it is important to know the complex ways in which mice are similar to humans and, crucially, the ways in which they differ. in this chapter, an in-depth analysis of these similarities and differences is provided to allow researchers to use mouse models of human disease and primary cells derived from these animal models under the most appropriate and meaningful conditions. although there are considerable differences between mice and humans, particularly regarding genetics, physiology, and immunology, a more thorough understanding of these differences and their effects on the function of the whole organism will provide deeper insights into relevant disease mechanisms and potential drug targets for further clinical investigation. using specific examples of mouse models of human lung disease, i.e., asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, this chapter explores the most salient features of mouse models of human disease and provides a full assessment of the advantages and limitations of these models, focusing on the relevance of disease induction and their ability to replicate critical features of human disease pathophysiology and response to treatment. the chapter concludes with a discussion on the future of using mice in medical research with regard to ethical and technological considerations. although the genetic lineages of mice and humans diverged around 75 million years ago, these two species have evolved to live together, particularly since the development of agriculture. for millennia, mice (mus musculus) were considered to be pests due to their propensity to ravenously consume stored foodstuff (mush in ancient sanskrit means "to steal" [1] ) and their ability to adapt to a wide range of environmental conditions. since the 1700s, domesticated mice have been bred and kept as companion animals, and in victorian england, "fancy" mice were prized for their variations in coat color and comportment; these mouse strains were the forerunners to the strains used in the laboratory today. robert hooke performed the first recorded inquiry-driven experiments on mice in 1664, when he investigated the effects of changes in air pressure on respiratory function [2] . more recently, with data from the human genome project and sequencing of the mus musculus genome showing remarkable genetic homology between these species, as well as the advent of biotechnology and the development of myriad knockout and transgenic mouse strains, it is clear why the mouse has become the most ubiquitous model organism used to study human disease. in addition, their small size, rapid breeding, and ease of handling are all important advantages to scientists for practical and financial reasons. however, keeping in mind that mice are fellow vertebrates and mammals, there are ethical issues inherent to using these animals in medical research. this chapter will provide an overview of the important similarities and differences between mus musculus and homo sapiens and their relevance to the use of the mouse as a model organism and provide specific examples of the quality of mouse models used to investigate the mechanisms, pathology, and treatment of human lung diseases. we will then conclude with an assessment of the future of mice in medical research considering ethical and technological advances. as a model organism used to test hypotheses and treatments related to human disease, it is important to understand the complex ways in which mice are similar to humans, and crucially, the ways in which they differ. a clear understanding of these aspects will allow researchers to use mouse models of human disease and primary cells derived from mice under the most appropriate and meaningful conditions. in 2014, the encyclopedia of dna elements (encode) program published a comparative analysis of the genomes of homo sapiens and mus musculus [3] , as well as an in-depth analysis of the differences in the regulatory landscape of the genomes of these species [4] . encode, a follow-up to the human genome project, was implemented by the national human genome research institute (nhgri) at the national institutes of health in order to develop a comprehensive catalog of protein-encoding and nonproteincoding genes and the regulatory elements that control gene expression in a number of species. this was achieved using a number of genomic approaches (e.g., rna-seq, dnase-seq, and chip-seq) to assess gene expression in over 100 mouse cell types and tissues; the data were then compared with the human genome. overall, these studies showed that although gene expression is fairly similar between mice and humans, considerable differences were observed in the regulatory networks controlling the activity of the immune system, metabolic functions, and responses to stress, all of which have important implications when using mice to model human disease. in essence, mice and humans demonstrate genetic similarity with regulatory divergence. specifically, there is a high degree of similarity in transcription factor networks but a great deal of divergence in the cis-regulatory elements that control gene transcription in the mouse and human genomes. moreover, the chromatin landscape in cell types of similar lineages in mouse and human is both developmentally stable and evolutionarily conserved [3] . of particular relevance regarding modeling human diseases involving the immune system, in its assessment of transcription factor networks, the mouse encode consortium revealed potentially important differences in the activity of ets1 in the mouse and human genome. although conserved between the two species, divergence in ets1 regulation may be responsible for discrepancies in the function of the immune system in mouse and human [4] . certainly, the biological consequences of these differences in gene expression and regulation between human and mouse invite further investigation. the anatomical and physiological differences between model organisms and humans can have profound impacts on interpreting experimental results. virtually every biological process under investigation in experimental studies involves at least one anatomical structure. to aid in interpretation, many anatomy compendia have been developed for model organisms; the most useful organize anatomical entities into hierarchies representing the structure of the human body, e.g., the foundational model of anatomy developed by the structural informatics group at the university of washington [5] . although an analysis of the myriad differences between mouse and human anatomy is beyond the scope of this chapter, a few of the most critical issues that have an impact on the interpretation of data from mouse experiments should be mentioned. the most obvious difference between mice and humans is size; the human body is about 2500 times larger than that of the mouse. size influences many aspects of biology, particularly the metabolic rate, which is correlated to body size in placental mammals through the relationship bmr ¼ 70 â mass (0.75), where bmr is the basal metabolic rate (in kcal/day). thus, the mouse bmr is roughly seven times faster than that of an average-sized human [6] . this higher bmr has effects on thermoregulation, nutrient demand, and nutrient supply. as such, mice have greater amounts of metabolically active tissues (e.g., liver and kidney) and more extensive deposits of brown fat [6] . furthermore, mice more readily produce reactive oxygen species than do humans, which is an important consideration when modeling human diseases involving the induction of oxidative stress (i.e., aging, inflammation, and neurodegeneration) [6] . the lung provides an excellent example of the similarities and differences between human and mouse anatomy. similar to the human organ, the mouse lung is subdivided into lobes of lung parenchyma containing a branching bronchial tree and is vascularized by the pulmonary circulation originating from the right ventricle. there are a number of subtle variations in this general structure between species, i.e., the number of lobes on the right and left, the branching pattern, and the distribution of cartilage rings around the large airways, but the most important differences between the mouse and human lung are related to the organism's size (airway diameter and alveolar size are naturally much smaller in the mouse) and respiratory rate. moreover, there are important differences in the blood supply of the large airways in humans versus mice [7] . specifically, the bronchial circulation (a branch of the high-pressure systemic circulation that arises from the aorta and intercostal arteries) supplies a miniscule proportion of the pulmonary tissue in mice (the trachea and bronchi) compared to humans; the majority of the lung parenchyma is supplied by the low-pressure, high-flow pulmonary circulation. in the mouse, these systemic blood vessels do not penetrate into the intraparenchymal airways, as they do in larger species [8] . this difference, although subtle, has important ramifications regarding the vascular supply of lung tumors which, in humans, is primarily derived from the systemic circulation [9] . these differences may also have profound consequences when modeling human diseases involving the lung vasculature. the adaptive immune system evolved in jawed fish about 500 million years ago, well before the evolution of mammals and the divergence of mouse and human ancestral species [10] . many features of the adaptive immune system, including antigen recognition, clonal selection, antibody production, and immunological tolerance, have been maintained since they first arose in early vertebrates. however, the finer details of the mouse and human immune systems differ considerably, which is not surprising since these species diverged 75 million years ago [6] . while some have claimed that these differences mean that research into immunological phenomena in mice is not transferable to humans, as long as these differences are understood and acknowledged, the study of mouse immune responses can continue to be relevant. research on mice has been vital to the discovery of key features of both innate and adaptive immune responses; for example, the first descriptions of the major histocompatibility complex, the t cell receptor, and antibody synthesis were derived from experiments performed on mice [6] . the general structure of the immune system is similar in mice and humans, with similar mediators and cell types involved in rapid, innate immune responses (complement, macrophages, neutrophils, and natural killer cells) as well as adaptive immune responses informed by antigen-presenting dendritic cells and executed by b and t cells. however, due to the anatomical and physiological differences between these species as described above, divergence in key features of the immune system, such as the maintenance of memory t cells (related to the life span of the organism) and the commensal microbiota (related to the lifestyle of the organism), has arisen [11] . similar to what has been discovered regarding the genetics of mice and humans, i.e., broad similarities in structure but considerable differences in regulation, there are a number of known discrepancies in the regulation of innate and adaptive immunity in mouse models of human disease mice versus humans, including the balance of leukocyte subsets, t cell activation and costimulation, antibody subtypes and cellular responses to antibody, th1/th2 differentiation, and responses to pathogens (described in detail in table 1 ). in addition to these differences in immune cell functions, the expression of specific genes involved in immune responses also differs, particularly those for toll-like receptors, defensins, nk inhibitory receptors, thy-1, and many components of chemokine and cytokine signaling; additionally, differences between mouse strains are known to exist for many of these mediators [12] . another important consideration when using mice to perform immunological research (with a view to translating these findings to human medicine) is the availability of hundreds of strains of genetically modified mice that have enabled exquisitely detailed studies on immune cell function, regulation, and trafficking. many of these strains involve the expression of inducible cre or cas9 that allow for targeted knockdown or overexpression of key immune function-related genes in specific cell types at specific moments in time. however, it is important to note that drift between mouse colonies has long been known to occur. in fact, a recent report described the fortuitous discovery of a point mutation in the natural cytotoxicity receptor 1 (ncr1) gene in the c57/bl6 cd45.1 mouse strain, resulting in absent ncr1 expression. this mutation was found to have profound effects on the response of mice to viral infection, i.e., the mice were resistant to cytomegalovirus infection but more susceptible to influenza virus [23] . this cautionary tale highlights the importance of understanding the genetic evolution of laboratory strains of mice, the effect of these genetic and immunological changes on mouse biology, and the impact on the translation of these results to human medicine. in addition to the differences between mouse and human genetics, physiology, and immunology highlighted above, several factors must also be taken into account when performing in vitro assays using isolated mouse cells and applying these findings to our understanding of human disease. particularly with regard to stem cell research, it should be noted that the telomeres of mouse cells are five-to tenfold longer than human telomeres, resulting in greater replicative capacity [24] . there are also important differences in the regulation of pluripotency and stem cell differentiation pathways in humans and mice [25] . moreover, there are considerable species differences in the longevity of cultured cells; for example, mouse fibroblasts are capable of spontaneous immortalization in vitro, whereas human fibroblasts become senescent and ultimately fail to thrive in culture [26] . in summary, although there are considerable differences between mice and humans, constant improvement in the analytical techniques used to delineate these differences and their effects on whole organism and cell function have provided vital information and contributed to our understanding of both murine and human biology. experimentation employing mouse models of human disease will continue to provide key insights into relevant disease mechanisms and potential drug targets for further clinical investigation. however, several important considerations must be taken into account when selecting a mouse model of human disease, as described in the following section, using mouse models of human lung disease to illustrate this point. the two most salient features of a mouse model of human disease are the accuracy of its etiology (it employs a physiologically relevant method of disease induction) and its presentation (its ability to recapitulate the features of human disease). the relevance of any given mouse model can be judged on the basis of these two criteria, and there is considerable variation within mouse models of human disease in this regard. as a full assessment of the advantages and limitations of all currently available mouse models of human disease would be prohibitively long and complex, here we have elected to assess the accuracy of currently available models of human lung diseases, i.e., asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, focusing on the relevance of disease induction in these models and their ability to replicate critical features of human disease pathophysiology and response to treatment. the first and foremost notion when modeling human disease in mice is to acknowledge the species differences, which are significant [27] . as described above, genetics, anatomy, physiology, and immunology differ between mice and humans, but despite these differences, mouse models of human disease are useful and necessary, as long as data interpretation is performed appropriately. an elegant example of differences between mice and humans that must be considered when designing a mouse model of human inflammatory lung disease is the key effector cell type in human asthma, i.e., mast cells. these leukocytes differ in granule composition as well as localization in the mouse and human airways [28] . mice mostly lack mast cells in the peripheral lung [29] , whereas humans have numerous mast cells of multiple subpopulations in the alveolar parenchyma [30] . another example is anatomy: in contrast to humans, mice lack an extensive pulmonary circulation, which may have significant effects on leukocyte adhesion and migration, and subsequently inflammation [31] . still, as long as these differences are taken into consideration, mouse models can be powerful tools in the discovery and exploitation of new targets for the treatment of human disease. the world health organization (who) defines asthma as a chronic disease characterized by recurrent attacks of breathlessness and wheezing, which may vary in severity and frequency from person to person. the disease is characterized by airway hyperresponsiveness, airway smooth muscle thickening, increased mucus secretion and collagen deposition, as well as prominent inflammation affecting both large and small airways [32] . nowadays, it is recognized that asthma is not a single homogenous disease but rather several different phenotypes united by similar clinical symptoms [32, 33] . only a few animal species develop asthma naturally, including cats and horses [34, 35] , whereas mice do not [31] . however, mice can be manipulated to develop a type of allergic airway inflammation, which is similar in many ways to the human disease, in response to different aeroallergens [36] . importantly, these models are capable of recapitulating only the allergic type of human asthma and have less relevance for other types of asthma (i.e., endotypes induced by medication, obesity, and air pollution). as with many human diseases, asthma has a complex and multifaceted etiology, where environmental factors, genetic susceptibility, and microbial colonization all contribute; thus, it is important to take strain differences into consideration. generations of inbreeding have created mouse strains that differ not only in coat color and disposition but also from a physiological, immunological, and genetic perspective. different strains may be more susceptible to allergic airway inflammation or pulmonary fibrosis, whereas others are more or less resistant. choosing the right strain to model a specific disease or pathologic event is thus essential. the most widely used strains for models of allergic airway inflammation are balb/c and c57bl/6. these strains differ regarding the type of immune response mounted to an inhaled allergen: c57bl/6 is generally considered a t h 1-skewed strain, whereas balb/c is regarded as a t h 2-skewed strain [36] . due to their strong t h 2response, and subsequent development of robust asthmatic responses, balb/c has been commonly used to model asthma [37] . however, most humans do not express such a strongly t h 2-skewed immune system, suggesting this strain may not be the best model of human disease; instead, c57bl/6 may be more suitable as immune responses in this strain are more similar to those of atopic human subjects [37] . furthermore, as c57bl/6 is the most commonly used strain for the development of genetically manipulated mice, using these mice allows for very specific investigations into disease pathology; thus, this strain is increasingly used in models of human lung disease. besides the genetic differences in the mouse strains used in these models, the etiology (the method of disease induction) of commonly used models of asthma is highly variable. in humans with allergic asthma, environmental allergen exposure occurs at the airway mucosa; the immune response is coordinated in the bronchopulmonary lymph nodes, and the t cells, macrophages, and eosinophils recruited as part of this response travel to the lung where they mediate the cardinal features of asthma: airway inflammation, structural remodeling of the airway wall, and airway hyperreactivity [38] . ideally, these features should be found in a physiologically relevant mouse model of asthma. however, for the sake of cost and convenience, early mouse models of asthma used the surrogate protein ovalbumin (ova) [31] rather than an environmental allergen to induce an immune response, which also requires the use of a powerful t h 2-polarizing adjuvant such as alum delivered via the intraperitoneal route, followed by ova nebulization-a clear divergence from the etiology of human asthma [36] . in terms of disease presentation, mice develop some hallmarks of asthma, including airway eosinophilic inflammation, goblet cell metaplasia, and increased airway smooth muscle density [31] . after the cessation of ova exposure, most of the remodeling resolves, although some structural alterations remain up to 1 month after the last challenge [39] . based on these attributes, the ova model is primarily a model to investigate the initiation of inflammation, rather than the chronic progression and maintenance of inflammation [31] . a clear advantage with the ova model is the number of studies where it is used; both the pros and cons are familiar. it is easy to find a suitable protocol, and the model is readily accessible and flexible regarding the number of sensitizations and allergen doses. the model is relatively easy to reproduce, as ova and different adjuvants are easily obtained. however, the resolution of remodeling following the cessation of allergen provocations is a disadvantage, as is the practical problem with the nebulization of an allergen-it ends up in the mouse's coat and is ingested during grooming, potentially resulting in systemic exposure (this is particularly relevant in models employing systemic, intraperitoneal sensitization). in addition, concerns have been raised against the use of adjuvants to induce the immunological response, as well as the clinical relevance of ova as an allergen, which have driven the development of more clinically relevant allergens and models [31] . the common environmental aeroallergen house dust mite (hdm) extract is increasingly used to initiate disease in mouse models of allergic airway inflammation, as it is a common human allergen (around 50% of asthmatics are sensitized to hdm [40] ) that evokes asthma attacks and other allergic responses in susceptible individuals. in addition, hdm has inherent allergenic properties, likely due to components with protease activity [40] , so there is no need to use an adjuvant, thus improving the etiological similarity of these models with the clinical situation [41] . in contrast to ova, prolonged exposure of hdm (up to 7 weeks) induces asthma-like severe airway inflammation with prominent eosinophilia, severe hyperreactivity to methacholine, and robust remodeling of the airway wall [41] , i.e., the presentation of chronic respiratory hdm exposure in mice effectively recapitulates the key features of human allergic asthma. importantly, the airway structural changes induced by chronic hdm exposure, such as increased collagen deposition, airway smooth muscle thickening, and microvascular alterations, persist for at least 4 weeks after the cessation of hdm exposure [42] , another commonality with human asthma in which airway remodeling is currently considered to be irreversible. thus, the advantages of using hdm as the allergen in mouse models of asthma are the clinical relevance of the allergen [43] and the route of delivery via the respiratory tract. moreover, studies have shown that the type of inflammation and characteristics of tissue remodeling are relatively similar to those seen in human asthmatics [35, 41, 43] . one disadvantage is the complexity of hdm extract; as a consequence of this complexity, variations exist in some components between batches, particularly regarding the content of lipopolysaccharide, so reproducibility in these studies may be problematic. with similarity to hdm, these models were developed to be as clinically relevant as possible, as many patients suffer from allergy toward cockroach allergen, molds, and other environmental irritants. a common feature of these allergens is their complex nature, as they commonly consist of a mix of different allergic epitopes and fragments. this complexity is most likely why the immunological reaction in mice is relatively similar to that seen in asthmatics [44] . cockroach allergen (cra) is a common allergen, known to induce asthma in susceptible individuals; thus, it shares with hdm the advantage of being highly clinically relevant [45] . cra induces peribronchial inflammation with significant eosinophilic inflammation and transient airway hyperresponsiveness, both of which can be increased by repeated administrations of the allergen [45] . colonization of the airways with aspergillus fumigatus is the cause of allergic bronchopulmonary aspergillosis (abpa), a disease where the lungs are colonized by the fungus, but allergens from aspergillus fumigatus can also induce asthma similar to other allergens [46] . the reaction to aspergillus allergens is robust, and often no adjuvants are needed to elicit inflammation [46] . in addition to aspergillus, other fungi such as penicillium and alternaria can also induce asthma in humans and have been used to model disease in mice [47] . a common difficulty with these allergens is the method of administration, as the physiological route is believed to be the inhalation of dry allergens; mimicking this route with a nebulizer introduces the risk of the animals ingesting the allergen and thus causing systemic responses [47] . exacerbations of asthma are defined as the worsening of symptoms, prompting an adjustment in treatment, and are believed to be associated with increased inflammation in the distal airways. clinically, exacerbations are believed to be induced by infections (most common), allergen exposure, or pollutants, which can be modeled in different ways [48, 49] : 1. infections with viruses and bacteria or exposure to proteins/ dna/rna derived from these microbes. 2. administration of a high dose of allergen in a previously sensitized animal. 3. exposure to environmental pollutants, such as diesel exhaust or ozone. modeling exacerbations adds a layer of complexity, as robust ongoing allergic airway inflammation needs to be established first, before challenge with the exacerbating agent. both the ova and hdm models are used in this respect, and in both cases chronic protocols extending for several weeks before triggering an exacerbation have been used [48] . chronic obstructive pulmonary disease (copd) is characterized by chronic airway obstruction, in contrast to asthma where the obstruction is reversible (particularly in response to bronchodilator treatment). clinically, in copd, chronic bronchitis and emphysema can occur either separately or in combination. copd is almost always associated with either first-or secondhand tobacco smoking or in rare cases with a deficiency in the production of α1antitrypsin (a serpin that prevents elastin breakdown as a result of neutrophil degranulation) [50] . the etiology of copd is highly complex and is believed to develop after many years of smoking in combination with other known factors such as genetic susceptibility or environmental factors [51] . in similarity to asthma, inflammation is a major component in copd, but the leukocyte profile is very different: the most prominent players in copd-related inflammation are neutrophils and, to some degree, macrophages [51] . due to the complex etiology of copd, it is difficult to recapitulate all aspects of this disease in a single model, so in most cases, the aim is to induce copd-like lesions by exposing mice to tissue-damaging substances (usually cigarette smoke) or to mimic emphysema by the administration of tissue-degrading enzymes [27, 51] . clearly, mice do not smoke cigarettes on their own, so to model copd by cigarette smoke (cs) inhalation, the mice need to be exposed to unfiltered cs in an induction chamber; moreover, in an attempt to better model the chronic aspects of copd, this needs to be performed for a prolonged period of time. mice are very tolerant to cs, but eventually (over a period of several weeks), cs induces pulmonary neutrophilic inflammation that is associated with some degree of tissue degradation and destruction [51] . an important advantage of this model is the fact that cs is the actual irritant responsible for disease in humans, and mice develop several features similar to the clinical disease, making this model highly clinically relevant [27] . a significant drawback is the self-limitation of the model-the pathological changes do not progress after the cessation of cs exposure [51] . furthermore, the exposure time needed for mice to develop copd-like pathology is extensive, i.e., studies have shown that an exposure protocol of 5 days per week for a minimum of 3 months is needed to generate robust structural changes to the lung [52]. the pathological image in copd is complex and varies greatly between patients, commonly encompassing chronic bronchitis and bronchiolitis, emphysema, fibrosis, and airway obstruction. although mice develop some of these symptoms when exposed to cs, they do not develop all the symptoms of human disease; thus, cs has advantages as a model but fails to mimic the complexity of the clinical situation and disease presentation [27] . other models of copd rely on the administration of proteases (protein-degrading enzymes) that are believed to be involved in the pathology of this disease in a subset of patients, such as elastindegrading elastase. this approach mimics the emphysematous changes seen in copd, but the pathological process underlying tissue destruction is likely very different compared to the clinical situation [51] , as very few patients show evidence of elastase dysregulation [27] . however, if the aim of the study is to investigate the general effect of protease-induced tissue destruction and regeneration, then this is a highly relevant method [51] . some studies on copd have also used genetically modified animals, such as mice overexpressing collagenase, which results in tissue destruction without inflammation or fibrosis with an end result fairly similar to the type of emphysema observed in copd [53] . pulmonary fibrosis, the accumulation of fibrotic tissue within the alveolar parenchyma, is merely a symptom of disease, and the etiology of this pathology in humans varies greatly [54] . the most enigmatic class is perhaps the idiopathic interstitial pneumonias, especially idiopathic pulmonary fibrosis (ipf). ipf is a debilitating and progressive disease with a grave prognosis, characterized by progressive fibrosis believed to reflect aberrant tissue regeneration [55] . as the reason behind this defective repair is unknown, although a combination of immunological, genetic, and environmental factors are suspected, it is very difficult to model disease in a clinically relevant fashion [56] . the most common method used to model pulmonary fibrosis in mice is administration of the chemotherapeutic agent bleomycin; this agent is known to cause pulmonary fibrosis in humans as well, but this may not accurately reflect the true etiology of most cases of human disease. the strain of choice is c57bl/6, as it is prone to developing pulmonary fibrosis, whereas balb/c is relatively resistant, a feature believed to reflect the cytokine response following cellular stress and damage [57] . bleomycin administration can be performed locally or systemically, producing very different results. the most common model of pulmonary fibrosis is a single intranasal or intratracheal administration of bleomycin, with analysis 3 to 4 weeks later. during this time, the drug causes acute tissue damage in a restricted area of the lung (where the solution ends up during administration), followed by intense inflammation in this area and subsequent fibrosis, which gradually resolves within weeks. however, if older mice are used, the fibrosis will persist longer than in younger mice, which is in accordance with clinical ipf, where the majority of the patients are 65 years of age or older [56, 58] . a great advantage of this model is how well-characterized it is. in addition, local administration is labor-effective, as only one administration is required and the result is highly reproducible. the fibrosis is robust, only affects the lungs, and the accumulation of extracellular matrix can be easily measured using standard techniques [58] . furthermore, as it is used throughout the world, studies performed in different labs and by different groups can be compared relatively easily. unfortunately, the intense pulmonary inflammation may be lethal, and fatalities are to be expected with this model [59] , representing an important ethical limitation. furthermore, fibrosis is heterogeneous-it develops where the bleomycin solution is deposited. the solution usually deposits within the central lung, a localization that is not in agreement with the clinical situation where fibrosis is located in the more distal regions of the lung parenchyma. in addition, the fibrosis that develops as a result of severe tissue damage is self-limiting and reversible, unlike what is observed clinically [58] . the severe degree of tissue damage induced by bleomycin may in fact be more relevant for modeling acute lung injury (ali) or acute respiratory distress syndrome (ards). bleomycin can also be administered systemically, through intravenous or subcutaneous injection. in contrast to local administration, this route requires multiple administrations and is thus more laborintensive [58] . some studies have described the usage of osmotic mini-pumps, where bleomycin is slowly administered over a short period of time, and then fibrosis continues to develop over subsequent weeks [60] . irrespective of the route of delivery, systemic administration results in more homogenous fibrosis, affecting the entire lung through the pulmonary endothelium and persisting much longer than following local administration [61] . the main advantages of systemic administration are that inflammation is limited, while the fibrosis is more apparent and displays a more distal pattern, all of which mimics the clinical situation relatively well. the multiple administrations allow for lower doses with each injection; this is less stressful to the animals and results in little to no mortality [61] and is thus more ethically acceptable. a major disadvantage with this model is that it takes time for fibrosis to develop [58] , which may be the reason it is used relatively scarcely, and thus the pathological development is less well-understood. in addition, as ipf is a local disease, local administration of the etiologic agent may better mimic the clinical reality [56] . the administration of fluorescein isothiocyanate (fitc) induces focal inflammation, primarily involving mononuclear cells and neutrophils, and localizes in areas where the fitc solution is deposited [58] . antibodies against fitc can be detected after 1 week, and the fibrosis persists for up to 5 months after instillation [58] . the benefits of this model are mainly related to the persistent fibrosis that does not appear to be self-limiting, thus reflecting the clinical situation, and it is also very easy to determine which part of the lung has been exposed to fitc, as the molecule is fluorescent [58] . it is also an advantage that both c57bl/6 and balb/c mice are susceptible and develop fibrosis following fitc administration [56] . the disadvantages of this model include profound variability due to differences between batches of fitc, as well as in the method used to prepare the solution before instillation. importantly, given the characteristics of the etiologic agent used to induce this model of ipf, this model is considered a very artificial system with limited clinical relevance [56] . adenovirus vectors have been used to overexpress the pro-fibrotic cytokine transforming growth factor (tgf)-β, which results in pulmonary fibrosis. as tgf-β overexpression in the lungs is known to be crucial in the development of fibrosis in humans [62] , this model mimics an important feature of disease etiology. however, the delivery system has some drawbacks, as the virus itself initiates an immune response. moreover, adenoviruses display significant tropism for epithelial cells and rarely infect other cell types such as fibroblasts [58] , which are the cells meant to be targeted in this model. as tgf-β has major effects on fibroblast biology, the main feature of this model is the effect of epitheliumderived tgf-β on fibroblasts and myofibroblasts, resulting in the deposition of ecm proteins and areas of dense fibrosis [63] . an advantage of this model is the relatively low degree of inflammation, as well as what appears to be a direct effect on fibroblasts/ myofibroblasts [63] , which is in accordance with the clinical situation (as we understand it today). silica administration induces a similar pathology in mouse lungs as in humans exposed to silica, and as is also observed in human silicainduced fibrosis, structural remodeling persists when administration is halted [56] . following the administration of silica particles, fibrotic nodules develop in mouse lungs, with considerable resemblance to the human lesions that develop after exposure to mineral fibers [56] . the fibrotic response is accompanied by a limited inflammatory response, and different pro-fibrotic cytokines such as tgf-β, platelet-derived growth factor, and il-10 are involved in disease development, which is in accordance with the clinical situation [56] . another advantage is that nodules develop around silica fibers, and these fibers are easy to identify by light microscopy. the response in this model is strain-dependent, with c57bl/6 mice being the most susceptible. the main drawbacks are the time required to establish disease, i.e., 30-60 days, and the need for special equipment to aerosolize the silica particles. however, since the route of administration, the driving etiologic agent, and the resulting pathobiology are all similar to the characteristics of this subtype of pulmonary fibrosis [56, 58] , the silica exposure model can be considered to have very good clinical relevance. 7 what does the future hold for mouse models of human disease? medical research using experimental animals (not only mice but other animals including rats, guinea pigs, zebrafish, and fruit flies) has greatly contributed to many important scientific and medical advances in the past century and will continue to do so into the near future. these advances have contributed to the development of new medicines and treatments for human disease and have therefore played a vital role in increasing the human life span and improving quality of life. despite the acknowledged benefits of performing research using experimental animals, a number of considerations must be made before embarking on this type of research. of course, the financial aspects of conducting this type of work are an important limitation, as the costs of purchasing and housing mice can be prohibitive, especially when genetically modified mice and colony maintenance are required for the study. the practicalities of working with animals such as mice may also be an issue, as this type of work requires specialized facilities, equipment, and staff to ensure studies are carried out in a manner that is safe for both the researchers and the animals. moreover, as discussed in detail in this chapter, the relevance of the selected animal model to human disease must be carefully evaluated to ensure that these experiments provide robust results that are translatable to human health and disease. another important and demanding aspect of biomedical research using animals is the ethics of imposing pain and suffering on live animals. although there has been a considerable reduction in the numbers of animals used in research in the last 30 years, animal research remains a vital part of biomedical research. however, no responsible scientist wants to cause unnecessary suffering in experimental animals if it can be avoided, so scientists have accepted controls on the use of animals for medical research. in the uk, this ethical framework has been enshrined in law, i.e., the animals (scientific procedures) act 1986. this legislation requires that applications for a project license to perform research involving the use of "protected" animals (including all vertebrates and cephalopods) must be fully assessed with regard to any harm imposed on the animals. this involves a detailed examination of the proposed procedures and experiments, and the numbers and types of animal used, with robust statistical calculations to support these numbers. the planned studies are then considered in light of the potential benefits of the project. both within and outside the uk, approval for a study involving protected animals also requires an internal ethical review process, usually conducted by the research institution where the work is taking place, with the aim of promoting animal welfare by ensuring the work will be carried out in an ethical manner and that the use of animals is justified. additionally, the uk has a national animal use reduction strategy supported by the national centre for the replacement, refinement and reduction of animals in research (nc3rs; london, uk). this consortium was established in 2004 to promote and develop high-quality research that takes the principles of replacement, refinement, and reduction (the 3rs) into account. replacement strategies often involve the use of alternative, non-protected species (e.g., zebrafish, fruit flies, flatworms) and in vitro correlates (two-dimensional cell culture or threedimensional organoids containing multiple cell types) to test hypotheses and assess the effects of therapeutic interventions. the main obstacle with studies on non-protected animals is the difficulty of accurately mimicking the complex physiological systems involved in human health and disease, as described in detail above. for example, the fruit fly drosophila melanogaster is an excellent model organism for studies on genetic diseases, aging, and pathogen-borne illnesses but may be less relevant for studies on complex lung diseases. importantly, model organisms such as fruit flies, zebrafish, and flatworms do not possess lungs, which somewhat limits the translatability of research on these animals in the field of respiratory disease. as such, it is likely that rodents will remain the model organism of choice for studies into lung disease for some time to come. there has been considerable progress recently in imitating single organs such as the liver, lung, and brain in vitro using multiple cell types and a physical scaffold. as an important advantage, these in vitro tests have replaced a large number of rodents in initial drug discovery experiments, while also speeding up the process [64] . these studies still require further refinement and validation to establish them as suitable models for an entire organ; importantly, these in vitro organoids cannot take into account interactions between organ systems in complex, multisystem diseases such as copd. refinement involves selecting the most clinically relevant model for the disease available, informed by the discussion above on closely recapitulating the etiologic agent and disease pathobiology associated with clinical cases. another important factor is refining the management of pain. an assessment of the procedures used and the effects of the substance on the animal, as well as the degree of handling, restraint, and analgesia, are other important aspects of refinement. this standard of animal care is achieved through strict regulations and controls on how personnel are trained to carry out experiments on live animals. adequate training is an important aspect of refinement and should be reviewed and improved on an ongoing basis. moreover, refinement can be achieved by improving animal housing by environmental enrichment, e.g., providing a place for mice to hide in the cage and housing social animals such as mice in appropriate-sized groups. these simple changes can improve the physiological and behavioral status of research animals; this not only increases animal well-being but also contributes to the quality of the experimental results by reducing stress levels. the 3rs aspect of reduction focuses on the statistical power of experiments and by following the animal research: reporting of in vivo experiments (arrive) guidelines, originally published in plos biology in 2010. these guidelines provide a framework to improve the reporting of research performed on live animals by maximizing the quality of the scientific data and by minimizing unnecessary studies. the arrive guidelines provide a checklist of aspects that must be considered in good quality research using live animals. the guidelines are most appropriate for comparative studies involving two or more groups of experimental animals with at least one control group, but they also apply to studies involving drug dosing in which a single animal is used as its own control (within-subject experiments). the guidelines provide recommendations on what should be considered when preparing to report on the results of experiments involving live animals, i.e., by providing a concise but thorough background on the scientific theory and why and how animals were used to test a hypothesis, a statement on ethical approvals and study design including power and sample size calculations, a clear description of the methods used to ensure repeatability, objective measurements of outcomes and adverse effects, and interpretation of the results in light of the available literature and the limitations of the study. in addition to the positive impact of the arrive guidelines on reducing the number of animals used in experiments, this checklist provides an easy-tofollow roadmap on what is required for good quality reporting of experimental results. in conclusion, the use of animals in research will continue to be an important aspect of medical research, and these procedures can be ethically justified provided the proper controls are in place. the benefits of animal research have been vital to the progress of medical science; abandoning these studies would have severe negative consequences on human health. by considering aspects such as the 3rs and the arrive guidelines in planning experiments involving live animals, the number of animals used and suffering of these animals for the benefit of human health can be minimized. this requires a strong regulatory framework such as that found in the uk and many other countries, as well an ongoing public debate on the advantages and limitations of animal experimentation. use of house mice in biomedical research the laboratory mouse a comparative encyclopedia of dna elements in the mouse genome principles of regulatory information conservation between 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cord_uid: 780dcjo1 the astounding capacity of pluripotent stem cells (pscs) to differentiate and self-organize has revolutionized the development of 3d cell culture models. the major advantage is its ability to mimic in-vivo microenvironments and cellular interactions as compared to the classical 2d cell culture models. recent innovations in generating embryo-like structures (including blastoids and gastruloids) from pscs have advanced the experimental accessibility to understand embryogenesis with immense potential to model human development. taking cues on how embryonic development leads to organogenesis, pscs can also be directly differentiated to form mini-organs or organoids of a particular lineage. organoids have opened new avenues to augment our understanding of stem cell and regenerative biology, tissue homeostasis, and disease mechanisms. in this review, we provide insights from developmental biology with a comprehensive resource of signaling pathways that in a coordinated manner form embryo-like structures and organoids. moreover, the advent of assembloids and multilineage organoids from pscs opens a new dimension to study paracrine function and multi-tissue interactions in vitro. while this led to an avalanche of enthusiasm to utilize organoids for organ-transplantation studies, we examine the current limitations and provide perspectives to improve reproducibility, scalability, functional complexity, and cell-type characterization. taken together, these 3d in vitro organ-specific and patient-specific models hold great promise for drug discovery, clinical management, and personalized medicine. stem cells have a remarkable capacity to not only self-renew and differentiate but can also selfassemble and self-organize into complex and functional tissues and organs. an initially homogenous population of stem cells can self-organize and undergo an in-vivo like morphogenesis to give rise to three-dimensional (3d) mini-organ like structures in culture, called organoids (sasai et al., 2012) . they retain both functional and structural complexity of the tissue of origin and enable long-term growth of genetically stable cells. organoids can be generated from embryonic stem cells (escs) or induced pluripotent stem cells (ipscs) derived from patients, that can be used for studying organ development, modeling human diseases, and for autologous organ replacement. tissue-specific adult stem cells can also generate organoids and several protocols have been optimized for the same (clevers, 2016) . here in this review, we limit our discussion to how pluripotent stem cells (pscs, including escs and ipscs) form 3d models of the embryo and organs in culture. this process involves recapitulation of implantation, gastrulation, and organogenesis, which, altogether guide the key events during embryonic development when pscs progressively commit to particular lineages. the crosstalk between key signaling pathways during these developmental processes is the source of cellular diversity, behaviors, and forms that generates complex organ structure across the animal kingdom (perrimon et al., 2012) . these signaling pathways form the basis for directed differentiation of pscs to form "miniorgans" (sasai et al., 2012) or embryo-like structures in vitro (shahbazi et al., 2019) . while many reviews have addressed the principles of bioengineering to develop 3d cell culture models and protocols to generate organoids from adult tissues, here we present insights to the state-of-the-art knowledge on the self-organization property of pscs to generate embryo-like structures and organoids, its advantage to model human diseases and challenges for optimum clinical management. self-organization is a physical property observed in many biological phenomena ranging from collective behavioral traits to embryonic morphogenesis. embryonic stem cells (escs) derived from preimplantation embryos are pluripotent and can clonally divide and differentiate into all cell types. escs can aggregate together into 3d embryoid bodies (eb) with the presence of rudimentary cell types. although ebs can differentiate into eye cups, neural cortical structures, and even cell types from endodermal and mesodermal lineage, they lack proper axial organization, a characteristic of mammalian embryo. recent studies demonstrate the capability of escs to generate self-organizing embryo-like structures that can recreate early embryonic morphogenesis (shahbazi et al., 2019; simunovic and brivanlou, 2017) . the mammalian conceptus comprises of the placenta, the fetus, the extra-embryonic tissues to establish feto-maternal interaction, and the "embryo proper" which forms the main body (hyun et al., 2020) . the formation of extra embryonic tissues and the generation of 4 embryonic germ layers are the key stages of mammalian embryogenesis which progress through a series of events from implantation of the conceptus to gastrulation and followed by organogenesis (fig. 1a) . the relatively small size of the conceptus and lack of protocols to culture, along with ethical limitations to acquire human embryos inspired the generation of accessible stem cell-derived embryo models to understand key stages of embryogenesis. recent innovations have led to the generation of embryo-like structures that can recapitulate the interaction between embryonic and extra-embryonic tissues (harrison et al., 2017; rivron et al., 2018b; shao et al., 2017a shao et al., , 2017b sozen et al., 2018; zheng et al., 2019) . for example, human escs (hescs) grown in microfluidic chambers can generate epiblastlike structures known as pace, consists of extra embryonic tissue like the amniotic ectoderm, embryonic sac and amniotic cavity, resembling early post-implantation human embryonic landmarks (shao et al., 2017a (shao et al., , 2017b zheng et al., 2019) . mouse stem cells can also self-organize into preimplantation blastocyst-like structures called blastoids. blastoids were formed by combining escs and extra-embryonic trophoblast stem cells (tsc) (rivron et al., 2018b) , escs/extended pluripotent stem cells (epscs) and tscs (sozen et al., 2019) , or epscs alone (li et al., 2019b) . upon in vitro development, the escs (rivron et al., 2018b; sozen et al., 2019) and the epscs (li et al., 2019b; sozen et al., 2019) produced the primitive endoderm cells thus forming a pre-implantation conceptus comprising the three founding cell types. this was confirmed by independent scrna seq analysis and also showed that blastoids formed with epscs only, do not form cells with a proper trophoblast and epiblast transcriptome signature (posfai et al., 2020) . additional work is needed to capture totipotent stem cells in a dish and form blastoids from only one cell type. although blastoids are transcriptionally similar to blastocysts and can also trigger the formation of deciduae, they don't support de novo embryonic development beyond few days (similar to all embryo models currently available) (li et al., 2019b; rivron et al., 2018b; sozen et al., 2019) . similarly, self-organizing mescs in the presence of wnt agonist and constant agitation can undergo spatial morphogenesis with distinct body axes, germ-layer specification, and spatiotemporal gene expression, very much similar to a gastrulating embryo (beccari et al., 2018; van den brink et al., 2014; warmflash et al., 2014) . these "gastruloids" can be a complementary system to study early developmental events and recent scrna-seq datasets provide evidence on the emergence of neural tube and somites recapitulating early organogenesis (van den brink et al., 2020; veenvliet et al., 2020) . culturing mouse gastruloids with an underlying extracellular matrix led to the formation of structures resembling somites and neural tube (van den brink et al., 2020; veenvliet et al., 2020) while another report suggested mixing gastruloids and extra endodermal cells (xen) can led to the formation of the neural tube (bérenger-currias et al., 2020) . recently, gastruloids are generated from self-organizing human escs, that can also differentiate to three germ layers . somite formation is a rhythmic process that involves molecular oscillation and the expression of certain genes that pattern the embryo, collectively known as the molecular segmentation clock that have been widely characterized in several model system and pluripotent stem cells (diaz-cuadros et al., 2020; matsuda et al., 2020; pourquie, 2003) . the presence of an active segmentation clock in gastruloids similar to an in vivo embryo extends the power of this model system j o u r n a l p r e -p r o o f 5 to characterize mammalian development (van den brink et al., 2020; veenvliet et al., 2020) . nevertheless, the advent of blastoids and gastruloids from mescs has opened new avenues to understand embryogenesis with wide application in biomedical discoveries especially for understanding developmental malformations. the advantage is its reproducible culture conditions that can be scaled up for any high throughput studies and its key features strongly resembling an embryo (both in gene expression and axial organization) (fig. 1b) [ table 1 ]. the generation of embryo-like structures and organoids are recent breakthroughs of stem cell self-organization with many fundamental questions still unanswered. this will open new avenues to understand germ layer specification in embryos with the possibility to generate organ structures inside embryo models. a collective understanding of how embryonic stem cells can be manipulated in vitro to mimic developmental stages of the embryo (shahbazi and zernicka-goetz, 2018 ) is influential to generate more complex organ type in vitro. for example, the presence of an extracellular matrix, similar to a developing embryo led to the formation of somites in gastruloids (van den brink et al., 2020; veenvliet et al., 2020) . the role of the laminin-rich extracellular matrix (also called matrigel) in the growth and proliferation of intestinal tubules and mammary glands (li et al., 1987; montesano et al., 1991) led the foundation to initiate self-organizing structures from escs. seminal discoveries include the generation of bilayered optic cup-like structures from self-organizing mouse and human escs (eiraku et al., 2011; kuwahara et al., 2015) that can also differentiate in vivo into other retinal cell types after engraftment (shirai et al., 2016) . this triggered the concept that escs can be potentially utilized for organogenesis (murry and keller, 2008; sasai et al., 2012) . organoid generation utilizes the spatial and temporal gradients of morphogens that normally orchestrate embryonic development. one of the crucial aspects is the time and precise control of lineage promoting growth factors and expression of tissue-specific transcription factors (tfs). while organoids from tissue-specific adult stem cells (ascs) rely on the activation of growth factors necessary for the maintenance of tissue type, the sequential activation of signaling pathways in pscs leads to the desired organoid type. the precise control of these early developmental processes is still poorly understood and imposes a major challenge for directed differentiation. here, we present an overview of concerted efforts of key signaling factors that promote psc differentiation to self-organize into an organoid of a particular lineage ( fig. 2a) and unique features associated with each organoid (table 2) . j o u r n a l p r e -p r o o f 6 during gastrulation, the primitive streak (ps) forms at the midline, where epiblast cells undergo epithelial-to-mesenchymal transition and ingress to form the mesoderm and endoderm. the cells that don't ingress forms the ectodermal layer which patterns to form epidermal cells and most of the central nervous system. neuro-epithelium is induced in self-organizing pscs cultured in serum-free media, which evaginates to form neural retina but a timed notch signaling generates the photoreceptors (eiraku et al., 2011; völkner et al., 2016) . inhibiting tgf-β and bmp signaling (blocks mesendodermal commitment) leads to definitive ectoderm formation and neuroectoderm commitment. furthermore, an additional wnt activation leads to the formation of cranial neural crest and a pulse of bmp4 with a block of fgf signaling lead to the formation of non-neural ectodermal fate (tchieu et al., 2017) . following neuroectodermal fate, pscs can self-organize into embryoid bodies that can be guided to form distinct areas of the brain or can even recapitulate the whole brain containing discrete brain regions with cortical neurons (lancaster et al., 2013) . this has been extended to generate cerebral organoids from hpscs to recapitulate different brain regions including the hippocampus, midbrain, hypothalamus, cerebellum, and choroid plexus (reviewed in (lancaster and knoblich, 2014; di lullo and kriegstein, 2017; qian et al., 2019) ). choroid plexus organoids derived from pscs can secrete cerebrospinal fluid and has the ability to form blood-brain-barrier (pellegrini et al., 2020) . the interaction of the hypothalamic neuroectoderm along with non-neural ectoderm also led the path to generate anterior pituitary organoid expressing multiple endocrine lineages (suga et al., 2011) . non-neural ectoderm also gives rise to inner-ear and the skin appendages. a combination of bmp activation and tgfβ-inhibitor can differentiate pscs into non-neural ectoderm with the formation of more specialized pre-placoidal cells and a distinct population of mechanosensitive hair cells (koehler et al., 2013 (koehler et al., , 2017 . skin organoids also utilize a similar differentiation strategy, where individual hair follicles grow with prominent hair-shaft, adipocytes, melanocytes and, sebaceous glands . scrna-seq analysis of human esc-derived skin organoids also validated the presence of 16 cell clusters that resemble human skin, keratinocytes. interestingly grafting these organoids under the skin can generate hair follicles , a breakthrough in regenerative medicine that can be used to treat skin diseases and rejuvenating burnt-skin. hair follicle development shares a close analogy to the development of other skin appendages like the dental and, the mammary gland (mikkola, 2007) . although 3d in vitro culture method for culturing teeth (nakao et al., 2007) and mammary glands (jamieson et al., 2017) have been developed, generating them from pscs is still not successful. a deeper investigation of the above growth factors can be used to generate organoids from teeth, salivary glands, and the mammary gland. understanding the differentiation hierarchy of mammary, salivary and dental stem cells (jussila and thesleff, 2012; myllymäki and mikkola, 2019; visvader and stingl, 2014) will be important to generate psc-derived mammary organoids. although a mosaic mammary gland is generated by reprogramming dental epithelial stem cells mixed with mammary epithelial cells (mec) (jimenez-rojo et al., 2019) or by mixing mouse escs with mecs (boulanger et al., 2013) , generating organoids are not reported. human ipscs can be directed to generate mammary 7 organoids, however, they failed to reconstitute in vivo (qu et al., 2017) , suggesting a fine-tuning of growth factors and timing is critical to recapitulate the mammary microenvironment in organoids. organoids of mesoderm and endodermal lineage requires an intermediate primitive streak (ps) induction in pscs. ps formation is a major event during gastrulation, anterior-ps gives rise to definitive endoderm and paraxial mesoderm (source of bone, cartilage, and fibroblasts) while the mid-ps generates the cardiac and limb bud progenitors. dual smad signaling (bmp4 and tgf-β) activation leads to primitive streak formation in escs while blocking ectodermal commitment. bmp4 in the presence of fgf2 is the critical regulator that leads to the upregulation of mesoderm-related genes (brachyury, cdx2, tbx3, and gata4) in hescs (gunne-braden et al., 2020) while tgf-β inhibition accelerates mesoderm formation from ps and blocks endoderm specification (loh et al., 2014) . this strategy was used to differentiate escs into hematopoietic precursor (pearson et al., 2008; zhang et al., 2008) and endothelial progenitors (goldman et al., 2009 ) that was instrumental in the formation of cardiac organoids resembling human fetal hearts . however, differentiated cardiomyocytes don't recapitulate the structural complexity of a mammalian heart (devalla and passier, 2018; mummery et al., 2012) despite showing cardiac electrophysiological response . this is partly due to the complex architecture of the heart requiring multilineage interactions and paracrine signaling from adjacent tissues. a recent study utilized co-emerging cardiac-like mesoderm and gut-like endoderm from ipscs that can attain adult heart-type complexity with noncontractile myocytes and blood vessels surrounding it (silva et al., 2020) . human pscs can intrinsically self-organize to form cardiac mesoderm without exogenous extra-cellular matrix proteins and consists of separate myocardial and endothelial layers. the cardiac organoids (cardioids) can also form cavities resembling the heart chambers which overall opens new avenues to understand cardiogenesis during embryonic development (hofbauer et al., 2020) . insights from 2d culture suggests, cardioids can be further enhanced to form regionalized cell types by inhibiting ra, wnt signaling to generate ventricular cells, or activating them to generate atrial-like cells (devalla et al., 2015; zhang et al., 2011) . organoids resembling blood vessel can also be generated that can integrate with the host vasculature (wimmer et al., 2019) which brings hope to develop organoids of particular lineage coupled with endothelial cells or fused with blood vessels organoids to recapitulate complex organ structure, as recently evidenced in vascularized cortical organoids shi et al., 2020) . human pscs can be differentiated into ureteric epithelium or metanephric and mesonephric mesenchyme in vitro that recapitulates embryonic kidney, (taguchi and nishinakamura, 2017; taguchi et al., 2014; takasato et al., 2014 takasato et al., , 2015 or even generating kidney organoids from epiblast spheroids morizane et al., 2015) . one of the major feats of kidney-like organoids from pscs is its incredible ability to vascularize into glomerulus-like structures with interconnecting renal tubules and collecting ducts 8 (bantounas et al., 2018; van den berg et al., 2018; sharmin et al., 2016; tsujimoto et al., 2020) . a reproducible and a scalable 3d organoid culture strategy is developed that supports vascularization in an animal model and therefore brings great promise to regenerative medicine (koike et al., 2019; takebe et al., 2015) . activating tgf-β signaling at the ps stage leads to the definitive endodermal lineage that gives rise to the internal lining of the lungs, and intestinal tracts and other vital organs like thyroid, thymus, liver, and pancreas. during embryonic development, the endoderm layer gives rise to a primitive gut tube broadly divided into fore, mid and hindgut, from where other vital organs bud off. this is been achieved by using a nodal-related tgf-β signaling molecule, activin-a which promotes the differentiation of pscs to the definitive endoderm expressing sox17 and foxa2 (d'amour et al., 2005) . dual inhibition of bmp and tgf-β signaling generates anterior foregut endoderm (afe) which selfassembles to generate organoids of the esophagus, lung, parathyroid, and thymus. the sox2+ p63+ progenitors that are responsible for esophageal organoids block the wnt-bmp signaling required for ventral foregut to generate nkx2-1+ lung progenitors. (trisno et al., 2018; zhang et al., 2018) . however, lung progenitors can also be induced by the addition of fgf10, fgf7, and a low concentration of bmp4 (huang et al., 2014; wong et al., 2012) or simultaneous stimulation of wnt and fgf along with high hedgehog signaling (dye et al., 2015) . notably, the nkx2-1 progenitors give rise to basal, secretory cells leading to the formation of the airway and alveolar cells, characteristic of the human lung (huang et al., 2014; jacob et al., 2017; mccauley et al., 2017) but they mostly resemble a fetal lung, thereby raising the concern of organoid maturity in vitro. nkx2-1+ cells are considered to be a bipotent progenitor population and wnt-bmp-fgf signaling differentiates them from being lung versus thyroid (dame et al., 2017; kurmann et al., 2015; serra et al., 2017) . the thyroid progenitors can also form organoids and can maintain th level after xenotransplantation in the mouse model (kurmann et al., 2015) . thymic organoids are still not successful but thymic progenitors can be generated by activating bmp4, ra, wnt3a, fgf8b and inhibiting hedgehog signaling can differentiate into t cells in vitro but failed to support prolong thymogenesis in vivo (parent et al., 2013; sun et al., 2013) . the posterior foregut endoderm specifies the formation of the liver, stomach, and pancreas. fgf and bmp4 are pre-hepatic cues that induce the formation of liver progenitors from activin-induced endoderm (gouon-evans et al., 2006) . simultaneous inhibition of hedgehog signaling while activating ra results in pancreatic fate (d'amour et al., 2006) . another strategy utilized the mixing of endothelial and mesenchymal cells that can instruct human ipsc derived hepatic endoderm to self-organize into liver bud organoids (takebe et al., 2013 (takebe et al., , 2017 . the liver buds can further differentiate into bile duct cells (activating notch and tgf-β) or to hepatocytes (blocking notch and tgf-β) (ang et al., 2018) . a 9 complex architecture can be established by mixing biliary and hepatic cells to generate hepato-biliary organoids that can form bile ducts and hepatic epithelium after transplantation in mice (wu et al., 2019) . moreover, fusing anterior and posterior gut spheroids from ipsc-derived definitive endoderm can also assemble into multi-endodermal domains to form hepato-biliary-pancreatic organoids (koike et al., 2019) . the generation of the complex organoid system holds tremendous potential as models for studying organogenesis. while considerable progress has been made in generating multi-organ structures, compartmentalization of organ structures is also evident. for example, gastric organoids can be regionalized into the antral part (green et al., 2011; mccracken et al., 2014) which upon wnt and fgf10 activation leads to fundus (mccracken et al., 2017) , pancreatic organoids can generate beta cells (β) using keratinocyte growth factor and also resembles ductal and acinar morphology of human fetal pancreas (hohwieler et al., 2017) . the pancreatic progenitors can form insulin-producing β cells and can maintain glucose levels after transplantation in diabetic mice (pagliuca et al., 2014) . multiple in vitro differentiation protocols have resulted in pancreatic progenitors but the critical goal to derive functional β cells is of high biomedical value. however, insulin production is taken as a gold-standard to mark β cells, these functional β cells often co-express other hormones leading to the co-occurrence of contaminating lineages (shahjalal et al., 2018) . while unprecedented progress has been made towards generating β cells in vitro, the existence of unwanted lineages imposes significant concerns that need to be addressed for successful therapeutic application. scrna seq on psc-derived pancreatic organoids compared to the human pancreas will be instrumental to identify the correct lineage to its closest approximation. a similar level of compartmentalization is evident in in intestinal organoids with characteristic villus and crypt-like structures (spence et al., 2011; watson et al., 2014) which upon short exposure to fgf4 and wnt activation recapitulates duodenum and longer exposure generates ileum . the colon organoids are generated from the hindgut endoderm in the presence of bmp signaling (crespo et al., 2017; múnera et al., 2017) and, embryologically the caudal extension leads to the prostate, bladder, and penile urethra. not much is known regarding prostate, urethral and bladder organoid generation from pscs. while directed differentiation of human escs using wnt10b and fgf express prostate-specific transcription factor nkx3.1 and androgen-regulator tmprss2 (calderon-gierszal and prins, 2015) , this study focused entirely on nkx3.1 expression as a definitive factor for prostate organoid. several studies on knockout mice have revealed the function of hoxb1 and sox9 in prostate commitment irrespective of nkx3.1 status and detailed characterization of esc-derived prostate organoids is required with characteristic molecular markers (toivanen and shen, 2017) . overall, the focus has been on generating organoids from a single lineage in vitro that provides insights to organ development, but it fails to recapitulate multi-tissue interactions necessary for organ function. the development of multi-lineage organoids opens new avenues to obtain personalized microphysiological models for modeling developmental organogenesis in vitro. few examples include the fusion of neural spheroids or cortical organoids (bagley et al., 2017; birey et al., 2017; xiang et al., 2017 xiang et al., , 2019 , generating hybrid neuromuscular organoids (faustino martins et al., 2020) , fusing anterior and foregut endoderm for hepatic-biliary and pancreatic organoid structures (koike et al., 2019) , intestinal organoids with a functional enteric nervous system (workman et al., 2017) . multilineage organoids generated from co-emerging cardiac and gut lineage specification from ipsc can be useful to model heart complexity better than conventional cardiac organoids. these organoids consist of mature cardiomyocytes, stromal epicardial cells, and blood-vessel like structures along with the primitive gut that resembles the intestine (silva et al., 2020) . the emergence of multilineage organ models will be useful to map paracrine interaction and also understand the role of adjacent organ tissues required for organ maturation. collectively, these studies present a common set of signaling factors (wnt, bmp, tgfβ, hedgehog, notch) that works in a coordinated and chronological action to decide the cell fate (kojima et al., 2014) . a thorough identification of the appropriate cocktail of growth factors is crucial to generate correct cell types and validation using a variety of tools ranging from microscopy to scrna seq is necessary (fig 2 b) . embryo-like structures and organoids derived from pscs have diverse scientific applications ranging from modelling mammalian development to understanding disease outcomes (clevers, 2016; lancaster and huch, 2019; rossi et al., 2018; sharma et al., 2020) . while in vitro and in vivo models including 2d coculture, microfluidics, tissue engineering, humanized animals, and chimeras are used for decades to model human diseases and drug screening, they have their advantages and associated challenges. here we present a comprehensive overview of the current applications of psc derived 3d culture systems, along with strategies to overcome the challenges to make it accessible for regenerative medicine and cell-based therapeutics (fig. 2c) . ipscs can be differentiated to form gastruloids and blastoids that can help to understand rare human diseases. "rare diseases" collectively affect a significant population that has led to developmental malformations in infants. despite efforts that have been made to understand the genetic underpinnings of rare diseases, we still have limited clinical progress and lack of therapeutic strategies (anderson and francis, 2018; tambuyzer et al., 2020) . given the robustness of ipscs generation from a variety of genetic disorders including parkinson, down syndrome, and huntington disease, understanding the defects in early embryogenesis will be helpful from ipsc-derived embryo like structures. while there is a limitation in the number of patients, the patient-ipsc derived organoids/gastruloids will be particularly useful to model a genetic disorder, perform drug screening, and understand embryonic development. mutations in genes associated with the segmentation clock lead to defects in vertebrae. several other clinically relevant mutations exist that can be useful to study in gastruloids to model developmental anomalies and also perform functional screening. the optimized culture conditions allow in vitro cultures of embryos beyond implantation and provide a broader understanding of pregnancy loss and improve contraception technologies. a major advantage of using stem cell-based embryo models is to utilize them for studying human embryonic development as they represent an ethical alternative to replace the use of embryos. these models should only be used to better understand embryonic development and the origin and treatment of diseases in a dish (hyun et al., 2020; rivron et al., 2018a) . organoids have been widely used to model both congenital and acquired diseases including several age-related disorders and infectious diseases (lancaster and huch, 2019) . brain organoids from patient-derived ipscs are utilized to model neurodegenerative disorders and also recapitulating human brain development (di lullo and kriegstein, 2017). to name a few, different types of brain organoids have been used to model microcephaly, alzheimer's disease, and neuro-progenitors abnormality after zika virus infection (reviewed in (amin and paşca, 2018) ). similarly, gastric organoids are used to model helicobacter pylori infection (mccracken et al., 2014) . a recent study utilized a wide spectrum of organoids generated from human pscs to understand the viral transmission in different human organs. however, the lack of immune system in organoids may be a limiting factor in these studies to accurately understand the viral-immune response (yang et al., 2020) . the generation of thymus epithelial progenitors from pscs opens another avenue to the ever-growing organoid community. thymus is one of the specialized organs that plays a major role during infection and other age-related disorders. thymic organoids capable of t-cell differentiation will be particularly applicable for treating several autoimmune diseases (parent et al., 2013; sun et al., 2013) . on the other hand, a major population globally experiences hearing disorder, and inner ear organoids bring hope to utilize them for personalized medicine. although transplanting retinal organoids to macular degeneration in primates were successful, the development of esc-derived inner ear organoids is still in its infancy and need some fine-tuning before being utilized for regenerative therapies. furthermore, these organoids can be used for high-throughput drug screening to identify novel targets that promote survival/regeneration of sensory hair cells or show ototoxicity. it also holds great promise to gain insights into genetic mutations affecting inner ear development (roccio and edge, 2019; tang et al., 2019) and model hearing loss defects. skin organoids are able to regenerate hair follicles under the skin can be used as bioengineered skin-grafts in regenerative surgery (lee et al., 2019) . organoids can 12 be a powerful tool to functionally characterize genetic variants and pancreatic organoids are recently used to study diabetes-causing gene variants (maxwell et al., 2020) and similar strategies should be implemented to understand other disease-causing mutations. around 4.6 million missense variants are reported for disease-causing mutations in the genome aggregation database (gnomad), of which 99% are rare, and only 2% have a clinical interpretation in clinvar (claussnitzer et al., 2020; starita et al., 2017) . much of our understanding of clinical management comes from clinvar but the majority are still considered to be "variants of uncertain significance" (vus) (landrum et al., 2014; rehm et al., 2015) (fig. 3a) . most of the disease-causing mutations reported in clinvar are point mutations and currently, a broad understanding of single nucleotide variants (snvs) comes from generating transgenic mice which is often very laborious and time-consuming. to this end, several in vitro functional assays have been developed to screen diseasecausing missense variants, but it often fails to recapitulate the disease outcome (monteiro et al., 2020) . although, high-throughput screenings have accelerated the identification of pathogenic and neutral variants of several disease-causing genes (findlay et al., 2018; hanna et al., 2020) , it fails to comment on its tumorigenic potential in patients. it is not trivial to generate transgenic model organisms to understand the function of individual variants and its disease-causing potential. an alternative approach could be the use of base editors and prime editors (anzalone et al., 2020) to generate snvs in escs, that can be differentiated to organoids of interest (as described previously) (fig. 3b ). this will leverage its potential for in vivo functional studies and reduce the generation of genetically engineered mouse models (gemms). multiple high-throughput screening (hts) platforms have been developed using ipscs, that have advanced our understanding of drug development and toxicity studies (grskovic et al., 2011) . it has just started to be applied in organoids for drug screening (czerniecki et al., 2018) . these screening platforms are very cost-effective and reduce any manual error thereby generates uniform organoids more successfully in clinical drug development. during the past decade, tissue engineering and bioprinting technologies have revolutionized our understanding of simulating 3d organ complexity. this allows multiple cell types to be integrated and grown together in 3d culture systems that are compartmentalized by membranes or microchannels to mimic the in vivo environment. (bhatia and ingber, 2014) . developing an "organ-on-a-chip" concept to generate vascularized multi-lineage organoid along with high-throughput platforms will play a critical role to improve the scalability and uniformity of organoid generation. as it simulates to its maximum capability, these novel technological insights surely improve our understanding of organ development in vitro but not all microfluidics can recapitulate the organ complexity. breakthroughs have been made to integrate multi-organs in a chip (wu et al., 2020) and coupling these with multilineage organoids is of high biomedical relevance. generating organoid biobanks are crucial for personalized medicine, as it brings the ability to perform high-throughput drug screening, epigenomic and transcriptomic analysis, copy-number variations of individual patients at a large scale. while biobanks have been generated for breast cancer, gastric cancer, pediatric kidney cancer, and glioblastoma (calandrini et al., 2020; jacob et al., 2020; sachs et al., 2018; yan et al., 2018) , these can be extended to the development of organoid biobanks with individual disease variants generated in escs or, from ipscs of rare diseases. we hope to see patientvariant organoid biobank in the future that will accelerate personalized drug development. recent advances in genome editing, especially with the advent of crispr-cas9 (clustered-regularly interspaced short palindromic repeats-crispr associated protein 9), has revolutionized our understanding of human disease and have been a powerful tool to correct genetic mutations in human organoids (geurts et al., 2020; schwank et al., 2013) . while genome editing is a routine technique in 2d cell lines, it requires optimizations to improve its efficiency. characterizing correctly edited organoids is a major challenge and some strategies rely on the removal of culture media constituents, antibiotic selection, or facs sorting after transfection (artegiani et al., 2020; driehuis and clevers, 2017; sasselli et al., 2017) . for example, removal of wnt and r-spondin from culture medium selects survival of apc ko intestinal organoids and further addition of nutlin3 allows selection of apc and p53 double ko organoids (drost et al., 2015) . crispr and other nucleases have been classically used to understand the function of individual genes by making knockouts or tracking particular cell types using reporter organoids (artegiani et al., 2020; matano et al., 2015) . while, this "one-gene-at-a-time" approach was crucial to establish the power of genome editing in organoids, it fails to validate a large number of genomic threats underlying a disease. genome sequencing studies have identified several infrequent mutations that hit together with commonly mutated genes. some driver mutations that are often considered to be rare can form aggressive cancers and is the guiding force behind genetic heterogeneity in tumors (nussinov et al., 2019; vogelstein et al., 2013) . to better understand these rare events, genome wide crispr screens are a powerful tool to get an overview of less frequent but actionable genes (fig. 3c) . pooled crispr screens are increasingly popular to model these synergistic interactions and identify tumor suppressors and other novel cancer driver genes. the generation of cas9-knock-in mice was instrumental to extend the in vivo genome editing toolbox that accelerated high-throughput crispr screens in organoids (platt et al., 2014) . this led to the initial reports on crispr loss of function screens in biliary epithelial cell organoids to identify genes necessary for liver regeneration (planas-paz et al., 2019) and also for identifying co-occurring mutations in colorectal cancer (takeda et al., 2019) . these studies looked into a specific pool of genes as whole-genome-wide screens in organoids were not successful due to several technical limitations. while, growth factor selection assay is a powerful method to identify mutated organoids (drost et al., 2015; matano et al., 2015) , similar niche factor dependence strategies need to be devised for studying other gene perturbations. moreover, the heterogeneous growth of organoids from transduced single organoid cell suspension introduces variability in guide-rna (grna) representation and downstream sequencing analysis. to overcome these shortcomings, the development of barcoded sequencing of individual organoids compared to bulk sequencing has proven to be a good alternative to perform genome-wide knockout screens (ringel et al., 2020) . furthermore, pooled screens coupled with xenotransplantation of organoids in vivo can be used to perform tumorigenesis studies (fig. 3c) . largescale mouse mutagenesis screens are a valuable tool for functional genomics but require large research infrastructure and can be time consuming and laborious (gondo, 2008) . transplantation of human colonic organoids transduced with a pool of grnas led to the identification of recurringly mutated tumor suppressor genes and some novel mediators that were not previously associated with colorectal cancer (michels et al., 2020) . organoid xenografts in mouse models to study cancer metastasis can be a powerful alternative to mouse mutagenesis screens (o'rourke et al., 2017; roper et al., 2017) . while most of the forward genetic screen studies are performed on adult stem cell-derived organoids, there are no studies reported on crispr screens on psc-derived organoids. generating missense variants in escs or patient ipsc-derived organoids can be utilized in the future to study synergistic gene interactions and also for selective drug response. several synthetic-lethal relationships have been reported that forms the basis of drug development to treat several cancers, however, multiple studies have also reported on its low efficacy or selective survival (huang et al., 2020; nijman, 2011) . for example, parp and atr inhibitors that have revolutionized cancer treatment, have shortcomings and crispr activation and knockout screens have identified the genetic basis behind resistance and ways to improve sensitivity. however, 2d cell lines fail to recapitulate the interaction of stem cells and the associated microenvironment which plays a major role in drug sensitivity. future studies on genomewide crispr screens will be very useful to understand a particular drug response in patient-derived organoids for personalized medicine (fig. 3c) . apart from disease modeling, forward genetic screens in organoids can be utilized for optimizing niche growth factors, stem cell differentiation strategies,and identifying conditions for organoid maturation (li et al., 2019a) . despite paramount success in generating organoids from esc and ipscs, the technologies are still in its infancy for use in the clinic. genome instability and loss of heterozygosity during extensive rounds of in vitro culture and the introduction of de novo mutations during somatic reprogramming impose serious concerns on utilizing ipscs. moreover, the epigenetic memory and dna methylation status of the parent somatic cells from where ipscs are generated also affects cell fate (gore et al., 2011; hussein et al., 2011; lister et al., 2011) . collectively these limitations in ipscs are potential threats to utilize them for robust organoid generation, especially for therapeutics. recent advances to correct disease-causing mutations using different crispr based genome editing tools in ipscs followed by organoid generation is a promising avenue for regenerative medicine, however, they need to be tested for crispr-offtarget events that may have other harmful consequences. in vitro differentiation of pscs into organoids often resembles the embryonic stage and lacks proper maturation, another hurdle to utilize them for functional studies. these organoids can differentiate into mature cell types after orthotopic transplantation suggesting cues from the in vivo microenvironment enhances maturity and tissue functionality. recent advances in organoid protocols have addressed this issue using bioengineering platforms to obtain adult-like tissues (holloway et al., 2019) . the generation of interconnected vasculature has just begun using a mesenchyme-driven self-condensation method. organ buds are reported from multiple tissue types including the liver, intestine, lung, kidney, heart, and brain (takebe et al., 2015) . the lack of vasculature generates a hypoxic environment leading to increased necrosis or apoptotic cells in organoids. endothelial cells are mixed with lineage progenitors to generate vascularization in organoids. injectable organoids have recently been developed in hydrogels that can also generate perfusive vasculature in mice in vivo (rossen et al., 2020) . while improved vasculature is currently used, engineering organoids with other surrounding tissue types are necessary. to this end, assembloids have emerged to model the structural interactions between subdomains of an organ that opens the opportunity to couple multi-lineage differentiation. organ systems are mostly associated by surrounding muscles, nerves, and immune cells that are critical for organ function, engineering organoids containing a neuronal network along with blood vasculature is something to foresee in the future. another major challenge is the considerable amount of ambiguity that exists in identifying the correct lineage progenitors which leads to cell heterogeneity and inter-organoid variability. spontaneous differentiation due to long term culture can generate unwanted lineages (tang et al., 2019) and the high frequency of contaminating progenitors compared to the desired cell types during in vitro differentiation of pscs can affect organoid reproducibility (veres et al., 2019; yao et al., 2017) . therefore "separating the wheat from the chaff" is important to generate organoids, as a minor population of undifferentiated hpscs has a higher chance to give rise to teratoma (tumor) that often out-competes organ reconstitution in vivo (fowler et al., 2020) . heterogeneity of both differentiated and undifferentiated pscs is a common problem and therefore using specific cell-surface markers can efficiently generate the desired lineage. fluorescence activated cell sorting (facs) is a powerful method to sort the correct cell type based on surface-markers but can often affect the cell population thereby reduces its efficiency (kelly et al., 2011) . a current gold-standard to draw an analogy in organ recapitulation is to perform bulk transcriptomic sequencing or correlating protein expression in pscderived organoids with respective desired tissue. however, this often outnumbers the exact cell type and fails to detect the presence of a minor population of contaminating lineage. the development of scrna-seq is instrumental to get a comprehensive view of cell-type composition in an organoid. while limitations to scrna-seq exist with overarching challenges of not able to detect lowly expressed gene clusters (kharchenko et al., 2014) , the high-omics dataset can assess any inter-organoid variability and gives high confidence to utilize them to model a particular organ type. a large-scale transcriptomic profiling study at a single-cell level in brain organoid has identified six distinct clusters to neuroectodermal type and one cluster of cells of mesodermal origin (quadrato et al., 2017) . a stepwise affirmation with scrna-seq at different phases of differentiation could better mimic organ development in vitro. this led loh et al, to generate a nearly-pure cell population of primitive streak that differentiates to other mesodermal cell types (loh et al., 2016) . therefore, understanding cell types and state in a particular organoid using diverse technologies ranging from transcriptomics profiling to lineage tracing at single cell level (mckinley et al., 2020) will be crucial to establish pscderived organoids as robust and consistent models for functional studies (fig. 2c) . not all stem cell lines have similar potency to generate organoids of a particular lineage as observed in the ability to generate retina, inner ear, and skin organoids from escs osafune et al., 2008; völkner et al., 2016) . analysis of 162 differentiation strategies from 61 psc lines identified wnt/b catenin signaling leads to the varied differentiation ability and its activation could alleviate the outcomes (strano et al., 2020) . future strategies should focus on empirically investigating the organoid-forming efficiency for each es-cell-line before initiating functional studies. some common factors that decide the efficiency are self-assembling property of pscs, effects of matrigel concentration, precise timings of morphogens, and variable epigenetic status. matrigel being a biological material may have batch effects and alternative synthetic scaffolds have just started to be explored (aisenbrey and murphy, 2020) . the cytotoxicity of the chemical inhibitors may also affect cellcycle/growth, giving inconsistencies in cellular differentiation (laco et al., 2018) . thus, overexpressing tissue-specific transcription factors (tf) can be used as an alternative to induce differentiation in pscs as evident in generating hepatocyte-like cells in vitro (guye et al., 2016) , thyroid follicular organoids (antonica et al., 2012) , thymus progenitors (su et al., 2015) ), and intestinal organoids from pscs (sinagoga et al., 2018) . a collaborative effort to fine-tune the timing and concentration of biochemical and biophysical cues will increase the success and reproducibility. the advent of genome editing to rectify a faulty gene with personalized organoids is what we foresee for precision medicine in a personalized era. while the "bench-to-bed-side" transition of organoids will still take some time, but early pre-clinical studies are very promising with the increasing trend for commercializing organoids for disease modeling and regenerative medicine (choudhury et al., 2020) . research on stem cell-based embryo models (blastoids and gastruloids) and organoids will be increasingly popular in the coming years with more opportunity to utilize them for regenerative medicine. these 3d models have already proven to be an exciting platform for personalized therapies, the above challenges need to be overcome, and efforts have to be made to increase the yield and reproducibility. future research will look into expanding these platforms to other animal model systems. some examples include the generation of brain organoids from other non-human primates to study the evolution of the nervous system and brain maturation (kanton et al., 2019; pollen et al., 2019) , generation of snake venom gland organoids for anti-venom production (post et al., 2020) , bat intestinal organoids for modeling viral infection (zhou et al., 2020) . most of our fundamental understanding of signal transduction pathways that we exploit to generate "mini-organs in a dish" comes from studying animal development. generating self-organizing organoid-like structures from stem cells in other animal models will provide deep molecular evolutionary insights and a detailed appreciation of developmental networks. the conservation of gene regulatory networks will help to understand the evolution of the structure and function of organs. the disease-causing variants are catalogued in several public clinical database and are used for functional studies. (b) different variants can be generated in pscs using the crispr-based genome editing toolbox, which can further be differentiated to the organoid of the desired lineage. the organoids represent each patient/variant and high throughput drug screening and cytotoxicity studies can be performed to develop a personalized drug. (c) schematic showing the use of organoids in high throughput genetic screens using crispr to identify novel gene targets that get frequently mutated to cause cancer. furthermore, these organoids can be xenotransplanted to develop an in vivo model for mouse tumorigenesis studies. the resulting tumors can be further cryopreserved as organoids for drug screening and mutagenesis studies. • pluripotent stem cell-derived embryo-like structures as model system for developmental studies. • self-organization of pscs to generate multilineage mini-organs or organoids. • organoids as preclinical models of human diseases. • use of organoids for drug development and targeted therapy. • functional evaluation of disease-associated genetic variants. synthetic alternatives to matrigel building models of brain disorders with three-dimensional organoids modeling rare diseases with induced pluripotent stem cell technology a roadmap for human liver differentiation from pluripotent stem cells generation of functional thyroid from embryonic stem cells genome editing with crispr-cas nucleases fast and efficient generation of knock-in human organoids using homology-independent crispr-cas9 precision genome editing fused cerebral organoids model interactions between brain regions generation of functioning nephrons by implanting human pluripotent stem cell-derived kidney progenitors multi-axial self-organization properties of mouse embryonic stem cells into gastruloids renal subcapsular transplantation of psc-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo microfluidic organs-on-chips assembly of functionally integrated human forebrain spheroids embryonic stem cells are redirected to non-tumorigenic epithelial cell fate by interaction with the mammary microenvironment symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells engineering of human brain organoids with a functional vascular-like system directed differentiation of human embryonic stem cells into prostate organoids in vitro and its perturbation by low-dose bisphenol a exposure commercialization of organoids a brief history of human disease genetics modeling development and disease with organoids colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing high-throughput screening enhances kidney organoid differentiation from human pluripotent stem cells and enables automated multidimensional phenotyping efficient differentiation of human embryonic stem cells to definitive endoderm production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells thyroid progenitors are robustly derived from embryonic stem cells developmental stage-specific overexpression of nkx2-1 cardiac differentiation of pluripotent stem cells and implications for modeling the heart in health and disease atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology in vitro characterization of the human segmentation clock crispr/cas 9 genome editing and its applications in organoids sequential cancer mutations in cultured human intestinal stem cells in vitro generation of human pluripotent stem cell derived lung organoids self-organizing optic-cup morphogenesis in three-dimensional culture self-organizing 3d human trunk neuromuscular organoids accurate classification of brca1 variants with saturation genome editing a critical look: challenges in differentiating human pluripotent stem cells into desired cell types and organoids modelling kidney disease with crispr-mutant kidney organoids derived from human pluripotent epiblast spheroids efficient genetic engineering of human intestinal organoids using electroporation crispr-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank a boost of bmp4 accelerates the commitment of human embryonic stem cells to the endothelial lineage trends in large-scale mouse mutagenesis: from genetics to functional genomics somatic coding mutations in human induced pluripotent stem cells bmp-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells induced pluripotent stem cellsopportunities for disease modelling and drug discovery gata3 mediates a fast, irreversible commitment to bmp4-driven differentiation in human embryonic stem cells genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using gata6 massively parallel assessment of human variants with base editor screens assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro cardioids reveal self-organizing principles of human cardiogenesis human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling biologically inspired approaches to enhance human organoid complexity synthetic lethality as an engine for cancer drug target discovery efficient generation of lung and airway epithelial cells from human pluripotent stem cells copy number variation and selection during reprogramming to pluripotency toward guidelines for research on human embryo models formed from stem cells differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells a patient-derived glioblastoma organoid model and biobank recapitulates inter-and intra-tumoral heterogeneity derivation of a robust mouse mammary organoid system for studying tissue dynamics dental epithelial stem cells as a source for mammary gland regeneration and milk producing cells in vivo signaling networks regulating tooth organogenesis and regeneration, and the specification of dental mesenchymal and epithelial cell lineages organoid single-cell genomic atlas uncovers human-specific features of brain development cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells bayesian approach to single-cell differential expression analysis generation of inner ear sensory epithelia from pluripotent stem cells in 3d culture generation of inner ear organoids containing functional hair cells from human pluripotent stem cells modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary timing of developmental events in the early mouse embryo regeneration of thyroid function by transplantation of differentiated pluripotent stem cells generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue unraveling the inconsistencies of cardiac differentiation efficiency induced by the gsk3β inhibitor chir99021 in human pluripotent stem cells disease modelling in human organoids generation of cerebral organoids from human pluripotent stem cells cerebral organoids model human brain development and microcephaly clinvar: public archive of relationships among sequence variation and human phenotype hair follicle development in mouse pluripotent stem cell-derived skin organoids hair-bearing human skin generated entirely from pluripotent stem cells hair-bearing human skin generated entirely from pluripotent stem cells influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells genome-scale screens identify jnk-jun signaling as a barrier for pluripotency exit and endoderm differentiation generation of blastocyst-like structures from mouse embryonic and adult cell cultures hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations mapping the pairwise choices leading from pluripotency to human bone the use of brain organoids to investigate neural development and disease modeling colorectal cancer using crispr-cas9-mediated engineering of human intestinal organoids recapitulating the human segmentation clock with pluripotent stem cells gene-edited human stem cell-derived β cells from a patient with monogenic diabetes reverse preexisting diabetes in mice efficient derivation of functional human airway epithelium from pluripotent stem cells via temporal regulation of wnt signaling modelling human development and disease in pluripotent stem-cell-derived gastric organoids wnt/β-catenin promotes gastric fundus specification in mice and humans tools and concepts for interrogating and defining cellular identity pooled in vitro and in vivo crispr-cas9 screening identifies tumor suppressors in human colon organoids genetic basis of skin appendage development functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle 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screening a human pluripotent stem cell-based platform to study sars-cov-2 tropism and model virus infection in human cells and organoids a single-cell roadmap of lineage bifurcation in human esc models of embryonic brain development shortterm bmp-4 treatment initiates mesoderm induction in human embryonic stem cells direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals 3d modeling of esophageal development using human psc-derived basal progenitors reveals a critical role for notch signaling controlled modelling of human epiblast and amnion development using stem cells infection of bat and human intestinal organoids by sars-cov-2 we sincerely apologize to any authors whose work we were not able to include here due to space restriction. we would like to thank drs. kajal biswas, manisha jalan, suhas kharat, satheesh sengodan and terry sullivan for comments and suggestion. the figures in this review are made using a paid subscription to biorender.com, schematics of organoids and embryo models are drawn based on the appropriate references explaining them. the authors contributed equally to all aspects of the article. ss wrote the first draft of the manuscript. the authors declare no competing or financial interests. the sharan laboratory is funded by the intramural research program, center for cancer research, national cancer institute, u.s. national institutes of health.j o u r n a l p r e -p r o o f (dye et al., 2015; jacob et al., 2017; mccauley et al., 2017 (spence et al., 2011; tsai et al., 2017; watson et al., 2014; workman et al., 2017) key: cord-005068-3ddb38de authors: meslin, eric m.; garba, ibrahim title: biobanking and public health: is a human rights approach the tie that binds? date: 2011-07-15 journal: hum genet doi: 10.1007/s00439-011-1061-2 sha: doc_id: 5068 cord_uid: 3ddb38de ethical principles guiding public health and genomic medicine are often at odds: whereas public health practice adopts collectivist principles that emphasize population-based benefits, recent advances in genomic and personalized medicine are grounded in an individualist ethic that privileges informed consent, and the balancing of individual risk and benefit. indeed, the attraction of personalized medicine is the promise it holds out to help individuals get the “right medicine for the right problem at the right time.” research biobanks are an effective tool in the genomic medicine toolbox. biobanking in public health presents a unique case study to unpack some of these issues in more detail. for example, there is a long history of using banked tissue obtained under clinical diagnostic conditions for later public health uses. but despite the collectivist approach of public health, the principles applied to the ethical challenges of biobanking (e.g. informed consent, autonomy, privacy) remain individualist. we demonstrate the value of using human rights as a public health ethics framework to address this tension in biobanking by applying it to two illustrative cases. at first blush, the ethical foundations guiding public health and genomic medicine are at odds: whereas public health practice adopts collectivist principles that emphasize utilitarian and population-based benefits, genomic (and especially personalized) medicine is squarely grounded in an individualist ethic that emphasizes autonomous decisionmaking for personal benefits. one definition of public health illustrates its breadth and focus: the promotion of health and the prevention of disease and disability; the collection and use of epidemiological data, population surveillance, and other forms of empirical quantitative assessment; a recognition of the multidimensional nature of the determinants of health; and a focus on the complex interactions of many factors -biological, behavioral, social, and environmental -in developing effective interventions (childress et al. 2002) . lawrence o. gostin (2001) further highlights the critical role of collective entities like communities and governments in ensuring the public's health because although individuals, given the means, can do many things to protect their own health, there are health benefits such as a healthy environment, safe roads, potable water and clean air that require ''organized and sustained community activities''. in short, public health programs deliver to populations health benefits that cannot be effectively secured on an individual or small group basis (childress et al. 2002) . in contrast, genomic medicine-sometimes conflated with personalized medicine-has been described as an endeavor that ''will provide a link between an individual's molecular and clinical profiles, allowing physicians to make the right patient-care decisions and allowing patients the opportunity to make informed and directed lifestyle decisions for their future well-being'' (ginsburg and mccarthy 2001) . it envisions medical care in which ''drugs and drug doses are made safer and more effective because they are chosen according to an individual's genetic makeup'' (lesko 2007) . others, such as the ickworth group (burke et al. 2010) , characterize personalized medicine as any medical ''care that is tailored to the individual or stratified by the population subgroup''. common to all of these definitions is the emphasis on customizing therapy to the individual patient. indeed, for as long as clinicians have been caring for patients, medicine has been personalized (ramsey 1961) , but it is the accelerant of genetic technology that has led some to think that today's medicine has the potential to be even more ''personalized'' than its historical predecessors. of course, with the benefit of further reflection, the contrast between personalized medicine and public health is not so stark. for instance, the collectivist approach of public health does not preclude a role for clinical interventions and choices at the individual level. moreover, the claim that the treatment of a sick individual improves the health of the population of which she is a member is all but tautologous. vaccination is an example that fits both conditions. seen this way, personalized medicine and public health are not mutually exclusive, but rather incompletely overlapping. the goals of public health practice certainly include the impact on the health of individuals, and included in the potential value of a genomic approach to medical care is its generalizability to the public's health, for example through better screening and prevention programs (burke et al. 2010 ). recognition of this potential for demonstrating the relationship between public health and genomics is evident in a new area of study complete with its own journal, public health genomics that hopes to address some of these very issues. it has been noted, for instance, that a better understanding of what lies between the genes that make up the genome, the role of the environment on gene expression and the role of the interaction between genes will help us to know why some individuals remain healthy while others are more susceptible to genetic diseases. this understanding will also benefit the public health sector where the prevention and expression of communicable and infectious diseases, for example, is related in part to understanding genetic susceptibility… ). the ickworth group recently examined the potential for genomics and personalized medicine to inform public health practice and concluded that much still needs to be done before the promise can be realized (burke et al. 2010) . in particular, they made six recommendations: 1. efforts to integrate genomics into public health and practice should continue. 2. an appropriate research infrastructure for generating an evidence base for genomic medicine needs to be established and maintained. 3. model public health genomics programs and clinical services need to be developed, implemented and evaluated. 4. international collaborations should be promoted. 5. appropriate genetic services and genome-based research should be fostered within low and middle income countries. 6. programs, research and strategies in public health genomics should be informed by accepted ethical principles and practices. such qualified support for the potential for genomic impact on public health is not surprising, as others have commented on the status of promises made and kept (evans et al. 2011; hall et al. 2010) . biobanking is a useful case study to unpack issues at the intersection of genomics and public health. the storied history of the many uses of biological materials that help to improve the understanding, clinical diagnosis and treatment of human disease is long and impressive with detailed reports of the clinical value of banked specimens dating to the early eighteenth century (ackerknecht 1967; korn 2000) . without access to stored specimens of blood, urine, tumors, body tissues, dna and other human biological materials, important advances in cancer, infectious disease, cardiovascular care and mental disorders would not have been possible (nat'l bioethics adv. comm. 2000) . for example, the pap smear would not have been developed (younge et al. 1949 ) and the nonsteroidal estrogen hormone, diethylstilbestrol (des), would not have been found to be carcinogenic (herbst 1981) . without the knowledge gained from autopsies of korean war veterans, science would have known less about the age of onset for atherosclerosis (enos et al. 1955) . moreover, the cdc would not have been able to isolate and understand the hantavirus (wrobel 1995) and researchers would not have been able to make progress on certain brain tumors . no doubt researchers hoping to understand the impact of radiation leaks on residents near the fukushima nuclear plant in japan will make use of the chernobyl tissue bank established in 1998 to study the effects from (until this point) the world's foremost nuclear plant disaster (http://www. chernobyltissuebank.com). the completion of the human genome sequence (and other genomes) greatly expanded the capacity of science to use and obtain greater value from both previously collected biological specimens and those still to be collected (meslin and quaid 2004) . for example, the international community, led by canadian researchers, was able to rapidly sequence the sars virus from obtained specimens (marra et al. 2003) . others used similar technology for the h1n1 virus (graham et al. 2011; zhang and chen 2009 ), dramatically shortening the time it took to understand the nature of the threat and prepare a public health response. moreover, the prospect of using genome technology on already stored specimens for enhanced genetic diagnostics, drug development, and even domestic and international security threat analysis (meslin 2003; bugl et al. 2007; atlas 2002 ) offers a glimpse into the future of a genetically-informed public health capacity for nation-states. indeed, it is the fortuitous combination of genomics and pharmacology that gives rise to the most promising example of personalized medicine-the field of pharmacogenomics (evans 2006; evans et al. 2004; desta and flockhart 2007) . just as the past benefits to human health from using banked human biological materials stand on their own merit, any future benefits will need to be assessed over time. for us, the important challenge is whether the ethical and legal basis for using banked materials is sufficient to support its expanded use in more areas of public health practice and research. in other words, while we acknowledge that the boundary between the two domains is by no means a stark one; the failure to appreciate what makes them different may prevent productive engagement between these two domains of health care to serve the health interests of society. several explanations have been offered for why public health approaches to health and disease differ from clinical medical approaches, each of which have ethical valence. one theory credits medicine's increasing focus early in the twentieth century on treating the biological causes of disease, and public health's contrasting occupation with the social and environmental causes of illness, resulting in efforts geared toward health promotion and prevention (khoury et al. 2007 ). the vectors of medicine and public health diverged further when schools of medicine and public health in the united states were officially separated in 1916 (khoury et al. 2007 ), in part due to the conflicting goals of professionals in the fields (porter 2006) . additional ideas include ''the rise of medical authority with the expansion of hospital-based specialist practices'' (porter 2006) as well as a corresponding split between individualist and collectivist modes of analysis in the social sciences (arah 2009 ). this disciplinary, professional and institutional dissociation between the two fields has been blamed for the current gap between personal medical care and public health (arah 2009 ). the public health approach presupposes that an exclusive focus on the treatment of individuals is not sufficient to protect, promote and sustain effectively the health of a population. this is evident in the work and writings of public health practitioners such as the sanitarians (susser and susser 1996a) , thomas mckeown (szreter 2002) , geoffrey rose (marmot 2001) , dan e. beauchamp (kass 2004) , marc lappe (kass 2004) , marvin susser (susser and susser 1996b) , ezra susser (march and susser 2006) , norman daniels (kass 2004) , paula braveman (braveman et al. 2004 ) and the world health organization (who) commission on the social determinants of health among numerous others (marmot 2005) . whatever the historical source of the ''schism'' between clinical medicine and public health (khoury et al. 2007 ), the gap between them translates directly into the ethical plane. the individuating drive of personalized medicine could make the breach felt all the more keenly, especially when values of individual and population health conflict. for instance, genomics research has focused on ''individually rare single gene disorders,'' prompting warnings that such investments redirect limited resources from ''efforts to address the social and environmental causes of ill health'' (khoury et al. 2007) . moreover, the challenge of ethical analysis is exacerbated by a disparity in the maturity of ethical frameworks governing medicine and public health. whereas early bioethics scholarship often focused on the individual patient receiving care and to ethical principles supporting this relationship, a similar comprehensive and widelyaccepted ethical framework for public health is yet to be established (nixon and forman 2008; mann 1998; callahan and jennings 2002) . tellingly, nancy e. kass (2004) observes that the language of public health was conspicuously absent among the early bioethicists, despite some achievements with implications for public health ethics. daniel callahan and bruce jennings (2002) likewise point out the focus in bioethics on novel medical technologies in clinical settings at the expense of social and economic inequities. another reason an individualist outlook has prevailed in bioethics is that some public health interventions are conducted on the individual rather than the population level. for instance, postwar antismoking campaigns in great britain set a trend that involved educating and influencing individual behavior and lifestyles (porter 2006) . the approach, later adopted to combat heart disease, obesity and cancer, helped solidify the individualist and behavioral model already prevalent in clinical medicine (beauchamp 1985; porter 2006) . hence, the population perspective implicit in public health ethics was at times at odds with the individualist methods employed to serve the public's health. a further rationale for the individualist bias of hum genet (2011) 130:451-463 453 bioethics is the backlash against the misuse of populationbased policies in the field of eugenics, resulting in an understandable suspicion of collectivist bioethical analysis (pernick 1997; kirkman 2005; lombardo 2011 ). these factors have combined to generate a rich framework for ethical analysis, but one that has remained individualist in orientation. the inadequacy of the framework was noted by bioethicists such as dan e. beauchamp who argued, against the prevailing political valorization of individual autonomy, that a framework that privileged ''individual interests'' and ''market justice'' was detrimental to public health (kass 2004 ). beauchamp suggested that public health might require its own ''ethic,'' a proposal taken up by marc lappé (1986) who differentiated medical ethics from public health ethics. as the new millennium unfolded, several efforts were undertaken to establish frameworks for public health ethics. among these was the american public health association's (apha) adoption of the public health code of ethics in early 2002. the apha was the first national organization to adopt the code (thomas et al. 2002) , which is based on the public health leadership society's principles of the ethical practice of public health. the code is relatively narrow in scope, catering primarily to an audience in traditional public health institutions such as public health departments and schools of public health (thomas et al. 2002) . moreover, it focuses on public health practice rather than research, and has in view the united states' public health system. meanwhile, efforts were underway to mainstream another and more comprehensive ethical framework for public health ethics in the form of human rights. the appeal and promise of human rights as an ethics framework for public health was articulated by the late jonathan mann: given that the major determinants of health status are societal in nature, it seems evident that only a framework that expresses fundamental values in societal terms, and a vocabulary of values that links directly with societal structure and function, can be useful to the work of public health. for this reason, modern human rights, arising entirely outside the health domain, and seeking to articulate the societal level preconditions for human well-being, seems a more useful framework, vocabulary, and template for public health efforts to analyze and respond directly to the societal determinants of health than any framework inherited from the past biomedical or public health tradition. (mann 1998) apart from the capacity of human rights to speak in ''societal terms,'' a crucial part of mann's argument was his identification of the goals of human rights as virtually inseparable from those of health, i.e., human well-being (mann 1997) . although a human rights perspective has the practical advantage over other frameworks of being realized in (mostly international) law, it also benefits from being rooted in an established and fertile ethical vision. human rights can be traced back to the ancient world, but we describe here the prevailing view, which has origins in the writings of such philosophers as hugo grotius, thomas hobbes, jean jacques-rousseau and john locke. modern human rights assume that all persons possess inherent dignity and certain inalienable rights by the simple fact of their being human. the words ''inherent'' and ''inalienable'' mean these things belong to them naturally and are not granted to them by any political authority. to advance their individual and common well-being, however, people give up certain rights to set up a government that serves their needs. a functioning human rights framework is based on the proposition that a government should not take more rights from people than people give to the government in the first place. on this view, the government exists to ensure the well-being of the individuals who give up certain rights in exchange for certain protections and benefits from the government. the same applies to the community they jointly establish. from this analysis, the traditional roles of government include such things as collective security, the administration of justice, the protection of property and, relevant for our purposes, the promotion of the public's health. seen in this way, a human rights perspective provides an ethical framework for describing the conditions under which the government can protect and promote both individual and community well-being. with the onset of the cold war, however, rights that were part of a single ethical vision in the universal declaration of human rights (1948) were gradually split into two categories. the two classes of rights reflected the ideological priorities of the contending sides and were enshrined in two separate treaties in the 1960s. the international covenant on civil and political rights (1966) (iccpr) reflected the capitalist and liberal emphasis on such rights as free speech, freedom of movement, freedom of religion, the right to vote and the right to privacy. these civil and political rights required governments to refrain from interfering with the liberties of their individual citizens. on the other hand, the international covenant on economic, social and cultural rights (1966) (icescr), spearheaded by the communist eastern bloc, focused on such priorities as the right to work, the right to housing, the right to education and the right to health-rights that require governments to take some kind of action for the benefit of the whole society. in part due to their being costlier than civil and political rights and also because of their questionable justiciability (i.e., their enforcement in courts of law) (tarantola 2008) , social and economic rights were not given the same priority as civil and political rights by governments. the main result of this focus on individualist civil and political rights is that many governments have not invested as heavily in addressing issues at societal or population level-issues such as housing, education and health. hence, human rights norms in the twentieth century have developed along broadly individualist rather than collectivist lines. roberto adorno (2009) describes the potential for human rights as a framework for biomedicine and public health in the global context. he notes that ''[a]s our world becomes increasingly interconnected and threats to the global public health continue to proliferate, it is hard to see how the global governance of health could be managed without assigning an integral role to human rights''. the reasons he provides in support of a human rights framework include the fact that much biomedical activity has clear human rights implications (e.g., the rights to life and physical integrity); human rights have developed into a transcultural ethical discourse with the potential for setting common standards; and there are few if any other viable mechanisms that can serve as a ''global normative foundation''. considering the then incipient unesco universal declaration on bioethics and human rights, t. a. faunce (2005) noted the increasing application of human rights to address challenges traditionally considered within the sole purview of bioethics and medical ethics. in the narrower context of genomics, knoppers (2000) has argued that benefit-sharing in the context of genetic research ''is an aspect of fundamental human rights and serves to counterbalance the effects of commercialization and patenting''. she has also proposed human rights as a compelling model for policy governing new genetic technologies (knoppers 2004) . these developments notwithstanding, commentators have been quick to point out the limitations of adopting human rights approach for public health and genome-based medicine. meier and mori (2005) criticize the ''limited, atomized right to health'' contained in the icescr, a provision that establishes neither a robust individual right to health nor an effective means of ensuring public health. similarly, adorno (2009) acknowledges the criticism ''that human rights are conceived as excessively individualist for non-western mentalities and lack a significant concern for personal duties and for the common interest of society''. with particular reference to the field of genomics, iles (1996) points to two specific shortcomings of human rights as an ethical framework, both of which are traceable to the individualist orientation of the current system. his first criticism is that such a framework pays inadequate attention to the structural and social effects of genetic information. he argues that because economic, racial, ethnic and power disparities already exist between groups in societies, genetic information used without ethical oversight can exacerbate these differences and result in discrimination and exclusion. iles infers that human rights may adequately protect individuals facing genetic profiling in employment or insurance contexts, but it is questionable whether the framework's individualist lens can monitor the effects of genetic information on relations between and among groups. iles' second criticism of the applicability of human rights as a foundation for ethical uses of genomics is that individual freedom of choice regarding the use of genetic information can have an aggregate population-wide effect. for example, the choice parents make to have a ''normal'' child rather than one with a ''comparatively inert and tolerable'' disorder is not only heavily influenced by society's values but also determines eventually the society's constitution (iles 1996) . a narrow focus on individual choice, therefore, may obscure the effects of the uses of genetic information on a society. the preceding discussion demonstrates that even human rights as a framework for public health ethics are not immune from the individualist approach that characterized early bioethics. toward the end of the cold war, however, there were renewed efforts to reintegrate the individualist civil and political rights with the community-oriented economic and social rights (meier and fox 2010) . we outline three of these developments below. the first development is the increasing recognition of a category of rights known as ''solidarity'' or third-generation rights (wellman 2000) . the phrase ''third-generation'' distinguishes solidarity rights from the more individualist civil and political rights (''first-generation'' rights) and the more collectivist social, economic and cultural rights (''second-generation'' rights). like the other two generations of rights, solidarity rights were a response to a particular set of problems facing the international community. these included ''securing peace after the first and second world wars, achieving freedom for colonial peoples, reducing the gross economic inequalities between developed and underdeveloped countries, and preserving a healthy environment when the technologies in one nation seriously damage an environment shared by all nations'' (wellman 2000) . solidarity rights, in other words, are aimed at conditions that can be addressed only by global efforts rather than the laws of any single country. the classic examples of solidarity rights are the rights to peace, development, a healthy environment, self-determination, humanitarian intervention, communication and ownership of the common heritage of humankind (wellman 2000; monshipouri et al. 2003) . apart from requiring the concerted efforts of all countries, solidarity rights have two other criteria: first, that the rights belong to peoples (i.e., groups), not just individuals; second, that obligations apply to all actors on the international scene, not just governments. more recently, solidarity has been described as a key ethical foundation for biobanks (chadwick and berg 2001) . from an ethical perspective, solidarity rights complement first-and second-generation rights. whereas firstgeneration rights protect individuals from the abuses of their governments (e.g., no torture or arbitrary arrests), and second-generation rights enable individuals to claim benefits from their governments (e.g., education, housing), solidarity rights recognize that individuals cannot reach their full potential without ''cooperative participation in the social life of the various communities to which they belong'' (wellman 2000) . hence, solidarity rights further establish in human rights the ethical principle that human well-being has a communal dimension that goes beyond an individual citizen's relationship with her government. the second development emphasizing a collectivist approach in human rights is growth in the area of indigenous peoples' rights. the united nations general assembly adopted the declaration on the rights of indigenous peoples in 2007. what makes this declaration unique is that it explicitly recognizes a category of ''collective'' rights. until the declaration's adoption, human rights were concerned primarily with ''the rights of the individual against the state, without much attention to the collective and associational dimensions of human existence beyond the state'' (anaya 2006) . in an historic shift, the declaration recognizes rights to indigenous peoples as groups rather than merely as individual members of their communities. it is a particular instance of the ethical principle underlying solidarity rights, which proposes that community is not an elective component of human well-being. this development, moreover, has significant ethical implications for the involvement of indigenous peoples in research and in access to health benefits, and exemplifies the relevance of indigenous perspectives on genomics research generally (dodson and williamson 1999) . the third and final development pertains to regional human rights instruments. the major global regions are encouraged to adopt their own treaties, thereby customizing global human rights norms to their particular situations for more effective implementation. of particular relevance is the african charter on human and peoples' rights (also known as the banjul charter), which was adopted by the organization of african unity (now the african union) in 1981, and which includes ''a mixture of all three generations of rights'' (shepherd 1985) . as its official title suggests, the banjul charter includes the concept of peoples' rights, which, like the collective rights of indigenous peoples, is a version of group rights. the banjul charter deliberately omits a definition of the term ''people,'' thereby leaving the term open to several interpretations, e.g., persons struggling to gain political independence, persons living in a territory and sharing certain characteristics, or simply all people living in a country (kiwanuka 1988) . whatever their precise legal definition, peoples' rights in the banjul charter are based on the african philosophical belief that a human being is not ''an isolated and abstract individual, but an integral member of a group animated by a spirit of solidarity'' (kiwanuka 1988) . the kinship between this african principle and the ethical norms undergirding solidarity rights and the rights of indigenous peoples discussed above is evident. they all recognize the importance of community to human wellbeing and reject an approach to human rights that focuses exclusively on the individual. these three developments demonstrate how human rights have been finding ways to complement the protection of individual rights with approaches that recognize the ethical importance of community. these attempts to expand the vision of human rights beyond the individual are analogous to the efforts of public health ethicists to develop a population perspective that transcends the clinical encounter between a single patient and her caregiver. this similarity makes the human rights framework a compelling candidate for analyzing the ethics of biobanking and public health. as with early debates in medical ethics and bioethics generally, much of the ethical and legal attention in biobanking has been individualistic, focusing on informed consent (beskow and dean 2008; brekke and sirnes 2006) , privacy protections (chen et al. 2011; evans 2009) , and risks of exploitation, especially in vulnerable populations (lo 2004; bernhardt et al. 2003; dodson and williamson 1999) . important as these topics are, some now believe the time has come to update the ethical/legal dialog about biobanks to accommodate broader social and political perspectives (meslin and cho 2010; kaye 2004; caulfield et al. 2007) . it is against this backdrop that our analysis is set. a human rights approach may offer two advantages over other potential public health ethics frameworks. first, it may avoid having to resolve the seemingly interminable debate about the proper approach to obtaining individual informed consent for research using human biological materials. in situations in which groups may be consulted, approached and from which permission to participate in biobanks may be sought, informed consent may be necessary but not a sufficient mechanism for engaging a community. second, it recognizes the institutionalization and application of human rights discourse at international forums by providing tools for discussing the values of public health across national borders. this is important in light of observations by recent commentators of a linguistic shift with both practical and ethical implications: the gradual transition of the term ''international health'' to ''global health.'' ''international health'' was used to describe a technical endeavor conducted jointly by developing countries and their partners in the industrialized world through such large institutions as the world health organization (who) and care international (elmendorf 2010) . it was useful in this context to distinguish between ''international'' and ''domestic'' health. in contrast, the term ''global health'' reflects an acknowledgment that intensifying interaction between countries through trade and travel renders national borders increasingly immaterial for health challenges (elmendorf 2010) . the shift in terms represents the change from health conceived as an issue for diplomacy and knowledge transfer between countries to health conceived as a common asset and concern of the international community. importantly, the terminological shift from ''international'' to ''global health'' is also reflected in the bioethics literature (chadwick et al. 2011) . a specific example of the application of ''global'' rather than ''international'' health is the ''one world, one health'' initiative, a framework that builds on efforts to contain the avian influenza outbreak (fao et al. 2008) . the initiative is built on the premise that infectious diseases have potentially national, regional and international effects, thus requiring approaches that are not only ''interdisciplinary'' and ''cross-sectoral'' but indeed global. the changes signified by the term ''global health'' have implications for biobanking in many ways (burke et al. 2010) . public health genomics research is becoming ''increasingly international and collaborative'' resulting from the need for larger and more diverse datasets to evaluate genetic differences within groups (ickworth 2010). aided by more robust bioinformatics, genotypic and phenotypic data will be employed with greater frequency to study the significance of genetic variation (mendoza 2010 ). this will involve the use of larger databases and the consolidation of samples from sites around the globe (meslin and goodman 2010; ickworth 2010) . this raises the obvious challenge of harmonizing norms concerning privacy and confidentiality across jurisdictions and, beyond that, consideration of the varied cultural norms guiding data sharing particularly when information moves between developed and lower and middle income countries (lmic) (chalmers 2007; holman et al. 1999; asslaber and zatloukal 2007) . biobanking in the global public health arena is also faced with the challenge of determining research priorities given the different health problems facing populations in developed and lmic. although both regions face the complex diseases of urbanization (e.g., cancer, heart disease, diabetes), environmental factors like climate change and resource scarcity are likely to affect lmic more profoundly than their developed country counterparts. this is especially troubling given that a research imbalance exists between the regions: although african populations are ''the 'root and branch of genetic variability''' the bulk of genomic research is conducted by developed countries and among european populations (ickworth 2010). fortunately, new initiatives such as h3africa may begin to redress this historic injustice (nordling 2011) . these challenges confirm the need for an ethical framework that can be understood and implemented at global forums. s. h. e. harmon (2006) echoes the need for global frameworks ''given the rise of predictive medicine (involving genetic research and clinical genetics), which is driven by private global operators, thereby suggesting a need for regulatory responses which are similarly global''. although a 2003 who report on genetic databases concludes that biobanks are based more on ''communal value'' than on ''individual gain,'' the reality is that the ethics of biobanking has been analyzed predominantly in the traditional individualist bioethical categories of confidentiality, autonomy and informed consent (knoppers and chadwick 2005) . the fact has not been lost on some commentators. garrath williams, for instance, discusses the daunting task of developing ethical principles for large-scale biobanks. he attributes the difficulty in part to an excessive focus on the individual research subject's right to informed consent, an emphasis he finds inconsistent with the inevitably collective nature of large-scale biobanking (williams 2005) . williams maintains that this conceptual incongruity obscures important ethical questions about how research priorities are set and how to accommodate the diverse motives of actors in health care systems. he warns that ignoring analyses that transcend individualist frameworks may, paradoxically, end up harming the interests of individuals (williams 2005) . human rights can make no original contributions to the ethics of biobanking if they are incapable of transcending their individualist biases. the second challenge of a human rights framework for biobanking involves developments in global politics. the observation by knoppers and chadwick (2005) that genetic research has compelled ''a public and therefore a political examination of personal and social values'' illustrates the close connection between politics and ethics in biobanking. therefore, ethical analyses of international biobanking and public health that omit the global political context will likely remain deficient. the developments in global politics that pose the greatest challenge to human rights as an ethical framework for biobanking are efforts, in the context of globalization, to entrench policies that entail an increasing delegation of governmental responsibilities to private actors. in a publication on health and human rights, who (2002) notes that [w]ithin the human rights community, certain trends associated with globalization have raised concern with respect to their effect on states' capacity to ensure the protection of human rights, especially for the most vulnerable members of society. located primarily in the economic-political realm of globalization, these trends include: an increasing reliance upon the free market; a significant growth in the influence of international financial markets and institutions in determining national policies; cutbacks in public sector spending; the privatization of functions previously considered to be the exclusive domain of the state; and the deregulation of a range of activities with a view to facilitating investment and rewarding entrepreneurial initiative. these trends serve to reduce the role of the state in economic affairs, and at the same time increase the role and responsibilities of private (non-state) actors, especially those in corporate business, but also those in civil society. this transfer of responsibilities from governments to private actors is critical because the operation of international law depends both on governments assuming legal obligations by signing agreements and on these governments being held accountable for fulfilling the responsibilities they undertake. generally speaking and despite recent changes in international criminal law, private actors are not accountable under public international law, the branch of international law to which human rights belong (jessberger 2010) . hence, the transfer of governmental responsibilities such as health provision to private actors removes a growing number of issues from the direct supervision of human rights. governments retain the duty to ensure that private actors such as transnational corporations do not violate human rights, but monitoring and enforcing the norms remains a major challenge (gruskin et al. 2007; tarantola 2008) . we conclude this discussion with two examples of key ethical issues raised by the prospect of expanding international biobanking: the first addressing differences in national laws governing biobanks, and the second addressing ethical obligations of transnational corporations operating in lmic. various commentators have discussed the problem for international biobanking arising from the absence of common regulations applying across country borders. the regulatory terrain has been depicted as ''a patchwork of national laws, regulations and ethics advisory body guidelines'' (maschke 2005) , and comparisons have proven ''laborious and defy generalizations'' (helgesson et al. 2007 ). the discrepancies in ethical rules governing such issues as consent and secondary uses raise obvious barriers to the principled collection of tissue samples and the development of personalized medicine. adopting human rights as a public health ethic is not an ideal guide for drafting specific rules governing individual focused biobanking issues such as consent, privacy and secondary uses. however, such an ethic can inform efforts to determine the general principles that should govern the activity of biobanking as a broader societal undertaking. human rights can do this by integrating three concepts: (1) collective rights (from international human rights); (2) global public goods (from economics); and (3) the common heritage of humanity (from international environmental law). we have discussed above the welcome and increasing recognition of community-oriented socio-economic rights as well as solidarity rights in international human rights toward the end of the cold war. we noted also how the change was reflected in the explicit recognition of ''collective'' rights in the 2007 united nations declaration on the rights of indigenous peoples. these rights ''operate at an international level to assure public goods that can only be enjoyed in common with similarly-situated individuals and thus cannot be realized through individual rights claims against the state'' (meier and fox 2010) . the premise grounding the recognition of collective rights is that the realization of some human rights is simply not reducible to their exercise by an aggregate of individuals. harmon (2008) writes that social solidarity has been incorporated, even if implicitly, into unesco's major instruments on genomic research, namely the universal declaration on the human genome and human rights (1997) and the universal declaration on bioethics and human rights (2005) . he maintains that the emergent notion of social solidarity mitigates the excesses of modern individualism and is ''grounded in the recognition that individuals are socially embedded''. his analysis of the unesco documents describes a solidarity based on the fundamental unity of all humans, a focus on ''the collective, the observance of duties and the creation and preservation, through personal and collective action, of a just and decent society''. the notion that the human genome is the ''common heritage of humanity'' has been eloquently defended (knoppers 2005a ), but has not avoided the disquiet among some commentators, some of whom suggest that the human genome be classified as a common resource rather than the common heritage of humanity (spectar 2001; resnik 2005) . developed in the context of international law governing the management of resources in outer space and the high seas, this concept is founded on three basic principles: ''(1) absence of private property rights i.e. the right [usually of governments] to use resources but not to own them; (2) international management of all uses of the common heritage; and (3) sharing of benefits derived from such use'' (white 1982) . also included in the concept is an obligation to use the resource in a peaceful and responsible way, keeping the resource accessible to all and considering the interests of future generations (knoppers 2005a) . in economic terms, a global public good is a good ''for which the cost of extending the service to an additional person is zero and for which it is impossible or expensive to exclude individuals from enjoying'' (nordhaus 2005) . a global public good is marked by two criteria: that the good be non-excludable and non-rivalrous. stated differently, ''[a] good is non-excludable if persons cannot be excluded from accessing it, and non-rivalrous if one person's use of the good does not diminish the supply of that good'' (chadwick and wilson 2004) . a classic example is a lighthouse that lights the sea and which is not diminished in its use by multiple sailors (chadwick and wilson 2004) . other examples include a global positioning system (gps) whose value is not compromised by multiple users, or the eradication of an infectious disease, the benefits of which cannot be diverted from any susceptible persons (nordhaus 2005) . it has been argued that both genetic information (knoppers and fecteau 2003; chadwick and wilson 2004) and public health (meier and fox 2010) should be classified as global public goods in this same way. these three concepts have been integrated by several commentators in efforts to develop ethics frameworks for public health and biobanking. meier and fox (2010) consider public health a public good and make a case for its recognition in international law as a collective right. knoppers (2005a) notes growing support in international normative documents for the human genome to be classified as the common heritage of humanity, and argues, as do chadwick and wilson (2004) , that genetic databases should be considered a global public good (knoppers and fecteau 2003; knoppers 2005b; chadwick and wilson 2004) . the combination of features from all three concepts can provide the basic constituents of a human rights public health ethic for international biobanking. first, collective rights, premised conceptually on the fact that certain rights can be protected only in groups, is virtually analogous to the population perspective of public health, which presumes that certain health challenges require society-wide, rather than individual, interventions. the kinship of the two perspectives is highlighted in the argument made by meier and fox (2010) that public health be recognized as a collective right. second, the classification of genetic databases as the common heritage of humanity, which precludes private ownership while requiring shared uses and benefits, buttresses the view that biobanks should be managed under principles that consider the whole of humanity rather than narrower interests, no matter how seemingly benign. again, these principles would share an affinity with the principles of public health that target the health of the whole population. third, the arguments for the status of genetic information as a non-rivalrous and non-excludable global public good also support an approach to managing biobanks that recognizes the public character of the resource. together, these features ground the management of international biobanking in a framework that keeps foremost the population perspective of public health. biobanking and developments in personalized medicine entail the involvement of private investors. commentators have pointed out the costs associated with this infusion of private funding. they raise concerns that such involvement may influence the type of research, distort the process by restricting the direction of research, prevent collaboration, and restrict the sharing of the raw data generated by the research. it also might prevent the results of the research being disseminated effectively or cause publication bias. most importantly, it may serve to reduce public trust in the research process. some evidence suggests that potential participants may be less willing to engage in research if this is privately funded (as they perceive themselves to be more exposed to potential exploitation) (ickworth 2010). the risks expand significantly when, as projected, biobanking expands globally. most lmics have vulnerable populations and lax to minimal research regulation. but even where lmic governments have the ability to regulate research activity, we have noted above the growing trend under globalization for governments to delegate traditional responsibilities to private actors. this constitutes a major administrative and ethical challenge in the regulation of biobanks because, as a rule, governments rather than private actors assume international obligations (ratner 2001). the situation requires an ethical framework for protecting vulnerable populations living under governments either unwilling or incapable of protecting their interests. in 2005, john ruggie was appointed the united nations special representative of the secretary general (srsg) on business and human rights for an initial term of 2 years. ruggie's primary charge was to clarify the human rights obligations of companies operating internationally and the responsibilities of host governments to regulate such businesses (u.n. comm. on human rights 2005). in extending the srsg's mandate another 3 years in 2008, the human rights council 1 observed that weak national legislation and implementation cannot effectively mitigate the negative impact of globalization on vulnerable economies, fully realize the benefits of globalization or derive maximally the benefits of activities of transnational corporations and other business enterprises and that therefore efforts to bridge governance gaps at the national, regional and international levels are necessary… (u.n. human rights council 2008) the appointment of the srsg underscores the ethical implications of international trade and politics. it also testifies to the potential of human rights as a framework for addressing global governance challenges. the srsg fulfills his mandate through research, consultations and workshops that lead to recommendations, standards and tools for the use of businesses and other stakeholders. in the course of his mandate, the srsg has developed a human rights framework for business in the global economy. the framework (known as the ''un framework'') has three pillars: the duty of governments to protect their citizens from human rights violations by third parties (particularly international businesses); the responsibility of businesses to respect human rights (typically contained in corporate codes of conduct); and the establishment of remedies for people whose human rights have been violated (u.n. spec. rep. of the sec. gen. 2008). the un framework provides a useful tool for helping mitigate the regulatory hazards associated with privatelyfunded biobanking enterprises in lmics. by further clarifying the responsibilities of both host governments and foreign investors, the un framework increases the chances that clear laws regulating biobanking will be passed by lmic governments. effective biobanking governance models (kaye and stranger 2009 ) are necessary if biobanking is to benefit public health as governments remain the primary actors in public heath practice. moreover, by ensuring the availability of remedies for violations, the un framework reduces the incentive of foreign investors to take advantage of weak and/or corrupt governments unwilling to implement existing biobanking regulations. the un framework was endorsed by the human rights council in june 2011, thereby enhancing its credibility as a global ethical standard for regulating international business activity. this endorsement ensures that the un framework will help guarantee that the projected extension of especially privately financed biobanking to lmics will take into account the public health interests of lmic populations. we have taken the view that one of the ethical challenges raised by genomic medicine reflects an enduring problem in public health: the appropriate balancing of individual and collective values, rights and interests. biobanking in the context of public health genomics reflects a unique case study in this classical problem because it must accommodate both individual and community interests (including multiple types of affected communities). while no single ethical-legal framework has been accepted to bridge 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projects 25 questions and answers on health and human rights serendipity, science and a new hantavirus a study of 135 cases of carcinoma in situ of the cervix at the free hospital for women possible origin of current influenza a h1n1 viruses key: cord-016990-ot1wi3xi authors: zaki, sherif r.; paddock, christopher d. title: viral infections of the lung date: 2008 journal: dail and hammar&#x02019;s pulmonary pathology doi: 10.1007/978-0-387-68792-6_11 sha: doc_id: 16990 cord_uid: ot1wi3xi the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states.2,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918–1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the u.s.4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths5 and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states. 2 ,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918-1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the us. 4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths s and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. although viruses are commonly identified causes of pneumonia of infants and young children, they are relatively infrequently recognized as agents of communityacquired pneumonia in adults. 6 in several large series that investigated a microbiologic cause of community-acquired pneumonia, viral etiologies were identified in only 9% to 18% of cases (table 11 .1).7-12 however, it is likely that viral pneumonias are underrecognized and underdiagnosed for various reasons. although some agents may cause distinct cytopathology or inclusions (e.g., adenoviruses, herpesviruses, and paramyxoviruses), many important pathogens (e.g., influenza viruses) do not, and none of these agents are resolved specifically in tissue by routine histologic stains. viruses require live cells for cultivation, and are generally more difficult than bacteria to isolate from clinical samples. for some viral pneumonias, 426 the pathogen appears to initiate a cascade of destructive host responses that continue or progress even in the absence of the specific infectious agent, and in these patients the etiologic agent may be absent from host tissues at the time of autopsy.13 thirty to sixty percent of community-acquired pneumonias are etiologically undetermined, 14 and it is entirely possible that viruses directly cause more episodes of pneumonia than currently appreciated. because viral infections of the lower respiratory tract often precede bacterial pneumonias, viruses may indirectly exert considerable influence on the cumulative morbidity and mortality of infectious pneumonias. 15 , 16 the mechanisms by which viruses may facilitate bacterial invasion of the respiratory tract are complex and varied. certain viruses cause the death of ciliated respiratory epithelium and thereby disrupt normal ciliary activity. viruses may also inhibit the phagocytic or bactericidal activities of neutrophils, t lymphocytes, and alveolar macrophages, and predispose the host to secondary infections. certain gram-positive and gram-negative bacteria adhere to and colonize virus-infected epithelium more readily than to noninfected cells by various hypothetical mechanisms, including alteration and induction of receptors at the host-cell surface and changes in the extracellular environmenty-19 finally, viral infections of the lung may exacerbate noninfectious pulmonary conditions (e.g., asthma and chronic obstructive pulmonary disease) and indirectly contribute to aggregate morbidity and mortality associated with these conditions. 20 although influenza viruses remain the most frequently identified cause of viral pneumonia in adults (table 11 .1), the diversity of agents identified as causes of viral pneumonias has expanded considerably. several newly recognized viral pneumonias have been identified since 1992 that are among the most feared and lethal of all emerging infections, including those caused by hantaviruses, nipah virus, and sars corona virus (co v). !3,21-23 certain causes "rubella (2), rhinovirus (1) , mixed viral infections (3) . source: adapted from greenberg. 6 of viral pneumonia, particularly those that occur in vulnerable patient cohorts, have diminished during this same interval. by example, the u.s. incidence of varicella pneumonia has dropped by two thirds since universal childhood vaccination for varicella was implemented in 1995, 24, 25 and advances in the clinical management of transplant recipients have reduced the incidence of cytomegalovirus (cmv) pneumonia?6 also occurring during the last decade has been the development and use of powerful molecular techniques that have unveiled the identity of historic pathogens (e.g., the hini "spanish" influenza a virus),27.28 and have facilitated the rapid characterization of emerging agents (e.g., sars-co v). 13 it should be noted that the disease manifestations of several of these agents (e.g., human parainfluenza virus [hpiv] , respiratory syncytial virus [rsv] , and influenza) are often confined to the upper airway and are not invariaorb rsv hpiv adeno cmv varicella other' 10 0 3 0 0 3 27 1 4 3 0 0 0 7 2 0 0 1 0 19 5 5 0 0 1 7 8 13 15 0 0 0 37 5 11 5 0 0 3 21 (11) 32 (16) 30 (15) 1 (0.5) 1 (0.5) 6 (3) ably associated with pneumonia. with some of these pathogens (e.g., influenza viruses, rsv, hantaviruses, and hpiv viruses) respiratory disease is the primary manifestation. for other agents, such as measles, nipah virus, and herpesviruses, typically the lungs are involved as part of a multisystem syndrome. the diagnosis of viral pneumonia, suspected by patient history and clinical manifestations, also can be supported histopathologically, and the general pattern of histopathologic lesions may suggest a specific diagnosis. many viruses can be identified in lung by examining the tissue response and cytopathic changes. some of these viruses cause recognizable tissue reaction patterns including necrotizing tracheobronchitis, bronchiolitis, and interstitial pneumonia. a summary of the key diagnostic histopathologic and ultrastructural features for the most common viral pathogens that cause a majority of pulmonary infections is provided in tables 11.2 and 11.3. yes (cytoplasmic; ill-defined) yes (nuclear and cytoplasmic) pulmonary tissue reaction necrotizing bronchiolitis; smudge cells; dad severe edema, early dad interstitial pneumonia; dad; occasional multinucleation interstitial pneumonia; dad; cytomegaly dad; necrosis and rare multinucleation dad; necrosis and rare multinucleation dad; necrotizing bronchiolitis interstitial pneumonia with multi nucleation dad dad; interstitial pneumonia with occasional multinucleation necrotizing bronchiolitis, interstitial pneumonia with occasional multinucleation recently recognized; human pathology not well described interstitial pneumonia with multinucleation; dad only certain viruses can cause cytopathic changes that are sufficiently distinct to enable the pathologist to recognize a specific diagnosis on routine histologic examination of lung specimens. with the availability of special figure 11 .1. negative stain electron microscopic images of different viruses that can cause pulmonary infections. a. adenovirus. adenoviruses are protein-shelled icosahedral-shaped nonenveloped viruses that measure approximately 70 to 90nm in diameter. two of the viruses are stain penetrated revealing the dna-containing nucleoprotein. b. sin nombre virus. sin nombre virus, the causative agent of hantavirus pulmonary syndrome, belongs to the genus hantavirus in the family bunyaviridae. the envelopes of hantaviruses are checkerboard in appearance, and particles measure 90 to 150nm in diameter. c. herpes simplex virus. the stain has penetrated the envelope of several of these herpesvirus particles, delineating the icosahedral-shaped nucleocapsids, which measure 90 to 100nm in diameter. d. cytomegalovirus. another herpesvirus; one virus particle (left) is intact while two nearby particles are stainpenetrated and show the viral nucleocapsids. the nucleocapsid of the upper right particle shows a central core that harbors the dna of the virus. e. influenza virus. influenzaviruses belong to the family orthomyxoviridae; viral particles are pleomorphic s.r. zaki and cd. paddock diagnostic techniques, such as immunohistochemistry (ihc) and in-situ hybridization (ish), many viruses can be detected in formalin-fixed, paraffin-embedded tissue samples even if specific viral inclusions cannot be found in histologic examination of tissue sections. among the techniques, ihc utilizing specific antibodies can be routinely performed on formalin-fixed tissue and can enhance the pathologist's accuracy in identifying organisms in tissue specimens. in addition to histologic pattern recognition, ihc, and ish in tissue, several other diagnostic tests are available to aid the pathologist. cell culture techniques, serology, polymerase chain reaction (pcr), and electron microscopy (em) all play vital roles in the diagnosis of these infections. while histologic techniques can be an excellent means of demonstrating organisms, tissue culture isolation remains essential for definitive identification of the virus. when a viral pneumonia is suspected, samples of lung tissues should be evaluated by cell culture, which has the advantage of being a nonbiased method for screening purposes that does not rely on the availability of specific antibodies or probes. electron microscopy offers the same utility as a broad scope diagnostic tool and has been especially critical in outbreaks of unknown etiology. it played a critical role during the hendra and nipah virus outbreaks in 1994 and 1999, respectively, and more recently, in the early recognition of a novel coronavirus associated with sars in 2003 and in the diagnosis of emerging transplant-associated infections. 13.22.29-33 the advantage of this approach is that viral particles may be demonstrated by negative stain or thin section em, either directly in clinical material or after amplification in cell culture. like culture, em is not limited by narrow specificity of reagents or by prior clinical bias ( fig. 11 .1). and can be filamentous or spherical in shape. the evenly spaced spikes cover the entire virus surface and contain both the hemagglutinin and neuraminidase surface glycoproteins. f. human metapneumovirus. these paramyxoviruses are heterogeneous in size and shape, and range in size from 150nm to 1 j.lm in diameter. g. parainfluenza virus. another paramyxovirus, the viral nucleocapsid, with its typical herringbone appearance, can be seen both within the stain-penetrated particle as well as partially extruded from the virion. an important feature that can help distinguish between paramyxovirinae (parainfluenza and measles viruses) and pneumovirinae (human metapneumovirus and respiratory syncytial virus) is the diameter of the nucleocapsids, which measure 18nm and 14nm, respectively. h. severe acute respiratory syndrome (sars) coronavirus. these 80-to 100-nm particles are named for the characteristic crown-like fringe on the surface. scale bars, 100nm. (a,b,d,f,h: courtesy of c. humphrey; c,g: courtesy of e. palmer; e: courtesy of ea. murphy, all at centers for disease control and prevention, atlanta, ga.) adenoviruses were first cultured and identified during the early 1950s by investigators searching for etiologic agents of acute respiratory infections. the initial adenovirus isolate was made serendipitously from adenoid tissues obtained from children during efforts to establish a primary human adenoid cell line. 34 a related virus was identified the following year by investigators studying respiratory disease in military recruits?5 these agents were subsequently named adenoviruses after the original source of tissue from which the prototype strain was identified. 36 adenoviruses are non enveloped viruses with a single, linear, double-stranded dna genome that is contained within an icosahedral capsid that measures 70 to 90nm in diameter (fig. ilia) . the capsid is comprised of seven known polypeptides, including the hexon capsomere, which contains group-specific antigenic determinants. 37 adenoviruses are a ubiquitous and diverse group of viruses found naturally in the upper respiratory tracts and gastrointestinal systems of humans, other mammals, and birds. most adenoviruses infect mucosal epithelium, although some pathogens of animals are trophic for endothelial cells, and endothelial infection has been identified in some immunocompromised humans. 38 adenoviruses are represented by at least 51 serotypes on the basis of resistance to neutralization by antisera to other known adenovirus serotypes, and comprise six subgroups or subgenera (a through f) that are distinguished by differential hemagglutination with erythrocytes from various animal species. 37 ,39ao more than 50% of the known adenovirus serotypes are associated with human diseases of the upper and lower respiratory tract, conjunctiva, urinary tract, intestine, and occasionally heart, liver, and central nervous system. the others are rarely encountered and mayor may not act as pathogens in recognizable disease. 37 it is estimated that approximately 5% to 10% of all pneumonias in infants and young children are caused by adenoviruses. 41 ,42 most pediatric cases of adenovirus pneumonia occur between 6 months and 5 years of age, and serotypes 3, 7, and 21 (all members of the b subgenus), are the most common causes of pneumonia in this patient cohort. 43 -45 serotypes 3 and 7 are particularly pathogenic adenoviruses that can cause disseminated and often fatal disease in previously healthy children. 46 in adults, pneumonia is generally associated with serotypes 3,4, and 7.47 periodic epidemics of adenovirus pneumonia in young adults have been identified, particularly among military recruits. 48 ,49 in a manner similar to other pathogens, adenoviruses take advantage of impaired or destroyed immune systems to establish persistent and disseminated infections in immunocompromised hosts. in this patient cohort, the s.r. zaki and cd. paddock case fatality rate of adenoviral pneumonia approaches 60%, compared with an approximately 15% mortality in immunocompetent patients. 37 immunocompromised patients are also susceptible to a broader range of different adenovirus serotypes. by example, the commonly recognized serotypes in normal children account for only about 50% of the adenovirus serotypes reported for children with congenital immune deficiencies. 37 .4 6 because some adenoviruses establish latency in lymphoid tissues and the kidneys of their host, it is believed that many, possibly most, cases of clinical disease caused by adenoviruses in immunocompromised patients are reactivated infections. 37 the lungs of patients with adenovirus pneumonia are typically heavy and edematous, and the bronchi are generally filled with mucoid, fibrinous, or purulent exudates. the histopathologic findings ( fig. 11 .2) include necrotizing bronchitis and bronchiolitis with extensive denudation ofthe surface epithelium,particularly in medium-sized (1 to 2mm in diameter) intrapulmonary bronchi ( fig. 11 .2a). affected airways may be occluded by homogeneous eosinophilic material, mixed inflammatory cells, detached epithelium, and cellular debris. the lamina propria of bronchi and bronchioles is typically congested and infiltrated by predominantly mononuclear inflammatory cell infiltrates. bronchial serous and mucous glands are also often involved and show necrosis and mixed infiltrates. 5o as the infection progresses, there is involvement of the pulmonary parenchyma, forming bronchocentric necrosis with hemorrhage, neutrophilic and mononuclear cell infiltrates, and karyorrhexis. these findings generally occur against a background of exudative diffuse alveolar damage, with filling of the air space by macrophages, fibrin, and detached pneumocytes, and hyaline membrane formation. 51 patients with fatal pneumonia may develop disseminated intravascular coagulopathy and demonstrate fibrin thrombi in vessels of the lungs, kidney, heart, adrenals, and central nervous system (see fig. 4 .20 in chapter 4). 48 adenoviruses form intranuclear inclusions in respiratory epithelial cells of the trachea, bronchi, and bronchioles, in the acinar cells of bronchial glands, and in alveolar pneumocytes, and are generally most abundant at the viable edges of necrotic foci. by using the hematoxylin and eosin (h&e) stain, early inclusions appear as small, dense, amphophilic structures surrounded by a cleared zone and peripherally marginated chromatin, similar to herpetic inclusions. as the cellular infection progresses, the inclusion becomes larger (as large as 14/-lm in some cells) and more basophilic, and the margins of the nuclear membrane become blurred to form the characteristic "smudge cell" (fig. 11 .2b,c).5o,51 tracheal aspirates of patients with adenovirus pneumonia may show distinctive features on cytologic preparations that include cells with fine strands of chromatin that radiate from a central inclusion to the marginated chromatin at the nuclear membrane ("rosette cells"), and cells with foamy, "honeycomb" nuclei, as well as typical smudge cells (see fig. 7 .45 in chapter 7). 52 various methods can be used to diagnose adenovirus infections that include antigen detection, cell culture, electron microscopy, molecular assays, and serology. direct detection techniques that identify the common group-reactive hexon antigen in tissues or body fluids include fluorescence antibody assays and enzyme immunoassays.53 immunohistochemistry staining methods have been used successfully to detect adenovirus-infected cells in formalin-fixed, paraffinembedded tissues using various commercially available, adenovirus group-specific antibodies ( fig.11 .2e,f).38,51 electron microscopy of adenovirus-infected tissues reveals a paracrystalline array of virions ( fig. 11 .2d ). 54, 55 most adenoviruses can be isolated in cell culture from bronchial washings, tracheal aspirates, or lung biopsy specimens during the early stage of the illness and grow well in various cell lines, including human embryonic kidney, hela, and hep-2 cells. 47 cell cultures infected with adenoviruses exhibit a relatively characteristic cytopathologic effect, described as a "cluster of grapes," within 3 to 5 days after inoculation. serotyping of the isolate is accomplished by using hemagglutination inhibition and neutralization tests with hyperimmune type-specific animal antisera. 56 molecular assays, particularly gene amplification using pcr, and ish methods, have been developed to detect adenovirus nucleic acid in respiratory secretions and in formalin-fixed, paraffin-embedded tissues. 51 ,57,58 broad-range, sensitive assays that can detect any adenovirus amplify common genomic sequences (e.g., the hexon gene region). other more specific assays detect specific adenovirus types with unique genomic sequences. 59 serologic assays include tests for groupspecific antibodies (e,g., complement fixation and enzyme immunoassays), or type-specific antibodies (e.g., neutralization and hemagglutination-inhibition assays). pitfalls associated with serologic testing for adenoviruses include occasional rises in heterotypic antibodies when typespecific assays are used, and relatively low sensitivity demonstrated by complement fixation assays. 59 human hantaviral diseases are caused by a group of closely related, trisegmented, negative-sense rna viruses of the genus hantavirus, of the family bunyaviridae. 60 -62 members of the genus hantavirus have similar morphologic features. 63 ,64 virus particles are 70 to 130nm in diameter and generally appear spherical to ovoid, although pleomorphic forms may be seen. a lipid envelope containing glycoprotein spikes surrounds a core consisting of the genome and its associated proteins (nucleocapsids) s.r. zaki and cd. paddock arranged in delicate tangles of filaments showing occasional granulation. the presence of characteristic inclusion bodies in thin section electron microscopy and a unique grid-like pattern on negative-stain electron microscopy differentiate these viruses from other members of the family bunyaviridae ( fig. 11.1b) . 65, 66 the severity and disease type largely depends on the viral serotype. two categories of hantavirus-associated illnesses are described: hemorrhagic fever with renal syndrome (hfrs) for disease in which the kidneys are primarily involved, and hantavirus pulmonary syndrome (hps) for disease in which the lungs are primarily involved. 67 -69 the isolation of the first recognized hantavirus (hantaan virus, named for the river in south korea), and its subsequent identification as the causative agent of hfrs was reported in 1978. 70 in 1993, the deaths of several previously healthy individuals due to a rapidly progressive respiratory disease in the southwestern united states were etiologically linked to a previously unrecognized hantavirus. clinically, the disease differs from hfrs in its pronounced pulmonary involvement and higher mortality rates and is known as hps. 23.69,71.72 hantavirus-associated diseases primarily affect blood vessels and result in different degrees of generalized capillary dilatation and edema. 73 in contrast to severe hfrs where abundant protein-rich, gelatinous retroperitoneal edema fluid is found, all hps patients have large bilateral pleural effusions and heavy edematous lungs. 7 in fatal far eastern hfrs, a distinctive triad of hemorrhagic necrosis can be seen in the renal medullary junctional zone, cardiac right atrium, and anterior pituitary. 75, 78 however, in patients with hps, hemorrhages are rare, and ischemic necrotic lesions, except those attributed to shock, are not seen. 72, 74 histologically, morphologic changes of the endothelium are uncommon but, when seen, consist of prominent and swollen endothelial cells. vascular thrombi and endothelial cell necrosis are rare. in hfrs, the most severe and characteristic microscopic lesions involve the kidney; however, an interstitial pneumonitis can also be seen in some fatal cases. in contrast, the microscopic changes of north and south american hps are principally seen in the lung and spleen. 72 ,74 the lungs show a mild to moderate interstitial pneumonitis characterized by variable degrees of edema and an interstitial mononuclear cell infiltrate comprised of a mixture of small and enlarged mononuclear cells with the appearance of immunoblasts ( fig.11 .3a). focal hyaline membranes composed of condensed proteinaceous intraalveolar edema fluid, fibrin, and variable numbers of inflammatory cells are observed ( fig. 11 .3b). typically, neutrophils are scanty and the alveolar pneumocytes are intact with no evidence of cellular debris, nuclear fragmentation, or hyperplasia. in fatal cases, with a prolonged survival interval, tissues show features more characteristic of the exudative and proliferative stages of diffuse alveolar damage. lung biopsies taken from patients who survive their illness appear similar with proliferated reparative type ii pneumocytes, severe edematous and fibroblastic thickening of the alveolar septa, and severe air-space disorganization with distorted lung architecture (see chapter 4) . other characteristic microscopic findings in hps cases include variable numbers of immunoblasts within the splenic red pulp and periarteriolar white pulp, lymph nodal paracortical zones, hepatic portal triads, and peripheral blood ( fig. 11.3d,e) . similarly, in severe hfrs cases, large mononuclear cells can be present in the spleen, lymph nodes, blood, and hepatic portal triads. 75 . 76 electron microscopic studies of hps lung tissue demonstrate infection of endothelial cells and macrophages. 69 . 72 the virus or virus-like particles observed are infrequent and extremely difficult to identify in autopsy tissues because of the considerable degree of viral pleomorphism and the postmortem deterioration of tissues. however, typical hantaviral inclusions are seen more frequently and their identity can be confirmed by immunolabeling ( fig. 11 .3f,g). similar inclusions are observed in epithelial cells in hfrs and are considered to be ultrastructural markers of hantavirus-infected cells. 63 .79.8o using immunohistochemistry, viral antigens are found primarily within capillary endothelium throughout various tissues in both hps and hfrs. in hps, marked accumulations of hantaviral antigens are in the pulmonary microvasculature and in splenic and lymph nodal follicular dendritic cells (fig. 11 .3c).72 despite the extensive endothelial cell accumulations of hantaviral antigens, there is little ultrastructural evidence of cytopathic effect. hantavirus pulmonary syndrome should be suspected in cases of adult respiratory distress syndrome (ards) without a known precipitating cause among previously healthy individuals. the level of suspicion should be particularly high when patients have a known exposure to rodents in areas where peromyscus maniculatus or other reservoirs of hantavirus are found. physicians need to differentiate hps from other common acute respiratory diseases, such as pneumococcal pneumonia, influenza virus, and unexplained ards. the diagnosis of hps, suspected by patient history and clinical manifestations, can also be supported histopathologically. although there is no single pathognomonic lesion that would permit certain histopathologic diagnosis of hps, the overall constellation of histopathologic hematologic findings suggests the diagnosis.72.7 4 diseases that need to be distinguished pathologically from hps include a relatively large number of different viral, rickettsial, and bacterial infections, as well as various noninfectious disease processes. virus-specific diagnosis and confirmation can be achieved through serology, pcr for hantavirus rna, or ihc for hantaviral antigens. 2 1,72 serologic testing can detect hantavirus-specific immunoglobulin m or rising s.r. zaki and cd. paddock titers of immunoglobulin g in patient sera and is considered the method of choice for laboratory confirmation of hps. immunofluorescent assays and enzyme-linked immunosorbent assays (elisas), which demonstrate the presence of specific antihantaviral antibodies, are currently used as rapid diagnostic tests and provide results within a few hours. recently, synthetic hantaviral nucleocapsid proteins have been used to improve the sensitivity and specificity of serologic assays. these proteins are more available than inactivated hantaviral antigens. 81 . 82 polymerase chain reaction detects viral rna in blood and tissues and is extremely useful for diagnostic and epidemiologic purposes. hantaviral rna can also be detected in formalin-fixed, paraffin-embedded archival tissue by reverse-transcriptase (rt)-pcr.83 immunohistochemistry testing of formalin-fixed tissues can be used to detect hantavirus antigens, and is a sensitive method to confirm hantaviral infections. 72 it has a unique role in the diagnosis of fatal hps cases when serum samples and frozen tissues are unavailable but formalinfixed autopsy tissues are obtainable. 84 • 85 severe acute respiratory syndrome the causative agent of sars is an enveloped, positivestranded rna virus that is a member of the genus coronavirus, of the family coronaviridae. corona viruses have the largest genomes of all rna viruses and replicate by a unique mechanism that results in a high frequency of recombination. maturation of sars coronavirus (sars-co v) is similar to features previously described for other coronaviruses. 8 6-9 0virions form by alignment of the helical nucleocapsids along the membranes of the endoplasmic reticulum or golgi complex and acquire an envelope by budding into the cisternae. the cellular vesicles become filled with virions and progress to the cell surface for release of the virus particles; large numbers of particles remain adherent to the plasma membrane at the cell surface. severe acute respiratory syndrome was recognized during a global outbreak of severe pneumonia that began in late 2002 in guangdong province, china, and gained prominence in early 2003 as cases were identified in more than two dozen countries in asia, europe, north america, and south america. the disease causes an influenza-like illness with fever, cough, dyspnea, and headache, and in severe cases it can cause death in humans. person-toperson transmission, combined with international travel of infected persons, accelerated the worldwide spread of the illness. 5 • 13 . 91 several reports have described diffuse alveolar damage with various levels of progression and severity as the main histopathologic findings in sars patients ( fig. 11 .4a,b)y·92-98 lungs typically show changes described for the proliferative phase of diffuse alveolar damage, with hyaline-membrane formation, desquamation of epithelial cells, fibrin deposit in the alveolar space, and hyperplasia of type 2 pneumocytes. increased mononuclear infiltrate in the interstitium can be seen in some cases. other findings identified in some patients included focal intra alveolar hemorrhage, necrotic inflammatory debris in small airways, and organizing pneumonia. in addition, multinucleated syncytial cells may be seen in the intraalveolar spaces of some patients who died 14 days or more after onset of illness ( fig. 1l .4c). infection with some coronaviruses, including sars-co v, is known to induce cell fusion in culture producing syncytial cells similar to those sometimes observed in lungs of patients who die from sars. these cells contain abundant vacuolated cytoplasm with cleaved and convoluted nuclei, but without obvious intranuclear or intracytoplasmic viral inclusions. the ish and ihc studies of tissues from sars patients have identified corona virus infection of upper airway bronchiolar epithelium ( fig. 11 .4d_f).92,99-101 infected ciliated columnar epithelial cells can be seen focally in lining epithelium of trachea and larger bronchi ( fig. 11 .4e). many of these infected cells slough from the epithelium and can be observed by using ish within the bronchial lumen. abundant viral antigens can also be found distributed focally in parenchyma of lungs of some patients and are seen predominantly in the cytoplasm of pneumocytes, in occasional macrophages, and in association with intra alveolar necrotic debris and fibrin (fig. ll.4d). double-stain studies indicate that most sars-co v-infected cells are type 2 pneumocytes. double-stain studies also detected viral nucleic acids with a distribution similar to that seen in ihc studies, mainly in pneumocytes and in some macrophages.looelectron microscopic examination of lung tissues selected from areas with abundant ihc staining shows numerous coronavirus particles and nucleocapsid inclusions. virions are seen in cytoplasmic vesicles and along the cell membranes of pneumocytes, in phagosomes of macrophages, and associated with fibrin in alveolar spaces (fig.ll.4g-i). because corona virus particles may be confused morphologically with other non viral cellular components, definitive ultrastructural identification can be achieved by using immunogold labeling electron microscopy. the primary histopathologic lesions seen in the lungs of patients who die from sars are somewhat nonspecific and can also be seen in acute lung injury cases caused by infectious agents, trauma, drugs, or toxic chemicals. 102 multinucleated syncytial cells similar to those seen in some sars patients can also be found in a number of virus infections, including measles, parainfluenza viruses, rsv, and nipah virus infections. 102 -104 in an early study of four human sars patients,13 viral antigens were not detected in the lung by ihe. the most likely explanation is that all patients in the study had a clinical course aver-s.r. zaki and cd. paddock aging more than 2 weeks. for many virus infections, viral antigens and nucleic acids are cleared within 2 weeks of disease onset by the host immune response. it is also possible that the pulmonary damage associated with sars is not caused directly by the virus, but represents a secondary effect of cytokines or other factors induced by the virus infection. similarly, in influenza virus infections, viral antigens are seen predominantly in respiratory epithelial cells of large airways and are only rarely identified in pulmonary parenchyma despite concomitant and occasionally severe interstitial pneumonitis. !os in recent reports by shieh et al. 100 and chong et al.,92 the temporal relationship between the duration of illness and clearance of sars-co v in human lung tissue was examined. viral antigens and nucleic acids were detected only in pulmonary tissues of patients who died early in the disease. the development of specific ihc, ish, and immunoelectron microscopy (iem) assays to identify sars-co v in formalin-fixed, paraffin-embedded samples has facilitated the assessment of the cellular tropism of sars-co v infection in human lung tissues. localization of sars-co v in the lung occurs mainly in the cytoplasm of pneumocytes, primarily type 2, and occasionally in alveolar macrophages ( fig. ll.4f ). type 2 pneumocytes are known to secrete pulmonary surfactant, resulting in reduced surface tension and preservation of the integrity of the alveolar space. these cells also play an important role in tissue restitution following lung damage. moreover, there is mounting evidence to support their contribution to the development of acute inflammatory lung injury following exposure to biological or chemical agents. additional studies are needed to further define the role of type 2 pneumocytes and alveolar macrophages in sars-co v infection. cynomolgus macaques inoculated with sars-cov develop pathologic findings of pneumonia and have been proposed as an animal model. 106 haagmans et a1. 107 showed extensive sars-co v antigen expression in experimentally infected macaques 4 days after infection. the antigens were mainly in alveolar lining epithelial cells with morphologic characteristics of type 1 pneumocytes, indicating type 1 pneumocytes are the primary target for sars-co v infection early in the disease. type 1 pneumocytes normally represent 90% of the alveolar epithelial cell volume and are easily damaged during pulmonary infections or other types of injury. in a more recent study on nonhuman primates,108 evidence of infection of type 1 pneumocytes in addition to some type 2 pneumocytes and macrophages was found. small animal models, such as rodents, would be very useful for evaluating vaccines, immunotherapies, and antiviral drugs, and we have recently identified the mouse as an animal model for this purpose. 10 9 in those studies, microscopic examination of trachea, bronchus, lung, thymus, and heart on day 2 after infection revealed mild and focal peri bronchiolar mononuclear inflammatory infiltrates with no significant histopathologic change in other organs. viral antigens and nucleic acids were focally distributed in bronchiolar epithelial cells, and virions were found in these same areas by ultrastructural analysis. data suggest that sars-co v replicates in mice to a titer sufficient to evaluate vaccines and antiviral agents. the mouse and other small animal models l1o might also be used to test the ability of the virus to replicate and cause disease and facilitate identification of host-immune mechanisms that contribute to the resolution of sars-co v infection. cytomegaloviruses (cmv) comprise a distinct and ancient group of herpesviruses that are widely distributed in nature, share similar growth characteristics in cell culture, and cause cellular enlargement and form distinctive inclusions in infected cells. these cytopathic changes, identified by early pathologists in the salivary glands of children dying from various unrelated diseases,111-113 led to the early designation of cytomegalic inclusion disease many years before the causative agent was isolated in the mid-1950s. the name cytomegalovirus was proposed in 1960 to reflect the cytopathic changes caused by these viruses. 114 cytomegaloviruses are highly host-specific, and various mammalian hosts, including nonhuman primates, rodents, and domesticated animals, are infected with their own distinct cmy. in this context, human cmv is stringently species-specific and, with rare exception, only infects cells of human origin. ll5 several cell types are permissive for cmv replication, including alveolar pneumocytes, vascular endothelium, fibroblasts, monocytes, dendritic cells, and exocrine and endocrine glandular epithelial cells.ll6 cytomegalovirus is a ~-herpesvirus with the largest genome (230 kilobase pair [kbp]) of all the herpesviruses known to infect humans. the double-stranded linear dna genome is contained within a 90 to 100nm icosahedral capsid and is surrounded by an amorphous material known as the tegument. these components are enclosed in a lipid bilayer envelope that is derived from the host cell nuclear or golgi membranes and contains several vir ally encoded glycoproteins necessary for infection of other cells. mature enveloped virions range from 150 to 200nm, making cmv one of the largest viruses that infect humans (fig. i1.1d ).ll7 the structure of cmv is typical of other human herpesviruses, but demonstrates some subtle ultrastructural differences from other viruses in this group including greater pleomorphism of the lipid envelope and dense body inclusions in the cytoplasm of infected cells.ll8 437 cytomegalovirus is a ubiquitous human pathogen, and in north america infects approximately 50% to 90% of the population.ll7 most of these infections are inapparent, although some cases of primary infection in otherwise healthy individuals result in a self-limited mononucleosis syndrome similar to that caused by epstein-barr virus; it is estimated that 20% to 50% of cases of heterophile-negative mononucleosis, and 8% of all cases of mononucleosis, are caused by cmy' 119 pulmonary involvement in cmv mononucleosis is infrequent and occurs in approximately 6% of these casesyo congenitally acquired cmv infection has various deleterious effects on the fetus, including mental retardation, neurologic abnormalities, sensorineural hearing loss, and retinitis, and in one series pulmonary involvement occurred in 64 % of symptomatic infants. 121 like all herpesviruses, cmv remains with its host for life after primary infection and establishes latency in various cell types, including vascular endothelial cells, monocytes and macrophages, neutrophils, and renal and pulmonary epithelial cells. 122 activation of viral replication occurs in persons with severely compromised immunity. patients with advanced hiv disease and recipients of hematopoietic stem cell or lung transplants are particularly at risk of developing cmv pneumonia. before the use of cmv screening and effective antiviral prophylaxis regimens, 10% to 30% of all patients undergoing allogeneic bone marrow transplantation for leukemia, and 15% to 55% of solid organ transplants, developed cmv pneumonia with case fatality rates of greater than 80% in some series. 26 ,123,124 the relatively high frequency of cmv pneumonia in lung transplant recipients may be a correlate of animal model data that indicate the lungs are a major site of latent cmv infection. 125 before the use of ganciclovir as therapy for cmv disease in aids patients, the case fatality rate for cmv pneumonia in this patient cohort was 75% when cmv was the only pathogen identified. mixed infections with cmv and another pathogen had an even worse prognosis, and the case fatality rate for patients with pulmonary disease caused by cmv and pneumocystis jiroveci (formerly carinii) was 92%. 126 cytomegalovirus pneumonia can show various histopathologic patterns (fig. 11 .5). extensive intra alveolar hemorrhage with scattered cytomegalic cells and relatively few inflammatory cell infiltrates may occur ( fig. 11 .5a).127 in a similar manner, extensive involvement of the alveolar epithelium with minimal inflammation or overt evidence of parenchymal injury has also been described. 128 other patterns include multifocal lesions with mixed inflammatory cell infiltrates, hemorrhage, necrosis, and cytomegalic cells, or a diffuse, predominantly mononuclear cell infiltrate, interstitial pneumonitis with intraalveolar edema and fibrin deposition, and diffusely distributed cytomegalic cells (fig. 11 .5e).129-131 the cytomegalic changes of cmv-infected cells are evident by standard h&e staining and are virtually pathognomonic of active cmv infection. the cells are enlarged (25 to 40!j,m) and contain amphophilic to deeply basophilic intranuclear and intracytoplasmic inclusions ( fig. 11 .5b,e). the single intranuclear inclusion is composed of viral nucleoprotein and assembled caps ids, and is a large (up to 20 !j,m), round to ovoid body with a smoothly contoured border that is generally surrounded by a clear halo that gives the inclusion a distinctive owl'seye appearance. the host cell nucleolus is often retained in the inclusion. 131 cytoplasmic inclusions are small (1 to 3!j,m), granular bodies that appear after the intranuclear inclusion is well developed and are not uniformly present in all cmv-infected cells (fig. 11 .5b). these inclusions represent a mixture of virions and various cellular organelles, and increase in size and number as the infection progresses. 132 unlike the intranuclear inclusion, the cytoplasmic inclusions stain with periodic acid-schiff stain and are deeply argyrophilic with gomori's methenamine silver stain.!33 cytomegalovirus pneumonia is defined by the presence of signs or symptoms of pulmonary disease combined with the detection of cmv in bronchoalveolar lavage (bal) fluid or lung tissue samples. in this context, detection methods that support this definition include virus isolation, histopathologic observation of cytomegalic cells, ish, or ihc stains (fig. 11 .5f). detection by pcr alone is considered too sensitive for the diagnosis of cmv pneumonia and is insufficient for this purpose. 134 cytomegalovirus is most often cultured in human diploid fibroblasts such as human embryonic lung and human foreskin fibroblasts. it grows slowly in conventional cell culture, and the cytopathic effect is generally not detected until the second week or longer after inoculation. for this reason, a shell vial method using centrifugation to enhance infectivity has become the standard isolation technique and can usually yield diagnostic results within 48 hours. 135 for centuries, the term herpes, derived from the greek erpein (to crawl), was used in medicine to describe any spreading cutaneous lesion. by the end of the 19th century, investigators surmised that the herpetic lesions of the lips and genitalia were manifestations of a single infectious agent, and recognized that it was a disease distinct from herpes zoster. 136.137 as with all human herpesviruses, hsv is a large, enveloped, double-stranded dna-containing virion with an icoshedral nucleocapsid approximately 100 to 1l0nm in diameter, composed of 162 cap somers. the nucleocapsid is surrounded by an amorphous, sometimes asymmetric material (the tegument) that is surrounded by a thin, trilaminar envelope that contains numerous glycoprotein spikes (fig. 11.1c ). the assembly of hsv begins in the nucleus of its host cell and the virus acquires its envelope as the capsid buds through the inner lamella of the nuclear membrane.120.l38 two serologic types are recognized and each is most frequently associated with particular disease syndromes; however, either serotype may cause any of the aggregate clinical syndromes. herpes simplex virus-1 causes gingivostomatitis, pharyngitis, esophagitis, keratoconjunctivitis, and encephalitis, and is the serotype most commonly associated with adult hsv pneumonia. herpes simplex virus-2 typically infects genital sites such as the penis, urethra, vulva, vagina, and cervix, and is the serotype associated with approximately 80% of disseminated disease and pulmonary infections in newborn infants. 136 all herpesviruses have the ability to persist in an inactive state for varying periods of time and then recur spontaneously following undefined stimuli associated with physical or emotional stress, trauma to nerve roots or ganglia, fever, immunosuppression, or exposure to ultraviolet radiation. 138 during the primary infection, hsv replicates at the portal of entry (typically oral or genital mucosae), and infects sensory nerve endings. the virus is transported centripetally along peripheral sensory nerves to central axons and finally to nerve cell bodies in the trigeminal, sacral, and vagal ganglia, where it replicates briefly before becoming latent.139-141 antiviral drugs have no effect on latent infection with hsv. following cues that initiate viral reactivation, hsv replicates in sensory ganglia and is transported centrifugally along sensory nerves to epithelial cells on mucosal surfaces. 138 reactivation of hsv from the trigeminal ganglion is associated with asymptomatic excretion of virus in saliva, and with the development of herpetic ulcers on the vermillion border of the lip, oral mucosa, or external facial skin. 142 newborn infants, severely immunosuppressed or burned patients, and patients with severe trauma are at greatest risk of developing hsv pneumonia.143-146 lower respiratory tract disease in neonates is most commonly associated with disseminated herpetic infections. disseminated hsv infection in the newborn was first described in 1935 as "hepatoadrenal necrosis"147 because of the prominent and frequent necroses that occur in the livers and adrenal glands of affected neonates. 148 most cases of neonatal disease represent primary hsv infections and are acquired during parturition from hsvinfected mothers. the incidence of neonatal hsv infection is approximately 1 in 3200 deliveries, and disseminated disease develops in approximately 25% of infected neonates. 149 in disseminated infections, signs and symptoms appear a mean of 5 days after birth (range, 0 to 12 days), and approximately 40% to 50% of these patients develop pneumonia. in the pre antiviral era, 85 % of neonates with disseminated disease died from the infection. with early diagnosis and high-dose acyclovir therapy, mortality has been reduced to approximately 30%?6,138,149 the disease can be exceedingly difficult to diagnose in a timely manner as only 10% of mothers of affected infants have clinically apparent hsv infection at the time of delivery.15o neonates that survive severe disseminated disease may develop hepatic and adrenal calcifications evident on abdominal radiographs. 151 in adults, infection of the respiratory tract with hsv may be associated with disseminated herpetic infection, but is more commonly identified as an isolated disease manifestation resulting from reactivation of latent herpetic infections in the oropharynx. herpetic tracheobronchitis is an ulcerative process characterized by large areas of denuded mucosal epithelium and fibrinopurulent exudate containing necrotic cells with densely eosinophilic cytoplasm. despite extensive tissue damage, cells with intranuclear inclusions may be sparse, and, when identified, are found most often at the margins of the ulcerated epithelium or occasionally in the mucous glands subjacent to ulcerated surfaces. ls2 aspiration of viruscontaining secretions into the lower respiratory tract is believed to be the most frequent cause of pulmonary infection with hsv; however, oral lesions may be absent in patients with herpetic laryngotracheobronchitis and bronchopneumonia. 153 disease can be also associated with airway trauma caused by tracheal intubation or from hematogenous dissemination of hsv. 144 ,1s2,1s4 chest radiographs of hsv pneumonia generally show ill-defined nodular or reticular densities of various sizes scattered in both lung fields. during the early stages of disease, these nodules measure 2 to 5 mm and are best seen in the periphery of the lungs. as the disease progresses, these lesions coalesce and enlarge to form more extensive infiltrates. 137 herpetic infections of the airways and lung are characteristically difficult to diagnose clinically and hsv pneumonia was not described as a distinct clinical entity until 1949.155 several studies attest to the relative infrequency with which this diagnosis is considered in patients with respiratory disease. for example, none of the 15 cases of hsv disease of the middle and lower respiratory tract identified in a review of autopsies at brooke army medical center during 1965 to 1968 were suspected prior to autopsy. 143 in a 1982 review of 20 culture-confirmed cases of hsv pneumonia, none of the patients had been diagnosed prior to death and all 16 had oral herpetic lesions at the time of death.144 recent investigations also suggest that lower respiratory tract disease caused by hsv may be more common than currently appreciated. in a study from sweden, hsv was cultured from proa diagnosis of tracheobronchitis and pneumonia is best established histologically (fig. 11.6 ). because lower respiratory tract hsv infections are often focused in the tracheobronchial tree, open lung biopsy may be less sensitive than bronchoscopy.154 herpetic lesions show extensive necrosis and karyorrhectic debris and are associated with hemorrhage and a sparse-to-moderate neutrophilic infiltrate (fig. 11.6a,b) . intranuclear inclusions are best appreciated in cells at the leading edge of necrotic foci ( fig. 11 .6b,c). inclusions appear either as homogeneous, amphophilic, and glassy (cowdry type b inclusions), or as eosinophilic with a halo separating the inclusion from the nuclear membrane (i.e., cow dry type a inclusions). 158 cowdry type b inclusions contain actively replicating virus. type a inclusions, considered noninfectious and devoid of viral nucleic acid or protein, represent the nuclear "scar" of hsv infection. 136 ,141 other changes associated with hsv, including multi nucleation and nuclear molding, and ballooning degeneration of the cytoplasm, are more frequently associated with squamous epithelium and are seldom encountered in the lung. because of the high frequency of hepatic and adrenal involvement with disseminated hsv infection in young children ( fig. 11 .6f,g), liver biopsy has been suggested as a diagnostic technique in this patient cohort. 148 commercially available antibodies exist for ihc detection of hsv in tissues (fig. 11.6d ).ls9 virus isolation remains an important diagnostic method; however, because hsv can be isolated from oropharyngeal secretions and occasionally from the lower respiratory tract of patients who lack overt pulmonary disease, virologic cultures must be interpreted in the context of complementary clinical, radiographic, and histopathologic findings as much as possible. cell culture systems susceptible to hsv include vero cells and foreskin fibroblasts. cytopathic effects generally develop within 24 to 48 hours after cultures are inoculated with infectious specimens. suitable specimens include scrapings made from mucocutaneous lesions, tracheobronchial aspirates, or bal specimens. in infants with evidence of hepatitis, it may also be useful to obtain duodenal aspirates for hsv isolation. 138 polymerase chain reaction methods that amplify hsv dna from clinical specimens, including tissue and blood, can be particularly useful by specifically distinguishing between hsv-l or hsv-2 infections.138,149 varicella-zoster virus (vzv), also known as human herpesvirus 3 (hhv-3) , is a human a-herpesvirus most closely related to hsv. it has a linear, double-stranded dna genome with approximately 125 kbp that encodes more than 70 proteins. the icosahedral nucleocapsid is indistinguishable in appearance from other herpes viruses. the nucleocapsid and the tegument are surrounded by a lipoprotein envelope derived from the host cytoplasmic membranes. the enveloped viral particle is pleomorphic to spherical in shape and 180 to 200nm in diameter. 16o the primary infection is initiated by inoculation of respiratory mucosa by the virus through infectious aerosols or by direct contact of skin lesions of patients with varicella or herpes zoster. after a primary viremia in the reticuloendothelial system, and secondary viremia in circulating mononuclear cells, the virus is disseminated to the skin, where it initiates a vesicular rash, and back to mucosal sites in the lungs. the release of infectious virus into respiratory droplets is a pathogenic characteristic that distinguishes vzv from other human herpesviruses. the attack rate for previously uninfected household contacts exposed to varicella is approximately 90%. for less sustained exposure, it is estimated to be approximately 10% to 30%. 160 during primary infection with vzv, viral replication in keratinocytes (fig. 11.7 a) and vascular and lymphatic endothelial cells of the superficial dermis produces the generalized, pruritic, vesicular rash of varicella commonly known as chickenpox. varicella-zoster virus also establishes latent infection within satellite cells and neurons of the trigeminal and dorsal root ganglia and can reactivate under various conditions to cause herpes zoster, a painful vesicular rash commonly referred to as shingles. 161 the origin of the term chickenpox is speculative, but believed to derive from gican, an old english term for itching. the term shingles originates from the medieval latin cinguls, or girdle, and alludes to the partial encircling of the trunk by the rash of herpes zoster. 137 ,160 varicella-zoster virus is ubiquitous in human populations around the world, and humans are the only known host. during the prevaccine era in the u.s., approximately 4 million cases, 4000 to 9000 hospitalizations, and 50 to 140 deaths were reported annually.25.162 the risk of severe illness during primary or recurrent vzv infection appears to depend more on host factors rather than a particular viral strain. chickenpox is considered a relatively benign infection in children, but adult patients are approximately 25 times more likely than children to develop pneumonia. the greatest risk of severe disease and pneumonia occurs in those patients with chronic lung disease, immunesuppressing conditions, neonates, and pregnant women. 163 varicella-zoster virus-related deaths have declined sharply in the u.s. since universal childhood vaccination was implemented in 1995. 24 ,25 s.r. zaki and cd. paddock varicella pneumonia was first described in the medical literature in 1942 164 and is the most frequently reported complication of chickenpox in adult patients. 24 ,165 pneumonia occurs in approximately 10% to 15% of adults infected with vzv. 166 ,167 the occurrence of pneumonia during herpes zoster is rare, and limited primarily to profoundly immunosuppressed patients, particularly bone marrow transplant recipients. m varicella-zoster virus pneumonia generally develops within 2 to 7 days following the onset of rash and may be characterized by fever, cough, tachypnea, chest pain, and hemoptysis. 26 .168 hypoxemia is common and may be severe. radiographically, the lungs show multiple, scattered, defined, nodular densities. untreated adult varicella pneumonia is fatal in approximately 10% of cases, but mortality is as high as 25% to 40% in certain high-risk cohorts, including pregnant women, transplant recipients, and neonates. 137 ,lfj9-171 massive pulmonary hemorrhage is a frequent terminal event. gross examination reveals lungs that are generally two to three times heavier than normal, firm, and plumcolored. there are often multiple necrotic and hemorrhagic lesions on the visceral pleura that resemble the pox lesions of skin. 169 ,172,173 pox may also be seen on the parietal pleura, although pleural effusions are uncommon and rarely prominent. 163 ,173 the trachea and bronchi are generally edematous and erythematous with occasional vesicles on the mucosal surfaces, and there may be lobular consolidation of the lungs. microscopically, pulmonary involvement consists primarily of interstitial pneumonitis and diffuse miliary foci of necrosis and hemorrhage in the pulmonary parenchyma that involve alveolar walls, blood vessels, and bronchioles ( fig. 11.7b,d) . 172 other findings may include intraalveolar collections of edema, fibrin, or hemorrhage, diffuse alveolar damage, and septal edema. 1m ,168.173 virally infected cells with intranuclear inclusions may be identified in respiratory epithelial cells, pneumocytes, interstitial fibroblasts, or capillary endothelium ( fig. 11 .7e,f).167 eosinophilic intranuclear inclusions and multinucleated syncytial cells may be difficult to locate but are best identified at the edges of necrotic foci (fig. 11.7c ). in cases of disseminated disease, similar necrotizing hemorrhagic lesions and occasional viral cytopathic changes in epithelial cells or fibroblasts may be observed in other tissues and organs, including esophagus, pancreas, liver, renal pelves, ureters, urinary bladder, spleen, bone marrow, thymus, lymph nodes, adrenal glands, and brain. 172,174 in those patients who recover from severe vzv pneumonia, some necrotic parenchymal foci may mineralize, and can be identified by chest radiograph years later as miliary, 1 to 5 mm, nodular opacities. microscopically, the lesions are characterized as discrete collections of dense fibrous connective tissue that surround multiple, small, calcified bodies. the periphery may include a cellular zone of fibroblasts and occasional giant cells.175 a radiographic survey of 16,894 persons identified pulmonary calcifications in eight (1.7%) of 463 patients who had chickenpox during adulthood compared with only eight (0.05%) of the remaining 16,431 who did not report having varicella as an adult. 176 a positive history of varicella predicts immunity in >95% of persons. 160 because pulmonary symptoms most often occur several days following the onset of the characteristic rash of varicella, a pathologic diagnosis is seldom required for a real-time diagnosis of vzv pneumonia. however, hematopoietic cell transplant recipients may present with signs of visceral dissemination and pneumonia 1 to 4 days before the localized cutaneous eruption of herpes zoster appears, and lower respiratory tract disease has been described in the absence of skin lesions, particularly in neonates and bone marrow transplant recipients. 26 ,137,177 commercially available antigen detection kits can be used for rapid diagnosis of cutaneous vzv infection. epithelial cells are scraped from the base of a newly formed vesicle, applied to a slide, and stained by using fluorescein-conjugated vzv monoclonal antibodies to detect specific viral proteins in the specimen. in a similar manner, the tzanck test uses wright-giemsa stain to demonstrate multinucleated giant cells in these specimens; however, this test does not differentiate between hsv and vzv, and false-negative results are common. commercially available antibodies are also available for ihc detection of vzv in tissue specimens ( fig. 11 .7e,f); however, relatively few laboratories are able to provide well-validated assays. some commercial laboratories offer pcr amplification to detect viral nucleic acid in clinical specimens. isolation of the virus in cell culture remains the reference standard for the diagnosis of vzv. in human melanoma cells, an excellent substrate for vzv isolation, the average time for visible cytopathic effect from the virus is 3 to 5 days.178 infectious vzv is usually recoverable from the clear fluid of cutaneous vesicles of varicella for approximately 3 days after the appearance of these lesions and for approximately 1 week from herpes zoster lesions. the lungs are the most common organ from which vzv is isolated at autopsy, but isolates have also been obtained from heart, liver, pancreas, gastrointestinal tract, brain, and eyes. 160 influenza is derived from the term influentia, meaning epidemic in the italian form of latin, originally used because epidemics were thought to result from astrologic or other occult influences. influenza is a highly contagious, acute respiratory illness with a spectrum of clinical illness ranging from asymptomatic or mild disease with rhinitis or pharyngitis to primary viral pneumonia with s.r. zaki and cd. paddock fatal outcome. influenza may also be associated with a broad range of other disorders affecting the heart, brain, kidneys, and muscle. influenzaviruses belong to the orthomyxoviridae family, which consists of four genera that include the two important influenza viruses types a and b associated with significant human disease. 179 ,180 influenza a viruses are further classified into subtypes based on the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. only one type of ha and one type of na are recognized for influenza b. influenza a occurs in both pandemic and interpandemic forms. fortunately, pandemics, defined as worldwide outbreaks of severe disease, occur infrequently. interpandemic influenza, although less extensive in its impact, occurs virtually every year. the epidemiologic pattern of influenza in humans is related to two types of antigenic variation of its envelope glycoproteins, namely antigenic drift and antigenic shift. during antigenic drift, new strains related to those circulating in previous epidemics evolve by accumulation of point mutations in the surface glycoproteins. this enables the virus to evade the immune system leading to repeated outbreaks during interpandemic years. antigenic shift occurs with the emergence of a "new" potentially pandemic, influenza a virus that possesses a novel ha alone or in combination with a novel na. there are 16 recognized ha subtypes and nine na subtypes of influenza a virus. viruses from all ha and na subtypes have been recovered from aquatic birds, but only three ha subtypes (hi, h2, and h3) and two na subtypes (nl and n2) have established stable lineages in the human population since 1918. since 1997, widespread avian infection with influenza a (h5n1) and associated clusters of human disease have aroused concern about the threat of a pandemic, and attention has been appropriately focused on control measures to deal with such an event. all influenzaviruses have a segmented, negative-sense rna core surrounded by a lipid envelope. influenzavirus particles are pleomorphic. among isolates that have undergone a limited number of passages in cell culture or eggs, more filamentous than spherical particles are seen. spherical morphology becomes dominant when the virus is extensively passaged in the laboratory. a 10-to 12-nm layer of ha (rod-shaped) and na (mushroomshaped) spikes project radially on the surface of the influenza a and b viruses. hemagglutinin facilitates entry of virus into host cells through its attachment to sialic-acid receptors. because neutralizing antibodies are directed against this antigen, it is a critical component of current influenza vaccines. neuraminidase, the second major antigenic determinant, catalyzes the cleavage of glycosidic linkages to sialic acid and the release of progeny virions from infected cells. accordingly, it has become an important target for drug inhibitors such as oseltamivir and zanamivir. the m2 surface component and channel of influenza a (not present in influenza b virus) regulates the internal ph of the virus and is blocked by the antiviral drug amantadine. influenzaviruses are spread person-to-person primarily through the coughing and sneezing of infected persons. the typical incubation period for influenza is 1 to 4 days, with an average of 2 days. adults can be infectious from the day before symptoms begin through approximately 5 days after illness onset. children can be infectious for ~1o days, and young children can shed virus for several days before their illness onset. severely immunocompromised persons can shed virus for weeks or months. uncomplicated influenza illness is characterized by the abrupt onset of constitutional and respiratory signs and symptoms (e.g., fever, myalgia, headache, malaise, nonproductive cough, sore throat, and rhinitis). among children, otitis media,nausea,and vomiting are also commonly reported with influenza illness. respiratory illness caused by influenza is difficult to distinguish from illnesses caused by other respiratory pathogens on the basis of symptoms alone. influenza typically resolves after 3 to 7 days in most patients, although cough and malaise can persist for >2 weeks. among certain persons, influenza can exacerbate underlying medical conditions (e.g., pulmonary or cardiac disease), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a co-infection with other viral or bacterial pathogens. young children with influenza infection can have initial symptoms that mimic bacterial sepsis. more than 20% of children hospitalized with influenza can have febrile seizures. influenza has also been associated with encephalopathy, transverse myelitis, reye syndrome, myositis, myocarditis, and pericarditis. the risks for complications, hospitalizations, and deaths from influenza are higher among persons aged ~65 years, young children, and persons of any age with certain underlying health conditions than among healthy older children and younger adults. influenza-related deaths can result from pneumonia or from exacerbations of cardiopulmonary conditions and other chronic diseases. the histopathologic features of nonfatal and fatal influenza have been well described and include necrotizing bronchitis, thrombosis, interstitial inflammation, hemorrhage, hyaline membrane formation, and intra alveolar edema (figs. 11.8a,b, 11 .9a,c, and 11.10a). 105, [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] the pathology is more prominent in larger bronchi, and inflammation may vary in intensity in individual patients, viral inclusions cannot be identified by light microscopy (fig, 11 .8d), secondary bacterial infections with organisms such as streptococcus pneumoniae (group a streptococcus [gas]), staphylococcus aureus, and haemophilus influenzae may occur as a complication in about 50% to 75% of fatal cases and make it difficult to recognize the pathologic changes associated with the primary viral infec-445 tion ,190,192,193 the histopathologic features in other organs may include myocarditis, cerebral edema, rhabdomyolysis, and hemophagocytosis (figs, 11.8h and 11.9e,f), immunohistochemistry and ish assays demonstrate that viral antigens and nucleic acids are usually sparse and are primarily seen in the bronchioepithelial cells of larger bronchioles (figs. 11.8c,e,f and 11.9d) . 105.189,190 antigens are more readily identified in patients who die within 3 to 4 days of onset of illness. recent studies suggest that unlike human influenza viruses, avian virus h5n1 preferentially infects cells in the lower respiratory tract of humans, resulting in extensive damage of the lungs with minimal pathology in the upper respiratory tract (fig. 11.10a,c) . this may help explain why the h5n1 avian influenza virus is so lethal to humans but so difficult to spread from person to person. these studies show that the avian virus preferentially binds to the a-2,3-galactose receptors, which are found only in and around the alveoli. this is in contrast to the human influenzaviruses that preferentially bind to the a-2,6-receptors, which are found throughout the respiratory tract from the nose to the lungs. 194.195 in birds and other animals, viral antigens can be detected in the lung as well as a variety of extrapulmonary tissues (fig. 11.10b) . the diagnosis of influenza, suspected by history and clinical manifestations, can also be supported histopathologically. however, because of the absence of any characteristic viral inclusions and because the overall pathologic features of influenza may resemble other viral, rickettsial, and certain bacterial infections, an unequivocal diagnosis can be made only by laboratory tests such as viral culture, direct fluorescent antibody and rapid antigen assays, serology, and ihc. 190 ,196,197 measles measles (rubeola) is an infectious, acute febrile viral illness characterized by upper respiratory tract symptoms, fever, and a maculopapular rash. the causative agent, a member of the genus morbillivirus, of the family paramyxoviridae,198,199 is an enveloped virus that contains a negative sense, single-stranded rna genome of 16,000 nucleotides. other human pathogens in this family include parainfluenza, mumps, and respiratory syncytial viruses. measles virions are pleomorphic, generally spherical, enveloped particles from 120 to 250 nm in diameter. the virus is morphologically indistinguishable from other members of the paramyxoviridae family when viewed by negative contrast electron microscopy. a lipid envelope surrounds a helical nucleocapsid composed of rna and protein. two transmembrane glycoproteins, hemagglutinin (h) and fusion (f), are present in the envelope and appear as surface projections. these proteins mediate viral attachment and figure 11.8. influenza a. a. alveolar damage in a patient with fatal influenza a showing prominent congestion with intraalveolar macrophages and fibrin deposits. b. extensive ulceration of respiratory epithelium in a large airway of a patient with fatal influenza a. the lamina propria shows florid vascular congestion and focal hemorrhage, and predominantly mononuclear inflammatory cell infiltrates. c-e. immunohistochemical staining (c,e) of influenza virus a hemagglutinin antigens in the cytoplasm of residual respiratory epithelial cells of a large airway. this same focus of extensively infected respiratory epithelium (d) demonstrates that, unlike many other viral respiratory pathogens, influenza viruses do not elicit specific cytopathic effects in infected cells. f. in-situ hybridization assay demon-• fusion with respiratory epithelium. they are also believed to playa role in virus maturation through their interaction with the matrix (m) protein, which, in turn, is thought to interact with the nucleocapsid structure. 2oo measles is a highly communicable disease of worldwide distribution. before the introduction of measles vaccines, epidemics occurred about every 2 to 5 years when the percentage of nonimmune members of a population reached critical levels. recently, epidemics have occurred in cycles of about 10 years. in small and isolated communities, measles circulation may cease altogether unless it is reintroduced. if introduced in non immune populations, the disease tends to be more severe and may involve more than 90% of the population because of the highly infectious nature of the virus. although still a significant problem in underdeveloped countries, measles infection became uncommon in the us. after the development and widespread use of an effective measles vaccine. however, a recrudescence of measles infection occurred in several large us. urban centers in recent years, associated with reduced use of the vaccine among children and young adults. during the peak of this activity (i.e., between 1989 and 1991), greater than 50,000 measles cases and approximately 150 measlesassociated deaths were reported. 20l measles virus is highly contagious, spread by aerosols and droplets from respiratory secretions of acute cases.202-204 less frequently, contaminated fomites are involved in transmission. a person with acute measles is infective from just before onset of symptoms to defervescence of fever. in developed countries, likely settings for exposure to measles virus are infectious disease clinics, pediatric emergency rooms, and physicians' offices. 20s children are usually infected by 6 years of age, resulting in lifelong immunity, and almost all adults are immune. clinical infection in children younger than 9 months of age is generally uncommon because of passive protection afforded the infant by the transfer of maternal antibodies. however, with the resurgence of measles in the us. came the realization that most women of childbearing age 447 strating active replication of influenza a in respiratory epithelium in a large airway. g. electron micrograph of influenza a particles (arrows) attached to the cilia of a rodent tracheal epithelial cell. viral ribonucleoproteins are evident in the central aspect of the particles. the hemagglutinin and neuraminidase surface glycoproteins make up the peripheral spike layer. (courtesy of f.a. murphy.) h. rhabdomyolysis in a patient with fatal influenza a. bar, 100nm. a,b,d,h, h&e; c,e, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain; f, digoxigenin-iabeled probe followed by immunoalkaline phosphatase staining, naphthol fast-red. and hematoxylin counterstain; g, uranyl acetate, lead citrate stain. acquired immunity to measles through vaccination and not through natural infection. lower levels of maternal antibodies were found to be transferred from an immunized mother to her infant; thus, a substantial number of measles cases occurred in children younger than 1 year of age in the us. in recent years. after an incubation period of about 1 to 2 weeks, the prodromal phase of measles begins with fever, rhinorrhea, cough, and conjunctivitis. koplik's spots, which are small, irregular red spots with a bluish-white speck in the center, appear on the buccal mucosa in 50% to 90% of cases shortly before rash onset. an erythematous maculopapular rash begins on the face 3 to 4 days after prodromal symptoms and usually spreads to the trunk and extremities. the symptoms gradually resolve, with the rash lasting for approximately 6 days, fading in the same order as it appeared. although recovery is rapid and complete in most cases, complications can arise as a result of continued and progressive virus replication, bacterial or viral superinfections, or abnormal host immune response. 204.206.207 the most common complications are secondary bacterial pneumonia and otitis media. 206 other complications include febrile convulsions, encephalitis, liver function abnormalities, chronic diarrhea, and sinusitis. several pulmonary and central nervous system (cns) syndromes that are often fatal have been described. death occurs in about 1 of every 1000 measles cases; however, the risk of death and other complications is substantially increased in infants, adults, malnourished and immunocompromised individuals, persons with underlying illnesses,and nonimmunized populations in underdeveloped countries.208-212 the first step in measles infection is attachment of the virus to cd46 cell surface receptors on the respiratory epithelium.213 adhesion and fusion of the virus to the respiratory epithelium is mediated by both the hand f viral glycoproteins. 214 this stage is followed by local replication in respiratory mucosa and draining lymph nodes. a primary viremia follows, with dissemination two types of multinucleated giant cells have been described in patient tissues during measles infection. 215 ,216 the reticuloendothelial giant cell (warthin-finkeldey) appears first during the incubation period and is seen in 449 the nucleus of a pneumocyte in the lung of a patient with fatal avian influenza. unlike other influenza viruses that cause disease in humans, h5n1 preferentially infects alveolar epithelial cells, and causes relatively minimal pathology in the upper airway. a, h&e; b,c, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain. different lymphoid tissues throughout the body. the second type is the epithelial giant cell, which has been observed in the epithelium of essentially every major organ. the onset of the rash temporally coincides with the appearance of detectable serum antibody to measles virus. interestingly, virus replication and giant cell formation cease with the appearance of rash. t-cell immunity is essential in the process of viral clearance from lymphoid tissue and respiratory tract. while children with congenital agammaglobulinemia respond normally to measles virus infection, patients with cell-mediated immunodeficiency develop severe disease that presents as giant-cell pneumonia or encephalopathy in the absence of an exanthem. [208] [209] [210] [211] 217, 218 immunity to the f surface glycoprotein is necessary to prevent the spread of measles infection. 219 an atypical measles syndrome characterized by pulmonary consolidation with pleural effusions and hilar adenopathy has been reported in children exposed to wild-type measles virus who had previously received the killed-measles virus vaccine.220-222 recipients of the formalin-inactivated vaccine have a good antibody response to the h protein, but antibodies to the functional region of the f protein and to the nucleoprotein are weak or absent. it has been suggested that the lack of a functional f antibody response may playa role in virus spread. 223 the pathologic features of measles have been well described and several references containing detailed morphologic descriptions are recommended. 13 1,209.224-230 the typical morbilliform skin lesions, koplik's spots, and measles lymphadenitis are seldom seen by the surgical pathologist since the clinical diagnosis is usually apparent. histopathologic changes in the skin include mild congestion, edema, and a predominantly mononuclear infiltrate surrounding small vessels of the dermis, as well as other nonspecific features. occasional diagnostic multinucleated epithelial giant cells with eosinophilic cytoplasmic and nuclear inclusions are observed. 228 .231 pathognomonic reticuloendothelial multinucleated giant cells can be observed in appendix specimens from patients mistakenly operated on for acute appendicitis before the emergence of diagnostic koplik's spots and rash. these cells, which have been reported in various lymphoreticular tissues throughout the body, are typically large and contain from a few to occasionally up to 100 nuclei. these cells do not usually contain viral inclusions. the lymphoid tissues are typically hyperplastic, and the architecture is partially or totally obliterated by diffuse proliferation of immunoblasts. [232] [233] [234] a focal or generalized interstitial pneumonitis, similar to that seen in many other viral infections, is seen in the lungs of measles patients. histopathologic features seen include various degrees of peribronchial and interstitial mononuclear cell infiltrates, squamous metaplasia of bronchial endothelium, proliferation of type ii pneumocyte alveolar lining cells, and intraalveolar edema with or without mononuclear cell exudates and hyaline membranes. secondary changes, such as bacterial or viral superinfection, or organizational changes may alter the original pathology. the hallmark of the disease is the formation of multinucleated epithelial giant cells (fig. ll.lla,b) . these cells, which are often numerous, are formed by fusion of bronchiolar or alveolar lining epithelial cells (fig.ll.l1a) . in contrast to the reticuloendothelial giant cells, these cells generally contain characteristic nuclear and cytoplasmic inclusions. the intranuclear inclusions are homogeneous, eosinophilic, and surrounded by a slight indistinct halo (fig. 11.11c,d) . the cytoplasmic inclusions are deeply eosinophilic, vary in size, and some form large masses with a "melted tallow" appearance ( fig. 11.11d ). these giant cells may undergo degenerative changes with progressive loss of cytoplasm, increasing basophilia, and shrinkage of nuclei. the presence of measles virus in these giant cells may be demonstrated by immunofluorescent,235.236 ihc,21o.237 and ish techniques (fig.ll.lle,f) . these giant cells can also be seen in extra pulmonary tissues (fig. 11.11 g,h) . the diagnosis of typical cases of measles can usually be made on the basis of clinical signs and symptoms. other causes of a similar rash, but without other features of measles, include rubella, dengue virus, enteroviruses, and drug reactions, especially to ampicillin. the typical case of measles giant-cell pneumonia generally presents little diagnostic difficulty for the surgical pathologist. the presence of giant cells with both intranuclear and intracytoplasmic inclusions in a setting of interstitial pneumonitis is highly specific for measles infection. however, multinucleated giant cells are not seen in all cases of measles pneumonia and their absence should not exclude the j diagnosis. other viral and rickettsial agents may also cause a similar interstitial pneumonitis, but without the typical giant cells, and should be differentiated. 229 as previously noted, the histopathologic features in measles pneumonia can be somewhat variable,226 and secondary bacterial and viral infections may modify the histology, further complicating the pathologic diagnosis ( fig. 11.5d ).207.229 other viral pathogens, such as respiratory syncytial virus,238 parainfluenza,239.240 vzv,241 and a recently discovered hendra virus,242 as well as granulomatous diseases of the lung, may give rise to pneumonia with giant cells and should also be considered in the differential diagnosis. however, these clinical entities can be distinguished by history, histopathologic features, and laboratory tests. immunohistochemistry237.243,244 or ish 243 ,244 tests demonstrate viral antigens or nucleic acids in the majority of cases. laboratory confirmation is useful to avoid possible confusion with other rash-causing illnesses. diagnostic laboratory procedures consist of direct detection of either the virus or the viral antigens, usually by indirect immunofluorescence or by serologic methods using hemagglutination inhibition, neutralization, or enzyme immunoassay. specimens for serologic testing consist of acute-and convalescent-phase serum pairs. antibody appears within 1 to 2 days after onset of rash, and titers peak approximately 2 weeks later. alternatively, the presence of specific immunoglobulin m (igm) antibody can be used to diagnose recent infection. 245 human parainfluenza viruses (hpivs) are second only to rsv as a cause of lower respiratory tract disease in young children. human parainfluenza viruses are negative-sense, nonsegmented, single-stranded, enveloped rna viruses that possess fusion and hemagglutinin-neuraminidase glycoprotein "spikes" on their surface (fig. 11 .ig). the four serotypes of hpiv belong in the family paramyxoviridae, subfamily paramyxovirinae, and genera respirovirus (hpiv-l and -3) and rubulavirus (hpiv-2 and -4). the virions are variable in shape and size, ranging from 150 to 300nm. 246 human parainfluenza viruses are spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects. infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. human parainfluenza viruses are unstable in the environment (surviving a few hours on environmental surfaces), and are readily inactivated with soap and water. they are ubiquitous, and infect most s,r. zaki and cd. paddock people during childhood. the highest rates of serious hpiv illnesses occur among young children. serologic surveys have shown that 90% to 100% of children aged 5 years and older have antibodies to hpiv-3, and about 75% have antibodies to hpiv-l and -2. the different hpiv serotypes differ in their seasonality, with hpiv-l causing biennial outbreaks of croup in the fall and hpiv-2 causing annual or biennial fall outbreaks. human parainfluenza virus-3 peak activity occurs during the spring and early summer months each year, but the virus can be isolated throughout the year. similar to rsv, hpivs can cause repeated infections throughout life, usually manifested by an upper respiratory tract illness (e.g., cold and sore throat). human parainfluenza viruses can also cause serious lower respiratory tract disease with repeat infection (e.g., pneumonia, bronchitis, and bronchiolitis), especially among the elderly, and among patients with compromised immune systems. each of the four hpivs has different clinical and epidemiologic features. the most distinctive clinical feature of hpiv-l and hpiv-2 is croup (i.e., laryngotracheobronchit is ); hpiv-l is the leading cause of croup in children, whereas hpiv-2 is less frequently detected. both hpiv-l and -2 can cause other upper and lower respiratory tract illnesses. human parainfluenza virus-3 is more often associated with bronchiolitis and pneumonia. human parainfluenza virus-4 is infrequently detected, possibly because it is less likely to cause severe disease. the incubation period for hpivs is generally from 1 to 7 days. 247 most hpiv infections cause a mild, self-limited illness; however, hpiv-3 infections are an important cause of bronchiolitis, croup, and pneumonia that may be lifethreatening in infants and newborns. 247 human parainfluenza virus infections are also increasingly being recognized as an important cause of severe morbidity and mortality in immunocompromised patients. 20 ,248--252 the mortality of bone marrow transplant patients with hpiv-3 infection has been reported to be as high as 60%. 249, 253, 254 in patients with severe hpiv infection, multinucleated giant cells derived from the respiratory epithelium may be seen in association with an interstitial pneumonitis and organizing changes (fig. 11.12a,b ) . 239,240.249,255-260 these giant cells, which may contain intracytoplasmic eosinophilic inclusions, (fig. 11.12c) , have also been reported in extrapulmonary tissues such as kidney, bladder, and pancreas. 258 other viral causes of giant cell pneumonia, including measks, rsv, vzv, and hsv, should be considered in the histopathologic differential and laboratory testing, including ihe, can be useful in making this differentiation possible ( fig. 11.12d) . diagnosis of infection with hpiv scan also be made by virus isolation, direct detection of viral antigens py enzyme-linked immunoassay (eia) or immunofluorescent assay (ifa) in clinical specimens, detection of viral rna by rt-pcr, demonstration of a rise in respiratory syncytial virus (rsv) is the most common cause of bronchiolitis and pneumonia among infants and children under 1 year of age. the causative agent is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size and ranges from 120 to 300 nm. respiratory syncytial virus is a member of the family paramyxoviridae, subfamily pneumovirinae, in the genus pneumovirus, and can be 453 ing poorly defined cytoplasmic eosinophilic inclusions. d. parainfluenza virus antigens in giant cells localized by using immunohistochemistry. a-c, h&e; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. further distinguished genetically and antigenically into two subgroups, a and b. the subgroup a strains are usually associated with more severe infections. two surface glycoproteins, g and f, are present in the envelope and mediate attachment and fusion with respiratory epithelium. the f protein also mediates coalescence of neighboring cells to form the characteristic multinucleated syncytial giant cells for which the virus name is derived. 265 respiratory syncytial virus is spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects?66 infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. respiratory syncytial virus is unstable in the environment, surviving a few hours on environmental surfaces, and is readily inactivated with soap and water. in temperate climates, rsv infections usually occur during annual community outbreaks, often lasting several months, during the late fall, winter, or early spring months. the timing and severity of outbreaks in a community vary from year to year. respiratory syncytial virus spreads efficiently among children during the annual outbreaks, and most children will have serologic evidence of rsv infection by 2 years of age. illness begins most frequently with fever, runny nose, cough, and sometimes wheezing. during their first rsv infection, between 25% and 40% of infants and young children have signs or symptoms of bronchiolitis or pneumonia, and 0.5% to 2% require hospitalization. most children recover from illness in 8 to 15 days. the majority of children hospitalized for rsv infection are under 6 months of age. respiratory syncytial virus also causes repeated infections throughout life, usually associated with moderate-to-severe cold-like symptoms; however, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. the major histopathologic changes described in fatal rsv infections include necrotizing bronchiolitis and interstitial pneumonia (fig. 11.13a,b) .238,267-272 bronchiallumina and airways are usually filled with necrotic debris and inflammatory cells. these findings may be accompanied by various degrees of diffuse alveolar damage (fig. 11.13e ), organizational changes, and secondary bacterial superinfection. giant cell pneumonia is a feature seen in some cases ( fig. 11.13c,d) . the multinucleated giant cells contain irregular, intracytoplasmic, eosinophilic inclusions surrounded by a clear halo. these inclusions are extremely difficult to identify with any degree of certainty and are only seen in about half the cases (fig. 11.13d) . other viral causes of giant cell pneumonia should be considered in the histopathologic differential and laboratory testing, including ihc,271,273,274 can be useful in making this differentiation possible ( fig. 11.13b,f) . diagnosis of rsv infection can also be made by virus isolation, direct detection of viral antigens in clinical specimens by eia or ifa, detection of viral rna by rt-pcr, demonstration of a rise in rsvspecific serum antibodies, or a combination of these approaches (see also fig. 7.43, chapter 7) .264,275-281 in 2001, van den hoogen et a1. 282 described the identification of this new viral agent from clinical specimens obtained from patients with respiratory illness, which 455 they designated human metapneumovirus (hmpv). it is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size, ranging from 150 to 300nm (fig. 11.1f ). it has been categorized in the family paramyxoviridae, subfamily pneumovirinae, genus metapneumovirus, based on genomic sequence and gene constellation. human metapneumovirus can be further distinguished genetically and antigenically into two subgroups, a and b. similar to rsv, hmpv infection is ubiquitous and occurs during infancy and early childhood, with annual epidemic peaks occurring in the winter and spring months in temperate regions. seroprevalence studies reveal that 25% of all children aged 6 to 12 months have antibodies to hmpv; by age 5 years, 100% of patients have evidence of past infection. like rsv, hmpv has been associated with a wide spectrum of respiratory illnesses.283-287 the patient may be asymptomatic, or symptoms may range from mild upper respiratory tract illness to severe bronchiolitis and pneumonia. although rsv, hpiv-l, and hpiv-3 have been definitively linked to cases of lower respiratory tract disease in infants and young children, the relative contribution of hmpv remains undetermined. like rsv and the hpivs, studies suggest that hmpv may also contribute to respiratory disease in elderly adults and the immunocompromised. 288-29o histopathologic descriptions of features of hmpv infections are few and have not been well described. [291] [292] [293] [294] this is partly related to interpreting the clinical significance of virus detection in context with the ubiquitous nature of the virus. virus detection in such cases is usually made by culture isolation of the virus from upper airways or by pcr assays performed on nasopharyngeal aspirates or bal washings. in nonhuman primates viral antigens are observed in ciliated epithelial cells, type 1 pneumocytes, and alveolar macrophages. this distribution is associated with mild, multifocal, erosive, and inflammatory changes in airways, and an increased number of foamy macrophages in alveoli. 291 the bal specimens collected from patients within a few days of a positive hmpv assay show degenerative changes and cytoplasmic inclusions within epithelial cells, multinucleated giant cells, and histiocytes. the intracytoplasmic inclusions are ill-defined, eosinophilic structures that measure 3 to 4 ~m. 293 lung biopsy or autopsy tissue obtained and examined later in the disease show chronic airway inflammation, intraalveolar foamy and hemosiderin-laden macrophages, and acute and organizing lung injury including areas of diffuse alveolar damage with hyaline membrane formation and foci of a bronchiolitis obliterans/organizing pneumonia like reaction ( fig. 11 .14). in such cases, typical multinucleated giant cells or viral inclusion cannot be identified. 292 -294 in-situ hybridization studies on limited number of human cases human metapneumovirus is difficult to identify with commonly used viral diagnostic procedures. the virus replicates slowly in primary and tertiary monkey kidney cell lines, and the cytopathic effect can be difficult to discern. commercial monoclonal antibody reagents to hmpv are not widely available. most hmpv studies have been conducted using rt-pcr assays or by demonstration of a rise in hmpv-specific serum antibodies. two novel paramyxoviruses, hendra and nipah, have been recently identified in australia and malaysia; these viruses have been associated with acute febrile encephalitis and respiratory tract disease. both infections are zoonotic. hendra was first identified in 1994 when patients who came in close contact with sick horses developed an influenza-like illness. two patients died with pneumonitis and multiorgan failure. 30 the closely related nipah virus was identified during an outbreak in malaysia and singapore during 1998-1999 that included more than 250 patients. patients presented with a severe acute encephalitic syndrome, but some also had significant pulmonary manifestations. 22 ,295-302 most of the patients had a history of contact with pigs, most of them being pig farmers. in bangladesh in 2001 and 2003, outbreaks of nipah encephalitis occurred. 303 ,304 similar to the malaysian outbreak, the most prominent symptoms were fever, headache, vomiting, and an altered level of consciousness. respiratory illness was much more common in the bangladesh cases, however, with 64 % having cough and dyspnea. the reason for increased involvement of the respiratory tract in this outbreak is not known. epidemiologic and laboratory investigations identified fruit bats of the pteropus genus as asymptomatic carriers of hendra and nipah viruses and possible animal reservoirs. [305] [306] [307] [308] [309] hendra and nipah viruses belong to the recently designated genus henipavirus within the family paramyxoviridae, subfamily paramyxovirinae. both viruses are nonsegmented, negative-stranded rna viruses composed of helical nucleocapsids enclosed within an envelope to form roughly spherical, pleomorphic virus particles.3io-312 the structure of their genome is consistent with the other members of the subfamily. 30 histopathologic findings in fatal cases of hendra and nipah infections are similar with varying degrees of cns and respiratory tract involvement. 104 ,313-315 findings include a systemic vasculitis with extensive thrombosis, endothelial cell damage, necrosis, and syncytial giant cell formation in affected vessels ( fig. 11.15a ,b,f). plaques with various degrees of necrosis, in association with inclusion-bearing neurons, can be found in both the gray and white matter of the cns (fig. 11.15e ). multinucleated giant cells with intranuclear inclusions can occasionally be seen in lung, spleen, lymph nodes, and kidneys ( fig. 11 .15c,g). in the lung, vasculitis and fibrinoid necrosis can be seen in majority of cases. fibrinoid necrosis often involves several adjacent alveoli and is frequently associated with small vessel vasculitis. the multinucleated giant cells with intranuclear inclusions are usually noted in alveolar spaces adjacent to necrotic areas. histopathologic changes of bronchiolar epithelium are uncommon; rarely, the large bronchi may show transmural inflammation and ulceration. widespread presence of nipah virus antigens can be seen by ihc in endothelial and smooth muscle cells of blood vessels as well as in various parenchymal cells (fig. 11.15d ). the diagnosis of nipah virus infection, suspected by patient history and clinical manifestations, can be supported by characteristic histopathologic findings. from a diagnostic standpoint, perhaps the most unique histopathologic finding is the presence of syncytial and parenchymal multinucleated endothelial cells. this feature 457 occurs in only approximately one fourth of the cases and cannot be used as a sensitive criterion for the diagnosis of henipah virus infections; furthermore, these cells can also be seen in measles virus, rsv, hpiv, herpesviruses, and other infections. unequivocal diagnosis can be made only by laboratory tests such as ihc, cell culture isolation, pcr, or serology.l04,304,316-319 the parvoviruses are small (18 to 26nm) naked viruses that possess a single-stranded dna genome and require actively dividing cells to complete the viral replication cycle. in 2005, allander et a1. 320 identified a new parvovirus (genus bocavirus) associated with lower respiratory tract infections in children; however, there is no information as yet that describes specific pulmonary pathology attributed to this newly identified agent. on the other hand, human parvovirus b19, a member of the genus erythrovirus, has long been known to cause human disease and has been well studied. 321 ,322 the most commonly recognized manifestation of b19 infections is erythema infectiosum, and approximately one third of the cases of maternal parvovirus infections result in intrauterine parvovirus b19 infections. this places the fetus at increased risk for severe anemia, hydrops, and death. hydrops fetalis is the most commonly recognized complication of intrauterine parvovirus infection, accounting for 4% to 18% of all cases of nonimmune hydrops. cases tend to be clustered during community outbreaks of erythema infectiosum. 321 . 3 22 pathophysiologic effects and histopathologic findings are a result of the tropism of b19 parvovirus for erythroid precursor cells. villi from placentas from patients with bl9-associated nonimmune hydrops are edematous, and fetal capillaries show numerous nucleated erythroid precursors, some containing parvovirus inclusions. the infected cells with eosinophilic "ground glass" intranuclear inclusions and ring-like margination of nuclear chromatin are easily recognized and in the context of a hydropic fetus are pathognomonic of b19 infection. the liver is the major site of blood production in the fetus and the principal organ affected by intrauterine b19 infection. inclusion-bearing nucleated erythrocytes can also be frequently identified in the lung, making histopathologic examination of this organ a worthwhile endeavor for confirming the diagnosis of intrauterine bi9 infection ( fig. il.i6a,b) .323-332 however, these cells may be infrequent and irregularly distributed, requiring examination of multiple sections and use of ihc to confirm the diagnosis (fig.l1.16c,d) . the combination of fever and hemorrhage can be caused by different viruses, rickettsiae, bacteria, protozoa, and fungi. however, the term viral hemorrhagic fever (vhf) is usually reserved for systemic infections characterized by fever and hemorrhage caused by a special group of viruses transmitted to humans by arthropods and rodents. the vhfs are febrile illnesses characterized by abnormal vascular regulation and vascular damage and are caused by small, lipid-enveloped rna viruses. this syndrome can be caused by viruses belonging to four different families that differ in their genomic structure, replication strategy, and morphologic features (table 11 .4). arenaviruses, bunyaviruses, and filoviruses are negative-stranded, 459 in the nucleus and cytoplasm of erythroid precursors by using immunohistochemistry (ihe) (same patient as in a,b). a,b, h&e; c,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. whereas fiaviviruses are positive-stranded rna viruses. hemorrhagic fever viruses are distributed worldwide, and the diseases they cause are traditionally named according to the location where they were first described. the oldest and best known is yellow fever virus; others include lassa fever, lymphocytic choriomeningitis, ebola, and dengue viruses. viral hemorrhagic fevers share many common pathologic features, although the overall changes vary among the different diseases. the similar pathologic and immunopathologic findings in cases of vhf suggest that microvascular involvement and instability is an important common pathogenic pathway leading to shock and bleeding in many instances. infection of the mononuclear phagocytic system and endothelium are thought to play c s.r. zaki and cd. paddock 11 .17. ebola virus hemorrhagic fever. a. pulmonary congestion and lack of inflammation. b. numerous filamentous ebola virus inclusions are seen within hepatocytes in association with hepatocellular necrosis. c. ebola virus-infected intra alveolar macrophages as seen by colorimetric in-situ hybridization using digoxigenin-labeled probes. d. viral anti-gens are seen in endothelial cells and other interstitial cells in this lung section. a,b, h&e; c, digoxigenin-labeled probes followed by immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. figure 11.18 . yellow fever. a. pulmonary congestion in fatal case of yellow fever associated with vaccination. b. yellow fever antigens in the pulmonary interstitium of the same patient. in natural infections, yellow fever antigens are usually not seen outside the liver in fatal cases. in contrast, in vaccine-associated cases viral antigens can be found in a variety of extrapulmonary a critical role in the pathogenesis of vhfs through the secretion of physiologically active substances, including cytokines and other inflammatory mediators (figs. 11.17c,d, 11.18b, 11.19b,c, and 11.20b). at autopsy common findings include widespread petechial hemorrhages and ecchymoses involving skin, mucous membranes, and internal organs. however, in many hf patients manifestations of bleeding may be minimal or absent. effusions, occasionally hemorrhagic, are also frequently seen. widespread, focal, and sometimes massive necrosis can be commonly observed in all organ systems and is often ischemic in nature. necrosis is usually most prominent in the liver and lymphoid tissues. the most con-461 organs, including heart, lung, and spleen. c. extensive midzonal hepatic necrosis characteristic of yellow fever. d. abundant antigens of yellow fever virus are observed in midzonal area of hepatic lobule in this immunohistochemical preparation. a,c, h&e; b,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. sistent microscopic feature is found in the liver and consists of multifocal hepatocellular necrosis with cytoplasmic eosinophilia, councilman bodies, nuclear pyknosis, and cytolysis (figs. 11.17b and 11.18c). inflammatory cell infiltrates and necrotic areas are usually mild and, when present, consist of neutrophils and mononuclear cells. commonly observed histopathologic changes in the lung include various degrees of hemorrhage, intra alveolar edema, interstitial pneumonitis, and diffuse alveolar damage (figs. 11.17 a, 11.18a, 11.19a, 11.20a, and 11.21a). several references containing detailed pathologic descriptions in human cases are recommended. 29.72.333-35o .' a. pulmonary hemorrhage and diffuse alveolar damage in a patient who was infected through organ transplantation. note that, unlike this case, which occurred due to immunosuppression, lcmv infections are rarely fatal and usually resolve with no specific treatment. b. abundant lcmv antigens in areas of the diagnosis of vhf should be suspected in patients with appropriate clinical manifestations returning from an endemic area, particularly if there is travel to rural areas during seasonal or epidemic disease activity. the diagnosis suspected by history and clinical manifestations can also be supported histopathologically, and the overall pattern of histopathologic lesions may suggest a specific diagnosis. however, because of similar pathologic features seen in vhf and a 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west nile virus in the united states acknowledgments. the authors gratefully acknowledge the invaluable assistance of gillian genrich, of the cdc, for her comments and help with organization of the references, cynthia goldsmith for her comments, and mitesh patel, of the cdc, for his help with preparation and assembly of the images. key: cord-015613-ls9qus8y authors: macdonald, david w.; laurenson, m. karen title: infectious disease: inextricable linkages between human and ecosystem health date: 2006-06-06 journal: biol conserv doi: 10.1016/j.biocon.2006.05.007 sha: doc_id: 15613 cord_uid: ls9qus8y nan intellectual ghettos are passé . there was a time when the control of wildlife diseases was the domain of veterinarians while conservation was that of biologists. that false dichotomy has long since passed as infectious disease has become a central issue in biological conservation, which itself has become enmeshed in an inter-disciplinary web that embraces the health of ecosystems and people (e.g. riordan et al., 2006) . indeed, these issues are set to become more entwined, as globalisation, climate change, human population expansion and the natural and unnatural movements of species interact to catapult emergent and mobile diseases to prominence beyond either the public health or conservation agendas, to land firmly on the global political table. infectious diseases have passed in both directions between people and wildlife since time immemorial, but today the enormous rise in human populations, their penetration to every corner of wilderness with concomitant land-use changes, and their transportation of organisms around the world creates an explosive mix of risks. is infectious disease generally a matter for concern by conservation biologists? one general answer might be that infectious diseases are hazards to ecosystems when they affect keystone species such as top predators, or when they undermine ecosystem support systems (foresight, 2006) . however, infectious disease is a natural phenomenon, and a general tenet of biological conservation might be not to meddle where natural processes operate naturally. compassion might prompt the rescue, or even euthanasia, of a sick animal, but such an intervention could be said to have little relevance to conservation, much of which is focused on the viability of populations and ecological communities. of course, even before since it was formalised in the models of anderson and may (1978) , ecologists have realised not only that parasites (in the widest sense of pathogens) were not merely a source of morbidity and mortality in nature, but could also limit, even regulate, some populations. in that sense, pathogens are clearly relevant to conservation biologists, as part of natural processes, but that certainly does not qualify them as a problem, nor does it constitute a justification for meddling in population processes insofar as these processes are natural. this line of thought and it has merit, leads to the conclusion that infectious diseases may often not be of concern to conservation. it also leads sometimes to conservationists being disquieted by a too ready eagerness to intervene when disease afflicts wildlife. on the other hand, there are clear and pressing cases where infectious disease in wildlife conspicuously affects, or is affected or caused by, humans, and human involvement is an operational definition of topics within the ambit of conservation . so, as is characteristic of conservation issues, the decision of when an infectious disease justifies intervention is not always straightforward, and indeed the position of infectious disease within conservation is both technically and philosophically challenging. it was discussions of such topics that prompted us to convene a conference to explore the diverse ways in which infectious disease threaded through issues in mammalian conservation. to say that this special issue of biological conservation is merely the proceedings of a conference would not be correct. nonetheless, it was catalysed by a two-day event held on 21st-22nd may 2005: the mammals trust uk's international conference on wild mammals and disease, and the ensuing think tank hosted at tubney house by oxford university's wildlife conservation research unit. from these events grew the papers that comprise this volume. our starting point was that infectious diseases can have profoundly damaging consequences for mammal populations, particularly those that are already small or isolated. some of the diseases affecting wildlife also pose potential threats to human health. of 1415 known pathogens that have infected humans historically, 62% had zoonotic origin . furthermore, emergence of human pathogens is associated with the ability to infect wildlife for bacteria, fungi and viruses (cleaveland et al., 2001) . it is estimated that 75% of all diseases emerging over the last two decades have been zoonoses; these include sars, avian influenza, ebola, monkey pox, and the west nile virus (brown, 1999; hart et al., 1999) . a brief scan of the major issues in conservation biology worldwide reveals infectious disease as a recurrent linking thread between them. for example, alien species bring with them a host of other, less obvious, creatures that can cause disease: ticks and fleas, intestinal worms and protozoans, viruses and bacteria. in britain, an obvious case is the grey b i o l o g i c a l c o n s e r v a t i o n 1 3 1 ( 2 0 0 6 ) 1 4 3 -1 5 0 a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c o n squirrel, sciurus carolinensis; more than half a century of research and puzzlement passed before the breakthrough that their impact on native red squirrels, sciurus vulgaris, was mediated by a virus. habitat loss and modification, the most important issue for biodiversity conservation, which is also due to the expansion of human populations and activities, has widespread ramifications for infectious disease transmission to and from wildlife. worldwide, the expansion, both in range and density, of domestic dogs and cat populations with their human masters has led to an apparent increase in incidence of outbreaks in wild carnivores, particularly canids . for example, rabies spread by dogs threatens canids around the world, including highly endangered ethiopian wolves, canis simensis, and african wild dogs, lycaon pictus (laurenson et al., 2004) . the few recent isolated cases of rabies transmitted to humans by daubenton's bats, m. daubentonii, have given a new impetus to studies of the prevalence of this group of viruses in bat populations in the uk and its implications for human health (harris et al., 2006) . these were some of the issues we sought to explore as we began planning the weekend of brain-storming that gathered together experts from the cutting-edge of wildlife disease studies from around the world. and from their energetic deliberations we identified a series of seven themes which we propose as the key topics in this field of conservation biology, and around which this special issue is constructed. while biological conservation is not solely about rarity, and seeks to solve problems involving species that span the pestilential to the imperilled, extinction risk is the ultimate indicator. our first theme, therefore, was to explore the circumstances in which infectious diseases can threaten extinction. several papers, including those on rabies in ethiopian wolves, canis simensis (randall et al., 2006) , and african wild dogs, lycaon pictus (vial et al., 2006) , disease in island foxes, urocyon littoralis (clifford et al., 2006) , squirrel parapox virus (sqpv) in red squirrels, sciurus vulgaris (gurnell et al., 2006) , and devil facial tumour disease (dftd) in tasmanian devils, sarcophilus harrisii (hawkins et al., 2006) examine this theme. wild canids are particularly susceptible to generalist pathogens transmitted from domestic dog reservoirs, such as rabies and canine distemper virus . the three papers on canids, case reports and population viability analyses, illustrate how dramatic die-offs of around 70% can threaten the persistence of such rare populations, particularly when combined with other causes of population decline, as in the case of island foxes. as canids generally exhibit rapid population growth rates, only population viability analyses can estimate population persistence and determine the effectiveness of disease control strategies. pathogen-mediated competition, which can lead to unviable populations, is starkly illustrated by analyses of the dynamics of sqpv in red and grey squirrels. gurnell et al. (2006) describe how sqpv causes disease with high mortality in red squirrels but appears non-pathogenic in grey squirrels. however, not all populations of introduced grey squirrels carry the virus -those in scotland and italy do not -but the rate of red squirrel replacement by grey squirrels is some twenty times faster in those areas where grey squirrels carry the virus. the conservation of red squirrels will depend on minimising contact between these species. these examples illustrate that diseases has the potential drive extinction when a pathogen infects a variety of host species and is maintained, often with low pathogenicity, in a more numerous reservoir host, spilling over into a less numerous and sometimes isolated threatened host (lyles and dobson, 1993 ). yet diseases can also decimate large populations of endemic species. hawkins et al. (2006) record the extraordinary case of devil facial tumour disease (dftd) that now constitutes a serious threat to the tasmanian devil sarcophilus harrisii. the tasmanian devil is the world's largest extant marsupial carnivore. dftd is a cancerous disease found exclusively in wild devil populations, and hawkins et al. report that it is consistently fatal to afflicted individuals. the tumours were first reported in 1996 and have subsequently been histologically confirmed in individuals from 41 separate sites, covering 51% of tasmania (with a maximum of 83% of individuals infected). on the basis of the threat posed by dftd, the devil has been listed as a threatened species in tasmania. a recurring issue in infectious disease epidemiology, from public health, conservation and livestock management perspectives, is the difficulty in identifying the reservoir of infection for multi-host pathogens (e.g. courtenay et al., 1994; haydon et al., 2002; mathews et al., 2005) . reservoir identification can aid disease management, ensuring that the correct host is targeted with appropriate and effective disease control tools. when disease control measures in wild species of conservation concern are difficult to implement, due to complicated logistics or limited finances, identification of a reservoir domestic host may offer potential control measures that might be both effective and feasible. the importance of reservoir identification is classically illustrated by a range of papers in this special issue, for example the ongoing dilemma facing bovine tuberculosis control , the diseases emerging from bats (breed et al., 2006) , phocine distemper virus (pdv) in northern seal population (hall et al., 2006) and the canid pathogens threatening island foxes (clifford et al., 2006) . in the southwest of england bovine tuberculosis has proved increasingly recalcitrant and is currently increasing in cattle at an annual rate of 18%. macdonald et al. report on studies of the possible role of various wild mammals and conclude that if any wildlife species plays a significant role in the epidemiology of btb in cattle, it is likely to be badgers, meles meles, although the role of deer, particularly fallow deer, dama dama, in a local context is still unclear. other species simply do not occur in sufficiently large numbers or transmission does not appear sufficiently frequent to allow m. tuberculosis persistence and thus constitute potential reservoirs. identifying the reservoirs of the emergent henipaviruses, which caused mortality in humans and domestic animals (hendra virus in horses, nipah virus in pigs) in the 1990s has been challenging, but these viruses have now both been identified in fruit bat species (breed et al., 2006) . this paper highlights the role of bats as reservoirs of these and other emerging diseases. behavioural and physiological characteristics of species such as the fruit bats, including the ability to cover long distances between islands and continents, close association at roosts and their mammalian physiology, ensure a large effective population size for pathogen persistence and adaptation. identification of fruit bats as reservoirs for henipaviruses means that control can be directed at minimising their contact with domestic species. ecological communities are complex and elucidating the reservoir host, when situations cannot be experimentally manipulated is challenging. hall et al. (2006) illustrate this with the case histories of two outbreaks of phocine distemper that have severely affected harbour seal (phoca vitulina) populations in european and uk waters. both outbreaks were detected on the danish island of anholt, the first in 1988 and the second in 2002. harbour seals were highly susceptible to infection while sympatric grey seals are more resistant. in this phocid community the most likely reservoir of the virus are arctic species of seals, but grey seals, halichoerus grypus, could be important asymptomatic carriers that link these reservoir hosts to the harbour seal populations further south. hall et al. emphasise that understanding of the determinants of the host range remains poor, and that development of more realistic epidemiological models should be combined with studies into the factors controlling species and individual susceptibility. only then will further understanding of reservoirs hosts and transmission routes be built up. clifford et al. (2006) paper on island foxes also illustrates the approach that must be taken when reservoirs cannot be identified. serological studies suggest that feral cats are not putative reservoirs for the canid pathogens under study, but have not revealed whether the pathogens can be maintained in the small island fox populations or are introduced from domestic dogs. in this situation, the practical solution seems to be targeted vaccination programs against the most virulent pathogens and continued intensive disease surveillance. using models to improve disease management for conservation it is clear from this special issue that modelling is a powerful tool for understanding and planning the management of infectious disease. recent stochastic, mixed models offer novel predictions about the role of culling, fertility control, and oral rabies vaccination in rabies control and are reviewed by sterner and smith (2006) . furthermore, both vial et al. (2006) and randall et al. (2006) illustrate how alternative management strategies might affect disease persistence and spread in african wild dogs and ethiopian wolves, respectively. targeting only a viable minimal 'core' of the population, through oral or even parenteral vaccination, is likely to be effective as well as more affordable and logistically less demanding. even if only 40% of animals were protected with a vaccine lasting only two years, this would ensure persistence of even small populations through suppression of the largest outbreaks of disease, that reduce populations to below minimum viable population sizes. similarly, modelling of the spread of both grey squirrels and their sqpv, by gurnell et al. (2006) has identified four main corridors whereby grey squirrels will reach kielder forest (one of the red squirrel's last strongholds in england), initially within two years and in large numbers within 10 years. assuming that greys will not settle within kielder because of the unfavourable nature of the spruce habitat, the authors predict that sqpv disease will burn out at the edges of the forest, although many red squirrels will die. this burn-out is unlikely to be the scenario in other refuge areas where the habitat is more favourable to greys. gurnell et al. conclude that the conservation of red squirrels will therefore depend on minimising contact between red and grey squirrel populations. this will necessitate monitoring grey squirrels as they approach refuge areas, and removing them. morgan et al. (2006) illustrate both the power and the limitations of a modelling approach, through their study of the pathogens of the saiga antelope (saiga tatarica) and domestic ruminants in central asia. for both foot and mouth disease and gastrointestinal nematodes, they reveal that the main risk is associated with infection of saigas from livestock, the putative reservoir host and subsequent geographical dissemination of infection through saiga migration. their discussion on the trade-off between adding biological reality to models, and thereby increasing mathematical complexity and intractability, highlights that the main contribution of modelling is probably to force researchers to formalise understanding in a logical way, highlighting the areas in which too little is known. these areas of uncertainty can then be made the focus of further research, and sound a warning not to accept too readily the predictions of simpler models. sterner and smith (2006) go on to adopt a further multidisciplinary approach and integrate models of disease spread with economic analyses of medical, public health, and veterinary costs. this reveals how post-exposure prophylaxis and increased pet vaccinations have been major costs during and after epizootics in north america. they recommend that this approach should be expanded when considering disease control options in biodiversity conservation. disease surveillance as a cornerstone in disease and conservation management disease surveillance and monitoring self-evidently provide crucial evidence to underpin management decisions. the advances in recent years in molecular techniques have been rapid and have provided powerful tools for the investigation of wildlife disease. this is forcefully illustrated by leendertz et al. (2006) who describe approaches and techniques that can be used in the field when investigating baseline health status and disease outbreaks in great apes, focusing in particular on non-invasive sample collection and mortality investigations. this provides the very basis for diagnosis and surveillance and can be extrapolated to other species. this paper also demonstrates how synergies can also be obtained through close collaboration between the fields of public health and conservation (see below). harris et al. (2006) , also report on exemplary programmes of both passive and active surveillance in the united kingdom to investigate the prevalence of european bat lyssaviruses (eblv) a rabies-related virus, in bats. since 1977, 784 cases of eblv have been recorded in 12 bat species in europe. some 5000 bats have been tested for lyssaviruses for surveillance, and an antibody prevalence level of 3-8% of eblv-2 has been found in daubenton's bat. however no cases of live lyssavirus infection or lyssavirus viral rna have been detected through active surveillance. the authors emphasise how research and monitoring regarding prevalence, transmission, pathogenesis and immunity is required to ensure that integrated bat conservation continues throughout europe, whilst enabling informed policy decision regarding both human and wildlife health issues. disease surveillance is also a cornerstone of diagnosis and control when considering the effect of reintroductions and translocations. however, as mathews et al. (2006b) argue, despite guidelines recommending health-screening and surveillance of translocated individuals and in re-established populations, few reports are available and best practice is rarely observed. these authors present two case studies of health surveillance in wildlife reintroduction programmes -in water voles (arvicola terrestris) in the uk, and in marsupial dibblers (parantechinus apicalis) in australia. these illustrate the potential importance of even basic screening strategies in helping to avoid disease transfer and identifying predictors of survival. building on improved techniques for surveillance, one would expect strong parallels between disease in humans and wildlife, and hence that advances in new research fields such as comparative genomics and molecular genetics would hold lessons for conservation biology. o'brien et al., 2006 argue exactly this case, using examples drawn from wild species of the cat family, felidae. they conclude that resolving the interaction of host and pathogen genomes can shed new light on the process of disease outbreak in wildlife and in people, by reviewing a highly virulent feline coronavirus epidemic in african cheetahs and a disease model for human sars, which illustrate the critical role of ancestral population genetic variation. furthermore, widespread prevalence of species specific feline immunodeficiency virus (fiv), a relative of hiv-aids, occurs with little pathogenesis in felid species, except in domestic cats, suggesting immunological adaptation in species where fiv is endemic. o'brien et al. conclude that conservation management may benefit greatly from advances in molecular genetic tools developed for human biomedical research to assay the biodiversity of both species host and emerging pathogens. leendertz et al. (2006) provide a further illustration of how these synergies can be built up, by demonstrating how collaboration between human laboratories, veterinarians and field biologists can successfully tackle emerging diseases in great ape and human populations. successful pathogen detection in wild great apes has been achieved several times thanks to approaches and techniques that have been developed for human pathogen detection, focusing in particular on investigation of deaths and non-invasive sample collection. most of the host-pathogen systems reported in this special issue concern situations where pathogens have been defined as 'emerging', that is they have recently increased in incidence, impact or geographic range; have recently moved into new populations; or are caused by recently-evolved pathogens (morse, 1995; dazak et al., 2000; lederberg et al., 1992) . a variety of reviews have highlighted the situations and risk factors for emergence in human and domestic animals populations (morse, 2004; taylor et al., 2001; cleaveland et al., 2001) . the expansion of human populations is paramount, influencing agricultural development, urbanisation, deforestation and habitat fragmentation. this in turn influences disease emergence by changing the densities and ecology of disease hosts vectors and pathogens, and altering human interaction with them (mcmichael, 2004) . for example, the penetration of remote forest areas through logging or bushmeat hunting is rapidly escalating the contacts between people and primates (zommers and macdonald, 2006) . not only does this have important implications for direct persecution through the harvest of bushmeat, but it also raises sinister opportunities for disease transmission as described by leendertz et al. (2006) . they describe recent outbreaks of disease in great apes, including ebola, and indications of cross-transmission of ebola and other viruses between primates and humans. there is no doubt that research that integrates infectious disease with primate ecology provides insights to emerging diseases in humans and the role of disease in primate evolution. international travel and global trade hugely increase the capacity for disease spread and are thus other factors that determine disease emergence. a prime example was apparently rabbit haemorrhagic disease virus (rhdv) which was first identified after thousands of domestic rabbits died suddenly in china in 1984. similar epidemics subsequently occurred in other regions of asia, the middle east, europe and north america, suggesting that the virus had dispersed widely following its emergence in china. however, forrester et al. (2006) report that rhdv had circulated apparently harmlessly for many years before the first recognised epidemic in china. they have therefore studied the evolution, emergence and dispersal of this virus in relation to its impact on conservation of wildlife species. using phylogenetic analysis they show that the chinese epidemic virus represents a relatively recent lineage derived from more divergent european viruses that circulated for many years prior to 1984. they show that the genetic lineages of the pathogenic viruses that emerged in the uk in the early 1990s, are distinct from and pre-date those of the 1984 chinese virus. in short, several other divergent pathogenic european strains of rhdv emerged from apparently harmless strains to cause epidemic outbreaks, independently of the chinese 1984 epidemic virus. forrester et al. also illustrate the complexity of conservation issues surrounding the rabbit in the uk -originally an invasive species, now an agricultural pest, yet a source of food for rare native predators and an important tool for habitat conservation. contact with wildlife has also been identified as a risk factor for emerging human pathogens . this is illustrated in this special issue by papers describing high profile emerging viruses that have caused significant disease in domestic animals and humans from wild fruit bats in asia and australia (breed et al.,) and european bats (harris et al.) hendra virus has caused disease in horses and/or humans in australia every five years since it first emerged in 1994. nipah virus has caused a major outbreak of disease in pigs and humans in malaysia the late 1990s and has also caused human mortalities in bangladesh annually since 2001. emergence may have been precipitated by unsustainable hunting of fruit bats and deforestation which have changed the distribution and thus contact rates between fruit bats and humans or their domestic animals, combined with an increase in domestic pig production in asian countries. animal behaviour, as the central factor, more often than not, in determining transmission of infectious disease between infected and susceptible individuals, is unquestionably important in wildlife epidemiology and conservation. this point was originally made in the context of failing attempts to control rabies in european red foxes, vulpes vulpes, which largely ignored their behavioural ecology (macdonald, 1980) , and in this volume it is made stridently by randall et al. and vial et al., who illustrate how knowledge of, respectively, ethiopian wolf and african wild dog social systems is important to understanding and managing the extinction threat they both face from rabies. further emphasise this in the context of attempted control of bovine tuberculosis (btb) in cattle. the worsening situation of btb in cattle has occurred despite a succession of government schemes involving killing badgers with the intention of reducing transmission of btb to cattle. macdonald et al. discuss the perturbation hypothesis which postulates that killing individuals may affect the survivors in ways (behavioural, physiological, immunological) that cause a disproportionate, and perhaps counterproductive, effect. they conclude that the perturbation hypothesis is supported by the data and does provide one plausible mechanism to explain why culling badgers has not generally achieved control of btb in cattle. macdonald et al. draw on various studies to argue that to have any prospect of contributing significantly to controlling btb in cattle, a badger cull would have to be undertaken over a very large area. considering the likely very important role of cattle-to-cattle transmission, and the opportunities for solutions in terms of farm management and surveillance, they judge it would be inappropriate (and probably impractical) to undertake such a cull now (especially in the context of revised agricultural payments which increasingly put a premium on custody of the countryside and its biodiversity (mathews et al., 2006a) ). remarkable satellite tracking studies have revealed long distance migrations by the fruit bats (breed et al., 2006) . this behaviour clearly has far-reaching implications for disease transmission within and across continents, particularly for migratory species. saiga movements, that determine the timing and scale of contacts with domestic animals, also crucially affect disease transmission and persistence. morgan et al. (2006) explain how for both foot and mouth disease and gastrointestinal nematodes, the main risk is associated with infection of saigas from livestock, and subsequent geographical dissemination of infection through saiga migration. the chance of this occurring for foot and mouth disease is predicted to be highly dependent on saiga population size and on the time of viral introduction. for nematodes, the level of risk and predicted direction of transmission are affected by key parasite life history traits, such that prolonged off-host survival of marshallagia in autumn enables infection of saigas and transfer northwards in spring. these seven themes have been in the news regularly fzs1 since our weekend workshop, as the world anxiously watches the progress of h5n7 avian flu, west nile virus and sars on the front-pages. sars, appearing in southern china in late 2002, illustrates that issues at the heart of biodiversity conservation and ecosystem health are fundamental to human wellbeing. sars may have emerged in humans from sars-like coronaviruses (cov) in himalayan palm civets (paguna larvata) and other small carnivores in the wet markets of asia. specimens collected from animals found in live wild-game markets in guangdong china have yielded a sars cov-like virus and several of the early sars patients in guangdong province worked in the sale or preparation of wildlife for food (bell et al., 2004; peiris et al., 2004) and half of 10 civet dealers at the market were found to have antibodies that cross reacted to sars (bell et al., 2004) . recent research suggests that the disease may have jumped to civets from rhinolophid bats in the marketplace, since sars-like coronavirus has been detected in three species from china, australia and the usa lau et al., 2005) . greater genetic variation between these bat strains (as revealed by nucleocapsid protein sequences) than seen in the human or civet sars indicates that the viruses and bats have had time to co-adapt, and hence that bats are probably the origin of sars. bats find themselves along with civets and people in wildlife markets, to which live mammals are brought from increasingly remote areas into contact with an increasingly large human population with global links. bats are also important pollinators and dispersal agents, and many are endangered species and thus their conservation is a priority for the continuing function of many ecosystems. this blend of factors links the concerns of public health, agriculture, biodiversity conservation, animal welfare, third world development and global economics. a salient reality shrieks from the sars story and is touched on elsewhere in this volume: the inter-connectedness of global populations of humans and wildlife. hunters, farmers, market vendors and consumers experience direct risk of zoonotic disease transmission from bushmeat and animals (karesh et al., 2005) . however, human to human transmission then spreads the risk to other individuals around the world and can turn a local outbreak into a global crisis. the local sars outbreak in hong kong and southern china, quickly spread to 25 countries across five continents, through human air travel (peiris et al., 2004) . the fact that over 700 million people travel by air annually (karesh et al., 2005) means that the risk of global epidemics has never been higher and is the factor that could accelerate the spread of a global flu pandemic, ahead of vaccine production, should the mutations for human-human transmission occur. all these studies bring repeatedly to the fore the inextricable linkages that ensure that no wildlife disease issue is the preserve of any one discipline. ecologists to economists, virologists to veterinarians, philosophers to politicians, are all enmeshed in understanding the linkages, and in working together to find solutions. the need for an interdisciplinary approach has been reiterated widely in recent years and is highlighted again in the recently published uk office of science and innovation foresight report ''infectious disease: preparing for the future''. this report brought together diverse experts to consider the threats from disease for wildlife and for humans. their conclusions emphasise the pressing nature of the issue, not least because of advancing climate change which may affect host and vector abundance and distribution and thus contact networks and transmission rates, but also because of the huge costs of disease outbreaks that affect human or livestock health. for example, bse in the united kingdom in 1996-1997 is estimated to have cost £2.3 billion whereas avian influenza in vietnam in 2003/2004 is costed at £0.32 billion (foresight, 2006) . against this background, progress forward must be made. but which way is forward? whilst evidence-based policy is surely essential, and thus science must underpin disease management, we live in a rapidly changing, unpredictable world where a complex web of risk factors can give rise to new disease-related problems at any turn. science thus may not always have complete answers prepared in anticipation but, with investment, it can prudently look ahead. one delphic circle of the wise recently identified the following priority areas (foresight, 2006) for investment (i) novel information technology for capture, analysis and modelling of data for the early detection of infectious diseases, (ii) early detection and characterisation of new or newly resistant/virulent pathogens using genomics, (iii) improving technology for the rapid identification and characterisation of infectious diseases in the field and (iv) high-throughput screening for infectious diseases of people, animals and plants using surrogate, noninvasive markers in airports, sea/road containers and livestock markets. others could add to this list. for example, this, and all other branches of conservation biology, must urgently develop inter-disciplinary syntheses, must achieve alignment with other major guests around the table of environment, sustainability and development, and must not forget the underpinning importance of a deep understanding of natural history . some of the roads leading to these new initiatives are charted in the papers gathered in this special issue. biological conservation stands to benefit from the anticipated advances in new diagnostic, monitoring and control tools. given that the vast majority of endangered species occur in the developing world, these tools and technologies must also be cheap and practical to use in areas where infrastructure is poor. moreover, although we are easily seduced by the new technology, we must keep an eye on the fact that old, lo-tech methods might frequently be best in the developing world and we must keep at eye on the fact that new technology is seductive and that in many instances, old methods might still be best. several spectres loom as infectious diseases, the exploding human population and the biodiversity and extinction crises travel together into the 21 century. some wild populations that were once sufficiently abundant to withstand epizootic disease are now so reduced that an outbreak could tip the balance towards extinction. many of these find themselves encircled and infiltrated by burgeoning hoards of infectious domestic species. some wildlife diseases that once smouldered in isolated wilderness now challenge people that have penetrated their isolation and, through remarkably few links, populate a transmission chain that spans the globe. on the bright side, however, the papers in this volume reveal how the problems are starting to be understood, in some cases sufficiently to achieve solutions. furthermore, biodiversity conservation has taken its place, along with others concerned with infectious diseases, at the table where environmental futures will be decided. one conservation truth is particularly, and perilously, clear when it comes to infectious disease: biodiversity and humanity are in it together. through the kindness of professor john beddington and jill nelson, the mammals trust uk sponsored the weekend of conference and think-tank that spawned this volume, and professor rob mars agreed to publish this special issue of biological conservation. dr. andrew pullin, as the editor guiding us, has been both wise and helpful, and at elsevier julie millard has been unwaveringly supportive and unflustered. all the papers in this volume have been reviewed by at least three reviewers, and by both of us; the result is that all the authors have had to forebear writing at least two, and sometimes more, revisions. we thank both the reviewers and authors for their hard work, and tolerance of our pernicketiness. this paper, which serves as a preface, benefited from the comments of angela mclean, gus mills and zinta zommers. emerging henipaviruses and flying foxes -conservation and management perspectives. biological conservation, this volume emerging disease of animals diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence the role of pathogens in biological conservation pathogen exposure in endangered island fox (urocyon littoralis) populations: implications for conservation management epidemiology of canine leishmaniasis: a comparative serological study of dogs and foxes in amazon brazil emerging infectious diseases of wildlife-threats to biodiversity and human health unravelling the paradox of the emergence of rabbit haemorrhagic disease virus using phylogenetic analysis squirrel poxvirus; landscape scale strategies for managing disease threat phocine distemper virus in the north and european seas -data and models, nature and nurture. biological conservation, this volume european bat lyssaviruses: distribution, prevalence and implications for conservation emerging disease and population decline of an island endemic, the tasmanian devil sarcophilus harrisii. biological conservation, this volume identifying reservoirs of infection: a conceptual and practical challenge wildlife trade and global disease emergence severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats assessing and managing infectious disease threats to canids emerging infections: microbial threats to health in the united states pathogens as rivers of population declines: the importance of systematic monitoring in great apes and o other threatened mammals. biological conservation, this volume bats are natural reservoirs of sars-like coronaviruses infectious disease and intensive management: population dynamics, threatened hosts and their parasites rabies and wildlife: a biologist's perspective principles, practice and priorities: the quest for 'alignment biological hurdles to the control of tb in cattle: a test of two hypotheses to explain the failure of control. biological conservation, this volume bovine tuberculosis (mycobacterium bovis) in british farmland wildlife: the importance to agriculture health surveillance in wildlife reintroductions. biological conservation, this volume keeping fit on the ark: assessing the suitability of captive-bred animals for release environmental and social influences on emerging infectious disease: past, present and future assessing risks of disease transmission between wildlife and livestock: the saiga antelope as a case study factors in the emergence of infectious diseases factors and determinants of disease emergence plagues and adaptation: lessons from the felidae models for severe acute respiratory syndrome integrated disease management strategy for the control of rabies in ethiopian wolves modelling wildlife rabies: transmission, economics, and conservation. biological conservation, this volume risk factors for human disease emergence development of vaccination strategies for the management of rabies in african wild dogs. biological conservation, this volume infectious disease in the management and conservation of wild canids the wildlife trade and global disease emergence. foresight. infectious diseases: preparing for the future laurenson frankfurt zoological society, p.o. box 14935, arusha, tanzania and royal (dick) school of veterinary studies all rights reserved key: cord-255137-utg8k7qs authors: yinda, claude kwe; vanhulle, emiel; conceição-neto, nádia; beller, leen; deboutte, ward; shi, chenyan; ghogomu, stephen mbigha; maes, piet; van ranst, marc; matthijnssens, jelle title: gut virome analysis of cameroonians reveals high diversity of enteric viruses, including potential interspecies transmitted viruses date: 2019-01-23 journal: msphere doi: 10.1128/msphere.00585-18 sha: doc_id: 255137 cord_uid: utg8k7qs diarrhea remains one of the most common causes of deaths in children. a limited number of studies have investigated the prevalence of enteric pathogens in cameroon, and as in many other african countries, the cause of many diarrheal episodes remains unexplained. a proportion of these unknown cases of diarrhea are likely caused by yet-unidentified viral agents, some of which could be the result of (recent) interspecies transmission from animal reservoirs, like bats. using viral metagenomics, we screened fecal samples of 221 humans (almost all with gastroenteritis symptoms) between 0 and 89 years of age with different degrees of bat contact. we identified viruses belonging to families that are known to cause gastroenteritis such as adenoviridae, astroviridae, caliciviridae, picornaviridae, and reoviridae. interestingly, a mammalian orthoreovirus, picobirnaviruses, a smacovirus, and a pecovirus were also found. although there was no evidence of interspecies transmission of the most common human gastroenteritis-related viruses (astroviridae, caliciviridae, and reoviridae), the phylogenies of the identified orthoreovirus, picobirnavirus, and smacovirus indicate a genetic relatedness of these viruses identified in stools of humans and those of bats and/or other animals. these findings points out the possibility of interspecies transmission or simply a shared host of these viruses (bacterial, fungal, parasitic, …) present in both animals (bats) and humans. further screening of bat viruses in humans or vice versa will elucidate the epidemiological potential threats of animal viruses to human health. furthermore, this study showed a huge diversity of highly divergent novel phages, thereby expanding the existing phageome considerably. importance despite the availability of diagnostic tools for different enteric viral pathogens, a large fraction of human cases of gastroenteritis remains unexplained. this could be due to pathogens not tested for or novel divergent viruses of potential animal origin. fecal virome analyses of cameroonians showed a very diverse group of viruses, some of which are genetically related to those identified in animals. this is the first attempt to describe the gut virome of humans from cameroon. therefore, the data represent a baseline for future studies on enteric viral pathogens in this area and contribute to our knowledge of the world’s virome. the studies also highlight the fact that more viruses may be associated with diarrhea than the typical known ones. hence, it provides meaningful epidemiological information on diarrhea-related viruses in this area. d iarrhea is the second most common cause of death worldwide and accounts for about 8 to 9% of the 5.9 million yearly deaths in children under the age of 5 (1, 2) . most of these deaths occur in southeast asia and sub-saharan africa (3, 4) . the chances of infection with enteric viruses are higher in developing countries than developed countries, probably due to suboptimal sanitation and hygienic conditions and low quality of drinking water, especially in rural areas (5) . in cameroon, a limited number of studies have investigated the prevalence of enteric pathogens as the cause of gastroenteritis in humans. these studies mainly focused on the epidemiology of a limited number of pathogens such as rotavirus, norovirus, and enteroviruses, revealing significant differences in the prevalence of these viruses in different settings and time periods (4, 6, 7) . in parts of cameroon, a high prevalence of several enteric viruses such as enterovirus, norovirus, rotavirus, and adenovirus was found in children and adults (8) . generally in africa, many episodes of gastroenteritis remain unexplained as no etiological agent is determined (9, 10) . a proportion of the unexplained gastroenteritis cases are likely due to other known viruses, for which no tests were performed. however, a part of these gastroenteritis cases could also be caused by novel viral agents. transmission of these enteric viruses is predominantly fecal-oral, and humans are constantly exposed to these viruses through various routes (11) . one of these routes is zoonosis from reservoirs in wild or domestic animals, either by insect vectors or by exposure to animal droppings or tissues. one rich but, until recently, underappreciated reservoir of emergent viruses is bats. of the ϳ5,500 known terrestrial species of mammals, about 20% are bats (12) . several viruses pathogenic to humans are believed to have originated in bats over the last several years, including severe acute respiratory syndrome (sars)-and middle east respiratory syndrome (mers)-related coronaviruses, as well as filoviruses, such as ebola and marburg viruses, or henipaviruses, such as nipah and hendra viruses (13) (14) (15) (16) (17) (18) . in the southwest region of cameroon, bats are hunted and eaten. such close interactions provide ample opportunity for zoonotic events to occur (19) . previously, we identified a plethora of known and novel eukaryotic viruses in cameroonian fruit bats using a viral metagenomics approach, including viruses known to cause gastroenteritis in humans (sapovirus, sapelovirus, and rotaviruses a and h) and those not yet associated with gastroenteritis (bastrovirus and picobirna-like viruses) (20) (21) (22) (23) . in the current study, we metagenomically screened 221 human fecal samples collected in the same region (where bats are hunted and eaten), to assess (i) if any viruses of animal origin could be identified and (ii) which known human gastrointestinal viruses were present. these fecal samples were collected from children less than a year old to adults of more than 60 years who had gastroenteritis and/or were in contact with bats. additionally, since the gut virome typically contains both eukaryotic and prokaryotic viruses (phages), of which the latter usually represents the largest fraction of the gut virome, we also analyzed the phageome of these samples. sample characterization. a total of 221 human fecal samples (131 from kumba and 90 from lysoka) were collected from two hospitals in the southwest region of cameroon, for viral metagenomics screening. from these fecal samples, a total of 63 pools were constituted in categories based on age, bat contact status, and location (see table s1 in the supplemental material). illumina sequencing of all the 63 human pools generated in total approximately 708 million raw paired-end (pe) reads (between 4.3 and 53.4 million reads per pool). after trimming, 67% of the reads (471 million) were retained and 86% of these retained trimmed reads (405 million) were annotated using diamond. of these, 18% (74 million) could be attributed as viral. ngs viral read distribution/abundances. in each of the categories of pools, phages make up at least 84% of the total number of viral reads while the maximum proportion of eukaryotic viral reads is 16%. a similar annotation profile was observed for pools of patients in different age groups, different locations, and different bat contact statuses (fig. s1 ). further analysis of eukaryotic viral reads revealed that at least 70% of the reads mapped to viruses of the families astroviridae, reoviridae, and anelloviridae (fig. 1a) . other viruses were also present, particularly those that are known to cause gastroenteritis belonging to the families adenoviridae, caliciviridae (sapovirus and norovirus), and picornaviridae (of which about 60% were enteroviruses [fig. 1b and fig. s2] ). also, reads from viruses known to cause other human diseases (parvoviridae) or other animal diseases (circoviridae) or not associated with any diseases at all (picobirnaviridae) were present in variable numbers in the different groups ( fig. 1b to e) . the rest of the viral families were either plant-or insect-associated viruses. notably, in age groups a to d, the percentage of pools in which picobirnaviridae viruses were present increased with age with low percentages in age groups a and b (fig. 1c) . also, the percentages of pools positive for anelloviruses differed with respect to age, with higher percentages in young children and the elderly. further, there were no observable trends in the percentage of eukaryotic viral presence with respect to bat contact status or location ( fig. 1d and e) . figure 1f shows a heat map of the percentage of pools in which eukaryotic viral families were present in human and bat pools, while fig. s3 presence in human and bats at the genus level (23) . astroviridae (mamastrovirus), calciviridae (sapovirus), picornaviridae (parechovirus), and reoviridae (rotavirus), viral families known to cause gastroenteritis in humans, were identified in both bat and human pools from the same region. also, mammalian viruses not yet established to cause gastroenteritis (picobirnaviridae, circoviridae, and parvoviridae [bocaparvovirus]) were also common in both bats and humans from the same regions ( fig. 1f and fig. s3 ). phylogeny of eukaryotic viruses. in this study, we focused on viruses from which near-complete genomes were obtained, particularly those that are known to cause viral gastroenteritis (belonging to the astroviridae, caliciviridae [norovirus and sapovirus] , picornaviridae [enterovirus, parechovirus, cosavirus] , parvoviridae, reoviridae, and adenoviridae [human mastadenovirus]). furthermore, we also looked at other viruses not fully proven to cause gastroenteritis in humans but which have only sporadically been associated with gastroenteritis, like picobirnaviridae and small circular singlestranded dna viruses. phylogenetic analysis was done for each of the selected viruses using the protein or nucleotide sequences of suitable conserved regions and representative members of their viral family, genus, or species. reoviridae. reoviridae is a large viral family of segmented dsrna viruses with a wide host range. they are further divided into two subfamilies and 15 genera. genomes of viruses belonging to the reoviridae contain 9 to 12 segments (24) . in total, reoviridae reads were found in 6 pools, and (nearly) complete genomes of 2 viruses of the family reoviridae were obtained from pool hp55. samples in this pool were from two diarrheic children (less than 5 years), originating from kumba and without contact with bats. mammalian orthoreovirus. mammalian orthoreoviruses (morvs) contain 10 segments, l1 to l3, m1 to m3, and s1 to s4, coding for 12 to 13 proteins (24, 25) . a morv strain was identified represented by 16,913 reads (0.4% of all viral reads of the pool). phylogenetic analysis based on the nucleotide sequences of each of the 10 segments of this morv ( fig. 2 and fig. s4 ) showed topological incongruence with four distinctive patterns. based on segments l2 and s1, this strain clustered with bat strains wiv3 and wiv5 from china with 86% and 70% nucleotide (nt) identity, respectively ( fig. 2a and b ). for the l1 and s2 segments, the human strain clustered with the ndelle murine strain, also from cameroon, with 95% and 92% nt identity, respectively (fig. 2c and d) . on the other hand, segment s3 of the cameroonian morv strain clustered with a human strain and a civet morv strain from china (88% and 89% nt identity, respectively [fig. 2e] ). the rest of the segments (l3, m1 to m3, and s4) did not cluster together clearly with any of the abovementioned strains (fig. s4) . rotavirus a. rotavirus a (rva) contains 11 segments coding for 11 or 12 proteins: vp1 to vp4, vp6, vp7, and nsp1 to nsp6 (26, 27) . we identified a near-complete rva sequence which made up 99% (4.3 million) of the eukaryotic viral reads of that pool. the nsp3 segment was not identified in the sample. the vp7 gene of this strain was genetically most related to rva/human-tc/usa/wa/1974/g1p1a [8] and rva/human-tc/usa/rotarix/2009/g1p [8] (nt identity of 92 and 97%, respectively) while the vp4 gene was 90% identical to the same strains. the phylogenetic trees of the remaining segments shared the same clustering pattern (fig. 3a and b and fig. s5 ). according to the rotavirus classification scheme, this strain is a typical wa-like g1p [8] named rva/ human-wt/cmr/cmrhp55/2014/g1p [8] . cmrhp55 was distantly related to bat rva strains identified from the same regions (only 69 to 71% nt identity). picornaviridae. the picornaviridae represent a large family of small, cytoplasmic, nonenveloped icosahedral ssrna viruses consisting of 80 species, grouped into 35 genera. they have a genome of 7.1 to 8.9 kb in size and are most often composed of a single orf encoding a polyprotein flanked by a 5= and 3= utr (28) . the members of the family picornaviridae can cause gastroenteritis, meningitis, encephalitis, paralysis (nonpolio and polio-type), myocarditis, hepatitis, upper respiratory tract infections, and diabetes (29, 30) . out of the 63 pools, 41 contained picornaviridae reads, making the picornaviridae the eukaryotic viral family of which reads could be identified in the highest number of pools. enterovirus. the genus enterovirus (ev) consists of 15 species: enterovirus a to l and rhinovirus a to c. ev a, b, c, and d are found in humans; e and f in cattle; g in pigs; h, j, and l in monkeys; k in rodents; and species i in dromedary camels (http://www .picornaviridae.com). in this study, eighteen (nearly) complete genomes of evs were obtained. the strains were named ev/human/cmrhpxx/cmr/2014, here referred to as ev-cmrhpxx. all eighteen genomes were found in pools of age groups a and b (ͻ3 and 3 to 20 years, respectively). eight of these were identified in age group a, three (ev-cmrhp1, 5a, and 5b) of which were pools consisting of samples of infants who had indirect contact with bats while the rest (ev-cmrhp14, 45, 52a, 52b, and 55) were those that had no contact with bats. the ten other strains were identified in pools belonging to age group b, three of which had direct contact with bats (ev-cmrhp8a, 8b, and 9), 5 indirect contact 4, 35a, 35b, and 39) and two with no contact . based on the phylogenetic analysis of the vp1 nucleotide sequences, the evs found in this study were quite divergent from each other, belonging to three different species of enterovirus, a, b, and c (fig. 4a ). most of the strains belonged to the enterovirus c clade (ev-cmrhp1, 3, 4, 8a, 8b, 9, 14, 18, 35a, 52a, and 55) , while ev-cmrhp35b, 39, and 45 clustered within the enterovirus b genotype, and evcmrhp5a, 5b, 52b, and 58 in the genogroup enterovirus a. some pools had multiple strains of ev present, and some of these clustered together (cmrhp8a and 8b: vaccine type pv-3), whereas other pools contained distinct ev species (ev-cmrhp35a and 35b; 52a and 52b). the presence of vaccine strains in pool hp8 probably indicates recent vaccination events of the infants in this pool. apart from ev-cmrhp39 (which clustered with 11c52_cmr), all the ev strains identified here were distantly related to those previously identified in the far north region of cameroon (31) . furthermore, none of the human strains from cameroon were related to any of the animal ev strains (from chimp or gorilla). a summary of the detailed classification of these evs using an online typing tool (32) is shown in table 1 . parechovirus. the genus parechovirus is comprised of two species, parechovirus a (human parechovirus [hpev] ) and parechovirus b (ljungan virus, isolated from bank voles) (33) . hpev is subdivided into 19 types (hpev1 to -19) . hpev is associated with mild gastrointestinal or respiratory illness; however, severe disease conditions, such as meningitis/encephalitis, acute flaccid paralysis, and neonatal sepsis, may occur (34) (35) (36) . here, three (nearly) complete hpevs were identified in pools hp2, hp46, and hp48 with sequence lengths of 7,142 bp, 7,202 bp, and 7 ,219 bp, respectively, collected from children less than 3 years old (age group a). in terms of bat contact status, they were in pools of those either in indirect contact with bats (hp2 and hp48) or without contact (hp46). they were all distantly related to each other, with hpev-cmrhp46 and hpev-cmrhp48 having the highest identity (76% and 86% nt and aa identity, respectively). phylogenetically, hpevs in hp46 and in hp48 fell into a clade of type 1 hpevs (fig. 4b ). the hpev in hp46 clustered together with hpev1/harris strain with 76% nt identity, while cmrhp48 clustered closely with japanese and norwegian strains a1086-99 and no-3694 (84 to 90% nt identity) . furthermore, hpev-cmrhp2 clustered distantly with type 16 hpevs from china and bangladesh with only 70 to 71% nt identity. considering the 75% identity demarcation for hpev types (37, 38) , this strain potentially represents a novel type. cosavirus. the genus cosavirus consists of five species (cosavirus a, b, and d to f), which have been associated with gastroenteritis in children (39) . six near-complete [8] strains. red, cameroonian human rva strain identified in this study; blue, cameroonian bat rva strains. trees were constructed human cosavirus (hcosv) genomes were identified: 1 from children less than 3 years old (hp49), 3 from those between 3 and ͻ20 years old (hp6a and hp6b, hp57), and 2 from pools of individuals between 20 and ͻ60 years old (hp44, hp24). some of these pools had direct or indirect contact with bats (hp6, hp24, and hp44), while others had no contact with bats (hp49 and hp57). phylogenetic analysis (fig. 4c ) showed that cosaviruses from hp6b, hp49, and hp57 formed a clade with two other strains from australia and nigeria (hcosv/e1/aus and hcosv/ng385/nga) in species hcosv e. meanwhile the strains in hp6a, hp24, and hp44 clustered with hcosv in species a, d, and b, respectively. therefore, it seems that humans in cameroon host a diverse range of cosaviruses. cardiovirus. the genus cardiovirus consists of three species, cardiovirus a to c. species b includes saffold virus (safv) infecting humans. it has been found in cases with acute flaccid paralysis, respiratory tract infections, and diarrhea in china (40) (41) (42) . here, we found a near-complete genome of a safv in one pool (hp35) belonging to the age group between 3 and ͻ20 years old who had indirect contact with bats. the vp1 phylogenetic trees were based on the nucleotide sequences of the vp1-p2a region for the species hepatovirus a and the vp1 region for the rest of the genera. all the trees were constructed using the gtrϩgϩi nucleotide substitution model using raxml, with the automre flag, which enables a posteriori bootstrapping analysis. only bootstrap values greater than 70% are shown. bars indicate nucleotide substitutions per site. red, novel strains from this study; blue, human cameroonian enterovirus strains from other studies; green, animal enterovirus strains from cameroon. segment of the identified safv was 72 to 74% and 78 to 80% identical (on nt level) to safv strains in types 5 and 6, respectively. phylogenetic analysis based on the vp1 region confirmed the clustering of the novel strain between types 5 and 6 with more phylogenetic relatedness to type 6 ( fig. 4d) . hence, this novel safv strain may be a distant member of type 6 or represent a new type. hepatovirus a. hepatitis a virus (hav), now hepatovirus a, belongs to the genus hepatovirus, which consists of nine species (hepatovirus a to i). the hepatovirus a species is comprised of a single serotype, hav, subdivided into human and simian viruses (43) . it causes acute hepatitis throughout the world (44) . there were three (nearly) complete hav genomes in pools hp2, hp4, and hp6, all of which were pools from those in direct (hp6) or indirect (hp2 and hp4) contact with bats. these strains were either from infants less than 3 years old (hp2) or from children between 3 and ͻ20 years old (hp4 and hp6). based on the vp1-p2a region, the nt identity between these strains was 98 to 99%. strains in hp4 and hp6 were 99% identical to brab13, isolated from a patient from the netherlands in 2001, who was staying in a hippie community with visitors from all over the world and under primitive living conditions (45) . on the other hand, the hav strain in hp2 was closely related to strain g2b1-vp from france (98% nt identity). therefore, all strains identified here are genotype iia (fig. 4e) , increasing the number of completely sequenced genotype ii strains to five (the other two strains are ba/ita/2012 and cf53/berne). astroviridae. astroviridae is a family of nonenveloped, spherical viruses with a linear ssrna(ϩ) genome of 6.8 to 7kb, containing three overlapping orfs. the family is divided into two genera: genus mamastrovirus (mastvs) and genus avastrovirus (aastvs). the genera are further divided into 33 and 7 species, respectively (46) . fourteen out of the sixty-three human pools contained astroviridae reads, and we were able to obtain eight near-complete genomes of mastvs (hp2, 3, 6, 34, 35, 43, 45, and 46) . additionally, these pools were either from children less than 3 years old (hp2, hp45, and hp46), age group 3 to ͻ20 (hp3, hp6, and hp35), or between 20 and ͻ60 (hp34 and hp43). phylogenetic analysis of the rdrp and capsid regions ( fig. 5a and b) depicted clustering of the novel mastvs in species 1 (cmrhp2, 3, 34, 35d, 43, and 46) , 6 (cmrhp45), and 9 (cmrhp6). in the mastvs1 clade, there seems to be topological inconsistency in the different phylogenetic trees. strain astv8_yuc8 (af260508) clustered with the novel strains cmrhp2, 3, and 35d in the capsid tree, while in the rdrp tree it clustered with the chinese strain v4-guangzhou, suggesting a recombination event between these strains in the past. bat astrovirus identified in cameroon (23) phylogenetic trees based on the nucleotide sequences of the rdrp (a) and capsid (b) genes of the astvs identified in this study and representative strains from genbank. trees were constructed using the gtrϩgϩi nucleotide substitution model using raxml, with the automre flag, which enables a (continued on next page) formed a clade (in the rdrp tree) with other bat astroviruses from guangxi but was distantly related to the human astvs from the same region. caliciviridae. caliciviridae are a family of nonenveloped viruses with a linear ss-rna(ϩ) genome of 7.3 to 8.3 kb, containing two or three orfs. the family contains five genera (47, 48) . in total, caliciviridae reads were found in 16 pools belonging to either the norovirus or sapovirus genus. norovirus. this genus consists of a single species, norwalk virus (nv), divided into 5 genogroups. genogroups i, ii, and iv infect humans, whereas genogroup iii infects bovine species and genogroup v has been isolated from mice (49) . three nearcomplete nvs were present in the 16 pools that contained caliciviridae reads (hp1, hp18, and hp59), from people who had indirect (hp1 and hp59) or no (hp18) contact with bats, and from age group a (hp1), b (hp18), or c (hp59). the phylogenetic tree (fig. 6a) showed that the four nvs belonged to two genogroups: i (nv_cmrhp18, genotype i.3) and ii (nv_cmrhp1 and nv_cmrhp59, genotypes ii. 12 and ii.13, respectively) . the novel strain nv_cmrhp18 was more than 98% similar to strain c13/ 2009cmr_gi.3 (a partial sequence [jf802509]) isolated from the littoral region of cameroon in 2009, whereas strains of genogroup ii from the same study (ii.4, ii.8, ii.17) were distantly related to those identified here (ii.12 and ii.13) (7) . strains from this previous study were not included in the phylogenetic analysis because only 200 to 300 nt of the capsid region was available in databases. sapovirus. the genus sapovirus (sav) consists of a single species, sapporo virus. it has been detected in humans, pigs, minks, dogs, sea lions, bats, chimpanzees, rodents, and carnivores (50, 51) . three near-complete sav genomes were present in pools hp4 (age group b), hp15 (age group a), and hp22 (age group d) from people who were in indirect contact, were not in contact, and were in direct contact with bats, respectively. phylogenetic analysis (fig. 6b) showed that sav from hp22 could be classified as a giv genotype, and the savs hp4, hp53, hp46, hp56, and hp15 belonged to genotype gii. the phylogenetic tree showed that the bat savs found in cameroon (in blue) (22) clustered together and formed a clade with other bat savs from china and hong kong but divergent from these human savs, indicating no evidence of interspecies transmission of savs in this region. picobirnaviridae. picobirnaviruses (pbvs) belong to the family picobirnaviridae, genus picobirnavirus, and are small bisegmented dsrna viruses with a total genome size of about 4 kb. segment one encodes a polyprotein, containing the capsid protein, and segment two encodes the rdrp. based on the rdrp gene, pbvs are classified into two genogroups. although pbv is genetically highly diverse and has been found in stool samples of a broad range of mammals, its true host(s) remain(s) enigmatic. the disease association is unclear, but pbv infection has been associated with gastroenteritis in both animals and humans (52, 53) . up to 28 out of the 63 pools contained reads annotated as picobirnaviridae with most of the positive pools from individuals in age groups above 20. we could obtain 37 (near-complete) rdrp sequences of pbvs from these 28 pools. phylogenetic analysis based on rdrp (fig. 7) revealed the clustering of the novel strains in four different clades: in genogroup i (26 strains) , in genogroup ii (9 strains), and in 2 clades (3 strains) of uncharacterized picobirna-like viruses that use an alternative mitochondrial invertebrate genetic code (lysoka picobirna-like virus cmrhp9 and cmrhp10b and kumba picobirna-like virus cmrhp21a). interestingly, a wolf pbv strain from portugal (ans53886) from genogroup i clustered together with human strains from cameroon with an aa identity of 76% with strains cmrhp26a and cmrhp35. likewise, in genogroup ii, strains cmrhp34a, cmrhp63b, and cmrhp26c clustered closely (75 to 76% aa identity) with a portuguese feline strain (agz93689). intriguingly, the cameroonian human picobirna-like viruses cmrhp9 and cmrhp10b were 99% identical to a cameroonian bat strain picobirna-like virus, p11-300, suggesting a possible interspecies transmission. however, their true host has not yet been determined. it could be that the true hosts of pbvs are found in both humans and bats and that this therefore explains their presence in both. small circular, rep-encoding, ssdna (cress-dna) genomes. (i) smacovirus. smacovirus (scv) is a relatively recently described virus with a small circular dna genome with a size of about 2,529 bp. it belongs to the smacovirus group and is an unclassified eukaryotic virus of unknown origin (54) . in this study, we identified two scv sequences, one complete genome (huscv-cmrhp10) and a near-complete genome (huscv-cmrhp03). they were identified in pools of patients belonging to age group b, coming from lysoka and having direct (hp3) or indirect (hp10) contact with bats. these strains shared 99% amino acid identity. their replicase genes were 94% and 95% identical to chimpanzee (kp233190) and human (huscv3, kt600069) strains from the united states, respectively. based on the capsid region, these novel cameroonian strains were 98 to 99% identical to the chimp strain and only 85% identical to the human strain huscv3. the close genetic relatedness of human strains to a strain found in a chimpanzee sample suggest that these viruses infect a host shared between chimps and humans, and if indeed smacovirus infects mammals, this could be a case of interspecies transmission (55) . phylogenies of the replicase (fig. 8a ) and the capsid genes ( fig. 8b) indeed showed a cluster of these cameroonian strains with a human genogroup i and pecovirus (c and d) of the replicase and capsid genes, respectively. the trees were constructed using the lgϩgϩi substitution model using raxml, with the automre flag, which enables a posteriori bootstrapping analysis. only bootstrap values greater than 70% are shown. bars indicate amino acid substitutions per site. red, cameroonian human strains; blue, previously known human smacoviruses or pecovirus. and a chimpanzee strain from the united states. however, the topological inconsistency in the replicase and capsid trees may suggest a recombination event between these strains in the (distant) past. (ii) pecovirus. pecoviruses (peruvian stool-associated circo-like viruses [pecvs] ) are cress-dna genomes that were first identified in the feces of a patient during an outbreak of acute gastroenteritis in the netherlands and later in samples of peruvian children (55, 56) . subsequently, they were identified in other humans, pigs, a dromedary camel, and a seal (55) (56) (57) (58) . here we identified a genome sequence (hupecv-cmrhp60) of 2,937 bases made up of two orfs that code for a capsid (372 aa) and a replicase protein (336 aa). unlike other human pecvs, the cameroonian strain shared the same canonical nonamer (nantattac) atop the predicted stem-loop structure with seal, dromedary, and porcine pecv strains. the rep showed 31 to 42% aa sequence identity to all other rep genes, and a rep-based phylogenetic analysis (fig. 8c) showed that hupecv-cmrhp60 clustered together with pecovirus genomes from a seal and 3 human strains. based on the cap protein (fig. 8d) , the cameroonian strain was only 22 to 42% identical to all other pecoviruses and clustered only distantly from the seal strain, a porcine strain, and the human strains. this demonstrates the existence of a high level of genetic diversity within this group of circular dna genomes, pointing to the possible existence of multiple species in this clade. furthermore, we identified 2 incomplete sequences related to sewage-associated circular dna molecules recovered from a sewage treatment oxidation pond in new zealand (59), with only 38% aa identity on the rep protein, further expanding the great diversity of cress-dna genomes in the cameroonian population. bacteriophages. bacteriophages are viruses that infect and replicate within bacteria. their presence therefore reflects the gut microbiota of the patients. because most of the obtained viral reads were classified as bacteriophages, we further investigated the bacteriophage composition of the human samples. with virsorter (60), a tool developed to identify highly divergent dsdna phages from metagenomics data, 5,905 of the contigs in our data set were identified as phages. from these, the tool diamond (61) annotated 2,647 as bacterial, 21 as metazoan, and only 606 (ϳ10%) as viral, while 1,309 contigs remained unannotated. from the contigs annotated as viral by diamond, most were phages belonging to the myoviridae (236 contigs), podoviridae (95 contigs), siphoviridae (145 contigs), and microviridae (36 contigs) families. to get insight into the differences in the bacteriophage communities, we compared the virsorter-identified bacteriophage richness between the different age groups (fig. s6a) , locations (fig. s6b, ) and bat contact status (fig. s6c ), all of which showed no significant differences. to identify the potential bacterial hosts of these phages, we searched for bacterial crispr spacer sequences in the phage contigs, to identify its potential host. the search revealed that the most likely hosts of these phages are bacteria of the families bacteroidaceae, bifidobacteriaceae, enterobacteriaceae, enterococcaceae, erysipelotrichaceae, eubacteriaceae, lactobacillaceae, odoribacteraceae, streptococcaceae, and veillonellaceae (table s2) . network analysis of human and bat phageomes. in order to visualize the genetic relatedness between the human and bat gut phageome, a recently developed bioinformatics tool (vcontact) was used. it groups phages based on their genome sequences into viral clusters which correlate rather well with viral genera as defined by the international committee of taxonomy of viruses (ictv) (62) . a total of 30,875 protein clusters were predicted using the prokaryotic and archaeal refseq combined with the proteins predicted from the phage contigs identified from the human and bats pools using virsorter. using a network analysis approach (fig. 9) , 792 viral genome clusters were predicted of which 173 contained reference phages together with bat or human phage contigs, whereas the rest contained only bat, only human, or bat-human clusters. figure 9 shows that both cameroonian human and bat phage contigs identified in our studies are spread across the known phage sequence space. however, several of the phage contigs constituted completely new clusters (indicated by filled gray ovals), completely unconnected to phages in the reference database. also, the genetic diversity of several previously known phage subclusters was significantly expanded (as indicated by open ovals) while some clusters (in brown ovals) were made up of only bat and human phages identified in this study. recently, we thoroughly investigated the gut virome of fruit bats from cameroon (20) (21) (22) (23) 63) and showed the presence of many novel and divergent eukaryotic viral families, including viruses known to cause gastroenteritis in humans. the aim of the current study was to investigate the gut virome of humans (n ϭ 221) from cameroon and to further determine if bat viruses are possible causative agents 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virsorter_node_3496_length_1469_cov_3_86782-cat_2 riemerella~phage~rap44 virsorter_node_185_length_4953_cov_6_15402-cat_1 virsorter_node_1708_length_1576_cov_4_20547-cat_2 virsorter_node_44_length_7557_cov_10_863-cat_2 virsorter_node_233_length_4482_cov_10_1639-cat_2 pseudomonas~phage~af virsorter_node_552_length_2485_cov_4_35756-cat_2 virsorter_node_192_length_3929_cov_3_8676-cat_1 virsorter_node_322_length_4472_cov_38_5126_gc021836_gc021836-cat_1 virsorter_node_538_length_2956_cov_5_02813-cat_1 virsorter_node_992_length_1573_cov_4_92848-cat_2 virsorter_node_16_length_23516_cov_138_53-cat_2 virsorter_node_151_length_1878_cov_116_108_id_301_gc022959_gc022959-cat_1 virsorter_node_85_length_8370_cov_15_7844-cat_1 virsorter_node_15_length_11161_cov_176_621_gc021830_gc021830-cat_1 virsorter_node_2781_length_1390_cov_3_66794-cat_1 virsorter_node_1540_length_1662_cov_4_40252-cat_2 virsorter_node_14_length_17500_cov_14_0744-cat_2 virsorter_node_300_length_4134_cov_17_8536-cat_2 virsorter_node_6_length_27203_cov_21_9593-cat_2 enterobacter~phage~tyrion virsorter_node_5_length_48144_cov_42_8117-circular-cat_2 virsorter_node_10_length_41314_cov_88_2574-circular-cat_2 virsorter_node_559_length_1988_cov_2_74359-cat_2 virsorter_node_536_length_1343_cov_4_53318-cat_2 clostridium~phage~phicd146 bordetella~virus~bpp1 virsorter_node_27_length_13648_cov_10_3894-cat_1 virsorter_node_16_length_5962_cov_37_6794_id_31_gc022959_gc022959-cat_2 virsorter_node_29_length_18268_cov_15_4076-cat_1 virsorter_node_110_length_5017_cov_16_0623-cat_1 cyanophage~natl1a-7 virsorter_node_51_length_6048_cov_17_5609-cat_1 virsorter_node_218_length_2204_cov_4_58862-cat_2 virsorter_node_3_length_38384_cov_31_6491_gc021830_gc021830-cat_2 virsorter_node_27_length_8525_cov_17_6017-cat_2 virsorter_node_2_length_41612_cov_24_7299-cat_1 escherichia~phage~tl-2011b virsorter_node_361_length_3416_cov_6_31087-cat_1 virsorter_node_91_length_5972_cov_36_3963-cat_2 virsorter_node_376_length_1948_cov_3_65526-cat_2 synechococcus~phage~s-cbs3 virsorter_node_48_length_6491_cov_22_8788-cat_2 virsorter_node_116_length_5103_cov_33_1303-cat_2 virsorter_node_28_length_11201_cov_16_625-cat_2 pseudomonas~phage~phipsa2 virsorter_node_253_length_2462_cov_8_22893-cat_2 virsorter_node_810_length_1414_cov_2_10995_gc021842_gc021842-cat_1 virsorter_node_285_length_4031_cov_5_88012-cat_2 virsorter_node_245_length_3176_cov_5_55082-cat_2 virsorter_node_11_length_22035_cov_75_4007-cat_2 xanthomonas~phage~xp15 virsorter_node_399_length_2511_cov_12_2247-cat_2 virsorter_node_51_length_6198_cov_78_8288_id_1234693-cat_1 virsorter_node_1872_length_1092_cov_1_13005-cat_2 virsorter_node_1511_length_1484_cov_3_12722-cat_2 virsorter_node_1195_length_1353_cov_8_8895-cat_2 virsorter_node_69_length_7221_cov_10_606-cat_2 virsorter_node_555_length_1364_cov_3_66667_gc021836_gc021836-cat_2 virsorter_node_3_length_41166_cov_1412_67-circular-cat_2 virsorter_node_23_length_13188_cov_34_6875-cat_1 virsorter_node_9_length_34390_cov_51_9275-cat_2 virsorter_node_20_length_21130_cov_32_3079-cat_2 virsorter_node_10_length_37636_cov_94_1194-cat_2 virsorter_node_253_length_5676_cov_8_48509-cat_1 virsorter_node_174_length_3625_cov_24_6435-cat_2 virsorter_node_63_length_10962_cov_10_2398-cat_1 virsorter_node_4_length_95574_cov_53_2034-cat_2 virsorter_node_44_length_12771_cov_25_968-cat_2 virsorter_node_93_length_5511_cov_39_8675-cat_1 virsorter_node_1_length_63229_cov_49_3797-cat_2 virsorter_node_1_length_62610_cov_351_397-cat_2 virsorter_node_428_length_1474_cov_31_388-cat_2 virsorter_node_10_length_8122_cov_44_0481_gc021842_gc021842-cat_2 virsorter_node_84_length_7309_cov_13_3547-cat_2 virsorter_node_113_length_3110_cov_6_34454-cat_1 virsorter_node_156_length_3846_cov_90_3208-cat_2 virsorter_node_3_length_52558_cov_119_096-cat_2 virsorter_node_58_length_2924_cov_7_11907-cat_1 virsorter_node_136_length_1738_cov_8_4401-cat_2 virsorter_node_119_length_3091_cov_3_65196-cat_2 virsorter_node_71_length_5867_cov_6_41969-cat_2 virsorter_node_3_length_41933_cov_233_011-cat_2 virsorter_node_258_length_3503_cov_8_13689-cat_2 virsorter_node_321_length_1425_cov_3_60831-cat_1 virsorter_node_3_length_47484_cov_53_1945-cat_2 virsorter_node_6_length_18914_cov_22_0195-cat_2 virsorter_node_58_length_6232_cov_20_7313-cat_2 family were identified most frequently (fig. 1b) . this is partly because it is one of the largest viral families and is made up of at least 29 genera, many of which are transmitted through the fecal-oral or respiratory route (28) . most of these infections were in pools of individuals less than 20 years of age (fig. 1c) . this finding is consistent with previous findings from cameroon, where a high prevalence of ev in children was reported using pcr-based approaches (65) . furthermore, most of the evs here were of genotype c, also supporting a recent study that identified a high rate of ev cs in the northern regions of cameroon (31) . therefore, the high prevalence of ev c is probably national. however, the absence of genetic relatedness between the cameroonian human ev strains and animal strains (from chimp and gorilla [66] ) does not indicate interspecies transmission of evs from animals. additionally, we report for the first time (nearly) complete genomes of picornaviruses of the genera parechovirus, cardiovirus, hepatovirus a, and cosavirus from cameroonian patients. this broadens the range of picornaviruses found in the cameroonian population, indicating that picornaviruses might be playing a vital role in gastroenteric viral infection in the cameroonian population, especially given that most of these were from samples of sick children. rotavirus a (rva), a common viral gastroenteritis-causing agent, was identified only in a limited number of pools. this was previously observed in cameroon, and possible reasons for the low prevalence could include the acute nature of rotavirus infections or seasonal changes in rotavirus infections (6) . of note, rotavirus vaccination was introduced in cameroon in april 2014, coinciding with the period of sample collection of this study (february to september 2014); however, the vaccination campaign had not started in the sampling locations within this period, and therefore, the result represents a prevaccination rotavirus prevalence status. the identified rotavirus strain showed 3% nt differences with the vaccine strain, further suggesting that this was a wild-type rva strain, rather than a vaccine-derived strain. uncommon human gastroenteric virus: mammalian orthoreovirus (morv). this first morv strain from cameroon showed topological incongruence in its phylogeny, thereby pointing to possible reassortment events in the past. the phylogenetic clustering of some segments to strains from animals (rodents and bats) could be an indication of a zoonotic event or could also be due to the absence of related strains in databases from an unknown host. given that this strain was from a pool of samples from two children suffering from severe diarrhea, it is not unlikely that this strain might have contributed to the disease. therefore, morv might be playing a greater role in diarrheal diseases in this region than was previously known. hence, extensive epidemiological studies in different regions and in different hosts are required to fully delineate the prevalence, genomics, and interspecies transmissibility of morv. viruses not (yet) associated with gastroenteritis: picobirnaviridae, smacovirus, and anelloviridae. apart from the above-mentioned gastroenteritis-related viruses, several other viruses with unelucidated gastroenteric roles were also identified in this study. first, we observed fewer reads of picobirnaviruses (pbvs) in pools from children than in adults. previous studies also detected a relatively low percentage of children with pbvs (67, 68) . this therefore adds up to the notion that pbvs are likely to be absent in infants and young children and only start to increase with age and potentially a changing diet, though this needs to be further proven (69, 70) . interestingly, the genetic relatedness of a human picobirna-like virus with one that was found in a bat pool from the same region suggests an interspecies transmission. however, these picobirna-like sequences are translated using an alternative mitochondrial codon, indicating that their hosts may not be mammals. a principal component analysis of the codon usage bias of different known mitochondrial genome sequences, mitoviruses, and pbvs seems to suggest that they may have the same lifestyle as mitoviruses known to infect fungal mitochondria (71) . however, the recent identification of a bacterial ribosomal binding site in pbv genomes suggests prokaryotes as a potential host (72) . given that the mitochondria have descended from ancient eubacterial endosymbionts (73) , this may explain the clustering of these pbvs with mitoviruses. therefore, the question about the true host of pbvs remains controversial. second, for the first time, two strains of african smacovirus (scv) were identified in cameroonian samples. their genetic relatedness to a chimpanzee strain (isolated from a captive chimp in a zoo in san francisco) and a strain from a child from the united states (54, 55) indicates either an interspecies transmission event or the presence of a shared viral host in both humans and chimps. although the role of smacovirus in gastroenteritis has not been elucidated, their presence in cases of unexplained diarrhea in french patients seems to indicate a potential role in gastroenteritis (54) ; hence, these could be instances of interspecies transmissions. the percentages of pools positive for anelloviruses were higher in age categories of children and the old and lower for the middle-aged groups. given the well-established notion that infants and the elderly have reduced immunity (74, 75) , this could be in line with previous studies that suggest a link between the burden of anelloviruses and host immune competence (76) (77) (78) . despite their ubiquity, anelloviridae have an undefined implication in hosts' health and are thought to be probably asymptomatic (harmless) or even beneficial. however, they have been associated with hepatitis, pulmonary diseases, hematologic disorders, myopathy, and lupus, but it is not clear if their presence is the cause or the result of disease progression (79) (80) (81) (82) . human viruses and interspecies transmission from bats. in bats from the same area, we were able to identify gastroenteritis-related and nonrelated viruses. here, the corresponding viruses identified from the families astroviridae (astrovirus), caliciviridae (sapovirus), and reoviridae (rva) are genetically diverse from those identified in bats from the same region, indicating no evidence of recent interspecies transmissions between bats and humans (63) . however, genetic relatedness of human morv to animal strains showed the possibility of zoonosis between humans and not only bats but animals in general. additionally, the presence of some cameroonian strains of scv and pbv in bats or other animals would indicate interspecies transmissions if their infectivity in these animals is fully elucidated. human and bat phageome. in this study, we detected a huge phage community with a great diversity beyond the range of known bacteriophages in reference databases, potentially representing the gut microbiome diversity in the patients (83, 84) . overall, this further supports the idea that the full phageome richness is still to be completely elucidated (85) . furthermore, network analysis indicates the presence of completely novel phage groups and that phage genera in the gut microbiota might be shared between humans and bats. conclusion. several diverse viruses were discovered in the gut virome of cameroonians. some of these were already known to be the causative agent of gastroenteritis, whereas others are likely to be the cause of gastroenteric problems in the patients. further screening of patients for these viruses will be needed to establish their prevalence in the population, allowing for more appropriate measures and treatment and prevention of viral gastroenteritis. also, to be able to completely elucidate the role of the novel viruses like pecovirus and smacovirus, more studies are required. further attention should also be given to newly identified viruses (for example, morv) and their potential as emerging pathogens in the human population. ethical authorization. ethical authorization for the use of human samples was obtained from the cameroon national ethics committee, yaoundé. all human experiments were performed in accordance with the ministry's national ethics committee guidelines. sample collection and preparation. human fecal samples were collected between february and september 2014, after informed consent was obtained from patients in two different hospitals (lysoka health district and kumba district hospital of the southwest region of cameroon). this region was chosen because here bats are hunted, sold, and eaten. diarrheic patients and/or people who came into contact with bats directly (by eating, hunting, or handling) or indirectly (if a family member was directly exposed to bats) were eligible for sampling. a total of 221 samples were collected from subjects between age 0 and ͻ3 years (age group a, 80 samples), 3 and ͻ20 (age group b, 63 samples), 20 and ͻ60 (age group c, 65 samples), and 60 and older (age group d, 13 samples). all the samples were from people who had symptoms of gastroenteritis, except 2 from age group c who had contact with bats. samples were then placed into labeled tubes containing universal transport medium (utm), placed on dry ice, and stored at ϫ20°c, until being shipped to the laboratory of viral metagenomics, leuven, belgium. the samples were stored at ϫ80°c until used (63) . fecal samples were first diluted using utm, and equal volumes of the dilutions were pooled based on the location, age, and bat contact status (direct, indirect, or none). each pool contained two to five samples, and for the different age groups (a to d) we had 22, 17, 20, and 4 pools, respectively . the pools were then treated according to the netovir protocol (86) . briefly, the pools (10% [wt/vol] fecal suspensions) were homogenized for 1 min at 3,000 rpm with a minilys homogenizer (bertin technologies) and filtered using an 0.8-m pes filter (sartorius). the filtrate was then treated with a cocktail of benzonase (novagen) and micrococcal nuclease (new england biolabs) at 37°c for 2 h to digest free-floating nucleic acids. total nucleic acids (both rna and dna) were extracted using the qiaamp viral rna minikit (qiagen) according to the manufacturer's instructions but without addition of carrier rna to the lysis buffer. first-and second-strand synthesis and random pcr amplification for 17 cycles were performed using a slightly modified whole-transcriptome amplification (wta2) kit procedure (sigma-aldrich). wta2 products were purified with msb spin pcrapace spin columns (stratec), and the libraries were prepared for illumina sequencing using a slightly modified version of the nextera xt library preparation kit (illumina), which is described in detail in reference 86. samples were pooled in an attempt to obtain an average of approximately 10 million paired-end reads per pool. sequencing was performed on a nextseq 500 high-output platform (illumina) for 300 cycles (2 ϫ 150-bp paired ends). genomic and phylogenetic analysis. ngs reads were analyzed as described in the work of yinda et al. (20, 63) . briefly, raw reads were trimmed using trimmomatic (parameters: headcrop: and fastuniq to remove identical reads. the de novo assembly or reads and annotation of reads were performed using spades (with the meta flag) and diamond (with the sensitive option using the genbank nonredundant database), respectively (61, 87, 88) . open reading frames (orfs) of contigs of interest were identified and further analyzed for conserved motifs in the amino acid sequences using ncbi's conserved domain database (cdd) (89) . nucleotide and amino acid alignments of viral sequences were done with muscle implemented in mega7 (90) or mafft (91) . substitution models were determined using modelgenerator (92) , and phylogenetic trees were constructed using raxml (93) , with the automre flag, which enables a posteriori bootstrapping analysis. all trees were visualized in figtree (http://tree.bio.ed.ac.uk/software/figtree/) and midpoint rooted for purposes of clarity. phageome analysis. contig annotation with diamond is dependent on the accuracy of the database used, and in most databases, phages are poorly annotated. however, virsorter uses a manually curated database of virus reference genomes augmented with metagenomic viral sequences sampled from freshwater, seawater, and human gut, lung, and saliva. hence, for further identification of bacteriophages, scaffolds ͼ1 kb were classified using virsorter (decontamination mode [60] ). only scaffolds assigned to categories 1 and 2 were considered bacteriophage contigs and were filtered for redundancy at 95% nucleotide identity over 70% of the length using cluster genomes (94) . then, trimmed reads from each pool were mapped using bowtie 2 (95) to the bacteriophage contigs, and the generated bam files were filtered to remove reads that aligned at ͻ95% identity using bamm (http://ecogenomics.github .io/bamm/). abundance tables were obtained and normalized for total number of reads of each sample. for the richness comparison, mann-whitney tests were used, and for the clustering, an adonis test was performed. all downstream analyses were done in r (96) using the vegan package (97) . furthermore, to identify the potential corresponding bacterial host, a database of these contigs was made to which a nucleotide blastn search (100% identity without gaps) was performed using a fasta file of crispr sequences (98) as query. these sequences correspond to different bacterial hosts, and their presence in the phage genome highlight the potential host of the phage. to see if the phage community of these humans is related to those of the bats from the same locality, a visualization of the network of both human and bat phageomes was performed using vcontact (62) . initially, proteins were predicted using prodigal (99) , and combined with the viral refseq of archaeal and prokaryotic predicted proteins. a database was generated from the contigs of bat pools, human pools, and viral refseq proteins, and blastp was performed against the combined proteins. the output of blast was used to run vcontact, and the output network was visualized in cytoscape (100). data availability. all sequences were deposited in genbank under the following accession numbers: mh608285 to mh608287 and mh933752 to mh933860 (details in table s3 ). raw reads were submitted to the ncbi's short read archive (sra) under the project id prjna491626. supplemental material for this article may be found at https://doi.org/10.1128/ msphere.00585-18. ef051629_hpev-1/bni-788st/deu/xx jn867764/hcosv/a13-np6/npl gu985458_mink_astv-sms ay720892_humanastv5-goiania/go/12/94/brazil fj890351_sea_lionastv1 human-astv_cmrhp35d hq889774_pigeonanv-sh10 human-astv_cmrhp6 z25771_humanastv-newcastle human-astv_cmrhp46 human-astv_cmrhp3 fj973620_humanastv-va1 ita fj402983_humanastv-mlb1-wd0016 jx857869_humanastv-va4/human/nepal/s5363 hq916317_bovineastv/b76-2/hk fj890352_sea_lionastv2 z25771_humanastv-newcastle hq916313_bovineastv/b18/hk 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assumptions for choice of matrix are not justified raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies clustering viral genomes in ivirus fast gapped-read alignment with bowtie 2 r: a language and environment for statistical computing. r foundation for statistical computing community ecology package the crisprdb database and tools to display crisprs and to generate dictionaries of spacers and repeats prodigal: prokaryotic gene recognition and translation initiation site identification integration of biological networks and gene expression data using cytoscape g31_rva/bat-wt/cmr/batli08/2014/g31p [42] g30_rva/bat-wt/cmr/batli10/2014/g30p [42] g3_rva/bat-wt/chn/myas33/2013_g3p [10] g25_rva/bat-wt/ken/ke4852/07/2007/g25p [6] g21_rva/cow-wt/jpn/azuk-1/2006/g21p [29] g13_rva/horse-tc/gbr/l338/1991/g13p [18] g3_rva/human-tc/jpn/au-1/1982/g3p [39] g15_rva/cow-tc/ind/hg18/1995/g15p [21] g30_rva/bat-wt/cmr/batli09/2014/g30p [42] g22_rva/turkey-tc/deu/03v0002e10/2003/g22p [35] g8_rva/human-tc/ind/69m/1980/g8p [4] 10g18_rva/pigeon-tc/jpn/po-13/1983/g18p [17] g20_rva/human-wt/ecu/ecu534/2006/g20p [28] g26_rva/pig-wt/jpn/tj4-1/2010/g26p [x] g14_rva/horse-tc/usa/fi23/1981/g14p [12] g1_rva/human-wt/cmr/cmrhp55/2014/g1p [8] g7_rva/turkey-tc/irl/ty-3/1979/g7p [17] g3_rva/bat-tc/mslh14/2012/g3p [3] g6_cow g25_rva/bat-wt/cmr/batly03/2014/g25p [43] g19_rva/chicken-tc/gbr/ch-1/197x/g19p [17] g1_rva/human-tc/usa/wa/1974/g1p[1]a8 g10_rva/human-tc/gbr/a64/1987/g10p [14] g9_rva/human-tc/usa/wi61/1983/g9p[1]a8 g30_rva/bat-wt/cmr/batly17/2014/g30p [47] g27_rva/sugarglider-tc/jpn/sg385/2012/g27p [36] g24_rva/cow-tc/jpn/dai-10/2007/g24p [33] g11_rva/pig-tc/mex/ym/1983/g11p[9]7 g16_rva/mouse-tc/usa/ew/xxxx/g16p [16] acknowledgments c.k.y. was supported by the interfaculty council for development cooperation (iro) from the ku leuven. n.c.-n. and l.b. were supported by the flanders innovation & entrepreneurship (vlaio). this work was supported by ku leuven grant ejx-c9928-stg/15/020bf. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.c the authors declare no competing financial interests. key: cord-010570-ytv7dwr0 authors: casadevall, arturo; scharff, matthew d. title: return to the past: the case for antibody-based therapies in infectious diseases date: 1995-07-17 journal: clin infect dis doi: 10.1093/clinids/21.1.150 sha: doc_id: 10570 cord_uid: ytv7dwr0 in the preantibiotic era, passive antibody administration (serum therapy) was useful for the treatment of many infectious diseases. the introduction of antimicrobial chemotherapy in the 1940s led to the rapid abandonment of many forms of passive antibody therapy. chemotherapy was more effective and less toxic than antibody therapy. in this last decade of the 20th century the efficacy of antimicrobial chemotherapy is diminishing because of the rapidly escalating number of immunocompromised individuals, the emergence of new pathogens, the reemergence of old pathogens, and widespread development of resistance to antimicrobial drugs. this diminishment in the effectiveness of chemotherapy has been paralleled by advances in monoclonal antibody technology that have made feasible the generation of human antibodies. this combination of factors makes passive antibody therapy an option worthy of serious consideration. we propose that for every pathogen there exists an antibody that will modify the infection to the benefit of the host. such antibodies are potential antimicrobial agents. antibody-based therapies have significant advantages and disadvantages relative to standard chemotherapy. the reintroduction of antibody-based therapy would require major changes in the practices of infectious disease specialists. the antibiotic era is about 60 years old. as we approach the 21st century, the progress made in controlling infections is threatened by the growing numbers of immunosuppressed individuals; the emergence of new pathogens; the reemergence of old pathogens; and the widespread resistance of organisms to antimicrobial drugs. each development poses its own set of challenges; in combination, these developments are already undermining the efficacy of antimicrobial chemotherapy. there is a worldwide epidemic of immunosuppression due to malnutrition, aids, medical therapies for cancer and autoimmune diseases, and organ transplantation. immunosuppressed hosts provide ecological niches for low-virulence microorganisms [1] . in addition, antimicrobial drugs are less effective and sometimes are unable to eradicate infection in individuals with impaired immunity. classical pathogens such as mycobacterium tuberculosis and treponema pallidum have reemerged and are difficult to treat in immunosuppressed individuals [2, 3] . the increasing frequency of drug-resistant organisms is an equally alarming development, and resistance may be developing faster than new antibiotics can be introduced [4] . for example, in one new york city hospital, the percentage of vancomycinsusceptible enterococcus faecium decreased from 86% to 26% in 2 years [5] . the problem of resistance is not limited to bacteria; there are numerous reports of fluconazole-resistant candida albicans [6] . we briefly review the use of antibody-based therapy in the early 20th century and make the case for reintroducing passive antibody administration for the treatment ofinfectious diseases. advances in monoclonal antibody (mab) technology provide new opportunities for developing antibody-based therapies. in the case of pathogens for which antimicrobial chemotherapy already exists, antibody-based therapies could be used with existing chemotherapy to improve outcome and possibly reduce the development of resistance. in the case of pathogens for which no effective chemotherapy exists, antibody-based therapies could be used for primary therapy. given the diminishing efficacy of existing antimicrobials because of widespread resistance and the difficulties of treating infections in immunosuppressed individuals, the reintroduction of antibody-based therapies is an option that should be given serious consideration. this reintroduction would require major changes in the practice of infectious disease medicine. the administration of immune serum (usually animal) for the treatment of infections was called serum therapy and was introduced in the 1890s for the treatment of diphtheria. by the 1930s, serum therapy was widely used for the treatment of table 1 . infectious diseases that were treated with antibody-based therapies in the preantibiotic era. bacterial and viral infections (table 1) . for example, in the late 1930s, 86% of patients with type i pneumococcal pneumonia admitted to boston city hospital were treated with type-specific serum [36] . from 1927 to 1932 at bellevue hospital (new york), > 3,000 patients with erysipelas were treated with serum [37] (figure 1). the efficacy of serum therapy varied with the type or severity of infection. several large controlled studies revealed that type-specific serum reduced the mortality ofpneumococcal pneumonia [7] . serum therapy also appears to have significantly reduced mortality due to meningococcal meningitis in some epidemics [7, 38] . serum therapy reduced mortality due to haemophilus irif/uenzae meningitis, but the effect was small [8] [9] [10] . serum therapy for erysipelas reduced mortality in comparison to historical controls [37, 39] . serum therapy reduced mortality in diphtheria, and antibody therapy continues to be used today to treat this disease [40] . the efficacy of serum therapy for whooping cough, anthrax, dysentery (shigella dysenteriae), and gas gangrene was uncertain. human convalescent serum was effective for prophylaxis ofmeasles, which had a mortality rate of 6%-7% in some populations [29] [30] [31] . the effectiveness of serum in the pre-paralytic stage of poliomyelitis was uncertain [31, 32] . no consistently effective sera were developed against many pathogens, including staphylococcus [41] , mycobacterium, and salmonella species [42] . the historical use of serum therapy provides lessons for the effective use and development of antibody-based therapies. accurate microbiological diagnosis was essential for successful treatment, and serum therapy was most effective when used for prophylaxis or for therapy early in the course of infection. note. this is not a complete list. [7] [7] [8] [9] [10] nevertheless, it was possible to treat established bacterial infections such as meningococcal meningitis and pneumococcal pneumonia if antibody was administered shortly after symptoms began [7] . antibodies functioned as direct antibacterial agents (e.g., pneumococcal antisera) or antitoxins (e.g., diphtherial antisera). nonphysiological and nontherapeutic animal models of infection allowed investigators to successfully identify clinically useful antibody reagents [7] . a considerable amount of basic immunologic and microbiological research was required to develop each serum, and many fundamental discoveries were made in the search for better sera. (the contributions of pneumococcal research to basic science are discussed in [43] .) sulfonamides were introduced in 1935 and rapidly became the standard therapy for many infections [44] . because antimicrobial chemotherapy had significant advantages over serum therapy, the latter was largely abandoned. systemic administration of animal sera caused fevers, chills, and allergic reactions [23, 45] ; "serum sickness," a self-limited syndrome characterized by rash, proteinuria, and arthralgias, occurred in 10%-50% of patients because of immune complex disease. in addition, strain typing was necessary for choosing pneumococcal antisera; there was significant lot-to-lot variation in serum efficacy [46] ; and dosing was based on clinical experience. inadequate dosage, delays in treatment, errors in typing, mixed infections, and complicated infections (e.g., empyema) could result in failure of serum therapy [47] . serum was expensive because of the costs of animal husbandry, antibody purification, refrigeration, and reliance on mouse protection tests for standardization. chemotherapy was less toxic and more effective than serum therapy. treatment with type-specific serum reduced the mortality of pneumococcal pneumonia from 30%-40% to 10%-20% [7] , whereas the mortality rate among patients treated with sulfonamide was 7% [48] . serum therapy reduced the mortality rate of erysipelas from 10% to 7% [37] , whereas the mortality rate for patients treated with sulfonamides was 1%-2% [49] . for meningococcal meningitis, the efficacy of serum depended on the epidemic. in the mid-1930s, waghelstein [50] reported that the mortality rate for patients with meningococcal meningitis who were treated at sydenham hospital (baltimore, md) with sulfonamide was 15.3% and for those treated with serum it was 27%. in controlled trials, the superiority of chemotherapy over serum therapy was lessevident. for example, the mortality among patients with meningococcal meningitis who were treated early with adequate serum therapy was 16.7% vs. 11.6% for sulfanilamide therapy [50] . the mortality among patients with pneumococcal pneumonia treated with antimicrobial drugs or serum was 11% and 16.7%, respectively, but there was no difference in the mortality rates among younger .. literature sent on request. is used at bellevue hospital. it is supplied in packages of one therapeutic dose of approximately ten cubic centimeters. the data cited were published in [37] . patients, and younger patients who received serum recovered faster than those who received antimicrobials [51] . in the early days of the antibiotic era there was interest in using combination therapies with antibiotics and serum. sulfonamides and serum had a synergistic or additive effect against streptococcus pneumoniae [52] [53] [54] [55] , streptococci [56] , and meningococci [57] . the finding that sulfonamides made pneumococci more susceptible to antibody-mediated phagocytosis supported the idea of combination therapy [58] . it was similarly recognized that sulfonamides, unlike antibody, did not neutralize bacterial toxins or modify the course of disease caused by diphtheria or tetanus toxins [59] . combination therapy was recommended for scarlet fever [60] , pneumococcal pneumonia [44, 54, 61] , whooping cough [20] and meningitis due to pneumococcus [8, 62] , meningococcus [8, 50, 63, 64] , or h. influenzae [8] . however, the side effects of serum made the potential benefits of combination therapy marginal [50] . in summary, serum therapy was abandoned because of toxicity, difficulty in administration, narrow specificity, lot-to-lot variation, and expense. chemotherapy was less toxic and easier to use, lots were more consistent, and it was more effective in eradicating infection. in addition, there was no need for strain typing with chemotherapy. however, it is ironic that the broad antimicrobial activity of these drugs used for chemotherapy might have contributed to widespread resistance, and today susceptibility testing is essential to the selection of appropriate antimicrobial drugs. although antibodies are no longer used directly as antibacterial drugs, they continue to be used in infectious diseases (for recent reviews see [65] [66] [67] ). the majority of antibody preparations are now human. antibody administration reduces infection in individuals with immunodeficiencies [65] [66] [67] [68] and is effective for postexposure prophylaxis against measles, hepatitis a and b, rabies, and varicella viruses. specific antibody is used for the treatment of botulism [22] , diphtheria [40] , tetanus [22] , snake bites [69] , and spider stings [70] . passive antibody administration has been used for prophylaxis and/or therapy of several viral infections, including cytomegalovirus (cmv) [71, 72] , rotavirus [73] , lassa virus [74] , varicella [75] , enterovirus [76, 77] , and parvovirus b19 infections [78] . in recent years, there has been renewed interest in the use of antibody preparations to prevent infection in high-risk groups. intravenous administration of polyclonal antibody has been reported to reduce the number of infections in patients with aids [79] [80] [81] , patients in surgical intensive care units [82] , organ transplant recipients [83] , and neonates [84, 85] (for critical reviews and commentary on this practice see [86] [87] [88] [89] ). antibody therapy is used for a variety of illnesses that may or may not be infectious, including autoimmune thrombocytopenia, kawasaki disease, and autoimmune neuromuscular diseases [65, 66] . passive antibody administration is used for prevention of rh-hemolytic disease [90] . equine lymphocyte antiserum [91] and murine mabs to lymphocytes are used to prevent organ rejection [92] . digitalis-binding antibodies are useful for the treatment of digoxin toxicity [93] . thus, antibody therapy is still widely used in medicine, but its role in the treatment of infections is limited largely to viral and toxin neutralization and replacement therapy in patients with immunoglobulin deficiencies. in reconsidering the antibody option it is clear that heterologous antibody (i.e., serum therapy) is a poor choice because of toxicity. human immune sera have fewer side effects, but there are concerns about availability, potency, and consistency. a major disadvantage of all immune sera is that specific antibodies make up a small minority of the antibody present. there is significant variation in pathogen-specific opsonic activity of commercially available immunoglobulin preparations [94] . there is also concern about the potential of human sera to transmit infectious agents [95] . the discovery of hybridoma technology in 1975 [96] and recent advances in mab technology, including the generation of humanized antibodies [97] , make mab-based therapies a more attractive therapeutic option. in contrast to polyclonal sera, mabs are homogeneous and reproducible reagents that can be generated in large amounts. generation of mabs from mice and rats is still easier and more efficient than production of human mabs. however, several technologies are available for the humanization of murine mabs and generation of human mabs [97] . mouse-human chimeric antibodies can be constructed by linking the genes expressing the mouse variable region to human constant region genes [97] . the result is a molecule that is mostly human and has a longer half-life than the murine precursor [98] . as an alternative, the antigen binding regions of murine mabs can be grafted into human antibody frameworks by molecular techniques [97] , resulting in molecules that are almost completely human in origin. other strategies to generate human antibodies are the transformation of human b cells, the generation ofrecombinant antibody libraries from human b cells, and the use of transgenic mice that have the human immunoglobulin locus. recent setbacks for mab-based therapies: historical perspective clinical trials of antiendotoxin mabs for gram-negative sepsis have produced inconclusive results [99, 100] . when the difficulties encountered in developing antiendotoxin mab strategies are considered in the context of the development of serum therapy, they do not seem unusual or unexpected. for example, passive protection with pneumococcal antisera was demonstrated in 1891 by the klemperers [101] , but 30-40 years elapsed before serum therapy for pneumococcal pneumonia was widely accepted. for pneumococcal antiserum therapy to be consistently successful, several developments were necessary. these included the appreciation of antigenic variation in pneumococci [102] , the need for type-specific sera [103] , the development of rapid in vitro assays to establish antigenic type (i.e., capsular swelling and agglutination reactions) [104] , the development of methods to standardize potency (mouse protection test) [46] , and improved purification techniques that would reduce the toxicity of serum. for meningococcal antisera the situation was reversed; flexner's serum significantly reduced mortality in early epidemics of meningitis [38] but was subsequently less effective, possibly as a result of antigenic changes in neisseria meningitidis [7] . research into better meningococcal antisera was then hampered by the lack of useful animal models, loss of strain virulence in vitro, and poor understanding of the antigenic diversity of n meningitidis [105, 106] . a lesson from the preantibiotic era is that a considerable amount of basic research on the immunology and pathogenesis of each infection is usually necessary before antibody therapy can be developed that will be successful in clinical practice. spectrum ofactivity. antibodies can modify bacterial, fungal, parasitic, and viral infections and are a class of biological agents with broad antimicrobial activity against diverse pathogens. antibody-based therapies are usually pathogen-specific and have the theoretical advantage that they should not affect the normal flora of the patient or select for resistance in nontargeted microbes. nevertheless, narrow specificity is a disadvantage because mixed infections may not be treated by a single antibody preparation. pneumonia due to s. pneumoniae with more than one serotype was recognized as a reason for the failure of type-specific serum therapy [107] . one solution is to use cocktails of mabs active against common antigenic types. cocktails may be designed to include mabs to multiple serotypes and mabs with multiple isotypes to enhance antibody effector function. pathogen-specific drugs (such as mabs) are less attractive to the pharmaceutical industry because the narrow specificity reduces the potential market for the drug. however, the increasing incidence of resistant organisms resulting from the use of broad-spectrum antibiotic regimens could make pathogen-specific drugs attractive to drug companies. precedents for the development of pathogen-specific drugs already exist in the area of antiviral drug research. mechanisms ofaction. antibodies mediate protection by a variety of mechanisms. direct antibody mechanisms of action include inhibition of attachment, agglutination (and immobili-cm 1995;21 (july) zation), viral neutralization, toxin neutralization, antibodydirected cellular cytotoxicity, complement activation, and opsonization [108] . antibody therapy has the potential to enhance immune function in immunosuppressed hosts. however, since the mechanism of action of many antibodies involves promoting microbial clearance through nonspecific cellular immunity, antibody-based therapies may be less effective in individuals with defective macrophage, neutrophil, and natural killer cell function. in this regard, it is encouraging that antibodies are effective against pseudomonas aeruginosa in neutropenic mice [109] and that they can reduce the number of infections in pediatric patients with aids [79] . antibodies are usually considered to be "protective" effector molecules of the immune system. however, not all antibody responses to pathogens are protective and some may be deleterious to the host. for example, some viral-specific antibodies are capable of enhancing infection [110] . thus, antibody molecules being considered for clinical development will require extensive testing in vitro and in vivo. studies of the mechanisms of antibody action are important for understanding the mode of protection and for designing clinical trials. pharmacokinetics. the pharmacokinetics of human igg suggests several useful characteristics for its role as an antiinfective agent. these include a long half-life (~20 days [111]), good tissue penetration [112] , and, depending on the isotype, the ability to either cross the placenta to provide antibody protection to fetuses and newborns [113] or to be excluded from the placenta if fetal toxicity is a concern. heterologous mabs (i.e., murine mabs) have shorter half-lives in humans, but chimeric and humanized mabs have longer half-lives than their murine precursors. mabs with a longer half-life can, in principle, be engineered by altering the regions of the constant domain that regulate clearance. a disadvantage of antibody-based therapies is the need for systemic administration. oral administration is unlikely to be effective, with the possible exception of therapy for enteric pathogens [73, 114] . the blood-brain barrier is a potential obstacle for antibody therapy for infections of the brain. however, in infections of the brain in which there is inflammation, there is increased antibody penetration, and intravenous administration of antibodies appears to have been successfully used for therapy of meningococcal meningitis [115] . antibodybased therapies can also be administered directly into the subarachnoid space, as has been done for the treatment ofmeningococcal and h. influenzae meningitis [8, 38] . enhanced penetration of the brain can be achieved by modifying the charge of the molecule [116] or by linking it to carrier proteins that cross the blood-brain barrier [117] . antibody therapy for brain infections may be feasible if the blood-brain barrier is leaky, if antibody is administered directly into the subarachnoid space, or if antibodies with greater brain penetration are used. additional research into the pharmacokinetics of antibodies in infection is likely to be required for optimal use of antibody-based therapies. studies of antibody binding to tumors have revealed complex pharmacokinetics [112] . there may be problems with antibody penetration into abscesses as have been found with tumors. toxicity. immunoglobulins are generally safe drugs [67] . nevertheless, serious side effects have been reported with highdose (0.5-2 g/kg) antibody therapy, including rare cases of renal failure [118] , aseptic meningitis [119] , and thromboembolic events [120, 121] . high-dose immunoglobulin therapy is unlikely to be required in antiinfective mab therapy. in the past, serum therapy was effective against various pathogens despite the fact that immune sera contained only small amounts of specific antibody. mabs have significantly higher specific activity than polyclonal preparations. for example, 0.7 mg of two anti-tetanus toxin human mabs provides the same activity as 100-170 mg of immune globulin [122] . thus, the toxicities described for high-dose immunoglobulin therapy may not be relevant in antiinfective therapies with mabs. until fully human mabs are available, rodent, mousehuman chimeric, or humanized mabs are therapeutic options. for over a decade, murine mabs directed against t cells have been used to prevent organ graft rejection in humans [92] . a murine igm to endotoxin (e5) was tested in 247 patients with sepsis and appeared to be a safe treatment [123] . although administration of murine mabs is generally well tolerated in humans, allergic reactions occur in 1%-2% of patients [123] and most patients develop antibody responses to the murine mabs that may interfere with their therapeutic function [43, 124] . experience with human-mouse chimeric antibodies and humanized mabs is accumulating rapidly as clinical trials with several compounds progress. a chimeric anti-cd4 mab for the treatment of rheumatoid arthritis has been well tolerated [125] , but approximately half the patients had infusion-related side effects (headaches, nausea, fever, and chills), which were diminished by slowing the infusion rate [125] . mouse-human chimeric and humanized mabs are less immunogenic than their murine precursors [98, 126] , but antiidiotypic responses occur after repeated treatments [127] . overall, the experience with chimeric and humanized mabs suggests that they are relatively safe compounds [98, [125] [126] [127] [128] . antigenic variation and antibody resistance. the efficacy of antibody-based therapies may be diminished by antigenic changes in the pathogen. this could be minimized by antigenic surveillance systems, as is presently done for influenza virus. antibody-resistant mutants can be generated in the laboratory [129] , and it is likely that the same can occur in patients. mechanisms of antibody resistance include mutations that change the antigenic site and protease production. antibody use may select for antigenically distinct variants. evidence for the horizontal transfer of iga protease genes exists in neisseria gonorrhoeae and it is conceivable that widespread antibody use would result in selection of protease-producing strains for many pathogens [130] . thus, large-scale use of antibody-based therapies may result in rapid emergence of antibody resistance in a manner analogous to that of antibiotic resistance. however, one advantage of antibody-based therapies is the versatility of antibody compounds. for example, emergence of an antibodyresistant strain could be countered by a new antibody directed toward the mutated epitope or another antigenic target. emergence of protease-producing strains could be countered by designing antibodies without protease cleavage sites (although these may then be recognized as foreign and be immunogenic) or by addition of antiprotease mabs. selection of antibodyresistant organisms could be minimized by using cocktails of mabs directed at multiple antigenic targets or by using a combination of antibodies and antimicrobials. cost. cost effectiveness is a significant concern that is likely to be a major obstacle for the development of passive antibody therapies. antibody prophylaxis for cmv infections can cost $4,000 to $9,000 per patient [71] . the cost effectiveness of antibody therapy for prevention of infection in leukemic patients has been questioned [88, 131] . however, in selected populations such as premature infants, antibody therapy may be cost effective [85] . furthermore, the cost of a drug when first introduced may not reflect its long-term costs given the potential for improvements in technology and production. mabs are presently made in tissue culture, so the cost of production is high. mabs can be made in bacteria or yeast, and less costly means of production might be developed [97] . in the past, serum therapy was used despite its high cost because it was believed to be effective. new antibody-based therapies are likely to be used if they prove to be effective. for pathogens such as s. pneumoniae, n. meningitidis, and h infiuenzae, there is a large body of experimental and clinical evidence to support the development of passive antibody therapy. however, for many pathogens, antibody immunity has not been proven to be important. for example, cryptococcus neoformans is a fungus for which the importance of antibody immunity is uncertain. however, administration of preformed antibodies can modify the course of cryptococcal infection in various animal models [132] [133] [134] [135] [136] , and the combination of antibody and amphotericin b is a more effective treatment than the use of either agent alone [137] [138] [139] . studies with mabs have shown that there are protective, nonprotective, and deleterious antibodies to the c. neoformans capsule polysaccharide [136] . heavy chain isotype [136, 140] and epitope specificity [141] are important determinants of protective efficacy. the existence of protective and nonprotective antibodies against c. neoformans, a fungus for which antibody immunity mayor may not be important, provides a paradigm that may be applicable to other pathogens. we propose the hypothesis that for every pathogen there exists an antibody that will modify the course of infection to the benefit of the host; such antibodies are candidates for development as antimicrobial drugs. our proposal includes the use of antibodies for intracellular pathogens; for example, anti-bodies may exist that could prevent cell entry of intracellular bacteria and/or shift the intracellular location of such bacteria by promoting internalization through fe receptors. an mab that inhibits intracellular toxoplasma gondii infection has recently been described [142] . in addition, antibodies have been described that can neutralize viruses intracellularly [143] or penetrate the nuclear membrane [144] . our proposal is not limited to those pathogens for which antibody immunity has been demonstrated to be protective. conclusions on the importance of antibody immunity are usually based on observations made with polyclonal sera (i.e., passive protection or correlation of immunity with the presence of antibody). since polyclonal sera might contain protective antibodies, nonprotective antibodies, and disease-enhancing antibodies, the existence of protective antibodies cannot be ruled out on the basis of absence of protection in experiments involving passive transfer of polyclonal sera or immunizations. conversely, experiments that demonstrate that polyclonal sera can mediate protection indicate the existence of protective antibodies. for pathogens for which polyclonal antibody has shown no protection, the question of whether useful antibodies exist must be reevaluated with mabs since the presence of nonprotective or deleterious antibodies in polyclonal sera could create confounding variables. a corollary of our proposal is that antibody-based therapies may be developed against pathogens for which antibody immunity is not considered to be important. infections that are difficult to treat and that can be modified by antibody immunity provide a logical starting point for the development of antibody-based therapies. antibodies have historically been more effective in prophylaxis than in therapy. antibody-based therapies have traditionally been most effective in infections where viral and toxin neutralization modifies the course of the disease. however, most of the historical experience was gained with polyclonal preparations of uncertain composition and mayor may not be applicable to mab preparations with high activity. opportunistic infections. infections with low-virulence organisms in immunosuppressed individuals are often difficult to treat and are sometimes incurable. since the problem is deficient immunity, antibody therapy is an attractive option because antibodies can enhance immune function. table 2 lists examples of opportunistic pathogens for which antibody can modify the course of infection. antibiotic resistance. drug-resistant organisms are targets for development of antibody-based therapies. the spread of penicillin-resistant s.pneumoniae, methicillin-resistant staphylococcus aureus, and multiply-drug-resistant e. faecium has limited the useful antibiotic arsenal against these pathogens [4, 6, 153] . for pneumococcus, antibody therapy has been shown to be useful [7] . antibody may be useful against staphylococcal infection [154, 155] . for e.faecium, the role of antibody immu-ern 1995;21 (july) table 2 . opportunistic pathogens for which experimental data suggest a potential role for antibody-based therapies. nity is uncertain, but some patients make opsonic antibodies, which might be protective [156] . significant work has already been done in the development of pseudomonal antibodies for resistant gram-negative organisms (table 2). the addition of antibody-based therapies to chemotherapy may slow the development of resistance and improve outcome. toxin neutralization. the ability to neutralize toxins is a unique characteristic ofantibody-based therapies. antitoxin antibodies are useful for the treatment of diphtheria, tetanus, botulism, snake bites, and black widow bites (see above). recent attempts to develop antibody therapy for gram-negative sepsis have focused on mabs to neutralize endotoxin [123, 157, 158] and cytokines [159] . pseudomonas aeruginosa sepsis, combination therapy with mabs to endotoxin and tumor necrosis factor was superior to therapy with a single mab [160] . examples ofpotential targets for antibody-based therapy include the toxins and proteases associated with toxic shock syndromes [161, 162] . combination therapy with antibodies to exotoxins could improve outcome, but cocktails of mabs may be necessary because of toxin heterogeneity [162] . intravenous immunoglobulin administration has been associated with dramatic clinical improvement in a patient with streptococcus pyogenes toxic shock syndrome [163] . antivirals. virus neutralization is an established property of antibodies. in the preantibiotic era, serum was used for prophylaxis ofmeasles, chickenpox, mumps, and poliomyelitis. recently, the potential of antibody therapy against many viruses, including hepatitis b [164] , hiv [165] , respiratory syncytial virus [166] , cmv [150] , and parvovirus [167] , has received considerable attention. newly identified viral illnesses are targets for antibody drug research. combination ofantibody therapy and chemotherapy. antibody-based therapies are unlikely to be used alone unless they are the only available therapy or are being used for prophylaxis of infection. combinations of antibody therapy and chemotherapy offer theoretical advantages. antibodies promote microbial killing directly by a complement-mediated lytic process or indirectly by enhancing nonspecific immune mechanisms. table 3 lists examples of bacterial, fungal, and viral pathogens for which combination therapy has shown promise. combination therapy could reduce the amount of either agent required to table 3 . pathogens for which the combination of chemotherapy and antibody based therapy has shown some promise. pathogen chemotherapy antibody [57, 168] neisseria meningitidis sulfanilamide horse immune sera [55] streptococcus pneumoniae rabbit immune sera [8, 9] haemophilus infiuenzae sulfonamide rabbit, horse immune sera [169] staphylococcus aureus sparftoxacin human igg1 mab [172] p. aeruginosa human igm mab [173] p. aeruginosa murine e5 lps antibodies [170] p. aeruginosa human gamma globulin [172] escherichia coli murine e5 lps antibodies [174] shistosoma mansoni praziquantel rabbit immune sera [175] candida albicans amphotericin b human gamma globulin [137] cryptococcus neoformans amphotericin b rabbit immune sera [138, 139] c. neoformans murine igg1 mab [176] c. neoformans murine igg1 mab [177] lassa virus ribavirin monkey immune sera [72, 178] cytomegalovirus ganciclovir murine immune sera [179] herpes simplex virus adenine arabinoside rabbit and human sera [180] herpes simplex virus acycloguanosine human immune globulin note. this is not a complete list. achieve a therapeutic effect. for example, some antibiotics are quite toxic, and the ability to reduce doses could lessen side effects. in a similar vein, the addition of chemotherapy to antibody therapy could reduce the amount of antibody necessary to achieve a therapeutic effect, which would lessen the cost of therapy. the availability of broad-spectrum antibiotics has diminished the need for making exact microbiological diagnoses. for example, gram-negative sepsis or presumed fungal infections can be treated with empirical therapy without identifying the pathogen. this situation is in contrast to the practice of infectious diseases in the 1930s when identification of the pathogen (and its serotype) was necessary for choosing the correct antiserum. for antigenically diverse pathogens such as s. pneumoniae, rapid protocols were developed for typing strains. the present practice of infectious diseases is not unlike a gambling strategy where the probability of infection by a given microbe is matched to the likelihood of activity by the available antibiotics. this has resulted in great emphasis on broad-spectrum antibiotic "coverage" and less emphasis on making an exact microbiological diagnosis. although the relative merits of this practice are beyond the scope of this article, widespread antimicrobial resistance is decreasing the effectiveness of existing antibiotics, and increased caution is warranted when designing broad-spectrum combinations. a 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infection protection of mice against meningococcus infection by polyvalent antimeningococcic serum combined protective action of human gamma globulin and antibiotics when administered simultaneously in experimental staphylococcal infections synergism between human gamma globulin and chioroamphenicol in the treatment of experimental bacterial infections combined serum and sulphanilamide in the treatment of streptococcal infections in mice monoclonal antibodies for treatment of gram-negative infections efficacy of anti-endotoxin monoclonal antibody e5 alone or in combination with ciprofloxacin in neutropenic rats with pseudomonas sepsis praziquantelinduced exposure of schistosoma mansoni alkaline phosphatase: drugantibody synergy which acts preferentially against female worms. parasite immuno11994 effects of immunoglobulin g and low-dose amphotericin b on candida albicans infections in burned mice combination of 5-flucytosine and capsule-binding monoclonal antibody in therapy of cryptococcus neoformans infection enhanced treatment of lassa fever by immune plasma combined with ribavirin in cynomolgus monkeys -3-dihydroxy-2-propoxymethyl) guanine prevents death but not immunity in murine cytomegalovirus-infected normal and immunosuppressed balb/c mice combined effects of acycloguanosine and humoral antibodies in experimental encephalitis due to herpesvirus hominis synergistic antiviral effects of adenine arabinoside and humoral antibodies in experimental encephalitis due to herpesvirus hominis unequalled but unobtainable key: cord-009792-e2vvi8qo authors: pandit, sb; balaji, s.; srinivasan, n. title: structural and functional characterization of gene products encoded in the human genome by homology detection date: 2008-01-03 journal: iubmb life doi: 10.1080/15216540400006105 sha: doc_id: 9792 cord_uid: e2vvi8qo availability of the human genome data has enabled the exploration of a huge amount of biological information encoded in it. there are extensive ongoing experimental efforts to understand the biological functions of the gene products encoded in the human genome. however, computational analysis can aid immensely in the interpretation of biological function by associating known functional/structural domains to the human proteins. in this article we have discussed the implications of such associations. the association of structural domains to human proteins could help in prioritizing the targets for structure determination in the structural genomics initiatives. the protein kinase family is one of the most frequently occurring protein domain families in the human proteome while p‐loop hydrolase, which comprises many gtpases and atpases, is a highly represented superfamily. using the superfamily relationships between families of unknown and known structures we could increase structural information content of the human genome by about 5%. we could also make new associations of domain families to 33 human proteins that are potentially linked to genetically inherited diseases. iubmb life, 56: 317‐331, 2004 an important event in the history of humankind has been made at the turn of the 21st century in the form of the draft version of human genome data (1, 2) . this monumental effort provides us with an opportunity to better understand the various biological processes and possible cognition. the functional characterization of the gene products encoded in the human genome could provide insights into such complex biological process. there are various experimental endeavors to understand the functions of gene products encoded in the human genome. in addition to these experimental efforts, computational analyses of human proteins could form an important step in the functional inference of the genome data. the computational approaches for the prediction of functional features of proteins encoded in genomes relies on establishing relationships to homologues that are experimentally studied. there have been several attempts, using various sophisticated homology search tools, to assign functions to gene products encoded in various proteomes (see for example references 3 -8) . such functional predictions could be used as a guiding tool in order to direct the relatively time consuming, more difficult and expensive experimental methods for exploring protein functions. furthermore, functional inferences of human proteins that are implicated in diseases could provide valuable insights on the molecular basis of human diseases. such an understanding could aid identification of effective drug targets and rational design of lead compounds to combat the diseases. the most commonly used computational approach for genome-wide association of functions to proteins is by identification of well-characterized homologues using sequence-based search procedures such as blast (9) and fasta (10) . but, pairwise sequence alignment based search procedures are unlikely to be able to identify related proteins with low sequence similarity. however, these distantly related proteins could often be identified with the use of threedimensional (3-d) structural information (11) as the structure is conserved better than sequence during evolution (12, 13) thus, use of structural information could potentially enhance the functional assignments (14 -17) . moreover, structure prediction with relevant biochemical motifs can provide more detailed functional insights than sequence comparisons alone (18 -20) . the search methods for such an analysis could be improved by the use of multiple sequence alignment of the homologues in a family, which can indicate structurally/ functionally important positions. the information in these multiple sequence alignments can be converted into position specific scoring matrices (pssm) usually referred as profiles (21) or into a probabilistic model called the hidden markov model (hmm) (22) . the use of profile-based search methods is known to improve sensitivity of detection of remotely related homologues (23 -28) . hence, use of structure and profile-based method would enable detection of remote homologues and thus enrich the functional assignments. some of the commonly used profile-based search methods include psi-blast (25) , impala (29) , rps-blast and hidden markov model (hmmer2)-based (30) procedures. these methods have been shown to detect remote and subtle similarities (31, 32) between proteins that were previously possible only by structure comparison procedures, which obviously demand the knowledge of 3-d structures. although the profile-based annotation methods are among the widely used procedures to detect remote similarities, there are other procedures such as genthreader (33) and environment-based profiles (34) , which are fold recognition methods for assigning the structural domains to amino acid sequences. furthermore, the comparison of proteins across various genomes could also aid in enhancement of the assignment of functions to the proteins (34 -41) . the comparative genomics methods are based on the functional characterization of the gene products by detecting the orthologous proteins in the closely related organisms where the experimental functions of the proteins have been proposed. however, the effectiveness of this approach is dependent on at least two factors: (1) ability to identify homologues of a given protein in other organisms. (2) the extent of divergence of amino acid sequences of the homologues across the organisms and its implication on the similarity of functions between the two proteins. in order to understand the biological function of the human proteins we have associated functional or structural domains using sensitive profile-matching procedures. association of functional domain would provide clues to biochemical role of the protein. the structural domain association could provide enhanced abilities to assign function and also provide the molecular basis of action of proteins. furthermore, we have enhanced the structural information content of human genome by relating families apparently with unknown structures to known structural families, as in the supfam database, which was developed by us earlier (26, 27) . the functional domain information was considered from pfam database (http://www.sanger.ac.uk/software/pfam) (42) and structural domain from pali (http://pauling.mbu.iisc. ernet.in/*pali) (43, 44) databases. pali contains structurebased sequence alignment and phylogeny of proteins in the families derived from the scop database (45) , which is a hierarchical structural classification database. the profiles for pfam and pali families have been generated as described by pandit et al (26) . these profiles were searched using rps-blast. we have also used hmm-based search procedure against the hmm libraries of pfam to associate functional domain to human proteins. profiles of transmembrane sequences have been associated with human proteins in order to predict membrane localisation of the proteins. using sensitive profile-matching procedures, we could make a comprehensive compilation of functional/structural domains to gene protein encoded in human genome. similar attempts have been made in the past by muller et al. (38) . they have used psi-blast for much of their analysis, apart from impala, to assign structural/functional domains. in their approach psi-blast has been scanned against the nonredundant database of protein sequences augmented with scop domain sequences. in the present review we will discuss the current status of large-scale function association, using various computational procedures, of various gene products encoded in the human genome. furthermore, human proteins potentially involved in diseases have been specifically analyzed by associating functional/structural domain to these protein sequences. the amino acid sequences of the open reading frames (orfs) that correspond to the gene products encoded in human genome have been obtained from the ensembl database (46) (release 22.34d.1, http://www.ensembl.org). the total number of gene products predicted in this release is 29,031. the omim database (47) is a comprehensive collection of genes and genetic disorders in humans. in our analysis the protein sequences corresponding to the entries in the omim database have been derived from the swissprot database (48) . it is also possible to obtain details of genes involved in genetic disorders through the genelink table provided at ensembl database (46) . the genelink table indicates the association of human proteins to omim identifiers. we were able to associate 6257 unique protein sequences, which have one or more reference, to the omim database. the number of omim entries referenced in swissprot is 6770, as in march 2002 release of the swissprot database. a possible reason for the difference in the numbers of genes could be that more than one genetic disorder entry in omim database is associated with a given protein. in the data set used by muller et al. (38) there were 5856 proteins linked to omim database entries. we have used profile-matching method rps-blast that matches a sequence to sequence-profile obtained from structural (pali -release 2.2) and functional domain (pfam -version 10.0) families. we used stringent e-value cut off of 3 6 10 75 in our search methods to ensure reliability of the domain association. this e-value cut-off has been extrapolated from the one reported by schaffer et al. (1999) (29) as well as based on the benchmarking (n. s. mhatre, b. anand and n. srinivasan, unpublished results) using the database of structure-based sequence alignments of similarly folded proteins. we have used hmmer based procedure against pfam hmm profiles with an e-value cut off of 10 72 to extract reliable domain association. subsequent to functional/ structural domain identification, all the sequences were subjected to tmhmm2.0 (49) in order to assign transmembrane helical regions. the functional and structural domain assignments for human proteome along with other organisms are made publicly available at: http://hodgkin.mbu.iisc.ernet.in/*human. the following sections discuss some of the interesting results derived by analysing this large dataset of human proteins. we could assign a total of 52,297 functional/structural domains to 21,835 (75%) human proteins out of 29,031 proteins encoded in the human genome. we also surveyed for transmembrane regions in human proteins, since this would suggest putative localization of these proteins to the membrane hence, could aid in function prediction. using tmhmm (49) we could identify transmembrane regions in 6777 gene products. of these, 5424 are found to be present in combination with extracellular or intracellular functional/ structural domains. the total number of residues covered in structural/ functional domain and transmembrane region assignments are about 42% of the proteome. these functional/structural domain assignments would indicate probable biochemical functions for the assigned proteins, which could be useful for biological function prediction. a total of 7196 human proteins with no domain assignment, hence with no function or cellular localization information, could form an interesting set for experimental exploration for their properties and biological roles. the association of gene products with structure can give valuable insights, since structural information provides molecular details of the function of a protein. the structural domain assignment will also help in prioritizing the target for structural genomics consortium by indicating gene products with no structural predictions. with a view to enhance structural information present in human genome, we have used structural information as in pali profiles that is generated using structure-dependent sequence alignments of a large number of protein domain families, since the incorporation of 3-d structural information could aid in effective detection of remotely related proteins. using pali profiles alone, we could associate additional 1191 structural domains to 1076 human proteins that are remotely related. furthermore, we tried relating families with unknown structures to known structural families as in supfam database, which was developed by us earlier (26, 27) in order to enhance information on the structural content of human proteome. the supfam database relates two or more homologous protein families, of either known or unknown structure, using profiles derived from structure-based sequence alignments. integrating the relationships derived in supfam we could provide structural information for an additional *5% of domain families (fig. 1 ). these family assignments would increase known structural content in the genome. a total of 2669 pfam families are assigned in human genome, of which 1195 pfam families, have structural information documented in pfam. out of 1474 pfam families, apparently with no structural information, 129 families could be related to a family of known structure in supfam. there are now 1324 families (*50%) with structural information known directly or indirectly through relationships present in sup-fam (fig. 1 ). these 1324 unique families with structural information are present in 40,947 domains, hence would provide further insights into their functions. a total number of 52,082 functional domains could be assigned to the human proteins. these assigned functional domains belong to 2669 sequence/functional families of the pfam database (42) . we have surveyed for the most commonly occurring pfam families in human genome. fig. 2 shows the most frequently occurring functional domain families in the human genome. the most frequently occurring family is the zf-c2h2 family, which is a classical zinc-finger domain with very short length (typically 25 residues). identification of such a family with short motifs using bioinformatics tools could be unreliable. hence, we did not consider them in our analysis. the other most frequently occurring globular protein family is protein kinase. it was previously shown that protein kinases occur with typical and atypical combination of domain families in the gene products encoded in human genome. these kinase domain-containing proteins are involved in a wide variety of biological roles (50) . among the other most frequently occurring pfam families, the majority are involved in or in part responsible for protein-protein interactions (immunoglobulin, ankyrin repeat, tpr domains), cell attachment adhesion function (fibronectin, collagen, cadherin domains), signalling function (ph, sh3, c2 domains), nucleic acid binding function (zf-c2h2, homoeobox, rrm domains). a considerable number of human proteins are characterized by short lengths, although they match significantly with protein domain families which are typically much longer. there are 129 functional families, associated in 1144 human proteins, apparently with no structural information but could be associated to distantly related families of known structures using relationship described in supfam. these 1144 proteins have 1184 number of domains. most of these 129 families correspond to enzymes. from the structural genomics perspective, structure association for 129 pfam families meant that clues about structure and function could be extended for 1144 proteins. some of the pfam protein families are known to be characteristic of prokaryotic organisms or viruses only. however members of some of these families from human genome could be identified from the current analysis and these families are referred to as atypical families. we could associate 16 bacterial specific families and 18 viral specific families to 41 and 188 human proteins respectively. the list of bacterial and viral specific families, identified in human, along with associated gene products in human genome are listed in table 1a and 1b respectively the complete list of proteins with the region of pfam domain assignment is made available at http:// hodgkin.mbu.iisc.ernet.in/*human. the assigned domain family includes for example cobalamin biosynthesis protein, minor capsid protein, bacteriophage lambda head decoration protein. such functions have not been shown before to be present in humans. there are two possible explanations that could be drawn in the context of occurrence of the bacterial and viral specific families in human genome. first explanation is that the superfamily relationship exists between the assigned bacterial or viral domain families and the corresponding eurkaryotic domain families as the sequence similarity of these domains with human proteins is low, while significant. these regions in the human proteins could have diverged significantly and sequence data corresponding to these families in other eukaryotes is currently lacking. an alternative possibility is horizontal gene transfer of these bacterial/viral specific families to humans. we surveyed the human genome for the occurrence of specific pfam families, which are known to be present only in eukaryotes. such eukaryote specific families are known to be involved in specific functions in eukaryotes. out of 2229 eukaryote specific pfam families, we could not associate 1054 eukaryotic specific families to the human proteome. further, we assessed reasons for the absence of these eukaryotic specific families in human genome. most of these families are organism or lineage specific. some of them have no known functions and other, as mentioned below, are involved in functions not required or present in humans, hence not identified in the human proteome. the probable reasons for absence of eukaryotic specific pfam families in human genome are: (1) class of toxin families (which also includes snake and scorpion toxins) (2) families that are unique to the plant kingdom, like seed storage class of proteins, potato inhibitor and plant disease resistance response protein. the zf-c2h2 has been excluded from this histogram due to low reliability in assignments of short domains. protein srb, c. elegans sre g protein-coupled chemoreceptor and c. elegans srg family integral membrane protein. this analysis showed that human proteome has eukaryotic specific families (1175) which are involved in eukaryotes-like functions. however, absence of some of the eukaryotic specific families could be explained from the observations that such biochemical functions are undesirable for human or they are highly specific to lower eukaryotes. the pfam families, without known 3-d structure, could be clustered into sequence superfamilies and such superfamily relationships are documented in the supfam database. in the current release of supfam, 96 of the 3904 pfam families, with no structural information, could be clustered into 39 new potential superfamilies. it is expected that members of all the families in each new potential superfamily would share the same fold and might have gross similarity in their functional properties. these relationships could help in prioritizing the target for structural genomics, since the 3-d structural determination of one of the representative member in each superfamily would result in 39 structures that can serve as framework models. using these sequence superfamilies information we could identify 18 of the 39 sequence superfamilies in the human genome. the list of these new potential superfamilies with their constituent families identified in human genome is listed in table 2 . the 18 sequence superfamilies identified in human genome consist of 25 pfam families, with no known 3-d structure for any of their members. there are 371 domains belonging to these 18 new potential superfamilies that could be assigned to the 367 unique gene products in human genome. hence, an experimental structure for 18 domains or proteins one each from these superfamilies could provide templates for interpreting the functions of other members in the superfamily. this results in substantial reduction (from 371 to 18) in the number of 3d-structures to be determined experimentally in order to get clues about their functions experimentally. these superfamilies may be considered as priority targets for structural genomics initiatives in order to improve the coverage of structural information for the human proteins. the nature of the superfamily relationships for some of the new potential sequence superfamilies that are identified in human genome is discussed further. this superfamily consists of three families namely patched domain, acr_tran and secd_secf domain families. the acr_tran family is an integral membrane protein family whose members are known to be involved in drug resistance in bacteria (51) . the other family in this superfamily, patched domain, is a receptor for the morphogene sonic hedgehog and transduces hedgehog signals (52) . this secd and secf family consists of various prokaryotic secd and secf protein export membrane proteins (53) . we could identify 16 human proteins with patched family domain assigned. the functional and structural elucidation of other two families could be could be extended to patched domain because of superfamily relationships. this superfamily has four families cluster together viz. sugar_tr, oatp_c, duf791 and duf894. the sugar_tr family is single-polypeptide capable only of transporting small solutes, such as sugar, in response to chemiosmotic ion gradients and lies in uniporter-symporter-antiporter family (54) . oatp_c is eukaryotic organic-anion-transporting polypeptides that vary in tissue distribution and substrate specificity (55) . the functions of dufs (domains of unknown function) are not known. we could associate sugar_tr and oatp_c domains to 85 and 16 gene products respectively. this superfamily constitutes methyltransf_4 and trna_u5-meth_tr pfam families. both families have methyltransferase activity, however, the trna_u5-meth_tr family is involved in methylation of t-rnas (56) . we could identify 1 and 5 human homologues of methyltransf_4 and trna_u5-meth_tr respectively. the gde_c and duf608 pfam families could be clustered in this superfamily. the gde_c family is glycogen branching enzyme and has glucosidase activity (57) . we could identify gde_c and duf608 in 3 and 2 human proteins respectively. from, this relationship it could be suggested that duf608 might have glucosidase-like activity. the 3-d information provides precise molecular details about the function of the protein. the association of gene products encoded in human genome to 3-d structures would assist in providing further insights into their function. the databases of protein structures in which domains with similar 3-d architecture are grouped together could be used for such structural analysis. we have used pali database derived from scop for the present analysis. scop classifies protein domain having high sequence and structural similarity into families. the families are grouped in superfamilies when they share similar functional features and have an evolutionary common ancestor. superfamilies are grouped in fold when major secondary structures are topologically equivalent with similar topological connectivity. the assignment of structural domains to the proteins would aid in the investigation of the preponderance of superfamilies and fold in the human genome. using the various search procedures we could associate 38,017 structural domains to 16,459 human proteins either directly or by using the sequence superfamily relationships as described in supfam. further, we classified these domain assignments at the level of fold or superfamilies to understand the most commonly used function present in human genome. we analyzed the most commonly occurring superfamilies in human proteome. the figure 3 shows the top few superfamilies along with their extent of representation in the human genome. this distribution of superfamilies is similar to the one obtained by muller et al. (38) . the most commonly occurring superfamily is c2h2 zinc finger, followed by immunoglobulin domain. because of the short length of c2h2 zinc-finger domain and associated low complexity region, there is bias in identification of these domains. hence, all the gene products having this domain might not have zinc-finger like function and we excluded them from our present analysis. p-loop containing nucleotide triphosphate hydrolases domain is the next most represented superfamily and it is involved in many different critical biological functions such as cell growth, differentiation, repair and modification of dna, transcription, etc. this superfamily comprises various atpases and gtpases that are essential for cell survival. for example gtpases include elongation factors, ga subunit of the heterotrimeric g-proteins that are absolutely critical in major cellular processes. the other superfamilies among frequently occurring superfamily are involved in various functions in the cell as cellular signalling (protein kinase-like, ph domainlike), cell adhesion (cadherin, fibronectin type iii), nucleic acid binding function (rna-binding domain). interestingly, 'family a-g protein-coupled receptor-like' superfamily that consists of many receptors as other most populous superfamilies. the complete list of structural superfamilies that occur in human genome with their respective frequency of occurrence in human genome is provided at http://hodgkin.mbu.iisc.ernet.in/*human. figure 4 shows population distribution of few most populated folds, which occur in the human proteome. figure 5 shows the 3-d folding patterns in the most populated folds. the c2h2 and c2hc zinc finger is the most frequent occurring fold in human genome. for the reasons mentioned before, we have excluded c2h2 and c2hc zinc finger from this analysis. ferredoxin-like fold has the highest number of superfamilies in the human proteome as well as in scop. however, 16 of the superfamilies occur in the human proteome out of the currently known 36 superfamilies in the ferredoxin fold. ribonuclease h-like motif fold has six out of currently known seven superfamilies in the human proteome. except the superfamily of hypothetical protein mth1175 from methanobacterium, all other superfamilies of ribonuclease h-like motif occur in the human proteome. these superfamilies are actin-like atpase domain, creatinase/ prolidase n-terminal domain, ribonuclease h-like, translational machinery components, dna repair protein muts domain ii and methylated dna-protein cysteine methyltransferase domain. this could be expected as the nucleic acid binding/related superfamilies are highly represented in the human proteome. the complete list of protein structure folds that occur in human genome with their respective frequency of occurrence in human genome is provided at http://hodgkin. mbu.iisc.ernet.in/*human. the sequence to profile matching procedure described in the methods section resulted in the association of at least one functional domain family in pfam database to the 4864 proteins of swissprot database linked to omim entries (77.8% of the total of 6257 proteins in the omim database). the remaining 1393 disease-related proteins could not be associated to any functional or structural domain family. hence these proteins could be high priority targets in structural genomics to provide further insights into the molecular basis of the function of these proteins. these 4864 proteins contain 8431 functional and structural domains from 1288 pfam families. it is important to note that 6491 domains out of 8431 domains could be linked to 802 pfam families with known structural information. in terms of the amino acids coverage in these domains about 51% of the amino acids in the proteins are in the functional or structural domain (58) assigned regions in these 4864 proteins. figure 6 shows the distribution of the domains in the top 15 most populous families in the proteins, these families contain 2551 domains which is about 30% of all assigned domains. protein kinase is the most frequently occurring domain family in the human disease proteins. among the top 15 most populous families 14 have known structural information. the most populous structural superfamily that is assigned to the proteins is p-loop containing nucleotide triphosphate hydrolases and this has 361 domains in it. the largest representations in the p-loop superfamily come from the domain families like ras, helicase_c, and dead. the list of highly populated superfamilies has much in common with the analogous list generated by muller et al. (38) . much of these highly represented superfamilies are associated with regulatory roles in development, differentiation and proliferation. further analysis revealed that there are 33 proteins that have been assigned additional functional domains apart from previously assigned functional domains. these 33 proteins are listed in table 3 . these newly assigned domains may play a significant role in furthering our understanding of overall functions of these proteins. using various methods of domain association we could associate at least one domain to about 75% of gene products in the human genome. interestingly, the assignments of remote homologues to the human proteins revealed the occurrence of some of the viral and bacterial specific proteins in the human genome. among most commonly occurring functional family, protein kinases is one of the most frequently occurring domains, and the p-loop containing nucleotide triphosphate hydrolases is the one of the most represented superfamily. the assignment of 1184 domains to families with apparently no structural information to structural families would aid in the prioritization of targets for structural genomics of human genome. the assignment of new domains in addition to previously identified domains to the proteins possibly linked to genetically inherited human diseases could form a basis for the experimental verification of the roles of these domains as well as the molecular basis of disease. initial sequencing and analysis of the human genome the sequence of the human genome predicting function: from genes to genomes and back structural assignments to the mycoplasma genitalium proteins show extesive gene duplication and domain rearrangements the cath extended protein-family database providing structural annotations for genome sequences trends in protein evolution inferred from sequence and structure analysis studying genomes through the aeons: protein families, pseudogenes and proteome evolution predicting protein function by genomic context: quantitative evaluation and qualitative inferences basic local alignment search tool improved tools for biological sequence comparison distant homology recognition using structural classification of proteins the relation between the divergence of sequence and structure in proteins protein evolution. how far can sequences diverge? how representative are the known structures of the proteins in a complete genome? a comprehensive structural census homology-based fold predictions for mycoplasma genitalium proteins the relationship between protein structure and function a comprehensive survey with application to the yeast genome enhanced genome annotation using structural profiles in the program 3d-pssm predicting structures for genome proteins from protein structure to function genomic-scale comparison of sequence-and structure-based methods of function prediction: does structure provide additional insight? profile analysis: detection of distantly related proteins hidden markov models in computational biology. applications to protein modeling fold and function predictions for mycoplasma genitalium proteins applying motif and profile searches gapped blast and psi-blast: a new generation of protein database search programs supfam-a database of potential protein superfamily relationships derived by comparing sequence-based and structure-based families: implications for structural genomics and function annotation in genomes supfam: a database of sequence superfamilies of protein domains enhanced functional and structural domain assignments using remote similarity detection procedures for proteins encoded in the genome of mycobacterium tuberculosis impala: matching a protein sequence against a collection of psi-blast-constructed position-specific score matrices profile hidden markov models benchmarking psi-blast in genome annotation identification of related proteins on family, superfamily and fold level genthreader: an efficient and reliable protein fold recognition method for genomic sequences a method to identify protein sequences that fold into a known three-dimensional structure comparative genome and proteome analysis of anopheles gambiae and drosophila melanogaster the identification of functional modules from the genomic association of genes genecensus: genome comparisons in terms of metabolic pathway activity and protein family sharing structural characterization of the human proteome protein fold recognition using sequence profiles and its application in structural genomics functional and structural genomics using pedant genome sequences and great expectations the pfam protein families database pali-a database of phylogeny and alignment of homologous protein structures integration of related sequences with protein three-dimensional structural families in an updated version of pali database scop: a structural classification of proteins database for the investigation of sequences and structures the ensembl genome database project omim passes the 1000-disease-gene mark the swiss-prot protein sequence database and its supplement trembl in 2000 a hidden markov model for predicting transmembrane helices in protein sequences the repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations acrab efflux pump plays a major role in the antibiotic resistance phenotype of escherichia coli multiple-antibiotic-resistance (mar) mutants the drosophila patched gene encodes a putative membrane protein required for segmental patterning secd and secf are required for the proton electrochemical gradient stimulation of preprotein translocation major facilitator superfamily. microbiol molecular identification and characterization of novel members of the human organic anion transporter (oatp) family dual function of the trna (m(5)u54) methyltransferase in trna maturation identification of the catalytic residues of bifunctional glycogen debranching enzyme the human serum paraoxonase/arylesterase gene (pon1) is one member of a multigene family setor: hardware lighted three-dimensional solid model representations of macromolecules this research is supported by the award of senior fellowship to n.s. by the wellcome trust, london as well as by the computational genomics initiative supported by the department of biotechnology, new delhi. s.b. and s.b.p. are supported by the wellcome trust, london and csir, new delhi respectively. key: cord-006127-rl7rur2j authors: brown, nik; faulkner, alex; kent, julie; michael, mike title: regulating hybrids: ‘making a mess’ and ‘cleaning up’ in tissue engineering and transpecies transplantation date: 2006-02-08 journal: soc theory health doi: 10.1057/palgrave.sth.8700062 sha: doc_id: 6127 cord_uid: rl7rur2j this paper explores the institutional regulation of novel biosciences, hybrid technologies that often disturb and challenge existing regulatory frameworks. developing a conceptual vocabulary for understanding the relationship between material and institutional hybrids, the paper compares human tissue engineering (te) and xenotransplantation (xt), areas of innovation which regulators have sought to govern separately and in isolation from one another. contrasting definitional boundaries and regulatory mechanisms partition them socio-institutionally. but despite these attempts at purification, te and xt have proven increasingly difficult to tell apart in practical and material terms. human and animal matters, cell cultures and tissue products have much greater corporeal connection than has been institutionally recognized, and are therefore a source of acute instability in the regulation of implants and transplants. this paper tells the story of how the messy worlds of te and xt have leaked into one another, calling into question the abilities of regulation to adequately control hybrid innovations. globally mobilized in a worldwide traffic of scientific, medical and commercial transactions. while human implant technologies have a long history, contemporary technoscience has thrown up an ever-wider range of boundary-crossing possibilities for both the body politic and corporeal (franklin and lock, 2003; brown and webster, 2004) . medicine is now at the heart of an array of combinatorial human, animal and mechanical materials. while the troubled nature of transplantation has been relatively well documented (eg swazey, 1978, 1992) , less well understood are new forms of innovation that cut across machines, humans and animals raising regulatory concerns about material and cultural risk (brown and michael, 2004; faulkner et al., 2004; kent et al., 2005) . at the same time, such hybrids are powerful sources of hope -new treatments for large populations throughout the aging societies, and new sources of wealth for countries seeking a place in the emerging tissue economies. hybridity takes many forms and contemporary developments in the manipulation of tissues have extended these profoundly. hybrids signal the breach of various socio-material categories, indicating inconsistencies that disorder routines and accepted mores. it is no accident that concepts of pollution and contamination have had an increasingly important place in sociological and anthropological accounts of the life sciences lately. these disruptions are frequently framed around questions of new standards for the purity of cell lines, the cleanliness of animal tissues, new rules to secure safety and avoid hazard. often falling outside existing frames of institutional and disciplinary understanding, hybrids are messy/disorderly creatures. they are 'matters in wrong places' (douglas, 1966) and for regulation they have consequently become 'matters of concern' (latour, 2004) . this paper compares the regulatory ordering of human tissue engineering (te) and xenotransplantation (xt) and the changing ways in which both have been defined and classified 1 for regulatory purposes. crucially, they are areas of innovation which regulators have sought to govern separately and in isolation from one another. that is, contrasting regulatory mechanisms and definitional boundaries partition them socially and institutionally. but despite these attempts at purification, te and xt have proven increasingly difficult to tell apart in practical and material terms. human and animal matters, cell cultures and tissue products have much greater corporeal connection than has been institutionally recognized. this paper tells the story of how the messy worlds of te and xt have leaked into one another, calling into question the abilities of regulation to adequately control hybrid innovations. clinical xt has been subject to forceful regulatory prohibition over recent years with the relatively stable consensus that risks outweigh benefits, particularly in respect to transpecies disease. but recently the regulatory grip on xt has been called into question as various -at first seemingly unrelated -areas of te have come to regulatory attention. this has resulted in significant attempts to 'clean up' the definition of xt, broadening its regulatory identity. 'te' on the other hand signifies a far more diverse set of practices whose identity in regulation has been more plastic than that of xt. as such, regulators have sought to draw a clean distinction between different aspects of implant innovation, steering te clear of potential contamination by more problematic hybrids like xt and stem cell therapy. the paper speculates on whether such distinctions can be sustained. first the paper sets out a conceptual terrain -particularly in respect to notions of regulatory ordering, technological zoning, hybridity, and pollution -resources that are analytically useful in making sense of innovative regulation and bioscience. we then take a much closer look first at xt and then human te drawing on data and fieldwork from two social science research projects. 2 to conclude we consider the implications of these comparisons for understanding the variability of governance in human implant technologies. novel natures -like te and xt -are enmeshed in the production of equally novel/hybrid regulatory orders and institutions, processes whereby nature and the social are made available to each other in what rabinow (1992) describes as 'biosociality'. here we are interested in various forms of 'institutional biosociality' (brown and michael, 2004) through which scientific and regulatory actors -in te and xt -configure one another materially, culturally and institutionally. in other words, regulatory bodies form particular representations of corporeal bodies and in turn subject corporeality to the innovativeness of regulation. hybrids present regulation with the need to alter the boundaries between existing institutional arrangements. stem cells traverse the borders between regulated reproduction and transplantation (waldby, 2002; franklin, 2001) . pharmacogenomics newly combines the regulation of genetic diagnostics and medicines (hedgecoe and martin, 2003) . te and xt are similarly hybrid, falling into a 'regulatory vacuum' (faulkner et al., 2003) between drugs, devices, human implants and animal research. hybrid regulatory capacities are therefore 'risky creatures' (brown and michael, 2004) . innovation occurs at the limits of conventional organizational arrangements (gibbons et al., 1994; nowotny et al., 2001) challenging the taken for granted and presenting novel risks. new regulatory bodies can be seen as institutional interpretations of the composition and materialities of these novel risks. various regulatory ingredients are assembled together in such a way that they reflect, often imperfectly, new regulatory objects, producing intricate connections between 'natural' and 'institutional' hybrids, or as martin puts it 'how governance arrangements are being challenged and transformed cannot be detached from these other sociotechnical changes' (martin, 2001, p. 158) . more radically, regulatory work may also be seen as a powerful force in the very conception and conceptualization of innovative technology (bud, 1995, p. 297) . here we employ the concept of the 'regulatory order' (faulkner et al., 2004) and 'regulatory ordering' to draw together strands of theory relevant to the innovativeness of hybrids. 3 firstly, we can regard corporeal and institutional hybridity in terms of cleanliness and dirtiness (douglas, 1966) . pollution is a fundamental axis in the dynamics of everyday life. 'dirt is essentially disorder ' (1966, p. 12) , 'matter out of place' which must be excluded if order is to be maintained, insights into boundary-work and categorization that have proven highly influential in the analysis of medical and scientific knowledge (bloor, 1978; gabe, 1995; carter, 1995; mody, 2001) . but arguably the douglasian perspective, while bringing into view the value-laden partitioning of material and social boundaries, assumes relatively static categories -pollution as the expression of an underlying structuralist order. by contrast we look to a second set of theoretical perspectives that are more dynamic and indeed even celebrate transgressive intermixing (ansell pearson, 1999) . the two views might be articulated in terms of deleuze and guattari's (1988) conception of 'being and becoming'. between these extremes lies a more ambivalent position that can be identified with, among others, haraway (eg haraway, 1991a, b; prins, 1995) . myerson has unpacked this ambivalence in relation to haraway's (1997) writing on oncomouse, a profoundly ambiguous figure with whom 'we can acknowledge our kinshipyeither as victims or as heroes' (myerson, 2000, p. 73 ). haraway's hybrid, the cyborg, also has political potential in terms of holding out the prospect of couplings that ultimately demolish oppressive dichotomies operating across genders, races, species and machines. and for latour (1993) hybrids are perhaps even more ambivalent. they are often dangerous, even catastrophic -the ozone hole, climate change, bse -and are evidence of the underlying hidden connections between humans and non-humans, and especially between 'objective' science and 'subjective' politics. hybrids challenge representational order, disturbing the very basis of modernity's sorting. and of course, they can be highly hazardous unless recognized as such in political process, a 'parliament of things' (ibid). hybrids have agency in as much as they can undermine attempts to conceal connection. regulatory re-ordering, therefore, implies important concepts of cleaning/dirtying, pollution, purification and decontamination. equally, it suggests that hybridity is much more highly unstable and volatile, constantly challenging systems of classification and the material boundaries of technology. the re-ordering of socio-material hybrids therefore recognizes the importance of constructed boundaries and of the transgression and re-formation of new boundaries, often contested normative processes. the jurisdictional fields of socio-technology that regulation attempts to define can be usefully conceptualized as 'zones' (barry, 2001) or 'territories' (sharp, 2002) . it is part of the regulatory construction of such zones that the technologies that we will discuss here have come to be known conventionally as 'te' and 'xt'. jurisdictional structures can be difficult to establish in processes of regulatory ordering, but are 'meant to invoke order and to demarcate boundaries' (hogle, 2002, p. 243) . processes of regulatory ordering engage with a fluid governance jigsaw -a web of interlinked laws, regulations, guidance and surveillance interacting with the negotiation of technology zones. regulation exhibits, par excellence, societies' attempts to establish links between innovative technologies and the social management of their opportunities and risks, stabilized in institutions and patrolled through standard-setting and surveillance. as we illustrate in the two cases that follow, the ordering of the regulatable zones of bioscience is in the view of many stakeholders -though not all -severely challenged by human and animal tissue-based technologies. in this discussion we will see that both xt and te have been subject to important contestations over their definitional, regulatory identities -that is, what is or is not to be considered inside or outside their leaky borders. we now present these two cases as examples of the workings of 'regulatory re-ordering'. xt has hovered controversially on the horizons of biomedicine for decades but remains firmly locked within a whole range of presently prohibitive dangers. nevertheless, some research has continued together with corresponding developments in regulation and public consultation. one of the foundational problems for regulation in this area has been how to define xt as separate and distinguishable from other developments in bioscience. that is, what are its limits? what is and is not xt? how might regulators 'clean up' the messy worlds of bioscience such that they have a precise understanding of their regulatory object? in 2001, the us food and drug administration (2001) revised its definition of xt as it became aware of an apparently 'unrelated' medical procedure which had until then not been considered relevant to xt regulation. since 1987 genzyme had been marketing epicelt a method for culturing human skin for treating severe burns. the problem was that the production method requires base layers of irradiated mouse cells on which to culture human epidermal grafts. xt regulators saw in this the very same dangers that concerned them about transpecies transplants, particularly human/non-human disease transmission. more importantly, many of these diseases are difficult to detect especially in the case of endogenous retroviruses, viruses embedded deep within the dna of a species, which are entirely harmless to the host animal. however, when introduced into an unrelated species they can reactivate, becoming highly infective and harmful. years earlier, epicelt had been originally regulated as a 'medical device', though its connection to the risks associated with xt were not foreseen at the time, nor included in the definition of xt that emerged during the early and mid-1990s. but with a now increased regulatory focus on transpecies disease the status of epicelt as a 'device' began to collapse, as did the existing definition of xt. the previous definition used by the fda and the british regulatory body for xt (the united kingdom xenotransplantation interim regulatory authority, 2003) did not account for production methods whereby human and animal tissues may be subject to 'ex-vivo contact' as is the case with epicelt. while they foresaw that there would be the need to regulate clinical practices involving ex vivo 'perfusion' (for example, pig livers used temporarily outside of the body to support a patient in hepatic failure), ex vivo contact remained out with the duties of responsibilities of regulators. the amended fda definition now reads: yany procedure that involves the transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs (fda, 2001) . this realization in the late 1990s and the fda's later redefinition, undermined the adequacy of existing regulation and also the belief held by many authorities that they had produced a precautionary regulatory process. regulators had been fundamentally committed to the principle that recipients of xenografts should be registered within a programme of life-long surveillance, monitoring potential cross-species infection. but in testimony to the fda, representatives of genzyme acknowledged that while many thousands of patients had been treated internationally with epicelt, there was no registry of recipients and no means of checking for cross-infection. awareness of epicelt and other products suggested that the xeno-horse had indeed already long since bolted. so these questions of definition and what is or is not the object of regulatory action, and by which regulatory institution, are therefore far from trivial. and in this case there are issues about whether a new clinical practice is a device/appliance or a medicine, or more crucially whether it is human or animal, alive or dead, inert or animate, benign or dangerous. as one respondent from the uk department of health commented: ythe mice who provided the cells were long dead. years and years ago, so it's an established cell line that's being used, really without people thinking about it as being particularly mousy (doh member 1 -june 2001). questions of species were crucial here -a device's 'mousiness' or not. initially, the 'radar' of regulation was focused on the species that looked most likely to be the primary donor source, pigs. therefore, characterizing the pig and its potential donor-host relationship to recipient humans occupied most of the regulatory workload. non-human primates too were at the centre of sustained discussion, particularly in respect to their welfare as proxy humans in preclinical trials. plus the focus was initially skewed towards large organ transplants and perfusion techniques from pigs rather than cellular applications involving mice, and perhaps other slightly more mundane technologies including epicelt. in all, cells and mice were arguably peripheral in the regulatory consciousness: first it was all aboutywhole organsyconcern as a committee tended to shift more towards the use of animal tissuesy. infusion devices, transplanting foetal cells, this kind of thingythis arose out of an american experience where their definition included any tissues which had been in contact with animal body fluids, and this then became relevant in certain thingsynotably the growth of tissue cells, using at one stage mouse cells as a base, and that this was not xenotransplantation according to our definition because no cells or vital animal tissues were transplanted. but human cells have been in contact with mouse cells. and this created quite a difficult problemybecause there was a possibility that we might have inadvertently created a lot of xenotransplant patients simply by changing the definition (ukxira member 2, oct 2001). people felt the definition was pretty comprehensiveyright from the startymedia attention and the excitement was always focused round the idea of the kidneys and the heartsyukxira's always been aware that things like liver assist devices, for example, were as likely or more likely to come along and there's the potential cell therapies that parkinson's or diabetes or something; those have always been possible developments. but somehow that's never impinged on the public interest (doh member 1 -june 2001). as these definitions shift so to does the ground on which regulators understand transpecies risk. as one of our respondents put it, there are differing degrees of risk between whole organs that are transplanted and irradiated mouse cells that are not. in this case, there is the difficulty of balancing the fact that there are now many more various forms of xt and differing degrees of contact, but 'contact' nonetheless. an expert witness from genzyme at an fda committee hearing acknowledged that contact may be sufficient cause for regulatory concern: i think the assumption that one takes using any mouse cell line is that there is the potential for expression of a xenotropic retrovirus from that line. the fact that these tests are negative is comforting at the post-production level, and the fact that they're radiated prior to their use as a feeder cell line is comforting to some extent in that they're not actively replicating. but there is always a potential with any mouse line the assumption being that there is potentially endogenous retrovirus there (d moore, genzyme, fda hearing, april, 2000) . this account shows how the once 'black boxed' (latour, 1987) definition of xt was opened up to controversy, how the 'zoning' of xt has expanded, and evolved in relation to institutional assumptions about species difference, and about whether something is an inert device or an active biological agent. the focus on pigs distracted the regulatory gaze from other xenogeneic hybrids including the living cells of long dead mice and the many thousands of human skins cultured on them. the broader point is that the disjunction between the apparent hybridity of a body like ukxira and its actual (species) rigidity generates apparently new risks. in this sense then, hybrid corporealities are in profound tension with hybrid institutional bodies -what we described earlier as a form of 'institutional biosociality'. these processes are strongly linked to questions of species boundary change, institutionally and corporeally. that is, institutions -like other areas of specialization within the biosciences such as professions, disciplines, royal societies, etc -have a certain species identity or emphasis. in this way they may be aptly described as 'institutional animals', each resonating with particular elements in nature. in the uk, two regulatory bodies in particular emerged as crucial to the management of human and non-human risk in xt; ukxira and also the home office's animals procedures committee (apc). ukxira is an interesting illustration of a hybrid or transpecies institutional capacity. its terms of reference include oversight of applications to undertake clinical trials in humans and also consideration of the welfare of source animals from whom tissues and organs might be derived. its remit, its routes of consultation and even its membership criss-cross various 'relevant' regulatory capacities that feed into its role as an intermediary focus in the uk regulatory system for xt: the authority's role as a focal point for xenotransplantation issues is important given the number of interests which xenotransplantation brings together -animal and human welfare and ethics, industry, public health, and the other regulatory systems which exist for medicines and medical devicesy (ukxira, 2003, p. 5) . ukxira is answerable to the secretary of state for health and its secretariat is located within the department of health. the important point here is that it is, we might say, a predominantly human-medical regulatory animal. on the other hand, we also have the home office's apc, responsible for preclinical experimental procedures on animals, a non-human-welfare regulatory animal. while both of these committees are crucial regulatory bodies for xt they have been entrenched in institutional arrangements that inhibit good joint responsibility for 'interspecies' regulatory problems. a crucial issue here is that the apc is bound by clause 24 of the animal (scientific procedures) act 1986 making it a criminal offence for members of the apc to disclose information to third parties,'otherwise than for the purpose of discharging his functions under the act'. this also applies to the disclosure of information to other regulatory authorities, even when their terms of reference may directly apply. one of our respondents sat on both of these committees, finding themselves at the centre of critical institutional tensions. the apcyis all tied up with confidentialityywhen somebody applies to the apc to do a project on xenotransplantation involving primates, the logical thing to do would be to refer back to the body that regulates xenotransplantation [ukxira]ybut you can't do that because ukxira cannot have sight of the project licence applicationy (ukxira/apc member 1, april 2002). another respondent was a member of ukxira but not the apc. they expressed their frustration at being consulted by the apc about licence applications, the contents of which they were forbidden from seeing: ythe apcydoesn't allow the dissemination of information to anybody outside the committee. i mean the sort of issues which arise are whether a series of experiments on a group of primates would be of sufficient importanceyto justify the sufferingy and we would get a question about the health of the animals from the home office but we couldn't get details of the experiments, and we couldn't get details of previous experiments to put them in contexty (ukxira member 2, oct 2001). there is then a legal firewall here in the governance of humans and animals, reflected in the structure of government departments and regulatory bodies. these species divisions represent potential weaknesses in the risk management of innovations like this that cut across institutions and natures. so despite its hybrid character, ukxira clearly has a species identity. its institutional location reflects a stronger alignment with networks of humanmedical governance than it does with those of animal-welfare governance. this is not unusual -novel natures and innovative hybrids routinely present difficult challenges to institutional structures. increasingly, clause 24 has come under greater scrutiny but is likely to remain in place until legal process has taken its long and circuitous institutional course through processes of consultation, lobbying and parliamentary timetabling. in the mean time, hybrid bodies like ukxira remain pragmatic and rely on a variety of strategies with which to extend their hybridity and to better meet their terms of reference. as ukxira's fifth annual report notes (2003): ythe relationship between the ukxira, the ho and the apc is very important. it had previously been agreed that a member of the authority would be co-opted onto the apc's primates subcommittee to review applications to undertake xenotransplantation research on primates if and when these are receivedyit may [also] be possible for the ukxira to be given verbal overviews of current areas of work and research directions by ho personnel and the ukxira will be seeking authority for this from the ho (p. 9). this story illustrates the limits to institutional hybridity, that the capacity of regulatory bodies to move smoothly across long established institutional structures is inherently partial. more importantly, this partiality articulates particular institutional representations of the boundaries that are seen to exist in nature, and of course those boundaries traversed or innovated in biotechnology. xenografts, as with other biotechnological innovations, often belie both 'natural' and 'institutional' classification, as we could see in the debates about the definition of xt, challenging the way in which routes of responsibility are organized. but as we will see next in our discussion of te, these problems of 'making a mess' and 'cleaning up' are endemic as novel natures are generated and the definitional and institutional alignments of regulation become unsettled. the discussion of xt highlights a number of key features of regulatory re-ordering that are important to consideration of human tissues and te including the purification of the regulatory object, a strong alignment of regulatory institutions with one rather than another substantive fields of governance, and the dynamic tension between institutional hybrids and novel natures. we now draw upon recent research -principally in the uk and europe -elaborating te as a regulatable zone and in relation to xt. more heterogeneous than xt, te includes cultured cell implants for cartilage repair, bone substitutes, 'living' skin tissues (like epicel tn discussed above). future developments are expected to include vascular prostheses, organ-assist devices (liver, kidney), whole organs, structures (heart valves, joints), neurological tissues and stem cell therapies. one influential definition describes te as the 'regeneration of biological tissue through the use of cells, with the aid of supporting structures and/or biomolecules' (scmpmd, 2001a) . they are often conceived of in regulatory policy communities themselves as 'borderline' or 'hybrid' products -occupying a 'regulatory vacuum' at the borders of existing regulatory frameworks. the notion of a regulatory vacuum has been the starting-point for much of the recent development of regulatory policy for human tissues, and the boundaries between pharmaceuticals, medical devices and te are crucial areas of negotiation in re-ordering regulation. some te products have already been regulated as pharmaceuticals while some parts of combination products (eg using synthetic scaffolds) need to gain approval as medical devices. in the face of this complexity there has been a widely, although not unanimously, perceived need for 'new regulation' for human tissues and te, and here we refer to two distinct pieces of regulation. 4 te embraces two closely related regulatory fields whose boundaries are themselves unclear and overlapping. on the one hand, 'human tissues and cells' (htcs) are defined and covered in europe by the tissue and cells directive (tcd) on the sourcing, storage, processing (eg cleaning) and distribution of a wide range of human materials (excluding, as we shall see, matters like blood and blood products and whole organs). on the other hand, the proposed 'human tissue engineering regulation' (ter) refers to manufacturing and market approval, excluding the accreditation of safety and quality of sourcing and storage covered by the tcd, and distinguishes between autologous (donor is also patient) and allogeneic (multiple recipient) applications. these two jurisdictions were already separated in the united kingdom, whose regulatory work in the early 2000s included a tightening of standards and accountability through a code of practice for tissue banking (department of health, 2001) , and then a code of practice for 'human-derived therapeutic products', voluntary guidance for manufacturers in the te field (mda, 2002) . importantly, the european directive concerned with sourcing and storage is nevertheless framed, as discussed below, in terms of transplantation and therapeutic application. a te manufacturer or tissue establishment engaged in significant tissue manipulation would have to meet requirements of both fields. these ambiguities are characteristic of the hybrid aspects of tissuebased therapies, pointing to a distinction between the traditional tissue banking for transplantation and the emerging activity of engineering tissues in implants. this is an increasingly troubled distinction as tissue banks engage in manipulation, and industry engages in tissue 'banking'. it has been important for both commercial and regulatory interests that a clearly delineated zone of activity can be identified. european commission directorate-general sanco negotiated the tcd in seeking to fulfil its requirements to protect public health, prefacing this directive that 'the transplantation of human tissues and cells is a strongly expanding field of medicine offering great opportunities for the treatment of as yet incurable diseases' and 'as tissue and cell therapy is a field in which an intensive worldwide exchange is taking place, it is desirable to have worldwide standards'. note here the inclusion of 'cell therapy' in their definition. biomedical zones are matters of negotiation, with national and sectoral interests playing an important role. unlike xt it is not the case with te and htcs that an initially stabilized definition has been changed by a later revision, but that a range of definitions have been proposed and continue to be negotiated, taking the form of partitioning and cleaning with attempts to distinguish te and htcs from xenografts. in what follows we also illustrate further attempts to cleanse te, variously also including or excluding embryonic stem cells, whole organs and blood products. we saw above how the definition of xt had been formed in isolation from regulated 'devices', and how, given the production method used (irradiated cultures of non-human cells) that zone began to collapse, overwhelmed by hybrid linkages. now, while xt regulators have been slowly expanding the definition of xt, just the opposite has been taking place in human te. regulatory efforts have been directed, with mixed success, at distancing te and human tissues/cells from the allied worlds of xt. one respondent spoke of how regulators sought to maintain a firm distinction between te/htcs and xt: q: what about the use of animal ory? a: animal? for me it's a separate areayxenograftingywe should have a centralised authorisation system for that. q: if you look at apligraf [te wound treatment]yit makes use of bovine serum during the culturing process. a: oh, we have a problem of definition here again because for us a xenograft is the use of the animal part in the body or in the perfusion extracorporeal. but it's a direct use. we have a specific category of products we call produits thérapeutique annexe which means additives you could say. and we have a specific authorisation for additives. q: including? a: ybovine serum in many culture processes. yand our regulation of xenograft existed since '96ythat only the health minister can authorise a xenograft trialy . you can see that it's been placed at the most important level (a-eu4, 2003) . essentially, however, the respondent is describing a national regulatory boundary te and xt that no longer exists in the us and will cease to exist in those regulatory arenas that take their lead. the distinction of two regulatory jurisdictions will no longer be tenable, with both defined as xt. this clearly signifies the increasing reach of cleansing policies, with 'problems of definition' potentially jeopardizing both the political and material cleanliness of te and htcs. critical clinical commentary also recognizes this: in ex-vivo corneal stem cell expansionythere is a need for accreditation of laboratories conducting such work. the use ofyof co-culture system must be in consultation [with regulators]. without such stringency, there will be a risk of cross-animal contamination such as the one we witness (sic) recently in outbreaks of sars (kong y then, 2003) . this writer recognizes the extension of xt and the role of the tcd in providing a framework for inter-national accreditation. these processes of cleaning and re-organizing are as much material as they are institutional and regulatory, calling to mind what rheinberger describes as the 'intracellular representation of extracellular projects' (rheinberger, 2000, p. 19 ). the quotation further illustrates the ambiguous boundaries around htcs and te, where adult stem cell therapy may be regarded as a form of te. in what follows, a respondent in the academic-commercial te sector talks about their attempt to materially re-engineer the methods used to culture skin cells, seeking to replace the use of animal 'feeder layers' with human equivalents, reflecting similar attempts across the te industries. we are very keen to develop a methodology that doesn't use any bovine materials. personally i'm more concerned about using bovine than mouse cells as a feeder layer. y[xt regulators in the uk] are very concerned with our groups using mouse fibroblasts, but when i ask them 'aren't you concerned about bovine material?', they say that's not part of our remit because the cells are not alive. at which point you put your head in your hands and cry. we are working very hard toy[develop] a methodology for using the patients' own fibroblasts to substitute for bovine serumy (s5, 2003) . the statement highlights a further classificatory distinction in te regulation, that of viability/non-viability. here we see a hybrid 'consumer' of regulation troubled by the codified distinction between xt and medical device, and the regulatory vacuum for human te products. (european medical device directives cover the use of non-viable animal materials in medical device manufacture.) industry has sought to maintain the limited relevance to final te products of 'ancillary' viable xt in production processes, as we will see below. the following statement from the european parliamentary debate on the draft tcd concedes some serious ambivalence about the exclusion of xt in the new legislation for htcs: organs, tissues and cells of animal origin for human therapy are still in the research phase, but nevertheless pose different regulatory problems that will need to be addressed in due course' (from the explanatory memorandum to the proposal for a directive on quality and safety of human tissues and cells (eu commission/dg sanco, 2003) . animal tissues and cells were in fact excluded from the final directive. the ec here is thus acting to preserve the integrity of htcs as a regulatable zone uncontaminated by animal matters. the ec's dg enterprise (eu commission dg enterprise, 2003 , 2004a found in its consultations on ter -the te-specific regulation in 2002 and 2004 -that most stakeholders favoured a separate regulatory framework for xt products. however, some attempt to address the question about contact with animal materials during production was suggested. responding, industry associations proposed the following text: hteps containing not intentionally small quantities or traces of material of animal origin (used during the manufacturing process) which do not perform any function in the finished product are not, for the purpose of this regulation, regarded as xenogenic products (europabio et al., 2004; author italics) . thus attempting to preserve te production processes against the incursion of the extended definition of xt as discussed above in the case of the us and, possibly, the uk. such products should be regarded as te products in spite of viable xt elements in the production process. the uk has a dedicated xt regulatory body but the european union does not. the uk's code of practice for human-derived therapeutic products (mda, 2002) is the basis of interim guidance for manufacturers of te technologies while the eu ter is being formulated. the uk code states that 'where cell culturing techniques use cell lines that are not of human origin, for example, murine fibroblasts used for co-culture, guidance should be sought from ukxira'. the code specifies appropriate measures to avoid material contamination and to provide for regulatory accountability: documentation shall be obtained that demonstrates the application of appropriate quality assurance measures by suppliers of biological material, including origins and veterinary certificates for the animals used in the preparation of the material (eg bovine serum albumin). and: culture media, reagents and processing materials derived from animals shall be evaluated for the risk of contamination with micro-organisms, particularly viruses and agents of transmissible spongiform encephalopathies,y verification shall be obtained that all primary raw materials of animal origin originated from animals that had been subject to veterinary inspection, certification, an effective surveillance system and comprehensive sourcing controls. thus it is clear the uk te policy group assumed that the uk system, including ukxira, should and will adopt the extended definition of xt, and allows for a linkage between regulatory zones with parallels to that seen above between medical and animal welfare domains. returning to the clean ordering of htcs, whole organs were also excluded from the tcd, in the face of strong dissension. as one commissioner noted: yi remain convinced that it is not appropriate to include organs in the scope of this directive. the problems to solve in this area are quite differenty requiring a different policy approachy as organ transplantation is a highly specialised subject in its own right, the commission is currently conducting a scientific evaluation of the available optionsyfollowing the example of the blood directive and this proposal on tissues and cells, we would like to get the science right first, before tabling a legal instrument in this sensitive area (byrne, ec, 2003; author italics). we see here 'getting the science right' as a rhetorical device with the effect of proliferating separate though linked regulatory jurisdictions -blood, organs and human tissues. these distinctions between biosocial matters are artefacts of regulatory political process. but paradoxically, purifications aimed at defining discrete fields at the same time increase the overall complexity. on the exclusion of organs again: we should not include organs in this measure on cells and tissues. organs are for another day. equally, this is not the time to permit cloned human embryos or hybrid human-animal embryosythis is a very young area of scienceyleaving aside the ethical issues, one that should not be permitted now' (mep bowis, 2003) . this remark nicely points to the complexity of the human tissue terrain and the inevitable difficulties of policing its fragile borders. it is worth remembering here the way the regulation of xt had 'mistakenly' focused on whole organs, for years neglecting cell-based 'xeno-like' practices in te. there is then a curious patterning in the regulation of tissues, human and xenogeneic. while htc regulators would like not to have to embrace whole organs, xt regulators would rather not have had to embrace te. the fact that cells (particularly ex vivo contact) are now more central in the regulation of xt poses yet more problems for htcs and te legislation. it is possible that the exclusion of whole organs raises similar questions about the longer-term tenability of the tcd directive's boundaries. thus the leakiness of distinctions between types of human tissue and their methods of 'production' means that the isolation or segregation of particular zones of a regulatory order is difficult to achieve. hybrids have the potential to overwhelm purification because rhetorical and material connections constantly reference new associations. turning to consider institutional hybridity accompanying the contestation of biomedical boundaries, we note that in the united kingdom the current regulatory authority, the medicines and healthcare products regulatory agency (mhra), was formed from a merger of the medicines control agency (mca) and the medical devices agency (mda) avowedly as a response to the increase in materially hybrid or combination products. products deemed 'tissue-engineered' are assessed in the mhra on a case-bycase basis in the absence of te-specific regulation. in a subsequent recent development, the uk has established a dedicated human tissue authority, which will shortly assume responsibility for oversight of all tissue establishments and their sourcing and related activities. various models for an institutional regulatory agency have been proposed in europe to satisfy conflicting interests in the te zone. the high status scientific committee for medicinal products and medical devices (scmpmd, 2001a) proposed a separate te regulatory authority. opinion then appeared to favour founding a structure located within europe's existing agency for the evaluation of medicinal products (emea). this drew criticism from those stakeholders who preferred to see te products as 'more devicey' (as one informant put it) than pharmaceutical. the hybridity of the technology was further highlighted by apparent territorial disputes within the ec's dg enterprise between the medicines and medical devices jurisdictions, which in a further twist resulted in a proposal to re-classify te within the 'biotechnology' division of emea -neither pharmaceutical nor device. following a europe-wide guidance document (cpmp, 2001) in 2003 the ec medicinal product directive (mpd) was augmented by an annex on 'advanced therapy medicinal products' (eu commission, 2003) which in effect extended the definition of the regulatory field of medicines (as opposed to devices, or biologics, or te) to include somatic cell therapy products, human and xenogeneic. the overlap of a future european te legislation with the mpd definition of cell therapy medicinal products, and the possibility that some products would be both te and medicinal, had been criticized in responses to the eu dg enterprise consultation on the need for te-specific regulation (eu commission dg enterprise, 2002) . and it is at this point that we can return to a consideration of the chequered regulatory history and ambiguous regulatory identity of epicel tm, currently described by its manufacturer genzyme as an 'autologous cell therapy product' that is 'co-cultured with mouse cells to form cultured epidermal autografts', and uses 'a cell culture medium containing bovine serum'. thus were epicel tm and allied products to be submitted for authorization in europe now their regulatory status may be unclear, in spite of meeting the main criteria of te, and even though in the case of epicel it is regulated as a device in the us. while the ec does not have an xt regulatory authority it nevertheless does provide some oversight via the advanced therapy medicinal regulation just described. this contains a 'specific statement on xt medicinal products' that, interestingly, includes requirements in respect of animal sourcing and animal husbandry. the definition of xt used is the extended criteria adopted by the us and embraced in the uk code of practice on human derivedtherapeutic products. given that it is highly likely that the eu will consider a specific xt regulatory body (as recommended by the scmpmd (2001b)) this prior alignment of xt aspects toward medicines and thus by implication the centralized emea approval system sets the scene for a governance configuration with parallels to that described for the uk in which a humanmedical emphasis predominates over the animal welfare domain. to summarize, the links between htcs, te products and the institutional hybridity of regulatory authorities in the uk and europe are highly complex. like xt, we see attempts to construct and align pure regulatable fields across the hybrid materiality of human tissue-derived therapies in order to control various politico-material risks. also like xt, we see these attempts at partitioning undermined by changes in the sociotechnical definition of regulatable therapeutic materials. the xt/device divide could not be sustained, nor could the cell therapy/medicines divide, nor could the te/medicines/ devices divides as overarching distinctions. in an admission of the difficulty of assigning stable classifications to capture novel te therapies, ec proposals for te regulation allow for a 'lex specialis' function to adjudicate on products that are not clearly either te or medicinal or medical device,'to minimize the risk of grey areas for borderline products' (ec dg enterprise, 2004) . so we also see tensions between different organizational and indeed ontological claims at the very heart of the social constitution of human therapeutic materials. unmistakable connections cut across the regulatory and material practice of life science innovation generally, particularly evident in the context of the two innovative areas discussed here. that is, engineered human and non-human tissues are inherently messy and liable to 'leakiness' (hogle, 2002) . their edges, their boundaries, are for regulators annoyingly variegated, and a source of frustration in their attempts at definition, cleanliness and purity. te/htcs and xt both illustrate new capacities of isolation and mobilization in life science innovation. here, processes of 'purification' render matters isolatable, manipulable, and legible in laboratory-based science (knorr-cetina, 1999, p. 27) . cells, tissues and bodies, as waldby points out, are increasingly caught up in 'biotechnical fragmentation ' (2002, p. 239) . crucially, at stake here are regulatory processes of 'territoriality' or 'political ecology' (sharp, 2002) . that is to say, natural objects are delegated to various arms of regulatory order, institutionally enacted readings of biological risk that subsequently order cells, tissues, embryos. there are important lessons to be learnt from hybrids and dirt. crucially, mess is a consequence of purification and not a cause, a 'by-product' of ordering and for latour, it is the very act of purification that proliferates the production of hybrids. boundary-making is intended to deny connection, to foreclose the production of hybrids, and so paradoxically acts to facilitate their manufacture. and all too often, regulatory ordering systematically obscures the complex interplay of regulated matters. risks flourish, it seems, when practices of regulatory purification continue to be applied, in ignorance and denial of evident associations between technical and social considerations. this prompts crucial questions of regulatory activity in the areas of htcs and te and xt -especially the sustainability of regulating them separately when based upon political or commercially pragmatic differences. this is not to suggest that acts of purification and cleaning-up are bad, even avoidable. they are not, especially in a context where transpecies innovations depend upon strong and rigorous regulatory ordering to lessen the chances of potentially devastating population-wide risks. as barad puts it, 'boundaries are not our enemies' and we can hardly expect to do without them, they are: ynecessary for making meanings, but this does not make them innocent. boundaries have real material consequencesy. our goal should not be to find less false boundaries for all spacetime, but reliable, accountable, located temporary boundaries, which we should anticipate will quickly close in against us (barad, 1998, p. 187) . biotechnological innovations are reciprocally enabled by regulatory structures that facilitate particular sorts of research regimes and interactions out of which emerge biotechnological innovation -what we describe above as a form of 'institutional biosociality'. these are highly heterogeneous interactions between multiple forms of social, biological and institutional participants who jointly constitute innovation (callon et al., 1986; bijker, 1995; hughes, 1983) . indeed, it might be useful to think of this as an institutional form of 'intercorporeality' (waldby, 2002; weiss, 1999) , the connections of identification and disidentification between bodies that are as 'inter-institutional' as they are inter-embodied. that is, various innovated corporealities (stem cells, growth media, pigs, mice, primates, plants, viruses, patients, etc) are distributed between regulatory bodies each participating in a complex process of exchange and interaction, potentially embodied in both donors and recipients of transplantable tissues. in summary, the messy material hybridity of biomedical regulatory objects highlights societal attempts to introduce clear partitions, jurisdictions and stable regulatory orders in highly complex socio-political zones. we have illustrated how the formations of xt and te have inter-acted with shifting regulatory terrains. we have seen both successful and unsuccessful attempts to maintain boundaries, particularly between xt and human tissues. in both fields we have seen the strength of biomedical and pharmaceutical 'institutional animals' in shaping the discourse in which novel technological governance is constructed. and we have seen the shifting definitions of scientific and social appraisal of public health risk being refracted through the composition of the regulatory bodies that governance activity produces. thus in order to understand the variability of governance in different innovative technological fields, it is necessary to develop accounts of the hybridity of their material forms, to bring into view the detailed social, cultural and material shaping that produce regulatory orderings. and it is here perhaps that we can return to the link between regulatory ordering and deeper categorizing of nature, the human, animal and the moral. for the messy work of regulating is also an inalienably social and moral process of seeking benefit and minimizing risk in highly pluralistic, technologically inventive societies. hybrid technologies highlight the manufactured disturbance of foundational categories and societies' attempts to manage this disruption, a partitioning and aligning process that, in turn, re-distributes the productive elements for the continuing hybridization of biomedical technologies. understood to mean the transplantation into one species of cells or tissues derived from another species. te is commonly taken to involve a combination of cell culturation and chemical engineering to create implantable cells, skin, bone, cartilage and other body parts. 2 note on data and fieldwork: 'medical device governance: regulation of tissue engineering in the uk and eu' (funding: uk economic and social research council (esrc)). this project involved data collection consisting of a literature review and 65 interviews with respondents from regulatory agencies, treatment centres, manufacturers and engineers; 'xenotransplantation: risk identities and the human/nonhuman interface' (funding: uk esrc). data collection for this project consisted of a literature review plus 25 interviews and 11 focus groups with a range of respondents including regulation, public and commercial research, clinical centres, ngos, community and healthcare organizations. 3 'regulatory regime' is frequently used by analysts of formal regulatory activity (eg hood et al., 2001) but perhaps conveys greater connotations of planned, systematic, rational design than is often warranted. the verb 'ordering' (law, 1994) adds process to the sociological concept of 'social order'. 4 in europe, two main regulatory instruments are most directly relevant for te activity. these are the directive on setting standards of quality and safety for the donation, procurement, testing, processing, storage and distribution of human tissues and cells (adopted by the european parliament in 2004) (we refer to this as the tissues and cells directive (tcd) henceforth; (eu commission/dg sanco, 2003) ; and a proposed ec regulation on human tissue engineered products (under the auspices of dg enterprise (european commission dg enterprise, 2004). we will refer to this as the tissue engineering regulation (ter)). germinal life. routledge: london getting real: technoscientific practices and the materialization of reality of bicycles, bakelite and bulbs: toward a theory of sociotechnical change polyhedra and the abominations, leviticus risky creatures: institutional species boundary change in biotechnology regulation. health new medical technologies and society -reordering life resistance to new technology: nuclear power, information 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actors are cyborg, nature is coyote, and the geography is elsewhere: postscript to 'cyborgs at large modest_witness@second_millenium.femaleman meets_oncomouse: feminism and technoscience. routledge: london the drugs don't work: expectations and the shaping of pharmacogenetics claims and disclaimers: whose expertise counts? the government of risk: understanding risk regulation regimes networks of power: electrification in western society towards governance of human tissue engineered technologies in europe: framing the case for a new regulatory regime epistemic cultures: how the sciences make knowledge the need to regulate human tissue engineered products: ophthalmology perspective science in action we have never been modern why has critique run out of steam? from matters of fact to matters of concern organising modernity genetic governance: the risks, oversight and regulation of genetic databases in the uk a code of practice for the production of human-derived therapeutic products a little dirt never hurt anyone: knowledge-making and contamination in materials science donna haraway and gm foods. cambridge icon books: cambridge re-thinking science: knowledge and the public in an age of uncertainty the ethics of hybrid subjects: feminist constructivism according to donna haraway artificiality and enlightenment: from socio-biology to biosociality beyond nature and culture: modes of reasoning in the age of molecular biology and medicine scientific committee on medicinal products and medical devices opinion on the state of the art concerning xenotransplantation bodies, boundaries, and territorial disputes: investigating the murky realm of scientific authority united kingdom xenotransplantation interim regulatory authority stem cells, tissue cultures and the production of biovalue body images: embodiment as intercorporeality. routledge: london the findings presented here draw on two esrc-supported projects: 'medical device governance: regulation of tissue engineering' (l218 25 2058) and 'xenotransplantation: risk identities and the human/nonhuman interface' (l218 25 2044). we thank in particular the work of ingrid geesink for her contribution the research on tissue engineering. dr julie kent is a reader in sociology of health technology at the university of west of england. she has written on social aspects of pregnancy and childbirth, genetic technologies, medical devices, and human tissue engineering. her current research focuses on abortion and stem cell technologies. mike michael is professor of sociology of science and technology at the department of sociology, goldsmiths college, university of london. his interests have included public understanding of science, cultural aspects of xenotransplantation and the role mundane technology in social ordering. current research topics include technoscience and everyday life, and stem cells and practical ethics. key: cord-003629-xogzl1lv authors: alsuheel, ali mohammed; ali, abdelwahid saeed; al-hakami, ahmed musa; shati, ayed abdullah; chandramoorthy, harish c.; al-qahtani, saleh mohammed title: human metapneumovirus in pediatric patients with acute respiratory tract infections in the aseer region of saudi arabia date: 2019-04-12 journal: saudi j med med sci doi: 10.4103/sjmms.sjmms_72_18 sha: doc_id: 3629 cord_uid: xogzl1lv background: human metapneumovirus (hmpv) is a paramyxovirus known to cause acute respiratory tract infections in children and young adults. to date, there is no study from the aseer region of saudi arabia determining the proportion and severity of hmpv infection among pediatric hospitalized patients with respiratory infections. objectives: the objective of this study is to determine the presence of hmpv antigens in the nasopharyngeal secretions of pediatric patients hospitalized with respiratory tract infections in the aseer region of saudi arabia. materials and methods: this prospective, serological hospital-based study included all pediatric patients who were admitted to aseer central hospital, abha, saudi arabia, from july 2016 to november 2017 with upper and/or lower respiratory tract infections. basic demographics of patients and their clinical data on and after admission were recorded. direct fluorescent antibody assay was used to detect the presence of hmpv antigens in the obtained nasopharyngeal secretion specimens. results: during the study, 91 pediatric patients were hospitalized due to upper and/or lower respiratory tract infections, of which 9.9% were positive for hmpv. these patients were aged 9 months to 16 years, were from abha city or its surrounding localities and were mostly (77.8%) hospitalized during autumn or winter. the most common diagnosis on admission was bronchopneumonia (55.5%) and aspiration pneumonia (22.2%), and some patients also had underlying chronic conditions such as chronic heart disease (22.2%) and bronchial asthma (11.1%). conclusions: the results obtained indicated that hmpv is a potential etiologic factor for the commonly occurring acute respiratory infections in hospitalized children from the aseer region of saudi arabia. hmpv infection was also found to be associated with complicated respiratory conditions such as bronchopneumonia, chronic heart disease and bronchial asthma. human metapneumovirus (hmpv) is a single-stranded rna-enveloped virus, recently classified in the order mononegavirales, family pneumoviridae, genus metapneumovirus and species hmpv. it was first isolated in the netherlands by van den hoogen et al., [1, 2] and is now a known causative agent of upper and lower respiratory tract infections in children and adults. hmpv infections have been reported in australia, [3] canada, [4] the united states, [5] the united kingdom, [6] hong kong, [7] south africa, [8] mexico, [9] spain [10] and peru. [11] however, in the middle east, there have only been few reports of hmpv infections, mainly as sporadic infections in egypt, [12] jordan, [13] kuwait [14] and saudi arabia. [15] along with respiratory syncytial virus (rsv) infections, hmpv is now recognized as a primary etiologic agent for acute upper and lower respiratory tract infections in pediatrics. [16, 17] in a study from mexico, it was found that the number of hmpv infections increased in children aged 24-36 months as compared with those in younger age groups, whereas rsv infections were inversely proportional to increase in age. [9] co-infection with both viruses can also occur, resulting in a more complicated and serious clinical disease. [3, 18] in addition to the pediatric population, studies have also found hmpv to infect adults [19] and elderly people. [20] in terms of transmission, hmpv spreads through contact with contaminated secretions, i.e., droplet, aerosol or fomites. hospital-acquired hmpv infections have also been reported. [5] in young children, infections are usually not asymptomatic, and bronchiolitis, with or without pneumonia, is the most common associated presentation. [18, 20, 21] other reported conditions include bronchial asthma exacerbation, otitis media, pneumonitis, flu-like illness and community-acquired pneumonia. [9] in adults, hmpv has been associated with bronchitis, pneumonia and exacerbations of both bronchial asthma and chronic obstructive pulmonary disease. [22, 23] in terms of detection, reverse transcriptase-polymerase chain reaction (rt-pcr) is a sensitive and commonly used method to detect hmpv. [5] real-time rt-pcr is also commonly used for detecting hmpv in clinical specimens with many genomic target sequences. [24, 25] a touch-down genomic amplification protocol for the diagnosis of acute viral respiratory tract infections has also been used previously. [26] the enzyme-linked immunosorbent assay for hmpv diagnosis is a simple and specific serological test for anti-hmpv antibodies detection. [27] immunofluorescence using specific antibodies is routinely used for detecting hmpv antigens, particularly in epidemiological studies. [28] however, cell culturing techniques have a low sensitivity in detecting hmpv from respiratory tract secretions, as the virus exhibits extremely limited types of cell tropism. [2, 10, 29, 30] in saudi arabia, there is a paucity of data regarding the occurrence of hmpv and its role in complicated clinical cases of commonly reported respiratory infections. therefore, the current study aimed to determine the role of hmpv in the respiratory tract infections' severity and complications among hospitalized children in the aseer region, where no such study has previously been conducted. the study was conducted after obtaining approval from the ethical committee of college of medicine, king khalid university, saudi arabia (kku research ethics committee meeting no. rec # 2014-01-08; dated january 5, 2014) this prospective, serological study included pediatric patients who were admitted to aseer central hospital, abha, kingdom of saudi arabia, from july 2016 to november 2017 with upper and/or lower respiratory tract infections. aseer central hospital is the largest tertiary care referral hospital in the aseer region, and thus its sample is representative of the area. data such as age, gender, clinical presentation and current medications were collected using an objectively prepared questionnaire. informed consent was obtained from the parents/guardians of all patients before sample collection. nasopharyngeal secretions were collected from all hospitalized patients included in this study using the standard collection method. briefly, physicians collected the nasopharyngeal secretions with the help of a sterile feeding tube connected to a vacuum pump. following the vacuum application, the tip of the tube was cut and placed into a sterile container labelled with the patient's name and identification number. the container was then transported to the virology laboratory at the department of microbiology and clinical parasitology, college of medicine, king khalid university, abha, saudi arabia, and either processed on the same day or stored at −70°c. specimens were processed according to the manufacturer's instructions for the direct fluorescent antibody (dfa) kit (oxoid ltd., cambridge, uk) with minor modifications. samples were transferred to an eppendorf tube containing 1 ml of phosphate-buffered saline (pbs; ph 7.5). the specimens were gently vortexed for 30 s to reduce the viscosity and dilute the mucus. samples were then centrifuged at 3000 rpm for 5 min to separate the cells from the mucus. the supernatant was removed, and the cells in the pellets were used for dfa staining. the authors chose to use dfa because it has been found to be a useful technique for wider hmpv epidemiological studies. [31] preparation of cells the cell suspension was washed several times with pbs and the final cell deposit was resuspended in 2 ml pbs (ph 7.5). the cells were then gently agitated by pipetting up and down until the cellular material was released from the mucus. additional pbs was added until a smooth suspension was obtained, and any visible flecks of mucus were removed. after the cell separation process was completed, the obtained cell suspension was centrifuged at room temperature (15°c-30°c) for 10 min at 3500 rpm and the supernatant was discarded. the final cell deposit was resuspended in pbs to dilute any remaining mucus and maintain high cell density. a volume of 25 µl of the resuspended cell deposit was placed in slides with 6-mm-diameter wells. the specimens were then allowed to air dry thoroughly and fixed with fresh acetone at room temperature (15°c-30°c) for 10 min. the slide was air-dried after fixation. a volume of 25 µl of imagen™ hmpv reagent (oxoid ltd., cambridge, uk), which contains monoclonal antibodies against hmpv conjugated to fluorescein isothiocyanate, was added to the fixed cell preparation on the slide to cover the wells. the same amount was also added to the positive control slide. the slides were then incubated with the reagent in a moist chamber for 15 min at 37°c. following incubation, excess reagent was washed off with pbs, and the slide was gently washed in an agitating bath containing pbs for 5 min. the pbs was drained off, and slide was allowed to air dry at room temperature (15°c-30°c). one drop of imagen™ hmpv mounting fluid was added to the center of each well, and cover-slip was placed over the mounting fluid and specimen to ensure that there are no trapped air bubbles. the stained slides were immediately examined under epifluorescence microscope at ×400 and then ×1000. apple-green fluorescence was observed in the cells infected with hmpv, whereas non-infected cells appeared as red color because they were stained with the evans blue counterstain. images of these cells were captured using a microscopic camera (nikon-ds-fi1, nikon corp., tokyo, japan) and archived. during the study, a total of 91 pediatric patients were hospitalized based on upper and/or lower respiratory tract infections. of these 9 (9.9%) patients tested positive for hmpv antigens, as demonstrated by dfa from the nasopharyngeal secretions [ figure 1 ]. table 1 provides the demographic and clinical presentation data of all hmpv-positive patients. the age of these patients ranged from 9 months to 16 years and all were saudi nationals except one infant, who was a jordanian by nationality but was born and raised in saudi arabia. from the patient's demographics, it was observed that hmpv antigens were detected not only in patients from abha but also among those from its bordering areas, namely, algahama, bilahmar, ahud rufida, sarat abeedah, khamis mushait and bilasmer. three of the nine positive cases were found from abha (33.3%), and one positive case from each of the previously mentioned six cities was reported (11.1%). of the nine hmpv-positive patients, seven (77.8%) were hospitalized during the autumn and winter of 2016-2017. in the hmpv-positive patients, the symptoms included fever (77.8%), cough (77.8), shortness of breath (66.7%), nasal congestion (11.1%), cyanosis (11.1%) and stridor (11.1%). these patients also had underlying chronic illnesses such as chronic heart disease (22.2%) and bronchial asthma (11.1%), and most had tachypnea (88.8%). on physical examinations, bilateral crepitation and wheezing were found to be the major findings along with bronchopneumonia (55.5%) and aspiration pneumonia (22.2%). in saudi arabia, although few studies have reported the incidence, epidemiological elements and genetic diversity of hmpv in some regions, [15, 32, 33] there are no reports on the association between hmpv infections and the clinical presentations of respiratory tract infections among pediatrics. the current study found that in the aseer region of saudi arabia, about 10% of hospitalization among pediatrics with respiratory tract infections between july 2016 and november 2017 was due to hmpv infections. in addition, this study also found that hmpv infections were associated with presentation of acute respiratory symptoms, thereby highlighting the role of the infection in complicating the course of the disease. the results of the present study concur with several other studies demonstrating an association between hmpv infection and acute respiratory conditions such as bronchopneumonia and pneumonia. [3, 5] a previous study implicated hmpv as a causative agent for severe and acute respiratory infections among pediatric patients, [34] which is similar to the findings of the current study. another study found that children with hmpv infection are likely to have immunodeficiency; however, the current study was not able to substantiate these findings as none of the patients were found to be immunocompromised. [13] the prevalence of hmpv infection of the current study (about 10%) was similar to that reported in studies from saudi arabia and kuwait. [14, 15] however, other similar studies that used direct immunofluorescence assays for the detection of hmpv antibodies in sera of patients revealed much higher prevalence rates. [35] this suggests that different results can be obtained with different diagnostic techniques for hmpv infection. in the current study, we used dfa with monoclonal antibody, which has previously been shown to have 100% specificity, thereby indicating the reliability of our results. [31] the current study did not find any gender predilection in terms of the infections; these findings are in accordance with that of bastien et al. [4] and kahn. [29] in this study, all nine hmpv-positive children were from abha or its surrounding areas. these areas are known for their high altitude, low temperature and low oxygen tension. it was well known that the prevalence of viral respiratory infections is high in cold and dry areas. [36] in addition, almost 78% of the hmpv-positive cases were in children who had been admitted during the autumn and winter seasons. it is also known that the majority of the viruses responsible for bronchiolitis and bronchopneumonia have their peak infectivity during winter and late autumn, with only sporadic cases through other seasons. [37, 38] the hmpv-positive patients in the current study had fever, cough, nasal congestion, cyanosis, stridor and shortness of breath, while some also had underlying chronic illnesses such as chronic heart disease and bronchial asthma; similar hmpv-associated illnesses were observed in another study. [39] physical examination revealed wheezing and bilateral crepitation and clinically, the most common presentations were bronchopneumonia and aspiration pneumonia. these findings are in line with those of williams et al., [40] who found significant association between hmpv and wheezing exacerbations and/or bronchiolitis in infants and young children. in the aseer region of saudi arabia, hmpv was found to be responsible for about one-tenth of hospitalizations in children with acute respiratory tract infections. this study also confirmed that hmpv infection is associated with presentation of acute respiratory symptoms. ictv virus taxonomy profile: pneumoviridae a newly discovered human pneumovirus isolated from young children with respiratory tract disease evidence of human metapneumovirus in australian children human metapneumovirus infection in the canadian population human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus in severe respiratory syncytial virus bronchiolitis children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus infection in hospital referred south african children human metapneumovirus infections in mexico: epidemiological and clinical characteristics human metapneumovirus infections in hospitalised infants in spain human metapneumovirus study of human metapneumovirus-associated lower respiratory tract infections in egyptian adults human metapneumovirus in hospitalized children in amman human matapneumovirus in patients with respiratory tract infection in kuwait human matapneumovirus and human coronavirus infection and pathogenicity in saudi arabia children hospitalized with acute respiratory illness viral respiratory infections in the neonatal intensive care unit-a review comparison of clinical characteristics of human metapneumovirus and respiratory syncytial virus infection in hospitalized young children a case series on common cold to severe bronchiolitis and pneumonia in children following human metapneumovirus infection in sri lanka human metapneumovirus in adults. viruses 2013:5;87-110. 20. van den hoogen bg. respiratory tract infection due to human metapneumovirus among elderly patients human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children the role of human metapneumovirus in the critically ill adult patient clinical and radiologic characteristics of human metapneumovirus infections in adults detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in india real-time pcr probe optimization using design of experiments approach a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time rna pcr diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the north-east of england epidemiology of human metapneumovirus human metapneumovirus: review of an important respiratory pathogen evaluation of a commercial direct fluorescent-antibody assay for human metapneumovirus in respiratory specimens viral etiology of respiratory infections in children in southwestern saudi arabia using multiplex reverse-transcriptase polymerase chain reaction molecular epidemiology of human metapneumovirus in riyadh province, saudi arabia clinical impact and diagnosis of human metapneumovirus infection detection of human metapneumovirus by direct antigen test and shell vial cultures using immunofluorescent antibody staining seasonality of viral respiratory infections in southeast of brazil: the influence of temperature and air humidity seasonal occurrence of human metapneumovirus infections in croatia genetic diversity, seasonality and transmission network of human metapneumovirus: identification of a unique sub-lineage of the fusion and attachment genes human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults human metapneumovirus infection in children hospitalized for wheezing there are no conflicts of interest. key: cord-016293-pyb00pt5 authors: newell-mcgloughlin, martina; re, edward title: the flowering of the age of biotechnology 1990–2000 date: 2006 journal: the evolution of biotechnology doi: 10.1007/1-4020-5149-2_4 sha: doc_id: 16293 cord_uid: pyb00pt5 nan the significance of developing genetic and physical maps of the genome, and the importance of comparing the human genome with those of other species. it also suggested a preliminary focus on improving current technology. at the request of the u.s. congress, the office of technology assessment (ota) also studied the issue, and issued a document in 1987 -within days of the nrc report -that was similarly supportive. the ota report discussed, in addition to scientific issues, social and ethical implications of a genome program together with problems of managing funding, negotiating policy and coordinating research efforts. prompted by advisers at a 1988 meeting in reston, virginia, james wyngaarden, then director of the national institutes of health (nih) , decided that the agency should be a major player in the hgp, effectively seizing the lead from doe. the start of the joint effort was in may 1990 (with an "official" start in october) when a 5-year plan detailing the goals of the u.s. human genome project was presented to members of congressional appropriations committees in mid-february. this document co-authored by doe and nih and titled "understanding our genetic inheritance, the u.s. human genome project: the first five years" examined the then current state of genome science. the plan also set forth complementary approaches of the two agencies for attaining scientific goals and presented plans for administering research agenda; it described collaboration between u.s. and international agencies and presented budget projections for the project. according to the document, "a centrally coordinated project, focused on specific objectives, is believed to be the most efficient and least expensive way" to obtain the 3-billion base pair map of the human genome. in the course of the project, especially in the early years, the plan stated that "much new technology will be developed that will facilitate biomedical and a broad range of biological research, bring down the cost of many experiments (mapping and sequencing), and finding applications in numerous other fields." the plan built upon the 1988 reports of the office of technology assessment and the national research council on mapping and sequencing the human genome. "in the intervening two years," the document said, "improvements in technology for almost every aspect of genomics research have taken place. as a result, more specific goals can now be set for the project." the document describes objectives in the following areas mapping and sequencing the human genome and the genomes of model organisms; data collection and distribution; ethical, legal, and social considerations; research training; technology development; and technology transfer. these goals were to be reviewed each year and updated as further advances occured in the underlying technologies. they identified the overall budget needs to be the same as those identified by ota and nrc, namely about $200 million per year for approximately 15 years. this came to $13 billion over the entire period of the project. considering that in july 1990, the dna databases contained only seven sequences greater than 0.1 mb this was a major leap of faith. this approach was a major departure from the single-investigator-based gene of interest focus that research took hitherto. this sparked much controversy both before and after its inception. critics questioned the usefulness of genomic sequencing, they objected to the high cost and suggested it might divert funds from other, more focused, basic research. the prime argument to support the latter position is that there appeared to be are far less genes than accounted for by the mass of dna which would suggest that the major part of the sequencing effort would be of long stretches of base pairs with no known function, the so-called "junk dna." and that was in the days when the number of genes was presumed to be 80-100,000. if, at that stage, the estimated number was guessed to be closer to the actual estimate of 35-40,000 (later reduced to 20-25,000) this would have made the task seem even more foolhardy and less worthwhile to some. however, the ever-powerful incentive of new diagnostics and treatments for human disease beyond what could be gleaned from the gene-by-gene approach and the rapidly evolving technologies, especially that of automated sequencing, made it both an attractive and plausible aim. charles cantor (1990) , a principal scientist for the department of energy's genome project contended that doe and nih were cooperating effectively to develop organizational structures and scientific priorities that would keep the project on schedule and within its budget. he noted that there would be small short-term costs to traditional biology, but that the long-term benefits would be immeasurable. genome projects were also discussed and developed in other countries and sequencing efforts began in japan, france, italy, the united kingdom, and canada. even as the soviet union collapsed, a genome project survived as part of the russian science program. the scale of the venture and the manageable prospect for pooling data via computer made sequencing the human genome a truly international initiative. in an effort to include developing countries in the project unesco assembled an advisory committee in 1988 to examine unesco's role in facilitating international dialogue and cooperation. a privately-funded human genome organization (hugo) had been founded in 1988 to coordinate international efforts and serve as a clearinghouse for data. in that same year the european commission (ec) introduced a proposal entitled the "predictive medicine programme." a few ec countries, notably germany and denmark, claimed the proposal lacked ethical sensitivity; objections to the possible eugenic implications of the program were especially strong in germany (dickson 1989) . the initial proposal was dropped but later modified and adopted in 1990 as the "human genome analysis programme" (dickman and aldhous 1991) . this program committed substantial resources to the study of ethical issues. the need for an organization to coordinate these multiple international efforts quickly became apparent. thus the human genome organization (hugo), which has been called the "u.n. for the human genome," was born in the spring of 1988. composed of a founding council of scientists from seventeen countries, hugo's goal was to encourage international collaboration through coordination of research, exchange of data and research techniques, training, and debates on the implications of the projects (bodmer 1991) . in august 1990 nih began large-scale sequencing trials on four model organisms: the parasitic, cell-wall lacking pathogenic microbe mycoplasma capricolum, the prokaryotic microbial lab rat escherichia coli, the most simple animal caenorhabditis elegans, and the eukaryotic microbial lab rat saccharomyces cerevisiae. each research group agreed to sequence 3 megabases (mb) at 75 cents a base within 3 years. a sub living organism was actually fully sequenced and the complete sequence of that genome, the human cytomegalovirus (hcmv) genome was 0.23 mb. that year also saw the casting of the first salvo in the protracted debate on "ownership" of genetic information beginning with the more tangible question of ownership of cells. and, as with the debates of the early eighties, which were to be revisited later in the nineties, the respondent was the university of california. moore v. regents of the university of california was the first case in the united states to address the issue of who owns the rights to an individual's cells. diagnosed with leukemia, john moore had blood and bone marrow withdrawn for medical tests. suspicious of repeated requests to give samples because he had already been cured, moore discovered that his doctors had patented a cell line derived from his cells and so he sued. the california supreme court found that moore's doctor did not obtain proper informed consent, but, however, they also found that moore cannot claim property rights over his body. the quest for the holy grail of the human genome was both inspired by the rapidly evolving technologies for mapping and sequencing and subsequently spurred on the development of ever more efficient tools and techniques. advances in analytical tools, automation, and chemistries as well as computational power and algorithms revolutionized the ability to generate and analyze immense amounts of dna sequence and genotype information. in addition to leading to the determination of the complete sequences of a variety of microorganisms and a rapidly increasing number of model organisms, these technologies have provided insights into the repertoire of genes that are required for life, and their allelic diversity as well as their organization in the genome. but back in 1990 many of these were still nascent technologies. the technologies required to achieve this end could be broadly divided into three categories: equipment, techniques, and computational analysis. these are not truly discrete divisions and there was much overlap in their influence on each other. as noted, lloyd smith, michael and tim hunkapiller, and leroy hood conceived the automated sequencer and applied biosystems inc. brought it to market in june 1986. there is no much doubt that when applied biosystems inc. put it on the market that which had been a dream became decidedly closer to an achievable reality. in automating sangers chain termination sequencing system, hood modified both the chemistry and the data-gathering processes. in the sequencing reaction itself, he replaced radioactive labels, which were unstable, posed a health hazard, and required separate gels for each of the four bases. hood developed chemistry that used fluorescent dyes of different colors for each of the four dna bases. this system of "color-coding" eliminated the need to run several reactions in overlapping gels. the fluorescent labels addressed another issue which contributed to one of the major concerns of sequencing -data gathering. hood integrated laser and computer technology, eliminating the tedious process of information-gathering by hand. as the fragments of dna passed a laser beam on their way through the gel the fluorescent labels were stimulated to emit light. the emitted light was transmitted by a lens and the intensity and spectral characteristics of the fluorescence are measured by a photomultiplier tube and converted to a digital format that could be read directly into a computer. during the next thirteen years, the machine was constantly improved, and by 1999 a fully automated instrument could sequence up to 150,000,000 base pairs per year. in 1990 three groups came up with a variation on this approach. they developed what is termed capillary electrophoresis, one team was led by lloyd smith (luckey, 1990) , the second by barry karger , and the third by norman dovichi. in 1997 molecular dynamics introduced the megabace, a capillary sequencing machine. and not to be outdone the following year in 1998, the original of the species came up with the abi prism 3700 sequencing machine. the 3700 is also a capillary-based machine designed to run about eight sets of 96 sequence reactions per day. on the biology side, one of the biggest challenges was the construction of a physical map to be compiled from many diverse sources and approaches in such a way as to insure continuity of physical mapping data over long stretches of dna. the development of dna sequence tagged sites (stss) to correlate diverse types of dna clones aided this standardization of the mapping component by providing mappers with a common language and a system of landmarks for all the libraries from such varied sources as cosmids, yeast artificial chromosomes (yacs) and other rdnas clones. this way each mapped element (individual clone, contig, or sequenced region) would be defined by a unique sts. a crude map of the entire genome, showing the order and spacing of stss, could then be constructed. the order and spacing of these unique identifier sequences composing an sts map was made possible by development of mullis' polymerase chain reaction (pcr), which allows rapid production of multiple copies of a specific dna fragment, for example, an sts fragment. sequence information generated in this way could be recalled easily and, once reported to a database, would be available to other investigators. with the sts sequence stored electronically, there would be no need to obtain a probe or any other reagents from the original investigator. no longer would it be necessary to exchange and store hundreds of thousands of clones for full-scale sequencing of the human genome-a significant saving of money, effort, and time. by providing a common language and landmarks for mapping, sts's allowed genetic and physical maps to be cross-referenced. with a refinement on this technique to go after actual genes, sydney brenner proposed sequencing human cdnas to provide rapid access to the genes stating that 'one obvious way of finding at least a large part of the important [fraction] of the human genome is to look at the sequences of the messenger rna's of expressed genes' (brenner, 1990) . the following year the man who was to play a pivotal role on the world stage that became the human genome project suggested a way to implement sydney's approach. that player, nih biologist j. craig venter announced a strategy to find expressed genes, using ests (expressed sequence tag) (adams, 1991) . these so called ests represent a unique stretch of dna within a coding region of a gene, which as sydney suggested would be useful for identifying full-length genes and as a landmark for mapping. so using this approach projects were begun to mark gene sites on chromosome maps as sites of mrna expression. to help with this a more efficient method of handling large chunks of sequences was needed and two approaches were developed. yeast artificial chromosomes, which were developed by david burke, maynard olson, and george carle, increased insert size 10-fold (david t. burke et al., 1987) . caltech's second major contribution to the genome project was developed by melvin simon, and hiroaki shizuya. their approach to handling large dna segments was to develop "bacterial artificial chromosomes" (bacs), which basically allow bacteria to replicate chunks greater than 100,000 base pairs in length. this efficient production of more stable, large-insert bacs made the latter an even more attractive option, as they had greater flexibility than yacs. in 1994 in a collaboration that presages the snp consortium, washington university, st louis mo, was funded by the pharmaceutical company merck and the national cancer institute to provide sequence from those ests. more than half a million ests were submitted during the project (murr l et al., 1996) . on the analysis side was the major challenge to manage and mine the vast amount of dna sequence data being generated. a rate-limiting step was the need to develop semi-intelligent algorithms to achieve this herculean task. this is where the discipline of bioinformatics came into play. it had been evolving as a discipline since margaret oakley dayhoff used her knowledge of chemistry, mathematics, biology and computer science to develop this entirely new field in the early sixties. she is in fact credited today as a founder of the field of bioinformatics in which biology, computer science, and information technology merge into a single discipline. the ultimate goal of the field is to enable the discovery of new biological insights as well as to create a global perspective from which unifying principles in biology can be discerned. there are three important sub-disciplines within bioinformatics: the development of new algorithms and statistics with which to assess relationships among members of large data sets; the analysis and interpretation of various types of data including nucleotide and amino acid sequences, protein domains, and protein structures; and the development and implementation of tools that enable efficient access and management of different types of information. paralleling the rapid and very public ascent of recombinant dna technology during the previous two decades, the analytic and management tools of the discipline that was to become bioinformatics evolved at a more subdued but equally impressive pace. some of the key developments included tools such as the needleman-wunsch algorithm for sequence comparison which appeared even before recombinant dna technology had been demonstrated as early as 1970; the smith-waterman algorithm for sequence alignment (1974); the fastp algorithm (1985) and the fasta algorithm for sequence comparison by pearson and lupman in 1988 and perl (practical extraction report language) released by larry wall in 1987. on the data management side several databases with ever more effective storage and mining capabilities were developed over the same period. the first bioinformatic/biological databases were constructed a few years after the first protein sequences began to become available. the first protein sequence reported was that of bovine insulin in 1956, consisting of 51 residues. nearly a decade later, the first nucleic acid sequence was reported, that of yeast alanine trna with 77 bases. just one year later, dayhoff gathered all the available sequence data to create the first bioinformatic database. one of the first dedicated databases was the brookhaven protein databank whose collection consisted of ten x-ray crystallographic protein structures (acta. cryst. b, 1973) . the year 1982 saw the creation of the genetics computer group (gcg) as a part of the university of wisconsin biotechnology center. the group's primary and much used product was the wisconsin suite of molecular biology tools. it was spun off as a private company in 1989. the swiss-prot database made its debut in 1986 in europe at the department of medical biochemistry of the university of geneva and the european molecular biology laboratory (embl). the first dedicated "bioinformatics" company intelligenetics, inc. was founded in california in 1980. their primary product was the intelligenetics suite of programs for dna and protein sequence analysis. the first unified federal effort, the national center for biotechnology information (ncbi) was created at nih/nlm in 1988 and it was to play a crucial part in coordinating public databases, developing software tools for analyzing genome data, and disseminating information. and on the other side of the atlantic, oxford molecular group, ltd. (omg) was founded in oxford, uk by anthony marchington, david ricketts, james hiddleston, anthony rees, and w. graham richards. their primary focus was on rational drug design and their products such as anaconda, asp, and chameleon obviously reflected this as they were applied in molecular modeling, and protein design engineering. within two years ncbi were making their mark when david lipman, eugene myers, and colleagues at the ncbi published the basic local alignment search tool blast algorithm for aligning sequences (altschul et al., 1990) . it is used to compare a novel sequence with those contained in nucleotide and protein databases by aligning the novel sequence with previously characterized genes. the emphasis of this tool is to find regions of sequence similarity, which will yield functional and evolutionary clues about the structure and function of this novel sequence. regions of similarity detected via this type of alignment tool can be either local, where the region of similarity is based in one location, or global, where regions of similarity can be detected across otherwise unrelated genetic code. the fundamental unit of blast algorithm output is the high-scoring segment pair (hsp). an hsp consists of two sequence fragments of arbitrary but equal length whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score. this system has been refined and modified over the years the two principal variants presently in use being the ncbi blast and wu-blast (wu signifying washington university). the same year that blast was launched two other bioinformatics companies were launched. one was informax in bethesda, md whose products addressed sequence analysis, database and data management, searching, publication graphics, clone construction, mapping and primer design. the second, molecular applications group in california, was to play a bigger part on the proteomics end (michael levitt and chris lee). their primary products were look and segmod which are used for molecular modeling and protein design. the following year in 1991 the human chromosome mapping data repository, genome data base (gdb) was established. on a more global level, the development of computational capabilities in general and the internet in specific was also to play a considerable part in the sharing of data and access to databases that rendered the rapidity of the forward momentum of the hgp possible. also in 1991 edward uberbacher of oak ridge national laboratory in tennessee developed grail, the first of many gene-finding programs. in 1992 the first two "genomics" companies made their appearance. incyte pharmaceuticals, a genomics company headquartered in palo alto, california, was formed and myriad genetics, inc. was founded in utah. incyte's stated goal was to lead in the discovery of major common human disease genes and their related pathways. the company discovered and sequenced, with its academic collaborators (originally synteni from pat brown's lab at stanford), a number of important genes including brca1 and brca2, with mary claire king, epidemiologist at uc-berkeley, the genes linked to breast cancer in families with a high degree of incidence before age 45. by 1992 a low-resolution genetic linkage map of the entire human genome was published and u.s. and french teams completed genetic maps of both mouse and man. the mouse with an average marker spacing of 4.3 cm as determined by eric lander and colleagues at whitehead and the human, with an average marker spacing of 5 cm by jean weissenbach and colleagues at ceph (centre d'etude du polymorphisme humaine). the latter institute was the subject of a rather scathing book by paul rabinow (1999) based on what they did with this genome map. in 1993, an american biotechnology company, millennium pharmaceuticals, and the ceph, developed plans for a collaborative effort to discover diabetes genes. the results of this collaboration could have been medically significant and financially lucrative. the two parties had agreed that ceph would supply millennium with germplasm collected from a large coterie of french families, and millennium would supply funding and expertise in new technologies to accelerate the identification of the genes, terms to which the french government had agreed. but in early 1994, just as the collaboration was to begin, the french government cried halt! the government explained that the ceph could not be permitted to give the americans that most precious of substances for which there was no precedent in law -french dna. rabinow's book discusses the tangled relations and conceptions such as, can a country be said to have its own genetic material, the first but hardly the last franco-american disavowal of détente (paul rabinow, 1999) . the latest facilities such as the joint genome institute (jgi), walnut creek, ca are now able to sequence up to 10mb per day which makes it possible to sequence whole microbial genomes within a day. technologies currently under development will probably increase this capacity yet further through massively parallel sequencing and/or microfluidic processing making it possible to sequence multiple genotypes from several species. nineteen ninety-two saw one of the first shakeups in the progress of the hgp. that was the year that the first major outsider entered the race when britain's wellcome trust plunked down $95 million to join the hgp. this caused a mere ripple while the principal shake-ups occurred stateside. much of the debate and subsequently the direction all the way through the hgp process was shaped by the personalities involved. as noted the application of one of the innovative techniques, namely ests, to do an end run on patenting introduced one of those major players to the fray, craig venter. venter, the high school drop out who reached the age of majority in the killing fields of vietnam was to play a pivotal role in a more "civilized" but no less combative field of human endeavor. he came onto the world stage through his initial work on ests while at the national institute of neurological disorders and stroke (ninds) from 1984 to 1992. he noted in an interview with the scientist magazine in 1995, that there was a degree of ambiguity at ninds about his venturing into the field of genomics, while they liked the prestige of hosting one of the leaders and innovators in his newly emerging field, they were concerned about him moving outside the nind purview of the human brain and nervous system. ultimately, while he proclaimed to like the security and service infrastructure this institute afforded him, that same system became too restrictive for his interests and talent. he wanted the whole canvas of human-gene expression to be his universe, not just what was confined to the central nervous system. he was becoming more interested in taking a whole genome approach to understanding the overall structure of genomes and genome evolution, which was much broader than the mission of ninds. he noted, with some irony, in later years that the then current nih director harold varmus had wished in hindsight that nih had pushed to do a similar database in the public domain, clearly in venter's opinion varmus was in need of a refresher course in history! bernadine healy, nih director in 1994, was one of the few in a leadership role who saw the technical and fiscal promise of venter's work and, like all good administrators, it also presented an opportunity to resolve a thorny "personnel" issue. she appointed him head of the ad hoc committee to have an intramural genome program at nih to give the head of the hgp (that other larger than life personality jim watson) notice that he was not the sole arbitrator of the direction for the human genome project. however venter very soon established himself as an equally non-conformist character and with the tacit consent of his erstwhile benefactor. he initially assumed the mantle of a non-conformist through guilt by association rather than direct actions when it was revealed that nih was filing patent applications on thousands of these partial genes based on his ests catalyzing the first hgp fight at a congressional hearing. nih's move was widely criticized by the scientific community because, at the time, the function of genes associated with the partial sequences was unknown. critics charged that patent protection for the gene segments would forestall future research on them. the patent office eventually rejected the patents, but the applications sparked an international controversy over patenting genes whose functions were still unknown. interestingly enough despite nih's reliance on the est/cdna technique, venter, who was now clearly venturing outside the ninds mandated rubric, could not obtain government funding to expand his research, prompting him to leave nih in 1992. he moved on to become president and director of the institute for genomic research (tigr), a nonprofit research center based in gaithersburg, md. at the same time william haseltine formed a sister company, human genome sciences (hgs), to commercialize tigr products. venter continued est work at tigr, but also began thinking about sequencing entire genomes. again, he came up with a quicker and faster method: whole genome shotgun sequencing. he applied for an nih grant to use the method on hemophilus influenzae, but started the project before the funding decision was returned. when the genome was nearly complete, nih rejected his proposal saying the method would not work. in a triumphal flurry in late may 1995 and with a metaphorical nose-thumbing at his recently rejected "unworkable" grant venter announced that tigr and collaborators had fully sequenced the first free-living organism -haemophilus influenzae. in november 1994, controversy surrounding venter's research escalated. access restrictions associated with a cdna database developed by tigr and its rockville, md.-based biotech associate, human genome sciences (hgs) inc. -including hgs's right to preview papers on resulting discoveries and for first options to license products -prompted merck and co. inc. to fund a rival database project. in that year also britain "officially" entered the hgp race when the wellcome trust trumped down $95 million (as mentioned earlier). the following year hgs was involved in yet another patenting debacle forced by the rapid march of technology into uncharted patent law territory. on june 5, 1995 hgs applied for a patent on a gene that produces a "receptor" protein that is later called ccr5. at that time hgs has no idea that ccr5 is an hiv receptor. in december 1995, u.s. researcher robert gallo, the co-discoverer of hiv, and colleagues found three chemicals that inhibit the aids virus but they did not know how the chemicals work. in february 1996, edward berger at the nih discovered that gallo's inhibitors work in late-stage aids by blocking a receptor on the surface of t-cells. in june of that year in a period of just 10 days, five groups of scientists published papers saying ccr5 is the receptor for virtually all strains of hiv. in january 2000, schering-plough researchers told a san francisco aids conference that they have discovered new inhibitors. they knew that merck researchers had made similar discoveries. as a significant valentine in 2000 the u.s. patent and trademark office (uspto) grants hgs a patent on the gene that makes ccr5 and on techniques for producing ccr5 artificially. the decision sent hgs stock flying and dismayed researchers. it also caused the uspto to revise its definition of a "patentable" drug target. in the meantime haseltine's partner in rewriting patenting history, venter turned his focus to the human genome. he left tigr and started the for-profit company celera, a division of pe biosystems, the company that at times, thanks to hood and hunkapillar, led the world in the production of sequencing machines. using these machines, and the world's largest civilian supercomputer, venter finished assembling the human genome in just three years. following the debacle with the then nih director bernine healy over patenting the partial genes that resulted from est analysis, another major personality-driven event in that same year occurred. watson strongly opposed the idea of patenting gene fragments fearing that it would discourage research, and commented that "the automated sequencing machines 'could be run by monkeys.' " (nature june 29, 2000) with this dismissal watson resigned his nih nchgr post in 1992 to devote his full-time effort to directing cold spring harbor laboratory. his replacement was of a rather more pragmatic, less flamboyant nature. while venter maybe was described as an idiosyncratic shogun of the shotgun, francis collins was once described as the king arthur of the holy grail that is the human genome project. collins became the director of the national human genome research institute in 1993. he was considered the right man for the job following his 1989 success (along with lap-chee tsui) in identifying the gene for the cystic fibrosis transmembrane (cftr) chloride channel receptor that, when mutated, can lead to the onset of cystic fibrosis. although now indelibly connected with the topic non-plus tout in biology, like many great innovators in this field before him, francis collins had little interest in biology as he grew up on a farm in the shenandoah valley of virginia. from his childhood he seemed destined to be at the center of drama, his father was professor of dramatic arts at mary baldwin college and the early stage management of career was performed on a stage he built on the farm. while the physical and mathematical sciences held appeal for him, being possessed of a highly logical mind, collins found the format in which biology was taught in the high school of his day mind-numbingly boring, filled with dissections and rote memorization. he found the contemplation of the infinite outcomes of dividing by zero (done deliberately rather than by accident as in einstein's case) far more appealing than contemplating the innards of a frog. that biology could be gloriously logical only became clear to collins when, in 1970, he entered yale with a degree in chemistry from the university of virginia and was first exposed to the nascent field of molecular biology. anecdotally it was the tome, the book of life, penned by the theoretical physicist father of molecular biology, edwin schrodinger, while exiled in trinity college dublin in 1942 that was the catalyst for his conversion. like schrodinger he wanted to do something more obviously meaningful (for less than hardcore physicists at least!) than theoretical physics, so he went to medical school at unc-chapel hill after completing his chemistry doctorate in yale, and returned to the site of his road to damascus for post-doctoral study in the application of his newfound interest in human genetics. during this sojourn at yale, collins began working on developing novel tools to search the genome for genes that cause human disease. he continued this work, which he dubbed "positional cloning," after moving to the university of michigan as a professor in 1984. he placed himself on the genetic map when he succeeded in using this method to put the gene that causes cystic fibrosis on the physical map. while a less colorful-in-your-face character than venter he has his own personality quirks, for example, he pastes a new sticker onto the back of his motorcycle helmet every time he finds a new disease gene. one imagines that particular piece of really estate is getting rather crowded. interestingly it was not these four hundred pound us gorillas who proposed the eventually prescient timeline for a working draft but two from the old power base. in meetings in the us in 1994, john sulston and bob waterston proposed to produce a 'draft' sequence of the human genome by 2000, a full five years ahead of schedule. while agreed by most to be feasible it meant a rethinking of strategy and involved focusing resources on larger centers and emphasizing sequence acquisition. just as important, it asserts the value of draft quality sequence to biomedical research. discussion started with the british based wellcome trust as possible sponsors (marshall e. 1995) . by 1995 a rough draft of the human genome map was produced showing the locations of more than 30,000 genes. the map was produced using yeast artificial chromosomes and some chromosomes -notably the littlest 22 -were mapped in finer detail. these maps marked an important step toward clone-based sequencing. the importance was illustrated in the devotion of an entire edition of the journal nature to the subject. (nature 377: 175-379 1995) the duel between the public and private face of the hgp progressed at a pace over the next five years. following release of the mapping data some level of international agreement was decided on sequence data release and databases. they agreed on the release of sequence data, specifically, that primary genomic sequence should be in the public domain to encourage research and development to maximize its benefit to society. also that it be rapidly released on a daily basis with assemblies of greater than 1 kb and that the finished annotated sequence should be submitted immediately to the public databases. in 1996 an international consortium completed the sequence of the genome of the workhorse yeast saccharomyces cerevisiae. data had been released as the individual chromosomes were completed. the saccharomyces genome database (sgd) was created to curate this information. the project collects information and maintains a database of the molecular biology of s. cerevisiae. this database includes a variety of genomic and biological information and is maintained and updated by sgd curators. the sgd also maintains the s. cerevisiae gene name registry, a complete list of all gene names used in s. cerevisiae. in 1997 a new more powerful diagnostic tool termed snps (single nucleotide polymorphisms) was developed. snps are changes in single letters in our dna code that can act as markers in the dna landscape. some snps are associated closely with susceptibility to genetic disease, our response to drugs or our ability to remove toxins. the snp consortium although designated a limited company is a nonprofit foundation organized for the purpose of providing public genomic data. it is a collaborative effort between pharmaceutical companies and the wellcome trust with the idea of making available widely accepted, high-quality, extensive, and publicly accessible snp map. its mission was to develop up to 300,000 snps distributed evenly throughout the human genome and to make the information related to these snps available to the public without intellectual property restrictions. the project started in april 1999 and was anticipated to continue until the end of 2001. in the end, many more snps, about 1.5 million total, were discovered than was originally planned. by 1998 the complete genome sequence of mycobacterium tuberculosis was published by teams from the uk, france, us and denmark in june 1998. the abi prism 3700 sequencing machine, a capillary-based machine designed to run about eight sets of 96 sequence reactions per day also reached the market that year. that same year the genome sequence of the first multicellular organism, c. elegans was completed. c. elegans has a genome of about 100 mb and, as noted, is a primitive animal model organism used in a range of biological disciplines. by november 1999 the human genome draft sequence reached 1000 mb and the first complete human chromosome was sequenced -this first was reached on the east side of the atlantic by the hgp team led by the sanger centre, producing a finished sequence for chromosome 22, which is about 34 million base-pairs and includes at least 550 genes. according to anecdotal evidence when visiting his namesake centre, sanger asked: "what does this machine do then?" "dideoxy sequencing" came the reply, to which fred retorted: "haven't they come up with anything better yet?" as will be elaborated in the final chapter the real highlight of 2000 was production of a 'working draft' sequence of the human genome, which was announced simultaneously in the us and the uk. in a joint event, celera genomics announced completion of their 'first assembly' of the genome. in a remarkable special issue, nature included a 60-page article by the human genome project partners, studies of mapping and variation, as well as analysis of the sequence by experts in different areas of biology. science published the article by celera on their assembly of hgp and celera data as well as analyses of the use of the sequence. however to demonstrate the sensitivity of the market place to presidential utterances the joint appearances by bill clinton and tony blair touting this major milestone turned into a major cold shower when clinton's reassurance of access of the people to their genetic information caused a precipitous drop in celera's share value overnight. clinton's assurance that, "the effort to decipher the human genome will be the scientific breakthrough of the century -perhaps of all time. we have a profound responsibility to ensure that the life-saving benefits of any cutting-edge research are available to all human beings." (president bill clinton, wednesday, march 14, 2000) stands in sharp contrast to the statement from venter's colleague that " any company that wants to be in the business of using genes, proteins, or antibodies as drugs has a very high probability of running afoul of our patents. from a commercial point of view, they are severely constrained -and far more than they realize." (william a. haseltine, chairman and ceo, human genome sciences). the huge sell-off in stocks ended weeks of biotech buying in which those same stocks soared to unprecedented highs. by the next day, however, the genomic company spin doctors began to recover ground in a brilliant move which turned the clinton announcement into a public relations coup. all major genomics companies issued press releases applauding president clinton's announcement. the real news they argued, was that "for the first time a president strongly affirmed the importance of gene based patents." and the same bill haseltine of human genome sciences positively gushed as he happily pointed out that he "could begin his next annual report with the [president's] monumental statement, and quote today as a monumental day." as distinguished harvard biologist richard lewontin notes: "no prominent molecular biologist of my acquaintance is without a financial stake in the biotechnology business. as a result, serious conflicts of interest have emerged in universities and in government service (lewontin, 2000) . away from the spin doctors perhaps eric lander may have best summed up the herculean effort when he opined that for him "the human genome project has been the ultimate fulfilment: the chance to share common purpose with hundreds of wonderful colleagues towards a goal larger than ourselves. in the long run, the human genome project's greatest impact might not be the three billion nucleotides of the human chromosomes, but its model of scientific community." (ridley, 2000) 6. gene therapy the year 1990 also marked the passing of another milestone that was intimately connected to one of the fundamental drivers of the hgp. the california hereditary disorders act came into force and with it one of the potential solutions for human hereditary disorders. w. french anderson in the usa reported the first successful application of gene therapy in humans. the first successful gene therapy for a human disease was successfully achieved for severe combined immune deficiency (scid) by introducing the missing gene, adenosine deaminase deficiency (ada) into the peripheral lymphocytes of a 4-year-old girl and returning modified lymphocytes to her. although the results are difficult to interpret because of the concurrent use of polyethylene glycol-conjugated ada commonly referred to as pegylated ada (pgla) in all patients, strong evidence for in vivo efficacy was demonstrated. ada-modified t cells persisted in vivo for up to three years and were associated with increases in t-cell number and ada enzyme levels, t cells derived from transduced pgla were progressively replaced by marrow-derived t cells, confirming successful gene transfer into long-lived progenitor cells. ashanthi desilva, the girl who received the first credible gene therapy, continues to do well more than a decade later. cynthia cutshall, the second child to receive gene therapy for the same disorder as desilva, also continues to do well. within 10 years (by january 2000), more than 350 gene therapy protocols had been approved in the us and worldwide, researchers launched more than 400 clinical trials to test gene therapy against a wide array of illnesses. surprisingly, a disease not typically heading the charts of heritable disorders, cancer has dominated the research. in 1994 cancer patients were treated with the tumor necrosis factor gene, a natural tumor fighting protein which worked to a limited extent. even more surprisingly, after the initial flurry of success little has worked. gene therapy, the promising miracle of 1990 failed to deliver on its early promise over the decade. apart from those examples, there are many diseases whose molecular pathology is, or soon will be, well understood, but for which no satisfactory treatments have yet been developed. at the beginning of the nineties it appeared that gene therapy did offer new opportunities to treat these disorders both by restoring gene functions that have been lost through mutation and by introducing genes that can inhibit the replication of infectious agents, render cells resistant to cytotoxic drugs, or cause the elimination of aberrant cells. from this "genomic" viewpoint genes could be said to be viewed as medicines, and their development as therapeutics should embrace the issues facing the development of small-molecule and protein therapeutics such as bioavailability, specificity, toxicity, potency, and the ability to be manufactured at large scale in a cost-effective manner. of course for such a radical approach certain basal level criteria needed to be established for selecting disease candidates for human gene therapy. these include, such factors as the disease is an incurable, life-threatening disease; organ, tissue, and cell types affected by the disease have been identified; the normal counterpart of the defective gene has been isolated and cloned; either the normal gene can be introduced into a substantial subfraction of the cells from the affected tissue, or the introduction of the gene into the available target tissue, such as bone marrow, will somehow alter the disease process in the tissue affected by the disease; the gene can be expressed adequately (it will direct the production of enough normal protein to make a difference); and techniques are available to verify the safety of the procedure. an ideal gene therapeutic should, therefore, be stably formulated at room temperature and amenable to administration either as an injectable or aerosol or by oral delivery in liquid or capsule form. the therapeutic should also be suitable for repeat therapy, and when delivered, it should neither generate an immune response nor be destroyed by tissue-scavenging mechanisms. when delivered to the target cell, the therapeutic gene should then be transported to the nucleus, where it should be maintained as a stable plasmid or chromosomal integrant, and be expressed in a predictable, controlled fashion at the desired potency in a cell-specific or tissue-specific manner. in addition to the ada gene transfer in children with severe combined immunodeficiency syndrome, a gene-marking study of epstein-barr virus-specific cytotoxic t cells, and trials of gene-modified t cells expressing suicide or viral resistance genes in patients infected with hiv were studied in the early nineties. additional strategies for t-cell gene therapy which were pursued later in the decade involve the engineering of novel t-cell receptors that impart antigen specificity for virally infected or malignant cells. issues which still are not resolved include nuclear transport, integration, regulated gene expression and immune surveillance. this knowledge, when finally understood and applied to the design of delivery vehicles of either viral or non-viral origin, will assist in the realization of gene therapeutics as safe and beneficial medicines that are suited to the routine management of human health. scientists are also working on using gene therapy to generate antibodies directly inside cells to block the production of harmful viruses such as hiv or even cancer-inducing proteins. there is a specific connection with francis collins, as his motivation for pursuing the hgp was his pursuit of defective genes beginning with the cystic fibrosis gene. this gene, called the cf transmembrane conductance regulator, codes for an ion channel protein that regulates salts in the lung tissue. the faulty gene prevents cells from excreting salt properly causing a thick sticky mucus to build up and destroy lung tissue. scientists have spliced copies of the normal genes into disabled adeno viruses that target lung tissues and have used bronchioscopes to deliver them to the lungs. the procedure worked well in animal studies however clinical trials in humans were not an unmitigated success. because the cells lining the lungs are continuously being replaced the effect is not permanent and must be repeated. studies are underway to develop gene therapy techniques to replace other faulty genes. for example, to replace the genes responsible for factor viii and factor ix production whose malfunctioning causes hemophilia a and b respectively; and to alleviate the effects of the faulty gene in dopamine production that results in parkinson's disease. apart from technical challenges such a radical therapy also engenders ethical debate. many persons who voice concerns about somatic-cell gene therapy use a "slippery slope" argument. it sounds good in theory but where does one draw the line. there are many issues yet to be resolved in this field of thorny ethics "good" and "bad" uses of the gene modification, difficulty of following patients in long-term clinical research and such. many gene therapy candidates are children who are too young to understand the ramifications of this treatment: conflict of interest -pits individuals' reproductive liberties and privacy interests against the interests of insurance companies or society. one issue that is unlikely to ever gain acceptance is germline therapy, the removal of deleterious genes from the population. issues of justice and resource allocation also have been raised: in a time of strain on our health care system, can we afford such expensive therapy? who should receive gene therapy? if it is made available only to those who can afford it, then a number of civil rights groups claim that the distribution of desirable biological traits among different socioeconomic and ethnic groups would become badly skewed adding a new and disturbing layer of discriminatory behavior. indeed a major setback occurred before the end of the decade in 1999. jesse gelsinger was the first person to die from gene therapy, on september 17, 1999, and his death created another unprecedented situation when his family sued not only the research team involved in the experiment (u penn), the company genovo inc., but also the ethicist who offered moral advice on the controversial project. this inclusion of the ethicist as a defendant alongside the scientists and school was a surprising legal move that puts this specialty on notice, as will no doubt be the case with other evolving technologies such as stem cells and therapeutic cloning, that its members could be vulnerable to litigation over the philosophical guidance they provide to researchers. the penn group principal investigator james wilson approached ethicist arthur caplan about their plans to test the safety of a genetically engineered virus on babies with a deadly form of the liver disorder, ornithine transcarbamylase deficiency. the disorder allows poisonous levels of ammonia to build up in the blood system. caplan steered the researchers away from sick infants, arguing that desperate parents could not provide true informed consent. he said it would be better to experiment on adults with a less lethal form of the disease who were relatively healthy. gelsinger fell into that category. although he had suffered serious bouts of ammonia buildup, he was doing well on a special drug and diet regimen. the decision to use relatively healthy adults was controversial because risky, unproven experimental protocols generally use very ill people who have exhausted more traditional treatments, so have little to lose. in this case, the virus used to deliver the genes was known to cause liver damage, so some scientists were concerned it might trigger an ammonia crisis in the adults. wilson underestimated the risk of the experiment, omitted the disclosure about possible liver damage in earlier volunteers in the experiment and failed to mention the deaths of monkeys given a similar treatment during pre-clinical studies. a food and drug administration investigation after gelsinger's death found numerous regulatory violations by wilson's team, including the failure to stop the experiment and inform the fda after four successive volunteers suffered serious liver damage prior to the teen's treatment. in addition, the fda said gelsinger did not qualify for the experiment, because his blood ammonia levels were too high just before he underwent the infusion of genetic material. the fda suspended all human gene experiments by wilson and the university of penn subsequently restricting him solely to animal studies. a follow-up fda investigation subsequently alleged he improperly tested the experimental treatment on animals. financial conflicts of interest also surrounded james wilson, who stood to personally profit from the experiment through genovo his biotechnology company. the lawsuit was settled out of court for undisclosed terms in november 2000. the fda also suspended gene therapy trials at st. elizabeth's medical center in boston, a major teaching affiliate of tufts university school of medicine, which sought to use gene therapy to reverse heart disease, because scientists there failed to follow protocols and may have contributed to at least one patient death. in addition, the fda temporarily suspended two liver cancer studies sponsored by the schering-plough corporation because of technical similarities to the university of pennsylvania study. some research groups voluntarily suspended gene therapy studies, including two experiments sponsored by the cystic fibrosis foundation and studies at beth israel deaconess medical center in boston aimed at hemophilia. the scientists paused to make sure they learned from the mistakes. the nineties also saw the development of another "high-thoughput" breakthrough, a derivative of the other high tech revolution namely dna chips. in 1991 biochips were developed for commercial use under the guidance of affymetrix. dna chips or microarrays represent a "massively parallel" genomic technology. they facilitate high throughput analysis of thousands of genes simultaneously, and are thus potentially very powerful tools for gaining insight into the complexities of higher organisms including analysis of gene expression, detecting genetic variation, making new gene discoveries, fingerprinting strains and developing new diagnostic tools. these technologies permit scientists to conduct large scale surveys of gene expression in organisms, thus adding to our knowledge of how they develop over time or respond to various environmental stimuli. these techniques are especially useful in gaining an integrated view of how multiple genes are expressed in a coordinated manner. these dna chips have broad commercial applications and are now used in many areas of basic and clinical research including the detection of drug resistance mutations in infectious organisms, direct dna sequence comparison of large segments of the human genome, the monitoring of multiple human genes for disease associated mutations, the quantitative and parallel measurement of mrna expression for thousands of human genes, and the physical and genetic mapping of genomes. however the initial technologies, or more accurately the algorithms used to extract information, were far from robust and reproducible. the erstwhile serial entrepreneur, al zaffaroni (the rebel who in 1968 founded alza when syntex ignored his interest in developing new ways to deliver drugs) founded yet another company, affymetrix, under the stewardship of stephen fodor, which was subject to much abuse for providing final extracted data and not allowing access to raw data. as with other personalities of this high through put era, seattle-bred steve fodor was also somewhat of a polymath having contributed to two major technologies, microarrays and combinatorial chemistry, the former has delivered on it's, promise while the latter, like gene therapy, is still in a somewhat extended gestation. and despite the limitations of being an industrial scientist he has had a rather prolific portfolio of publications. his seminal manuscripts describing this work have been published in all the journals of note, science, nature and pnas and was recognized in 1992 by the aaas by receiving the newcomb-cleveland award for an outstanding paper published in science. fodor began his industrial career in yet another zaffaroni firm. in 1989 he was recruited to the affymax research institute in palo alto where he spearheaded the effort to develop high-density arrays of biological compounds. his initial interest was in the broad area of what came to be called combinatorial chemistry. of the techniques developed, one approach permitted high resolution chemical synthesis in a light-directed, spatially-defined format. in the days before positive selection vectors, a researcher might have screened thousands of clones by hand with an oligonucleotide probe just to find one elusive insert. fodor's (and his successors) dna array technology reverses that approach. instead of screening an array of unknowns with a defined probe -a cloned gene, pcr product, or synthetic oligonucleotide -each position or "probe cell" in the array is occupied by a defined dna fragment, and the array is probed with the unknown sample. fodor used his chemistry and biophysics background to develop very dense arrays of these biomolecules by combining photolithographic methods with traditional chemical techniques. the typical array may contain all possible combinations of all possible oligonucleotides (8-mers, for example) that occur as a "window" which is tracked along a dna sequence. it might contain longer oligonucleotides designed from all the open reading frames identified from a complete genome sequence. or it might contain cdnas -of known or unknown sequence -or pcr products. of course it is one thing to produce data it is quite another to extract it in a meaningful manner. fodor's group also developed techniques to read these arrays, employing fluorescent labeling methods and confocal laser scanning to measure each individual binding event on the surface of the chip with extraordinary sensitivity and precision. this general platform of microarray based analysis coupled to confocal laser scanning has become the standard in industry and academia for large-scale genomics studies. in 1993, fodor co-founded affymetrix where the chip technology has been used to synthesize many varieties of high density oligonucleotide arrays containing hundreds of thousands of dna probes. in 2001, steve fodor founded perlegen, inc., a new venture that applied the chip technology towards uncovering the basic patterns of human diversity. his company's stated goals are to analyze more than one million genetic variations in clinical trial participants to explain and predict the efficacy and adverse effect profiles of prescription drugs. in addition, perlegen also applies this expertise to discovering genetic variants associated with disease in order to pave the way for new therapeutics and diagnostics. fodor's former company diversified into plant applications by developing a chip of the archetypal model of plant systems arabidopsis and supplied pioneer hi bred with custom dna chips for monitoring maize gene expression. they (affymetrix) have established programs where academic scientists can use company facilities at a reduced price and set up 'user centers' at selected universities. a related but less complex technology called 'spotted' dna chips involves precisely spotting very small droplets of genomic or cdna clones or pcr samples on a microscope slide. the process uses a robotic device with a print head bearing fine "repeatograph" tips that work like fountain pens to draw up dna samples from a 96-well plate and spot tiny amounts on a slide. up to 10,000 individual clones can be spotted in a dense array within one square centimeter on a glass slide. after hybridization with a fluorescent target mrna, signals are detected by a custom scanner. this is the basis of the systems used by molecular dynamics and incyte (who acquired this technology when it took over synteni). in 1997, incyte was looking to gather more data for its library and perform experiments for corporate subscribers. the company considered buying affymetrix genechips but opted instead to purchase the smaller synteni, which had sprung out of pat brown's stanford array effort. synteni's contact printing technology resulted in dense -and cheaper -arrays. though incyte used the chips only internally, affymetrix sued, claiming synteni/incyte was infringing on its chip density patents. the suit argued that dense biochips -regardless of whether they use photolithography -cannot be made without a license from affymetrix! and in a litigious congo line endemic of this hi-tech era incyte countersued and for good measure also filed against genetic database competitor gene logic for infringing incyte's patents on database building. meanwhile, hyseq sued affymetrix, claiming infringement of nucleotide hybridization patents obtained by its cso. affymetrix, in turn, filed a countersuit, claiming hyseq infringed the spotted array patents. hyseq then reached back and found an additional hybridization patent it claimed that affymetrix had infringed. and so on into the next millennium! in part to avoid all of this another california company nanogen, inc. took a different approach to single nucleotide polymorphism discrimination technology. in an article in the april 2000 edition of nature biotechnology, entitled "single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips," nanogen describes the use of microchips to identify variants of the mannose binding protein gene that differ from one another by only a single dna base. the mannose binding protein (mbp) is a key component of the innate immune system in children who have not yet developed immunity to a variety of pathogens. to date, four distinct variants (alleles) of this gene have been identified, all differing by only a single nucleotide of dna. mbp was selected for this study because of its potential clinical relevance and its genetic complexity. the samples were assembled at the nci laboratory in conjunction with the national institutes of health and transferred to nanogen for analysis. however, from a high throughput perspective there is a question mark over microarrays. mark benjamin, senior director of business development at rosetta inpharmatics (kirkland, wa), is skeptical about the long-term prospects for standard dna arrays in high-throughput screening as the first steps require exposing cells and then isolating rna, which is something that is very hard to do in a high-throughput format. another drawback is that most of the useful targets are likely to be unknown (particularly in the agricultural sciences where genome sequencing is still in its infancy), and dna arrays that are currently available test only for previously sequenced genes. indeed, some argue that current dna arrays may not be sufficiently sensitive to detect the low expression levels of genes encoding targets of particular interest. and the added complication of the companies' reluctance to provide "raw data" means that derived data sets may be created with less than optimum algorithims thereby irretrievably losing potentially valuable information from the starting material. reverse engineering is a possible approach but this is laborious and time consuming and being prohibited by many contracts may arouse the interest of the ever-vigilant corporate lawyers. over the course of the nineties, outgrowths of functional genomics have been termed proteomics and metabolomics, which are the global studies of gene expression at the protein and metabolite levels respectively. the study of the integration of information flow within an organism is emerging as the field of systems biology. in the area of proteomics, the methods for global analysis of protein profiles and cataloging protein-protein interactions on a genome-wide scale are technically more difficult but improving rapidly, especially for microbes. these approaches generate vast amounts of quantitative data. the amount of expression data becoming available in the public and private sectors is already increasing exponentially. gene and protein expression data rapidly dwarfed the dna sequence data and is considerably more difficult to manage and exploit. in microbes, the small sizes of the genomes and the ease of handling microbial cultures, will enable high throughput, targeted deletion of every gene in a genome, individually and in combinations. this is already available on a moderate throughput scale in model microbes such as e. coli and yeast. combining targeted gene deletions and modifications with genome-wide assay of mrna and protein levels will enable intricate inter-dependencies among genes to be unraveled. simultaneous measurement of many metabolites, particularly in microbes, is beginning to allow the comprehensive modeling and regulation of fluxes through interdependent pathways. metabolomics can be defined as the quantitative measurement of all low molecular weight metabolites in an organism's cells at a specified time under specific environmental conditions. combining information from metabolomics, proteomics and genomics will help us to obtain an integrated understanding of cell biology. the next hierarchical level of phenotype considers how the proteome within and among cells cooperates to produce the biochemistry and physiology of individual cells and organisms. several authors have tentatively offered "physiomics" as a descriptor for this approach. the final hierarchical levels of phenotype include anatomy and function for cells and whole organisms. the term "phenomics" has been applied to this level of study and unquestionably the more well known omics namely economics, has application across all those fields. and, coming slightly out of left field this time, the spectre of eugenics needless to say was raised in the omics era. in the year 1992 american and british scientists unveiled a technique which has come to be known as pre-implantation genetic diagnosis (pid) for testing embryos in vitro for genetic abnormalities such as cystic fibrosis, hemophilia, and down's syndrome (wald, 1992) . this might be seen by most as a step forward, but it led ethicist david s. king (1999) to decry pid as a technology that could exacerbate the eugenic features of prenatal testing and make possible an expanded form of free-market eugenics. he further argues that due to social pressures and eugenic attitudes held by clinical geneticists in most countries, it results in eugenic outcomes even though no state coercion is involved and that, as abortion is not involved, and multiple embryos are available, pid is radically more effective as a tool of genetic selection. the first regulatory approval of a recombinant dna technology in the u.s. food supply was not a plant but an industrial enzyme that has become the hallmark of food biotechnology success. enzymes were important agents in food production long before modern biotechnology was developed. they were used, for instance, in the clotting of milk to prepare cheese, the production of bread and the production of alcoholic beverages. nowadays, enzymes are indispensable to modern food processing technology and have a great variety of functions. they are used in almost all areas of food production including grain processing, milk products, beer, juices, wine, sugar and meat. chymosin, known also as rennin, is a proteolytic enzyme whose role in digestion is to curdle or coagulate milk in the stomach, efficiently converting liquid milk to a semisolid like cottage cheese, allowing it to be retained for longer periods in a neonate's stomach. the dairy industry takes advantage of this property to conduct the first step in cheese production. chy-max™, an artificially produced form of the chymosin enzyme for cheese-making, was approved in 1990. in some instances they replace less acceptable "older" technology, for example the enzyme chymosin. unlike crops industrial enzymes have had relatively easy passage to acceptance for a number of reasons. as noted they are part of the processing system and theoretically do not appear in the final product. today about 90% of the hard cheese in the us and uk is made using chymosin from geneticallymodified microbes. it is easier to purify, more active (95% as compared to 5%) and less expensive to produce (microbes are more prolific, more productive and cheaper to keep than calves). like all enzymes it is required only in very small quantities and because it is a relatively unstable protein it breaks down as the cheese matures. indeed, if the enzyme remained active for too long it would adversely affect the development of the cheese, as it would degrade the milk proteins to too great a degree. such enzymes have gained the support of vegetarian organizations and of some religious authorities. for plants the nineties was the era of the first widespread commercialization of what came to be known in often deprecating and literally inaccurate terms as gmos (genetically modified organisms). when the nineties dawned dicotyledonous plants were relatively easily transformed with agrobacterium tumefaciens but many economically important plants, including the cereals, remained inaccessible for genetic manipulation because of lack of effective transformation techniques. in 1990 this changed with the technology that overcame this limitation. michael fromm, a molecular biologist at the plant gene expression center, reported the stable transformation of corn using a high-speed gene gun. the method known as biolistics uses a "particle gun" to shoot metal particles coated with dna into cells. initially a gunpowder charge subsequently replaced by helium gas was used to accelerate the particles in the gun. there is a minimal disruption of tissue and the success rate has been extremely high for applications in several plant species. the technology rights are now owned by dupont. in 1990 some of the first of the field trials of the crops that would dominate the second half of the nineties began, including bt corn (with the bacillus thuriengenesis cry protein discussed in chapter three). in 1992 the fda declared that genetically engineered foods are "not inherently dangerous" and do not require special regulation. since 1992, researchers have pinpointed and cloned several of the genes that make selected plants resistant to certain bacterial and fungal infections; some of these genes have been successfully inserted into crop plants that lack them. many more infection-resistant crops are expected in the near future, as scientists find more plant genes in nature that make plants resistant to pests. plant genes, however, are just a portion of the arsenal; microorganisms other than bt also are being mined for genes that could help plants fend off invaders that cause crop damage. the major milestone of the decade in crop biotechnology was approval of the first bioengineered crop plant in 1994. it represented a double first not just of the first approved food crop but also of the first commercial validation of a technology which was to be surpassed later in the decade. that technology, antisense technology works because nucleic acids have a natural affinity for each other. when a gene coding for the target in the genome is introduced in the opposite orientation, the reverse rna strand anneals and effectively blocks expression of the enzyme. this technology was patented by calgene for plant applications and was the technology behind the famous flavr savr tomatoes. the first success for antisense in medicine was in 1998 when the u.s. food and drug administration gave the go-ahead to the cytomegalovirus (cmv) inhibitor fomivirsen, a phosphorothionate antiviral for the aids-related condition cmv retinitis making it the first drug belonging to isis, and the first antisense drug ever, to be approved. another technology, although not apparent at the time was behind the second approval and also the first and only successful to date in a commercial tree fruit biotech application. the former was a virus resistant squash the second the papaya ringspot resistant papaya. both owed their existence as much to historic experience as modern technology. genetically engineered virus-resistant strains of squash and cantaloupe, for example, would never have made it to farmers' fields if plant breeders in the 1930's had not noticed that plants infected with a mild strain of a virus do not succumb to more destructive strains of the same virus. that finding led plant pathologist roger beachy, then at washington university in saint louis, to wonder exactly how such "cross-protection" worked -did part of the virus prompt it? in collaboration with researchers at monsanto, beachy used an a. tumefaciens vector to insert into tomato plants a gene that produces one of the proteins that makes up the protein coat of the tobacco mosaic virus. he then inoculated these plants with the virus and was pleased to discover, as reported in 1986, that the vast majority of plants did not succumb to the virus. eight years later, in 1994, virus-resistant squash seeds created with beachy's method reached the market, to be followed soon by bioengineered virus-resistant seeds for cantaloupes, potatoes, and papayas. (breeders had already created virusresistant tomato seeds by using traditional techniques.) and the method of protection still remained a mystery when the first approvals were given in 1994 and 1996. gene silencing was perceived initially as an unpredictable and inconvenient side effect of introducing transgenes into plants. it now seems that it is the consequence of accidentally triggering the plant's adaptive defense mechanism against viruses and transposable elements. this recently discovered mechanism, although mechanistically different, has a number of parallels with the immune system of mammals. how this system worked was not elucidated until later in the decade by a researcher who was seeking a very different holy grail -the black rose! rick jorgensen, at that time at dna plant technologies in oakland, ca and subsequently of, of the university of california davis attempted to overexpress the chalcone synthase gene by introducing a modified copy under a strong promoter.surprisingly he obtained white flowers, and many strange variegated purple and white variations in between. this was the first demonstration of what has come to be known as post-transcriptional gene silencing (ptgs). while initially it was considered a strange phenomenon limited to petunias and a few other plant species, it is now one of the hottest topics in molecular biology. rna interference (rnai) in animals and basal eukaryotes, quelling in fungi, and ptgs in plants are examples of a broad family of phenomena collectively called rna silencing (hannon 2002; plasterk 2002) . in addition to its occurrence in these species it has roles in viral defense (as demonstrated by beachy) and transposon silencing mechanisms among other things. perhaps most exciting, however, is the emerging use of ptgs and, in particular, rnai -ptgs initiated by the introduction of double-stranded rna (dsrna) -as a tool to knock out expression of specific genes in a variety of organisms. nineteen ninety one also heralded yet another first. the february 1, 1991 issue of science reported the patenting of "molecular scissors": the nobel-prize winning discovery of enzymatic rna, or "ribozymes," by thomas czech of the university of colorado. it was noted that the u.s. patent and trademark office had awarded an "unusually broad" patent for ribozymes. the patent is u.s. patent no. 4,987,071, claim 1 of which reads as follows: "an enzymatic rna molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded rna in a separate rna molecule, and causing cleavage at said cleavage site by a transesterification reaction." although enzymes made of protein are the dominant form of biocatalyst in modern cells, there are at least eight natural rna enzymes, or ribozymes, that catalyze fundamental biological processes. one of which was yet another discovery by plant virologists, in this instance the hairpin ribozyme was discovered by george bruening at uc davis. the self-cleavage structure was originally called a paperclip, by the bruening laboratory which discovered the reactions. as mentioned in chapter 3, it is believed that these ribozymes might be the remnants of an ancient form of life that was guided entirely by rna. since a ribozyme is a catalytic rna molecule capable of cleaving itself and other target rnas it therefore can be useful as a control system for turning off genes or targeting viruses. the possibility of designing ribozymes to cleave any specific target rna has rendered them valuable tools in both basic research and therapeutic applications. in the therapeutics area, they have been exploited to target viral rnas in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. most notably, several ribozyme gene therapy protocols for hiv patients are already in phase 1 trials. more recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. however, targeting ribozymes to the cellular compartment containing their target rnas has proved a challenge. at the other bookend of the decade in 2000, samarsky et al. reported that a family of small rnas in the nucleolus (snornas) can readily transport ribozymes into this subcellular organelle. in addition to the already extensive panoply of rna entities yet another has potential for mischief. viroids are small, single-stranded, circular rnas containing 246-463 nucleotides arranged in a rod-like secondary structure and are the smallest pathogenic agents yet described. the smallest viroid characterized to date is rice yellow mottle sobemovirus (rymv), at 220 nucleotides. in comparison, the genome of the smallest known viruses capable of causing an infection by themselves, the single-stranded circular dna of circoviruses, is around 2 kilobases in size. the first viroid to be identified was the potato spindle tuber viroid (pstvd). some 33 species have been identified to date. unlike the many satellite or defective interfering rnas associated with plant viruses, viroids replicate autonomously on inoculation of a susceptible host. the absence of a protein capsid and of detectable messenger rna activity implies that the information necessary for replication and pathogenesis resides within the unusual structure of the viroid genome. the replication mechanism actually involves interaction with rna polymerase ii, an enzyme normally associated with synthesis of messenger rna, and "rolling circle" synthesis of new rna. some viroids have ribozyme activity which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates. it has been proposed that viroids are "escaped introns". viroids are usually transmitted by seed or pollen. infected plants can show distorted growth. from its earliest years, biotechnology attracted interest outside scientific circles. initially the main focus of public interest was on the safety of recombinant dna technology, and of the possible risks of creating uncontrollable and harmful novel organisms (berg , 1975) . the debate on the deliberate release of genetically modified organisms, and on consumer products containing or comprising them, followed some years later (nas, 1987) . it is interesting to note that within the broad ethical tableau of potential issues within the science and products of biotechnology, the seemingly innocuous field of plant modification has been one of the major players of the 1990's. the success of agricultural biotechnology is heavily dependent on its acceptance by the public, and the regulatory framework in which the industry operates is also influenced by public opinion. as the focus for molecular biology research shifted from the basic pursuit of knowledge to the pursuit of lucrative applications, once again as in the previous two decades the specter of risk arose as the potential of new products and applications had to be evaluated outside the confines of a laboratory. however, the specter now became far more global as the implications of commercial applications brought not just worker safety into the loop but also, the environment, agricultural and industrial products and the safety and well being of all living things. beyond "deliberate" release, the rac guidelines were not designed to address these issues, so the matter moved into the realm of the federal agencies who had regulatory authority which could be interpreted to oversee biotechnology issues. this adaptation of oversight is very much a dynamic process as the various agencies wrestle with the task of applying existing regulations and developing new ones for oversight of this technology in transition. as the decade progressed focus shifted from basic biotic stress resistance to more complex modifications the next generation of plants will focus on value added traits in which valuable genes and metabolites will be identified and isolated, with some of the later compounds being produced in mass quantities for niche markets. two of the more promising markets are nutraceuticals or so-called "functional foods" and plants developed as bioreactors for the production of valuable proteins and compounds, a field known as plant molecular farming. developing plants with improved quality traits involves overcoming a variety of technical challenges inherent to metabolic engineering programs. both traditional plant breeding and biotechnology techniques are needed to produce plants carrying the desired quality traits. continuing improvements in molecular and genomic technologies are contributing to the acceleration of product development in this space. by the end of the decade in 1999, applying nutritional genomics, della penna (1999) isolated a gene, which converts the lower activity precursors to the highest activity vitamin e compound, alpha-tocopherol. with this technology, the vitamin e content of arabidopsis seed oil has been increased nearly 10-fold and progress has been made to move the technology to crops such as soybean, maize, and canola. this has also been done for folates in rice. omega three fatty acids play a significant role in human health, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), which are present in the retina of the eye and cerebral cortex of the brain, respectively, are some of the most well documented from a clinical perspective. it is believed that epa and dha play an important role in the regulation of inflammatory immune reactions and blood pressure, treatment of conditions such as cardiovascular disease and cystic fibrosis, brain development in utero, and, in early postnatal life, the development of cognitive function. they are mainly found in fish oil and the supply is limited. by the end of the decade ursin (2000) had succeeded in engineering canola to produce these fatty acids. from a global perspective another value-added development had far greater impact both technologically and socio-economically. a team led by ingo potrykus (1999) engineered rice to produce pro-vitamin a, which is an essential micronutrient. widespread dietary deficiency of this vitamin in rice-eating asian countries, which predisposes children to diseases such as blindness and measles, has tragic consequences. improved vitamin a nutrition would alleviate serious health problems and, according to unicef, could also prevent up to two million infant deaths due to vitamin a deficiency. adoption of the next stage of gm crops may proceed more slowly, as the market confronts issues of how to determine price, share value, and adjust marketing and handling to accommodate specialized end-use characteristics. furthermore, competition from existing products will not evaporate. challenges that have accompanied gm crops with improved agronomic traits, such as the stalled regulatory processes in europe, will also affect adoption of nutritionally improved gm products. beyond all of this, credible scientific research is still needed to confirm the benefits of any particular food or component. for functional foods to deliver their potential public health benefits, consumers must have a clear understanding of, and a strong confidence level in, the scientific criteria that are used to document health effects and claims. because these decisions will require an understanding of plant biochemistry, mammalian physiology, and food chemistry, strong interdisciplinary collaborations will be needed among plant scientists, nutritionists, and food scientists to ensure a safe and healthful food supply. in addition to being a source of nutrition, plants have been a valuable wellspring of therapeutics for centuries. during the nineties, however, intensive research has focused on expanding this source through rdna biotechnology and essentially using plants and animals as living factories for the commercial production of vaccines, therapeutics and other valuable products such as industrial enzymes and biosynthetic feedstocks. possibilities in the medical field include a wide variety of compounds, ranging from edible vaccine antigens against hepatitis b and norwalk viruses (arntzen, 1997) and pseudomonas aeruginosa and staphylococcus aureus to vaccines against cancer and diabetes, enzymes, hormones, cytokines, interleukins, plasma proteins, and human alpha-1-antitrypsin. thus, plant cells are capable of expressing a large variety of recombinant proteins and protein complexes. therapeutics produced in this way are termed plant made pharmaceuticals (pmps). and non-therapeutics are termed plant made industrial products (pmips) (newell-mcgloughlin, 2006) . the first reported results of successful human clinical trials with their transgenic plant-derived pharmaceuticals were published in 1998. they were an edible vaccine against e. coli-induced diarrhea and a secretory monoclonal antibody directed against streptococcus mutans, for preventative immunotherapy to reduce incidence of dental caries. haq et al. (1995) reported the expression in potato plants of a vaccine against e. coli enterotoxin (etec) that provided an immune response against the toxin in mice. human clinical trials suggest that oral vaccination against either of the closely related enterotoxins of vibrio cholerae and e. coli induces production of antibodies that can neutralize the respective toxins by preventing them from binding to gut cells. similar results were found for norwalk virus oral vaccines in potatoes. for developing countries, the intention is to deliver them in bananas or tomatoes (newell-mcgloughlin, 2006) . plants are also faster, cheaper, more convenient and more efficient than the principal eukaryotic production system, namely chinese hamster ovary (cho) cells for the production of pharmaceuticals. hundreds of acres of protein-containing seeds could inexpensively double the production of a cho bioreactor factory. in addition, proteins can be expressed at the highest levels in the harvestable seed and plant-made proteins and enzymes formulated in seeds have been found to be extremely stable, reducing storage and shipping costs. pharming may also enable research on drugs that cannot currently be produced. for example, croptech in blacksburg, va., is investigating a protein that seems to be a very effective anticancer agent. the problem is that this protein is difficult to produce in mammalian cell culture systems as it inhibits cell growth. this should not be a problem in plants. furthermore, production size is flexible and easily adjustable to the needs of changing markets. making pharmaceuticals from plants is also a sustainable process, because the plants and crops used as raw materials are renewable. the system also has the potential to address problems associated with provision of vaccines to people in developing countries. products from these alternative sources do not require a so-called "cold chain" for refrigerated transport and storage. those being developed for oral delivery obviates the need for needles and aspectic conditions which often are a problem in those areas. apart from those specific applications where the plant system is optimum there are many other advantages to using plant production. many new pharmaceuticals based on recombinant proteins will receive regulatory approval from the united states food and drug administration (fda) in the next few years. as these therapeutics make their way through clinical trials and evaluation, the pharmaceutical industry faces a production capacity challenge. pharmaceutical discovery companies are exploring plant-based production to overcome capacity limitations, enable production of complex therapeutic proteins, and fully realize the commercial potential of their biopharmaceuticals (newell-mcgloughlin, 2006) . nineteen ninety also marked a major milestone in the animal biotech world when herman made his appearance on the world's stage. since the palmiter's mouse, transgenic technology has been applied to several species including agricultural species such as sheep, cattle, goats, pigs, rabbits, poultry, and fish. herman was the first transgenic bovine created by genpharm international, inc., in a laboratory in the netherlands at the early embryo stage. scientist's microinjected recently fertilized eggs with the gene coding for human lactoferrin. the scientists then cultured the cells in vitro to the embryo stage and transferred them to recipient cattle. lactoferrin, an iron-containing anti-bacterial protein is essential for infant growth. since cow's milk doesn't contain lactoferrin, infants must be fed from other sources that are rich in iron -formula or mother's milk (newell-mcgloughlin, 2001) . as herman was a boy he would be unable to provide the source, that would require the production of daughters which was not necessarily a straightforward process. the dutch parliments permission was required. in 1992 they finally approved a measure that permitted the world's first genetically engineered bull to reproduce. the leiden-based gene pharming proceeded to artificially inseminate 60 cows with herman's sperm. with a promise that the protein, lactoferrin, would be the first in a new generation of inexpensive, high-tech drugs derived from cows' milk to treat complex diseases like aids and cancer. herman, became the father of at least eight female calves in 1994, and each one inherited the gene for lactoferrin production. while their birth was initially greeted as a scientific advancement that could have far-reaching effects for children in developing nations, the levels of expression were too low to be commercially viable. by 2002, herman, who likes to listen to rap music to relax, had sired 55 calves and outlived them all. his offspring were all killed and destroyed after the end of the experiment, in line with dutch health legislation. herman was also slated for the abattoir, but the dutch public -proud of making history with herman -rose up in protest, especially after a television program screened footage showing the amiable bull licking a kitten. herman won a bill of clemency from parliament. however, instead of retirement on a comfortable bed of straw, listening to rap music, herman was pressed into service again. he now stars at a permanent biotech exhibit in naturalis, a natural history museum in the dutch city of leiden. after his death, he will be stuffed and remain in the museum in perpetuity (a fate similar to what awaited an even more famous mammalian first born later in the decade). the applications for transgenic animal research fall broadly into two distinct areas, namely medical and agricultural applications. the recent focus on developing animals as bioreactors to produce valuable proteins in their milk can be catalogued under both areas. underlying each of these, of course, is a more fundamental application, that is the use of those techniques as tools to ascertain the molecular and physiological bases of gene expression and animal development. this understanding can then lead to the creation of techniques to modify development pathways. in 1992 a european decision with rather more far-reaching implications than hermans sex life was made. the first european patent on a transgenic animal was issued for a transgenic mouse sensitive to carcinogens -harvard's "oncomouse". the oncomouse patent application was refused in europe in 1989 due primarily to an established ban on animal patenting. the application was revised to make narrower claims, and the patent was granted in 1992. this has since been repeatedly challenged, primarily by groups objecting to the judgement that benefits to humans outweigh the suffering of the animal. currently, the patent applicant is awaiting protestors' responses to a series of possible modifications to the application. predictions are that agreement will not likely be forthcoming and that the legal wrangling will continue into the future. bringing animals into the field of controversy starting to swirl around gmos and preceding the latter's commercialization, was the approval by the fda of bovine somatotropin (bst) for increased milk production in dairy cows. the fda's center for veterinary medicine (cvm) regulates the manufacture and distribution of food additives and drugs that will be given to animals. biotechnology products are a growing proportion of the animal health products and feed components regulated by the cvm. the center requires that food products from treated animals must be shown to be safe for human consumption. applicants must show that the drug is effective and safe for the animal and that its manufacture will not affect the environment. they must also conduct geographically dispersed clinical trials under an investigational new animal drug application with the fda through which the agency controls the use of the unapproved compound in food animals. unlike within the eu, possible economic and social issues cannot be taken into consideration by the fda in the premarket drug approval process. under these considerations the safety and efficacy of rbst was determined. it was also determined that special labeling for milk derived from cows that had been treated with rbst is not required under fda food labeling laws because the use of rbst does not effect the quality or the composition of the milk. work with fish proceeded a pace throughout the decade. gene transfer techniques have been applied to a large number of aquatic organisms, both vertebrates and invertebrates. gene transfer experiments have targeted a wide variety of applications, including the study of gene structure and function, aquaculture production, and use in fisheries management programs. because fish have high fecundity, large eggs, and do not require reimplantation of embryos, transgenic fish prove attractive model systems in which to study gene expression. transgenic zebrafish have found utility in studies of embryogenesis, with expression of transgenes marking cell lineages or providing the basis for study of promoter or structural gene function. although not as widely used as zebrafish, transgenic medaka and goldfish have been used for studies of promoter function. this body of research indicates that transgenic fish provide useful models of gene expression, reliably modeling that in "higher" vertebrates. perhaps the largest number of gene transfer experiments address the goal of genetic improvement for aquaculture production purposes. the principal area of research has focused on growth performance, and initial transgenic growth hormone (gh) fish models have demonstrated accelerated and beneficial phenotypes. dna microinjection methods have propelled the many studies reported and have been most effective due to the relative ease of working with fish embryos. bob devlins' group in vancouver has demonstrated extraordinary growth rate in coho salmon which were transformed with a growth hormone from sockeye salmon. the transgenics achieve up to eleven times the size of their littermates within six months, reaching maturity in about half the time. interestingly this dramatic effect is only observed in feeding pins where the transgenics' ferocious appetites demands constant feeding. if the fish are left to their own devices and must forage for themselves, they appear to be out-competed by their smarter siblings. however most studies, such as those involving transgenic atlantic salmon and channel catfish, report growth rate enhancement on the order of 30-60%. in addition to the species mentioned, gh genes also have been transferred into striped bass, tilapia, rainbow trout, gilthead sea bream, common carp, bluntnose bream, loach, and other fishes. shellfish also are subject to gene transfer toward the goal of intensifying aquaculture production. growth of abalone expressing an introduced gh gene is being evaluated; accelerated growth would prove a boon for culture of the slowgrowing mollusk. a marker gene was introduced successfully into giant prawn, demonstrating feasibility of gene transfer in crustaceans, and opening the possibility of work involving genes affecting economically important traits. in the ornamental fish sector of aquaculture, ongoing work addresses the development of fish with unique coloring or patterning. a number of companies have been founded to pursue commercialization of transgenics for aquaculture. as most aquaculture species mature at 2-3 years of age, most transgenic lines are still in development and have yet to be tested for performance under culture conditions. extending earlier research that identified methylfarnesoate (mf) as a juvenile hormone in crustaceans and determined its role in reproduction, researchers at the university of connecticut have developed technology to synchronize shrimp egg production and to increase the number and quality of eggs produced. females injected with mf are stimulated to produce eggs ready for fertilization. the procedure produces 180 percent more eggs than the traditional crude method of removing the eyestalk gland. this will increase aquaculture efficiency. a number of experiments utilize gene transfer to develop genetic lines of potential utility in fisheries management. transfer of gh genes into northern pike, walleye, and largemouth bass are aimed at improving the growth rate of sport fishes. gene transfer has been posed as an option for reducing losses of rainbow trout to whirling disease, although suitable candidate genes have yet to be identified. richard winn of the university of georgia is developing transgenic killifish and medaka as biomonitors for environmental mutagens, which carry the bacteriophage phi x 174 as a target for mutation detection. development of transgenic lines for fisheries management applications generally is at an early stage, often at the founder or f1 generation. broad application of transgenic aquatic organisms in aquaculture and fisheries management will depend on showing that particular gmos can be used in the environment both effectively and safely. although our base of knowledge for assessing ecological and genetic safety of aquatic gmos currently is limited, some early studies supported by the usda biotechnology risk assessment program have yielded results. data from outdoor pond-based studies on transgenic catfish reported by rex dunham of auburn university show that transgenic and non-transgenic individuals interbreed freely, that survival and growth of transgenics in unfed ponds was equal to or less than that of non-transgenics, and that predator avoidance is not affected by expression of the transgene. however, unquestionably the seminal event for animal biotech in the nineties was ian wilmut's landmark work using nuclear transfer technology to generate the lambs morag and megan reported in 1996 (from an embryonic cell nuclei) and the truly ground-breaking work of creating dolly from an adult somatic cell nucleus, reported in february, 1997 (wilmut, 1997) . wilmut and his colleagues at the roslin institute demonstrated for the first time with the birth of dolly the sheep that the nucleus of an adult somatic cell can be transferred to an enucleated egg to create cloned offspring. it had been assumed for some time that only embryonic cells could be used as the cellular source for nuclear transfer. this assumption was shattered with the birth of dolly. this example of cloning an animal using the nucleus of an adult cell was significant because it demonstrated the ability of egg cell cytoplasm to "reprogram" an adult nucleus. when cells differentiate, that is, evolve from primitive embryonic cells to functionally defined adult cells, they lose the ability to express most genes and can only express those genes necessary for the cell's differentiated function. for example, skin cells only express genes necessary for skin function, and brain cells only express genes necessary for brain function. the procedure that produced dolly demonstrated that egg cytoplasm is capable of reprogramming an adult differentiated cell (which is only expressing genes related to the function of that cell type). this reprogramming enables the differentiated cell nucleus to once again express all the genes required for the full embryonic development of the adult animal. since dolly was cloned, similar techniques have been used to clone a veritable zoo of vertebrates including mice, cattle, rabbitts, mules, horses, fish, cats and dogs from donor cells obtained from adult animals. these spectacular examples of cloning normal animals from fully differentiated adult cells demonstrate the universality of nuclear reprogramming although the next decade called some of these assumptions into question. this technology supports the production of genetically identical and genetically modified animals. thus, the successful "cloning" of dolly has captured the imagination of researchers around the world. this technological breakthrough should play a significant role in the development of new procedures for genetic engineering in a number of mammalian species. it should be noted that nuclear cloning, with nuclei obtained from either mammalian stem cells or differentiated "adult" cells, is an especially important development for transgenic animal research. as the decade reached its end the clones began arriving rapidly with specific advances made by a japanese group who used cumulus cells rather than fibroblasts to clone calves. they found that the percentage of cultured, reconstructed eggs that developed into blastocysts was 49% for cumulus cells and 23% for oviductal cells. these rates are higher than the 12% previously reported for transfer of nuclei from bovine fetal fibroblasts. following on the heels of dolly, polly and molly became the first genetically engineered transgenic sheep produced through nuclear transfer technology. polly and molly were engineered to produce human factor ix (for hemophiliacs) by transfer of nuclei from transfected fetal fibroblasts. until then germline competent transgenics had only been produced in mammalian species, other than mice, using dna microinjection. researchers at the university of massachusetts and advanced cell technology (worcester, ma) teamed up to produce genetically identical calves utilizing a strategy similar to that used to produce transgenic sheep. in contrast to the sheep cloning experiment, the bovine experiment involved the transfer of nuclei from an actively dividing population of cells. previous results from the sheep experiments suggested that induction of quiescence by serum starvation was required to reprogram the donor nuclei for successful nuclear transfer. the current bovine experiments indicate that this step may not be necessary. typically about 500 embryos needed to be microinjected to obtain one transgenic cow, whereas nuclear transfer produced three transgenic calves from 276 reconstructed embryos. this efficiency is comparable to the previous sheep research where six transgenic lambs were produced from 425 reconstructed embryos. the ability to select for genetically modified cells in culture prior to nuclear transfer opens up the possibility of applying the powerful gene targeting techniques that have been developed for mice. one of the limitations of using primary cells, however, is their limited lifespan in culture. primary cell cultures such as the fetal fibroblasts can only undergo about 30 population doublings before they senesce. this limited lifespan would preclude the ability to perform multiple rounds of selection. to overcome this problem of cell senescence, these researchers showed that fibroblast lifespan could be prolonged by nuclear transfer. a fetus, which was developed by nuclear transfer from genetically modified cells, could in turn be used to establish a second generation of fetal fibroblasts. these fetal cells would then be capable of undergoing another 30 population doublings, which would provide sufficient time for selection of a second genetic modification. as noted, there is still some uncertainty over whether quiescent cells are required for successful nuclear transfer. induction into quiescence was originally thought to be necessary for successful nuclear reprogramming of the donor nucleus. however, cloned calves have been previously produced using non-quiescent fetal cells. furthermore, transfer of nuclei from sertoli and neuronal cells, which do not normally divide in adults, did not produce a liveborn mouse; whereas nuclei transferred from actively dividing cumulus cells did produce cloned mice. the fetuses used for establishing fetal cell lines in a tufts goat study were generated by mating nontransgenic females to a transgenic male containing a human antithrombin (at) iii transgene. this at transgene directs high level expression of human at into milk of lactating transgenic females. as expected, all three offspring derived from female fetal cells were females. one of these cloned goats was hormonally induced to lactate. this goat secreted 3.7-5.8 grams per liter of at in her milk. this level of at expression was comparable to that detected in the milk of transgenic goats from the same line obtained by natural breeding. the successful secretion of at in milk was a key result because it showed that a cloned animal could still synthesize and secrete a foreign protein at the expected level. it will be interesting to see if all three cloned goats secrete human at at the identical level. if so, then the goal of creating a herd identical transgenic animals, which secrete identical levels of an important pharmaceutical, would become a reality. no longer would variable production levels exist in subsequent generations due to genetically similar but not identical animals. this homogeneity would greatly aid in the production and processing of a uniform product. as nuclear transfer technology continues to be refined and applied to other species, it may eventually replace microinjection as the method of choice for generating transgenic livestock. nuclear transfer has a number of advantages: 1) nuclear transfer is more efficient than microinjection at producing a transgenic animal, 2) the fate of the integrated foreign dna can be examined prior to production of the transgenic animal, 3) the sex of the transgenic animal can be predetermined, and 4) the problem of mosaicism in first generation transgenic animals can be eliminated. dna microinjection has not been a very efficient mechanism to produce transgenic mammals. however, in november, 1998, a team of wisconsin researchers reported a nearly 100% efficient method for generating transgenic cattle. the established method of cattle transgenes involves injecting dna into the pronuclei of a fertilized egg or zygote. in contrast, the wisconsin team injected a replication-defective retroviral vector into the perivitelline space of an unfertilized oocyte. the perivitelline space is the region between the oocyte membrane and the protective coating surrounding the oocyte known as the zona pellucida. in addition to es (embryonic stem) cells other sources of donor nuclei for nuclear transfer might be used such as embryonic cell lines, primordial germ cells, or spermatogonia to produce offspring. the utility of es cells or related methodologies to provide efficient and targeted in vivo genetic manipulations offer the prospects of profoundly useful animal models for biomedical, biological and agricultural applications. the road to such success has been most challenging, but recent developments in this field are extremely encouraging. with the may 1999 announcement of geron buying out ian wilmuts company roslin biomed, they declared it the dawn of an new era in biomedical research. geron's technologies for deriving transplantable cells from human pluripotent stem cells (hpscs) and extending their replicative capacity with telomerase was combined with the roslin institute nuclear transfer technology, the technology that produced dolly the cloned sheep. the goal was to produce transplantable, tissue-matched cells that provide extended therapeutic benefits without triggering immune rejection. such cells could be used to treat numerous major chronic degenerative diseases and conditions such as heart disease, stroke, parkinson's disease, alzheimer's disease, spinal cord injury, diabetes, osteoarthritis, bone marrow failure and burns. the stem cell is a unique and essential cell type found in animals. many kinds of stem cells are found in the body, with some more differentiated, or committed, to a particular function than others. in other words, when stem cells divide, some of the progeny mature into cells of a specific type (heart, muscle, blood, or brain cells), while others remain stem cells, ready to repair some of the everyday wear and tear undergone by our bodies. these stem cells are capable of continually reproducing themselves and serve to renew tissue throughout an individual's life. for example, they continually regenerate the lining of the gut, revitalize skin, and produce a whole range of blood cells. although the term "stem cell" commonly is used to refer to the cells within the adult organism that renew tissue (e.g., hematopoietic stem cells, a type of cell found in the blood), the most fundamental and extraordinary of the stem cells are found in the early-stage embryo. these embryonic stem (es) cells, unlike the more differentiated adult stem cells or other cell types, retain the special ability to develop into nearly any cell type. embryonic germ (eg) cells, which originate from the primordial reproductive cells of the developing fetus, have properties similar to es cells. it is the potentially unique versatility of the es and eg cells derived, respectively, from the early-stage embryo and cadaveric fetal tissue that presents such unusual scientific and therapeutic promise. indeed, scientists have long recognized the possibility of using such cells to generate more specialized cells or tissue, which could allow the generation of new cells to be used to treat injuries or diseases, such as alzheimer's disease, parkinson's disease, heart disease, and kidney failure. likewise, scientists regard these cells as an important -perhaps essential -means for understanding the earliest stages of human development and as an important tool in the development of life-saving drugs and cell-replacement therapies to treat disorders caused by early cell death or impairment. geron corporation and its collaborators at the university of wisconsin -madison (dr. james a. thomson) and johns hopkins university (dr. john d. gearhart) announced in november 1998 the first successful derivation of hpscs from two sources: (i) human embryonic stem (hes) cells derived from in vitro fertilized blastocysts (thomson 1998 ) and (ii) human embryonic germ (heg) cells derived from fetal material obtained from medically terminated pregnancies (shamblott et al. 1998) . although derived from different sources by different laboratory processes, these two cell types share certain characteristics but are referred to collectively as human pluripotent stem cells (hpscs). because hes cells have been more thoroughly studied, the characteristics of hpscs most closely describe the known properties of hes cells. stem cells represent a tremendous scientific advancement in two ways: first, as a tool to study developmental and cell biology; and second, as the starting point for therapies to develop medications to treat some of the most deadly diseases. the derivation of stem cells is fundamental to scientific research in understanding basic cellular and embryonic development. observing the development of stem cells as they differentiate into a number of cell types will enable scientists to better understand cellular processes and ways to repair cells when they malfunction. it also holds great potential to yield revolutionary treatments by transplanting new tissue to treat heart disease, atherosclerosis, blood disorders, diabetes, parkinson's, alzheimer's, stroke, spinal cord injuries, rheumatoid arthritis, and many other diseases. by using stem cells, scientists may be able to grow human skin cells to treat wounds and burns. and, it will aid the understanding of fertility disorders. many patient and scientific organizations recognize the vast potential of stem cell research. another possible therapeutic technique is the generation of "customized" stem cells. a researcher or doctor might need to develop a special cell line that contains the dna of a person living with a disease. by using a technique called "somatic cell nuclear transfer" the researcher can transfer a nucleus from the patient into an enucleated human egg cell. this reformed cell can then be activated to form a blastocyst from which customized stem cell lines can be derived to treat the individual from whom the nucleus was extracted. by using the individual's own dna, the stem cell line would be fully compatible and not be rejected by the person when the stem cells are transferred back to that person for the treatment. preliminary research is occurring on other approaches to produce pluripotent human es cells without the need to use human oocytes. human oocytes may not be available in quantities that would meet the needs of millions of potential patients. however, no peer-reviewed papers have yet appeared from which to judge whether animal oocytes could be used to manufacture "customized" human es cells and whether they can be developed on a realistic timescale. additional approaches under consideration include early experimental studies on the use of cytoplasmic-like media that might allow a viable approach in laboratory cultures. on a much longer timeline, it may be possible to use sophisticated genetic modification techniques to eliminate the major histocompatibility complexes and other cell-surface antigens from foreign cells to prepare master stem cell lines with less likelihood of rejection. this could lead to the development of a bank of universal donor cells or multiple types of compatible donor cells of invaluable benefit to treat all patients. however, the human immune system is sensitive to many minor histocompatibility complexes and immunosuppressive therapy carries life-threatening complications. stem cells also show great potential to aid research and development of new drugs and biologics. now, stem cells can serve as a source for normal human differentiated cells to be used for drug screening and testing, drug toxicology studies and to identify new drug targets. the ability to evaluate drug toxicity in human cell lines grown from stem cells could significantly reduce the need to test a drug's safety in animal models. there are other sources of stem cells, including stem cells that are found in blood. recent reports note the possible isolation of stem cells for the brain from the lining of the spinal cord. other reports indicate that some stem cells that were thought to have differentiated into one type of cell can also become other types of cells, in particular brain stem cells with the potential to become blood cells. however, since these reports reflect very early cellular research about which little is known, we should continue to pursue basic research on all types of stem cells. some religious leaders will advocate that researchers should only use certain types of stem cells. however, because human embryonic stem cells hold the potential to differentiate into any type of cell in the human body, no avenue of research should be foreclosed. rather, we must find ways to facilitate the pursuit of all research using stem cells while addressing the ethical concerns that may be raised. another seminal and intimately related event at the end of the nineties occurred in madison wisconsin. up until november of 1998, isolating es cells in mammals other than mice proved elusive, but in a milestone paper in the november 5, 1998 issue of science, james a. thomson, (1998) a developmental biologist at uw-madison reported the first successful isolation, derivation and maintenance of a culture of human embryonic stem cells (hes cells). it is interesting to note that this leap was made from mouse to man. as thomson himself put it, these cells are different from all other human stem cells isolated to date and as the source of all cell types, they hold great promise for use in transplantation medicine, drug discovery and development, and the study of human developmental biology. the new century is rapidly exploiting this vision. when steve fodor was asked in 2003 "how do you really take the human genome sequence and transform it into knowledge?" he answered from affymetrix's perspective, it is a technology development task. he sees the colloquially named affychips being the equivalent of a cd-rom of the genome. they take information from the genome and write it down. the company has come a long way from the early days of venter's ests and less than robust algorithms as described earlier. one surprising fact unearthed by the newer more sophisticated generation of chips is that 30 to 35 percent of the non-repetitive dna is being expressed as accepted knowledge was that only 1.5 to 2 percent of the genome would be expressed. since much of that sequence has no protein-coding capacity it is most likely coding for regulatory functions. in a parallel to astrophysics this is often referred to in common parlance as the "dark matter of the genome" and like dark matter for many it is the most exciting and challenging aspect of uncovering the occult genome. it could be, and most probably is, involved in regulatory functions, networks, or development. and like physical dark matter it may change our whole concept of what exactly a gene is or is not! since beadle and tatum's circumspect view of the protein world no longer holds true it adds a layer of complexity to organizing chip design. depending on which sequences are present in a particular transcript, you can, theoretically, design a set of probes to uniquely distinguish that variant. at the dna level itself there is much potential for looking at variants either expressed or not at a very basic level as a diagnostic system, but ultimately the real paydirt is the information that can be gained from looking at the consequence of non-coding sequence variation on the transcriptome itself. and fine tuning when this matters and when it is irrelevant as a predicative model is the auspices of the affymetrix spin-off perlegen. perlegen came into being in late 2000 to accelerate the development of high-resolution, whole genome scanning. and they have stuck to that purity of purpose. to paraphrase dragnet's sergeant joe friday, they focus on the facts of dna just the dna. perlegen owes its true genesis to the desire of one of its cofounders to use dna chips to help understand the dynamics underlying genetic diseases. brad margus' two sons have the rare disease "ataxia telangiectasia" (a-t). a-t is a progressive, neurodegenerative childhood disease that affects the brain and other body systems. the first signs of the disease, which include delayed development of motor skills, poor balance, and slurred speech, usually occur during the first decade of life. telangiectasias (tiny, red "spider" veins), which appear in the corners of the eyes or on the surface of the ears and cheeks, are characteristic of the disease, but are not always present. many individuals with a-t have a weakened immune system, making them susceptible to recurrent respiratory infections. about 20% of those with a-t develop cancer, most frequently acute lymphocytic leukemia or lymphoma suggesting that the sentinel competence of the immune system is compromised. having a focus so close to home is a powerful driver for any scientist. his co-founder david cox is a polymath pediatrician whose training in the latter informs his application of the former in the development of patient-centered tools. from that perspective, perlegen's stated mission is to collaborate with partners to rescue or improve drugs and to uncover the genetic bases of diseases. they have created a whole genome association approach that enables them to genotype millions of unique snps in thousands of cases and controls in a timeframe of months rather than years. as mentioned previously, snp (single nucleotide polymorphism) markers are preferred over microsatellite markers for association studies because of their abundance along the human genome, the low mutation rate, and accessibilities to high-throughput genotyping. since most diseases, and indeed responses to drug interventions, are the products of multiple genetic and environmental factors it is a challenge to develop discriminating diagnostics and, even more so, targetedtherapeutics. because mutations involved in complex diseases act probabilisticallythat is, the clinical outcome depends on many factors in addition to variation in the sequence of a single gene -the effect of any specific mutation is smaller. thus, such effects can only be revealed by searching for variants that differ in frequency among large numbers of patients and controls drawn from the general population. analysis of these snp patterns provides a powerful tool to help achieve this goal. although most bi-alleic snps are rare, it has been estimated that just over 5 million common snps, each with a frequency of between 10 and 50%, account for the bulk of the dna sequence difference between humans. such snps are present in the human genome once every 600 base pairs or so. as is to be expected from linkage disequilibrium studies, alleles making up blocks of such snps in close physical proximity are often correlated, resulting in reduced genetic variability and defining a limited number of "snp haplotypes," each of which reflects descent from a single, ancient ancestral chromosome. in 2001 cox's group, using high level scanning with some old-fashioned somatic cell genetics, constructed the snp map of chromosome 21.the surprising findings were blocks of limited haplotype diversity in which more than 80% of a global human sample can typically be characterized by only three common haplotypes (interestingly enough the prevalence of each hapolytype in the examined population was in the ratio 50:25:12.5).from this the conclusion could be drawn that by comparing the frequency of genetic variants in unrelated cases and controls, genetic association studies could potentially identify specific haplotypes in the human genome that play important roles in disease, without need of knowledge of the history or source of the underlying sequence, which hypothesis they subsequently went on to prove. following cox et al. pioneering work on "blocking" chromosome 21 into characteristic haplotypes, tien chen came to visit him from university of southern california and following the visit his group developed discriminating algorithms which took advantage of the fact that the haplotype block structure can be decomposed into large blocks with high linkage disequilibrium and relatively limited haplotype diversity, separated by short regions of low disequilibrium. one of the practical implications of this observation is as suggested by cox that only a small fraction of all the snps they refer to as "tag" snps can be chosen for mapping genes responsible for complex human diseases, which can significantly reduce genotyping effort, without much loss of power. they developed algorithms to partition haplotypes into blocks with the minimum number of tag snps for an entire chromosome. in 2005 they reported that they had developed an optimized suite of programs to analyze these block linkage disequilibrium patterns and to select the corresponding tag snps that will pick the minimum number of tags for the given criteria. in addition the updated suite allows haplotype data and genotype data from unrelated individuals and from general pedigrees to be analyzed. using an approach similar to richard michelmore's bulk segregant analysis in plants of more than a decade previously, perlegen subsequently made use of these snp haplotype and statistical probability tools to estimate total genetic variability of a particular complex trait coded for by many genes, with any single gene accounting for no more than a few percent of the overall variability of the trait. cox's group have determined that fewer than 1000 total individuals provide adequate power to identify genes accounting for only a few percent of the overall genetic variability of a complex trait, even using the very stringent significance levels required when testing large numbers of dna variants. from this it is possible to identify the set of major genetic risk factors contributing to the variability of a complex disease and/or treatment response. so, while a single genetic risk factor is not a good predictor of treatment outcome, the sum of a large fraction of risk factors contributing to a treatment response or common disease can be used to optimize personalized treatments without requiring knowledge of the underlying mechanisms of the disease.they feel that a saturating level of coverage is required to produce repeatable prediction of response to medication or predisposition to disease and that taking shortcuts will for the most part lead to incomplete, clinically-irrelevant results. in 2005 hinds et al. in science describe even more dramatic progresss. they describe a publicly available, genome-wide data set of 1.58 million common singlenucleotide polymorphisms (snps) that have been accurately genotyped in each of 71 people from three population samples. a second public data set of more than 1 million snps typed in each of 270 people has been generated by the international haplotype map (hapmap) project. these two public data sets, combined with multiple new technologies for rapid and inexpensive snp genotyping, are paving the way for comprehensive association studies involving common human genetic variations. perlegen basically is taking to the next level fodor's stated reason for the creation of affymetrix, the belief that understanding the correlation between genetic variability and its role in health and disease would be the next step in the genomics revolution. the other interesting aspect of this level of coverage is, of course, the notion of discrete identifiable groups based on ethnicity, centers of origin and such breaks down and a spectrum of variation arises across all populations which makes the perlegen chip, at one level, a true unifier of humanity but at another adds a whole layer of complexity for hmos! at the turn of the century, this personalized chip approach to medicine received some validation at a simpler level in a closely related disease area to the one to which one fifth of a-t patients ultimately succumb when researchers at the whitehead institute used dna chips to distinguish different forms of leukemia based on patterns of gene expression in different populations of cells. moving cancer diagnosis away from visually based systems to such molecular based systems is a major goal of the national cancer institute. in the study, scientists used a dna chip to examine gene activity in bone marrow samples from patients with two different types of acute leukemia -acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all). then, using an algorithm, developed at the whitehead, they identified signature patterns that could distinguish the two types. when they cross-checked the diagnoses made by the chip against known differences in the two types of leukemia, they found that the chip method could automatically make the distinction between aml and all without previous knowledge of these classes. taking it to a level beyond where perlegen are initially aiming, eric lander, leader of the study said, mapping not only what is in the genome, but also what the things in the genome do, is the real secret to comprehending and ultimately curing cancer and other diseases. chips gained recognition on the world stage in 2003 when they played a key role in the search for the cause of severe acute respiratory syndrome (sars) and probably won a mcarthur genius award for their creator. ucsf assistant professor joseph derisi, already famous in the scientific community as the wunderkind originator of the online diy chip maker in pat brown's lab at stanford, built a gene microarray containing all known completely sequenced viruses (12,000 of them) and, using a robot arm that he also customized, in a three day period used it to classify a pathogen isolated from sars patients as a novel coronavirus. when a whole galaxy of dots lit up across the spectrum of known vertebrate cornoviruses derisis knew this was a new variant. interestingly the sequence had the hottest signal with avian infectious bronchitis virus. his work subsequently led epidemiologists to target the masked palm civet, a tree-dwelling animal with a weasel-like face and a catlike body as the probable primary host. the role that derisi's team at ucsf played in identifying a coronavirus as a suspected cause of sars came to the attention of the national media when cdc director dr. julie gerberding recognized joe in march 24, 2003 press conference and in 2004 when joe was honored with one of the coveted mcarthur genius awards. this and other tools arising from information gathered from the human genome sequence and complementary discoveries in cell and molecular biology, new tools such as gene-expression profiling, and proteomics analysis are converging to finally show that rapid robust diagnostics and "rational" drug design has a future in disease research. another virus that puts sars deaths in perspective benefitted from rational drug design at the turn of the century. influenza, or flu, is an acute respiratory infection caused by a variety of influenza viruses. each year, up to 40 million americans develop the flu, with an average of about 150,000 being hospitalized and 10,000 to 40,000 people dying from influenza and its complications. the use of current influenza treatments has been limited due to a lack of activity against all influenza strains, adverse side effects, and rapid development of viral resistance. influenza costs the united states an annual $14.6 billion in physician visits, lost productivity and lost wages. and least we still dismiss it as a nuisance we are well to remember that the "spanish" influenza pandemic killed over 20 million people in 1918 and 1919, making it the worst infectious pandemic in history beating out even the more notorious black death of the middle ages. this fear has been rekindled as the dreaded h5n1 (h for haemaglutenin and n for neuraminidase as described below) strain of bird flu has the potential to mutate and recognise homo sapiens as a desirable host. since rna viruses are notoriously faulty in their replication this accelerated evolutionary process gives then a distinct advantage when adapting to new environments and therefore finding more amenable hosts. although inactivated influenza vaccines are available, their efficacy is suboptimal partly because of their limited ability to elicit local iga and cytotoxic t cell responses. the choices of treatments and preventions for influenza hold much more promise in this millennium. clinical trials of cold-adapted live influenza vaccines now under way suggest that such vaccines are optimally attenuated, so that they will not cause influenza symptoms but will still induce protective immunity. aviron (mountain view, ca), biochem pharma (laval, quebec, canada), merck (whitehouse station, nj), chiron (emeryville, ca), and cortecs (london), all had influenza vaccines in the clinic at the turn of the century, with some of them given intra-nasally or orally. meanwhile, the team of gilead sciences (foster city, ca) and hoffmann-la roche (basel, switzerland) and also glaxowellcome (london) in 2000 put on the market neuraminidase inhibitors that block the replication of the influenza virus. gilead was one of the first biotechnology companies to come out with an anti-flu therapeutic. tamiflu™ (oseltamivir phosphate) was the first flu pill from this new class of drugs called neuraminidase inhibitors (ni) that are designed to be active against all common strains of the influenza virus. neuraminidase inhibitors block viral replication by targeting a site on one of the two main surface structures of the influenza virus, preventing the virus from infecting new cells. neuraminidase is found protruding from the surface of the two main types of influenza virus, type a and type b. it enables newly formed viral particles to travel from one cell to another in the body. tamiflu is designed to prevent all common strains of the influenza virus from replicating. the replication process is what contributes to the worsening of symptoms in a person infected with the influenza virus. by inactivating neuraminidase, viral replication is stopped, halting the influenza virus in its tracks. in marked contrast to the usual protracted process of clinical trials for new therapeutics, the road from conception to application for tamiflu was remarkably expeditious. in 1996, gilead and hoffmann-la roche entered into a collaborative agreement to develop and market therapies that treat and prevent viral influenza. in 1999, as gilead's worldwide development and marketing partner, roche led the final development of tamiflu, 26 months after the first patient was dosed in clinical trials in april 1999, roche and gilead announced the submission of a new drug application to the u.s. food and drug administration (fda) for the treatment of influenza. additionally, roche filed a marketing authorisation application (maa) in the european union under the centralized procedure in early may 1999. six months later in october 1999, gilead and roche announced that the fda approved tamiflu for the treatment of influenza a and b in adults. these accelerated efforts allowed tamiflu to reach the u.s. market in time for the 1999-2000 flu season. one of gilead's studies showed an increase in efficacy from 60% when the vaccine was used alone to 92% when the vaccine was used in conjunction with a neuraminidase inhibitor. outside of the u.s., tamiflu also has been approved for the treatment of influenza a and b in argentina, brazil, canada, mexico, peru and switzerland. regulatory review of the tamiflu maa by european authorities is ongoing. with the h5n1 birdflu strain's relentless march (or rather flight) across asia, in 2006 through eastern europe to a french farmyard, an unwelcome stowaway on a winged migration, and no vaccine in sight, tamiflu, although untested for this species, seen as the last line of defense is now being horded and its patented production right's fought over like an alchemist's formula. tamiflu's main competitor, zanamivir marketed as relenza™ was one of a group of molecules developed by glaxowellcome and academic collaborators using structure-based drug design methods targeted, like tamiflu, at a region of the neuraminidase surface glycoprotein of influenza viruses that is highly conserved from strain to strain. glaxo filed for marketing approval for relenza in europe and canada. the food and drug administration's accelerated drug-approval timetable began to show results by 2001, its evaluation of novartis's gleevec took just three months compared with the standard 10-12 months. another factor in improving biotherapeutic fortunes in the new century was the staggering profits of early successes. in 2003, $1.9 billion of the $3.3 billion in revenue collected by genentech in south san francisco came from oncology products, mostly the monoclonal antibody-based drugs rituxan, used to treat non-hodgkin's lymphoma, and herceptin for breast cancer. in fact two of the first cancer drugs to use the new tools for 'rational' design herceptin and gleevec, a small-molecule chemotherapeutic for some forms of leukemia are proving successful, and others such as avastin (an anti-vascular endothelial growth factor) for colon cancer and erbitux are already following in their footsteps. gleevec led the way in exploiting signal-transduction pathways to treat cancer as it blocks a mutant form of tyrosine kinase (termed the philadelphia translocation recognized in 1960's) that can help to trigger out-of-control cell division. about 25% of biotech companies raising venture capital during the third quarter of 2003 listed cancer as their primary focus, according to online newsletter venturereporter. by 2002 according to the pharmaceutical research and manufacturers of america, 402 medicines were in development for cancer up from 215 in 1996. another new avenue in cancer research is to combine drugs. wyeth's mylotarg, for instance, links an antibody to a chemotherapeutic, and homes in on cd33 receptors on acute myeloid leukemia cells. expertise in biochemistry, cell biology and immunology is required to develop such a drug. this trend has created some bright spots in cancer research and development, even though drug discovery in general has been adversely affected by mergers, a few high-profile failures and a shaky us economy in the early 2000's. as the millennium approached observers as diverse as microsoft's bill gates and president bill clinton predicted the 21st century wiould be the "biology century". by 1999 the many programs and initiatives underway at major research institutions and leading companies were already giving shape to this assertion. these initiatives have ushered in a new era of biological research anticipated to generate technological changes of the magnitude associated with the industrial revolution and the computerbased information revolution. complementary dna sequencing: expressed sequence tags and human genome project basic local alignment search tool high-tech herbal medicine: plant-based vaccines asilomar conference on recombinant dna molecules potential biohazards of recombinant dna molecules hugo: the human genome organization chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice the human genome: the nature of the enterprise orchestrating the human genome project separation and analysis of dna sequence reaction products by capillary gel electrophoresis nutritional genomics: manipulating plant micronutrients to improve human health helping europe compete in human genome research genome project gets rough ride in europe construction of a linkage map of the human genome, and its application to mapping genetic diseases separation of dna restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields preimplantation and the 'new' genetics a history human genome project it aint necessarily so: the dream of the human genome and other illusions high speed dna sequencing by capillary electrophoresis a strategy for sequencing the genome 5 years early expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain fv epitopes in tobacco plants generation and analysis of 280,000 human expressed sequence tags national academy of sciences. introduction of recombinant dna-engineered organisms into the environment: key issues functional foods and biopharmaceuticals: the next generation of the gm revolution in let them eat precaution biotechnology: a review of technological developments, publishers forfas vitamin-a and iron-enriched rices may hold key to combating blindness and malnutrition: a biotechnology advance french dna: trouble in purgatory genome: the autobiography of a species in 23 chapters harper collins derivation of pluripotent stem cells from cultured human primordial germ cells production of correctly processed human serum albumin in transgenic plants high-yield production of a human therapeutic protein in tobacco chloroplasts the common thread: a story of science, politics, ethics and the human genome capillary gel electrophoresis for dna sequencing. laser-induced fluorescence detection with the sheath flow cuvette production of functional human alpha 1-antitrypsin by plant cell culture genetic modification of oils for improved health benefits, presentation at conference, dietary fatty acids and cardiovascular health: dietary recommendations for fatty acids: is there ample evidence? stable accumulation of aspergillus niger phytase in transgenic tobacco leaves antenatal maternal serum screening for down's syndrome: results of a demonstration project viable offspring derived from fetal and adult mammalian cells key: cord-001427-qw1e5cof authors: cantas, leon; suer, kaya title: review: the important bacterial zoonoses in “one health” concept date: 2014-10-14 journal: front public health doi: 10.3389/fpubh.2014.00144 sha: doc_id: 1427 cord_uid: qw1e5cof an infectious disease that is transmitted from animals to humans, sometimes by a vector, is called zoonosis. the focus of this review article is on the most common emerging and re-emerging bacterial zoonotic diseases. the role of “one health” approach, public health education, and some measures that can be taken to prevent zoonotic bacterial infections are discussed. key points: a zoonotic bacterial disease is a disease that can be very commonly transmitted between animals and humans. global climate changes, overuse of antimicrobials in medicine, more intensified farm settings, and closer interactions with animals facilitate emergence or re-emergence of bacterial zoonotic infections. the global “one health” approach, which requires interdisciplinary collaborations and communications in all aspects of health care for humans, animals, and the environment, will support public health in general. new strategies for continuous dissemination of multidisciplinary research findings related to zoonotic bacterial diseases are hence needed. zoonotic diseases are those infections that can be transmitted between animals and humans with or without vectors. there are approximately 1500 pathogens, which are known to infect humans and 61% of these cause zoonotic diseases (1) . the unique dynamic interaction between the humans, animals, and pathogens, sharing the same environment should be considered within the "one health" approach, which dates back to ancient times of hippocrates (2, 3) . bacterial zoonotic diseases can be transferred from animals to humans in many ways (4): (i) the transfer may occur through animal bites and scratches (5) ; (ii) zoonotic bacteria originating from food animals can reach people through direct fecal oral route, contaminated animal food products, improper food handling, and inadequate cooking (6) (7) (8) ; (iii) farmers and animal health workers (i.e., veterinarians) are at increased risk of exposure to certain zoonotic pathogens and they may catch zoonotic bacteria; they could also become carriers of the zoonotic bacteria that can be spread to other humans in the community (9) ; (iv) vectors, frequently arthropods, such as mosquitoes, ticks, fleas, and lice can actively or passively transmit bacterial zoonotic diseases to humans. (10) ; (v) soil and water recourses, which are contaminated with manure contains a great variety of zoonotic bacteria, creating a great risk for zoonotic bugs and immense pool of resistance genes that are available for transfer of bacteria that cause human diseases (11, 12) . bacterial zoonotic infections are one of the zoonotic diseases, which can, in particular, re-emerge after they are considered to be eradicated or under control. the development of antimicrobial resistance due to over-/misuse of antibiotics is also a globally increasing public health problem. these diseases have a negative impact on travel, commerce, and economies worldwide. in most industrialized countries, antibiotic resistant zoonotic bacterial diseases are of particular importance for at-risk groups such as young, old, pregnant, and immune-compromised individuals (13) . almost 100 years ago, prior to application of hygiene rules and discovery of neither vaccines nor antibiotics, some bacterial zoonotic diseases such as bovine tuberculosis, bubonic plague, and glanders caused millions of human deaths. the spread and importance of some bacterial zoonoses are currently globally increasing. that is precisely why most of the developing countries are sparing more resources for a better screening of animal products and bacterial reservoirs or vectors for an optimal preventative public health service (14) . improvements in surveillance and diagnostics have caused increased recognition of emerging zoonotic diseases. herein, changes in our lifestyles and closer contacts with animals have escalated or caused the re-emergence of some bacterial infections. some studies lately have revealed that people have never been exposed to bacterial zoonotic infection risks as high as this before (15) . it is probably due to closer contact with adopted small animals, which are accepted and treated as a family member in houses. on the other hand, more intensified animal farms, which have a crucial role in the food supply, are still one of the greatest sources of food-borne bacterial zoonotic pathogens in today's growing world (4, 8) . people who have closer contact with large numbers of animals such as farmers, abattoir workers, zoo/pet-shop workers, and veterinarians are at a higher risk of contracting a zoonotic disease. members of the wider community are also at risk from those zoonoses that can be transmitted by family pets. the immune-suppressed people are especially at high risk for infection with zoonotic bacterial diseases. people can be either temporarily immuno-suppressed owing to pregnancy, infant age, or long-term immuno-suppressed as a result of cancer treatment or organ transplant, diabetes, alcoholism or an infectious disease (i.e., aids). this manuscript reviews the most common bacterial zoonoses and practical control measures against them. companion animals are increasingly treated as family members, and pets have many bacteria that may infect their owners. the human population of the european union (eu) was approximately 500 million 1 in 2012. the number of pet owning households was estimated at around 70 million in 2010 2 . the most commonly suffered zoonotic bacterial infections in humans are transmitted via animal bites and scratches. various dog breeds have been characterized for their role in killing dog bite attacks, such as pit bull breeds, malamutes, chows, rottweiler, huskies, german shepherds, and wolf hybrids (16) (17) (18) . in usa, pit bull breeds accounted for almost half of the dog bite-related zoonotic infections, three times more than german shepherds (17) . the oral cavity of healthy dogs and cats contains hundreds of different pathogenic bacteria including pasteurella sp. (19) . only 20% of dog bites get infected overall compared with 60% in cats. there are 10 times higher pasteurella multocida infection risks after a cat bite than a dog bite (20, 21) . p. multocida infected bite wounds appear usually within 8 h. it is estimated that approximately 20% of animal bites or scratches get infected in humans (5) . bacterial culturing from pet bite infections in humans is found to be smilar to the oral microbiota of the pets. infections in dog bite wounds are usually dominated by aerobic bugs: p. multocida (50%), alpha-hemolytic streptococcus (46%), staphylococcus (46%), neisseria (32%), and corynebacterium (12%). however, following anaerobic bacteria are also isolated from infected wounds: fusobacterium nucleatum (16%), prevotella heparinolytica (14%), propionibacterium acnes (14%), prevotella intermedia (8%), and peptostreptococcus anaerobius (8%) (22) . normal human skin bacteria or other environmental microorganisms are scarcely isolated from infected wounds in bitten person (22) (23) (24) . usually, infection occurs within 8-24 h after the animal attack, with variable pain on the site of the injury. the cellulitis might be followed by discharge that contains pus, which can sometimes be foul-smelling. immuno-suppressed patients with diabetes or liver dysfunction are frequently predisposed to develop serious infections after animal bites. in those cases, they may develop bacteremia faster and pass away in a shorter period of time (5) . a penetrating bite close to the joints and bones may cause septic arthritis and osteomyelitis. knowing the microbial composition of dental plaque biofilm formation in pets' mouth is a key factor in wound chronicity in humans (5, 25) . cat-scratch disease is a clinical syndrome that has been reported in people for over 100 years. yet, the etiological agent bartonella henselae, which was transmitted by cat scratches and bites, was only identified in 1992 (26) . however, contact with cat saliva on broken skin or sclera can also cause bartonellosis. a person who has had a cat scratch may show papules and pustules at the site of injury (the first initial sign). the disease may progress with a chronic non-healing wound, fever (sometimes), weak regional lymph circulation, and abscession. cat owners and veterinarians are most at risk (27) . systematic medical treatment is usually needed in people with suppressed immune systems. otherwise, encephalopathy, osteomyelitis, and granulomatous conjunctivitis might develop. horses and humans have always shared a close relationship due to recreation, sporting, and occupational reasons, for over thousands of years. in europe, the number of horses per capita remained relatively stable during the past decade. germany and great britain have the largest horse populations in the eu, whereas sweden has the highest number of horses per capita. the frequency of infected horse bite wounds is estimated to be 3-5% in europe (28, 29) . however, it has been roughly estimated that the horse bites account for as high as 20% of overall animal bites in turkey, which comes after dog bites (70%) (30) . more extensive muscle damage may develop in most of the horse attacks, which is different from small animal bites. a mixture of aerobic and anaerobic organisms has been isolated from horse bites in humans, which are frequently dominated by actinobacillus lignieresii (31, 32) . escherichia coli and bacteroides species have also been isolated from foul-smelling infections and pus drainage after horse bites in humans (33) . infectious diarrhea in companion animals is caused by salmonella sp., escherichia coli, shigella sp., and campylobacter sp. can also be transmitted to people through fecal oral route. it is difficult to estimate the distribution of these ubiquitous microorganisms. but it is known that they can be isolated from many healthy animals, which can be shed in their feces for long periods of time. campylobacteriosis were the most frequently reported zoonotic bacterial diseases in 2009 among the eu member countries in humans (34) . like many other enteropathogens, they can cause gastroenteritis (diarrhea, vomiting), headaches, and depression, sometimes even leading to death. it is obvious that raw food diets for pets dramatically increase the risk of human exposure to such zoonotic bacterial enteropathogens, which cause gastrointestinal diseases. although pet birds, also called songbirds (e.g., canaries, finches, sparrows) and psittaciformes (e.g., parrots, parakeets, budgerigars, love birds) are a small fraction of adopted pets, they are widely popular in europe and they are potential carriers of zoonotic diseases (35) . some of them could have an important impact on human health, such as chlamydophilosis (36) , campylobacteriosis (37) , and salmonellosis (38) . parrot fever (chlamydophilosis), which is caused by intracellular bacteria, chlamydia psittaci, lives within the frontiers in public health | infectious diseases respiratory system of birds. inhalation of dust, dander, and nasal secretions of infected birds is the main way of transmission to humans (39, 40) . the mild to severe flu-like illnesses may develop and infected people might be misdiagnosed as influenza. there is unfortunately a lack of quantitative research into the antimicrobial susceptibility of bacterial zoonotic organisms isolated from bite/scratch wounds or companion animal associated gastroenteritis. zambori et al. (5) revealed an increased prevalence of drug resistance in animal bite isolates from people. furthermore, methicillin-resistant staphylococcus aureus (mrsa) or extended-spectrum beta-lactamases (esbl) producing enterobacteriaceae, which are known as nosocomial infections have been frequently isolated in companion animals (41) , including horses (42) . it might be one of the main reasons for the rising prevalence of these potential zoonotic pathogens in human clinical samples. food producing animals in stock has reached a total of more than 200 million (cattle, pigs, sheep, goats, and chicken) living on farms in europe (see text footnote 1). it has been demonstrated that farm animals are reservoirs of many zoonotic pathogens to humans (34, 43) . however, annually, a large amount of drugs are being used worldwide to sufficient quantities of food to feed a rapidly growing world human population (44) (45) (46) (47) . the farm animals consume worldwide approximately eight million kilograms of antibiotics annually (70% of which is used for non-therapeutic purposes such as growth promotion; forbidden in the eu from january 2006, and disease prevention) compared with only approximately one million kilogram per year used in human medicine (7) . antibiotics are routinely fed to livestock as growth promoters to increase profits and to ward off potential bacterial infections in the stressed and crowded livestock factory environment (48) (49) (50) (51) (52) . despite large differences in methodology, most results demonstrate that not so long after the introduction of an antibiotic in veterinary practice, resistance in pathogenic zoonotic bacteria and/or the fecal flora increases. in particular, the wide-spread use of antibiotics in animals has resulted in an increased emergence of bacterial resistance to antibiotics, in zoonotic organisms such as salmonella, campylobacter, shigella, yersinia, listeria, and enterococcus genera, as well as the e. coli species. these zoonotic bacterial organisms are propagated primarily among animals and subsequently infect people (53) (54) (55) (56) . humans can be infected by contact with animals and their dung or droppings or consumption of infected food. severe diarrhea may develop, sometimes with blood in the feces. at all ages, but especially in children under 5 years and adults over 65 years, very serious illnesses often occur. these complications can result in loss of life or permanent kidney damage. according to the latest epidemiological trends, salmonellosis and campylobacteriosis are indicated as the most frequent food-borne bacterial zoonoses in europe. the main food sources were eggs and mixed foods (57) . furthermore, the recent emergence of esbl-producing and carbapenemase positive enterobacteriaceae bacteria in animal production (58) , the emergence of farm associated mrsa st398 (the main pig associated clone) (59) (60) (61) , and of plasmid-mediated quinolone resistance in animal isolates and food products (62, 63) are great threat for public health. unfortunately, these antimicrobial resistant "superbugs" are not only confined to hospital environments where antimicrobial use was high and many pathogens were prevalent. they are already widespread in the european community and animal populations that have a great hazard on public health (64, 65) . the causative agent of bovine tuberculosis, mycobacterium bovis (m. bovis) has been identified worldwide. thanks to decades of disease control measures that the occurrence of the infection has been greatly reduced. but there are still hundreds of new cases of human tuberculosis reported in the usa (66) . experience in europe and the usa, has shown that m. bovis can be controlled when it is restricted in livestock; however, eradication is almost impossible once it has spread into wildlife as follows; the european badger in the united kingdom (67), elk in canada (68) and white tailed deer in the usa (69) . in the last decade, q fever caused by coxiella burnetii was one of the most devastating farm animal originated bacterial zoonotic bacteria in europe. the netherlands, in particular, has experienced several outbreaks from 2007 to 2010 following identification of a q fever outbreak on various dairy farms in 2007. infected humans were mainly located within the intensive dairy goat farms (<5 km) (70) . the infection is spread by ticks, inhalation of the organism from the placental fluids, urine, and consumption of unpasteurized milk -meat products of sheep, goats, and cattle. the clinical signs in humans might be severe flu-like syndrome that may last for months (71) . in the eu, many vector-borne zoonotic diseases are considered as emerging infectious diseases, which either appear in a population for the first time or may have existed previously but spreading rapidly. the ecology of vector-borne zoonotic bacterial diseases is complex where climate and weather may influence the transmission cycles. milder winters, earlier start of spring or long intervals between winters cause extended seasonal tick activity and hence pathogen transmission between hosts in new regions of the world (72, 73) . many vector-borne infections occurred in new regions in the past two decades, while many endemic diseases have increased in incidence (74) . the following bacterial pathogens were most frequently identified as the causes of emerging vector-borne infections in the last decades in the eu: rickettsiae spp., anaplasma phagocytophilum, borrelia burgdorferi, bartonella spp., and francisella tularensis (75, 76) . rickettsia rickettsii causes rocky mountain spotted fever and spreads to humans by ticks. the signs of this disease are fever, headache, muscle pain, and spots with very dark rash. hiking in an area with many infested ticks is a great risk factor. a tick bite of <20 h is usually not enough to transfer these bacteria to a person (77) . ehrlichiosis (anaplasma phagocytophilum) and lyme disease (borrelia burgdorferi) have emerged as an important vector-borne zoonotic disease since 1980s (78, 79) . hard ticks are principal vectors, whereas small rodents are known as their natural vertebrate reservoir. a wide variety of signs including rash, joint pains, fever, enlarged lymph nodes, and some neurological signs may www.frontiersin.org develop. the trend of house buildings in woodlots where humans share the same ecology with reservoirs and vectors was found to be correlated with the increased frequency of such diseases in humans (79) . bartonella spp. is transferred to humans via fleas, lice, and sand flies (80) . however, recent studies have shown the importance of tick exposure in human bartonellosis (81) . as previously mentioned elsewhere in this article, bartonellosis are usually associated with cat-scratch diseases. lately, researchers have revealed that bartonella spp. can be transmitted via cat fleas without any scratches to humans (82) . symptoms include fever, enlarged lymph nodes (after 1-3 weeks), and a papule at the inoculation site. etiological agent of tularemia, f. tularensis, is a rare disease in europe (83) . bacteria are usually transferred by slaughtering (hunters are at a higher risk), eating of infected hares, respiration of dust, or drinking of contaminated water (84) . the prevalence of f. tularensis was found to be 1-5% from dog ticks in north america (85) . clinical symptoms depend on how the organism is acquired: erythematous papule at inoculation side within 48 h, pneumonia (the most serious form), endotoxemia, which gives continuous fever, acute pharyngotonsillitis, mucopurulent conjunctivitis (rarest form) (86) . among many others, brucellosis, which is not an emerging disease, has a significant impact on the endemic southern european countries with sporadic outbreaks. fortunately, the impact on humans has not increased since 2000 (87) . however, the cross border tracing of some brucella strains isolated in germany revealed concordance with sheep isolates originating from eastern anatolian, turkey. it is a characteristic example for the global spread of such diseases, in that case most probably by turkish immigrants living in germany (88) . plague, caused by yersinia pestis, is the most important reemergent bacterial wild rodent borne disease. the current case reports of plague are primarily limited to africa. however, it is a great potential public health hazard for europe due to increased traveler mobility or a potential bioterrorist attack (89) . bacterial zoonoses have a major impact on global public health. both emerging and re-emerging bacterial zoonoses have gained increasing national and international attention in recent years. the closer contact with companion animals and rapid socioeconomic changes in food production system has increased the number of animal-borne bacterial zoonoses. animal bite injuries in daily human-animal contact are not surprising, especially for the school-aged children. most of these wounds are medicated by patients as first aid and not registered in health systems. in more developed countries, most of the victims with moderate to severe bite injuries will seek professional medical treatment. regardless, all bites should be treated as serious, especially if the skin is broken. prompt diagnostic and treatment can prevent wound complications. the possibility to form biofilms by previously mentioned wound microorganisms is quite high, may cause severe tissue damage and protect the bacteria from innate-immune response and antimicrobials. the most of the commercial topical agents and wound dressings are ineffective against the biofilm matrix. surgical repair (for example, co 2 surgical laser techniques, leon cantas, personal research notes 2014), which is usually used to obtain a better cosmetic result might be needed to remove biofilm formed bite infections. this mechanical debridement is essential in the eradication of a wound biofilm. antimicrobials may be more effective in the treatment of the wound after debridement in the prevention of biofilm reformation. despite the use of currently optimal culturing methods, approximately 7% of infected wounds yield no bacterial growth. in such cases, some other fastidious pathogens, i.e., chlamydia spp., mycoplasma spp., and even viruses should be investigated. new advanced molecular diagnostic techniques are needed. prevention strategies for animal bites include close supervision of child-animal interactions, stronger animal control laws, better reporting of animal bites, and public education for better ownership of pets. regular nail trimming, routine oral examinations under annual health checks and comprehensive dental treatments of the companion animals (i.e., routine removal of the teeth tartar and plaques) by veterinarians will reduce the bacterial mass exposure to humans in case of direct contacts or animal bites. it is important to realize that enteropathogenic zoonoses may be contracted from both clinically sick and apparently healthy companion animals. feeding of pets with raw food diets is a potential source of salmonella, campylobacter, and other important bacterial zoonoses; however, some recalls of commercial pet food diets have also occurred as a result of contamination with those microorganisms. pig ear dog treats, in particular, have been implicated as an important source of salmonella infection for dogs, which can also serve as a source of infection to humans. nevertheless, it can be said that easy-to-use personal hygiene rules should be applied by companion animal owners. thorough hand washing with soap after handling of a companion animal and before eating or drinking, avoiding mouth-to-mouth contact, avoiding aerosolization of dusty fecal matter will help to prevent transmission of the zoonotic disease to humans. the animals with diarrhea should be isolated immediately and veterinary advice should be sought. the household should be cleaned with agents and kept as clean as possible. on the other hand, the prevalence of antimicrobial resistance in small animal pathogens is increasing globally due to overuse of broad spectrum antibiotics by veterinarians. there is an immediate need for worldwide smarter use of antimicrobials that have some positive effect on the recovery of animals from life threatening diseases. national veterinary antimicrobial treatment guidelines should be established by the local authorities according to the updated routine surveillance results. chronic diarrhea, dermatitis, ear and eye infections of pets caused by microbes demand longer durations of antimicrobial remedies at home. more frequent use of advanced laboratory tests, such as; feed/insect/mould allergy tests and differential diagnosis of the other relevant auto-immune disorders may help to investigate the main underlying cause of the such reactions which can be managed in various alternative treatment methods (i.e., hypoallergenic diets) rather than antibiotics solely. herein, pet specific auto-immune vaccines against allergens and auto-lactobacillales (auto-lac, leon cantas, personal research notes, 2011-2014) as dietary supplements can also be more frequently administered within the preventative veterinary practice measures. owners should be encouraged to insure their family animals to afford such costly veterinary services contradictory to the cheaper and sometimes life-long medical (i.e., antibiotic) treatment demanding options. veterinarians should also spear more time to educate the pet owners under consultations to handle infected-antimicrobial treated animals with precaution due to irreversible consequences of the antimicrobial resistance development and its spread in households. proper hand washing and use of gloves are strictly recommended while handling antimicrobial in veterinary clinics. veterinarians should prescribe broad spectrum and synthetic antimicrobials preferably after culturing with extreme precautions (i.e., dosage, dosing intervals and length of the treatment). reduced antibiotic use will hinder the development of antibiotic resistance in animal microbiota which might cause zoonotic infections in humans (50, 52) . food-borne zoonoses are an important public health concern worldwide and every year a large number of people affected by diseases due to contaminated animal originated food consumption. food hygiene education of the consumers is an important competent of food-borne diseases prevention. however, main prevention of food-borne zoonoses must begin at the farm level with in the concept of "one health." herein, control of the production stress especially in intensive livestock industry, with the development of better animal health management routines (i.e., routine vaccinations, immune stimulants: pre-, probiotic feed additives) and the increased animal welfare programs, will contribute eventually to an optimal production of animal health. increased antimicrobial resistance among emerging and re-emerging farm-borne bacterial pathogens in crowded settings (i.e., poultry, pig farms) is a growing problem. restrictive antimicrobial choice with better animal welfare managements are needed to control the spread of antibiotic resistance elements. in the eu, the use of avoparcin was banned in 1997 and the use of spiramycin, tylosin, and virginiamycin for growth promotion were banned in 1998. all other growth promoters used in feeding of food producing animals were banned from january 1, 2006 after a few national bans the years ahead 3 . in the u.s., politicians are still discussing to introduce a similar ban (s-742, 109th u.s. congress (preservation of antibiotics for medical treatment act). despite the ban on the use of all antibiotics as growth promoters in the eu and a ban on the use of quinolones as growth promoters in the poultry feed in the us medical, important antibiotics are still routinely fed to livestock prophylactically to increase profits and to ward-off potential bacterial infections in the stressed and crowded livestock and aquaculture environments in some parts of the world (50, 90, 91) . because stress lowers the immune system function in animals, antibiotics are seen as especially useful in intensive animal confinements (92) . the non-therapeutic use of antibiotics involves low-level exposure in feed over long periods -an ideal way to enrich resistant bacterial population (93, 94) . moreover, antibiotic resistance has been detected in different aquatic environments (95) . fish pathogenic bacteria often produce devastating infections in fish farms where dense populations of fish are intensively reared. bacterial infections in fish are regularly treated with antibiotics in medicated feed. so far, most of the fish pathogenic bacteria with a 3 http://europa.eu history in diseased fish farms have developed drug resistance (96) . modern fish farming relies increasingly on vaccination procedures and improved management to avoid infections (97) . for example, the norwegian aquaculture industry has produced over one million tons farmed fish 4 by using improved vaccines, management techniques, and only 649 kg of antimicrobials in 2011 (98) . vector-borne and zoonotic bacterial pathogens are a major source of emerging diseases, and since the time of hippocrates, weather and climate are linked to the incidence of such infectious diseases. complexity of epidemiology and adoptive capacity of microorganisms and the arthropods make the vector-borne disease almost impossible to eradicate. insect repellants, routine tick checks after outdoor activity in risk regions, prompt-proper tick removal, use of long sleeves and trousers (light-colored), and routine insecticide treatment of pets are recommended as general preventative measures (99) . herein, lyme disease, tick-borne illness, is vastly underestimated over past decades and clearly the urgent prevention is needed. besides individual awareness of such vector-borne diseases, better national surveillance and reporting programs will contribute to improved the disease control strategies. clinicians have an important role in the effective management of vector-borne zoonotic diseases, with enhanced differential diagnostic skills based on clinical symptoms and rapid molecular identification techniques (100) (101) (102) (103) . most of the time, the clinicians are on the first line of detection of these epidemics due to large group of patients with novel sets of similar symptoms. increased medical networking via online databases offer a broad overview to followers with regard to changes in temporal patterns of illness in real time, which helps faster detection of new epidemics (104) . identification and control of emergent zoonotic bacterial diseases require a "one health" approach, which demands combined efforts of physicians, veterinarians, epidemiologists, public health workers, and urban planners. collaborative international routine surveillance strategies, prompt -reliable agent identification techniques, and optimization of the treatment regiments will ensure the prevention and management of such infections. leon cantas defined the review theme, manuscript design, established the coordination and the collaborations, designed the manuscript, contributed to the data collection, data analysis, and drafting, writing and editing of the manuscript. kaya suer contributed to drafting and editing of the manuscript. all authors have seen, and approved the manuscript. risk factors for human disease emergence professor emeritus of veterinary epidemiology the components of 'one world -one health' approach animal associated opportunistic infections among persons infected with the human immunodeficiency virus biofilms in oral cavity of dogs and implication in zoonotic infections sensitive populations: who is at the greatest risk? monitoring and identifying antibiotic resistance mechanisms in bacteria emerging foodborne diseases: an evolving public health 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a potential conflict of interest. key: cord-016499-5iqpl23p authors: mackay, ian m.; arden, katherine e. title: rhinoviruses date: 2014-02-27 journal: viral infections of humans doi: 10.1007/978-1-4899-7448-8_29 sha: doc_id: 16499 cord_uid: 5iqpl23p picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide. hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under 5 years and occur in all seasons. aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing and with significant direct and indirect healthcare expenditure. ari incidence is highest in the first 2 years of life, with up to thirteen episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month. picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide [ 1 ] . hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under 5 years and occur in all seasons [ 2 , 3 ] . aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing [ 4 ] and with signifi cant direct and indirect healthcare expenditure [ 5 , 6 ] . ari incidence is highest in the fi rst 2 years of life, with up to 13 episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month [ 3 , 7 -9 ] . in preschool-aged children, nearly 50 % of general practitioner visits are for ari [ 10 ] , many of which are self-limiting. aris can often be managed in the community with supportive care from parents, but complications can arise that require a medical visit for management of asthma, otitis media, or sinusitis [ 11 ] . hrvs replicate in nasal cells, sinus cells, bronchial epithelial cells (becs) [ 12 , 13 ] , and smooth muscle cells [ 14 ] but not in monocytes [ 15 ] or dendritic cells (dcs) [ 16 ] . the infl ammatory immune response they trigger very soon after infection has its greatest impact in the young, the elderly, those with asthma or chronic obstructive pulmonary disease (copd), and in the immunocompromised. first infections usually elicit a stronger response. antiviral interventions have been under development for decades; to date most have met with varying degrees of failure or unacceptability. vaccines have been considered unachievable because of the large number of diverse and distinct viral types. there are 100 classically defi ned and recognized hrv serotypes grouped into two species, hrv-a and hrv-b, and a recently defi ned third species, hrv-c, containing more than 60 genotypes identifi ed and characterized entirely by molecular means. their cousins, the four enterovirus species (ev-a, ev-b, ev-c, and ev-d), are also found in the airways at times. most systematic and mechanistic studies of hrv etiology and pathogenesis have been informed by studies in adults, mostly prior to the discovery of hrv-cs. adults exhibit reduced symptoms from hrv infections because of prior exposure and the resultant protective immune memory which that imparts (see sect. 7.3 ). furthermore, many modern studies (1) draw conclusions about lower respiratory tract (lrt) disease using urt specimens and (2) infrequently sample, doing so across small cross sections of time. these limitations have hampered attempts to associate virus detection and disease. current thinking is that hrv-cs may be key players in asthma exacerbations although our inability to culture them routinely has hindered our progress in understanding their role. the impact of the hrvs has been underestimated for decades, and the concept of the hrvs as a very large assemblage of genetically, immunogenically, antigenically, and temporally distinct and stable viral entities remains rare; they are more commonly considered a single variable virus, a view that science does not support. the disease most commonly associated with the airways and resulting from hrv infection is the common cold, a selflimiting coryzal illness [ 17 -19 ] . the term dates back to ancient greece, but evidence that the syndrome and asthma, another disease most frequently due to hrv infection, has been with us since ancient times can be viewed in writings on the ebers papyrus, a medical document written in the sixteenth century bc [ 20 , 21 ] . in 1930 the common cold was considered either to be due to exposure to the elements or to infection by bacteria [ 22 ] . it was later understood to be largely due to something in bacteria-free fi ltrates, and so the search for viral causes began [ 23 , 24 ] . the common cold unit (ccu) was established in salisbury, uk, to seek solutions to the mysteries of the common cold, mostly through adult volunteer infection studies and careful systematic science [ 23 ] . the ccu functioned for 44 years (1946-1990) , and it was here in 1953 that the fi rst in vitro culture of an hrv was achieved using lung tissue from a particular embryo ( fig. 29 .1 ) [ 25 , 26 ] . propagation failed once this tissue was expended [ 22 , 33 ] . once hrv isolation was possible, viral serotyping developed and culture techniques were further refi ned. this leads to an international effort to characterize and name the hrvs [ 27 -30 ] . in 2006 renewed interest in hrv research was triggered by the description of a distinct clade of hrv types [ 31 ] found using molecular typing. the resultant fl urry of hrv research raised questions about many earlier paradigms of rhinovirology and of the role of established respiratory viruses in aris. the novel clade was proposed as a new species, hrv-c, which was taxonomically confi rmed in 2009 [ 32 -34 ] . prior to the discovery of the hrv-cs, the genus rhinovirus had been abolished and the hrv-a and hrv-b species assigned to the genus enterovirus within the family picornaviridae [ 35 ] . the hrv-cs have been assigned a new naming scheme based on genetic sequence in the absence of antigenic or serological data. while the sequencing of all serotyped hrv genomes was completed in 2009, few of the hrv-cs or apparently novel hrv-as or hrv-bs have been similarly characterized, so the full spectrum of hrv genomes, the rhinovirome, remains incomplete. in this chapter we have described individual serotyped hrvs as the "classical" types, a type being the description for a single, genetically stable, stand-alone hrv. methods for epidemiologic analysis the original clinical defi nition of an hrv infection was written using data from cell and tissue culture and adult human infection studies. after 1953 in vitro isolation methods employed a virus interference test to more easily determine successful isolation; cultures suspected of infection with an uncharacterized hrv prevented infection by another, readily titratable virus [ 36 ] . later, price (1956 ; the jh strain) and then pelon and co-workers (1957; 2,060 strain) developed culture systems that permitted hrv replication to be more easily identifi ed [ 37 , 38 ] . the early hrvs were initially classifi ed as echoviruses (echo 28; later hrv-1) [ 39 ] . at the same time, propagation of the hgp (hrv-2) strain resulted from using increased acidity, lowered cultivation temperatures, and constant motion (rotation) [ 40 , 41 ] . despite the challenges [ 42 ] , virus isolation was a more sensitive indicator of infection than an antibody rise in paired sera [ 43 ] . it was found that several cell lines and methods were required to encompass virus concentrations ranging from 10 1 to 10 5 tcid 50 /ml [ 44 -47 ] and growth differences among the different virus types. additionally, cell age after plating (<72 h), inoculum volume (relevant to the culture vessel), medium ph (6.8-7. 3), and cell density were important factors for the reproducible appearance of hrv-induced plaques and for higher virus yields [ 48 -51 ] . the hrvs can grow at temperatures above 35 °c (some prefer that under certain conditions) [ 52 ] , but rolling at 33 °c, preceded by a 2-4-h stationary incubation period [ 41 ] , has historically provided the highest yield and fastest in vitro hrv growth [ 36 , 50 , 53 , 54 ] . serodiagnosis grew increasingly impractical as the number of serotypes increased [ 49 , 55 ] . however, antibody-based methods were essential for type-specifi c neutralization of infection [ 56 ] from which early epidemiology data were derived and around which the hrv nomenclature system evolved in 1967 [ 28 ] . the fi rst classical strains were officially named in 1967 [ 57 ] , the last in 1987 [ 30 ] . today we know that cell culture-based methods are unreliable for accurately representing respiratory virus epidemiology; although enhanced by immunofl uorescence, they are still used [ 58 ] . the hrv-cs have not been successfully cultured in any cell lines or primary cell culture, although many attempts have been described [ 32 , 59 -62 ] . in 2011 hrv-c15 and w23 (another hrv-c) were shown to grow using organ culture [ 63 ] . sinus tissue hosted increasing levels of viral rna, as did adenoid, tonsil, and nasal polyp tissue, but much less effectively, as measured by in situ hybridization [ 63 ] . the sinus organ culture system also allowed testing of the fi rst reverse engineered hrv-c (pc15) [ 63 ] . isolation identifi ed hrvs in ~23 % of adults with aris, associated with 0.5 illnesses per year [ 64 ] . because culture is ineffi cient and subjective and requires expertise, even for the culturable hrv types, it is becoming an art lost to clinical laboratories the world over. it is unsurprising that pcr-based methods now prevail, providing a much improved understanding of the nature and scope of hrv infections. the virological and immunobiological cost of this improvement is a paucity of low passage "wild" hrv isolates to work with; thus, many research fi ndings from recent years have employed easy to grow highly passaged and adapted hrv isolates. the impact of virus adaptation on the reliability of data from use of such viruses is unknown. pcr-based assays have dramatically increased the frequency of hrv detection [ 65 -70 ] . the improved sensitivity and reduced turnaround time have shown that hrvs, as a group, are usually the predominant viruses in ari cases [ 71 -73 ] . with reliable detection levels that extend from as few as 10 2 tcid 50 /sample to well above clinically relevant loads, pcr can detect virus levels which are commonly shed during all stages of experimental infection studies [ 74 , 75 ] . the common understanding of the systemic [ 76 -78 ] or symptomatic [ 79 , 80 ] context of hrv detections was established during the era of culture detection, and pcr has challenged these paradigms by detecting virus more often than culture. hrvs are sometimes found in "healthy controls"; however, it is likely that with more thoughtful defi nitions of "healthy," these detections would reduce. it is not uncommon to experience a feeling that one is "coming down" with something that never develops further. this is likely due to a transient infection or reinfection by an hrv or other respiratory virus that is eliminated quickly by the host response. it is possible to correlate viral nucleic acid load at the sampling site with disease severity; however, this is made diffi cult by the highly variable sampling effi ciency of respiratory tract specimens which only permit the generation of reliable quantitative pcr (qpcr) data if serial specimens are available [ 81 ] . the 5′ untranslated region (utr; figs. 29.2 and 29.3 ) is the most common target for diagnostic oligonucleotides since the fi rst hrv rt-pcr in 1988 [ 82 ] , and the region has retained relevance for virus detection by its adaptation to reverse transcriptase real-time methods (rt-rtpcr) [ 53 , 65 , 66 , 69 , 74 -76 , 79 , 83 -103 ] . the 5′utr is comprised of a number of conserved sequence "islands" (fig. 29 .2 ) that permit the robust detection of the majority of hrvs and those "respiratory evs" which can be regularly detected in the respiratory tract [ 104 , 105 ] . the detection of respiratory evs in no way detracts from the importance of supporting clinical decision making using these assays. however, repositioning [ 68 ] . the pcr primers of broadly reactive conventional rt-pcr [ 82 , 113 ] and rt-rtpcr [ 75 , 114 ] assays are shown these primers or changing the method of employing them [ 106 -109 ] may undermine assay performance, as evidenced by predicted hybridization mismatches, uncommonly low detection frequencies [ 110 ] , and by comparison of multiple primer sets using the same specimens [ 111 ] . the addition of an oligoprobe rtpcr method increases amplicon detection sensitivity and specifi city, identifying 100-fold fewer tcid 50 /ml or 10 fold fewer genome copies than agarose gel detection of amplicon [ 75 , 79 , 112 ] . other molecular tools, capable of detecting multiple targets, have evolved in recent years [ 58 , 70 , 115 -121 ] , and some have gone on to be approved for clinical laboratory use [ 122 ] . microarrays can detect thousands of viral targets, but are expensive for routine use (usd30-300 per sample) and not sensitive enough to avoid a pre-hybridization pcr amplifi cation when using clinical specimens. at their most robust, microarrays, like pcr, rely on the existence of conserved regions of sequence to detect unknown viruses allowing them to detect previously unknown hrv types [ 123 ] . highthroughput or "deep" sequencing platforms have become less expensive and more readily available, and they have succeeded in fi nding new diversity within the hrv species [ 124 ] . the experiments remain costly so have not yet found a place for regular screening tasks and remain coupled to a need for pre-pcr steps. rapid protein-or virion-based assays are not (yet) adequately sensitive [ 125 , 126 ] . because of the high number of hrvs and the high frequency of infections, genotyping methods have become an essential accompaniment for understanding hrv epidemiology. nucleotide sequencing of the vp1, 5′utr+vp4+vp2 (called hereafter vp4/vp2), or 5′utr region has replaced traditional serological methods, because of its speed and need for fewer specialized reagents compared to serotyping. vp1 yields the most comprehensive subgenomic genotyping information and is essential for the minimal defi nition of a new hrv type [ 127 ] . the vp4/vp2 region (fig. 29. 3 ) is considered easier to use because it encompasses suffi cient genetic diversity to confi rm the identity of a clinical hrv type while also providing broad enough sensitivity to amplify the ~160 hrvs from a challenging biological substrate, clinical specimens [ 128 ] . screening of airway specimens for hrvs is not routine [ 111 ] due to factors including cost and the perceived low clinical relevance of detection. genotyping is mostly relegated to research facilities. because of this, hrv molecular epidemiology studies tend to be smaller and focused on a specifi c disease or research question. most in-depth molecular studies of hrv replication have focused on a single hrv type. generally, it is presumed that results can be extrapolated to the other hrv types and to the in vivo situation. hrvs replicate in the cytoplasm (fig. 29 .4 ) [ 129 ] with membrane-associated replication structures containing double-stranded rna (dsrna) replicative intermediates (ri) which are formed in cells 4 h after infection [ 52 , 130 ] . single-stranded infectious rna forms after ris start to accumulate [ 130 ] . genomic rna (plus strand) is the template for complementary minus strand synthesis which in turn is the template for new genomic plus strands that become incorporated into virions [ 131 ] . virions are synthesized from 4 to 7 h after infection and reach maximum release levels at 10-18 h [ 131 ] . hrv replication in epithelial cells may shut off host cell transcriptional activity via direct cleavage of transcription factors and nuclear pore complex components. protease 2a (2a pro ) of hrv-b2 may directly cleave eukaryotic initiation factor 4g (eif4g) when bound to eif4e [ 132 , 133 ] . the eifs have key roles in initiation and rate control of host cell translation [ 132 ] . host cellular protein production is virtually replaced by hrv-b14 proteins after only 6 h of infection [ 134 ] . hrv-b14-infected cells also display reduced nuclear importing and degraded nuclear pore complex (npc) components [ 135 ] . this may represent another hrv strategy for limiting the host response by preventing or reducing key signaling pathway molecules (e.g., irf-3, stat1, nf-κb) and shutting down host cell protein synthesis. protease 3c (3c pro ) from hrv-a16 targets the nucleus and can disrupt active and passive nucleocytoplasmic transport [ 129 , 136 ] . recombinant 2a pro protein from hrv-a16,hrv-a89, hrv-b4, hrv-b14, hrv-c2, and hrv-c6 exhibited differing specifi cities and kinetics against eif4g as well as npc components demonstrating functional diversity between hrv types [ 137 ] . this fi nding underscores the functional diversity within the hrv species and the risk of extrapolating too greatly from the study of single hrv types. it is apparent from a wealth of immunobiological data that hrvs still effi ciently trigger a proinfl ammatory immune response that has considerable clinical impact among at-risk groups, and that their putative interruption of host cell machinery does little to hinder this. the virion encapsulates an approximately 7 kb positive sense rna genome (fig. 29. 3 ), which tends to be more adenine and uracil (a+u) rich than the ev genome [ 139 ] . in particular, a+u more frequently occupies the third or "wobble" genome replication in association with membranes produces the viral polyprotein which is co-and posttranslationally processed by 2a pro and 3c pro into the proteins ( p1 -p3 ) and structural peptides ( vp1 -vp4 ; vp2 and vp4 derive from the vp0 precursor protein) that assemble into protomers, pentamers, and fi nally capsids. nonstructural proteins are also released in these cleavages as well as through autoproteolytic cleavage. mature hrv virions packaged with an ssrna genome escape by cell lysis (adapted with permission from arden et al. [ 138 ] ) codon position. the single rna "gene" acts as messenger rna to encode the single multi-domain, proteolytically processed "polyprotein." the coding region is bracketed by utrs which perform regulatory functions necessary for genome duplication [ 140 ] . these are very similar genomic, transcriptional, and translational features to those of their close cousins, the evs. most of the information currently required for virus identifi cation by the international committee on taxonomy of viruses (ictv) can be found through analysis of the genetic features of hrvs ( fig. 29.3 ). there are 158 complete hrv polyproteins on the genbank database ( fig. 29 .5 ). the fi rst complete hrv genome sequence (hrv-b14) was described in 1984 [ 141 ] followed by hrv-a2 in 1985 [ 142 ] and hrv-a1b in 1988 [ 143 ] (fig. 29 [ 145 ] . sequencing of the vp4/vp2 region was completed for all classical strains in 2002 [ 146 ] , and the complete set of 1d regions were available in 2004 [ 147 ] . currently there are at least 50 named hrv-c vp1 regions the current spectrum of 168 complete hrv complete polyprotein amino acid sequences available on the genbank database. the alignment was conducted using mafft within geneious pro v5.6 [ 148 ] . the phylogenetic and molecular evolutionary analyses were con-ducted using mega version 5 (poisson model, 500 bootstraps with consensus support shown at the nodes where space permitted [ 149 ] ) (reprinted with permission from miller and mackay [ 150 ]) available and 20 complete hrv-c genomes. many more genomes are appearing as part of the rhinovirus consortium's efforts to complete and study the rhinovirome using highthroughput sequencing technologies to genetically characterize hrvs from their combined clinical specimen stores ( http://www.international-rhinovirus-consortium.org/ ). many 5′utr and vp4/vp2 sequences reside on the genbank database, most of which are labeled using in-house laboratory schemes rather than an approved nomenclature. analysis of the full-length genomes supports the use of 5′utr, vp1, and vp4/vp2 subgenomic regions for useful representation of hrv species and types [ 144 , 147 ] . recombination, the process of genetic exchange which results in a chimeric genome [ 151 ] , can only be detected in mature viruses after the fact, and it must therefore be inferred indirectly through genomic analysis and comparison. predictions of infrequent recombination among the hrvs [ 83 ] have been made based on examination of the available set of hrv coding and noncoding regions [ 152 ] . intensive analyses reported that recombination is not a driving force for the evolution of hrv types [ 144 , 153 , 154 ] . some discrepancies are likely because of the different number of sequences used, the different origins of the viruses used for sequencing, and the analysis methods employed. hrv-c evolution seems to have been more affected by prior recombination, than is apparent for members of hrv-a or hrv-b. this is similar to the ev species but with far fewer predicted recombination events than for ev evolution [ 114 , 151 , 155 , 156 ] . most of the recombination proposed to have affected the hrvs occurred between hrv-c and hrv-a and is often found within the 5′utr or at the 5′utr/vp4 junction [ 83 , 157 , 158 ] but rarely in coding sequence (2a [ 158 ] or 3c [ 83 ] ). the high sequence diversity among the individual hrv polyprotein coding sequences may keep recombination events to a minimum in order to retain viral fi tness [ 158 ] . the ability of hrvs to recombine in practice awaits empirical evidence; the extent of recombination among all hrv or ev types and the frequency with which viable recombinants arise are entirely unquantifi ed. the 28-30 nm hrv virion has been visualized for only a handful of hrv-a and hrv-b types (including hrv-a1a, hrv-a2, hrv-b3, hrv-b14, and hrv-a16), but no hrv-c structures have been empirically determined to date. the fi rst, hrv-b14, was described in 1985 [ 159 ] followed by hrv-a1a in 1989 [ 160 ] , hrv-a16 in 1993 [ 161 ] , hrv-a3 in 1996 [ 162 ] , and hrv-a2 in 2000 [ 163 ] . hrv-c structure has only been predicted using computer modeling, but their basic structure seems to be that expected of an hrv ( fig. 29.6 ) [ 33 ] . the hrv capsid shell is composed of 60 protomers, each comprising one copy of the viral proteins vp4, vp3, vp2, and vp1. vp1, vp2, and vp3 (each ~30 kda) are to some extent exposed on the capsid surface, whereas vp4 (~7 kda) is internalized and associated with viral rna. five protomers come together at a point around a fi vefold axis, and this cluster is called the pentamer. the fi vefold axis is circumscribed by a cleft referred to as the "canyon." vp1, vp2, and vp3 are each formed by a convoluted set of protein sheets and loops [ 159 ] . the loops protrude beyond the external capsid surface and contain discontinuous antigenic sites. of the hrv types studied, four neutralizing antibody immunogenic (nim) regions have been identifi ed on hrv-b14 and hrv-a16: nim-1a (located in vp1), nim-1b (vp1), nim-ii (vp2 and vp1), and nim-iii (vp3 and vp1) [ 159 ] . antigenic sites identifi ed on hrv-a2 are called a, b, and c [ 164 ] . the scope and location of antigenic and immunogenic moieties among the hrv-cs is unknown. using known receptor binding sequence as a guide for computer modeling ( fig. 29 .6 ), it has been predicted that when discovered, the receptor for the hrv-cs will differ from the major and minor receptors defi ned for the hrv-as and hrv-bs [ 33 ] . the three hrv species within the genus enterovirus are a genetically, immunogenically, and antigenically diverse assemblage of >160 viral types (table 29 .1 ). this accounts for the combination of hrv-a1a and -a1b, exclusion of hrv-87, which is actually ev-d68 despite confusion over acid liability [ 169 -171 ] and combination of hrv-hanks which is actually hrv-a21 [ 147 ] . serological studies indicate that some hrv-a and hrv-b types may not be distinct enough to deserve a unique identity [ 147 ] . species within the genus share >70 % amino acid (aa) identity in the polyprotein and in 2c+3cd and >60 % aa identity in p1 ( fig. 29 .3 ) as well as their host cell receptors, a limited natural host range, a genome base composition (g+c) that varies by no more than 2.5 %, and a similar compatibility of proteolytic processing, replication, encapsidation, and genetic recombination [ 172 ] . a variant of the same hrv type shares 87-88 % aa identity or more in vp1 [ 129 ] . much of the nongenetic criteria remain undefi ned for the hrv-cs. in 2008 the genera enterovirus and rhinovirus were offi cially combined, retaining the former genus name enterovirus with the human enterovirus c as the prototype species. a genus in the order picornavirales , family picornaviridae , is at least 58 % different in its amino acid identity from any other genus. in 2009 a proposal establishing the species human rhinovirus c was ratifi ed by the ictv. formal hrv-c numbering commenced in 2010, and type numbers were initially assigned based on the date of submission of relevant sequences to genbank (hrv-c1, formerly nat001; hrv-c2, f. nat045; hrv-c3, f. qpm; hrv-c4, f. c024, etc.; table 29 .1 ) [ 127 ] . a clinical detection of an hrv-c can be considered a novel type principally based on its vp1 sequence or provisionally ("c_pat," table 29 .1 ) based on vp4/vp2 [ 146 ] and could be confi rmed as a variant of a previously characterized hrv-c by identity thresholds to either region. the 5′utr can be and still is used [ 173 , 174 ] for hrv genotyping, but it is a more problematic region than vp1 or vp4/vp2 because of the recombination activity that affects this region, especially among the hrv-cs [ 175 ] . this is presented as phylogenetic intermingling of some hrv-a and hrv-c types [ 114 ] . nonetheless, careful application of sequence identity thresholds when comparing clinical sequences to the genbank database (≥96 % identity required before assigning a clinical detection to a particular type) succeeds in characterizing hrv species and types [ 9 ] . there are currently 50 types within hrv-c (which includes the types once grouped together under hrv-"a2," hrv-x, and hrv-ny clades), 78 hrv-a types, and 25 hrv-bs. the most up-to-date information on current taxonomic trends can be found at the ictv picornaviridae study group website ( http://www.picornastudygroup.com/ ). ( c ) hrv-c3 versus hrv-a2 simplot data projected onto the hrv-c3 pentamer. the domains of interest are mostly shown within a single asymmetric unit. ( d ) a minor group pentamer (hrv-a2, gray ) including antigenic sites (sites a -c , green ) and very-low-density-lipoprotein receptor (vldlr) footprint ( red ) [ 167 ] . attachment of the vldl-r involves adjacent vp1 molecules. magnifi ed vp1 area represents one half of a vldl-r footprint [ 168 ] . amino acid substitutions ( arrowed ) contributed to the differences between minor group sites b and c (adapted with permission from mcerlean et al. [ 33 ] ) historically a key feature distinguishing the hrvs from the evs was the instability of the hrv capsid in the presence of acid and their lower preferred laboratory propagation temperature (33-34 °c versus 37 °c for evs). over time hrvs have been subclassifi ed in different ways. the fi rst was based on tissue tropism and host range. hrvs that preferred growth using monkey cells were called "m" strains and those (the majority) that grew only in human cell cultures, "h" strains [ 56 , 176 -180 ] . these two groups correlate with receptor usage [ 131 ] (table 29 .1 ) and possibly with the titer of the inoculum employed [ 181 ] . in 1962 it was proposed to abandon this terminology in favor of a sequential numbering system [ 177 ] . picornaviruses recognize a variety of cellular receptors [ 169 , 182 , 183 ] . hrv types are also subdivided into major and minor groups defi ned by use of one of the two main receptor molecules [ 184 , 185 ] . the capsid of the majority of classical hrvs ( n = 89) [ 184 ] interacts with the amino-terminal domain of the 90 kda intercellular adhesion molecule (icam-1; cd54) [ 186 -189 ] . receptor binding destabilizes the hrv capsid, probably by dislodging the "pocket factor," and initiates uncoating [ 164 , 182 , 190 ] . icam-1 interacts with its receptor, leukocyte function antigen-1 (lfa-1), and plays a role in recruitment and migration of immune effector cells [ 191 ] . the minor group [ 184 ] of classical viruses employ members of the low-density lipoprotein receptor (ldlr) family to attach to cells [ 167 ] . binding of vldl-r occurs outside of the canyon employing a different destabilizing and uncoating mechanism. heparan sulfate may act as a receptor under specifi c conditions [ 183 , 192 , 193 ] . in 1990 andries et al. defi ned, and laine et al. refi ned, two "antiviral groups" (a and b) based on their susceptibility to a panel of antiviral molecules [ 165 , 194 ] . these groupings refl ected the nature of the amino acid (and hence nucleotide) sequence of the region interacting with the antiviral molecules. these antiviral groups can also be visualized using phylogeny [ 194 ] . when sequences from other subgenomic regions, including p1, 2c, and 3cd, were examined by phylogeny, the species were found, in most cases, to inversely correlate with antiviral grouping labels (table 29 .1 ). m and h indicate early cell tropism-based classifi cation (monkey, human) abandoned in favor of a sequential numbering system [ 177 ] . hrv types were later divided into the major and minor groups defi ned by receptor tropism [ 184 , 185 ] . receptor-designated minor group hrv types are underlined, and major group types are shown in bold. antiviral groups (a and b) are labeled [ 165 , 194 ] . hrv-a8 and hrv-a95 are also likely the same serotype [ 147 ] . a full list of genetically close serotype pairings was presented by ledford et al. [ 147 ] hrv-c nomenclature was defi ned in 2010 and currently includes a number of p rovisionally a ssigned t ypes (pat) which are confi rmed once preliminary vp4/vp2 data can be confi rmed with vp1 sequence and the provisional number removed (e.g., c_pat1 to c_pat13 have already been reassigned) today, sequencing and phylogeny play a central role in species classifi cation within the genus, and together, they are surrogates for the important biological classifi cation criteria [ 146 , 147 , 165 , 195 -197 ] . for the hrv-cs, fi rst described as the "hrv-a2" clade (not to be confused with the single virus, hrv-a2, this naming scheme appeared after the hrv-c clade's name was proposed) of viruses in 2006 [ 31 ] , sequencing of 5′utr and vp4/vp2 has provided the bulk of hrv information from clinical studies. while culture in primary sinus tissue has been reported [ 63 ] , no receptor is yet defi ned. hrvs are the most numerous and frequently detected of all the "respiratory viruses," so-called because of their predominant detection in and tropism for the human urt or lrt ( fig. 29 .7 ). the circulation of hrvs varies with population age, underlying disease, immunocompromise, over time, and across distance. circulation is infl uenced by the nature, strength, distinctiveness, and memory of the immune response hrvs trigger and by the nature and prevalence of other concurrently circulating respiratory, and perhaps nonrespiratory, viruses. with the recent discovery of the unculturable hrv-cs came the realization that previous hrv epidemiology was only reliable if conducted by one or more suitably broad-spectrum hrv pcr assays [ 111 ] ; hence, prior to 1988, detection of the full spectrum of ≥160 hrvs did not occur. after 1988, the ability to detect all types very much depended on the nature of the pcr primers and detection methods used. the great number of distinct hrv types has burdened the search for answers to epidemiology-related questions. however, as for other important respiratory viruses including human respiratory syncytial virus (hrsv) and the infl uenza viruses (ifvs), the virus types within a species show evidence of being both distinct and discrete viruses that are independently recognized by their host and consequently independently infect their hosts. each hrv type is also genetically stable [ 144 ] . the hrv species circulate variably from year to year with evidence of epidemics of distinct types. a prospective longitudinal cohort study over 6 months examined hrv frequency and diversity in 272 specimens from 18 healthy children (0-7 years of age) [ 198 ] . a median of three hrvs and a maximum of six were detected per child. a similar outcome resulted from an australian cohort study [ 9 ] . genotyping reveals more of the hrv diversity at a single site than culture ever could with molecular studies fi nding between 34 and 70 distinct hrvs at a single location [ 9 , 128 , 199 ] . the number of additional hrv cases that occur in children outside of specifi cally defi ned symptomatic periods remain to be defi ned, with current studies indicating that a much higher number of hrv infections may occur. more comprehensive investigation of hrv type and illness will be undertaken during analysis of data from the australianbased observational research in childhood infectious diseases (orchid) study ( http://clinicaltrials.gov/show/ nct01304914 ). interestingly, the hrv-bs are often underrepresented, even when accounting for the smaller number of known hrv-b types [ 128 ] . a number of studies have not found any robust patterns between the circulating hrv types or species and clinical outcome, but the majority of studies seeking this information are short and sample infrequently, limiting their ability to fi nd the patterns they seek [ 128 ] . studies into the relative sensitivities of nasopharyngeal aspirates (npa) and swab sampling methods produce differing results, but generally, if seeking the best diagnostic yield for as many respiratory viruses as possible (i.e., seeking a laboratory diagnosis to support clinical decision making), npas are the sample of optimal choice. one study reported similar clinical sensitivities between swabs and npas for human coronaviruses (hcovs), ifvs, and hrsv, but reduced sensitivities using swabs for hrvs, human adenoviruses (hadvs), human metapneumovirus (hmpv), or parainfl uenza viruses (hpivs) [ 201 ] . a second study reported no difference in sensitivities for hrvs, hadvs, and hpivs but a reduced sensitivity for hrsv and ifvs when using swabs [ 202 ] . nasopharyngeal washes also yield more viral culture success than either nasal or pharyngeal swabs. nonetheless, many studies use nasal swabs as the sample of choice because they allow self-collection and involve much less discomfort than npas, and pcr has meant that infectious virus is not required, only viral nucleic acid which relaxes some limitations imposed by the need for rapid, careful, temperaturecontrolled, and expensive transport requirements [ 64 , 203 , 204 ] . bronchoalveolar lavage samples are best for seeking lrt etiologies, especially in adults where nasal wash viral loads can be low compared to those in children, but this is an invasive method with some risk attached [ 205 ] . hrvs infect all people, all around the globe. spread of hrvs is most obvious and frequent from child to child and from child to parent [ 206 ] . in populations of mixed age, the majority of hrv detections occur in children [ 128 ] . among 272 specimens from 18 healthy children, over a third (37 %) were hrv positive. children less than 5 years of age (44 % of whom were hrv positive) were shown to have more hrv infections and a wider diversity of hrv types than children more than 5 years old (28 % hrv positive) [ 198 ] . healthy adults in the military [ 54 , 207 ] , at university [ 208 ] , at home [ 209 -212 ] , and in the workplace [ 209 ] have also featured prominently in historical, culturebased, and volunteer infection studies and heavily infl uenced our view of hrv infection outcomes [ 64 , 206 ] . although studies of children in hospital-based populations usually report more signifi cant clinical outcomes (relating to the lrt) [ 213 ] than community-based studies, these data are still broadly applicable. hospital populations originate from the community and refl ect the more serious and perhaps fi rst exposures to the virus. hospital-based populations defi ne the potential of a virus to cause severe clinical outcomes. disease at this end of the spectrum has the strongest infl uence on future prioritization of therapeutic research and developments [ 214 ] . modern air travel contributes to the rapid spread of respiratory viruses as seen in their often frequent detection among travelers [ 215 ] including those with febrile illnesses [ 216 ] . apart from children, hrvs are found with the great clinical impact in the elderly (described as 60-90 years of age) with 50 % of aris positive for an hrv, sometimes with a greater burden of disease than ifvs [ 217 ] . those with asthma or copd are also affected by the ari triggering exacerbations of wheezing illness (see sect. 8.2 ). it is thought that this is not a different type of infection but rather a different response to infection by the host. wheezing can also result from infection in atopic people who do not have underlying asthma or copd. hrvs cause signifi cant impact in the immunocompromised, and this group is the only population to date that has been found to host truly persistent hrv infections (see sect. 5.7 ). because the hrvs are the largest group of viruses to infect humans, it is not surprising that they confuse differential diagnoses during pandemics and have key roles in co-detections and asymptomatic disease. the study of hrvs is the study of all respiratory viruses; while each can be considered in isolation, this will likely be detrimental to a greater understanding of respiratory virus pathogenesis. hrvs circulate throughout the year but usually with a bimodal peak in temperate locations in both hemispheres. the highest peaks, mostly defi ned using adult populations, are in the autumn (fall) and spring [ 64 , 66 , 211 ] (and, peculiarly, on a monday [ 218 ] ). the major winter dip in hrv prevalence closely coincides with the peaks of other respiratory viruses, particularly ifvs [ 219 ] and hrsv [ 66 ] . one hypothesis states that a miasma exists in the school classroom, of particular relevance to those who suffer asthma exacerbations, and this miasma maintains immune stimulation, which subsequently wanes among school children during holidays, to be challenged anew upon return to school [ 220 ] . it is clear that an interplay or interference takes place between viruses at the population level, particularly evident among rna viruses. there is a correlation between spiking spring and autumnal hrv case numbers and an asthma exacerbation "season" 10-24 days after return to school from holidays, in a range of climates [ 220 -223 ] . this was particularly obvious among asthma hospitalizations of children (5-15 years of age) in ontario, canada, which peaked at weeks 37-39 across a decade [ 223 ] . upon investigation, hrvs were the most prevalent of the viruses found in a 1-year analysis of emergency room presentations in ontario [ 223 ] . hrvs also predominate during "hay fever season" [ 172 ] . although a defi ned seasonality is not always found in the tropics [ 224 ] , this may sometimes be due to testing that does not include hrvs [ 222 , 225 ] or only some hrvs [ 226 ] . all the hrv types continue to circulate today, including those named in the earliest of the nomenclature assignments. at a single site during 12-24 months, 70 or more types can co-circulate [ 8 , 9 ] [ 174 ], dropping [ 198 , 227 ] if the study time frame at the site is shortened. a recurring hrv type, defi ned using molecular tools, accounted for 1.6 % of any virus detected in a birth cohort followed for 12 months [ 8 ] and, in another cohort, occurred twice in two children, within a 6-month period [ 198 ] . within a given year and across different years, it is apparent that hrv species exchange predominance [ 9 , 36 , 60 , 227 -229 ] . no evidence exists to satisfactorily explain this; however, herd immunity may be a factor. the use of cell and tissue culture underestimated the frequency of multiple infections in patients, most likely because the dominant virus out-replicated any others, or due to viral load differences, specimen quality issues, differing cell tropisms, or the triggering of an antiviral state by the fi rst virus. when the majority of respiratory viruses are sought using pcr techniques, multiple virus-positive specimens can comprise a third of those tested [ 230 ] , dropping to around a fi fth of ari episodes when fewer viruses are sought [ 217 ] . there is sometimes an emphasis on the high number of hrv cases that are identifi ed in the presence of another virus, and including hrv testing does raise the frequency of pathogen detection above one per sample [ 231 ] . coinfections, or, more correctly for pcr-based studies, co-detections (since pcr cannot determine infectivity), have been found to either increase [ 71 , 232 -236 ] or have no impact on the clinical outcome in their host [ 237 -241 ] , and so the issue of clinical relevance of co-detections is still uncertain. in extreme cases, half of all hrv detections can be found concurrently with another virus. on the surface, this is a signifi cant fraction, and yet 80 % or more of hrsv, hmpv, ev, and ifv detections and 71 % of hcov-nl63 detections can be found in the company of another virus [ 242 ] . other studies fi nd different, but still higher proportions of co-detections involving non-hrvs [ 217 ] . whether co-detections represent a particular synergism between the involved viruses, a differential capability to manipulate the host immune response, a sign of innocuousness for the most frequently involved virus [ 243 ] , or a chance due to overlapping seasons remains unclear. it is clear, however, that co-detections are not an anomaly or an error due to "overly sensitive" pcr tests; they are evidence of further biological complexity that, until recently, remained hidden from us. recent studies have shown that the initial impression of hrvs being overrepresented in these cases was incorrect. closer analysis of viral co-detections has revealed patterns [ 231 , 244 ] . these became clear when codetections were examined bidirectionally, not just how many hrvs were positive for virus x but also how many of virus x cases were positive for an hrv. whether in a hospital or a community setting, hrvs more often occur as the sole virus detected in aris [ 9 , 244 ] . considering their ubiquity, it is interesting that relatively low numbers of concurrent detections occur [ 245 , 246 ] , supporting the concept that hrvs have a direct role in the clinical outcome of their infection [ 247 ] . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens, while the host provides a vessel for hrv replication and transmission. studies of single respiratory viruses without being in the context of the respiratory virome are of limited value in drawing conclusions about clinical impact. much of the longitudinal epidemiology data previously relied upon to form assessments of hrv signifi cance was acquired using culture-based techniques. with improved and more comprehensive testing, patterns can be seen among the interactions of hrvs and other respiratory viruses. virus interference is a type of virus-virus interaction (vvi) that has been known for decades. vvi has recently been categorized into types [ 248 ] . at the population level, it has been noted that during trials of live attenuated ifv (laiv) vaccines, an interferon (ifn) response was triggered that protected vaccinees against off-target viruses for 7 days postvaccination [ 249 ] . this 1970 study went so far as to suggest such effects could be maintained for a prolonged period using a regime of consecutive schedule vaccinations, each separated by 7 days or more, during times of a prolonged epidemic [ 249 ] . a similar effect was produced using live ev vaccines (lev) to replace pathogenic ev types and interrupt outbreaks [ 250 ] . orally administered levs succeeded in their principal task but also reduced the incidence of aris during epidemics by 50 % overall [ 250 ] . this shows that immune activation in the gastrointestinal system generates an anatomically distinct protective effect and there may be a similar effect on the gut's infl ammatory status after respiratory virus infection. in contrast to the laiv results, the offtarget protective effect was reversed in a study using a trivalent inactivated ifv vaccine [ 251 ] . the mechanism underneath these opposing outcomes is unclear. during the heyday (1960s) of tissue culture for virus studies, a common biological assay for infection with hrv involved attempted infection of the culture with an enterovirus (ev) or hpiv-1 [ 252 , 253 ] . failure of the superinfecting virus to grow heralded the likely presence of a noncytopathogenic hrv. virus interference has been used to measure ifn in specimens through its inhibition of hrv growth [ 254 ] . more recently hrv-hadv dual pcr-positive cases were found less often than expected and harbored lower viral loads of hrv than did specimens from cases of sole hrv infections [ 255 ] . signifi cantly, the majority of these instances of vvi involve rna viruses [ 244 ] . it has been shown that dual infections of peripheral blood mononuclear cells (pbmcs) with viruses other than hrsv (including hrvs) induced immune responses similar to those of single infections, but coinfections including an hrsv resulted in reduced ifn-γ responses [ 71 ] . vvis are affected by the ability of each to moderate the host response against them. virus interference has also been identifi ed in virus positives as a series of patterns among respiratory specimens tested for up to 17 respiratory viruses (fig. 29.8 ) [ 9 , 244 ] . statistical analyses supported that many of the co-detections occurred in patterns, in particular that fewer co-detections involved an hrv than would have been expected by chance alone ( p ≤ 0.05). for some period, rna virus infection, especially the hrv group, may render the host less likely to be infected by other viruses and, by extrapolating to the community level, help constrict the epidemic periods of other viruses by reducing the number of fully susceptible hosts. virus interference as a feature of respiratory virus epidemiology can also be seen in results of other studies [ 256 ] . during an 8-week period that spanned peak 2009 h1n1 pandemic infl uenza season in wisconsin, it was infl uenza a virus (ifav) that seemed to dominate hrv in children with asthma who were sampled weekly [ 236 ] . whether this refl ects all ifv-hrv interactions or just those involving a novel ifv such as 2009 h1n is unclear. it was found that pbmcs from these children exhibited normal immune responses [ 236 ] . reports of subjects with continuous and extended (greater than 2-3 weeks) periods of hrv positivity [ 3 , 257 ] increased as pcr methods replaced cell culture for hrv detection. this had only rarely been recorded using culture [ 54 ] . hrv rna has been detected days prior to symptoms commencing and for as long as 5 or more weeks after they cease [ 3 , 258 -261 ] . studies that only defi ne the period between aris in children as that time when specimens are rt-pcr negative [ 3 ] will not detect overlapping serial infections (fig. 29.9 ). epidemiology that incorporates hrv typing generally does not fi nd chronic shedding [ 204 ] . hrv shedding normally ceases within 11-21 days, after signs and symptoms have stopped [ 3 , 9 , 44 , 75 , 85 , 260 ] . thus, the perception of persistence is probably due to serial or overlapping infections by multiple untyped strains [ 8 , 54 , 210 , 262 ] . few studies [ 263 ] have suitably addressed persistence in hrv infections involving healthy subjects since pre-and post-sampling clinical data are rarely described [ 80 , 264 ] . to date, true persistence-an ongoing detection of a single confi rmed hrv type-has been limited to individuals with underlying immunosuppression or immune dysfunction [ 260 ] . hrv-cs were detected more than three times longer in immunocompromised young patients than in immunocompetent children, with a mean of 16 versus 53 days [ 265 ] . multiple detection of the same hrv type (100 % identical hrv-1a sequence in each patient over time) extended to 4 months in hematopoietic stem cell transplant recipients. the proof of causality is as diffi cult to achieve as the proof of innocuousness when it comes to respiratory viruses and aris. the defi nition of "well" subjects prior to or at the time of sampling or inoculation is sometimes not clear, especially for young children who cannot reliably report symptoms [ 3 , 96 , 204 ] . often parents notice a symptomatic illness before an infection is detected in the laboratory [ 3 ] , supporting the importance of diaries in longitudinal home-based community studies. nonetheless, even with the support of telephone interviews and home visits, milder cold symptoms may be missed. it is not uncommon for an asymptomatic control to subsequently become symptomatic or have been symptomatic before sampling [ 8 , 266 ] . some studies employ sensitive symptom scoring systems [ 267 ] , but the criteria for being symptomatic are usually designed to describe and clearly discriminate overt or more "severe" illnesses, those with obvious and measurable signs. strict defi nitions help improve patient management and the commencement or better direction of treatment or cohorting. however, in research studies the arbitrary degree of severity required for reporting a symptomatic event often overlooks very simple changes in host biology due to a virus's replication. these changes to the norm are mild but nonetheless represent disease (a disorder of structure or function that produces specifi c symptoms or that affects a specifi c location and is not simply a direct result of physical injury) in the literal sense. such minor or short-lived, often unrecorded [ 3 ] , indications of infection include sinus pain, headache, sore throat, earache, watery eyes, fatigue, muscle aches and pains, and mood changes. within families, hrvs are frequently transmitted from vsig activated "shields up" a b children who are usually symptomatic [ 204 ] . infants frequently exposed to other children have more asymptomatic viral infections [ 8 ] . among infected adult family members, asymptomatic infections are more likely [ 204 ] . among older parents, whether their children live at home or not, asymptomatic infections are more frequent following hrv challenge than among adults without children or in younger parents [ 268 ] . in a study of viral species in age-stratifi ed cases and controls, signifi cantly lower viral loads were found in those without the required symptoms [ 269 ] . qpcr may prove useful to determine viral load cutoffs to address this issue in the future, although the respiratory tract is a diffi cult tissue for qpcr [ 200 ] . the high sensitivity of pcr-based methods has raised concerns over the clinical relevance of a virus-positive result [ 269 ] . it is clear that a proportion, around fi ve to 28 % of study-defi ned asymptomatic control populations [ 90 , 91 , 269 ] , are virus positive using sensitive pcr-based methods. this may vary up to nearly 50 % of cases when stratifi ed by age, virus, and season or when including highrisk populations [ 8 , 269 ] . every respiratory virus, even ifvs and hrsv, can be found in cases without symptoms at the time of specimen collection even after specifi c inoculation of adults [ 137 , 269 , 270 ] . this is a complex and incomplete story in need of more research, and so it is frustrating that positivity in asymptomatic people is often used to rank viral importance. better data are required from asymptomatic controls for any conclusion to be drawn about causality [ 266 ] , but this requirement often disregards the memory of a normal functioning protective host immunity. it is the host response that defi nes the degree of clinical severity for the infl ammatory disease that is the hallmark of an ari [ 271 ] . it is well known that previous exposure to a virus affords protection from the full clinical spectrum of disease upon repeat exposure to that virus. it should come as no surprise then that hrvs, which usually cause brief infection anyway, could well produce only minor signs and symptoms upon reinfection. the unique and extremely personal infection history of each member of a control group cannot be determined unless they are part of a longitudinal cohort. so, what do cohort studies, supported by comprehensive pcr-based testing, tell us about asymptomatic virus infections? some cohort studies do not look in asymptomatic children, seeking samples only at times of symptomatic illness [ 66 , 246 , 272 ] . a birth cohort of children enrolled and sampled when ill and every 6 months for 24 months identifi ed hrvs 14-28 % of infants and toddlers who had no nasal symptoms (defi ned solely by the presence of rhinorrhea) [ 273 ] . the childhood origins of asthma (coast) birth cohort followed 285 infants at high risk for allergies and asthma for 12 months and identifi ed hrv infections as preceding (mean age of fi rst detection, 4 months) those of hrsv (mean age at least 6 months), and hrvs were found in 35 % of asymptomatic versus 61 % of moderately to severely ill patients; the most frequently symptomatic children also had the greatest proportion of asymptomatic infections [ 8 ] . in a study of 58 children with asthma sampled weekly for 5 weeks during each of two peak hrv seasons, nearly two-thirds who were virus positive but not sensitized to at least one allergen showed no asthma symptoms, and nearly half showed no ari symptoms; in the children who were sensitized, less than one-third showed no asthma symptoms, and only a fi fth had no ari symptoms [ 227 ] . a convenience population of 15 healthy children (1-9 years old) without asthma were followed during at least three seasons, and picornaviruses were detected in 5 % of 740 specimens (21 % of infections) not associated with symptoms, the impact of hrv typing and of sampling based only on symptoms. the example provided here diagrammatically represents a single, hypothetical monitoring period, starting at time = 0, for a single individual. the period of potentially detectable hrv is indicated by an open box. if sampling occurred at each time point ( 0 -6 ) and hrv positives were genotyped, it would be apparent that three different strains infected the individual, although discerning hrv-x from hrv-z at time point 3 would require a molecular cloning approach. illness, in different forms, may have continued over the entire period depending on the symptoms required/recorded and the period of time represented by the monitoring period. in this case a clinical diagnosis may record only a single symptomatic episode. genotyping may not be performed, and sampling may be intermittent, and so association between viral type or species and disease is impossible. in the study examples indicated by ( a ) start and fi nish sampling or ( b ) symptomatic sampling, ( asterisks mark sampling times in fi lled bars), the laboratory data would have made only one or two identifi cations, respectively. in the third example, ( c ) frequent sampling of this type has previously led to conclusions of hrv persistence or chronic shedding; when combined with genotyping, it becomes apparent that different hrv types are present although 9 of the 25 infections came from households with an infected sibling [ 3 ] . in summary, there is clear evidence for the presence of hrvs in asymptomatic controls. a precise proportion cannot yet be defi ned. some study controls show signs of a "lead-in" period where rna positivity precedes an ari defi ned on follow-up, while others may have been defi ned as symptomatic if more symptoms had been accounted for. mechanisms and routes of transmission hrvs have been found at extra-respiratory sites. viremia was determined in the blood of children with lrt infection or pericarditis [ 274 , 275 ] , and hrv-c was more commonly associated with viremia than was hrv-a, supporting possible increased pathogenicity [ 274 ] . blood was also positive for hrv rna and infectious virus from infants at necropsy [ 276 , 277 ] , and hrv rna was detected in the plasma of children with asthma, bronchiolitis, or common cold [ 76 ] . an hrv was once isolated from feces [ 203 ] , and more recently higher than expected loads of hrvs were detected in fecal specimens from children with suspected meningitis and fever of unknown origin [ 77 ] , with gastroenteritis [ 278 ] , and in a child with pericarditis [ 275 ] . nonetheless, the nasopharynx is still considered the main site of focal virus production [ 279 ] , regardless of inoculation route [ 280 ] , and most studies of transmission routes have centered on the urt. in contrast to ifv and hrsv, hrv infection involves less destruction of tissue. ciliated epithelial cells are sloughed off in proportion to the severity of an hrv ari, but this damage is minimal and does not occur during the viral incubation period or with subclinical infections [ 137 , 281 ] . the incubation period between infection and onset of virus shedding into nasal secretions is 1-4 days with shed viral titers peaking in adults between days 2 and 10 [ 44 , 282 ] . the time until successful hrv transmission among adults in a childless family setting is usually 5-8 days and requires the donor to be shedding at least 10 3 tcid 50 at some stage, to have recoverable virus on the hands and in the nares, enough shared time, and a moderate to severe ari [ 283 ] . the lungs have been shown to host replicating hrv [ 260 ] , and the reader of such reports may be left with the perception that detection of hrv replication in the lrt explains all lrt symptoms. however, relatively few studies seek or identify true hrv replication in the lrt. while the overwhelming majority of lrt cases detect hrv from the urt, a correlation between urt positivity and lrt disease does exist [ 284 ] . it is well known from experimental inoculation studies that hrv infection can result from inoculation of the conjunctival sac after virus is moved through the nasolacrimal duct [ 280 ] . in these studies virus was commonly delivered by aerosol or intranasal instillation of 0.25 ml to 5 ml of suspension [ 43 -46 , 280 , 285 -287 ] . in the laboratory, hrvs can retain infectivity for hours to days on suitable, nonporous solid surfaces, especially if the inoculum remains damp [ 47 , 287 ] , which supports direct self-inoculation especially in the family setting and indirect inoculation via fomites [ 288 ] . in a trial to defi ne the movement of virus from a contaminated donor to a recipient via multiple surfaces or by hand-to-hand contact, 13 % (donor to objects to recipient) and 6 % (donor to recipient fi ngers) of the virus recoverable from the donor's fi ngertips were recoverable from the recipients' [ 289 ] . even under observation, eye rubbing (0.37 h −1 -2.5 h −1 ) and nosepicking (0.33 h −1 -5.3 h −1 ) occur frequently [ 47 , 290 ] , suggesting self-inoculation could outpace personal hygiene, particularly in the young. it was once thought strange that aris were so common, but isolation rates for the expected viruses were so low [ 36 , 291 ] . with a better understanding of the importance of preexisting antibody (something common among the predominantly adult volunteers used by many studies), the discovery of a third, unculturable species of hrv (still causing aris but impossible to isolate or detect using antibody-based systems for which no reagents existed), and a vastly improved diagnostic sensitivity, this is much less confounding. in the past, household cross infection, determined by ari, was low, about fi ve exposures to infected members required for infection [ 17 ] despite viral loads in nasal washings peaking at 1.6 × 10 5 tcid 50 /ml [ 44 ] . experimental transmission was also reportedly ineffi cient [ 45 ] . in contrast, "naturally" close-quartered military populations, interacting over 1-4 weeks, experienced rapid spread of hrvs to >50 % of the group [ 54 ] . the use of pcr recently clarifi ed this discrepancy, confi rming that frequent transmission in families is more common than culture-based studies had identifi ed, often resulting in asymptomatic infection among older siblings and parents [ 204 ] . pcr has helped defi ne the scope of viral rna, if not actual infectious virus, survival, and spread. transmission studies require infectious hrv, and so the hrv-cs do not contribute to the historical data. under crowded or intimate conditions and with more severe colds, transmission reaches 38-100 % [ 283 , 292 ] . in some studies, both large-and small-particle aerosols proved ineffi cient, supported by a low isolation rate from saliva (39 % compared to 65 % of hand washes and 50 % of nasal swabs) [ 44 , 47 , 293 ] and from only 8.3 % of participants exposed to large-particle aerosols [ 293 ] . in other human donor-recipient model studies however, aerosol proved to be the main transmission route among antibody-free adults [ 46 , 282 ] . the discrepancy may have been due to insuffi ciently long or intense exposure in the earlier aerosol experiments [ 45 , 267 ] . apart from particle size, spread of virus by aerosol is affected by existing nasal obstruction which can divert secretions from the nares to contaminate saliva, the presumptive source of virus in coughs and sneezes [ 44 ] . when exposed to 10 liters of a small-particle aerosol, 10 1 tcid 50 of hrv-15 was associated with fever and prominent tracheobronchitis in antibody-free (<1:2) adult volunteers but not when delivered via nasal drops or a coarse aerosol [ 46 ] . it has also been found that simple breathing releases hrv rna (the same type was also identifi ed from nasal mucous) from at least a third of adults and children with symptomatic aris and infectious hrv could be isolated from a fi fth [ 294 , 295 ] . it is apparent that hrvs accumulate at sites with heavy human traffi c, potentially forming a secondary source of infection. hrv rna can be detected from 32 % of ~47-hourold fi lters placed to sample air in offi ce buildings [ 296 ] . in aircraft, high effi ciency particulate air (hepa) fi lters have been found to harbor hrv rna more than 10 days after they were removed for servicing [ 297 ] . hrv infections trigger a vigorous proinfl ammatory immune response that is thought to drive the symptoms experienced as illness [ 271 , 298 , 299 ] , but they do not seem to actively prevent or interfere with the host's immune response the way most other viruses have evolved to do. there may be a role for repeated challenge by hrvs and other respiratory viruses leading to infl ammation and tissue remodeling. the host response to hrv infection can be broadly broken into the innate (very fast, encoded in the germ line, nonadaptive) and adaptive (slower to develop, reliant on t cells, b cells, and the generation of antibody) responses. while the innate system is "always watching," it is signifi cantly amplifi ed by virus infection. the adaptive response is initiated by the host's fi rst infection with a particular virus and then functions to limit subsequent infections through the production of neutralizing antibodies and amplifi cation of existing cell-mediated immunity. after virus-receptor binding and internalization, the earliest host cell immune response to an hrv infection is elicited by the innate immune system (fig. 29.10 ). epithelial cells represent the front line against hrv invasion although alveolar macrophages and dcs are better equipped to respond [ 300 ] and do so despite not hosting hrv replication directly [ 16 ] . virus detection is mediated by pattern recognition receptors (prrs) that have evolved to recognize conserved molecular structures shared among diverse pathogens. internal-or surface-mounted prrs include sentinels that specifi cally recognize picornavirus rna and protein and, in doing so, trigger an immune circuit that results in the production of ifns and subsequently hundreds of ifn-stimulated gene products. the innate response to viral infection hinges on inducing two type i ifns (initially ifn-ß then ifn-α), secreted cytokines that produce antiviral, antiproliferative, and immunomodulatory outcomes [ 301 ] . the type iii ifns (ifn-λ1 or il-29, ifn-λ2 or il-28a, and ifn-λ3 or il-28b) are also produced in response to viral infection in a range of cells, although their receptor is not as widespread [ 302 ] . the type ii ifn, ifn-γ, is produced by activated t cells and natural killer cells rather than in direct response to virus [ 303 ] . detection of viral components triggers protein signaling cascades that regulate ifn synthesis through the activation of viral stress-inducible genes (vsigs) [ 301 , 304 ] . these are sometimes expressed constitutively but upregulated after ifn induction following hrv infection [ 305 ] . released ifn-ß binds to the ifn-α/ifn-ß receptor in an autocrine (the same cell) and paracrine (neighboring cells) manner, starting a positive feedback loop for type i ifn production, the "second wave." vsigs include the antiviral proteins protein kinase r (pkr), 2′5′oas/rnasel, and the mx proteins [ 306 ] . ifn-α upregulates expression of mxa, 2′4′-oas, and pkr [ 307 ] . the mx pathway is also induced after virus infection but is not constitutively expressed [ 307 ] . depending on the sentinel system stimulated, there are different pathways to vsig activation. those vsigs with antiviral properties (e.g., mxa, pkr, 2′5′oas/rnasel) inhibit different stages of virus replication and strengthen an antiviral state in the host. while this state is well known, the nature of its induction by different respiratory viruses and the impact of induction upon the replication of other respiratory viruses are topics for considerable ongoing research. one pathway to ifn induction relies on the ifn-upregulated cytosolic sentinels retinoic acid inducible gene rig-i-like receptors (rlrs) rig-i (specifi c for ifav and others) and melanoma differentiation-associated gene 5 (mda5, specifi c for picornaviruses and others) [ 306 , 308 ] . these rna helicases recognize either rna with a 5′-triphosphate or distinct dsrnas, which results in activation of nf-κb leading to "classical" type i ifn induction [ 301 , 306 ] . studies into the innate response to hrv infection have been limited to the use of a very few easily cultured types. it is presumed that the result can be extrapolated to most if not all types. this is yet to be tested. rig-i is degraded by hrv-a16 [ 309 ] , ifn regulatory factor (irf)-3 homodimerization is interfered with hrv-b14 which limits ifn-β induction [ 310 , 311 ] , and mda5 is degraded by hrv-a1a but not hrv-a16 [ 312 ] . another pathway for recognizing hrv infection involves the toll-like receptors (tlrs), transmembrane prrs that terminate in an intracellular signaling region. the endosomally localized tlr3, tlr7, tlr8, and tlr9 recognize nucleic acids and are also involved in innate antiviral responses. tlr7 and tlr8 identify g/u-rich ssrna from endocytosed viruses, while tlr9 recognizes unmethylated cpg dna present in dna viruses [ 301 , 318 ] . tlr2 and tlr4 are found on the cell surface and recognize hrv or hrsv proteins, respectively [ 318 , 319 ] , and tlr3 recognizes dsrna. tlrs operate mainly, but not exclusively, in plasmacytoid dc [ 301 ] . the particular tlr that notifi es of an hrv incursion may depend on the method of virus approach [ 319 ] . tlr7 activation can reduce 2′5′oas and mxa mrna expression and ip10 protein in adolescents with asthma compared to healthy controls [ 320 ] . tlr3 activation did not result in a similar disparity [ 320 ] . it has been suggested that hrvs may have evolved with humans to such an extent that their symbiotic relationship serves to help train the human immune system [ 321 ] . intriguingly, within the hrv species, there are differences in the type and level of host response induced [ 322 ] which may refl ect receptor usage, route of entry and cell type infected, hrv species, or the degree of laboratory-adapted virus used during in vitro studies. after initial hrv infection, the innate response results in production of proinfl ammatory cytokines, vasoactive peptides, and chemokines that attract leukocytes, granulocytes, dcs, and monocytes (table 29. 2 ) [ 321 , 323 , 324 ] . the t-lymphocyte response to viral intrusion can be broadly categorized as t h -1-like and t h-2-like. other t-cell subsets exist, but most work in relation to hrv has been conducted on the earliest defi ned subsets. the t h -1 cellular response is important in managing cellular immunity and producing interleukin (il)-2 and ifn-γ. the t h -2 cellular response manages humoral immunity and stimulates b cells via il4 (initiating production of ige), il5 (infl uencing eosinophils), and il13 (crucial component of allergen-induced asthma). these two t-cell responses act in concert with epithelialderived chemokines (e.g., eotaxin) to promote the recruitment and activation of eosinophils and mast cells, contributing to chronic airway infl ammation and the hyperresponsiveness of airways to a variety of nonspecifi c stimuli [ 325 ] . t h-2 lymphocytes, opposing t h-1 lymphocytes, contribute to an allergic infl ammatory cascade, akin to what occurs to rid humans of parasites [ 326 ] . the t h-1 response can also be repressed by binding of microrna, which leads to an altered balance favoring a t h-2 state in mice and probably in humans [ 327 ] . regulatory t cells (t reg ) suppress allergic infl ammatory pathways and are therefore fundamental in protecting the airway from allergen sensitization [ 326 ] . considerable immunobiological research has focused on asthma exacerbation, with which hrvs are intimately involved. although upregulated by hrv infection, the t h -1 response is comparatively defi cient in people with asthma [ 328 , 329 ] . this is problematic as an increased t h -1-like cytokine response, deduced from higher sputum mrna ifn-γ/il5 values, speeds clearance of hrv and symptom amelioration [ 85 ] . one possible cause of the t h -1 defi ciency in people with asthma is inadequate maturation of type i and iii ifn responses due to reduced exposure to infections early in life [ 330 ] . the "hygiene hypothesis" [ 331 , 332 ] posits a pathway for an asthma etiology described [ 325 ] in terms of the young, unchallenged immune system, dependent on infections to stimulate the development of its t h-1-like functions. one theory suggests that hrvs play a central role in developing that effi cacious antiviral immunity, particularly in infancy, via their frequent, usually mild self-limiting infections [ 333 ] . genome-wide expression analysis of becs from healthy and asthmatic adult subjects after hrv-a1a infection revealed some signifi cant differences that were found between cell types and response to infection [ 61 ] . these included immune response genes (il1b, il6, il8, il1f9, il24) and airway remodeling genes (loxl2, mmp10, fn1) and an overall proinfl ammatory response and metabolic slowdown consistent with proteolytic cleavage of transcription factors by some hrvs [ 133 , 334 -336 ] in the infected cells. this study further noted some similarities to gene expression changes observed in brushings from people with mild asthma after allergen exposure and in bal cells from subjects with corticosteroidresistant asthma [ 61 ] . overall, hrv replication and the host transcriptional response to it were similar in normal or asthmatic bec cells [ 61 ] . this indicated, at least in adults, that something beyond the epithelial cell is an important contributor to more severe clinical outcomes in asthma. the application of inactivated hrv-b14 was found to promote release of il10 from monocytes (an immunosuppressive cytokine) and to inhibit the stimulation of il12 (drives t h -1 development) [ 337 ] . however, neither il10 nor il12 was signifi cantly induced in asthmatic adult volunteers in response to hrv-a16 compared to healthy subjects [ 338 ] . while ifn-α was detected after transfection of dcs with hrv-b14 ssrna, low tnf-α and il12 levels were also noted [ 16 ] . it was posited that the reduced il12 could indicate negatively affected local immunity possibly predisposing to secondary infections [ 337 ] . infection of stromal lung cells by hrv-b14 triggered exaggerated levels of the pleiotropic il11 (an il6type cytokine), akin to those triggered by hrsv, which were also detected in nasal secretions from children with wheezing [ 339 ] . other cytokine changes have been identifi ed in atopic adult volunteers challenged with hrv-a16. g-csf and il8 (chemo-attractant for neutrophils) levels rose in the urt (as examined by protein detection in nasal lavage) and lrt (mrna detection in sputum) with concomitant rises in blood and nasal neutrophil numbers [ 85 , 203 , 340 ] . the nasal epithelial cells of atopic individuals, especially in season, express more icam-1 than those of nonatopic adults [ 341 ] as do normal subjects infected by the major group hrv-b14 [ 341 ] . by contrast, ifn-γ and il8, which appear later postinfection, downregulate icam-1 expression in infected cells [ 191 ] and encourage infi ltration of neutrophils [ 342 ] , respectively. changes in icam-1 levels may modify participates in creation of an antiviral state; produced by and infl uences the maturation of dcs il-1β proinfl ammatory properties; enhances adhesion molecule expression including icam-1; induces il-2 receptor gm-csf a granulocyte and monocyte growth factor il-2 stimulates growth and differentiation of t and b lymphocytes and cytotoxic activity of nk cells and monocytes il-4 t h 2 differentiation, promotes ige synthesis il-6 activation, differentiation, and proliferation effects on t and b lymphocytes; induces c-reactive protein stimulating pyrexia il-8/cxcl-8 neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and degranulation activity il-10 anti-infl ammatory factor produced by monocytes that acts by inhibiting proinfl ammatory cytokines il-1, il-6, and tnf-α irf7 a master hub, regulating antiviral immunity ip10/cxcl10 chemoattractant for activated t h 1 and nk cells tnf-α proinfl ammatory activity similar to il-1 β; activates neutrophils; induces vascular permeability mpc-1 a monocyte attractant bradykinin potent infl ammatory mediator, increases vascular permeability tslp 3 an il-7-like cytokine that activates myeloid dcs to induce naive t cells into t h 2 cells producing il-4, il-13, and tnf-α; induced by hrv in the presence of il-4 bec bronchial epithelial cells, dc dendritic cell, irf interferon regulatory factor, ifn-γ inducible cytokine protein, nk natural killer, pbmc peripheral blood mononuclear cells, il interleukin, tnf tissue necrosis factor, tslp thymic stromal lymphopoietin t-lymphocyte-mediated cytotoxic or t h interactions with hrv-infected cells, upregulating receptor expression and encouraging eosinophil and t-cell infi ltration into the lower airways of asthmatic individuals [ 187 , 343 ] . before an hrv can enter a cell, it must pass through a defensive barrier of secreted anti-hrv antibody, mostly iga. the ease with which this passage occurs is proportional to the progression of clinical disease. healthy adult volunteers were found to develop iga by at least 3 days to 2 weeks after inoculation-about the same time as serum antibody-and retain peak levels for at least 8 weeks [ 178 , 344 -346 ] , falling faster than serum levels [ 347 , 348 ] . there is also some evidence for a degree of nasal immune memory [ 347 ] . volunteers with pre-study serum antibody could still be infected in some studies [ 46 , 210 , 292 ] , but not in others [ 349 ] . infection is more clear in volunteers without preexisting nasal antibody to experimental challenge virus; they become infected, exhibit more severe ari, and shed more virus for longer [ 292 , 347 ] . iga does not seem to modify illness severity or virus shedding, but high levels prevent reinfection by the initiating virus type. low levels or absence of iga does not prevent reinfection by the same hrv type, which may manifest as symptomatic or asymptomatic disease [ 349 ] . older children, adolescents, and adults have greater amounts of hrv-neutralizing antibody than young children [ 42 ] , accompanying a trend toward decreasing numbers of symptomatic aris with increasing age [ 218 , 350 ] . this feature raises an issue: did the use of older subjects in many common cold studies underplay the pathogenic potential of the hrvs because protective or partially cross-protective antibodies moderated the impact of infection? consequently, quantifying levels of type-specifi c serum antibody became routine practice prior to some studies. adult volunteer studies determined that no infections resulted if preexisting neutralizing antibody titers ≥1:16 existed; as levels grew from 0, so did levels of resistance to infection [ 43 , 210 ] . adults were protected by serum titers of 1:3-1:8 [ 209 , 210 ] . the trend was interrupted by adults in the 20-39 year age group, presumably because they had begun families and their young children acquired and amplifi ed currently circulating types from the community and transmitted them into a household that was either immune naïve or lacking suffi cient antibody or cell-mediated memory for protection [ 351 ] . traditional vaccine strategies were quickly ruled out as a prophylactic intervention for hrv illness because of the extensive antigenic variability that is a hallmark of the genus enterovirus [ 178 , 325 ] . however, if it were possible to identify "master" strains [ 352 ] that exhibit suffi cient antigenic cross-reactivity to induce broad heterotypic responses against many other hrv strains, then an effective vaccine could still be possible. in fact, boosting host immunity to an hrv type by repeat infection does heighten immunity to one or more other types [ 44 , 353 ] . the highest of these heterotypic antibody titers develop against those types with the highest preexisting antibody levels [ 352 ] . the fi rst description of a unifying hrv numbering system recounted the appearance of minor serological cross-reactions, which were removed by modifi cation of the technique [ 57 ] . subsequently, cross-reactions were better defi ned during experimental inoculation when multiple hrv immunogens and antigens were used to deduce the extent of heterotypic responses [ 56 , 210 , 352 , 354 ] . less promising for hrv vaccinology was the description of antigenic variation within hrv types which suggested that immunity to one variant of the type might not protect against infection by other variants [ 355 , 356 ] . the "prime strain" is a specifi c antigenic variant of a prototype hrv type that is neutralized to a lesser extent by antisera from the prototype, while yielding antisera that effectively neutralize both itself and the prototype [ 357 ] . another form of this cross-neutralization is ascribed to the "intertypes," which are hrv isolates that share a lower-level serological relationship with a pair of hrv strains, which themselves share neutralizing reactivity, e.g., hrv-a36 and hrv-a58 [ 358 ] . the low-level reciprocal neutralizing activity was not equivalent in both directions; anti-hrv-a36 sera had a higher titer for hrv-a58 than anti-hrv-a58 sera did for hrv-a36 [ 358 ] . over 40 strains were linked directly by such one-or two-way cross-reactions or indirectly through two or more strains. hrv-a67 and hrv-a28 are linked via hrv-a11, hrv-a13, and hrv-a32 (anti-a11 serum neutralizes hrv-a28, anti-a13 neutralizes hrv-a11, and anti-a32 neutralizes both hrv-a13 and hrva-a67 [ 358 , 359 ] ). a surrogate molecular method which provided insight into these interrelationships, perhaps expanding upon them to identify useful patterns for vaccine immunology purposes, would be most welcome. in summary, heterotypic immunity and hrv intertypes might be exploitable features of hrv immunobiology that could confer maximum protection upon the host from the minimum number of hrv types [ 358 ] . hrvs circulate in great numbers, and any specifi c roles for distinct hrv types in initiating disease remain to be defi ned. the relatively inconsequential common cold is the most frequent manifestation of viral infection in humans, with 30 to >80 % of colds positive for an hrv [ 69 , 360 , 361 ] . furthermore, aris due to hrv infection can exacerbate or result in a much greater burden of disease in those with asthma, copd, or cystic fi brosis. other complications include otitis media, pharyngitis, and wheeze in atopic people without asthma. the role of viruses in the origin of some of these diseases or their exacerbation is still unresolved. the lrt disease may mask the urt nature of the infection, favoring clinical diagnosis of an lrt illness. interestingly, during the 2009 h1n1 pandemic, much of the parentinitiated healthcare visits from a birth cohort in the united states were not due to pandemic virus but hrv and hrsv [ 362 ] . there is no known natural murine rhinovirus on which to base a small animal model of hrv infection, and mice are not natural hosts for hrvs. a recently developed model of airway disease using mengovirus (a picornavirus infecting rodents) may yield valuable in vivo airway infection and infl ammation data [ 363 ] . hrvs are often detected in neonates and infants with lrt signs and symptoms because the very young have narrow, immature airways and are more signifi cantly affected by airway swelling, excessive secretions, and smooth muscle contraction [ 364 ] . this may also be due to the relatively naive immunity of very young children. much of the more severe disease in hrv-positive children occurs in the youngest of them. some key examples are addressed below. for the common cold, as for any illness, accurate epidemiology and burden of disease data underpin the prioritization of preventing, treating, and further researching the etiological agent. to assign funds for researching the agent, health policy makers also need to understand how effi cacious and costeffective the development of an intervention will be [ 365 ] . the host immune response to hrv replication is the main cause of the signs (quantifi able fever, rhinorrhea) and symptoms (feeling of fever, myalgia, headache, fatigue, and mood change) of a cold that the host experiences [ 321 , 366 , 367 ] . a feature of common colds is increased vascular permeability which, enhanced by kinins, results in increased plasma protein (albumin and immunoglobulin [ig] g) levels in mucus, approaching the levels in serum [ 368 ] . histamine levels do not rise in nasal secretions of otherwise healthy cold sufferers [ 368 ] . during the resolving phase of the ari, glandular proteins (lysozyme, siga) predominate [ 346 ] . the common cold syndrome is also described as rhinosinusitis (the agglomeration of rhinitis and sinusitis since they frequently clinically coexist) [ 369 , 370 ] . this consists of nasal discharge or rhinorrhea, nasal obstruction, sore throat, sinus pain, headache, sneezing, watery eyes, cough, fever, fatigue, muscle aches and pains, and mood changes [ 367 , 371 ] . these are caused directly or indirectly by viral infection; cough is the result of vagus nerve irritation by mucus; sneeze results from trigeminal nerve irritation; sore throat is likely due to the action of prostaglandins and bradykinins; and fever, psychological effects, fatigue, and myalgia are mediated by cytokines [ 367 ] . hypertrophic adenoids have also been found to have a high proportion of viral, especially hrv, occupation regardless of host symptomatic state [ 372 ] . observation of natural culture-confi rmed hrv colds in adults noted that cough usually started by day 1 and was more persistent up to 9 days later [ 264 , 373 ] . rhinorrhea, sneezing, and sore throat were reported by half or more of patients and headache by at least a quarter of cases [ 207 , 373 ] . as neutrophils accumulate at the site of primary urt infection, the myeloperoxidase in their azurophilic granules creates the yellow-green coloration of nasal mucus that was once considered a sign of bacterial superinfection [ 271 , 367 ] . a common cold caused by an hrv cannot be clinically distinguished from one that caused by any of the other respiratory viruses [ 207 , 371 ] . as is likely for a single hrv type, once the host has been infected by an hmpv, hpiv, ifv, etc., a secondary exposure to that same virus type will produce less severe clinical outcomes due to pre-primed host immunity. asthma is a clinical diagnosis made on the basis of patient history, physical examination, assessment of airway obstruction or reversibility, and response to bronchodilators [ 374 ] . it is a complex chronic respiratory disease involving airway infl ammation, airfl ow obstruction, and airway hyperresponsiveness, which manifests as recurrent reversible attacks with deteriorating asthma control that are generated by interactions between infectious agents and other environmental and genetic factors that remain incompletely characterized [ 375 ] . the mechanistic role for hrvs in asthma inception and exacerbation is not yet defi ned [ 364 , 376 ] but is being revealed as the extremely complex interplay between infl ammation due to virus versus that due to atopy is explored [ 315 ] . possible virus-host interactions include (i) severe hrv infection of healthy infants which may result in subsequent development of asthma; (ii) hrvs may trigger asthma in children with a genetic predisposition toward atopy; (iii) repeated mild infections may protect against more asthmogenic/cytopathic viruses or the overdevelopment of the t h 2type response; and (iv) hrvs may simply exacerbate that which already exists [ 377 ] . it is unclear if the risk of atopic asthma during infancy is increased by aris which affect the development of the immune system, or whether aris lead to asthma development in children with a genetic predisposition to more severe responses to infection [ 325 , 343 , 378 ] , or a mix of both. in children with asthma, viruses have been detected in at least 77 % of exacerbations (65 % picornaviruses, probably hrvs [ 379 ] ) and in 50 % of adults [ 380 ] . acute wheezing episodes (including bronchiolitis and acute asthma) are a frequent, epidemic, and seasonal lrt manifestation of urt respiratory virus infection of children from all ages, especially during the fi rst year of life [ 214 , 381 -385 ] . bacteria are not major factors in wheezing exacerbations [ 386 ] . wheezing is blamed for high socioeconomic and healthcare costs, overuse of antibiotics, being the primary cause of hospitalization among children, and, rarely, for death [ 109 , 387 , 388 ] . traditionally hrsv infection has most often been the virus causally associated with expiratory wheezing, wheezy bronchitis, or asthma exacerbations because of the virus's well-known ability to infect the lrt, its more frequent detection in some studies [ 386 ] , and the low perceived likelihood of urt viruses such as hrvs replicating in the warmer lrt. nonetheless, periods of epidemic wheezing in the absence of high rates of hrsv detection are common [ 383 , 389 ] . hrvs even predominated in some culture-based studies of wheeze [ 384 , 390 ] . the coast study used sampling criteria that were intentionally designed to investigate the role of hrsv in illness, but instead indicated that hrvs were the most important predictor of subsequent wheezing in early childhood, and this is supported worldwide [ 224 , 391 , 392 ] . the asthmatic airway is characterized by an infi ltration of eosinophils and th2-type t cells (th2 cells) [ 393 ] . in those with an atopic background, eosinophilia was more common, and the virus isolation rate was higher than in the nonatopic group [ 394 ] . the cytokine and eosinophil activation profi les for hrsv-induced wheezing differ from those induced by hrv in which il5 is signifi cantly higher in serum and nasal aspirates than for hrsv [ 118 ] . ip10 was the only cytokine signifi cantly elevated in all symptomatic wheezing groups [ 118 ] . signifi cantly higher rates of hrv detection with more obvious lrt symptoms are more common in children with asthma than in non-asthmatic populations [ 380 , 388 , 395 , 396 ] . exacerbations of asthma are often preceded by a symptomatic rather than asymptomatic hrv infection [ 17 , 379 , 388 , 397 -399 ] although, in some instances, an exacerbation is the only sign of infection [ 400 ] . reduced peak expiratory volume in children is especially associated with detection of respiratory picornaviruses [ 379 ] . severe "wheezy bronchitis," a historical term describing an acute illness with preceding ari and characterized by cough, wheezing, breathlessness, and mucous production, was more often positive for a virus than mild disease [ 394 ] . even the use of culture found that hrvs predominated in both urt and lrt (sputum containing becs) or combined respiratory tract samples [ 394 ] . bacteria were often present with ifv, but not with hrvs [ 394 ] . the airway epithelial cells form a physical barrier in addition to their roles in immune surveillance and regulatory control [ 393 ] . however, the asthmatic bronchial epithelium is compromised by incomplete tight junctions that are more sensitive to airborne pollutants [ 401 ] and most likely to allergens and respiratory viral infections. this is further specifically disturbed by hrv infection which reduces expression levels of tight and adherens junction proteins [ 402 ] . in those with asthma, the presence of an hrv can induce illness that, while often more severe than in non-asthmatics, has been associated with signifi cantly different hrv load or duration of hrv rna detection in people with asthma compared to those without [ 403 ] . hrv-c types are often detected in more serious clinical outcomes than hrv-a or -b [ 265 ] although hospitalizations may be fewer for hrv-cs than the other species [ 404 ] . aom is diagnosed by middle ear effusion (otorrhea) with simultaneous signs and symptoms of ari including fever, earache, rhinitis, cough, sore throat, chest wheeze, nocturnal restlessness, irritability, poor appetite, diarrhea, and vomiting. transient abnormal (negative) ear pressure upon tympanometry occurs in two-thirds to three quarters of uncomplicated colds among healthy children [ 405 , 406 ] . aom is a frequent reason for outpatient antibiotic therapy which can reduce the time to resolution of symptoms in infants and has been attributed to reducing the overall hospital burden of aom [ 407 -412 ] . since a longitudinal day-care study in 1982, the association between aom and viral urt infection has been coalescing, and it is now clear that aom often occurs with or shortly after a viral ari, most frequently in the young and occurring more often during winter than summer [ 413 , 414 ] . the use of infl uenza vaccines reduced aom occurrence by a third during an epidemic period [ 415 ] , but the use of pneumococcal vaccine did not reduce the occurrence of aom overall, just that relatively small fraction (6 %) due to the target bacteria [ 416 ] . the isolation by culture and pcr detection of viruses from middle ear fl uids and the refractory nature of some aom cases to antibiotic therapies confi rmed that viruses play an important role in this illness [ 409 , 414 , 417 ] . studies relying on underperforming culture-based techniques underestimated the role for viral aris [ 414 , 418 ] , but other studies using pcr techniques and including hrvs found them to be the most frequently detected virus in middle ear fl uids and nasopharyngeal secretions [ 409 , 417 ] . the use of pcr has identifi ed respiratory viruses, most often hrvs, in nasal secretions of 50-70 % of children with aom [ 66 , 413 ] . because virus is often detected in the nasopharynx at the same time as the middle ear fl uid, the question of the relevance of a pcr positive is a valid one [ 419 ] . picornaviruses have been detected in 30 % of nasopharyngeal swabs taken during cold season from aom-prone infants and young children, and large quantities of hrv rna have been detected by in situ hybridization of adenoid tissues from 65 % of children with recurrent aom and/or adenoid hyperplasia [ 259 , 420 ] . in a cohort of children followed from 2 to 24 months and using culture-rt-pcr, hrvs in the urt were the second most frequent pathogens associated with aom, after hrsv [ 66 ] . viruses, most often hrvs (30.8 % of aom with ari), were also detected concurrently with non-ari periods associated with aom episodes (14 % of aom without ari) [ 418 ] suggesting that aom may be the only manifestation of some hrv aris, just as wheezing sometimes is. in the united states, 180 subjects were enrolled and followed in a birth cohort until the fi rst aom episode or between 6 and 12 months of age 362 ]. hrvs accounted for 55 % of viruses detected and 69 % of specimens with a single virus detected. this dominance was maintained even through the 2009 h1n1 infl uenza pandemic [ 362 ] . in the day-care aom study mentioned above, primary acquisition of streptococcus pneumoniae or haemophilus infl uenzae had minimal importance as an initiation factor for aom with effusion, but nasopharyngeal colonization was important [ 413 ] . animal studies have shown that virusbacteria interactions have a role in nasopharyngeal colonization and aom development [ 414 ] . positive correlation has been made between hrv detection in aom-prone children and moraxella catarrhalis infection as well as a tendency toward the copresence of streptococcus pneumoniae [ 259 ] . the presence of hrv-b14 was shown to increase adherence of s. pneumoniae in human tracheal epithelial cell cultures [ 421 ] . it is believed that these three bacterial pathogens can colonize without symptoms until a viral ari shifts the balance toward a cytokine-mediated infl ammatory state [ 419 ] . other diseases in which hrvs are often detected this disorder of older patients encompasses emphysema (alveolar destruction) and chronic bronchitis (large airway infl ammation with chronic mucous production) and describes a long-term obstruction to airfl ow in the lung (compared to asthma which is a reversible obstruction with normal fl ow between exacerbations). while bacteria are found in half of all exacerbations, antibiotic therapies have often yielded poor outcomes [ 422 ] . hrv infections result in more copd exacerbations (~66 % of cases [ 92 ] ) than any other virus identifi ed to date [ 422 , 423 ] . an experimental human model of hrv infection in copd provided preliminary evidence that hrvs cause exacerbations [ 286 ] . viral culture associated symptomatic hrv infections with exacerbations among chronic bronchitics, including cases of isolation from sputum (lrt sample) in the absence of hrv in the urt [ 424 ] . adding the measurement of an infl ammatory marker in the serum, like il-6, further improves the speed of predicting an infectious etiology for exacerbations of copd [ 425 ] . pneumonia is a disease that often occurs early in life, is responsible for millions of deaths each year [ 426 ] , and is caused by viral and/or bacterial infections. a diagnosis of pneumonia requires a radiologically confi rmed infl ammatory infi ltration of the lung tissue. childhood communityacquired pneumonia (cap) is common in developing countries [ 427 ] . cap also complicates existing chronic medical conditions and takes advantage of immunosenesence [ 428 ] . the role of hrvs in contributing to the development of bacterial pneumonia is likely underestimated [ 426 , 429 ] . determining an etiology is confounded by the rarity of obtaining lrt specimens, by short-term studies, and by the complex milieu of viruses and bacteria involved. less invasive sampling of the urt permits more routine sampling and screening, and so convenience and reduced risk have led to the detection of putative pathogens in the urt with the general assumption that they account for lrt disease, especially in children under the age of 5 years [ 430 ] . pneumonia studies are complicated by the lack of a suitable control group; sputum is not produced from the healthy lower airway and needle aspiration, while a gold standard is also a hospital procedure with some risk [ 431 ] . studies that are comprehensive and use sensitive molecular testing are also rare for the study of cap etiology. when used for cap investigations, pcr methods almost double the microbiological diagnoses over conventional culture and serology techniques, especially improving the identifi cation of mixed infections and fastidious viruses [ 432 ] . rapid diagnosis aids management and helps make decisions about treatment, while prolonged searching for an etiological agent leads to further invasive testing [ 256 , 433 ] . at least a quarter of clinical cap cases remain unsupported by microbiological fi ndings [ 241 , 432 ] . infections causing pneumonia vary with age and vaccination status [ 433 ] . viruses can be detected in up to 90 % of infants (1-12 months of age) with pneumonia, and these cases follow a seasonal pattern [ 427 , 433 ] . bacteria can also be detected in over 90 % of infants and older children, the elderly, and those with severe cap [ 432 , 434 ] . studies that predated the use of pcr pronounced hrsv, followed by hrvs, the major viral contributors to cap, with viruses comprising 27-72 % of childhood pneumonia cases [ 429 , 434 ] . in the pcr age, the role of hrvs has received increasing attention, and they are increasingly the major viral group detected from both urt and lrt (sputum) specimens of children with cap. this holds true even when studies extend across 1 or more years, which presumably would account for seasonal variation in virus prevalence [ 241 , 256 , 426 , 434 ] . it is suspected that viruses such as hrv prepare the way for subsequent bacterial infection in some direct or indirect fashion [ 65 , 259 , 420 , 435 ] . there are laboratory data which support this [ 436 ] as well as observational data showing a high proportion of hrvbacterial co-detections [ 256 , 437 ] . mixed infections including viruses are a possible cause of antibacterial treatment failure and sometimes a puzzle for physicians. mixed infections occur frequently in lrt diseases such as pneumonia, which is not surprising since new techniques make it clear that the lungs are not the sterile environments we once thought [ 427 , 434 , 438 , 439 ] . viral-bacterial coinfections can comprise 15 % of patients, while viral-viral (2-30 %) and bacterial-bacterial (1-7 %) are much less common [ 230 , 241 , 256 , 434 , 437 ] . hrsv or hrv is often co-detected with s. pneumoniae in urt samples [ 256 , 437 , 440 ] . hrv detections dominate in younger children with pneumonia during peak hrv seasons, although frequently in co-detections with other viruses [ 230 ] . acute bronchitis (less than 4-week duration in children) is defi ned as a sudden cough that often results from large airway infection and frequently involves viruses. croup or laryngotracheobronchitis (viral or recurrent [ 441 ] ) is a common lrt illness in children that includes the trachea and larynx as well as the larger airways, resulting in a barking cough. patients with croup most often have a viral infection with some role for hrvs, although the extent of this is unclear [ 441 , 442 ] . despite testing, a third of cases remain without a viral etiology [ 441 ] . tracheobronchitis resulted from some hrv-a15 infection of volunteers [ 46 ] . chest pain and cough have been reported in half or more of adults with hrv infection [ 207 ] as well as in children and adults with hrvs detected during exacerbations of bronchitis, with or without an associated ari [ 443 , 444 ] . bronchiolitis occurs seasonally, especially in winter, in infants (1-12 months of age), affecting the small peripheral bronchioles. winter is the peak season for hrsv circulation, but not usually for hrv. bronchiolitis is a clinical diagnosis encompassing various disease entities and is most often reported in association with detection of hrsv, a winter virus [ 445 , 446 ] . however, hrvs make up the majority of hrsv-negative bronchiolitis cases [ 128 ] , and hrvs are co-detected with hrsv for which hospitalization is prolonged compared to cases positive for either virus alone [ 447 ] . those children positive for an hrv during a clinically diagnosed bout of bronchiolitis have a signifi cantly higher risk of recurrent wheezing in the subsequent year than those in whom another virus is detected [ 446 ] . hrvs were reported in over fi vefold more cases of bronchiolitis than hrsv among patients in a 2-year prospective cohort of very low birth weight infants in buenos aires, argentina [ 448 ] . after a viral ari, some proportion of infections may be complicated by sinusitis (infl ammation of the sinus mucosa), the extent of which may be underestimated in children if the ari is mild and unattended by parents [ 11 ] . symptoms may include sinus pain, headache, facial pain, discolored nasal discharge, postnasal drip, cough, sore throat, malaise, and sometimes fever (more so in children) [ 367 , 449 ] . the precise role for viruses and bacteria in sinusitis is still unclear [ 450 ] . sinusitis is a common comorbidity in those with asthma [ 451 ] . the in situ presence of hrv-b14 rna in maxillary sinus epithelium was reported in seven of 14 adults with acute sinusitis [ 452 ] . hrvs were also detected by pcr in half of adults with acute maxillary sinusitis; half of the hrv positives were negative for any bacteria [ 65 ] . the common cold is often associated with computed tomographically confi rmed sinus cavity occlusion or abnormality in adults with self-diagnosed aris [ 367 , 453 ] . magnetic resonance imaging identifi ed reversible abnormalities of the paranasal sinuses in a third of healthy adult volunteers following challenge with hrv-a39 [ 454 ] . further evidence of the tropism of hrvs for sinus tissue comes from it being, so far, the only successful host for in vitro hrv-c replication [ 63 ] . culture-and serology-based testing has shown that virus infections in cystic fi brosis (cf) patients occur with the same prevalence as the general community, but the consequences of infection are more obvious or severe. these include deterioration of lung function, cough, increased expectoration and weight loss, and a synergistic increase in bacterial growth or acquisition of new bacterial infections [ 107 , 455 -458 ] . the mechanism behind the acquisition of new bacteria is still unknown and not always observed [ 459 ] , but may involve a reduction in the host's immune response or viral damage to the respiratory epithelium. there is circumstantial evidence that hrv infections have been associated with respiratory exacerbations in cystic fi brosis patients [ 459 , 460 ] , albeit in very low numbers by nonmolecular studies [ 461 ] and without a significantly different clinical outcome from non-hrv aris in these patients [ 107 ] . molecular methods have not yet been applied regularly, thoroughly, and systematically, but they generally fi nd hrvs to be prominent among cf children with ari-associated respiratory exacerbation and involved in mixed viral-bacterial infections [ 459 ] . hand washing and disinfectant wipes have been shown to be effective methods of interrupting transfer from fomites to the nose or to conjunctivae [ 87 , 288 , 293 ] . however, with eye rubbing, face touching, and nose-picking occurring frequently [ 47 , 290 ] , self-inoculation often outpaces personal hygiene, particularly in the young. hand disinfection is frequently recommended for prevention of hrv infection but has not been supported by controlled clinical trials in a natural setting [ 462 ] despite good results in experimental tests [ 463 ] . ethanol-containing disinfectants were more effective than simple hand washing with soap and water for removal of hrv-a39 inoculum, as assessed by culture, and the inclusion of organic acids afforded a residual antiviral effect [ 463 -465 ] . however, continual hand washing with extra ingredients resulted in skin irritation [ 462 ] . the experimental testing [ 463 , 464 ] may have been biased by short study periods, the absence of a mucus carrier to mimic natural surface deposition and overly stringent control over virus application/hand disinfection compared with the natural study. additionally, the natural setting study used pcr [ 462 ] which detects hrvs more often than culture. the disparity between outcomes may also refl ect the contribution of airborne hrv transmission. because of the absence of a vaccine or specifi c antiviral, the most popular method of intervention in uncomplicated hrv aris is treatment of the symptoms. this is achieved using analgesics, decongestants, antihistamines, and antitussives. due to a lack of studies, data are limited on the effectiveness of over-the-counter common cold medications for children [ 466 ] . anticholinergic agents have proven useful to reduce rhinorrhea [ 467 ] . for controlling symptoms in those with exacerbated asthma, most of which do not require hospitalization, bronchodilators and oral corticosteroids are the main treatments [ 468 ] . the interruption of proinfl ammatory immune responses or specifi c signaling pathways using steroids, or other novel therapeutics, may prove to be a more robust approach for treating hrv infections; they have not been successful for hrsv [ 325 ] . when initiated early in the illness, a combination of antiviral (ifn-α2b) and anti-infl ammatory (chlorpheniramine) components showed promise for interrupting nasal viral replication and symptoms [ 469 ] . antiviral agents (table 29. 3 ) require early application to effectively precede the pathogenic immune response to hrv infection [ 325 ] , but they often fail to reproduce their in vitro successes in vivo. most antirhinoviral drugs are based on capsid-binding agents (fig. 29.11 ) . additionally, oral delivery can complicate drug safety because this route increases the risk of systemic side effects compared to a nasal or topical route, but these risks must be considered alongside the disease to be treated; drug side effects are disproportionately severe compared to a common cold than to a severe asthma exacerbation. a systemic route is benefi cial if an effect is sought on hrv replication sites that are otherwise inaccessible, such as those not associated with respiratory tract illness [ 470 ] . the recent discovery of the new species, hrv-c, has shone a bright light on how little was known about the hrvs. the hrv-cs and also the newly discovered hrv-as and hrv-bs are fastidious in culture, with a single report of hrv-c growth in primary sinus tissue, and the identity of a cellular receptor still unknown. thus, it is diffi cult to proceed in many areas, including basic virology, seroepidemiology, immunobiology, and antiviral testing. determination of the receptors for these new hrvs would aid the search for a more accessible culture system. there would be great interest in a vaccine for some or all of the hrvs, but with increasing evidence of the interactions between hrvs, their hosts, and other respiratory viruses, it may not be wise to interfere before we fully understand what the impact of losing a constantly circulating hrv challenge would be. antivirals specifi cally targeting the hrvs may be a better bet, but routine hrv testing and genotyping will fi rst need to be more widespread as surveillance for antiviral resistance will be an important component of monitoring the success of any intervention. studies to determine whether there are differences in clinical and immunobiological impact between the many different types are lacking but would greatly improve our ability to plan future routine testing, understand all the clinical responses to the diverse hrvs and to outbreaks of ari, and improve hrv epidemiology. it is interesting to note that the hrv-bs are signifi cantly underrepresented 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the cold viruses do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? human rhinoviruses: the cold wars resume proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types cold wars: the fi ght against the common cold acknowledgments we wish to sincerely thank the following for valuable discussions: john upham, anne chang, danielle wurzel, michael nissen, ron turner james gern, and stephen b. liggett. we are grateful for the extreme patience of corin and ronan mackay, slightly less so for their frequent provision of our fi rsthand experience in hrv clinical symptoms. key: cord-186405-f9m3e31q authors: ortenzi, valerio; cosgun, akansel; pardi, tommaso; chan, wesley; croft, elizabeth; kulic, dana title: object handovers: a review for robotics date: 2020-07-25 journal: nan doi: nan sha: doc_id: 186405 cord_uid: f9m3e31q this article surveys the literature on human-robot object handovers. a handover is a collaborative joint action where an agent, the giver, gives an object to another agent, the receiver. the physical exchange starts when the receiver first contacts the object held by the giver and ends when the giver fully releases the object to the receiver. however, important cognitive and physical processes begin before the physical exchange, including initiating implicit agreement with respect to the location and timing of the exchange. from this perspective, we structure our review into the two main phases delimited by the aforementioned events: 1) a pre-handover phase, and 2) the physical exchange. we focus our analysis on the two actors (giver and receiver) and report the state of the art of robotic givers (robot-to-human handovers) and the robotic receivers (human-to-robot handovers). we report a comprehensive list of qualitative and quantitative metrics commonly used to assess the interaction. while focusing our review on the cognitive level (e.g., prediction, perception, motion planning, learning) and the physical level (e.g., motion, grasping, grip release) of the handover, we briefly discuss also the concepts of safety, social context, and ergonomics. we compare the behaviours displayed during human-to-human handovers to the state of the art of robotic assistants, and identify the major areas of improvement for robotic assistants to reach performance comparable to human interactions. finally, we propose a minimal set of metrics that should be used in order to enable a fair comparison among the approaches. object handovers: a review for robotics v. ortenzi 1, 4 , a. cosgun 2, 4 , t. pardi 3 , w. chan 2 , e. croft 2,4 , and d. kulić 2, 4 abstract-this article surveys the literature on human-robot object handovers. a handover is a collaborative joint action where an agent, the giver, gives an object to another agent, the receiver. the physical exchange starts when the receiver first contacts the object held by the giver and ends when the giver fully releases the object to the receiver. however, important cognitive and physical processes begin before the physical exchange, including initiating implicit agreement with respect to the location and timing of the exchange. from this perspective, we structure our review into the two main phases delimited by the aforementioned events: 1) a pre-handover phase, and 2) the physical exchange. we focus our analysis on the two actors (giver and receiver) and report the state of the art of robotic givers (robot-to-human handovers) and the robotic receivers (humanto-robot handovers). we report a comprehensive list of qualitative and quantitative metrics commonly used to assess the interaction. while focusing our review on the cognitive level (e.g., prediction, perception, motion planning, learning) and the physical level (e.g., motion, grasping, grip release) of the handover, we briefly discuss also the concepts of safety, social context, and ergonomics. we compare the behaviours displayed during human-to-human handovers to the state of the art of robotic assistants, and identify the major areas of improvement for robotic assistants to reach performance comparable to human interactions. finally, we propose a minimal set of metrics that should be used in order to enable a fair comparison among the approaches. index terms-human-robot interaction, object handover. r ecent years have witnessed the industry progressing towards a more direct collaboration between humans and robots. the current trend of industry 4.0 envisions completely shared environments, where robots act on and interact with their surroundings and other agents such as human workers and robots [1] , [2] , enabled by technological advances in robot hardware [3] . the recent covid-19 pandemic has also increased the demand for autonomous and collaborative robotics in environments such as care homes and hospitals [4] , [5] . accordingly, human robot interaction (hri) is featured prominently in the robotics roadmaps of europe, australia, japan and the us [6] [9] . the advantages of human-robot teams are multifaceted and include the better deployment of workers to focus on high manipulation and cognitive skill tasks, while transferring repetitive, low skill, and ergonomically unfavourable tasks to robot assistants. effective deployment of robotic assistants can improve both the work quality and the experience of human workers. the structured nature of traditional industrial settings has eased the use of robots in work cells. however, a similarly successful presence of robots is yet to occur in unstructured fig. 1 . example of a direct handover where a robot partner passes a bottle of mustard to a human. as both hands are in contact with the object, this picture shows the physical exchange phase of a handover. we review the literature on the cognitive and physical aspects of object handovers. environments (i.e., in factories without work cells, in households, in hospitals). for such places, robots need a better understanding of the tasks to perform, a robust perception system to detect and track changes in the surrounding dynamic environment and smart, adaptive action and motion planning that accounts for the changes in the environment [10] . human-robot collaboration and human-robot interaction are frequent keywords in our research community. we refer the reader to [3] , [11] for reviews on physical collaboration and to [12] , [13] for an overview of the cognitive aspects. our community has seen an increasing focus on collaborative manipulation tasks [14] [16] . in this context, robots must be capable of exchanging objects for successful cooperation and collaboration in manipulation tasks, as in fig. 1 . for example, consider an assembly task where a human operator has to assemble a complex piece of furniture and requires a tool. the robot assistant should be able to fetch and pass the tool to the human operator. or consider a service robot handing out flyers to passersby [17] or serving drinks [18] . a further example can be a mechanic asking for a tool while under a car: in this scenario, the motion range of the mechanic is extremely limited and extra care is needed to pass the tool [19] . the action of passing objects is usually referred to as an object handover. more formally, an object handover is defined as the joint action of a giver transferring an object to a receiver. despite being a frequent collaborative action among humans, a handover is a concerted effort of prediction, perception, action, learning, and adjustment by both agents. the implementation of a human-robot handover that is as efficient and fluent as the exchanges among humans is an open challenge for our community. in this paper, we review the state of the art of robotic object handovers. in particular, we investigate the aspects of the handover interaction that in our opinion require the most effort to enable a more useful and successful functioning of robots, especially in unstructured environments. we start this paper with a review of the main findings about human-human handovers in section ii. then, in the following two sections, we refer to each of the two phases of a handover: pre-handover, and physical handover. in section iii we focus on the reasoning and actions of the giver and receiver before the physical exchange of the object, analysing aspects such as communication, grasping, and motion planning and control. section iv describes the physical exchange of the object, focusing on aspects such as grip modulation and object release. section v reports a comprehensive list of quantitative and qualitative metrics that are commonly used for assessing handovers. we conclude this review with a discussion identifying directions for future work in section vi. we further propose a minimal set of metrics to adopt in experimental protocols in order to enable a fair comparison among the different approaches. a handover is a joint action between a giver and a receiver. joint actions are formally defined as [20] any form of social interaction whereby two or more individuals coordinate their actions in space and time to bring about a change in the environment . . . successful joint action depends on the abilities (i) to share representations, (ii) to predict actions, and (iii) to integrate predicted effects of own and others actions. joint actions are much more complicated than individual actions. social context is shown to modify the plans of actions of an agent [21] . while there is still much to understand and learn about how humans coordinate to meet their final goals, a number of scientific results shed some light on how humans behave during such actions. a minimal architecture for a joint action should include representations, processes like monitoring (feedback) and prediction (feedforward), and coordination [22] . humans tend to form representations of their own goals and tasks, and potentially also of their partners' goals and tasks. then, two processes use those representations: monitoring and prediction. monitoring is a process to check the advancement of those tasks and goals. such feedback can be on one's own task, on the task of the other agent [23] and on the overall goal. predicting the outcome of one's own actions and possibly, the other agent's actions, helps the coordination between the agents. agents are interested in predicting: the what, i.e., the actions of the other and their goal; the when, i.e., the temporal coordination [24] ; and the where, i.e., the spatial distribution of common space [25] . shared representations help to predict the other's actions and achieve higher coordination, integrating the what, when and where. coordination is also increased through joint attention (thus sharing perceptual inputs) [20] . in particular, research has shown that there seem to be similar eye motor programs when performing and observing the same scene [26] , [27] , thus reinforcing the link between perception and action. more recently, a dyadic motor plan was proposed in [28] . this plan highlights the possibility of joint actions to be based on active prediction of not only the actions of the partner, but also of the effects of the actions of the partner, in a deeper effort of prediction. to summarise, during joint actions humans tend to plan their motions considering the partner's needs and representing and predicting the partner's actions and their outcomes [28] , [29] . for this reason, scientists argue that humans form shared representations of the task to better predict each other's movements and to act accordingly [30] . efficiency and social cohesion are also listed as reasons to adopt such shared representations. coordination is extremely important for the success of a joint action. there are two types of coordination [31] : planned and emergent. planned coordination emerges from the representations of the desired outcomes and one's own tasks and goals. emergent coordination is independent of joint plans, and emerges from perception-action couplings. entrainment is an example of emergent coordination: two people in rocking chairs involuntarily synchronise [32] , [33] . note that, entrainment requires adaptation by both agents to happen. it was found that such synchronisation does not emerge in the interaction of a human with a non-adaptive robot [34] . considering these two types of coordination mechanisms, a joint action such as a handover requires the synergetic harmony of planned coordination for the final goal, and emergent coordination for the real-time aspects of the interaction. from this perspective, an object handover is a joint action where two agents collaborate to accomplish the transition of the object from one agent, referred to as giver, to a second agent, referred to as receiver. while the two agents share the overall goal of the object transfer, the objectives of the two agents differ during the interaction [35] . the giver aims to: most appropriately present the object to the partner; hold the object stably till the completion of the physical handover; and finally, release the object to the receiver as safely as possible. conversely, the receiver aims to: acquire the object by grasping; stabilise the grasp on the object; and finally, following the handover, perform the task the object was required for. it is crucial to remember that, in most cases, the object is passed in order to have the receiver perform a certain task. this task might be as simple as to place the object on a table (thus imposing loose constraints on the use of the object); or it might be more complicated, such as turning a key in a keyhole or cutting a piece of paper with a pair of scissors. while these tasks are frequently actualised in our everyday life, they require an appropriate utilisation of the object, i.e., they impose severe constraints on the use of the object. in the case of cutting with scissors, the scissors have to be grasped by the rings of the handles, and the blades must be used to perform the cutting. the giver should consider the subsequent task that the receiver would perform with the handed over object, in order to facilitate the task of the receiver [36] . a handover can be divided into two phases [35] , [37] , [38] . we use the tactile events, control discontinuities, and transitions that characterise any manipulation, to detail each phase [37] . pre-handover phase includes the explicit and implicit communication between agents, as well as the grasping and transport of the object by the giver. the first contact of the receiver's hand on the object begins the physical handover. this phase comes to an end when the giver removes their hand from the object and the object is fully in the hold of the receiver. therefore, we divide a handover into two phases: a pre-handover phase, and the physical handover phase. during these phases, the agents display different levels of activity, with respect to their own tasks and objectives, as shown in fig. 2 . two conditions define the start and the end of a handover. a handover can be initiated by the need of an agent to obtain an object to perform a certain task (handover by object request). this agent becomes the receiver and requests the object from the giver. the mechanic under a car asking for a tool is a typical example of this type of initiation. another example is a cook that asks the sous chef for a kitchen tool. alternatively, a handover can be initiated by an agent asking another to perform a certain task with an object (handover by task request). this agent becomes the giver and gives the object to the receiver. for example, while tidying up a room, an agent can pass an object to another agent in order for the latter to place the object in a certain location; another example is a chef asking the sous chef to stir some sauce on a pan by offering the appropriate kitchen tool. once the exchange is initiated, the giver offers the object to the receiver. the physical exchange of the object can be direct or indirect. the object is passed from the hand of the giver to the hand of the receiver during a direct handover. in a number of situations, as in the example of the mechanic asking for a tool from under a car, a direct handover is also the most immediate solution to pass the requested tool. alternatively, the object might be placed by the giver on a surface, e.g., on a table, during an indirect handover. indirect handovers allow a greater flexibility to the receiver in terms of the timing and of the grasp used to obtain the object. however, direct handovers can reduce the effort of the receiver in terms of motions required to obtain the object [39] . in this paper, we focus on direct handovers because almost all the works in this field belong to this kind of exchange type. the physical phase of the handover terminates when the receiver has fully obtained the control of the object. at this stage, the receiver progresses to performing the task that initiated the handover. the next two sections focus on action and cognition during each of the two phases of the exchange: the pre-handover, and the physical handover phases. in particular, we will bring attention to aspects such as motion planning and control, prediction and communication, object grasping and offering, and modulation of grip forces. as we discussed in the previous section, a handover is initiated either by the request for an object or by the request for a task. in both circumstances, the request for an object or the request for the task must be communicated to the other agent. communication is a foundation for every joint action, and it can occur in various manners. humans display a wide array of communication skills to help coordinate the what, when and where of a handover. gaze and oral cues are two common ways for the agents to communicate during this phase. communication does not happen only directly, as in for example voicing the intent to pass an object; but also during the action: such as motions or gestures during the pre-handover phase, where a giver clearly displays their intent to hand over the object. similarly, the way an object is grasped and offered often presents cues on the intent to hand over. in section iii-a we will review the most important findings on communication. once initiated, a handover enters the preparation phase that leads to the physical exchange of the object. in preparing to offer the object, the giver predicts how the receiver would perform the task the object is being passed for, and given its predictions, how the receiver would want to grasp the object. using these predictions, the giver plans motions to obtain (grasp) the object if not yet grasped, or (if needed) to re-grasp the object to best prepare for the exchange, and then to offer it to the receiver. the giver relies on visual and tactile feedback to perceive and track the object as well as the state of the receiver, i.e., both the position and whether they are ready to receive. during this time, communication signals are constantly exchanged between giver and receiver. the giver then uses this sensor feedback to adjust their motion plans, coupling this feedback with updated predictions of the receiver's behaviour. these updates and adaptations aided by prediction, perception and learning are used to control the motions realised to grasp the object and to offer the object to the receiver. we delve deeper into grasping and motion planning in sections iii-b and iii-e, respectively. the receiver shows lower activity in this phase. however, the receivers actions and communication are perceived by the giver, therefore influencing the givers actions. attention and state of preparedness of the receiver are important as they communicate the readiness to receive. similarly to the giver, the receiver also predicts the behaviour of the giver, and forms a plan of action. the receiver may move their hand towards the predicted handover location in anticipation of the handover. the receiver's plan and actions are updated using sensor feedback such as vision, touching and hearing. the receiver's plan and actions are also dependent on the subsequent task that the receiver would perform with the object. at the end of the pre-handover phase, the receiver has reached for and made contact with the object. communication is crucial in any joint action. signalling strategies (i.e., communication) aid coordination by improving the partner's prediction of one's actions (thus minimising uncertainty) [40] . in particular, communication is used to initiate the action, i.e., to show the intent to start with the action; and then to coordinate the action once it has started [22] . humans are extremely skilful in communicating their intent (the what, i.e., the action to perform and the object to pass) and expressing cues about the when and the where of a handover [41] . communication is so important that a handover can be thought of as a physical process (approach, fig. 2 . timeline of an object handover. for each phase of the handover, this figure describes the giver's and receiver's activities; and the giver's and receiver's tasks. the initiation occurs either by task request by the giver, or by an object request by the receiver. during the pre-handover phase, both agents prepare for the physical handover. after the receiver has taken full control of the object, the task that initiated the object transfer can be performed. reach, transfer) and a cognitive process to establish what, when and where to pass [41] . these findings indicate that robots also require such communication skills and adaptation capacity in order to match human performance during interaction with a human partner. speech 1 can be used to express the intent to hand over 1 interestingly, there is evidence for the embodiment of language, i.e., that the motor system is activated during the comprehension of the language [42] . moreover, there is further evidence of the involvement of the motor system in processing action words such as "kick", "pick". however, it is not clear yet if this activation is due to the real processing of the action words or rather it is a by-product of imagining the action [43] . an object as well as to coordinate the actions during the exchange. as we discussed earlier in this section, speech can be used to initiate the action by either one or both of the agents, and language use could be considered as a form of joint action per se [44] . thereby, much information can be conveyed to a partner through speech. for example, a robot and a human could have a dialogue to decide their roles during an interaction, and then to coordinate actions [45] . however, the use of speech can also degrade coordination during a joint action when the partners' attention is divided between multiple modalities of sensory communication (visual and auditory in [46] ). in addition to speech, humans use a number of other ways such as body stance and position, arm pose, gestures (with arm and/or hand), and gaze to communicate their intent to hand over an object and when/where the handover will take place. the presentation of an object, such as an extended arm and offering the object such that the free part is towards the receiver and tilting the object towards the receiver, are configurations that convey intent to pass an object [47] , [48] . cakmak et al. [48] claim that such anticipation in the behaviour of the agents makes the interactions more fluent. an analysis of kinematic features could lead to an automatic detection of the intent to hand over an object, for example using machine learning classifiers [49] . another learningbased approach presented in [50] posits that the orientation of a person and joint attention (on the object or on the position where the handover will happen) are important cues for physical interaction. similarly, statistical models have been used to model the physical aspect of a handover, and then endowed with a higher-level cognitive layer that uses nonverbal cues (head orientation) to better understand the intent of a human receiver to grasp the object [51] . alterations of more common movements and arm trajectories can also be used to communicate during joint actions as well as a deviation in the normal pronunciation of a word [40] . trajectories of motion can be altered in order to communicate to one's partner [40] . taking this to the extreme, some movements can be fabricated altogether in order to mislead one's opponent in a competitive joint action, e.g., a footballer's feint move [52] . similarly, robots can devise deceptive motions too [53] . moreover, the initial pose of a robot receiver can inform the human giver about the geometry of a handover [54] . we will focus more on the gestures and motions as action in section iii-e. gaze is also a very powerful tool for communicating the intent to act and for coordinating the action. gaze is the ensemble of eyes, head, and body orientation that reacts to the joint action [55] . human gaze supports the planning of actions of object manipulation, spotting positions (contact points) to which to direct a grasp [56] . furthermore, there seems to be a link between action perception and execution. in other words, humans are able to read other people's action intentions by observing their gaze [57] . therefore, it is not surprising that during a handover, the use of gaze by a robot positively impacts the interaction, resulting in faster object reaching and a more natural perception of the interaction by the human receivers [58] [60] . similarly, gaze can have an effect on cooperation also in terms of faster human response times [61] . interestingly, a deliberate delay in releasing the object by the robot results in an increase of attention to the robot's head, and also an increase of the compliance with the robot's suggestions (actualised with the robot's head motions) [62] . a closely related concept is turn-taking, which helps humans communicate their understanding and control of the turn structure to a conversation partner by using speech, eye gaze, and body language. turn-taking has been explored in human-robot interaction [63] , and it would especially be beneficial for handovers if the scenario involves handovers in both directions: robot-to-human and human-to-robot. we have previously considered that during a handover, the giver plans their motions considering the task of the receiver. in particular, the giver considers how to grasp the object so as to offer it to the receiver in the best way possible, i.e., whenever possible, to minimise object manipulation by the receiver before using the object for its intended use [64] . this is an example of second-order planning for object manipulation, which is defined as: ... altering one's object manipulation behaviour not just on the basis of immediate task demands but also on the basis of the next task to be performed [65] . if the planning takes into account more than two steps, then it is termed higher-order planning. in the case of a handover, the grasp of the giver could also account for the task to be performed by the receiver [36] . in effect, the grasp of the giver influences the grasp of the receiver, as the latter can only grasp the object on the unencumbered portion of the object. the grasp choice of the giver can influence whether a receiver can directly use the object for their task or must re-manipulate the object to be able to use it. factors such as object shape, object function and safety are important to consider when planning a grasp for a handover [66] . in a human user study, it was shown that when participants are handing over objects to each other, they tend to orient some objects differently when they were explicitly asked to consider the presentation that is most convenient to the receiver [67] . similarly, object constraints and the receiver's task are highlighted to be key factors in the choice of grasp by the giver [68] . in particular, grasp type and grasp location change to facilitate the grasp of the receiver on the object. similar reasoning was already adopted for robot to human handovers in [69] , [70] . however, the robotic giver acted knowing a priori the 'appropriate' parts of the objects and the human receiver did not have to perform any subsequent task with the objects. learning by demonstration was proposed by the same authors as a possible method to further explore the semantic segmentation of objects for grasping [71] . similar to this work, learning handover grasp configurations through observation of human behaviour has been shown to be a viable solution [72] . using the concept of affordance axis, a method has been proposed for selecting good handover observation sets to learn grasp configurations [73] ; however, while this works well with objects with one main grasp configuration, it is a more challenging problem when the object can be presented in multiple orientations, as the robot needs to see a larger set of possible configurations and then decide which is best in a given situation. the grasping adaptation performed by the giver is in line with theories that consider grasping an inherently task-oriented or purposive action in humans [74] [76] , that involves both sensory and motor control systems [77] , [78] . a human study shows that when participants took hold of a vertical cylinder to move it to a new position, grasp heights on the cylinder were inversely related to the height of the target position [79] , which is a clear example of adaptation of the grasp to the task. humans display a wide range of grasps, and several taxonomies have been proposed to categorise human grasps based on specific aspects such as hand shape on the object, contact points, and pressure [80] [82] . humans choose their grasp considering many factors [80] , [83] [85] : object constraints (e.g., shape, size, and function), gripper constraints (e.g., the human hand or gripper kinematics and the hand or gripper size relative to the object to be grasped), habits of the grasper (e.g., experience and social convention), and environmental factors (e.g., the initial position of the object and environmental constraints [86] ). while a successful grasp is usually characterised by stability [87] and/or speed [88] , one aspect of robotic grasping that is often overlooked is the task to perform [89] and its requirements in terms of force and mobility [90] . there is further evidence that the reaching movement of the arm and the grasping movement of the fingers may also be influenced by the graspers goal [91] [94] . from this perspective, it is not surprising that the intention to cooperate influences the grasp choice during an interaction like an object handover. as objects are passed to accommodate the receiver's needs, the functional parts of the objects play a decisive role [95] [101] . gibson [95] coined the term "affordances" to define the possibilities for action offered by objects and their environment. norman [102] added a perceptual dimension to the concept of affordance, associating it not only to the agent's capabilities, but also to their tasks to perform. however, a clear functional part of an object, such as a handle of a screwdriver, can elicit different behaviours in single-agent scenarios and cooperative tasks [68] , [103] , [104] . for example, a single agent having to tighten a screw will grasp a screwdriver from the handle, whereas a giver wanting to hand over the screwdriver, should grasp it from the metal rod, thus offering the handle to the receiver. while this adaptation is natural to humans (having developed it through understanding and the repetitive use of the object), such understanding is somewhat still to be achieved in robots. a concerted effort in perception and action [105] is needed in order to endow robots with such capabilities [106] [113] . as already established, givers do reason about how to grasp the object and where to place their hand on the object. givers consider which area of the object can afford the receiver's subsequent task and adapt their grasp strategy accordingly. so much so that when the task of the receiver has fewer performance constraints, i.e., when the task of the receiver is as simple as placing the object on a table, there are less stringent constraints to perform the task and thus the exchange of the object can be more relaxed [68] . however, when the task of the receiver requires the use of the object in a very specific way (i.e., cutting a sheet of paper with a pair of scissors), then the grasp of the giver usually accounts for the constraints of the task of the receiver [68] . similarly to the considerations about the grasp of the giver, different tasks and objects elicit different levels of constraints on the grasp that the receiver has to use. a planner for interactive manipulation tasks between robots could potentially account for both the grasp of the robotic giver and the grasp of the robotic receiver, thus enabling both robots to grasp successfully [114] . this approach is hardly extendable to human-robot handovers, as the human behaviour is more difficult to model with certainty. to overcome this problem, one option is to probabilistically model the behaviour of the human receiver, accounting for the ergonomic cost of the receiver, and thus influencing the grasp of the receiver [115] . a big challenge in handovers is a reliable perception of the object, the hand (self and partner) and the partner's fullbody motion. some approaches try to track object and hand to plan for the grasp [116] [118] , leveraging large datasets for training and physical relationships between hand and object. while grasping, the hand and objects can become severely occluded, thus harder to track with vision sensors. differently, this problem can be addressed as a grasp classification problem [119] , in which common human grasps for the task of humanrobot handover are divided into categories such as "waiting" or "lifting", inspired by the human grasp taxonomy [82] . the grasp class information can then be used by a planner to devise the most appropriate approach and grasp strategy for the robot receiver. however, the classification of grasps suffers the drawback of detecting only a relatively small subset of grasps, thus failing to detect the richness of behaviours displayed by humans. the human body can also be tracked in addition to the object and the human hand in order to improve safety [120] . we will discuss the safety aspects of handovers in more detail in section iii-e2. while the perception of the human partner's hand and body is critical real-time feedback, there have been efforts also in predicting the human partner's motion. dynamic movement primitives (dmps) [121] , [122] have been used successfully to predict human motion (point attractor and time scale, which mean handover location and time), coupled with an extended kalman filter [123] . real-time estimation of human motion can also leverage the concept of minimum jerk trajectories [124] . the minimum jerk model can be used in conjunction with regressors to predict when and where a human giver will transfer an object [125] . the minimum jerk model is used with a semi-adaptable neural network to predict human arm motion in [126] . gaussian processes can also be used for proactively estimating human motion for handovers [127] . luo et al. [128] propose a 2-layer framework using gaussian mixture models and gaussian mixture regressor to represent and predict human reaching motions. the handover must occur in a location that is reachable by both agents. thereby, an aspect that deserves thorough analysis is the handover location. human-human handovers have been shown to occur roughly midway between giver and receiver [129] . thus, the interpersonal distance between the agents has a fundamental influence on the location of the handover, and on the height of the point of exchange [130] . conversely, the object mass seems not to affect the location of the exchange, but rather the duration of the exchange. leveraging on this notion, a task-specific interaction workspace can be built as the intersection of the spaces that can be accessed by robot and human [131] . information such as the effort needed by the human to reach a certain location can be used in an on-line manner to shape the interaction workspace, in order to plan the robot's movements. similarly, handover locations can account for biomechanical properties of the human receiver, such as height, weight, strength and range of motion [132] . these considerations of the biomechanical properties of the human partners are especially critical when there are environmental or task constraints to limit the motion of the human (like in the case of the mechanic under the car) and when the human is motor-impaired. furthermore, optimising the robot's motions over safety, acceptability and task constraints could help improve the posture of the human receiver [133] , thus decreasing the chances of musculoskeletal disorders and discomfort [134] . the human mobility could also be accounted for while planning, to devise different paths for the robot to the handover location [135] , [136] . incorporating models of the kinematics and the dynamics of the body of the human receiver can effectively devise handover locations that are more acceptable to the human partner [137] . finally, the human arm manipulability could also be embedded in an optimisation framework to reduce muscular strain [138] , [139] . all these considerations point to the fact that the handover location should not be fixed a priori, which would force the human to adapt to the robot. instead, such location should be planned for considering all the factors mentioned above, and then potentially modified in an on-line fashion if need be, for example, if the environment is dynamic and the human agent moves, thus adapting to the human. during a joint action, the movements of the agents simultaneously actualise the physical joint action and signal important information for the coordination. however, movements are importantly the actualisation of a handover. movements during human-human handovers are generally smooth rather than being separate and successive phases [140] . for example, receivers usually start the reaching movement toward the givers while the giver reaches out for the receiver (in a concurrent motion) [141] , [142] . as such, the dominant aspects of successful movements in the context of a joint action like a handover are: legibility, predictability, safety, robustness, reactivity, and context awareness. 1) legibility and predictability: legibility and predictability relate to how easy it is for one agent to understand and predict the other agent's movements. albeit similar, legibility and predictability are not synonyms [143] . using a psychological interpretation of actions, legibility is a characteristic of motion that enables an observer to infer the goal (actionto-goal). on the other hand, predictability is a characteristic of motion that matches what an observer expects given the knowledge of the goal (goal-to-action). by this definition, motions of collaborative robots must be legible, thus allowing the partner to quickly and reliably predict the goal of the actions of the robot. interestingly, humans prefer robot configurations that are more natural or human-like as they are more readable [144] . inverse kinematics algorithms mapping cartesian motions to the robot's joint space can also aim at devising overall movements for the robot that are legible to the human partner [145] , [146] . 2) safety: the safe planning of motions while approaching a human partner is also a critical aspect during a joint action. in the context of a handover, safety is a multi-faceted concept that revolves around the physical safety of the human partner. safety can be ensured (or achieved) through software and/or hardware [147] , [148] . more generally, safety is a pivotal topic in the whole human-robot interaction field of research. research 2 has led to the standard iso/ts 15066:2016 that regulates collaborative robots and contains the norms of appropriate behaviour during physical human-robot interaction. collaborative robots promise an intrinsic safety by ensuring the hardware is safe to interact with. furthermore, safety can also be increased through software [147] , [148] . motion planning and control should be framed in a way that safety risk is minimised throughout the entire interaction. some approaches proposed danger criteria and attempted at minimising this metric. for instance, in [149] the robot is kept in low inertia configurations in case of unanticipated collisions; moreover, the chance of collision is reduced by distancing the robot's centre of mass from the human. similarly, a metric of distance from the operator is used in the optimisation in [150] , and safety barrier functions are built around the robot links to allow collision-free planning [151] . motion planning should devise safe, reliable, effective and socially acceptable motions [152] , [153] . frontal approach versus lateral approach by the robot towards the human receiver is discussed with some contrast in [154] , [155] . such considerations are further used to develop the planner in [152] , which is composed of three components: spatial reasoning to account for the human receiver (perspective placement [156] ), path planning optimising over costs that account for safety, visibility and human arm comfort (humanaware manipulation planner [157] ), and trajectory control to ensure minimum-jerk motions at the end effector (soft motion trajectory planning [158] ). humans minimise jerk in order to realise well-behaved trajectories for arm movements [159] . minimum-jerk motions by a robotic giver also result in shorter reaction time and faster adaptation for human receivers [160] . further, to better match the human trajectories of minimum jerk, a decoupled minimum jerk trajectory could be used, using different time constants in the gravity axis z (thus decoupling the motion in the x-y plane to the motion in the z plane) [161] . 3) robustness, reactivity and context awareness: the robot's motions should be flexible to accommodate changes in the environment, and to accommodate behaviours of different partners. to this end, principles such as robustness, reactivity, and context awareness should guide the design of human-robot interaction systems [162] . from this perspective, a fully preplanned motion falls short of general adaptability. in other words, a fully deterministic approach to planning is only possible if the environment is fully known, as in the case of robot-to-robot handovers [163] . instead, a mixture of planned motions and control over sensory feedback aids to modify the motions and adapt to the partner. a switching planning mechanism that mixes global and local planning can help to overcome the drawbacks of fixed planned motions [164] . fast responsiveness of the robot giver is particularly important as it increases the positive impression of the interaction [19] . interestingly, a human study suggests that the speed of the interaction might be more important than the spatial accuracy of the robot for the subjective experience of a human receiver [165] . when the robot acts as a receiver, adaptive reaching displays better performance compared to a fully pre-planned reaching motion in terms of predictability and aggressiveness, both being important quality measures of a handover [166] . humans adapt their actions to account for the workload of their partner [167] . similarly, a robot should be aware of the task status [168] . for example, a more proactive robot giver could increase the speed of the handovers, negatively impacting the user experience. on the contrary, coordinating a reactive robot could be perceived as a better user experience, even if the performance deteriorates [167] . while pure planning usually devises a feedforward trajectory to follow, control architectures provide the means to use sensorial feedback and change the behaviours of the robot. impedance control and admittance control are two common strategies to use in physical human-robot interaction [169] [173] . variants of classical approaches include using redundancy and null space [174] [176] , modelling the interactive forces [177] , [178] and parameter adaptation [179] , [180] . early work on control proposes to use fuzzy logic on three aspects: relevance, confidence and effect [181] . human-human handovers show a smooth and fluid continuum of motion. for this reason, rather than switching control paradigms between handover phases, a phaseless controller (no distinction between reaching, passing and retracting) could be based on insights about the human behaviour, e.g., existence of motion during the passing and existence of coupling between the movements of the giver and those of the receiver [142] . however, one specific implementation of such a controller in [142] assumes that the object mass is known, in order to best modulate the grip forces. dynamic movement primitives (dmps) [121] , [122] represent an alternative to both pure feedforward and pure feedback control during an interaction. to specifically target a handover, the feedforward part can be weighted more at the start of the motion (shape-attraction), and subsequently the feedback (goal-attraction) can be weighted more as the interaction nears the physical exchange of the object [182] . in order to generate a wider range of behaviours during interactions, interaction primitives (ips) build on the framework of dmps and maintain a distribution over their parameters [183] . probabilistic motion primitives [184] are shown to allow a robot to recognise human intent (task) and at the same time, generate commands for a robot according to the observed human motions, achieving coordination [185] . in this way, planning is replaced by inference on the probabilistic model. learning from human feedback might also improve the adaptability of handovers. for example, in a contextual policy search, a robot could learn a reward function from human preference feedback [186] . this phase encompasses the physical interaction between giver and receiver and the object transfer. during this phase, both players are physically and cognitively engaged. entering this phase, the giver possesses the object thus controls its stability. after the occurrence of the physical contact, the giver can couple vision and force feedback to understand to which extent the receiver has grasped the object. at this point, the giver starts releasing the object in order to allow the full transition of the object to the receiver. the timing must be coordinated as an early release can cause the object to fall; and a late release can cause higher interaction forces [187] . the receiver approaches this phase by planning a grasp on the object given the visual feedback from the actions of the giver. given the presentation of the object, the receiver then acts and places the hand on the object to maximise the stability of the grasp and also in the most appropriate way to be able to perform their task afterwards. the transition ends when the giver entirely releases the object to the receiver, who then acquires the object in full. in this phase, the success of a handover is dictated by the coordination of the when and where of the joint action. for this reason, the most crucial aspect of this phase is the modulation of the grip force to complete a safe transfer of the object. in line with literature in neuroscience and psychology, the joint action of the physical handover is an interplay of anticipatory control and somatosensory feedback control [35] . visual feedback augments the anticipatory control in starting the release of the object, by predicting and detecting the collision created by the hand of the receiver on the object [188] . visual feedback is also used to adapt predictions to different speeds of the receivers reaching out movements. from this perspective, the speed of the grip force release seems to be correlated with the reaching velocity of the receiver (i.e., the faster the approach, the faster the giver releases the object) [188] . giver and receiver show similar strategies for controlling their grip forces with respect to the evolution of the load forces generated by the object and the exchange. compared to other schema, forces arising during the release are different when a robot acts as receiver. in fact, the faster the retraction of the robot after grasping the object (still in the partial hold of the human giver), the larger the interaction forces. this might be explained as the giver does not have enough time to withdraw [54] . all of these findings point to the fact that the giver is in charge of the safety of the object, while the receiver modulates the efficiency of the object exchange [35] , [187] . the task of the giver is shown to resemble the evolution of a picking up task [187] , [189] in that the giver, like the picker, typically will use excess grip force to ensure that the object does not slip or drop. moreover, in [187] a linear relationship between grip force and load force is observed, except when either actor is supporting very little of the object load [187] . an analysis of these grip forces reinforces the idea that the giver is responsible for the safety of the object during the transfer, while the receiver is responsible for the timing of the transfer. the same control strategy can also be applied to an under-actuated hand, using linear models leveraging force readings from the elbow of the robot [190] . moreover, the feedback from a force sensor mounted on the robot's wrist can be robustly used to modulate the release of an object [191] . another task for both the giver and the receiver is the handling of errors and disturbances during the handover. for example, there might be cases where the receiver makes unwanted contact with the object and the giver should not release the object. the contact forces exerted by the receiver should then be recognised as disturbances and should be compensated to maintain a stable grasp on the object. the tactile information from a shadow robot hand is used in [192] to build probabilistic models to detect these disturbances and feed them back to an effort controller. another threat to safety is a potential fall of the object. it has been found that human givers tend to primarily rely on vision rather than haptic sensing to detect the fall of the object during handovers [193] . thus, the object acceleration measured with an optical sensor at the gripper can be used as an indicator of handover failure (object dropping) [194] . more recently, force control and fuzzy control were similarly used [195] , [196] . there is a general consensus on the need for standardised measurement tools and metrics in the human-robot interaction and collaboration communities [197] , [198] . however, the spectrum of aspects to cover is so broad that finding a set of metrics and tools to adopt in every situation is very difficult. nevertheless, such common and codified metrics would allow for an easier and fairer comparison among the proposed techniques, and would possibly help to build new frameworks. metrics should aim to assess a handover qualitatively and quantitatively [197] . along the same lines, a survey on metrics for human-robot interaction [199] reports productivity, efficiency, reliability, safety and co-activity to be the areas to assess for an interaction. furthermore, there is a wide range of literature analysing metrics for human-robot interaction and collaboration, such as for human-robot teams [200] [203] and for social and physical interaction [204] [206] . in this section we analyse three different types of metrics: 1) task performance metrics which provide a measure of success, 2) psycho-physiological metrics to measure the human partner's physiological responses, and 3) subjective metrics in the form of user questionnaires. these metrics are represented graphically in figure 3 . we also analyse the variety of the test objects used in handover experiments. task performance metrics are often utilised in hri experiments to evaluate a measure of success quantitatively and the choice of such metrics is highly dependent on the task. 28 out of 38 papers surveyed reported a measure of task performance for the handover experiments; the full list can be seen in the "task performance" column in table i . the performance of a handover can be coarsely described using the success rate: number of successful handovers divided by the total number of trials. the success rate metric is the most popular task performance metric for human-robot handovers [17] , [19] , [39] , [41] , [48] , [51] , [119] , [120] , [166] , [182] , [185] , [207] . even though the overall success rate of an implementation is important, it only reports a statistical view of the handovers rather than the quality of the interaction, and by itself it does not explain why and how the errors have occurred. besides, different experimental protocols used by each research team make it difficult to compare the success rate metrics directly. the interaction force is another measure that has been commonly used to evaluate the success of the interaction [54] , [142] , [189] , [191] , [192] , [194] . considerations of performance also include the task completion time. from this perspective, fluency is an important characteristic of an interaction such as the handover. to evaluate fluency, objective metrics should include percentage of concurrent activity, human idle time, robot idle time and robot functional delay [208] [210] . these concepts are also related to task effectiveness and interaction effort [211] . moreover, time considerations can include the reaction time of the human, and also task completion time and overall handover time. among the surveyed handover papers, several have reported time-related metrics, including the wait time of the robot and the human [48] , total handover time [18] , [41] , [51] , [166] and timing of different phases of the handover [59] , [69] , [70] , [119] , [188] . other task performance metrics used in handovers include defining and minimising a cost function [115] , [135] , [185] , relating either to the trajectory or to the interaction. aside from the task performance metrics, another way to gather quantitative data from user studies is to measure the physiological responses of the human partner during the interaction. in hri, psycho-physiological measures can be used to identify and evaluate the human partner's responses to the interaction with the robot [212] . physiological signals such as electromyography (emg) can be used to measure the human's motor activity during the handover. physiological signals can also be used to estimate the affective state of a human partner during an interaction. furthermore, physiological responses can be exploited when evaluating responses to a safe planner (less anxiety and surprise, reported feeling more calm) [ pre-planned 1 2020 rosenberger [120] pre-planned 13 2006 kulic [213] 2009 bartneck [216] 2019 hoffman [210] heart rate variability (hrv), which can be used as a quantitative index to assess mental fatigue [217] . the psychophysiological measures to assess anxiety and stress in response to the interaction include, but are not limited to: eye movement; heart rate and heart rate variability; blood pressure; electroencephalography; skin conductance response; urinary tests; pupillary dilation; respiratory rate and amplitude; muscular activity; corrugator muscle activity; electromyography. our survey shows that only a few works used psychophysiological metrics in human-robot handover experiments. [138] and [132] utilised emg to measure the muscle activity in the human arm during the handovers while [214] , in addition to emg, also made use of galvanic skin resistance and eyetracking. subjective metrics assess aspects such as the subjective perception of the human regarding the perceived difficulty of the task, the cooperation and alliance of the robot, trust in the robot and contribution of the robot [210] . additional concepts that are recurrent in a qualitative evaluation of an interaction include anthropomorphism, animacy, likeability, perceived intelligence, and perceived safety [216] . the robotic social attribute scale (rosas) framework proposes measuring the subjective and social perception of robots using three dimensions: warmth, competence and discomfort [218] . legibility, safety and physical comfort are also key criteria to consider [214] . furthermore, ad-hoc questionnaires and the nasa-tlx 3 can be utilised to provide additional instruments to assess the cognitive workload of humans. the majority of the surveyed papers with a real-world handover implementation (22 out of 38) have conducted a user study (the list can be seen in the "subjective" column of table i ). the most common vehicle for user studies were poststudy surveys, in which the participants rated different aspects of their interaction in a likert scale. the most commonly asked questions in the questionnaires relate to the fluency of the interaction (i.e., natural, legible, predictable robot motions) [41] , [59] , [119] , [144] , [161] , [166] , [188] , [210] , [214] , how safe [69] , [70] , [161] , [182] , [214] and comfortable [19] , [54] , [69] , [70] , [161] , [165] , [182] , [214] the participants felt during the interaction, whether participants were satisfied with the experience [19] , [39] , [115] , [165] , [182] , [188] , the ease of use of the interface [19] , [39] , [115] , [165] , [182] , [194] , the competence of the robot [54] , [115] , [119] , [144] , [186] , the appropriateness of the robot's timing [41] , [59] , [166] , [188] , the perceived aggressiveness of the robot [41] , [166] , the trust in the robot [115] , [119] , [210] and whether the robot acted in a human-like manner [70] , [161] . in addition, for some papers the main subjective evaluation was the indication of preference and/or subjective opinions and comments from the participants [48] , [59] , [62] , [115] , [189] , [194] . there have been recent efforts in the grasping community to create physical benchmarks and experimental protocols in 3 https://humansystems.arc.nasa.gov/groups/tlx/ order to facilitate the replication of research results [219] [222] . towards the same goal, object datasets have been generated for grasping, such as ycb: an object dataset [223] [225] ; dexnet: a synthetic dataset of 6.7 million point clouds, grasps, and analytic grasp metrics [110] and egad: a dataset with procedurally generated objects with varying grasp difficulty [226] . the choice of objects used in human-robot handovers usually depends on the target application; for example it differs for industrial and domestic environments. the last column of table i shows how many classes of test objects were used for the experiments in the surveyed papers. we found that 26 out of 38 papers used only a single object class for the experiments. the most commonly used objects were cylindrical objects such as bottles [18] , [19] , [48] , [59] , [62] , [115] , [120] , [132] , [141] , [142] , [144] , [152] , [165] , [166] , [182] , [185] , [186] , [190] [192] , [214] , followed by rectangular objects such as boxes [119] , [120] , [161] , [166] , [167] , [185] , [191] , [192] , [194] , [207] . while some researchers opted for custom-designed objects with sensors mainly for measuring grip and load forces [51] , [54] , [189] , [190] , some have chosen application-specific objects such as handing out flyers [17] . in this paper, we analyse in detail the two phases of a handover, reviewing the results from human studies and the corresponding state of the art in the robotic literature. we summarise the salient features of the papers implementing physical human-robot handovers in table i . for each paper we report: the paradigm (robot-to-human or human-to-robot); what the authors investigated (communication, grasping, motion planning and control, and perception during the prehandover phase; grip force and error handling during the physical handover); whether the handover location was fixed, pre-planned accounting for aspects such as the ergonomics, or adapted online to the human partner; whether the experimental protocol included a post-handover task for the receiver; the metrics used to assess the task performance and the user experience; and finally the number of different objects used in the real robot experiments. studies in neuroscience, physiology and psychology highlight that a handover is an intricate joint action that requires a physical level and a cognitive coordination. in particular, it seems apparent that the cognitive level of the interaction is as important as the physical level [227] , for a robot to be considered as a partner, and not only as a tool [228] . to match the human skills of understanding and adaptation [207] , it is preferable that robots also display adaptation and understanding. in fact, human givers can control the object's position and orientation to facilitate the robotic receiver's grasping of the object [207] . however, fatigue of the human worker having to accommodate the robot repeatedly and for a long time can become an issue [139] . from this perspective, robots that are able to adopt different behaviours adapting fig. 3 . mind map of the metrics. metrics can assess not only the overall performance of a handover, with measures such as timing and success rate; but also the user experience, with psycho-physiological measures, such as heart rate variability, eye movement etc., and subjective measures, such as trust in the robot, working alliance and safety. to their partner could assist their human counterpart [19] , [167] . in other words, it is crucial to account for the feedback coming from the human partner during the interaction when controlling the robot. however, as can be seen in table 1 , to date most approaches focus on fixed or pre-planned handover locations, with far fewer attempts at adapting to the human partner online. in many human-robot handover scenarios, the handover location is kept fixed, i.e., the robot is either going always to the same position for the object transfer, or the handover location is pre-planned based on several criteria (including ergonomics, safety, etc) and not updated in realtime with the perceptual feedback. this is far from ideal, as the human has to potentially adapt to the robot and could incur cognitive and physical fatigue. the ergonomics of the interaction should be accounted for, as the transfer should happen in the comfort zone of the human, i.e., the range of positions (and tasks to perform in) reachable (and doable) with little or no compensatory movements [229] . in humans, an optimisation principle over a muscle stress index is shown to determine the arm motions and postures (selected over the infinite possibilities of motion) and also the perceived comfort [230] . we believe that while pre-planning such a location accounting for the ergonomics and the physical characteristics of the human partner is appropriate, more effort is needed in order to adjust to changing circumstances (adapting in realtime to the needs of the human partner). communication is a key factor to achieve a successful coordination during a joint action. humans use speech, gaze, and body movements to communicate intent and coordinate during the execution of the joint action. we observe that robots have displayed a general lack of communication skills for object handovers. most of the effort in the literature so far has been put on the physical aspects of the interaction, focusing on motion planning and control, grasping and perception. on the other hand, effort in communication is less prevalent, as only 37% of the papers in table i include an element of communication in their implementation. there are also only a few papers that focus on grip release and how to handle potential falls of the object. while the literature in human studies continues to investigate how both agents modulate their grip force on the object and how the different sensory modalities (vision and tactile) come into play, most of the reviewed work has adopted a simplistic approach, i.e., robotic givers completely release the object whenever a pull by the receiver is detected. conversely, robotic receivers need to modulate their pulling force as too little force could be unsafe for the object transfer and too much force could be dangerous for the human partner. we believe that grip force modulation is a key component that needs further investigation and effort. there are many additional open research directions, such as: (i) the use of different hardware (under-actuated vs fully actuated, soft vs rigid, parallel jaw gripper vs multifingered hands); (ii) the use of different grasping strategies (grasp type and location on the object); and (iii) the use of objects varying their size and weight. such exploration could thus give rise to various options to modulate the grip. in terms of the paradigm, robot-to-human (r2h) handovers have been more frequently investigated (87% of surveyed papers in table i ), compared to human-to-robot (h2r) (29%), while only 16% reported experiments in both directions. we speculate that the idea of having a robot assistant that can fetch objects and give them to humans when needed, has driven the deeper investigation of the r2h paradigm. the r2h paradigm is particularly representative of the cases where the human receiver will then perform a cognitively challenging task with the object, a task that robots are not yet able to perform. however, it is our opinion that h2r handovers are worth exploring more and represent an open area of research. one of the biggest challenges in human-to-robot handovers is safety [120] , as the robot should be careful to not contact the human giver. for this to happen, perception systems should be able to robustly discriminate the human giver (hand and arm) from the object [119] , [120] . moreover, in the h2r paradigm grasp planning becomes another critical issue, as the robot will have to perform a task with the handed-over object, i.e., at the very least need to put the object down in a pose preferable to humans [231] . from the robot's higher-level behaviour standpoint, there is a critical need for improvement in the integration of cognitive and physical reasoning [10] in both paradigms (h2r and r2h). in other words, robots currently lack a vision and understanding of the general goal of such an action. such understanding is the key contributor to enabling higher-order planning [36] , [64] , [65] , [68] . for example, robotic grasping has achieved peaks in performance [232] [233] [234] [235] ; however, the ultimate goal of the grasp is rarely taken into account [90] . as a result, robots can manage to grasp objects but seldom these grasps would allow the execution of a task with the objects. considering a handover, a successful grasp should account for the interaction partner and this is a key feature that a robot should display in order to be fully collaborative. following a similar reasoning, we believe that any experimental protocol should include a task to perform by the receiver with the handed-over object, as proposed in [68] . this is a critical consideration because the object exchange is normally initiated in order for the receiver to perform a task with the handed-over object. a complete experimental procedure should consider the capability of the receiver to use the object directly following the handover. if the receiver can grasp the object in a way that its subsequent use does not require any further in-hand or bi-manual re-manipulation, then the receiver can start the task straight away after the physical exchange of the object. conversely, the receiver might need to re-adjust their grasp of the object in case their temporary grasp (realised during the exchange) is not an ideal grasp to correctly use the object for the specific task. however, this post-handover grasp adjustment could decrease the quality of the handover in objective terms (longer task performance time, higher strain when the handover happens multiple times) and in subjective terms (the giver could be perceived as a lesser partner, and the task could be perceived as more cognitively difficult). these quality evaluations are pivotal in establishing the degree of success of a handover, as considered in the section v on metrics. however, very few experimental protocols include a posterior task for the receiver (18% of the surveyed papers). even though it might be argued that a handover can be considered finished after the object transfer, we believe that such task performance is important to effectively assess the overall performance of the dyad and gauge the experience of the human partner, particularly for the r2h paradigm. our survey has revealed a need for standardisation in the choice of metrics and objects for real robot experiments. most of the surveyed papers in table i report results using task performance metrics (e.g., success rate and timings) and subjective metrics on the experience of the human partner (often in the form of likert-scale post-experiment questionnaires). the last three rows of the table report papers that focus purely on metrics that have been used for handovers. we believe that a minimal set of metrics should be defined in order to enable a fairer and more direct comparison among the different approaches. to this end, we propose the following combination of metrics that assess the most common aspects of a handover: 1) task performance (objective): success rate, total handover time, receiver's task completion time. 2) experience of the human (subjective): fluency, trust in robot, working alliance. this minimal set includes metrics which are clearly defined, thus reproducible, and which are easy to measure. for these reasons, the set does not include psycho-physiological measurements as they require sensors placed on the body of the human participant, and thus are difficult to standardise and deploy in a variety of contexts. the experience of the human participant should be assessed administering the following questionnaire (the following set of questions includes a subset of questions from [210] ): 1) human-robot fluency • the human-robot team worked fluently together. • the human-robot team's fluency improved over time. • the robot contributed to the fluency of the interaction. • i trusted the robot to do the right thing at the right time. • the robot was trustworthy. • the robot accurately perceives what my goals are. • i understand what the robot's goals are. • the robot and i are working towards mutually agreed upon goals. all questions should be evaluated on a likert scale. we believe that this set of questions covers a broad set of important general aspects of the interaction, namely fluency, trust and working alliance. furthermore, additional questions can be added to this minimal set in order to investigate additional specific aspects of a handover, such as preference between different approaches, and learning/improvement over time. 68% of the surveyed papers in table i used only a single object class and only 10% of the papers used four or more object classes. this observation shows that generalisation of handovers to a variety of objects has not been the main focus of a majority of the papers until very recently [120] . the most commonly used test objects have been either cylindrical objects such as bottles (55% of the surveyed work) and rectangular objects such as boxes (26% of the surveyed work). this is likely because these object shapes are easier to grasp and many everyday objects belong to those categories. we support the practice of a broader set of objects, as different objects generate different behaviours and can be used to address different manipulation tasks. we propose the use of objects that elicit all three grasp macro-types in [80] , i.e., power, intermediate and precision grasps. the three macro-types offer sufficient opportunities to explore different behaviours, investigating aspects such as different object offering and reception; different post-handover tasks; and handover of objects with different weights and shapes. nevertheless, we acknowledge that the choice of objects might depend on the specific focus of each study. for example, studies on the reaching motion might place their focus on the motion and not on the objects, so three objects evoking the three 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ai challenges working collaboratively with humanoid robots the postural comfort zone for reaching gestures optimization principle determines human arm postures and "comfort learning to place objects onto flat surfaces in human-preferred orientations learning robust, real-time, reactive robotic grasping model-free and learning-free grasping by local contact moment matching computation of independent contact regions for grasping 3-d objects learning hand-eye coordination for robotic grasping with deep learning and large-scale data collection the authors want to thank the australian research council (centre of excellence for robotic vision (project number ce140100016)). elizabeth croft acknowledges support from australian research council (project number dp200102858) and the natural sciences and engineering research council of canada (project number rgpin-2017-04450). tommaso pardi is supported by a doctoral bursary of the uk nuclear decommissioning authority. the authors want to thank: peter corke for his invaluable feedback; alessandro de luca for the good advice about safety in physical human-robot interaction; marco controzzi for the discussion on the role of the task in experimental protocols. key: cord-018017-c8myq6bi authors: iversen, patrick l. title: the threat from viruses date: 2018-09-30 journal: molecular basis of resilience doi: 10.1007/978-3-319-98164-2_3 sha: doc_id: 18017 cord_uid: c8myq6bi infectious disease represent the most significant threat to human health. significant geologic cataclysmic events have caused the extinction of countless species, but these “wrath of god” events predate the emergence of homo sapiens. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic and are here to stay marking their presence with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but are never prepared. the long-term consequences often remain unrecognized and are always inconvenient including cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. viral success is based on rapid replication resulting in large numbers. single-stranded rna viruses with their high replication error rate represent a paradigm for resilience. contemplating recent career paths, i reviewed a broad range of scientific questions. most prominent was, why study an area of science that has no significant impact? in fact, why study anything that is not the most highly impactful area? this question demands a definition, what constitutes high impact? i decided to create a definition that impact and threat to life are related. further, threat to human life is the highest impact and it is likely that things threatening human life may also threaten all life. what presents the greatest threat to human life today? history should provide critical insights to answer this question. a comprehensive look would seek causes of mass extinctions over the past 3.5 billion years of life on earth. this perspective has been a trending subject in science with focus on five mass extinctions. there are inherent biases in focusing on mass extinctions such as these events fail to appreciate small living things such as single celled organisms or bacteria. another bias is in order to appreciate life in the past, the life form must occasionally produce a fossil when it dies. those concerns aside-geologic events have threatened the survival of living things (table 3 .1). the actual events causing these extinctions are frequently debated, for example, the asteroid impact 66 million years ago off the coast of mexico was accompanied by a massive tsunami that was responsible for mass extinctions in montana, the impact may have also triggered an enormous volcano in india resulting in ash and lava flow responsible for regional species extinctions, and the collective dust resulted in prolonged climate change which was probably responsible for even more species extinctions. the lesson remains the same, volcanic eruptions; climate change and asteroid impact threaten life in a highly significant way and are responsible for extensive selection pressure. unfortunately, these events are unpredictable and so enormous little can be done about them. a mass extinction may not represent a legitimate threat or selection pressure because large numbers of species are completely eradicated leaving few survivors to drive evolution. finally, outside of removing dinosaurs so that mammals could thrive, what impact did these events have on human life; humans were not yet on the earth when these events took place. refining the search to dramatic changes in human populations moves the period up to the last 100,000 years. mass migrations took place about every 23,000 years, which is in line with the precession of the earth leading to a possible linkage between earth's precession and human migration. this may have been due to regional climate change like ice ages in the northern hemisphere. however, human population changes on this timescale are not easily documented so estimates of climate and human population dynamics will be limited. time to refine the search. consider the exponential growth, outbreak, of tent caterpillars, malacosoma disstria, in montana reported by david quammen in his book spillover (quammen 2012) . the extensive caterpillar population consumed all of the leaves from the local elm and cottonwood trees in the summer of 1993. the caterpillar activity produced a crackle sound, "like a distant brushfire." the city attacked these insects with a broad arsenal of modern countermeasures but to no effect. however, a nuclear polyhedrosis virus (npv), uses the caterpillar host density like a critical mass in a nuclear weapon to explode destroying the caterpillars in an epic battle. the caterpillar population retreated to undetectable levels in a single season as a result of npv. human populations changed over the past 20,000 years with particular emphasis on the most recent 7000 years. a human population curve over this segment of time shows an exponential growth period interrupted in the middle ages by the plague. hypothesis: the plague and other pandemic infectious disease events appear to be the greatest threat to human life. infectious disease kills 15 million people each year, 26 percent of the total 57 million annual deaths in the global population (fauci and morens 2012) . a brief review of pandemic infections over the past 3000 years illuminates several events that reduced human populations. the plague of athens 430 bce killed 25 percent of the human population (table 3 .2). both bacterial and viral pandemic yersinia pestis is a bacterium causing plague. fleas can be infected with y pestis which transmit the bacterium to rodents, the primary hosts. changes in the environment may lead to the movement of rats into populated areas where humans become infected. homer points to such an infection in the iliad in his description of the trojan war in 1190 bce. plague has returned several times since the trojan war imposing enormous loss of human life (table 3 .2). the most recent plague epidemic killed over ten million people in india in the early 20th century. y pestis is still out there ready for favorable conditions to pounce on human populations but outcomes are likely to be less dramatic due to understanding of sanitation practices, quarantine, and availability of antibiotics. if infections are the greatest threat to human life, they should be critical drivers of evolution? clearly infections pose selection pressure on the human populations. origins of evolutionary thought did not include infection as darwin established key evolution concepts on the galapagos islands. these islands are isolated and an unlikely place for the spread of infections. the concepts speciation point to geographical separation of populations so infections would most likely be restricted to isolated populations. in many cases the survival selection pressure is not identified or ascribed to insufficient sources of food. unfortunately, common single-stranded rna viruses are so unstable that there are limited data for a viral fossil record. pandemic infections remain a threat to human survival in the presence of the information revolution, daily medical breakthroughs, and global travel. the human retrovirus hiv currently a global infection that infects up to 25% of the population in southern and eastern africa with a projected death toll of up to 100 million by 2025. measles killed 200 million people in the last 150 years and the development of an effective vaccine in 1963 reduced concerns for this infection but there were 777,000 deaths in the year 2000. vaccination programs are frequently disrupted due to complacence resulting from vaccine success, conflicts that shift healthcare focus, and social crisis such as the recent ebola outbreak in west africa. smallpox is also an ancient infection causing fever, skin lesions, and at times death. king ramses v of egypt is thought to have died from smallpox around 1200 bce. introduced into mexico in 1520, smallpox killed 3.5 million aztec indians or about half of the population in a period of 2 years and then proceeded to decimate the population of south america. variola is a highly infectious virus killing 300-500 million people during the 20th century inspiring the eradication campaign in 1967. variola was eradicated by december of 1979 (de cock 2001 , a rare triumph of public health. the who deserves acknowledgment for this unprecedented accomplishment and proof of concept that human suffering is not inevita-ble. however, variola is a dna virus with limited rate of mutation and a narrow host range so that animal reservoirs do not exist. the eradication of other viral infections will be more challenging. the world continues to confront a broad array of microbial threats. progress and preparedness make our engagement a likely success for those microbes that resurface and infections for which we have experience. medical and epidemiological uncertainties surround emerging infectious disease, those that challenge us with their novelty. pandemics dominate the infectious disease "fear factor" but each pandemic began as a much more frequent occurrence, an epidemic. most but not all epidemics come from emerging infectious agents, the most significant problem facing life on earth today. the concept of emerging is a human centric term as most of these infections are endemic in an animal host that serves as a viral reservoir. a 2005 report from the university of edinburgh identified 1407 human pathogens and 177 are emerging or re-emerging of which 75% are zoonotic, that is jump from an animal host to human. numerous emerging infections caused by viral agents have imposed high impact on human survival (table 3 .3). all the viral agents in table 3 .3 have genomes based on single-strands of rna except hbv which should focus scientific attention on rna. there are numerous questions that strike investigators as they ponder a collection of viral agents like those in table 3 .3. the viral polymerase errors in replicating single-stranded rna genomes are not corrected so the species are constantly changing. the apparent success of these viruses is that as they move from reservoir hosts to humans and as humans become immune to the initial infection, the population of diverse genomes offers multiple chances to adapt by finding a "fit" genome version which can propagate until the next transition requiring adaption. acquired immunodeficiency syndrome (aids) is caused by human immunodeficiency virus 1 (hiv-1), a retrovirus. these viruses have a single-stranded rna genome that is converted into dna, a paradigm shift in the flow of genetic information from dna to rna. the 36,000,000 human deaths caused by hiv-1 (table 3.3) is accompanied by a spectrum of clinical signs; eg. fever, diarrhea, peripheral neuropathy, pelvic inflammatory disease, cervical cancer, cytomegalovirus retinitis, kaposi's sarcoma, lymphoma, mycobacterium avium infection, recurrent pneumonia, and wasting syndrome. hiv-1 genome diversity includes base substitution, insertion, deletion, recombination, and gain or loss of glycosylation sites which all arises from the limited fidelity of the viral reverse transcriptase. these mutations are found in clusters or hypervariable regions indicating the fit virus is selected for from vast numbers of less-fit genome sequences. hiv-1 emphasizes key observations: (1) eid is a significant contemporary concern, (2) we are not prepared for the novel characteristics introduced by eid, (3) clinical signs can be diverse and often mimic symptoms of other diseases, and (4) the replication mechanisms are often error prone resulting in an array of fit viruses. dengue is a flavivirus with a positive sense single-stranded rna (+ssrna) genome carried by mosquitos to man. dengue has been a tropical disease for hundreds of years, typically a disease of young children but in 1953 an emerging severity was recognized in manila (table 3 .3), hemorrhagic fever (dhf) and dengue shock syndrome (dss). more than 2 billion people are at risk of dengue infection but only a small fraction of those infected will develop dhf or dhs. infected people develop antibodies that can lead to antibody-dependent enhancement (ade) in subsequent infections and more severe dhf or dss outcomes. it appears ade events occur in people that produce immunoglobins (iggs) with enhanced affinity to the activating fc receptor due to the igg1 subclass and lack of a fucose glycan modification of the igg (wang et al. 2017) aedes aegypti is the primary vector transmitting dengue but a new vector, aedes albopictus, now carries dengue to the southern united states. emergence of dengue in the southern united states is likely due to used tires imported from japan which provided a place for the asian tiger mosquito to live. outbreaks of dengue fever have become more numerous and more severe over the past three decades. viral "fitness" is constrained by the requirements imposed by the natural host so that it is a low probability event for a virus to move from the natural host to a human. while an insect frequently plays the role of vector carrying a virus from an animal reservoir to humans, several zoonotic viruses are transmitted by placing humans near rodents. several arenaviruses, minus sense single-stranded rna viruses, jump from their rodent natural hosts directly to humans through contact with rodent urine or saliva. notable arenaviruses are named for the hemorrhagic fever (hf) region of their zoonosis; bolivian hf is caused by machupo (macv), argentine hf is caused by junin (junv) and venezuelan hf is caused by guanarito (gotv). these south american outbreaks are caused by new world arenaviruses in contrast to lassa hf (lasv), an african or old world arenavirus. lasv is an endemic disease of west africa (table 3 .3) and can be confused with dhf or ebola infections. viruses that are transmitted by arthropod vectors are called arboviruses. in 1930, only yellow fever of six known arboviruses caused disease in humans. the discovery of arbovirus caused human disease expanded beginning in the late 1930s with western and eastern equine encephalomyelitis (weev, eeev) and st. louis encephalitis. both chickungunya and zika (table 3 .4) are arboviruses that have emerged to become global infections in the twenty-first century. zika not only became infamous for causing microcephaly in newborns of infected mothers and guillain-barre syndrome but is now sexually transmitted between humans. i worked on a therapeutic for the treatment of ebola infections from 2007 to 2013. the genome sequence recovered from each outbreak from 1976 to 2013 has been different. studies were conducted at usamriid in their bsl4 facility by expert ebola investigators. rhesus monkeys (macaca mulatta) were injected with 1000 plaque forming units (pfu) of zaire ebolavirus (zebov) kikwit into their thigh muscle. all of the untreated control monkeys died between days 8 and 10 following viral injection. we measured viral burden by quantitative polymerase chain reaction (qrt pcr) a measure of genome copies per milliliter (copies/ml) of plasma and plaque formation a measure of infectious viruses per ml (pfu/ml) plasma. we found an average of 1.7x10 8 genome copies per ml on day 8 post infection and 1.2x10 6 pfu/ml on day 8. nearly 100 genomes present for each successful virus in a nonhuvwas not observed, the dominant population of viral genome sequence did not change from one individual to another or from one time to another within the same individual. the existence of defective viral genomes may offer potential for rapid change but in a stable host, change was not rapid. when one considers the magnitude of differences in conditions as a virus jumps from a reservoir host to a human, rapid adaptability is a great advantage. a virus that kills all the hosts is not a successful virus. perhaps defective viral particles facilitate host immune responses giving them a chance to catch up to the rapidly proliferating virus. the ebola outbreak of 2014 illuminated the collateral damage that accompanies eid outbreaks. first, the first responders are local physicians and care givers are killed leaving a population lacking doctors and nurses. even physicians trained to exercise appropriate caution when interacting with patients such as dr. sheik humarr khan died of the ebola infection (bausch et al. 2014) . second, women are the main caregivers in their families and 75 percent of ebola deaths in the 2014 outbreak were women. this leaves families lacking traditional structure. third, the outbreak disrupts production, labor markets and trade resulting in scarcity and inflation of food prices. food security and nutrition are diminished which preferentially affects poor people as they spend 50 to 70% of their income on food. finally, public health measures are discontinued. before the 2014 outbreak, 97 percent of children in west africa were receiving routine vaccinations but that figure fell below 27 percent. the west africa loss of measles vaccinations was followed by measles out coronaviruses are unique in their large single-stranded rna genome (20,000 bases) and are often associated with mild disease, bronchitis and gastroenteritis. a 2003 outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in china rapidly spread to 8098 cases in 37 countries (table 3 .4). retrospective analysis of this outbreak linked zoonosis to a culinary trend in which people sought out exotic animals to eat. these nontraditional food animals appeared in street markets in urban areas bringing people close to the reservoir host triggering the outbreak. the civet cat was identified as the source of the first human cases but this is not the natural host. the natural host is the fruit bat, which infects a variety of small mammals including the civet cat. a point of intervention has been removal of the civet cat from markets but the natural bat host remains in the environment maintaining the virus for the next outbreak. a second coronavirus outbreak in 2014 of middle east respiratory syndrome coronavirus (mers cov) rapidly involved 23 countries including africa, western europe, and southeast asia. the number of cases is relatively low but the case fatality ratio is of great concern (table 3.4). the camel is reservoir host in this case but it may not be the natural host since camels often get sick as well. transition from animal-to-human transmission to efficient human-to-human transmission was observed quickly in the case of both sars-cov and mers-cov. this rapid adaptability of a highly infectious virus group should raise concern over endemic coronaviruses in companion animals since these viruses can clearly become pathogenic, can jump from animal to human and then become transmitted from human-to-human. an ongoing outbreak of yellow fever in africa (table 3 .4) is a striking reminder that eid can re-emerge into virtually naïve populations. some people recover from the acute symptoms but liver damage causes yellow skin, the reason for the name for the disease. the liver damage leads to bleeding and kidney damage and occasionally death. yellow fever is a flavivirus that originated in africa but was distributed to the world on barges and sailing ships to tropical ports. the virus arrived in the americas on slave ships. this virus interrupted the building of the panama canal which attracted the attention of walter reed, the military physician responsible for demonstrating transmission by mosquitos. an effective vaccine is used worldwide with immunity lasting over 10 years. the limitation of this vaccine like most is that populations need to be vaccinated regularly which in a world constantly changing due to political and economic drivers leads to vaccination gaps. cancer is one of the leading causes of human death but estimates are that 20 percent of all human cancers are directly caused by viruses (table 3 .5) (morales-sanchez and fuentes-panana 2014). the first tumor virus was identified by peyton rous in 1911. he took cell lysates from a chicken sarcoma which he passed through a filter known to hold back bacteria and then injected the filtered lysate into chickens. a tumor developed at the injection site. the virus is now known as rous sarcoma virus (rsv), a single-stranded rna virus. rous won a nobel prize in physiology or medicine for the discovery in 1966. rsv is a retrovirus that captured a tyrosine kinase, src gene, that triggers uncontrolled growth in infected host cells. the avian leukemia virus (alv) was discovered in denmark early in the twentieth century capable of causing disease in blood forming tissues of chickens. a strain of alv, avian myeloblastosis virus (amv) was described in 1952 that provided a convenient source of tissue for biochemical studies. during the 1930s a strain of inbred mice was found to develop leukemia between 6 and 18 months of (burkitt's lymphoma 2014) . after that he saw a second case at the jinja district hospital on the shores of lake victoria this became more than a curiosity. a review of hospital case notes confirmed the prevalence of these tumors of the jaw and they were accompanied by swellings in kidneys, ovaries, adrenal glands and liver. he assembled data from 38 patients and histological examination led to description of "lymphoma syndrome" or the "african lymphoma." burkitt received a grant in 1961 for ₤250 to visit 57 hospitals in eight african countries; uganda, kenya, tanzania, malawi, mozambique, zimbabwe, zambia, and republic of south africa to investigate african lymphoma. the tumor was found everywhere at the equator in areas where year-round temperature was above 60 °c but not in areas over 5000 feet in elevation. burkitt further determined the tumors only occurred where the annual rainfall was above 20 inches and not seen in the dry savannah of nigeria. burkitt contacted doctors in papua new guinea to discover these tumors were the most common childhood tumor in that country but only in wet coastal regions and not in the dry highland areas. burkitt's working hypothesis was that an insect transmitted virus infection was responsible for the lymphoma syndrome. he then observed adults with the lymphoma but only in individuals that moved into the "african lymphoma belt." these tumors were then observed in the united states and europe at a rate of 1-3 per million or about 100x less frequently than in africa. biopsy samples did produce viruses but none that came from insects so the working hypothesis was abandoned. a new hypothesis emerged between 1963 and 1966 in which p falciparum (a parasite causing malaria) was the causative agent for the lymphoma. it is believed that severe malaria does play a role in the development of burkitt's lymphoma. at the advice of peter clifford, burkitt administered methotrexate to treat patients which produced encouraging results. in 1961 burkitt gave a lecture at middlesex hospital, "the commonest children's cancer in tropical africa: a hitherto unrecognized syndrome." anthony epstein attended the lecture which changed his life and provides a punctuation mark in the history of science. epstein introduced himself after the lecture and the two sat down for tea. burkitt agreed to send tumor specimens to epstein and epstein received a grant from the us national institutes of health in 1963 for $45,000 which allowed him to employ two research assistants. yvonne barr was one of the research assistants that was able to propagate a virus after 26 tries with tumor specimens. the other research assistant was bert achong, an electron microscopist, who was first to identify the virus as a member of the herpes virus family. the virus bears the name epstein barr virus or ebv and is a causative agent of burkitt's lymphoma. an interesting historical note involves werner and gertrude henle at the children's hospital of philadelphia. they created antibodies to ebv infected cells and their survey of blood samples from american adults revealed over 90% to be ebv positive in 1966. they also showed that normal human lymphocytes could be made immortal by infecting them with ebv in 1967. we now know ebv silently infects children in the western world but in those infected a bit later in life become susceptible to infectious mononucleosis (the kissing disease) as adolescents. in 1972, a collaboration between george klein and the manolovs led to a discovery of a chromosome change that was specific to burkitt's lymphoma. this chromosome change was a translocation of 8:14 and rarely 8:2 or 8:22 (zech et al. 1976 ). the break points in chromosomes 2, 14, and 22 contained immunoglobin genes that become active in b lymphocytes as they produce antibodies during infection. a nobel prize in 1989 to michael bishop and harold varmus provided the significance to the break point in chromosome 8, bringing active immunoglobin genes near the oncogene c-myc. in 1970, a second cancer common in north and east africa as well as southern china was found to be ebv positive, carcinoma of the post-nasal space (nasal pharyngeal carcinoma or npc). the tumor is observed in epithelial cells not b-cells and is not geographically linked to endemic malaria regions. in this case, it appears that aerosol exposure to environmental carcinogens (possibly in preserved fish) may introduce mutations in nasal epithelial cells. the npc may be the result of silent ebv infection, carcinogen induced cellular mutations, and people carrying hla a*0207 and b*46 antigens. in 1975, several reports describing "fatal infectious mononucleosis" appeared. david purtilo described x-linked proliferative syndrome (xlp) based on an international registry of boys with fatal ebv infections (purtilo et al. 1977) . using genetic marker analysis from the international registry they narrowed xlp to a three million base pair segment on the x-chromosome. in 1998, a 384-base pair segment encoding the xlp protein of 128 amino acids was found to be lost or damaged in al xlp patients (nichols et al. 1998) . curiously, the xlp protein loss leads to a defect in nk and t lymphocytes which are necessary for recognition of ebv infected b lymphocytes. these individuals cannot make antibodies to ebv because their b cells require help from t lymphocytes. the development of potent immune suppressing drugs like cyclosporine a was linked to lymphoma after organ transplantation. two girls with acute lymphoblastic leukemia received bone marrow transplants from their hla-match brothers. the grafts were successful but they developed lymphoblastic leukemia from the transplant (male cells). these lymphomas do not present chromosomal translocation like burkitt lymphomas revealing a new path from ebv to lymphoma. the immune suppression upsets the ebv host homeostasis crippling t-cells that are required for keeping ebv under control. another situation where the virus can replicate without control is in hiv/aids patients and aids-lymphoma is yet another ebv induced casualty. ebv provides key insights into tumors associated with viral infections. first, the same virus is linked to multiple tumors in different populations including burkitt's lymphoma, hodgkin lymphoma, and nasopharyngeal carcinoma. second, tumorigenesis involves more than one mechanistic pathway but interest has focused on the latent membrane protein-1 (lmp1), ebv nuclear antigen 1 (ebna1), and bamh1-a reading frame-1 (barf1) genes. third, dissecting the association between infection with ebv and cancer has unfolded over a period of 30 years and led to a greater understanding of cancer, the immune system, and methods for immortalizing cells to study specific cell clonal populations. ebv has been a boon to scientific research while imposing a challenge to human survival. approximately 2 billion people have been infected with hbv and 360 million suffer from chronic infection. acute hbv infections are usually self-limiting associated with 5.2 million cases a year. chronic hbv infections lead to complications in 15-40 percent of cases resulting in 0.5 to 1.2 million deaths each year. hb v causes 60-80 percent of the world hcc cases which represents 5 percent of all cancers in the world. the hbx gene is considered key to oncogenesis. vaccination programs are effective in reducing mortality in infants and have reduced emerging prevalence of the disease. dietary exposure to aflatoxin enhances hbvrelated hcc (sun et al. 1999 ) as well as co-infection with hcv and excessive alcohol consumption. hepatitis b virus (hbv) was named due to differences in transmission: hepatitis a type virus follows fecal-oral transmission while b type virus is parenteral. the genome is circular dna that is not fully double-stranded with the end of the fulllength strand linked to dna polymerase. the genome is 3020-3320 nucleotides long (full length strand) and 1700-2800 nucleotides for the short strand. four genes are encoded including: (1) c the core protein (hbcag) synthesized by uorf aug to make pre-core protein, (2) p is the dna polymerase, (3) s is the surface antigen (hbsag) which has three aug start sites that divide the gene into pre-s1, pre-s2, and s, and (4) x is a gene that is not fully understood but may be a transcriptional transactivator. non-coding rna include hbv prealpha, hbv prebeta, and hbv rna encapsidation signal epsilon. there are 10 known genotypes which differ by 8 percent in sequence with distinct geographical distributions and are labeled a to j and at least 24 subtypes. type f is the most divergent form and found in central and south america, predominantly brazil. human hbv has a narrow host range infecting humans and higher primates, eg. chimpanzees. in the 1970s a woodchuck hepatitis virus (whv) was discovered which will not infect rodents. a ground squirrel form was identified (gshv) that is a distant relative of the marmot and woodchuck. indeed, dhbv infects ducks but not all species of duck, grey herons are infected with hhbv, the ross' goose with rghbv, the snow goose with sghbv, white storks with sthbv, and cranes with chbv. there are no hepadnaviruses in arthropods or insects. human t-cell lymphotropic virus (htlv-1) htlv-1 is a single-stranded rna retrovirus, defined by their use of reverse transcriptase, a polymerase, that makes a dna copy of the rna 7 kb viral genome. the dna viral genome is integrated into the host genome where it is referred to as a provirus and is replicated along with the host genome during cell division. only 1 percent of infected individuals will develop leukemia and this is observed 20 to 30 years after asymptomatic infection. the htlv-1 tax protein is likely to initiate cell transformation through interactions with transcription activators and cell cycle regulators. hepatitis c virus (hcv) hcv has infected approximately 3 percent of the world's population (~210 million) but screening of the blood supply has reduced prevalence. hcv is a flavivirus composed of a 9.4 kb single-stranded, positivesense rna. hcv is characterized by a single serotype but at least 6 major genotypes. genotype 1b is the most common genotype seen in the united states and taiwan. hcv becomes a chronic infection by evading host immune defenses through a combination of: (1) high replication rate (10 12 virions/day) and (2) lack of error proofreading by the viral polymerase leading to mutations in response to immune pressure. the genetic variability of hcv has limited efforts to design an effective vaccine. the polyomaviruses are small (3.4 kbp), double-stranded dna viruses. early studies with simian virus 40 (sv40) led to identification of the large tumor antigen (large t; lt). lt is also found in bk and jc viruses which are more suspect human tumor viruses and merkel cell polyomavirus (mcv) which is now well established as a human tumor virus (feng et al. 2008) . the n-terminus of lt contains an lxcxe motif that interacts with the retinoblastoma protein rb while the c-terminus contains an atpase/dna helicase domain that can inactivate p53. the ls-p53 complex activates insulin-like growth factor i (igf-i) which alone is capable of cell transformation. a recent report of morbidity and mortality reveals heart disease and cardiovascular events are the number one killer with neoplasia and infections following close behind. however, infections frequently cause neoplasia and cardiovascular disease leading to death. if we combine cardiovascular events and neoplasia caused by infection, then infectious disease is the most significant threat to human life and qualifies as the area of greatest impact. the picornavirus family are small (pico-), single-stranded, positive sense rna genome (7.4 kb) viruses that synthesize a single polyprotein that is cut into a small collection of functional proteins by virally encoded (2a and 3c) and cellular proteases. poliovirus is a picornavirus that has served as the prototype for the viral family. the enteroviruses are a group of picornaviruses that have been associated with cardiac disease. coxsackievirus b (cvb), coxackievirus a (cva), and echovirus infections lead to cardiac signs 3.2% of the time. about one-third of patients with acute cardiac disease (inflammation of the heart) are antibody positive for enteroviruses. while acute myocarditis is often self-limiting, chronic cardiac disease often leads to dilated cardiomyopathy (dcm) which is present with no heart inflammation. dcm is associated with heart failure which can be lethal (table 3 .6). these chronic infections lead to 50 percent of all cardiac transplants worldwide. the enteroviral 2a protease can also degrade dystrophin in the heart leading to cardiac necrosis, reduced ejection fraction, and then to dilated cardiomyopathy. transmission of these viruses is from contaminated food and water. the human herpesviruses are large double-stranded dna genome viruses. the human herpesvirus-5 (hhv5) or cytomegalovirus (cmv) has a 235 kbp genome encoding over two hundred genes. one problem in finding associations with cmv infections is that it is not an emerging disease, 50 to 99% of the population has been infected making comparisons to a control group challenging. most infections are observed in children and in newborns serious clinical findings can be observed. hence, most adults carry latent infections that are reactivated when individuals become immune suppressed following solid organ transplantation, malignant hematological disease, and aids. reactivation in immune suppressed individuals is associated with increased mortality. the association with cardiovascular disease has been demonstrated in two recent studies. in one, cmv infections were detected in 14.5 percent of coronary artery samples from bypass operations compared to 4 percent in of patients who needed cardiac surgery for reasons other than atherosclerosis hebar et al. 2015) . in the other, cmv reactivation (viremia) was detected in 16.5% of immunocompetent patients admitted for major heart surgery (roa et al. 2015) . the incidence of coronary artery disease is the major contributor to death from heart disease and if 14 to 16.5% of this disease is associated with cmv infections, this infection is a significant human health hazard. recognizing an emerging infectious disease involves well established strategies of surveillance; (1) identify unusual clusters of disease, (2) evaluate the spread of an outbreak, (3) estimate the magnitude of the problem, and (4) if possible identify the atheroscleotic plaque infectious agent. the strategy has proven valuable for known infectious and noninfectious diseases but has limited capacity to detect emerging infectious diseases. however, deviation from the traditional approach to surveillance is not likely to gain support. al smith, a veterinary virologist, approached me after a seminar i presented in 1997 in the college of veterinary medicine at oregon state university. i had just joined avi biopharma as the head of their research and development program. al was a veterinarian and professor and had devoted his career to the caliciviridae family of viruses dating back to his time in the naval research station in california. he had isolated the nonhuman vesivirus group of caliciviruses not only from suffering sea lions in the channel islands, but from reptiles in the san diego zoo and whales held in captivity. al and his capable technician, doug skilling successfully propagated the virus isolates in cell culture, an accomplishment not shared by other laboratories at the time. al developed nucleic acid probes and induced antibodies to these viral isolates. over decades of research he assembled an extensive collection of vesiviruses which were held in redundant −70° freezers. his singular vesivirus focus gave the appearance of a zealot but his ability to cultivate viral isolates, his extensive collection of isolates, and his one of a kind detection reagents made him a one of a kind virologist. al was well beyond retirement age and was an "old school" virologist ready at a moment's notice to take his bag into the real world and collect swabs from ailing animals. he maintained careful records of the condition of his patients, their location, and the setting of the animal in the community. every field trip led to work in the lab propagating virus from his swabs. al wanted to know if i would be interested in finding an antiviral agent for these viruses. my prime directive at avi biopharma was to explore the capabilities of our proprietary antisense technology and i had yet to investigate targeting a virus. the caliciviruses have positive sense single-stranded rna genomes which express three genes from a single polyprotein. we identified an active agent following an investigation with small collection of candidates (stein et al. 2001) . the vesivirus group of caliciviruses are considered animal only and are not believed to infect humans but al began exploring human samples with his collection of detection reagents. after several years making incremental progress, we found an association between human blood samples seropositive for vesivirus and markers of liver disease (smith et al. 2006) . we felt this evidence of an emerging infectious disease in humans and its potential to cause liver disease would be welcomed by the medical community. al and i made a trip to washington dc at our own expense to relate the findings in person. we meet with virologists at the national institutes of health but were met with judicious skepticism. harvey alter had been instrumental in the discovery of hepatitis c virus (hcv) and felt all viral liver disease is already accounted for by hepatitis viruses. hence, no interest in our findings. we met with the american red cross blood banking group in shady grove maryland and were met with concern. blood supplies are scrutinized by the nucleic acid test (nat) to eliminate viral contamination and any further elimination of blood samples would threaten an already limited inventory. our findings simply add complication to the blood supply finding an emerging infectious disease business. they asked for more compelling and more extensive data to add robustness to our claims. the only problem is that they were not interested in providing financial support for the recommended studies. the conundrum is all too common, you need more data to convince granting agencies to support the work but there is no support to add to the limited data. life on the cutting edge is frequently discouraging. our strategy took two paths: one to seek commercial support and the other to create proof of concept data for an antiviral solution. the most logical commercial solution was to meet with michael houghton at chiron. he was the driving scientific force in finding hcv and chiron might like adding to their reputation by finding another previously unrecognized hepatitis virus. indeed, dr. houghton invited us to bay area to discuss the project. he reviewed our data and the quality of al's detection reagents. chiron provided modest support and their in-house laboratory help to confirm our observations, a glimmer of hope. al created a company, calicitech, so that he could accept support and license his reagent patents from the university. testing an antiviral agent in humans would be expensive but in the absence of natural history of the infection would make human proof of concept impossible. however, al had been contacted by a cat rescue facility in atlanta, mommy cat, describing an outbreak of feline calicivirus (fecv) in their cattery. these cats had all been vaccinated with an approved fecv vaccine which failed to protect these cats. a veterinarian can decide to use alternative medicines if there are no alternatives and the owner oc the cats can sign the equivalent of informed consent. we provided our fecv specific antiviral to mommy cat along with a detailed protocol for treatment. the infected kittens we treated survived while those not treated died providing encouraging data. we then discovered a similar outbreak in the humane society facility in eugene oregon, our neighbors. again, we reached an agreement to treat infected kittens and were successful ). this was the first time we treated an infection based on symptoms in an "outbread" population of cats. with over 100 kitten patients and remarkable survival differences between treated and untreated, we had our proof of concept. there is no disease cluster to link to human vesivirus. norovirus is a calicivirus and is known to infect humans, in fact it is well known on cruise ships, day care centers, and extended care retirement facilities. our efforts to have human vesivirus recognized as an emerging infectious disease have failed. chiron was not impressed with our antiviral and no longer were interested in evaluating the detection reagents. if we are lucky, human vesivirus infections will remain mild clinical oddities. al smith has an interesting way of responding, "that virus is out there infecting humans. it won't go away." the existence of non-pathogenic viral infections led to the emergence of the study of the immune system, vaccination, gene therapy, and concern for future pandemics. we carry evidence of ancient retroviral infections in our genome from integration events that became vertically transmitted making up as much as 8 percent of the human genome (meyer et al. 2017) . these genomic fossils are called endogenous retroviral genomes (erv). most erv sequences accumulate sufficient mutations over evolutionary time that horizontal transmission is unlikely. these viral genome segments are trapped but can still be transcribed and encode some viral proteins. the significance of these integrated genomes is a current topic of investigation. adeno-associated virus (aav) is a single stranded dna virus that infects humans but are not known to cause disease. the lack of pathology has led to their use as viral vectors for human gene therapy. aav can infect dividing and quiescent cells and will persist extra chromosomally without integrating into the genome of the host cell. aav is a member of the parvoviridae family in the genus dependoparvovirus. is an arenavirus closely related to lassa virus and shares reservoir hosts. mv is naturally attenuated and nonpathogenic in humans. infection of humans with mv protects against lethal challenge with lv (wulff et al. 1977) . flaviviridae) named after g. barker, a surgeon, first identified in 1995. hgv infects one sixth of the world's population but does not cause human disease. a metaanalysis of 15 publications investigating gbv-c infections in hiv-positive individuals indicate coinfection with gbv-c slows the progression of hiv disease in individuals that have been seropositive for 2 years or more (zhang et al. 2006a) . two of the studies investigated gbv-c 5 years after documented hiv seroconversion estimate the hazard ratio of 0.36 (95% ci). is a retrovirus first identified in 1971 from a lymphoblastoid cell culture from a kenyan patient with nasopharyngeal carcinoma. hfv is homologous to primate foamy viruses and is most closely related to the chimpanzee foamy virus (sfvcpz). early studies raised alarm for association with autoimmune diseases but more extensive studies with more precise diagnostic reagents fail to find a disease associated with hfv. hfv is a rare human infection and concerns parallel sfv infections in humans. simian foamy virus (sfv; spumavirus-retroviridae) is a retrovirus infecting most primates born in captivity and people making contact with infected primates can become infected. human infections frequently occur in males probably requiring a bite from infected nonhuman primates but are harmless. the infected cells often fuse to form syncytia of giant foamy cells, which gives the virus its name. the error rate in sfv genomes is exceptionally low, 1.7 x 10 −8 substitutions per site per year, compared to hiv 10 −3 substitutions per site per year. since so-called crossspecies, infections have only been observed for a little over a decade the long term consequences are not known. these infections are watch and wait for everything from a zoonotic epidemic to identified disease clusters. perhaps this is exactly the sort of infection that will emerge as a significant human concern in the future. torque teno virus (ttv; alphatorquevirus) is a single-stranded, positive sense dna genome virus about 3.8 kb in size in the anelloviridae family. nearly 100 percent of even healthy individuals are infected in some countries. the virus was discovered in 1997 as the "transfusion transmitted virus (ttv)" in a japanese patient. it is often found in patients with liver disease but does not cause hepatitis on its own. closely related torque teno mini virus (ttmv) were isolated in 2007 and found to have smaller genomes of 2.8-2.9 kb. ttmv infections are also common but do not cause any described human disease. human adenovirus type 5 (rad5) is used to create an ebolavirus (ebov) vaccine encoding zaire ebolavirus glycoprotein failed to protect animals immune to ad5. a replication defective chimpanzee-derived adenovirus (chad3-ebo-z) provided protection against lethal ebov challenge in macaques but protection wane over several months. they boosted with a modified vaccinia ankara (mva-bn-filo) that led to durable protection (stanley et al. 2014 ). this vaccine progressed through phase i, single-blind, randomized human trials in mali between 2014 and 2015. a single dose of the chad3-evov-z is efficient as a prime vaccine strategy followed by mva-bn-filo as a boost was well tolerated in humans tapia et al. 2016) . is a 5229 base double-stranded dna virus infecting less than 5 percent of the human population. wu, named after washington university, is found as a co-infection in various respiratory infections but wu does not cause disease on its own. wu is closely related to ki virus that also is not known to cause clinical disease. however, related polyomaviruses that are clinically relevant include bk virus associated with nephropathy, jc virus associated with progressive multifocal leukoencephalopathy, sv40 virus associated with mesothelioma, and merkel cell polyomavirus associated with cancer. vaccinia virus (orthopoxvirus) is a large double stranded dna virus closely related to smallpox. edward jenner, the father of immunology, found the milkmaids exposed to cowpox (vaccinia) were immune to smallpox in 1798. this was the first vaccine (named after vaccinia) leading to the modern vaccine that has allowed for the eradication of smallpox. viral sequences are constantly mutating with no purpose other than seeking a survival/infectivity benefit. this means viruses with no current pathology represent a pre-mutation reservoir for the next catastrophic human pandemic. the popularity of rnaseq is likely to expand our catalog of nonpathogenic viral infections. however, the management of such information is in question. technology used to counter viral infections has resulted in over 90 approved drugs for the treatment of nine different human viral infections in just 50 years (de clercq and li 2016) . several different antiviral drug groupings have been reported, but the following arise from review of the mechanisms of action of antiviral drugs assembled in table 3 .7: (1) inhibition of viral attachment and entry, (2) inhibition of viral uncoating, (3) viral polymerase inhibitors, nucleotide analogues (ntrti) and non-nucleotide reverse transcriptase inhibitors (nnrti) and dna polymerase inhibitors, nucleic acid synthesis inhibitors and nucleotide pool size agents, (4) latency reversal agents, (5) integrase inhibitors, (6) protease inhibitors for both hiv and hcv, (7) neuraminidase inhibitors, (8) immune response modifiers, and (9) antisense inhibitors. administration of hyperimmune sera from immunized animals or human donors was the first effective treatment for infectious diseases. the practice has limitations but is still used to treat bacterial toxins and viral infections caused by cmv, rsv, hav, hbv, rabv, vzv and mev (keller and stiehm 2000) . the development of human or humanized monoclonal antibodies (humabs) has created a feasible way to rapidly generate novel antiviral therapeutics. humabs have advantages over serum therapy in that they are chemically defined reagents with minimal variability, greater activity per mass of protein, and they have no immunological consequences related to serum sickness. several mabs have been approved for treatment of infectious diseases including viral and bacterial pathogens (table 3 .8). the antiviral mab discovery field is exploding with activity particularly for hiv and hcv infections. a humanized mab targeting lymphocyte ccr5 receptors called pro140 has demonstrated potent and prolonged anti-hiv-1 activity and a large margin of safety (jacobson et al. 2010a) . administration of pro 140 by the subcutaneous route offers patients a way to self-administer the mab but importantly the mab is transported in the lymphatics providing enhanced access to binding to the cellular target (jacobson et al. 2010b) . the next generation of antiviral therapeutics are likely to be dominated by mabs. perhaps the only way to clear a viral infection involves a host immune response (table 3 .9). the innate immune response is particularly effective centered on a type 1-interferon pathway. unfortunately, many viruses carry mechanisms to evade host innate responses and innate immune effectors do not have the capacity for memory. the adaptive immune response can mitigate infection with antibodies, generally to surface antigens, which prevent the spread of the virus and t-lymphocytes, which can clear virus-infected cells. the first strategy will be to use an existing drug designed for another virus offlabel. this seems likely for antiviral drugs like cidofovir, foscarnet, and ganciclovir particularly for double-stranded dna viruses like ebv, hpv, and hhv6. ribaviran has been used for a number of single-stranded rna viruses including polio, junin, and lassa fever. secondary strategies will require investment of time and effort beginning with vaccine development and creation of monoclonal antibodies. platform technology rna-based therapeutics offer rational design for an expansive number of new antiviral strategies. this advantage is superimposed on the theoretical advantages of selectivity, specificity and affinity provided by watson-crick base pairing. rna-based therapeutics are expected to provide a substantially more narrow range of pharmacokinetic properties and toxicities thus are easier to compare to each other and ultimately combine into multi-agent cocktails. however, the mechanism of action may vary from rnase h or risc mediated degradation of the targeted rna or steric inhibition of rna function. the objectives of our antiviral program have been to exploit the broad understanding of rna-based therapeutics for antiviral activity with a common chemical type, the phosphorodiamidate morpholino oliogmer (pmo) and their enhanced derivatives. in this way the mechanism of action is common to all agents, which is steric blockade of rna function. studies reported by zamecnik and stevenson introduced the first approach to identification of an antisense antiviral agent stephenson and zamecnik 1978) . they used a 13-mer targeted to rous sarcoma virus. since the rous sarcoma virus pioneering efforts, antiviral rna based therapeutics have involved multiple oligomer chemistries with a variety of different mechanisms of action. chemical approaches to oligomers directed to hiv have been plentiful. a nonionic methylphosphonate oligonucleotide targeted to the splice acceptor site of hiv tat inhibited splicing of viral rna inhibiting syncytia formation and p24 synthesis at 3um concentration. poor aqueous solubility limited the utility of the methylphosphonate chemistry. phosphoramidate chemistry was investigated for inhibition of the splice-donor and splice-acceptor of hiv tat agrawal et al. 1988 ) and were more potent but these agents were cytotoxic and poorly water soluble. phosphorothioate chemistry targeting hiv-rev (matsukura et al. 1987 ) and hiv-tat were shown to be effective in inhibiting hiv replication, were not cytotoxic and were very soluble. further, the hiv-rev phosphorothioate oligodeoxynucleotide was stable in vivo with an acceptable pharmacokinetic profile (iversen et al. 1994) . a 25-mer phoshorothioate called gem91 targeting the initiation site of hiv-gag was evaluated in clinical trials (agrawal 1998 ) but the trials were discontinued. i focused on phosphorodiamidate morpholino oligomer chemistry which is both stable and net-neutral in charge at physiological ph. single stranded rna viruses with positive sense (ssrna(+)) these viruses are the most simple in terms of genome size, number of potential translated viral proteins, their genomes are all linear and they enter the cell ready for translation. the design of steric blocking rna-based therapeutics involves preventing translation, disrupting rna secondary structure and masking recognition sites for rna dependent rna polymerase (rdrp). the targeting of either the 5′-terminus and the first orf-aug are active. further, efficacy in animal challenge studies is observed with high fidelity when the most effective agent identified in vitro is employed. the family astroviridae with six different human astroviruses (huastv) responsible for 2-17% of all gastroenteritis. we found the 5′-terminus 1 to be the most effective site to target. this was also the optimal site for caliciviridae including vesivirus (vev; martin-alonso et al. 2005; stein et al. 2001), norovirus (nov; bok et al. 2008) , and feline calicivirus (fcv; smith et al. 2008) ; the flaviviridae including dengue (den; kinney et al. 2005; holden et al. 2006) , and west nile virus (wnv; deas et al. 2007; zhang et al. 2008) , arteriviridae including agriculturally important equine arterivirus (eav; van den born et al. 2005) , and porcine respiratory and reproductive virus (prrsv; zhang et al. 2006a, b) ; and the togaviridae exemplified by venezuelan equine encephalitis virus (veev; paessler et al. 2008 ). the family coronaviridae revealed a new active target site in the transcription regulatory sequence (5'-cgaac-3′) in both mouse hepatitis virus (mhv; neuman et al. 2004 ) and the severe acute respiratory syndrome virus (sars; neuman et al. 2005) . the picornaviridae active target site involved a highly conserved sequence in the internal ribosomal entry site (ires) in polio virus (pv; stone et al. 2008) , foot and mouth disease virus (fmdv; vagnozzi et al. 2007) , and the coxsackievirus (cvb3; yuan et al. 2006) . single stranded rna viruses negative sense (ssrna(−)) these viruses are generally more complex with respect to genome size, number of potential translated viral proteins, and multiple genome segments. the genome must be replicated prior to translation of viral proteins. the design of steric blocking rna-based therapeutics is similar to the positive sense rna genomes in that targets involve preventing translation and masking recognition sites for rna dependent rna polymerase (rdrp). we investigated 11 different targets in measles virus (mev), a member of the paramyxoviridae, finding the translation initiation start site of n the optimal target (sleeman et al. 2009 ). studies with the human respiratory syncytial virus (hrsv) found the translation site for l to be most active (lai et al. 2008) . the orthomyxoviridae studies investigated influenza a virus probing each of the 8 viral segments finding translation of pb1 active as well as the 5'terminal of vnp for h3n2 (ge et al. 2006 ) and h3n8 (lupfer et al. 2008 ) but a combination of targets was required in animal studies with a high pathogenic viral h7n7 isolate (gabriel et al. 2008 ). more extensive influenza a studies revealed a new target, the m-segment splice donor site. this target was evaluated in phase i clinical trials. the antisense platform technology has limitations in targeting viral sequences. inhibiting virally encoded proteins or blocking viral replication by interfering with the polymerase does not always work. the arenaviridae family proved difficult. we experienced some success with junin and lymphochoriomeningitis virus (lcmv) in cell culture observing 4 log 10 reductions in viral titer with a pmo targeting the highly conserved viral genome terminus. however, we failed to provide survival benefit to guinea pigs challenged with either junin or lassa virus. we then tried the mouse challenged with lcmv and failed again but we observed hemorrhagic disease in the mouse, a severe consequence of infection that mimics the worst aspects of arenaviral infection. we decided to try targeting host genes to mitigate disease in the mouse and found targeting il-17 provided a survival benefit (schnell et al. 2012) . the success in the face of failure puts targeting host genes at the center of attention for dealing with emerging infectious diseases. the antisense platform technology represents an excellent approach to rapid drug discovery for emerging infectious disease. rapid discovery demonstrated repeatedly but of course, the cost of advanced development is daunting. the application is best for immediate treatment of index cases, close contacts, and healthcare workers. we have an 800-pound gorilla in the room and everyone in the room considers it someone else's problem. the current course is to ignore the majestic creature until it starts tearing limbs from people and then the consensus is to kill the gorilla. agencies like the world health organization (who), the centers for disease control (cdc) and the national institutes of health (nih) can assemble highly skilled personnel and can confer with some of the greatest minds in the world. unfortunately, they are all aware of the potential problem that an emerging pandemic is likely to take us by surprise. significant speculation that a single-stranded rna virus will emerge killing tens of million people, costing hundreds of billions of dollars, and changing the course of human history. there is a high probability that the virus will be influenza a (h5n1 or h7n9) that will jump from a reservoir population of birds and establish human-to-human transmission. the pandemic will be a global event by the time an effective vaccine is available. neuraminidase (na) inhibitors as therapeutic and prophylactic agents in the setting of pandemic influenza a (flua) were called in to doubt in the past decade (michiels et al. 2013; jefferson et al. 2014) . indeed, 100% of isolates in the 2008/2009 a/h1n1 pandemic were found to be resistant to adamantanes, and resistance to oseltamivir (tamiflu; osl) was observed in virus recovered from individuals taking osl therapeutically or prophylactically (dharan et al. 2008; cdc mmwr 2009) , while effect on duration of shedding was not impacted. a recent outbreak of an influenza a (h7n9) virus caused 137 cases and 45 deaths in china revealed a novel na mutation r292k resulting in high level resistance to osl (wang et al. 2014) . therapeutic options for treatment of individuals with complicated influenza a are severely limited, perhaps no options. pandemic strains such as a/h1n1pdm09 carried significant morbidity and mortality, particularly in those who had not experienced h1-strain influenza in their lifetime. we are also witnessing more rapidly emerging highly pathogenic avian influenza strains that are resulting in human infection; some such as a/h5n1 and a/h7n9 appear to becoming more efficient in person-to-person transmission, and reports suggest osl resistance develops during treatment (de jong et al. 2005; lam et al. 2015) . we are not ready for an outbreak of avian flu or any other emerging single-stranded rna virus. why not? an estimate for the time and cost to develop a new drug is 10 years and $1 billion. the commercial use of a drug for an emerging infection is hard to estimate since by definition when you start development the infection has not emerged. most of us would be unlikely to use our retirement savings to invest in a drug development project with no reliable way to expect a return on our investment. it is a poor business model. when you consider there may be hundreds of emerging infectious diseases each times 10 years and $1 billion each the task is daunting. on which disease should we focus? consider the ebola drug avi-7537. the ebola therapeutic project began in 2004 following a laboratory accident at usamriid. we identified three compounds each with activity and when combined we observed unprecedented efficacy in a lethal challenge primate animal model (warfield et al. 2006 ). hundreds of experiments over the next 3 years optimized these agents (swenson et al. 2009 ). we were able to obtain research grants from the transformational medical technologies (tmt) division of the defense threat reduction agency (dtra) within the department of defense (dod) and we completed proof of efficacy studies (warren et al. 2010) . this led to submission of an investigational new drug application (ind) to the food and drug agency (fda) and phase i safety and tolerability studies were conducted i healthy human volunteers (heald et al. 2014) . after 11 years of continuous effort, we completed key aspects related to the fda approval process under the "animal rule" and we streamlined our treatment to a single agent, avi-7537 (warren et al. 2015) . however, shortly before the ebola outbreak in western africa, the us government "budget sequester" cancelled our project and the most advanced therapeutic on the planet was not deployed to treat those infected during the outbreak. as the outbreak continued, we found no viral resistance of avi-7537 unlike the monoclonal antibody therapy in use (khiabanian et al. 2014) . avi-7537 sits on a shelf, a political casualty and an unfavorable business model. the global virosphere may contain up to 10 31 virus/virus-like particles (suttle 2005) , the greatest reservoir of genetic diversity. the earth's atmosphere transports viruses all over the planet. viruses are found in soil at 1.5 x 10 8 to 6.4 x 10 8 particles per gram of dry soil (kimura et al. 2008) . the surface oceans carry approximately ten million viral particles in each milliliter of seawater, most of which are bacteriophages (phage). the small viral particles are easily carried into the upper environmental viral reservoirs atmosphere by up drafting winds. bacteria are deposited from the atmosphere at a rate of 0.3 to 8 x 10 7 per meter each day and viral deposition rates are 9-461 times greater (reche et al. 2018) . these phages influence bacterial lifecycles and play a role in natural energy and nutrient cycles fundamental to life on earth. the dynamics of phage-bacterial evolution drive changes in photosynthesis, phosphate, and nitrogen balance (breitbart 2012) . human accidental release of radioactive waste (discussed in chap. 2) and disposal of chemicals including potent antiviral and antibacterial compounds (discussed in chap. 7) may alter the eco-evolutionary dynamics producing unanticipated environmental consequences. the objective of this chapter has been to provide convincing evidence that infectious disease is the most significant threat to human health. the focus has been on viral infections because they rely on host ribosomes to produce their proteins, recent emerging infections have been from single-stranded rna genome viruses, and replication of rna viruses is error prone. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but have never prepared for these inevitable catastrophies. the long-term consequences often remain unrecognized and are always inconvenient such as cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. infectious disease will certainly continue to pose the most significant threat to human health in the age to cell phones, artificial intelligence, and global commerce. rapid replication of viral genomes combined with low fidelity polymerases provide the foundation for an unending source of new emerging infectious agents. these traits also make viral genomes sensitive to environmental contaminants in a way that may expand probabilities for zoonosis. infectious disease as part of our environment is not appreciated. the study of infectious disease is not a part of the curricula of students in environmental science/management. textbooks in in environmental studies do not include chapters in infectious disease. the integration of research at superfund sites focused on chemical contamination with infection and zoonosis would result in valuable insights into threat analysis. viruses with rna genomes lack sequence proofreading quality control during replication. the cumulative mutations in their genomes limits the genome size to under 30,000 bases. essentially, a larger genome would evolve out of existence, so called catastrophe evolution. the limited genome size makes these viruses exceptionally resilient to a changing environment. the virus must economize by combining functions. this means evolution and resilience are the same thing in the rna genome viruses. a unique insight is that in human evolution is restricted to the dna genome and resilience limited to rna, as it is in the rna genome viruses. antisense oligonucleotide-based therapy for hiv-1 infection from laboratory to clinical trials oligodeoxynucleotide phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus a tribute to sheik humarr khan and all the healthcare workers in west africa who have sacrificed in the fight against ebola virus disease: mae we hush inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of 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placebo-controlled trial inhibition of foot-and-mouth disease virus in cell cultures with antisense morpholino oligomers antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture pcr for detection of oseltamivir resistance mutation in influenza a(h7n9) virus igg antibodies to dengue enhanced for fcγriiia binding determine disease severity gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced antisense therapies for postexposure protection against lethal filovirus infections single component avi-7537 antisense compound provides greater protection than double component avi-6002 against lethal ebola virus infection in rhesus monkeys isolation of an arenavirus closely related to lassa virus from mastomys natalensis in south-east inhibition of coxsackievirus b3 in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosomal entry site inhibition of rous sarcoma virus replication and transformation by a specific oligodeoxynucleotide characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with burkitt and non-burkitt lymphomas effect of early and late gb virus c viremia on survival of hiv-infected individuals: a meta-analysis suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions key: cord-016070-e9ix35x3 authors: perret pérez, cecilia; ferrés garrido, marcela title: pneumonia caused by emerging viral agents date: 2020-02-01 journal: pediatric respiratory diseases doi: 10.1007/978-3-030-26961-6_34 sha: doc_id: 16070 cord_uid: e9ix35x3 emerging viruses that cause pneumonia in humans are agents which normally circulate in the animal population but can move to human hosts under certain circumstances, which determines the occurrence of a new type of disease. the middle east respiratory syndrome (mers) is caused by a coronavirus. the disease has a wide symptomatic spectrum that can range from asymptomatic infections to fulminant respiratory failure. diagnostic confirmation is achieved through viral isolation. severe acute respiratory syndrome (sars), also produced by a coronavirus, is capable of producing a serious pulmonary disease outbreak with no reappearance. the clinical presentation includes fever, malaise, cough, and headache followed by diarrhea. other coronaviruses (hcov-nl63 and hcov-hku1) can cause serious lower respiratory infections in small children, the elderly, and immunosuppressed patients. influenza virus is widespread in nature, and avian virus may spread to humans, as has been reported with h7n9, h5n1, h10n8, and h6n1. cardiopulmonary hantavirus syndrome, a feverish disease characterized by respiratory insufficiency and shock, is produced by andes virus. other emerging viruses are enterovirus d68 and polyomavirus. emerging viruses that cause pneumonia in humans have a common trait: they are all zoonoses. normally, these agents circulate in the animal population, often without causing morbidity, but under certain circumstances they can move to human hosts, which determines the occurrence of a new type of disease. nature holds many examples of such diseases, including avian influenza, ebola, and severe acute respiratory syndrome (sars) . in this chapter we review agents that have provoked major concern outside their country of origin, but also hanta virus, because of the endemic nature it has acquired in chile. the middle east respiratory syndrome (mers) is caused by mers-cov, a recently identified coronavirus. several coronaviruses can cause upper respiratory tract infections, but in some cases they can also produce lower respiratory tract infections and flu-like states. the sars coronavirus and mers-cov are two pathogens from the coronavirus family that predominantly cause serious lower tract respiratory infections with a high mortality rate, but they are genetically different viruses. mers-cov was first identified in 2012 following the death of a patient in saudi arabia with a serious respiratory infection. this finding led to the retrospective diagnosis of the first cases of an inhospital outbreak during 2012 in jordan. most cases have been recorded in the middle east, with more than 75% of the cases in saudi arabia and some cases outside this region affecting travelers. the mortality rate of mers is approximately 30%. information about the mers mechanism of transmission is still limited, but it is likely to occur through droplets and by direct and indirect contact with infected respiratory secretions. aerosol transmission has not been ruled out. currently, transmission between humans is limited and occasional, with a low secondary attack rate. isolated cases have been recorded, consisting of nosocomial and household outbreaks, but transmission is not sustained over time. this was the situation in the major south korean outbreak, which originated through a case in saudi arabia and affected more than 180 people. adequate infection control measures in healthcare rapidly limit in-hospital transmission. genetic analysis shows that the human mers-cov is quite similar to the virus found in bats and essentially identical to that observed in camels. the virus appears to have originated in bats, transitioning through camels, probably in africa, and afterward being transmitted to humans. serological studies do not show its presence in humans before 2012, but it has been observed in camels since the 1990s. this observation suggests that camels are the reservoirs of the virus, which can be transmitted to humans through direct contact with these animals or through consumption of their milk: 1599 cases had been diagnosed by july 2015, with 574 deaths [world health organization (who)]. of these patients, 63% are male, with an average age of 48 years ( fig. 34.1 ). in children, the disease tends to be milder or asymptomatic, with severe cases resulting from a comorbidity. incubation, defined as the period between the primary and the secondary case, is estimated to last an average of 5 days (2-14 days). clinical presentation is characterized by fever, cough, myalgia, and diarrhea. the disease has a wide symptomatic spectrum, which can range from asymptomatic infections to fulminant respiratory failure, which is related to high mortality. patients evolve rapidly (5-day period on average) to respiratory and kidney failure, requiring ventilation support and intensive care management in more than 60% of cases. diagnostic confirmation is achieved through viral isolation in laboratories with biosecurity clearance level 3; alternatively, the virus is detected in respiratory samples through molecular biology techniques (polymerase chain reaction, pcr), which are only available in reference laboratories. in patients who have displayed symptoms for more than 14 days, the determination of specific antibodies is recommended. there is no specific treatment, because no antiviral therapy is available, only support measures in intensive care for the most serious cases. there is no specific preventive vaccine. this infection should be suspected in every traveler who reports having traveled to the arabian peninsula during the past 14 days and who also presents with respiratory symptoms and fever. sars is the first identified coronavirus capable of producing a serious pulmonary disease, unlike those previously known (hcov-229e and hcov-oc 43), which cause the common cold. sars emerged in late 2002 in china and quickly disseminated through southeast asia, europe, africa, and america. bats were its presumed reservoir, and it was transferred to humans through civets. this virus caused great international alarm because of its rapid progression and its seriousness, resulting from extensive and rapidly progressing pneumonia with a mortality rate around 50%. health workers were particularly affected. the most constant symptoms during admissions were high fever, malaise, cough, and headache, followed by diarrhea and prolonged fever. more than 8000 cases were noted, causing more than 700 deaths. thanks to international coordination under who leadership, the epidemic was controlled in july 2003. there were five clear objectives, which were achieved in record time: identification of the etiological agent, development of diagnostic tests for virus detection, creation and evaluation of epidemiological treatment protocols to reduce morbidity and mortality, definition of key epidemiological parameters to control transmission, and formulation of appropriate public health measures. sars demonstrated that coronaviruses can cause serious lower respiratory infections, which would later be observed for hcov-nl63 and hcov-hku 1. to date, no new circulation of this agent has been proven. coronavirus nl 63, which belongs to coronavirus group i, was discovered for the first time in a child with bronchiolitis in the netherlands in 2004, and then ratified in small children hospitalized for serious respiratory infections. hku1, which belongs to the coronavirus group ii identified in 2005, was also shown to be capable of causing a lower respiratory tract infection. both have been described throughout the world, proving their ubiquity. this virus is detected in up to 10% of acute respiratory disease cases, and its symptoms range from upper respiratory disease, including flu-like disease, fever, rhinitis, odynophagia, and cough, to serious conditions with a rapidly progressing respiratory disease. infection by hcov-n63 in children manifests as an obstructive laryngotracheitis in up to 45% of cases in comparison with children not infected by this virus. the first hcov-hku1 viruses were described in elderly patients with preexisting conditions that caused their death. some later studies, which considered children as well as adults, have confirmed that infection caused by this coronavirus may worsen the health status of individuals with underlying diseases, so that they need to be hospitalized more often. as with hcov-n63, infection causes upper respiratory tract symptoms, such as fever, coryza, odynophagia, and coughing. wheezing, pneumonia, and bronchiolitis may also be present. in children a greater fre-quency of feverish convulsions has been observed in comparison with children who were not infected by hcov-hku1. hcov-nl63 and hcov-hku1 are viruses that tend to manifest as a common cold, just as the usual coronaviruses hcov-229e and hcov-oc43; nevertheless, in small children, elderly patients, and immunosuppressed patients, they can cause serious respiratory disease with a high mortality rate. the influenza a virus is widespread in nature: its main reservoir is domestic and feral birds. several types exist, according to their hemagglutinin (h) and neuraminidase (n) makeup, only two of which have recently circulated among humans and are causing seasonal outbreaks: h1n1 and h3n2. the animal reservoir of the virus is large and varied. several subtypes have been described, which include 17 hemagglutinin and 9 neuraminidase types. these zoonotic viruses may cause diseases and infections in several animals, generally pigs and poultry. these zoonotic viruses in particular adapt very poorly to humans, so they seldom cause human diseases, which are limited to outbreaks circumscribed in terms of time and population. nevertheless, human cases of a h5n1 were observed in hong kong for the first time in 1997 and have continued being reported to date. more than 700 cases have been notified, all in asia and northern africa, with 413 deaths. most cases are isolated, with some intrafamilial clusters being described. human-to-human transmission is rare, which reflects the poor ability of the virus to adapt to human respiratory mucosa. the persistence of the circulation of these viruses and the great genetic variability of the influenza a virus make us fear that the avian virus may adapt to humans, which would ease its transmission among the human population, potentially causing a pandemic: this is what happened with the h1n1 influenza a, which had a porcine origin and adapted to humans, creating a pandemic in 2009. the first cases of human infection caused by h7n9 influenza a were reported in china during 2013, and most of these were related in one way or another to contact with poultry, whether direct or environmental in markets where live birds are commercialized. by 2015 there had been 488 confirmed h7n9 influenza cases in china since the beginning of the outbreak, with 185 deaths. this virus does not seem to spread easily among humans, and there has been no proof of sustained human transmission, although its transmission potential seems to be more effective than that of the h5n1 influenza a virus. a couple of cases have been reported outside china: a traveler in malaysia who stayed in china and a man and his wife who were diagnosed in canada in january 2015 after traveling to china (who). there are no recorded secondary cases in these countries, which confirms its low probability of human-tohuman transmission. cases have been observed during the coldest seasons in china. from december 2014 to february 2015, 83 new cases were diagnosed, with an average age of 56 years and 19 deaths. of these newly diagnosed patients, 72% are male, and 93% of patients have had direct contact with poultry markets. the incubation period is 4 days (2-8 days). even though mild cases have been described, most diagnoses have been serious, with a mortality rate close to 35%. hospitalized patients have a febrile disease, with temperatures above 102.2 °f and cough. the disease progresses swiftly from moderate to severe. in contrast to human seasonal influenza, most patients do not report rhinorrhea or odynophagia. in a set of hospitalized patients, the disease progressed swiftly for an average of 3 days after its onset, counting from the beginning of the symptoms until hospitalization. almost 70% required invasive mechanical ventilation, with a death rate of 30%. deaths from respiratory failure reached 38%, and 62% were caused by septic shock. most deaths corresponded to elderly patients and with a underlying disease, as well as to the use of systemic steroids. diagnosis is performed by identifying viral rna through rt-pcr in respiratory tract samples, obtained through swabbing or nasal suctioning. as well as other avian influenza a virus, this virus is sensitive to oseltamivir and is resistant to adamantines. oseltamivir is indicated for hospitalized or ambulatory patients, whether they have been confirmed or are under suspicion of being infected by h7n9 influenza, even if more than 48 h have passed since the onset of symptoms. the dosage and timeframe of therapy are not clearly established for serious patients, but a longer timeframe is suggested, around 10 days and in higher doses. in patients with mild and noncomplicated infections, therapy must continue for 5 days. patients with mild infection, who require ambulatory treatment and whose only exposure factor is travel to an area where there are recorded cases, in humans or birds, have no empirical indication for oseltamivir. isolated cases of avian origin in humans caused by the influenza h10n8 virus and h6n1 have been observed in china. these facts prove that in these zoonotic influenza cases vigilance is extremely important, given its pandemic potential, so it is crucial to pay close attention to travelers and enforce local vigilance. in 1993, a new virus from the the bunyaviridae family was identified in the united states of america. it was named "virus with no name" and was deemed responsible for what is now known as cardiopulmonary hantavirus syndrome (síndrome cardiopulmonar por hantavirus, scph), a feverish disease characterized by respiratory insufficiency and shock. this discovery, which was a new zoonosis, in practice extended over the next years across the whole american continent. in this process, new clinical manifestations were recognized and new agents identified, including the andes, laguna negra, araquara, and choclo viruses, which are prevalent in chile, argentina, paraguay, brazil, and panama, respectively. their natural reservoir are sigmondontinae rodents, which belong to the muridae family. these animals develop a chronic infection with an intermittent viral and asymptomatic excretion. stress situations, such as lack of food during birthing periods, cold, or habitat interventions such as logging, have been associated with greater virus excretion in these rodents. mice excrete the virus through their feces, urine, and saliva, contaminating the environment. the most representative virus-carrying rodents are peromyscus maniculatus or "deer mouse" (which carries the no-name virus), oligoryzomys longicaudatus or "long-tailed mouse," and calomys laucha, among others. humans acquire the infection through the inhalation of secretions (stools, urine, saliva) of infected rodents. the andes virus, predominant in southern argentina and the single causal agent in chile, is only transmitted through close human-to-human contact. hantaviruses are spherical viruses with a trisegmented rna genome with a lipid envelope through which two glycoproteins (gn and gc) protrude. these three segments code through proteins such as rna polymerase (l segment); glycoproteins gn and gc (m segment), which are important in the recognition of β3 integrins that the virus use as receptors; and nucleoprotein (s segment), a highly conserved protein used for the laboratory diagnosis of these agents. the lipid envelope is sensitive and is destroyed by detergents, chloride, desiccation, and sun exposure. all these actions form the basis of the prevention and control recommendations for hantavirus infections. the agent enters the respiratory tract through inhalation of the aerosolized virus in the environment or through contaminated human secretions. after an incubation period that ranges from 1 to 6 weeks (18 days on average), nonspecific symptoms begin, including fever, myalgias, and headache, plus digestive symptoms (more common in children). this stage is followed by progressive respiratory disease and finally by respiratory failure, which is the most serious manifestation of this infection. in 100% of the cases of acute infection, the virus is present in all the white cells and, in variable proportions, in plasma, respiratory secretions, saliva, and urine. also, viral rna has been detected in white cells up to 15 days before and 90 days after the first symptoms. interaction with β3 integrins and the replication of viral endothelial cells of various tissues appear to alter the modulation functions of permeability in these cells, especially increasing the permeability in small lung vessels, which favors arterial hypotension, thrombocytopenia, and hypoxia from plasma flooding into alveolar spaces. protein n and superficial glycoproteins stimulate the production of specific and neutralizing antibodies, which have been associated with better survival outcomes when they increase prematurely. patients who progress to the cardiopulmonary phase, where respiratory failure sets in, require supplementary oxygen; in addition, more than two thirds need mechanical ventilation, and 50% of patients develop cardiogenic shock, which constitutes a poor prognosis factor. the increased vascularity rate explains the pulmonary edema observed during this stage. this functional alteration is transitory, lasting from 48 to 72 h; afterward, pulmonary function is quickly recovered following a brief period of noticeable diuresis. cardiogenic shock is difficult to manage and is the main cause of death. the lethality of hanta cardiopulmonary syndrome is about 35%, and most deaths occur during the first 48 h of evolution. the hemogram is the most useful general laboratory test for hypothesizing a diagnosis, because it can, from an early stage, reveal manifest reductions in the number of platelets as well as the presence of lymphocytes, which take the shape of immunoblasts. a late onset of hematocrit increase has been observed, which is concomitant with the beginning of the cardiopulmonary phase of the disease. it is also helpful to test for ldh and transaminases, which increase nonspecifically. a chest x-ray may change in a matter of hours from a nonspecific interstitial pattern to diffuse pulmonary edema. the virological diagnosis is confirmed through rt-pcr in white cells or through the elisa detection of specific igm/igg for each regional virus. currently, there is no specific treatment for hantavirus infection other than cardiopulmonary support. patient should be monitored closely, preferably in an intensive care unit, to provide measures such as mechanical ventilation for breathing support, restriction of liquids, and vasoactive drugs. the use of an antiviral such as ribavirin has not been shown to be an effective treatment. extracorporeal membrane oxygenation (ecmo) has been used as a rescue therapy in some cases of serious cardiopulmonary failure that do not respond to conventional ventilation and vasoactive drugs. in chile, two therapeutic options have been investigated for hantavirus: methylprednisolone in high doses and immune plasma with high neutralizing antibody titers. only the latter strategy has shown promising results relative to mortality rate reduction when used immediately after symptom onset. as a control measure in hospitalized patient management, and considering that hantavirus has been described as capable of causing nosocomial transmission, standard precautions must be taken: ideally, interning the patient in an individual room and wearing protection equipment [apron, gloves, face mask (no. 95) with a high efficiency filter, and security glasses], especially in procedures during which there is close contact with the patient's fluids, such as intubation, secretion suctioning, and retrieval of samples for laboratory tests. this viral agent, enterovirus d68, has been known since 1962, when it was isolated in children suffering from bronchiolitis and pneumonia. because no widely available trials have been conducted, not much is known about its epidemiology and clinical manifestations. during the fall of 2014, missouri and illinois hospitals reported an unusual rise in the number of serious cases of children, with or without a background of obstructive disease, who presented with acute respiratory infection. enterovirus d65 was detected in these patients by applying molecular biology techniques to respiratory samples. the infection is more frequent in school-age children, whose most relevant clinical antecedent was the presence of previous persistent coughing, asthma, or wheezing episodes that may have required intensive care unit (icu) management. the virulence of this agent is more impressive than that of other enteroviruses, considering that it was also identified as a causal agent of obstructive episodes in previously healthy children who, when treated with antiasthma therapy, did not respond adequately and had to be hospitalized in intensive care because of hypoxemia and, with some exceptions, had to receive mechanical ventilation. the most common laboratory findings were high total neutrophils and chest x-ray showing peribronchial interstitial infiltrations, hyperinsufflation, and atelectasis. as diagnostic tests improve in sensitivity and specificity, and their use becomes more widespread in pediatric centers, our understanding of the epidemiology and pathogeny of this viral agent will increase. two polyomaviruses with respiratory tropism were discovered in 2007 through deep sequencing of samples taken from respiratory secretions of symptomatic patients: polyomaviruses ki (kipyv) and wu (wupyv). these viruses can be found in the lower and upper respiratory tract of immunocompetent as well as immunocompromised patients. their pathogenic role is not completely clear, because they usually occur at low frequency, have a low viral load, and are related to pathogens whose morbidity is better characterized. diagnosis requires molecular techniques (real-time pcr), whose inclusion in future research will allow us to better characterize the epidemiology and clinical spectrum of the infections caused by these agents. clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection confirmed human cases of avian influenza a (h7n9) reported to who prospective evaluation of household contacts of persons with hantavirus cardiopulmonary syndrome in chile hantaviruses and cardiopulmonary syndrome in south america prospective population-based study of viral lower respiratory tract infections in children under 3 years of age (the pride study) human infection with a novel avian-origin influenza a (h7n9) virus human hantavirus infections: epidemiology, clinical features, pathogenesis and immunology person-to-person household and nosocomial transmission of andes hantavirus middle east respiratory syndrome coronavirus disease in children clínica de prevención, diagnóstico y trata-miento del sindrome cardiopulmonar por hantavirus web site minsal the novel human coronaviruses nl63 and hku1 update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities hantavirus immunology of rodent reservoirs: current status and future directions summary of current situation, literature update and risk assessment novel avian-origin influenza a (h7n9) in critically ill patients in china key: cord-002337-8v907g24 authors: lipsitch, marc; barclay, wendy; raman, rahul; russell, charles j; belser, jessica a; cobey, sarah; kasson, peter m; lloyd-smith, james o; maurer-stroh, sebastian; riley, steven; beauchemin, catherine aa; bedford, trevor; friedrich, thomas c; handel, andreas; herfst, sander; murcia, pablo r; roche, benjamin; wilke, claus o; russell, colin a title: viral factors in influenza pandemic risk assessment date: 2016-11-11 journal: nan doi: 10.7554/elife.18491 sha: doc_id: 2337 cord_uid: 8v907g24 the threat of an influenza a virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. to date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. the goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. we describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin ph of activation, and polymerase complex efficiency) that contribute to pandemic risk. doi: http://dx.doi.org/10.7554/elife.18491.001 aquatic birds are the main reservoir of influenza a viruses in nature (krauss and webster, 2010) . influenza viruses from aquatic birds sporadically enter terrestrial bird and mammalian host populations and achieve sustained circulation in these new hosts (vandegrift et al., 2010) , sometimes after reassortment with influenza viruses already circulating in the new host (webster et al., 1992) . adaptation of viruses from aquatic birds to mammals involves a change in tissue tropism from intestinal to respiratory epithelia (hinshaw et al., 1979; hénaux and samuel, 2015) . multiple influenza a subtypes-defined by the patterns of antibody recognition of two surface proteins, hemagglutinin (ha) and neuraminidase (na)-circulate in avian species and swine at any given time. among these, a number are known to cause sporadic zoonotic infections in humans (peiris, 2009 ). more than one thousand human infections with avian influenza viruses were detected in the last decade, for example h5n1 and h7n9 (qin et al., 2015) as well as swine influenza viruses, e.g. an h3n2 variant that spilled over into humans attending agricultural shows in the early 2010s, h3n2v (jhung et al., 2013) . in addition, zoonotic infections with other viruses from poultry or wild birds have occurred, including for example h7n7 (fouchier et al., 2004) , h10n8 (wohlbold et al., 2015) , h6n1 (wei et al., 2013) , h9n2 (butt et al., 2005) , and h5n6 ; for more examples and a fuller discussion see (short et al., 2015) . the severity of zoonotic influenza a infections ranges from clinically inapparent (gomaa et al., 2015; to et al., 2016) to fatal (de jong et al., 2006; gao et al., 2013) . although secondary transmission can occur following some of these spillover events (kucharski et al., 2014) , only a very small proportion of them-four in the last hundred years, which seems to be close to the historical average (patterson, 1986 )-led to sustained person-to-person transmission with global spread (box 1). there are 18 known ha types and 11 known na types (tong et al., 2013) , which could theoretically be found in any combination. so far, sustained spread in humans has been limited to the h1n1, h2n2, and h3n2 subtypes , though it is possible that other subtypes circulated prior to 1918, the year of the first pandemic from which viruses are available for study (worobey et al., 2014) . multiple virus-host interactions are necessary for replication and onward transmission; the differences in the genetic requirements to accomplish each of these interactions in humans versus other animals provide a barrier to sustained transmission following spillover . experiments in ferrets have been used to measure viral transmissibility via respiratory droplets (in this review we use this term to refer to any transmission through the air between ferrets that are not in direct or indirect physical contact). droplet transmission in ferrets is a useful, albeit imperfect, correlate of the potential of influenza strains to transmit efficiently in human populations (buhnerkempe et al., 2015) . for this reason, some have argued that there is a box 1. steps in pandemic emergence. for an avian-adapted strain of influenza a to become a pandemic strain, several events are required: 1. the avian-adapted strain must become sufficiently widespread in wild or domestic birds, swine or other reservoir species to expose at least one human to infection. 2. one or more humans must acquire infection from the reservoir species. 3. the infection must replicate sufficiently in a zoonotic case to produce infectious virus in respiratory or other secretions. 4. the infection must be transmitted to additional humans, avoiding an "early" termination of the transmission chain due to chance. such early termination is a significant risk given the relatively low infectiousness of influenza and the moderate degree of overdispersion in the number of secondary cases infected by each case, both of which contribute to the probability that a transmission chain will terminate by chance (lloyd-smith et al., 2005; lipsitch et al., 2003) . it must also avoid extinction due to deliberate control efforts put in place by public health authorities (ferguson et al., 2006; merler et al., 2013) . 5. finally, the infection must spread beyond the local area to infect members of distant populations, a process accelerated by modern global travel (cooper et al., 2006) . this step and the one before are enhanced if the level of population susceptibility is high, as occurs when the surface proteins of the new strain are dissimilar to those on any currently or recently circulating human influenza a strains. we know from serologic studies and human infections that several different influenza a viruses have achieved steps 1 and 2 at any given time (gomaa et al., 2015; to et al., 2016) . steps 3 and/or 4 appear to be the rare, rate-limiting steps; that is, the conditional probability of achieving step 3 and 4 given the previous steps is low, so that sustained human-to-human transmission of a novel strain occurs a few times per century while zoonotic infections must occur thousands or more times per year. no case is known in which an influenza a strain has reached stage 4 but failed to reach stage 5, although it may have happened undetected. the appearance -by mutation or reassortment -and selection of genetic changes that encode human-adaptive viral traits may be seen as a process that can accelerate or increase the probability of one or more of these steps (though there is no guarantee that a given change that enhances one of these steps will enhance all of them. this is why the detection of phenotypes associated with human adaptation in avian or zoonotic isolates of novel influenza a viruses is thought to correlate with increased risk of pandemic emergence. as we describe throughout the paper, the process of human adaptation need not be complete to initiate a pandemic, so it may continue to occur at various stages throughout this progression. doi: 10.7554/elife.18491.002 general phenotype of 'transmissibility by respiratory droplets in mammals' such that experiments to select for such transmission in droplets in ferrets are important models of the process of adaptation to human transmission (imai et al., 2012; herfst et al., 2012) . this view is not universally shared . starting from an zoonotic highly pathogenic avian influenza isolate from a human case of infection (or a reassortant of the ha from a different zoonotic h5n1 highly pathogenic avian influenza isolate, with the other segments from the 2009 pandemic h1n1 strain), it was shown that certain specific traits that had been previously associated with mammalian host adaptation were required to achieve respiratory droplet transmission. these ferret-transmission phenotypes in turn were associated with certain genetic changes relative to the original avian viruses (imai et al., 2012; herfst et al., 2012; linster et al., 2014) . these specific changes occur both in ha and in polymerase-complex proteins. the rationale for these experiments was that, because the ferret model recapitulates many features of human infection, changes identified in adaptation to ferret transmission would also be important for adaptation to sustained transmission in humans schultz-cherry et al., 2014) , though this can never be known with certainty . notably, viruses isolated from humans who were infected by contact with birds show some of these changes (russell et al., 2012; bi et al., 2015) , particularly the change at amino acid 627 of the pb2 gene (jonges et al., 2014; fonville et al., 2013) , which often affects polymerase complex efficiency (see below). this indicates that even the first generation of human infection from nonhuman hosts can initiate a process of host adaptation. it also indicates that not all the human-adaptive changes must be in place in the avian reservoir to initiate this process. some human infections, including some zoonotic cases (de jong et al., 2006; chen et al., , 2006 sha et al., 2016) and some cases early in a pandemic (rogers and d'souza, 1989; connor et al., 1994; glaser et al., 2005; pappas et al., 2010) , involve viruses that are not yet fully human-adapted (see below and tables 2-4). the interpretation of some of these isolates is complicated by uncertainty about whether they were passaged in hen's eggs at some point in their history. certain types of countermeasures against an influenza pandemic are effective only against one lineage of viruses -for example, creating stockpiles or seed stocks of vaccines against a particular subtype, or culling poultry infected with that subtype. it is not currently feasible to invest in such countermeasures against all viruses circulating in avian or other reservoirs, or even against all those causing known zoonotic cases. therefore, there would be value in an accurate system to assess the relative pandemic risks posed by each virus and prioritize them for the development of such strain-specific countermeasures, while directing fewer resources to strains of lower concern (kaplan et al., 2016) . this consideration has motivated calls for comprehensive analysis of all available data to assess the threat to public health posed by these strains. one response is the cdc's influenza risk assessment tool (irat) (trock et al., 2015) , which incorporates elements including properties of the virus, field and epidemiological findings, and attributes of the human population to provide a framework to differentiate among novel influenza viruses thought to possess pandemic potential. such risk assessments can help focus pandemic prevention and response efforts on the viruses thought to pose the highest risk of pandemic spread , in the most worrisome cases providing a rationale for costly measures such as poultry culling or vaccine seed stock development, or even stockpiling of large quantities of vaccine. a guiding question of this article is to examine the degree to which it is justified to rely on measurements and predictions of viral genetic and phenotypic traits in prioritizing responses to particular viral subtypes and within-subtype lineages. there are several hurdles to evaluating the accuracy of such predictions . factors limiting our ability to identify high-risk viruses and predict the risk they pose include: . limited surveillance of nonhuman influenza viruses, such that high-risk viruses may not be detected and hence cannot be assessed (butler, 2012) . limitations include both the number, geographic and species diversity of hosts sampled, and the difficulty in sampling all genetic variants present in a given infection (poon et al., 2016; varble et al., 2014) ; . failure to fully characterize some viruses that are detected (hoye et al., 2010) ; . imperfect public health systems lacking capacity to detect zoonotic infections presenting in patients (sanicas et al., 2014; simonsen et al., 2013) ; . epistasis and other complexities that prevent straightforward prediction of viral traits from genotype kryazhimskiy et al., 2011; gong and bloom, 2014; bloom et al., 2010; wu et al., 2016; raman et al., 2014; tharakaraman et al., 2013; gong et al., 2013) ; . technological limitations in molecular modeling and phenotypic assays that limit confidence in predicting and measuring viral traits (russell, 2014) ; . uncertainties about the taxonomic level at which risk predictions should be made (box 2); . practical, ethical and cost limitations of animal transmission experiments, as well as some exceptions to the correlation between human transmissibility and droplet transmissibility in nonhuman animal models (buhnerkempe et al., 2015) ; . lack of data on immediate animal precursors of viruses that caused previous pandemics; . multiple scales at which viral strains compete and hence experience selection (i.e. replication within hosts, transmission between hosts). evolutionary theory for such multi-scale selection is incomplete. viral fitness components are rarely measured at both scales for the same strain and are imperfectly correlated across scales (gog et al., 2015; park et al., 2013; beauchemin and handel, 2011) ; . the role of stochastic events in the ecology and evolution of influenza viruses during and after host-switching to humans (gog et al., 2015; lloyd-smith et al., 2015) , including the potential for transmission bottlenecks to either promote or inhibit emergence of human-adapted viruses (varble et al., 2014; moncla et al., 2016; wilker et al., 2013; zaraket et al., 2015) . these difficulties are exacerbated by the fact that influenza pandemics are rare events, and that risk assessments are not yet made with enough quantitative precision to formally evaluate their practical application. even perfect information about the viral determinants of pandemic risk might only be enough to distinguish between strains with a low risk of causing a pandemic (say, 0.1% per year) and those with an extremely low risk (say, less than 0.01% per year), with unpredictable ecological or evolutionary contingencies determining which of these low-probability events will actually occur. one such contingency is that an avian influenza virus could acquire one or more of the determinants of pandemic potential by reassorting with a human seasonal influenza virus. with only one pandemic every few decades, the data set for testing the prediction of such rare events is inadequate, a problem that challenges predictions in many fields beyond infectious diseases (king and zeng, 2001; hansson, 2006) . evolutionary events in which a strain increases human-to-human transmissibility, but not enough to spark a pandemic, are extremely hard to observe, but if we could do so it would increase our ability to characterize the process of adaptation (kucharski et al., 2015) . despite these challenges, there has been tremendous interest and investment in making and using such predictions, and a number of new ideas to improve predictions are in various stages of development (box 3). building on the findings of a previous workshop , we considered in detail the present state of knowledge concerning three phenotypic traits: ha receptor binding specificity, and ha ph of activation, and polymerase complex activity, (figure 1 ). these were chosen from a longer list of candidate traits (table 1 ) because they span the viral life cycle ( figure 1 ) and their role in host adaptation has been extensively studied. all three are believed to be required for an influenza virus to cause a pandemic; consistent with this assumption, all three traits have been present to some degree in the earliest viruses isolated in pandemics since the 20th century, though some have been enhanced by subsequent evolution during seasonal transmission in humans. moreover, for each of these three traits, viruses isolated from avian hosts typically do not show the mammalian-adapted phenotype, reflecting divergent selection pressures in the two classes of hosts (tables 2, 3, and 4). all three traits changed in the adaptation of zoonotic h5 influenza viruses to droplet transmission in ferrets (imai et al., 2012; linster et al., 2014) . we emphasize that each of these traits is quantitative, and that human-adaptation is not a threshold criterion but a continuum; in this review when we speak of human adaptation we mean a tendency to be better adapted to humans, rather than an absolute yes-or-no property. this review starts with a summary of our knowledge about the role of each of the three functional traits in conferring pandemic potential on a virus strain. following these case studies, we draw some generalizations about the prospects of predicting pandemic risk from virus genotype or from assays of particular viral traits. for each trait we present a table showing the degree to which the sequence changes or phenotypic properties associated with avian or human adaptation are present in isolates from birds and humans, respectively. if the avian traits were always found in avian isolates and human traits always in human isolates, only the shaded cells on the main diagonal would be filled. in such a case, however, it is hard to see how viruses would ever make the jump from birds to humans, since so many traits would have to change simultaneously, and indeed the off-diagonal cells are not empty. finding avian-adapted traits in viruses isolated from humans most often occurs in zoonotic cases, showing that not all human-adapted traits are required for the first human infection. in some cases there are also box 2. granularity of pandemic risk prediction -for what taxonomic level does it make sense? determining the appropriate taxonomic level for influenza virus risk assessment is a challenging endeavor. influenza virus subtype is a convenient classification but there can be substantial variation in estimable risk within subtype. for example, h5n1 viruses can be roughly segregated into high pathogenicity and low pathogenicity phenotypes with the high pathogenicity variants generating substantially greater concern for both human and animal populations. even within the high pathogenicity h5n1 variants, risk to animal populations and potential for adaptation to humans is likely to vary by phylogenetic lineages or clades of viruses. much of the difficulty for predicting the threat posed by subtypes or coarse grained concepts of virus variants stems from two factors: first, a lack of understanding of how genetic context affects the ability of a virus to adapt for efficient spread in humans; and second, the critical, and geographically variable, role of ecology in determining likelihood of cross species transmission. phylogenetic clade is a practical unit for risk prediction. however, in species where reassortment is frequent, phylogenetic clade must be considered on a gene by gene basis. the definition of phylogenetic clades can be challenging and arbitrary, but recent efforts to develop a unified nomenclature for highly pathogenic h5n1 viruses may offer a transferrable framework for the classification of other viruses (smith and donis, 2015 ; who/oie/fao h5n1 evolution working group, 2008) . clades of viruses circulating in poultry, swine or other domestic animals with extensive human interactions should be prioritized for surveillance and further study. foundational efforts are required to assess the diversity of viruses present in these animal populations, particularly for low pathogenic avian influenza viruses. further study will then be required to assess the abundance and prevalence of different virus subtypes and clades, along with geographic spread and overlap with ecological risk factors (hill et al., 2015; gilbert et al., 2014) , e.g. live animal markets, cohabitation of humans and animals, and biosecurity in animal processing facilities. antigenic characterization of animal influenza viruses should form part of a comprehensive risk assessment, particularly of viruses from swine and possibly dogs. swine influenza virus diversity is driven in large part by introductions of viruses from humans to swine (nelson et al., 2015 lewis et al., 2016) . the substantial antigenic diversity of viruses circulating in swine and antigenic differences with viruses circulating in humans poses an ever increasing risk for re-introduction into humans. much of the antigenic variation in swine has a strong relationship to phylogenetic clade (lewis et al., 2016) . similarly, the high contact rates between humans and dogs, combined with increased circulation of h3n2 canine influenza viruses, may present increasing opportunities for reassortment (na et al., 2015) and for zoonotic infections (flanagan et al., 2012) . doi: 10.7554/elife.18491.003 viruses isolated from humans after a pandemic starts that retain some degree of avian-like traits, and we discuss these in more detail in the text -these represent the greatest challenge to use of genotypic or phenotypic information for pandemic prediction because they run the risk of false negatives. the other off-diagonal cell, which represents avian isolates with some human-like traits, simply shows that some circulation of viruses in birds is possible without the classical 'avian' phenotypes. how this happens is a phenomenon worthy of further study. we conclude with some recommendations for future research and for the practice of pandemic risk assessment. trait 1: hemagglutinin receptor binding specificity attachment of an influenza virus to a host cell requires binding of the viral ha to a sialylated glycan receptor (sialic acid) on the surface of the host cell. cells of the avian gut and a minority of cells in deep lung in mammals predominantly express receptors terminated with an a2,3linked sialic acid: hereafter, a2,3 glycans or avian receptors gambaryan et al., 1997; van riel et al., 2006; shinya et al., 2006) . by contrast, in humans and other mammals, upper respiratory epithelial cells express mainly glycan receptors terminated by a2,6-linked sialic acid: a2,6 glycans or human receptors shinya et al., 2006; chandrasekaran et al., 2008) . the human upper respiratory epithelium is the primary target site for infection of human-adapted viruses, and infection at this site is thought to be a prerequisite for efficient human-to-human transmission via respiratory droplets. thus, it appears that human adaptation of an ha is associated with a switch in its binding preference from avian to human receptors. receptor binding is not either-or; human-adapted influenza virus ha may show some binding to avian receptors, and vice versa. receptor binding preference is defined as the ratio of affinity (or avidity) of an ha molecule for an a2,6 glycan relative to that for an a2,3 glycan, with higher values associated with greater human adaptation. the evolution of receptor binding specificity is driven by the host environment, with selection for specificity during the infection process within a host and during the process of transmission. the error-prone replication of influenza genomes can facilitate rapid emergence of viruses with amino acid substitutions that alter the receptor binding characteristics of the ha (lakdawala et al., 2015) . increased transmissibility may result from mammalian receptor adaptation, either because the virus shedding form the infected donor host is increased, or because the ability of virus to infect the recipient host at a low dose is enhanced, or for both of these reasons. recent experimental evidence in ferrets implicates the soft palate as an important site of selection for a2,6 specificity (lakdawala et al., 2015) . preference for binding human or avian glycan receptors is determined by the structure of the viral ha. except for a few conserved amino acids in the sialic acid receptor binding pocket, the influenza ha has considerable structural plasticity to evolve variation at the rim of the pocket to engage different sialic acid linkages. importantly, antigenic regions of the ha are located nearby regions that determine receptor-binding preference, meaning that selection for antigenic escape may be constrained by the need to maintain receptor preference (koel et al., 2013) . more speculatively, selection for changes in receptor preference might also alter recognition of the ha by host antibodies. conformation of hemagglutinin as a determinant of receptor binding preference. although the co-crystal structures of ha and sialylated glycans have not been solved for all pairs, there is evidence that avian-or human-adapted ha bind to different conformations of the avian and human receptors: the cis conformation of human receptors and the trans conformation of avian receptors ha et al., 2001 ha et al., , 2003 gamblin et al., 2004; liu et al., 2009; xu et al., 2010; yang et al., 2010; lin et al., 2012; yang et al., 2012; zhang et al., 2013) . this finding has led to the concept of "hallmark" residues within the receptor-binding site of avianand human-adapted has. avian-adapted has typically carry glu at position 190, gln at position 226, and gly and position 228 (h3 numbering), and the gln226->leu, gly228->ser substitutions have been associated with a switch to human receptor preference in has of h2, h3 (matrosovich et al., 2000) , and h5 (chutinimitkul et al., 2010) viruses. in h1 ha, glu190fiasp and gly225fiasp have been considered as hallmark amino acid changes to switch receptor specificity leading to greater human box 3. novel approaches to identifying genomic predictors of traits and transmission phenotypes. the advent of inexpensive, large-scale sequencing, combined with improved computing power and novel algorithms to interpret nucleotide and protein sequences, have generated new approaches to characterizing the genotype-trait and genotype-transmission phenotype maps in influenza viruses. some are well-established, while others are under active development. they include: protein structural analysis to identify properties of individual amino acid residues and pairs of residues. a number of approaches have been devised to make use of databases of genome sequences and inferred protein sequences of influenza virus isolates, alone or in combination with metadata on the source (species), date of isolation and passage history of the isolates. characterizing the predictors -at the level of individual amino acid residues within a proteinof variability or conservation can assist in identifying the major selection pressures on that protein. evolutionary analysis of the predictors of high rates of nonsynonymous substitutions within ha showed solvent accessibility and proximity to the sialic acid receptor binding site are the strongest predictors of high nonsynonymous evolutionary rates (meyer and wilke, 2015) . comparisons of residue-specific evolutionary rates in avian and human lineages can help to assess which sites are specifically involved in human adaptation and which may be evolving in avian reservoirs with potential consequences for human adaptation (meyer et al., 2013) . innovative use of metadata associated with sequences deposited in databases will be required to ensure that computational inferences from these databases are reliable. for example, methods that aim to identify sites under positive selection in the ha protein frequently find regions or sites that seem to contradict experimental evidence (meyer and wilke, 2015; tusche et al., 2012; kratsch et al., 2016) . several of these apparent contradictions can be resolved by accounting for viral passaging. for example, passaging in regular mdck cells produces a strong signal of positive adaptation underneath the sialic-acid binding site; this signal is entirely absent in unpassaged virus or virus passaged in siat1 mdck cells (mcwhite et al., 2016) . at the same time, passage bias mutations are assumed to increase fitness of the strain in the respective species and are often necessary to grow in culture at all. therefore, sites associated with isolates passaged in mammalian cultures vs. those passaged in embryonated hen's eggs have the potential to further identify sites associated with mammalian or human adaptation. metadata can also help to point to individual amino acids associated with human adaptation. for example, one proposed computational approach is to find potentially zoonotic human-isolated sequences when the majority of their database hits from preceding years were of animal origin. this serves, on one hand, as systematic survey to derive lists of times and places of likely zoonotic events and, on the other hand, provides close sequence pairs of zoonotic human and their putative animal precursors. in those pairs, common sites that repeatedly changed from the animal to the human zoonotic isolates could be reasoned as being involved in human adaptation. combining these sites with those from passage changes, provides strong evidence for the involvement of a particular site in host adaptation. network analysis of the level of sequence covariation of pairs of residues among protein sequences in the database has led to the identification of groups of mutually covarying sites, which have been used to define features of the ha protein that play a role in determining glycan receptor usage . complementary to such covariation analysis is the analysis of predicted molecular interactions. using x-ray co-crystal structures or modeled structural complexes of ha-glycan receptors, molecular features have been defined as distinct networks of inter-residue interactions involving key residues that make contacts with the different glycan receptor topologies. these features go beyond hallmark residue analyses and more accurately predict how amino acid variations in the receptor binding site impact the inter-residue interactions and glycan receptor binding specificity (raman et al., 2014) . similarly, network analysis of amino acid residues predicted to have significant interactions has shown that antigenic sites on the ha interact with residues controlling glycan receptor binding specificity, and that changes in these antigenic residues can then lead to changes in receptor-binding affinity (soundararajan et al., 2011) . it seems likely that as these different lines of evidence -structural location, biophysical interaction, sequence covariation, sequence evolutionary rates, association with zoonotic or in vitro adaptation, etc.-begin to be understood at the resolution of individual amino acids within an influenza protein, such overlapping approaches will yield clearer understanding of the genetic and structural bases of host adaptation to human infection and transmission. a significant step toward such integration is the recent release of the flusurver online tool which automatizes influenza sequence and structure analysis and highlights mutations that could alter the discussed traits based on extensive literature-derived genotype to phenotype lists, structural visualization of the mutation positions and their geographic and temporal frequency of occurrence and cooccurrence for epidemiological relevance (http://flusurver.bii.a-star.edu.sg and directly from within gisaid http://www.gisaid.org). in particular, the tool has been successful in picking up mutations affecting host receptor binding as well as ph dependency (cotter et al., 2014; maurer-stroh et al., 2010) . however, also in this approach, annotations of the effects of mutations are based on inference from similarity to mutations studied in specific sequence contexts, which in most cases will not be identical to the investigated input sequences. association studies. understanding the genetic basis of adaptive phenotypic change is a central goal in biology, and influenza poses special challenges and advantages relative to other organisms. association studies have begun mapping the genomes of arabidopsis thaliana to over 107 quantitative traits and the genomes of humans to over 100,000 (bergelson and roux, 2010; leslie et al., 2014) . these studies often investigate genetic variation at the scales of single nucleotide polymorphisms, alleles, and loci. motif-based approaches have already proven useful in influenza (e.g., the insertion of multiple basic amino acids indicates highly pathogenic h5 and h7), and such simple, robust correlations simplify the prediction of phenotypic traits. recent investigations of influenza (thyagarajan and bloom, 2014; ashenberg et al., 2013; pinilla et al., 2012) have shown that many mutations have roughly consistent impacts across diverse backgrounds. a complication of all association studies is confounding from genetic linkage and diverse environmental selective pressures. although influenza's genes might be tightly linked over short time scales, the virus evolves quickly, and many traits can be assumed to be under stabilizing selection. thus, association studies that appear statistically impractical now may be feasible with a few more years of expanded surveillance. as reviewed here, however, influenza often breaks simple genetic rules, perhaps due to epistasis (e.g., [bloom et al., 2010] ). high-dimensional genotype-phenotype relationships obscure simple correlations from association studies. a relevant lesson comes from the cancer genome atlas (tcga), which amassed sequences from thousands of diverse tumors to investigate the mutations leading to different cancer types. although metastatic cancers are typically conceptualized as possessing six main phenotypic traits (hanahan and weinberg, 2011) , tcga revealed that the genetic commonality among tumors of any given type is shockingly low (kandoth et al., 2013; ledford, 2015) . human genomes are much larger and more complex than influenza's, however, and so it is possible that an influenza atlas might reveal more patterns, which could inspire hypothesis-driven experiments (weinberg, 2010) . binding of upper-respiratory-tract glycans by the influenza virus hemagglutinin is one of the best-understood ingredients in making a virus capable of efficient human transmission. yet the viral sequence determinants of this trait have been mapped only for a limited number of variants. a systematic screening strategy to scan the genetic "landscape" for sequences with a preference for human glycan receptors might include four components: (1) selection of viral genetic background, (2) large-scale mutagenesis, (3) screening and selection, and (4) confirmatory assays. because both mutations near and far from the sialic-acid-binding site on hemagglutinin have been shown to alter glycan specificity, this should be based on a minimally biased approach to mutagenesis: screening combinations of all possible substitutions at all hemagglutinin residues that are not absolutely conserved across known subtypes. critical considerations include choice of viral genetic background (both subtype and strain identity), extent of combinatorial screening (if conserved sites are omitted, every mutant containing changes at up adaptation (glaser et al., 2005; tumpey et al., 2007) . the determinants of specificity are reviewed in much more detail in (paulson and de vries, 2013) . additional structural features involved in receptor binding preference. the cis and trans definition of glycan conformation does not fully describe ha binding to a range of structurally diverse glycans displayed on human respiratory cells and tissues (chandrasekaran et al., 2008) . this limitation motivated studies that revisited the definition of glycan conformation, extending the conformational analysis beyond the terminal sialic acid linkage to describe overall topology and dynamics of the glycan receptor upon binding to the receptor-binding site of avian and human-adapted has (chandrasekaran et al., 2008; xu et al., 2009) . ha sequence determinants of preference for the "cone"-like topology of avian receptors, versus the "umbrella"-like topology of human receptors, are still being defined (raman et al., 2014). c. experimental assays to measure hemagglutinin receptor binding specificity experimental evidence on differential binding of avian and human viruses to sialic acid receptors in avian and human conformations, respectively, was first obtained by hemagglutination assays with erythrocytes whose surfaces had been chemically modified to display glycans terminating with either homogeneous a2,3 or homogeneous a2,6-linked sialic acids (paulson and rogers, 1987) . subsequent analysis of the repertoire of glycan structures in erythrocytes of various animal species informed the use of cells from different species as probes of ha receptor binding preference in hemagglutination assays (ito et al., 1997) . greater precision and reproducibility has been achieved with the use of purified sialylated glycans to create solid-phase binding assays with fluorogenic or enzymatic detection (gambaryan et al., 2006; gambaryan and matrosovich, 1992) . with these assays, it is possible to characterize the relative direct binding of whole virions or recombinant trimeric ha oligomers to glycans attached to a solid phase or the competition of such glycans with binding to a generic glycoprotein attached to the solid phase (gambaryan and matrosovich, 1992) . in recent work, biosensor interferometry and thermophoresis have been used to measure glycanbinding avidities and affinities in a more precise manner and to relate the two (xiong et al., 2013a) . the development of glycan microarrays represented a turning point in the analysis of influenza virus receptor binding specificity, because it allowed simultaneous evaluation of virion or recombinant ha binding to a large repertoire of sialoglycans blixt et al., 2004; childs et al., 2009) . several measures of preference for an ha molecule or whole virus have been defined, including the ratio of the number of a2,6 to a2,3 glycans bound (stevens et al., 2006a (stevens et al., , 2006b or the corresponding ratio of binding affinity or avidity (imai et al., 2012; xiong et al., 2013a) . a limitation to predictive power is that glycans tested on current arrays may not match those present in the human respiratory tract (walther et al., 2013) . these arrays may also not present glycans in the same fashion as respiratory epithelial cells, so strategies such as measuring the binding of labelled viruses to human respiratory tissues (chutinimitkul et al., 2010) or explant cultures ) may be promising alternatives, although challenges remain in standardization and quantification of such assays. structural studies of wild-type and mutant ha in complex with representative sialoglycans provide the ultimate level of detail by characterizing interactions at the atomic level. x-ray to 4 simultaneous sites could be screened with substantial effort), and design of highly parallel screening, selection, and confirmatory assays. the mutagenesis and screening involved would be extremely large in scope: (before eliminating conserved residues, all 4-site mutnats~[550 residues x 20 amino acids] 4 = 1.4 x 10 16 variants for each subtype tested). however, some computational pre-screening to narrow the set of residues tested combined with contemporary mutagenesis and screening technologies such as deep scanning codon mutagenesis (thyagarajan and bloom, 2014; bloom, 2015; fowler and fields, 2014 ) make such an endeavor feasible. doi: 10.7554/elife.18491.004 crystallography advances in recent years have accelerated structural determination, and similar progress in recombinant protein purification techniques combined with robotic crystal screening have reduced the amount of protein and labor required. in summary, genetic and protein sequence analysis, glycan arrays, and x-ray crystallography studies provide complementary data towards understanding the sialoglycan interactions of emerging viruses, with tradeoffs of equipment and reagent costs and throughput against level of precision and detail provided. at present, estimating the contribution of receptor specificity to the pandemic risk posed by a novel virus relies primarily on the similarity between the receptor binding characteristics of the emerging virus and that of the most closely related ha with known transmissibility among humans or a surrogate animal model. as noted above, hallmark residues have substantial predictive power. these distinct sets of hallmark residues in the h1, h2 and h3 subtype (paulson and de vries, 2013) correlate with human-adaptation in known sequences collected from birds or humans (connor et al., 1994; paulson and de vries, 2013) ; they induce changes in receptor-binding specificity when introduced experimentally (chen et al., 2012; leigh et al., 1995) ; and experimental selection for receptor binding in vitro (chen et al., 2012) or in ferrets (imai et al., 2012) cause these changes to appear. however, hallmark residue predictions of receptor-binding specificity are imperfect, as evidenced by a failure to switch in vitro receptor-binding preference from avian to human when changes observed in h5n1 strains after selection in ferret gain-of-function experiments were introduced to other h5n1 viruses (tharakaraman et al., 2013) . the involvement of other features in human adaptation, such as the topology of the bound ha-receptor complex, further complicate the genetic prediction of human adaptation, as the residues involved in these features are less well characterized (shriver et al., 2009) . in principle, phenotypic assays that directly measure the receptor-binding preference of ha -if performed under realistic conditions that capture the interaction of the ha trimer with the receptor (gambaryan et al., 1997; takemoto et al., 1996; collins and paulson, 2004 )-may better capture the trait of interest than genetic predictions of this preference. however, even here, a simple equivalence between binding preference for a2,6-linked glycans and pandemic risk could be misleading. several viruses circulating in humans during the early phase of previous pandemics were found to show either a preference for avian receptors (rogers and d'souza, 1989; connor et al., 1994) or a mixed preference for both human and avian receptors (rogers and d'souza, 1989; glaser et al., 2005; childs et al., 2009 ). in the case of early 2009 pandemic viruses, findings are mixed (childs et al., 2009; chen et al., 2011) . some of the findings of dual or avian specificity may reflect artifacts introduced when human isolates were passaged in eggs before receptor specificity was assayed; alternatively, they may genuinely reflect a transitional stage in the evolution of ha genes in human populations after transmission from other species (connor et al., 1994; glaser et al., 2005; stevens et al., 2010) , ( table 2) . consistent with this latter possibility, an h5n1 virus isolated from a human zoonotic case in vietnam displayed strong avian receptor preference . this preference changed in the course of experiments to adapt it to respiratory droplet transmission in ferrets (imai et al., 2012) . taken together, these findings confirm that there is a strong correlation between measured receptor preference and the host from which a virus is isolated. however, they raise questions about the predictive value of human receptor binding preference. indeed, the examples of mixed receptor preference in human isolates from the early phase of the h1 or h2 pandemics suggest that the ability to evolve human receptor specificity over a chain of human infections, which may be present in many avian-receptor-adapted viruses, may be sufficient for pandemic emergence. in summary, detection of a human receptor preference in a spillover virus may be an indication of increased risk, but exclusive human receptor preference is probably not necessary for an influenza a virus to initiate a pandemic. with several possible exceptions noted above, most viruses isolated to date fall within the shaded cells in table 2 , which indicates concordance between the source of the isolate and the virus trait. thus, prioritizing pandemic countermeasures against virus lineages with inferred or measured human receptor preference will likely lead to better targeting of such figure 1 . key phenotypic traits for the adaptation of avian influenza viruses to replicate efficiently in humans. (a) a switch in receptor binding preference from avian-like (a2,3-linked sialic acid) to human-like (a2,6-linked sialic acid) receptors. the human form on the left shows the typical distribution of human adapted influenza viruses determined by their receptor binding preference for a2,6, linked sa that is predominantly expressed in the upper respiratory tract but also in the lungs. the human form on the right shows that infection with avian influenza viruses is concentrated in the lungs where their preferred a2,3 linked sa receptor is expressed. (b) lower ha ph of activation and increased polymerase complex efficiency. free-floating viruses that enter the human respiratory tract (upper part of figure) encounter mucus and a mildly acidic extracellular environment that act as innate barriers to virus infection. if na is able to desialylate decoy receptors on mucus and ha has a sufficiently low ph of activation, then the virus particle may reach the apical surface of the respiratory epithelium intact. there through a multiplicity of interactions between ha and cell-surface sialic acid, the virus enters the target cell. after the virus is internalized, it passes through the endosomal pathway where the ph is progressively decreased. the low ph of the endosomal environment triggers an irreversible conformational change in ha that fuses the viral and endosomal membranes and ultimately results in the release of virus genetic material in the form of the viral ribonucleoprotein complex (vrnp) into the cell cytoplasm. the eight vrnps are subsequently imported into the cell nucleus by interactions between the vrnps and cellular nuclear import machinery. inside the nucleus the virus polymerase complex replicates the virus genome in conjunction with co-opted cell proteins. doi: 10.7554/elife.18491.005 table 1 . influenza virus adaptations that appear to be required for human-to-human transmission. preference for a2,6-linked mammalian sialic acid receptors over a2,3-linked avian ones ha ph of activation ha avoids extracellular inactivation and undergoes conformational changes leading to membrane fusion at appropriate ph for human cells (5.0-5.4 or perhaps 5.5) (russell, 2014) polymerase complex efficiency efficient replication in human cells (cauldwell et al., 2014; naffakh et al., 2008) virus morphology filamentous morphology associated with several adaptations to mammals (seladi-schulman et al., 2014; seladi-schulman et al., 2013; campbell et al., 2014; beale et al., 2014) length of na stalk longer stalk of na required to penetrate human mucus and deaggregate virions (blumenkrantz et al., 2013) antagonism of interferon production species-specific binding of the ns1 protein to host factors (rajsbaum et al., 2012) ha-na "balance" substrate selectivity and catalytic rate of na are calibrated to "balance" avidity of ha for the cell-surface glycan receptor (zanin et al., 2015; baum and paulson, 1991; yen et al., 2011; handel et al., 2014) doi: 10.7554/elife.18491.006 glaser et al., 2005) ; most human h2 and h3 seasonal isolates (connor et al., 1994; matrosovich et al., 2000) *these anomalous results are speculated by the authors to be possibly, or even probably the result of laboratory adaptation to egg passage and may not reflect the properties of the primary isolate. a possible counter to this interpretation is that it is seen only in the earliest isolates from human pandemic viruses, while nearly all isolates from after the pandemic year, which should also have been passaged in eggs, show human-adapted phenotypes. avian h1-h4, h11 isolates (galloway et al., 2013; russier et al., 2016; dubois et al., 2011; reed et al., 2010) avian h5, h8, h9,h10,h14,h15 isolates (galloway et al., 2013) found in human isolates h5n1 (imai et al., 2012; linster et al., 2014) and h7n9 (schrauwen et al., 2016) human zoonotic isolates with ph 5.6. one human h1n1 (2008) * the role of amino acids 590 and 591 in adaptation was not recognized until after the 2009 strain had already emerged (mehle and doudna, 2009) ; it has the residues associated with avian adaptation at sites 627 and 701 that were known at that time (herfst et al., 2010) . ** complete sequence information not given in the paper *** the rarity of these raises questions about possible sequencing errors. doi: 10.7554/elife.18491.009 greater acid stability (lower ph of activation) is associated with greater human adaptation. the ha is synthesized and folded such that the fusion peptide is buried and inactive until specific activation signals are provided. the structural changes that expose the fusion peptide and lead to fusion have been described in detail (skehel and wiley, 2000) . if the virion is exposed to sufficiently low ph outside of a host or host cell, the ha protein undergoes irreversible structural changes too early and is unable to mediate virus entry; such virions become inactivated. thus the term acid stability is more broadly used to define the threshold for acidification that triggers membrane fusion (in the endosome) or inactivation (if triggered outside of the cell for an ha that is not sufficiently stable). during endocytosis, an influenza virion is exposed to sequentially lower ph values in early endosomes (ph 6.0-6.5), late endosomes (ph 5.0-5.5), and lysosomes (ph 4.6-5.0) (mellman et al., 1986) . if the ha is too stable, and fusion is not triggered in the acidic endosome of the host cell, further traffic into lysosomes results in virus inactivation by lysosomal proteases (skehel and wiley, 2000) . based on surveillance studies, human-transmissible influenza isolates appear to have ha proteins that are more acid stable (have a lower activation ph) than avian influenza viruses (russell, 2014). the ha activation ph values for h1n1, h2n2, and h3n2 seasonal viruses during box 4. ferret model: validity and limitations in pandemic risk assessment. the use of small mammalian models in influenza virus pathogenesis and transmission has proven invaluable for the study of these complex, polygenic traits. the ferret model is particularly valuable, as ferrets are highly susceptible to most influenza a viruses without the need for prior host adaptation. however, even this gold-standard model is not a true substitute for humans. below, we summarize the benefits, drawbacks, and alternatives to the ferret model for the study of influenza. validity. influenza is a respiratory pathogen in humans, and employing mammalian models that possess comparable lung physiology permits a greater extrapolation of results from the laboratory. importantly, the linkage types and distribution of sialic acids throughout the ferret respiratory tract are generally comparable to humans (jayaraman et al., 2012; jia et al., 2014) : like humans, ferrets express the sialic acid n-acetylneuraminic acid ( neu5ac), but not the sialic acid n-glycolylneuraminic acid ( neu5gc), on respiratory epithelia. as a result, ferrets are uniquely suited for the study of influenza viruses compared with other small mammalian models which express neu5gc (ng et al., 2014) . furthermore, human and avian influenza viruses exhibit comparable binding to upper and lower respiratory tract tissues in ferrets and humans (van riel et al., 2006; shinya et al., 2006) . secondly, ferrets infected with influenza viruses demonstrate numerous clinical signs and symptoms of infection associated with human disease. ferrets infected with human influenza viruses often exhibit transient weight loss, transient fever, and sneezing, whereas infection with selected hpai viruses in this species can lead to pronounced weight loss, sustained fever, lethargy, dyspnea, and neurological complications (belser et al., 2009 ). thus, ferrets represent a preclinical model to assess the ability of novel vaccine and antiviral treatments to mitigate influenza virus. as ferrets are a suitable model for the coincident study of pathogenesis and transmission, this model allows for a greater understanding of virus-host interactions and the interplay between both of these parameters. finally, the ferret model can yield valuable insights about the potential human-to-human transmissibility of influenza viruses -the critical determinant of pandemic risk. a recent meta-analysis showed that estimates of transmissibility derived from ferret respiratory droplet transmission studies could explain 66% of measured variation in human transmissibility, for influenza subtypes that have been detected in humans (buhnerkempe et al., 2015) . furthermore, there is a strong statistical relationship between the attack rates measured in particular ferret experiments and the probability that the influenza strain in question is capable of sustained transmission among humans: if two-thirds or more of contact ferrets become infected via respiratory droplets, then the strain is likely to have pandemic potential (see figure) . however, extrapolation of this relationship to novel strains is inherently risky, and variable outcomes observed for h7n9 influenza transmission in ferrets highlight the potential for false alarms. further analysis of ferret transmission experiments, ideally in concert with molecular and virological research, could raise their sensitivity and specificity for identifying pandemic threats. limitations. there is no 'perfect' small mammalian model for influenza. a longstanding challenge of the ferret model has been limited availability of ferret-specific commercial reagents compared with other models, though recent sequencing of the ferret genome should improve this situation . ethical considerations, and the size and cost of ferrets, necessitate generally small sample sizes in ferret experiments, limiting statistical power (belser et al., 2013) . like other vertebrate models, the ferret is not appropriate for high-throughput screens, so research in the ferret model is most potent when complemented with in vitro and computational approaches. finally, ferrets are not well suited to model the multiple influenza exposures over several years that may be experienced by humans and may mold their immune responses in ways that affect the infection risk with subsequent viruses (andrews et al., 2015) . studies of first influenza infection in ferrets may thus overestimate infection and/or transmission risk relative to that in populations with a history of prior infection with related viruses (belser et al., 2016) . the 20th century range from ph 5.0 to 5.4 (galloway et al., 2013) . in 2009, emerging pandemic h1n1 viruses had ha activation ph values of approximately 5.5, but numerous subsequent isolates have acquired mutations that lower the activation ph to the range of the 20th century human influenza viruses (cotter et al., 2014; maurer-stroh et al., 2010; russier et al., 2016) . broad surveys of avian and swine influenza isolates have shown that ha activation ph can vary substantially with a range from ph 4.6-6.0 (galloway et al., 2013; scholtissek, 1985) . among avian viruses, low-pathogenic duck viruses appear to range in acid stability from ph 5.3-6.0 and highly pathogenic avian viruses range from 5.6-6.0 (galloway et al., 2013) . consistent with observed patterns in natural isolates, some experimental evidence indicates that within the range of natural variation, lower activation ph is adaptive for mammalian replication while higher activation ph is adaptive for replication in avian hosts. for isolates of h5n1 alternatives. the ferret is but one of several well-characterized mammalian models for influenza virus. mice are widely used in the field as they offer a greater availability of commercially available species-specific reagents, permit studies with greater statistical power due to larger sample sizes, and offer the advantage of transgenic animals. however, not all human influenza viruses replicate well in mice without prior adaptation due to a predominance of avian-like receptors in the murine respiratory tract; also mice do not display clinical signs and symptoms of influenza that mimic humans, and are not a reliable model for virus transmission studies. the guinea pig is another model, and offers several comparable advantages to ferrets, including generally similar lung physiology to humans and potential for transmission studies. experiments in guinea pigs are often less expensive than in ferrets, because of lower husbandry costs and reduced drug costs when dosing is based on body weight (lowen and palese, 2007) . however, guinea pigs do not exhibit clinical signs and symptoms of infection similar to humans, and do not exhibit severe disease following infection with hpai or pandemic influenza viruses, limiting their utility for viral pathogenesis studies. box 4-figure 1. ferret respiratory droplet transmission experiments predict the potential for sustained human-to-human transmission of influenza viruses. the solid line shows the weighted logistic regression relationship predicting the probability that a given strain is supercritical (i.e. capable of sustained spread among humans), and the dashed lines show the 95% confidence interval for the prediction. filled circles show the measured secondary attack rates (sar) in ferrets for influenza subtypes that are known to be subcritical (blue) or supercritical (red) in humans. the filled pink area shows the range of sar for which the virus is significantly likely to be supercritical. reprinted from (buhnerkempe et al., 2015) . box 5. role of seroepidemiology in pandemic risk assessment. pandemic threat assessment can also be enhanced by immunological surveys of human populations in geographical area where strains of concern are known to be circulating (van kerkhove et al., 2011) . serological surveys can help to estimate the frequency of spillover infections from non-human to human hosts and also to assess the degree of cross reactivity arising from endemic human strains that share recent genomic ancestors with non-human strains of concern (figure for box 4). attempts have been made to use serological surveys to estimate the rate of spillover infections to humans for recent strains of concern van kerkhove et al., 2012) . sometimes blood samples are obtained from the general population (chao et al., 2011) and other times only high risk individuals are tested. inherent measurement error and cross-reactivity between human and non-human strains make the measurement of low rates of incidence problematic (van kerkhove et al., 2012) . confidence that serologic responses truly reflect zoonotic transmission, rather than cross-reactivity with antibodies generated in response to human influenza infection, may be enhanced by comparison of high-risk persons to those without known exposure to zoonotic sources (huang et al., 2015; gomaa et al., 2015) . although there is evidence of exposure of poultry workers to h5n1 influenza viruses in china, rates are much lower than for other endemic non-human influenza viruses (kim et al., 2011) , such as h9n2 (blair et al., 2013) . more recent studies of exposure of high risk workers to the h7n9 lineage suggest even higher rates of exposure to this new strain than has been observed in similar studies of h5n1 or h9n2 . even when rates of spillover can be estimated accurately, the use of such information in pandemic threat assessment is not obvious. clearly, the first detected presence of human infections for a given strain is of concern because the degree of transmissibility among humans is unknown. should the emergent strain fail to achieve sustained transmission, it is not immediately clear how best to use further information on the frequency of human spillover infections. for example, should we interpret high sustained levels of human spillover as evidence of increased risk because of the number of human infections, or as evidence of decreasing risk because of the number of times the strain has failed to achieve sustained transmission? cross reactivity between non-human and human influenza strains has implications beyond the measurement of spillover infections. often levels of cross reactivity in humans may indicate some degree of reduced population susceptibility (worobey et al., 2014) . all else equal, such evidence of lower population susceptibility should reduce our level of concern about a pandemic threat from a particular virus, because even if it gains efficient human-to-human transmissibility, its effective reproductive number and the proportion of the population at risk will be less than for a virus to which there is no cross-reaction in the population. for example, older individuals are thought to have been far less susceptible to pandemic h1n1 than were younger individuals, because they had previously been exposed to similar strains early in life (yu et al., 2008) . the low average age of infection with a swine variant form of h3n2 (h3n2v) in north america (jhung et al., 2013) is likely driven by reduced susceptibility in adults because of early exposure to similar strains. such immunological overlaps are likely to be a general feature of influenza emergence because human strains frequently emerge into swine populations (nelson et al., 2015) . data on reduced human susceptibility due to cross-reactivity must be synthesized with other data used for threat assessment. in some cases, the aging of the part of the population with prior exposure to a closely related strain could be the most important known factor increasing the risk of an emergence event. mechanistic models could be used to estimate the degree of increased risk of emergence due to the aging of partially immune cohorts. highly pathogenic avian influenza virus, an increase in ha activation ph within the range of 5.2-6.0 has been associated with increased replication and pathogenicity in chickens (dubois et al., 2011) . conversely, a mutation that decreased the ha activation ph of a/ chicken/vietnam/c58/2004 (h5n1) from 5.9 to 5.4 has been shown to attenuate virus growth and prevent transmission in mallard ducks (reed et al., 2009 ) but increase virus growth in the upper respiratory tracts of mice and ferrets (zaraket et al., 2013a (zaraket et al., , 2013b . therefore, for h5n1 viruses, a higher ha activation ph (5.6-6.0) has been associated with a component of fitness in birds, and a lower ha activation ph (ph 5.0-5.4) has been linked to greater replication in the mammalian upper respiratory tract. two h5n1 viruses were adapted to transmit by the airborne route between ferrets (imai et al., 2012; herfst et al., 2012) . after a switch in receptor-binding specificity from avian to human receptors (as described above) and deletion of a glycosylation site, in both studies a final mutation that decreased the ha activation ph was shown to be necessary for airborne transmissibility in ferrets. however, these and other studies have shown that this acid stability change is not sufficient in the absence of human receptor-binding specificity (zaraket et al., 2013a; shelton et al., 2013) . recently, an ha protein whose activation ph was 5.5 or lower was shown to be required for the pandemic potential of 2009 ph1n1 influenza virus . nearly 100 mutations have been described to alter the ha activation ph values of various influenza a virus subtypes (russell, 2014; mair et al., 2014) . these acid stabilizing/destabilizing residues are located throughout the ha1 and ha2 subunits and tend to be positioned in regions of the molecule that undergo large-scale changes in structure during ph-activated protein refolding (russell, 2014; bullough et al., 1994; wilson et al., 1981) . mutations that modify the activation ph do not appear to alter the prefusion ha protein box 5-figure 1. transmission genomics of non-human transmission (top), spillover transmission (middle) and sustained human transmission (bottom). haemagglutinin and neuraminidase gene segments have been color-coded to show an example shared infection history in humans who are current spillover hosts for h7n9 and h9n2. these shared evolutionary histories make it challenging to interpret serological studies of human spillover infections. humans infected by h2n2 or h3n2 will likely have cross-reactive antibodies to h9n2, because of the similarity between the neuraminidase in those viruses. because incidence of spill-over infection is likely to be low, even low-levels of cross-reactivity can make the interpretation of serological studies of the general population challenging. doi: 10.7554/elife.18491.014 doi: 10.7554/elife.18491.013 backbone in x-ray crystal structures (dubois et al., 2011; weis et al., 1990; de vries et al., 2014) . therefore, an experimental determination or modeling of intermediate structures may be required in order to reliably predict ha ph of activation. further complicating genetic prediction of ha activation ph values are observations that the na and m proteins can also modulate ha acid stability in some cases (huang et al., 1980; su et al., 2009; reed et al., 2010; o'donnell et al., 2014) . a variety of experimental techniques have been developed to measure the activation ph of the ha protein, quantified as the highest ph at which the ha protein is activated to undergo the irreversible structural changes that mediate membrane fusion (hamilton et al., 2012) , or alternatively the highest ph at which, in the absence of a membrane with which to fuse, the ha protein is inactivated (inactivation ph). classical membrane fusion assays have measured the property in bulk (hoekstra et al., 1984) . the ph of inactivation can be measured using aliquots of virions that are exposed to buffers of progressively lower ph and, after restoration to neutral ph, assayed for retention or loss of infectivity (scholtissek, 1985) . in many classical fusion assays, fluorescent probes are used to label virions, ha-expressing cells, and/or target liposomes or cells. in these in vitro assays, habound target cells are typically exposed to buffers of various ph values and then lipid and/or contents mixing are measured by fluorescence (loyter et al., 1988; hoekstra and klappe, 1993) . alternatively, cell monolayers expressing cleaved ha proteins can be pulsed by low-ph buffers and then incubated to readout hamediated cell-to-cell fusion either microscopically by syncytia formation or by reporter gene expression. if ha conformation-specific monoclonal antibodies are available for the subtype being studied, ha-expressing cells can be pulsed with low ph and then analyzed for conformational changes by flow cytometry (reed et al., 2009 ). if such antibodies are lacking, ha-expressing cells can be assayed for trypsin susceptibility after low-ph exposure, with prefusion ha being resistant and postfusion ha susceptible to trypsin degradation (steinhauer et al., 1996) . recently, methods have been developed to study ha activation and membrane fusion by individual virions, including single virion fusion using total internal reflection fluorescence microscopy (hamilton et al., 2012) . although the biological trigger for ha's conformational change is a drop in ph, ha refolding can also be triggered by other destabilizing agents such as heat and urea (scholtissek, 1985; ruigrok et al., 1986; carr et al., 1997) . stability at a lower ph is associated with stability at higher temperatures and higher urea concentrations, permitting the use of these agents instead of, or in addition to, ph in assays of stability. thermal stability has been determined by measuring the threshold temperature at which denatured ha protein loses its ability to bind erythrocytes and cause hemagglutination (linster et al., 2014) . many questions remain regarding whether ha activation ph plays a similar role in all influenza subtypes isolated from a wide variety of avian species. for early isolates of the h1n1pdm lineage in 2009, the ha protein has an activation ph of 5.5, which appears intermediate between the canonical human (lower) and avian (higher) ranges. subsequent h1n1pdm isolates have ha activation ph values ranging from 5.2-5.4, suggesting ph 5.5 may be the upper limit for human pandemic potential and a lower value may be preferred. indeed, a destabilizing ha mutation in the background of h1n1pdm results in a lossof-function of airborne transmissibility in ferrets and has been reported to be followed by regain-of-function by mutations that lower the ha activation ph to 5.3, a value representative of human-adapted h1n1pdm viruses . for the moment, it appears that while ha ph of activation that is shown experimentally to be suitable for human infection is highly typical of isolates from human pandemic and seasonal influenza ( table 3 , bottom right) (galloway et al., 2013) , it is possible for humans to have symptomatic infection with (though not extensively transmit) viruses with activation ph closer to the range associated with terrestrial birds ( table 3 , bottom left). conversely ( table 3 , top right), there are avian h9, h10, h14, and h15 isolates that display activation ph typical of human viruses (galloway et al., 2013) . the existence of these human-like avian viruses is perhaps unsurprising, as they may lack other essential adaptations for human transmission. as in the case of receptor binding, reliance on this trait to prioritize pandemic prevention measures should consider this property in conjunction with other properties associated with pandemic potential and will likely enrich the coverage of truly high-risk strains on average. systematic assessment of the predictive value of ha activation ph will require broad empirical testing, since nearly 100 residues throughout the ha molecule have been implicated in regulating ha ph of activation. predicting activation ph from sequence will therefore require more extensive data. to address this issue, sequencing studies combined with phenotypic assays could be performed on a large range of ha variants to determine the effects of ph-altering mutations in different ha subtypes. high-resolution determination of ha structural intermediates may assist in developing molecular modeling approaches to calculate ha stability from sequence. in the interim, there is a pressing need to develop high-throughput assays for ha ph of activation, along with other properties believed important to interspecies adaptation, in the thousands of surveillance samples obtained annually. the heterotrimer of influenza polymerase subunits (pa, pb1, pb2 gene products, together forming the rna-dependent rna polymerase) and the nucleoprotein (np gene product) is required to transcribe and replicate the viral genome (huang et al., 1990) . the polymerase genes of viruses isolated from avian hosts show a number of genetic differences from their counterparts in viruses isolated from humans (chen et al., 2006) , and avian virus polymerase typically performs inefficiently in replicating the viral genome in human cells (cauldwell et al., 2014; naffakh et al., 2008) . adaptation to efficient human-to-human transmission requires efficient activity of this complex of proteins, which we refer to as the polymerase complex, in human cells (cauldwell et al., 2014; naffakh et al., 2008) . some mutations in pb2 are consistently associated with efficient function of the polymerase complex in mammalian cells ( figure 2 ). as long ago as 1977, it was shown that an avian influenza virus could achieve efficient replication in mammalian cells by acquiring mutations solely in the pb2 subunit of the viral polymerase (spooner and barry, 1977) . the most famous of these mutations was later described as pb2 residue 627 (subbarao et al., 1993) , which is a glutamic acid (glu) in avian influenza viruses but a lysine (lys) in human-adapted viruses, including those that emerged in the pandemics of 1918, 1957 and 1968 , and their seasonal descendants. an important exception is the virus that sparked the pandemic of 2009. in this virus, the pb2 segment had been introduced from an avian precursor into swine viruses in the 1990s, and mammalian adaptation had been achieved by a different set of pb2 mutations including changes at residues at 271, 590 and 591 (mehle and doudna, 2009) . now that the 3-dimensional structure of the viral polymerase has been elucidated, we can see that residue 627, 271, 590 and 591 lie on the same external surface. mammalian-adapting mutations increase the positive charge of this domain, suggesting that they either adapt the virus for interaction with an enhancing host factor or enhance its ability to repel a restriction factor (mehle and doudna, 2009) . recently a host factor, anp32a, that differs between mammals and flighted birds was shown to be a cofactor of the influenza polymerase, and the species specific difference could explain the inefficient function of avian virus polymerase and the stringent selection for the 627glu->lys adaptive mutation in mammals (long et al., 2016) . another residue implicated in mammalian adaptation of the polymerase is residue 701 of pb2, which lies close to but is distinct from the 627 cluster. it has been suggested that this mutation and others in this domain at residues 702 and 714 affect the interaction between pb2 and importin-alpha isoforms either in a way that enhances nuclear import of newly synthesized pb2 or that affects polymerase function once inside the nucleus, the site of viral rna replication (resa-infante et al., 2008; gabriel et al., 2005; sediri et al., 2015) . other mutations have been described that adapt pb2 for the mammalian nucleus (for example the triplet threonines at positions 147, 339 and 588) but whether they affect interaction with anp32a, importins or as yet unidentified host factors is not yet elucidated. the adaptive value of these mutations is shown by experimental or observational data in which a mammalian host is infected with a virus whose pb2 is not adapted for efficient mammalian replication, but such a mutation becomes common in the virus population over the course of infection. such evolution has been observed in a fatal human case of influenza a/h7n7 (jonges et al., 2014) and in mouse experiments following serial lung passage using an isolate from this outbreak . lys at position 627 has also been associated with greater severity in zoonotic h7n9 (sha et al., 2016) and h5n1 (de jong et al., 2006) cases however, reverse genetics experiments show that certain strains of avian influenza may be less able to accept these mutations than others (long et al., 2013) . polymerase complex efficiency in human cells can be measured by an in situ assay in which the influenza polymerase is reconstituted from cloned cdnas in plasmids and then coexpressed with "minigenome," a viral-like rna encoding a reporter, such as luciferase. by measuring the rate of reporter accumulation in the transfected human cell line, specific combinations of rna sequences for the polymerase-complex viral genes can thereby be screened directly for their efficiency in producing the mrna encoding the reporter gene product, providing a measure of human adaptation of the polymerase complex (moncorgé et al., 2010) . the original form of the in situ reconstituted polymerase assay requires expression of just the minimal set of four viral proteins to replicate the minigenome rna: pb1, pb2, pa and np. however, recent work showed an important additional role for another protein, the nuclear export protein (nep), which is translated from a spliced mrna derived from rna segment 8 (that also encodes the major interferon antagonist ns2) (robb et al., 2009) . in human h5n1 isolates that do not contain pb2 host-adapting mutations, the inefficient activity of these avian polymerases in human cells could also be compensated for by certain mutations in nep (mänz et al., 2012) . it appears that nep is an important regulator of the balance between transcription and replication (chua et al., 2013paterson and , and can thus enhance fitness in viruses containing otherwise inefficient polymerases. the mechanism of this is as follows: the polymerase-enhancing domain of nep is masked when nep is folded in one conformation. however, mutations that increase the ability of nep to rescue avian polymerase function allow more ready unfolding of the protein, unmasking the "activating" domain at the lower temperature of the mammalian respiratory tract. interestingly, nep overexpression in cells in which human-adapted polymerase is reconstituted is inhibitory because excess complementary rna accumulates at the expense of messenger rna and further viral rna replication (robb and fodor, 2012) . thus although a short-term adaptation of avian virus polymerase to mammalian cells can be achieved in this way, it may be that further compensatory changes rebalance nep function in the face of polymerase adaptation during continued circulation in humans, although direct evidence for this selection is lacking. indeed, although the rescue of low polymerase activity by nep may explain the human infections by h5n1 viruses that lack other polymerase adaptations, it is not clear that such rescue is sufficient to create a level of transmissibility consistent with pandemic spread. nonetheless, this finding shows that the minimal polymerase assay is not always sufficient to predict viruses that have functionally adapted polymerase activity to human cells and that a role for other viral proteins including at least nep should also be considered in assessment of polymerase function. alternatively, polymerase activity could be measured in the context of viral infections (although this will require proper containment). this could be achieved by measuring intracellular levels of viral transcripts using transcriptomics or qrt-pcr. such experiments would provide important information if they are performed using appropriate cell lines (or respiratory explants) at the temperature of the human airway (33˚c). it has been suggested that plaque size at 33˚c can be used as a surrogate measure of polymerase function but plaque size is a mutligenic trait. the predictive value of such assays for transmissibility is limited. ultimately, it would be valuable to develop a simple screen to assess the ability of a viral polymerase to support replication and transmission in humans. this phenotype is influenced by at least 4 different viral genes and involves interactions with several different human host factors. if all the relevant host factors were enumerated, one could imagine quickly converting sequence information into an assay that tested for interactions that should support activity. along these lines the recent description of a host factor, anp32a that differs between flighted birds and mammals and explains the poor activity of avian polymerase in mammalian cells is a step forward (long et al., 2016) . the inefficient polymerase of avian influenza viruses in mammalian cells is one of the hostrange barriers that likely diminishes pandemic risk. unlike the requirement for adaptive mutation in the novel ha, this polymerase barrier can be rather readily overcome by reassortment in which an avian virus with novel antigenicity can acquire one or more polymerase genes from mammalian-adapted viruses. in addition, adaptation of avian virus polymerase by accumulation of adaptive mutations in either the polymerase genes or possibly in other viral genes such as the ns segment encoding nep can enhance avian virus polymerase function sufficiently to support a host range jump. many h5n1 viruses that circulate today in the avian reservoir already have mutations in pb2 at 627 (long et al., 2013) or 701 (de jong et al., 2006) , likely resulting from the reintroduction of mammalian-adapted strains back into the wild bird reservoir. these have been associated in human infections with more severe cases (de jong et al., 2006) . the fact that these strains have not achieved sustained human-tohuman transmission demonstrates that while polymerase adaptations to humans are likely necessary, they are not sufficient for a strain to spark a pandemic. moreover, the absence of the signature pb2 627k mutation in the 2009 h1n1 pandemic strain demonstrates the limitations of relying on any single mutation for risk prediction (herfst et al., 2010) ; viruses with the avian-like residue have also been isolated from zoonotic human cases of h5n1, h7n9, and h9n2 infections ( table 4 , bottom left). on the other hand, the concept that adaptation of the polymerase is necessary for sustained human transmission is validated by findings that the 2009 pandemic strain had adapted to replication in human cells by changes other than e627k within the polymerase (mehle and doudna, 2009) . identification of biophysical mechanisms common to mammalian-adaptive mutations may in the future provide the basis for new biological or biophysical assays of polymerase function to inform risk predictions. in summary, no single polymerase mutation appears to be predictive of pandemic risk for all viruses, but the concept that the polymerase must adapt to human cells before it can cause extensive human-to-human transmission appears consistent with the four pandemic jumps that have occurred in modern times. there has been tremendous progress in understanding the traits involved in the adaptation of avian influenza viruses for efficient human-tohuman transmission and the genetic and structural basis of each of these traits. while the ability to use virus sequence data to inform risk assessment of pandemic potential is improving, it remains essential to consider these data alongside other experimental and epidemiological data. for example, in 2013 there was a substantial increase in the number of human infections with a/h5n1 viruses in cambodia. the increase in infections was cause for substantial concern by itself. enhancing the level of concern was the finding that some of the viruses collected from infected humans contained previously identified genetic mutations suggestive of human adaptation . these findings led to extensive epidemiological and experimental investigations and then to the decision to produce a candidate vaccine virus from a virus representative of the 2013 cambodian outbreaks. while predictions of virus phenotypes from sequence data can be informative, they are not infallible, for several reasons, notably the large number of sites involved in determining such traits (raman et al., 2014; russell, 2014; cauldwell et al., 2014; mehle and doudna, 2009; mänz et al., 2012; herfst et al., 2010) , the important role of epistasis (dependence of a mutation's effect on the genetic background in which it appears) in determining these traits kryazhimskiy et al., 2011; gong and bloom, 2014; bloom et al., 2010; wu et al., 2016; raman et al., 2014; tharakaraman et al., 2013; gong et al., 2013) , and the consequent imperfections in our ability to map single sequence polymorphisms to a trait value. for example, the hallmark ha amino acid residues 190, 226 and 228 are important to human adaptation, but "human-adapting" mutations at these residues do not always change receptor-binding specificity; it depends on the genetic background. similarly, amino acid residues 627 and 701 of the pb2 protein are often involved in human adaptation, but both carried the "avian-adapted" residue in the 2009 h1n1 pandemic strain. when these changes were introduced individually or together in the laboratory, the resulting polymerase showed greater activity in a minigenome assay, but replication was unchanged or attenuated in vitro, in mice, and in ferrets (herfst et al., 2010; jagger et al., 2010) . after the pandemic strain was identified and its anomalous residues at these sites noted, other sites within pb2 were identified and found to be responsible for human adaptation (mehle and doudna, 2009; yamada et al., 2010) . based on the evidence to date, it seems clear that all three of the traits considered in this review, and possibly others in table 1 , must be simultaneously present at least to some degree for a strain to cause a pandemic. yet with only a few instances of pandemic strains emerging per century, it should not be surprising that a new pandemic strain would violate an apparent rule of human adaptation that applied perfectly to previous pandemic strains, as in the case of the pb2 residues associated with human adaptation in 2009, or as in the case of activation ph of ha in early 2009 isolates , which had a value outside the range previously seen in human influenza viruses. it is unclear whether the list of sites and phenotypic traits associated with human adaptation is nearly complete or will continue to grow as we experience additional pandemics. at least for the traits of receptor binding (childs et al., 2009) (table 2) and acid stability (table 3) , full human adaptation may not be required to initiate a pandemic in a virus that is otherwise well-adapted for humans. thus, whether or not the list of traits required for pandemic is now complete, our understanding of where the threshold lies for being sufficiently humanadapted continues to change. there are three complementary approaches to address these limitations: improving genetic prediction of biological traits, improving assays of these traits, and improving animal models of human transmission; all approaches are currently progressing in parallel simon et al., 2011) . the first approach aims to further refine our understanding of sequence-totrait relationships by continued studies of diverse, naturally or artificially produced mutations and their effects on the traits of interest. such research could use all of the approaches described above and higher-throughput assays that could be developed with improved technology, for example as described in box 3. this will include generating mutations not found in known strains in nature to probe for those that could be involved in human adaptation in the future thyagarajan and bloom, 2014) . the goal would be to identify classes of functionally equivalent substitutions, sufficient individually or in defined combinations to confer a trait of interest when introduced into a defined, avian-adapted genetic background. use of in vitro approaches with noninfectious viruses or viral components, or infectious viruses containing surface proteins to which there was already population-wide immunity would reduce the possible biosafety and biosecurity risks of such studies (lipsitch and inglesby, 2014) . the second approach is to develop and improve the throughput and accuracy of biochemical and cell-biological assays of these traits, so that virus isolates can be characterized phenotypically in a routine manner, reducing reliance on sequence-based predictions. it seems feasible to develop high-throughput versions of many of the assays described in this review for each of the three traits discussed, which could then be routinely run on surveillance isolates to contribute to risk prediction. for none of these three traits is there a single gold standard assay, and different assays may provide different estimates of risk. the third approach is to improve animal models to more precisely study phenotypes that are important for human adaptation, and to clarify whether the notion of 'mammalian adaptation' is in fact a valid category. ferrets are the closest known model for human transmission (see box 4). respiratory-droplet transmission studies in ferrets, and potentially in other animal models, have shown a remarkably strong correlation with human transmissibility of influenza a strains (buhnerkempe et al., 2015) . while these assays are not perfectly predictive, they may be the most reliable way at present to assess the transmission potential of a virus in human populations. here a partial counterexample to their overall strong predictive value is h7n9 avian influenza isolates from human zoonotic cases. these viruses transmit in ferrets, albeit less efficiently than human seasonal strains, yet humanto-human transmission has been extremely rare in the hundreds of human zoonotic cases caused by h7n9 (buhnerkempe et al., 2015) . a challenge is the expense and practical challenge of using large enough numbers of ferrets (buhnerkempe et al., 2015; belser et al., 2013; nishiura et al., 2013) to assess transmissibility; this will remain a technique of limited throughput for the foreseeable future. nonetheless, the value of ferret testing for risk assessment can be enhanced in at least two ways: first, by standardizing the conditions for ferret transmission experiments, so these can be more confidently compared between laboratories; and second, by continuing to combine ferret studies with studies of viral traits and sequence/structural studies to further identify correlates of transmissibility in ferrets. while the biological properties of a virus certainly play a large role in determining the pandemic risk posed by a strain, it is possible that even a virus perfectly adapted for human-tohuman transmission might fail to transmit extensively, due to ecological factors, chance, or both. initiation of a pandemic requires not only a well-adapted virus but ecological opportunity to spill over into humans (perhaps multiple times if the first introduction is not "successful" [mills et al., 2006] ), as well as a human population that is immunologically susceptible and sufficiently connected to establish ongoing transmission (box 5). additional complexity arises from the fact that selection pressures for within-host proliferation and competition may diverge from those needed for efficient transmission (park et al., 2013) . genetic bottlenecks at the time of transmission (poon et al., 2016; varble et al., 2014; zaraket et al., 2015) may further enhance the role of chance, as a highly adaptive variant arising in a host may not get transmitted in a particular event. however, selective bottlenecks, which have been observed in experimental transmission of h5n1 and avianlike h1n1 viruses in ferrets, could lower the barrier to emergence of human-adapted viruses (moncla et al., 2016; wilker et al., 2013) . both ecological and host factors are considered in the cdc's irat (trock et al., 2015) . evolutionary factors also play a role in pandemic risk evaluations. even with excellent surveillance, we may never isolate exactly the virus that is destined to cause a pandemic from an animal reservoir or a zoonotic human case; rather, we may isolate its evolutionary precursor. understanding the potential of a strain to produce pandemic-capable progeny is yet a further scientific challenge. perhaps the most startling finding of the gain-of-transmissibility experiments with h5n1 avian influenza viruses was that so few mutations were required to convert a strain circulating in birds to mammalian transmission. this concern was reinforced by a finding that many of these mutations, including combinations of some of them, were already present in strains isolated during surveillance (russell et al., 2012) . the interpretation of the latter finding, however, is complicated by the problem of epistasis: the effect of these mutations in the genetic background of field strains may or may not be the same as in the strain studied in the laboratory. it seems clear that a pandemic risk assessment informed by genetic sequence data is better than one uninformed by such sequence data, but the thought experiment of considering the 2009 swine-origin virus, had it been seen prior to initiating the pandemic, shows that such efforts may fail to identify the risk posed by strains that subsequently cause a pandemic. according to the knowledge at the time, early human isolates of 2009 (and presumably their swine precursors) would have had an ha intermediate between human and avian adaptation in terms of receptor binding. they had an activation ph outside the range previously seen in human viruses and more typical of avian viruses. moreover, these viruses lacked the amino acid residues then thought to confer human adaptation for the polymerase complex. we must imagine that had this strain been detected in swine surveillance prior to the pandemic emergence, genetic as well as phenotypic considerations would have marked it as low risk, creating a false-negative risk assessment. given that this virus did in fact create a pandemic, it is evident that failure of a nonhuman influenza virus to fully meet the three criteria discussed in this review does not disqualify it from posing a significant risk of a pandemic. whether false positive predictions of high pandemic risk are also possible is more difficult to determine, because even a strain that is truly high risk may fail to cause a pandemic for any number of reasons; thus it is challenging to prove that an assessment of high risk for a particular strain was erroneous. from a decisionmaking perspective, a false positive is perhaps less worrisome than a false negative, as a false positive may motivate expenditure on prevention measures directed at a strain that would not have caused a pandemic, while a false negative may lead to a failure to respond to a strain that would. as we seek to improve our understanding of genetic and phenotypic bases of efficient human transmission of influenza viruses, there are multiple possible approaches. one approach that has received considerable attention recently is to perform gain-of-transmissibility studies in highly pathogenic avian viruses; this has been controversial because of concerns about the unusual biosafety and biosecurity risks entailed in such studies (for contrasting perspectives on these risks, see the exchange in 2014-5 between lipsitch and inglesby, and fouchier inglesby, 2014, 2015; fouchier, 2015] ). there are alternative approaches to ferret gain-of-transmission experiments in highly pathogenic avian influenza viruses, though disagreement remains about the level of evidence such alternatives provide. one alternative is to perform similar experiments starting from avian viruses that are not highly pathogenic in mammals, and/or are related to currently circulating human seasonal viruses, so that immunity would already be present in the population. such experiments can provide the same degree of causal rigor as gain-of-function in highly pathogenic avian viruses with novel surface antigens and can elucidate general principles of mammalian adaptation, but they cannot confirm that the same changes would be observed in other strains that are not used in the experiment. a recent report shows a related way forward: the recreation of the steps of mammalian adaptation using viruses whose ha and na are already circulating in humans (lakdawala et al., 2015) . such loss+gain-of-transmissibility experiments reconstruct the properties of naturally occurring seasonal human strains, from laboratory-generated, avian-adapted (or at least human-deadapted) precursors. reconstructing such seasonal strains should pose a risk similar to that of working with the seasonal strains themselves, less than that of a novel subtype. a 2011 report employed a similar strategy, demonstrating the importance of ha activation ph in mouse adaptation by selection experiments on a live attenuated h5n1 vaccine strain lacking the ns1 gene (krenn et al., 2011) . more recently, a 2009 h1n1 pandemic virus was modified to express a mutation that increased ph of ha activation, then selected in ferrets for droplet transmission, and it was found that a second site mutation partially restored the lower ph of activation of the selected virus . one limitation of de-adaptation strategies is that the acquisition of transmissibility is perhaps most likely to evolve by reversion of the de-adaptation changes. as with all gain-of-function and loss-offunction studies, epistatic effects of other loci in the genetic background of the viruses used in such studies set limits to the generalizability of such experiments. another kind of alternative is simple characterization of ferret transmissibility of naturally occurring highly pathogenic strains without selection for airborne transmission. this approach can provide correlative evidence for the importance of genetic differences but cannot prove the mechanistic role of any particular change. even if strain-based assessment methods were much better, surveillance would be a key rate-limiting step for pandemic risk assessment to direct countermeasure development. if a virus about to cause a pandemic is not found in surveillance it cannot be assessed. the fact that we have yet to identify a pandemic strain in nonhuman hosts or in human spillover cases before the pandemic starts indicates there is much work to be done. although surveillance has expanded since the 2009 pandemic, it has not been designed to optimize the chances of detecting a pandemic strain before it becomes pandemic; indeed, how to do so is not clear at present. some possible considerations would be to maximize the diversity of isolates collected, to preferentially sample strains that are known to cause human infections, and to feed back information from risk assessments to inform choice of sampling. rapid sequencing and phenotypic characterization of strains and dissemination of this information, along with interpretations of the risk profiles implied, is also important to maximizing the value of surveillance. further thought should be given to the possibility of using high-throughput sequencing as a screen for which viruses should be subjected to phenotypic testing, which for the moment is typically more costly, slower, and lower-throughput than sequencing. more deliberate approaches to the design of surveillance systems would also depend on answering the question addressed in box 2: how different must a virus be from previously characterized viruses to merit separate evaluation of its pandemic risk? the uncertainties noted above about the phenotypic characteristics of viruses isolated from previous pandemics (which may have been present in the primary isolate or may have arisen during egg passage in the laboratory) underline the need for careful attention to passage histories of surveillance isolates to avoid altering their genotype and phenotype post-isolation (bush et al., 2000) . expanding and rationalizing surveillance in this way would require overcoming political, logistical and financial constraints that vary between countries and regions. even with all of the foregoing suggestions in place, it may be improbable that we can reliably identify the 'needle in the haystack' that is the next pandemic influenza strain. ultimately, the goal is not risk assessment for its own sake, but preparedness and early response to pandemic threats. in other areas where security is at stake, it has been argued that making and improving predictions should be accompanied by a systematic effort to design responses that will not fail even if the predictions are wrong (danzig, 2011) . in the influenza context, the value of some countermeasures is strongly reliant on our ability to identify truly high-risk prepandemic threats: notably, preparation of seed vaccine stocks for candidate pandemic strains, stockpiling of subtype-specific vaccines, and culling of poultry infected with such strains. other types of countermeasures, ranging from strengthening local public health departments to stockpiling antivirals or ventilators to developing faster processes for vaccine manufacture to universal vaccines that should be effective against any influenza a strain, should provide benefits whether or not we have advance notice of the strain causing the next pandemic. a comprehensive assessment of priorities to prevent or mitigate the next influenza pandemic should consider the balance between improving our risk assessment capacity and developing responses robust to the possibility that we will once again be caught by surprise. we thank jesse bloom, ruben donis, judith fonville, scott hensley, and benjamin tenoever for contributing valuably to the workshop on which this paper is based and ruben donis for significant assistance in improving the manuscript. we thank james hay for the figure for box 5. this work and the workshop from which it originated were supported in part by the research and policy for infectious disease dynamics (rapidd) program of the science and technology directorate, u.s department of homeland security, and the fogarty international center, nih. the findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the centers for disease control and prevention, the department of health and human services or its components, or the nih or its institutes. this paper is dedicated to the memory of ellis mckenzie, cofounder of rapidd and senior scientist at the fogarty international center of nih, who was a valued friend and colleague and who enthusiastically supported the workshop that produced his paper. immune history profoundly affects broadly protective b cell responses to influenza mutational effects on stability are largely conserved during protein evolution the n2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity a lc3-interacting motif in the influenza a virus m2 protein is required to subvert autophagy and maintain virion stability a review of mathematical 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influenza a(h5n6) virus. infection molecular basis of the receptor binding specificity switch of the hemagglutinins from both the 1918 and 2009 pandemic influenza a viruses by a d225g substitution competing interests: ml: reports the following financial disclosures for topics unrelated to this manuscript: consulting income from pfizer and affinivax (both donated to charity) and research funding from pfizer and path vaccine solutions. these entities had no role in the preparation of this work or in the decision to submit the work for publication. reviewing editor for elife. car: during the prepartion of this manuscript, caab received funding through a research contract with astrazeneca inc., but for distinct, unrelated research. astrazeneca inc. had no role in the preparation of this work or in the decision to submit the work for publication. the other authors declare that no competing interests exist. key: cord-004222-z4butywi authors: joyce, collin; burton, dennis r.; briney, bryan title: comparisons of the antibody repertoires of a humanized rodent and humans by high throughput sequencing date: 2020-01-24 journal: sci rep doi: 10.1038/s41598-020-57764-7 sha: doc_id: 4222 cord_uid: z4butywi the humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. assessment of the baseline antibody repertoires of unimmunized model animals will be useful as a benchmark for future immunization experiments. we characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the omnirat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. multiple differences were found in both the heavy and kappa chain repertoires between omnirats and humans including gene segment usage, cdr3 length distributions, class switch recombination, somatic hypermutation levels and in features of v(d)j recombination. the inference and generation of repertoires (igor) software tool was used to model recombination in vh regions which allowed for the quantification of some of these differences. diversity estimates of the omnirat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. despite variation between the species repertoires, a high frequency of omnirat clonotypes were also found in the human repertoire. these data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies. rag1, rag2 and artemis (among others). p and n nucleotides are added in the vh-dh and dh-jh junctions by artemis and tdt, dramatically increasing sequence diversity. after successful pairing of this newly formed heavy chain with surrogate light chain (slc), recombination of a light chain from v and j gene segments of the kappa or lambda loci occurs and the b cell swaps the slc for this new light chain. unless the immature b cell is autoreactive or anergic and undergoes receptor editing or clonal deletion, it matures into a naïve b cell and migrates to the periphery whereupon it can become activated by encountering antigen and form germinal centers with help from t-cells. sequence diversity is again enhanced in the germinal center by somatic hypermutation (shm) and/or class switch recombination (csr), two processes that depend on activation induced cytidine deaminase (aid). the omnirat was created by genomic integration of human immunoglobulin (ig) loci on a background of inactivated endogenous rat ig loci. it expresses chimeric heavy chains (i.e. human v, d, and j genes and rat constant genes) that pair with fully human light chains 11, 12 . we sought to characterize the circulating antibody repertoire diversity in this animal and make comparisons to humans. high throughput antibody sequencing has been used to describe the circulating antibody repertoire of organisms, including more recently at unprecedented depth in humans 13 . reverse transcription of antibody rna and combined tagging with unique molecular identifiers (umis) have allowed us 14 and others 15, 16 to correct for error and bias in antibody sequencing. using these methods to gain insight into the antibody repertoire of omnirats, we ask whether or not it accurately represents that of humans, and by extension allows for usefulness in the approximation of the human antibody response. we postulate that there are major differences in the repertoires due to distinctness in the ig loci genomic structure and genes that shape antibody diversity between species. here, we provide the most thorough description of humanized transgenic rodent antibody repertoires to date and leverage a novel extremely deep human dataset to make comparisons with implications of immediate use as a reference for omnirat immunization studies. we individually separated total rna from spleens and lymph nodes of three unimmunized omnirats and pcr amplified the heavy and kappa chain antibody v gene segments. the resulting amplicons were subjected to high throughput sequencing in conjunction with preprocessing and annotation by the abstar analysis pipeline 17 (methods) which resulted in a mean of ~3 × 10 6 processed heavy chain sequences and ~1.5 × 10 6 processed kappa chain sequences per transgenic animal (table s1 ). two previously published datasets 13,18 of the same 10 humans which together contain a mean of ~3.6 × 10 7 processed heavy chain sequences and ~1.5 × 10 6 processed kappa chain sequences per individual were used for comparison. we started by making intra-animal comparisons, intra-species comparisons and inter-species comparisons of the immunoglobulin gene segment usage frequencies for each antibody repertoire by performing hierarchical clustering ( fig. 1 ) and linear regression analysis (figs. s1 and s2). repertoires were found to cluster by species and tissue when variable heavy (vh) (fig. 1a) , diversity heavy (dh) (fig. 1b) , joining heavy (jh) (fig. 1c) and variable kappa (vk) (fig. s5a ), but not joining kappa (jk) (fig. s5b ) gene usage was examined. differences between the lymph node and spleens of individual omnirats were next investigated. vh gene, dh gene and jh gene usage frequencies between these tissues were highly correlated (fig. s1) , although a few vh gene segments were overrepresented in spleen as compared to lymph nodes including vh4-34, vh4-59 and vh5-1 (figs. 1a and s3a). dh gene and jh gene usage remained highly correlated with minor differences in specific genes (figs. 1b,c, s1 and s3b,c). inter-animal spleen gene usage was highly correlated for all three heavy chain gene segments (fig. s2 ). inter-human comparisons yielded similar, albeit slightly less correlated results (fig. s2 ). intra-species vh and dh usage comparisons made show much weaker correlations with lower r-squared values than any other previous comparison, while surprisingly jh gene usage was highly correlated (fig. s2 ). species specific gene usage biases were more predominant in variable genes (vh and vk) than in joining genes (jh and jk) (fig. s3a-e) . we hypothesize that this may be due to species specific differences in variable gene order, but not joining gene order at the genomic loci 11 , although no significant correlation between variable gene order and gene usage in the omnirat was found (data not shown). omnirats show a preference for dh gene families of shorter average length such as dh1, dh5, dh6 and dh7 as compared to humans which show a higher representation of longer dh genes from dh2, dh3, and dh4 families with the exception of dh3-9 which appears at similar frequencies between each species (figs. 1b and s3b). vk and jk gene usage frequencies were very similar for all comparisons made (figs. s1-3 and s6). differences in cdr3 length distributions of each repertoire were next determined. the mean heavy chain cdr3 (cdrh3) length in humans is 14.8 amino acids, while in the omnirat we observed a mean cdrh3 length that is shorter with a mean length of 12.1 amino acids (fig. 2a) . there are minor differences in the kappa light chain (cdrl3) lengths between species with near identical average lengths of 9.0 and 9.1 for omnirats and humans respectively (fig. s6c ). the frequency of light chains with a cdr3 of 5 amino acids in length is an important consideration when choosing a model animal for vaccination experiments involving the germline targeting immunogen eod-gt8 which is in human clinical trials 3, 14, 19 . this frequency of 5-amino acid cdrl3s was lower in omnirats (0.02%) than in humans (0.56%) i.e. a factor of 28 (fig. s6c ). after observing a tendency for shorter cdrh3 lengths in the omnirat as compared to humans, we wanted to know if the number of n and p nucleotide additions in the heavy chain v-d and d-j junction sites were different. figure 2b ,c shows average v-d and d-j junction nucleotide addition lengths in the omnirat are indeed shorter as compared to humans. nucleotide additions in the v-j junctions of kappa chains are also shorter on average as compared to humans (fig. s6e) . the longest dh gene segments are found in the dh3 family and the longest jh gene segments come from the jh6 gene family. gene segments from these families are important contributors to the generation of unusually long cdrh3s in humans and are consistently found in certain broadly neutralizing antibodies (bnabs) that bind to the human immunodeficiency virus (hiv) envelope (env) glycoprotein, indicating the importance of these rearrangements in hiv vaccine studies 20 . on average, the frequency of antibodies with d3-j6 rearrangements in omnirats is 0.012 with little variation, while in humans the frequency of these antibody species is more variable between subjects with a higher mean of 0.028 (fig. 2d) . the preference of omnirats for shorter cdrh3 lengths and dh gene segments can be placed in the context of shorter dh gene lengths in the wild-type rat (rattus norvegicus) as compared to human dh genes (fig. 2e) , indicating a possible biologically intrinsic bias. we used igor 21 to infer recombination models for each individual repertoire from 100,000 unmutated sequences allowing for the quantification of differences in features of heavy chain vdj recombination and generated 1,000,000 synthetic sequences per model. cdrh3 length, vd insertion length and dj insertion length distributions from the synthetic sequence data (fig. s4a-c) were found to be very similar to the observed data columns are antibody repertoires and rows are gene segments. data was scaled by calculating the z-score for each gene (row) and hierarchical clustering (euclidean distance metric) was done. a dendrogram representation of clustering is shown and indicates uniqueness in gene segment usage between the lymph node and spleen repertoires of the omnirat and between the omnirat and human repertoires. red and blue indicate high and low z-scores respectively (legend shown in a), and since it is calculated per gene it represents differences between repertoires and not the relative frequencies of gene segment usage in each repertoire. ( fig. 2a-c) . kullback-leibler (kl) divergence is a measure of how different two probability distributions are. kl divergence between models (fig. s4d ) and model 'events' (fig. s4e) were computed as previously described 13 . kl divergence between pairs of omnirat models was found to be lower than kl divergence between pairs of human models for both complete and all event level calculations. the average pairwise omnirat model versus human model complete kl divergence calculation was found to be much greater than that of pairwise inter-animal calculations and more than twice that of pairwise inter-human calculations. "d-gene", "v-gene trim (3')", and "d-gene trim (3')" were among the events computed to have the mean highest kl divergence from pairwise inter-species event level model comparisons. class switch recombination and somatic hypermutation in omnirats. in supplementary fig. s5a , the frequency of antibody isotypes is shown. the human repertoire contains average frequencies of 0.84 and 0.16 for igm and igg respectively as previously published 13 , while in the omnirat antibody repertoire we observe mean frequencies of 0.15 and 0.003 for lymph node and spleen igg respectively and means of 0.85 and 0.997 for lymph node and spleen igm respectively. mean numbers of variable gene mutations in igm (fig. s5b) , igg (fig. s5c) and kappa (fig. s6d) sequences of the omnirat were about half of those found in the human repertoire. the observed increase in shm of class-switched igg sequences as compared to igm sequences in the omnirat demonstrates the ability of the animal to generate memory b cells. we first examined clonotype (defined as identical vh gene, jh gene and cdrh3 amino acid sequence) diversity of the heavy chain repertoire for each individual animal. all sequences from lymph nodes and spleens were collapsed into unique clonotypes, separately for each tissue and animal. any clonotype found in both tissue compartments must have originated from different b cells, allowing for the measurement of repeatedly observed clonotypes. rarefaction curves for each animal were www.nature.com/scientificreports www.nature.com/scientificreports/ generated (fig. 3a) and indicate a low frequency of repeatedly observed clonotypes. we estimated diversity using chao 2 22, 23 and recon 23,24 as previously described 13 . diversity estimates were similar between the two estimators, (4.8 × 10 6 -7.4 × 10 6 ) for chao and (9.4 × 10 6 -1.9 × 10 7 ) for recon (fig. 3b) . we next estimated heavy chain sequence diversity for each animal (fig. 3c) and again found that both estimators broadly agreed, giving similar values of (5.0 × 10 7 -8.1 × 10 7 ) for chao and (5.4 × 10 7 -1.0 × 10 8 ) for recon. previously published estimates of both clonotype and sequence diversity in individual humans 17 only exceed that in the omnirats by a maximum two orders of magnitude. this is surprising given that the omnirat is more restricted in cdrh3 length. for each combination of two or more animals, we computed the frequency of shared unique heavy chain clonotypes (fig. 4a) . there was on average 9.32% of clonotypes shared between each combination of two omnirats. surprisingly, we found that 4.90% of clonotypes were shared between all three of the animals. next, we pooled unique heavy chain clonotypes from all ten human subjects and measured the percentage of clonotypes in each animal that could be found in the total human pool (fig. 4b) . we found that (11.9-13.7%) of each omnirat clonotype repertoire and 13.8% of clonotypes combined from all animals could be found in the total human clonotype pool. shared clonotypes have shorter cdrh3 lengths than unshared clonotypes on average (fig. 4c) which is expected given that sequence diversity is expected to increase as the number of amino acids increases giving less of a chance for sharing. vh gene family usage between shared and unshared clonotypes indicates no major differences (fig. 4d) . sequence logos for 8 amino acid long (fig. 4e) and 13 amino acid long (fig. 4e) cdhr3s from both shared and unshared fractions were made and indicate broad similarity between the two fractions. we set out to determine commonalities and differences between omnirat and human antibody repertoires to be used as a reference for vaccine studies. our results show that there exists substantial variation in gene usage frequencies and elements of recombination, indicating specific limitations of this animal model for predicting the human immune response. we found that by performing hierarchical clustering on gene segment usage, repertoires clustered together by both species and tissue. differences in gene segment usage between transgenic animal models and humans, as well as between tissues are expected. for example, multiple human ig loci transgenic rodents are reported to have gene usage profiles that slightly vary from that of humans 25, 26 . furthermore, antibody repertoires from separate human tissues are known to deviate strongly enough to be clustered by hierarchical clustering 27 . in our case, the lymph node repertoire from the omnirat was most likely able to be distinguished from that of the spleen due to the increased presence of antigen-experienced b cells in the latter as shown by somatic hypermutation and class-switched transcript frequencies. this indicates that tissue selection will affect the outcome of an antibody discovery campaign and reinforces evidence for normal b cell development by suggesting the existence of affinity maturation. investigation into cdr3 length distributions revealed that the omnirat prefers shorter cdrh3s as compared to humans. interestingly, the mechanisms of this preference are due to decreased n additions, and a tendency to incorporate shorter dh gene segments. this result has also been seen in multiple other transgenic and wild-type rodents 25, 26, 28, 29 . the specific reasons remain unclear, although the observation that wild-type rat germline d gene segment lengths are shorter suggests intrinsic species-specific mechanisms of selection as well as differences in tdt expression during bone marrow b cell development. we further speculate that another possible reason for intra-species variation can be attributed to distinct prior antigen exposure and divergent gut microbiomes 30, 31 . the diversity of the omnirat heavy chain repertoire was shown to be slightly lower than that in humans. our results indicate biased gene usage and decreased junctional diversity are the primary reasons for the resulting repertoire diversity estimate comparisons. we also showed that there is a much higher frequency of 'public clonotypes' or clonotypes shared between members of this species than previously reported in humans 13, 32 . lower sequence diversity combined with identical genetic background and highly similar gene usage are possible reasons for this result. in summary, we have determined specific differences between the omnirat and human antibody repertoires which must be taken into careful consideration when evaluating an antibody response in order to make predictions for human subjects. we have also shown that this animal's antibodies show signs of class switch recombination, somatic hypermutation and large diversity supporting its value for the discovery of monoclonal antibodies to targets that may not be immunogenic in other models. even though a high degree of variation exists, we still found many clonotypes to be shared between the species pools. finally, more studies will need to be done in order to characterize omnirat serum and memory b cell responses to immunogens. next-generation sequencing of omnirat antibody repertoires. total rna from spleens and lymph nodes was extracted (rneasy maxi kit, qiagen) from each unimmunized heavy chain and kappa chain only transgenic rat (omnirat, open monoclonal technology inc., palo alto, ca, usa) and antibody sequences were amplified as previously described 13 except for different primers used during reverse transcription (table s2) . we chose to interrogate the antibody repertoires found in secondary lymphoid organs as opposed to peripheral blood due to the higher number of b cells. correct pcr product sizes were verified on an agarose gel (e-gel ex; invitrogen) and quantified with fluorometry (qubit; life technologies), pooled at approximately equimolar concentrations and each sample pool was re-quantified before sequencing on an illumina miseq (miseq reagent kit v3, 600-cycle). all animal experiments were conducted in accordance with the institutional animal care and use committee of scripps research and approved by the institutional research boards of scripps research. (d) distribution of vh gene family usage for unshared clonotypes from the animal pool (black) or clonotypes shared by both species pool (red). (e,f) sequence logos of the cdrh3s encoded by shared and unshared clonotypes of length 8 (e) or 13 (f). head-region amino acid coloring: polar amino acids (gstycqn) are green; basic amino acids (krh) are blue; acidic amino acids (de) are red; and hydrophobic amino acids (avlipwfm) are black. all torso residues are gray. murine antibody responses to cleaved soluble hiv-1 envelope trimers are highly restricted in specificity rational hiv immunogen design to target specific germline b cell receptors priming hiv-1 broadly neutralizing antibody precursors in human ig loci transgenic mice bacterially derived synthetic mimetics of mammalian oligomannose prime antibody responses that neutralize hiv infectivity a repertoire of monoclonal antibodies with human heavy chains from transgenic mice antigen-specific human monoclonal antibodies from mice engineered with human ig heavy and light chain yacs antigen-specific human antibodies from mice comprising four distinct genetic modifications human antibody production in transgenic animals transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies mechanisms of central tolerance for b cells high-affinity igg antibodies develop naturally in ig-knockout rats carrying germline human igh/igκ/igλ loci bearing the rat ch region human antibody expression in transgenic rats: comparison of chimeric igh loci with human vh, d and jh but bearing different rat c-gene regions commonality despite exceptional diversity in the baseline human antibody repertoire clonify: unseeded antibody lineage assignment from next-generation sequencing data genetic measurement of memory b-cell recall using antibody repertoire sequencing toward a more accurate view of human b-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding massively scalable genetic analysis of antibody repertoires rapid and focused maturation of a vrc01-class hiv broadly neutralizing antibody lineage involves both binding and accommodation of the n276-glycan hiv-1 vaccines. priming a broadly neutralizing antibody response to hiv-1 using a germline targeting immunogen identification of common features in prototype broadly neutralizing antibodies to hiv envelope v2 apex to facilitate vaccine design high-throughput immune repertoire analysis with igor estimating the population size for capture-recapture data with unequal catchability how many different clonotypes do immune repertoires contain? robust estimates of overall immune-repertoire diversity from high-throughput measurements on samples complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery mechanisms that shape human antibody repertoire development in mice transgenic for human ig h and l chain loci tissue-specific expressed antibody variable gene repertoires intrinsic bias and public rearrangements in the human immunoglobulin vλ light chain repertoire immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire microbial symbionts regulate the primary ig repertoire b cell superantigens in the human intestinal microbiota high frequency of shared clonotypes in human b cell receptor repertoires moderated estimation of fold change and dispersion for rna-seq data with deseq. 2 the authors thank all the study subjects for their participation. c.j., d.r.b. and b.b. planned and designed the experiments. c.j. performed experiments. c.j. analyzed data. c.j. wrote the manuscript. all authors contributed to manuscript revisions. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-57764-7.correspondence and requests for materials should be addressed to d.r.b. or b.b.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-104317-t30dg6oj authors: parker, michael t. title: an ecological framework of the human virome provides classification of current knowledge and identifies areas of forthcoming discovery date: 2016-09-30 journal: yale j biol med doi: nan sha: doc_id: 104317 cord_uid: t30dg6oj recent advances in sequencing technologies have opened the door for the classification of the human virome. while taxonomic classification can be applied to the viruses identified in such studies, this gives no information as to the type of interaction the virus has with the host. as follow-up studies are performed to address these questions, the description of these virus-host interactions would be greatly enriched by applying a standard set of definitions that typify them. this paper describes a framework with which all members of the human virome can be classified based on principles of ecology. the scaffold not only enables categorization of the human virome, but can also inform research aimed at identifying novel virus-host interactions. the term "microbiome" was coined by whipps, lewis, and cooke in 1988, defined as "a characteristic microbial community occupying a reasonably well defined habitat which has distinct physico-chemical properties" [1] . many attribute the popularization of the term to nobel laureate joshua lederberg, where he (anthropocentrically) defined the microbiome as "the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease" [2] . the community of microorganisms referenced in these definitions includes viruses, bacteria, fungi, protozoa, and archaea. within this community there is an inherent plasticity arising from the interaction of organisms with one another and with their environment. such meta-interactions lead to complex repercussions for each level of life, and this review will focus on the consequences that have implications for human health. the viral component of the microbiome, termed the "virome" [3] is a poorly understood facet of the microbiome, despite having the potential to significantly impact human health. from a philosophical perspective, viruses were likely integral to the very existence of man as the primordial precursor to all current life on earth [4] . more tangibly, during the course of human evolution, the viruses in and around humans not only drove evolution via selective pressure, but also contributed novel genetic material. in fact, approximately 42 percent of the human genome is composed of viral sequence [5] . this role as a major agent driving horizontal (i.e., non-reproductive) gene transfer between biomes [6] [7] [8] [9] and the fact that the diversity, complexity, and abundance of viruses surpasses any other biological entity make it apparent that understanding viromes will impart unparalleled understanding of the organisms they inhabit [10] . however, the obvious importance of viruses in the composition of all biomes has not (yet) been met with an appropriate fervor for the characterization of the viral review recent advances in sequencing technologies have opened the door for the classification of the human virome. while taxonomic classification can be applied to the viruses identified in such studies, this gives no information as to the type of interaction the virus has with the host. as follow-up studies are performed to address these questions, the description of these virus-host interactions would be greatly enriched by applying a standard set of definitions that typify them. this paper describes a framework with which all members of the human virome can be classified based on principles of ecology. the scaffold not only enables categorization of the human virome, but can also inform research aimed at identifying novel virus-host interactions. component of microbiomes. the original explosion of microbiome information was spurred by the utilization of 16s sequencing technology, which can give vast information about microbial communities using universal primers [11] . however, this technique is specific to organisms with ribosomes, so early research focused on analysis of bacterial sequences rather than much more technically challenging viral sequences [12] . thankfully, recent advances in next generation sequencing technologies are making virome characterization more technologically and financially possible and will ensure an explosion of virome description in the near future [13, 14] . in an effort to facilitate such classification of the human virome, it is prudent to use basic ecological interactions both to organize current knowledge as well as identify areas where new information may be found. this review will build a framework of the human virome from an ecological perspective, identify currently underappreciated and possibly undiscovered roles of the virome, and apply these principles to analysis of the role of the virome in human health. such a systematic conception of the structure of these ecological interactions can also facilitate the application of similar conventions to the other components of the microbiome. while appreciation of the possible benefits of the human microbiome accumulated in the latter half of the 20th century, a concordant acknowledgement for the human virome was not so quickly realized [15] . it is likely this was because human viruses rely on invasion of host cells to replicate and this provokes an overwhelmingly negative impression. however, all interactions of viruses with their hosts are symbioses in the classical sense [16] and fall along a spectrum from exclusively antagonistic to exclusively mutualistic [17] . in general, these interactions are facultative for the human and obligate for the virus, but cases like the presence of viral sequences in human genomes exemplify where this simplistic view breaks down [5] . while the position of a particular virus along this spectrum is not fixed, it is obvious that many viruses have varied, intimate relationships with their hosts. botanists and entomologists were among the first to notice the intricacy of virus-host interactions, which challenged the purely parasitic dogma of virus-host interaction (reviewed in [17] [18] [19] [20] ). for example, parasitoid wasps harboring polydnavirus genomes utilize virally packaged host genes to prevent rejection of wasp eggs introduced into the parasitized caterpillar [21] . in a more benign example, the white clover suppresses nodulation in conditions of sufficient nitrogen in a manner dependent on viral coat proteins of a persistently infecting cryptic virus [22] . while the breadth of knowledge of interactions types is surely incomplete, their presence in other organisms al-ludes to the possibility of the existence of similar interactions between humans and viruses. recent reviews have addressed the issue of the current, primarily negative, view of the human virome [18, [23] [24] [25] [26] . virologists are beginning to realize that the viruses within us may be more important than previously appreciated. the discovery of intimate interactions of viruses with humans, like the role of endogenous retrovirus (erv †) syncytins in placentation [27] , are categorically dissimilar to the classical view of viruses only as parasites and brings to issue how scientists are approaching the study of the virome. a lack of structure in this endeavor has arguably hindered the true understanding of non-pathogenic consequences of human-virome interactions. to address this issue, an ecological framework can be constructed for more informative classification of the virome based on how viruses fill niches in humans. here, i describe a conception of the human virome with three general classifications of virus types: parasitic, commensal, and mutualistic ( figure 1 ). within these groupings are subcategories containing more specific characteristics germane to different viral groups. table 1 illustrates how representative members of the human virome spanning a vast array of families, replication schemes, genome architectures, and tropisms can be succinctly classified with this scheme. this arrangement provides functional information about the nature and plasticity of virus-host interaction, the most critical defining characteristic of a virus in the context of human health, in a way absent in the more classical types of organization mentioned above. in the rest of this review, i will characterize each grouping and provide examples from the literature supporting this classification. the application of this scaffold will not only deepen the understanding of known virus-host interactions in the ecological context of the virome, but will also identify logical next steps and gaps in current knowledge that are tantalizing areas for future exploration. the canonical classification of viruses is as parasites. in general, parasitic viruses are transmitted horizontally from host to host, and this process is necessary for the virus' survival. their obligate life cycles necessitate manipulation of cellular processes and have been classically linked to disease manifestation. while the role of viruses in human disease was not appreciated until the 19th century [28] , throughout human history, the diseases caused by particularly pathogenic viruses have been some of the most feared and deadly. currently, there are 129 known human viral pathogens [29] and at least 219 viruses that can infect humans [30] , with more being discovered every year [31, 32] . the apparent bias toward pathogenic species is undeniably because phenotype (read: pathogenesis) has traditionally been a major factor in the discovery of new viruses. while parasitic viruses are a morose facet of human history, they have contributed to man's evolution as a species. the biologist's viewpoint includes the selective pressure on man in which viruses kill susceptible hosts, leading to the emergence of fitter genotypes. some viruses play a similar role by limiting an infected host's reproductive fitness [33] . the presence of deadly viruses has also supplied pressure for behavioral and psychological adaptation via influence on practices in mate selection, food preparation, and sanitation [34] . a modern example of such behavioral adaptation is the development of vaccination, kick-started by edward jenner's famous work with cowpox [35] . however, not all parasitic viruses are deadly. in fact, excessive pathogenicity is an evolutionarily poor strategy for a virus' survival unless balanced with a number of other trade-offs such as transmission rate and recovery rate [36] . most parasitic infections occur horizontally (though there are rare exceptions [37] ) and the equilibrium between pathogenicity and other trade-offs is crucial to ensuring continuation of the viral lineage. while parasitic viruses by definition have some level of pathogenic effect on their host, it is not the intent of the virus to do so, because viruses don't have intentions. viral genomes (particularly those of rna viruses) exist as a pool of non-identical, related sequences, termed quasispecies, and are constantly adapting in response to selective pressures [38] . in some cases, the virus' environment can select for changes that create a balance of the above-mentioned trade-offs where pathogenicity may be a successful, or at least not detrimental, strategy [39] . in this light, i will define types of parasitic viruses according to both their degree of pathogenesis and the way in which they interact with their host. the key characteristic of pathogenic viruses is that they cause an acute disease in which the host either lives or dies, with the virus simply using it as a vessel to move to the next host. in these interactions, hosts typically mount strong immune responses and literally fight for their lives. this is not to say that all pathogenic infections are deadly or resolved immediately, since the degree of pathogenicity and time to resolution vary widely, as will be discussed in following sections. rather, the virus-host interactions classified here as "pathogenic" represent perturbations of homeostasis for which the only resolution is that one side must prevail. either the host completely clears its body of the virus, or it succumbs to infection. while this sounds like a poor transmission strategy, history has shown it can be quite effective, as long as the proper trade-offs are satisfied (see the parasitic virome, above). influenza virus is a prime example of the success of a pathogenic strategy. most humans can expect to be infected with at least one influenza virus per year, and in general they will observe a fairly robust set of symptoms including, but not limited to, fever, chills, headache, nasal congestion, and coughing [40] . while infection can be severe and even cause death, this is typically limited to hosts in an immunocompromised state, as selective pressure typically drives virus evolution to less virulence [41] . in general, virus is shed one to two days after infection, with symptoms usually presenting one day later, ensuring that a person shedding virus will be out and about among currently uninfected individuals [42] . severe symptoms may be due to viral replication and destruction of tissue; however, over-reaction of the immune system is often the cause [41] . this is exemplified by secondary bacterial pneumonia where blooms of pathogenic and/or commensal bacteria can result in severe disease or death [38] [39] [40] [41] . however, by the time a person has become ill, they have likely spread the virus by direct contact with an uninfected individual, contaminated fomites, or aerosolized respiratory droplets [46] . the fact that many pathogenic viruses have circulated in humans for hundreds to thousands of years, despite our best efforts to eliminate them, speaks to their success. as far back as the 16th-11th centuries b.c., there is evidence of poliomyelitis caused by poliovirus in egypt [47] . a more recent example is measles virus, which appears to have diverged from rinderpest sometime in the 11th or 12th century b.c. [48] . the long histories of human disease caused by these and other pathogenic viruses made them low hanging fruits for study in the new field of microbiology in the early 19th century [47] . this can explain much of the bias of current knowledge toward pathogenic viruses. while it can be argued that in the majority of human history these viruses were the most formative in terms of social, cultural, and behavioral characteristics of humans, it cannot be overlooked that this point may also be similarly biased. the notion that some viruses could live in relative homeostasis with the organisms they infect is very recent, and such interactions may prove to be a renaissance in the thinking of the importance of different portions of the virome. in fact, some of the most clinically important viruses of the last century were not classically pathogenic, but rather, persist within the host in relative silence for long periods of time. the characteristic strategy of persistent viruses is one of immune evasion and long term interaction with the host. typically, these viruses exhibit mild and short-lived acute phase infections followed by establishment of a long term niche within a host. in this niche, the virus may go through stages of dormancy and reactivation, or slow replication for a long period of time [50] . this ability to progress to persistence for months, years, or the entire life of the host clearly delineates these viruses from the pathogenic viruses [51] . these relationships straddle the border somewhere between symbiosis and pathogenesis, traditionally making it difficult to classify them. in this context, i will typify long-term infections with well-described detriment to the host as persistent, and other longterm interactions as either commensal or mutualistic (see respective sections, below). while the virus has a safe home during persistence, the question arises that if a virus is not replicating, or is present at very low levels, how is it transmitted to the next host? the answer lies in the spread of virus via close intrahost interactions that may occur many times during the host's life. the most conventional method by which persistent viruses are transmitted is contact between mucous membranes. this includes kissing and sexual intercourse, but can also include transmission vertically from mother to child, in rare cases [37] . the prototypical example is herpes simplex virus 1 (hsv-1) which, after an initial acute or subclinical infection, moves into a sensory neuron and enters a period of latency [52] . later in life the virus can break latency, move down the neuron to the skin, and begin to replicate, the prototypical presentation of which is a cold sore. these lesions release infectious virus that can be transmitted to a new host. after a short time, the virus returns to latency and the lesion will heal [52] . the success of this strategy of latency and mild pathogenic effect is made obvious by the fact that approximately two-thirds of the world's population is infected with hsv-1 [53] . non-conventional methods of inoculation have arisen concomitant with certain human tendencies and interactions. the most common examples include intravenous drug use, blood transfusion, and organ transplantation, all of which break typically impassable barriers [54] . for viruses like hepatitis c virus (hcv) and human immunodeficiency virus (hiv) that circulate in the blood of their host, these disruptions of natural barriers created a heyday for transmission. an important development of modern medicine was the implementation of standard blood screening for these and other persistent viruses and has led to a decrease in medically acquired disease [55, 56] . because of the longevity of asymptomatic interaction, those who are infected with persistent viruses may not know for years. this has consequences not only for transmission to others, but also for eventual progression to disease, as treatment is delayed until symptoms emerge when the damage is often irreparable. for hiv, the condition of acquired immune deficiency syndrome (aids) weakens the immune system drastically, allowing opportunistic microbes to cause fatal disease [57] . hcv is the most common cause of chronic liver disease, cirrhosis, and liver cancer in the form of hepatocellular carcinoma, all caused by years of persistent viral presence [58, 59] . the delayed onset and long-term homeostasis of persistent viruses within their hosts makes them a unique class within the human virome. understanding the biology of these viruses and the development of methods to control and eliminate them is an area of much importance in future medical science. additionally, further characterization of the human virome is likely to uncover more viruses that persistently infect humans [31] , and such discoveries could pave the way for the treatment of diseases of currently unknown etiology. as will be discussed in later sections, not all viruses are pathogenic. herein, i classify viruses that impact host physiology without directly eliciting a disease state as atypically pathogenic viruses. for example, some viruses infect humans without ever causing disease while others inhabit and change the composition of the human microbiome without ever infecting human cells. since our immune system can't tell the difference between harmful or benign viral signatures, the mere presence of these viruses harbors potential to cause disease. so far, this type of interaction has not been defined as a facet of the virome, but in this section i will review examples from the scientific literature that hint at such scenarios. endogenous retroelements (eres) represent a unique class of human-colonizing viral material that is resident in the germ line and include endogenous retroviruses (ervs), retrotransposons, and retrotranscripts [60] . while the general classification of these genetic elements will be defined in this work as mutualistic (see the mutualistic virome, below), it is evident from a number of studies that they can sometimes contribute to pathogenesis. in the autoimmune disease multiple sclerosis (ms), human endogenous retrovirus (herv) syncytin expression is upregulated in lesions and leads to an increase of cellular protein oxidation and destruction of oligodendrocytes [61] . another possible autoimmune contribution of eres is by their engagement of nucleic acid pattern recognition and downstream immune activation during both their rna and cdna synthesis [62, 63] . carcinogenesis may also occur, as exemplified by the rec protein of herv-k(hml2), which can facilitate tumorigenesis when expressed in mice [64] . a final example of deleterious consequences conferred by eres is their ability to destabilize the genome via insertion, rearrangement, and deletion [65] . continued exploration is necessary to expand upon these data to elucidate the mechanisms behind the negative impacts of eres on human health. bacteriophages (from here, referred to as phages), which are canonically classified as commensals (see the commensal virome, below), may also play an atypically negative role. while these viruses do not infect human cells, the estimated 10 15 phages present in the human gut [66] and those elsewhere on the body are not likely to be completely benign. while it is known that phages can be co-opted for benefit to the host [66] , i postulate that the presence of this amount of genomic and proteinaceous material is unlikely to be immunologically inert. an example of how a phage could affect human health is as an endogenous ligand for immune activation. it is known that humans harbor antibodies to phage proteins [67] [68] [69] [70] and that phagocytic cells of the immune system can ingest phages [71] , but whether this has any negative impact on health is unknown. toll-like receptors (tlrs) -7 and -9 play key roles in the production of autoantibodies, presumably via sensing of nucleic acid containing immune complexes (reviewed in [72] ). these autoantibodies could be derived from any number of endogenous sources, including those of phages. interestingly, identifying the particular nucleic acids responsible for this activation in vivo has been challenging, though functional work has vetted this mechanism with immune complexes containing nucleic acids [73, 74] . it is possible that b-cell receptor (bcr)-mediated endocytic or plasmacytoid dendritic cell (pdc) phagocytic internalization of phages could deliver ligands necessary for these responses. in this light, i would argue that correlating phage localization and expression with disease state is a particularly intriguing area of research moving forward. such research may also elucidate mechanisms by which specific bacteria (and their viral cargo) are linked to autoimmune, allergic, and pathogenic phenotypes. protozoan parasite-associated viruses have emerged as another source of atypically pathogenic effects in humans [75] . such interactions are reminiscent of the uses of viruses as agents for forwarding their host's survival in parasitoid wasps and plants (see an ecological framework of the human virome, above). the recognition of the leishmania-infecting lrv1 virus by human macrophages can promote inflammation, subvert the immune response, and ultimately promote persistence of the parasite [76] . trichomonas vaginalis harbors a trichomonasvirus that triggers ifn responses via tlr-3, leading to inflammatory sequelae [77] . at present, there is insufficient data as to whether these are evolutionarily adapted mechanisms meant to perturb host immunity to benefit the parasite, but the extensive literature of such interaction in insects and plants makes this a tantalizing conjecture. additional characterization should explore the nature of the interactions of protozoan viruses with humans. while all the above postulates are conjecture on the limited knowledge base regarding atypically pathogenic virus-host interactions, it must be noted that these are certainly not overlying principles. associated pathologies likely require particular genetic dispositions and/or external stressor states. it cannot be understated, however, that a better understanding of the viruses that typically inhabit humans will likely lead to a concomitant rise in the cases of viruses identified as atypically pathogenic. the term commensal was originally coined to describe an organism that benefitted from being on or in a host but did not have any direct detriment or benefit for the host [78] . as the field of microbiome research blossomed, it became clear that many of the microbes that had originally been considered commensal truly did have direct effects on host health (reviewed in [79] ). these organisms may be more accurately defined as atypically pathogenic or mutualistic (see sections above and below, respectively) if these effects are common or stable, but in general, scientists have followed the practice of innocence until proven guilty. however, it is likely that many commensals exist that either rarely or never have effects on the host. while at the local level some commensal viruses may have detrimental effects on individual cells, many eukaryotic and prokaryotic viruses are associated with healthy human tissues [80] [81] [82] [83] [84] [85] [86] [87] . the prototypical examples of commensal viruses are phages that infect bacteria [88] . no phages are known to infect human cells and therefore their presence may be primarily innocuous. the effect of phages on the composition of the microbiome, in that they may kill or inhibit growth of some bacteria, could be considered a possible detriment or benefit depending on the situation, albeit indirect. in fact, the targeted utilization of phages to eliminate unwanted bacterial pathogens (phage therapy) was introduced in the early 1900s and has many potential benefits including, but not limited to, low toxicity, treatment of antibiotic resistant infections, and rapid discovery/generation [89] . however, concern over unknown health implications and the rise of antibiotics stalled the field, though a renaissance has been building since the 1990s [90] . many different levels of this application exist, such as treatment of animals and plants with phages to ensure better yield as well as to kill human pathogens that may grow on or in these food sources [91] . phages are also known to be able to stimulate and/or modulate immune action in humans at multiple levels including antibody generation, development of adaptive immune cells, and innate pattern recognition (reviewed in [88] ), and the possible applications of phages in these contexts are intriguing. in the future, sections of this group may be able to re-classified as atypically pathogenic or mutualistic if their presence can be linked to roles in specific disease phenotypes, but for now they are most appropriately classified as commensal. viruses can also infect fungal members of the human microbiome (mycoviruses) and protozoa of the human macrobiome [77, [92] [93] [94] [95] [96] [97] [98] . while much less is known about these particular types of viruses, similar conjectures have been made as for phages in regard to their potential as both atypical pathogens and mutualists. direct effects on human health are likely rare, though they have been observed for protozoan parasite viruses (see atypically pathogenic viruses, above). the presence of these viruses may ultimately have an indirect effect on human health of unknown consequence. investigation of the breadth and impact of such viruses is an interesting and important area of future research, particularly for the benefit of the immunocompromised and those in developing countries who are most affected by diseases caused by fungi and protozoa. there is also an abundance of prototypically planttropic viruses found in the human gut [99] . it is likely these viruses have been acquired via agricultural crops in the diet and the effect of their presence is unknown, but assembly of plant viral particles in escherichia coli has been exhibited, so it is possible that these viruses may interact with gut bacteria [100] . some commensal viruses can infect human cells and use the human as a vessel for transmission. these viruses establish asymptomatic infections that do not result in a change in host health or behavior. this represents a veritable antithesis to the strategy of parasitic human viruses (see the parasitic virome, above), wherein viruses have prioritized trade-offs other than pathogenicity to ensure their transmission to a new host. the most well documented examples of asymptomatic infection of humans by commensal viruses are the rhinoviruses and other infections of the nasopharynx and upper respiratory tract [80, [101] [102] [103] [104] . many of the human papilloma viruses (hpvs) interact with human cells as an asymptomatic infection of mucous membranes or skin [105] . anelloviruses are also well described human commensals [106] [107] [108] [109] [110] . while immune detection of non-human tropic commensal viruses is avoided by physical separation, the human-tropic commensal viruses assuredly engage with host innate and adaptive immune responses, and so must avoid or subvert these mechanisms to maintain their immunological silence. description of the viral techniques enabling this evasion is an important direction for future research on these viruses, as this may point to novel avenues of therapeutic intervention for suppression or activation of immune responses. while commensal viruses do not have any direct effect on human health during homeostatic equilibrium, perturbation of homeostasis may cause them to contribute to pathogenesis (see atypically pathogenic viruses, above). contrarily, the presence of viruses from the final major group of the human virome, mutualistic viruses, exhibit positive effects on host health via an assortment of mechanisms. perhaps the most underappreciated portion of the virome are those viruses that have a positive health benefit for humans. a mutualistic interaction is one that benefits both organisms involved [111] . it is reasonable to assume that it may be beneficial for a virus to encourage and aid in its host's health to facilitate a longer lasting and amenable environment for survival. but this is a bit counterintuitive considering that above i discussed that viruses are intrinsically parasitic. this can be rationalized with the assertion that nearly all mutualistic interactions were probably at some point a form of parasitism, then passed through a commensal stage, and finally adjusted to mutualistic harmony through co-evolution. this stipulation is based on the reasoning that the amount of evolutionary change necessitated in the virus and/or the host to switch interaction types requires significant adaptation and thus is more likely to proceed stepwise [112] . a relationship must become sufficiently non-pathogenic to allow interaction without rejection as well as develop benefit for both sides, and an intermediate state seems a logical platform for this. indeed, this postulation seems bolstered by the spectrum of mutualistic relationships that exist, spanning obligate to conditional mutualistic benefits [18] . a classic non-viral example that illustrates this point is the integration of the mitochondrion into the cells of ancient prokaryotes or eukaryotes. this kind of relationship is considered symbiogenic, meaning that once separate organisms have now fused into a distinct new species [18] . either by phagocytosis or infection, an endosymbiosis was established in which the mitochondrial bacterial progenitor supplied some benefit to its host. millions of years later, mitochondria are part of all eukaryotic organisms with functions spanning energy production, innate immunity, and membrane potential, to name a few [113, 114] . it is now appreciated from data on the microbiome that it would be to the host's benefit if the power of the genetic information in and around them could be harnessed as an evolutionary (and medical) tool for self-betterment [115] [116] [117] . this could occur in a number of ways, either through encouraging colonization by beneficial microbes or even by integrating the genetic material of these microbes into the human genome. a small body of literature characterizing virus-human mutualism has given a glimpse into this facet of the virome. in the next sections, i will review two levels of symbiotic relationships and discuss important gaps in knowledge important for the understanding and possible utilization of these interactions for human health. symbiotic viruses that are transmitted vertically from parent to child in the germ line are considered primary symbionts [118] . these relationships are often ancient and obligate for both parties [119] . the most obvious example is the eres that have integrated themselves into human genomes at many points throughout millions of years of evolution. while some have been inactivated by recombination, deletions, and random mutations, many still appear to be expressed [65] . it is also obvious the control of these elements is an important challenge for the host, as many strategies have evolved to control replication and reinfection of inserted eres [120, 121] . to date, the role of most eres in the human genome is unknown, but a few human and mammalian examples indicate that their presence confers a number of benefits. some of the most important effects of ere colonization come in the form of genomic diversity and plasticity. eres can not only insert new genetic material, including protein coding genes, but also play roles in genetic mobility and control. the recombinatorial ability of eres is conferred upon insertion and has facilitated recombination and duplication of large swathes of genomic material [122, 123] . where an ere inserts itself can be an important factor in how it affects the genome, as insertion into a protein coding gene could be innocuous or catastrophic. alternatively, insertion in or near promotion and regulatory elements can result in changes in transcriptional control [124] , as can the native regulatory sequences in the eres themselves [125, 126] . of course, all of these benefits can have similarly damaging consequences, as destabilization of the genome or particular genes could have profound negative effects on the host (see atypically pathogenic viruses, above). however, natural selection dictates such effects will not successfully continue in the host lineage. eres also have important immunologic and developmental roles. building off of the transcriptional control effects mentioned in the last paragraph, eres appear to have a role in gene upregulation via pattern recognition during the initiation of adaptive immune responses [127, 128] . adaptive responses to eres can also play a role in restricting viruses by neutralizing incoming exogenous virus via antibody development against ere surface glycoprotein antigens [129] . examples from mice also indicate that the expression of the erv glycoprotein env can competitively bind surface receptors important for exogenous virus entry and that a short product of an erv gag gene can inhibit the nuclear translocation of incoming capsids. [130, 131] . developmentally, the acquisition of erv syncytins has been a critical step in the evolution of placentation in humans [27, 132, 133] . the presence of retroviral nucleic acids in human cells also suggests that ere reverse transcription may be able to activate host innate immunity [62] . whether humans can harness the beneficial properties of eres is of much scientific import. for years it has been debated whether gene therapy using retroviral vectors for insertion of genes into an individual would be of more help than harm. overall the utility of such methods have been tempered by the observed oncogenic potential of such therapies [134] . recently, studies with retroviral-modified chimeric antigen receptor t-cells (car tcells) have shown impressive application for the activation of adaptive immunity against cancers, though there have also been morbid failures in this field [135] . the application of retroviral-modified car t-cells could presumably be expanded to infectious disease as well. these progressive areas of medical science will continue to be explored and debated well into the future, and better understanding the relationship of eres with their human hosts will be indispensable for these studies. similar to the primary symbionts, secondary symbionts form mutualistic relationships with their host, but are instead acquired via non-germline vertical transmission or horizontally during the post-natal life of the host. a popular example is that of phages. beginning with the microbial colonization of a child during birth, phages hitch a ride with their host bacteria. phages can regulate bacterial species in the gut, as it is known that phages can be coopted into mucosal surfaces for this purpose [136] . free phages may also serve a similar role as sentinels maintaining a balanced microbiota. these benefits have been studied extensively as possible medical tools in phage therapy (reviewed in [137, 138] ) and there has recently been a resurgence of interest as the problem of antimicrobial resistance has become more dire [139] . these same principles can likely be extended to viruses that parasitize other microbes and macrobes as well. other examples of secondary symbiotic viruses are isolated, but numerous. in general, these viruses likely compete for receptors, cellular resources, or niches within hosts to confer protection. hepatitis g virus (hgv) is known to slow development of hiv infection to aids pathogenesis [140, 141] and human cytomegalovirus (hcmv) infection can suppress hiv infection by limiting availability of the coreceptor ccr5 on macrophages [142] . infection with hepatitis a virus (hav) can suppress infection with hcv and may actually promote clearance [143] . evidence from mice supports the assertion that viruses can confer resistance to non-viral disease as well, as infection with murine gammaherpesvirus results in protection from challenge with listeria [144] and type 1 diabetes can be prevented in mice infected with lymphotropic viruses [145] . oncolytic viruses, which specifically target, infect, and kill tumor cells are also notable in both their novelty and their clinical utility (reviewed in [146] [147] [148] ). viruses may even be able to provide developmental and immunological benefits not unlike that of the bacterial microbiota, as evidenced by the establishment of gut homeostasis in mice colonized with norovirus [149] . endogenous retroelements could also presumably be included here in the case of their population of a nongermline subset of cells within an individual and conferral of benefit to the host. one of the main functions of secondary symbionts may be to help maintain a baseline of immune activation in order to give the host a head start upon challenge with incoming pathogens. it has been estimated that healthy humans are infected with > 10 viral infections at any given time [51] . this constant barrage of immunogenic material presumably drives a smoldering activation of both innate and adaptive immunity. from this, it may be concluded that a state of continual challenge may be integral for the immune system to be fortified against incoming pathogens. while there is a plethora of research about secondary symbiotic bacteria, similar attention must to be paid to the virome. the examples outlined above point to incredible benefit derived from the interaction of humans with these mutualists, and significant effort must be made to discover and harness such potentially impactful relationships. moving forward, consideration of the effects of the human virome will be crucial in disease treatment and discovery of disease etiologies. from purely pathogenic viruses to those mutualists helping humans adapt to their world, the human virome has the potential to lend insight into the basic and applied biology of human health. in light of this, further exploration into the composition, tropism, and evolution of the human virome will undoubtedly make way for innovations in the treatment of human disease. throughout this manuscript, i have assembled the components of the human virome into broad classifications of parasitic, commensal, and mutualistic. while these definitions are logical, the intermixing of the groups and exceptions to rules drives home the point that the virome has inherent plasticity. disruption of the yin and yang of virus-host interactions can contribute to this transitory nature and may blur the lines that delineate the three main groups. this acknowledgement of convention and the departures from it gives both structure and flexibility to this conception of the human virome. the culmination of these ideas provides a template that identifies gaps in our current knowledge, postulates interactions that are currently unknown, and describes future areas of research, the results of which can then be classified in this same manner. i look on with anticipation to the continuation of the scientific soul search that is the 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and may lead to clearance of hcv herpesvirus latency confers symbiotic protection from bacterial infection prevention of type i diabetes in nonobese diabetic mice by virus infection recent progress in the battle between oncolytic viruses and tumours gene therapy progress and prospects cancer: oncolytic viruses apoptin, a tumor-selective killer an enteric virus can replace the beneficial function of commensal bacteria any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the national science foundation. key: cord-002933-zmx4k46v authors: stabell, alex c; meyerson, nicholas r; gullberg, rebekah c; gilchrist, alison r; webb, kristofor j; old, william m; perera, rushika; sawyer, sara l title: dengue viruses cleave sting in humans but not in nonhuman primates, their presumed natural reservoir date: 2018-03-20 journal: nan doi: 10.7554/elife.31919 sha: doc_id: 2933 cord_uid: zmx4k46v human dengue viruses emerged from primate reservoirs, yet paradoxically dengue does not reach high titers in primate models. this presents a unique opportunity to examine the genetics of spillover versus reservoir hosts. the dengue virus 2 (denv2) encoded protease cleaves human sting, reducing type i interferon production and boosting viral titers in humans. we find that both human and sylvatic (reservoir) dengue viruses universally cleave human sting, but not the sting of primates implicated as reservoir species. the special ability of dengue to cleave sting is thus specific to humans and a few closely related ape species. conversion of residues 78/79 to the human-encoded ‘rg’ renders all primate (and mouse) stings sensitive to viral cleavage. dengue viruses may have evolved to increase viral titers in the dense and vast human population, while maintaining decreased titers and pathogenicity in the more rare animals that serve as their sustaining reservoir in nature. dengue viruses cause clinical disease in approximately 100 million individuals each year and are found in over 100 countries (bhatt et al., 2013) . yet, to date no vaccine exists that conveys crossprotection against all human dengue viruses (scherwitzl et al., 2017) . dengue viruses are positive sense rna viruses in the family flaviviridae, and are related to yellow fever virus, zika virus, and west nile virus (best, 2016) . these viruses are primarily transmitted between humans in highly populated areas by aedes aegypti and aedes albopictus mosquitoes, in what are referred to as human (or 'urban') transmission cycles (diamond and pierson, 2015; hanley et al., 2013; vasilakis et al., 2011) . sylvatic (i.e. forest) dengue virus transmission cycles, which are separate from the human transmission cycles, exist in asia and africa and involve nonhuman primates and forest-dwelling aedes mosquitos (vasilakis et al., 2011; wang et al., 2000; rico-hesse, 1990) . while the exact nonhuman primate species that serve as the sustaining natural reservoirs for sylvatic dengue viruses are unknown, the global distribution of both dengue viruses and their transmitting mosquitoes could be consistent with a significant number of primate species being involved (figure 1-figure supplement 1) (hanley et al., 2013; vasilakis et al., 2011) . primarily, dengue viruses have been associated with monkeys (rather than apes) found in africa and asia (figure 1 ). human dengue viruses cluster into four phylogenetically distinct clades referred to as denv1, 2, 3, and 4 . these clades have sylvatic dengue virus isolates at their bases, supporting zoonotic origins of the four dengue viruses that now circulate in humans (wang et al., 2000; pyke et al., 2016; weaver and vasilakis, 2009) . human dengue viruses have now become uncoupled from the sylvatic reservoir and require only humans and mosquitoes to be sustained (mayer et al., 2017) . in side-by-side experiments, sylvatic and human dengue viruses replicate similarly in human cells (vasilakis et al., 2007; . these results have been interpreted to mean that there is little or no adaptive barrier for the emergence of sylvatic dengue viruses into human populations, and the view that dengue viruses are generalists capable of infecting a wide range of primate species including humans. thus, a paradox exists in understanding why human dengue viruses are so difficult to model in nonhuman primates. chimpanzees (pan troglodytes) (scherer et al., 1978) , rhesus macaques (macaca mulatta) (halstead et al., 1973; hickey et al., 2013) , marmosets (multiple callithrix species) (moi et al., 2013; ferreira et al., 2014) , and other nonhuman primate species (althouse et al., 2014) have been explored as possible primate models for studying dengue virus pathogenesis and for vaccine challenge. in general, it has been observed that dengue does not replicate to high titers in these models, and little or no overt disease pathology is observed (cassetti et al., 2010; zompi and harris, 2012) . if human and sylvatic viruses are the same in their properties, we speculated that there must instead be something special about the replication of these viruses in the human host. sting is a multi-pass transmembrane protein found in the endoplasmic reticulum, and functions as a critical component in the innate immune sensing pathway for intracellular pathogens (ishikawa and barber, 2008; zhong et al., 2008; ishikawa et al., 2009; jin et al., 2008; sun et al., 2009; burdette and vance, 2013) . although originally described as part of the response to cytosolic dna sensing (zhang et al., 2011) , sting is also activated upon rna virus infection (holm et al., 2016) . underscoring this, several rna viruses encode proteins that antagonize or elife digest dengue viruses are found in over 100 countries and cause the tropical disease known as dengue fever. dengue viruses affect around 100 million people per year and can -in severe cases -lead to death. unlike many other deadly diseases, there is currently no vaccine that completely prevents dengue fever. it is thought that dengue viruses that circulate in human populations were derived from monkey versions of that same virus. however, research suggests that both human and primate variations of dengue viruses appear to multiply much better in humans than in other species. scientists believe that this is because some animals, including primates, have defense mechanisms that are ineffective in humans. to explore this idea, stabell et al. looked at a protein called sting in humans and in three different primates: the chimpanzee, the rhesus macaque, and the common marmoset. sting plays an important role in the immune system and helps to fight infections caused by viruses and other microbes. during replication -the process by which a virus spreads through an organism's cells -the dengue virus cuts and inactivates the human sting protein, and so helps the virus spread. stabell et al. discovered that in most primates, dengue viruses cannot inactivate sting. this was found to be reliant on a small region in the sting protein that differed between humans and primates. this small difference may, in part, explain why dengue viruses replicate better in humans than other primates. stabell et al. then searched for other animals whose sting protein would be susceptible to dengue virus inactivation. using a database with genetic information of over 5,000 mammals, stabell et al. identified sting proteins of three types of apes and three types of rodents that could also be deactivated by dengue viruses. to develop a vaccine or antiviral drug scientists generally need to study the disease in living animals. since dengue viruses replicate more successfully in humans than they do in other animal models, it makes it more challenging to find an effective treatment. the results from stabell et al. may help to identify animals that could be strong candidates for future research into dengue viruses, potentially paving the way for further therapeutic development. (perelman et al., 2011) . the primate species from which sting is tested in this study are shown with purple arrows. possible primate reservoir hosts for sylvatic dengue viruses, based on virus isolation from sentinel monkeys, or antibody detection, are shown in red (africa) and green (asia). the current evidence for these primate reservoir hosts is reviewed in the discussion section. (b) 293t cells were cotransfected with plasmids encoding sting-ha, and the ns2b3-flag protease complex with or without the s135 inactivating mutation. whole cell lysate isolated 24 hr post transfection was run on a protein gel and immunoblotted with anti-flag or anti-ha antibodies. the encoded ns2b-ns3-flag polyprotein auto-processes into the ns2b3 protease complex if the protease is active, as seen in the anti-flag blot where in some samples the ns3-flag protein has been liberated through cleavage. we sometimes see lower bands underneath the full-length mouse sting, but conclude that they are endogenous degradation products since they are equal in intensity in the presence of the active or dead protease. doi: https://doi.org/10.7554/elife.31919.003 the following figure supplement is available for figure 1: degrade sting (sun et al., 2012; nitta et al., 2013; ding et al., 2013; aguirre et al., 2012; yu et al., 2012) . for instance, the ns2b3 protease of one human dengue virus, denv2, has been shown to target human sting for cleavage yu et al., 2012) . through the cleavage of sting, denv2 renders the host unable to induce the phosphorylation of interferon regulatory factor 3 (irf3), therefore decreasing production of type i interferon and increasing viral titers (green et al., 2014) . mouse sting is resistant to cleavage by the denv2 protease yu et al., 2012) . this at least partially explains why mice mount an effective immune response against dengue viruses, protecting them against infection and compromising their utility as model organisms (cassetti et al., 2010; zompi and harris, 2012; ashour et al., 2010) . dengue viruses are known to mute the host interferon response in other ways as well, with the other predominant mechanism being the degradation of stat2 (ashour et al., 2009; jones et al., 2005; mazzon et al., 2009; best, 2017; morrison et al., 2012) . in this study, we show that the ns2b3 proteases of human (denv1-4) and sylvatic dengue viruses universally cleave human sting. however, none of these proteases can cleave the sting proteins of chimpanzees, macaques, or marmosets, three primate species that have been pursued as model organisms. we show that an 'rg' motif at positions 78/79 of sting is critical for susceptibility to cleavage, and conversion of these residues to 'rg' renders all nonhuman primate sting proteins tested, as well as mouse sting, sensitive to dengue virus proteases. out of the entire genbank database, along with our sequencing of sting from 16 additional primate species, we identify only a small number of apes (gorillas, orangutans, and gibbons), and three small rodent species (chinchillas, naked mole rats, and desert woodrats) as encoding a functional dengue virus cleavage determinant in sting. this may, in part, explain why modeling dengue virus in animal models has been so difficult. the protease of human dengue virus, denv2, cleaves only human sting to begin, we cloned sting from chimpanzee (pan troglodytes, genbank xm_016953921), rhesus macaque (macaca mulatta, genbank mf622060), and the common marmoset (callithrix jacchus, genbank mf622061). these species have been explored as animal models of dengue infection, and also represent the three major clades of simian primates: apes (represented by chimpanzee), old world monkeys (represented by macaque), and new world monkeys (represented by marmoset; figure 1a ). most suspected dengue virus reservoir hosts belong to the old world monkey clade (red and green type in figure 1a ). on the other hand, new world monkeys (such as marmosets), which reside exclusively in the americas, have presumably never been exposed to sylvatic dengue viruses since sylvatic cycles do not exist in the new world. we also included human (genbank mf622062) and mouse (mus musculus, genbank mf622063) sting in our studies as positive and negative controls, since it was previously shown that human but not mouse sting is sensitive to denv2 ns2b3 cleavage yu et al., 2012) . the dengue virus ns2b3 protease complex is composed of the viral non-structural proteins ns2b and ns3 (preugschat et al., 1990; zhang et al., 1992; falgout et al., 1991) . in the dengue virus genome, the ns2b and ns3 genes sit adjacent and are cotranslated as part of a single long viral polyprotein (perera and kuhn, 2008; chambers et al., 1990) . when the ns2b -ns3 region is expressed from a plasmid, the region is translated into a small polyprotein that then auto-cleaves itself to become the functional protease complex (yusof et al., 2000; bera et al., 2007) . we used a plasmid expressing the ns2b-ns3 region, including a 3x flag tag at the c-terminus of ns3, from the new guinea c isolate of denv2 (see methods). as a control, a mutation was created at the activesite serine, changing it to an alanine (s135a), which renders the protease inactive (rodriguez-madoz et al., 2010) . we then used a previously established cotransfection assay yu et al., 2012) to determine if the dengue virus protease could cleave primate sting orthologs. plasmids encoding primate or mouse sting, and either active or s135a (dead) ns2b3 dengue proteases, were cotransfected into 293t cells. sting cleavage was assessed 24 hr later by western blot. the inactivity of the s135a protease can be seen in the anti-flag blot, where the ns2b-ns3 polyprotein does not self-cleave when this mutation is present ( figure 1b) . we see only a fraction of the human sting being cleaved, but this is consistent with previous publications and is presumably exacerbated by the overexpression of sting achieved in transfection experiments yu et al., 2012) . unexpectedly, none of the nonhuman primate stings tested were susceptible to cleavage ( figure 1b) . remarkably, the denv2 protease could not even cleave chimpanzee sting, which differs from human sting at only three amino acid positions. the dengue virus cleavage site in sting was previously mapped to between the 95th and 96th residues yu et al., 2012) . some uncertainty existed, though, because in the previous studies it was noted that the human residues around 95/96 were not sufficient to convey cleavage susceptibility to mouse sting. indeed, human and chimpanzee sting proteins have the exact same amino acid sequence at these positions ( figure 2a ). human and chimpanzee sting (wu et al., 2014) . an alignment of human and chimpanzee sting in the region of the newly identified cleavage determinant (78/79) and the one previously determined (95/96) yu et al., 2012) . (b) site-directed mutagenesis was performed on either human or chimpanzee sting at position 78, substituting the residue at this position in human (r) with that in chimpanzee (w) and vice versa. plasmids encoding the nsb3 protease complex and sting were cotransfected into 293t cells, and 48 hr later lysates were collected and analyzed by anti-flag western blot. in this experiment, both the protease and sting are tagged with flag. data presented are representative of at least two experiments. (c) (bottom) 293t cells were transfected with plasmids expressing the denv2 ns2b3 protease and wildtype (wt) or mutated (78w or 78r) sting. irf3 and phosphorylated irf3 (pirf3) were detected by western blot in lysates harvested 48 hr later. (top) the identical experiment, but performed in biological triplicate and with the addition of plasmids encoding a firefly luciferase gene driven by the interferon beta (ifnb) promoter, and a renilla luciferase gene driven by a cmv promoter. the relative luciferase activity (y-axis) was calculated by normalizing the luciferase signal to the renilla signal in each replicate. a welch's t-test was used to compare the levels of luciferase produced in the presence of active versus dead protease. data is representative of at least two experiments. doi: https://doi.org/10.7554/elife.31919.005 the following figure supplements are available for figure 2: differ at only three amino acid positions, residues 78, 230, and 232. we found that mutating the human sting to encode the chimpanzee residue at site 78 (78w) caused it to become resistant to cleavage by denv ns2b3 ( figure 2b ). likewise, mutating the chimpanzee sting at residue 78 to the human amino acid (78r) rendered the chimpanzee sting susceptible to cleavage ( figure 2b ). we saw no effect of mutations at a second site, 230, either alone or in combination with residue 78 ( figure 2b ). previously, it had been shown that sting site 78 may be important for retention in the endoplasmic reticulum (er) (sun et al., 2009) . to ensure that er retention was not disrupted by the mutations that we tested, we disrupted both copies of sting in a549 cells using crispr-cas9 targeting, and then stably re-complemented them with wildtype or 78w (cleavage resistant) human sting (figure 2-figure supplement 1). both the wildtype and mutant sting similarly localized to the er (figure 2-figure supplement 2). it is logical that the 78w substitution would not affect erlocalization of sting, since 78w is naturally occurring in the chimpanzee sting protein. therefore, we can conclude that position 78r is a critical determinant for dengue virus cleavage, either as a binding site or a cleavage site for the dengue protease. it was previously estimated that sting is cleaved in a way that divides the protein into approximately 25% and 75% of its original molecular weight, with the n-terminus of the protein representing the smaller portion (yu et al., 2012) . this would place the cleavage site in the vicinity of the 78th residue. in addition, the 78/79 'rg' motif is . these cells were recomplemented by retroviral transduction with no gene (plpcx-empty), wildtype human sting, cleavage-resistant human sting (human 78w), wildtype chimpanzee sting, or cleavage-susceptible chimpanzee sting (chimp 78r). these cell lines were infected at moi of 0.3 with dengue virus 2 (denv2 16681). after 24 and 48 hr the virus supernatant was removed and titrated on bhk21 cells. at the same time, cells were collected in ripa buffer, lysed, and run on a gel for western blotting using antibodies against sting, dengue virus ns3, and gapdh (loading control). a tukey's multiple comparisons test indicated significant differences in infectious virus in the presence of each mutant sting compared to wildtype sting, as shown (****=p < 0.0001), after significant one-way anova. data are representative of at least two independent experiments. doi: https://doi.org/10.7554/elife.31919.008 the following figure supplement is available for figure 3: in good agreement with what is known about the preferences of ns2b3, where glycine (g) often lies directly downstream of the peptide cleavage site, and an arginine (r) directly upstream (li et al., 2005a) . next we wished to ensure that the cleavage of sting alters its ability to signal in the interferon induction pathway. transfection of plasmids encoding sting into cells is sufficient to activate the interferon induction pathway (ishikawa and barber, 2008) . we again performed cotransfection of plasmids encoding sting and the dengue virus protease. 48 hr after transfection, cell lysates were probed in western blots for phosphorylated irf3 (pirf3) and for total irf3. we found that pirf3 was reduced when human or chimpanzee sting was susceptible to ns2b3 cleavage, and not reduced when sting was resistant to cleavage ( figure 2c , bottom). we also monitored the activation of the interferon-beta (ifnb) promoter. we performed an identical cotransfection assay with plasmids encoding sting and ns2b3, only in triplicate, and with two additional plasmids: one encoding a firefly luciferase reporter gene downstream of the ifnb promoter, and another encoding a renilla luciferase reporter gene downstream of a cmv promoter (used to normalize transfection efficiencies between samples, by taking the ratio of firefly:renilla luciferase). with human sting and the version of chimpanzee sting rendered sensitive to cleavage (78r), there was a significant reduction in firefly luciferase production in the presence of active ns2b3, in comparison to the catalytically dead version of the protease ( figure 2c , top). this reduction is not observed with chimpanzee sting, or with human sting rendered resistant to cleavage by the 78w mutation. we then verified these results with infection experiments. we stably re-complemented our a549 sting knockout cells, using retroviral transduction, to express various forms of sting: chimpanzee or human 78w (both cleavage resistant), human or chimpanzee 78r (both cleavage susceptible), or cells were complemented with an empty vector (figure 2-figure supplement 1). these cells were infected with dengue virus 2 (strain 16681) at moi 0.3. at 24 and 48 hr post infection, supernatant was harvested and viral content was quantified by plaque assay on bhk21 cells, and at the same time cells were harvested and lysed for western blot. we found that a549 cells re-complemented with sting, regardless of the version, produced less dengue virus than the sting knockout cell line that was not re-complemented ( figure 3) . however, cells re-complemented with a cleavage-resistant sting produced less virus than those re-complemented with a cleavage-susceptible sting (figure 3) . in fact, cell lines in this experiment that differ by only a single amino acid in sting demonstrate as much as a 176-fold change in infectious virus produced at 24 hr post-infection, according to the titration experiments (human versus human 78w sting). the difference remains significant at 48 hr post-infection. this suggests that cleavage of sting is critically important for dengue virus replication, and has a large impact on viral titers. the sting cleavage product was not visible in the western blots performed during these experiments. this cleavage product is typically only detectable when cells are treated with mg132 proteasome inhibitor for several hours before cell lysis. while our transfection-based cleavage assays typically incorporate mg132 treatment (see materials and methods), it was not used in the infection experiments shown here in order to not perturb infectious virus produced. in a separate experiment performed in the presence of mg132, we do see the cleavage of sting during infections (figure 3-figure supplement 1) . further, the cleavage of endogenous sting during dengue infection was previously demonstrated under other conditions yu et al., 2012) . we next wanted to determine if our newly identified cleavage determinant could explain the resistance to sting cleavage seen in other species. the dengue protease also cannot cleave rhesus macaque, marmoset, or mouse sting ( figure 1b) , all of which deviate from the 'rg' motif found in human sting (highlighted green in figure 4a ). we next performed site-directed mutagenesis to alter either the 78th or 79th residue in sting of these species. we found that, in all cases, mutations that restored this motif to the human 'rg' restored susceptibility to cleavage ( figure 4b) . consistent with previous studies yu et al., 2012) , mutation of residues 93-96 in mouse sting to match the human 'lrrg' did not confer susceptibility to cleavage by ns2b3 ( figure 4b ). overall, these results further support the conclusion that sites 78 and 79 are critical determinants for cleavage by the denv ns2b3 protease. an 'rg' motif at these two positions is both necessary and sufficient to make primate and rodent sting susceptible to cleavage by the denv2 protease. to test whether these results are generalizable to other dengue viruses endemic in humans, we cloned the region encoding the ns2b3 protease complex from three additional denv isolates (one from each endemic human virus): denv1 (hawaii), denv3 (philippines/h887/1956), and denv4 (h241). while some of these proteases expressed better than others, all were able to cleave wildtype human sting far more efficiently than human sting bearing the 78w mutation ( figure 5a ). this data indicates that the 78/79 rg motif of sting is recognized (i.e. bound or cleaved) by the ns2b3 proteases of all endemic human dengue viruses. if residues 78/79 in fact constitute the actual cleavage site for the protease, this would be in line with biochemical studies showing that the proteases of all four endemic human dengue viruses have similar cleavage motif preferences (li et al., 2005a) . we next cloned the ns2b3 protease from a sylvatic dengue strain (dakar-141069). this virus was first isolated from an ae. luteocephalus mosquito in senegal in 1999 . we find that this viral protease also cleaves human sting, but not the sting of chimpanzee, rhesus macaque, or marmoset ( figure 5b) . further, the restoration of the 'rg' motif at positions 78/79 again renders all of these sting proteins susceptible to cleavage ( figure 5b) , indicating that the sylvatic protease is targeting (i.e. binding or cleaving) the same cleavage determinant as the proteases from human dengue viruses. this is consistent with the high degree of similarity between human and sylvatic proteases, as can be seen in alignment of the two ( figure 5-figure supplement 1) . it is curious to find that a sylvatic dengue virus does not cleave nonhuman primate sting. since we don't know the exact species that constitute the viral reservoir, we next considered the question of whether any nonhuman primates encode the correct cleavage determinant at positions 78/79 in sting. to address this, we harvested mrna from cell lines derived from 16 different nonhuman primate species (see materials and methods). from these mrna pools, we made cdna libraries and sequenced the sting cdna using sanger sequencing. we also gathered sting sequence for 14 additional primate species from genbank. an alignment of the eight amino acid region in sting surrounding the 78/79 cleavage determinant (downward arrow in figure 6a ) is shown for all 30 of these primate species (a full alignment of primate sting sequences is provided in supplementary file 1). with the exception of chimpanzees and bonobos, all apes encode the same amino acids as human in this motif, constituting the correct cleavage determinant for dengue virus. in contrast, no monkey species encodes an 'rg' at positions 78/79. instead, old world monkeys all encode 'rd,' which is the same motif found in the macaque clone that we have tested. also, no monkeys from the americas encode an 'rg' at these residues, and instead these species encode a 'qg' at positions 78/79, just like the marmoset clone tested herein. finally, we queried the entire genbank database for sting sequences available from placental mammals. mice and pigs, two current models for dengue virus infection (cassetti et al., 2010) , also do not have the correct rg residues at sting 78/79 ( figure 6a) . out of the entire database, only two other mammals were identified that share the exact same sequence as humans in the eight amino acid region surrounding the newly identified dengue virus cleavage determinant in sting: chinchilla and naked mole rat, both of which are rodents ( figure 6a) . a third rodent species, the desert woodrat, has the rg at positions 78/79, but encodes two amino acid substitutions just downstream of these residues, compared to human sting ( figure 6a ). the fact that only a small handful of mammals encode an rg at position 78/79, out of the entire database, may in part explain why modeling dengue viruses in animals has been so difficult. we next wished to determine if sting is in fact cleaved by dengue in these rodent species, since all three already serve as animal models for biological research (keane et al., 2014; nathaniel et al., 2013; shimoyama et al., 2016; campbell et al., 2016; skopec et al., 2013) . we synthesized ha-tagged sting genes for the rodent species discussed in figure 6a , as well as an additional rodent (13-lined ground squirrel) which does not have the 'rg' motif at sting 78/79, as a negative control. cleavage assays were performed using the co-transfection assay described previously. we see that the sting of naked mole rat and desert woodrat is clearly cleaved by the denv2 protease, in that the cleavage product is evident ( figure 6b ). we do not see a cleavage product for chinchilla sting, even under long exposure, but we do see the sting band disappear. it's possible that, in this case, the cleavage product is too unstable to be detected. the identification of animal models encoding sting proteins that can be cleaved by dengue might be important; the advantage to using such species as models is that, unlike in sting knockout mice, the sting pathway would be intact in these animals. in humans cells, sylvatic and human dengue viruses replicate similarly (vasilakis et al., 2007 . these results have been interpreted to mean that there is little or no adaptive barrier for the emergence of sylvatic dengue viruses into human populations. our data agree with, but add a new element to, this model. rather than there being critical differences between human and sylvatic viruses, our data suggest that there are critical differences between human and monkey hosts. this difference tracks, at least in part, to sting, revealing one way in which dengue viruses are reaching higher titers in humans than in monkey models. collectively our data suggest that all dengue viruses cleave human sting, but not the versions of sting found in most other mammals. we have used the sting proteins of closely related primate species to map the determinant of cleavage in sting. we find that the viral protease is recognizing (i.e. cutting or binding) residues around positions 78/ 79 of sting. we show that an 'rg' motif at these two residues is necessary and sufficient for cleavage by the proteases of all four human epidemic dengue viruses, and one sylvatic dengue virus. yet, only some apes and three rodent species, out of all of the mammalian sting sequences in genbank, encode an rg at positions 78/79. why do dengue viruses universally cleave human but not monkey sting? it's possible that what we have uncovered is a brilliant method for balancing alternate host species, one of which is dense and abundant (humans), versus others that are spare and exist in smaller populations (primates in nature). in this scenario, dengue viruses have evolved to suppress innate immunity in humans in order to increase viral titers and spread, even though this trait comes at the cost of increased pathogenicity in some individuals. this might be a good strategy in our abundant and dense host population, where the fitness cost of severe disease in a fraction of individuals would be outweighed by excellent spread. remarkably, though, dengue viruses have achieved this by evolving to recognize a portion of human sting that is not conserved in the sting of the wild and more rare animals that serve as their sustaining reservoir in nature, allowing the viruses to maintain decreased pathogenicity in these species. the evolution of the viral proteases to cleave human sting and simultaneously to avoid cleavage of monkey sting would be expected to reduce virus titers in monkeys, as the interferon pathway would be at least partially enabled. this may be beneficial for many reasons, one of which is that the production of a low-level innate immune response may allow the virus to replicate in reservoir host species without inducing high titers and strong adaptive immune responses. alternately, a second possibility is that sylvatic dengue viruses do cleave the sting of monkeys, but that the sylvatic virus (dakar-141069) that we tested is not representative. however, we find this unlikely. because denv1-4 also cannot cleave monkey sting, and all derive from the sylvatic reservoir, this supports the finding that sylvatic viruses do not cleave monkey sting. third, a final possibility is that apes are critical reservoirs for dengue viruses in nature. gorillas encode 'rg' at 78/79 and are found in africa, while orangutans and gibbons are found in asia and also encode the correct cleavage motif for the dengue virus protease. in fact, wild orangutans have previously been found to have neutralizing antibodies against dengue virus (wolfe et al., 2001) . while apes could be playing a role as sylvatic hosts, the highly endangered and rare status of most apes makes it hard to believe that they are playing a major role in sustaining sylvatic dengue virus currently (geissmann, 2007; walsh et al., 2003) . the specific primate species that serve as the sustaining reservoir for sylvatic dengue viruses in nature are unknown (vasilakis et al., 2011) . various old world monkey species in both asia and africa are suspected hosts (vasilakis et al., 2011; rodhain, 1991; diallo et al., 2003) . for instance, sylvatic dengue viruses have been isolated directly from macaques (macaca fascicularis) and leaf monkeys (presbytis obscura) that were placed as sentinels in forest canopies (rudnick, 1986 ). other primates have been shown to have antibodies to dengue virus, including macaques (macaca fascicularis and macaca nemestrina), leaf monkeys (presbytis cristata) (rudnick, 1965) , african green monkeys (chlorocebus sabaeus) (diallo et al., 2003) , and one ape species, the bornean orangutan (pongo pygmaeus) (wolfe et al., 2001) . but these results are not definitive due to the cross-reactivity of antibodies directed against various flaviviruses (calisher et al., 1989; tesh et al., 2002; mansfield et al., 2011) , and the possibility that some primates might be accidental, rather than sustaining reservoir hosts (vasilakis et al., 2011) . instead of being directly isolated from primates, most sylvatic dengue viruses have been obtained from forest mosquitoes (diallo et al., 2003; rudnick, 1986) , or from humans that contracted the virus in the forest (pyke et al., 2016; cardosa et al., 2009; franco et al., 2011) . therefore, there are many deficiencies in our understanding of the natural reservoir for dengue viruses. interestingly, though, we have not identified any monkey species with an 'rg' at positions 78/79 in sting. our results would indicate that dengue viruses, in general, cannot cleave the sting of monkey hosts. our data suggests that even a sylvatic dengue virus, which we find targets the same residues in sting, would not be able to cleave sting of monkeys. there are also implications of these findings to our understanding of dengue virus model organisms. if dengue proteases do not cleave most nonhuman forms of sting, this may at least partially explain why it has been so difficult to model dengue infection in immune-competent animals. nonhuman primates infected with dengue virus generally don't develop clinical signs of disease, consistent with enhanced control of the virus compared to humans (cassetti et al., 2010) . in fact, when human dengue viruses have been observed to replicate robustly in primate cell lines, these experiments have typically been done in cells such as vero rossi et al., 2012; vasilakis et al., 2009) which are deficient in the type i interferon response (osada et al., 2014; desmyter et al., 1968) . human dengue viruses also cannot cleave mouse sting ( yu et al., 2012) and herein), consistent with the heightened control of this virus in mice as well (cassetti et al., 2010) . dengue virus will replicate to high titers in mice lacking key genes important for the interferon response, but for many reasons it is desirable to develop animal models in immune competent hosts (cassetti et al., 2010) . sting now adds to a growing list of host proteins that regulate viral infection differently even in closely related host species (for example, [stabell et al., 2016; lou et al., 2016; rowley et al., 2016; kerr et al., 2015; meyerson et al., 2015; demogines et al., 2012; stremlau et al., 2004; demogines et al., 2013; ng et al., 2015; hueffer et al., 2003; radoshitzky et al., 2008; patel et al., 2012; elde et al., 2009; martin et al., 2013; miller et al., 2012; sawyer and elde, 2012; meyerson et al., 2017] ). the identification of such genes is critical to our understanding of viral adaptation during host switching, and to the development of animal models in which to study human viruses. it is possible that the identification of small mammals that have a cleavage-susceptible sting would facilitate the development of better animal models for studying dengue virus. our work suggests the identity of three such species: the naked mole rat, the common chinchilla, and the desert woodrat. all three of these small rodents are already used as animal models in biomedical research, and the genomes of all three have been sequenced (keane et al., 2014; nathaniel et al., 2013; shimoyama et al., 2016; campbell et al., 2016; skopec et al., 2013) . these species could be figure 6 continued [cassetti et al., 2010] ), several small rodent species which encode the correct cleavage motif at 78/79 (naked mole rat, common chinchilla, desert woodrat), and one that does not (13 lined ground squirrel). genbank accession numbers of sequences shown: mouse (xp_017173483), pig (xp_005661761), 13-lined ground squirrel (xm_005327275), naked mole rat (jao02071), chinchilla (xp_005382124), and desert woodrat (obs58238). (b) sting-ha genes were synthesized for the rodent species discussed in panel a. cleavage assays were performed by co-transfecting plasmids encoding the dengue protease (dead or active) as well as each sting, and then performing immunoblotting as described in the methods. the data presented are representative of at least two independent experiments. doi: https://doi.org/10.7554/elife.31919.013 superior to sting knockout mice, in that the sting pathway would be intact and the cleavage of sting by the virus would be naturally modeled rather than just bypassed. these species may also be superior to future models where mouse (mus musculus) sting would be replaced with human sting in transgenic animals. in this case, it is unknown if human sting would perform all of its functions the same in mouse as it does in humans. the advantage of using a rodent model with a sting that is naturally susceptible to dengue virus cleavage would be that the sting pathways would all be fully functional and intact. it is important to point out that, in addition to cleaving sting, dengue viruses modulate the interferon response in other ways as well. for instance, dengue viruses also bypass the type i interferon response by binding and degrading host stat2 via the viral ns5 protein (ashour et al., 2009; jones et al., 2005; mazzon et al., 2009; best, 2017) . ideally, human dengue viruses would also be able to bind and degrade stat2 in newly developed models, as they do in humans. further, dengue viruses neutralize both the type i and type ii interferon responses in other ways as well (perry et al., 2011; shresta et al., 2004; . other known host-virus interactions would also need to be characterized in any potential new model organism. it is notable that chimpanzees and bonobos encode stings that are resistant to cleavage, while stings of all other apes are susceptible. these two species differ from other apes in encoding a 'wg' at 78/79 of sting rather than the 'rg' encoded by all other apes ( figure 6) . remarkably, it was previously found that the 'w' at position 78, destroying the dengue cleavage determinant, was fixed by positive natural selection in wild chimpanzee populations (mozzi et al., 2015) . chimpanzees are not one of the suspected natural reservoirs of dengue virus, but chimpanzee ranges do co-occur with known human outbreaks and with sylvatic cycles (figure 1-figure supplement 1) . one model is that, as dengue virus spread through africa, a snp in chimpanzee sting (or the sting of the chimpanzee/bonobo ancestor) at position 78 started to experience strong selection because it provided protection against cleavage by dengue viruses. this would have driven a selective sweep in chimpanzee populations, causing this species to become less susceptible to viral infection. it has previously been proposed that evolutionary pressure imposed by flavivirus proteases can drive selection at cleavage sites. for examples, the hepatitis c protease cleaves mavs, another host signaling protein in the interferon induction cascade (li et al., 2005b; meylan et al., 2005) . mavs has experienced positive selection at a residue in the cleavage site for the hepatitis c virus protease . the authors of this study speculated that ancient viruses may have exerted selective pressure on primate genomes to acquire mutations in the cleavage site. like other viruses, dengue viruses remodel their host cellular environment in numerous ways, including the cleavage of sting and degradation of stat2. using the rich information that exists on how dengue viruses accomplish this, the genetic susceptibility of both suspected reservoir hosts, and potential new animal models, can be systematically assessed. characterizing how host-virus interactions play out uniquely in different host species will help us to understand dengue virus in critical ways. for instance, it will reveal how dengue viruses do (or do not) need to evolve their genomes as they transmit to humans from nonhuman primates in nature. also, understanding the genetics of host tropism will help identify better laboratory animals that can be used to study dengue virus pathogenesis and to develop drugs and vaccines. : tgcttagtcgacactttcttccactggcaaactccttg) to amplify the ns2b + ns3 genomic region in one fragment. in this experiment, the protease from denv2 was re-cloned so that the structure of the four protease clones was identical in all four cases. the pcr templates were cdnas created from rna obtained through the world reference center for emerging viruses and arboviruses (wrceva) (cat# nr-32847). denv1 (hawaii, nr-4287), denv2 (new guinea c, nr-4288), denv3 (philippines/h87/1956, nr-2771), and denv4 (h241, nr-4289). the pcr products, and the plasmid containing the denv2 protease mentioned above (gift from yi-ling lin), were both digested with hindiii and sal1. the pcr products were ligated into this plasmid and transformed into dh5a chemically competent e.coli. the sylvatic ns2b3 (dakar141069) was synthesized (without an epitope tag) using the sequence information deposited on ncbi (genbank accession ef105389). sting genes used for functional analysis were amplified from cdna libraries constructed from the following cell lines: human (a549), chimpanzee/bonobo (sting sequence identical for these two species, clone amplified from coriell, pr00748), rhesus macaque (mm265-95, a gift from welkin johnson), marmoset (coriell, pr07404), and mouse (generated from rna extracted from whole liver). either an ha or 3xflag tag were engineered onto the 3' end of the gene sequences, separated from the coding sequence by a 3xglycine-alanine (gagaga) linker region (nucleotide sequence = ggtgc tggtgctggtgct). these sequences were cloned into the pcdna3.1 expression vector with a 5' kozak sequence (gccacc). rodent sting constructs were synthesized (quintarabio) to include a kozak sequence, c-terminal ha-tag, and flanking linkers that were used for gibson cloning into the plpcx mammalian expression plasmid. 293 t cells (mycoplasma negative) were grown at 37˚c in dmem supplemented with 10% fbs, pen/ strep, and l-glutamine. 24 hr prior to transfection, cells were plated at a density of 4.5 â 10 5 cells per well in a 12-well dish in antibiotic free media. wells were transfected with 800 ng plasmid encoding sting and 800 ng plasmid encoding ns2b3 using transit 293 reagent (mirus mir 2704). for most experiments ( figures 1b, 3b, 5a and b) , cells were treated with 10um mg132 for 8 hr prior to harvesting for western blot. cells were lysed in ripa buffer supplemented with protease inhibitor (roche, 4693159001). protein concentration was calculated using the bradford method. 10% 37.5:1 acrylamide/bisacrylamide gels were used to run 30 ug of whole cell lysate for each sample. protein was transferred overnight at 30 volts onto a polyvinyl membrane. blocking was performed with a 10% milk solution in tris-buffered saline supplemented with 0.1% tween20. primary antibodies used were used against ha (3f10 clone sigma 11867423001), flag (m2 clone sigma f3165), gapdh (cellsignaling 14c10), sting (abcam 92605), actin (santa cruz biotech sc47778), and dengue virus ns3 (mouse polyclonal antibody raised against purified full-length ns3 from dengue 2 strain 16681 [heaton et al., 2010] ). secondary antibodies used were goat-anti-mouse-hrp (thermo 62-6520) and goat-anti-rabbit (thermo 65-6120). blots were developed using ecl prime (amersham rpn2232) and imaged using imag-quant las 4000 (amersham 28-9558-10). crispr-cas9 mediated disruption of sting, and stable recomplementation with primate orthologs a549 cells (mycoplasma negative) were transfected with the pspcas9(bb)-p2a-egfp (px458) with the guide rna sequence 5' agagcacactctccggtacc 3'. gfp-positive cells were single-cell sorted into a 96-well dish and colonies were grown up. cloned a549 cells were screened for homozygous mutations that disrupted the coding sequence of sting as follows. 10,000 cells were used to prep whole genomic dna. the region surrounding the guide rna was amplified using the following primers: 5' gtccccaagggttcttggtt 3' and 5' aaccagtcccactcccagta 3'. amplified genomic dna was sanger sequenced to determine the nature of the crispr-cas9-mediated genomic disruption. a cell line with confirmed homozygous disruption of sting (figure 2-figure supplement 1) was then re-complemented with primate orthologs of sting. four different c-terminally ha-tagged versions of sting were cloned into the plpcx retroviral vector: wildtype human sting, r78w human sting, wildtype chimpanzee sting, and w78r chimpanzee sting. these were packaged into retroviral particles by cotransfecting into 293t cells (mycoplasma negative) each plpcx-sting construct with plasmids expressing nb-tropic murine leukemia virus (mlv) gag-pol and vsv-g. as a control, we also made virus to complement with an empty plpcx vector. supernatants were collected and used to transduce 10^5 a549 cells in the presence of 10 ug/ml polybrene. 24 hr post transduction, cells were selected in 0.75 ug/ml puromycin. immunofluorescence 24 hr after plating, cells were fixed with 4% paraformaldehyde and permeabilized with 1% tri-tonx100 in pbs. blocking was performed with 3% bsa solution in pbs. primary antibodies used were rabbit-anti-grp78 (bip) (abcam ab21685) and mouse-anti-ha (clone 16b12 abcam ab130275). secondary antibodies used were donkey-anti-rabbit conjugated to alexafluor594 (invitrogen a21207) and donkey-anti-mouse conjugated to alexafluor488 (invitrogen a21202). cells were mounted using vectashield hardset mounting media (vectorlabs h-1400). the indicated sting knockout and re-complemented cell lines were plated out in f-12k media with 10% fbs, after 24 hr the cells were counted. an moi of 0.3 was calculated for each well and dengue virus 2 (16681) was allowed to attach to cells for 1 hr at room temperature. unattached virus was then removed from cells, 2%serum in f-12k media was added to cells and they were maintained at 37˚c with 5% co 2 . after 24 and 48 hr the virus supernatant was removed for downstream titration on bhk21 cells. at the same time, cells were removed for downstream western blotting. sequencing sting from other primate species the following sting sequences were collected from genbank: chimpanzee (pan troglodytes, xm_016953921.1), gorilla (gorilla gorilla gorilla, xm_004042612.1), sumatran orangutan (pongo abelii, xm_002815952.2), golden snub-nosed monkey (rhinopithecus roxellana, xm_010388119.1), black snub-nosed monkey (rhinopithecus bieti, xm_017895026.1), african green monkey (chlorocebus sabaeus, xm_008014636.1), pigtail macaque (macaca nemestrina, xm_011716377.1), rhesus macaque (macaca mulatta, xm_015141010.1), sooty mangabey (cercocebus atys, xm_012090448.1), drill (mandrillus leucophaeus, xm_011997224.1), marmoset (callithrix jacchus, xm_00898588.2), capuchin monkey (cebus capucinus imitator, xm_017536735.1), black-capped squirrel monkey (saimiri boliviensis, xp_003933962.1). the remaining sting gene sequences were obtained by direct sequencing of cdna libraries produced from the following primary or immortalized primate fibroblast cell lines: bonobo (pan paniscus, coriell pr00748), bornean orangutan (pongo pygmaeus, coriell pr00650), white-handed gibbon (hylobates lar, coriell pr01131), agile gibbon (hylobates agilis, coriell pr00773), siamang (symphalagus syndactylus, coriell pr00722), white-cheeked gibbon (nomascus leucogenys, coriell pr01037), leaf monkey (trachypithecus francoisi, coriell pr01099), colobus monkey (colobus guereza, coriell pr00980), wolf's guenon (cercopithecus wolfi, coriell pr01241), talapoin (miopithecus talapoin, coriell pr00716), crab-eating macaque (macaca fasicularis, 103-06, gift from welkin johnson), olive baboon (papio anubis, coriell pr00978), grey-cheeked mangabey (lophocebus albigena, coriell pr01215), bolivian red howler monkey (alouatta sara, coriell pr00708), red titi monkey (callicebus (or plecturocebus) cupreus, coriell pr00793), common squirrel monkey (saimiri sciureus, coriell pr00603). briefly, cells were grown in dmem (cellgro) supplemented with 15% fbs (gibco) at 37˚c and 5% co2. rna was extracted using the allprep dna/rna extraction kit (qiagen). cdna libraries were generated using super-script iii first strand synthesis kit (invitrogen). pcr was performed using pcr supermix high fidelity (invitrogen). pcr products were directly sequenced. each primate sequence was used as a query to search the human genome, and human sting gene was returned as the top hit. sting gene sequences generated in this study have been deposited in genbank (accession numbers mf616339-mf616355). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. collateral damage during dengue virus infection: making sense of dna by cgas dengue virus ns2b protein targets cgas for degradation and prevents mitochondrial dna sensing 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virus type 2 polyprotein precursor ns3-ns4a-ns4b-ns5 the helicase ddx41 senses intracellular dna mediated by the adaptor sting in dendritic cells the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation animal models of dengue virus infection we thank kartik chandran, nels elde, emily feldman, maryska kaczmarek, michael smallegan, cody warren, and qing yang for thoughtful comments on this study. this work was supported by national institutes of health grant r01-gm-093086 to sls. acs was supported by an m.d./ph.d. training fellowship from the national institutes of health (f30-ai-112277). nrm was supported by a graduate fellowship from the national science foundation, and a pdep award from the burroughs wellcome fund. rcg and rp were supported by the department of microbiology, immunology and pathology at csu. sls is a burroughs wellcome investigator in the pathogenesis of infectious disease. key: cord-035138-7v92aukg authors: tognoni, gianni; macchia, alejandro title: health as a human right: a fake news in a post-human world? date: 2020-11-10 journal: development (rome) doi: 10.1057/s41301-020-00269-7 sha: doc_id: 35138 cord_uid: 7v92aukg based on a synthetic overview that embraces the evolution of the ‘health’ concept, and its related institutions, from the role of health as the main indicator of fundamental human rights—as envisaged in the universal declaration of human rights—to its qualification as the systems of disease control dependent on criteria of economic sustainability, the paper focuses on the implications and the impact of such evolution in two model scenarios which are centred on the covid-19 pandemia. the article analyses covid-19 both in the characteristics of its global dynamics and in its concrete management, as performed in a model medium income country, argentina. in a world which has progressively assigned market values and goods an absolute strategic and political priority over the health needs and the rights to health of individual and peoples, the recognition of health as human right is confined to aspirational recommendations and rather hollowed out declarations of good will. the title of an article where a question mark follows a provocative statement could easily sound as a rhetoric artefact rather than the formulation of a real research hypothesis. to provide a clearer answer to this doubt, and a full justification of the legitimacy and relevance of the question mark, let's start with a few preliminary considerations and explanatory notes, which we deem necessary as a general conceptual and methodological framework. here they come, in this logical sequence: • the two domains associated and confronted in the question posed in the title-public health and human rights-have been the long-term professional field of the authors. both of them have been engaged mainly in intense research activity related to field projects and thematic areas where they have directly crossed, and experienced, the often conflicting tension and interaction between the point of view of 'health' as a fundamen-tal human and peoples right, and that of 'health' as an increasingly dominant component of market rules. since the seventies, model health areas have included drug policies such as the who essential drugs, innovative population clinical trials, critical epidemiological use of large databases, normative regulations of accessibility to health technologies (tognoni et al. 2019) . a parallel line and a different type of research that both authors have conducted has focussed on the causes and consequences of massive violations of peoples' rights. 1 their cumulative experience, derived from the insides of these most diverse scenarios, has provided them with a solid confirmation of what has emerged with a growing consensus also in the most prestigious 'scientific' literature, in the last 10 years: structural inequality is the direct product and the expected outcome of the mainstream models of development, which trigger a highly visible impact on the rights to health and life, and prove to be a systemic source of in-human levels of inequity (evans 2020 ). • the title of the important book from indian academic baxi (2007) which has inspired the question mark of our contribution has proved to be neither a pessimistic forecast nor a philosophical theoretical generalization of trends on the eve of this century's first financial crisis. health, the most sensitive indicator of the right to human dignity, has become the arena of a most impressive cultural transformation. it is the terrain that best documents the level of inequalities-iniquities generated by assuring the privilege of priority attributed to goods over humans in economic profit oriented legislations. this terrain is also an inevitable object of observation and analyses, where humans and their lives are normal, expected, quantifiable 'victims', and not inviolable subjects (for a very careful documentation of the broader evolution in this direction of juridical and institutional international scenarios (dentico 2014 ). • an independent and enriching integration of baxi's view (with an impressive documentation from real life sociological contexts, populations, outcomes) is provided in saskia sassen's book which gives a most effective definition of the whys and hows of the post-human qualification. the globalization project hinges on rigid logistic and economic supply chains for whatever may be considered a market good-anything that generates profits in the selling and buying universes, including humans. the expulsion of whatever claims to be, or may be assumed to be, a 'subject' endowed with inviolable life and dignity rights (as opposed to a marketable object), is a mandatory and automatic reaction of the algorithms guiding the decisions (sassen 2014 ). • the last viewpoint that concludes this introductory framework is the most unexpected, hence the most significant one: for its peculiar origin, its world vision, and indeed its language. we refer to pope francis's two last encyclical documents, 2 widely recognized as the expression of a truly 'universal' conversion, not confined by religious walls or political frontiers. the pope's letters envisage humankind's re-positioning in its one and common home, that being creational nature. his approach marks an innovative recuperation of the principles of fundamental rights, which cannot be but oriented to the full inclusion of all humans (migrants, marginalized, etc.) as subjects entitled to the same dignity of life and wellbeing-which accessible health is a concrete indicator of. our attempt here is to try and provide a synoptic view with some interpretative lights on the events and/or documents that have given substance to a long history. in table 1 we intend to portray the chronology and the core themes that allow a comprehensive understanding of the challenges that we need to face in today's political, economic, conceptual scenarios while looking for an answer to the questions about the state of the right to health raised in the title of our article. the narrative of the long list's contents aims to underline the continuity and the articulation of a process where the role of the same actors (international agencies, national governments, representatives of civil society and people's movements), and their reciprocal hierarchy of power, have mutually evolved to shape the time we are living. as the first un operational agency created even before the proclamation of the universal declaration of human rights (udhr), the who was regarded as the immediately available, concrete and easy-to-grasp instrument to advance the political agenda of the new era, after the plight of two world wars: the human right to life and dignity could be seen and promoted as truly universal, if accessibility to the right to health could be secured. the vision then was pinning down the world to translating a declared universal principle-the right to health-into effective policies and visible practices in the context of the different countries, whose national constitutions were meant to convert the udhr principles into a daily experience. this horizon did not last long. less than 30 years after the udhr, the perspective of the linear development of a democratically organized society had substantially changed already. the udpr turned out to be promoted as an independent charter to reflect and clearly denounce the dramatic limitations of the existing international law. it did not take long for the international community of states to progressively become the expression and the vehicle of 'strategic'-economic, military, political-interests, a scenario in which the multinational corporations had been playing an increasing role while denying any loyalty or accountability to the udhr principles, and their implications. the world health organization (who) soon became well aware of the mounting threats to its authority and field of competences, under the guise of a rapidly growing health market deprived of any control. the agency did not stay idle, in the face of what was coming. a very restricted list of 'essential drugs', based on the consensus of the scientific community, was formulated and proposed for approval to the who member states. the idea was to equip governments with a tangible as well as symbolic tool to resist the pressure stemming from the neoliberal development models that mainly the international monetary fund (imf) and the world bank (wb) had started to impose. the first half of the 1990s coincides with a major cultural and institutional translation, in the arena of health, of the shifts that had occurred to the original udhr paradigm. the newly born world trade organization (wto) declares its exclusive competence on health goods, technologies and structures. in a nutshell, the trade agenda asserts itself as the new overarching normative regime. in alliance with the who, the wb draws a new map of health priorities where investments should concentrate. the map does not set its priorities on the assessment of real unmet needs. it is designed on the tricky notion of the global 'economic burden' of the individual diseases (gbd), now the main-if not exclusive-term of reference of health market costs and provisions, accessibility trends, international procedures. contrary to many expectations, the newly established international criminal court (icc) declares its juridical incompetence and lack of mandate on the 'new' crimes against individual and peoples' rights committed in compliance with the new economic and trade laws. in the narrative of the acronyms for the new millennium, health appears as one of the main goals to be pursued. yet, the health goal is placed in a generic framework of socioeconomic variables and featured with the profile of an insurance scheme for which somebody should pay. isn't this what the acclaimed universal health coverage (uhc) approach ultimately aims for? the ambivalence, the limits, and the remarkable failures of these top-down global goals are too well known across the research reports or the administrative reviews of concerned agencies and disciplines, to require any further comment. conversely, the silence of the scientific and institutional literature on the who-sponsored report on the social determinants of health (sdh), is indeed very meaningful. the sdh report was supposed to remind the world, renew the memory, relaunch the era of the 'health for all' (hfa) culture and its mobilizing manifesto, the alma ata declaration. this reticence is a confirmation of how deeply the normative and epistemic changes have been interiorized. no space is left for a terminology reminiscent of the 'human' in the global reporting or goal setting, anymore. the semantic oblivion had been knowingly experimented through the gbd methodology, whose main focus is the geo-mapping of health economic burdens. the iteration of strictly descriptive and repetitive data on the growing notavoidable levels of inequalities-inequities has paved the way to the planned disappearance even from the dictionaries of health rights disciplines (abassi 2020) . the often announced but substantially unexpected covid-19 pandemia has produced a more than foreseeable confirmation: the statistics of mortality made available for all the affected countries reproduce similar gradients of excesses of contagion and deaths which overlap and add to the underlying conditions of inequalities. a structural and pandemic characteristic featuring high income countries (hics) just as well (swenor 2019) . it is not the purpose of this article to either summarize or comment the rapidly changing data related to covid-19. the main issue is rather the degree of credibility the multiple actors in the pandemic landscape-the un agencies, the european commission, religious leaders, academia, ngos representing civil society, the concerned corporate actors, the science community-have vehicle through their institutional positions. the uniquely symbolic power of what has happened, its extension and consequences, cannot leave reality as it is. radical changes in development modelling must be prepared, different hierarchies of values need to be adopted. unfortunately, as often if the case, power balance evolution does not seem oriented to seriously engage with the challenge of radical modifications. the field of health appears to be a case in point, for its specific perfect reproduction of 'wishful thinking' strategies. a tentative check list of challenges and priorities made visible by covid-19 would be useful. the first and most impressive 'discovery' triggered by covid-19 is the structural fragility of a system of marketdriven supply chains-of goods, knowledge, information, data. in the first two decades of the twenty-first century these supply-chains appeared to be the solid and untouchable protagonists of the global development models. until the arrival of a biological agent, doomed to be under global knowledge's tight control, has successfully revealed the inherent ignorance and deep fragmentation defining the various branches of the most advanced disciplines of basic sciences: public health, epidemiology, health technologies. possibly, the unpreparedness to ignorance, in a global culture increasingly confident on the power of technology and the linearly conceived algorithmic decision-making, has produced a decisive worsening of the pandemic impact. a world proud of its digital capacity of 'linking' and 'sharing' has discovered its communication failure when it comes to real risks and potential solutions, as well as its cultural carelessness, its political and scientific lack of foresight when it comes to sharing data on unmet needs and possible solutions. globalization's top-down approach has proved highly ineffective, 'lost in translation', when devising the needed strategies for collaborative involvement of human beings. classical health security measures (certainly needed) have been the only remedy put in place with great variability, and unmeasurable/incomparable results in different countries and health systems. which has entailed a recognized negative impact on the democratic and health rights of ordinary citizens, not to mention the enhanced precariousness of migrants, refugees, uninsured, homeless, simply 'poor' or marginalized people, across the world. the acclaimed capacity of handling and relying on big data to monitor, anticipate, measure the yield of the (often more than controversial) interventions has produced an international situation of confusion and uncertainty in the epidemiological understanding, and evaluation, of the pandemic evolution. scientists and health authorities have simply failed to agree on reliable and applicable criteria for comparing their data. the retraction of scientific reports from major specialized journals, the breach of fundamental rules and practices of independent results' evaluation, even in regulatory agencies, the virtual lack of any formal coordination of public health efforts months after the inception of the 'emergency' are known elements of the daily chronicle we have familiarized with, and abundantly so. in this scenario, the only recognized exception concerns health professionals. confronted with the human face of the pandemic, close to the solitude of human bodies unable to breathe, medical doctors, nurses, health personnel have assured an impressive quality of presence and intensity and care across the spectrum of the different given settings. they have done so mooring their unexpected and extraordinary experience to the safe bay of the old robust-albeit tragically neglected-value of medicine as a science and practice that is grounded on a rigorous combination of professionalism, solidarity, ethics. it is reasonable to say that this 'care for humans'-often institutionally unplanned, hardly politically supported, at risk now of being forgotten in terms of participation to the overall planning for the future-has been the only positive, authentic, understandable, credible form of communication to the public opinion. the other significant consensus is that health systems had suffered greatly during the pandemic crisis due to the sharp reductions of investments in human resources and community strategies, imposed by 'economic sustainability' criteria. the scenario we have tried to summarize, therefore, bear witness to the fact that the global challenge introduced by the covid-19 is only very partially a health problem. the ongoing economic, political, juridical 'wars' currently surrounding the narratives about the development, distribution and accessibility of the covid-19 vaccines are, yet again, a most worrying confirmation of the real question for our societies today: namely, what model of civilization will be chosen after covid-19 (bloom et al. 2020; nowak et al. 2020 ). humans-their lives and their values of democracy, solidarity, and hope for a better future-have played their role as victims, rather than as subjects. the viral pandemic has disclosed the structural pandemic impact of the social and economic viruses that have infected the global chains of knowledge production, denied the possibility of delivering the common goods, seconded the vested priorities of private powerful minorities over the vast majorities of peoples. the most recent reports from the gbd groups have 'discovered' that the global data which consistently document the extent and the severity of the impact of the inequalities worldwide should evolve from a strategy of neutral description into one approach of looking into the avoidability of such inequalities-iniquities. it seems an obvious cultural and methodological change, but it could represent quite a revolutionary step forward from the tragically impressive analytical model illustrated by burstein et al. in a top scientific journal (2019). a substantial proportion of the 123 million neonatal, infant, paediatric deaths occurred in the twenty-first century could be considered avoidable with simple, mainly non-sanitary measures, the study says. nothing else. ultimately, there in an urgent need for re-establishing human lives and deaths as the measure of needs and outcomes, against the persistently dominant focus on disease burdens. repeating the usual soliloquy of 'international' literature, an abundant production of medical articles has flooded physicians and public health professionals working in the global south with the intent of explaining them what is happening in their countries, what their needs are, what lessons they should learn from the covid-19 pandemic. while the flow of information is meant to be educational, the appropriate term we can use is, possibly, indoctrination (daniels 2020; hogan et al. 2020; walker et al. 2020) . with covid-19, a new wave of top-down, complex and unverifiable mathematical models keep coming from the center of the world foreseeing when the peripheries will run out of respirators, how many people will die in the next semester, how the pandemic will impact tuberculosis and hiv infected people (walker et al. 2020) , and how all this knowledge should inform a model of healthcare organization (walker et al. 2020) . all data must be noted and lessons learnt. this dynamic was not brought in by covid. for decades, centrally produced guidelines have indicated how to invest mostly borrowed money for the 'proper' administration of health resources. to verify the sparkle of the truth hidden in the lies, a small, basic technical frame of reference could be useful to discuss concepts related to solidarity, rights and autonomies of real populations. for decades now (world bank 1993; murray and lopez 1996; cooper et al. 1998; jamison et al. 2006; mathers and loncar 2006) , gbd reports have informed us that in argentina things are going reasonably well. the increase in life expectancy, the decrease in infant mortality and the overall improvement in access to healthcare (haq index) indicate that things have improved significantly. the institute of health metrics (its logo explicitly states: 'measuring what matters') incorporates comparisons with other countries to provide a reference aimed at defining the magnitude of trends and problems. this approach makes argentina indistinguishable from bosnia and herzegovina when it comes to deaths from cirrhosis, although it would profile it better than china in terms of self-inflicted injuries and significantly worse than the mauritius islands of east africa in neonatal conditions. this is a description of what is published. beyond the international metrics and their grotesque comparisons, these estimates lead to an important conceptual distortion. when analyzing argentina's factual data, we see that the average improvement occurs at the expenses of a fraction of the population. inequity increases and access to services worsens among those who need it most (macchia et al. forthcoming). women, for example. their health situation is dramatic. available data from the last 2 years show that in 2016-2017 argentinian women have the same standardized death rate as in 1990-1991. premature mortality increases significantly among the poorest segments of society, and does so with particularly harsh trends among women over 60 (macchia et al. forthcoming ). yet, these populations disappear completely from the health metrics analysis. their eclipse is not accidental. the categorization of a wide, polychromatic and heterogeneous universe into smaller, measurable, homogeneous fractions recording an average improvement, manages to reduce the world to estimates showing some improvement but leaving out immense population groups (sassen 2014) . the resulting disappearance of humans portions shrinks the world to a selected group of inhabitants, those who coincide with as many given categories or clusters. the covid is a chapter written with the same letters. the grotesque comparisons and 'rankings' among countries circulated by the media on a daily basis, arithmetically counting the number of sick and dead, only represent the further trivialization of a culture that does not pretend to learn or listen but to talk. in a world where many could know so much, the number of people who know something seriously is alarmingly low. again, a few technical data to reflect on this. in buenos aires, a city of 3.1 million people, the cumulative incidence rate (ic95%) of covid-19 was 1890 covid-19 was (1832 covid-19 was -1948 per 100,000 people, as of august 2020. in the slums it was 5916 (5542-6289) corresponding to 13,829 people, while in the city areas not inhabited by proximity to the slums it was 1559 (1505-1612) corresponding to 44,314 people. despite their younger age, slum dwellers have a significantly higher mortality rate. even disaggregating by age, sex and risk factors, the mortality rate of slum population doubles that of people living outside the slums (macchia et al. submitted) . ultimately, covid-19, like coronary disease, tuberculosis, malnutrition, obesity and cancer (marmot 2005 ) is socially conditioned. additionally, the possibility of confining cases and close contacts in overcrowded housing is close to zero. in buenos aires more than 60% of covid-related cases among slums dwellers had to be confined to alternative care sites (hotels). 3 what's the point of describing covid in argentina merely counting the number of cases, without telling people's stories, without understanding the different human groups, their identities and challenges? it seems that global world standards do not need this information. after all, slum dwellers are not part of the global picture, just like the over one billion world inhabitants who live undocumented (byass et al. 2013) , and who spend their lives in daily statistical and political anonymity. health has ceased to be a public matter since years. it was regarded as such for a brief period of political history, a time that cannot return it seems, at least not in the short term. but it is not only the administration of health care that has been transferred into private hands. public health research and its priorities setting is in private hands, too. philanthrocapitalism, particularly through the bill & melinda gates foundation, finances the research agenda and modulates collective health's decisions at the level of the multilateral community. members of 'philanthropic' foundations have close ties to pharmaceutical companies, so they sail from private interest to public policymaking with no restraints. philanthropic foundations' staff sits on corporate entities' boards and public health organizations alike, as if this were a normal circumstance (birn 2014) . in covid's case, the relationship between the director of the 'warp speed' programme and his relationship with one of the candidates to produce the vaccine is one point in case. example. 4 the relationship between health, collective rights and market interests has become increasingly blurred. some latin american countries hailed the vaccine production in their own territories, announcing it as the exercise of state responsibility for the benefit of collective health. but this narrative is nowhere close to reality, at least in argentina. the production of the astrazeneca and the oxford university vaccine was entrusted by the consortium to an argentine businessman who owns a group of transnational companies. the business group quickly clarified that this is an 'agreement between private parties' and that the argentinian state has nothing to do with it. in fact, the argentinian state does have a role. it has secured the advanced purchase of tens of millions of doses without even negotiating the price of the product. it takes the risk of vaccinating early, in the absence of long-term safety monitoring results. but there's more. in the past, the government has financed the vaccine production plants. the state is indeed playing a role with a variety of interventions, including that of a consumer. 5 the race to win the market competition encouraged by ignorant politicians with no scruples is doomed to produce even greater damage (krause et al. 2020) . obtaining a poorly effective vaccine (as it will most likely be the case for the first generation of products) will generate a gold standard that other follow-on trials will adopt as their benchmark. in 1935, martin heidegger envisaged what did not exist then, but we must consider today: 'when the farthest corner of the globe has been conquered technologically and can be exploited economically; when any incident you like, in any place you like, at any time you like, becomes accessible as fast as you like; when you can simultaneously 'experience' an assassination attempt against a king in france and a symphony concert in tokyo; when time is nothing but speed, instantaneity, and simultaneity, and time as history have vanished from all being of all peoples; when a boxer counts as the great man of a people; when the tallies of millions at mass meetings are a triumph; then, yes then, there still looms like a specter over all this uproar the question: what for?-where to?-and what then?' the substantial convergence between the initial question of our title and the questions that heidegger formulates in the quote we have chosen could be seen as the most coherent and certainly worrying conclusion. the historical parenthesis when the universality of human rights was politically adopted not as an utopia, but as an innovative (first in history!), concrete, long-term programme of a post-war (never again!) world order is radically threatened, if nor formally declared obsolete. the ongoing covid-19 event is the symbolic-even more importantly than the real-expression of a time when unmet needs and questions come forcefully to the fore as protagonists, higher and above the accepted challenge. health was the first ideal and realistic proposition of that historical parenthesis (table 1) : the life with dignity of human beings (no one left behind!) was assumed to be the mandatory, reasonable, long term outcome. but the amazing successes of life sciences in the last decades have moved in parallel with the progressive transformation of the economy from a valuebased, competent science that guided the use of resources as a key component to support human development, into an autonomous uncontrolled power. the legitimate and binding indicators of human and peoples' rights were marginalized, to the advantage of development models and economic strategies, whereby market goods acquired the status of free and untouchable subjects of legal contractual laws. on the other hand, most juridical sciences and institutions, satisfied with the formal solidity of their principles, conventions and constitutions based on universality paradigms, became confined to a role of arbiters and controllers of the new global order 's compliance. in this scenario, the universality of human and peoples' rights was progressively transformed into a dependent variable of economic sustainability and of the impunity needed by the new political and 5 on 18 october 2020, the minister of health has assured that in march 2021 a massive vaccination campaign will be carried out in argentina with the astra zeneca vaccine, the production of which is planned in argentina available at https ://www.lanac ion.com.ar/polit ica/coron aviru s-argen tina-gines -gonza lez-garci a-en-marzo -nid24 83167 . economic powers, active at the national and international level. the 2020 pandemic, triggered by one of the oldest companions-enemies of human life and history, could become a determinant of a cultural, social, mental lockdown; a test of the limits of resistance of societies, more than a powerful stimulus of resilience. as representatives and disenchanted actors of disciplines where uncertainties and failures compel to imagine, promote, experiment answers 'to care for the right of life'-this indeed has been the only recognized 'light' of engagement in the dark tunnel of the pandemicwe have the duty to give our last question an horizon. something that does not exist but has a very important role, as eduardo galeano showed in his imagined upside-down world. the role of the horizon is simply to oblige us to move on and go ahead. let's at least be the custodians, let's at least preserve the memory of the utopia of the first acronyms in table 1 . the universal right to life in dignity is assumed in a bottom-up mobilization by the subjects of history, the 'discarded', the expelled majorities, as a priority area of high cultural and social conflicts, to become the political terrain of struggle and confrontation with the economic and institutional powerful minorities, in the quest for a new, human, parenthesis. it is our horizon. not an answer. health inequalities: death by political means philanthrocapitalism, past and present: the rockefeller foundation, the gates foundation, and the setting(s) of the international/global health agenda when will we have a vaccine? understanding questions and answers about covid-19 vaccination mapping 123 million neonatal reflections on the global burden of disease 2010 estimates disease burden in sub-saharan africa: what should we conclude in the absence of data? covid-19 cases surge in colombia nutrition, pathologies of power and the need for health democracy health equity: are we finally on the edge of a new frontier? potential impact of the covid-19 pandemic on hiv, tuberculosis, and malaria in low-income and middle-income countries: a modelling study disease control priorities in developing countries covid-19 vaccine trials should seek worthwhile efficacy doval. forthcoming press. an analysis of death trends in argentina covid-19 among the inhabitants of the slums in the city of buenos aires: a population based study social determinants of health inequalities projections of global mortality and burden of disease from the global burden of disease: a comprehensive assessment of mortality and disability from diseases, injuries and risk factors in 1990 and projected to 2020 pandemic influenza vaccines: communication of benefits, risks, and uncertainties expulsions: brutality and complexity in the global economy meeks lm, disability inclusion. moving beyond mission statements embedding patient-and public health-oriented research in a national health service: the gissi experience the impact of covid-19 and strategies for mitigation and suppression in low-and middle-income countries key: cord-256543-7kfi2yvu authors: de graaf, miranda; beck, relja; caccio, simone m; duim, birgitta; fraaij, pieter la; le guyader, françoise s; lecuit, marc; le pendu, jacques; de wit, emmie; schultsz, constance title: sustained fecal-oral human-to-human transmission following a zoonotic event date: 2016-11-23 journal: curr opin virol doi: 10.1016/j.coviro.2016.11.001 sha: doc_id: 256543 cord_uid: 7kfi2yvu bacterial, viral and parasitic zoonotic pathogens that transmit via the fecal-oral route have a major impact on global health. however, the mechanisms underlying the emergence of such pathogens from the animal reservoir and their persistence in the human population are poorly understood. here, we present a framework of human-to-human transmission of zoonotic pathogens that considers the factors relevant for fecal-oral human-to-human transmission route at the levels of host, pathogen, and environment. we discuss current data gaps and propose future research directions. in recent years there have been many examples of pathogens crossing the species barrier and infecting humans, although the vast majority of these zoonotic events did not result in sustained human-to-human transmission [1] [2] [3] . nevertheless, the continuing emergence of zoonotic pathogens is a cause of concern globally, especially due to the high morbidity and mortality of pathogens like mers-cov and a/h5n1 influenza virus [4, 5] . humanto-human transmission of microorganisms generally occurs via one or multiple transmission routes, including the fecal-oral, airborne, direct contact, or vector-borne route. whilst pathogens including bacteria, parasites and viruses have very different biological properties, they can employ similar routes of transmission and emergence. identification of the mechanisms underlying the effective human-to-human transmission of emerging zoonotic pathogens and their commonalities across different pathogens, may help design of interventions aimed at reducing the risk of sustained human-to-human transmission after a zoonotic event. as part of the activities of the antigone consortium on the emergence of zoonotic pathogens, an expert opinion meeting was organized. using a comparative approach including parasites, bacteria and viruses that transmit via the fecal-oral route, the meeting aimed at identifying the key drivers of sustained human-to-human transmission after a zoonotic event, taking into account the host, the pathogen and the interface (transmission amplifiers). in addition, major knowledge gaps were identified that require future research in order to better control emerging zoonotic pathogens that potentially are transmitted through a fecal-oral route. the main conclusions of this meeting are presented in this perspective. enteric pathogens can be transmitted between humans by the fecal-oral route via direct contact or indirect contact via contaminated fluids, including surface water, food, and carriers such as fomites ( figure 1 ). the risk of a zoonotic pathogen becoming human-to-human transmissible depends on its adaptation to the human host and the environment. to analyze this process, we considered fecal-oral transmission of a zoonotic pathogen between two human hosts as follows; the human host that is infected with a zoonotic pathogen after a zoonotic event is defined as the donor while the susceptible human host that is subsequently infected by the first human host is considered the recipient. the transmission interface is the environment that the pathogen encounters after release from the donor and before infecting the recipient. for sustained fecal-oral human-to-human transmission certain elements in this transmission cycle, which we will refer to as transmission amplifiers, appear essential whereas other elements are not an absolute requirement, but increase the likelihood of transmission. transmission amplifiers may interact and their presence may or may not depend on conditions under which transmission occurs, including for example socio-economic conditions and cultural and behavioral variation. we designed a framework of human-to-human transmission that includes the transmission amplifiers relevant for the fecal-oral transmission route at the levels of host, pathogen, and environment ( figure 2 ). several key transmission amplifiers are specific for fecaloral transmission (figure 2 ), such as the intestinal microbiomes of the donor and recipient hosts. individuals with a healthy intestine are less likely to become infected or colonized by opportunistic pathogens, although the resistance provided by a healthy colonization (microbiome) can, in principle, be disrupted by a pathogenic species depending on its pathogenic potential (virulence) [6, 7] . the composition of the human intestinal microbiome is, amongst others dependent on the presence of a functional immune system [8] [9] [10] . changes in microbiome composition, in addition to the impaired immunity itself, may impact the outcome of infection and subsequent transmission. clinical symptoms such as diarrhea and vomiting can increase the likelihood of fecal-oral transmission as they can facilitate the spread of a pathogen into the fecal-oral transmission between humans. after shedding from the host enteric pathogens can be transmitted between humans by the fecal-oral route via direct contact between humans, or via indirect contact via contaminated fluids, including surface water, food, and carriers such as fomites. environment and onto fomites [11] . remarkably, most pathogens that transmit via the fecal-oral route are very stable and can survive under various conditions, which may be related to the fact that these pathogens have to pass the hostile conditions of the gastrointestinal tract. zoonotic pathogens need to adapt to factors specific to this niche, such as the acidic conditions in the stomach and low oxygen in the large intestine, the temperature and the availability of specific sugars and nutrients. for example, comparative genomics of cryptosporidium parvum genotype iic suggests that the ability to establish an infection in a particular host species may depend in part on the presence of transporters controlling the exchange of metabolites between the host cell and the pathogen [12] . fecal shedding of a pathogen does not necessarily require replication in the intestine. for example, the hepatitis e virus (hev) is shed via the feces despite its liver tropism [13] . however, the presence of receptors and the tissue distribution of these receptors is a crucial element for tropism of infection, the shedding of microorganisms in stool and subsequent human-to-human transmission. in addition, it should be noted that not all pathogens that are shed via the feces transmit via the fecal-oral route. several respiratory viruses of zoonotic origin, that are capable of human-to-human transmission, are shed in feces. during sars-cov, mers-cov and influenza a infection, viral rna can be detected in stool. however, for these pathogens there is currently no evidence of fecal-oral transmission resulting in disease [14] [15] [16] [17] [18] [19] . similarly, the enteric pathogen campylobacter subspecies jejuni was transmitted from human-to-human via sexual contact following a zoonotic event [20] . once a donor is shedding the pathogen, environmental factors at the transmission interface can have a large impact on transmission efficiency. contamination of the surface water after flooding can magnify the size of an outbreak via waterborne and foodborne routes [21] . food sources can be contaminated by irrigation with sewage-contaminated water or the use of manure that contains traces of human feces, or on site by food-handlers. anecdotally, even food preservation measures can impact transmission as some additives to preserve lettuce were shown to also increase the stability of hepatitis a virus [22] . transmission via food can have a major impact on the global spread of pathogens. in 2011 nearly human-to-human transmission following a zoonotic event de graaf et al. 3 framework for human-to-human transmission after a zoonotic event showing the key transmission amplifiers from the host (triangle), pathogen (blue) and environmental transmission amplifiers (green), respectively. the transmission amplifiers that are specific to the fecal-oral route are indicated with a red star. 4000 people were infected during an escherichia coli o104:h4 outbreak in europe, resulting in 54 deaths. epidemiological and trace-back investigations pointed to salad sprouts as the possible contaminated food source [23] . transmission amplifiers not specific for fecaloral transmission route several factors that are important transmission amplifiers of the likelihood of fecal-oral transmission are generic to most human-to-human transmission routes. the immune system of the human host is an important factor that has to be confronted for sustained human-tohuman transmission. most pathogens that successfully transmit, have acquired genes that can counteract or evade the adaptive and or innate immune responses. pathogens may have adapted through altered domains that are recognized by the cellular or humoral immune system, to evade pre-existing immunity based on previously infecting pathogens. loss of genes or gene function may also be associated with adaptation to the human host. a salmonella enterica serotype typhimurium clone which causes bloodstream infection amongst children and hiv-infected adults in sub-saharan africa, has adapted to these immuno-compromised hosts through loss of gene functions enabling bacterial survival outside the host, whilst retaining the ability to cause enteritis in multiple host species [24 ] . with the general population aging and technologies becoming more invasive, medical interventions can become an amplifier of human-to-human transmission. although medical interventions do not necessarily select for human-to-human transmissible pathogens, they can increase the duration of infection, and thereby the likelihood of evolution and adaptation [25] . for instance, the application of extracorporeal membrane oxygenation prolongs and increases survival in patients with otherwise acute fatal infectious disease [26] . the use of immune modulatory and suppressive drugs has created a human population that is more susceptible to prolonged pathogen proliferation and shedding [27] [28] [29] [30] [31] . an immunodeficient individual was found to excrete a vaccine-derived poliovirus for twenty years. during this time the virus became virulent and changed antigenically [32] . the unrestrained use of antimicrobial drugs in medical and veterinary care and in agriculture in low-income, middleincome, and high-income countries creates an unprecedented selective pressure that may select for pathogens that are more transmissible. salmonella enterica serotype typhimurium has the ability to develop a super shedder phenotype, that can be induced by antibiotic treatment in mice [33] . a human reservoir for non-typhoid salmonella (nts) transmission of multiple serotypes was demonstrated in a study of nts-infected patients who continued to shed nts for months up to years, and strains of these patients acquired antimicrobial resistance genes and virulence genes that possibly affected host-pathogen interactions [34 ] . the infectious dose, replication kinetics and the number of pathogens being shed can have a major impact on the efficiency of fecal-oral transmission. for example, norovirus and shigella spp. require a low infectious dose and can be transmitted via hands and fomites [35, 36] , whereas listeria monocytogenes infections require a high infectious dose [37] , making these transmission routes less likely. however, surprisingly little is known about the infectious dose for many fecal-orally transmitted human pathogens. as for shedding in the environment, several strategies can be successful, such as shedding of lower amounts of microorganisms over a long period (chronic/persistent infection) and thus a long period of transmission associated with mild clinical symptoms , or shedding of high loads of microorganisms for a relatively short period with significant clinical symptoms [36] . receptor usage is a key element for successful human-tohuman transmission. not only are the site of the expression of these receptors in the host and the receptor specificity of the pathogen of importance, but also the prevalence of these receptors in the human population can potentially contribute to the likelihood of efficient transmission. for group a rotaviruses attachment to histobloodgroup antigens is an essential step for infection. interestingly, strains of the p [9] , p[14] and p [25] subtypes that are generally found in cattle but can also infect humans, attach to the blood group a epitope [38] . however, since the frequency of blood group a in human populations ranges from 20% to 40%, the majority of individuals is expected to be resistant to infection by these strains, which would constitute a barrier to transmission. sustained human-to-human transmission for a virus with a relatively low r 0 would thus require an adaptation of the glycan binding capacity to the human hbga genetic polymorphism [39] . hev genotype 1 and 2 exclusively infect humans while genotypes 3 and 4 infect pigs but occasionally infect and transmit between humans [40] . the high conservation of hev attachment and entry factors may explain the observed cross-species transmission while factors limiting efficient human-to-human transmission are thought to include regulation of subgenomic translation and specific virus-host receptor interactions [41, 42] . surprisingly few differences exist between the basic requirements of the different types of pathogens to become human-to-human transmissible. however, bacteria can replicate in the environment whereas viruses and parasites cannot. as the transmittable stages of parasites are environmentally very resistant, and can withstand water treatment processes, parasites are probably more likely to be transmitted via food and water compared to direct fecal-oral-transmission [43,44 [ 1 _ t d $ d i f f ] ]. genome plasticity is an important factor for all pathogens but while parasites, viruses and bacteria can all adapt by mutations, recombination and lateral gene transfer, viruses can also acquire genes by genome rearrangements and bacteria can acquire mobile genetic elements that carry genes that may encode determinants that facilitate increased fitness in certain conditions. 'for a zoonotic pathogen the risk of becoming human-tohuman transmissible depends on further adaptation to the human host. for efficient fecal-oral transmission amplifiers in the transmission interface appear crucial.' the focus of the public health and emerging infectious disease communities on emerging viruses causing severe infections, has resulted in the discovery of several possible determinants of sustained human-to-human transmission of zoonotic viruses. however, the likelihood of sustained human-to-human fecal-oral transmission after a zoonotic event of any pathogen type is difficult to assess as these events are hardly described in current literature. one could conclude that zoonotic pathogens rarely become human-to-human transmissible through the fecal-oral route because there may be need for dual adaptation, i.e. to the harsh conditions in the environment in addition to the human host, and therefore these events are rare. however, we cannot exclude that we may be missing some of these events, because it can be difficult to distinguish between strictly human and zoonotic pathogens once the latter have established themselves in the human population or because they remain undistinguished with the use of current clinical microbiology tools. for example, recent results strongly suggest that a pig roundworm can act as an important source of human ascariasis [45] [46] [47] but this can go unnoticed as the human and pig parasite population show minor phenotypic and genotypic differences. zoonotic pathogens that transmit via the fecal-oral route appear to cause similar clinical symptoms compared to other (related) enteric pathogens and further research is not pushed due the relative lack of (more) serious disease. we thus may neglect relevant pathogens and even if we do study these pathogens, the results may have undesired economic consequences for agriculture and food sectors. in addition, until recently we lacked tools for studying important zoonotic events; whilst the small genomes and rapid evolution of viruses allow identification of novel causative agents with limited sequencing effort, such analysis is much more complicated for bacterial and parasitic enteric pathogens which have relatively large genomes. some experimental models for fecal-oral transmission between hosts have been described, such as for norovirus [48 ] , but there is a general lack of suitable animal models to study fecal-oral transmission. in fact, most research on host-pathogen interactions is focused on mechanisms of pathogenicity rather than on mechanisms of transmission, whilst the latter is crucial for the development of intervention strategies to prevent further spread and to prevent sustained human-to-human transmission of zoonotic pathogens. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest one health, multiple challenges: the inter-species transmission of influenza a virus zoonotic aspects of rotaviruses shiga toxin-producing escherichia coli o157, england and wales sars and mers: recent insights into emerging coronaviruses intestinal microbial communities associated with acute enteric infections and disease recovery salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota the intestinal microbiota and susceptibility to infection in immunocompromised patients the intestinal microbiome in early life: health and disease gut microbiota composition correlates with diet and health in the elderly makison booth c: vomiting larry: a simulated vomiting system for assessing environmental contamination from projectile vomiting related to norovirus infection comparative genome analysis of two cryptosporidium parvum isolates with different host range hepatitis a: old and new viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection influenza virus infection among pediatric patients reporting diarrhea and influenza-like illness enteric involvement of severe acute respiratory syndrome-associated coronavirus infection long-term sars coronavirus excretion from patient cohort extra-pulmonary viral shedding in h7n9 avian influenza patients long-lasting outbreak of erythromycin-and ciprofloxacinresistant campylobacter jejuni subspecies jejuni from 2003 to 2013 in men who have sex with men quantitative assessment of infection risk from exposure to waterborne pathogens in urban floodwater survival of hepatitis a virus on modified atmosphere-packaged (map) lettuce the enemy within us: lessons from the 2011 european escherichia coli o104:h4 outbreak loss of multicellular behavior in epidemic african nontyphoidal salmonella enterica serovar typhimurium st313 strain d23580 the potential for respiratory droplet-transmissible a/ h5n1 influenza virus to evolve in a mammalian host year in review 2015: extracorporeal membrane oxygenation noroviruses as a cause of diarrhea in immunocompromised pediatric hematopoietic stem cell and solid organ transplant recipients high prevalence of prolonged norovirus shedding and illness among hospitalized patients: a model for in vivo molecular evolution persistent spiking fever in a child with acute myeloid leukemia and disseminated infection with enterovirus infectious complications and vaccinations in the posttransplant population prolonged influenza virus shedding and emergence of antiviral resistance in immunocompromised patients and ferrets twentyeight years of poliovirus replication in an immunodeficient individual: impact on the global polio eradication initiative host transmission of salmonella enterica serovar typhimurium is controlled by virulence factors and indigenous intestinal microbiota this study showed that nts strains of persistently infected humans acquired antimicrobial resistance and virulence genes inoculum size in shigellosis and implications for expected mode of transmission norwalk virus shedding after experimental human infection an outbreak of gastroenteritis and fever due to listeria monocytogenes in milk cell attachment protein vp8* of a human rotavirus specifically interacts with a-type histoblood group antigen noroviruses and histoblood groups: the impact of common host genetic polymorphisms on virus transmission and evolution hepatitis e: an emerging awareness of an old disease molecular biology and replication of hepatitis e virus hepatitis e virus genotype 1 infection of swine kidney cells in vitro is inhibited at multiple levels human cryptosporidiosis in europe effect of sanitation and water treatment on intestinal protozoa infection: a systematic review and meta-analysis this systemic review and meta-analysis shows how lack of clean water is associated with increased risk of intestinal protozoa infection pig ascaris: an important source of human ascariasis in china assessing the zoonotic potential of ascaris suum and trichuris suis: looking to the future from an analysis of the past from the twig tips to the deeper branches: new insights into evolutionary history and phylogeography of ascaris prophylactic treatment with the nucleoside analogue 2 0 -c-methylcytidine completely prevents transmission of norovirus the authors developed a murine fecal-oral transmission model the authors would like to thank all participants of the dahlem 'inter human barriers' workshop for their contributions and ryan kissinger (niaid, nih) for designing the figures. this work was supported by antigone (grant numbers 278976); edw is supported by the intramural research program of the national institute of allergy and infectious diseases, us national institutes of health; pf receives funding from the eu fp7 project prepare (grant number 602525); mdg is supported by the eu grant compare (grant number 643476) and the virgo consortium, funded by the dutch government (project number fes0908). key: cord-020756-d9f5fd7x authors: de jong, menno douwe title: avian influenza viruses and pandemic influenza date: 2007 journal: new and evolving infections of the 21st century doi: 10.1007/978-0-387-32830-0_9 sha: doc_id: 20756 cord_uid: d9f5fd7x nan global outbreaks with high death tolls. these pandemic strains can certainly be regarded as (re)emerging pathogens. the "athen's plague" described by hippocrates' contemporary thucydides is believed by some to constitute the first account of such a devastating influenza epidemic. since the 16th century, many large-scale outbreaks of influenza-like illnesses have been described in europe. in 1580, one of such outbreaks spread from europe into africa and asia, possibly making it the first recorded influenza pandemic. the most devastating influenza pandemic in modern recorded history, known as the "spanish flu," occurred in 1918-1919, killing up to 100 million people worldwide. other less destructive pandemics during the past century occurred in 1957 and 1968. avian influenza a viruses are key to the emergence of human influenza pandemics. the virus strains implicated in the 20th century's influenza pandemics originated directly from avian influenza viruses, either through genetic reassortment between human and avian influenza strains (1957, 1968) or possibly through adaptation of purely avian strains to humans (1918) . it was long thought that the host range of avian influenza viruses precluded direct transmission to humans and that the emergence of pandemic strains required genetic reassortment between avian and human strains. however, occurrences of direct bird-to-human transmission of avian influenza viruses have increasingly been reported in recent years, culminating in the ongoing outbreaks of influenza a (h5n1) among poultry and wild birds in several asian, european and african countries with continuing instances of human infections. these unprecedented developments have resulted in increasing global concerns about the (re)emergence of pandemic influenza a strains and the role of avian influenza viruses in this. influenza viruses are pleomorphic, enveloped rna viruses belonging to the family orthomyxoviridae. protruding from the lipid envelope are two distinct glycoproteins: the hemagglutinin (ha) and neuraminidase (na) . ha attaches to cell-surface sialic acid receptors, thereby facilitating entry of the virus into host cells. because it is the most important antigenic determinant to which neutralizing antibodies are directed, ha represents a crucial component of current vaccines. na is the second major antigenic determinant for neutralizing antibodies. by catalyzing the cleavage of glycosidic linkages to sialic acid on host cell and virion surfaces, this glycoprotein prevents aggregation of virions thus facilitating the release of progeny virus from infected cells. inhibition of this important function represents the most effective antiviral treatment strategy to date. a third membrane protein, the m2 protein, is present in small quantities in influenza a viruses. by functioning as an ion channel, this protein regulates the internal ph of the virus, which is essential for uncoating of the virus during the early stages of viral replication. this function is blocked by the antiviral drugs amantadine and rimantadine. the genome of influenza viruses is segmented, consisting of eight single-stranded, negative-sense rna molecules that encode 10 proteins. the rna segments are contained within the viral envelope in association with the nucleoprotein (np) and three subunits of viral polymerase (pa, pb1, and pb2), which together form the ribonucleoprotein (rnp) complex responsible for rna replication and transcription. additional proteins contained within the virion include m2 and the viral nuclear export protein (nep), which function in assembly and budding and in export of rnp from the nucleus, respectively. the only nonstructural protein of influenza a viruses is ns1, which has multiple functions in viral replication and is also thought to counteract interferon activity of the host thereby evading the immune response. based on antigenic differences in np and m proteins, influenza viruses are classified as types a, b, and c. influenza b and c viruses are not divided into subtypes. all avian influenza viruses are classified as type a. further subtyping of influenza a viruses is based on antigenic differences between the two surface glycoproteins ha and na. to date, 16 ha subtypes (h1-h16) and 9 na subtypes (n1-n9) of influenza a viruses have been identified (fouchier et al., 2005) . the standard nomenclature for influenza viruses include the influenza type, the host of origin (excluding humans), the place of isolation, the strain number, the year of isolation, and finally the influenza a subtype in parentheses (e.g., a/duck/vietnam/11/04 [h5n1]). the natural reservoir of influenza a viruses are aquatic birds, in which the viruses appear to have achieved an optimal level of host adaptation and do not cause disease . from this principal reservoir, viruses are occasionally transmitted to other animals, including mammals and domestic poultry, causing transitory infections and outbreaks. through adaptation by mutation or genetic reassortment, some of these viruses may establish species-specific permanent lineages of influenza a viruses and cause epidemics or epizootics in the new host. in the human population, the establishment of these lineages in the 20th century was preceded by influenza pandemics. transmission of viruses and transitory infections may also occur among the new hosts (e.g., between humans and pigs or chickens and humans). although all ha and na subtypes are found in aquatic birds, the number of subtypes that have crossed the species barrier and established stable lineages in mammals is limited. only three ha and two na subtypes (i.e., h1n1, hin2, h2n2, and h3n2) have circulated in humans since 1918. in horses, only two influenza a subtypes (h7n7 and h3n8) are found, while, despite susceptibility to all avian subtypes in experimental settings, the only subtypes recovered from pigs in nature are h1, h3, n1, and n2. the molecular, biological, or ecological factors determining the apparent subtype-specific ability of viruses to cross species barriers and spread among a range of hosts remain largely unresolved. although interspecies transmission does occur at times, there certainly are host-range restrictions. for example, avian influenza viruses usually do not replicate efficiently in humans and vice versa (hinshaw et al., 1983; beare and webster 1991) . relatively little is known about the viral and host factors governing the host range of influenza viruses and the mechanisms by which species barriers are crossed. however, in view of their role in entry of the virus, the viral ha glycoproteins and their sialic acid receptors on host cells clearly are important determinants of host-range restrictions. human influenza strains preferentially bind to sialic acid residues linked to galactose by the α2,6 linkage, and avian and equine influenza strains recognize sialic acid linked to galactose by α2,3 linkage rogers and d'souza 1989; connor et al., 1994; gambaryan et al., 1997; matrosovich et al., 1997 matrosovich et al., , 2004 . correspondingly, human respiratory epithelial cells predominantly contain α2,6 sialic acid-galactose linkages, whereas the host cells in birds and horses mainly contain α2,3 linkages (couceiro et al., 1993; ito et al., 1998; matrosovich et al., 2004) . interestingly, in contrast with the human respiratory tract, epithelial cells in the human eye predominantly contain α2,3-linked sialic acid receptors, which may explain why conjunctivitis is a common symptom of human infections with avian influenza viruses (paulsen et al., 1998; terraciano et al., 1999; diebold et al., 2003) . it has been hypothesized that, by serving as the main port of entry and site of initial replication, the eye may play a role in the adaptation of avian influenza viruses to humans (olofsson et al., 2005) . the presence of α2, 3-linked sialic acid receptors has also recently been demonstrated in the lower respiratory tract of humans, i.e. on bronchiolar and alveolar cells, which may explain the propensity of avian h5n1 viruses to cause pneumonia and not upper respiratory illnesses, in humans (shinya et al., 2006; van riel et al., 2006) . respiratory epithelial cells in the pig contain both α2,3 and α2,6 linkages, which explains why this animal is susceptible to both human and avian influenza viruses . because of this trait, the pig is widely regarded as a potential source of new pandemic strains, because it could serve as a nonselective host in which mixed infection of avian and human strains efficiently occurs, potentially resulting in new reassortant viruses, or in which purely avian strains can adapt to human receptor recognition (figure 9 .1). the receptor specificity of ha for either of the two sialic acid-galactose linkages is determined by the structure of the receptorbinding site of ha. although several residues have been implicated, the amino acids at positions 226 and 228 particularly seem to determine ha receptor specificity, that is, glu-226 and gly-228 are predicted to have affinity for avian and equine receptors, whereas leu-226 and ser-228 confer specificity for human receptors (wilson et al., 1981; naeve et al., 1984; weis et al., 1988; suzuki et al., 1989) . albeit less important, substrate specificity of na for either α2,3or α2,6-linked sialic acid also contributes to the efficiency of viral replication in different hosts (hinshaw et al., 1983) . this is illustrated by the fact that during its evolution in humans, the na of h2n2 viruses, which were of avian origin and therefore highly specific for hydrolization of α2,3-linked sialic acids, acquired high affinity for the human α2,6-linked sialic acids (baum and paulson, 1991) in addition to the surface glycoproteins, laboratory experiments with reassortant viruses suggest that the genes encoding internal proteins, such as m, np, pb1 and pb2, may also play a role in determining the host range (almond 1977; scholtissek et al., 1978a; snyder et al., 1987; subbarao et al., 1993) . however, because most of these experiments evaluated reassortant viruses with different constellations of gene segments, it remains difficult to interpret whether the proteins themselves contribute to host-range restrictions or whether certain combinations of gene segments from different origins are incompatible. manipulation of the genome using reverse genetics approaches will undoubtedly provide more definitive insight in the role of other host range determinants. antigenic variation of influenza a viruses can occur gradually by accumulation of point mutations (antigenic drift) or drastically by genetic reassortment (antigenic shift). antigenic drift, driven by immunological pressure on ha and na, allows the virus to evade the immune response and is the reason that influenza viruses manage to cause yearly epidemics. it is also because of antigenic drift that periodic replacements of human vaccine strains are needed. in contrast with human and other non-avian influenza strains, antigenic drift in avian viruses is very limited despite similar mutation rates (austin and webster, 1986; kida et al., 1987; liu et al., 2004) . most likely, this reflects optimal adaptation of these viruses to the host resulting in limited immunological pressure and consequent evolutionary stasis of these viruses in their natural reservoir. drastic changes in antigenicity can occur through the acquisition of completely new surface proteins by genetic reassortment . the segmented nature of the influenza virus genome facilitates the exchange of genes between two viruses (e.g., human and avian strains) that coinfect a host cell. although such exchange can result in 256 possible combinations of the eight different genomic segments of the virus, antigenic shift only arises when the reassortment at least includes the ha gene. provided that the reassortant virus is efficiently transmissible from infected to noninfected hosts, such an antigenically novel virus strain has pandemic potential when introduced in a population that completely lacks immunity against the new surface protein (figure 9 .1a). the pig is regarded as the ideal host for reassortment in view of its equal susceptibility for human and avian influenza strains . however, the increasing reports of bird-to-human transmissions of avian viruses indicate that coinfections, and consequently reassortments, could also take place in humans. beside genetic reassortment, antigenic shift is also caused by direct transmission of non-human influenza viruses to humans, as occurred or is still occurring on a relatively large scale in hong kong in 1997 (h5n1), in the netherlands in 2003 (h7n7), and in asia, the middle east, europe and africa since 2004 (h5n1) (yuen et al., 1998; fouchier et al., 2004; hien et al., 2004a) . as is true for reassortant viruses, these viruses are of pandemic potential when acquiring the ability for efficient transmission between humans through adaptation in either humans or an intermediate host (figure 9 .1b). finally, antigenic shift can occur when a previously circulating human influenza virus reemerges after an extended period of time. this happened in 1977 when h1n1 virus, which circulated in the 1950s, reappeared in the human population ("russian flu"), possibly after escaping a laboratory (nakajima et al., 1978; scholtissek et al., 1978b) . the reemergence of this virus gave rise to a relatively mild pandemic affecting mainly young persons who were still immunologically naive to this subtype. the same could have happened in 2005, when h2n2 virus, which had disappeared from the human population after the emergence of h3n2 viruses in 1968, was inadvertently sent to more than 3000 laboratories worldwide as part of an external quality assurance scheme (enserink, 2005) . avian influenza viruses can infect a wide range of domestic and wild birds, including (but not restricted to) chickens, ducks, turkeys, geese, quail, pheasants, seabirds, shore birds, and migratory birds. in these natural hosts, influenza viruses replicate in the gastrointestinal tract and are secreted in large amounts into the feces (webster et al., 1978) . transmission between birds occurs directly or indirectly through fecally contaminated aerosols, water, feed, and other materials. the spectrum of disease in birds ranges from asymptomatic infection, to mild respiratory illness, to severe and rapidly fatal systemic disease. most avian influenza viruses isolated from birds are avirulent (i.e., result in asymptomatic infection or only mild disease). avian influenza viruses capable of causing outbreaks of severe disease (fowl plague) in chickens or turkeys are classified as highly pathogenic and are currently restricted to h5 and h7 subtypes. typically, these highly pathogenic strains do not cause disease in ducks or geese. infection of poultry by highly pathogenic avian influenza viruses is characterized by disseminated infection and clinically manifested by decreased egg production, respiratory signs, excessive lacrimation, edema of the head, diarrhea, neurological symptoms, and death. the knowledge concerning the viral factors that determine the pathogenicity of influenza viruses is limited and is primarily derived from studies of highly pathogenic avian influenza viruses. a broad tissue tropism and the ability to replicate systemically are the hallmarks of these viruses. the most important and well-studied molecular correlate of these properties resides in the cleavability of the ha precursor glycoprotein (webster and rott, 1987; garten and klenk, 1999; steinhauer, 1999) . in the viral life cycle, post-translational cleavage of the precursor ha molecule into two subunits (ha1 and ha2) by host proteases is essential for infection to proceed. this cleavage generates a fusogenic domain at the amino terminus of ha2 that mediates fusion between the viral envelope and the endosomal membrane. has of avirulent avian influenza strains are cleaved only in a limited number of cell types, resulting in localized respiratory or gastrointestinal infections and mild illness. in contrast, has of highly pathogenic h5 and h7 strains can be cleaved in several different host cells, resulting in a broad cell tropism and the ability of causing systemic infection (klenk and garten, 1994; senne et al., 1996) . this apparent promiscuity of ha for a broad range of cellular proteases is determined by the structure of the ha cleavage site: has with high cleavability (i.e., from highly pathogenic strains) have multiple basic amino acid residues immediately upstream of the cleavage site, whereas has from avirulent subtypes usually have only a single arginine residue at this site (bosch et al., 1981; walker and kawaoka, 1993; senne et al., 1996; chen et al., 1998) . evidence for the correlation between a multibasic cleavage site, susceptibility for proteases and virulence has been provided by experiments in which viruses were generated with altered cleavage sites in otherwise unchanged genetic backgrounds (ohuchi et al., 1991; horimoto and kawaoka, 1994) . the reason why multibasic cleavage sites seem restricted to the has of h5 and h7 subtypes is unclear but may suggest that the number of basic residues is limited by structural features of ha. analyses of nucleotide sequences of h5 and h7 ha genes has shown the occurrence of direct repeats of purine-rich sequences (aagaaa) at the cleavage site in many cases . such repeats may arise because of pausing of the transcriptase-complex at a region of secondary structure, resulting in slippage of the complex and insertion of a short repeat sequence. additionally, recombination events between two genes of the same virus (e.g., from m or np to ha) may result in the insertion at the cleavage site of short sequences that code for multibasic amino acid residues (orlich et al., 1994; suarez et al., 2004) . in addition to the presence of multiple basic amino acids, susceptibility to ubiquitous proteases is also determined by the loss of a glycosylation site in the vicinity of the cleavage site (deshpande et al., 1987; webster, 1988, 1989) . although ha clearly is an important determinant of viral pathogenicity, animal studies indicate that virulence in mammals is a polygenic trait involving a constellation of other genes that can vary with the specific virus strain and host . however, besides ha, two genes have specifically been implicated in viral pathogenicity in mammals; that is, pb2 and ns. by reverse genetics experiments, it has been shown that a lysine residue at position 627 (lys627) of pb2 seems essential for high virulence and systemic replication in mice of highly pathogenic influenza h5n1 viruses responsible for the outbreak among poultry and humans in hong kong in 1997 (h5n1/97) (hatta et al., 2001) . the presence of lys627 in pb2 of h5n1/97 viruses appears to determine the viral replicative efficiency in mouse cells (and not avian cells) but does not increase the tissue tropism of the virus in mice . lys627 has also been found in pb2s of some, but not all highly pathogenic h7n7 and h5n1 viruses isolated from humans during the outbreaks of these viruses among poultry and humans in 2003 (the netherlands) and 2004 (viet nam, thailand), respectively li et al., 2004) . interestingly, a lysine residue at position 627 of pb2 is also present in all human influenza subtypes (h1, h2, h3) (webster, 2001) . furthermore, single-gene reassortant viruses carrying a pb2 gene of avian origin and all other genes from a human virus showed efficient replication in avian but not mammalian cells (subbarao et al., 1993) . this host cell restriction could be traced to a glutamic acid residue at position 627 of the avian pb2 instead of a lysine residue at the same position in the human virus (subbarao et al., 1993) . these observations suggest that an amino acid change to lys627 in pb2 may help avian viruses to adapt to efficient replication in mice and possibly other mammals, thereby increasing the virulence in these hosts. reverse genetics experiments with highly pathogenic avian influenza h5n1 and h7n7 viruses have indicated that other members of the viral polymerase complex beside pb2, i.e. pa and pbi, likely also play a role in adaptation of avian viruses to the mammalian host, suggesting that host factors are important for viral polymerase activity (gabriel et al., 2005; salomon et al., 2006) . in addition to pb2, the ns gene seems to play a role in the pathogenesis of avian and human influenza virus infections. this gene encodes two proteins: the nuclear export protein (nep) and the only nonstructural protein of influenza viruses, ns1. the ns1 gene or its product contribute to viral pathogenesis by allowing the virus to evade the interferon response of the host (garcia-sastre, 2001 krug et al., 2003) . this evasion may occur through multiple mechanisms, including interference with the activation of cell-signaling pathways and protein kinases involved in interferon induction or interference with the maturation of cellular pre-mrna at the post-transcriptional level. the ns gene has also been implicated in determining the high pathogenicity of influenza h5n1/97 viruses in mammals. experiments in pigs using recombinant viruses showed that the presence of the ns gene of h5n1/97 viruses greatly increased the pathogenicity of an h1n1 virus, possibly by escaping the antiviral effects of interferons and tumor necrosis factor alpha (tnf-α) seo et al., , 2004 . this enhanced virulence in pigs required the presence of glutamate instead of aspartate at position 92 (glu-92) of the h5n1 ns gene, but this amino acid change has not been found in all highly pathogenic h5n1 viruses isolated from humans or animals . beside the apparent cytokine resistance of h5n1/97 viruses, in vitro studies in human macrophages and respiratory cells showed that these viruses also seem to induce the transcription of proinflammatory cytokines, in particular tnf-α and interferon-β, and that the ns gene contributes to this induction (cheung et al., 2002; chan et al., 2005) . similar results were obtained in mice, in which infection with a recombinant h1n1 virus containing the h5n1/97 ns gene caused a cytokine imbalance in mouse lungs, characterized by increased concentrations of proinflammatory cytokines and decreased levels of anti-inflammatory cytokines (lipatov et al., 2005a) . cytokine dysregulation by h5n1/97 viruses is also suggested by observations in human infections. pathological examination of patients who died of influenza h5n1 infection during the 1997 outbreak in hong kong showed reactive hemophagocytic syndrome, which is believed to be a cytokine-driven condition, as the most prominent feature . in addition, exceptionally high levels of certain chemokines were observed in the serum of human cases with avian influenza h5n1 . together, these observations may suggest that a combination of increased resistance against, and high induction of cytokines by the virus synergistically lead to a profound cytokine dysregulation that may play a role in explaining the severity of illness in mammals, including humans. although the ns gene seems to play a crucial role in this, it is likely that other particular gene constellations involving different internal genes also contribute. the virulence of highly pathogenic avian influenza viruses is clearly influenced by the specific host. two variants of the h5n1/97 virus, one of which was isolated from a human patient with mild repiratory illness and the other from a fatal human case, displayed similar differential pathogenicity in mice (zitzow et al., 2002) . however, in ferrets, both variants caused indistinguishable severe systemic disease (zitzow et al., 2002) . conversely, experimental infection with h5n1/97 viruses exhibiting high virulence in mice caused localized respiratory illness without systemic spread in primates and only viral replication in the respiratory tract without clinical illness in pigs rimmelzwaan et al., 2001) . the host factors determining the clinical outcome in animals are unclear. the clinical outcome of human influenza is influenced by factors such as the patient's age, the level of preexisting immunity, immunosuppression, comorbidities, pregnancy, and smoking habits, indicating that host-related factors certainly contribute to pathogenesis in humans. most of the above factors may be explained by differences in local, innate, or specific immunity at different stages of life or under specific circumstances, but other factors likely also play a role. for example, the observation that influenza-related encephalopathy seems well-recognized in japan but less so in other countries may suggest that there are differences in proneness for certain disease manifestations among populations, possibly related to genetic differences (morishima et al., 2002; sugaya, 2002) . although evidence is lacking at present, it is not unlikely that host factors also play a role in the the susceptibility and pathogenesis of human infections with avian influenza viruses. introduction of an influenza a virus with a novel ha gene in a population that lacks immunity to this ha has the potential to cause a pandemic when the virus posesses the ability to spread efficiently among humans (figure 9 .1). during the 20th century, this has happened three times, in 1918, 1957, and 1968 , killing millions of people worldwide. in all three pandemics, the viruses originated from avian influenza viruses. the virus strains responsible for the influenza pandemics of 1957 and 1968 both first emerged in southeastern asia, and both arose through reassortment of genes between avian viruses and the prevailing human influenza strain (scholtissek et al., 1978c) . the "asian influenza" pandemic of 1957 was caused by an h2n2 virus that had acquired three genes (h2, n2, and pb1) from avian viruses infecting wild ducks, in a backbone of the circulating h1n1 human influenza strain. as the asian flu strain emerged and established a permanent lineage, the h1n1 strains soon disappeared from the human population for unclear reasons. similarly, the h3n2 virus causing the "hong kong influenza" pandemic of 1968 (a) (b) figure 9 .1. mechanisms for generation of a pandemic influenza a strain. pandemic influenza a strains could result from genetic reassortment involving the hemagglutinin gene between avian and human strains in coinfected pigs or humans, followed by adaptation to human receptors in either host and human-to-human transmission (a); or through adaptation to humans of a purely avian influenza strain, either in humans or in an intermediate host such as the pig (b). mechanism a was implicated in the "asian" (1957) and "hong kong" (1968) influenza pandemics. the h1n1 virus that caused the "spanish flu" influenza pandemic of 1918 likely resulted from mechanism b. consisted of two genes from a duck virus (h3 and pb1) in a background of the human h2n2 strain circulating at that time. the latter virus disappeared with the emergence of the h3n2 virus and since then has not been detected in humans. sequence analysis of the hypothetical precursor strain that immediately preceded the pandemic h3n2 virus suggested that fewer than six amino acids in ha had changed during the avian-to-human transition . interestingly, a number of these changes may reflect adaptation to the new host because they modified the area surrounding the receptor-binding pocket of ha, including a glu to leu change at position 226, which is particluarly implicated in determining specificity for human receptors (see section 9.2.4). the fact that beside one or two novel surface glycoproteins, both pandemic strains also posessed a pb1 gene of avian origin is intriguing and may suggest a role of this gene in interspecies transmission . although millions of people died during the 1957 and 1968 pandemics, the viruses involved did not appear particularly virulent, suggesting that lack of immunity was the main reason for the excess mortality. this was different during the "spanish flu" pandemic of 1918, in which lack of immunity in the human population was combined with an apparent extremely high virulence of the virus, resulting in the demise of up to 100 million people worldwide. because the 1918 pandemic occurred before viruses were identified as the causative agents, no intact virus has been available for analysis. this and the similar lack of available human and animal influenza strains circulating before 1918 has made it difficult to determine the exact origin of the pandemic h1n1 virus and the reason for its extreme virulence. however, valuable insight has been provided by the recovery of fragments of viral rna isolated from archived autopsy specimens and tissue from alaskan flu victims buried in the permafrost (taubenberger et al., 1997) . this enabled sequence analysis of all eight genes of the virus (reid et al., 2004; taubenberger et al., 2005) . phylogenetic analyses of these genes suggest that the 1918 h1n1 virus may not have arisen by the same mechanism as the 1957 and 1968 pandemic viruses (i.e., by reassortment of avian and human influenza viruses) but perhaps by direct transmission from an avian source after adaptation in humans or another permissive mammalian host, such as the pig (reid et al., 2004; taubenberger et al., 2005) . this is supported by the observation that human h1n1 strains, including the 1918 pandemic strain, have retained the amino acid residues at positions 226 and 228 of ha predictive for binding to avian receptors (see section 9.2.4) (taubenberger et al., 1997) . recent crystallographic studies showed that structural changes in the h1 ha allowed the virus to recognize human receptors despite the presence of these avian-like residues, which may explain why the virus could nevertheless efficiently infect and spread among humans (gamblin et al., 2004; stevens et al., 2004) . the possibility that the 1918 strain had retained the structure and biological properties of its avian ancestors while acquiring the ability to recognize and efficiently infect human cells may explain the high virulence of this virus. mathematical modeling studies have suggested that the transmissability of the 1918 virus was not remarkably different than regular human influenza strains, indicating that extremely efficient spread did not account for the high morbidity and mortality (mills et al., 2004) . although part of the high mortality of the 1918 pandemic could be explained by the lack of antibiotics to treat secondary bacterial pneumonia and poor living conditions, the extremely rapid and severe clinical course implies high pathogenicity of the virus as the major cause. the molecular basis for this high virulence remains unclear. the 1918 h1 ha lacks the multibasic cleavage site characteristic of highly pathogenic avian influenza viruses (see section 9.3.2) (taubenberger et al., 1997; reid et al., 1999) . there are conflicting observations concerning the role of the ns gene in the 1918 pandemic strain. in mice, the presence of the complete ns or only the ns1 segment seemed to confer decreased, rather than enhanced pathogenicity of reassortant h1n1 viruses (basler et al., 2001) . in contrast, in vitro experiments in human lung cells suggested more efficient inhibition of interferon-regulated genes by h1n1 virus in the presence of the 1918 ns gene (geiss et al., 2002) . the most convincing evidence implicates ha as an important determinant of the high virulence. the presence of ha of the 1918 virus conferred high pathogenicity in mice to human strains that were otherwise non-pathogenic in this host . furthermore, these infections were associated with severe hemorrhagic pneumonia and the induction of high levels of macrophage-derived cytokines and chemokines, strikingly reminiscent of clinical observations in humans during the spanish flu pandemic, as well as of recent in vitro and in vivo observations of infections with highly pathogenic avian influenza h5n1 viruses (oxford 2000; cheung et al., 2002; peiris et al., 2004 ). before the year 2003, a few isolated cases of human infections with highly pathogenic h7n7 viruses were reported. these infections concerned laboratory accidents involving exposure to viral cultures or infected animals, and in one case presumed exposure to infected waterfowl in the absence of an overt outbreak (delay et al., 1967; campbell et al., 1970; taylor and turner, 1977; webster et al., 1981; kurtz et al., 1996) . all reported cases, except one, were clinically characterized by selflimiting conjunctivitis. influenza h7n7 virus was isolated from blood of one patient wih hepatitis, but the clinical relevance of this finding was unclear as was the source of this infection (delay et al., 1967; campbell et al., 1970) . in 2003, a large-scale outbreak of h7n7 viruses decimated the poultry industry in the netherlands and was associated with several human infections koopmans et al., 2004) . after diagnosing the first case of human infection with h7n7 virus, active case finding among exposed persons and their close contacts identified a total of 89 laboratory-confirmed infections in humans . this amounted to approximately 2% of the estimated number of people potentially exposed to the virus. highest infection rates were observed in veterinarians and chicken cullers. of the 89 h7n7 cases, 83 persons presented with conjunctivitis, of whom 5 also complained of influenza-like symptoms koopmans et al. 2004 ). it cannot be excluded that the remaining six patients also had conjunctivitis. although two individuals reported an influenza-like illness only, one had suffered a previous eye injury that precluded evaluation of conjunctivitis, while the other had chronic blepharitis. four individuals did not fit any case definition, either because of missing data or because they complained of red eyes only and therefore did not meet the criteria for a diagnosis of conjunctivitis. the prominence of conjunctivitis as the presenting syndrome in these and other reported human cases of influenza h7n7 is striking and may be explained by the presence of α2,3-linked sialic acid receptors in the eye, which are preferentially recognized by ha of avian influenza viruses, including h7n7 subtypes (see section 9.2.4) (olofsson et al., 2005) . the observation that, in contrast with human influenza strains, viral loads in conjunctival specimens of the dutch h7n7-infected individuals seemed higher than in respiratory specimens, supports the notion that h7n7 virus may replicate efficiently in cells in or near the eye and not in the respiratory tract . six of the seven cases of influenza-like illnesses were mild. one patient, a previously healthy 57-year-old veterinarian, complained of fever and headache 2 days after visiting an infected farm . he subsequently developed pneumonia complicated by acute respiratory distress syndrome and multiorgan failure, of which he died 13 days after the onset of illness. autopsy revealed pathologic changes in the lungs similar to those found in influenza h5n1-infected humans and no significant abnormalities in other organs. direct human-to-human transmission of h7n7 virus during the dutch outbreak is suggested by the fact that three individuals with confirmed infections had not been in direct contact with infected poultry but were family members of poultry workers with h7n7 conjunctivitis . the h7n7 virus causing the outbreak in the netherlands most likely evolved from a low pathogenic virus from wild ducks after the introduction of this virus into the poultry population . in agreement with its classification as a highly pathogenic avian influenza virus, the ha contained multiple basic amino acids at the cleavage site. sequence comparison of virus isolates from chickens and humans, including those implicated in human-to-human transmission, revealed virtually no differences, indicating no significant accumulation of mutations on bird-to-human or human-to-human transmission. the only exception was the virus isolated from the fatal case, which showed a total of 14 amino acid substitutions not seen in the other isolates. most of these substitutions involved the ha, na, and pb2 genes, which have all been implicated as determinants of host range and pathogenicity. intriguingly, the mutations in pb2 included a glutamine to lysine change at positions 627, associated with high virulence of h5n1 viruses in mice (see section 9.3.2) . the h7n7 outbreak in poultry was effectively contained by the culling of approximately 30 million chickens, which amounts to about 28% of the total chicken population in the netherlands . after the first human infections were identified, individuals exposed to potentially infected chickens were vaccinated with the available human vaccine to prevent possible dual infection with human and avian strains and the resulting risk of reassortment. as the outbreak progressed, the recommendation for vaccination was extended to all poultry farmers in a 3-km radius of infected farms and to persons suspected of h7n7 infection. in addition, a prophylactic regimen of the neuraminidase-inhibitor oseltamivir was started for all people handling potentially infected poultry to prevent bird-to-human transmission and human-to-human transmission of avian viruses. prophylactic treatment was to be continued for 2 days after the last exposure. these control measures may serve as a model for the control of emerging influenza viruses because they, at least theoretically, minimize the possibility that the virus spreads among the human population. in early 2004, an outbreak of highly pathogenic h7n3 viruses occurred in poultry farms in british columbia, canada . the causative virus probably had evolved by homologous recombination between the ha and m genes in a low pathogenic h7n3 virus . this recombination event resulted in the introduction of a multibasic sequence at the cleavage site of ha. surveillance among potentially exposed people identified two laboratory-confirmed cases of h7n3 infection. in these cases, conjunctivitis and mild influenza-like symptoms (coryza, headache) developed 1-3 days after exposure . both were treated with oseltamivir and recovered fully. no secondary cases were identified. the outbreak among poultry was contained by extensive culling. control measures in potentially exposed people were similar to those during the dutch h7n7 outbreak. in 1999, human infections with h9n2 viruses were reported in two unrelated children from hong kong, aged 1 and 4 years (peiris et al., 1999) . both children had a mild influenza-like syndrome, associated with mild lymphopenia in one, and slightly raised transaminase levels in the other child. neither child developed pneumonia and both recovered uneventfully within 3-7 days. there was a history of probable contact with live chickens in one of the patients, but otherwise the source of transmission was unclear. no serological evidence of h9n2 infection was found in the children's family members or health care workers. three serum samples from 150 volunteer blood donors in hong kong showed the presence of neutralizing antibodies against h9n2 virus, suggesting that additional infections had occurred in hong kong (peiris et al., 1999) . around the same time as the infections in hong kong, five additional, similarly mild cases of human h9n2 in humans were reported from mainland china . the human infections in hong kong and mainland china were caused by non-highly pathogenic h9n2 viruses of two disinct lineages. the hong kong virus was related to a quail h9n2 virus (a/quail/hk/ g1/97 [h9n2]) and possessed internal genes similar to the h5n1 virus that caused the outbreak in poultry and humans in 1997 (guan et al., 1999 lin et al., 2000) . this may suggest that the quail h9n2 virus has been the donor of all internal genes to the h5n1 outbreak strain (guan et al., 1999) . the strains isolated from humans in mainland china were related to a different lineage of h9n2 viruses found in ducks and chickens (a/duck/hk/y280/97 [h9n2] and a/chicken/hk/g9/97 [h9n2]) (guo et al., 2000) . most h9n2 strains isolated since 1999 seem to be related antigenically to the latter virus but posess a variety of internal gene constellations, including those of h5n1/97-like origin (choi et al., 2004; lipatov et al., 2004) . interestingly and perhaps concerning, h9n2 viruses isolated from poultry have acquired a preference for binding to the human-like α2,6 sialic acid-galactose linkages, which may suggest that certain species of poultry could act as an intermediate host in the zoonotic transmission of influenza viruses from their natural reservoir in acquatic birds to mammals, including humans (matrosovich et al., 2001) . indeed, h9n2 viruses have also been isolated from pigs in southeastern china, indicating widening of the host range (peiris et al., 2001) . in addition, contemporary human h3n2 strains are cocirculating in southeastern chinese pigs, providing ideal circumstances for potential genetic reassortment leading to the emergence of viruses with pandemic potential (peiris et al., 2001 ). in recent years it has become clear that, in contrast with the usually mild illnesses caused by h7 and h9n2 viruses, human infections with highly pathogenic influenza h5n1 viruses are associated with severe, often fatal disease. in may 1997, after outbreaks of influenza h5n1 among poultry on three farms in the new territories of hong kong, an influenza h5n1 virus was isolated from a 3-year-old boy in hong kong, who died of severe pneumonia complicated by acute respiratory distress syndrome and reye syndrome (subbarao et al., 1998) . in november and december of the same year, concomittant with outbreaks of influenza h5n1 among chickens in poultry markets and on farms in hong kong, 17 additional cases of human h5n1 infections were identified, five of which were fatal (yuen et al., 1998; chan 2002) . the outbreak was contained after the slaughtering of all 1.5 million chickens in hong kong. in response to the outbreak, influenza surveillance in poultry was intensified permitting early recognition of other outbreaks of avian influenza in 2001 and 2002. no further human h5n1 infections were reported until february 2003, when two laboratory-confirmed cases and one probable case were identified in one family from hong kong . the daughter died of an undiagnosed respiratory infection while visiting fujian province in mainland china. upon their return to hong kong, the father and son developed severe respiratory illnesses of which the father died. h5n1 virus was isolated from both patients. in december 2003, an outbreak of highly pathogenic h5n1 virus was identified among poultry in the republic of korea (lee et al., 2005) . subsequently, outbreaks by antigenically related viruses were reported among poultry in thailand, viet nam, japan, china, cambodia, laos, malaysia, and indonesia. the reason for this apparent simultaneous occurrence of h5n1 outbreaks in many asian countries remains unclear. however, h5n1 viruses have also been found in dead migratory birds, which may suggest a role of wild birds in the spread of h5n1 viruses in the region .since 2005, migratory birds indeed have also been responsible of spreading the virus to regions outside asia, including several countries in the middle east, europe and africa. human infections during the southeast asian outbreaks were first reported in early 2004 from viet nam and thailand, (hien et al., 2004a; chotpitayasunondh et al., 2005) . since then, concurrent with the spread of the virus by migrating birds and consequent poultry outbreaks elsewhere, human h5n1 infections have been reported in several other countries in asia (china, cambodia, indonesia), eurasia and europe (azerbaijan, iraq, turkey), and africa (egypt, djibouti). at the time of this writing (may 2006) more than 200 human infections have been reported worldwide of which more than half were fatal (who, 2005) . it cannot be excluded and may even be likely that additional cases have gone unnoticed in affected countries due to a lack of clinical awaress, active surveillance, or diagnostic facilities (hien et al., 2004b) . although many countries initially affected by poultry outbreaks in 2004 have been declared free of the virus, h5n1 virus seems to have reached endemic levels in poultry and aquatic birds in several asian countries, despite attempts to contain the outbreak by extensive culling of poultry. this is also suggested by the establishment of multiple geographically distinct sublineages of h5n1 influenza viruses in asia . continuing occurrences of bird-to-human transmissions increase the opportunity of the virus to adapt to humans and acquire the ability to spread between humans. in addition, continuing cocirculation of avian and human viruses in countries, where humans live in close proximity with poultry and pigs, increases the risk of reassortment between both in coinfected humans or other mammalian hosts, such as the pig. the isolation of h5n1 viruses from pigs in china and indonesia is concerning in this respect . for all these reasons, the current developments in asia and other regions in the world seem to justify the global concern that, similar to 1957 and 1968, a new pandemic influenza strain may emerge in the near future. at presentation, most cases of human h5n1 infections were characterized by a severe influenza syndrome, clinically indistinguishable from severe human influenza, with symptoms of fever, cough, and shortness of breath, and radiological evidence of pneumonia (yuen et al., 1998; hien et al., 2004a; chotpitayasunondh et al., 2005) . abnormalities on chest radiographs at presentation included extensive, usually bilateral infiltration, lobar collapse, focal consolidation, and air bronchograms (figure 9 .2). radiological evidence of pulmonary damage could still be observed in surviving patients several months after the illness (t.t. hien, personal communication). beside respiratory symptoms, a large proportion of patients also complained of gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain, which are common in children with human influenza, but not in adults. in some cases, diarrhea was the only presenting symptom, preceding other clinical manifestations . unlike human infections with h7 or h9 viruses, conjunctivitis was not prominent in h5n1infected patients. the clinical course of the illness in severe cases was characterized by rapid development of severe bilateral pneumonia necessitating ventilatory support within days after onset. complications included acute respiratory distress syndrome, renal failure, and multiorgan failure. evidence that the clinical spectrum of human h5n1 infections is not restricted to pulmonary symptoms was provided by a reported case of possible central nervous system involvement in a vietnamese boy who presented with diarrhea, followed by coma and death. influenza h5n1 virus was isolated from throat, rectal, blood, and cerebrospinal fluid specimens, suggesting widely disseminated viral replication . his sister had died of a similar illness 2 weeks earlier, but no diagnostic specimens were obtained. although highly virulent h5n1 viruses have shown neurotropism in mammals such as mice and cats (lipatov et al., 2003; tanaka et al., 2003; keawcharoen et al., 2004) , these cases may be similarly rare as central nervous system manifestations associated with human influenza (morishima et al., 2002; sugaya, 2002) . genetic predisposition of the host to such manifestations may play a role. striking routine laboratory results in h5n1-infected patients, especially in severe cases, were an early onset of lymphopenia, with a pronounced inversion of the cd4+/cd8+ ratio, thrombocytopenia, and increased levels of serum transaminases (yuen et al., 1998; hien et al., 2004a; chotpitayasunondh et al., 2005) . high levels of cytokines and chemokines have been observed in several h5n1-infected patients, suggesting a role of immune-mediated pathology in the pathogenesis of h5n1 infections (see section 9.3.2) peiris et al., 2004) . this was supported by pathological examination in two patients who died during the outbreak in hong kong, which showed reactive hemophagocytosis as the most prominent feature . other findings included diffuse alveolar damage with interstitial fibrosis, hepatic central lobular necrosis, acute renal tubular necrosis, and lymphoid depletion. although the gastrointestinal, hepatic, renal, and hematologic manifestations could suggest wider tissue tropism, there was no evidence of viral replication in organs outside the respiratory tract . although many laboratory-confirmed h5n1 infections were associated with severe, often fatal disease, milder cases have also been reported, especially during the outbreak in hong kong (yuen et al., 1998; chan 2002 ). an increasing number of milder cases also seemed to occur in viet nam, as the outbreak progressed in 2005 (who, 2005) . although increased clinical awareness and surveillance may account for such observations, progressive adaptation of the virus to humans is the dreaded alternative explanation. detailed monitoring of virus evolution during outbreaks is obviously important and may help to distinguish between both possibilities. the occurrence of mildly symptomatic and asymptomatic infections have also been suggested during the outbreak in hong kong by seroepidemiological studies in household members of h5n1infected patients and health care workers. in these studies, 8 of 217 exposed and 2 of 309 nonexposed health care workers were seropositive for h5n1-specific antibodies (bridges et al., 2002) . seroconversion was documented in two exposed nurses, one of whom reported a respiratory illness 2 days after exposure to an h5n1-infected patient. more importantly than showing the occurrence of asymptomatic infections, these data indicated that nosocomial person-to-person transmission had occurred, albeit limited to a few cases. an additional case of possible human-tohuman transmission during the hong kong outbreak was suggested by h5n1-seropositivity in a household contact of a patient, who had no history of poultry exposure . seroepidemiological studies in health care workers involved in the care of h5n1-infected patients in thailand and viet nam in 2004 have not shown evidence of person-toperson transmission, despite the absence of adequate infection control measures in the vietnamese cohort at the time of study liem and lim, 2005; schultsz et al., 2005) . during the outbreak in thailand in 2004, extensive epidemiological investigations have suggested person-to-person transmission from a child, who died of presumed h5n1 infection, to her mother who had no history of exposure to poultry and had provided prolonged unprotected nursing care to her daughter . an aunt of the child may have been infected by the same route because her last exposure to poultry before infection had been 17 days, considerably longer than the estimated incubation period of 2-10 days. there have been several similar family clusters of h5n1 cases in other affected countries, which have all ignited concerns about the possibility of human-to-human transmission, but most of which could be explained by common exposure to poultry. although there has been no evidence of efficient transmission of influenza h5n1 virus between humans to date, caution and detailed investigations obviously remain warranted in case of any cluster of infections, especially in view of the relatively rapid evolution h5n1 viruses have exhibited in recent years. 9.4.5.3. the evolution of h5n1 viruses, 1997 in 1996, an h5n1 virus was isolated from geese during an outbreak in guangdong province in china (influenza a/goose/guangdong/1/96 [a/g/gd/96]) (xu et al., 1999) . this virus proved to be the donor of the ha gene of the reassortant h5n1 viruses causing the outbreak among poultry and humans in hong kong in 1997. the internal genes of the hong kong h5n1 viruses were closely related to those of an h9n2 virus isolated from quail (see section 9.4 .3) (guan et al., 1999) . the origin of the na gene remains unclear but was notable for a 19-amino-acid deletion in the stalk region (subbarao et al., 1998) . such deletions may be associated with adaptation of influenza viruses to land-based poultry (matrosovich et al., 1999) . the ha gene contained multibasic sequences at the cleavage site, in accordance with its classification as a highly pathogenic strain (claas et al., 1998; matrosovich et al., 1999) . the role of other genes potentially involved in its pathogenicity is reviewed in section 9.3.2. after the eradication of the 1997 hong kong strain, the goose precursor viruses continued to circulate in geese in southeastern china (cauthen et al., 2000; webster et al., 2002) . through reassortment between this virus and other avian viruses, multiple antigenically similar genotypes, which were highly pathogenic in chickens but not in ducks, emerged and again were eradicated in hong kong in 2001 and 2002 . then, in late 2002, h5n1 strains isolated from wild migratory birds and resident waterfowl in two hong kong parks showed marked antigenic drift and exhibited high pathogenicity in ducks sturm-ramirez et al., 2004) . the latter property is rarely found in nature and had not been observed in strains isolated during previous years. an antigenically and molecularly similar virus caused the two confirmed human infections in early 2003 in a family from hong kong (see section 9.4.5.1) peiris et al., 2004) . h5n1 influenza viruses isolated from healthy ducks in southern china between 1999 and 2002 were all antigenically similar to the precursor influenza a/g/gd/96 virus . it is thought that these ducks played a central role in the generation of the virus responsible for the outbreaks in southeast asia since 2003. detailed genetic analyses of h5n1 strains isolated during the period 2000-2004 from poultry and humans in china, hong kong, indonesia, thailand, and viet nam demonstrated that a series of genetic reassortment events, all traceable to the a/g/gd/96-precursor virus, ultimately gave rise to a dominant h5n1 genotype (genotype z) in chickens and ducks . this genotype is implicated in the human cases in hong kong in 2003 and the outbreaks among poultry and humans since 2004. the evolution of h5n1 viruses in recent years has been associated with increasing virulence and an expanding host range, which beside terrestrial poultry and wild birds also includes mammals. although all h5n1 viruses isolated from ducks in china between 1999 and 2002 were highly pathogenic in chickens, an increasing level of pathogenicity was observed in mice with the progression of time: virus isolated in 1999 and 2000 were less pathogenic than those isolated in 2001 and 2002 . it has been suggested that the increasing ability to replicate in mammals has resulted from transmission between ducks and pigs the expanding host range is also illustrated by successful experimental infection of domestic cats and natural infections of cats, tigers and leopards with recent h5n1 strains (keawcharoen et al., 2004; kuiken et al., 2004; songserm et al., 2006) . in summary, continued evolution of h5n1 viruses since 1997, involving multiple genetic reassortment events between a/g/gd/96-like viruses and other avian viruses and perhaps transmission between birds and pigs or other mammalian hosts, has resulted in a highly virulent genotype with an expanded host range that is causing widespread outbreaks among wild birds, poultry and humans affecting several regions in the world. although transmission between birds and humans at present still seems inefficient, as does transmission between humans, this may change when the virus is allowed to continue its evolution through adaptation and reassortment. in clinical trials, human influenza has been clinically diagnosed correctly in approximately two-thirds of adults with influenza-like symptoms, despite the lack of pathognomomic features (monto et al., 2000) . although virus isolation remains the gold standard of diagnosis and indispensable for virus characterization, rapid laboratory confirmation of suspected human influenza in routine diagnostic laboratories is usually performed by immunochromatographic or immunofluorescent detection of influenza virus antigens or by reverse transcriptase (rt)-pcr detection of viral nucleic acids in respiratory specimens. in addition, serological evidence of human influenza a virus infection can be obtained by commercially available elisa kits that detect antibodies to conserved viral antigens, such as the nucleoprotein. in the absence of cocirculating avian influenza strains in the human population, further subtyping of influenza viruses or detection of subtype-specific antibodies are usually not done by routine diagnostic laboratories but are restricted to reference laboratories involved in epidemiological analyses and planning of vaccine strains. however, in case of an outbreak of avian influenza, efforts to further subtype the virus (e.g., by subtype-specific rt-pcr methods) should be made by routine laboratories because immediate knowledge about the infecting influenza subtype is essential for infection control and timely epidemiological investigations. dependence on reference laboratories, which in the case of many southeast asian countries affected by avian influenza outbreaks are situated abroad, potentially results in unacceptable delays and hampers timely recognition of outbreaks and institution of adequate control measures (hien et al., 2004b) . however, the reality is that diagnostic facilities in many affected countries are scarce and often not sufficiently equipped for virological diagnostics, let alone subtyping of influenza viruses. global efforts to improve diagnostic capacity in resource-poor countries may prove an important step toward the prevention and control of pandemic influenza (hien et al., 2004b ). similar to human influenza viruses, avian viruses can be isolated in embryonated eggs or in cell culture, using permissive cells such as madin darby canine kidney (mdck) cells or rhesus monkey kidney (llc-mk2) cells. unlike human strains and avirulent avian strains and in accordance with their promiscuity for cellular proteases, highly pathogenic avian viruses do not require the addition of exogenous trypsine for efficient replication in cell culture. for safety purposes, the isolation of highly pathogenic avian influenza virus requires biosafety level 3 laboratory facilities or higher. cytopathic effects in cell culture are nonspecific. initial identification of influenza a virus can be performed by immunofluorescence staining with monoclonal antibodies against the nucleoprotein. further ha and na subtyping is performed by subtype-specific rt-pcrs of culture supernatant or hemagglutination inhibition and neuraminidase inhibition assays using a panel of reference antisera against various subtypes. in human infections, avian influenza viruses have mostly been isolated from conjunctival swabs and respiratory specimens such as throat or nasal secretions or washings (yuen et al., 1998; fouchier et al., 2004; hien et al., 2004a) . in one case of h5n1 infection, virus was also isolated from serum, cerebrospinal fluid, and a rectal swab . detection of influenza a viral antigens in clinical specimens by direct immunofluorescence or by rapid immunochromatographic assays is widely used for diagnosis of human influenza because of their ability for rapid diagnosis. however, in patients with avian influenza, the usefulness of these assays seems limited due to low sensitivity, possibly because of lower viral loads than during human influenza (yuen et al., 1998; peiris et al., 2004) . in addition, some rapid antigen detection kits do not distnguish between influenza types a and b, and none of the currently available immunofluoresence and immunochromatographic assays distnguishes between influenza a subtypes. however, developments of h5n1-specific rapid antigen detection tests are ongoing . rt-pcr methods allow for sensitive and specific detection of viral nucleic acids and have shown to increase the diagnostic sensitivity for many viral pathogens when compared with culture or antigen detection methods. during the h5n1 outbreaks in hong kong and southeast asia, rt-pcr methods for specific detection of h5n1 viral nucleic acids proved valuable and seem to be the diagnostic methods of choice in case of an outbreak of avian influenza (yuen et al., 1998; hien et al., 2004a; chotpitayasunondh et al., 2005) . especially when using real-time pcr technology, a reliable subtype-specific diagnostic result can be generated within a few hours after specimen collection. a disadvantage of rt-pcr methods is its proneness for contamination and the consequent risk of false-positive results, which should be minimized by proper precautions, including physical separation of laboratories for pcr preparation and amplification. in addition, the inclusion of an internal control in rt-pcr assays is highly desirable to monitor for false-negative results due to inefficient nucleic acid extraction, cdna synthesis, or amplification. during outbreaks of avian influenza, the detection of subtypespecific antibodies is particularly important for epidemiological investigations. hemagglutination inhibition (hi) assays are the gold standard for detection of antibodies against human influenza viruses. however, their usefulness for detection of antibodies against avian viruses in mammalian species, including humans, seems limited (hinshaw et al., 1981; beare and webster, 1991; kida et al., 1994) . several studies have shown a failure to detect hi antibodies against avian viruses in mammals, even in cases where infection was confirmed by virus isolation. possible reasons for this failure include poor immunogenicity of some avian viruses and lack of sensitivity to detect low-titered or less avid antibodies induced by avian viruses (hinshaw et al., 1981; lu et al., 1982; kida et al., 1994; rowe et al., 1999) . it has been demonstrated that hi testing with subunit ha, but not with intact virus, could detect antibodies against an avian h2n2 virus (lu et al., 1982) . however, neutralizing antibodies against this virus could readily be detected with intact virus. a direct comparison of hi testing with a microneutralization assay in h5n1-infected persons from the 1997 hong kong outbreak indeed showed the latter to be more sensitive . although an indirect elisa assay using recombinant ha from h5n1/97 showed at least equal sensitivity as the microneutralization assay, the specificity in adult sera was inferior, most likely due to the presence of cross-reactive epitopes common to all has . based on these observations, neutralization assays are the methods of choice for detection of antibodies against avian viruses in humans. using these assays, it has been shown that the kinetics of the antibody response against h5n1 virus in patients infected during the hong kong outbreak are similar to the primary response to human influenza viruses . neutralizing antibodies were generally detected 14 or more days after the onset of symptoms, and titers equal to or higher than 1:640 were observed 20 or more days after onset. using neutralization assays, antibodies against h9n2 could be detected in a small number of blood donors from hong kong (peiris et al., 1999) . however, in two laboratory-confirmed h7n3-infected patients with conjunctivitis, no neutralizing antibodies could be detected in sera obtained more than 20 days after onset of the illness . similarly, no hi antibodies could be detected in an h7n7-infected patient with conjunctivitis . although the reason for this apparent failure to mount an antibody response remains unclear, it has been suggested that this could be secondary to the highly localized nature of the infection in these cases . currently, two classes of drugs are available with antiviral activity against influenza viruses: inhibitors of the ion channel activity of the m2 membrane protein, amantadine and rimantadine; and inhibitors of the neuraminidase, oseltamivir and zanamivir. the therapeutic efficacy of amantadine in human influenza is unclear due to a paucity of reliable clinical studies, but reductions of fever or illness by 1 day have been observed in adults and children (nicholson et al., 2003) . major disadvantages of amantadine include neurotoxicity and a rapid development of drug resistance during treatment. resistance is conferred by single nucleotide changes resulting in amino acid substitutions at positions 26, 27, 30, 31, or 34 of the m2 protein. rates of resistance against amantadine in human influenza viruses has increased from less than 0.5% in 1994-1995 to more than 12% in 2003-2004 . particularly high resistance frequencies of up to 61% were observed in viruses isolated in asia (bright et al., 2005) . rimantadine causes less neurological side effects but is not available in most parts of the world. although several h5n1-infected patients have been treated with amantadine during the 1997 h5n1 outbreak in hong kong, the numbers were too small to draw any meaningful conclusions concerning its activity against this virus (yuen et al., 1998) . in vitro sensitivity testing of virus isolated from the first patient during this outbreak showed normal susceptibility to amantadine (subbarao et al., 1998) . strikingly, the sublineage of genotype z h5n1 viruses prevalent in thailand, viet nam, cambodia and malaysia in 2004 invariably showed an amantadine-resistance conferring amino acid substitution at position 31 of the m2 protein, while this mutation was mostly not present in sublineages of h5n1 viruses isolated in other geographic regions puthavathana et al., 2005; cheung et al., 2006) . both oseltamivir and zanamivir have proven efficacy in the treatment of human influenza when started early during the course of illness and are particularly effective as seasonal or postexposure prohylaxis (nicholson et al., 2003) . zanamivir has poor oral availability and is therefore administered by inhalation, which has limited its use in the elderly and may induce bronchospasm. oseltamivir can be given orally. the development of drug resistance during treatment has been reported for both drugs and is associated with mutations in the active site of neuraminidase or in the hemagglutinin. the latter mutations decrease the affinity of ha for the cellular receptor, thereby obviating the need for neuraminidase to escape the cells. data on the efficacy of neuraminidase inhibitors in avian influenza virus infections are scarce. the h5n1 strains implicated in the 1997 hong kong outbreak were susceptible in vitro to oseltamivir and zanamivir (leneva et al., 2000; govorkova et al., 2001) . oral oseltamivir and topical zanamivir also showed therapeutic and protective activities against hong kong h5n1 isolates in murine animal models (gubareva et al., 1998; leneva et al., 2001) . recent murine studies suggest that, perhaps due to higher virulence, higher doses of oseltamivir and longer durations of treatment are necessary to achieve antiviral effects in mice against h5n1 strains causing the southeast asian outbreak since 2004, when compared with the 1997 hong kong h5n1 strain (yen et al., 2005) . in vitro sensitivity testing of h7n7 isolates during the 2003 outbreak of this virus in the netherlands showed normal susceptibility to zanamivir and oseltamivir . h7n7 infection was detected in 1 of 90 persons who reportedly received prophylactic treatment with oseltamivir during that outbreak, compared with 5 of 52 persons who had not taken oseltamivir prophylaxis . oseltamivir treatment has been given to several patients infected with avian influenza viruses, including h7n7, h7n3, and h5n1 subtypes, but no conclusions can be made concerning its efficacy. however, the timing of antiviral treatment may not have been optimal in many human cases of avian influenza so far. beneficial effects of antiviral treatment in human influenza are optimal when started within 48 h after onset of the illness. during the h5n1 outbreak in viet nam in 2004, h5n1infected patients were admitted 5 days or later after onset of symptoms (hien et al., 2004a) . earlier recognition of avian influenza in humans may improve the efficacy of antiviral treatment. nevertheless, favourable virological responses associated with a beneficial clinical outcome have been reported in h5n1-injected patients despite late initiation of treatment . the emergence of drug-resistant h5n1 variants during prophylaxis or treatment with oseltamivir has also been reported, and may be associated with clinical failure of treatment le et al., 2005) . treatment strategies which minimize the risk of resistance development, such as antiviral combination treatment, deserve attention. in addition, parenteral formulations of antiviral drugs may be desirable to guarantee systemic drug levels in h5n1 patients with severe disease. a novel intravenously administered neuraminidase inhibitor, peramivir, is currently in clinical development. although several h5n1-infected patients have received steroids in addition to oseltamivir, the potential benefits of this need formal evaluation in clinical studies (hien et al., 2004a) . considering the observed cytokine dysregulation in h5n1-infected animals and humans, a beneficial effect of immunomodulating agents could be hypothesized and perhaps requires further study. finally, neutralizing monoclonal antibodies have been shown effective in treating established influenza a virus infection in mice with severe combined immunodeficiency (palladino et al., 1995) . although mice are not men, this strategy deserves attention in the treatment of a severe illness such as influenza h5n1. birds infected with avian influenza excrete large amounts of virus in feces and other secretions, which contaminate the direct environment, such as dust, soil, water, cages, tools, and other fomites. avian influenza virus may remain infectious in soil, water, or contaminated equipment for weeks to months, depending on the temperature and humidity (i.e., longer in colder climates). illness in birds caused by highly pathogenic avian influenza viruses results in systemic replication and the presence of infectious virus in their eggs and many tissues and organs. transmission of avian influenza viruses between birds occurs directly or indirectly through contact with fecally contaminated aerosols, water, feed, and other materials. bird-to-human transmission likely occurs via the same route (i.e., direct contact with birds or contaminated fomites). most, but not all human infections with avian influenza viruses involved handling of affected poultry or direct exposure to live poultry in the week before onset of the illness (mounts et al., 1999; hien et al., 2004a; koopmans et al., 2004) . case-control studies during the 1997 h5n1 outbreak in hong kong identified visiting a stall or market selling live poultry during the week before the illness as a risk factor, whereas eating or preparing poultry products were not risk factors (mounts et al., 1999) . in cases in which no apparent direct exposure to poultry could be identified, contact with contaminated environment, such as water, has been suggested . of note, it has been shown that ducks infected by the currently circulating h5n1 strain in southeast asia remain healthy but excrete large amounts of virus for prolonged periods of time (hulse-post et al., 2005) . because water in ponds and canals in which large flocks of ducks reside is widely used for bathing and drinking in rural areas of many southeast asian countries, it may not be unlikely that such water represents a source of transmission when contaminated by infected ducks. in fact, contact with contaminated water is regarded as the most important mode of transmission between aquatic birds. a limited number of possible human-to-human transmissions have been reported, which involved prolonged, close, and unprotected contact with infected patients koopmans et al., 2004; ungchusak et al., 2005) . similar to human influenza, droplet and contact transmission are probably the most effective means of transmission of avian influenza virus between humans, should the the virus acquire the ability for efficient spread, but airborne transmission remains a possibility. the occurrence of diarrhea in h5n1-infected patients, which may contain infectious virus, represents a potential nonrespiratory route of transmission that needs to be considered in infection control practices hien et al., 2004a; . data concerning excretion patterns and periods of potential infectivity are lacking for human infections with avian influenza viruses. based on exposure histories, the incubation time for human h5n1-infections has been estimated at 2-10 days, but it is not known whether excretion of virus occurs during this time (yuen et al., 1998; hien et al., 2004a) . based on the current (lack of) knowledge, infection control measures during contact with potentially infected birds or environment or with patients with suspected or confirmed infection should prevent contact, droplet, and airborne transmission. these measures include mask (preferably high-efficiency masks, with surgical masks as a second alternative), gown, face shield or goggles, and gloves. the efficacy of neuraminidase inhibitors as seasonal or postexposure prohylaxis against human influenza is high (nicholson et al., 2003) . offering prophylactic treatment to potentially exposed people in the setting of a poultry outbreak of avian influenza, as has been done during h7outbreaks in the netherlands and canada tweed et al., 2004) , is rational but hardly feasible during the ongoing outbreak in asia and africa for logistical and financial reasons. postexposure prophylaxis to unprotected health care workers and close contacts of infected patients needs serious consideration. the potential use of specific monoclonal antibodies for prophylaxis warrants further investigation. eliminating the source of infection (i.e., infected birds) remains the most effective infection control measure. culling of all infected poultry has proved succesful during avian influenza outbreaks in hong kong, the netherlands, and canada (chan, 2002; koopmans et al., 2004; tweed et al., 2004) . however, considering the geographic extensiveness of the outbreak, the different farming practices in affected regions, and the occurence of infection in migratory birds, it is doubtful whether culling of poultry will be able to contain the outbreaks in the various regions. the bulk of human influenza vaccines are produced from inactivated viruses grown in embryonated eggs. vaccine production against highly pathogenic avian influenza viruses is complicated because of the requirement for high biosafety containment facilities and the difficulty, in some cases, to obtain high virus yields in embryonated eggs because of the virus' pathogenicity (stephenson et al., 2004; wood and robertson, 2004) . several other approaches have been used in an attempt to overcome these obstacles, including the use of reverse genetics techniques, generation of recombinant hemagglutinin, dna vaccination, and the use of related apathogenic h5 viruses with and without different adjuvants (nicholson et al., 2003; stephenson et al., 2004; webby et al., 2004; wood and robertson, 2004) . experimental h5n1 vaccines in which important virulence determinants were altered using plasmid-based reverse genetics have shown protective efficacy to homologous and heterologous h5 strains in animal models and may prove an attractive approach takada et al., 1999; lipatov et al., 2005b) . studies in humans using an h5n3 vaccine developed from a 1997 apathogenic avian virus showed high rates of seroconversions to the vaccine strain and heterologous h5n1 strains after 3 doses, but only when the vaccine was given with the adjuvant mf59 . in animal models, baculovirus-derived recombinant h5 vaccines were immunogenic and protective, but results in humans were disappointing even when using high doses (crawford et al., 1999; treanor et al., 2001) . in a study using a subvirion influenza h5n1 vaccine, neutralizing antibody responses were observed in approximately half of the subjects receiving the highest dose of the vaccine (two intramuscular injections of 90 micrograms) (treanor et al., 2006) . h5 dna vaccines protected mice from infection by homologous, but not by heterologous h5n1 viruses (kodihalli et al., 1999; epstein et al., 2002) . the increasing frequency of outbreaks with highly pathogenic avian influenza viruses among poultry and wild birds, and direct transmission of these viruses to humans, has ignited grave concerns about an imminent influenza pandemic. indeed, two of three prerequisites for a human pandemic have been met in the h5n1 outbreaks since 1997: the emergence of an antigenically novel strain to which the population has no immunity, and the transmission of this strain to humans in whom it can cause severe disease. to date, there fortunately is no evidence of efficient spread of h5n1 virus between humans, but continued circulation of this strain, which now has reached levels of endemicity among poultry in several asian countries, increases the opportunity to adapt to humans through mutation or genetic reassortment in humans or intermediate mammalian hosts. as suggested by the "spanish flu" pandemic of 1918, extremely high transmissibility is no prerequisite for a severe pandemic killing tens of millions of people, and as shown by the severe acute respiratory syndrome (sars) virus epidemic in 2003, viruses can rapidly spread across the globe in the current age of intense global travel. as a consequence of all this, pandemic preparedness has become an increasingly important issue, and pandemic plans are being developed by an increasing number of countries worldwide. control measures based on case identification (e.g., contact tracing and quarantine) were essential for the control of sars. however, during an influenza epidemic, such measures may not be as effective because of short incubation periods and the potential infectivity before the onset of case-defining symptoms. much of the preparedness therefore will rely on clinical management and vaccination. mathematical modelling studies have suggested the possibility of containing an influenza pandemic at the source by antiviral prophylaxis and other preventive measures (ferguson et al., 205; longini et al., 2005) . many developed countries are now stockpiling antiviral drugs for initial management of illness or prophylaxis during the first months of a pandemic, when vaccines are in development and not yet available. in case of an influenza pandemic, there will be limitations on the timeliness and availability of vaccines (stohr and esveld, 2004) . it has been estimated that it could take at least 6 months for the first vaccine doses to be produced after identifying a pandemic strain. currently, global production capacity for influenza vaccines is insufficient for worldwide coverage in case of a pandemic, especially because vaccination for the novel influenza strain likely requires two doses, and interruption of annual production of the human influenza vaccine is undesirable (schwartz and gellin, 2005) . in response to the pandemic threat by the h5n1 outbreak in southeast asia, plans have been made to stockpile candidate h5n1 vaccines based on the currently circulating strain. however, there is no certainty whether the next pandemic will indeed be caused by h5n1 virus, and if so, whether antigenic drift will not have rendered the stockpiled vaccine less effective by the time pandemic spread occurs. in the latter event, such vaccines may still mitigate the illness, which is beneficial for vaccinated people but may also carry a risk of prolonged excretion and increased spread of the virus. similar worries exist when poultry would be vaccinated with a suboptimal vaccines. in case of an influenza pandemic, all possibilities for rapid production of vaccines, as well as potential methods to reduce doses without affecting immunogenicity should be considered. this would require the use of alternative, currently not officially approved methods for vaccine production, such as reverse genetics techniques and cell culture-based vaccine production, and the use of alternative adjuvants that may enable dose reduction (webby and webster, 2003; stephenson et al., 2004; wood and robertson, 2004; schwartz and gellin, 2005) . in addition, vaccine doses may be spared by alternative administration routes. it has been shown that intradermal, instead of intramuscular vaccination for human influenza may require less antigen by recruiting efficient antigen-presenting cells present in the dermis (belshe et al., 2004; kenney et al., 2004) . notwithstanding the importance of current efforts to prepare for a possible h5n1 pandemic, more structural and longer term global efforts are needed to allow for early recognition of emerging novel influenza viruses in the future. in 2002, a who global agenda for influenza surveillance and control has been adopted, of which the main objectives are to strengthen surveillance, improve knowledge of the disease burden, increase vaccine use, and accelerate pandemic preparedness (stohr, 2003) . it is essential that these objectives are increasingly focused on the southeast asian region, which has been the source of previous pandemics and is the epicenter of the current pandemic threat. however, many southeast asian countries currently lack the expertise, financial means, and infrastructure for human and animal surveillance. global investments to improve public health care infrastructures and laboratory facilities and to transfer clinical, epidemiological, and technical knowledge to these countries are much needed (hien et al., 2004b) . the window of opportunity in the era of global travel is narrow. local capacity, and less dependence on foreign laboratories and expertise, will allow for earlier recognition and quicker responses to epidemics. in addition, local availability of clinical, scientific, and laboratory capacity facilitates and expedites clinical, virological, and epidemiological analyses needed to optimize outbreak control, infection control, and clinical managment and guarantees the timely availability of virus strains for monitoring virus evolution and planning of vaccines by reference laboratories. in response to the 1997 h5n1 outbreak in hong kong, influenza surveillance in poultry was intensified, which permitted early recognition of outbreaks by other avian influenza strains and timely interventions and has helped to keep hong kong free of h5n1 influenza despite the outbreak of this virus in many other countries in the region. the hong kong response may serve as a model, but wider implementation of this approach will require global efforts. a single gene determines the host range of influenza virus atypical avian influenza (h5n1) seroprevalence of anti-h5 antibody among thai health care workers after exposure to avian influenza (h5n1) in a tertiary care center antigenic mapping of an avian h1 influenza virus haemagglutinin and interrelationships of h1 viruses 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containing an emerging influenza pandemic in southeast asia avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome characterization of a novel influenza a virus hemagglutinin subtype (h16) obtained from black-headed gulls the viral polymerase mediates adaptation of an avian influenza virus to a mammalian host specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: nonegg-adapted human h1 and h3 influenza a and influenza b viruses share a common high binding affinity for 6′-sialyl(n-acetyllactosamine) the structure and receptor binding properties of the 1918 influenza hemagglutinin inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses mechanisms of inhibition of the host interferon alpha/beta-mediated antiviral responses by viruses understanding influenza virus pathogenicity cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza comparison of efficacies of rwj-270201, zanamivir, and oseltamivir against h5n1, h9n2, and other avian influenza viruses molecular characterization of h9n2 influenza viruses: were they the donors of the "internal" genes of h5n1 viruses in hong kong? h9n2 influenza viruses possessing h5n1-like internal genomes continue to circulate in poultry in southeastern china emergence of multiple genotypes of h5n1 avian influenza viruses in hong kong sar h5n1 influenza: a protean pandemic threat characterization of influenza a/hongkong/156/97 (h5n1) virus in a mouse model and protective effect of zanamivir on h5n1 infection in mice characterization of the pathogenicity of members of the newly established h9n2 influenza virus lineages in asia molecular basis for high virulence of hong kong h5n1 influenza a viruses avian influenza a (h5n1) in 10 patients in vietnam avian influenza-challenge to global health care structures replication of avian influenza a viruses in mammals altered tissue tropism of human-avian reassortant influenza viruses novel avian influenza h7n3 strain outbreak reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza a virus role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia molecular basis for the generation in pigs of influenza a viruses with pandemic potential antibody response in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts molecular mechanism of acquisition of virulence in influenza virus in nature interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics avian influenza h5n1 in tigers and leopards dose sparing with intradermal injection of influenza vaccine antigenic and genetic conservation of h3 influenza virus in wild ducks potential for transmission of avian influenza viruses to pigs host cell proteases controlling virus pathogenicity enhanced virulence of influenza a viruses with the haemagglutinin of the 1918 pandemic virus dna vaccine encoding hemagglutinin provides protective immunity against h5n1 influenza virus infection in mice transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands intracellular warfare between human influenza viruses and human cells: the roles of the viral ns1 protein avian h5n1 influenza in cats avian influenza virus isolated from a woman with conjunctivitis avian flu: isolation of drug-resistant h5n1 virus characterization of highly pathogenic h5n1 avian influenza a viruses isolated from south korea the neuraminidase inhibitor gs4104 (oseltamivir phosphate) is efficacious against a/hong kong/156/97 (h5n1) and a/hong kong/1074/99 (h9n2) influenza viruses efficacy of zanamivir against avian influenza a viruses that possess genes encoding h5n1 internal proteins and are pathogenic in mammals genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia recombinant influenza a virus vaccines for the pathogenic human a/hong kong/97 (h5n1) viruses lack of h5n1 avian influenza transmission to hospital employees avian-to-human transmission of h9n2 subtype influenza a viruses: relationship between h9n2 and h5n1 human isolates neurovirulence in mice of h5n1 influenza virus genotypes isolated from hong kong poultry in 2001 influenza: emergence and control pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice: the role of cytokines and b-and t-cell responses efficacy of h5 influenza vaccines produced by reverse genetics in a lethal mouse model genetic conservation of hemagglutinin gene of h9 influenza virus in chicken population in mainland china containing pandemic influenza at the source failure to detect hemagglutination-inhibiting antibodies with intact avian influenza virions avian influenza a viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the ha receptor-binding site the surface glycoproteins of h5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties h9n2 influenza a viruses from poultry in asia have human virus-like receptor specificity human and avian influenza viruses target different cell types in cultures of human airway epithelium transmissibility of 1918 pandemic influenza clinical signs and symptoms predicting influenza infection encephalitis and encephalopathy associated with an influenza epidemic in japan case-control study of risk factors for avian influenza a (h5n1) disease, hong kong mutations in the hemagglutinin receptorbinding site can change the biological properties of an influenza virus recent human influenza a (h1n1) viruses are closely related genetically to strains isolated in 1950 human influenza virus hemagglutinin with high sensitivity to proteolytic activation avian influenza and sialic acid receptors: more than meets the eye? nonhomologous recombination between the hemagglutinin gene and the nucleoprotein gene of an influenza virus influenza a pandemics of the 20th century with special reference to 1918: virology, pathology and epidemiology virus-neutralizing antibodies of immunoglobulin g (igg) but not of igm or iga isotypes can cure influenza virus pneumonia in scid mice functional anatomy of human lacrimal duct epithelium human infection with influenza h9n2 cocirculation of avian h9n2 and contemporary "human" h3n2 influenza a viruses in pigs in southeastern china: potential for genetic reassortment? re-emergence of fatal human influenza a subtype h5n1 disease molecular characterization of the complete genome of human influenza h5n1 virus isolates from thailand origin and evolution of the 1918 "spanish" influenza virus hemagglutinin gene evidence of an absence: the genetic origins of the 1918 pandemic influenza virus pathogenesis of influenza a (h5n1) virus infection in a primate model receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h3 hemagglutinin based on species of origin single amino acid substitutions in influenza haemagglutinin change receptor binding specificity receptor binding properties of human and animal h1 influenza virus isolates detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays the polymerase complex genes contribute to the high virulence of the human h5ni influenza virus isolate a/vietnam/1203/04 host range recombinants of fowl plague (influenza a) virus genetic relatedness between the new 1977 epidemic strains (h1n1) of influenza and human influenza strains isolated between 1947 and 1957 (h1n1) on the origin of the human influenza virus subtypes h2n2 and h3n2 avian influenza h5n1 and health care workers vaccination strategies for an influenza pandemic survey of the hemagglutinin (ha) cleavage site sequence of h5 and h7 avian influenza viruses: amino acid sequence at the ha cleavage site as a marker of pathogenicity potential tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells lethal h5n1 influenza viruses escape host antiviral cytokine responses the ns1 gene of h5n1 influenza viruses circumvents the host anti-viral cytokine responses avian flu: influenza virus receptors in the human airway pb2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of hong kong h5n1 influenza a viruses in mice characterization of avian h5n1 influenza viruses from poultry in hong kong the avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza a/pintail/79 reassortant viruses for monkeys avian influenza h5n1 in naturally infected domestic cat role of hemagglutinin cleavage for the pathogenicity of influenza virus confronting the avian influenza threat: vaccine development for a potential pandemic cross-reactivity to highly pathogenic avian influenza h5n1 viruses after vaccination with nonadjuvanted and mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a potential priming strategy structure of the uncleaved human h1 hemagglutinin from the extinct 1918 influenza virus the global agenda on influenza surveillance and control public health. will vaccines be available for the next influenza pandemic? reemerging h5n1 influenza viruses in hong kong in 2002 are highly pathogenic to ducks recombination resulting in virulence shift in avian influenza outbreak a single amino acid in the pb2 gene of influenza a virus is a determinant of host range characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness influenza-associated encephalopathy in japan single-amino-acid substitution in an antigenic site of influenza virus hemagglutinin can alter the specificity of binding to cell membrane-associated gangliosides avirulent avian influenza virus as a vaccine strain against a potential human pandemic neurotropism of the 1997 hong kong h5n1 influenza virus in mice initial genetic characterization of the 1918 characterization of the 1918 influenza virus polymerase genes a case report of fowl plague keratoconjunctivitis pathology of fatal human infection associated with avian influenza a h5n1 virus safety and immunogenicity of an inactivated subvirion influenza a (h5n1) vaccine safety and immunogenicity of a recombinant hemagglutinin vaccine for h5 influenza in humans human illness from avian influenza h7n3, british columbia probable person-to-person transmission of avian influenza a (h5n1) h5n1 virus attachment to lower respiratory tract importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza a virus are we ready for pandemic influenza? responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines intestinal influenza: replication and characterization of influenza viruses in ducks conjunctivitis in human beings caused by influenza a virus of seals molecular mechanisms of variation in influenza viruses influenza virus a pathogenicity: the pivotal role of hemagglutinin evolution and ecology of influenza a viruses virology. a molecular whodunit characterization of h5n1 influenza viruses that continue to circulate in geese in southeastern structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution from lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza genetic characterization of the pathogenic influenza a/goose/guangdong/1/96 (h5n1) virus: similarity of its hemagglutinin gene to those of h5n1 viruses from the 1997 outbreaks in hong kong latex agglutination test for monitoring antibodies to avian influenza virus subtype h5n1 virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/1203/04 (h5n1) influenza virus in mice clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus pathogenesis of avian influenza a (h5n1) viruses in ferrets key: cord-016782-aods92rf authors: lessenger, james e. title: diseases from animals, poultry, and fish date: 2006 journal: agricultural medicine doi: 10.1007/0-387-30105-4_27 sha: doc_id: 16782 cord_uid: aods92rf nan a key problem is the lack of foot protection so that the unprotected feet of workers come in contact with feces of the animals. the fecal-hand route of transmission is also critical. perhaps the most insidious and difficult to control is the consumption of raw poultry and meat products by workers in farms and processing plants. many people in agriculture are living on subsistence or below-subsistence wages and consume products off the processing lines. many of these products are not fully processed and may contact pathogens that have not been killed through cooking or irradiation (see chapter 2) (table 27 .1) (3, 4) . the improper handling of manure is a major source of disease, including the use of manure on food crops, the discharge of manure into community water sources, and the spread of manure onto areas where children play. in canada, an outbreak of escherichia coli o157:h7 was traced to organic growers who contaminated their produce with cow manure containing e. coli. also in canada, an outbreak of citrobacter freundii infections was associated with parsley originating from an organic garden in which pig manure was used. other documented infections of humans from manure-contaminated foods includes listeria monocytogenes in cabbage contaminated by sheep waste, cryptosporidium spread by municipal water contaminated by cattle, salmonella hartford in food prepared by contaminated water from a shallow well polluted with poultry manure, and pleisomonas shigelloides infection associated with well-water contaminated by poultry manure (5) . 368 j.e. lessenger guan and holley (5) , and weber and rutala (6) . workers, visitors, inspectors, veterinarians, and people who live on or adjacent to farms, ranches, feedlots, processing plants, and other agricultural endeavors are at risk for contracting diseases from animals, poultry, or fish. one needs only to follow the animals from the farm to the feedlots, slaughter house, processing and sorting lines, and packaging plants to appreciate the large number of people who are at risk due to contact with animals and animal products. physicians and other health care professionals are also at risk as they visit farms and plants for inspections or orientations (6) . methods of preventing the transmission of infectious material from animals and poultry to agricultural workers mirror in many ways the safety techniques for protection from chemicals, trauma and other hazards (see chapter 6) . the methods are summarized in table 27 .2. key to the prevention of the transmission of animal disease to humans is the proper processing of food products. this includes proper cook times and temperatures, adequate refrigeration, and appropriate transportation, processing, and stocking in stores. personal protective equipment includes hats or head coverings and protective coats or uniforms that can be laundered and left at the plant or farm. boots should also be cleaned and left at the farm or plant. especially in poultry operations, protective particulate masks may be necessary. in some 27 . diseases from animals, poultry, and fish 369 protective physical barriers in farm, ranch, or plant design allow for the raising or processing of food products without actual contact of humans with the animals or products. built-in barriers, changing rooms, boot baths, and hand-free handling techniques allow for the safe and efficient handling of food. in british chicken hatcheries, an aggressive combination of egg sanitization and handling methods was successful in decreasing zoonotic infections and diseases spread through flocks. procedures included: 1. design changes in incubators 2. whole building ventilation systems 3. control of dust, fluff, and aerosol production 4. disinfection of surfaces and equipment 5. improved handling of wastes (7). policies and procedures to limit or prevent physical contact with animals, feces, or urine prevent transmission. rules prohibiting the consumption of food products on farms and ranches or on production lines are especially important. not only can the production food product be infectious to workers, but food brought in by workers can become contaminated, which mandates eating areas for workers away from the livestock (7) . aggressive veterinary monitoring of livestock can detect early evidence of disease outbreaks in herds. similarly, public health monitoring of disease in humans can detect and appropriately treat epidemics of food-borne disease in humans and trace the source to the food-processing breakdown that caused the disease. hazard analysis of critical control points (haccp) is crucial to the prevention of infections in herds. low cost, ease of performance, and rapidity of results are the key criteria for the tests, and are sometimes more important than the performance characteristics of sensitivity, specificity, and reproducibility. field test kits are available for bacterial, protozoa, antibiotic residue, and other parameters of animal health (8, 9, 10) . medical monitoring can detect early disease and prevent its spread to other employees, the food product, and family members. pre-placement medical monitoring can identify people who are susceptible to infection, for example people with diabetes or immune diseases. in parts of the world where bovine tuberculosis is common, tb skin test monitoring can detect early infections and allow early treatment (8, 9) . immunizations are expensive, unavailable in many parts of the world, and only recommended for areas of high infectivity or occupations of high risk such as veterinarians. three critical immunizations are tetanus, rabies, and influenza (see chapter 25) . vaccines against salmonella, shigella, and other pathogens are in development or testing. training and education in proper handling techniques are important. proper ways of herding, handling, and caring for animals and poultry can prevent infection and the transmission of infectious material. see chapter 5 for details of education and training. research and the development of new techniques to prevent transmission are critical. for example, airborne dust has been discovered to be a carrier of pathogens in broiler breeder pullets (chicken pens). the use of an electrostatic space charge system has decreased the particle concentration and, in the process, decreased the potential of disease transmission to other chickens and to poultry workers (11) . hygiene, both in the person and in the workplace, is essential in preventing the transmission of disease. for example, in many german piggeries workers must shower and change clothing when they enter and leave the buildings. this technique prevents the infection of the pigs with outside pathogens, the transfer of pathogens from one piggery to another, and the transfer of pathogens to the home environment. especially important are the cleaning of machinery and the timely cleaning of animal and poultry urine and feces. not only can urine and feces be infectious but they can attract insects that can spread pathogens. as in medicine, the most important hygiene procedure is aggressive hand washing for all persons handling food products. in louisiana, for example, alligator farmers must wear rubber boots and waders to protect themselves from pathogens (but not from bites, which can go right through the protective ensembles). each day, the pens must be flushed and hosed off to remove the wastes that could harbor pathogens dangerous to the alligator colonies and workers. governmental regulations and oversight are important in providing standardization and systemization of methods and procedures to reduce the risk of infection to agricultural workers. good regulations and oversight are evidence-based and consistent with sound agricultural methods (see chapter 4). it is not enough to have rules, regulations, equipment and techniques to prevent the spread of pathogens from animals and poultry to workers. fair and consistent supervision by knowledgeable managers is critical to see that the proper equipment and supplies are used and that handling and hygiene rules and regulations are carried out. game are mammals killed or captured in the field for human consumption or for their hides, including elk, boars, bison, and deer. production animals include cattle, pigs, goats, sheep, horses, dogs, deer, and other animals grown in small to large farms and ranches for human consumption. typically the animals are slaughtered and dressed in various cuts made from the different parts of the animal. in addition, many animals are raised and kept as pets. rabies is a common viral infection in children who live in rural areas and in people who handle un-immunized mammalian animals. the prophylaxis for rabies is discussed in chapter 31. with the exception of four cases where the disease was treated with intensive therapy, the disease is considered universally fatal. therefore, immunizations and prophylaxis are critical. during may and june 2003, the first cluster of human monkeypox cases in the united states was reported. most patients with this febrile, vesicular rash illness presumably acquired the infection from prairie dogs. monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with lesser amounts in adjacent macrophages, fibroblasts, and connective tissues. viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus among rodents and to humans. prairie dogs can be studied for insights into transmission, pathogenesis, and vaccine and treatment trials, because they are susceptible to severe monkeypox infection (12) . chronic wasting disease (cwd) in north american deer and elk has been associated with creutzfeldt-jakob disease (cjd) in 3 hunters who killed, prepared, and ate their own game. an absolute association was not established, but further monitoring is ongoing (see chapter 29) . creutzfeldt-jakob disease does not appear to be a problem with workers who raise cattle or dairy cows (13) . champylobacter chlamydophila abortus is a well recognized pathogen causing abortions in cattle and goats. a recent report from germany cites a case where a pregnant woman became infected from farm animals and aborted. this rare zoonotic infection underlines the insidious and widespread problem of zoonotic infections on farms (14, 15) . campylobacter jejuni and c. coli have recently become recognized as common bacterial causes of diarrhea. infection can occur at any age. sources of infection are typically mammalian and avian hosts. the usual incubation period of campylobacter enteritis is 2 to 5 days. fever, diarrhea and abdominal pain are the most common clinical features. the stools frequently contain mucus and, a few days after the onset of symptoms, frank blood. significant vomiting and dehydration are uncommon. a rapid presumptive laboratory diagnosis may be made during the acute phase of the illness by direct phase-contrast microscopy of stools. isolation of the organism from stools requires culture in a selective medium containing antibiotics and incubation under reduced oxygen tension at 42˚c. the organism persists in the stools of untreated patients for up to 7 weeks following the onset of symptoms. erythromycin may produce a rapid clinical and bacteriologic cure and should be used to treat moderately to severely ill patients as well as patients with compromised host defenses (14) . salmonellosis is one of the most important public health disease problems, affecting more people and animals than any other single disease in agriculture. in canada, for example, there were 7,138 cases of food-borne salmonellosis in humans during 2003. the native habitat of members of the genus salmonella is the intestinal tract of warm-blooded and many coldblooded vertebrates. in humans, the incubation period is 6 to 48 hours and produces headache, malaise, nausea, fever, vomiting, abdominal pain, and diarrhea (with and without blood). salmonella is also capable of invading the intestinal mucosa, entering the blood stream, and causing septicemia, shock, and death. the diagnosis is made through the clinical presentation and confirmation with blood and stool cultures and serology. treatment is first started empirically pending culture results and then adjusted if necessary. multi-drug resistant s. typhimurium bacteria have been documented to be present in milk after pasteurization (16, 17) . listeria monocytogenes is a zoonotic food-born pathogen that is responsible for 28% of food-related deaths in the united states annually and that is a major cause of food recalls worldwide. agricultural exposure is through drinking unpasteurized milk or direct contact with the animal or manure. the disease pattern is similar to salmonella (18). tuberculosis (tb) continues to be a worldwide infectious problem for humans. while human-to-human infection is of greatest concern, one infected dairy herd can infect hundreds, if not thousands, of people. potentially, tuberculosis can infect any mammal, although production cattle, especially dairy cattle, are at greatest risk. complicating efforts to combat the disease is the fact that deer, badgers, elk and other wild species have been found to harbor the mycobacterium. in england, badgers were found to be spreading the infection to herds of cattle. also, in england and ireland, herds of sheep were found to be infected. in new zealand, wild brush tail possums (trichosurus vulpecula) were discovered to be the main source of infection in livestock, including deer herds. in tanzania, tuberculosis-infected herds were found more often in small, pastoral farms that have little veterinary monitoring, as opposed to the large, commercial enterprises (19) (20) (21) (22) . in a los angeles zoo, tb was found in two asian elephants, three rocky mountain goats, and one black rhinoceros. an investigation found no active cases of tuberculosis in humans; however, tuberculin skin-test conversions in humans were associated with training the elephants and attending an elephant necropsy (23) . human-to-animal transmission of tb has been documented. in an exotic animal farm in illinois, three elephants died of mycobacterium tuberculosis and a fourth tested culture-positive. twenty-two handlers were screened for tb; eleven had positive reactions to intradermal injection with purified protein derivative. one had a smear-negative, culture-positive active tb. dna comparisons by is6110 and tbn12 typing showed that the isolates from the four elephants and the handler with active tb were the same strain, thus documenting that the infection of the elephants came from the handler (24) . mycobacterium (tuberculosis) can infect agricultural workers in a number of ways: 1. human-to-human contact with co-workers through the inhalation of respiratory droplets 2. drinking contaminated, unpasteurized milk 3. direct contact with infected animals 4. direct contact with the secretions of infected animals such as respiratory droplets, milk, manure, urine, semen, and vaginal secretions 5. direct contact or inhalations of respiratory droplets during necropsy, slaughter, or processing of meat or dairy products (20) (21) (22) (23) (24) . the clinical presentation is that of weight-loss, night sweats, a chronic cough, and hemoptysis. asymptomatic workers are typically discovered through public health surveys. diagnosis is through the purified protein derivative (ppd) skin test, smears of respiratory secretions demonstrating acid-fast bodies, cultures of respiratory secretions and other body fluids, radiographs demonstrating caseating granulomas, and other typical findings. treatment is by multidrug therapy, complicated by regional drug resistance patterns (20) (21) (22) (23) (24) . giardia infections have been associated with contaminated sewage and water in agricultural environments, producing gastroenteritis. in the sierra foothills of california, cattle drink water contaminated by infected beavers. beaver-and cattle-contaminated water is then consumed by unsuspecting tourists who develop crampy abdominal pain, fevers, and a profuse bloody diarrhea. the giardia infections are easily treated with metronidazole (5). fowl are birds that grow in the wild. nearly every bird found in the wild can be prepared for human consumption. poultry are birds grown in farm environments for human consumption. common poultry include: chickens, turkeys, ducks, pigeons, game hens, geese, doves, and peacocks. avian influenza a (h5n1) first infected humans in 1997, in hong kong. the virus was transmitted directly from birds to humans. eighteen people were admitted to hospitals, and 6 died. in 2003, 2 cases of avian influenza a (h5n1) infection occurred among members of a hong kong family, 3 of whom had traveled to mainland china. one person died. how or where these 2 people became infected was not determined. influenza a has the potential to cross species and has been implicated in the 3 flu pandemics in the 20th century (1918, 1957 and 1968 ). pandemics occur when 3 conditions are met: 1. the emergence of influenza a virus with a hemagglutinin subtype is completely different from that of strains circulating in humans for many preceding years. 2. there is a high proportion of susceptible people in the community (i.e., a population with low antibody titers to the new strain). 3. efficient person-to-person transmissibility of the new virus is possible with accompanying human disease (25) (26) (27) . the reported signs and symptoms of avian influenza in humans include: 1. typical flu-like symptoms such as fever, cough, sore throat, and muscle aches 2. eye infections 3. pneumonia 4. acute respiratory distress syndrome (ards) 5. multiple organ failure 6. lymphopenia 7. elevated liver enzyme levels 8. abnormal clotting profiles. physicians are advised to isolate the patient, initiate droplet precautions, and contact their local medical officer for further discussions if an epidemiological link is suspected. the world health organization (who) is moving to rapidly produce a new influenza vaccine capable of protecting people against the h5n1 strain of avian influenza a. preliminary genetic tests conducted in cdc laboratories in atlanta, london, and hong kong suggest that the h5n1 strain is resistant to amantadine and rimantadine but is believed to be susceptible to neuraminidase inhibitors. the who has recommended urgent, rapid culling of infected and exposed bird populations to eliminate the reservoir of the h5n1 strain. in addition, who has discouraged the practice of marketing live poultry directly to consumers in areas currently experiencing outbreaks of avian influenza a (h5n1). some countries have introduced trade restrictions to protect animal health. however, available data do not suggest that processed poultry products (i.e., refrigerated or frozen carcasses and products derived from them) or eggs from affected areas pose a public health risk. the virus is killed by cooking (25) (26) (27) . newcastle disease is caused by virulent strains of apmv. death rates among naive bird populations can exceed 50%. the virus responsible for newcastle disease has been known to cause conjunctivitis and upper respiratory infections in humans since the 1940s. the disease is self-limiting and does not have any permanent consequences (28) . in 2002, wisconsin public health officials were notified of two cases of febrile illness in workers at a commercial turkey breeder farm. a high prevalence of west nile virus antibody was found among workers and turkeys. an associated high incidence of febrile illness among farm workers also was observed. possible non-mosquito transmission among birds and subsequent infection of humans was postulated, but the mode of transmission was unknown (29) . avian tuberculosis was diagnosed in two mature rheas on different ratite farms over a 2-year period. both birds died after weight loss and development of granulomas in the lungs of one bird and bilaterally in the cubcutis cranial to the shoulder in the other. smears and cultures of the granulomas were positive for acid-fast bacilli and tuberculosis (30) . chlamydophila (chlamydia) psittaci, c. trachomatis, and c. pneumoniae can be passed from birds of all species to humans. wild pigeons and pheasants have been demonstrated to be a source. wild birds in captivity, pets (usually cockatiels, parakeets, parrots, and macaws), and production animals can infect workers, and there are reports of customs and health inspection workers becoming infected. infection is through contact with feces, urine, and oral secretions (31) . mild infection produces a tracheobronchitis with flu-like symptoms of cough, congestion, myalgias, fatigue, and fever. in severe infections, untreated workers, and immunocompromised workers, pneumonia, sepsis, shock, and death can occur. radiographs reveal a lobar infiltrate (31) . diagnosis is by detection of the 16s rrna gene of c. psittasi in sputum with a pcr analysis, and a typical radiographic appearance and culture. tetracyclines and erythromycin are effective for treatment. prevention is through close monitoring and culling flocks and pet birds and personal protection equipment (32). raising poultry at home is common in low-income countries. studies demonstrate that proximity to free-range domestic poultry increases children's risk of infection with diarrhea-causing organisms such as campylobacter jejuni. corralling might reduce the risk, but research on the socioeconomic acceptability of corralling is lacking. many people report that home-grown poultry and eggs taste better and are more nutritious. they enjoy living around animals and want to teach their children about raising animals. to prevent theft, some residents shut their birds in provisional enclosures at night but allege that birds are healthier, happier, and produce better meat and eggs when let loose by day. many rural peoples view bird feces in the house and yard as dirty, but few see a connection to illness. residents consider chicks and ducklings more innocuous than adult birds and are more likely to allow them inside the house and permit children to play with them. additional food and water costs with corralling are a significant obstacle for some. adequate space and corral hygiene must also be addressed to make this intervention viable. developing a secure, acceptable, and affordable corral remains a challenge for rural populations (33, 34) . although approximately 95% of disease caused by non-typhoidal salmonella is transmitted by food-borne vehicles, four documented salmonella outbreaks in the 1990s have been traced to contact with young poultry. no environmental studies of source hatcheries were completed. a case-control study was performed by comparing culture-confirmed salmonella infantis in michigan residents, identified between may and july 1999, with two age-and neighborhood-matched controls. eighty environmental and bird tissue samples were collected from an implicated hatchery; all salmonella isolates underwent pulsed-field gel electrophoresis (pfge) analysis. the study included 19 case-patients sharing the same pfge subtype and 37 matched controls. within 5 days before illness onset, 74% of case-patients resided in households raising young poultry compared with 16% of controls (matched or 19.5; 95% ci 2.9, 378.1). eight hatchery samples yielded s. infantis with pfge subtypes matching the patients' isolates. this investigation identified birds from a single hatchery as the source of human illness and confirmed the link by matching pfge patterns from humans, birds and the hatchery environment. subsequent public health interventions reduced, but did not eliminate, transmission of poultry-associated salmonellosis. five additional pfge-linked cases were identified in spring 2000, necessitating quarantine of the hatchery for depopulation, cleaning and disinfection (35) . fish farming, or aquaculture, for fish and shellfish is becoming more common and more internationalized with every passing year. in the united states, more than half the seafood consumption is imported, much of it from fish farming. the world's seafood trade is very complex, and if is often difficult or impossible to determine where the seafood is raised or harvested. for example, the united states imports salmon from switzerland and panama though neither country is known for large salmon fisheries (36) . in general, farmed fish is as safe and nutritious as wild-caught species, but there are public health hazards associated with ignorance, abuse, and neglect of aquaculture technology. numerous small fish ponds increase the shoreline of ponds causing higher densities of mosquito larvae and cercaria, which can increase the incidence and prevalence of lymphatic filariasis and schistosomiasis. especially dangerous is the use of human waste draining to fertilize or create ponds. technology abuse includes the misuse of therapeutic drugs, chemicals, fertilizers and natural fish habitat areas. technology neglect includes the failure to pay attention to mosquito habitats and the concomitant increase in malaria, as well as the propagation of other organisms (36) . human exposure can be through direct skin contact with fish or the consumption of contaminated fish or shellfish products or contaminated water. the main pathogens acquired topically from fish (through spine puncture or open wounds) are aeromonas hydrophila, edwardsiella tarda, erysipelothrix rhusiopathiae, mycobacterium marinum, streptococcus iniae, vibrio vulnificus, and vibrio damsela. s. iniae has recently emerged as a public health hazard associated with aquaculture, and m. marinum often infects home aquarium hobbyists. common zoonoses contracted through the consumption of contaminated products or water include salmonella, leptospirosis, yersiniosis, and tuberculosis (37) . salmonellae species have been found associated with all of the poikilothermic vertebrate species studied, as well as the mollusks and crustaceans (38) . leptospirosis does occur in the poikilothermic vertebrates, as evidenced by positive serological reactions and by the isolation of pathogenic leptospiral serovars. the finding of leptospirosis species in fish, mollosks and other aquatic species are of special importance in view of the increased worldwide interest in aquaculture farming. since 1975, 24 of the 101 (23.7%) reported human cases of leptospirosis in hawaii have been associated with aquaculture industries (taro farms, prawn farms and watercress farms) (39) . species of yersinia are a particular problem in fish and in people involved in fish farming. workers who wade in fish ponds or drink drainage water are especially at risk. yersinia enterocolitica has been demonstrated to be a causative agent in acute diarrhea illness in humans after workers become infected through the feces-hand-oral route (19) . tuberculosis has also been reported in freshwater and marine fish species (piscine tuberculosis), especially in those grown on fish farms. mycobacterium marinum and m. celonae have been demonstrated in fish farms (30) . turtles, lizards, snakes, green iguanas (iguana iguana), alligators, and crocodiles are grown from eggs in farms for their hides and meat. some species are also grown for sale as pets. salmonella infections in persons who had contact with reptiles usually cause gastroenteritis but can result in invasive illness, including septicemia and meningitis, especially in infants and immunocompromised persons. for decades, reptiles have been known to be a source for salmonellosis; however, numerous reptile owners remain unaware that reptile contact places them and other household members, including children, at greater risk for infection. (40) captive reptiles (such as iguanas) are routinely identified as reservoirs of salmonella and the number of reports about reptile-associated salmonellosis is increasing. in germany and austria, salmonella was detected in 54.1% of fecal reptile samples cultured. the percentage of salmonella-positive samples was significantly lower in turtles as compared with lizards and snakes, as salmonella was only detected in one sample from a single turtle out of 38 turtles investigated. in all, 42 different salmonella serovars were found. all isolated salmonella belonged to the species enterica, predominantly to the subspecies i (n = 46) and iiib (n = 30) but also to subspecies ii (n = 3), iiia (n = 6), and iv (n = 2). all isolates were sensitive to the antimicrobials examined. a significantly higher percentage of salmonella-positive reptiles was detected in the group of owners who purchased reptiles in comparison with pure breeders. the high percentage of salmonella in reptiles in the study confirms the risk for the transmission of the infection to humans (41) . zoonoses as a risk when associating with livestock or animal products social and environmental risk factors in the emergence of infectious diseases public health implications related to spread of pathogens in manure from livestock and poultry operations occupational bio hazards: current issues pathogen survival in swine manure environments and transmission of human enteric illness: a review zoonotic infections an approach to reduction of salmonella infection in broiler chicken flocks through intensive sampling and identification of cross-contamination hazards in commercial hatcheries maff statuatory incident reports in surveillance, prevention, and control of human salmonella typhimurium infection surveillance and control of emerging zoonoses testing to fulfill haccp (hazard analysis critical control points) requirements: principles and examples effect of an electrostatic space charge system on airborne dust and subsequent potential transmission of microorganisms to broiler breeder pullets by airborne dust veterinary monkeypox virus working group. monkeypox transmission and pathogenesis in prairie dogs wild game feasts and fatal degenerative neurological illness abortion in humans caused by chlamydophila abortus chlamydophila abortus infection in a pregnant woman associated with indirect contact with infected goats human salmonellosis associated with exotic pets multidrugresistant salmonella typhimurium infection from milk contaminated after pasteurization dairy farm reservoir of listeria monocytogenes sporadic and epidemic strains a study of the foodborne pathogens: campylobacter, listeria and yersinia, in faeces from slaughter-age cattle and sheep in australia factors influencing the incidence and scale of bovine tuberculosis in cattle in southwest england diagnostic strategies and outcomes on three new zealand deer farms with severe outbreaks of bovine tuberculosis prevalence of bovine tuberculosis in cattle in different farming systems in the eastern zone of tanzania human exposure following mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants avian influenza: recent developments human health implications of avian influenza viruses and paramyxoviruses avian influenza outbreak: update phylogenetic relationships among virulent newcastle disease virus isolates from the 2002-2003 outbreak in california and other recent outbreaks in north america center for disease control and prevention. west nile virus infection among turkey breeder farm workers: wisconsin tuberculosis in farmed rheas (rhea americana) a flu like syndrome a woman with a lobar infiltrate due to psittacosis detected by polymerase chain reaction campylobacter, from obscurity to celebrity campylobacter enteritis human salmonellosis associated with young poultry from a contaminated hatchery in michigan and the resulting public health interventions public, animal, and environmental health implications of aquaculture topically acquired bacterial zoonoses from fish: a review epidemiologic aspects of salmonellosis in reptiles, amphibians, mollusks and crustaceans-a review leptospirosis in poikilothermic vertebrates. a review reptile-associated salmonellosis-selected states salmonella enterica in reptiles of german and austrian origin systemic infection with alaria americana (trematoda) amphibians include frogs, toads, newts, and salamanders that are caught in the wild or grown on farms for use as food or as pets. frogs are caught in the wild and grown in farms for their meat, primarily frog legs. eating inadequately cooked frog legs can lead to an infection of alaria americana, a trematode. increasing evidence suggests that amphibians can pose risks for salmonellosis in humans (42) . key: cord-270911-z637eh2z authors: zhou, jie; li, cun; sachs, norman; chiu, man chun; wong, bosco ho-yin; chu, hin; poon, vincent kwok-man; wang, dong; zhao, xiaoyu; wen, lei; song, wenjun; yuan, shuofeng; wong, kenneth kak-yuen; chan, jasper fuk-woo; to, kelvin kai-wang; chen, honglin; clevers, hans; yuen, kwok-yung title: differentiated human airway organoids to assess infectivity of emerging influenza virus date: 2018-06-26 journal: proc natl acad sci u s a doi: 10.1073/pnas.1806308115 sha: doc_id: 270911 cord_uid: z637eh2z novel reassortant avian influenza h7n9 virus and pandemic 2009 h1n1 (h1n1pdm) virus cause human infections, while avian h7n2 and swine h1n1 virus mainly infect birds and pigs, respectively. there is no robust in vitro model for assessing the infectivity of emerging viruses in humans. based on a recently established method, we generated long-term expanding 3d human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. we report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. in addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. we also established improved 2d monolayer culture conditions for the differentiated airway organoids. to demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3d and 2d differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, h7n9/ah versus h7n2 and h1n1pdm versus an h1n1 strain isolated from swine (h1n1sw). the human-infective h7n9/ah virus replicated more robustly than the poorly human-infective h7n2 virus; the highly human-infective h1n1pdm virus replicated to a higher titer than the counterpart h1n1sw. collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. these differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human. novel reassortant avian influenza h7n9 virus and pandemic 2009 h1n1 (h1n1pdm) virus cause human infections, while avian h7n2 and swine h1n1 virus mainly infect birds and pigs, respectively. there is no robust in vitro model for assessing the infectivity of emerging viruses in humans. based on a recently established method, we generated long-term expanding 3d human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. we report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. in addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. we also established improved 2d monolayer culture conditions for the differentiated airway organoids. to demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3d and 2d differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, h7n9/ah versus h7n2 and h1n1pdm versus an h1n1 strain isolated from swine (h1n1sw). the humaninfective h7n9/ah virus replicated more robustly than the poorly human-infective h7n2 virus; the highly human-infective h1n1pdm virus replicated to a higher titer than the counterpart h1n1sw. collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. these differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human. airway organoid | proximal differentiation | influenza virus | infectivity i nfluenza a viruses (iavs) can infect a diversity of avian and mammalian species, including humans, and have the remarkable capacity to evolve and adapt to new hosts (1) . the segmented rna genomes of iavs and the low fidelity of rna polymerase allow antigenic shift and drift, which drive the viral evolution. thus, novel viruses from birds and pigs cross the species barrier and infect humans, leading to sporadic infections, epidemics, and even pandemics (1, 2) . despite the tremendous progress made in virology and epidemiology, which subtype or strain of iav will cause the next outbreak remains unpredictable. a novel reassortant h7n9 influenza virus from poultry has led to recurrent outbreaks of human infection in china since 2013 (2, 3) . more than 1,500 laboratory-confirmed cases of h7n9 human infections were reported by october 2017, with a case-fatality rate higher than 35% (4). in 2009, the first influenza pandemic of the 21st century was caused by a novel pandemic h1n1 (h1n1pdm), which originated via multiple reassortments of "classical" swine h1n1 virus with human h3n2 virus, avian virus, and avian-like swine virus (5). while swine viruses infect humans only sporadically, this novel strain of swine-derived h1n1pdm virus has infected a large proportion of the human population, establishing sustained human-to-human transmission and circulating globally as a seasonal virus strain since then. proteolytic cleavage of viral glycoprotein ha is essential for iav to acquire infectivity, since only the cleaved ha molecule mediates the membrane fusion between the virus and the host cell, a process required for the initiation of infection. ha proteins of low-pathogenic avian iavs and human iavs carry a single basic amino acid arginine at the cleavage site (6, 7), recognized by trypsin-like serine proteases. productive infection of significance influenza virus infection represents a major threat to public health worldwide. there is no biologically relevant, reproducible, and readily available in vitro model for predicting the infectivity of influenza viruses in humans. based on the longterm expanding 3d human airway organoids, we developed proximal differentiation and further established a 2d monolayer culture of airway organoids. the resultant 3d and 2d proximal differentiated airway organoids can morphologically and functionally simulate human airway epithelium and as a proof of concept can discriminate human-infective influenza viruses from poorly human-infective viruses. thus, the proximal differentiated airway organoids can be utilized to predict the infectivity of influenza viruses and, more broadly, provide a universal platform for studying the biology and pathology of the human airway. these viruses in the human airway thus requires serine proteases such as tmprss2, tmprss4, hat, and others (8) . however, ha proteins of highly pathogenic avian viruses, such as h5n1, contain a polybasic cleavage site which is activated by ubiquitously expressed proteases. current in vitro models for studying influenza infection in the human respiratory tract involve shortterm cultures of human lung explants and primary airway epithelial cells. human lung explants are not readily available on a routine basis. in addition, the rapid deterioration of primary tissue is a major problem in infection experiments, since there is no protocol to maintain tissue viability in vitro. under air-liquid interface conditions, basal cells isolated from human airway can polarize and undergo mucociliary differentiation. however, this capacity is lost within two or three passages (9) . collectively, these primary tissues and cells hardly constitute a convenient, reproducible model to study human respiratory pathogens. although various cell lines, e.g., a549 and madin-darby canine kidney (mdck) cells, have been commonly used to propagate influenza viruses and to study their virology, they poorly recapitulate the histology of human airway epithelium. in addition, due to the low serine protease activity, most cell lines do not support the growth of the influenza viruses with a monobasic ha cleavage site unless the culture medium is supplemented with an exogenous serine protease, trypsin treated with n-tosyl-l-phenylalanine chloromethyl ketone (tpck). thus, a biologically relevant, reproducible, and readily available in vitro model remains urgently needed for studying the biology and pathology of the human respiratory tract. recent advances in stem cell biology have allowed the in vitro growth of 3d organoids that recapitulate the essential attributes of their counterpart organs in vivo. these organoids can be grown from pluripotent stem cells (pscs) or tissue-resident adult stem cells (ascs) (10) . asc-derived organoids consist exclusively of epithelial cells and can be generated from a variety of human organs, the first being the human gut (11) . these human intestinal organoids represent the first model for in vitro propagation of norovirus and have allowed the study of other viruses (12, 13) . we very recently described the initial conditions for expanding human asc-derived airway organoids (aos) for >1 y (14) . of note, protocols also have been established for generating lung organoids from human pscs and embryonic lung (15, 16) . characterization of the human aos. based on the recently reported protocol (14) , we established several lines of aos from small pieces of normal lung tissue adjacent to the diseased tissue from patients undergoing surgical resection for clinical conditions. these aos, 3d cysts lined by polarized epithelium, accommodated the four major types of airway epithelial cells, i.e., ciliated cells (acctub + or foxj1 + ), goblet cells (muc5ac + ), basal cells (p63 + ), and club cells (cc10 + ) (fig. 1a) . apical acctub clearly indicated the orientation of polarization. most organoids are orientated towards the lumen (fig. 1a , acctub left), while a small proportion of the organoids are inverted (fig. 1a , acctub right). beating cilia were visible (movie s1). no type i and type ii alveolar epithelial cells were present. thus, these organoids resembled the pseudostratified ciliated airway epithelium. the aos were inoculated with human iav h1n1pdm, the low-pathogenic avian virus h7n9/ah, and the highly pathogenic avian virus h5n1. the intracellular (cell lysate) viral loads of all three virus strains increased over 2 log 10 units (fig. 1b) . the extracellular (supernatant) viral loads, especially the viral titers, were elevated by 2-3 log 10 units. ciliary beating plays an essential role in human airway biology and pathology, and 50-80% of airway epithelial cells are ciliated (17) . however, immunostaining (fig. 1a ) and flow cytometry indicated that ciliated cells were apparently underrepresented in these aos. therefore, despite the discernible multilineage differentiation and the ability to support replication of iavs, further improvements in morphology and differentiation appeared required. pd of the aos. to improve proximal differentiation (pd), we tried various protocols and variations thereof and settled on a medium described by konishi et al. (18) for inducing ciliary differentiation in human psc-derived airway epithelial spheroids. the ascderived organoids in the original ao medium gradually enlarged, while those in pd medium became more compact ( fig. 2a) . after 16 d of culture, the organoids in ao medium grew approximately two times larger, while the organoids in pd medium remained basically unchanged in size (fig. 2b) . from day 7, the number of ciliated cells increased markedly in pd medium. at day 16, beating cilia were observed in a minority (<10%) of the organoids in ao medium, while abundant beating cilia were present in every pd organoid ( fig. 2c and movies s2 and s3). the synchronously beating cilia drove the cell debris within the organoid lumens to swirl unidirectionally (movie s3). the dramatically increased abundance of ciliated cells in the pd organoids was verified by immunofluorescence staining (fig. 2d) . consistently, the transcriptional levels of the ciliated cell markers foxj1 and sntn were strongly up-regulated in the pd organoids compared with the organoids in ao medium. the expression levels of the basal cell markers p63 and ck5 and the goblet cell marker muc5ac also increased, whereas the levels of club cell markers cc10 and scgb3a2 were substantially down-regulated in the pd organoids (fig. 3a) . importantly, we observed globally elevated expression of serine proteases including tmprss2, tmprss4, tmprss11d (hat), and matriptase, which are essential for the activation and propagation of human iavs and low-pathogenic avian iavs (8) . we also performed flow cytometry analysis to measure the percentages of the four cell types in the organoids cultured in two distinct media at day 16. it was shown that the percentage of ciliated cells increased remarkably, by about threefold, after pd, to over 40% in the pd organoids, while the ciliated cells invariably constituted a minority of the cells in the organoids in ao medium (fig. 3 b and c). goblet cells also increased marginally, while club cells consistently decreased after pd (fig. 3 b and c) . collectively, we successfully induced mucociliary differentiation and developed pd aos which morphologically and functionally simulate human airway epithelium. pd aos can identify human-infective virus. one of the most important and challenging issues for influenza research is to predict which animal or emerging influenza virus can infect humans. as mentioned above, the novel reassortant avian h7n9 viruses have caused continuing poultry-to-human transmission since 2013. in fact, other subtypes of avian iavs, including h7n2, h9n2, and h9n9, have been cocirculating with the h7n9 viruses in domestic poultry. these viruses are highly similar in internal genes but differ in neuraminidase (na) or ha and na (19) . however, very few human infections by h7n2, h9n2, and h9n9 virus have been reported in the same territory and time frame, although people were exposed equivalently to these viruses and the h7n9 viruses (20) ; these findings suggest that these viruses are less infective to humans than the h7n9 viruses. we isolated, plaque-purified, and genotyped these cocirculating viruses. we chose to compare the infectivity of h7n2 with that of h7n9/ah in the pd organoids as a proof of concept, to verify that the differentiated aos can indeed simulate human airway epithelium in the context of influenza virus infection. fig. 4 shows that viral loads in the cell lysate and medium of h7n9/ ah-infected organoids increased gradually after inoculation; the viral titer increased more than 3 log 10 units within 24 h, indicating a robust viral replication. the addition of the serine protease inhibitor aebsf significantly restricted the active replication of h7n9/ah virus, highlighting the importance of elevated serine proteases for viral replication. in contrast, h7n2 propagated modestly, with viral titer 2-3 log 10 units lower than h7n9/ah. thus, the distinct efficiencies of h7n9/ah and h7n2 to infect and replicate in the pd aos can recapitulate the infectivity of these viruses in humans. establishing a 2d airway monolayer from aos to assess the infectivity of iavs. a limitation of 3d organoids for studying microbial infections is the inaccessibility of the apical surface to pathogens, since most organoids are orientated inwards, while receptors for most respiratory viruses are distributed in the apical surface. for virus inoculation, organoids must be sheared to enable sufficient apical exposure to virus inoculum (21) . as reported previously, primary bronchial epithelial cells cultured in transwell inserts with the defined medium undergo mucociliary differentiation (22) . this prompted us to transform 3d aos into a 2d monolayer using transwell culture. to this end, 3d aos were enzymatically dissociated into a single-cell suspension, seeded in transwell inserts, and then cultured in pd medium. the transepithelial electrical resistance (teer) in the 2d monolayers stabilized in the second week after seeding (si appendix, fig. s1 ). in addition, the dextran penetration assay performed at day 12 indicated that an intact epithelial barrier was formed across the 2d monolayers (si appendix, fig. s1 ). the intense signal of acctub indicated that the 2d monolayers developed appreciable pd (fig. 5a) . the productive infection of h7n9/ah was clearly shown by the virus nucleoprotein-positive (np + ) cells at 8 h post infection (hpi) in the 2d pd organoids (fig. 5a) . to verify the ability of 2d pd organoids to assess the zoonotic potential of animal viruses and identify the human-infective virus, we compared the relative replication capacities of h7n9/ah and h7n2 viruses and of another pair of viruses, the highly human-infective h1n1pdm and a swine h1n1 isolate (h1n1sw). the higher replicative capacity of h7n9/ah over h7n2 virus was more pronounced in the 2d pd organoids than in the 3d pd organoids; the viral titer of h7n9/ah in the apical medium was 3-4 log 10 units higher than that of the h7n2 virus (fig. 5b) . consistently, h1n1pdm replicated dramatically, with a viral titer in the apical medium 1-2 log 10 units higher than that of h1n1sw. due to the epithelial barrier formed in the 2d monolayers and the preferential release of virus from the cell apical side, the viral titers in the basolateral medium were consistently lower than those in apical medium at the corresponding time points. nevertheless, the differences in replicative capacity between h7n9/ ah versus h7n2 and h1n1pdm versus h1n1sw were even more prominent in basal medium than in apical medium at most time points. in this study, we describe the pd of human asc-derived ao culture for investigating a major animal and human pathogen, influenza virus. in particular, our differentiation conditions increase the numbers of ciliated cells (fig. 2) , the major cell type in the human airway epithelium. the pd medium induces ciliated cell numbers to a nearly physiological level, with synchronously beating cilia readily discernible in every organoid (movies s2 and s3). in addition, the expression levels of serine proteases (fig. 3) , known to be important for productive viral infection, were elevated after pd. among the up-regulated ha-activating serine proteases, the dramatically increased expression of hat in the differentiated aos can very likely be attributed to the increased abundance of ciliated cells, since ciliated cells are the main source of hat in the human respiratory tract (23) . thus, the differentiated aos can simulate the human airway epithelium morphologically and functionally. as a further improvement, we developed 2d pd airway monolayers with an intact epithelial barrier to allow exclusively apical exposure to viruses (fig. 5a and si appendix, fig. s1 ), the natural mode of iav infection in the human respiratory tract. we then utilized two pairs of viruses with known infectivity to demonstrate, as a proof of concept, that these organoids indeed show significantly higher susceptibility to the human-infective viruses than to the poorly human-infective viruses. these 3d and 2d differentiated aos support the active replication of human-infective h7n9/ah and h1n1pdm. in contrast, the h7n2 virus, which has been temporally and spatially cocirculating with h7n9 viruses in domestic poultry and contains internal genes similar to those in h7n9 viruses, replicated much less efficiently in both models. similarly, the swine h1n1 isolate showed a substantially lower growth capacity than its counterpart, human-adapted h1n1pdm (fig. 5b) . the avian iav h7n2 subtype viruses have been circulating in the bird market from 1994-2006 and have caused poultry outbreaks in the united states. sporadic human infections have been reported in the united states and europe. fortunately, all reported cases of human infection experienced mild influenzalike symptoms (24) . while pigs are considered the intermediate hosts for interspecies transmission of iavs, swine influenza viruses lacking human adaptation markers rarely infect humans. sporadic human infections documented in the literature or reported by public health officials are generally mild or subclinical (25) . the ability of the pd aos to differentiate avian h7 subtype virus and swine h1 subtype virus from the counterpart human-infective viruses suggests that these models could be used to assess the cross-species transmission potential of emerging influenza viruses in humans. in this study, only four strains of virus were assessed in the differentiated aos as a proof of concept. more virus strains should be evaluated to further understand the strengths and limitations of this pd ao model. in summary, we believe that these differentiated aos significantly extend the current armamentaria of the influenza research toolbox. asc-derived organoids, once established, can be expanded indefinitely while displaying remarkable phenotypic and genotype stability. they thus overcome the limitations in reproducibility and availability of the current in vitro model systems. aos can be easily established from human bronchiolar lavage material, allowing interindividual comparisons; aos can also be readily modified by lentiviruses and crispr technologies and can be single-cell cloned (14) . in combination with the molecular toolbox of the influenza virologist, the human differentiated ao model system offers great opportunities for studying virus and host factors that define the characteristics of this major animal and human pathogen. establishing asc-derived human aos. the generation of four lines ascderived human aos was based on the previous protocol (14) with a success rate of 100%. briefly, with ethical approval by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw 13-364) and informed consent of the patients, small pieces of normal lung tissue (â�¼0.2-0.6 cm 2 in size) adjacent to the diseased tissues were obtained from patients who underwent resection. the tissues were minced and digested with 2 mg/ml collagenase (sigma aldrich) for 1-2 h at 37â°c, followed by shearing using a glass pasteur pipette (drummond) and straining over a 100-î¼m cell strainer (falcon). the resultant single cells were embedded in 60% matrigel (growth factor reduced basement membrane matrix; corning) and were seeded in 24-well suspension culture plates (greiner bio-one). after solidification, matrigel droplets were maintained with ao culture medium (si appendix, table s1 ) at 37â°c in a humidified incubator with 5% co 2 . the organoids were passaged every 2-3 wk. the bright-field images of the organoids were acquired using a nikon eclipse ts100 inverted routine microscope. pd of human aos. the aos were split and maintained in ao medium for 3-7 d. the culture medium in half of the organoids was changed to pd medium, pneumacult-ali medium (stemcell technologies) supplemented with 10 î¼m dapt (tocris). the organoids were then cultured in ao or pd medium for 16 d. bright-field images were taken every 3 d. diameters of individual organoids were measured with imagej (nih). the movies of organoids were acquired using a total internal reflection fluorescent (tirf) microscope (movie s1) and a nikon eclipse ti2 inverted microscope system (movies s2 and s3). at the indicated times, the organoids in the different media were harvested for detection of cellular gene expression or for flow cytometry analysis. establishing 2d differentiated aos with transwell culture. transwell culture of aos was performed as described elsewhere (24, 25) with modifications. briefly, the organoids were dissociated into single-cell suspensions after digested with 10ã� tryple select enzyme (invitrogen) for 1-5 min at 37â°c, sheared using a pasteur pipette, and strained over a 40-î¼m filter. approximately 3.5 ã� 10 5 cells were seeded into each transwell insert (product no. 3494; corning). the cells were cultured in ao medium at 37â°c in a humidified incubator with 5% co 2 for 1-2 d. when cells reached >90% confluence, the ao medium was changed to pd medium in both the apical and basal chambers. the medium was changed every other day, and the cells were maintained for 14 d. teer was measured every other day using a millicell ers-2 volt-ohm meter (emd millipore). to assess the integrity of the 2d organoid monolayer as an epithelial barrier, at day 12 after seeding, fitc-dextran with an average molecular weight of 10,000 (sigma aldrich) was added to the medium in the upper chamber at a concentration of 1 mg/ml followed by incubation at 37â°c for 4 h. subsequently, the culture media were harvested from the upper and bottom chambers to detect the fluorescence intensity using the victor xiii multilabel reader (perkinelmer). eggs at 37â°c for 36 h. the eggs were chilled overnight at 4â°c; then the virus-containing allantoic fluid was harvested, aliquoted, and stored at â��80â°c. virus titer was determined by plaque assay. iav infection in human aos. the 3d aos were sheared mechanically to expose the apical surface to the virus inoculum. the sheared organoids were then incubated with viruses at a multiplicity of infection (moi) of 0.01 for 2 h at 37â°c. after washing, the inoculated organoids were re-embedded in matrigel and then were cultured in the pd medium. in the h7n9/ah and h7n2 infection in the 3d pd organoids, one set of h7n9/ah-inoculated organoids was treated with the serine protease inhibitor aebsf (0.125 mm; merck millipore) during inoculation and after inoculation. at the indicated times, organoids, dissolved matrigel, and culture medium were harvested for detection of viral load. the cell-free matrigel and the culture medium from each droplet were pooled together as one sample, referred as the "supernatant." the supernatant samples were also used for viral titration. the 2d pd aos were inoculated with the indicated viruses at an moi of 0.001 from the apical side by adding the virus inoculum to the apical chamber followed by incubation for 2 h at 37â°c. at the indicated times, cell-free media were collected from the apical and basolateral chambers for viral titration. the membranes seeded with 2d organoids were incised from the transwell inserts and fixed, and immunofluorescence staining was applied as described previously (13) . rna extraction and rt-qpcr. to evaluate the differentiation status of aos cultured in pd medium versus those in ao medium, the organoids were harvested at the indicated times, and rna was extracted using minibest universal rna extraction kit (takara). to quantify virus replication, the organoids and supernatant samples were lysed for rna extraction using the minibest universal rna extraction kit and minibest viral rna/dna extraction kit (takara), respectively. cdna was synthesized with the transcriptor first strand cdna synthesis kit (roche) with oligo-dt primer. qpcr was performed with lightcycler 480 sybr green i master mix (roche) using genespecific primers (si appendix, table s2 ) to detect cellular gene-expression level and viral gene copy number. the mrna expression levels of cellular genes were normalized with that of gapdh. viral gene copy number was determined by absolute quantification using a plasmid expressing a conserved region of the iav m gene. plaque assay. a plaque assay was performed to determine titers of the virus stocks and supernatant samples as described elsewhere (26) , with minor modifications. briefly, mdck cells were seeded in 12-well plates. confluent monolayers were inoculated with 400 î¼l of 10-fold serial dilutions of samples and were incubated for 1 h at 37â°c. after the inoculum was removed, the monolayers were overlaid with 1% low melting point agarose (invitrogen) supplemented with eagle's minimal essential medium (mem) and 1 î¼g/î¼l tpck-treated trypsin and were further incubated for 2-3 d. the monolayers were fixed with 4% paraformaldehyde (pfa) and were stained with 1% crystal violet to visualize the plaque after the agarose plugs were removed. virus titers were calculated as plaque-forming units per milliliter. immunofluorescence staining. to identify the indicated cell types and the virusinfected cells, immunofluorescence staining was applied to the 3d and 2d aos. briefly, the organoids were fixed with 4% pfa, permeabilized with 0.1-5% triton x-100, and blocked with protein block (dako). then the organoids were incubated with primary antibodies (si appendix, table s3 ) diluted in antibody diluent buffer (dako) overnight at 4â°c, followed by incubation with secondary antibodies (si appendix, table s3 ) for 1-2 h at room temperature. nuclei and actin filaments were counterstained with dapi (invitrogen) and phalloidin-647 (sigma aldrich), respectively. the confocal images were acquired using a carl zeiss lsm 780 or 800 confocal microscope. flow cytometry analysis. to assess the percentages of four types of cells, the aos were analyzed by flow cytometry. briefly, the organoids were dissociated with 10 mm edta (invitrogen) for 30-60 min at 37â°c, fixed with 4% pfa, and permeabilized with 0.1% triton x-100. subsequently, the cells were incubated with primary antibodies (si appendix, table s3 ) for 1 h at 4â°c followed by staining with secondary antibodies. a bd facscanto ii system was used to analyze the samples. statistical analysis. student's t test was used for data analysis. p < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01. ***p < 0.005. influenza viruses en route from birds to man the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities human infections with the emerging avian influenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome human infection with avian influenza a(h7n9) virus china novel swine-origin influenza a (h1n1) virus investigation team (2009) emergence of a novel swine-origin influenza a (h1n1) virus in humans proteolytic 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embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids airway epithelial cell cilia and obstructive lung disease directed induction of functional multi-ciliated cells in proximal airway epithelial spheroids from human pluripotent stem cells cocirculation of three hemagglutinin and two neuraminidase subtypes of avian influenza viruses in human infection with an avian influenza a/h9n2 virus in guangdong in 2016 enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner human and avian influenza viruses target different cell types in cultures of human airway epithelium localization of human airway trypsin-like protease in the airway: an immunohistochemical study avian influenza a(h7n2) virus in human exposed to sick cats swine influenza virus infections in man. swine influenza d225g mutation in hemagglutinin of pandemic influenza h1n1 (2009) virus enhances virulence in mice delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus key: cord-017364-d9zmdm23 authors: crowe, james e.; williams, john v. title: paramyxoviruses: respiratory syncytial virus and human metapneumovirus date: 2014-02-27 journal: viral infections of humans doi: 10.1007/978-1-4899-7448-8_26 sha: doc_id: 17364 cord_uid: d9zmdm23 human respiratory syncytial virus (rsv) and human metapneumovirus (mpv) are members of the family paramyxoviridae of the mononegavirales order, comprising the nonsegmented negative-strand rna viruses. paramyxoviridae has two subfamilies: paramyxovirinae, which includes the parainfluenza viruses 1–4 and measles and mumps viruses, and pneumovirinae, which includes rsv and mpv. pneumovirinae has two genera: pneumovirus, which includes human rsv, bovine respiratory syncytial virus, and pneumonia virus of mice, and metapneumovirus, which includes human mpv and avian metapneumovirus, sometimes called avian pneumovirus. human respiratory syncytial virus human respiratory syncytial virus (rsv) and human metapneumovirus (mpv) are members of the family paramyxoviridae of the mononegavirales order, comprising the nonsegmented negative-strand rna viruses. paramyxoviridae has two subfamilies: paramyxovirinae , which includes the parainfl uenza viruses 1-4 and measles and mumps viruses, and pneumovirinae , which includes rsv and mpv. pneumovirinae has two genera: pneumovirus , which includes human rsv, bovine respiratory syncytial virus, and pneumonia virus of mice, and metapneumovirus , which includes human mpv and avian metapneumovirus, sometimes called avian pneumovirus. rsv was isolated fi rst in 1956 from an ill chimpanzee in a laboratory setting that had an illness similar to the common cold. a virus causing a similar cytopathic effect in cultured cells was recovered from infants with respiratory illness shortly after, and studies of human antibodies in the serum of infants and children indicated that infection was common early in life [ 1 , 2 ] . now it is known that rsv is the most common viral agent of serious pediatric respiratory tract disease worldwide. in most areas of the world, rsv is the most common cause of pneumonia and bronchiolitis in infants less than 1 year of age. rsv causes severe disease in young infants, but disease is not restricted to the early life period. the virus can cause severe lower respiratory tract illness in large numbers of elderly subjects and also in subjects who are severely immunocompromised such as hematopoietic stem cell transplant recipients [ 3 -7 ] . mortality in infants and children caused by rsv is uncommon in developed countries with modern critical care units. estimates of the mortality rate are about 0.3 % of hospitalized children or less. mortality has been dropping over the last several decades, and by the late 1990s the estimated number of deaths in the usa was several hundred children a year or less [ 8 , 9 ] . large epidemiologic studies report that the us mortality in children may be only about 100 cases a year. interestingly, while many providers think of rsv infection as principally a pediatric illness, there are over 17,000 deaths per year in the elderly, making them the highest risk population for death [ 9 ] . in less developed countries, however, infant deaths due to rsv infection may be more common. infants with underlying illness are at highest risk among young children for morbidity and mortality from rsv infection. infants with chronic lung disease requiring supplemental oxygen following treatment for prematurity, due to bronchopulmonary dysplasia, are perhaps at the highest risk for prolonged, severe, or fatal illness due to rsv [ 10 ] . infants with severe congenital pulmonary or cardiac disease have been reported to be at risk of death in 3-4 % of cases when hospitalized, although this rate is likely decreasing in the usa [ 11 ] . both children and adults with primary immunodefi ciency or medically induced immunosuppression are at high risk of mortality due to rsv infection. the most severely immunocompromised, and thus those at highest risk of mortality, are hematopoietic stem cell transplant patients of any age [ 12 ] . in some settings, the mortality rate of rsv infection in hematopoietic stem cell transplant patients with severe immunosuppression verges on 100 % [ 12 ] . hospitalization rates of infants for rsv disease vary with the setting, probably due to variations in exposure, genetics, socioeconomic, and other risk factors and due to the local practice style of medical providers. many developed countries report hospitalization rates of about 1 in 100-200 infected infants during the fi rst year of life [ 13 , 14 ] . studies of rsv disease in developed countries suggest that of those infants hospitalized, about 9 % require mechanical ventilation [ 15 , 16 ] . there are certain populations at extraordinarily high risk of hospitalization with rsv, for example, alaskan native infants younger than 1 year have been reported to have a hospitalization rate of 53-249 per 1,000 infants [ 16 ] . low socioeconomic status is a risk factor for higher rate of hospitalization in most areas. rsv also is one of the most common viral causes of serious lower respiratory tract illness in the elderly, especially in institutionalized subjects [ 17 ] . exacerbations of chronic obstructive pulmonary disease (copd) are frequently associated with rsv infection [ 18 , 19 ] . the elderly do not exhibit a remarkably diminished level of antibodies to rsv [ 20 ] . decreased levels of t cell memory in the elderly and specifi cally in patients with (copd) may contribute to the increased susceptibility to rsv infection in these populations [ 21 ] . many think of infl uenza virus as the principal viral respiratory pathogen in this population, but in a hospitalized cohort, infl uenza a virus and rsv infection resulted in similar mortality, lengths of stay, and rates of use of intensive care [ 17 ] . rsv infection accounted for over 10 % of hospitalizations for pneumonia. seroepidemiology studies suggest that virtually all children are infected in the fi rst 2 years of life, and early infection is especially common in infants attending group day care. serological methods are helpful in epidemiology and vaccine studies, but serologies are not often used for diagnosis in clinical settings. because of the transfer of maternal rsvspecifi c antibodies across the placenta, and the high prevalence of early infection, it is unusual to fi nd infants who are rsv seronegative. in older children and adults, a fourfold rise in serum antibodies is often used as evidence of rsv infection, but asymptomatic infections in which viral shedding is low in titer often are not accompanied by serum antibody rises. serological tests in infants are even less sensitive, because young infants may not exhibit a large or durable rise in antibodies. neutralizing antibody tests are considered the best functional marker of infection, but sensitivity is much higher in antigen binding assays using individually purifi ed rsv f or g proteins [ 22 ] . isolation of the virus in cell culture provides a defi nitive test for diagnosis of active infection. various methods of obtaining respiratory virus secretions for testing have been compared. most studies suggested that aspiration or gentle fl ushing of nasal secretions using a solution like saline is best, though some types of nasal swab have given reasonable results. the virus is more thermolabile than most, and thus samples should be transported on wet ice to the diagnostic laboratory and processed rapidly. prolonged times of transport to remote reference laboratories reduce the effectiveness of isolation. after inoculation onto susceptible cell culture substrates, highly trained staff can recognize cytopathology in the cell monolayers by visual inspection and conventional bright-fi eld microscopy after about 3-7 days of incubation. detection may be more effi cient when using shell vial cultures and immunofl uorescence [ 23 ] . various cell lines have been used for rsv detection, such as hep-2 epithelial cells, mrc-5 fi broblasts, and rhesus monkey kidney cells, but the r-mix commercial mix of human and mink lung cells may perform better for detection of rsv [ 24 ] . culture is expensive and requires highly trained staff and therefore is not usually available at the point of care, which is often an outpatient clinic or emergency department. therefore, rapid diagnostic methods were developed for the detection of viral proteins or rna in respiratory secretions. rsv antigen tests mostly rely on direct immunofl uorescent assays (dfa) on exfoliated cells in secretions or enzyme immunoassay (eia). nucleic acid detection assays based on rt-pcr are now widely available for rsv, often in a multiplexed panel for detection of multiple respiratory virus pathogens. these tests are typically more sensitive than any of the virus isolation or protein-based detection assays discussed above. enhanced sensitivity is especially helpful when testing adults, who often shed virus in very low titers. positive rt-pcr tests need to be interpreted in the context of the clinical scenario, since the tests can remain positive for prolonged periods of time after infection, well beyond the period during which infectious virus can be isolated. since children may experience symptomatic respiratory infections every few weeks during the winter, caution must be used in interpretation of positive pcr tests, especially when multiple viruses are detected simultaneously in a sample. some instances of multiple pcr test positivity likely represent residual rna from a previous virus infection and a second rna type representing live virus from the active current infection. rsv typically is propagated in monolayer cell cultures of continuous cell lines of human epithelial cell origin, such as hep-2 cells. monkey kidney cells of various types are also used commonly for propagation in the laboratory. in fact, the virus replicates to some extent in most cell lines of mammalian origin. in non-polarized epithelial cells, the virus often causes a typical cytopathic effect in which multinucleated giant cells form due to cell-cell fusion, termed cell syncytia. this in vitro effect is the origin of the virus name, although it is not clear that rsv causes syncytia in vivo. in polarized epithelial cells in culture, the virus assembles and buds from the apical surface of cells, mimicking to some extent the budding of virus into the lumen of the airway. the virus has a negative-sense single-stranded rna genome with 10 genes encoding 11 proteins. figure 26 .1 compares the genomes of rsv and mpv, which are similar in many respects. the replication proteins are common to both of the viruses, as are the matrix (m) protein and the surface fusion (f) and glycosylated attachment (g) glycoproteins. the gene order differs slightly, and rsv possesses two nonstructural (ns) genes ns1 and ns2 that are not present in mpv. the functions of these genes are not fully defi ned, but involve interactions with the host response machinery, especially interferons. the presence of these host response-modifying genes may explain in part why rsv appears to cause severe disease more commonly than mpv. many of the gene sequences exhibit some clear global sequence relatedness; however, the extent of the relatedness of many of the sequences of corresponding proteins is relatively low. based on sequence homology, it is not expected that there is a signifi cant amount of immunologic cross-reactivity in responses to the two viruses. the rsv virion buds from airway epithelial cells, incorporating host cell membrane as the lipid bilayer that forms the envelope of the particle. since the virus is enveloped, chemicals that disrupt lipid bilayers (detergents) inactivate the virus, leading to the strong recommendations for healthcare provider hand washing following patient contact. the genome is a single strand of rna, which forms a helical complex with the nucleoprotein (n). the fi nal nucleocapsid structure likely is formed by the complete set of replication proteins, which also include the phosphoprotein (p) and the large rna-dependent rna polymerase (l). it is suspected that the m protein helps the particle to form by bridging the nucleocapsid and the lipid envelope with its incorporated surface proteins. the surface of the particle incorporates three integral membrane surface glycoproteins, the highly glycosylated rsv g protein suspected to be the attachment protein, the fusion protein f, and the small hydrophobic protein (sh). rsv f (a type i integral membrane protein) and rsv g (a type ii integral membrane protein) form oligomers on the surface of the particles, which appear like small spikes with globular heads when seen in electron microscopic (em) images by negative stain. the morphology of particles in em images or in fl uorescent microscopy images labeled by conjugated antibodies suggests that the virion particles are fi lamentous. however, spherical particles, fi laments, and more pleomorphic forms have been observed; therefore, it is uncertain what the morphology of infectious particles in vivo during natural infection is. the f protein is critical for entry into cells, by breaching the barrier of the cell lipid bilayer. it is thought that binding of virion particles to susceptible cells at physiologic ph triggers a conformational change of the f protein from a metastable pre-fusion state [ 25 ] to an altered post-fusion conformation [ 26 , 27 ] in which the hydrophobic fusion peptide in the protein is exposed and extended to insert into the host cell membrane. this membrane insertion event accomplishes a fusion of the viral and host membranes, allowing delivery of the nucleocapsid to the cytoplasm where viral replication occurs. this event is termed "fusion from without" when the particle enters a cell. an alternative fusion event ("fusion from within") occurs when newly expressed f protein on the surface of infected cells causes fusion of an infected cell to an adjacent cell in culture causing "syncytia." it is not certain whether this latter form of fusion (cell-cell fusion) occurs during natural infection and contributes to pathogenesis or if the formation of syncytia is a tissue culture phenomenon only. although there are many animal forms of rsv, there is no known animal reservoir of human rsv; close contact with infected humans is the only source of rsv infection. early prospective studies showed that approximately half of infants in the usa are infected during their fi rst year of life; most were infected after two winter epidemics [ 28 ] . about a quarter of infants exhibit signs and symptoms of lower respiratory tract disease (wheezing and/or pneumonia) during a primary rsv infection [ 14 , 28 -31 ] . rsv is the most common virus associated with hospitalization for respiratory illness and in fact is one of the most common of all causes of infant hospitalization. for example, rsv caused 13 hospitalizations per 1,000 us children younger than 1 year in one large study [ 32 ] . during winter rsv epidemics, over 75 % of the children hospitalized for acute lower respiratory tract infection are infected with rsv [ 33 , 34 ] . the rate of very severe disease in hospitalized infants is high, with about 2-5 % of hospitalized infants requiring mechanical ventilation for respiratory failure [ 35 ] . although primary infection of infants is probably most efficient, rsv can infect subjects of any age [ 28 , 36 , 37 ] . some adult infections are asymptomatic, and most are limited symptoms related to infection of the upper respiratory tract, such as the common cold [ 28 , 36 , 38 ] . since otherwise healthy adults all possess measurable rsv serum antibodies and rsv-specifi c t cells, it is clear that prior exposure and induction of immune responses may not prevent infection. however, disease severity is usually reduced after one or several infections early in life, thus immune effectors such as serum neutralizing antibodies do prevent severe disease during reinfections. unlike infl uenza virus, rsv does not exhibit a progressive antigenic drift. although rsv antigenic diversity is observed in fi eld strains, diversity of the antigenic proteins is not required for rsv to cause reinfection [ 39 -41 ] . most experts believe that serum neutralizing antibodies protect against severe lower respiratory tract disease but do not result in sterilizing immunity against infection at the respiratory mucosa. thus, healthy adults show signs and symptoms of the common cold in about half of cases of natural or experimental infections, even though they have experienced numerous previous rsv infections [ 39 ] . there is a single serotype of rsv, but two antigenic subgroups of rsv, with about 25 % antigenic relatedness, have been defi ned using immune sera; the subgroups are designated a and b. antigenic dimorphism for rsv had been noted in an early study [ 42 ] and subsequently was delineated using mabs, which identifi ed extensive differences in the g protein and less extensive differences in f and other proteins [ 43 ] . the two subgroups exhibit a three-to fourfold reciprocal difference in neutralization by polyclonal convalescent serum. analysis of glycoprotein-specifi c responses in experimental rodent models or human infants by enzymelinked immunosorbent assay (elisa) with purifi ed f and g glycoproteins showed that the f proteins are 50 % related antigenically and the g proteins are 1-7 % related [ 44 ] . consistent with this level of antigenic relatedness, f protein expressed by a vector immunization was equally protective in small animals against challenge with the homologous or heterologous subgroup virus, whereas the g protein was 13-fold less effective against the heterologous subgroup virus [ 45 ] . thus, the f protein is responsible for most of the observed cross-subgroup neutralization and protection. in some communities a pattern of infection with viruses of alternating subgroups has been described, but this is not a universal phenomenon. rsv subgroup b virus is more diffi cult to isolate and propagate in culture, so subgroup b viruses are less commonly associated with severe disease in some studies. however, clearly viruses of both subgroups can cause severe disease leading to hospitalization [ 46 -50 ] . infants exhibit the highest risk of severe lower respiratory tract disease. this elevated risk is explained by a myriad of physiologic, immunologic, and other factors. first, the highest point of resistance in the airways is the bronchioles, and the resistance of airways is inversely proportional to the airway radius to the fourth power (resistance ~ 1/radius 4 , from poiseuille's law). the bronchioles of infants are narrow, and infl ammation and secretion in the bronchioles leads to turbulent airfl ow, and further reduction of the airway lumen size. these physical factors lead to the clinical signs of wheezing (a sign of outfl ow obstruction) and air retention. increased respiratory rate can compensate to some extent for respiratory compromise, but prolonged tachypnea can lead to fatigue and eventual respiratory failure. also, during primary infection infants do not possess active immunity to infection, which allow this effi cient virus to replicate in the airway to titers as high as 10 7 infectious particles per ml of secretion. mothers pass rsv antibodies to infants across the placenta during the third trimester, but premature infants do not obtain maternal antibodies prior to 32 weeks' gestation. also, the airways of premature infants are smaller than those of term infants. another factor leading to respiratory diffi culty is dehydration. obstruction of the nasal passages with thick secretions can impede the feeding of infants, who are obligate nose breathers. rsv also is an important cause of serious infection in elderly adults; in fact rsv appears to cause substantially more fatalities in elderly adults than in children [ 17 , 51 ] . as above, a large study of the us population showed that rsv was associated with approximately 17,000 all-cause deaths per year among persons of all ages in the usa [ 9 ] , with most of those deaths in the elderly. virtually any serious medical or genetic condition is associated with some increased risk of severe diseases [ 32 ] . certain particular categories of subjects are at highest risk for severe rsv disease, including infants with congenital heart or chronic lung disease, immunodefi cient subjects of any age and the elderly [ 9 , 10 , 17 , 33 , 52 -57 ] . these latter subjects are thought to have reduced competency of rsv-specifi c t cells. prematurity increases risk to a small extent, but more importantly chronic lung diseases are important factors [ 10 , 33 , 56 , 58 -62 ] . infants who are born prematurely and then suffer persistent chronic lung disease, especially those needing oxygen supplementation, are at very high risk of hospitalization with rsv. children with cystic fi brosis are at high risk of severe disease [ 63 , 64 ] . in children younger than 2 years with cystic fi brosis, the consequence of rsv infection may be prolonged respiratory morbidity [ 65 ] . children with congenital heart disease also are at increased risk [ 33 , 56 , 58 , 62 , 66 , 67 ] . rsv is a common cause of severe respiratory disease in immunocompromised patients, including lower respiratory disease [ 68 ] . mortality rates in some populations of immunocompromised patients verges on 50 % [ 7 , 69 ] . infants with congenital severe combined immunodefi ciency are at special risk [ 57 , 70 ] , but any acquired immunosuppressive condition such as cancer or transplantation puts patients at risk, especially when t cell function is compromised [ 68 , 71 , 72 ] . rsv infection can cause severe lung disease in recipients of lung transplants, sometimes with a long-term outcome of obliterative bronchiolitis [ 73 -75 ] . children with hiv infection shed rsv for extended periods, but disease is not especially severe in hiv-infected children prior to onset of aids [ 76 -78 ] . interestingly, although most immunocompromised subjects appear to be at risk because of t cell problems, infants with phagocytic cell defects including those with interferon-γ receptor defi ciency or chronic granulomatous disease also are at risk of severe rsv disease. transmission of rsv in the hospital setting can lead to serious disease, especially in critical care units with neonates or other high-risk infants [ 79 -86 ] . nosocomial outbreaks in inpatient transplantation facilities are sometimes severe and unit outbreaks can be diffi cult to terminate because transplant patients can shed rsv for many months [ 57 , 58 , 69 , 84 , 87 , 88 ] . theoretically, transmission in inpatient healthcare settings should be preventable through strict compliance with infection control practices, especially hand washing and contact precautions, which are universally recommended for rsv patients. a high level of compliance with precautions is diffi cult to achieve in busy care settings but is needed to prevent transmission by healthcare providers. the use of the prophylactic monoclonal antibody palivizumab has been studied to interrupt an outbreak in a neonatal intensive care unit setting [ 89 ] , but currently palivizumab use is not recommended for this purpose. bacterial otitis media is a common complication of rsv upper respiratory tract infection. in fact, rsv infection is probably the most common precipitating factor associated with otitis media. rsv antigens and nucleic acids have been reported in middle ear fl uids [ 90 , 91 ] . the disease is predominantly due, however, to eustachian tube dysfunction, resulting in bacterial stasis in the middle ear and subsequent otitis media. rsv regularly occurs in annual epidemics. the us national respiratory and enteric virus surveillance system (nrevss) considers that the rsv season starts when the fi rst of two consecutive weeks during which the mean percentage of specimens testing positive for rsv antigen is ≥10 %. the rsv season is considered to have ended in a community when the mean percentage of positive specimens is ≤10 % in reference laboratories for two consecutive weeks. rsv infection occurs in infants and adults worldwide, in yearly epidemics. the virus has been isolated in every area of the world in which surveillance studies have been conducted. the principal season varies depending on the climate and region, but infection is ubiquitous. virtually all children in the world are infected within the fi rst few years of life. epidemics occur in the winter and early spring in the usa. onset of the annual epidemic varies by region of the country and by year but typically begins in the usa in october or november and lasts through late spring. within a region, the timing of rsv season changes slightly each year. florida often has the earliest onset of rsv epidemic and the longest lasting season in the usa. near the equator, infections may be more common during rainy season. during community outbreaks of rsv, the venues with the highest level of young infants and children exhibit the highest rates of transmission of virus, especially large families and day-care settings with large numbers of children per room. hospital infection control practices must be used to prevent rsv spread, using measures including careful hand hygiene, contact precautions for patient isolation, gowns and gloves [ 92 , 93 ] , and, when direct coughing occurs, facemasks and goggles [ 94 , 95 ] . rsv rapid diagnostic testing can be used in hospital infection control practice to identify rsv-infected patients during the admission process [ 96 , 97 ] . rsv is shed for prolonged periods. it has been reported that 92-100 % of hospitalized children are still shedding infectious virus after 7 days [ 98 , 99 ] . exposure to tobacco smoke and poor nutrition increase the incidence of disease [ 100 -102 ] . low socioeconomic status increases the risk of severe disease for uncertain reasons. lower income populations exhibit a fi ve-to tenfold increased risk of hospitalization in many studies [ 14 , 32 , 103 ]. breast-feeding may confer some protective benefi t against rsv disease, but the extent of the benefi t of breast-feeding has been controversial. the results of epidemiologic studies on benefi t have been confl icting, although recent studies suggest that breast-feeding may have a strong protective effect only in girls [ 104 ] . the sex of the infant modulates the severity of rsv infection for additional reasons that are not fully understood. even though the same high proportion of both male and female infants become infected, males have a higher incidence of rsv lower respiratory tract disease than girls [ 30 , 32 , 105 -108 ] . ethnic and genetic factors appear to play a role. alaska and other native american children are at increased risk of severe respiratory disease during rsv infection [ 109 ] . direct contact with secretions from an infected human, usually by fomites (contaminated objects, including hands), allows transmission of virus. in some cases, infected subjects may inoculate contacts at short distance by coughing via large-particle droplets. however, the virus is not spread efficiently by small-particle aerosols [ 110 , 111 ] . the nasal and conjunctival mucus membranes are probably the most common portals of entry [ 112 ] . rsv is one of the most infectious viruses spread by contact, and transmission is very effi cient even among subjects who possess partial immunity due to prior infection. spread among family members and day-care contacts is especially common. the infectious doses for humans are probably only a few infectious particles per ml of respiratory secretions, but infants routinely shed at least a millionfold higher concentration of virus during the peak of illness. the incubation period is not known defi nitely but is about 3-6 days and likely varies according to the intensity of exposure and the amount of virus in the inoculum. the period of viral shedding is many days; infants can shed infectious virus for weeks [ 98 , 99 ] . rsv can survive on hard surfaces for greater than 24 h. infection occurs by inoculation of the nasal or conjunctival mucosa, often by self-inoculation with infected secretions from a close contact. adult volunteers were infected experimentally if virus was inoculated onto the conjunctival sac or into the nose, but not following introduction into the mouth [ 112 ] . the incubation period from time of inoculation to onset of illness for rsv is likely about 4-5 days [ 113 ] . virus replication initiates in the nasopharynx and rapidly can reach concentrations of over a million particles per ml in the upper airway in children. adults, who are partially immune, typically shed much lower amounts of virus. virus spreads quickly to the lower respiratory tract, often causing symptoms within days of onset of upper respiratory symptoms. virus may spread from cell to cell, but also it is likely that small aspirations of upper respiratory secretions with virus inoculate the lower tract. higher titers of virus in respiratory secretions usually are associated with increased severity of disease, in prospective studies of natural infection [ 114 ] or of clinical vaccine trials [ 115 ] . rsv infection is an acute infection and virus shedding usually resolves within days to weeks. rt-pcr tests for virus nucleic acid may remain positive for prolonged periods. some animal studies suggest that a negligible amount of infectious virus may persist in airways far after apparent resolution of shedding, as evidenced by the recovery of low amounts of infectious virus during immunosuppression several months after infection [ 116 ] . the virus infects respiratory epithelial cells in the lung and airway. it is not clear whether rsv also replicates productively in macrophages and dendritic cells in the airway or not. rsv protein antigens have been detected in circulating mononuclear cells [ 117 ] , and viral genomic rna and mrna have been detected by rt-pcr in blood cells [ 118 ] , but this probably represents cells from the airway recirculating, as infectious virus in the blood (viremia) is not detected. rsv replicates in the cells at the luminal surface of the respiratory epithelium and virus both enters and is shed from the apical surface of infected epithelial cells [ 119 , 120 ] . studies of polarized, differentiated respiratory epithelial cells in vitro show that rsv infection preferentially infects ciliated cells at the luminal face [ 121 ] , but it is not clear if infection is restricted to such cells in humans. histopathology studies of infected humans are very limited, but show rsv antigens in the superfi cial cells of the airway in a patchy distribution, with antigen-positive cells and debris in the airway lumen. pathology caused by rsv infection during infant bronchiolitis includes necrosis and proliferation of the bronchiolar epithelium and destruction of ciliated epithelial cells [ 120 , 122 ] . there is an infl ux of a large variety of immune cell types including neutrophils, lymphocytes, and macrophages. the respiratory tissues become edematous. mucous secretion, sloughing of dead cell debris, and the infl ux of apparent infl ammatory cells obstruct the lumen of the narrow bronchioles and alveoli. the small diameter of infant bronchioles is easily obstructed in the presence of dead cells and edema of the airway tissues [ 123 ] . virus-infected cells can be identifi ed in the epithelium of the bronchi, bronchioles, and alveoli [ 120 ] . it is surprising, given the extent of disease, that rsv antigen staining is usually patchy or focal, and even in some cases of fatal rsv bronchiolitis, antigen is present only in small amounts [ 119 , 120 ] . the number of cases that have been collected is limited, however, and there is virtually no histopathology from milder cases. rsv upper respiratory infection is complicated frequently by otitis media caused by bacteria. it is unusual to observe frank bacterial pneumonia or sepsis as a complication of rsv infection, in contrast to some other respiratory viruses like infl uenza. although some clinicians may empirically use antibiotics during infant pneumonia, there is no evidence that antibiotic therapy alters the course of rsv bronchiolitis or pneumonia. antimicrobial therapy should not be used in most cases of rsv bronchiolitis or pneumonia because of the lack of benefi t and risk of selection of antibiotic-resistant colonizing organisms. nevertheless, there is some suggestion that bacterial-viral interactions may affect the overall rate of disease in the population. it is interesting that annual rsv and infl uenza virus epidemics correlate directly with the time of peak incidence of invasive pneumococcal disease in many population studies [ 124 ] . a double-blind, randomized, placebo-controlled trial of pneumococcal conjugate vaccine showed a vaccine-attributable reduction in rates of childhood viral pneumonia requiring hospitalization, caused by any of seven respiratory viruses, including rsv [ 125 ] . the immune mechanisms responsible for resolution of infection and protection against reinfection by rsv are not fully defi ned. antibodies . most experts agree that high levels of serum neutralizing antibodies are associated with relative protection against severe lower respiratory tract disease in otherwise healthy subjects. this idea is supported strongly by the observation that prophylaxis of high-risk infants with a neutralizing monoclonal antibody prevents about half of hospitalizations in that group of patients. many population studies suggest that infants born with high levels of transplacental rsv-neutralizing maternal antibodies develop milder illness or illness at an older age than infants with low maternal antibody levels [ 15 ] . most infants and children in whom maternal antibodies have declined to a low level make their own serum and secretory antibodies to both the f and g surface glycoproteins in response to rsv infection [ 126 ] . antibody responses in neonates are particularly low in quality and magnitude due to immunologic immaturity and the suppressive effect of passively acquired maternal antibodies [ 126 , 127 ] . antibody-mediated immune suppression by passive antibodies primarily affects humoral rather than cellmediated immunity [ 128 , 129 ] . high levels of serum antibodies do not appear to provide solid immunity against disease in the upper respiratory tract. mucosal secretory iga appears to contribute to local protection against reinfection in the airway, although potent protective iga responses are likely relatively short lived. in human infants, the decrease in virus shedding in nasal secretions was associated with the appearance of rsv-specifi c iga antibodies [ 130 ] . t cells . t cells clearly play a major role in resolution of active infection. rsv-specifi c t cells with cytolytic activity, thought to be cd8+ t cells, have been detected in peripheral blood mononuclear cells from infants with rsv disease [ 131 ] . immunodefi cient children, especially those with t cell defects, often fail to clear rsv infection and can shed virus for many months [ 57 ] . adults with leukemia or hematopoietic stem cell transplant also have a very high incidence of prolonged rsv infection leading to severe disease and sometimes death. patterns of host response rsv infection usually causes upper respiratory tract symptoms during primary infection in otherwise healthy term infants; asymptomatic primary infection is not common. there is often profuse rhinorrhea, and the upper tract disease is very often complicated by otitis media. symptoms of lower respiratory tract involvement occur in about a third of primary cases [ 28 ] . the principal diagnoses are bronchiolitis (manifested by tachypnea and wheezing) and pneumonia. these entities are probably not discrete processes but more likely represent a continuum of disease involving increasing tissue distribution. the typical illness starts with nasal congestion, followed in a few days by cough. the infection is sometimes associated with fever, which is usually low grade. after several days of upper tract symptoms, infants may wheeze. many infants suffer mild wheezing that resolves, but some cases progress with tachypnea, diffuse inspiratory crackles, and expiratory wheezes. most children recover in 1-2 weeks with supportive care and observation. if expiratory obstruction becomes severe, however, hyper-expansion of the chest occurs due to air retention, and the compliant nature of the infant chest wall leads to intercostal and subcostal retractions during tachypnea. with prolonged tachypnea, fatigue may occur with poor oxygenation and co 2 retention (measured by pulse oximeter or arterial blood gas measurement), markers of respiratory failure. intubation and mechanical ventilation is used in this setting to manage the respiratory failure. infection during the fi rst day or weeks of life may just be characterized by temperature instability or fever, irritability, and lethargy even in the absence of overt respiratory signs or symptoms. in very young infants, especially those born prematurely, apneic spells may occur in response to rsv infection. apnea may be the fi rst reported evidence of infection in some cases, and apneic spells may recur during the acute infection. these events thankfully are usually self-limited and rarely cause neurologic damage. apneic events are an indication for hospitalization and careful medical supervision with respiratory monitoring. because of the association with apnea, some have considered whether rsv is associated with sudden infant death syndrome (sids). although rsv has been detected in the lung tissues of some cases of sids, there is no statistically signifi cant association between rsv and sids. the reported cases likely refl ect a temporal association caused by the high prevalence of rsv in this age group, the prolonged pattern of shedding, and the simultaneous peak incidence of both rsv infection and sids in winter months [ 132 ] . it is not clear whether infection with rsv causes prolonged abnormal pulmonary function during childhood or whether children with underlying predisposition to lower respiratory tract disease of all causes manifest their susceptibility fi rst to rsv because of the young age of rsv infection. certainly measureable pulmonary function abnormalities are common after rsv lower respiratory tract disease, and these fi ndings may persist for a decade or more [ 133 ] . recurrent wheezing is common during subsequent viral infections after severe rsv bronchiolitis or pneumonia, with an incidence of 10-50 % [ 134 ] . a large case-control study of 200 children hospitalized for bronchiolitis or pneumonia in which rsv was the most common cause found that 7 years later there was a strong predisposition in these subjects toward decreased pulmonary function, recurrent cough, wheezing, and bronchitis [ 135 ] . seminal prospective studies by martinez et al. involved measurement of pulmonary function in infants at birth and then found a strong correlation between prior lower pulmonary function and the development of wheezing during rsv infection [ 136 ] . this correlation persisted in children during the fi rst 3 years of life [ 136 ] . even some individuals who do not typically exhibit recurrent wheezing have postexercise or pharmacologically induced bronchial reactivity [ 137 ] , which may be responsive in part to bronchodilators [ 134 ] . symptomatic upper respiratory tract rsv infections manifested by common cold symptoms are common in otherwise healthy adults, especially in those with frequent exposure to small children [ 80 ] . in the elderly, particularly those with underlying medical diseases, severe pneumonia may occur, leading to hospitalization or even death. astute clinicians can often make a presumptive diagnosis of infection based on the clinical signs of wheezing or pneumonia in an infant during a local epidemic. laboratory testing of nasal or lower airway secretions by antigen test (elisa) or nucleic acid detection (by rt-pcr) provides rapid diagnosis of the presence of virus in many cases. the gold standard for diagnosis is isolation of the virus in cell culture, but this test is typically only available in referral laboratories because of the need for extensive equipment and a high level of technical expertise. control and prevention primary treatment is supportive care, which includes oral or intravenous hydration; monitoring of respiratory status, especially of oxygen saturation during tachypnea; use of supplemental oxygen; removal of secretions from the upper airway; and, in the case of respiratory failure, intubation and mechanical ventilation. advances in support care in pediatric critical care units have caused a major decrease in morbidity and mortality from rsv in the developed world. infants hospitalized for rsv disease should be monitored for apnea. investigators have studied nitric oxide [ 138 , 139 ] mixtures of helium and oxygen [ 140 , 141 ] and surfactant treatment [ 142 ] in clinical experimental studies in the support of infants with severe rsv disease. nitric oxide treatment does not appear to mediate a bronchodilator effect during rsv infection [ 139 ] . therapy of rsv disease by any antiviral agent is challenging because it is a rapid acute infection, and by the time the onset of disease is recognized, it may be too late to alter the course of disease by reducing viral load. the guanosine (ribonucleic acid) analog ribavirin is a nucleoside inhibitor that inhibits viral rna synthesis and viral mrna capping. the drug has in vitro antiviral activity against rsv, and aerosolized ribavirin therapy has been associated with a small but statistically signifi cant increase in oxygen saturation during the acute infection in several small studies. decreases in mechanical ventilation and duration of rsv-associated hospitalization have not been proven (reviewed in [ 143 ] ). ribavirin was approved in 1986 in the usa for treatment of rsv infection [ 144 ] . clinically, the drug usually is administered as a small-particle aerosol using a tent, mask, or mechanical ventilator, delivered for 6-18 h daily for a period of 3-7 days. the drug now is not recommended for routine use because follow-up studies have not shown a major benefi t. the drug may be considered for use in select patients with documented, potentially life-threatening rsv infection. over a dozen other experimental small molecule inhibitors of rsv fusion to cells have been described and tested in preclinical studies for inhibition of rsv, but none have progressed in development to date. a third approach employs short interfering rnas (sirnas), taking advantage of an ancient host cell regulatory system. single-stranded and double-stranded rna molecules that exhibit rsv-specifi c small interfering rnas have been developed for treatment, which cause rna interference activity against rsv, destroying the corresponding rsv rna. these novel compounds have shown promising results in preclinical studies [ 145 ] and have been tested in small clinical trials. human immune globulin with a high titer of rsv antibodies and the rsv monoclonal antibody palivizumab have been tested as therapy of acute rsv disease, but they were not effective for treatment of established disease. anti-infl ammatory strategies have been investigated. no benefi t of corticosteroid therapy on disease severity or length of hospital stay has been demonstrated, despite studies in over a dozen randomized clinical trials of outpatients or hospitalized infants with rsv bronchiolitis. since the drug is of no benefi t on its own [ 146 ] and may prolong virus shedding, it is not recommended. in the future, a possibility might be to combine an effective antiviral treatment with an antiinfl ammatory agent [ 147 ] . intravenous antimicrobial therapy is not appropriate in hospitalized infants with rsv bronchiolitis or pneumonia unless there is clear evidence of secondary bacterial infection. otitis media occurs very often in infants with rsv bronchiolitis; oral antimicrobial agents can be used for therapy of otitis media if necessary. it is intuitive to think of using beta-adrenergic agents, commonly used for the treatment of asthma, to treat the wheezing associated with rsv infection. these agents are not usually recommended for routine care of fi rst-time wheezing associated with rsv bronchiolitis. short-term improvements in oxygenation and clinical scores can be achieved by these therapies, but it has not been established that their use results in improvements in duration or severity of illness or disease outcomes. studies in this area have been confl icting, but systematic reviews of randomized clinical trials of nebulized beta-agonist therapy for treatment of bronchiolitis suggest that they offer little benefi t [ 148 , 149 ] . alpha-adrenergic receptor stimulation results may decrease interstitial and mucosal edema [ 150 ] , and use of nebulized epinephrine (with combined alpha-and beta-adrenergic activity) has been studied with confl icting results [ 151 , 152 ] . alphaagonist stimulation of the sympathetic nervous system is expected to reduce capillary leakage by constricting precapillary arterioles, reducing hydrostatic pressure and consequently bronchial mucosal edema [ 150 ] . racemic epinephrine treatment relieves some respiratory distress but does not affect length of stay [ 153 ] . the usefulness of such agents in the management of rsv bronchiolitis is not clear. the most effective mode of prevention is to avoid contact with infected subjects. in the hospital setting, careful adherence to infection control practices is important for the protection of high-risk patients from rsv infection. careful hand washing may reduce transmission in family and daycare settings. pharmacologic intervention is indicated to prevent hospitalization for the highest risk infants, however. passive rsv immunoprophylaxis with antibodies has proven a costly but relatively effective intervention. parenteral infusion of rsv-neutralizing antibodies into experimental animals was shown early on to confer substantial resistance in the respiratory tract to a subsequent rsv virus challenge [ 154 ] . signifi cant reductions in rsv-associated hospitalizations and disease severity in high-risk human infants were fi rst accomplished with prophylactic administration of human immunoglobulin with high rsv-neutralizing activity given by the intravenous route (rsv-ivig; fda licensed in 1996) [ 155 , 156 ] . monthly intravenous infusions during the rsv season reduced the frequency of pediatric hospitalization and duration of stay by approximately 55 % and decreased the number of days spent in intensive care by 97 %. the use of rsv-ivig was superseded by the use of a monoclonal antibody (mab) that was developed subsequently that could be given by intramuscular route, and production of the former has been discontinued. several mabs were developed for immunoprophylaxis against rsv. the most successful of these was based on murine mab 1129 [ 157 ] , which is specifi c to the f protein and effi ciently neutralizes viruses of both rsv subgroups a and b. this mab was humanized by recombinant methods by transferring its variable regions onto a human igg1 backbone, resulting in a recombinant antibody now named palivizumab [ 158 ] . this mab is 50-100-fold more effective for in vitro neutralization on a per weight basis than was rsv-ivig, and thus the total amount of immunoglobulin administered could be reduced to an amount that could be given im. palivizumab (trade name synagis) was licensed in 1998 for rsv prophylaxis of high-risk infants, following studies demonstrating its safety and effi cacy [ 159 -162 ] . palivizumab is administered monthly through the rsv season and has been widely used in high-risk patients with prematurity, chronic lung disease, and hemodynamically signifi cant heart disease [ 163 ] . more potent derivatives of this recombinant antibody have now been developed [ 164 ] ; however, the lead candidate from these affi nity maturation efforts exhibited increased side effects in a large effi cacy study. prevention of severe disease probably will best be accomplished by development and use of an effective vaccine. vaccine development for rsv has proven exceptionally diffi cult, however. first, young infants are a diffi cult population to immunize. obstacles to immunization at this early age include immunologic immaturity and immunosuppression by maternal antibodies, as already noted [ 165 ] . also, severe adverse events occurred in early rsv vaccine trials. a formalin-inactivated rsv vaccine candidate (fi-rsv) was developed and evaluated in infants and children in the 1960s [ 166 , 167 ] . this vaccine suspension was made by mixing concentrated, inactivated virus with alum adjuvant and was delivered by the intramuscular (im) route. this inoculation did not protect against infection or disease, but rather during subsequent natural infection vaccinees experienced more frequent and severe disease. most fi-rsv vaccinees (80 %) required hospitalization during subsequent natural infection, compared to 5 % in the control group. autopsies of two fatalities showed evidence of rsv replication and pulmonary infl ammation [ 167 ] . this event put a chilling effect on rsv vaccine development efforts. therefore, rsv protein vaccines have been problematic for use in infants given their possible potential for disease enhancement, together with the poor immunogenicity in this population. however, an rsv protein vaccine might be useful in boosting immunity in rsv-experienced older children and adults who are at increased risk of severe rsv disease due to underlying disease or advanced age. protein vaccines for rsv have been evaluated clinically for use in such rsvexperienced individuals, in whom they appear to be safe. one experimental subunit vaccine consisted of purifi ed f protein (pfp) isolated from rsv-infected cell culture. this purifi ed protein vaccine candidate was evaluated in adults, in older children with and without underlying medical diseases, and in the elderly [ 168 ] . the pfp vaccine candidate was well tolerated and moderately immunogenic in these settings. a large multicenter study in children 1-12 years of age with cystic fi brosis did not provide evidence of signifi cant protection against rsv infection [ 169 ] . pfp also has been evaluated for maternal immunization in the third trimester of pregnancy. in the single study to date, the increase in antibody titers was only minimal [ 170 ] . maternal immunization studies are being pursued currently with newer non-replicating vaccine candidates such as emulsion vaccines [ 171 ] and nanoparticle protein preparations [ 172 , 173 ] . live-attenuated vaccines represent an attractive strategy for preventing rsv, since live infection induces a balanced immune response that is not associated with enhanced disease on subsequent natural infection. many live-attenuated rsv vaccine candidates have been developed over several decades. it has proven diffi cult to identify a candidate that is satisfactorily attenuated while remaining satisfactorily immunogenic in the youngest infants. clinical trials of a safe, live-attenuated rsv vaccine for intranasal administration have shown restriction of viral replication in infants following administration of a second dose and have been encouraging [ 174 ] , and additional attenuated vaccine candidates are being developed [ 175 ] . despite over 50 years of research on rsv, many challenges and questions remain. there are many unanswered fundamental questions about the biology of the organism and the pathogenesis of disease. why does reinfection occur throughout life? what are the defi nitive mechanisms of immunity in humans? is there a genetic basis for susceptibility to severe disease? does severe rsv disease cause asthma? what drives the seasonality of rsv? the mortality in infants has been greatly reduced in the usa through advances in critical care, but little rsv-specifi c intervention is available. currently, there is little to offer for therapy except for supportive care. prophylaxis of high-risk infant with a mab prevents some hospitalizations but is expensive and is not always effective. there are no licensed vaccines. given the disaster of early fi-rsv trials, it is not clear that a non-replicating vaccine can be proven safe enough in preclinical models to absolutely assure that enhanced disease will not occur. on the other hand, the explosion of new technologies for generation of recombinant rsv strains, the determination of pre-and post-fusion antigen structures, and new tools for the detailed study of the molecular and genetic basis of human immune responses suggest that much progress will be made in the rsv research fi eld in the coming years. mpv was discovered by investigators in the netherlands who cultivated specimens from children with respiratory infection on a variety of cell types [ 176 ] . a hitherto unknown virus produced cytopathic effects (cpe) in tertiary monkey kidney cells, but could not be identifi ed by antibody staining or rt-pcr for common viruses. electron microscopy of infected cells revealed pleomorphic enveloped particles with surface projections suggesting protein spikes, while biochemical experiments showed that the virus contained a lipid envelope and did not hemagglutinate avian or mammalian red blood cells. elegant randomly primed rt-pcr experiments yielded multiple fragments of genome, which sequence analysis identifi ed as related to avian metapneumovirus (ampv). ampv (formerly turkey rhinotracheitis virus), identifi ed in 1979, is an important global pathogen of poultry including turkeys and chickens [ 177 ] . there are four serotypes of ampv (a-d), and mpv is most closely related genetically to ampv-c [ 178 ] . phylogenetic analysis shows that mpv likely diverged from ampv-c ~200 years ago and thus mpv is of zoonotic origin [ 179 , 180 ] . however, mpv exhibits extremely restricted or no replication during experimental infection of chickens or turkeys and thus is a true human pathogen [ 176 ] . human poultry workers exhibit serological evidence of asymptomatic infection with ampv, providing evidence for the feasibility of an original trans-species transmission event [ 181 ] . while recently identifi ed, mpv is not truly a new virus. studies using archived sera collected in the 1950s revealed a 100 % seroprevalence in humans greater than 5 years old [ 176 ] . nasal washes collected prospectively during the 1970s from children with ari had detectable mpv rna upon retrospective testing by rt-pcr 25 years later [ 182 ] . the specialized cell culture requirements, slow growth, and limited cpe of mpv likely prevented the earlier discovery of this common respiratory pathogen. limited mortality data are available and consist of sporadic case reports, case series, or the identifi cation of fatal cases of mpv infection in research studies. mpv is not a reportable infection and there is no specifi c icd-9 diagnostic code. thus, an accurate estimate of the mortality associated with mpv is not feasible. however, lower respiratory infections are a leading cause of death in children worldwide, primarily in developing nations. mpv is a common cause of severe lower respiratory infection and thus likely accounts for a substantial number of deaths globally. a substantial body of literature has accumulated in the last decade describing the epidemiology, disease burden, and clinical features of mpv. many groups have used standard techniques to provide some estimate of the burden of mpv in diverse populations. most reports have been crosssectional studies of selected populations, usually based on convenience samples of patients with acute respiratory illness. these studies are thus limited by potential selection bias, narrow time periods, and incomplete demographic or clinical data and often lack controls. nonetheless, the broad application of these studies to sizable global populations has illuminated the frequency of mpv infection. many of these studies have focused on special populations, such as patients with asthma, immune compromise, or chronic obstructive pulmonary disease (copd), and thus offer valuable information about mpv among these persons. a number of prospective, well-designed studies in adults and children have been published and offer the best estimates of the population-based incidence of mpv infection. some of these used preexisting prospective data and samples collected prior to the discovery of mpv for retrospective analysis. classical methods including active day care and clinic surveillance, as well as newer approaches such as home surveillance with parent-collected swabs, have been used. these studies have been built upon the foundations of seminal longitudinal studies conducted to investigate other viruses including infl uenza, parainfl uenza viruses, and rsv. taken together, these reports provide a broad survey of mpv epidemiology across diverse geographic environments, socioeconomic populations, and high-risk groups. seroprevalence studies using diverse methods have been performed in different populations. most have used enzymelinked immunosorbent assay (elisa) techniques against whole virus or purifi ed proteins to detect igm or igg. a few have used immunofl uorescent detection of mpv-specifi c antibodies or measured serum virus-neutralizing antibodies. these data have mainly been useful in determining the ubiquity of infection with mpv. some studies have measured acute and convalescent sera to diagnose mpv infection, while others have attempted to establish a serum neutralizing titer that correlates with susceptibility to infection. inherent limitations of serological surveys include the potential for cross-reactive antibodies and the lack of standardized reagents for mpv. most studies have used rt-pcr to detect mpv due to the diffi culty in cultivating the virus. the original isolation of mpv in tertiary monkey kidney cells was possible because the investigator had access to a monkey kidney cell source that was free of the endogenous simian foamy virus (a.d.m.e. osterhaus, personal communication). primary monkey kidney cells commercially available in the usa all contain sfv, and even the addition of anti-sfv antisera cannot prevent the growth of this endogenous virus prior to the slow emergence of mpv. further, the fusion protein of mpv requires cleavage by exogenous trypsin for robust in vitro growth. trypsin is added by most clinical virology laboratories only to cultures of madin-darby canine kidney (mdck) cells for the isolation of infl uenza virus; however, mdck cells are very poorly permissive for mpv even in the presence of trypsin. finally, primary isolation of mpv often requires one or more passages prior to visible cpe and few laboratories routinely follow this procedure. unlike rsv, mpv is not particularly labile to freeze-thaw cycles [ 183 ] and thus can be retrospectively isolated from pcr-positive specimens. fluorescent antibody staining of patient specimens or shell vial cultures can facilitate more rapid identification [ 184 -187 ] . thus, molecular diagnostic techniques have been used for virtually all studies of mpv epidemiology. a number of sensitive and specifi c real-time rt-pcr assays have been described [ 188 -195 ] . many early assays were based on limited sequence data and subsequently were found to be suboptimal for detecting multiple diverse strains [ 195 ] . both individual and multiplexed rt-pcr assays offer more sensitive detection of mpv than culture; however, multiplex assays sometimes balance decreased sensitivity for a single agent with the convenience of detecting multiple viruses simultaneously [ 196 ] . another limitation of molecular detection for all viruses is the ability to detect low levels of viral nucleic acid in the absence of infectious virus. it has become common to detect more than one virus in a single specimen, and the interpretation of these data is far from clear. community respiratory viral infections are frequent in childhood, and the likelihood of detecting viral genome prior to the onset of illness or for prolonged periods after illness resolution complicates the assignment of causation to one of several co-detected viruses. mpv is an enveloped pleomorphic virus ranging in size from 150 to 600 nm, containing a single-stranded negative-sense rna genome [ 178 ] . complete genomic sequences of numerous mpv strains have been published [ 178 , 180 , 197 ] . the genome comprises eight separate open reading frames encoding nine distinct proteins (fig. 26.1 ) . mpv genes are analogous to those of rsv (though ns1 and ns2 are absent in mpv), but the organization of genes differs. ampv and mpv have been taxonomically classifi ed in the separate metapneumovirus genus based on the gene order. phylogenetic analysis of mpv genes consistently identifi es four genetic clades, two major groups designated a and b, each with two minor groups designated a1, a2, b1, and b2 [ 180 , 198 -203 ] . one group has suggested further sublineages based on partial f sequence diversity [ 204 ] , but there is no evidence that this further genetic distinction is of any antigenic or immunologic importance. the two major surface proteins are the fusion (f) and attachment (g), with a third integral membrane short hydrophobic (sh) protein. f is the target of neutralizing antibodies in animal models, f-only vaccines induce protection in animals, and f-specifi c monoclonal antibodies provide passive protection [ 205 -213 ] . in contrast, g-specifi c antibodies do not neutralize virus and g-only vaccines induce neither neutralizing antibodies nor protection [ 206 , 209 , 214 ] . thus, it appears that mpv is unique among human paramyxoviruses in that the attachment protein does not contribute to protective antibodies. further, the g protein exhibits a high degree of genetic variability between subgroups, with as low as 29 % amino acid identity between the major a and b subgroups and a minimum 60 % identity within subgroups [ 202 , 215 -218 ] . the selective pressure for this diversity is unclear. in contrast to g, the f protein is conserved, with a minimum 94 % amino acid identity between a and b subgroups and a minimum 98 % identity within subgroups [ 179 , 202 , 203 ] . presumably there are functional constraints on the diversity of f, since the mutation rate of mpv is high, similar to other rna viruses. the major question regarding the diversity between major or minor subgroups is whether it contributes to antigenic variation or escape in human populations. cross-neutralization against heterologous virus from the a and b lineages was tested using experimental infection of ferrets [ 202 ] . this study found relative neutralization of homologous to heterologous virus ranging from 12 to 96-fold difference, thus providing some evidence for antigenic serotypes. however, subsequent experiments using hamsters, african green monkeys, chimpanzees, and rhesus macaques found that the a and b groups were 64-99 % related antigenically [ 219 ] . infected animals developed neutralizing antibodies that were highly effective against heterologous virus, and previously infected primates were protected against challenge with heterologous virus. cynomolgus macaques infected with a or b subgroup viruses or with candidate vaccines exhibited only a 6-16-fold difference in neutralizing titer against homologous and heterologous viruses [ 220 , 221 ] . taken together, these data show that while mpv f exhibits some antigenic diversity, the virus does not have truly distinct serotypes. the potential implications for human epidemiology are discussed further below. descriptive epidemiology numerous studies document the fact that mpv infection is ubiquitous and that reinfection is common. serosurveys testing large sample collections in canada, china, croatia, germany, israel, japan, the netherlands, taiwan, thailand, the usa, and uruguay show that 95-100 % of children have antibodies against mpv by the age of 5 years [ 222 -231 ] . in many of these studies, 50-75 % of children are seropositive by age 2 years, suggesting that most acquire primary mpv infection early. most identify a decrease in serum mpv antibody titer from birth to 6-12 months, presumably due to the expected decline of maternally derived antibodies. studies in japan and india that compared mpv and rsv titers in the same cohort found that after the expected nadir during early infancy, rsv titers began increasing at an earlier age than mpv [ 232 , 233 ] . this fi nding is interesting in light of epidemiologic data suggesting that primary mpv infection peaks between 6 and 12 months of life compared with the peak of rsv at 2-3 months (discussed below). longitudinal studies in adults and children have documented reinfection by a fourfold rise in serum antibody titer [ 222 , 230 , 231 , 234 -237 ] . the serological data show that mpv infection is nearly ubiquitous during the fi rst years of life. further, reinfection occurs throughout life. in children, primary mpv infection is associated commonly with lower respiratory illness, while reinfection is associated with upper tract disease [ 182 , 238 ] . most epidemiologic studies of mpv in children show that the virus is the second leading cause of lower respiratory infection after rsv. the prevalence of mpv in studies of children with lri is 5-25 %. a 25-year prospective study of otherwise healthy children <5 years old detected mpv in 12 % of children with lri; several children experienced recurrent infection [ 182 ] . a 2-years multicenter study of inpatient and outpatient japanese children with ari identifi ed mpv in 57/637 (8.9 %) [ 239 ] . a 5-years observational study of otherwise healthy korean children <5 years old found mpv in 24/515 (4.7 %), similar to the rates for infl uenza and parainfl uenza virus type 3 (piv-3) [ 240 ] . a very large observational study in queensland, australia, tested specimens obtained from patients of all ages with lri from 2001 to 2004; mpv was detected in 707/10,025 (7.1 %). ninety-two percent of patients with mpv were <5 years old, and mpv was the second most common virus after rsv in these children [ 241 ] . a south african group tested specimens from children hospitalized with ari who were subjects in a prospective pneumococcal vaccine trial; mpv was present in 126/1,409 (8.9 %) and was the most common virus after rsv. the estimated incidence of mpv-associated hospitalization in hiv-negative children was 29/1,000; a number of children had repeat infections [ 242 ] . a prospective study of hospitalized children in hong kong over a 13-month period found mpv in 32/587 (5.5 %); the estimated incidence of mpv-associated hospitalization was 4.4 per 1,000 children <6 years old [ 243 ] . a chinese group conducted a prospective 2-years study of children hospitalized with ari and identifi ed mpv in 227/878 (25.9 %), with most <6 years old [ 244 ] . a large, population-based, prospective surveillance study conducted in three us cities over 6 years found that the incidence of hospitalization for mpv in children <5 years old was 1 per 1,000, lower than the rate of rsv-associated hospitalization in the same cohort (3/1,000) but similar to the rates for infl uenza (0.9/1,000) and piv-3 (0.5/1,000) [ 245 ] . respiratory disease is among the leading causes for hospitalization of adults in the usa, and "infl uenza and pneumonia" ranks among the top 10 causes of deaths annually. although the data are limited, mpv appears to be associated with a substantial burden of ari in adults, primarily those with comorbidities. a prospective study in rochester, ny, enrolled four cohorts during four winters: healthy adults >65, high-risk adults >65 with comorbidities, healthy adults 19-40 years old, and adults hospitalized for acute cardiopulmonary illness [ 246 , 247 ] . overall, mpv infection was detected in 8.5 % of ari in the cohort. the rate of mpv infection was highest in young adults at 13 %, though many of these were asymptomatic and detected only serologically. of note, this group had a mean age of 33, was predominantly female, and had daily exposure to children. of the hospitalized patients, the incidence of mpv annually ranged from 4.4 to 13.2 %. more than 85 % of the hospitalized mpvinfected subjects had underlying conditions, chiefl y cardiopulmonary disease or diabetes mellitus. there were six deaths in this study, all with comorbidities; one had concomitant bacteremia with streptococcus pneumoniae . interestingly, the incidence of mpv infection was similar to the annual average infection rate for rsv (5.5 %) and infl uenza a (2.4 %) in these cohorts during the same study period. a prospective, population-based study in nashville, tn, recruited adults hospitalized for ari at several county hospitals over three winters [ 248 ] . of 508 subjects, 23 (4.5 %) had mpv, 33 (6.5 %) infl uenza, and 31 (6.1 %) rsv. notably, mpv-infected subjects were signifi cantly older than infl uenza-infected subjects (mean 76 vs. 60 years) and had higher rates of chronic cardiopulmonary disease (78 % vs. 52 %). the overall population-based rates of hospitalization for the three viruses were similar, at 1/1,000 for mpv, 1.5/1,000 rsv, and 1.2/1,000 for infl uenza. however, for subjects ≥65 years, hospitalization rates were much higher for mpv and rsv at 2.2/1,000 for mpv and 2.5/1,000 for rsv compared to 1.2/1,000 for infl uenza, likely refl ecting the use of infl uenza vaccine for older adults. a prospective study of community-acquired pneumonia in canada found mpv in 4 % of hospitalized cases during an 18-month period, all with underlying conditions [ 249 ] . a dutch group detected mpv in 2 % of bronchoalveolar lavage specimens from intensive care unit patients. all were >50 years old with comorbid conditions and 83 % died [ 250 ] . similarly, a retrospective study in north ireland found mpv in only 0.8 % of residual respiratory specimens from adults, but 33 % of these died [ 251 ] . together, these data show that mpv is an important cause of acute respiratory disease in adults, primarily older adults or those with underlying comorbid conditions. mpv is associated with acute asthma exacerbations [ 252 -254 ] . mpv was detected in 10 of 132 hospitalized finnish children with acute wheezing [ 255 ] . similarly, mpv was isolated from 7 % of adults hospitalized for acute asthma exacerbation, only one of whom tested positive 3 months later [ 256 ] . premature infants who developed mpv bronchiolitis within the fi rst year of life had decreased lung function at 1 year of age [ 257 ] . a prospective, case-control study of children with mpv bronchiolitis during infancy compared to infants with acute gastroenteritis found that mpv infection early in life was signifi cantly associated with a later diagnosis of asthma and recurrent wheezing at 5 years of age [ 258 ] . mpv causes severe disease in children with comorbid conditions such as cardiac and pulmonary disease or prematurity [ 259 -263 ] . a prospective 1-year study of hospitalized children found that 34 % of patients with mpv had a history of prematurity, chronic lung disease, complex congenital heart disease, or immunodefi ciency [ 264 ] . vicente et al. detected mpv by rt-pcr in 6 % of adults >64 years old with acute exacerbations of copd; none had other pathogens identifi ed by culture or pcr [ 265 ] . a canadian study found that 4 % of hospitalized adults with communityacquired pneumonia or copd exacerbations tested positive for mpv, all with comorbid conditions; one also had infl uenza a and s. pneumoniae [ 266 ] . rsv was present in 9 % and infl uenza a in 6 % of the cohort. mpv was detected in 12 % of adults hospitalized for copd exacerbation during one winter in connecticut, none with other viruses codetected; rsv was present in 8 % and infl uenza a in 4 % of the entire cohort [ 267 ] . severe and fatal mpv disease can occur among immunocompromised individuals, including solid organ and stem cell transplant recipients, hiv-infected persons, and chemotherapy patients [ 268 -275 ] . mpv is associated with morbidity and mortality in adults with hematologic malignancies and stem cell transplant; [ 270 , 276 ] mpv was detected in bronchoalveolar lavage specimens from 5/163 (3 %) episodes of acute respiratory infection in stem cell transplant recipients, and four died [ 270 ] . hiv-positive south african children with mpv were signifi cantly more likely to receive a diagnosis of pneumonia and experience longer hospitalization, lower mean oxygen saturation, and bacteremia; further, hiv-positive children were fi vefold more likely to be infected with mpv than hiv-negative children [ 242 ] . mpv has been implicated in several hospital and institutional outbreaks leading to mortality [ 277 , 278 ] . kim and colleagues [ 279 ] report the transmission of mpv to pediatric hematology-oncology patients during a nosocomial outbreak. the incubation period was between 7 and 9 days. standard, but not droplet, precautions were used. in laboratory studies, infectious virus persists on metal and nonporous surfaces for up to 8 h [ 183 ] . due to the signifi cant morbidity and mortality of mpv in high-risk children, isolation precautions are important. mpv is associated with both upper and lower respiratory tract disease [ 182 , 280 -282 ] . rhinorrhea and cough are the most frequent symptoms, while hoarseness, laryngitis, sore throat, and croup are less common [ 238 -241 , 243 , 282 ] . a large prospective study of children with uri detected mpv in 5 % of patients, similar to rsv, infl uenza, and piv but less frequent than adenovirus and rhinovirus. in these children with uri associated with mpv, fever was present in 54 %, coryza in 82 %, cough in 66 %, pharyngitis in 44 %, hoarseness in 8 %, and conjunctivitis in 3 % [ 280 ] . mpv is associated with acute otitis media and has been detected in nasal secretions and middle ear fl uid [ 182 , 280 , 283 -285 ] . signs and symptoms of lri with mpv include cough, wheezing, and rhonchi. a large chinese study of children with acute respiratory infections found that wheezing was more common in children with mpv than with rsv [ 282 ] . in that study, children with mpv were diagnosed with pneumonia signifi cantly more than children with rsv, 47 % versus 31 % ( p = 0.002). conversely, a larger percentage of children with rsv were diagnosed with bronchiolitis compared to mpv, 62 % versus 42 %, respectively ( p < 0.001). other studies also note the trend toward a higher percentage of children with mpv and pneumonia compared to rsv [ 182 , 238 , 245 ] although this is not always statistically signifi cant [ 243 ] . mpv has been associated rarely with neurologic complications, including febrile seizures and altered mental status, but there is no conclusive evidence for direct neural infection. one case report describes a patient who died and had mpv isolated by rt-pcr from brain and lung tissues [ 286 ] . mpv was detected by rt-pcr in nasal specimens from 4 of 1,570 persons with encephalitis of unknown etiology [ 287 ] . several other reports describe the detection of mpv in a respiratory specimen from patients with encephalitis [ 286 , 288 -290 ] . only one case reports detection of mpv in cerebrospinal fl uid [ 291 ] . the incidence of viral infection varies between countries and years, but mpv circulates in every year [ 176 , 182 , 239 -241 , 243 , 282 , 292 -296 ] . epidemiologic studies have verifi ed the presence of mpv worldwide. mpv is present during all months in temperate regions, although predominant in late winter-early spring, often following the peak of rsv (fig. 26.2 ) [ 176 , 182 , 191 , 239 , 241 , 246 , 264 , 280 , 282 , 294 , 297 ] . in subtropical climates such as hong kong, a springsummer season similar to rsv occurs [ 243 ] . biannual peaks of seasonality have been described in some european studies [ 298 , 299 ] . annual rates of mpv-associated ari are lower than rsv and comparable to parainfl uenza virus types 1-3 combined and infl uenza [ 240 , 241 , 243 , 282 , 294 , 300 ] although mpv does on occasion surpass rsv in incidence [ 293 ] . multiple outbreaks have been reported in nursing homes and other long-term care facilities (ltcf). mpv was the only pathogen identifi ed in an outbreak of ari that occurred over a 6-weeks period at a quebec ltcf, with 6 pcr-confi rmed and 96 epidemiologically linked probable cases. there were three deaths among the confi rmed and nine deaths among the probable cases [ 278 ] . a california study described an outbreak of ari in 26/148 (18 %) residents and, importantly, 13 staff of a ltcf; mpv was confi rmed in 5 residents, 2 of whom were hospitalized, and no other viruses were detected in any case [ 301 ] . attendance in out-of-home day care, breast-feeding, and passive smoke exposure are not signifi cantly associated with mpv infection [ 245 ] . mpv infection is not associated with lower socioeconomic status. viral coinfection has been suggested with mpv; dual infection with rsv and mpv increased the risk of picu admission compared to rsv alone in one small study [ 302 ] . however, this fi nding was refuted by subsequent larger reports [ 303 -305 ] . other studies demonstrate no signifi cant difference in children with coinfections, including adenovirus, bocavirus, coronavirus, infl uenza virus, parainfl uenza viruses, or rsv [ 240 , 241 , 282 , 293 , 306 , 307 ] . mpv has four distinct genetic lineages or subgroups: a1, a2, b2, and b2 [ 202 , 203 , 218 ] . the predominant subtype varies by year and location [ 241 , 280 , 293 , 297 , 308 ] . in italy, over a 3-years period, all four subtypes were identifi ed each season but the predominant subtype changed. in 2001-2002, a1 accounted for 59 % of all strains, the following year b1 and b2 were present equally, and in 2003-2004, 72 % of strains were a2 [ 308 ] . similar variation was observed over 20 years in a us study, with multiple subgroups present in most seasons (fig. 26. 3 ) [ 280 ] . it is unclear whether viruses from different subgroups differ in virulence. a study in spain reported that children with group a infection more frequently had pneumonia and higher disease severity [ 309 ] , while in canada, group b was associated with more severe disease in hospitalized patients [ 261 ] . patients with group b strains in a french study were more likely to have abnormal chest radiographs but did not have signifi cant differences in oxygen saturation, hospitalizations, or clinical severity scores [ 310 ] . other studies have found no major distinctions in disease severity [ 239 , 241 , 2 0 0 1 2 0 0 0 1 9 9 9 1 9 9 8 1 9 9 7 1 9 9 6 1 9 9 5 1 9 9 4 1 9 9 3 1 9 9 2 1 9 9 1 1 9 9 0 1 9 8 9 1 9 8 8 1 9 8 7 1 9 8 6 1 9 8 5 1 9 8 4 1 9 8 3 1 9 8 2 311 ], laboratory abnormalities [ 228 ] , or symptoms [ 240 ] between subgroups. group a viruses replicate more efficiently in animal models, suggesting some meaningful biological differences between groups [ 219 , 220 ]. mpv is an enveloped virus and thus inactivated by soap, disinfectants, or alcohols. spread is thought to occur by direct or close contact with contaminated secretions. infectious virus can persist at room temperature, especially nonporous surfaces, for up to 8 h [ 183 ] . contact precautions are recommended as for rsv with meticulous hand hygiene. cohorting of patients and caregivers should be considered during outbreaks in care facilities. human data are limited; studies in rodents and nonhuman primates reveal mild erosive and infl ammatory changes in the mucosa and submucosa of the airways, with viral replication observed in ciliated epithelial cells in the respiratory tract [ 312 -314 ] . infl ammatory infi ltrates with a lymphocytic and monocytic predominance are present in perivascular and peribronchial areas. sumino and colleagues [ 315 ] reviewed the lung pathology of fi ve adults with mpv infection. histopathology in three patients demonstrated acute, organizing lung injury with diffuse alveolar membrane formation and the presence of smudge cells. the fourth patient had no evidence of lower respiratory tract infection, and the fi fth patient had nonspecifi c acute and chronic infl ammation. a similar study in children revealed chronic infl ammatory changes of the airways with intra-alveolar macrophages [ 316 ] . a major limitation of reports of human pathology is that patients had been mechanically ventilated prior to death, making it diffi cult to distinguish virus-induced pathology from barotrauma and nonspecifi c infl ammation. mpv lacks genes present in other paramyxoviruses that inhibit interferon responses; nevertheless, mpv is capable of blocking type i interferon responses by an unknown mechanism [ 317 , 318 ] . mpv and other respiratory viruses induce pulmonary cd8+ t cells that fail to secrete ifnγ or exhibit cytotoxic degranulation in response to viral peptides; these impaired cd8 t cells resemble exhausted cd8 t cells induced by chronic infections such as hiv and hepatitis c virus [ 319 ] . humans develop neutralizing antibodies to mpv, and passive antibodies alone can protect in animal models. however, immunity wanes over time and likely provides limited cross-protection between subgroups, since reinfections occur in children and adults [ 246 , 248 ] with genetically different strains [ 182 , 239 , 240 , 268 , 320 ] as well as strains from the same subgroup [ 280 ] . early protection against reinfection following primary infection was confi rmed in macaques; however, when challenged 12 weeks later, virus replication was detectable despite the presence of serum antibodies [ 220 ] . eleven months later, antibody levels had waned still further, and all macaques challenged with heterologous virus and two of three animals reinfected with homologous virus had no evidence of protection [ 220 ] . a prospective study in humans noted that baseline mpv antibodies were lower in patients who subsequently became infected versus those who did not become infected [ 321 ] . thus, cross-protection and duration of antibody responses are important issues for vaccine development. patterns of host response mpv causes both upper and lower respiratory tract signs and symptoms clinically indistinguishable from disease associated with rsv and other respiratory viruses [ 182 , 239 , 300 , 322 , 323 ] . fever is present in most cases, especially children [ 239 -241 , 243 , 264 , 280 , 293 ] . transient maculopapular rash has been described in a minority of patients [ 241 , 243 , 293 ] , and vomiting or diarrhea are described with low frequency [ 182 , 239 , 243 ] . laboratory abnormalities are uncommon, although one study identifi ed 27 % of patients with elevated alt and ast values [ 293 ] . white blood cell count and c-reactive protein were not signifi cantly different between rsv and mpv [ 238 , 282 ] . mpv is rarely detected in asymptomatic persons [ 182 , 191 , 324 -326 ] , though in otherwise healthy young adults, mpv infection can be subclinical [ 246 ] . the duration of shedding in healthy individuals is approximately 7-14 days [ 239 , 327 ] . detection in cell culture requires prolonged incubation and is both insensitive and often impractical. shell vial culture offers increased sensitivity over traditional culture [ 184 , 328 ] . ifa has demonstrated a sensitivity of 73 % and specifi city of 97 % with rt-pcr as the gold standard [ 329 ] . dfa has shown similar results [ 330 ] . commercial antibody kits for immunofl uorescent detection of mpv are available. rt-pcr is most commonly used for detection of the virus in epidemiologic studies and is becoming more common in clinical laboratories [ 176 , 320 , 331 , 332 ] . real-time rt-pcr targeting the conserved n gene has high sensitivity for detection of all four subgroups [ 189 , 195 ] . control and prevention the primary therapy for mpv infections is supportive care, including oral or intravenous hydration, monitoring of respiratory status and oxygen saturation, supplemental oxygen, and mechanical ventilation for frank respiratory failure. there are no licensed antivirals for mpv. reports of pharmacologic treatment of mpv are limited to severely ill or immunocompromised patients. ribavirin, an antiviral agent used in severe rsv infection, reduced infl ammation and viral replication in mice with mpv infection [ 333 ] . commercial intravenous immunoglobulin (ivig), ribavirin, and nmso3 (a sulfated sialyl lipid) effectively inhibited mpv in vitro [ 334 , 335 ] . ribavirin, with and without ivig, has been used in immunocompromised adults [ 336 ] . bonney and colleagues [ 337 ] reported successful treatment of an immunocompromised child with mpv using iv ribavirin and ivig. subsequently, oral ribavirin and inhaled ribavirin with ivig have been used [ 275 , 338 ] . however, no randomized, controlled trials have been conducted, and these data should be regarded as purely anecdotal. human and murine monoclonal antibodies exhibit therapeutic effi cacy in rodent models and thus offer potential for immunoprophylaxis [ 210 , 211 , 213 ]. a number of vaccine approaches for mpv have been investigated. the f protein is conserved between subgroups, immunogenic, and the only target of neutralizing antibodies; in contrast to rsv, the mpv g protein does not induce neutralizing antibodies and is not a protective antigen [ 206 , 209 , 214 ] . a recombinant parainfl uenza virus encoding the mpv f protein demonstrated protection against mpv [ 339 ] , and soluble f protein vaccines reduced viral titers in cotton rats and hamsters [ 207 , 208 ] . reverse genetics technology has been developed for mpv and has been used to produce recombinant strains for vaccine development [ 197 , 340 ] . viruses lacking the g, m2-1, m2-2, or sh proteins or with point mutations are attenuated and immunogenic in rodent and primate models [ 197 , 221 , 341 -344 ]. the major unresolved problems in mpv epidemiology and research involve understanding mechanisms of disease and developing therapeutic or preventive strategies. abundant evidence shows that mpv is a signifi cant cause of acute respiratory disease, especially in the very young, 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patients: a recent experience physiologic risk factors for respiratory viral infections and immunoprophylaxis for respiratory syncytial virus in young children with congenital heart disease respiratory viral infections in immunocompetent and immunocompromised persons community respiratory virus infections in bone marrow transplant recipients: the m.d. anderson cancer center experience ribavirin treatment of viral pneumonitis in severe combined immunodefi ciency disease respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience community respiratory viruses: organ transplant recipients community respiratory viral infection in adult lung transplant recipients respiratory syncytial virusassociated infections in adult recipients of solid organ transplants infectious complications in heart-lung transplantation. analysis of 200 episodes respiratory syncytial virus infection in human immunodeficiency virus-infected children differing manifestations of respiratory syncytial virus-associated severe lower respiratory tract infections in human immunodeficiency virus type 1-infected and uninfected children increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodefi ciency virus type-1 nosocomial respiratory syncytial virus infections: the "cold war" has not ended nosocomial respiratory syncytial virus infections control of nosocomial respiratory syncytial viral infections neonatal respiratory syncytial virus infection nosocomial respiratory syncytial virus infection in canadian pediatric hospitals: a pediatric investigators collaborative network on infections in canada study nosocomial transmission of respiratory syncytial virus in immunocompromised adults rsv outbreak in a paediatric intensive care unit medical and economic impact of a respiratory syncytial virus outbreak in a neonatal intensive care unit maternal immunization against viral disease respiratory syncytial virus infections in pediatric liver transplant recipients use of palivizumab to control an outbreak of syncytial respiratory virus in a neonatal intensive care unit presence of respiratory syncytial virus genomic sequences in middle ear fl uid and its relationship to expression of cytokines and cell adhesion molecules identifi cation of respiratory virus antigens in middle ear fl uids of children with acute otitis media prospective controlled study of four infection-control procedures to prevent nosocomial infection with respiratory syncytial virus prevention of nosocomial respiratory syncytial virus infections through compliance with glove and gown isolation precautions respiratory syncytial virus (rsv) infection rate in personnel caring for children with rsv infections. routine isolation procedure vs routine procedure supplemented by use of masks and goggles the use of eye-nose goggles to control nosocomial respiratory syncytial virus infection rapid identifi cation of respiratory viruses: impact on isolation practices and transmission among immunocompromised pediatric patients nosocomial respiratory syncytial virus infections: prevention and control in bone marrow transplant patients respiratory syncytial virus infections in infants: quantitation and duration of shedding quantitative shedding patterns of respiratory syncytial virus in infants environmental and demographic risk factors for respiratory syncytial virus lower respiratory tract disease casecontrol study of the risk factors linked to respiratory syncytial virus infection requiring hospitalization in premature infants born at a gestational age of 33-35 weeks in spain the pediatric investigators collaborative network on infections in canada study of predictors of hospitalization for respiratory syncytial virus infection for infants born at 33 through 35 completed weeks of gestation respiratory syncytial virus in infants and children differential gender response to respiratory infections and to the protective effect of breast milk in preterm infants risk factors for respiratory syncytial virusassociated lower respiratory illnesses in the fi rst year of life epidemiologic patterns of acute lower respiratory disease of children in a pediatric group practice epidemiology of acute lower respiratory disease in children clinically useful method for the isolation of respiratory syncytial virus bronchiolitis-associated hospitalizations among american indian and alaska native children modes of transmission of respiratory syncytial virus possible transmission by fomites of respiratory syncytial virus infectivity of respiratory syncytial virus by various routes of inoculation an outbreak of febrile illness and pneumonia associated with respiratory syncytial virus infection respiratory syncytial virus load predicts disease severity in previously healthy infants illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus latency and persistence of respiratory syncytial virus despite t cell immunity respiratory syncytial virus infection of human mononuclear leukocytes in vitro and in vivo respiratory syncytial virus rna in cells from the peripheral blood during acute infection speculation on pathogenesis in death from respiratory syncytial virus infection demonstration of respiratory syncytial virus in an autopsy series respiratory syncytial virus infection of human airway epithelial cells is polarized, specifi c to ciliated cells, and without obvious cytopathology pathological changes in virus infections of the lower respiratory tract in children age as a factor in the distribution of lower-airway conductance and in the pathologic anatomy of obstructive lung disease seasonality of invasive pneumococcal disease: temporal relation to documented infl uenza and respiratory syncytial viral circulation vaccine trialist g. a role for streptococcus pneumoniae in virus-associated pneumonia effect of age and preexisting antibody on serum antibody response of infants and children to the f and g glycoproteins during respiratory syncytial virus infection passive transfer of respiratory syncytial virus (rsv) antiserum suppresses the immune response to the rsv fusion (f) and large (g) glycoproteins expressed by recombinant vaccinia viruses microplaque immunoperoxidase detection of infectious respiratory syncytial virus in the lungs of infected mice infl uence of maternal antibodies on vaccine responses: inhibition of antibody but not t cell responses allows successful early prime-boost strategies in mice the immunologic response to infection with respiratory syncytial virus in infants type 1-like immune response is found in children with respiratory syncytial virus infection regardless of clinical severity detection of respiratory syncytial virus nucleic acid in archival postmortem tissue from infants effect of pneumonia in childhood on adult lung function association of radiologically ascertained pneumonia before age 3 yr with asthmalike symptoms and pulmonary function during childhood: a prospective study outcome of acute lower respiratory tract infection in infants: preliminary report of seven-year follow-up study initial airway function is a risk factor for recurrent wheezing respiratory illnesses during the fi rst three years of life. group health medical associates study of 8-year-old children with a history of respiratory syncytial virus bronchiolitis in infancy treatment of respiratory failure with inhaled nitric oxide and high-frequency ventilation in an infant with respiratory syncytial virus pneumonia and bronchopulmonary dysplasia effect of inhaled nitric oxide on respiratory mechanics in ventilated infants with rsv bronchiolitis helium-oxygen improves clinical asthma scores in children with acute bronchiolitis heliox therapy in infants with acute bronchiolitis exogenous surfactant supplementation in infants with respiratory syncytial virus bronchiolitis ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children inhibition of respiratory viruses by nasally administered sirna glucocorticoids for acute viral bronchiolitis in infants and young children prospects of antiviral and anti-infl ammatory therapy for respiratory syncytial virus infection effi cacy of bronchodilator therapy in bronchiolitis. a meta-analysis bronchodilators for bronchiolitis the pharmacologic mechanism by which inhaled epinephrine reduces airway obstruction in respiratory syncytial virus-associated bronchiolitis short term effects of adrenaline in bronchiolitis: a randomised controlled trial effect of racemic epinephrine and salbutamol on clinical score and pulmonary mechanics in infants with bronchiolitis racemic epinephrine compared to salbutamol in hospitalized young children with bronchiolitis; a randomized controlled clinical trial quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. the respiratory syncytial virus immune globulin study group respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease. the cardiac study group neutralization epitopes of the f glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function development of a humanized monoclonal antibody (medi-493) with potent in vitro and in vivo activity against respiratory syncytial virus safety and pharmacokinetics of an intramuscular humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. the medi-493 study group the impact-rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants safety, tolerance and pharmacokinetics of a humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. medi-493 study group palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically signifi cant congenital heart disease committee on f, newborn. revised indications for the use of palivizumab and respiratory syncytial virus immune globulin intravenous for the prevention of respiratory syncytial virus infections ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization immunology of viral respiratory tract infection in infancy an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with lot 100 or a newly prepared reference vaccine safety and immunogenicity of a respiratory syncytial virus subunit vaccine (pfp-2) in the institutionalized elderly immunogenicity of a new purifi ed fusion protein vaccine to respiratory syncytial virus: a multi-center trial in children with cystic fi brosis safety and immunogenicity of respiratory syncytial virus purifi ed fusion protein-2 vaccine in pregnant women a novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without th2 associated vaccine-enhanced disease safety and immunogenicity of a sf9 insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats identifi cation of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants the interferon antagonist ns2 protein of respiratory syncytial virus is an important virulence determinant for humans a newly discovered human pneumovirus isolated from young children with respiratory tract disease metapneumoviruses in birds and humans analysis of the genomic sequence of a human metapneumovirus evolutionary dynamics of human and avian metapneumoviruses genomic analysis of four human metapneumovirus prototypes serologic evidence of avian metapneumovirus infection among adults occupationally exposed to turkeys human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children studies of culture conditions and environmental stability of human metapneumovirus detection of human metapneumovirus in clinical samples by immunofl uorescence staining of shell vial centrifugation cultures prepared from three different cell lines simultaneous detection and typing of human metapneumovirus strains in nasopharyngeal secretions and cell cultures by monoclonal antibodies detection of human metapneumovirus by direct antigen test and shell vial cultures using immunofl uorescent antibody staining evaluation of a commercial direct fl uorescent-antibody assay for human metapneumovirus in respiratory specimens molecular assays for detection of human metapneumovirus real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages detection and quantification of human metapneumovirus in pediatric specimens by realtime rt-pcr detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness molecular epidemiology of human metapneumovirus in ireland clinical evaluation of nuclisens magnetic extraction and nuclisens analytespecifi c reagents for real-time detection of human metapneumovirus in pediatric respiratory specimens detection and characterisation of human metapneumovirus from children with acute respiratory symptoms in north-west england real-time reverse transcriptase pcr assay for improved detection of human metapneumovirus real-world comparison of two molecular methods for detection of respiratory viruses recovery of human metapneumovirus from cdna: optimization of growth in vitro and expression of additional genes sequence analysis of the n, p, m and f genes of canadian human metapneumovirus strains genetic diversity between human metapneumovirus subgroups global genetic diversity of human metapneumovirus fusion gene use of the p gene to genotype human metapneumovirus identifi es 4 viral subtypes antigenic and genetic variability of human metapneumoviruses genetic diversity and evolution of human metapneumovirus fusion protein over twenty years novel human metapneumovirus sublineage effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfl uenza virus type 3 on virus replication and immunogenicity individual contributions of the human metapneumovirus f, g, and sh surface glycoproteins to the induction of neutralizing antibodies and protective immunity human metapneumovirus fusion protein vaccines that are immunogenic and protective in cotton rats immunization of syrian golden hamsters with f subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains an alphavirus repliconbased human metapneumovirus vaccine is immunogenic and protective in mice and cotton rats isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo a recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo the prophylactic administration of a monoclonal antibody against human metapneumovirus attenuates viral disease and airways hyperresponsiveness in mice prophylactic and therapeutic benefi ts of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model soluble recombinant human metapneumovirus g protein is immunogenic but not protective genetic variability of the g glycoprotein gene of human metapneumovirus sequence polymorphism of the predicted human metapneumovirus g glycoprotein genetic heterogeneity of g and f protein genes from argentinean human metapneumovirus strains human metapneumovirus g protein is highly conserved within but not between genetic lineages the two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (f) protein is a major contributor to this antigenic relatedness experimental infection of macaques with human metapneumovirus induces transient protective immunity immunogenicity and effi cacy of two candidate human metapneumovirus vaccines in cynomolgus macaques estimates of individuals exposed to human metapneumovirus in a community-based taiwanese population in 1999 seroepidemiology of human metapneumovirus (hmpv) on the basis of a novel enzyme-linked immunosorbent assay utilizing hmpv fusion protein expressed in recombinant vesicular stomatitis virus seroprevalence of human metapneumovirus (hmpv) in the canadian province of saskatchewan analyzed by a recombinant nucleocapsid proteinbased enzyme-linked immunosorbent assay mlinaric galinovic g. serosurvey of human metapneumovirus infection in croatia large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in beijing, china high seroprevalence of neutralizing capacity against human metapneumovirus in all age groups studied in bonn clinical impact of human metapneumovirus genotypes and genotype-specifi c seroprevalence in yamagata serologic evidence of human metapneumovirus circulation in uruguay human metapneumovirus reinfection among children in thailand determined by elisa using purifi ed soluble fusion protein high seroprevalence of human metapneumovirus among young children in israel seroepidemiological study of human metapneumovirus comparison of the seroprevalence of human metapneumovirus and human respiratory syncytial virus development and validation of an enzyme-linked immunosorbent assay for human metapneumovirus serology based on a recombinant viral protein human metapneumovirus infection among children association between seroprevalence of human metapneumovirus and c-reactive protein level and apolipoprotein e-epsilon4 allele in elderly inpatients in japan development and evaluation of a whole virus-based enzyme-linked immunosorbent assay for the detection of human metapneumovirus antibodies in human sera population-based incidence of human metapneumovirus infection among hospitalized children human metapneumovirus infection in japanese children the association of newly identifi ed respiratory viruses with lower respiratory tract infections in korean children human metapneumovirus seasonality, incidence, and repeat human metapneumovirus lower respiratory tract infections in an area with a high prevalence of human immunodefi ciency virus type-1 infection children with respiratory disease associated with metapneumovirus in hong kong acute lower respiratory tract infections by human metapneumovirus in children in southwest china: a 2-year study burden of human metapneumovirus infection in young children human metapneumovirus infections in young and elderly adults human metapneumovirus infections in adults: another piece of the puzzle rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and infl uenza virus in older adults human metapneumovirus pneumonia in adults: results of a prospective study human metapneumovirus in bronchoalveolar lavage fl uid of critically ill patients with suspected pneumonia human metapneumovirus in adults: a short case series respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection prevalence of viral respiratory tract infections in children with asthma metapneumovirus and acute wheezing in children human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults lung function in prematurely born infants after viral lower respiratory tract infections human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 the impact of infection with human metapneumovirus and other respiratory viruses in young infants and children at high risk for severe pulmonary disease severe human metapneumovirus pneumonia in a child with chronic illness comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children clinical course of hospitalised children infected with human metapneumovirus and respiratory syncytial virus comparison of human metapneumovirus infection with respiratory syncytial virus infection in children a 1-year experience with human metapneumovirus in children aged <5 years human metapneumovirus and chronic obstructive pulmonary disease human metapneumovirus infection in adults with community-acquired pneumonia and exacerbation of chronic obstructive pulmonary disease human metapneumovirus and exacerbations of chronic obstructive pulmonary disease respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodefi ciency virus type 1 (hiv-1)-infected and hiv-1-uninfected african infants brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients fatal pneumonia associated with human metapneumovirus (hmpv) in a patient with myeloid leukemia and adenocarcinoma in the lung human metapneumovirus infection in immunocompromised child respiratory failure associated with human metapneumovirus infection in an infant posthepatic transplant human metapneumovirus infection in hematopoietic stem cell transplant recipients the human metapneumovirus: a case series and review of the literature a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections an outbreak of human metapneumovirus infection in hospitalized psychiatric adult patients in taiwan an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility molecular epidemiological investigation of a nosocomial outbreak of human metapneumovirus infection in a pediatric hemato-oncology patient population the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus in children with acute respiratory tract infections in suzhou viral load and acute otitis media development after human metapneumovirus upper respiratory tract infection frequency of human metapneumovirus in the upper respiratory tract of children with symptoms of an acute otitis media detection of human metapneumovirus from children with acute otitis media human metapneumovirus rna in encephalitis patient beyond viruses: clinical profi les and etiologies associated with encephalitis a fatal case of encephalopathy possibly associated with human metapneumovirus infection seasonal distribution and phylogenetic analysis of human metapneumovirus among children in osaka city human metapneumovirus associated with central nervous system infection in children human metapneumovirus in the cerebrospinal fl uid of a patient with acute encephalitis characterization of human metapneumovirus infections in israel human metapneumovirus-associated lower respiratory tract infections in korean infants and young children community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens evidence of human metapneumovirus in children in argentina human metapneumovirus in hospitalized children in amman, jordan epidemiology and genetic variability of human metapneumovirus during a 4-yearlong study in southeastern brazil biennial spring activity of human metapneumovirus in austria human metapneumovirus infections-biannual epidemics and clinical fi ndings in children in the region of basel, switzerland human metapneumovirus infection among children hospitalized with acute respiratory illness a summer outbreak of human metapneumovirus infection in a long-term-care facility dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection human metapneumovirus and severity of respiratory syncytial virus disease viral respiratory infections in hospitalized and community control children in alaska co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in japan co-circulation of human metapneumovirus and sars-associated coronavirus during a major nosocomial sars outbreak in hong kong changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children human metapneumovirus genotypes and severity of disease in young children (n = 100) during a 7-year study in dijon hospital, france genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness experimental human metapneumovirus infection of cynomolgus macaques (macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract t lymphocytes contribute to antiviral immunity and pathogenesis in experimental human metapneumovirus infection the cotton rat (sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment pathology of human metapneumovirus infection: insights into the pathogenesis of a newly identifi ed respiratory virus human metapneumovirus glycoprotein g inhibits innate immune responses human metapneumovirus inhibits ifn-alpha signaling through inhibition of stat1 phosphorylation viral acute lower respiratory infections impair cd8+ t cells through pd-1 characterization of human metapneumoviruses isolated from patients in north america humoral immunity to human metapneumovirus infection in adults human metapneumovirus infections in hospitalized children respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin, china prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus in the community detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in india detection of multiple respiratory viruses by real-time polymerase chain reaction in infants attending an outpatient clinic comparison of different cell lines and incubation times in the isolation by the shell vial culture of human metapneumovirus from pediatric respiratory samples detection of human metapneumovirus antigens in nasopharyngeal secretions by an immunofl uorescent-antibody test rapid detection of human metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal antibodies virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups comparative evaluation of real-time pcr assays for detection of the human metapneumovirus effect of ribavirin and glucocorticoid treatment in a mouse model of human metapneumovirus infection comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by nmso3 in tissue culture assays human metapneumovirus infection in a hematopoietic transplant recipient successful treatment of human metapneumovirus pneumonia using combination therapy with intravenous ribavirin and immune globulin treatment with oral ribavirin and ivig of severe human metapneumovirus pneumonia (hmpv) in immune compromised child a host-range restricted parainfl uenza virus type 3 (piv3) expressing the human metapneumovirus (hmpv) fusion protein elicits protective immunity in african green monkeys recovery of human metapneumovirus genetic lineages a and b from cloned cdna infection of nonhuman primates with recombinant human metapneumovirus lacking the sh, g, or m2-2 protein categorizes each as a nonessential accessory protein and identifi es vaccine candidates deletion of m2 gene open reading frames 1 and 2 of human metapneumovirus: effects on rna synthesis, attenuation, and immunogenicity generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters deletion of human metapneumovirus m2-2 increases mutation frequency and attenuates growth in hamsters key: cord-022226-qxp0gfp3 authors: meager, anthony title: interferons alpha, beta, and omega date: 2007-09-02 journal: cytokines doi: 10.1016/b978-012498340-3/50026-9 sha: doc_id: 22226 cord_uid: qxp0gfp3 interferon alpha (ifn-α) is a mixture of closely related proteins, termed “subtypes,” expressed from distinct chromosomal genes. interferon β (ifn-β) is a single protein species and is molecularly related to ifn-α subtypes, although it is antigenically distinct from them. ifn omega (ifn-ω) is antigenically distinct from ifn-α and ifn-β but is molecularly related to both. the genes of three ifn subtypes are tandemly arranged on the short arm of chromosome 9. they are transiently expressed following induction by various exogenous stimuli, including viruses. they are synthesized from their respective mrnas for relatively short periods following gene activation and are secreted to act, via specific cell surface receptors, on other cells. ifn-α subtypes are secreted proteins and as such are transcribed from mrnas as precursor proteins, pre-ifn-α, containing n-terminal signal polypeptides of 23 hydrophobic amino acids (aa) mainly. pre-ifn-β contains 187 aa, of which 21 comprise the n-terminal signal polypeptide and 166 comprise the mature ifn-β protein. ifn-ω contains 195 aa—the n-terminal 23 comprising the signal sequence and the remaining 172, the mature ifn-ω protein. at the c-terminus, the aa sequence of ifn-ω is six residues longer than that of ifn-α or ifn-β proteins. ifn-α, as a mixture of subtypes, and ifn-ω may be produced together following viral infection of null lymphocytes or monocytes/macrophages. the biological activities of ifns are mostly dependent upon protein synthesis with selective subsets of proteins mediating individual activities. ifns can also stimulate indirect antiviral and antitumor mechanisms, depending upon cellular differentiation and the induction of cytotoxic activity. the phenomenon of viral interference was first described nearly 60 years ago when hoskins (1935) described the protective action of a neurotropic yellow fever virus against a viserotropic strain of the same virus in monkeys. although viral interference was further investigated in the 1940s and 1950s, the underlying mechanism was not discovered until 1957 when isaacs and lindemann, working at the national institute for medical research (london, uk), isolated a biologically active substance from virally-infected chicken cell cultures that, on transfer to fresh chicken cell cultures, produced a protective antiviral effect (isaacs and lindemann, 1957) . the word interferon (ifn) was coined for this substance. its discovery aroused considerable scientific and medical interest since by 1957 antibiotics were widely available to control bacterial infections, but, in stark contrast, viral diseases such as influenza, measles, polio, and smallpox were virtually untreatable. interest was further heightened by many subsequent studies that demonstrated that ifn could be produced by human cells and was active against a broad spectrum of viruses (see schlesinger, 1959 , for an early review). at that time, ifn was being hailed by the media as a wonder drug, but it soon became clear that ifn was being produced naturally in too small quantifies for that extravagant claim to be immediately confirmed. in fact, the low production of ifn was to bedevil attempts both to characterize it molecularly and evaluate it clinically for many years following its discovery. although the protein nature of ifn was recognized at an early stage in its development (see fantes, 1966 , for an early review), it was only following the introduction of large-scale production methods in the 1970s (cantell and hirvonen, 1977) and the simultaneous development of efficient purification procedures (knight, 1976; rubinstein et al., 1978) that sufficient amounts of partially pure ifn protein became available for characterization and clinical use. gradually, it became apparent that ifn was not a single protein and that there were likely to be different types of ifn molecules. however, despite progress in the area of purification and in initial characterization by sequencing n-terminal polypeptides, ifn proteins all but defied full characterization until the advent of recombinant dna (rdna) technology in the late 1970s. this technology, spurred on by the pharmaceutical industry's desire to produce pharmacologically active proteins cheaply, revealed that one type of human ifn, now designated ifn-cx, was a mixture of several closely related proteins, termed subtypes, expressed from distinct chromosomal genes . second and third types of ifn, designated ifn-i3 and ifn-y respectively, have subsequently been "cloned" (taniguchi et al., 1980; gray et al., 1982) but, unlike ifn-0q are single protein species. ifn-13 is molecularly related to ifn-~ subtypes but is antigenically distinct from them, whereas ifn-y is both molecularly and antigenically distinct from either ifn-cx subtypes or ifn-i3. (for this reason, ifn-y is considered separately elsewhere in this volume.) finally, a fourth type of ifn, antigenically distinct from ifn-0~ and ifn-i3, but molecularly related to both, has more recently been cloned and characterized. rather untypically, this new ifn type has been designated ifn omega (ifn-00) (adolf, 1987) . the genes for ifn-0~ subtypes, ifn-13 and ifn-03 are tandemly arranged on the short arm of chromosome 9. they are only transiently expressed following induction by a variety of exogenous stimuli, including viruses. ifn-0~, ifn-i3 and ifn-00 proteins are synthesized from their respective mrnas for relatively short periods following gene activation and are secreted to act, via specific cell surface receptors, on other cells. early studies on the characterization of ifn receptors indicated that ifn-0~ and ifn-i3 were likely to share a common receptor, but it has only been comparatively recently that such receptors have been cloned (uz~ et al., 1990; novick et al., 1994) . ifn actions are initiated by activated receptors and cytoplasmic signal transduction pathways, which are now well characterized for the ifn-~/i3 receptor, and manifested following expression of a number of ifnspecific inducible genes. induction of the antiviral state, which is dependent on such protein synthesis, may now be viewed as just one of the many activities attributed to ifn in general; these activities include inhibition of cell proliferation and immunomodulation (see pestka et al., 1987 , for a review). in the 1960s, two types of lfn were defined on the basis of the capacity of their antiviral activity to withstand acidification to ph 2. these were termed type i ifn for acid-stable ifn and type ii ifn for acid-labile ifn. type i ifn included ifn produced by virally infected leukocytes, alternatively known as leukocyte ifn, and ifn produced by virally infected human diploid fibroblasts, alternatively known as fibroblast ifn. type ii ifn, which was only produced by antigenically or mitogenically stimulated human peripheral blood mononuclear cells (pbmc), has often been referred to as immune ifn (stewart, 1979) . antigenic differences were described for leukocyte and fibroblast ifn and these were put on a molecular basis when knowledge of their respective n-terminal amino acid sequences became available (allen and fantes, 1980; knight et al., 1980; levy et al., 1980; zoon et al., 1980) . at that time, an international nomenclature committee (stewart et al., 1980) reviewed the growing evidence for the existence of distinct molecular forms of ifn and introduced the greek alphabetical system to apply to the then known antigenically distinct types of ifn. leukocyte ifn was designated ifn-~, fibroblast ifn was designated ifn-13, and immune ifn became ifn-y. however, complications immediately arose when it was revealed, following the cloning of several different leukocyte ifn complementary dnas (cdnas) brack et al., 1981; goeddel et al., 1981; streuli et al., 1980) , that leukocyte ifn was heterogeneous and contained many different, molecularly and antigenically related species, now commonly referred to as subtypes. the research group at hoffmann-la roche labeled the subtypes produced in e. coli ra, 0~b, 0~c, 0cd, etc. (evinger et al., 1981; rehberg et al., 1982) , distinguishing them from natural components of leukocyte ifn , while the biogen group labeled these recombinant subtypes ix1, 0~2, 0c3, 0~4, etc. , regrettably without an appropriate alphabeticalnumerical correspondence: for example, ~k -~2, 0cd -0~1. however, the numerical system now prevails. when an ifn preparation is a mixture of ifn-~ subtypes, e.g., leukocyte ifn, lymphoblastoid ifn, this is often designated ifn-o~n. the later cloning of cdnas encoding ifn-0~-like proteins (capon et al., 1985; hauptmann and swetly, 1985) initially led to the naming of this new ifn as ifn-o~ subclass ii, with all of the earlier-characterized ifn-0~ subtypes being reclassified as ifn-tx subclass i. this large, unwieldy nomenclature system has been superseded by the renaming of ifn-~ ii as ifn-03; this has been generally accepted with the finding that ifn-03 is antigenically distinct from ifn-~ and ifn-i] proteins and thus qualifies as a separate type of ifn (adolf, 1987) . fortunately, the nomenclature for ifn-i] has remained straightforward since there is only one protein species, at least in humans (derynck et al., 1980 taniguchi et al., 1980) . from the initial cloning of ifn-o~ cdnas, there has been a plethora of reports on the cloning of new, and sometimes distinct, genomic and edna clones, and fairly disparate nomenclatures have arisen. diaz and allen (1993) therefore undertook the considerable task of compiling the ifn genes and genomic and cdna clones from the literature and introduced an arabic-alphabetical system for naming ifn genes to enable their distinction from ifn proteins. thus, ifn-oc genes became ifna genes with the addition of a numeral to denote subtype, i.e., ifna1, ifna2, etc. (table 25 .1). the ifn-~ gene became ifnb and, since there is only one gene in humans, it is referred to as ifnb 1. for ifn-03 genes, w has been used; hence ifnw1 (table 25 .1). besides genes that are capable of being expressed and translated into ifn proteins, there are a number of pseudogenes which are unable to give rise to ifn proteins, and which in this new nomenclature system are designated by a p, e.g., ifnap22, ifnwp2, or simply ifnp1 where pseudogenes are clearly ifn-like but cannot be definitely included in any one of the ifna, ifnb or ifnw gene families (table 25 .1). the ifn genomic and edna clones have been designated in a variety of ways, as illustrated in table 25 .1. pseudogenic or non-translatable clones are normally prefixed with a greek ~. for the purposes of this chapter, and to reduce the complexity of naming ifn clones and proteins, the nomenclature system adopted by weissmann and colleagues will be adhered to: ifn-0q, %, {x4, o~, etc. 2.1 numbers, structure, and localization in humans, there are 14 nonallelic ifna genes, one of which, ifnap22, is a pseudogene (table 25 .1). in addition, there are a further four nonallelic pseudogenes that possibly also belong to the ifna gene family. probable allelic variants of certain ifna genes, e.g., ifna2, are also known to exist goeddel et al., 1981; dworkin-rastl et al., 1982; emanuel and pestka, 1993) . this extensive family of ifna genes are tandemly arranged on the short-arm of chromosome 9 (9p23) and span a region of approximately 400 kb (owerbach et al., 1981; shows et al., 1982; slate et al., 1982; ullrich et al., 1982) . the ifnb gene and the ifnw gene/pseudogene family are also located in the same region of chromosome 9 (meager et al., 1979a,b; owerbach et al., 1981; henry et al., 1984; capon etal., 1985) . there is a high degree of homology among the ifna genes, but these show much less homology to either ifnb or ifnw genes. nevertheless, all of these ifn genes share the common feature of being intron-less (taniguchi et al., 1980; goeddel et al., 1981; houghton et al., 1981; capon et al., 1985) , suggesting a very ancient origin of their common ancestral gene. it has been proposed that the primordial ifn gene arose some 500 million years ago, with the first split occurring around 400 million years ago to yield the first ifna and ifnb genes (wilson et al., 1983) . since then the ifna gene has evolved and duplicated many times to give rise to the multiple ifna genes found in present-day animals and man (mij~ita and hayashida, 1982; gillespie and carter, 1983) . around 100 million years ago, an ifna gene appears to have diverged sufficiently from the main group to give rise to the ifnw gene family, which is present in most mammals except mice and dogs (himmler et al., 1987; roberts et al., 1992) . it is not clear what characteristic of ifna and ifnw genes enabled the numerous reduplication events to occur in comparison to the nonexistent or more limited (some mammals have more than one ifnb gene, e.g., bovines (wilson et al., 1983) ) reduplication of the ifnb gene (ohlsson et al., 1985) . it is apparent that gene conversion, as a result of mismatch repair and unequal crossover, contributed significantly to the creation of distinct, but highly homologous, nonallelic ifna (and ifnw) genes (de maeyer and de maeyer-guignard, 1988) . in most cases, both coding and noncoding regions have diverged, but in the case of ifna13 the coding region has remained identical to that of ifna1, although its 5' and 3' flanking regions have diverged (todokoro et al., 1984) . the structures of ifna, ifnb, and ifnw genes are similar. each gene has a 5' regulatory promoter region upstream from the transcriptional start (cap) site, a ifna10 ifna13 ifna14 ifnals ifna~ ifna21 ifnap~ ifn-oq ifn-o h, lelf-d ifn-oq (d) ;~a 2, ifn-a ifn adapted and modified from diaz and allen (1993) and allen and diaz (1996) , which see for specific references for genomic clones and cdnas. reproduced with permission of mary ann liebert, inc., new york, usa. coding region containing a nucleotide sequence encoding a signal polypeptide of 21-23 mainly hydrophobic amino acids, which is typical for secreted proteins, and consecutively the sequence encoding the mature ifn protein, followed by the 3' flanking noncoding region, which can vary in length up to 450 base pairs (bp) (figure 25 .1) (derynck etal., 1980 (derynck etal., , 1981 nagata et al., 1980; streuli et al., 1980; taniguchi et al., 1980; degrave etal., 1981; goeddel etal., 1981; gross et al., 1981; lawn et al., 1981a,b; gren et al., 1984; capon et al., 1985; henco et al., 1985) . the 5' flanking region contains a tata or hogness box, which delineates the boundary of the upstream promoter, approximately 30 bp from the cap site. farther upstream are found a number ofhexameric repeat sequences gaaann, where n can be any base, which in their dimeric or multimeric forms act as binding sites for nuclear transcription factors and repressor molecules (fujita et al., 1985; ryals et al., 1985) . (this area is covered in more detail in section 2.2, inducers and transcriptional control). the 3' flanking regions vary in length and contain several polyadenylation sites and thus can give rise to mrnas of different lengths (mantei and weissmann, 1982; henco et al., 1985) . they contain above-average numbers of the sequence motifs atta or ttatttat. such sequences are common, however, in many other cytokine genes and other genes, such as protooncogenes, that are inducibly and transiently expressed. it has been proposed that these sequences contribute to the relative instability and short half-lives of ifn and cytokine mrnas (caput et al., 1986; shaw and kamen, 1986 ). all ifn genes are normally silent and thus require some sort of stimulus to induce expression. a wide range of inducers, including viruses, bacteria, mycoplasma, endotoxins, double-stranded polynucleotides or rna (dsrna), and some cytokines, have been shown to efficiently activate transcription of ifn genes (stewart, 1979; de maeyer and de maeyer-guignard, 1988 ). such inducers have in general the potential to induce expression of all ifna, ifnb, and ifnw genes; however, there appears to be cell-and inducer-specific selectivity that governs the type and numbers of ifn genes expressed. for instance, virally induced human diploid fibroblasts produce mainly ifn-13 and only a minor amount of ifn-~ (havell et al., 1978) , whereas virally induced pbmc produce mainly ifn-a plus ifn-o3 and only a minor amount of ifn-~ (cantell and hirvonen, 1977; adolf et al., 1990) . this has to some extent been confirmed at the mrna level (shuttleworth et al., 1983; hiscott et al., 1984) . differences have also been reported in the proportions of individual ifn-0~ subtypes produced by different cell types (goren et al., 1986; finter, 1991; greenway et al., 1992) , suggesting that ifna genes may be differentially expressed. however, the way in which such differential expression is regulated is presently not understood. transcriptional control of ifna genes resides in their 5' flanking region, upstream from the cap site. nucleotide deletions outside of position-117 from the cap site have little impact on transcriptional control, but deletions farther in eliminated induction, indicating that this region,-117 to-1, contained promoter regulatory elements (ragg and weissmann, 1983; weidle and weissmann, 1983) . these have been further delineated as a purine-rich nucleotide tract between-109 and-64, containing hexameric repeats of gaaann (gaaa g/c t/c), which appears to be necessary for inducible transcription and which has been termed the "virusregulating element" (vre) (ryals et al., 1985) . similar studies involving 5' deletions have been carried out with the ifnb gene, and it has been found that 5' sequences within-110 to -1 contain regulatory elements that are required for induction by viruses and dsrna. the minimum vre has been localized to-74 to-37 with respect to the cap site (goodbourn et al., 1986; goodbourn and maniatis, 1988) and contains two positive virus-inducible elements, termed positive regulatory domains (prdi,-77 to-64; prdii,-66 to -55), and a negative regulatory domain (nrd,-57 to -37) (figure 25 .1) (fujita et al., 1985; fan and maniatis, 1989; whittemore and maniatis, 1990; nourbakhsh et al., 1993) . in addition, the hexameric repeat sequences gaaann (also present in ifna genes) spanning from -110 to-65 contain variants of the prdi sequence and two further regulatory elements, prdiii (-90 to-78) and prdw (-104 to-91) have been identified that are required for a functional vre in ifnb gene expression in mouse l cells (dinter and hauser, 1987; du and maniatis, 1992) . prdi and prdiii act as binding sites for a nuclear transcription factor, designated "interferon regulatory factor-l" (irf-1) , whose expression is transiently increased by virus infection and which appears to mediate the activation of transcription of the ifnb gene (fujita et al., 1988; harada et al., 1989; xanthoudakis et al., 1989) . a second virus-inducible factor, designated "interferon regulatory factor-2" (irf-2), also binds to prdi but suppresses, rather than activates, transcription (harada et al., 1989 (harada et al., , 1990 . prdii is a binding site for the nuclear transcription factor nfra3 (clark and hay, 1989; fujita et al., 1989a; hiscott et al., 1989; lenardo et al., 1989; visvanathan and goodbourn, 1989) , which interacts with the major groove of the dna (thanos and maniatis, 1992) . additionally, another protein, highmobility group y/l, also binds to prdii, interacting with the minor groove of the dna (thanos and maniatis, 1992) . both factors appear to be necessary for virus induction of the ifnb gene promoter. prdiv contains a binding site for a protein of the camp response element binding protein (atf/creb) family of transcription factors (du and maniatis, 1992) . viral induction of the ifnb gene is thought to occur following activation of pre-existing nftcb and by de novo synthesis of irf-1; these nuclear transcription factors bind to the tandemly arranged prdi and prdii and act cooperatively to initiate/activate transcription (leblanc et al., 1990; lenardo et al., 1989; visvanathan and goodbourn, 1989; fujita et al., 1989b; watanabe et al., 1991) . reporter constructs containing prdi supported by a simian virus 40 (sv40) enhancer, or (gaaagt)4 , which contains the functional equivalent of dimeric prdi (n~if et al., 1991) are activated not only by virus but also by overexpression of irf-1 macdonald et al., 1990) . however, in most cell lines, overexpression of irf-1 has led to poor induction of ifnb (and ifna) genes (harada et al., 1990; fujita et al., 1989b) or none at all (macdonald et al., 1990; reis et al., 1992) . this has been attributed to the repressive effect of irf-2, a homologue oflrf-1, which also binds to prdi (harada et al., 1989) . in the undifferentiated murine embryonal carcinoma (stem) cell line p19, which is refractory to viral induction oflfnb (and ifna) genes and which expresses neither irf-1 nor irf-2, overexpression of an introduced irf-1 construct leads to activation of endogenous ifn genes and to activation of reporter plasmids with the ifnb promoter (harada et al., 1990) . in addition, cell lines permanently transformed with an antisense irf-1 expression plasmid exhibited strongly reduced ifnb gene inducibility that nevertheless could be restored by transient transformation with an irf-l-overproducing expression plasmid (reis et al., 1992) . however, the role oflrf-1 in virus-induced activation of the ifnb promoter remains controversial (whiteside et al., 1992; pine et al., 1990) and ruffner and colleagues (1993) have shown that in murine embryonal stem cells devoid of both irf-1 gene alleles (irf-1 ~ ) viral induction of ifnb was only slightly higher in control irf-1 0/+ differentiated stem cells than that in irf-1 ~ differentiated cells. this suggests that while irf-1 at high levels may elicit or enhance induction of ifnb under certain circumstances, it is not essential for viral induction. in cultured mouse fibroblasts devoid of irf-1, ifnb induction by the synthetic dsrna molecule poly(i):poly(c) was absent, whereas induction by newcastle disease virus (ndv) was normal (matsuyama et al., 1993) . however, ifn induction in vivo by either virus or dsrna has been found to be unimpaired in irf-1 ~ mice, indicating that irf-1 is not essential . it has also become clear recently that the prdi site can bind factors other than irf-1 and irf-2, and these may be more important for regulating ifnb gene activation (whiteside et al., 1992; keller and maniatis, 1991) . in contrast, targeted disruption of the irf-2 gene to yield mouse fibroblasts deficient in the repressor irf-2 has been found to lead to upregulated induction of ifnb following ndv infection (matsuyama et al., 1993) . this suggests that irf-2 negatively regulates or represses ifnb gene induction. the induction of the ifna1 gene appears to be regulated differently from that of the ifnb gene. irf-1 is bound poorly by the equivalent prdi site in the ifna1 promoter and this promoter also lacks an nf~cb site ( figure 25 .1) macdonald et al., 1990) . the ifna1 gene vre does contain a hexameric repeat nucleotide sequence (gaaatg)4, designated a "tg-sequence" (macdonald et al., 1990 ), which appears to mediate virus inducibility when supported by an sv40 enhancer, but which does not respond to irf-1 (n~if et al., 1991) . it has, however, been reported that overexpression of irf-1 can induce ifna genes, at least under special circumstances (harada et al., 1990; au et al., 1992) . the ifnw gene, like ifna and ifnb genes, is virus inducible and has structural features in its 5' promoter region similar to those in ifna/b promoters ( figure 25 .1). in particular, hexameric repeat units are present, but are organized differently from those present in ifna/b genes (hansen et al., 1991; roberts et al., 1992) . however, the regulation of transcription of the ifnw gene has not been studied in detail. the 14 ifn-(x subtypes are secreted proteins and as such are transcribed from mrnas as precursor proteins, pre-ifn-(x, containing n-terminal signal polypeptides of 23 mainly hydrophobic amino acids (figure 25 .2). the signal polypeptide is cleaved off before "mature" ifn-(x molecules are secreted from the cell. from their cerna sequences, mature ifn-(x subtypes have been predicted to contain 166 amino acids (except ifn-%, 165 amino acids) nagata et al., 1980; goeddel et al., 1981; lawn et al., 1981a,b; gren et al., 1984) . the calculated molecular mass of recombinant ifn-(x subtypes is approximately 18.5 kda, although apparent molecular masses of leukocyte-derived ifn-(x subtypes in sodium dodecyl sulphate (sds)-polyacrylamide gels vary between 17 and 26 kda, possibly owing to variable processing of c-terminal amino acids (le w et al, 1981) and post-translational modifications. the amino acid sequences of ifn-(x subtypes are highly related, with complete identity at 85 of the 166 amino acid positions (langer and pestka, 1985; de maeyer and de maeyer-guignard, 1988) . this is illustrated in figure 25 .2, where the amino acid sequences of the subtypes are compared to an idealized consensus sequence. many of the positions where amino acids differ from subtype to subtype are conservative substitutions. interestingly, ifn-(x subtypes contain four cysteine residues whose positions (1, 29, 99, and 139) are highly conserved (figure 25 .2). these four cysteines form disulfide bridges (1-99, 29-139) which induce folding of the ifn-(x molecule (figure 25 .3) and whose integrity is essential for biological activity (morehead et al., 1984) . ifn-(x subtypes are predicted to contain a high proportion (-60%) of (x-helical regions and are folded to form globular proteins (zoon and wetzel, 1984) . it has not yet proved possible to apply x-ray crystallographic techniques to ifn-(x subtypes, or to human ifn-~3, but the three-dimensional crystal structure of recombinant mouse ifn-[3, which is approximately 60% related in amino acid sequences to its human counterpart, has been solved (senda et al., 1992) . this has revealed that mouse ifn-[3 has a structure which consists of five (x-helices folded into a compact (x-helical bundle ( figure 25 .4). from comparative sequence analysis it is predicted that in all mammalian ifn-(x and ifn-]3 proteins these five (x-helical domains are conserved (korn et al, 1994; horisberger and di marco, 1995) and thus show similarities with many other cytokines, which also have (x-helical bundle structures (bazan, 1990) . with one exception, ifn-(x subtypes do not contain recognition sites (asn-x-ser/thr) for n-linked glycosylation (henco et al, 1985) ; only ifn-(x14 contains two of these sites (figure 25 .2). nevertheless, o-linked glycosylation may be possible in other ifn-(x subtypes. for example, it has been found that natural ifn-%, purified from human leukocyte ifn, contains the disaccharide galactosyl-n-acetylgalactosamine in olinkage to thr-106 (adolf et al, 1991a) . however, since ifn-% is the only ifn-(x subtype with a theonine at position 106, it may represent the only o-glycosylated ifn-(x protein (figure 25 comprise the mature ifn-]3 protein (derynck et al., 1980 taniguchi et al., 1980; houghton et al., 1981) . although ifn-] 3 is the same length as the majority of ifn-a subtypes, it shows only approximately 30% amino acid sequence relatedness with them ( figure 25 .2) and is antigenically distinct. the ifn-~ protein lacks the n-terminal cys-1 residue present in ifn-a subtypes, but contains three other cysteines at positions 17, 31, and 141, the latter two corresponding to the disulfide bond pairing 29-139 in ifn-a subtypes. replacement of cys-17 by serine does not result in any loss of biological activity, whereas serine substitution of cys-141 does (mark et al., 1981; shepard et al., 1981) . as mentioned previously, on the basis of the threedimensional structure of recombinant mouse ifn-~ ( figure 25.4) , human ifn-[3 is predicted to contain five a-helices and to fold up into an a-helical bundle structure (senda et al., 1992) . human ifn-[3 has one potential n-glycosylation site at asn-80 (taniguchi et al., 1980) and n-linked oligosaccharides, primarily of the biantennary complextype, are known to be attached to this site in natural ifn-13 (hosoi et al, 1988) . however, these may vary considerably depending on the producer cell type (utsumi et al., 1989) . from cdna sequence data, it was predicted that pre-ifn-m contains 195 amino acids, the n-terminal 23 comprising the signal sequence and the remaining 172 the mature ifn-m protein (capon et al., 1985; hauptmann and swetly, 1985) . the amino acid sequence of ifn-m is therefore six residues longer at the cterminus than ifn-r or ifn-[3 proteins. however, it has been found that natural ifn-m is heterogeneous at the n-terminus owing to variable cleavage of pre-ifn-m; about 60% of mature ifn-m molecules carry two additional n-terminal amino acids (adolf, 1990; shirono et al., 1990) . it is approximately 60% related to ifn-cx subtype sequences, but only 30% related to that of ifn-[3 ( figure 25 .2), and is antigenically distinct from both ifn-~ and ifn-[3 (adolf, 1990) . nevertheless, the four cysteines occur in the same notional positions, 1, 29, 99, and 139, as they do in ifn-~ subtypes and it is likely that ifn-m will have a similar cx-helical bundle structure to those predicted for both ifn-cx and ifn-[3 proteins (senda et al., 1992) . ifn-m has one potential site at asn-78 for n-linked glycosylation and natural ifn-m has been demonstrated to be a glycoprotein with biantennary complex oligosaccharides (containing neuraminic acid) attached at this site (adolf, 1990; adolf et al., 1991 b) . type i ifns (ifn-0t,-[3, and-m) are produced by a variety of normal cell types responding to extracellular or intracellular stimuli (stewart, 1979) . ifn-~, as a mixture of subtypes, and ifn-m may be produced together following viral infection of null lymphocytes or monocytes/macrophages (cantell and hirvonen, 1977; adolf, 1990) . the proportions of ifn-0t subtypes may vary according to the type of virus used as inducer (hiscott, 1984; finter, 1991) . however, production of ifn-[3 is usually restricted to double-stranded polynucleotide, e.g., poly-inosinic, poly-cytidylic acid, or it is well established that the biological activities of ifns are mostly dependent upon protein synthesis with selective subsets of proteins mediating individual activities. antiviral, antiproliferative and immunomodulatory activities have been ascribed to ifn-~/[3/c0 (reviewed in pestka et al., 1987; de maeyer and de maeyer-guignard, 1988) . the proteins and mechanisms involved in these activities are described below. virally-induced normal fibroblasts and other tissue cell types, e.g., epithelial cells (meager et al., 1979; stewart, 1979) . in all the above cases, the amount of ifn secreted is dependent on the dose of the inducer. besides normal cells, a range of transformed and tumor-derived cell lines are known ifn producers, e.g., mg63 human osteosarcoma cell line (meager et al., 1982) , and the namalwa b-lymphoblastoid cell line (phillips et al., 1986) . generally speaking, adherent fibroblastic cell lines produce mainly ifn-[3 and only a minor quantity of ifn-~ (havell et al., 1978) , whereas nonadherent myeloid or lymphoid cell lines produce mainly ifn-c~ and only a small amount of ifn-i3 (cantell and hirvonen, 1977; shuttleworth et al., 1983; zoon et al., 1992) . actual production of ifns lasts only a matter of a few hours following induction. this is due to ifn mrna instability and the rapid shut-off of ifn gene transcription (caput et al., 1986; shaw and kamen, 1986) . under conditions where ifn mrna stability is increased, e.g., by blocking protein and rna synthesis following induction, ifn production has been shown to be "superinduced" once the block on protein synthesis is removed (meager et al., 1979; stewart, 1979) . despite there being vast numbers of viruses with different replication strategies, it appears that many viruses can be countered by relatively few ifn-inducible "antiviral" proteins (samuel, 1987) . one of the bestcharacterized of these is a family of enzymes collectively known as "2-5a synthetase" which, in the presence of dsrna (often an intermediate of viral rna synthesis) catalyses the formation of an unusual oligonucleotide, ppp (a2'p) na (2-5a), which in turn activates an ifninduced latent endonuclease, rnase l (williams and kerr, 1978; wreschner et al., 1981; ghosh et al., 1991; hovanessian, 1991; lengyel, 1982; zhou et al., 1993) . when activated, the rnase l degrades viral (and cellular mrna) and therefore inhibits viral protein synthesis. small rna viruses (picornaviridae), e.g., mengo virus and murine encephalomyocarditis virus (emcv), whose replication is cytoplasmic are most inhibited by the induction of2-5a synthetase (lengyel, 1982; rice et al., 1985; chebath et al., 1987; kumar et al., 1988) . a further important ifn-induced "antiviral" protein is a dsrna-dependent protein kinase, now designated pkr, which in the active form phosphorylates the peptide initiation factor, eif2, involved in polyribosomal translation of mrna (miyamoto and samuel, 1980; gupta et al., 1982; samuel, 1987) . phosphorylated eif2 is inactive and thus viral protein synthesis is inhibited. this inhibition has been associated with the loss of replicating capacity of reoviruses and rhabdoviruses such as vesicular stomatitis virus (vsv). the 2-5a synthetase and pkr antiviral mechanisms are rather general and potentially could affect a wide range of viruses. however, ifn can induce certain proteins that inhibit specifically one class of virus. for example, the ifn-inducible mx proteins block the replication of influenza virus, probably by inhibiting the nuclear phase of viral transcription (mouse cells) or later cytoplasmic phases (human cells), without affecting the replication of many other viruses (staeheli, 1990; mel6n et al., 1992; ronni et al., 1993) . in addition, since ifns can impair various steps of viral replication, including penetration, uncoating and assembly of progeny virions as well as transcription and translation, there are likely to be several other antiviral proteins and mechanisms (de maeyer and de maeyer-guignard, 1988) . for instance, some viruses, e.g., herpes virus and certain retroviruses, appear to be inhibited at the relatively late stage of virus particle maturation and budding (aboud and hassan, 1983) . in the course of evolution, many viruses have developed countermechanisms by which they can disrupt the antiviral mechanisms induced by ifn. such countermechanisms often point to the significance of particular "antiviral" proteins. one of the main "targets" for several different viruses is the ifn-inducible pkr. the action of this kinase is overcome in adenovirus or epstein-barr virus (a member of the herpes virus family) by the production of small viral rna molecules, vaiand eber-rnas, respectively, which bind to pkr and block its activation by dsrna (clarke et al., 1991; ghadge et al., 1991; mathews and shenk, 1991) . reoviruses and vaccinia virus (a member of the pox virus family) produce viral proteins (sigma3 and ski, respectively), that bind to dsrna and thus reduce activation of pkr (sen and lengyel, 1992) . interestingly, if ifn-treated vsv-infected cells are coinfected by vaccinia virus, vsv replication is rescued, presumably partly by the inhibitory effect of ski on pkr (whitaker-dowling and youngner, 1983) . vaccinia virus also produces a nonfunctional protein analog of elf2 which competes with the real eif2 for phosphorylation by pkr and thus dilutes out the antiviral effect of activated pkr (beattie et al., 1991) . other viruses, such as influenza, may activate latent cellular inhibitors of pkr activity, e.g., a 58 kda protein (p58) (lee et al., 1992) . the 2-5a synthetase-rnase l system can also be subverted. for example, emcv, a picornavirus, can inactivate rnase l in several cell lines, but this inactivation is usually blocked by ifn treatment (lengyel, 1982) . herpes viruses, in contrast, appear to inhibit rnase l activation by producing competing analogs of2-5a (cayley et al., 1984) . some viruses even .have the ability to block the transcription of ifn-inducible genes. the "early" ela regulatory proteins of adenoviruses prevent the activation of isgf3 by ifn, probably by inhibiting the transcription of the isgf37 subunit gutch and reich, 1991; kalvakolanu et at., 1991; nevins, 1991) . in the case of hepatitis b virus-infected cells, the so-called virus-specified "terminal protein" inhibits ifn-inducible gene expression . the antiviral mechanisms induced by ifn are mediated by enzymes, e.g., 2-5a synthetase and picr, whose activities have broad implications for cell growth and proliferation. viral replication may be regarded as a form of pathological growth of a foreign, "cell-like", entity at the expense of a living cell. in the presence of ifn, enzymes are activated which curtail protein synthesis in general, but because viral protein synthesis is normally rapid, the inhibitory effect on viral replication appears more dramatic than on the slower and more complex cellular growth. possibly, the "ifn system" was evolved more as a part of a complex, interactive network of intercellular mediators of cell growth and proliferation than as one for antiviral mechanisms. recent investigations tend to support the role of ifns in regulating cell growth. for example, if a mutant form of pkr that is unable to phosphorylate eif2 is introduced into cells, they undergo neoplastic transformation (koromilas et al., 1992; lengyel, 1993; meurs et al., 1993) . this suggests that pkr normally acts as a "tumorsuppressor"' gene product. therefore, one of the mechanisms by which ifn inhibits cell proliferation could be through its capacity to induce enhanced expression/activity of pkr. ifn-~ has also been reported to inhibit cyclin-dependent cdk-2 kinase, which is responsible for phosphorylation of retinoblastoma (rb) protein, and this could contribute to antiproliferative activity (kumar and atlas, 1992; resnitzky et al., 1992; zhang and kumar, 1994) . the 2-5a synthetase-rnase l system may also have antiproliferative and tumor suppressor activities. for instance, the levels of these two enzymes are high in growth-arrested cells: introduction of 2-5a-like oligoadenylates into proliferating cells also causes growth impairment (sen and lengyel, 1992; lengyel, 1993; zhou et al., 1993) . the ifn-stimulated increases in synthesis of pkr and 2-5a synthetase are dependent on ifn-inducible transcription factors, such as irf-1 (isgf2) pine et al., 1990; williams, 1991; reis et al., 1992) . the latter has a short half-life and thus transcription of ifn-inducible genes is rapidly repressed by the longer-lasting, inhibitory irf-2 (harada et al., 1989) . if irf-2 is overexpressed, cells become transformed as even low level constitutive production of pkr and 2-5a synthetase, which can regulate normal cell growth, is abrogated. this transformation was reversed by overexpressing irf-1 (harada et al., 1993) , indicating that irf-1 can be viewed as a pivotal player in the growth, regulatory, and tumor suppressor machinery. a variety of other ifn-induced mechanisms, including suppression of oncogenes (contente et al., 1990) , depletion of essential metabolites (sekar et al., 1983) , and increased cell rigidity (e. wang et al., 1981) , could also contribute to its antiproliferative activity. the antiproliferative effects of ifns in different tumor cell lines cultured in vitro is highly variable. besides tumor cell lines, ifn-0t/[3 have antiproliferative activity in hematopoietic precursor cells, e.g., of the myeloid lineage (rigby et al., 1985; de maeyer and de maeyer-guignard, 1988 , for review). they are also potent inhibitors of angiogenesis, the process whereby blood capillaries are formed to envasculate tissues (sidky and borden, 1987) . besides activating intracellular processes, ifns can also activate intercellular activities, especially within the immune system, which are an essential part of host defense against infectious and invasive diseases. thus, ifns can stimulate indirect antiviral and antitumor mechanisms, which in the main rest upon cellular differentiation and the induction of cytotoxic activity. for example, in the presence of antigen-specific antibodies, macrophages can effect cell-mediated cytotoxicity. such antibody-dependent cell-mediated cytotoxicity (adcc) is enhanced by ifn, possibly through an augmentation of immunoglobulin g (igg)-fc receptor (fcr) expression (hokland and berg, 1981; vogel et al., 1983; de maeyer and de maeyer-guignard, 1988 ). in addition, another category of leukocytes comprising large granular lymphocytes and known as natural killer (nk) cells are activated, by unknown mechanisms, to kill virally-infected or tumor cell targets independently of major histocompatibility complex (mhc) antigen expression (trinchieri and perussia, 1984; rager-zisman and bloom, 1985; de maeyer and de maeyer-guignard, 1988) . ifns can stimulate increased expression of class i mhc antigens, i.e., hla-a, -b, -c, which are crucial for recognition of foreign antigen by cytotoxic t lymphocytes (ctl, cd8+); recognition of virally infected cells by ctl depends on class i mhc antigen presentation of viral antigens at the cell membrane (heron et al., 1978; fellous et al., 1979) . ifn-(x/[3 have sometimes been observed to increase class ii mhc antigen expression, which is necessary to trigger both humoral and cell-mediated immunity, but probably play a lesser role than ifn-y, which is the major class ii mhc antigen inducer (baldini et al., 1986; rhodes et al., 1986; de maeyer and de maeyer-guignard, 1988 ). all of the biological activities so far described (see above) for ifns have followed from in vitro experimentation. here it is possible to pick and choose conditions that favor particular outcomes, e.g., the antiviral response, by adjusting doses of ifn, times of incubation, levels of virus challenge, and so on. such experiments illustrate the range of biological activities of ifns but cannot define their physiological roles. that ifns have the potential for inducing antiviral and antitumor activity suggests their main role in vivo is to act as regulators of host defense mechanisms, and to prevent pathophysiological events occurring. investigations in experimental animals have supported this likelihood. for example, injection of mice with anti-ifn-~/]3 antibody has been shown to increase their susceptibility to a range of virus infections (virelizier and gresser, 1978; gresser, 1984) . the earliest evidence for an antitumor effect of ifn-tx/13 came from inoculation of murine l1210 cells into mice. l1210 cells are sensitive to the antiproliferative action of ifn-~/13 in vitro and in vivo, ifn~/[3 prevented tumor growth by these cells. however, when a clone of l1210 was isolated that was resistant to the antiproliferative action of lfn-~/[3, there occurred a similar retardation of tumor growth upon ifn-c~/]3 treatment to that observed with "sensitive" l1210 cells, suggesting that ifn was acting indirectly in vivo by a host-mediated mechanism (gresser et al., 1970 (gresser et al., , 1972 . a similar conclusion was reached more recently using ifn-resistant b-cell lymphoma cells . in the intervening years, many studies have been conducted confirming that ifn can act directly (e.g., human ifn-a 2 against a range of human tumor xenografts in nude mice where human ifn-0~ 2 has no activity on the murine immune system) and indirectly (reviewed by balkwill, 1989) . although antitumor activity has been clearly demonstrated by the application of exogenous ifns, it is not certain that endogenously produced ifns are involved in countering tumor growth. however, some experimental evidence that endogenous ifn could play a role in host resistance to cancer or its spread has been obtained by treating mice with anti-ifn antibodies. under these conditions, the intraperitoneal transplantability of six different experimental murine tumors was observed (gresser, 1984) . ifn-a/~ can inhibit the growth of hematopoietic progenitor cells in vitro (rigby et al., 1985) and this is also likely to occur in vivo. such an occurrence is undesirable in most instances, but suppression of proliferation of bone marrow hematopoietic cell precursors has been turned to advantage in protecting tumor-bearing mice against the cytotoxicity of chemotherapeutic agents such as 5-fluorouracil (5-fu) (stolfi et al., 1983) . ifns exercise their actions in cells via ifn-specific cell surface receptors. these receptors bind ifns with high affinity (aguet, 1980) and transduce the signal occasioned by ligand (ifn) binding across the cell membrane into the cytoplasm. ifn-cq ifn-[3 and ifn-co share the same binding sites (aguet et al., 1984; flores et al., 1991) , but ifn-y binds to different sites (branca and baglioni, 1981; aguet et al., 1988) . the binding of ifn-c~ and ifn-~ to lymphoid cells and fibroblasts has been studied extensively and has been reviewed (rubinstein and orchansky, 1986; branca, 1988; langer and pestka, 1988; grossberg et al., 1989) , and results have generally demonstrated the presence of up to a few thousand complex, high-affinity receptors per cell. chemical cross-linking studies with ~2si-labeled ifn-~ or ifn-[3 to receptor-bearing cells have led to the identification of various ifn-receptor complexes, their molecular masses ranging from 80 to 300 kda (joshi et al., 1982; eid and mogensen, 1983; faltynek et al., 1983; raziuddin and gupta, 1985; thompson et al., 1985; hannigan et al., 1986; vanden broecke and pfeffer, 1988; colamonici et al., 1992) . such results have suggested that there are either multiple binding sites for ifn-cx, ifn-[3, and ifn-m or that there are complex multichain receptors (colamonici et al., 1992; hu et al., 1993) . although on human cells all the ifn-c~, ifn-~, and ifn-m proteins compete for common binding sites, individual ifn-c~ subtypes show different levels of activities on cells (streuli et al., 1981; week et al., 1981; rehberg et al., 1982) which appear to correlate with their binding behavior to the cell surface (uzd et al., 1985) . in particular, ifn-% shows a much lower binding for human membrane receptors than either "ifn-0~ 2 or ifn-% (uz6 et al., 1988) . interestingly, this differential binding of human ifn-~ subtypes is not manifested in bovine cells, and all of the subtypes exhibit high specific activities (yonehara et al., 1983; shafferman et al., 1987) . ifn-i3 and ifn-m are also active in bovine cells (capon et al., 1985; adolf et al., 1990) , but this cross-reactivity does not extend to mouse cells, a feature that has provided experimental systems in which to characterize ifn-receptors. thus, somatic cell genetic studies with human • rodent hybrid cells containing various combinations of human chromosomes have provided evidence that the presence of human chromosome 21 confers sensitivity of such hybrid "rodent" cells to human ifn-~, ifn-i3 and ifn-m (tan et al., 1973; slate et al., 1978; epstein et al., 1982; raziuddin et al., 1984) . further, it was demonstrated that antibodies raised against human chromosome 21encoded cell surface proteins were able to block the binding and action of human ifn-~ to human cells, indicating that this chromosome contained a gene(s) specifying the human ifn cell surface receptor . the elucidation of the full complement of components of the ifn-~/[3/m receptor has long been sought. one methodology used to isolate receptor cdnas involves transfecting mouse cells with total human dna and then selecting for cells sensitive to human ifn-cz. after several attempts, this approach led successfully to the isolation of a 2.7 kb edna from a library constructed from human lymphoblastoid (daudi) cells, which encoded an ifn-cz binding protein (uzd et al., 1990) containing 557 amino acids (molecular mass 63485 da) including a signal sequence of 27 mainly hydrophobic amino acids. this protein has a structure typical of a transmembrane glycoprotein: a large n-terminal extracellular domain, which potentially could be highly glycosylated owing to a preponderance of n-linked glycosylation sites, a short hydrophobic transmembrane domain, and an intracellular or cytoplasmic tail (figure 25.5) . its amino acid sequence shows little homology with any currently available sequences of proteins, including the sequence of the human ifn-7 receptor (aguet et al., 1988) ; however, the extracellular domain has been predicted to show structural similarities with the latter receptor and to a lesser extent with the so-called hematopoietin receptor supergroup (bazan, 1990a,b) . the gene coding for this putative ifn-cz receptor has been mapped to chromosome 21.q22 , in confirmation of the earlier rodent x human hybrid cell data (tan et al., 1973; slate et al., 1978; epstein et m., 1982; raziuddin et al., 1984) . although the cloned "ifn-cz receptor" could be shown to confer sensitivity to human ifn-% in transfected mouse cells (uzd et al., 1990) , such cells were relatively insensitive to human ifn-% and human ifn-[3. these findings, together with those from anti-ifn-cz receptor antibody blocking studies (colamonici et al., 1990; revel et al., 1991; uz~ et al., 1991) and affinity cross-linking studies with ifn-% (colamonici et al., 1992) , have suggested that a second ifn-cz receptor exists or another component is required besides the cloned ifn-cz 8 binding protein, to complete the receptor complex. this hypothesis is further supported by a study in which introduction of a yeast artificial chromosome (yac) containing a segment of human chromosome 21 into chinese hamster ovary (cho) cells conferred a greatly increased response to both ifn-0~ 2 and ifn-0~8, as well as an increased response to ifn-i3 and ifn-m, whereas the expression of the ifn-~ 8 binding protein alone did not confer sensitivity (revel et al., 1991) . however, these increased responses can be "knocked out" by disruption of the ifn-~ 8 binding protein gene in the yac, and then reconstituted by expression of the cdna encoding the ifn-% binding protein (cleary et al., 1994) , suggesting that cell surface expression of this protein is required for a fully functional receptor (see also hertzog et al., 1994; constantinescu et al., 1994) . a second human ifn-~/i3 receptor, which is probably the additional component of the receptor complex referred to above, has been cloned (novick et al., 1994) . the 1.5 kb cdna encodes a 331-amino-acid protein, including a signal sequence, which has the predicted structure of a transmembrane glycoprotein. the nterminal ectodomain (217 amino acids) corresponds in sequence to a soluble 40 kda ifn-(x/[3 binding protein, p40, isolated from urine. this domain is linked to a transmembrane segment (21 amino acids) and a relatively small cytoplasmic domain of 67 amino acids ( figure 25 .5)i overall, the primary sequence shows little ifn-a/]3 binding protein bind ifn-a 2 but are insensitive to its effects, suggesting that an accessory protein, possibly the cloned ifn-c~ 8 binding protein, is required for signaling (novick et al., 1994; constantinescu et al., 1994) . the findings that anti-p40 antiserum and a particular monoclonal antibody to the ifn-cx 8 binding protein (benoit et al., 1993) both block the biological activity of ifn-0q3co indicate that the ifnhomology with that of the previously cloned ifn-a 8 . c~/13 and ifn-a 8 binding proteins are in close proximity, binding protein (uzd et al., 1990) , but when the extracellular domains are compared, 23.4% relatedness is found (novick et al., 1994) , suggesting that both of these ifn binding proteins belong to the same so-called class ii cytokine receptor family (uz6 et al., 1995) . two classes of cytokine receptor (class i and class ii) have been proposed by bazan (1990a,b) , these being distinguished by the positions of cysteine pairs in the extracellular domain. the latter is comprised of fibronectin type iii-like units containing around 200 amino acids and designated d200 (uzd et al., 1995) . a schematic drawing of both ifn-a/[5 receptor chains is shown in figure 25 .5. subsequently, it has been found that alternative splicing of the ifn-a/13 receptor gene can produce a transcript encoding a long form of the receptor protein containing a larger cytoplasmic domain of 251 amino acids . mouse cells transfected with the cdna encoding the and thus probably interact to form a high-affinity ifna/]3/co receptor complex. the most likely scenario on present evidence is for a two-chain ifn-c~/13/co receptor, comprising the cloned ifn-c~ 8 binding protein (uz6 et al., 1990) and the long form of the ifn-c~/[3 binding protein lutfalla et al., 1995) , each of which binds to some extent particular ifn types or ifn-(x subtypes but which together more strongly bind all ifn-~, -13, and -co species and function to transmit signals across the cell membrane. however, it is not completely ruled out that other cell surface components, e.g., membrane glycosphingolipids, are required for fully functional receptors (colamonici et al., 1992; platanias et al., 1994; ghislain et al., 1995; uz4 et al., 1995) or, possibly, that there are alternative ifn receptors, e.g., the epstein-barr virus/complement c3d receptor as an ifn-c~ receptor on b-lymphocytes (delcayre et al., 1991) . vaccinia virus and other orthopoxviruses contain a gene b 18r encoding a soluble type i ifn receptor which, unlike the class ii cytokine type receptors, belongs to the immunoglobulin superfamily (symons et al., 1995; colamonici et al., 1995) . 7.1 the intracellular domains of the two cloned ifn-binding proteins are unrelated to the tyrosine kinase class of receptors, e.g., epidermal growth factor receptor (egf-r) and platelet-derived growth factor-receptor (pdgf-r), and are not predicted to have kinase activity of any sort (uzd et al., 1990; novick et al., 1994) . however, it appears that the cytoplasmic domain of the ifn-0~ 8 and a/13 binding proteins associate with nonreceptor tyrosine kinases tyk2 and janus kinase 1 (jak1), respectively, known to be involved in the signal transduction pathway of ifn-a/[3 and other cytokines (novick et al., 1994; ghislain et al., 1995; ihle, 1995; ihle and kerr, 1995; velasquez et al., 1995) . the current understanding of this pathway is as follows. after binding of ifn-a/[3/c0 to their cognate receptors, the intracellular domains are phosphorylated by tyk2 and jak1. these phosphorylated domains act as docking sites for the cytoplasmic stat (signal transducers and activators of transcrilstion ) proteins p84/p91 (statla/b) and p113 (stat 2) (ihle, 1996) . the latter undergo tyrosine phosphorylation mediated by receptor-associated tyk2/jak1, dimerize, translocate to the nucleus, and combine with a dna binding protein, p48, to form the ifn-stimulated gene factor-3 (isgf3) transcription factor complex (schindler et al., 1992; velasquez et al., 1992; mtiller et al., 1993; platanias et al., 1994; shuai et al., 1994; gupta et al., 1996; yan et al., 1996) . both tyk2 and jak1 need to be reciprocally activated for signal transduction to occur, since cell mutants lacking either tyk2 or jak1 are unresponsive to ifn-0~ (ihle, 1995; ihle and kerr, 1995) . isgf3 binds to cis-acting ifn-stimulated response elements (isre), present in the promoter regions of ifn-inducible genes, to initiate their transcription (williams, 1991) . targeted disruption of the stat 1 gene in mice has shown that stat 1 has an obligatory role in ifn-cx and ifn-y signaling (durbin et al., 1996; meraz et al., 1996) . the jak1/tyk2-isgf3 pathway may not be the only "receptor-to-cell nucleus" signaling mechanism activated in ifn-stimulated cells. there has been some evidence to implicate protein kinase c (pkc) pathways as well (reich and pfeffer, 1990; pfeffer et al., 1991; c. wang et al., 1993) . however, this remains controversial owing to the lack of specificity of kinase inhibitors used (kessler and levy, 1991; james et al., 1992) . ifn-inducible genes have a common regulatory nucleotide sequence (g/a)ggaaan(n)gaaact in their 5' flanking region and this type of sequence, which resembles the vre sequences (ryals et al., 1985; present in ifn genes, is designated interferonstimulated response element (isre) (williams, 1991) . the resemblance between isre and vre sequences probably accounts for the finding that many ifninducible genes are transcriptionally activated by virus infection or dsrna, which also activate the transcription of ifn genes (hug et al., 1988; wathelet et al., 1988) . as mentioned previously (see section 5.1.3), ifn-receptor occupation activates cytoplasmic isgf-3 and this complex is translocated to the nucleus and binds to isre of ifninducible genes as a transcriptional activator. in addition, a second factor, isgf2, forms complexes with isre in ifn-stimulated cells. isgf2 is a single, inducible phosphoprotein that has been shown to be identical to irf-1 pine et al., 1990; williams, 1991; reis et al., 1992) . the role of a third transcription factor, isgf1, which is constitutively produced and requires only the central 9 bp core of isre for binding, remains to be fully defined (kessler et al., 1988 ) . a number of other negative regulatory factors, including irf2 (harada et al., 1989) and the isgf2 (irf1)/isgf3yrelated "human interferon consensus sequence binding protein" (icsbp) (weisz et al., 1992; bovolenta et al., 1994) , which also bind to isre, are also probably involved in the regulation of transcription of ifn-inducible genes. it is clear that the regulation of expression of ifninducible genes is complex and that the mechanisms that control their selective expression are not fully understood (see taylor and grossberg, 1990, for review) . ifninducible proteins, whose number probably exceeds 20, include both those proteins induced early after ifn stimulation and those proteins that may be produced at later times, often in response to the actions of "early" ifn-inducible proteins (sen and lengyel, 1992) . the full set of ifn-inducible proteins is probably not known, but several have been identified and characterized. table 25 .2 shows an incomplete list of ifn-inducible proteins together with their likely functions. some of these proteins are not exclusively induced by ifn-0~/[3/c0; ifn-y and certain other cytokines, e.g., tumor necrosis factor-~ (tnf-~) often induce spectra of proteins that overlap with the set induced by ifn-~/13/~ (revel and chebath, 1986; rubin et al., 1988; wathelet et al., 1992) . it should be noted that ifn-inducible proteins tend also to be cell type-specific and thus not all proteins listed in table 25 .2 will be expressed in all cell types. in some cases, ifn-inducible proteins are completely absent from a cell before ifn stimulation, but in other cases revel and chebath, 1986; sen and lengyel, 1992; samuel, 1987; staeheli, 1990. revel and chebath, 1986; lengyel, 1992. de maeyer and de maeyer guignard, 1988; heron et al, 1978 . schwemmle and staeheli, 1994 . ronni et al, 1993 . revel and chebath, 1986 . c. wang et al., 1993 . kumar and atlas, 1992 . loeb and haas, 1992 . aiidridge et al, 1989 . fellous et al, 1982 . sen and lengyel, 1992 . sen and lengyel, 1992 . bange et al., 1994 . chebath et al, 1983 . choubey and lengyel, 1992 . lawn et al, 1981a constantoulakis et al, 1993 . porter and itzhaki, 1993 . hokland and berg, 1981 . thomas and linch, 1991 . revel and chebath, 1986 they are being constitutively produced, their synthesis augmented by ifn. the genes for mouse ifn-~ subtypes and mouse ifn-[3 (no functional mouse ifn-c0 gene has been found) are located on mouse chromosome 4 (dandoy et al., 1984 (dandoy et al., , 1985 de maeyer and de maeyer-guignard, 1988, for review) . these genes are, like their human counterparts, intronless and of comparable structure. twelve mouse ifn-~ genes or pseudogenes have been identified, of which the cdnas for 10 different genes have been cloned and expressed (langer and pestka, 1985; de maeyer and de maeyer-guignard, 1988) . mouse ifn-~ subtype proteins contain 166 or 167 amino acids, or exceptionally 162 (mouse ifn-~8) , and the four cysteines at positions (langer and pestka, 1985) . there is only a single-copy mouse ifn-[3 gene and this encodes the 16i-amino-acid mature mouse ifn-[3 protein (higashi et al., 1983; de maeyer and de maeyer-guignard, 1988) . mouse ifn-13 contains only one cysteine and thus cannot form intramolecular disulfide bonds. it has three potential n-linked glycosylation sites and is heavily glycosylated when secreted from mouse fibroblasts; the molecular mass of the native glycoprotein is approximately 34 kda compared to the predicted 17 kda for the nonglycosylated counterpart (de maeyer and de maeyer-guignard, 1988 ). the amino acid sequence of mouse ifn-i3 is about 4:8% related to that of human ifn-i3. the threedimcnsional structure of mouse ifn-i3 has been solved (senda et al, 1992 ) (see section 3.1.1 ) and the protein has been shown to comprise five s-helices folded into a compact s-helical bundle (figure 25.4) . induction of transcription of mouse ifn-0~ subtype genes and the mouse ifn-i3 gene is probably regulated by transcription factor-binding nucleotide sequences present in the 5' noncoding promoter region, in a similar way to that of human ifn-~ and ifn-[3 genes (see section 2.2). for. example, repeated gaaa-rich sequences are present in the 5' flanking regions of most mouse ifn-cz subtype genes and these are likely to be important for virus-inducible transcription (shaw et m., 1983; zwarthoff et al., 1985) . inducers of mouse ifn-~ and ifn-i3 synthesis, which include a number of viruses and double-stranded polynucleotides, are similar to those which induce human ifn-c~, ifn-[3, and ifn-60 production (stewart, 1979; de maeyer and de maeyer-guignard, 1988) . similarly, the type of ifn produced follows the pattern found among different human cell types: fibroblastic and epithelial cell lines produce mainly ifn-~, whereas leukocytes produce mainly ifn-0~ subtypes (de maeyer and de maeyer-guignard, 1988) . the biological properties of mouse ifn-0~ and ifn-~ are similar to those of human ifn-0~, ifn-j3 and ifn-03 (see section 5). since mouse and human ifn-~ subtypes are only 40% homologous, there is considerable species preference in biological activity, i.e., mouse ifn-cx is weakly active in human cells and vice versa. mouse ifn-13 is also not active in human cells (stewart, 1979) . rather less is known regarding receptors for mouse ifn-~ and ifn-13, than for the human counterparts, but it is probable that they comprise two or more chains, as is the case for the human ifn-~/j3/00 receptors (uzd et al., 1995) . the mouse equivalent receptor chain to the ifn-0~ 8 binding protein (uzd et al., 1990) has been cloned (uz~ et al., 1992) . the gene for this mouse ifn-~/13 receptor has been located to mouse chromosome 16 (cheng et al., 1993) . the mouse ifn-~/13 receptor is 564 amino acids long and is divided into a large nterminal extracellular domain (403 amino acids), a short hydrophobic transmembrane segment (20 amino acids), and a cytoplasmic domain (141 amino acids) (uz~ et al., 1992) . the extracellular domain contains eight potential n-linked glycosylation sites and is predicted to exhibit the two-d200 domain structure of the human ifn-~ 8 binding protein extracellular domain (figure 25 .5) (uzd et al., 1995) . further mouse ifn-0~/]3 rceptors or components thereof await identification and characterization. signal transduction via mouse ifn-~/]3 receptors is expected to involve the jak1/tyk2-isgf3 (stat 1/2) pathway as outlined for human ifn-0~/]3/60 receptors (see section 7.1). in stat 1 genedeleted mice there are no overt developmental abnormalities, but they display a complete lack of responsiveness to mouse ifn-0~ and ifn-~, (durbin et al., 1996; meraz et al., 1996) . as a consequence, stat 1 -/-mice are highly susceptible to infection by viruses and microbial pathogens. stat 1 is therefore an obligatory mediator in the signal transduction pathway triggered by ifns. targeted disruption of the cloned mouse ifn-0~/]3 receptor gave rise to a knockout with a similar phenotype (miiller et al., 1994) . such mice, lacking the ifn-cx/13 receptor, developed normally but were unable to respond to mouse ifn-0~/]3 and thus unable to cope with viral infections. the potent antiviral activity of ifn-~/]3/c0 together with their potential antitumor actions provided the impetus for large-scale manufacture of ifns for the purpose of clinical evaluation in a variety of viral and malignant diseases. in the early 1970s, ifn production depended on pooled, human buffy coats (leukocytes) and thus only limited quantities could be made (cantell and hirvonen, 1977) . later in that decade, human lymphoblastoid cells (e.g., namalwa), which could be grown to large culture volumes, became available for ifn production. by the 1980s, following the cloning of ifn-~ and ifn-j3, these ifn species were massproduced by recombinant rdna technology, leading to abundant availability of certain ifn-0~ subtypes, e.g., ifn-0~ 2 and "stabilized" ifn-]3 ser 17. there followed production of ifn-7 and ifn-c0 by this means. clinical usage of ifn-~ preparations far exceeds that of ifn-[3 and ifn-c0 because of early production difficulties with the latter types, though these are now solved. at the beginning of the 1980s there was tremendous enthusiasm, both from manufacturers of ifns and from clinicians, to evaluate the therapeutic potential of ifns. however, early clinical trials had been poorly devised, were not "blinded", and often yielded only anecdotal evidence of success. it was only after many controlled, randomized studies had been conducted that it became apparent that ifns in general, administered as a single agent, were not beneficial for the treatment of the majority of malignant diseases, including the major cancers (lung, breast, colon) of the developed world. the initial optimism all but vanished and was replaced in the mid-to-late 1980s by a more sober and realistic appreciation of the potential therapeutic value ofifns. a number of general conclusions have been drawn, as follows. (i) ifn-0~ and ifn-]3, and to a lesser extent ifn-3', have antitumor activity in a small number of cancers, particularly in those that are relatively slow-growing and well-differentiated. (ii) there is no indication that heterogeneous ifn-~ preparations containing mixtures of ifn-0~ subtypes (e.g., leukocyte ifn, lymphoblastoid ifn) have different clinical effects from those of homogenous, recombinant ifn-0~ subtype or ifn-]3 preparations. (iii) continuous or intermittent high dosing appears to be required for antitumor efficacy. (iv) ifns probably work best in patients with a minimal tumor burden (balkwill, 1989) . a major concern that has emerged from clinical studies is that ifns all generate a considerable number of undesirable, clinically observable, side-effects, including fever, chills, malaise, myalgia, headache, fatigue, and weight loss, and in certain cases these have been severe enough for treatment to be halted (bottomly and toy, 1985; rohatiner et al., 1985; goldstein and laszlo, 1986) . in addition, a variable proportion (1-40%) of patients treated with ifn-0~ or ifn-]3, especially recombinant ifn-~ 2 and recombinant ifn-]3 ser 17, develop neutralizing antibodies to the ifn species used (rinehart et al., 1986; antonelli et al., 1991) , that in some instances have been associated with clinical "resistance" to ifn (steis et al., 1988; oberg et al., 1989; freund et al., 1989; fossa et al., 1992) . a further important, but generally unrecognized, side-effect of ifn-~ treatment is the possible induction of certain types of autoimmune disease (feldmann et al., 1989; gutterman, 1994) , probably mediated via ifn-induced upregulation of mhc antigen expression and generalized immunosuppression. the most responsive cancer to ifn-a therapy is a very rare form of b-cell leukemia, known as "hairy cell" leukemia (hcl), in which a response rate up to 80% has been reported (gutterman, 1994; baron et al., 1991; vedantham et al., 1992) . in hcl patients, the "hairy cells" invade the spleen and bone marrow and the disease takes an indolent course. it has been shown convincingly that ifn-~ therapy continued over several months leads to a clearance of "hairy cells" and in some patients a long-term remission is achieved. ifn-~ ser 17 or ifn-y were less effective against hcl (saven and piro, 1992) . the ifn-~-induced mechanisms whereby clearance of "hairy cells" is achieved are not fully understood, but it is believed that a direct action of ifn-~ leading to differentiation of "hairy cells" to a nonproliferating phenotype is involved (vedantham et al., 1992; gutterman, 1994) . not all patients benefit greatly from ifn-~ treatment and some develop neutralizing antibodies, particularly when ifn-0~ 2 is used (steis et al., 1988) . when such neutralizing antibodies cause resistance to further ifn-~ 2 treatment, clinical responses can be "rescued" by switching to a heterogeneous ifn-0~ preparation, e.g., leukocyte ifn-c~ (von wussow et al., 1991) . however, on the whole, ifn-~ therapy of hcl appears at least as effective and durable as chemotherapy with the drug pentostatin (2-deoxycoformycin) (saven and piro, 1992) . ifn-0~ therapy has also been shown to slow down the progression of chronic myelogenous leukemia (cml) (baron et al., 1991; gutterman, 1994) . in this malignant disease, leukemic cells grow slowly in the initial chronic, but benign, phase and persist for 2-4 years, but there follows a dramatic "blast crisis" producing rapidly proliferating myeloid leukemia cells and a fatal outcome. cml patients treated with ifn-~ in the chronic phase often achieve durable remissions, associated with the elimination of leukemic cells bearing the so-called "philadephia chromosome", sometimes lasting up to 8 years. other malignancies in which ifn-a therapy seems to work, although with generally a lower percentage of patients responding than in hcl and cml, include lowgrade non-hodgkin lymphoma, cutaneous t cell lymphoma, carcinoid tumors, renal cell carcinoma, squamous epithelial tumors of the head and neck, multiple myeloma, and malignant melanoma. in most of these cancers, complete responses are low compared to partial responses, but ifn-~ may help with maintenance therapy of diseases in some cases, e.g., multiple myeloma (mandelli et al., 1990; johnson and selby, 1994) . the neovascularization of primary tumors is a crucial step in their development and thus the anti-angiogenic activity of ifn-~/i3 (sidky and borden, 1987) may have therapeutic value in certain early malignancies, e.g., primary melanoma (gutterman, 1994) . kaposi sarcoma, often found in aids patients, has been regarded as an angiogenic tumor or angioproliferative disease, which may explain why ifn-c~ treatment can lead to regression of lesions in up to 40% of patients with this condition (de wit et al., 1988; groopman and scadden, 1989) . in preclinical systems, the combination oflfn therapy and conventional chemotherapy has appeared to offer greater chances of producing effective treatment of many cancers, but in clinical trials this strategy has produced mostly disappointing results (see wadler and schwartz, 1990, for review) . this may be due to (i) the inability of preclinical models accurately to predict the clinical situation; (ii) the lack of understanding of the biochemical interactions and biological consequences of combining ifns and chemotoxic agents; (iii) a failure to incorporate information on dose, scheduling, and sequence of administration of ifns and chemotoxic agents into clinical trials. despite having proven antiviral activity in vitro, ifns have not proved the hoped-for panacea for most common viral infections in man. ifn-0~/~ prevent the replication of common cold viruses (rhinoviruses and coronaviruses) in the test tube and when administered to volunteers in the form of a nasal spray, but cannot "cure" colds once they are established (scott r al., 1982; r.m. douglas et al., 1986; turner et al., 1986) . ifn-ix is only partially effective in preventing influenza virus infections (treanor et al., 1987) . topical applications of ifn-~/~ in the form of creams or ointments to herpes virus lesions, e.g., in herpes zoster (chickenpox), and genital warts (condyloma acuminatum) caused by papilloma viruses have been investigated, but have given limited beneficial effects. however, when administered parenterally, i.e., by intramuscular or intravenous injection, greater beneficial effects of ifn-(x/6 on virally caused lesions and warts have been found, although not to an extent that ifn therapy has become the treatment of choice (schneider et al., 1987; j.m. douglas et al., 1990; baron et al., 1991; gutterman, 1994) . another wart-like disease, juvenile laryngeal papilloma (jlp), which can severely obstruct the airways of young children, caused by the same papilloma virus types (6 and 11) as cause genital warts, has also been found to respond beneficially to ifn-~ therapy. disappointingly, ifn-cz therapy appears neither curative nor of substantial value as an adjunctive agent in the long-term management ofjlp (healy et al., 1988) . probably the most successful application of ifn-~ therapy to viral disease is in the treatment of chronic active hepatitis, caused by either hepatitis b or c viruses (baron et al., 1991; gutterman, 1994) . up to about 40% of chronic active hepatitis b patients respond to ifn-~ therapy; viral infectivity markers disappear and seroconversion and cure follow. it is interesting in the case of hepatitis b virus that viral activity is responsible for inhibiting the endogenous ifn system , and thus the administration of exogenous ifn-cz constitutes a replacement therapy. in hepatitis c virus infection, some serotypes of the virus are apparently more sensitive to ifn-~ therapy than others and prolonged treatment may be necessary (>6 months) to prevent relapses occurring (gutterman, 1994) . both ifn-~ and ifn-[3 have been shown to inhibit human immunodeficiency virus-1 (hiv-1) replication in vitro (hartshorn et al., 1987) . however, in vivo, there is little evidence showing that ifn-c~ therapy has any longterm beneficial effect in asymptomatic hiv-l-positive individuals or mds patients (friedland et al., 1988; lane et al., 1990) , except for limited regressions in kaposi sarcoma lesions (de wit et al., 1988; groopman and scadden, 1989) . combination therapies for hiv-1infected individuals involving ifn-~ and antiviral drugs such as zidovudine (azt) have also proved to be ineffective (berglund, 1991 ) . as mentioned earlier, ifn-0~/~ inhibits hematopoiesis and therefore induces leukopenia in patients. this effect has generally been thought to be undesirable and it can lead to immunosuppression; however, it has proved useful for the treatment of diseases in which there is uncontrolled leukocytosis, e.g., thrombocytosis (markedly elevated platelet numbers), associated with various myeloproliferative diseases (gisslinger et al., 1989) . resistance to ifn-~ 2 therapy has occurred in such patients when neutralizing antibodies to ifn-~ 2 have developed, but successful retreatment with a heterogeneous ifn-~ preparation, lymphoblastoid ifn (ifn-0~n1) has been reported (brand et al., 1993) . the findings that production of ifn-~ and ifn-7 was deficient in multiple sclerosis (ms) patients (neighbor and bloom, 1979) stimulated clinical trials to evaluate ifns in ms. rather unexpectedly, it has repeatedly been found that ifn-]3, either natural fibroblast-derived or the later recombinant ifn-j3 ser 17 (ifn-j3-1b), injected intrathecally, subcutaneously, or intramuscularly in patients with relapsing/remitting disease leads to a reduced rate of exacerbations of the disease and thus is possibly of clinical benefit in some patients (jacobs et al., 1981 (jacobs et al., , 1987 (jacobs et al., , 1993 the ifnb multiple sclerosis study group, 1993; paty et al., 1993) . the ifn-[3-induced mechanisms that contribute to this beneficial outcome are not known, but probably immunomodulatory actions are involved, e.g., suppression of growth and activity of autoreactive t lymphocytes in the central nervous system (goodkin, 1994) . it is unclear whether ifn-c~ would have a similar effect. however, the results with ifn-[3 treatment have been encouraging so far, although more follow-up of patients will be necessary to monitor any effects on the clinical progression of ms (ebers, 1994) . accumulation and breakdown of rna-deficient intracellular virus particles in interferon-treated nih 3t3 cells chronically producing moloney murine leukaemia virus inhibition of the cellular response to interferons by products of the adenovirus type 5 eia oncogene antigenic structure of human interferon 0~1 (interferon alpha ii): comparison with other human interferons monoclonal antibodies and enzyme immunoassays specific for human interferon (ifn) c01: evidence that ifn-c01 is a component of human leukocyte ifn purification and characterisation of natural interferon c01: two alternative cleavage sites for the signal peptidase natural human interferon-~2 is o-glycosylated human interferon c01: isolation of the gene, expression in chinese hamster ovary cells and characterization of the recombinant protein high affinity binding of 12si-labelled mouse interferon to a specific cell surface receptor various human interferon ~ subclasses cross-react with common receptors: their binding activities correlate with their specific biological activities molecular cloning and expression of the human interferon-7 receptor interferon [3 increases expression of vimentin at the mrna and protein levels in differentiated embryonal carcinoma (psmb) cells nomenclature of the human interferon proteins a family of structural genes for human lymphoblastoid (leukocyte-type) interferon neutralizing antibodies to interferon-cx: relative frequency in patients treated with different interferon preparations distinct activation of murine interferon-~ promoter region by irf-1/isgf-2 and virus infection human recombinant interferon c~-2c enhances the expression of class ii hla antigens on hairy cells cytokines in cancer therapy ifp35 is an interferon-induced leucine zipper protein that undergoes interferon-regulated cellular redistribution the interferons: mechanisms of action and clinical applications shared architecture of the hormone binding domains in type i and ii interferon receptors haemopoietic receptors and helical cytokines vaccinia virus-encoded eif-2 alpha homolog abrogates the antiviral effect of interferon a monoclonal antibody to recombinant human ifn-c~ receptor inhibits biologic activity of several species of human ifn-c~, ifn-[3 and ifn-m combined treatment of symptomatic human immunodeficiency virus type 1 infection with native interferon c~ and zidovudine clinical side effects and toxicities of interferon molecular interactions between interferon consensus sequence binding protein and members of the interferon regulatory factor family molecular analysis of the human interferon-cx gene family the interferon receptors evidence that types i and ii interferons have different receptors successful retreatment of an anti-interferon resistant polycythaemic vera patient with lymphoblastoid interferon-czn1 and in vitro studies on the specificity of the antibodies preparation of human leukocyte interferon for clinical use two distinct families of human and bovine interferon-c, genes are coordinately expressed and encode functional polypeptides identification of a common nucleotide sequence in the 3'-untranslated region of mrna molecules specifying inflammatory mediators activation of the ppp(a2'p)na system in interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(a2'p)na-dependent rnase interferon-induced 56,000 m r protein and its mrna in human cells: molecular cloning and partial sequence of the edna constitutive expression of (2'-5') oligo a synthetase confers resistance to picornavirus infection gart, son, ifnar, and crf-24 genes cluster on human chromosome 21 and mouse chromosome 16 interferon action: nucleolar and nucleoplasmic localization of the interferoninducible 72-kd protein that is encoded by the if: 204 gene from the gene 200 cluster sequence requirement for specific interaction of an enhancer binding protein (ebp1) with dna binding of epstein-barr virus small rna eber-1 to the double-stranded rna-activated protein kinase dai knockout and reconstitution of a functional human type i interferon receptor complex characterization of three monoclonal antibodies that recognize the ifna2 receptor multichain structure of the ifn-cx receptor on hematopoietic cells vaccinia virus b18r gene encodes a type i interferon-binding protein that blocks interferon transmembrane signaling role of interferon cz/[3 receptor chain 1 in the structure and transmembrane signalling of the interferon ~/[3 receptor complex inhibition of revmediated hiv-1 expression by an rna binding protein encoded by the interferon-inducible 9-27 gene expression of gene rrg is associated with reversion of nih 3t3 transformed by ltr-c-h-ras linkage analysis of the murine interferon-0~ locus on chromosome 4 segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine ifn-[3 gene to the murine ifn-cx genes nucleotide sequence of the chromosomal gene for human fibroblast (131) interferon and of the flanking regions epstein barr virus/complement c3d receptor is an interferon c~ receptor interferons and other regulatory cytokines isolation and structure of a human fibroblast interferon gene isolation and characterisation of a human fibroblast interferon gene and its expression in escherichia coli clinical and virological effects of high-close recombinant interferon alpha in disseminated aids-related kaposi's sarcoma nomenclature of the human interferon genes cooperative interaction of multiple dna elements in the interferon-j3 promoter cloning and expression of a long form of the 13 subunit of the interferon 0q3 receptor that is required for signalling a randomized trial of combination therapy with intralesional interferon alpha 2b and podophyuin versus podophyllin alone for the therapy of anogenital warts prophylactic efficacy of intranasal alpha-2 interferon against rhinovirus infections in the family setting an atf/creb binding site protein is required for virus induction of the human interferon ]3 gene targeted disruption of the mouse stat 1 gene results in compromised innate immunity to viral disease molecular cloning of human alpha and beta genes from namalwa cells treatment of multiple sclerosis isolated interferon alpha-receptor complexes stabilised in vitro human interferon-~a, -~2 and -~2 (arg) genes in genomic dna direct evidence that the gene product of the human chromosome 21 locus, ifrc, is the interferon-cx receptor recombinant human leukocyte interferon produced in bacteria has antiproliferative activity characterization of an interferon receptor on human lymphoblastoid cells two different virus-inducible elements are required for human ]3-interferon gene regulation purification, concentration and physico-chemical properties of interferons enhanced expression of hla antigens and ~2-microglobulin on interferon-treated human lymphoid cells modulation of tubulin mrna levels by interferon in human lymphoblastoid cells why are there so many subtypes of alpha-interferons human interferon omega (co) binds to the ~/~ receptor recombinant interferon c~-2a combined with prednisone in metastatic renal-cell carcinoma: treatment results, serum interferon levels and the development of antibodies expression of the terminal protein region of hepatitis b virus inhibits cellular responses to interferons c~ and 3' and double-stranded rna recombinant human interferon (ifn) alpha-2b in chronic myelogenous leukaemia: dose dependency of response and frequency of neutralizing anti-interferon antibodies a randomized placebo-controlled trial of recombinant human interferon alpha 2a in patients with aids delimitation and properties of dna sequences required for the regulated expression of human interferon evidence for a nuclear factor(s), irf-1, mediating induction and silencing properties to human ifn-j] gene regulatory elements involvement of a cis-element that binds an h2tf-1/nf-~cb like factor(s) in virus-induced interferon-[3 expression induction of the transcription factor irf-1 and interferon-j3 mrnas by cytokines and activators of second messenger pathways induction of endogenous ifn-~ and ifn-j3 genes by a regulatory transcription factor, irf-1 binding of the adenovirus vai rna to the interferon-induced 68-kda protein kinase correlates with function configuration of the interferon-cx/[3 receptor complex determines the context of the biological response cloning, sequencing and expression of two murine 2'-5'-oligoadenylate synthetases concerted evolution of human interferon ~ genes long-term interferon therapy for thrombocytosis in myeloproliferative diseases human leukocyte interferon produced by e. coli is biologically active the structure of eight distinct cloned human leukocyte cdnas interferon therapy in cancer: from imagination to interferon overlapping positive and negative regulatory domains of the human [3-interferon gene the human ~-interferon gene enhancer is under negative control interferon beta-lb human monocytes and lymphocytes produce different mixtures of s-interferon subtypes expression of human immune interferon edna in e. coli and monkey cells selective production of interferon-alpha subtypes by cultured peripheral blood mononuclear cells and lymphoblastoid cell lines novel human leukocyte interferon subtype and structural comparison of ~ interferon genes role of interferon in resistance to viral infection in vivo interferon and cell division. i. inhibition of the multiplication of mouse leukaemia l1210 cells in vitro by interferon preparations mechanism of antitumour effect of interferon in mice injection of mice with antibody to interferon enhances the growth of transplantable murine tumours interferon therapy for kaposi's sarcoma associated with the acquired immunodeficiency syndrome (aids) the structure of a thirty-six kilobase region of the human chromosome including the fibroblast interferon gene ifn-~ interferon receptors and their role in interferon action interferon action against reovirus: activation of interferon-induced protein kinase in mouse l929 cells upon reovirus infection the sh2 domains of stat 1 and stat 2 mediate multiple interactions in the transduction of ifn~ signals regression of the interferon signal transduction pathway by the adenovirus e1a oncogene cytokine therapeutics: lessons from interferon ~. proc. natl acad. sci. usa 91 differential human interferon alpha receptor expression on proliferating and non-proliferating cells the genes for the trophoblast interferons and the related interferon ~ii possess distinct 5'-promoter and 3'-flanking sequences structurally similar, but functionally distinct factors, irf-1 and irf-2, bind to the same regulatory elements of ifn and ifn-inducible genes absence of type i ifn system in ec cells: transcriptional activator (irf-1) and repressor (irf-2) are developmentally regulated anti-oncogenic and oncogenic potentials of interferon regulatory factors 1 and 2 activity of interferons alpha, beta, and gamma against human immunodeficiency virus replication in vitro a novel class of human type i interferons synthesis of two distinct interferons by human fibroblasts treatment of recurrent respiratory papillomatosis with human leukocyte interferon structural relationship of human interferon-~ genes and pseudogenes the gene for human fibroblast interferon (ifb) maps to 9p21 enhanced expression of 132-microglobulin and hla antigens on human lymphoid cells by interferon a gene on human chromosome 21 located in the region 21q22.2 to 21q22.3 encodes a factor necessary for signal transduction and antiviral response to type i interferons structure and expression of a cloned cdna for mouse interferon-[5 structure and expression in escherichia coli of canine alpha-interferon genes differential expression of human interferon genes induction of human interferon gene expression is associated with a nuclear-factor that interacts with the nf~cb site of the human immunodeficiency virus enhancer interferon enhances the antibody-dependent cellular cytotoxicity (adcc) of human polymorphonuclear leukocytes interferon-alpha hybrids a protective action of neurotropic against viserotropic yellow fever virus in macacus rhesus structural characterization of fibroblast human interferon-beta the absence of introns within a human fibroblast interferon gene interferon-induced and double-stranded rna-activated enzymes: a specific protein kinase and 2',5'-oligoadenylate synthetases evidence for multiple binding sites for several components of human lymphoblastoid interferon-cx interferon betal b is effective in relapsing-remitting multiple sclerosis. i. clinical results of a multicenter, randomized, double-blind, placebo-controlled trial cytokine receptor signalling stats: signal transducers and activators of transcription jaks and stats in signaling by the cytokine receptor superfamily virus interference. i. the interferon intrathecal interferon reduces exacerbations of multiple sclerosis intrathecally administered natural human fibroblast interferon reduces exacerbations of multiple sclerosis: results of a multicenter, double-blind study a phase iii trial of intramuscular recombinant beta interferon as treatment for multiple sclerosis: current status role of protein kinase c in induction of gene expression and inhibition of cell proliferation by interferon cx the treatment of multiple myelomaman important mrc trial interferon receptors. crosslinking of human leukocyte alpha-2 to its receptor on human cells inhibition of interferon-inducible gene expression by adenovirus e1a proteins: block in transcriptional complex formation identification and characterisation of a novel repressor of interferon-~ expression protein kinase activity required for an early step in interferon-~ signalling two interferon-induced nuclear factors bind a single promoter element in interferon-stimulated genes interferon: purification and initial characterisation from diploid cells human fibroblast interferon: amino acid analysis and amino terminal amino acid sequence three-dimensional model of a human interferon alpha consensus sequence malignant transformation by a mutant of the ifn-inducible dsrna-dependent protein kinase studies on the role of the 2'-5'-oligoadenylate synthetase-rnase l pathway in l-interferon-mediated inhibition of encephalomyocarditis virus replication interferon c~ induces the expression of retinoblastoma gene product in human burkitt lymphoma daudi cells: role in growth regulation interferon alpha in patients with asymptomatic human immunodeficiency virus (hiv) infection: a randomized, placebo-controlled trial structure of interferons interferon receptors dna sequence of two closely linked human leukocyte interferon genes dna sequence of a major human leukocyte interferon gene synergism between distinct enhanson domains in viral induction of human beta interferon gene characterization and regulation of the 58,000-dalton cellular inhibition of the interferon-induced, dsrna-activated protein kinase the involvement of nf-~cb in 13-interferon gene regulation reveals its role as a widely inducible mediator of signal transducfion biochemistry of interferons and their actions tumor-suppressor genes: news about the interferon connection amino-terminal amino acid sequence of human leukocyte interferon amino acid sequence of a human leukocyte interferon molecular analysis of a human interferoninducible gene family the interferon-inducible 15-kda ubiquitin homolog conjugates to intracellular proteins the structure of the human interferon c~/[3 receptor gene mutant u5a cells are complemented by an interferon-a[3 receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster different pathways mediate virus inducibility of the human ifn-~i and ifn-~ genes maintenance treatment with recombinant interferon alpha 2b in patients with multiple myeloma responding to conventional induction chemotherapy controlled transcription of a human m-interferon gene introduced into mouse l-cells the nucleotide sequence of a cloned human leukocyte interferon cdna site-specific mutagenesis of the human fibroblast interferon gene adenovirus virus-associated rna and translational control targeted disruption of irf-1 or irf-2 results in abnormal type i ifn gene induction and aberrant lymphocyte development involvement of a gene on chromosome 9 in interferon production interferon production: variation in yields from human cell lines somatic cell genetics of human interferon production in human-rodent cell lines the effect of hypertonic salt on interferon and interferon mrna synthesis in human mg63 cells interferon-induced mx proteins form oligomers and contain a putative leucine zipper targeted disruption of the stat 1 gene in mice reveals unexpected physiologic specificity in the jak-stat signaling pathway tumor suppressor function of the interferon-induced double-stranded rna-activated protein kinase recent divergence from a common ancestor of human ifn-~ genes mechanism of interferon action. interferon mediated inhibition of reovirus mrna translation in the absence of detectable mrna degradation but in the presence of protein phosphorylation regulated expression of a gene encoding a nuclear factor, irf-1, that specifically binds to ifn-[3 gene regulatory elements roles of the 29-138 disulfide bond of subtype a of human interferon in its antiviral activity and conformational stability the protein tyrosine kinase jak1 complements defects in the ifn-a/[3 and -~, signal transduction functional role of type i and type ii interferons in antiviral defense multimerization of aagtga and gaaagt generates sequences that mediate virus inducibility by mimicking an interferon promoter element the structure of one of the eight or more distinct chromosomal genes for human interferon-c~ absence of virus-induced lymphocyte suppression and interferon production in multiple sclerosis transcriptional activation by viral regulatory proteins interferon-j3 promoters contain a dna element that acts as a position-independent silencer on the nf-~cb site the human interferon ~/j3 receptor: characterisation and molecular cloning treatment of malignant carcinoid tumors with recombinant interferon alfa-2b: development of neutralizing interferon antibodies and possible loss ofantitumor activity close linkage of ~ and ~ interferons and infrequent duplication of ]3 interferon in humans leukocyte and fibroblast interferon genes are located on human chromosome 9 interferon beta-lb is effective in relapsing-remitting multiple sclerosis. ii. mri analysis results of a multicenter, randomized, double-blind, placebo-controlled trial interferons and their actions transmembrane signalling by interferon ~ involves diacylglycerol production and activation of ~ isoform of protein kinase c in daudi cells large scale production of human interferon from lymphoblastoid cells purification and cloning of interferon-stimulated gene factor 2 (isgf2): isgf2 (irf-1) can bind to the promoters of both beta interferon and interferon-stimulated genes, but is not a primary transcriptional activator of either tyrosine phosphorylation of the ~ and ~ subunits of the c~ and [3 subunits of the type i interferon receptor gene targeting in human somatic cells: complete inactivation of an interferoninducible gene interferons and natural killer cells not more than 117 base pairs of 5'-flanking sequence are required for inducible expression of a human ifn-~ gene receptors for human interferon-alpha: two forms of interferon-receptor complexes identified by chemical cross-linking receptors for human cx and [3 interferon but not for 7 interferon are specified by human chromosome 21 specific molecular activities of recombinant and hybrid leukocyte interferons evidence for involvement of protein kinase c in the cellular response to interferon ~ a single dna response element can confer inducibility by both ~-and 7-interferons enhanced in vivo therapeutic response to interferon in mice with an in vitro interferon-resistant b-cell lymphoma mice devoid of interferon regulatory factor (irf-1) show normal expression of type i interferon genes critical role of a common transcription factor, irf-1, in the regulation of ifn-~ and ifn-inducible genes interferons and interleukin-6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells interferon-activated genes components of the human type antigen presentation by human monocytes: effects of modifying major histocompatibility complex class ii antigen expression and interleukin 1 production by using recombinant interferons and corticosteroids double-stranded rna-dependent protein kinase and 2-5a system are both activated in interferon-treated, encephalomyocarditis virus-infected hela cells the effect of recombinant-dna-derived interferons on the growth of myeloid progenitor cells phase i/ii trial of human recombinant ~-interferon serine in patients with renal cell carcinoma interferons as hormones of pregnancy central nervous system toxicity of interferons control of ifn-inducible mxa gene expression in human cells tumour necrosis factor and ifn induce a common set of proteins the interferon receptors human leukocyte interferon purified to homogeneity human leukocyte interferon: isolation and characterisation of several molecular forms induction of type i interferon genes and interferon-inducible genes in embryonal stem cells devoid of interferon regulatory factor i a 46-nucleotide promoter segment from an ifn-cx gene renders an unrelated promoter inducible by virus interferon induction of the antiviral state proteins induced by interferons and their possible roles in the antiviral mechanisms of action treatment of hairy cell leukaemia interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor interference between animal viruses interferon treatment of human genital papilloma virus infection: importance of viral type the interferoninduced 67-kda guanylate-binding protein (hgbp1) is a gtpase that converts gtp to gmp prevention of rhinovirus colds by human interferon alpha-2 from e. coli inhibition of ornithine decarboxylase in human fibroblast cells by type i and type ii interferons the interferon system: a bird's eye view of the biochemistry three-dimensional crystal structure of recombinant murine interferon-[3 specific residues within an amino-terminal domain of 35 residues of interferon-~ are responsible for recognition of the human interferon-a cell receptor and for triggering biological effects structure and expression of cloned murine ifn-a genes a conserved au sequence from the 3' untranslated region of gm-csf mrna mediators selective mrna-degradation a single amino acid change in ifn-[31 abolishes its antiviral activity existence and unique n-terminal sequence of alpha ii (omega) interferon in natural leukocyte interferon preparation clustering of leukocyte and fibroblast genes on human chromosome 9 interferon activation of the transcription factor star91 involves dimerization through sh2-phosphotyrosyl peptide interactions antibodies to chromosome 21 coded cell surface components block binding of human ~ interferon but not 7 interferon to human cells expression of interferon-~ and interferon-j3 genes in human lymphoblastoid (namalwa) cells inhibition ofangiogenesis by interferons: effects on tumour-and lymphocyte-induced vascular responses presence of human chromosome 21 alone is sufficient for hybrid cell sensitivity to human interferon chromosomal location of a human cx interferon gene family expression of a functional human type i interferon receptor in hamster cells: application of functional yeast artificial chromosome (yac) screening interferon-induced proteins and the antiviral state resistance to recombinant interferon alfa-2a in hairy-cell leukaemia associated with neutralizing anti-interferon antibodies the interferon system interferon nomenclature modulation of 5-fluorouracil-induced toxicity in mice with interferon or with the interferon inducer, polyinosinic-polycytidylic acid at least three human type a interferons: structure of a 2 target specificity of two species of human interferon-a produced in escherichia coli and of hybrid molecules derived from them vaccinia virus encodes a soluble type i interferon receptor of novel structure and broad species specificity the linkage of genes for the human interferon-induced antiviral protein and indophenol oxidase-b traits to chromosome g-21 human leukocyte and fibroblast interferons are structurally related recent progress in interferon research: molecular mechanisms of regulation, action, and virus circumvention the high mobility group protein hmgi (y) is required for nf-~cb-dependent virus induction of the human ifn-13 gene an intracellular 50kda fc~,-binding protein is induced in human cells by 0~-ifn characterization of human beta-interferon-binding sites on human cells two non-allelic human interferon a genes with identical coding regions intranasally administered interferon as prophylaxis against experimentally induced influenza a virus infection in humans human natural killer cells: biologic and pathologic aspects prevention of experimental coronavirus colds with intranasal alpha-2b interferon nucleotide sequence of a portion of human chromosome 9 containing a leukocyte interferon gene cluster characterization of four different mammalian-cell-derived recombinant human interferon-[31s receptor dynamics of closely related ligands: "fast" and "slow" interferons electrostatic interactions in the cellular dynamics of the interferon-receptor complex genetic transfer of a functional human interferon 0~ receptor into mouse cells: cloning and expression of its cdna murine tumor cells expressing the gene for the human interferon 0q3 receptor elicit antibodies in syngeneic mice to the active form of the receptor behaviour of a cloned murine interferon alpha beta receptor expressed in homospecific or heterospecific background 0~ and ]3 interferons and their receptor and their friends and relations characterization of interferon-alpha binding sites on human cell lines mechanism of interferon action in hairy cell leukaemia: a model of effective cancer biotherapy a protein tyrosine kinase in the interferon a/j5 signalling pathway distinct domains of the protein tyrosine kinase tyk2 required for binding of interferon-a/j3 and for signal transduction role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type 3 infection of susceptible and resistant strains of mice double-stranded rna activates binding of nf-~cb to an inducible element in human [3-interferon promoter interferon-induced enhancement of macrophage fc receptor expression: [3-interferon treatment of c3h/hej macrophages results in increased numbers and density of fc receptors effective natural interferon-~ therapy in recombinant interferon-m-resistant patients with hairy cell leukaemia antineoplastic activity of the combination of interferon and cytotoxic agents against experimental and human malignancies: a review interferon c~ induces a protein kinase c-e (pkc-e) gene expression and a 4.7kb pkc-8 related transcript interferon increases the abundance of submembranous microfilaments in hela-s3 cells in suspension culture activation of ifn-13 element by irf-1 requires a translational event in addition to irf-1 synthesis regulation of two interferon-inducible human genes by interferon, poly(ri):poly(rc) and viruses regulation of gene expression by cytokines and virus in human cells lacking the type-i interferon locus antiviral activities of hybrids of two major human leukocyte interferons the 5'-flanking region of a human ifn-c~ gene mediates viral induction of transcription human interferon consensus sequence binding protein is a negative regulator of enhancer elements common to interferon-inducible genes vaccinia rescue of vsv from interferon-induced resistance: reversal of translation block and inhibition of protein kinase activity identification of novel factors that bind to the prd1 region of the human [3-interferon promoter postinduction repression of the 13-interferon gene is mediated through two positive regulatory domains treatment of malignant carcinoid tumors with recombinant interferon alfa-2b: development of neutralizing interferon antibodies and possible loss of antitumour activity transcriptional regulation of interferon-stimulated genes inhibition of protein synthesis by 2'-5' linked adenine oligonucleotides in intact cells a comparison of vertebrate interferon gene families detected by hybridisation with human interferon dna ribosomal rna cleavage, nuclease activation and 2-5a (ppp(a2'p)na) in interferon treated cells multiple protein-dna interactions with the human interferon-[3 regulatory element phosphorylated interferon-~ receptor 1 subunit (ifnar1) acts as a docking site for the latent form of the 113 kda stat 2 protein different binding of human interferon-c~l and-c~2 to common receptors on human and bovine cells. studies with recombinant interferon produced in escherichia coli interferon-0~ inhibits cyclin eand cyclin dl-dependent cdk-2 kinase activity associated with rb protein and e2f in daudi cells expressing cloning of 2-5a-dependent rnaase: a uniquely regulated mediator of interferon action comparative structures of mammalian interferons amino-terminal sequence of the major component of human lymphoblastoid interferon purification and characterization of multiple components of human lymphoblastoid interferon-c~ organization, structure and expression of murine interferon c~ genes key: cord-017112-5men6dfk authors: gupta, varsha; sengupta, manjistha; prakash, jaya; tripathy, baishnab charan title: biosafety and bioethics date: 2016-10-23 journal: basic and applied aspects of biotechnology doi: 10.1007/978-981-10-0875-7_24 sha: doc_id: 17112 cord_uid: 5men6dfk the advancement in technology is likely to tame several life forms present on earth. microorganisms are posing a big challenge due to difficulties encountered to control the diseases caused by them. working with deadly disease-causing microorganisms for their characterization, diagnostics or therapeutics and vaccine development purposes are posing increasingly potential biosafety problems for laboratory workers. thus, an appropriate biosafe working environment may protect workers from laboratory-induced infections. biotechnology has the ability to solve the upcoming problems of the world’s increasing population. however, there is often reluctance among the public to accept and support biotechnological products in medicine, industry, or agriculture. there are many safety and ethical issues raised for gm crops and human cloning. raising transgenic animals and plants has fueled ethical concerns, and the scientists have faced a lot of resistance where genetically modified crop plants or reproductive cloning research of human beings is involved. thus, biosafety and bioethics are continuously being expanded to combine the rationale of ever-increasing scientific knowledge in biotechnology that is often in conflict with the long-standing social and moral value system of our society. the advancement in technology is likely to tame several life forms present on earth. microorganisms are posing a big challenge due to diffi culties encountered to control the diseases caused by them. working with deadly diseasecausing microorganisms for their characterization, diagnostics or therapeutics and vaccine development purposes are posing increasingly potential biosafety problems for laboratory workers. thus, an appropriate biosafe working environment may protect workers from laboratory-induced infections. biotechnology has the ability to solve the upcoming problems of the world's increasing population. however, there is often reluctance among the public to accept and support biotechnological products in medicine, industry, or agriculture. there are many safety and ethical issues 24 7 raised for gm crops and human cloning. raising transgenic animals and plants has fueled ethical concerns, and the scientists have faced a lot of resistance where genetically modifi ed crop plants or reproductive cloning research of human beings is involved. thus, biosafety and bioethics are continuously being expanded to combine the rationale of ever-increasing scientifi c knowledge in biotechnology that are often in confl ict with the long-standing social and moral value system of our society. however, biotechnological tools have resulted in high-yielding crop plants, more nutritive values of food grains, longer shelf life, and resistance to insects and pests, but the public acceptance to these biotechnological products is rather low. for example, gm foods have low support in europe and india. with the passing years, there is further decline in public support, probably due to media focus and the visible debates on gm crops for fear of their long-term effects and unknown risks and environmental safety issues. the controversies from nongovernment organizations (ngos), scientists, and media have led to a negative impact on the gm crops. gm crop usage has been controversial with fears being expressed on flavr savr tomato and many others. gm food again went into controversy due to labeling issues. the bt crops were challenged with the development of resistance in insects. so-called terminator technology and a gene use restriction technology (gurt) met with a lot of resistance due to introduction of negative traits of sterility in the seeds and never came to the market. the development of cloned animals and its impact on other wild-type animals and the environment also triggered many safety and ethical concerns. whether or not animals should be used in research, their welfare, sufferings, and wellbeing were debated throughout the world. in india many plants and animals are linked to religious beliefs and are respected and worshipped for their contribution in the life of human beings. likewise usage of embryonic stem cells faced controversies and concerns. protestants agree to have stem cell research under strict public regulations. many however are opposed to embryonic stem cell research as it destroys the embryo. the debates and issues surrounding the stem cell are all centered on the potential source of these cells. embryonic stem cell usage either is prohibited or is under strict public regulation. somatic stem cells and dedifferentiated somatic cells can be used for therapy. the public support for xenotransplantation was low due to safety issues with fear of spread of unknown contaminants. however, some xenogeneic tissue-engineered products are now available to treat dermal wounds and burn patients. the usage of biological weapons (use of living organism or its product for human killing) in war is presently banned. the biotechnology has tremendously evolved including the technical aspects of medical sciences like diagnostics and therapeutics. with all these, we are also witnessing rapid and dangerous adaptations in microorganisms especially for developing resistance to antibiotics. microbial pathogens are causative agents of many diseases and due to mutation in their genes, they have developed multiple drug resistance. it is gradually becoming diffi cult to control these multidrug-resistant infectious pathogenic microorganisms. to search for solutions to overcome mdr of these pathogenic agents, several scientists and medical workers are handling these pathogens . this poses enormous biohazards and raises serious "biosafety" issues, i.e., scientifi c practices, methods, and use of appropriate equipment in a biosafe environment. the biosafety aspects have become very important in various conditions and require many precautions in health-care systems as hospitals, diagnostic laboratories, animal care systems, biological laboratories, and so on. the precautions, which can be taken to reduce or nullify the risk, associated with samples by continuously monitoring and recognizing potential hazards, their risk assessment, and preventive measures to avoid exposure which might result in infection. individual worker should be appropriately trained and must understand the conditions like containment (conditions where infectious agent can be safely manipulated) and good laboratory practices which can minimize exposure to pathogens . [ 4 ] biorisk risk is the likelihood of the occurrence of an adverse event, thus biorisk is the likelihood of the occurrence of serious infection due to exposure to pathogenic microorganism or biohazards. upon exposure there may be mild to severe infections, allergies, or other clinical problems associated with the pathogen . the biorisk can be managed by risk assessment, effective biosafety measures, and biocontainment . the fi rst report on laboratory-associated infections was published at the beginning of the twentieth century. however, 4,079 infections associated with laboratory were reported with 168 deaths somewhere between 1930 and 1978. the major pathogenic agents responsible for these infections were brucella spp., blastomyces dermatitidis , chlamydia psittaci , coccidioides immitis , coxiella burnetii , hepatitis b virus ( hbv ), salmonella typhi , francisella tularensis , mycobacterium tuberculosis , and venezuelan equine encephalitis virus [ 23 , 29 , 30 , 38 ] . after this 1978 report, many more laboratories associated infections and deaths were reported [ 31 , 32 ] . in these cases also the infectious agents were arboviruses , brucella spp., coxiella burnetii , cryptosporidium spp., hantavirus , mycobacterium tuberculosis , hbv , salmonella spp., shigella spp., and hepatitis c virus . the processes used for risk assessment are (1) identifi cation of the hazardous properties of a familiar infectious agent or material, (2) the activities responsible for pathogen exposure to the person, (3) the chances of the exposure becoming a laboratory-associated infection, and (4) the ultimate consequences of infection. risk assessment needs very alert judgments. if risks are underestimated, they might result in seri-ous and adverse consequences. overestimation of risks can result in undue protective measures, which may be a burden for the laboratory. assessment of risk also needs to evaluate the factors responsible for infections i.e., whether it is due to hazardous agents or due to hazards of laboratory procedure, or the working attitude of the laboratory staff. the staff's capability can be improved by appropriate training and inculcating good laboratory practices with training of accidental spillage/inoculations. biohazards may be the microbial infectious agent or other biological materials posing a risk for human health, parasites, viruses, prions , or biologically derived toxins, allergens, venoms, or recombinant dna that can adversely affect human or animal health and environment [ 24 ] . the properties of an agent which make it potentially hazardous are [ 40 ] (1) infection capability, (2) ability to cause diseases in human or animal host, (3) its minimum infective dose, (4) severity of the disease, (5) availability of vaccine, (6) availability of therapeutic modality to control it, (7) the probable route followed by it for transmission, (8) stability in the environment, and (9) host (animal-human or only human). the probable routes of pathogen transmission may be: 1. through exposed part of the body such as the skin, eye, or mucous membrane 2. through inhalation 3. through needle or other sharp objects 4. through accidental ingestion 5. through bites of mosquito the world health organization (who) has recommended the classifi cation based upon the risks and route of transmission of the pathogenic agents in a laboratory environment. according to their disease-causing capacity and preventive measures available, the human pathogenic agents have been divided into four groups (according to who and nih) [ 44 , 45 ] . national biosafety guidelines came into being because of efforts of microbiological and biomedical communities. these involved the recognition of hazards, assessment of risk, and appropriate use of measures including biocontainment to prevent hazards. they promoted safe laboratory practices and usage of safety equipments along with occupational health programs to reduce laboratory-induced infections while handling microorganisms [ 5 , 6 ] . preventive measures also require personal protective equipment like gloves, mask, eye protection, laboratory coat, shoe covers, and respiratory protective measures for prevention of exposure. the biosafety level (bsl) indicates the kinds of precautions of biocontainment for handling dangerous microbial pathogens . the bsl-1 indicates lowest safety measures while bsl-4 indicates highest possible safety measures. the containment of laboratory refers to: current biosafety and biocontainment practices and procedures are designed to reduce the exposure of laboratory personnel, the public, agriculture, and the environment to potentially hazardous biological agents [ 2 ] . biosafety levels (bsl) are laboratory designations, which are based upon the degree of risk. they are designated as bsl-1, bsl-2, bsl-3, and bsl-4. these safety levels ensure different levels of protection when working with virulent microbial strain. these laboratories are very sophisticated and have very good design, engineering control, and safe work practices. their different levels are used depending upon the pathogen properties like vaccine preventability, infectivity and its contagious nature, severity of the diseases, and risk assessment in case of infection. every level of containment has its own safety aspects: any kind of activity that includes usage of potential hazardous human pathogens , zoonotic agents ( rabies virus, infl uenza virus , trypanosomes (sleeping sickness)), toxins, and agricultural threats which may pose danger to human civilization is recommended for use only in high biosafety levels . the various biosafety levels appropriate for these works are biosafety levels 3 and 4, animal facility/vivarium (absl-3 and absl-4), and biosafety level 3 agricultural facilities (bsl-3-ag). high containment refers to the highest level of biosafety. good biosafety and biocontainment practices can help in effective laboratory biosecurity. there is an urgent requirement to provide adequate safety for all life forms in case of natural emergency. natural emergency can be the outbreak of any disease or other conditions requiring attention due to attempt for spreading bioterrorism. thus federally funded research programs are being developed to protect all life forms. in the measures the products were designed and developed which can protect public health, are called medical countermeasures. these include public health and hygiene, diagnostics, vaccines, therapeutics, and development of biosafety level laboratories. for the purpose of biosafety, the general laboratory workers (students, scientists, and laboratory staff), along with non-laboratory workers as electricians, plumbers, and sweepers, should also be given appropriate training. the trainings are required in the areas of: every scientifi c revolution brings with it a host of ethical and social questions. the so-called genetics revolution is no exception, giving rise to a broad international debate on how the undoubted benefi ts of progress in this area can be reconciled with certain core human values. (unesco 2002a : 1) present-day biotechnology opens many opportunities in research and development, addressing medical issues and new ways to explore things; improving human health conditions, fi ght food, and feed problems; and so on. it has greatly infl uenced medicine and agriculture. it has tremendously affected the thinking process also like consuming vegetables and fruits without pesticides, using biofertilizer in place of chemical fertilizer, using renewable sources of energy, and switching to products which are biodegradable. it has also advocated the sustainability of systems (agricultural, environmental), knowledge sharing, and brought many products in the market. as the techniques involved gene manipulations in many life forms (plants, animals, microbes), thus with these advancements came the concerns in the form of ethical issues giving rise to bioethics. in our routine life also, we encounter ethical questions in terms of right or wrong or ethical or unethical; thus, in science also things are judged as being ethical or unethical. bioethics addresses policy and ethical issues arising by researches and products targeted for human applications. bioethics addresses the ethical issues in all the streams of life sciences like health care, genetics, and medical research by applying the principles of morality and philosophy [ 37 ] . bioethics has evolved from medical ethics and moral philosophy. the ethical concerns for patient well-being were in use in the form of the hippocratic oath. it starts right from the laboratory to industry and government and affects a very wider society. the study of the social and moral responses arising due to scientifi c invention or experimentation is "bioethics." thus it led to granting of ethical clearance for any proposed research projects requiring animal or human experimenta-tions. the project is fi rst reviewed by research/ institutional ethics committee (rec/iec). the rec evaluates the risks, benefi ts, animal sufferings, utility of work and then only grants approval for conducting research projects and proposals. nuremberg code (1947) this code surfaced in the response of human rights abuses that had taken place under the nazis during world war ii. the war captives were subjected to abuses through experiments on human subjects. this propounded the use of informed consent and non-malefi cence, benefi cence, justice, and autonomy. these provided normative framework used by researchers and medical practitioners. the ethical principles consist of certain virtues such as autonomy, non-malefi cence, beneficence, and justice. now the area has gained so much of momentum that before even small experimental work, one needs to justify its rightness or wrongness. analyzing the ethical issues before starting any new experimental work [ 11 ] would depend upon the assessment of these points: • consequences : what would be effect of this experiment? who is going to be benefi ted? what would be harms associated with this work? thus weighing the possible outcomes and their effects. • rights and responsibilities : are we exploiting somebody else's rights? do those rights need protection? who would protect these? these are questions whose justifi cation would have to be weighted. • autonomy : if one feels it is right, should things move on? or else someone feels it is wrong, should things stop? alternatively, who would decide things should be or should not be? these questions would again need to be addressed. • virtue ethics : is this the best thing to do? what is good about it? • animal rights : one big question is "are we authorized to use animals the way we want?" knowing that they too can think, they are aware of family members, they feel pain (at different levels), and they are alive. animals like primates and whales resembling human brain features, family behavior, and some sensitivity like humans should not be manipulated but others should be, why? animals have been in use since long. they are used for manipulation of their genes, as model system for study of human diseases, and as transgenics for production of pharmaceuticals. is inflicting pain in any form, which may be by creating a disease model or studying mutations in animals, ethical or not? • morality : it is a system of moral values and conduct, which govern our decision of right and wrong or good and bad. • empathy : the ability of one to understand and share the feelings of the other. • euthanasia : it refers to painless killing of any terminally ill patient suffering from painful and incurable disease on demand of the patient and the court. removing all life support system from a patient in irreversible coma is also termed as euthanasia. • autonomy is freedom from external control or infl uences, independence. • justice : it is the treatment or behavior, which is genuine, right, or just according to the prevailing laws. • equality : state of being at par or equal in status or ideology or opportunities. • benefi cence : it is related to kindness, good, or charity for good. • non-malefi cence : an act done to avoid harm or any act which would not harm or violate the trust of others. in the case of physicians, it is their act which will not harm the patient. • accountability : the condition where somebody is held responsible, answerable, or accountable for an act. philosophical ethics can be divided into descriptive ethics and normative ethics . it explains the actual moral viewpoints of the people; thus, it gives an accurate estimation of people's ethics. opinion polls or questionnaire evaluation are used to build a consensus of the view of the people. however, these are not affected by facts and reasons so they may be right or wrong [ 17 , 20 , 21 ] . it explains the viewpoint, which people should have, and accordingly the actions, which people should undertake (according to what ought to be). cult situations about what should be done [ 3 , 37 ] . some of the important ethical theories are discussed: 1. ethical egoism : this theory states that morality is mere fulfi lling our self-interest. it criticizes the views associated with morality that are in the way of personal self-interest. it advocates that the actions are entirely dependent on desire and self-interest of the people. this theory advocates the selection of moral action, which in its consequence brings greatest happiness for the large number of people. it works to decide the result or consequences of good action, which would maximize happiness. thus it deals with consequences of action, in that happiness is judged because of some decision. however, greatest good for an individual or society, human race, or animals is debated. their supporters say that the theory aims to do good for the human race as a whole including nonhuman animals. for example, terminally ill patients should be allowed for physician-assisted mercy killing to avoid pain and sufferings. utilitarians also believe that abortion and infanticide may be possible in case the baby has severe disability. research on human embryo and genetic enhancement, which can benefi t humans, may be made possible. which states that morality arises from inner sense of duty, was infl uenced by the german philosopher immanuel kant (1724-1804). according to this all people have rights and duties and they should follow these. it states that the decision should come from inner sense of duty without considering the consequences of action. thus according to kant any act that follows categorical imperative is moral. kantian theory requires the use of morality based on rationality with respect to persons and human dignity. kantian views refrain from hiding the truth from patients who have terminal cancer. kant's categorical imperative-"act in such a way that you treat humanity, whether in your own person or in the person of any other, never merely as a means to an end, but always at the same time as an end" (kant 1785/1968) [ 19 ] . it has been used to avoid abuses in research experiments on human subjects. this theory has been propounded and supported by a number of philosophers [ 1 ] . main features of his theory are as follows: (a) respect : dignity of each and every human being should be respected, and one person should not use the other person as a means to fulfi ll one's own desire. (b) universality : if something ethically right is applicable to us as an individual, then it applies to others also, making it a universal right. it should be respected and followed by everyone. (c) the formula of ends : rational agents should be treated as ends. virtue ethics there are certain ethical virtues and any decision or action is considered morally correct when taken considering the virtues. these promote fl ourishing and well-being. thus, any action with a right motive based upon good character or virtue is considered morally right, for example, an action where treatment of a patient is sponsored to gain publicity and honor. the action is right but morally it is not good. jansen ( 2000 ) had highlighted ethical issues with virtue ethics: (1) problem of content, vague virtues are unable to give proper guidance, and (2) problem of pluralism, competing conceptions of the good life complicate a sound solution. the social and political background of feminist bioethics is feminism and feminist theory with its major social and political goal to end the oppression of women and to empower them to become an equal gender. the use of transgenic animals has sparked signifi cant controversy in many areas. some groups see the generation of genetically modifi ed organisms as interfering with natural biological states or processes. they feel that these natural biological states have evolved over long periods which should not be altered. a few other groups are concerned with the limitation of modern science to fully comprehend the potential negative ramifi cation and unforeseen possible effects of genetic manipulation . despite the importance of transgenic animals in biomedical research, there are some concerns raised about their use in research. transgenic animals suffer more abnormalities than regular research animals. the introduction of dna into an animal can be very complex and the possible side effects can be diffi cult to predict. possible harm might arise from surgical techniques used to harvest and reimplant embryos, the collection of tissue from the tip of the tail for genotyping, and nonspecifi c effects caused by damage to genes adjoining the altered area. in addition, reduced fertility and/or oversize fetuses may result from this technology. in most cases the mutations highly affect specifi c metabolic processes or cell receptors without actually resulting in disease but causing discomfort, pain, or malfunctioning in the animals. transgenic animals not expressing foreign dna or not containing a particular gene modification are destroyed. because transgenesis is a complex science, it is not 100 % effi cient. however, new methods are being developed to increase the accuracy in transgenesis. again, it should be remembered that such genetic alteration could only be attempted if the authorities are persuaded that there is no other way to pursue important research. the potential risks of transgenics to animals, humans, and the environment are too great to justify their use. the genetic modifi cation of organisms regulations and the environmental protection act (1990) address the risks to those working with animals and the impact on the environment of accidental or planned releases . the intrinsic worth of animals may be devalued and their integrity violated by genetic modification. transgenic animals have not chosen to have foreign dna or other genetic modifi cations. however, this potential "cost" to the animals is routinely assessed under the ethical review of proposed procedures and weighed against the potential benefi ts. medical researchers only employ this technology when no alternative research avenue exists but with appropriate breeding facility of animals. as the royal society concluded in its 2001 report "the use of genetically modifi ed animals", the use of transgenic animals is fundamentally little different from the use of other animals in biomedical research. however, the technology has opened new frontiers in biomedical applications and has provided new opportunities for exploring the organization, biological pathway, regulation, and pathological function of molecular processes. contractarianism : it says that the animals are important as many people feel their importance. humanity is the virtue of morality. thus, humans have obligations toward other animals. utilitarianism : it says that as animals can also suffer; thus, they are morally relevant. it chooses the action, which aims to give happiness to all. thus, its goal is to maximize the total sum of happiness in the world and is impartial, that is, the pain and happiness of everyone count. animal activists view : according to these whatever is the motif there is no justifi cation of harming animals for human purposes. ruthless view : this has no concern about animals, whichever way they are being used. however, principles governing animal rights are the 3rs, which mean replace , reduce , and refi ne . it involves ethical and humane approach while using animals. the alternatives of animals in the research were also explored. the 3rs are replacement of conscious living animals with nonsentient animals or materials, reduction of the number of animals used in an experiment or procedure, and refi nement of the techniques used in order to decrease the incidence or amount of animal pain and distress [ 34 ] . genetically modifi ed crops or gm crops have given new dimensions to agriculture. with the advancements in the technology, the agricultural productivity is increased, the food's nutritive value is enhanced, the requirement of pesticides and insecticides is greatly reduced, and pharmaceutical substances (vaccines) are being produced in the plants. however, gm foods have become the target of public concern due to unknown and unseen fears of their effects on the ecosystem and risks to human health. the gm foods are also not labeled leading to distrust and fear in the customers regarding the safety of food. the ethical issues raised are: • what about evolution? • each living being has evolved to grow in one condition, and if we put human gene or fi sh's gene in a plant or animal, what would be its effects? • what would happen if the gene can fi nd an escape route to introduce itself in other members of that species or other species? • what would happen if the herbicide resistance gene could transfer itself in weeds, generating superweeds? • if we have all gm crops of any grain or pulses, then occurrence of any disaster would lead to complete loss of the crop, as there is no biodiversity where one is sensitive and the other one is resistant. this would lead to disaster and damage of whole of the crop. • would the monogenetic crops, if not able to react appropriately to environmental stress , again face a situation like "ireland's potato famine"? • what if gm crops breed with other crops, would the biodiversity be lost? • what are their risks to other animals like birds, insects, and other higher animals (like monarch butterfl y and bt crops)? • there is tremendous fear of gmos on human health. • if the gm can induce allergy due to insertion of unrelated gene what would happen, because gm foods are not labeled. for example, a brazil nut gene was transferred to a soybean variety; this nut gene product was a source of allergy for many individuals. as gm products are not labeled, therefore, the consumers may consume soybean without being aware of nut gene product in them. this might lead to allergy causing major allergic reactions in the consumers. however resultant modifi ed crop was never released to the public . • human food supply may have gm products as evidenced by the settlement between syngenta and the us government over the accidental sale of unapproved gm (bt10) corn seed to farmers. • these seeds can only be afforded by big farmers and landowners, thus negatively affecting small-scale farmers. • the farmers need to buy seeds every year, as they cannot collect, store, and replant these seeds. • there would be huge differences in the farming practices in one country only, and that gap would be widened much between developed and developing nations [ 17 ] . • as gm food is not labeled, thus, public trust and acceptance of gm food would be a big challenge . • the gm crops have higher productivity as they are resistant to pests; thus, loss due to diseases is prevented. • they are engineered to survive in high salt and frost, thus utilizing the land, which was unused for agricultural purposes. • they also help to reduce contaminants as insecticides and pesticides, thus preventing their entry in soil, water, and organic matter. • all these effects would ultimately affect the environment and consumers. • biotechnology can fulfi ll the increasing requirements of food, with maximum utilization of land. • biotechnology can help reduce the environmental contaminants as insecticides and pesticides preventing their entry in food chain. • it can help maintain clean environment. • it can enhance nutritive values of the food, for example, golden rice can help prevent blindness due to nutritional defi ciency of vitamin a. • the longer shelf life of fruits and vegetables can decrease their wastage and increase their availability with simple storage conditions. • the genes for allergins can also be engineered so that the allergins can be effectively removed from the food crops. • thus they are benefi cial for humans and animals. • it can also eliminate trans fats, producing healthier foods. • nowadays, plants are also being explored and used for production of biopharmaceuticals like vaccines, scfv, antibodies, and so on. there is reduced risk of adverse reactions in terms of animal pathogen transmission, and the protein undergoes posttranslational modifi cations. these genetically modifi ed varieties faced tremendous resistance in europe and india. their public acceptance has also met with resistance in terms of fears, controversies, and acceptance. the controversy from ngos, media, and scientists 24.5 genetically modifi ed crops and bioethics has led to tremendous negative impacts on the gm crops. the environmental council of the european union further augmented the controversy by halting regulatory approval of gm crops. the ethical issues and safety issues in gm cops can be addressed by evaluating their safety and other benefi ts and risks. the risks associated with humans, animals, and plants should be well considered along with the intention to do good. the consideration of well-being of all (humans, animals, plants, and environment) is very important for the sustainability of life, along with moving forward with positive aspects of technology. the ultimate aim of the technology should be improvement and sustainability in the future of all life forms with minimal adverse effects on our surroundings and other plants and animals. assisted reproductive technology (art) techniques are used in the case of male or female infertility . the technique is detailed in chap. 15 . the process of art involves ovulation, artifi cial 5. aren't we imparting sufferings for the plants and animals by using these tools and technology on them? 6. rights about the well-being of other plants and animals. thus, there is a requirement of welldefi ned guidelines when using gene manipulations [ 39 ] as well as labeling of the products produced by gene transfer technologies. there is no doubt that apart from genetically modifi ed plants and animals, the gene manipulation techniques are well accepted in production of recombinant pharmaceuticals for production of life-saving drugs. the technique has enormous potential for solving food problems and sustainable agricultur al practices and preventing and treating diseases. there are many concerns regarding reproductive cloning of humans. thus, the concerns should be there whether the technology is being used for what is worth or we simply want to see the extremes of what technology can do. each achievement of science has both negative and positive aspects; nuclear power when used judiciously can help us solve energy problems but can also result in disaster like what happened in hiroshima and nagasaki during world war ii. the gene manipulation technology is with tremendous potential but should be used just for benefi t of mankind, the environment, and animals. the experiments being done to create transgenics have left the people with many concerns regarding the health and life. the changes which unnatural gene transfer might be doing to the environment and the society? there have been concerns regarding the transfer of animal genes into plants (for vegetarians) and food animals. this has raised not only safety issues but also ethical issues: although many people are in favor of ivf and surrogacy , a survey shows that people are against posthumous sperm procurement or reproductive cloning. the technology is surrounded by many issues of creation and destruction of embryos [ 13 , 33 ] . bioethical issues are for seeking justice, non-malefi cence, and beneficence of all involved in any issue. adequate ethical guidelines and moral evaluations of new technologies are essential so that laws can be framed for national regulations and restrictions of unacceptable practices [ 12 ] . stem cells for use as therapy have given new dimensions to medicine. due to capacity of these cells to differentiate into almost all different kinds of cells, they are being looked as potential alternative for the cure of many diseases. probably sometime in the future, scientists would be able to generate whole organs by stem cells and tissue engineering to fulfi ll the increasing demand for organs. depending upon their potency and differentiation capabilities, they can be: • adult stem cells: these are present in each tissue of the human body, where they help in replenishing cells for regular wear and tear of the cells. • embryonic stem cells are located in the human embryo at the blastocyst stage (5-6 days of age). leftover embryos at this age are often unwanted in assisted reproductive technology, and some parents can donate them for research. in many countries very strict laws are there for their usage. some of the nations recommend their use when no other option is available. • cord blood stem cells are derived from the umbilical cord, which is often still routinely discarded at birth. the ethical issues raised for stem cell research is the usage and destruction of human embryos for obtaining embryonic stem cell [ 14 ] . as the life begins with the embryo, thus destruction of life is immoral; therefore, its usage and the extraction of embryonic stem cells are unethical. however, using adult stem cells or umbilical cord blood stem cells has been considered ethically superior alternative to the use of embryonic stem cells. their rejection and uncontrolled growth would be big challenges to fully explore their therapeutic potential. human cloning is a very controversial issue [ 22 , 43 ] to discuss and its cloning can be "reproductive" cloning and "therapeutic" or "research" cloning. though both the terms are not scientifically accurate, they are widely used. in somatic cell nuclear transfer (scnt) , the nucleus from the somatic cell is transferred into the enucleated egg. the resulting embryo can either be implanted into a mother or surrogate mother for development (the cloned sheep dolly was the outcome of scnt ) or the embryo can be used for research purposes [ 7 ] . implantation of embryo for gestation is referred as reproductive cloning, whereas in therapeutic cloning embryo is harvested for embryonic stem cells [ 16 ] . the stem cells obtained from embryo are embryonic stem cells , which can be induced to form different cells [ 8 , 25 ] . • people and scientifi c community in favor of human cloning say that it can combat infertility and very useful for therapeutic purposes. • many people are against the creation and usage of embryo for research purposes; some are concerned about the risks like ovarian hyperstimulation syndrome in egg donors. • many more object to the destruction of embryos as they represent the initial phase of human life thus considered morally equivalent to humans; therefore, they consider therapeutic cloning at par to reproductive cloning. • human reproductive cloning is unacceptable and unethical for many scientists and philosophers [ 9 ] . most of the countries have banned all kinds of human cloning. • in the universal declaration on the human genome and human rights (udhghr) by unesco in 1997 , it says that the "practices which are contrary to human dignity, such as reproductive cloning of human beings, shall not be permitted." • the resolution in this line was also passed by the who saying that human reproductive cloning is against human dignity and urged member states to prohibit it. biotechnology has had a tremendous impact on human race [ 26 ] . the technology has potential to change what humans are [ 28 ] . humans may come up as transhumans. transhumanism is the international movement which is aimed to transform humans by the use of technology to augment brain function and enhance human intellect, physical potential, and psychological capabilities [ 27 ] . their goal is to extend human life, enhance the brain and its capacities, delay aging, and improve physical conditions (socalled superhumans). attempting technological augmentation on the normal human function [ 17 , 36 ] can take us far away from our own species to "superhuman" function. thus, technological interventions can shift us from what we called as homo sapiens to "superhuman" homo sapiens technologicus -a species that uses, fuses, and integrates technology to enhance its own function [ 46 , 47 ] . we are living in the best of times as exhibited by the fast-paced economic growth of many countries around the world. various initiatives that were taken in the fi elds of molecular biology, genetics, and recombinant dna technology are beginning to show fruits, strengthening the basis of research. the technological advances have made our life easier. the problems encountered in the form of ethical dilemma need appropriate attention with a view of the interests of society. our ultimate aim should be to generate thorough knowledge on all aspects of modern biology and garner excellence in the making of an economically, socially, and morally sustainable society [ 18 ] . 6 . which of the following procedures is comkant and applied ethics: the uses and limits of kant's practical philosophy control of communicable diseases manual science and society: different bioethical approaches towards animal experimentation. presentation at a symposium "use of animals in research: a science-society controversy? fatal cercopithecine herpesvirus 1 (b virus) infection following a mucocutaneous exposure and interim recommendations for worker protection studies on release and survival of biological substances used in recombinant dna laboratory procedures health care rights and responsibilities: a review of the european charter of patients' rights goodbye dolly?" the ethics of human cloning after-birth abortion: why should the baby live what exactly is an exact copy? and why it matters when trying to ban human reproductive cloning in australia applying the four-principle approach the future of human nature conventional vs unconventional assisted reproductive technologies: opinions of young physicians human genetic data: preliminary study of the ibc on their collection, processing, storage and use, rumball s and smith am legal and ethical approaches to stem cell and cloning research: a comparative analysis of policies in latin america, asia, and africa the virtues in their place: virtue ethics in medicine human reproductive cloning and reasons for deprivation the imperative of responsibility: in search of an ethics for the technological age ethics and biogenetic art grundlegung zur metaphysik der sitten (groundwork for the metaphysics of morals) [1785] akademie-ausgabe kant werke iv life, liberty & the defense of dignity: the challenge for bioethics a companion to bioethics negotiating bioethics: the governance of unesco's bioethics programme human infection with venezuelan equine encephalomyelitis virus common sense in the laboratory: recommendations and priorities proceedings of a conference held at the asilomar conference center cloning mice and men: prohibiting the use of ips cells for human reproductive cloning transhumanism, medical technology and slippery slopes your destiny, from day one laboratory-associated infections: summary and analysis of 3921 cases past and present hazards of working with infectious agents laboratory-associated infections: incidence, fatalities, causes, and prevention continuing importance of laboratory acquired infections monitoring stem cell research the principles of humane experimental technique. methuen london, altex (alternatives to animal experimentation) offi cial publication of the johns hopkins center for alternatives to animal the technological world picture and an ethics of responsibility the paradox of choice: why more is less survey of laboratoryacquired infections nih guidelines for research involving recombinant dna molecules airborne q fever universal declaration on the human genome and human rights human genetic data: preliminary study of the ibc on their collection, processing, storage and use who, fifty-fi rst world health assembly agenda item 20: ethical, scientifi c and social implications of cloning in human health world health report, world health organization laboratory biosafety manual, 3rd edn. world health organization inventing iron man: the possibility of a human machine the potential transformation of our species by neural enhancement guidelines for biosafety laboratory competence european union, charter of fundamental human rights • biotechnology has made tremendous progress in providing therapeutics, new ways to diagnose diseases, regenerative medicines, food and feed solutions, food with nutritive values, and other countless achievements. • biosafety is a very important aspect as the health-care and laboratory workers are continuously exposed to highly contagious and infectious pathogenic agents. they work with these agents either for the purposes of diagnostics or research. • many laboratory-induced infections were reported among the workers and several of them were serious and resulted in death of workers. these laboratory-induced infections were due to organisms in biorisk-3 and biorisk-4 groups. • thus, it becomes very important to do appropriate risk assessment, monitoring, and installation of appropriate preventive measure like biosafety levels and biocontainment facility and training to the laboratory workers for prevention. • bioethical issues are there with all the major achievements of biotechnology . the issues were being raised because of rightness or wrongness of the experiment, to ensure experiments are conducted in a way that it ensures safety for all, the humans, animals, and the environment. • bioethical issues related with genetically modifi ed crops are related to development of resistance in insect-resistant crops and their impact on the environment, animals, and humans. many ethical issues were raised for genetically modifi ed animals and their sufferings. • embryonic stem cell therapy and assisted reproductive technologies along with genetically modifi ed crops faced too much of ethical discussions and resistance. human reproductive cloning created tremendous furor; thus, human cloning in all forms was banned in most of the countries. • as with any other technological advancements, each technology is faced with ethical and social issues and so is biotechnology . however, it is important that before we keep on proceeding, we should ask some basic questions to ourselves: (1) why am i doing this? (2) would it be benefi cial for anyone? key: cord-016292-o4cw5ufy authors: horby, peter w.; hoa, ngo thi; pfeiffer, dirk u.; wertheim, heiman f. l. title: drivers of emerging zoonotic infectious diseases date: 2014-07-19 journal: confronting emerging zoonoses doi: 10.1007/978-4-431-55120-1_2 sha: doc_id: 16292 cord_uid: o4cw5ufy this chapter discusses drivers of emerging infectious diseases (eid) of humans that have an origin in other vertebrate animals (zoonoses). this is a broad topic, worthy of a book in its own right. this chapter will therefore provide only an overview of key concepts of drivers of the emergence of zoonotic diseases, and particularly infectious diseases with a major disease burden in humans. as the authors mainly work in asia, the focus of this chapter is asia, but many of the lessons learned in this region are likely to apply elsewhere. more than 60 % of the world population live in asia, a region with some of the fastest developing economies in the world. yet, despite tremendous advances, infectious diseases still remain a major burden for the human population in asia. of the estimated 2.1 million deaths in children aged less than 5 years in southeast asia in 2010, 47 % are attributable to infectious causes (liu et al., lancet 379:2151–2161, 2012). as such, asia is both vulnerable to imported eids and a global focus of major social and environmental change that may facilitate the emergence and dissemination of new pathogens. however, it would be too simplistic to present the extensive changes in asia as inevitably increasing the risk of eids. some aspects of socio-economic change might serve to reduce the overall risk of infectious disease emergence, but all ecosystem changes have the potential to provide new opportunities for microorganisms to spill-over into human populations. the conditions or events (the 'drivers') that result in the successful cross-over of an animal microbe into humans are not well characterised, but emergence is often precipitated by changes to ecological or biological systems (wilcox and colwell 2005) . such changes include altered patterns of contact between wild and domestic animals (e.g. nipah virus), of direct human and wild animal contact (e.g. hiv, ebola), and changes in species abundance or diversity (e.g. hantavirus; lyme disease). species diversity, including the diversity of insect vectors and pathogenic microorganisms, increases towards the equator (guernier et al. 2004) , and jones et al. found a correlation between the emergence of zoonotic pathogens and the diversity of mammalian wildlife species (jones et al. 2008) . whilst high animal host and pathogen species diversity may be associated with a high burden of infectious diseases and an increased risk of disease emergence, biodiversity loss may, perhaps counter-intuitively, be associated with increased disease transmission. biodiversity loss directly disrupts the functioning and stability of ecosystems, producing effects that can extend well beyond the particular lost species (hooper et al. 2012) . changes in species abundance and diversity may favour pathogen amplification and 'spill-over' through a variety of mechanisms, including reduced predation and competition resulting in increased abundance of competent hosts, and the loss of 'buffering species' leading to increased contact between amplifying host species and compatible pathogens (keesing et al. 2010) . tropical regions with a rich pool of existing and potential pathogens that are increasingly connected, but also experiencing high rates of ecosystem disruption and biodiversity loss, may therefore be at a particularly high risk of disease emergence. human pathogens occasionally re-emerge as a result of dynamics that are beyond the control of humans. for example the hantavirus outbreaks in the southwestern u. s. in 1991-1992 and 1997-1998 have been attributed to changes in the abundance of infected rodents following periods of heavy snow and rainfall and vegetation growth leading to abundant production of rodent food. however, many ecological disturbances resulting in an eid seem to originate in the direct actions of humans. a wide range of anthropogenic factors have been linked to infectious disease emergence, including changes in land-use, travel, trade, and demographics. notably, most of these associations are speculative and supported (wolfe et al. 2007 ). stage 1: the pathogen is still exclusively infecting animals. stage 2: the pathogen has been transmitted from animals to humans under natural conditions but not yet from human to human. stage 3: there is limited transmission from animals to humans and between humans. these are often severe and lethal diseases due to for instance filoviruses (e.g. ebola). stage 4: animal disease that have sustained transmission between humans (e.g. influenza). stage 5: a microbe that exclusively infects humans (e.g. measles, syphilis) by little hard data because ecological and biological systems are highly complex and multi-layered (woolhouse 2011) . demonstrating or predicting the impact of particular conditions or events on the functioning of a system is difficult, with further inference of the impact of any changes on the risk of pathogen emergence posing a formidable challenge. socioeconomic development is associated with large increases in demand for natural resources. the demand for water, wood, pulp, agricultural land, living space, roads, minerals and power has had an enormous impact on the landscapes of asia. deforestation occurred throughout the 1990s and the area of primary forest in asia has continued to decline (fao 2012) . deforestation, forest fragmentation, and afforestation are all alterations in habitat, which change species composition and the interaction between wild animals, domestic animals, insect vectors and humans, providing new opportunities for microbial transmission and potential emergence. there are well-documented examples of deforestation and forest encroachment resulting in increases in infectious diseases, such as yellow fever, mayaro, and chagas disease in the americas (saker et al. 2004 ). the clearing of forest and planting of large cacao plantations was linked to the emergence of oropouche virus in brazil. in asia there are already very high pressures on productive land, and the peak in land-use change in asia has probably passed. many areas are now in an era of increasing intensification of land productivity. this intensification is driven largely by demographic pressures, which are predicted to result in a 70 % increase in food production by 2050, with decreased consumption of grains and increased demand for meats, fruits and vegetables (fao 2011a, b) . the increased demand for food, and meat in particular, when combined with demands for natural resources from industry and domestic consumers, and river damming for hydroelectric power, is resulting in a large increase in stress on water resources (fao 2011b). the consequences of intensified agricultural production include the depletion and degradation of river and groundwater, reduced soil quality, and biodiversity loss. a direct and predictable effect of reduced access to clean water for low-income families is an increase in the risk of water-washed diseases (diseases that increase when the availability of water for personal hygiene is limited e.g. diarrhoeal and respiratory infections, trachoma), and water-borne diseases such as typhoid and hepatitis e. however, unquantified risks arising from the intensification of agriculture are pollution of freshwater with pesticides and fertilizers, loss of biodiversity, and land abandonment by small-scale farmers. the potential consequences of these changes on the risk of emergence of zoonotic infections have not been assessed. wild animals are an important source of food (bush meat) in some developing countries and bush meat has been implicated in the emergence of hiv, and the spill over of monkeypox, nipah and ebola virus (brashares et al. 2011) . whilst the reservoir of the sars coronavirus is thought to be bats, wild civet cats traded for food are thought to have acted as an intermediate host, transmitting the sars virus to humans through live animal markets (li et al. 2005) . wild animal products are popular in asia as traditional medicines, tonics, delicacies, or as symbols of wealth. although all ten countries in the association of southeast asian nations (asean) are signatories to the convention on international trade in endangered species of wild fauna and flora (cites), asia continues to host the largest illegal wildlife trade in the world (rosen and smith 2010) . the smuggling of h5n1-infected birds of prey into europe, the frequent smuggling of bush meat from africa into the u.s., and the importation into the u.s. of pet rodents infected with monkeypox show that both the legal and illegal trade in wild animals and wild animal products is a potential conduit for the international spread of zoonotic pathogens (bair-brake et al. 2013; van borm et al. 2005) between 2011 and 2050 the world population is expected to increase by 2.3 billion (a 32 % increase), and the increase will be concentrated in urban areas of developing countries (un-desa 2012). whilst mega-cities (cities with a population of at least ten million) receive a lot of attention, most urban dwellers live in small cities, with half of the global urban population in 2011 living in cities of less than 500,000 people (un-desa 2012). this can be perceived positively as cities generally offer better economic opportunities, better educational opportunities, better living conditions, better nutrition, better sanitation, and therefore better health than underdeveloped rural areas. at the same time it is likely to mean that in many low to middle income countries, health and veterinary infrastructure in rural areas will not improve, or may even deteriorate, adversely affecting the likelihood of the early detection of eid. the demand for food in urban centres will increase and result in livestock and their products being transported over large distances from a wider catchment area, and thereby increase risk of spread and amplification of eid. urbanisation is one facet of changing human sociocultural systems, which also includes changing consumer demands and dietary habits (janes et al. 2012 ). the consequence is a spatial concentration of people and animals; not necessarily co-located, but connected through increasingly complex networks of rural and peri-urban farms and markets, distributors, agricultural workers, and consumers. due to the increase in global human population and economic development, demand for livestock products has risen dramatically over the last 50 years, with the per capita consumption of meat in developing countries more than tripling since the early 1960s and egg consumption increasing fivefold (fao 2011a, b) . the increased demand for meat has been met by more intensive and geographically concentrated production of livestock, especially pigs and poultry (steinfeld et al. 2006 ). much of this has been through expansion of both the number of small-scale production units and large commercial farms. high-density monoculture of domestic animals is a form of low biodiversity that poses a particular threat for the spread of infectious diseases from farmed animals to humans. where domesticated animals are a conduit of spread from wild animals to humans, high density livestock production may promote spread of zoonotic diseases. genetic diversity within an individual host species is important since genetic diversity limits the potential for devastating epidemics (king and lively 2012) . the nipah virus outbreak in malaysia and singapore in 1998-1999 is a good example. once nipah virus crossed from wild bats to domestic swine, an explosive outbreak in high-density swine farms resulted in widespread exposure of humans and over 250 human cases of encephalitis (pulliam et al. 2012) . other examples where intensified livestock production practices may have led to emergence of a zoonosis include: • streptococcus suis causes severe sepsis and meningitis in humans and is associated with areas of intensive pig production (see fig. 2 .2). risk factors for human infection include swine slaughtering and the eating of undercooked pig products (wertheim et al. 2009 ). outbreaks in swine herds of porcine reproductive and respiratory syndrome virus also potentially increases the rate of invasive s. suis infection in swine, which in turn leads to an increased risk of s. suis infection in humans (hoa et al. 2013 ). • highly pathogenic avian influenza a subtype h5n1 crossed-over from wild aquatic birds (the natural reservoir of influenza a viruses) to humans via massive amplification in domestic poultry. • the human q-fever epidemic in the netherlands during 2007-2010 caused by the bacterium coxiella burnetii is thought to have arisen when economic drivers led to an increased density of dairy goat farming, which resulted in amplification of coxiella burnetii prevalence and consequent increased spill-over to humans (roest et al. 2011; georgiev et al. 2013 ). the classical foodborne diseases such as e. coli, campylobacteriosis and salmonellosis associated with livestock products have been a significant problem in highincome countries for some time (cdc 2011; painter et al. 2013 ). one of the key factors, in particular in the case of campylobacter, has been the integration of highly intensive poultry production with poultry processing systems facilitating cross-contamination of meat (moore et al. 2005; nyachuba 2010) . it is notable that some foodborne pathogens cause subclinical infections in their animal hosts (e.g., campylobacter in poultry); therefore there is no direct incentive for farmers to control them. in addition, subsequent attribution of the source of foodborne disease in an affected human is notoriously difficult. it is likely that the global incidence of foodborne illness will rise as a result of increased industrial production of poultry in the emerging economies. the situation will be exacerbated by the emergence of antimicrobial resistant pathogens of foodborne pathogens (sahin et al. 2012; luangtongkum et al. 2009; altekruse and tollefson 2003) . despite the examples above, the intensification of livestock farming often entails more effective separation of domestic and wild animals, improved standards of animal health and welfare, reduced movement and species mixing: all of which reduce the risk of eids. reducing contact between domestic and wild animals, whether the wild animals are in their natural habitat or captive, is a key strategy of the food and agriculture organization (fao) to reduce risk to human health, and is part of the wider fao strategy of enhancing 'biosecurity'. improving biosecurity in farms is a major challenge since a large proportion of farming in asia is based on small-scale backyard production, and there is often a mix of commercial and backyard farming in any one location. achieving improvements in biosecurity without adversely affecting the livelihoods of small-scale farmers requires an approach to risk management that is adapted to their socio-economic context. the longer-term vision is to restructure the livestock production sector towards a more commercialised and controlled system, where controls benefit animal health, human health, and commercial profitability. but it has also been recognised that small-scale producers will continue to have a key role in providing food security in developing economies, and in fact their importance may increase due to their more efficient use of natural resources compared with large-scale industrial production (sjauw-koen-fa 2012; quan 2011). food production has become a complex national, regional and global network of food value chains (ercsey-ravasz et al. 2012; dickson-hoyle and reenberg 2009). many countries have begun to more tightly regulate live animal trade in the wake of bovine spongiform encephalopathy (bse), sars and avian influenza a/h5n1, but complex and poorly regulated food manufacturing and distribution chains still offer ample opportunities for disease outbreaks. in endemic regions, live bird markets are frequently contaminated with highly pathogenic avian influenza a subtype h5n1 (davis et al. 2010; samaan et al. 2011; wan et al. 2011) . contaminated markets can become reservoirs and the source of infection for poultry and humans. measures to reduce the risk in live animal markets include the separation of species, not allowing animals to remain in the market more than 24 h, not allowing poultry to exit the market alive, improved cleaning and disinfection, and weekly rest-days, when all animals are removed and the market is thoroughly cleaned. the re-emergence of brucellosis, one of the world's most common zoonoses, is thought to be the result of higher risk of transmission through increased within-and between country trade of susceptible livestock and their products (seleem et al. 2010) . the development of denser regional road transport networks may be especially important since, compared to air travel, roads offer a more egalitarian form of connectivity that includes animals and goods as well as humans. this provides opportunities for pathogens to disperse beyond their traditional niche by increasing opportunities for informal trade in live animals and their products (eisenberg et al. 2006) . veterinary medical authorities often struggle to enforce compliance with regulations, and it is common practice for farmers and traders to adapt their production and trading behaviors to avoid adverse economic consequences of regulations aimed at controlling zoonotic diseases. as a result, informal trade networks may intensify and re-structure in unpredictable ways during disease outbreaks. increasing intensification of animal husbandry in asia may be a trade-off between a lower risk of emergence events, as animals will be 'healthier' and better isolated, but should an eid event occur, an increased risk of massive amplification in large, naïve monocultures. perhaps the greatest risk arises when a limited number of intensive livestock production units are surrounded by substantial backyard farming with little or no biosecurity, thereby linking extensive and weakly regulated value chains with global food systems. livestock production practices can have major unintended and unanticipated impacts on the risk of zoonotic infections. perhaps the most notable example is bse. the feeding of ruminant-derived meat and bone meal to cattle, and possibly changes in practices for rendering animal tissue, led to an epidemic of bse in cattle, that was followed by an epidemic of a fatal neurological disease (variant creutzfeldt-jacob disease) in humans (taylor and woodgate 2003) . in contemporary livestock production, the use of antibiotics is a significant concern. almost 40 % (23/59) of eids identified in asia since 1940 represent the emergence of a new pattern of antimicrobial resistance (jones et al. 2008) . bacteria in some areas show alarmingly high rates of resistance to anti-bacterial agents, and the recent emergence of the new-delhi metallo-beta-lactamase-1 (ndm-1) resistance gene, conferring resistance to a last resort antibiotic, and its rapid dissemination to other regions highlights the serious threat to human health of antimicrobial resistance (khan and nordmann 2012) . antibiotics are used extensively in the livestock and aquaculture sector to treat or prevent infections, or as growth promoters. non-therapeutic use of antibiotics as growth promoters involves the prolonged administration of sub-therapeutic doses. this practise has a demonstrable effect on the emergence and prevalence of resistant microorganisms in food animals and their environment, as well as resulting in the excretion of antibiotics into the environment, where environmental bacteria may be subject to antibiotic selection pressures (marshall and levy 2011) . synthetic antibiotics such as quinolones are quite stable in the environment over long periods of time. heavy metal contamination of animal food may also play a role in selection of antimicrobial resistance. whilst there remains some debate about the overall impact of these findings on human health, it is clear that the continued use of non-therapeutic antibiotics in an agriculture industry that is rapidly increasing in scale and intensity, has potential for becoming a very real threat through the inability to prevent/cure disease in production animals and the consequences for human food security as well as the transmission, for example, of resistant food-borne bacterial pathogens to humans. some recent examples concern transmission of methicillin resistant staphylococcus aureus (mrsa) from pigs to humans and esbl (extended spectrum betalactamase, an important resistance mechanism against most beta-lactam antibiotics) positive e. coli on chicken meat for human consumption (verkade et al. 2012; kluytmans et al. 2013) . swine-associated mrsa is now contributing significantly to invasive disease in patients in the netherlands (verkade et al. 2012) . antibiotic production and consumption continues to increase, often in an uncontrolled way, accelerating the evolution of antibiotic resistance, which then spreads rapidly across the globe. since the 1990s few new antibiotics have been developed and we are on the cusp of returning to an era of untreatable infections. the consequences of antibiotic use in animals therefore require better surveillance, research and regulation. infectious diseases continue to be an important cause of human and animal morbidity and mortality worldwide. whilst important health advances (e.g. hygiene and vaccination) have been made, infectious diseases are dynamic and resilient, and continue to challenge local, national and global public health systems (fauci and morens 2012). the recognition of the linkage between anthropogenic changes, animals, and disease emergence has resulted in repeated calls for a more holistic, interdisciplinary, and integrated approach to the study of infectious diseases. the one health approach is one initiative aimed at realizing this aspiration. the task ahead is to understand how social, economic, and environmental changes are altering the landscape of infectious disease risks for both animals and humans, and how future emerging risks may be mitigated. a more analytical approach to these emergence events requires characterisation of the attributes of natural and artificial ecological systems before and after disturbance, and the functional relationship between changes in system attributes and pathogen emergence (woolhouse 2011) . human campylobacteriosis: a challenge for the veterinary profession is that a rodent in your luggage? a mixed method approach to 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agriculture organisation of the united nations fauci as, morens dm (2012) the perpetual challenge of infectious diseases q fever in humans and farm animals in four european countries ecology drives the worldwide distribution of human diseases streptococcus suis and porcine reproductive and respiratory syndrome a global synthesis reveals biodiversity loss as a major driver of ecosystem change emerging infectious diseases: the role of social sciences global trends in emerging infectious diseases ecology of zoonoses: natural and unnatural histories impacts of biodiversity on the emergence and transmission of infectious diseases spread of carbapenemase ndm-1 producers: the situation in india and what may be proposed does genetic diversity limit disease spread in natural host populations extended-spectrum beta-lactamaseproducing escherichia coli from retail chicken meat and humans: comparison of strains, plasmids, resistance genes, and virulence factors bats are natural reservoirs of sars-like coronaviruses global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since antibiotic resistance in campylobacter: emergence, transmission and persistence food animals and antimicrobials: impacts on human health atlas communicable diseases vietnam from attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data agricultural intensification, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis a future for small-scale farming the q fever epidemic in the netherlands: history, onset, response and reflection summarizing the evidence on the international trade in illegal wildlife molecular evidence for zoonotic transmission of an emergent, highly pathogenic campylobacter jejuni clone in the united states globalization and infectious diseases; a review of the linkages. world health organization critical control points for avian influenza a h5n1 in live bird markets in low resource settings brucellosis: a re-emerging zoonosis framework for an inclusive food strategy. cooperatives -a key for smallholder inclusion in valeu chains rendering practices and inactivation of transmissible spongiform encephalopathy agents highly pathogenic h5n1 influenza virus in smuggled thai eagles recent emergence of staphylococcus aureus clonal complex 398 in human blood cultures indications that live poultry markets are a major source of human h5n1 influenza virus infection in china streptococcus suis: an emerging human pathogen emerging and reemerging infectious diseases: biocomplexity as an interdisciplinary paradigm origins of major human infectious diseases how to make predictions about future infectious disease risks key: cord-255181-du6rqc6i authors: louz, derrick; bergmans, hans e.; loos, birgit p.; hoeben, rob c. title: cross‐species transfer of viruses: implications for the use of viral vectors in biomedical research, gene therapy and as live‐virus vaccines date: 2005-06-29 journal: j gene med doi: 10.1002/jgm.794 sha: doc_id: 255181 cord_uid: du6rqc6i summary all living organisms are continuously exposed to a plethora of viruses. in general, viruses tend to be restricted to the natural host species which they infect. from time to time viruses cross the host‐range barrier expanding their host range. however, in very rare cases cross‐species transfer is followed by the establishment and persistence of a virus in the new host species, which may result in disease. recent examples of viruses that have crossed the species barrier from animal reservoirs to humans are hantavirus, haemorrhagic fever viruses, arboviruses, nipah and hendra viruses, avian influenza virus (ai), monkeypox virus, and the sars‐associated coronavirus (sars‐cov). the opportunities for cross‐species transfer of mammalian viruses have increased in recent years due to increased contact between humans and animal reservoirs. however, it is difficult to predict when such events will take place since the viral adaptation that is needed to accomplish this is multifactorial and stochastic. against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and for live‐vector vaccines is a cause for concern. this review addresses a number of potential risk factors and their implications for activities with viral vectors from the perspective of cross‐species transfer of viruses in nature, with emphasis on the occurrence of host‐range mutants resulting from either cell culture or tropism engineering. the issues are raised with the intention to assist in risk assessments for activities with vector viruses. copyright © 2005 john wiley & sons, ltd. is known as a zoonosis in the case where the virus is transmitted from non-human hosts to humans and causes disease [6] . crossing the species barrier is an unpredictable event that involves complex interactions between the virus and the newly adopted host [7] . the hiv virus and contemporary human influenza viruses are prominent examples of viruses that have crossed the species barrier and established themselves permanently in the human population without further dependence on the presence of the original animal host reservoir. fortunately, natural adaptation of a new virus leading to permanent establishment and dissemination within the human population is a rare event [8] . often, the virus is not readily adapted to infect and spread efficiently from human to human. the emergence of new viral infections often follows environmental, ecological and technological changes caused by human activities [9] . these activities may lead to an increased contact between humans and animal hosts acting as reservoirs of zoonotic viruses. agricultural development, an increased exploitation of environmental resources, growth and increase in the mobility of the human population and trade and transportation of food and livestock, have been identified as important factors contributing to the introduction and spread of a number of new viruses in the human population [10] [11] [12] [13] . the road map for cross-species transfer may differ for individual viruses. however, some common underlying factors that affect the probability of zoonotic events can be identified ( table 1 ). the potential of viruses to adapt to new or changing cellular environments or ecological niches via genetic variation appears to be a key feature [8, 14] . the advent of novel technologies for genetic modification of viruses offers new opportunities for biomedical research. often, these activities involve handling of viruses and their genetically modified variants in large quantities. the use of viral vectors in experimental gene therapy or as live vaccines may be also a cause for concern. indeed, such activities meet the primary requirements for cross-species mutations that facilitate the use of alternative receptors in the newly adopted host species feline parvovirus [29] transmission to humans after occupational exposure hendravirus [135] intensified contact between natural and new host species due to climatic changes hantavirus [136] immune evasion by genetic variation (e.g. antigenic drift and shift) influenza virus [55] introduction in new geographic areas by migrating birds west nile virus [137] initial close contact between natural and newly adopted host due to changes in natural infrastructures nipah virus [135] transfer, i.e. contact between infectious viruses and a potential new host species. therefore, it is important to identify those activities that have a finite risk of leading to new viral infections and to practice appropriate biosafety measurements. in this review risk factors associated with activities with viral vectors will be addressed from the perspective of emerging viruses that have crossed the host barrier in nature. the processes that underlie cross-species transfer through host-range expansion and establishment of viruses in new host species depend on the accumulation of genetic changes [7, 15] . these are likely to differ for various viruses and may affect virtually every aspect of the viral life cycle. this process of adaptation can occur by a variety of mechanisms including mutation, recombination and reassortment. mutations occur in the genomes of dna as well as rna viruses. in general, mutations occur slower in dna viruses than in rna viruses because of the proofreading function of many dna polymerases. this corrects mistakes made by the polymerase during replication. rna genomes are replicated by rna polymerases that lack proofreading. therefore, mutations in rna viruses can occur up to a million-fold more frequently than in dna viruses [16, 17] . as a consequence, rna viruses generally evolve more rapidly, and lead to genetic heterogeneity and the presence of so-called viral quasispecies [18] . the concept of quasispecies states that in an infected host, the virus exists as a population of genetically related but divergent variants defined by a master sequence and a complex and dynamic series of mutant sequences. while the master sequence remains the predominant sequence present within the population, the spectrum of mutants may shift in response to selective pressures. from the quasispecies population a variant may be selectively expanded [19] [20] [21] . although the major mechanism that drives adaptation is based on accumulation of point mutations, evolution of viruses also occurs through recombination. recombination occurs in both dna and rna viruses leading to the exchange of parts of genomes. this may result in the emergence of new virus variants. for recombination to take place, at least co-infection of a cell by two different virus variants is required. recombination plays an important role in evolutionary changes of dna viruses. in some cases, recombination between viruses and cellular nucleic acid can lead to the capture of cellular coding sequences [22] . reassortment is another important evolutionary mechanism in rna viruses with a segmented genome, such as influenza viruses and reoviruses. after co-infection of a cell with different strains or subtypes, genomic segments may be shuffled and rearranged in progeny virus particles resulting in the generation of new viruses with different biological properties. this offers viruses a large adaptive potential by facilitating evolutionary leaps in response to changing cellular environments without the need for gradual accumulation of favourable mutations [23] . in this section a number of examples of animal viruses that have crossed the species barrier are chosen to illustrate how they have evolved in nature through genetic changes. the examples illustrate that under the right environmental conditions host-range variants evolve that may establish themselves in the newly recruited host as new viruses causing disease. after the initial cross-transfer to the new host species, a period of further adaptation may be required. the examples also show how the evolutionary processes continue while an epidemic evolves. in the late 1970s, a new syndrome of viral enteritis and myocarditis emerged in dogs and subsequently swept rapidly across the world, killing thousands of dogs within a few years after its initial appearance. the virus was named canine parvovirus type 2 (cpv-2). phylogenetic analysis revealed that this virus was remarkably similar to feline parvovirus-like viruses such as feline panleukemia virus (fpv) which infects cats, mink, and raccoons, but not dogs [24] . therefore, cpv-2 presumably emerged as a natural host-range mutant of a feline parvovirus [25, 26] . host-range properties are determined by the capsid protein for both cpv-2 and fpv. although cpv-2 is capable of infecting feline cells in culture, the virus does not replicate in cats. fpv, on the other hand, is able to replicate in dogs in a restricted fashion, i.e. in bone marrow and thymus, but not in cultured dog cells [27] . cpv-2 differs from fpv by only two nucleotide substitutions within the capsid gene resulting in two amino acid substitutions [28] . these changes are associated with the ability of cpv-2 to bind to the canine transferrin receptor with high affinity [29] . as a result, cpv-2 acquired the capacity to infect canine intestinal tissue. it is thought that subsequently this virus acquired transmissibility between dogs and further adapted to replicate more efficiently in dogs as it became pandemic. interestingly, within three years after its initial appearance in 1978, cpv-2 was replaced worldwide by an antigenically and genetically variant virus cpv-2a [25] . this indicates that cpv-2a had a strong selective advantage over cpv-2. another antigenic variant derived from cpv-2a, cpv-2b which differs by only two amino acids, arose in 1984. this implies that variants of cpv gradually arose due to further adaptation and selection in dogs, in reaction to selective, most likely immunological, pressure. the two new variants (types 2a and 2b) differ at 5 or 6 amino acid from cpv-2 isolates [30] . at present, the two variants are still endemic in the canine population. these two variant viruses have an expanded host range compared to the original cpv-2 since they replicate in cats in both experimental settings and in the wild although with no or relatively low pathogenicity [31, 32] . remarkably, the prevalence of cpv-2a and cpv-2b and new antigenic variants (cpv-2c) has now been demonstrated in a wide range of feline populations worldwide (reviewed in [33] ). the emergence of cpv-2 serves as an example of rapid global distribution and establishment of host-range mutants in an immunologically naï ve new host. whereas cpv-2 most likely arose as a natural host-range mutant derived from cats, some other scenarios on its emergence have been suggested [34] . it was suggested that cpv-2 may have emerged after cross-transfer from a yet unidentified animal host to dogs, or cpv-2 may have arisen under selective growth conditions during fpv livevirus vaccine production in canine cells and subsequently spread via vaccination. the high titers of the virus shed in faeces and its resistance to inactivation may explain its initial rapid dissemination, also into countries with strict quarantine regulations for dogs. human activity may have stimulated the spread through mechanical transport, presumably aided by long-distance air travel [25, 35] . the aids pandemic is now generally accepted to stem from a viral zoonosis. human immunodeficiency viruses 1 and 2 (hiv-1 and hiv-2) emerged separately around the same time in distinct geographically areas as the result of multiple zoonotic transmissions from simian immunodeficiency virus (siv)-infected non-human primates to humans [36] . based on their genomic organization and phylogenetic analyses it is clear that hiv-1 and hiv-2 fall into two different siv lineages [37] . this implicates that both viruses must have had distinct origins. both phylogenetic and epidemiologic evidence indicate that hiv-1 evolved as a consequence of sivcpz transmission from chimpanzees to humans in central africa [38, 39] . however, to date, no serological or genetic evidence of widespread prevalence of hiv-1-related strains exists in chimpanzees in the wild in africa. transmission of siv from sooty mangabeys in west africa most probably caused the emergence of hiv-2 since siv strains derived from sooty mangabeys are phylogenetically closely related to hiv-2. hiv-2 is found at a high prevalence in sooty mangabeys [40, 41] . both hiv-1 and hiv-2 show enormous genetic diversity. hiv-1 comprises three genetically distinct virus groups (m, n, and o) of which the predominant group m consists of 11 subtypes or clades of which all but two have spread throughout the world. in contrast, hiv-2 is mainly confined to the african continent and comprises seven distinct phylogenetic lineages, subtype groups a through g, which can be categorized in epidemic subtypes a and b and non-epidemic subtypes c through g [37, 42] . it has been estimated from phylogenetic and epidemiological data that initial cross-species transfers of both the m group of strains of hiv-1, and the progenitor group of subtypes of hiv-2, may have taken place around 1930 in west africa [43, 44] . hiv-1 may have initially started to spread in africa at the beginning of the 1960s [45, 46] . therefore, more than two decades of 'silent' human-tohuman transmission may have occurred in africa before aids became apparent and hiv was identified as its causative agent in the early 1980s. in the advent of the hiv epidemic early siv strains initially may have crossed the species barrier as a result of an increase of contact between humans and siv-infected simian species. apparently, activities such as hunting, handling and consumption of contaminated uncooked simian meat led to direct exposure to animal blood and body fluids [47] . the following years passaging between infected humans of partially adapted siv strains may have resulted in series of cumulative mutations and genetic changes. the large genetic differences that exist between siv and hiv indicate that the initial siv strains that crossed the species barrier must have undergone adaptation in humans in a relatively short period of time. this suggests the involvement of some modern iatrogenic event. massive vaccination programs carried out at that time using non-sterile injection needles may have provided opportunities for transmission and further adaptation of the virus to humans in africa [48, 49] . the genetic and phenotypic evolution of the hiv virus still proceeds at a high pace not only between individuals worldwide, but also within infected individuals. during the time-course of infection the extensive genetic diversity originates from the rapid viral turnover and replication errors caused by reverse transcriptase [50, 51] . in addition, among the globally pandemic hiv-1 m group, several circulating recombinant forms (crfs) exist. these crfs result from recombination events between two different strains within the same individual and now constitute 10-20% of newly characterized circulating strains [52] . this diversity allows the hiv virus to rapidly adapt under selective pressure generated by antiretroviral drugs and host immune responses. selection of hiv variants has been implicated in the use of different co-receptor molecules and selection for different cell types and tissues and body compartments such as lymph nodes and the brain during late stages of infection and manifestation of different disease patterns [53, 54] . the emergence of hiv exemplifies how multiple independent cross-species transmissions of simian viruses that are not associated with disease in their natural hosts eventually resulted in the establishment of two types of hiv in the human population. while adapting to its new host the virus underwent a myriad of molecular changes. changes in social behaviour of humans may well have offered opportunities for newly evolved hiv strains to become pandemic. pandemic influenza a is a zoonotic disease caused by cross-species transfer of influenza a viruses from animal reservoirs. the twentieth century has witnessed three influenza pandemics, spanish influenza (1918), asian influenza (1957), and hong kong influenza (1968), that killed millions of people worldwide. although influenza a viruses have been isolated from a variety of vertebrates, including pigs, horses, seals, and whales, birds serve as the main reservoir and are a potential source for new pandemic strains [55] . influenza a viruses contain eight negative-sense rna segments that code for at least ten polypeptides of which eight are structural viral proteins and two nonstructural proteins. influenza a viruses are divided into subtypes based on both serological and genetic differences between the surface proteins and their encoding genes, respectively. to date 15 hemagglutinin (ha) subtypes (h1-h15) and nine subtypes (n1-n9) of the neuraminidase (na) proteins have been identified. influenza a viruses containing all different combinations of the ha and na subtypes have been identified in aquatic birds. in humans only influenza a viruses of hemagglutinin subtypes h1 through 3 and neuramidase subtypes n1 and n2 have established permanent lineages. these viruses are considered human influenza a viruses [56] . in nature new influenza a viruses emerge via two mechanisms of antigenic variation. the first, antigenic drift, is caused by accumulation of point mutations in both the na and ha surface proteins enabling new antigenic variant viruses to evade the human immune system and emerge via selection. influenza a viruses that emerge via antigenic drift are responsible for the yearly epidemics in the human population. antigenic shift occurs when a variant influenza a virus arises that is antigenitically completely distinct from former circulating influenza a viruses. the new virus is a reassortant that is characterized by the presence of a novel hemagglutinin gene segment alone or in combination with a complete novel neuraminidase gene segment. influenza a variants that emerge through antigenic shift are potentially capable of causing novel pandemics in an immunologically naï ve human population [55] . the avirulent nature of avian influenza virus infections in ducks and waterfowl results from adequate adaptation to their hosts [55] . avian influenza viruses do not replicate efficiently in humans. similarly, human influenza a viruses do not replicate efficiently in birds [57, 58] . the trachea tissue of the pig contains receptors for both avian and human influenza a viruses. therefore, they are permissive to both human and avian viruses and thought to function as a 'mixing vessel' for reassortment of not only human and avian, but also swine influenza a viruses [59] . pigs are therefore also considered to be ecological niches important for the emergence of new influenza a viruses in humans. in pigs, newly reassorted viruses may further evolve by accumulation of additional mutations, further adapt to a mammalian host and eventually be transmitted to humans. ample data indicate that further adaptation to the human cellular environment is necessary for replication and efficient transmission in humans. adaptation has a polygenic basis and may involve multiple viral gene segments. it should be noted, however, that only in rare cases will cross-species transmission lead to permanent establishment of new lineages of influenza a viruses in humans [56, 60] . analyses of the viruses that caused the asian and hong kong pandemics revealed that these were caused by reassortants that contained a mixture of avian and human genome segments [61, 62] . genetic analysis of the 1918 spanish influenza virus initially suggested that the epidemic originated from a whole avian influenza virus that had been transmitted from infected pigs to humans [63] . however, the origin of the 1918 pandemic strain still remains an enigma since the presence of its ha molecule did not originate from any known avian strain. in addition, there was no evidence of adaptation to a mammalian host [64] . it was not until the 1997 hong kong epidemic that direct transmission of whole avian viruses to humans was observed. analysis of the virus that caused this epidemic revealed that a reassorted influenza a virus (h5n1) of entirely avian origin had crossed the species barrier, apparently without adaptation to a mammalian host. interestingly, the virus was able to replicate in humans but had not acquired human-tohuman transmissibility, preventing efficient spread and, potentially, a global epidemic (reviewed in [65] ). receptor specificity is considered to be a major determinant of the host range of influenza a viruses. the ha protein plays a pivotal role in host-cell receptor recognition and attachment. it binds sialic acid (sa) on the host cells. avian influenza a viruses preferentially bind to terminal sa which is joined by an alpha2,3-linkage to the sugar chain of the glycoprotein or glycolipid in the gut. however, human influenza a strains bind to terminal sa through an alpha2,6-bond to cells in the respiratory tract as a result of acquired mutations [66] [67] [68] . the 1997 hong kong avian h5n1 strain, however, possessed avian binding properties [69] . this indicates that receptor specificity alone is not an absolute requirement for birdto-human transmission. the host range of influenza a viruses is determined by a complex interplay of multiple factors [70] . the influenza a virus illustrates the unpredictability of virus variation as well as the virus's great potential for adaptation. regular close contact between birds, pigs and humans offers opportunities for reassortment and crossspecies transfer. hence, the live-bird markets in south-east asia are considered a risk [71] . a variety of experimental conditions are applied in the laboratory for propagation and isolation of viruses and their genetically modified derivatives. as a result, these viruses are subject to selective forces that are likely to differ from those experienced in nature. although conditions may be well defined and controlled, various selective pressures are generated in culture due to, e.g., changes of concentrations of nucleotide substrates, the addition of mutagenic substances, the use of different incubation temperatures, incubation with antibodies, or a change of host cells. these different selective forces have unpredictable influences on the virus. since cell culture conditions can have profound effects on the composition of viral populations, viral stocks consist of genetically heterogeneous populations (reviewed in [20, 72] the following examples demonstrate that upon persistent infection and passage in cell culture, cross-species transmissibility may be promoted by selection of virus variants with an altered host range. adaptation in cell culture may result in changes in receptor specificity and tropism, and leads to the emergence of host-range mutant viruses. the mouse hepatitis virus (mhv) is characterized by a narrow host-range and tissue specificity, both in vivo and in vitro. this specificity is primarily determined by the virus's surface spike (s) glycoprotein, which is responsible for attachment to specific host-cell receptors [73] . mhv virus variants with both an altered receptor specificity and a broadened host range were selected during continued passaging in murine or mixed cultures consisting of murine and non-permissive hamster cells. here mhv acquired the ability to infect human, hamster and monkey cells. mhv host-range expansion has been attributed to the presence of virus variants recognizing homologues of the normal receptor. adaptation required mutations in the surface s protein and selection of host-range mutants for the use of the alternative cellular receptor [74] [75] [76] [77] . selection of variants with a changed receptor specificity resulting from passage in cell culture has also been demonstrated for foot-and-mouth disease virus (fmdv). host-cell specificity of the parental virus is based on an rgd motif-dependent integrin-mediated entry pathway [78] . the rgd motif is an arginine-glycine-aspartic protein sequence within the virus capsid that recognizes and binds to some integrins on the cell surface of the host cell [79] . yet, upon multiple passages in bhk-21 cells, fmdv variants emerged that acquired the ability to infect several initially non-permissive human and animal cell lines via an alternative entry pathway. analysis of these variants revealed that adaptation of fmdv in cell culture led to an enhanced affinity for heparan sulfate as a receptor, independent of the rgd motif. interestingly, these host-range variants were able to maintain infectivity in cell culture not only independent of an rgd motif, but also without the requirement to bind to heparin sulfate [80, 81] . this implies the use of alternative receptors. selection of the fmdv host-range mutants was associated with amino acid substitutions in or near the capsid rgd motif [82] [83] [84] . recently it was demonstrated that passaging of fmdv in bhk-21 cells led to an expansion of the host-cell tropism to non-human primate and human cell lines. selection of these host-range variants was also associated with amino acid substitutions in the viral capsid proteins [85] . there is less data on mutation frequencies and adaptation of dna viruses in cell cultures compared to rna viruses. however, there are a number of illustrative examples. adaptation of sv40 and polyomavirus to different cellular environments resulted in the emergence of host-range mutants in cell cultures (reviewed in [86] ). random mutagenesis of human adenovirus followed by repeated passaging in certain cell lines allowed isolation of adenovirus host-range mutants [87, 88] . human adenovirus 2 (hadv2) mutants with altered specificity resulting from reduced binding affinity of the adenovirus penton-base protein for the integrins on the cells were selected in persistently infected cell lines [89] . more recently, upon passaging in cell culture, adaptation led to the emergence of herpesvirus host-range mutants. these viruses use alternative receptors and replicate in different cell types in the natural host, and in cells from different species that were previously nonpermissive. several glycoproteins are essential for the entry of alphaherpesviruses such as pseudorabies virus (prv), herpes simplex virus (hsv) and bovine herpesvirus 1 (bhv-1) (reviewed in [90] ). interaction between, e.g., the viral glycoprotein c (gc) and heparan sulfate mediates primary attachment. for infection, however, a secondary interaction between glycoprotein d (gd) and one of several entry receptors is required. single amino acid substitutions in the gd glycoprotein of herpes simplex 1 (hsv-1) as well as complete ablation of this glycoprotein in the swine pseudorabies virus (prv) led to a gd glycoprotein-independent entry mode using alternate receptors [91] [92] [93] : at least three classes of cell-surface proteins are now thought to be involved in alphaherpesvirus entry [94] . also, cell culture adaptation of human cytomegalovirus (hcmv), a betaherpesvirus, resulted in the selection of phenotypic variants that had lost their endothelial tropism [95] . in cell culture viruses may readily adapt by mutation, selection and competition. these processes are stochastic in nature. adaptation is therefore an unpredictable process, strongly influenced by the experimental setting, e.g. the multiplicity of infection used, the number of passages employed, and the type of selection employed. one should be aware of the potential adaptation when working with virus-infected cell cultures. recombinant dna technology, including 'reverse genetics' and the availability of complete (infectious) clones for a large number of rna and dna viruses, allows genetic modification of viral genomes and generation of recombinant [90, 90] viruses in vitro. these technologies offer the possibility to deliberately change the tropism or host range of the viruses. some of the latest technologies are discussed in the context of two important virus groups, i.e. influenza a viruses and coronaviruses. the advent of reverse genetics systems now allows the generation of recombinant infectious influenza a viruses entirely from cloned cdnas in cell culture. these systems are based on transfection of at least eight plasmids, each containing a copy of one of the eight influenza a virus genomic segments [96, 97] . the technology permits the generation of custom-made recombinant influenza a viruses (reassortants) containing specific (heterologous) gene segments of interest and offers the possibility to study their biological properties in cellular and animal model systems. in addition, reverse genetics can be used in the development of vaccine strategies. the use of reverse genetics allows the deliberate introduction of specific mutations in viral genes allowing selective evaluation of the contribution of individual genes or segments to, e.g., the virus's virulence/pathogenicity, transmissibility and host range. this approach has already been shown to be pivotal for the generation and characterization of reassortants containing heterologous influenza a segments from, e.g., highly pathogenic avian influenza (hpai) h5n1 strains [98] or the 1918 pandemic strain [99, 100] . in addition, this technology allowed the generation from cloned segments of the hpai h5n1 strain that caused the deadly 1997 hong kong outbreak [101] . reverse genetics systems can also be used as an alternative for the production of both live attenuated and inactivated vaccines in preparing for pandemic influenza a virus threats. conventional annual (inactivated) vaccine production is based on simultaneous infection of chicken eggs with two different influenza a strains followed by selection of the desired vaccine virus. this reassorted virus then contains the na and ha segments of the relevant circulating influenza a virus against the background of six complementary segments derived from an attenuated reference strain (e.g. a/puerto rico/8/34 h1n1), which is safe for humans [102] . to overcome the difficulties of selecting such reassortants and subsequent laborious time-consuming passaging of these viruses, plasmidbased reverse genetics can be used for fast and directed generation of vaccine strains [103] . for vaccines based on hpai viruses the use of reverse genetics has another important advantage. such highly pathogenic viruses are lethal to chicken embryos and cannot be grown in large quantities in this way. the virus's high pathogenicity is associated with the presence of basic amino acids adjacent to the cleavage site within the ha molecule [104] . by using recombinant dna technology this sequence can be eliminated. plasmid-based reverse genetics can then be used to generate the desired vaccine strain containing the attenuated ha molecule [105, 106] . in conclusion, plasmid-based reverse genetics enables the generation of defined reassorted influenza a viruses consisting of, e.g., human and avian viral gene segments of interest. however, it is usually not possible to predict the biological properties from the gene constellation of such variant viruses. such activities therefore pose potential risks, in particular when the gene constellation is not based on characterized isolates. recombinant dna technology has allowed the construction of infectious cdna clones of large rna viruses such as coronaviruses, including the sars-associated coronavirus (sars-cov) [107] [108] [109] . these reverse genetics systems can now be used as tools for the production of defined genetically modified coronaviruses [110] . this allows the introduction of specific mutations into the genome of coronaviruses, and, e.g., the exchange of specific viral genes between different coronaviruses to study their pathogenesis, replication strategy, and cross-species transmissibility. the possibility to engineer tissue and host tropism using these technologies makes coronaviruses potential vectors for vaccine development and possibly for gene therapy [111] [112] [113] . host-range specificity of coronaviruses is primarily determined at the virus entry level. several studies have demonstrated that sequence changes in the gene encoding the coronavirus surface spike (s) glycoprotein can lead to a change of tropism and host-range specificity [73] . this is illustrated by the generation of a chimeric coronavirus by targeted recombination, in which the ectodomain of the s glycoprotein of mouse hepatitis virus (mhv) was replaced by the ectodomain of the s glycoprotein of feline infectious peritonitis virus (fipv). this substitution conferred specific tropism for feline cells, while the ability to infect murine cells was lost [114] . vice versa, a reverse genetics strategy for fipv was developed conferring the ability to infect murine cells [115] . similar techniques may aid the studies on the pathogenicity of the sars-cov. the examples mentioned above indicate that the use of recombinant dna technology now provides for powerful systems to generate and modify, e.g., highly pathogenic viruses such as pandemic influenza a viruses and sars-cov. live attenuated virus vaccines are among the most successful viral vaccines known to date. traditionally, attenuation is achieved by the 'jennerian approach', i.e. serial passaging in cell culture [116] . by this method a number of useful vaccines currently in use have been generated. still, the mechanism by which the attenuated phenotype evolves is largely unknown. for instance, the nature and degree of genetic variation present at different stages of the attenuation process is usually not known. the presumed mechanism of attenuation is based on host-range restriction due to accumulation of changes in surface (glyco)proteins [117] . thus selection of variants seems inherent to the process of generating the desired level of attenuation and genetic stability to prevent reversion to the wild-type virus therefore, dependent on the passage history, diversity within the virus population is likely to represent adaptations to growth in cell culture. as a result, genetic variants with different host-range phenotypes may be present in the vaccine strain of the virus. examination of substrains of live attenuated vaccine lots based on the yellow virus 17d strain and measles virus edmonston strain demonstrate that these virus stocks indeed consist of a heterogeneous population of variants [118] [119] [120] [121] . a number of adverse consequences of the use of such virus stocks have been reported and associated with possible selective growth advantage of host-range variants in the recipient [122, 123] . therefore, caution should be taken before releasing live attenuated viral vaccines based on non-human animal viruses. significant progress has been made in approaches to genetically modify the tropism of vector viruses. such strategies have been used in the development of cancer gene therapy. initially, replication-deficient vectors were used for this purpose. however, to improve efficacy, tumor-targeted replication-competent viruses have been developed for the use of viral therapy of cancer (virotherapy) [124, 125] . here we discuss some of the developments with human adenovirus type 5 (hadv5). hadv5 has been widely used for a number of vector applications [126] . however, the use of genetically modified adenoviral vectors has some limitations [127] . their efficacy relies on the presence of its receptor, car (coxackievirus and adenovirus receptor for hadv5), on target cells. primary binding of the virus to car is mediated by the knob domain of the adenoviral fiber protein. subsequent internalization is mediated by the interaction between the rgd motif in the penton base of the virus and secondary host-cell integrin receptor molecules [128, 129] . to achieve cell-type specificity and a high efficacy in the absence of the car receptor different strategies have been developed. these include redirecting adenoviral binding to alternative cellular receptors by genetic modification of genes coding for the capsid proteins fiber, hexon and penton base [130] . this may result in either an expanded tropism or in abolishment of the adenoviral native tropism. table 2 summarizes a number of properties contributing to the relative risk for the use of vector viruses. for this purpose a numerical hazard score was assigned to each property. table 3 summarizes a number of adenoviral vectors with altered properties and their relative risks. such modified viruses are now being evaluated in a clinical setting for experimental gene therapy. table 3 illustrates that a change of cell tropism, tissue tropism, or host range of a viral vector should be considered as factors in risk assessment for activities with genetically altered vector viruses. in general, the use of replicationcompetent viral vectors poses special concerns with regard to unintended spread to new and undesired cell types, as well as horizontal transmission of the vector [131, 132] . a replication-competent vector virus with an altered tropism or host range virtually constitutes a new viral pathogen with the potential of a new disease manifestation. in nature many factors may contribute to the emergence of a new zoonotic viral disease. these factors consist of viral evolutionary processes such as mutation, natural selection and competition, host determinants, e.g., immune status and physiological factors, and environmental determinants such as ecological and climatological circumstances. as highlighted by the emergence of new viral diseases in the last two decades, the process of adaptation often involves the acquisition of an altered cell tropism or host range. against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and for live-vector vaccines is a cause for concern. this review highlights the importance of identifying and evaluating the risks and consequences of activities that may generate host-range mutants with the capacity of cross-species transmission. the use of such viruses may lead to inadvertent introduction of vector viruses with a changed cell or tissue tropism and/or host range through an immunologically naï ve and non-adapted hosts. interactions between the virus and the cellular receptor often determine the host range of the virus and therefore constitute a species barrier [133] . minor mutations in the viral capsid or surface glycoproteins may already result in profound changes in cell tropism or host range of a virus. in this review we have therefore focused on the level of virus entry to address some implications for activities with viral vectors, and in particular with hostrange mutants. this could contribute to a rational and reasoned inventory of factors that should be considered in risk assessments of activities with viral vectors. in considering possible risks involved in handling replication-competent vector viruses in the laboratory, [134] . this implies that concepts such as host-range barrier and hostcell specificity may be rather flexible than rigid. if the host range and completion of the viral life cycle is exclusively restricted to the level of entry, forced entry may bypass important discriminatory host-cell restriction steps. this may result in distinct pathological phenotypes and new disease manifestations. therefore, precaution should be taken to avoid inadvertent release and spread of 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thank prof. alex j van der eb and dr. huub schellekens for critical review of the manuscript. in addition the authors wish to acknowledge the useful commentaries of the anonymous reviewers. key: cord-256903-8lyw27gh authors: guzman, efrain; montoya, maria title: contributions of farm animals to immunology date: 2018-12-06 journal: front vet sci doi: 10.3389/fvets.2018.00307 sha: doc_id: 256903 cord_uid: 8lyw27gh by their very nature, great advances in immunology are usually underpinned by experiments carried out in animal models and inbred lines of mice. also, their corresponding knock-out or knock-in derivatives have been the most commonly used animal systems in immunological studies. with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. from the identification of b cells to the realization that γδ t cells can function as professional antigen presenting cells, farm animals have contributed significantly to a better understanding of immunity. great advances in immunology are usually supported by experiments carried out in animal models and by far, inbred lines of mice and their corresponding knock-out or knock-in derivatives, are the most commonly used animal systems in immunological studies. though with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. also, some answers provided in mouse models are not applicable to other species of animals or humans. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. the humble cow, the underestimated pig and the unassuming chicken have greatly influenced our current understanding of human immunology. for most immunologists dedicated to fundamental and applied research, it is easy to forget that b cells were first identified in chickens and vaccination first occurred because of a cow. although there are far too many important events to discuss in this paper, we have chosen to highlight a few of the most important contributions of farm animals to the current understanding of immunology ( table 1) . edward jenner published in 1798 a booklet entitled "an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england, particularly gloucestershire and known by the name of cow pox" (1, 2) and although strictly speaking jenner did not discover vaccination, he was the first person to use scientific rigor to prove protection from disease through targeted intervention. the english dairy farmer benjamin jesty (1737-1816) was the first person known to vaccinate against smallpox (3) protecting his family against the virus even after numerous exposures (3) . however, the idea and indeed the term vaccination, only came into the light spot 100 years later thanks to louis pasteur. this time the chicken takes a privileged position and the story was beautifully explained by pasteur himself (4, 5) and has been romanticized in paul de kruif 's book "microbe hunters" (6) . in 1878 pasteur inoculated chickens with "stale" cultures of pasteurella multocida. the chickens became sick but recovered so he decided to re-inoculate them with a fresh culture. the chickens that had received the "stale" culture recovered whereas chickens that had not been pre-exposed to the stale cultures died. pasteur recognized the similarities between his studies in chickens and what jenner had published with smallpox. he coined the term "vaccine" (4, 5, 7) in honor of jenner. by the early 1880s, william smith greenfield in the uk (8, 9) and pasteur working with henri thullier, charles chamberland and pierre paul émile roux in france (10, 11) had begun developing and testing vaccines against anthrax in sheep and cattle. a decade later, the german scientists friedrich loffler and paul frosch identified the first ever filterable infectious agent in mammals: foot and mouth disease virus (fmdv) and developed a fully protective heat-inactivated vaccine against it (12, 13) ; however an effective long-lasting and broadly protective vaccine against fmdv remains elusive. pigs also played an important role in early vaccinology studies. by the late 1800s swine plague or hog cholera (later discovered to be caused by a virus now called classical swine fever virus, csfv (14) was killing hundreds of thousands of pigs across the word and was particularly of concern to the us pig producing industry, causing an impressive us$15 million a year in losses in 1875 (15) and us$20 million by 1878 1 . once again, pasteur and thullier developed a vaccine to what is now thought to be the first ever vaccine against a viral infectious disease (16) and the first mass-vaccination campaign in history. in addition, it is rarely recognized that csfv was the first animal virus ever to be cultured in vitro (17) and the techniques developed by carl tenbroek continue to be used today. horses have also contributed to the understanding of fundamental immunological mechanisms. in a series of experiments, emile roux working with alexandre yersin and followed by emil von behring and shibasaburo kitasato immunized horses to produce an "antidote" or immune sera against the diphtheria toxin that was eventually used to treat humans, an important step in understanding antibodies and humoral immunity (18) . behring won the nobel prize for medicine in 1901 for this work. another milestone in vaccine development was the generation in the 1970's of vaccines to control marek's disease (md), a naturally occurring neoplastic disease in chickens caused by an oncogenic herpesvirus (19) . md vaccines are the first examples of the use of vaccination to protect against cancer (20, 21) . with the discovery of molecular biology techniques in the 1960's and 70's, the race was on to develop recombinant vaccines against numerous infectious diseases. the first report of a biosynthesized polypeptide vaccine was published in 1981 (22) . the structural protein vp3 of fmdv was cloned 1 (1881). swine plague. science 2, 121 and expressed in e. coli and the purified protein used to vaccinate six cattle and two swine, which developed neutralizing antibodies and were protected against challenge with fmdv (22) . and new technologies have only helped to highlight the importance of farm animals in vaccine development: using a computational approach to assess protein-protein stability, kotecha and colleagues (23) used molecular dynamic ranking to predict fmdv capsid stabilities and produced stabilized fmdv capsids based on these predictions, assessed their stability using x-ray crystallography and demonstrated their improved immunogenicity in vivo by vaccinating cattle. this demonstrates the potential value of structure-based design of vaccines to develop stabilized vaccine antigens for animals and humans alike. although the innate immune system of animals is largely conserved, there are significant variations in the pattern-recognition-receptor (prr) structures of various species (24) . it has been suggested that laboratory mice have not been subjected to the selective pressures that other animals have and so innate immune studies carried out in laboratory animals do not accurately inform human biology (25) . it has been demonstrated that human and farm animal prr responses to their ligands (24, 26) are more similar to each other than human-mouse prr responses (26) (27) (28) . because prr recognition is associated with adaptive immunity, a better understanding of these molecules in farm animals is likely to better inform on their effect in these animals as well as humans. a major contribution of the chicken to fundamental innate immunity was the description in 1957 of the first interferon. chicken embryos were exposed to influenza virus by alick issacs and jean lindenmann (29) and they identified an immune soluble element responsible for regulating virus infection. this discovery was certainly one of the scientific landmarks in cell biology in the twentieth century and one which opened the doors of what we now know as innate immunity. perhaps the most recognizable contribution of the chicken to science, and immunology in particular, was in the definition of the two elements of adaptive immunity: the b-dependent and the t-dependent immunity. the avian bursa of fabricius, named after hieronymus fabricius of aquapendente (30) is a sac-like structure located in the cloacal passage of the bird and its function remained elusive until well into the twentieth century. bruce glick and timothy chang working at the poultry science department at ohio state university (30, 31) described how following the surgical removal of the bursa, chickens injected with salmonella typhimurium "o" antigen failed to develop bacteria-specific antibodies. glick and chang wrote a paper entitled: "the role of the bursa of fabricius in antibody production" and was rejected by leading scientific journals (30) and eventually published in poultry science (32) . several years later, the bone marrow in mammals was shown to be the equivalent of the bursa in birds (33) , and so the term "b-lymphocyte" originated from "bursa-derived lymphocyte". several years later, cooper et al. published a fundamental paper on the demarcation of the thymic and bursal and systems in birds and proposed the existence of equivalent systems in mammals (34) . the cannulation of lymphatic vessels was developed in the early twentieth century in rats to study the lymphatic system but due to the complexity of the surgical procedure, sheep and then cattle were used extensively in the 1960s and 70s in lymphatic cannulation studies (35) . in a series of adoptive transfers of lymph-migrating cells and fluid, hall and colleagues first identified in sheep that antibody-secreting cells (acs) encounter antigens and are activated in the lymph nodes (36), then migrate via the efferent lymphatics to the circulatory system, and that the immune response depends on an intact lymphatic system. cattle have also contributed to fundamental b cell immunology and the generation of a highly diverse antibody repertoire. most vertebrates encode a large number of variable (v), diversity (d), and joining (j) gene segments and antibody diversity is achieved by recombination of these 3 segments. in contrast, cattle only express a limited number of v genes and so it is thought that antibody diversity is achieved recombination events and endogenous mutation mechanisms in the cdr3 region (37) . another unusual feature of bovine antibodies is their exceptionally long cdr3 regions (38) . these long cdr3 and unusual mutation mechanisms result in "microfolds" within the cdr3 region that allow bovine antibodies to bind antigens that would normally be inaccessible (38) . a recent report demonstrated that cows can be immunized with a single hiv env trimer and this results in potent hivspecific nabs which are dependent on the length of the cdr3 loops of bovine ig (39) . it has been suggested that this could be an efficient way of producing super-antibodies against other human pathogens. transchromosomal cows have been engineered to produce large amounts of full human igg molecules with pathogen specificity: mers-cov (40) , hanta virus (41), veev (42) , and ebola virus (43) . this technology has the potential to generate prophylactic antibodies against emerging viral diseases. on the other hand, chickens have serum igm and iga both of which are homologs of their mammalian counterparts; in addition, they express igy, not found in mammals but thought to be an evolutionary ancestor for mammalian igg and ige. chickens however do not have either ige nor igd but instead use a distinct process for generating antibody diversity that is distinctly different to mammals (44) . engineers frequently look to nature for inspiration. antibody engineers are no exception, modeling new therapeutics on molecules found in animals such as camels and cows. indeed, 10% of bovine antibodies have unusually long heavy-chain cdr3s as part of their antigen-recognition sites. stanfield et al. (45) have solved crystal structures of three new bovine fab fragments and analyzed the five known structures to show that their ultra-long cdr h3s all adopt similar architectures composed of a knob domain containing a small conserved β-sheet connected by diverse disulfide-bonded loops that is separated from the antibody surface by a long conserved stalk. they propose that varying the length of the stalk and the positions and number of disulphide links in the knob may help drive antibody diversity. these structural insights could be leveraged to tailor antibody-based therapeutics. in contrast to all other mammals, camelids (dromedaries, camels, llamas, etc) also have an unique antibody type similar igg but with identical heavy chains lacking the ch1 domain and which do not pair with their corresponding light chains. these "heavy-chain antibodies" (hcabs) display antigenspecific variable domains or "vhh" which are structurally and functionally similar to an igg fv but have only three cdr loops defining the antigen biding sites. camel vhh domains, also called "nanobodies, " have been of great interest because of their stability and small size and strong affinity to their corresponding antigens. in fact, several camel vhh domain antibodies are in early preclinical development in oncology, infectious, inflammatory, and neurodegenerative diseases (44), the most recent example being the generation of broadly neutralizing antibodies to influenza in llamas (46) . cytotoxic and helper t cells are generally considered to be phenotypically different due to the mutually exclusive expression of the co-receptors cd8αβ and cd4 and differences in mhcrestriction (class i vs. class ii). however, between 3 (in normal individuals) and 60% (in certain pathologies) of human peripheral blood lymphocytes have been shown to be cd4/cd8 double positive (dp) t cells (47) . thymic and extra-thymic development of t cells has been studied mainly in mice and because the expression of cd8 and cd4 in mouse t cells for the most part mutually exclusive, cd4/cd8 dp lymphocytes have generally been ignored. nevertheless, the presence of cd4/cd8 dp t cells in many animals makes it impossible to ignore these cells. studies in pigs have shown that cd4/cd8 dp are a distinct subset of activated and/or memory t helper cells (48) and in humans the increase in circulating cd4/cd8 dp t cell frequency has been identified in autoimmune and chronic inflammatory diseases (49) (50) (51) (52) (53) suggesting the importance of this particular t cell population in human health. most circulating t cells in humans and mice are conventional t cells expressing the αβ t cell receptor (tcr) and either cd4 or cd8. unlike mice, other species like cattle, pigs and chickens possess a substantial proportion of t cells expressing the γδ tcr cells in the circulation suggesting that circulating γδ tcr t cells have a more important role immunity than previously thought (54) . the fact that the phenotype and frequency of circulating and tissue resident t cells is so vastly inconsistent in different species suggests that immune responses to (vaccine) antigens are also distinct. it is assumed that that all animal species have a similar immune response to a particular antigen, but this is a statement to be reviewed in light of each host particularities. in addition, the th1/th2 polarization of t cells observed in response to particular antigens is a phenomenon of certain strains of laboratory mice and not of outbred mammals including farm animals and humans (55, 56) . in fact, it has been shown that cytokine profiles defining th1/th2 responses to antigens in cattle are more similar to human responses than those observed in mice (57). dendritic cells (dc) as such, and their role in immunity were first described in the 1970s and in 1995 ralph steinman published a series of papers describing that a cellular receptor called "dec-205" (now cd205) was expressed on mouse dc, was involved in antigen processing (58, 59) and was detected by the monoclonal antibody nldc-145. in fact, it was 2 years earlier in 1993, that chris howard, a bovine immunologist working at the then called "institute for animal heath" in the uk published a series of papers identifying an important and until then uncharacterized marker expressed on pseudoafferent lymph veiled cells (also called aldc) detected by the monoclonal antibody wc9 (now cc98) (60-63). although steinman's identification of mouse cd205 helped characterize the binding of cc98 to bovine cd205 (64), the importance of cd205 in identifying dc was first evident in cattle. as mentioned above, steinman's seminal work in characterizing dc using the mouse system has been one of the most important developments in cellular immunology of the twentieth century, and one which lead to his nobel laureate. however, the idea that a component of the immune system was involved in antigen processing and presentation had been proposed many times before. as mentioned above, cannulation of the lymphatic vessels is more practical in large than small animals, and this technique has been used to investigate dc biology. afferent or peripheral lymph dc were first described in sheep in 1972 (65) as "very phagocytic dendritic macrophages that are involved in long term immunological reactions" that are very potent antigen presenting non-lymphoid cells (66) and that their phagocytic and antigen presentation capacities differed from "classical" peritoneal macrophages (67) , therefore indicating that dc and macrophages were different cell types (67) several years before steinman's observations (68) . in addition, lymphatic cannulation of sheep has revealed important ontologic, phenotypic and functional characteristics of dc subsets that are relevant in other mammals, particularly humans (69, 70) . similitudes and differences between swine and human dc/macrophage populations have recently been described (71) . in one striking example and in contrast to studies performed in mice, swine and human cdc2, which are associated with th2 responses, both express fcεriα and are localized in or next to the tracheal and bronchial epithelia. these observations have been proposed to imply that swine and humans have similar allergen responses as opposed to mice. this theory is supported by the fact that localization of cdc2 helps them access antigens such as airborne allergens, and fcεriα expression on these cells might help proliferation observed in allergic responses. as mentioned before and unlike mice, horses and humans, most other animals have a large γδ t cell compartment. for example, up to 70% of all blood lymphocytes in young calves are t cell expressing the γδ t cell receptor (tcr). although the reasons for the enlarged t cell compartment in cattle, pigs and chickens is still unknown, their large numbers and ease of collection has resulted in great advances in γδ t cell biology knowledge not only for farm animals, but also for humans. for example, apcs were shown to influence γδ t cell proliferation (72, 73) . cynthia baldwin's lab has defined antigen-specific bovine γδ t cell responses in various systems (74) (75) (76) and adrian smith's lab has done similar observations in chickens (77, 78) . it has also been shown that bovine γδ t are potent regulatory t cells (79) , an observation that is also true for a subset of human γδ t cells (80, 81) . these results in farm animals have and continue to enhance our understanding of human γδ t biology (82) . perhaps the most important one was the realization that a subset of bovine γδ t cells expressed mhc class ii and co-stimulatory molecules on their surface, a characteristic normally attributed to macrophages, b cells and dc but not t cells (83) . bovine γδ t cells were also shown to phagocyte antigens and of mhc iirestricted presentation to cd4+ t cells (83) . this function of bovine γδ t cells was subsequently reported in pigs (84) and much later in mouse (85) and human (86) (87) (88) γδ t cells. perhaps the best known contribution of any farm animal to scientific progress was the somatic cell nuclear transfer that gave origin to dolly, the sheep (89) . although nuclear transfer itself is not a direct contribution to immunology, nuclear transfer technology has directly influenced many immunological concepts underpinned by technologies such as induced pluripotent stem (ips) cells and crispr-cas systems. clustered regularly interspaced short palindromic repeats (crispr) is a rna-guided endonuclease used both in vivo and in vitro. genetically modified animals becoming more common and their availability can be exploited in many applications such as comparative immunology, physiology and disease, to generate in vivo bioreactors to produce complex proteins, or to produce genetically modified organs for transplantation in humans (90) . although the majority of pharmaceutical research is performed in laboratory mice models, it is clear that humans are not "large mice." by a large extent, studies in laboratory mice have been the victim of over interpretation; for example, by extrapolating successful pre-treatment in mice to therapeutic treatment in men. the weakness of the mouse model in pharmaceutical research was recently highlighted in a study showing that inflammatory responses in mouse models do not correlate with human inflammatory disease (91) . an additional study showed a close similarity in expression profiles of immune-related genes between humans and pigs (92) . cattle, pigs, and chickens, are useful, valid, and valuable models to study human infectious diseases and important clinical targets in their own right. both humans and cattle are the natural hosts for tuberculosis (being infected with the geneticallyrelated mycobacterium bovis and mycobacterium tuberculosis, respectively) and the bovine and human diseases share many similarities in terms of immunity and pathology (93) , whereas the mouse model of tuberculosis does not provide a faithful representation of the disease in humans (94) . similarly, bovine respiratory syncytial virus (brsv) is closely related to human (h) rsv and the pulmonary pathology, immune responses and epidemiology seen in young calves and children are very similar (95) . swine have been shown to be a more faithful model for human influenza infection and immunity studies and the same strains of influenza infect both humans and pigs because the distribution of influenza virus receptors and physiopathology are similar in both species. the transfer of maternal-derived antibodies (mda) to new born pigs enables fancy vaccine study design to elucidate the role of mda in immunity (96, 97) , vaccine efficacy and in enhancement of respiratory disease (98) . gnotobiotic piglets have been used to study various human gastrointestinal pathogens. for example, human noroviruses are antigenically and genetically related to swine noroviruses and unlike mice, humans and pigs show genetic susceptibilities to noroviruses depending on their histoblood group antigen phenotypes and the virus strain. similarly, gnotobiotic pigs have been used in rotavirus research to study disease pathogenesis and identify virus-specific iga and asc as correlates for protection and vaccine efficacy in children (99) . pigs have also been proposed to be better models than mice for many other infectious diseases including female genital infection with chlamydia trachomatis, helycobacter pylori, neisseria meningitides, and nipah virus among others because of the natural susceptibility of pigs to these pathogens (100) . endogenous retroviruses were first discovered in pig kidney cell lines in 1971 (101) and are now known to be present in most, if not all, mammalians. the presence and potential reactivation of endogenous retroviruses has very important consequences in both allo-and xeno-transplantation. immunotherapy is becoming more popular in clinical trials and vaccine efficacy studies. the success of immune cell therapies partially depends on the effective delivery of cells to target organs, a process that invariably involves the lymphatic system. dc migration in mice has not proven to be very informative, however, dc migration in pigs may be able to answer several question on dc migration that cannot be addressed otherwise. these studies demonstrate that using large animals to investigate immune cell trafficking will help improve immunotherapies in humans (102) . in 1906 the french surgeon mathieu jaboulay (1860-1913) implanted a pig's kidney into one woman and a goat's liver into another thus starting the idea of xenotransplantation; unsurprisingly, both women died (103) . the acceptance or rejection of a donor's organ or cells is fundamentally an immunological event. cellular rejection involves nk and t cells that recognize foreign antigens on the grafted tissue. using xenotransplantation models (pig-to-rat, pig-to-primate, and pigto-human), the main mechanism for organ and tissue rejection has been proposed to involve arteriosclerosis, or thickening of the arterial walls. this process if thought to be caused by activated and allo-reactive lymphocytes that migrate over time to the transplanted organ (104) . arteriosclerosis is a major cause of chronic organ rejection (103) . studies in laboratory mice have underpinned many concepts of immune tolerance and the generation of immune responses in the neonate. however, the peripheral immune system in mice remains unpopulated during pregnancy and it is only after birth that b and t cells begin to emmigrate to the periphery. in contrast, lymphoid cells circulate through the fetus in humans and large animals well-before birth and specialized lymphoid tissues are also well-developed and populated by the time of birth and are able to respond to a number of antigens (105, 106) . certainly the immune system in neonate humans and large animals is not matured, but calves, lambs and piglets can be more useful than mice in understanding immune responses during pregnancy and in new borns and these studies can be used to better inform human developmental immunology. this advantage over mice has recently been used to develop extracorporeal support technologies using neonatal lambs with the ultimate objective to use these technologies in premature children (107) . perhaps one of the most common uses of large animal models is in the development of vaccines with several advantages over mice. the serial collection of peripheral blood from animals such as pigs, cattle, chickens, and horses allows for immunokinetic studies to be possible in response to vaccination or infection at the level of the individual. these immunokinetic studies can be used to correlate immune responses generated with protection after challenge with the relevant pathogen. in vaccinology studies using mice, the typical approach would be to sacrifice groups of mice sequentially and harvest spleen and blood, so the immune response to vaccination at the individual level is not normally achieved. large animals also provide several advantages over mice when investigating mucosal immunity. when mice are vaccinated or inoculated intranasally, it is common for the inoculum to be digested because anesthetized mice can both swallow and inhale the material placed on their nose. in addition, the structure of the mucosal associated lymphoid tissue (malt) differs significantly in mice from that of large animals and humans; for example mice do not have tonsils but instead have undefined networks of malt, whereas cattle, pigs and sheep have well-defined tonsils (108) (109) (110) . in the case of vaccine delivery through the skin, cattle, and pigs appear to be better suited than mice for these studies. skin thickness, structure of the epidermis and the presence and distribution of langerhan's cells are among many characteristics that humans and cattle and pigs have in common and which are practically relevant in transcutaneous immunizations (111) . the process of selecting a relevant and appropriate animal model is a balanced and complicated exercise due to the diversity in vertebrate physiology, adaptive and innate immunity. studies in mice, for example, have shown the efficacy of vaccines against fmdv, however these efficacy studies have failed to be translated to the target species (cattle and pigs), presumably due to fundamental differences in the immune systems of model organisms and target species and the ability of the virus to mutate in these animals (112) . it has recently been shown that because immunoglobulin subclass diversification occurred after speciation (113, 114) a particular immunoglobulin subclass in one species bears no functional homology to one of the same name of another species (115) . thus, our knowledge of the functions of igg1 in mice cannot be extrapolated to other mammals. characterizing generating reagents for each animal model hinders the development and usefulness of any of these models and therefore limiting the usefulness of cows, cattle or chickens as models for human immunology. mice and rats are and will probably continue to be the chosen model organisms over farm animals. mice can be readily mutated (knock in or knock out) to study immunological pathways; so far this has been proven to be very difficult-and expensive-in large animals. as mentioned above, the availability of reagents to study immune cells and processes in mice far out competes the availability of these reagents for large animals. pharmacokinetic and toxicology studies would be prohibitively expensive in pigs, horses or cattle, so small rodents and rabbits are the best organisms to use in these studies. in addition, studies in mice have been fundamental in the discovery of antibiotics, chemotherapy agents and more recently car-t cell therapies that can be directly applied to humans. genetic homogeneity, low cost, the availability of biologically-relevant mutants and reagents make the mouse the optimal animal model for many academic and industrial researchers. farm animals have historically contributed and continue to contribute to fundamental and applied immunology. the use of these animals in research is not difficult as long as the appropriate facilities and reagents are available. dedicated housing, cost, biosecurity, and genetic variability are some of the many disadvantages confronted when using farm animals in research. however, selecting an appropriate animal model should be more than just a matter of accessibility and common practice (116) but should be based on the scientific question to be addressed and its relevance. not just a country doctor: edward jenner, scientist edward jenner and the eradication of smallpox new light in the dawn of vaccination on the germ theory on chicken cholera: study of the conditions of non-recidivation and of some other characteristics of this disease microbe hunters the life and works of louis pasteur lectures on some recent investigations into the pathology of infective and contagious diseases professor superintendent, the brown animal sanatory institution concerning the priority due to him for the production of the first vaccine against anthrax remarks on anthracic vaccination as a prophylactic of splenic fever summary report of the experiments conducted at pouilly-le-fort, near melun, on the anthrax vaccination, 1881 summarischer bericht uber die ergebnisse der untersuchungen der kommission zur erforschung der maul-und klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fur bakteriologie berichte der kommission zur erforschung der maulund klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fuxr bakteriologie the survival of the hog-cholera virus in laboratory animals, particularly the rat swine plague, or hog cholera correspondence of pasteur and thuillier concerning anthrax and swine fever vaccinations cultivation of the hog cholera virus remembering emil von behring: from tetanus treatment to antibody cooperation with phagocytes studies on the epidemiology of marek's disease herpesvirus in broiler flocks effect of vaccination with herpesvirus of turkeys (hvt) on horizontal spread of marek's disease herpesvirus protective efficacy of marek's disease vaccines cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design toll-like receptors in domestic animals variation matters: tlr structure and species-specific pathogen recognition human but not murine toll-like receptor 2 discriminates between tripalmitoylated and tri-lauroylated peptides human toll-like receptor 4 recognizes host-specific lps modifications identification of full length bovine tlr1 and functional characterization of lipopeptide recognition by bovine tlr2/1 heterodimer virus interference. i the interferon a landmark contribution to poultry science-immunological function of the bursa of fabricius growth of the bursa of fabricius and its relationship to the adrenal gland in the white pekin duck, white leghorn, outbred new-hampshire, and inbred new-hampshire the bursa of fabricius and antibody production cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells restoration of gamma globulin production in agammaglobulinemic chickens surgical techniques for the collection of lymph from unanaesthetized sheep the ultrastructure and function of the cells in lymph following antigenic stimulation maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection reshaping antibody diversity rapid elicitation of broadly neutralizing antibodies to hiv by immunization in cows human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo dna vaccine-derived human igg produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy against venezuelan equine encephalitis virus production of potent fully human polyclonal antibodies against ebola zaire virus in transchromosomal cattle structural and genetic diversity in antibody repertoires from diverse species conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin coexpression of t4 and t8 on peripheral blood t cells demonstrated by two-color fluorescence flow cytometry phenotypic maturation of porcine nk-and t-cell subsets evaluation of t cell subsets in myasthenia gravis using anti-t cell monoclonal antibodies circulating cd3+ cd4+ cd8+ t lymphocytes in multiple sclerosis circulating cd4+cd8+ t lymphocytes in patients with kawasaki disease cd4+ cd8+ double positive (dp) t cells in health and disease peripheral cd4cd8 double positive t cells with a distinct helper cytokine profile are increased in rheumatoid arthritis porcine gammadelta t cells: possible roles on the innate and adaptive immune responses following virus infection the relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species differences in immune responses against leishmania induced by infection and by immunization with killed parasite antigen: implications for vaccine discovery type 1 and type 2 responses in regulation of ig isotype expression in cattle the receptor dec-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing dec-205, a 205-kda protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody nldc-145: purification, characterization, and n-terminal amino acid sequence summary of workshop findings for cattle (tables 1 and 2) leukocyte antigens of cattle and sheep. monoclonal antibodies submitted to the second workshop studies of monoclonal antibodies identifying two novel bovine lymphocyte antigen differentiation clusters: workshop clusters (wc) 6 and 7 phenotypic variation and functional differences within dendritic cells isolated from afferent lymph dec-205 expression on migrating dendritic cells in afferent lymph the cells of lymph and their role in immunological reactions. handbuch der allgemeinen pathologie the properties and functional activity of non-lymphoid cells from bovine afferent (peripheral) lymph some properties of dendritic macrophages from peripheral lymph identification of a novel cell type in peripheral lymphoid organs of mice. morphology, i., quantitation, tissue distribution plasmacytoid dendritic cells migrate in afferent skin lymph existence of cd8alpha-like dendritic cells with a conserved functional specialization and a common molecular signature in distant mammalian species the respiratory dc/macrophage network at steady-state and upon influenza infection in the swine biomedical model monocytes control gamma/delta t-cell responses by a secreted product bovine gamma/delta t-cell proliferation is associated with self-derived molecules constitutively expressed in vivo on mononuclear phagocytes protective killed leptospira borgpetersenii vaccine induces potent th1 immunity comprising responses by cd4 and gammadelta t lymphocytes the role of bovine γδ t cells and their wc1 co-receptor in response to bacterial pathogens and promoting vaccine efficacy: a model for cattle and humans the bovine model for elucidating the role of γδ t cells in controlling infectious diseases of importance to cattle and humans an alphabeta t-cell-independent immunoprotective response towards gut coccidia is supported by gammadelta cells γδ t cells 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cell lines in vivo tracking and immunological properties of pulsed porcine monocyte-derived dendritic cells xeno's paradox: why pig cells are better for tissue transplants than human cells xenoislet rejection following pig-to-rat, pig-to-primate, and pig-to-man transplantation expanding the role of peyer's patches in b-cell ontogeny induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector an extra-uterine system to physiologically support the extreme premature lamb of mice and not men: differences between mouse and human immunology revisiting the b-cell compartment in mouse and humans: more than one b-cell subset exists in the marginal zone and beyond the lymphoid system: a review of species differences transcutaneous immunization of domestic animals: opportunities and challenges laboratory animal models to study foot-and-mouth disease: a review with emphasis on natural and vaccine-induced immunity antibody repertoire development in fetal and neonatal piglets. xiii hybrid vh genes and the preimmune repertoire revisited genomic organization of the immunoglobulin light chain gene loci in xenopus tropicalis: evolutionary implications therapeutic administration of broadly neutralizing fi6 antibody reveals lack of interaction between human igg1 and pig fc receptors model organisms: there's more to life than rats and flies all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors were funded by the uk's bbsrc grants bbs/e/i/00002067 and bbs/e/i/00002014. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2018 guzman and montoya. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-253548-izya7nws authors: catchpole, ken; bowie, paul; fouquet, sarah; rivera, joy; hignett, sue title: frontiers in human factors: embedding specialists in multi-disciplinary efforts to improve healthcare. date: 2020-09-09 journal: int j qual health care doi: 10.1093/intqhc/mzaa108 sha: doc_id: 253548 cord_uid: izya7nws despite the application of a huge range of human factors (hf) principles in a growing range of care contexts, there is much more that could be done to realize this expertise for patient benefit, staff wellbeing and organizational performance. healthcare has struggled to embrace systems safety approaches, mis-applied or misinterpreted others, and has stuck to a range of outdated and potentially counter-productive myths even has safety science has developed. one consequence of these persistent misunderstandings is that few opportunities exist in clinical settings for qualified hf professionals. instead, hf has been applied by clinicians and others, to highly variable degrees – sometimes great success, but frequently in limited and sometimes counter-productive ways. meanwhile, hf professionals have struggled to make a meaningful impact on frontline care and have had little career structure or support. however, in the last few years, embedded clinical hf practitioners have begun to have considerable success that are now being supported and amplified by professional networks. the recent covid-19 experiences confirm this. closer collaboration between healthcare and hf professionals will result in significant and ultimately beneficial changes to both professions and to clinical care. human factors (hf) perspectives are familiar to many in healthcare, and thousands of people are exploring or benefiting from the value those perspectives bring to a multitude of care contexts. hf has been applied in acute care 1 , primary care 2,3 , emergency care 4 , home care 5 , device design 6 , health it 7 , health systems design 8 , architecture 9 , simulation 10 , education 11, 12 , quality improvement 13 , safety 14 , and reliability 15 . studies have analyzed systems of work 16 , teamwork 17 , decision making 18 , displays 19 , device interactions 20 , risks 21 , threats 22 , performance shaping factors 23 , environmental and organizational approaches 24, 25 , and regulatory influences 26 . the methods of direct observation, interviews, task analysis, usability studies, human reliability analysis, and incident analysis have been frequently, reliability and creatively applied. however, there is much more that could be done to realize the full potential of hf principles and expertise for patient benefit, staff wellbeing and organizational performance 27 . this has raised a number of questions about how hf knowledge and expertise could be integrated better into clinical work. prior to the application in healthcare, hf knowledge and expertise had arisen mostly from either consumer products and software, or heavily engineered industries 28 . in these industries, human actions are often carried out through the technology, with the hf focus on integrating humans, processes and technologies into a 'top-down' work system with known inputs, goals and constraints. clinical systems are not necessarily like those other industries, though there remains a need design for human use. healthcare is more complex, with multiple different goals and outcomes, a huge variation in inputs, work contexts, procedures and necessary variation in process (to account for individual patient needs). new procedures and technologies are continually being introduced. it was never purposely engineered but has grown organically over thousands of years to meet a range of human needs. every healthcare organization is different, and procedures can vary from provider to provider and patient to patient. how the system works is opaque and it can be difficult to understand what is, or what should be, happening. the particular, and arguably unique, constraints and demands in healthcare mean that while the principles of systems safety science in general, and hf in particular, are similar, their application and mechanisms of effect can be very different. while healthcare also contains many different groups who have a strong interest in improving human work (e.g. hr professionals, organisational development specialists, clinical risk, patient safety and quality improvement advisors), the hf professional can provide much-needed 'added-value' to multidisciplinary team efforts to jointly optimise overall organisational performance and human wellbeing. this paper explores the application of hf in relation to those that apply it; what we need to do to be more effective; how this might fundamentally change both healthcare and hf professions; and the opportunities for service transformation it might present. despite much progress in the application of systems thinking to clinical work, there remains a focus on "error" as a clinical-professional issue to be "fixed" rather than signifying on the deeper causes of error, within the clinical system. instead, the psychology, engineering, sociology and other expertise necessary to address the deep systems problems associated with accidental harms has often been simplified to lookalike principles that sadly missed important details 29 . what might be termed, pseudo-hf approaches have arisen 30, 31 where systems-level thinking is eschewed for well-meaning but limited training, or other attempts at direct behavioral change that apply hf tools but do not consider systems complexity. the hf profession focuses on integrating humans and systems, and brings with it knowledge and experience of a range of concepts, principles, standards and methods. these are arguably better suited to understanding and resolving many of the problems and issues routinely experienced in highly complex, dynamic healthcare systems. consequently, hf professionals can spend significant amounts of time and energy challenging the myths and misunderstandings which continue to prevail, particularly amongst key groups and leaders, and trying to persuade some to unlearn what they think they know about hfe. among the widely-held and stubbornly persistent myths are: • the myth of the individual practitioner. instead of clinical outcomes being driven by a single clinician, they are driven by the collaboration of many individuals across a wide number of disciplines. this myth is perpetuated in, for example, physician league tables, despite clear evidence around the importance of teamwork, and organizations in outcomes 32 . • the myth of the perfect system. when harm happens, the tendency is to believe that this was an exception in an otherwise safe system. however, extensive observational evidence demonstrates that clinical systems are far from perfect 33 and require a wide range of work-arounds and trade-offs every day to be successful 34 .. • the myth that reliable care processes lead to safe systems. safety in complex care situations is an emergent property of the whole system, while reliability is a property of individual system components. every system component can function exactly as intended (reliability) but the complex interactions between components can lead to an unsafe system. 35 . • the myth of 'human error'. as people are always implicated in accidents, there has been an assumption that "removing" human error (or humans), would prevent harm. again, multiple safety scientists demonstrate that this a simplistic view of accident causation as it fails to acknowledge the everyday trade-offs humans in the system are required to make. indeed, it is the ability for humans to vary what they do that is responsible for everyday effective systems function. • the myth of standardization and control of variation. a further perturbation of these myths is the need to control individual human variation, and the ubiquitous call for "standardization". however, given that every patient is different, every unit, every specialty, and every organization is also different, variation is not only required, but is an essential part of patient-centered care. • the "zero" myth. though "zero" harms ("never events") may be useful goals, they are, by their nature, unattainable 29 . instead, risks, costs and the number of patients we treat need to be traded against each other. a system where zero harm could be achieved would have such a huge financial burden, and would have such a profound effect on care delivery, that it would fail in a range of other ways. the risk / benefit considerations and outcomes in response to the recent coivd-19 pandemic are an excellent example of how finances and treatment needs need to be carefully balanced with risks to patients and staff, often in the absence of clear data. • the myth of linear determinism. a further fallacious assumption is that process and outcome are directly related; thus a good outcome means everything went well (and no further attention is needed), while bad outcome must mean something went wrong, which requires investigation. again, safety science demonstrates the inadequacy of these assumptions. many things can and do go wrong in the care of a patient, but few affect outcomes, because the people in the system are always adapting to moment-to-moment demands in ways that technologies, policies and processes do not.. instead of understanding healthcare systems as complex and adaptive, where trade-offs judgements are being made between safety, cost and throughput all the time 36 , the pervading myths have led to the rigid application of approach or tools regardless of the context. the use of quality processes derived from highly engineered systems such as fmea and lean/6 sigma suit some contexts and not others, and at least has required adaptation for healthcare 13, 37 . checklists are a complex socio-technical intervention requiring understanding of the tasks, risks, design and use, so evaluating a checklist only on patient outcomes does not help understand the task, team, organizational or clinical specialty variations necessary for it to be adopted and effective 38, 39 . crew resource management or non-technical skills training is often delivered in the absence of appropriate organizational support, and without a sufficient understanding of the underlying engineering that made those approaches successful in aviation. there is no doubt that all are valuable but fail when applied without consideration of the wider systems in which they will be used. learning from other industries benefits from translation, based on an understanding of the mechanism-of-effect and differing constraints. this is where hf expertise can help. seeing hf as a set of 'tools' that can be 'applied' in a rule-based way, or trained in short course will have narrow benefits, and vastly under-appreciates the value that the discipline can bring. hf principles can be applied across highly diverse contexts, organizations and units, informing both improvement in complex systems, and the methods to hypothesize and evaluate them. the expertise of hf is in understanding the potential effects of interventions, and the selection of the appropriate evaluation from a wide range of methods. some clinicians who practice hf have embraced this more nuanced understanding of hf; some have not; and a few have been deeply resistant to this broader systems perspective. we need to think carefully about how to enhance the enthusiasm for hf, while challenging the range of misperceptions that limit its value. the use of hf tools by clinicians is an excellent place to start and should be encouraged, and supported with higher level hf. then, we need to find ways in which hf professionals and clinicians can work together to bring the right hf principles to bear in the right place, at the right time. despite a growing recognition of the need and value of hf in clinical work across the world, one consequence of the persistent misunderstanding of systems safety science is that few opportunities exist in clinical settings for qualified hf professionals, limiting the availability of appropriately sophisticated hf expertise 27 . until the fda mandated hf as part of device design in 2011 40 , hf healthcare work was funded through research, with much of it based at universities rather than hospitals, on a by-project basis rather than being applied full-time alongside and in support of clinicians. given that return-on-investment can be difficult to calculate, and effect on outcomes difficult to measure in a non-linear system, a direct business case is still hard to make. however, this did not prevent the expansion of quality improvement and patient specialists, who are mostly unqualified in their subject matter area, and lack appropriate professional development and regulation, unlike their hf counterparts. while the fda regulatory demands have vastly increased the number of hf experts within the healthcare industry, and there is much that could be learned from this about the development of and spread of hf expertise, device design is often far removed from clinical practice. professional healthcare hf practice outside of device development, has therefore been disparate, ad-hoc and precarious, with no formal career structure. these professional and financial constraints have also limited the ability of hf professionals to understand "work as done", the complexity of healthcare systems, or the new language and approaches required to address clinical needs. this has created a chicken-and-egg problem, where hf professionals have not been employed in care organizations, because there has been a limited understanding of what they can do, no clear and immediate application, no business case, no clear evidence base, and without day-to-day frontline experience of work-as-done they have not always been effective. a "simple checklist" or " training" are easier to package and demonstrate value, no matter how simplistic (and occasionally thoroughly erroneous 41 ), than a multitude of more appropriate approaches to improvement in complex adaptive systems 42 . this is a significant barrier to progress, but it has begun to change. in the last few years, hospitals across the world have begun to employ clinically embedded hf professionals -that is, fully qualified hf practitioners who work alongside clinicians every day to apply hf methods and perspectives. countries that have embedded hf practitioners include the usa, canada, australia, uk, and portugal, and most likely many others. positions have also been appearing in in regulatory roles. effective applied clinical practice thus requires both the technical demands of understanding when and how to apply hf approaches; and the understanding of the complexity of clinical context of a given application. healthcare organizations need to move beyond the underlying fixation on human errors and a lack of systems thinking. they need to know how they can employ hfe specialists and upskill key parts of the workforce through accredited routes at comparative low cost. they also need to know what sort of hf expertise to employ, and when. while there is formal route for hf professional accreditation (through professional societies such as hfes, ciehf, iea), few clinicians, patient safety administrators or quality improvement specialists who practice have any formal qualifications, training or appropriate cpd around risk/safety/hfe when compared with many of their equivalents in other industrial sectors. clinicians who champion hf causes would benefit from appropriate accreditation, and access to and wider support from qualified hf professionals. they may also benefit from understanding the limitations of and adaptions for popular but limited paradigms such as checklists, teamwork training, or non-technical skills. for example, effective teamwork skills in cardiac surgery are likely to be very different from the teamwork skills in robotic surgery 43 . surgeons or anesthesiologists are often well placed to lead this type of approach, but do not always appreciate how these skills fit into the broader clinical systems picture. broader and equally powerful principles -such as safety-ii, user-centered design, task analysis, selection and training, controls and displays, situational awareness, anthropometry -need to be applied in different ways to solve new problems and / or translate to a new context. they are also continually updating as our theoretical and methodological knowledge improves. in order to be effective at a local level, expertise needs to distributed rather than centralized. rather than working independently, we need to support the application of hf locally with national and international networks. often, clinicians and embedded hf practitioners work alone, or in small groups, often on the same problems as others in other organizations (for example, on clabsi, or failure-to-rescue, or retained foreign objects). there may be common themes, but solutions might look very different in different organizations, or even across different units. the ability to share learning and best practices, many of which will not end up in clinical publications, could have a powerful effect in spreading -and especially translating -good practices for different work contexts. such a network would also support embedded hf professionals who may be otherwise isolated from their peers and from new safety science, practice, or methodological development. there have also been a number of successful organizational approaches to support both clinicians applying hf and hf professionals working in healthcare. most notably the clinical human factors group (www.chfg.org) that originally worked to introduce aviation-style approaches but has subsequently begun to embrace the wider systems view. meanwhile, the uk's chartered institute (www.ciehf.org) has been increasingly bringing together clinicians and professionals work on specific projects, and has defined a set of skills, competence levels and training appropriate for clinical practice. in the usa, the human factors and ergonomics society (www.hfes.org) runs an annual conference devoted to healthcare human factors, while also supporting a specific healthcare technical group (www.hctg.hfes.org). meanwhile, the human factors transforming health network (hfth) (www.hfthnetwork.org) has been developed specifically to support embedded clinical hf practice, through a network of practitioners, clinical champions, and organizations aiming to deploy professional hf expertise. we anticipate increasing alignment of professional hf and clinical hf groups as the two fields continue to integrate. thus, despite the challenges of embedding appropriate, professional expertise into frontline clinical work, there is much to be positive about in the future. the rapid response to the covid-19 pandemic has demonstrated this. the spread of covid-19 exposed a range of challenges with the development, design, dissemination, education, assurance and integration of safe, effective healthcare delivery for a range of clinical contexts. this required substantial adaptation based on complex trade-off decisions between care demands, resources, risks and finances, often without clear authoritative evidence, but from continually evolving information. this can be seen as a 'micro systems' problem, where training based on the availability of specific personal protective equipment (ppe) required day-to-day adaptation by staff, based on varying threats and availability; and it can be seen as "macro" systems issue demonstrating close coupling of acute health, population health, society, commerce and politics. at a micro level, the availability of embedded practitioners in acute care demonstrated a range of ways in which hf expertise can be rapidly applied to help clinicians deal with the problem. it was possible to quickly disseminate this information via the hfth professional network. this work has included training in the use of ppe; designing icons to help with donning and doffing; working to support staff new to icu through decision support tools; prone-positioning protocols and checklists; covid-19 specific daily briefing checklists; supporting virtual visits; developing transportation plans; limiting exposure risks from suspected or confirmed patients; developing new protocols for emergency responses. the uk ciehf rapidly produced guidance on the design of ventilators and the design of work procedures for care teams in all sectors (https://covid19.ergonomics.org.uk/). several professionals have taken key leadership roles in the responses in their organizations. others were invited to be involved, including, importantly, in field hospitals where an influx of excess ventilated patients were/are expected. in most cases, it required the hf professional to actively insert themselves into the work, where their perspectives were valued. at a macro level, practices contributed both to the adaptive flexibility required for resilient systems (for example, by taking the load associated with protocol designs off already stretched clinicians), and to the wider understanding of how resilience can be built into future healthcare systems. this includes information sharing; risk perceptions; risk, cost, and throughput trade-offs; the need for adaptive skills rather than invariant process following; cross-training; planning for adaptability; and even the use of social media in the absence of other clinical guidance. indeed, this demonstrated exactly why the linear deterministic view of healthcare systems -where tasks are standardized, variability is controlled, and deviations are "errors" -is a deleterious over-simplification of healthcare work, even in otherwise 'normal' function. it is people that hold complex systems together; and the goal of is to understand and support the people working in the system to be adaptable and effective, rather than seeing them as a hazard 44 . in short, covid-19 demonstrates that the "new normal" has benefitted hugely from embedded hf expertise, but the key question is how to build on and sustain this welcome development beyond a pandemic situation. the ultimate goal of everyone involved in the application of hf principles in healthcare should be to improve the care we deliver to our patients and the working lives of our healthcare professionals. we need to understand and accept that there are multiple routes in, each with different requirements, and each need to be addressed with appropriate professional and practice support. areas where clinicians and others have applied hf techniques have been most notably in teamwork and teamwork training, simulation training, checklists, education and training, hazard identification and risk assessment, safety culture measurement and discussion, and team-based learning from events (table 1 contains links to some practical resources in these areas). however, a focus on these particular interventions are also symptomatic of the limited nature of application and the persistence of debunked safety science myths. hf professionals, on the other hand, have often struggled to bring their expertise to bear for meaningful clinical benefit. however, organizational, financial and evidence-based pressures are bringing about positive practice changes that is enabling embedded hf professional practice, and closer collaboration with clinical professionals, who now have access to appropriate professional certification and supportive professional networks dedicated to the application of healthcare hf. clinical systems offer vast potential for both specific improvement for patient and staff benefit, and as a natural laboratory for systems safety methodological and theoretical innovation. the interaction of hf and clinical work has already changed both professions. healthcare is at the forefront of hf frontiers; and hf is at the forefront of clinical frontiers. this is an exciting possibility that everyone involved is keen to realize. usa) www.hfes.org healthcare technical group www.hctg.hfes.org human factors and ergonomics society of australia www.ergonomics.org.au international ergonomics association www.iea.cc resilience engineering association www.resilience-engineering-association.org clinical support networks clinical human factors group www.chfg.org human factors transforming healthcare network www twelve tips for hf human factors in healthcare white paper systems thinking safety culture discussion cards 2dlb6c2 from safety i to safety ii: a white paper a human factors subsystems approach to trauma care human factors and ergonomics for primary care human factors in general practiceearly thoughts on the educational focus for specialty training and beyond human factors and simulation in emergency medicine health care human factors/ergonomics fieldwork in home and community settings human factors in medical device design: methods, principles, and guidelines a usability and safety analysis of electronic health records: a multi-center study resilient health care: turning patient safety on its head using an integrative mock-up simulation approach for evidence-based evaluation of operating room design prototypes leveraging health care simulation technology for human factors research: closing the gap between lab and bedside a uk perspective on human factors and patient safety education in pharmacy curricula twelve tips for embedding human factors and ergonomics principles in healthcare education human factors and ergonomics and quality improvement science: integrating approaches for safety in healthcare human factors systems approach to healthcare quality and patient safety building high reliability teams: progress and some reflections on teamwork training work systems analysis of sterile processing: decontamination the effect of teamwork training on team performance and clinical outcome in elective orthopaedic surgery: a controlled interrupted time series study impact of robotic surgery on decision making: perspectives of surgical teams evaluation of user-interface alert displays for clinical decision support systems for sepsis the mixed blessings of smart infusion devices and health care it human factors-based risk analysis to improve the safety of doffing enhanced personal protective equipment system factors affecting patient safety in the or: an analysis of safety threats and resiliency a work systems analysis of sterile processing: sterilization and case cart preparation. structural approaches to resilience and resilience engineering in health care enhancing the effectiveness of significant event analysis: exploring personal impact and applying systems thinking in primary care will human factors restore faith in the gmc? human factors in healthcare: welcome progress, but still scratching the surface world war ii and other historical influences on the formation of the still not safe: patient safety and the middle-managing of american medicine the science of human factors: separating fact from fiction spreading human factors expertise in healthcare: untangling the knots in people and systems the relationship between patient safety culture and patient outcomes: a systematic review improving patient safety by identifying latent failures in successful operations learning from positively deviant wards to improve patient safety: an observational study protocol engineering a safer world -systems thinking applied to safety resilient health care: turning patient safety on its head effect of a "lean" intervention to improve safety processes and outcomes on a surgical emergency unit the problem with checklists participatory design of a preliminary safety checklist for general practice applying human factors and usability engineering to medical devices: guidance for industry and food and drug administration staff changes in safety attitude and relationship to decreased postoperative morbidity and mortality following implementation of a checklist-based surgical safety intervention complexity science in healthcare -aspirations, approaches, applications and accomplishments: a white paper integration of robotic surgery into routine practice and impacts on communication, collaboration, and decision making: a realist process evaluation protocol overcoming covid-19: what can human factors and ergonomics offer? key: cord-016839-cqtpj3m0 authors: ramcharan, robin title: intellectual property and human security date: 2012-08-17 journal: international intellectual property law and human security doi: 10.1007/978-90-6704-900-9_2 sha: doc_id: 16839 cord_uid: cqtpj3m0 this chapter discusses the interrelatedness between intellectual property and human security. there are two sides of this interrelationship. in the first place, ip issues are closely related to the hard security of nations. in the second place, the application of the regime of international intellectual property laws can help promote economic and social development and, at the same time, can result in major hardships when it comes to protection of the right to life and realization of the rights to health, food, and education. in the pages that follow, different aspects of these issues are explored. this chapter takes an in-depth look at the relationship between iprs and human security. it examines the nature of security and the contemporary understanding of the term "security", which now encompasses "human security". whereas the term security had been applied to states traditionally it now encompasses the individual as an object of security. iprs are discussed in the framework of human security, which has placed emphasis on fundamental human rights and the right to development. this chapter discusses the interrelatedness between intellectual property and human security. there are two sides of this interrelationship. in the first place, ip issues are closely related to the hard security of nations. in the second place, the application of the regime of international intellectual property laws can help promote economic and social development and, at the same time, can result in major hardships when it comes to protection of the right to life and realization of the rights to health, food, and education. in the pages that follow, different aspects of these issues are explored. the term "security" is widely accepted as encompassing three levels: individual or human, national and international. 1 the nature of threats have moved well beyond cold war era geo-political concerns of soviet-usa balance of power and 1 see buzan 1983 and ramcharan 2002. no longer can state security be limited to protecting borders, institutions, values, and people from external aggressive or adversarial designs. the spread of deadly infectious diseases, massive forced population movements, human rights violations, famine, political oppression and chronic conditions of deprivation threaten human security and, in turn, state security. 8 a debate has been raging on the confines of the human security concept, since its popularization by the undp's human development report of 1994, about the utility of an expansive definition of human security for theorizing about security. one the one hand, an expansive definition has seen the human security paradigm being applied to a wide range of contemporary problems affecting individuals, communities, states, and global society. these include environmental problems, humanitarian intervention, underdevelopment, small arms proliferation, and so on. on the other hand, theory-inclined scholars have questioned the utility of an expansive definition for the purposes of theorizing about security. some scholars have warned against "overstretch". from a policy perspective, taylor owen, has warned that this was corroding the impact of human security on the un landscape. 9 three approaches to human security have emerged since 1994: (1) a rightsbased approach anchored in the rule of law and treaty-based solutions to human security, that believes that new human rights norms and convergent national standards can be developed by international institutions; (2) a humanitarian conception of human security, according to which the safety of peoples is the paramount objective, and links human security to preventive and post-conflict peace building; and (3) a sustainable human development conception, which draws on the undp's 1994 report. 10 kaldor has distinguished between the canadian government's approach, namely "security of the individual as opposed to the states'" but with primary emphasis on security in the face of political violence 11 and the undp approach. the latter has emphasized the importance of development as a security strategy. 12 a japanese commission on human security (chs) initiated discussions on the "responsibility for development"-freedom from want and human security as development became a topic of the reform agendas at the un and in regional organizations (eu). 13 owen has warned that there has been a failure to distinguish clearly between human development and human security and that there is a lack of distinction between human rights and human security, both of which are detrimental on the un landscape. 14 sorpong peou has warned that we must not make the human security concept too elastic and amorphous. from a political science theory perspective, he has cautioned that scholars must not carelessly combine competing insights from different theoretical perspectives, rendering our arguments unintelligible. "there are limits to eclecticism or pluralism. if possible, clear theoretical statements should be made to allow us to test our theoretical insights against empirical evidence or to keep critically evaluating our normative commitment to human security." 15 in order for human security to be more useful, mary kaldor has argued for a "global conversation" about human security, "the transformation of the social relations of warfare and the character of threats we face." 16 the key to dealing with "new wars is the reconstruction of political legitimacy around the ideas about human rights and global civil society that were reinvented in the last decades of the cold war." 17 kaldor noted that millions of people live in daily fear of violence and new wars were increasingly intertwined with global risks-disease, natural disasters, poverty, and homelessness. her work sought to develop new proposals to address gaps in understanding of "war", which is still influenced by the example of world war i and world war ii. for kaldor, human security is about the security of individuals and communities rather than the security of states, and it combines both human rights and human development. 18 mcfarlane and khong agree with the notion that the individual's security is not subordinate to that of the state and that this pre-dates the 1994 undp report. indeed, they have shown that it is pervasive throughout the international human rights instruments that were drafted during the cold war. 19 however, they limit their definition of human security to protection from violence. this reflects a concern among scholars and policymakers that human security remain relevant and useful for policy making, just as the concept of "national security" has been. "this concern is reflected in glasius and kaldor's attempt to reconcile internal and external security", now held to be inseparable. they sought to define a global security agenda for europe, nato, and the us. 20 they drew upon amartya sen's work on development as freedom and focus on the "downside risks", that is "the insecurities that threaten human survival or the safety of daily life, or imperil the natural dignity of men and women, or expose human beings to the uncertainty disease and pestilence, or subject vulnerable people to abrupt penury." 21 they have contrasted these to an expansive view of human security as human rights as 14 owen 2009 , 3. 15 peou 2009 , 7. 16 kaldor 2007 macfarlane and khong 2006, 10. 20 glasius and kaldor 2006, 3-4. 21 id., 7. suggested by bertrand ramcharan, who served as un high commissioner for human rights, and note that violations of the right to food, health and housing, even grave and massive ones, are not commonly recognized as belonging to the category of jus cogens norms like genocide, large-scale torture, inhuman and degrading treatment, disappearances, slavery, crimes against humanity, and war crimes as defined by icc. 22 the moral case for europe's interest in human security outside its borders was founded simply on 'our common humanity', which posits that human beings have a right to live with dignity and security, and a concomitant obligation to help each other when that security is threatened. it was also founded on the legal consideration that articles 55 and 56 of the un charter enjoin states to promote universal respect for, and observance of human rights. the development and human rights perspectives were two sides of the same coin: both were rooted in the philosophical approach that privileges the search for substantive equality and justice. these stood at the heart of the human rights movement and the attendant international legal regime that guarantee such rights. the commission on human security, in 2003, defined human security as the protection of the vital core of all human lives in ways that enhance human freedoms and human fulfillment. human security meant protecting fundamental freedoms-freedoms that were the essence of life. it meant protecting people from critical (severe) and pervasive (widespread) threats and situations. it meant using processes that build on people's strengths and aspirations. it meant creating political, social, environmental, economic, military and cultural systems that together give people the building blocks of survival, livelihood, and dignity. human security reinforced human dignity. human security complemented state security in four respects: its concern was the individual and the community rather than the state. menaces to people's security included threats and conditions that had not always been classified as threats to state security. the range of actors was expanded beyond the state alone. achieving human security included not just protecting people but also empowering people to fend for themselves. 23 the commission on human security proposed a new framework-a human security framework-to address the conditions and threats people face at the start of the twenty-first century. human security was 'people-centred', focusing the attention of institutions on human beings and communities elsewhere. by placing people at the center, the human security approach called for enhancing and redirecting policies and institutions. human rights and human development had reoriented legal, economic and social actions to consider their objectives from the perspective of their effect of people. recognizing the interdependence and interlinkages among the world's people, the human security approach built on these efforts, seeking to forge alliances that could wield much greater force together than alone. 22 ibid. 23 commission on human security 2003, 4. human security, the commission added, was also concerned with deprivation: from extreme impoverishment, pollution, ill health, illiteracy, and other maladies. catastrophic accident and illness ranked among the primary worries of the poorand understandably, because of their toll on human lives-causing more than 22 million preventable deaths in 2001. educational deprivations were particularly serious for human security. without education, men and especially women were disadvantaged as productive workers, as fathers and mothers, as citizens capable of social change. without social protection, personal injury or economic collapse could catapult families into penury and desperation. all such losses affected people's power to fend for themselves. each menace, terrible on its own, justified attention. yet to address this range of insecurities effectively demanded an integrated approach. 24 human security, in the view of the commission, was deliberately protective. it recognized that people and communities are deeply threatened by events largely beyond their control: a financial crisis, a violent conflict, chronic destitution, a terrorist attack, hiv/aids, underinvestment in health care, water shortages, and pollution from a distant land. to protect people-the first key to human security-their basic rights and freedoms must be upheld. to do so, required concerted efforts to develop national and international norms, processes and institutions, which must address insecurities in ways that are systematic not makeshift, comprehensive not compartmentalized, and preventive not reactive. human security helped identify gaps in the infrastructure of protection as well as ways to strengthen or improve it. 25 as many as 800 million people in the developing world and at least 24 million people in developed and transition countries lived without enough food. these people suffered daily hunger, malnutrition, and food insecurity even though most national food supplies are adequate. the problem was lack of entitlement to food and access to adequate food supply. food insecurity and hunger undermined a person's dignity and well-being. 26 human security, the commission urged, should be mainstreamed in the agendas of international, regional, and national security organizations. 27 the growing inequity between and within countries affected displacement patterns. as long as inequity and imbalances between labor demand and supply were growing among countries, people would continue to seek every opportunity to better their livelihoods. 28 measures to ensure that there was adequate social protection for all, including the working poor and those not in paid work are critical. 29 disease and poverty went hand in hand. so, too, do disease and conflict. 30 good health was both essential and instrumental to achieving human security. it was essential 24 id., 6. 25 id., 11. 26 id., 14. 27 id., 33. 28 id., 44. 29 id., 85. 30 id., 95. because the very heart of security was protecting human lives. health security was at the vital core of human security-and illness, disability and avoidable death are critical pervasive threats to human security. health included not just the absence of disease, but also a state of complete physical, mental, and social well-being. health was both objective physical wellness and subjective psychosocial wellbeing and confidence about the future. 31 one may ask: why 'securitise' intellectual property? this is a logical and natural consequence of the human security agenda of the international community that places individuals at the center of security. objections may come from academics who long for a concept of security that allows for the development of neat theories of national and international security. but the complexity of security studies no longer allows for this, a point made amply clear by the field of critical security studies. 32 the term 'security' injects a sense of urgency into the inquiry and securitization may also perhaps serve as a guide to policy making and allocation of resources. jonathan ban has suggested two analytical tools for thinking about national (threats to the state, national interests, and state power), international (interconnectivity of states' security), and global security (social development, public health, environmental protection human rights, and other such global issues). 33 first, threats can be characterized as either direct or indirect to determine the immediacy or tangential concern for security planners. second, a risk-based approach could provide a framework to characterize the degree to which problems like health concerns represent threats to security. securitization also serves to bring intellectual property into the mainstream of the field of international relations, which is increasingly characterized by feuds over knowledge. in an increasingly globalized world, spearheaded by revolutions in communications technology as exemplified by global internet communication, geo-economic competition between nation-states have become as important or perhaps even more important as trade relations between nations deepen. 34 paradoxically, while freer trade between nations is touted as a means of ensuring that wars become a phenomenon of the past, the deepening of trade relations between nations often leads to ferocious competition between economies as each seeks to preserve its competitive advantage or to protect particular industries. moreover, in the so-called knowledge economy, where information is a prized asset, nations seek to maintain a stranglehold on information, which they perceive as vital to their economic well-being. the protection of intellectual property thus takes on a different dimension when viewed in this light, as it is not only an asset in and of itself, but the protection of state and privately owned intellectual property assets may provide significant competitive advantages to nations. where the well being of one 31 id., 96. 32 see peoples and vaughan-williams 2010 and baylis et al. 2010 for an overview of the field of security studies. 33 ban 2003 , 19-20. 34 sorensen 1990 , bergsten 1990 nation depends on access to technology in another, ip is of vital importance. sadako and cels have noted the fact that many of the poorest countries and people are excluded from technological and knowledge-based advances. in order to meet "the challenges that the current intellectual property rights regime poses to health security requires new thinking about the ownership of knowledge, health as a human right, and effective market and institutional structures to protect incentives as well as lives." 35 clearly, the concept of security has 'broadened' (to include non-military threats) and has 'deepened' (to include security of individuals and groups). the study of security, therefore, encompasses many aspects of human activity. the founding editors of the journal international security (is) noted in the first issue in 1976 that the view of international security taken then was one which embraced "all of those factors which have a direct bearing on the structure of the nation state system and the sovereignty of its members, with particular emphasis on the use, threat and control of force." 36 steven miller, editor in chief of is, noted that he and his predecessors had aspired "to reflect the inherently multidisciplinary character of the field." 37 what then, is the relationship between ip and the security of the individual, the state, and the international community? the concern with national and human security is apparent in some intellectual property treaties. article 27 (1) of the trips agreement stipulates that "patents shall be available for any inventions, whether products or processes, in all fields of technology, provided that they are new, involve an inventive step and are capable of industrial application." according to para 2: members may exclude from patentability inventions, the prevention within their territory of the commercial exploitation of which is necessary to protect ordre public or morality, including to protect human, animal or plant life or health or to avoid serious prejudice to the environment, provided that such exclusion is not made merely because the exploitation is prohibited by their law. carvalho has noted that the rationale for exclusion of patentability on grounds of ordre public or morality is often misunderstood to mean "that patentability should be excluded whenever the technology puts health at risk or offends public morality." 38 following this logic, it would appear that there is a line beyond which research should not cross. the fallacy of this line of reasoning is exposed when 35 one considers that "patents alone are not sufficient to promote technology". indeed, technology will evolve with our without patents. the term "order public or morality" was borrowed from article 53(a) of the european patent convention (epc). 39 the european board of appeals has understood the term to mean "not whether certain living organisms are excluded [from patentability] as such but rather whether or not the publication or exploitation of an invention relating to a particular organism is to be considered contrary to "ordre public" or morality". 40 rather, the board defined the concept of ordre public "as covering the protection of public security and integrity of individuals as part of society. it also encompassed the protection of the environment". accordingly inventions, that would likely seriously prejudice the environment were to be excluded from patentability as being contrary to ordre public. 41 the latter term "is linked to a notion of security, both collective and individual". carvalho has noted that trips article 73, titled "security exceptions", has acknowledged the same concept of security in the light of which "exclusions from patentability do not require any sort of justification or objective test (such as the necessity to prevent the invention's commercial exploitation)". 42 article 73 states that nothing in the trips agreement shall be construed: (a) to require a member to furnish any information, the disclosure of which it considers contrary to its essential security interests; or (b) to prevent a member from taking any action which it considers necessary for the protection of its essential security interests; (i) relating to fissionable materials or the materials from which they are derived; (ii) relating to the traffic in arms, ammunition and implements of war and to such traffic in other goods and materials as is carried on directly or indirectly for the purpose of supplying a military establishment; (iii) taken in time of war or other emergency in international relations; or (c) to prevent a member from taking any action in pursuance of its obligations under the united nations charter for the maintenance of international peace and security. in the same context, a "security exception" is mentioned in article 4 of the patent law treaty (plt) of june 2000, which stipulates that "[n]othing in this treaty and the regulations shall limit the freedom of a contracting party to take any action it deems necessary for the preservation of essential security interests". in the context of the wider scope of national and international security concerns, article 8 of the trips agreement is noteworthy in that it takes into account public health concerns. it stipulates that: 1. members may, in formulating or amending their laws and regulations, adopt measures necessary to protect public health and nutrition, and to promote the public interest in 39 ibid. 40 however, article 8 (2) calls for "appropriate measures" consistent with trips, to be taken to "prevent the abuse of intellectual property rights by right holders or the resort to practices which unreasonably restrain trade or adversely affect the international transfer of technology". a significant aspect of transfer of technology is the publication of technical details of an invention. article 29 (1) of the trips agreement set forth that: members shall require that an applicant for a patent shall disclose the invention in a manner sufficiently clear and complete for the invention to be carried out by a person skilled in the art and may require the applicant to indicate the best mode for carrying out the invention known to the inventor at the filing date or, where priority is claimed, at the priority date of the application. among the genuine and urgent security concerns in recent times is the threat of aids (acquired immune deficiency syndrome). persons afflicted by this and other deadly viruses cannot wait for compulsory licensing schemes or for contracts to be negotiated on favorable pricing schemes as their lives hang in the balance. the commission on human security recognized that the burden of hiv/aids is overwhelmingly concentrated among the poorest people in the poorest regions. hiv/aids decreases the ability of affected individuals to work and increases their health care costs, resulting in greater financial strain on their households. 43 national disease surveillance and control systems should be strengthened and then networked into a global system. health empowerment and protection depend on reliable and up-to-date data and analysis and a capacity to act in response to information. central to health and human security, therefore, are systems to collect and deploy information for detecting disease threats, monitoring their changes, and guiding control efforts. all surveillance and control activities ultimately depend on people and local communities, but national and international systems are needed to empower people and communities. 44 health and human security are central matters of human survival in the twentyfirst century. knowledge and technology can make a difference. the challenges are to make tools and knowledge accessible while promoting incentives and 43 commission on human security 2003, 99. 44 id., 104. structures for the production of new knowledge. social action was needed to deploy that knowledge for health and human security. 45 education and knowledge may enable groups to identify common problems and act in solidarity with others. four priorities for action are promoting a global commitment to basic education; protecting students' human security at and through school; equipping people for action and democratic engagement; teaching mutual respect. 46 access to information and skills allowed people to learn how to address concerns that directly affect their security. knowledge, education, and democratic engagement were inseparable-and essential. free and diverse information media can provide individuals with the knowledge required to exercise their rights and to influence-or challenge-the policies of the state and other actors. 47 there is an urgent need for institutional arrangements to make inexpensive and affordable generic drugs available to the developing countries that need them most. community-based health initiatives, community-based health care, and selfinsurance schemes are fundamental to this progress. the world urgently needs primary health services and national disease surveillance systems. it is important to develop an efficient and equitable system for patent rights. global flows of knowledge and technology are increasing under the wto. in november 2001, the wto's doha ministerial declaration recognized the challenges facing developing countries. a number of important drugs do not have patent limitations. but for those that do, current international rules governing intellectual property leave many of the poorest people in the world unable to use the drugs. because so many lives were at stake there was an urgent need for institutional arrangements to make inexpensive and affordable generic drugs available to the developing countries that need them most. developing countries that currently export generic medicines-such as brazil, china, and india-were obliged to comply by january 2005 with the wto requirements that generic medicines be used domestically only. they cannot be exported, even to other countries with similar emergencies that may not be able to produce medicines on their own. if a country has insufficient manufacturing capacity to produce medicines domestically, it will have to rely on expensive patented medicines for health needs-unless the rules are changed. on the positive side, the wto has recognized public health emergencies as requiring special provisions. the doha round affirmed the rights of governments to grant 'compulsory licenses' allowing the domestic production of essential medicines, when they are covered by patent, and to purchase 'parallel imports' from legitimate international sources during national emergencies, including the hiv/ aids pandemic. further the ministers at doha agreed that the least developed countries would not be required to offer patent protection on pharmaceutical products 45 id., 109. 46 id., 116. 47 id., 120. 2.3 balancing public and private rights: intellectual property and human security until 2016. because many poor countries did not have sufficient manufacturing capacity, their exercise of compulsory licensing and parallel imports depends on international sources. if other developing countries cannot export essential emergency medicines and vaccines under the wto, the exercise of emergency measures will be nominal, not real. the doha round of trade talks is not yet completed 10 years on. moreover, matthew kennedy noted the slow pace of acceptance of the protocol amending the trips agreement (2005) that would allow the doha agreements to come into effect. 48 according to the commission on human security, three challenging issues that needed to be resolved were the following: clarifying the definition of "insufficient manufacturing capacity"; allowing companies in one country to export inexpensive generic drugs still under patent to other countries; and deciding on the measures necessary to prevent the re-export of drugs manufactured under compulsory licenses back to the developed world. a major objective was to have intellectual property rights systems that advance human security through the efficient development of appropriate drugs and the facilitation of their extensive use. any resolution of the current impasse should involve favoring flexibility and overcoming import and export controls on the drugs and vaccines needed for emergencies. a balance was required in order to provide incentives for research and development for both profitable products and technologies to fight diseases of the poor. that balance should also provide equitable access to life saving essential drugs and vaccines for people unable to purchase technologies from the global marketplace. the balance should recognize the very large public investments in basic research that underlie product development by all manufacturers, including private ones. 49 in the context of such concerns, it is not surprise that some developing countries have enacted laws to deal partly with such situations. in egypt, article 25 of the patent law stipulates that the state may expropriate a patent for national security reasons and in cases of extreme urgency. 50 in tunisia, its patent law of august 2000 has provided in article 78, para 5, that the state may avail itself of an ex-officio license for defense and national security reasons for the exploitation of an invention. 51 such exploitation may be undertaken by a third party on behalf of the state. in morocco, a law on the protection of industrial property sets forth in article 75, that the state may be granted an ex-officio license for the exploitation of an invention for national defense and that third parties may undertake such exploitation for the state. given the expansive definition of human security that is found in the literature and recognition that national and human security are interconnected, one may take note of the direct or indirect linkages between intellectual property and national and global security, which have been explored by this author in an earlier work. 53 for example, in an age when weapons of mass destruction and their potential use by non-state actors has become a major concern, we argued that careful attention must be paid to the patent regime and the information that is available through the same. information contained in a patent application enters the public domain once the patent is granted, and thus becomes an invaluable source of information on the state-of-the-art in any given field. these documents are easily searchable by any government, corporate entity, or individual and they constitute an important means/source of transfer of technology. transfer of technology is defined as a "matter of how items used in one area of activity or in one place, can be applied and used in others". 54 such a transfer refers to products but also includes, according to molas-gallart, "a broader concept encompassing the social relations and the "mode of production" in which the development and production of artifacts occur". information can be retrieved through the international patent classification (ipc) system, which is based on the strasbourg agreement concerning the international patent classification, a wipo-administered international treaty concluded in 1971, that entered into force in 1975. the ipc is a hierarchical classification system covering all fields of technology that is indispensable for efficient retrieval of patent information. wipo has promoted the use of the ipc since: the amount of information contained in patent documents is immense. they contain practically everything that represents an advance in the knowledge of mankind in the field of technology. it is therefore extremely important that this information be accessible to anyone who needs it. such accessibility exists in theory because the patent documents are published, that is, are made available to any member of the public. 55 in relation to trade secrets, it was argued that in light of concern over the national and international security implications of trade secrets (confidential information which is the object of economic espionage) a balance must be struck between the legitimate public concern for security and the legitimate rights of the inventor. this calls for an honest distinction between genuine security concerns and non-genuine security concerns. in a climate of concern for terrorism and the threat of wmd, excessive controls on the publication of information may inadvertently serve the cause of terrorists who seek to disrupt normal commercial, economic, social, and political intercourse in society. other global security vulnerabilities may be added to this discussion, including social development (poverty and its impact on state security), human rights and 53 ramcharan 2005. 54 molas-gallart 1998. 55 wipo 2000. environmental challenges, and transborder public health issues. these are addressed briefly in chap. 4 and elsewhere in this work. climate change scientists have called attention to a fast-approaching point of no return that would herald catastrophic consequences for the earth's climate, and thus human life in the next 50-100 years. in terms of 'immediacy' one may highlight the global nature of the security challenges posed by health. the un secretary general's agenda for peace, which took stock of "new risks for stability", had explained that "drought and disease can decimate no less mercilessly than the weapons of war". 56 jonathan ban has argued that the question is not whether some health challenges generate risks that have implications for security but, rather, to what degree do the various health challenges pose risks and have security implications. using the 'direct' versus 'indirect' categorization scheme, he has noted that direct security involves risks that relate more to traditional aspects of security, such as biological attacks, attacks on medical personnel facilities and supplies by combatants in a conflict, and threats to the health of military personnel, peacekeepers or deployed contingents because of infectious diseases. indirect threats, such as hiv/ aids and sars (severe acute respiratory syndrome, which led to international crisis response in 2001 and 2002, may carry less risk than direct threats). they nevertheless "have the potential to impact national and international security and should not be excluded from traditional national security considerations". 57 the un security council convened a meeting in january 2000 to discuss aids. the us national intelligence council produced a report on "the global infectious disease threat and its implications for the united states" in january 2000. in april 2000, the clinton administration announced that it formally recognized aids as a threat to us national security. this was later enshrined in the us national security strategy of 2007. 58 security is as much real as it is about perceived threats. the nature of the threats faced by individuals, nations and the international community, has changed dramatically. the end of the bipolar cold war superpower rivalry has seen greater economic interdependence as more parts of the world are effectively integrated into the world economy. in an increasingly technologically and economically interconnected world, interdependence causes occurrences in one part to impact directly upon individuals and nations in another, and sometimes the impact is immediate and devastating. the national security of a state exists symbiotically with its economic well-being. nations seek to protect scarce resources of which intellectual property assets are a key component. for technologically advanced states it is the specter of lost capital, jobs, and especially military advantage, which are worrisome. in the post-cold war era, the quest for technological and economic supremacy is raging among china, the eu, 56 boutros-ghali 1993 . 57 ban 2003 see national intelligence council, the next wave of hiv/aids: nigeria, ethiopia, russia, india and china. ica 2002 -04 d, september 2002 , footnote 14, ban 2003 india, japan, and the usa while russia was trying to regain its soviet-era grandeur. a larger strategic competition between big powers is evidenced, for example, in the close monitoring by the us of transfers of sensitive technologies. of special concern to the us is china. 59 for the less technologically advanced states and especially the world's least developed countries the success of their quest to acquire knowledge and new technologies that they can absorb into their economies may make the difference between life and death. in this chapter, we have reviewed the literature on human security and noted instances in which there is a direct relationship with international intellectual property laws. we would conclude this chapter with a simple point: it must be right to argue that international intellectual property laws should seek to protect human security and advance human welfare across the globe. this is the basic thrust of this book that we take forward next by looking at the fundamentals of the international intellectual property law regime. national security and international affairs division. gao/t-nsiad-98-211 us commercial technology transfers to the people's republic of china key: cord-021770-zn7na974 authors: slifka, mark k.; amanna, ian j. title: passive immunization date: 2017-07-17 journal: plotkin's vaccines doi: 10.1016/b978-0-323-35761-6.00008-0 sha: doc_id: 21770 cord_uid: zn7na974 nan passive immunization, passive immunity, and passive immunotherapy all refer to the transfer of antibodies to an unprotected individual for the prevention or treatment of disease. the first formal demonstration of passive immunization for successfully treating diphtheria and tetanus dates back to animal studies published in deutsche medizinische wochenschrift (german medical journal) in 1890. 1 the technique was quickly adapted to clinical use and as early as the mid-1890s, diphtheria-specific antitoxin was used successfully in the hospital setting to reduce mortality during diphtheria outbreaks. [2] [3] [4] indeed, in 1901 emil von behring was awarded the first nobel prize for physiology or medicine for the discovery of this important medical intervention. 5 the significance of this clinical advance cannot be overstated; behring estimated that 45,000 lives were saved each year using diphtheria-specific passive immunotherapy in germany alone. 6 in the 1890s, the mortality rate of hospitalized cases ranged from 47% to 60%, 7 and the work of emil von behring and his colleague, shibasaburo kitasato, provided the only hope for diphtheria patients in the preantibiotic era. according to behring, the discovery of passive immunization would not have occurred if it were not for his earlier work that focused on characterizing the protective mechanisms of active immunization against diphtheria 5, 8 and through the work of his collaborator, kitasato, on the mechanisms of vaccine-mediated immunity against tetanus. 1 when guinea pigs were infected with corynebacterium diphtheriae, the animals routinely died of the disease. however, when behring vaccinated animals and they mounted neutralizing antibodies to diphtheria toxin, he found that they were protected from a normally lethal dose of c. diphtheriae. to determine if protection was now an intrinsic property of the immune host that could be transferred to a susceptible host, he injected naïve guinea pigs with diphtheria toxin and then successfully treated them with immune serum from vaccinated animals. likewise, injection of clostridium tetani or purified tetanus toxin was typically lethal, but through a method developed by paul ehrlich, 5 animals could eventually become immune to high doses of tetanus toxin by sequentially inoculating them with lower, nonlethal doses of tetanus toxin. kitasato used this approach to demonstrate that the blood of vaccinated, tetanusimmune rabbits could be transferred to naïve mice and fully protect them from a normally lethal dose of virulent c. tetani or from filtered c. tetani culture supernatant containing tetanus toxin. 1 behring and kitasato may have said it best in the final sentence of their landmark 1890 study, "the result of our experiments remind us forcibly of these words: blut ist ein ganz besonderer saft [blood is a very unusual fluid]." 1 technology has advanced substantially in the more than 125 years since behring and kitasato's first formal demonstration of protective passive immunotherapy. 1 in those early days, it was infeasible to use human immune serum to treat diphtheria, so the first large-scale production of polyclonal diphtheria-immune serum was prepared by vaccinating dairy cows. 5 to this day, commercial antisera used to treat a broad range of toxins are still produced in animals (table 8 .1). passive immunotherapy with animal-derived antibody preparations should only be used under close medical supervision 9 or the resulting host immune response to the foreign 8 immunoglobulins and serum proteins may trigger serum sickness, urticaria, and/or anaphylaxis following administration. fortunately, the advent of several innovative technologies that reduce the need for animal-derived antibodies have forged new paths in terms of safety, feasibility, and the protective efficacy afforded by passive immunization. following the discovery of monoclonal antibody technology, 10, 11 further refinements have been made, including use of various display techniques (e.g., phage display, yeast display) to screen large antibody libraries. 12 other technological advances include the development of chimeric monoclonal antibodies in which the murine antibody is "humanized" by genetically replacing the heavy chain region of the molecule with the human immunoglobulin counterpart and the use of transgenic mice in which the endogenous murine immunoglobulin genes have been replaced by human immunoglobulin genes. 12 this latter approach has the advantage that hybridomas from immunized transgenic mice produce fully human monoclonal antibodies without requiring further genetic modifications. recently, development of epstein-barr virus (ebv)-transformed human memory b cells for the production of monoclonal antibodies has led to yet another surge in the production of new human monoclonal antibodies with rare antigenic specificities to uncommon pathogens and these can be produced directly from immune human subjects. 12, 13 before the era of antibiotics, antibodybased therapy was the only option available for combating many bacterial diseases. even today, there are only a handful of antiviral drugs available and no therapeutic options exist for most viral diseases. however, new antibody-based therapies are continuing to be developed with the potential to provide protection against a broad array of bacterial and viral pathogens. in this chapter, we describe the role of passive immunity in the protection of the naïve host, discuss the parameters involved with successful immunotherapy, and provide examples of protective efficacy in animal models as well as in human clinical studies. age who were born to mothers who received pertussis vaccination during pregnancy, 30,31 thus lending further support to the current recommendations for the vaccination of pregnant mothers against b. pertussis. 32 the age limit of younger than 2 months was chosen as this is the age at which primary pediatric vaccination is recommended and analysis beyond this age might be confounded by the protective effects of direct vaccination of the child. nevertheless, the protection afforded by maternally derived igg against respiratory infections involving viral (e.g., influenza) or bacterial (e.g., b. pertussis) pathogens together demonstrate the broad impact that maternal vaccination and the subsequently increased transfer of maternal antibodies can have on the health of young infants. before vaccines and antibiotics revolutionized modern medicine, antibody-based therapies represented the only effective medical treatment for many life-threatening diseases including diphtheria, scarlet fever, bacterial meningitis, and bacterial pneumonia. 33,34 today, most commercial forms of antibodybased immunotherapy for infectious disease still rely on polyclonal antibodies of human or animal origin, with the notable exceptions of the monoclonal antibodies, palivizumab and raxibacumab (see table 8 .1). the main advantage of using polyclonal antibodies for passive immunotherapy is that this approach will include antibodies to multiple epitope specificities that may work in an additive or synergistic manner with the potential contribution of multiple immunoglobulin isotypes and subclasses that have different biological functions (table 8. 2). 35 on the other hand, there are several potential challenges to using polyclonal antibodies for immunotherapy including low antigen-specific activity, supply limitations (especially for rare diseases), variability between manufacturing lots, and safety as well as quality control issues that are often associated with the use of human blood products. in ungulates. these differences also indicate that care should be taken when choosing an appropriate animal model for studying the role of maternal antibodies against infectious disease as the mechanisms may be more species-specific than typically realized. in a comprehensive study involving the analysis of antibodies to 16 viruses using samples from 58,500 patients, the relationship between maternal immunity and infant immunity is clear (fig. 8.1 ). 15 the prevalence of antibodies to each viral antigen among infants less than 1 month old is remarkably similar to those observed in the 20-to 40-year old adults who represented the main age group of the mothers. for instance, immunity to common childhood diseases such as measles and mumps was comparable between newborns and their mothers. immunity to less-common viral pathogens, such as influenza b, was relatively low among infants and adults in the cohorts examined in 1971-1972, but higher among those sampled in 1973-1974,1975-1976, and 1977-1978 , coinciding with an influenza b epidemic that had occurred in 1974. 15 this shows that the prevalence of maternal antibodies is dynamic and that recent outbreaks involving a specific pathogen will result in a higher frequency of pathogenimmune mothers and a concomitant increase in the number of infants who are likewise bestowed at least transient immunity to that particular microbe. as expected, maternal antibodies wane rapidly during the first 6 months of life and then exposure to pathogens over the following months and years results in an accumulation of different antibody specificities as children reach adulthood (see fig. 8 .1). the overall protective efficacy of maternal antibodies is perhaps most pronounced among children with genetic immunodeficiencies such as severe combined immunodeficiency (scid), resulting in the lack of functional t and b cells or agammaglobulinemia, in which patients lack functional b cells while still having the ability to mount pathogen-specific t-cell responses. the clinical presentation of scid is not apparent at birth but relatively uniform diagnosis occurs at a mean of 6.59 months of age, 16 which is also about the age that maternal antibodies have reached their lowest levels 15 (see fig. 8 .1). likewise, agammaglobulinemic patients also begin to present with symptoms of immunodeficiency around this same age. 17 maternal antibodies represent an immunological "doubleedged sword" in the sense that they are known to interfere with live attenuated virus vaccines such as the mmr (measles, mumps, rubella) [18] [19] [20] and rotavirus vaccines, 21, 22 whereas direct immunization of mothers in the third trimester of pregnancy can significantly increase protection of infants against common respiratory viruses such as influenza. [23] [24] [25] indeed, maternal vaccination may result in a 45% to 91% reduction in influenzarelated hospitalizations among infants younger than 6 months of age. [23] [24] [25] likewise, the importance of maternal vaccination against bordetella pertussis (i.e., whooping cough) was recognized as early as the 1930s to 1940s with studies showing higher antibacterial antibody responses and potential protection from exposure to whooping cough among infants born to vaccinated mothers. [26] [27] [28] [29] recent studies verify these earlier results, demonstrating a 90% to 91% vaccine efficacy against whooping cough among infants younger than 2 months of nonlymphoid tissues and to penetrate mucosal sites of infection is likely to explain why it is often considered the best immunoglobulin isotype for routine passive immunization and has shown clinical benefit ranging from reduced clinical symptoms to nearly complete protection from lethal infection in a number of infectious disease models (table 8 .3). over the last century, it has been well established that high specific antibody titers and early timing of antibody transfer in relation to disease onset are the two most important parameters involved with determining the protective efficacy of passive immunization ( fig. 8.2 ). in one account of the early days of clinical diphtheria-specific immunotherapy developed by behring and ehrlich, 5 initial failures in patients after treatment with weak or unstandardized diphtheria-immune serum brought ehrlich to describe three points that he believed were important for successful immunotherapy: (a) treatment has to be initiated at the onset of disease; (b) the more the disease has progressed, the higher the serum quantities necessary for cure; and (c) depending on the severity of the case, certain minimal doses can be specified. later studies confirmed these results: if diphtheria immunotherapy was initiated on the first day of disease, there was 0% mortality (n = 183). 49 however, if therapy was delayed to 2, 3, or 4 days after disease onset, then the accompanying diphtheria case-fatality rate subsequently increased to 1.6% (n = 905), 4.4% (n = 632), and 6.9% (n = 436), respectively. 49 these results are similar to those observed during antibiotic-based therapy of bacterial sepsis. in an ideal setting, it is recommended that antibiotics be administered within 1 hour of diagnosis of severe sepsis or septic shock as these drugs provide clinical benefit only if administered early in the course of disease and are generally ineffective during late-stage disease. 50 the importance of high-dose immunotherapy given at the earliest sign of disease is not unique to bacterial anti-toxin therapy. the same rules apply to preventing or treating viral infections as well. during a measles epidemic in 1931-1932, 72% of exposed individuals (n = 32) who received no passive immunization contracted measles. if convalescent serum was administered within 10 days of exposure, then the attack rate was reduced to 16% (n = 219) whereas if therapy was not initiated until 12 to 16 days postexposure, approximately 80% of contrast, monoclonal antibodies are, by definition, limited to a single epitope specificity but they have several advantages over polyclonal antibodies since they can be manufactured in vitro at large scale, with inherently high specificity and lot consistency (table 8 .2). for example, the combination of 0.7 mg of two tetanus-specific human monoclonal antibodies has the same neutralizing capacity observed with administration of 100 to 170 mg of polyclonal tetanus immunoglobulin. 36 likewise, administration of 0.023 mg of a vaccinia virus-specific monoclonal antibody provides the same level of protection afforded by 5 mgs of vaccinia immunoglobulin (vig). 37 although neutralization escape mutants are a valid concern when using monoclonal antibody therapy, 38,39 this has not yet been a major problem during clinical use of palivizumab for respiratory syncytial virus (rsv). initially, sequencing of 371 rsv isolates demonstrated that there were no mutations in the neutralizing epitope of the f protein. 40 subsequent studies identified rsv escape mutants in approximately 5% of 146 breakthrough cases, indicating that selective pressure for escape mutations is still relatively uncommon under current conditions of use. 41 this suggests that monoclonal antibodies can remain effective when used clinically in the long-term, as long as they are specific for a stable epitope for that particular pathogen. the functional characteristics of the immunoglobulins used for passive immunization is an important consideration in determining protective efficacy in vivo. 35 for example, serum igg molecules equilibrate into extravascular space whereas igm is largely confined to intravascular space. 14 igm molecules also have a short half-life (5 days 14 ) and are typically of low affinity, which is why igm is not an optimal choice for passive immunotherapy. serum iga is monomeric and, although it also equilibrates into extravascular space, 14 it has only a 6-day half-life 14 and does not appear to contribute significantly to functional iga in the lungs of mice. 42,43 human igg on the other hand, has an average half-life of approximately 21 days (except igg 3 , which has a 7-day half-life), 14, 44 is typically of high affinity, and transudation across mucosal barriers can protect against pathogens that invade through mucosal routes. interestingly, serum igg (and serum iga) responses elicited in response to vaccination against neisseria meningitidis correlate strongly with the levels of antibacterial antibodies present in the saliva at 1 month and 1 year after vaccination, 45 indicating that circulating serum antibodies may be an important contributor to the antibodies released in mucosal secretions. indeed, after intravenous administration of an hiv-specific monoclonal antibody into rhesus macaques, serum antibody titers of 690 to 725 µg/ml resulted in mucosal antibody titers of 17 to 30 µg/ml in vaginal fluids and provide complete protection against intravaginal challenge with shiv (chimeric simian immunodeficiency virus expressing hiv envelope). 46 influenza virus is another mucosal pathogen with strict tropism to the respiratory tract, but influenza-specific serum antibody titers correlate with protection in humans. 47 in mice, the relative roles of influenza-specific polymeric iga and igg were compared in terms of antiviral protection in the upper respiratory tract versus the lung after influenza challenge. 42 when polymeric iga was transferred 4 hours prior to influenza infection, this prevented pathology in the upper respiratory tract but was not effective in the lung, whereas transfer of igg prevented pathology in the lung, but required higher doses to protect against infection of the upper respiratory tract. the authors concluded that different antibody isotypes may function preferentially at different anatomical sites in vivo. these results are in contrast to experimental influenza infection in humans in which inactivated influenza vaccinederived igg is believed to be a major contributor to protection of the nasal compartment. 48 overall, the ability of igg to enter efficacy of passive immunity decreases with disease progression. full protection from symptomatic disease is best achieved through prophylactic administration of antibody therapy prior to exposure or infection. however, antibody therapy may also be highly effective at early points postexposure, prior to the onset of disease symptoms. passive immunity is generally less effective when administered after the onset of symptomatic disease, and typically shows little to no clinical benefit once severe late-stage disease has occurred. to 16 iu/ml, the postexposure incidence of measles increased from 17% to 57% despite either lot being administered within 5 days of exposure. 53 likewise, the timing of passive immunotherapy is also important for enteric (e.g., polio) and respiratory pathogens (e.g., rsv). an outbreak in 1934 involving 2992 polio patients showed that if convalescent serum was administered within 0 to 2 days of meningitis, then paralysis was reported in 5.4% of patients (n = 2367). if treatment was delayed until 3 to 6 days after meningeal disease onset, 15.5% reported paralysis (n = 536), and if treatment was delayed for more than 6 days, then paralysis was noted in 30.3% of polio patients (n = 89). 54 for rsv, polyclonal rsv-immunoglobulin reduced the incidence of rsv-associated hospitalization by contacts subsequently contracted measles (n = 5). 51 in a study published in 1945 involving 1024 cases of measles exposure, 36% of the individuals who received immunotherapy within 0 to 2 days of exposure contracted measles compared to 48% for those whose treatment was delayed to 6 to 8 days postexposure. 52 the dose used in these studies was also critical: 67% of patients who received 0.01 ml/kg of gammaglobulin contracted measles whereas only 16% of patients who received 0.06 ml/kg of gammaglobulin contracted the disease. the titer of virus-specific antibodies will often differ between lots of polyclonal immunoglobulin preparations (see table 8 .2). in another study, when the measles-specific titer of gammaglobulin from different lots decreased from 33 iu/ml for animal studies, prophylaxis is defined as antibody administration prior to experimental infection and treatment is defined as antibody administration after infection. for clinical studies, prophylaxis is defined as antibody administration prior to disease onset and treatment is defined as antibody administration after disease onset. b evidence provided through maternal immunization studies. c anecdotal results are defined as small studies that indicate passive immunization may provide clinical benefit but are too limited in scope to be conclusive. d not available, although two studies 433,434 demonstrated prophylaxis by direct mixing of mumps virus and antibody prior to inoculation. confirmed lassa fever who received immune serum within 10 days of hospitalization survived (4 of 4; 100%). however, if treatment was not initiated until more than 10 days after hospitalization, then only 1 of 4 (25%) patients survived, similar to the untreated group in which only 1 of 5 (20%) patients with virologically confirmed lassa fever survived. although passive immunity against toxins and systemic infections such as measles 51,53,80-84 and smallpox [64] [65] [66] is well established, the impact of this approach for the prevention or amelioration of disease caused by respiratory and enteric pathogens may not be as well recognized. however, several studies support the role of passive immunity against mucosal pathogens (see table 8 . 3) , including examples such as influenza (respiratory virus), haemophilus influenzae (respiratory bacterium), rotavirus (enteric virus), and escherichia coli (enteric bacterium). influenza is a significant cause of morbidity and mortality throughout the world, including both seasonal transmission and pandemic outbreaks. [85] [86] [87] [88] the clinical correlation between homotypic influenza immunity and vaccine-associated protection was recognized early in the development of the influenza vaccine. [89] [90] [91] early animal studies confirmed this result, with passive transfer of antibodies (both systemic and mucosal delivery) able to protect naïve animals against subsequent challenge, or provide therapeutic benefit when administered postexposure. [92] [93] [94] more recent animal studies with defined monoclonal antibodies continue to support and extend these earlier results. 95,96 passive immunization against influenza in humans has also been successful. in a comprehensive retrospective metaanalysis of eight passive immunization studies performed during the spanish influenza outbreak (1918) (1919) (1920) (1921) (1922) (1923) (1924) (1925) , a significant 21% decrease in mortality (95% confidence interval [ci], 15-27%; p < .001) was observed. 97 subset analysis of studies that recorded early (treatment initiated within 4 days of pneumonia complications) versus late intervention (>4 days) showed a significant advantage for early treatment, with mortality decreasing from 59% (49 of 83) to 19% (28 of 148) with earlier intervention, consistent with general considerations for effective passive immunity against infectious diseases (see fig. 8 .2). in a recent double-blinded, randomized controlled study during the 2009 influenza pandemic, the use of hyperimmune intravenous immunoglobulin (ivig) (from recovered convalescent donors) was compared to normal ivig in the treatment of severe infection in 34 subjects. 98 the hyperimmune treated group (n = 17) demonstrated more rapid viral clearance than the control group (n = 17), with a greater than 90% drop in viral loads by day 5 posttreatment. in those patients receiving immunoglobulin within 5 days of symptom onset (n = 22), all 12 who received hyperimmune ivig survived (12 of 12), whereas only 60% of patients receiving normal ivig survived (6/10, p = .02). h. influenzae type b (hib) is an extracellular gram-negative bacterium that initially infects the host via the respiratory tract and represents another important human pathogen that can be controlled through passive immunization. several early reports described the use of concentrated rabbit immune serum as a successful adjunct therapy to sulfonamide treatment for patients suffering from hib meningitis. 99-101 indeed, a full course of serum therapy (in addition to antibiotics) was able to reduce mortality to 14% (3 of 19) when compared to 78% mortality rate (7 of 9) in those patients only receiving sulfonamides. 101 a more recent study established the prophylactic 41% among children with a history of prematurity or bronchopulmonary dysplasia. 55 prophylactic administration of a neutralizing monoclonal antibody, palivizumab, was shown to significantly improve clinical outcome by reducing rsvassociated hospitalizations of children with congenital heart disease by 45%. 56 among premature infants or those with bronchopulmonary dysplasia, rsv-associated hospitalizations were reduced by 55%. 57 a third palivizumab study confirmed these results by showing a 70% reduction in hospitalizations among premature infants and infants with chronic lung disease. 58 another monoclonal antibody, motavizumab, 59 demonstrated a further 26% relative reduction in rsv hospitalizations compared with patients receiving palivizumabbased prophylaxis. 60 in contrast, once rsv infection has been established, the use of palivizumab, 61 motavizumab, 59 or rsvimmunoglobulin 62 shows no clinical benefit, although rsvimmunoglobulin may provide limited protection in the most severe cases. 62 passive immunotherapy can be highly successful for severe, even life-threatening human diseases such as smallpox, or hemorrhagic fever caused by arenaviruses including junin or lassa fever virus (see table 8 .3). successful intervention, however, typically requires initiating treatment before or very shortly after symptom onset. when convalescent serum from smallpox survivors was administered to smallpox patients during the late stages of confluent or hemorrhagic smallpox, there was no clinical benefit observed in comparison to untreated controls (80% vs. 72% mortality, respectively). 63 when vaccinia-immune gammaglobulin (vig) was administered to smallpox contacts prior to disease onset in addition to postexposure vaccination (i.e., standard of care), the number of smallpox cases was reduced by 70% compared to contacts who received postexposure smallpox vaccination alone. 64 likewise, administration of vacciniaimmune serum of animal origin along with postexposure vaccination resulted in 0 of 13 cases (0%) of smallpox among close contacts compared to 13 of 29 cases (45%) among controls who received smallpox vaccination alone. 65 during a smallpox outbreak in 1941, 3 of 10 patients (30%) died while undergoing standard clinical care. 66 to determine if addition of passive immunotherapy would reduce mortality after smallpox diagnosis, 250 cases of smallpox were treated with convalescent serum or blood, with no smallpoxassociated deaths reported (0 of 250). approximately 75 patients were described as having severe or hemorrhagic smallpox at the time of treatment and yet all survived. this appears to be the result of using convalescent serum obtained at the peak of the humoral immune response shortly after recovery from smallpox and the use of an optimized dosing schedule with higher doses administered to patients with the more severe disease manifestations. 66 argentine hemorrhagic fever is caused by infection with the junin virus and untreated cases result in 15% to 40% mortality. [67] [68] [69] convalescent serum is protective in animal models of junin infection [70] [71] [72] and when administered within 8 days of symptom onset, the mortality rate among human cases drops to 1% to 3%. 68, 69 likewise, in 35% to 50% of hospitalized cases, lassa fever virus causes severe disease including diffuse capillary leakage and hemorrhagic diathesis. 73 prophylactic administration of immune serum protects guinea pigs 74, 75 and nonhuman primates 76,77 from subsequent lethal challenge, indicating that antibodies play a clear role in protection against this virulent viral pathogen. in one small clinical study, if passive immunotherapy was administered within 0 to 5 days after admission to the hospital, 4 of 4 (100%) patients survived whereas if immunotherapy was initiated 7 to 9 days after hospitalization, 0 of 3 (0%) patients survived. 78 in another study, 79 patients with virologically with any new scientific advance, there is controversy. in 1890, when behring demonstrated that immune serum therapy could protect against diphtheria, it went against the current dogma at that time in which the cellular theory of phagocytosis was believed to be the primary mechanism of host protection. 5 there were also skeptics who, as early as 1896, discussed why antibody immunotherapy would not work. 114 however, the science not only prevailed but today a number of passive immunotherapy products are in clinical use (see table 8 .1) and an ever-increasing number of human diseases benefit from the use of this technology (see table 8 .3). some believe that antibody plays a more important role in protection against cytopathic viruses and extracellular bacteria, but that t cells must be required for protection against infection by noncytopathic viruses and other intracellular pathogens. 115 although this is partially refuted by the protective efficacy of maternal antibodies and ivig therapy in scid patients who do not have functioning t cells, it is important to bear in mind that antibody-mediated protection by passive immunotherapy in immunocompetent individuals does not function in isolation, but instead works best in conjunction with other immune defenses, including host t cells, b cells, natural killer (nk) cells, etc. although the role of antibody-mediated protection against intracellular bacteria and chronic viral infections was thought to be relatively minor, there are examples in each of these instances in which passive immunity provides substantial clinical benefit. as noted previously, prior to the advent of antibiotics, passive immunotherapy was the only option for clinical treatment of most bacterial infections including francisella tularensis, a facultative intracellular bacterium that causes tularemia, a severe disease associated with up to 30% mortality in untreated cases. 116,117 when streptomycin became available, a comparative study in 1946 was performed with 542 tularemia patients who received only symptomatic treatment, 832 who received immune equine serum, 60 who received hyperimmune equine serum, and 9 who received streptomycin. 118 the untreated tularemia cases required an average of 3.78 months to recover and only three modes of therapy showed substantial improvement-treatment with immune serum within 9 days of disease onset (2.41 months until recovery), treatment with hyperimmune serum (2.15 months until recovery), and treatment with streptomycin (2.40 months until recovery). two clinical cases were extensively described, with the following summary: "the clinical responses to each agent [i.e., immune serum, and streptomycin] were similar, prompt amelioration of the symptoms of intoxication-headache, mental dullness or lethargy, sense of prostration and severe malaise; reduction of fever and of the sizes of the buboes, acceleration in the healing of ulcers and in the resolution of pulmonary exudates." in other words, passive immunotherapy appeared in many ways to mimic antibiotic therapy in terms of protective efficacy. however, it was noted that treatment with equine serum caused serum sickness in 51% of the patients and had a more variable outcome than the antibiotic approach, leading to the recommendation that streptomycin would be the agent of choice for future treatment of this disease. 118 with the recent development of polyclonal and monoclonal antibodies that show protective efficacy against tularemia in animal models, [119] [120] [121] it may be possible to incorporate both passive immunotherapy and antibiotic treatment into clinical practice not only for tularemia, but for other bacterial diseases, especially in cases in which antibiotic resistance is becoming more widespread. 122, 123 mycobacterium tuberculosis is another intracellular bacterium that, despite the availability of antibiotics, remains one use of human immunoglobulin in at-risk populations. 102 santosham and colleagues administered hyperimmunoglobulin (n = 353), or saline placebo (n = 350) to infants at 2, 6, and 10 months of age and examined the rates of invasive hib. for the first 90 days following the passive immunization protocol, none of the treated infants experienced invasive hib (0% incidence), compared to 7 of 350 placebo-treated children (2.0% incidence, p = .007). 102 rotavirus represents an enteric viral pathogen wherein protective passive immunotherapy has been demonstrated. [103] [104] [105] [106] [107] [108] in one example of postexposure treatment in infants, oral administration of hyperimmune antibody (in addition to standard supportive care) was able to efficiently reduce rotavirus shedding compared to placebo controls; treated patients (n = 26) exhibited no evidence of viral shedding by day 8 posttreatment as compared to 25% of controls (n = 26). 105 in a separate study, prophylactic passive immunity using orally administered bovine colostrum from immunized animals was tested in a blinded and randomized trial among infant children (3-15 months old) admitted to a hospital, typically for respiratory conditions. 103 following admission, infants were given a 10-day course of the bovine colostrum or placebo. infants who received placebo contracted symptomatic rotavirus at a rate of 14% (9 of 65) whereas no symptomatic rotavirus disease was observed in the colostrum-treated infants (0 of 55; p < .001). analysis of rotavirus vaccine failures also indicates that maternally derived antibodies play a role in passive immunity to rotavirus infection. in a study involving 177 vaccinated infants, a strong inverse correlation was observed between maternally derived rotavirus antibodies and the ability of infants to seroconvert following vaccination with a live rotavirus vaccine. 21 this is an important demonstration not only of passive immunity to an enteric pathogen, but also has broader implications on the timing of vaccine administration, especially in developing countries where preexisting immunity is relatively high, and rotavirus vaccine immunogenicity appears impaired. 109 e. coli is a significant enteric pathogen wherein prophylaxis through passive immunity has been demonstrated in several clinical studies. 110-113 tacket and colleagues were able to passively protect human subjects against experimentally induced e. coli diarrhea with specific bovine antibody. 110 using heatinactivated or glutaraldehyde-inactivated e. coli for vaccination, pregnant cows were hyperimmunized with a large number of enterotoxigenic o serogroups. milk collected during the first 10 days of lactation was purified, concentrated, lyophilized, and formulated for oral administration. as a control, a similar preparation was made using rotavirus as the immunizing antigen. subjects received daily treatment (3 times daily) for 7 days, with e. coli challenge administered 3 days into the treatment regimen. of the 10 subjects who received the e. coli antibody prophylaxis, all remained diseasefree following challenge, compared with clinical diarrhea in 9 of 10 placebo subjects (p < .0001). using a closely related clinical protocol, otto and colleagues also demonstrated good efficacy with hyperimmune bovine colostrum tablets. 111 in the first study conducted in this trial, 11 of 15 (73%) of placebo subjects contracted diarrhea following challenge, but this was reduced to only 1 of 15 (7%) in treated subjects (p = .0005). in a second study investigating the impact of omitting buffer to the oral prophylaxis, the authors also examined dose sparing. in these studies, the standard dose still conferred significant protection with 3 of 15 (20%) treated subjects contracting diarrhea, compared with 12 of 14 (86%) of controls. interestingly, if the dose was reduced by one-half then disease incidence increased to 5 of 14 subjects (36%), indicating a key role played by treatment dose in achieving successful passive immunotherapy. developed at similar rates among all three groups (p = .97). however, because administration of immunoglobulin is typically only performed during the first 4 months after transplantation and antiviral antibody half-life is estimated to be approximately 25 days, 134 it is not surprising that the protective effects of passive immunotherapy were only maintained through the first year. nevertheless, the inadvertent discovery of the protective role of antibodies in preventing ebv-induced non-hodgkin lymphoma represents a potential breakthrough in clinical management of this vulnerable patient population. despite decades of research aimed at finding a vaccine or a cure for hiv infection, this virus remains a scourge of global proportions. early attempts at passive immunotherapy using first-generation hiv-specific monoclonal antibodies were not highly effective [135] [136] [137] and this approach was not further pursued until a new generation of highly potent and broadly neutralizing antibodies were identified. 138, 139 in particular, a recent phase i clinical trial 140 involving a single administration of a broadly neutralizing antibody, 3bnc117, has renewed interest in the study of passive immunotherapy for hiv prevention and therapeutic intervention. 3bnc117 is an anti-cd4 binding site antibody that neutralizes 195 of 237 hiv strains comprising six different clades and was tested in a doseescalation study among hiv-positive patients with different levels of viremia. at a dose of 10 or 30 mg/kg, patient viral load was reduced by up to 2.5 log 10 (average decline: 1.48 log 10 ) in 10 of 11 individuals. the subject that did not respond to antibody treatment at 10 mg/kg was infected with a resistant strain of hiv. although the effect of antibody therapy on viremia was mainly transient after a single administration, the viral load remained lower than their preexisting set point in 3 of 10 patients at 56 days and one subject exhibited viremia levels that remained near the limits of detection throughout the 56-day study. it is currently unclear if hiv viremia in these patients will eventually rebound to their original levels. similar results were observed during antibody-based therapy of shiv-infected rhesus macaques in which most animals showed a rebound in viral replication after the transferred monoclonal antibodies declined to undetectable levels but a subset of animals maintained virological control in the absence of further infusions. 141 combinations of antiretroviral drugs are currently the standard of care for treatment of hiv infection and it is unlikely that one dose of a single monoclonal antibody will be sufficient to have a long-term clinical benefit among a broad patient base. however, there is growing optimism that combining a cocktail of potent, broadly neutralizing monoclonal antibodies with antiretroviral drugs and/ or agents that activate latent virus reservoirs could theoretically provide long-term reduction in viral load and reduce the rates of transmission. with substantial advances in monoclonal antibody technologies and an increasing appreciation for the role of antibodies in the control of infectious disease, the development of sophisticated new passive immunotherapies is likely to continue at an accelerated pace. antibiotic resistance among clinically relevant bacteria including multidrug-resistant (mdr) and xdr m. tuberculosis, methicillin-resistant staphylococcus aureus (mrsa), and dominant strains of antibiotic-resistant salmonella typhi and other gram-negative bacterial species is a growing concern. 122, 125, [142] [143] [144] this, coupled with the knowledge that fewer new antibiotics are moving through the drug pipeline, 122,123 may further motivate research into the development of antibody-based therapies to overcome these challenges to clinical intervention against microbial disease. one drawback to passive immunization is that antibody half-life in vivo of the most common human diseases and it is estimated to infect up to one-third of the world's population. 124 the development of strains of extensively drug-resistant (xdr) tuberculosis (tb), 125 some of which are resistant to all current antibiotic therapies, 126, 127 is also a growing concern, especially as there are few antibiotic drugs in the pipeline. 122, 123 there is considerable debate over the role of antibodies in controlling tb, with many believing that antibody plays little or no role in protective immunity (reviewed in references 124 and 128). in a comprehensive historical review by glatman-freedman and casadevall, 128 the clinical benefit of antibody-mediated immunotherapy, albeit quite variable, provides evidence to suggest that antibody plays a role in protection against tb. in studies reported by paquin in 1895, a group of patients with pulmonary tb confirmed by the presence of bacterium in their sputum showed clinical benefit. after 2 months of passive immunotherapy, 82% of patients showed reduced cough, reduction in bacterial load in sputum, clearance of pulmonary infiltrates, reduction in hemoptysis, improved appetite, and weight gain. 128, 129 at 6 months after initiating treatment, all the treated patients were alive and more than half were discharged from the hospital. in contrast, more than 30 untreated tb patients from another ward in the hospital had died within 4 months of starting the study. experimental proof of antibody-mediated protection against tb was also published in 1897 by fisch. 128, 130 after lethal tb challenge of guinea pigs, administration of immune serum was performed on days 4, 7, and 10, with further doses administered every other day for 4 weeks and once a week after that. fisch reported that 16 of 18 treated animals were alive after 2.5 months (89% survival). if treatment was delayed until day 14 postchallenge, then 2 of 3 (66%) animals survived but showed signs of illness. if no antibody treatment was performed, then 0 of 3 (0%) of the animals survived past day 28. the same approach was used to treat 50 patients with pulmonary tb. 131 all of the 19 patients treated at the earliest stages of disease improved rapidly after passive immunotherapy and were tuberculin negative at the end of the study. of the 11 patients treated at the "incipient" stage of disease, 36% no longer had bacilli in their sputum and were considered cured and 64% showed substantial improvement in disease symptoms. the 20 patients with advanced tb showed only modest or no improvement after therapy and it was concluded that immune serum was only beneficial in early but not advanced cases of disease. 131 ebv is a common human pathogen that causes a chronic infection and is a leading cause of posttransplant non-hodgkin lymphoma resulting from the uncontrolled proliferation of ebv-infected b lymphocytes in patients undergoing immunosuppressive therapies. 132 in a large retrospective study involving 44,828 kidney transplant patients, the effect of prophylactic treatment for cytomegalovirus (cmv) on posttransplant incidence of non-hodgkin lymphomas was examined. 133 the standardized incidence ratio (sir) for non-hodgkin lymphoma was expressed as the number of lymphoma cases per 100,000 persons and calculated after normalizing for age, sex, and geographical origin. the 30,255 patients who did not receive cmv prophylaxis had a sir = 26.4, which remained unchanged (sir = 24.2, p = .62) among the 12,470 patients who received antiviral drugs (acyclovir or ganciclovir). in striking contrast, the 2103 patients who received anti-cmv immunotherapy showed a complete absence of lymphomas during the first year after transplantation (sir = 0, p = .016 vs. antiviral treatment). the most common anti-cmv immunoglobulin products were shown to contain antibodies against ebv and it is believed that this is the mechanism of action for the protection afforded during the first year posttransplantation. 133 in the subsequent 5 years of follow-up, new cases of lymphoma sufficient for protection or therapeutic intervention of acute or remittent disease, active immunization through improved vaccine design may still be needed to train the host immune system to maintain long-term levels of protective immunity. importantly, examples of successful passive immunization approaches may provide a useful framework for developing new and improved vaccines that elicit the most protective antibody responses. references for this chapter are available at expertconsult.com. often provides only transient protection unless repeated administrations are performed. this may change as new technologies that increase the half-life of monoclonal antibodies are employed. for example, the fc region of an anti-rsv monoclonal antibody, motavizumab, was mutated to increase its binding to the neonatal fc receptor (fcrn), resulting in serum antibody pharmacokinetics in human subjects that increased from a typical 19-to 34-day half-life to up to a 100-day half-life while still retaining virus-specific neutralizing activity. 144a ueber das zustandekommen der diphtherie-immunitat und der tetanus-immunitat bei thieren (on the realization of immunity in diphtheria and tetanus in animals) variations in child mortality in the past 100 years ueber die behandlung diphtheriekranker kinder mit "diphtherieheilserum" (concerning the treatment of children suffering from diphtheria with "diphtheria serum") behring's discovery of diphtheria and tetanus antitoxins emil von behring and serum therapy obituary on emil von behring emil von behring: infectious disease gesammelte abhandlungen zur atiologischen therapie der ansteckenden krankheiten (collected treaties of aetiologic therapy of infectious diseases) feigin and cherry's textbook of pediatric infectious diseases continuous cultures of fused cells secreting antibody of predefined specificity monoclonal antibodies: the story of a discovery that revolutionized science and medicine the growth and potential of human antiviral monoclonal antibody therapeutics an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus metabolism of immunoglobulins age-specific prevalence of complement-fixing antibodies to sixteen viral antigens: a computer analysis of 58,500 patients covering a period of eight years severe combined immunodeficiencies (scid) x-linked agammaglobulinemia: an analysis of 96 patients persistence of maternal antibody in infants beyond 12 months: mechanism of measles vaccine failure measles vaccine failure. a survey of causes and means of prevention appropriate age for measles vaccination in the united states transplacental rotavirus igg interferes with immune response to live oral rotavirus vaccine orv-116e in indian infants rotavirus-specific igg antibodies from mothers' serum may inhibit infant immune responses to the pentavalent rotavirus vaccine effectiveness of maternal influenza immunization in mothers and infants influenza vaccine given to pregnant women reduces hospitalization due to influenza in their infants smallpox and sulphonamide the use of vaccinia hyperimmune gammaglobulin in the prophylaxis of smallpox the use of hyperimmune antivaccinia gammaglobulin for the prevention and treatment of smallpox bulletin de l'institut d'hygiène treatment of argentine hemorrhagic fever efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome acción de los inmunosueros en la fiebre hemorrágica experimental treatment of junin virus-infected guinea pigs with immune serum: development of late neurological disease protection of guinea pigs against experimental argentine hemorrhagic fever by purified human igg: importance of elimination of infected cells diagnosis of lassa fever and the isolation and management of patients endemic lassa fever in liberia. iv. selection of optimally effective plasma for treatment by passive immunization protection of lassa virus-infected guinea pigs with lassa-immune plasma of guinea pig, primate, and human origin passive antibody therapy of lassa fever in cynomolgus monkeys: importance of neutralizing antibody and lassa virus strain enhanced treatment of lassa fever by immune plasma combined with ribavirin in cynomolgus monkeys lassa immune serum the use of lassa fever convalescent plasma in nigeria convalescent whole blood plasma and serum in prophylaxis of measles the serum prophylaxis of measles: (section of epidemiology and state medicine) post-exposure passive immunisation for preventing measles chemical, clinical, and immunological studies on the products of human plasma fractionation. xii. the use of concentrated normal human serum gamma globulin (human immune serum globulin) in the prevention and attenuation of measles the prevention and modification of measles global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis mortality associated with influenza and respiratory syncytial virus in the united states immunity to influenza the role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza a2 and b viruses immunity to influenza as related to antibody levels prophylactic and therapeutic antiviral effect of human gamma globulin influenza: the preparation of immune sera in horses passive immunization of mice against human influenza virus by the intranasal route a comparative study of the treatment of tularemia with immune serum, hyperimmune serum and streptomycin passive protection of mice against lethal francisella tularensis (live tularemia vaccine strain) infection by the sera of human recipients of the live tularemia vaccine role of antibody to lipopolysaccharide in protection against low-and high-virulence strains of francisella tularensis generation and characterization of hybridoma antibodies for immunotherapy of tularemia therapeutic strategies to combat antibiotic resistance the drug push antibody-mediated immunity against tuberculosis: implications for vaccine development multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis first tuberculosis cases in italy resistant to all tested drugs. euro surveill emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in iran serum therapy for tuberculosis revisited: reappraisal of the role of antibody-mediated immunity against mycobacterium tuberculosis the treatment of tuberculosis by injections of immunized blood serum the antitoxic and bactericidal properties of the serum of horses treated with koch's new tuberculin a further report on the use of "anti-phthisic serum t.r." (fisch) in tuberculosis malignant lymphomas in transplantation patients effect of cytomegalovirus prophylaxis with immunoglobulin or with antiviral drugs on post-transplant non-hodgkin lymphoma: a multicentre retrospective analysis pharmacokinetics of viral antibodies after administration of intravenous immunoglobulin in patients with chronic lymphocytic leukaemia or multiple myeloma passive immunization with the anti-hiv-1 human monoclonal antibody (hmab) 4e10 and the hmab combination 4e10/2f5/2g12 delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies adjunctive passive immunotherapy in human immunodeficiency virus type 1-infected individuals treated with antiviral therapy during acute and early infection antibodies in hiv-1 vaccine development and therapy structural insights on the role of antibodies in hiv-1 vaccine and therapy viraemia suppressed in hiv-1-infected humans by broadly neutralizing antibody 3bnc117 therapeutic efficacy of potent neutralizing hiv-1-specific monoclonal antibodies in shiv-infected rhesus monkeys phylogeographical analysis of the dominant multidrug-resistant h58 clade of salmonella typhi identifies inter-and intracontinental transmission events recurrent challenges for clinicians: emergence of methicillin-resistant staphylococcus aureus, vancomycin resistance, and current treatment options current and future treatment options for infections caused by multidrugresistant gram-negative pathogens a novel investigational fc-modified humanized monoclonal antibody, motavizumab-yte, has an extended half-life in healthy adults monoclonal antibody therapies against anthrax raxibacumab for the treatment of inhalational anthrax the serum treatment of anthrax septicaemia the local and general serum treatment of cutaneous anthrax protection against botulinum toxins provided by passive immunization with botulinum human immune globulin: evaluation using an inhalation model botulinum neurotoxin neutralizing activity of immune globulin (ig) purified from clinical volunteers vaccinated with recombinant botulinum vaccine (rbv a/b) antibody protection against botulinum neurotoxin intoxication in mice equine antitoxin use and other factors that predict outcome in type a foodborne botulism human botulism immune globulin for the treatment of infant botulism early antitoxin treatment in wound botulism results in better outcome infant botulism: a 30-year experience spanning the introduction of botulism immune globulin intravenous in the intensive care unit at childrens hospital los angeles identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication the therapeutic effect of homologous and heterologous antitoxins in experimental diphtheria and tetanus importance of intravenous injection of diphtheria antiserum the recent epidemic of diphtheria in the johns hopkins hospital and medical school: general procedures adopted use of diphtheria antitoxin in the treatment and prevention of diphtheria diphtheritic polyneuropathy: a clinical study and comparison with guillain-barre syndrome mice are actively immunized after passive monoclonal antibody prophylaxis and ricin toxin challenge conformation-dependent highaffinity potent ricin-neutralizing monoclonal antibodies characteristics of toxin-neutralization by anti-tetanus human monoclonal antibodies directed against the three functional domains [a], [b] and [c] of the tetanus toxin molecule and a reliable method for evaluating the protective effects of monoclonal antibodies observations with antitetanus serum in the monkey tetanus: analysis of 1458 cases, which occurred in home military hospitals during the years 1914-1918 intrathecal vs. intramuscular administration of human antitetanus immunoglobulin or equine tetanus antitoxin in the treatment of tetanus: a meta-analysis the treatment of tetanus with antitoxin: an analysis of the outcome in sixhundred forty-two cases protective activities in mice of monoclonal antibodies against pertussis toxin protective effects of pertussis immunoglobulin (p-igiv) in the aerosol challenge model the therapeutic effect of sulfadiazine and immune rabbit serum in experimental murine pertussis prophylaxis of whooping cough use of convalescent blood in whooping cough with a review of the literature specific immunoglobulin for treatment of whooping cough active and passive immunity against borrelia burgdorferi decorin binding protein a (dbpa) protects against infection passive immunization of hamsters against experimental infection with the lyme disease spirochete protective monoclonal antibodies to chlamydia trachomatis serovar-and serogroup-specific major outer membrane protein determinants monoclonal immunoglobulin a antibody to the major outer membrane protein of the chlamydia trachomatis mouse pneumonitis biovar protects mice against a chlamydial genital challenge protective role of serum antibody in immunity to chlamydial genital infection human monoclonal antibodies directed against toxins a and b prevent clostridium difficile-induced mortality in hamsters mechanisms of protection against clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab treatment with monoclonal antibodies against clostridium difficile toxins chicken egg yolk antibodies against f18ab fimbriae of escherichia coli inhibit shedding of f18 positive e. coli by experimentally infected pigs prevention and therapy of experimental escherichia coli infection with monoclonal antibody stx2-specific human monoclonal antibodies protect mice against lethal infection with escherichia coli expressing stx2 variants field trial of an infant formula containing anti-rotavirus and anti-escherichia coli milk antibodies from hyperimmunized cows treatment of enterotoxigenic and enteropathogenic escherichia coli-induced diarrhoea in children with bovine immunoglobulin milk concentrate from hyperimmunized cows: a double-blind, placebo-controlled, clinical trial a summary of certain aspects of the disease including methods for early diagnosis and the results of serum treatment in 600 patients efficacy of human hyperimmune globulin in prevention of haemophilus influenzae type b disease in infant rats monoclonal anti-lps inner core antibodies protect against experimental hematogenous haemophilus influenzae type b meningitis protection from infection with haemophilus influenzae type b by monoclonal antibody to the capsule serum treatment of influenzal meningitis a mycobacterial lipoarabinomannan specific monoclonal antibody and its f(ab′) fragment prolong survival of mice infected with mycobacterium tuberculosis therapeutic efficacy of high-dose intravenous immunoglobulin in mycobacterium tuberculosis infection in mice passive serum therapy with polyclonal antibodies against mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in scid mice passive immunization with neisseria meningitidis pora specific immune sera reduces nasopharyngeal colonization of group b meningococcus in an infant rat nasal challenge model protective efficacy of monoclonal antibodies to class 1 and class 3 outer membrane proteins of neisseria meningitidis b:15:p1.16 in infant rat infection model: new prospects for vaccine development experimental meningococcal infection in the mouse experimental cerebro-spinal meningitis and its serum treatment protection against infection with neisseria meningitidis group b serotype 2b by passive immunization with serotype-specific monoclonal antibody the results of the serum treatment in thirteen hundred cases of epidemic meningitis a novel anti-pcrv antibody providing enhanced protection against pseudomonas aeruginosa in multiple animal infection models a multifunctional bispecific antibody protects against pseudomonas aeruginosa in vitro and in vivo properties of a fully human igg1 monoclonal antibody that combats multidrug resistant pseudomonas aeruginosa pseudomonas hyperimmune globulin passive immunotherapy for pulmonary exacerbations in cystic fibrosis intravenous immune globulin treatment of pulmonary exacerbations in cystic fibrosis safety and pharmacokinetics of an anti-pcrv pegylated monoclonal antibody fragment in mechanically ventilated patients colonized with pseudomonas aeruginosa: a randomized,double-blind, placebo-controlled trial immunoprotection by monoclonal antibodies to the porins and lipopolysaccharide of salmonella typhimurium a new antigen of b. typhosus: its relation to virulence and to active and passive immunisation oral passive immunization against experimental salmonellosis in mice using chicken egg yolk antibodies specific for salmonella enteritidis and s. typhimurium clinical trials with a new antitiyphoid serum multi-serotype outer membrane vesicles of shigellae confer passive protection to the neonatal mice against shigellosis passive immunity with multiserotype heat-killed shigellae in neonatal mice protection against keratoconjunctivitis shigellosa induced by immunization with outer membrane proteins of shigella spp efficacy of bovine milk immunoglobulin concentrate in preventing illness after shigella flexneri challenge hyperimmune bovine colostrum in the treatment of shigellosis in children: a double-blind, randomized, controlled trial protein a-neutralizing monoclonal antibody protects neonatal mice against staphylococcus aureus a human monoclonal antibody targeting the conserved staphylococcal antigen isaa protects mice against staphylococcus aureus bacteremia functional antibodies targeting isaa of staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy a blinded, randomized, multicenter study of an intravenous staphylococcus aureus immune globulin antistaphylococcal immunoglobulins to prevent staphylococcal infection in very low birth weight infants multicenter study to assess safety and efficacy of inh-a21, a donor-selected human staphylococcal immunoglobulin, for prevention of nosocomial infections in very low birth weight infants phase ii, randomized, multicenter, double-blind, placebo-controlled trial of a polyclonal anti-staphylococcus aureus capsular polysaccharide immune globulin in treatment of staphylococcus aureus bacteremia. antimicrob agents chemother phase ii, randomized, double-blind, multicenter study comparing the safety and pharmacokinetics of tefibazumab to placebo for treatment of staphylococcus aureus bacteremia human monoclonal antibodies to group b streptococcus. reactivity and in vivo protection against multiple serotypes monoclonal antibodies in the therapy of experimental neonatal group b streptococcal disease monoclonal antibodies targeting different cell wall antigens of group b streptococcus 95.e6 section 1 general aspects of vaccination mediate protection in both fc-dependent and independent manner immunological fingerprinting of group b streptococci: from circulating human antibodies to protective antigens maternal antibody at delivery protects neonates from early onset group b streptococcal disease correlation of maternal antibody deficiency with susceptibility to neonatal group b streptococcal infection pro tection against pneumococcal pneumonia in mice by monoclonal antibodies to pneumolysin human antibodies to phtd, pcpa, and ply reduce adherence to human lung epithelial cells and murine nasopharyngeal colonization by streptococcus pneumoniae preclinical evaluation of the pht proteins as potential crossprotective pneumococcal vaccine antigens effective combination therapy for invasive pneumococcal pneumonia with ampicillin and intravenous immunoglobulins in a mouse model studies on experimental pneumonia: vii. treatment of experimental pneumococcus type i pneumonia in monkeys with type i antipneumococcus serum the treatment of lobar pneumonia with concentrated anti-pneumococcus serum bacterial polysaccharide immune globulin for prophylaxis of acute otitis media in high-risk children the serum treatment of lobar pneumonia defense from the group a streptococcus by active and passive vaccination with the streptococcal hemoprotein receptor active and passive immunizations with the streptococcal esterase sse protect mice against subcutaneous infection with group a streptococci mechanism of protection induced by group a streptococcus vaccine candidate j8-dt: contribution of b and t-cells towards protection scarlet fever prophylaxis: use of blood serum from persons who have recovered from scarlet fever the prevention of scarlet fever intravenous immunoglobulin g therapy in streptococcal toxic shock syndrome: a european randomized, double-blind, placebo-controlled trial clinical efficacy of polyspecific intravenous immunoglobulin therapy in patients with streptococcal toxic shock syndrome: a comparative observational study intravenous immunoglobulin therapy for streptococcal toxic shock syndrome-a comparative observational study. the canadian streptococcal study group antitoxin versus no antitoxin in scarlet fever the antitoxin treatment of erysipelas passive immunity in experimental cholera passive oral immunization by egg yolk immunoglobulin (igy) to vibrio cholerae effectively prevents cholera experimental cholera in infant mice: protective effects of antibody further investigation of a new anti-cholera serum treatment of cholera with a new anti-cholera serum: preliminary note human anti-plague monoclonal antibodies protect mice from yersinia pestis in a bubonic plague model synergistic protection of mice against plague with monoclonal antibodies specific for the f1 and v antigens of yersinia pestis la peste bubonique (duexiéme note) treatment of plague: promising alternatives to antibiotics protection of mice from fatal bubonic and pneumonic plague by passive immunization with monoclonal antibodies against the f1 protein of yersinia pestis the scid/beige mouse as a model to investigate protection against yersinia pestis human immune response to a plague vaccine comprising recombinant f1 and v antigens administration of antibody to the lung protects mice against pneumonic plague passive immunity to yersiniae mediated by anti-recombinant v antigen and protein a-v antigen fusion peptide roles of v antigen in promoting virulence and immunity in yersiniae les épidémies de peste en extrême-orient. xiiie congrès international de médecine sur la peste bubonique (sérothérapie) prophylaxis and therapy for chikungunya virus infection chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes case reports of neuro-chikungunya in southern thailand antigenic sites of coxsackie a9 virus inducing neutralizing monoclonal antibodies protective in mice a murine model of coxsackievirus a16 infection for anti-viral evaluation effect of intravenous immunoglobulin for neonates with severe enteroviral infections with emphasis on the timing of administration vaccination for the prevention of maternal and fetal infection with guinea pig cytomegalovirus rat cytomegalovirus vaccine prevents accelerated chronic rejection in cmvnaive recipients of infected donor allograft hearts complete protection of mice against lethal murine cytomegalovirus challenge by immunization with dna vaccines encoding envelope glycoprotein complex iii antigens gh, gl and go passive immunization during pregnancy for congenital cytomegalovirus infection prophylaxis of primary cytomegalovirus disease in renal transplant recipients. a trial of ganciclovir vs immunoglobulin cmv-hyperimmune globulin for preventing cytomegalovirus infection and disease in solid organ transplant recipients: a meta-analysis the development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1 epitope determinants of a chimpanzee dengue virus type 4 (denv-4)-neutralizing antibody and protection against denv-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody development of a humanized antibody with high therapeutic potential against dengue virus type 2 ebola gp-specific monoclonal antibodies protect mice and guinea pigs from lethal ebola virus infection successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies therapeutic intervention of ebola virus infection in rhesus macaques with the mb-003 monoclonal antibody cocktail treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee preventive effect of igg from ebv-seropositive donors on the development of human lympho-proliferative disease in scid mice a mouse monoclonal antibody against epstein-barr virus envelope glycoprotein 350 prevents infection both in vitro and in vivo active and passive vaccination against hantavirus pulmonary syndrome with andes virus m genome segment-based dna vaccine dna vaccine-derived human igg produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome a non-randomized multicentre trial of human immune plasma for treatment of hantavirus cardiopulmonary syndrome by andv the use of human immune serum globulin (gamma globulin) in infectious (epidemic) hepatitis in the mediterranean theater of operations i. studies on prophylaxis in two epidemics of infectious hepatitis prevention of infectious hepatitis with gamma globulin the prevention and attenuation of infectious hepatitis by gamma globulin gamma globulin in the prevention of infectious hepatitis; studies on the use of small doses in family outbreaks preclinical evaluation of two human anti-hepatitis b virus (hbv) monoclonal antibodies in the hbv-trimera mouse model and in hbv chronic carrier chimpanzees hepatitis b immune globulin as a prophylactic measure for spouses exposed to acute type b hepatitis liver transplantation in hbsag-positive hbv-dna-negative cirrhotics: immunoprophylaxis and long-term outcome liver transplantation in hbs antigen (hbsag) carriers. prevention of hepatitis b virus (hbv) recurrence by passive immunization human monoclonal antibody hcv1 effectively prevents and treats hcv infection in chimpanzees sexual transmission of the hepatitis c virus and efficacy of prophylaxis with intramuscular immune serum globulin. a randomized controlled trial human monoclonal antibody mbl-hcv1 delays hcv viral rebound following liver transplantation: a randomized controlled study immunotherapeutic potential of neutralizing antibodies targeting conserved regions of the hcv envelope glycoprotein e2 clinical evaluation (phase i) of a human monoclonal antibody against hepatitis c virus: safety and antiviral activity successful passive and active immunization of cynomolgus monkeys against hepatitis e role of immune serum globulins in pregnant women during an epidemic of hepatitis e passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein d and monoclonal antibodies to glycoprotein b effect of antibody alone and combined with acyclovir on neonatal herpes simplex virus infection in guinea pigs l or more. effect on viral, opportunistic, and bacterial infections. the national institute of child health and human development intravenous immunoglobulin clinical trial study group intravenous immunoglobulins suppress the recurrences of genital herpes simplex virus: a clinical and immunological study antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv-1 infection in a transgenic mouse model antibody-based protection against hiv infection by vectored immunoprophylaxis hiv therapy by a combination of broadly neutralizing antibodies in humanized mice hiv-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice passive immunotherapy in aids: a double-blind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus 1 antibodies vs. transfusions of seronegative plasma delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies safety and efficacy of hiv hyperimmune globulin for prevention of motherto-child hiv transmission in hiv-1-infected pregnant women and their infants in kampala passive immunotherapy in the treatment of advanced human immunodeficiency virus infection a murine genital-challenge model is a sensitive measure of protective antibodies against human papillomavirus infection in vivo mechanisms of vaccine-induced protection against hpv infection the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory metaanalysis protection of mice against japanese encephalitis virus by passive administration with monoclonal antibodies passive protection of mice, goats, and monkeys against japanese encephalitis with monoclonal antibodies protection of monkeys against machupo virus by the passive administration of bolivian haemorrhagic fever immunoglobulin (human origin) protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the h protein mimicked by peptides which are not recognized by maternal antibodies correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their hemagglutination inhibiting activity smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus a protective therapy for mumps mumps; use of convalescent serum in the treatment and prophylaxis of orchitis serum prophylaxis of epidemic parotitis use of human blood in protection against mumps intravenous immunoglobulin therapy for pure red cell aplasia related to human parvovirus b19 infection: a retrospective study of 10 patients and review of the literature guidelines on the use of intravenous immune globulin for hematologic conditions the relation of the meninges and choroid plexus to poliomyelitic infection passive immunity in poliomyelitis. iv. protection of rhesus monkeys against cerebral challenge experimental poliomyelitis in monkeys seventh note: active immunization and passive serum protection evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis. iv. final report of results based on clinical diagnoses evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis. 5. reanalysis of results based on laboratory-confirmed cases resume of the results of therapy with convalescent serum in poliomyelitis intramuscular use of convalescent serum in treatment of poliomyelitis preparalytic poliomyelitis: observations in one hundred and six cases in which convalescent serum was used recherches sur la vaccination antirabique evaluation of human rabies immune globulin and homologous and heterologous antibody antiserum in the prophylaxis of rabies comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin use of hyperimmune anti-rabies serum concentrates in experimental rabies laboratory data supporting the clinical trial of anti-rabies serum in persons bitten by a rabid wolf management of rabies in humans development of a humanized monoclonal antibody (medi-493) with potent in vitro and in vivo activity against respiratory syncytial virus quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats studies of passive immunotherapy for infections of respiratory syncytial virus in the respiratory tract of a primate model isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (fab rsvf2-5) that exhibits therapeutic efficacy in vivo respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high risk children active and passive immunization against rift valley fever virus infection in syrian hamsters topological mapping of antigenic sites on the rift valley fever virus envelope glycoproteins using monoclonal antibodies passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp3 and vp7 serum igg mediates mucosal immunity against rotavirus infection a gastrointestinal rotavirus infection mouse model for immune modulation studies rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection prophylaxis of german measles with immune serum globulin prevention of rubella by gamma globulin during an epidemic in barrow, alaska, in 1964 trial of high-titre human rubella immunoglobulin rubella epidemic, 1964: effect on 6,000 pregnancies passive immunization against rubella: studies on the effectiveness of rubella-immunoglobulin after intranasal infection with rubella vaccination virus human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection passive immune globulin therapy in the siv/macaque model: early intervention can alter disease profile passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies fc receptor but not complement binding is important in antibody protection against hiv protection of macaques against pathogenic simian/human immunodeficiency virus 89.6pd by passive transfer of neutralizing antibodies determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies highly potent hiv-specific antibody neutralization in vitro translates into effective protection against mucosal shiv challenge in vivo antibody-mediated immunotherapy of macaques chronically infected with shiv suppresses viraemia passive transfer of modest titers of potent and broadly neutralizing anti-hiv monoclonal antibodies block shiv infection in macaques neutralizing antibody directed against the hiv-1 envelope glycoprotein can completely block hiv-1/siv chimeric virus infections of macaque monkeys pre-and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model passive immunization of mice with monoclonal antibodies raised against tickborne encephalitis virus efficiency of use of immunoglobulin preparations for the postexposure prevention of tickborne encephalitis in russia (a review of semi delayed humoral immunity in a patient with severe tick-borne encephalitis after complete active vaccination combination therapy of vaccinia virus infection with human anti-h3 and anti-b5 monoclonal antibodies in a small animal model disparity between levels of in vitro neutralization of vaccinia virus by antibody to the a27 protein and protection of mice against intranasal challenge protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies postexposure prevention of progressive vaccinia in scid mice treated with vaccinia immune globulin prophylactic effect of antivaccinia gamma-globulin against post-vaccinal encephalitis experience of anti-vaccinia immunoglobulin in the united kingdom complications of smallpox vaccination. effects of vaccinia immune globulin therapy prevention of varicella by zoster immune globulin the use of serum gamma globulin antibodies to control chicken pox in a convalescent hospital for children congenital varicella syndrome: the evidence for secondary prevention with varicella-zoster immune globulin a humanized murine monoclonal antibody protects mice either before or after challenge with virulent venezuelan equine encephalomyelitis virus antibody prophylaxis and therapy against west nile virus infection in wild-type and immunodeficient mice efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of west nile virus encephalitis in hamster model using high titer west nile intravenous immunoglobulin from selected israeli donors for treatment of west nile virus infection development of a humanized monoclonal antibody with therapeutic potential against west nile virus possible benefit of intravenous immunoglobulin therapy in a lung transplant recipient with west nile virus encephalitis treatment of west nile virus encephalitis with intravenous immunoglobulin lethal 17d yellow fever encephalitis in mice. i. passive protection by monoclonal antibodies to the envelope proteins of 17d yellow fever and dengue 2 viruses the duration of passive immunity in yellow fever persistence of yellow fever immunity on the use of immune serum at various intervals after the inoculation of yellow fever virus into rhesus monkeys notes on laboratory infections with yellow fever observations on laboratory and hospital infections with yellow fever in england immunoprotection against systemic candidiasis in mice rats clearing a vaginal infection by candida albicans acquire 95.e10 section 1 general aspects of vaccination specific, antibody-mediated resistance to vaginal reinfection a vaccine and monoclonal antibodies that enhance mouse resistance to candida albicans vaginal infection vaccine and monoclonal antibody that enhance mouse resistance to candidiasis immunoglobulin g3 blocking antibodies to the fungal pathogen cryptococcus neoformans human immunoglobulin g2 (igg2) and igg4, but not igg1 or igg3, protect mice against cryptococcus neoformans infection more is not necessarily better: prozone-like effects in passive immunization with igg phase i evaluation of the safety and pharmacokinetics of murine-derived anticryptococcal antibody 18b7 in subjects with treated cryptococcal meningitis enteral human serum immunoglobulin treatment of cryptosporidiosis in mice with severe combined immunodeficiency efficacy of monoclonal antibodies against defined antigens for passive immunotherapy of chronic gastrointestinal cryptosporidiosis prophylactic effect of bovine anti-cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with cryptosporidium parvum demonstration of passive immunity in experimental monkey malaria model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum the importance of human fcgammari in mediating protection to malaria gamma-globulin and acquired immunity to human malaria antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes monoclonal antibodies to toxoplasma cell membrane surface antigens protect mice from toxoplasmosis generation of a neutralizing human monoclonal antibody fab fragment to surface antigen 1 of toxoplasma gondii tachyzoites protection against experimental toxoplasmosis by adoptive immunotherapy impact of trough igg on pneumonia incidence in primary immunodeficiency: a meta-analysis of clinical studies high-vs low-dose immunoglobulin therapy in the long-term treatment of x-linked agammaglobulinemia history of immunoglobulin replacement use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology effect of intravenous gammaglobulin therapy in igg2 deficient and igg2 sufficient children with recurrent infections and poor response to immunization with hemophilus influenzae type b capsular polysaccharide antigen intravenous immune globulin for the prevention of bacterial infections in children with symptomatic human immunodeficiency virus infection. the national institute of child health and human developments intravenous immunoglobulin study group polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: a systematic review and metaanalysis supplemental immune globulins in sepsis: a critical appraisal prevention of infection in multiple trauma patients by high-dose intravenous immunoglobulins the role of intravenous immunoglobulin for the prevention and treatment of neonatal sepsis immunity in mumps: i. experiments with monkeys (macacus mulatta). the development of complement-fixing antibody following infection and experiments on immunization by means of inactivated virus and convalescent human serum an experimental study of parotitis key: cord-272955-kkkrkgg1 authors: belsy, acosta; odalys, valdés; alexander, piñón; clara, savón; angel, goyenechea; grehete, gonzalez; guelsys, gonzalez; luis, sarmiento; pedro, más; guadalupe, guzmán maría; alina, llop; pilar, perez breña ma; inmaculada, casas title: molecular characterization of adenoviral infections in cuba: report of an unusual association of species d adenoviruses with different clinical syndromes date: 2009-03-12 journal: arch virol doi: 10.1007/s00705-009-0338-4 sha: doc_id: 272955 cord_uid: kkkrkgg1 adenoviruses are common pathogens that are responsible for a wide variety of infectious syndromes. the objectives of this study were to identify and characterize members of different adenovirus species at the molecular level and to describe the correlation between viruses and clinical syndromes during a period of 4 years. between 2002 and 2006, 45 of 512 respiratory specimens (8%) from patients with acute respiratory tract infection tested positive for adenovirus. four adenovirus isolates from samples sent for enterovirus isolation were also analyzed. this research identified 49 confirmed cases of human adenovirus infection by pcr and/or viral culture. the most common diagnosis was upper respiratory infection (44%). human adenovirus d was the major species found (59%), followed by human adenovirus c (36%) and human adenovirus b (4%). human adenovirus 5 was the major serotype found producing bronchiolitis, followed by human adenovirus 6. in patients with upper respiratory infection, the major serotype found was human adenovirus 17. viruses of the species human adenovirus d were identified in seven (77%) cases of acute febrile syndrome. four isolates from clinical materials obtained from patients with encephalitis, acute flaccid paralysis and meningoencephalitis were identified as belonging to the species human adenovirus d. our data demonstrate a surprising result about the identification of an unusual association of viruses of the species human adenovirus d with different clinical syndromes. this observation could be evaluated as a possible indicator of the emergence of a novel strain but further studies are required. human adenoviruses (hadvs) are members of the family adenoviridae, whose members infect hosts across a broad spectrum of vertebrates. there are 51 serotypes of hadv in the genus mastadenovirus, distributed in six species, a-f (formerly subgroups or subgenera) on the basis of their physicochemical, biological and genetic properties. a new serotype isolated recently (52) has been proposed to represent a new species g [21] . human adenoviruses are associated with sporadic infection, and community and institutional outbreaks. these viruses cause a variety of clinical manifestations, such as conjunctivitis, pneumonia, gastroenteritis, and hemorrhagic cystitis. some serotypes can occasionally infect tissues of the central nervous system (cns) and cause aseptic meningitis, meningoencephalitis, and encephalitis. they can cause especially severe disease in infants, young children, immunocompromised persons, and transplant recipients [2, 5, 14, 15, 25, 30, 31] . serosurveys suggest that virtually all people are exposed to hadv during childhood [22, 35] . they can remain in an asymptomatic carrier state until at least young adulthood [18] , and virus may be actively shed long after symptomatic infection [16] . over the past decades, neutralization tests, elisa, and virus isolation had been used for the detection and identification of adenovirus serotypes. however, these methods are relatively complicated, labour-intensive, and timeconsuming, and they have low sensitivity. [26, 39, 42] . these disadvantages have limited their use. amplification of the viral genome by pcr has been introduced as a convenient and powerful alternative for molecular diagnosis. additionally, genome amplification allows further characterization of the adenovirus serotype by sequence analysis [1, 7, 34] . the objectives of this study were the identification and molecular characterization of different hadv isolates and to describe the correlation between viruses and clinical syndromes during a period of 4 years. between october 2002 and september 2006, 512 respiratory specimens (nasopharyngeal swabs and pharyngeal washes) from patients with acute respiratory tract infection (arti) were sent specifically to the national reference laboratory of respiratory viruses for testing respiratory viruses, including influenza virus a, b and c, human respiratory syncitial virus (hrsv), human parainfluenza virus 1-4, hadv, human coronavirus, human rhinovirus and human metapneumovirus (hmpv). routine virological testing for respiratory pathogens was performed using a combination of direct immunofluorescence, isolation in cell culture and pcr assays [9, 10, 27] . in addition, four samples (three stools and one cerebrospinal fluid) from the enterovirus (ev) laboratory with previous viral isolation without identification were analyzed. nasopharyngeal swabs and pharyngeal washes were collected in 3 ml of virus transport medium (mem, gibco-brl, life technologies, paisley, scotland; penicillin 200 u/ml, and streptomycin 200 lg/ml, biowhittaker, ma; mycostatin 200 u/ml, sigma; bovine serum albumin 0.25%, merck, darmstadt, germany) the specimens were frozen and stored at -70°c until the analysis was carried out. a human embryonic fibroblast cell line was used for primary isolation of ev. tubes with 80% confluent monolayers were inoculated with 0.2 ml of homogenized samples. cells were fed with 2 ml of 2% fetal calf serum in basal medium eagle and visualized for cytopathic effect (cpe) twice a week. when a cpe that was not typical of ev was observed, the monolayer was scraped and tested for adenovirus antigen by immunofluorescence with a specific monoclonal antibody (chemicon, temecula, ca). total viral rna/dna from 200-ll aliquots from clinical samples or supernatant of infected cell culture was extracted using the guanidinium thiocyanate method as described previously by casas et al. [6] . the lysis buffer included 100 copies of the cloned, amplified product of the internal control described by coiras et al. [9] . it was used for checking the extraction process, the amplification efficiency, and the presence of inhibitors in the clinical specimens. after processing, the dried pellet was resuspended in 15 ll of rnase-free sterile water. negative controls consisting of rnase-free sterile water (sigma) were treated following the same procedure. for each assay, known positive controls, derived from infected viral cells, were added. in all cases, nucleic acid extracts (5 ll) were examined for influenza virus a, b and c, hadv, hrsv a and b [9] , human parainfluenza virus 1-4, coronaviruses, rhinoviruses, and ev [10] , using nrt-pcr. in addition, a multiplex npcr assay for human herpesvirus was used in the case of a patient with encephalitis [40] . the protocols employed have been published previously and used for clinical diagnosis. three different biosafety cabinets were used for master mix preparation, sample handling and primary reaction product handling. different laboratory wear and coats were used for each cabinet. amplicon detection was done in a different room. specimens that were positive for hadv were processed by two independent nested reactions with 20 pmol of adhex2f, nt 20485 to 20503; (5 0 cccittyaaccacc accg3 0 ) and adhex1r, nt 20836 to 20854; (5 0 katg gggtaragcatgtt3 0 ) or 20 pmol of adhex1f, nt 20380 to 20400; (5 0 caacacctaygastacatgaa3 0 ) and adhex2r, nt 20632 to 20652; (5 0 acatccttbc kgaagttcca3 0 ) degenerate primers as described previously [7] . these nested primer pairs were designed to bind inside the hexon protein coding region of the adenovirus genome [1] . the pcr products were purified with qiaquick pcr purification kit (qiagen) according to the manufacturers's protocol and were sequenced in both directions using the primer pairs described above. sequences were obtained using an automatic dna sequencer (abi prism 3700; applied biosystems) and a big dye terminator cycle sequencing kit version 3.1 (applied biosystems). the fragment size sequenced corresponded to 122 amino acids, from amino acid position 540-662 of the hexon protein. this fragment is located at the external surface l2 of the hexon protein monomer but does not overlap with the hrv7 domain [11] . the nucleotide sequences obtained were first analysed using the chromas software (version 1.3). the forward and reverse sequences were combined using bioedit and were compared and aligned with the corresponding previously published sequences of prototype viruses available from genbank, using the clustal x (version 1.83) program. specimens that yielded identity scores c90%, were considered to be good genotype matches. phylogenetic trees were inferred using programs from the mega package (version 4) and reconstructed using the neighbourjoining method. the evolutionary distances were estimated using kimura's two-parameter method [24] . the statistical significance of a particular tree topology was evaluated by 1,000 replicates of boostrap re-sampling. the criteria for serotype assignation were the shortest distance from a prototype strain, as deduced from the phylogenetic tree ( fig. 1) , and the highest similarity value obtained after sequence comparisons. the genbank accession numbers of the nucleotide sequences presented in this study are: eu179755-eu179763, eu179765, eu179768-eu179785 and eu439569-eu439589. in the present report, the nested pcr method used was able to detect different hadvs in clinical samples and supernatant culture with a sensitive internal control system to assure the quality of reaction conditions in each individual tube. in this investigation, 49 confirmed cases of hadv infection were identified by pcr and/or viral culture ( table 1) . the monthly collection of clinical samples is shown in table 1 . human adenovirus was detected throughout the year; however, 38 samples (77%) were collected between june and september. two significant outbreaks of hadv infection were identified, one in july of 2005 and the other in september of 2006. more than half of the positive samples (53%) were from children younger than 5 years of age, and 50% of them were under 6 months of age. thirty-two percent were from patients between 5 and 20 years, and 14% were from adults between 21 and 64 years of age. no difference in gender distribution was observed. the most common diagnosis was upper respiratory infection (44%). medical records documented the following symptoms at initial presentation: nasal congestion (98%), sore throat (83%) and cough (78%). all patients were febrile. fourteen (28%) patients had bronchiolitis. acute febrile syndrome (18%) was defined as the concurrence of fever, cold symptoms, headache, weakness, vomiting and diarrhoea, a common but non-specific symptom. in these cases, the results of diagnostic assays for the detection of other potentially pathogenic enteric microorganisms were negative. an interesting finding was the detection of hadv in a patient 64 years of age who was admitted with a diagnosis of encephalitis. no human herpesvirus, influenza virus, ev or flavivirus genomes were found in the cerebrospinal fluid of this patient. human adenovirus dna was detected in the supernatant of a cell culture infected with viruses obtained from fecal specimens taken from a patient with acute flaccid paralysis (afp), as well as in two cases of meningoencephalitis. overall, 73% of hadv positive patients were hospitalized. partial hexon nucleotide sequences for 49 different hadv types were obtained. a blast search in the genbank database for all of the amplicon sequences determined in the present study was done. a 100% agreement with existing genbank sequences for serotypes 1, 3, 5, and 6 was obtained. an agreement ranging from 99 to 100% was obtained for prototype strains of human adenovirus d. an alignment of the hexon gene sequences was computed by using sequences from representative serotypes of species b (hadv 3), c (hadvs 1, 5, and 6), and d (hadvs 9, 10, 13,15,17, 19, 23, 25, 28, 30, 32, 33, 37-39, 43, 44, and 47-49) . in order to evaluate the phylogenetic relationships between individual prototype viruses and positive cases of hadv infection by pcr and/or viral culture, phylogenetic trees were constructed using a phylogeny reconstruction algorithm (the neighbour-joining method) and a nucleotide substitution method . phylogenetic and sequence similarity analysis permitted accurate species classification and serotype identification except for species d hadv, for which serotypes could not be clearly defined. the resulting phylogenetic tree showed three different clusters, represented by the species b, c and d. the bootstrap values ( fig. 1) were, in general, quite high, and especially at nodes between species. however, these values were less good for some nodes within species d. the assignment of a serotype to a clinical isolate was done by calculating the shortest distance between the isolate and a prototype strain. a total of 31 (63%) hadvs were serotyped, and typing results yielded some interesting observations. the major species found was hadv-d, with 29 clinical cases (59%), followed by hadv-c, with 18 cases (36%), and hadv-b, with two cases (4%). in bronchiolitis cases, hadv-5 (71%) was the major serotype, followed by hadv-6 (14%) and single cases of hadv-1 and hadv-d. in the group of patients with upper respiratory infection, the major serotype found was hadv-17 (77%). hadv-5 (22%) was the second-most common serotype, and five specimens were typed as hadv-d; one of them recovered in 2003 and four in 2005. lastly, two specimens recovered in 2003 were hadv-3. hadv-d was the species identified in nine (100%) cases detected during an atypical outbreak of acute febrile syndrome in july of 2005. four isolates from clinical materials obtained from patients with severe disease such as encephalitis, afp and meningoencephalitis were identified as hadv-d. hadvs are categorized by species (hadv-a, hadv-b , hadv-c, hadv-d, hadv-e and hadv-f), and further by serotype (ad1-ad51), and cause a wide spectrum of illnesses in both children and adults, including those involving the cns. a new recently isolated serotype (52) has been proposed to belong to a new species g [3, 13, 21] . more than half of the positive samples (53%) were from children younger than 5 years of age, and 50 % of them were under 6 months of age. the highest incidence of adenoviral infection occurs in children from 6 months to 5 years of age, although outbreaks have been noted when susceptible hosts, such as military recruits, adolescents, visitors to summer camps, and sometimes nursing home residents, congregate together. explanations for the relative lack of illness in the youngest or in older individuals include the presence of transplacentally acquired maternal antibody in young infants and the development of neutralizing antibody to the most common adenoviral strains in the majority of children older than 5 years old. the age distribution of hadv in developing countries appears to be similar to that in developed countries [8] . the results in this study showed that adenovirus infections were more common in children younger than 5 years of age, and 50% of them were under 6 months of age. most patients were hospitalized (73%); nevertheless, none of these had severe respiratory infections. in cuba, infants are considered special patients by pediatricians. this report also describes the seasonal variation of adenovirus infections in cuba. respiratory viral agents usually have characteristic seasonal patterns in temperate and tropical climates. in temperate climates, the majority of isolations of respiratory viruses are in the winter. in central america, there are few studies published about the epidemiological characteristics of adenoviruses. the cuban island is located in the caribbean sea at the entrance of the gulf of mexico. the climate of cuba is semitropical, and two seasons are generally recognized: a rainy season from may to october and dry season from november to april. cuba is often hit by hurricanes from june to november, resulting in great economic loss and temporarily interrupted sanitary conditions. the average minimum temperature is 21°c (70°f), and the average maximum is 27°c (81°f). the warmest month is july with 30°c (86°f). in this study, adenovirus was detected throughout the year; however, two significant outbreaks of hadv infection were identified, the first in july of 2005, when hurricane dennis hit severely the western part of cuba, and the second in september of 2006. we believe that the effect of temperature and rainfall appears to be a determinant of the timing of those outbreaks. figure 1 displays a phylogenetic tree built from an alignment of the nucleotide sequences of the amplicons obtained from clinical samples and each serotype prototype. as shown in fig. 1 , each serotype is clearly distinct. serotypes belonging to the same species cluster together. in cuba, human adenovirus circulates in the pediatric population throughout the year. furthermore, members of adenovirus species c of are commonly involved in respiratory diseases in the pediatric population. two previous studies have been reported on the associations between specific clinical syndromes and hadv serotypes. the analysis of the occurrence of hadv-c revealed that hadv-1 and hadv-6 were the predominant serotypes in children with acute respiratory diseases, and hadv-37 was the most prevalent one associated with conjunctivitis [36, 41] . human adenovirus respiratory infections have usually been associated with species b, c, and e [8] . hadv-c includes hadv-1, hadv-2, hadv-5, and hadv-6. these serotypes are commonly associated with febrile respiratory illness in children and are noted to be endemic in certain regions or in epidemics [32] . the significant proportion of infection with hadv-1, hadv-3, hadv-5 and hadv-6 among patients with respiratory infection is consistent with previous reports [7, 20, 23, 32] . however, we detected hadv-d in a child with bronchiolitis, and in 15 cases of upper respiratory infection. hadv-d are rarely isolated in respiratory illness surveys, and strong causal correlations are generally lacking. nevertheless, this association was previously described by casas et al. [7] . on the other hand, hadv-d was identified in a group of patients with acute febrile syndrome and in four isolates from clinical materials obtained from patients with encephalitis, afp and meningoencephalitis. sporadic cases or small outbreaks of neurological disease following adenovirus infection are well documented [12, 17, 25, 38] . the high prevalence of infection with hadv-d in previously healthy patients is not consistent with previous reports. this association was unexpected, and a detailed analysis of this sample set will be published in a separate report. human adenovirus is unique among the common respiratory viruses in that it can spread to organs other than the respiratory system, resulting in conjunctivitis, gastroenteritis, acute hemorrhagic cystitis, and meningoencephalitis. the liver, spleen, pancreas, kidney, or heart may also be involved in disseminated infections, both in previously healthy people and in immunocompromised patients (e.g., infants, patients with aids, transplant recipients) [33] . hadv-d has been frequently associated with severe adenovirus infection in these patients. in our study, we reviewed the medical records and did not find that the patients suffered from aids or another type of immunodeficiency. however, we thought that is important to take into account that the highest percentage of hadv-d was identified in children. the identification of such a large number of hadv-d infections is extremely interesting and a major and unique finding. we recognize that the limited sequence data analyzed (370 nt in a relatively conserved region of the hexon) for typing did not generate enough results. the sequencing of the hypervariable regions of the hexon gene (hvr7 alone or hvr1-6) and fiber gene of select strains will certainly provide a solid basis for confirming the species/serotype identification [29, 37, 43] . we used the method previously published by casas et al. [7] , to facilitate sequencing and typing of hadvs using the hexon protein coding region of the hadv genome. this method was extensively validated using clinical samples and prototype strains, and we also noted unusual associations between specific serotypes and clinical presentation. the primers and pcr conditions have also been extensively validated. some years ago, adenovirus infection was considered to have little consequence. however, much has changed since these early epidemiological studies were conducted. in contrast to the modest number of hadvs recognized 35 years ago, 51 unique serotypes are now recognized, and a new serotype isolated recently (52) has been proposed to belong to a new species g. different serotypes have been found to have different tissue tropisms that correlate with different clinical manifestations of infection. partial epidemiological investigations have revealed that, among some specific serotypes, multiple genetic variants exist that often have quite different geographical distributions and associated virulence [19, 21, 28, 30] . our study expands the range of hadv serotypes that have been reported elsewhere in association with major clinical syndromes. further independent testing is needed to verify these associations. however, future studies of hadv should not exclude these rare serotypes, and they should be kept in mind. unfortunately, because adenoviruses can be asymptomatically shed for prolonged periods of time, recovery of hadv from the upper respiratory tract or stool samples by culture does not confirm it as the cause of a specific disease. accordingly, recovery of hadv should always prompt an effort to identify any additional or alternate potential explanations for symptoms present at the time of a positive viral culture [4, 16] . additional research is needed for the development of better rapid methods to detect and to quantify hadv in various body fluids (blood, stool, and throat). finally, our findings, combined with the existence of several reports concerning the association of hadv with different syndromes confirm that members of the family adenoviridae, mainly the serotypes belonging to hadv-c are common causal agents of respiratory infection. in contrast, serotypes of hadv-d could be involved in the aetiology of acute respiratory infection and neurological disorders in previously healthy people. however, this observation needs to be confirmed in a larger study. our identification of hadv-d associated with different syndromes may signify the emergence of new genomic variants that have the potential to spread globally. further studies of such typing with other molecular typing methods are necessary. these reports show that surveillance, serotyping and molecular characterization methods need to be improved in order to identify emerging adenovirus variants. authors wish to thank all of the physicians who provided samples and clinical data. we also acknowledge all technicians and researchers of the virology deparment of the instituto de medicina tropical pedro kourí, la habana, cuba, who collaborated with this investigation. thanks to dr enrique tabarés, from universidad autónoma de madrid, spain, for his help. rapid and sensitive diagnosis of human adenovirus infections by a generic polymerase chain reaction outcome and clinical course of 100 patients with adenovirus infection following bone marrow transplantation family adenoviridae infections in 18, 000 infants and children in a controlled study of respiratory tract disease. ii. variation in adenovirus infections by year and season intravenous cidofovir therapy for disseminated adenovirus in a pediatric liver transplant recipient new method for the extraction of viral rna anddna from cerebrospinal fluid for use in the polymerase chain reaction molecular identification of adenoviruses in clinical samples by analysing a partial hexon genomic region molecular and clinical characteristics of adenoviral infections in taiwanese 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new respiratory threat? molecular typing of human adenovirus by pcr and sequencing of a partial region of the hexon gene pcr analysis of egyptian respiratory adenovirus isolates, including identification of species, serotypes, and coinfections hemorrhagic cystitis caused by adenovirus type 34 after allogeneic bone marrow transplantation molecular epidemiology of human adenovirus isolated from children hospitalized with acute respiratory infection in sao paulo, brazil disseminated adenovirus disease in immunocompromised and immunocompetent children development of a novel protocol for rt-multiplex pcr to detect diarrheal viruses among infants and children with acute gastroenteritis in eastern russia the distribution of adenovirus antibodies in normal children isolation and identification of adenovirus in hospitalized children, under five years, with acute respiratory disease comprehensive detection and serotyping of human adenoviruses by pcr and sequencing a syndrome of transient encephalopathy associated with adenovirus infection serotyping of adenoviruses on conjunctival scrapings by pcr and sequence analysis detection and typing of human herpesviruses by multiplex polymerase chain reaction the incidence of adenoviruses in viral conjunctivitis characterization of fastidious adenovirus type 40 and 41 by dna restriction analysis and by neutralizing monoclonal antibodies species-specific identification of human adenoviruses by a multiplex pcr assay acknowledgment this work was supported in part by the cuban national program of surveillance and control of acute respiratory infection. the nucleotide sequence was performed in the instituto de salud carlos iii, madrid, spain, and was funded by the proposal mpy-1177/06 entitled caracterización molecular de hadv detectados en muestras clínicas de pacientes con infección respiratoria. the key: cord-268378-tcuv255v authors: hood, ernie; jenkins, kristin p. title: evolutionary medicine: a powerful tool for improving human health date: 2008-02-13 journal: evolution (n y) doi: 10.1007/s12052-008-0036-9 sha: doc_id: 268378 cord_uid: tcuv255v modern evolutionary research has much to contribute to medical research and health care practices. conversely, evolutionary biologists are tapping into the rapidly expanding databases of medical genomic information to further their research. these two fields, which have historically functioned in almost complete isolation, are finding mutual benefit in the exchange of information. the long-term benefits of this synthesis of two major areas of research include improved health care. recently, efforts to catalyze this relationship have brought together evolutionary biologists, medical practitioners, anthropologists, and ethicists to lay the groundwork for further collaboration and exploration. the range of overlap is surprisingly broad and potentially invaluable. evolution has not traditionally been considered to be an important aspect of medicine, and medical practitioners and researchers have not traditionally approached their work from the perspective offered by evolutionary biology. medical practice tends to focus on the immediate, or proximate, causes of disease, including genetics and lifestyle, to identify a potential treatment. an evolutionary viewpoint pushes the focus out farther to look at long-term ecological relationships, including symbiotic bacteria, parasites and pathogens, historical lifestyles, and the genetics of populations. conversely, evolutionary biologists have not traditionally paid much attention to the medical implications or applications of their findings, with a few exceptions such as the study of emerging infectious diseases. other evolutionary studies have seemed far removed from contemporary human health issues. recently, however, these traditions have been giving way to a new approach, as researchers and medical practitioners discover connections based on evolutionary biology that are leading them to new conclusions about their respective fields. evolution is providing clues about puzzling medical results, and studies of human health are giving us new information about the rate and driving forces of evolution. initiatives such as the human genome project and the haplotype map have provided new tools for studying evolution and human health. genes are important, environment is important, the interaction between the two is important-but so is human evolution, as it exerts an ongoing influence on characteristics of the human population. in may 2007, evolutionary and medical researchers, doctors, ethicists, and anthropologists met at the national evolutionary synthesis center to catalyze the emerging scientific discipline known as evolutionary medicine. although the participants work on different aspects of human health, they all realize the value of evolution in their work. as the title of the meeting-evolution in contemporary human populations: medical, genetic and behavioral implications (govindaraju 2007 )-suggests, this group of scientists clearly share the belief that evolution is going on in contemporary human populations. in fact, a recent study presents evidence that evolution may have been occurring even faster in human populations than expected over the past 10,000 years (hawks et al. 2007) . the field of medicine has developed to address the wide array of environmental and lifestyle-induced diseases. over time, doctors have learned more about the relationship between environment and disease, including factors such as hygiene and lifestyle. more recently, genomic studies have opened the door to studying genetic differences between populations, bringing us the possibility of more effective individualized medicine. all in all, medicine seems to be making great progress in preventing, treating, and curing disease without including evolutionary information. so what does understanding our evolutionary history add to our ability to improve human health? it has been shown that human evolution, in the form of natural selection for particular traits, can occur over the short term-just a few generations, "the blink of an eye to evolutionary biologists," according to yale university evolutionary biologist stephen stearns, one of the nescent meeting organizers. continued lactose tolerance in adults is an example of a trait that has evolved in certain populations within the last 10,000 years, since the domestication of milkproducing livestock (tishkoff et al. 2006; swallow 2003) . "if a doctor can become aware of how all these different traits actually interact with each other, then you begin to see your individual patient as something that isn't just the symptom sitting in front of you in the office that day," stearns says. "if you treat that symptom, then the perspective of evolutionary biology will tell you what all of the possible associated reactions are. the point is, they're a bit broader, and they may contain more surprises than a strictly medical orientation would give you. evolution gives you a broader view of the whole way an organism reacts, and why it does sobecause it's been shaped by evolution in certain ways." psychiatrist randolph nesse of the university of michigan, a strong advocate of evolutionary medicine, agrees: "once a doctor starts understanding how the body came to be, instead of seeing the body as a machine-which it's not-he or she starts seeing the body as a product of natural selection, where everything in the body is pretty good, because it's got to be, and nothing is perfect….this perspective is needed for literally every aspect of the body that's vulnerable to disease. why hasn't natural selection made it better? there are always good reasons." among those reasons is the idea that evolution involves an array of trade-offs, compromises in selection that favor one trait at the inevitable expense of another. stearns, nesse, and david haig of harvard university eloquently explain the importance of evolutionary trade-offs in the initial chapter of evolution in health and disease (stearns and koella 2007): no trait is perfect. every trait could be better, but making it better would make something else worse. our vision could be as acute as that of an eagle, but the price would be a decreased capacity to detect color, depth, and movement in a wide field of vision. if the bones in our wrists were thicker they would not break so readily, but we would not be able to rotate our wrists in the wonderful motion that makes throwing efficient. it the stomach made less acid we would be less prone to ulcers, but more prone to gi infections. every trait requires analysis of the trade-offs that limit its perfection. consideration of these compromises that have evolved over time can guide medical decisions about altering our physiology and behaviors. the evolutionary trade-off between survival and reproductive success of the individual is an interesting example. reproduction is expensive, on many fronts. just looking at mating success, high testosterone levels may provide a man with a competitive advantage in securing a mate, but will also decrease his resistance to pathogens and parasites (muehlenbein and bribiescas 2005) . a woman who has regular and frequent menstrual cycles has more opportunities to become pregnant, but is also at increased risk of cancer (strassmann 1999) . exposure to higher hormone levels, either incidentally or by design, can have unexpected side effects because of this evolutionary trade-off. reproductive success is ultimately the more important factor in evolution, as one's evolutionary fitness is measured by the number of offspring that survive to reproductive age themselves. the compromise results from the fact that we produce only one or two offspring at a time, and human offspring require extended parental care. humans who threw everything into mating but did not survive long enough to raise the few resulting offspring would be unsuccessful because their offspring would not survive either. radically changing traits that have been shaped over time runs the very real risk of tipping the balance in unexpected, and potentially unpleasant, directions. homo sapiens has been around for about 100,000 years, and both fossils and genomic studies present evidence of ongoing evolution in h. sapiens. as a species, h. sapiens has experienced many lifestyle changes-some generated by culture, others by new or changing environments. anthropology can provide valuable insights into our past, and this information can improve our application of evolutionary medicine (trevathan 2007) . we learn from studies of ancient societies, but also from the few remaining traditional hunter-gatherer societies and by comparisons between different societies. a major shift in lifestyle was the development of agriculture. agriculture led to many changes, including dietary alterations and exposure to zoonoses from domesticated animals. agriculture required groups to abandon a mobile hunter-gatherer lifestyle and live in sedentary populations, such as farming villages. ultimately, agriculture provided the ability to support large urban populations. sedentary groups encounter issues such as accumulation of wastes and transmission of epidemic diseases (armelagos et al. 2005) , issues that increase with population size. even more recently, increases in expected life span and change in diets have led to a rise in the frequency of chronic diseases (armelagos et al. 2005; leonard 2007 ). these changes in the environment and the increase in population size have the potential to create a surge in the rate of evolutionary change (hawks et al. 2007 ). clinicians and biomedical researchers need to incorporate an understanding of this mismatch to modernity into their explorations of pathophysiology. in simple terms, many aspects of our modern environment and lifestyle, including diet, exercise, exposure to chemicals, hygienic practices and circumstances, and a variety of other elements, are mismatched to our bodies' evolutionary state. for example, if-thanks to our prehistoric ancestors-our physiology is evolutionarily hardwired for low caloric intake and intense physical activity, our rich modern diets and sedentary lifestyles mismatch our innate metabolism (neel 1962; wang and mariman 2008) . evolutionary medicine tells us that many of today's so-called "diseases of civilization" that are so highly prevalent and growing rapidly in incidence, such as diabetes, obesity, and cardiovascular disease, may well be fueled by a metabolic mismatch (leonard 2007) . this phenomenon can be readily observed in contemporary populations that have undergone a "nutrition transition" in which rapid socioeconomic, demographic, and technological changes have brought profound changes in diet and activity patterns, often resulting in sudden and widespread increases in metabolic disorders. for example, a recent paper examined the changes in diet and health in the rapidly urbanizing black south african population. the study found that the change in lifestyle was accompanied not only by an improvement in micronutrients but also by an increase in obesity and chronic diseases (vorster et al. 2005 ). characterizing such mismatches through the use of today's analytical tools promises to shed important new light on the etiology of these and many other common, complex disorders (e.g., autoimmune, inflammatory, and mental/behavioral conditions) and offers an innovative approach to the identification of novel interventions and therapies. continuing to look at mismatches between what we are prepared for evolutionarily and what we experience with a modern lifestyle, we find puzzling results that require an understanding of our evolutionary history to solve. for example, recent studies have demonstrated a connection between decreased intestinal parasite load and increased incidence of autoimmune diseases such as asthma (lau and matricardi 2006) . in one study, children who had been dewormed showed an increase in the occurrence of allergic reactions to house mites. why? what was the link between removing worms and developing allergies? one proposed explanation is the "hygiene hypothesis" (strachan 1989) , which takes an evolutionary view and considers that the historically "normal" condition for humans is a constant barrage of parasites and that the immune response evolved in response to these conditions. an over-reactive immune system is a recognized cause of allergies, and it turns out that the presence of helminthes, common intestinal parasites, actually increases regulation and control of the immune response. when these children were dewormed, regulation of their immune response decreased, allowing an allergic response to house mites. an interesting application of this research is the experimental use of intestinal parasites in the treatment of autoimmune diseases such as irritable bowl syndrome, crohn's disease, and multiple sclerosis (wickelgren 2004) . evolution can also shed light on normal physiological responses such as vomiting and fever and guide medical interventions (nesse and williams 1998) . in terms of evolution, a trait will be selected for if it provides some advantage. an example is "morning sickness"-the nausea and vomiting suffered by 75% of pregnant women (badell et al. 2006) . nausea and vomiting are commonly experienced in the first and second trimesters, and generally, foods such as meat, fish, poultry, and eggs elicit the strongest responses. clearly, feeling nauseated and vomiting frequently are unpleasant experiences at the very least, and hospitalization for rehydration is frequently required. occasionally, more serious complications arise. however, there is significant evidence that this physiological process is associated with successful pregnancies (weigel and weigel 1989) . one tested hypothesis posits that this response is beneficial to both mother and embryo because it reduces the mother's intake of, and embryo's exposure to, potentially toxic chemicals and pathogens during key developmental stages (flaxman and sherman 2000) . in addition, the foods most commonly avoided are animal products which tend to harbor more pathogens than plant foods. the immune system of pregnant women is depressed to avoid rejection of the fetus, so both mother and embryo are at higher risk from pathogens. the benefits of this response may well justify the discomfort and risks-or at least provide some psychological comfort to suffering women. evolution is not something that happened long ago and then stopped. it remains an active force in the modern world not only in human populations but also in the pathogens and parasites with which we live. one need look no farther than the local news or health clinic for proof. the evolution of antibiotic-resistant strains of tuberculosis made the news in spring 2007 when a man with extremely drugresistant (xdr) tb made multiple international flights (cnn 2007) . fortunately, no one else was infected in this instance, but the recent evolution of drug-resistant tb has revived old fears, especially in places where the complicated medical treatment required for these strains is not available. evolutionary medicine can help physicians take into account the effects of people's changing environments and lifestyles. human behaviors such as conflict, poverty, and urbanization provide opportunities for infectious diseases to spread (halstead 1996) . today, more people live in areas of high population density-a change from the more traditional small community. high population density urban centers are ideal for transmission of pathogens, particularly when coupled with high rates of poverty (armelagos et al. 2005) . old antagonists are not the only concern. behaviors that bring humans into contact with pathogen reservoirs in wildlife, such as logging and the exotic animal and bush meat trades, also create opportunities for the transmission of "zoonoses"-pathogens normally found in animal hosts but capable of infecting humans (patz et al. 2004) . for example, logging brings roads to new areas, providing easy access for local inhabitants who come to hunt for food. in the process, hunters either become prey themselves as they are infected by novel pathogens or bring the pathogens back to populated areas in the form of bush meat (chomel et al. 2007 ). sometimes, the infection is relatively benignfor example, children often pick up worms from household pets-but sometimes, the transfer is deadly. the ancestor of the human immunodeficiency virus (hiv) is the simian immunodeficiency virus, which is found in a wide range of primate species and rarely causes disease in the primate host (hahn et al. 2000) despite the fact that hiv is devastating in humans. taking an evolutionary approach to medicine can help us deal with novel pathogens by increasing our understanding of the pathogen and facilitating the development of appropriate responses. when the mysterious severe acute respiratory syndrome (sars) virus emerged on the world scene, researchers quickly gathered information about its physical structure, life cycle, and genetics. by comparing its characteristics with those of known viruses, researchers were able to quickly identify the novel sars virus as a coronavirus (who 2003) . that crucial piece of information allowed health care professionals to develop appropriate diagnostic tools, preventive measures, and treatments based on knowledge of how other coronaviruses behave. additionally, the information about viral type allowed field researchers to identify the source of the infection based on known hosts of coronaviruses. civet cats, sold live in meat markets, were frequently found to be infected with a sars virus. further research indicated that horseshoe bats, which were also sold in these markets, had a more ancestral variant of the virus (britigan 2005; lau et al. 2005; wendong et al. 2005) . the virus was also found to be more widespread in bats. the civet cats probably act as an intermediate host, or "amplifier." understanding the lifestyle and environment in which the disease arose helped to rapidly control it, preventing a feared worldwide epidemic. recently, new technologies such as improved genome sequencing and microarrays are generating huge quantities of genomic information. bioinformatic tools for analysis of this ever-expanding volume of raw data are developing apace, and genomics, the study of whole genomes, has become an independent biological discipline. the medically relevant value of this data is the potential to identify the genes underlying disease for the purpose of developing treatment or even prevention. evolutionary medicine plays a critical role in the interpretation and application of this information, since human evolutionary history has shaped the genome. humans as a species are relatively young with a mere 100,000-year history. the pattern of our relatively recent global dispersal from our home in africa is reflected in our genes. different populations demonstrate our common ancestry and recent isolation during dispersal (kidd and kidd 2007) . interpretation of genomic data must take our evolutionary history into account to yield useful applications. one of the most informative analytical approaches is comparison between sequences, either of small genomic regions or entire genomes. by examining differences and similarities between the genomes of two organisms, researchers learn about ancient genes that have deep evolutionary roots and about genetic differences between organisms that make them unique. in comparing the human genome with those of closely related primates, we learn what minor differences may be important keys to our human condition. comparisons between the human genome and more distantly related organisms provide information about ancient genes that play important roles in fundamental processes such as development and physiology. this can be done on various levels. microarrays allow comparisons between sets of genes or two entire genomes. bioinformatic tools allow comparisons of multiple genes, sections of genomes, or entire genomes. a new method that exploits both microarray technology and genome sequencing is the genome-wide association (gwa) study. in a gwa, a microarray of all of the known human single nucleotide polymorphisms (which are simply a change in one nucleotide) is screened for differences between diseased and healthy individuals. to give the study statistical significance, samples from thousands of individuals are used. in a recent gwa, researchers identified genomic polymorphisms potentially associated with seven different diseases (wellcome 2007). this information can be used to guide future research on the genetics of these diseases. the gwa is yet another method based on enormous data sets that may lead to significant advances in medicine. resources to promote this kind of megaanalysis include the human genome project and the hapmap, which uses collections of single-nucleotide differences as markers for larger genetic regions. the hapmap is important because genomes of any two humans differ only by 0.1%. researchers can use the general markers from the hapmap to narrow down their searches to areas of variation. an important key to drawing useful information from genomic data is the ability to link a genotype to a phenotype. this is a difficult problem for several reasons: genomic data are not usually collected from a specific individual with an extensive health history; family medical histories are rarely complete; environmental conditions vary between populations, etc. one approach to deal with this issue is the use of large, long-term studies of specific populations. the framingham heart study (fhs) is the quintessential large cohort, long-term study. initiated in 1948 to investigate the causes of heart disease and stroke, the project collected data on more than 5,000 people living in framingham, massachusetts. in addition to birth, marriage, reproduction, and death data, the participants were given physical exams and lifestyle interviews. followup physicals were given every 2 years. adult children of the original cohort and the children's spouses, as well as the original cohort's grandchildren, also participated in the study. this study allowed researchers to identify the major environmental risk factors for cardiovascular disease, such as smoking and high blood pressure. because of the extensive data collected for the project and the virtually unprecedented length of the study, the fhs is incredibly valuable to medical researchers. the data are now being mined by groups researching other diseases such as obesity and eye and lung diseases. other studies of large or multigenerational cohorts are also under way using different populations. diddahally govindaraju is the director of the fhs genetics laboratory at the boston university school of medicine and was one of the organizers of the nescent meeting. he is enthusiastic about the concept of applying evolutionary principles to cohort data such as that generated over nearly six decades in the fhs. "i was essentially sitting on a wealth of medical data that has been collected for the last 50 years," he says. "having been raised on evolutionary biological thought in the first part of my professional life, i looked at this and realized that the doctors were not looking at these data from an evolutionary biology perspective." dr. govindaraju and his colleagues have organized a working group to explore the fhs from an evolutionary viewpoint. the group proposes to look for microevolutionary changes in the study population and to use the extensive medical data to correlate differences in genomic inheritance and phenotypic outcomes. given the vast quantities of data available because of the genomics revolution and multi-generational, longitudinal clinical cohorts such as the framingham heart study, it has become increasingly clear that the analysis of new and existing data from an evolutionary perspective promises to yield important insights into long-standing questions about human physiology and pathophysiology. combined with the development of new technologies that have given us entire genomes and the tools with which to study them, these vast data sets have the potential to launch an evolutionary medicine revolution. it should come as no surprise that evolutionary medicine comes with a unique set of ethical issues. a field that encompasses so many aspects of who we are as individuals and has the power to provide better health to so many people also has the potential to be abused in a variety of ways. positive applications such as personalized medicine, in which an individual would receive the most beneficial medical treatment based on his or her genomic makeup, hold great promise for improving the efficacy of medical care. however, it is no great stretch to imagine that genetic information about individuals could be misused outside of the medical realm. communication of complex evolutionary medicine concepts to the general public will be fraught with cultural perspectives, biases, and historical antagonism, and miscommunication could have serious consequences for individuals and society. privacy concerns are a major issue. when so much data, including genetic information, are being gathered on so many people, how can a participating individual's privacy be protected while simultaneously making the information as widely available as possible (nhgri 2007) ? also, the use of particular populations in studies is a double-edged sword. in some cases, researchers can learn a great deal by studying a particular group whose members share a lifestyle or ancestry. but how can that group be certain that the information will not be used against them, by insurance companies, for example? on the other hand, if beneficial information is gleaned from one group, how is that benefit made available to other groups who were not studied? another important issue to consider is the concept of race (kittles and weiss 2003) . particularly in the usa, the sensitive nature of this issue cannot be overstated. the genetic concept of race and the social concept of race are not the same. in colloquial use, the term race designates a group of people based on their appearance and culture. among the research community the definition of race has not been consistent or well defined. scientifically, race correlates roughly with shared genetic ancestry usually based on geographic proximity, but even these parameters fail to clearly delineate groups. there is some debate as to whether race is a legitimate biological concept at all. social concepts of race also influence how research is done by biasing selection of populations to be sampled, methodologies, and other aspects of research. paradoxically, some diseases are associated with socially defined races, such as tay-sachs disease in the ashkenazi jews, making it necessary to take into account these social groupings in conducting research. the exciting potential of evolutionary medicine to yield important new insights into long-standing questions about human physiology and pathophysiology was summed up by stearns: "when you see how evolution illuminates those questions, you see there's the potential for a new way of looking at them to save literally hundreds of millions of lives. that is an enormous payoff, and it's the kind of thing that people in medical research have felt all of their lives, but it's a new motivation for evolutionary biologists, and it's a revelation to them to realize that they could bring that to the table." evolutionary, historical, and political economic perspectives on health and disease treatment options for nausea and vomiting during pregnancy journal watch infectious diseases exotic pets, and emerging zoonoses man knew he had tb before flying to europe morning sickness: a mechanism for protecting mother and embryo evolution in contemporary human populations: medical aids as a zoonosis: scientific and public health implications human factors in emerging infectious diseases recent acceleration of human adaptive evolution human genetic variation of medical significance race, ancestry, and genes: implications for defining disease risk worms, asthma, and the hygiene hypothesis severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats lifestyle, diet and disease: comparative perspectives on the determinants of chronic health risks testosterone-mediated immune functions and male life histories genetic discrimination diabete mellitus: a "thrifty" genotype rendered detrimental by "progress evolution and the origins of disease members of the working group on land use change disease emergence. unhealthy landscapes: policy recommendations on land use change and infectious disease emergence (eds) evolution in health and disease hay fever, hygiene, and household size menstrual cycling and breast cancer: an evolutionary perspective genetics of lactase persistence and lactose intolerance convergent adaptation of human lactase persistence in africa and europe evolutionary medicine the nutrition and health transition in the north west province of south africa: a review of the thusa (transition and health during urbanization of south africans) study insulin resistance in an energy-centered perspective nausea and vomiting of early pregnancy and pregnancy outcome. a meta-analytical review wellcome trust case control consortium. genome-wide association study of 14,000 cases of seven common disease and 3,000 shared controls bats are natural reservoirs of sars-like coronaviruses can worms tame the immune system coronavirus never before seen in humans is the cause of sars acknowledgments the authors would like to thank the organizers and participants of the evolution in contemporary human populations: medical, genetic and behavioral implications meeting at nescent for providing access to the meeting and supporting information, and reviewers for their helpful comments. key: cord-000261-ip32y0j5 authors: becker, pablo d.; legrand, nicolas; van geelen, caroline m. m.; noerder, miriam; huntington, nicholas d.; lim, annick; yasuda, etsuko; diehl, sean a.; scheeren, ferenc a.; ott, michael; weijer, kees; wedemeyer, heiner; di santo, james p.; beaumont, tim; guzman, carlos a.; spits, hergen title: generation of human antigen-specific monoclonal igm antibodies using vaccinated “human immune system” mice date: 2010-10-04 journal: plos one doi: 10.1371/journal.pone.0013137 sha: doc_id: 261 cord_uid: ip32y0j5 background: passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. however, the ‘humanization’ of murine monoclonal antibodies (mabs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. the immortalization of human b cells represents an alternative for obtaining human mabs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. in this work we describe a novel approach to generate fully human mabs by combining a humanized mouse model with a new b cell immortalization technique. methodology/principal findings: after transplantation with cd34(+)cd38(−) human hematopoietic progenitor cells, balb/c rag2(−/−)il-2rγc(−/−) mice acquire a human immune system and harbor b cells with a diverse igm repertoire. “human immune system” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis b surface antigen. sorted human cd19(+)cd27(+) b cells were retrovirally transduced with the human b cell lymphoma (bcl)-6 and bcl-xl genes, and subsequently cultured in the presence of cd40-ligand and il-21. this procedure allows generating stable b cell receptor-positive b cells that secrete immunoglobulins. we recovered stable b cell clones that produced igm specific for tetanus toxoid and the hepatitis b surface antigen, respectively. conclusion/significance: this work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mabs against a wide range of antigens. hyper-immune sera containing polyclonal immunoglobulins (igs) have been widely used in both therapeutic and prophylactic clinical settings [1] . however, the use of polyclonal sera was associated with several problems, such as the stimulation of allergic reactions, low reproducibility between clinical batches and high off-label use, which finally caused a decline in their use [2] . the advent of technologies to make monoclonal antibodies (mabs) derived from animals, especially mice, has overcome many of the problems associated with the use of polyclonal sera. the technology to make monoclonal cell lines of antibody-producing cells by fusing antibody producing plasma cells with myeloma cells was described for the first time in 1975 by milstein and kohler [3] . the therapeutic potential of mabs was immediately recognized and in 1980 the first mab, okt3, was approved for therapeutic applications. this antibody inactivates t cells, thereby preventing rejections of organ transplants [4] . however, because of the animal origin of the first generation of mabs that were used in clinical trials, human subjects treated with these antibodies developed vigorous immune reactions against the animal proteins, which were thereby eliminated preventing their therapeutic actions [5] . to overcome these problems technologies were developed to diminish the immunogenicity of mouse antibodies by replacing part or the complete mouse antibody backbone by its human equivalent, first generating chimeric, and subsequently fully humanized antibodies [6] . in a parallel approach transgenic mice bearing the human ig region were created to obtain fully human antibodies following immunization. the use of these mice obviates the elaborate molecular engineering of antibodies that is needed to humanize antibodies generated in wild-type mice, however, the maturation process of the mouse b cells expressing human igs is different from that of fully human b cells [7] . immortalization of b cells from immune humans seems to be the logical strategy to avoid these problems. however, the methods to achieve this goal have showed low efficiencies, although some progress has recently been reported [8, 9] . nevertheless, the major disadvantage of human b cells immortalization is the need for cells from either vaccinated individuals or patients who had recovered from an infection. thus, to fully exploit the ig repertoire of human b cells in an in vivo setting, we explored the possibility to raise mabs following de novo induction of human b cell responses in mice carrying elements of the human immune system (his). his mice are generated by engrafting immunodeficient mice with human hematopoietic stem cells (hsc) with or without human lymphoid tissues from fetal origin [10, 11, 12] . in particular, mice deficient for the recombinase activating gene-2 (rag2) and the common gamma chain of the il-2 receptor (il2rg) on a balb/c or a non-obese diabetic (nod) background are permissive for human hsc xenografts. inoculation of newborn mice from these strains with human hsc of fetal or umbilical cord blood origin gives rise to robust engraftment of a number of immune cells, including t, b, nk and dendritic cells. in this work, we describe a convenient approach to generate fully human mabs based on the immunization of balb/c rag2 2/2 il-2rcc 2/2 engrafted with human cd34 + cd38 2 hsc [13, 14] . to this end, his mice were immunized with commercial vaccines against hepatitis b virus (hbv) and tetanus. following immunization, human cd19 + b cells were sorted based on surface cd27 expression, as a marker of memory phenotype, and the isotype of surface igs. the sorted b cell populations were immortalized in vitro by retroviral transduction with human b cell lymphoma (bcl)-6 and bcl-xl genes and antigen-specific b cell clones were established and characterized. the obtained results provided the proof-of-concept for the usefulness of this generic approach based on his mice combined with immortalization of human b cells for the rapid and inexpensive development of human mabs against a wide range of antigens. the use of fetal liver tissue obtained from elective abortions with gestational age ranging from 14 to 20 weeks was approved by the medical ethical committee of the amc-uva and was contingent on informed written consent. balb/c rag2 2/2 il-2rcc 2/2 mice were bred and maintained in individual ventilated cages, and fed with autoclaved food and water. his mice were generated as previously described [13, 14, 15, 16] , with the approval of the animal ethical committee of the amc-uva (permit number dhl-100970). in brief, human fetal livers were obtained from elective abortions with gestational age ranging from 14 to 20 weeks. magnetic enrichment of cd34 + cells (.98% pure) was performed by using the cd34 progenitor cell isolation kit (miltenyi biotech), after preparation of single cell suspensions and isolation of mononuclear cells by density gradient centrifugation over lymphoprep (axis shield). finally, newborn (,5 days old) sub-lethally irradiated (3.5 gy) balb/c rag2 2/2 il-2rcc 2/2 mice were injected via intra-hepatic route with 5-10610 4 sorted cd34 + cd38 2 human fetal liver hematopoietic stem cells in 30 ml. all manipulations of his mice were performed under laminar flow. cell suspensions were prepared in rpmi medium supplemented with 2% fetal calf serum (fcs). twelve to sixteen weeks after cd34 + cd38 2 hsc engraftment, his mice were killed and single cell suspensions of splenocytes were prepared. red cells lysis was performed in 1 ml of red cell lysis buffer (sigma) for 10 min. splenocytes were washed, resuspended in 600 ml of rlt lysis buffer (qiagen) and homogenized by passing through a 21-gauge needle several times using rnase free syringes. rna was prepared using rneasy mini kits (qiagen) according to manufactures instructions. bcr v h immunoscope was performed as previously described [17] . briefly, cdna was prepared and real-time pcr performed by combining primers for the different v h chains (v h 1-7) and specific fluorochrome-labeled probes against the different constant regions (c h m, c h a and c h c). an additional four pcr cycles 'run-off reactions' were then performed on the pcr products using fluorescent primers specific for the constant regions (fcm, fca and fcc). products were gel separated to determine cdr3 lengths. analysis of six individual his-mice containing greater than 30% human chimerism in the spleen was performed. the number of human cd19 + b cells in chimeric spleens ranged from 5-12610 6 . eight weeks after hsc transplantation, blood was taken from his mice to verify the level of engraftment by flow cytometry, as described elsewhere [18] . his mice with a good level of human reconstitution (.20% hcd45 + cells) were immunized by intramuscular route (biceps femoris) using a 29g needle, three times on weeks 14, 16 and 18 with either 100 ml of the hbv vaccine (engerix-b, glaxosmithkline) or 50 ml of tetanus toxoid (tt) containing vaccine (tetanus vaccine, the netherlands vaccine institute). these amounts correspond to 1/10 of the normal human dose. negative controls received the same volume of pbs buffer. two weeks after the last immunization, his mice were exsanguinated under isofluran/oxygen narcosis. spleens and mln were removed aseptically and cellular suspensions were prepared. the bm cells were isolated from the femur and tibia. from beckman coulter; cd3 (sk7), cd4 (sk3), cd8 (sk1), cd19 (hib19), cd38 (hit2), cd45 (2d1 and hi30), cd45ra (hi100), cd138 (mi15), igm (g20-127), igd (ia6-2), igg (g18-145) and ccr7 (3d12) from bd biosciences; cd27 (lt27) from abd-serotec; cd27 (lg.7f9) from ebioscience. tt-specific b cells were also occasionally stained with pe-coupled tt, kindly provided by dr. andreas radbruch (german rheumatism research center, berlin, germany). dead cells were excluded based on dapi incorporation. all washings and reagent dilutions were done with pbs containing 2% fcs and 0.02% nan 3 . stained cells were analyzed with an lsr-ii interfaced to a facs-diva software system (bd biosciences). cell sorting of b cell subsets were performed on his mouse bm and spleens using a facs-aria cell sorter interfaced to a facs-diva software system (bd biosciences). for these experiments, all washings and reagent dilutions were done with 2% fcs supplemented pbs without nan 3 . the human bcl6 [19, 20] and bcl-xl [21] encoding cdnas were further cloned in a lzrs retroviral expression vector, around a t2a cleavage-promoting peptide sequence and upstream a cassette containing an internal ribosome entry site (ires) and the gene encoding gfp. we therefore obtained a lzrs vector in the following configuration: bcl6-t2a-bclxl-ires-gfp [9] . transfection of phoenix-galv packaging cells and virus production were performed as previously described [22] . before retroviral transduction, memory b cells were activated on c-irradiated (50 gy) mouse l cell fibroblasts stably expressing cd40l (cd40l-l cells) in the presence of 25-50 ng/ml recombinant mouse interleukin-21 (rmil-21, r&d systems) for 36 h [19] . the b cells were washed, mixed with retroviral supernatants in retronectin-coated plates (takara), centrifuged at room temperature for 60 min at 360 g, and subsequently incubated with the retroviruses at 37uc, 5% co 2 for 6-8 h. transduced b cells were maintained in co-cultures using cd40l-l cells (10 5 cells/ml) and in standard imdm (gibco) culture medium supplemented with 8% fetal bovine serum (fbs; hyclone), penicillin/streptomycin (roche) and 25 ng/ml rmil-21. the analysis of human ig-v h sequences was performed as follows. total rna was isolated from approximately 5610 5 monoclonal b cells with trizol (invitrogen). the cdna was generated and subjected to pcr with primers specific to the different v h family members. pcr products were sequenced to determine the cdr3 region of the different clones. sequence analysis was performed using bigdye terminator chemistry (applied biosystems inc.) and codoncode aligner software. the plasma harvested from his mice (7 days after the first and second immunization; 10 days after the third immunization) and b cell clone culture supernatants were screened by elisa for the presence of total human igm, total human igg and antigenspecific antibodies. measurement of total igm and igg was performed by coating 96-well plates either with affinipure f(ab') 2 fragment goat anti-human igm (fc5m-specific, jackson immu-noresearch) or affinipure goat anti-human igg (fcc fragmentspecific; jackson immunoresearch). control human serum protein calibrator (dako) with known igm (0.8 mg/ml) and igg (10.4 mg/ml) concentrations was used as a standard to be compared to the samples. for the detection of antigen-specific antibodies, 96-well plates were coated either with tetanus vaccine (nederlands vaccin instituut) or engerix b (glaxosmithkline) (106 diluted in pbs) for 1 h at 37uc or overnight at 4uc. alternatively, ridascreen tetanus igg elisa plates (biopharm) were also used to screen for tt-specific antibodies. after coating, the plates were washed in elisa wash buffer (pbs, 0.5% tween-20). a pbs solution containing 4% of milk was used as a blocking agent, before adding serial dilution of his mouse plasma (starting at a dilution of 1:5) or cell culture supernatants (starting at a dilution of 1:2). enzyme-conjugated detection antibodies were added at a dilution of 1:2500 for hrp-conjugated anti-igg and a dilution of 1:5000 for hrp-conjugated anti-igm (both from jackson immunoresearch). then, tmb substrate/stop solution (biosource) was used for the development of the elisa assay. statistical analyses were performed using graphpad prism version 5.02 for windows (graphpad software). data were subjected to two-tailed unpaired student t test analysis. the obtained p values were considered significant when p,0.05. we have generated his mice by transplanting human hsc into alymphoid balb/c rag2 2/2 il-2rcc 2/2 newborn mice ( figure 1a ). as reported previously, multilineage human hematopoietic reconstitution is observed in his mice, which demonstrate human thymopoiesis, b cell differentiation, nk cell and plasmacytoid dendritic cell development, and myelopoiesis [10, 13, 14, 15, 16] . human immune cells accumulate in lymphoid tissues, and several b cell subsets are observed in his mice ( figure 1b) . we analyzed the human b cell repertoire present in naive his mice by using b cell receptor (bcr) immunoscope analysis based on quantitative pcr of ig variable (v h ) and constant (c h ) region gene segments [17] . due to the lack of human spleen samples, the cells isolated from his mouse spleens, which contained sufficient numbers of human b cells to perform the immunoscope analysis, were compared to control human peripheral blood mononuclear cells (pbmc) samples, which were considered acceptable for the purpose of the performed comparison. we observed that igm-expressing b cells as well as ig isotype-switched b cells are found in naive his mice ( figure 1c) . the vast majority of b cells of his mice expressed an igm (97.961.0%), whereas igg (1.861.0%) and iga (0.0760.04%) expressing b cells represented minor populations. only the frequency of iga-expressing b cells was found significantly higher in control human pbmc samples (p,0.0001). at 10-14 weeks post-transplantation (i.e. in steady state conditions), the human ig concentrations in the blood were 12268 mg/ml (igm) and 143612 mg/ml (igg) ( figure 1d ), as previously reported [8, 16] . in comparison, the normal range for ig concentration in healthy humans is 400-3100 mg igm/ml and 7200-14700 mg igg/ml. in brief, despite a low frequency of igg-expressing cells, both human igm and igg accumulated in the plasma of ,3 month-old his mice to levels representing around 10% and 1% of adult human igm and igg concentrations, respectively. we further examined the antigen receptor repertoire diversity in his mice, by determining the length of cdr3 hypervariable regions for each ig-v h gene family. the analysis of cdr3 length distribution of individual his mouse splenocytes showed that igm repertoires are undistinguishable from normal human pbmc igm repertoires, as measured by the bcr immunoscope analysis ( figure 1e ). this observation suggests that his mice contain a broad variety of naive igm + b cell clones. the v h -family usage was large and similar to control human pbmc ( table 1) . the bcr immunoscope analysis was also performed for igg and iga repertoires and we observed more restricted repertoires, as expected from b cells undergoing clonal selection and ig class switch recombination ( figure s1 ). immunization of his mice with hbv and tetanus vaccines results in the generation of antigen-specific antibody responses since his mice contained broad naïve b cell repertoires, we analyzed the induction of human antigen-specific b cell responses after immunization with commercially available human vaccines. we designed a vaccination protocol based on repeated intramuscular immunizations (3 injections with 2-week intervals) of 10-14-week old his mice with vaccines containing hepatitis b surface antigen (hbsag) or tt. seven days after the last immunization mice were sacrificed, the blood and the lymphoid organs were harvested, and the phenotype and function of human cells was analyzed. all his mice showed human reconstitution (.20% hcd45 + cells) in the blood before starting the immunization protocol, which correlated with human engraftment in lymphoid organs. overall, 42% of hbsag-vaccinated (8 out of 19 vaccinated animals) and 40% of tt-vaccinated (6 out of 15) his mice showed significant production of antigen-specific igm antibodies, as detected by elisa (figure 2a) . we performed a kinetic monitoring of antigen-specific plasma ig levels in individual hbsag-vaccinated responder his mice and we observed that after the first immunization antigen-specific igs were rarely detected. in contrast, after the second immunization antigen-specific igm was detected, which steadily increased after the third immunization with approximately 25-40% of responder mice also showing an antigen-specific igg response (figure 2a) . this suggests that repeated vaccination leads to enhanced antigen-specific antibody production. the responder mice exhibited higher total igm (173641 mg/ml) and total igg (4596140 mg/ml) concentrations in their plasma, as compared to pbs-injected (igm: 37612 mg/ml; igg: 191658 mg/ml) and non-responder vaccinated (igm: 44611 mg/ml; igg: 192667 mg/ml) animals ( figure 2b) . at the end of the immunization protocol, vaccinated animals showed significantly higher numbers of hcd45 + cells in all organs (i.e. spleen, bone marrow (bm) and mesenteric lymph nodes (mln)) in comparison to mock-injected control mice. responder his mice exhibited higher numbers of human t and b cells in the spleen, as well as t cells in the bm ( table 2; table s1 ), suggesting that the vaccination protocol had a positive impact on the accumulation of human b and t cells. moreover, the mln isolated from vaccinated his mice contained 4 to 5-fold more hcd45 + cells than those of control animals ( figure 2c ), suggesting that the mln structure might play a role in eliciting an immune response in the his mice. in humans, the cd27 + memory b cell population contains the majority of antigen-experienced b cells [23, 24] , and we reasoned that the same should be true in vaccinated his mice. we therefore cell sorted several different cd19 + cd27 + b cell subsets from individual his mice. we used two strategies to isolate the following human b cell (cd45 + cd19 + ) subsets from bm and spleens of vaccinated his mice: (i) cd27 hi cd38 hi , (ii) cd27 + cd38 lo/int igd + , and (iii) cd27 + cd38 lo/int igd 2 on the one hand ( figure 3 , although the number of cells was increased for each of these subpopulations in the vaccinated animals, as expected from the enhanced number of total b cells ( table 2) . we only observed a significant increase in the frequency of igg + b cells within the cd27 hi cd38 hi plasmablast population of vaccinated responder his mice, as compared to pbs-injected animals (13.262.7% and 4.361.8% of cd27 hi cd38 hi b cells, respectively; p = 0.0311). in order to identify, isolate and immortalize the antigen-specific antibody-producing b cells, the aforementioned b cell subsets were transduced immediately after cell sorting with a retroviral vector encoding both human bcl6 and bcl-xl [9, 19] . by ectopically expressing bcl6 and bcl-xl in splenic or peripheral blood memory b cells and culturing them with factors produced by follicular helper t cells (cd40l and il-21), we generated highly proliferative, bcr positive b cell lines that secrete igs. since these cells express bcl6, the differentiation of memory b cells to terminal plasma cells is blocked [28, 29, 30] . therefore, the resulting b cells can expand extensively in vitro for long periods of time in presence of cd40l and il-21, and provide a tool to generate antigen-specific human bcr-positive, antibody-secreting b cell lines. the number of isolated cells from spleen and antigen-specific b cell clones that were generated with the bcl6/bcl-xl transduction approach is provided in the table s2 . since the frequency of antigen-specific b cell clones was unknown, we started with microcell cultures ranging from 0.6 to 640 cells per well. the wells containing antigen-specific b cells -as determined by hbsagspecific or tt-specific elisa -were subsequently cultured by limiting dilution to obtain monoclonal b cell lines. overall, we generated 15 anti-hbsag igm + b cell clones from 3 his mice vaccinated with hbsag, and 18 anti-tt igm + b cell clones from 3 his mice vaccinated with tt ( table s2 ). the estimated frequency of hbsag-specific b cells (clones) in the his mice after vaccination was 1/350. the igm secretion level of the b cell clones were in the range of 1 mg per 10 5 cells over 3 days in culture, which was in a similar range of secretion (0.6-5 mg/10 5 cells/3 days) to what was previously reported for b cell clones generated from human blood [9] . the igm v h regions of the bcr of the antigen-specific igm + b cell clones were sequenced. overall, the bcr of hbsag-specific and tt-specific b cell clones exhibited a v h sequence close to the germ-line sequence, although limited frequencies of somatic hyper-mutations were observed (table s3 and table s4 ). somatic hyper-mutations were occasionally detected in all framework regions (fr) and complementary determining regions (cdr), and most of the bcr diversity was the result of nadditions in the cdr3 region. based on the bcr sequence, we observed that 12 out the 15 anti-hbsag igm + b cell clones were unique, as well as 5 out the 18 anti-tt igm + b cell clones ( table s2, table s3 and table s4 ). the supernatants of tt-specific b cell clones were further tested for their capacity to recognize different antigens by elisa. we observed that igm mabs did not cross-react with unrelated antigens (i.e., hbsag and respiratory syncytial virus (rsv) antigens) ( figure 4a) . the tt-specific b cell clones were also screened by flow cytometry for direct binding of the tt antigen labeled with a fluorochrome ( figure 4b) . interestingly, three types of clones that produced antibodies that gave a similar signal in elisa were detected, with high, intermediate and low binding of the fluorescent tt antigen. in the present work we established a new approach to generate fully human mabs. we immortalized b cells from vaccinated his mice by transduction with bcl6 and bcl-xl followed by expansion in presence of il-21 and cd40l. antigen-specific b cell clones were obtained that expressed the bcr on their cell surface and secreted antigen-specific antibodies. similarly to methods based on the immortalization of human memory b cells from individuals that were either vaccinated or exposed to pathogens, our strategy exploits the antibody repertoire of human b cells which is likely to be different from that of b cells of mice expressing human ig gene segments. naïve his mice display an extensive human igm-expressing b cell repertoire. based on the analysis of the length of the cdr3 regions, this igm b cell repertoire is similar to the repertoire of healthy individuals. thus, his mice have no obvious limitations for the generation of human igm mabs against any possible antigen. upon intramuscular vaccinations with either tt or hbsag, approximately 40% of the his mice were able to mount an antigen-specific antibody response. human igm-producing b cell lines against both antigens were obtained after isolation of memory b cells followed by ex vivo differentiation into plasmablastlike cells. it is important to highlight that the selection of the antigen-specific human b cell clones relied on relevant bioassays (e.g., elisa or neutralization test). in contrast to ebv-based approaches, human b cell immortalization using transduction with bcl-6 and bcl-xl preserves the expression of the bcr at the surface and antigen-specific b cell clones can also be selected by binding of labeled antigen to the bcr of immortalized memory b cells (e.g. by using a labeled antigen). even when igg was used as a selection criterion, we were unable to establish antigen-specific igg + human b cell clones. the reason for this might be that t cell help in this system is suboptimal as indicated by the absence of antigen-specific t cell responses after vaccination (not shown). we also observed that the bcr of the b cell clones had a close to germ-line sequence, suggesting that also the induction of somatic hyper-mutation is sub-optimal in his mice. in our hands the great majority of the vaccinated his mice showed a defective formation of germinal centers [14, 16] , which further explains the absence of antigenspecific ig-class switched b cells. so far, humanized mouse models based on the transplantation of human hsc only -i.e. without additional human tissues -share these limitations, and immunization strategies result in the limited generation of class-switched antigen-specific b cell responses [14, 31, 32] . similar patterns are observed in human hsc-transplanted immunodeficient mice infected with lymphotropic pathogens, such as hiv [33] or ebv [34] , although dengue virus infection in his mice was reported to induce an igg response in a majority of the responder animals [35] . it is not clear why igg antigen-specific responses are limited while serum igg can accumulate efficiently, considering the low frequency of igg + b cells in his mice. it remains to be determined whether this apparent discrepancy might be explained by the conjunction of particularly effective igg production on a cell basis by igg + b cells (which might occur in a t cell independent manner, such as in the case of the igg3 subclass), long-term stability of human igg in the his mouse serum as compared to human igm, and/or defective survival of igg + b cells under specific conditions (e.g. after antigen-specific triggering of the bcr). although igm mabs might already be useful for some specific applications or could be modified by ig class swapping to obtain igg mabs [36] , optimized humanized mouse models with improved b cell function are highly desirable. one reason for the suboptimal interaction of t and b cells may be the poor survival resulting in a high turnover of human t cells (discussed in [10, 37] ), making it very likely that procedures leading to improved accumulation of human t cells may promote b cell responses and isotype switching. it was already shown that human b cells undergoing isotype switching can be obtained in humanized mice, provided that a human environment supporting this process is present, e.g. in scid mice transplanted with human fetal bones, thymus and lymph nodes [38] . consistent with this notion, enhanced human peripheral t cell accumulation was observed in nod/scid mice transplanted with human bone marrow hsc, fetal liver and fetal thymus tissues (referred to as blt mice), as compared to conventional humanized mouse systems [39] . interestingly, blt mice consistently generated an antigen-specific igg response after hiv-infection [40] . although it is yet unknown whether the isotype switch observed in blt mice is truly t cell dependent, those data might support the idea that improved t cell homeostasis has a positive impact on b cell responses. to obtain humanized mouse models with improved b and t cell homeostasis, alternative strategies not relying on the transplantation of human fetal tissues -which are not necessarily easy to access to, for ethical, legal or practical reasons -will likely be favored in the future. the replacement of mouse genes involved in the hematopoietic system by their human equivalent is a valuable strategy to improve development, maintenance and/or function of several hematopoietic cell subsets in humanized mouse models, as shown with cytokines, such as il-7 and il-15 [15, 21, 41, 42] , or mhc molecules (n.d.h and j.p.d., manuscript submitted) [43, 44] . the fact that the human cd47 was shown to be unable to properly interact with the mouse sirpa indicates that reintroducing a functional phagocyte inhibition mechanism via the cd47/sirpa signaling axis is another strategy of potential interest [45] . in conclusion, our results show using two standard vaccine antigens the general applicability of an innovative b cell immortalization method in combination with the his mouse model to generate human mabs. similarly to methods based on the immortalization of human memory b cells from vaccinated or convalescent individuals [9] , our approach exploits the broad antibody repertoire of human b cells, overcoming the potential limitations of conventional humanized murine mabs such as laboriousness or impaired biological properties, synthetic antibody libraries that require a known target antigen, and transgenic mice bearing the human ig locus that have limited b cell repertoires. in addition, our method enables to exploit experimental infection models and immunization regimes that would be unethical or untenable in humans. considering the upcoming advances in his mice models [37] , this new approach will provide a powerful tool to generate human mabs for either diagnostic or therapeutic purposes. figure s1 igg/iga b cell repertoire in naïve his mice. similarly to figure 1e , the naive igg (a) and iga (b) b cell repertoires of his (balb-rag/c) mice were evaluated on splenocytes by performing a bcr immunoscope for each v h family. the profiles obtained with control human pbmc are also shown. found at: doi:10.1371/journal.pone.0013137.s001 (1.61 mb tif) figure 3 . limited dilutions of b cells transduced with bcl-6 and bcl-xl were performed with 6.4 and 0.64 cells/well. after sub-cloning of the positive wells, we generated 15 igm + anti-hbsag mabs, of which 13 are unique (as determined by ig-v h sequence, see table s3 ), and 18 igm + anti-tt mabs, of which 5 are unique (see table s4 ). in the case of hbsag vaccination, the number of screened b cells was ((192*6.4)+(96*0.64))*3 = 3870, which eventually suggests that the frequency of hbsag-specific b cells is at least 1/350 b cells. found at: doi:10.1371/journal.pone.0013137.s003 (0.13 mb doc) igm v h amino-acid sequence of generated hbsagspecific b cell clones. the germ-line sequence is given for each v h family, with indication of framework regions (fr) and complementary determining regions (cdr). highlighted amino-acids correspond to n-additions (in the cdr3 region) and somatic hyper-mutation events, whether it results in a silent mutation (green) or not (red). clones with identical bcr sequences are grouped together. found at: doi:10.1371/journal.pone.0013137.s004 (0.02 mb pdf) the mechanism of diphtheria immunity and tetanus immunity in animals immunogenicity of therapeutic monoclonal antibodies continuous cultures of fused cells secreting antibody of predefined specificity upping the ante on antibodies mice with a human touch molecular engineering and design of therapeutic antibodies from xenomouse technology to panitumumab, the first fully human antibody product from transgenic mice an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus generation of stable monoclonal antibody-producing b cell receptorpositive human memory b cells by genetic programming experimental models to study development and function of the human immune system in vivo human-hemato-lymphoid-system mice: opportunities and challenges humanized mice in translational biomedical research monitoring the effect of gene silencing by rna interference in human cd34+ cells injected into newborn rag2-/-gammac-/-mice: functional inactivation of p53 in developing t cells development of a human adaptive immune system in cord blood celltransplanted mice il-15 trans-presentation promotes human nk cell development and differentiation in vivo t cellindependent development and induction of somatic hypermutation in human igm+ igd+ cd27+ b cells many human peripheral vh5-expressing igm+ b cells display a unique heavy-chain rearrangement experimental model for the study of the human immune system: production and monitoring of ''human immune system'' rag2-/-gamma c-/-mice stat3-mediated up-regulation of blimp1 is coordinated with bcl6 down-regulation to control human plasma cell differentiation a senescence rescue screen identifies bcl6 as an inhibitor of anti-proliferative p19(arf)-p53 signaling il-7 enhances thymic human t cell development in ''human immune system'' rag22/2il-2rgammac2/2 mice without affecting peripheral t cell homeostasis stat5 regulates the self-renewal capacity and differentiation of human memory b cells and controls bcl-6 expression cd27: a memory bcell marker human b cell subsets rapid cloning of high-affinity human monoclonal antibodies against influenza virus human immunoglobulin m memory b cells controlling streptococcus pneumoniae infections are generated in the spleen human blood igm ''memory'' b cells are circulating splenic marginal zone b cells harboring a prediversified immunoglobulin repertoire control of inflammation, cytokine expression, and germinal center formation by bcl-6 bcl-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control the bcl-6 protooncogene controls germinal-centre formation and th2-type inflammation antigen-specific antibody production of human b cells in nog mice reconstituted with the human immune system antigen-specific human t-cell responses and t cell-dependent production of human antibodies in a humanized mouse model disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2-/-gamma c-/-mice a new humanized mouse model of epstein-barr virus infection that reproduces persistent infection, lymphoproliferative disorder, and cell-mediated and humoral immune responses dengue virus infection and immune response in humanized rag2(2/2)gamma(c)(2/2) (rag-hu) mice potent antibody therapeutics by design humanized mice for modeling human infectious disease: challenges, progress, and outlook generation of primary antigen-specific human t-and b-cell responses in immunocompetent scid-hu mice humanized mice mount specific adaptive and innate immune responses to ebv and tsst-1 intrarectal transmission, systemic infection, and cd4+ t cell depletion in humanized mice infected with hiv-1 lentiviral vector delivery of human interleukin-7 (hil-7) to human immune system (his) mice expands t lymphocyte populations human lymphoid and myeloid cell development in nod/ltsz-scid il2r gamma null mice engrafted with mobilized human hemopoietic stem cells generation of functional human t-cell subsets with hla-restricted immune responses in hla class i expressing nod/scid/il2r gamma(null) humanized mice priming of protective t cell responses against virus-induced tumors in mice with human immune system components polymorphism in sirpa modulates engraftment of human hematopoietic stem cells we thank berend hooibrink for expert cell sorting and maintenance of the amc-uva flow cytometry facility. we acknowledge the bloemenhove clinic (heemstede, the netherlands) for providing fetal tissues and the staff of the animal research institute amsterdam for animal care. access to technologies available at the centre d'immunologie humaine of the institut pasteur was greatly appreciated. key: cord-022544-7jn4ns6x authors: lawrence, robert m. title: host-resistance factors and immunologic significance of human milk date: 2010-12-27 journal: breastfeeding doi: 10.1016/b978-1-4377-0788-5.10005-7 sha: doc_id: 22544 cord_uid: 7jn4ns6x nan some of the most dramatic and far-reaching advances in the understanding of the immunologic benefits of human milk have been made using newer techniques to demonstrate the specific contribution of the numerous "bioactive factors" contained in human milk (table 5 -1). the multifunctional capabilities of the individual factors, the interactive coordinated functioning of these factors, and the longitudinal changes in the relative concentrations of them for the duration of lactation make human milk unique. the immunologically active components of breast milk make up an important aspect of the host defenses of the mammary gland in the mother; at the same time, they complement, supplement, and stimulate the ongoing development of the infant' s immune system. [107] [108] [109] the explosion of research on all the immunologic properties and actions of breast milk in the last 10 years makes it impossible to summarize all the important aspects of what we now know about the immunologic benefits of breast milk. the recently developed technologies of genomic studies using microarrays and proteomics promise to continue this rapid expansion of knowledge on the biology of the mammary gland, human milk, and the infant's developing immune system. this chapter emphasizes the important concepts of these immunologic benefits and refers the interested reader to the most recent literature for more extensive information on the many specific components. the immunologic benefits of human milk can be analyzed from a variety of perspectives: 1. reviewing the published information on the protection of infants from specific infections that compares breastfed and formula-fed infants. developing immune systems and the actions of bioactive factors provided in breast milk. 3. examining the proposed function of the active components contained in human milk: antimicrobial, antiinflammatory, and immunomodulating. 4. considering the nature of the different factors: soluble, cellular, and hormone-like. 5. examining the contribution of breast milk to immune function of mammary glands and infants as an evolutionary process. 6. determining the site of the postulated action of the specific factors (e.g., in the breast or in the infant) at the mucosal level (respiratory tract or gastrointestinal [gi] tract) or at the systemic level. 7. classifying the factors relative to their contribution to the constitutive defenses (innate immunity) versus the inducible defenses (adaptive immunity) of the infant' s immune system. 8. clarifying the mechanism of action of the proposed immunologic benefit (the mucosalassociated lymphoid tissue formation of the chapter 5 bioactive factors at the level of the mucosa and their subsequent action at the breast or in an infant). 9. considering the contribution of human milk to the development of an infant' s immune system relative to potential long-term immunologic benefits, such as protection against allergy, asthma, autoimmune disease, or inflammatory bowel disease. the protective effect of breast milk against infection was documented as early as 1892 in the medical literature by data proving that milk from various species, including humans, was protective for offspring, containing antibodies against a vast number of antigens. 271 veterinarians have long known the urgency of offspring receiving the early milk of the mother. death rates among human newborns not suckled at the breast in the third world are at least five times higher than among those who receive colostrum and mother' s milk. the evidence that a lack of breastfeeding and poor environmental sanitation have a pernicious synergistic effect on infant mortality rate has been presented by habicht 101 after studying 1262 women in malaysia. the evidence that breastfeeding protects against infections in the digestive and respiratory tracts has been reported for several decades. 269 however, many of the older studies were criticized for flawed methodology and because they were performed in "developing countries," where the risk for infection due to poor sanitation was expected to be higher. 13, 101, 116 various researchers have proposed specific criteria for assessing the methodology of studies reporting on the protective effects of breast milk, clearly identifying measurable outcomes and the definition of breastfeeding, with other methods to limit bias and to control for confounding variables. 13, 46, 147, 149 more recent studies, which have incorporated many of the proposed methodologic criteria, continue to document that breastfeeding protects infants against diarrhea, respiratory infections, and otitis media.* individual papers report protection against urinary tract infections and neonatal sepsis. 6, 109, 220, 275 several papers document the decreased risk for dying in infancy associated * references 7, 15, 43, 51, 55, 122, 167, 199, 208, 223, 226, 243, 278. with exclusive or predominant breastfeeding in pakistan, peru, ghana, india, nepal, and bangladesh. 4, 6, 9, 62, 179 a systematic review by the bellagio child survival study group predicted that exclusive breastfeeding for 90% of all infants through 6 months of age could prevent 13% of the childhood deaths occurring younger than 5 years of age. 133 a recent review on human breast milk documents the evidence for protection against infectious diseases from breastfeeding for resource-rich and resourcepoor countries. 153 one of the important considerations relative to measuring the immunologic benefits of breast milk is the exclusivity and duration of breastfeeding. the basic concept is identifying a dose-response relationship between the amount of breast milk received by an infant during the period of observation and the immunologic benefit gained with greater exclusivity and duration equaling greater volume of breast milk or "dose." dr. labbok and krasovec 149 have carefully defined breastfeeding in terms of the patterns of breastfeeding relative to the amount of supplementation with formula or other fluids or foods (full/nearly full, medium or equal, low partial, or token) to standardize the use of equatable terms in different studies. box 5-1 outlines these definitions of the "amount" of breastfeeding. 153 raisler et al 227 referred to a doseresponse relationship when they studied the effect of "dose" of breast milk on preventing illness in more than 7000 infants. "full breastfeeding" was associated with the lowest rates of illness (diarrhea, cough, or wheeze) and even children with "most" or "equal" breastfeeding had evidence of lower odds ratios of ear infections and certain other illnesses. a number of other long-term studies demonstrated greater protection from infection with increased exclusivity of breastfeeding and durations of at least 3 months.* a couple papers demonstrated a "dose" effect relative to decreased occurrence of late onset sepsis in very low-birth-weight infants 73 and premature infants 245 associated with the infants receiving at least 50 ml/kg per day of mother' s milk compared with receiving other nutrition. the current recommendations from the american academy of pediatrics reinforce the importance of the doseresponse relationship between breastfeeding and the benefits of breastfeeding when it recommends exclusive breastfeeding for the first 6 months of life and at least partial breastfeeding after the introduction of solid foods for an additional 12 months or longer. 1 another important consideration relative to exclusive breastfeeding is the potential effect of other foods and fluids in an infant' s diet that could negatively influence immunologic benefits and infection-protective effects at the level of the gi mucosa. the human immune system begins forming and developing in the fetus. newborn infants' immune systems are immature and inadequate at birth. immune systems rapidly adapt in the postnatal period related to the natural maturation of the skin and mucosal barriers and in response to the exposure of infants to inhaled and ingested antigens and microbial agents in the extrauterine environment. infants' immune systems develop throughout at least the first 2 years of life. overall, infants have limited abilities to respond effectively and quickly to infectious challenges, which explains infants' ongoing susceptibility to infections. † box 5-2 lists most of the better understood deficiencies in infants' immune systems. an extensive discussion of these developmental immune deficiencies affecting infants is presented by lawrence and pane. 153 the b-lymphocytes and immunoglobulin production are deficient in the amount and specificity of antibodies produced. there is limited isotype switching and slow maturation of the antibody response to specific antigens (polysaccharides). 114, 177 the systemic cell-mediated immune response, including complement components, and both the "classical" and alternative pathways have limitations for complement activation. 2, 63, 251, 277 numerous immune components are produced in limited amounts in infancy, including complement, interferon-γ, secretory immunoglobulin (ig) a, interleukins (il-3, il-6, il-10), tumor necrosis factor (tnf)-α, lactoferrin, and lysozyme. 45, 87 relative to these various immune deficits in infants, one can find various bioactive and immunomodulating factors in breast milk that are potentially capable of complementing and enhancing the development of infants' mucosal and systemic immune systems. 87, 110 this concept of bioactive and immunomodulating factors in breast milk is an important area of evolving research that has been extensively reviewed in the literature. 87, 88, 110, 144 the most intense focus of this research centers on the effects of human milk on infants' gi tract. 85, 201 the bioactive factors being studied are as diverse as proteins (lactoferrin, lysozyme, etc.), hormones (erythropoietin, prolactin, insulin, etc.), growth factors (epithelial growth factor, insulin-like growth factor, etc.), neuropeptides (neurotensin, somatostatin, etc.), cytokines (tnf-α, il-6, etc.), antiinflammatory agents (enzymes, antioxidants, etc.), and nucleotides (see table 5 -1). in the past, it was adequate to point to the lists of factors (especially immunoglobulins) to "explain" the immunologic benefit of breast milk. today, it is necessary to understand the multifunctional and dynamic action of these individual factors, their specific mechanisms of action on the innate, the adaptive, and the mucosal immune system, and their role in direct infection protection, in the normal development of infants' immune systems, and their contribution against potentially harmful inflammation. from an evolutionary perspective, maternal antibody is transmitted to the fetus by different pathways in different species. 87, 159, 261 an association has been recognized between the number of placental membranes and the relative importance of the placenta and the colostrum as sources of antibodies. by this analysis, horses, with six placental membranes, pass little or no antibodies transplacentally and rely totally on colostrum for protection of foals. humans and monkeys, having three placental membranes, receive more of the antibodies via the placenta and less from the colostrum. the transfer of igg in humans is accomplished by active transport mechanism of the immunoglobulin across the placenta. secretory iga (siga) immunoglobulins are found in human milk and provide local protection to the mucous membranes of the gi tract. other investigations have established that the mammary glands and their secretion of milk are important in protecting the infant not only through the colostrum but also through mature milk from birth through the early months of life. although the predominance of iga in human colostrum and milk had long been described, the importance of this phenomenon was not fully appreciated until the discovery that iga is a predominant immunoglobulin present in mucosal secretions of other glands in addition to the breast. mucosal immunity has become the subject of extensive research. 21, 22, 120, 201 it is clear that considerable traffic of cells occurs between mucosal epithelia and secretory or lymphoid tissue sites. the data support the concept of a general system of mucosa-associated lymphoid tissue (malt), which includes the gut, lung, mammary gland, salivary and lacrimal glands, and the genital tract ( figure 5 -3). through the immune response of malt, a reaction to an immunogen at a mucosal site may be an effective means of producing immunity at distant sites. antibodies against specific antigens found in milk have also been found in the saliva, evidence for transfer of protection to two different distant sites simultaneously. evidence suggests that the mammary glands may act as extensions of the gut-associated lymphoid tissue (galt) and possibly the bronchiole-associated lymphoid tissue. the ability of epithelial surfaces exposed to the external environment to defend against infectious agents has been well documented for the gi, genitourinary, and respiratory tracts. 142 siga and secretory igm (sigm) produced through the adaptive response of the mucosal-lymphoid immune system act by blocking colonization with pathogens and limiting the passage of harmful antigens across the mucosal barrier. activated b cells and cytokines pass to the mammary gland where they contribute to the production of siga in breast milk. direct contact between the antigen and the lymphoid cells of the breast is unlikely. 202 peyer patches, tonsils, and other malt structures appear to be well developed at birth. 24 nevertheless, the actual effective production of siga to various antigens presented to infants' mucosal surfaces (respiratory and gi tracts) is still inadequate to protect against infection. a breastfeeding infant, as part of the maternal-infant dyad exposed to the same antigens via their mucosal services, can receive protective siga and sigm in the mother' s breast milk produced by the mother' s malt ( figure 5-1) . the protective properties of human milk can be divided into cellular factors and humoral factors for facility of discussion, although they are closely related in vivo. a wide variety of soluble and cellular components and hormone-like agents have been identified in human milk and colostrum (see table 5 -1). although the following discussion separates these elements, it is important to emphasize that the constituents of human milk are multifunctional and their functioning in vivo is interactive and probably coordinated and complementary. cells are an important postpartum component of maternal immunologic endowment. more than 100 years ago, cell bodies were described in the colostrum of animals. as with much lactation research, further study of colostral corpuscles was undertaken by the dairy industry for commercial reasons in the early 1900s. this research afforded an opportunity to make major progress in the understanding of cells in milk. initially, it was thought that these cells represented a reaction to infection in the mammary gland and were even described as "pus cells." it has become clear that the cells of milk are normal constituents of colostrums in all species. cells include macrophages, lymphocytes, neutrophils, and epithelial cells, and they total approximately 4000/mm 3 . cell fragments and epithelial cells were examined by electron microscope in fresh samples from 30 women by brooker. 27 he found that the membrane-bound cytoplasmic fragments in the sedimentation pellet outnumbered intact cells. the fragments were mostly from secretory cells that contained numerous cisternae of rough endoplasmic reticulum, lipid droplets, and golgi vesicles containing casein micelles. secretory epithelial cells were found in all samples and, after the second month postpartum, began to outnumber macrophages. ductal epithelial cells were about 1% of the population of cells for the first week or so and then disappeared. all samples contained squamous epithelial cells originating from galactophores and the skin of the nipple. living leukocytes are normally present in human milk. 142 the overall concentration of these leukocytes is of the same order of magnitude as that seen in peripheral blood, although the predominant cell in milk is the macrophage rather than the neutrophil. macrophages compose about 90% of the leukocytes, and 2000 to 3000/mm 3 are present. lymphocytes make up about 5% to 10% of the cells (200 to 300/mm 3 ), which is a much lower concentration than in human blood. 91 the number of cells found in human milk increases with mastitis. both large and small lymphocytes are present. by indirect immunofluorescence with anti-t-cell antibody to identify thymus-derived lymphocytes, it has been shown that 50% of human colostral lymphocytes are t cells, and in human milk up to 80% of the lymphocytes are t cells. 276 immunofluorescence procedures to detect surface immunoglobulins characteristic of b lymphocytes identified 34% as b lymphocytes. the number of leukocytes and the degree of mitogenic stimulation of lymphocytes sharply decline during the first 2 or 3 months of lactation to essentially undetectable levels, according to ogra and ogra ( figure 5 -2). 92 enumeration of the total cell numbers in milk has been difficult, but when various techniques are compared (coulter electronic particle counter, visual cell counting with special stains, filter trapping with fluorescent detection, and automated fluorescent cell counting), stains for deoxyribonucleic acid (dna) were superior to the other techniques. macrophages are large-complex phagocytes that contain lysosomes, mitochondria, pinosomes, ribosomes, and a golgi apparatus. the monocytic phagocytes are lipid laden and were previously called the colostral bodies of donne. they have the same functional and morphologic features as phagocytes from other human tissue sources. these features include ameboid movement, phagocytosis of microorganisms (fungi and bacteria), killing of bacteria, and production of complement components c3 and c4, lysosome, and lactoferrin. other milk macrophage activities include the following 219 : phagocytosis of latex, adherence to glass secretion of lysozyme, complement components c3b-mediated erythrocyte adherence igg-mediated erythrocyte adherence and phagocytosis bacterial killing inhibition of lymphocyte mitogenic response release of intracellular iga in tissue culture giant cell formation interaction with lymphocytes data suggest these macrophages also amplify t-cell reactivity by direct cellular cooperation or by antigen processing. the colostral macrophage has been suggested as a potential vehicle for the storage and transport of immunoglobin. a significant increase in iga and igg synthesis by colostral lymphocytes, when incubated with supernatants of cultured macrophages, has been reported. 221 the macrophage may also participate in the biosynthesis and excretion of lactoperoxidase and cellular growth factors that enhance growth of intestinal epithelium and maturation of intestinal brush-border enzymes. the mobility of macrophages is inhibited by the lymphokine migration inhibitor factor, which is produced by antigen-stimulated sensitized lymphocytes. the activities of macrophages have been demonstrated in both fresh colostrum and colostral cell culture, and certain functions are altered compared with their counterpart in human peripheral blood. the highest concentration of cells occurs in the first few days of lactation and reaches more than a million per milliliter of milk. colostrum (1 to 4 days postpartum) contains 10 5 to 5 × 10 6 leukocytes/ml, and 40% to 60% are polymorphonuclear cells (pmns). mature milk (i.e., after 4 days) has fewer cells (see figure 5 -3), approximately 10 5 /ml with 20% to 30% pmns. after 6 weeks, few pmns are present. the functions of the pmns normally include microbial killing, phagocytosis, chemotactic responsiveness, stimulated hexose monophosphate shunt activity, stimulated nitroblue tetrazolium dye reduction, and stimulated oxygen consumption. 33 when milk pmns are compared with those in the serum, their activity is often less than that of serum pmn cells. whether milk pmns actually perform a role in protection of the infant has been studied by many investigators using many techniques. briefly, animal studies have shown that (1) the mammary gland is susceptible to infection in early lactation, (2) a dramatic increase in pmns occurs with mammary inflammation, and (3) in the presence of peripheral neutropenia during chronic mastitis, severe infection of the gland occurs. this implies, according to buescher and pickering, 33 that the primary function of milk pmns is to defend the mammary tissue per se and not to impart immunocompetence to the newborn. this may explain the presence of large numbers of pmns that are relatively hypofunctional early and then disappear over time. evidence shows that neutrophils found in human milk demonstrate signs of activation, including increased expression of cd11b (an adherence glycoprotein), decreased expression of l-selectin, spontaneous production of granulocyte-macrophage colony-stimulating factor (gm-csf), and the ability to transform into cd1 + dendritic cells (dcs). 125 human milk macrophages have the morphology and motility of activated cells. the movement of these cells in a three-dimensional system is greater than that of monocytes, their counterparts in peripheral blood. such activated neutrophils may play a role in phagocytosis at the level of the mucosa of the gi tract, supplementing infants' poor ability to recruit phagocytes to that site. 137 both t and b lymphocytes are present in human milk and colostrum and are part of the immunologic system in human milk. t cells are 80% of the lymphocytes in breast milk. human milk lymphocytes respond to mitogens by proliferation, with increased macrophage-lymphocyte interaction and the release of soluble mediators, including migration inhibitor factor. cells destined to become lymphopoietic cells are derived from two separate influences, the thymus (t) and the bursa (b) or bursal equivalent tissues. the population of the b cells makes up the smaller part of the total. they synthesize iga antibody. the term b cell is derived from its origination in a different anatomic site from the thymus; in birds, it has been identified as the bursa of fabricius. the b cells can be identified by the presence of surface immunoglobulin markers. the b cells in human milk include cells with iga, igm, and igg surface immunoglobulins. b cells transform into plasma cells and remain sessile in the tissues of the mammary gland. more rapid mitotic activity occurs in the thymus gland than in any other lymphatic organ, yet 70% of the cells die within the cell substance. the thymus is the location for much of the t-cell differentiation and selection and plays a major role in the development of infants' immune systems. thymosin has been identified as a hormone produced by thymic epithelial cells to expand the peripheral lymphocyte population. after emergence from the thymus gland, t cells acquire new surface antigen markers. the t cells circulate through the lymphatic and vascular systems as long-lived lymphocytes, which are called the "recirculating pool." they then populate restricted regions of lymph nodes, forming thymic-dependent areas. 276 it is interesting to note that exclusively breastfed infants have a significantly larger thymus than formula-fed infants at 4 and 10 months. 111 the significance of the lymphocytes in human milk in affording immunologic benefits to breastfed infants continues to be investigated. it is suggested that lymphocytes can sensitize, induce immunologic tolerance, or incite graft-versus-host reactions. according to head and beer, 115 lymphocytes may be incorporated into sucklings' tissues, achieving short-term adoptive immunization of the neonate. studies of the activities of lymphocytes have been carried out by a number of investigators who collected samples of milk from lactating women at various times postpartum, examined the number of cell types present, and then studied the activities of these cells in vitro. 138, 142 ogra and ogra 203 collected samples from 200 women and measured the cell content from 1 through 180 days (see figure 5 -3). they then compared the response of t lymphocytes in colostrum and milk with that of the t cells in the peripheral blood. t-cell subpopulations have also been shown by surface epitopes to be similar to those in the peripheral blood. the greatest number of cells appeared on the first day, with the counts ranging from 10,000 to 100,000/mm 3 for total cells. by the fifth day, the count had dropped to 20% of the first day' s count. in addition, the number of erythrocyte rosette-forming cells was determined by using sheep erythrocyterosetting technique. the erythrocyte rosette formation lymphocytes constituted a mean 100/mm 3 on the first day and 1 ⁄10 of that by the fifth day. at 180 days, total cells were 100,000/mm 3 , lymphocytes were 10,000/mm 3 , and erythrocyte rosette formation lymphocytes were 2000/mm 3 . the investigators compared the values with those in the peripheral blood of each mother; the levels remained essentially constant. 202 in a similar study, bhaskaram and reddy 18 sampled milk over time from 74 women and found comparable cell concentrations. they examined the bactericidal activity of the milk leukocytes and found it to be comparable with that of the circulating leukocytes in the blood, irrespective of the stage of lactation or state of nutrition of the mother. ogra and ogra 203 also studied the lymphocyte proliferation responses of colostrum and milk to antigens. their data show response to stimulation from the viral antigens of rubella, cmv, and mumps. analysis of cell-mediated immunity to microbial antigens shows milk lymphocytes are limited in their potential for recognizing or responding to certain infectious agents compared with cells from the peripheral circulation. this is thought to be an intercellular action and not caused by lack of external factors. in contrast, the t cells and b cells have been shown to have unique reactivities not seen in peripheral blood. colostral lymphocytes are derived from mature rather than immature t-cell subsets. the distribution of t-cell subsets in colostrum includes both cd4 + and cd8 + cells. 231 the distribution of cd4 cells in colostrum and human milk is lower than in the serum, and fewer cd4 cells exist than cd8 cells. 276 the percentage of cd4 cells is higher than in the serum of either postpartum donors or normal control subjects. no correlation exists with length of gestation and number of cells (normal blood usually contains twice as many cd4 + as cd8 + lymphocytes). 134 parmely et al 212 partially purified and propagated milk lymphocytes in vitro to study their immunologic function. milk lymphocytes responded in a unique manner to stimuli known to activate t lymphocytes from the serum. the authors found milk lymphocytes to be hyporesponsive to nonspecific mitogens and histocompatibility antigens on allogenic cells in their laboratory. they found them unresponsive to candida albicans. significant proliferation of lymphocytes occurred in response to k 1 capsular antigen of escherichia coli. 118 lymphocytes from blood failed to respond to the same antigen. this supports the concept of local mammary tissue immunity at the t-lymphocyte level. more recent experiments in rodents have provided evidence that t lymphocytes that are reactive to transplantation alloantigens can adoptively immunize a suckling newborn. foster nursing experiments performed in rodents have shown that newborn rats exposed to allogenic milk manifested alterations in their reactivity to skin allografts of the foster mother' s strain. in animals, mothers may give their suckling newborn immunoreactive lymphocytes. the influence of maternal milk cells on the development of neonatal immunocompetence has been demonstrated in several different immunologic contexts. congenitally, athymic nude mice nursed by their phenotypically normal mothers or normal foster mothers had increased survival. the mothers contributed their t-cell-helper activity to the suckling newborn. colostral lymphocytes proliferate in response to various mitogens, alloantigens, and conventional antigens. colostral cells survive in the neonatal stomach and in the gut of experimental animals, some remaining viable in the upper gi tract for a week. no evidence, however, indicates that transepithelial migration takes place when neonatal mice are foster-nursed by newly delivered animals whose colostral cells were tagged with 3 h-thymidine. 33 cells in human milk have been studied using the same markers employed with cells in the peripheral blood; 80% of the lymphocytes are t cells that are equally distributed between cd4 + and cd8 + subpopulations, and their t-cell receptors are principally of the α/β type. cd4 + cells are common leukocyte cells of the helper and suppressorinducer subsets, and cd8 + cells are leukocytes of the cytotoxic and noncytotoxic subsets. t cells in human milk are presumed activated because they display increased phenotypic markers of activation including hla-dr and cd25 (il-2 receptor). the majority of t cells in human milk are cd45ro + , consistent with effector and memory t cells. 236, 276 these cells are effective producers of interferon-γ, which is consistent with their phenotypic features. here again, human milk may supplement the infant with a functioning immune cell to compensate for an identified deficiency in the infant, a paucity of memory t cells. juto 134 studied the effect of human milk on b-cell function. cell-free, defatted, filtered colostrum as well as mature breast milk showed an enhancing effect on b-cell proliferation and generation of antibody secretion. this was not seen with formula. juto suggested that this could represent an important immunologic mechanism. goldblum et al 82 were able to show a b-cell response in human colostrum to e. coli given to the mother orally, which was not accompanied by a systemic response in the mother. this suggests that the breast and breast milk reflect sites of local humoral or cell-mediated immunity, which were initially induced at a distant site such as the gut and transferred via reactive lymphoid cells migrating to the breast. head and beer 115 provided a scheme to describe this mechanism (see figure 5 -1). the diagram depicts the progeny of specifically sensitized lymphocytes that originated in galt, specifically peyer patches, as they migrate to the mammary gland. as they infiltrate the mammary gland and its secretion, they supply the breast with immune cells capable of selected immune responses. ogra and ogra 202, 203 suggest that the cells may selectively accumulate in the breast during pregnancy. the responses of milk cells and their antibodies are not representative of an individual' s total immunity. 212 most of these immunocompetent cells, initially stimulated in galt, recirculate to the external mucosal surface and populate the lamina propria as antibody-producing plasma cells. a substantial number of these antigen-sensitized cells selectively home-in to the stroma of the mammary glands and initiate local iga antibody synthesis against the antigens initially encountered in the respiratory or intestinal mucosa. 18 more recent work on human-milk-derived b cells demonstrates that breast milk contains activated memory b cells, different than those in the blood. these cells express mucosal adhesion molecules (α 4 β 7 -/+ , α4β 1 + , cd44 + , cd62l -) suggesting origin in the mammary gland, but similar to galt-associated b cells. 265 the mucosae-associated epithelial cytokine ccl28 may contribute to migration of and retention of these cells in the mammary gland. 274 this information supports the concept of the mammary gland as an effector site of the mucosal immune system. the accumulated epidemiologic research support the concept that colostrum and milk provide human infants with immunologic benefits. both t and b lymphocytes found in breast milk are reactive against organisms invading the intestinal tract. however, the proof of specific viral or bacterial protection secondary to the action of immunologically active b cells has not been demonstrated. although it is clear that cells are provided in the colostrum and milk, the effectiveness and impact of these cells on the neonate depend on their ability to survive in the gi tract. it has been demonstrated in several species, including humans, that the ph of the stomach can be as low as 0.5, but the output of hydrochloric acid is minimal for the first few months, as is the peptic activity. immediately after a feeding begins, the ph rises to 6.0 and returns to normal in 3 hours. the cells from milk tolerate this. studies in rats have also shown that intact nucleated lymphoid cells are found in the stomach and intestines. 16 these cells, when removed from rat stomachs, are capable of phagocytosis. lymphoid cells in milk have been shown to traverse the mucosal wall. when human milk is stored, however, the cellular components do not tolerate heating to 63° c (145.4° f), cooling to −23° c (−9.4° f), or lyophilization. although a few cells may be identified in processed milk, they are not viable. 83 cregan et al 49 have reported the presence of mammary stem cells in human breast milk based on the demonstration of the cytokeratin 5 mammary stem cell marker. additionally cells from breast milk were analyzed after culturing which showed both a multipotent stem cell marker, nestin, and the cytokeratin 5 marker. although human milk may serve as a source of mammary stem cells in the future, no evidence of an immunologic role for these cells in developing infants currently exists. all classes of immunoglobulins are found in human milk. the study of immunoglobulins has been enhanced through the techniques of electrophoresis, chromatographics, and radioimmunoassay. more than 30 components have been identified; of these, 18 are associated with proteins in the maternal serum, and the others are found exclusively in milk. the concentrations are highest in the colostrum of all species, and the concentrations change as lactation proceeds. 173 iga, principally siga, is highest in colostrum. although postpartum levels fall throughout the next 4 weeks, substantial levels are maintained throughout the first year, during gradual weaning between 6 and 9 months, and even during partial breastfeeding (when infant receives solid foods) in the second year of life (figures 5-4 and 5-5 and table 5 -2). specific siga antibodies to e. coli persist through lactation and may even increase (see figure 5 -4). the main immunoglobulin in human serum is igg; iga content is only one fifth the level of igg. in milk, however, the reverse is true. iga is the most important immunoglobulin in milk, not only in concentration but also in biologic activity. siga is likely synthesized in the mammary alveolar cells 256 or by lymphocytes that have migrated from peyer patches in the gi tract or from lymphoid tissue in the respiratory tract via the lymphatics to the breast. cytokines cause isotype switching of local igm + b cells to become iga + b lymphocytes. 85, 248, 272 these isotype switched cells travel to the breast where they are transformed into plasma cells producing secretory, dimeric iga. it is through this "enteromammary" pathway that the mother provides increased amounts of siga to the infant against the microorganisms present in the mother' s and infant' s environment. 261 brandtzaeg et al 22 have proposed a model for the transport of iga (polymeric) and igm (pentameric), produced by plasma cells, across the secretory epithelium with the formation of siga and igm through binding with the secretory component attached to the epithelial membrane. this occurs in the membrane of mammary epithelial cells during lactation. 23, 93 quantitative determinations of immunoglobulins in human milk were made from milk collected at birth to as long as 27 months postpartum by peitersen et al 213 iga is the predominant immunoglobulin in breast milk, constituting 90% of all the immunoglobulins in colostrum and milk. ogra and ogra [202] [203] [204] studied the serum of postpartum lactating mothers and nonpregnant matched control subjects and noted that the individual and mean concentrations of all ig classes were lower in the postpartum subjects. the levels were statistically significant for igg; they were 50 to 70 mg higher in the nonpregnant women. immunoglobulin levels, particularly iga and igm, are very high in colostrum and drop precipitously in the first 4 to 6 days, but igg does not show this decline. the volume of mammary secretion, however, increases dramatically in this same period; thus the absolute amounts of immunoglobulins remain more nearly constant than it would first appear. local production and concentration of iga and probably igm may take place in the mammary gland at delivery. ige and igd have also been measured in colostrum and milk. using radioimmunoassay techniques, colostrum was found to contain concentrations of 0.5 to 0.6 iu/ml ige in 41% of samples and less in the remainder. 10 igd was found in all samples in concentrations of 2 to 2000 mg/dl. plasma levels were poorly correlated. the findings suggest possible local mammary production rather than positive transfer. the question of whether ige or igd antibodies in breast milk have similar specificities for antigens as the iga antibodies in milk remains unanswered. 171 keller et al 139 examined the question of local mammary igd production and its possible participation in a mucosal immune system by comparing colostrum and plasma levels of total igd with specific igd antibodies. from their work comparing colostrum/plasma ratios for igg, igd, and albumin and measuring igd against specific antigens, the authors reported evidence for igd participation in the response of the mucosal immune system, with increases in total igd and igd against specific antigens found in colostrum. to address the question of total quantities of immunologic components secreted into human milk per day and available to an infant, butte et al 36 lysozyme, in contrast, rose during the same period in total amount available and amount per kilogram per day. the authors 36 suggest that production and secretion of these immunologic factors by the mammary gland may be linked to the catabolism of the components at an infant' s mucosal tissues. when the concentrations of siga, igg, igm, α 1antitrypsin, lactoferrin, lysozyme, and globulins c3 and c4 were compared in relationship to parity and age of the mother, no consistent trend was observed. when maturity of the pregnancy was considered, however, mean concentrations of all these proteins were higher, except for iga, when the delivery was premature. because several proteins in human milk have physiologic function in infants, davidson and lönnerdal 53 examined the survival of human milk proteins through the gi tract. crossed immunoelectrophoresis showed that three human milk proteins transversed the entire intestine and were present in the feces: lactoferrin, siga, and α 1 -antitrypsin. miranda et al 176 reported on the effect of maternal nutritional status on immunologic substances in human colostrum and milk. maternal malnutrition was characterized as lower weight-to-height ratio, creatine/height index, total serum proteins, and igg and iga. in malnourished mothers, the colostrum contained one third the normal concentration of igg, less than half the normal level of albumin, and lower iga and complement c4. lysozyme, complement c3, and igm levels were normal. levels improved with development of mature milk and improvement in maternal nutrition. according to one report in 2003, moderate exercise during lactation does not affect the levels of iga, lactoferrin, or lysozyme in breast milk. 165 immunologic components contained in human milk during the second year of lactation become a significant point as more infants are nursed longer. for a longitudinal study of lactation into the second year by goldman et al, 89 women were included who had fully breastfed their infants for 6 months to a year, and were continuing to partially breastfeed. samples were collected by fully emptying the breast by electric pump. table 5 -2 summarizes the concentrations of the measured factors. no leukocytes were detected. concentrations of total iga and siga, lactoferrin, and lysozyme were similar to those 7 to 12 months postpartum and during gradual weaning. siga antibodies to e. coli were produced in the second year, demonstrating significant immunologic benefit to the infant with continued breastfeeding. 89 iga, igm, and igg were measured in nursing women from the beginning of lactation and simultaneously in the feces of their children by jatsyk et al 130 at the academy of medicine in moscow. they reported iga to be very high in the milk and rapidly increasing in the feces. igg and igm levels, however, were low in both milk and feces. in normal full-term bottle-fed infants, iga appeared in the feces at 3 to 4 weeks of age but at much lower levels than in breastfed infants. koutras 146 reported that in the first 8 weeks of life increased amounts of siga are found in the stools of breastfed infants compared with formula-fed infants. the authors ascribed this phenomenon to the presence of siga in human milk and a stimulation of the local gi production of immunoglobulin. savilahti et al 242 measured serum levels of igg, iga, and igm in 198 infants at 2, 4, 6, 9, and 12 months of age. by 9 months, the exclusively breastfed infants had igg and igm levels significantly lower than those who had been weaned early (before 3.5 months) to formula. six infants were still exclusively breastfed at 12 months, and their iga levels had also lowered to levels found at 2 months with bottle feeders. infection rates were similar. two months after the children were weaned to formula, the igg and igm levels were comparable. iron and zinc levels were the same in all children. siga antibodies have been identified in human milk that recognize a large variety of microorganisms. the siga antibodies that recognize bacteria, viruses, parasites and fungi are listed in table 5 85, 196 the list of viruses for which siga antibodies exist in human milk is equally long including enteroviruses (poliovirus, coxsackie, and echovirus), cytomegalovirus (cmv), herpes simplex virus, human immunodeficiency virus (hiv), semliki forest virus, respiratory syncytial virus (rsv), rubella, reovirus type 3, rotavirus, measles, norovirus, and porcine coronavirus. igg and igm antibodies also exist in human milk against cmv, rsv, and rubella as well as ige antibodies against parvovirus b19. noguera-obenza and cleary 196 reviewed the role of breast milk siga in providing protection for infants against various agents specifically causing bacterial enteritis. preservation of human milk at −20° c for up to 3 months does not decrease significantly the levels of iga, igg, igm, c3, c4, lactoferrin, or lysozyme. 65, 70, 160, 205 the preservation of siga, il-6 and tnf-α with freezing at 4° c or −20° c was recently confirmed by hines et al. 117 a variety of different heat treatments have been applied to milk to protect against bacterial contamination or to protect against infection with specific infectious agents (especially hiv and cmv). heat treatments include low-temperature, short-time 56° c for 15 minutes; holder pasteurization 62.5° c for 30 minutes; high-temperature, short-time 70° to 73° c for 15 seconds; boiling 100° c for greater than 1 minute; sterilization, variable time periods, pretoria pasteurization 56° to 62.5° c for approximately 15 minutes 131 ; flash heating 56° c for approximately 6 minutes with a peak temperature at 72° c 127, 128 ; and microwave heating, with milk temperatures of 20° to 77° c for 30 seconds. 225 boiling or sterilization essentially destroys 100% of immunologic activity. siga and lysozyme activities drop by 20% with holder pasteurization and by 50% at 65° c. neither low-temperature, short-time nor high-temperature, short-time reduces the siga or lysozyme content markedly. igg and igm are greatly reduced by holder pasteurization. siga differs antigenically from serum iga. iga can be synthesized in the nonlactating as well as in the lactating breast. it is a compact molecule and resistant to proteolytic enzymes of the intestinal tract and the low ph of the stomach. siga present in human milk is primarily manufactured by plasma cells in the mammary gland, modified in its translocation across the mammary epithelia and only minimally produced by the cellular lymphocytes in milk. levels in milk are 10 to 100 times higher than in serum. levels in cow milk are very low, that is, a tenth of the level in mature human milk (0.03 mg/dl). later in life, the human intestinal tract's subepithelial plasma cells secrete iga. the intestinal secretion of siga does not occur in the neonatal period but increases between 4 to 12 months of life. discussion continues as to whether any antibodies are absorbed from the intestinal tract, although probably 10% are absorbed. almost 75% of ingested iga from milk survives passage through the intestinal tract and is excreted in the feces. all immunoglobulin classes have been identified in the feces. 230 a large body of evidence demonstrates the activity of the immunoglobulins, especially iga, at the mucosal level of the gi and respiratory tracts. these antibodies provide local intestinal protection against microorganisms, which may infect the mucosa or enter the body through the gut or respiratory tract. it is well established that the predominant bacteria found in breastfed infants are bifid bacteria. bifid bacteria are gram-positive, nonmotile, anaerobic bacilli. many observers have shown the striking difference between the flora of the guts of breastfed and bottle-fed infants. györgy 100 demonstrated the presence of a specific factor in colostrum and milk that supported the growth of lactobacillus bifidus. bifidus factor has been characterized as a dialyzable, nitrogen-containing carbohydrate that contains no amino acid. in vitro studies by beerens et al 17 showed the presence of a specific growth factor for bifidobacterium bifidum in human milk, which they called bb. other milks, including cow milk, sheep milk, pig milk, and infant formulas, did not promote the growth of this species but did show some activity supporting b. infantis and b. longum. this growth factor was found to be stable when the milk was frozen, heated, freeze-dried, and stored for 3 months. growth-promoting factors were present for the six strains studied, which varied in their resistance to physical change. because all these factors were active in vitro, they did not require the presence of intestinal enzymes for activation. it has not been possible to show the presence of this growth factor in other mammalian milks; thus it may contribute to the implantation and persistence of b. bifidum in a breastfed infant' s intestine. lactobacillus has been described as one of a number of probiotic bacteria, which provide an immune protective benefit to their host. lactobacillus reportedly stimulates antibody production and improves phagocytosis by blood leukocytes. 135, 215 the use of probiotic bacteria has reportedly produced benefits in a variety of situations associated with infections. the addition of such bacteria to formula is another example of trying to make formula better by making it more like breast milk. hatakka et al 112 examined the possible effect of adding probiotic bacteria to formula on the occurrence of infection in children attending day care. they reported modest reductions in the number of children with complicated respiratory infections or lower respiratory tract infections as well as the number of children receiving antibiotics for a respiratory infection in the group of children receiving formula supplemented with lactobacillus rhamnosus gg compared with children receiving unsupplemented formula. it was well known in the preantibiotic era that human milk protects human infants throughout lactation against staphylococcal infection. györgy 100 identified the presence of an "antistaphylococcal factor" in experiments with young mice that had been stressed with staphylococci. this factor, with no demonstrable direct antibiotic properties, was termed resistance factor and described as nondialyzable, thermostable, and part of the free fatty acid part of the phosphide fraction, probably c18:2, but distinct from linoleic acid. human milk contains a nonspecific antimicrobial factor, lysozyme, which is a thermostable, acidstable enzyme. this enzyme is a 130-amino-acidcontaining glycoprotein that can hydrolyze the 1-4 linkage between n-acetylglucosamine and n-acetylmuramic acid in bacterial cell walls. it is found in large concentrations in the stools of breastfed infants and not in stools of formula-fed infants; thus it is thought to influence the flora of the intestinal tract. goldman et al 92 describe an initial fall in lysozyme levels from 85 to 90 mg/ml to 25 mg/ml at 2 to 4 weeks and then an increase during 6 months to 250 mg/ml (see . lysozyme levels show an increase over time during lactation; this finding is more apparent in indian women than in those of the western world. reddy et al 229 studied the levels of lysozyme in well-nourished and poorly nourished women in india and found no difference between them (table 5-4) . as shown in this study, lysozyme levels increase during lactation. levels in human milk are 300 times the level in cow milk. lysozyme is bacteriostatic against enterobacteriaceae and gram-positive bacteria. 219 it is secreted by neutrophils and some macrophages and is present in many body secretions in the adult. in a study of immunologic components in human milk in the second year of lactation, goldman et al 89 reported that concentrations of lysozyme, lactoferrin, and total and siga were similar to those in uninterrupted lactation and in gradual weaning at 6 to 9 months. siga antibodies to e. coli were also produced during the second year. the authors state that "this supports the idea that the enteromammary lymphocyte traffic pathway, which leads to the development of lymphoid cells in the mammary gland that produce iga antibodies to enteric organisms, operates throughout lactation." 89 when cow milk formula is added to human milk, it reduces the effect of lysozyme; however, powdered human milk fortifier (enfamil) did not inhibit the antiinfective properties. 141 lactoferrin is an iron-binding protein closely related to the serum iron transport protein, transferrin, and is part of the larger transferrin protein family. lactoferrin is found in mucosal secretions (tears, saliva, vaginal fluids, urine, nasal and bronchial secretions, bile, gi fluids) and notably in milk and colostrum. a bacteriostatic effect of lactoferrin is well established for a wide range of microorganisms, including gram-positive and gram-negative aerobes, anaerobes, viruses, parasites, and fungi. the original proposed mechanism of action for its bacteriostatic effect was depriving the microorganism of iron. a second antibacterial action involving direct action with bacterial surfaces; binding negatively charged molecules (lipoteichoic acid) on the surface of gram-positive bacteria neutralizing the surface charge allowing the action of other antibacterial factors like lysozyme or binding lipid a on gram-negative bacteria, releasing the lipid, producing damage to the cell membrane. another antibacterial action is binding bacterial adhesions blocking host cell interaction. 94 lactoferrin can kill candida albicans and c. krusei by changing the permeability of the fungal cell surface. lactoferrin now is considered a multifunctional, immunoregulatory protein. the biologic role of lactoferrin has been reviewed in several studies. 163, 164, 198, 241 they point out that lactoferrin reversibly binds two ferric ions and that its affinity for iron is 300 times greater than that of transferrin, retaining iron down to a ph of 3. human lactoferrin is strongly basic. lactoferrin is normally unsaturated with iron, 35 and it is usually less than 10% saturated with iron in human milk. 72, 241 oral iron therapy for an infant can interfere with the bacteriostatic action of lactoferrin, which depends on its unsaturated state for some portion of its bacteriostatic function. reddy et al 229 showed that giving iron to the mother did not interfere with the saturation of lactoferrin in the milk or thus its potential bacteriostatic effect. protein energy malnutrition, rather than iron supplies, influences lactoferrin synthesis in the mammary gland. malnourished but non-iron-deficient mothers are lactoferrin deficient. the concentration of lactoferrin is high in colostrum-600 mg/dl-then progressively declines over the next 5 months of lactation, leveling at about 180 mg/dl. breast milk also contains small amounts of transferrin (10 to 15 mg/ml). lactoferrin is 10% to 15% of the total protein content of human milk. 163 lactoferrin is resistant to proteolysis, especially in its iron-saturated form. intact lactoferrin is detectable in the stool of infants, with higher proportions of lactoferrin measurable in the stool of premature infants. 56 both intact lactoferrin and fragments have been detected in the urine of premature infants, although absorption is less likely in full-term infants. 107 the absorption of iron from breast milk is directly enhanced by lactoferrin. 164 many bacteria require iron for normal growth, and one bacteriostatic effect of lactoferrin has been ascribed to its iron-binding action. in neutrophils, lactoferrin within neutrophilic granules tightly binds iron, but neutrophils with excessive iron are inefficient at destroying bacteria. lactoferrin does not limit the growth of all microorganisms; helicobacter pylori and neisseria, treponema, and shigella species all have receptors for lactoferrin, directly binding iron and allowing adequate growth. some evidence supports various other proposed mechanisms of action for lactoferrin' s antimicrobial effect. lactoferrin has been shown to limit the formation of biofilms by specific organisms, inhibit adhesion to host cells by other organisms and to directly bind to viral particles of herpes simplex virus, hiv, and adenovirus. a proteolytic action of lactoferrin appears to inactivate virulence factors of some organisms. separately, lactoferrin binds directly to glycosaminoglycans (gags) and integrins interrupting the binding of various viruses (herpes simplex virus, hiv, adenovirus, cmv, hepatitis b virus [hbv]) to host cells. pepsin hydrolysate products of lactoferrin (b or h) may exert a direct bactericidal effect by binding to lipopolysaccharide of gram-negative organisms and disrupting bacterial membranes. 263 lactoferrin may cause an increased release of cytokines by cells including antiinflammatory cytokines such as il-10. 50, 157 others have shown that lactoferrin suppresses the release of il-1, il-2, il-6, il-8, and tnf-α, all proinflammatory cytokines, which would be more of an immune-modulating effect. 157 other investigators using a recombinant human lactoferrin (talactoferrin) demonstrated evidence of lactoferrin causing increased maturation of dcs 252 and talactoferrin causing the recruitment and activation of neutrophils and macrophages 233 as other examples of how lactoferrin affects the innate immune protection of the growing infant. several other effects have been proposed for lactoferrin, including inhibition of hydroxyl radical formation, decreasing local cell damage; lipopolysaccharide binding, also leading to a diminished inflammatory response; and dna binding, affecting transcription and possibly regulation of the production of cell products. 198 activation of natural killer (nk) cells, modulation of complement activity, and blocking of adhesion of enterotoxigenic e. coli and shigella flexneri 98 are other proposed actions of lactoferrin. a specific region of lactoferrin, near the n terminus of the molecule, is strongly basic and is reported to mediate some of lactoferrin' s antimicrobial activity. "lactoferricins," small peptides containing this basic region, produced by proteolytic cleavage reportedly bind to lipopolysaccharide, leading to disruption of the bacterial cell wall and cytoplasmic membrane. 263 in another area of immune protection, lactoferrin may limit cancer development. 157 the proposed mechanisms of its anticancer effects includes increasing nk cell cytotoxicity, increased production of il-18 and inhibition of angiogenesis, augmented apoptosis of cancer cells and initiation of cell cycle arrest in growing tumor cells. 157 the multiple roles and proposed mechanisms of action of lactoferrin in breastfed infants continue to be more specifically elucidated. colostral cells in culture have been shown to be stimulated to secrete an interferon-like substance with strong antiviral activity up to 150 national institutes of health units/ml. 219 this property has not yet been identified in the supernatant of colostrum or milk. interferon-γ has been produced by t cells from human milk when stimulated in vitro. 219 the t cells isolated from human milk were the cd45ro phenotype and have been identified as a source of interferon. srivastava et al 254 have measured low levels of interferon-γ in not only colostrum but also transitional and mature milk. they postulated that the low level of interferon-γ (0.7 to 2 pg/ml) might be adequate to protect against infection without hyperactivation of t cells. interferon is produced by nk cells and by t cells, phenotypically thy0 and thy1. it can cause increased expression of major histocompatibility complex molecules, increase macrophage function, inhibit ige and il-10 production, and produce antitumor and antiviral activity. the exact role of interferon-γ in breast milk has not been delineated. the c3 and c4 components of complement, known for their ability to fuse bacteria bound to a specific antibody, are present in colostrum in low concentrations compared with their levels in serum. igg and igm activate complement. c3 proactivator has been described, and iga and ige have been identified as stimulating the system. activated c3 has opsonic, anaphylactic, and chemotactic properties and is important for the lysis of bacteria bound to a specific antibody. no functional role for complement in breast milk has been identified. unsaturated vitamin b 12 -binding protein of high molecular weight has been found in very high levels in human milk and in the meconium and stools of breastfed infants compared with its levels in infant formulas and infants who are formula fed. the protein binding renders the vitamin b 12 unavailable for bacterial growth of e. coli and bacteroides. 99 glycans are complex carbohydrate structures attached to various other structures (a lactose moiety, a lipid component, peptides, proteins, or aminoglycans) that are present in large amounts in human milk. 192 they include glycoproteins, glycolipids (gangliosides), glycosaminoglycans, mucins, and oligosaccharides. oligosaccharides are composed of a basic core structure derived from glucose, galactose, or n-acetylglucosamine and are linked to a variety of terminal fucose linkages or sialic acid linkages to create numerous different compounds. oligosaccharides compose the major portion of glycoconjugates in milk and are present in the milk-fat globule membrane and in skim milk. 188, 192 gangliosides are glycolipids found in the plasma membrane of cells, especially in cells in the gray matter of the brain. more specifically, gangliosides are glycosphingolipids that contain sialic acid, hexoses, or hexose amines as the carbohydrate component and ceramide as the lipid component of the molecule. the predominant gangliosides in human milk are gm1, gm2, gm3, and gd3, as reported by newburg. 190 a diverse abundance of these complex carbohydrates are synthesized by the many glycosyltransferases contained in the mammary gland. mucin and lactadherin are two glycoproteins included in this group that have antimicrobial effects. 238 some of these carbohydrate molecules are structurally similar to glycans on the surface of small intestine epithelial cells that act as receptors for microorganisms. one proposed mechanism for the antimicrobial effect of these soluble substances is direct binding with the potential pathogenic organisms. 189, 190 schroten et al 247 proposed that mucins contained in the human milk fat-globule membrane can block bacterial adhesion throughout the intestine after studying the adhesion of s-fimbriated e. coli to buccal epithelial cells. gangliosides appear to be responsible for blocking the activity of heat-labile enterotoxin from e. coli and the toxin from vibrio cholerae in rat intestinal loop preparations. 207 another toxin from campylobacter jejuni, with similar binding specificity, also seems to be inhibited by gm1. 151, 235 globotriaosylceramide, another glycolipid in human milk, is the natural cell surface receptor for the toxin from shigella dysenteriae and verotoxin released by enterohemorrhagic e. coli. 191 the proposed mechanism of action of these glycolipids is that by binding to the toxin they form a stable complex that prevents the toxin from binding to the appropriate receptors on intestinal cells; however, crane et al 48 proposed from their studies that the oligosaccharide binds to the toxin receptor to block the action of the heatstable enterotoxin of e. coli. human milk gangliosides may be important in protecting infants against toxin-induced diarrhea, but this has not been specifically demonstrated in vivo in controlled trials. 191, 207 evidence exists that human milk glycans inhibit a broad range of pathogens (table 5-5) . [188] [189] [190] [191] [192] [193] newberg et al 191 document the constitutive expression of various fucosylated glycans in human milk and secretions and present "typical" concentrations of these active agents in human milk from the literature. their secretion is related to the "secretor" and lewis genes, which control the individual differences in expression of lewis blood group types. chaturvedi et al 44 have recently examined the survival of oligosaccharides from human milk in infants' intestines. they demonstrated that the concentrations of oligosaccharides were higher in the infants' feces than in mothers' milk and higher in feces than urine. the profile of oligosaccharides found in the infants was similar to that found in their mothers' milk. the formula-fed infants had lower concentrations of oligosaccharides and the profiles of the oligosaccharides were different from those found in the breastfed infants. the oligosaccharides remained intact passing through the intestine. a small percentage are absorbed and excreted intact in the urine. the oligosaccharides were available at these sites to block intestinal and urinary pathogens. two other groups of researchers have documented variation of the composition of glycans in human milk over the first 4 months of lactation 47 and variations in the composition of glycans in diverse populations. 64 therefore a diverse repertoire of glycans are present in large amounts in human milk, which persist intact in the intestine and reach the urine, and have demonstrated inhibitory effects on a variety of pathogens. these components constitute a major contribution of human milk to innate immunity at the level of an infant' s gut. ganglioside gm 1 cholera toxin other authors propose that the gangliosides gd3 and gm3 may play an immunomodulatory role early in lactation by affecting dcs decreasing the production of interleukins (il-10 and il-12) and suppressing the expression of various cluster designation (cd) markers and major histocompatability complex class ii on dcs. 26 interleukins (ils) are considered a "subgroup" of cytokines. 161 originally, when cytokines were first hypothesized, it was thought that they were primarily produced by leukocytes and acted on other leukocytes, and therefore they could be called ils. although much of their effect is on lymphocyte activation and differentiation, it is now known that ils act on and are produced by a variety of cells. 91 goldman et al 91 identified il-1β, il-6, il-8, and il-10 in breast milk (table 5-6). srivastava et al 254 reported measuring moderate amounts of il-6, il-8, and il-10 in the different stages of breast milk. very low amounts of il-1β were detected, especially in comparison with the amount of il-1 receptor antagonist (ra), which presumably could block the activity of the small amount of il-1. hawkes et al 113 reported on the amount of cytokines in breast milk over the first 12 weeks of lactation. the proposed "proinflammatory" cytokines, il-1β, il-6, and tnf-α, were present in only 7 of 36 mothers who donated samples at each point throughout the study. a broad range of concentrations of each of these cytokines was seen during the course of the study. the "antiinflammatory" cytokines, transforming growth factor (tgf) -α 1 and tgf-β 2 , were present in significant amounts in all samples. il-2 has also been reported in breast milk in 81% of the mothers tested, with milk (aqueous) levels correlating with plasma il-2 levels. il-2 was constitutively produced from 57% of milk cell samples and il-2 production was markedly increased by stimulation of the cells with con a. 29 il-6 has been identified in breast milk by other investigators, especially in the first 2 days of life. 209, 234 the authors suggest that il-6 in human milk may augment the newborn' s immune functions before the body can begin full production of cytokines. specifically, this is accomplished by increasing antibody production, especially iga; enhancing phagocytosis; activating t cells; and increasing α 1 -antitrypsin production by mononuclear phagocytes. il-7 is a chemokine known to improve thymic output in animals and appears related to the proliferation and survival of t cells in all stages of development. 194 ngom et al 194 have described improved thymic function in exclusively breastfed infants associated with higher il-7 concentrations in the mother' s breast milk. the breast milk of gambian mothers contained variable levels of il-7, but the geometric mean levels were higher in the first 8 weeks postpartum in mothers whose infants were born in the "harvest-season" (january to june) compared with those mothers whose infants were born in the "hungry-season." the authors postulate that il-7 in breast milk enhances t-cell proliferation and survival and overall thymic development in the infant, leading to long-term benefits in protection from infection. il-8 is a chemokine capable of attracting and activating neutrophils and attracting cd45ra + t cells. il-8 is produced by mammary epithelial cells. 209 srivastava et al 254 also detected messenger ribonucleic acid (mrna) for il-8, suggesting that cells in breast milk were capable of producing il-8. the exact function of il-8 in breast milk remains to be elucidated. il-10 is thought to have antiinflammatory effects, including decreasing the production of interferon-γ, il-12, and other proinflammatory cytokines. it has been reported to enhance iga, igg, and igm synthesis. il-18 has been identified in colostrum, early milk, and mature milk with the highest levels occurring in colostrum and in association with preterm deliveries and complications of pregnancy in the mothers. 260 the levels of il-18 were correlated with soluble fas ligand in colostrum. il-18 was detected by immunohistochemical staining in actively secreting epithelial cells in a lactating breast. il-18 has been shown to be produced by intestinal epithelial cells and activated macrophages. it leads to the production of other chemokines (gm-csf, il-2, tnf-α). it induces the expression of fas ligand on lymphocytes. the authors suggested that il-18 present in colostrum may play a role in stimulating a systemic t h 1 response and causing nk cell and macrophage activation in neonates. the interaction and the direct effect of these ils in breast milk must be clarified. the amount of t cells bearing markers of recent activation is increased in human milk compared with the results in peripheral blood of adults. wirt et al 276 of the many bioactive substances that have been identified in human milk, cytokines are some of the most recently identified and investigated agents, although their existence has long been suspected in attempts to explain certain immunologic and protective effects of breast milk on infants. more than 40 cytokines have been described, 166 and more than 10 of these have been identified in human milk. 91, 254 cytokines are small proteins or glycoproteins that, through binding to receptors on immune and nonimmune cells, produce a broad range of effects (many still unidentified) through autocrine, paracrine, and endocrine actions. cytokines are produced predominantly by immune cells and function in complex associations with other cytokines to stimulate and control the development and normal functioning of the immune system. the nomenclature and abbreviations used are complicated and confusing. newer systems of classification have been established according to which cells produce them or what their general functions are 161 or based on the relative position of their cysteine residues or their receptor types (ccr, cxcr, cx3cr). 166 box 5-3 provides a simplified list with abbreviations. little evidence demonstrates specific in vivo activity of the different cytokines. based on general information on the function and interaction of the particular cytokines, as well as consideration of as yet unexplained effects of breast milk, proposed functions of the cytokines include initiation of development of host defense, stimulation of host defenses, prevention of autoimmunity, antiinflammatory effects in the upper respiratory tract and gi tract, and stimulation of the development of the digestive system, especially the mucosal immune system of the alimentary tract and the proximal respiratory tract. the maternal breast may respond to feedback stimulation or suppression by secreted cytokines, influencing the growth, differentiation, and secretory function of the breast. as shown in other situations, cytokines may enhance receptor expression on cells in the respiratory and gi tracts for major histocompatibility complex molecules or immunoglobulins. various cell types in the mucosal immune system may be activated or attracted to specific sites in the gi tract by the action of cytokines. beyond these proposed beneficial effects of cytokines, newer studies are identifying specific leukocyte inhibitory factor lif immunologic and protective roles for different cytokines in developing infants. for example, extensive work has been done on epidermal growth factor (egf) and other growth factors (hb-egf, g-csf, epo, and epo-like growth factors) have been studied relative to their role in preventing necrotizing enterocolitis (nec) and gut homeostasis. 183 a number of potential roles for egf in gut homeostasis have been proposed and studied, including intestinal development, proliferation and adaptive response to damage, repair and regeneration and diminishing inflammatory responses to various stimuli. tgf-β has been studied for its role in initiating and stimulating iga production early on in infancy. 200 the actual measurement of cytokines in breast milk has been complicated by a number of factors, including different assays used (bioassays, enzyme-linked immunosorbent assay [elisa], radioimmunoassay), binding to proteins, their existence in monomeric or polymeric forms, 8 the presence of antagonists, and their varying presence in colostrum, early milk, or mature milk. goldman et al 91 reported on the bioactivity and concentration of cytokines in breast milk from their own work and that of others (see table 5 and at lower levels in mature milk, as well as high levels of soluble tnf-α receptor i (stnf-αri), stnf-αrii, and il-1ra. also, they identified that most tnf-α did not exist "free" in breast milk but was associated with tnf receptors. the in vivo significance of these findings remains to be assessed. given the complex interaction and regulation of cytokine production and cytokines' relation to coordinated inflammatory and antiinflammatory responses in tissues, one should assume that the interaction of cytokines in breast milk and the effect of cytokines, cytokine receptors (soluble and expressed on various cell types), and cytokine antagonists on the infant will be equally complex. a new methodology, antibody-based protein arrays has been applied to identify cytokines in human milk. 148 kverka et al 148 analyzed colostrums and milk samples from the first 4 days postpartum using two different arrays capable of detecting 42 and 79 cytokines. three cytokines (egf, il-8/cxcl8, gro/cxcl1-3) were detected in all of the tested samples. nineteen cytokines were present in more than 50% of the samples. an additional 32 cytokines were identified in human milk for the first time. the concentration of cytokines varied in the different women and varied over time. continued investigation with this and other assays will be essential to understanding the significance and specific effects of these substances in breast milk. nucleotides, nucleosides, nucleic acids, and related metabolic products are essential to many biologic processes. although they are not essential nutrients because they can be synthesized endogenously and recovered from in vivo "salvage" sources, their presence in the diet may carry significant benefits under various conditions (i.e., "conditionally essential"). 42, 184, 266 in situations of disease, stress, rapid growth, or limited dietary intake, supplementation of the diet with nucleotides may decrease energy expenditure to synthesize or salvage nucleotides, which optimizes the host response to these adverse situations. nucleotides exist in relatively large amounts in human milk, 15% to 20% of the nonprotein nitrogen, suggesting that they have some nutritional significance, although no clinical syndromes have been associated with nucleotide deficiency to date. nucleotides are present in the natural milk of different species in varying amounts and composition. the nucleotide content and composition of bovine milk are particularly less and different from human milk. infant formulas supplemented with nucleotides contain roughly the same amounts of nucleotides as human milk, from 20 to 70 mg/l. 39, 40, 154 unsupplemented formulas contain less and different amounts of nucleotides. mammalian cells contain a large variety of nucleotides and related products, which have many metabolic functions, including the following 40-42 : 1. energy metabolism: adenosine triphosphate is a major form of available cellular energy. 2. nucleic acid precursors: the monomeric units for rna and dna are present. 3. physiologic mediators: cyclic adenosine monophosphate and cyclic guanosine monophosphate serve as "messengers" for cellular processes; adenosine diphosphate is necessary for platelet aggregation; and adenosine has been shown to affect vasodilatation. 4. related products function as coenzymes in metabolic pathways: nicotinamide-adenine dinucleotide, flavin adenine dinucleotide, and coenzyme a. rying molecules in synthetic reactions: uridine diphosphate glucose in glycogen synthesis and guanosine diphosphate mannose, guanosine diphosphate-fucose, uridine diphosphate-galactose, and cytidine monophosphate sialic acid in glycoprotein synthesis. 6. allosteric effectors: the intracellular concentrations of nucleotides influence the progression of certain steps of metabolic pathways. 7. cellular agonists: extracellular nucleotides influence intracellular signal transduction (e.g., cyclic adenosine monophosphate and inositol-calcium pathway). nucleotide concentrations in cells and tissues are maintained by de novo synthesis and salvage from intermediary metabolism and diet ( figure 5-11 ). 224 nucleosides are the predominant product absorbed in the small intestine. nucleosides are probably transported by passive diffusion and a carrier-mediated process; purines and pyrimidines are transported by passive diffusion at high concentrations and by a sodium-dependent active mechanism at low concentrations ( figure 5-12) . 224 the digestion and absorption of nucleotides, nucleosides, and pyrimidines and purines also involve polymeric and monomeric nucleotides and other adducts (nucleosides in a biologically active moiety). in early reports on the nucleotide and nucleoside content of milk, various methods of measurement were used, and the amounts were described as either the monomeric fraction of nucleotides or the total rna. leach et al, 154 recognizing the complex nature of digestion and absorption of nucleotides and related products, attempted to measure the total potentially available nucleosides (tpans) in human milk using solid-phase extraction, highperformance liquid chromatography analysis, and enzymatic hydrolysis of the various fractions. they analyzed breast milk samples at various stages throughout lactation (colostrum; transitional, early, and late mature milk) from 100 european women and 11 american women. they used an aqueous tpan-fortified solution containing ribonucleosides, 5′-mononucleotides, polymeric rna, and nucleoside-containing adducts to estimate the accuracy of their process. the mean ranges of tpan values were similar for european women from different countries and american women, although broad ranges were seen and the composition of individual nucleotides varied. 154 the mean tpan value was lowest in colostrum but did not show a consistent upward or downward trend in transitional, early, or late mature milk. the mean ranges of tpan values were 82 to 164 mmol/l for colostrum, 144 to 210 mmol/l for transitional milk, 172 to 402 mmol/l for early mature milk, and 156 to 259 mmol/l for late mature milk (table 5-7) . monomeric and polymeric nucleotides were the predominant forms of tpan in pooled samples. cytidine, guanosine, and adenosine were found mainly in these fractions, whereas uridine was found primarily as free nucleotide and adduct ( 154 concluded that their process of estimating tpans, including sequential enzymatic hydrolyses, and measuring the entire nucleotide fraction provides a reasonable estimate of the in vivo process and the nucleotides available to the infant from human milk. proposed effects of dietary nucleotides include effects on the immune system, iron absorption, intestinal flora, plasma lipoproteins, and growth of intestinal and hepatic cells. effects on the immune system, related to nucleotide supplementation to the diet, have mainly been reported from animal studies and include increased mortality rate from graft-versus-host disease, improved delayed-type cutaneous hypersensitivity and alloantigen-induced lymphoproliferation, reversal of malnutrition and starvation-induced immunosuppression, increased resistance to challenge with s. aureus and c. albicans, and enhanced t-cell maturation and function. 218 spleen cells of mice fed a nucleotide-free diet produce lower levels of il-2, express lower levels of il-2 receptors, and have decreased nk cell activity and macrophage activity. 40, 41 presumably these nucleotide-associated changes are related to t-helper/inducer cells and the initial phases of antigen processing and lymphocyte proliferation. 41, 42, 266 in vitro and in vivo experiments have documented that ingested nucleotides increased iron absorption, perhaps affecting xanthine oxidase. 224 although in vitro studies showed that added nucleotides enhanced the growth of bifidobacteria, conflicting results have been obtained on the influence of dietary nucleotides on the fecal flora of infants receiving breast milk or nucleotide-supplemented formula. 11, 224 clinical studies in infants receiving nucleotide-supplemented formula demonstrated increased high-density lipoprotein cholesterol, lower very-low-density lipoprotein cholesterol, increased long-chain polyunsaturated fatty acids, and changes in red blood cell membrane phospholipid composition. 11 supplementation studies in animals have shown enhanced gi tract growth and maturation, improved intestinal repair after diarrhea, stimulation of hepatic growth, and augmented recovery from hepatectomy. 218 a recent review discusses the effects of dietary nucleotides on the immune system and protection against infection reported in studies in the literature. 244 carver et al 41 †adducts are of the form nucleoside-phosphate-phosphate-x, where x is a biologically relevant moiety (e.g., uridine diphosphate-galactose or nicotinamide-adenine dinucleotide). 4 months, nk cell activity and il-2 production were higher in the breastfed and nucleotidesupplemented groups compared with those receiving formula without nucleotide supplements. infections occurred infrequently in all groups, but slightly less in the breastfed group. no differences were noted in hematologic profiles and plasma chemistry values, and no toxicity or intolerance was associated with nucleotide supplementation. the sample size was small, marked variability was seen in the il-2 measurements, and the differences noted at 4 months were less than at 2 months. therefore the authors concluded that dietary nucleotides may contribute to improved immunity in breastfed infants. brunser et al 28 examined the effect of a nucleotide-supplemented formula on the incidence of diarrhea in 392 infants in chile, studied through 6 months of age. although the infants receiving the supplemented formula (20 mg/l) experienced less diarrhea, the difference in the duration of diarrhea was small. the numbers were too small to comment on the causative agents of diarrhea, although no apparent protection against any one agent was seen. the beneficial effect of nucleotides against diarrhea was proposed to be secondary to enhanced immune response to intestinal pathogens or improved intestinal integrity or a combination of both. in a larger study of 3243 infants younger than 6 months of age, the severity of the diarrhea (duration and number of bowel movements) as well as the incidence of diarrhea was lower in the nucleotide supplemented group. 152 two groups of premature infants fed either nucleotide supplemented (20 mg/l) or unsupplemented formula were followed, measuring the concentration of plasma immunoglobulins throughout the first 3 months of life. 184 igg plasma concentrations were not different in the two groups during the study period. igm plasma levels were higher in the nucleotide supplemented group at 20 to 30 days and 3 months of life, while iga plasma levels were significantly higher at 3 months of age in the supplemented group. pickering et al 218 published a 12-month, controlled, randomized study of 311 infants to examine the effect of added nucleotides at levels comparable to human milk on infants' immune responses to various vaccine antigens; 103 nonrandomized infants received breast milk for at least 2 months and then either human milk or a standard infant formula. another 208 infants were randomized to receive either a standard infant formula or one supplemented with nucleotides. the amount and actual nucleotide content added were based on tpans, as measured by leach et al, 154 equaling 72 mg/l. overall growth and nutrition tolerance were similar in each group. the nucleotide group had significantly higher geometric mean titers of h. influenzae type b antibody and diphtheria antibody than the control group or the breastfed infants. no significant difference was seen between the nucleotide and control groups for the igg response to oral poliovirus vaccine or tetanus. infants who are breastfed for longer than 6 months had significantly higher antibody responses to oral poliovirus vaccine than children breastfed for less than 6 months or either of the two formulafed groups. no significant differences were found between the different groups with respect to total igg, iga, or ige. differences were seen in the number of children who experienced at least one episode of diarrhea: the nucleotide group (4/27, 15%) versus the control group (13 of 32, 41%, p <0.05) and the breastfed group (6 of 27, 22%) . notably, the breastfed group was heterogeneous relative to the amount of breast milk received and the duration of feeding, whereas the nucleotide group received supplementation for the entire 12 months. questions that remain concerning nucleotides and their proposed beneficial effects in an infant' s diet include the following: • what are the proven mechanisms of action of these proposed benefits? • what form and concentration of nucleotides are necessary to effect these benefits? • is adequate information available to justify using nucleotides in infant formula in higher amounts and different compositions than are currently used? debate and research to answer these and other questions concerning nucleotides will continue. a primary function of each of the body' s different mucosal surfaces is immunologic. each distinct mucosal surface has multiple other physiologic functions including gas exchange (in the lungs), nutrient absorption (in the gut), sensory detection (in the eyes, nose and mouth) and reproduction (in the uterus and vagina). the thin, permeable nature of these barrier mucosal surfaces, their large surface area and the constant exposure to microorganisms, foreign proteins, and chemicals predisposes the mucosal membranes to damage and infection. during the first year(s) of life, when the infant' s immune system is developing and maturing, it is doing so on a systemic and a mucosal basis as well as involving both innate and adaptive immune mechanisms. that development must include the ability to respond to and protect against invasive pathogens at the same time as "tolerate" or "ignore" the multitude on commensal organisms that reside at these surfaces. during this early development, breast milk contains numerous bioactive factors that supplement the immune protection at the mucosal level while limiting inflammation and contribute to the immune modulation and growth stimulation of infants' mucosal and systemic immune defenses. the mucosal immune system involves both innate mechanisms and adaptive immune mechanisms functioning in concert. the development of the mucosal immune system occurs in the prenatal period and continues in the postnatal period. the functional mucosal barrier includes the action of enzymes, chemicals, acidity or ph, mucus, immune globulins and indigenous flora. in as early as 8 weeks of gestational age, researchers have identified changes in the intestinal barrier with the development of enterocytes, goblet cells and enterochromaffin cells along with evidence of development of tight junctions between the epithelial cells. 211, 222 mucus production, which can block adherence of pathogens to epithelial cells, demonstrates both pre-and postnatal development beginning with evidence of expression of the muc2 gene as early as 12 weeks' gestational age. 34 this is approximately the same time that paneth cells appear in intestinal crypts. these cells secrete various products, including α-defensin, lysozyme, secretory phospholipase a 2, and tnf-α, which contribute to protection from pathogens, stem cell protection within the epithelia layer and influence the selection and number of commensal organisms. 180, 240 secretory immune globulins siga and igm act at the epithelial surface, largely without inflammation, by limiting adherence and transmigration and facilitating phagocytosis of potential pathogens. the well-recognized malt is present in localized areas beneath the mucosal surfaces: tonsils and adenoids in the nasopharynx and peyer patches and isolated lymphoid follicles in the intestine. overlying the isolated lymphoid follicles of the gut are specialized epithelial cells called m-cells. m-cells (membrane, microfold, or multifenestrated cells) come in direct contact with microorganisms and antigens due to a lack of a surface glycocalyx covering. these remarkable cells endocytose, phagocytose and transcytose molecules, and antigens from their luminal surface to their basal surface. antigenpresenting cells and lymphocytes process the trancytosed molecules, presenting them to submucosal aggregates of lymphocytes. the activated lymphocytes that have responded to the specific presented antigens migrate via the lymphatics to the thoracic duct and into the blood. these lymphocytes circulate in the blood, until they return to mucosal tissues, predominantly the same ones they originated from, where they now function as effector lymphocytes in the lamina propria. this process of "directed migration" to specific sites occurs due to the influence of cytokines and adhesion molecules, such as chemokine ccl28 (mucosal epithelia chemokine) expressed in the colon and salivary glands and ccl25 (thymus-expressed chemokine) which effects the site-specific migration. 217 the immune response of lymphocytes in the submucosa and the subsequent directed migration to the same and other mucosal sites produces a focused response to a selected repertoire of antigens at those sites. the lactating mammary gland is an essential component of malt. a mother' s mature effective immune response to microorganisms in her and her infant' s environment through this antigenic stimulation of malt in the mother' s gut and respiratory mucosa and the directed migration of cells to the breast results in activated lymphocytes and antibodies in the breast milk providing protection to the infant against those microorganisms. this is a wellrecognized example of how breast milk can provide additional immune protection to the infant. it is also one of the reasons to continue breastfeeding when a mother or the infant have a possible infection. the mucosal immune system undergoes significant postnatal development, in part due to the dramatic exposure of the mucosa to large numbers of microorganisms in early postnatal life. peyer patches are rudimentary, and few immunoglobulinproducing intestinal plasma cells are present until several weeks after birth. 24 after several weeks, germinal centers within the lymphoid follicles develop, and the number of igm-and iga-producing cells in the intestine increase. immunoglobulin-producing intestinal plasma cells (primarily iga-producing cells) in the lamina propria increase in number from 1 to 12 months of age. 257 with normal maturation of the mucosal immune system, large numbers of immunoglobulin-producing cells locate in the intestinal lamina propria. the monomeric iga produced by these plasma cells is transported through epithelial cells to the mucosal lumen. attachment of an epithelial glycoprotein, the membrane secretory component to two iga molecules leads to the formation of a dimeras; the siga molecule is "secreted" at the mucosal surface. igm, in the form of a pentamer, contains a polypeptide j-chain and is transported by the same mechanism. 25 a portion of the secretory component remains attached to the siga and igm, which protects these molecules against proteolysis and contributes to their stability. large amounts of siga and igm are produced, in a similar fashion, by the mammary glands and delivered to the infant via breast milk. the siga and igm remain stable in saliva and feces 105 and provide specific protection by blocking adherence and entry and facilitating inactivation, neutralization, and agglutination of a wide variety of microorganisms. distinct from the action of immunoglobulins, a large number of bioactive factors in breast milk act at the mucosal level to supplement the innate defenses. 103 these include lactoferrin, lysozyme, casein, oligosaccharides, glycoconjugates and lipids. mucin-1, lactadherin, and a glycosaminoglycan are antimicrobial components, which are part of the milk-fat globule. free-fatty acids and monoglycerides, digested components of the milk-fat globule, can cause lysis of enveloped viruses, bacteria, fungi, and protozoa. lauric and linoleic acids, which constitute a large percentage of the ffe in human milk, are two such acids produced by lipolysis in the stomach. 104 additional factors contained in breast milk with demonstrated activity at the level of the mucosa include cytokines, hormones and growth factors. il-10 and ifn-γ act by influencing epithelial barrier integrity. 74 other factors that are considered to contribute to mucosal growth and development are tgf-α, egf, and hormones (insulin and insulinlike growth factor). 56 many other factors contained in breast milk have the potential for activity at the level of the mucosa, including nutrients, vitamins, nucleotides, enzymes, and soluble molecules with receptor-like structures (soluble cd14, soluble toll-like receptor 2) 150,155,267 toll-like receptors (tlrs) and the complex interaction between indigenous bacterial flora and the intestine is an important aspect of research into the development of the mucosal immune system. forchielli and walker 69 have reviewed many of these immune mechanisms acting at the mucosal level. tlrs are transmembrane receptors (pattern recognition receptors) that are capable of detecting and discriminating among various groups of potential pathogens and initiate different immune responses to them. tlrs "recognize" pathogenassociated molecular patterns, conserved features in the pattern of molecules expressed by pathogens and commensal organisms. specific tlrs recognize a particular repertoire of patterns: tlr2 identifies bacterial lipoproteins and peptidoglycan molecules; tlr3 recognizes double-stranded dna and tlr4 identifies lipopolysaccharide. ten tlrs are recognized in humans to date; some have identified legends (pathogen-associated molecular patterns from viruses, bacteria, and protozoa) to which they bind. tlrs are present on some epithelial cells, but are predominantly expressed on macrophages and dcs. 228 intestinal epithelial cells are influenced by gut flora and local immune response to express specific tlrs. the recognition of specific antigens by epithelial cell tlrs stimulates different intracellular signal pathways that lead to different t-lymphocyte immune responses. it has been postulated that the ongoing immune stimulation elicited by the microbial flora in the gut "programs" the host to predominately express different t-helper cell responses: t h 1-like, t h 2-like and t h 3-like. this is referred to as "crosstalk" between the indigenous intestinal flora and the body' s immune system. the t h 1-like response is described as delayed-type hypersensitivity or cellular immunity; characterized by the predominant release of il-2, il-12 and interferon-γ. the t h 2-like is primarily humoral immunity, antibody production (especially ige) associated with ils: il-4, il-5, and il-6. the t h 3-like response is related to oral tolerance and antiinflammatory effects in association with the release of il-10 and tgf-10. a theoretical "ideal" for this system is the ability of the host to respond to various stimuli with balanced protection against the microbial invasion without excessive inflammation or damage to the host. an imbalanced (or poorly regulated) response of this system could result in an allergic reaction against food proteins (t h 2 excess) or an autoimmune inflammatory response against self-antigens (t h 1 excess). 69 ongoing research continues to explore these molecular mechanisms, their potential contribution to allergy, autoimmune disease and normal immune function development within a fetus, infant, and young child. the role of breast milk in the development of the systemic and mucosal immune systems takes on new significance when considering these concepts and mechanisms. this is especially true when examining the role of breast milk in adding to the innate and adaptive immune response at the level of the mucosa, the effect breast milk has on the respiratory and intestinal microbiota, the purported antiinflammatory effects of breast milk, and the proposed influence of breast milk on the maturational development of the intestine. vorbach, capecchi, and penninger 270 postulated that the mammary gland evolved from a protective immune gland as part of the innate immune system. they present a list of various protective molecules that are part of both mucosal secretions and human milk. they discuss how specific nutritional factors in human milk have dual functions: nutritional and protective. this highlights the dual role of the breast as a nutritional and immune organ and should stimulate further research into the breast' s role in innate immunity as a component of the mucosal immune system. investigation into the microbial colonization of the intestinal tract has exploded. much of this investigation has been driven by new molecular techniques involving the analysis of ribosomal rna sequences of microbes that might not have been identified by traditional culture techniques. the diversity of the microbiota (all the microbes which colonize the gi tract) can be viewed from different perspectives based on the technical methods used. 255 probiotics have been broadly defined as microorganisms that can exist within a host while affording benefits for the organisms and the host. prebiotics are substances that (through different mechanisms) increase the growth and survival of probiotic bacteria within the host. commonly recognized probiotic bacteria are lactobacillus rhamnosus gg, bifidobacteria infantis, streptococcus thermophilus, bacillus subtilis, saccharomyces boulardii, and bifidobacteria bifidus. many more organisms are considered to be probiotic, some of which are commercially available. 178 prebiotics are predominantly nondigestible oligosaccharides that ferment within the colon changing the ambient ph and producing small-chain fatty acids. breast milk with its significant composition of oligosaccharides functions as a prebiotic source for an infant, facilitating the growth of bifidobacteria and lactobacilli. 52, 249 ongoing research is exploring the potentially mutually beneficial relationship between the microbes and the host with particular attention to nutrition (the availability of nutrients, energy sources, and synthesis of vitamins as influenced by the microbes), the developing gi tract (including angiogenesis and mucosal barrier repair), 75, 186, 210 the maturation of mucosal immunity, 140,216 both the innate system 119 and adaptive system, 170 and the bioavailability and metabolism of drugs and chemicals in the gi tract. 57, 121 specific proposed mechanisms of how probiotic bacteria and prebiotic substances contribute to an infant' s developing immune system include competition with pathogenic bacteria for colonization, strengthening the tight junctions to enhance the mucosal barrier, producing antimicrobial bacteriocidins, stimulating mucus production, stimulating peristalsis, influencing the secretion of siga, stimulating the crosstalk interaction between intestinal cells and colonizing bacteria to affect the mucosal immune development, and increasing the production of certain cytokines (il-10 and interfer on-γ). 75, 140, 214, 216 the microbial colonization of an infant' s intestinal tract begins at birth, with organisms from the maternal flora being the first colonizers. numerous additional factors directly influence the composition of the intestinal microbiota in early infancy, including gestational age, ingestion of breast milk or formula, initiation of solid foods, mode of delivery, the route of delivery of food, the time of onset of feeding, exposure to other microbes through contact (with family, animals, persons from other environments), antibiotics, and intercurrent or chronic illness. 66, 215 the predominant flora of breastfed infants are lactobacillus bifidus and bifidobacterium spp., which constitute up to 95% of the culturable organisms. the remaining minority of bacteria include streptococcus, bacteroides, clostridium, micrococcus, enterococcus, e. coli, and other uncommon organisms in small numbers. 178, 186, 282 lactobacillus bifidus metabolizes milk saccharides, producing large amounts of acetic acid, lactic acid, and some formic and succinic acids, which create the low ph of the stool of breastfed infants. l. bifidus also produces short-chain fatty acids in the course of colonization. large numbers of bifidobacteria can lower the ph of the intestine, which limits the growth of some pathogens such as e. coli, bacteroides, and staphylococci. the flora of bifid bacteria is inhibitory to certain pathogenic bacteria. substantial clinical evidence is available to demonstrate protection against intestinal infections from s.aureus, shigella, salmonella, vibrio cholerae, e. coli, rotavirus, campylobacter, and protozoa. 81 two facilitory actions of breast milk are apparent. the first encourages the growth of l. bifidus and thus crowds out the growth of other bacteria. in the second, the number of pathogens is also kept low by the direct action of lysozyme and lactoferrin. when the number of pathogenic bacteria is kept low, immune antibodies can keep the growth of potentially pathogenic bacteria under control and limit the invasion of bacteria through the gut wall into the bloodstream. the intestinal flora of formula-fed infants is made up of predominantly gram-negative bacteria, especially coliform organisms, bacteroides, and including clostridium, enterobacter, and enterococcus. 282 studies have demonstrated that potentially four distinctly different "microhabitats" for microflora within the gi lumen, within the mucus layer, separately within crypt mucus, and directly on the surface of the intestinal epithelium. the significance of the microhabitats and the effect of specific microorganisms has yet to be determined. 75 at weaning, the facultative anaerobes decline in number and obligate anaerobes (bacteroides) become the predominant organisms in the intestine. preterm infants are colonized with different types and numbers of bacteria from full term infants. the environment of neonatal intensive care units (nicus), including incubators, widespread use of antibiotics and parenteral nutrition, and illness influence the microbial colonization. the short-and long-term effects of the different and changing gi microbiota are a concern. 186,206 this is particularly true when one considers the contributing factors or causes for sepsis, nec, chronic lung disease, or poor neurologic outcome in nicus. the question of a causal role of intestinal microflora and the development of nec in premature and very-low-birth-weight (vlbw) infants has been proposed. 123, 174 gewolb et al 79 suggested that a low percentage of bifidobacterium and lactobacillus in the stool of vlbw infants within the first month of life is a risk factor for infection. some studies on the use of probiotics and the occurrence of nec have demonstrated a lower incidence of nec in infants receiving probiotics. 19, 124, 162 here again, the use of mothers' breast milk for premature infants and vlbw infants decreases the risk to the infant of sepsis, nec, and infection-related events. 73, 245 in a controlled prospective study of high-risk, low-birth-weight infants in india using donor human milk, significantly fewer infections and no major infections were found in the group receiving human milk, although the control infants experienced diarrhea, pneumonia, septicemia, and meningitis. 185 the properties of human milk do appear to control or limit infections in infants. hundreds of articles 168, 185 have been written about the protective effect of breastfeeding, including the recent agency for healthcare research and quality publication on breastfeeding and maternal and infant health outcomes in developed countries from 2007. 126 using evidence-based analyses, the report documents the decreased risk for acute otitis media, gi infections, and lower respiratory tract diseases in breastfed infants in developed countries. 126 breast milk iga has antitoxin activity against enterotoxins of e. coli and v. cholerae that may be significant in preventing infantile diarrhea. antibodies against o antigen of some of the most common serotypes of e. coli were found in high titer in breast milk samples collected from healthy mothers in sweden. the infants who had consumed reasonable amounts of breast milk with high titers of e. coli antibodies had antibodies in their stool. 106 protection against cholera in breastfed children by antibodies in breast milk was studied by glass et al. 81 a prospective study in bangladesh showed cholera antibody levels to vary in the colostrum and milk. the correlation among colonization, disease, and milk antibodies led the authors to conclude that breast milk antibodies against cholera do not protect children from colonization with v. cholerae, but they do protect against disease. salmonella infection was similarly studied by france et al 71 to evaluate the immunologic mechanisms in host colostrum and milk specific for salmonellae. vigorous responses of colostral and milk cells against these organisms and nonspecific opsonizing capacity of the aqueous phase of colostrum and milk were demonstrated. gothefors et al 95 showed that e. coli isolated from stools of breastfed infants differed from strains found in formula-fed infants in two respects. first, e. coli strains were more sensitive to the bactericidal effect of human serum. second, and more often, spontaneously agglutinated bacteria from other sites, such as the prepuce or periurethral area, were less sensitive in breastfed infants. these findings support the theory that breast milk favors proliferation of mutant strains, which have decreased virulence. this mutation of bacterial strains is another way breastfeeding may protect against infection. it has been suggested that "milk immunization" is a dynamic process because a mother' s milk has been found to contain antibody to virtually all her infant' s strains of intestinal bacteria. the mother exposed to the infant' s microorganisms through either the breast or the gut responds immunologically to those microorganisms and thus directly provides protection for her immunologically immature infant. the orderly review of data on the presence of antibodies in human milk has produced a substantial list of affected organisms. in addition to e. coli, antibodies to bacteroides fragilis, clostridium tetani, haemophilus pertussis, diplococcus pneumoniae, corynebacterium diphtheriae, salmonella, shigella, chlamydia trachomatis, v. cholerae, s. aureus, and several strains of streptococcus (table 5-9 ) have been identified. noguera-obenza and cleary 196 have summarized the contribution of siga in breast milk in protecting infants from bacterial enteritis. a study in oslo by hanson 106 of an outbreak of severe diarrhea caused by e. coli strain 0111 showed that six severely ill children were formula fed. two infants who were breastfed had e. coli strain 0111 in their stools but showed few symptoms. their mothers had no detectable antibodies for strain 0111 in their milk, which would suggest that other factors in human milk protect the infant from serious illness when no antibodies are in the milk. hanson 106 also reported the results of another study in which after colonization with a specific strain of e. coli, mothers had large numbers of lymphoid cells in their milk with antibodies to that e. coli. the mothers' serum showed no such response. this supports the concept that antigen-triggered lymphoid cells from peyer patches seek out lymphoid-rich tissue, producing iga in the mammary gland. the mother is immunized in the gut at the same time as her milk. it has also been shown that e. coli enteritis can be cured by feeding human milk. schlesinger and covelli 246 studied possible cell-mediated immunity in breastfed infants. they showed that tuberculin-positive nursing mothers had reactive t cells in their colostrum and early milk. furthermore, 8 of 13 infants nursed by tuberculin-positive mothers had tuberculinreactive peripheral blood t cells after 4 weeks. cord blood had no such activity. no clinical or research data suggesting a protective effect of this apparently induced tuberculin reactivity in infants are available. protection against viruses has been the subject of similar studies. breast milk contains antibodies against poliovirus, coxsackievirus, echovirus, enterovirus, influenza virus, reovirus, rsv, rotavirus, and rhinovirus. 187, 237 it has been confirmed that human milk inhibits the growth of these viruses in tissue culture. nonspecific substances in human milk are active against arbovirus and murine leukemia virus, according to work by fieldsteel. 67 a high degree of antiviral activity against japanese b encephalitis virus as well as the two leukemia viruses has been found in human milk. the factor was found in the fat fraction and was not destroyed by extended heating, which distinguishes it from antibodies. may 168 believes the nonimmunoglobulin macromolecule antiviral activity in human milk is caused by specific fatty acids and monoglycerides (table 5-10) . it is important to recognize other factors besides immmunoglobulins are contained in breast milk that can play a role in protection of the breastfeeding infant from viral infections. 102, 104 specimens of human colostrum have been found to contain neutralizing activity against rsv. rsv has become a major threat in infancy and is the most common reason for hospitalization in infancy in some developed countries. it has a high mortality rate. epidemics have occurred in special care nurseries. statistically significant data collected by downham et al 58 showed that, compared with uninfected control subjects who were breastfed (46 of 167), few breastfed babies (8 of 115) were among the infants hospitalized for rsv infection. fishaut et al 68 studied the immune response to rsv prospectively in 26 nursing mothers during several months. antiviral igm and igg were rarely found in colostrum or milk. rsv-specific iga, however, was identified in 40% to 75% of specimens. two mothers with the disease had specific igg, igm, and iga antibody in serum and nasopharyngeal secretions, but only iga was found in their milk. this confirms that iga antibody to specific respiratory tract pathogens is present in the products of lactation. because rsv appears to replicate only in the respiratory tract, the authors suggest that viral-specific antibody activity in the mammary gland may be derived from the bronchioleassociated lymphoid tissue. in human milk, bile salt-stimulated lipase has been found to be the major factor inactivating protozoans (table 5-11 ). 168 the mechanism by which lipase acts is not known, although it may generate fatty acids and monoglycerides, which inactivate enveloped bacteria, viruses, or protozoa. a nonimmunoglobulin, nonlipase, heat-stable factor has been identified in human milk that can inactivate giardia lamblia. human milk protects against many intestinal and respiratory pathogens with minimal evidence of inflammation. goldman et al 90 hypothesize that human milk is poor in initiators and mediators of inflammation but rich in antiinflammatory agents. several major biochemical pathways of inflammation, including the coagulation system, the fibrinolytic system, and complement, are poorly represented in human milk. box 5-4 outlines the antiinflammatory properties of various constituents and the paucity of certain proinflammatory mediators in breast milk. the interaction of factors in the milk with one another or with host defenses cannot be entirely predicted by examining each factor separately. when the decreased response of human milk leukocytes to chemoattractant peptides was demonstrated by thorpe et al, 262 the failure of the response of human milk leukocytes was not caused by alterations in maternal peripheral blood leukocytes. this suggests that inhibitors are in the milk and that human milk leukocytes may be modified in the mammary gland to protect through noninflammatory mechanisms. 262 only low numbers of basophils, mast dells, eosinophils, and cytotoxic t-cells are present in breast milk. many other studies have documented the decreased function of milk polymorphonuclear leukocytes and macrophages in both colostrum and mature milk. 30 the antioxidant properties of human colostrum were demonstrated by buescher and mcilheran 31 using aqueous human colostrum on human pmns. the colostrum significantly interfered with pmn oxygen metabolic and enzymatic activities that are important in the mediation of acute inflammation. antioxidants in breast milk can also contribute to the overall antiinflammatory effect of breast milk. demonstrated antioxidants contained in breast milk include an ascorbate-like compound, uric acid, α-tocopherol, β-carotene, and l-histidine, all of which scavenge oxygen radicals. blood levels of α-tocopherol and β-carotene are higher in breastfed than unsupplemented formula-fed infants. catalase, glutathione peroxidase, and lactoferrin are functionally antioxidants. antioxidant activity has been demonstrated in colostrum and at lower levels in mature human milk. additionally, specific cytokines that can exhibit antiinflammatory effects have also been identified in human colostrum and milk: tgf-β 1 and -β 2 195,239,259 and il-10. 74 a cytokine antagonist, il-1ra, and soluble receptors for tnf-α are also found in colostrum and milk. 32, 254 palkowetz et al 209 have reported that il-1ra can decrease the action of il-1β. both human colostrum and milk cause a diminished influx of polymorphonuclear cells to a local site of inflammation in two different in vivo models of inflammation in rats. 38, 96, 181 the inflammatory response can be protective for the host at the same time as it can produce the symptoms of clinical illness. breast milk contains a large variety of antimicrobial factors that exert their protective effect without causing significant inflammation (e.g., siga, oligosaccharides, lactoferrin, nucleotides). many other cells and factors in breast milk participate in a complex interaction to both protect the infant and limit the potential damaging effects of an uncontrolled inflammatory response. further study into the dynamic interplay of the many factors in breast milk with developing infants' mucosal barriers and immune systems is needed to fully understand the protective immune response and the antiinflammatory benefits of human milk. (see chapter 17 on human milk as prophylaxis in allergy) in discussing the allergic protective properties of human milk, it is difficult to identify specific protective factors that are proved to protect against allergy. it is equally difficult to discuss the proposed mechanisms of protection because the exact mechanism of "oral tolerance" remains theoretic and the relative importance of contributing factors to hypersensitivity must still be adequately defined. some of the important variables concerning tolerance and sensitization are genetic background of the host, nature and dose of the antigen, frequency of exposure, timing (age) at first and subsequent exposures, immunologic status of the host, and route of exposure. during the neonatal period the small intestine has increased permeability to macromolecules. infants have more serum and secretory antibodies against dietary proteins than children or adults. production of iga in the intestinal tract is delayed until 6 weeks to 3 months of age. iga in colostrum and breast milk prevents the absorption of foreign macromolecules when an infant' s immune system is immature. mucin, oligosaccharides, and other factors within breast milk may affect antigen presentation. protein of breast milk is species specific and therefore nonallergic for human infants. no antibody response has been demonstrated to occur with human milk in infants. it has also been shown that macromolecules in breast milk are not absorbed. indirect evidence can be inferred from a demonstration of an infant' s response to cow milk protein. within 18 days of taking cow milk, the infant will begin to develop antibodies. since the advent of prepared formulas, in which the protein has been denatured by heating and drying, the incidence of cow milk allergy has been considered to be 1%. the most reliable means of diagnosing cow milk allergy is by challenging with isolated cow milk protein. although circulating antibodies and coproantibodies have been identified, these are not reliable techniques for a clinician involved in patient care. the allergic syndromes that have been associated with cow milk allergy include gastroenteropathy, atopic dermatitis, allergic rhinitis, chronic pulmonary disease, asthma, eosinophilia, failure to thrive, and sudden infant death syndrome, or cot death, which has in some cases been attributed to anaphylaxis to cow milk. 132, 156 gi symptoms have received the greatest attention and include spittingup, colic, diarrhea, blood in the stools, frank vomiting, weight loss, malabsorption, colitis, and failure to thrive. cow milk has been associated with gi protein and blood loss. the diagnosis is best made by elimination of cow milk from diet and, when appropriate, challenge tests. cutaneous testing is of little help. cow milk allergy has been described in breastfed infants, and exclusive breastfeeding alone is not sufficient to protect an infant at high risk to become sensitized to cow milk proteins. 129 the incidence of cow milk allergy in exclusively breastfed infants has been estimated as 0.4% to 0.5% compared with the overall incidence ranging from 1.9% to 7.5% in population-based studies. 129 murray 182 showed the association of nasal secretion eosinophilia with infants freely fed cow milk or solid foods compared with eosinophilia in strictly breastfed infants. in infants receiving cow milk, 32% had high eosinophilic secretions, and only 11% of breastfed infants had eosinophils present in nasal secretions. not surprisingly, many different antigenic specificities are recognized when the colostrum or milk of one species is fed to or injected into another species. cow milk is high on the list of food allergens, particularly in children. sensitivity to cow milk is responsible for at least 20% of all pediatric allergic conditions, according to gerrard. 78 evidence indicates that iga antibodies play an important role in confining food antigens to the gut. food antigens given to bottle-fed infants before they can make their own iga, and when they are deprived of that in human milk and the plasma cells, may be expected to be more readily absorbed. glaser 80 first made the association between the drop in breastfeeding and the rise in allergy. he pioneered the theory of prophylactic management of allergy. allergy in infancy is associated with a familial history of atopic disease and elevated cord blood ige levels. the introduction of "foreign" proteins to an infant' s diet and even to the mother' s diet in the breastfeeding dyad can lead to allergic symptoms in the infant. exclusive breastfeeding does not protect high-risk children from allergic symptoms unless the mother also adheres closely to a restrictive diet that excludes common allergens. 5, 12 a large body of literature examines whether breastfeeding protects against atopic disease. in 1988, kramer 148 defined 12 standards for methodology and the study of allergy and breastfeeding. the standards clarified the definitions of breastfeeding, measurable outcomes, and the diagnostic criteria for specific allergic syndromes, defined children at high risk for atopic disease, and addressed methods to decrease bias and control for confounding variables. several recent large meta-analyses have been performed assessing the protective effect of breastfeeding against allergic rhinitis, atopic dermatitis, and asthma. 76, 77, 175 exclusive breastfeeding during the first 3 months of life protected against allergic rhinitis (summary odds ratio 0.74; 95% confidence interval 0.54 to 1.01) with or without a family history of atopy. 175 exclusive breastfeeding for at least 3 months was associated with lower rates of atopic dermatitis in children with a family history of atopy. 76 exclusive breastfeeding in the first months of life was protective against asthma during childhood (odds ratio 0.70; 95% confidence interval 0.60 to 0.81). 77 chapter 17 discusses the prophylactic management of the potentially allergic infant. the major elements in human milk related to the infant' s immune system are direct-acting antimicrobial factors, antiinflammatory factors, and immunomodulating bioactive compounds. 108 epidemiologic studies have produced compelling information that suggests that breastfeeding for 4 months or longer can provide some immunologic protection against some childhood-onset diseases. 85, 91, 169 in 1991, viirtanen et al 268 reported a prospective long-term study among children in finland that showed a significantly lower incidence of type 1 diabetes in those at-risk children who had been breastfed for 4 months or longer. other epidemiologic studies have demonstrated a decreased incidence of type 1 insulin-dependent diabetes mellitus in breastfed children. 20, 169, 197 these clinical observations have been supported in the laboratory by studies of diet control in diabetic mice. the isolation of a bovine albumin peptide as a possible trigger of type 1 insulin-dependent diabetes mellitus makes further study imperative. 136 based on limited data, the recent agency for healthcare research and quality report cautiously concluded that breastfeeding for at least 3 months reduced the risk for type 1 diabetes compared with breastfeeding for less than 3 months. for type 2 diabetes, the same report concluded that breastfeeding in infancy produced a decreased risk compared with not breastfeeding. 126 the review of the national perinatal collaborative study by davis et al 54 showed a protective effect against development of childhood cancer by being breastfed for 4 months or longer for children followed for 10 years. the effect was greater for acute leukemia and lymphoma. the role of infant feeding practices showed a similar effect of breastfeeding as protective in postponing or decreasing the occurrence of inflammatory bowel disease in childhood. 145, 232 greco et al 97 reported a decreased risk for celiac disease in breastfed infants. the ahrq report concluded that an association exists between breastfeeding for at least 6 months and a decreased risk for developing acute lymphocytic leukemia and acute myelogenous leukemia. 126 maternal renal allografts have a better survival rate in individuals who were breastfed in infancy compared with those who were not breastfed. 37, 143 the mechanism of these apparent long-term immunologic benefits remains unclear, although theories abound. 85 given the potential for confounding factors and bias in large long-term studies, confirmation of these proposed benefits by additional carefully controlled trials is required. an increasing amount of accumulated epidemiologic literature, utilizing improved methodology and statistics, demonstrates the protective benefits of human milk for infants. a large number of bioactive factors have been identified and measured in breast milk during the period of lactation. additional research is needed to clarify the interactions and the mechanisms of action of the many bioactive factors in human milk and then correlate these immunomodulatory actions with specific protective benefits for the infant. section on breastfeeding: breastfeeding and the use of human milk activity of the alternative pathway of complement in the newborn infant a prospective cohort study on breast-feeding and otitis media in swedish infants exclusive breastfeeding reduces acute respiratory infection and diarrhea deaths among infants in dhaka slums sensitization to common allergens and its association with allergic disorders at age 4 years: a whole population birth cohort study breast feeding and protection against neonatal sepsis in a high risk population breastfeeding and the capital risk of hospitilization for respiratory disease in infancy human chemokines: an update infant feeding patterns and risks of death and hospitalization in the first half of infancy: multicentre cohort study ige and igd in human colostrum and plasma diet and faecal flora in the newborn: nucleotides influence of dietary manipulation on incidence of atopic disease in infants at risk studies of breastfeeding and infections. how good is the evidence? relation between infant feeding and infections during the first six months of life positional otitis media immunologic benefits and hazards of milk in maternal-perinatal relationship influence of breast-feeding on the bifid flora of the newborn intestine bactericidal activity of human milk leukocytes oral probiotics prevent necrotizing enterocolitis in very low birth weight neonates relation between breast-feeding and incidence rates of insulin-dependent diabetes mellitus: a hypothesis development and basic mechanisms of human gut immunity mucosal immunity: integration between mother and the breast-fed infant mucosal and glandular distribution of immunoglobulin components: differential localization of free and bound secretory component in secretory epithelial cells ontogeny of the mucosal immune system and iga deficiency the b-cell system of human mucosae and exocrine glands milk derived gm 3 and gd 3 differentially inhibit dendritic cell maturation and effector fuctionalities the epithelial cells and cell fragments in human milk effect of dietary nucleotide supplementation on diarrhoeal disease in infants interleukin-2 in human milk: a potential modulator of lymphocyte development in the breastfed infant bioactive components of human milk antioxidant properties of human colostrum soluble receptors and cytokine antagonists in human milk polymorphonuclear leukocytes in human colostrum and milk developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. ii. duodenum and liver, gallbladder, and pancreas iron-binding proteins and other factors in milk responsible for resistance to escherichia coli daily ingestion of immunologic components in human milk during the first four months of life breast feeding and maternal-donor renal allografts: possibly the original donor-specific transfusion endotoxin and hypoxiainduced intestinal necrosis in rats: the role of platelet activating factor advances in nutritional modifications of infant formulas dietary nucleotides: cellular immune, intestinal and hepatic system effects dietary nucleotide effects upon immune function in infants the role of nucleotides in human nutrition full breastfeeding duration and associated decrease in respiratory tract infection in us children bioactive components of human milk decreased interleukin-10 production by neonatal monocytes and t cells: relationship to decreased production and expression of tumor necrosis factor-alpha and its receptors changes in carbohydrate composition in human milk over 4 months of lactation oligosaccharides from human milk block binding and activity of the escherichia coli heat-stable enterotoxin (sta) in t84 intestinal cells identification of nestin-positive punitive mammary stem cells in human breastmilk regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin breastfeeding reduces risk of respiratory illness in infants role of oligosaccharides and glycoconjugates in intestinal host defense persistence of human milk proteins in the breast-fed infant infant feeding and childhood cancer differences in morbidity between breast-fed and formula-fed infants partition of nitrogen intake and excretion in low-birth-weight infants maternal exposure to endocrine-active substances and breastfeeding breast-feeding protects against respiratory syncytial virus infections the effects of infant feeding on rotavirus-induced gastroenteritis: a prospective study exclusive breastfeeding protects against bacterial colonization and day care exposure to otitis media exclusive breast-feeding for at least 4 months protects against otitis media delayed breastfeeding initiation increases risk of neonatal mortality deficient classical complement pathway activity in newborn sera variability of human milk neutral oligosaccharides in a diverse population effect of storage and heat on antimicrobial proteins in human milk intestinal microflora in early infancy: composition and development nonspecific antiviral substances in human milk active against arbovirus and murine leukemia virus bronchomammary axis in the immune response to respiratory syncytial virus the role of gut-associated lymphoid tissues and mucosal defense influence of the heat treatment of human milk on some of its protective constituents breast-feeding and salmonella infection iron in human milk the effect of maternal milk on neonatal morbidity of very low-birth-weight infants expression of functional immunomodulatory and anti-inflammatory factors in human milk impact of the intestinal microbiota on the development of mucosal defense breastfeeding and the onset of atopic dermatitis in childhood: a systematic review and meta-analysis of prospective studies breast-feeding and the risk of bronchial asthma in childhood: a systematic review with meta-analysis of prospective studies sensitization to substances in breast milk: recognition, management and significance stool microflora in extremely low birthweight infants the dietary prophylaxis of allergic disease in infancy protection against cholera in breast-fed children by antibodies in breast milk antibodyforming cells in human colostrum after oral immunisation rapid hightemperature treatment of human milk immunologic system in human milk the immune system of human milk: antimicrobial, antiinflammatory and immunomodulating properties modulation of the gastrointestinal tract of infants by human milk: interfaces and interactions-an evolutionary perspective evolution of immunologic functions of the mammary gland and the postnatal development of immunity spectrum of immunomodulating agents in human milk immunologic components in human milk during the second year of lactation anti-inflammatory properties of human milk cytokines in human milk: properties and potential effects upon the mammary gland and the neonate immunologic factors in human milk during the first year of lactation immunologic protection of the premature newborn by human milk lactoferin structure, function and applications breast feeding and biological properties of faecal e. coli strains antiinflammatory effects of human milk on chemically induced colitis in rats case control study on nutritional risks in celiac disease free secretory components and lactoferrin of human milk inhibit the adhesion of enterotoxigenic escherichia coli possible influence of vitamin b 12 -binding protein in milk on the intestinal flora in breast-fed infants. ii. contents of unsaturated b 12 -binding protein in meconium and faeces from breast-fed and bottle-fed infants a hitherto unrecognized biochemical difference between human milk and cow' s milk mother' s milk and sewage: their interactive effects on infant mortality antiviral activity of purified human breastmilk mucin bioactive factors in human milk protective function of 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authors: maritz, julia m.; land, kirkwood m.; carlton, jane m.; hirt, robert p. title: what is the importance of zoonotic trichomonads for human health? date: 2014-06-18 journal: trends parasitol doi: 10.1016/j.pt.2014.05.005 sha: doc_id: 261466 cord_uid: b9r4cyp7 trichomonads are common parasites of many vertebrate and invertebrate species, with four species classically recognized as human parasites: dientamoeba fragilis, pentatrichomonas hominis, trichomonas vaginalis, and trichomonas tenax. the latter two species are considered human-specific; by contrast, d. fragilis and p. hominis have been isolated from domestic and farm mammals, demonstrating a wide host range and potential zoonotic origin. several new studies have highlighted the zoonotic dimension of trichomonads. first, species typically known to infect birds and domestic mammals have been identified in human clinical samples. second, several phylogenetic analyses have identified animal-derived trichomonads as close sister taxa of the two human-specific species. it is our opinion, therefore, that these observations prompt further investigation into the importance of zoonotic trichomonads for human health. the trichomonad lineage in phylum parabasalia trichomonads are anaerobic, flagellated protists belonging to the large and diverse groups trichomonadea and tritrichomonadea of phylum parabasalia [1] . they are characterized by the presence of three to five anterior flagella, hydrogenosomes -hydrogen-producing organelles corresponding to anaerobic versions of mitochondria [2] , a parabasal body (a large golgi), and a complex cytoskeleton. a few species have been isolated from environmental samples and may represent free-living species; however, the majority of species form symbiotic interactions (see glossary) with various animal hosts. among the parasitic trichomonads, several species inhabit the oral, digestive, and urogenital tracts of invertebrate and vertebrate hosts, including livestock, pets, and humans. historically, phylum parabasalia was divided into two groups based on morphological characteristics; however, the recent inclusion of molecular data recovered six groups: trichomonadea, tritrichomonadea, hypotrichomonadea, cristamonadea, spirotrichonymphea, and trichonymphea [3] . the trichomonadea, tritrichomonadea, and hypotrichomonadea are of primary concern to parasitologists; however, the evolutionary relationships within and between these groups are unclear [4] . several molecular phylogenies have attempted to resolve these evolutionary relationships using phylogenetic markers such as ribosomal rna (rrna) and protein coding genes (figure 1 ), which give inconsistent phylogenies [4, 5] . four species of trichomonad are considered human parasites: trichomonas vaginalis (found in the urogenital tract) [6] , trichomonas tenax (localized to the oral cavity) [7] , and pentatrichomonas hominis and dientamoeba fragilis (located in the digestive tract) [8, 9] . only one species has wellestablished pathogenic potential: t. vaginalis, the cause of the most prevalent non-viral sexually transmitted infection in humans, trichomoniasis [10] . only t. vaginalis and t. tenax are considered human-specific, with the former characterized by the richest, although still limited, epidemiology data [11] , but very little is known about the latter. p. hominis and d. fragilis can cause gastrointestinal symptoms in some patients, such as abdominal pain and diarrhea [8, 12] , d. fragilis has also been proffered as a potential causative agent of irritable bowel syndrome (ibs) [13, 14] , but debate surrounds its pathogenicity, infection route, and epidemiology [15] . in addition, several trichomonad species are of veterinary importance, such as the avian pathogens trichomonas gallinae, tetratrichomonas gallinarum, and histomonas meleagridis [16] [17] [18] [19] , and tritrichomonas foetus, the causative agent of a venereal disease in cattle [20] . this extensive host range, along with the isolation of d. fragilis [21] and p. hominis [22] from various animal hosts, suggests that certain species of trichomonads may exhibit the characteristics of zoonoses. although the question of zoonotic trichomonads has been considered for some years (e.g., [23, 24] ), recent results from several different sources have highlighted this potential. here we summarize the clinical and phylogenetic studies that suggest a zoonotic potential for trichomonads, discuss their implications for human health, and the next steps required for investigation into their epidemiology, pathobiology and evolution. new evidence supports the zoonotic potential of trichomonads human trichomonad infections are not body site-specific the four trichomonad species recognized as human parasites were initially thought to be site-specific [25] (table 1) . however, various clinical studies have shown that they can also be found in atypical locations. for example, t. tenax, a commensal of the human mouth found in patients with poor oral hygiene [7] , has been identified by microscopic and molecular methods in the upper and lower respiratory tracts [26, 27] . one possibility that could account from this 'aberrant' location is inhalation of the parasite from the oral cavity into the respiratory tract. however, in some cases where t. tenax was identified in the respiratory tract, no parasites were found in the mouth [26] . other human trichomonad species have also been identified in the respiratory tract including the sexually transmitted species t. vaginalis [28, 29] and the gut parasite p. hominis [30] , which suggests that these species too can proliferate outside their usual body sites. at least five species of trichomonad, including p. hominis, t. tenax, t. vaginalis, t. foetus, and t. gallinarum, have been identified in the human respiratory tract and as causative agents of pulmonary trichomoniasis (table 1) . they have been found in up to 60% of patients with pneumocystis pneumonia (pcp) and in up to 30% of patients with acute respiratory distress syndrome (ards) [31] . because trichomonads are microaerophilic it is unlikely that they initiate and cause these diseases themselves, but may represent secondary and opportunistic infections that could exacerbate symptoms and prolong illness [23] . these trichomonad respiratory infections seem to depend upon: (i) the presence of bacteria on which to feed and (ii) local anaerobic conditions caused by pcp or ardsassociated infections [32] but not necessarily upon immunosuppression, because drugs against pcp consistently cure patients of pulmonary trichomonosis and, in one study, treated ards patients were not found to be immunocompromised [25] . thus, the presence of an increasing number of distinct trichomonads in a broader range of clinical samples from patients with diverse diseases, such as aids, rheumatoid arthritis, prostate cancer, pulmonary infections (empyema and pneumonia in addition to pcp and ards), and digestive conditions such as diarrhea and ibs [33] [34] [35] , is becoming increasingly apparent. indeed, the frequency of pulmonary trichomonosis infections may be higher than reported because transformation of parasites from the motile, pear-shaped stage to the amoeboid stage renders microscopic identification in clinical samples difficult [31] , highlighting the importance of molecular data to identify such infections [25] . non-human species of trichomonad have been isolated from clinical samples trichomonads were thought to have strict host specificity [25] ; however, trichomonad parasites not previously reported as infecting humans have recently been found in human clinical samples (table 1) . for example, parasites belonging to the genus tritrichomonas can be isolated from the reproductive tract of cattle (tritrichomonas foetus), the nasal mucosa and intestine of pigs (tritrichomonas suis), and the intestine of non-human primates (tritrichomonas mobilensis) [20] . another example is t. foetus, historically considered specific to cattle [25, 36] . nonetheless, experimental cross-infections of the parasites between pigs and cattle in addition to analysis of molecular data suggest that these three species should be considered glossary commensal: : a form of symbiosis between two organisms where one derives benefit, whereas the other is unaffected. some gut trichomonads are thought to represent commensals. disease incidence: : the number of new disease cases that occur in a population for a given time period (typically per year). dysbiosis: : an imbalance of the microbiota (the microbial populations at a particular body site of an animal host) that leads, or predisposes, the host to disease conditions [60] . emerging infectious disease: : outbreaks of previously unknown diseases or known diseases that show an increase in incidence, expansion in geographical range, or spread to a new population. emerging infections can be caused by previously unknown or undetected infectious agents, newly evolved strains, environmental changes, and changes in human demography [51] . a recent review found that over 60% of human emerging infectious diseases are zoonotic in origin [53] . examples include influenza, hiv/aids, and severe acute respiratory syndrome (sars) coronavirus. intermediate host: : a host in or on which a pathogen spends a part of its life, usually a transition period, but does not reach sexual maturity. mutualism (mutualist): : a form of symbiosis between two organisms in which both benefit from the relationship. some gut parabasalids from termites are thought to represent mutualists [80] . opportunistic: : a potential pathogen that typically does not cause disease in a healthy host, but can in particular situations cause disease, for example, owing to the compromised immune system (e.g., attributable to aids, chemotherapy, or malnutrition) of the host. lung trichomonads represent probable opportunistic infections -see main text. parasitic (parasite): : a non-mutual symbiosis between two species where one, the parasite, benefits at the expense of the other, the host. parasites typically do not kill their hosts but exploit them for resources necessary for their survival. obligate parasites cannot complete their life cycles and reproduce without a suitable host. pathogenic (pathogen): : a broad term that refers to the ability of an organism to cause disease. it is typically used to describe an infectious agent or microorganism, such as a bacterium, protist, virus, etc., that causes disease in its host. some pathogens, for example, protists acanthamoeba spp. and naegleria fowleri and fungi aspergillus spp. are free-living species thriving in the environment and occasionally infect humans, often in an opportunistic manner in compromised hosts [59] . pathogenicity: : the ability of a pathogen to overcome host defenses and cause disease. re-emerging infectious disease: : the reappearance of a historically known infectious disease after a significant decline in incidence. acquired resistance of pathogens to antimicrobial medications is an important factor in the reemergence of many diseases. examples include west nile virus, cholera, mrsa (methicillin-resistant staphylococcus aureus). reservoir: : the habitat or host that harbors an infectious agent, where it can live, grow, and multiply. reservoirs can include humans, animals, and serve as a source of potential disease outbreaks. symbiosis: : a close and often long-term relationship between two or more different biological species. these relationships can be obligate or facultative, mutualistic, commensalistic, or parasitic. transmission: : the passing of an infectious agent from one host to another host. direct transmission routes include: physical contact, contact with a contaminated environment or surface, airborne transmission, and fecal-oral transmission. indirect transmission routes involve another organism such as an insect vector or intermediate host. vector: : an organism that carries and transmits a pathogen from an infected individual to another individual. virulence: : a property of a pathogen, such as specific structural elements or biochemical compounds commonly called virulence factors that cause a reduction in host fitness or damage to the host. it is now recognized that virulence is multifactorial and involves characteristics of both the pathogen and its host, which influence the outcome of their interaction and hence the observed virulence (e.g., an opportunistic pathogen in immunocompromised patients) [59] . zoonosis: : an infectious organism, such as a bacterium, virus, parasite, or fungus, transmissible between wildlife or domesticated animals and humans. examples include: (i) the lyme disease bacterium borrelia transmitted to humans by ticks from a natural reservoir in rodents; (ii) the malaria parasite plasmodium knowlesi transmitted by anopheles vectors that causes malaria in monkeys and humans; and (iii) cryptosporidium parvum, a parasite found in cats, dogs, and farmed animals and transmitted as a cyst in contaminated water, food, or through the fecal-oral route. zoonoses are the leading cause of emerging infectious diseases worldwide, responsible for devastating disease outbreaks, mortality, and serious socioeconomic consequences [21, 56, 79] . zoonotic potential: : the potential for infectious diseases of wildlife or domestic animals to be transmitted to humans. trends in parasitology july 2014, vol. 30, no. 7 strains of the same species [20, 37] . in addition, several different genotypes of t. foetus have been identified as causing diarrhea in cats in 12 countries [38, 39] and have also been isolated from dogs with diarrhea [40] . moreover, in several new clinical cases, t. foetus or t. foetus-like organisms have unexpectedly been identified in the lungs of human patients [41] . such findings suggest that t. foetus is a zoonotic parasite capable of colonizing an extensive range of hosts and body sites. other examples of species of non-human trichomonads recently found to infect humans are members of the genus tetratrichomonas, currently the largest genus in phylum parabasalia. tetratrichomonas species are found in the small intestine of a wide spectrum of invertebrate and vertebrate hosts, such as leeches, birds, and rodents [42] . indeed, some species of tetratrichomonad are known to infect a wide range of unrelated hosts, such as tetratrichomonas prowazeki, which has been found in species of amphibians and reptiles [42] . another example, tetratrichomonas gallinarum, is primarily thought of as an avian parasite of the digestive tract in domestic and wild birds [43] , although its pathogenicity is not well established [17] . however, several recent studies have identified tetratrichomonas strains isolated from human lungs or the human the primary host may not represent the true natural history of the species, which may have a broader host range. c pulmonary infections include pcp, ards-associated infections, pneumonia, and can lead to empyema. trends in parasitology july 2014, vol. 30, no. 7 oral cavity as t. gallinarum or t. gallinarum-like organisms [24, 44, 45] . studies have also shown that genus tetratrichomonas is much more diverse than previously thought and that t. gallinarum comprises at least three cryptic species with variable host specificity, some that represent human isolates [24, 43] . notably, experiments failed to transmit two tetratrichomonas of human origin to birds, although the authors suggest this result could be explained by either adaptation of the t. gallinarum-like trichomonads to the human host or extensive in vitro culturing, so that infection of birds was no longer biologically achievable [24] . molecular phylogenies reveal close relationships between human and avian trichomonads recent molecular phylogenetic analysis of trichomonads using rrna and protein coding genes (e.g., rpb1) has begun to answer important questions regarding trichomonad phylogeny. rpb1 is a ubiquitous eukaryotic gene coding for the largest subunit of rna polymerase ii and is present as a single copy in many eukaryotes. a recent analysis of rpb1 generated a fully resolved phylogeny of trichomonadea, tritrichomonadea, and hypotrichomonadea, and species and isolates within these groups (figure 2 ) [5] . interestingly, the phylogeny recovered some avian isolates of trichomonas spp. as sister taxa to t. vaginalis, and t. tenax as closely related to t. gallinae; these findings are consistent with previous phylogenies based upon rrna and other protein coding genes [24, [46] [47] [48] . the common ancestor to this complex is also related to the avian t. gallinarum (e.g., [42] ). these new phylogenies complicate the inferred relationship between t. vaginalis and t. tenax, which on the basis of host specificity might be expected to be sister taxa [47] . in this scenario, an ancestor of both species infected humans and subsequently differentiated into two distinct species with different body site preferences. however, the lg+g+i model, 100 bootstrap replicates, based on 387 unambiguously aligned sites (the alignment is available upon request)], based on rpb1 illustrating the relationship between human-and animal-specific trichomonad species and one isolated from the environment (pseudotrichomonas keilini). note the high similarity of sequences derived from the human trichomonas vaginalis isolates and trichomonas sp. (hmo16231) isolated from a dove. this sequence and other trichomonas gallinae sequences are clearly distinct from the recently defined trichomonas stableri (kf233590) species isolated from band-tailed pigeons [49] . taxa in red were isolated from humans and those in blue isolated from birds; accession numbers of each sequence are shown. adapted from [5] . trends in parasitology july 2014, vol. 30, no. 7 phylogenetic data suggest the zoonotic transfer of trichomonad parasites from humans to birds, and/or vice versa, on at least two occasions, or a combination of these events. other studies investigating outbreaks of avian trichomonosis in wild birds, primarily attributed to t. gallinae infections, have also confirmed the close relationship between avian and human trichomonads [48] . epidemic infections by t. gallinae of passerine species in europe have recently been described and associated with high mortality and population decline [18] . this dramatic case of disease emergence demonstrates the potential for a trichomonad to jump host species (columbiform to passerine) and spread rapidly through populations. the disease is thought to have been initiated and spread through transfer of the parasite via contaminated water and bird feeders, as well as through direct contact between passerines [17] . furthermore, the recent isolation and preliminary characterization of trichomonas stableri associated with epidemic mortality in california band-tail pigeons suggest that avian trichomonosis may be caused by pathogens other than t. gallinae and h. meleagridis [17, 49] . genetic analysis at multiple loci indicated t. stableri to be more closely related to t. vaginalis than to the bird-associated t. gallinae [49] (figure 2 ), further supporting the hypothesis that trichomonads are crossing host boundaries. generating genetic markers and whole genome sequences of t. stableri and other t. vaginalis-like isolates derived from cases of epidemic avian trichomonosis will provide important insight into the evolution and origins of these pathogens. for example, does the trichomonas sp. isolate from a whitewinged pigeon extremely closely related to isolates of t. vaginalis represent a case of human to bird transfer (accession hm016231 in figure 2 )? the implications of zoonotic trichomonads for human health according to current disease dynamic models, the zoonotic emergence of parasitic diseases in humans is typically associated with several characteristics, including broad host range, genetic variability, presence of genotypes better suited to the parasitism of humans, and modified pathogenic potential [50] . emergent zoonoses are thought to appear through a number of different stages, for example, some develop as animal 'parasitoses' that are newly transmissible to humans, although the source of the disease remains the animal reservoir [50, 51] . in other cases, parasites are able to cross the species barrier, modify their specificity, and become sustainably transmissible from human to human [50, 52, 53] . the evolution of these emergent parasitoses is not linear, and an explanation for such a complex process requires consideration of the multi-host ecology and complex dynamics of zoonotic infections [54] . based on these models and the clinical and molecular evidence discussed previously, it may be that several trichomonads are at different stages of zoonotic emergence ( figure 3 ). these observations raise important questions regarding the implications of the potential widening pathological spectrum of trichomonads in humans and suggest that owing to links with other diseases these parasites may be of greater medical importance than previously thought. historically, trichomonads have not been considered as emerging infections because of their site-and host-specific occurrence. nonetheless, the presence of trichomonads in a diverse array of clinical disorders suggests that they may exhibit a form of opportunism and multiply when local conditions are favorable. for example, the diseases in which trichomonads are found as co-infecting agents in respiratory infections are probably not limited to pcp and ards-associated infections, but may include other pulmonary diseases such as cystic fibrosis [31, 32] . overall, the high prevalence of pulmonary diseases globally [55] combined with the higher burden of both lung conditions and zoonotic diseases among people in resource-limited settings [55, 56] suggest that only the 'tip of the iceberg' of pulmonary trichomoniasis may currently be known. digestive tract infections by trichomonads are also increasingly recognized as being common. although the exact clinical profile of d. fragilis is still poorly understood some consider this species to have pathogenic capabilities [12] , and recent studies have associated the rise of ibs with a high prevalence (40%) of d. fragilis in europe [35] . however, the pathogenicity of d. fragilis has been questioned owing to the asymptomatic nature of many infections, and it is considered by some as a commensal of the intestinal flora [57] . indeed, treatment of d. fragilis-infected children with metronidazole was not associated with better clinical outcomes [58] . because association is not evidence for causality, additional data are required to establish the pathogenicity of trichomonads in the digestive tract and 'aberrant' body sites such as the lungs. in this context, it is important to consider characteristics of both host and parasite with regard to the outcome of their interactions. for example, one extreme is represented by severely immune-compromised patients that are more susceptible to a wider range of microbial infections compared with immunocompetent hosts [59] . when studying the outcome of human-microbe interactions, a complex interplay among viruses, bacteria and archaea, microbial eukaryotes, and animal parasites influence the health status of the human host, with mucosal microbiota playing a key role influencing health and disease status [60, 61] . based on these considerations and examples, trichomonads may be more prevalent and have a wider pathological spectrum in humans than currently recognized, influencing human health through direct pathologies but also indirectly through dysbiosis of the mucosal microbiota and local inflammation, facilitating transmission of pathogens -a prime example being t. vaginalis infection and bacterial vaginosis contributing to hiv transmission [61, 62] . the potential influence of gut trichomonads to human health will also have to consider their potential impact on the gut microbiota, which might explain observations of the association between d. fragilis and ibs through inducing gut dysbiosis [35] . indeed, the ability of trichomonads to live on a variety of mucosal tissues may be the key to their wide host range and ability to develop infections at different body sites, as well as contribute directly or indirectly to pathologies. once the capacity to thrive on vertebrate mucosal surfaces has developed, there may be less of a barrier to cross both species and mucosal sites. for example, in the case of review trends in parasitology july 2014, vol. 30, no. 7 t. foetus, this sexually transmitted species may represent a recent transfer from the digestive to the urogenital tract, with a capacity of the parasite to thrive in the gut in different species (e.g., pigs, cats, and dogs). trichomonads provide a unique system for the study of the origins and pathobiology of zoonotic and emerging infectious diseases. in addition, they have attracted interest as model systems for evolutionary biology and comparative genomics [63, 64] , and for biochemical, molecular, and cell biology investigations [2, 65, 66] . the t. vaginalis genome sequence published in 2007 was the first species of trichomonad to be sequenced [67] , and others are currently underway including isolates of t. foetus, p. hominis, t. gallinae, and t. tenax. these sequences will enable comparative analysis of common and unique parasitic modes of life cycle, and possible adaptive mechanisms. for example, t. vaginalis and t. foetus appear to have evolved independently to colonize the urogenital tracts of different mammalian hosts [68] . in addition, t. foetus has been isolated from the digestive tract of cats and dogs, indicating that it is capable of colonizing a broad range of hosts and environments [69] . these two species of sexually transmitted trichomonad probably represent cases of convergent evolution and provide an opportunity to compare the derived similarities and the origins of these traits that coincide with a shared niche. species of trichomonad exhibit a range of genome sizes, from 94 mb for the p. hominis genome to 177 mb for the t. foetus genome [70] . the t. vaginalis genome sequence revealed the 160-mb genome to be a result of expanded transposable elements and protein coding gene families, . speculative models of zoonoses caused by trichomonads.trichomonads are listed on the right and colored according to primary hosts assigned historically in the literature. unbroken lines represent known infections or transmission routes, and broken lines represent speculative infections or transmission routes for which data are lacking. the relationships are represented as follows: (blue box) trichomonads identified in wild bird species (e.g., green finch [16] and toucan [81] ) in partially domesticated species (rock dove) and in fully domesticated species (chicken) circulate within these populations with variable host specificity [17] (blue unbroken circle with arrow). two of the four avian trichomonads listed (tetratrichomonas sp. and tetratrichomonas gallinarum) have been identified in human lungs [24] , and trichomonas gallinae and trichomonas stableri are also included owing to their close relationship to trichomonas tenax and trichomonas vaginalis [5, 49] . (red box) t. vaginalis and t. tenax are the two species considered human-specific, with known human-to-human infections (unbroken red circle). the close genetic relationship of the human and avian trichomonads ( figure 2 ) suggests either independent zoonotic acquisitions from avian sources (broken blue arrow) or transfer of the parasites from humans to birds through environmental contamination (broken red arrow). (green box) tritrichomonas foetus has been isolated from a variety of pets and farm animals, with the same strain known to infect cattle and pigs (unbroken green arrow) [26] , but different genotypes infecting cattle and cats [29, 31] ; the origins of dog infections remain unclear [32] . thus, there are at least two t. foetus genotypes capable of colonizing an extensive range of hosts, including humans [41] (broken green circle and arrow). the lack of precise epidemiological data is indicated by '?'. (purple box) pentatrichomonas hominis has been isolated from a variety of pets and farm animals [22] , but little is known about its infection route and epidemiology; the same strain could be circulating between all identified hosts (broken purple circle). (orange box) dientamoeba fragilis has been isolated from farm animals (pigs) and non-human primates (gorillas), with the same strain known to infect pigs and humans [21] (unbroken orange arrow). recent evidence suggests that household pets do not play a role in transmission [82] ; however, the origins remain unclear and multiple strains could be circulating in animal hosts (broken orange circle and arrow). additionally, given recent prevalence and transmission data it seems unlikely that transmission from non-human hosts represents a significant proportion of infections. contaminated surfaces and water [83] , uncooked meat, or direct contact with pets and farm animals could lead to animal-to-human transmissions of trichomonads. initial infections were presumably through the digestive tract (via oral ingestion) with further progression to the lungs for some (various) species or the urogenital tract (t. vaginalis). trends in parasitology july 2014, vol. 30, no. 7 including those responsible for interaction of the parasite with its immediate environment [71] [72] [73] . it has been hypothesized that the large genome size may be a recent event that occurred when the most recent common ancestor of t. vaginalis underwent a population bottleneck during its transition from the digestive tract to the urogenital tract. because genome size is positively associated with cell volume, an increase in genome size and concomitant increase in cell size might have increased its phagocytic potential as well as surface area for the interaction of the parasite with host cell tissue [67, 73] . in this context, it will be particularly interesting to compare the genomes of t. vaginalis with its closely related isolates and species derived from birds ( figure 2 ) to gain detailed insight into correlations between genome evolution and how this might relate to parasite pathobiology in humans and birds. lateral gene transfers from bacterial donors sometime in the evolutionary past have also importantly influenced the evolution of t. vaginalis protein coding genes [63, 67] . for example, the parasite has gained an almost complete pathway for the degradation of complex glycans present in host mucosal secretions, factors which may contribute to its adaptive potential and pathogenicity [61, 63] . comparison of a broad range of trichomonad genomes will help test this hypothesis and may pinpoint gene families whose acquisition and/or expansions correlate with pathogenicity and facilitated or mediated transitions from: (i) an ancestral animal-only stage to human-inclusive infections or (ii) from the digestive to urogenital tracts. trichomonads also provide a unique system to study the features of a zoonotic lifestyle via comparative examination of molecular and cellular characteristics. for example, successful t. vaginalis infections are probably favored by virulence mechanisms such as cytoadherence and phagocytosis [72, 73] . the 60 000 predicted protein coding genes of t. vaginalis [67] includes a plethora of candidate genes for surface molecules mediating interaction with host tissues and membrane trafficking and signaling, important processes involved in parasite pathobiology [72, 73] . cysteine proteases in particular have been identified as virulence factors central to the host-pathogen interface in t. vaginalis [61, [72] [73] [74] [75] . transcriptomic studies have shown upregulation of some of these t. vaginalis virulence factors in response to contact with host cells in vitro [76] and have also documented their expression under in vitro growth conditions in t. foetus [69] . similar to t. vaginalis, recent studies have shown the presence of cysteine proteases in the cell-free filtrate of t. gallinae and demonstrated their involvement in its in vitro cytopathogenic effects [77] . mining other trichomonad genome data to identify important virulence proteins will improve our understanding of the molecular and cellular basis of infections and can be used to test hypotheses, such as whether zoonotic organisms show greater diversity in key virulence proteins underlying their capacity to parasitize a variety of host species and mucosal sites [78] . concluding remarks and future perspectives although we have discussed several recent studies that provide strong evidence for the zoonotic origin and potential of trichomonads, regular and sustained zoonotic transmission of these microbes has yet to be definitively established. to improve our knowledge of the zoonotic origins of trichomonads, detailed investigations including systematic surveys of trichomonads in humans and animals will be required. molecular methods have been instrumental in our understanding of the biology and complexity of trichomonads so far; however, more data and novel approaches are needed to resolve evolutionary relationships and to improve diagnostic tools. wide sampling and whole genome sequencing of trichomonads, with subsequent comparative genomic investigations, will facilitate identifying the closest relatives of human trichomonad pathogens, providing a solid evolutionary framework for how these diseases have emerged and forming a basis for epidemiological studies across both animal (wild and domestic) and human hosts. environmental studies such as the 'microbes, sewage, health and disease' metagenomics project in new york city (http://www.nyu.edu/ about/news-publications/nyu-stories/video-mapping-nycs-metagenome.html) will provide important information on potential transmission routes and the patterns, nature, and occurrence of trichomonad infections in humans, animals, and birds; and establish the importance of trichomonads zoonotic transmissions -as has been established for species of trypanosoma, cryptosporidium, and toxoplasma [79] . furthermore, studies investigating the potential pathogenicity of these parasites in various mucosae (respiratory, digestive, and urogenital) are needed to determine the clinical significance and public health implications of trichomonads. accumulation of these different types of data will advance our understanding of parasite biology and infection mechanisms, and provide approaches towards developing drug targets and vaccine candidates for species of this increasingly recognized medically and veterinary important lineage. the revised classification of eukaryotes biochemistry and evolution of anaerobic energy metabolism in eukaryotes. microbiol critical taxonomic revision of parabasalids with description of one new genus and three new species molecular phylogeny and evolution of parabasalia with improved taxon sampling and new protein markers of actin and elongation factor-1a phylogeny of parasitic parabasalia and freeliving relatives inferred from conventional markers vs rpb1, a singlecopy gene trichomoniasis and hiv interactions: a review salivary trichomoniasis. a case report of infestation of a submaxillary gland by trichomonas tenax molecular identification of 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elements and a cognate transcription factor in the divergent unicellular eukaryote trichomonas vaginalis the actin-based machinery of trichomonas vaginalis mediates flagellate-amoeboid transition and migration across host tissue draft genome sequence of the sexually transmitted pathogen trichomonas vaginalis the evolution of infectious agents in relation to sex in animals and humans: brief discussions of some individual organisms functional profiling of the tritrichomonas foetus transcriptome and proteome comparative analysis of trichomonad genome sizes and karyotypes getting trichy: tools and approaches to interrogating trichomonas vaginalis in a post-genome world the effects of environmental factors on the virulence of trichomonas vaginalis trichomonas vaginalis pathobiology new insights from the genome sequence identification of trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells involvement of the gp63 protease in infection of trichomonas vaginalis deep sequencing of trichomonas vaginalis during the early infection of vaginal epithelial cells and amoeboid transition cysteine peptidases, secreted by trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture data-mining approaches reveal hidden families of proteases in the genome of malaria parasite oxford textbook of zoonoses symbioses of flagellates and prokaryotes in the gut of lower termites molecular confirmation of trichomonas gallinae and other parabasalids from brazil using the 5.8s and its-1 rrna regions evaluation of the easyscreen enteric parasite detection kit for the detection of blastocystis spp., cryptosporidium spp., dientamoeba fragilis, entamoeba complex, and giardia intestinalis from clinical stool samples persistence of two trichomonas gallinae isolates in chlorinated and distilled water with or without organic material we thank four anonymous referees for their constructive comments that allowed us to improve the manuscript. j.m.m. is supported by the maccracken program in the graduate school of arts and science, and by a new york university grand challenge project. r.p.h. acknowledges past wellcome trust funding for his work on trichomonas vaginalis. key: cord-017188-d3xg05ty authors: swartz, h.m.; mason, r. p.; hogg, n.; kalyanaraman, b.; sarna, t.; plonka, p.m.; zareb, m.; gutierrez, p. l.; berliner, l. j. title: free radicals and medicine date: 2005 journal: biomedical epr, part a: free radicals, metals, medicine, and physiology doi: 10.1007/0-387-26741-7_3 sha: doc_id: 17188 cord_uid: d3xg05ty epr has been employed in attempts to understand the basis of specific pathophysiologies in which free radicals have a postulated role. examples described include pulmonary free radical damage, free radicals and sickle cell disease, free radicals in amyotrophic lateral sclerosis, melanin and free radicals and the potential role of oxidative stress in the induction of cancer. the final section of the chapter describes the use of nmr as the spectroscopic measure of spin-trapped radicals, after they have reacted further to form diamagnetic species. this chapter is a joint effort that aims to provide an illustrative view of the current understanding of the relationship between free radicals and disease. this is a vast subject and while epr has an important role in its study‚ the role of epr is far from dominant. therefore‚ instead of trying to provide a comprehensive review of the role of free radicals in medicine‚ we have chosen to provide an overview through some general remarks‚ followed by more detailed considerations of several illustrative examples in which epr has been employed in attempts to understand the basis of specific pathophysiologies in which free radicals have a postulated role. the first author has drafted the overview section and the primary author(s) for each specific section are indicated in those sections. the terms epr and esr are used in accordance with the preference of the authors of each section. the theoretical basis for expecting free radicals to be related to disease has evolved over the years. initially the hypothesized links between free radicals and disease were based on loosely reasoned considerations that because free radicals are so reactive‚ and cells are so well organized‚ that the occurrence of free radicals in vivo would likely lead to disease. this type of reasoning became somewhat more focused on the role of free radicals in cancer (swartz‚ 1979) . the current status of concepts of the role of free radicals in cancer is illustrated in the section drafted by gutierrez. the next phase of theories potentially linking free radicals and disease was based on the specific potential of free radicals to initiate strong oxidizing reactions. the fundamental rationale comes from the fact that living organisms include many highly oxidizable components‚ yet usually require the presence of oxygen in order to carry out basic metabolic functions. interest in these aspects became greatly increased with the discovery of the natural occurrence of large amounts of superoxide dismutases in both the blood and cells. subsequently a role of free radicals in almost every conceivable disease has been proposed on various theoretical grounds related to the potential for uncontrolled oxidations. such theories have general support by observations that the extent of many pathologies can be modified by changing the availability of superoxide (especially by modifying the activity of superoxide dismutases) or the strongly oxidizing functions of macrophages. but the observations did not provide rigorous proof of causality‚ and the chemical reactions through which the superoxide caused the observed effects has remained obscure. until very recently‚ it has been assumed that the damage caused by free radicals occurred because of direct reactions with key molecules‚ damaging sufficient numbers of the key molecules to alter function. this has been puzzling when looked at critically‚ because when considering the specific free radicals that are expected to be involved‚ it has been difficult to construct plausible reaction schemes that would account for the observed effects. the hydroxyl radical and closely related species (e.g. the perferyl radical)‚ are so reactive that they must be generated on or immediately adjacent to the target molecules‚ because their diffusion distance is usually the distance to the next molecule. therefore it would take very special circumstances for such radicals to be important in a disease. on the other hand‚ the superoxide radical is so unreactive that it is difficult to understand how it can lead to the very profound effects that clearly are associated with excess or inappropriate production of superoxide. because of recent advances in molecular biology‚ however‚ it now seems reasonable to expect that within the next few years we will have‚ finally‚ an adequate theoretical basis for understanding the roles of free radicals in biology. then it will be much more feasible to develop methods to control or ameliorate the undesirable processes that they can cause. the keys to the potential of much more effective progress have been the discoveries that oxidizing species and the amount of oxygen can have profound effects on gene activation and cell signaling. this has‚ finally‚ provided a potentially satisfactory mechanism for understanding the association of free radicals (and other oxidizing species) with pathophysiology. it has become clear that there are a large number of activators and modulators of genes and cytokines that respond to the presence of oxidizing species. currently the understanding of the role of free radicals and other oxidizing species in cell signaling is still at an early stage‚ but rapid progress is likely as the cascades of effectors that are activated or inactivated by the amount of oxidizing species and/or oxygen are delineated. it seems likely that in many cases the origin of the free radicals will be from inappropriate responses of the naturally occurring defense systems‚ mediated through cell signaling. the old paradigm of deleterious actions of free radicals occurring through direct damage from the free radicals is likely to be applicable only in exceptional circumstances (e.g. ionizing radiation). thus the experimental approaches in which roles of free radicals are likely to be important aspects for understanding probably will be in the arena of molecular biology‚ and the role of epr in these studies may be critical. this is perhaps even more exciting than the vague postulates of free radicals being "bad" and leading to disease‚ because the responses of cells to the presence of oxidizing species are probably very fundamental properties of cellular systems. these mechanisms have enabled life to evolve in an environment with an abundant potential for deleterious oxidations‚ due to an atmosphere that is 20% oxygen. with this understanding‚ the value of epr and related techniques to follow free radical reactions is likely to increase‚ as the techniques are applied in experiments that are more appropriately framed. it is not surprising then‚ that recently theories suggesting links between free radicals and pathophysiological‚ physiological‚ and therapeutic processes have become more specific‚ linked to specific intermediates and/or specific cell signaling pathways. these are areas where it seems more likely that important and useful links will be found. epr is likely to have an important role in elucidating what already has been shown to be a very complex set of processes. initially investigators sought evidence for the role of free radicals in disease by direct observations with epr‚ reasoning that there should be increased amounts of free radicals in the related diseases. in general increases were not found‚ although in a few cases in studies of cancer some unexpected epr signals were seen (swartz‚ 1986). as noted in the review‚ more thorough consideration of this approach leads to the conclusion that even if free radicals are involved‚ there are seldom specific reasons for there to be increased amounts of free radicals that can be detected by epr. the role of free radicals could be critical but transient. and even if there was a continuing role of free radicals‚ the more reactive that they are‚ the less likely it would be that they could be detected directly by conventional epr spectroscopy. hence it is not surprising that there has been little concrete evidence for a general link between the amount of free radicals and pathological or physiological processes. subsequently epr has been used more specifically to detect free radical intermediates associated with a wide range of physiological‚ pathophysiological‚ and therapeutic processes that are potentially related to medicine. these studies have ranged from serially observing tissues in which tumors are expected to development in models of carcinogenesis‚ to looking for free radical intermediates in drugs. the more specific the hypothesis and the rationale for anticipating the occurrence of free radicals‚ the more likely it is that such approaches will be productive. in virtually all situations‚ it is unlikely that measurements of free radicals will be sufficient to test a hypothesis or reach a firm conclusion pertinent to a disease or its treatment. appropriate additional studies‚ including tests of factors that may affect the amount or type of free radicals usually will be needed. the later sections of this chapter provide some illustrative examples of studies in which the role of free radicals in disease have been studied under appropriately rigorous conditions. a wide and effective range of approaches have been used for studies in animal models relevant to clinical medicine. the available approaches are quite wide because unlike the situation in human subjects‚ there is much less concern about the long term effects of materials that are administered to carry out the studies and/or the invasiveness of the approach. recently‚ as described in much more detail in ch. 9 on in vivo studies it has become possible to carry out studies in intact animals as well as in model systems and cells. sometimes it is possible to have direct detection of free radicals‚ if they occur in high enough concentrations and have sufficient stability. this approach is especially applicable when substantial amounts of a substance are administered‚ such as with therapeutic drugs or toxins. the occurrence of free radical intermediates of drugs is among the most likely situations where direct observations with epr are likely to be productive. sometimes it may be possible to have sufficient amounts of radicals to observe them directly in aqueous systems under physiological conditions‚ including normal temperatures for the organism (fujii et al.‚ 1994; mader et al.‚ 1995) . often this will not provide sufficient sensitivity and rapid freezing will be required. while the use of frozen samples often results in the loss of resolvable structure in the epr spectrum‚ the ability to have a much larger sample (because of the much lower dielectric loss in ice‚ the sample volume can be much larger) and the enhanced sensitivity of lower temperatures usually result in very significant increases in sensitivity versus aqueous samples. for most studies‚ however‚ it is likely that there will be a need to employ spin trapping or other techniques to observe the free radicals. several different experimental approaches using spin traps have been successfully employed with cells and experimental animals including: a) use of model systems (in vitro) . this is the most widely used approach. the concentration of spin traps can be made as high as needed when live organisms are not used. when the studies are carried out in cell systems careful attention has to be paid to effects of the spin traps on the cells (toxicity) and effects of the cells on the spin adducts (stability of the adduct) (khan et al.‚ 2003) . b) ex vivo approaches in which the trapping occurs in vivo and samples are obtained for study in vitro. this approach has been used productively, especially by mason and colleagues (ch. 5) . it involves the administration of the spin trap to the animal and then obtaining samples through biopsies or removal of biological fluids. it can provide excellent evidence for the occurrence of free radical reactions in vivo, but considerable care is needed to avoid artifacts or misleading results. the potential problems include loss of spin adducts during the extraction steps and generation of spin adducts during the processing of the samples. c) direct spin trapping in vivo with measurements made directly in the organism under physiological conditions. in principle this is a very desirable approach, providing direct evidence without intervening processing steps and, also, the potential to provide kinetic data under physiological conditions. this approach, however, has lower sensitivity than ex vivo methods, because the latter can study the samples at x-band or even higher frequencies‚ while direct in vivo studies require the use of l-band or even lower frequencies‚ with the consequent power sensitivity of such frequencies. both ex vivo and direct in vivo approaches share the problem of potential instability of the spin adducts‚ which often are converted to non-paramagnetic products in the presence of functioning cells (khan and swartz‚ 2002; timmins et al.‚ 1999) . spin trapping using combined nmr and epr. this is a new approach whose utility has not been fully evaluated. in principle it provides a means to use the widespread availability of in vivo nmr‚ and that is very attractive. the section by berliner in this chapter provides an excellent summary of this approach. indirect assays based on reactions of free radicals with nitroxides or other paramagnetic labels. this has been especially advocated by utsumi's group‚ using the rate of disappearance of nitroxides. while in principle this could be a very productive approach‚ there are some potential fundamental limitations because of the non-specificity of the observed parameter. d) e) in the near future it may be possible to develop techniques for studies in human subjects. the potential limiting factors for such studies include the technical problems of carrying out epr measurements in human subjects and‚ for techniques that involve the administration of spin traps or other substances‚ the complex and difficult process for obtaining permission to administer substances to human subjects (swartz‚ 2003) . these also are discussed in some detail in ch. 9. here we will briefly summarize those aspects that are especially pertinent to measurements of free radicals in human subjects. the technical problems for using epr in human subjects revolve around accommodating the human body in the system and obtaining sufficient sensitivity. the geometric constraints can readily be overcome and at least one specifically designed whole body clinical epr spectrometer already is in operation. this is at dartmouth where a conventional but large-sized permanent magnet has been specifically built by sumitomo special metals for the facility. it provides a 400 gauss field with a gap that can accommodate human subjects on a cart‚ or sitting or standing within the gap of the magnet. for studies in which the direct observable is a free radical form of a drug‚ etc.‚ the only potential limitations are the sensitivity that can be achieved and the pertinence of the radical to the processes under investigation. unfortunately‚ there appears to be a very limited number of drugs or toxins that generate sufficient amounts of free radicals to be observed directly. all other approaches‚ e.g. spin traps‚ interactions with nitroxides‚ require the administration of a compound. this adds the requirement that the administered compound has been demonstrated to be safe for use in humans. this is potentially a very limiting aspect‚ because of the time and expense involved in obtaining the data for achieving such clearance. there are a few drugs that may be of use that already have been cleared for use in human subjects. in particular‚ one of the usual epr responsive trapping agents for nitric oxide‚ the dithiocarbamates‚ have been approved for use in humans as chelators of potentially toxic metals and as the alcohol avoidance agent ("antabuse"). there are some possibilities for the approval of some spin trapping agents and nitroxides as the result of initiatives to use them as therapeutic agents. the nitrone spin traps are being evaluated for use in humans because they appear to have potential therapeutic benefits‚ probably through their effects on the induction of nitric oxide synthesizing enzymes. the nitroxides have been put forth as potential therapeutic agents for the dismutation of superoxide and/or as protective gents against damage by ionizing radiation. if these initiatives result in the approval of nitrones or nitroxides for use in humans‚ then it would be much more feasible to obtaining permission to use them as spin trapping agents in humans. pulmonary free radical damage (r. mason) the herbicide paraquat serves as a model for investigating free radicalmediated pulmonary toxicity because it has no known metabolism other than the free radical metabolism. in microsomal systems‚ the enzymatic reduction of paraquat to its cation radical is catalyzed by the flavoenzyme nadphcytochrome p-450 reductase. the paraquat radical is stable in the absence of oxygen. in the presence of oxygen‚ paraquat is re-formed‚ and superoxide is generated in a catalytic fashion with no net change occurring to the paraquat molecule. this process has been termed futile metabolism (mason 1979‚ 1982 . the mechanism of paraquat poisoning in man is a superoxidemediated toxicity that is completely analogous to the herbicide mode of action. the lung is the site of injury by paraquat because it accumulates there (rose and smith‚ 1977) . the energy-dependent uptake of paraquat and the subsequent free radical formation are cell-specific. paraquat free radical formation occurs with clara cells and alveolar type 2 cells‚ but not with alveolar macrophages (horton et al.‚ 1986) . diquat‚ morfamquat‚ and other bipyridylium compounds do not affect the lung as seriously‚ but these compounds do cause liver damage and are reduced by rat hepatocytes to their respective radical cations (degray et al.‚ 1991). in early investigations of the mechanism of rat hepatic mitochondria! and microsomal nitroreductase (mason and holtzman‚ 1975 a) ‚ esr and kinetic evidence demonstrated that the first step in these nitroreductase reactions is the transfer of a single electron to nitro compounds to give the corresponding nitro anion free radical. for instance‚ in the case of nitrofurantoin‚ the interaction of the free electron with the nitrogens and protons gives a complex hyperfine pattern that has been analyzed to demonstrate that the free radical is simply nitrofurantoin plus an extra electron (rao et al.‚ 1987‚ 1988a . nitrofurantoin increased the nadph-supported oxygen consumption by pulmonary microsomes sevenfold over the basal rate‚ and this stimulation was partially reversed by superoxide dismutase. the presence of superoxide anion radical strongly suggested reductase under aerobic conditions (mason and holtzman‚ 1975b) . as expected‚ the disproportionation of hydrogen peroxide by catalase also decreased the nitrofurantoin-stimulated oxygen uptake. the effect of superoxide dismutase and catalase on the nitrofurantoinstimulated oxygen consumption by microsomes is consistent with the formation of nitroaromatic anion radicals under aerobic conditions and with the rapid air oxidation of these radical intermediates‚ which results in the catalytic generation of superoxide and the well-known oxygen inhibition of nitroreductases. the nitrofurantoin-catalyzed reduction of oxygen to superoxide and the hydrogen peroxide that forms from this superoxide may be responsible for some of the toxic manifestations that occur during nitrofurantoin therapy (mason and holtzman‚ 1975b) . for instance‚ the occasional cases of pulmonary edema and fibrosis caused by nitrofurantoin therapy are similar to the effects of paraquat poisoning. subsequent work with animal models supported this proposal (peterson et al.‚ 1982; boyd et al.‚ 1979) . boyd and coworkers (1979) have shown that the acute toxicity of nitrofurantoin is markedly increased by vitamin e deficiency‚ an oxygenenriched atmosphere‚ or a diet high in highly polyunsaturated fats. further studies by peterson and colleagues (1982) showed that decreasing the activity of selenium-dependent glutathinone peroxidase in 8-day-old chicks enhanced the acute toxicity of nitrofurantoin. both cigarette smoke and the smoke from burning buildings form free radicals even after combustion stops. both the in vitro effects of this smoke and the use of esr in these studies have been extensively reviewed (pryor 1992) . smoke inhalation by rabbits formed unidentified pbn radical adducts in plasma (yamaguchi et al.‚ 1992; murphy et al.‚ 1991) . cotton smoke formed radical adduct concentrations similar to those caused by 2.5 atm of oxygen (yamaguchi et al.‚ 1992) . formation of a burst of pbn radical adducts by a brief exposure to cigarette smoke required pretreatment by bacterial endotoxin (murphy et al.‚ 1991) . the spin trapping technique detected free radicals produced in vivo by ozone exposure (kennedy et al.‚ 1992) . when rats were exposed for 2 hr to either 0‚ 0.5‚ 1.0‚ 1.5‚ or 2.0 ppm ozone with 8% to increase their respiratory rate‚ a six-line 4-pobn/radical spin adduct signal was detected by esr in lipid extracts from lungs of rats treated with 4-pobn. only a weak signal was observed from rats exposed to 0 ppm ozone (air with only). a correlation was observed between the radical adduct concentration and the lung weight/body weight ratio‚ an index of lung damage. these results demonstrate that ozone induces the production of free radicals in rat lungs during inhalation exposure and that radical production may be involved in the induction of pulmonary toxicity by ozone. it has been postulated that the in vivo toxicity of asbestos results from its catalysis of free radical generation. we examined in vivo radical production using esr and the spin trap 4-pobn; 180 day-old rats were intratracheally instilled with either crocidolite asbestos or saline. twenty-four hours later‚ histologic examination revealed a neutrophilic inflammatory response. esr spectroscopy of the chloroform extract from lungs exposed to asbestos gave a spectrum consistent with a carbon-centered radical adduct‚ while those spectra from lungs instilled with saline revealed a much weaker signal. this same radical formation persisted and‚ even one month after instillation‚ could be detected in the lungs of rats exposed to asbestos. exposure to air pollution particles can be associated with increased human morbidity and mortality. lung exposure to oil fly ash (an emission source air pollution particle) causes in vivo free radical production as evidenced by esr analysis of chloroform extract from lungs of animals exposed to oil fly ash‚ which gave a spectrum consistent with a carboncentered radical adduct (kadiiska et al.‚ 1997) . this signal was also reproduced by instilling animals with the soluble fraction of the oil fly ash‚ which contains soluble metal compounds. the same signal was observed after instillation of either a mixture of vanadium‚ nickel‚ and iron sulfates or alone‚ metals which are prevalent in oil fly ash. therefore‚ this generation of free radicals appears to be associated with the soluble metals in the oil fly ash. intratracheal instillation of lipopolysaccharides (lps) activates alveolar macrophages and infiltration of neutrophils‚ causing lung injury/acute respiratory distress syndrome. free radicals are a special focus as the final causative molecules in the pathogenesis of lung injury caused by lps. in vivo free radical production by rats was detected after intratracheal instillation of lps. esr spectroscopy of lipid extract from lungs exposed to lps for 6 h gave a spectrum consistent with that of a pobn/carbon-centered radical adduct tentatively assigned as a product of lipid peroxidation. to further investigate the mechanism of lps-initiated free radical generation‚ rats were pretreated with the phagocytic toxicant which significantly decreased the production of radical adducts with a corresponding decrease in neutrophil infiltration. nadph oxidase knockout mice completely blocked phagocyte-mediated‚ esr-detectable radical production in this model of acute lung injury‚ demonstrating that superoxide formation by nadph oxidase is the root cause of this free radical formation. there has been a long association between sickle-cell disease and the formation of oxidants from the partial reduction of molecular oxygen. in general this association has focused on either the deposition of redox active iron as a mediator of biomolecule oxidation‚ or on accelerated autoxidation of hbs as opposed to hba‚ leading to enhanced superoxide formation. in both cases increased oxidative stress in sickle cell disease results in the oxidation of biological macromolecules which contributes to the pathogenesis of the disease (hebbel et al.‚ 1982; klings et al.‚ 2001; natta et al.‚ 1992; repka and hebbel‚ 1991; schacter‚ 1986; sheng et al.‚ 1998) of free radicals and oxidants in the control of vascular function (as opposed to oxidative damage). endothelial dysfunction‚ which is most often indicated by loss of endothelial-dependent relaxation‚ can be regarded as a disease of altered free radical balance. for example the scavenging of nitric oxide by superoxide has long been thought to be contributing factor to hypertension and it is becoming apparent that this same mechanism may be responsible for altered endothelial signaling in atherosclerosis‚ diabetes‚ and other cardiovascular pathologies‚ and may also be a control mechanism of normal cardiovascular responses such as the modulating effects of shear stress on vessel relaxation. recently two nitric oxide scavengers have been identified in the lumen of patients with sickle cell disease. the first of these‚ xanthine oxidase‚ has long been understood to be a physiological source of superoxide‚ however‚ the recently discovered elevation of vessel wall xanthine oxdase may be an important component of endothelial dysfunction in sickle cell disease. aslan et al. (2001) have demonstrated that liver injury in both sickle-cell patients and a mouse sickle cell model can result in the release of xanthine oxidase into the blood stream. accumulation and/or uptake of this enzyme by the vascular endothelium can lead to enhanced superoxide production and impairment of endotheliumdependent relaxation. another molecule that has only recently been implicated as a nitric oxide scavenger in sickle cell disease is plasma hemoglobin. a major paradox of nitric oxide function has been the presence of high (10 mm) concentrations of hemoglobin in the vascular lumen. calculations have clearly shown that this level of hemoglobin should preclude nitric oxide radical from having any appreciable steady-state concentration in the vessel wall. this paradox has been recently addressed by the discovery that compartmentalized hemoglobin reacts with nitric oxide at significantly slower rates (up to 1000 times) than cell-free hemoglobin. the effects of increased levels of cell-free hemoglobin on vascular function have only recently been realized. as recently demonstrated by reiter et al. (2002) ‚ the levels of plasma cell free hemoglobin are substantially elevated in patients with sickle cell disease‚ averaging and as high as the hemoglobin appears to be in the ferrous form as the plasma from sickle cell patients exhibits a greater ability to scavenged nitric oxide. measurements of blood flow in the forearm indicate that plasma hemoglobin can severely limit nitric oxide-dependent vasodilation. an important corollary of these studies is that pharmacologically administered nitric oxide (for example by inhalation) can act to 'scavenge the scavenger' by oxidizing plasma hemoglobin to its met or ferric derivative. ferric hemoglobin then has limited ability to scavenge peripheral nitric oxide‚ thus potentially restoring no bioavailablility. the level of methemoglobin in plasma (as demonstrated by electron paramagnetic resonance spectroscopy) is increased when sickle-cell patients inhale nitric oxide‚ together with the level of nitrosylhemoglobin (reiter et al.‚ 2002) . cell-free hemoglobin circulating through the pulmonary circulation is a much more amenable target for nitric oxide therapy than guanylyl cyclase and it may be the case that the effects of nitric oxide inhalation in other clinical conditions are at least in part mediated through plasma hemoglobin oxidation. it is interesting to speculate that the ability of hydroxyurea‚ recently identified as an no congener‚ to relieve sickle cell disease may also be at least in part related to its ability to oxidize hemoglobin in the extracellular compartment (glover et al.‚ 1999; huang et al.‚ 2002; jiang et al.‚ 1997) . motor neuron disease or als (also known as lou gehrig's disease) is a fatal illness that progressively causes the degeneration of the nerve cells that control the muscles‚ paralyzing the body but sparing the mind (price et al.‚ 1994) . approximately 10% of als cases are familial with the rest being sporadic. the genetic defect in familial als (fals) has now been linked to sod 1‚ the gene encoding the cytosolic cu‚ zn‚ superoxide dismutase (sod1) (brown‚ 1995‚ 1998 . at present‚ approximately 70 or so missense fals mutations in sod 1 at 26 different amino acid positions have been identified. the mechanisms by which fals-linked sod 1 mutants cause selective degeneration of motor neurons of the spinal cord‚ brain stem‚ and cerebral cortex remain unclear (brown‚ 1995‚ 1998 . of the various mechanisms proposed‚ the hypothesis suggesting that the fals-linked mutants acquired a cytotoxic "gain-in-function" has found increased acceptance (brown‚ 1998) . the "gain-in-function" of sod 1 mutants had been postulated to arise from an increased peroxidase or hydroxyl radical activity. the epr technique has played a key role in identifying the structure of the oxidant formed from the peroxidase activity of sod 1 (singh et al.‚ 1998) . nearly 30 years ago‚ hodgson and fridovich demonstrated that the copper-bound hydroxyl radical a putative oxidant generated in the reaction between sod1 and could oxidize several anionic ligands (e. g.‚ formate‚ azide‚ and nitrite anion) (hodgson and fridovich‚ 1975) . in addition to these anions‚ several other molecules (which are too bulky to find access to the active site of cu‚ zn and sod) were also oxidized. these experiments were performed in a bicarbonate buffer. the x-ray crystal structure of sod1 indicates that access to the active site is via a narrow channel that restricts the entry of large molecules. however‚ a relatively small anion like could reach the active site of sod1 and undergo oxidation to the carbonate anion radical by is a diffusible oxidant that could leave the active site and cause oxidation of various substrates in free solution. bicarbonate thus effectively exports oxidation from the sterically hindered active site to large molecules in bulk solution (liochev and fridovich‚ 1999) . this model which gives a new perspective on the peroxidative mechanism of sod1‚ is pivotal for explaining the published discrepancies in spin trapping reports using sod1 and fals sod1 mutants (goss et al.‚ 1999; zhang et al.‚ 2002) . evidence for increased peroxidase activity from fals sod1 mutants (e. g.‚ a4v with alanine substituted by valine at amino acid 4‚ g93a with glycine replaced by alanine at amino acid 93) was first obtained by monitoring the oxidation of nitrone spin trap 5‚ 5'-dimethyl-1-pyrroline noxide (dmpo) to its hydroxylated adduct (dmpo-oh) (wideau-pazos et al.‚ 1996) . the epr spectrum of dmpo-oh is characterized by an equivalent hyperfine interaction (=15 g) from the nitroxide nitrogen and the atom. investigators attributed the formation of dmpo-oh to trapping of hydroxyl radical (see zhang et al.‚ 2000 for references). significant differences in dmpo-oh signal intensity were observed between the wild type sod1 and fals mutant sods. however‚ the fact that bicarbonate was needed for dmpo-oh formation in this system has been previously ignored. we reinvestigated the mechanism of formation of dmpo-oh using oxygen-17 labeled water and hydrogen peroxide. results from these experiments unambiguously demonstrated that nearly all of the oxygen in dmpo-oh originated from water. we proposed that either a nucleophilic addition of to the dmpo-carbonate radical intermediate or an addition of to the dmpo cation radical intermediate. independent evidence for the intermediacy of was obtained from photolysis studies using the pentammine carbonate complex of co (iii). the uv photolysis of this cobalt complex has been shown to generate the authentic radical. this system yielded the same type of radical adducts in the presence of substrates (e. g., azide, ethanol, and formate) as detected in the system (zhang et al., 2000 (zhang et al., , 2002 . bicarbonate-dependent peroxidase activity of sod1 was found to be responsible for hydroxylation and oxidation of azulenyl nitrone to an aldehyde. aldehyde formation was attributed to trapping of hydroxyl radicals by the azulenyl nitrone (gurney et al., 1998) . recently, we showed that in the absence of bicarbonate, oxidation of azulenyl nitrone to azulenyl aldehyde was negligible in phosphate buffers containing sod1, and the chelator dtpa (zhang et al., 2002) . photolysis of solutions containing the cobalt complex that generates the carbonate radical anion and azulenyl nitrone yielded azulenyl aldehyde. based on these results, we proposed that is responsible for mediated oxidation of azulenyl nitrone. bicarbonate-dependent sod1 peroxidase activity is, thus, inhibitable by nitrones. bicarbonate-dependent sod1 peroxidase activity was shown to be higher in fals mutants (brown, 1995 (brown, , 1998 . the first in vivo spin trapping evidence for increased free radical formation was provided using the sod1-g93a transgenic mouse model for fals (gurney et al.‚ 1998) . the investigators found that when azulenyl nitrone was administered to the nontransgenic‚ wild-type transgenic‚ and mutant transgenic mice of different ages (30‚ 60‚ and 90 days)‚ increased levels of azulenyl aldehyde were detected in spinal cord extracts of mutant mice but not in transgenic wild-type and nontransgenic mice. concomitantly‚ azulenyl nitrone treatment prolonged the survival of fals overexpressing mice. more recently‚ it was shown that treatment of mutant g93a-sod1 transgenic mice with nitrone trap dmpo significantly delayed paralysis and prolonged survival (li et al.‚ 2002) . however‚ dmpo-derived products were not analyzed. in conclusion‚ in vitro and in vivo experiments suggest that nitrone spin traps can potentially mitigate oxidative stress in fals mutant overexpressing cells and mice and protect against progressive motor neuron death. melanins are virtually the only naturally occurring stable free radicals in mammals. epr spectroscopy can be used as a unique non-destructive tool for studying melanin in in vitro in cultivated cells (pilas and sarna‚ 1985; cieszka et al.‚ 1995; hill et al.‚ 1997) ‚ in isolated tissues and organs ex vivo (enochs et al.‚ 1993a; slominski et al.‚ 1994; plonka et al.‚ 2002) ‚ and even under in vivo conditions (lukiewicz and sarna‚ 1972; katsuda et al.‚ 1990) . the putative function of the pigment is protection against light-induced damage‚ perhaps mediated by free-radical damage (woods et al.‚ 1999) . the interactions of melanin probably involve both radical and nonradical aspects of the quinone-hydroquinone equilibria that underlie many of its properties (sarna and swartz‚ 1993) . because of their redox activity and ability to bind charged materials‚ including metal ions and drugs‚ melanins potentially can affect many physiological‚ pathophysiological‚ and therapeutic processes (enochs et al.‚ 1994; larsson‚ 1993; mars and larsson‚ 1999) . in addition‚ due to the high reactivity of some of the intermediates‚ melanin synthesis has also been linked to cytotoxicity (halaban and lerner‚ 1977; pawelek and lerner‚ 1978) . because of these complex and diverse properties‚ the study of melanins with epr has been a large and productive field that has pertinence to the subject of this chapter: free radicals and medicine. as an example of such studies‚ we consider here two aspects in which the esr center at the medical college of wisconsin has been especially involved‚ ocular melanins and neuromelanins. specialized cells that synthesize melanin in humans are found not only in the skin epidermis and in the hair follicles‚ they are also present in the eye‚ ear and in restricted regions of the brain (boissy‚ 1998). although populations of these extracutaneous melanin-synthesizing cells have some morphological and functional similarities to the various cutaneous melanocytes‚ it is expected that significant differences between extracutaneous and cutaneous melanocytes should exist (boissy‚ 1998). melanin synthesis usually occurs in specific organelles‚ the melanosomes‚ and is controlled by specialized enzymes (orlow‚ 1998; pawelek and chakraborty‚ 1998)‚ but the formation of neuromelanin (nm) in dopaminergic and noradrenergic neurons of the substantia nigra (sn) and locus coeruleus (lc)‚ appears to be enzyme-independent and the neuromelanin granules show significant similarities to inactivated lysosomes (barden and brizzee‚ 1987) . in primates‚ two different cell types are involved in ocular melanogenesis -melanocytes of the uveal tract and neuroepithelial cells of the retinal pigment epithelium (rpe). all true melanocytes‚ including ocular melanocytes‚ originate from the neural crest; in contrast‚ the rpe cells are derived from neuroepithelial cells of the developing forebrain of the embryo (noden‚ 1991; mann‚ 1964) . both types of the melanin-synthesizing cells of the eye express melanocyte-specific proteins (boissy‚ 1998). allthough biological functions of ocular melanin have not been unambiguously determined‚ a growing body of experimental evidence and epidemiological data suggest that both the iridial (uveal) and rpe melanin may have important photoprotective and antioxidant properties (sarna‚ 1992; sarna and 1994) . clearly‚ ocular pigmentation‚ particularly in the uveal tract‚ protects the retina from overexposure by absorbing and scattering the impinging light. melanin in the choroid and rpe contributes to visual acuity by preventing light reflection from the fundus. it has long been recognized that the severity of the visual abnormalities in albinism correlates with the degree of hypopigmentation (kinnear et al.‚ 1985) . however‚ even with corrective tinted lenses that help photophobia and‚ in a minor way‚ vision by increasing contrast‚ the results in albinos are rather disappointing (taylor‚ 1978) . it has been postulated that the albino visual system may represent a case of arrested development (wilson et al.‚ 1988) . thus‚ melanin synthesis in the rpe appears to be of crucial importance for normal development of the visual system of an organism‚ and the presence of rpe is not only responsible for the maintenance of the neural retina but also for its successful embronic development and organization (boissy et al.‚ 1993; raymond and jackson‚ 1995) . age-related macular degeneration (amd) is the major cause of late onset blindness in developed countries (evans and wormald‚ 1996; klein et al.‚ 1992) . there is currently no established etiology that could serve as a basis for preventive medicine and no cure for this disease. light can damage photoreceptor cells in the mammalian retina (organisciak and sarna‚ 2001; rapp‚ 1995; ham et al.‚ 1980) and population-based studies indicate an association between advanced amd and a patients' exposure to blue or visible light over the preceding 20 years (cruickshanks et al.‚ 1993; mccarty and taylor‚ 1999; taylor et al.‚ 1992) . the primary lesion in amd may occur in the rpe (zarbin‚ 1998) and result from oxidative damage (beatty et al.‚ 2000; cai et al.‚ 2000) . due to the close structural and functional association between the rpe and photoreceptors‚ it is generally agreed that any oxidative damage to the rpe‚ which causes rpe dysfunction‚ could contribute to the photoreceptor degeneration that characterizes amd. thus‚ the antioxidant status of the rpe may be a key factor in determining the tissue's health‚ and the health of the photoreceptors that the rpe supports. an rpe antioxidant that may be particularly relevant when photic stress is implicated in tissue damage is the light-absorbing pigment melanin (sarna‚ 1992). it has been postulated that rpe melanin can protect the rpe from oxidative stress by sequestering redox active metal ions‚ quenching electronically excited states of certain photosensitizing dye molecules and by scavenging reactive free radicals (sarna et al.‚ 1998; rozanowska et al.‚ 1999) . significantly‚ melanin content of the human eye seems to be inversely correlated with the incidence of amd (weiter et al.‚ 1985; young‚ 1988 )‚ and the risk of suffering from amd is about 40 times higher in whites than in blacks (pauleikhoff and holz‚ 1996) ‚ even though the inter-racial differences in the amount of ocular melanin mainly concern the uveal pigmentation. however‚ it is important to realize that antioxidant abilities of melanin may change with age. indeed‚ a recent epr study has shown that the amount of rpe melanin decreases by a factor of 2.5 between the first and ninth decade of life (sarna et al.‚ 2002) . in an independent study‚ the melanin content of rpe melanosomes from human donors of different age was analyzed by epr after drying the pigment granules (bilinska et al.‚ 2002) . surprisingly‚ the content of melanin‚ when normalized to a single pigment granule‚ was found independent of age. on the other hand‚ rpe melanosomes from younger individuals could contain significantly higher percentage of strongly bound water‚ that those from older individuals (bilinska et al.‚ 2002) . rpe melanosomes from older human donors exhibit increased photoreactivity: they induce faster oxygen photouptake and accumulation of superoxide anion spin adducts (rozanowska et al.‚ 2002) . thus with age‚ rpe melanin may contribute to oxidative stress in the outer retina both as a result of a decreased antioxidant efficiency and an increased aerobic photoreativity. interestingly‚ w-band epr spectroscopy may provide a sensitive measure for differentiating rpe melanin from human donors of different age and monitoring possible chemical changes of the melanin that are likely to be aggravated with aging (rozanowska et al.‚ 1993) . in this preliminary study a striking pattern in the changes of magnetic parameters of melanin radicals with age was observed. although the molecular nature of these changes is not yet understood‚ they may result from life-long oxidative modifications of the rpe melanin. neuromelanin (nm) is an insoluble brown-black or greyish in appearance pigment that accumulates with age in the midbrain of primates‚ most notably of humans (van woert et al.‚ 1966) . based on standard histochemical tests and various physicochemical analysis‚ including epr spectroscopy‚ nm has been classified as a true melanin (barden and brizzee‚ 1987; enochs et al.‚ 1993b) . characteristic degradation products of nm indicate that this melanin has chemical properties similar to both eumelanins and pheomelanins (odh et al.‚ 1994) . it appears that the synthesis of nm occurs via oxidative polymerization of dopamine and its sulfur containing derivatives. as a synthetic model of nm‚ a polymer obtained by autooxidation of dopamine and cysteine is often used (shima et al.‚ 1997) . biological functions of nm remain unknown. unlike cutaneous and ocular melanins‚ neuromelanin is never exposed to environmental light. therefore‚ no photoprotective role of nm is likely. it has been postulated that under normal physiological conditions nm may have a cytoprotective function by the sequestration of redox-active metal ions (swartz et al.‚ 1992; zareba et al.‚ 1995; korytowski et al.‚ 1995) . on the other hand‚ the presence of nm may also be responsible for the vulnerability of pigmented neurons observed in parkinson's disease (pd). this neurological disorder is characterized clinically by akinesia‚ rigidity‚ and tremor (stern‚ 1990) . histological analysis of parkinsonian brains shows extensive degeneration of the pigmented dopaminergic neurons of the substantia nigra. in pd‚ the neuromelanin containing cells of the sn appear to be more vulnerable to degeneration than the non-pigmented neurons. however‚ there seems to be no strict correlation between the vulnerability of the neurons and their melanin content (kastner et al.‚ 1992) . the formation of nm in the human brain‚ probably is a by-product of oxidative metabolism of dopamine‚ known to occur at high rate in catecholaminergic neurons of the sn and lc. while both autooxidative reactions and enzymatic oxidation of dopamine are accompanied by the generation of hydrogen peroxide‚ the former process can also lead to the formation of potentially cytotoxic semiquinones and quinones (cohen‚ 1983; graham 1984) . therefore‚ the dopaminergic neurons of the sn and lc are intrinsically at risk of oxidative stress. despite the presence of antioxidative mechanisms in catecholaminergic neurons for maintaining their neurotransmitters in a reduced state‚ the system is not perfect and some semiquinones and quinones may escape from reduction and randomly form products that cannot be catabolized and gradually accumulate in lysosomes‚ eventually becoming neuromelanin granules (enochs et al.‚ 1994) . it can be argued that when formed‚ nm can exert its antioxidant action by binding of advantitious multivalent metal ions‚ such as iron and copper (swartz et al.‚ 1992) . there is evidence that sn neurons in patients with pd exhibit an elevated level of oxidative stress (jenner‚ 1991). thus the ratio of reduced to oxidized glutathione is decreased in parkinsonian brains‚ compared to normal brains (riederer et al.‚ 1989; sofic et al.‚ 1992) . in addition‚ a decreased level of polyunsaturated fatty acids and increased levels of malondialdehyde‚ a product of lipid peroxidation‚ were found in pd brains (dexter et al.‚ 1989) . whether these changes are the cause of the disease or they result from an increased dopamine turnover in the remaining neurons of a pd patient‚ remains unclear. one of the most important properties of nm that may determine its biological functions is the ability of nm to bind metal ions (enochs et al.‚ 1993b) . high levels of metal ions‚ particularly iron; were found in the human sn (zecca and swartz‚ 1993; gerlach et al.‚ 1995; shima et al.‚ 1997) . although ferritin is known to be responsible for accumulation of iron in many tissues‚ it is believed that in sn iron is bound to nm zecca and swartz‚ 1993; gerlach et al.‚ 1995) . indeed‚ recent measurements suggest that ferritin is not present in sn neurons (moos‚ 2000)‚ which implies that neuromelanin may be a key buffering system for iron in pigmented neurons (zecca et al.‚ 2002) . the antioxidant efficiency of neuromelanin was studied in model systems. the study has clearly shown that the yield of hydroxyl radicals generated via iron ion-catalyzed free radical decomposition of hydrogen peroxide dramatically decreased in the presence of synthetic nm (zareba et al.‚ 1995) . both synthetic and natural nms were able to inhibit peroxidation of lipids induced by iron/ascorbate or thermolabile azo-compounds . a hypothesis‚ about the role of nm in the multifactioral etiology of pd was proposed by enochs et al. (1994) : oxidative degradation of nm that can occur with aging or as a result of an intense or chronic oxidative stress in the pigmented neurons of sn could‚ via positive feed-back mechanism‚ further increase the level of the oxidative stress. oxidatively modified nm is expected to have a decreased binding affinity for iron and other metal ions. it would‚ therefore‚ not only bind fewer metal ions compared to "native" nm‚ but even raise the cytoplasmic level of potentially cytotoxic species by release of the accumulated ones. the genetic basis of human cancer has been well documented. widespread genomic instability is a hallmark of tumor cells (jeong‚ 2003; nowell‚ 1976 ). an average of about 1/3 of all tumor types appear to exhibit a genomic instability manifested by subtle nucleotide sequence alternations that activate proto-oncogenes or inactivate tumor suppressor genes. chromosomal rearrangements are likely to be a result of aberrant recombination repair that occurs during normal cellular mitotic growth (przybytkowski‚ 2003; friedberg‚ 1985; friedberg et al.‚ 1995; kucherlapati and smith‚ 1988) . a very large number of genes has been identified as tumor promoter genes or tumor suppressor genes (cheng‚ 2003; stanbridge‚ 1990; weiberg‚ 1989) . as far back as 1993‚ genetic analysis of tumor cells has suggested that between 6 to 12 of these genes are altered during the development of a tumor (renan‚ 1993). the number of altered genes far exceeds the number of mutations that would be predicted by the spontaneous mutation rate in human cells. of the several theories to account for this discrepancy‚ the "mutator hypothesis" for tumorigenesis has gained much attention (loeb‚ 1991‚ 1994 . in this hypothesis‚ an early spontaneous mutation in one of many genes that maintain the genetic information in chromosomes becomes altered. this leads to genetic instability‚ or a mutator phenotype‚ either by accumulation of replicative errors or by gross chromosome rearrangement. a higher-than-normal mutation rate then leads to the combination of mutations in genes that regulate growth‚ invasion‚ or metastasis. post-replication mismatch repair systems enhance the fidelity of dna replication by correction of replicative errors. it has recently been shown that mutations in mismatch repair genes are associated with cancers (e.g. jeong‚ 2003; worrillow‚ 2003) . oxidative stress caused by reactive oxygen species (ros) can lead to genomic instability. oxygen is metabolized inside the cell by a series of one electron reductions with the generation of reactive and potentially damaging ros which include superoxide radical anion‚ and the hydroxyl radical (halliwell and gutteridge‚ 1989) . a pro-oxidant status in the cell can lead to the accumulation of ros. humans are exposed to substantial amounts of oxidative stress from a variety of sources. halliwell has estimated levels of up to in the urine of individuals ingesting such foods as instant coffee (long et al.‚ 1999a‚b) . oxygen and its metabolites are known to function as intracellular signals‚ playing a role in apoptosis (butts et al‚ 2003; liu et al.‚ 2003; barr and tomei‚ 1994; sandstrom‚ 1994‚1995; corcoran et al.‚ 1994; davies‚ 1995; forrest et al.‚ 1994; greenspan and aruma‚ 1994; hockenbery‚ 1995; sandstrom et al.‚ 1994) and metabolizing enzymes (anderson et al.‚ 1994; burdon‚ 1995; crawford et al.‚ 1988; curran and morgan‚ 1995; janknecht et al.‚ 1995; khan and wilson‚ 1995; koong et al.‚ 1994 a‚b; chen and giaccia‚ 1994; nose et al.‚ 1991; piechaczyk et al.‚ 1994; remade et al.‚ 1995; schreck et al.‚ 1991; yao et al.‚ 1994; lambert et al.‚ 1994; vile et al.‚ 1994; firth et al.‚ 1994) . ros are believed to play a causative role in the degenerative diseases of aging including cancer peterszegi et al.‚ 2003; cantuti-castelvetri et al.‚ 2003) . endogenously‚ activated neutrophils and macrophages generate large quantities of no‚ as part of the inflammatory response (chanock et al.‚ 1994; densen et al.‚ 1995‚ ginsburg and kohen‚ 1995; laskin and pendino‚ 1995) ‚ and a variety of other normal cells can also be stimulated to produce low levels of superoxide and hydrogen peroxide (burdon‚ 1995). ames has estimated that one third of the cancers in developing countries can be attributed to chronic infections . when oxidative stress due to these reactive species from whatever source exceeds the capacity of the defense systems of the cells to intercept and neutralize them (i.e. prooxidant status)‚ toxicity (mytar et al.‚ 1999) ‚ genomic instability and mutations in cells result. for example‚ tumor promoters stimulate the production of reactive oxygen species which may play a critical role in the progression to malignancy (beckman et al.‚ 1994; gopalakrishna et al.‚ 1994; hu et al.‚ 1995; marnett and ji‚ 1994; lin and shih‚ 1994; panandiker et al.‚ 1994) . also‚ an african green monkey kidney cell line (cv-1) transfected with the enzyme peroxisomal fatty acyl-coa oxidase. when exposed to the enzyme substrate linoleic acid for 2-6 weeks‚ 4% of the cells became tumorigenic (chu et al.‚ 1995) . ros targets are many‚ including membranes and proteins. the most critical potentially carcinogenic targets of ros are probably deoxynucleotides and dna‚ which generate in dna 7‚8-dihydro-8hydroxy-2'-deoxyguanine (8-ohdg)‚ 7‚8-dihydro-8-hydroxy-2'deoxyadenine (8-ohda)‚ 2‚6-diamino-4-hydroxy-5-formamidopyrimidine (fapy-g)‚ and 4‚6-diamino-5-formamidopyrimidine (fapy-a) (bertoncini and meneghini‚ 1995; breen and murphy‚ 1995; dizdaroglu‚ 1993; feig et al.‚ 1994; giese et al.‚ 1995; halliwell and aruoma‚ 1991; steenken‚ 1989) . guanine is the most easily oxidized of the nucleic acid bases (an electronloss center created in a system containing the four nucleic acid bases will eventually migrate to guanine) (steenken‚ 1989). the keto and enol forms of this damaged guanine are found in equilibrium. thus‚ the interchangeable use of 8-oxodg and 8-ohdg found in the literature. we will use the 8-oxog terminology in this discussion. since the last step in formation of 8-oxog and 8-oxoa is an oxidation while the last step in the formation of fapy-g and fapy-a is a reduction (breen and murphy‚ 1995; steenken‚ 1989 )‚ it has been suggested that 8-oxog formation is favored under the more oxidizing (pro-oxidant) conditions found in cancer cells (malins et al.‚ 2003; malins‚ 1993; malins et al.‚ 1993) ‚ or alternatively‚ that a pro-oxidant condition exists in cells destined to become malignant favoring the accumulation of 8-oxog. for instance‚ the dna of hepatitis b virus infected mouse liver cells destined to become tumorigenic had elevated levels of 8-oxog (hagen et al.‚ 1994) . whatever the reason‚ 8-oxog is considered to be a major stable product generated by ros attack on dna and it is excreted in the urine of humans where it is used as a biological marker of in vivo oxidative dna damage (halliwell‚ 1993; shigenaga et al.‚ 1994) . the formation of 8-oxog in dna‚ if not repaired‚ leads to misincorporation of da opposite to the 8-oxog lesion. the misincorporated da leads to an a/8-oxog mispair resulting in g·c t· a transversions moriya‚ 1993; cheng et al.‚ 1992; moriya et al.‚ 1991) . the a/8-oxo g mispair also interferes with dna-processing enzymes such as methylases (weitzman et al.‚ 1994) and restriction enzymes (turk and weitzman‚ 1995) . it is interesting to note that g·c t·a transversions occur frequently as mutations of the p53 tumor suppressor gene in human lung‚ breast‚ and liver cancers (e.g. hollstein et al.‚ 1991) . more importantly‚ in p53‚ induces a g to t transversion at both g-residues of codon 249 (agg) and a c to a transversion at codon 250 (ccc) (hussain et al.‚ 1994) . in order to minimize the deleterious effects of reactive oxygen species‚ all aerobic organisms possess extensive defense systems. the primary defense is achieved by decomposing superoxide and hydrogen peroxide‚ which thus minimizes the formation of the ensuing hydroxyl radical (·oh). this primary antioxidant defense system includes various enzymatic and nonenzymatic mechanisms such as superoxide dismutase‚ catalase‚ glutathione peroxidase‚ and ascorbic acid (e.g. halliwell and gutteridge‚ 1989) . in addition‚ although controversial‚ data indicate that bcl-2‚ the antiapoptotic protein also has antioxidant powers (amstad et al.‚ 2001) . the reactive oxygen species that escape from the primary defense system have a significant chance of damaging dna. several repair enzymes in both prokaryotes and eukaryotes have been shown to remove 8-oxog residues from dna providing a secondary line of defense. the mammalian repair enzyme hogg1‚ the human homolog of the yeast ogg1 is able to repair c/8-oxog dna damage back to cg in cells due to its 8-oxog glycosylase /ap lyase activity (gu et al.‚ 2001; aburatani et al.‚ 1987; arai et al.‚ 1997; friedberg et al.‚ 1995; hazra et al.‚ 1998; nash et al.‚ 1996; radicella et al.‚ 1997; ramotar and demple‚ 1993; roldan-arjona et al.‚ 1997; shibutani et al.‚ 1991; van der kemp et al.‚ 1996) . the mammalian mth1 enzyme‚ like the e. coli mutt homolog eliminates 8-oxodgtp damage from the deoxynucleotide pool (maki and sekiguchi‚ 1992) . based on sequence homology with yeast ogg1‚ hoog1 has been cloned (aburatani et al.‚ 1997; arai et al.‚ 1997; lu et al.‚ 1997; radicella et al.‚ 1997; roland-arjona et al.‚ 1997; rosenquist et al.‚ 1997; shibutani et al.‚ 1991) . if the 8-oxog lesion is not removed by the hogg1 protein before dna replication‚ then misincorporation of da opposite to the 8-oxog lesion occurs (hazra et al.‚ 1998; moriya et al.‚ 1991; nghiem et al.‚ 1988; shibutani et al.‚ 1991) . the mammalian hmyh enzyme‚ like e. coli muty‚ removes this misincorporated adenine from an a/8-oxog mispair‚ with the formation of c/8-oxog‚ which is then a substrate for hogg1. this is considered the third level of defense (au et al.‚ 1989; mcgoldrick et al.‚ 1995; slupska et al.‚ 1996; tsai-wu et al.‚ 1991; yeh et al.‚ 1991) . the muty gene has been cloned and sequenced (boiteux et al.‚ 1987; michaels et al.‚ 1990; michaels et al.‚ 1991; slupska et al.‚ 1996) . in addition‚ it has been shown that expression of the e. coli mutm gene which expresses fpg‚ an enzyme with the same activity as hogg1 in mammalian cells‚ reduces the mutagenicity of x-rays (laval‚ 1994). furthermore‚ germ line mutations in human genes hmsh2‚ or hmlh1‚ which express proteins with the ability to correct dna mismatches and misalignments lead to genetic instability in microsatellite repeat sequences in hereditary nonpolyposis colon cancer (jeong et al.‚ 2003; bronner et al.‚ 1994; papadopoulos et al.‚ 1994) ‚ it thus seems likely that the hogg1/hmyh repair pathways are linked to protection against certain cancers. an elevation of oxidative modifications in dna has recently been reported in human cancers of the breast‚ prostate (malins and haimanot‚ 1991; malins et al.‚ 1993‚ malins et al.‚ 2003 and other tissues (lepage et al.‚ 2000; olinski et al.‚ 1992) . the 8-oxog lesion has been suggested to be a putative link to cancer development (malins et al.‚ 2003; malins‚ 1993 ) and a predictor for breast cancer (malins et al.‚ 1995) . this modification occurs by the attack of hydroxyl radicals that arise from mediated by trace metal ions such as and (luo et al.‚ 1994) . since diffuses readily across the nuclear membrane‚ hydroxyl radicals are formed near dna and modify it by attacking the purine and pyrimidine bases (meneghini and martins‚ 1993) . breast cancer is not commonly associated with pre-existing chronic inflammatory conditions‚ unlike bowel cancer where a causal relationship between ulcerative colitis and bowel cancer has been suggested (weitzman and gordon‚ 1990) . breast lesions (benign or malignant) are‚ however‚ commonly infiltrated with macrophages‚ which lead to oxidative stress. this in turn could lead to the progression of some cells from benign to malignant and/or facilitate the transformation to a more aggressive metastatic tumor. there are a number of tumorigenic cell lines including one from breast that chronically elaborate large quantities of (szatrowski and nathan‚ 1991) that can lead to chromosome mutations and cancer (emerit‚ 1994) . in addition‚ the most lethal form of locally advanced breast cancer is termed inflammatory breast cancer due to the acute inflammatory changes observed in patients with this type of malignancy (jaiyesimi et al.‚ 1992) . as mentioned above‚ humans are exposed to considerable oxidative stress as judged by measurements of up to in urine (long et al.‚ 1999a‚b) . these levels vary with a normal diet. halliwell concluded that urine contains "autooxidizable proteins" which upon exposure to oxygen result in the generation of to generate (long et al.‚ 1999a) . exposing cells to can shift the cell to a prooxidant state‚ which facilitates ros damage to a variety of targets. we have shown this to be true in the benign human breast epithelial cell line mcf-10a (gu et al.‚ 2001) . mcf-10a is a spontaneously immortalized human breast cell line‚ which has characteristics of human breast epithelium such as: lack of tumorigenicity in nude mice and lack of anchorage independent growth (soule et al.‚ 1990) . we treated these cells with 5 separate time after allowing for growth (p5 line) or gradually at increments until reaching (gp line). the peroxide decay and the concomitant production of oh radicals was measured by epr using dmpo as mentioned earlier in this chapter (figure 1 ). after exposure to hydrogen peroxide‚ the cells became resistant to this oxidant and went on to exhibit different degrees of some of them being able to grow in immunodeficient mice (gp line). this transformation towards malignancy can be related to the cell's ability to bypass apoptosis to ensure survival‚ cell cycle anomalies modulated by oxidative stress-induceable genes and the lack of repair of 8-oxog in dna (gu et al.‚ 2001) . with respect to the latter damage‚ the role of hogg1 and hmyh repair enzymes in mammalian tumor progression is to maintain genetic stability by avoiding g . c t . a transversions. this is based on the work on e. coli muty and mutm gene mutants. cells with a single mutation in these genes are moderate mutators; however‚ cells with a double mutation in muty and mutm genes have mutation rates three orders of magnitude higher than the wild-type cells cabrera et al.‚ 1988) . overexpression of these genes will reduce the transversion mutations in tumor promoter genes or tumor suppressor genes and thus reduce the risk of tumorigenesis. thus‚ if indeed the role of 8-oxog in breast cancer tumorigenesis is established‚ one application to the treatment of cancer would be elevating the enzymes that repair such lesion over the endogenous levels present in healthy tissue. with respect to cell responses to we found that induced p53 in mcf-10a but not in the line p5. in addition‚ all transformed lines overexpress bcl-2 when compared to mcf-10a (data not shown). it has been recently shown that the transfection of mcf-10a cells with the bcl-2 gene resulted in a 5 fold increase in bcl-2 protein expression over the parental mcf-10a line (upadhyay et al.‚ 1995) and that apoptosis did not occur in the transfectants at concentrations of in contrast‚ this concentration of induced apoptosis in the parental non-transfected line (upadhyay et al.‚ 1995) . this apoptosis in mcf-10a cells is p53 dependent (upadhyay et al.‚ 1995) . the other contributors to this book have presented excellent approaches and examples to the use of epr for observing radical species in biological systems. however‚ there are many further potential applications that currently cannot be done because of limited radical concentration or stability. this section describes alternate approaches to overcoming some of these obstacles‚ combining the two magnetic modalities that jim hyde has used so productively‚ often in combination with the use of spin traps. spin traps have the potential advantages of being able to accumulate ("trap") highly reactive radicals that could not be otherwise observed directly‚ due to sensitivity limits and kinetic considerations. this is clearly the case for the hydroxyl radical‚ which may be directly observed only by fast freeze quenching at liquid nitrogen temperatures and quickly undergo secondary radical reactions and interconversions. obviously‚ quickly immersing a mouse into liquid nitrogen negates an in vivo experiment! many of the applications‚ successes and pitfalls of in vivo spin trapping are reviewed in this volume by mason (ch. 5) . a major problem that is difficult to overcome is the tendency of the living cell to reduce radicals; consequently‚ the nitroxyl product of a nitrone radical adduct is normally bioreduced to the diamagnetic hydroxylamine. this section discusses potential alternatives utilizing nmr as the ultimate spectroscopic probing method. it also considers the use of more conventional nmr techniques to further characterize the spin traps and their metabolism. many years ago selinsky et al. (1989) suggested the use of nmr to study free radical reactions with fluorinated spin traps. they monitored organic free radical reactions in the test tube by both epr and nmr. in later work outlined below‚ this technique was coined as "nmr spin trapping." the success of this approach depends on the following: 1. do the diamagnetic (decomposition or transformed) products of a spin trap adduct accumulate to sufficient levels for detection by nmr? 2. does the resultant nmr spectrum allow extrapolation back to a specific radical adduct? the definition encompasses not only the spin trap chemistry described below‚ but also "mri spin trapping" described in the last section below‚ in which the weighted nmr images of water protons are measured in the presence of stable‚ paramagnetic‚ biologically generated radical adducts or complexes. the enhanced relaxation of the water protons at the site of the radicals effectively 'localizes' the site of radical generation. tordo and coworkers (frejaville et al.‚ 1995) described a series of nitrone spin traps‚ based initially on depmpo (5-diethoxyphosphoryl-5-methyl-1pyrroline-n-oxide)‚ a phosphorus containing analog of dmpo (2‚2dimethyl-pyrroline-n-oxide). the advantages were that oxygen radical adducts of this trap showed a somewhat longer half life compared with dmpo. despite these improvements‚ the in-vivo "stability" of paramagnetic spin trap adducts is sharply reduced relative to stable nitroxyl radicals‚ probably due to biological reduction of the resulting nitroxide to the hydroxylamine‚ but perhaps also by other destructive chemical processes. it seems prudent to take advantage of the phosphorus moiety of depmpo by utilizing to probe its chemistry during and after radical reactions to understand the pathways of its radical adduct degradation. as described below, a series of biologically relevant depmpo adduct reaction products for and as well as methyl radical and hydroxymethyl radical were characterized by figure 2 and esr spectra of and reaction products. a) spectra obtained from 0.1 m depmpo in 0.1 m cacodylate buffer, 20 mm dtpa, ph 7.0, 10% dmso, detected 2 h after addition of 5 mm and 5 mm at t=300k. inset: corresponding esr spectra observed at 10 min; b) the same reactions but in the presence of 10% methanol instead of dmso. inset: corresponding esr spectra observed at 10 min.. experimental spectra were in a good agreement with simulations using the calculated parameters for and for adducts, which are in agreement with literature data. from khramtsov et al. (1999) with permission. concentration calculated from the integral intensity of the peak at 27.05 ppm; filled circles: the same integral intensities of the peak multiplied by 2.0 to reflect the mechanism in scheme b. the bent curve was calculated proposing bimolecular decay of depmpo-oh from initial concentration equaling the concentration of fenton reagents with the rate constant (namely where t=10 min. was the time at which the epr spectra were obtained). the straight line was inserted as a guide to evaluate how closely the experimental data reflected a 1:1 stoichiometry between hydroxyl radical produced and product formed. note that both the esr data (at concentrations lower than 50 mm) and the adjusted nmr data show a linear concentration dependence on the fenton concentration reagents. from khramtsov et al. (1999) with permission. the reaction of depmpo with radical, as shown in scheme a, emphasizes the fact that two stereoisomers are obtained since the spin trap contains an asymmetric center (yielding, in effect, two pairs of epr spectra). however, the radical signals decay within two hours, yielding the nmr spectra depicted in figure 2 . for example, in figure 2a there is evidence of several new, unique peaks at 24.54 ppm with additional peaks at 30.83 and 32.52 ppm. (a small resonance at 27.05 ppm also occurs since the reaction conditions involve fenton chemistry (khramtsov et al., 1999) . these peaks originate from bimolecular disproportionation chemistry as outlined in scheme b, where both reaction products were diamagnetic: a new nitrone (na) and the hydroxylamine of the spin adducts, and this accounts for the three nmr peaks, one of which is 50% total intensity, two of which are 25% each (the ratio depends slightly on the stereospecificity of the initial radical addition). since the concentrations of spin trap adduct are significantly lower in vivo, disproportionation would not be appreciable; however, direct reduction of the depmpo adduct to the hydroxylamines and is highly facile. hence, when ascorbic acid was added to the radical reaction system, direct conversion to and occurred. consequently the two new lines at 32.31 and 30.83 ppm reflected the direct "history" of spin adduct formation of the methyl radical adduct of depmpo and its subsequent disproportionation or in vivo bioreduction. at high concentrations the bimolecular decay of the adduct spectrum reaction is relatively rapid. the adduct remains for only a few minutes unless balanced by extremely high steady state levels of new radical production timmins et al., 1999) . as shown in figure 3 , the maximum intensity of the new line at 27.05 ppm never exceeded 50% of the theoretical yield of the diamagnetic product from an adduct. scheme c suggests that the nitrone product of disproportionation (depicted in scheme b) is in tautomeric equilibrium with the more stable n-hydroxypyrrolidone, na-oh, to which the nmr line at 27.05 ppm was attributed. in addition, as shown in the scheme, the hydroxylamine can spontaneously eliminate regenerating depmpo. similar results were found with superoxide radical where, in fact, the nmr peaks were identical to those observed after reaction with suggesting a conversion of to (or transformation of their diamagnetic products). in vivo, however, this is unlikely to be important because the local concentrations of spin adduct are unlikely to high. from a sensitivity viewpoint, nitrones that contain trifluoromethyl groups might be more promising than those containing other stable isotopes. this was borne out with new spin trap, fdmpo (4-hydroxy-5,5-dimethyl-2trifluoro methylpyrroline-1-oxide). the parent nitrone yields a single nmr resonance at -66.0 ppm, which yields a net ten-fold higher sensitivity than with nmr of depmpo. figure 4 shows simulated and observed epr spectra for the and adducts, respectively (khramtsov et al., 2001) . the epr signals were stable for over a day since bimolecular decay (as noted in scheme b) is much less probable with the absence of an alpha proton. in order to observe the products by nmr, it was necessary to reduce the paramagnetic adduct(s) with ascorbic acid. however, despite the significantly increased in vitro stability of the paramagnetic adducts, it is likely that bioreduction would occur fairly rapidly. figure 5 depicts results for the adduct of fdmpo in a system containing ascorbate. the inset shows the precursor epr spectrum. spectra of adduct products: (a) in a fenton "iron recycling" system in 0.1 m k-phosphate buffer, ph 7.0, 2 mm dtpa, 20 mm 50 mm fdpmpo, 10% v/v of dmso, 20 mm of ascorbate, 30 min after addition of 0.5 mm feso4 solution; number of scans, 208; inset: kinetics of product accumulation. the symbols and denote integral intensities of the 19p-nmr peaks of reduced products at -78.6 ppm and-80.0 ppm, respectively; (b) 19f-nmr spectra of 3 mm of synthesized in scheme b, in 0.1 m phosphate buffer, ph 7.0, before and after addition of 10 mm ascorbate; number of scans, 120; inset: epr spectrum of 0.1 mm adduct; spectrometer settings were microwave power 20 mw; modulation amplitude, 0.63 g; sweep time, 1 min. from khramtsov et al. (2001) with permission. the adduct gave identical nmr spectra to that with however, overall, the fluorine containing spin traps are less likely to undergo destructive metabolism assuming toxicity levels will not be a major problem. overall the reactivity of fdmpo was comparable to that of dmpo as measured by competitive kinetics. consequently, the trifluoromethyl nmr spin traps have greater potential than traps containing other stable isotopes. the remaining biological isotope, is at ca 1% natural abundance. in order to distinguish a particular molecule of interest, one must synthesize it enhanced with in a novel application by bose-bsau et al. (2001) . (mnp) was employed, in the presence of as an nmr spin trap for tyrosyl radical in met-myoglobin. relatively stable paramagnetic adducts were detectable by epr; however, the direct identification and characterization of the final adduct on the protein and the specific chemistry involved was possible only after reduction of the paramagnetic adduct by ascorbate and the use of 2d high resolution nmr to assign the relevant tyr residue and the chemistry of the modification. previous spin trapping studies on myoglobin had shown the presence of tyrosyl radicals that could be trapped with mnp (barr et al., 1996) , but the epr results were ambiguous as to which residue was preferentially labeled. these recent results were definitive in identifying tyr 103 in equine myoglobin and that the adduct formation occurs at the c-3 carbon of the amino acid. figure 6 depicts the reaction scheme that most likely occurs. in addition, the studies identified the nearby protons of phe 106. the direct localization and imaging of free radical distributions is much more challenging with epr due both to aspects of stability discussed above as well as linewidth limitations. despite almost three orders of magnitude higher sensitivity (eg, a free electron vs a proton) the much broader linewidths usually encountered in epr places limitations on both resolution and overall sensitivity. there are additionally other limits to distinguishing tissue boundaries with radical distributions; these are simply more difficult to visualize by epr. on the other hand, mri, which is a mature, commercialized technology, offers exquisite anatomical resolution based on the protons of water (i.e. 100 m). several physical aspects, such as nuclear relaxation times, diffusion rates, oxygen magnetic susceptibility effects, etc., allow multifaceted approaches to epr and mri image resolution. on the other hand, paramagnetic compounds serve as excellent contrast agents in mri by inducing proton (nuclear) relaxation enhancement from rapidly exchanging water molecules. consequently, mri spin trapping potentially allows visualization of free radical biology at the site of its generation, by using the spin adducts as "contrast agents". no may be 'spin trapped' with dithiocarbamate compounds and fe(ii), which form a stoichiometric 2:1 complex for axial coordination of no. the water soluble trap, mgd, forms the no complex where a diagnostic epr spectrum is found with septic shock mice (which has been almost the sole example with sufficient sensitivity in vivo). recently kaneko et al. (2002) demonstrated in vivo observations of no in the brain of epileptic mice models. the relaxivity of this complex is relatively frequency independent for both and at 20 and 85 mhz, respectively. uncomplexed mgd, fe(ii) or show essentially negligible relaxivity while the no complex yields appreciable relaxation enhancement (fujii et al., 1999) . consequently, in mri measurements, only the no complex will yield image contrast enhancement, frequently at the site of no generation. as shown in figure 7 , 1.5t mri images of septic-shock rats showed increased enhancement in the liver with time. this was suppressed by injecting the animals with a specific inos inhibitor, l-nmma. consequently, both the localization and "biochemistry" of no generation can be visualized at high-resolution in vivo. figure 7 . transverse mr images of the liver in lps-doped rats. mr images were measured at the times indicated for two different slices of the liver. six hours after lps injection, the no spin-trap (3 ml of mgd: 100 mm, fe: 20 mm) was administered i.p. the slice thickness was 2 mm, and each slice was separated by 1 mm. the image enhancement was drastically reduced upon treatment with the nos inhibitor, l-nmma. adapted from fujii et al. (1999) with permission. most of the new approaches outlined here are still in their infancy. the future, however, looks promising feasibility studies have been quite successful. most of the stable isotopes incorporated in the spin traps described above are easily resolvable in the presence of other biological milieu. sensitivity is particularly promising with nmr, a technique that has never been exploited to its full capacity in vivo. of particular note is the use of paramagnetic relaxation enhanced mri. the power of paramagnetic contrast agents to mri has been proven time and again. several sophisticated pulse sequences exist which take advantage of small susceptibility/relaxation effects. in addition, the development of new, targeted paramagnetic spin traps and spin probes promise to provide the requisite localization. the ability to observe biological radical production at the site of generation offers the ultimate advantage in diagnosis. there clearly are many areas in which free radicals are intrinsically involved in both physiology and pathophysiology. there also are many situations where the involvement of free radicals is minimal or not significant. epr spectroscopy, combined with thoughtful experimental approaches can be a powerful method for resolving many of the questions that arise regarding the role of free radicals in disease. the national biomedical esr center at mcw has had, and will continue to have an important role in providing critical epr spectroscopy tools and original experimental results that will provide an understanding of the role of free radicals in disease. there appear to be sufficient important problems to make this a productive field for many years. cloning and characterization of mammalian 8-hydroxyguaninespecific dna glycosylase/apurinic, apyrimidinic lyase, a functional mutm homologue oxidants are a major contributor to cancer and aging oxidants, antioxidants, and the degenerative diseases of aging separation of oxidant-initiated and redox-regulated steps in the signal transduction pathway cloning of a human homolog of the yeast ogg1 gene that is involved in the repair of oxidative dna damage bcl-2 is involved in preventing oxidant-induced cell death and in decreasing oxygen radical production oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease escherichia coli muty gene encodes an adenine glycosylase active on g/a mispairs the histochemistry of lipofuscin and neuromelanin esr spin trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide apoptosis and its role in human disease the role of oxidative stress in the pathogenesis of age-related macular degeneration phospholipid peroxidation in tumor promoter-exposed mouse skin dna strand breaks produced by oxidative stress in mammalian cells exhibit 3'-phosphoglycolate termini formamidopyrimidine-dna glycosylase of escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors the pigmentary system. physiology and patophysiology ocular pathology in mice with a transgenic insertion at the microphthalmia locus protein nmr spin trapping with application to the tyrosyl radical of equine myoglobin free rad acute pulmonary injury in rats by nitrofurantoin and modification by vitamin e, dietary fat, and oxygen reactions of oxyl radicals with dna mutation in the dna mismatch repair gene homologue hmlhi is associated with hereditary nonpolyposis colon cancer amyotrophic lateral sclerosis: recent insights from genetics and transgenic mice sod1 aggregates in als: cause, correlate, or consequence superoxide and hydrogen peroxide in relation to mammalian cell proliferation redox regulation of programmed cell death in lymphocytes oxidative stress as a mediator of apoptosis elevated basal reactive oxygen species and phospho-akt in murine keratinocytes resistant to ultraviolet b-induced apoptosis ) mutm, a second mutator locus in escherichia coli that generates transversions oxidative damage and protection of the rpe dopamine neurotoxicity: agedependent behavioral and histological effects the respiratory burst oxidase 8-hydroxyguanine, an abundant form of oxidative dna damage causes g t and a c substitutions monochromosome transfer provides functional evidence for growth-suppressive genes on chromosome 14 in nasopharyngeal carcinoma transformation of mammalian cells by overexpressing peroxisomal fatty acyl-coa oxidase growth and pigmentation in genetically related s91 cloudman melanoma cell lines treated with ibmx and msh monoamine oxidase and oxidative stress at dopaminergic synapses the pathobiology of parkinson's disease: biochemical aspects of dopamine neuron senescence apoptosis: molecular control point in toxicity oxidant stress induces the protooncogenes c-fos and c-myc in mouse epidermal cells sunlight and age-related macular degeneration. the beaver dam eye study fos: an immediate-early transcription factor in neurons the bcl-2 family of proteins, and the regulation of neuronal survival principles and practice of infectious diseases reduction of paraquat and related bipyridylium compounds to free radical metabolites by rat hepatocytes basal lipid peroxidation in substantia nigra is increased in parkinson's disease chemistry of free radical damage to dna and nucleoproteins reactive oxygen species, chromosome mutation, and cancer: possible role of clastogenic factors in carcinogenesis. free rad a standardized test for the identification and characterization of melanins using electron paramagnetic resonance (epr) spectroscopy purified human neuromelanin, synthetic dopamine melanin as a potential model pigment, and the normal human substantia nigra: characterization by electron paramagnetic resonance spectroscopy the roles of neuromelanin, binding of metal ions, and oxidative cytotoxicity in the pathogenesis of parkinson's disease: a hypothesis is the incidence of registrable age-related macular degeneration increasing? brit reverse chemical mutagenesis: identification of the mutagenic lesions resulting from reactive oxygen species-mediated damage to dna spin trapping of superoxide and hydroxyl radical: practical aspects oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase a genes: similarities with the erythropoietin 3' enhancer oxidative stress-induced apoptosis prevented by trolox. free rad dna repair dna repair and mutagenesis (diethoxyphosphoryl)-5-methyl-1-pyrroline -oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals in vivo epr studies of the metabolic fate of nitrosobenzene in the mouse in vivo imaging of spin-trapped nitric oxide in rats with septic shock: mri spin trapping in vivo evidence of free radical formation after asbestos instillation: an esr spin trapping investigation. free rad the chemistry of single-stranded 4'-dna radicals: influence of the radical precursor on anaerobic and aerobic strand cleavage cell damage in inflammatory and infectious sites might involve a coordinated "cross-talk" among oxidants, microbial haemolysins and ampiphiles, cationic proteins, phospholipases, fatty acids, proteinases and cytokines mossbauer spectroscopic studies of purified human neuromelanin isolated from the substantia nigra detection of nitrosyl hemoglobin in venous blood in the treatment of sickle cell anemia with hydroxyurea tobacco smoke tumor promoters, catechol and hydroquinone, induce oxidative regulation of protein kinase c and influence invasion and metastasis of lung carcinoma cells bicarbonate enhances the peroxidase activity of cu, zn-superoxide dismutase oxidative stress and apoptosis in hiv infection: a role for plant-derived metabolites with synergistic antioxidant activity catecholamine toxicity: a proposal for the molecular pathogenesis of manganese neurotoxicity and parkinson's disease mutant cu, zn,-superoxide dismutase in motor neuron disease alteration of dna base excision repair enzymes hmyh and hogg1 in hydrogen peroxide resistant transformed human breast cells tyrosinase and inhibition of proliferation of melanoma cells and fibroblasts oxidative dna damage: meaning and measurement dna damage by oxygen-derived species. its mechanism and measurement in mammalian systems free radicals in biology and medicine extensive oxidative dna damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma the nature of retinal radiation damage: dependence on wavelength, power level and exposure time the presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation spontaneous oxygen radical generation by sickle erythrocytes comparative action spectrum for ultraviolet light killing of mouse melanocytes from different genetic coat color backgrounds bcl-2, a novel regulator of cell death the interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme mutations in human cancers paraquat uptake into freshly isolated rabbit lung epithelial cells and its reduction to the paraquat radical under anaerobic conditions redox-active chalcogen-containing glutathione peroxidase mimetics and antioxidants inhibit tumor promoter-induced downregulation of gap junctional intercellular communication between wb-f344 liver epithelial cells iron nitrosyl hemoglobin formation from the reactions of hemoglobin and hydroxyurea oxy-radical induced mutagenesis of hotspot codons 248 and 249 of the human p53 gene signal integration at the c-fos promoter inflammatory breass cancer: a review (1992) iron-melanin complex in substantia nigra of parkinsonian brains:an x-ray microanalysis oxidative stress as a cause of parkinson's disease microsatellite instability and mutations in dna mismatch repair genes in sporadic colorectal cancer in vivo evidence of free radical formation in the rat lung after exposure to an emission source air pollution particle consequences of nitric oxide generation in epileptic-seizure rodent models as studied by in vivo epr is the vulnerability of neurons in the substantia nigra of patients with parkinson's disease related to their neuromelanin content? electron spin resonance imaging of mouse b16 melanoma application of the epr spin trapping technique to the detection of radicals produced in vivo during inhalation exposure of rats to ozone measurements of in vivo parameters pertinent to ros/rns using epr spectroscopy reactive oxygen species as cellular messengers spin traps: in vitro toxicity and stability of readical adducts. free rad nmr spin trapping: detection of free radical adducts using a phosphorus containing nitrone spin trap nmr spin trapping: detection of free radical reactions using a new fluorinated dmpo analog, free rad role of free radicals in the pathogenesis of acute chest syndrome in sickle cell disease prevalence of age-related maculopathy. the beaver dam eye study hypoxia causes the activation of nuclear factor through the phosphorylation of on tyrosine residues hypoxia activation of nuclear factor is mediated by a ras and raf signaling pathway and does not involve map kinase (erk1 or erk2) antioxidant action of neuromelanin: the inhibitory effect on lipid peroxidation genetic recombination the ord1 gene encodes a transcription factor involved in oxygen regulation and is identical to ixr1, a gene that confers cisplatin sensitivity to saccharomyces cerevisiae interaction between chemicals and melanin macrophages and inflammatory mediators in tissue injury expression of the e. coli fpg gene in mammalian cells reduces the mutagenicity of xrays transcription coupled repair of 8-oxoguanine: requirement for xpg, tfiih, and csb and implications for cockayne syndrom on the role of bicarbonate in peroxidations catalyzed by cu, zn, superoxide dismutase. free rad inhibitory effect of curcumin on xanthine dehydrogenase/oxidase induced by phorbol-12-myristate-13-acetate in nih3t3 cells increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant sodl-mediated motor neuron cell death and increased amyotrophic lateral sclerosis-like transgenic mouse survival evaluation of depmpo as a spin trapping agent in biological systems. free rad gd(3+) and yb(3+) induced changes in mitochondrial structure, membrane permeability, cytochrome c release and intracellular ros level mutator phenotype may be required for multistage carcinogenesis microsatellite instability: marker of a mutator phenotype in cancer hydrogen peroxide in human urine: implications for antioxidant defense and redox regulation generation of hydrogen peroxide by "antioxidant" beverages and the effect of milk addition. is cocoa the best beverage? free radio electron spin resonance studies on living cells. iii. development of single individuals in triturus alpestris three chemically distinct types of oxidants formed by iron-mediated fenton reactions in the presence of dna a mammalian dna repair enzyme that excises oxidatively damaged oxoguanine maps to a locus frequently lost in lung cancer in vivo detection of anthralin-derived free radicals in the skin of hairless mice by low-frequency electron paramagnetic resonance spectroscopy mutt protein specifically hydrolyzes a potent mutagenic substrate for dna synthesis identification of hydroxyl radical-induced lesions in dna as structure biomarkers with a putative link to cancer development major alterations in the nucleotide structure of dna in cancer of the female breast the etiology of breast cancer: characteristic alterations in hydroxyl radical-induced dna bas lesions during oncogenesis with potential for evaluating incidence risk cancer-related changes in prostate dna as men age early identification of metastasis in primary prostate tumors the etiology and prediction of breast cancer the development of the human eye modulation of oxidant formation in mouse skin in vivo by tumor-promoting phorbol esters pheomelanin as a binding site for drugs and chemicals phosphorylated nitroxides in the pyrrolidine series: reduction by ascorbate. free radic free radical metabolites of foreign compounds and their texicological significance free radical intermediates in the metabolism of toxic chemicals the mechanism of microsomal and mitochondrial nitroreductase. electron spin resonance evidence for nitroaromatic free radical intermediates the role of catalytic superoxide formation in the inhibition of nitroreductase light and risk for age-related eye diseases characterization of a mammalian homolog of the escherichia coli hydrogen peroxide and dna damage mutm, a protein that prevents g:c to t:a transversions, is formamidopyrimidine-dna glycosylase muty, an adenine glycosylase active on g:a mispairs, has homology to endonuclease iii single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in dna induces targeted g-c transversions in simian kidney cells site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in e. coli absence of ferritin protein in substantia nigra pars compacta neurons. a reappraisal to the role of iron in parkinson disease pathogenesis direct detection of free radical generation in an in vivo model of acute lung injury induction of reactive oxygen intermediates in human monocytes by tumor cells and their role in spontaneous monocyte cytotoxicity cloning of yeast 8-oxoguanine dna glycosylase reveals the existence of a base-excision dna-repair protein superfamily antioxidant status and free radicalinduced oxidative damage of sickle erythrocytes the muty gene: a mutator locus in escherichia coli that generates g:c to t:a transversions vertebrate craniofacial development: the relation between ontogenic process and morphological outcome transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line the clonal evolution of tumor cell populations dna base modifications in chromatin of human cancerous tissues neuromelanin of the human substantia nigra: a mixed-type melanin genetic-, age-and light-induced degeneration of the retina and retinal pigment epithelium the biogenesis of melanosomes dose-response effects of malachite green on free radical formation, lipid peroxidation and dna damage in syrian hamster embryo cells and their modulation by antioxidants mutation of a mutl homolog in hereditary colon cancer age-related macular degeneration. 1. epidemiology, pathogenesis and differential diagnosis chapter 28. the enzymology of melanogenesis 6-dihydroxyindole is a melanin precursor showing potent cytotoxicity effect of selenium and vitamin e deficiency on nitrofurantoin toxicity in the chick studies on skin aging. preparation and properties of fucose-rich oligo-and polysaccharides. effect on fibroblast proliferation and survival ) c-fos proto-oncogene regulation and function nitrone spin traps and their pyrrolidine analogs in myocardial reperfusion injury: hemodynamic and esr implications quantitative determination of melanin in pigmented cells by electron spin resonance spectroscopy transplantable melanomas in gerbils (meriones unguiculatus). ii -melanogenesis motor neuron disease and animal models biological effects of cigarette smoke, wood smoke, and the smoke from plastics: the use of electron spin resonance. free rad widespread bimodal intrachromosomal genomic instability in sporadic breast cancers associated with 13q allelic imbalance cloning and characterization of hogg1, a human homolog of the ogg1 gene of saccharomyces cerevisiae enzymes that repair oxidative damage to dna generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate generation of nitro radical anions of some 5-nitrofurans, and 2-and 5-nitroimidazoles by rat hepatocytes chapter 32. retinal phototoxicity the retinal pigmented epithelium is required for development and maintenance of the mouse neural retina cell-free hemoglobin limits nitric oxide bioavailability in sicklecell disease low levels of reactive oxygen species as modulators of cell function how many mutations are required for tumorgenesis? implications from human cancer data hydroxyl radical formation by sickle erythrocyte membranes: role of pathologic iron deposits and cytoplasmic reducing agents transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains molecular cloning and functional expression of a human cdna encoding the antimutator enzyme 8-hydroxyguanine-dna glycosylase tissue uptake of paraquat and diquat cloning and characterization of a mammalian 8-oxoguanine dna glycosylase quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphoshporyl)-5-methyl-1-pyrroline-n-oxide photoreactivity of aged human rpe melanosomes: a comparison with lipofuscin or7 -photooxidation of retinal pigment melanin -a model for melanin aging free radical scavenging properties of melanin interaction of eu-and pheo-melanin models with reducing and oxidising radicals. free rad inhibition of activation-induced death in t cell hybridoma by thiol antioxidants: oxidative stress as a mediator of apoptosis properties and function of the ocular melanin--a photobiophysical view interactions of melanin with oxygen (and related species) loss of melanin in human rpe with aging photobiology of the eye retinal melanin and lipofuscin: possible role in photoprotection and phototoxicity of human rpe in vivo lipid-derived free radical formation by nadph oxidase in acute lung injury induced by lipopolysaccharide: a model for ards generation of superoxide anion and hydrogen peroxide by erythrocytes from individuals with sickle trait or normal hemoglobin reactive oxygen intermediates as apparently widely used messengers in the activation of the transcription factor and hiv-1 development of fluorinated, nmr-active spin traps for studies of free radical chemistry comparative oxidation of hemoglobins a and s insertion of specific bases during dna synthesis past the oxidation-damaged base 8-oxodg assays of oxidative dna damage biomarkers 8-oxo-2'-deoxyguanosine and 8-oxoguanine in nuclear dna and biological fluids by high-performance liquid chromatography with electrochemical detection methods binding of iron to neuromelanin of human substantia nigra and synthetic melanin: an epr spectroscopy study. free rad reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated cu,zn superoxide dismutase mutants and h2o2 cloning and sequencing a humal homolog (hmyh) of the escherichia coli muty gene whose function is required for the repair of oxidative dna damage reduced and oxidized glutathione in the substantia nigra of patients with parkinson's disease isolation and characterization of a spontaneously immortalized human breast epithelial cell line, mcf-10 human tumor suppressor genes purine bases, nucleosides, and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical cations and and oh adducts free radicals in cancer use of electron spin resonance spectroscopy to study cancer potential medical (clinical) applications of epr: overviews and perspectives in in vivo epr(esr): theory and applications modulation by neuromelanin of the availability and reactivity of metal ions production of large amounts of hydrogen peroxide by human tumor cells visual disabilities of oculocutaneous albinism and their alleviation the long-term effects of visible light on the eye trapping of free radicals with direct in vivo epr detection: a comparison of 5,5-dimethyl-1-pyrroline-n-oxide (dmpo) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (depmpo) as spin traps for and free rad nucleotide sequence of the escherichia coli mica gene required for a/g-specific mismatch repair: identity of mica and muty free radical dna adduct 8-oh-deoxyguanosine affects activity of hpa ii and msp 1 restriction endonucleases ) bcl-2 suppresses expression of in breast epithelial cells cloning and expression of escherichia coli of the ogg1 gene of saccharomyces cerevisial, which codes for a dna glycosidose that excises 7,8-dihidro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-n-methylformamido pyrimidine spectroscopic studies of substantia nigra pigment in human subjects heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts oncogenes, antioncogenes, and the molecular basis of multistep carcinogenesis relationship of senile macular degeneration to ocular pigmentation inflamation and cancer: role of phagocyte-generated oxidants in carcinogenesis free radical adducts induce alterations in dna cytosine methylation altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis albino spatial vision as an instance of arrested visual development what's the use of generating melanin? an intron splice acceptor polymorphism in hmsh2 and risk of leukemia after treatment with chemotherapeutic agents analysis of c-fos and c-jun redox-dependent dna binding activity the redox and dna-repair activities of ref-1 are encoded by nonoverlapping domains measurement of free radicals from smoke inhalation and oxygen exposure by spin trapping and esr spectroscopy activation of ap-1 and of a nuclear redox factor, ref-1, in the response of ht29 colon cancer cells to hypoxia two nicking enzyme systems specific for mismatch-containing dna in nuclear extracts from human cells solar radiation and age-related macular degeneration age-related macular degeneration: review of pathogenesis the effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems total and paramagnetic metal ions in human substantia nigra and its neuromelanin the neuromelanin of human substantia nigra and its interaction with metals bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase bicarbonate enhances peroxidase activity of cu, zn-superoxide dismutase-role of carbonate anion radical and scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme mimetics key: cord-288119-3zq8l5z0 authors: dijkman, ronald; van der hoek, lia title: human coronaviruses 229e and nl63: close yet still so far date: 2009-04-30 journal: journal of the formosan medical association doi: 10.1016/s0929-6646(09)60066-8 sha: doc_id: 288119 cord_uid: 3zq8l5z0 hcov-nl63 and hcov-229e are two of the four human coronaviruses that circulate worldwide. these two viruses are unique in their relationship towards each other. phylogenetically, the viruses are more closely related to each other than to any other human coronavirus, yet they only share 65% sequence identity. moreover, the viruses use different receptors to enter their target cell. hcov-nl63 is associated with croup in children, whereas all signs suggest that the virus probably causes the common cold in healthy adults. hcov-229e is a proven common cold virus in healthy adults, so it is probable that both viruses induce comparable symptoms in adults, even though their mode of infection differs. here, we present an overview of the current knowledge on both human coronaviruses, focusing on similarities and differences. located between the orf1b and s gene. expression of the non-structural replicase proteins is mediated by translation of the genomic rna that gives rise to the biosynthesis of two large polyproteins, pp1a (encoded by orf1a) and pp1ab (encoded by orf1a and orf1b), which is facilitated by a ribosomal frameshift at the orf1a/1b junction. in contrast, the structural proteins are translated from subgenomic mrnas. these subgenomic mrnas are the result of discontinuous transcription, a hallmark of cov gene expression. the structural gene region also harbors several orfs that are interspersed along the structural protein coding genes. the number and location of these accessory orfs vary between the cov species. in animals, cov infections can lead to a variety of syndromes, e.g. bronchitis, gastroenteritis, progressive demyelinating encephalitis, diarrhea, peritonitis and respiratory tract disease. 1 the first reports on human covs (hcov) appeared in the mid-1960s. the human viruses were isolated from persons with the common cold, and two species were detected: hcov-229e and hcov-oc43. 2, 3 almost 40 years later, a cov was identified as the causative agent of the severe acute respiratory syndrome (sars). 4,5 a highly effective global public health response prevented further spread of this virus, and as a result, sars-cov was eradicated from the human population. soon thereafter, it became clear that there are more hcovs. hcov-nl63 was identified in 2004 and hcov-hku1 in 2005. 6, 7 both viruses are not emerging viruses like sars-cov but were previously unidentified. in fact, infections caused by these viruses are as common and widespread as hcov-229e and hcov-oc43 infections. 8 the sars outbreak intensified research on the unknown animal covs. as many as 16 new animal cov species have been identified in the last 3 years. [9] [10] [11] [12] [13] [14] [15] [16] there are currently 29 complete reference genome sequences available in genbank of the various viruses, and three phylogenetically distinct groups exist ( figure 2 ). 17, 18 hcov-229e and hcov-nl63 belong to the group 1 covs, together with various covs isolated from pigs, cats and bats. as shown in figure 2 , hcov-229e and hcov-nl63 are the only two human viruses that have a relatively close relationship. hcov-oc43 is a group 2 virus and clusters tightly with bovine, porcine and equine covs. hcov-hku1 is not part of that cluster, although the virus clearly belongs to the group 2 covs. sars-cov is of animal origin, with civet cat sars-cov and bat sars-cov as very close relatives. 19 discovery of group 1 covs the first described cov of group 1 was porcine transmissible gastroenteritis virus (tgev), which was isolated in 1946 from pigs suffering from gastroenteritis. 20 almost two decades later, one research group located in the uk identified a human respiratory tract pathogen from nasal washings of persons with the common cold. 2 this novel pathogen, hcov-229e, was later characterized morphologically by electron microscopy and compared with the already-well-known avian infectious bronchitis virus. 21 the viruses exhibited a typical crown-like appearance (from latin corona). during the following years, another group 1 member, canine coronavirus, was isolated from sentry dogs with diarrhea and mild gastroenteritis. 22 similar clinical symptoms were later observed in pigs during a diarrheal outbreak in 1978 on four separate swine breeding farms. 23 the recovered pathogen, now known as porcine epidemic diarrhea virus (pedv), was first mistyped as a member of the rotavirus family, yet it soon became clear that the virus shared the morphological characteristics of cov but was serologically distinct from tgev. 23 two cat-associated cov species were identified in 1981. feline enteric coronavirus and feline infectious peritonitis virus (fipv) shared serological characteristics, but differed in clinical outcome. 24 in 1986, another porcine cov was isolated, porcine respiratory coronavirus, a close relative of tgev. 25 hereafter, no new group 1 members were discovered for more than 15 years. in 2004, we isolated hcov-nl63 from a 7-month-old child with bronchiolitis. 6 shortly thereafter, fouchier et al independently described the same virus from a clinical sample collected in 1988. 26 in 2005, it became clear that several bat species can harbor covs that belong to group 1. 10, 14, 16 most of these viruses cluster with hcov-229e, hcov-nl63 and pedv, although none of them is a very close relative to any of these viruses. there are notable differences in the genome composition that divides the group 1 viruses into two separate branches, named 1a and 1b ( figure 1 ). all group 1a members contain several short accessory protein-coding genes between the s and e genes and one or two accessory protein genes on the 3' side of the n gene. in contrast, all group 1b members carry only one accessory protein gene, between the s and e genes, with the exception of some bat covs. three bat covs carry an additional orf at the 3'-side of the n gene. the function of the accessory proteins from the group 1 covs is unknown. reverse genetic analyses of fipv and extensive cell culture adaptation of pedv, tgev and hcov-229e suggest that they are not required for in vitro virus replication. [27] [28] [29] [30] [31] moreover, deletion of fipv, pedv and tgev accessory genes results in attenuation of the virus, which indicates that the group 1 accessory proteins represent pathogenicity factors. [27] [28] [29] [30] the discovery timeline of the group 1 covs illustrates that this group has grown only recently into a more mature form in which its members can infect a diversity of mammalian hosts. it is not unlikely that additional members will be identified in the near future. hcov-229e was the first hcov to be fully sequenced; 32 however, it is striking that the sequence information of circulating strains is very poor. only one study has described the variability of the s and n genes over time, which suggests that genetic drift shapes hcov-229e evolution. 33 fortunately, the sequence information allows calculation of the evolution rate of the virus. with this evolutionary rate, the time to the most common recent ancestor of hcov-nl63 and hcov-229e could be calculated. 34 as many as 1000 years ago, the viruses evolved from a common ancestor. 34 for hcov-nl63, many more sequences of circulating strains are now available. four full genomes have been sequenced, and 312 sequences of other regions are available in genbank (compared to 123 for hcov-229e). the full-length hcov-nl63 sequences have shown that two types of viruses exist, but recombination between hcov-nl63 strains occurs frequently. 34 unfortunately, it is unknown whether different types of hcov-229e strains exist and recombination occurs, since only the first full-length sequence of the laboratory-adapted strain vr-740 is available thus far. 32 full-length sequences of clinical isolates are urgently needed to address this question. the limitation of having just one laboratory-adapted strain sequence is exemplified by our analysis of the orf4 region of hcov-229e. 31 the laboratory-adapted vr-740 strain contains orf4a and orf4b genes, and it was assumed that clinical isolates would follow the same gene order. we have sequenced the region from several clinical samples and revealed that hcov-229e in patients always contains an intact orf4 gene that encodes one putative orf4 accessory protein, whereas laboratory-adapted strains are very prone to mutations in this region. cell tropism of hcov-229e and hcov-nl63 the s glycoproteins of hcov-229e and hcov-nl63 are both class i fusion proteins that mediate infection of target cells. 35, 36 the proteins share 56% amino acid identity, but do not use the same receptor. 37 the receptor-recognition regions within s are, for both viruses, not well-defined linear binding sites. 38 for hcov-nl63, the region between amino acids 476 to 616 is important for binding, whereas for hcov-229e, amino acids 417 to 547 are involved in receptor recognition. 39, 40 hcov-229e utilizes cd13 (also known as aminopeptidase n) as a receptor, whereas hcov-nl63 uses angiotensin-converting enzyme 2 (ace2) for cellular entry. [41] [42] [43] cd13 is a zinc-binding metalloprotease that is ubiquitously expressed in various cell types, including small intestinal and renal tubular epithelial cells, the granulocytic and monocytic lineage, synaptic membranes from the central nervous system, and respiratory epithelial cells. [44] [45] [46] cd13 functions in digestion, angiogenesis and synaptic activity, and cleaves peptides bound to major histocompatibility complex molecules of antigenpresenting cells. 46 ace2 belongs to the same protease family as cd13, and the protein is expressed in testicular, renal, cardiovascular, gastrointestinal and airway tissue. 47 both metalloproteases are involved in the renin-angiotensin system, which regulates blood pressure. ace2 plays a role in vasodilatation by c-terminal cleavage of angiotensin ii into angiotensin 1-7, and angiotensin i into angiotensin 1-9, whereas cd13 functions at another level by n-terminal cleavage of angiotensin iii into angiotensin i, and angiotensin iv into angiotensin 4-8. 48 besides hcov-229e, cd13 is used by pedv, tgev and fipv to enter the cell, [49] [50] [51] [52] whereas hcov-nl63 is the sole group 1 virus that uses ace2. only sars-cov uses the same protein for entry. 53 it has been suggested that sars-cov pathogenicity is related to the downregulation of ace2 upon infection. 54 ace2 protects against lung damage and the lack of ace2 on the cell surface may account for the damage during infection. 54 whether hcov-nl63 induces a similar downregulation during infection is unknown. hcov-229e can be cultured on various types of cells derived from the human nervous system, cells of granulocytic and monocytic lineage, airway tract cells and hepatocytes. 44, 45, 55, 56 hcov-nl63 in vitro replication can be achieved by culturing upon monkey-kidney-derived cell lines, tertiary monkey kidney cells and hepatocytes. 6, 26, 43, 57 on pseudostratified human primary lung epithelial cell cultures, cd13 and ace2 proteins are expressed on the apical surface. 45, 58 the release of newly produced hcov-229e viral particles exhibits the same polarization as the receptor, and therefore, apical release, whereas for hcov-nl63, this is still unknown. 45 unfortunately, to date, no permissive animal models have been reported that can be utilized as in vivo models to further characterize hcov-229e-or hcov-nl63-induced pathogenicity. [59] [60] [61] prevalence of hcov-nl63 and hcov-229e an accumulating number of reports has revealed that hcov-229e and hcov-nl63 infections occur without gender, age or geographic boundaries. 8, [62] [63] [64] [65] all children encounter their first hcov-229e and hcov-nl63 infection during early childhood. 43, 66, 67 in most children, these infections do not lead to severe clinical symptoms, but for some, the severity of the upper or lower respiratory tract infections can require hospitalization. hcov-nl63 and hcov-229e infections can account for 5% of all acute respiratory infections in the hospital, especially during the winter. 68, 69 very often, these severe infections are accompanied by a second respiratory virus infection. 70 at a later age, reinfection with the viruses occurs, but only in frail persons does the infection require hospital admission. 6, 64, 65 studies with hcov-229e infection of volunteers have shown that reinfection with common cold symptoms occurs when the level of antibodies directed against the virus is low. 71 the decrease in titers of hcov-229e antibodies is observed as soon as 1 year after infection, which indicates that every individual probably encounters numerous infections by hcov-229e during a lifetime. whether reinfection of hcov-nl63 in healthy adults occurs is still unknown. until 1989, clinical infection trials with hcov-229e in healthy volunteers were performed by researchers at the medical research council (mrc) in salisbury, uk. hcov-229e was administered nasally to volunteers. 72 among the infected volunteers, 50% developed the common cold. the observed symptoms included malaise, headache, nasal discharge, chills, cough and sore throat. one fifth of the volunteers developed fever. the incubation period ranged from 2 to 5 days, with a mean of just over 3 days. the duration of symptoms that were induced by hcov-229e varied between 2 and 18 days, with a mean of 7 days. during the trials, researchers also noticed the high daily amount of disposable handkerchiefs used. from this, it was concluded that nasal discharge is one of the main symptoms of hcov-229e infection. the number of handkerchiefs used ranged from 8 to 120, with a mean of 23 per day, a high number compared to other common cold viruses, such as rhinoviruses. in addition, the mean incubation period of hcov-229e was significant longer than that of rhinoviruses, whereas the duration of the illness was somewhat shorter. similar symptoms were observed with nine different hcov-229e strains, thus, no indications that various strains of hcov-229e induce different symptoms. 73 the most frequently observed clinical manifestations in hcov-nl63-infected patients are fever, cough, coryza, sore throat, bronchiolitis, bronchitis, pneumonia and croup. 8 as mentioned above, hcov-nl63 infections in the hospital are frequently accompanied by infection with other respiratory viruses. therefore, association of hcov-nl63 with a certain disease remains difficult to establish. we investigated a large group of 949 children with lower respiratory tract infections and found that, among those infected with hcov-nl63, a large percentage had croup (24%). 70 focusing only on single hcov-nl63 infections revealed a very strong association (43%, p < 0.0001). a second study confirmed this finding. five hundred and thirty-nine taiwanese children were tested and hcov-nl63 was the most common pathogen (14.7%) in children who had croup. 74 also, two korean studies observed the association of hcov-nl63 with croup. 62, 75 one study found three (50%) cases of croup among hcov-nl63-infected children, and the other found 64.2% of croup among 14 children with hcov-nl63 infection. we hypothesize that hcov-nl63 is responsible for croup, since in most studies, no other pathogen has been detected. still, it cannot be ruled out that laryngotracheitis facilitates hcov-nl63 replication, but the virus is not involved in causing the disease. whether hcov-229e is involved in croup is unknown. hcov-229e testing of the above-mentioned 949 children (tested previously for hcov-nl63) will shed more light on this matter. therefore, it is of interest to determine the prevalence of hcov-229e infection among children with croup. there has been one study that has linked hcov-nl63 infection to kawasaki disease, 76 one of the most common forms of childhood vasculitis. 77 however, no subsequent study has been able to confirm this association. [78] [79] [80] [81] [82] hcov-229e has been suggested as the causative agent of multiple sclerosis. [83] [84] [85] [86] some research groups have found a higher frequency of hcov-229e in the brains of patients with multiple sclerosis compared to a control group. however, the high frequency might have been influenced by the increased susceptibility of these patients, as a result of damage to the blood-brain barrier. common cold virus infections have a large impact on the economy because of the reduced productivity of the working population. therefore, effective viral treatment against the common cold may limit this economic impact. additionally, effective treatment can modulate severe respiratory disease among children or elderly and immunocompromised patients. currently, there are no treatments available for any of the hcovs, including hcov-nl63 and hcov-229e. however, some candidate drugs have been investigated and might provide options for treatment in the future. the viral replication cycle of hcov-229e and hcov-nl63 can be tackled theoretically by synthetic or natural antiviral compounds at various stages, including receptor binding, membrane fusion, transcription, rna biosynthesis and posttranslational processing. for hcov-nl63 and hcov-229e, there are no inhibitory neutralizing monoclonal antibodies available. however, hcov-nl63 replication can be inhibited in vitro by pooled intravenous immunoglobulins from healthy adult donors, which probably contain neutralizing antibodies. 87 whether this also relates to hcov-229e remains to be investigated, although it is not unlikely since many healthy adults carry antibodies directed against hcov-229e. 88 treatment with intravenous immunoglobulins is beneficial in numerous (auto)immune diseases, such as multiple sclerosis, but also severe respiratory diseases and kawasaki disease. 89 type i interferon (ifn-î± and ifn-î²) modulate the viral permissiveness and replication efficiency by toggling infected and neighboring cells into their antiviral state. 90 for hcov-229e, it is known that ifn-î± exhibits a potent antiviral activity towards hcov-229e in vitro and in vivo. 91, 92 however, prolonged intranasal administration of ifn-î± to hcov-229e-infected volunteers gave rise to blood-stained nasal discharge, a side effect which is perhaps worse than the common cold that is caused by hcov-229e. 92 other novel means to inhibit viral replication are rna interference 87, 93 and broad-spectrum protease inhibitors. 87, 91 nevertheless, the in vivo efficacy and safety of these inhibitors remain to be established. to date, there is a lot known about hcov-229e and hcov-nl63, but there are several areas of research that are underrepresented. for instance, the sequence information on hcov-229e is very limited, and an animal model for both hcovs is urgently needed. furthermore, the implication of the receptor usage of hcov-229e and hcov-nl63 on the renin-angiotensin system remains to be established. future research will hopefully reveal the mechanism by which these viruses cause disease. understanding of the pathogenesis may eventually lead to a simple, non-hazardous treatment that can be used with acute respiratory infections not only in the hospital, but also at home to cure common colds. fields virology, 5 a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome identification of a new human coronavirus phylogenetic and recombination analysis of coronavirus hku1, a novel coronavirus from patients with pneumonia human coronaviruses: what do they cause? bats are natural reservoirs of sars-like coronaviruses prevalence and genetic diversity of coronaviruses in bats from china genomic characterization of equine coronavirus comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern china complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome identification of a novel coronavirus from a beluga whale by using a panviral microarray genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats clustal w and clustal x version 2.0 molecular evolutionary genetics analysis (mega) software version 4.0 bats, civets and the emergence of sars a transmissible gastroenteritis in pigs the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture recovery and characterization of a coronavirus from military dogs with diarrhea a new coronavirus-like particle associated with diarrhea in swine characterization of a feline infectious peritonitis virus isolate isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis a previously undescribed coronavirus associated with respiratory disease in humans the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf7a/7b transcription unit of different biotypes live, attenuated coronavirus vaccines through the directed deletion of groupspecific genes provide protection against feline infectious peritonitis differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 efficacy of a transmissible gastroenteritis coronavirus with an altered orf-3 gene human coronavirus 229e encodes a single orf4 protein between the spike and the envelope genes infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus analysis of human coronavirus 229e spike and nucleoprotein genes demonstrates genetic drift between chronologically distinct strains mosaic structure of human coronavirus nl63, one thousand years of evolution characterization of hcov-229e fusion core: implications for structure basis of coronavirus membrane fusion core structure of s2 from the human coronavirus nl63 spike glycoprotein molecular characterization of human coronavirus nl63 highly conserved regions within the spike proteins of human coronaviruses 229e and nl63 determine recognition of their respective cellular receptors identification of residues in the receptor-binding domain (rbd) of the spike protein of human coronavirus nl63 that are critical for the rbd-ace2 receptor interaction identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e human aminopeptidase n is a receptor for human coronavirus 229e human angiotensin-converting enzyme 2 (ace2) is a receptor for human respiratory coronavirus nl63 human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry characterization of functional domains in the human coronavirus hcv 229e receptor human coronavirus 229e infects polarized airway epithelia from the apical surface the moonlighting enzyme cd13: old and new functions to target tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis enzymatic pathways of the brain renin-angiotensin system: unsolved problems and continuing challenges identification of a putative cellular receptor 150 kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes porcine aminopeptidase n is a functional receptor for the pedv coronavirus aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury involvement of aminopeptidase n (cd13) in infection of human neural cells by human coronavirus 229e comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness identification of cell lines permissive for human coronavirus nl63 severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs development of a transgenic mouse model susceptible to human coronavirus 229e studying human pathogens in animal models: fine tuning the humanized mouse cells of human aminopeptidase n (cd13) transgenic mice are infected by human coronavirus-229e in vitro, but not in vivo human coronavirus-nl63 infections in korean children new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia human coronavirus nl63 infection in canada frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction human coronavirus nl63 and 229e seroconversion in children seroepidemiology of group i human coronaviruses in children human respiratory coronavirus hku1 versus other coronavirus infections in italian hospitalised patients human (non-severe acute respiratory syndrome) coronavirus infections in hospitalised children in france croup is associated with the novel coronavirus nl63 the time course of the immune response to experimental coronavirus infection of man effects of a "new" human respiratory virus in volunteers isolation from man of "avian infectious bronchitis virus-like" viruses (coronaviruses) similar to 229e virus, with some epidemiological observations clinical manifestations of human coronavirus nl63 infection in children in taiwan the association of newly identified respiratory viruses with lower respiratory tract infections in korean children association between a novel human coronavirus and kawasaki disease kawasaki syndrome lack of association between infection with a novel human coronavirus (hcov), hcov-nh, and kawasaki disease in taiwan blinded case-control study of the relationship between human coronavirus nl63 and kawasaki syndrome human coronavirus-nl63 infection is not associated with acute kawasaki disease human coronavirus nl63 is not detected in the respiratory tracts of children with acute kawasaki disease lack of association between new haven coronavirus and kawasaki disease two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients detection of coronavirus rna and antigen in multiple sclerosis brain neuroinvasion by human respiratory coronaviruses coronaviruses in brain tissue from patients with multiple sclerosis inhibition of hcov-nl63 infection at early stages of the replication cycle community-wide outbreak of infection with a 229e-like coronavirus in tecumseh, michigan efficacy of various intravenous immunoglobulin therapy protocols in autoimmune and chronic inflammatory disorders pathogenic viruses: smart manipulators of the interferon system rapid identification of coronavirus replicase inhibitors using a selectable replicon rna the efficacy and tolerance of intranasal interferons: studies at the common cold unit rna interference against viruses: strike and counterstrike key: cord-256615-gvq8uyfk authors: rosenberg, ronald title: detecting the emergence of novel, zoonotic viruses pathogenic to humans date: 2014-11-22 journal: cell mol life sci doi: 10.1007/s00018-014-1785-y sha: doc_id: 256615 cord_uid: gvq8uyfk rna viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. despite great advances made in diagnostic technology since the 1950s, the annual rate at which novel virulent viruses have been found has remained at 2–3. most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. an analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations. in common with all organisms, pathogens evolve. every year brings reports of previously unrecognized human pathogens or of pathogens extending their geographic range, becoming less susceptible to treatment or prevention, or displaying unprecedented epidemic tendencies. as i write this an unprecedented ebola virus epidemic threatens west africa [1] and chikungunya, a mosquito-borne virus, which first appeared in the western hemisphere in november 2013, has already infected nearly 1,000,000 people there [2] . for those of us with responsibility for preventing or controlling infectious diseases, the speed with which new battles must be fought can be disconcerting. zaire ebola virus was first identified as a human pathogen only in 1977 and chikungunya in 1956 but neither reached pandemic magnitude until decades later. how can we be better prepared to identify emerging pathogens early? i will try to briefly examine some of the factors that influence our success in finding and characterizing previously unrecognized human viruses. the concept of emerging diseases is relatively recent [3] , even if the phenomenon is not. the definition used by the world health organization [4] is representative: ''an emerging disease is one that has appeared in a population for the first time or that may have existed previously but is rapidly increasing in incidence or geographic range''. in practice, determining if a disease is increasing in incidence or geographic range sometimes requires interpretation that might be considered arbitrary. for example, using this broad definition a recent paper [5] claimed to have identified about 400 emergent ''events'' between 1940 and 2012, most of which were examples of antimicrobial resistance. a more limited and less ambiguous subset of emerging pathogens will be described here: virus species first recognized to cause human illness. before proceeding, it might be worth describing how three recently described pathogens were discovered and their disease characteristics. all three were first reported during the last six years and all three are generally accepted as distinct pathogenic entities causing serious human illness. in early september 2008, a 36-year-old female resident of lusaka, zambia developed fulminant symptoms of an acute infection, beginning with headache and myalgia, and progressing over the next 10 days to extensive rash, facial swelling and severe sore throat [6, 7] . by the time she was airlifted to a hospital at johannesburg, south africa, she had developed cerebral edema, acute respiratory distress, and renal failure. despite intensive care, including hemodialysis, she died 14 days after her initial symptoms. five of those who cared for her during transport to south africa or at the johannesburg hospital-a paramedic, two nurses and a cleaner -subsequently developed symptoms and four of these died. a previously undescribed arenavirus, lujo virus (a conflation of lusaka and johannesburg) was isolated from the index case and all four secondary cases [6] . the arenaviruses, which have bisegmented, single-stranded, negative-sense rna genomes, are broadly divided phylogenetically into new world and old world groups. lujo belongs to the old world group, as does lassa virus. typically arenaviruses have rodent reservoirs but the specific host for lujo virus has yet to be determined and how the index case was infected is unknown [7] . there have been no further cases reported. during summer, 2009, two men, aged 57 and 67 years, were admitted within a few weeks of each other to heartland regional medical center, st. joseph, missouri, usa, with similar symptoms of fever, fatigue, anorexia, nausea and non-bloody diarrhea. the two men were farmers who lived approximately 100 km distant from each other in northwestern missouri. both men had histories of frequent tick bite and were initially suspected to be infected with ehrlichia chaffeensis, a tick-borne rickettsia endemic to the area. serological and molecular testing of both, however, were negative for ehrlichia and neither responded to antibiotics. while in hospital both men developed precipitous thrombocytopenia and leukopenia. symptoms resolved with supportive care and both men were released from hospital 10 and 12 days after admission. culture of specimens indicated the presence of virus, which was confirmed by electron microscopy, and subsequently a unique bunyavirus, in the group phlebovirus, was sequenced from both patients [8] . phleboviruses are singlestranded, negative-sense rna viruses with tripartite genomes, all of which appear to be transmitted by biting arthropods. heartland virus is the first pathogenic phlebovirus described from the western hemisphere and has a 75 % nucleotide homology with the severe fever with thrombocytopenia syndrome (sfts) virus, reported from china in 2011 [9] . heartland virus has since been isolated from ticks and antibodies to it have been found in a variety of wild animals, including white-tailed deer (odocoileus virginianus) and raccoons (procyon lotor) [10] . there is no evidence for direct human to human transmission of heartland although a number of mostly nosocomial cases have been reported for sfts. between april, 2012 and late july, 2014, middle east respiratory syndrome coronavirus (mers-cov) was definitively diagnosed in 837 people, 291 of whom died [11] . the focus of cases has been in saudi arabia, united arab emirates and other middle eastern countries; the few cases detected in europe and north africa appear to be travelers from the middle east. the index case was a 60-year-old male admitted to hospital at jeddah, saudi arabia in june 2012 with a recent history of fever, cough and shortness of breath [12] . at the time of admission his laboratory blood results were generally unexceptional but by 10 days post-admission his white blood cell count had increased to 23,800/cu mm and his platelets fallen to 78,000. antibiotic-sensitive strains of klebsiella pneumoniae and staphylococcus aureus were cultured from his respiratory tract but he did not respond to antibiotic therapy. despite being on intensive support from the second day of admission, the patient died 11 days post-admission of respiratory deterioration and renal failure. coronaviruses, which include severe acute respiratory syndrome coronavirus (sars-cov) and some agents of the common cold, have positive-sense, single-stranded rna genomes and are predominately transmitted between humans by fomites. many of the mers-cov cases have been nosocomial or appear to have been transmitted within families. neutralizing antibodies to mers-cov have been widely found in dromedary camels [camelus dromedarius] from the arabian peninsula and africa [13] and virus has been isolated from them, strengthening the evidence that they are the immediate link to emergence in humans. despite the differences in clinical presentation and geographical location, these three pathogens share three characteristics: all were unknown before found infecting humans, all are rna viruses, and all have proven or putative non-human, animal sources. animal rna viruses are the most common source of emerging pathogens in a seminal study, woolhouse et al. [14] tabulated 87 pathogens first reported to be pathogenic to humans during 1980-2005. two-thirds of these were viruses, 85 % of which had single-stranded rna (ssrna) genomes. the predominance of rna viruses mostly owes to two characteristics. first, the rate of error during rna replication (* 10 -4 ) is an order of magnitude greater than that of dna (* 10 -5 ). rna replication does not benefit from the proofreading capabilities of dna polymerase or post-replication mismatch repair; consequently the potential for mutation per replication cycle is high [15] and the lack of fidelity may have limited the size of rna genomes, many of which are in the range of 10,000-15,000 nucleotides. second, most rna viruses are zoonoses, that is, they were transmitted, at least initially, to humans from non-human mammal or avian hosts. examples of rna viruses retaining the capacity to be directly transmitted from animals to humans include influenza, nipa, and sars viruses, but even some viruses commonly transmitted exclusively between humans, such as hiv and hepatitis c, have likely animal origins [16] . all arthropod-borne viruses (arboviruses) are zoonoses, although some, like dengue, yellow fever, and chikungunya, have adapted to efficient vectorborne transmission between humans. humans have been in contact with infectious animals since prehistory but their exposure accelerated with the development of livestock husbandry beginning about 15,000 years ago [17] . the growing global population has not only increased the demand for domesticated meat in the 21st c but has increased encroachment on areas once wild, both are trends that increase human exposure to animals and animal products [18] . by the end of 2010, there had been, by one tabulation [19] , 213 virus species from 25 virus families incriminated as causes of human disease. more than two-thirds of these viruses (68 %) are known or presumed zoonoses. more than a quarter (28 %) were first described from non-human mammals, birds or blood-feeding arthropods 1-77 years before being recognized as human pathogens. indeed, the first vertebrate virus described, the cause of foot and mouth disease, was isolated from a cow in 1897 [20] but conclusively shown to cause human disease only in 1954 [21] . the dates of discovery, regardless of host, are plotted in fig. 1a . the rate at which virulent viruses have been discovered has been governed by two equally important factors: the ability of existing technology to detect and discriminate between viruses, and the ability to collect specimens potentially containing novel viruses. initially, the lack of methods for the laboratory cultivation of viruses, which require cells for replication, prevented their isolation for study. early characterization as a virus depended mainly on demonstration that a filterable agent smaller than bacteria was responsible for transmissible disease. until the late-1930s, when embryonated chicken eggs and suckling mice began to be used commonly to culture animal viruses, only 26 of the viruses now known to be pathogenic had been described. the rate of discovery again accelerated after the introduction of in vitro cell culture in 1949 (fig. 1a) . the mean annual rate of virus discovery during 1950-1959 was 3.3. during this period methods for antigentically typing viruses using panels of antibodies were refined and came into wide use. there was a striking increase in the number of novel viruses described during 1960-1969 to 4.9/year. this was followed, however, by a sudden deceleration in the rate of discovery to only about 2/year, which persisted through 2010, despite the availability of increasingly powerful methods for genomic characterization, such as polymerase chain reaction (pcr) from the mid-1980s and, more recently, high-throughput, parallelized (''next-generation'') sequencing. vertebrate viruses can be sorted into two broad categories: those directly transmissible between humans or between animals and humans, and the arboviruses, which require the mediation of blood-feeding arthropod vectors, such as mosquitoes or ticks; 39 % (83) of pathogenic viruses are transmitted to humans only by arthropod vectors. the sudden, transitory increase in rate during 1960-1969 has been shown [19] to be because the rates of discovery of these two classes differed (fig. 1b) . before 1950 the rates for the two were the same and each comprised about half the pathogenic viruses. after 1950, however, the trends of the two classes of virus diverged. while about two non-arboviruses were discovered each year between 1950 and 2010, the arboviruses dramatically increased during 1960-1969, only to fall equally dramatically to nearly zero by 1980. during 1960-1969 twice as many arboviruses (32) were discovered than non-arboviruses (15); by contrast, during 1981-2010, only 2 arboviruses were discovered compared to 57 non-arboviruses. the difference in the rates for the two classes highlights the important role that strategies for specimen collection play in the recognition of novel pathogens [19] . [22] . all the field stations were located in tropical or sub-tropical countries and all carried out an integrated strategy that attempted to discover viruses from humans, vertebrate animals and biting arthropods. of the 83 arboviruses discovered by the end of 2010, 35 (42 %) were discovered by rf staff, 23 of those during 1951-1969. in comparison, the single most successful institutional discoverer of non-arboviruses, the united states national institutes of health, described 12 of 130 (9 %). the rf protocol, which was the model for several other institutions, including the institut pasteur, was directly responsible for two additional characteristics differentiating arbovirus from non-arbovirus discovery. although 68 % of all non-arboviruses were discovered in europe or the usa, 67 % of all arboviruses were discovered in sub-saharan africa, latin america/caribbean, or egypt/india/ near east (fig. 2) . second, 33 % of arboviruses were first isolated from arthropods, a consequence of systematic vector collections. the predetermined cessation, by 1970, of most rf support for international arbovirus researchincluding sponsorship of reference collections, conferences, new technology and reagents-was soon followed by a rapid, worldwide decline in arbovirus discovery (fig. 1b) . the east african virus research institute, for example, which was founded by rf in 1935, isolated 5 arboviruses after direct rf administration ended in 1952, but none after the general rf program closed in 1965. the disproportionate productivity of rf resembles that of especially effective individual discoverers of plant species [23] . those botanical ''big hitters'' combined technical expertise and persistence over many years with concentration in a limited geographic area where they had gained deep knowledge. the rf was committed to a long-term strategy founded on five components. first, it chose study sites where it had evidence that arbovirus diversity would be high. these were mostly tropical and contained forested and rural areas. they chose countries where a professional work force could be recruited and where it was hoped the work would be sustained after the rf departed. second, the program concentrated only on one subclass of pathogens, the arboviruses. despite the wide competence of the professional staff, few reports were published dealing with local diseases other than arboviral. third, the research strategy called for long-term commitment. rf staff, including expatriates, typically lived on site and implemented projects for years, allowing for continuity of not only research but training. fourth, the program was integrative. human, animal and vector investigations were simultaneously pursued. because all scientists worked in a single unit connections between human virus isolates and those from animals or vectors were readily made. on the other hand, for many viruses isolated from animals or arthropods there has yet to be a link to human infection [24] . and fifth, each unit was self-contained. each was capable of conducting both specimen collection and sophisticated laboratory analyses. how many pathogenic viruses remain to be discovered? mathematical methods for extrapolating from historical rates of discovery to estimate the pool of yet to be discovered organisms in a given taxon tend to be accurate only after most species have already been discovered [25] . the fundamental weakness of these computations, which generally rely on analysis of cumulative frequency curves, is the assumption that the numbers of organisms known at a given time are the result of methods of discovery that have been consistent everyplace and throughout time. the increased rate of virus discovery during 1955-1970 was largely due to the temporary efforts of the rf, a ''big hitter'' [23] , whose combination of active surveillance, geographic specialization, and integrated approach remains atypical. in contrast to sophisticated computations, a recently published prediction that a minimum of 320,000 mammalian viruses of 9 families remain to be discovered was based on a simple arithmetical calculation using data from a single study [26] . considering how few new, virulent viruses are found every year, the potential for any of 320,000 viruses jumping to humans and being discovered would then be very low (6.25 9 10 -6 ). the authors used degenerate, virus-family-level primers to amplify genomic segments from specimens of feces, urine, and throat swabs collected from the bat species pteropus giganteus in bangladesh. amplicons were as short as 250 bp and no biological information was obtained. they found 55 viruses (and statistically surmised an additional three existed), some of which might be novel, belonging to seven virus families. in calculating a number for the universe of viruses yet to be found they speculated that each of the known 5,486 mammal species will host an average 58 unique viruses, unshared with other species. the authors concede that there is little evidence to support these presumptions. a single subtropical bat species hardly represents all mammal species and indeed many viruses are known to infect more than one species; they tested for only 9 of the 25 virus families pathogenic to humans. ultimately, the number of viruses remaining to be discovered is irrelevant if, as expected, there are many and they continue to rapidly evolve. the discovery of a virus can long predate its emergence as a recognized public health threat. the discovery of zika virus in a monkey in uganda preceded its first incrimination as the cause of a human epidemic-12,000 km distant in micronesia-by 70 years [27] . as noted above, the availability of ever more powerful molecular techniques is substantially increasing the catalog of distinct viruses found in nature but the number found annually to be pathogenic to humans rarely exceeds a few each year. is it feasible to predict which animal viruses have the potential to cause disease in humans? steps to emergence the steps by which a virus might emerge from exclusively animal hosts are schematically depicted in fig. 3 . at the most preliminary level (tier 1) viruses circulate within mammals and birds, not necessarily causing disease, before some opportunistically infect humans (tier 2). typical human-animal contact includes husbandry, capture of wild animals for food, and exposure to animal fomites or waste, as may happen in bat infested environments. indirect exposure via arthropods must be frequent, as evidenced by the large proportion of pathogenic viruses that are vectorborne [19] . in most instances these tier 2, opportunistic infections are dead ends or remain rare events because the pathogen is not well adapted to transmission between humans (e.g., crimean-congo hemorrhagic fever virus) or because the type of contact between infected animals and humans is uncommon (e.g., sealpox virus). a small but fig. 2 comparison of regions in which arboviruses and nonarboviruses were discovered fig. 3 schematic of the emergence of zoonotic viruses as human pathogens. in tier 1, viruses are only transmitted among sub-human animals. in tier 2, viruses infect humans, but only directly from animals. some animal viruses (solid arrows), like west nile, can fuel zoonotic epidemics. others, like hantaviruses, are frequent but subepidemic causes of human illness (dashed black arrows), while many, like sealpox, are rare (dashed red arrows). in tier 3, zoonotic viruses have acquired the ability to be transmitted between humans without the contribution of the animal host. in some cases (w) a virus might leap directly to tier 3 or transition through tier 2 significant group of zoonotic viruses, including rift valley fever, nipah and west nile viruses, are capable of instigating human epidemics without ever adapting to humanhuman transmission. theoretically even sub-epidemic (r o \ 1) transmission can favor mutations that will enhance future transmission [28] . in rare instances (tier 3) pathogens do evolve to allow human-human transmission (e.g., hiv) or appear to already posses that capacity (e.g., mers-cov, lujo). some viruses maintain animal-animal and animal-human cycles but are mostly propagated by human-human transmission (e.g., chikungunya, zika). the geographically limited sylvatic cycles of dengue in west africa and southeast asia account for a tiny percentage of the estimated 390 million infections annually [29] . human contact with an animal virus does not ensure infection. among the biological barriers for the virus are finding a route of entry, evading general immune defenses, invading host cells, replicating sufficient numbers before specific immune responses are mounted, and finding a route to the next host. aerosol delivery, for example, greatly enhances transmissibility but efficiency depends on the anatomical site within the respiratory system of the invaded cells [30, 31] . arbovirus transmission, in which the vector amplifies, transports and inoculates the virus into humans, can be enhanced by viral mutations that increase the potential for successfully infecting the vector [32, 33] or animal host [34] without altering its virulence to humans. much recent research has focused on identifying determinants essential for viral invasion of host cells and how modification of the viral ligands might increase their ability for interspecies infectivity [34] . it is not yet possible on the evidence of sequence alone to predict with confidence the probability of an animal virus transitioning to humans. in general, vertebrate specificity greatly limits the ability of viruses adapted to one species to invade similar cells in another, distant species. influenza a is the most studied and best understood of the few viruses that frequently jump from animals to humans. a number of mutations have been identified that enhance infectivity. these include substitutions in the hemagglutinin (ha) protein receptor binding sites that enable the virus to exploit sialylated glycan receptors on respiratory cells belonging to other species [35] . for example, two nonsynonymous base changes in the ha receptor binding sites of avian h2 and h3 viruses, which converted their specificity from the avian a2,3-sa to human a2,6-sa, led to the 1957 pandemic of h2n2 and the 1968 pandemic of h3n2 [36] . much current research on the determinants of influenza specificity is experimental and its extension to complex natural transmission of other virus families remains to be tested. understanding how such adaptability works could focus our attention on those virus families or species with the greatest chance of infecting humans but how this knowledge could be used more specifically to identify potential threats to humans among animal viruses, as has recently been proposed [5] , is unclear. geographical bias, human behavior and likelihood of contact species richness of mammals and birds is greatest at the equator, thinning toward the poles [37] ; mosquitoes [38] and ticks [39] appear to follow a similar latitudinal species diversity gradient. pathogen species are also richer in the tropics than in temperate zones [40] although, as has been ruefully pointed out, ''the fact that warbler species distributions are better understood than the distribution of human pathogens is a gap that clearly deserves research attention'' [41] . there is a strong association between mammal and pathogen richness but mammal diversity appears to be an indicator rather than the cause of pathogen diversity [41] . pathogens that maintain external life cycles, for example vector-borne and helminthic, which are directly susceptible to variability in precipitation, tend to be more geographically restricted to the tropics than those directly transmissible between people, such as influenza [40] . environmental barriers to dispersion can be circumvented. monkeypox virus, whose natural transmission is largely restricted to parts of equatorial africa by the range of its natural rodent hosts, has demonstrated the ability to make use of new hosts in temperate zones [42] and a number of arboviruses, such as chikungunya, dengue and zika viruses, have widely expanded their natural ranges as their principal vectors have [43] . the rf selection of field sites in 1950 was based on the relatively greater diversity of arboviruses found during the preceding 35 years in tropical countries and it can be argued that their success in discovering new viruses owed as much to this factor as their choice of integrated, active surveillance. as would be expected, there appears to be a correlation between zones of species richness and frequency of reported vector-borne and zoonotic emerging disease events [44] . the species richness of the tropics suggests that human populations there are exposed to greater risk and that they are fertile grounds for virus mutation. certain behaviors common to some regions, such as the harvesting of wild animals for food, aggravate that risk. the lack of housing barriers to rodents, bats and arthropod vectors are major vulnerabilities to pathogens carried by them. environmental and sanitation deficiencies also increase risk to enteric and vector-borne viruses; in the absence of dependable water supply, many people, for example, are forced to store containers of water that provide breeding for the virus-carrying mosquitoes. most importantly, as dunn et al. [41] have shown, countries with the highest pathogen richness spend the least per capita on health care, and there is an inverse correlation between investment in public health and pathogen prevalence, independent of species richness. because of the obvious link between the abundance of novel viruses and the tropics, geospatial modeling could help target areas for surveillance. there is a tradition of developing and using models to identify those areas of the world whose species richness is most in need of conservation [37, 45] and recently attempts have been made to use models to identify areas most liable to spawn emerging diseases [5, 44] . considering the association between mammal and pathogen species richness there can be expected to be some overlap between the two sets of ''hot spots''. the predictive robustness of a model depends not only on the algorithms used but in the choice and weighting of variables, and the representativeness and validity of the data. for example, a widely cited model [5, 44] , which chooses ''the original case or cluster of cases representing an infectious disease emerging (during 1940-2004) in human populations for the first time'', lists only 61 of 168 viruses first described as infecting humans during that period [19] . on the other hand it credits the first occurrence of a number of viruses described years earlier to the period 1940-2004; these include measles, influenza a, and rabies, assigning to each a single, arbitrary origination location (us, hong kong, and costa rica, respectively) for modeling geospatial associations. because this model also lumps a variety of emerging disease types, including many examples of antimicrobial resistance, population density is a major factor, which might explain the higher likelihood for emergent events it assigns to india and java than to the amazon or equatorial africa, where so many novel viruses have been discovered. modeling has been more successful for single pathogens for which large amounts of specific data have been collected, such as for dengue [46] or malaria [47] . associating disease prevalence in a limited area with well-characterized environmental attributes, as has been done for plague bacteria (yersinia pestis) in uganda, can be used to predict areas potentially at risk that would be difficult to collect data from, such as large plague-prone tracts of the neighboring democratic republic of congo [48] . because many of the arbovirus species are known only from places where long-term field operations were established by the rf, institut pasteur, and others, and because those sites were selected for logistical and political realities as well as scientific interest, their usefulness in modeling is still limited. surveillance for emerging pathogens: the problem of knowing the unknown successful surveillance depends on how and where one looks. ideally, an emerging virus will be detected at its source and contained before spreading. this ideal requires, however, extensive networks of alert health care providers, adequate laboratory resources, and an effective method for communicating results to an authority capable of responding. it also assumes that a zoonotic virus will not be spread by animal hosts impossible to control, as was the case with the avian arbovirus, west nile. in practice, human disease surveillance raises an alarm only after an arbitrary number of seemingly related, serious cases are reported and arouse attention. generally, number of cases and length of time to detection are least for anticipated pathogens with distinct presentations, such as poliovirus presenting with acute flaccid paralysis. in countries with rudimentary public health systems that threshold might be reached for unexpected pathogens only after the number of cases reaches epidemic proportions impossible to ignore, as the recent epidemic of ebola virus in west africa demonstrates. many viral disease cases present as clinically indistinguishable acute febrile illnesses (afi) or with symptoms so mild the patient does not seek attention. cases with neurological involvement or systemic bleeding can also be difficult to diagnose clinically without adequate laboratory support. in those tropical areas most likely to spawn emerging viruses, afi will be misdiagnosed or undiagnosed in at least 50 % of patients [49] [50] [51] [52] . overlooking novel pathogens as the cause of nonspecific symptoms is not confined to developing countries: it is likely heartland virus was a cause of illness in the usa long before it was characterized in 2012. nevertheless, the probability that more undescribed viruses infect humans in the tropics seems to be greater. it is likely, therefore, that many emerging diseases due to novel viruses will be overlooked, especially at tier 2, until they become epidemic, are transported to countries with more sensitive surveillance, or are discovered by chance. laboratory support for clinicians is critically deficient nearly everywhere in the rural tropics. the first step in determining if an illness might be caused by a rare or unknown virus is to eliminate the possibility of pathogens known to be endemic. simple, relatively accurate rapid diagnostic tests (rdts) are available for a few common causes of afi, notably malaria, but tests for most viruses require not only equipment, such as elisa readers, but modest, dependable infrastructural support-electric power, clean water, cold storage-rarely available outside major cities. poor roads often make the timely, proper transport of specimens to centers with laboratory capacity impractical. even in cities, many hospitals and government laboratories do not have the basic equipment, fresh reagents or accurate testing protocols to assay for most common endemic pathogens. should laboratory capability be available to eliminate most known etiologies for the disease observed, description of a novel pathogen typically requires both biological and molecular characterization [53] . recovery of viable virus for culture and histological evidence of pathology remain fundamental steps establishing causality. the increasing power and availability of rapid, next-generation sequencing has made whole-genome analysis an increasingly routine and important part of describing novel viruses but because of the large number of commensal species found in the human virome [54] , linking a novel genome with virulence will be tentative without supporting biological evidence. for example, wu and ki polyomaviruses, isolated in the mid-2000s from children suffering from acute respiratory infections and tentatively included in the list of human pathogens [19] , have yet to be proven causal of illness [55] . our ability to detect and characterize novel pathogenic viruses in hot spot locations lags behind global systems that have arisen to report and respond to unusual occurrences. the international health regulations (ihr) of the world health organization (who) binds 196 countries to plans to improve their ability to detect and respond to outbreaks, and the global outbreak alert and response network (goarn) of who helps organize international response to public health emergencies. open source networks for reporting outbreaks include the program for monitoring emerging diseases (promed-mail), a free, internet-based system for disseminating information posted by 40,000 professional contributors in 185 countries, and healthmap, which collects and continuously updates disease outbreak data from a variety of public sources, including news services. many of these reports seem never to be investigated or resolved. the full value of these systems can only be attained if provided with accurate information. considering the barriers to obtaining human surveillance data, it has been proposed [5] that monitoring animal populations at sentinel locations could alert us to risk from viruses with pandemic potential. for such a plan to be feasible for emerging viruses it would be necessary to judge the potential risk posed by a virus not yet known to infect humans. epizootic disease in livestock or wild animals is used as a threat indicator for some known zoonotic viruses, such as influenza, rift valley fever virus, and west nile virus, but there is no assurance that an agent potentially pathogenic to humans will cause noticeable disease in animal hosts. coronaviruses closely related and putatively ancestral to sars virus, for example, seem not to cause disease in host bats [56] . the extent of animal disease surveillance is also far less in the tropics than even the poorest human clinical networks so the likelihood of recognizing an unusual event is less. periodic sampling of animals can discover novel viruses but is too infrequent and limited to be surveillance. considerable attention has been given to human contact with bush meat, or animals captured for food [5] . while harvesting and slaughtering wild animals appear to have provided the mechanism by which some important pathogens have emerged, such as hiv and sars, it has not played a role in the emergence of many others, including the three examples discussed in this paper: lujo, heartland and mers-cov. vectors obviate the need of direct human-mammal or human-bird contact and can move viruses across ecological zones. sequencing and cataloging the viruses of animals in selected areas can provide valuable insight to transmission dynamics and phylogenetics but, as discussed, cannot yet be used to predict. one must wonder if the us $6.3 billion proposed to catalog mammalian viruses not yet known to be pernicious [26] would not be better spent on developing more suitable diagnostic tests for humans in remote areas most liable to emerging pathogen risk or on conducting sentinel human surveillance. ultimately, the best indication that a pathogen has the ability to jump to humans is finding it in humans [57] . although there is not a strong rationale for conducting autonomous searches for potentially pathogenic viruses in animal populations, there is much value in animal investigations in support of surveillance for infectious diseases in selected, sentinel human populations. the discovery of lujo virus in a human, for example, should direct our attention to its epidemiology and ecology in the area where we suspect exposure occurred. had lujo been discovered in an animal instead, its significance as a pathogen would have been speculative until the detection of the first human case. the integrative, long-term approach of the rf can serve as a model but with primary focus on conducting population-based surveillance for acute illness. concomitant ecological profiling and virological studies in arthropods, mammals and birds can more quickly clarify the epidemiology of any novel viruses discovered in the human population. the zoonotic viruses pathogenic to humans represent a small but unknown proportion of those infecting mammals and birds. from this constantly evolving universe of vertebrate viruses two or three are recognized every year to have broken the species barrier, a remarkably small number considering the frequent contact between humans and animals, and the high adaptability of rna viruses. while most of these novel, emergent viruses have inconsequential public health significance, some, such as mers-cov or lujo, have obvious destructive potential. what is the best strategy for identifying and limiting the menace from novel, zoonotic viruses? identifying potential pathogens before they leap to humans (tier 1) would seem ideal but is impractical. the determinants of pathogenicity are complex and poorly understood, while a system for wildlife or livestock surveillance in those areas with conditions most conducive to emergence cannot anytime soon reach a scale or effectiveness to be pragmatic. it is likely that yet to be recognized viruses already infecting humans will be sources of disease outbreaks of varying magnitude in the future. these tier 2 infections can be uncovered as part of comprehensive, investigative surveillance in human populations at risk. in the near term this would be best accomplished in most places through specially designed sentinel surveillance sites. modeling might at some point provide guidance for site selection but too narrow a definition for target sites (e.g., bush meat markets) will be self-defeating. by identifying and eliminating poorly appreciated endemic agents, investigations can then focus on illnesses with unresolved etiologies. unlike investigations directed at animal populations there would be a tangible, immediate improvement in the health of the subject communities. complimentary studies of vectors and animals, as pioneered by the rockefeller foundation, would prepare for epidemiological investigations of those zoonoses uncovered but the primary focus must be on humans. ultimately, one hopes, surveillance for emerging zoonoses will be a part of improved health 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and cancer bats and their virome: an important source of emerging viruses capable of infecting humans overviews of pathogen emergence: which pathogens emerge, when and why? key: cord-010977-fwz7chzf authors: myserlis, pavlos; radmanesh, farid; anderson, christopher d. title: translational genomics in neurocritical care: a review date: 2020-02-20 journal: neurotherapeutics doi: 10.1007/s13311-020-00838-1 sha: doc_id: 10977 cord_uid: fwz7chzf translational genomics represents a broad field of study that combines genome and transcriptome-wide studies in humans and model systems to refine our understanding of human biology and ultimately identify new ways to treat and prevent disease. the approaches to translational genomics can be broadly grouped into two methodologies, forward and reverse genomic translation. traditional (forward) genomic translation begins with model systems and aims at using unbiased genetic associations in these models to derive insight into biological mechanisms that may also be relevant in human disease. reverse genomic translation begins with observations made through human genomic studies and refines these observations through follow-up studies using model systems. the ultimate goal of these approaches is to clarify intervenable processes as targets for therapeutic development. in this review, we describe some of the approaches being taken to apply translational genomics to the study of diseases commonly encountered in the neurocritical care setting, including hemorrhagic and ischemic stroke, traumatic brain injury, subarachnoid hemorrhage, and status epilepticus, utilizing both forward and reverse genomic translational techniques. further, we highlight approaches in the field that could be applied in neurocritical care to improve our ability to identify new treatment modalities as well as to provide important information to patients about risk and prognosis. electronic supplementary material: the online version of this article (10.1007/s13311-020-00838-1) contains supplementary material, which is available to authorized users. translational genomics represents a diverse collection of research approaches that leverage human genomics and model systems to identify new approaches to treat and prevent disease and improve healthcare (1, 2) . rooted by the central dogma of dna to rna to protein, genomic research examines the entire genome concurrently, and may include analyses of dna variants in association with traits of interest as well as the impact of genomic variation on gene transcription and translation. genomic research has been enabled by technological advances to accurately and cost-effectively study variation across the genome at scale, as well as computational techniques to store and analyze genomic data quickly and efficiently (3) . while translational research is often defined in terms of the traditional "bench to bedside" techniques that advance discoveries from model systems through biomarkers and mechanisms ultimately to clinical applications, genomic research offers a strong use-case for an alternative approach. termed "reverse translation," this approach starts with humans as the model system, utilizing genomic associations to derive new information about biological mechanisms that can be in turn studied further in vitro and in animal models for target refinement (fig. 1) . both of these approaches possess advantages and drawbacks (4, 5) . forward translation depends on the relevance of the model system to human disease, both in terms of the physiologic responses to disease or insult, as well as the approach taken to perturb the system. for instance, the human applicability of genomic studies of the response to traumatic brain injury (tbi) in a mouse model require that the mouse's response to tbi is analogous to a human's, and that the approach taken to pavlos myserlis and farid radmanesh contributed equally to this work. create a tbi in the mouse provokes a similar pattern of injury seen in human tbi (6) (7) (8) (9) . as such, a great deal of careful work is required to demonstrate the validity of these model systems before the results arising from them can be judged relevant to human disease. the challenges of bridging this divide are illustrated by the universal failure of neuroprotection mechanisms that reached human trials in the last several decades, essentially all of which had promising model system data in preclinical development (10) (11) (12) (13) (14) . reverse genomic translation, in contrast, begins with humans ( fig. 1 ). as such, there are few concerns as to the relevance of the system for discovery of biomarkers and mechanisms of disease. however, this approach carries a new series of challenges in study design and data acquisition (4) . compared to isogenic cell lines or carefully bred animals in a controlled setting, humans are highly variable in both their environmental and genetic exposures. this is advantageous in identifying genetic susceptibility to disease risk and outcomes, but teasing out these small genetic effects from highly variable non-genetic exposures requires both careful computational techniques as well as large sample sizes. furthermore, because genomic data is both identifiable and can potentially lead to discrimination, human genomic studies require complex consent and data management procedures (15) . in neurocritical care, the relative rarity of many of the diseases we encounter, coupled with the challenges of critical illness and surrogate consent make human genomic studies all the more difficult to execute effectively (16) (17) (18) (19) . neurointensivists routinely encounter diseases and complications for which there are a dearth of effective treatments, or even foundational knowledge of their underlying pathophysiologic mechanisms (20, 21) . in this review, we will highlight some of the approaches being taken to apply translational genomics to the study of diseases commonly found in neurocritical care, utilizing both forward and reverse genomic translational techniques. further, we will highlight some of the best practices in the field that could be applied in neurocritical care to improve our ability to identify new treatment modalities as well as risk and prognosis information to patients and their families. in advance of the human genome project and the hapmap consortium, genetic studies were confined to the study of candidate genes and lower-resolution genome-wide techniques such as categorization of restriction fragment length polymorphisms (rflp), tandem repeats, and microsatellites (22) . these genomic features enabled early efforts to perform linkage analyses in families with related traits and disorders, as well as selected populations of unrelated individuals. careful work in this arena led to validated discoveries that have survived replication in the common era, such as chromosome 19 in late-onset alzheimer disease (ad), ultimately mapped to the apoe locus that has become a target for a great deal of genetic research in ad, as well as many other diseases including tbi and intracerebral hemorrhage (ich) (23) (24) (25) (26) . still, much of the pregwas era was characterized by candidate gene studies that suffered from low statistical power and multiple sources of confounding that led to a failure to replicate many reported associations in the gwas era that followed (27, 28) . the most substantial source of confounding in candidate gene analyses is population stratification, in which differences in allele frequency due to ancestral imbalance between cases and controls introduces spurious associations (positive or negative) between genotype and trait based solely on these cryptic ancestral imbalances (29, 30) . even in studies of apoe in european ancestry populations, uncontrolled variation in the percentages of individuals of northern vs. southern european ancestry between cases and controls can mask true associations between apoe and ich, for instance (31) . the gwas era, in which variants across the genome could be reliably genotyped and mapped to a common reference template by chromosomal location, ushered in a new system of best practices that could minimize the contribution of many of the sources of confounding in describing associations between genomic variation and traits or diseases. the international hapmap consortium obtained genotypes on individuals across 11 ancestral populations around the globe, creating a resource that described the patterns of allele frequency variation across diverse populations (32) . with these breakthroughs and a number of landmark evolutions that followed, case/control and population-based gwas have led to the identification of over 11,000 associations with human diseases and other traits (https://www.ebi.ac.uk/gwas/). obviously there is an enormous disconnect between the discovery of genetic loci and leveraging of this information for human benefit, which is where the translational genomic work that serves as the topic of the present review becomes relevant (32) . post-gwas, in addition to functional and translational efforts, the movement has been towards so-called "next-generation sequencing" methodologies consisting of whole exome sequencing (wes) and whole genome sequencing (wgs). using these approaches, each nucleotide in the exome or genome is ascertained with high reliability, permitting the identification of rare and de novo variants that escape detection in traditional gwas (33) . wes captures within-gene coding variation only, offering detection of variants that may more directly impact protein structure and function than non-coding variation detected by wgs (34) . because the coding exome is only~2% of the overall genome, it is more cost-effective than wgs, but debate continues as to which is the more appropriate tool for large-scale study of the human genome (35) . regardless, both wes and wgs remain orders of magnitude more expensive than traditional gwas approaches at this time, and as such well-powered sequencing studies remain unreachable for many diseases in the current pricing models. less common diseases and conditions that one may find in a neurocritical care unit are doubly disadvantaged, as even larger sample sizes are required for sequencing analyses than gwas, due to the need for many observations to identify rare exonic or intronic variants associated with disease (17, 36) . as pricing models improve and larger and larger community or hospital-based cohorts receive sequencing through clinical or biobanking efforts, it is hoped that even uncommon conditions such as subarachnoid hemorrhage or status epilepticus will benefit from the insights achievable through sequencing analysis, where case/control and smaller sequencing studies have shown promise (37, 38) . obviously genomic research need not be limited solely to human studies. a wealth of information about disease pathogenesis and response to injury can be gleaned from model systems of human conditions using genomic and transcriptomic approaches. because animal models and isogenic tissue cultures are specifically designed to limit genetic differences between individual animals or plated cells, dna-based association tests typically do not offer insight in the same way that they do in humans. as such, many model system studies start with rna, examining how the genome responds to perturbation through the transcriptome. however, there are substantial genomic differences between model systems and humans, as coding sequences are not necessarily conserved, promoter and enhancer control of gene expression can vary, and in the case of immortalized tissue and cell-based assays, the chromosomal architecture itself can be quite different from the organism from which it was derived (39, 40) . these differences can be highly relevant when determining whether observed transcriptomic and proteomic results from model systems are likely to be shared in humans. with those caveats, the dynamic nature of the transcriptome in model systems offers opportunities to assess the way in which the genome responds to noxious insults or drug exposures, and in animal models this can even be done across specific organs or tissues of interest (40) . as one example, traumatic brain injury researchers have obtained insight into both the initial injury cascade as well as brain response to potential injury modulators such as valproate using animal models and transcriptional microarrays, in which rna expression patterns in brain tissue can be rapidly and replicably assessed across the transcriptome (41) . using more recent technological advancements such as drop-seq, rna expression can be assessed in single cells, as has been done in individual hippocampal neurons in a mouse model of tbi (42) . at a minimum, these elegant studies can help to identify relevant cell types important in the response to injury, highlighting testable hypotheses that may be important in human conditions, all with access to tissues and control over experimental conditions that would never be possible in human-based research. given that diseases common to the neurocritical care population so rarely afford access to brain tissue for pathologic or genomic analysis antemortem, model system genomic studies offer an important adjunct for translational research. forward genomic translation begins with model systems with the goal of using the measured associations in these models to derive insight into biological mechanisms that may also be relevant in human disease. forward translation requires wellcharacterized models that are often designed to mimic the human exposures of interest as closely as possible. this is often challenging given the natural differences between humans and many of the animals chosen to serve as models. in this section, we will highlight several model systems in current use for translational genomics relevant to neurocritical care, but the field of translational modeling in neurologic disease is suitably large to prevent an exhaustive review herein. malignant cerebral edema is a highly lethal complication of ischemic stroke, with mortality of 60-80% (43) . currently, hemicraniectomy is the only available option to prevent death and yet it does not address the underlying pathophysiology. hyperosmolar therapy is potentially useful as a bridge to surgery. preclinical data based on a forward translation approach has been useful in highlighting mechanisms underlying postinfarct edema as potential targets for therapeutic manipulation. the sulfonylurea receptor 1 (sur1) is encoded by the abcc8 gene that is upregulated after cns injury, forming an ion channel in association with transient receptor potential melastatin (trpm4). continuous activation of this complex can lead to cytotoxic edema and neuronal cell death, which has been demonstrated in both animal and human models (44, 45) . sur1 is also found in pancreatic beta cells, constituting the target for the oral hypoglycemic agent, glyburide. studies of rodent and porcine stroke models demonstrated that in the first few hours after an ischemic insult, both sur1 and trpm4 are upregulated (46, 47) . limited case series of human postmortem specimens also demonstrated upregulation of sur1 in infarcted tissue (48) . therefore, intravenous glyburide has been proposed for treatment of malignant cerebral edema. targeting sur1 in rat models of ischemia have consistently resulted in reduced edema and better outcomes (49) . in particular, glyburide infusion starting 6 h after complete middle cerebral artery occlusion resulted in decreased swelling by two thirds and 5% reduction in mortality (50) . one desirable characteristic of glyburide is that it cannot penetrate intact blood-brain barrier, but that is facilitated following brain injury (51) . the effect of glyburide for treatment of cerebral edema has also been studied in tbi with promising data obtained from animal studies (52) . limited randomized trials in human using oral glyburide have shown promising results; however, use of oral formulation and study design limitations prohibit generalizability of results (52, 53) . building on this preclinical data, the phase 2 randomized clinical trial (games-rp) showed that the iv preparation of glyburide, glibenclamide, is associated with reduction in edema-related deaths, less midline shift, and reduced rate of nih stroke scale deterioration. however, it did not significantly affect the proportion of patients developing malignant edema (54) . the phase 3 charm trial, sponsored by biogen, is currently enrolling patients with large hemispheric infarction to determine whether iv glibenclamide improves 90-day modified rankin scale scores. if this trial proves successful, this vignette will represent a dramatic success story for the forward translation paradigm in genomic research. in the light of recent advances in revascularization therapy, the national institute of neurological disorders and stroke has supported an initiative aiming to develop neuroprotective agents to be used as adjunctive therapy to extend the time window for reperfusion and to improve long-term functional outcome. this stroke preclinical assessment network (span) supports late-stage preclinical studies of putative neuroprotectants to be administered prior to or at the time of reperfusion, with long-term outcomes and comorbidities constituting the endpoint. the goal is to determine if an intervention can improve outcome as compared to reperfusion alone and/or extend the therapeutic window for reperfusion. span directly applies to forward translation efforts in preclinical models of neuroprotection after stroke and is an outstanding opportunity to stimulate research efforts in a field more remembered for its past failures than the promise it holds for the future of therapeutic development in the area. other societies have also begun to endorse more comprehensive modeling approaches in areas with few therapeutic options with the hope of implementing a paradigm shift. for example, the neurocritical care society has initiated "curing coma" campaign with the 10-to 20-year mission to improve the understanding of the mechanisms and to ultimately develop preventative and therapeutic measures. traumatic brain injury (tbi) is among the leading causes of disability and death worldwide, particularly in the young. the type of tbi is in part determined by the attributes of mechanical forces, including objects or blasts striking the head, rapid acceleration-deceleration forces, or rotational impacts. following the primary injury, an intricate cascade of neurometabolic and physiological processes initiates that can cause secondary or additional injury (55, 56) . intensive care management has improved the prognosis of tbi patients; however, specific targeted treatments informed by pathophysiology could have a tremendous impact on recovery. the period of secondary tissue injury is the window of opportunity when patients would potentially benefit from targeted interventions, given that in tbi, the primary injury cannot be intervened upon by the neurologist or intensivist. the goal of therapy is therefore to reduce secondary damage and enhance neuroplasticity. the utility of animal models of tbi primarily depends on the research question, as each model emulates specific aspects of injury and has selective advantages and disadvantages. these include biomechanics of initial injury, molecular mechanisms of tissue response, and suitability for high-throughput testing of therapeutic agents, to name a few. although phylogenetically higher species are likely more representative models for human tbi, rodent models are more commonly used given the feasibility to generate and measure outcomes, as well as ethical and financial limitations of higher-order models. table 1 summarizes some common and representative tbi models [ table 1 ]. in contrast with the rodent models described in table 1 , other model systems in tbi have been selected specifically to study other aspects of the physiologic response to tbi. for example, a swine model of controlled cortical impact offers the opportunity to readily monitor systemic physiologic parameters such as tissue oxygen and acid-base status while investigating therapeutic interventions, which is argued to provide greater insight into human response to injury (66) . translation of preclinical studies using these animal tbi models to humans is inherently challenging. differences in brain structure, including geometry, craniospinal angle, gyral complexity, and white-gray matter ratio, particularly in the rodent models, can result in different responses to trauma (65) . the limitation of extrapolating animal studies to human is also manifested at the genetic level, as differences in gene structure, function, and expression levels may suggest genetic mechanisms that are incompletely correlated with humans. as an example, female sex may be associated with better outcome through the neuroprotective effect of progesterone in animal models, but these observations did not carry over to humans in the protect-iii trial (67, 68) . variable outcome measures, including neurobehavioral functional tests, glasgow outcome scale correlates, and high-resolution mri have been used in attempts to correlate animal responses to injury with those of humans. the lack of a large cache of standardized tools further limits comparison or pooling the results of different studies that use variable models of tbi or outcome measurement. transcriptomics, a genomic technique in which global rna expression is quantified through either expression microarrays or rna sequencing, has been employed to characterize specific inflammatory states following tbi. many studies have assessed the transcriptome in the acute post-tbi interval within 1-2 days after injury, with some showing upregulation of inflammation and apoptosis genes. gene ontology analysis at 3 months post-tbi have shown similar changes, with upregulation of inflammatory and immune-related genes (69) . importantly, late downregulation of ion channel expression in the peri-lesional cortex and thalamus suggests that this delayed examination of the transcriptome could be valuable for revealing mechanisms relevant to chronic tbi morbidities, including epileptogenesis and prolonged cognitive impairment (70) . in addition, tissue-specific analysis of gene diffuse axonal injury reproduces human tbi needs standardization, e.g., location of animal within shock tube and heard immobilization (65) expression across cell types in brain could provide useful insight into cell-specific pathways. for example, temporal trending of microglial expression profile indicates a biphasic inflammatory pattern that transitions from downregulation of homeostasis genes in the early stages to a mixed proinflammatory and anti-inflammatory states at subacute and chronic phases (71) . the list of antiepileptic drugs has expanded significantly in the past decade, reflecting substantial investment in the search for new therapeutics with better efficacy and tolerability. however, the list of options with demonstrated efficacy in status epilepticus (se) has remained limited. the utility of benzodiazepines, often deployed in the field as a first-line agent, decreases with increasing duration of se. in addition, 9-31% of patients with se develop refractory se when they fail to respond to first-and second-line therapy, posing a significant management and prognostic challenge (72) . the development of aeds has relied substantially on preclinical animal models to establish efficacy and safety prior to proceeding to human trials. different epilepsy models exist that are each useful for different aspects of drug development and no model is suitable for all purposes. the majority of animal models induce epilepsy using electroshock or chemical seizure induction. nearly all recent aeds have been discovered by the same conventional models, and the reliance on these common screening models has been implicated as one of the reasons for the low yield of drugs with efficacy in refractory epilepsy (73) . the pros and cons for each epilepsy model are discussed in detail in several excellent reviews (74, 75) . some of the chemicals used include kainic acid, pilocarpine, lithium, organophosphates, and flurothyl (76) . sustained electrical stimulation to specific sites, including the perforant path, the ventral hippocampus, the anterior piriform cortex can induce se (77) . the latency, length, and mortality of convulsive se are more variable in chemoconvulsant as compared to electrical models, which are in turn determined by the drug and route of administration, species, sex, age, strain, and genetic background among other factors (78) . it should also be noted that the presence of behavioral convulsion does not correlate fully with the electrographic data and vice versa. this can have c r i t i c a l i m p l i c a t i o n s w h e n s t u d y i n g d r u g s f o r pharmacoresistant se. therefore, it has been suggested that electroencephalographic quantification be used to measure the severity of se (78) . furthermore, the genetic background and expressivity of animals can have a significant effect on seizure susceptibility, even between batches of inbred mice (79) . proteomic and transcriptomic approaches have been utilized for assessment of alterations in expression profile following se, demonstrating that certain subsets of genes are upregulated at each timepoint following the onset of se. specifically, upregulation of genes regulating synaptic physiology and transcription, homeostasis and metabolism and, cell excitability and morphogenesis occur at immediate, early, and delayed timepoints. in addition, related studies have demonstrated changes in expression of micrornas related to epileptogenesis, including mirna-124 and mi-rna-128 following se (80, 81) . selective rna editing post-transcription is yet another potential source of proteomic diversity in preclinical models of se, and merits further investigation as a modulator of protein levels that may be less closely tethered to gene expression (82) . aneurysmal subarachnoid hemorrhage (sah) has an earlier age of onset and is associated with higher morbidity compared with other stroke subtypes. the pathophysiology of insult has traditionally been studied under two time-intervals, early brain injury (ebi) and, cerebral vasospasm (cv) and delayed cerebral ischemia (dci). the prime goal of translational research in this arena is to identify the mechanisms and targets related to the risk, severity, evolution and outcome. about 15% of patients die immediately following sah (83) . thereafter, early brain injury within the first 3 days, followed by dci are the most feared complications. cv is the phenomenon with strongest association with the development of dci, which 30-70% of patients experience between day 4 to 14 (84) . the underlying mechanisms leading to cv remain poorly understood and have therefore been a prime focus of preclinical studies. the majority have used rodent models, but primate, swine, and dog models have also been employed (85) . cerebral aneurysms are difficult to model and hence two common approaches to modeling sah use alternative strategies. the first is direct injection of blood into the subarachnoid space, specifically into either the prechiasmatic cistern or cisterna magna, to generate sah predominantly in the anterior or posterior circulation territories, respectively (86) . the second model, endovascular suture, passes a suture or filament through the internal carotid artery, creating a hole in one of the major branches resulting in egress of variable amount of blood into the subarachnoid space (87) . variations in some parameters of the first method, including injected blood volume, csf removal prior to injection to prevent egress of blood into the spinal canal, and replenishing intravascular volume to keep cerebral perfusion pressure constant through maintenance of mean arterial pressure, as well as the rapidity of injection have raised questions about comparability and biofidelity of the results (88) (89) (90) (91) . the latter model appears to remove some of the mentioned confounding factors, as the hemorrhage occurs at physiologic mean arterial pressure (map) and intracranial pressure (icp), but is limited by variable puncture site and ultimate hemorrhage volume. another potential drawback is the period of ischemia caused by the intraluminal suture, although the occlusion period is typically not judged to be long enough to cause significant ischemia. the missing element in these models is the absence of aneurysm formation and rupture, and consequently the vascular processes intrinsic to the aneurysm itself that influence dci. as such, some studies have used combinations of interventions to generate aneurysms, including induced hypertension via unilateral nephrectomy and administration of angiotension ii or deoxycorticosterone acetate, as well as elastase injection. the downside of these models is that the timing of aneurysm rupture cannot be reliably predicted, which limits close monitoring and physiologic assessments in the early phase following sah, blurring the timing of dci (92) (93) (94) . the immediate hemodynamic changes following the hemorrhage are monitored via a variety of methods. regardless of the method chosen, reports on the direction and range of values of cpp, cbf, and map can be quite variable, both within the same model and between different models. a common technique to measure blood flow is laser doppler flowmetry that provides a continuous measure of cortical perfusion. although it does not measure global cerebral blood flow and has spatial limitation, it appears to be relatively reliable and technically reproducible. other methods of flow measurement include radiolabeling methods and mri with the latter has the advantage of capturing the dynamic nature of the condition, as well as global and region-specific blood flows. as noted, cv and dci are responsible for delayed morbidity and mortality. given that these manifestations typically occur while patients are inpatient for care of their sah, therapeutic interventions are more feasible compared to the hyperacute phase when the processes leading to initial damage may have already occurred. however, monitoring for cv in animal models is not straightforward. one method of identifying cv is measuring the intraluminal diameter of vessels on histological samples. in addition to being an end-measure and therefore precluding measurements at different time points in the same animal, varying degrees of tissue desiccation among samples may yield numbers different from actual in vivo values. digital subtraction angiography and magnetic resonance angiography can provide a real-time evaluation, but the severity of cvand its timing, as well as neuronal cell death varies depending on the model and the affected vessels (86) . the foundational molecular pathways that orchestrate cv are complex and remain incompletely elucidated. however, translational research using many of the above models has demonstrated that endothelin-1, nitric oxide, and an inflammatory cascade ignited by breakdown of blood products play predominant roles. endothelin-1 is a potent vasoconstrictor produced by infiltrated leukocytes, and based on this notion, clazosentan was developed as an endothelin-1 receptor antagonist to combat cv. in human trials, clazosentan was found to significantly reduce the incidence of the dci without improving the functional outcome, and this or a related approach could ultimately prove beneficial if off-target drug effects, including pulmonary complications, hypotension, and anemia can be mitigated (95, 96) . hemoglobin and its degradation products are also a strong stimulus for cv through direct oxidative stress on arterial smooth muscle, decreased nitric oxide production and, increasing endothelin and free radical production (97) . this suggests that facilitating clearance of hemoglobin degradation products from the csf may be a potential therapeutic target. modulating the intense inflammatory response is also intuitive and while preclinical results support this notion in general, the evidence has thus far not been judged adequate to justify clinical trials. for example, il-1 receptor antagonist (il-1ra) reduces blood-brain barrier (bbb) breakdown, a biomarker that is itself correlated with the severity of brain injury, and work continues to determine whether this or related pathways mediating bbb permeability might have therapeutic promise (98) . given these numerous and likely interconnected mechanisms of delayed brain injury, further research is needed to understand their relative applicability to humans, and whether targeting a single pathway or a number of pathways simultaneously is likely to be the most adaptive strategy to reduce cv and dci in humans. the results of genome-wide rna sequencing analysis have supported the primary role of neuroinflammation in the pathogenesis of early brain injury. some studies have specifically found a key role for long non-coding rna (lncrna), a type of rna without protein-coding potential that are particularly abundant in the brain, in modulating the inflammatory behaviors of microglial cells (82) . high-throughput mass spectrometry has also been utilized in demonstration of differential expression of proteins in the cerebral vessels after sah, as well as for monitoring the effect of experimental therapeutics (99). we will not cover these proteomic studies in detail here, as they typically fall outside the rubric of what is classically considered "genomics", but their approach, which leverages global protein signatures rather than restricting observations to specific compounds, shares many similarities with genomics. as mentioned above, reverse genomic translation refers to an approach to the study of a disease by starting with humans using either cohort-based or case/control genomic studies. the observations made through the course of these studies then inform on the best approach for target validation and refinement to prioritize candidate mechanisms and related endophenotypes for therapeutic development. it has been shown that candidate compounds with independent confirmation of their therapeutic target via human genomics are more than twice as likely to prove effective in clinical trials (100) . therefore, the reverse translation approach would seem an adaptive strategy to identify disease-associated mechanisms and therapeutic targets with the best chance of impacting clinical care in the near term. however, the approach to reverse translation requires large sample sizes with well-characterized patient data in order to achieve a statistically confident result. these large sample sizes raise the issue of variability in risk and treatment exposures between participants, which could impact patient outcomes independently of genomic effects and therefore erode power to detect genetic risk. the utility of reverse translation in target refinement and mechanism exploration in model systems can be highlighted using an example from the stroke community. recent gwas and subsequent meta-analyses of ischemic stroke and stroke subtypes in very large case/control datasets have validated the histone deacetylase 9 (hdac9) region in chromosome 7p21.1 as a major risk locus for stroke due to large artery atheroembolism (laa). this locus was also previously discovered in association with coronary artery disease (cad) (101, 102) . based on these findings, azghandi et al. sought to investigate the role of the leading single nucleotide polymorphism (snp) in this genomic region (rs2107595) in increasing laa stroke risk (103) . they found that rs2107595, both in heterozygotes as well as in homozygote human carriers, is associated with increased expression of hdac9 in peripheral blood mononuclear cells on a dose-dependent manner, suggesting that the effect of this locus in stroke risk may be mediated by increased hdac9 expression. additionally, they demonstrated that hdac9 deficiency in mice is associated with smaller and less advanced atherosclerotic lesions in the aortic valves, curvature, and branching arteries, suggesting that hdac9 may increase atherogenesis and therefore represents a novel target for atherosclerosis and laa stroke prevention. notably, recent studies have suggested that both nonspecific (e.g. sodium valproate) as well as specific hdac9 inhibitors can have a positive impact on both stroke recurrence risk, as well as other phenotypes, including cancer. this highlights the central role that reverse translation can have in therapeutic target investigation and refinement, with potential beneficial off-target properties (104, 105) . while acute stroke care is a vital component of neurocritical care at many institutions, reverse genomic translation successes in other relevant traits also merit mention. acute respiratory distress syndrome (ards) is a frequent complication of severe neurologic injury due to sah or neurotrauma. in a recent gwas by bime et al., variation in the selectin p ligand gene (selpg), encoding p-selectin glycoprotein ligand 1 (psgl-1) was found to be associated with increased susceptibility to ards (106) . the most significant snp in this locus, rs2228315, which results in a missense mutation, has been successfully replicated in independent cohorts. further functional analyses have demonstrated that selpg expression was significantly increased in mice with ventilator (vili)-and lipoprotein (lps)-induced lung injury, and that psgl-1 inhibition with a neutralizing polyclonal antibody led to an attenuation of inflammatory response and lung injury. in selpg knockout mice, inflammatory response as well as lung injury scores were significantly reduced compared to wild-type mice (106) . these results highlight the value of reverse genomic translation in first identifying human-relevant genetic risk factors for disease, and using model systems to understand the pathways impacted by their introduction to select rationally-informed modalities for potential treatment. intracranial aneurysms (ia) are commonly encountered in the neurocritical care setting, albeit most commonly after rupture. even so, inroads leading to a better understanding of aneurysm formation may ultimately reveal opportunities for treatments to prevent acute re-rupture or prevent future aneurysm formation after sah. the strongest associations with ia have been reported in the region near cdkn2a/cdkn2b in 9p21.3 as well as in a nearby intragenic region known as cdkn2bas or anril (107, 108). anril is a long noncoding region responsible for the regulation of cdkn2a and cdkn2b and has also been implicated in the pathogenesis of cad and atherosclerosis, among other traits (109) . overexpression of anril in mouse models of cad has been associated with negative atherosclerosis outcomes including increased atherosclerosis index, unfavorable lipid profiles, thrombus formation, endothelial cell injury, overexpression of inflammatory factors in vascular endothelial cells, increased apoptosis of endothelial cells, and upregulation of apoptosis-related genes. notably, reduced anril expression has been associated with reduced inflammatory, biochemical and molecular markers of atherosclerosis, indicating a potential target for atherosclerosis and ia prevention (110) . when utilizing the reverse translation approach in genomic studies, the aforementioned examples highlight two distinct but equally important considerations for a successful implementation of such approaches. the first major consideration is that large populations of well-characterized individuals must be selected to ensure adequate statistical power to detect meaningful associations. thorough and standardized phenotyping of study subjects is one of the main predictors of the success of a gwas (111, 112) . careful assignment of cases based on strict phenotypic criteria permits well-executed gwas even in diseases with heterogeneous presentations and multiple pathogenic features, such as multiple sclerosis (ms) and stroke (113) . in neurocritical care populations where subtle characteristics of disease presentation and intermediate outcomes may represent important phenotypes for genomic investigation, such as sah, these traits should be closely defined and recorded to the greatest degree possible in all participants. this initial step is critically important in the greater scheme of reverse translational genomics, as these associations with subclasses and endophenotypes of disease often provide the biological insights needed to continue translational efforts using model systems tailored to refine observations. the second major consideration is that the execution of genomic studies needs to be comprehensive and thorough so as to permit association testing in a hypothesis-free environment. at the moment, gwas array-based studies seem to remain a favorable option of the genome, considering the lower cost associated with their utilization and proven track record in discovery, but over time, wes and wgs studies will become reachable even on more modest research budgets. for the transcriptome, rna sequencing and rna microarrays both offer unbiased surveys of global transcriptional variation, but because gene expression varies substantially by tissue it is critical that rational choices are made regarding the suitability of specific tissues for specific conditions. in uncommon conditions with necessarily small sample sizes, including neurocritical care-relevant diseases like se, sah, and ards, external validation studies can strengthen associations from an initial small discovery dataset, and in many cases these follow-up studies can make use of freely available resources. for example, in a recent expression-based gwas (egwas), microarray data for ards from the gene expression omnibus (geo) were collected and combined in an effort to identify novel genetic targets (114) . the study not only validated previously known lung injury-and ardsrelated genes, but also discovered 14 new candidate genes that may prove to be useful in future translational work. identifying loci, variants, expression patterns, and genenetworks with the use of human genomic studies is only the initial step in the reverse translation process. these discoveries must inform and guide the research to further understand and refine the phenotypic effects of these variants in model systems, including some of those described above. there are several techniques with which we can utilize the discoveries made from case/control genomic studies to build or modify model systems. one approach is transgenesis, in which a larger dna sequence including a human gene containing a mutation of interest, called a transgene, is injected into the pronucleus of a mouse fertilized egg. the fertilized egg is then inserted into the oviduct of a pseudopregnant female mouse, which is a female who has been mated with vasectomized male in order to achieve the hormonal profile of a pregnancy state. the offspring produced from this female can create an animal line that contains the human gene and allele of interest (115) . however, because the transgene is inserted randomly at one or more genetic locations as either one or more copies, the level of expression and regulatory influences of the gene of interest may not initially be well-controlled across animals. as such, there are several intermediate steps that can allow more specific genetic alteration using transgenesis, involving embryonic stem cells (escs). the first step is the introduction of regulatory sequences (such as expression cassettes) into escs. then, by injecting the transgene first into these modified escs, gene expression can be more closely controlled. the escs with the transgene can then be inserted into blastocysts and give rise to new strains, using the same methods previously described (116) . there are multiple variations on the transgenic approach which are uniquely suited to the model system being employed and can give rise to models that express transgenes in response to a particular stimulus, or in particular tissues of interest. a newer method utilizing programmable endonucleases has allowed researchers to bypass more traditional escbased methods for direct and precise gene editing. endonucleases are enzymes that cause double stranded dna (dsdna) breaks that can further be repaired either with non-homologous end-joining (nhej), an imprecise method for rejoining the dna breaks that involves various enzymes and may result in inactivating mutations, or with homologydirected repair (hdr), in which the dna breaks are repaired based on a co-injected template. four categories of programmable endonucleases have been used for direct and precise gene editing: homing endonucleases (he), zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens) and the clustered regularly interspaced short palindromic repeats/crispr-associated 9 (crispr/cas9) system. the common characteristic of these enzymes is that they possess sequence-specific nuclease activity, allowing researchers to cleave dsdna at desired, pre-specified sites. the crispr/ cas9 system has proven to be the most successful so far, in terms of efficiency, cost, and simplicity of use. perhaps the most important advantage of this approach is that programmable endonucleases do not require the use of escs and can directly be inserted into one-or two-cell stage embryos, thus allowing more specific and direct gene-editing in a single step (116) . drawbacks include enzymatic limitations as to where dna breaks can be reliably introduced, as well as off-target endonuclease activity at other sites across the genome which can disrupt gene activity in unintended ways. work is ongoing to refine these tools, improving the number of sites where gene editing can occur while also improving the specificity of the system (117). one illustrative example of human genomic studies being used to refine models to understand disease processes is the case of human ich-associated mutations in col4a1 and col4a2. col4a1 and col4a2 are the most abundant proteins in basement membranes. they form heterotrimers consisting of one col4a1 and two col4a2 peptides and are produced and modified in the endoplasmic reticulum (er). after their production, they are packaged into vesicles in the golgi apparatus and transferred to vascular endothelial basement membranes (118) (119) (120) . the initial identification of mutations in this region in familial forms of cerebral small vessel disease, coupled with the subsequent detection of common col4a1/col4a2 variants associated with sporadic deep ich led to the development of animal model systems to explore their effects (121) (122) (123) (124) . through mouse models, representative col4a1/col4a2 mutations were found to recapitulate human disease phenotypes, with multifocal ich in subcortical regions of the forebrain and the cerebellum, as well as porencephaly, small vessel disease, recurrent intraparenchymal and intraventricular hemorrhages, agerelated ich, and macro-angiopathy (125, 126) . using cellular assays and tissue derived from mouse models, mutations in col4a1/col4a2 have been associated with decreased ratio of intracellular to extracellular col4a2, retention of abnormal collagen proteins in the er, er stress, and activation of the unfolded protein response (126) (127) (128) , suggesting that the intracellular accumulation and er-stress could be an important molecular mechanism underlying ich related to col4a1 and col4a2 mutations. notably, treatment with the molecular chaperone sodium 4-phenylbutyrate resulted in decreased intracellular accumulation and significant decrease of ich severity in vivo, which could point the way towards eventual forms of treatment for both familial and sporadic col4a1and col4a2-associated ich (125) . another recent example of model system refinement for neurocritical care-relevant disorders is status epilepticus (se). pyridoxal phosphate binding protein (plpbp) variants have been associated with a rare form of b6-dependent epilepsy, which, if left untreated can lead to se. in a recent study, johnstone et al. utilized crispr/cas9 to create a zebrafish model lacking its encoded protein (129) . they observed that plpbp-deficient zebrafish experienced significantly increased epileptic activity compared to their wild type counterparts, in terms of physical activity (high-speed movements), biochemistry (c-fos expression) and electrophysiologicallyrecorded neuronal activity. additionally, treatment of plpbp â��/ â�� larvae with plp and pyridoxine led to an increase in their lifespan, and a decrease in their epileptic movements and neuronal activity. lastly, in these plpbp-deficient zebrafish, systemic concentrations of plp and pyridoxine were significantly reduced, as well as concentrations of plp-dependent neurotransmitters. collectively, these results provide insights for biomarker development and preclinical target refinement in b6-dependent epilepsy. understanding how novel treatments might impact rare disease presentations could ultimately lead to new insights for common forms of disease as well, just as the discovery of rare pcsk9 variants in patients with very low cholesterol ultimately led to pcsk9-inhibitors to treat more common forms of familial hypercholesterolemia. however, the use of animal models is not always the ideal approach to describing the effects of genetic variation, as the phenotypic alterations may be too subtle to observe or require impractical prolonged observation in late-life animals to ultimately exhibit relevant phenotypes. in these cases, tissuebased systems can provide a useful tool to study these effects. for example, ia formation, as previously described, has been associated with variants in anril. although the direct impact of these variants in human tissue or animal models is difficult to discern, work with mutations of anril in endothelial models have provided valuable insight. specifically, upregulation of anril has been associated with increased expression of inflammatory and oxidative markers in the vascular tissue such as il-6, il-8, nf-îºb, tnf-a, inos, icam-1, vcam-1, and cox-2 (130, 131) . these observations provide vital information about cellular mechanisms impacted by human disease-associated genetic risk factors without requiring the expense and time investment of creating, validating, and studying animal models. ultimately such models may still be required, but prior knowledge about cellular phenotypes associated with genetic variation may be highly valuable in choosing the right model system and selecting efficient approaches to validate these systems. the aforementioned examples highlight significant contributions of the field of translational genomics in identifying novel therapeutic targets, developing biomarkers of disease severity and elucidating disease-relevant pathophysiology. undoubtedly, these contributions are valuable in application to existing model systems of disease, or through refinement of models informed by the reverse translation process. given that many of our current models have proven to be ineffective in many cases, the reverse translation approach offers a significant advantage in that the translational discoveries arising in established or refined model systems have already been proven to be relevant to human disease. this advantage provides us with reasonable expectation that observed effects in model systems will also remain relevant to human disease, providing a substrate for therapeutic development. certainly, the ultimate goal of translational genomics is to be able to transfer the discoveries found from experimental models into clinically useful information in order to improve human health. this aim, with regard to the translational genomic approach, can be satisfied with two distinct approaches. one is concerned with improving our understanding of the mechanisms of disease, providing novel targets for therapeutic development. the other is concerned with leveraging the conclusions of translational genomics through more direct applications to clinical care. we will discuss these in order. once genomic discovery and translational exploration have confirmed the mechanism and relevance of a particular genomic association, translational genomics offers the opportunity to use these same translational approaches to derive highthroughput assays for screening of compound libraries, which are collections of small molecules useful for early-stage drug-discovery (132) . the same in vitro assays used to identify cellular phenotypes associated with genetic risk factors can be tested for amelioration or "rescue" of wild-type features after exposure to library compounds. this is particularly advantageous for the reverse genomic translation approach, as these assays are often critical components of the overall discovery cycle, and with optimization to provide ideal readouts, screening can proceed quickly. an example of success here is the identification of molecular chaperones that can ameliorate the unfolded protein response detrimental to cell survival in col4a1-mediated cerebrovascular disease (125) . identifying hits in these assays has the potential to accelerate drug-discovery, provided that the mechanism can be targeted by a small molecule and not a designed biologic entity such as a monoclonal antibody. while screening can be performed using novel compound libraries, it can also be accomplished using libraries containing already-approved drugs, providing an innovative way for compound repurposing based on genetic interactions. numerous tools already exist for in silico evaluation of existing compounds based on known mechanisms, so this step can begin even in the genomic discovery phase prior to translational validation (133) . the second approach in which translational genomics has proven to be of great potential is the rapidly evolving and highly anticipated field of precision medicine. the observations arising from translational genomics, even when not informing us about the specific mechanisms associated with the phenotype in question, may be of predictive value. this finds application in two relevant translational genomics tools: polygenic risk scores (prs) and biomarker development based on rna expression profiles. while common genetic variation can provide valuable information about disease-relevant mechanisms and help refine disease models, they are relatively weak in explaining a significant proportion of the genetic basis of complex polygenic disorders, such as cad, diabetes, stroke, or sah (111) . by summarizing the impact of many variants of small effect across the genome simultaneously, a polygenic risk score (prs) can be developed which explains far more of the genetic risk of a disease than any common variant can individually (fig. 2) . application of these prs in independent clinical populations as a predictive tool represents a novel translational approach. in a recent study examining stroke, a prs combining snps associated with atrial fibrillation (af) was found to be significantly associated with cardioembolic (ce) stroke risk and no other stroke subtypes, paving the way for a potentially useful tool to discriminate ce stroke from other etiologies without reliance on expert adjudication or longitudinal monitoring (134) . another recent study compiled a prs of cad, demonstrating that individuals in the highest quantiles of the prs exhibited cad risk on par with known mendelian cardiac diseases (135) . these studies highlight the potential uses of prs as a genetic biomarker of disease, capturing orthogonal risk information compared to clinical risk factors alone. much work is still needed in this arena, ranging from derivation of readily accessible clinical genomic testing, dissemination of prs results in an interpretable format, disclosure of off-target results that may be clinically meaningful in their own right, and, critically, the validation of prs in ancestrally-diverse populations (136) . despite these challenges, utilization of polygenic risk data to directly inform patient risk independent of our understanding of the underlying mechanisms is an exciting and rapidly evolving use-case for translational genomics. development of biomarkers is another approach in translational genomics that focuses more on predictive utilization than on elucidating mechanisms, and critical care has seen some early potential applications of this approach. in sepsis, where clinical prs percentile across the population distribution. plotting percentiles by disease risk, patients in higher prs percentiles (red dots) are at correspondingly highest risk for the disease outcomes are highly heterogenous, tools that might identify patients who are more likely to respond to certain treatments or identify individuals at highest risk for morbidity and mortality would be highly useful. in a recent study by scicluna et al., the authors categorized sepsis patients based on peripheral bloodderived genome-wide expression profiles and identified four distinct molecular endotypes (mars1-4) (137) . the mars1 expression profile was the only category that was significantly associated with 28-day and 1-year mortality. in addition, combination of the apache iv clinical score with this genetic scoring system resulted in significant improvement in 28-day mortality risk prediction, compared to apache iv alone. to further aid translation to clinical application, the authors used expression ratios of combinations of genes to stratify patients to the four molecular endotypes. bisphosphoglycerate mutase (bpgm): transporter 2, atp binding cassette subfamily b member (tap2) ratio reliably stratified patients to mars1 endotype; while other protein ratios were able to assign individuals to the other three mars categories. using this approach, not only could bpgm and tap2 transcripts potentially be used to identify patients with increased risk of mortality, but if these categorizations can be demonstrated to be causal, these molecular pathways could also be explored for therapeutic target identification and validation. further work is required to extend these findings across clinical populations, but this approach could ultimately yield new tools for prognostication in sepsis. in ischemic stroke, tissue plasminogen activator (t-pa) response and risk for hemorrhagic transformation (ht) are highly correlated with functional outcomes, and biomarkers to predict each of these would have obvious clinical utility. in a recent study, del rio-espinola et al. found that two genetic variations (rs669 and rs1801020) were associated with increased risk for ht and mortality after t-pa administration in stroke patients (138) . specifically, rs669 in a2m was associated with ht and rs1801020 was associated with in-hospital mortality. in a subsequent validation study, researchers created a genetic-clinical regression score that was successfully used to stratify stroke patients treated with t-pa based on risk for ht and parenchymal hemorrhage (ph) (139) . while in the current clinical landscape the vast majority of patients do not have readily accessible genome-wide genotypes prior to events like acute stroke, increasing uptake of clinical genomics and genomically-enabled electronic health record systems could soon enable real-time risk prediction calculations incorporating both clinical and genetic information, providing more accurate tools for clinicians to incorporate into medical decision-making. a separate set of tools that could potentially become diagnostically useful in the clinical setting is the transcriptomic approaches to identify biomarkers, using array-based screening or rna sequencing. in a recent systematic review, a total of 22 mirnas were reported to be differentially expressed in the blood cells of patients with acute ischemic stroke within 24 h after stroke (140) . some studies reported the area under the curve (auc) ranging from 0.76 to 0.987, indicating a potential for clinical utility as early diagnostic markers when neuroimaging is not immediately available or is limited by feasibility. subsequent studies were able to partially replicate these findings, showing three mirnas (mir-125a-5p, mir-125b-5p, and mir-143-3p) that were upregulated in the acute poststroke period, an effect independent of stroke pathophysiology and infarct volume (141) . these transcripts were associated with an auc of 0.90 in differentiating ischemic stroke and healthy controls, a metric that significantly outperformed computed tomography, as well as previously reported bloodbased biomarkers. in ich, a recent report identified up to 489 and 256 transcripts from whole blood that are differentially expressed between ich and controls and, ich and ischemic stroke, respectively (142) . when comparing ich and ischemic stroke transcriptomes in the first 24 h, 2667 and 311 transcripts were differentially expressed compared to controls, respectively. ich transcriptome was over-represented by t cell receptor genes compared to none for ischemic stroke and underrepresented by non-coding and antisense transcripts. t cell receptor expression successfully differentiated between ich, ischemic stroke, and controls. similarly, rna-seq of whole blood rna successfully differentiated between not only ich, ischemic stroke and controls, but also between different stroke subtypes (143). the list of genetic mutations that can cause se is extensive with most genes are associated with infantile-onset or childhood epilepsy syndromes. only a minority are seen in adultonset status epilepticus (144) . the patients in the former group usually have accompanying intellectual disability related to their epilepsy syndromes. however, the evidence supporting a genetic etiology in the latter group may be absent, posing a diagnostic challenge. the available options include gene panel sequencing, whole exome sequencing, or whole genome sequencing. sequencing a pre-selected panel of genes is more common, but with decreasing cost, exome and genome sequencing are being used with increasing frequency. bioinformatic filtering and genotype-phenotype correlation are the main challenges, particularly with the large number of genetic variants identified during whole exome or genome sequencing. the yield of sequencing studies depends on pretest probability that is determined by early age of onset, consanguinity, or affected siblings. as such, to date, only a few genes associated with adult-onset se have been identified, posing a practical limitation that predominantly limits next-generation sequencing to pediatric patients at present (144) . as clinical tools for determination of putative functional significance and deleteriousness of variants identified through sequencing are refined, it is hoped that sequencing approaches find a home in the armamentarium of the clinicians treating refractory or recurrent se in the neurocritical care unit. translational genomics undoubtedly represents an important component to overall efforts to improve our understanding of the diseases we treat, and in principle should improve our ability to identify therapeutic approaches to improve outcomes and, in some cases, prevent disease altogether. given the inherent complexity and inaccessibility of the human brain and its tissues, combined with the relative infrequency of the conditions we treat at the overall population level, progress has been modest when compared to conditions such as hyperlipidemia (145) , coronary artery disease (146) , or atrial fibrillation (147) . nevertheless, the observation-based, hypothesisfree experimental process inherent to translational genomics lends itself well to conditions such as stroke and tbi in which the search for the "master regulator" that governs response to injury has remained elusive despite carefully designed and executed hypothesis-driven studies. an important component to future translational genomic studies in neurocritical care is the pressing need for collaboration across centers with access to large, well-characterized patient populations. the success of the international stroke genetics consortium, and the track-tbi and center-tbi consortia in amassing large human populations with stroke and traumatic brain injury, respectively, is a proven model to accelerate the human genomic studies that serve as the basis for reverse genomic translational research (112, (148) (149) (150) . similar efforts through the critical care eeg monitoring research consortium and other partners could lead to biorepositories of specific conditions relevant to neurocritical care that could provide sample sizes sufficient to drive unbiased genomic discoveries (151) . close alliances with model systems researchers are another critical component to accelerating translational genomics in neurocritical care. as characterization of human disease through multimodal and continuous physiological monitoring, electrophysiology, medical imaging, and biomarker sampling continues to evolve, it is imperative that this information is shared and explored with allied model systems researchers to ensure that models are re-evaluated for their correlation with these endophenotypes, and potentially for dedicated exploration of how these human-derived phenotypes inform on the utility of specific model systems to investigate disease. finally, building relationships with biotechnology and pharmaceutical industry partners will be essential to efforts to extend therapeutic targets arising from translational genomic discoveries towards drug development (152) . while repurposing existing drug compounds for new indications is an important consideration, small molecule and biologic targets are likely to require extensive research and development in the preclinical and clinical space, and industry partners are often optimized for these phases of the therapeutic development process (153, 154) . relatedly, development of polygenic risk scores for assessment of risk, prognosis, or treatment response will also require commercial investment and infrastructure, as few academic environments exist that can manage cliacertified genotyping, quality control, and result reporting and interpretation for on-target and clinically relevant secondary results (155, 156) . particularly in rarer or particularly challenging disease indications like those commonly encountered in neurocritical care populations, academic-industry partnerships are important to raise awareness of and interest in important clinical indications where investment could yield a large impact on a relatively small population of patients. translational genomics, in which genomic associations with risk, outcome, or treatment response are systematically identified and explored for functional relevance in humans or model systems of disease, is a valuable tool for identification of mechanisms, risk factors, therapeutic targets, and risk estimates in multiple diseases that are highly relevant to clinicians and scientists operating in the neurocritical care space. while there are undoubtedly challenges to studying some of the most complex diseases that affect the most complex organ in the body, translational genomic approaches may be uniquely suited to this task. coordinated investments in the collaborations, consortia, and infrastructures that enable these studies are likely to contribute to 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proteomic expression changes in large cerebral arteries after experimental subarachnoid hemorrhage in rat are regulated by the mek-erk1/2 pathway the support of human genetic evidence for approved drug indications genetic risk factors for ischaemic stroke and its subtypes (the metastroke collaboration): a meta-analysis of genome-wide association studies genome-wide association study identifies a variant in hdac9 associated with large vessel ischemic stroke deficiency of the stroke relevant hdac9 gene attenuates atherosclerosis in accord with allele-specific effects at 7p21.1 sodium valproate, a histone deacetylase inhibitor, is associated with reduced stroke risk after previous ischemic stroke or transient ischemic attack identification of hdac9 as a viable therapeutic target for the treatment of gastric cancer genome-wide association study in african americans with acute respiratory distress syndrome identifies the selectin p ligand gene as a risk factor genome-wide association study of intracranial aneurysms confirms role of anril and sox17 in disease risk genome-wide association study of intracranial aneurysm identifies three new risk loci anril: a lncrna at the cdkn2a/b locus with roles in cancer and metabolic disease effect of circular anril on the inflammatory response of vascular endothelial cells in a rat model of coronary atherosclerosis. cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology genome-wide association studies recommendations from the international stroke genetics consortium, part 1: standardized phenotypic data collection genes associated with multiple sclerosis: 15 and counting. expert review of molecular diagnostics identification of new biomarkers for acute respiratory distress syndrome by expression-based genome-wide association study technical approaches for mouse models of human disease generating mouse models for biomedical research: technological advances. disease models & mechanisms potential pitfalls of crispr/cas9-mediated genome editing the role of secretory granules in the transport of basement membrane components: radioautographic studies of rat parietal yolk sac employing 3h-proline as a precursor of type iv collagen monoclonal antibodies against chicken type iv and v collagens: electron microscopic mapping of the epitopes after rotary shadowing basement membrane (type iv) collagen is a heteropolymer col4a1 mutations as a monogenic cause of cerebral small vessel disease: a systematic review common variation in col4a1/col4a2 is associated with sporadic cerebral small vessel disease review: molecular genetics and pathology of hereditary small vessel diseases of the brain genome-wide association study of cerebral small vessel disease reveals established and novel loci molecular and genetic analyses of collagen type iv mutant mouse models of spontaneous intracerebral hemorrhage identify mechanisms for stroke prevention col4a2 mutations impair col4a1 and col4a2 secretion and cause hemorrhagic stroke abnormal expression of collagen iv in lens activates unfolded protein response resulting in cataract col4a1 mutation causes endoplasmic reticulum stress and genetically modifiable ocular dysgenesis. human molecular genetics plphp deficiency: clinical, genetic, biochemical, and mechanistic insights interfering with long chain noncoding rna anril expression reduces heart failure in rats with diabetes by inhibiting myocardial oxidative stress the interplay of lncrna anril and mir-181b on the inflammation-relevant coronary artery disease through mediating nf-kappab signalling pathway compound libraries: recent advances and their applications in drug discovery. current drug discovery technologies multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes atrial fibrillation genetic risk differentiates cardioembolic stroke from other stroke subtypes genome-wide polygenic scores for common diseases identify individuals with risk equivalent to monogenic mutations predicting polygenic risk of psychiatric disorders classification of patients with sepsis according to blood genomic endotype: a prospective cohort study. the lancet respiratory medicine a predictive clinicalgenetic model of tissue plasminogen activator response in acute ischemic stroke validation of a clinical-genetics score to predict hemorrhagic transformations after rtpa journal of stroke and cerebrovascular diseases : the official journal of national stroke association rna-seq identifies circulating mir-125a-5p, mir-125b-5p, and mir-143-3p as potential biomarkers for acute ischemic stroke the intracerebral hemorrhage blood transcriptome in humans differs from the ischemic stroke and vascular risk factor control blood transcriptomes intracerebral hemorrhage and ischemic stroke of different etiologies have distinct alternatively spliced mrna profiles in the blood: a pilot rna-seq study genetic mutations associated with status epilepticus association of genetic variants related to cetp inhibitors and statins with lipoprotein levels and cardiovascular risk pcsk9: from basic science discoveries to clinical trials rare truncating variants in the sarcomeric protein titin associate with familial and early-onset atrial fibrillation recommendations from the international stroke genetics consortium, part 2: biological sample collection and storage investigators t-t. outcome prediction after mild and complicated mild traumatic brain injury: external validation of existing models and identification of new predictors using the track-tbi pilot study the center-tbi core study: the making-of comparison of machine learning models for seizure prediction in hospitalized patients developing interactions with industry in rare diseases: lessons learned and continuing challenges. genetics in medicine : official journal of the american college of medical genetics drug repurposing from the perspective of pharmaceutical companies insights into computational drug repurposing for neurodegenerative disease clinical providers' experiences with returning results from genomic sequencing: an interview study recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (acmg sf v2.0): a policy statement of the american college of medical genetics and genomics publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-265857-fs6dj3dp authors: liu, yu-tsueng title: infectious disease genomics date: 2010-12-24 journal: genetics and evolution of infectious disease doi: 10.1016/b978-0-12-384890-1.00010-8 sha: doc_id: 265857 cord_uid: fs6dj3dp the history and development of infectious disease genomics are discussed in this chapter. hgp must not be restricted to the human genome and should include model organisms including mouse, bacteria, yeast, fruit fly, and worm. the completed or ongoing genome projects will provide enormous opportunities for the discovery of novel vaccines and drug targets against human pathogens as well as the improvement of diagnosis and discovery of infectious agents and the development of new strategies for invertebrate vector control. the polysaccharide capsule is important for meningococci to escape from complement-mediated killing. with the completion of the genome sequence of a virulent menb strain, a “reverse vaccinology” approach was applied for the development of a universal menb vaccine by novartis. the indispensable fatty acid synthase (fas) pathway in bacteria has been regarded as a promising target for the development of antimicrobial agents. through a systematic screening of 250,000 natural product extracts, a merck team identified a potent and broad-spectrum antibiotic, platensimycin, which is derived from streptomyces platensis. vector biology network was formed to achieve three goals (1) to develop basic tools for the stable transformation of anopheline mosquitoes by the year 2000; (2) to engineer a mosquito incapable of carrying the malaria parasite by 2005; and (3) to run controlled experiments to test how to drive the engineered genotype into wild mosquito populations by 2010. the most immediate impact of a completely sequenced pathogen genome is for infectious disease diagnosis. the history and development of infectious disease genomics are tightly associated with the human genome project (hgp) (watson, 1990) . a series of important discussions about the hgp were made in 1985 and 1986 (dulbecco, 1986; watson, 1990) , which led to the appointment of a special national research council (nrc) committee by the national academy of sciences to address the needs and concerns, such as its impact, leadership, and funding sources. the committee recommended that the united states begin the hgp in 1988 (nrc, 1988) . they emphasized the need for technological improvements in the efficiency of gene mapping, sequencing, and data analysis capabilities. in order to understand potential functions of human genes through comparative sequence analyses, they also advised that the hgp must not be restricted to the human genome and should include model organisms including mouse, bacteria, yeast, fruit fly, and worm. in the meantime, the office of technology assessment (ota) of the us congress also issued a similar report to support the hgp (ota, 1988) . in 1990, the department of energy (doe) and the national institutes of health (nih) jointly presented an initial 5-year plan for the hgp (dhhs and doe, 1990) . in october 1993, the sanger center/institute (hinxton, uk) was officially open to join the hgp. the cost of dna sequencing was about $2à5 per base in 1990, and the initial aim was to reduce the costs to less than $0.50 per base before large-scale sequencing (dhhs and doe, 1990) . the sequencing cost gradually declined during the subsequent years. in 2004, the national human genome research institute (nhgri) challenged scientists to achieve a $100,000 human genome (3 gb/haploid genome) by 2009 and a $1000 genome by 2014 to meet the need of genomic medicine. the first complete genome to be sequenced was the phix174 bacteriophage (5.4 kb) by sanger's group in 1977 (sanger et al., 1977 . the complete genome sequence of sv40 polyomavirus (5.2 kb) was published in 1978 (fiers et al., 1978; reddy et al., 1978) . the human epsteinàbarr virus (170 kb) genome was determined in 1984 (baer et al., 1984) . the first completed free-living organism genome was *e-mail: ytliu@ucsd.edu haemophilus influenza (1.8 mb), sequenced through a whole-genome shotgun approach in 1995 (fleischmann et al., 1995) . the second sequenced bacterial genome, mycoplasma genitalium (600 kb), was completed in less than a month in the same year using the same approach (smith, 2004) . the doe was the first to start a microbial genome program (mgp) as a companion to its hgp in 1994 (doe, 2009 . the initial focus was on nonpathogenic microbes. along with the development of the hgp, there was exponential growth of the number of completely sequenced freeliving organism genomes. the fungal genome initiative (fgi) (fgi, 2010) was established in 2000 to accelerate the slow pace of fungal genome sequencing since the report of the genome of saccharomyces cerevisiae in 1996 (goffeau et al., 1996) . one of the major interests was to sequence organisms that are important in human health and commercial activities. as of september 2009, 1100 completed genome projects, a 1.7-fold increase from 2 years ago, were documented (liolios et al., 2010) . these include 914 bacterial, 68 archaeal, and 118 eukaryotic genomes. in addition, more than 4000 other ongoing sequencing projects were reported. the genomes of human malaria parasite plasmodium falciparum and its major mosquito vector anopheles gambiae were published in 2002 (gardner et al., 2002; holt et al., 2002) . the effort to sequence the malaria genome began in 1996 by taking advantage of a clone derived from laboratory-adapted strain (hoffman et al., 1997) . many parasites have complex life cycles that involve both vertebrate and invertebrate hosts and are difficult to maintain in the laboratory. currently, a few other important human pathogenic parasites, such as trypansomes el-sayed et al., 2005) , leishmania (ivens et al., 2005) , and schistosomas (berriman et al., 2009; consortium, 2009) , have been either completely or partially sequenced (brindley et al., 2009; aurrecoechea et al., 2010) . in the meantime, the genome sequence of aedes aegypti, the primary vector for yellow fever and dengue fever, was published in 2007 . the genome size (1376 mb) of this mosquito vector is about 5 times larger than the previously sequenced genome of the malaria vector anopheles gambiae. approximately 50% of the genome consists of transposable elements. in 2010, the genome sequence of the body louse (pediculus humanus humanus), an obligatory parasite of humans and the main vector of epidemic typhus (rickettsia prowazekii), relapsing fever (borrelia recurrentis), and trench fever (bartonella quintana), was reported (kirkness et al., 2010) . its 108 mb genome is the smallest among the known insect genomes. genome sequencing projects for other important human disease vectors are in progress megy et al., 2009 ). these include culex pipiens (mosquito vector of west nile virus), ixodes scapularis (tick vector of lyme disease, babesia, and anaplasma), and glossina morsitans (tsetse fly vector of african trypanosomiasis). the challenge to sequence the genome of an insect vector is much greater than a microbe. for example, the genomes of ticks were estimated to be between 1 and 7 gb and may have a significant proportion of repetitive dna sequences, which may be a problem for genome assembly (pagel van zee et al., 2007) . furthermore, the evolutionary distances among insect species may also affect homology-based gene predictions. it is as important to understand the sequence diversity within a species as to perform a de novo sequencing of a reference genome from the perspective of human health. this is true for both hosts and pathogens (feero et al., 2008; alcais et al., 2009) . the goal of the 1000 genomes project is to find most genetic variants that have frequencies of at least 1% in the human populations studied (kaiser, 2008) . one of the similar efforts for human pathogens is the nih influenza genome sequencing project. when this project began in november 2004, only seven human influenza h3n2 isolates had been completely sequenced and deposited in the genbank database (fauci, 2005; ghedin et al., 2005) . as of may 2010, more than 5000 human and avian isolates have been completely sequenced, including the 1918 "spanish" influenza virus (taubenberger et al., 2005) . databases for human immunodeficiency virus (hiv) and hepatitis c virus have also been established. while most human studies of microbes have focused on the disease-causing organisms, interest in resident microorganisms has also been growing. in fact, it has been estimated that the human body is colonized by at least 10 times more prokaryotic and eukaryotic microorganisms than the number of human cells (savage, 1977) . it was suggested to have "the second human genome project" to sequence human microbiome (relman and falkow, 2001) . highly variable intestinal microbial flora among normal individuals has been well documented (eckburg et al., 2005; costello et al., 2009; turnbaugh et al., 2009) . therefore, the human microbiome project (hmp) was initiated by the nih to study samples from multiple body sites from each of at least 250 "normal" volunteers to determine whether there are associations between changes in the microbiome and several different medical conditions, and to provide both standardized data resources and new technological approaches (peterson et al., 2009) . the completed or ongoing genome projects (table 10 .1) will provide enormous opportunities for the discovery of novel vaccines and drug targets against human pathogens as well as the improvement of diagnosis and discovery of infectious agents and the development of new strategies for invertebrate vector control. specific examples will be provided to illustrate how the information provided by various genome projects may help achieve the goal of promoting human health. meningococcal isolates produce 1 of 13 antigenically distinct capsular polysaccharides, but only 5 (a, b, c, w135, and y) are commonly associated with disease (lo et al., 2009) . the polysaccharide capsule is important for meningococci to escape from complement-mediated killing. while conventional vaccines consisting of the conjugation of capsular polysaccharides to carrier proteins for meningococcus serogroups a, c, y, and w-135 have been clinically successful, the same approach failed to produce clinically useful vaccine for serogroup b (menb). the capsule polysaccharide (α2-8 n-acetylneuraminic acid) of menb is identical to human polysialic acid and therefore is poorly immunogenic (finne et al., 1987) . alternatively, vaccines consisting of outer membrane vesicles (omv) have been successfully developed to control menb outbreaks in areas where epidemics are dominated by one particular strain (bjune et al., 1991; sierra et al., 1991; boslego et al., 1995; jackson et al., 2009) . the most significant limitation of this type of vaccine is that the immune response is strain-specific, mostly directed against the porin protein, pora, which varies substantially in both expression level and sequence across strains (martin et al., 2000; pizza et al., 2000) . with the completion of the genome sequence of a virulent menb strain, a "reverse vaccinology" approach was applied for the development of a universal menb vaccine by novartis (pizza et al., 2000; tettelin et al., 2000; giuliani et al., 2006) . through bioinformatic searching for surface-exposed antigens, which may be the most suitable vaccine candidates due to their potential to be readily recognized by the immune system, 570 open reading frames (orfs) were selected from a total of 2158 orfs of the mc58 genome. eventually, five antigens were chosen as the vaccine components based on a series of criteria including the ability of candidates to be expressed in escherichia coli as recombinant proteins (350 candidates), the confirmation of surface exposure by immunological analyses, the ability of induced protective antibodies in experimental animals (28 candidates), and the conservation of antigens within a panel of diverse meningococcal strains, primarily the disease-associated menb strains (pizza et al., 2000; giuliani et al., 2006; rinaudo et al., 2009) . the vaccine formulation consists of an fhbp-gna2091 fusion protein, a gna2132-gna1030 fusion protein, nada, and omv from the new zealand menzb vaccine strain, which contains the immunogenic pora. initial phase ii clinical results in adults and infants showed that this vaccine could induce a protective immune response against three diverse menb strains in 89à96% of subjects following three vaccinations and 93à100% after four vaccinations (rinaudo et al., 2009) . in 2010, a phase iii trial for this vaccine (4cmenb) has met primary endpoint. targeting an essential pathway is a necessary but not sufficient requirement for an effective antimicrobial agent (brinster et al., 2009) . identification of essential genes in a completely sequenced genome has been actively pursued with various approaches (hutchison et al., 1999; ji et al., 2001) . the indispensable fatty acid synthase (fas) pathway in bacteria has been regarded as a promising target for the development of antimicrobial agents (wright and reynolds, 2007) . the subcellular organization of the fatty acid biosynthesis components is different between mammals (type i fas) and bacteria (dissociated type ii fas), which raises the likelihood of host specificity of the targeting drugs. comparison of the available genome sequences of various species of prokaryotes reveals highly conserved fas ii systems suggesting that the antimicrobial agent can be broad spectrum (zhang et al., 2003) . in addition, through computational analyses, new members of the fas ii system have been discovered in different bacterial species (heath and rock, 2000; marrakchi et al., 2002) . one of the protein components in this system, fabi, is the target of an anti-tuberculosis drug isoniazid and a general antibacterial and antifungal agent, triclosan (banerjee et al., 1994; levy et al., 1999; zhang et al., 2006) . through a systematic screening of 250,000 natural product extracts, a merck team identified a potent and broad-spectrum antibiotic, platensimycin, which is derived from streptomyces platensis and a selective fabf/b inhibitor in fas ii system (wang et al., 2006) . treatment with platensimycin eradicated staphylococcus aureus infection in mice. platensimycin did not have cross-resistance to other antibiotic-resistant strains in vitro, including methicillin-resistant s. aureus, vancomycin-intermediate s. aureus, and vancomycin-resistant enterococci. no toxicity was observed using a cultured human cell line. the activity of platensimycin was not affected by the presence of human serum in this study. however, the fas ii system appears to be dispensable for another gram-positive bacterium, streptococcus agalactiae, when exogenous fatty acids are available, such as in human serum (brinster et al., 2009; balemans et al., 2010) . the susceptibility to inhibitors targeting the fas ii system indicates heterogeneity in fatty acid synthesis or in acquiring exogenous fatty acids among gram-positive pathogens (balemans et al., 2010) . comparative genomic approaches may be useful to identify and develop a strategy to target the salvage pathway for streptococcus agalactiae. alternatively, similar approaches as described earlier for menb vaccine may also be applied for streptococcus agalactiae (group b streptococcus) (maione et al., 2005) . an early mathematical model for malaria control suggested that the most vulnerable element in the malaria cycle was survivorship of adult female mosquitoes (macdonald, 1957; enayati and hemingway, 2010) . therefore, insect control is an important part of reducing transmission. the use of ddt as an indoor residual spray in the global malaria eradication program from 1957 to 1969 reduced the population at risk of malaria to b50% by 1975 compared with 77% in 1900 (hay et al., 2004; enayati and hemingway, 2010) . engineering genetically modified mosquitoes refractory to malaria infection appeared to be an alternative approach (curtis, 1968) given the environmental impact of ddt and the emergence of insecticide-resistant insects. the vector biology network (vbn) was formed in 1989 and proposed a 20-year plan with the world health organization (who) in 2001 to achieve three major goals: (1) to develop basic tools for the stable transformation of anopheline mosquitoes by the year 2000; (2) to engineer a mosquito incapable of carrying the malaria parasite by 2005; and (3) to run controlled experiments to test how to drive the engineered genotype into wild mosquito populations by 2010 (alphey et al., 2002; morel et al., 2002; beaty et al., 2009) . while some proof-of-concept experiments were achieved for the first two aims in 2002 when the anopheles gambiae genome was completely sequenced (catteruccia et al., 2000; ito et al., 2002) , the progress has been relatively slow (marshall and taylor, 2009) . genomic loci of the anopheles gambiae responsible for plasmodium falciparum resistance have been identified through surveying a mosquito population in a west african malaria transmission zone (riehle et al., 2006) . a candidate gene, anopheles plasmodium-responsive leucine-rich repeat 1 (apl1), was discovered. subsequently, other resistant genes have also been identified (blandin et al., 2009; povelones et al., 2009) . studying the genetic basis of resistance to malaria parasites and immunity of the mosquito vector will be important to control malaria transmission. perhaps the most immediate impact of a completely sequenced pathogen genome is for infectious disease diagnosis. the information may be of great importance to the public health when a newly emerged or re-emerged pathogen is discovered. the 2009 swine-origin influenza a virus (s-oiv) (dawood et al., 2009) and 2003 sars (severe acute respiratory syndrome) coronavirus rota et al., 2003) are the two most recent examples. s-oiv emerged in the spring of 2009 in mexico and was also discovered in specimens from two unrelated children in the san diego area in april 2009 (cdc, 2009; dawood et al., 2009) . those samples were positive for influenza a but negative for both human h1 and h3 subtypes. the complete genome sequence and a real-time pcr-based diagnostic assay were released to the public in late april. the outbreak evolved rapidly and the who declared the highest phase 6 worldwide pandemic alert on june 11, 2009. s-oiv has three genome segments (ha, np, ns) from the classic north american swine (h1n1) lineage, two segments (pb2, pa) from the north american avian lineage, one segment (pb1) from the seasonal h3n2, and most notably, two segments (na, m) from the eurasian swine (h1n1) lineage (dawood et al., 2009) . with the available influenza genome database, diagnostic assays to distinguish previous seasonal h1n1, h3n2, and s-oiv can be easily accomplished (lu et al., 2009) . a comprehensive pathogen genome database is not only useful for infectious disease diagnosis but also for novel pathogen discovery (liu, 2008) . homologous sequences within the same family or among different family members are important for new pathogen identification even with the advent of third-generation sequencing technology (munroe and harris, 2010) . de novo pathogen discovery may be also complicated by coexisting microorganisms, such as commensal bacteria in the human body. without prior knowledge of these microorganisms, one may be misled. in 2003, a microarray-based assay, designated virochip, was used to help discover the sars coronavirus (wang et al., 2003) . the virochip contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in genbank. the computational search for conservation was performed across all known viral families. a microarray hybridized with a reaction derived from a viral isolate cultivated from a sars patient revealed that the strongest hybridizing array elements belong to families astroviridae and coronaviridae. alignment of the oligonucleotide probes having the highest signals showed that all four hybridizing oligonucleotides from the astroviridae and one oligonucleotide from avian infectious bronchitis virus, an avian coronavirus, shared a core consensus motif spanning 33 nucleotides. interestingly, it had been known previously through bioinformatic analyses that this sequence is present in the 3 0 utr of all astroviruses, avian infectious bronchitis virus, and an equine rhinovirus (jonassen et al., 1998) . therefore, a new member of the coronavirus was identified through the unique hybridizing pattern and subsequent confirmations. the finding of the seventh human oncogenic virus, merkel cell polyomavirus (mcv) (feng et al., 2008) in 2008 is another example of why conserved sequences are important for novel pathogen discovery. mcv is the etiological agent of merkel cell carcinoma (mcc), which is a rare but aggressive skin cancer of neuroendocrine origin. two cdna libraries derived from mcc tumors were subjected to high-throughput sequencing by a next-generation roche/454 sequencer. nearly 400,000 sequence reads were generated. the majority (99.4%) of the sequences derived from human origin were removed from further analyses. only one of the remaining 2395 cdna was homologous to the t antigen of two known polyomaviruses. one additional cdna was subsequently identified to be part of the mcv sequence when the complete viral sequence was known. later analyses showed that 80% (8/10) of the mcc had integrated mcv in the human genome. monoclonal viral integration was revealed by the patterns of southern blot analysis. only 8à16% of control tissues had low copy number of mcv infection. while we can expect that the efforts of a variety of genome projects may improve human health, the socioeconomic issues that are not discussed in this chapter may be substantial. in addition, the tremendous amount of information derived from these projects will also be a challenge for scientists as well nonscientists to follow and understand. human genetics of infectious diseases: between proof of principle and paradigm malaria control with genetically manipulated insect vectors eupathdb: a portal to eukaryotic pathogen databases dna sequence and expression of the b95-8 epsteinàbarr virus genome essentiality of fasii pathway for staphylococcus aureus inha, a gene encoding a target for isoniazid and ethionamide in mycobacterium tuberculosis the influenza virus resource at the national center for biotechnology information from tucson to genomics and transgenics: the vector biology network and the emergence of modern vector biology the genome of the african trypanosome trypanosoma brucei the genome of the blood fluke schistosoma mansoni effect of outer membrane vesicle vaccine against group b meningococcal disease in norway dissecting the genetic basis of resistance to malaria parasites in anopheles gambiae efficacy, safety, and immunogenicity of a meningococcal group b (15:p1.3) outer membrane protein vaccine in iquique, chile. chilean national committee for meningococcal disease helminth genomics: the implications for human health type ii fatty acid synthesis is not a suitable antibiotic target for gram-positive pathogens stable germline 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mc58 a core gut microbiome in obese and lean twins viral discovery and sequence recovery using dna microarrays platensimycin is a selective fabf inhibitor with potent antibiotic properties the human genome project: past, present, and future antibacterial targets in fatty acid biosynthesis the application of computational methods to explore the diversity and structure of bacterial fatty acid synthase inhibiting bacterial fatty acid synthesis key: cord-264255-q5izs39f authors: chieochansin, thaweesak; samransamruajkit, rujipat; chutinimitkul, salin; payungporn, sunchai; hiranras, thitikul; theamboonlers, apiradee; poovorawan, yong title: human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization date: 2007-12-31 journal: j infect doi: 10.1016/j.jinf.2007.11.006 sha: doc_id: 264255 cord_uid: q5izs39f objective: human bocavirus (hbov), a novel virus, which based on molecular analysis has been associated with respiratory tract diseases in infants and children have recently been studied worldwide. to determine prevalence, clinical features and perform phylogenetic analysis in hbov infected thai pediatric patients. methods: hbov was detected from 302 nasopharyngeal (np) suctions of pediatric patients with acute lower respiratory tract illness and sequenced applying molecular techniques. results: the incidence of hbov infection in pediatric patients amounted to 6.62% with 40% co-infected with other respiratory viruses. there were no clinical specific manifestations for hbov; however, fever and productive cough were commonly found. generalized rales and wheezing were detected in most of the patients as well as perihilar infiltrates. the alignment and phylogenetic analysis of partial vp1 genes showed minor variations. conclusion: our results indicated that hbov can be detected in nasopharyngeal aspirate specimens from infants and children with acute lower respiratory tract illness. acute respiratory tract infection is a major cause of pediatric morbidity and mortality worldwide. in most cases, viruses including influenza a and b, parainfluenza viruses, adenoviruses, respiratory syncytial virus (rsv), and human metapneumovirus (hmpv) are the causative agents. recently, a new respiratory tract virus of the parvoviridae family, human bocavirus (hbov), has been discovered applying molecular analysis on pooled respiratory tract aspirations taken from children in sweden. this virus is closely related to the bovine parvovirus and canine minute virus, which have been classified as members of the genus bocavirus. the virus comprises two major open reading frames (orfs) encoding a nonstructural protein (ns1) and at least two capsid proteins (vp1 and vp2), respectively. moreover, the hbov genome also contains a third middle orf encoding a nonstructural protein (np1) of unknown function. 1 the most conserved region of this virus is the ns1 and np1 gene whereas the vp1/vp2 gene constitutes the variable region. 2 the induction of respiratory illness by hbov is not clearly defined due to lack of propagation techniques in cell culture or animal models. 1 however, many studies have reported this virus infection to be associated with acute respiratory illness. 1, 3, 4 upon discovery of hbov in respiratory pools, its global prevalence has been reported to range from 1.5% to 19% and co-infection with other viruses was commonly found. 1,3e24 moreover, in few studies were shown negative results for hbov infection in nasopharyngeal (np) swabs from healthy volunteers. 20, 25, 26 additional epidemiological and clinical investigation will be essential in order to elucidate what exactly engenders hbov related illness. therefore, in the present study we applied polymerase chain reaction to detect hbov from np suctions collected from infants or children who had been admitted with respiratory tract illness. nasopharyngeal suction specimens were collected from 302 individual infants or children (age range: 5 days to 14 years) who were admitted and diagnosed as having acute lower respiratory illness during the period february 14, 2006 to february 28, 2007 . all of the clinical samples were provided by the department of pediatrics, king chulalongkorn memorial hospital, thailand. nasopharyngeal suction samples were collected in transport medium with antibiotics (0.5% bsa, penicillin g (2 â 10 6 u/l), streptomycin 200 mg/l) and stored at à70 c until tested. the study was conducted after receiving approval by the ethics committee of the faculty of medicines, chulalongkorn university. prior to enrolment, all participating parent gave their written informed consent. dna and rna were extracted from 150 ml of np suction using tri reagent â ls (molecular research center inc., cincinnati, oh) and solubilized in 20 ml of 8 mm naoh or 12 ml of depc treated water for dna or rna, respectively. for hbov detection we amplified the np1 gene by conventional pcr modified from a previous study. 1 the reaction mixture contained 2 ml dna, 0.5 mm 188f primer and 0.5 mm 542r primer, 10 ml 2.5â eppendorf mastermix (eppendorf, hamburg, germany), and nuclease-free water to a final volume of 25 ml. the amplification reaction was performed in a thermocycler (eppendorf) under the following conditions: initial denaturation at 94 c for 3 min, followed by 35 amplification cycles consisting of 94 c for 30 s (denaturation), 55 c for 30 s (primer annealing), and 72 c for 1 min (extension), and concluded by a final extension step at 72 c for 7 min. another set of primers specific for the vp1 gene, vpf2 forward primer (5 0 -ttcagaatggt cacctctaca-3 0 : nt 3639e3659) and vpr2 reverse primer (5 0 -ctgtgcttccgttttgtctta-3 0 : nt 4286e4266), were used in a separate pcr reaction to exclude false positives. after 2% agarose gel electrophoresis stained with ethidium bromide, the expected products of 354 and 648 bp representing the np1 and vp1 gene, respectively, were visualized on a uv transilluminator. influenza a virus detection was performed by conventional pcr using 1 ml of cdna, 0.5 mm of flua_m_f: 5 0 -rggccccctcaaagccga-3 0 (nt 76e93), 0.5 mm of flua_m_r: 5 0 -actgggcacggtgagygt-3 0 (nt 235e218), 10 ml of 2.5â eppendorf mastermix, and nuclease-free water to a final volume of 25 ml. the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) of each sample was amplified in one additional reaction mixture of identical volume with primers gapdh_f: 5 0 -gtg aaggtcggagtcaacgg-3 0 (nt 112e131) and gapdh_r: 5 0 -gttgtcatggatgaccttggc-3 0 (nt 603e583) at a 0.5 mm concentration, each. the amplification reaction was performed in a thermocycler (eppendorf) under the following conditions: initial denaturation at 94 c for 3 min, followed by 40 amplification cycles consisting of 94 c for 30 s (denaturation), 55 c for 30 s (primer annealing), and 72 c for 1 min (extension), and concluded by a final extension step at 72 c for 7 min. after 2% agarose gel electrophoresis, the expected products were influenza a virus (160 bp) and the housekeeping gene (492 bp). parainfluenza virus was detected by real-time pcr using sybr green i carried out in a rotor-gene 3000 (corbett research, new south wales, australia). the reaction mixture contained 1 ml of dna sample, 10 ml 2.5â eppendorf mastermix, 0.5 mm paraf: 5 0 -gctaaatactgtcttmah tggagat-3 0 (nt 11,254e11,278), 0.5 mm parar: 5 0 -gtaagg atcaccwacatadawtgta-3 0 (nt 11,392e11,370), 2 ml 1â sybr green and nuclease-free water to a final volume of 20 ml. the amplification reaction consisted of a preincubation step at 95 c for 3 min followed by 35 cycles of amplification including 95 c for 15 s, 55 c for 15 s and 72 c for 30 s. the fluorescent signal was detected once per cycle upon completion of the extension step. after amplification, melting curve analysis was performed by heating to 95 c then cooling to 60 c for 15 s, followed by a temperature increase to 95 c, while continuously collecting the fluorescent signal data. adenovirus, influenza b virus, respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) were detected by the method described by krafft et al., 27 chi et al., 28 samransamruajkit et al. 29 and thanasugarn et al., 30 respectively. all hbov positive samples were subjected to vp1 gene sequencing. the partial vp1 gene at the 5 0 end of hbov was amplified into two segments with two primer sets, vpf1 (5 0 -gataactgacgaggaaatgct-3 0 : nt 3009e3029) and vpr1 (5 0 -agtatgtccatggagttgtga-3 0 : nt 3731e3711) for the first segment and vpf2 and vpr2 for the second. the expected product sizes after 2% agarose electrophoresis were 723 and 648 bp, respectively. pcr conditions have been described elsewhere. 2 the pcr products were purified using the perfectprep gel cleanup kit (eppendorf). dna sequencing was performed using the gene amp pcr system 9600 (perkineelmer, boston, ma). the sequencing products were subjected to a perkin elmer 310 sequencer (perkine elmer) for subsequent sequence analysis. the dna sequences were analyzed with the blast (http://www. ncbi.nlm.gov/blast) program and the phylogenetic analyses and genetic comparisons between hbov strains were performed using the molecular evolutionary genetics analysis (mega) version 3.1 program. we applied pcr and rtepcr to detect hbov, other respiratory viruses and gapdh. of 302 specimens, 20 (6.62%) were positive for hbov. all specimens had been collected throughout the year and plotting of seasonal hbov detection was shown in fig. 1 . among the 302 specimens we also detected co-infection with other respiratory viruses such as rsv in 48 (15.89%) samples, influenza a virus in 33 (10.92%) samples, hmpv in 28 (9.27%) samples, adenovirus in 18 (5.9%) samples, parainfluenza in 14 (4.63%) samples, and influenza b virus in 1 (0.33%) sample. hbov-positive samples co-infected with other respiratory viruses comprised altogether 40% (table 1) . the clinical manifestations of hbov infected patients are summarized in table 1 . the majority of infants positive for hbov were male (14/20) , between 4 and 36 months old (median age 12 months). on average, they were hospitalized for 6.15 days (range 2e22 days). the most common clinical symptom in all hbov infected infants was fever with a mean duration of 3.47 days and other common symptoms such as runny nose (55%) and productive cough (50%) were found. generalized rales were the most common (70%) and wheezing the second most common lung signs (35%). acute bronchiolitis was diagnosed in 9 out of 20, and 11 samples had viral pneumonia. perihilar infiltration was ubiquitously present on the chest x-ray of hbov positive patients (except for cu6 and cu15 where no chest x-ray result was available) (data not shown). additional diseases of some patients with hbov infection included congenital heart disease (cu33 and cu74), chronic lung disease (cu 31 and cu171), asthma (cu71) and cow milk protein sensitive enteropathy (cu157). the nucleotide sequences obtained from this study have been submitted to the genbank database under accession numbers: ef690641eef690657 (table 1 ) except for cu6 (ef203920), cu49 (ef203921) and cu74 (ef203922) that were subjected to analysis for complete coding sequences as described elsewhere. 2 the 5 0 terminal 1182 base pairs (bp) of the vp1 gene were sequenced in all hbov positive samples for phylogenetic analysis. other vp1 sequences from several areas were available at the genbank database and used for phylogenetic analysis including sequences from china: dq778300, ef441262, ef584447, dq457413, dq987595, dq987596, dq677523, dq494203, dq457415, dq988933, dq988934, dq987597, dq987598 and dq494201, japan: ef035488, usa: dq340570, and sweden: dq000495 and nc_007455. the constructed phylogenetic trees of the vp gene (nt 2986e4167) are shown in fig. 2 . alignment of these partial vp1 genes showed minor variations in that the percent identity with the st1 swedish prototype strain ranged from 98.6% to 99.5%. a phylogenetic tree of the hbov vp gene was constructed based on neighbor-joining (nj) trees. confidence values for the tree topologies were evaluated by bootstrap analysis of 1000 pseudo-replicate datasets that showed the low genetic diversity. in the past few years, applying molecular techniques has led to the discovery of hbov as a novel virus apparently associated with respiratory tract illness in humans. 1 however, due to lack of suitable culture systems and animal models to propagate the virus previous studies could not meet with koch's postulates in order to clarify that hbov can cause respiratory illness. it can be concluded from epidemiology data that hbov has been distributed throughout every continent with a different incidence rate (1.5% to 19%). in this study, the np suctions had been collected from pediatric patients hospitalized with acute lower respiratory tract illness. hbov has been detected in 20 (6.62%) out of 302 np suction samples, whereas the previous study in the rural area of thailand had the prevalence of 4.5%. 18 co-infection with other respiratory viruses was found in 40% of the samples tested. rsv and parainfluenza virus were frequently detected as co-infecting viruses. however, some of the respiratory viruses were not included in this study, for examples; rhinoviruses and coronaviruses. therefore, the percentage of co-infection may be higher than this report. thailand has a tropical climate country and seasonal detection of hbov can not be deduced from the few (1e3) hbov-positive samples found in each month. rsv and hmpv were shown the seasonal distribution that peaked in july to september which correlated to the number of samples collection (data not shown). moreover, the study of mannig et al. reported a similarity in seasonal appearance between hbov and rsv 12 whereas weissbrich et al. did not. 16 the clinical features commonly found in hbov positive patients were fever and productive cough. bilateral rales and wheezing were among the most common abnormal lung signs observed and almost equally found in these patients (table 1) . these findings might indicate both lung parenchyma and airway involvement by this pathogen. furthermore, the detection of this virus in specimens aspirated from the nasopharynx together with the significant lower respiratory tract illness indicated the lung pathology in these patients. in conclusion, we detected hbov infection in 6.62% and co-infection with other respiratory viruses in 40% of the np suctions obtained from infants and children with acute lower respiratory tract illness with non-specific clinical features and age distribution. seasonal appearance of hbov was not significant. based on phylogenetic analysis of the vp1 gene, the low genetic diversity was defined. the present study has investigated associations of hbov with clinical manifestations and performed genome analysis but in order to completely describe this novel virus, further studies on serology, tissue and animal culture systems or clinical manifestation inductions will be required. cloning of a human parvovirus by molecular screening of respiratory tract samples complete coding sequence and phylogenetic analysis of human bocavirus (hbov) human bocavirus: prevalence and clinical spectrum at a children's hospital detection of human bocavirus in cannadian children in a 1 year study frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infection human bocavirus infection the association of newly identified respiratory viruses with lower respiratory tract infections in korean children bocavirus infection in hospitalized children human bocavirus in french children human bocavirus infection among children detection of human bocavirus in japanese children with lower respiratory tract infection epidermiology profile and clinical associations of human bocavirus and other human parvovirus real-time pcr assays for detection of bocavirus in human specimens evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus in hospitalized children frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus and acute wheezing in children human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol human bocavirus in italian patients with respiratory diseases human bocavirus in febrile children, the netherlands human bocavirus in iranian children with acute respiratory infections human bocavirus infection, people's republic of china human bocavirus dna detected by quantitative real-time pcr in two children hospitalized for lower respiratory tract infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus detection of bocavirus dna in nasopharyngeal aspirates of a child with bronchiolitis evaluation of pcr testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification detection and characterization of new influenza b virus variants in 2002 plasma endothelin-1 in infants and young children with acute bronchiolitis and viral pneumonia human metapneumovirus infection in thai children this study was supported by the thailand research fund (senior research scholar), royal golden jubilee ph.d. program, the thailand research fund and the center of excellence in clinical virology, chulalongkorn university. we would like to thank the entire staff of the pulmonary unit, department of pediatrics, faculty of medicine, chulalongkrong university for their assistance in collecting the specimens. we also would like to thank ms petra hirsch for reviewing the manuscript. key: cord-281957-1p54k8it authors: kaplan, bruce; kahn, laura h; monath, thomas p; woodall, jack title: 'one health' and parasitology date: 2009-08-12 journal: parasit vectors doi: 10.1186/1756-3305-2-36 sha: doc_id: 281957 cord_uid: 1p54k8it nan one health is a concept that proposes that a paradigm shift in approaching diseases of humans and animals is essential to meet the challenges of the 21 st century. human and veterinary medicine, as well as all other scientific-health related disciplines, must begin forging coequal, all-inclusive collaborations. physicians, veterinarians, other health scientists, and their respective educational institutions, organizations and health agencies must work together. in the past, this approach has resulted in more rapid, efficacious, synergistic achievements in advancing health. unfortunately, such an approach has been relatively rare during the 20 th century. since ancient times the concept that animal health and the environment influence human health has been around. one health began in the late 19 th and 20 th centuries with physician leaders in medicine like rudolf virchow, known as the "father of comparative medicine, cellular pathology, and veterinary pathology" and william osler, called the "father of modern medicine." they embraced the concept that human and animal health were inextricably linked. virchow conducted experimental animal studies on trichinella spiralis in porcine muscular tissue and cysticercosis and tuberculosis in cattle; he coined the term "zoonosis" and stated, "between animal and human medicine there are no dividing lines -nor should there be." osler, who studied in berlin with, and was influenced by virchow, helped promote "one health" as he taught veterinary pathology at montreal veterinary college, and established veterinary pathology as an academic discipline in north america. the physician and veterinarian research team of theobald smith, and f. l. kilborne, discovered the etiology of cattle fever, babesia bigemina, and that it was transmitted by tick vectors, in 1893. their important work helped set the stage for the discovery of the mosquito vector transmission of yellow fever by walter reed and colleagues. in october 1976, the virus causing ebola hemorrhagic fever was identified and named by the u.s. centers for disease control and prevention (cdc) physician virologist, karl johnson, in collaboration with veterinarian pathologist-virologist, fred murphy. they collaborated on zoonotic viruses, their pathogenesis, epidemiology, and ecology for many years. the late 20 th , and particularly the early 21 st century, have been significantly subject to the risks from emerging deadly zoonotic diseases such as human immunodefi-ciency virus/acquired immune deficiency syndrome (aids), severe acute respiratory syndrome (sars), west nile virus and others. this phenomenon demands the urgent need for all human medical and veterinary medical scientific professionals to renew and increase collaborative efforts. one health has accelerated biomedical research discoveries and expanded scientific knowledge in clinical health care. some clinical health examples are elaborated upon in the one health initiative website articles including cancer, orthopedic biomechanical prosthetics, diabetes, obesity, cardiovascular diseases, heart-valve advances, and vaccine development. the late renowned 20 th century veterinary epidemiologist, parasitologist, and global authority on zoonoses, calvin w. schwabe at the university of california coined the term "one medicine" (now commonly referred to as "one health") which was aimed at unifying human medical and veterinary medical disciplines against zoonotic diseases occurring in the public health arena. two examples of recently emerging zoonotic disease epidemics include the avian influenza a h5n1 strain that primarily affects poultry (with some linked human occurrences) and the most recent human pandemic of h1n1 influenza that has spread across asia, africa, europe and the united states. included among many current and previous online topics from parasites and vectors is the zoonotic protozoan parasite responsible for african trypanosomiasis (sleeping sickness). an excellent history has been provided by steverding [2] . an interesting common parasite of dogs, i.e. dirofilaria immitis (heartworm) is also a rare zoonotic disease in humans. aspects of this parasitic species and others are described by otranto et al. [3] . parasitologists, of all the health professional scientists, are generally most familiar with the long list of parasitic zoonoses that affect humans via animals as well as specific details pertaining to each. hence, the critical need for one health research collaborations and cooperation. veterinary medical school students usually receive considerably more exposure to parasitology than do human medical school students. this is largely due to the much greater volume of endoparasite (and ectoparasite) infections and infection rates for animals vis-à-vis humans. thus, epidemiologic prevention, diagnosis, control and treatment needs of animals exceed those for humans. yet, there remain significant research requirements for understanding ideal management in human and animal species utilizing one health principles. the official report of the american public health association's control of communicable diseases manual, 19 th edition, edited by physician david l. heymann, includes a staggering number of zoonotic parasites. heymann, a one health supporter/advocate, is currently chairman of the health protection agency (hpa) with sites across the united kingdom. he is also chairing an australian scientific advisory committee for a one health symposium entitled, "the 1 st international one health congress: human health, animal health, the environment and global survival", tentatively proposed for 14-16 february 2011 in melbourne. one health supporter/advocate veterinarian, martyn jeggo director, australian animal health laboratory (aahl), chairs the organizing committee responsible for developing a framework for the symposium; aahl will also be working in conjunction with a number of colleges. one health initiative will unite human and veterinary medicine the history of african trypanosomiasis. parasites & vectors changing distribution patterns of canine vector borne diseases in italy: leishmaniosis vs. dirofilariosis. parasites & vectors in addition to african trypanosomiasis and dirofilariasis mentioned above, the global list of parasites of human concern includes amebiasis, angiostrongyliasis, anisakaiasis, ascariasis, babesiosis, balantidiasis, capillariasis, clonorchiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, giardiasis, hookworm disease, hymenolepiasis, leishmaniasis, malaria, shistosomiasis, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinellosis, and american trypanosomiasis (chagas disease).in a press release of april 24, 2009, sanaria inc. announced that with support from the path malaria vaccine initiative (mvi) it has initiated a phase 1 clinical trial of its unique malaria vaccine candidate. sanaria's approach "deploys a weakened form of the whole malaria parasite harvested from irradiated mosquitoes instead of small portions of the parasite." ... "while most malaria vaccines in clinical development consist of recombinant or genetically engineered proteins that represent small portions of the parasite, sanaria's plasmodium falciparum sporozoite vaccine candidate contains a weakened form of the entire malaria parasite." the sanaria vaccine currently requires liquid nitrogen transport and storage, which poses challenges for widespread use in africa.an example of "one health in action" was a recent outreach to the veterinary medical community by monath, one of the co-authors of this editorial in an effort to ascertain methods for vaccine storage and transport. the idea was to investigate and compare how veterinary vaccines were handled and if utilizing or incorporating some of these methods might be utilized and provide better transportation of the human malaria vaccines. a request for information on the distribution of veterinary vaccines using liquid nitrogen placed on the one health initiative website resulted in many potentially useful responses from industry and academia.medical professionals and health scientists must include parasitologists, microbiologists, physiologists, pathologist, physicians, osteopaths, veterinarians, dentists, nurses, biomedical engineers, physicists, biochemists, plant pathologists and others. anyone capable of contributing should be considered important and co-equal without reservations.one health has indeed become the "rosetta stone" for a health enlightening paradigm shift revolution. it is the critical key that translates difficult problem solving into less difficult models. it presents a means for the health scientific communities to move towards a more panoramic view, a sustainable revolution (described as an environmental definition for our civilization to survive) and the pursuit of altruistic excellence, notwithstanding respecta-ble status quo advancements of the past. one health works! the authors declare that they have no competing interests. all authors contributed equally to this work key: cord-276110-zztp61pj authors: sætra (sætra is the family name), henrik skaug title: a shallow defence of a technocracy of artificial intelligence: examining the political harms of algorithmic governance in the domain of government date: 2020-06-08 journal: technol soc doi: 10.1016/j.techsoc.2020.101283 sha: doc_id: 276110 cord_uid: zztp61pj artificial intelligence (ai) has proven to be superior to human decision-making in certain areas. this is particularly the case whenever there is a need for advanced strategic reasoning and analysis of vast amounts of data in order to solve complex problems. few human activities fit this description better than politics. in politics we deal with some of the most complex issues humans face, short-term and long-term consequences have to be balanced, and we make decisions knowing that we do not fully understand their consequences. i examine an extreme case of the application of ai in the domain of government, and use this case to examine a subset of the potential harms associated with algorithmic governance. i focus on five objections based on political theoretical considerations and the potential political harms of an ai technocracy. these are objections based on the ideas of ‘political man’ and participation as a prerequisite for legitimacy, the non-morality of machines and the value of transparency and accountability. i conclude that these objections do not successfully derail ai technocracy, if we make sure that mechanisms for control and backup are in place, and if we design a system in which humans have control over the direction and fundamental goals of society. such a technocracy, if the ai capabilities of policy formation here assumed becomes reality, may, in theory, provide us with better means of participation, legitimacy, and more efficient government. artificial intelligence (ai) has proven to be superior to human decision-making in certain areas. this is particularly the case whenever there is a need for advanced strategic reasoning and analysis of vast amounts of data in order to solve complex problems. few human activities fit this description better than politics. in politics we deal with some of the most complex issues humans face, short-term and long-term consequences have to be balanced, and we make decisions knowing that we do not fully understand their consequences. one step at a time, ai has conquered realms of great complication, such as chess, go and the game of starcraft. while these are games, some now suggest that the same techniques of machine learning used to conquer such games could also be used to conquer politics. one example of this is the suggestion that an 'ai economist' based on reinforcement learning and multilayered agent-based simulations could make better tax policies, improving productivity while simultaneously reducing income inequality (zheng et al., 2020) . in this paper i examine an argument in favour of involving ai in political decision-making. this idea is based on the premise of ai's stipulated ability to make better decisions than humans in certain areas, and the premise that we ought to implement the best policies possible. this is a hypothetical situation in which technologies such as the ai economist just discussed are mature, ready for real life applications, and functional on a large scale. this is not a technical treatise about ai but an analysis of the political theoretical implications of ai in politics. as such, i introduce a thought experiment called the magical decision box, which is used to test a set of political theoretical arguments for and against an ai technocracy. there are several approaches to the analysis of the effects of computer decision-making in politics. algorithmic governance is not an entirely new phenomenon, and various harms following the use of ai to govern various aspects of human affairs have been examined in much detail. there are, amongst other issues, concerns related to issues of privacy and surveillance, bias and inequality, transparency and procedure, and freedom and autonomy (danaher, forthcoming; sadowski and selinger, 2014) . sadowski & selinger (2014) provides a taxonomic tool for technocracy, in which they propose categories for the domain, means, and harms of technocracy. i limit my examination to the political harms of technocracy. furthermore, i consider only a subset of the phenomena that are analysed in the literature on algorithmic governance, as i focus on the technocracy in the domain of government, and mainly through the means of mandates. in addition, my case is one in which the technocracy of ai is based on systems with low transparency and high degrees of automation (katzenbach & ulbricht, 2019) . this is a consideration of an extreme case of a technocracy of ai, and this case serves well to highlight the political harms to which i limit my analysis. these choices make this article a partial contribution to the overall consideration of the dangers of technocracy. it is, however, a central part, as these dangers have not been sufficiently examined, particularly not in the setting of an extreme case of ai technocracy as i consider here. the political harms i examine are mainly based on ideas from the discipline of political theory. the first is that human beings might require political activity in order to thrive. if that is the case, the drawbacks of technically 'better' decisions might outweigh the benefits. the next problem is that legitimacy might suffer if people are taken out of politics. i will here examine several approaches to this problem, and how it might be possible to overcome it through regulation and popular participation in guiding computer politics. we could also argue that computers should not make decisions that affect citizens' lives and wellbeing. the fourth problem is that we lack transparency when we rely on decisions that are beyond the capacity of human reasoning. without such transparency we will be left to conduct politics on the basis of trust in machines. a final issue is that of accountability when ai is involved in making political decisions. however, none of these objections are strong enough to fully debunk the argument in favour of employing ai in politics, if a technocracy of ai is implemented with a satisfactory system of control, mechanisms of backup, and a participatory element. such a technocracy, if the ai capabilities of policy formation here assumed becomes reality, may, in theory, provide us with better means of participation, fair and impartial political outcomes, and more efficient government resulting in benefits for most individuals and society in general. should we actually choose to implement a technocracy of ai, politics will, and must, change in ways that might both shift its focus and, perhaps paradoxically, revitalise democracy and popular participation. in an age where algorithms are increasingly governing human action, there is a need to openly and carefully consider a) whether we as societies wish to allow the various forms of algorithmic governance and b) how such forms of governance lead to a need to discuss fundamental political institutions and arrangements. my main goal with this article is to highlight the implications of developments already underway. as sadowski & selinger (2014) point out, governments are increasingly opening the door for a) private companies solving public problems, and b) the use of ai in government. de sousa et al. (2019) also shows how ai is now employed in all areas of governments, while recommending that implementation of ai in the public sector should be preceded by a 'debate with society' about such applications. ai creep -in the sense that the ai slowly and almost unnoticeably is applied to ever new areas -if left unchecked, might take us to a situation in which we have technocracy of ai without either political design or democratic input. i propose that we examine the key questions in advance, and prepare our societies for a future in which ai has the role we desire it to have in politics. i will first develop the concept of traditional technocracy, in order to make sense of the logic and potential benefits involved, and the problems associated with traditional technocracies of humans. i then show how ai can, and in many cases already have, taken the role of a technocrat. such use of ai is associated with somewhat similar, but not identical, risks, to those of traditional technocracy. the combination of the concept of technocracy and the use of ai lets me propose a set of objections to an ai technocracy based on the idea of political harms, and these are first presented and discussed before i move on to the discussion of the implications they have, and the potential for a legitimate and workable technocracy of ai. 2. technocracy, expert rule and democracy schumpeter (2003) argues that if we want results that are satisfactory to people at large, and judge our governments by such a criterion, government by the people 'would often fail to meet it'. for us to understand what an ai technocracy means, we must understand the basic ingredients of technocracy. sadowski and selinger (2014) are right to call for an improved understanding of what technocracy is, and they intend for their taxonomy to be a 'conversation starter' on the topic of technocratic uses of technology. while they argue that it is an ill-defined concept, i posit that the solution to this problem is to draw upon the existing literature on the subject form the field of political science. furthermore, i reject their implicit position that technocracy is by definition a bad thing, associated with harms only, and no benefits. this is based on one of the fundamental points in this article, which is that democracy, and specific forms of democracy, cannot be assumed to be ultimate goals at the outset of an evaluation of the effects of democracy. if we start with such an assumption, anything that challenges democracy is by necessity bad. a more fruitful approach is to take an agnostic stance to the value of democracy. firstly, this lets us perform an honest and open evaluation of alternatives. secondly, it enables us to strengthen and revitalize democracy by showing why it is good, and not merely stating that it is. technocracy is a term that in daily parlance refers to the rule of experts. more specifically, i use the term to refer to a state whereby experts in science and technology -not experts in politics -wield power. technocracy is thus differentiated from other forms of rule by the few, such as epistocracy and meritocracy. epistocracy is a form of government whereby those with a particular knowledge of politics rule, and is often considered a rule of the wise -those with knowledge of 'politics, history, economics' (estlund, 2003 (estlund, , 2008 . this idea is old, and famous political philosophers such as plato (1968) and john stuart mill (1985) have proposed variations of it. meritocracy, on the other hand, is often linked to mental and cognitive abilities, regardless of specific knowledge of a particular subject (arrow, bowles, durlauf, 2000; young, 2017) . technocracy has been defined in various ways, one being meynaud's (1969) description of 'a system of governance in which technically trained experts rule by virtue of their specialist knowledge and position in dominant political and economic institutions' (meynaud, 1969) . the experts, then, become technocrats, and have authority based on technical expertise (bell, 1973) . putnam (1977) uses the word technocrat to describe proponents of technocracy, whilst i use it to describe technicians in power. there are several possible arguments in favour of technocracy, and i split them into positive and negative arguments. the negative arguments mainly focus on the shortcomings of people and democracy -usually implying that the people are unfit to rule, or that democracy itself produces bad outcomes. the positive arguments chiefly focus on the benefits that could be gained from expert rule, e.g. efficiency, rationality and optimisation. it is worth noting that technocracy usually flourish in times of panic and great crises, as the need for efficiency and optimal policies in such times tend to be more pressing, and obvious (sadowski & selinger, 2014) . the negative arguments are mostly based on the idea that human beings are the main problem of politics, and that they are unfit to rule. people have personal interests, and they tend to pursue them, even when they are tasked with pursuing the public interest (mueller, 2003) . furthermore, they have limited capabilities, in terms both of how much they can do and how well they can do things. some of them have a greater capacity than others, however, and this is the foundation of elitist arguments for technocracy, for example as seen in schumpeter (2003) . another class of negative arguments focuses on democracy as a system, as it is portrayed as inefficient, unjust and prone to instability. the positive arguments are mainly based on achieving some form of rational optimisation in politics, and partly the belief that most problems, when properly understood, are technical problems amenable to the logic of statistical analysis and optimisation. however, there are also arguments in favour of non-democratic systems based on, for example, the idea that markets are the best way to organise everything. at least almost everything. and if we do need some form of government, it had better be small and kept out of the way (hayek, 1960) . a point of great importance is that the technicians do not have all power in a technocracy. while a technocratic society delegates much authority to their technicians, they are not given the power to determine the goals of society. the technocrats are not political or moral experts, and i do not consider a situation in which morality and the question of political values are reduced to technical questions amenable to the calculus of mathematics and logic. this means that the technocracy i discuss have a political apparatus in place for providing the direction for society, while the technocrats have the authority to determine and effectuate the policies deemed necessary to proceed in the desired direction. this is akin to the process described by peters (2014) , where technical and political decisions are separated, and the technical issues are transferred to "independent agencies, bureaucracies and technocratic elites". technical issues are also political, however, which is why i argue that we have a technocracy of ai when we delegate power to make technical decisions to computers. this is the argument made by naess (1989) , who urges us to see the political nature of all things technical and to submit the technical to an evaluation in light of our ultimate values. fischer (1993) writes that technocracy revolves around the desire to use experts with specific knowledge in positions of political power, for the sake of building the 'good society'. this, then, is not the technocracy i discuss, as politicians -or the people -are charged with determining what the good society is, while the technocrats are charged with making it reality in the most effective manner possible. this is in line with sadowski & selinger's (2014) picture of technocrats as influential, but still largely controlled by elected politicians. this implies a break with the understanding of technocracy as an approach in which 'political realities play no role' (janssen & kuk, 2016) . technocracy may have a bad reputation, but it is here given a blank slate and the chance to prove any worth it may have. while sadowski & selinger (2014) aim to arm us to 'combat technocracy', i claim that we must first understand if, and why, it should be combatted. before moving on the use of ai in politics, i present the main traditional arguments against technocracy. however, as some of these arguments become less relevant if human technocrats are replaced by ai, i do not examine these in great detail, unless they remain relevant, and thus become part of the objection in section 4. the arguments against technocracy are also based on various principles, and i briefly consider arguments based on the inherent values of democracy, human beings' needs and finally the inherent flaws of technocratic government itself. estlund (2003) discusses objections to epistocracy, which are partly applicable to technocracy as well. the demographic objection is based on the fact that giving those with, say, university degrees more voting power means giving privilege to certain groups. the second objectionthe bias objection -is a version of the former, adding the claim that the privileged classes, races etc. will exert a certain bias that is unacceptable (estlund, 2003; lippert-rasmussen, 2012) . these arguments constitute the first class of arguments against technocracy, and i label them the technocrats are people, too arguments. one of the arguments in favour of technocracy is that people are flawed, but some are less flawed than others. no-one is free of flaws, however, and people can never be assumed to have totally overcome irrationality and bias. but can computers? i return to this issue in section 5. it is also worth noting at the outset that problems of bias and discrimination are not exclusive to the use of algorithms, and in evaluating algorithmic governance, we must examine whether algorithmic bias is in fact worse than existing problems associated with human bias. a second argument against technocracy is based on a concern for legitimacy. it posits that technocracy is bad because the process itself does not provide legitimacy (only participatory politics does so). this constitutes one of the objections discussed in section 4. thirdly, people might object to technocracy because they believe a deliberative process will provide better outcomes. this particularly comes from the deliberative-democracy camp (elster, 2005) . opposed to these arguments would be ones inspired by janis (1982) and hobbes (1946) , who both emphasise the dangers inherent in democratic, or group, decisionmaking. it must be added that human technocrats are also liable to group-think. the fourth argument against technocracy might be called a decentralisation argument. it is the hayekian notion of dispersed knowledge (hayek, 1960) . no human being or small group of human beings can ever amass the kind of knowledge that each individual has about their own affairs. the only effective way to utilise such knowledge, hayekians would argue, is to give power to the markets (hayek, 1960) . the problems of technocracy are not insignificant, but as this examination shows, there is reason to ask if a technocracy of ai may in fact be better than a technocracy of human beings. this stems from the fact that several of these arguments more easily applies to human beings than to computers. however, as janssen & kuk (2016) shows, human faults are often refound in the workings of our algorithms, in the form of, for example, bias and discrimination. the title of this article promises a shallow defence of a technocracy of ai. this refers to the argument i will construct in the first part of the article, which consists of three proposition that lead to the conclusion that we should employ ai in politics, and erect a technocracy of ai. this argument will then be subjected to a set of objections to such a technocracy in section 4. the first two components of the shallow defence follows form the preceding consideration on technocracy and politics. first of all, politics can be understood as a process aimed at implementing the best possible policies. but what policy is considered best can, of course, only be decided once we know what criteria to apply. thus, the first and fundamental purpose of politics is to develop and elucidate what fundamental moral values a society is based on. only then can politics as we know it in the day-to-day workings of society take place. and only then can we properly assess the value of technology (naess, 1989) . this is the first building block in the defence of an ai technocracy: policies should be evaluated on the basis of the fundamental moral values of the society in question, and finding these values is the first purpose of politics. furthermore, if politics revolves around the question of finding the best policies, as schumpeter (1946) implies, we also have a second premise in the establishment of what will become the defence: the best policies in accordance with the evaluation discussed in the first premise should be implemented. artificial intelligence is superior to human intelligence for analysis of large and complex problems involving the need for strategy, prediction of long-term effects and analysis of vast amounts of data. one example is playing chess and go (campbell et al., 2002; kurzweil, 2015; chouard, 2016; google, 2018) . however, ai can beat humans at more than fun and games. politics is complex, and it involves many considerations of notoriously uncertain short-and long-term consequences. as the literature on algorithmic governance shows, ai is already employed in many facets of government, as i show in section 3.1. i will note at the outset that the potential benefits from ai in politics is heavily contested (katzenbach & ulbricht, 2019) . i agree with the idea that ai as of today is not some silver bullet that can be employed in order to create flourishing societies. however, as the many existing applications clearly show, there is undeniably a potential for the beneficial use of ai in politics. i will assume that some of this potential can be realised, and that we will continue to see certain improvements in the technologies involved. this means that i consider possible near-future technologies, and not speculative developments related to some form of singularity, etc. (kurzweil, 2015) . i will not discuss the technologies involved in great detail, but rely on the assumption that much of the future applications of ai is based on the technologies currently employed. de sousa et al. (2019) chart the technologies most often employed in public sector ai; various forms of machine learning are used, while artificial neural networks (ann) is the most popular technique in terms of usage. artificial neural networks (anns) imitate biological information-processing systems, and consist of artificial neurons that transmit signals to each other in reinforcement systems, for example (bishop, 2006) . deep learning is our term for deeply layered neural networks, and this is the machine learning approach used by the ai economist i use as an example, as well as by google's alphago and alphazero (sutton and barto, 2018; zheng et al., 2020; google, 2020a google, , 2020b . algorithmic governance is a term describing the utilisation of algorithms both in ordering human action in general, and in traditional political structures (danaher et al., 2017; katzenbach & ulbricht, 2019) . as such, it is a very broad term, and as discussed in the introduction, i only focus on a subset of what is referred to in general as algorithmic governance. using the taxonomic tools of sadowski & selinger (2014) , i limit my discussion to the use of ai in the domain of government, and mainly to a situation in which ai is given the authority to govern by mandates (not more informal nudging, or even less direct governing through technological mediation). according to the same typology, i mainly focus on a specific set of harms, labelled political harms. these are distinguished from existential and discursive harms (sadowski & selinger, 2014) . existential harms relate to the reduction of individuals through the datafication and behaviouristic approach to the control of human beings. discursive harms involve the restriction of discourse through a domination by technocratic ideas. in this article, i do not focus on harms related to privacy and surveillance or bias, unfairness and inequality. a potential loss of freedom is another potential harm that falls beyond the scope of the article (danaher, forthcoming) . issues of privacy and surveillance are of great importance, but i will in the following assume that ai can be built on data which is not individualised, and that it does not govern by individualized nudges or micro-directives, etc., based on detailed personality profiles (saetra, 2019; ma, 2020) . hypothetically, policy could also be based on machine learning in combination with simulations, such as in the example of the ai economist (zheng et al., 2020) . related to issues of privacy are issues of agency, autonomy and freedom (katzenbach & ulbricht, 2019; danaher, forthcoming) . another point is that increased used of data, and the translation of all aspects of human action into data is that such quantification by necessity involves multiple stages of human interpretation, challenging the objective veneer of big databased decision making (janssen & kuk, 2016; saetra, 2018; ma, 2020) . such issues are only partially considered in this article, and will thus remain important philosophical issues for further research. some of the potential harms that i do not focus on, fall in the categories of unforeseen and unintended consequences (katzenbach & ulbricht, 2019) . one such problem is algorithmic bias and resulting issues of fairness and inequality (de sousa et al., 2019) . furthermore, algorithms, partly because of problems associated with the training data, could also favour particular political ideologies, and they could reinforce discriminatory and other undesirable practices (janssen & kuk, 2016) . these problems are real, and important, and any technocracy of ai must implement mechanisms of algorithmic oversight in order to address such problems. however, i do not consider these challenges in detail in the present article, and employing the thought experiment presented in section 3.2 enables me to isolate the political harms i do focus on from the issues of algorithmic bias. according to sadowski and selinger (2014) , political harms relate to people being disenfranchised and deprived of political power and influence. this relates to the potential depoliticisation effects of algorithmic governance (katzenbach & ulbricht, 2019) . in addition to this, questions of transparency and procedure are relevant to the analysis of political harms (danaher, forthcoming) . these concerns are examined in more detail in section 4. with regard to the applications of ai, i posit that we have already moved a long way towards ai decision making, and this is an important point to keep in mind when we consider the technocracy of ai. as i will show, a condemnation of the technocracy of ai that i propose will in many respects simultaneously involve a condemnation of many current practices. ai is now used in all areas of society: driving cars, guiding our missiles, trading stocks, helping us navigate new places, playing chess, recognising speech, speaking to us, finding our mates, forecasting the weather, etc. (danaher, 2016) . ai is even used to determine who gets bail and who gets loans and identify likely tax evaders (tashea, 2018; danaher, 2016) . all these applications of ai are relevant to an examination of algorithmic governance in general, but not all of them are in the domain of government. de sousa et al. (2019) provide a thorough account of current applications of ai in the public sector. they find that it is employed in all areas of government already, while general public service, economic affairs and environmental protection is the areas most emphasized in the literature on ai in government. examples of current applications are found in most functions of the public sector, such as public health, transportation, education, security, communications, and the actual examples involve, amongst others, systems of identification of risks, optimization systems, systems for response, analysis and prediction (de sousa et al., 2019) . predictive policing is another area that has gotten a lot of attention, and ai is also used in, for example, the administration of immigration and in calculating social benefits (katzenbach & ulbricht, 2019; janssen & kuk, 2016) algorithmic control of traffic lights and speed limits is one low level example in the domain of transportation, where the utility of having dynamic systems based on machine learning could increase efficiency and utility (ma, 2020) . if the objective of our traffic lights and speed limits is to a) optimise traffic flow while b) minimising casualties, ai could most likely improve a static system of pre-determined speed limits -allowing for increased speed in low danger situations and not having drivers wait unnecessarily at red lights. the idea is that similar benefits could be reaped in other domains of law and politics, such as tax policies (zheng, 2020) . another example would be the use of ai to implement optimal strategies for pandemic response, which could involve using ai to predict and identify outbreaks and dynamically apply the optimal response based on a society's tolerance for risks weighed against other factors, such as economic costs. in general, when we face optimisation problems, ai could have the potential to aid us. most problems of politics are highly complicated optimisation problems. as long as we have a goal, ai can in theory take us there in an effective manner. furthermore, it could continuously experiment, and could update its policies. policies would be dynamic, always responding to changes in human preferences and behaviour. one recent example is the ai economist which can make tax policies, in a simulation, that both improve productivity and decrease inequality (zheng et al., 2020) . another is the use of webuildai, which through participatory algorithmic design managed to increase efficiency of a food donation transportation service while simultaneously improving the perceived fairness of the outcome -adjudicating equity and efficiency (lee et al., 2019) . such examples are limited and hypothetical, but, in combination with the many existing applications, they suggest that we could in time find us in a situation where such systems are applicable to real-world large-scale situations. one way to approach this issue without getting lost in the technical aspects of ai is to consider an analogy. i invite you to replace ai as we know it with a hypothetical device called the magical decision box (mdb). this thought experiment requires you to accept that a society has discovered a box -origins unknown -that makes decisions. the box accepts input and constraints, it accepts goals and it provides answers. through rigorous tests the society in question has found that the box significantly outperforms human experts in making policies that optimise the goals it has been given. thus far they have found no major errors. despite their very best efforts, they have not been able to understand how the box works, and they cannot recreate (even in retrospect) the ways in which the box reasons. the decisions, however, are good, so should they employ it? the mdb is, of course, different from ai in many ways. however, there are certain similarities that might elucidate the core issues involved in the objections to ai in politics. like the mdb, ai is a 'black box' that we do not always fully understand. even if we understand the principles involved in making advanced machine learning models, they will produce outcomes that we often cannot predict (janssen & kuk, 2016) . i will later return to the notion of explainable, or explicable, ai -also referred to as xai (gunning, 2017; robbins, 2019) . the realist theory of democracy and both cognitive and behavioural science have shown many of the shortcomings of human decision-making. this brings me to the next proposition in the argument leading to the preliminary conclusion that artificial intelligence should be used to improve political decisions. this could come about in several ways, ranging from computers aiding human decision-makers to their replacing them. i here examine the latter possibility, which i call an ai technocracy. we now have the option of letting computers make expert judgements for us. with the use of advanced machine learning, computers could make better decisions than human beings. vogl et al. (2019) argue that computers might improve decision-making by overcoming the human limitations of bounded rationality and information processing. machines are better than us at chess and go, and if this ability also applies to other well-defined strategic problems, should we not let computers decide for us? if we had the mdb, and knew that its results were good, would we use it? 3.2.1. the third premise -ai supremacy the possibility of ai superiority in solving complex issues related to human beings and their societies is not out of the question. as my goal is to analyse the possible future impact of ai on politics, the exact probability of this happening, or the exact scope of ai superiority, is of less importance. for these reasons, we can consider ai to be the mdb for our present purposes. what matters is that for now we should accept the premise that ai might in the future play the part i am proposing here. if this does not happen, or until it happens, this paper can be considered null and void, and no harm should have occurred. to be clear, i do not believe ai can currently be the basis of a full technocracy such as the one i here describe. with this, the shallow defence of an ai technocracy is complete. firstly, the purpose of politics is to find the best policies, in accordance with some set moral basis. secondly, we should enact the best policies we have identified. thirdly, ai is better than human beings at creating and identifying the best policies. all this leads to the preliminary conclusion that ai should be given the power to discover and enact our policies. it seems non-controversial to assume that such a technocracy could in certain areas give us better decisions in terms of efficiency, utility or whatever other criteria we set as the computer's goal. lee et al. (2019) and zheng (2020) provides proofs of concept for the idea that ai could provide both efficiency and still be amenable to popular control and direction. however, there are also several potential downsides, which will have to be weighed up against these benefits. i will here provide a schematic overview of some of the obvious objections to an ai technocracy, based on the political harms it may cause. they are: a) people might need politics, b) legitimacy is linked to democracy, c) ai is not capable of morality, d) we have an issue with transparency related to ai and e) ai decision-making involves problems assigning responsibility. note that i am not considering the objection that such a technocracy would not lead to better policies, as this very premise is part of the argument i am testing. if this premise fails, the argument in favour of technocracy crumbles, and there is little need for any of these objections. accepting the first three premises for now, i proceed to the political theoretical objections against the shallow argument in favour of an ai technocracy. in this brief examination of the objections i will first present each objection, and then some considerations concerning its possible shortcomings. according to aristotle (1981) , humans are political animals. the best that they can achieve is to exercise their political capabilities and live a life of self-determination in a political society. only then will they realise their true nature -their telos. this is, however, only one of the interpretations of aristotle's view of humans and politics. mulgan (1990) shows that aristotle can also be understood as supporting 'the withdrawal from politics, or at least reluctant participation'. in both the politics and ethics the philosopher's lifestyle is portrayed as being superior to the life of the statesman (mulgan, 1990, p. 195) . another immediate objection would be to ask: are people politically active in today's political systems? what will really change if ai makes our decisions, instead of bureaucrats? the 'political animal' argument is against any move from direct democracy towards democracy by representation or republicanism, and this move could be said to be a necessary evil if we are to have large-scale, complex societies. this objection also relies on an agreement with aristotle's hierarchical ordering of human activities. other philosophies, such as hedonism, could easily portray a life of increased wealth and leisure as a potential improvement to a life material hardship and political activity. philosophers such as f. a. hayek have proposed that involvement in politics is not that important, and that economic liberty is what is essential to good societies (hayek, 1960) . following this argument, he states that democracy is not necessarily the best way to rule, and that '... a democracy may well wield totalitarian powers, and it is conceivable that an authoritarian government may act on liberal principles' (hayek, 1960) . however, a crucial point of this article is that a technocracy of ai may in fact lead to a revitalization of popular participation and the accessibility of the political domain. through new ways of participation, such as algorithmic co-creation, we could find ourselves in a situation in which most people have, and experience, more political power than they do today. this is examined in more detail in the next objection. another objection is that people need to be involved, and that they need deliberation to take place if political systems are to have legitimacy (rousseau, 1968; manin, 1987) . even if the best decisions were made by a dictator, his decisions would not be legitimate if the citizens had no political power. we thus distinguish between legitimate authority and effective authority. ai might be extremely effective, but might still lack legitimacy. we can see this objection as consisting of two parts: a) policy should be decided by the populace, and b) all citizens should have equal political rights and a realistic chance of exercising them. the objection to technocracy would be that legitimacy is lost when the population is deprived of crucial political rights and the ability to take part in decision-making. this objection is based on a procedural view of legitimacy (peter, 2017) . according to such a view, due process is the source of legitimacy, and deliberation is one aspect of such a process (manin, 1987) . people will not deem a government in which they do not participate to be legitimate the counterobjections are similar to those regarding the previous objection. we could first argue that an ai technocracy makes people more equal than they would have been in any democracy -equally free to participate in the moral debates about the direction of society, and equally deprived of participation in policy formation. we might also argue that such a system would enable people to understand more about politics, and that the barrier to understanding politics and taking part would be lowered. when human politics only revolves around fundamental moral values, it is easier to both understand and take part in this process. furthermore, not everyone agrees that legitimacy is gained through participation. for hobbes (1946) , legitimacy is achieved through the social contract, which ensures that a governmentby one, few, many or all -is created to satisfy everyone's fundamental need, this need being security -sometimes labelled liberty (manin, 1987) . könig (2019) even discuss how algorithmic governance bears some resemblance to hobbes's apolitical leviathan. if democracy contributes to better political outcomes, it is conducive to legitimacy. if it does not, it is not necessary (raz, 1994) . the latter argument is the instrumental view of democracy. even in today's societies participation in politics is restricted to a select few, despite everyone having a theoretical chance of taking part. the size and complexity of our societies have led to the growth of bureaucracies, expert rule and indirect democracy. going from this to an ai technocracy is perhaps not as radical as it might seem at first sight. one obvious potential benefit of digitalisation and the increased capacity to communicate using digital tools is the possibility of directly involving far more people in political processes. the pirate party is an example of an actual attempt at this, and they have done very well in several countries (beyer, 2014) . if a technocracy of ai is to overcome this objection, participation in some form must be ensured. one way to implement this is to promote co-creation and 'democratizing algorithm' (janssen & kuk, 2016) . one such example is the webuildai example, which shows several methods of popular participation in the creation and design of algorithms, and how this could lead to both increased participation and increased algorithmic awareness (lee et al., 2019) . this also addresses a potential objection based on the idea that only democracy has the pedagogical effects ensuing from participation in politics. people learn from taking part in politics (rousseau, 1968; tocqueville, 2004) . according to this argument, the beneficial effects participation has on people could outweigh the negative effects of having sub-optimal policies. however, if systems such as webuildai allows for participation and efficiency, there is little reason to choose sub-optimality. another possible counterargument against the use of ai in politics is based on the argument that politics always involves questions of values and morality. this is not necessarily a problem per se, but it creates an obstacle for ai as soon as we run into people arguing that morality is solely a human endeavour. according to some people moral machines do not exist, and human qualities such as compassion and wisdom are crucial prerequisites for important decisions (weizenbaum, 1976) . resolving this question entails coming to some sort of agreement about how we define morality, and whether or not our definitions exclude machines. what we are concerned with here is whether or not machines can act morally and make moral decisions. in the terminology of ethics: whether or not they can be moral agents (nolt, 2015) . some people believe that we can train a machine to become moral, e.g. by having a lot of people answer various questions on the morally superior decision in various instances of the trolley problem. the moral machine is one such endeavour that has attracted much attention (awad et al., 2018) . it is an attempt to crowd-source the morality of people around the world in order to teach a computer to distinguish right from wrong, in order to find ethical principles to guide machine behaviour. this is an approach which could be related to the approach of webuildai, in which social choice theory and popular participation is used to train algorithms (lee et al, 2019) . kurzweil (2015) predicts that machines will gain human proficiencies and capabilities, including morality-related emotions. maldonato and valerio (2018) and scheutz (2016) also discuss related topics, e.g. machines with value systems and the creation of machines with moral competences. even if they do not gain anything akin to human morality, machines are becoming better at understanding human morality, as it can quantify, track and compare moral bias, for example, 'across cultures and over time' (schramowski et al., 2020) . however, we could also approach this objection more pragmatically. if we decide that decisions that affect people's wellbeing require moral capabilities, we are already employing ai in situations where it does not belong. self-driving cars could not then be allowed. robots trading in markets would become highly problematic. use of ai to determine whether or not criminal offenders should receive bail would be highly dubious. ai is already involved in moral decisions in countless ways, and our current approach to this issue is to make someone other than the computer itself morally responsible for the decisions made. we will include the premise in the tentative discussion, but i will argue in the conclusion that this objection is better understood by way of handling objection 5 and the attribution of ai decisions. another obvious counterobjection would be that if machines cannot be moral, neither can they be immoral. to some people this situation would appear to be a huge advantage compared with the capacities of today's politicians. if indeed power corrupts, we should see it as an advantage that ai has no morally corruptible nature (dalberg-acton, 1907) . a different kind of objection would be that the important issue is not morality but whether or not machines are able to act ethically. if morality is right action based on some internal value system, then ethics are actions based on some external system. machines' ability to act in accordance with humans' codes of ethics might be sufficient. following this objection, one could also question the nature of human morality itself, and portraying morality as some vague and unscientific idea, plagued by no consensus, which is thus of little interest. we might even object that we have no test for the morality of our human politicians, so why should we implement one in order to keep robots away from power? human experts can explain their choices to the populace in a way that computers cannot -at least not yet. and even if they become better at explaining why certain choices are made, will we be able to understand? the reason for giving computers the authority to decide is that they are able to make evaluations and calculations that are beyond human capacity. transparency would then be an issue, and we might be left guessing why our ai technocrat made the decisions it made, in the same way as chess experts guess why the computer calls moves good or bad when analysing the world's best players. santiago (2019) emphasises that humans must have the last say in decisions made by ai. however, if we cannot understand the reasoning behind the decisions, transparency and control are hard to achieve (janssen & kuk, 2016) . opacity is one of danaher's (2016) main objections to algorithmic governance. pressure is mounting, wachter et al. (2017) state, to make sure that ai becomes explainable, and thus transparent. explainable ai (xai) is the quest for machines that can make us understand their decisions and reasoning. such a development could make us trust, understand and manage ai decision makers (gunning, 2017) . this is a field that receives a lot of attention, but until we have achieved this goal we could imagine other ways of dealing with the problem. like with the magical decision box, which is by definition unfathomable, we must perhaps rely on control by goal achievement and observable result parameters with ai technocracy. if ai or the mdb make decisions that take us closer to our goals, do we really need to understand how they manage to do so? similarly, if the bureaucrats make decisions beyond politicians' understanding, do we complain? we might simply recognise that the very reason politicians rely on experts, or ai technocrats, is because the latter make decisions that are unfathomable, owing to their being too advanced. lack of understandability is the price for better decisions. in the words of robbins (2019), 'a principle of explicability for ai makes the use of ai redundant'. janssen & kuk (2016) is right in arguing that a full understanding of how complex algorithms operate is most likely restricted to the 'happy few'. however, so is a full understanding of how many current systems of government operates. danaher (2016) does not see this counterobjection as a valid reason for ignoring transparency issues. the fact that we have similar problems today, he argues, is not a reason to allow them (in more serious form) in algorithmic governance. i am, however, not arguing that an ai technocracy is an ideal system. i merely argue that it could be argued to be better than what we currently have. as such, the pragmatic counterarguments pointing out that an ai technocracy does not lead to a worsening compared to current affairs is of a great interest. if ai technocracy leads to a pareto improvement where efficiency is improved, while other aspects of political life remain the same, i will consider this desirable. a related point is that opacity could provide certain benefits. if the exact workings of a system is not known, it is difficult to game the system (janssen & kuk, 2016) . however, the ai economist of zheng et al. (2020) allows for such gaming of the tax system, and find their policy maker capable of coping with it. the final objection is based on the problem of accountability. if a computer makes a decision, who is held accountable for this decision? until we are willing to consider robots legal persons with rights and obligations, someone else must be held accountable for their actions. but who? their authors, or perhaps their owners, who are responsible for using them? the accountability gap is a term used to describe such issues, and it is a problem that becomes increasingly prominent as our technologies become more advanced and capable (wachter et al., 2017) . this complexity creates situations in which the creator of a machine cannot foresee the actions of his creation (matthias, 2004) . when this happens, some perceieve a gap between who acts (for example, a complex machine), and who is perceived as responsible (the creator). santiago (2019) states that when businesses employ ai in decision-making, humans must have the final say. but which humans, when we are discussing ai technocracy? we are not discussing a political system in which bureaucracy is simply replaced by ai, but one in which the technocrat machines are given greater autonomy in terms of policy formation. thus, the current system, whereby politicians are responsible for the decisions of their bureaucracies, will not work, as they will not be assumed to be in control of their technocrats as they now are of their bureaucrats. we might not have any perfect solutions to this problem, but one might also argue that neither are popular modern political systems perfect. we do, however, have several options. one is to hold the producers of ai legally responsible for their artificial technocrats. another possibility could be to create a human council for machine control, tasked with making sure our technocrats do as we wish. algorithmic 'constituents', binns (2018) argues, have a right to scrutinise and hold someone accountable for the exercise of algorithmic governance. as janssen & kuk (2016) point out, few are able to exercise such control. educating the population and organising such a council for machine control is a vital precondition for an ai technocracy, as i will return to later. the shallow argument in favour of an ai technocracy is based on three premises. where, then, does this lead us? firstly, if o1 or o2 is true, the argument in favour of technocracy is weakened. we might, however, introduce a counterobjection, to remove some of the sting of these two objections. if politics is really about deciding which moral values are important, and what we see as the good society, having a policy process whereby human beings take part in the formulation of these issues is compatible with a technocracy. i will even argue that it is necessary in order to avoid a technocracy with no goal or direction. if this counterobjection is accepted o1 and o2 can be disregarded. however, o1 states that people require full participation, and this condition might not be satisfied by my proposed policy process involving only fundamental values. however, objection 1 could conceivable also strengthen the initial argument in defence of an ai technocracy. while danaher (2016) argues that algorithmic governance will tend to limit and reduce the possibilities of participation, my argument in favour of reorienting the goal of politics to fundamental values could make it more accessible. o3 is an objection that sounds both true and reasonable, but let us consider the implications before accepting it. if we take it as it stands, ai must not be used in any area where human wellbeing is affected -not in determining loans, not in determining bail, and not even in helping us identify criminals, or in areas such as financial fraud. furthermore, positive use of ai affects people's wellbeing just as much as use that we more often perceive to be problematic. this means that autonomous vehicles are most certainly out of the question, and even brake assist etc. must be got rid of. the implications are absurd, which implies that we must subject the objection to close scrutiny. some use of technology is unavoidable if we are not to reject all the assistance it currently provides us with. when we pursue this issue, we find that all technologies have moral and political implications, which means that the issue at hand is not really restricted to the use of ai. the important issue, i argue, is accountability, which will be discussed shortly. the proposition that ai should not make decisions because the decisions are not transparent (o4) is a universal problem associated with letting those with the most knowledge make decisions. it is, however, possible to imagine a situation whereby decisions are explained by virtue of the results they lead to. how economists arrive at a new and better tax system is perhaps of less interest than their proposal showing what a new tax system will lead to. politicians will not understand the technical aspects of the reasoning, just as they will not understand the processes that lead the ai from input to ideal policies. this problem is not restricted to the use of ai. i argue that ai is transparent, but that we simply have a hard time understanding how known methods and known input lead to decisions that are better than those we could make ourselves. if nothing is hidden or secret, a decision is by definition transparent, and whilst further work on explainable ai will be beneficial, objection 4 does not appear to be particularly strong. this issue of transparency is a problem in two respects. it is a challenge to the legitimacy of an ai technocracy, and people might not be willing to take the risk involved in being ruled by something one cannot understand. with human politicians we realise we may be taking a degree of risk, as human fallibility is well known. it is, however, harder to identify the risks involved in computer decisions, and the fear that it will err in ways we will not be able to uncover, or correct, is different from the risk of being ruled by fallible leaders who are just like us. furthermore, if we develop and apply methods of participation and co-creation, we could increase general algorithmic awareness. this might hypothetically lead to most people having a better understanding of how our societies function than they do today. accountability issues are the fifth objection, and i have proposed that either a council for oversight or the producers of the ai systems could be held accountable. it is not a problem per se if we refuse to give ai moral agency, and thus moral responsibility and accountability. an ai technocracy need not be unlimited and arbitrary. it could be a constitutionally limited ai whereby a council -small or large -of men is tasked with giving the ai both a direction and a goal. ai is in certain respects like a modern-day version of leibniz's god -the good architect who must be assumed to have created the best possible version of the universe (leibniz, 1989) . had we known what god knows, we would realise that the universe is as good as it can be, despite occurrences of what we perceive as evil. the argument works as long as you believe in an almighty and good god, and this kind of trust must thus be transferred to ai. this takes us back to the magical decision box. if we knew it had always produced good decisions in the past, should we employ it, even if we did not understand how it worked? we would be in a situation similar to the one leibniz considers when examining whether or not the world is good. god is good, and god created it, thus the world must be good, even if it may not always seem that way to us. if god created the magical decision box, i argue that we should employ it as our technocrat. however, as my analysis suggests, we should perhaps not discard the use of ai as our technocrat even if it is not a magical and perfect decision box. there are, however preconditions that must be satisfied for such a technocracy to be able to overcome the objections i have presented. furthermore, the defence of a technocracy of ai here presented is shallow also in the sense that not all objections and considerations have been considered. the future capabilities of ai are somewhat speculative, i have not gone into detail on issues of bias, discrimination, agency, and liberty, and it remains to be seen if the world of wicked political problems is truly amenable to the optimising hand of artificial intelligence (head, 2008) . nevertheless, i do propose a defence that is not without value, as it suggests that the political harms of an ai technocracy are not insurmountable. one of the preconditions for a technocracy of ai is that there are backup and control mechanisms in place, in case the ai systems should fail, or if we desire to scale back the use of ai. control systems are necessary in order to supervise the performance of the systems, and particularly to monitor potential issues related to bias, discrimination, and various other unintended consequences. furthermore, times of crises and rapid change have shown that machine learning systems can easily become confounded, and in such cases, there must be mechanisms in place for human intervention. this was shown clearly during the covid-19 situation that led to problems for many machine learning models trained on 'normal behaviour' (heaven, 2020) . putting ai in power involves, in a certain sense, putting the companies and individuals that create these systems in power (danaher, 2016) . some people might argue that the creators are simply creating algorithms -just doing maths and creating abstract and valueless systems. dwork & mulligan (2013) , however, point out that any such system is bound to reflect some of the implicit ideas and values of its creators. whilst this might occur by pure accident, despite the intentions of the designers, gillespie (2010) points out that most of the creators of such systems have clear profit-maximisation goals. such goals and accompanying incentives might make it difficult for us to regard ai as an equivalent to leibniz's god. griffy-brown et al. (2018) notes the profit-driven technocratic ideology, which is different from the disinterested and scientific technocratic ideal i previously described. these considerations show that a robust system of algorithmic supervision is required whenever the ai in question is a human creation, and not a magical decision box. another precondition is some form of political control of the core values and direction of society. one possibility is to simply scale back, but largely keep, the current system of representative democracy. this involves a limitation of the scope of politics, as the technocrats have authority over many issues of policy. another, and more radical possibility, is to pursue some of the new modes of popular participation and popular government provided by new technologies. the shaping of algorithms and the potential of increasing popular understanding of ai (lee et al., 2019) . this latter option, while more radical, also shows how a technocracy of ai could in fact increase people's opportunities for political participation, rather than the opposite. while doing so, it could also meet the demands for involving various stakeholders and social groups in political decision making. if this is not enough, increasing broad participation in political also allows us to reap the benefits of dispersed knowledge and decentralised markets, as emphasised by hayek (1960) . the benefits of better political decisions are considerable. i have shown that ai is already being employed in the public sector in order to improve political analysis and decisions, and that it will become ever more capable. if we agree that politics should have good policy as a goal, and if we do not consider our current democratic order as a given and inviolable good, we start opening the door to the idea that ai should have political power. in this article i have focused on the potential widespread application of ai in the domain of government, and i have examined an extreme case in which ai is given the authority to form policy. this is done through systems characterised by low transparency, high complexity and high degrees of automation. as such, the situation is rigged to trigger the alarms of those concerned with democracy, participation and the political harms of algorithmic governance. i have chosen to focus mainly on a set of harms perceived as vital from a political science stance, while acknowledging that other important harms are both important and necessary for acquiring a complete picture. however, all the harms from and objection to an ai technocracy cannot not be discussed in the required detail in one article. as such, my account is a partial one which must be seen in combination with detailed accounts of the harms related to bias, discrimination, autonomy, etc. however, the political harms are vital, and not as often discussed in detail as some of the other harms, which makes this an important part of a full understanding of the consequences of algorithmic governance. through five objections, based on participation, legitimacy, nonmorality of machines, transparency and accountability, i have reached the conclusion that an ai technocracy is not derailed by such objections. if, that is, certain preconditions are met. an ai technocracy capable of overcoming the objections discussed must involve some way of making sure that humans retain the power to define the fundamental goal of politics -that of deciding what our fundamental values are, and how we imagine the good society. if humans retain this role, and make sure that the process is a democratic one, both participation and legitimacy might be preserved. furthermore, i have argued that the non-morality of machines is not a problem if we assign responsibility either to the proposed democratic council of values and ai control or to the producers of ai. ai is in some respects like the magical decision box. if we had such a device, we could employ it, and over time we would might to trust that its decisions were good -like leibniz's god. if the mdb were a mystical device, not one created by humans, there would be, i argue, no reason for any particular group of people to object to its rule. while there are many reasons to be wary of an ai technocracy of human origins, it is time we take the possibility seriously. firstly, because we are already seeing the application of ai in many areas of the public sector, without a full and open debate about the desirability and implications of such a development. secondly, we must take the possibility seriously, as i have shown that it has the potential to revitalise democracy, increasing public participation in politics, and provide more efficient outcomes. the moral machine experiment the coming of post-industrial society the emergence of a freedom of information movement: anonymous, wikileaks, the pirate party, and iceland algorithmic accountability and public reason pattern recognition and machine learning deep blue the go files: 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equality and productivity with ai-driven tax policies much research focuses on the possibilities of using technology to facilitate participation, and janssen & kuk (2016) calls for democratizing algorithms. i have focused on the webuildai example in this article, which points toward the potential for massive popular participation in acknowledgements i wish to thank the anonymous reviewers for their invaluable comments and recommendations that greatly improved the article. aristotle ( henrik skaug saetra: all parts of the article. key: cord-276039-nqqwnmwc authors: rua, rejane; gessain, antoine title: origin, evolution and innate immune control of simian foamy viruses in humans date: 2015-02-17 journal: curr opin virol doi: 10.1016/j.coviro.2014.12.003 sha: doc_id: 276039 cord_uid: nqqwnmwc most viral pathogens that have emerged in humans have originated from various animal species. emergence is a multistep process involving an initial spill-over of the infectious agent into single individuals and its subsequent dissemination into the human population. similar to simian immunodeficiency viruses and simian t lymphotropic viruses, simian foamy viruses (sfv) are retroviruses that are widespread among non-human primates and can be transmitted to humans, giving rise to a persistent infection, which seems to be controlled in the case of sfv. in this review, we present current data on the discovery, cross-species transmission, and molecular evolution of sfv in human populations initially infected and thus at risk for zoonotic emergence. most viral pathogens that have emerged in humans during the last century are thought to have originated from various animal species and are thus of zoonotic origin [1] . while the modes of viral dissemination within the human population are generally well characterized, the initial steps at the interspecies interface that lead to viral emergence remain poorly understood. epidemiological field studies conducted in certain specific high-risk populations are thus crucial to obtain new insights into these early events of the emergence process. human infections by simian viruses represent an increasing public health concern. indeed, non-human primates (nhps) are considered to be the likely sources of viruses that infect humans and thus may pose a significant threat to human population [2] . this is well illustrated by some retroviruses, which have the ability to cross-species, possibly adapt to a new host and sometimes spread within this new species. it is now clear that the emergence of human immunodeficiency virus type 1 (hiv-1) and hiv-2 in humans has resulted from several independent interspecies transmissions. different siv types from chimpanzees and gorillas in the western part of central africa as well as sooty mangabeys in west africa, gave rise to hiv-1 and hiv-2 respectively, probably during the first part of the last century [2] . similarly, the origin of most human t cell lymphotropic virus type 1 (htlv-1) subtypes appears to be linked to interspecies transmission between stlv-1-infected monkeys and humans, followed by variable periods of evolution in the human host [2] . in this brief review, we will present the current available data on the discovery, cross-species transmission and molecular evolution of the simian foamy viruses (sfv) present in different human populations at risk for zoonotic emergence. since the initial description of foamy virus (fv) in rhesus monkey kidney cells in 1954 [3], such viruses have been isolated from several animal species, including numerous nhp species. the prevalence of fvs in naturally infected animals is generally high, but varies widely according to species [2]. among nhp populations, sfv seroprevalence can reach up to 75-100% in adults [2] . in african green monkey and macaques, oral mucosa tissue is an important site for viral replication, explaining why foamy viral rna is found at a high concentration in the saliva of such primates [4 ]. saliva-based means of transmission, such as bites, have been thus strongly suggested. infection by sfv itself does not seem to cause any disease in infected nhps, but studies have not been conducted to address this question specifically. however, when considering co-infections in macaques, sfv can increase the pathogenicity of simian immunodeficiency virus [5] . in 1971, the first human foamy virus was isolated from the cell culture of a kenyan patient suffering of nasopharyngeal carcinoma [6] . phylogenetic studies demonstrated that this virus originated from the east african chimpanzee subspecies (pan troglodytes schweinfurtii). this strain is now considered to be the 'prototype foamy virus' (pfv). however, its exact origin remains unclear (in vivo crossspecies transmission from a chimpanzee to the african patient, or cell culture contamination) [7] . in the 1970/ 80s, several papers showed conflicting results on the presence of sfv in human populations and in different patients [8, 9] . these findings reflected the high percentage of non-specific serological reactivity and the lack of specific confirmatory tests at that time. in 1995, based on specific serological and molecular assays, the first clear evidence of sfv in humans was reported among 3 laboratory and monkey house personnel [10 ]. since then, other groups have reported similar findings in a series of workers occupationally exposed to nhps in the usa and canada and more recently in personnel from zoos or primate centers in gabon and china [8] . infection by sfv in a more natural setting was then demonstrated in villagers from cameroon [11] . they were mostly hunters who reported direct contacts with blood and/or body fluids from wild nhps. we extended such studies into different areas and populations of this central african country and found the presence of sfv infection in at least 50 persons [12 ] . the great majority of them (bantus or pygmies) were men who had been bitten by an ape (mostly gorillas but also chimpanzees) or a small monkey (mostly cercopithecus nictitans) during hunting activities. a recent report from gabon confirmed such frequent cross-species transmission in hunters after severe bites from mostly gorilla [13] . infected women were also recently found in the democratic republic of congo [14] . in south and southeast asian countries, sfv zoonotic infection has also been detected in various contexts of interspecies contact including 'monkey temple' workers, pet owners and people living around freeranging macaques in south and southeast asia [15] . it is interesting to note that the human/nhp interface in asia differs greatly from that in africa [16] . human and macaques sympatry in southeast asia dates back as far as 25,000 years. human-macaque commensalism is frequent in many monkey temples of these regions each year putting a very large number of persons, including tourists, at risk for macaques bites [17] . indeed, human subjects in some south asian countries came into contact with rhesus macaques mostly in the context of their daily lives, sharing a geographical area that is the 'home range' of both primate populations [16] . such frequent interactions can lead to possible viral interspecies transmission. the situation is very different in central africa where bushmeat hunting (including apes such as gorillas and chimpanzees) is the major risk factor associated with sfv interspecies transmission [12 ] . furthermore, in central africa, the number of contacts between humans (mostly hunters and their wives and butchers) and potentially infected nhps has probably greatly increased during the last century. this is believed to be due to increased hunting activities, resulting from a combination of urban demand for bush-meat, greater access to nhp habitats provided in part by logging roads, easier accessibility to fire arms, an increase in populations living in forest areas, and the associated increase in local food needs [2]. while nowadays, humans are not considered to be a natural host of sfv, more than 100 cases of sfv infection have been reported so far in individuals in close contact with nhps [8]. among them, no specific pathology has been yet demonstrated. however, the selection bias inherent in the enrollment of healthy persons in the very few performed studies greatly limits the current ability to identify any potential associated disease. the sfv genome comprises the retroviral gag, pol and env genes, and two regulatory genes tas and bet. an internal promoter allows basal transcription of tas and bet ( figure 1 ). the transactivator tas then activates a second promoter located in the long-terminal repeat, which induces the synthesis of the gag, pol and env structural proteins. sfvs can also go through a late reverse transcription step, before the release of sfv particles. thus, sfv particles can contain sfv rna and sfv dna genomes ( figure 2a ) [18] . 48 emerging viruses: interspecies transmission in vitro, sfv can infect most cell types. heparan sulfates represent attachment factors [19] . however, an sfv receptor has not yet been discovered and is probably ubiquitous. once productively infected, cells usually form syncitia with a 'foam-like' cytopathic effect, before cell death occurs (figure 3 , a to d). however, some cell lines as well as primary cells of the myeloid or lymphoid lineage can remain chronically infected [20, 21] . in nhps, sfv is latent in blood cells and replicates in the superficial epithelial layer of the oral mucosa [4 ,22] , which explains the mode of transmission of sfv to humans, mainly through bites [12 ] . viral genomes in the buccal swab usually reflects those found in the blood [23]. in humans, sfv is also present in pbmcs and saliva. however, no active replication, as monitored by sfv rna, has been detected in the pbmcs nor in the saliva [24]. in the blood, the cellular targets of sfv remain poorly studied. preliminary studies, performed mostly in small series of chimpanzees, african green monkeys, and in few humans, have shown sfv dna in lymphocytes, especially cd8+ t cells [25, 26] . by studying a larger cohort of humans infected with a gorilla strain of sfv in cameroon, we demonstrated that cd8+, cd4+ t cells and cd19+ b lymphocytes were preferentially infected, compared to monocytes and nk cells (table 1) . sfv dna was detected in both memory and naive cd4+ t lymphocytes and sfv dna levels in cd4+ t cells were positively correlated with the duration of the infection [27] . sfv genomes display a high evolutionary conservation among all the species infected and sfv genetic variability within one infected animal is very low over time (<1% variation over 13 years) [28] . this genetic stability might be explained by the long co-evolution with their host [29 ] and their subsequent efficient adaptation. sfv genetic stability could also prevent or delay infection of new hosts as molecular changes may be required to infect cells from a different species and/or evade host-specific immune responses [30] . genetic stability was also found in sfv-infected humans, with less than 1% divergence in the nucleotide sequence over several years [13, 24] . several groups also reported genetic modifications including mutations in the bet accessory gene [25, 31] , deletion in the u3 sequence of the ltr which improves fv replication in vitro [31, 32] and deletions in the tas sequence, which might be linked with the chronicity of the infection [31] . however, most of these modifications are not a unifying trait of zoonotic strains, and can also be found in nhp or experimentally infected animals [31] . this suggests that viral strains remain mainly stable, even decades after cross-species transmission. this is probably related to the apparent low level of sfv replication in infected humans, which could be linked with their efficient immune control. observation of sfv and sfv-infected cells. bhk-21 cells were infected with a chimpanzee sfv strain isolated from an sfv-infected hunter [31] . a large syncytium with a 'foamy' aspect is visible using light microscopy (a). an immunofluorescent staining was performed as previously described [62] . sfv antigens are revealed in the green channel and nuclei are stained with dapi (b). the transactivator tas then activates a second promoter located in the long-terminal repeat, which induces the synthesis of the gag, pol and env structural proteins. after assembling, viral particles bud essentially from the endoplasmic reticulum, with a late reverse-transcription step giving rise to both dna and rna particles. (b) sfv is detected by plasmacytoid dendritic cells (not exclusively) which triggers ifn-i production. several antiviral factors that can be induced by ifn-i are active against sfv. trim5a are antiviral proteins that prevent sfv decapsidation [50] . apolipoprotein b-editing catalytic polypeptide-like subunit (apobec) enzymes act on the negative strand dna produced by the viral reverse transcriptase, resulting in g-to-a mutations in the viral genome, with potential deleterious consequences. fv bet protein has been shown to partially prevent apobec action [40] [41] [42] [43] . n-myc interactor (nmi) as well as a member of the interferon-induced protein (ifp) family, ifp35, can inhibit the replication of the prototype and the bovine fv respectively, by direct interaction with the viral transactivator tas [46, 47] . tetherin is known to block many different types of enveloped viruses by tethering the budding virus at the cell membrane and is also efficient to prevent fv release [48, 49] . virology 2015, 10:47-55 www.sciencedirect.com origin, evolution and innate immune control of simian foamy viruses in humans rua and gessain 51 table 1 sfv tropism and viral load in the blood of sfv-infected humans and nhps. the proportion of sfv dna positive samples among leukocyte populations in sfv-infected nhp and sfvinfected humans is indicated. infecting strain hum. sfvgor nb of sfv dna + samples/ind. tested 10/11 9/11 7/11 2/11 1/11 nt [27] sfv dna copies/number of cells quantitative pcr 6-274 sfv dna/10 5 cells 6-61 sfv dna/10 5 cells 10-91 sfv dna/10 5 cells 4-5 sfv dna/10 5 cells 5 sfv dna/10 5 cells na [27] agm: african green monkey; cpz: chimpanzee; hum.: human; nt: not tested; nb.: number; ind.: individuals; na: not applicable. sfv-hu1 is a strain close to sfv-3. a in the study from von laer et al. [26] , the population tested is actually non-cd4+ non-cd8+ lymphocytes, and is thus likely enriched in both cd19+ b and cd56+ nk lymphocytes. the proportion of positive samples are 7/9 agm, 3/4 cpz and 0/2 humans. sfv dna loads in this sorted cell population is 1/10 5 cells (in the 7/9 positive samples from agm) and 1/10 5 cells (in the 3/4 samples from cpz). b in the study from callahan et al. [25] , only one dilution of leucocytes dna was shown, corresponding to a viral load of >70 sfv dna copies/10 5 cells, assuming the usual sensitivity of 1 sfv dna copies/10 5 cells. current opinion in virology 2015, 10: [47] [48] [49] [50] [51] [52] [53] [54] [55] in vivo super-infection and recombination of sfv some groups have reported cases of co-infection by two different sfvs in apes, monkeys, as well as in humans. some chimpanzees infected by sfvcpz strains were coinfected with sfv from colobus monkeys [33] , or cercopithecus monkeys [34] . more frequently, chimpanzees and macaques can harbor distinguishable sfvcpz and sfvmac strains respectively, likely the result of multiple independent infections that accumulate with age [16, 35, 36] . sfvcpz infection appears to first occur via vertical transmission but is followed in adult life by the acquisition of further infections, possibly stemming from aggressive interactions [35] . in some of these studies, as well as in a recent one, some sfv appears to be recombinant strains [34, 36, 37] . we, and others, have found different clones of sfv in single individuals [12 , 16, 24] , suggesting a co-infection with multiple sfv strains. whether they were acquired at a single time or through several contacts with nhp is not known. interferons are potent antiviral molecules and usually represent one of the first lines of defense to pathogens. in vitro, sfvs were first thought to be low interferoninducers [38] but this actually depends on the cell type. indeed, we found that sfvs are efficiently sensed by human hematopoietic cells and induce the production of high levels of ifn-i [39] . the main producers of ifn-i in the blood are the plasmacytoid dendritic cells, which detect sfv genome after uptake of sfv particles, through the endosomal toll-like receptor 7 [39] . in culture systems, the addition of type i ifn impairs sfv replication, suggesting that interferon-inducible genes might be involved in the control of sfv replication (figure 2a and b): -apolipoprotein b-editing catalytic polypeptide-like subunit (apobec) enzymes are a family of antiviral cytidine deaminases that act on the negative strand dna produced by the viral reverse transcriptase, resulting in g-to-a mutations in the viral genome, with potential deleterious consequences. however, viruses often produce proteins that counteract cellular defense systems. fv bet protein has been shown to prevent apobec action [40] [41] [42] [43] . despite the action of bet, it was reported that sfv genomes found in humans displayed some g-to-a mutations [24, 44] . matsen et al. found that the frequence and the type of hypermutations were different in humans and macaques, with a majority of hypermutations leading to stop codons in humans, suggesting active restriction of the infecting strain in humans and not only passive acquisition of previously mutated strain [45 ] . additional studies have been performed in vitro to elucidate the mechanisms involved in the control of sfv replication, although their relevance in vivo has not been tested so far: -two homologous proteins, n-myc interactor (nmi) as well as a member of the interferon-induced protein (ifp) family, ifp35, can inhibit the replication of pfv and bfv, respectively, by direct interaction with the viral transactivator tas [46, 47] . -tetherin is known to block many different types of enveloped viruses by tethering the budding virus at the cell membrane and is also efficient to prevent fv release [48, 49] . -trim5a are antiviral proteins that prevent viral decapsidation [50] . they act in a species-specific manner: pfv and sfvmac are restricted by trim5a from most new world monkeys, but not from other primates including humans. this indicates that trim5a is probably not involved in the restriction of sfv after cross-species transmission from chimpanzees and macaques to humans, at least nowadays [51] . of note, some known antiretroviral factors are not potent against sfv. for instance, as reverse transcription occurs predominantly before entry into target cell, these viruses are resistant to the dntp levels reduction by samhd1 [52] . few studies have addressed the question of adaptative immune response to sfv. first, neutralizing antibodies present in the serum of infected animals inhibit sfv transmission and infection in rhesus macaques [53] , indicating a role of the adaptive immune response in the control of sfv in vivo. this is further supported by the fact that antibodies titers in feces of chimpanzees infected with sfv are inversely correlated with sfv viral load [34] . however, very few data are available concerning the adaptative immune response to sfv in humans. antibodies against sfv are detected in the blood of infected individuals and were also found in saliva and urine samples in four sfv-infected individuals tested. however, antibody titers were lower than in infected chimpanzees, which could be linked to lower levels of sfv replication in humans [54] . neutralization of sfv by sera from three sfvagm-infected individuals was reported in 1983 and 1997 [55, 56] . secondly, neutralization of ifn-gamma in activated pbmcs infected with sfv, increases viral expression [57] and sfv up-regulates mhc-i in vitro [58] , suggesting that adaptative immunity might be elicited and efficient in the control of sfv in infected individuals. although virus-specific t lymphocytes are key effectors that control the outcome of htlv and hiv infection [59] , no report has addressed their presence in sfvinfected nhps or humans. human infection by sfv constitutes a unique natural model to study the various parameters involved in restriction of retroviral emergence. the future of sfv emergence in humans remains an open question. however, the current increase in nhp-to-human contacts (in part due to hunting activities and animal handling) may favor such cross-species transmission [2] . furthermore, other factors such as sfv co-infection with hiv, already reported in cameroon [60] , may increase the risk of sfv pathogenicity and will require further investigation. nevertheless, any prediction concerning viral emergence remains very difficult and hazardous, as recently exemplified by ebola and mers coronavirus outbreaks in west africa and saudi arabia respectively [61] . in this regard, intensive research on mechanisms of the first steps of viral emergence should be further developed. the authors declare no competing financial interests. cross-species transmission of simian foamy virus to humans in rural gabon, central africa novel simian foamy virus infections from multiple monkey species in women from the democratic republic of congo diverse contexts of zoonotic transmission of simian foamy viruses in asia zoonotic simian foamy virus in bangladesh reflects diverse patterns of transmission and co-infection primateto-human retroviral transmission in asia molecular biology of foamy viruses heparan sulfate is an attachment factor for foamy virus entry in vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus productive persistent infection of hematopoietic cells by human foamy virus expanded tissue targets for foamy virus replication with simian immunodeficiency virus-induced immunosuppression persistent zoonotic infection of a human with simian foamy virus in the absence of an intact orf-2 accessory gene lymphocytes are the major reservoir for foamy viruses in peripheral blood in vivo cellular tropism of gorilla simian foamy virus in blood of infected humans genetic stability of foamy viruses: longterm study in an african green monkey population this important study sets the date of cospeciation between foamy viruses and primates as far as 30 my. the recent discovery of endogenous viruses, and fv in fishes overviews of pathogen emergence: which pathogens emerge, when and why? genetic characterization of simian foamy viruses infecting humans long terminal repeat u3 length polymorphism of human foamy virus interspecies transmission of simian foamy virus in a natural predator-prey system molecular ecology and natural history of simian foamy virus infection in wild-living chimpanzees mother-offspring transmission and age-dependent accumulation of simian foamy virus in wild chimpanzees population dynamics of rhesus macaques and associated foamy virus in bangladesh. emerg microbes infect identification of recombination in the envelope gene of simian foamy virus serotype 2 isolated from macaca cyclopis in vitro studies on interferon-inducing capacity and sensitivity to ifn of human foamy virus innate sensing of foamy viruses by human hematopoietic cells foamy virus bet proteins function as novel inhibitors of the apobec3 family of innate antiretroviral defense factors the antiretroviral activity of apobec3 is inhibited by the foamy virus accessory bet protein species-specific inhibition of apobec3c by the prototype foamy virus protein bet prototype foamy virus bet impairs the dimerization and cytosolic solubility of human apobec3 g restriction of foamy viruses by apobec cytidine deaminases this interesting study provides evidence of distinct effects of apobec in humans and monkeys, suggesting that mutated strains found in humans are not passively acquired from macaques but are efficiently targeted by anti-viral mechanisms in humans n-myc interactor inhibits prototype foamy virus by sequestering viral tas protein in the cytoplasm ifp35 is involved in the antiviral function of interferon by association with the viral tas transactivator of bovine foamy virus tetherin inhibits prototypic foamy virus release feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection speciesspecific inhibition of foamy viruses from south american monkeys by new world monkey trim5{alpha} proteins restriction of foamy viruses by primate trim5 alpha restriction of diverse retroviruses by samhd1 role of neutralizing antibodies in controlling simian foamy virus transmission and infection mucosal and systemic antibody responses in humans infected with simian foamy virus characterization of a foamy virus isolated from cercopithecus aethiops lymphoblastoid cells simian foamy virus isolated from an accidentally infected human individual gamma interferon is a major suppressive factor produced by activated human peripheral blood lymphocytes that is able to inhibit foamy virus-induced cytopathic effects human foamy virus infection activates class i major histocompatibility complex antigen expression ctl quality and the control of human retroviral infections coinfection with hiv-1 and simian foamy virus in west central africans emerging infectious diseases and pandemic potential: status quo and reducing risk of global spread simian foamy virus transmission from apes to humans, rural cameroon origin, evolution and innate immune control of simian foamy viruses in humans rua and gessain 55 www.sciencedirect.com current opinion in virology rr was supported by the bourse de l'ecole normale supã©rieure from the facultã© paris diderot. this work was supported by the institut pasteur in paris, france, by programme transversal de recherche 437 from the institut pasteur, and by the french government program investissement d'avenir (grant anr-10-labx-62-ibeid). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. betsem e, rua r, tortevoye p, froment a, gessain a: frequent and recent human acquisition of simian foamy viruses through apes' bites in central africa. plos pathog 2011, 7:e1002306. this study elucidates modes of transmission of sfv from apes and monkeys to humans, mainly by bites. key: cord-021552-6jbm869r authors: hurst, christon j.; adcock, noreen j. title: relationship between humans and their viruses date: 2007-05-09 journal: viral ecology doi: 10.1016/b978-012362675-2/50015-x sha: doc_id: 21552 cord_uid: 6jbm869r nan many of the viruses that can infect humans should not be considered as viruses of humans, but rather as zoonotic. zoonotic viruses are those viruses of animals that can cross boundaries such that they occasionally infect humans. some examples of diseases induced in humans by zoonotic viruses are dengue and ebola fever, the equine encephalitids (i.e., eastern, st. louis, venezuelan, and western), hantavirus pneumonia, lassa fever, marburg fever, rabies, and yellow fever. additionally, it should be noted that the zoonotic category includes most, if not all, of the human illnesses induced either by arboviruses (viruses whose transmission is vectored by arthropods) or by the hemorrhagic fever viruses. with respect to the zoonotic viruses humans are, at best, alternate hosts. humans do in fact usually represent dead-end hosts for these zoonotic viruses, meaning that subsequent transmission of those viruses either to new humans or back to the natural host is not sustained. there is a subgroup of zoonotic viruses that, although principally remaining viruses of animals, seem to have adapted themselves to use humans as natural hosts. this adaptation is indicated by the fact that these viruses have demonstrated an ability to sustain a chain of transmission among humans. examples of zoonotic viruses that have shown this ability to adapt themselves to become viruses of humans are those members of the family flaviviridae (genus flavivirus) that induce the human diseases known as dengue and yellow fever. all of the above-mentioned zoonotic viruses contrast with the viral agents that clearly are known by their nature to be viruses of humans. examples of viruses of humans are those that induce the diseases known as acquired immunodeficiency syndrome, fever blisters, measles, mumps, polio, rubella, smallpox, t-cell leukemia, t-cell lymphoma, and type a influenza. the aim of this chapter is to address those viruses that are considered to be viruses of humans. those viruses of terrestrial mammals that are considered to be zoonotic are addressed by calisher and fenner in chapter 13. every virus species needs to have a successful overall approach for sustaining its existence. that overall approach must enable the virus to attain its two principal goals, namely, that the virus be able to reproduce itself within a host and that the virus then be transmitted onward to a new host. those mechanisms that any given virus species employs for achieving its sustainment, of course, have been developed through a process that involved an initiation of events by random chance followed by evolutionary selection. the most successful overall approaches may be those that subsequently evolve into the types of relationships between a virus and its host species that will allow the virus to persist without eliminating the host population. this latter point is very important, because the virus may in turn become extinct if it kills off the host population. it is for this reason that excessive virulence will be detrimental to the virus, and an interesting side point may be that, if an individual host cannot successfully surmount the infection, then the death of that individual host may be seen as an altruistic defense mechanism for the host population as a whole. achieving self-reproduction is the first principal goal of the virus. the processes involved can be divided into three aspects. the first aspect is the virus's overall approach with regard to the course of the infection it establishes within the host. this involves the time course of the infection and the extent to which infectious progeny viral particles are produced during the course of the infection. the second aspect is the replication strategy employed by the virus. this involves the issues of where the virus begins its march through the host body and the physical trajectory followed until the virus exits in search of a new host. the third aspect involves which approaches, if any, the virus uses to avoid the host defensive mechanisms. as mentioned in chapter 1, the goal of establishing an effective course of infection is an aspect of viral reproduction that can be attained in many ways. we can summarize the strategies that viruses use as following four basic patterns. three out of the four patterns of the course of viral infection are considered to be productive. the infection will be acquired in the form of infectious viral particles, and subsequently produced progeny viral particles then serve to infect future hosts. productive, in this context, means that the number of progeny viral particles produced during the course of an infection is sufficient for the particles to transmit the infection to a new host with some reasonable probability. the productive approach involves an evolutionary decision as to whether there will be either a very short initial but highly productive course of infection (short term-initial), a course of infection that is prolonged but only intermittently productive (recurrent), or a productive though very slow course of infection whose severity progressively increases with time to reach a dramatic end-stage (increasing to end-stage). these three patterns can be described as follows. a. short term-initial. viral production only occurs during a short time course near the time of initiation of the infection, which then abruptly ends. the human host may or may not survive beyond the course of this short infection. host survival depends on the type of virus involved, the extent to which the involved virus and humans have had time to coevolve as species, and whether or not the ancestral humans of that particular subgroup of the human host population previously have had contact with the causative virus. coevolution usually will tend to make the outcome of this pattern of viral infection sufficiently mild as to be associated with a fairly low incidence of mortality in an otherwise healthy population of human hosts. some examples of this pattern would be the infections caused by the human caliciviruses (family caliciviridae), human influenza viruses (family orthomyxoviridae), human polioviruses and human rhinoviruses (family picornaviridae), and the human rotaviruses (family reoviridae). b. recurrent. viral production, often very pronounced initially, is recurrent when the virus persists in a latent state within the host body and viral particle production periodically recurs but is not life threatening. some examples of this pattern would be the infections caused by the human herpesviruses (family herpesviridae) and the human papillomaviruses (family papovaviridae). c. increasing to end-stage. viral infection normally is associated with a slow, almost innocuous, start, followed by a gradual progression, associated with an increasing level of viral production and eventual host death. death of the host may relate to destruction of host immunological defense systems, which then results in death by secondary infections. this pattern of infection may take from l0 to 40 years to kill a human host. the infections caused by the human immunodeficiency viruses and the human t-lymphotropic viruses (all belonging to the family retroviridae) represent examples of this pattern. the fourth basic pattern of viral infection is considered to be nonproductive. a nonproductive infection is one in which the production of infectious virus particles is so limited that the virus must transmit itself through other means, usually by transferring a copy of only the nucleic acid genome of the virus. in these instances, the viral infection is normally acquired by direct transfer of the viral genetic material from the human parents to their developing fetuses, such transfer occurring via the egg and sperm cells. there may be no apparent health effects associated with such an infection. an example of this pattern is the infections caused by the endogenous retroviruses (family retroviridae), whose genomes are incorporated into the chromosomal material of every cell in the human body (villareal, 1997) . the nonproductive pattern of infection seems to suggest the highest degree of coevolution between a virus and its host, since a nonproductive virus has no means of transmitting itself to a new host without some very active, albeit perhaps unwitting, participation on the part of the present host. this section addresses the questions of where and how the virus begins its march through the host body, and how the virus then continues the course of this attack, leading ultimately to the concept of viral reproduction strategies at the host population level. discussion of the strategy of viral replication within the body of a host organism begins at the most basic level, which is attachment of the virus to a particular molecule present on the surface of host cells. such a molecule is said to be the "virus's receptor," and will be some cellular protein or lipid component naturally produced by those cells. the virus's choice of receptor is a product of viral evolution. after binding to its receptor, the virus gains entrance to the interior of the cell and viral replication begins. viruses whose genomes are composed of dna generally replicate mainly within the nucleus. in contrast, those viruses with rna genomes generally focus their center of replication in the cytoplasm. during the course of replication, the virus must decide which cellular systems and machinery it will employ. some large viruses carry the genomic coding capacity for many of their own enzymes, while others may rely almost completely on the enzymatic machinery possessed by the host cell. many viruses, including those belonging to the genus enterovirus of the family picornaviridae, are said to be highly cytopathogenic, meaning that they usually quickly kill the host cell as a result of infection. other viruses, such as those that occupy the genus rubivirus of the family togaviridae, may cause prolonged and severe crippling of the cell rather than killing it outright. a further discussion of these issues can be found in chapter 3. viruses vary greatly with respect to the tissues they tend to target for infection. on a larger scale, this then leads to identification of those organs that the viruses are affecting. this selective targeting is referred to as a "tropism." viral tropisms can be divided into those considered primary and those considered secondary. primary tropisms will be associated with production of those viral particles that subsequently contribute to transmission of the viral infection to a new host. as such, the primary tropisms tend to be related to those sites (termed "portals") through which the virus either enters or exits the host body. secondary tropisms may represent accidents. some of these accidents may come about as a result of the molecule that a virus uses as its receptor, existing on the cells of tissues that are unrelated to those that the virus must employ in order to achieve transmission. nevertheless, secondary tropisms may contribute greatly to the types and severity of the illnesses associated with infection of humans by any particular virus. when considered at the host population level, the strategy of viral replication includes the ease with which or likelihood that a virus is transmitted to new hosts plus the severity of infection and accompanying likelihood of death (including the age-related likelihood of death) for any given host individual. through the course of evolution, many viruses have developed mechanisms for either countering or evading the human immune and non-immune defenses as a means for enhancing the probability of viral success. the human immune system includes both humoral (antibody-mediated) and cellular components. the cellular components can include granulomatous reactions, which play a role in defense against protozoans, though their possible role in antiviral defenses has been incompletely explored. those mechanisms that viruses use to either avoid or minimize attack by the host immune system can be divided into the four following groups. the use of these types of mechanisms seems particularly critical in association with those viral infections that persist within a human host for very long periods of time, often up to decades. a. antigenic mimicry. the produced antigens are similar to those of the host, as with prions. b. rapid viral mutation. this mechanism includes both antigenic drifting and shifting. some viral types demonstrate rapid viral mutation during the course of an infection, as occurs with the human immunodeficiency viruses of the family retroviridae. other virus types, such as the influenza viruses of the family orthomyxoviridae, demonstrate rapid viral mutation between reinfections of the same host. c. low antigenicity. some viruses inherently seem to provoke little, if any, immune response. this often occurs because the virus persists in a latent state within host cells, during which time either little or no viral antigenic material is produced. examples include the endogenous retroviruses of the family retroviridae and the human herpesviruses of the family herpesviridae. d. infect the immune cells! the most direct attack may be the most effective. exceptionally notorious examples of this approach are the genus rubivirus of the family togaviridae and the genus lentivirus of retroviridae. aside from the above groupings, some viruses such as the norwalk virus of the family caliciviridae seem to be antigenic but provoke an immune response that is minimally effective. the body has non-immune defense mechanisms that help to protect against viral infections. these mechanisms are associated with the portals through which viruses can enter the body of a host. examples of non-immune defenses include the enzymes secreted as a part of pancreatic fluid, saliva, and tears. various glands associated with mucosal tissues secrete antimicrobial compounds into the mucus produced by those tissues. some mucosal tissues also possess cilia whose movement helps to expel both the mucus and any foreign materials, including pathogens, that become entrapped within the mucus. an additional example of a non-immune defense is the stomach acid produced to aid digestion of organic compounds. many gastroenteritis viruses, such as the rotaviruses of the family reoviridae and the astroviruses of astroviridae, have evolved such an effective resistance to attack by proteolytic enzymes that those viruses virtually need partial proteolysis to facilitate their infectivity. the influenza viruses of the family orthomyxoviridae are known for their ability to paralyze the activity of the mucosal cilia located within the respiratory tract. one of the defining characteristics for the enteroviruses of the family picornaviridae is their resistance to acidic exposure. the task of achieving viral transmission between hosting individuals involves two aspects. the first is the type of infectious material in which a virus will leave its present host, while the second involves the route by which the virus can encounter its proximate host. the types of bodily materials within which viruses can be released include substances that exit during the course of normal body functions. among these substances are feces and a variety of liquids, including menstrual blood, respiratory secretions of the upper and lower tracts, saliva, semen, tears, urine, and vaginal fluid. sweat is another fluid that is naturally released from the body; however, it is not known to contain viruses. viruses can also be found in blood released from wounds in the skin; from blood acquired by blood-consuming parasitic insects, among which are the fleas and several groups of flies, ticks, and mosquitoes; and blood leaked from swollen or ruptured capillaries into mucosal tissue and skin pores. those natural routes by which viruses are transferred to and between humans are the same routes associated with all surface-dwelling terrestrial vertebrates. these routes are tightly associated with the portals of entry and exit that any particular virus family uses as it tries to survive and find its way from one host to the next. viral transmission routes can be subdivided into two broad groups. the first is transmission by direct contact (also known as direct transfer) between two members of those species that host the virus. this includes both the possibility of transmission between two members of the principal host species and the possibility of transmission between a member of that principal host species and some alternate host species, and the latter may represent a vectoring species. the second group is transmission by indirect contact (also known as indirect transfer). these routes have been described in detail by hurst and murphy (1996) and are represented in figures 9 and 12 of chapter 1 herein. as explained in chapter 1, there are some routes of viral transmission that are considered unnatural vehicular routes. such routes represent the use of unnatural vehicles as a means to evade the host defenses associated with natural portals of entry. these unnatural routes involve invasive medical devices (such as syringes, endoscopes, and other surgical instruments) and transplanted tissues, including transfused blood and blood products. the remainder of this section describes the natural routes of viral transmission between hosts. the direct contact approach offers one major advantage and one major drawback to the virus. the advantage is that those viruses transmitted by direct contact need not be stable when exposed to ambient environments. the drawback is that the number of new hosts to which they have potential access may be smaller than for viruses transmitted by indirect contact. viruses that are endogenous by their nature will survive for as long as the host survives. although endogenous viruses can only be transmitted to host progeny, these viruses neither have to adapt themselves to nor coevolve with any other hosting species. an example of this type of endogenous agent would be the endogenous retroviruses of the family retroviridae. viruses that are venereal in nature, that is, transmitted in semen and vaginal secretions during sexual activity, have a somewhat greater potential for contacting new hosts. represented among the venereal viruses of humans are some species of the genera simplexvirus (family herpesviridae) and papillomavirus (family papovaviridae). once these venereal viruses infect a host, they remain associated with the host for the rest of the host's life in the form of a permanent infection. thus, although the frequency with which endogenous and venereal viruses can find a new host is restricted, the viruses compensate for this to some degree by remaining with the host for a very long time. the next step on the scale of host access is represented by those viruses transmitted via direct contact with insect vectors. these viruses have greatly increased access to new hosts and tend not to remain with their present host for the remainder of the host's life. those viruses transmitted by biting insects are commonly referred to as "arboviruses," which is an abbreviation of "arthropod-borne viruses." included among those arboviruses that infect humans are members of the genera alphavirus (family togaviridae); bunyavirus, nairovirus, and phlebovirus (all of the family bunyaviridae); and flavivirus (family flaviviridae). viruses transmitted by way of saliva may be perceived as bridging the categories of direct and indirect contact. if any particular type of virus that is secreted into saliva has either no stability when exposed to ambient environments in oral secretions or only limited stability under those conditions, then the virus will have to be transmitted by saliva that is transferred during oral contact between hosts. conversely, if the particular virus type has good stability when exposed to ambient environments in oral secretions, then that virus can be transmitted on shared food or in association with fomites. some of the viruses transmitted in saliva do remain associated with the host as a permanent infection, and these often are the viruses that possess limited stability in ambient environments, such as members of the family herpesviridae. many of the viruses that are secreted into saliva and can be transferred to a new host in association with fomites do not remain associated with the host as a permanent infection, such as members of the family picornaviridae. in general, those viruses transmitted by indirect contact between hosting individuals tend to produce only transient infections of their individual hosts rather than remain associated with the individual host as a permanent infection. these several latter points suggest that there may be some evolutionary relationship between ease of viral transmission to a new potential host individual or the frequency of opportunities for viral transmission, and the length of time that a virus must be capable of remaining with its present host to have a reasonable chance of achieving eventual transmission. the indirect contact approach also offers one major advantage and one major drawback to a virus. viruses transmitted by indirect contact have the advantage of potential access to a far greater number of hosting individuals than is the case for viruses transmitted by direct contact. the drawback is that the viruses must have evolved stability when they are exposed to the ambient environment. the vehicles that viruses utilize to achieve transmission between hosting individuals by indirect contact are subdivided into the following four categories: food, water, air (in actuality, this refers to aerosols), and fomites. transmission by any of these four categories of vehicles will usually be associated with some specific physical activity on the part of the present host, and will always be associated with some physical activity on the part of the proximate host. of course, foods are items intentionally ingested for their caloric or nutritional value. food contamination can occur by way of the food being a virally infected animal consumed by the proximate host. in such cases, there is no specific physical activity on the part of the present host (the one being eaten) that can be identified as having caused the proximate host to be ingesting contaminated food (indeed, perhaps it is a lack of physical activity on the part of the present host that is to blame). otherwise, viral contamination of foods can result from fecal material being transferred via contact with unwashed hands or if contaminated aerosols fall into the food. a particularly notable example of a virus of humans that is transmitted via foods is the hepatitis a virus of the genus hepatovirus (family picornaviridae). water usually serves as a vehicle after it has been contaminated with fecal material. acquisition of a viral infection from water usually results from the proximate host ingesting contaminated water. physical contact of the skin or mucosa of the proximate host with contaminated water, as may occur during recreational activities or body washing, can result in acquisition of infection. notable examples of viruses transmitted by these waterborne routes are those belonging to the viral families astroviridae and caliciviridae, and the genera enterovirus and hepatovirus of the family picornaviridae. viral contamination of air can occur by two principal mechanisms. the first, and most significant, involves release of aerosols that contain droplets of respiratory secretions (i.e., nasal, oral, or pulmonary mucus). this type of transmission route is referred to as being the route of droplet aerosols. notable examples of viruses transmitted in this manner are those belonging to the viral families coronaviridae, orthomyxoviridae, and paramyxoviridae, and members of the genus viral contamination of fomites (defined as solid environmental surfaces that can serve in transmission of infections) can occur in many ways. the variety of things that represent fomites include items used to conserve warmth (e.g., blankets, clothing), items used while eating (e.g., cups, dinner plates, and utensils), tables on which diapers are changed, doorknobs, medical devices, toilet seats, and toys. the ways by which these environmental items become contaminated include projection of droplet aerosols onto environmental objects during sneezing or coughing, aerosols falling onto objects, and unintended surface contamination (including children's clothing, blankets and toys) by blood, feces, fluid from skin lesions (rashes), nasal secretions, saliva, or urine. the task of achieving viral transmission via this route is accomplished when these objects are subsequently handled or used by a potential proximate host. among the viral genera whose members can be transmitted via fomites are orthopoxvirus (family poxviridae) and rhinovirus (family picornaviridae). twenty of the viral families contain members capable of infecting humans. together, they cause a broad range of illnesses in humans. the terminology used in describing these illnesses is given in table i . the rest of this section summarizes the ecology of these viral families. figure 1 schematizes the manner in which the different aspects of the ecology of viral infection fit together. the literature sources used for to compile this summary include hurst and murphy (1996) , ictv (1995), evans and kaslow (1997) , and white and fenner (1994) . dae) in association with a natural host. transmission of this virus between hosts occurs when an infected animal bites an uninfected animal, with the virus being transferred by saliva into the bite wound. subsequent movement of viral infection into the nervous system and salivary glands of the newly bitten host animal is considered to represent primary tropisms, as infection at these sites is directly related to movement of virus into the body of the current host and subsequent transfer of virus to the next host. infection of the adrenal cortex is considered a secondary tropism, since those viruses produced in the adrenal cortex will not be transferred to a subsequent host animal. however, infection of the adrenal cortex may play a role in the viral ecology by augmenting the aggressiveness of this newly infected host, thereby increasing the likelihood that this animal will then bite other potential host animals. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. adenopathy (the origin of the family name), conjunctivitis, coryza, encephalitis, gastroenteritis, keratoconjunctivitis, pharyngitis, pharyngoconjunctival fever, and pneumonia. infection course ~ productive, both short term-initial and recurrent. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the cervix, conjunctiva, pharynx, small intestine, and urethra; the secondary tissue and organ tropisms are toward the brain, kidney, lungs, and lymph nodes; at the host population level, these viruses generally are endemic and initially acquired at a very early age, with the infections very often asymptomatic in young children. evasion of host defenses ~ uncertain. and indirect (vehicle-borne) contact via fecally contaminated water, food, fomites, and fomites contaminated by respiratory secretions. genus affecting humans: arenavirus. familial nature with respect to members affecting humans" zoonotic. natural hosts: rodents, including commensal voles and mice, as well as commercial colonies of hamsters and nude mice. arthralgia, carditis, encephalomyelitis, encephalopathy, facial edema, fetal loss, focal necrosis of liver, gastritis, hemorrhagic fever, hepatitis, inhibition of platelet function (causes the fatal bleeding associated with this virus family), malaise, meningitis, myalgia, nerve deafness, and pneumonia. infection course ~ productive, short term-initial. at the individual host level, the primary tissue and organ tropisms presumably are toward the liver and lungs; the secondary tissue and organ tropisms are toward the brain, fetus, heart, joints, and nerves; at the host population level, these viruses can be extremely devastating to individual hosts but are poorly transferred between humans, which usually represent dead-end hosts. evasion of host defenses ~ uncertain. predominant routes of transmission between hosts: indirect (vehicle-borne) contact via inhalation of particulate aerosols bearing dried rodent urine or acquisition of infectious materials through skin abrasion (a form of surface contact). genus affecting humans: astrovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. types of illnesses induced in humans: enteritis, gastroenteritis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the small intestine; the secondary tissue and organ tropisms presently are unknown; at the host population level, these viruses are endemic, principally causing a mild enteritis seen in young adults. avoids host non-immune defenses by resistance to proteolytic attack (their infectivity is actually increased by proteolytic attack). via fecally contaminated water, food, and fomites. genera affecting humans: bunyavirus, hantavirus, nairovirus, phlebovirus. familial nature with respect to members affecting humans: zoonotic. natural hosts: largely rodents, but also hares and rabbits, and some ungulates. types of illnesses induced in humans: arthralgia, encephalitis, hematemesis, hematuria, hemolytic uremia, hemorrhagic fever, hemorrhagic pneumonia, hemorrhagic (petechial) skin rash, hepatitis, melena, myalgia, pneumonia, renal dysfunction, retinitis, retroocular pain. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the kidneys, liver, and lungs; the secondary tissue and organ tropisms are toward the brain and eyes; at the host population level, these viruses are not well sustained within human populations, and humans usually represent dead-end hosts. evasion of host defenses ~ uncertain, but may include avoiding host immune defenses by infecting immune cells. direct host-to-vector contact by gnats, midges, mosquitoes, sandflies, and ticks for the genera bunyavirus, nairovirus, and phlebovirus, and indirect (vehicle-borne) contact via particulate aerosols containing dried rodent urine, or contact with rodent excreta or contaminated fomites for the genus hantavirus. genus affecting humans: calicivirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: fish, terrestrial as well as marine mammals (especially swine). gastroenteritis, hepatitis (nonprogressive, but extraordinarily high fatality rate-10 to >25% --for women if contracted during the third trimester of pregnancy), myalgia. infection course ~ productive, short term-initial. ~ral replication ~ at the individual host level, primary tissue and organ tropisms are toward the small intestine; secondary tissue and organ tropisms are toward the liver; at the host population level, these tend to be epidemic within human populations; for the hepatitis e virus it seems that acquisition occurs from swine, with the result being epidemics (often very widespread) of human disease; some acquisition from animals may come from eating infected animals; subsequent transmission of all caliciviruses within human populations is by fecally contaminated waste and thus can be very widespread. evasion of host defenses ~ avoids host non-immune defenses by resistance to proteolytic attack. indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites. genera affecting humans: coronavirus, torovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: some terrestrial ungulates and carnivores. coryza, gastroenteritis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the intestines, lungs (possibly), nasopharynx, and sinuses; at the host population level, these viruses are very widespread and essentially nonfatal. predominant routes of transmission between hosts: indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites. familial nature with respect to members affecting humans: generally zoonotic. natural hosts: unknown, but may include bats and rodents, with primates serving as intermediary hosts leading to human exposure. conjunctivitis, hemorrhagic fever (frequently fatal, death possibly resulting from extreme inflammatory response), hepatic necrosis, myalgia, pharyngitis. infection course ~ productive, short term-initial. viral replication w at the individual host level, primary tissue and organ tropisms are toward the immune cells and liver (possibly); secondary tissue and organ tropisms are toward the adrenal glands, kidneys, liver, and spleen; at the host population level, these viruses are transferred between humans but seem unable to be sustained in human populations; humans usually represent deadend hosts. predominant routes of transmission between hosts: direct contact via host-tohost transfer of contaminated bodily fluids. genera affecting humans: flavivirus, hepatitis c-like viruses. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: members of the genus flavivirus cross-infect a variety of birds and terrestrial mammals via mosquitoes or ticks (depending on the viral species) and most clearly are zoonotic, although those that cause yellow fever and the four that cause dengue may have become viruses of humans without time to coevolve; one species of the genus hepatitis c-like viruses affects humans and seems naturally limited to humans. arthritis with rash, encephalitis, hemorrhagic fever, hepatitis (chronic, which may lead to hepatocellular carcinoma). infection course ~ short term-initial for the genus flavivirus, increasing to end-stage for the hepatitis c virus. viral replication m at the individual host level, primary tissue and organ tropisms are toward the immune cells (principally monocytes and macrophages) and liver; secondary tissue and organ tropisms are toward the brain and liver; at the host population level, most of these viruses are zoonotic, with humans representing dead-end hosts; however, some can be sustained within human populations and occasionally have high lethality rates. evasion of host defenses ~ avoids host immune defenses by infecting immune cells. for flaviviruses, direct host-tovector contact; for hepatitis c virus, presumably direct contact via host-to-host transfer of contaminated bodily fluids. genera affecting humans: orthohepadnavirus. the hepatitis d virus (hdv) is a member of the floating genus deltavirus; it is a defective satellite virus which can coinfect humans, but only in association with the hepatitis b virus (hbv) because hdv encapsidates itself with proteins encoded by the genome of the coinfecting hbv. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: one species of viral family hepadnaviridae (hepatitis b virus) is known to infect humans, and it seems naturally limited to humans. type of illness induced in humans: hepatitis, which may become chronic in adults. infection course ~ productive, short term-initial, and increasing to end-stage. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the liver; secondary tissue and organ tropisms are toward the bile duct epithelium, circulating immune cells, and pancreatic acinar cells; at the host population level, when acquired by adults and older children, these viruses generally cause an acute but short-term illness that sometimes can be fulminant; when acquired by neonates or younger children, initially tends to be subclinical but becomes chronic, and the tendency to be chronic can be racially associated (chinese, possibly also black african). evasion of host defenses ~ avoids host immune defenses by infecting immune cells. direct contact via host-to-host transfer of contaminated bodily fluids and perinatally from contaminated maternal blood. genera affecting humans: cytomegalovirus, lymphocryptovirus, roseolovirus, simplexvirus, varicellovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans, but may pass to primates. carcinoma, carditis, chronic gastrointestinal infection, encephalitis, hepatomegaly, keratoconjunctivitis, lymphoma, myelitis, neuralgia, papular rash of skin and mucosa, paralysis, retinitis, splenomegaly. infection course ~ productive, recurrent. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the genital and oral mucosa, pharynx, and salivary glands; secondary tissue and organ tropisms are toward the eyes, kidneys, liver, lymph nodes, nervous system including brain, and spleen; at the host population level, these viruses are ubiquitous, tend to be acquired in childhood or early adulthood, and seldom directly result in host death. evasion of host defenses ~ avoids host immune defenses by infecting immune cells. direct contact via host-to-host transfer of fluid from viral-induced lesions of skin or mucosa and by saliva contaminated by chronically infected salivary glands; plus transmission to offspring either transplacentally, intrapartum (during the birth process), or via breast milk. genera affecting humans: influenzavirus a, influenzavirus b, and influenzavirus c. familial nature with respect to members affecting humans: generally viruses of humans. alternate hosts: birds (possibly), swine. types of illnesses induced in humans: coryza, malaise, myalgia, nasopharyngitis, pneumonia, retroocular pain, tracheobronchitis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the ciliated columnar epithelium of the respiratory tract (the exact tissue tropism is directly related to the virus hemagglutinin [ha] serotype); at the host population level, these viruses constantly undergo antigenic drift and antigenic shift and cause wide-scale seasonal epidemics in humans, although infection-related fatality is usually limited to humans aged 65 or older (most notably, age 75 or older). evasion of host defenses ~ avoids host immune defenses by antigenic mimicry and by rapid viral mutation. indirect (vehicle-borne) contact via droplet aerosols (from sneezing and coughing) and aerosol-contaminated fomites. genera affecting humans: papillomavirus, polyomavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. types of illnesses induced in humans: benign tumors of skin and mucosa that may progress to malignancy, progressive demyelinating encephalopathy. infection course ~ productive, recurrent. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the mucosa and skin (genus papillomavirus) and toward the upper respiratory tract (genus polyomavirus); secondary tissue and organ tropisms are toward the brain and kidneys (genus polyomavirus); at the host population level, these viruses are ubiquitous and almost never directly responsible for host death. evasion of host defenses ~ avoids host immune defenses by antigenic mimicry. host-to-host or indirect (vehicle-borne) contact by way of fomites (genus papillomavirus); indirect (vehicle-borne) contact via aerosols (genus polyomavirus). genera affecting humans: morbillivirus, paramyxovirus, pneumovirus, rubulavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. bronchiolitis, conjunctivitis, coryza, encephalitis, glandular enlargement (especially salivary glands), immunosuppression (morbillivirus causes an immunosuppression that is temporary, but which is arguably the most severe induced by a virus of humans, and can result in death by other coinfecting pathogens, such as enteric protozoans, that normally would not cause fatality), macular rash, nerve deafness, orchitis, pneumonitis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the epidermis and mucosa (including conjunctival, oral and respiratory); secondary tissue and organ tropisms are toward the brain, breasts, circulating immune cells, and testicles; at the host population level, these viruses tend to be acquired at a young age and are almost never directly responsible for host death, although severe sequelae can result if acquired beyond early childhood. evasion of host defenses avoids host immune defenses by infecting immune cells. indirect (vehicle-borne) contact via aerosols. genus affecting humans: parvovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. anemia, arthralgia, erythema, myalgia. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the throat; secondary tissue and organ tropisms are toward the circulatory system, erythrocyte precursor cells in bone marrow, possibly reticulocytes in blood, and skin; at the host population level, these viruses usually cause a disease of childhood; parvoviral disease is either mild or self-limiting in otherwise healthy children or adults. evasion of host defenses ~ uncertain. uncertain, but potentially direct host-to-host contact, including transplacental, and indirect (vehicle-borne) contact via aerosols and fecally contaminated water, food, and fomites. genera affecting humans: enterovirus, hepatovirus, rhinovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans, but may pass to primates and canines. diabetes, encephalitis, macular and maculopapular rashes of skin and mucosa, meningitis, myocarditis, otitis media, paralysis of skeletal muscles (occasionally including the diaphragm), pericarditis, retroocular pain, sinusitis (genus enterovirus); hepatitis (nonprogressive) (genus hepatovirus); coryza (genus rhinovirus). infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the nasopharynx and small intestine; secondary tissue and organ tropisms are very genus and species specific and toward the beta cells of the pancreas, conjunctiva, liver, meninges, muscles (including the heart), neurons (including those of the central nervous system), and skin; at the host population level, infections caused by members of the genus enterovirus usually are nonfatal, and both enterovirus and hepatovirus tend to result in asymptomatic infections if acquired in infancy, though the likelihood of severe symptomatology increases with age at acquisition; infections caused by members of the genus rhinovirus generally are symptomatic but essentially nonfatal regardless of host age. evasion of host defenses m members of genus enterovirus avoid host non-immune defenses by resistance to low ph (resistant to stomach acid) and to moderate alkalinity. indirect (vehicle-borne) contact via aerosols and fecally contaminated water, food, and fomites. genera affecting humans: molluscipoxvirus, orthopoxvirus, parapoxvirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. alternate hosts: one species affecting humans (smallpox) seems naturally limited to humans; monkeypox is a very notable but rare zoonotic exception and is presumably acquired from monkeys; several other species may cycle with domesticated bovines and ovines appearing as lesions on teats and udder. types of illnesses induced in humans: necrotic lesions of abdominal organs and skin, nodules and tumors in skin, papular rash. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the skin; secondary tissue and organ tropisms are toward the internal organs and lymph nodes; at the host population level, these viruses have very low transmissibility but have prolonged survivability on fomites due to extreme resistance to desiccation. evasion of host defenses ~ avoids host immune defenses by antigenic mimicry. direct contact via host-to-host contact with skin lesions and indirect (vehicle-borne) contact via lesion-contaminated fomites (very notably blankets and other bedding items). genera affecting humans: coltivirus, orthoreovirus, rotavirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: those species of the genus coltivirus infecting humans seem zoonotic with terrestrial mammals (notably rodents and squirrels) serving as their natural hosts; species of the genus orthoreovirus cross-infect nearly all known terrestrial mammals (especially rodents); those species of the genus rotavirus affecting humans seem naturally limited to humans. hemorrhagic fever, meningoencephalitis (genus coltivirus); upper respiratory symptoms (possibly associated with genus orthoreovirus); gastroenteritis (genus rotavirus). infection course m productive, short term-initial. viral replication-at the individual host level, primary tissue and organ tropisms are highly genus specific, and are toward the immune cells (genus coltavirus), possibly upper respiratory area (genus orthoreovirus), and the small intestine (genus rotavirus); secondary tissue and organ tropisms are toward the brain and meninges; at the host population level, these viruses have high transmissibility, especially among newborns, for whom they usually produce asymptomatic infections; in older children and adults, these viruses likewise have a tendency to produce asymptomatic infections; although rarely fatal in wellnourished children, members of the genus rotavirus are estimated to cause a million deaths every year in undernourished children. evasion of host defenses ~ avoids host immune defenses by infecting immune cells (genus coltivirus), avoids host non-immune defenses by resistance to heat, low ph, and proteolytic attack (infectivity actually increased by proteolytic attack) (members of the genera orthoreovirus and rotavirus). indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites with the orthoreovirus possibly also being spread by aerosols. genera affecting humans: blv-htlv retroviruses, lentivirus, spumavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. carcinoma, encephalitis, leukemia (adult t-cell), lymphoma (adult t-cell), progressive chronic immunosuppression and immunodepletion (including acquired immunodeficiency syndrome), progressive myelopathy, sarcoma. infection course m productive, short term-initial, often followed by increasing to end-stage; also may seem nonproductive in the case of some endogenous retroviruses. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the immune cells (largely t-cell populations); secondary tissue and organ tropisms are toward the brain and intestines; at the host population level, those viruses considered transmissible (i.e., excluding endogenous retroviruses) have a very low transmissibility rate, produce infections whose incubation times are very long (10--40 years), and may pass through breast milk; the endogenous retroviruses are permanently integrated into the human genome and are passed genetically to all offspring. evasion of host defenses ~ avoids host immune defenses by rapid viral mutation and by infecting immune cells. direct contact via host-to-host transfer of contaminated bodily fluids. genera affecting humans: lyssavirus, vesiculovirus. familial nature with respect to members affecting humans: zoonotic. natural hosts: foxes, skunks, and vampire bats (genus lyssavirus), cattle and horses (genus vesiculovirus). neuronal infections leading to encephalitis which appears invariably fatal (genus lyssavirus); myalgia (genus vesiculovirus). infection course m productive, short term-initial. viral replication m at the individual host level, primary tissue and organ tropisms are toward the neurons, including those in the spinal cord and limbic system of the brain, and the salivary glands (genus lyssavirus); and toward either the muscles or nerves (genus vesiculovirus); secondary tissue and organ tropisms are toward the adrenal cortex and pancreas (genus lyssavirus); at the host population level, these viruses are essentially nontransmissible. evasion of host defenses ~ avoids host immune defenses by limited antigenic exposure within the host because the virus largely remains within neuronal cells until near end-stage (genus lyssavirus). direct contact via host-to-host contact associated with deposition of contaminated saliva into a bite wound and possibly associated with contamination of skin or mucosal wounds by other types of bodily fluids; in the case of the genus vesiculovirus, vesicular fluids. genera affecting humans: alphavirus, rubivirus. familial nature with respect to members affecting humans: viruses of human and zoonotic. natural or alternate hosts: species of the genus alphavirus cross-infect a wide variety of terrestrial vertebrates, mostly via mosquitoes and ticks; one species of the genus rubivirus affects humans and it seems restricted to humans. arthralgia, arthritis, diabetes, encephalitis, fetal developmental abnormalities (cardiological, diabetic, and neurological including auditory, encephalitic, and visual k caused by rubivirus if contracted during the first trimester of pregnancy), macular rash of skin, myalgia, myositis. infection course m productive, short term-initial. viral replication m at the individual host level, primary tissue and organ tropisms are toward the immune cells (specifically monocytes and macrophages in bone marrow, liver, lymph nodes, and spleen) and oropharynx; secondary tissue and organ tropisms are toward the beta cells of the pancreas, muscles, neurons of the central nervous system including the brain, skin, and synovial cells of joints; at the host population level, most members of the genus alphavirus seem poorly transmitted between humans, and humans probably represent a dead-end host; infection by the genus rubivirus is seldom fatal but highly transmissible via aerosols and usually causes a trivial exanthema of childhood or mild symptoms in adults, although infection during the first trimester of pregnancy can result in extremely severe developmental abnormalities. evasion of host defenses avoids host immune defenses by infecting immune cells. to-vector (genus alphavirus), host-to-host (genus rubivirus), or indirect (vehicleborne) contact via aerosols (genus rubivirus). there are many types of viruses that afflict humans. we have managed to coevolve with some of these to lessen our misery. the struggle will continue as new viruses appear and as the existing ones reshuffle their genes or change their antigenicity by mutation. in the end, the contest is a struggle of biology versus biology, and the basic biology of the viruses is the same as ours. viral infections of humans: epidemiology and control modeling disease transmission and its prevention by disinfection virus taxonomy: sixth report of the international committee on taxonomy of viruses on viruses, sex and motherhood key: cord-017527-ylng1us2 authors: herman, philippe; pauwels, katia title: biosafety recommendations on the handling of animal cell cultures date: 2014-11-05 journal: animal cell culture doi: 10.1007/978-3-319-10320-4_22 sha: doc_id: 17527 cord_uid: ylng1us2 the first steps in tissue culture are dating back to the beginning of the nineteenth century when biosafety measures did not yet exist. later on, animal cell culture became essential for scientific research, diagnosis and biotechnological activities. along with this development, biosafety concerns have emerged pointing to the risks for human health and in a lesser extent for the environment associated to the handling of animal cell cultures. the management of these risks requires a thorough risk assessment of both the cell cultures and the type of manipulation prior the start of any activity. it involves a case-by-case evaluation of both the intrinsic properties of the cell culture genetically modified or not and the probability that it may inadvertently or intentionally become infected with pathogenic micro-organisms. the latter hazard is predominant when adventitious contaminants are pathogenic or have a better capacity to persist in unfavourable conditions. consequently, most of the containment measures primarily aim at protecting cells from adventitious contamination. cell cultures known to harbour an infectious etiologic agent should be manipulated in compliance with containment measures recommended for the etiologic agent itself. the manipulation of cell cultures from human or primate origin necessitates the use of a type ii biosafety cabinet. the scope of this chapter is to highlight aspects relevant for the risk assessment and to summarize the main biosafety recommendations and the recent technological advances allowing a mitigation of the risk for the handling of animal cell cultures. specified-pathogen-free tse transmissible spongiform encephalopathies biosafety is a concept that refers to the need to protect human health and the environment from the possible adverse effects of pathogenic and/or genetically modified organisms and micro-organisms used in basic research, research and development (r&d) and modern biotechnology. to this end, a case-by-case risk assessment is conducted which consists in the identification and characterisation of potential effects, which may be intended or unintended, together with an assessment of the likelihood and consequences should any effect occur. depending on the risks identified, risk management measures can be proposed. the use of cell cultures may fall within the scope of one or several regulatory provisions which consider an assessment of biological risks. for example in europe, tissue culture work will in many cases involve the use of genetically modified cell lines as well, in which case a risk assessment should be made in accordance with the provisions of the directive 2009/41/ec related to the contained use of genetically modified micro-organisms (european commission 2009). cell culturing activities aiming at manufacturing biopharmaceuticals are covered by the regulation (ec) no 726/2004 and its amending acts laying down procedures for the authorisation and supervision of medicinal products for human and veterinary use (european commission 2004a), whereas activities that involve the use of human cells and tissues for application to the human body falls within the scope of the directive 2004/23/ec and its amending acts (european commission 2004b) . the manipulation of animal cell cultures also exposes the worker to potential biological risks which are considered under the provision of the european directive 2000/54/ ec (european commission 2000) . it should be pointed out that guidelines aiming at mitigating the biological risks for the laboratory workers, public health and the environment have been issued by some scientific advisory bodies or competent authorities (world health organization 2004; centers for disease control and prevention 2009a; swiss expert committee for biosafety 2011). while biosafety recommendations (as outlined hereafter) are principally aimed at providing maximal protection of human health (including laboratory workers) and the environment, it is recognised that many of the precautionary measures will also directly benefit the quality of research activities involving animal cell cultures. indeed, cross-contamination (lucey et al. 2009; capes-davis et al. 2010; stürzl et al. 2013; jäger et al. 2013; johnen et al. 2013; macleod et al. 2013) or inadvertent contamination with infectious micro-organisms (bacteria, fungi, yeasts, virus, and prion) are plaguing many researchers, often leading to unproductive data, misinterpretation of results and a considerable waste of time and energy (mahy et al. 1991; drexler and uphoff 2000; mirjalili et al. 2005; cobo et al. 2007; pinheiro de oliveira et al. 2013) . it should also be emphasized that even if good manufacturing process (gmp) aim at protecting the product, some of the gmp measures are compatible with biosafety measures and reveal to be complementary. the objective of this chapter will be to address and review biorisk assessment and management considerations of diagnostic and research activities involving cell cultures. from an historical perspective, the assessment of biological risks has an empirical basis and has resulted from the awareness of the scientific community with regards the risks associated with the handling of pathogenic organisms, as demonstrated through many reported cases of laboratory-acquired infections, followed by the potential risks associated with experiments involving recombinant dna. in the 1970s, some initiatives for implementing measures guaranteeing the safe use of recombinant dna were linked to the safety measures which were at that time already successfully applied in microbiology for the containment of pathogenic organisms (national institute of health 1979). the conjunction of these two aspects constitutes two pillars of biosafety and has led to a classification system of organisms into risk classes or risk groups. so far, many risk assessments have been carried out among the scientific community on the use of pathogenic organisms (genetically modified or not), regardless of the scale or the purpose of the activity. the basis for this risk assessment methodology takes into account the most recent scientific, technical data and uses a scientifically sound approach. the methodology of biological risk assessment of contained use activities involving pathogenic and/or genetically modified organisms (gmo) identifies and takes into account the probability of occurrence and the severity of a potential negative effect on public health (including the exposed workers) and/or the environment. as a result of this methodology, a well characterised risk will lead to the choice of appropriate preventive measures encompassing the adoption of an appropriate containment level, the use of safety equipment including personal protective equipment, work practices and waste management. the risk assessment methodology is commonly used and its 4-step approach is described in fig. 22 .1. the first step (1) takes into account the characteristics of the organism(s) used and, in the case of genetic modification, the genetic material introduced and the resulting gmo. based on information relative to their harmful characteristics, natural pathogenic micro-organisms can be categorised in several classes of risk or risk groups. this classification takes into account the severity of the disease that pathogenic organisms may cause to human or animal health, their ability to spread amongst the population and the availability of prophylaxis or efficient treatment (world health organization 2004) . for zoopathogens, the classification system is mainly based on the definitions of the world organisation of animal health (oie), which categorises animal pathogens into four groups according to their risk to animal health, and since 2008, their risk to human health as well (world organisation for animal health 2009). micro-organisms that are unlikely to cause disease are classified into class of risk 1 while etiologic agents responsible for severe diseases with a high potential of transmissibility and for which no prophylaxis or treatment is available are assigned to class of risk 4. as such, pathogenic organisms will be categorised from class of risk 2 to class of risk 4. some periodically revised reference lists issued by international and national authorities or advisory committees classify natural biological agents (not genetically modified) into risk groups or assign the biosafety level under which these should be manipulated ( in the second step (2), the magnitude of the identified negative effects such as human diseases, including allergenic or toxic effects or the transfer of genetic material to other organisms are characterized. in a third step (3), an assessment is performed of the exposure of the laboratory worker, the population and/or the environment to the considered organism and the consequences of each negative effect should it occur. in the fourth step (4), a characterization of the risk is performed resulting in the assignment of the risk level associated with the contained use involving the use of the organism(s). on this basis, the containment measures and other protection measures (e.g. safe work practices, safety equipment and biological waste management) to be adopted are determined. there are four levels fig. 22 .1 flow diagram summarizing the biorisk assessment and management methodology of risk to which contained uses can be assigned, with level of risk increasing from 1 to 4. the final step consists in definitively classifying the contained use activity by conducting a re-assessment of the whole procedure before starting the research, diagnosis or production activity. in this chapter, the risk assessment methodology applied to animal cell cultures is developed and illustrated by examples. it is important to mention that such a risk assessment is always performed on a case by case basis by the scientist (s) responsible for the activity and the biosafety officer (or biosafety professional) in compliance with local guidance and regulatory requirements. the risk assessment applied to animal cell cultures relies on a thorough evaluation of both the intrinsic properties of the cell culture -including subsequent properties acquired as a result of genetic modification(s) -and the possibility that the cell culture may inadvertently be contaminated or deliberately infected with pathogenic micro-organisms. it also includes an exposure assessment which means that the type of manipulation carried out with the cell cultures is taken into account. the assessment of cell cultures harbouring pathogens follows the same principles as the assessment of the pathogens itself. first, the main organism characteristics (a comprehensive description of the pathogen) is considered by taking into account the following parameters (not by order of importance): (1) the pathogenicity and, when available, the infectious dose (2) the mode of transmission, (3) the host range, (4) the epidemiology (assignment of appropriate risk group may depend on the geographic localisation), possible reservoir and vector(s), and the ability to zoonosis (5) the stability and the persistence of the organism in the environment (i.e. survival outside the host). in addition, information related to the physicochemical properties of the pathogenic organism is considered such as: (1) susceptibility to disinfectants, (2) physical inactivation and (3) drug susceptibility (e.g. sensitivity and known resistance to antibiotics or antiviral compounds). finally, aspects related to the disease caused by the pathogen are also to be taken into consideration. this includes (1) the availability of an effective prophylaxis, (2) the availability of an efficient therapy and (3) any reported case of laboratory-acquired infection (s) (lais). although underestimated, many cases of lais related to the handling of cell cultures and/or containing virus suspension have been reported. among them are the reported laboratory worker's exposure to (recombinant) vaccinia viruses amplified in cell culture resulting into infections openshaw et al. 1991; mempel et al. 2003; moussatché et al. 2003; wlodaver et al. 2004; lewis et al. 2006 ; centers for disease control and prevention 2009b). recommendations to work safely with vaccinia virus have been reviewed recently together with an overview on the reported cases of lais involving this virus (isaacs 2012) . the risk assessment of cell cultures that are genetically modified basically follows a comparative approach: the characteristics of the gmo are compared to those of the non-modified (wild-type) organism from which it is derived under corresponding situations of use. the distinctive feature of the risk assessment of genetically modified cell cultures which consists of the evaluation of the recipient cell, the vector, the donor organisms and the inserted genetic material (insert) is developed in sect. 22.2.5. good knowledge and characterisation of the intrinsic properties of cells are key to successful and safe cell culturing. with respect to the biological risks and the risk assessment associated with the manipulation of animal cell cultures, three properties intrinsic to cell cultures should be considered : the species of origin, the cell type or type of tissue, organ from which the cell line is derived and the status of the culture ( fig. 22 .2). with respect to the species of origin and based on the fact that pathogens usually have specific species barriers, it is considered that the closer the genetic relationship of the cell culture is to humans, the higher the risk is to humans. the incidence to harbour organisms that could cause harm to human health is therefore considered higher in human or primate cells compared to cells of non-human origin (brown 1997) . accordingly, mammalian cells other than human or primate cells are considered to represent less risk, followed by avian and invertebrate cells. however, it should be kept in mind that some infectious agents are able to cross the species barrier and to persist in new host species, leading to zoonotic diseases. it is acknowledged since many years that more or less 70 % of the emerging infectious diseases are zoonotic (chomel et al. 2007) . well documented cases of viruses that have crossed the species barrier from animal reservoirs to humans include hantavirus (murine reservoir), haemorrhagic fever viruses (ebola, marburg) (peters et al. 1992 ), avian influenza virus (reperant et al. 2012) and severe acute respiratory syndrome (sars) associated coronavirus (ksiazek et al. 2003; herman et al. 2004) . these examples show that incidences of cross-species transfer can occur and that occupational risks related to exposure to infected animal tissues or cell cultures should not be underestimated (mahy and brown 2000; louz et al. 2005) . cells may dramatically differ in their in vivo half-life depending on the cell type or type of tissue from which these are derived. for example, intestinal and certain leukocytes have a half-life of a few days, human erythrocytes have approximately a 55-60-day half-life, healthy liver cells have a life span of several months, whereas, in adults, there is a slow loss of brain cells with little replacement. partly due to this fact, some cell lines can be more readily obtained than others. the establishment of cell lines is often obtained by a series of (generally uncontrolled) mutations that occur by culturing cells for a longer period. it is known that cells cultured for extensive periods of time display changing growth properties. a reduction of the doubling time, as a result of transformation, may give cells the ability to overgrow the rest of the population and to survive for a large (infinite) number of passages compared to primary cells with a finite life span. therefore, the establishment of cell cultures of a certain cell type upon extensive passage relies on the positive selection for cells that have a growth advantage. these transformed cells can have an increased tumorigenic potential and may present more risks of becoming/being fully neoplastic upon accidental (gugel and sanders 1986) or deliberate introduction into the human body. therefore, taking the tumorigenic potential into account, the following cell types may be ranked in increasing order of risk: epithelial and fibroblast cells, gut mucosa, endothelium, neural tissues, haematogenous (e.g. blood, lymphoid) cells and tissue. a third inherent property to consider is the status of cell culture. diagnostic and research activities involve the manipulation of primary cultures or cell lines as well as continuous cell lines derived from primary cultures. primary cell cultures and cell strains are produced directly from organs or tissues and are often the most accurate in vitro tool for reproducing typical cellular responses observed in vivo. however, as they are characterised by a finite life span, the time available for characterisation and detection of contaminating agents remains limited. also, because typical cell characteristics are often lost during the passage of cells, primary cell cultures are repeatedly obtained from fresh tissue, resulting in increasing risks for potential contaminating pathogens. a feature that distinguishes continuous cell lines from primary cell cultures is the ability to survive if not infinitely, at least for a great number of passages. these immortalised cells are obtained by isolating cells from tumours, by mutating primary cells with mutagens, by using viruses or recombinant dna to generate indefinitely growing cells or by cell fusioning of primary cells with a continuous cell line. due to their increased life span, the time left for thorough characterisation and detection of contaminating agents is considerably increased. within this respect, well-characterised cell lines present the lowest risks compared to primary cultures or less characterised cell lines as the origin, the source and suitability are well-known and well-defined. for cell lines obtained from external sources (e.g. different laboratory), crosscontamination of cell-lines and/or a lack of proof of identity is actually a widespread problem (buehring et al. 2004; capes-davis et al. 2010) . in order to have at least evidence of the species of origin of a cell line and to be able to conduct a thorough risk assessment, it may be necessary to fully characterise the used cell lines. for this purpose, a number of techniques are available such as cytogenetic analysis, dna fingerprinting, pcr, flow cytometry and isoenzyme analysis. (matsuo et al. 1999; cabrera et al. 2006 ). many micro-organisms benefit from a cell's machinery to complete their life cycles and to disseminate. hence the study of a pathogens' lifecycle or immunity escape mechanism requires the intentional in vitro infection of animal (or human) cells. the identification of potential hazards associated with infected cell cultures requires a consideration of the intrinsic cell properties and the inherent properties of the infecting pathogen. the latter implies an assessment of a number of pathogen specific criteria along with aspects such as the existence of effective treatment or prophylaxis. on the basis of these criteria the who defines a classification system that enables the categorisation of micro-organisms into four risk groups (world health organization 2004) . a fundamental rule is that the biological risk of infected cell cultures will depend on the infecting pathogen(s) class of risk. for example, cell cultures deliberately infected with hepatitis c virus (hcv) in order to produce virus particles are assigned to class of risk 3, as hcv is a class of risk 3 virus. human cells infected with an airborne pathogen like species of the mycobacterium tuberculosis complex are also assigned to class of risk 3 and are requiring the adoption of biosafety level 3 containment. however, as discussed below, the class of risk to which the infected cell cultures are assigned will not necessarily indicate the level of containment to be implemented as the latter will also be determined by the nature of the work carried out with these cells. an example is the infection of bovine leukocytes with theileria parva, a tick-transmitted, intracellular protozoan of veterinary importance and the causative agent of east coast fever among domestic livestock. it is an animal pathogen of risk group 3, which is not pathogenic to humans. the sporozoite form (infective form) invades bovine lymphocytes where it develops into a non-infective form (shizonts) and induces host cell transformation and clonal expansion of the cell. these infected bovine leucocytes may be categorised under class of risk 2 for animals, while the biosafety level (bsl1 or 2) appropriate for handling is determined by the presence or absence of the infectious form of the parasites. cell culture can also be coupled with electron microscopy to identify viral diseases of unknown cause as shown in a recent study published by the centers for disease control and prevention (goldsmith et al. 2013) . in case of outbreaks the harvested tissues from dead or living infected patients are inoculated to a permissive cell line (generally on vero e6 cells) and eventually subjected to electron microscopy for morphological analysis of the causal virus. although alternatives methods such as high throughput dna sequencing are available to identify a microorganism without a prior in vitro expansion, cell culture followed by electron microscopy remains the complementary approach of choice to molecular methods for the unbiased diagnosis of ill-defined infectious disease. some of the diagnostic activities presented by the authors required the adoption of bsl3 measures to handle the cell cultures. it illustrates how activities involving the in vitro amplification of unknown virus may represent a risk for the laboratory personnel and requires the adoption of an appropriate containment level and work practices. adventitious contamination of cell cultures is a major drawback for any activity that involves cell culturing (langdon 2004) . causative agents of cell contamination include bacteria, fungi, mycoplasms, parasites, viruses, prions and even other animal cells. beside the fact that contamination of cell cultures may place experimental results in question or may lead to the loss of cell cultures, one of the main biosafety concerns when manipulating animal cell cultures for research, diagnosis or production purposes is the fact that they may provide a support for contaminating agents that cause harm to human health. generally, bacterial or fungal contamination can be readily detected because of their capacity to overgrow cell cultures. typically, these organisms cause increased turbidity, ph shift of media (change in media colour), slower growth of the cells and cell destruction. antibiotics may be used to prevent cell bacterial contamination, however, continuous use of antibiotics in cultures may lead to development of resistant organisms with slow growing properties, which are much more difficult to detect by direct visual observation. compared to bacterial or fungal infections, mycoplasma contamination gives more problems in terms of incidence, detectability, prevention and eradication. mycoplasma, an intracellular bacterium, is one of the most common cell culture contaminants. it may go unnoticed for many passages and can change several cell properties such as growth, metabolism, morphology and genome structure (paddenberg et al. 1996; mcgarrity and kotani 1985) . it has also been reported to influence the yield of virus production in infected cells (hargreaves and leach 1970) . mycoplasmal contamination is also a biosafety concern, because some of the contaminating mycoplasma spp. belong to risk group 2. together with m. arginini, m. orale, m. pirum and m. fermentans, pathogenic organisms like m. gallisepticum (risk group 3 for animals), m. hyorhinis (risk group 2 for animals), m. pneumoniae and m. hominis (risk group 2 for humans) account for more than 96 % of mycoplasma contaminants in cell cultures. primary sources of contamination with m. orale, m. fermentans, and m. hominis in the laboratory are infected people who handle cell cultures and suspensions of viruses. sources of m. argini and m. hyorhinis are usually animal donors of tissues and biological constituents used for cell culture, e.g. calf serum and trypsin (razin and tully 1995; pinheiro de oliveira et al. 2013) . it was already reported that the contamination of cell cultures by mycoplasma occurs via aerosols (o'connell et al. 1964 ). viral contamination merits particular attention because infected cells may pose a serious harm to human health, especially when infected cells are able to release infectious particles. human cells may be infected by various viruses like hepatitis viruses, retroviruses, herpes viruses or papillomaviruses. although cell cultures from non-human origin may pose less risk, it should be emphasised that many viruses have a broad host range and can cross species barriers. since a number of non-human viruses are capable of infecting and/or replicating in human cells in vitro, their possibility to infect human cells in vivo if human exposure occurs should be carefully considered. well-known viral contaminants of primate tissues or cells from non-human origin that can cause human disease are listed in table 22 .1. while contamination with some viruses may be associated with changes in cell morphology or behaviour -such as the formation of syncytia (hiv, herpes viruses), swelling of cells (adenoviruses) or haemagglutination or haemadsorption -viral contamination may be harder to detect when cytopathic effects remain absent. viral contamination could also trigger adverse effects as a result of recombination events or phenotypic mixing between contaminating components and experimentally introduced agents, creating agents with new properties. for example, experimental results suggested that htlv-i or htlv-ii undergo phenotypic mixing with hiv-1 in htlv/hiv-1 co-infected cells, leading to an increase of the pathogenicity of hiv-1 by broadening the spectrum of its cellular tropism to cd4 negative cells (lusso et al. 1990 ). another example is the contamination of murine cell cultures by lymphocytic choriomeningitis virus (lcmv). lcmv is an arenavirus that establishes a silent, chronic infection in mice but causes aseptic meningitis, encephalitis or meningoencephalitis to humans. the significance of lcmv contamination has been reinforced by the description of cases of laboratory-acquired lcmv infections arising from contaminated murine tumour cell lines (mahy et al. 1991) . manipulation of lcmv infected material or material with an increased likelihood of lcmv contamination necessitates the whitley (2001) papovaviruses 2 butel ( adventitious contamination with parasites may be an issue when handling primary cell or organ cultures originating from a donor organism that is known or suspected to be infected with a specific parasite. as the life cycle of most parasites comprises distinct developmental stages, transmission and survival of the parasite will strongly depend on the ability of the invasive stage to recognize and invade specific host cells. but even with cells developing the non-infectious form of parasites, possible harmful effects remain to be considered since natural modes of transmission could be bypassed during the manipulation of infected cells. it is recognised that most of the parasitic laboratory acquired infections are caused by needle stick injuries (herwaldt 2001) . finally, the use of bovine-derived products as tissue culture supplements may also lead to the contamination with unconventional agents that cause transmissible spongiform encephalopathies (tse), the so-called prions (solassol et al. 2003; cronier et al. 2004; vorberg et al. 2004 ). contrary to the majority of the infectious agents, tse agents are resistant to most of the physical and chemical methods commonly used for decontamination of infectious agents. it has been shown that neuroblastoma cell lines, primary cultured neurons and astrocytes can serve as hosts (butler et al. 1988) . although many studies have suggested that the risk of propagation of tse agents in tissue culture cells cultivated in the presence of bovine serum potentially contaminated with tse was restricted to neurons or brain-derived cell cultures, it has been shown recently that non-neuronal cells can also support tse infection, suggesting that any cell line expressing normal host prion protein could have the potential to support propagation of tse agents (vilette et al. 2001; vorberg et al. 2004) . while the understanding of the transmission of prions is still in progress (natural transmission seems mainly to take place via oral route in human and animals), investigators using cell cultures need to take into account different routes by which these agents may be transmitted experimentally. mouse scrapie aerosol transmission has been successfully obtained in mice haybaeck et al. 2011) . in cervids, chronic wasting disease (cwd) was already proposed as a natural airborne pathogen (denkers et al. 2010 ). in the case of creutzfeldt jacob disease (cjd), there is to date no proof of release of prion into aerosols. animal cell cultures can also harbour unknown pathogens or whose tropism has not been defined yet. examples described in the literature include viruses such as hepatitis g (linnen et al. 1996) , hhv8 (moore et al. 1996) , tt virus (nishizawa et al. 1997) or human pneumovirus (van den hoogen et al. 2001) . cell cultures can be contaminated by different sources. infected organisms or infected animal cells or tissues from which a cell line has been established are the primary source of contamination. an accidental contamination can also occur through the material used for cell culturing including glassware, storage bottles and pipettes due to incorrect maintenance or sterilisation. before the use of disposable material the lip of the culture flask and the outside of the used pipette were important sources of contamination with mycoplasma (mcgarrity 1976). nowadays, the use of disposable and sterile pipettes has significantly decreased the likelihood of adventitious contamination. a third source of contamination resides in culture media and its components such as serum, basic culture media and salt solutions and enzymes (trypsin, pronase and collagenase). for example, media and additives derived from bovine sources are often contaminated with bovine viral diarrhoea virus (bvdv) (levings and wessman 1991) . as mentioned above the relative resistance of tse agents may also be an issue when using bovinederived products as tissue culture supplements. finally, non-filtered air supply, clothing, personnel and floor can be a source of airborne contamination (hay 1991) . genetically modified (gm) animal cell cultures are employed for a number of different activities. for example, the expression of transgenes and production of proteins of interest whose function depends on methylation, sulfation, phosphorylation, lipid addition, or glycosylation may necessitate the capacity of higher eukaryotic cells to perform post-translational modifications. gm animal cell cultures may also be chosen for the replication of defective recombinant or even wild type viruses. the risk assessment of gm cells should follow the five-step methodology as outlined in fig. 22 .1, which means that an evaluation of each individual aspect in the process of genetic modification should be performed. this includes an evaluation of the recipient cell, the vector, the donor organism properties and an assessment of the characteristics of the inserted genetic material. a comprehensive risk assessment of genetically modified cells expressing transgenes should also take into account the risk associated with the transgene products. a gene product may be intrinsically harmful (e.g. toxic properties) or could induce hazardous properties via its expression in gm cells, dependent upon the genome integration site, promoter activity and expression of regulatory sequences governing expression. the risk assessment for transgenes is not straightforward and demands appropriate consideration. comprehensive reviews have specifically addressed this topic (bergmans et al. 2008; van den akker et al. 2013 ). genetic modification may confer an expanded life-span, immortalisation or increased capacity for tumour induction. however, it is unlikely that recombinant properties obtained by genetic modification may have an adverse effect upon release of the recombinant animal or human cells into the environment. cells (genetically modified or not) have difficulties to survive in a hostile environment where control of temperature and osmolality is lacking or where cell-specific nutrients (e.g. glucose, vitamins, lipids) are not balanced or missing. hence, the survival of such primary cells or cell lines outside of proper conditions is unlikely to occur. apart from the fact that gm cell cultures may harbour pathogens and pose serious biological risks to human health (as discussed above), recombinant cells are more likely to cause harm when entering the body of animals or humans. however, the extent of the harmful effect remains hard to predict. it should be kept in mind that the lack of histocompatibility between recombinant cells and the host organism remains a major obstacle for these cells to survive and to multiply as the natural immune response of the healthy (immunocompetent) host will recognise foreign cells and eventually destroy them. this is also one of the main reasons why the culturing of cells originating from the laboratory worker is not allowed for research and diagnostic activities (risk associated with autologous cells). particular attention should be paid to the use of packaging cell lines. these are established cell lines that are deliberately and stably transfected with "helper constructs" to ensure the replication and packaging of replication deficient viral vectors. for example, in case of retroviral packaging cell lines, the expression of "helper genes" allows high-level constitutive production of viral proteins (e.g. gag, pol and env proteins), which are missing in the genome of the replication deficient viral vector but are crucial for viral replication. one of the main biosafety issues related to the use of packaging cell lines is the fact that replication-competent viruses may be generated as a result of (homologous) recombination between the replication deficient viral vector and viral sequences present in the packaging cell. these events could result in the formation of viruses with novel yet unwanted properties such as the generation of replication-competent viruses. one of the strategies to engineer safer generations of packaging cell lines consists in minimising the likelihood of generating replication-competent viruses by separating viral functional elements into different expression plasmids, thereby increasing the number of recombination events necessary to generate replication-competent viruses (dull et al. 1998) or by reducing or eliminating the sequence homology between the viral vector and the helper sequences. however, endogenous retrovirus genomes expressed in safer generations of retroviral packaging cell lines may still give rise to unwanted recombination events (chong et al. 1998) . this means that the possibility to generate replication-competent viruses cannot be ruled out. clearly, the risk group of the transfected packaging cell line will depend on the risk group of the viral vector itself. consequently, risk assessment of packaging cell lines should be based on the biosafety of the produced viral vectors, including an evaluation of their infectivity, spectrum of host range, capacity of integration (insertional mutagenesis), stability and physiological role of the transgene(s) if expressed (baldo et al. 2013 ). aside from the identification and characterisation of hazards intrinsic to the cell culture, a thorough risk assessment must also consider the exposure pathways through which cell cultures may present a risk to human health or the environment. this necessitates an evaluation of the type of manipulation, because processes, methods and/or equipment involved may increase or decrease the likelihood of exposure and hence the resulting potential risks. for instance, while established cell lines inherently present low risks, large scale operations involving the culturing of large volumes (from 10 to 1,000 l) are prone to contamination when inadequate containment measures are applied. this is exemplified by continuous processes such as cell cultivation in bioreactors where an appropriate design of seals, valves, pumps and transfer lines is required to guarantee long-term sterility of the operation to avoid inadvertent contamination. at the opposite, the handling of cell cultures belonging to risk group 2 may present less risk once they have been fixed by glutaraldehyde or formaldehyde/acetone for immunostaining and may therefore require less stringent containment measures manipulations that are common to research and diagnostic activities and warrant consideration with respect to the risk assessment of animal cell cultures are described hereunder: -procedures generating aerosols: pipetting, vortexing, centrifugation, opening of wet cups, etc.; -handling cells outside of a class ii bsc: flow cytometric analysis and cell sorting constitute a special case of cell manipulation in which cells are handled outside of a bsc. the use of a fixative is in many cases not appropriate (e.g. viable cell sorting for subsequent further cell culturing) and the risk of aerosol formation can be particularly high, especially during sorting experiments and upon instrument failure such as a clogged sort nozzle. all scientists in the field of flow cytometry must be aware of the potential hazards associated with their discipline and only experienced and well-trained operators should perform potentially biohazardous cell sorting. general recommendations approved by the international society of analytical cytology should help to set a basis for biosafety guidelines in flow cytometry laboratories (schmid 2012) . some standard operating procedures and methods have also been described for ensuring the cell sorting under optimal biosafety conditions even under bsl3 conditions (lennartz et al. 2005; perfetto et al. 2011 ). -altering culture conditions: changing the availability of cell-specific nutrients, growth factors, signal molecules or adopting co-culture techniques may have significant effects on animal cell cultures as it may result in altered neoplasia (stoker et al. 1990 ), altered expression of (proto)onco-genes or cell surface glycoproteins and release of endogenous viruses (cunningham et al. 2004) . as a consequence, changing culture conditions may lead to altered susceptibility of cultured cells to biologic agents such as viruses (anders et al. 2003; vincent et al. 2004 ). -manipulations involving use of needles or sharps: due to injuries cell material may be accidentally transferred directly to an operator's tissue and/or blood stream. -in vivo experiments involving animals: major risks are self-inoculation (needlestick injury) and exposure to aerosols. laboratory workers handling infected rodents or cell cultures originating from infected animals expose themselves at risk by directly exposing cuts, open wounds or mucus membranes with infected body fluids or by inhaling infectious aerosolized particles of rodent urine, faeces or saliva. the risk can be minimised by utilising animals or cell cultures from sources that are regularly tested for the virus. finally, the purpose of cell culturing should be taken into consideration as many clinical approaches such as stem cell therapy, gene therapy, xeno-or allotransplantation involve cell culturing ex vivo for therapeutic purposes. this latter clearly justifies more careful consideration regarding safety, ethical, social and regulatory issues, which will not be addressed in this chapter (food and drug administration; european medicines agency; ich 1997). the assessment of biological risks related to animal cell cultures and the type of manipulation allows the determination of an adequate containment level in order to optimally protect human health and the environment. the implementation of an appropriate containment level includes a list of general and more specific work practices and containment measures. table 22 .2 lists precautionary measures that should be applied whenever handling animal cell cultures. much of these measures focus on reducing the risk of contamination with adventitious agents. it should be emphasised that the drawn-up of an appropriate set of standard operating procedures and adequate training of the staff is of crucial importance. as a general rule, cell cultures known to harbour an infectious etiologic agent should be manipulated in compliance with containment measures recommended for the etiologic agent. when cell cultures are not known to harbour infectious agents, cells may be considered free of contaminating pathogens as long as a number of conditions are fulfilled. this implies the use of well-characterised cell lines or controlled cell sources for primary cells such as specified-pathogen-free (spf) animals. if no well-characterised cell lines or spf are available, tests for detection of likely contaminating agents should be negative. second, whenever cell cultures are manipulated, media sources should be pathogen free and appropriate containment measures should be adopted to reduce potential contaminations during sampling or subsequent manipulation of cells (re-feeding and washing steps). as the history of a cell culture may be poorly documented when a given cell culture is manipulated for the first time in the laboratory, it often remains unclear whether all appropriate measures have been implemented regardless of the fact that it may have been manipulated for years in another laboratory facility. in this case, cell cultures should be considered to be potentially infectious and should be manipulated in a class ii bsc. if the presence of adventitious agents of a higher risk group is considered likely, the cell line should be handled under the appropriate containment level until tests have proven the absence of such organisms. good documentation of the history of cell cultivation is mandatory. the extent to which cell cultures should be controlled on the likelihood of contaminants strongly depends on the nature of activity. for example, guidelines have been issued aiming at minimising any potential risk for transmission of infectious agents with respect the use of animal cell cultures for industrial production of biopharmaceuticals (european medicines agency; food and drug administration; ich 1997; world health organization 1998). hardly any guidance has been provided for the extent of detecting possible contaminants in case animal cell cultures are used for in vitro research or diagnostic activities, or for purposes other than therapeutics or production of biopharmaceuticals. the choice of the detection technique depends on the contaminating pathogen and often a combination of methods is recommended for important samples such as master cell banks. the implementation of bsl2 measures is adequate for most of the work carried out with cell cultures from human or primate origin. bsl1 measures may be considered provided that all manipulations occur in a class ii bsc and the cell culture is a well-characterised and certified cell line that presents no increased risk resulting from genetic modification or contaminating pathogen. the contained use to be in compliance with good cell culture practice (gccp), especially in industrial settings for vaccine production. to avoid opening of culture vessels or contact with culture fluid through a defective culture vessel, stopper or poor technique because of the ever present likelihood of contamination with airborne pathogens. to treat each new culture that is manipulated for the first time in the laboratory facility as potentially infectious. to clean up any culture fluid spills immediately with an appropriated and validated disinfection protocol. to work with one cell line at a time and disinfect the work surfaces between two operations involving cell lines. to aliquot growth medium and other substrates so that the same vessel is not used for more than one cell line. to mitigate cross-contamination by avoiding pouring actions. to proceed to the use of the biosafety cabinet (bsc) by adequately trained staff, t.i. turn on for a period before and after use, thoroughly disinfect bsc surfaces after each work session and do not clutter the bsc with unnecessary materials. to restrict the use of antibiotics in growth media. to quarantine new cell cultures to a dedicated bsc or separate laboratory until the culture has been shown negative in appropriate tests. to carry out a quality control of cells demonstrating the absence of likely contaminating pathogens on a regular basis or whenever necessary. to operate cell cultures from undefined sources as risk group/class of risk 2 organism. if the presence of adventitious agents of higher risk class is expected, the cell line should be handled under appropriate containment level until tests have proven safety. adapted from pauwels et al. (2007) in a bsl1 of viral infected cells could be envisaged if no viral particles are detectable in the supernatant of the infected cells. however, it should be emphasized that procedures for viral clearance are virus-host specific and that variables such as vector titre and the infection protocol can influence the degree and rate of clearance (bagutti et al. 2012) . since the implementation of good laboratory practices and the use of a bsc is usually the norm in most laboratories dealing with cell culturing, we think that bsl1 laboratories can relatively easily be upgraded to bsl2 facilities by implementing a restricted number of simple additional safety measures. it is important to notice that horizontal laminar air flows and clean benches minimise the risk of adventitious contamination of the cell cultures, however they offer no protection for the manipulator or the environment. bearing in mind that biosafety measures intend to provide a maximal protection of human health and the environment, it is important to note that the sole use of a horizontal laminar "clean bench" should be prohibited. based on, but not limited to, key features of risk assessment and the type of manipulation performed as discussed in former paragraphs, we developed a flow diagram providing the cell culture users a schematic guidance for the assignment of an appropriate containment level when manipulating human or primate cells in vitro (fig. 22.3) . this flowchart is indicative and should be applied and/or reconsidered according to case specific conditions and risk assessments proper to the activities performed. whenever possible approaches developed to ab initio reduce the risk associated with the handling of animal cell cultures should be favoured. such approaches involve the use of dedicated instruments or safer biological material. the examples hereunder illustrate how the choice to apply one or several of these approaches is determined on a case-by-case basis, depending on the intrinsic characteristics of the biological material and the intended use. during the last couple of years instruments enabling automated mechanical passaging and nutrient supply for in vitro cell expansion have been developed for primary cells, adherent and non-adherent mammalian cells (kato et al. 2010; thomas and ratcliffe 2012) . some of these developments also provide a safer approach with respect to biosafety considerations by including a biosafety module in the design of such automated platforms. for example a long-term cell culture device ensuring both the protection of the product and the operator has been designed for the culturing of embryonic stem cells in an antibiotic-free medium by means of an integrated automation platform using a class ii bsc confining the microwells (liquid handling robot contained in a bsc) (hussain et al. 2013) an automation system has also been deployed for the production (140-1,000 ml) of hiv-1 pseudovirus for hiv vaccine trials in compliance with gclp. this robust automated system for cultivation of 293 t/17 cells is contained in a class ii bsc that guarantees the protection of the workers, environment and the product. this system can be implemented to produce other biological reagents under standardised large-scale conditions (schultz et al. 2012) . lowering the hazards associated with the cell cultures handled is another approach that was applied in the characterisation of pandemic influenza viruses under emergency situations. while such activities typically require bsl3 conditions, inactivation protocols applied to influenza virus culture allows for performing the virological and immunological assays under bsl2 conditions (jonges et al. 2010) . another example where the assignment of a less stringent containment level was enabled relates to the use of insect cells and gm bacteriophage lambda capsids in cases where usually the maintenance of infectious stocks of highly pathogenic or emerging influenza viruses is involved (domm et al. 2014) . the bacteriophages are "decorated" by the viral glycoproteins of interest in an insect cell-derived system so as to use hemagglutinin displaying bacteriophages in hemagglutination-inhibition assay on pathogenic influenza viruses. the same approach was already applied for hiv-envelope protein (mattiacio et al. 2011) . with regards to hiv-1 in vitro testing, a non-infectious cell-based assay to assess the hiv-1 susceptibility to protease inhibitors was also developed recently (buzon et al. 2012) . here again, activities that normally require relatively high containment level can be performed in facilities with less expensive infrastructure. it also shows that careful consideration of the material handled can lead to costeffective choice while maintaining the same objectives. biosafety is an internationally recognized concept referring to the maximal protection of laboratory workers, public health and the environment from the possible adverse effects associated to the use of organisms and microorganisms (genetically modified or not). within this respect activities involving the use of animal cell cultures for fundamental research, r&d or in vitro diagnosis purposes pose biosafety considerations as well. before starting any of such activities biosafety considerations should be addressed by performing a biological risk assessment allowing the identification and characterisation of any potential adverse effect together with an evaluation of the likelihood and consequences should any effect happen. such an approach is made on a case-by-case basis, taking into account the type of manipulation and the type of cell culture handled. a risk assessment will result in biosafety recommendations in view of the implementation of an adequate containment level. considering the very limited persistence capacity of animal and human cells outside non-optimised culture conditions and the fact that many cell lines have a long history of safe use, it is generally considered unlikely that cell cultures may inherently cause harm to humans or the environment. the main hazard associated with the handling of animal cell cultures resides in the presence of adventitious pathogenic micro-organisms, which are often difficult to detect and hence less controllable. in contrast to their host cells, adventitious organisms can persist in more hostile conditions and may present risks for human health or the environment in case they are pathogenic (bean et al. 1982; walther and ewald 2004; kallio et al. 2006; kramer et al. 2006) . for this reason, a risk assessment of cell cultures will frequently lead to a risk assessment of the potential adventitious contaminants, the organisms used for cell's immortalisation (viruses, viral sequences, etc.) and/or the microorganisms intentionally used to experimentally infect them. though the assignment of biosafety containment level requirements cannot be generalised and should be performed on a case-by-case basis, it is recognised that most of the containment measures primarily aim at protecting cells from adventitious contamination in order to mitigate potential risks for the laboratory worker. except for authenticated cell lines proved to present no risk, the activities involving cell cultures from human or primate origin should generally be performed under containment level 2 involving the use of a class ii biosafety cabinet and good laboratory practice. whilst the occurrence of adverse effects related to the handling of cell cultures cannot be excluded, a thorough risk assessment and the implementation of the appropriate containment level offer an optimal protection for the laboratory worker and by extension, public health and the environment. continuous efforts have been made to mitigate the risk associated with the handling of cell culture in laboratory settings. these biorisk management (continued) measures (i.e. adoption of the appropriate containment level) can be coupled with the implementation of automation platforms allowing the reduction of cell culture cross-contamination and accidental contamination with infectious agents. it is anticipated that future efforts contributing to the improvement of the quality of the data necessary for the risk assessment, the containment measures and the increased awareness of biosafety considerations within the scientific community will also directly benefit the quality of research activities involving animal cell cultures. authentication is the process by which the true origin and identity of cell lines are determined and should form an essential part of any cell culture operation. biosafety in the context of this chapter, biosafety relates to the evaluation of the potential risks to human health and the environment associated with the use of genetically modified organisms (gmos) or pathogenic organisms. biosafety cabinet (class ii) safety cabinet with a front aperture through which the operator can carry out manipulations inside the cabinet and which is constructed so that the laboratory worker is protected, the product and cross contamination is low. the escape of airborne particulate contamination generated within the cabinet is controlled by means of an appropriate filtered internal airflow and filtration of the exhaust air (hepa filters). contained use any activity in which (micro)-organisms are genetically modified or in which such organism (pathogenic or not) are cultured, stored, transported, destroyed, disposed or used in any other way, and for which specific containment measures are used to limit their contact with the general population and the environment. culture type: primary cell cultures, diploid cell lines, continuous cell lines primary cell cultures are established directly from tissues of animals and are often the most appropriate in vitro tool for reproducing typical cellular responses observed in vivo. however, as typical cell characteristics are lost during passaging, these cultures must be obtained from fresh tissue that may contain or may become inadvertently contaminated with pathogens. consequently, primary cell cultures may potentially present increased risks compared to continuous, established cell lines. diploid cell lines are similar to primary cells, are considered non-tumourogenic and have a finite capacity for serial propagation. they are used for the preparation of viral vaccines and are from human or monkey origin. continuous cell lines are immortalized cells that may survive almost infinite serial passages. these cells are obtained by either isolating cells from tumours (neoplastic origin), primary cells treated with mutagens, oncogenic viruses or recombinant dna (oncogenes) or by cell fusioning of primary cells with a continuous cell line. as a consequence, the class of risk of these cell lines is often correlated to the class of risk of primary cells of whom they are derived. due to the "immortality" of the continuous cell lines, their ability to induce tumours has also to be considered. transfection gene transfer of dna in eukaryotic cells using non-viral delivery methods. transduction gene transfer of dna in eukaryotic cells using viral vectors. infection process occurring when a virus (wild type) that typically replicates and spreads into neighbouring cells mediates a gene transfer belonging to its own genome or extra genes it may carry. viral vector is a protein particle derived from a replicative virus that contains genetic information in the form of rna or dna. a viral envelope may be present as well. the approved list of biological agents, 3rd edn. health and safety executive disruption of 3d tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor washout kinetics of viral vectors from culture mammalian cells general considerations on the biosafety of virus-derived vectors used in gene therapy and vaccination survival of influenza viruses on environmental surfaces belgian classifications for micro-organisms based on their biological risks identification of potentially hazardous human gene products in gmo risk assessment detection of human t-cell lymphoma/leukemia virus type i dna and antigen in spinal fluid and blood of patients with chronic progressive myelopathy threat to humans from virus infections of non-human primates cell line cross-contamination: how aware are mammalian cell culturists of the problem and how to monitor it? in vitro cellular and developmental biology scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins a non-infectious cell-based phenotypic assay for the assessment of hiv-1 susceptibility to protease inhibitors identity tests: determination of ell line cross-contamination check your cultures! a list of cross-contaminated or misidentified cell lines biosafety in microbiological and biomedical laboratories (bmbl) 5th edn laboratory-acquired vaccinia virus infection, united states (virginia) 2008. morbidity and mortality weekly report wildlife, exotic pets, and emerging zoonoses a replication-competent retrovirus arising from a splitfunction packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences isolation of a new human retrovirus from west african patients with aids microbiological contamination in stem cell cultures prions can infect primary cultured neurons and astrocytes and promote neuronal cell death activation of primary porcine endothelial cells induces release of porcine endogenous retroviruses polio vaccines, simian virus 40, and human cancer: the epidemiologic evidence for a causal association b virus infection in man foamy viruses -a world apart aerosol and nasal transmission of chronic wasting disease in cervidized mice nonhuman primate-associated viral hepatitis type a. serologic evidence of hepatitis a virus infection use of bacteriophage particles displaying influenza virus hemagglutinin for the detection of hemagglutination-inhibition antibodies mycoplasma contamination of cell cultures a third-generation lentivirus vector with a conditional packaging system simian herpesviruses and their risk to humans on the protection of workers from risks related to exposure to biological agents at work of the european parliament and of the council of 31 march 2004 laying down community procedures for the authorization and supervision of medicinal products for human and veterinary use and establishing a european medicines agency. off j 30.04 directive 2004/23/ec of the european parliament and of the council of 31 march 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells on the contained use of genetically modified micro-organisms food and drug administration (fda). centers for biologics evaluation and research cell culture and electron microscopy for identifying viruses in diseases of unknown cause correspondence. needle-stick transmission of human colonic adenocarcinoma aids as a zoonosis: scientific and public health implications the influence of mycoplasma infection on the sensitivity of hela cells for growth of viruses operator-induced contamination in cell culture systems aerosols transmit prions to immunocompetent and immunodeficient mice biosafety risk assessment of the severe acute respiratory syndrome (sars) coronavirus and containment measures for the diagnostic and research laboratories laboratory-acquired parasitic infections from accidental exposures outbreak of lymphocytic choriomeningitis virus infections in medical center personnel chronic neurodegenerative disease associated with htlv-ii infection encephalomyelitis due to infection with herpesvirus simiae reproducible culture and differentiation of mouse embryonic stem cells using an automated microwell platform step 4 consensus guideline, quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin, cpmp/ich/295/95. the european agency for the evaluation of medicinal products, human medicines evaluation unit working safely with vaccinia virus: laboratory technique and review of published cases of accidental laboratory infections hiding in plain view: genetic profiling reveals decades old cross contamination of bladder cancer cell line ku7 with hela cross-contamination of a urotsa stock with t24 cells-molecular comparison of different cell lines and stocks accidental human vaccination with vaccinia virus expressing nucleoprotein gene influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling prolonged survival of puumala hantavirus outside the host: evidence for indirect transmission via the environment a new subtype of human t-cell leukemia virus (htlv-ii) associated with a t-cell variant of hairy cell leukemia a compact, automated cell culture system for clinical scale cell expansion from primary tissues brief report, infections of a laboratory worker with simian immunodeficiency virus how long do nosocomial pathogens persist on inanimate surfaces? a systematic review a novel coronavirus associated with sars cell culture contamination: an overview improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet bovine viral diarrhea virus contamination of nutrient serum, cell cultures and viral vaccines ocular vaccinia infection in laboratory worker a tale of two clades: monkeypox viruses molecular cloning and disease association of hepatitis g virus: a transfusion-transmissible agent infection of laboratory workers with hantavirus acquired from immunocytomas propagated in laboratory rats cross-species transfer of viruses: implications for the use of viral vectors in biomedical research, gene therapy and as live-virus vaccines henrietta lacks, hela cells, and cell culture contamination cd4-independent infection by human immunodeficiency virus type i after phenotypic mixing with human t-cell leukemia viruses where all the cell lines gone? zoonosis and haemorrhagic fever emerging zoonoses: crossing the species barrier virus zoonoses and their potential for contamination of cell cultures efficient dna fingerprinting method for the identification of cross-culture contamination of cell lines dense display of hiv-1 envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody response to hiv-1 env spread and control of mycoplasmal infection of cell cultures cell culture mycoplasmas laboratory acquired infection with recombinant vaccinia virus containing an immunomodulating construct microbial contamination of cell cultures: a 2 years study primary characterization of a herpesvirus agent associated with kaposi's sarcoma accidental infection of laboratory worker with vaccinia virus laboratory safety monograph: a supplement to the nih guidelines for recombinant dna research. national institute of health a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology aerosols as a source of widespread mycoplasma contamination of tissue cultures accidental infections of laboratory worker with recombinant vaccinia virus internucleosomal dna fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases animal cell cultures: risk assessment and biosafety recommendations standard practice for cell sorting in a bsl-3 facility filovirus contamination of cell cultures detection of contaminants in cell cultures, sera and trypsin detection and isolation of type c retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous t-cell lymphoma detection, isolation, and continuous production of cytopathic retroviruses (htlv-iii) from patients with aids and pre-aids molecular and diagnostic procedures in mycoplasmology adaptative pathways of zoonotic influenza viruses: from exposure to establishment in humans how to develop a standard operating procedure for sorting unfixed cells an automated hiv-1 env-pseudotyped virus production for global hiv vaccine trials marburg and ebola virus infections in laboratory non-human primates: a literature review the origin and evolution of hepatitis viruses in humans prion propagation in cultured cells unintended spread of a biosafety level 2 recombinant retrovirus aerosols: an underestimated vehicle for transmission of prion diseases the embryonic environment strongly attenuates v-src oncogenesis in mesenchymal and epithelial tissues, but not in endothelia kaposi's sarcoma-derived cell line slk is not of endothelial origin, but is a contaminant from a known renal carcinoma cell line classification of organisms recommendation on the safe handling of human and animal cells and cell cultures automated adherent human cell culture (mesenchymal stem cells) environmental risk assessment of replication competent viral vectors applied in clinical trials: potential effects of inserted sequences a newly discovered human pneumovirus isolated from young children with respiratory tract disease persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent emergent human pathogen simian virus 40 and its role in cancer ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells susceptibility of common fibroblast cell lines to transmissible spongiform encephalopathy agents pathogen survival in the external environment and the evolution of virulence biology of b virus in macaque and human hosts, a review herpes simplex viruses laboratory-acquired vaccinia infection world health organization (1998) who requirements for the use of animal cells as in vitro substrates for the production of biologicals. who technical report series laboratory biosafety manual manuel of diagnostic tests and vaccines for terrestrial animals. chapter 1.1.2 biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities acknowledgments the authors are grateful to their colleagues dr. didier breyer and dr. aline baldo (scientific institute of public health wiv-isp, brussels, belgium) for their useful review of this manuscript. key: cord-018834-4ligp4ak authors: farag, ehab; ebrahim, zeyd y. title: the perioperative use of albumin date: 2016-06-23 journal: perioperative fluid management doi: 10.1007/978-3-319-39141-0_9 sha: doc_id: 18834 cord_uid: 4ligp4ak human serum albumin (hsa) is the predominant product of hepatic protein synthesis and one of the more abundant plasma proteins. hsa is a monomeric multidomain macromolecule, representing the main determinant of plasma oncotic pressure and the main modulator of fluid distribution between body compartments. hsa displays an essential role in maintaining the integrity of the vascular barrier. hsa is the most important antioxidant capacity of human plasma, in addition to its ability to protect the body from the harmful effects of heavy metals such as iron and copper and reduce their ability to produce reactive oxygen radicals. hsa is the main depot for nitric oxide (no) transport in the blood. hsa represents the main carrier for fatty acids, affects pharmacokinetics of many drugs, and provides the metabolic modification of some drugs and displays pseudo-enzymatic properties. hsa has been widely used successfully for more than 50 years in many settings of perioperative medicine including hypovolemia, shock, burns, surgical blood loss, sepsis, and acute respiratory distress syndrome (ards). recently, the use of hsa has shown a promising neuroprotective effect in patients with subarachnoid hemorrhage. the most recent evidence-based functions and uses of hsa in the perioperative period are reviewed in this chapter. human serum albumin (hsa) is the most abundant protein in the human plasma (40-50 g/l). hsa has many functions; it is the main regulator of the vascular barrier, antioxidant in the plasma, and transporter of nitric oxide (no) and fatty acids and drugs. hsa infusions have been used successfully for more than 50 years since world war ii in many perioperative settings such as shock, volume expansion, burns, cardiopulmonary bypass, acute liver failure, sepsis, and many more. recently, its use has been questioned following a widely publicized meta-analysis in 1998 that reported increased mortality in patients who received albumin solutions; the role of albumin administration in critically ill patients became highly controversial. however, the results of this meta-analysis have been challenged by several metaanalyses, randomized controlled trials that not only proved the safety of hsa but its benefi t especially in patients with sepsis, liver failure, hypoalbuminemia, and burns [ 1 -4 ] . the most recent evidence-based functions and uses of hsa in the perioperative settings are reviewed in this chapter. human serum albumin (hsa) is a non-glycosylated, negatively charged plasma protein. hsa is a single polypeptide chain of 585 amino acids and has a molecular mass of 66.5 kda. hsa consists of α(alpha)-helix but no β(beta)-sheet, and it consists of three homologous domains (i-iii) that assemble to form a heartshaped molecule. each domain is composed of two subdomains (a & b) with distinct helical folding patterns connected by fl exible loops. the center of the molecule is made up of hydrophobic radicals, which are binding sites for many ligands, while the outer part of the molecule is composed of hydrophilic ligands ( fig. 9 .1 ) [ 5 ] . hsa is a member of the albumin superfamily, which also includes α(alpha)fetoprotein, vitamin d-binding protein, and afamin (α[alpha]albumin). hsa synthesis is governed by a single copy gene lying on the long arm of chromosome 4, near the centromere for the long arm, at position 4q11-13. the mrna for hsa encodes a precursor protein (preproalbumin) of 609 amino acid residues. cleavage of the single peptide of 18 residues and the propeptide (proalbumin) of six residues yields the mature protein of 585 residues [ 6 ] . hsa plays an integral role in maintaining the integrity of the vascular barrier. hsa enhances the integrity by electrostatic binding to the negatively charged heparin sulfate side chains of core glycoproteins such as syndecan-1 and glypican-1 of the endothelial glycocalyx via its positively charged arginine residues and enhances the availability of sphingosine-1-phosphate (s1p) produced by red blood cells (rbcs). extracellular sphingosine is taken up and phosphorylated by rbcs sphingosine kinases (sk) into s1p that is stored in the cell membrane of rbc. s1p is extracted from the rbc membrane by apo lipoprotein m (apom) of high-density lipoprotein (hdl) (apo lipoprotein m is the principal partner of s1p in hdl) and hsa, and this ensures a constant supply of receptor-available s1p for cellular signaling purposes. in contrast to the bond formed between s1p and hdl, hsa facilitates the solubility of s1p in the aqueous solution but not in physical bond to hsa. this unbound s1p is the active form of s1p. it is worth mentioning that one s1p molecule is extracted by 500 serum albumin molecules, indicating that hsa does not physically bind s1p [ 7 -9 ] . s1p activates the g protein-coupled s1p1 receptor, which rapidly activates the rho family small gtpase rac1 in the endothelial cells, leading to peripheral localization of cytoskeletal effectors (cortactin and nonmuscle myosin light chain kinase). this localization promotes adherents' junction (including vascular endothelial-cadherin and associated catenins) and tight junction (occluding, zonula occludens proteins and claudins) formation. therefore, s1p improves the vascular barrier and stabilizes the endothelial glycocalyx. s1p has been found to reduce matrix metalloproteinase activation, thereby attenuating the loss of endothelial cell surface glycocalyx components. both actions appear to involve signaling via the s1p receptor [ 10 ] . oxidative stress is defi ned as a disturbance in pro-oxidant and antioxidant balance leading to damage of lipids, proteins, and nucleic acids. according to halliwell and whiteman, an antioxidant is a substance that, when present at low concentrations compared with those of an oxidizable substrate, signifi cantly delays or prevents oxidation of that substrate [ 11 ] . human serum albumin represents a major antioxidant agent in human plasma. the antioxidant activity of has results from the redox properties of the cysteine 34 (cys 34) and from metal-binding abilities. among the metal ligands, copper (cu) and iron (fe) are very important, as they are able to generate reactive oxygen species (ros) after a reaction with oxygen. free cu (i) and fe (ii) ions can react with h 2 o 2 leading to the formation of the deleterious hydroxyl radical via the fenton reaction. cu(i) and fe(ii) binding to hsa promotes their oxidation to cu(ii) and fe (iii), thereby limiting their ability to participate in fenton reaction. copper ions bind to hsa with high affi nity at the n-terminal tripeptide asp-ala-his. the fi rst four amino acids of the n-terminus of hsa, asp-ala-his-lys (dahk), form a tight binding site for cu(ii) ions. dahk/cu has a superoxide dismutase activity, which thereby reduces the ros generation. by trapping cu(ii), hsa prevents low-density lipoprotein (ldl) lipid peroxidation. moreover, hsa and the tetrapeptide (dahk) were shown to prevent neuronal death in murine cell cultures exposed to oxidative stress generated by h 2 o 2 /cu(i)/ascorbic acid reagent [ 12 ] . therefore, the binding of cu ions with albumin is considered one of the most important antioxidant functions of albumin as cu can react with h 2 o 2 to hydroxyl radicals 60 times faster than fe. hsa is important for heme-fe scavenging, providing protection against free heme-fe oxidative damage. during the fi rst seconds after heme-fe appearance in plasma, more than 80 % of this powerful oxidizer binds to hdl and ldl, and only the remaining 20 % binds to hsa and hemopexin (hpx). then, hsa and hpx remove most of the heme-fe from hdl and ldl. afterward, heme-fe transits from hsa to hpx, which releases it into hepatic parenchymal cells after internalization of the hpx-heme-fe complex by cd91 receptor-mediated endocytosis. it should be mentioned that kinetics of heme-fe transfer from hdl and ldl to hsa and hpx is faster than the heme-fe-induced lipoprotein oxidation [ 13 , 14 ] . albumin-bound bilirubin confers an antioxidant effect by inhibiting lipid peroxidation. bilirubin bound to albumin was shown to protect α(alpha)-tocopherol from damage mediated by peroxyl radicals and to prolong the survival of human ven-tricular myocytes against in situ-generated oxidative stress [ 15 , 16 ] . cholesterol undergoes oxidation in vitro and in vivo, forming biologically active derivatives known as oxysterols. oxysterols bind to albumin with high affi nity. oxysterols carried by albumin are less rapidly released to cells than cholesterol. by this, albumin could limit detrimental effects of oxysterols on cells. furthermore, binding homocysteine by hsa protects from atherosclerosis as elevated plasma homocysteine is a well-known risk factor for atherosclerosis (figs. 9.2 and 9.3 ) [ 17 ] . physiologically, has exists predominately in a reduced form (i.e., with free thiol, hsa-sh) and is known as mercapto-albumin. however, a small but significant proportion of albumin pool exists as mixed disulfi des (hsa-s-s-r); where r represents low-molecular-weight, thiol-containing substances in plasma -chiefl y cysteine and glutathione [ 18 ] . mixed disulfi de formation increases as part of the aging process and during disease processes characterized by oxidative stress that enhances endothelial cell damage through oxidative stress and increase in apoptosis levels. cysteine 34 (cys 34) represents the largest fraction of free thiol in human plasma, has being the most abundant protein in plasma. cys34 is located at the surface of has, close to aspartate 38 (asp38), histidine 39 (his39), and tyrosine 84 (tyr 84). these three residues affect the ionization state of cys34, thus modulating its reactivity [ 13 ] . in healthy adults, about 70-80 % of the cys34 in albumin contains a free sulfhydryl group, whereas about 25-30 % of the hsa molecules have cys34 forming a mixed disulfi de with either cysteine or homocysteine or glutathione, thus affecting the cys34 redox potential. oxidation of cys34 leads to the formation of sulfenic acid (rsoh), which is further oxidized to sulfi nic (rso 2 h) or sulfonic acid form (rso 3 h). sulfenic acid constitutes a central intermediate in both the reversible and irreversible redox modulation by reactive species. reactive nitrogen species (rns) constitute nitrogen-centered species analogous to ros. rns such as nitric acid (no) contribute to various biological processes. hsa acts as a no depot and a no transducer. moreover, 82 % of no in blood (~7 μ[mu] m) is transported as an s-nitrosothiol bound at the hsa residue cys34. s-nitrosylated hsa may represent a circulating endogenous reservoir of no and may act as an no donor. s-nitrosylated hsa acts primarily as a vasodilator in vivo and represents a stable reservoir of no that can be released when the concentrations of lowmolecular-weight thiols are elevated [ 19 ] . s-nitrosylated hsa has been shown to reduce either ischemia or reperfusion injury in pig and rabbit hearts after unprotected warm ischemia through long-lasting release of no [ 13 ] . other rns, such as peroxynitrite (onoo − ), constitute powerful oxidants and nitrating species [ 20 ] . the -sh group of albumin represents an important antioxidant against peroxynitrite as the thiol group was oxidized to a sulfenic acid (hsa-soh). subsequently, hsa-soh can be converted to a disulfi de and then back to mercapto-albumin (hsa-sh). hsa administration favorably infl uences plasma thiol-dependent antioxidant status, as well as levels of protein oxidative damage in patients with sepsis and acute respiratory distress syndrome (ards) [ 21 , 22 ] . moreover, hsa is able to scavenge strongly oxidant compounds such as hypochlorous acid (hoci) and hypothiocyanous acid. cys34 is oxidized preferentially by hypochlorous and hypothiocyanous acid with the corresponding sulfenyl derivative. hsa is able to scavenge hoci, preventing alteration of its preferential biological target α(alpha) 1 -antiprotease [ 23 ] . interestingly, west nile virus is neutralized by hypochlorous acid-modifi ed hsa that binds to domain iii of the viral envelope protein e [ 24 ] . during its long life (~3 weeks), an hsa molecule makes 15,000 passes through the circulation, incurring some damages that affect its ligand-binding and antioxidant properties. diabetes mellitus is one of the main pathological conditions that impairs the antioxidant functions of albumin. in this disease, albumin undergoes increased glycation. the level of glycated hsa in normal humans is about 10 %, and increased to 20-30 % in hyperglycemic patients. glycation corresponds to the nonenzymatic attachment of glucose molecule to a free amine residue. hsa glycation is associated with oxidation of his and trp residues, main chain fragmentation, and loss of both secondary and tertiary structure. both the use of diclofenac, a nonsteroidal anti-infl ammatory drug (nsaid) and aspirin reduces the levels of advanced glycation [ 25 ] . the glycation of hsa impairs its antioxidant activity and its copper-binding ability. glycation of hsa induced a marked loss of its antioxidant activity to copper-mediated oxidation of ldl, probably by the generation of superoxide. moreover, the fe(iii)-binding antioxidant capacity of hsa is markedly reduced in diabetic patients. finally, the hsa transport of tryptophan (trp), which is the largest and essential amino acid, is reduced after its glycation. hsa glycation alters the binding of endogenous and exogenous ligands; in particular, glycation of lys 199 enhances warfarin binding, but decreases bilirubin affi nity [ 13 ] . several receptors for advanced glycation end products initiate intracellular signaling and enhance ros formation in the cells through recognition and binding of glycated (macro) molecules including hsa. moreover, hypochlorous acid-mediated carbonylation of lys residues of glycated hsa represents a major antigenic advanced glycation end product in hyperglycemia and in infl ammation [ 26 ] . glycated albumin was shown to impair vascular endothelial no synthase activity in vivo in aortas of rabbits [ 27 ] . glycated hsa displays a toxic effect on microglial cells associated with impairments in cellular proteolytic systems, possibly refl ecting the role of advanced glycation end products in neurodegenation. hsa may protect other proteins including hemoglobin, insulin, and immunoglobin from glycation in the early stages of diabetes due to its long half-life and its high concentrations compared to other proteins [ 28 ] . the irreversible damages associated with diabetes such as retinopathy, nephropathy, neuropathy, and coronary artery disease could be attributed to reduced antioxidant properties of glycated hsa. alterations in antioxidant properties of hsa were very recently identifi ed in vivo in patients with obstructive sleep apnea syndrome. this refl ects the impaired antioxidant hsa activity, which is associated with the enhanced glycation level of hsa in patients with obstructive sleep apnea syndrome. that might have increased the perioperative risks in those patients [ 29 ] . hsa has anticoagulant and antithrombotic functions. these functions may in part be mediated by the hsa capacity to bind no forming s-nitrosothiols, thereby inhibiting the rapid inactivation of no and allowing prolongation of its antiaggregatory effects on platelets [ 30 ] . therefore, the use of hsa might be very benefi cial in cases with hypercoagulable conditions such as during the perioperative period. the interaction between hsa and another molecule results in enzymatic activity. this property of hsa is called an enzyme-like or a pseudo-enzymatic activity. the esterase activity involving lysine (lys) 199 is able to split acetylsalicylic acid (aspirin) into salicylic acid, which is released and the acetyl group is transferred to especially lys 199. therefore, aspirin but not other salicylates induce the aspirin resistance syndrome, as the acetylation of albumin molecule can be allergic. asthma, rhinitis, and nasal polyps characterize aspirin resistance syndrome. moreover, lys 199 and penicillins can covalently bind via an aminolysis, generating a penicilloylcontaining peptide. the covalent labeling of lys 199 can have clinical consequences. the penicilloyl-hsa complex has no antibacterial activity; however, it represents the major antigenic determinant of penicillin allergy. hsa acts as a phosphotriesterase activity, which thereby inactivates organophosphorus compounds. hsa can catalyze rna phosphodiester bond cleavage; therefore, it participates in the degradation of endogenous extracellular rna and of circulating pathogenic nucleic acids. hsa possesses enolase activity toward dihydrotestosterone, converting it from the 3-keto to the 3-enol form. in addition, hsa facilitates the isomerization and the stereoselective hydrolysis of glucuronide conjugates and the removal of glucuronide conjugates, thereby reducing their plasma levels by reversible and/or irreversible binding. finally, hsa seems to have a signifi cant role in both the biosynthesis and the elimination the prostaglandins. hsa has no enzymatic effects on leukotrienes or thromboxanes. however, it binds and thereby stabilizes thromboxane a 2 . binding could play a major role for the inactivation of these potent compounds, diminishing the biological activities of substances that may be harmful for the body if present in too large amounts [ 6 , 13 ] . hypoalbuminemia is generally defi ned as serum albumin concentration ≤30 g/l and is usually very common in critically ill patients. the albuminemia could result from increased loss of hsa into the gastrointestinal tract, increased capillary permeability leading to redistribution from the intravascular to the interstitial space, and reduced hepatic synthesis of hsa caused by cytokines and stress of critical illness. hypoalbuminemia is considered an independent risk factor for worse outcomes in critically ill patients. hsa levels <20 g/l were associated with higher mortality risk in burn patients with 84 % sensitivity and 83 % specifi city [ 31 ] . in surgical septic patients, every 1 g/l decrease in albumin below 23 g/l was associated with a 19.4 % increase in hospital mortality and 28.7 % increase in the incidence of multiple organ failure [ 32 ] . moreover, in a meta-analysis of 90 cohort studies that evaluated hypoalbuminemia as a prognostic biomarker in acutely ill patients, each 10 g/l in serum albumin was associated with a 137 % increase in morbidity, and a 71 % increase in length of hospital stay [ 33 ] . preoperative low serum albumin (<4.0 g/dl) was shown to be an independent risk factor for acute kidney injury (aki) following off-pump coronary artery bypass surgery (opcab). aki was associated with prolonged stay in the intensive care unit (icu) and hospital and a high mortality rate [ 34 ] . hsa circulates from the blood across the capillary wall into the interstitial compartments, including cerebrospinal fl uid, and returns to the blood through the lymphatic system with a circulation half-life of approximately 16 h. the movement of hsa across the capillary wall is defi ned as the transcapillary escape rate (5 % per hour), which indicates the percentage of intravascular hsa leaving the intravascular compartment per hour [ 13 ] . in its long half-life of ~2-3 weeks, 1 hsa molecule could make about 15,000 passes through the circulation. hsa is mainly synthesized in the liver. in healthy young adults, about 12-25 g of hsa per day is synthesized in polysomes bound to endoplasmic reticulum of hepatocytes. hsa is not stored hepatically and there is therefore no reserve for release on demand [ 30 ] . under physiological circumstances, only 20-30 % of hepatocytes produce hsa and its synthesis can be increased up to 200-300 % on demand. hsa synthesis is regulated by colloid osmotic pressure and the osmolality of the interstitial liquid around the hepatocytes. insulin plays an important role in stimulating hsa synthesis; therefore, diabetic patients could suffer hypoalbuminemia. estrogens do not affect hsa transcription, but act by modifying the stability of the hsa mrna. hsa synthesis can be enhanced by corticosteroids, insulin, and amino acids administration. hsa synthesis can be rate-limited by amino acid defi ciencies, but these are rarely seen clinically, except in states of extreme starvation and malnutrition [ 30 ] . in acutephase reactions, such as in trauma and the perioperative period, the synthesis of hsa is depressed by hepatic cytokines such as interleukin-6 and tumor necrosis factor-α(alpha). immunoglobulin g (igg) and albumin, despite their disparate forms and functions, have long been known to share two unique characteristics, namely, their lengthy life spans and inverse relationship between their serum concentrations and half-lives. the long half-lives are attributed to the effi cient receptor-mediated recycling pathway involving the neonatal fc receptor (fcrn). fcrn is a heterodimer of a nonclassical major histocompatibility class i (mhc i) α(alpha)-chain and β(beta) 2 microglobulin (β[beta] 2 m) that binds the two abundant serum proteins igg and albumin in the body. fcrn binds both igg and albumin simultaneously on the opposite sides of the receptor, where the net transport can be basolateral to apical, apical to basolateral, or apical to apical (endothelial cells). fcrn interacts with igg and albumin in a strictly ph-dependent manner; therefore, it binds them at acidic ph and not at physiological ph. pinocytosed igg and albumin bound by the receptor within acidifi ed endosomes are transported back to the cell surface where physiological ph of the blood triggers release of the ligands into the blood circulation. the intracellular nonbound fractions are targeted for lysosomal degradation. fcrn is also largely responsible for transporting the igg across the placenta whereby the igg concentration in newborns at term normally exceeds that of the mother. animals defi cient in fcrn catabolize igg and albumin more rapidly than normal animals and manifest low plasma concentrations of both molecules. familial hypercatabolic hyoproteinemia, where defi ciency of fcrn is due to mutation in β (beta) 2m results in hypercatabolism and low plasma concentrations of both albumin and igg. however, patients with myotonic dystrophy (dm) exhibit plasma defi ciency only in igg but not albumin caused by reduced affi nity of fcrn to igg [ 35 -37 ] . the catabolism of hsa takes place in several organs at a rate of about 14 g per day in a 70 kg healthy adult, or 4 % of whole body protein turnover. the rate of has catabolism is increased by protein and caloric deprivation as hsa is used as a source of energy. the mechanism of hsa breakdown involves protein uptake into endocytotic vesicles, which fuse with lysosomes of endothelial cells. circulating hsa is also lost into the intestinal tract (about 1 g each day), where digestion releases amino acids and peptides that are reabsorbed. there is minimal urinary loss of hsa in healthy subjects. it is worth mentioning that of the 70 kg of hsa that passes through the kidneys each day, only a few milligrams are secreted from kidney tubules [ 13 ] . the use of human albumin in critically ill and septic patients has been through much controversy in the last two decades. in 1998, a cochrane meta-analysis for albumin administration in critically ill patients was published in the british medical journal [ 38 ] . the average sample size of the selected 32 studies in this meta-analysis was just 46 patients. the results of this meta-analysis showed increased mortality of almost 70 % in patients given albumin. the results of this cochrane report changed the practice rapidly around the world with dramatic reduction in albumin use especially in europe. the validity of this meta-analysis has been disputed for several methodological reasons, such as omission of relevant trials, small trials bias, and combination of heterogeneous trials, which included adults and high-risk neonates, inadequate assessment of the effect of methodological quality on outcome, and the absence of a plausible mechanism to explain albumin-associated excess mortality [ 39 , 40 ] . moreover, the meta-analysis did not include burns trials in which the mortality rate was lower in albumin [ 41 ] . finally, the crossover pattern in which the most seriously ill patients in the control group were switched to albumin as a rescue measure, therefore, would bias the pooled estimates of relative risk in favor of the control group [ 40 ] . only a few years later, this meta-analysis was followed by an updated meta-analysis, in which 55 trials involving 3,504 randomly assigned patients had been included and 525 deaths occurred [ 40 ] . pooled relative risk estimates among trials with blinding and those with 100 or more patients were 0.73 (ci, 0.48-1.12) and 0.94 (ci 0.77-1.14), respectively. the relative risk was also consistently less than 1.0 for trials that had two or more of the four attributes indicating higher methodological quality such as blinding, mortality as an endpoint, no crossover, and 100 or more patients. these observations suggest that albumin therapy reduces mortality. overall, the results of this meta-analysis supported the safety of albumin use in critically ill patients. in 2004, the results of the saline versus albumin fluid evaluation (safe) randomized control trial (rct) in 7,000 critically ill patients were published, showing that a 4 % albumin solution was as safe as normal saline as resuscitative fl uid in critically ill patients [ 42 ] . furthermore, the subgroup analysis of the safe study showed benefi t of using albumin in patients with severe sepsis, with an adjusted odds ratio (or) for death of 0.71 (95 % ci, 0.52-0.97; p = 0.03) for albumin compared with saline. therefore, the authors concluded that administration of albumin compared to saline did not impair renal function or organ function and may have decreased the risk of death in patients with severe sepsis [ 1 ] . moreover, guidet and colleagues assessed the cost-effectiveness of albumin, as given in the safe study on patients with severe sepsis and septic shock, who were admitted to 1 of 35 french icus. based on a presumed 4.6 % reduction in mortality associated with albumin therapy as shown in the safe trial, 513 lives were saved among the 11,137 patients included, with an estimated life expectancy for each life saved of 9.8 years. therefore, the authors suggested that albumin administration was a cost-effective intervention in patients with severe sepsis or septic shock [ 4 ] . in a subsequent meta-analysis that included 17 studies with randomized 1,977 participants, there were eight studies that included only patients with sepsis and where patients were a subgroup of the study population. the use of albumin for resuscitation of patients with sepsis was associated with a reduced mortality, with the odds ratio of 0.82 % (ci 95 % 0.67-1.0, p = .047) [ 1 ] . caironi and colleagues randomized 1,818 patients with severe sepsis in 100 icus to receive either 20 % albumin and crystalloid solution or crystalloid solution alone. during the fi rst 7 days, patients in the albumin group had a higher mean arterial pressure and lower net fl uid balance ( p < 0.001). at 28 days the mortality rate was 31.8 % in the albumin group and 32.0 % in the crystalloid group. at 90 days the mortality rate was 41.1 % in the albumin group and 43.6 % in the crystalloid group. however, there was improved survival associated with albumin in patients with septic shock (1,121 patients; 90-day mortality, 43.6 % in the albumin group vs. 49.9 % in the crystalloid group; relative risk 0.87; 95 % ci, 0.77-0.99; p = 0.03) [ 43 ] . the results of a recent meta-analysis, which included 14 studies (18,916 patients with sepsis), showed that resuscitation with balanced crystalloids or albumin in patients with sepsis seems to be associated with reduced mortality [ 44 ] . furthermore, the improved survival associated with albumin in patients with septic shock was confi rmed in a recent metaanalysis. in this meta-analysis, 3,658 with severe sepsis and 2,180 with septic shock patients were included in the analysis [ 45 ] . compared with crystalloid, a trend toward reduced 90-day mortality was observed in severe sepsis patients resuscitated with albumin (or 0.88; 95 % ci, 0.76-1.01; p = 0.08). however, in septic shock patients the use of albumin for resuscitation signifi cantly decreased 90-day mortality (or 0.81; 95 % ci, 0.67-0.97; p = 0.03) [ 45 ] . albumin resuscitation in sepsis has a unique feature compared to crystalloid as its effectiveness as a plasma-volume expander does not change in pathophysiological conditions associated with increased microvascular permeability as sepsis. in addition, in severe sepsis the ratio of albumin to crystalloid for equal plasma volume expansion is approximately 1-4.5 [ 46 ] . the use of intravenous albumin in addition to antibiotics in patients with cirrhosis and spontaneous bacterial peritonitis reduced the incidence of renal impairment, death, and paracentesis-induced circulatory collapse in comparison with treatment with an antibiotic alone [ 47 , 48 ] . in patients with acute respiratory distress syndrome, the use of albumin improved oxygenation but did not affect mortality [ 49 ] . the only exception for the benefi t of using albumin in patients with sepsis was shown in the fluid expansion as supportive therapy (feast) trial as evidenced in increasing mortality with the use of albumin and saline boluses compared to no bolus (control group) in pediatric patients infected with malaria in eastern african countries. the bolus-therapy-induced hypervolemia by albumin and saline boluses in those patients could explain the increased mortality in this study compared to control group [ 50 ] . human serum albumin is a unique pleiotropic protein with neuroprotective properties. rats received 2-h middle cerebral artery occlusion (mcao) and were treated with human albumin or saline after 30 min of recirculation. the cortical blood vessels were examined afterward by laser-doppler perfusion imaging (ldpi). albumin therapy resulted in signifi cant increases in arteriolar diameter, and reversing stagnation, thrombosis, and corpuscular adherence within cortical venules in the reperfusion phase after focal ischemia [ 51 ] . in a rat model of acute ischemic stroke induced by mcao, rats received 1.25 g/kg intravenously at 2, 3, 4, or 5 h after onset of mcao. albumin therapy markedly improved neurological function, and reduced infarction volume and brain swelling [ 51 ] . the neuroprotective effects of albumin have been confi rmed in a study with permanent mcao in rats, where albumin treatment led to 48 % increases in cortical perfusion ( p < 0.002), but saline in the control group caused no change [ 51 ] . moreover, functional magnetic resonance imaging (fmri) was used to assess the albumin treatment during stroke recovery in rats. albumin treatment was associated with restoration of fmri response magnitudes and temporal profi les [ 52 ] . rats underwent subarachnoid hemorrhage by endovascular perforation. albumin of either 0.63 or 1.25 g/kg was injected immediately after the surgery. albumin at lowto-moderate doses markedly improves long-term neurobehavioral sequelae after subarachnoid hemorrhage [ 53 ] . there are only two large published trials for the use of albumin after acute ischemic stroke and subarachnoid hemorrhage (sah). in a randomized, double-blind, parallel-group multicenter trial in patients with acute ischemic stroke with a baseline national institutes of health stroke scale (nihss), 422 patients were randomly assigned to receive 25 % albumin (2 g [8 ml] per kg; maximum 750 ml) and 419 to receive an equivalent volume of isotonic saline. the primary outcome was favorable, defi ned as either a modifi ed rankin scale score of 0 or 1, or an nihss score of 0 or 1, or both, at 90 days. the rate of favorable outcome did not differ between the groups. however, the patients in the albumin group had more mild-to-moderate pulmonary edema and symptomatic intracranial hemorrhage [ 54 ] . the reason for the negative outcome of this well-designed study was the high dose of albumin given as a single bolus, which might have induced those unfavorable effects and obscured the neuroprotective effect of albumin. albumin in the dose of 1.25 g/kg/day/7 days was tolerated by the patients with sah without major complications and may be neuroprotective. albumin in the dose of 1.25 g/kg/day/7 days had lower rates of cerebral vasospasm measured by transcranial doppler (tcd), delayed cerebral ischemia (dci), and cerebral infarctions. the main physiological effects of albumin treatment were elevation of the serum albumin concentration and mean arterial blood pressure. in addition, serum albumin remained elevated 7 days after treatment, which might be benefi cial throughout the critical period of dci [ 55 , 56 ] . the mechanisms of the neuroprotective effects of albumin could be explained by its ability to attenuate brain edema and inhibit the endothelia cell apoptosis [ 57 , 58 ] . albumin administration may improve microcirculatory blood fl ow, increase organ perfusion, decrease leukocyte rolling and adherence, and reduce the infl ammatory response [ 59 ] . albumin preserves the blood brain barrier (bbb) by abolishing the hyperactivation of metalloproteinases −2 and −9 (mmp-2/9) following subarachnoid hemorrhage, suggesting mmp-2 and mmp-9 are key mediators for the albumin-induced neurovascular protection [ 53 ] . moreover, albumin is considered the major antioxidant agent in the body. albumin functions as an endogenous nitric oxide (no) reservoir via binding of its sulfhydryl moiety of cysteine 34 residue with no to form s-nitrosothiols (rsno). it is worth mentioning that 82 % of no in blood is preserved in stable form as rsno [ 13 ] . therefore, albumin is able to neutralize the excessive circulating no so as to prevent the nitro-oxidative stress and, on the other hand, to continue to release no when the concentrations of lowmolecular thiols are elevated. thereby, albumin via rsno-adducted no can relax blood vessels, inhibit platelet aggregation, and increase aortic blood fl ow [ 53 ] . in the safe trial, patients with traumatic brain injury (tbi) treated with albumin had worse outcomes than saline, most probably because the hypo-osmolar (4 %) albumin solution with mean measured osmolarity of 266 (266-267) mosm/kg h 2 o used in the study induced increases in intracranial pressure but not the use of albumin per se [ 60 , 61 ] . however, the use of 4 and 20 % solutions in 93 patients with severe tbi and glasgow coma score ≤ 8 in addition to a neutral or to a slightly negative fl uid balance was associated with low mortality in those patients [ 62 ] . therefore, the correct conclusion should be hypo-osmolar solutions should not be used in patients with tbi [ 63 ] . the activation of systemic infl ammatory and hemostatic systems that takes place during cardiopulmonary bypass (cpb) results in fi brin formation, platelet activation/consumption, and endothelial damage. however, the use of 5 % albumin in priming the cpb machine has many advantages, such as preservation of oncotic pressure, preventing fi brinogen and platelet adhesion, and endothelial glycocalyx protection. in addition, it maintains the vascular barrier competency, prevents interstitial edema, and keeps the integrity of the microcirculation [ 64 ] . oliver et al. compared 5 % albumin priming with fresh frozen plasma (ffp)based priming in pediatric patients [ 65 ] . patients in the 5 % albumin group had signifi cantly lower administration of blood products. it was shown that using albumin for the priming volume, a dilution of coagulation factors is accepted during cpb. this will lead to less thrombin generation and consumption of coagulation factors and the ffp will be supplemented after protamine administration. however, the use of ffp as a priming solution will result in enhancing the thrombin formation during cpb, thereby more heparin is needed and more consumption of coagulation factors is triggered. the use of albumin in priming the adult cpb may compete with fi brinogen in the formation of the protein layer coating the circuit and the oxygenator, and the preadsorption of albumin prevents fi brinogen adsorption and platelet adhesion. russell et al. have shown in their meta-analysis that albumin compared with crystalloids as a priming solution exerts a number of benefi cial effects, including platelet count and colloid osmotic preservation [ 66 ] . the use of albumin in the postoperative period after cardiac surgery has resulted in the preservation in clot formation time and maximum clot fi rmness. however, the use of low molar hydroxyethyl starch solutions (hes) (6 % 200/0.5 or 130/0.4) resulted in prolongation in clot formation time and reduction in maximum clot fi rmness [ 67 ] . moreover, the use of old high-molar hes and gelatin solutions correlated with the amount of postoperative bleeding after cardiac surgery, but the use of 4 % albumin solution did not [ 68 ] . the same results have been confi rmed in a metaanalysis comparing the use of hes solutions with albumin. hemodynamics were similar in both groups, but the use of albumin decreased blood loss, the amount of blood products transfusions, and the need for reoperation postoperatively [ 69 ] . the presence of hypoalbuminemia (cutoff 18 g/l) after cardiac surgery was found to be a better predictor for mortality after cardiac surgery -even better than euroscore [ 70 ] . in a recently published prospective, randomized, double-blind, placebocontrolled trial, the preemptive correction of a low preoperative albumin level by administering hsa in patients undergoing off-pump coronary artery bypass (opcab) is associated signifi cant reduction in the incidence of aki, from 26 % in the control group to 13.7 % in the albumin group. the editorial that accompanied the study has suggested that restoring the target level is associated with reduction in aki in amplitude greater than that of any known intervention in patients undergoing opcab [ 71 , 72 ] . edwin cohn's development of stable albumin solution during world war ii was based on a fractionation scheme, which was rapidly adopted by a number of pharmaceutical companies. the pasteurization technique used in albumin solutions production is very effective in eliminating the risk for viral and bacterial infections. moreover, the recent introduction of ion exchange chromatography in the production of albumin is very effective in reducing the risk of prion disease transmission by albumin solutions [ 73 ] . the use of albumin is considered safe practice; in a study evaluating adverse event reporting between 1998 and 2000, the incidence of all reported serious nonfatal and fatal adverse events was just fi ve per million doses, and no patient death was classifi ed as probably related to albumin administration [ 74 ] . currently available human albumin solutions may differ in protein content and composition, binding capacity, metal ion content, antioxidants prosperities, and capacity to bind drugs [ 75 ] . it is noteworthy to mention that cysteine 34 -the most important antioxidant residue in hsa -is oxidized in 23 % of healthy human volunteers versus 54-60 % in commercial preparations [ 76 ] , which may infl uence the properties and hence the clinical impact of albumin solutions [ 77 ] . albumin solutions are available in a variety of concentrations, mainly 20-25 % or 4-5 %. iso-oncotic preparations of hsa are more effective than crystalloids solutions in maintaining the intravascular volume (>80 % vs. <20 %) [ 78 ] . hypertonic albumin (20-25 %) is used in patients with edema as it avoids excessive sodium and chloride loads [ 75 ] . nevertheless, hypotonic 4 % solutions should not be used in patients with traumatic brain injuries. the excessive need for the hsa solutions has encouraged its production using recombinant dna technology in both prokaryotic and eukaryotic hosts. hsa molecule structure is quite complicated; with 35 cysteine residues, 34 of them form disulfi de bonds. such complicated structure in this large recombinant protein could be a burden in both protein synthesis and folding system, which could result in the low expression or incorrect folding of recombinant hsa (rhsa). recently, transgenic rice oryza sativa has been used successfully as a novel bioreactor to produce suffi cient quantities of safe rhsa. however, to establish appropriate impurity removal and detection methods in rhsa manufacturing remains a challenge ( fig. 9.4 ) [ 79 , 80 ] . hsa has many physiological and biochemical properties that render its use relevant to many aspects of the disordered vascular and cellular functions. hsa has not yet showed all its secrets, and its benefi ts can only be realized by conducting clinical trials appropriately powered to relevant clinical endpoints. the role of albumin as a resuscitation fl uid for patients with sepsis: a systematic review and meta-analysis biologically active peptides from bothrops jararacussu venom albumin for bacterial infections other than spontaneous bacterial peritonitis in cirrhosis. a randomized, controlled study the coasst study: cost-effectiveness of albumin in severe sepsis and septic shock albumin usage in clinical medicine: tradition or therapeutic human serum albumin isoforms: genetic and molecular aspects and functional consequences erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1-phosphate the effects of native and modifi ed bovine serum albumin on the permeability of frog mesenteric capillaries sphingosine 1-phosphate in blood: function, metabolism, and fate sphingosine-1-phosphate protects endothelial glycocalyx by inhibiting syndecan-1 shedding measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? human serum albumin and its n-terminal tetrapeptide (dahk) block oxidant-induced neuronal death human serum albumin: from bench to bedside the antioxidant properties of serum albumin free and albumin-bound bilirubin are effi cient co-antioxidants for alphatocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage vascular oxidant stress and infl ammation in hyperhomocysteinemia albumin: biochemical properties and therapeutic potential s-nitroso-albumin carries a thiol-labile pool of nitric oxide, which causes venodilation in the rat nitric oxide and peroxynitrite in health and disease administration of albumin to patients with sepsis syndrome: a possible benefi cial role in plasma thiol repletion albumin infl uences total plasma antioxidant capacity favorably in patients with acute lung injury albumin -an important extracellular antioxidant? west nile virus is neutralized by hoclmodifi ed human serum albumin that binds to domain iii of the viral envelope protein e glycation of human serum albumin: inhibition by diclofenac hypochlorous acid generates n epsilon-(carboxymethyl)lysine from amadori products impairment of vascular endothelial nitric oxide synthase activity by advanced glycation end products albumin competitively inhibits glycation of less abundant proteins impairment of serum albumin antioxidant properties in obstructive sleep apnoea syndrome review article: albumin as a drug--biological effects of albumin unrelated to oncotic pressure serum albumin level as a risk factor for mortality in burn patients risk factors and prognosis of hypoalbuminemia in surgical septic patients hypoalbuminemia in acute illness: is there a rationale for intervention? a meta-analysis of cohort studies and controlled trials preoperative hypoalbuminemia is a major risk factor for acute kidney injury following off-pump coronary artery bypass surgery extending half-life by indirect targeting of the neonatal fc receptor (fcrn) using a minimal albumin binding domain kinetics of fcrn-mediated recycling of igg and albumin in human: pathophysiology and therapeutic implications using a simplifi ed mechanism-based model the multiple facets of fcrn in immunity human albumin administration in critically ill patients: systematic review of randomised controlled trials beautiful small: misleading large randomized controlled trials? the example of colloids for volume resuscitation patient survival after human albumin administration. a metaanalysis of randomized, controlled trials fluid resuscitation of burn patients comparing a crystalloid with a colloid containing solution: a prospective study a comparison of albumin and saline for fl uid resuscitation in the intensive care unit albumin replacement in patients with severe sepsis or septic shock fluid resuscitation in sepsis: a systematic review and network meta-analysis comparison of the effects of albumin and crystalloid on mortality in adult patients with severe sepsis and septic shock: a metaanalysis of randomized clinical trials plasma volume expansion with 5% albumin compared to ringer's acetate during normal and increased microvascular permeability in the rat albumin reduces paracentesis-induced circulatory dysfunction and reduces death and renal impairment among patients with cirrhosis and infection: a systematic review and meta-analysis effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis albumin versus crystalloid solutions in patients with the acute respiratory distress syndrome: a systematic review and meta-analysis mortality after fl uid bolus in african children with severe infection albumin therapy of transient focal cerebral ischemia: in vivo analysis of dynamic microvascular responses fmri of delayed albumin treatment during stroke recovery in rats: implication for fast neuronal habituation in recovering brains human albumin improves long-term behavioral sequelae after subarachnoid hemorrhage through neurovascular remodeling high-dose albumin treatment for acute ischaemic stroke (alias) part 2: a randomised, double-blind effect of human albumin on tcd vasospasm, dci, and cerebral infarction in subarachnoid hemorrhage: the alisah study the albumin in subarachnoid hemorrhage (alisah) multicenter pilot clinical trial: safety and neurologic outcomes albumin treatment reduces neurological defi cit and protects blood-brain barrier integrity after acute intracortical hematoma in the rat serum albumin is a specifi c inhibitor of apoptosis in human endothelial cells early albumin infusion improves global and local hemodynamics and reduces infl ammatory response in hemorrhagic shock albumin resuscitation for traumatic brain injury: is intracranial hypertension the cause of increased mortality? saline or albumin for fl uid resuscitation in patients with traumatic brain injury fluid therapy and the use of albumin in the treatment of severe traumatic brain injury fluid resuscitation in patients with traumatic brain injury: what is a safe approach? albumin-beyond fl uid replacement in cardiopulmonary bypass surgery: why, how, and when? semin cardiothorac vasc anesth blood loss in infants and children for open heart operations: albumin 5% versus fresh-frozen plasma in the prime albumin versus crystalloid for pump priming in cardiac surgery: meta-analysis of controlled trials rapidly degradable hydroxyethyl starch solutions impair blood coagulation after cardiac surgery: a prospective randomized trial gelatin and hydroxyethyl starch, but not albumin, impair hemostasis after cardiac surgery effect of hydroxyethyl starch on bleeding after cardiopulmonary bypass: a meta-analysis of randomized trials post-operative hypoalbuminaemia and procalcitonin elevation for prediction of outcome in cardiopulmonary bypass surgery albumin supplementation as a therapeutic strategy in cardiac surgery: useful tool or expensive hobby? effect of exogenous albumin on the incidence of postoperative acute kidney injury in patients undergoing off-pump coronary artery bypass surgery with a preoperative albumin level of less than 4.0 g/dl prion-removal capacity of chromatographic and ethanol precipitation steps used in the production of albumin and immunoglobulins safety of human albumin -serious adverse events reported worldwide in 1998-2000 albumin administration in the acutely ill: what is new and where next? heterogeneity and oxidation status of commercial human albumin preparations in clinical use pharmacological aspects of albumin as a niche product in the intensive care unit changes in intravascular volume during acute normovolemic hemodilution and intraoperative retransfusion in patients with radical hysterectomy human serum albumin from recombinant dna technology: challenges and strategies large-scale production of functional human serum albumin from transgenic rice seeds albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles redox properties of serum albumin the thiol pool in human plasma: the central contribution of albumin to redox processes key: cord-018613-83r6lhpo authors: norman, robert a.; paul, sharad p. title: the last natural brain date: 2017-03-21 journal: the last natural man doi: 10.1007/978-3-319-42217-6_7 sha: doc_id: 18613 cord_uid: 83r6lhpo the work of the brain would be easy if we knew what we needed to remember or understand in the future. life is full of surprises – new people whom we need to know, names we have to remember, problems we try to solve. the work of the brain would be easy if we knew what we needed to remember or understand in the future. life is full of surprises -new people whom we need to know, names we have to remember, problems we try to solve. to a creationist, the brain as god's masterpiece seems self-evident: a summation of motor synapses and sensory stimuli, infused with the most important emotions of all -faith, hope and love; the least copy-able of god's own artworks. to a computer-programmer, this vision may seem a gimmick, a concept that has the blitheness of ignorance. in an interview published in the guardian 2 , the celebrated scientist stephen hawking said: i regard the brain as a computer which will stop working when its components fail. there is no heaven or afterlife for broken down computers; that is a fairy story for people afraid of the dark. the idea that the brain, once its blood supply has ceased, simply degenerates into nothingness is both stirring and frightening. for human beings drunk with an engorged quest for immortality, the search for a 'human computer' to replace or replicate our brain is ongoing. but is it possible? about, two decades ago, in 1994, penrose wrote 3 : as yet, no computer-controlled robot could begin to compete with even a young child in performing some of the simplest of everyday activities: such as recognizing that a colored crayon lying on the floor at the other end of the room is what is needed to complete a drawing, walking across to collect that crayon, and then putting it to use. for that matter, even the capabilities of an ant, in performing its everyday activities, would far surpass what can be achieved by the most sophisticated of today's computer control systems. scenes from films like 'blade runner' and 'terminator' featured intelligent bioengineered 'replicants' whose brains are indistinguishable from human thought, but academics have been long frustrated by the inability to replicate a human brain's analytical power. i met garry kasparov, considered one of the greatest chess grandmasters at an international ideas conference called think, when we were both asked to sign copies of our books at the store they had set up for speakers' books -garry was signing copies of how life imitates chess: insights into life as a game of strategy 4 and i was autographing copies of my skin: a biography 5 . that was the biggest book-signing i've ever had to do -250 copies one after the other. 'you like chess?' i was lured out of the monotony by garry's voice. he had been leafing through my book and found a passage featuring bobby fischer, the late chess prodigy. bobby fischer, towards the end of his life, became reclusive, neurotic, paranoid and 'stateless', and iceland had offered him residence after the us refused to allow him entry post a trip to japan. in his prime, fischer had been obsessive about chess and had admired marcel duchamp, a french artist, who gave up art for chess. duchamp had described an opening called trebuchet or 'the trap'. but his favourite chess position was of an endgame called the lasker-reichelm position: a rare and unique position where black cannot win, but at best delay events. 'my dad used to play tournaments,' i replied. 'i played as a child with him, but not later on.' in his prime as world chess champion, kasparov had famously played against an ibm supercomputer, named 'deep blue'. a team of computer and chess experts recalibrated the machine between games, while kasparov reprogrammed his own brain. the first match was played in february 1996 in philadelphia, pennsylvania. kasparov won 4-2, losing one game, drawing in two, and winning three. a rematch took place in 1997, with deep blue winning 3½-2½. i asked garry about what it was like to play a computer at chess -surely a computer was capable of calculating possible moves faster than a human? 'the best way to beat a computer is to play unlike a computer,' gary said. 'which is why carlsen will win this year's chess championship . . . and why fischer was so good.' i opened garry's book and read: "… ordered systems lose less energy than chaotic systems." if our pieces work together they can better transform one advantage into another without losing quality. this theory of 'ordered systems' conserving energy and 'pieces working together' to create an advantage is a good analogy to explain the functioning of the human brain and memory. one of the problems thus far has been that in medicine, we have simplified the brain into modules -physicians have insisted in dividing up the brain into well-defined anatomical regions -frontal lobe for personality, temporal lobe for memory etc., theories that we know are problematic when we survey the evidence. just because computers need memory, it seems convenient to locate a 'memory module' in the brain. megan erickson puts it elegantly 6 : the brain is not a storage dump, and consciousness is not a place. synapses are also far more complex than electrical circuits. neither processing speed nor short term memory capacity are fixed, whereas ram is. we measure computers based on storage and speed, yet even an average human brain shades a computer when if comes to efficiency. writing in the scientific american, mark fischetti notes 7 : the world's most powerful supercomputer, the k from fujitsu, computes four times faster and holds 10 times as much data. and of course, many more bits are coursing through the internet at any moment. yet the internet's servers worldwide would fill a small city, and the k sucks up enough electricity to power 10,000 homes. the incredibly efficient brain consumes less juice than 6 megan erickson. "the electronic brain? your mind vs. a computer." http://bigthink. com/re-envision-toyota-blog/the-electronic-brain-your-mind-vs-a-computer accessed dec 25, 2015. a dim lightbulb and fits nicely inside our head. biology does a lot with a little: the human genome, which grows our body and directs us through years of complex life, requires less data than a laptop operating system. even a cat's brain smokes the newest ipad-1,000 times more data storage and a million times quicker to act on it. how does the ancient brain beat the power of modern computers? it is the battle of the analog vs. digital -humans brain store information as varying threads and 'learn' and evolve based on previous experiences; a computer by contrast sees memory as a binary collection of ones and zeroes. scientists working in this space of artificial intelligence now realize that storing memory as varying threads of information, rather than as binary digits is the key to making computers 'more human. ' memory is everything when we choose a computer, yet the capacity to remember is the least important in selecting a mate as a human companion. when we go to the computer store, devices tout memories of 4gb or 16gb of ram (random access memory). ram may help a computer multi-task but any arrest in the power supply leads to a rapid unraveling of memory (at least until the last 'auto-save'!). enter memory-resistors, or 'memristors' -electronic components where electrical resistance is not constant as in standard resistors, but is determined by the history of the electric current that has previously flowed through it. in other words, a memristor "remembers" like a human brain. researchers working at the royal melbourne institute of technology (rmit) and the university of california in santa barbara claim to have constructed the world's first electronic memory cell that effectively mimics the analog process of the human brain -and with it comes the possibility and peril of a truly bionic brain. however, there exist some significant hurdles -when it comes to memory, speed alone isn't the problem -artificial intelligence (ai) will need to come up with new ways to match the 2c's of brain function -complexity and consciousness. *** perhaps medical neuroscience has been slow to move towards 'singularity' due to the constraints of a medical model, or the differences between biological and technological concepts of the brain. for a start, if the human brain could be re-created, then medicine would have to be more inclusive, and couldn't inflict the prices it does on the premise of citizenship of a guild or cartel. the world of computer science is inherently less bureaucratic by nature, but also it couldn't care less about things like difficulty or danger that an artificially intelligent world would bring. the concept of singularity was first espoused by vernon vinge, from the department of mathematical sciences of san diego state university, to mean 'the imminent creation by technology of entities with greater than human intelligence.' somewhat chillingly (for natural humans), in this essay written in 1993, vinge wrote 8 : within thirty years, we will have the technological means to create superhuman intelligence. shortly after the human era will have ended. however, people like ray kurzweil, proponents of 'singularity' theorize that computers shall exceed human capacity for thought fairly soon. in his book, the singularity is near: when humans transcend biology (viking press, 2005) kurzweil creates a vision of intelligent nano-robots integrated into our bodies, our brains, and our environment, with a capability to eradicate pollution and poverty, offering the new artificial man extended longevity, while enjoying the sensory stimulation of a full-immersion virtual reality (think movies like "the matrix" or "being john malkovich"). kurzweil sets the date for this dramatic and disruptive transformation into a new human species as 2045, a hundred years after the end of the second world warduring that year, the non-biological intelligence created will be one billion times more powerful than all human intelligence today. indeed, the world of nanotechnology is progressing rapidly. microscopic robots can be placed inside human organs to give us an inside-knowledge of both brains and blood vessels. in his book, kurzweil feels that for singularity to occur, there has to be a confluence of three scientific streams -the three great overlapping technological revolutions that go by the letters "gnr," that stands for genetics, nanotechnology, and robotics. genetic sequencing has also become faster and cheaper -sequencing the hiv virus took over a decade; recently sars virus was sequenced in a month. this is about the last natural man playing god -creating a new species in man's own image, using the power of science with one difference from the last time around -the new bionic humans will no longer be in the shadow of their creators. kurzweil does not see hardware requirements as a barrier. in fact, in an interview 9 about his book on singularity 10 , kurzweil describes the computing power of the brain and computers thus: we can break this down further into hardware and software requirements. in the book, i show how we need about 10 quadrillion (1016) calculations per second (cps) to provide a functional equivalent to all the regions of the brain. some estimates are lower than this by a factor of 100. supercomputers are already at 100 trillion (1014) cps, and will hit 1016 cps around the end of this decade. several supercomputers with 1 quadrillion cps are already on the drawing board, with two japanese efforts targeting 10 quadrillion cps around the end of the decade. by 2020, 10 quadrillion cps will be available for around $1,000. there is not much dissent about the concept of singularity anymore; the controversies are more focused on the actual algorithms. speaking of algorithms, it is this ability of computer-scientists to view the human brain as a generator of various algorithms that creates the possibility of treatment of various disabilities. at think, i also met moran cerf, my good friend, who is now a professor of neuroscience and business at the kellogg school of management and heads the neuroscience program at northwestern university. additionally, he holds a position at the department of neurosurgery, where he studies patients undergoing brain-surgery to examine behavior, emotion and decision-making. moran sees the brain differently to me. i trained in medicine and have an interest in evolutionary biology and cutaneous oncology. moran had a background as a computer hacker, before he studied neuroscience, and sees the brain as a generator of patterns -all emotions or dreams or maladies can be interpreted by studying brainwaves -once you've studied enough patterns, the 'big data' seems to make sense -for example, as we discussed the ability to mimic a human brain, moran spoke to me about the interpretation of dreams. using electrodes implanted in the brain he was able to decipher dreams -he could for example tell if you were thinking of an elephant (at the time we spoke, the technology was not refined to determine if the elephant was african or asiatic). in his lecture, moran spoke about an experiment that featured a chimpanzee and a robotic arm. the scientists implanted electrodes onto the motor cortex, over an anatomical region known to signal arm and hand movements. these electrodes, each connected to neurons that were wired to the brain, were also linked to a computer. there were plenty of food treats in the rooms -grapes, bananas etc. it didn't take a lot of time before the ape realized that the robotic arm was able to read brain signals, and therefore could be controlled by thought. within a few days, the monkeys needed no help. they sat stationary in a chair, repeatedly manipulating the arm with their brains to reach out and grab grapes or nuggets of food dangled in front of them. imagine the useful of thought-control for physically-disabled patients. writing about a similar experiment in nature 11 , dr. john f. kalaska, a neuroscientist at the university of montreal, feels that as this technique is refined further, scientists might even discover areas of the cortex that allow more intimate, subtle control of prosthetic devices: such controllers would allow patients with severe motor deficits to interact and communicate with the world not only by the moment-to-moment control of the motion of robotic devices, but also in a more natural and intuitive manner that reflects their overall goals, needs and preferences for example someone paralyzed or in an institution because of mobility issues could order a robot to provide food or coffee by using thought-waves. the technology is here. now. *** in the past, drosophila, the fruit fly, was the most studied 'model organism' in genetics and biology. this was because it is hardy, easy to care for, breeds quickly and lays many eggs. in 1965, sydney brenner, mooted caenorhabditis elegans, the non-parasitic nematode worm, as a new model organism for studying organ development, cell death, behavior and many other biologic processes. brenner had obtained a culture of the bristol strain of this worm from ellsworth c. dougherty (then in the department of nutritional sciences at the university of california at berkeley), who had worked extensively with the organism. brenner, originally from south africa and then director of the laboratory of molecular biology (lmb) at cambridge had written: "nearly all the 'classical' problems of molecular biology have either been solved or will be solved in the next decade." for his work on this worm, in 2002, the nobel prize committee honored sydney brenner with the nobel prize for physiology or medicine (along with john sulston and robert horvitz). this soil worm had excited scientists as it had many attributes that made it a natural model for scientific study -for a start, it is barely over 1 mm in length, lives for approximately three days, feeds on easily cultivated bacteria such as e. coli and a single petri-dish can hold over 10,000 of these critters. in its hermaphrodite form, it is even capable of reproducing itself. it is anatomically simple, with a body containing just over 1000 cells including a 302-cell nervous system. c. elegans was the first multicellular organism to have its genome completely sequenced. the genome of this worm contains 97 million base pairs. this is about 3 % the size of the human genome, which has three billion base pairs. drawing a connectome is basically mapping all neural connections in the brain, and can be thought of as being similar to an electrical "wiring diagram". in 1986, the entire connectome of c. elegans was mapped, and as the genome had been mapped, science was much closer to completing the gnr triad needed to create an artificial brain. therefore in 2011, a team of scientists from the us, europe and russia began the openworm project -an attempt to build a complete artificial nervous system of this worm with the capacity to stimulate various actions i.e. an artificial c. elegans. researchers on the openworm project soon reached a philosophical crossroad. if they could replicate the nervous system of this creature and implant that 'brain' into another body, would that body behave like a worm? this was the very experiment they undertook -scientists took the connectome of a c. elegans worm and transplanted it as software into a lego mindstorms ev3 robot -what happened next? the lego model robot began to behave exactly like the worm! stimulation of the nose stopped forward motion. touching the anterior and posterior touch sensors made the robot move forward and back accordingly. stimulating the food sensor made the robot move forward. they now had a creature that behaved like a worm but was a lego model (and therefore not as squishy). slowly but surely science is wiggling towards singularity. it's all very well messing about with a tiny nervous system, but if we were to build a truly human brain, could we control this being? after all human beings have 13 billion neurons with trillions of possible interactions. many scientists indeed voice a fear of artificial intelligence taking over the world. the other possibility for an artificial brain is both frightening and exciting. think about this: if the openworm project could be replicated, we would no longer be constrained by our organic bodies -physical forms that were the result of four billion years of evolution by natural selection. life could become inorganic, powered by exact replicas of our own brains. this possibility was made more probable by funding from the european union -in april 2013, it selected the human brain project to be its scientific flagship, giving it a grant of more than €1 billion. even before the openworm project model, henry markram, a south african neuroscientist led the blue brain project at the école polytechnique fédérale de lausanne in switzerland. using a similar technique (as in the c. elegans research), his team began studying the neural networks of rat brains -with a goal of computer-based models that mirror the rat brain's complex biological networks. "technologically, in terms of computers and techniques to acquire data, it will be possible to build a model of the human brain within 10 years," markram has been quoted saying. 12 yuval harari, debates the transition or rather our progress as a species from evolution by natural selection to evolution by intelligent design. he calls this 'the greatest revolution in thousands of years of history, but also the greatest revolution in billions of years of biology.' harari, in sapiens: a brief history of humankind writes about the many positive aspects of intelligent design 13 : the replacement of natural selection by intelligent design could happen in any of three ways. the first way is through biological engineering. biodesigners could re-engineer the shapes, abilities and desires of organisms, in order to realize some preconceived cultural idea. there is nothing new about biological engineering, per se. people have been using selective breeding, castration and other forms of bio-engineering for at least 10,000 years. but recent advances in our understanding of how organisms work, down to the cellular and genetic levels, have opened up previously unimaginable possibilities. for example, scientists can today take a gene from a jellyfish that glows in a green florescent light, and implant it in a rabbit or a monkey, which starts glowing in a green florescent light. e. coli bacteria have been genetically engineered to produce human insulin and bio-fuel. a gene extracted from an arctic fish has been inserted into potatoes, making the plants more frost resistant. harari has a very hopeful view of intelligent design. but as a medical doctor, i am aware that medicine is often unpredictable, phenomena like the 'placebo effect' cannot be easily explained by computers. a computer can beat me hands down in predicting what percentage of my patients would respond to a particular medication, but could it deal with the imperfections and frailties of the human mind? max pemberton, the author of the doctor will see you now (hodder and stoughton, 2012) writes about an experiment conducted by the psychologist ellen langer in 1979 14 . a group of elderly men were taken to a 'reminiscence retreat' outside boston. the first group was the 'control group' and spent a week simply reminiscing about the fifties. the second group, however, was taken back in time and surrounded by objects from the fifties and asked to behave as if it really was 1959. over the week, something astonishing happened. the men stopped using their walking sticks, they began to walk faster and their posture improved. what this experiment demonstrated is that as humans we are more than genes and brains. we have a collective consciousness. pemberton is skeptical that computer code could replace medicine. "computers are a useful tool in treatment, but medicine is far too complex to be reduced to code," he says 14 . like me, pemberton is a medical doctor and this may only turn out to be wishful thinking. technically it will soon be possible to create artificial versions of our brains and implant them into all kinds of inorganic models. given a choice, you could take on any form that you wish. the question more to the point would be: what do you want to become? but with your brain and without your existing form, would that really be you? when i visit oxford university, i usually stay at keble college. it is a lovely college and the last time i was there, i stayed in the lewis room, named after c.s lewis. in 1945, lewis penned that hideous strength: a modern fairy-tale for grown-ups 15 , as the final book in his theological sci-fi space trilogy, which was recently reviewed in salvo magazine 16 . in its plot, a philosophy professor, mark studdock, gets caught up in a diabolical scheme to take over the world. the leader of the cult is the severed head of a criminal genius, kept alive by tubes and wires. "don't you see," says one character, inviting mark into the fellowship, "that we are offering you the unspeakable glory of being present at the creation of god almighty?" this book was lewis' foray into transhumanism -transhumanists speculate freely about becoming immortal by "uploading" their consciousness into computer brains with robotic bodies, something that is now becoming closer and closer to scientific reality. *** in my view, there are two requirements for a brain to be truly human: the 2c's of complexity and consciousness. the complexity of the human brain has been a barrier in facilitating rapid evolution. chimpanzees, the apes closest to humans weigh about 20 % less than the average human, but the human brain is 250 % larger than that of a chimp. given this, it was assumed that human genes that regulate brain development would evolve at a much faster rate than that of apes. in actual fact, the reverse is true. this is all to do with complexity. you see in primitive creatures, the larger the brain, the more rapid the evolution -protein sequences in chimpanzees changed faster than genes from monkeys, which changed faster than those from mice. writing in the influential scientific journal plos biology, a team from the university of chicago found that gene evolution in human beings has slowed. on the basis of individual neurons of the brain, humans may indeed have a far more active, or even more complex, transcription profile than chimpanzee …we suggest that such abundant and complex transcription may increase gene-gene interactions and constrains coding-sequence evolution. this means that natural selection is no longer enough to code new advanced genetic sequences. for human populations to improve their "fitness", intelligent design needs to design new codes. necessity may indeed be the mother of new inventions. let's examine consciousness for a bit. at a basic level, being conscious means 'being awake.' therefore if your computer cannot wake itself up every morning, it cannot be conscious, right? michael graziano is a professor of neuroscience at princeton university in new jersey. his latest book is consciousness and the social brain (oxford university press, usa, 2013). in an essay titled, 'build-a-brain' he describes how human brains perceive objects. for example, if we were to look at a tennis ball bouncing, we would note its color, shape, how high it bounced, the variability of the bounce etc. our brain constructs something like a dossier, a dataset that is constantly revised as new signals come in. this is what scientists refer to as the "internal model." however, if we were to ask a robot a few questions regarding the tennis ball, graziano suggests that the conversation would go something like this 18 : singularity will not be achieved. but what if humans could 'lend' a computer some conscious awareness? this has led scientists to develop braincomputer interfaces (bci). in a first-of-its-kind study, engineers and neuroscientists in india and france recently demonstrated the viability of direct brain-to-brain communication in humans. they successfully transmitted information (words like 'hola' and ciao') via the internet between intact scalps of two human subjects -located 5,000 miles apart. explaining their methods in plos one, they say 19 : by using advanced precision neuro-technologies including wireless eeg and robotized tms (trans-cranial magnetic stimulation), we were able to directly and noninvasively transmit a thought from one person to another, without them having to speak or write one of the reasons consciousness has been elusive for otherwise intelligent robots is that consciousness (or to the philosopher, the psyche) is everywhere. neuroscientist christof koch, chief scientific officer at the allen institute for brain science, talks about the theory of panpsychism -put simply, we are just a mind (or soul) in a world of minds. this was a theory espoused by thinkers like plato and spinoza, only to be sidelined by modern society's quest for empiric science. but a failure to infuse computers with consciousness has reignited this debate. according to koch, consciousness arises within any sufficiently complex, information-processing system and the solution to singularity may lie in us being connected. in an interview with wired magazine 20 (rather appropriate, don't you think), he noted: the internet contains about 10 billion computers, with each computer itself having a couple of billion transistors in its cpu. so the internet has at least 10 19 transistors, compared to the roughly 1000 trillion (or quadrillion) synapses in the human brain. that's about 10,000 times more transistors than synapses. but is the internet more complex than the human brain? it depends on the degree of integration of the internet… for instance, our brains are connected all the time. on the internet, computers are packet-switching. they're not connected permanently, but rapidly switch from one to another… therefore, consciousness = connectivity. therein lies the future. technology now has the capability of making humans communicate via computers without using any of their traditional five senses. genomes have been mapped. artificial brains have been created for worms and mice that transmit their behaviors to inorganic hosts, even lego models. we also have the capability to use our thoughts to control robotic bodies. even if singularity isn't here yet, we have reached a new level of human-machine interaction as a halfway measure towards achieving the ultimate goal. for now, this human-computer seems the immediate future and one that will help computers 'learn' our way. will the future be dystopian and utopian? time will tell. however, i must say this -when i read utopia i felt uneasy about the sameness of society portrayed therein. i am not worried about singularity as long as artificial intelligence can be varied, moody, altruistic and joyful. isn't life about celebrating differences? over half a century ago, i.j. good wrote about the dangers of artificial intelligence 21 : let an ultra-intelligent machine be defined as a machine that can far surpass all the intellectual activities of any man however clever. since the design of machines is one of these intellectual activities, an ultra-intelligent machine could design even better machines; there would then unquestionably be an "intelligence explosion," and the intelligence of man would be left far behind. thus the first ultra-intelligent machine is the last invention that man need ever make, provided that the machine is docile enough to tell us how to keep it under control. if you consider evolution by its dictionary meaning 22 -'a gradual process in which something changes into a different and usually more complex or better form' -then the move from natural selection to intelligent design as the architect of biological progress is no different. the problems for human brains will be coping with mathematics and robotics with no specific purpose in mind. in a brave new world of independent thought, some human, some not -there is bound to be a conflict with the most human tendency of all: the need to control other beings. as vinge noted in his essay 23 right through evolution, the next life form does not pay its dues to previous models: 'follow the money' may be an adage when it comes to solving complicated crimes, but it often gives us an idea where science is leading us. recently, darpa, the us defense advanced research projects agency announced a $60 million challenge to researchers to create connections between the human brain and computers. successful teams must create a neural engineering design system -with 'full-duplex interaction with at least 1,000 neurons -initially in regions of the human auditory, visual, and somatosensory cortex' 8 . the future of the computerized brain is here. one concern is however, security of such a system. humans can be influenced; computers can be hacked. therein lies another challenge. intelligent machine of the sort he (good) describes would not be humankind's "tool" --any more than humans are the tools of rabbits or robins or chimpanzees speculations concerning the first ultralntelligent machine american heritage® dictionary -english language, fifth ed proceedings of a symposium cosponsored by the nasa lewis research center and the ohio aerospace institute electronic memory may bring bionic brain one step closer the singularity is near: when humans transcend biology artificial brains: the quest to build sentient machines a worm's mind in a lego body'. iprogrammer sapiens: a brief history of humankind how computers will think. siri-ous business darpa challenges researchers to link human brains with computers we ask: 'what's there?' it says: 'a ball. ' we ask: 'what are the properties of the ball?' it says: 'it's green, it's round, it's at that location.' it can answer that because the robot contains that information. now we ask: 'are you aware of the ball?' it says: 'cannot compute.' it is this lack of 'awareness' that differentiates a computer from a human. it is the inability to understand the abstract or imperfect relationships. however, people developing artificial intelligence view this as a technical or engineering problem rather than an emotional one.giulio tononi at the university of wisconsin-madison has been trying to solve this problem of the mathematical theory of consciousness, dubbed the "integrated information theory." tononi feels one of the main aspects of human consciousness is the ability to learn from experience; the second is the ability to solve mysteries -everyday relationships are mysteries are they not (men are from mars, women from venus)? tononi reckons that unless these problems are solved, a computer cannot be truly conscious and key: cord-016403-id6fjgye authors: djikeng, appolinaire; nelson, barbara jones; nelson, karen e. title: implications of human microbiome research for the developing world date: 2011-10-11 journal: metagenomics of the human body doi: 10.1007/978-1-4419-7089-3_16 sha: doc_id: 16403 cord_uid: id6fjgye the human microbiome refers to all of the species that inhabit the human body, residing both on and in it. over the past several years, there has been a significantly increased interest directed to the understanding of the microorganisms that reside on and in the human body. these studies of the human microbiome promise to reveal all these species and increase our understanding of the normal inhabitants, those that trigger disease and those that vary in response to disease conditions. it is anticipated that these directed research efforts, coupled with new technological advances, will ultimately allow one to gain a greater understanding of the relationships of these species with their human hosts. the various chapters in this book present a range of aspects of human microbiome research, explain the scientific and technological rationale, and highlight the significant potential that the results from these studies hold. in this chapter, we begin to address the potential and long-term implications of the knowledge gained from human microbiome research (which currently is centered in the developed world) for the developing world, which has often lagged behind in the benefits of these new technologies and their implications to new research areas. new high-throughput sequencing and data analysis approaches (costello et al., 2009; turnbaugh et al., 2009) , along with novel diversity screens and even more intrinsic single cell approaches to isolating new species (lasken, 2009) , have presented the sciences with a unique opportunity to investigate and interrogate the microorganisms that are associated with the human body, all at a greater depth than previously appreciated. from the earliest studies, the greater scientific community has recognized a high level of microbial diversity in nature beyond imaginations. this includes observations on the oceans, soils, and on animals. with respect to humans, it became increasingly apparent that the species on and in our bodies make significant contributions to our health and development. these species maintain normal cell function in the gastrointestinal tract (for example, see eckburg et al., 2005; bik et al., 2006; gill et al., 2006) . we are also dependent on these species for the efficient digestion of food components, such as plant material and xenobiotics , and to ward off certain diseases. in parallel, these microbes have been associated with, and can result in, many common diseases such as cavities, stomach ulcers, bacterial vaginosis (bv), and irritable bowel syndrome (foster et al., 2008; dorer et al., 2009) . most of the initial studies on the microbial diversity associated with the human body focused either on traditional culturing approaches or on sequencing and phylogenetic analysis of the 16s ribosomal (r)rna genes derived from microbial samples taken from the human body (eckburg et al., 2005; bik et al., 2006) . the limit to culturing or 16s rrna sequencing was primarily a reflection of the availability of molecular tools and approaches, and the cost associated with earlier versions of available sequencing technologies. the 16s rrna sequencing and analysis invariably revealed a higher level of microbial diversity than that seen with conventional culture techniques (gao et al., 2007; gao et al., 2008) . from the human stomach, for example, although the highly acidic environment was thought to only contain helicobacter types, bik et al. (2006) used 1,833 16s rrna sequences obtained from 23 gastric endoscopic biopsy samples to identify 128 phylotypes of bacteria that potentially reside in the human stomach. the majority of these phylotypes was shown to belong to the proteobacteria, firmicutes, actinobacteria, bacteroidetes, and fusobacteria. this work also described that 10% of the clones represented organisms that were previously uncharacterized. subsequent ongoing studies continue to reveal high levels of diversity from the microbial species that inhabit the human body, with high levels of both intra-and interspecies diversity (costello et al., 2009; turnbaugh et al., 2009) . most of these studies that have investigated the diversity of the microbial species associated with the human body have however left important questions unanswered such as the identity of the nondominant community members and their biological roles, and which metabolic processes the populations that are present encode and carry out. in addition, the past 15 years of genomic research have made it clear that closely related species, and even species that are identical at the 16s rrna level, can have wide variation in gene content (perna et al., 2001; kudva et al., 2002) . terms such as pan-genome and core-genome have been coined over the years to address the variations that are apparent in closely related species and have now been applied in a similar fashion to metagenomic populations (callister et al., 2008; bentley, 2009) . the initial publication of a shotgun sequencing of the human microbiome focused on the analysis of fecal samples from the human gastrointestinal tract . this study along with subsequent applications of shotgun techniques to the study of the human microbiome again highlighted the extent of microbial diversity associated with the human body (grice et al., 2009) . gordon and co-workers, for example, investigated the interplay between the gastrointestinal ecology and energy balance of animals on a western diet. here they found that obesity that was induced by the diet resulted in an increased proportion of one class of the firmicutes and that this same population could be reduced by manipulation of the diet. transplantation of the microbial populations from the obese mice to lean germ-free mice resulted in a higher level of the deposition of fat than when these microbial populations were taken from lean donors (turnbaugh et al., 2008) . more recently, gordon's group presented the results of a monozygotic and dizygotic twin pair study, where twins were concordant for leanness or obesity, and their mothers (turnbaugh et al., 2009) . the aim of this study was to address how host genotype, environmental exposure, and host adiposity influence the gut microbiome (turnbaugh et al., 2009) . the comparative analysis of fecal samples that were derived from 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16s rrna sequences. in addition, 2.14 gb of metagenomic data was obtained from their microbiomes. the results from this analysis suggest that the gastrointestinal microbiome is shared among family members, but variations are present within each individual with respect to the lineages that can be observed. this variation was evident in both the monozygotic and dizygotic twin pairs. the results however suggest that there is a core functional microbiome that can vary depending on physiological states. the genomic era created the possibility of studying the detailed genetics of many microbial species. these include pathogens and nonpathogens and species that have both positive and negative impacts on the environment. the developments in the genomics arena have taken advantage of emerging and improving sequencing technologies, as well as improved assembly algorithms and approaches coupled with reduced costs for generating genomic data. the developments also placed the greater scientific community in positions to ask in-depth gene-based questions and get real answers. the advent of metagenomics was a natural progression of the genomics field, and in particular took advantage of the ability to sequence dna that was derived from a mixed community and assemble this genetic information to reconstitute metabolic and physiological information of any community of choice. metagenomic approaches have by now been successfully applied to environments as diverse as soils, the oceans rusch et al., 2007; yooseph et al., 2007; yutin et al., 2007) , and the human body costello et al., 2009; turnbaugh et al., 2009) in an attempt to describe and decipher the microbial species that are present in these niches. entire systems can now be studied with respect to viral, microbial, and fungal diversity, over varying time courses, and before and after perturbation (costello et al., 2009 ). on the human side, when coupled with 16s rrna analyses for detailed estimates of microbial diversity, metagenomics approaches present the perfect opportunity to address questions related to human health and associated problems. this is particularly relevant in the developing world, which presents its own series of challenges, some of which are presented below. one of the most valuable examples to date of the potential benefits from knowledge gained with human microbiome studies comes from a series of studies performed by dore and colleagues at inra. the significance of the studies conducted by this group relates to how the evolution of initial studies focusing on the microbiome can result in recommendations to improve human health conditions. their results evolved from initial metagenomic studies on human gastric samples using fosmid libraries from six healthy patients, and six patients with crohn's disease (cd). the group was able to identify 125 nonredundant ribotypes mainly represented by the phyla bacteroidetes and firmicutes, of which 43 distinct ribotypes were identified in the healthy microbiota, and only 13 in cd. this metagenomic approach that was initially published gave the first insight into the reduced microbial diversity in patients with cd. their work continued to focus on microbiology of cd sokol et al., 2006; sokol et al., 2007; seksik et al., 2009) . they most recently compared fecal samples of 22 patients active for cd (a-cd) patients, 10 cd patients in remission (r-cd), 13 active ulcerative colitis (a-uc) patients, four uc patients in remission (r-uc), eight infectious colitis (ic) patients, and 27 by 16s pcr and found that members of firmicutes (clostridium leptum and c. coccoides groups in particular) were less represented in a-ibd patients compared to healthy subjects (hs) with faecalibacterium prausnitzii species (a major representative of the c. leptum group) in lower abundance in a-ibd and ic patients compared with hs. as a result of the initial work, the group proposed that f. prausnitzii was important for gut homeostasis. in 2008 members of the same group had published on the composition of the mucosa-associated microbiota of cd patients at the time of surgical resection and 6 months following using fish analysis (sokol et al., 2008) , and again found reduced abundance of f. prausnitzii being correlated with an increased risk of postoperative recurrence of ileal cd. they further studied the anti-inflammatory effects of f. prausnitzii and showed that the bacterium exhibited anti-inflammatory effects on cellular and tnbs colitis models. the us national institutes of health (nih) initiated a roadmap program focused on the human microbiome (peterson et al., 2009) . the project has been described as a community resource, with overarching aims to help determine the core human microbiome, to understand the changes in the human microbiome that can be correlated with changes in human health, to develop new technological and bioinformatics tools to support these goals, and to address the ethical, legal, and social issues raised by human microbiome research (http://nihroadmap.nih.gov/hmp/). the project has a heavy sequencing and data analysis component that currently is underway at the four large nhgri/niaid-funded sequencing centers (j. craig venter institute (jcvi), baylor college of medicine, the broad institute and washington university). the current sequencing focus includes generating at least 3000 reference genomes at various levels of finishing (chain et al., 2009) , coupled with significant 16s rrna sequencing and metagenomic sequencing from 15 to 18 body sites on 300 individuals some of which would be repeat sample donors (http://nihroadmap.nih.gov/hmp/). a number of "investigator"-driven demonstration projects have also been awarded. these demonstration projects aim to understand the implications of a number of diseases including cd, bv, psoriasis, and esophageal cancer to name a few (peterson et al., 2009) . it is anticipated that the results from these demonstration projects will give additional insights into the relationship between human health and disease and changes in the human microbiome. finally, the human microbiome project (hmp) roadmap initiative has awarded funds to investigate new technologies for improving knowledge of the human microbiome, as well as for the development of computational tools that will increase the value of metagenomic data (http://nihroadmap.nih.gov/hmp/fundedresearch.asp), and the ethical, legal, and social implications of this work. in summary, and as captured in the recent publication by peterson et al. (2009) , the goals of the hmp are to demonstrate the characterization of the human microbiome with population, genotype, disease, age, etc., and also catalog the influence of disease. the aim also is to present a standardized data resource and technological benefits. the project will go over 5 years at a funding level of close to 157 million us dollars. since the launch of this roadmap initiative in 2007, a number of other hmps have been described. an overview of available projects as of mid-2008 was presented in an editorial (mullard, 2008) . projects beyond the large nih usa based human microbiome efforts include work in europe, china, australia, and canada. in 2007, the european commission committed close to 31 million us dollars to a 4-year initiative called the metagenomics of the human intestinal tract (metahit) where the primary focus is the microorganisms that inhabit the gut, and how they contribute to obesity and inflammatory bowel disease (mullard, 2008) . a review of this effort is presented in another chapter written by ehrlich and colleagues. we are all cognizant of the fact that age, diet, and geographical location contribute to variations in the human microbiome. consequently, the more initiatives that we have in diverse parts of the world, the better positioned we will be to fully understand the key components of the microbiome and their interactions with the host under various environmental and physiological cues. because of a slow rate of progress in the areas of scientific research, along with low levels of available funding and investment in sciences in most developing countries, there has been very little scientific contribution toward solving major problems that hinder their global development. as coloma and harris (2009) nicely put it, "researchers in many developing countries will not be participating in genomics research, mainly because of their technological isolation and their limited resources and capacity for genomics research combined with the urgency of many other health priorities." areas such as public health, emerging infectious diseases, and agricultural development, which are key to long-lasting and sustainable national development, still lack the funding and innovation required to mitigate their inability to contribute to global development. the global health sector is of particular importance given the increasing number of diseases that plague the developing world (some of which are making a comeback after several years under effective control). examples of some of these are detailed below. consequently, in most developing countries throughout the world, and specifically in sub-saharan africa, south america, and asia, there is a serious need to improve public health. in these countries, communicable diseases caused by known and even unknown pathogens (see below) remain a leading cause of mortality. emerging infectious diseases are a major cause for alarm, and malnutrition and associated effects are also major issues that need to be effectively addressed. if one takes emerging infectious disease as an example, this captures many viral and bacterial agents. severe acute respiratory syndrome (sars) was the first infectious disease to emerge in the 21st century, and other emerging viral infectious diseases according to the world health organisation (who) include but are not limited to ebola and marburg hemorrhagic fevers. in addition, in an earlier publication by who ("new who office to help developing countries control emerging diseases"; j environ health 63, 2001) it was stated that in 1998 alone, communicable diseases caused the death of over 13 million people worldwide, mainly among the poorest populations of developing countries. since then, more than 30 new communicable diseases have been identified, and this list includes several diseases that were thought to be almost extinct that apparently have come back into the human population. certain food-borne diseases are also considered to be emerging as they now occur more often, and that list includes outbreaks of salmonellosis, which have increased significantly in the past 25 years. listeria monocytogenes also falls into this category as its major role in food-borne diseases has become more recently appreciated, and some food-borne trematodes are also emerging as a serious public health concern. although food-borne infections with escherichia coli serotype o157:h7 were first described in 1982, it has rapidly emerged to be a leading cause of infections, which in turn result as a major cause of bloody diarrhea and acute renal failure, with an infection that is sometimes fatal. finally, while cholera devastated much of asia and africa for years, its introduction for the first time in almost a century on the south american continent in 1991 makes it another example of an emerging infectious disease. in addition, very little to none of the successful metagenomics stories in understanding the human microbiome and its role in aspects of human health have been conducted or duplicated in developing countries. notwithstanding ongoing efforts focusing on vaccines, better diagnostics, and improved treatment of many of these diseases, it is becoming increasingly essential to complement such approaches with an investigation of the role of the human microbiome on human health. several areas of anticipated important contribution of the human microbiome include zoonotic diseases and other emerging and re-emerging infectious diseases, sexually transmitted diseases, diarrheal diseases, respiratory diseases, eukaryotic diseases, malnutrition, and the integration of probiotics for improving human health. in addition to the translation of findings into practical approaches for improving human health, other opportunities offered by the human microbiome initiative are related to the transfer of technology to developing countries and the associated long-term benefits to training local populations in these developing sciences so that nations can retain the benefits. this will further strengthen capacity in genomics and bioinformatics and all the associated downstream applications that come with these areas of research. it is now appreciated that there is a resident (which constitutes the core microbiome) and a nonresident microbiota. the nonresident microbiota contains known and unknown microbes that cause a wide range of human diseases, most of which remain to be effectively controlled in both the developed and the developing world. human losses in the developing world in terms of mortality and contributions to economic development appear to be greater, however. currently, for example, communicable diseases caused by eukaryotic parasites such as plasmodium spp., leishmania spp., trypanosoma spp., and various viruses, among others, remain serious public health concerns in the developing world and affect more than 1.2 billion people (mahmoud and zerhouni, 2009) . in this context, scientific challenges that include genomics, metagenomics, proteomics, and metabolomics-related activities need to be expanded to ultimately encompass system and ecological understanding of communities of microbes and their interactions with humans. it has in fact been anticipated that the control of these diseases may be accelerated by the complete understanding of the genomes of these species, coupled with an understanding of the changes of the human microbiome that favor or reduce/eliminate their virulence and/or transmissibility. the adaptation processes, for example, by which zoonotic microorganisms that enter the human population adapt to become pathogens overtime can also be accelerated by longitudinal studies that focus on the populations on the bodies of both healthy and diseased individuals. as with most advanced technological and scientific approaches, and as is evident from the developing countries reports presented above, the development and applications of technological advances probably will take a significant amount of time to trickle to the developing world. in the realm of genomics and metagenomics as applied to human health, there is limited evidence that this will happen soon enough to allow developing countries to actively participate and shape research in these new fields. however, a recent award from the bill & melinda gates foundation (bmgf) to dr. jeffrey gordon at the washington state university in st. louis to study childhood malnutrition in developing countries (http://www.gatesfoundation.org/pressreleases/pages/child-malnutrition-microbial-cells-study-090331.aspx) suggests that there will be more movement in the direction of applying these technologies to problems in the developing world. for the above-mentioned study, gordon's group will investigate the microbes in severe malnutrition with a major focus on severely malnourished infants living in malawi and bangladesh, and whether their microbial flora varies from that found in healthy infants who live in the same environment. it is anticipated that as a result of these studies, we will be better positioned to develop effective treatment regimes for these disease conditions. this award is part of an initiative by the bmgf to fund research on malnutrition (http://mednews.wustl.edu/news/page/normal/13840.html?emailid=23653). in addition to that award and the anticipated outcome, there have been a significant number of plant and microbial genome projects initiated and conducted in the developing world. the range of microbial species that have been sequenced includes human, plant, and animal pathogens, as well as organisms that have potential benefit to the environment. some of these species include actinobacillus pleuropneumoniae serovar 3 str. jl03 that causes fibrinous and necrotizing pleuropneumonia in swine, and that was sequenced by the huazhong agricultural university in china and haemophilus parasuis sh0165 also sequenced by the huazhong agricultural university/institute of pathogen biology/chinese academy of medical sciences/peking union medical college. chromobacterium violaceum atcc 12472 was sequenced by the lncc (national laboratory of scientific computing in rio de janiero, brazil); this bacterium carries the bacteriocidal pigment violacein and can also cause diarrhea and septicemia in humans. ehrlichia ruminantium str. welgevonden was sequenced at the university of pretoria, south africa. leifsonia xyli subsp. xyli str. ctcb07, the causative agent of ratoon stunting disease in sugar cane, was sequenced by the sao paulo state (brazil) consortium and leptospira interrogans serovar copenhageni str. fiocruz l1-130 and xylella fastidiosa were also sequenced by the same group. leptospira interrogans serovar lai str. 56601 sequenced by the chinese national hgc, shanghai, and lysinibacillus sphaericus c3-41 sequenced by the chinese academy of sciences/beijing genomics institute, chinese academy of sciences. the developing world has also been involved in the sequencing and analysis of some of the major eukaryotic parasites such as trypanosoma brucei, trypanosoma cruzi, leishmania major, and theileria parva (nene et al., 2000; berriman et al., 2005; bishop et al., 2005; el-sayed et al., 2005; gardner et al., 2005) . there have also been several initiatives that have looked at host genotyping in many developing countries. according to coloma and harris (2009) , thailand, south africa, indonesia, brazil, mexico, and india have all devoted resources to studies on human genetics and variation in human populations. as a result of many of these initiatives in developing countries, a limited capacity of tool development for genomics and bioinformatics approaches has occurred. however, much more remains to be achieved in technology and knowledge transfer, particularly in sub-saharan africa and latin america. the main focus should be on developing genomics platforms leveraging on the next-generation sequencing approaches that remain to be established in much of the developing world. events of emerging and reemerging infectious diseases in the human population remain constant occurrences in sub-saharan africa, southeast asia, and south america. emerging infectious disease events such as sars (field, 2009) and the most recent pandemic of h1n1 illustrate and confirm the constant flow of pathogens from wild and domesticated animals, and other reservoirs into the human population. chikungunya fever, which is an arboviral infection, reemerged in asia in 2005-2006 after a long period of quiescence (bhatia and narain, 2009) . it is thought that factors including microbial, climatic, social, and economic aspects influenced the reemergence of the disease and the pace at which it spread, eventually resulting in high death rates (bhatia and narain, 2009) . indeed, there are many microorganisms (viral, bacterial, and eukaryotes) that have moved into the human population and remain part of the human microbiota, which, when able to effectively survive, can cause either new diseases or disease with a much higher severity. such cases of unknown and potentially pathogenic microorganisms in circulation in the human population are usually favored by factors related to (1) the ability of microorganisms to adapt in new hosts, (2) human actions (interactions with wild animals, hunting and effects on the environment leading to ecological disturbances), and (3) human movements as a result of global world travel (field, 2009) . consequently, at any particular time, there could be a set of known and unknown microorganisms present in a given individual or a population as a result of their presence in and interaction with a specific environment or organisms therein such as animal reservoirs (i.e., bats, mice, and rats) of known and unknown microorganisms. the main challenge in the context of forecasting, and better yet preventing emerging and reemerging infectious diseases has been early detection and genetic identification of such known and unknown microorganisms. global human microbiome studies using metagenomics analysis of known and unknown microorganisms provide unique but powerful opportunities to uncover the near-complete composition of the microbial content of an individual or a population at any given time, thus setting the stage for a comprehensive inventory of the genetic characteristics of potential human pathogens. studies of the human microbiome in the developing world will likely contribute significantly to the discovery of emerging pathogens (viruses, bacteria, and others) in circulation in humans. in fact, in both developed and developing countries the issues of early identification of emerging pathogens have been an impediment for the prevention of emerging and reemerging infections. based on recent studies, human metagenomics coupled with the next-generation dna sequencing provides an opportunity for early detection of microbial organisms even when there is significantly low abundance (relman, 2002) . because of the extreme importance of monitoring zoonotic infections, metagenomics studies should in principle be extended from humans to domesticated and nondomesticated animals. in fact, based on the technologies already available for human metagenomics studies, there has been increasing interest in launching animal metagenomics initiatives. such initiatives will not only provide insights into the resident and transient microbial populations but also, in the case of natural reservoirs of given microorganisms, provide an opportunity to pinpoint the genetic changes that must occur for their adaptation to a new host -the human body. this is applicable in particular to invertebrate vectors and bats that are known to be host to a number of highly pathogenic viruses that pose significant public health problems to humans. developing countries are particularly affected by the impact of mycobacterium tuberculosis virulence and tb drug resistance. this has been primarily because of genome plasticity in the causative agent. unfortunately, available microarray-based platforms to identify strain diversity have not been fully implemented with the greatest tb incidence largely due the hiv/aids epidemic. the renewed interest and funding for top infectious diseases has recently revamped efforts to accelerate tb research, with a particular focus on the use of integrated approaches to find better control measures. in this context, it is proposed and highly anticipated that key aspects such as the integration of large-scale "omics," datasets focusing on parasite genetic determinants, host genetics, and host-parasite interactions will be crucial for this quest for better control measures. in addition, and given recent reports, the human microbiome would be a great addition to this integration of data in the context of systems biology. to this end, the evaluation of the human microbiome in cases of latent, nonlatent tb, and drug-resistant tb infections will provide insights into the role of the human (resident and nonresident) flora in various aspects of tb infections. such information would most likely contribute to improving diagnosis, control, and spread of tb infection. another example of the potential to come from using human metagenomic research and approaches in the developing world relates to another emerging infectious pathogen that causes leptospirosis. the leptospires cause an infection that is associated with very high levels of mortality annually, but have received relatively little attention, probably because the infection is concentrated in the tropical regions and in the developing world. more than half a million cases are reported annually, and majority of these cases are associated with human exposure to pathogenic leptospira species in the environment. mortality rates as high as 25% have been recorded. the genus leptospira is serologically divided into two species: l. interrogans, which is pathogenic to humans and animals, and l. biflexa, a free-living nonpathogenic species found in water and wet soil. more than 16 pathogenic and saprophytic species have been recognized. many animals including rodents and dogs are known to be reservoirs of leptospira, and humans are considered to be the accidental hosts of this pathogen. transmission of the pathogen is primarily from soil and water to mammalian tissues (often noticed following on large-scale flooding), with the infection occurring via penetrating leptospires through mucosa or open skin. symptoms of leptospirosis include meningitis, pneumonitis, hepatitis, nephritis, pancreatitis, erythema nodosum, and death. no human vaccine against leptospirosis is available, and mild leptospirosis is treated with doxycycline, ampicillin, or amoxicillin. for severe leptospirosis, the primary therapy is penicillin g. the molecular diagnosis of leptospirosis has been with traditional approaches such as restriction enzyme analysis, nucleic acid probes and hybridization, pulse field gel electrophoresis (pfge), and varying ribotyping approaches. genome sequences from at least six leptospiras have become available in the past few years, and these genomes are providing insight on the diversity of these species. in addition, the availability of these genomes is allowing for the identification of novel virulence factors, and ultimately will facilitate vaccine development. recently, the genome sequence of the free-living l. biflexa was completed (picardeau et al., 2008) and shown to contain 3,590 protein-coding genes distributed across three circular replicons. in the current study, it has been estimated that 2,052 genes (61%) represent a progenitor genome that existed before divergence of pathogenic and saprophytic leptospira species. basically, nearly one-third of the l. biflexa genes are absent in pathogenic leptospira. in addition, 1,431 pathogen specific genes that are found in the pathogenic leptospires are not present in l. biflexa. of these, 893 genes have no known function suggesting that there are mechanisms that are unique to leptospira and that the pathogenic specific genes need further study. the resulting genome studies suggest that there is still a significant amount of information that is not understood about the leptospiras, particularly as it relates to how the species adapts to new environments and how the genomes mutate. metagenomic studies of samples derived from infected populations will present an opportunity to study the pathogen without repeated passage where it has been shown to have genome rearrangements. in parallel, the pathogen can be studied directly in the environment when it is in transition from its natural host to humans (the accidental host). interestingly, there is a large niaid-funded project underway at the jcvi to sequence the genomes of an additional 400 leptospira isolates (joe vinetz, personal communication; http://gsc.jcvi.org/). leptospirosis is another example of an emerging infectious disease that is prevalent in tropical environments and has not received as much attention as the major diseases in the developed world although the causative organisms result in a high mortality rate. genomics and metagenomics approaches have the potential to increase the understanding of these species and their impact on human health. diarrheal diseases remain one of the leading causes of deaths worldwide (culligan et al., 2009) . specifically, diarrheal diseases are the second most common cause of child deaths worldwide, and more than 80% of child deaths due to diarrhea occur in africa and south asia. worldwide, 88% of deaths from diarrhea are due to unsafe water and poor sanitation or hygiene. three-quarters of all deaths from diarrhea in children younger than 5 years occur in 15 countries. there are about 2.5 billion cases of diarrhea among children each year, in addition to those who die from the disease. the un reports that vaccines and better hygiene could decrease the number of deaths from diarrhea among children. since the 1970s, oral rehydration therapy has been the cornerstone of treatment programs. this therapy prevents dehydration that is associated with diarrhea. giving zinc supplements with oral rehydration salts has also been shown to reduce the length of the illness and also the risk of more diarrhea episodes. sixty percent (60%) of children in developing countries do not get the recommended treatment for diarrhea, which is vaccination against rotavirus, the leading cause of the disease. in fact, rotavirus causes about 40% of hospital admissions of children below 5 years suffering from diarrhea. current therapies are focused on rehydration therapies but the studies from a human microbiome approach, coupled with the development of novel antibiotics and/or probiotics holds significant potential (culligan et al., 2009) . many diarrhoeal diseases have been associated with viruses (ramani and kang, 2009) . recent results suggest that viruses are present in as much as 43% of diarrheal samples in the developing world (ramani and kang, 2009 ). there are however a significant number of cases of diarrhea without obvious causes, thus making it difficult to control them. in addition and specifically in the case of rotaviruses, because of their high genetic diversity, the emergence of new genotypes, and the reassortment between different genotypes (matthijnssens et al., 2009) , there is constant need for surveillance of circulating strains. human metagenomics studies hold the promise for increasing our understanding of the diversity of rotavirus and other etiological agents of diarrheal diseases. based on previous studies, gastrointestinal tract metagenomics studies in both healthy and diarrheal patients in developing countries may lead to the identification and association of additional microorganisms (bacteria, viruses, and eukaryotes) with various cases of diarrheal diseases . as an example, recent human microbiome studies have led to the discovery of a novel virus of the cosavirus genus and its association with acute diarrhea in a child in australia (holtz et al., 2008) . regular and comprehensive metagenomics analyses focusing on acute and difficult-to-cure cases of diarrhea and diarrhea cases with known and unknown causes primarily in developing countries may provide opportunities for (1) a constant assessment of the diversity of known causative agents of diarrhea and (2) identification of new microorganisms as they relate to cases of diarrheal diseases. sexually transmitted diseases (stds) are common infections throughout the developed and the developing world. stds can result in premature birth, stillbirth, and neonatal infections (de schryver and meheus, 1990) . many ongoing studies on bv aim to understanding the microbial populations that are present in the vaginal ecosystem and how they vary under health and disease conditions. recent studies that are focused on 16s rrna gene analysis have suggested that the extent of microbial diversity in the vaginal tract is not fully understood, which in turn has implications for current treatment regimes. this has potentially significant implications for asymptomatic disease conditions for example. additional results show a lack of homogeneity within the vaginal tract, highlighting a complex ecosystem (kim et al., 2009) . metagenomic approaches to studying this environment promise to give additional insights into the extent of diversity within this niche. ongoing studies in several parts in sub-saharan africa reveal that there is some relationship between the population of microbes that exists in the vaginal tract and stds. recently, van de wijgert et al. (2008) described a study in which they investigated the relationships among bv, vaginal yeast, and vaginal practices, mucosal inflammation, and hiv acquisition. from a cohort of 4,531 hiv-negative women, they observed that women who were positive for bv or vaginal yeast had a higher likelihood to acquire hiv, and they concluded that bv and yeast may contribute more to the hiv epidemic than previously appreciated (van de wijgert et al., 2008) . similar observations have been made in a review of all available literature on the extent to which bv may increase the risk of hiv acquisition (atashili et al., 2008) . earlier, in 2000 , van de wijgert et al. (2000 studied 169 zimbabwean women to determine if intravaginal practices could be associated with changes in the vaginal flora and acquisition of stds. in this study, they found that some disturbances of the flora could be associated with increased likelihood of stds and hiv; the absence of lactobacilli from the vaginal flora was associated with being positive for hiv (van de wijgert et al., 2000) . martin et al. (1999) had similarly looked at a cohort of sex workers in kenya and demonstrated that although only 26% of these women were colonized with lactobacillus species at baseline, follow-up studies showed that the absence of culturable vaginal lactobacilli could be associated with the increased likelihood of acquiring hiv-1. abnormal vaginal flora on grams-stain was associated with increased risk of both hiv-1 acquisitions. this group proposed that the treatment of bv and the use of lactobacilli to colonize the vaginal cavity should be evaluated for reduce risk of acquiring hiv-1, gonorrhea, and trichomoniasis (martin et al., 1999) . how the microbial populations in the vaginal cavity can contribute to reduce chances of hiv infection is one of those major areas that need attention, and that will undoubtedly benefit from human microbiome research. according to the world health organisation (who, http://www.who.int/ mediacentre/factsheets/fs094/en/), every 30 seconds a child dies of malaria, a disease that can be prevented and cured. in 2006 there were 247 million cases of malaria, and these resulted in nearly 1 million deaths mostly among african children. in fact, 90% of all malaria deaths occur in sub-saharan africa. people who live in lower-income communities, i.e., approximately half of the world's population, are at risk of the disease. the who reports that in 2006 malaria was present in 109 countries and territories. the disease, however, can be eradicated, says bill gates. in an interview with the bbc world services world today program in january 2010, gates said "we have a vaccine that's in the last trial phase -called phase three." he added that "a partially effective vaccine could be available within 3 years." a vaccine that is fully effective against malaria would take 5-10 years to develop. although most cases of malaria are found in sub-saharan africa, there are other countries, including in asia, latin america, the middle east, and parts of europe, that are also affected. key interventions include prompt and effective treatment with artemisin-based combination therapies; people at risk using insecticide nets; and indoor residual spraying with insecticide to control the vector mosquitoes. genomics approaches have already been used to elucidate the genomes of several of the plasmodium species (gardner et al., 2002; carlton et al., 2008; pain et al., 2008; mitsui et al., 2009 ), but new metagenomics approaches present opportunities to monitor the impact of the parasite of the microbial communities that reside on and in the human body, with a longer-term potential to develop novel probiotic approaches to supplement nutrition of infected individuals while the parasite runs its course. in countries that have a high rate of malaria, economic growth rates may be cut by as much as 1.3%. in addition, genomic studies on the environments, in which the mosquitoes reside and breed, are being and will continue to allow for an increased understanding of the communities that they require for their survival (ponnusamy et al., 2008a (ponnusamy et al., , b, 2009 ). this is particularly relevant since mosquitoes breed in areas where there are wet conditions, and the transmission of the disease can differ according to local factors such as rainfall, proximity of mosquito breeding sites to people, and the mosquito species in the area. a november 2009 report from susan anyangu in nairobi, kenya, carried by inter press service (ips) states that the rts.s vaccine being developed is to be used specifically in africa. it will be for infants and children aged less than 5 years (the most vulnerable to malaria). the vaccine could be ready for use in 5 years time. supplementing nutrition of people with malaria with probiotic solutions that have been derived from a metagenomic approach to understand the human microbiome holds significant promise. the fao/who defines probiotics as "live microorganisms which, when administered in adequate amounts confer a health benefit on the host." probiotics have become more and more valuable over the past few years and are available in a number of food sources, including yogurts and other milk products, fermented and unfermented milk, and some juices. these live microorganisms are in most cases bacteria that are similar to beneficial microorganisms found in the gastrointestinal tract. each species that is present in the gut environment would seem to hold some potential for use as a probiotic and therefore in human health. probiotics have been shown to be effective in treating irritable bowel syndrome (ibs), childhood and traveler's diarrhea, prevention and treatment of vaginal yeast infection and urinary tract infection, preventing and treating inflammation of the colon after surgery, reduction of the recurrence of bladder cancer, shortening the time of intestinal infections, and preventing eczema in children. although the benefits of probiotics are evident, they have yet to be adapted extensively in the developing world (reid and devillard, 2004) . other ideas on the use of probiotics for reducing the morbidity and mortality associated with hiv/aids have been explored and proposed (reid et al., 2005) where it has been proposed that lactic acid bacteria could play a role in maintaining the health of the human gut. we can only hope that as a result of the initiatives of the human microbiome project, new probiotics for a range of human health conditions may be developed based on baselines for people in different geographic locales. the efficient implementation of human microbiome research relies on the advanced instrumentation necessary for the processing of collected clinical samples, preparation and amplification of nucleic acid, and dna sequencing. in addition, dna sequence analysis also requires advanced bioinformatics resources. all genomicsrelated technologies developed over the past 10 years remain very expensive to be acquired by developing countries. this is usually justified by low-use volume and high costs of equipment and maintenance (coloma and harris, 2009) . therefore, as suggested by these authors, involvement of laboratories and institutions in developing countries should take advantage of "north-south" and "south-south" collaborations. previous examples of successful "north-south" collaborations could be leveraged to initiate new ones in the context of human microbiome studies. for the past several years, there have been numerous initiatives in developing countries to reduce the technological divide and hence begin to actively contribute to genomics research. in this context, activities have included training and capacity building in genomics and bioinformatics. in addition, there has also been an emphasis on the development of "centers for excellence" to provide resources and a critical scientific mass at regional levels. four such regional "centers of excellence" are currently being established in eastern and central africa, southern africa, west africa, and north africa. one of the most advanced "centers for excellence," biosciences for eastern and central africa (beca), located at the international livestock research institute (ilri) in nairobi, kenya, has established facilities (with advanced genomics and bioinformatics resources) to support and accelerate research in a wide range of disciplines, including plant/crop sciences and animal sciences. such infrastructure would ideally be poised for use as a focal point for the implementation of a regional initiative on the human microbiome. the existence of such facilities would normally be used to engage various african institutions in south-south collaborations. the "south-south" collaborations indeed provide opportunities to strengthen the scientific capacity of institutions in developing countries, which would be translated into their effective participation in north-south initiatives. genomics and metagenomics initiatives are usually quite expensive, and obviously, most institutions in the developing world would not be able to fund such activities independently. however, given the existence of several human microbiome projects in the united states, canada, europe, china, japan, singapore, and australia, components in developing countries could easily be justified. for example, an african component of the human microbiome would provide elements to answering important outstanding microbiome questions, among which are included: (1) is there a core human microbiome? (2) does the composition of the human microbiome vary from one geographical region to another? given the anticipation of such interesting outcomes, existing initiatives could further provide seeds to launch other initiatives in the developing world. furthermore, in the context of the use of biosciences for africa's development, a strong case should be made to various stakeholders such as the african union and other regional organizations to fund the african component of the human microbiome. this next wave of genomics research will not be without its own set of challenges. recent studies, for example, show that many diseases present with similar observations, and as such initial surveys into the human microbiome under health and disease may give unexpected outcomes (yazdanbakhsh and kremsner, 2009) . bacterial vaginosis and hiv acquisition: a meta-analysis of published studies sequencing the species pan-genome the genome of the african trypanosome trypanosoma brucei re-emerging chikungunya fever: some lessons from asia molecular analysis of the bacterial microbiota in the human stomach analysis of the transcriptome of the protozoan theileria parva using mpss reveals that the majority of genes are transcriptionally active in the schizont stage comparative bacterial proteomics: analysis of the core genome concept comparative genomics of the neglected human malaria parasite plasmodium vivax molecular genomic approaches to infectious diseases in resourcelimited settings bacterial community variation in human body habitats across space and time probiotics and gastrointestinal disease: successes, problems and future prospects epidemiology of sexually transmitted diseases: the global picture helicobacter pylori's unconventional role in health and disease diversity of the human intestinal microbial flora comparative genomics of trypanosomatid parasitic protozoa bats and emerging zoonoses: henipaviruses and sars metagenomic analysis of human diarrhea: viral detection and discovery application of ecological network theory to the human microbiome molecular analysis of human forearm superficial skin bacterial biota substantial alterations of the cutaneous bacterial biota in psoriatic lesions genome sequence of theileria parva genome sequence of the human malaria parasite plasmodium falciparum from where did the 2009 'swine-origin' influenza a virus (h1n1) emerge? metagenomic analysis of the human distal gut microbiome topographical and temporal diversity of the human skin microbiome identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea heterogeneity of vaginal microbial communities within individuals strains of escherichia coli o157:h7 differ primarily by insertions or deletions, not singlenucleotide polymorphisms genomic dna amplification by the multiple displacement amplification (mda) method neglected tropical diseases: moving beyond mass drug treatment to understanding the science reduced diversity of faecal microbiota in crohn's disease revealed by a metagenomic approach vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition rotavirus disease and vaccination: impact on genotype diversity phylogeny of asian primate malaria parasites inferred from apicoplast genome-encoded genes with special emphasis on the positions of plasmodium vivax and p. fragile microbiology: the inside story metagenomics and the global ocean survey: what's in it for us, and why should we care? theileria parva genomics reveals an atypical apicomplexan genome the genome of the simian and human malaria parasite plasmodium knowlesi genome sequence of enterohaemorrhagic escherichia coli o157:h7 the nih human microbiome project genome sequence of the saprophyte leptospira biflexa provides insights into the evolution of leptospira and the pathogenesis of leptospirosis species composition of bacterial communities influences attraction of mosquitoes to experimental plant infusions identification of bacteria and bacteria-associated chemical cues that mediate oviposition site preferences by aedes aegypti diversity of bacterial communities in container habitats of mosquitoes viruses causing childhood diarrhoea in the developing world probiotics for mother and child probiotics for the developing world new technologies, human-microbe interactions, and the search for previously unrecognized pathogens the sorcerer ii global ocean sampling expedition: northwest atlantic through eastern tropical pacific the role of bacteria in onset and perpetuation of inflammatory bowel disease incidence of benign upper respiratory tract infections, hsv and hpv cutaneous infections in inflammatory bowel disease patients treated with azathioprine temperature gradient gel electrophoresis of fecal 16s rrna reveals active escherichia coli in the microbiota of patients with ulcerative colitis molecular comparison of dominant microbiota associated with injured versus healthy mucosa in ulcerative colitis faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of crohn disease patients diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome a core gut microbiome in obese and lean twins intravaginal practices, vaginal flora disturbances, and acquisition of sexually transmitted diseases in zimbabwean women bacterial vaginosis and vaginal yeast, but not vaginal cleansing, increase hiv-1 acquisition in african women influenza in africa the sorcerer ii global ocean sampling expedition: expanding the universe of protein families assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the atlantic and pacific oceans using the global ocean sampling expedition metagenomes the authors wish to acknowledge the invaluable information found on the world health organisation (who) website and on the mayo clinic website. key: cord-270940-acwkh6ed authors: kallio-kokko, hannimari; uzcategui, nathalie; vapalahti, olli; vaheri, antti title: viral zoonoses in europe date: 2005-06-29 journal: fems microbiol rev doi: 10.1016/j.femsre.2005.04.012 sha: doc_id: 270940 cord_uid: acwkh6ed a number of new virus infections have emerged or re-emerged during the past 15 years. some viruses are spreading to new areas along with climate and environmental changes. the majority of these infections are transmitted from animals to humans, and thus called zoonoses. zoonotic viruses are, as compared to human-only viruses, much more difficult to eradicate. infections by several of these viruses may lead to high mortality and also attract attention because they are potential bioweapons. this review will focus on zoonotic virus infections occurring in europe. during the past 15 years a number of new virus infections have emerged or re-emerged. most of them, such as sin nombre and andes hantaviruses, sars coronavirus, avian influenza, nipah and hendra viruses, have appeared in subtropical or tropical regions. dengue is spreading to new areas and west nile virus has reached the new world. infections by several of these viruses may lead to high mortality and also attract attention because they are potential bioweapons. some viruses such as tick-borne encephalitis virus are spreading to new areas along with climate and environmental changes. most of these infections are zoonoses and clearly viruses shared by animals and humans are, unlike human-only viruses, much more difficult to eradicate. here, we review zoonotic virus infections occurring in europe. the infections like lassa fever and dengue that are imported to europe but are not indigenous to european nature will not be discussed in detail in the review. we have divided the virus infections into two categories, those that are transmitted to humans directly from vertebrate animals (like rodents, foxes, bats and birds) and those that are primarily transmitted by arthropods (mosquitoes, ticks, sandflies). the latter class is formed by arboviruses but notably they have vertebrate hosts in nature. hantaviruses are enveloped viruses with a tri-segmented negative-stranded genome and belong to the family bunyaviridae [1, 2] . the 6.4 kb l (large) segment rna encodes the 250 kda rna polymerase, the 3.6 kb m (medium) segment the two glycoproteins 68-76 kda gn and 52-58 kda gc, -formerly known as g1 and g2; and the 1.7 kb s (small) segment the 50-54 kda nucleocapsid protein (n) ( table 1 , fig. 1 ). in addition, the s segment of some hantaviruses has another open reading frame named ns but its product or function remains to be discovered. viral messenger rnas of the members of the bunyaviridae are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. messenger rnas have 5 0 -methylated caps and 10-18 nontemplated nucleotides which are derived from host cell mrnas. the termini of all three segments are conserved and complementary to each other, a feature that has assisted in cloning and discovery of new hantaviruses. unlike most other bunyaviridae, hantaviruses are not arthropod-borne (arboviruses), but are rodent-borne, roboviruses. each hantavirus is primarily carried by a distinct rodent/insectivore species although a few host switches seem to have occurred during the tens of millions of years of their co-evolution with their carrier animals [3] . we now know that the genetic diversity of hantaviruses is generated partly by (i) genetic drift (accumulation of point mutations and insertions/deletions) leading to quasispecies [4, 5] , (ii) genetic shifts (reassortments of genome fragments within the same virus genotype/species), and (iii) according to recent findings [6, 7] , by homologous recombination, a mechanism not previously observed for negative-strand rna viruses. hantaviruses, which cause hemorrhagic fevers with renal syndrome (hfrs) in eurasia and hantavirus cardiopulmonary syndrome (hcps) in the americas, are prime examples of emerging and re-emerging infectious agents. like most of these infections hantaviral diseases are zoonoses. with the exception of the south-american andes virus, which can be transmitted directly from human to human, hantavirus infections are thought to be transmitted to humans primarily from aerosols of rodent excreta. only some [9, 10] . topografov hantavirus isolated from siberian lemmings (lemmus sibiricus) has not been detected in north european lemmings (lemmus lemmus) although it can grow in them [11] . infections in rodents are mainly asymptomatic and persistent. hantavirus infections are quite common in europe [8] (table 3) , puumala virus is common in northern europe, european russia and parts of central-western europe (fig. 2) . dobrava virus is found mainly in the balkans (fig. 2) . saaremaa virus has been detected in eastern and central europe but its epidemiology is not well defined (fig. 2) . apart from laboratory infections [12, 13] seoul virus has been detected in wild rats, only in france [14] . it is also apparent that many parts of europe, such as britain, poland and byelorussia, remain ''white'' on the european hantavirus map [8] . this means either that hfrs is rare or nonexistent in these regions or is not widely recognized and diagnosed by the biomedical community. in northern europe hfrs as well as the carrier rodents exhibit peaks in 3-4 year cycles [15] while in central europe the hfrs incidence follows the fluctuations of ''mast years'', i.e. the availability of beech and oak seeds for the hantavirus-carrying rodents. in central europe hfrs peaks in the summer while in northern europe most cases occur in late autumn and early winter, from november to january. risk factors to catch hantavirus infections and hfrs include professions such as forestry, farming, and military, or activities such as camping, and the use of summer cottages. males are more likely to be exposed than females [15, 16] . puumala and dobrava viruses both cause hfrs but the infections differ considerably in severity [17] : both are characterized by acute-onset fever, headache, abdominal pains, backache, temporary renal insufficiency -first oliguria, proteinuria and increase in serum creatinine and then polyuria -and thrombocytopenia but the extent of hemorrhages (hematuria, petecchiae, internal hemorrhages), requirement for dialysis treatment, hypotension and mortality are much higher in [19] . this is important since the infection is so common in many areas of europe [8] (table 3 , fig. 2 ). in addition, in some patients puumala virus infection may invade the pituitary gland and lead to mortality or at least hypophyseal insufficiency requiring hormone-replacement therapy [20] . the pathogenesis of hfrs is poorly understood [17, 21] . however, it is known that b3 integrins can mediate the entry of pathogenic hantaviruses [22] and that hantaviruses can regulate apoptosis [23] [24] [25] [26] 28] . also there is evidence [17, 21] that increased capillary permeability is an essential component in the pathogenesis of both hfrs and hcps, although different target tissues, kidneys and lungs are affected in the two diseases. hfrs patients show locally increased levels of tnf-a in the plasma and kidneys [27, 28] and high levels of urinary secretion of the proinflammatory cytokine il-6 [29] . studies with a monkey model mimicking human puumala virus infection [30] may assist in elucidating the mechanism of pathogenesis. of the four structural proteins, both in humoral and cellular immunity, the n protein appears to be the principal immunogen [31] . cytotoxic t-lymphocyte (ctl) responses are seen [32] and may be important both for protective immunity and pathogenesis of hantavirus infections [21] . the diagnosis of acute hfrs is primarily based on serology, since viral rna cannot be regularly detected in the blood or urine of patients [33, 34] . both immunofluorescence tests and enzyme immunoassays are widely used for detection of specific igm or low-avidity igg antibodies, characteristic of acute infection [35] [36] [37] . in addition, immunochromatographic 5-min igm-antibody tests [38, 39] have been developed. vaccines against hantavirus infections have been used for years in china and korea, but not in europe or the americas [40] . no specific therapy is used in europe, although both ribavirin and interferon-a have been successfully used in trials in china [41, 42] . a major problem is that at the time hfrs patients are hospitalized, virus replication is already disappearing. members of the genus lyssavirus within the family rhabdoviridae are bullet-shaped, enveloped viruses approximately 60 nm in diameter and 200 nm in length. the 12 kb non-segmented negative-strand genome encodes five proteins (starting from the 3 0 end): the 58-62 kda nucleoprotein (n), the 35-40 kda phosphoprotein (p), the 22-25 kda matrix protein (m), the trimeric 65-80 kda glycoprotein (g) and the 190 kda polymerase protein (l) ( table 1 , fig. 1 ). proteins are separately transcribed in cascade by a special mechanism from a single 3 0 -end promoter which results in a decreasing transcription and expression gradient for proteins encoded from the 3 0 to 5 0 end. the major antigen with neutralizing epitopes and pathogenetic determinants is the glycoprotein, which is responsible for receptor recognition and membrane fusion. after endocytosis the viral envelope fuses with endosomal membranes provoking the release of the internal viral nucleocapsid in the cytoplasm where transcription and replication takes place. the helically wound nucleocapsid results from the intimate association of the nucleocapsid protein and the rna genome. it serves as template for the polymerase (l protein and p cofactor) for transcription and replication. it buds to intracytoplamic membranes in infected neurons, but on plasma membranes in salivary gland epithelial cells. the lyssaviruses currently consist of 7 established genotypes, or lineages, of rabies(-like) viruses [56] of which the classical rabies virus, found throughout the world and associated with terrestrial mammalian hosts and american bats, forms genotype 1 ( table 2 , fig. 3 ). other lineages, or genotypes, are found in bats, of which mokola and lagos bat viruses seem to be less pathogenic, and towards which the vaccines based on the classical genotype 1 rabies virus are not protective [43a,43b] . in addition to the 7 genotypes, new bat lyssavirus genotypes have been recently found, e.g. from russia [43c]. during the past 100 years or more in europe, classical rabies virus has made two major host shifts, firstly from the dog to red foxes, and then to racoon dogs brought from east asia to be raised for fur; this species then became widely established in the wild (fig. 3) . phylogenetic data also suggest that westand southward spread of rabies virus occurred during the last century [44a] . although in europe the most important carriers of classical rabies virus are foxes and racoon dogs, the virus can be transmitted to secondary hosts such as domestic animals (dog, cat, cattle, horse, sheep) or e.g. deer -practically any mammal species could be a potential carrier. bats are a special case; lyssaviruses are maintained in bats, even in the absence of classical rabies in carnivores; thus in countries where classical rabies has been eliminated, bat rabies has become the dominating or only source of rabies virus and retains the potential for host-switching into the carnivore reservoir which constitutes a more direct threat for public health [44b]. as many bats are protected species, the detection or ''absence'' of bat rabies is dependent also on the intensity of screening efforts [45, 46] . as of the beginning of 2004, the following countries in europe were declared ''rabies-free'' by who (meaning no indigenous cases occurred during the last two years): belgium, cyprus, finland, greece, iceland, ireland, italy, luxembourg, norway, portugal and sweden (fig. 3) . in 2003, rabies virus was detected in europe in 7095 wild animals (excluding bats), 3951 domestic animals; 33 bat and 6 human rabies cases were diagnosed (see table 4 ). the countries where rabies was circulating include (in diminishing order of cases) russia, ukraine, lithuania, belarus, latvia, estonia, croatia, poland, slovakia, serbia-montenegro and turkey with hundred(s) to thousands of (animal) cases, romania, bosnia-herzegovina, germany, moldova and bulgaria with dozens of cases, and individual cases were registered (some imported and not affecting the rabies-free status) in slovenia, the netherlands, denmark, france, albania, finland, austria and switzerland ( members of 6 out of the 7 lyssavirus genotypes (except lagos bat virus, i.e. genotype 2) have caused rabies disease in man. the infection is inevitably fatal in humans or other mammals unless immune intervention (vaccination and administration of antibodies) is used. transmission may occur though the bite of an animal delivering the virus deep in to striated muscle or connective tissue, but infection may also occur after abrasion of the skin or licking of mucous membranes. the bite of a bat with small teeth may go unnoticed; on the other hand the bat lyssaviruses may infect human skin more easily than has been recognised. the incubation period is 20-60 days, but has been shown in some cases to be months, even years. the classical picture of rabies includes prodromal illness with fever and non-specific symptoms, as well as itching and local paresthesia. this is followed by neurological signs, consisting of either encephalitic ''furious rabies'' or paralytic ''dumb rabies''. in the former, episodic hyperactivity and excitation of the cns manifest as, e.g. hydrophobia, hypersalivation and convulsions. the patient finally develops paralysis, coma and cardiorespiratory failure. in the paralytic form, no excitation is seen, but paralytic disease develops to coma and death [45,52a,52b] . after entry of the virus to the body, the virus must gain access to peripheral nerves and to be transported towards the cns. rabies virus components are attached either directly or by encapsulated vesicles to the dynein motor carrying the ''cargo'' along the axonal microtubular system towards the cell stroma, approximately at a speed of 25 mm/day [45, 53, 54] . it is transmitted after replication on neuronal membranes transsynaptically to adjacent neurons and finally invades the cns where it first disturbs the limbic system associated with the excitation, and later neocortex, with little histopathological changes in neurons. centrifugal spread of the virus from the cns to many tissues through somatic and autonomic nerves also occurs, where the salivary glands are especially important for the spread of the virus to the next victim. immunological responses do not occur before cns involvement. all the viruses in phylogroup i are also pathogenic to mice by i.m. injection, and can cross-neutralize each other. this has practical implications, as fortunately the vaccine strains (of genotype 1) also appear to protect against the ebvl viruses [55] . a pathogenic determinant common to phylogroup i viruses seems to be amino acid r333 in the glycoprotein [56] ; substitution of this amino acid also abolishes the retrograde transport [57] . when symptoms develop, no cure is available. however, after exposure to a bite or scratch of a potentially rabid animal, rabies must and can effectively be prevented by post-exposure prophylaxis, originally developed by louis pasteur. the treatment includes in a non-vaccinated person (a) washing (with water and detergent) and disinfecting the wound to minimize the amount of cell-free virus (this alone can increase survival 50%), (b) starting a post-exposure vaccine regimen which includes (in europe) five doses of cell-culture derived vaccine intramuscularly into the deltoid muscle on days 0, 3, 7, 14, 28 and (c) in case of severe or deep injury additional passive anti-rabies immunoglobulin, which should be administered principally to the wound area. if the person was pre-vaccinated or the animal can be caught and studied for the presence of rabies, the protocol can be adjusted accordingly [45,52a] . clinical suspicion of rabies in a case of encephalitis of unknown origin is the starting point. ante mortem diagnostics can be achieved most easily with rt-pcr from saliva. in addition, rabies antigen can be detected in brain or nuchal skin biopsies, and in some cases antibodies in serum or csf may be found. post-mortem diagnostics is most rapid with antigen detection or rt-pcr from the brain, virus isolation is also possible. in addition to post-exposure prophylaxis, vaccines in humans can be used for pre-exposure prophylaxis (3 doses) in risk groups (veterinarians, wildlife workers, travellers to endemic areas; especially small children who may be unable to explain a potential exposure to a rabid animal) [45,52a,58] . the primary method for control of rabies is vaccination of dogs (and cats). this practice was established at the beginning of the last century in most of europe, but has not yet been achieved in many developing countries, where annually 60 000 people still die of rabies. in most of europe (excluding eastern europe) rabies has been eradicated from terrestrial wildlife species (carnivores) by aerial distribution of vaccine baits across the countryside. the vaccines used to protect wildlife species include attenuated vaccines as well as a recombinant vaccinia virus carrying the rabies virus glycoprotein (the latter has in one case caused a skin infection in man). although rabies in terrestrial animals can be controlled efficiently, eradication from the bat reservoir is not currently feasible. arenaviruses are the only members of the rna virus family arenaviridae. these viruses are enveloped, lipid solvent-sensitive, pleomorphic particles with a mean diameter of 120 nm (ranging between 50 and 300 nm). host cell-derived ribosomes are present in the virions, and give the virus particles a ''sandy'' appearance under the electron microscope, hence the name arenavirus (arena: sand in latin). the virion structure of arenaviruses is quite simple, virus particles contain two rna segments (s and l) linked to nucleocapsid proteins and viral polymerase molecules, and these nucleocapsidpolymerase complexes are surrounded by a lipid envelope into which two glycoproteins (g1 and g2) are linked protruding on the outside of the virion. arenaviral rna segments have an ambisense coding arrangement, the s segment (3.5 kb) encoding a 63-kda nucleocapsid protein (n) in the viral complementary sequence, and in the viral-sense 5 0 -end sequence a 75-kda glycoprotein precursor (gpc) which is posttranslationally cleaved to two glycosylated proteins 34-44 kda g1 and g2; and the l segment (7.2 kb) encoding a 180-kda viral polymerase (l) in the viral complementary sequence, and in the viral-sense 5 0 -end of the sequence a 10-14 kda ring finger z protein, which has been shown to have a role in arenavirus budding [59a] (table 1 , fig. 1 ). initiation of transcription may involve cap-snatching, although the transcription mechanism is not yet fully elucidated. arenaviruses include 23 viruses all carried by different rodent hosts (except tacaribe virus which has been isolated only from fruit bats) [59b]. arenaviruses are capable of causing chronic infections in their rodent hosts, and infectious virus is present in the blood and is also secreted into body fluids (saliva, urine, semen), which is presumably the route of transmission to humans. the appearance and incidence of arenaviral infections are closely associated with the distribution of the rodent host species and the rodent population dynamics. arenaviruses have co-evolved with their specific host species during millions of years, and have been divided into old world and new world groups first on a serological bass, and later into evolutionary lineages (new world group) using genetic analysis [60a,60b,60c]. both groups contain viruses that are included in the category a pathogen list (defined by cdc, usa), which means that the propagation of these agents is allowed only in biosafety level 4 laboratories. these highly pathogenic arenaviruses include the south american junin, machupo, guanarito, and sabia viruses from the new world group, and the african lassa virus from the old world group. all these viruses can cause hemorrhagic fevers in humans, and are considered potential bioterrorism agents being thus included in biohazard preparedness programs. in europe, these viruses occur only rarely as imported cases. the only arenavirus endemic in europe is lymphocytic choriomeningitis virus (lcmv), shown to circulate in mus musculus populations (table 2) , and associated with pet hamster derived epidemics [59b,61a]. relatively few epidemiological data are available concerning the actual distribution of lcmv in europe, but in addition to serological evidence from mus sp. material from spain [61b], antibodies against lcmv have been detected in rodent species other than mus musculus (our unpublished data), which indicates that other yet unknown arenaviruses may circulate in europe. at least ten arenaviruses have been reported to be able to cause disease in humans. as mentioned above, five arenaviruses (lassa, junin, machupo, guanarito, and sabia viruses) are even capable of causing a lifethreatening viral hemorrhagic fever [62a,62b] . none of these five viruses are endemic in europe, but a few imported lassa virus infections have been diagnosed in germany and great britain during the last few years with no secondary infections detected [63] [64] [65] . increased travelling increases also the risk for transmission of exotic arenaviruses to non-endemic areas such as europe. lcmv is thus far the only known endemic arenavirus in europe [62b]. in humans lcmv infections are mostly either asymptomatic or influenza-like diseases. in some cases aseptic meningitis or meningoencephalomyelitis is seen. lcmv is also capable of causing congenital infections manifested by hydrocephalus, microcephalus, chorioretinitis, and mental or psychomotor retardation [66,67a,67b] . lcmv infections are rarely fatal for humans. the diagnosis of arenaviral infections is based on serology and/or direct detection of the virus [68] . for serodiagnosis methods using immunofluorescence assay (ifa) as well as enzyme immunoassay (eia) have been described. either a four-fold rise in igg antibody titers or presence of igm antibodies is considered indicative of acute infection. the antibodies that appear first in the acute phase of infection are directed against the nucleocapsid protein; neutralizing antibodies against the glycoproteins appear later in the convalescent phase (if at all). this means that typing of the causative agent is difficult on a serological basis at the early stage of infection, and is actually possible only in the convalescent phase due to the slow rise of virus typespecific neutralizing antibodies. for direct detection of the virus, antigen detection assays are useful in the early diagnosis of lassa fever especially. also reverse transcriptase (rt)-pcr tests have been developed to detect arenaviral rna in patient samples [69a,69b,70a,70b,70c]. virus isolation attempts can also be successful. the supportive treatment of arenaviral infections includes ensuring the fluid, electrolyte and osmotic balance. in severe hemorrhagic fever-cases early diagnosis is important because the use of ribavirin has been found effective if the treatment commences within the first six days after onset of symptoms [71] . immune plasma containing neutralizing antibodies has also been useful in some cases. for prevention of arenaviral diseases, several attempts to develop vaccines have been made [72a] . one vaccine, candid 1, has been successfully used in the prevention of argentine hemorrhagic fever caused by junin virus with a clear reduction in the number of infections observed in humans [72b,73]. the genus orthopoxvirus in the family poxviridae consists of large 220-450 nm brick-shaped viruses, with a double-stranded dna genome (160-220 kb) ( table 1) , that are serologically cross-reactive and -protective. the middle part of the genome is very conserved among orthopoxviruses encoding structural proteins and replication machinery whereas the ends are more variable comprising genes involved with host-specificity and counteracting the immune response [74] . replication of orthopoxviruses occurs in the cytoplasm and includes translation of early mrnas (such as dna polymerases and immune defense molecules), dna replication, translation of intermediate mrnas (for late transcription factors), and late mrnas encoding structural proteins and late enzymes, respectively. altogether, e.g. the cowpox virus genome encodes nearly 200 open reading frames. intracellular non-enveloped virions are first formed, comprising the majority of the infectious viral progeny; some particles develop in er/golgi into enveloped, either cell-attached or extracellular viral particles, often motile due to attached actin tails [75] . homologous recombination occurs readily between orthopoxvirus sequences which has raised some concerns about the use of vaccinia virus-based vaccines in wild animals [76] . some orthopoxviruses are host-specific, whereas some are more promiscuous but have a distinct reservoir. their nomenclature may be misleading; e.g. monkeypox is not carried by monkeys, neither is cowpox carried by cows. smallpox or variola virus, now eradicated and historically the cause of one of the most feared human diseases, was specific to man. many other orthopoxviruses circulate in wildlife species and are often zoonotic. examples include the vaccinia virus, the modern smallpox vaccine, the origins of which are unclear but was originally described as cowpox by edward jenner [77] in late 18th century england, and which later has also re-escaped to nature in other parts of the world [78] ; monkeypox virus, pathogenic to primates, including humans, causing a smallpox-like disease with secondary transmission and with a likely reservoir in small rodents in central africa; cowpox virus, which is the main orthopoxvirus in europe and may be transmitted to man either directly from rodents or from a secondary carrier, typically cat. cowpox virus has been detected in western eurasia and in europe, voles of clethrionomys and microtus species and apodemus mice are the main reservoir hosts [79] ( table 2) . shedding of the virus from rodents is apparently transient. infection of cattle is rare; domestic cats relatively frequently present with clinical disease, but infection of zoo animals, e.g. elephants, has also been reported [80] . following the cessation of smallpox vaccination, more than 25 years ago, the number of humans susceptible to cowpox has increased. more than 60 human cowpox cases have been reported in the literature since 1969 [81, 82] . all age groups may acquire cowpox, but most cases have been in girls under 12 years of age, who have had a cat or e.g. a field mouse as a pet. infection probably occurs through abrasions in the skin; persons with atopic eczema are more prone to the infection. [81] [82] [83] [84] . the incubation period is 5-7 days, after which papules develop into lesions 1-3 cm in diameter which proceed through pustular, ulceral and eschar stages over a period of a about two weeks. they may be painful and vary in number, size and severity. local lymphadenopathy, pyrexia and nausea may occur; secondary bacterial infections are common. typically, solitary lesions are found, located mainly in fingers, hands or face (e.g. eyelid) [81, 85] . in 6-8 weeks the lesions heal gradually, some residual scars may remain. in some cases severe generalized skin infection occurs [83] , especially in atopic and in immunocompromised individuals and may in extreme cases lead to death [86] . cidofovir (a phosphorylated nucleoside analog of cytosine) may have potential as an antiviral against cowpox virus [87] . man-to-man transmission of cowpox virus (unlike for monkeypox) has not been reported. it is usually possible to detect orthopoxvirus particles directly from the skin lesions by electron microscopy. the virus can also be readily isolated in e.g. vero cells or chorioallantoic membrane of chicken embryos from the lesions and subsequently characterised [80] . several sensitive pcr approaches have been described, some related to the bioterrorism (smallpox) preparedness [88] [89] [90] ; in each case further typing at the species level is needed. in addition, during acute cowpox infection, igm antibodies and low-avidity igg antibodies have been detected [83] . following the cessation of smallpox vaccination approximately 30 years ago, the younger age groups are the most susceptible population, both to smallpox and to cowpox, which is more closely related to vaccinia virus. recent estimates indicate as low as 40% protection levels among europeans. for instance in finland, in the age group over 50, everybody had orthopoxvirus antibodies as measured by immunofluorescence assay. the seroprevalence decreased gradually towards younger age groups reflecting the gradual cessation of smallpox vaccination, with the last vaccinations in finland occurring in 1977 [83] . smallpox was finally declared to be eradicated from the world in 1980 [91] after which few people in europe have received the vaccine. in addition to wild rodents carrying cowpox, import of exotic pets may also pose a risk for orthropoxvirus transmission, as was seen in a recent outbreak of monkeypox virus in the usa [92] . orthomyxoviruses are enveloped, negative-strand rna viruses with 6-8 genome segments, of which avian influenza, (i.e. influenza a) viruses may cause severe disease in domestic poultry and cause zoonotic infections. the influenza viruses in wild aquatic birds are the source of these epidemics in chickens as well as providing a gene pool for reassortants with human influenza a viruses which then may become established in humanto-human transmission resulting in influenza pandemics. in addition, influenza a viruses are known pathogens of pigs, horses, mink, seals and whales [93, 94] . influenza a virus is 80-120 nm in diameter and has 8 genome segments varying in size from 0.89 to 2.3 kb. the three largest segments 1-3 encode the polymerase subunits pa (83 kda), pb1 (87 kda) and pb2 (84 kda), respectively; whereas the segments 4-6 each encode one viral protein, namely the 63 kda hemagglutinin (ha), the 56 kda nucleoprotein (np), and the 50 kda neuraminidase (na), respectively (table 1 , fig. 1 ). the two smallest segments, 7 and 8, encode each two proteins, the 28 kda matrix protein (m1) and the 11 kda membrane protein (m2), and the 27 kda ns1 and the 14 kda ns2 proteins, respectively (table 1 , fig. 1 ). in common with the bunyaviridae, the genomic rna is packed in the nucleoprotein which carries polymerase subunits, and the 5 0 and 3 0 -ends are conserved and complementary to each other and thus able to form panhandle structures in which the promoter regions reside. ''cap-snatching'' from cellular mrnas and an oligo-u motif are used to create viral mrnas starting with a cap structure and ending in a poly-a region. the virus enters the cells after binding to the sialic acid receptors by endocytosis, which is followed by acidification of the endocytic vesicle and, mediated by the ion channel forming m2 protein, of the interior of the virus, which leads then to fusion of the viral and endosomal membrane and release of the viral nucleocapsids to the cytoplasm, respectively. however, untypically for an rna virus, the replication, transcription and nucleprotein assembly occur in the nucleus. the envelope proteins are processed to the plasma membrane, where budding of the virions finally occurs. the envelope proteins are also the most important antigenic determinants. the homotrimeric hemagglutinin (ha), defines the ''h type'' which is responsible for the binding to the sialic-acid containing host cell receptors and membrane fusion properties. the neuraminidase (na) defines the ''n type'', and cleaves terminal sialic acid residues from glycoconjugates enabling the virus to reach target cells in the mucin-rich epithelium and facilitating release of the virus from the cells [93, 94] . the catalytic site of the neuraminidase is a target for antivirals oseltamivir and zanamivir, and the m2 protein is a target for amantadine and rimantadine. to become active, the hemagglutinin protein needs to be cleaved by trypsin-like proteases found in respiratory and gastrointestinal epithelia. human-adapted influenza viruses replicate in the respiratory tract, whereas in avians the virus replicates primarily in the gut. when transmitted to and within poultry, the normal hemagglutinin of influenza a virus h5 or h7 of wild aquatic birds of the ''low pathogenic type (lpai) may be mutated. accumulation of basic residues at the cleavage site makes the ha cleavable by most proteases of cells, such as furin, and results in ''highly-pathogenic avian influenza'' (hpai) virus able to replicate in most tissues killing rapidly up to 100% of chickens [93] [94] [95] . hpai, also known as ''fowl plague'' was first described in 1878 and the virus was first isolated in 1901 suggesting that humans have been directly exposed to avian influenza a viruses for centuries [96] . influenza a virus gene pools reside in wild aquatic birds where at least 15 ha types and 9 na types are found, as compared to 3 ha (h1-h3) and 2 na (n1, n2) types circulating in man. also, the genes in aquatic birds are in evolutionary stasis without undergoing changes due to selective pressures. influenza a viruses in man are under constant selective pressure imposed by population immunity causing the hemagglutinin of influenza a virus to change its antigenic properties by accumulation of mutations (genetic drift). however, through double infection and reassortment (genetic shift), novel (e.g. hemagglutinin) genes from aquatic birds may become established in human influenza viruses giving rise to pandemics due to lack of adaptation to and immunity in, humans. previously, it was thought that the different receptor specificity -favoring different side chains of sialic acid -of human and avian influenza viruses would make direct transmission of avian viruses to humans unlikely, but both could be expected to infect swine, which carry receptors for both. thus pigs are considered to be potential reservoirs for generating new influenza virus variants. it has only recently been discovered that viruses considered unique to avian species may also infect man, although this may involve change in receptor specificities [97] . in 1996 conjunctivitis in uk caused by avian influenza viruses (and previously, by seal influenza a viruses) was reported [98, 99] (table 2) . direct zoonotic transmission of avian influenza viruses to man resulting in human respiratory illness, was not known to occur or had not been diagnosed before the outbreak of h5n1 avian influenza virus in hong kong in 1997 where 6/18 patients died of lower respiratory tract infection [100] . after this, in europe, an outbreak of h7n1 hpai avian influenza was encountered in italy (1999) (2000) without reported transmission to humans [101] . in 2003, a major h7n7 hpai avian influenza outbreak occurred in the netherlands [102, 103] . during this epidemic, veterinarians and people who culled infected poultry were at greatest risk of infection. it was noted by active surveillance that human h7 infections (and simultaneous h3 infections) were occurring, and consequently prophylactic treatment with oseltamivir was started. the h7 virus was confirmed to be transmitted to 85 humans of which the majority had conjunctivitis, seven had an influenza-like illness; one veterinarian (who did not receive prophylactic oseltamivir) died. his symptoms started with high fever and headache two days after visiting a farm with infected chickens. one week later, he was admitted to hospital with pneumonia, where his status deteriorated to multi-organ failure, with death due to respiratory insufficiency 2 weeks after onset of symptoms. in addition, in three cases household primary contacts were shown to have the disease by human-to-human transmission [102] . both na inhibitors, zanamivir and oseltamivir inhibited virus obtained from humans during this outbreak and a significant difference was found in avian influenza virus detection between oseltamivir users (1/38) and those who had not taken prophylactic medication (5/52) [102] . outbreaks of avian influenza have continued to occur in other parts of the world, especially the devastating ongoing h5n1 epidemic in south-east asia since late 2003 with as of june 2005 a total of 54 deaths/107 cases in viet nam, thailand and cambodia. the epidemic has had a dramatic impact on poultry farming and industry with million birds dying or being destroyed to reduce virus dispersal. this ongoing epidemic clearly has ''pandemic potential''. avian influenza viruses may be detected by virus isolation (in cell culture or embryonated eggs) or rt-pcr which may be targeted at the specific ha subtype or may be generic to influenza a viruses (e.g. targeting the matrix protein gene) [104] . also, serological tests such as. hemagglutination inhibition may be used. typing may be based on serological methods (such as hemagglutination inhibition), specific primers/probes or sequencing. many commercial influenza a antigen tests detect the nucleoprotein or na activity and should be applicable to the avian viruses, although this is poorly studied and documented. furthermore, it should be noted that rt-pcr from throat swabs of the lethal case in the netherlands were negative [102] . avian influenza, or ''fowl plague'' outbreaks usually arise following contact with wild aquatic birds, such as mallards and ducks, in which the virus replicates as a rule without causing symptoms. the virus that is excreted in the gut can typically be isolated (or detected by rt-pcr) from cloacal swabs. hinshaw et al. [105] studied over nine thousand birds and detected 30% prevalence in young and 11% prevalence in adult birds. influenza virus is readily transmitted in cold environments and is stable for 126-207 days at +17â°c, or in wet faeces (as shown for h5n1 virus) for at least 4 days at 25â°c and more than 40 days at +4â°c [106, 107] . the main preventive and control measures include proofing the chicken breeding facilities against wild birds. raising chickens and turkeys in the open is a risk, minimizing secondary spread of outbreaks by stamping out the infected poultry, followed by cleaning, disinfection and controlling movements of humans and animals, trade embargoes and reporting the outbreaks (''fowl plague'' is in the top priority ''list a'' of the international animal health code of the office international des epizooties). selling poultry live, a common practice in south-east asia, is a definite risk factor. vaccination of poultry has been used as an additional control measure but may lead to undetected shedding and transmission of mutant virus selected under the pressure imposed by the vaccine [94] . to prevent further transmission to humans, rapid measures are needed due to the short incubation period. the dutch experience suggests that results can be achieved by personal protection (e.g. protective eye glasses, masks) for all workers who screen and cull poultry, vaccination with regular inactivated influenza virus vaccine and prophylactic oseltamivir for those handling potentially infected poultry, to be continued for 2 days after last exposure [102] . for humans, specific vaccines containing h5 and h7 antigens would be welcome. the only known zoonotic agent of the togaviridae family to cause human disease in europe is the mosquito-borne sindbis virus (sinv), in the genus alphavirus. sinv is distributed throughout the old world and australia and causes rashes and polyarthritis outbreaks in northern europe, similar to chikungunya, oã�nyong-nyong virus and ross river virus in far east asia, africa and australia, respectively, whereas other alphaviruses causing encephalitic infections in man (venezuelan, western and eastern equine encephalitis virus) are found in the new world. alphaviruses are enveloped, positive-strand rna viruses with an 11 kb genome. the non-structural proteins (nsp1-4) are encoded from the 5 0 -terminus of the genome, and a separate subgenomic 26s rna from the 3 0 -end is used as a messenger for the structural proteins: the 30-33 kda capsid protein (c), and the 45-58 kda envelope glycoproteins (e1 and e2) [94] (table 1 , fig. 1 ). sinv was first isolated in the nile delta in the 1950s from a pool of mosquitoes without knowledge of any disease association. it is now known to be the causative agent of mosquito-borne epidemic polyarthritis with accompanying rashes and when described in northern europe it was given the names ockelbo disease, pogosta disease, or karelian fever when found in sweden, finland and russia, respectively. most infections occur during august-september, and larger outbreaks tend to appear with a seven-year interval (e.g. in finland, 1282 serologically verified cases occurred in 1995, 600 cases in 2002; and in sweden 50-65 cases are reported during peak years) [108, 109] , with a peak incidence (56%) in 50-year old females. the association of sinv with pogosta disease was first discovered in the early 1960s and it is believed that the virus may have been distributed throughout northern europe around this time. this conclusion is based on the evidence that thousands of human and bird sera collected during the early 1960s in finland, were negative for sinv antibodies, whereas in the 1990s 2-5% of the population were sinvantibody positive [108] . antibodies to sinv without polyarthritis outbreaks have been recorded in italy, romania, greece and the former yugoslavia [109] . however, human disease due to sinv has been recorded in south africa, from where it may have originated. sinv was isolated from mosquitoes in sweden, norway and russia in the early 1980s [109] [110] [111] and sinv antibodies are found in wild tetraonic and migratory birds, most commonly (in sweden) from passeriformes such as redwing (turdus iliacus), fieldfare (turdus pilaris), blue tit (parus caeluleus), chaffinch (frinigilla coelebs), songthrush (turdus philomelos); thrushes have been suggested to be a major candidate as an amplifying host [109, 112] . in addition, antibodies are commonly found in galliformes [109, 108] . phylogenetically, sinv strains are similar in northern europe and south africa (in the north to south dimension), but differ considerably from strains circulating in asia and australia, where sinv-associated rash-arthritis is not recorded. this is consistent with the notion of a north-south dispersal of sinv strains by migratory birds [113] . several bird species can be infected experimentally [114] . it is evident that sinv cycles between birds and ornithophilic (culex and culiseta) mosquitoes (table 2) . however, it may spill over to other hosts (evidence from many vertebrates from a frog to a bear) and vectors (including ticks and aedes mosquitoes) [109] . recently, during an outbreak in finland in 2002, the causative agent of pogosta disease was isolated for the first time in europe from skin biopsies and a blood sample of patients [115] ; the virus strains were most closely related to sinv strains isolated from mosquitoes in sweden and russia 20 years previously. the incubation period for the disease is about one week and the onset is accompanied by arthritis/arthralgia and itchy rash as the dominant symptoms, and also fatigue, mild fever, headache and muscle pain. hematological laboratory parameters are within the normal range and levels of c-reactive protein (crp) are not elevated. the rash is usually located on the trunk and thighs and lasts for a couple of days. one third to a half of patients, suffer from joint pains for more than 12 months [116, 117] . usually several joints are affected, the most common being the ankle, wrists, knee, and finger joints (50% or more of patients), as well as hip, shoulder and elbow joints [118,119a,119b ]. diagnosis is based on serology using enzyme immunoassay, immunofluorescence assay or hemagglutinationinhibition, and detection of seroconversion or a 4-fold rise in titre between two samples, or positive igm in a single sample. the first sample is usually taken during the first week after onset of illness, another sample is required to diagnose or exclude sinv infection; igm antibodies are detectable until approximately 6 days post-onset, and igg antibodies can be detected approximately 10 days after onset of illness [119b,120] . in some cases persisting igm antibody can be detected years after infection [117] . for research purposes, the virus can be detected by 121] or isolated from skin biopsies [115] . no specific preventive measures are available, apart from avoiding mosquito bites. flaviviruses comprise a diverse group of pathogens that have been traditionally classified as arthropodborne viruses. they are linear positive single-stranded rna viruses with a monopartite genome (10-11 kb) that encodes 3 structural proteins: the 13 kda capsid protein (c), the 51-59 kda major envelope protein (e) and the 8.5 kda glycoprotein m (in mature virions); and 7 non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5) (table 1, fig. 1 ). noncoding or untranslated regions flank the infectious rna genome. when seen in the electron microscope flaviviruses appear as uniform spherical particles, 40-60 nm in diameter. the virus particles consist of a lipid envelope that has a surface covered by protrusions that contain envelope (e) and membrane (m) structural proteins, organized as dimers. this envelope surrounds an isometric capsid protein of approximately 30 nm in diameter [122] [123] [124] . there are currently about 70 members in the family flaviviridae [125] which have been found infecting a wide variety of organisms including mammals, arthropods, avian and amphibians. many of these viruses are major pathogens of humans, domestic and farmed animals as well as wildlife species. with the possible exception of the dengue viruses, the flaviviruses are zoonotic, depending almost entirely for their existence on wildlife vertebrate and in many cases, invertebrate species. the type species of the genus is yellow fever virus (yfv), hence the term ''flavi'' from the latin word flavus, which in turn describes the yellowish color of the skin in yellow fever infections [126] . the classification, and serological and phylogenetic studies of flaviviruses reflect the importance of the vector on the biology and evolution of this genus. there are essentially three groups of flaviviruses: tick-borne, mosquito-borne and non-vectored flaviviruses, although this grouping is, to some extent, arbitrary since some mosquito-borne viruses are also transmitted by ticks and vice versa [127] (table 2 ). phylogenetic analysis also shows very strong correlations between genetic relationships, epidemiology and ecology of these viruses [128, 129] . some flaviviruses are responsible for a significant proportion of the morbidity and mortality that is registered annually worldwide. they cause epidemic outbreaks that involve encephalitis and/or haemorrhagic fever, often fatal and involving millions of humans or in some case birds or mammals. important flaviviruses affecting humans are the dengue viruses, yellow fever virus, west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus, saint louis encephalitis virus, and murray valley encephalitis virus among others. dengue virus alone causes more than 50-100 million cases worldwide each year and some 2500 million people are now at risk from dengue infections [130] . the most important flavivirus in europe is tbev, which is endemic in many european countries, and also in russia, ( table 5 , fig. 4 ) northern china and northern japan [126,131a] . it affects thousands of people annually and has a significant impact on public health. the virus is transmitted to humans mainly through a tick bite, however, the infection has also been reported to occur by drinking unpasteurised goat milk from viraemic animals [131b,131c]. the virus is maintained in nature in a cycle involving ticks and wild vertebrate hosts and also by transovarial and transstadial transmission in its vector [126, 132] . serological evidence and viral isolations, as well as sequence similarities have suggested that migratory birds could also play a role in the transmission of tbev from central europe to scandinavian countries; moreover, endemic areas of tbev are regions of high migratory bird activity [133] . tbev is classified taxonomically into three subtypes: european subtype, far eastern subtype, and siberian subtype. the first subtype is transmitted mainly by ixodes ricinus, and the last two by ixodes persulcatus [134, 135] . the distribution of tbev is well coordinated with the distribution of its vector, furthermore, different genotypes are located in distinct geographical areas and associated with specific vector hosts. recent data have shown the co-circulation of all three subtypes of tbev in the same geographical region, specifically in latvia, where the two vector ixodes species habitats meet [136] . however, the endemic region in europe is patchy and covers only part of the geographical range of e.g. ixodes ricinus. there has been an increase of the incidence of tbev in many of its endemic areas but not in austria where a countrywide successful vaccination campaign was established reducing the disease incidence to lower levels [131c,131d,137a]. tbev affects principally the nervous system and can cause several clinical features of different severity table 5 tick-borne enchephalitis viral infections in europe per country through time year 1990 year 1995 year 2000 year 2002 austria 89 109 60 60 belarus -a 66 23 18 croatia 23 59 18 30 czech r. 193 744 719 647 denmark --3 1 estonia 37 175 272 90 finland 9 23 41 38 france 2 6 0 2 germany -226 133 including meningitis, meningoencephalitis, meningoencephalomyelitis and meningoradiculoneuritis. hospitalization varies from days to months and in some cases years of treatment and rehabilitation are necessary as sequelae occur in approximately 1/3 of the patients. the incubation period of tbe is between 7 and 14 days. the disease is characteristically biphasic. the first phase (day 2-4 of onset of symptoms) is viraemic and can be either asymptomatic or fever, malaise, headache, anorexia, nausea, and muscle pains may be present. the second phase occurs in up to 30% of the patients after about 8 days lag period (approximately 21 days after the tick-bite) as a neurological disease of which about 0.5% has a fatal outcome [137b,137c,137d]. the neurological symptoms and severity vary: the clinical picture includes meningitis (in about half of the patients), meningoencephalitis, meningoencephalomyelitis and meningoradiculoneuritis. hospitalization varies between days and months and in some cases years of treatment and rehabilitation are necessary in case of e.g. paresis. altogether, neuropsychiatric sequelae occur in approximately 1/3 of the patients. louping ill virus (liv) is endemic in ireland, in the northern region of great britain and in norway (fig. 4) affecting principally sheep with a disease that is known as ovine encephalomyelitis, infectious encephalomyelitis of sheep, or trembling-ill. liv is transmitted to sheep by the tick vector ixodes ricinus. the natural life cycle of liv resembles that of tbev, it can be sustained in the natural environment through non-systemic transmission of virus between ticks cofeeding on rodents and other wild animals, which in turn infect grazing animals such as sheep and goat as a zoonotic disease. louping ill virus has also been observed in a bird-tick-bird cycle involving the red grouse (lagopus lagopus scoticus), ptarmigan bird species and the ixodes tick [138] . infection with liv in sheep is characterised by a biphasic fever, depression, ataxia, muscular in-coordination, tremors, posterior paralysis, coma, and death. there is evidence for infection by liv in other domestic species and wildlife, i.e. cattle, horses, pigs, dogs, deer, shrews, wood-mice, voles, and hares [139,140a] . it is generally believed that the majority of avian infections occur through a tick bite, however laboratory experiments and field observations have demonstrated that the red grouse can also become infected by feeding on infected ticks indicating that the vector bite is not the only route of infection [140b] . humans are also susceptible to infection with liv. however, the majority of the cases reported are accidentally acquired infections in laboratory workers. the second most frequent infection results from handling infected animal carcasses. infection with liv in humans can cause a neurological disease resembling the clinical picture observed for tbev infections, i.e. biphasic encephalitis, influenza-type illness, fever, articular pain, meningitis, myagia and poliomyelitis-like illness [141] . other possible transmission routes for liv infection to humans include drinking contaminated milk from goat or sheep in an acute phase of infection [142] , tick-bite, exposure to infective material, or through skin abrasions or wounds. antigenic and phylogenetic studies have shown that liv is most closely related to strains of the western european subtype of tbev and it is estimated to have emerged from this lineage approximately 800 years ago [143] . west nile virus (wnv) was first discovered in 1937 in uganda [144] . it occurs throughout africa, the middle east, europe, russia, india and indonesia and was recently introduced into north america (new york) [126, 145] . in the old world wnv is primarily considered to be an african virus which annually disperses northwards out of africa when birds migrate to europe, the middle east and asia [145] . in humans, the majority of wnv infections cause a nonsymptomatic or mild febrile illness; however some infections can cause encephalitis and in most severe cases can lead to death, particularly in elderly patients. the incubation period is between 3 and 15 days after a mosquito bite (http://www.cdc.gov/ncidod/ dvbid/westnile/wnv_factsheet.htm). west nile virus is an illustrative example of the human impact on the dispersal and evolution of flaviviruses. the virus appeared for the first time in the usa, in new york in 1999 causing sixty-two confirmed human infections and seven deaths [146] . the virus successfully over-wintered and during the next years dispersed widely throughout north america and now more recently also to central america and the caribbean. in north america the virus infects a very wide range of mosquito and animal species. the exact mechanism of introduction into north america is not known [146-148a] . to date, more than 14 000 human cases and 586 deaths from wnv have been reported in the united sates of america (http:// www.cdc.gov/ncidod/dvbid/westnile). in europe, outbreaks caused by wnv have been recorded since the early 1950s, especially in mediterranean countries, romania and southern russia (fig. 4) . larger outbreaks (over 800 cases) have been reported since the mid 1990s in urban settings, especially a large outbreak in bucharest, romania in 1996 [145a,145b,145c] . in southern russia, specifically in volgograd, astrakhan and krasnodar regions, wnv has caused large epidemics of approximately 1000 human cases, with 4% mortality rate of reported cases. molecular epidemiological studies have shown that the latest large outbreaks in volgograd were caused by strains genetically similar to that of romania-1996 , kenya-1998 and newyork-1999 reflecting the widespread distribution capacity of these epidemic viral strains [149b,151b]. the incidence of wnv in europe is largely unknown. phylogenetic studies have showed that wnv has diverged into two main lineages, which form internal clusters or clades [149c,150a,150b,150c]. more recently, a novel virus (rabensburg virus), antigenically and genetically closely related to wnv was isolated from a culex pipens mosquito pool in czech republic. this new virus could represent a third lineage of wnv, however more studies are needed to confirm this hypothesis or to determine whether or not this could represent a new member of the flaviviridae family [151a] . usutu virus (usuv) was first isolated from a mosquito in africa in 1959 [148c]. it was characterised serologically and classified within the japanese encephalitis virus serocomplex. usutu virus had rarely been isolated since then, with only one reported human case and being present only in two regions of africa. however, in the late summer of 2001, usuv was found in vienna in austria during a study of an avian epidemic characterized by fatal encephalitis that was thought to be caused by wnv. the virus caused die-off among the bird population, especially blackbirds, in vienna (fig. 4) and when isolated from birds and mosquitoes it showed 97% genetic identity to the african usuv [148b]. this is the first time usuv has been observed outside africa and also the first time it has been associated with fatal disease in animals. the virus re-appeared in the late summer of 2002 in the same region of vienna [149a] . most recently, in 2003 the virus has been isolated from humans (associated with rash in one patient) and from birds in austria and evidence exists as to the virus being able to overwinter establishing itself in this geographical area (nowotny, n. second european congress of virology, eurovirology 2004). as mentioned above the distribution of flaviviruses is worldwide, and we have described the zoonotic flaviviruses endemic to europe. however, it is important to note the increasing incidence of imported flaviviruses totaling over 500 cases mainly due to changes in human behavior, such as increased travel to non-european destinations (http://www.eurosurveillance.org). the dengue viruses (denv) are a world-wide public health problem. they are transmitted to man by mosquitoes, particularly aedes aegypti and cause a wide range of different clinical outcomes [151c,152a] . it is estimated that 100 million cases of dengue fever (df) and 250 000 of dengue hemorrhagic fever (dhf) cases occur annually [126] . in a study based on the epidemiology and clinical course of 294 travellers with symptoms of dengue infection it was found that the incidence of df among european travellers is underestimated, since diagnostic procedures, in general, for febrile patients often do not include tests for tropical arthropod-borne diseases, such as dengue virus [152b] . there have also been some imported cases of yfv to europe mainly by tourists travelling from endemic areas. however, the incidence of yfv as an imported disease is much lower than that of dengue infections (http://www.eurosurveillance.org) presumably because of the different nature of these diseases, denv having spread worldwide throughout the tropics and yfv occurring only in its endemic regions in africa and south america [152c]. traditionally, diagnosis of flaviviruses in europe has been restricted to detecting tbev infections through serological assays; igm-capture enzyme immunoassay, hemagglutination inhibition-, and immunofluorescence assay. igm antibodies in serum, and in some cases in cerebrospinal fluid during the neurological disease usually provide the diagnosis. rt-pcr is rarely positive at the second phase of the disease [153a]. because of the close antigenic relatedness of flaviviruses, laboratory findings can easily lead to misdiagnosis [153b]. in a study conducted in hungary, tbev-positive serum panels were tested retrospectively against wnv and some sera were found to show higher titres to wnv than to tbev when compared in serological assays (e. ferenczi, personal communication). as mentioned above, a study including travellers returning to europe from tropical destinations proved that many cases remain undetected. there is a need to improve the diagnosis for flaviviruses in order to determine the true incidence and prevalence of these infectious diseases in europe, and to study the ecology of imported viruses to determine which viruses have been able to establish themselves in the continent and which are still continuously being imported from africa or asia. meanwhile anti-vector campaigns and vaccination for travellers to endemic areas are the only measures to prevent and control these infectious diseases [146] . there are effective vaccines for tbev and yfv but a protective immunogen does not exist for denv or wnv. tbev vaccines from two commercial manufacturers (baxter and chiron) are available and widely used, especially in austria and germany, both are based on formalin-inactivated virions, three injections are needed for full protection. booster immunizations are recommended every 3-5 years for people living or spending holidays in endemic areas. 3.3. nairoviruses: crimean-congo hemorrhagic fever virus the genus nairovirus (family bunyaviridae) is composed of 34 predominantly tick-borne viruses that have been divided into seven serogroups [154] including several associated with severe human and livestock diseases (especially crimean-congo hemorrhagic fever virus (cchfv) and nairobi sheep disease virus). of the pathogenic viruses only cchfv causes significant human morbidity and mortality and is found in europe. like other members of the bunyaviridae family nairoviruses possess a tripartite single-stranded rna genome of negative polarity consisting of large (l), medium (m) and small (s) segments. the 12 kb l segment encodes an rna-dependent rna polymerase (deduced size 448 kda in cchfv), the 4.9 kb m segment encodes the precursor for the two envelope glycoproteins gn (75 kda in cchfv) and gc (37 kda in cchfv), and the 1.7 kb s segment the viral nucleocapsid n (50 kda) ( table 1 , fig. 1 ). like with other members of the bunyaviridae viral messenger rnas (mrna) are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. messenger rnas have 5 0 -methylated caps and 10-18 nontemplated nucleotides which are derived from host cell mrnas. the nairovirus l segment encoding the rna polymerase is conspicuously large, almost twice the size of many other members within the family bunyaviridae, and may thus provide other functions such as viral helicase and a papain-like cysteine protease activity predicted from the nucleotide sequence [155, 156] . viral protein analysis has suggested that gn and gc may be derived from 85 and 140 kda precursors, of which the latter contains an n-terminal region with a highly o-glycosylated mucin-like domain [157] . the cchfv nucleocapsid protein colocalizes and interacts with human mxa protein; this interaction may explain the antiviral effect of interferons on cchfv [158] . similar to the hantavirus-rodent association, the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts [159] . within cchfv isolates, comparison between m and s segment phylogenetic groupings suggests that reassortment events have occurred in some virus lineages [160] . reverse genetics, recently established for cchfv [161] , provides a unique opportunity to study the biology of nairoviruses and tailor optimal therapeutic and prophylactic measures against cchfv infections. cchfv is transmitted most efficiently by hyalomma ticks, followed by rhipicephalus, dermatocentor spp and many other species of ixodid (hard) and some argasid (soft) ticks (table 2) . among ticks cchfv is capable of transmitting infection both transovarially and transstadially. the life cycle of cchfv also includes a tick-vertebrate host cycle involving both wild and domestic animals. the virus or antibodies against it have been detected in rodents, hares, hedgehogs and some birds but human infections seem to be principally from contacts with livestock (mainly cattle, sheep, goats) including in africa farmed ostriches and less often ticks [162, 163] . thus cchfv is more commonly seen in persons exposed to blood and tissues of infected animals during occupational activities, such as farming, herding, veterinary examination and abattoir work. nosocomial infection is common and often results in small outbreaks. yet, outdoor and recreational activities also represent a risk factor. in addition, horizontal transmission of cchfv from mother to child may occur [164] . the global distribution of cchf follows closely that of hyalomma spp. in the middle east, asia, africa and southeast europe [162, 163] . sequence analysis of cchfv s rna segments gives patterns following links between different geographic locations, in some cases suggesting links originating from trade in livestock and long-distance carriage of virus or infected ticks during bird migration [165] . in europe, cchf occurs in the balkan peninsula (albania, bulgaria, greece, turkey, yugoslavia) but also in southern russia, hungary, france and portugal [162, 166, 167] . the symptoms and signs of crimean-congo hemorrhagic fever are similar to those of other viral hemorrhagic fevers [162, 163] . the incubation period is generally short ranging usually from 2 to 9 days. there is typically a very sudden onset of illness with fever, rigors, chills, intense headache and backache or leg pains, myalgia, nausea, and vomiting. patients may also present with photophobia, somnolence and menigism with confusion or aggression. hemorrhages in the form of petecchiae, ecchymoses, epistaxis, melena and bleeding from various organs usually begin a few days later. tachycardia is common and lymphadenopathy is seen occasionally. the case-fatality rate is 20-35%. the pathology consists of hemorrhagic and necrotic lesions in various organs as well as fibrin deposits. the methods used for detection of cchfv infection in humans or livestock include indirect immunofluorescence on virus-infected or n-expressing transfected cells. both igm and igg antibodies reactive for cchf appear within a week in patients [162, 168] . alternatively a recombinant nucleocapsid protein-based enzyme immunoassay [169] [170] [171] may be used. rapid detection and quantification of cchfv rna is possible by real-time reverse transcription pcr [172] . ribavirin is clearly effective in treatment of crimean-congo hemorrhagic fever [173, 174] but supportive treatment (blood, thrombocytes, coagulation factors) is equally important. immune plasma from recovered patients has also been used to treat patients but not in controlled studies with a follow-up of virus-neutralizing activities [162] . the most effective method of preventing infections is to take measure to avoid exposure to ticks (protective clothing, tick repellent, frequent body searches to remove ticks). inactivated virus prepared in mouse brains has been used on a limited scale as a vaccine in southeastern europe and the former ussr but the sporadic and unpredictable occurrence of the disease renders it difficult to identify target populations and has slowed development of a safe and modern vaccine [162] . orthobunyaviruses belong to the family bunyaviridae. these enveloped viruses have a three-segmented negative-strand rna genome. the 0.97 kb s segment encodes the 19-25 kda nucleocapsid protein (n) and a 10-13 kda nonstructural protein (ns); the 4.5 kb m segment encodes the polyprotein precursor of two glycoproteins, g1 and g2 (108-210 kda and 29-41 kda, respectively), and a nonstructural protein nsm (15) (16) (17) (18) ; and the 6.9 kb l segment encodes the rna polymerase (260 kda) ( table 1 , fig. 1 ). viral messenger rnas are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. messenger rnas have 5 0 -methylated caps and some nontemplated nucleotides which are derived from host cell mrnas. orthobunyaviruses are transmitted to humans by a variety of mosquito species, and in europe two known human pathogenic representatives of the genus circulate, tahyna virus and inkoo virus (mostly transmitted by aedes spp.) [109, 175, 176] (table 2 ). these viruses belong to the california serogroup, and their incidence is associated with the distribution of the carrier mosquitoes. the natural cycle of these viruses involve also mammal hosts. tahyna virus circulates throughout most of europe, whereas inkoo virus is mainly reported from northern europe [109,177a] . tahyna virus causes an influenza-like disease which may also present as a meningitis. the infection caused by inkoo virus is mostly sub-clinical or a mild disease, although encephalitis has been reported. the most typical symptoms associated with encephalitis or meningitis caused by californian serogroup viruses include fever, headache, nausea, vomiting, convulsions and confusion. in europe the seroprevalence for californian serogroup viruses varies from 1% to 69%, depending on the geographic area [109] . in finland the seroprevalence against inkoo virus varies from 2% to 6% (ã� land islands) to 61-69% (finnish lapland) [109, putkuri et al, unpublished results] . in disease-endemic areas the seropositivity of the population increases with age [177b]. the diagnosis of orthobunyaviruses is based on serology, either as a rise in igg-antibody titers, or the presence of igm antibodies. also rt-pcr methods are under development to detect viral rna in cerebrospinal fluid samples of patients with encephalitis. phleboviruses are one of the five groups within the family bunyaviridae. they have a negative singlestranded rna tripartite genome, namely: l (6.4 kb), m (3.2-4.2 kb) and s (1.7-1.9 kb). the l segment encodes a single protein of 240 kda (rna polymerase) in the complementary sense. the m segment encodes a protein precursor of 130 kda in the complementary sense which is cleaved into two glycoproteins g1 (56 kda) and g2 (59 kda). the s segment has an ambisense coding arrangement, and it encodes the nucleocapsid protein (n) of 27 kda in the complementary sense and a non-structural protein (nss) of 30 kda in the virion sense [178, 179] (table 1, fig. 1 ). viral messenger rnas (mrna) are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. mrnas have 5 0 -methylated caps and some nontemplated nucleotides which are derived from host cell mrnas. when seen under the electron microscope the virions are spherical and enveloped and approximately 80-110 nm in diameter. glycoprotein projections of 5-10 nm are also seen embedded in the membrane. there are between 200 and 1500 spikes per virion [178, 179] . phleboviruses are arthropod-borne viruses and are transmitted mainly by phlebotomine sandflies, hence the derivation of its name ''phlebo'' (table 2) . however, mosquitoes and ticks can also transmit phleboviruses. phleboviruses are distributed throughout most of the world but have not been reported in australia [180] . the first documented record of a disease resembling those caused by phleboviruses dates back to the 1900s in the mediterranean countries of europe [178] . however, the first virus isolation occurred for rift valley fever virus in 1930 in kenya [181] . retrospective serological studies have indicated that outbreaks caused by phleboviruses have occurred since 1912 in the sub-saharan africa region [182] . phleboviruses are major causal agents of encephalitis in humans, especially among children, in mediterranean countries. they also have an important impact in livestock especially in african endemic areas. infection of livestock is characterised by a high rate of abortions and pathologies associated with hemorrhagic illness (leukopenia, thrombocytopenia, fibrin thrombi, intravascular coagulopathy, etc.) [183] [184] [185] . the distribution of the disease follows that of its vector. however the natural history of phleboviruses is largely unknown and for many species the amplifying host has not been identified [185] [186] [187] . in general, phlebovirus infections cause a 2-4 days illness characterised by an incubation period of 2-6 days followed by fever, general malaise, headache, photophobia, and back and joint pain [188] . the illness occurs during the summer months when the activity of phlebotomine flies is high [178] . there are three recognised members of the genus phlebovirus, associated with disease in humans. rift valley fever virus (rvfv), which is the type species of the genus and is transmitted by mosquitoes, causing an influenza-like disease that affects domestic animals and humans. the majority of human infections are asymptomatic; however, in 0.5% of cases it can cause severe haemorrhagic fever, encephalitis and death. the geographical distribution of rvfv includes africa, egypt, saudi arabia and yemen and occurs as epizootics involving aedes spp. mosquitoes and domestic animals [180] . toscana virus (tsv) causes a mild influenza-like disease that can occasionally develop into an acute neurological disease, such as meningitis or meningoencephalitis in italy and possibly other mediterranean countries. a similar virus (granada virus) was recently isolated in spain [193] . tsv is the number one cause of encephalitis among children in italy. the distribution of the virus follows that of its vector, phlebotomus perniciosus, found in italy, spain, portugal, and cyprus. human cases have also been reported from southern france and greece. the virus replicates in the sandfly vector population; however, experimental evidence suggests that an amplifying vertebrate host is needed to sustain the virus in the arthropod population [189] [190] [191] [192] [193] [194] . sandfly fever virus (sfv) sicilian and naples viruses can cause a mild-influenza like disease. the distribution of sfv also follows that of its vector, phlebotomus papatasii, found in the mediterranean basin extending to the middle east and arabian peninsula, the caucasus mountains, pakistan and india [185] [186] [187] . diagnostics of phlebovirus is performed mainly by igm-capture eia and ifa. the virus can be isolated using intracranial inoculation in suckling mice or using a susceptible tissue culture cell line (vero, llc-mk2, bhk-21) [178,195a] . toscana virus and naples sandfly fever virus group together and are distinct antigenically and phylogenetically from sicilian sandfly fever virus [195b] . prevention of infections by phleboviruses varies depending on the type species. immunising livestock with the formalin-inactivated or live-attenuated vaccines can prevent rvfv by avoiding epizootics and hence human infections [178, 196] . in the case of tsv and sfv, prevention is limited to controlling the vector population through insecticides and repellents [197, 198] . what are the reasons for the appearance of all these emerging and re-emerging viruses? have changes taken place in the viruses themselves? apparently not. the principal reasons are changes in the environment and human activity that have created new attacks with nature. factors such as climate change, increasing intensity of agriculture, the building of dams and introduction of other irrigation measures which generate new breeding sites for mosquitoes, crises and wars which bring rodents and arthropods closer to humans, population growth and especially in suburban slums, and global air traffic are important factors generating such new contacts, or may affect the ecological cycles of these viruses. many epidemics may be unexpected outcomes of such changes. moreover, the new molecular biology techniques facilitate detection of new viral agents. many prevention and control measures, such as the anti-aedes campaigns, have been abandoned in tropical countries. the yellow-fever vaccination campaigns have 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structure and comparison with hantaan virus using negative stain electron microscopy phylogenetic relationships among members of the genus phlebovirus (bunyaviridae) based on partial m segment sequence analyses climate and satellite indicators to forecast rift valley fever epidemics in kenya evaluation of a new rift valley fever vaccine: safety and immunogenicity trials immunization against rift valley fever virus. studies of the immunogenicity of lyophilized formalin-inactivated vaccine zoonotic transmission of hepatitis e virus from deer to human beings key: cord-016095-jop2rx61 authors: vignais, pierre v.; vignais, paulette m. title: challenges for experimentation on living beings at the dawn of the 21(st) century date: 2010-06-08 journal: discovering life, manufacturing life doi: 10.1007/978-90-481-3767-1_5 sha: doc_id: 16095 cord_uid: jop2rx61 “we can talk endlessly about moral progress, about social progress, about poetic progress, about progress made in happiness; nevertheless, there is a type of progress that defies any discussion, and that is scientific progress, as soon as we judge it within the hierarchy of knowledge, from a specifically intellectual point of view.” introduction of new exploratory methods such as biocomputing or bioinformatics and high-throughput screening, which involves the simultaneous processing of hundreds or even thousands of samples. this approach is in contrast with traditional biology, in which the research strategy is based upon the observation of effects obtained as a function of experimental parameters that are modified one by one. another aspect of modern times is that, with the irresistible trend in genetic manipulation towards a focus on human beings, certain areas of fundamental research are finding themselves locked into philosophical dilemmas that are matter for ethical and sociocultural consideration, and the subjects of fierce debate. instead of setting out to discover unknown mechanisms by analyzing effects that are dependent on specific causes, with some uncertainty as to the possible success of the enterprise being undertaken, which is the foundation stone of the bernardian paradigm of the experimental method, many current research projects give themselves achievable and programmable objectives that depend upon the means available to them: sequencing of genomes with a view to comparing them, recognition of sequence similarities in proteins coded for by genes belonging to different species, with the aim of putting together phylogenetic trees, synthesis of interesting proteins in transgenic animals and plants, analysis of the three-dimensional structure of proteins, in order to find sites that are likely to fix medicinal substances, and synthesis of molecular species able to recognize pathogenic targets. the facilities that are called into play include instruments that are often sophisticated, the performance of which, in terms of miniaturization, computerization and robotization, is far beyond that of apparatus that was in use a few decades ago. these facilities, applied to research into living beings, have entered the framework of a methodology that has been given the label biotechnology. proceeding handin-hand with applications that have become more and more meaningful in the domains of medicine, pharmacology, agronomy and animal husbandry, the biotechnological process has come to the fore as a new paradigm for the experimental method as applied to living beings. in addition to new discoveries, the driving forces behind biotechnologies are related to economic imperatives as well as the interest and support they receive from the political powers-that-be. the academic spirit that presides over fundamental science gives way to the entrepreneurial spirit that implements a rational programming of facilities and an efficient organization of scientific collaborations. as an example, the sequencing of the human genome, which includes three billion nucleotide base pairs, required the coordination of several dozen scientific teams around the world and the matching of several tens of thousands of results. research on dna provides a typical illustration of the way in which research has become divided, over the last few decades, between an approach and an interest that had previously been purely academic, and the increasing role of technology, which can be justified by the results that arise in the life of society at large, but which, because of these results, also gives rise to questions concerning how wellfounded some of these results are, particularly in the health domain. the experimental method, which had been confined to the laboratory, is now a matter for public debate. before it won acclaim, dna, which was isolated under the name of nuclein by johann friedrich miescher (1844 -1895), at the end of the 19 th century, had to undergo a series of structural evaluation tests that were spread out over the first five decades of the 20 th century. an overall conclusion then came to the fore. dna is a polydeoxyribonucleotide that carries four cyclic bases, adenine, thymine, cytosine and guanine. each base is involved in the structure of a mononucleotide where it is itself associated with a sugar, deoxyribose, which is associated with a phosphate residue. dna was compared to a ladder, the rungs of which (mononucleotides) were linked by ester bonds between an acid group of a phosphate residue of a nucleotide and the free hydroxyl group of the deoxyribose of the following nucleotide. research committed them to this path. it was the curiosity of each, a new way of considering old problems, that led a few men and women to solve the problems of heredity." the middle of the 20 th century saw an accumulation of experimental evidence showing that dna carries genetic information, and because of this, that it controls the transmission of hereditary characteristics: the proof provided in 1944 by oswald avery (1877 -1955) , colin macleod (1909 -1972 and maclyn mccarthy (1911 -2005 of the transforming power of dna in pneumococcus, the highlighting by alfred hershey (1908 -1997) and martha chase (1927 -2003 , in 1952, of the role played by bacteriophage dna as an infectious agent for bacteria, the revelation by erwin chargaff at the beginning of the 1950s of the equivalence of molar concentrations of adenine (a) and thymine (t), on the one hand, and of cytosine (c) and guanine (g), on the other hand, in dnas arising from a multitude of sources, animal, plant and microbial, thus suggesting a complementary pairing of adenine and thymine, and cytosine and guanine. based on the pairing of a/t and c/g bases, the model of the double helix structure of dna, formulated in 1953 by james watson and francis crick, made it possible to understand the identical synthesis of double strands of dna by replication during cell division (figure iv.1 ) and, as a consequence, the conservation of hereditary characteristics in descendants. afterwards, it was found that the information contained in the dna base sequence determines the amino acid sequence in proteins. then the roles played by messenger rna and transfer rnas were elucidated, the former acting as a carrier of information between dna and the proteins being synthesized and the latter acting as double-headed adaptors, able to recognize nucleotide triplets (codons) in messenger rna and to specifically fix amino acids in order to position them on the ribosomes, the final result being the synthesis of a protein chain. in 1966, the genetic code was deciphered. the veil of mystery that had covered the mechanism of the synthesis of proteins was lifted, and the decisive role played by nucleic acids in this synthesis was shown. later on, there were a few adjustments. although, in bacteria, proteins are coded for by a continuous sequence of nucleotide triplets in dna, in the 1970s the surprising discovery was made that in eukaryotic organisms, genes are discontinuous and made up of coding dna sequences (exons) interrupted by non-coding sequences (introns). from the end of the 1950s, françois jacob and jacques monod had postulated the existence of a dual determinism for protein synthesis and shown that, next to structural genes expressed as proteins, there are regulatory genes able to control the expression of the structural genes. the importance of the differential regulation of gene expression in cell differentiation in higher organisms was quickly recognized. from this point on it was possible to explain why a particular species of protein is more specifically expressed in a given tissue and another species of protein is more particularly expressed in another tissue, each type of tissue finding its specificity in its molecular components. this fantastic framework of knowledge, which was built up over a couple of decades, has been used as the foundation stone for the so-called central dogma of molecular biology, which explains the transcription of dna sequences into messenger rnas and the translation of messenger rnas into proteins and which, with only a few variants, is the same throughout the living world (figure iv.1) 1 . 1 the fascinating history of molecular biology was well described by james d. watson in molecular biology of the gene (3 rd edition 1976). there are now many works concerning this subject and how it has progressed, including two well-documented books in french: the secrets of the gene by françois gros (1986) and histoire de la biologie moléculaire by michel morange (1994) . new strand a -double helix structure of dna and simplified representation of its self-replication. each strand of the parent molecule of dna acts as a matrix for the synthesis of a daughter molecule of complementary dna, in conformity with the rules of pairing: adenine (a) with thymine (t) and guanine (g) with cytosine (c). the double strands that appear are identical to each other and identical to the parent dna molecule. b -transcription of dna into messenger rna and its translation into an amino acid chain. diagram of gene expression in a eukaryotic cell. one of the strands of dna (the coding strand) has coding sequences (exons) and non-coding sequences (introns). it is said that the gene is split. the transcription of the exons, accompanied by their splicing leads to the formation of a messenger rna, the codons (nucleotide triplets) of which are translated into amino acids that are linked to each other by covalent bonds in order to form a protein chain. in prokaryotic cells (bacteria), the genes do not contain introns and are not split. a: adenine; c: cytosine; g: guanine; t: thymine; u: uracil. met: methionine; his: histidine; tyr: tyrosine; gly: glycine; phe: phenylalanine. interlinked with the epic rise of molecular biology, there was a succession of technical innovations that led to the synthesis of dna by chemical or enzymatic means, and to its being cleaved at specific locations, with the pieces that were obtained being joined together again 2 . in 1962, in geneva, werner arber (b. 1929) 3 and daisy dussoix highlighted the restriction phenomenon, which involves the degradation of bacteriophage dna by a recipient bacterium. they discovered that an extract of e. coli has a restriction activity, and that this activity is of an enzymatic nature, caused by a nuclease that breaks the phosphodiester bonds in dna. in 1970, the americans hamilton smith (b. 1931) and kent wilcox 4 purified the first restriction enzyme from a strain of haemophilus influenzae. in 1971 , daniel nathans (1928 -1999 and kathleen danna 5 (b. 1945) at johns hopkins university in baltimore (usa) drew up the first restriction map based on the circular dna of the monkey sv40 virus, using a restriction enzyme that was named hindiii and a follow-up of the sequential appearance of shorter and shorter fragments resulting from the partial digestion of dna. in the following years, dozens of restriction enzymes were isolated, all of them endowed with a surprising specificity with respect to specific base sequences in dna ( figure iv .2). these enzymes were to be indispensable tools in genetic recombination experiments. the transformation of rna back into dna was observed by howard temin (1934 -1994) and s. mizutani 6 , in experiments on the rous sarcoma virus, a virus with rna that, when it proliferates in host cells, is able to synthesize a dna that is complementary to its rna. the enzyme responsible, reverse transcriptase, was purified by both h. temin and david baltimore (b. 1938) 7 . starting with a determined messenger rna, it then became possible to work back to the dna, i.e., to the gene, by a simple enzymatic reverse transcription operation. dna that has been synthesized in this way is called complementary dna (cdna). in eukaryotic organisms, reverse transcription has proved to be all the more useful as a technique in that all cdna is coding, unlike the situation in vivo in which the genes are divided up into portions that are coding (exons) and portions that are non-coding (introns). the ability to cleave dna and to join together the fragments obtained in a deliberately chosen order, or, in other words, to manufacture previously unseen dna sequences by making new combinations, led to the dawning of recombinant dna technology and caused scientists to come to the sudden realization that the pandora's box that contains the secrets of life had been opened, that uncontrollable catastrophes might arise from this, and that there was a potential danger of causing tumorigenic viruses to reproduce in commensal bacteria such as the enterobacterium escherichia coli. in 1975, around a hundred molecular biologists gathered together at the asilomar conference center 8 near monterey in california, in order to discuss the dangers of the new dna technology. they proposed strict regulation to govern genetic manipulation. time and experience have shown that the risks being run were very low. in 1977, dna sequencing methods were published. one of them made use of chemical techniques 9 , the other made use of enzymatic techniques 10 . applications were not slow in appearing. from 1977, the team led by frederick sanger (b. 1918) in cambridge (uk) determined the first sequence of a genome, that of bacteriophage phix174, which is 5 375 nucleotides long. this was the beginning of an audacious adventure, the apparently senseless challenge being met with unbelievable rapidity thanks to the innovative methods of bioengineering, resulting, during the first years of the 21 st century, in the sequencing of the human genome. analysis of the human dna sequence involved the participation of two rival groups, one of them being academic, coordinated by francis collins (b. 1950) , and bringing together dozens of laboratories around the world, and the other being a private californian company directed by craig venter (b. 1946) . at the beginning of the 1980s, when everyone was persuaded that the rnas could be placed into three well defined categories, messenger rnas, transfer rnas and ribosomal rnas, it was with great surprise that it was learned that there were rnas that have catalytic properties (see thomas cech 11 [b. 1947] ). these rnas, called ribozymes, have, in keeping with enzymatic proteins, structured catalytic sites that are able to catalyze rna or dna cleavage or ligation reactions. recently, engineering techniques have been used to obtain artificial ribozymes that have been found to be able to catalyze reactions as varied as oxidations or the synthesis of peptides and nucleotides, thus opening up wide-ranging possibilities of applications in molecular therapeutics, and, in addition, reinforcing the famous theory of the "world of rna" at the beginning of the appearance of life on earth 12 . another discovery of the 1980s was the role of methylation of dna bases, cytosine and adenine, and its deregulation in a certain number of pathologies: fragile x syndrome, scapulohumeral dystrophy, certain forms of cancer 13 … in the past decade or so, basic proteins known as histones that are associated with the nuclear dna of eukaryotes in the form of a complex called chromatin and which had previously been assigned a structural role, have now acquired the status of functional partners. thanks to specific modifications of certain amino acids (acetylation, methylation, phosphorylation), histones control the state of condensation of the chromatin and the efficacy of transcription of dna contained in the chromatin, to such an extent that we now speak of the "histone code". the development of our understanding of histones is a good illustration of the complexification of a concept, the dna code, into an entity that comes closer to living reality, the dna code in partnership with the histone code. there has also been the discovery of interfering micrornas, small polymers made up of around twenty nucleotide units, the role of which is to control protein synthesis (chapter iv-1.2.2). methylation of dna, structural modifications of the chromatin histones, and blocking of transcriptional activity by interfering micrornas are a few of the major areas of research in a scientific domain that is in full expansion, epigenetics, which could be said to have "pipped the science of genetics at the post," and which explains the plasticity of the functions of living beings. with the arrival of restriction enzymes and reverse transcription, the foundation stones of genetic engineering have now been laid, and are ready to be used, this being all the easier in that synthetic chemistry is now able to manufacture dna chains that are several hundreds of nucleotides long, and progress in robotics and computing techniques made it possible for chemists to avoid carrying out tedious routine tasks by using completely-programmable machines. the hope that it would be possible to experiment on living beings by means of the manipulation of dna became a reality when the american researchers paul in the first work carried out on the expression of foreign genes, the use of plasmids as vectors was preferred, particularly that of plasmid pbr322, because of its considerable replication capacity. in 1978, a first success was obtained by herbert boyer and his co-workers, with the expression of the gene for somatostatin, a peptide hormone comprising twelve amino acids that negatively regulates the secretion of growth hormone, in the bacterium e. coli. because of its small size, the somatostatin gene was synthesized by chemical means. the expression of somatostatin in e. coli was verified using immunological and physiological criteria, thus demonstrating the validity of the procedure that was used. the following year, human insulin was produced in e. coli. fairly soon, yeast was substituted for this bacterium because, as a eukaryotic organism, it has enzyme systems that are able to carry out chemical finishing operations on neosynthesized proteins that bacteria are unable to do, for example the formation of disulfide bridges in insulin. genetic recombination is used in order to cause bacteria to manufacture a foreign protein of animal or plant origin. this involves the insertion of the fragment of animal or plant dna that codes for this foreign protein into a plasmid. the plasmid, a small ring of bacterial dna, acts as a vector for the foreign dna. in order to carry out insertion, plasmid dna is cleaved by an appropriate restriction enzyme. the foreign dna is obtained by reverse transcription from a useful messenger rna. its duplication is catalyzed by a dna polymerase. the s1 nuclease makes it possible to break the covalent bond between two strands of dna. in the following step, a terminal transferase is used to add four nucleotides for which the base is a cytosine (c) to each of the two dna strands. the same lengthening operation is carried out on the bacterial plasmid, but, in this case, the addition involves a sequence of four nucleotides for which the base is a guanine (g) (complementary to the cytosine c). the bacterial plasmid is hybridized in vitro with foreign animal or plant dna and then introduced into the bacterium which, using its own machinery, perfects the junction between the integrated dna and the plasmid dna. a very small volume of dna (2.10 -9 ml) is injected under the microscope into eukaryotic cells (hela cells in inset) using a micropipette with a very fine end that pierces the cell membrane. the swelling of the cells at the moment of injection can be seen (inset). supported by these successes, genetic engineering started to come to the fore as an application-oriented discipline. levels of performance that would never have been imagined half a century before were achieved, such as the production of growth hormone, interferons, blood coagulation factors and vaccines. in the final decades of the 20 th century, phenotype transformations using genetic modifications that had previously been carried out in bacteria and yeasts were successfully attempted in animals and plants. it was observed that a mutated dna integrated into a plasmid and introduced into a fertilized mouse egg (by micromanipulation) modifies the mouse's genetic inheritance, which affects first the embryo and then the adult mouse with phenotype modifications. such mice, which are said to be transgenic because of the stable integration of a foreign dna into their genome, are now widely used as animal models in studies that aim to understand the mechanisms involved in high-incidence human pathologies such as cancer, diabetes, and rheumatoid conditions. in 1982, two american researchers 16 , ralph brinster (b. 1932) and richard palmiter (b. 1942) carried out a spectacular transgenesis experiment in mice. using microinjection, they introduced the growth hormone gene (obtained from the rat) into oocytes of mice from a "little" germ line. once the transgeneic mice had reached adulthood, they were giants. at present, the transgenesis technique is being applied both to the animal kingdom and to the plant kingdom. in the 1980s, kary mullis 17 (b. 1944) perfected an ingenious technique, the polymerase chain reaction (pcr), which makes it possible to produce several tens of thousands of copies of a fragment of dna. using this technique, it is possible to detect traces of a fragment of dna of a given sequence down to an attomolar concentration, i.e., one billion billion (10 -18 ) times smaller than molar concentration. by the end of the 20 th century, genetic engineering had become well-established and wide-spread, thanks to a mastery of techniques involving the manipulation of dna such as the accurate cleavage of a gene into fragments using commercially available restriction enzymes, the covalent assembly of two fragments of dna by ligases, the automated chemical synthesis of fragments of dna of more than one hundred nucleotides and the possibility of manufacturing a complementary dna (cdna) from a messenger rna by using a reverse transcriptase and automated dna sequencing. given this particularly well-equipped toolbox, the molecular biologist is now able to manipulate dna, that is to say, the chemical material that contains the information that is central to the functioning of living structures (microorganisms, plant and animal organisms), and thus to modify, at will, the genotype of these structures that the selective pressure of evolution has previously favored. genomics has produced enormous quantities of data that are stored in databanks. automated procedures have been invented to make the information contained in these data intelligible, these procedures forming the basis for a new discipline, biocomputing, or bioinformatics, which develops programs, or algorithm-based strategies, that are able to solve specific problems, of which the annotation of genomes, i.e., the identification of coding and non-coding sequences. while the annotation of the prokaryotic genomes is relatively easy, because of the absence of introns, that of the eukaryotic genomes is considerably more difficult because of the alternating exons and introns and the small proportion of coding exons (fewer than 2% in the case of the human genome). this explains why, at the time of writing, several hundred genomes of prokaryotes (around 300 at the beginning of 2006) have been sequenced, as opposed to only a few dozen genomes of eukaryotes. annotation was carried out manually at first, but it has become automated and it is now possible to analyze thousands of items of genomic data. the comparison of nucleotide base sequences in dnas and of amino acids in proteins of different origins involves biocomputing. the identification of similar or identical regions that provide information about functional similarities and phylogenetic proximity involves the use of alignment methods. one of these methods, which is in current use, is called blast (base local alignment search tool). comparison of protein sequences has been particularly instructive in the science of evolution. it has highlighted evolutive processes in the phylogenesis of proteins and linked these processes to precise functions. it has been possible to deduce that, over time, different families of proteins with similar functions appeared independently and evolved along different routes. this is the case for membrane proteins whose polypeptide chain crosses the thickness of the membrane six or twelve times; thus, the mitochondrial membrane proteins that transport metabolites are formed by triplication of an element with two transmembrane segments, while proteins located in other membranes of the cell are derived from duplication of an element with three transmembrane segments. from this academic context arose the study of paleogenetics, a new discipline that compares dna sequences extracted from fossils and amplified by pcr with dna sequences of current species. in addition to being of immense interest to fundamental biology, genetic bioengineering has led to innumerable industrial applications making use of genetically modified microorganisms that are able to synthesize molecules with a high added value that can also be used in xenobiotic depollution operations. so much data has already been deposited, equivalent to the sequencing of more than one hundred billion nucleotides, that it is inevitable that there have been errors, some of which might prove prejudicial for future use (comparison of sequences, screening of drugs…). nevertheless, the ever-increasing numbers of genome sequencing projects for animal, plant and microbe species show the interest that is shown in understanding the genetic information present in different types of cells, in order to be able to exploit their potential. dna chips appeared in the last decade of the 20 th century, and came to the fore as part of a new technical revolution, the "high throughput" revolution ( figure iv.5 ). an article written by the group headed by ronald davis and patrick brown (b. 1954 ) at the university of stanford 18 gives a precise description of the hybridization technique used in dna chips. thus, around a hundred short dna strands, corresponding to portions of genes of the plant arabidopsis thaliana, commonly known as mouse-ear cress, a small plant in the brassicaceae (formerly cruciferae) family, are synthesized. a robot is used to deposit microquantities of these dnas in solution in a dot pattern on a small glass slide coated with poly-l-lysine, thus comprising a "chip" on which the covalently fixed dnas act as probes for specific molecules. a later step involves both the use of reverse transcription to produce complementary dnas (cdnas) from messenger rnas arising from the expression of genes in the same plant and the labeling of these cdnas with fluorescent ligands for use in screening. once these fluorescent cdnas have been denatured, i.e., after separation into single strands, they are brought into contact with the dna chip. the unhybridized molecules are removed by washing. a -the term dna chip corresponds to a small, chemically-treated glass (or sometimes silicon) plate on which a robot has deposited dna strands of known sequence in a pre-determined order. b -the dna chip may be used for different types of diagnostic procedures. in the differential diagnosis experiment represented here, messenger rnas are prepared from two cell samples that have been treated in parallel, the control sample (normal cell) and the experimental sample (pathological cell). these messenger rnas are reverse transcribed into complementary dnas (cdnas) by means of a reverse transcriptase. each of these two types of cdna, corresponding to the two types of messenger rna, is labeled by a chemical reaction with a specific fluorescent ligand (cy3, which emits at 568 nm and cy5, which emits at 667 nm). they are then hybridized with chip dna strands. after hybridization and then washing, the fluorescence emitted at 568 nm and 667 nm under laser irradiation are analyzed using an appropriate detection system. the differential expression of the genes in the control cell sample and the experimental sample (shown by a color difference) can thus be analyzed. hybridization between the fluorescent dnas called targets and the complementary nucleotide probes fixed to the dna chip is detected by means of an automated fluorescence detection system. at the beginning of the 1990s, stephen fodor 19 and his group, who were working at the affymax research institute (palo alto, california), developed an ingenious procedure for microphotolithography that led to the synthesis of a network of a thousand peptides on chemically pre-treated glass microscope slides. the resolution of the network was shown by epifluorescence microscopy after fixation of specific antibodies labeled with fluorescent probes. soon after this, microphotolithography was used for the manufacture of dna molecule networks on solid supports. from then on, two competing techniques for the preparation of dna chips became well-established, either the depositing on a solid support of cdna obtained by gene amplification (technique used by davis and brown), or the synthesis in situ of oligonucleotides carried out directly on a solid support (technique used by the american affymetrix company, arising from affymax). one considerable advantage of dna chip technology is that it provides information on the level of transcription of thousands of genes into messenger rnas (mrnas), in a simultaneous manner and in a relatively short lapse of time. experiments that previously required weeks, months or even years to be completed can now be carried out in a matter of hours. we therefore have a sort of instantaneous, precise, freeze-frame picture of the state of a cell at a given moment, with a great number of parameters explored in a semi-quantitative manner. dna chips are a typical example of the application of high throughput technology to the study of living beings. the panoply of mrnas produced by the transcription of dna is called the transcriptome. the method by which transcriptomes are obtained is called transcriptomics. it should be added here that there is not necessarily a correlation between the abundance of a mrna, evaluated on a dna chip, and the functionality of the corresponding protein, which depends on multiple factors that particularly involve post-translational modifications: phosphorylation, glycosylation, hydroxylation, and so on. at the end of the 1990s, dna chips were being used extensively in the research programs of many biology laboratories. they are used in a variety of domains: human pathology, to differentiate between forms of cancer linked to multiform mutations; microbiology, to identify pathogenic germs; comparative genomics, to look at model eukaryotic organisms that have in common a certain number of genes; or even in populations genetics, to detect polymorphisms linked to a change in a single base in a dna sequence. as a complement to the dna chip technique, the fish (fluorescent in situ hybridization) method holds a key position in the study of cytogenetics. this method is based on hybridization between fluorescent nuclear probes of known nuclear sequence and of complementary motifs located in the dna of the chromosomes. it allows the detection of chromosomal modifications with a gain or loss of genetic material, such as those that are found in certain tumors. it is used in prenatal diagnostics to diagnose such modifications. protein chips, which are used to characterize the reactivity of proteins with respect to specific ligands, are another example of the application of high throughput technology. dozens of proteins of different types (antibodies, for example), as well as derivatives of nucleic acids or even molecules capable of being ligands of proteins that might arise from combinatorial chemistry (chapter iv-3.3), are arranged in a network on small glass plates that are chemically treated to act as hooks to entrap specific proteins present in a tissue extract or serum ( figure iv .6a). this procedure, which is essentially analytical in nature, is complemented by a functional study in which proteins that have been isolated in their native form, i.e., those that are capable of expressing the same functions that they posses in vivo, are deposited on a glass microplate. this type of biochip makes it possible to analyze the reactivity of the proteins that are fixed to it with respect to a multitude of targets (proteins, nucleic acids or pharmaceutical substances) (figure iv.6b). antigens peptides a -biochip for the identification of proteins. different types of ligands, antibodies, antigens, dna or rna, small molecules with a high affinity and specificity, are deposited on a reactive surface. these biochips can be used to determine the level of expression of proteins and the type of proteins expressed in cell extracts. they can be used for clinical diagnostics. b -biochip for the functional study of proteins. native proteins or peptides are arranged in micronetworks on a reactive medium. biochips produced in this way are used to analyze the activities of proteins and their affinities as a function of posttranslational modifications. they are useful for identifying drug targets. analytical proteomics, which was still in its infancy in the last decades of the 20 th century (chapter iii-6.2.3) has become a vigorous discipline. the association of liquid nanochromatography and of mass spectrometry allows the identification of peptides obtained by the trypsin hydrolysis of samples of proteins of around one picomole in size. applied to fundamental and pathological cell biology, the aim of analytical proteomics is not only to decipher the list of proteins on the scale of the cell, but also to highlight variations in the abundance of synthesized proteins as a function of environmental conditions. it also aims to determine the posttranslational modifications that are undergone by the proteins inside the cells, which, for a large part, control the specificity of their operation. in parallel with the study of proteomics, the study of peptidomics, or the study of all of the peptides (peptidome) present in animal and plant cells and in the fluids that bathe these cells, has developed. for example, several hundred different peptides have been found in the cerebrospinal fluid. structural proteomics, which deals with the three-dimensional structure of proteins, has also become a domain in which the activity has been increasingly dominated by high-throughput techniques. this is partly due to the fact that the pharmaceutical industry has given considerable, sustained attention to the understanding of the structure of proteins that could play the role of therapeutic targets. this is the case for protein kinases that catalyze, via atp, the phosphorylation of endocellular proteins, of proteases involved in hydrolysis reactions, or even of cell surface receptors that are able to combine with ligands such as hormones. the use of automated crystallization systems has become common in structural biology. from the classical technique of the hanging drop, in plates comprising 24 wells, we have moved on to plates with 96, 384 and, recently, 1536 wells. obviously, this increase in dimensions requires the use of an automated system that includes a robot in charge of transferring microaliquots of the protein solution into the wells and adding media that differ according to their ph, ionic force and molecular composition to these wells. the crystallization process is followed by an automated microscopic examination coupled with video photography. making use of the recent development of genomics and of proteomics, and a detailed inventory of the structures and functions of the different protein species of living beings, contemporary biology is now able to sketch out a scheme of molecular systematics, including a classification into phylla, families, and classes that echoes those of the zoological and botanical systematics of the 17 th and 18 th centuries. however, modern systematics does not tell us how protein macromolecules interact within dynamic networks. there is still an enormous amount of work to be done in order to achieve an understanding of the meaning of the dialogue between macromolecules in a normal or pathological cell context. this work will require a detailed analysis of metabolic pathways and of how they are controlled, and their evaluation in kinetic and thermodynamic terms. it will be accompanied by modeling (chapter iv-4). there is no doubt that it will be successful. making use of subtle differences in the qualitative and quantitative expression of genes, it will become possible to understand the molecular principles that modulate differences in morphology and in function between neighboring animal, plant or microbial species. the science of evolution should benefit from this. in medicine, the forecasting of predispositions for certain diseases should be made easier (chapter iv-3.2), opening up the perspective of prevention strategies. using recombinant dna technology, metabolic engineering applied to microorganisms and plants should make it possible to improve the production of molecules that are of economic interest or can be used for drugs. the diversity of bacteria is amazing, much greater than might be supposed by looking at the number of bacterial species identified by culturing on appropriate media. in fact, the number of bacterial species that can be cultivated only represents 1% of the total number of existing bacterial species on the surface of the earth. there are two major reasons for this: we do not know the appropriate conditions for culturing these bacteria; a certain number of environmental bacteria live in symbiosis, acting as commensal organisms that benefit from the products secreted by other organisms. nevertheless, the study of the bacterial genome, without any clonal culture, has been carried out, and comprises a branch of genomics known as metagenomics. instead of looking at an isolated, well-identified bacterial species, in order to analyze the sequence of its dna, as has been done traditionally, researchers look at a heterogeneous bacterial sample from which the dna is extracted, amplified, and then sequenced by high throughput methods. computer processing of the data provides information about individual germs. craig venter, who had already gained notoriety with the sequencing of the human genome, recently applied "metagenomic" procedures to the study of the sequence of the "metagenome" of the bacterial species of the sargasso sea 20 . he came up with nucleotide sequences corresponding to approximately 1 million kilobases of non-redundant nucleotides, attributable to more than two thousand different genomes. the challenge to be met by metagenomics is to connect a function to its phylogenetic source and to extend this information to specific species within a bacterial community. group 21 . in human biology, a metagenomics approach has been applied to the study of the population of bacteriophages present in the intestinal flora. approximately 1 200 genotypes have been identified, a number that greatly exceeds the 400 bacterial species of this flora 22 . this result leads us to think that the luxuriant community of bacteriophages which cohabits with that of the intestinal bacteria may influence the diversity of the latter by selective bacterial lysis and also by promoting the exchange of genes between bacteria. a rapid overview of the history of the exploration of genomic dna over the last fifty years shows the rapidity with which a traditional experimental paradigm can move thanks to modern computing and robotics procedures. in less than twenty years, we have moved from the manual sequencing of dna that was developed at the end of the 1970s to automated high throughput sequencing. at the turn of the 21 st century, the sequencing of communities of genomes (metagenomics) has been substituted for the sequencing of individual genomes. dna and protein chips have become objects of everyday use in fundamental and applied biology. transgenesis is widely practiced. dna, a molecule that remained mysterious for a long time after it was discovered, delivered some of its secrets during the second half of the 20 th century. the purpose of the first experiments on dna was to understand how dna, detector of the genetic code, transmitted its message. after having questioned dna, researchers moved on to manipulating it. the current aim is to use oligonucleotides to build nanoscale constructions with original and, if possible, useful, properties. in addition, the possibility that has recently become available of being able to interfere with the expression of the genome in living cells, with the intervention of small rna molecules, allows the programmed manipulation of the genome. another challenge, the extending of the coding power of the genetic code, now appears to be achievable. from the fact that each of the strands of the double helix overhangs in one direction, and in the other the strand with which it is paired, thus leaving a few bases free ( figure iv.7) . if two strands of dna with sticky ends are brought into contact, when the bases of these ends are complementary, a branched structure will appear spontaneously. using this principle as a basis, cube-shaped nanometric constructions that make it possible to encage molecules of interest have been built. the opening of the cage by appropriate devices liberates the encaged molecules, which can act as substrates in specific reactions. the cutting of a double strand of dna using restriction enzymes able to create fragments with cohesive ends (a) has been used to "build" an artificial construction (b), which, in this case, is a cube (c), but which could be an object of a different geometrical type. a dna nanomachine that is capable of movement is becoming a reality. one dna nanomachine, which is admittedly still rudimentary, has been put together based on the structural difference that exists between b-dna, the classical double helix that twists to the right, and z-dna, a double helix that twists to the left. a propensity to adopt the z-form is triggered when there is an alternating sequence of cytosine (c) and guanine (g) (cg sequence) in the dna. the experiment illustrated in figure iv.8 makes use of a duplex formed of b-double helices. the dna nanomachine constructed by n.c. seeman comprised a duplex of double strands of dna. one of the double strands, of the classical b-form of dna (right-hand twist), has been cleaved in such a way as to fix fluorescent molecular probes onto the cleavage zones. facing this cleavage zone, a short nucleotide sequence, in which the cytosine (c) guanine (g) motif is repeated, can be found in the other dna double strand, which is also of b-form. the addition of cobaltihexammine induces the transition of the cg segment from a right-hand twist (b-dna) to a left-hand twist (z-dna), which leads to a rotation of this segment and to a rotation of the assembly, which can be detected using fret (fluorescence resonance energy transfer) spectroscopy. one of the double helices has a short cg segment. facing the cg segment, the other double helix is interrupted, and its ends, where the interruption is, carry fluorescent molecular probes. the simple fact of adding a cationic substance such as cobaltihexammine, which neutralizes the negative charges of the phosphate groups, triggers a conformational transition, with the cg segment taking the z-form, causing a rotational movement of the assembly that is detected by the movement of the probes. there is no doubt that the use of dna strands in order to build nanomolecular constructions that are capable of programmed movement marks the beginning of an adventure that we may imagine will be rich in outlets for domains such as computer technology, nanomechanics and even the life sciences. in addition, the discovery that dna conducts electrical current gives rise to dreams of a revolutionary technology in which dna may be used in the design of electrical circuits, in competition with classical electronics 24 . interfering rnas are non-coding rnas of around twenty nucleotides that control gene expression at post-transcriptional level. as with many discoveries, that of rna interference was the result of serendipity. it began during the 1980s with observations made by two american research groups, that of victor ambros 25 now at darmouth medical school, hanover, and that of gary ruvkun 26 (b. 1951) at boston's massachusetts general hospital, that a gene named lin-4, which is involved in the post-embryonic development of the nematode c. elegans, did not code for a protein, but for a small size rna that played an antisense role. this odd discovery was supported and made more explicit a few years later by the research groups of andrew fire (b. 1959) at baltimore's carnegie institute and craig c. mello (b. 1960 ) at the university of massachusetts in worcester 27 . in order to block the production of certain proteins in the nematode c. elegans, the researchers used synthetic antisense rnas. the control involved the use of sense rnas according to a classical protocol. unexpectedly, protein synthesis was blocked in both cases, suggesting that a contaminant was present in the sense and antisense rna preparations. this contaminant was identified as a double strand rna (dsrna -double strand) that is, an rna that is folded back on itself in a "hairpin" loop because of the pairing of complementary bases (adenine vs uracil and guanine vs cytosine). in order to verify the mechanism by which the translation of messenger rnas into proteins is silenced, the nematode c. elegans was injected with a synthetic dsrna, part of the sequence of which was complementary to that of the gene unc-22, known to code for a protein involved in muscular contraction. within a few hours, the worm was making disordered movements, suggesting that the dsrna interferes with the production of proteins in the process of muscular contraction. the mechanism of action of dsrna was quickly unraveled: dsrna gives rise to two single strand rnas after cleavage by a specific enzymatic mechanism. one of the single strand rnas (sirna -small interfering rna) is paired thanks to a complementarity of bases with a short sequence of messenger rna transcribed from the gene unc-22. the result is a blockage of the translation of messenger rna into a protein, followed by the destruction of messenger rna. this phenomenon was named rna interference ( figure iv the dicer cleavage enzyme, which has a ribonuclease activity, cuts the double strand rna into two strands. in the presence of the risc (rna-induced silencing complex) protein complex, one of the rna strands finds a complementary nucleotide sequence in a messenger rna (mrna) and associates itself with this rna, making it unable to be translated into protein. we now know that eukaryotic cells from animals and plants produce and host interfering rnas that are said to be "natural" 28 . natural interfering rnas, of around twenty nucleotides, are called micrornas (mirnas). although a few details differ between the modes of formation and action of natural interfering rnas and those of synthetic interfering rnas, in particular the fact that messenger rnas are not destroyed by mirnas but blocked in their translation, the effect of negative regulation on the production of specific proteins comes to the same thing ( figure iv.9 ). there is a far from negligible number of genes that code for mirnas. already, several hundred mirnas have been identified in the genomes of plants and animals. the amount of interest that they arouse, and the feverishness of the research being carried out on them, are in keeping with the major mechanisms that they control: embryogenesis, hematopoiesis, neuronal differentiation, etc. given an understanding of the genome sequence in man, the rat and the mouse, trials have begun that aim to achieve an understanding of how the expression of mammal genes of known sequence might be manipulated by the interplay of interfering rnas (chapter iv-3.1). the treatment of viral infections such as aids or hepatitis b, which are worrying public health problems, could benefit from this new technology. it appears that interfering rnas have much more to give to us in the near future than they have taught us up until now. the deciphering of the genetic code in the middle of the 1960s was the end of a first step in elucidating the mechanism by which a sequence of nucleotides in dna is translated into a sequence of amino acids in a protein (chapter iv-1.1). during the years that followed, the subtleties of the transcription of dna into messenger rna and of the translation of messenger rna into protein via transfer rnas were explored in hundreds of laboratories around the world. particular attention was paid to the understanding of how a given amino acid is activated and bound to a transfer rna (trna) after being picked up by an aminoacyl-trna synthetase. nevertheless, the idea remained of a code in which triplets of purine and pyrimidine bases of messenger rnas are translated into natural amino acids. recently, methods have been developed that give more flexibility to the action of the aminoacyl-trna synthetases, or, in other terms, relax their specificity 29 . synthetases that have been manipulated in this way are able to recognize non-natural amino acids and to incorporate them into proteins by working together with the ribosomal machinery. it is in this way that, at the time of writing, around thirty non-natural amino acids, obtained by insertion of different types of chemical residue (photoactivable, fluorescent or radioactive residues capable of acting as probes for structural and functional analyses) (figure iv.10) have been incorporated into protein structures. with such an innovation, an unexpected field of exploration has opened up to research in domains as far apart as pharmacology and the science of evolution, giving rise to burning questions: could such non-natural proteins have therapeutic properties? could they give a selective advantage to the organisms that host them? with the addition of non-natural amino acids to the genetic code and the demonstration that proteins containing such amino acids can function in living cells, in sum, with the transgression of the potentialities of the natural genetic code, the experimental method appears to challenge the order of living beings. the triumph of genetic engineering via the study of dna is not unique to biology. many other sectors are undergoing changes in their type of experimental approach, dictated by the technosciences and making use of computer sciences, robotics and high-throughput screening. however, given the many questions that its operation continues to raise, and its central position at the heart of scientific ethics, the study of dna remains a typical example of the way in which the experimental life sciences and the techniques that underlie them are evolving nowadays. "i perfectly agree that when physiology is sufficiently advanced, the physiologist will be able to make new animals or plants, as the chemists produces substances that have potential, but do not exist in the natural order of things." more than a century after claude bernard predicted a genetically manipulated world, it has come to pass. the molecular biologist, having original, highperformance methods for "tinkering" with dna, has moved on to the application and use of his technical expertise for utilitarian ends. during the 1970s, with transgenesis, research on bacteria (chapter iv-1.1.2) opened up a new biological domain, that of genetically modified organisms (gmos). results led to predictions that it would be possible to transfer a fragment of dna corresponding to a gene of a certain species into the genome of another species and have this foreign gene express itself as a protein in the host cell. in 1983, the successful trans-genesis of a gene for resistance to an antibiotic, kanamycin, in tobacco plants, signaled the beginning of the technology of the first plant-type genetically modified organisms, still called gmps (genetically modified plants). in 1996, the birth of dolly the ewe unveiled the era of reproductive cloning in mammals, i.e., the identical reproduction of an already-existing organism. an additional step was taken with the first tests on the differentiation of embryonic stem cells towards different types of lines that are characteristic of well-defined tissues, such as nerve tissue, cardiac tissue or the hepatic parenchyma, thus opening up promising perspectives in regenerative medicine. the frontiers of the experimental method continue to be pushed back to the limit of what is feasible and sometimes into the realm of fiction, as in immunology, for example, with the idea of xenotransplantation, using "humanized" animal organs. given the universality of the genetic code, any gene that is introduced into the genome of a plant, whether that gene is of animal, plant or microbial origin, is able to replicate itself and be expressed as a specific protein. thus plant gmos or genetically modified plants (gmps) are able to express specific foreign proteins from another plant, a bacterial microorganism or an animal organism. in the 1990s, a short time after fundamental research had revealed the feasibility of plant transgenesis, the first transgenic plant, the flavr savr tomato, was marketed in the usa. since this time, numerous other plant gmos have been cultivated on a large scale and become available on the world market, including corn, soya, rice, cotton and the poplar. one of the desired aims is to produce modified plants that are able to resist destruction by the herbicides that are commonly used to eliminate weeds, while another is to prevent predation by harmful insects. in the first case, transgenesis involves the insertion of a herbicide-resistant gene, and in the second case, the inserted gene codes for an insecticidal toxin. recently, plant gmos that produce proteins with a therapeutic effect have appeared, ranging from antibiotic peptides to antibodies or proteins as unexpected as human hemoglobin. current projects aim to create plants that are resistant to adverse conditions such as the dryness of arid climate zones. the preferred procedure for producing a plant gmo is to use a bacterium, agrobacterium tumefaciens, a microorganism that is able to insert fragments of its own dna into plant cells ( figure iv .11). the useful gene that we wish to transfer into the plant may be a gene for resistance to a pesticide such as glyphosate, marketed under the name of roundup, phosphinothricin (basta) or glufosinate (liberty). the plasmid is reintegrated into a. tumefaciens. during infection of a plant cell by a. tumefaciens the t-dna carrying the gene of interest is inserted into one of the chromosomes of this cell. the ti plasmid is isolated and cut using a restriction enzyme. a foreign gene, known as a gene of interest, is inserted into the t-dna of the plasmid. a plant is generated from a modified clone. all of its cells carry the foreign gene. transgene of interest in the case of the fight against insect predators, the useful gene is carried by a fragment of dna contained in the genome of the bacterium bacillus thuringiensis. this gene, called bt, expresses a toxin responsible for the insecticidal capability of b. thuringiensis. a current application involves the protection of bt corn with respect to the corn-borer, a devastating insect whose caterpillars are particularly destructive. another, more direct, gene transfer method, known as biolistics, involves bombarding plant cells with tungsten microbeads covered with modified dna. with the implementation of large-surface-area experimental fields and the first marketing of gm soya, in 1996, the question of whether or not the advantages achieved with respect to crop yields are counter-balanced by risks for the environment and for consumers came to the fore. food risks could arise from the toxicity or allergenic power of artificially synthesized proteins. at the time of writing, this question remains unanswered, due to the lack of epidemiological studies carried out rationally over several years. when the first creations of gmos took place, the transfer of the gene of interest was carried out by means of the co-transfer of an antibiotic resistance gene. the transformed cells were selected according to the criterion of their resistance to this antibiotic, which involved a risk of dissemination of the resistance gene. this selection technique has been abandoned. in practice, it is difficult to evaluate the theoretical ecological risk of wild plants being invaded by genes that have been inserted artificially into gmos. as a precaution, zones used for experimentation of plant gmos are now surrounded by refuge zones, i.e., fields in which the same species of plants, in non-gmo form, are cultivated. there has been a much fiercer and completely legitimate debate concerning the presence of the terminator gene in seed from the first gmos marketed by the monsanto company in the usa. the terminator gene blocked germination of the seed from the cultivated plant, so it was necessary for the farmer to buy more seed from the company each season, thus creating a state of dependency. this technique is no longer in use, but the fact remains that most transgenic seed is patented, and therefore farmers who use such seed are dependent on the companies that posses this genetic know-how. the culture of plant gmos has spread around the world, covering more than a billion hectares of our planet, more than half of which are in the united states of america. this type of culture is used on a large scale for soya and in a less extensive way for corn, rape seed and cotton, but there are many other applications of plant transgenesis. among the countries that are actively involved we may mention argentina, brazil, canada and china, and more recently india, paraguay and south africa. while the policies of these countries are based on the fact that gmo products do not differ fundamentally from non-gmo products with respect to checks carried out a posteriori, and that there is thus no reason to prohibit them, european policy has taken refuge behind a principle of precaution, and it remains basically restrictive. although the moratorium on the culture and marketing of plant gmos that was put in place in 1999 was lifted in 2004, mandatory labeling for any consumable product containing more than 0.9% gmo remains dissuasive. the united states of america has refused to use such labeling. the worries that are aroused by plant transgenesis, which are often exacerbated by the diktats of ecology groups, must be analyzed in a reasoned manner. common sense and lucid thought dictate that the debate should be situated within a scientific perspective in which the main role is played by the experimental method in long-term applications. simple reflection leads us to think that with time parasites and self-propagating plants will develop a resistance to the most drastic treatments, as was the case for bacteria confronted with antibiotics. the perspective of an acquisition of uncontrolled resistances, which gives rise to so much passionate debate, is, in fact, only the first stage of a technology with promising applications. the mastery of plant transgenesis that was acquired through the first experimentations should, in fact, allow the emergence of plant gmos that are assigned to the production of molecules with therapeutic effects (drugs, vaccines, human proteins, vitamins…). in this domain, there have already been creations that include golden rice, which carries β-carotene, the precursor of vitamin a, banana plants that express a vaccine against hepatitis b and tobacco that produces human lactotransferrin and hemoglobin. if we just look at the production of golden rice as a palliative for vitamin a deficiency, it should be remembered that, in certain countries of our planet, this deficiency affects people's sight and is a frequent cause of blindness, that it generates problems with development and the immune response to infections, that it affects more than a hundred million children around the world and that it is responsible for the death of three million of them each year. if these plants are considered to be a material of choice for the production of proteins with a therapeutic effect, this is partly due to the yield of such crops over large surface areas, and also partly due to the low risk of transmission of viral pathogens to man, because of the species barrier, a risk that is less negligible when animal productions are involved. genetically modified plants are also potential factories for the manufacture of chemical products with an industrial impact, for example lubricants, perfumes and aromas. given the unpredictable outlets that plant gmos may have in human medicine and the different domains of the economy, plant gmo technology should be considered in a manner that is free of any pressure or passion, and, as far as the political authorities are concerned, it should be subject to appropriate measures to surround and protect certain strategic experiments. when looking at the worries being expressed by the european society, it should be remembered that the genetic inheritance of plants has never ceased changing, not only in the most of natural of manners, over millions of years, particularly with the mobility of transposable elements located in the genome, but also artificially, at the hands of farmers from ancient times onwards, with their methods of hybridization and selection. the nervousness of european authorities, showing an ignorance of basic scientific ideas, with the pretext of a principle of precaution, and sometimes political compromises that are exemplified by fluctuating and contradictory positions, runs the risk, in the short term, of causing their countries to lag disadvantageously behind the united states of america, which holds the majority of plant biotechnology patents. the principle of gene therapy is simple: the introduction of an appropriate gene into the cells of a patient who carries a mutation can correct the phenotypical consequences of this mutation, or, in other terms, cure the disease affecting the patient, or at least slow down its evolution. the technical difficulty involved in gene therapy is that of finding an appropriate vehicle or vector for the transfer of the gene and addressing it to an appropriate location in the genome of the host cell. the most commonly used vectors in human gene therapy are viral. a certain number of criteria are necessary for a transfer to be efficacious, including a high concentration of viral particles carrying the gene to be transferred (more than a billion viral particles per milliliter) and a good capability on the part of the foreign gene to be integrated into the host's genome. the patient's immune response remains a major worry in the use of viral vectors: at cell level it often leads to a proliferation of cytotoxic lymphocytes and, especially at humoral level, to the synthesis of antibodies directed against the viral proteins. in order to minimize its immune response, the genetic material of the viral vectors is modified. for ethical reasons, gene therapy is currently only applied to somatic cells, germinal gene therapy being rejected. somatic gene therapy has been experimented in the treatment of hereditary illnesses linked to hematopoiesis. one of the technical reasons for this choice is easy access to the progenitor cells of the bone marrow, with the aim of transfection. it was with this in mind that mouse gene therapy models were developed a few years ago. the sickle cell mouse is one of these models. human drepanocytosis (sickle cell anemia) is a serious disease that is caused by a mutation in the β protein chain of normal human hemoglobin a. the molecules of sickle cell hemoglobin s tend to aggregate and form fibers that obstruct the blood capillaries of the microcirculation. somatic gene therapy has been applied to these sickle cell mice. this involves an autograft of bone marrow hematopoietic cells transfected with a retrovirus hosting the gene coding for the β subunit of normal hemoglobin. encouraging results have shown the validity of this approach. in 2000, a gene therapy protocol that had been applied with success to man was described by the group of alain fischer (b. 1949) and marina cavazzana-calvo at the necker hospital in paris 30 (science, vol. 288, pp. 669-672). the purpose of this therapy was to bring about a long term remission in the case of an immune disease known as scid-x1 (severe combined immunodeficiency linked to a mutation on the x chromosome). because of their susceptibility to microbial and viral infections, babies who are affected can only survive in sterile rooms. they are known as bubble babies. in this illness, the hematopoietic progenitor cells of the bone marrow are unable to differentiate into t and nk (natural killer) lymphocytes because of a mutation that affects a cytokine receptor. previous experiments carried out on model mice show that scid can be corrected by in vivo transfer of the cytokine receptor gene into hematopoietic progenitors. the transfer of the gene of interest paired with a retroviral vector was carried out first in march 1999, in two babies, one of them eleven months and the other two months old. progenitor cells from their own bone marrow, cultured and modified genetically, were injected into them. these were therefore autografts, without any risk of immune rejection. a remission of symptoms over a period of nearly a year, shown by the almost normal behavior of the babies' immune cells, encouraged the application of the same therapy to other babies. in total, ten babies were given this therapy. the enthusiasm that greeted the successes that were recorded was nevertheless tempered by fact that in the spring of 2002, and again in the following year, a child who had undergone the gene therapy developed a leukemia characterized by an anarchical proliferation of lymphocytes, necessitating chemotherapy. these two occurrences were explained by the random character of the insertion of the gene of interest into the patients' genomes: insertion into a site close to a proto-oncogene had led to activation of this proto-oncogene and the proliferation of the lymphocytes. while the trial carried out at the necker hospital gave rise to great hopes, it nevertheless showed that there is still a long way to go before we achieve a targeted transfection of genes so that no undesirable consequences follow. here we have a typical example of the limits of an experimental method that is based on an in-depth technological know-how, but also on a still imperfect understanding of the complex arcana of the mechanisms that regulate the positioning and interaction of genes in the chromosomes of eukaryotic cells. this example highlights a harrowing ethical dilemma: should we not treat a patient whose illness is likely to be fatal, or attempt a therapy that may save the patient, without having any formal assurance of its success? an experimental medicine that has the power to modify the human organism via its genetic material is now able to take over from the experimental method that up until now operated on animals and plants. we can easily understand, given the progress that has already been accomplished and that which is to come in the domain of gene therapy, that the temptation will be great, in the future, to consider manipulations of the human germ cell genome as being licit, insofar as such manipulations make it possible to eradicate a handicapping defect in our descendents. at present, the idea of any attack on the germinal genetic inheritance has been rejected unconditionally on the basis of ethical considerations. nevertheless, the history of science shows that prohibitions that were once considered to be untouchable finish by being contravened. this was the case for abortion. in a text entitled why genetic engineering should continue its battle 31 , james watson writes of his confusion when faced with a choice that is likely to become more and more insistent over the years: "dare we be entrusted with improving on the results of several million years of darwinian natural selection? or do the human germ cells represent on the contrary rubicons that geneticists will never dare to cross?" a mastery of the differentiation of stem cells and of cloning are two essential weapons in the biotechnological arsenal, the use of which for utilitarian ends, particularly in human medicine, gives rise to hope and disquiet, agreement and disapproval. at the beginning of the 1960s, experiments carried out by the canadian biologists ernest mcculloch (b. 1926) and james till (b. 1931) attracted attention to the particular properties of cells in the bone marrow, the stem cells, which would subsequently be found in other tissues 32 . the experimental protocol is simple. bone marrow cells from a mouse are injected into another mouse that has previously been irradiated in order to destroy its stem cells. the injected cells go to the spleen where they divide and form colonies that take the form of nodules of different sizes. the researchers realized that the cells of these nodules present differences in their potential for renewal, which is more or less rapid. they reinjected the nodule cells into mice from a second batch. the reinjected cells showed themselves capable of multiplying and generating several types of blood line. these observations suggest the presence in the nodules of progenitor cells that have a strong potential for self-renewal and self-differentiation. in the following years, these observations were confirmed and explained by two characteristic criteria of stem cells; self-renewal and differentiation into multiples cell lines with specific characteristics. from this point on, it was possible to understand the enigma of the amputated hydra in the experiments carried out by trembley, two centuries beforehand (chapter ii-3.4.2). we now understand why, like the hydra, organisms like the flatworm, the salamander, the starfish and the zebrafish are able to recreate an amputated or damaged part of their bodies. the hydra mobilizes stem cells that it has preserved since its birth. in the case of the salamander, regeneration involves the reprogramming of cells that have already been differentiated. like all stem cells, embryonic stem cells (or es cells) are able to self-renew and differentiate into the different types of known adult cell line, giving rise to different types of cell such as neurons, cardiac cells that are able to contract, or hepatocytes ( figure iv .12). this potential has led to the hope that es cells could be used in regenerative medicine. in a fertilized egg that has developed to the blastocyst stage, it is possible to distinguish a cell mass (inner cell mass, icm) which protrudes inside the blastocyst. the icm cells are removed and placed on a mat of irradiated (and thus unable to divide) fibroblasts that provide them with a support and nutrients (steps 1 and 2) so that they can proliferate. the stem cells arising from the icm cells, placed in a medium that has been specifically conditioned to provide cytokines and other biomolecules, are able to differentiate into various cell types (step 3). at what stage of embryo development is it possible to remove es cells for experimental purposes? after fertilization by a sperm cell, the ovum undergoes a series of divisions that give rise to a microstructure, the blastocyst, the cells of which are called blastomeres. each isolated blastomere remains capable of producing an entire organism of fetus and placenta, by division and differentiation. at this stage, blastomeres are totipotent. five days after fertilization, the embryo has the form of a hollow sphere. an external layer of cells, the trophoectoderm, surrounds a cavity, the blastocele, inside which a small mass of cells, the inner cell mass, protrudes. from the beginning of the implantation of the blastocyt in the uterus, the trophoectoderm evolves to form the placenta. the cells of the inner cell mass take part in the process of differentiation that generates all of the tissues of the future adult organism. these are called embryonic stem cells (es cells). es cells are said to be pluripotent. isolated, they have lost their ability to give rise to a complete individual, but they have maintained the possibility of differentiating, according to their environment, into any of the two hundred cell types that make up animal tissues. during their division, es cells evolve from a stage of being pluripotent to a stage of being unipotent, passing through a stage of multipotency beyond a hundred cells. a state of multipotency characterizes cells that give rise to a restricted number of cell lines in the tissues in which they nest. this is the case for of the hematopoietic stem cells of the bone marrow that form the red blood cells and the white blood cells. the term unipotent refers to the progenitors, which give rise to a single type of cell, for example the hepatocyte of the liver or the cardiomyocyte of the heart. when es cells are cultivated for 4 to 7 days in a conventional nutritive medium, they multiply and aggregate. if the culture medium is supplemented with certain biomolecules such as insulin, retinoic acid, transferrin or fibronectin, the differentiation of the es cells is oriented towards cells of different types, such as neuron cells, glial cells or muscle cells. there are many publications about experiments concerning the grafting of differentiated stem cells in the mouse or the rat. for example, neuron precursors derived from the spinal cord or the brain are grafted into rats whose spinal chords have been injured. five weeks after the grafts are carried out, the transplanted cells have filled the area of the injury and differentiated into oligodendrocytes, astrocytes and neurons. what is more, after about twelve weeks, locomotive function has been partially restoredirradiated 33 . other experiments involving the grafting of differentiated stem cells have been carried out on rats in which the dopaminergic neurons of the "substantia nigra" of the brain that secrete the neurotransmitter dopamine have been selectively destroyed by injection of 6-hydroxydopamine. the problems found in the rat as a result of this neuronal degenerescence mimic those found in man in patients suffering from parkinson's disease. dopaminergic neurons obtained by the differentiation of mouse es cells are grafted into the striatum of each of these rats, a region of the brain whose neurons communicate with those of the substantia nigra and play a fundamental role in the control of movement. this results in a significant improvement in the motor deficit, coupled with the establishment of functional synapses between the injected neurons and those of the host 34,35 . a recent publication bringing together the results of two french research teams, that of michel pucéat (b. 1961) in montpellier and that of philippe menasché (b. 1950) in paris, provides interesting information about how mouse embryonic stem cells, grafted into sheep cardiac tissue where an infarctus has been artificially induced, are able to colonize the infarct zone and regenerate cardiac contraction in a functional manner. moving from the mouse to the sheep constitutes a considerable species leap, and the absence of any immune rejection leads us to say that embryonic stem cells have an "immune privilege" 36 . the use of es cells in regenerative medicine necessarily requires that their differentiation be regulated in an exhaustive manner into well-defined pathways, in order to produce homogeneous cell lines with a view to implanting them in damaged tissues. in fact, contamination with non-differentiated es cells is likely to cause tumors (teratomas) over the long term. the mastering of the use of es cell culture and differentiation, as well as of cloning, in such a way as to overcome problems of histocompatibility, is still in its infancy. for a long time, the mouse was the preferred animal model for experimental studies on the differentiation of es cells. in 1981, the first es cells from mouse blastocysts were isolated and successfully cultured by two groups of researchers in great britain and the usa. it was only in 1998 that human embryonic stem cells (hes) were isolated for the first time 37 and held in culture, on a nutritive layer of fibroblasts from irradiated mice. this delay with respect to the ability to culture animal es cells can be explained by the fact that the molecular machinery that activates replication and cell differentiation programs is not completely identical in man and the mouse 38 . for example, a cytokine called lif (leukemia inhibitory factor), which is indispensable for the renewal of es cells in an undifferentiated state in the mouse, has no effect on human es cells. there are several other differences concerning the control of proliferation and differentiation in human and murine es cells by growth factors. briefly, the conclusions obtained from experiments carried while there is a highly promising future for the use of es cells, this future is littered with obstacles, and rigorous checks and balances need to be put in place. nevertheless, research on such cells is mandatory if we wish to move on to a regenerative medicine that aims to be a new frontier in the art of healing. after specific differentiation, hes cells could provide unlimited quantities of the tissues needed to replace damaged tissues responsible for handicapping illnesses (dopaminergic neurons in parkinson's disease, cardiomyocytes in myocardial infarction, pancreatic islets of langerhans cells in diabetes, fibroblasts in skin grafts, chondrocytes in rhumatoid arthritis). in addition, metabolic analysis of hes cells carrying defective genes whose phenotypical expression is known in human pathology should improve our understanding of the perturbed mechanisms, and could lead to pharmacological advances. as well as the technical difficulties involved, which have not yet been adequately overcome, the handling of hes cells is subject to much ethical debate in many countries, with those who object to it holding to their prejudices, which are linked to religious or cultural traditions. this is the case in france, where, nevertheless, a few timid dispensations had begun to appear at the time of writing. in contrast, in great britain, the law authorizes the isolation of hes cells for therapeutic purposes, using embryos of less than one hundred cells, produced by in vitro fertilization, and surplus to requirements. the british response to the burning question of whether an isolated hes cell may be considered as a potential human embryo is clearly "no", for, in order to be able to develop in utero, such hes cells would need to have the placental progenitor cells. an alternative to the use of es cells is to make use of adult stem cells. however, the proliferation capacity of adult stem cells is considerably lower than that of their embryonic homologues. the hematopoietic stem cell is the paradigm of the adult stem cell. it can differentiate into all known types of cells. in the last decade of the 20 th century, several publications concerning the plasticity of the adult stem cell awakened a hope that these cells could transform the treatment of degenerative illnesses. certain of these publications stated that adult bone marrow stem cells, implanted into different types of tissues, differentiate into hepatocytes, cardiomyocytes or neurons, depending on the specific environment. careful re-examination of the techniques used revealed that, in certain cases, interpretation of the results as showing cell transdifferentiation was an erroneous one, and that the fusion of the bone marrow stem cells with cells from other tissues was a more plausible explanation. in any case, while not ignoring the use of adult stem cells, experimentation on hes cells remains a judicious choice, given our current state of understanding. in france, the 2004 law application decree that was issued on the 7 th of february 2006, revising the restrictive bioethical standards of 1994, opens up the possibility of using human embryonic stem cells for scientific purposes, with certain ethical reserves being maintained. one of the obstacles to the stabilization over time of a stem cell graft in a receiver involves the phenomenon of rejection for reasons of histocompatibility. considered to be foreign by the receiver (host), grafted stem cells coming from a donor are rejected. this obstacle could be overcome by using the technique of cloning. based on experiments on several animal species, it is now accepted that the transfer of the nucleus of an adult somatic cell from a host into an enucleated oocyte makes it possible to obtain from this oocyte, which is once again nucleated, and which is the equivalent of a zygote and able to divide, es cells whose genome is identical to that of the host. because of this, the es cells are immunologically compatible with the tissues of the host. in man, such cells could be directed by differentiation towards stable cell lines creating well-defined tissues and organs (liver, muscle…) that could be used in regenerative medicine. this is the principle of therapeutic cloning. in march 2004, korean veterinary researcher woo suk hwang (b. 1953) and his co-workers, who were recognized experts in animal cloning, announced in the american review science 39 that they had succeeded for the first time in obtaining around thirty human blastocysts by cloning, i.e., by the transfer of nuclei of somatic cells into enucleated ova. this first experiment involved autologous cloning (ovum nuclei and enucleated somatic cells taken from the same woman). hwang and his team used 176 ova, and the yield from the experiment was close to that obtained at that time for the cloning of mammals. using the inner cell mass of one of the blastocysts, they isolated a line of embryonic stem cells able to maintain a normal karyotype after several dozen divisions. this publication, which appeared in a highly prestigious scientific review, triggered an enthusiasm in the media that was in keeping with the spectacular nature of the team's exploit, tempered here and there by a few comments that were mainly linked to questions of medical ethics. in 2005, there were numerous other articles by the same team on the same subject, reinforcing the first results with a heterologous cloning technique (ovum nuclei and enucleated somatic cells taken from different people), thus giving rise to great hopes that the era of regenerative medicine was near. at the beginning of 2006, professor hwang's retractation of all his work, and a public confession of a spectacular fraud, were even more dramatic, offering certain media an occasion for a disproportionate level of fury against therapeutic cloning. however, despite such rear-guard combats, it is obvious that one day these technical difficulties will be overcome. human cloning, in order to obtain stem cells for therapeutic purposes, cannot escape the future. once this aim has been achieved, it will be spoken of as the outcome of a long story. the adventure of animal reproductive cloning began in 1960. in developmental biology 40 , two american researchers, robert briggs (1911 -1983) and thomas king (1921 -2000 described experiments involving the transfer of cell nuclei of embryos from a frog (rana pipiens), at the blastula and gastrula stages, into enucleated eggs of the same species. a high percentage of the clones obtained in this way were able to reach the tadpole stage when the transferred nuclei came from the early blastula stage, but only mediocre success was achieved when the nuclei came from the later gastrula stage. these experiments emphasized both the totipotency of the embryo somatic cells and the equivalency of the somatic cell nucleus and the nucleus of the fertilized egg in cell division and differentiation. briggs and king's publication did not arouse any particular interest. it is true that the 1960s were dominated by the saga of molecular biology, which would reach its culmination in the deciphering of the genetic code. from the 1980s onward, the first attempts to clone mammals (rat, mouse, pig) began. moving from the amphibian egg, which was a millimeter wide, to a mammal egg that was one hundred times smaller, presented a technical difficulty that would be overcome by a technique of cell-to-cell electrofusion. cloned embryos were thus obtained by nuclear transfer and then implanted into the uterus of a surrogate female. however, in all cases, the nucleus came from embryo cells. in february 1997, the announcement made by ian wilmut (b. 1944), keith h. campbell (b. 1954) and their collaborators at edinburgh's roslin institute 41 of the birth of the cloned lamb dolly had an immediate effect in the media. in fact, this was not only the cloning of a higher mammal, but, above all, the cloning by insertion of an adult somatic cell, in this case a mammary tissue cell, into an enucleated oocyte. this went far further than the experiment carried out by briggs and king, which essentially involved the transfer of embryo cell nuclei into enucleated frog eggs. the trick that gave wilmut and campbell their success was to bring the cells providing the nuclei to a quiescent state corresponding to the interphase stage of the cell cycle, by impoverishing their culture medium, before electrofusion with enucleated oocytes. although we should be aware that 434 attempts were made before a positive result was achieved, this does not make it any less astonishing that the nucleus of a cell in its adult state, i.e., completely differentiated, was able to behave as if it were totipotent. despite being committed to a program of differentiation that is considered to be more-or-less irreversible, and which will give it a specific identity, the nucleus of an adult cell can be reprogrammed and become totipotent. since dolly, many other mammals have been cloned from nuclei of adult cells; mice, cows, goats, pigs, rabbits, cats, dogs, rats and horses. as far as ethical discussion about cloning is concerned (chapter iv.5), it is essential to note that the demarcation line between reproductive cloning and therapeutic cloning is situated where decisions are made concerning the destiny of the cloned blastocyst ( figure iv .13). the transfer of the nucleus of a somatic cell (liver, epidermis, muscle) containing 2n chromosomes into an enucleated oocyte gives rise to an egg (2n chromosomes) that is able to divide and to produce a blastocyst. the cells of the blastocyst inner cell mass (icm) can be used as stem cells that can differentiate into different types of cell line (therapeutic cloning). on the other hand, if the whole blastocyst is implanted into a uterus, it will produce an embryo which, after birth, will grow into an adult animal (reproductive cloning). reproductive cloning and therapeutic cloning therefore differ because of the fact that in reproductive cloning, the whole blastocyst is used, while in therapeutic cloning, only certain cells, corresponding to the inner cell mass (icm) of the blastocyst, are used. the structural and functional identity of the cells of a given tissue in an adult organism involves a basic mechanism: while each cell has the same set of genes, only some of the genes are expressed as proteins and the genes that are expressed differ according to the tissue involved. the key to the mechanism responsible is in the epigenetic type chemical modifications of cell dna, for example methylations, which repress the expression of certain genes without altering the expression of others. these modifications of the dna, which control cellular specificity (muscle, liver, brain…) are not very reversible, but, in certain circumstances, they can become so. this is what happens from time to time when the nucleus of an adult cell is inserted into an enucleated oocyte. we are thus able to assume that in the molecular arsenal of the oocyte cytoplasm there are substances that can cancel the epigenetic modifications of the dna present in the nucleus of an adult somatic cell and recreate a state of pluripotency in this nucleus, or, in other words, provoke the reprogramming of the somatic cell nucleus. in the long term, it is to be hoped that biochemical technology will be able to find and purify the molecules responsible for the nuclear reprogramming of somatic cells. the use of human oocytes for the purpose of therapeutic cloning is still subject to severe criticism. certain groups wish it to be prohibited, because of a fear of a drift towards reproductive cloning. to obviate this risk, the idea has been to make use not of human oocytes but of those of animals, transferring the nuclei of human somatic cells into them. even supposing that the technical difficulties involved could be overcome, the cells that would result, a sort of man-animal chimera, would also be the subject of an ethical debate, even if the purpose of this type of cloning were to be solely therapeutic. some japanese researchers 42 have succeeded in creating mice according to a parthenogenetic process that involves adding the nucleus of an oocyte that is haploid (1n chromosomes) to another haploid oocyte, the result being the equivalent of a fertilized egg (2n chromosomes). this exploit is achieved by the invalidation of one of the genes (h19) involved in the control of the parental imprint. it is known that sexual reproduction in mammals involves a phenomenon called the parental imprint, which, by means of the methylation of dna and perhaps also of histones, allows the expressing or silencing of certain genes in male and female gametes. a single copy of a given gene, originating either from the oocyte or from the sperm cell, is therefore expressed, while the other is inactive. in the japanese experiment, if the mouse h19 gene had not been invalidated, the result would have been an anarchical development of the responsible genes involved in the parental imprint with overexpression in the case of some and an absence of expression in the case of others. these disturbances would have been incompatible with the viability of the embryo. however limited its application might be, the manipulation of the germinal genome poses the problem of the mechanism by which the parental imprint intervenes in the viability of the egg, a parameter that at the time of writing is still not completely understood, but is being actively explored. in boston, massachusetts, in 1954, a kidney was transplanted from a healthy boy into his twin brother, who was suffering from a fatal renal anomaly. the success of this graft ushered in the era of transplantations of such organs as the heart, liver and kidney in man. in order to try to prevent the rejection of grafts, caused by an immune incompatibility between the receiver and the graft from the donor, different immunosuppressing treatments were tried, one by one, involving corticosteroids or cytostatic agents such as 6-mercaptopurine. in the 1980s, a decisive step forward was made with the fortuitous discovery of the powerful immunosuppressive effect of the cyclosporin a, a cyclic polypeptide isolated from the mold tolypocladium inflatum. each year, human organ transplants into patients make it possible to save many lives. however, for some time now, organ transplantation has been suffering from penury of donors. one alternative to the homograft is the grafting of animal organs, or the xenograft, and, at the dawn of the 21 st century, this type of graft has entered an active, promising phase, with the creation of pigs that have been partially "humanized" and are thus, as a consequence, immunocompatible. for reasons of genetic and physiological similarity, the first choice for such grafts was to use apes or monkeys. however, this idea was quickly abandoned, for several reasons; a non-negligible risk of viral infection due to the phylogenetic kinship of human and simian species; a slow growth rate; a low reproduction rate and, finally, laws that protect primates. these disadvantages are not found, or are at least minimized, in the pig: the risk of a viral infection passing from the pig to man should be low because of the species barrier (but nevertheless, it ought to be evaluated), the pig growth rate is relatively rapid, pig litters are large and pig organs are of a size close to those of man. hyperacute rejection of grafts is the critical obstacle that must be overcome before it is possible even to envisage the feasibility of xenotransplantation. hyperacute rejection is caused by the presence in man of natural antibodies (xenoantibodies) that accumulate throughout a lifetime and are directed against antigenic motifs carried by the products of the digestion of food or dust that is breathed in. xenoantibodies are mobilized when a xenograft occurs, and when they combine with xenoantigens brought by the graft this activates immune proteins such as the complement proteins. the catastrophic effect of this xenoantibody/xenoantigen combination is a vascular thrombosis followed by necrosis and rejection of the graft. the pig xenoantigen that is considered to be the one mainly responsible for the phenomenon of rejection in man is a sugar molecule, galactose α-1,3-galactose, located on the plasma membrane of endothelial cells. synthesis of this molecule requires the enzyme α-1,3-galactosyltransferase, which is present in most mammals, but absent in man and the primates. this enzyme disappeared in man around twenty million years ago, following a double mutation of the gene. in 2002, cloning by nuclear transfer, associated with the invalidation of the gene coding for galactosyltransferase, made it possible to create pigs without galactose α-1,3-galactose 43 . this performance shows that the xenotransplantation objective, although it can only be envisaged over the long term, is not based on false hopes. plant gmos, gene therapy, embryonic stem cells, therapeutic cloning, and xenotransplantation are a few of the many examples that show how far experimentation on living beings has progressed in just a few decades, from inquiries into the operating mechanism of an organ or a cell, in the interests of pure understanding, to a programmed process, planned with an objective in mind, the chances of success of this objective being analyzed and counted in terms of impact and cost-effectiveness. during the renaissance, ecclesiastical authorities, worried by the libertarian forces that were assailing them, applied the brakes to audacious questioning of dogma such as the circumterrestrial revolution of the sun that had, since ancient times, placed man at the center of the universe. nowadays, civil authorities, conscious of the potential but also of the possible misuses of genetic manipulation, insist on having the right to oversee such procedures. in truth, since the 19 th century, governments have been interesting themselves in research on living beings and encouraging it, as long as its applications have allowed improvements in human health. this has been the case for vaccinations against infectious diseases or for prevention of microbial infections by means of aseptic or antiseptic methods. with the breakthroughs made in genetic manipulation at the end of the 20 th century, it was more than just the results of experiments on living beings that attracted the attention of the political authorities, it was, above all, the manner in which the experimental method, with all its hazards, made use of living material, sometimes of human origin, in order to unlock mysteries. conscious of the social impact of emerging discoveries that are subject to considerable media coverage and are sensationalized in both the written and audiovisual media, the state, with the help of researchers and philosophers, has laid down a code of bioethics, applied through strict or even restrictive legislation. it remains to be seen whether the rules of this code will continue to be an inviolable absolute or will be modified according to the evolution of the moral codes and the cultures of nations. university teaching and the education of a society must now take into account not only the content of successive discoveries, but also the fallout of these discoveries, insofar as they concern man, and even the ethical justification of the methods that have allowed these discoveries. in "remaking" living beings according to imposed norms, and in scheduling, in a certain fashion, the manufacture of life according to new codes, certain questions move from the "how" to the "why", i.e., from the scientific domain that is accessible to human thought to the metaphysical sphere, with its problems of the limit of what is surmountable and tolerable in terms of ethics. in his birth of predictive medicine, jacques ruffié (1921 -2004) reminds us that medicine has evolved through three stages over the course of time: curative medicine, which has been practiced since ancient times and is still being practiced; preventive medicine, which is more recent, and is designed to prevent people from falling ill, either by vaccinating them, in the case of infectious diseases, or by recommending an appropriate diet and medication in the case of metabolic disorders such as diabetes or arterial hypertension that have been detected by means of systematic examination; and, finally, predictive medicine, a branch of medicine that is still in its early phases, and which is based on modern technology and is able to predict situations of risk because of anomalies detected in the genetic inheritance or because of exposure to environments that are reputed to be dangerous (for example, carcinogenic smoke, asbestos). about one and a half centuries ago, the publication of the introduction to the study of experimental medicine (1865) provided proof, based on scientific arguments, that the time had come to transfer the experience that had been acquired through the experimental method practiced on animal models to the ill person. after claude bernard, attentive to the progress made by ideas and techniques in the physical and chemical sciences, and making use of its own advances in the understanding of the living cell, both normal and pathological, experimental medicine was to live through a development that was without precedent in the history of humanity. to understand the causes of epidemics, nutritional deficiencies, metabolic deviations of hereditary origin and degenerative illnesses, and then to translate these causes in cellular and molecular terms, this was the process undertaken by medicine once it began to use the experimental method. in fact, for several decades, from the beginning of the 19 th century, medicine had already undergone some major revisions of outdated practices and had inaugurated a new era in diagnosis. for example, the differential diagnosis of pulmonary ailments became possible because of the invention of the stethoscope by rené laennec (1781 -1826) and the practice of auscultation and percussion by joseph skoda (1805 -1881), the uncontested master of the vienna school. in france, pierre louis (1787 -1872) used statistical methods to evaluate the efficacy of different treatments. armand trousseau (1801 -1867), a pupil of pierre bretonneau (1787 -1862) wrote a famous treatise on the hôtel-dieu medical clinic in paris. in great britain, chronic nephritis, with its identifying symptoms, was described by richard bright (1789 -1858), paralysis agitans by james parkinson (1755 -1824) and addison's disease, which affects the adrenal glands, by thomas addison (1793 -1860). during the 19 th century, many other famous names signaled the arrival of a medicine that was resolutely anatomoclinical in nature, in line with bernardian doctrine. "experimental medicine is thus a medicine that claims to understand the laws of the organism in sickness and in health, in such a way that it not only predicts phenomena, but also in such a way that it can regulate and modify them, within certain limits." in the introduction to the study of experimental medicine, claude bernard stigmatizes the relics of an empirical medicine that was still being practiced in his day and was forgetful of rationalism. the terms that he uses are without leniency: "i have often heard doctors who, when asked the reason for a diagnosis, reply that they don't know how they recognize such a case, but it is obvious, or who, when asked why they administer certain remedies, reply that they don't really know how to put it exactly, and that anyway they are not required to give a reason, because it is their medical tact and their intuition that guides them. it is easy to understand that doctors who reason in this way are denying science. what is more, it is impossible to be too forceful in rising up against such ideas, which are bad not only because they stifle any scientific seed in the young, but also, above all, because they favor laziness, ignorance and charlatanism." in order to evaluate the meaning of these words, it should be remembered that in claude bernard's time, the medical profession was far from considering the microscope as a useful instrument for the study of cell structures and that the cause and effect relationship between bacterial germs and infections was still to be shown. with the development of increasingly effective instruments for exploration, and of methods for microanalyses concerning a wide range of blood and humoral constants, throughout the 20 th century, medicine, which was once empirical, has now become scientific. claude bernard's dream, experimental medicine, is now operative. this medicine is no longer content simply to determine the cause of an illness and to locate the affected organ, which was the major objective of clinical medicine, but it seeks to detect the mechanisms of pathological processes by means of histological and physicochemical explorations. this medicine is no longer willing to passively monitor the evolution of an infectious disease. after having identified the responsible germ, it tries to target this germ with the chemical weapon that is able to selectively destroy it. this medicine is no longer content simply to find remedies, it aims to understand the mode of action. it sets itself the goal of meeting challenges such as finding the genetic cause of degenerative illnesses or of cancers and developing appropriate therapies. it is supported by statistical data. when a new drug is implemented, the results are now evaluated by the double blind method: neither any of the patients (treated and non-treated) nor any of the investigators are aware of who has been administered with the drug and who has been administered with a placebo. in the surgical domain, audacious techniques have also led to considerable progress, particularly in neurosurgery and in cardiovascular surgery. thanks to robotics and to computer technology, remote surgery or telesurgery has become practicable, although up until not that long ago, it was only to be found in fiction. faced with emerging problems in public health, the task undertaken by experimental medicine is immense. in the middle of the 20 th century, the spectacular recovery from high-incidence infectious diseases such as pneumococcal pneumonia, meningococcal meningitis or acute forms of tuberculosis, which that was brought about by antibiotics, gave rise to the idea that medicine had won a battle against the microbial world and that, from then on, it would be able to control the evolution of infectious diseases and to offer rational treatments. the gradual appearance of a microbial resistance to antibiotics has brought an end to this euphoric era. penicillin, for example, which was put on the market at the end of the 1940s, was active on practically all strains of staphylococcus aureus. sixty years later, more than 90% of the strains of this same microbe are resistant to penicillin. the incidence of nosocomial infections, which are contracted in health care facilities, never ceases to rise. at present, around 10% of the hospitalizations that take place are complicated by the patient developing a nosocomial infection. equally worrying are the re-emergence of diseases that were once considered to be under control, such as tuberculosis or poliomyelitis in africa, and the emergence of new diseases such as aids, whose hiv virus (human immunodeficiency virus), which was identified at the beginning of the 1980s, has generated a pandemic that has spread throughout the planet. infectious diseases are currently responsible for more than a quarter of human deaths. the koch bacillus responsible for tuberculosis and the pneumococcus kill three to four million people a year, around the world. in 2004, hiv killed more than three million people, and more than forty million people are infected. one person is infected every 30 seconds. in viral diseases, the role of vectors (insects, various animals) as well as the notions of contagiousness and aggressivity have been emphasized. we have only to remember the dreadful contagiousness and aggressivity of the spanish flu virus (influenzavirus ah1n1) which, in 1918 -1919, killed more human beings around the world than the first world war that preceded it. in contrast, the sars (severe acute respiratory syndrome) epidemic of 2003, the vector of which was doubtless the civet, a small carnivore raised in china and desired for its meat, was rapidly contained because of its low contagiousness and also because of the isolation measures that were taken. human behavior is not without its effect on the emergence of viral diseases. the growth in intercontinental travel and human migration, as well as intensive deforestation in africa and south america, which bring virus vectors into contact with man, are factors concerned in the emergence of viral diseases that risk being explosive and devastating. in this context, the history of the ebola virus and of the marburg virus, which cause violent hemorrhaging, is edifying. in 1967, in the german village of marburg, an epidemic of unknown origin broke out, the illness manifesting itself with brutal suddenness by vomiting, diarrhea, a high fever and an increased tendency to bleed. this pathology, which was contained rapidly by means of drastic isolation measures, was found to be of viral origin. the pathogen concerned was a filovirus (filiform virus). a brief enquiry showed that the origin of the epidemic was contact between technicians of a pharmaceutical company and monkeys that had been imported from uganda and that were carrying the virus. in 1976, two other epidemics, characterized by severe and often fatal hemorrhagic fevers, were reported in the sudan and the republic of the congo. here again, the illness was caused by a filovirus, the ebola virus. at the time of writing, only public health organizations, including the nih (national institutes of health) in the usa, have attempted to set up vaccination and therapeutic strategies. research on these dangerous viruses requires high security installations that are particularly costly, so that private companies are reticent about investing in work that is only targeted on poor regions and which concerns epidemics that have so far been contained successfully, although one day the ebola and the marburg virus could quite well escape their african niches. experimental medicine must also understand the colossal challenge of the five thousand hereditary diseases that are currently listed, the most handicapping of which are myopathies and neuropathies. given the means that are available to the contemporary clinician in order to assign each of these diseases to a genetic defect, one can only be amazed by the mass of information about them that has accumulated over a century, since the first diagnosis of a hereditary disease, alcaptonuria, which was made in 1902 by archibald garrod (1857 -1936), a doctor at london's st bartholomew's hospital. alcaptonuria is a non-serious genetic flaw that can be detected easily by a blackening of the urine. it is the result of a blockage caused by the mutation of an enzyme involved in the catabolism of an amino acid, tyrosine, this blockage leading to the accumulation of homogentisic acid, the polymerization of which gives rise to a brownish color. the patient examined by garrod was a young boy. investigation of the family history revealed that transmission of the flaw was correlated to cross-cousin marriages and followed mendel's laws for recessive traits. garrod demonstrated other hereditary-type anomalies, cystinuria, porphyria and pentosuria. in 1909, these observations were published in a work that became a classic: inborn errors of metabolism. in 1956, the specific molecular defect of a metabolic anomaly linked to a mutation was identified for the first time by the german-born british biochemist vernon ingram. this was the hemoglobin defect responsible for drepanocytosis or sickle cell anemia: a glutamic acid in the β chain is replaced by a valine. the consequence of this simple change is a modification of the structure of the hemoglobin, leading to a sickle-shaped deformation of the red blood cells, the increased fragility of these cells and also a tendency towards cell lysis. this discovery made use of the electrophoresis and chromatography techniques that had just been introduced in biochemistry (chapter iii-6.2.2): such a discovery would not have been possible without these techniques. because of the progress made in molecular biology, the nosological framework of hereditary diseases has been greatly enriched over the last twenty years. for example, at present, more than one hundred hereditary-type myopathies have been identified by accurately locating molecular lesions in the genomic dna and characterizing the structural and functional modifications of the mutated proteins. certain health problems present real challenges for experimental research. this is the case for the spongiform encephalopathy caused by a prion (proteinaceous infectious particle) , which has all the more impact on the imagination because its etiology remains a mystery. it is also the case for degenerescence of the central nervous system correlated with aging, alzheimer's disease being a striking example, although, as far as familial forms of this illness are concerned, i.e., those of the hereditary type, it has been possible to link the invasion of the brain by a so-called amyloid peptide, which accumulates in plaques, on the one hand, and, on the other hand, the absence, due to a mutation, of an enzyme, a peptidase, which normally degrades the amyloid peptide. contemporary scientific medicine sometimes acquires a revolutionary aspect. here again, as with other disciplines involved in the study of living beings, it arises from discoveries resulting from the principle of serendipity (chapter iii-2.2.3). this was the case when, in january 1987, a team in grenoble, france 44 , led by the neurosurgeon alim-louis benabid (b. 1942) and the neurologist pierre pollack (b. 1950) discovered by accident that in patients affected by parkinson's disease a beneficial effect was achieved by deep, high-frequency electrical stimulation of the brain. the three major symptoms of parkinson's disease are muscular rigidity, a tremor when at rest and a slowing down of the execution of movements. in the 1960s, the swedish team of arvid carlsson (b. 1923) , who won the nobel prize for physiology and medicine, demonstrated a relationship between the parkinson syndrome and a deficit in the secretion of a neurotransmitter, dopamine. a group of neurons that is limited to half a million (of the 100 billion contained in the brain) produces this neurotransmitter in a small structure located in the midbrain, called the substantia nigra. the neurons of the substantia nigra have elongations that interact with different nerve formations (called nuclei) including the subthalamic nucleus. in 1990, bergman et al. 45 published an article that describes a curious relationship between a provoked lesion of the subthalamic nucleus and the disappearance of the signs of parkinson's disease in a monkey which had been made parkinsonian by chemical treatment. this publication led the team in grenoble to target their electrical stimulation on the subthalamic nucleus. this was completely successful. this electrical stimulation procedure, which is now well-codified, involves using stereotactic neurosurgical techniques, controlled by magnetic resonance imaging, to implant an electrode into the subthalamic nucleus. the electrode is connected to a generator that is implanted under the patient's clavicle. the generator sends brief electrical impulses of frequencies from 100 to 200 hz. under the effect of this stimulation, the characteristic symptoms of the illness, particularly the static tremor and bradykinesis, regress in a spectacular manner. the mechanism by which this stimulation acts is not yet understood. no doubt this has to do with complex phenomena involving the inhibition of certain neuron relays near to the substantia nigra, which remain to be deciphered. here we have a typical case of a progression from an experimental fact, discovered by accident, towards the analysis of its cause. from the point of view of the experimental method, it is interesting to make parallels between this discovery by serendipitous means of the beneficial role of electrical stimulation of the midbrain in parkinson's disease and cartesian style programmed research that aims to graft into the brain of parkinson's disease sufferers embryonic stem cells differentiated into dopaminergic neurons 46 . civilian society and its armed force, the political authorities, have understood that experimental science has the tools, the method and the thought processes necessary to develop strategies for prevention and healing. immune cells of this person and induces the synthesis of immune proteins. finally, it seems relatively certain that hopes concerning gene therapy for hereditary diseases will be fulfilled within before too long (chapter iv-2.3.1). one of the traits that is characteristic of the period we live in, and which arises partly from the economic stakes involved, is the shortening of the time that elapses between a discovery being made and the application of that discovery. for example, interfering rnas, which were discovered in the 1990s (chapter iv-1.2.2) are already the subject of therapeutic investigation. more than a hundred biopharmaceutical companies around the world are using them with a view to producing drugs from them 47 . in mice, a certain number of synthetic interfering rnas have proved their efficacy in silencing genes which, following mutation, have acquired carcinogenic potential. however, the use of interfering rnas as therapeutic agents requires them to be stabilized, because they are fragile molecules. the group headed by achim aigner 48 (b. 1965 ), at the school of medicine in marburg, germany, managed to stabilize a synthetic interfering rna by complexing it with polyethyleneimine, and this interfering rna is able to block the expression of a receptor involved in cancerization (c-erbb2/neu(her-2) receptor). used in mice, such a drug appears promising. despite the undeniable progress that has been made, experimental medicine is still some way from finding solutions to some of the enigmas that it meets along the way, and which underline the complexity of living beings. some time ago, it was thought that after having invalidated a gene coding for a protein that is indispensable to a function, we would discover the secret of a cause-and-effect relationship. experimental practice has shown that, generally, this is far from being the case. another example of the complex relationships that exist in living beings is the interference of the mental and the organic. one experiment that suggests this interference was carried out on mice who had acquired a form of pathology similar to huntington's chorea, by transgenesis. mice from the same line were separated into two batches, one acting as a control, and the other being subjected to daily mental stimulation, including memorization tests. unexpectedly, the appearance of symptoms was noticeably slowed down in mice who had been subjected to mental gymnastics 49 , as if the brain, by intentionally mobilizing its neuron activity, was able to secrete substances able to alleviate its own defects. in short, by means of possible retroactive mechanisms that are called upon by the mind, the brain appears to act as actor and spectator. at the turn of the 21 st century, experimental medicine was being nourished by techniques inherited from experimental physics, chemistry, and even mathematics and computer technology, in the same way as the other sciences of living beings. the progress made in medical imaging techniques has been particularly impressive since the time, at the end of the 19 th century, when the x-rays discovered by wilhelm röntgen made it possible to view the structure of the human skeleton. the saga of x-radiation continued through the 20 th century (chapter iii-2.6.1). for the last few decades, new imaging techniques have come to the fore. they have spread rapidly, and been refined. ultrasound imaging, which is based on the principle of the reflection of ultrasound waves off of different kinds of surfaces, has become an everyday technique for viewing blood flow in blood vessels and the heart. however, it is mainly in the study of the brain that medical imaging has benefited from technical advances in the domains of physics and computer technology, and it has been innovative in assigning cognitive activities to well-identified anatomical structures. this functional neuroanatomy makes it possible, in a non-trauma-inducing manner, to monitor and locate the operation of neuron networks with great temporal and spatial precision, during various cognitive tasks such as reading and the written or oral expression of thought. the middle of the 20 th century saw the gradual development of two methods for exploring zones of cerebral activity, electroencephalography and magnetoencephalography. at present, these techniques are being taken over by mri (magnetic resonance imaging) ( figure iv.14) . the principle of mri is based on the detection of hydrogen nuclei and their differentiation according to their environment. functional mri leads to the location of the areas of the brain that are active during calculation exercises, the perception of sounds, language and objects, and memorization, with a resolution of just a few millimeters. its power of exploration is such that it has been possible to analyze the brain response, in sleeping or awake babies who are only three months old, to auditory stimuli from language that either makes sense or does not make sense 50 . the response, located in the left hemisphere and the prefrontal cortex, leads to the conclusion that, from the first months of life, there are zones of the brain that are potentially active before the first attempts at language appear. both in france (cea-saclay and the frederic joliot hospital at orsay) and abroad, recent mri performance has encouraged projects concerning the manufacture of instruments able to produce magnetic fields of around ten teslas, which allows an unequaled definition in the identification of areas of the brain assigned to specific cognitive functions and in the highly accurate determination of the location of pathological lesions. a technique that is complementary to mri is positron emission tomography (pet). this generally uses water labeled with oxygen 15 ( 15 o), a radioactive isotope of natural oxygen that has a very short lifetime (123 s), produced extemporaneously in a cyclotron by bombardment of an 14 n target with protons. the radiolabeled water is injected into the blood flow of the patient. it is found in greater concentration in the zones that are the most irrigated by blood capillaries. the positrons that it emits collide with the surrounding electrons and give rise to photons that can be detected by the appropriate apparatus. affected by a stimulus (whether this stimulus results from talking, writing or listening), the blood irrigation of the zones of the brain that have been specifically excited increases noticeably. the location of the positron emission provides information about the location of these zones. within a few dozen minutes, it is possible to locate a highly vascularized cerebral tumor. pet can use molecules other than water, such as organic molecules labeled with positron-emitting atoms, ( 18 f) fluorine (half life 110 min) and ( 11 c) carbon (half life 20 min). around twenty years ago, in canada, an analogue of l-dopa, the precursor of dopamine in the brain, 18 f-6-l-fluorodopa, was synthesized, and was found to be an excellent probe for determining the capture capability of the endings of the dopaminergic neurons in the striatum. in patients suffering from parkinson's disease, this capture capability is noticeably reduced. at present, pet involving 18 f-6-l-fluorodopa is being used to evaluate the survival of dopaminergic cells grafted into the striata of parkinson's disease sufferers 51,52 . nowadays, brain imaging techniques can be used to explore the electromagnetic anomalies of neurological or neuropsychiatric illnesses such as huntington's chorea, the different forms of alzheimer's disease or even autism, the genetic origin of which is in the process of being deciphered. a bridge has now been built between the molecular defects identified by genetics and the electromagnetic anomalies that result, analyzed by functional cerebral imaging. it was not so long ago that descartes considered that human thought was unconnected to a material support (chapter ii-3.4.3). we are not far from the era when broca located the language area in a specific zone of the brain after the autopsy of an aphasic patient (chapter iii-3.1), thus opening the door to another scientific domain, neuropsychology, which had previously only been the subject of speculation. the consequences, from the societal point of view, were far from being insignificant. thus autism, which was once suspected of being caused by errors in the mother's behavior with respect to her child, has been shown to be a disturbance in the development of the fetal nervous system, in the temporooccipital region. while the neurosciences occupy a preponderant position in the medicine of the beginning of the 21 st century, because of the development of techniques that aim to analyze even the functions of thought, emerging methodologies of another order, such as gene therapy (chapter iv-2.2), are in the process of completely modifying our ways of treating and curing a range of previously incurable human diseases, from incapacitating immune disorders to cardiovascular diseases and cancer. "it is in the domain of thought about the future that man is singled out. we are beings who have an imagination. not content to live in the present, to profit from past experience, we remain haunted by a future that we are conscious of constantly entering. this obsession with the future has been a powerful driving force in cultural evolution. we seek to predict in order to avoid the worst and to better prepare for our tomorrows." by predicting potential dangers in subjects who are in good health, predictive medicine aims to provide the means of avoiding these dangers. these dangers can be intrinsic in nature, being written, for example, into a certain genome dna sequence, or they can be extrinsic in nature, linked to an unsuspectedly deleterious environment. in each generation, mutations occur, certain of which can lead to so-called genetic diseases; between 3 and 4% of newborns are affected. besides these spontaneous mutations, there are also mutations arising from the genetic inheritance of the parents. the purpose of genetic counseling is to warn parents when the existence of a potentially serious genetic flaw is suspected. the highlighting of genes that give a predisposition for cancer (proto-oncogenes) is a convincing illustration of the power of predictive medicine. this involves genes that control the synthesis of growth factors, the activity of which is essential to embryogenesis and to the repair of damaged tissue. while they are normally subject to strict control by anti-oncogenes, proto-oncogenes are able to become active in an anarchical manner, under different influences, and to transform themselves into cancer-generating oncogenes. recently, mutations have been found in two genes, brca1 and brca2, these mutations giving a predisposition for cancers of the breast and of the ovary. thanks to genetic exploration, it will soon be possible to predict whether a cancer of the breast will have a rapid progression leading to uncontrollable metastases or a slow progression. depending on the case patients will be subject to heavy chemotherapy or to a less aggressive treatment. in this context, targeted therapy with monoclonal antibodies is a source of great hope. while genetic inheritance has a role in cancer, the environment plays a notinsignificant role as well. this is the case, for example, in lung cancer sufferers who smoke tobacco, cancer of oesophagus in those who drink alcohol and job-related cancers in those working in factories producing colorants or materials derived from asbestos or tars. cardiovascular diseases are the primary cause of death in the more developed countries, involving either an infarctus, or a stroke. many risk factors for these diseases are known, i.e., metabolic deviations affecting cholesterol or the blood serum proteins involved in the transport of lipids. these metabolic anomalies result in a syndrome known as atherosclerosis, which is characterized anatomically by the deposit of fats in the form plaques in the arteries. while genetic factors are at the origin of these metabolic problems, the latter are clearly amplified by an inappropriate diet. the role of predictive medicine is to recognize the genes that are responsible, warn individuals of the risks they are running and to advise them about the types of lifestyle and diet that do not increase these risks. being able to predict, predictive medicine should be able to prevent by means of targeted drugs. within this context, it gives rise to reflection upon polymorphism linked to variation in a single nucleotide in the dna of the genome of an individual. known as snp (single nucleotide polymorphism), this polymorphism has proved to be a very useful auxiliary in molecular medicine. hundreds of thousands of snps are present in the human genome and several tens of thousands in genes coding for the proteins. where they are located differs according to ethnic backgrounds. among these snps, some appear to be linked to certain pathologies, such as certain forms of cancer or degenerative illnesses such as alzheimer's disease. in addition, in a small number of patients, the location of certain snps has been connected with previously-inexplicable drug incompatibilities. in line with these observations, pharmacogenomics, a branch of pharmacology that deals directly with genome sequence data, is trying to evaluate the impact of "snp variants" on the efficacy and toxicity of drugs and to understand the genetic bases that explain the differences that are observed in the responses of different individuals to the same medication 53 . rather than using a standard drug that is not very efficacious or causes adverse side effects, it might be possible, depending on the genetic profile of the patient, to use a drug that is more appropriate to his or her genetic map. it is doubtless not just a fantasy to imagine that, in 20 or 30 years' time, a patient visiting the doctor will be offered a genetic map thanks to cells taken from the buccal mucosa. finding snp variants that are known to be responsible for drug incompatibilities in such a map will make a targeted prescription possible. it will allow the detection of genes for susceptibility to an illness, at the same time uncovering targets for new drugs. pharmacogenomics, which is still called new pharmacogenetics, contrasts with old pharmacogenetics in which, having found an adverse clinical response to a certain therapy, an attempt was made to identify the protein target of the incriminated drug, and then to go back to the gene coding for this protein, and to look for the mutation responsible for the aberrant response to the drug. the existence of customized predictive medicine, which would read the destinies of individuals in their genes, would not be without its consequences in the life of a citizen. by registering each citizen with a genetic map, matched with a named identity card, predictive medicine might begin to take on the aspect of a janus, with his beneficent face warning subjects of potential risks of metabolic problems, and guiding them towards the actions to be taken to lower the risks, but also with his evil face delivering each individual's intimate details to the indiscrete inquisitiveness of investigators who are operating towards their own ends (insurance companies, employers…). no less worrying would be the sly but predictable transformation of the individuality of the repaired or even doped human being within a system of imposed, docilely-accepted assistance. in the 19 th and 20 th centuries, the methodology for biological experimentation underwent a revolution caused by the progress made in the domain of chemistry, both analytical chemistry, with the deciphering of increasingly complex molecular structures, and also in synthetic chemistry, with the large-scale production of tens of thousands of new molecules. the effects of these molecules, which might eventually be used as drugs, were tested directly on animals. it was thus that in 1910 the german chemist paul ehrlich discovered salvarsan, a derivative of arsenic, which was active against a type of treponeme, the agent of syphilis. this was the result of a systematic analysis of the effect of synthetic products, aromatic derivatives of arsenic acid, on syphilis in rabbits. salvarsan was the 606 th derivative that was tested, and this is why it was called 606 for a long time before it was given the name salvarsan. sometimes, lucky chance shows surprising and unexpected properties in synthetic molecules. this was the case for chlorpromazine, which was initially used as an antihistamine. it was luck that led to its antipsychotic activity being discovered in 1950. a new era opened up in psychiatry with the arrival of synthetic narcoleptics like chlorpromazine. a new chemical science known as combinatory chemistry, which dates from the 1990s, has aroused an increasing amount of interest in pharmacology. this involves making two or more species of organic molecules that carry reactive functional residues react in solution or in the solid phase in such a way as to synthesize, by means of all possible combinations, a number of final and intermediary products that is situated in the hundreds or even the thousands, and which makes up chemical library or drug library. we can directly test all of the products formed on a sample of eukaryotic cells, in order to verify their effects (for example the inhibition of an anarchical proliferation of cancerous cells), or on microorganisms in order to evaluate an antibiotic capability. we can also proceed straight away with the fractioning of the reaction products and the testing of each of the fractions. if the response is positive, fractioning is continued until the molecular species responsible for the desired effect is obtained. other evaluation parameters for this molecule, such as its absorption, its toxicity and its metabolic future (distribution in the organs, chemical modifications and excretion) are then explored, first in cells, and then in animals (rats, mice), thus comprising pre-clinical tests. these screening operations, which are said to be high-throughput, require automation and robotization aided by powerful computer technology. each year, pharmaceutical companies screen tens of thousands of different molecules on hundreds of targets. complementary to combinatory chemistry, in silico chemistry works by molecular modeling and uses computer programs for the rational design of new drugs that are able to fix onto specific protein targets. the purpose of this is to provide a virtual follow up to modifications in the reactivity of a given drug molecule as a function of the modifications imposed on its structure, for example, the addition of residues that differ according to their electrophilic or hydrophilic properties, or according to the length of their side-chain. provided there is a chemical library and we know the three-dimensional structure of a macromolecule, for example an enzyme, as well as the nature of the residues that define its active site, we can hope to select and chemically modify a substance that is able recognize the active site of this enzyme and to make an almost perfect ligand out of it which is able to efficiently block the operation of the target enzyme. this method, which is based on computer-aided chemistry, is called "structure-based drug design", and has had some notable successes. it has made it possible to develop an inhibitor capable of blocking a protease involved in the replication of the aids virus. however, both in combinatory chemistry and in molecular modeling, the many successes that have been achieved remain modest in number compared to the means that have been deployed to achieve them. in terms of statistics, out of ten thousand molecules that are recognized as being efficacious for a given target in vitro, around one hundred are chosen for preclinical trials on animals, around ten are chosen for preclinical trials in man and only one will come out as a drug. the financial and economic effect is far from being negligible. it has even become a preoccupation in a system where merciless competition is the rule. in addition to synthetic chemistry, preparative chemistry, which is based on the isolation of natural molecules, is now the subject of renewed interest, due to the introduction of high-throughput techniques. high-throughput screening, which is an essential tool in combinatory chemistry, is also carried out to ensure the systematic detection and isolation of natural substances having interesting pharmacological activities such as antibiotic activities or anti-cancer activities, based on marine animals, microscopic fungi, prokaryotic organisms and various plants. for example, among the substances that have been isolated recently are cibrostatin, a specific cytostatic of melanoma cells, from a marine sponge, mannopeptimycin, a bacterial antibiotic from an actinobacterium streptomyces hydroscopicus and a whole set of alkaloids with a cytostatic activity with respect to human tumor cells from an exotic plant of the genus daphniphyllum. the molecular diversity of the living world is such that the reserves of natural products having pharmacological activities are far from being exhausted. so far, only a small percentage of the microbial species populating the earth have been listed. the depths of the oceans harbor many unknown species. thousands of insect species remain to be discovered in the canopies of tropical rainforests. exploration of the plant kingdom is far from being complete. the listing of natural molecules having a therapeutic activity has only just begun. the hunt promises to be a fruitful one, all the more so because the highthroughput screening methods that can now be used greatly increase the efficiency of the search. high-throughput screening, applied to natural molecules, has overturned the methodological procedures that were in use until recently, which progress through logical steps, using relatively simple artisanal analytical methods, from observation, often resulting from serendipity, to the isolation of the active substance. thus, in the 19 th century, using inherited traditional knowledge that a decoction of cinchona officinalis bark calms malaria crises, pierre joseph pelletier (1788 -1842) and joseph bienaimé caventou (1795 -1877) decided to isolate the active substance of this bark. from the raw extract, they purified an alkaloid, quinine, which proved to be the anti-malarial substance they were looking for. more recently, the starting point of florey and chain's isolation of penicillin from the microscopic fungus penicillium notatum was the fortuitous observation made by fleming that this penicillium secretes an antibiotic factor (chapter iii-2.2.5). there are many examples in which serendipity has been the principle factor involved in the discovery of a drug, and this will no doubt continue to be the case. the appearance of a lucky chance, after all, is not incompatible with highthroughput practices. also, it is not impossible that in the future there will be a conjugation of the discovery of new natural substances and the use of combinatory chemistry, with the aim of manufacturing derivatives having a much greater power of action and quality of specificity from these substances 54 . to sum up, the experimental method has caused contemporary medicine to take a giant leap forward, with the discovery of increasingly high-performance functional exploration techniques, the development of therapies using molecules that are already present in nature or are manufactured by synthesis and the more and more advanced understanding of molecular mechanisms that takes into account the basic idea of the pioneers of molecular biology was that the function of a macromolecule depended on its structure. thus, perutz's elucidation of the tetrameric three-dimensional structure of hemoglobin, and of its modifications depending on the degree of oxygenation, shed a considerable amount of light on the cooperative mechanism of the transition from the hemoglobin state to the oxyhemoglobin state (chapter iii-6.2.1). in the same way, an understanding of the structure of many enzymes, receptors and transporters of metabolites has shed light on their mechanisms. in a parallel manner to the exploration of the structures and functions of proteins, that of genomes has made remarkable progress. the subtle entanglements of genomics and proteomics that have become accessible to the experimental method are the order of the day. one major challenge for post-genomics is to understand how proteins, expressed by genes, interact with one another to generate functions that characterize cellular specificity. even more ambitious are attempts to understand the operation of organs or even of living organisms, based on mechanisms that are implemented at molecular level. these attempts lead straight to an integrated biology, that is, a biology that aims to understand the overall functioning of living beings. taking as its purpose the access to emerging functions, resulting from interactions between macromolecules, integrated biology first tries to invent methods that make it possible to detect these interactions. strengthened by the information obtained, it tries to integrate this information with a mathematized language into modules that attempt to simulate living beings. from the simplistic procedures of the middle of the 20 th century, which were justifiable within the reductionist context of this period, and which involved considering each species of proteins as an autonomous functional entity, we have moved on to the idea that the different species of protein that inhabit a cell have a dialogue with one other, and that they may move from one endocellular organelle to another, depending on post-translational modifications (for example, phosphorylations) that change their conformation and, at the same time, their reactivity and their behavior. thus, an enzyme protein is not only defined by its catalytic performance with respect to a given substrate, but also by its place in a metabolic network where it interacts in a dynamic and transitory manner with a multitude of other protein species (figure iv.15a) . the concept of cell signaling has also evolved. instead of considering that a cell membrane receptor, activated by fixation of an extracellular ligand (a hormone, for example), addresses the information received to an endocellular effector protein via a linear cascade of individual proteins, it has come to be postulated that communication between an activated receptor and its effector is mediated by proteins organized into interactive networks ( figure iv.15b) . this machinery provides more flexibility in the addressing of messages to effectors. the diagram on the right shows that besides its catalytic function, protein a interacts with other proteins in the cell. b -case of the transduction of a signal that is external to the cell (a hormone, for example). the diagram on the left refers to the classical idea of signaling from a receptor r according to a linear cascade of protein-protein interactions inside the cell, leading to an effector z. the diagram on the right shows that the signal is spread through proteins organized into interactive networks. another subject to be considered is the density of macromolecules of all types, such as proteins, nucleic acids, lipids and polysaccharides, contained by a microorganism or a eukaryotic cell, which reaches values of 300 to 500 g/liter, denoting a semi-solid state or a considerable degree of compacting. however, for technical reasons, kinetic studies carried out in vitro on isolated enzymes have been carried out with solutions that are 1 000 or 100 000 times more dilute. conscious of this difference in scale between information obtained from in vitro studies and the in vivo reality, the biology of today is trying to re-evaluate molecular dynamics within the context of a cell. this is why we are seeing the birth of an integrated (or integrative) biology of functions, which, using modeling procedures, aims to achieve an understanding of the temperospatial dynamics of the interactive components inside cells. this holistic conception of biological systems ("systems biology ") has been made possible by progress in technological expertise in domains as varied as biochemistry, molecular biology, physical optics, electronics, nanomechanics, physical and mathematical modeling and computer technology. it is a necessary complement to the classical experimental method based on bernardian determinism which, in order to connect an effect with a cause, explores living beings in a manner that is often monoparametric and is inevitably reductionist. this signals a change in paradigm in the experimental approach to living beings. a particularly effective investigative method used to explore the dialogue between proteins is the double-hybrid by genetic construction, two proteins, p 1 and p 2 , whose interaction is to be tested, are expressed in the form of fusion proteins in yeast. protein p 1 is fused with the binding domain (gal4-bd) to the dna of gal4, a protein that regulates the transcription of the β-galactosidase gene. protein p 2 is fused with the activation domain of gal4 (gal4-ad). insofar as p 1 interacts with p 2 (b), the gal4 transcription regulation activity is re-established, which is verified by the transcription of the reporter gene. if the opposite occurs, i.e., in the absence of any interaction between the two domains of gal4 (a), the reporter gene is not transcribed. the principle of this method is based on the modular nature of numerous transcription factors in eukaryotes. these factors contain both a dna-binding domain that includes a specific dna-binding site and a transcription activation domain that starts up the machinery for transcribing dna into messenger rna. these two domains can be dissociated and then re-associated in a functional manner, by forming hybrids with interacting proteins. a first protein, p1, is fused with the dna-binding domain of a transcription factor by genetic manipulation, and a second protein, p 2 , is fused with the activation domain of the same transcription factor. if p 1 is able to interact with p 2 , the transcription factor is reconstituted and the reporter gene upon which it depends can be expressed. the trapping technique, which is complementary to the double-hybrid system, was developed to make it possible to identify a set of interactive proteins within a cell. a protein that is included in this set (protein of interest) is fused by genetic engineering techniques to a short polyhistidine chain (called a tag). using this tag, the protein of interest is fixed to a solid medium containing nickel ions, a material that is reactive with respect to the polyhistidine chain. in the presence of a soluble cell extract, the protein of interest binds the cognate proteins of this extract, making it possible to retrieve a complex whose components, corresponding to interactive proteins, can be resolved after denaturing gel electrophoresis and then characterized ( figure iv .17). the techniques described above are backed up by cell imaging techniques that make use of confocal microscopy, which is more directly in keeping with living reality. the optical performance level of confocal microscopes has improved lately, with the arrival of biphotonic and multiphotonic lasers that illuminate precise points of the cell. as we have seen previously (chapter iii-6.2.1), it is possible to create a protein chimera made up of a protein of interest fused with a fluorescent protein, in this case gfp (green fluorescent protein), inside a cell. there are currently several variants of gfp that are able to emit fluorescent light at different wavelengths. this has allowed the development of a technique known as fret (fluorescence resonance energy transfer ) which explores the interaction between two fluorescent proteins. in practice, two gfp variants that have neighboring emission spectra are fused, by genetic engineering inside the cell, to two proteins of interest, p 1 and p 2 , that are suspected of being interactive. if this is the case, the fluorochromes that they carry are sufficiently close that the result is a modification in the intensity of the emission fluorescence of the donor fluorochrome (decrease) and of the receiver fluorochrome (increase), which is readily detectable. all of these studies, taken together, have given rise to the idea that endocellular proteins are organized into networks, that these networks are interactive and that their location in defined compartments of the cell is dependent on epigenetic events such as phosphorylations. two attributes can be found in integrated systems: firstly, the presence of modules, i.e., interactive motifs, which, like the pieces of a jigsaw puzzle, fit together to produce a complex, coherent structure, and, secondly, the emergence of functional properties due to the newly created interactions. the protein of interest p is expressed in the form of a protein fused to a protein "tag" t that is able to bind to a solid support with a specific affinity. the assembly is brought into contact with a cell extract. certain proteins of this extract, a, b and c, which are able to interact with the protein p, become fixed to the latter. in a second step, the tag t is freed from its attachment to protein p by means of a specific cleavage enzyme. the pabc complex that is recovered from the solid medium in soluble form is subjected to polyacrylamide gel electrophoresis, in order to separate and identify its components. given an analytical description of the basic building blocks that are used to construct living systems, and an understanding of their modes of association in defined circumstances, it is normal to try to reconstruct, in their entirety, mechanisms that show the functioning of these systems. this idea was first applied to the yeast saccharomyces cerevisiae for different reasons, such as cell homogeneity (in principle, and, in any case, statistically speaking, all yeast cells have the same genome and the same proteome), an in-depth understanding of the genome and the proteome and the presence of a vast directory of well-characterized mutants. the use of techniques for the detection of interactions between proteins has revealed the existence of a potential dialogue of unexpected richness between a multitude of proteins ( figure iv.18) , in the yeast cell. it is necessary to reflect upon this evidence, which leads to the postulate that, for a given protein, there are mechanisms that restrict and select the many partners able to react with it at a precise moment.faced with a situation in which chance has the upper hand, leading to uncontrollable anarchy, it is necessary to have regulation, which is underpinned by darwinian logic. example of an interaction network involving the yeast sup45 prion protein. the line of dashes refers to experimental data concerning the interaction of sup45 with another protein, sup35. the lines in bold refer to interactions taken from experimental data; while the fine lines refer to predictions, particularly phylogenetic ones. this logic arises from a choice of the most efficient reaction path, which is first of all dictated by the speed constants involved in the association and dissociation of molecular partners, without, however, neglecting any stochastic events that may arise. chemical modifications of proteins participate in this regulation, such as phosphorylation, glycosylation and acylation. the result at cell level is a coherent channeling of the information that is carried from a molecular signal. thus, fixation of a hormone onto a receptor induces a series of modifications to the intracellular proteins that channel information towards an effector terminal, for example an enzyme responsible for the production of a metabolite with a strategic function. how can the sum of the scattered experimental data that we have concerning the catalytic capabilities of a multitude of enzymes of cellular origin be integrated into the operation of a cell? how can we envisage the gene-enzyme relationship according to current evidence concerning the complexity of the genetic message? biocomputing, or bioinformatics, a science that emerged towards the end of the 20 th century, proposes to try to answer these questions. at the turn of the 21 th century, with the development of increasingly powerful computer microprocessors that are able to carry out complex operations with amazing rapidity, the hope arose that it would be possible to simulate processes as varied as the regulation of the cell cycle, molecular flow in metabolic pathways and the reception of molecular signals, for example from hormones by living cells, as well as the transmission of the messages that result. the dream of an in silico virtual biology has become achievable. the first mathematical theory of simple enzyme reaction kinetics was put forward approximately a century ago, by victor henri (1872 -1940). born in marseilles to russian parents, victor henri studied in saint petersburg and then spent time at the universities of göttingen and leipzig before becoming established in paris. having an eclectic mind, studying both psychology and physicochemistry, he had the wonderful intuition that enzyme catalysis arises from a specific mechanism, different from that implemented in a chemical reaction. the study carried out by henri concerned the cleavage of sucrose (table sugar) into fructose and glucose by the action of an enzyme called invertase (sucrase). the term invertase was used because during reaction there was a change in the rotatory power of the sucrose solution, shown by a polarimeter. analysis of the reaction suggested that an enzyme-substrate complex is formed, which breaks down to regenerate the enzyme and liberate the product of the reaction. this analysis gave rise to an equation for the speed of the enzyme reaction as a function of substrate concentration. henri published the results of his experiments both in his thesis, which he presented to the paris faculty of sciences in 1902, and in two articles that appeared in the reports of the academy of sciences 56,57 . in 1913, in biochemische zeitschrift (vol. 49, pp. 333-369), leonor michaëlis and maud menten (1879 -1960) confirmed the results of victor henri and formulated an equation that became a classic, describing the speed of formation of a product from a substrate in enzyme catalysis. since the period of these first studies, the concepts involved in enzyme kinetics have evolved considerably. the first metabolic pathway be deciphered was that of the degradation of glucose (glycolysis) , either into ethanol in yeast, or into lactic acid in muscle tissue. after this, researchers became aware that the activity of enzymes could be modulated as a function of covalent modifications of amino acid residues of their protein chain (phosphorylation, dephosphorylation, acylation…). metabolic flow analysis led to the idea of the limiting reaction. in the 1970s, the idea that there is a single limiting reaction in a chain or a cycle of reactions gave way to the idea that metabolic control is distributed over several reactions, and that each reaction has its own, more-or-less intense control force. another complexity factor came to light with the discovery of allostery 58 . allosteric enzymes have the particularity that they can fix reversibly onto a site that is different from the active site (allosteric site) molecules that are often the terminal products of a chain of reactions: the consequence of this is a conformational modification of the structure of the enzyme that has repercussions on the geometry of the active site and modifies its reactivity with respect to the substrate. in the 1980s, faced with the complexity of the tangle of listed metabolic and signaling networks, attempts were made to use mathematical modeling to show the progress of the traffic of molecules inside a cell in relatively simple metabolic pathways such as glycolysis. in the modeling procedure, the concentrations of the different molecular species are considered to be variables whose variations over time depend on their speed of production and their speed of disappearance, which leads to a set of paired differential equations. with this procedure, we entered the domain of virtual biology. thanks to the creation of increasingly powerful software, the aim of virtual biology is to simulate signaling and metabolic pathways. in the longer term, the aim is to understand the molecular and cellular processes that direct embryo development, or to test the effects of drugs of known target on the metabolic behavior of the cell. metabolic engineering (which is already well developed) comprises two types of models, stoichiometric models and kinetic models. stoichiometric models describe metabolic networks in the stationary state, based on analytical data. kinetic models combine stoichiometric information and that concerning the catalytic capabilities of the enzymes in a metabolic network. in canada, the cyber-cell project plans to model the overall functioning of the machinery which, in the bacterium, includes its metabolism and its proliferation. the aim of the afcs (alliance for cellular signaling), which was launched in the usa, is to understand how signaling occurs in cells such as the b lymphocyte, the macrophage or the cardiac cell in response to different types of stress. the techniques that are used range from identification of all signaling network proteins to the evaluation of the flow of circulating information and to the integration of the data acquired into theoretical models. the european nerve synapse project makes use of similar procedures, with its long-term hope of linking the functioning of nerve cells with the cognitive and behavioral functions of living beings. this is a sizable challenge. in fact, there is far from being a real consensus concerning the principle of a demarcation between, on the one hand, cognitive functions such as language or memory, which are located in precise zones of the brain, and which could be reduced to physicochemical processes, and, on the other hand, forms reflective thought that are expressed through the creative imagination or by judgements concerning ethics or esthetics, the notion of personal responsibility, or even pictorial, architectural or musical beauty. should we see the human soul as the programmer of a superb computer that never ceases to develop from the embryonic state onwards, like john eccles (1903 -1997) and others, or should we admit, like jean-pierre changeux (b. 1936), stanislas dehaene (b. 1965), daniel dennett (b. 1942) and others, that thought is not transcendent, and that it is intrinsically dependent on the brain, which is considered to be a neurochemical system, and thus look for the secret of the individuation of the human being in brain information storage mechanisms with retrocontrol loops associated with subtle neuron architectures, or, in short, refer to a sort of turing machine? whatever the case, in this domain, as in others, simple animal models are used in order to identify elementary processes that are able to explain easily-tested functions such as the memory in anatomical and physiological terms. this is the case for the sea slug or sea hare (chapter iii-6.1) which, despite its rudimentary cognitive capabilities, provides information that can be used to reconstruct higher cognitive functions, present in the brains of mammals. it is clear that the cognitive sciences have reached a stage in which they are emerging from their infancy (chapter iv-3.2). now, ingenious computing methods and a basis for reflection that has spread beyond the confines of psychology and philosophy, are available to them. they have set themselves the goal of producing an artificial intelligence, using ultrarapid computers as well as software that is able to model the operation of neural networks and to come close to the performance of human intelligence in terms of the power of their reactivity and their memorization. at present, many other biological systems are being subjected to multiparametric exploration, with the aim of producing models. this is the case, for example, with the program of the differentiation of certain white blood cells, the neutrophils (chapter iii-2.2.4), from precursors located in the bone marrow, a differentiation that leads to the emergence of functions such as phagocytosis that are implemented in the fight against microbial infections 60 . in a domain that is closer to mechanical science, hydrodynamics, the digital simulation of the cardiovascular system has already made it possible to represent the physical phenomena associated with the propagation of a wave in deformable arteries during a cardiac contraction, in the form of equations, with a good approximation 61 . in short, from a monoparametric approach that often began as being essentially and necessarily reductionist, the experimental method, applied to living beings, has become a "globalized", or synthetic, multiparametric approach, the aim of which is to understand the dynamics of molecular interactions in defined biological systems. making use of data obtained, the hope is to use mathematical processing to simulate the overall functioning of a cell, organ or organism. this new paradigm of the experimental method ("systems biology" 62 ) is not limited to a simple accumulation of observations concerning a given biological system and their abstraction in mathematical form. the originality of this approach is that it formulates predictions of changes in the behavior of a system as a function of the manipulation of parameters such as substrate concentration, the presence of inhibitors, and so on. the mathematical processing of experimental data, with a view to learning about the functioning of complex systems by modeling, is supported by the technosciences, particularly biocomputing. it is linked not only to the enormous sum of accumulated knowledge concerning the structures and functions of living beings in the post-genomic era, and to the notion that the life of a cell depends on multiple networks of molecular interactions and thousands of enzyme reactions located in its different organelles, but also to the information that comes to it from its environment. a first type of modeling is based on observations made or experiments carried out on an easy-to-study model system. laws are drawn up from this. this so-called "bottom-up" (or synthetic) procedure, which proceeds from the simple to the complex, makes it necessary to have a set of very precise biochemical data. this great precision is all the more imperative in that any deviation, even a minimal one, in the integration of an experimental result can generate a mathematical model that is apparently plausible but which is unconnected to the living reality. the reverse, "top-down" (analytic) procedure proceeds from the overall operation of an organ and its theoretical analysis towards the specific mechanisms of its components. it takes into account the functioning of complex integrated systems such as the nerve and endocrine systems, immune and reproduction systems and the system controlling homeostasis, descending in stages towards the cellular, molecular and genetic levels. in the end, an understanding of living beings involves the management of an amazing capital of experimental data. this makes it necessary to consider all of the genes (genome), all of the transcripts coding for the proteins (transcriptome), all non-coding rnas (rnaome), all proteins expressed in a particular cell type (proteome) and all metabolites (metabolome) as a function of the enzyme catalyzers expressed and the energetics that underlie the catalyzed reactions, and, finally, to connect upstream events (mutations of genes and interference of messenger rnas, chemical modifications to amino acid residues in the proteins) to phenotypical modifications on the scale of the whole organism (phenome) (figure iv.19) . the goal of integrated or integrative biology ("systems biology") is therefore to put living beings into equations, that is, to represent them in virtual systems for which the behavior, accessible by means of calculation, can be predicted as a function of modifying parameters. in addition to the possibilities that are opened up in terms of a deeper understanding of physiological mechanisms, such virtual systems could be used for the design of new drugs or for the manufacture of economically valuable biomolecules. the diagram illustrates the different levels of complexity in the pathway that goes from all the genes together (genome) to all of the expressed characteristics (phenome) in the living being, passing via coding rnas (transcriptome) and non-coding rnas (non-coding rnaome), all the proteins (proteome), all addressing systems in the cell compartments (localisome) and all of the metabolic pathways (metabolome). at a scientific meeting held in sheffield, england, in january of 2005 63 , with the theme systems biology: will it work?, an argumentative discussion of the advantages as well as the disadvantages of an integrated, mathematized biology was useful in that it included a reminder that most of the parameters used in "systems biology" come from studies that are carried out in vitro on purified enzymes, and that it is not sufficient to know the value of the michaelian parameters (v max and k m ) in order to reach biological reality. in fact, in vivo, many enzymes record variations in activity that are hard to control due to allosteric type regulation or interenzyme contact; several enzymes of a metabolic pathway being able to interact to form a metabolon. however, by compacting several enzymes that catalyze contiguous reactions in a metabolic pathway, a metabolon considerably increases the catalytic efficiency of this pathway. another element of uncertainty arises from the protein density of the cell medium, and also from the fact that covalent modifications of enzymes can introduce a change in endocellular location (nucleus, organelles of the cytoplasm…). nevertheless, an approximative approach could limit itself to dealing with biological systems in modular terms, i.e., to considering them as being made up of a number of black boxes, each black box containing a series of reactions being processed mathematically together, with an input and an output. there is still a long way to go if we place ourselves on the cellular scale, but the end of the pathway seems even further away if we envisage the organism as a whole, taking into account the remote interactions between organs involving the interplay of chemical mediators. the brain plays a critical role in the dialogue between different organs, and in the regulation of the energy equilibrium in higher animals. this equilibrium can be disturbed by fasting or intense, prolonged muscular activity, or by an overabundant diet. the corrective response comes from a deep region of the brain, the hypothalamus, via the secretion of different types of peptides, some of which stimulate the appetite and others of which suppress it 64 . while taking into account the multitude of parameters that affect the complexity of living beings on an individual level, the theoretical approach to the study of cell function by modeling has the advantage that it produces predictions and provides information about the validity of conclusions and of theories based on experiments that are old and accepted in the absence of contradictory elements. this was the case for the theory that stated that the state of activation of a gene is determined only by the presence in its environment of transcription factors. recent studies concerning the level of gene transcription in isolated cells have shown that there are probabilistic-type factors which mean that a given gene in a given cell can be activated at any moment. a review which came out in 2005 65 sums up this subject. in this review, the authors use modeling to analyze the behavior of cells in the process of differentiation during embryogenesis. their darwinian model, which associates contingency and selectivity, competes advantageously with the determinist (or instructive) model, based on an all-or-nothing logic, that has been implicitly accepted up until now. the darwinian model takes into account the occurrence of stochastic events at gene expression level, events that are partially linked to the structural modifications to the chromatin that depend on covalent modifications of an epigenetic nature (phosphorylation, methylation…). by basing itself on the existence of mutational fluctuations that arise by chance, associated with a selfregulation of gene expression, the model that is obtained shows that during embryogenesis a cell has a choice either to differentiate into another cell type or to remain in its initial state. differentiated cells stabilize their own phenotype and, in their surroundings, stimulate the proliferation of foreign cell phenotypes. a harmonious equilibrium between these two processes is the necessary condition for the setting up of the steps that lead to the arrangement of different cell types during organogenesis, which take place in an apparently inescapable order, in the absence of disturbances. a break in this equilibrium leads to an anarchical cell proliferation. generally speaking, from the point of view of experimental science, the lesson that can be drawn from current modeling experiments is that the bernardian determinism that has prevailed as the essential foundation stone of the methodology applied to the study of living beings may find itself being requalified by the taking into account of stochastic phenomena. this is the case when the number of reacting molecules is low and the probability of stochastic events is non negligible. the modeling of such systems necessitates having recourse to a complex mathematical formalism. it remains true that determinist models for simulation of the dynamics of living beings, represented by classical differential equations, are more-or-less valid when the number of reacting molecules involved is high and the reactions supposedly take place in a homogeneous medium. should "systems biology" be regarded as a resurgence of a physiology that has been somewhat neglected over the last few decades, but has been reinvigorated by a salutary hybridization of biologists and model-makers? in any case, this is the intention of the "physiome" project 66 which has recently been launched on an international scale. it is also doubtless due to this state of mind that a trend which had gone out of fashion, involving the simulation of the performance of living beings by very elaborate concrete models, robots, is being reborn. an immense distance has been covered in just over two centuries, since the time when vaucanson presented automata in the forms of human figures, moved by ingenious springs and cogs, and giving the illusion that their movements were controlled by an intelligence, to a marveling public (chapter ii-6.4). in the last decades of the 20 th century, considerable progress was made in the understanding of the operation of the nervous system and in the development of technologies in which miniaturized electronics have come to the aid of already high-performance micromechanics. the brain being considered as an information processing machine, the aim is to understand the logic of this information machine by means of simulations on computers and, based on the results obtained, to construct robots whose electrical circuits take their inspiration from the operation of animal neurons. these robots are called biorobots or animats. insects have been chosen as a reference for the construction of such creatures because of the relative simplicity of their nervous systems: several hundred thousand neurons, in comparison with the billions of neurons present in mammals (100 billion in man). the fly's system of vision has been favored as a subject of study because of the possibility it offers of being able to record the electrical response of neurons that can be identified one by one. in the middle of the 1980s, in france, this inspired the pioneering work in biorobotics carried out by nicolas franceschini (b. 1942) and his team 67,68 (figure iv.20) . their objective was to study how an animal can avoid obstacles by means of its ocular perception and its movement-detecting neurons, the operation of which the team just analyzed using microelectrodes and a microscope-telescope specially built for the purpose. the fly's composite eye has 3 000 elementary units or ommatidia, each carrying eight light receptor neurons. the electrical signals emitted by these neurons in response to captured light (at most a few dozen millivolts) are sent to subjacent neurons that are organized into three levels that correspond to the optical ganglions called the "lamina", "medulla" and "lobula". the lobula is a strategic decoding center which, because of the small number of neurons contained in it (sixty), has been the subject of in-depth electrophysiological investigation. each of the sixty neurons of the lobula operates as a signal integrator. the neurons of the lobula send their messages to motor neurons involved in the contraction of small muscles that control the guidance and stabilization of the fly's flight. based on an exhaustive study of the neuron wiring of the fly's eye, franceschini and his colleagues were able to reconstruct a facetted artificial eye that can retranscribe the light signals received optoelectronically. this artificial eye, the electronic components of which correspond to around one hundred movement detectors in the fly, was incorporated into the head of a robot. the recorded light signals were transmitted to the moving components of the robot. a -head of the blowfly, calliphora, seen from the front, showing the two compound eyes with their multifacetted array. each eye hides 40,000 photoreceptors that drive various image processors based on a few hundred thousand neurons. b -"elementary motion detector" (emd) neuron and its evolution over fifteen years: on the left, first generation (1989), using surface mounted device (smd) technology, compared to a one franc coin from that period; on the right, the 2003 version of the highly-miniaturized hybrid (analog + digital) emd circuit (mass 0.8 grams), compared with a one euro coin. c -autonomous vehicle (12 kg) able to move around in a field of obstacles that it does not know about in advance. its vision is based on a genuine compound eye, whose circuits are inspired by those of the fly. it includes a network of 114 "motion detecting neurons", transcribed electronically according to the principle analyzed in the fly's eye by means of microelectrodes and a specially-constructed microscope-telescope. this network is arranged around a ring that is about thirty centimeters in diameter. the recently-constructed roboflies, oscar and octave, only weigh around one hundred grams. d -routing of the electronic components (resistances, condensers, diodes and amplifiers that operate in their thousands) soldered onto the six-layer printed circuit-board that provides the connection between the sensors and the steering motor on board the autonomous mobile robot shown in (c). figure iv .20 illustrates the neuromimetic biorobofly constructed according to this principle. completely autonomous because of its on-board power supply, this robot was able to move around at high speed (50 cm/s) in a cluttered area, avoiding the obstacles. this first "terrestrial" robofly, which was completed in 1991, was followed by several much lighter brothers and sisters: fania, oscar and octave are aerial roboflys 69 . constructed in 1999, oscar is a captive robot that weighs around one hundred grams. it is equipped with an eye that reproduces the retinal microscanning of the fly's eye discovered by franceschini, oscar is able to rotate around a vertical axis because of its two diametrically opposed helices and can thus orient its view towards an object. if this object moves, oscar follows it with its eye, up to an angular speed comparable to the tracking speed of the human eye. produced in 2003, octave is another aerial robofly that is able not only to take off and distinguish a relief, but also to land automatically and to react sensibly to a contrary wind in a turbulent atmosphere. on board, it has an electronic visuomotive self-regulation system, the operation of which is based on the signal processing operations that, in the insect, carry out the automatic pilot functions 70 . the age of biorobotics, in which robots take their inspiration from animals, has only just begun 71 . if specimens are still so rare, this is because behaviors for which we have a good understanding of the underlying neuronal bases are also rare. at the time of writing, a robot rat named psikharpax, with artificial muscles and a vision system that enables it to perceive objects in three-dimensional space, is being developed at the university of paris vi. almost in the realm of science fiction, we find hybrid robots obtained by hybridization of the living and the non-living. this is the case for the hybrid robot produced by japanese researchers, based on the silkworm moth. control of the nervous system of this insect is spread throughout its body. if its head is cut off, it continues to fly, which gave rise to the idea of replacing the head with an electronic transistor system 72 . using a remote measurement device, it was possible to explore certain behavioral aspects of the insect. although the construction of hybrid robots may raise ethical objections, such technology is capable of giving rise to spectacular applications in the domain of prostheses. the neurological prostheses of the future will nevertheless require that a contact be made between living neurons and the electronic chips that are able to improve the inadequate processing of the physiological signal. such a contact was produced recently in a german laboratory directed by peter fromherz 73 (b. 1942) . a small network of snail neurons, chosen because of their large size, was cultured on the surface of a silicon chip. a signal emitted at one location of the chip was able to be transmitted to another location via the synapse connection between two neurons 73 ( figure iv.21) . on the molecular scale, mitochondrial atpase or atp synthase, with a size of around ten nanometers (chapter iii-6.2.1) was used recently for the manufacture of a biorobot that made its mark in the media as the smallest known rotating molecular motor. the membrane-type enzyme catalyzes the reversible reaction atp + h 2 o adp + pi. this enzyme therefore has a double function; hydrolysis and synthesis. for this reason it is called atpase or atp synthase depending on the physiological context in which it is involved. in the mitochondria that oxidize metabolites, the enzyme operates like atp synthase. it catalyzes the synthesis of atp coupled to oxidation reactions. in the absence of respiration or of oxidizable substrates, the enzyme operates like atpase; it catalyzes the hydrolysis of atp. for ease of language, the enzyme will be designated here by the term atpase. it should be remembered that mitochondrial atpase includes two sectors, a hydrophobic sector, fo, characterized as a proton channel located inside the mitochondrial membrane, and a hydrophilic sector, f1, carrying catalytic subunits that are arranged as if they were on a turret (see figure iii .18c). fo contains two master parts of the atpase motor, i.e., a rotor comprising an assembly of around ten so-called "c" subunits and a stator that corresponds to the "a" subunit. the "c" subunit assembly is attached to the "γ" subunit of the catalytic sector f1, which thus functions as a rotor. in 1961, the british biochemist peter mitchell (1920 -1992) showed that phosphorylative oxidation in the mitochondria is associated with a transmembrane transfer of protons. the mechanism involved is said to be chemiosmotic. the most important experiment involved an almost serendipitous observation, carried out with a simple ph meter. when a current of oxygen was passed through a suspension of mitochondria in an unbuffered saline medium, in the absence of adp and phosphate, an instantaneous acidification of the extramitochondrial medium occurred, shown by means of the ph meter electrode immersed in this medium. it was concluded that the sudden switch from anaerobiosis to aerobiosis, i.e., the start up of respiration, is correlated with an ejection of protons from the mitochondrial matrix to the extramitochondrial medium. afterwards, this fact was linked with several others, the whole leading to the formulation of the chemiosmotic theory. briefly, mitochondrial respiration generates a vectorial movement of protons from the interior to the exterior of the mitochondrion. because of this, a proton concentration difference is established on either side of the mitochondrial membrane. the electrical potential that is created in this way is used by the mitochondrial atpase in order to synthesize atp from adp and mineral phosphate. this process involves two correlated events: return movement of protons towards the inside of the mitochondrion across the fo sector of the atpase; rotation of the assembly of c subunits and the γ subunit that is interdependent with it. we have therefore moved from electrical to mechanical energy. during its rotational movement, the γ subunit establishes contacts with the three catalytic subunits of the f1 sector, in succession. one after the other, each of the three catalytic subunits in contact with the γ subunit undergoes a change in the conformation of its active site, which is at the origin of the synthesis of atp. in the absence of mitochondrial respiration, the reverse process occurs. the atp is hydrolyzed into adp and mineral phosphate, and the energy released at each of the three catalytic subunits is used to rotate the γ subunit in the reverse direction to that which accompanies the synthesis of atp. the existence of a rotational movement of mitochondrial atpase, which had been suggested on the basis of biochemical arguments 74 and of structural data 75 was authenticated by masasuke yoshida and his co-workers in japan in 1997, thanks to an imaging technique 76 . in a first step, the molecular system was simplified by being limited to the catalytic f1 sector of the enzyme. a methodological trick was employed: genetic engineering was used to modify the α and β subunits of this sector by fixing polyhistidine chains to them. because of the strong affinity between polyhistidine and nickel ions, the f1 sector α and β subunits were immobilized on a medium covered with nickel ions (carried by an organic molecule). an actin filament labeled with a fluorescent ligand was attached to the end of the f1 sector γ subunit. this assembly made it possible, under a fluorescence microscope, to display a rotational movement of the actin arm carried by the g subunit affected by the addition of atp and by its hydrolysis into adp and mineral phosphate. a similar rotational movement of the γ subunit carrying a metal microbar was observed by an american research group 77 . remarkably, in 2004, after having fixed a metal microbead onto the γ subunit, the japanese researchers 78 demonstrated synthesis of atp from adp and mineral phosphate by rotating the γ subunit by means of rotation of the magnetic bead, induced by magnets. thus, the experimental coupling of a mechanical force and a chemical synthesis was demonstrated. in 2005, the japanese research team 79 succeeded in photographing the rotational movement of the enzyme powered by atp under the microscope, this time looking at the entire atpase complex, f1fo. after having attached a gold microbead onto the "c" subunits of the fo sector, to act as a probe, the researchers were able to confirm that the rotational movement of these subunits depended on the hydrolysis of atp into adp and phosphate ( figure iv.22) . the whole of the mitochondrial atpase (atp synthase) does, in fact, function as a molecular rotational motor powered by a proton flow, rather like an industrial rotational motor powered by a fossil fuel or electricity. the analogy is a striking one; the γ subunit of the enzyme corresponds to the motor driveshaft and the "c" subunits correspond to the motor itself. because of its association with non-living structures, for example metal bars or gold beads, which are carried along in the rotational movement of the enzyme, it is possible to speak of molecular biorobots. this domain, in which nanomachines use macromolecules from the living world, has only just opened up, but its future is full of promise. the story of scientific progress made with respect to the mechanisms of phosphorylative oxidation via the functioning of mitochondrial atpase, from the time of mitchell's experiment with the ph meter until the time of the manufacture of yoshida's biorobots, is an exemplary one. it is typical of the way in which a mode of thought evolves over time, from a primary discovery resulting from serendipity or an experiment "to see what happens", leading to the proposal of the existence of a mechanism, to a carefully programmed project which, because of its inventive technicity, shows the validity of the proposed mechanism, and, in addition, demonstrates its future utilitarian value. nowadays, certain biotechnologists dream of being able to "synthesize life" 80 in terms of cells that are able to imitate the performance of living cells. the concept of the "lab-in-a-cell " is coming to the fore 81,82 . nevertheless, it would be necessary to design an artificial cell that is an authentic replica of a living cell, and which benefits from all the attributes of a living cell. this is not achievable at the moment. thus, the current aim of nanobiotechnology is limited to scheduling the construction of artificial cells that are relatively simple both in composition and in function, for example, a microvesicle edged with a lipid membrane, containing a system of protein synthesis expressed from a short sequence of dna, as well as a system of atp synthesis able to supply the energy necessary for this protein synthesis. demonstration of a rotational movement of f1fo mitochondrial atpase (atp synthase) , induced by atp. atpase or atp synthase (reversible catalysis enzyme that hydrolyzes or synthesizes atp) has two sectors (see figure iii .18). the membrane sector, fo, comprises an assembly of a dozen so-called c (rotor) subunits and an a (stator) subunit. the other, extra-membrane sector, f1, is catalytic. it comprises three β catalytic subunits and three α non-catalytic subunits arranged in a ring, in alternating order. at the center of the ring is the γ subunit which is attached to the c subunits of the fo sector. subunits δ, ε and b stabilize the whole of the molecular complex. in the experiment illustrated in this figure, subunits α and β of the f1 sector of the enzyme have been genetically modified to include polyhistidine chains (his-tag, artificial ligand). due to the interaction of these chains with nickel ions (linked to an organic molecule) covering a solid support medium, the α and β subunits are immobilized. in addition, a gold microbead is fixed onto the ring of the fo sector c subunits by means of a chemical device (streptavidin molecule, artificial ligand). following the addition of atp, rotation of the microbead attached to the ring of fo sector c subunits is observed by microscopy on a black background. this rotation is dependent on (and at the same time an indicator of) the rotation of the c subunits, itself led by the rotation of the γ subunit in contact with the catalytic β subunits. note the ejection of protons. when the enzyme functions as atp synthase, the proton movement takes place in the opposite direction. "progress in biology is possibly mainly tributary to the drawing up of concepts or principles […] . in the process of elaborating concepts, which marks scientific progress in biology, there is sometimes a crucial step, when we realise that a more-or-less technical term that we had previously considered to cover a given concept, in fact covers a mixture of two (or more) concepts." ernst mayr translated from a french translation entitled history of biology. diversity, evolution and heredity -1989 on the margins of the modeling and the difference in mathematized systems that comprise theoretical biology, particularly in silico biology, concepts are mental representations, often image-filled and idealized ones, of fundamental mechanisms that are deduced on the basis of experimental results. from the imaginary domain of the probable, they extrapolate constructions of the mind that are in phase with the facts and experimental data, within a reflective projection that gives them their meaning and makes it possible to make certain predictions. there are premonitory concepts. this was the case for the concept of the reflex arc that associates movement with sensation. this concept was already present in the ideas of descartes (chapter ii-3.4), but it took more than a century before the theory of the existence of a reflex arc was supported by bell and magendie's demonstration of the existence of relay centers for sensory and motor nerves in the spinal chord (chapter iii-1). there have been premonitory concepts that, while they were demolished at the time they were first proposed, were shown to be completely accurate a few decades later. in the middle of the 19 th century, the german pathologist jacob henle (1809 -1885) needed a healthy dose of imagination and audacity in order to oppose the theory of the "miasma", a theory that was taught as a dogma, with a new theory that not only explained the spread of contagious diseases by microscopic beings, but also formulated the criteria for validating this theory, i.e., isolation of the pathogenic agent and its development in culture away from the diseased organism, then reproduction of the original pathology after injection of the pathogenic agent, which has been isolated, characterized and multiplied in a culture, into a model animal. thirty years would go by before the formulation of koch's postulates, based on experimental evidence (chapter iii-4). we may ask ourselves whether or not history is currently repeating itself in the case of spongiform encephalopathies that affect humans and animals, for which, according to the thesis of stanley prusiner 83 (b. 1942) , the prion, as an infectious protein, is responsible. other evocative concepts hold the keys that open doors to domains that are unknown, but are potentially rich in information. it is thus that the double helix dna structure proposed by crick and watson, based on the complementarity of adenine-thymine and cytosine-guanine bases (chapter iv-1.1.1), led to the concept of dna replication with reconstruction of a double strand that is identical to the original double strand. the concept of dna replication spurred on matthew meselson (b. 1930) and franklin stahl (b. 1929) to develop an experimental protocol based on the labeling of the dna nucleotide bases of the enterobacterium e. coli with a heavy isotope of nitrogen, 15 n, and on the differentiation of monocatenary dna strands in the process of synthesis by measurement of their density, as analyzed by centrifugation in cesium chloride gradients. in the same vein, jacob and monod's discovery of regulatory genes (chapter iv-1.1.1) gave rise to the concept of the operon which, in the bacterium, defines a genetic unit comprising structural genes and regulatory genes. the concept of the regulation of gene expression, extended to higher eukaryotes, makes it possible to explain the phenomenon of differentiation in cells with specific activities (muscle cells, nerve cells, epithelial cells…) by the silencing of certain genes and the activation of others. within the framework of bioenergetics, the chemiosmotic theory put forward by mitchell, in order to explain the coupling of mitochondrial respiration with atp synthesis (chapter iv-4.3), gave rise to consideration of the concepts of transmembrane transport of metabolites and of vectorial metabolism. some generalizing concepts that carry a unifying virtue within them are known. one such is the concept of compartmentation. the cell is no longer considered to be a bag of enzymes, as used to be the case. it is now considered to be a compartmented structure in which each type of compartment corresponds to a type of organelle delimited by a membrane and characterized by specific functions. thus, because of the genetic material that is present in it, the nucleus of the cell holds the information necessary for the manufacture of proteins. the mitochondria, which are called cell power plants, are in charge of oxidizing the products of cell catabolism and using the resulting energy for the synthesis of atp from adp and mineral phosphate. the lysosomes are the garbage collectors of the cell. among the functions carried out by peroxisomes is the partial breakdown of very long chain fatty acids. the endoplasmic reticulum and the golgi apparatus are involved in the maturation and the secretion of proteins. the ribosomes represent the machinery upon which messenger rnas are displayed in order to be decoded into proteins. a sign of the extreme sophistication of this setup is that the membranes of the endocellular compartments are not sealed common walls. they contain proteins that act as selective transporters of metabolites or highly specific ion channels, allowing the exchange of messages throughout the cell. thus, each organelle, informed of the condition of the others, is able to adjust its own activity to ensure the greatest harmony of the whole. this conditioned compartmentation at cell level may be compared to the socialization of human communities. while endocellular organelles are compartments delimited by membranes, there are non-membrane-bound compartments in the cell, such as protein complexes in which two, three or even more proteins are closely linked. often, these are enzymes that catalyze reactions that are contiguous in a metabolic pathway. being compacted into a complex known as a metabolon results in an increased efficiency of the flow of metabolites by facilitating the channeling of this flow. concepts evolve, often adjusting their representations according to accumulated knowledge. a good example of this is the evolution of the concept of the gene since its formulation at the beginning of the 20 th century. the term "genetics" was created in 1906 by the english naturalist william bateson (1861 -1926) . the term "gene" was introduced three years later by the dane wilhelm johannsen. this term designated a principle which, in the chromosomes of fertilized egg, and in an intentionally vague manner, was supposed to have an influence on the phenotype of the progeniture. during the same period, the term "locus" appeared out of the experiments carried out by the american thomas hunt morgan on the drosophila, a locus being defined as a region of a chromosome which, when altered by a mutation, leads to a modification of the phenotype of the living organism. based on cross-breeding experiments carried out on hundreds of drosophila mutants, morgan and his co-workers drew up the first genetic maps. by chance, the salivary glands of the drosophila have a particular characteristic; the nuclei of their cells contain giant chromosomes called polytenes, which result from the association of a hundred replicate copies of chromosomes that, after staining, are visible under the optical microscope. on these chromosomes, it is possible to distinguish colored bands separated by clear bands. it was observed that specific mutations had specific effects on the arrangement and number of these bands. the material contained in the bands was therefore the site of mutations. in the middle of the 1930s, the listing of more than 3 500 bands made it possible to construct a cytological map that was already highly detailed. the concept of the gene, the material basis of inheritance, took root. the sporadic mutagenic effect of x-radiation in the drosophila, which was shown by the geneticist and biophysicist hermann müller (1890 -1967), led the austrian physicist erwin schrödinger to question the sporadic event which, at the level of a target of a few dozen atoms, determines a mutation. he postulated that the target is located in the chromatin of the chromosomes, organized as an aperiodic crystal. the chemical nature of this target was identified with dna, following bacterial transformation experiments (avery, macleod and mccarthy, 1944) and experiments concerning bacterial infection by the bacteriophage (hershey and chase, 1952) (chapter iv-1.1.1). this is how the idea that gene = dna was born. the simple and reassuring idea that one gene → one enzyme, which was deduced from mutation experiments carried out by beadle and tatum on the mold neurospora crassa (chapter iii-6.1), had only a limited lifetime. a first stumbling block appeared when it was shown that the activity of a gene, and in consequence its contribution to the phenotype, depends on nucleic elements outside the gene. the definition of the term "gene" was then extended to include promoting and regulatory sequences. in the case of the lac operon of escherichia coli, these sequences are located just upstream of the site where transcription begins. however, in eukaryotes, a regulatory sequence may be distant from the gene that must be transcribed and sometimes it may be involved in the regulation of several genes (chapter iv-1.1.1). in the 1970s, the idea of the existence of the mosaic gene in eukaryotes appeared. a gene was now thought of as an assembly of several exons that originally in the chromosome are separated by introns. the alternative splicing of these pieces of genes gives rise to numerous possibilities for reconstitution, i.e., many messages coding for many different proteins. thus, however useful the concept of the gene has been with respect to its ability to generate discussion and to provoke experimentation concerning the molecular machinery responsible for the transmission of the hereditary characteristics, we can see that the term itself has not ceased to be the subject of readjustments, since the time it was first formulated. certain concepts are matched with metaphors. while some metaphorical concepts, particularly those that make use of images designed to grab the imagination, and to be easy to understand, tend to take liberties with the realities of living beings, they can also shed light on unsuspected mechanisms in sectors that have been neglected. metaphorical concepts are not a current fashion. it should be remembered that in his passions of the soul (1649), descartes, when asked "how limbs can be moved by objects of the senses and by the mind without the help of the soul," responds that this takes place "in the same way as the movement of a watch is produced only by the force of its spring and the arrangement of its cogs." later on, with lavoisier's clear vision of the vital role of oxygen, and his comparison of respiration with combustion, the concept of the chemistry of life, combined with that of bioenergetics, came to the fore, and was at the heart of studies on the metabolism. chemical reactions that liberate and absorb heat were substituted for the cogs of cartesian mechanics. the second half of the 20 th century saw the birth and development of the concept of the program, a concept with computer technology connotations, which was destined to explain the phenomena of inheritance. this concept began to fill out from the moment when it became certain that, in its nucleotide sequence, dna contains the necessary information for the construction of the protein material of cells. for a certain period of time, the passion for molecular genetics eclipsed the interest that had previously been given to metabolic chemistry. the powerfulness of the metaphorical concept may be measured according to the effect it has in pushing scientific research in particular directions, with the results this has on society. thus, during the 17 th and 18 th centuries, the study of human pathology was impregnated with a strong iatromechanical current. physiological chemistry and its corollary, pathological chemistry, which emerged as disciplines in their own right in the 19 th century and achieved full expansion in the 20 th century, are our inheritance from lavoisier and the concept of discussion about concepts necessarily leads to a brief discussion of scientific semantics, as shown by the few examples given in the previous pages. as we have just seen, the word gene that was put forward by johannsen around one century ago did not have the same meaning at that time as it has now, a meaning that still remains fluid. the gmo, an acronym meaning genetically modified organism, which has been the subject of vehement diatribes over the last few years, becomes much less of an object of passion if it is considered within the context of evolution. after all, for the last two to three billion years, living organisms have been genetically modified constantly by spontaneous mutations, which is why the human beings that we are today are able to discuss them! the term cloning is another example of a semantic misunderstanding that leads to inaccurate interpretation and arouses the passions. the primary meaning of the term cloning is the multiplication and the identical reproduction of a living cell. the simplest and most unambiguous example is that of bacterial cloning, a bacterial cell producing millions of cells that are identical to the original cell by its multiplication in a nutritive medium. the term animal reproductive cloning does not carry exactly the same semantic weight. it should be remembered that, in eukaryotes, the preliminary act of the cloning procedure involves the injection of the nucleus (with 2n chromosomes) from a somatic cell into an enucleated oocyte (chapter iv-2.3.2; see also chapter iv-6.1). the somatic cell nucleus, by providing its genetic equipment, gives the being that will develop in the uterus a phenotype that is practically identical to that of the somatic cell donor, but nevertheless not completely identical, as the cytoplasm of the enucleated ovum, with its mitochondria, provides a small but non-negligible fraction of genes, the mitochondrial genes. as for therapeutic cloning (in the absence of uterine implantation), this is used for the manufacture of differentiated cells that may be grafted into the individual who has donated the original somatic cell, with no immune-related rejection occurring. this is non-reproductive cloning. the passionate argument that has arisen because the term cloning is bandied about in an ill-considered fashion illustrates the confusion that can result from a lack of precision in the use of certain terms with a high level of media impact. "all the major problems of the relations between society and science lie in the same area. when the scientist is told that he must be more responsible for his effects on society, it is the applications of science that are referred to […] . no government has the right to decide on the truth of scientific principles, nor to prescribe in any way the character of the questions investigated." the progress of science is linked to that of civilization. it is in keeping with the state of mind, the beliefs, the lifestyle and the thought patterns of societies. in ancient greece, where manual work was considered to be servile, science remained essentially theoretical, confined to logic and dialectics, and strongly attached to questions of philosophy. the birth of experimental science in the 16 th and 17 th centuries went hand-in-hand with the rehabilitation of manual work. the technical side dominates in modern biology, which seeks to solve problems concerning the "how", rather than to address philosophical problems concerning the "why". as ian hacking (b. 1936) says in representing and intervening (1983), nowadays engineering, and not theorizing, is the greatest proof of scientific realism, which leads to the minimization of philosophical thought. in a skeptical biochemist (1992) , the polish-born american biochemist joseph fruton (1912 fruton ( -2007 emphasizes the contrast between the 19 th century and the first half of the 20 th century, when eminent scientists were still interested in the ideas of the professional philosophers of the history of the sciences concerning the progress of experimental research and, in contrast, the end of the 20 th century, when philosophy and the experimental sciences pretended to ignore one another. this is doubtless partly because the history of biology has become the history of biotechnologies to such an extent that, according to some, the objects being explored are so familiar that they are now part of the life of society. in pandora's hope (1999), bruno latour (b. 1947) considers that the current confrontation between subject and object, in which the researcher-subject explores the structure and function of the object, is being transformed into a human-nonhuman dialogue, in which the nonhuman-object becomes "socialized". taking yeast as an example, latour writes that it has been "working for millenia in the brewing industry, but now it works in a network of thirty laboratories where its genome is mapped, humanized and socialized like a code, a book, or a program of action that is compatible with our ways of coding, counting and reading […] . non-humans have become automatons, admittedly without rights, but much more complex than material entities." latour visualizes the human-nonhuman associations in the form of collectives that are organized into strata that implement the technical, the political, the social, the ethical, the ecological… the technosciences correspond to one of these strata, the sociotechnical stratum that is directly linked to the stratum of political ecology. in the same spirit, the belgian philosopher gilbert hottois (b. 1946), in his philosophies of the sciences, philosophies of techniques (2004) remarks that "laboratories produce things that go off to live their lives in society and in nature." thus, bacteria, yeasts or genetically modified plants are able to produce drugs such as insulin, growth hormone and vaccines for human medicine. these drugs become part of and indispensable to life in society. they are evaluated according to their market value by the companies that patent, manufacture and sell them, and according to the comfort they bring to the patients to whom they are administered. the financial management that results from their consumption becomes a worry for those responsible for public health, while their manufacture by specialized companies generates industrial activity and economic growth which may be measured according to how fashionable they are and how they sell. for a long time, society, while benefiting from scientific progress, remained indifferent to the experimental method, that is to say, the way in which knowledge progresses. in the last decades of the 20 th century, society became aware, via information concerning the occasionally demonized exploits of genetic engineering, that science can "take liberties" with the human being. populations were well informed about the effects that genetic engineering could have on the mortality rates of pathologies such as cancer and diabetes or on degenerative illnesses of the nervous system, and about the closeness of possible solutions. however, they were also warned about the risks to which science was exposing humankind. remembering certain tragic episodes concerning hiv-contaminated blood transfusions, growth hormone and mad cow disease, and certain cassandra-like predictions, such as a catastrophic epidemic of spongiform encephalopathy that has happily yet to appear, society shows reservations when the media inform its members of new feats of modern technology. political authorities, for their part, afraid of potential problems, tend to follow the principle of precaution, which in fact hides a fear of risk. however, evaluating risk involves not being afraid of it but understanding it in a lucid and courageous fashion. informed by the media, which often use sensationalism, the citizen is increasingly calling into question whether certain practices involving the biosciences, such as cloning, or certain mercantile transactions such as the taking out of patents concerning gene sequences, or even experimentation on live animals, are well-founded. "the problem of experimentation on man is no longer a simple problem of technique. it is a problem of value. from the moment that biology concerns man no longer simply as a problem, but as instrumental to the search for solutions concerning him, the question arises of deciding whether the price of knowledge is such that the subject of the knowledge is able to consent to become the object of his or her own knowledge. we have no difficulty here in recognizing the still open debate concerning man as a means or an end; an object or a person. this is to say that human biology does not contain in and of itself the answer to questions concerning its nature and its significance." knowledge of life -1965 written at a time when people were far from imagining how molecular biology was going to expand, the prophetic words of georges canguilhem (1904 -1995) have maintained their philosophical validity. manipulation of the human embryo, whether this involves its creation by cloning or the modification of its genetic inheritance, obviously leads to the need to consider the societal, religious and political points that arise from the domain of bioethics and are a reflection of the period in which we are living. until recently, advances made in biology left moralists indifferent. this ceased to be the case when scientific experimentation began to look at the human embryo with a view to utilitarian ends in the health domain. the specter of cloning was brandished without any clear distinction being made between reproductive cloning and therapeutic cloning. biology became demonized. however, as biologist pierre chambon (b. 1931) said in an interview in the french journal biofutur: "in absolute terms, biology is unable to tell us whether the cloning of a human being is moral or immoral, it simply tells us whether it is biologically possible." the birth dolly the sheep in 1997 (chapter iv-2.3.2) triggered a virulent debate because now that the cloning of an animal had become possible, that of a human being became envisageable. the media sensationalized this debate all the more in that it was exacerbated by debate concerning gmos (chapter iv-2.1). the dolly affair became a problem of society. up until then, the biosciences had been happy just to try and understand the mechanisms that explained the functions of living beings, but now, with the advent of gmos and cloning, it became obvious that a forbidden barrier had been crossed and that man had the power not only to transform but also invent himself. faced with this desacralisation of nature, the need arose for some philosophical reflection. this was given the name of bioethics, which is the title of the book, bioethics, a bridge to the future, which was written by the american biologist van rensselaer potter (1911 -2001) in 1971. the term bioethics covers philosophical considerations that range from the biosphere to the human person. bioethics tries to give a wider meaning to the moral codes which, in human societies, depend on ancestral traditions. it aims to prescribe that which is desirable according to the kantian maxim of the categorical imperative. in his what is bioethics? (2004), the belgian historian gilbert hottois reminds us that bioethics are above traditional morals, the latter being a set of norms that are most often spontaneously respected as good habits, without any critical reflection being involved, while bioethics, on the other hand, arises out of critical thought, analysis, discussion and the evaluation of established mores. over the last few years, the problems that are targeted by bioethics have moved towards today's burning issues. human cloning is an example. while allegations of the transcendence of man in nature may lead to human reproductive cloning being considered as a crime, strictly scientific considerations lead to an emphasis on the lack of responsibility shown by a few zealots, given the hazards involved in cloning in animals, such as the need to use a large number of oocytes in order to achieve success in cloning, the very low viability of the cloned embryos and the development of serious functional anomalies in the clones that survive. even supposing that scientific progress will one day overcome these difficulties, human reproductive cloning will come up against an insurmountable obstacle, the cloned subject's fear of finding that he or she is identical to the relative from whom his or her genetic inheritance comes. after all, the notion of manipulation of the human ovule with the aim of serial reproduction has often haunted science fiction stories. in brave new world (1932), aldous huxley (1894 -1963) gives an apocalyptic vision of the budding of human eggs that produce hundreds of identical twins which are conditioned into classes and subclasses while being raised in jars, depending on the quality of the nutritive substances they are given. in the artificial uterus (2005), henri atlan (b. 1931) predicts that the raising of human fetuses in jars could well become an alternative to uterine gestation in a distant future. let it be understood that human reproductive cloning, which is no longer part of the domain of science fiction, as it has become feasible, must be considered as being reprehensible because it goes beyond the limits of reason, and is a denial of human transcendence. man as subject cannot be considered as an object. the problem of therapeutic cloning is quite different, although it leads to reticence and prohibition because the demarcation between therapeutic and reproductive cloning depends mainly on whether a cloned embryo is implanted in a uterus. while, at the time of writing, therapeutic cloning has been prohibited in france, germany and other countries, it is tolerated in great britain. in the usa, the prohibition only applies to publicly-financed researched, while each state has its own legislation, which is relatively flexible. the objective of therapeutic cloning is to provide patients with tissues that arise from their own selves, and are therefore immunocompatible and able to be grafted without there being any risk of rejection (chapter iv-2.3.2). it is based on the removal of somatic cells from the subject to receive the graft and the transfer of the nuclei of these cells into enucleated oocytes. the stem cells that are obtained after the first divisions are stimulated using appropriate growth factors. depending on the factor used, the stem cells differentiate to form a type of tissue (hepatic, muscular, nerve…) that can be used as a graft. such a procedure may be envisaged for patients who have suffered a serious, invalidating trauma, for example section of the spinal chord. a graft of immunocompatible nerve cells might make it possible to re-establish nerve continuity. a similar type of therapy has been considered for parkinson's disease, the cause of which is a degenerescence of certain cells of the encephalon (chapter iv-2.3.1). given the hopes that are raised by the possibility of such therapies, and the fact that, after all, such therapeutic cloning is the equivalent to an autograft, even if the ways in which the graft is obtained are slightly tortuous, the demonization and rejection of such practices should be reconsidered, calmly and coolly. another option for therapeutic cloning is the correction of mutations identified in the mitochondrial genome of a woman wishing to have children. it is, in fact, the mother's ovum that provides the fertilized egg with its complement of mitochondria that are indispensable for its viability. the manipulation involves inserting the nucleus of a fertilized ovum from the mother, obtained by artificial insemination, into an enucleated oocyte taken from a woman who is not suffering from the mitochondrial defect. the cytoplasm of the enucleated oocyte provides the stock of functional mitochondria that are indispensable to normal cell function in the future embryo. in a domain of the bioethics, in which rational objectivity comes up against deliberately technophobic religious and cultural considerations, it is useful to remember certain legal and legislative paradoxes. thus, in france, after having been considered to be a criminal act that was subjected to severe repression by the law up until 1975, the right to have an abortion before the end of the third month of pregnancy became not only authorized but also protected by law. it is interesting to note that in the 13 th century, thomas aquinas, the father of the church, had acknowledged that a fetus only becomes "animated" by the implantation of the soul by holy will in the third month after fertilization. another subject to be considered is pre-implantation genetic diagnosis (pgd), in which human embryos that have been fertilized in vitro are sorted in order to find those that are without defects, a practice which is on the verge of being a deviation in the direction of eugenics. nevertheless, pgd is the basis of a practice that is either already legalized or is in the process of being so in several european countries, the creation of so-called designer babies. a typical example is that of a designer baby arising from an embryo whose immune profile to that of an older sibling who is suffering from leukemia. in this case, there is good reason to hope that a graft of immunocompatible blood cells from the designer baby into the sibling who is suffering from leukemia will save the latter from death. out of the disharmony of opinions that arising from cultural tradition, religious conviction or simply scientific pragmatism, the american biologist and philosopher h. tristram engelhardt (b. 1941) , in the foundations of bioethics (1996) proposes a lay bioethics that is based upon the principle of permission. lay bioethics advocates tolerance while admitting that this tolerance in no way prevents anyone from taking up a personal position; it means that each human being has a moral sensitivity as well as the ability to reason and to choose within a defined limit of non-harmfulness and of justice. the individual is free to modify his or her destiny, or to manipulate his or her nature by genetic interventions because, adds engelhardt, "there is no lay moral foundation to prohibit such an intervention." when a researcher or the research organization that the researcher belongs to files for a patent for an invention with a patent office, it is necessary to demonstrate the novel and utilitarian nature of this invention. if a patent is accepted, this gives the person or body that filed it the exclusive right to make use of the invention over a pre-determined period of time, generally 20 years, which is a means of protection, or, if desired, to allow others to make use of the invention by issuing a license to do so. in the domain of living beings, there has sometimes been confusion between invention and discovery. in 1991, craig venter, known for his participation in the sequencing of the human genome, filed a demand for a patent covering the sequences of 2 700 fragments of recombinant dna (cdna) called est (expressed sequence tags) that are obtained by reverse transcription from human brain messenger rnas, in the name of the nih (national institutes of health) at bethesda (usa). the patent specified that ests could be used as probes to characterize genes that are potentially involved in neurological ailments. the resulting outcry led the nih to withdraw its patent demand. in fact, the patenting of living beings has a long history that goes back to the patent that was filed in 1895 in france by louis pasteur, and then in 1873 in the usa, for the use, in brewing, of a yeast culture that was free from pathogenic bacteria. from this historical perspective, the case of ananda chakrabarty (b. 1938) set a legal precedent. in 1994, chakrabarty filed a demand with the us patent office for a patent relating to a pseudomonas type bacterium which, by genetic modification, had acquired the ability to digest crude oil. his demand was refused. after an appeal and many legal battles, the united states supreme court overturned the patent demand refusal, the basis of the judgement being that any modified microorganism is a product of human ingenuity and has a specific name, characteristics and use. thus, from 1980 onwards, the arrival of an era of patents derived from genetic engineering was indicative of how this discipline was growing. in december of that year, stanley cohen and herbert boyer, acting on behalf of the university of stanford, patented a nucleic chimera comprising a recombinant dna carried by a vector. in 1982, a patent concerning the growth hormone gene was awarded to the university of san francisco. in 1984, the university of california at berkeley obtained a patent for the human insulin gene. in 1985, the american company pioneer hi-bred succeeded in patenting a variety of corn in which genetic modification has led to an increased synthesis of tryptophan, an amino acid that is indispensable for animal feed. in 1988, the genentech company acquired a patent for the gene coding for human gamma interferon. this was followed in japan by a patent for the gene coding for beta interferon. in the same year harvard university patented the oncomouse, a transgenic mouse whose susceptibility to cancer is greatly increased. after this, several species of transgenic animals were patented for utilitarian purposes, such as the production of human alpha-1-antitrypsin taken from the milk of transgenic goats and used for the treatment of cystic fibrosis. the frenetic patenting of living beings has reached the domain of natural products arising from the plant world in tropical regions, the immensely varied essences arising from these plants being full of pharmacological potential. the potential for producing drugs of a considerable commercial value from such plants is very high. here we return to the problem of the patenting of genetically modified, cultivatable plants (gmps) (chapter iv-2.1). thus, the experimental method, the principle of which is to acquire pure knowledge, finds itself led astray in its applications. whatever the motives that are given, particularly for manipulations that give rise to the manufacture of marketable products, the patenting of genomes for mercantile ends shows the regrettable, but unfortunately inevitable, direction in which the very spirit of a science, molecular biology, which half a century ago wished to be at the heart of an understanding of living beings, has drifted. the suffering of animals that are being experimented upon gives rise to a moral problem. the end of the 19 th century saw large-scale demonstrations against vivisection and repeated demands for it to be abolished. today, there is renewed vigor in the call for the abolition of vivisection, without any real coherent basis. this desire to stop experimentation on animals ignores the imperatives of contemporary medicine, which must meet the challenge of pathologies whose increasing incidence is worrying, such as cardiovascular diseases, diabetes, cancer, and the degenerative illnesses that are linked with aging or are of genetic origin. it is true that animal experimentation inevitably leads to questions. are the stakes involved in a particular experiment, in terms of the acquisition of new knowledge, worth the suffering of an animal used in that experiment? is it not necessary to ensure that the experimental protocol is well-documented, that it is not redundant, or even that it has been the subject of previous studies carried out on cells in culture? it is easy to see the size of the methodological chasm that separates contemporary physiology from that of the time of claude bernard, when cell culture techniques were not yet being used, when the main instrument used was the scalpel and the researcher, using his or her imagination and creativity, had to develop specific protocols that were able to validate or refute a working hypothesis. each period in history operates in its own way according to its moral laws and its technical capabilities. the bloody operations carried out by magendie and by claude bernard in the 19 th century, which were tolerated at this time despite criticisms from antivivisectionists, would not be permitted today. nevertheless, it is true that the physiologists of the 19 th century, by means of the results of their experiments, wove a tapestry of new knowledge on which contemporary biologists were going to work and without which the level of understanding the modern science would be much lower than it is. animal experimentation remains indispensable in many areas of physiological investigation, in genomics, in toxicology and in pharmacology. it is a precondition for clinical trials of any new drug, being used to test for the drug's efficacy, its metabolism and any toxicity. however, not all data arising from animal experi-mentation can be extrapolated to man. the margin of uncertainty can be reduced by means of comparative trials on several animal species. because of their phylogenetic proximity to man, primates may seem to be the solution for experimentation prior to the application of a drug in man. this was the case for the development of a vaccine against hepatitis b. it has been proposed the grafting of stem cells in man should be preceded by experimentation in apes, in order to ensure the absence of tumorization over the long term. however, the researcher is confronted with a dilemma: should he or she ensure the safety of man with respect to possible deleterious effects or respond to ethical demands that recognize the very great genomic similarities between man and the chimpanzee. a consideration of cloning, patenting and animal experimentation practices illustrates the excesses of the experimental method in domains where political authorities consider themselves able to legislate. administrative decisions, often made in the absence of any dialogue with scientific authorities, can have serious consequences. thus, given the pretext of strict obedience to the principles of bioethics, which are a matter of tradition, and while certainly respectable, are nevertheless arguable, and also given the pretext of a sickly and unconsidered fear of the risk involved in certain experimental practices, and the absence of an intelligent evaluation of this risk, research, which until recently took place in a motivating atmosphere of liberty, may, over the long term, be weighed down with a highly prejudicial handicap and a limitless sense of discouragement. in the 17 th and 18 th centuries, experimental research, which was still in an emergent phase, was mainly artisanal, and in the hands of rare scholars. it took form during the 19 th century in the west, particularly actively in germany, and became operational in the 20 th century, under the aegis of governmental authorities, with the creation of institutes, the programmed recruitment of researchers and the allocation of renewable budgets. modern science, based on the principles of the experimental method, came to the fore much later in the east than in the west. the globalization of knowledge has meant that at present experimental science, in all domains, including that of the life sciences, has spread throughout the world, with even those countries that had become relatively backward in these domains because of their isolation catching up rapidly. nevertheless, it is true that the progress of the experimental sciences in the usa and in the united kingdom has been distinguished by pragmatic management of these countries' science policies, based on the excellence and the high degree of autonomy of their universities and research institutes with respect to recruitment and choice of subjects of study. the efficacy of this policy in the life sciences may be judged by the number of researchers who have won nobel prizes since the second world war (at the time of writing, more than 80 in the usa and twenty or so in great britain as opposed to only 4 in france). in france, research on living beings is carried out in the laboratories of universities, in institutes connected with higher education and in laboratories that are run by large organizations such as the national scientific research center (cnrs), the national institute of health and medical research (inserm), the national institute of agronomic research (inra), the atomic energy commission (cea), the national institute of research in computer processing and automation (inria), the national center for space studies (cnes) and the french institute of research on the seas and oceans (ifremer). equivalent bodies exist in countries other than france, some of them being institutes that are dedicated solely to research, and some being university laboratories that associate research and teaching. at the beginning of the 20 th century, the function of researcher was most often associated with that of a professor occupying a chair at a university, surrounded by a few assistants, the professor directing the research work in his area of specialization. now, within a period of a few decades, the status of researcher has been modified greatly. today we talk of research careers classified according to level of expertise and technicality. management, or the supervision of career paths and the control of financing, is carried out by an administration that is itself highly hierarchical. the scientific process has undergone a metamorphosis, shown by changes in the behavior of researchers not only within the institutions in which they work but also in their relationships with the media, the political sphere and society. the teaching of the life sciences needs to take this into account. "long ago, there was a time when scientists recounted the exact circumstances of their discoveries, without shame, even when their recital showed up the fragility of their forecasts or an indecent collaboration on the part of every bit of luck. such times are past, and the researchers of today often like to make us believe that they only find what they are looking for. the thousands of pages pasteur's lab books provide an opportune reminder to us (and to program-makers or impatient users) that it is just as difficult to ask a question as to answer it, that a scientific discovery often occurs after a long, winding path, that rather than following the fashion, it is preferable to follow one's ideas, particularly if they are good ones, and are in advance of the fashion." jean jacques molecular dissymmetry, in "pasteur, workbooks of a scholar" -1995 current technological progress, the accumulation of the scientific knowledge, the institutionalization of the public research and many other factors are disrupting a ritual of the experimental process that had survived until the middle of the 20 th century, and even beyond. the experimental life sciences of the 21 st century will necessarily see themselves remodeled with respect to their objectives and procedures. faced as it is by an increasingly tough international competition, the scientific community is also subject to restrictions in terms of operation and prospectives. an organization into small teams of a few researchers gathered around a boss, working in friendly interaction, is increasingly giving way to large groupings that sometimes seem like consortiums. focused on research subjects that are deemed to be "cost-effective", these superstructures are encouraged, or even imposed, in the sadly illusive hope that the will lead to greater efficacy. the person in charge of such large groups is taken up with everyday management tasks and by maintaining good relations with the administrative bodies on which his or her organization's survival depends. he or she may become distanced from the experimentation and forget the intellectual motivations that in the past caused his or her competence to be recognized. it should be emphasized that the secret of future successes lies in situations where young researchers are in direct contact with their bosses, and where friendly interaction with a known master teaches the apprentice researcher how to learn, how to think and how to experiment in a critical fashion. preoccupied by the rapid expansion of the scientific population, accompanied by the creation of laboratories whose operation necessarily requires financing, often on a large scale, political authorities, giving way to the requirements of media-fed public opinion, are interfering more and more, via administrative relays, in the control of the objectives of experimental research. short-term objectives, considered to be "visible", are favored. a priori, the viability of a project is judged according to the scientific context of the period and its impact on society, insofar as the project looks at health problems with a high degree of media coverage (cancer, degenerative illnesses, viral infections…) and often in agreement with a consensus that avoids going against the orthodoxy of the moment. this leads to a rigid management of projects that are financed and controlled according to objectives that have been fixed in advance, and that are all the more easily accepted by state authorities when they are somewhat fantastic in character. however, fundamental research proceeds from a playful activity, and for this reason, its efficacy is dependent on the passion of the researcher for the problem that he or she is studying. in contrast to what is believed by the narrow-minded, the effectiveness of a researcher in terms of discoveries depends upon the liberty that is given to this researcher, assuming, of course, that this liberty is underpinned by criteria of confidence such as the researcher's scientific past, his or her motivation, and judgements made concerning the researcher by impartial peers. it should not be forgotten that the determination of the three-dimensional structure of hemoglobin by max perutz (chapter iii-6.2.1) took around twenty years of solitary, uninterrupted and untiring labor. the theoretical and technical tricks that led to this success helped to open up the domain of the structures of giant macromolecules, several dozen kilodaltons in size, which no-one had dared study before. anyone who uses the experimental method realizes that while fundamental research must be organized, it cannot be scheduled. such a person knows that the pathways to discovery are convoluted, and that an inexplicable observation that appears unexpectedly during an experiment can sometimes, if the researcher is sufficiently perspicacious, be the beginning of an adventure that leads to a discovery. it was to just such a convoluted path that the belgian biologist christian de duve (b. 1917) alluded in his speech when he received the nobel prize for medicine and physiology in 1974. after working at the university of saint louis in the usa, de duve, who had taken up a post at the university of louvain, belgium, decided to look at a research theme that had received a great deal of media coverage, diabetes and insulin. it was while operating on one of the subcellular fractions obtained from ground rat's liver, and analyzing certain of the enzyme activities of these fractions, that he was surprised to find, in one of them, enriched with mitochondria, a phosphatase activity that, paradoxically, increased with time, while the enzyme activities specific to the mitochondria declined. this was an activity belonging to organelles that were contaminating the mitochondria. dropping all research on diabetes, de duve set out to identify and characterize these unknown organelles. he discovered that they were involved in the breakdown (lysis) of molecules that are undesired by the cell and, for this reason, he called them lysosomes. the discovery of lysosomes helped to open a new chapter in cell biology and to attribute a molecular cause to diseases with serious prognoses whose etiology had remained a mystery up until then. these diseases were given the label lysosomal diseases. these diseases result from the absence of a lysosomal enzyme that is responsible for the breakdown of a given metabolite. the accumulation of this non-broken-down metabolite in the lysosomes leads to cell malfunction, which causes the lysosomal disease. as de duve said jokingly, if he had carefully followed the experimental process laid down in his diabetes research project, and if he had not given way to the temptation of "playing hooky" or "playing truant" he would never have mounted the podium in stockholm. in the same way, henri-gèry hers 84 (1923 -2008), a cell pathologist at the internationally renowned louvain school, remarked in an article published in the review médecine/sciences: "i believe we would obtain maximum value for the money devoted to research if we were willing to distribute it to those who have been shown to be productive, according to their needs, and without asking them for a program." hers concluded, in a tone that was deliberately playful, but thought-provoking, "such a simple system would lead to unemployment for a large number of administrators, which is why i suspect that it will never be adopted." research has its own set of ethics, driven by anticonformity and the creative imagination, capable of shaking up firmly-anchored ways of thinking and established hierarchies, and of leaving the researcher the freedom to express him or herself and to experiment off the beaten paths. as eccles says in evolution of the brain and creation of the conscience, it is important to distinguish between intelligence and imagination. intelligence is measured according to the rapidity and depth of understanding and clearness of expression. it may be measured and even given a numerical value. the same is not true for the imagination, a more subtle, unmeasurable phenomenon that cannot be learned. the imagination is one of the levers that is able to lift the boulder that hides scientific truth. the imagination is the ultimate weapon of research, which shakes up the knowledge acquired by the intelligence. nevertheless, the imagination must be tempered by a good critical sense that is able to perceive potential sources of artifacts, both in sophisticated instruments that act as so many black boxes from which already manufactured information emerges and in genetic or chemical cell exploration methods whose specificity must be carefully checked. the benefits that can sometimes be gained from prospective research that is far from dogma that is rooted in sterilizing tradition, the way in which knowledge progresses, most often by moving away from any orthodoxy, the way discoveries appear unexpectedly on the fringes of carefully put together projects, all of these points are matters for reflection for those in power in the worlds of politics, economics and industry. publication is an essential tool for communicating scientific knowledge, and is the judgement criterion for committees in charge of evaluating the creativity of a researcher. in order to have meaning, a publication must provide information that is sufficiently innovative with respect to parallel work carried out in other laboratories. here again, media coverage has quietly infiltrated the scene. its role is all the more perverse in that the rating of a publication is estimated according to its impact index, or, roughly speaking, the renown of the scientific journal in which it is published. curiously, it has happened that articles that would later be considered to be of primary importance have been rejected by highly prestigious journals, simply because the facts mentioned in the article and the conclusions made have not coincided with the orthodox opinions of the period and the traditionalist spirit of the journal's editorial committee. this was the case for an article which the biochemist hans krebs (1900 krebs ( -1980 submitted to the british journal nature in 1937. in this article krebs described a series of experiments showing that an endocellular metabolite, pyruvate, product of glycolysis, is completely degraded during a cycle of enzyme reactions. this degradation cycle would later be recognized as the central pivot of the intermediate metabolism. called upon to judge revolutionary scientific considerations, and unable to perceive their importance, nature's editorial committee rejected the article. krebs then sent his article to a journal with a relatively restricted audience, enzymologia. it was accepted and published in the two months that followed. the importance of the concept that was put forward in the article ensured that its author gained international recognition, leading to his winning the nobel prize for physiology and medicine in 1953. for the researcher, publication is a way of making his or her work known. it is also the way in which the researcher learns about the work of others. while the rhythm at which publications in the life sciences appeared increased slightly in the first half of the 20 th century, the second half of that century saw a great acceleration in this rhythm, leading to a difficult-to-manage proliferation of reviews and books. it has been estimated that in the last thirty years the volume of publications in the biological domain has increased five-fold; in the preceding twenty years it had already doubled. this accumulation of publications makes it harder for the researcher to judge the quality of the huge mass of published articles, even in the highly targeted domains that are within his or her area of expertise. the researcher, therefore, will deliberately choose a particular article according to the prestige of the journal in which it is published, which is not an inviolable criterion of quality. in addition, any judgement concerning the pertinence of a scientific article necessitates a dissection of the subtleties of the methodology, the well-groundedness of the experimental protocol and the validity of the results, by means of a careful examination of tables of results and graphs, and, finally, the logic of the discussion. this restrictive yet absolutely necessary requirement limits the number of articles that are likely to be screened. however, this is not the worse fault of publication today. there is another problem that is much more worrying. many documentation centers have reacted to this inflation in the scientific press by equipping themselves with computing facilities that are able to find, in data banks, articles that have been selected on the basis of a key word index, and to display them on screens. while acknowledging that this constitutes an inescapable change in the transmission of scientific know-how, it should be recognized that in browsing through the pages of a highquality scientific review, it is possible to come across an article containing an innovative idea or a useful technique, an advantage that is less available when using the on-line system of scientific publication that is most prevalent nowadays. mention should also be made of the requirement to publish frequently and within short time frames, for reasons of competitivity, when aspiring to obtain jobs or promotions, or even just to obtain recognition, this requirement being another factor that is prejudicial to fundamental research. it is the cause of worrying excesses, such as experiments that are hastily published and non-reproducible, or even the falsification of experimental results, occasionally within a context of considerable media coverage. although such practices, which are the exception rather than the rule, are rapidly detected and condemned in a scientific culture where information circulates freely, the publicity that they incite, which reaches society at large via the media, leads to an overall discrediting of experimental research. at present, one of the most noticeable trends in scientific publication is that of collectivism. while, in the 19 th century, scientific articles were usually published in the name of a single author, occasionally two authors, and very rarely more than two, nowadays publications are often co-authored by several people, and when the work involves the analysis of structures, or the sequencing of genomes, several dozen researchers may be co-authors. from being the work of individuals, research has become collective. in domains whose complexity requires a wide selection of techniques that may range from physics to genetics, the hybridization of specific areas of expertise is certainly indispensable, and this requires the collaboration on a particular project of researchers who are sometimes physically remote from one another. the downside for the researcher, particularly one who is young, is that this requires him or her to abandon individuality and creativity. both collectivism and inflation in scientific publication are facts that are an integral part of contemporary science, facts which reflect an irreversible trend that it would be difficult to obviate. over the last few years, scientific publication has been subject to a type of restraint, in that certain "sensitive" data in the domain of molecular biology might be used for the manufacture of biological weapons in a form of terrorism known as bioterrorism. thus, the means of synthesizing de novo viruses (influenza virus, poliomyelitis) and the possibility of modifying their tropism by "directed molecular evolution" (change from a sexual tropism to a respiratory tropism for the aids virus) have been the subject of publications in prestigious journals. given sufficient means, terrorist pharmacists could well make use of such data in order to carry out malicious actions with catastrophic consequences 85 . in order to please a public that is eager for progress and the sensational, politicians favor, by means of targeted financing, the types of organization that appeal to their sensibilities, such as the technological platforms. while recognizing that such platforms are now an integral part of the landscape of research on living beings, and that they must therefore be taken into account, and while acknowledging that projects which implement the latest technologies in different domains need to be federated, it is nonetheless vital not to underestimate the potential creativity of small groups of researchers, a point that was expressed by one of the greatest of contemporary biologists, arthur kornberg (1918 kornberg ( -2007 , winner of the nobel prize for physiology and of medicine, in a speech given in 1997: "as i view the steady growth of collective science and big science, the greatest danger i see is a dampen-ing of individual creativity and reversion to the old politics -the inevitable local politics that infects every group and institution." however, conscious of the metamorphosis that is occurring in the experimental method, and faced with a particularly inventive and all-conquering technology, fundamental research in the life sciences must come to terms. a century ago, fundamental research and technological research interacted all the more directly because they were both in their infancy. this is no longer the case. management of the ever-increasing amount of knowledge in the life sciences, and the degree of sophistication achieved by bioengineering techniques and instruments, is widening a gap that makes dialogue increasingly laborious. however, dialogue appears to be a guarantee of future progress. the solution can only come from an increase in cross-disciplinarity, which should begin with university teaching and the establishment of a recruitment policy that advocates the cohabitation of talents from different educational backgrounds in the same laboratory. fortified by such hybrid expertise, while maintaining its share of originality and liberty in the choice of problems to be studied, fundamental research on living beings can only be enriched by a marriage of reason with biotechnology. convinced of the necessity for such a marriage, stanley fields, the inventor of the double hybrid method (chapter iv-4.1), in an article entitled "the interplay of biology and technology" (proceedings of the national academy of sciences, usa, 2001, vol. 98, pp. 10051-10054), concludes,: "it is at the interfaces of biology and other sciences that many of the future discoveries will be made, at the interfaces of biology and engineering that these discoveries will come to be exploited, and at the interfaces of biology and ethics and law that their consequences for society will be decided." the desired dialogue between biology and technology also implies the breaking down of barriers that too often isolate fundamental research and so-called applied research, and the facilitating of consistent interaction between the discoveries made in the academic institutions and their application for utilitarian ends in private companies. this is where the twin demons of money and power raise their heads. already, at the turn of the 1980s, a. bartlett giamatti 86 (1938 -1989) , who was then president of yale university in the usa, commenting on american university policies, spoke of a "ballet of antagonisms" between, on the one hand, commercial companies that are interested in the rapid cost-effectiveness of any new therapeutic advance and, on the other hand, non-profit university laboratories. recently, james j. duderstadt 87 (b. 1973), emeritus president of the university of the michigan, argued that the university is a "counter-hierarchical" organism. in fact, its members are free to carry out the research that pleases them and to think in the ways that they wish to think, in any case within an academic norm that considers itself as being free from the constraints dictated by private interest groups. until recently, such behavior was considered as a sort of ethic which arose out of the university conscience and dignity. the crumbling away of this ethic in the final decades of the 20 th century coincided with the rise of biotechnologies and the large-scale filing of patents relating to molecular genetics techniques that could be applied to the manipulation of living beings, by researchers in the public sector. the intrusion of the american private sector into public research laboratories, in the form of collaborations with transfer of "sensitive" information from the public to the private, has become such a worrying problem that drastic control measures have had to be taken. within this context, the american federal government, in february 2005, issued a certain number of prohibitions targeting the national institutes of health (nih) of bethesda, particularly with respect to the retribution of researchers for services rendered to industry 88 . these stands call for thought concerning the place that is currently held in universities with respect to fundamental research. without arguing against the efficacy of major research institutes, it is nevertheless necessary to remember the part played by the university in this domain. the university is not only the place where knowledge, both as it is now, in its current state of advancement, and as it has been, it is also the place where knowledge must be created by fundamental research. for the last few decades, under pressure from state policies, and also as a function of an improvement in social status, the world of the university has opened up to a wider public, leading to an influx of students that is sometimes so enormous that the task of teaching them has become overwhelming. because of this, the share of their time that university researchers can, in practice, devote to their research tasks has shrunk. this situation is highly prejudicial to the mission to innovate, which should be a priority. it is, in fact, during their university studies that the thought patterns of young students are forged by contact with teachers who not only instruct them, but also educate them by inspiring in them a motivation and an enthusiasm that gives rise to hope. how could this be true if the teaching faculty did not itself participate in scientific creation? "what can teaching, ex cathedra, do to guide the researcher? nothing, obviously. the researcher is trained in the laboratory. and the first stroke of genius on the part of a future researcher is to find a good boss. such a find will open up the royal road to success. the road will be opened -but the researcher must travel along it. a researcher may be taught many things. he or she can become familiar with techniques and with equipment. she or he can be assigned a problem to resolve. however, what is essential for the researcher is to know how to understand relationships between phenomena that seem unrelated, and to be able to progress from the particular to the general. a boss may develop such qualities in a gifted young researcher, but intuition is a gift; it cannot be taught." while the bernardian style experimental method, based on a working hypothesis aroused by an observation, followed by implementation of an experimental protocol, is still extant in the life sciences, and while "serendipity" is still the origin of great discoveries, "big science" , underpinned by sophisticated biocomputing or bioinformatics procedures, is intruding more and more, while genomics and proteomics are not far behind. the methods and instruments developed by the biotechnosciences have led to profound modifications in the ways that the structures and functions of living beings are investigated. for example, by varying multiple parameters in dna chips or protein chips, at the same time, the experimenter is able to ask questions that lead to grouped all-or-nothing answers (chapter iv-1.1.3). in combinatory chemistry, screening makes it possible to detect a molecule that is active for a given pathology from among a multitude of molecules (chapter iv-3.4). the mathematical simulation of metabolic networks or of signaling chains is already well under way (chapter iv-4.2). given this new technological outlook and the hope that it can provide rapid solutions to health problems subject to considerable media coverage, the teaching of biology in universities must not be limited to a description of current advances, no matter how brilliant and promising they may be. this teaching should return to its origins, be a reminder of history, and should not hesitate to use examples to illustrate how a major discovery can arise from a long period of wandering in the wilderness. in practical terms, while being conscious of the extraordinary complexity of living nature, and carefully avoiding the dangers of simplification, it is important to remember that the reductionist method was a necessary path to an understanding of the integrated, modelized biology that is emerging nowadays. at present, certain people call reductionism naive, but this is only the case insofar as we have faith in recent advances in integrated biology 89 . with this in mind, it should be noted that the deciphering of the protein synthesis mechanism in prokaryotic microorganisms (chapter iv-1.1.1) was, along with the discovery of the genetic code, a jumping-off point for an inventory of similar, but noticeably more sophisticated, mechanisms in eukaryotic organisms. the reductionist "one gene, one enzyme" dogma, formulated on the basis of beadle and tatum's experiments on the mold neurospora crassa (chapter iii-6.1) was a necessary prerequisite to a considerably more elaborate understanding of the relationship between the genotype and the phenotype. the way in which the nucleic acid and protein units in the tobacco mosaic virus spontaneously organize themselves (chapter iii-7.3) acted as a basis for thought concerning the self-organization of macromolecular complexes in the cell. these few examples underline the fact that it is difficult to comprehend the scientific research process if we only refer to experiments carried out in the present, and if we do not have a clear idea not only of the way in which hypotheses, even false ones, were once formulated, but also of the way in which experimental work, which may have led to failures, was once carried out, or, in brief, if we do not look back at the past. let us add that it is occasionally good for us to show some humility when we take the trouble to examine the past. thus, the processes involved in the phagocytosis of bacteria by innate immune cells (neutrophils, macrophages), which are today studied in the greatest detail with particularly refined technical facilities, had already been perceived more than a century ago by metchnikoff, and even analyzed, admittedly with the clumsy means at his disposal, but with such accuracy that none of the conclusions formulated at that time have yet been disproved (chapters iii-2.2.4 and iii-6.2.5). the experimental method applied to the life sciences, the history of its birth and of its development, the way in which it is regarded by political and societal authorities, and, finally, the dependencies that are developing at present between the technosciences, human medicine and the different branches of the economic sector, all of these aspects should be covered by university teaching that includes not only the pure sciences, but also the human, political and economic sciences, as well as philosophy. the student should not be saturated with book-learning, but he or she should be taught to reason, to imagine and to criticize, not to accumulate knowledge in an indigestible catalogue, but to ask questions about the way in which certain, carefully chosen, items of knowledge have been acquired, and not to deliberately accept science in its current state without knowing what it was like in the past. he or she should understand what pathways of thought led to dogmas that were established and taught as truths being refuted, and favor experimentation, with its risks and questions, rather than well-smoothed, abstract theoretical presentations without rough edges. these should be the principles of teaching that is designed to open up young minds to creativity. in anglo-saxon countries, the worlds of industry and research that welcome the graduate manage to communicate with one another, but these worlds ignore one another in france, or at least remain reserved, a situation which is prejudicial from the economic point of view. if we look at the pharmaceutical industry in particular, we see that only half a century ago the pharmacopeia was limited to plant extracts or active agents isolated from these plants, with antibiotics quietly beginning to make their appearance. in the last decades of the 20 th century, a great technological leap forward was made, with completely new methods in bioengineering, combinatory chemistry, and the finding of therapeutic targets in macromolecules, and this created a hiatus that severely handicapped countries that were unprepared for it. france, with its biological fundamental research training that is out of phase with that of the anglo-saxon countries, fell behind, and continues to be behind, a situation that is prejudicial for its economy. the remedy for this does not lie in incantatory speeches. it requires a volontarist policy for the management of experimental research. generally speaking, the fact that the major engineering schools in france, which recruit the scientific intellectual elite, students being chosen by competitive exams that select for intelligence rather than imagination, are unable to impose upon their students an end-of-course thesis that would authenticate their engineering degree, should not be tolerated. in contrast to other countries, in france only a small percentage of engineers have received doctoral training or had to present a thesis before entering their careers. the french dual system of major engineering schools and universities, which, a century ago, made sense for the economy of that period, has become completely obsolete, and deserves a courageous revision. "there is a question, much older than modern science, which has never ceased haunting certain men of science: that of the conclusions that the existence of science and the contents of scientific theories can lead to concerning the relationships that humankind has with the natural world. such conclusions cannot be imposed by science as is, but they are an integral part of the metamorphosis of this science." the new alliance. metamorphose of science -1986 (2 nd edition) in the 1950s -1960s, the hybridization of the techniques of genetics, biochemistry and biophysics gave birth to molecular biology. with the resolution of the double helix structure of dna, the demonstration of its replication, the elucidation of the mode of expression of its nucleotide sequence as a sequence of amino acids in proteins and finally the deciphering of the genetic code, biology underwent a revolution of an amplitude similar to that which, at the end of the 19 th century, saw a blossoming of the seeds of cell biology. the last decades of the 20 th century represented the utilitarian era of molecular biology. the introduction of genetic engineering into biological experimentation dates to the beginning of the 1970s. it was at this time that techniques were developed that made it possible to transfer a fragment of genomic dna from one species into the genome of another species. genetic engineering now fills a predominant position in the life sciences, supported by increasingly effective biocomputing or bioinformatics techniques. it is easy to understand that expertise and a high degree of knowledge about fundamental research is necessary in order to be able to master or even invent the genetic engineering techniques that are indispensable if we are going to produce biomolecules with a therapeutic impact, such as those that are currently being used in the pharmaceutical domain: insulin, growth hormone, blood coagulation factors, vaccines, etc. the engineering sciences that make up the greater part of contemporary biotechnology have now come to the fore in many domains of the life sciences. it is thus that a modernistic and original way of investigating nature has come into being. a multiparametric model, in which biocomputing or bioinformatics and high-throughput screening reign, is added to, or even substituted for, the bernardian model for the experimental method, based on observation, an a priori hypothesis, and experimentation to verify this hypothesis by varying a single parameter at a time. the aim of this globalized approach is to integrate the multiple reactions that take place almost simultaneously in different locations of a cell into a coherent whole, to rationalize the interpretation of the dialogue that operates between the different endocellular organelles, and finally to discover how the exchanges of information between cells in an organ and between organs in multicellular organisms are set up. we are therefore witness to the emergence of an integrated biology that has been labeled "systems biology". its long-term objective is to model the functioning of living beings and to theorize them. its development is encouraged by the perspective of consequences that could revolutionize certain sectors of the human economy and of public health. today, concrete, mechanical models, in the form of biorobots and hybrid robots, and, very recently, molecular motors are added to abstract models that are based on the logic of mathematics and algorithms, ushering in the era of nanobiomachines. becoming more utilitarian, the life sciences are imperceptibly detaching themselves from traditional philosophical concepts that try to explain the modes of reasoning of the researcher, or even to impose a framework for thought that is likely to orient his or her way of doing research. looking at genetic inheritance, contemporary experimentation has shown that at all levels of the tree of nature, including man, this inheritance can be modified. aware of his or her ability to influence the functioning and the destiny of living beings, the researcher is confronted with the dilemma of a desire for knowledge versus a questioning of the use to which discoveries may be put. there has never been such a real divorce between the world of phenomena that are understood by the experimenter and the world of noumena whose intelligibility is foreign to our senses. there has never been such a wide gap between the biotechnosciences, whose possibilities are coming to be seen as limitless, and a reflective analysis of thought, which wanders between freedom of action and prohibition. as society becomes aware of the potential applications of discoveries made concerning living beings, problems of bioethics, particularly those involving reproduction, have become problems of public interest. cloning and the production of stem cells are subjects that give rise to diatribes and passions. in the near future, genotyping, which is the result of progress in pharmacogenetics, could usher in a new form of customized medicine. elsewhere, the cognitive sciences that are bringing together philosophy and psychology in the domains of computer technology and artificial intelligence, and which are tackling the processes of thought, the creative imagination and memory, will no doubt be the subject of the considerable questioning concerning research on living beings with which the experimental method will be confronted in the 21 st century. when faced with the way in which biotechnologies have erupted into the life of society, the mind travels back to the allegorical illustration that embellishes francis bacon's novum organum (see figure ii.19) , showing vessels returning from unknown lands, loaded with precious cargoes and returning to port having sailed past the pillars of hercules. at present, the challenge has been partially met, but a great deal remains to be done. innumerable cargoes have already reached port, but what will be the destiny of this precious merchandise? after all, the seeds of the idea of technoscience were already in place in the 17 th century, in the philosophy of francis bacon and robert boyle (chapter ii-6). bacon recommended that the governments of the time promote experimental science by the creation of laboratories equipped with high-performance instruments and libraries, by the organization of researchers into teams and by appropriate financing. the utilitarian ends of scientific research were underlined. boyle imagined a situation in which laboratories were open to society and researchers were able to accept criticism. given innovations that upset tradition, protestations arose. the pneumatic machine or vacuum pump was the subject of the fameuse diatribe between boyle and the philosopher hobbes (chapter ii-6.2). hobbes criticized the validity of boyle's conclusions, drawn from experiments that he qualified as doubtful. following his words, he came to see in the discoveries of experimental science a possible threat to the power of governments and the hierarchical layout of society. such overcautious opposition to the pursuit of knowledge is in no way anecdotal, it is still a reality, with the uprooting of genetically modified plants and the veto that has been placed in certain areas on stem cell research. this type of opposition is also shown when pressures or even vetoes are in operation that take into account more the opportunism of the moment than an in-depth understanding of science and of its history and that forget that freedom of the mind is a guarantee of its creativity, because, just as in the world of arts and letters, the world of scientific research is situated outside those norms that can be modulated by state decrees. the creativity of the researcher cannot be manufactured on demand. where it exists, it still needs to be detected and encouraged. the atp-synthase -a splendid molecular machine structure at 2.8 å of f1-atpase from beef heart mitochondria direct observation of the rotation of f1-atpase powering an inorganic nanodevice with a biomolecular motor mechanically driven atp synthesis by f1-atpase atp-driven stepwise rotation of fo-f1 atp synthase key: cord-277076-yvsyo4l9 authors: berger, a.; preiser, w. title: sars date: 2019-09-12 journal: encyclopedia of environmental health doi: 10.1016/b978-0-444-63951-6.00624-0 sha: doc_id: 277076 cord_uid: yvsyo4l9 severe acute respiratory syndrome (sars) emerged in southern china in late 2002. it first spread within guangdong province and then to other parts of china. via air travelers, it quickly reached various countries around the globe, causing several major hospital outbreaks. within weeks, the causative agent, a previously unknown coronavirus (sars-cov), was identified, thanks to an unprecedented international effort led by the world health organization (who). its origin was quickly traced to wild animals traded locally for culinary purposes. masked palm civet and some other species seem to have acted as intermediate hosts. since then, sars-like coronaviruses were found in different bat species in china and elsewhere, and bats are now regarded as the wildlife reservoir for sars-cov. fortunately, the sars outbreak could be contained within months. until july 2003, it had caused 8096 cases, with 774 deaths. once adequate measures such as isolating patients and quarantining their contacts were strictly adhered to, further transmission between human beings could be interrupted. sars is an example of how rapidly an infectious agent can spread in the modern world. at the same time, it should serve as a showcase of how international cooperation and modern science can help to combat the spread of infectious diseases. in an unprecedented move, who initiated a collaborative multicenter research project to identify the causative agent of sars. avian influenza was quickly ruled out. several laboratories diagnosed active infections with chlamydia or paramyxoviruses in somebut far from allpatients suffering from sars. however, there was good evidence that neither of these were plausible etiological agents for the novel infectious disease. the network comprised laboratories with access to sars patient samples and those with particular expertise on emerging or respiratory viruses. for several weeks, a spirit of cooperation rather than competition prevailed. samples were exchanged and results and findings shared continuously via daily telephone conferences and a password-protected website. within weeks, groups in hong kong, germany, canada, and the united states of america found a hitherto unknown member of the coronavirus family in sars patients. this was achieved through independent studies but certainly benefited greatly from the real-time sharing of interim results. although different approaches were employed, including cell culture, electron microscopy, and molecular techniques, sequencing data showed all laboratories had identified the same coronavirus, previously unknown to human and veterinary virology ( figure 1) . it was shown that sars patients seroconverted against this virus in the course of their illness; healthy, unexposed control individuals lacked antibody reactivity. however, it remained to be proven that this novel coronavirus was indeed the etiological agent for sars rather than an 'innocent bystander' newly discovered by thorough studies. after experimental infection of macaques with the newly isolated agent was shown to cause a sars-like illness, and subsequent reisolation of the agent, all of koch's postulates had been fulfilled. on 16 april 2003, who officially announced that the provisionally termed sars-associated coronavirus (sars-cov) was the causative agent of sars. based on this breakthrough, tests for the detection of viral sequences and specific antibodies were quickly developed and made available to affected countries. in addition, numerous scientists embarked on programmes to develop vaccines and drugs or antibodies for prophylactic or therapeutic use. within a few months, the sars outbreak was brought under control. on 5 july 2003, who declared that the last chain of person-toperson transmission had been interrupted. measures including source isolation of patientswho only became infectious after onset of clinical symptomsstrict infection control in health care facilities, timely identification and quarantining of exposed contacts, and perhaps also measures to increase social distance, such as travel warnings and screening of travelers, had led to this remarkable and remarkably rapid success. thorough and consistent implementation of these measures eventually brought an end to the sars outbreak even in the worst affected areas. in the meantime, however, several areasdifferent chinese provinces other than guangdong, most prominently the capital, beijing, but also toronto in canada, and taiwanpaid a high price for not implementing adequate countermeasures in a timely fashion. typically, a so-called 'superspreader,' that is, a highly contagious sars patient, would seek treatment at a poorly prepared facility, and by the time the danger was realized, scores of staff and patients had become infected and themselves become sources of spread. interestingly, despite the rapid identification of the agent and laboratory tests becoming available almost immediately, these formidable achievements did not contribute much to the containment of the outbreak. instead, it was the prudent and thorough use of 'old-fashioned' measures such as isolation and quarantine that proved to be the key to success. identification of suspected cases was based on clinical and epidemiological criteria: high fever (>38 c) plus symptoms of respiratory tract infection plus an exposure history, the details of which depended on each location's sars status at the time. an additional positive sars-cov test result or radiological or pathological evidence of pneumonia or respiratory distress syndrome would make it a probable case. the final case count from 1 november 2002 until 31 july 2003 is 8096, with 774 deaths. since mid-2003, sars has reappeared on four occasions. three involved laboratory-acquired infections, which demonstrates the dangers of breaching biosafety procedures and the risks of subsequent further spread in the community by secondary transmission outside of the laboratory. the fourth sars outbreak was due to reintroduction from the reservoir. to minimize the risk of reemergence, who has issued guidelines for the surveillance of possible sars cases. risk categories to guide adequate national surveillance strategies to guard against the possible (re-)emergence of sars are emergence from wildlife or other animal reservoirs, emergence or introduction from laboratories or via international travel, or low risk of sars-cov emergence or introduction. who also urges all countries to conduct an inventory of all laboratories working with or storing sars-cov and to ensure strict enforcement of biosafety procedures. before sars, only two coronaviruses (hcov-229 e and hcov-oc43) were known to infect humans. because they both cause only relatively minor disease and are difficult to propagate in the laboratory, they received relatively little attention by diagnostic and research laboratories. in contrast, several coronaviruses were recognized as animal pathogens, infecting pigs, dogs, rabbits, cats, mice, rats, turkeys and chicken. some of these had for some time played important roles in veterinary medicine. it was obvious that the majority of human sars cases were acquired through transmission from other sars sufferers (which proved key to its eventual control). however, it seemed highly unlikely that this was a previously existing disease entity that was only then being recognized; its propensity to cause nosocomial outbreaks, often in hospital settings, would not have been overlooked for long. the very first sars cases were identified retrospectively in various localities in guangdong province. the first of these early cases occurred in foshan in november 2002, and several more over the following weeks in nearby places. interest soon focused on these early index cases: was there any characteristic these patients had in common that could point to a source of their sars-cov infection? there was indeed; many of these early index patients either worked in kitchens or at markets or lived nearby where they were constantly exposed to a multitude of species of domestic and wild animals that were being traded, kept in captivity, and finally slaughtered and prepared for consumption. this provided the impetus for studying animals being sold at guangdong markets. infection with coronaviruses closely related to sars-cov was identified in different species, most commonly in the masked palm civet (paguma larvata) but also in the chinese ferret badger (melogale moschata) and the raccoon dog (nyctereutes procyonoides). a further, small sars outbreak occurred again in guangdong in late 2003/early 2004; molecular analysis of virus isolates from human cases and animals sampled at the same place and time confirmed that this was zoonotically acquired from paguma larvata. however, further investigations revealed that this widely consumed species is most likely not the animal reservoir, as most populations studied were uninfected. later research identified a multitude of coronaviruses in different species of bats in asia and elsewhere, among them what is probably the progenitor of sars-cov. studies by different groups demonstrate that sars was the result of spillover from a wildlife reservoirmost likely batsinto an intermediate host (or hosts; most importantly paguma larvata) and from there to the human population. rapid viral evolution was demonstrated and most likely was the pivotal factor that allowed sars-cov to rapidly adapt to nonreservoir species. table 1 shows the distribution of several different coronaviruses in humans and animals and their classification into different groups. disease caused by sars-cov may present with rather nonspecific clinical signs and symptoms. the differential diagnosis is therefore wide and may include various common respiratory pathogens, including influenza, parainfluenza, and respiratory syncytial viruses (rsv) the laboratory diagnosis of sars remains a challenge; in fact, despite the rapid identification of sars-cov as the etiological agent, testing contributed little to the successful control of the 2003 outbreak. insufficient test specificity on occasions caused false-positive results, leading to considerable confusion. in many viral diseases, virus shedding is greatest during the early symptomatic phase, that is, around, and immediately following the onset of symptoms. unfortunately, virus excretion is comparatively low during the initial phase of sars. it peaks in respiratory specimens and in stools at around day 10 after the onset of the clinical illness. in addition, there are currently no laboratory tests available to reliably diagnose sars in the first few days of illness. the highest test sensitivity is achieved with bronchoalveolar lavage fluid or lung biopsy tissue at the onset of illness. because of the invasiveness of such procedures and the associated risk of transmission, nasopharyngeal aspirates and throat washings, taken with respiratory precautions and preserved in viral transport medium, remain the most important diagnostic specimens. most commonly, viral genome detection, usually by reverse transcriptase-polymerase chain reaction (rt-pcr), is used diagnostically. nucleic acid amplification tests have been designed targeting the orf1b or nucleoprotein genes; it has not been clearly proven in clinical studies that they are superior. real-time rt-pcr of nasopharyngeal aspirates is the most sensitive and rapid method. furthermore, the determination of viral load in nasopharyngeal specimens or serum has been shown to be of clinical value, as it is an important prognostic factor. to avoid false-positive results, who stipulates that the following should be tested: at least two different clinical specimen types (e.g., nasopharyngeal aspirate and stool), or the same type of clinical specimen but collected on at least two occasions during the course of the illness (e.g., sequential nasopharyngeal aspirates), or the same original clinical sample but two different assays or by repeating the same assay but using a new rna extract for each test. sars-cov is cultivable on vero and caco2 cells not only from respiratory materials but also from fecal samples ( figure 2 ). once isolated, the virus must be identified as sars-cov using further tests. cell culture is a very demanding test, but currently (with the exception of animal trials) the only means to show the existence of a live virus. antigen detection in respiratory and fecal specimens using enzyme immunoassay (eia) is generally less sensitive than rt-pcr. however, antigen detection in serum specimens with monoclonal antibodies or monospecific polyclonal antibody against the viral n protein was found to be a sensitive and specific test for the diagnosis of sars. as serum antibody levels start to rise from day 7 after onset of illness, the sensitivity of the serum antigen assay progressively decreased to 0% at day 21. antibody testing allows the indirect diagnosis of sars-cov infection and is unsuitable during the acute illness. positive antibody test results indicate previous infection with sars-cov. seroconversion from negative to positive or a fourfold rise in the antibody titer from acute to convalescent serum indicates a recent infection. a negative antibody test result later than 21 days after the onset of illness is likely to indicate that no infection with sars-cov has taken place. virus-specific serum igg, igm, and iga antibodies against sars-cov appear at around the same time, between days 5 and 17 after the onset of symptoms. antibody testing is therefore generally not useful during the first week of illness. for antibody testing, the indirect immunofluorescent antibody test is more commonly performed than the neutralizing antibody test in cell cultures that again requires a biosafety level 3 laboratory (figure 3) . a recombinant nucleocapsid eia may be used as a rapid screening test and possesses a higher sensitivity, with detection as early as day 5 after onset of illness. single low-titer positive antibody results can be due to cross-reactivity with other human coronaviruses and require confirmation by more specific western blotting or neutralization assays. who regards seroconversion or an at least fourfold rise in antibody titer between an acute and a convalescent serum as proof of infection. the updated who guidelines for the global surveillance of sars of october 2004 replace all previous who guidance on sars surveillance and response. one key recommendation is that independent of the test used, who strongly recommends that during the interepidemic period all countries seek verification of laboratory-confirmed cases of sars ('preliminary positive' cases), preferably by an external laboratory that is part of the who sars international reference and verification laboratory network (figure 4) . human sars-cov are no longer circulating in the human population, nevertheless the virus is still present in the animal reservoir and virological laboratories and can reemerge anytime. there is no single test that can be used to diagnose sars with a reasonable degree of accuracy. diagnosis, therefore, continues to rely on the clinical examination, supported by case definitions that include the risk assessment according to the 2004 updated who guidelines (tables 2 and 3 ). the initial symptoms of sars are nonspecific, complicating the differential diagnosis. the mean incubation period is 5 days with the range of 2-10 days although there are isolated reports of longer incubation periods. unlike influenza virus, where the patients are most infectious in the first 2 days of illness, transmission from symptomatic sars patients usually occurred on or after the fifth day of onset of disease, which is in line with the rising viral load in nasopharyngeal secretions that peaked at around day 10. there have been no reports of transmission occurring before the onset of symptoms. the typical clinical presentation of sars is that of viral pneumonia with rapid respiratory deterioration. patients initially develop influenza-like prodromal symptoms. fever, malaise, myalgia, headache, rigors, and nonproductive cough are the major presenting symptoms, whereas rhinorrhea and sore throat are less frequently seen. clinical deterioration, often accompanied by watery diarrhea, commonly occurs 1 week after the onset of illness. although history of fever is the most frequently reported symptom, it may be absent on initial measurement. severe cases develop rapidly progressing respiratory distress and oxygen desaturation with approximately 20% requiring intensive care. chest radiographs typically show ground-glass opacities and focal consolidations, especially in the periphery and subpleural regions of the lower zones. progressive involvement of both lungs is not uncommon. features during the later stages have sometimes included spontaneous pneumothorax, pneumomediastinum, subpleural fibrosis and cystic changes. nosocomial transmission of sars-cov has been a striking feature of the sars outbreak. the majority of the cases have occurred in adults. children are less commonly affected than adults and usually have a milder illness. the overall mortality rate was approximately 10%. age and the presence of comorbidities are poor prognostic indicators. although the sars outbreak was still ongoing during the first half of 2003, significant funding was made available for sars-related research. immunomodulators (i.e., corticosteroids, intravenous immunoglobulins, thymosin, and anti-tnf) were empirically used for the treatment of sars during the initial epidemic. the correlation between viral load and clinical outcome suggests that suppression of viral replication by effective antiviral drugs should be the key to preventing morbidity and mortality. numerous potential antiviral agents have been identified using different approaches. in vitro susceptibility test results demonstrate that ifn-alpha and ifnbeta have some potential activity. ribavirin has good activity when tested in human caco-2 cells despite its lack of activity in vero cells. the viral proteases are important targets for the development of antiviral drugs. protease inhibitors like nelfinavir, glycyrrhizin, chloroquine, and many others as well as many herbal formulations have been found to possess some antiviral activity against sars-cov in vitro. in addition, the use of nitric oxide (s-nitro-n-acetylpenicillamine) inhalation as an experimental form of rescue therapy for sars appeared to have inhibitory activity against sars-cov. screening of chemical libraries has identified several inhibitors of the viral protease and helicase. identification of angiotensin-converting enzyme 2 (ace2) as an obligatory cellular receptor for table 2 risk categories for the emergence of sars emergence of sars-cov-like viruses from wildlife or other animal reservoirs -countries/areas identified as source(s) of the epidemic in 2002-03 in southern china or areas with an increased likelihood of animal-to-human transmission of sars-cov-like viruses from wildlife or other animal reservoirs. emergence or introduction of sars-cov from laboratories or international travel -countries/areas at potentially higher risk of sars-cov emergence or introduction due to the presence of laboratories in which sars-cov or sars-covlike viruses are being studied or in which clinical specimens infected with sars-cov are being processed or stored. or -countries/areas with entry of large numbers of persons from areas in which wildlife or other animal reservoirs of sars-cov-like viruses are found. low risk of sars-cov emergence or introduction -countries/areas that never reported cases or reported only imported cases during the 2002-03 epidemic, and that do not conduct research using live sars-cov-like viruses or store clinical samples from sars cases. sars-cov contributed to understanding of the sars-cov entry process, and helped to characterize two targets of antiviral therapeutics: the sars-cov spike protein and ace2. however, most of the chemicals or approaches have not been evaluated in human or animal models. various approaches toward producing a vaccine against sars have been pursued, including the use of inactivated sars-cov, plasmid dna, and adenovirus vectors. one obvious problem any vaccine would face is whom it should be given to, in the absence of sars-cov transmission. however, waiting until a renewed outbreak occurs before commencing vaccination means that precious weeks would be lost until individuals at risk become immune. the administration of preformed antibodies (obtained from human or animal donors or, more recently, produced in vitro) is effective in preventing a number of different infections, such as hepatitis b, varicella, and rsv. similarly, it could be shown that neutralizing antibodies against sars-cov can protect experimental animals from infection. using screening of a large naive antibody library by antibody phage display technology, neutralizing antibodies were identified and produced that were protective in vitro. there was no evidence of enhancement of sars-cov infection by subneutralizing concentrations of these antibodies, and immune escape mutants were not generated. in theory, this could be an ideal tool: if sufficient stocks of such human monoclonal antibodies could be procured, they would be ready for use when and wherever sars reemerges. one could then passively immunize anyone in contact with the source or patients, affording immediate protection. in summary, sars was a novel severe infectious disease that presumably originated in guangdong in southern china. large-scale wildlife trade and consumption favored the emergence of this zoonosis from a hitherto unrecognized animal reservoir. after its zoonotic origins, the new agent quickly spread within the human population, being transmitted mostly via the respiratory route through close human-to-human contact. sars was rapidly disseminated via the metropolis hong kong through international air travel. its important potential for nosocomial transmission in the community and in hospitals was soon recognized and allowed for appropriate measures to be instituted in most places. there was an unprecedented rapid gain of knowledge through global networking and international collaboration, led by who. unfortunately it took some major outbreaks, some of them affecting industrialized countries with modern health care systems, until the first sars epidemic was controlled. thus in the end rapid success was achieved through 'traditional' sanitary measures, mainly rapid identification of suspect cases and isolation of the diseased. fortunately, sars-cov hasat least so farnot established itself permanently in the human population. its relatively low transmissibility (with the exception of 'superspreaders') led to a low basic reproduction number r 0 ; together with the fact that infected individuals only become infectious themselves once they have developed clinical disease, this made 'traditional' public health measures very effective. is the fact that the first pandemic of the twenty-first century was quickly contained reason to take comfort in the knowledge that the history of humankind has reached a phase in which biomedical sciences and information technology are able to deal with such threats? probably not. first, the rapid success was remarkable. unfortunately, though, this does not reflect preparedness. in 1998, donald burke proposed the following criteria for identifying virus families with a high pandemic risk: (1) those that had caused pandemics in human populations in the recent past; (2) those with the proven ability to cause larger epizootics; and (3) those with an intrinsic propensity to rapidly undergo evolution on the basis of high mutation rate or genome organization favoring recombination ('intrinsic evolvability'). interestingly, even before the sars outbreak (which obviously fulfills the first criterion), the coronavirus family should clearly have been regarded as high risk: starting in the late 1970s, the porcine epidemic diarrhea coronavirus (pedv) caused a severe swine epizootic in europe and asia (second criterion). the high evolvability of the coronavirus family had also been demonstrated: the porcine respiratory coronavirus (prcv) evolved through a deletion mutation in the s gene from the transmissible gastroenteritis virus (tgev); the mutant has a different tissue tropism and is less virulent. second, infectious disease emergence from zoonotic sources was a well-recognized threat long before sars. nevertheless, it took anotherfortunately minor -sars outbreak before decisive steps were taken to control at least the trade in the directly implicated paguma larvata in southern china. starting shortly after sars had been controlled, another agent with pandemic potential has caused an ongoing epizootic of unprecedented proportions: highly pathogenic influenza a (h5n1) virus. transmission to humans occurs almost exclusively through close contact with infectedand sickpoultry. despite the possibility that this avian influenza virus will become fully 'humanized' and trigger the next influenza pandemic, even activities recognized as high risk, such as 'wet markets,' have mostly not been curtailed. finally, the lessons from sarsparticularly the experiences of beijing, toronto, and taiwan that suffered devastating sars outbreaks during the later phase of the outbreakemphasize over and over again the enormous importance of open and timely communication. at times, this may mean admitting to problems that (at least initially) reflect negatively on the country or territory concerned. in the medium to long term, however, such openness is the only way to prevent consequences that would be much worse. although this was widely accepted in the wake of the sars experience and has had a strong influence on the new international health regulations that entered into force in june 2007, the information policies of some countries during the h5n1 epizootic unfortunately do not attest to this at all. notwithstanding its grave consequences in humanitarian, political, and economic terms, sars should serve as an example what can be achieved through international cooperation, modern science, and rigorous use of 'traditional' approaches. even if prudent precautions were suddenly adopted, sars will certainly not have been the last highly pathogenic novel infectious agent that crosses the species barrier into the human population. although bats must be very high on the list of 'culprits' for emerging viral diseases, having been identified as reservoir hosts for a number of emerging viruses, there is no reason why other groups of animals should not figure as prominently in future. the mechanisms behind emergence have been studied. these may be linked to the agent, the new host species (i.e., human beings and in certain cases also the intermediate hosts) or to the connection between those. many of the factors and determinants of disease emergence are related to human activities. although this has accompanied humankind throughout history, and in fact may have helped shape human history, the speed and the extent of human-induced changes has accelerated markedly in recent times. although in the case of sars people were lucky in the end, this may be quite different next time round. it will be important to improve understanding of emergence to minimize the risks of what is a natural phenomenon but much aggravated by human behavior. severe acute respiratory syndrome (sars)ã�paradigm of an emerging viral infection the evolvability of emerging viruses development of antiviral therapy for severe acute respiratory syndrome severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome: identification of the etiological agent evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses viral zoonosesã�a threat under control? human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets bats, civets and the emergence of sars wet marketsã�a continuing source of severe acute respiratory syndrome and influenza? relevant websites www.cdc.gov/eid. emerging infectious diseases: (search for sars) health protection agency (uk) information and guidance on sars released by the world health organization (who) international office of epizootics key: cord-275796-4560i8cx authors: kumar, prashant; srivastava, mansi title: prophylactic and therapeutic approaches for human metapneumovirus date: 2018-10-20 journal: virusdisease doi: 10.1007/s13337-018-0498-5 sha: doc_id: 275796 cord_uid: 4560i8cx human metapneumovirus (hmpv) is an important pneumovirus which causes acute respiratory disease in human beings. the viral infection leads to mild to severe respiratory symptoms depending on the age and immune status of the infected individual. several groups across the world are working on the development of immunogens and therapy to manage hmpv infection with promising results under laboratory conditions but till date any virus specific vaccine or therapy has not been approved for clinical use. this minireview gives an overview of the prophylactic and therapeutic approaches to manage hmpv infections. respiratory viruses have been one of the major causes of concern among the public health authorities and scientists dealing with health issues. the threat posed by viruses like influenza a virus, respiratory syncytial virus (rsv) and severe acute respiratory syndrome (sars) virus is well understood and variety of strategies are under development to counter these viruses. human metapneumovirus (hmpv), which was identified in 2001 by scientists in netherland, is yet another respiratory virus having the ability to cause mild to severe disease in people of all age groups especially in children from 1 to 12 years of age, accounting for about 10% of all the lower respiratory tract infections (lrtis) [3, 27, 48, 70, 78, 107] . despite several researches, any vaccine or therapy has not yet been commercialized to take care of the hmpv infection. this review focuses on different prophylactic and therapeutic approaches that have been investigated till date against human metapneumovirus. hmpv belongs to metapneumovirus genus in the pneumoviridae family which was created in 2016 [86] . other members of the family include avian metapneumovirus (ampv), human respiratory syncytial virus (hrsv), bovine respiratory syncytial virus (brsv) and murine pneumonia virus (mpv). the enveloped virus has negative sense, single stranded, non-segmented rna genome of length between 13,280 and 13,378 nucleotides [77] . the genome contains eight genes encoding nine proteins in the order 3 0 n-p-m-f-m2-sh-g-l 5 0 which is further flanked by a leader sequence at 3 0 end and trailer sequence at the 5 0 end (fig. 1) [8, 52] . the leader and header sequence has a role in replication and transcription of the viral genome. the nucleoprotein (n), phosphoprotein (p), a matrix protein (m2-1) and rna dependent rna polymerase (l) get associated with rna genome to form rna polymerase complex to carry out viral replication and transcription [44] . the virion surface shows the presence of three glycoproteins viz. small hydrophobic (sh) protein, a heavily glycosylated g protein and fusion (f) protein (fig. 2) . while the g and f proteins have a role in fusion and entry of the virus in host cell through heparan sulphate proteoglycan receptors, the role of sh protein is not completely understood but some of the studies claim that it helps the virus in evading the host antiviral immune response by down-regulating the type i ifn signalling and also has a role in regulating host membrane permeability [41, 65] . the matrix (m) protein forms a layer beneath the virus envelop and have a role in virus assembly and budding. two proteins viz. m2-1 and m2-2 are encoded by the second m (m2) gene. the m2-1 protein enhances the processivity of rna dependent rna polymerase (rdrp) and is required for the full length synthesis of viral mrna [16] while the m2-2 protein maintains the balance between viral genome replication and transcription in the host cytoplasm [14, 91] . two genotypes of hmpv have been observed in the clinical isolates reported till date: a and b, which have been further divided into five genetic lineages: a1, a2a, a2b, b1 and b2. the classification has been done on the basis of variability in membrane glycoproteins of the virus. at protein level, f protein is highly conserved between the types of hmpv with the sequence identity of 94-98% while g protein is highly divergent with the sequence identity of only 30-35% between the subtypes [52, 110] . hmpv has a worldwide distribution with the peak of its activity coinciding with the peak of rsv activity. although the virus circulates throughout the year, it is predominant in late winter and spring [36, 63, 79, 93, 99, 105] . the disease severity also varies from mild respiratory symptoms to severe pneumonia which ultimately depends on other factors like age of child and presence of other chronic ailments [73, 74] . in a study done in 2017 at university hospital of trondheim, norway, approximately 10% of the children hospitalized with lrti were diagnosed with hmpv infection. it was observed that children who were born premature or had any other chronic airway disease like asthma were more prone to infection with hmpv than other related respiratory viruses like rsv. the age of child was also correlated with the disease severity. the child at the age between 12 and 23 months showed the most severe symptoms compared to other age groups and it was concluded that the maternal antibody plays a significant role in protecting a child below 6 months of age [68] . apart from children, hmpv infection is observed in people of all age groups, though at a lower rate, with only about 3.5% of adults with lrti being positive for hmpv infection [49, 71, 81, 92] . the sero-epidemiology studies show that almost 100% of adults across the world are seropositive with respect to one or the other strain of hmpv [48, 56] . similar observations were recorded in yet another study involving the subjects from both northern and southern hemisphere and it was observed that burden of hmpv was significantly higher in northern hemisphere as compared to southern hemisphere [3] . however, difference in disease severity due to strain variation is not clear and varies considerably. knowledge of host immune response to hmpv infection is required to understand the immunopathology induced by the virus and further development of vaccines or therapeutics. like any other virus, both innate and adaptive immune components are essential to clear the hmpv infection but it induces a weak memory response in the host [31, 38] . innate immune responses are the first line of defence against the viral infection and act by recognizing the pathogen associated molecular patterns (pamps) on the viral particles through the pattern recognition receptors (prrs) present on the immune cells in the respiratory tracts. the common prrs like membrane bound toll-likereceptors (tlrs), c-type lectin receptors (clrs), cytoplasmic rig-i like receptors (rlrs) and nucleotide-binding oligomerization domain (nod) like receptors (nlrs) recognize the viral pamps and initiate the inflammatory and immune response via the cytokines produced by the cells in respiratory tract [19] . myd88 (common adaptor of tlrs) mediated pathways has been shown to be essential for pulmonary immune response to hmpv infection by recruitment of immune cells to lungs [83] . however, hmpv g protein has an important role in inhibiting the innate immune responses by targeting rig-i in host cell cytoplasm [4, 55] . on the other hand, adaptive immunity is highly virus specific. during hmpv infection, the protective humoral immunity is mostly directed towards the f protein of the virus rather than g and sh proteins [35] . the cell mediated immune response is majorly responsible for immune surveillance in the infected host and clears the virus infection by activation of virus specific cytotoxic t lymphocytes (ctls) which induces the apoptosis of infected cells or by activating the th cells to further activate b cells and other immune cells like dcs and macrophages [19] . studies done on mice models indicate that during hmpv primary infection, cd4 ? as well as cd8 ? t cells are responsible for inflammation and body weight loss leading to severe lung disease but they also have a protective role of clearing the virus infection from the host's body which is evident by accumulation of hmpv specific ctls in the lungs of infected host at approximately 7 days post infection [2, 43, 54] . in the hmpv infected elderly hosts, th2 skewing response seems to be the cause of aggravated lung diseases [25] . however, if cd4 ? t cells are depleted, the infection is less severe and cd8 ? t cells alone can effectively clear the virus infection [54] . interestingly, it was also demonstrated in one of the recent study that the expression patterns of inhibitor receptor programmed death-1 (pd-1) and programmed death ligand-1 (pd-l1) during hmpv infection leads to impairment of cd8 ? t cell response which further contributes to hmpv re-infection [33, 34, 104 ]. an immunocompetent host shows antiviral immune response to eliminate virus infection and the virus, on the other hand, evades the host immune response by mechanisms which are specific for the virus. unlike rsv, hmpv doesn't have non-structural proteins and they evade the host innate immune response via their g, sh and m2-2 proteins [55] . the m2-2 proteins of hmpv are specifically responsible for immune evasion by blocking the interaction between the mitochondrial antiviral signalling proteins (mavs) and the downstream tnf receptor associated factors (trafs) [20] . although sero-epidemiological studies reveal that all the individuals are infected by hmpv by the age of 5 years, repeated infection by the virus is common throughout life which may be due to diminishing immunity against the viral antigen in the host and restricted cross-reactive antibodies. as with most of the viral infection, only symptomatic treatment is available for hmpv infection. several therapeutic strategies have been developed and evaluated by scientists across the world but any hmpv specific antiviral drug has not yet been brought under clinical trial. nucleoside analogues (na) are the antimetabolites which mimic physiological nucleosides to get incorporated into newly synthesized dna and blocks further dna synthesis leading to chain termination. several nas have been assessed as antiviral agents. ribavirin is one such na which is effective against the pneumoviruses both in vitro and in vivo and has been used to treat the cases of hmpv infections also [21, 32, 39, 53, 108] . apart from inhibiting the viral rna synthesis, ribavirin modulates the host immune system by up-regulating the secretion of t helper (th) 1 cytokines like tnfa, ifnc, il2 etc. by cd4 ? and cd8 ? t cells and down-regulating the secretion of th2 cytokines like il10 which further helps in restricting the viral pathogenesis [13, 89] . in absence of any experimental proof till date, it is suggested that ribavirin may be given to treat hmpv infection by aerosol at a dose that is recommended for containing rsv infection [96] . however, aerosolised drugs have their own limitations like high cost, teratogenicity and potential to weaken the respiratory functions. ribavirin has also been used through intravenous (i.v.) route in combination with intravenous immunoglobulin (ivig) to treat hmpv infections but the results have been quite contradictory. dokos et al. [30] and park et al. [75] observed that the effect of i.v. ribavirin combined with ivig was not significant in treating the hmpv infection while few other authors have reported that i.v. ribavirin/ oral ribavirin combined with ivig could successfully treat the infection in different category of patients [12, 50, 80, 95] . fusion between viral envelop and the host cell membrane via hmpv f protein is important for the initiation of viral infection [18, 22] . peptides capable of inhibiting this fusion event have the potential to reduce the virus replication in infected host. deffrasnes et al. identified few peptides derived from the heptad repeat domains of hmpv f protein and assessed their effectiveness in reducing the morbidity and mortality caused by the virus. one peptide, hra2, was found to be very effective in reducing the lung viral load, pulmonary inflammation, airway obstruction and levels of pro-inflammatory cytokines/chemokines in the treated balb/c mice [28] . the hra2 peptide, when expressed in nicotiana tabacum, has been shown to inhibit the binding of virus to hep2 cells under in vitro condition [64] . further studies need to be done to evaluate the efficacy of the peptide expressed in plant system in inhibiting the virus infection in animal models. recently, a fusion inhibitor, jnj-5371867, has been reported for rsv which is another pneumovirus closely related to hmpv [87] . there is a need to search such small molecule fusion inhibitors which may have the potential to bind to hmpv f protein also in its pre-fusion conformation and prevent the virus from infecting the host cells. nmso3, a sulfated sialyl lipid, has been shown to have potent antiviral activity against hmpv [109] . spetch et al. assessed the effect of nmso3 treatment on hmpv infection. treatment of balb/c mice with one dose of nmso3 at 50 mg kg -1 body weight at the time of infection significantly reduced the lung viral load and also reduced the recruitment of inflammatory cells in the lungs. the clinical illness and level of pro-inflammatory cytokines/chemokines in the lungs was also significantly reduced under the treatment with nmso3. it was speculated that in addition to having antiviral effect by inhibiting the viral entry and replication in host cells, nmso3 also has anti-inflammatory effect in the recipient host [97] . rna interference using small interfering rnas (sirnas) or short hairpin rnas (shrnas) has been used since a long time for specific silencing of target genes. as a therapeutic approach for hmpv, darniot et al. and deffrasnes et al. demonstrated the efficacy of sirnas specific for viral genes encoding nucleoprotein (n) and phosphoprotein (p). darniot et al. [26] showed that the 2 0 -o-methyl modified sirna targeted to the most conserved region of hmpv nucleocapsid mrna could partially inhibit the viral replication and confirmed that there was no off-target effect of this sirna and also that the cytokines did not contribute in this inhibitory effect. deffrasnes et al. [28] had previously identified two potent sirnas (sirna45 against n gene and sirna60 against p gene) and demonstrated their efficacy against the strains of all four subgroups of hmpv. the g gene of hmpv was also targeted by preston et al. but it's down-regulation did not result in reduction in viral growth, nor did any significant increase in type i interferon expression was observed. monoclonal antibodies directed against the most conserved epitopes of the immunogenic proteins of virus have the ability to protect or minimize disease severity in infected individuals. the efficacy of prophylaxis with palivizumab, a monoclonal antibody, has previously been shown for rsv infection in high risk groups. the fusion protein of hmpv is highly immunogenic and is conserved among various types and subtypes of the virus [24, 45, 76] . recently, 54g10, a human monoclonal antibody directed to a conserved epitope of hmpv fusion protein, was shown to be highly neutralizing and effective in decreasing the viral titre in lungs and nasal aspirates of infected hmpv permissive mice model. the prophylactic and therapeutic efficacy of 54g10 was comparable to palivizumab w.r.t. viral titre in nasal aspirates and better than palivizumab w.r.t. titre in lung homogenates [94] . prior to this, fab ds7, a recombinant human monoclonal antibody fragment generated using phage display technology against a fusion protein epitope was evaluated and found to be significantly effective in restricting the propagation of hmpv in the lungs of cotton rats [106] . even before this, ulbrandt et al. [102] generated a panel of monoclonal antibodies against the f protein in animal models, out of which two mabs viz. mab 338 and mab 234 were effective against almost all four subtypes of hmpv. it was speculated that humanization and optimization of the two mabs could give a better result in human individuals. in 2005, ma et al. [61] also generated two mouse monoclonal antibodies, 1g3 and 9b10, against f protein of hmpv and demonstrated their neutralizing capacity in the virus infected cells. recently, the replication and transcription of hmpv in bronchial epithelial-derived immortal cells was analyzed and it was deduced that like other filoviruses and rhabdoviruses, formation of cytoplasmic inclusion bodies is required for hmpv genome replication and transcription [69] . understanding the mechanisms and pathways for the formation of these inclusion bodies may give a crucial lead for development of new therapeutics. currently, we are also working on the development of ribozyme based therapeutics which is based on the conserved nature of fusion and nucleoprotein of the virus (data not published yet). vaccination is the most reliable strategy for managing any viral infection. several efforts have been made to develop hmpv specific vaccines and most of these vaccines are directed towards the fusion protein which is the most immunogenic protein of this virus. inactivated vaccines have been very successful in managing several viral infections like influenza, polio etc. where the vaccine is stable and biologically safe. however, the efficacy of this type of vaccine varies with the virus and method used for viral inactivation significantly affects the immune response [90] . it has been demonstrated that vaccination with formalin inactivated hmpv leads to enhanced disease severity upon challenge with wild type virus which could be attributed to enhanced pulmonary histopathology and imbalanced immune response with elevated level of th2 cytokine like il4 [111] . vaccination of animal models with heat inactivated hmpv has also been shown to be unsafe with enhanced level of th2 cytokines and eosinophil infiltration in lungs of vaccinated animals [40] . the outcome of vaccination with abovementioned inactivated virus suggest for the development of better strategies to generate hmpv specific vaccines. in past few years, nanoemulsion based inactivation of virus like rsv was demonstrated to yield safe vaccines which could induce effective humoral immune response and enhanced viral clearance from the host body [59] . this strategy along with other strategies like use of b-propiolactone (bpl) and hydrogen peroxide may also be used for safer vaccine development against hmpv [90] . vaccination with partial or full length viral proteins rather than whole virus has been shown to be sufficient to minimize the viral pathogenesis. viral vectors have been the vector of choice to introduce and express recombinant proteins in targeted host. a retroviral vector expressing the hmpv f protein has been shown to induce strong and protective immune response against different subtypes of hmpv when given through intra-peritoneal route in mice model but this was not the case with hmpv g protein [57] . expression of hmpv f protein via venezuelan equine encephalitis virus based viral replicon particle (vee-vrp) also demonstrated high level of immunogenicity and protective efficacy against hmpv infection in african green monkey [5] . likewise, recombinant sendai virus expressing the truncated fusion protein of hmpv could induce neutralizing antibodies in cotton rats indicating the potential of such a recombinant to protect the immunized host against hmpv infection [88] . prior to this, tang et al. [101] successfully demonstrated the immunogenicity and protective efficacy of bovine/human chimeric parainfluenza virus type 3 expressing the hmpv f protein in african green monkey. apart from the viral vector, bacterial systems have also been shown to act as carrier for viral genes. palavecino et al. demonstrated that recombinant bacillus calmette-guerin (rbcg) carrying the gene encoding hmpv p protein could successfully express the viral protein and immunization with the rbcg strain could induce a protective th1 immunity in mice model by activation of virus specific t cells producing ifnc and il-2 [72] . this was shown to be an effective strategy to manage the hmpv infections. use of soluble viral protein as vaccine can also induce the host immune response. intramuscular injection of iscom matrix adjuvanted soluble hmpv f protein in syrian golden hamster and cynomolgus macaque model was effective in inducing cellular as well as humoral immune response against different subtypes of hmpv but for a shorter duration of time as opposed to the viral vector based vaccines [45, 46] . prior to this, cseke et al. [24] showed that a recombinant f protein lacking the transmembrane domain, expressed from a dna plasmid construct, could induce a protective immune response in cotton rats and the response was better that the full length f protein without any lung pathology or th2 type response. later on baule et al. [7] observed the stimulation of inflammatory response by hmpv m protein and aerts et al. [1] demonstrated the effect of m protein as an adjuvant in enhancing the immunogenicity of hmpv f protein by elevating the cellular immune response. recently, it has been shown that purified hmpv fusion protein ectodomain stabilized in its pre-fusion or post-fusion states is sufficient to induce the production of neutralizing antibodies with a capacity to neutralize the virus infectivity [6] . virus-like-particles (vlps) have been successfully used as vaccine candidates against some important viral pathogens like human papilloma virus and rota virus. the nonpathogenic nature and ability of the vlps to expose the viral proteins in their native conformation makes it one of the best vaccination strategies. a hmpv vlp was developed by cox et al. [23] by expressing the hmpv fusion and matrix protein in suspension adapted hek293 cells and a protective immune response could be generated in mice model without any th2 skewed cytokine response. the vlp not only induced the antibody response but also cell mediated immune response against the hmpv f protein and the immunogenicity was enhanced by adjuvants like titermax gold or a-galactosylceramide. prior to this, levy et al. developed a hmpv vlp with retroviral core and hmpv surface proteins (f & g) and demonstrated its efficacy in activating the protective humoral immune response against homologous as well as heterologous strains of hmpv in mice model [57] . the observations suggest that the f protein based vlps can be a potent vaccine candidate to deal with the hmpv infections. live attenuated viruses are the live particles designed to provide protective immunity to vaccine recipients against specific viruses. these vaccines include viruses which are weakened enough to lose their virulence, maintaining their structural integrity so that its infection mimics the natural infection allowing the host immune system to recognize the virus as a whole but without causing any disease. these viruses, as a vaccine, activate both cellular as well humoral immunity and doesn't require any booster dose. though this type of vaccine has proven to be highly significant in eradicating some viral diseases but safety issues and efficacy is a matter of great concern in case of many viral infections [67] . these vaccines may be recombinant or non-recombinant. non-recombinant live attenuated viral vaccines are generated by virus culture under altered conditions when viral genome incorporates several mutations which make the virus avirulent under normal host physiological conditions. these altered conditions include low temperature conditions and chemical treatments [17, 37, 62] . though these non-recombinant attenuated viruses activate the immune system efficiently but they may tend to revert back to virulent viruses inside the host causing serious disease [113] . on the other hand, recombinant live attenuated viruses are generated by using reverse genetic techniques where the virulence factor is either eliminated or modified irreversibly [98] . sufficient attenuation is must for a successful live attenuated viral vaccine. non-recombinant cold adapted hmpv viral vaccine was developed and shown to be protective against the viral infection in hamster model [47] . several attempts have also been made to generate recombinant hmpv by transfecting the cultured animal cells with viral cdna and plasmids encoding the virus specific rdrp [9] . the generation of attenuated hmpv could be achieved by deleting the genes encoding g, sh, m2-2 or p protein [11] . the replication of virus lacking g protein was reduced by * 40-fold while that of virus lacking both g and sh protein was reduced by * 600-fold as compared to the wild type hmpv in rodent models [10] . maximum attenuation was achieved by the deletion of m2-2 gene and the attenuated virus could induce protective immunity in african green monkey model [11] . a modification in m2-1 gene of hmpv by substituting third cysteine and last histidine in the zinc binding motif also attenuates the virus and the attenuated virus has the ability to induce protective immune response in cotton rats [15] . mutation in s-adenosylmethionine binding motif of l protein and removal of n-linked carbohydrate in fusion protein also attenuates the virus and the attenuated virus provides protection to homologous as well as heterologous strains of hmpv [60, 112] . attenuation could also be attained by replacing the hmpv p protein with p protein of avian mpv and it was demonstrated that the recombinant virus having avian mpv p protein replicated very poorly in healthy adults but the mechanism is yet to be understood [51] . in 2013, a wild type recombinant hmpv having codon optimised sh protein was approved to be used as parent virus for the development of live attenuated hmpv specific vaccine candidate [100] . recently, ren et al. discussed the significance of m2-2 protein based live attenuated vaccine candidate which can not only induce cellular immune response via the ctl epitope but also suppress hmpv induced host innate immunity and regulate the expression of mirnas responsible for immune related gene expressions [29, 82, 84, 85] . with these strategies under development, there is a need for the validation of an efficient vaccine candidate through clinical trials. for the development of a successful vaccination strategy, it is necessary to identify the viral components required to stimulate the cellular and humoral immune response. host's adaptive immune response is targeted to either b cell or t cell epitope on a viral pathogen. several studies have been done to identify the immunogenic epitopes of hmpv [66, 94, 103] . herd et al. [42] identified major histocompatibility class i restricted ctl epitopes on n, g, m2-2 and sh protein of hmpv and demonstrated that administration of these epitopes as peptide vaccine elicits effector and memory ctl responses along with enhanced expression of th1 type cytokine (ifnc and il12) and significantly low level of th2 type cytokine response (il10 and il4) response resulting in reduced viral load and immunopathology in lungs of mice model. recently, a hmpv specific multi-epitope peptide was designed and evaluated for its protective efficacy in mice model. six b cell epitope, four ctl epitope and two th cell epitope of hmpv were predicted and linked using defined spacer sequences to design the multi-epitope peptide (mep) which was expressed in bacterial system. it was shown that vaccination with adjuvanted mep could induce strong humoral as well as cell mediated immune response in balb/c mice and the serum of treated mice had the capacity to neutralize the hmpv infection in vitro [58] . human metapneumovirus is an important respiratory pathogen having the capacity to cause serious and fatal disease in human beings. it has been easier to characterize this virus because of its similarity to another important respiratory virus, rsv. the approaches to manage the hmpv infection have also been quite similar to that rsv. though common antivirals like ribavirin and ivig are available, yet hmpv specific therapeutic approaches are necessary to effectively challenge the fatal cases. palivizumab, the monoclonal antibody generated against rsv, is effective against hmpv also, yet hmpv specific monoclonal antibodies like 54g10 and fab-ds7 have been developed and successfully tested in animal models. if tested and commercialized like palivizumab, these monoclonal antibodies may prove to be a boon for hmpv infected individuals showing serious symptoms. most of the therapies against hmpv are targeted to fusion protein owing to its conserved nature among various types and subtypes of the virus. certain peptide based fusion inhibitors like hra2 has been tested and found to be protective against hmpv. likewise, sirnas targeted to the conserved regions of genes encoding nucleoprotein and phosphoprotein of hmpv has also shown protective effects against the viral infection. among chemical inhibitors, a sulphated sialyl lipid, nmso 3 , has been shown not only to decrease the viral titre in infected individuals but it also modulates the host immune system by its anti-inflammatory effect. all the therapeutic approaches developed till date is under research but it's important that an effective therapy gets approved and made available for use by the patient's so that the morbidity and mortality due to hmpv infection is minimized. prophylaxis has always been better than therapy, and vaccination is the best approach to control or eliminate any pathogen from any population. hmpv, being a rna virus, is highly prone to mutation and development of an effective vaccine candidate is a challenge, yet research on the development of a universal vaccine candidate is underway. inactivated vaccine candidates against hmpv have not yet been successfully tested in animal models with most of them leading to enhanced disease severity upon subsequent natural infection. on the other hand, vaccination with subunit vaccine composed either of purified hmpv fusion protein or the fusion protein expressed via different viral or bacterial vectors viz. retroviral vector, alphavirus vector, rbcg etc. could induce protective immunity against both homologous as well as heterologous strains of the virus, though for a short duration. fusion protein based vlps in combination with certain adjuvants like titermax gold are also highly immunogenic and protective against the virus. though the inactivated or subunit vaccines are safe but the requirement of booster doses and lack of strong cellular immune response poses a limitation on its use. the live attenuated viruses overcome this limitation. recombinant approach to develop live attenuated viral vaccine candidate through reverse genetics technique can be considered as one of the best approach, provided a good level of attenuation is attained and immunogenicity is maintained. attempts to generate live attenuated hmpv vaccine candidates have been successful with good protective efficacy in rodents and non-human primates but none of them have yet entered the clinical trials. however, it can be said that though hmpv was discovered late, significant advances have been made to intervene the viral infection. conflict of interest there are no conflicts of interest. adjuvant effect of the human metapneumovirus (hmpv) matrix protein in hmpv subunit vaccines the immune response to human metapneumovirus is associated with aberrant immunity and impaired virus clearance in balb/c mice prevalence and characteristics of human metapneumovirus infection among hospitalized children at high risk for severe lower respiratory 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infection rna virus reverse genetics and vaccine design human metapneumovirus as a cause of community-acquired respiratory illness experimental infection of adults with recombinant wild-type human metapneumovirus a host-range restricted parainfluenza virus type 3 (piv3) expressing the human metapneumovirus (hmpv) fusion protein elicits protective immunity in african green monkeys isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo prophylactic and therapeutic approaches for human metapneumovirus 443 identification of antibody neutralization epitopes on the fusion protein of human metapneumovirus lung cd8t cell impairment occurs during human metapneumovirus infection despite virus-like particle induction of functional cd8t cells human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children a recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo population-based incidence of human metapneumovirus in hospitalized children comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by nmso3 in tissue culture assays genetic diversity and evolution of human metapneumovirus fusion protein over twenty years human metapneumovirus: enhanced pulmonary disease in cotton rats immunized with formalin-inactivated virus vaccine and challenged rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral mrna cap methyltransferase reversion of cold-adapted live attenuated influenza vaccine into a pathogenic virus key: cord-278195-1sle0d1j authors: castillo-huitrón, nathalia m.; naranjo, eduardo j.; santos-fita, dídac; estrada-lugo, erin title: the importance of human emotions for wildlife conservation date: 2020-06-24 journal: front psychol doi: 10.3389/fpsyg.2020.01277 sha: doc_id: 278195 cord_uid: 1sle0d1j animals have always been important for human life due to the ecological, cultural, and economic functions that they represent. this has allowed building several kinds of relationships that have promoted different emotions in human societies. the objective of this review was to identify the main emotions that humans show toward wildlife species and the impact of such emotions on animal population management. we reviewed academic databases to identify previous studies on this topic worldwide. an analysis of the emotions on wildlife and factors causing them is described in this study. we identified a controversy about these emotions. large predators such as wolves, coyotes, bears, big felids, and reptiles, such as snakes and geckos, promote mainly anger, fear, and disgust. this is likely due to the perceptions, beliefs, and experiences that societies have historically built around them. however, in some social groups these animals have promoted emotions such as happiness due to their values for people. likewise, sadness is an emotion expressed for the threatening situations that animals are currently facing. furthermore, we associated the conservation status of wildlife species identified in the study with human emotions to discuss their relevance for emerging conservation strategies, particularly focused on endangered species promoting ambiguous emotions in different social groups. since our origins, wildlife has always had a very important role in human life. the very diverse and continuous human-wildlife interactions can be seen from three main perspectives: (1) utilitarian, in which wild species provide goods for human well-being, such as food, clothing, transport, tools, raw materials, and companionship, among others; (2) affective, where human beings feel sympathy, admiration, and respect for animals because of religious, mystical, or philosophical reasons (kellert et al., 1996) , which has greatly contributed to cultural development worldwide (herzog and galvin, 1992; alves, 2012) ; and (3) conflictive, because of the real or potential damage that wild species may inflict on people and their interests (e.g., attacks on humans, livestock predation, damage on crops, and infrastructure, among others; lescureux and linnell, 2010) . human-wildlife conflicts have motivated animal killings for centuries, which in many cases continue nowadays (woodroffe, 2001) . human-wildlife relationships have relied on the uses, values, and meanings that animals represent for people through time and space in different cultures (driscoll, 1995; . societies have developed a cultural predisposition for emotional reactions toward wild animals (kellert and wilson, 1993) , causing either positive or negative effects depending on the species (york and longo, 2017) . fear, anger, and disgust are emotions generating attitudes and behaviors against the presence of some species (fritts et al., 2003; jacobs, 2012) . in contrast, emotions, such as happiness, which comes out when cherished species are seen in a given place, or sadness before the vulnerability of others, may generate positive attitudes for their conservation (prinz, 2004) . this relationship between human emotions and attitudes has an effect on the presence, absence, and recovery of wildlife populations (herzog and burghardt, 1988) . understanding the transcendence of the emotional factors triggered by animals on human beings would improve our knowledge on the human dimensions of wildlife conservation. in this paper, we offer an overview of the influence that emotions have had on the relationships between wildlife and people through time. a substantial amount of the literature reviewed consists of studies conducted on large carnivores in europe, such as the brown bear (ursus arctos) and the wolf (canis lupus), as well as on snakes around the world. we analyze and discuss relevant aspects that could be considered in further studies on threatened and culturally relevant animal species across latin american countries. darwin (1897) recognized that emotions are manifested by all persons throughout their lifetime, but they vary in an individual between different moments of its life span. frijda and mesquita (1998) mentioned the main points characterizing the emotions in their theoretical perspective: (1) emotions are considered individual responses to relevant events producing feelings of pleasure or pain; (2) they help to find solutions to concerns that cannot be treated routinely; (3) they are always about something, they are used to accept or decline the interaction with a real or imagined object, person, or wild animal in this case; (4) they tend to control behaviors and thoughts (e.g., angry impulses, behaviors, and thoughts); and (5) emotions are correlated with psychological, physiological, and social components establishing, changing, or maintaining a particular relationship with a specific object in a concrete situation. there is a wide array of studies analyzing human emotions, their origins, functions, and presence in human life (ekman, 1999; plutchik, 2001a; nummenmaa et al., 2014, among many others) . six basic emotions have been proposed: happiness, surprise, disgust, anger, fear, and sadness (ekman et al., 1969) . izard (2009) suggested classifying emotions into two groups: "positive, " representing interest and joy (happiness and surprise), and "negative, " including anger, disgust, fear, and sadness. this classification is an artifact of traditional psychology not informed about an evolutionary approach. here, we have focused on basic emotions to explain human-wildlife relationships because secondary emotions (the combination of basic emotions) are more useful for assessing social relationships among human beings (harelli and parkinson, 2008) . in this review, we consider two different approaches to explain the origins of basic emotions aiming to understand human-wildlife relationships through time. the first is the evolutionary approach, which suggests that emotions have evolved to solve adaptive problems in different environments (plutchik, 2001b) , such as social communication, reproduction processes, and mechanisms for information processing leading to behavioral responses to specific events or objects (al-shawaf et al., 2016) . predator presence could have been one of such events contributing to the evolution of human emotions and the development of physiological, psychological, and morphological responses for survival (öhman and mineka, 2001; prokop and randler, 2018) . in particular, fear and disgust are adaptive emotions helping to react toward something representing a risk for human life (ekman and cordaro, 2011) . fear and disgust, for instance, have been the most studied emotions due to their implications for human survival since the origin of our species . fear probably was a defense mechanism against dangerous animals, particularly large predators (öhman, 1986; dalgleish, 2004) . it is believed that potential alert signals emitted by human groups facing predators, with whom they coexisted and sometimes competed for space, water, prey, and other resources, triggered physiological reactions such as heart rate increase, profuse sweating, and pupil dilation, allowing the generation of alert responses. in that way, human beings have historically developed greater awareness toward potentially perilous animals, such as snakes and spiders öhman and mineka, 2003; lobue and deloache, 2008) . this adaptation mediated by fear has probably been genetically fixed throughout generations, provoking the innate physiological responses mentioned above when dangerous species are or could be present (öhman, 1986) . the amygdala is the brain region where fear-generating stimuli are processed into a strong reaction that in some cases may affect human vision (phelps et al., 2006) . on the other side, disgust can help protect the individual against infections and disease (curtis et al., 2011) . disgust is saved in memory to avoid future exposure to the subject, in this case with potentially threatening animals (al-shawaf et al., 2016) . the second approach explaining the origins of basic emotions is the cultural context, where people integrate their physical environment with individual and collective experiences, perceptions, meanings, attitudes, and animal-related traditions to construct emotional diversification (prinz, 2004; johansson et al., 2012) . in this view, it can be said that human emotions associated with wildlife have evolved over time and continue to be gradually built and rooted in our societies all over the world. under the cultural context approach, emotions can be understood on two levels: (1) the individual level, involving meanings, beliefs, attitudes, and behaviors based on personal experiences, knowledge, and perceptions, and (2) the social level, where emotions are determined by collective factors such as experiences, meanings, beliefs, and myths typical of a certain region or culture, which are transmitted among individuals throughout generations (ekman, 1999; prinz, 2004) . physical characteristics of wildlife species and their "personalities" created by humans have generated a variety of emotions (kellert et al., 1996; kruuk, 2002; prokop and randler, 2018) . emotions such as fear and anger may be induced by predators that are bigger and heavier than persons, as in the case of large carnivores (e.g., bears, wolves, and big cats) (røskaft et al., 2003) or by those species unattractive for most people, like worms, small carnivores, bats, and reptiles, which are often perceived as harmful (knight, 2008; prokop and tunnicliffe, 2008; prokop et al., 2009) . in contrast, beloved animals such as colorful birds or small herbivore mammals (e.g., rabbits) may cause happiness providing they are not noxious for people or their livelihoods (prokop and kubiatko, 2008) . however, these animals are sometimes perceived in different ways. for some social groups (e.g., farmers), small mammals such as rabbits as rodents may represent a threat due the damage they can inflict on crops, cattle, properties, and human health (morzillo and merting, 2011; breed and moore, 2016) . actual or potential damage can promote negative attitudes motivated by emotions of anger, disgust, and fear. animal body shape is another physical feature that has been found to be important for the expression of emotions such as fear and disgust. in the case of class reptilia, two groups could be recognized by people according with their similar morphotype (with legs and legless). reptiles with legs (lizard, turtle, and crocodile) tend to cause fear in many people; crocodiles, specially generate intense fear in many people, in part because of the number of attacks occurring worldwide (crocbite, 2020) . in contrast, legless reptiles (e.g., amphisbaenia and larutia) that have thin bodies, smooth textures, small eyes, and dull colorations generate disgust rádlová et al., 2019) . specifically, snakes have long bodies, scales with contrasting patterns, bright coloration, and silent, rolling movements that immediately calls up human attention deloache, 2008, 2011; rádlová et al., 2019) . it is likely that both fear and disgust can be simultaneously felt by a person observing a particular species . the ample diversity of snakes around the world makes it difficult to generalize emotions across cultures toward different taxa. species coloration has been an attribute to help identify dangerous animals (prokop and fančovičová, 2013) , allowing emotional responses in human beings (öhman, 1986) . striking color ("aposematic") combinations such as bright red and black in some snakes and spiders intensify fearful reactions (öhman and mineka, 2003; lobue and deloache, 2011; . on the other hand, it has been reported that striking coloration allowed perceiving snakes as beautiful animals in spite they are fearsome . it is noteworthy that aposematic species are simultaneously fearsome and attractive particularly for young persons between 10 and 20 years of age, promoting their interest in those animals (prokop and fančovičová, 2013) . on the other hand, animals' coloration could be attractive for humans and motivate "positive" feelings. in this sense, lišková et al. (2015) discovered that hues of blue and green in birds of the pittidae family promote human preference. psychologists have found that green is usually associated with happiness, relaxation, and comfort because it is related to nature, while blue elicit happiness, relaxation, and peacefulness, among other feelings (kaya and epps, 2004) . however, human affection for birds also represents a pressure for wild populations, especially for those charismatic species used as pets, promoting illegal trade (alves et al., 2013) . feeding habits of species may also influence emotions: large predators are usually regarded as hazardous and fearsome, while their prey provoke sadness (prokop and kubiatko, 2008) . large herbivores and omnivores in some places are often seen as less fearsome than strict carnivores. this is the case of the mainly vegetarian brown bear (ursus arctos) in some regions of europe (lescureux and linnell, 2010) . however, in other areas and cultures, large herbivores such as elephants (loxodonta africana) cause intense emotions of anger and fear because of the damage they inflict on crops and rural villages (lamarque et al., 2009) . although "dangerous" animals promote the attention of people (prokop and randler, 2018) , it is interesting to note that human emotions may vary depending on the life stage of the animal. for example, jaguar (panthera onca) cubs and lion (panthera leo) cubs are perceived as lovely and safe animals given their physical features, causing minor concern in societies, while adult jaguars and lions are generally considered less attractive and very dangerous, promoting fear (knight, 2008) . this trend is also reported for amphibians, for which people show more disgust toward the adult stage than for tadpoles (prokop and fančovičová, 2012) . venom in animal species is one of the most remarkable features triggering fear across cultural groups. as a consequence, snakes constitute an interesting case study in which most species produce fear all over the world, although particular species are in fact perceived as beneficial due to their role as controllers of agricultural pests, producing positive feelings in local farmers (ballouard et al., 2013) . in this regard, ballouard et al. (2013) observed different intensities of fear toward selected snake groups (cobras, vipers, and boas) depending on the nationality and cultural background of their interviewees. animal activity patterns constitute one more physical factor influencing human emotions toward wildlife. humans are not adapted for living in the darkness; they have a poor vision to act in this kind of environment, hence they may associate nocturnal species such as felines, some snakes, rodents, and bats with danger (buss, 2016) . in addition, these animals historically have been linked to "evil forces" damaging human beings worldwide (prokop et al., 2009) . contrastingly, many diurnal species (e.g., most of the birds and ungulates) are usually related to positive values such as peacefulness and wisdom that have inspired leaders and rulers to make better decisions (cano-contreras, 2009 ). physical characteristics have been useful to classify animals depending on the emotions they produce on people. in this sense, tarantulas, snakes, sharks, and mosquitoes have been categorized as perilous, generating agonistic emotions. contrastingly, large, charismatic species that have traditionally been regarded as dangerous but intelligent at the same time motivate emotions that may result in actions for their protection, as it has occurred for lions (panthera leo), tigers (panthera tigris), leopards (panthera pardus), and polar bears (ursus maritimus) (driscoll, 1995; landová et al., 2018) . these categories have emerged after the anthropomorphization of animals, a process in which cultural groups attribute human features and "personalities" to wildlife species (kruuk, 2002) . for instance, the panda bear (ailuropoda melanoleuca) inspires tenderness and happiness when it is observed, but those emotions are overcome by sadness after considering its high vulnerability to extinction. in this case, positive attributes facilitate particular species to become flagships for wildlife conservation (root-bernstein et al., 2013) . in rural communities where people frequently interact with wildlife, knowledge about the behavior of culturally relevant species develops better than in other areas. this facilitates the anthropomorphization of certain animals calling them "shy, " "noxious, " and "monstrous, " among other adjectives, which intensifies fear and rejection toward them (lescureux and linnell, 2010) . furthermore, if the presence of an animal implies economic losses for residents of a community, their predominant perception will be negative and will produce anger that may end in lethal management (naughton-treves, 1997). contrastingly, animals inspiring greatness and qualified as "kings" of the wilderness will likely motivate local people to feel happiness and pride because of their presence in the region (lescureux and linnell, 2010) . these examples help identifying the relevance of animal physical features in emotions, which transform throughout history according to the natural, social, and economic context of each human generation. in some cases, emotions produce attitudes against the conservation of unpopular species (knight, 2008) . therefore, we propose to highlight the ecological role of dangerous or disgusting species as a potential way to mitigate negative emotions toward them. emotions induced by wildlife differ among individuals according to variables such as their sex, age, cultural and natural environment, and perceived vulnerability to each species (johansson et al., 2012) . it has been shown that young children (under 3 years of age) of both sexes take more time to detect a snake and react toward it than their parents (lobue and deloache, 2011). that behavior was explained by deloache and lobue (2009), proposing that fear and alert signals in front of this kind of animals develop later, when individuals start to explore their environment and link adult behaviors with animal species. fear and disgust have been the most studied emotions between genders. in general, women tend to express stronger negative emotions (fear and disgust) toward invertebrates, amphibians, predatory mammals like bears, wolves, lynx (lynx lynx), and wolverine (gulo gulo) and toward snakes compared to men (öhman and mineka, 2003; røskaft et al., 2003; ballouard et al., 2013; bajwa et al., 2014; . this difference seems to be related to the female gender role taken since the start of human evolutionary history, where men developed skills for both hunting and escaping from predators (prokop and fančovičová, 2010) . likewise, men gradually reduced their fear of large animals, while women kept distance from those species in part because of their household activities and their care for children in safer places (røskaft et al., 2003; prokop et al., 2011) . however, differences within genders are usually present in different cultural and geographic contexts (kellert and berry, 1987; bjerke et al., 2001; de pinho et al., 2014) . in some societies, women, particularly adolescents, have a greater disposition to spend more time in wildlife related activities as compared to men (e.g., volunteer programs; kidd and kidd, 1997) . this information could be useful to direct conservation programs in spaces as zoos where experiences with uncharismatic and endanger animals could help to promote positive emotions and attitudes. age is a significant variable determining the presence and intensity of agonistic emotions toward animals, which may be related to personal experiences. childhood is the critical life stage when fear of predators starts and when attitudes and behaviors to avoid encounters with them develop (öhman, 1986) . it is likely that fear of predators intensifies with learning from parents, given that as the child gets older, his/her reactions become faster when facing species such as snakes (lobue and deloache, 2008) . in this regard, fear of animals may either decrease (kaltenborn et al., 2006) or increase (røskaft et al., 2003) with age. besides age, the natural and cultural environments in which an individual grows determine the knowledge, perceptions, and emotions related to animals (frynta et al., 2011) . for a person raised in close contact with nature, an encounter with a wild animal can induce happiness, while the same species may produce fear in an individual that has always lived far away from natural spaces (kellert, 1993; manfredo, 2008; almarcha, 2019) . the presence or absence of different species in human territories has a role in the generation of emotions. residents of rural areas who frequently interact with wildlife are usually less fearful of animals than city dwellers. this is because closeness with native animals promotes knowledge about their ecology and behavior, allowing for building better management strategies and reactions toward them (røskaft et al., 2003) . likewise, recreational activities involving contact with wildlife such as hiking, bird watching, fishing, and hunting have direct influence in emotions, facilitating the overcoming of fears and phobias by promoting learning through first-hand experiences, although in some cases, these activities decrease with age (bjerke et al., 2001; røskaft et al., 2003; prokop et al., 2011) . in particular, emotions produced by hunting deserve further discussion. subsistence hunting as a traditional practice in many rural areas of the world usually involves local regulations to avoid overexploitation and feelings of respect by the hunters toward their prey (e.g., santos-fita et al., 2015) . in contrast, sport hunting is more focused on the pleasure of the hunter for finding and killing his target species, which has been a motive social dispute in different contexts, generating anger in broad sectors of society considering this an unacceptable practice (nelson et al., 2016) . some of these recreational activities involve parents and their children, who get used to those practices at an early age (amiot and bastian, 2015) . this can be an important inter-generational strategy to avoid negative attitudes toward fearsome and disgusting animals and promote positive emotions (i.e., happiness and surprise), especially in areas where humanwildlife conflicts may arise. significant differences have been found among people with different levels of study with respect to fear of wildlife species: individuals with higher levels of education are generally less fearful of wild animals than those with lower degrees of studies (røskaft et al., 2003) . it is likely that individuals with higher education had more opportunities to receive information on the environment and wild animals in particular, which may have reduced their negative prejudices and perceptions about non-charismatic species, maximizing their perspectives on the ecological benefits provided by those animals. the geographic space where an event occurs triggers distinct emotions, which have varied according to the lifestyles of societies (mesquita and frijda, 1992) . this argument could be used to understand emotions historically induced by wildlife, considering the different worldviews of each culture. for example, snakes were regarded as deities in mesoamerican cultures, including quetzalcoatl or kukulkan (the feathered serpent), which was the most important deity for the aztecs and the maya, respectively (díaz, 2007) . snakes were also given high rankings among the deities of the ancient greek, egyptian, hindu, and roman civilizations, where some of these reptiles were associated with values of wisdom, justice, and power (stanley, 2008; al-rawi, 2012) . these reptiles have also starred countless stories and myths around the world (ménez, 2003) , but for christians, muslims, and jews, snakes have traditionally represented evil and death (gonzález, 2003; al-rawi, 2012) . nowadays, myths about the damage caused by snakes are important elements to promote and intensify fear in rural communities (fita et al., 2010) . the social fear could be learned, inherited, and used by societies across generations, driving particular attitudes toward wild species (öhman, 1986) . in this case, the relevant ecological role of snakes as predators and pest controllers has been largely neglected. another interesting example is that of wolves, which have been protagonists of many stories and myths worldwide. these carnivores have traditionally been portrayed as fearsome and dangerous animals, producing social rejection in most areas where they are present, nonetheless, in particular cases such as that of ancient rome (whose founders were suckled by a shewolf) and that of native north american cultures, for whom wolves were spiritual symbols related to power and intelligence (fritts et al., 2003; prokop et al., 2011) . beyond mythology, other elements that have facilitated the development of cultures (e.g., art, literature, symbolism, religion) have had their foundations in the relationships between humans and wildlife, involving emotions promoting respect and admiration (fritts et al., 2003; alves, 2012; almarcha, 2019) . these emotions frequently lead to attitudes favorable for animal care and conservation. other events that have always happened, but which have received special attention in recent decades because of the human population growth and expansion, are the attacks of large carnivores on people and livestock, and crop damage by large herbivores (inskip and zimmermann, 2009 ). these events make jaguars, tigers, lions, leopards (panthera pardus), hyenas (crocuta crocuta), african wild dogs (lycaon pictus), and african elephants (loxodonta africana), among others, be considered problems in rural communities, giving place to misunderstandings and false beliefs about their behavior (marchini and macdonald, 2012; dickman et al., 2014) . this situation has contributed to magnification of the actual damages of those species, stimulating even more fear, disgust, and rejection toward them (lescureux and linnell, 2010) . in this sense, the individual background and experiences of humans contribute to their emotions and behaviors. for example, the presence of large predators may produce fear and thoughts of escape in most people, while some others may feel encouraged to confront the danger (al-shawaf et al., 2016) . the context of the encounter with an animal may also be relevant for the emotions manifested. for a given person, the sighting of a carnivore such as a female puma with their offspring while hiking on a forest trail may produce fear and desire to escape. in contrast, the same person may feel surprised and delighted to have the same sighting from the safety of a car (narratives collected by the first author in chiapas, mexico). furthermore, local knowledge and the emotional links between people and wildlife could be useful to identify flagship species to foster interest in nature (bowen-jones and entwistle, 2002) . flagship species [e.g., giraffe (giraffa camelopardalis), elephants, and lions, among others] are usually charismatic and popular and may be relevant for promoting positive emotions in a public that has been distant from wild animals. differently, more complex sets of emotions (both positive and negative) are usually present where people are in constant interaction with these animal species (bowen-jones and entwistle, 2002; de pinho et al., 2014) . zoos represent spaces where emotional confrontations take place. for instance, marseille et al. (2012) observed visitors watching imposing and charismatic polar bears. the authors found that visitors felt happy in front of the bears, but at the same time they felt sad after recognizing the small size of the enclosures and the stereotyped behavior of the captive animals. interestingly, visitors' emotions transformed into fear and even greater sadness when they were told about observing polar bears in their natural habitat, which was associated with concerns about human safety and habitat vulnerability. another element that has an effect in the affection of children for wild animals is the presence of pets (bjerke et al., 2001) . pets can boost appreciation emotions, such as happiness, while naturalistic, ecological, humanistic, and moralistic attitudes may also be encouraged (prokop and tunnicliffe, 2010) . although knowledge about animals usually differs between urban and rural communities, the lack of accurate information about the species and their contribution to ecosystem services is persistent in both environments (gomes et al., 2017) . it promotes the intensification of emotions such as danger and disgust, especially for species that are unattractive to people. disgust has also been identified as one of the emotions inducing human rejection. it may arise when people perceive nasty odors in animals, or when unpleasant feelings emerge while touching (or thinking about) the fur of certain mammals (johansson et al., 2012) or the skins of amphibians such as frogs (lobue and deloache, 2011) . in other cases, disgust may be brought after linking animals such as spiders and rats with dirtiness, pollution, disease spreading, and potential crop damage (kellert, 1993; davey, 1994; prokop and tunnicliffe, 2010) . furthermore, animals that cause disgust are often perceived as ugly . contempt of human societies for amphibians and reptiles intensifies misinformation about them and favors negative attitudes toward them (manzanogarcía and martínez, 2017) . for example, it has been documented that non-venomous snakes are killed just because of their resemblance to poisonous species (breed and moore, 2016) . moreover, misinformation is an intensifier of disgust, for instance, when considering geckos (hemidactylus turcicus) as venomous animals or vectors of skin diseases (ceríaco et al., 2011) , or bats as a threat for fruit crops and responsible to infect people with parasites and viruses (musila et al., 2018) . in this sense, the case of bats and pangolins (pholidota) could be cited, which are considered the main transmitting agents of the novel coronavirus (covid-19; van staden, 2020). the respiratory illness has become a pandemic infecting million and killing many thousands of people around the world (nature, 2020). it is likely that the disease has a zoonotic origin as a result to the food and medicinal uses of animals (van staden, 2020). therefore, in some places there has been motivation to eliminate these animals (zhao, 2020) . this event might increase the negative perception and emotions of anger, disgust, and fear for this kind of animals and will encourage the eradication of populations without considering their importance in ecosystems. in this regard, it has been found that women and residents living near caves tend to believe in myths about bats more than men and people living far from caves (musila et al., 2018) . fearsome and disgusting species frequently induce rejection attitudes in social groups (öhman and mineka, 2001 ), a phenomenon known as "biophobia" that is used to express the feeling of panic, fear, and disgust in front of a particular non-human living being. phobia for animals (agrizoophobia) is one of the most frequently reported biophobias in the general population (antony and mccabe, 2005) , but there are actually around twenty-five documented phobias to particular animal groups, such as that for snakes (ophidiophobia), spiders (arachnophobia), insects (entomophobia or insectophobia), ants (myrmecophobia), bees (apiphobia or melissophobia), and birds (ornithophobia), among others (fredrikson et al., 1996; antony and mccabe, 2005; prokop and fančovičová, 2013) . however, there are no specific phobias for carnivores, probably because the coevolution between humans and these animals has been too short in comparison with other groups such as snakes (prokop and randler, 2018) . biophobia may promote persecution and extermination attitudes (zhang et al., 2014) . avoiding contact with animals or killing them are the most frequent reactions without considering their long-term impacts on ecosystems (antony and mccabe, 2005; al-shawaf et al., 2016) . orr (1993) mentioned that one of the causes of biophobia is social distancing from nature. in a parallel way, biophilia has a genetic basis and consists of the interest and empathy of humans for other living beings (wilson, 1993) . as industrialization and urbanization increase around the world, lifestyles change in human societies, sometimes in radical ways (steffen et al., 2008) . these processes have contributed to the distancing of people from their natural environment even in rural communities (louv, 2008; lescureux and linnell, 2010) . however, there are still spaces such as zoos and natural parks facilitating social approach and understanding of wildlife in most of the cities and large towns all over the world. in those spaces, visitors are generally safe in front of animals that otherwise would be considered dangerous or harmful, and they may feel sadness and even culpability after recognizing the impact of the human population on those species. in this sense, vining (2003) suggested that visiting zoos and natural parks may represent opportunities for reconnecting people and wildlife to enhance social cooperation in conserving biodiversity. human emotions transcend over time. a specific emotion is saved by the individual as an experience that may be used in future behavior and decision-making (izard, 2009) . protection attitudes toward spiders, insects, amphibians, and reptiles are milder than those shown for other groups, such as birds and mammals due the sentiments of danger or disgust that these animal groups provoke in humans (prokop and fančovičová, 2013; . in addition, emotional experiences may have an effect on wildlife management techniques (larson et al., 2015) . this has occurred during experiences of invasive species management. one example is that of the house sparrow (passer domesticus), which competes for food and space with native birds and generates anger or disgust when managed through nest and egg removal, repellents, and traps. in contrast, bluebirds (sialia sialis) stimulate happiness in people watching them and listening to their songs, who at the same time feel sadness for these birds due to the negative impact of human activity on their populations. these feelings motivate protection attitudes favoring the persistence of the liking bird species (larson et al., 2015) . it is important to recognize that fear impacts human attitudes and behaviors toward keystone species, particularly those regarded as dangerous or harmful (e.g., wolves, bears, and big cats). fear may limit the involvement of local communities in managing predator populations because of the high costs implied or because the social acceptance of certain techniques, such as reintroduction, may be difficult (johansson et al., 2012) . examples of this include reintroducing wolves in mexico and the united states, where emotions have played fundamental roles in the acceptance of new wolf populations (straka et al., 2019) . mexican wolves (canis lupus baileyi) were eradicated from the mexican territory in the 1960s because of conflicts with farmers and negative perceptions due to livestock predation (leopold, 1959; moctezuma et al., 2004) . wolf reintroduction projects have been started recently in northwestern mexico, where it has been clear that social acceptance is the primary limiting factor for their success (araiza et al., 2012; garcía, 2014; lara-díaz et al., 2015) . society's emotions toward wildlife may be key elements for decision-making on conservation issues. anger is one of the primary collective emotions that can lead to positive changes for natural resource management when social pressure is put on government leaders to improve and enforce environmental legislation. however, anger may have other implications and cause social fragmentation (buijs and lawrence, 2013) . in these cases, participation of wildlife management agencies is crucial given their social confidence. if the capacity of these agencies is not appropriate, collective distrust and fear of dangerous and disgusting animals may stimulate hostile environments for their proper management (johansson et al., 2012) . community confidence in environmental agencies is especially relevant where threatened species are under recovery, as is the case with wolves in different countries (swenson and andrén, 2005) , or where people take action by themselves, such as in the case of the killings of andean bears (tremarctos ornatus; figueroa, 2015) . it seems clear that some wildlife species are far more significant to humans than others (herzog and burghardt, 1988) , perhaps linked to their evolutionary closeness (e.g., primates, and particularly the great apes; gunnthorsdottir, 2001; miralles et al., 2019) or because of their cultural, aesthetic, or affective attributes favoring more interest and attention toward them. interest and attention favor people's attitudes for conserving these species, differently from others without a transcendental meaning for social groups. this idea highlights the relevance of designing conservation strategies fomenting interest for wildlife through generating affective links between humans and animals both in rural and urban areas. beautiful and attractive animals causing "positive" emotions (e.g., happiness and surprise) receive special attention driving in situ and ex situ conservation actions (gunnthorsdottir, 2001) . this could be a limitation for conservation efforts focused on species considered unattractive particularly in zoos. the preferences of human societies to watch specific animals have promoted that zoos keep attractive species more than those needing protection due to their conservation status (frynta et al., 2010 (frynta et al., , 2013 . mammals constitute the preferred group among zoo visitors around the world (moss and esson, 2010) . however, these spaces keep only 179,868 individuals belonging to 1,048 species (frynta et al., 2013) , which represent just 16.4% of known living species (burgin et al., 2018) . this preference is strongly biased toward large, attractive, and active mammals belonging to the families ailuridae, felidae, phascolarctidae, ursidae, giraffidae, elephantidae, equidae, macropodidae, mephitidae, and cervidae, among others (frynta et al., 2013) . the same correlation between human preference and species kept in zoos was found for large, colorful, and long-tailed parrot species (frynta et al., 2010) . in contrast, small and unpopular species do not motivate the same appreciation, even if they are endangered. as a consequence, zoos generally keep a few of those local species (frynta et al., 2013) . in this sense, zoos and other places keeping wildlife need to implement exhibition strategies to promote human interest on less attractive but highly relevant animal species of threatened ecosystems (bitgood and patterson, 1987; frynta et al., 2009) . considering this distinction in preference, it is relevant to spread information about the ecological importance of animals in ecosystems, especially regarding native and endangered species (conde et al., 2011) , messages to promote "positive" emotions in people could be a way to support the appropriation of endangered species by societies and improve their attitudes toward them in the long term. massive media communication may be of utmost importance for these purposes, especially if the appropriate images of and messages about target species are transmitted to the general public (gunnthorsdottir, 2001) . following breed and moore (2016) , successful conservation projects require focusing on promoting wide social empathy for wildlife species, particularly those that generate fear and disgust (e.g., large predators, venomous species, and many amphibians) motivating their killing or removal (bishop et al., 2012; prokop and fančovičová, 2012; . individual and collective idiosyncrasies have promoted a diversity of attitudes toward wildlife species (herzog and burghardt, 1988) motivated in part by a diversification of emotions built with dynamic biological and cultural elements. identifying and understanding diversified emotions and their local precursors (e.g., in areas where protected areas and human presence are relevant) would allow analyzing wildlife problems and their solutions through multidisciplinary strategies. considering that knowledge is a relevant element for the expression of emotions, we propose that regional strategies to integrate information on the biology, ecology, and management of culturally important animal species (particularly those regarded as fearsome, dangerous, harmful, and disgusting) should be included in national education systems and massive media campaigns throughout the neotropics (espinosa and jacobson, 2012) . these strategies must be carefully designed by taking into account the impact of mass media (e.g., news, television shows, documentaries, films, and public text books, among others) may have on the public about wildlife conservation (røskaft et al., 2003; knight, 2008; ceríaco et al., 2011; wieczorek, 2012) . when an animal species is projected as aggressive, a negative emotional experience can be produced in the public. this negative experience may in turn lead the individual to believe the species is a dangerous agent or threat to human life, bringing about attitudes against its conservation (prokop and fančovičová, 2017) . on the contrary, if wildlife species are positively seen by children through different media outlets, where the real facts about unpopular animals are shown, it is more likely that fear and disgust decrease, while empathy may grow (prokop et al., 2011) . ensuring the continuity of transmitting traditional ecological knowledge about animal species will be equally important to stimulate positive emotions and a long-term interest of the new generations in wildlife conservation (jacques-coper et al., 2019) . another strategy that could have a positive impact on emotions toward fearsome and disgusting animals is promoting physical interactions with them (e.g., touching snails, rays, amphibians, mice; randler et al., 2012; ; the new knowledge about the animals and physical contact with them could reduce the anxiety of danger. recognizing that emotions are culturally influenced, we propose developing outreach strategies by retrieving traditional aspects that formerly favored empathy with animal species, including the noncharismatic or unpopular ones, even if they are threatened. this review aimed to discuss the role of emotions in the conservation of species which a have been transcendent for the human species throughout history and that in many cases are currently threatened by extinction. in particular, we stress that the social component is of utmost importance in wildlife conservation across latin america, especially in megadiverse countries where ethnozoological studies have documented the relevance of human-wildlife relationships (jácome-negrete et al., 2013; sarukhán and dirzo, 2013; manzano-garcía and martínez, 2017) . nc-h wrote and edited the manuscript. en translated and edited the manuscript. ds-f and ee-l edited the manuscript. all authors read and approved the final manuscript. observando al lobo. un estudio antropológico sobre el lobo y el turismo en la 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biophobia and conservation attitude in china covid-19 drives new threat to bats in china mexico's national council of science and technology (conacyt) provided a scholarship to nc-h for her doctoral studies. we thank ph.d. angela may steward for reviewing and editing the manuscript. key: cord-025998-1qawjquv authors: lara, r.j.; islam, m.s.; yamasaki, s.; neogi, s.b.; nair, g.b. title: aquatic ecosystems, human health, and ecohydrology date: 2012-03-23 journal: treatise on estuarine and coastal science doi: 10.1016/b978-0-12-374711-2.01015-9 sha: doc_id: 25998 cord_uid: 1qawjquv this chapter treats two main topics: the relationship between human health, aquatic ecosystems, and water use; and the necessity of interdisciplinary approaches for the development of water management policies and disease control. main waterborne diseases, mostly affecting developing countries and relevant in terms of water management and changes in land use, such as malaria, schistosomiasis, or cholera, are discussed stressing links to the global water crisis. also, the role of artificial and natural wetlands in influenza epidemics is treated. the effects of increasing water use and scarcity on human health are discussed considering historical and contemporary incidence of diarrheal diseases in european and south asian megacities, relationships between dams and on waterborne diseases in asia and africa, and intensive agriand aquaculture resulting in man-made ecotones, fragmented aquatic ecosystems, and pathogen mutations. it is emphasized that the comprehension of the multiple interactions among changes in environmental settings, land use, and human health requires a new synthesis of ecohydrology, biomedical sciences, and water management for surveillance and control of waterborne diseases in basin-based, transboundary health systems. surveillance systems should monitor changes in water management, ecotones, and hydrological cycles and shifts in, for example, the outbreak timing of strongly seasonal diseases. these indicators would provide criteria for the development of innovative water management policies, combining methods of vector control and the safe creation of water reservoirs, irrigation systems, and wetland habitats. this chapter is not intended to provide an extensive review of water-borne diseases, since there are already excellent examples of these in the literature. rather, the intention is to call atten tion to particular aspects of the principal water-borne diseases and related water management issues. the emphasis is often on controversial aspects which it is hoped will stimulate an open, undogmatic, and fruitful discussion on possibilities and cur rent and future challenges for ecohydrology in relation to human health. ecological integrity is central to health (epstein, 1999) . pollution, disturbed environments, habitat loss, and climate change promote disease emergence in a number of ways. threats to human health arising from man's interaction with aquatic ecosystems can originate from multiple factors, which can be broadly grouped into the following major categories: • natural biological cycles in which humans can act as hosts of pathogenic microorganisms (protozoans, bacteria, etc.); • consequences of the management of aquatic resources (e.g., wetlands drainage or creation, aquaculture, and dam construction); • effects of water pollution (chemical, microbiological, radio active, and thermal) on man and on the physiology of individual organisms; and • the impact of global changes affecting climate and hydrolo gical cycles (e.g., habitat degradation, warming, increased rainfall, and storms). clearly, these are interlinked and specific case studies are likely to present evidence of more than one causal factor. thus, an effort has been made to integrate these different topics into sections covering the most relevant factors related to diseases that can be considered indicators of determined hydrological processes. globally, approximately 19% of deaths due to infectious dis eases are water related. this amounts to 3.4 million deaths per year, 75% of which are caused by diarrhea, a disease killing around 2 million people every year, mostly children in devel oping countries (wwd, 2001) . thus, it is crucial to develop an integrated water and health management system, as well as the tools required to identify and predict interconnected trends in the evolution of aquatic ecosystems and diseases. although the importance of integrating natural sciences with socioeconomic research is stressed and recommended in every forum dealing with sustainable use of natural resources, human health, one of the most valuable public goods, is seldom included as a significant issue in coastal or basin management programs. a frequent goal of cooperation between socioeconomic and natural sciences research is the evaluation of the sustainability of resource use and the vulnerability of the coastal zone. the seven step assessment framework of the international panel on climate change (ipcc, 1994) is the customary tool used for this purpose (figure 1) . similarly, the health map program from the world health organization (who) provides criteria and a software platform for linking epidemiological data with layers of geographic information (who and unicef, 1993) . however, these two types of research programs mostly lack any substantial interaction. ecohydrology can provide the conceptual framework for establishing such links. this involves a scale (the river basin and the coastal zone), an integrative process (flooding dynamics), and an integrative tool such as digital elevation models (dem) of the study area (figure 1) . high-resolution topography is essential for data integra tion in low-lying areas. for this purpose, a closer interaction of socioeconomic studies, climate, hydrology, ecology, micro biology, biogeochemistry, molecular biology, and medicine is required, as exemplified in the case of cholera (discussed in section 10.12.2.3.1). further, it is necessary to generate a common language to facilitate and promote inter-and transdisciplinary communication. several concepts related to disease dynamics, such as outbreak, epidemics, and pan demics, are commonly used in a qualitative way. yet, the creation of interdisciplinary databanks or the compilation of information from diverse sources (e.g., hydrological events and outbreak intensity) including historical ones would ben efit from objective category definitions. in the following sections, some examples are dealt with, which will appear in this chapter and in other related literature. it is considered that an epidemic occurs when new cases in a given human population, during a given period, sub stantially exceed what is expected, based on recent experience (the number of new cases in the population during a speci fied period of time is called the 'incidence rate'). an epidemic may be restricted to one locality (an outbreak), be more general (the usual 'epidemic'), or even global (pandemic). common diseases that occur at a constant but relatively low rate in the population are said to be 'endemic'. an example of an endemic disease is malaria in some parts of africa, where a large portion of the population is expected to contract malaria at some point in their lifetime. these somewhat subjective definitions require more precisionand a wider diffusion among the populationwhen, for aquatic ecosystems, human health, and ecohydrology 265 figure 1 integration of socioeconomic, ecological, and medical research within the ecohydrology framework. example, public funds have to be allocated for prevention, alleviation, or fight against determined diseases or for insur ance policies. an increasing number of direct or indirect cases of water-borne diseases are due to global warming, increasing frequency and intensity of storms, flood control, and environmental management (dam construction, irriga tion, use of fertilizers, etc.), and this has greatly widened the spectrum of stakeholders with an interest in the use of water and its impact on health. this underlines the need for agree ment on and diffusion of definitions and criteria between users and managers to facilitate the quantification of impacts of disease, ecological damage, and poor sanitation, as well as for cost-benefit analysis of proposed prevention or remedia tion measures. the global burden of disease analysis (who, 2009) pro vides a comprehensive and comparable appraisal of mortality and loss of health due to diseases and injuries, and risk factors for all regions of the world. the overall burden of disease is assessed using the disability-adjusted life year (daly), a timebased measure that combines years of life lost due to premature mortality and years of life lost due to time lived in states of less than full health. worldwide, about 82 million dalys per year are due to water-borne diseases (pruess et al., 2002) . a relevant specific example is the definition and evaluation of the impact of disease related to drinking water quality. this concerns the presence of chemicals or pathogenic microorganisms that are transmitted when contaminated drinking water is directly con sumed. if contaminated drinking water is used in the preparation of food, it can be the source of food-borne disease through consumption of the same microorganisms, often in the form of gastrointestinal illnesses. according to the who (who, 2004a) , diarrheal disease accounts for an estimated 4.1% of the total daly global burden of disease and is respon sible for the deaths of 1.8 million people every year. it is estimated that 88% of that burden is attributable to unsafe water supply, sanitation, and hygiene. at the national level, surveillance systems are the primary source of data concerning the scope and effects of water-borne diseases on persons. these data can be organized to provide information for political and administrative units such as counties or federal states, and/or geomorphological units, that is, basins. in the united states, since 1971, the centers of disease control and prevention (cdc), environmental protection agency, and the council of state and territorial epidemiologists have maintained a collaborative surveillance system which monitors the occurrence and causes of water borne-disease outbreaks (wbdos) (calderon et al., 2002 ). the surveillance system includes data for outbreaks associated with drinking water and recreational water. state, territorial, and local public health departments are primarily responsible for detecting and investigating wbdos and voluntarily reporting them to cdc on a standard form. the unit of analysis for the wbdo surveillance system is an outbreak, not an individual case of a water-borne disease. two criteria must be met for an event to be defined as a wbdo. first, more than two persons must have experienced a similar illness after either ingestion of drinking water or exposure to water encountered in recreational or occupational settings. this cri terion is waived for single cases of laboratory-confirmed primary amebic meningoencephalitis and for single cases of chemical poisoning if water-quality data indicate contamina tion by the chemical. second, epidemiologic evidence must implicate water as the probable source of the illness. the integration of national information on water-borne disease incidence into collaborative regional, transboundary basin databank networks of open access is crucial to allow a fair and efficient implementation of water management regula tions related to, for example, dam operation and international irrigation agreements. this is particularly relevant in tropical countries where large river basins (e.g., amazon, nile, mekong, indus, ganges, and yarlong-brahmaputra) represent the main source of income, water, and food for millions of people. the rivers originating in the tibetan plateau ( figure 2 ) are particu larly relevant as they flow through several, densely populated tropical and subtropical countries with quite different degrees of development. in general, water-borne disease can be caused by protozoa, viruses, or bacteria, many of which are intestinal parasites. in the following tables, some relevant examples of various disease types are grouped and summarized, including those which mostly affect developing countries, in many cases in tropical and subtropical regions. in the following sections, diseases have been selected that are relevant in terms of numerical incidence, water management, and changes in land use, parti cularly under the ecohydrological approach. these are malaria, schistosomiasis, lymphatic filariasis, onchocerciasis, cholera, spotted fever, and those illnesses induced by toxins produced during cyanobacteria blooms due to increased nutrient loads. we will provide a general overview of these diseases, stressing the links to the global water crisis, possible contributions of ecohydrology, and challenges to its classical approach. parasitic infections, particularly malaria and schistosomiasis, represent some of the most universal health problems. the who estimates that 200 million people are infected and another 600 million people are at risk of infection (who, 2007) . in fact, only malaria accounts for more cases of disease than schis tosomiasis, and both are closely related to water management (who, 1993) and will be extensively treated in this chapter. water scarcity and uncoordinated water management go hand in hand with poverty and disease. russel (1961) explains the links clearly: bilharziasis and malaria are both debilitating diseases and debility is not conducive to good farming. throughout history, malaria and bilharziasis have interfered with the use of arid land and indeed, calcified eggs of bilharzia worms were found in the kidneys of two mummies of the 20th dynasty -1250-1000 b.c. there is a crucial chain reaction that in some instances has contributed to the virtual abandonment of irrigation systems: the more debility, the less canal maintenance, therefore the more anopheles mosquitoes and the more snails transmitting more malaria and more bilharziasis, thus leading to more debility, and so on…. (russel, 1961: 253) . table 1 provides a summary of relevant water-borne diseases produced by protozoan parasites. in this section, we will con centrate on malaria, currently still the world's most important parasitic disease. today, malaria is one of the world's deadliest diseases and occurs mostly in tropical and subtropical countries, being transmitted from one person to another through the bite of female anopheles mosquitoes. the who estimates that there are 300-500 million cases of malaria, with over 1 million deaths each year (who, 2001a) . t he main burden of malaria (more than 90%) is in africa, south of the sahara. two-thirds of the remaining burden affects six countries: brazil, colombia, india, solomon islands, sri lanka, and vietnam. the ecology of the disease is closely associated with the availability of water, as the larval stage of mosquitoes develops in different kinds of water bodies. the mosquito species vary considerably in their water-ecological require ments (sunlit or shaded, with or without aquatic vegetation, stagnant or slowly streaming, and fresh or brackish; discussed in section 10.12.4.3.2.1), and this, to a great extent, determines the ecology of the disease. in many places, the nat ural habitat sustains intense malaria transmission; in others, the development of water resources (irrigation, dams, and urban water supply) has exacerbated the intensity of transmission and caused the disease to spread. in yet others, for example, the central asian republics of the commonwealth of independent states, malaria has returned as a result of a breakdown in water management and maintenance problems of local irrigation systems (who, 1999) . climate change (global warming) appears to be moving the altitudinal limits of malaria to higher elevations, for example, in the east african highlands and madagascar. further, in some of the tropical regions of the developing world, the incidence of malaria has increased in recent years as the mosquito and the malaria parasite it transmits have evolved more resistance to sewage, nontreated drinking water, flies in water supply (hand-to-mouth) untreated water, poor disinfection, pipe leaks, groundwater pollution, sharing of water source by humans and wildlife. beavers and muskrats create ponds that act as reservoirs for giardia (oral-fecal, hand-to-mouth) encephalitozoon intestinalis has been detected in groundwater chills, period fever attacks; debility, spleen and liver enlargement; anemia, jaundice; clogging of brain vessels can lead to death flu-like symptoms, watery diarrhea, loss of appetite, substantial loss of weight, bloating, increased gas, and nausea abdominal pain, fatigue, weight loss, diarrhea, bloating, and fever diarrhea, bloating abdominal discomfort, and flatulence diarrhea and wasting in immunocompromised individuals alternatives to dichlorodiphenyltrichloroethane (ddt) and to the medications used to prevent or treat the disease. in some regions (e.g., south africa), ddt is again increasingly used to control mosquitoes (thurow, 2001) . in general, land reclama tion for agriculture, deforestation, and changes in land use are probably the principal causes of the climatic and habitat changes responsible for these developments. yet, it should be borne in mind that it was the drainage of wetlands for agricul tural purposes in the nineteenth century which contributed most significantly to the eradication of malaria in europe (reiter, 2000) . these are crucial aspects to be carefully evaluated when wetland creation and management policy are being considered as an ecohydrological tool, for example, for sequestration of nutri ents in estuaries and for preventing toxic algal blooms. these issues will be discussed extensively in the following section (also discussed in section 10.12.5.2). it is often erroneously thought that larvae of malariatransmitting mosquitoes can only develop in freshwater. it is also frequently forgotten that malaria is not restricted to the tropics, and that only in 1975 did the who declare that europe was free of malaria. about 200 years ago, malaria was a leading cause of death in many marshland commu nities along the coast of southern england. there, extensive salt marshes provided high-quality grazing for sheep and cattle, but were also a favored habitat for anopheles atroparvus, a highly effective malaria vector, which prefers to breed in brackish water along river estuaries and in the presence of abundant algae. until the nineteenth century, malaria was a major mortality factor in the netherlands. however, by the end of that century transmission had dropped precipitously in the more prosperous countries of north europe. a major factor contributing to this decline was that the mosquito habitat had been eliminated by improved drainage and extensive land reclamation. major epidemics still occurred in russia and poland in the 1920s, with high death rates reaching regions near the arctic circle (wolanski et al., 2004 and references therein) . today, malaria is again common in many parts of central america, northern south america, tropical and subtropical asia, some mediterranean countries, and many of the republics of the former ussr. this spread of the disease has been attrib uted to, among other factors, forest clearance, irrigation, ecological change, population increase, deterioration of public health services, resistance of mosquitoes to insecticides, and resistance of the malarial parasite to antimalarial drugs (reiter, 2000) . thus, policies on wetland creation or restoration must take account of not only the benefits of the reestablishment of lost ecological services, but also the potential consequences of increased areas of slow-flowing or stagnant waters on disease vector proliferation, particularly under a scenario of increasing temperatures. the related ecohydrological concepts of system robustness and flushing dynamics can make a major contribu tion to the integrated analysis and handling of this issue, seeking equilibrium between the necessary water residence time, for example, efficient nutrient sequestration, and the minimization of mosquito reproduction. the choice cannot be formulated as 'mosquitoes in the basin' or 'toxic algal blooms in the estuary'. there are techniques such as 'runneling' that have been used with varying success to control mosquito proliferation in cre ated or natural wetlands. as mosquitoes can be vectors for several other diseases besides malaria, this will be treated in a separate section (discussed below in section 10.12.4.3.2.2). table 2 provides a summary of relevant water-borne diseases produced by worm infections. in this section, we will concen trate on schistosomiasis, lymphatic filariasis, and river table 2 main water-borne diseases produced by worms blindness because of their strong links to water management measures, such as irrigation and dam operation schemes. schistosomiasis is considered the second most important para sitic infection after malaria in terms of public health and economic impact. it is a chronic debilitating disease that is estimated to affect between 200 and 300 million people in 79 countries. as many as 600 million live in endemic areas. also known as bilharzia, bilharziosis, or snail fever, it is caused by several species of fluke (trematode) of the genus schistosoma. (gryseels et al., 2006; figure 3) . infection with any of the five species of schistosome worms is rarely fatal. although it has a low mortality rate, schistosomiasis is often a chronic illness that can damage internal organs and, in children, impair growth and cognitive development. the urinary form of schis tosomiasis is associated with increased risks for bladder cancer in adults (hodder et al., 2000) . human infections are most common in asia, africa, south america, or the middle east, especially in areas where the water contains numerous fresh water snails, which are intermediate hosts, that is, may carry the parasite. however, trematodes can be found anywhere human waste is used as fertilizer. the disease affects many people in developing countries, particularly children who may acquire the disease by swim ming or playing in infected water, and field workers in arid or semiarid regions where agriculture depends heavily on irrigation. first infection with schistosomiasis usually occurs during the early school years and is a frequent cause of absen teeism. it is not uncommon for 85% of a school's student population to be infected in some highly endemic areas in africa. in addition to its effect on children, schistosomiasis has a major impact on the agricultural workforce and on national economic productivity. in egypt, where 20% of the people are infected, economic losses due to lost work are estimated to exceed $500 million a year (anonymous, 2009a) . development, both planned and unplanned, has resulted in a number of changes in the epidemiology of the disease that threaten to increase its spread and the number of people infected, reduce economic productivity, and compromise development gains. water development schemes, including dam building and irrigation systems, with slow water flows and aquatic vegetation, have created new breeding sites for snails. intensive agriculture has encouraged people to migrate to urban and peri-urban areas that are ill-prepared to meet their needs for sanitation and water. in these areas, snail-infested streams and canals are often the most convenient water sources. new agricultural systems that emphasize irrigation, double cropping, and other intensive cultivation practices have increased farmers' exposure to infection. the emergence or reemergence of schistosomiasis as a result of large-scale hydroprojects has been reported from the egyptian aswan high dam (khalil, 1949; strickland, 1982) , the sudanese gezira-managil dam (amin, 1977; omer, 1978 (teklehaimanot and fletcher, 1990) . in china, the danling dam in the province of sichuan and the huangshi dam in the province of hunan have all had adverse effects by increasing local schistosome transmis sion (zhang and guo, 2006) . however, not all dam regions suffer from schistosomiasis. for example, when the ertan dam in the province of sichuan became operational, local schistoso miasis control centers collaborated with dam management offices and government ministries to actively monitor and pre vent the spreading of schistosome worms. as a result of these efforts, potential schistosome transmissions in the ertan region were successfully averted (gu et al., 2001) . schistosomiasis infection in humans, the definitive hosts, is caused mainly by three species of flatworm, namely schistosoma haematobium, s. japonicum, and s. mansoni. infection occurs when free-swimming larvae penetrate human skin. the larvae develop in freshwater snails, which serve as intermediate hosts for the parasite. humans are infected when they enter larvaeinfested water for domestic, occupational, and recreational purposes and the larvae of the parasite penetrate the unbroken skin. the life cycle is continued when people infected with schistosome worms deposit urine or fecally borne eggs into the water. the disease particularly affects children who may acquire the disease by swimming or playing in infected water (hodder et al., 2000) . the life cycle of s. mansoni provides a simplified example for all species of schistosomes and helps understand the potential contribution of water management to snail proliferation. after the eggs of the human-dwelling parasite are emitted in the feces, into the water, the ripe miracidium hatches out of the egg. the miracidium searches for a suitable freshwater snail to act as an intermediate host and penetrates it. following this, the parasite develops, via a so-called mother-sporocyst and daughter-sporocyst generation, into the cercaria. the purpose of the growth in the snail is the numerical multiplication of the parasite. a single miracidium can produce several thousand cercaria, each one of which is capable of infecting humans. the cercaria propel themselves in water with the aid of their bifurcated tail and actively seek out their final host. when they recognize human skin, they penetrate it within a very short time. following a migration through the body within the bloodstream, they develop into sexually mature adults. the larvae enter through the skin, migrate via the blood vessels, and mature in the lungs. from there they travel to the veins of the upper or lower intestine or bladder and, if they find a partner of the opposite sex, they reproduce. there are specific host/parasite combinations that lead to typical forms of bilhar ziasis in endemic areas in different regions of the world, frequently associated with a particular economic activity. there are also region-specific control measures with positive and negative consequences or side effects (mannesmann and fuchs, 2003) as discussed in section 10.12.2.2.1.1. in these regions, the snail bulinus truncatus, b. globosus, and b. forskali can be hosts of s. haematobium, which produces uro genital bilharziasis (lang, 1996) . the two clearest examples of large-scale irrigation systems spreading schistosomiasis are found in africa, especially in the nile valley. in upper egypt south of cairo, it has been known for a long time that the shift from basin irrigation by the floodwaters of the nile to perennial irrigation results in a dramatic increase in schistosomiasis. basin irrigation allows the land to dry out seasonally, killing almost all snails. under perennial irrigation, the land is wet all year round. soon after the construction of aswan low dam, four locations that changed irrigation methods in the mid-1930s experienced a surge in s. haematobium infections (khalil and abdel azim, 1938; khalil, 1949) . infection rates increased from 2-11% in 1934 to 44-75% in 1937. urinary schistosomiasis had a pre valence at 60% in areas of perennial irrigation in upper egypt and only 5% in areas with basin irrigation (scott, 1937) . in the 1950s, prevalence ranged from 56% to 68% in three districts with perennial irrigation, while in two districts with basin irriga tion, it was 9% and 13% (wright, 1973) . in the 1970s, after the construction of the aswan high dam, the nile delta became a major breeding habitat for the snail hosts of both urinary and intestinal schistosomiasis. irrigation canals and drains ( figure 4 ) harbored stable populations of these snails throughout the year. this resulted from the elimina tion in these canals of the so-called 'winter closure'. before the construction of the dam, the closure was enforced for about 40 days, during which the canals were closed and dried up, and the silt deposited on their beds during the nile flood was dredged out together with the snails and aquatic weeds (malek, 1975) . the other large-scale nile valley irrigation system impli cated in schistosomiasis transmission is the gezira irrigation scheme in the sudan. the major increase in prevalence of schistosomiasis in gezira came after 1960, when the cropping rotation changed to include 'winter' wheat. farmers kept the canals filled with water from march to may, when they were previously dry (fenwick, 1989) . another factor was the creation of the adjoining managil extension irrigation system which left tenants without adequate water supplies or sanitation facilities. also, there was an influx of migrant laborers in the original gezira scheme, who lived near irrigation canals, under very poor sanitary conditions, leading to the propagation of further water-borne diseases (tameim et al., 1985) . in north africa and the middle east, research has demon strated the association of even small-scale irrigation plans, decentralized at the village level, with increases in schistoso miasis transmission. malek (1962) reports that in sudanese villages along the nile, north of khartoum, prevalence of urin ary schistosomiasis in children was 11-12%, compared to an average of less than 2% in other areas. wright (1973) reports that in the rural area around baghdad, iraq, prevalence of schistosomiasis increased from 10% to 25% following the installation of lift pumps. 10.12.2.2.1.2 asia in east and southeast asia, intensification and expansion of irrigated rice production systems over the past decades have increased the habitats for snail and schistosoma. the host/para site combinations tricula aperta/s. mekongi and oncomelania hupensi/s. japonicum produce intestinal bilharziasis predomi nantly in rural communities living close to irrigation ditches. the association of asian schistosomiasis with rice-growing areas has been reported by different authors. however, in many cases, rice fields themselves did not seem to be breeding habitats; rather snails found in the rice fields appeared to have spread from nearby irrigation canals. cattle and water buffalo can also be important reservoir hosts. various methods have been applied to substantially reduce schistosomiasis in rice agriculture, which will be discussed later in section 10.12.4.3.1. in this part of the world, it is mainly the combination of s. mansoni with biomphalaria glabrata snails that causes bilhar ziasis. brazil, with 25 million people living in the endemic areas and 3 million infected, is the most affected country in the americas (chitsulo et al., 2000) . however, it is not obvious to what extent hydrological factors, including large dams or irrigation systems, contribute to spreading schistosomiasis in brazil. the african slave trade was probably responsible for the introduction of schistosomiasis in brazil soon after the country was discovered by european explorers, and internal migration was responsible for spreading it from seashore to interior. nowadays, schistosomiasis transmission occurs over a vast endemic region, from maranhão to espírito santo, and minas gerais, and there are further areas with a high risk of endemic expansion (araujo, 1986; paraense, 1986) . there are also iso lated foci in the federal district and in the states of pará, goiás, rio de janeiro, são paulo, paraná, and rio grande do sul. some examples from the literature point to patterns of labor and household migration as significant factors in the incidence of disease in brazil, probably more than irrigation schemes. cases of disease importation from endemic areas have been registered over almost the entire country, mainly in the states that are considered migration destination areas, such as rondônia (coura and amaral, 2004) . a study involving 23 irrigation projects in the semiarid region of five northeastern states of brazil (coutinho et al., 1992) found that schistoso miasis transmission was not a major problem in the areas studied. socioeconomic-sanitary analysis identified the pre sence of migrant farm workers coming from endemic areas of schistosomiasis and living in poor sanitary conditions in the irrigation areas as a main factor of epidemiological importance. however, continued epidemiological surveillance is essential in all irrigated areas of northeastern brazil if schistosomiasis control is to be maintained, as well as improvement in the water supply and sanitation measures for migrant workers (coutinho et al., 1992) . the potential association between irrigation levels and the occurrence and spread of s. mansoni infection was investigated (martins and barreto, 2003) in the state of bahia, where two forms of irrigation have been developed. the first is capital intensive and mechanized, requiring little manual labor. the second is labor intensive and characterized by limited mechan ization. according to the study, the municipalities with the largest irrigated areas are not the ones with the highest s. mansoni infection rates. in most of these counties, irrigation is capital-and technology intensive. according to these find ings, unlike africa, irrigation in the state of bahia has had little impact on the spatial profile of the schistosomiasis endemism. regarding the impact of schistosomiasis on labor-intensive irrigation schemes, some research points to the importance of the household in disease transmission, as a result of the cluster ing of domestic activities associated with water collection, storage, and usage. such activities can result in the sharing of water-contact sites and water-contact behavior, which expose all members of the household to an increased risk of infection. in previous studies in brazil (bethony et al., 2004) , it was deter mined that shared residence accounted for 28% of the variance in schistosoma fecal egg excretion rates. further, shared residence accounted for 30% of the variation in total water contacts per week. it also accounted for a large proportion of the variation in individual water-contact behavior: for example, agricultural con tacts (63%), washing limbs (56%), or bathing (41%). these results implicate the household as an important composite measure of the complex relationships between socioeconomic, environmental, and behavioral factors that influence water-con tact behavior and, therefore, the transmission of schistosomiasis. these results also support the idea of focusing on safe water supply and household density in the implementation of schis tosomiasis prevention and control measures. the role of aquatic animals in maintaining the schistosome life cycle in brazil requires further clarification. the influence of s. mansoni on a population of the water rat, nectomys squamipes, was studied at sumidouro, rio de janeiro, and brazil (d' andrea et al., 2000) . the population dynamics of parasites was studied. water contamination (i.e., the source of miraci dia), abundance of the intermediate host, and rodent migration were found to be related to schistosome prevalence. the n. squamipes population was not obviously influenced by the infection, as shown by the high number of reproductive infected females, high longevity of infected individuals, and the absence of a relationship between recruitment or survivorship rates and the intensity of schistosome infection. the data indicate that n. squamipes can increase transmission of s. mansoni in endemic areas and carry it to noninfected areas. furthermore, this rodent can be used as an indicator of trans mission foci. in the caribbean, b. glabrata can be found in shallow ponds with abundant vegetation or with fallen banana leaves. it has been also found in drains around banana plantations on the west indian island of st. lucia (sturrock, 1975) . this is a mosquito-borne parasitic worm infection, a debilitating parasitic disease, which affects 170 million people in the tropical and subtropical areas of southeast asia, south america, africa, and the islands of the pacific. while filariasis is rarely fatal, it is the second leading cause of permanent and long-term disability in the world. a person with the disease tends to have more bacterial infections in the skin and lymph system. this causes hardening and thickening of the skin, which in its most dramatic form is expressed in the symptoms of elephantiasis, the accumu lation of lymph, usually in legs. it is not a killer disease, but causes severe debilitation and social stigma. the who has named filariasis one of only six 'potentially eradicable' infectious diseases and has embarked upon a 20-year campaign to eradi cate the disease. in addition to consistent long-term treatment by oral medicines, eradication efforts focus on controlling the pro liferation of mosquitoes in aquatic environments, which will help to reduce the transmission of lymphatic filariasis, as well as that of malaria, which is prevalent in many of the same communities in africa (cdc, 2005) . onchocerciasis or river blindness is the world's second leading infectious cause of blindness. it is found in 36 countries in africa as well as in guatemala, southern mexico, some areas of venezuela, small areas in brazil, colombia, and ecuador, and in the arabian peninsula. a total of 18 million people are affected worldwide. the disease is caused by onchocerca volvulus, a parasitic worm that breeds in water and that can live for up to 14 years in the human body. controlling insect breeding sites in rivers is one of the pillars of prevention. the disease is trans mitted person to person by bite of a blackfly (simulium), which breeds solely in fast-flowing waters. symptoms of the disease in a person usually begin to show 1-3 years after infection. each adult female worm, which can be more than half a meter in length, produces millions of microscopic young worms (micro filaria). the microfilaria migrate through the skin and, upon death, cause intense itching and depigmentation of the skin (leopard skin), lymphadenitis, resulting in hanging groins and elephantiasis of the genitals, serious visual impairment, and blindness when they reach the eye. the disease blinds between 10% and 30% of its victims and seriously undermines the economic productivity of communities in endemic areas (who, 2001b; figure 5 ). figure 5 onchocerciasis also affects the development of exposed children. their school performance is affected by the unrelenting itching associated with this disease. many youth are deprived of their childhood as they are often forced to guide and look after elderly relatives blinded by the disease. photo credit: bill vanderdecker in: http://www.pqmd.org/cms/node/116 unlike malaria and schistosomiasis, transmission of river blindness is usually found along fast rivers or streams with white-water rapids and cascades. the species of blackflies, which transmit this blinding parasite, require well-aerated, high-velocity flow to deposit their eggs, usually on rocks or overhanging vegetation. the larval stages are filter feeders and need large flows passing their habitat to obtain sufficient food and oxygen for development. in many parts of africa, people living near rivers migrate out of the fertile river valleys because of the painful bites of the flies and the eventual blindness resulting from this parasite (kim and merritt, 1990) . river blindness has historically plagued the fertile valleys of west africa, but it was the arrival of europeans that unleashed the full force of the disease upon the region's inhabitants. traditional taboos had kept people from settling along river banks or visiting streams in broad daylight, when blackflies are most active. white colonists, however, insisted on recreating the riverside towns they remembered from home. by removing longstanding cultural prohibitions, they made onchocerciasis more prevalent than it had been before. by the 1970s, several hundred thousand people had been blinded by the disease. also tragically for the region, the most fertile farmlandan area roughly the size of michiganwas abandoned due to the risk of contracting the disease (wong, 2008) . currently, river blindness has been eliminated as a major public health problem in west african countries. despite success in west africa, 109 million people remain at risk of contracting river blindness in the 19 countries of central, eastern, and southern africa (anonymous, 2007) . due to the tight connec tions between the dynamics of this disease and changes in flow velocity, we will return to it in section 10.12.3.3 in the discus sion of the effect of dams on human health. table 3 provides a summary of relevant water-borne diseases produced by bacteria. we will concentrate on cholera and other vibrio illnesses for the reasons explained below. the case of cholera has been considered paradigmatic of the links between global climate change and infectious diseases. it offers an excellent example of how information about environ mental factors permits better understanding of disease virulence, transmission, and epidemiology. therefore, and due to the links to wetlands and other coastal ecosystems, aquaculture, and water management in megacities, this disease will be discussed in detail in this and the following sections, in relation to the dynamics of its causative agent in aquatic ecosystems. cholera is still an important cause of morbidity and mor tality in many countries in asia, africa, and latin america due to lack of safe water supply and poor hygienic practices (colwell, 1996) . cholera is endemic in the ganges and brahmaputra deltas. it was originally endemic to the indian subcontinent but spread worldwide along the trade routes, and mostly affects developing countries, particularly in coastal zones. in the last ∼200 years, there have been seven pandemics and there is evidence of accelerated change of vibrio and disease dynamics as consequence of environmental changes and increased global connectivity (faruque et al., 2003) . the cur rent pandemic, which started in 1961, is the most extensive in geographic spread and duration. cholera epidemics were reported from over 90 countries during 1994, the largest scale ever recorded in human history (who, 1998) . vibrio cholerae, a gram-negative comma-shaped gammapro teobacterium, is the causative agent of cholera ( figure 6 ). vibrios are aquatic bacteria of marine and estuarine origin but can survive and be pathogenic in freshwater ecosystems. apart from v. cholerae, some other vibrios (v. parahaemolyticus, v. vulnificus, etc.) are also responsible for incidences of diarrhea, gastroenteritis, necrotizing fasciitis, and septicemia, which afflict human populations (table 3 ) all over the world (chakraborty et al., 1997) . besides, many vibrios can also cause diseases in fish, shrimp, corals, and other aquatic organisms (thompson et al., 2004) . there are convergent approaches to the investigation of coral and human diseases. these include the research into the figure 6 the causative agent of cholera, vibrio cholerae, is a faculta tively anaerobic bacterium, 0.5-0.8 µm to 1.5-2.5 µm, and a natural inhabitant of temperate/tropical estuaries, salt marshes, mangroves, coastal waters, and reefs. photo credit: moredun animal health, ltd/ science photo library/photo researchers, inc. same bacteria genus (vibrio), responsible for coral bleaching and for cholera, the use of remote sensing of ocean-surface temperature, climate research, and an emerging common cur rent of epidemiological thinking. traditionally, the association between water temperature and coral bleaching has been stressed, and only recently it has been discovered that this is probably triggered by a vibrio bacterium, and that it could be transmitted by a coral-feeding worm, acting as 'vector'. it is the first time a vector has been found for a coral disease (rosenberg and falkovitz, 2004) . table 3 main water-borne illnesses produced by bacteria at the same time, the study of cholera disease has begun to encompass marine and estuarine research. the association of v. cholerae with plankton was established only recently, allow ing analysis of patterns of cholera epidemics, especially in those regions where it is endemic. the sporadic and erratic nature of cholera epidemics can now be related to climate-ocean coupling events, such as el niño (colwell, 1996) . since zoo plankton has been shown to harbor the bacterium and zooplankton blooms follow phytoplankton blooms, remote sensing can be employed to determine the relationship between cases of cholera and ocean chlorophyll concentration, as well as sea-surface temperature, ocean height, nutrient con centrations, salinity, and turbidity. during each pandemic, cholera has struck coastal regions before spreading inland. the principal agents responsible for cholera epidemics are the o1 and o139 serogroups of v. cholerae, of more than 200 serogroups so far identified. within the o1 serogroup, the classical biotype caused the pre vious six pandemics, while the el tor biotype is associated with the present seventh pandemic. a coupling of vibrio dynamics to that of aquatic, brackish ecosystems is strongly suggested by the marked cholera seasonality in coastal villages of the bay of bengal (colwell, 1996) . the role of coastal areas in maintain ing endemicity is clearly a significant feature of cholera ecology. however, it was mainly laboratory research that provided evi dence for the existence of aquatic environmental reservoirs where v. cholerae survives for long periods of time and from which a toxigenic form may emerge to support epidemic con ditions (miller et al., 1984 (miller et al., , 1985 (miller et al., , 1986 barua and greenough, 1992) . the o139 serogroup emerged in the coastal zone in 1992 through a natural genetic alteration of the o1 el tor biotype. a survey in bangladesh established that this strain was mainly found in southern coastal marshes (faruque et al., 2003) . however, systematic, interdisciplinary field studies on the relationship between habitat characteristics and vibrio diversity and virulence are scarce. little has been done to widely monitor v. cholerae and study its ecology from a basin perspec tive in different related coastal environments such as mangroves, marshes, and estuaries, despite clear evidence that this could be one significant step forward for early warning and understanding human vulnerability to the disease (collins, 2003) . as early as 1884, robert koch suggested that the bay of bengal, especially the sundarban mangrove forest, was the main source of cholera, noting that the combination of a brackish, organic-matter-rich environment, with a high den sity of human population represented the ideal conditions for the proliferation of v. cholerae (koch, 1884) . however, since then, there have been no extensive surveys of this transbound ary ecosystem (bangladesh and india), which represents the largest unitary mangrove and marsh system worldwide (akhtaruzzam, 2004) . difficult access, cyclones, floods, and the difficulty of obtaining funding for long-term research have precluded the systematic spatiotemporal monitoring of these habitats. most of the few available works are based on one-site samplings and do not provide information on seasonal trends. plankton is a significant marine reservoir of v. (huq and colwell, 1995) . in most cases, phytoplankton and zooplank ton are spatially and temporally associated (kiorboe and nielsen, 1994) and their abundance can be estimated by remote sensing, which has been related to cholera incidence in the bay of bengal region by lobitz et al. (2000) . living zooplankton can be a reservoir for vibrios, which attach them selves to the zooplankton's chitinaceous exoskeleton (kaneko and colwell, 1975) . nevertheless, watkins and cabelli (1985) found that growth and survival of v. parahaemolyticus was more stimulated by addition of pulverized chitin, than by living zooplankton, which however had a greater effect than the addition of sewage or other nutrients. in addition to salinity, nutrients, and plankton, suspended particle load and sediment resuspension also influence the vibrio amount in the estuary. recent investigations (lara, unpublished results) on seston size fractionation in sunderbans waters showed that the largest amounts of chitin and cultivable vibrio spp. were generally present in the fractions correspond ing to micro-, nanoplankton, microdetritus, and silt/clay particles, and not in zooplankton. it is still an open question whether the vibrio serotypes o1 and o139, as well as the viable but not cultivable, and nonviable forms are preferen tially associated with determined seston size classes or wetland ecosystem compartments. better understanding of vibrio diversity in aquatic environ ments such as estuaries, marshes, and mangroves can lead to new insights into their genetic expression and co-regulation in the environment where they interact with each other. however, most vibrio studies treat species of pathogenic importance (e.g., v. cholerae, v. parahaemolyticus, and v. vulnificus) and not the diversity of the genus itself. a further key issue is the compre hension of the seasonality, life cycle, and dormant phases of bacterial population in nature. the environmental persistence of v. cholerae may be facilitated by entering a dormant state in which it remains viable but becomes nonculturable (vbnc) in conventional laboratory media (colwell et al., 1985) . the v. cholerae cells attached to plankton enter into the vbnc state as survival strategy (colwell and huq, 1994) . therefore, the detection of the nonculturable state is crucial to understanding v. cholerae ecology. however, the mechanisms that cause the organism to associate with plankton or other particles, to form biofilms, or to enter dormant or free-swimming phases are not yet completely understood. in the karnaphuli estuary, bangladesh, recent studies (lara, unpublished) showed that suspended particulate matter (spm) other than zooplankton contained significant amounts of chitin, especially in the size class <20 µm. the quantitative contribution of respectively nano-or bacterioplankton, microdetritus of biological origin, or resuspended sediment particles to the chitin pool in that size class is still unknown. particle load together with salinity significantly influenced estuarine vibrio distribution. we compared the microbial landscape dur ing a pre-monsoon situation and after a strong cyclone: the amount of cultivable vibrio, and its relative contribution to total aerobic bacteria, increased dramatically after the cyclone. amounts of spm also increased and there were higher salinities along the estuary. sediment resuspension and salt intrusion can thus strongly influence the abundance and distribution of estuarine vibrio population. the above findings call attention on essential questions relating estuarine dynamics and human health: are vibrios in sediment part of a benthic community with its own character istics or do they basically consist of a fraction of a pelagic population reaching the sediment after sedimentation of the particles to which they are attached? nair et al. (1988) also addressed the role of sediments as a possible vibrio reservoir in freshwater environments in calcutta. in florida, there was a predominance of non-o1 v. cholerae infections at the time the organisms flourished in the sediment (williams and larock, 1985) , which was detected down to 15-cm depth. higher sedi ment vibrio concentrations were associated with organic matter flocs occurring after the seasonal phytoplankton productivity maximum and during the zooplankton decline. this suggested that the flourishing of vibrio in the sediments was related to the presence of organic matter input from plankton detritus. thus, although plankton itself is an important aquatic vibrio reser voir, its relevance for fueling benthic vibrio seasonal cycles has probably been overlooked. the relevance of seasonally driven sediment resuspension in relation to annual cholera cycles in endemic regions deserves more attention. the incorporation of particle-bound vibrios and porewater nutrients into the water column could favor a sharp vibrio increase, even at otherwise unfavorable salinities (singleton et al., 1982) . future studies should investigate the links between the spatiotemporal estuarine variability and the ecology, diversity, and spreading mechanisms of vibrios including v. cholerae. a key question is how and to what extent these microorganisms persist in the transition from a brackish to a freshwater envir onment, involving strong gradients in salinity, ph, inorganic nutrients, dissolved and particulate organic matter, turbidity, plankton, and wetland vegetation. further, an ecohydrological research approach should focus on how the distribution of the above factors influences the relative abundance of v. cholerae compared to the total vibrio population and other bacterial groups in aquatic environments of west bengal, including water and sediment compartments. there have been efforts to predict cholera outbreaks through models relating disease incidence and environmental variables such as seawater temperature, chlorophyll content, height of the ocean surface, and rainfall, as well as by remote sensing (e.g., lobitz et al., 2000) . recently, an empirical model relating multiyear data of the number of cholera cases, rainfall, and chlorophyll was able to successfully reproduce outbreaks of cholera in kolkata and matlab over the time span of the data set (magny et al., 2008 ). yet, these authors stated that a finer temporal resolution (submonthly) in environmental data col lection was needed to improve mechanistic models and account for short-term variability, especially for the kolkata region. there is also evidence that cholera cases increased following a rise in ocean-surface temperature. however, a direct correla tion exists only for the spring peak, while during the rest of the year there are lags and even an inverse correlation between the two variables (colwell, 1996) . simple (lag) correlations between the seasonality of cholera and that of climate variables such as monsoon rainfall merely confirm that cholera is seaso nal (bouma and pascual, 2001) . these authors hypothesized that there would be two different aquatic habitats: the marineestuary type and the inland water bodies, with potentially different driving factors. although much has been speculated about possible ocean/ land interactions based on empirical correlations, the causal links between ocean parameters and cholera incidence in ripar ian inland villages are tenuous, as discussed in the following. zooplankton in freshwater has been considered a main trans mission means of v. cholerae to humans . although tidal transport of vibrio-carrying marine zooplankton toward the inland is likely and has been proposed by these authors as a possible infection source, until now there have been no studies demonstrating that marine copepods survive long enough to represent an infection source in freshwater, which is what people drink finally, and not seawater. simultaneous variation of two parameters does not imply causality and, although the existing mechanistic models can be useful as predictive tools, they have not significantly contributed to explaining the reasons for cholera endemicity or the existence of bimodal and unimodal occurrence patterns in the indian subcontinent. for example, sea-surface tempera ture in the bay of bengal shows a bimodal cycle similar to the seasonal pattern of cholera in dhaka, bangladesh (colwell, 1996; figure 7) , and is therefore often used, directly or through covarying parameters, with success for such models. however, this seems to mask the fact that strong unimodal patterns are observed elsewhere in india (e.g., north bengal and vellore; jesudason et al., 2000; bouma and pascual, 2001) without any obvious relationship to the marine environment or even to temperature and rainfall. several studies found that the effect of rainfall on cholera incidence does not show any univocal pattern (glass et al., 1982; bouma and pascual, 2001; ruiz-moreno et al., 2007) . even in dhaka, which has a strong bimodal seasonality, there is no clear relationship between rainfall and cholera. the number of cases peaks before the monsoon (high rainfall period) and at its end, with a strong decrease in the middle of the monsoon. recently, hashizume et al. (2008) showed that the weekly number of cholera cases in the period 1996-2002 in dhaka did not show any direct relationship to rainfall, and suggested that river levelbelow flooding levelalso plays a role. river discharge is controlled not only by rainfall but also by snow melting in the himalayas and can control the survival of the bacterium through determining salinity and ph levels (bouma and pascual, 2001) . hashizume et al. (2008) stated that, because of the observed and potential effects of chemical changes in surface waters due to rainfall on vibrio survival and toxicity, it is necessary to quantify the level of v. cholerae in aquatic environments at the same time as measuring rainfall and river levels ( table 4) . this section deals with examples of diseases derived from or influenced by high nutrient loads, particularly those derived from toxic algal blooms, as well as with the general effect of global changes in nutrient cycles on parasitic infectious diseases according to toxin type and exposure: skin irritation, stomach cramps, vomiting, nausea, diarrhea, fever, sore throat, headache, muscle and joint pain, liver damage, and kidney disease in the body nitrate is reduced to nitrite, which reacts with hemoglobin forming methemoglobin, and reducing blood ability to carry oxygen; affects mostly infants and old persons (pids). although arsenicosis affects millions of people, parti cularly in bangladesh, the problem arises from using water from wells reaching layers where groundwater naturally con tains high as levels and there is, to date, no obvious way that ecohydrological methods could help solve this problem. for this reason, we will not discuss this disease further in this chapter. eutrophication in water reservoirs or semi-enclosed water bodies leads to the formation of intensive phytoplankton blooms. during recent years, both the incidence and intensity of such blooms appear to be increasing, when examined at the global scale. this increasing severity of algal blooms may be a consequence of increasing levels of nutrient enrichment as a result of sewage disposal, increased agricultural runoff, and changes in hydrological regimes potentially related to climate change (unep, 2002) . blooms caused by toxic cyanobacteria (harmful algal blooms) lead to outbreaks of disease and dete riorated recreational and aesthetic values, causing both economic losses and illness and death of both humans and animals. in evolutionary terms, cyanobacteria are one of the oldest organisms on earth, dating back to more than 3000 ma (schopf, 2000) . they are oxygenic photosynthetic prokaryotes possessing the ability to synthesize chlorophyll a as their photosynthetic pigment. the nitrogen-to-phosphorus ratio (n:p) has been frequently used as a key indicator in predicting algal biomass and compositions, and its seasonal succession in lentic systems (tilman, 1982; kilham, 1990) . smith (1983) pointed out that bloom-forming cyanobacteria had a tendency to dominate in a lake when the n:p ratio was less than 29. the significance of the n:p ratio as a critical factor, however, is still controversial, due to the variability of the other chemical char acteristics and phytoplankton composition within a geographic region. for example, cyanobacteria blooms can be induced by increases in the phosphorus concentration instead of by a decrease in the n:p ratio (trimbee and prepas, 1987; sheffer et al., 1997) . cyanobacteria ( figure 8 ) produce a variety of toxic com pounds known as cyanotoxins. the impacts of cyanotoxins on human health have been of increasing concern, as the impacts of cyanobacterial blooms on water supplies as well as nearshore marine ecosystems have become better understood. outbreaks of poisoning from cyanobacterial blooms can be catastrophic, such as the death of 50 dialysis patients following exposure to inadequately treated water from the tabocas reservoir in brazil (pouria et al., 1998) or the hospitalization of 140 children and 10 adults who drank cyanobacterially contaminated water impounded by the solomon dam in australia (hawkins et al., 1985) . cyanobacterial toxins have also been implicated in mortality in wild fisheries and terres trial mammals, and can accumulate in the ecosystem . toxins are classified by how they affect the human body (who, 2001c): hepatotoxins (which affect the liver) are produced by some strains of the cyanobacteria microcystis, anabaena, oscillatoria, nodularia, nostoc, cylindrospermopsis, and umezakia. neurotoxins (which affect the nervous system) are produced by some strains of aphanizomenon and oscilatoria. cyanobacteria from the species cylindroapermopsis raciborski may also produce toxic alkaloids, causing gastrointestinal symptoms or kidney disease in humans. not all cyanobacteria of these species form toxins and it is likely that there are other as-yet unrecognized toxins. further, cyanobacteria produce a wide range of secondary metabolites, among which new cya notoxins continue to be found. besides the recognized effects of the toxins mentioned above, a recent study opened up a discussion about possible new causes of known neurodegenerative illnesses. cyanobacterial strains were found to produce the neurotoxic nonprotein amino acid, β-n-methylamino-l-alanine (bmaa) (cox et al., 2003 (cox et al., , 2005 . the bmaa is considered a possible causative agent of human motor neuron disease, amyotrophic lateral sclerosis parkinsonism/dementia complex (als/pdc), and has been found to accumulate in brain tissues of patients with progressive neurodegenerative illness (murch et al., 2004) . this was first detected in humans as a consequence of dietary habits and biomagnification in terrestrial ecosystems in guam (cox et al., 2003) . however, sometime later, bmaa was detected in aquatic ecosystems, such as in seawater in the area of a trichodesmium bloom and in freshwater and brackish waterbodies (metcalf et al., 2008) . cox et al. (2005) isolated bmaa in several species of free-living cyanobacteria. possible implications of these findings (cox et al., 2005; ince and codd, 2005) are that bmaa of cyanobacterial origin might occur in diverse natural and controlled environments where massive cyanobacterial populations occur. because these environments can include waterbodies used for drinking and recreational use, bmaa in drinking water may contribute to chronic intoxication. this may involve incorporation of the neurotoxin into an endogenous protein reservoir with slow release (murch et al., 2004) and a possible lag between expo sure and effect of years or decades. since protein-associated bmaa can accumulate within food chains, it is possible that biomagnification of bmaa occurs in marine ecosystems in a way similar to that in terrestrial ecosystems (banack et al., 2007) . thus, production of bmaa by marine cyanobacteria may represent another route of human exposure to this neuro toxin. cox stated that the bmaa produced by the algae may act as a slow toxin. for these reasons, it would be advisable to monitor bmaa concentrations in drinking waters contami nated by cyanobacterial blooms and in fish and animals that may be ingesting the microbes, even at low environmental densities. this can be particularly relevant in semiarid or arid regions that are highly dependent for their drinking water supply on reservoirs, in which cyanobacteria can regularly occur on a seasonal basis, normally in summer. fertilizer use, widespread cultivation of leguminous crops, and fossil fuel burning have produced strong changes in the global n cycle (galloway et al., 2004) . more than half of all n fertili zers ever used in earth's history has been applied in the past two decades (howarth et al., 2002) . moreover, increased n loading in the environment often occurs together with that of phosphorus. in freshwater ecosystems in particular, a com bined increase in both n and p is highly likely to drive eutrophication and associated significant ecological change. mckenzie and townsend (2007) reviewed direct and indirect evidence that changing global nutrient cycles are influencing the dynamics and incidence of pids ( figure 9 ). some of the disease agents discussed earlier in this chapter could react particularly sensitively to increased n and p loads. although generalizations are difficult, the evidence presented by these authors, together with past reviews by lafferty (1997) and townsend et al. (2003) , suggests a trend in which most asso ciations between increased nutrients and disease are positive. a parasite or pathogen may respond to nutrients directly, that is, without requiring an intermediate or vector host; or an inter mediate or vector host may respond to nutrients, mediating the overall disease response. there can be an increase in vector host population density as a result of increased primary productivity, or a decrease in population due to some disturbance related to nutrient addition, in both cases resulting in changes in transmis sion success of the parasite or pathogen (anderson and may, 1978; dobson, 1990; arneberg et al., 1998) . besides the above-mentioned increase in cyanobacterial blooms, increased nutrients could particularly affect ecological processes associated with, for example, malaria, schistomiasis, cholera, and filariasis, leading to a higher occurrence of these diseases. for example, vector-borne pathogens that cause malaria and schistosomiasis involve intermediate hosts (mos quitoes and snails) whose growth and reproduction depend on algae or weed abundance in their respective environments. where n additions cause greater plant growth and/or changes in plant species composition, the density of these intermediate hosts is also likely to be affected, with cascading consequences for the risk of disease. lafferty (1997) reviewed parasitic responses to a suite of environmental changes and found that, in most cases, eutrophi cation caused an increase in parasite abundance. johnson and carpenter (2008) suggested that planorbid snails in nutrient-rich environments grow faster. larger individuals are able to produce significantly more trematode cercariae at a faster rate. the avail able evidence suggests that, in general, nutrient additions should favor trematode development and increase disease risks. in the case of cholera, the bacteria display a strong associa tion with marine plankton, and, therefore, factors that cause increases in plankton primary productivity can also increase the prevalence of v. cholerae. thus, nitrogen-based eutrophication of coastal regions (nrc, 2000) has been linked to increased cholera risks (epstein, 1993; colwell and huq, 2001) , prob ably as an indirect response to plankton dynamics. changes in the n cycle are not globally uniform (galloway et al., 2004) . the largest changes have occurred in industria lized countries but the focus of global change is shifting to tropical and subtropical countries, where greatest relative changes in the n cycle over the next 50 years are expected (galloway et al., 2004; dentener, 2006) . for example, the booming soybean agriculture in brazil in the last decade is leading to rapid increases in regional n deposition and nutrient loading to aquatic ecosystems (martinelli et al., 2006) . similar increases are occurring throughout tropical and subtropical portions of asia and central america. these same regions harbor the greatest diversity of human pids (guernier et al., 2004) , including those that currently cause the majority of pidrelated human deaths (who, 2004b) . thus, the next decades will probably bring an increase in the potential for nutrients, especially n, to affect parasitic and infectious diseases in these regions. the changes in temperature and rainfall regime in temperate regions and the changes in land use resultant from them or from market shifts can exacerbate that potential. diseases produced by viral infections can emerge, reemerge, and be transmitted by several different, but often interlinked, mechanisms involving tight interactions between man and domestic and wild animals sharing artificial or natural aquatic systems. table 5 summarizes some viral diseases of worldwide relevance. in the following section, we will focus on avian influenza due its multiple and complex connections with the dynamics and use of wetlands by man and animals. 10.12.2.5.1 influenza: human and animal links between artificial and natural wetlands nutrient reduction in wastewaters or surface waters is a prior ity for reducing risks to human health. nevertheless, from the point of aqua-and agriculture, the nutrients contained in wastewater are a valuable resource, in particular in arid and semiarid regions. wastewater form has been used for aquaculture, for example, in duck ponds, in several countries, mainly in asia, for centuries to produce human food. organizations such as who, food and agriculture organization (fao), etc., have developed guidelines for the safe use of wastewater, excreta, and gray water in agriculture and aquaculture in order to provide a basis for the develop ment and implementation of health risk assessment and management approaches, including standards and regula tions, to address hazards associated with human waste-fed aquaculture (edwards, 2008) . however, wastes and fecally polluted surface water are often used without any pretreat ment or assessment of the presence of pathogens. various hazards are associated with waste-fed aquaculture: excretarelated pathogens (bacteria, helminths, protozoans, and viruses), skin irritants, vectors that transmit pathogens, and toxic chemicals. wastewater systems were also developed independently in india from the 1930s, in china from the 1950s, and in vietnam from the 1960s onward, but they were designed primarily for aquaculture, not to treat wastewater. few engineered waste water-fed aquaculture systems have been developed recently. systems primarily engineered to treat wastewater that incorpo rated aquaculture were developed in germany from the end of the nineteenth century, but only the munich system remains, and only for tertiary wastewater treatment and currently as a bird sanctuary (edwards, 2008) . in this, the following aspects in the dynamics of bird popu lations are of ecohydrological and biomedical relevance. during migratory movements, birds carry pathogens that can be transmitted between species at breeding, wintering, and stopover places where numerous birds of various species are concentrated, such as wetlands. a study by jourdain et al. (2007) focused on bird migration routes to the camargue in relation to risk of pathogen dispersion into western mediterranean wetlands. they considered two pathogens clo sely associated with wild birds: avian influenza (ai) virus and west nile virus (wnv). the ai viruses have a water-borne transmission, and ducks are their main natural reservoirs (easterday et al., 1997; alexander, 2000) ; wnv has a vectorborne transmission, and passerines are believed to play a major role in the amplification cycle (hurlbut, 1956; malkinson and banet, 2002) . despite different transmission cycles and ecology, both viruses are known to be carried by reservoir birds during migration and have been associated with emer ging disease transmission risk for humans and domestic animals (rappole et al., 2000; reed et al., 2003; olsen et al., 2006) . environmental conditions, avifauna abundance, and diversity, as well as the interactions among birds from different species and departure sites in stopover wetlands, may be of key importance in terms of virus communication (hudson et al., 2002) . for water-transmitted pathogens such as ai viruses, the risk of transmission may be associated with the number of ducks congregated on the same water body, particularly in autumn and winter. this crowding of wintering species, in addition to the permanent presence of a transient population of birds using wetlands to stop off during migration, could provide the conditions for the circulation and rapid dissemi nation of ai viruses. for vector-transmitted pathogens such as wnv, transmission possibilities depend both on reservoir bird density and on the dispersion capability and activity periods of the arthropod vectors. the risk for interspecific transmission of disease is particularly problematic when wild and domestic species are involved. ducks are the aquatic birds most likely to come in contact with free-range poultry, especially because the presence of congeners can induce migrating wild ducks to make a stopover (jourdain et al., 2007) . the study by jourdain et al. (2007) showed that western mediterranean wetlands are a hub for birds from several differ ent origins in central asia, siberia, northern and eastern europe, western africa, and the mediterranean basin. as exam ple for the potential of wetlands for introduction or reemersion of these viruses, they state that wnv dispersion by birds migrat ing from sub-saharan africa might explain why an outbreak occurred in 2000 in the camargue, even though the virus had not been observed there since the 1960s. besides migration, breeding ducks in the aquaculture ponds can increase virus circulation between ducks, water, sediment, fish, and man. in a study (markwell and shortridge, 1982) of the occurrence and persistence of influ enza viruses (hong kong type) within domestic duck communities, the virus was isolated throughout the year from feces or pond water or both, indicating a cycle of water-borne transmission. infection was asymptomatic and virus persistence in the duck community appeared to be dependent upon the continual introduction of ducklings sus ceptible to infection onto virus-contaminated water, since the feces of ducks 70-80 days old were generally virus-free despite the ducks' exposure to the virus in pond water. the normal practice of raising ducks of different ages on the same farm, where the water supplies are shared (figure 10) , appears to be instrumental in maintaining a large reservoir of influenza viruses in the duck communities. domestic ducks may act as a silent reservoir for the h5n1 ai virus. the concern is greatest in rural areas of affected countries, where traditional free-range ducks, chickens, and wildlife frequently share the same water source. domestic ducks can harbor the virus for long periods and without showing any sign of illness. an altered role for domestic ducks is further supported by evidence that the h5n1 virus circulating in parts of asia has increased its virulence in chick ens and mice (a laboratory model for mammals), and has expanded its host range to include larger mammals (e.g., cats and tigers), not previously considered susceptible to infection (fao, 2004) . the assessment of respective roles of routes and timing of wild waterbird migration and poultry imports is of utmost importance in order to objectively identify the origin and pos sible evolution of an outbreak in a determined country. for example, the avian flu outbreak in nigeria in 2006 may have been caused by the supply of infected live poultry including day-old chicks from different sources, including east asia and turkey, and not by wild waterbirds. this is supported by sam plings of 5000 wild waterbirds in african wetlands, in which no evidence of the h5n1 virus was found, indicting that wild birds probably played a relatively minor role in the spread of ai in that region. northward migration of wild birds from africa to europe in the northern spring of 2006 did not cause any major outbreaks. nor do wild birds seem to play a role in indonesia, where h5n1 has been present for some years and several cases of human infections have been recorded. however, although not many major outbreaks took place in europe in 2006, there is evidence to suggest that wild birds did play a significant role in spreading the disease on the european continent (aiweb, 2006). useand scarcityon human health: some examples from aquaculture, megacities, dams, and intensive agriculture aquaculture in or close to wetlands is increasing worldwide. besides being responsible for massive wetland destruction, aqua culture itself faces serious problems, arising from several diseases that can affect shrimp ponds. among the groups of microorgan isms that cause serious losses in shrimp culture, the best known are bacteria because of the devastating economic effects they have on the affected farms. as mentioned before, vibrio organ isms attach themselves preferentially to chitin surfaces, such as in zooplankton and shrimp exoskeletons. bacterial diseases, mainly due to vibrio, have been frequently reported in penaeid shrimp culture systems. at least 14 vibrio species are implicated in disease outbreaks in shrimp, including vibrio harveyi, v. alginolyticus, v. vulnificus, v. parahaemolyticus, and v. cholerae (non-o1) (venkateswara, 2008 ; figure 11 ). vibrios can produce different chitinases to degrade various chitin types (svitil et al., 1997) in marine, estuarine, and figure 11 example of vibrio disease affecting shrimp. in such severe cases, extensively melanized black blisters can be seen on the carapace/ abdomen of the infected animals. copyright national institute of oceanography, dona paula, goa, india, 1998. pond and irrigation canal next to live poultry markets. in http://www.eastwestcenter.org/index.php?id=5518&print=1 freshwater environments. further, meibom et al. (2005) found that v. cholerae can acquire new genetic material by natural transformation during growth on chitin. thus, natural compe tence occurring in chitin-attached bacterial communities can act as a powerful driver of v. cholerae evolution, which could be accelerated by environmental events such as high nutrient input, giving rise to copepod blooms. it is still unclear whether growth on a determined type of chitin substrateand the production of the corresponding specific chitinasespromotes the capture of external genetic material by vibrios. this empha sizes the need for biogeochemical characterization of different aquatic microhabitats, such as different types of chitin-contain ing particulate matter, besides living zooplankton, in environmental studies of vibrio diversity and virulence. as organic-matter-rich aquatic environments contain multiple microbial strains and species and high concentrations of bac teriophage and free dna, horizontal gene transfer (hgt) provides the most likely explanation for why vibrionaceae have developed high levels of genomic diversity (meibom et al., 2005) . interestingly, the cholera toxin gene (ctx) is a phage-mediated mobile genetic element that is transferrable to genetically closely related bacterial strains. in the aquatic environment, vibriophages can regulate seasonal disease out breaks, for example, seasonal cholera epidemics in dhaka were inversely correlated with the prevalence of environmental cho lera phages (faruque et al., 2005) . the v. cholerae (non-o1) is frequently isolated from sewage, estuarine waters, and seafood in cholera-endemic and noncholera-endemic countries. it has been associated with sporadic episodes of diarrhea worldwide, but has not caused pandemics. interestingly, in 1990, there was the first report of an epidemic of diarrhea caused by v. cholerae non-o1 that produces heatstable toxin, affecting khmers in a camp in thailand. in con trast to the v. cholerae o1 isolated from the same camp, in 93% of the cases, the non-o1 were resistant to three or more anti biotics (bagchi et al., 1993) . this calls attention to several important aspects in relation to threats to human health. the fact that the toxin is heat stable could imply a higher diarrhea risk for humans consuming cultured seafood as cooking would not completely eliminate toxin activity. its higher resistance to antibiotics compared with v. cholerae o1 potentially increases the disease hazards associated with these non-o1 strains. further, as mentioned previously, the abundance of chitinac eous substrate can favor mutations. thus, just as v. cholerae o1 evolved from a nonpathogenic to a pandemic-causing form (faruque et al., 2003) , it cannot be excluded that the high individual density of aquaculturein the same way as megacitiescould be favoring the emergence of new and highly pathogenic vibrio types. in many of the world's cities, water management and sanitation are in crisis and will dramatically worsen with the continuing growth of cities and slums. sewage pollution is the largest and most common type of pollution and one of the most common causes of illnesses. illnesses caused by sewage pollution are estimated to affect the health of more than 120 million people at any one time. contaminated water, inadequate sanitation, and poor hygiene cause over 80% of all disease in developing countries; diarrhea is the world's second most serious killer of children, but paradoxically in 90% of cases it could be easily prevented or treated. pollution of water sources by sewage contributes to 4 billion cases of diarrhea in the world each year, killing some 2.2 million children under the age of 5. poor sanitation currently affects 2.5 billion people, 40% of the world's population, who lack access to even the most minimal toilet facilities. the number of people without sanita tion will double to almost 5 billion in 2025, as the world becomes more urbanized (anonymous, 2002) . some 160 000 people are moving to cities from the country side every day. at least 600 million people in africa, asia, and latin america now live in squatter settlements without any sanitation whatsoever. the pollution of rivers and groundwater by sewage spreads disease and causes environmental degrada tion. in latin america, as a whole, only 2% of sewage receives any treatment. in asia, the level of sewage in rivers is 50 times higher than the united nations (un) guidelines. levels of suspended solids in asia's rivers almost quadrupled since the late 1970s. every minute 1.1 million liters of raw sewage are dumped into the ganges river (vidal, 2002) . most megacities are in asia. dhaka's population rose from 250 000 in the early 1970s to ∼12 million today, including the metropolitan area, and will probably reach 23 million in 2015. in the same time, kolkata's population grew from 3 to 5 million; including the metropolitan region, its population is 13 million and is predicted to reach 17 million in 2015. pakistan experienced one of the highest growth rates of popu lation worldwide: it quadrupled in only 60 years to over 170 million in 2008 (prb, 2008) . according to the world bank, karachi is one of the fastest-growing megacities of the world and is expected to rank seventh by the year 2015 (kamal, 2005) . the country faces a serious situation in terms of water availability, depletion, and pollution of its water bodies and irrigation systems as well as a severe degradation of its coastal ecosystems. this complex and multifaceted setting will be clo sely analyzed in the following sections. it has been reported that at least 12 of the 18 towns of the city are supplied with water unfit for human consumption, in most cases infected with escherichia coli. only around 30% of the total sewerage generated by karachi at present is treated. the e. coli, found in human feces, and other bacteria found in drinking water could cause life-threatening diseases, including diarrhea and cholera. the bulk of the drinking water concerned is taken from the indus river. bacteria easily enter the drinking water as the pipelines are rusted and leaking (irin, 2004) . lowintensity seismic activity, though normally not felt by people, probably further damages worn-out pipelines. additionally, due to the water shortages, the pipelines remain empty for a considerable amount of time daily, during which time they develop negative pressure and absorb moisture and sewage that has leaked from the nearby, similarly worn-out sewers. additionally, after rains, rainwater mixes with sewage and gar bage can enter the pipelines through the leaks to contaminate the drinking water supply, making people vulnerable to numer ous health hazards (hasan, 2008) . karachi is situated in a desert. however, in a study by sheikh et al. (1997) the analysis of microbiological data for the period 1990-96 showed the permanent presence of cholerawith seasonal periodicityin karachi. cholera cases peak each year between may and august in both epidemic and nonepidemic years. both v. cholera o1 and o139 serogroups were involved in the outbreaks in 1993 and 1994; o139 disappeared in 1996. the role of rain in disease seasonality and incidence is not clear. rainfall is scarce and sporadic, usually only between june and august. in 1994, the city could have been flooded, with wide spread overflowing sewers, whereas, in 1993, there was virtually no rain at all. cholera appears each year before the rains, and epidemic years in the 1990-96 data set appear to have occurred independently of rainfall. like most enteric diseases in an endemic setting, cholera in karachi is a disease of young children. the mean age of patients with acute cholera in karachi closely resembles that in the rest of the indian subcontinent where social conditions are com parable. despite this association with poverty, 9% of the patients in the study were admitted to expensive private rooms in good hospitals. this shows that, in a city with a sanitary infrastructure like that of karachi, personal wealth affords no protection (sheikh et al.. 1997) . visitors and tourists are also at risk. in the same context, despite the common perception that bottled water was safe and pure, microbiologi cal tests showed that about 35% of 3500 samples of water supplied in many bottled brands tested all over the city was unfit for human consumption (hasan, 2008) . according to the same above-mentioned study, in 1993, the new strain of v. cholerae, serogroup o139, established itself in karachi and during 1993 and karachi experienced over lapping, but distinct epidemics of both strains. the serogroup o139 never wholly replaced serogroup o1, and by 1996 it had disappeared. at that time, it was still unclear whether the dis appearance would be permanent, or whether o139 would reemerge in subsequent epidemic years. karachi is in a semi desert area, and the strain may not be able to maintain itself outside the human host (sheikh et al., 1997) . this strain has also shown diminished ability to maintain its epidemic potential in bangladesh, and it has been suggested that one reason for this may be that it is less able to persist long term in the aquatic environment (faruque et al., 1996) . however, years later, in july 2002 and june 2003, cholera outbreaks were detected by a diarrhea surveillance system in a fisher village near karachi (siddiqui et al., 2006) . the first outbreak was caused by v. cholerae o139 and the second one by serotype o1. it would be erroneous to conclude that, because of the relatively small number of persons affected, these cases are not relevant in terms of public health. on the contrary, they should be considered extremely valuable indicators of environmental change, especially of aquatic systems, which provide an early warning of possible future trends for policymakers and sanitar ians. water source was a risk factor only in the first outbreak: a reservoir in the village containing brackish water was only used for washing utensils and clothes and for bathing. only illness caused by v. cholerae o139 was associated with the use of reservoir water, while o1 cases were not. washing the clothes of infected persons may have introduced the pathogen into the reservoir. this implies that v. cholerae o139 was probably able to survive for a time outside the body and in water long enough to infect other people. this may be partly because the water was salty and that v. cholerae is a salt-loving bacterium. in summary, fecal pollution, increased nutrients, turbidity, and sodium content create favorable conditions for the propagation of v. cholerae in pakistan's coastal zone, megacities, and irrigation systems. when the increasing pollu tantchemical and microbiologicalload reaches the coastal region, it encounters a disturbed wetland ecosystem (discussed in section 10.12.3.3.2), where vibrios could potentially multi ply and mutate to new pathogenic types. a close regular surveillance of vibrios at basin level, including in the coastal region, rivers, and channels, and in humans is essential in this region, which has the potential to become an epidemic center. further, restoration of riparian forests and wetlands habitat should be a priority to avoid further habitat loss, and potential host shifts, although salinization would probably preclude the reinstallation of the same species existent before dam building and population explosion. the hoogly river is the most important source for the water supply of kolkata. through an agreement with bangladesh, only a determined amount of water from the ganges can be diverted into the hooghly river during the dry season. although this does not increase the amount of pollution in the river, it does increase its concentration. during the mon soon, rubbish and feces are washed out from the city into the ground and into the river (karthe, 2002) . around 30% of the water supply is lost through leakages in the obsolete distribu tion network, reservoirs, and public water tap connections. however, the disposal of sewage is an even greater threat to human health. reduced capacity of the inadequate sewer net work, aggravated by obstruction caused by mud or garbage, as well as flooding during the monsoon produces pollution of surface and groundwater with enteric bacteria. as in karachi, interruptions or shortages in the water supply produce a negative pressure in the pipelines, which then absorb polluted water that had leaked from sewers and the surface, especially during the monsoon season. before and after the monsoon, water quality increases, that is, bacterial load decreases. following several cholera epidemics, the chlor ination of unfiltered water supply began in 1963. this reduced cholera incidence, but supply of potable water to many parts of the city is still insufficient. people in shanties frequently get untreated water from hydrants, or from the river (hensgens, 2005) . cholera seasonality in kolkata is further discussed in section 10.12.3.3.1. the situation in london 150 years ago resembled current conditions in many megacities in developing countries. by 1851, half of the population of britain was living in townsthe first society in human history to do so. over the previous 70 years, britain's population had risen at an unpre cedented rate. large towns were desperately unhealthy, with death from sickness at a level not seen since the black death (daunton, 2004) . london had a large scavenger class living off the refuse of the citya group so numerous that it could have formed the fifth largest city in england. new epidemics affected the citiescholera and typhoid were carried by polluted water, typhus was spread by lice, and 'summer diarrhea' was caused by swarms of flies feeding on horse manure and human waste. london suffered from recurring epidemics of cholera and in 1853-54 more than 10 000 londoners were killed by the disease (johnson, 2006) . the frequent occurrence of cholera in london gave impetus to legislation, enabling the metropolitan board to begin work on sewers and street improvements. by 1866 most of london was connected to a sewer network brilliantly devised by joseph bazalgette (bbc, 2009) . the flow of foul water from old sewers and underground rivers was intercepted and diverted along new, low-level sewers, built behind embankments on the river front and taken to new treatment works. by 1870, both the albert and the victoria embankments had been opened. the victoria embankment protected bazalgette's low-level sewer from the hydraulic pressure from the thames estuary. the chelsea embankment was completed in 1874. the public health act of 1875 required local authorities to implement building regulations, or bylaws, which insisted that each house should be self-contained, with its own sanitation and water. this change in the design of housing complemented the public investment in sewers and water supply. cholera never reappeared in london after that. london was the largest city on the planet in 1854, but now it is on the small side, in comparison to, for example, mexico city, são paulo, or mumbai. massive shantytowns have exploded at the margins of today's megacities. in such places, the water-borne diseasesincluding cholerathat plagued victorian london are still widespread, thanks to insufficient public health and sanitation resources. worldwide, up to a billion people live in shantytowns and according to some projections this will increase to a quarter of the world's popula tion by 2030 (johnson, 2006) . despite enormous progress in the molecular biology of v. cholera, still little is known about basic forces, such as spatial biogeochemical gradients, seasonal rainfall variations, or cyclones, driving its abundance, diversity, and virulence in the basins of rivers and estuaries of the indian subcontinent. the most recent detailed studies on seasonal variations of estuarine salinity and related urban cholera incidence are from the 1950s (chatterjee and gupta, 1959) . these compare the river systems kolkata-hoogly (a main branch of the ganges) and london-thames (data from the nineteenth century), and are discussed in miller et al. (1982) . an important aspect of the 1950s data is that minute but clearly delimited salinity oscillations (e.g., 0.01-0.15 and 0.05-1 ppt) in the hoogly river tightly correlate with cholera incidence in kolkata. several authors reported significantly higher salinity ranges for the growth and persistence of v. cholerae in the environ ment, for example, 2.5-30 ppt (miller et al., 1984) or 5-25 ppt (singleton et al., 1982; louis et al., 2003; randa et al., 2004) . salinities <1 ppt were considered suboptimal (miller et al., 1984) . however, kolkata's data clearly indicate cholera out breaks at much lower salinities. such oscillations could also be a proxy of other processes occurring at basin level, which were responsible for triggering the cholera outbreaks. vibrio survival at low salinities can be facilitated by adsorption onto algae, zooplankton, or by high nutrient concentrations. unfortunately, this information mostly originated from stag nant water bodies or short-term investigations (e.g., islam et al., 1994, 2007 and references therein) , but no studies are available about the seasonality of hydrology, biogeochemistry, and vibrio dynamics in flowing waters of the large rivers in this region through which vibrios most likely spread from the coastal zone toward inland habitats. since the 1950s, seven large dams for irrigation purposes have been constructed in india. the farakka barrage, complete in 1975, diverts the ganges river water into the hooghly river during the dry season to flush out the accumulating silt in the port of calcutta. it cuts off bangladesh's water supply, elevating salinity, and has affected fisheries, caused desertification, and hindered navigation, and poses a threat to water quality and public health (wolf, 2001) . there is evidence of changes in cholera seasonality due to hydrologic disturbances. before the 1970s, the peak cholera season in dhaka was november-february; now it is september-november. in kolkata, season ality has changed twice since the mid-1950s (niced, 1978 . these shifts may be related to changes in salinity, particle load, and associated estuarine biogeochemistry due to, for example, the construction of the farakka barrage on the river ganges (mirza, 1998) or increased melting of himalayan glaciers (unep, 2007) . more that 25 years ago, miller et al. (1982) postulated that dam construction in india could influence vibrio dynamics by salt intrusion. these aspects deserve further investigation; apart from the 1950s data, there are no other published data series systematically relating these parameters in the rivers of the region with cholera incidence. this informa tion is essential in order to evaluate the transboundary effects of dam construction and water management. dam construc tion in india has reduced riverine discharge in bangladesh, inducing desertification in its northern sector and facilitating salt intrusion into its estuaries, particularly in the southwestern region (wolf, 2001; adel, 2005) . this resulted in changes in land use from rice cultivation to shrimp farming in the southern bay of bengal (gebauer, 2007) (discussed in section 10.12.3.1). salinization of inland water bodies can facilitate the spreading of the halophilic vibrio organisms and affect drinking water availability. on the other hand, global warming increases glacier melting and associated riverine runoff. both factors, in a frame of increasing intensity and frequency of cyclones and flooding events, create an extremely complex situation in the coastal zone that could result in shifts in seasonal cholera patterns. despite the different overall climatic setting of pakistan as com pared to india and bangladesh, increasing water needs in pakistan are leading to a similar situation in the coastal zone, including increased cholera incidence. the common factors are the halophilic character of vibrio cholerae and other vibrios and the salinization of estuaries and inland waters. water needs for irrigation of desertic and semidesertic areas, as well as for drinking water supplies, mainly for karachi and islamabad, have led to the construction of several dams along the indus river. within the sindh province, there are three major barrages on the indus-guddu, sukkur, and kotri. severe reduction of water flow below the kotri barrage started affecting environments in the area from 1960s onward, with the following consequences in the river basin: (1) drying up and death of riparian forests, figure 12 the indus delta faces major degradation threats, whose major cause is the reduction in the flow of freshwater from the indus river. as the delta dries up and the mangrove forests decline, the sea is slowly sweeping in. which occurred soon after 1970; (2) reduction of the area under fruit and vegetable crops; (3) destruction of natural pastures causing a reduction in animal populations; and (4) desertifica tion leading to a shifting of human settlements. in the coastal region, intrusion of seawater in the river bed to a distance of 80 km upstream from the shore, with percolation of saline water from the riverine areas into groundwater of adjoining irrigated areas, has turned shallow water lenses brackish ( figure 12) . another reason for accelerated salinization in the indus river is the saline water discharges from the salinity control and reclamation program in the north-west frontier province, punjab, and india (hasan, 2008) . along the coast, the increase in salinity of seawater along the whole coastline of sindh has resulted in damage to man groves, colonization by other halophilic species, and the abandonment of ∼6500 ha of land reserved for shrimp farming by the government of sindh. sea shrimp can survive within salinity range of 17-27 of water, but in the space of 5 years , salinity rose beyond the tolerance limit. water salinity in sea creeks and estuaries increased from ∼25 to over 35 ppt, making estuaries inhabitable for some shrimp and other species of commercial interest. in conditions of low salinity, shrimp farming could have been established all along 320 km of seacoast. sea fish and prawn catch has declined considerably and severe erosion due to reduced sediment load occurs along the coast. in the coastal region, the livelihood of fishing communities and the fishing industry as a whole depends on ecosystem integrity. however, this has already been devastated by reclamation of former marine areas, and of mangrove marshes and mudflats. it desperately needs to be protected, for no city that destroys the ecology of the region where it is situated can be sustainable. the south asian tsunami gave ample proof of this and so did the flooding of karachi, much of which is the result of reclama tion from mangrove marshes, creeks, and natural drainage channels for elite real estate (hasan, 2008) . in addition to the deeply disturbed aquatic ecosystems described above, only 2% of cities with a population of over 10 000 have wastewater-treatment facilities. of the wastewater generated daily, 36% is used in agriculture and 64% is disposed of into rivers or the arabian sea (iwmi, 2003) . directly or indirectly, 90% of the people of sindh, in rural or urban regions, drink water from the indus. salt content at kotri reaches 0.60 ppt in winter months, and supplies to karachi range from 0.25 to 0.60 ppt (panhwar, 2000) . the combina tion of a riverine environment with increasing salinity, growing populations, lack of sanitation, input of untreated sewage, drinking water contaminated with enteric bacteria, and rela tively high sodium content is very similar to that in london during the cholera epidemics in the nineteenth century (see section 10.12.3.2.4). recently, a cholera outbreak in mirpur khas, a district of sindh province, has been reported (anonymous, 2008) . scientists at the university of health sciences found out that around 50% of the patients were affected by cholera instead, as was previously thought, by gastroenteritis. the authorities have been requested to pay particular attention to this outbreak of cholera, which could spread to the other areas of the province. mirpur khas has a population of ∼357 000, has successful agriculture, and is con nected to the indus via irrigation canals. the irrigation system of pakistan has been considered "one of the largest contagious systems of the world" by gachal et al. (2007) . the reservoir formed by the three gorges dam (tgd) is the largest in the world at over 600 km in length. studies about the impact of parasite dynamics have been restricted to medically important species such as schistosoma japonica (zheng et al., 2002) . these studies have focused on the distribution of the intermediate host oncomelania hupensis within the tgd area and associated downstream water bodies. before construction of the dam, neither o. hupensis nor s. japonica occurred in the reservoir region. however, it is widely predicted that the dam will lead to the introduction of s. japonica into the tgd region, while downstream both positive and negative effects on schistosomiasis transmission will occur (zheng et al., 2002) . nevertheless, a survey of the recently filled reservoir (jobin, 2005) concluded that because of the very steep shoreline along most of its length and subsequent narrow photic zone, which provides only a limited area for the growth of plants, there was little chance of o. hupensis, principally a marshland snail, becoming established. however, these unfavorable topo graphic conditions do not preclude other mollusk species colonizing the shoreline (morley, 2007) . for example, former gravel quarries converted into lakes have a steep-sided profile with a narrow discontinuous zone of plant life but support a diverse molluskan-trematode community (adam and lewis, 1993; morley et al., 2004) . river blindness is an important parasitic disease around tropi cal dams in africa, along the red sea in arabia, in central america, and in parts of south america. the rapids of the upper nile river used to be a classic focus of this blinding parasite, spread by the bite of a species of blackfly, which breeds in white-water habitats and on dam spillways. in uganda (jobin, 1999) , the history of river blindness can be traced to the owen falls dam in the upper nile river. this dam flooded out owen falls and also ripon falls near the outlet of lake victoria. the history of the impact from the owen falls dam on onchocerciasis over the last 50 years shows the importance of optimal water current velocity regulation for the avoidance of reduction of this and other water-borne dis eases such as schistosomiasis. prior to construction of the owen falls dam (figure 13 ), river blindness was endemic among the buganda people, downstream along the nile. in 1950, the prevalence of the parasite was 65%. to protect the workers, weekly applications of ddt were made at the outlet of lake victoria, treating the entire flow during the construction phase, and eliminating the blackflies for at least 50 km downstream. after dam completion in 1954 and discontinuation of ddt applications, the black flies did not return to their former habitats downstream of the dam. by 1974, the prevalence of the parasite had decreased to 0.2% among the populations along the river. the fact that blackfly populations did not return to former levels indicates that there must have been a change in the basic habitat condi tions in the river. the dam had two major hydraulic effects on the river: one effect of turbine operation is a reduction of velocities downstream. the preferred range of water velocity for breeding of the east african species of blackfly involved in river blindness is between 0.5 and 3 m s −1 . at present, the mean velocities downstream of owen falls dam are between 0.4 and 0.5 m s −1 , slightly less than the required velocities. prior to dam construction, the mean velocity was roughly twice the present, and thus highly suitable for blackfly breeding. yet, the major ecohydrologic effect of the dam reservoir was the complete submergence of owen falls and ripon falls upstream of the dam. these falls were preferential sites for blackfly breeding, and their permanent submergence eliminated the breeding completely. in west africa, dam construction has led to a significant switch in dominant water-borne diseases. the volta river basin extends over six west african countries (43% in burkina faso, 42% in ghana, and 15% in togo, benin, cote d'ivoire, and mali) and covers an area of about 400 000 km 2 . the volta lake is the largest man-made lake in the world, created after the construction of the akosombo dam in 1965. the primary purpose of the project was to supply cheap electricity to smelt aluminum, other significant uses being transportation, fishery, water supply (commercial and domestic purposes), tourism, and irrigation. construction of the volta lake led to the resettlement of about 80 000 people from several hundreds of villages. in the other riparian countries of the basin, small and larges dams have been built by governments, nongovernmental organizations (ngos), and local people to secure food produc tion after the severe droughts that occurred in the 1970s and 1980s. in the nakambe sub-basin (burkina faso) alone, more that 600 small dams have been built, mostly during that period (barry et al., 2005) . there have been serious health issues associated with the volta lake, in particular with two major diseases: schistosomia sis and river blindness. before the creation of the volta lake, schistosomiasis was endemic in ghana; but endemicity was low along the volta river. according to an epidemiological survey done in 1960-61 before the lake was formed, infection rates of schistosomiasis in the area had been 1-5%, mostly affecting children. in the asukwakwaw area, north of the akosombo dam, the prevalence of onchocerciasis is now almost 90%. principal public health impacts of the formation of the lake have included reduced prevalence of river blindness, but increased incidence of urinary schistosomiasis and a mas sive increase in malaria, as discussed in the following paragraphs. the dam virtually halted the rate of flow in the volta river, increasing stagnant water conditions and consequently creating ideal breeding grounds for carriers of water-borne diseases. in the period following the construction of the dam at akosombo, there has been a steady decline in agricultural productivity along the lake and the associated tributaries (gyau-boakye, 2001). the land surrounding lake volta is not nearly as fertile as the for merly cultivated land residing underneath the lake, and intensive agricultural activity has quickly exhausted the already inade quate soils. without the periodic floodings that brought nutrients to the soil, before the natural river flow was halted by the dam, upstream agricultural systems are also losing soil ferti lity (van de giesen et al., 2001) . the growth of commercially intensive agriculture has produced a rise in fertilizer runoff into the river. this, along with runoff from nearby cattle stocks and sewage pollution, has caused eutrophication of the river waters (gyau-boakye, 2001) . this nutrient enrichment, in combination with the low water movement, has allowed for the invasion of aquatic weeds (cerratophyllum) (fobil and attaquayefio, 2003) . these weeds, associated with the aquatic snail, the 'intermediate host', together with mass migration into the fishing commu nities from regions in which the disease was endemic, have led to a great increase in the prevalence of schistosomiasis in many localities around the lake. the presence of aquatic weeds along the lake and within tributaries has resulted in even greater devastation to local human health as they provide an excellent habitat not only for snails but also for mosquitoes (gyau-boakye, 2001) . before the construction of the akosombo and kpong dams, malaria was not much of a problem along the swift-flowing volta river, but, when it became a stagnant lake, it became a greater public health problem in lakeside villages. by 1979, urinary schisto somiasis had increased to become the most prevalent disease in the area, affecting some 75% of lakeside residents (gitlitz, 1993) and reaching a prevalence rate of 90% among children in certain localities. the problem of schistosomiasis in the lake basin must be seen as embracing both the lake and the volta delta. the migratory habits of the fishermen ensure the spread of the disease from endemic areas to other areas. in particular, resettlement villages have showed an increase in disease pre valence since the establishment of lake volta, and a village's likelihood of infection corresponds to its proximity to the lake. previously, the population in the basin generally lived away from the main watercourses because of the threat from water borne diseases. children and fishermen have been especially hard hit by this rise of disease prevalence (zakhary, 1997) . additionally, the degradation of aquatic habitat has resulted in the decline of shrimp and clam populations. the physical health of local communities has declined as a result of this loss of shellfish populations, as they provided an essential source of dietary protein (fobil and attaquayefio, 2003) . conversely, while leading to a dramatic increase in schisto somiasis, the lake has flooded out the riparian forests which constituted a breeding place for a species of tsetse fly, glossina spp. from the palpalis group, the vector of protozoan trypanosoma brucei gambiense, which causes the western african trypanosomiases (sleeping sickness) in people. the lake has also inundated and eliminated the major breeding sites of the onchocerciasis blackfly in rapidly flowing streams and rivers north of the akosombo dam. the construction of the second dam at kpong also eliminated the breeding sites downstream of akosombo and therefore stopped the transmis sion of river blindness in the vicinity. the main benefit to health because of the construction of the akosombo dam in 1965 and the kpong dam in 1981 is undoubtedly the reduc tion of the incidence of onchocerciasis in the volta basin. about 60 000 fishermen living mostly in isolated villages around the lake were exposed to the riverine disease and did not have access to health facilities (jobin, 2005) . increased global demand for biofuels has incentivized sugar cane plantation in brazil, giving rise to several social and environmental modifications (martinelli and filoso, 2008) . the widespread occurrence of generalist animal species in sugarcane areas has been associated with public health pro blems. for instance, the population increase of the semiaquatic rodent, capybara (hydrochoerus hydrochaeris), in the piracicaba river basin has led to the spread of brazilian spotted fever (bsf) (labruna et al., 2004) . the bsf is the most important tick-borne disease in brazil and is caused by the bacterium, rickettsia rickettsii, and transmitted by the tick, amblyomma cajennense, its main vector, and capybaras serve as host for the ticks (estrada et al., 2006) . r. rickettsii infections can cause a wide range of clinical manifestations, ranging from asympto matic or mild febrile illness to overwhelming and fatal disease. failure in diagnosis and delayed therapy have contributed to hidden mortality, frequently a result of atypical fulminant forms of the disease (gonçalves da costa et al., 2006 ) and physician's lack of knowledge about the disease, which is exa cerbated by the difficulty of adequate confirmatory laboratory tests during its acute phase. mortality can reach 40% of the infection cases. the bsf has been known in brazil since 1929. during the period between 1940 and the 1980s there was a marked drop in the number of reported cases of bsf in brazil, as well as in the united states (angerami et al., 2006) . however, since the 1980s an apparent reemergence of the disease has been observed with an increase in the number of reported cases in the southeast of brazil (angerami et al., 2006) . rickettsial diseases have been considered emerging zoo noses worldwide (raoult and roux, 1997) and should no longer be classified as rare diseases in brazil. despite the still moderate number of bsf cases, its increasing trend, together with high mortality rates, reflects and calls atten tion to deep changes in land use and habitat structure in brazil. increased fertilizer use, pollution, and soil erosion have caused deterioration of aquatic systems. as colluvium sediments are transported downhill across the landscape from sugarcane fields, they are deposited onto wetlands, and into small streams, rivers, and reservoirs. deposition affects water quality, and ecosystem biodiversity (politano and pissarra, 2005) and functions. high rates of n export into rivers draining watersheds heavily culti vated with sugarcane in brazil, such as the piracicaba and mogi river basins, have been reported (filoso et al., 2003) . the indus trial processing of sugarcane for production of sugar and ethanol is another source of pollution for aquatic systems with poten tially harmful effects for human health. waste products (vinasse) are rich in organic matter, and increase the biochemical oxygen demand (bod) of waters receiving these effluents, often causing anoxia (ballester et al., 1999) . with the boom of ethanol pro duction in brazil in the early 1980s, new legislation was enacted to ban the direct discharge of vinasse into surface waters. since then, nutrient and carbon-rich vinasse has been mixed with wastewater from washing sugarcane and is recycled back to sugarcane fields as organic fertilizer (gunkel et al., 2007) , although this practice is still far from generalized. as a conse quence, high nutrient concentrations in these effluents also contribute to the problem by enhancing algal blooms and pro moting eutrophication of surface waters (matsumura-tundisi and tundisi, 2005) . the increase and dispersion of the capybara population and the associated health risk is most likely due to a strong anthro pogenic habitat modification caused by extensive monoculture. paradoxically, this situation could be aggravated by current efforts to restore aquatic ecosystems. typical capybara habitat is com posed of two main components: water and a patch of forest or woodland. in são paulo state, capybaras and ticks share a habitat component, the riparian vegetation called gallery forest. these forests form as corridors along rivers or wetlands and project into landscapes that are otherwise only sparsely wooded, such as savannas, grasslands, or deserts. the boundary between gallery forest and the surrounding woodland or grassland is usually very abrupt, with the ecotone being only a few meters wide. in são paulo state, capybaras shelter in the gallery forest and feed in the sugarcane fields adjacent to the ecotone ( figure 14) . thus, the abundant food and lack of predators in this new habitat have led to a strong population increase (labruna, 2009 ). in the 1950s, the capybara was considered in danger of extinction in são paulo state, where population can reach densities 60 times higher than in natural environments in some areas, such as the extended wetlands of pantanal (verdade and ferraz, 2006) . this offers optimum conditions for the increase in the tick and rickettsia population. further, the capybara is a protected species and is gradually adapting to aquatic urban habitats with a larger spectrum of possible vertebrate hosts for ticks ( figure 15 ). capybaras have been observed in the outskirts of são paulo city along the highly polluted pinheiros river (labruna, 2009) , which flows through the fourth largest metropolitan area worldwide, with ∼11 million inhabitants. presently, a substantial cleanup pro gram for this river is underway and an increase of secondary vegetation and fragmented, regenerated systems is expected. with this, a further expansion of capybaras, ticks, and bsf toward anthropogenically modified habitats, including urban and suburban areas close to water sources such as rivers and lakes, is likely. in addition, it has recently been reported (meireles et al., 2007) that capybaras in the são paulo state were infected by cryptosporidium parvum, which is a protozoan pathogen that causes a diarrheal illness called cryptosporidiosis, an acute short-term infection that can become severe and chronic in children and immunocompromised individuals. despite not being identified until 1976 (meisel et al., 1976; nime et al., 1976) , it is one of the most common water-borne diseases and is found worldwide, being spread by direct ingestion of con taminated water or food and through recreational water activities. the finding of zoonotic c. parvum infection in this figure 14 aerial photo of a sugarcane plantation and a small water body with highly fragmented remains of riparian forest, brazil. photo credit: geraldo arruda, jr. semiaquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies in brazil may be contaminated with cryptosporidium oocysts from wildlife. cryptosporidiosis is the most significant water-borne dis ease associated with the public water supply in western europe. when contamination occurs, it has the potential to infect very large numbers of people. some notable outbreaks of cryptos poridiosis have been associated with heavy rainfall events. in this chapter and elsewhere (despommier et al., 2007 and references therein) , there are indications that the boundaries between ecological systems play a role in some of the most important emerging infectious diseases, with a correspondence between ecotonal processes and the ecological and evolution ary processes responsible for zoonotic and vector-borne infections. terrestrial ecotones include forest-edge habitats, fragmented forest landscapes, and forest-grassland interfaces. terrestrial-aquatic ecotones are found in riparian habitats, riverine landscapes, freshwater and estuarine wetlands, and in the coastal zone. anthropogenic ecotones can include crop land/pasture-natural habitat and settlement-natural habitat, and a combination of these. processes in ecotones can contribute to the shifts or changes in hosts, vectors, or pathogens that produce disease emergence. these dynamics are associated with changes in land cover/use and with the changing nature of the land-water interface. anthropogenic influences can intensify ecotonal processes by increasing their geographic extent and overlap. various ecotone features can contribute to disease emergence. animals congre gate in ecotones. populations of species that normally are members of distinct ecological communities from different habitats or ecosystems overlap in ecotones, facilitating patho gen spillover. host-vector hyperabundance increases the potential for pathogens to achieve critical threshold density. enhanced dispersal conditions facilitate dispersal at a higher rate or over longer distances, along linear habitats defined by habitat edges, such as riverine or gallery forest, and flowing water in streams or rivers themselves. cropland-forest-river transitions appear particularly relevant as sensitive indicators of change in this context. for example, the transition between fragmented riparian habitats such as degraded gallery forests and, for example, sugarcane fields seems to be a priority sector for the control of rickettsia-related diseases since capybaras must cross the ecotone to feed on sugarcane, as described else where in this article. further, nutrient pollution, degradation of riparian habitat, and the loss of ecological functions involving assimilation of nutrients and pathogens, combined with high concentrations of domestic fowl and their waste, are commonly associated with human settlement/aquatic-terrestrial ecotones. the emer gence of ai involved the mixing of three different communities: wild migratory waterfowl (wetlands), wild local birds, and domestic fowl (ponds), and, later, also pigs. like influenza, the emergence of japanese encephalitis has involved transmis sion in the spatial area of overlap of human settlements, agriculture, and natural habitat (despommier et al., 2007) . while waterbirds and wetland habitat are implicated in influ enza, nonaquatic wild birds and irrigation systems provide the vector habitat for encephalitis. the intensification and expan sion of irrigated rice production systems in southeast asia over the past 20 years have made an important contribution to the spread of this disease, which is produced by a mosquito-borne virus (see table 5 ). the flooding of the fields at the start of each cropping cycle leads to a sizable increase in the mosquito population. domestic pigs and wild birds are reservoirs of the virus and transmission to humans may cause severe symptoms. japanese encephalitis is a leading cause of viral encephalitis in asia with 30 000-50 000 clinical cases reported annually (who, 2001d) . in general, research in ecotoneparticularly terrestrialaquatic dynamicscan provide vital information about changes in climate, river hydrology, sea level (cohen and lara, 2003) , and land use (lara et al., 2002) . a strengthened integration of ecological and biomedical monitoring is essen tial for successfully restoring ecological functions and enhancing environmental and human health. globally, the surveillance of locally and regionally relevant ecotones could provide evidence of disease emergence related to environmen tal change. in this context, restoration of lost ecological functions, such as nutrient sequestration by wetland creation or regeneration of riparian vegetation, must be accompanied by careful monitoring of other changes in the surrounding land scape and in the connectivity between basin processes and land use. created or regenerated aquatic systemsindependently of their purposewill increment existing ecotonal processes or generate new ones. thus, surveillance of the created/recovered/ enhanced system ecological functions should include at least those disease agents (host, vectors, or pathogens) , which according to the present knowledge would have a higher prob ability of proliferation under modified or changing conditions (e.g., mosquito larvae in temperate wetlands and snails in tropical regions). floods and droughts will intensify with climate change and affect health through the spread of disease resulting from habi tat modification, with high risk of rapid increase in diarrheal and other diseases (lipp et al., 2002; ipcc, 2007) . there are many pathways through which hydrologically relevant events can affect health; notably when a river or stream bursts its banks producing changes in mosquito abundance (malaria, and dengue), or contamination of surface water with human or animal waste such as, for example, rodent urine (leptospiro sis). flooding may become more intense with climate change and can result in the spread of disease. conversely, droughts can produce changes in vector abundance if, for example, a vector breeds in ponds left in dried-up riverbeds (noji, 1997; menne et al., 1999) . coastal ecosystems and their basins are rapidly changing due to anthropogenic pressure and global warming, also inducing changes in patterns of resource use (lara et al., 2002) . integrative, comparative approaches are needed for the understanding of functional links between basin structure; morphology of different estuaries, marshes, and mangroves; flooding and biogeochemical regimes (lara and cohen, 2006) ; pathogen life cycles; and disease incidence (wolanski et al., 2004; lara et al., 2009) . for improved prediction of the dispersal of inundation waters, formation of drought ponds, or preventive identifica tion of vulnerable locations or sectors that could be used as drainage areas, it is crucial to have a detailed knowledge of the regional and local topography. the elaboration of highresolution topographic models (dem) of basins in connection with hydrology, meteorology, and biogeochemistry data will also allow the assessment of vulnerability descriptors such as soil moisture potential, salinity, or organic matter content, which can be crucial state parameters for the development of microorganisms and/or disease vectors. however, precisely in tropical coastal areas, where the impact of climate change on vector-transmitted diseases is of high concern, there is frequently a lack of topographic informa tion with an adequate resolution for low-lying sectors. in vulnerable regions, the combination of risks to both food and water can exacerbate the impact of even minor weather extremes (floods and droughts) on the households affected (webb and iskandarani, 1998) . a methodological approach including wetland basin microtopography and its relation with inundation dynamics and estuarine biogeochemistry is necessary for vulnerability assess ment and risk management. the use of geographic information systems (giss) provides an excellent basis for network coopera tion at the interfaces between environmental and biomedical research, adding a critical componenthuman healthto coastal management research. this is a major concern for the who, which also has set a priority on the link between gis and disease surveillance (who, 2000) . through the joint who/united nations children's fund (unicef) program health map, specific gis software was developed for that purpose, combining a standar dized geographic database, a data manager, and a mapping interface. however, although these concerns are closely linked to the subject matter of ecohydrology, they are not usually included in interdisciplinary research projects dealing with, for example, coastal wetlands. clearly, the only way to concretely reduce vulnerability is to ensure that infrastructure is in place for the removal of solid waste and wastewater and the supply of potable water. no sanitation technology is safe when covered by floodwaters, as fecal matter mixes with floodwaters and is spread wherever the floodwaters run (lara et al., 2009) . consideration should also be given to the deterioration of groundwater quality caused by salinity intrusion due to climate change and rising sea levels (e.g., sherif and singh, 1999) . thus, as stated in section 10.12.1.1, such health issues clearly require a basin approach, that is, considering basins as a natural unit of territorial management. however, a usual shortcoming of gis-derived vulnerability studies is that data sources from official institutions are most frequently based on municipalities or counties as an administrative unit, whose limits do not necessarily coincide with basin boundaries. thus, this vision of the relationship between climate and sealevel change and effect on human health converges with zalewski's (2002) statement that: as a consequence, the issue of water quality at the basin scale cannot be resolved without a profound understanding of the effects of hydrology on biotic processes and of biota on hydrology. the frame work for developing the principles of ecohydrology is logically the water basin scale. (zalewski, 2002: 825) creation of wetlands for nutrient sequestration from surface waters requires the inclusion of measures for control of locally major and regionally relevant disease vectors such as snails or mosquitoes. the latter are relevant for disease transmission in several climatic zones besides the tropics, and global warming is widening their habitats with severe consequences for human health (e.g., the dengue outbreak in argentina in 2009) and will therefore be treated with some detail. early methods of managing salt-marsh mosquitoes have primarily focused on maximizing the reduction of mosquito populations, with mini mizing environmental impact as a secondary consideration. however, in the last few decades, there have been attempts to apply diverse water management models to marsh systems, especially in terms of vector control and habitat modification (dale and hulsman, 1990; wolfe, 1996) using programs with minimal environmental impacts. the success of these programs requires a thorough knowledge of mosquito developmental conditions, as well as potential impacts on adjoining ecosys tems. a deep knowledge of the local microtopography and tidal regimes is critical. marsh drainage and hydrological linkage to the tidal source are essential in the determination of what type of wetland occurs where, and for the development of appro priate wetland management measures. elevation of a few centimeters is more critical in the coastal wetlands than that of hundreds of meters in the mountains. in this section we cover not only well-established methods for mosquito control, but a series of successful methods for snail control derived from good agriculture practice, basically from techniques for rice cultivation. ecohydrology can contri bute to and learn from these experiences. water management, molluskicides, and chemotherapy have been the main instruments for preventing or treating schisto somiasis. biological control of snails through predators such as ducks, fish, turtles, crustaceans, water rats, leeches, and aquatic insects have been also used, as yet with very limited success. an interesting lesson on the potential for improved water management to reduce vector proliferation from the 1960s can be drawn from the experience in the baluchi irrigation scheme in then tanganyika (sturrock, 1965) . this system was remark ably clear of snails, although several species occurred in small numbers. several factors may account for this. first, the water flow in the canal system was very rapid when it was in use, but the canals were completely dried out in the dry season. second, silt and vegetation were dug out of the canals twice a year. third, a complex system of rice husbandry was used in which the rice fields were ploughed, manured, and subjected to a program of alternate drying and flooding. consequently, neither the canal system nor the rice fields contained much in the way of snail habitats. snails were confined to temporary pool sites, filled with rain or seepage water. all these schemes were built on land with an appreciable slope and with relatively porous soils. even when irrigation was in progress, the field canals were not always in use and dried out rapidly. while snails are often able to withstand limited periods of drought, it is unlikely that any large snail populations could develop under these conditions in any one season. furthermore, on five of these schemes, field canals were often re-routed from season to season so that the establishment of suitable snail habitats was rendered even more unlikely. mobarak (1982) reported that after 1970 prevalence of urinary schistosomiasis in upper egypt reversed a previous downward trend and began to increase again because of the shift from basin to perennial irrigation. however, in any case, increased use of parenteral antischistosomal therapy (pat, injections of antimony-based drugs) was bringing schistoso miasis under control in egypt. in the northern part of upper egypt, prevalence dropped from 29.4% in 1977 to 11.5% in 1983, while further south, prevalence reportedly fell from 26. 4% in 1980 4% in to 16% in 1983 4% in (who, 1985 . most experts agreed that applying the proper combination of sanitary engi neering, water control management, snail control, infection surveillance, and treatment drugs can avert adverse effects of irrigation on schistosomiasis. moreover, there was an impres sion that even without water control measures and environmental sanitation, chemotherapy with or without treat ing water to kill snails would adequately control schistosomiasis transmission. fenwick (1989) noted that in the newer rahad irrigation scheme, east of the gezira plain in the sudan, because of the use of drugs and snail control, the incidence of schistosomiasis remained very low, despite very poor sanitary conditions. it was predicted, however, that relax ing control measures would cause a surge in schistosomiasis. although oral drugs started to be used in egypt in the 1970s, pat use continued into the mid-1980s. praziquantel, which also has a high cure rate for s. mansoni, became available in egypt in 1982 and has been the treatment of choice there since the late 1980s. although the massive pat campaigns were successful in strongly reducing disease incidence, a study by frank et al. (2000) concludes that the intensity, widespread geographical coverage, and duration of the campaigns, together with unsafe injection practices (inappropriate sterilization pro cedures), have been responsible for the nationwide spread of hepatitis c in egypt in recent decades. the authors state that the enormous dimension of egypt's schistosomiasis problem and the sheer size of the antischistosomiasis effort, combined with the characteristics of pat, provided an effective mechanism for a massive increase and establishment of hepatitis c virus in the egyptian population. according to these authors, this is "the world's largest iatrogenic transmission of blood-borne patho gens known to date." moreover, it is probable that the heavy reliance on an effective chemical treatment also allowed the continuation of water management schemes that were contri buting to the maintenance of large numbers of snails in aquatic environments. the evolution of the schistosomiasis problem in egypt and sudan described above highlights the importance of developing water management programs able to keep vector proliferation under control. this lesson is also highly relevant to the construction of large-scale reservoirs and irrigation facil ities as in the case of the three gorges dam. in israel, biomphalaria alexandrina was eradicated through a combination of factors including chemical applications. as in all cases, snails return some time after the application of mol luskicides. some combined measures used for the control of snail vectors have been successful, such as increasing water currents to over 20 cm s −1 , rapid emptying and drying up of water reservoirs, and weekly deflection of infested water courses along different routes (saliternik, 1979) . as stated previously, the success of chemotherapy in treating this disease and of molluskicides for eradicating snails does not imply that preventive measures should not be taken for avoid ing vector proliferation based on knowledge of their ecohydrological setting. all vector snails require water, at least for breeding. the management of water bodies is therefore a potentially powerful control method. for example, in the case of rice cultivation there are opportunities for vector control by changing the aquatic habitat of the snail in a way compatible with maximum crop production. infection of humans mostly takes place not in the rice field itself, but in irrigation canals and surrounding living quarters. different hydrological and rice husbandry approaches have been used in various countries. however, generalizations should be made with care because each snail species has its own preferences and tolerances regard ing shade, water velocity, the steepness of canal walls, and drought tolerance. rice cultivation by itself can be used as an environmental method of snail control in that it brings about ecological changes that can reduce snail habitat. the philippines has promoted more intensive scientific methods of rice cultivation to control schistosomiasis (hairston and santos, 1961) . snail control is achieved at different stages of rice growing in a number of different ways, for example, by deep plowing, which turns over the soil and buries the snails; or by draining the ricefield at harvest and keeping it dry until the next crop, which kills the snails and prevents them from breeding. in japan, s. japonicum eradication was accomplished by treatment, sanitation, control of animal reservoir host, educa tion, and elimination of most of the snail colonies (garcia, 1988) . the steepness of the walls of irrigation channels was increased and later they were lined with concrete and main tained clear of silt, vegetation, and debris to supplement the eradication of snails in the ricefields, which resulted from intensive cultivation. extensive rice-growing areas in china's mainland have been cleared of oncomelania snails (garcia, 1988) . the measures taken have included digging new water channels parallel to the existing snail-infested streams and using the excavated soil to fill the old ones, clearing streambeds, and removing vegetation. where the soil structure permits, the banks have been made steeper. in the philippines, similar measures have been taken as in china; in addition, converting undrainable swampy areas into fishponds and improving agricultural prac tices have successfully controlled snails in limited areas. thus, an integrated approach to drainage problems can result in increased production while reducing health risks. in the ecohydrology approach, it is essential to consider the whole basin in management policies, especially when a disease agent can be transmitted by different species of the same host (in this case a snail) that is differentially distributed along altitude gradients in river basins. both b. truncatus and planorbarius metidjensis are intermediate hosts of s. haematobium in southwestern morocco. a basin investigation (yacoubi et al., 2007) in five rivers identified sites colonized by these species and compared the habitats in which they were found. the p. metidjensis was observed in the upper valleys of three rivers, whereas b. truncatus was found in sites of lower altitude. a component analysis demonstrated that altitude (from 4 to 1380 m), water ph (from 5.9 to 9.2), and electric conductivity (from 120 to 6020 μs cm −1 ) were the main descriptors of environment. the p. metidjensis was associated to high altitude and low electric conductivity. however, b. truncatus was asso ciated to being found in lower altitude sites with medium electric conductivity in water. it has been mentioned above that periodic canal or field drying had been used successfully for snail control. nevertheless, before a control plan is adopted and implemen ted, the effects of drying out have to be monitored and thoroughly understood for each snail species: snails that are capable of undergoing diapause can circumvent unfavorable environmental conditions, including long periods of drought. cooper et al. (1992) found that diapause influenced the sus ceptibility of biomphalaria glabrata snails to s. mansoni infection. juvenile snails exposed just prior to diapause, or immediately following a diapause period of 3 weeks, were highly susceptible to infection by s. mansoni miracidia. however, snails that underwent diapause produced compar able or only slightly fewer cercariae than did nondiapausing snails. these studies indicate that diapause in b. glabrata does little to decrease a snail's ability to act as an intermediate host for s. mansoni or to interrupt the development of the parasite. for these reasons, great attention should be given to diapausing snail populations when planning programs for mollusk control. thus, for snail control, it is important that not only the agricultural field but also irrigation and drainage channels, as well as the water source, be considered part of the agroecosys tem. this parallels the ecohydrology approach that considers the whole basin, from the river source to the wetlands, estuar ine, and coastal zone as an integrated management unit. both agricultural and ecohydrological models should converge in a new synthesis integrating their own tools with practices based on traditional knowledge, socioeconomical cost-benefit analysis of vector eradication, and agri-and/or aquacultural production. the type of wetland management approach, particularly when restoration or creation is planned, will require previous surveys of the mosquito species and their habitat types, locally and at basin level. in the following sections, we present a summary of main species/habitat combinations for different hydrological settings extracted from anonymous (2009b) . relatively few mosquito species breed in running waters, such as streams. larvae can be flushed out when stream volume increases, and to remain in the stream requires a large amount of energy. the tropical genus, chagasia, and some anopheles species are stream breeders. stream breeders will find vegeta tion along banks with which to anchor themselves or attempt to remain away from the main flow of the stream by seeking isolated eddies. transient water sources, such as flooded areas and ditches, are used as breeding grounds for species whose eggs can with stand desiccation and whose life cycles require alternating periods of wet and dry, such as aedes and psorophora. the quality of transient water changes with time, which can result in a succession of different species using the same pool. transient waters include woodland pools created by spring rains (aedes stimulans), fresh floodwater (aedes canadensis), and tidal floodwater (aedes sollicitans). permanent or semipermanent waters support characteristic aquatic vegetation. cattail, rushes, and sedges are typical fresh water swamp vegetation. genera associated with permanent water are anopheles, culex, culiseta, coquillettidia, and uranotaenia, whose eggs are not desiccant resistant and must be laid directly on the water. aedes adults will oviposit near the edge of the swamp, or within tussocks of vegetation, requiring later flooding of the eggs for hatching. as with transient waters, there are seasonal changes in the vegetation, water quality, and mosquito species present. permanent waters can include fresh water swamps, such as, for example, tussock (aedes abserratus) or cattail swamps (coquillettidia perturbans), as well as brackish water swamps with salt marshes (culex salinarius). besides nat ural environments, polluted water with floating debris can be a habitat for species such as culex pipiens. container water habitat can be found in both natural set tings, such as water held by plants to artificial settings and water found in tires. container water is characteristically clear and many container species now also use artificial sites as they provide insulation against the weather and are more numerous (aedes aegypti and aedes albopictus). increasing dengue incidence in not only tropical but also subtropical countries requires a thorough elimination of such urban, man-made microhabitats. there are various techniques for mosquito control based on different principles. all involve modification of the hydrologi cal setting, ranging from total wetland drainage to an increase in their tidal flooding. a summary of these methods, including parallel grid ditching, open marsh water management, and runneling, is presented in the following paragraphs. parallel grid ditching consists in the physical removal of water from intertidal marshes and was one of the first largescale forms of mosquito control (lesser (2007) ; figure 16 ). parallel grid ditches were dug in salt marshes, spacing these ditches about 45 m apart to remove standing surface water where mosquitoes might breed. extensive ditching programs for mosquito control in north america were only moderately effective, since many breeding potholes were not drained dry, and there were long-term nega tive effects on wildlife and salt-marsh ecosystems. many nonmosquito breeding wetlands that provided wildlife habitat were unnecessarily drained and salt-marsh vegetation commu nities were changed into fragmented wetlands. birds were particularly affected through the draining of larger natural ponds. by the early 1960s, there was an increasing awareness of wetland values and functions, and the value of parallel grid ditching was questioned. understanding of the drawbacks of the parallel grid-ditching technique led to the development of a new mosquito control source-reduction technique called open marsh water manage ment (omwm). it started in the late 1960s and was further optimized until the early 1980s. the goals of omwm are: (1) control of salt-marsh mosquitoes; (2) reduction of insecti cide applications; and (3) habitat enhancement for salt-marsh fish and wildlife (ferrigno and jobbins, 1968; hruby et al., 1985) . the omwm method involves the selective installation of small, shallow ponds and interconnecting ditches superim posed on known mosquito-breeding habitats ( figure 17 ). this aims to eliminate wet-dry-wet cycles necessary for determined species and any newly created permanent water habitats are unattractive for mosquito egg deposition. this simultaneously improves habitats for mosquito-eating larvivorous fishes which can quickly invade, via tidal flooding, any newly created omwm pond or ditch. scattered mosquito breeding depressions and sheetwater habitats are connected through pond and ditch excavations to allow unimpeded water flow and predatory fish movement, while isolated potholes are often filled with natural soils to eliminate these smaller-sized breeding depressions (lesser, 2007 ; figure 17 ). the increase of tidal inundation frequency and predation by fish significantly reduce mosquito density. runneling is an effective method for controlling mosqui toes that breed in intertidal salt marshes through a type of habitat modification using shallow channels. this technique is based on omwm principles (wolfe, 1996) . it increases tidal frequency to a marsh and removes surface sheet water from low-lying areas high on the marsh. runnels are linked to the tidal source, promoting tidal exchange between graded regions of the marsh. they are conceived to allow transport of lowamplitude tides to areas of salt marsh in a way so that pools do not form, even after spring tides. runnels are shallow figure 17 the omwm system, involving the selective installation of small, shallow ponds and interconnecting ditches superimposed on known mosquito-breeding habitats. (<30 cm deep) spoon-shaped channels constructed along nat ural drainage lines on the salt marsh ( figure 18 ) to a maximum gradient of 1:1000 (hulsman et al., 1989) . due to the slight slope, runnels enable slow water movement even during lowamplitude tides. the net result is a reduction in mosquito breeding areas, the modification of pools and edges for egg conditioning (the process involving flooding and drying events that prepares mosquito eggs for hatching), and larval development. there are few apparent negative impacts at the modified site (hulsman et al., 1989; dale and hulsman, 1990; dale et al., 1993; latchford, 1997) . further, like omwm, they allow water to drain from trap pools and permit predatory fish to gain access to the mosquito larvae, at least during high tide. in comparison to grid ditching and omwm, runneling is an environment-oriented approach to salt-marsh management for mosquito control that aims to alter the salt marsh as little as possible, while causing significant reductions in mosquito numbers. the main difference between the three approaches lies in the magnitude of the habitat modification. ditching involves the greatest alteration to the marsh, and runneling the least. runneling has a lesser effect on the estuarine environ ment as a whole than does either ditching or omwm. 10.12.5 conclusions 10.12.5.1 some reflections on dams, water scarcity, ecohydrology, and health although the construction of reservoirs is controversial, the rising demand for water by an increasing human population makes more dams inevitable (jobin, 1999) . in a review, morley (2007) calls attention of the fact that most parasitological studies in relation to reservoir construction have been focused on schistosomiasis and other tropical diseases of humans (stanley and alpers, 1975; jobin, 1999) . in comparison, the impact of reservoir construction on indigenous aquatic parasite fauna of wildlife has been a subject largely ignored by the scientific community. however, reservoir formation can have profound effects on the parasite fauna of fish, birds in the reservoir, as well as up-and downstream of it. changes in host-parasite relationships and switches between animal and human hosts can occur (morley, 2007 and references therein) . the role of parasites in environmental monitoring is increas ingly recognized (lafferty, 1997; lafferty and kuris, 2005) . thus, the surveillance of the effects of reservoirs on parasite fauna of aquatic wildlife may provide important general infor mation on both short-and long-term changes that occur within and downstream of new reservoirs during the maturation pro cess of the reservoir. the above-described cases that show the effect of dam con struction on onchocerciacis and schistosomiasis call attention to the need to evaluate changes in flow power and velocity downstream of proposed dams, in order to assess their likely health impact. ecohydrological measures are required to improve the operation of existing dams to provide more effective control of disease vectors (see section 10.12.2.2.3). the modeling of the effect of controlled flooding pulses on the plankton dynamics in the guadiana estuary (wolanski et al., 2006) is an outstanding example of the potential of the appli cation of ecohydrological principles for the control of toxic algal blooms. in arid and semiarid regions, the quality of water in artificial reservoirs is essential to human health, particularly in climati cally unstable regions. for example, recent el niño/la niña-southern oscillation teleconnections have produced a pro longed drought of ∼3 years in south argentina. this has interrupted a period of about 30 years of rainfall significantly above the historical average and produced a decline to critical levels in the reserves of drinking water reservoirs, which like other water bodies in the region, have suffered from recurrent summer algal blooms (kopprio et al., 2008) . however, the perception that cyanobacteria can represent a threat to human health only in connection with acute events such as blooms should be widened to consider the possibility of neurologic damage as consequence of chronic exposition to toxins such as bmaa via long-term ingestion of water or aquatic fauna. this should be taken into account when developing schemes for preventing cyanobacterial blooms by manipulating natural predator communities. the reduction of the nutrient load input to the water reservoir probably remains the best preven tive measure to prevent harmful algal blooms. water treatment should be regularly checked and improved to remove the organisms and their toxins from drinking-water supplies, where appropriate. water treatment by flocculation and sedi mentation, followed by sand filtration, is supposed to remove live cyanobacterial cells and debris. however, there is evidence that plants that are not working properly can actually increase cell counts of algae with potential toxicity (in this case, anabaena circinalis and microcystis aeruginosa) in the treated water (echenique et al., 2006) . throughout this chapter, examples have been referred to of actual and potential conflicts of interest between alternative uses of aquatic systems, for example, between the desire to restore degraded wetlands and the need to protect health of the human population living nearby. a relevant example of policy conflicts in an industrialized country is the experience of the tennessee valley authority (tva), where health concerns in the past gave rise to management measures that conflict with modern recreational interests (bos, 1999) . the meeting report of the ninth meeting of the who/fao/united nations environment programme (unep) panel of experts on environmental management for vector control in 1989 includes the following passage: as national and regional priorities change, so do policies, and there must therefore always be a provision for their reconsideration and modification. sometimes, though, the acute problems that led to the original priority setting might have become latent rather than have disappeared completely, and while public awareness and political pressure favour a policy change, the original goals of such policies should not be ignored. this was well illustrated by the water man agement policies including mosquito control established by the tva in the 1930s. the standards of mosquito control maintained by tva equalled those maintained in privately owned river impoundments under prevailing public health regulations. the measures included the programmed fluctuation of water levels in the reservoirs, a practice that played a key role in reducing anopheles populations and eradicating malaria transmission from the valley. new uses of the reservoirs, including recreation and the promotion of nature conservation had led to a conflict of interests. for recreation, stable water levels during summer and early autumn were required; con servation of certain fish species and of waterfowl required higher water levels in spring to promote fish spawning and the rapid growth of aquatic vegetation for the fowl. such changes in water manage ment regimes would without doubt result in increased mosquito populations, yet the potential risk for the reintroduction of vectorborne disease was not appreciated after three generations of malaria free experience. in recent years much interest has been directed towards the protection and establishment of wetlands, without pay ing sufficient attention to their mosquito breeding potential. consequently, tva has been faced with a conflict of new policy directives concerning wetlands, existing mosquito control policies and state regulations for impounded water. the use of constructed (artificial) wetlands for the treatment of domestic waste water and its processing for reuse is of particular concern, since these could pro duce large quantities of potential disease vectors and they are often sited close to populated areas (peem/who, 1991: 17-18). this stresses the necessity of harmonizing policies of reservoir management for vector control with other policies concerned with land use patterns, to ensure that areas of potential risk, such as artificial wetlands, are planned away from areas of human habitation. presently we face a complex environmental situation that seems to be changing at a much faster rate than our current capacity to revise and renew our intellectual schemes or to generate integrated management structures capable to enhance both ecosystem and human health. take, for example, the case of influenza virus. the loss of wetlands around the globe may force many wild birds onto alternative sites like farm ponds and paddy fields, bringing them into direct contact with domestic fowl and humans and providing greater opportu nities for the spread of the h5n1 virus (anonymous, 2006) . poor planning in response to development pressures has led to the increasing loss or 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vincent resh has leveraged science and diplomacy to help bring an epidemic under control along 30,000 miles of west african rivers. breakthroughs magazin, college of natural resources geographical distribution of schistosomes and their intermediate hosts. epidemiology and control of schistosomiasis (bilharziasis) overview of disease fact sheets habitats of bulinus truncatus and planorbarius metidjensis, the intermediate hosts of urinary schistosomiasis, under a semiarid or an arid climate factors affecting the prevalence of schistosomiasis in the volta region of ghana ecohydrology -the use of ecological and hydrological processes for sustainable management of water resources impact of the three gorges dam construction on transmission of schistosomiasis in the reservoir area relationship between the transmission of schistosomiasis japonica and the construction of the three gorge reservoir key: cord-256537-axbyav1m authors: kimball, ann marie title: emergence of novel human infections: new insights and new challenges date: 2016-10-24 journal: international encyclopedia of public health doi: 10.1016/b978-0-12-803678-5.00153-3 sha: doc_id: 256537 cord_uid: axbyav1m novel human infections have continued to emerge over the past decade. their presentation, epidemiology, and microbiology have shifted the paradigms of traditional science. in particular insights into nongenetic or paragenetic mechanisms (plasmid mediated), modes of infection have challenged biology. in reviewing the new challenges posed by these emergent events, new technologies promise some answers; however, global health security against pandemic threats, particularly given the uneven distribution of global resources for prevention, detection, and response, remains a critical area of challenge. new human infections have continued to come forth over the last decade. this discussion will focus on the period from 2006 to 2016. given the importance of the severe acute respiratory syndrome (sars) as the harbinger of coronaviruses such as the middle east respiratory syndrome (mers), some discussion of sars is also included. the phenomenon of emerging infections has been constant with the majority of human infections reflecting the introduction into humans of zoonotic pathogens. over the last decade, great progress has been made in defining more fully how emergence occurs. in fact the emergence of new infections has expanded the paradigms of microbiology in a number of ways, which will be highlighted here. specifically: (1) it is now well appreciated that influenza can migrate directly from avian sources to humans, and the appreciation of the actual directness of 'species jumping' has moved forward; (2) new infections have also introduced uncertainty in transmission dynamics with emphasis on super-spreader events as well as nosocomial transmission; (3) infectious particles are not confined to those organisms which contain genetic material; (4) a new paradigm such as 'planetary health' may be necessary for defining these trends; and (5) global preparedness and response is not in place for the next pandemic. for further background information, the reader is referred to the original article on this phenomenon (kimball, 2008) . a general discussion of antimicrobial resistance and xenotransplantation is not included in this discussion as they were thoroughly covered in the original article. antimicrobial resistance will be covered in detail in another article of this volume. nonetheless they continue to be important considerations. while it has long been appreciated that the majority of human pathogens arise from zoonotic sources, the directness of the path from animal to human has become clear. ideally detecting pathogens in animals could be seen as a harbinger for human outbreaks. in fact this has been a working hypothesis of major us agency for international development (usaid) funding to their pandemic agents program. however, recent outbreaks of influenza, sars, and mers coronavirus (cov) demonstrate how emerging infections are shifting this longstanding paradigm. they lend further urgency to this area of research. in addition, these outbreaks have challenged our understanding of transmission dynamics. empirical observation has shown that there is not a single transmission force 'number' which completely characterized the spread of infectious diseases. this further complicates efforts to control infection within populations. in 2009, a cluster of high mortality influenza cases was detected in mexico. an observational study of 899 patients hospitalized in mexico between late march and 1 june 2009 showed that pandemic (h1n1) disproportionately affected young people. fifty-eight patients (6.5% of those hospitalized) became critically ill, with complications including severe acute respiratory distress syndrome and shock. among those who became critically ill, the mortality rate was 41% (domínguez-cherit et al., 2009) . the pandemic and its management have been the subject of an in-depth analysis commissioned by the world health organization (who), and that analysis proved prescient in more recent global outbreak events such as ebola (who, 2005) . this cluster heralded a global pandemic and the declaration by who of a public health emergency of international concern (phiec). it occurred in the context of a decade of planning for a potential h5n1 outbreak, which included who updating its pandemic guidance (pandemic influenza preparedness and response, 2009 ). in 1997 a small but lethal cluster of influenza occurred in children in a hong kong nursery. of the 18 cases, 6 died (mounts et al., 1999) . science shifted its understanding of influenza and recognized that flu could come directly from birds to humans. in fact h5n1, which emerged in 2003, acquired the popular moniker of 'bird flu.' the outbreak was curtailed through active surveillance, changes in practice, and poultry culling of live markets in hong kong. however, the outbreak proved a harbinger of more widespread recognition of 'bird flu.' h5n1 was identified through active surveillance of poultry and wild birds; waterfowl were particularly affected. human cases were sporadic but carried a very high mortality rate (estimated at 60%) (webster et al., 2006) . although the virus proved difficult to transmit to humans, case fatality was high. as of february 2011, about 500 laboratory-confirmed human cases had been reported to the who from 15 countries; about 60% of reported cases were fatal. the spread of h5n1 was geographically broad with extension into nigeria and throughout southern and se asia in waterfowl and in domestic fowl. extensive planning for a potential pandemic was put into place given the apparent proximity of the threat and the high mortality in humans. veterinary and human health collaboration was a central precept of this planning in the 'one health' concept (heymann and dixon, 2013) . the h5n1 threat did not materialize despite the high level of circulation in birds. preparedness in se asia in particular included tabletop exercises, culling of birds through rapid response, and at least one joint investigation carried out between laos and thailand. in this setting, h1n1 ran its course, with a milder clinical syndrome (although it is estimated that the global death toll was 284 000). the pandemic of h1n1 precipitated some confusion as well as competition for access to antivirals and vaccines. in the aftermath of these contentious discussions, new policy initiatives were put into place. this included the 'pandemic influenza preparedness' (pip) (who) plan which took some years to negotiate. it balances access to new viral strains for vaccine production with access to these vaccines for poor countries. the fineberg commission to examine an 'after action' performance of the international health regulations (ihr) was central to potential reform as well. this will be more fully discussed below. in 2013 a new influenza virus, h7n9, was isolated from patients in the people's republic of china. isolates were both from birds and from humans. although 571 cases occurred globally with 212 deaths, international spread was limited to two countries. the national response to this epidemic was very strong which is a key to control (discussed below). the 2009 influenza pandemic also followed another global respiratory emergency, sars. the etiology of sars (a new coronavirus) was not known in late 2002 when cases and deaths began to occur in guangdong province of the people's republic of china. initially misdesignated as a chlamydial pneumonia early in the course, public alarm mounted as antibiotics proved futile in treatment. between november 2002 and july 2003, a total of 8427 probable sars cases were reported from 29 countries with 813 deaths for a mortality rate of 9.6% (mmwr, 2003) . sars effectively demonstrated the potential mobility of respiratory pathogens. while sars was declared poorly transmissible, it effectively jumped continents in travelers and infiltrated populations through nosocomial spread. in contrast to influenza, which is highly transmissible, sars still requires direct exposure to bodily fluids for transmission. however, sars shifted the scientific paradigm of zoonotic transmission more fullywith incrimination of wet markets vending civet cats initially thought to be the source. this initial assessment has proved less robust in terms of 'reservoir' for new human pathogens as discussed below. sars also challenged our understanding of transmission dynamics. for reasons that remain unclear, a single infected person who spent a single night in a hotel in kowloon resulted in geographically broad transmission. the concept of a 'superspreader' was popularized (lloyd-smith et al., 2005) . this phenomenon was also described in a second scenario in beijing (shen et al., 2004) . traditionally infection has been characterized by a 'reproductive rate' which is essentially the rate at which an infectious case replaces itself. if the rate is 1, then there will be no spread, as the infection will simply stay the same absolute number as the index case recovers. however, if the rate is higher (i.e., >1), then spread into the population will occur as a single case infects multiple individuals so the number of cases increases. super-spreaders seem to have high infection rates although the overall rate for the pathogen in question may be low. this has, of course, changed our assumptions for modeling disease spread in populations. following the 2009 h1n1 pandemic (and the avian influenza emergence of 1997) and the sars pandemic of 2003, a new virus emerged on the arabian peninsula. mers-cov is a new human pathogen that also causes pneumonia and respiratory distress. the story of mers is less history and more present in terms of defining its epidemic potential. it reinforces the need for 'one health' collaboration between veterinarians, clinicians, and public health specialists, making use of their relative expertise. mers was first reported in 2012 as a case report of a patient in an icu with severe respiratory disease from jordan (hijawi et al., 2013) . at the time of this writing, 1321 cases and 466 deaths have been reported to the who, with an average case fatality ratio of 35%. in one epidemiological analysis, the case fatality ratio for primary cases was 74% (95% ci, 49-91), whereas for secondary cases, it was 20% (95% ci, 7-42) (alsolamy, 2015) . like its coronavirus cousin, sars, mers-cov has demonstrated agile spread within hospitals (oboho et al., 2015) and across continents. in 2015 a large outbreak occurred in south korea (cowling et al., 2015) . that outbreak was characterized by a mortality rate of about 20% among 186 cases, 33 were fatal. it also featured 'superspreading' events. in fact a single case housed in the emergency room with persistent cough was linked to 81 cases in one hospital (kucharski, 2015) . to summarize, the recent episodes of respiratory infectious diseases related to influenza, sars-cov, and mers-cov have demonstrated increasingly direct links between animal and human infections, agile intercontinental geographic spread, and complex transmission dynamics including 'superspreader' events. transmission within health-care settings has also been a prominent feature. these characteristics have challenged traditional assumptions about the pathogenesis and epidemiology of infectious diseases. traditionally microbiology has held that microbes (bacteria, viruses, protozoa, fungi, etc.) are organisms that replicate through genetic mechanisms. that replication is a major factor in the illness in humans these agents cause. recent emerging infectious events have brought an additional complexity into that decades' old assumption. a new form of a human neurodegenerative disease that emerged in britain in the 1990s was linked to the emergence of bovine spongiform encephalitis (bse) in cattle ('mad cow disease'). this link was demonstrated through multiple casecontrol studies. the biological proof of a common etiology came somewhat later (hill et al., 1997) . prions are not microbial life in the traditional sense. they are 'autocatalytic proteins' or proteins that make change. in the instance of 'transmissible spongiform encephalopathies' (tses) of mad cow disease (bse), sheep 'scrapie,' elk chronic wasting disease, as well as in the human diseases of creutzfeldt-jakob disease (cjd), new variant creutzfeldt-jakob disease (vcjd) and kuru, these changes occur in the central nervous system. while the new vcjd epidemic related to ingestion of infected beef from bse-affected cows has waned due to enhanced global surveillance and animal husbandry practices, research into prion disease continues. it appears that despite their lack of genetic material, prions do undergo mutation and strain diversification in response to selective pressures (collinge et al., 2007) . soberingly, it is believed that some humans (estimated 1 in 4000 britons) silently carry pathogenic prions for many years. this risk persists and creates safety concerns for blood transfusions and organ transplantation. the story of bse demonstrated the limitation of the traditional assumption that genetic reproduction of pathogens is necessary for infection as outlined above. the uk beef industry had historically been a relatively stable one when fragmented among many smaller farms across the british isles. to protect this industry, the government maintained a tariff on the imports of competing products from abroad. with the explosion of global trading in beef after world war ii, coincident with refrigerated transport, and the movement toward global free trade, the united kingdom negotiated a timetable under the general agreement on tariffs and trade to scale down tariffs on beef, which heightened competition in the uk beef industry and increased pressure for more efficient and less costly production methods. against this backdrop, innovation in rendering was introduced into the slaughterhouses of the united kingdom. the rendering or processing of carcasses of cows and other animals after the edible and usable bits of flesh and meat have been cut away has been done for centuries. and for decades, uk farmers used the meat and bone meal from rendering as a protein source for beef cattle. historically, the rendering process was similar to pressure cookingapplying very high temperatures for very long times so eventually even the bones broke down into powder. it was an expensive, fuel-consuming, and timeconsuming process. when a new cold vacuum extraction method of rendering was introduced requiring lower temperatures (i.e., less energy) and less time, it seemed a win-win situation considering the increasing pressure on the uk beef industry in the face of global competition. but sometime after the new rendering practice was introduced into the united kingdom, the prion disease known as mad cow disease emerged. the new process, discovered by uk beef industry, was not effectively disinfecting for prions. existence of prion disease was unknown prior to its dramatic emergence, first in cows and then in people. the context is important to appreciate. somehow the streamlining of the rendering process played a role. british scientists tested the new process by deliberately introducing animals with mad cow disease and assaying the resulting meat and bone meal product. they found that the newer rendering process does not remove the infection, whereas the older process did. zika virus emerged in sub-saharan africa in the late 1940s. it is a close cousin of dengue virus and in the same family as yellow fever. all of these viruses are transmitted to humans through the bite of a mosquito. all of these diseases are clinically mild, but occasionally severe causing fever, rash, and joint pain. unfortunately all of these diseases have become globally distributed. zika is the most recent arrival in the americas. it has circulated in asia for some time (chen and hamer, 2016) . while clinically relatively mild, epidemiologic studies in brazil have suggested a link between zika virus and severe birth defects in newborns. viral infection in early pregnancy appears to be associated with microcephaly, which is associated with profound brain injury in newborns. again, the emergence of a new infectious disease is pushing the boundaries of biomedical knowledge. in the absence of certain prevention or treatment options, the government of brazil has gone as far as advising women not to become pregnant (mcneil, 2015) . the emergence of antimicrobial resistance has continued to be a major concern. in 2008 a new enzyme in gram-negative bacteria was detected which caused broad antibiotic resistance. this enzyme was produced by mobile genes that travel on plasmids. plasmids are small circular dna (genetic) packages, which are distinct from the dna of the bacteria itself. these are mobile, with the ability to be taken in across classes of bacteria if and when they confer a selective advantage for survival, as in the case of antimicrobial resistance. the new enzyme ndm-1 (new delhi metalloproteinase-1) was traced to an infection that occurred in patients who had been treated in india. a more complete study of the epidemiology of this enzyme in gram-negative bacterial isolates from the subcontinent suggested a broad range of gram-negatives were affected (kumarasamy et al., 2010) . this new biology, which was first described for extended spectrum beta lactamase (esbl) resistance in the 1990s, has provoked renewed concern in antimicrobial resistance. 2011 was declared by the who as the year to address antimicrobial resistance. the pharmaceutical pipeline is bereft of new antibiotics to address this challenge. chillingly another new panresistant plasmid has been reported from china, which is resistant even to the last line of defense, the polymixin class of antimicrobials (liu et al., 2015) . this report included isolates from pigs at slaughter, retail pig and chicken meat, and humans. again, the specter of intensive agriculture fostering new biological threats calls for careful study. the isolates were largely from areas of intensive porcine agriculture in china. figure 1 indicates sites sampled for polymixin plasmid resistance. the question of where new infections lurk in nature has been an active area of research. as noted above, the initial reservoir for sars coronavirus was thought to be civet cats which are domestically raised for food in china. however, further investigation has suggested that there may be a single host involved in many emergent diseases. in an elegant demonstration of interdisciplinary research, han et al. (2015) have recently published a persuasive article on the role of bats in emerging infections. whether or not the theses of the article prove true with further research, the work provides additional evidence of how extremely powerful interdisciplinary research can be toward solving the puzzles of emergent diseases in humans. in discussing the spillover of viruses from bats to humans, the authors write: factors that contribute to the intrusion of bats into human living environment can be summarized into a 'push' and a 'pull' (brüssow, 2012) . a 'push' refers to the enormous demand for more space and resources brought by the human population explosion, which leads to the destruction of bat habitats and shortage of food. natural environmental changes, such as typhoons and droughts, can also place stresses on bats. a 'pull' involves the living environments built by humans, characterized by urbanization, intensive agriculture and food animal breeding, which attracts bats into human living environments for an abundant of food supply. so coming full circle, this discussion highlights the critical importance of further understanding these events of 'species jumping' or 'spillovers,' given the pressures will only augment rather than abate. infections potentially housed in bats as their natural reservoir (from han and colleagues) is shown in table 1 . it is an impressive array, which brings us to consider how indispensible further understanding in these interactions is to gain insight into emergent human infections. so what explains the apparent increased pace of emergence of new human infections? is it simply that we are more able, with our new technologies, to detect them? or are there forces at work that are fostering this trend? the stories of bse and nvcjd recounted above were outlined because the united kingdom, although a small country, has excellent epidemiologic, statistical, laboratory, and clinical acumen. while cases of bse and nvcjd were not confined to the united kingdom, the origin of emergence was tracked and described relatively efficiently. but, what of influenza? the potential contribution of intensive poultry and swine agriculture to the emergence of influenza a emergence was outlined in my earlier article for this publication. research over the past decade has continued to provide evidence of this risk. as intensive practices have spread to developing countries, the assurance of biosecurity has become less sure as noted in the discussion (above) of polymixin resistance through plasmids in pigs and humans. while many new strains of influenza arise in wild birds (particularly waterfowl), the link to human populations appears to be through domestic poultry (leibler et al., 2009) . however, other anthropogenic mechanisms are also at play. without the global 'mobile' environment of travel and trade, emergence would remain a largely local phenomenon. however, as we saw with the influenza discussion above that is not the case. nor was it the case with sars or with mers-cov. international tourism surpassed 1.1 billion arrivals in 2013 according to the world bank (world bank international tourism). after a slump in global trade during the global recession exports of food in 2014 reached nearly $1.5 trillion in value for selected countries where data were available. this, of course, does not include all of global trade, which has surpassed $3 trillion in value (merchandise trade by product). guarding against transcontinental transmission in food or through human travel is a complex undertaking, with safeguards at the source the most likely answer. however, research, testing, and demonstration of effective measures remain wanting. finally, climate change, largely attributed to human activity, seems to be readjusting the boundaries of mosquito borne diseases. the recent incursion of zika into brazil is attributed to the el niño weather pattern now in force in that geography. while el niño oscillation is not directly attributable to human activity, the shifting of such natural oscillations and their severity may well be. in his landmark 1993 book planetary overload: global environmental change and the health of the human species, anthony j. mcmichael outlines the human-generated stress on natural systems (mcmichael, 1993) . he posits that food will become increasingly scarce for the human community. the macroecologic effects of human activity on climate, water, food, agriculture, pollution, and human health are well described, but the systematic link between the macrolevel (what we can see) and what is occurring on the microlevel remains an important area of research. to address the emergence of new pathogens, we need more precise knowledge about the mechanisms that form the critical pathway to emergence. a follow-up report to the landmark iom report, emerging infections, microbial threats to health: emergence detection and response was published in 2003 (smolinski et al.) (microbial threats to health) . additional factors of emergence were examined in this report: human susceptibility to infection, climate and weather, changing ecosystems, poverty and social inequity, war and famine, the lack of political will, and intent to harm. thus the original 6 factors grew to 13. while enriching the discussion and description of emergence, this proliferation of factors also created overlapping domains within factors; for example, climate and weather are an integral physical science aspect of ecosystems, the failure of political will is integral to the neglect of public health systems, and so forth. from an analytic point of view, the need for in-depth study of factors and how they actually work has become critical for scientific insight into public health protection. mcmichael has suggested the term 'the anthropogenic epoch' to describe our contemporary situation. in other words, human kind is changing the nature of our environment in unprecedented ways. more recently the rockefeller-lancet commission has suggested the new scientific discipline of 'planetary health' as a unifying concept to bring the disparate strands of life sciences, and ecology more closely together to foster interdisciplinary research and insight (the rockefeller foundation, 2015). the key to success will require intense investment in transdisciplinary research which brings in disparate databases and talents into risk analysis. while a full discussion is beyond the scope of this discussion, a number of new tools have come into use that allow more rapid diagnosis and response to newly emergent outbreaks. a few will be highlighted here. in addition to formal disease surveillance reporting a number of informal diseases, surveillance networks have arisen among countries which share common borders or work within a common economic bloc. these networks facilitate the flow of information across borders and allow collaborative investigations, tabletop exercises, and resource sharing on an ongoing basis. in postconflict areas such as the mekong basin, they contribute to health security (gresham et al., 2013) . increasing sophistication of bringing disparate data sets together and creating models to understand possible scenarios has brought additional insight into prevention and control efforts for emerging diseases. prediction of where outbreaks are most likely to occur remains a very imperfect science (jones et al., 2008) . retrospective niche modeling has brought additional insight into how different factors may interact to foster outbreaks (daszak et al., 2013) . laboratory diagnosis of unknown agents has also advanced. during the sars outbreak, the who convened an informal network approach to fully sequence the new agent in 1-month time (david, 2004) . one group has put forward a vision of bringing full genomic sequencing into the laboratories of developing countries (aarestrup et al., 2012) . clearly the ability to quickly diagnose new agents without the necessity of culture would be an important advance. as importantly the integration of informatics linking genomic analyses to phylogenetic metadata would allow global tracking of agents. as the boundaries of microbiology are stretched with new insights from emergent diseases, the ability of the people of the world to protect against pandemics is more important than ever. global traffic and trade continue to grow, confounding national approaches with their international span. in 2005 (following sars) the who adopted the ihr (international health regulations). this is the only law with the force of an international treaty, which is in place to govern the conduct of countries during global security emergencies. the ihr outline 'core capacities' for national-level protection. it is not the optimal level for an emergent pandemic, but it is the minimum considered essential for global health security. the ihr implementation was to have been completed by all member countries by 2012. however, implementation has languished with only one-third of countries implementing and many not disclosing their status of implementation to the who in 2015. after the 2009 influenza pandemic, an independent expert panel was convened to assess how the ihr functioned. that panel, lead by dr harvey fineberg, then president of the institute of medicine, was explicit in outlining the gaps in global health security. the world is ill-prepared to respond to a severe influenza pandemic or to any similarly global, sustained and threatening public-health emergency. beyond implementation of core public-health capacities called for in the ihr, global preparedness can be advanced through research, reliance on a multisectoral approach, strengthened health-care delivery systems, economic development in low and middle-income countries and improved health status. fineberg (2014) . in 2013, ebola (a known infection) emerged in guinea. at the time of its appearance, none of the countries in sub-saharan africa had implemented the core capacities of the ihr (kimball and heymann, 2014) . the outbreak went on to create pandemonium in the three most affected countries (guinea, liberia, and sierra leone) killing over 11 000 people (and afflicting more than 28 000) (ebola-situation). infection was introduced in other countries, but onward transmission was limited. following ebola, the international systems are again under review. initial observations remain distressingly similar to those made in 2009. with reform of the un and who, once again underway, it will be important to follow through. of particular importance as highlighted above is the interdisciplinary (and in the case of the un interagency) nature of prevention, detection, and response to emergent threats. despite the new technological tools, the ecological factors in emergence are ever gathering force. clearly additional efforts are required. emerging infections remain an intersectoral challenge with every indication they will 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findings from a retrospective investigation the same prion strain causes vcjd and bse global trends in emerging infectious diseases ebola, international health regulations and global safety factors influencing the emergence of new (and "old") disease, international encyclopedia of pubic health superspreading in mers emergence of a new antibiotic resistance mechanism in india, pakistan and the uk: a molecular, biological and epidemiological study industrial food animal production and global health risks: exploring the ecosystems and economics of avian influenza emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in china: a microbiological and molecular biological study superspreading and the effect of individual variation on disease emergence planetary overload global environmental change and the health of the human species zika virus, a mosquito-borne infection, may threaten brazil's newborns global health microbial threats to health: emergence, detection, and response by committee on emerging microbial threats to health in the 21st century update: severe acute respiratory syndrome -worldwide u.s case-control study of risk factors for avian influenza a (h5n1) disease, hong kong mers-cov outbreak in jeddah-a link to health care facilities a who guidance document. world health organization the rockefeller foundation-lancet commission on planetary health safeguarding human health in the anthropocene epoch: report of the rockefeller foundation-lancet commission on planetary health h5n1 outbreaks and enzootic influenza report of the review committee on the functioning of the international health regulations the author wishes to thank ms kellie creamer for her assistance in preparing this manuscript.see also: antimicrobial resistance in a one health and one world perspective -mechanisms and solutions; arboviruses; ebola and other viral hemorrhagic fevers; global health law: international law and public health policy; influenza. key: cord-282878-8qgsq2km authors: fignani, daniela; licata, giada; brusco, noemi; nigi, laura; grieco, giuseppina e.; marselli, lorella; overbergh, lut; gysemans, conny; colli, maikel l.; marchetti, piero; mathieu, chantal; eizirik, decio l.; sebastiani, guido; dotta, francesco title: sars-cov-2 receptor angiotensin i-converting enzyme type 2 (ace2) is expressed in human pancreatic β-cells and in the human pancreas microvasculature date: 2020-10-23 journal: biorxiv doi: 10.1101/2020.07.23.208041 sha: doc_id: 282878 cord_uid: 8qgsq2km increasing evidence demonstrated that the expression of angiotensin i-converting enzyme type 2 (ace2), is a necessary step for sars-cov-2 infection permissiveness. in the light of the recent data highlighting an association between covid-19 and diabetes, a detailed analysis aimed at evaluating ace2 expression pattern distribution in human pancreas is still lacking. here, we took advantage of innodia network eunpod biobank collection to thoroughly analyse ace2, both at mrna and protein level, in multiple human pancreatic tissues and using several methodologies. using multiple reagents and antibodies, we showed that ace2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing β-cells. ace2 is also is highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. by using different ace2 antibodies we showed that a recently described short-ace2 isoform is also prevalently expressed in human β-cells. finally, using rt-qpcr, rna-seq and high-content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increases ace2 expression in the β-cell line endoc-βh1 and in primary human pancreatic islets. taken together, our data indicate a potential link between sars-cov-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct β-cell virus tropism. the expression of molecules that act as receptors for viruses determine tissue-specific tropism. sarscoronavirus 2 (sars-cov-2), that leads to the respiratory illness coronavirus disease 2019 (covid19) , uses its surface envelope spike glycoprotein (s-protein) to interact and gain access to host cells through the angiotensin-i converting enzyme-2 (ace2) receptor. as such, s-protein-ace2 binding is the key determinant for virus entry, propagation and transmissibility of covid-19-related disease (1, 2) . artificially induced ace2 de-novo expression in ace2-negative cell lines is a necessary step to sars-cov and sars-cov-2 infection (3, 4) . sars-cov-2 does not enter cells that do not express ace2 and does not use other coronaviruses receptors, such as aminopeptidase n (apn) and dipeptidyl peptidase 4 (dpp4), thus being fully dependent on ace2 presence in host cells (5) . additional host co-factors, such as transmembrane protease tmprss2, cathepsin b/l and furin protease, have been shown to enhance efficiency of sars-cov-2 cell entry by processing the s-protein and eliciting membrane fusion and syncytia formation (6) . the central role played by ace2 in sars-cov-2 infection has been further supported by evidence that sars-cov-2 infection is driven by ace2 expression level (6) . sars-cov-2 mainly targets cells of the nasal, bronchial and lung epithelium, causing respiratory-related symptoms; however, growing evidence shows that other tissues can also be infected. several reports indicate a wide, although variable, distribution of ace2 expression patterns among different tissues (7-9), thus underlining a potential different virus infection susceptibility among cell types. the fact that covid-19 disease may lead to multiple organ failure (10, 11) shows the crucial relevance for understanding the molecular mechanisms of host cell factors used by sars-cov-2 to infect their target tissues. 4 recent studies showed that older adults and those with chronic medical conditions like heart and lung disease and/or diabetes mellitus are at the highest risk for complications from sars-cov-2 infection. of importance, a yet unresolved conundrum relies on the recently hypothesized bidirectional relationship between covid-19 and diabetes mellitus (12, 13) . this concept is supported by reports in which impaired glycaemic control is associated with increased risk of severe covid-19. indeed, elevated blood glucose concentrations and deterioration of glycaemic control may contribute to increased inflammatory response, to abnormalities in the coagulation system and to impairment of ventilatory function, thus leading to severe covid-19 disease and to a worse prognosis (14) . interestingly, acute hyperglycaemia has been observed at admission in a substantial percentage of sars-cov-2 infected subjects, regardless of the past medical history of diabetes (15) (16) (17) (18) . the same observations were previously made in sars-cov-1 pneumonia during 2003 sars epidemic (19) . a recently published case report described autoantibody-negative insulin-dependent diabetes onset in a young patient who was infected by sars-cov-2 seven weeks before diabetes symptoms occurrence (20) . additional previous studies further support such observation (21, 22) . this indicates the possibility of a link between sars-cov-2 infection and new-onset diabetes through potential direct infection of pancreatic islets or additional indirect mechanisms. indeed, an in-vitro infection model of human pluripotent stem cells derived β-cells exposed to sars-cov-2 (23) showed permissiveness of these pre-β-cells to the virus. however, whether fully mature primary beta-cells or other cells of the human pancreas are indeed permissive to sars-cov-2 infection remains to be clarified. to address this question, we screened the ace2 expression pattern in human pancreata obtained from adult non-diabetic multiorgan donors and in the insulin-producing human β-cell line endoc-βh1, using different methodologies, multiple reagents, and publicly available or in-house generated rna sequencing datasets. our data indicate that ace2 is expressed by pancreas microvasculature, by scattered ductal cells and by a subset of human β-cells. these different cell types are thus potentially 5 prone to sars-cov-2 infection. we also identified a differential distribution of the two recently discovered ace2 isoforms (24, 25) . exposure of endoc-βh1 human beta-cell line and human pancreatic islets to pro-inflammatory cytokines significantly increased ace2 expression. taken together, our data suggest a potential link between sars-cov-2 infection and new onset diabetes, which deserves further investigation based on long-term follow up of patients recovered from covid-19 disease. 6 2 materials and methods human pancreatic sections analysed in this study were obtained from pancreata of brain-dead adult non-diabetic multiorgan donors within the european network for pancreatic organ donors with diabetes (eunpod), a project launched in the context of the innodia consortium (www.innodia.eu). whole pancreata were processed following standardized procedures at university of pisa. formalin fixed paraffin embedded (ffpe) pancreatic tissue sections and frozen oct pancreatic tissue sections were obtained from n=7 adult non-diabetic multiorgan donors, and from n=1 longstanding t1d donor pancreas (table s1 ). in innodia eunpod network, pancreata not suitable for organ transplantation were obtained with informed written consent by organ donors' next-of -kin and processed with the approval of the local ethics committee of the pisa university. human pancreatic islets were obtained from n=4 non-diabetic multi-organ donors (table s1) . briefly, purified islets were prepared by intraductal collagenase solution injection and density gradient purification, as previously described (26) . at the end of the isolation procedure, fresh human pancreatic islets preparations were resuspended in cmrl culture medium (cat. 11-530-037, thermofisher in order to evaluate the staining pattern of ace2 in human pancreatic tissues, we analyzed ffpe sections (7-μm thickness), prepared by using a microtome (cat. rm2125 rts -leica microsystems, wetzlar, germany) and baked overnight at 37°c, from two different portions of pancreatic tissue for each multiorgan donor (listed in table s1 ). after deparaffinization and rehydration through decreasing alcohol series (xylene-i 20min, xylene-ii coverslip allowing them to dry. a negative control with only secondary antibody incubation (no primary antibody control sample) was also included in order to exclude potential background artifacts generated by the secondary antibody or the enzymatic detection reaction ( figure s1a ). in order to further evaluate ace2 expression in pancreas sections, the same ace2 ihc protocol was applied to other 2 primary antibodies anti-human ace2: monoclonal rabbit anti-human ace2 (cat. all the 3 primary antibodies anti-human ace2, with respective secondary antibodies, were also used to perform a positive control staining in ffpe human lung sections (7-μm thickness), in order to double check the specificity of the primary antibodies. 12 immunofluorescence staining for ace2-insulin-glucagon and ace2-cd31 counterstained with dapi and then mounted as described above. cultured endoc-βh1 cells were immunostained for ace2 and insulin as follows. cytokines-treated or untreated cells were fixed in 4% pfa for 10 min, washed for 10 min in 0.1 mol/l glycine, permeabilized in 0,25% triton-x-100 for 5 min and blocked in 3% bsa+0.05% triton-x100 in pbs and were incubated with goat anti-mouse-488 1:500 in 1% bsa in pbs1x without ca 2+ and mg 2+ , or with goat anti-rabbit-488 diluted 1:500 in 1% bsa in pbs1x without ca 2+ and mg 2+ . cells were counterstained with dapi and then mounted as described above. images were acquired using leica tcs sp5 confocal laser scanning microscope system (leica microsystems, wetzlar, germany). images were acquired as a single stack focal plane or in z-stack 15 mode capturing multiple focal planes (n=40) for each identified islet or selected representative islets. sections were scanned and images acquired at 40× or 63× magnification. the same confocal microscope setting parameters were applied to all stained sections before image acquisition in order to uniformly collect detected signal related to each channel. colocalization analysis between ace2 and insulin and between ace2 and glucagon were performed using lasaf software (leica microsystems, wetzlar, germany). the region of interest (roi) was drawn to calculate the colocalization rate (which indicates the extent of colocalization between two different channels and reported as a percentage) as a ratio between the colocalization area and the image foreground. evaluation of the signal intensity of ace2 expression in human pancreatic islets of eunpod donors was performed using the lasaf software (www.leica-microsystem.com). this software calculates the ratio between intensity sum roi (which indicates the sum roi of the greyscale value of pixels within a region of interest) of ace2 channel and area roi (µm 2 ) of human pancreatic islets. both in colocalization and intensity measurement analysis, a specific threshold was assigned based on the fluorescence background. the same threshold was maintained for all the images in all the cases analysed. cultured endoc-βh1 cells were immunostained for ace2 and insulin as reported above. cytokinestreated or untreated cells were fixed in 4% pfa for 10 min, washed for 10 min in 0.1 mol/l glycine, permeabilized in 0,25% triton-x-100 for 5 min and blocked in 3% bsa+0.05% triton-x100 in pbs spot intensity" minus "spot background intensity") (31). total rna of endoc-βh1 cells and of pancreatic human islets exposed or not to ifnα or to il-1β + ifnγ for the indicated time points was obtained and prepared for rna sequencing as described (32) (33) (34) (38) . genes were considered significantly modified with a fdr < 0.05. total proteins from endoc-βh1 cells were extracted using a lysis buffer (20 mm chalfont, buckinghamshire, uk-rpn2232). spectrometer (thermofisher scientific), equipped with electrospray (esi) ion source operating in positive ion mode. the instrument is coupled to an uhplc ultimate 3000 (thermofisher scientific). the chromatographic analysis was performed on a column acquity uplc waters csh c18 130å (1 mm x 100 mm, 1,7 µm, waters) using a linear gradient and the eluents were 0.1% formic acid in water (phase a) and 0.1% formic acid in acetonitrile (phase b). the flow rate was maintained at 100 μl/min and column oven temperature at 50°c. the mass spectra were recorded in the mass to charge (m/z) range 200-2000 at resolution 35k at m/z 200. the mass spectra were acquired using a "data dependent scan", able to acquire both the full mass spectra in high resolution and to "isolate and fragment" the ten ions with highest intensity present in the full mass spectrum. the raw data obtained were analyzed using the biopharma finder 2.1 software from thermofisher scientific. the elaboration process consisted in the comparison between the peak list obtained "in silico" considering the expected aminoacidic sequence of human ace2 protein (uniprot id: q9byf1), trypsin as digestion enzyme and eventual modifications (carbamidomethylation, oxidation, etc.). ace2 proximal promoter sequence was retrieved from ensembl genome browser database the ncbi geo accession number for rna sequencing data reported in this paper are: gse133221, gse108413, gse137136. results were expressed as mean ± sd. statistical analyses were performed using graph pad prism 8 software. comparisons between two groups were carried out using mann-whitney u test (for nonparametric data) or wilcoxon matched-pairs signed rank test. differences were considered significant with p values less than 0.05. 21 3 results to determine the ace2 protein expression pattern in human pancreatic tissue, we first performed a colorimetric immunohistochemistry analysis to detect ace2 on formalin-fixed paraffin embedded (ffpe) pancreatic sections obtained from seven (n=7) adult non-diabetic multiorgan donors collected by the innodia eunpod biobank (table s1 ). to specifically detect ace2 protein in such context, we initially used a previously validated monoclonal anti-human ace2 antibody (r&d mab933) (7) which passed the validation criteria suggested by the international working group for antibody identified three main cell types positive for ace2 (figure 1, panel-a to -f ). in the exocrine pancreas there was a marked and intense staining in a subset of vascular components (endothelial cells or pericytes) found in inter-acini septa (figure 1, panel-a and -b) . we also identified ace2 positive cells in the pancreatic ducts, even though only some scattered cells with a clear ace2 signal were detected (figure 1, panel-c and -d) . of interest, we observed a peculiar ace2 staining pattern in the endocrine pancreatic islets, showing a diffuse ace2 signal in a subset of cells within islet parenchyma ( figure 1 , panel -e and -f; figure s2 ). however, the observed ace2 expression in islets was lower than the expression observed in microvasculature, the latter representing the main site for ace2 expression in the pancreas. in all cases analysed, including different blocks of the same case, a similar expression pattern of ace2 was observed, even though a certain degree of variability in terms of ace2 staining intensity within the islets was noted (figure s2 ). 22 the highest signal of ace2 within the pancreas was observed in putative association with microvasculature (figure 1, panel-a and -b) . of note, in such context, a lobular staining pattern of microvasculature associated ace2 was evident, as demonstrated by the presence of positive cells in certain lobules and low or null expression in other lobules of the same pancreas section (figure 2a) . ace2 staining pattern in inter-acini septa suggested an overlap with cells associated to microvasculature, most likely endothelial cells. in order to explore such possibility, we performed a double immunofluorescent staining on pancreas ffpe sections for ace2 and the endothelial cell specific marker cd31. the results showed that ace2 signal is associated, but not superimposed, to the cd31-specific one, thus resembling the tight association of pericytes to endothelial cells and strongly suggesting the presence of ace2 in microvasculature pericytes ( figure 2b) . in order to confirm the ace2 cellular distribution observed in the pancreas using mab933 antibody, we tested two additional anti-ace2 antibodies from abcam: ab15348 and ab108252 (see resources table) . according to the informations obtained by r&d and abcam, while mab933 and ab15348 are reported to recognize the c-terminus portion of ace2 (18-740aa and 788-805aa, respectively), ab108252 is specifically designed to react with a linear peptide located in the n-terminal ace2 protein sequence (200-300aa). it has been recently reported that a 459 aa short-ace2 isoform (357-805aa of ace2 + 10aa at n-terminus) can be co-expressed alongside the full-length ace2 protein (1-805aa) ( figure 3a ) (24, 25) ; the short-ace2 misses part of the n-terminal region targeted by ab108252 antibody. we observed ace2 islet-related signal using both mab933 ( figure 3b ) and ab15348 ( figure 3c ), but the ab108252 antibody did not show any positivity within the islet parenchyma ( figure 3d) , raising the possibility that the most prevalent ace2 isoform within pancreatic islets is the short one. of note, the three antibodies tested showed ace2 positivity in the microvasculature, thus suggesting a putative differential distribution of the two ace2-isoforms in the human pancreas. 23 as a positive control for our immunohistochemistry method and ace2 antibodies adopted, we evaluated ffpe lung tissue sections. as previously shown (7, 41, 42) , we observed scattered positive cells (putatively at2 pneumocytes) in the alveolar epithelium both using mab933 and ab15348 ( figure s3a and s3b) . in contrast, we did not observe any signal using ab108252 ( figure s3c) . collectively, these results indicate that the same staining pattern were obtained by using 2 out of 3 antibodies that may recognize both ace2 isoform (short-ace2 and long-ace2) thus confirming: (i) a high ace2 expression in microvasculature pericytes; (ii) rare scattered ace2 positive ductal cells; (iii) diffuse though weak ace2 positive staining in a subset of cells within human pancreatic islets. using both mab933 and ab15348 we observed ace2 signal in pancreatic islets which suggests that ace2 is expressed in endocrine cells. therefore, we sought to determine which pancreatic islet cell subset contributes to ace2 signal in such context. to do so, we performed a triple immunofluorescence analysis on the same set of ffpe pancreatic sections of non-diabetic multiorgan donors, aimed at detecting glucagon-positive α-cells, insulin-positive β-cells and ace2 signals (figure 4 and figure s4 ). using r&d mab933, ace2 preferentially overlapped with the insulin-positive β-cells (figure 4a, panel-a to -m), being mostly colocalized with insulin and low/not detectable in α-cells (figure 4a, panel-e, -f, -l , -m). such staining pattern was observed in all cases and was consistent between two different ffpe pancreas blocks of the same case ( table s2) . as expected, ace2-only positive cells within or around pancreatic islets were also observed ( figure 4a, panel g) , potentially indicating the presence of ace2-positive pericytes interspersed in the islet parenchyma or surrounding it. intriguingly, in the β-cells a major fraction of ace2 was observed in the cytoplasm and partially overlapped with insulin positive signal, while in a subsets of them only a minor fraction of the ace2 signal was attributable to several spots located on plasma membrane (figure 4a ; figure s5a red arrow, and figure s5b ). in microvasculature pericytes, the ace2-positive signal was mainly observed in plasma membrane ( figure 4a , panel g; figure s5 green arrow) as previously described (43) . there were also some ace2-negative β-cells (figure s5, white arrow) . colocalization rate analysis between ace2-insulin and ace2-glucagon, performed on a total of 128 single pancreatic islets from seven different adult non-diabetic cases, confirmed the significant preferential expression of ace2 in β-cells compared to α-cells (colocalization rate: ace2-ins 57.6±19.3% vs. ace2-gcg 6.8±5.4 % p<0.±0001) ( figure 4b and figure s6a ) the comparison of colocalization rates between ace2-insulin and ace2-glucagon among all cases analysed, confirmed the consistent preferential expression of ace2 in β-cells in comparison to α-cells ( figure 4b) . these results were confirmed when comparing different blocks of the same case (table s2) . there was however heterogeneity in terms of the ace2-insulin colocalization rate among different islets (ace2-ins colocalization rate range: 0.6 -91.4%). such heterogeneity was also highlighted by the presence of rare ace2-negative pancreatic islets in the same pancreas section. inter-islets heterogeneity was also clearly observed regarding ace2 islet-related signal intensity analysis ( figure 4d ). of note, some cases showed a lower ace2-insulin mean colocalization rate and islet-ace2 signal intensity compared to the other ones (figure 4d ), thus suggesting a high degree of heterogeneity among cases also in terms of islet-ace2 expression. no significant correlation between ace2-islets signal intensity and age, bmi or cold-ischemia time were observed in our donors cohort ( figure s6b ). 25 using western blot (wb) and immunofluorescence analysis, we explored the expression of ace2 in the human β-cell line endoc-βh1, a model of functional β-cells for diabetes research (27, 44) . to do so we used r&d mab933, abcam ab15348 and ab108252 antibodies, as previously done in the above described pancreas immunohistochemistry experiments. in wb analysis, mab933 revealed the presence of a prevalent 50 kda band corresponding to the short-ace2 isoform; use of this ab showed the brightest signal in immunofluorescence staining among the three antibodies tested (figure 5a ). abcam ab15348 worked better in wb for the recognition of both ace2 isoforms, and indicated that the most prevalent ace2 isoform present in human β-cells is the short-ace2 (50 kda, blue arrow) ( figure 5b ). in contrast, ab108252 recognized only the long-ace2 isoform (>110 kda-red arrow) ( figure 5c ). of note, the results obtained through wb analysis are in line with the immunofluorescence signal which revealed that ab108252 only stained a minor fraction of endoc-βh1 and the obtained signal was mainly found on the plasma membrane ( figure 5c, panel-b) . conversely, ab15348 and mab933, which recognized both ace2 isoforms, showed a higher signal and a different subcellular localization respect to ab108252 (figure 5a, 5b, panel-b) . mab933 ace2-insulin double immunofluorescence staining confirmed the main punctuate and likely granular cytoplasmic ace2 signal which also partially overlapped with insulin-positive secretory granules ( figure 5d, panel -a to panel -h) . in addition, we also observed some spots putatively localised on the plasma membrane ( figure 5d ). of note, the specificity of ace2 mab933 signal observed in endoc-βh1 was orthogonally tested in comparison to hela cells which showed very low/absent ace2 mrna expression ( figure s7a ) and resulted indeed negative for ace2 in immunofluorescence ( figure s7b ). an additional evidence of the presence of ace2 in endoc-βh1 was provided by the shotgun proteomic analysis, aimed at detecting specific peptides derived from ace2 protein independently of the use of 26 specific antibodies. by this independent approach, we observed the presence of both n-terminal and c-terminal unique ace2-derived peptides (figure s8) , which further confirmed the presence of the ace-2 protein in human β-cells (supplementary file 1a, 1b) . to confirm the ace2 expression in human islets, we also evaluated its transcriptional activity both in collagenase-isolated and in laser-capture microdissected (lcm) human pancreatic islets, by measuring its mrna expression using taqman rt-real time pcr. in order to avoid detection of genomic dna, we used specific primers set generating an amplicon spanning the exons 17-18 junction of ace2 gene, thus uniquely identifying its mrna ( figure s9a ). of note, the selected amplicon is shared between short and long-ace2 isoforms thus identifying total ace2 mrna. first, as a positive control we analysed total ace2 expression in rna extracted from a lung parenchyma biopsy tissue (figure 6a and 6e) . collagenase-isolated human pancreatic islets obtained from four different non-diabetic donors pancreata (table s1) showed ace2 mrna expression, as demonstrated by rt-real-time pcr raw cycle threshold (ct) values, reporting a ct range between 28-29 ( figure 6b and 6f) . since human pancreatic islets enzymatic isolation procedures may induce some changes in gene expression (45), we microdissected human islets from frozen pancreatic tissues obtained from five non-diabetic multiorgan donors recruited within innodia eunpod network (46) and evaluated ace2 mrna levels. the lcm procedure ( figure s9b ) allowed us to extract high quality total rna ( figure s9c ) from human pancreatic islets directly obtained from their native microenvironment, thus maintaining transcriptional architecture. ace2 mrna expression in lcm27 human pancreatic islets showed a consistent expression among cases, similar to isolated islets, as shown by ace2 mrna raw ct and normalized values (figure 5c and 5g) . finally, we analysed total ace2 mrna expression in the human β-cell line endoc-βh1. analysis of ace2 mrna expression in these cells demonstrated a similar expression level in comparison to human pancreatic islets (figure 3d and 3h) , with raw ct values ranging from 28 to 30. in order to determine whether metabolic or inflammatory stress conditions modify pancreatic endocrine β-cell expression of ace2, we exposed the human β-cell line endoc-βh1 and isolated human pancreatic islets to metabolic or inflammatory stressors and subsequently evaluated ace2 expression levels. exposure to palmitate (2mm palmitate for 24 h) did not significantly modulate ace2 expression ( figure 6a ). in line with these observations, neither primary human islets exposed to palmitate (47) nor human islets isolated from patients affected by type 2 diabetes (48) (figure 6a) . the same results were confirmed through immunofluorescence analysis aimed at measuring ace2 protein levels and subcellular localization in endoc-βh1 exposed or not to the same proinflammatory condition ( figure 28 6c). indeed, we observed a significant increase in ace2 mean intensity values upon cytokine treatment, confirming the upregulation of ace2 protein as well ( figure 6e) . these results were confirmed using an automated micro-confocal high content images screening system (31) which allowed us to measure ace2 intensity in cytokine-treated vs not-treated endoc-βh1 cells ( figure 6d and figure 6f ). in support of the observed increase of ace2 upon pro-inflammatory stress, rna sequencing data analysis of endoc-βh1 cells exposed to il-1β+ifnγ (48h) or to ifnα (18h) further confirmed such increase ( figure s10a ) expression in endoc-βh1 cells treated with il-1β+ifnγ or with ifnα, respectively. importantly, the same expression pattern was observed also in human pancreatic islets exposed to the same cytokines mix, as demonstrated by a 2.4 and 5.1 fold-increase in ace2 mrna expression following il-1β+ifnγ or ifnα treatment respectively (p<0.0001) (table s3 and figure s10b ). additionally, in order to strengthen such observations, we focussed on the ace2 gene promoter by analysing its upstream sequence (-1000 bp), from ace2 transcriptional start site (tss). using two different transcription factors (tf) binding motifs databases, we found several binding sites for tfs related to cytokine signalling pathways such as stat1 or stat3 (figure s11) , thus reinforcing our results of an association between inflammation and ace2 expression, and confirming what previously reported (65) . however, the analysis of ace2 expression distribution in ffpe pancreas sections from a t1d longstanding donor (see table s1 ) did not show remarkable changes in the levels or distribution of ace2 in infiltrated islets ( figure s12 ). this is in line with rnaseq analysis of whole islets from two t1d patients and four controls, which indicated similar ace2 expression (rpkm 1.2-1.7 in all cases) (49) . analysis of additional recent-onset t1d donors is needed to evaluate potential changes in ace2 expression in β-cells of highly infiltrated pancreatic islets. 29 collectively these results demonstrate that ace2 is upregulated upon in vitro exposure to early and acute inflammatory, but not metabolic, stressors both in endoc-βh1 and in human pancreatic islets. 30 in covid-19 disease, clinical complications involving the metabolic/endocrine system are frequently observed. these include critical alterations of glycaemic control in diabetic patients and new-onset hyperglycaemia at admission in individuals without previous clinical history of diabetes. although multiple causes have been indicated for covid-19-related hyperglycaemia, a recently published case report described autoantibody-negative insulin-dependent diabetes onset in a young patient who was infected by sars-cov-2 seven weeks before occurrence of diabetes symptoms. this suggests a potential effect (direct or indirect) of the sars-cov-2 infection on the pancreatic islet insulin producing β-cells, but additional evidence is needed to allow solid conclusions. previous studies suggested that ace2, the human host cell receptor for sars-cov-2 and sars-cov, which in other tissues has been shown to be a necessary component for infection permissiveness (6, 50) is expressed in pancreatic tissue (19) . however, an in-depth analysis aimed at evaluating ace2 expression pattern distribution in human pancreas is still lacking. here, we adopted multiple technologies and reagents to thoroughly analyse presence of ace2, both at mrna and protein level, in order to evaluate its expression and localization in pancreatic tissue samples obtained from adult non-diabetic multiorgan donors from the innodia eunpod biobank collection, in enzymatic-and lcm-isolated primary adult human pancreatic islets and in human β-cell line endoc-βh1. in human adult pancreas, we primarily observed ace2 expression in microvasculature component (endothelial cells-associated pericytes, both in endocrine and exocrine compartments). the expression of ace2 in the pancreatic microvasculature compartment was associated, but not superimposed, to the endothelial cells specific marker cd31 (or pecam-1). such staining pattern strongly suggests the presence of ace2 in pancreatic vascular pericytes which are tightly associated to endothelial cells. 31 of interest, although the exocrine pancreas and the pancreatic islets are highly vascularized (51) , only a subset of pancreatic pericytes cells markedly express ace2. additionally, ace2 expression in microvascular compartment is surprisingly lobular, resembling the heterogeneous staining pattern of several inflammatory markers. additional observations on multiple pancreatic lobules are required to confirm such heterogeneous lobular patterning observed. the presence of ace2 in pancreatic pericytes is of sure interest. as a matter of fact, the vascular leakage and endothelitis were reported as a typical sign of sars-cov-2 infection in various organs, driving early local inflammation and the subsequent exacerbation of immune responses (52, 53) . of note, multiple studies addressed the importance of an intact islet microvasculature in order to render pancreatic islets fully functional (54, 55) . therefore, a pancreatic islet local vascular damage and inflammation due to sars-cov-2 direct infection of ace2 + pancreatic pericyte cells is a potential contributory factor for islet dysfunction. of note, two recent preprint manuscripts also indicated the presence of ace2 expression in pancreatic microvasculature (56, 57) . our results indicate that ace2 is expressed also in the pancreatic islets and this expression is mostly located in β-cells as compared to α-cells. these results are in contrast with the two recent preprint manuscripts (56, 57) which failed to observe ace2 expression in pancreatic islet endocrine cells. such discrepancies may be explained by a resulting sum of differences in primary antibodies sensitivity, different epitopes targeted, tissue sections preparation and pre-treatment, as well as immunodetection methodology sensitivity (immunohistochemistry vs. immunofluorescence). such variables may be of critical importance when detecting a low-expressed protein and may generate different results. furthermore, it should be taken into consideration that ace2 expression may vary greatly among individuals due to genetic or environmental factors (58) . such intrinsic ace2 variability has been previously observed also in other cellular contexts, with some authors reporting high ace2 levels and others low or absent (59). 32 in our study, localization of ace2 in pancreatic islet endocrine cells was observed using two out of three different antibodies tested. surprisingly, we were not able to observe ace2 pancreatic islets positive signal using abcam monoclonal antibody ab108252, while signal was clearly evident in microvasculature and scattered ductal cells. however, these results are in line with kusmartseva et al 2020 (57) . of note, ab108252 monoclonal antibody specifically targets an epitope located in the nterminal domain of ace2 protein (200-300aa) which is missing in the recently discovered truncated ace2 isoform (short-ace2, 357-805aa) (24, 25) , thus being capable to recognize only the long-ace2 isoform. on the contrary, by using two different antibodies (mab933 and ab15348) -which can recognize the c-terminal domain shared between short-and long-ace2 -we obtained clear and concordant results, with identification of ace2 in pancreatic islet endocrine cells in addition to the microvasculature. as a positive control for the antibodies and our ihc procedure, mab933 and ab15348 were also tested in ffpe lung tissue sections, showing overlapping results with previously published studies (7, 41, 42) . overall, these results suggest that the short-ace2 isoform may be the prevalent one expressed in βcells. indeed, ace2 western blot analyses of the human β-cell line endoc-βh1 support this hypothesis by confirming that: (i) short-ace2 is prevalent over long-ace2, the latter being present but with low expressed in β-cells; (ii) abcam ab108252 cannot recognize the short-ace2 isoform. based on these results we suggest that in human β-cells both ace2 isoforms are present, with a predominance of the short-ace2. although the presence of the short-ace2 isoform, alongside with long-ace2, is clearly evident in human β-cells, its functional role remains to be established. a previous study suggested the ability of the short-ace2 isoform to form homodimers or heterodimer with the long-ace2 isoform, thus potentially being able to modulate both the activity and structural protein domains conformation of the long-ace2 (22) . significantly, short-ace2 is missing the aminoacidic residues reported as 33 fundamental for virus binding; however, due to the lack of detailed data regarding its function we cannot exclude that the short-ace2 may modulate sars-cov-2 susceptibility by interacting with long-ace2 or additional membrane proteins which may mediate the binding to sars-cov-2 spike protein [e.g. itga5, as previously observed (60)]. of interest, nasal epithelium, reported to represent the main reservoir of sars-cov-2 (61), showed higher levels of short-ace2 vs. long-ace2 (24). as described above, we report that in human pancreatic islets ace2 is enriched in insulin-producing (65), which usually detects only 1,000-3,000 genes/cell, thus representing a minor fraction (25-30%) of the total number of genes identified by bulk cells rna sequencing (>20,000 genes). another recent study analyzed sars-cov-2 host receptors expression using two different methods (microarray and bulk rna-seq) and further confirmed that ace2 is indeed expressed in human pancreatic islets, and also demonstrated that ace2 expression was higher in sorted pancreatic β-cells relative to other endocrine cells (66) . additional evidence of ace2 expression in endocrine pancreas and in β-cells derive also from mouse studies, which demonstrated ace2 expression in insulin-producing cells as well as its critical role in the regulation of β-cell phenotype and function (67) (68) (69) (70) . collectively, our in-situ expression data alongside with multiple published datasets and reports both in human and mouse show that ace2 is expressed in pancreatic islets, albeit at relatively low levels. ace2 expression in human β-cells may render these cells sensitive to sars-cov-2 entry (23). such hypothesis is consistent with the known sensitivity of these cells to infection by several enterovirus serotypes. indeed, multiple evidence from our and other groups (71) (72) (73) (74) showed that enteroviruses are capable to competently infect β-cells but less so α-cells (75, 76) ; these viruses are thus being considered 34 as one of the potential triggering causes of type 1 diabetes (t1d) (77) . of note, it has been previously demonstrated that human β-cells exclusively express virus receptor isoform coxsackie and adenovirus receptor-siv (car-siv), making them prone to infection by certain viruses (78) . therefore, it would not be surprising that, under particular conditions, human β-cells could be directly infected by sars-cov-2. importantly, a recent report showed that human pancreatic islets can be infected in vitro by sars-cov-2 (23), supporting our observations of a specific tropism of the virus due to ace2 expression. noteworthy, the subcellular localization of ace2 in β-cells recapitulates what was previously found for the virus receptor car-siv (78) . in our dataset, ace2 protein signal is mostly cytoplasmic/granular and partially overlaps with insulin granules. additional spots are also localized close to the plasma membrane, thus suggesting the existence of ace2 isoforms in multiple compartments within β-cells. such subcellular localization was observed both in-vitro in endoc-βh1 and ex vivo in β-cells of primary human pancreatic tissues. although ace2 has been primarily observed on cell surfaces (42, 79) , some studies also described ace2 granular localization in other cell types of epithelial origin (9, 80) . a similar intracellular localization and putative trafficking was observed for the viral receptor car-siv (78) , also expected to be mainly localized on the cell membrane. against this background, we speculate that: (i) upon activation, ace2 can be internalized through endosome/lysosome pathway (81); (ii) in β-cells, ace2 trafficking to cell membrane may be mediated by insulin granules; (iii) in β-cells the short-ace2 isoform could be mainly localized in cytoplasm, while long-ace2 in the plasma membrane; (iii) ace2 can be secreted and found in a soluble form or within exosomes (82, 83) . additional studies are needed to fully ascertain the subcellular localization and trafficking of ace2 isoforms in human β-cells, and to determine whether ace2 is indeed present in the secretome of β-cells. our data also indicate that in β-cells, total ace2 mrna expression is upregulated upon different proinflammatory conditions, but not following exposure to the metabolic stressor palmitate or to the t2d 35 environment. importantly, these observations were obtained both in the human β-cell line endoc-βh1 and in human primary pancreatic islet cells, as shown by qrt-pcr, rna-seq datasets and immunofluorescence. as a matter of fact, ace2 has been previously indicated as an interferonstimulated gene (isg) in a variety of cells (58) . of interest, a previous report suggested that the short-ace2 may represent the prevalent ace2 isoform upregulated upon inflammatory stress (25) . however, whether this is the case also in human β-cells it should be examined in depth in future studies. although total ace2 expression is increased upon inflammatory stress, in-situ analysis of ace2 in infiltrated pancreatic islets derived from ffpe sections of a pancreas from a longstanding t1d donor did not reveal significant changes of sars-cov2 receptor expression. however, we recognize that a high number of pancreatic islets with different degree of inflammation, and pancreata from recentonset t1d donors showing highly infiltrated islets are needed to adequately characterize ace2 expression in the early stages of the disease and to determine whether changes in ace2 expression contribute to: (i) the observed alteration of glycaemic control at admission in sars-cov-2 individuals without previous clinical history of diabetes; (ii) the increased severity of covid-19 in those subjects with previous inflammatory-based diseases. in conclusion, the presently described preferential expression of ace2 isoforms in human β-cells, the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor cell entry mechanisms of sars-cov-2 exogenous ace2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication functional 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ace2/ang-(1-7)/mas axis regulates the development of pancreatic endocrine cells in mouse embryos ace2 deficiency reduces β-cell mass and impairs β-cell proliferation in obese c57bl/6 mice angiotensin-converting enzyme 2 influences pancreatic and renal function in diabetic mice activation of ace2/angiotensin (1-7) attenuates pancreatic β cell dedifferentiation in a high-fat-diet mouse model coxsackie b4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients detection of a low-grade enteroviral infection in the islets of langerhans of living patients newly diagnosed with type 1 diabetes the prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes expression of the enteroviral capsid protein vp1 in the islet cells of patients with type 1 diabetes is associated with induction of protein kinase r and downregulation of mcl-1 viral infections in type 1 diabetes mellitus--why the β cells? the case for virus-induced type 1 diabetes prospective virome analyses in young children at increased genetic risk for type 1 diabetes unexpected subcellular distribution of a specific isoform of the coxsackie and adenovirus receptor, car-siv, in human pancreatic beta cells angiotensin-converting enzyme 2 (ace2), but not ace, is preferentially localized to the apical surface of polarized kidney cells sars-cov-2 receptor ace2 is expressed in human conjunctival tissue, especially in diseased conjunctival tissue angiotensin ii mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin ii type i receptor-dependent mechanism the insulin secretory granule as a signaling hub ace2-containing extracellular vesicles and exomeres bind the sars-cov-2 spike protein sars-cov-2 receptor angiotensin i-converting enzyme type 2 is expressed in human pancreatic islet β-cells and is upregulated by inflammatory stress we thank dr. r. scharfmann (institut cochin, inserm, université de paris) who provided the human pancreatic β-cell line endoc-βh1. we thank dr. laura salvini, dr. laura tinti and dr. vittoria key: cord-271979-a9u494tr authors: wolfe, nathan d.; daszak, peter; kilpatrick, a. marm; burke, donald s. title: bushmeat hunting, deforestation, and prediction of zoonotic disease date: 2005-12-17 journal: emerg infect dis doi: 10.3201/eid1112.040789 sha: doc_id: 271979 cord_uid: a9u494tr understanding the emergence of new zoonotic agents requires knowledge of pathogen biodiversity in wildlife, human-wildlife interactions, anthropogenic pressures on wildlife populations, and changes in society and human behavior. we discuss an interdisciplinary approach combining virology, wildlife biology, disease ecology, and anthropology that enables better understanding of how deforestation and associated hunting leads to the emergence of novel zoonotic pathogens. a pproximately three fourths of human emerging infectious diseases are caused by zoonotic pathogens (1). these include agents responsible for global mortality (e.g., hiv-1 and -2, influenza virus) and others that cause limited deaths but result in high case-fatality rates and for which no effective therapies or vaccines exist (e.g., ebola virus, hantaviruses, nipah virus, severe acute respiratory syndrome [sars]-associated coronavirus) (2) . despite the growing threat of zoonotic emerging infectious diseases, our understanding of the process of disease emergence remains poor. public health measures for such diseases often depend on vaccine and drug development to combat diseases once pathogens have emerged. indeed, many believe that predicting emergence of new zoonoses is an unattainable goal (3) . despite this, a growing trend in emerging disease research attempts to empirically analyze the process of emergence and move towards predictive capacity for new zoonoses. these studies track broad trends in the emergence of infectious diseases, analyze the risk factors for their emergence, or examine the environmental changes that drive them (4) (5) (6) . many new zoonoses are viruses that emerge as human and domestic animal populations come into increasing con-tact with wildlife hosts of potentially zoonotic pathogens (1) . the risk for emergence of new zoonotic agents from wildlife depends largely on 3 factors: 1) the diversity of wildlife microbes in a region (the "zoonotic pool" [5] ); 2) the effects of environmental change on the prevalence of pathogens in wild populations; and 3) the frequency of human and domestic animal contact with wildlife reservoirs of potential zoonoses. the first factor is largely the domain of virologists, particularly those analyzing evolutionary trends in emerging viruses (7) . the last 2 factors are studied by wildlife veterinarians, disease ecologists, wildlife population biologists, anthropologists, economists, and geographers (4, 8) . understanding the process of emergence requires analyzing the dynamics of microbes within wildlife reservoir populations, the population biology of these reservoirs, and recent changes in human demography and behavior (e.g., hunting, livestock production) against a background of environmental changes such as deforestation and agricultural encroachment. to fully examine zoonotic emergence, a multidisciplinary approach is needed that combines all of these disciplines and measures the background biodiversity of wildlife microbes. we use hunting and deforestation in cameroon as an example to discuss the complex interactions between human behavior, demography, deforestation, and viral dynamics that underpin the emergence of diseases. hunting of wildlife by humans is an ancient practice that carries a substantial risk for cross-species transmission. despite the discovery of cooking ≈1.9 million years ago (9), the risk of zoonotic diseases emerging from hunting and eating wildlife is still of global importance because of increases in human population density, globalized trade, and consequent increased contact between humans and animals. bushmeat hunting, deforestation, and prediction of zoonotic disease emergence nathan d. wolfe,* peter daszak, † a. marm kilpatrick, † and donald s. burke* deforestation of tropical forests is 1 cause of increasing contact between wildlife and hunters. however, the mechanics of disease emergence are complex. for example, clear-cut logging may be less likely to result in zoonotic emergence than selective extraction because of the relatively low contact rate between people and wildlife during clear-cutting. because of the high costs of extraction and transportation, logging in central africa generally involves selective extraction of high-value timber species. selective extraction is also more likely to sustain natural diversity of wildlife than clear-cutting (10) and therefore to sustain the diversity of potentially zoonotic pathogens available to hunters. selective logging generally involves constructing roads and transporting workers into relatively pristine forest regions. although roads can bring health care to rural communities, they also provide increased contact between low-density, remote human populations and urban populations with access to international travel, which allows localized emergence events the potential for rapid global spread (11, 12) . building logging roads also leads to habitat fragmentation as forest edges along roads are degraded, which lowers the movement of wildlife between forest patches. this process may have 3 counteractive effects. first, as patch size decreases, smaller, more discrete, less dense populations of reservoirs result, some of which may be lowered below the threshold density of some potentially zoonotic microbes (13) . in these cases, mathematical models of infectious diseases predict that the microbes will become extinct, lowering the risk for transmission to humans. second, in some cases, the loss of vertebrate reservoir host species richness may result in increased abundance of highly competent reservoirs of some zoonotic agents, increasing the risk for transmission to humans. although this phenomenon has only been demonstrated for 1 pathogen, borrelia burgdorferi, the causative agent of lyme disease (14) , it may be more widespread. in this case, fragmentation increases the relative abundance of the highly competent reservoir, the white-footed mouse (peromyscus leucopus) and results in a higher risk for infection to humans (14) . third, fragmentation due to road building may increase the functional interface between human populations and reservoir hosts. historically, hunting activities radiated in a circular fashion from isolated villages, with decreasing impact at the periphery of the hunting range. roads provide an increased number of points at which hunting activities can commence. roadside transport means that hunters can lay traps and hunt at the same distance from roads. this changes the pattern of human contact from a circular pattern to a banded pattern surrounding developed roads, increasing the area in which hunting can be conducted with economic returns. different activities associated with bushmeat trade will involve different levels of risk for microbial emergence. hunting (tracking, capturing, handling, sometimes basic field butchering, and transporting of the carcass) involves contact with potentially infected vectors, whereas distant consumption may not. particularly high risks may be associated with hunting nonhuman primates, and even greater risks in hunting species such as chimpanzee, which are phylogenetically closest to humans. butchering (opening, cutting, dressing, and preparing the carcass) is obviously more high risk for bloodborne pathogens than the transportation, sale, purchase, and eating of the butchered meat. research in medical anthropology has begun to examine indigenous theories of infectious disease (15) and the cultural contexts within which diseases emerge (16), but little data exist on local perceptions of health or other risks associated with hunting and eating bushmeat. humans as well as other animals employ behavioral adaptations to avoid exposure to infections, yet the type of protective strategies that hunters might use and the effectiveness of such strategies remain unknown. for this reason, anthropologic studies of bushmeat should include not only the details of hunting, but also the transportation of meat to the village, the market, the kitchen, and onto the table. these practices are often articulated along lines of gender and ethnicity and within cultural contexts. the demand for bushmeat in west and central africa is as much as 4 times greater than that in the amazon basin (10) . estimates of the extraction rate in the congo basin suggest that >282.3 g of bushmeat per person per day may be eaten there, with a total of 4.5 million tons of bushmeat extracted annually (17) . expanded demand for bushmeat will likely lead to changes in the exposure of humans to potentially zoonotic microbes. therefore, assessing the risk that bushmeat extraction and consumption poses to public health will include an assessment of the economy and geography of bushmeat demand and supply. a collaboration between johns hopkins university and the cameroon ministry of health and ministry of defense is exploring emergence of infectious diseases in cameroon (figure) . the ecologic diversity in cameroon and the range of new and changing land-use patterns make it an ideal setting to examine the impact of environmental changes on novel disease transmission. deforestation rates in cameroon are high, with a loss of 800-1,000 km 2 forest cover per year and corresponding increase in road-building and expansion of settlements (18) . finally, cameroon is representative of the region from which a range of notable emerging infectious diseases, including hiv/aids, ebola and marburg viruses, and monkeypox, have emerged (table) . a key factor driving the bushmeat trade in cameroon is the large and growing urban demand for bushmeat in conjunction with the opening up of logging concessions in the east province. the construction of the world bank-funded yaoundé-douala truck road in the mid-1980s and the european union-funded extension of this road to the border of the timber-rich east province in 1992 dramatically reduced the cost of extracting timber and increased access to these areas for bushmeat hunters. one of the most important non-timber forest product activities within this region is the poaching of bushmeat by market hunters. the bushmeat market among households for sauce preparation in yaoundé alone is estimated at ≈$4 million annually (international institute of tropical agriculture [iita], unpub. data). a recently conducted consumption study showed that bushmeat plays an important dietary role among poor households and is not a luxury product eaten mainly by the rich. across income classes, the poorest 2 quantiles spent 16% and 17%, respectively, of their meat budgets on bushmeat versus 7% for the richest quantile and 9% overall (iita, unpub. data). finally, our work in cameroon has shown that not only bushmeat hunters but also persons who keep various species of vertebrate pets or butcher and handle meat are at risk for zoonotic transmission due to bites, cuts, and other exposures to fluids or tissue (27) . the global emergence of a zoonotic pathogen such as sars or hiv-1 and -2 requires 3 steps. first, the pathogen must be successfully transmitted between a wild reservoir and humans or their domestic animals. several recently emerging zoonoses have achieved this stage without further transmission, e.g., hendra virus. second, the pathogen must be directly transmitted between humans. finally, the pathogen must move from a local epidemic into the global population. understanding and predicting the global emergence of pathogens require knowledge of the drivers of each of these steps or processes. these are, in fact, stages of emergence that have been described previously as invasion, establishment, and persistence of infectious diseases introduced into new host populations (8) . evidence suggests that many pathogens are transmitted between their animal reservoirs and humans but fail to be transmitted from human to human or do so at rates that do not allow pathogen establishment within the human population. for example, sequence data from hiv-1 and hiv-2 suggest that as many as 10 prior transmission events into human populations occurred over the last century before this virus emerged globally (23) . recent data from our own field sites suggest that simian foamy viruses infect bushmeat hunters regularly, so far without evidence of humanto-human transmission (26) . other pathogens, such as avian influenza and hendra viruses, which do not appear to be transmitted through bushmeat consumption, have also led to several small epidemics with little or no evidence of human-to-human transmission. we have termed this "viral chatter", a seemingly common phenomenon of repeated transmission of nonhuman viruses to humans, most of which results in no human-to-human transmission (28) . we hypothesize that this mechanism is common in viral emergence. high rates of viral chatter will increase the diversity of viruses and sequence variants moving into humans, increase the probability of transmission of a pathogen that can successfully replicate, and ultimately increase the ability of a human-adapted virus to emerge in a more widespread manner. in some cases this process may result in the evolution of a new viral strain (29) and may be a very common mechanism for viral emergence into the human population (23, 28) . monkeypox and nipah viruses are examples of the second stage towards global emergence. these viruses have shown limited human-to-human transmission in a number of relatively small epidemics before fading out (22, 30) . this phenomenon can be understood by using what mathematical modelers of disease dynamics refer to as the reproductive ratio (r 0 ), which measures a pathogen's ability to cause an outbreak. r 0 is the number of secondary cases in a population caused by a single case, assuming that all other members are susceptible (8) . when r 0 is >1, the pathogen will amplify within a population and cause an outbreak. in the environmental conditions in which monkeypox and nipah viruses emerged, r 0 was <1, and ultimately the epidemics faded out (22) . one of the crucial questions in disease emergence is: what environmental or evolutionary changes cause the r 0 of wildlife viruses to rise above 1 in human populations? in mathematical models for density-dependent transmission, r 0 is proportional to host density, so that there is a critical threshold of human population density (known as the threshold density, n t ), below which a pathogen will fade to extinction. increasing densities of human populations in urban centers close to bushmeat hunting areas and the increasing rates of movement of people between village, town, and city, will increase r 0 and the risk for new epidemic zoonoses. alternatively, changes to human behavior that increase the transmission of viruses between people (e.g., sexual contact, injected drug use, or fluid contact by means of medical procedures) will increase r 0 and may also assist in driving their emergence. in the final stage of emergence, increased travel or migration facilitate the global spread of new zoonoses. for example, increased movements between villages or cities and higher between-person contact rates through increased numbers of sexual partners appear to have facilitated the early emergence of hiv/aids in africa (12) . this disease became a global pandemic following the expansion of road networks, changes in workforce demography, and increases in international air travel to central africa and globally (12, 23) . our review suggests that predicting the emergence of new zoonoses will be a difficult but important task for future medical research. this goal has been described as challenging or impossible by some researchers (3) . however, we propose that it is now becoming possible to conduct the science of predicting emerging zoonoses and that far more attention should be paid to this approach than is currently given (31) . we have previously proposed 3 criteria that can be used to predict which microbes are most likely to emerge (6) . these include microbes that have a proven ability to 1) lead to human pandemics, 2) lead to panzootics in (nonhuman) animal populations, and 3) mutate at high rates and recombine with other similar or dissimilar microbes. the high mutation rates of rna viruses and their predominance within zoonotic emerging infectious diseases that are transmitted from human to human suggest that this group is a key candidate for future emergence (7) . simian foamy viruses are members of this group, and the high rates of viral chatter observed in cameroon suggest a strong potential for their emergence as a human-to-human transmitted pathogen. little is known about the complexity of this process, but with ≈75% of human emerging infectious diseases classified as zoonoses (1), understanding the process is critical to global health. we propose that more attention be given to multidisciplinary studies at all stages of the process. for example, understanding how the rates of viral chatter respond to anthropogenic land-use changes (e.g., deforestation, mining) that affect the density of wildlife species and the prevalence of viruses that affect them will be critical for predicting hotspots of disease emergence. second, understanding which viruses are likely to rapidly evolve in humans, rather than become dead-end hosts, will involve a combination of host immunologic and viral evolutionary traits (7, 32) . studies of the characteristics of the zoonotic pool (i.e., the biodiversity of yet-to-emerge wildlife viruses [5] ) may explain these events. some strains within viral quasispecies may be able to infect and be transmitted between humans far more readily than others. such complexity requires the collaboration of medical scientists with many other disciplines, including geography, ecologic and evolutionary biology, conservation biology, medical anthropology, and veterinary medicine. recent advances in a number of fields include some of direct relevance to predicting unknown zoonoses, among them modeling multihost disease dynamics in wildlife and humans (33) , modeling the evolutionary dynamics of pathogens (34) , insights into the phylogenetic characteristics of emerging pathogens (7, 32) , greater understanding of the environmental changes that drive emergence (4), risk assessments for pathogen transmission (35, 36) and introduction (37) , and major advances in the technology for microbial discovery (e.g., microarrays) and characterization (e.g., noninvasive sequencing) (38) . a number of collaborative initiatives between veterinary medicine, human medicine, and ecology have already begun (39, 40) , and our analysis suggests these should be strengthened by even wider collaboration. the fusion of these diverse, rapidly evolving fields will allow the first steps to be taken towards emerging disease research's ultimate challenge of predicting new zoonotic disease emergence. risk factors for human disease emergence microbial threats to health: emergence, detection and response. washington: the national academies press emerging zoonoses emerging infectious diseases of wildlife-threats to biodiversity and human health examining the origins of emerging viruses the evolvability of emerging viruses the population genetics and evolutionary epidemiology of rna viruses the invasion, persistence and spread of infectious diseases within animal and plant communities the raw and the stolen: cooking and the ecology of human origins impact of market hunting on mammal species in equatorial-guinea human immunodeficiency virus type 1 epidemic: date of origin, population history, and characterization of early strains the river detecting disease and parasite threats to endangered species and ecosystems the ecology of infectious disease: effects of host diversity and community composition on lyme disease risk indigenous theories of contagious disease cultural contexts of ebola in northern uganda bushmeat exploitation in tropical forests: an intercontinental comparison la conservation des ecosystèmes forestiers du cameroun gland. switzerland and cambridge, uk: international union for the conservation of nature first confirmed dengue-1 fever cases reported from cameroon epidemic of yellow fever in north cameroon in 1990-1st isolation of yellow fever virus in cameroon current topics in microbiology and immunology four generations of probable person-to-person transmission of human monkeypox aids as a zoonosis: scientific and public health implications transmissible drug resistance in shigella and salmonella isolated from pet monkeys and their owners b-virus from pet macaque monkeys: an emerging threat in the united states? naturally acquired simian retrovirus infections in central african hunters central african hunters exposed to simian immunodeficiency virus simian retroviral infections in human beings-reply simian retroviral infections in human beings nipah virus encephalitis reemergence conservation medicine and a new agenda for emerging diseases error thresholds and the constraints to rna virus evolution emerging infectious pathogens of wildlife large shifts in pathogen virulence relate to host population structure west nile virus risk assessment and the bridge vector paradigm climate and satellite indicators to forecast rift valley fever epidemics in kenya a quantitative risk assessment of the pathways by which west nile virus could reach hawaii hybrid origin of siv in chimpanzees conservation medicine: building bridges conservation medicine we thank the following researchers, who greatly assisted our study: mpoudi ngole eitel, jim gockowski, pia k. muchaal, christian nolte, a. tassy prosser, lisa m. schloegel, judith ndongo torimiro, and stephan f. weise. we thank the staff of the walter reed johns hopkins cameroon program for assistance with fieldwork and specimen processing, the government of cameroon for permission to undertake the study, and the us embassy, yaoundé for their support. key: cord-010046-7hlgjiqp authors: harvey, david j. title: analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: an update for 2003–2004 date: 2008-09-29 journal: mass spectrom rev doi: 10.1002/mas.20192 sha: doc_id: 10046 cord_uid: 7hlgjiqp this review is the third update of the original review, published in 1999, on the application of matrix‐assisted laser desorption/ionization (maldi) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings the topic to the end of 2004. both fundamental studies and applications are covered. the main topics include methodological developments, matrices, fragmentation of carbohydrates and applications to large polymeric carbohydrates from plants, glycans from glycoproteins and those from various glycolipids. other topics include the use of maldi ms to study enzymes related to carbohydrate biosynthesis and degradation, its use in industrial processes, particularly biopharmaceuticals and its use to monitor products of chemical synthesis where glycodendrimers and carbohydrate–protein complexes are highlighted. © 2009 wiley periodicals, inc., mass spec rev 28:273–361, 2009 this review is a continuation of the three earlier ones in this series on the application of matrix-assisted laser desorption/ionization (maldi) mass spectrometry to the analysis of carbohydrates and glycoconjugates (harvey, 1999 (harvey, , 2006 (harvey, , 2008 and brings the coverage of the literature to the end of 2004. the review is intended to illustrate both developments in technology and in the way in which analysis of carbohydrates contributes to scientific knowledge in general. matrix-assisted laser desorption/ionization (maldi) continues to be a major technique for the analysis of carbohydrates although electrospray, particularly with quadrupole-time-offlight (q-tof) instruments, is becoming increasingly popular. advantages of the maldi-q-tof combination for proteomics and glycomics have been stressed in a brief focus paper (huang, 2003) . figure 1 shows the year-by-year increase in papers reporting use of maldi ms for carbohydrate analysis for the period 1990-2004. as the review is designed to complement the earlier work, structural formulae, etc. that were presented earlier are not repeated. however, a citation to the structure in the earlier works is indicated by its number with a prefix (i.e., 1/x refers to structure x in the first review and 2/x to the second). other reviews and review-type articles directly concerned with, or including maldi analysis of glycoconjugates that have been published during the review period include general reviews by harvey (2003) , stuhler and meyer (2004) , and zaia (2004) , a review of capillary electrophoresis-mass spectrometry for glycoscreening (zamfir & peter-katalinic, 2004 ) and a review on new methods for mass spectrometry of bioorganic macromolecules (cristoni & bernsrdi, 2003) . more specific reviews include those on the glycosylation of caenorhabditis elegans , characterization of substituent distribution in starch and cellulose derivatives (richardson & gorton, 2003) , derivatization of carbohydrates for chromatographic, electrophoretic, and mass spectral structural analysis (lamari, kuhn, & karamanos, 2003) , structure of bacterial lipopolysaccharides (caroff & karibian, 2003) , analysis of post-translational modifications (cantin & yates, 2004; jensen, 2004; seo & lee, 2004) , the use of maldi ms to detect enantioselectivity in gas-phase ion-molecule reactions with carbohydrates such as cyclodextrins (speranza, 2004) , synthesis of heparan and heparin sulfate fragments , analysis of protein glycation products (horvat & jakas, 2004; kislinger, humeny, & pischetsrieder, 2004) , carbohydrate biosensors (jelinek & kolusheva, 2004) , synthesis and discovery of oligosaccharides and glycoconjugates for the treatment of disease (macmillan & daines, 2003) , dendrimers in drug research (boas & heegaard, 2004) , combinatorial carbohydrate synthesis (baytas & linhardt, 2004) , chemical tagging strategies for proteome analysis (leitner & lindner, 2004) , capillary electrophoresis of biopharmaceutical products (kakehi, kinoshita, & nakano, 2002) and the use of mass spectrometry to study congenital disorders of glycosylation type iix (mills et al., 2003b) . a comparison between spectra obtained with a conventional quadrupole ion trap (qit) (electrospray ionization) and a maldi-qit-reflectron-tof instrument has shown that the latter instrument produces significant amounts of metastable fragmentation due to the longer (msec) time span of the qit trapping process, an observation also made by harvey et al. (2004) with released n-glycans. glycopeptides were completely desialylated in the process. fragmentation (ms 2 ) of glycopeptides produced fragments exclusively from the carbohydrate with the esi-qit instrument with peptide fragmentation occurring in ms 3 , whereas the maldi-qit-reflectron-tof instrument yielded both types of fragmentation at the ms 2 stage (demelbauer et al., 2004) . coupling of both vacuum and atmospheric pressure maldi (ap-maldi) ion sources with ion-trap instruments has been reviewed (moyer et al., 2003) and the latter technique investigated as a method for obtaining fragmentation spectra from carbohydrates. high-pressure ion sources have been developed to cool ions in an attempt to reduce or eliminate fragmentation occurring either in-source or post-source. such fragmentation is the primary factor limiting the use of vacuum maldi for analysis of acidic glycans and can be considerably reduced with a high-pressure ion source but at the cost of diminished sensitivity. arabinosazone (1/40) has been used as the matrix for maltohexaose (a-d-glcp-(a-d-glcp-) 4 -(1 ! 4)-a-d-glcp, 1) in positive ion mode ([m þ na] þ ion) and for 6 0 -sialyllactose (2) in negative mode ([màh] à ). the spectrum of the latter compound contained two abundant fragments, an o,2 a 3 and a c-ion (domon and costello (1988) nomenclature, see first review, scheme 3) whereas the major glycosidic fragments in the positive ion spectrum were produced by b or y cleavages. because of the near physiological conditions attainable at atmospheric pressure with glycerol as the matrix, it has been possible to observe non-covalent complexes between carbohydrates and synthetic peptides that were designed to mimic the binding sites of three members of the siglec family. 3 0 -(3) and 6 0sialyllactose (2), were used as the carbohydrate ligands with 3sialyllactose maintaining its binding specificity for sialoadhesin (von seggern & cotter, 2004) . a new ap-maldi technique named atmospheric pressure infrared ionization from solution (ap-iris) that is claimed to be 16 times a sensitive as ap uv-maldi has been implemented on a qit mass spectrometer . the method operated in the absence of an extraction year 2004 year 2002 year 2000 year 1998 year 1996 year 1994 year 1992 year 1990 number of publications 0 100 200 300 400 500 figure 1. yearly totals of the number of publications containing work relating to the application of maldi mass spectrometry to carbohydrates and glycoconjugates. field and used a high power ir laser with aqueous glycerol as the matrix. spectra consisting of [m þ na] þ ions were obtained from several high-mannose and complex n-linked glycans with much better signal/noise ratios than with corresponding amounts ionized by uv-maldi from 2,4,6-trihydroxyacetophenone (thap, 1/44 matrix-assisted laser desorption/ionization (maldi) plates sprayed with the hydrophobic 3m product, scotchgard tm , a water repellent material for coating fabrics, has the effect of reducing the maldi spot size and improving sensitivity (owen et al., 2003) . for cleaning, the coating could easily be removed with methyl-t-butyl ether and the film reapplied to the clean plate. experiments with the carbohydrate-containing antibiotic, erythromycin a (4), showed increases in sensitivity of two-to threefold when ionized from the coated targets compared with signals from uncoated plates. a microdeposition device has been constructed which mixes effluent from capillary electrochromatography or microcolumn liquid chromatography with the maldi matrix and deposits the mixture onto the maldi plate. dextrin and n-linked glycans from ribonuclease b were successfully analyzed (tegeler et al., 2004 ). an array of 96 perforated nanovials manufactured by silicon microfabrication and filled with 40 nl of reversed-phase beads has been developed as a maldi target for peptide analysis. it was successfully used to examine peptides and glycopeptides from prostate-specific antigen (ekström et al., 2004) . liquid chromatography-mass spectrometry (lc-ms) has the disadvantage that the entire sample is consumed after exiting the hplc column. lochnit and geyer (2004) have used nano-lc-maldi-tof ms to examine tryptic peptides and glycopeptides, a technique that allows more detailed ms investigation of the effluent to be made. the technique also produced enhanced detection of glycopeptides in the presence of peptides and improved characterization of the attached glycans by optimizing the experimental conditions. investigations into the mechanism of proton transfer from dihydroxybenzoic acid (dhb) isomers continues. yassin and marynick (2003) have calculated the gas-phase acidities of the radical cations of all six dihydroxybenzoic acid (dhb) isomers using density functional theory and found excellent agreement with experimental measurements. the 2,5-isomer (1/26), the most effective matrix, was the least acidic (in this review the abbreviation dhb is used for the 2,5-isomer, unless stated otherwise). the results indicate that, as proposed earlier (gimon et al., 1992) , deprotonation occurs at the phenolic rather than the acidic site as the result of resonance stabilization of the resulting ion. this conclusion is consistent with the theory proposed by harvey (1993) that one of the reasons that the 2,5-isomer is so effective a matrix is that it is the only one that can form a p-benzoquinone-type (5) structure after photochemical decarboxylation. sugars, however generally ionize as [m þ na] þ ions for which the factors governing formation are much less clear. a recent study by antonopoulos et al. (2003) on the mechanism of attachment of sodium to glucose and its methylated derivatives found no simple correlation between ion yield and factors such as volatility and hydrophobicity. a study by luxembourg et al. (2003) using the high resolution capability of matrix-assisted secondary ion mass spectrometry has demonstrated crystal inhomogeneity among dhb crystals on maldi targets. some crystals were free of sodium whereas others contained sodium and sample. yet other areas of the target contained mostly sodium and little matrix. the results were thought to explain the analysis of carbohydrates and glycoconjugates & phenomenon of ''sweet spots'' frequently observed with maldi targets. proton-affinity values for 15 common matrices have been measured by a kinetic method. a-cyano-4-hydroxycinnamic acid (chca, 1/23) and dhb showed the lowest affinity whereas 2-(4 0 -hydroxyphenyl)azobenzoic acid (haba, 1/32) and norharmane (1/35) had the highest affinity. in general, the rank order was similar to that found by other investigators. with bcyclodextrin (6, cyclic-(1 ! 4)-a-d-glucose) 7 as the reference compound in negative ion mode, matrices with low proton affinity, such as dhb (204.4 ae 0.17), produced no [màh] à ions whereas those with high affinity, such as nor-harmane (233.0 ae 0.44), did (mirza, raju, & vairamani, 2004b) . gasphase potassium binding energies of several maldi matrices have also been published . cyclic-((1→4)-α-d-glc) 7 6. β-cyclodextrin fournier et al. (2003) have measured the ablation volume of dhb crystals under different laser powers and found that, although the ablation volume increased only slowly with increasing laser power, the signal rose dramatically. they interpreted their results as involving a very rapid ablation to produce a plume that was further ionized by the incoming laser beam. frankevich et al. (2003) have discussed the role of photoelectrons on maldi sensitivity. suppression of photoelectrons with a film of insulating material on the target; the authors used scotch magic tape; was found to produce an increase of up to two orders of magnitude in sensitivity and an improvement in resolution. bashir, mutter, and derrick (2003b) have investigated long-chain esters of dhb as matrices and found some improvement in performance, particularly after the addition of iodine to those compounds that contained a double bond in the chain. the mechanism producing this enhancement in performance by the addition of iodine was unclear but the reason for the improved performance with some of the long-chain compounds was thought to be the formation of micelles that enhanced the matrixanalyte interactions. 5-amino-2-mercapto-1,3,4-thiadiazole (amt, 7) has been described as a new matrix for carbohydrates (mirza et al., 2004a) . its performance was similar to that of dhb although slightly higher-mass compounds could be ionized from dextran-5000. kéki et al. (2004) have ionized simple organic molecules such as a peracetylated isoflavone glycoside (8) from common matrices such as dhb doped with silver trifluoroacetate to give [m þ ag 3 ] þ species. these ions were shown to be more stable than the corresponding [m þ ag] þ ions. the latter ions and their cu þ counterparts, fragmented in a similar manner to those from na þ and li þ (kéki et al., 2004) . carbon nanotubes, prepared from coal by an arc discharge have been reported as a novel matrix for low molecular weight peptides and b-cyclodextrin . spectra showed a very low background as the result of the absence of matrix peaks and ions produced by fragmentation. to obtain the spectrum, a well-dispersed ethanolic solution of nanotubes was deposited onto the maldi target and allowed to dry. the sample solution was then deposited onto the nanotubes and dried with hot air. two papers reporting the use of fine cobalt powder for the analysis of lepidimoic acid (9) from okra pectic polysaccharide (hirose et al., 2003) and of lepidimoide (10) from okra mucilage have appeared (hirose, endo, & hasegawa, 2004) . a mixture of dhb and chca has been shown to give enhanced performance for the ionization of glycopeptides than use of the individual matrices. the mixed matrix showed better tolerance towards the presence of salts and impurities and improved peptide sequence coverage (laugesen & roepstorff, 2003) . a thin layer of very homogeneous crystals has been produced by mixing thap with nitrocellulose and used to analyze pectin digests (mohamed, christensen, & mikkelsen, 2003) . atmospheric pressure maldi (ap-maldi) has the advantage over vacuum maldi of collisional stabilization of fragile molecules such as carbohydrates containing sialic acid residues. the technique is also compatible with the use of liquid matrices whose volatility presents problems under vacuum conditions. von seggern, moyer, and cotter (2003a) have compared water, glycerol, and nitrobenzyl alcohol (1/21) for ionization of sialylated glycans with an ir laser (er:yag, 2,490 nm) and have found that glycerol provides the longest lasting signal with the best signal/noise ratio. water was too volatile to survive for long under the heat produced by the laser. intact sialylated glycans were desorbed as [màh] à and [m þ na] þ ions under negative and positive conditions respectively. in a further study (von seggern, zarek, & cotter, 2003b) , the glycerol matrix was doped with alkali (li, na, k) alkaline earth (ca) or transition metal cations (ca, co) for production of adducts containing one or two singly charged cation or one doubly charged cation. adduction proved to be efficient with lithium or potassium being able to replace the more common sodium adduction when salts (cl à or i à ) containing these metals were used as the doping agents. cobalt adduction was achieved by use of cobalt powder added to the glycerol. fragmentation (see below) was obtained with an ion-trap instrument. the use of glycerol as the matrix with an ir laser under atmospheric pressure conditions has also enabled non-covalent sugar-sugar complexes to be observed (von seggern & cotter, 2003) . the stability of the complexes varied with different sugars potentially due to the strengths of the hydrogen bonding networks. fragmentation of the complexes was also a function of structure; some complexes, particularly those containing sialic acid, fragmented by loss of the acid before breakdown of the complex whereas complexes between neutral molecules tended preferentially to dissociate into monomer units. ionic liquid matrices appear to show great promise for carbohydrate analysis. the matrix dhb-butylamine (dhbb) was reported to give improved shot-shot reproducibility compared with solid matrices such as dhb, and reduced the relative standard deviation by 50%. it also produced much less fragmentation from compounds such as sialylated carbohydrates (mank, stahl, & boehm, 2004) . greatly improved quantitative performance has also been reported by zabet-moghaddam, heinzle, and tholey (2004) . derivatization techniques for analysis of carbohydrates have been reviewed (gao et al., 2003; suzuki et al., 2003b) . advantages of analytical derivatization have been emphasized by rosenfeld (2003) with, among others, examples from the reductive amination of carbohydrates for improving the interpretation of fragmentation spectra and for modifying behavior in chromatographic systems. maldi is frequently used to monitor reducing-terminal labeling, used to attach fluorophores or chromophores to carbohydrates for chromatographic detection as illustrated in a recent study of glycans from the tamm-horsfall glycoprotein (rohfritsch et al., 2004) and the use of 2-aaderivatives to monitor glycosylation of therapeutic glycoproteins (dhume et al., 2002) . benzylamine (2/9) derivatization was used by mainly for esi-ms/ms analysis of small and n-linked glycans. however, exoglycosidase sequencing was performed on a maldi target and it was observed that the derivative did not inhibit the enzymic reaction. an, franz, and lebrilla (2003a) have derivatized several sugars, including n-linked glycans released from ribonuclease b, with benzylamine and quaternized the product with methyl iodide as originally performed by broberg, broberg, and duus (2000) but with a procedure not involving the use of resins. the product allowed separation of isomers by capillary electrophoresis (ce) and also gave strong maldi spectra on account of the inbuilt charge. it is possible to remove labels attached by reductive amination; thus, 2-aminopyridine (2-ap, 1/52)-derivatized nglycans have been underivatized by a process that involved catalytic hydrogenation and hydrazinolysis using the method reported by hase (1992) to give the reducing-terminal amine which was converted to an aldehyde using the sommlet reaction (hexamethylenetetramine and acetic acid) (takahashi, nakakita, & hase, 2003a ). phenylhydrazine (11) has been used to form hydrazone derivatives of n-linked glycans for both maldi and electrospray ionization. because the derivatives were not reduced, as with reductive amination, no post-derivatization clean-up was necessary. derivatives could be prepared on-target by addition of a solution (0.5 ml), prepared by dissolving phenylhydrazine (1 ml) in 10 ml of water/methanol (4:1 v/v), directly to the glycanmatrix mixture on the maldi target and allowing the mixture to dry under ambient conditions for 45 min. no osazone formation was reported and the derivatives produced [m þ na] þ ions. postsource decay (psd) spectra were reported to be simpler than those of the underivatized glycans and to be dominated by b/y internal fragments (lattova & perreault, 2003a,b) . hn nh 2 several hydrazones containing either a constitutive cationic charge, such as those formed from girard's t reagent (1/55, quaternary ammonium) or from hydrazines carrying a guanidine group (e.g., 12), have been shown to produce increases in sensitivity and to suppress signals from peptides, thus allowing n-linked glycans to be detected in the presence or tryptic peptides without extensive clean-up . differences in sensitivity among the derivatives were observed. among those containing a charge, and which formed m þ ions, the pyridinium analysis of carbohydrates and glycoconjugates & derivative (13) was the most efficient, producing a detection limit of 1 fmol. the guanidine derivatives formed [m þ h] þ ions and some produced equal detection limits. it would appear that the most efficient derivatives possessed both good ion-forming properties and high lipophilicity. improved detection limits have also been achieved by use of hydrazide derivatives of the cyanine dyes or cy-5 (15) that contain a constituent positive charge . n-glycans from chicken ovalbumin could be detected with near independence from the type of matrix. psd spectra contained mainly y-type ions consistent with localization of the positive charge on the derivative. sialic acids can be stabilized for maldi analysis by formation of methyl esters to remove the very labile acidic proton on their carboxyl groups. migration of this hydrogen atom is largely responsible for lability of this carbohydrate. a recent example of methyl ester formation is in a study of the n-linked glycosylation of the glycoprotein, n-cadherin, from human melanoma cell lines (ciolczyk-wierzbicka et al., 2004) . the acids were first converted into their sodium salts with a dowex ag50 resin (na þ form) and these salts were then reacted with methyl iodide. other examples are included below. removal of contaminants such as salts, buffers, etc. is essential to obtain high quality spectra. a variety of methods are used, the majority being dialysis, use of various membranes such as nafion 117 (börnsen, mohr, & widmer, 1995) or various resins. methods for preparing samples for mass spectrometric analysis have been reviewed (perreault, 2004) . some examples of those during the review period are listed below. drop dialysis has been used in clean-up of lipooligosaccharide (los) from moraxelle catarrhalis by luke et al. (2003) and a nafion membrane for on-probe clean-up of o-acetylated glucomannans from birch and aspen (teleman et al., 2003) . dowex ag50 resins in various forms have been used for analysis of n-glycans from the moss physcomitrella patens (viëtor et al., 2003) (ag 50w-x2 form), deacylated lipopolysaccharide (lps) from vibrio parahaemolyticus strains (hashii et al., 2003a,b) (50 w â 8, h þ form to remove cations) and extracellular polysaccharide (eps) from burkholderia cepacia (cescutti et al., 2003) (50 w â 8-200) . ag50 combined with ag3 (anions) in a gel loader tip have been used for n-linked glycans (hoja-lukowicz et al., 2000; ciolczyk-wierzbicka et al., 2004) . porous graphitized carbon has been employed to desalt quaternized benzylamine derivatives (an, franz, & lebrilla, 2003a) and hypercarb columns have been used by mills et al. (2003b) for n-linked glycans released from human plasma glycoproteins proteins and by forno et al. (2004) (higai et al., 2003a) and sialyl lewis x antigen-expressing glycoproteins secreted by human hepatoma cell line (higai et al., 2003b) and wada, tajiri, and yoshida (2004) have employed both cellulose or sepharose for the separation of tryptic glycopeptides from tryptic peptides. c18-solid-phase extraction followed by carbograph column was used by ohl et al. (2003) to remove detergents from nglycans. salts, etc. ware eluted from the columns with water, neutral glycans with 25% mecn and acidic glycans with 25% mecn/0.05% trifluoroacetic acid (tfa). amberlite ir-120 (h þ ) was used by frirdich et al. (2003) to purify core oligosaccharide from e. coli. release of n-glycans with protein-n-glycosidase f (pngase f) is more efficient if the glycoprotein is pre-digested with trypsin or other protease. however, the resulting peptides can interfere with subsequent analysis and are difficult to remove with octadecyl (c 18 ) silica cartridges, cation exchange columns or graphitized carbon cartridges. to overcome this problem, nakano, kakehi, and lee (2003b) have modified the amino groups of the peptides with 2,4,6-trinitrobenzene-1-sulfonate (16) to render them more hydrophobic so that they can be more strongly retained on c 18 or graphitized carbon. 16. 2,4,6-trinitrobenzene-1-sulfonate affinity capture of glycoproteins to a maldi target has been reported by koopmann and blackburn (2003) . the target was coated with poly-l-lysine poly(ethylene glycol)-biotin polymer followed by tetrameric neutravidin. this surface then acted as a capturing surface for biotinylated proteins which could be adsorbed and desalted by washing. maldi spectra were obtained following addition of chca to the surface. proteinprotein interactions could also be studied with this treated target and, furthermore, it could be used to isolate glycoproteins by lectin binding. thus, biotinylated wheat germ agglutinin bound to the surface was used to further capture fetuin. the resulting maldi spectrum contained ions from the biotinylated lectin, neutravidin and fetuin. glycopeptides, such as those obtained from protease digests, frequently give weak signals in the presence of hydrophobic peptides. a simple method for separating hydrophobic from hydrophilic peptides on a maldi target has recently been devised (kjellström & jensen, 2003 ). an aqueous solution of the peptide mixture was placed on the target followed by an immiscible solvent, such as ethyl acetate with or without added matrix. the organic solvent extracted the hydrophobic peptides and was allowed to evaporate, after which, the residual aqueous phase was transferred to another part of the target and allowed to dry after addition of dhb. maldi spectra could then be acquired from both the hydrophobic and hydrophilic fractions. a paper on quantitation of glucose (1/4) in a study on enzyme kinetics has highlighted problems in the use of maldi-ms for quantitative work and how the technique can produce good quantitative results in spite of the popular belief that the technique is not quantitative. the inhomogeneity of the target and variations in the shot-to-shot laser intensity can be compensated for by averaging many shots from different target positions. it is also important to eliminate any spectrum in which the a/d converter is saturated. by use of a [u-13 c]-labeled internal standard and measurement of the [m þ na] þ ions, a linear calibration curve was obtained with a correlation coefficient of r ¼ 0.991. the mean standard deviation was 6% but the authors (bungert, heinzle, & tholey, 2004b) state that this could be improved if more spectra were averaged although this would have increased the analysis time. a maldi method for the quantification of glucose from hydrolyzed starch has been reported. sorbitol (1/42) was used as the internal standard and dhb provided the matrix even though it produced peaks in the same region, but not at the same mass as the analytes. calibration curves were linear over the range 0.1-10 pmol added to the target and the method compared favorably with the normal colorimetric method for measuring glucose. even though the maldi results showed a greater degree of variability, this was more than compensated for by the speed of analysis (grant et al., 2003) . a quantitative maldi-tof ms method as also been developed for screening of ten pyranose oxidase variants. the isotopic labeled internal standard, [u-13 c]-glucose and the ionic liquid matrix, dhb/pyridine, were used with aliquots of enzyme reaction mixtures without pre-purification steps (bungert et al., 2004a) . the ionic, liquid matrix enabled spectra to be recorded from most areas of the target (only 7 out of 200 spots failed to produce a signal). the mean standard deviation for glucosone was 11.8% and the results were in good agreement with hplc measurements. glycosidic and cross-ring cleavages produce most of the fragment ions from carbohydrates. a third type of fragmentation mechanism has now been reported involving hexacyclic hydrogen rearrangements. the products were proposed to consist of unsaturated sugar rings lacking two oxygen atoms as shown in scheme 1 (spina et al., 2004) . post-source decay (psd) fragmentation of per-acylated isoflavone glycosides (e.g., 8) cationized with various metals and hydrogen have shown that the number of fragment ions decreased in the order li þ =na þ > ag þ > cu þ > h þ > k þ > rb þ =cs þ , roughly in line with earlier studies of underivatized carbohydrates (kéki et al., 2003) . fragments were largely glucosidic and losses of acetic acid and ketene but, unusually, there were a few fragments in some of the metal-cationized spectra that had lost the metal. loss of acetic acid and ketene was highest for the lighter metals. the authors attributed this observation to elimination of metal acetates. neutral and some acidic carbohydrates have been shown to form stable adducts with chlorine that survive the maldi process to give [m þ cl] à ions from matrices such as harmine (1/36) that have gas-phase acidities lower than or close to that of hcl (cai, jiang, & cole, 2003) . such adducts open the way for the production of negative ion fragmentation spectra from carbohydrates that, in many cases, produce more informative spectra than their positive ion counterparts. in the above publication, the adduct of the disaccharide d-turanose (a-d-glcp-(1 ! 3)-d-fru, 17) was reported to fragment initially by loss of hcl to give a psd spectrum containing both glycosidic and ms/ms analysis of n-acetyllactosamine-6,6 0 -disulfate (18) and its 2 0 -epimer produce different fragmentation patterns under several mass spectrometric methods (fast-atom bombardment (fab), esi and maldi-psd). the psd spectra were characterized by an abundant ion at m/z 431 that was produced from the 2a-epimer by elimination of both the n-acetylamino-group and the 6-sulfate (ohashi et al., 2004) . muzikar et al. (2004) have found that many carbohydrates containing an amino group at the reducing terminus show enhanced psd fragmentation with production of more abundant cross-ring fragments. n-linked glycans are normally released enzymatically in this form but cross-ring fragmentation of these branched compounds was not enhanced as much as that from linear compounds and was inferior to the high-energy fragmentation obtained with a tof/ tof mass spectrometer. a novel method of ion isolation for cid has utilized ir-induced fragmentation and consequent removal of unwanted ions by use of a nd:yag laser that shone directly into the analytical cell of a modified fourier transform ion cyclotron resonance (ft-icr) mass spectrometer (xie, schubothe, & lebrilla, 2003) . the method differed from existing techniques in that it did not involve diverting the unwanted ions with magnetic or electric fields. the paper reported the isolation of g-cyclodextrin (1/65) from a mixture containing maltotetraose ((b-(1 ! 4)-linked d-glucose) 4 , 19) and maltohexaose both of which also produced fragments in the analytical cell prior to irradiation with the ir laser. infrared multiphoton dissociation (irmpd) of alkali metaladducted carbohydrates has also been studied in this instrument and compared with cid. although higher energy fragmentation could be obtained with cid, irmpd provided continuous energy transfer to the fragment ions with the result that more extensive fragmentation was achieved . fragmentation of maltohexaose and high-mannose glycans (positive ion) with a tof-tof mass spectrometer with air as the collision gas has been shown to give high-energy-type fragmentation with the production of abundant cross-ring cleavage ions and, in particular, x-type cleavages not seen in the low energy spectra. isomers of man 7 glcnac 2 (20) could be distinguished with respect to the antenna (3 or 6) to which the seventh mannose was attached by the o,4 a 3 and 3,5 a 3 ions (mechref, novotny, & krishnan, 2003) . similar results have been reported by with a series of smaller sugars. a study of the effect of dhb or chca on fragmentation patterns of native and permethylated oligosaccharides in a tof/tof mass spectrometer has shown that, whereas chca promoted mainly glycosidic cleavages, dhb initiated glycosidic, cross-ring and internal cleavages with x-type cross-ring cleavages among the most abundant ions. permethylation produced increased sensitivity and spectra that were easier to interpret. studies with avidin from a dhb matrix allowed most of its n-glycans to be identified (stephens et al., 2004a) . a comparison of matrices used for ionization of 2-ap-labeled complex n-linked glycans with a tof-tof mass spectrometer has shown that, whereas chca produced only sodiated fragments in ms 2 spectra from [m þ na] þ parent ions, dhb produced both sodiated and protonated ions. it was suggested that the two matrices produced parent ions in different excited states . however, chca was more effective than dhb in producing abundant psd ions. although providing additional information through the production of more cross-ring cleavages, acidic glycans showed extensive fragmentation before reaching the collision cell. sialylated glycans, however, can be stabilized by permethylation or methyl ester formation, as discussed above. negative ion fragmentation appears to be gaining more in popularity because of its ability to produce abundant cross-ring cleavages. the [màh] à ion generated by ap-maldi from 6 0sialyl-lactose (6 0 -sl, 1) using a glycerol matrix, for example, yields the o,2 a 3 ion as the most abundant fragment (von seggern, moyer, & cotter, 2003a) . c-ions were prominent in the spectrum of this and larger sugars such as lsta (neu5ac-aglc, 22) and the spectra easily allowed the location of sialic acids to be determined. cross-ring and c-type fragments were also prominent in the spectra of lithiated adducts and, in particular, the spectra of lithium salts of sialic acids (von seggern, zarek, & cotter, 2003b) . fragmentation of the [m þ li] þ and [m þ co] þ ions showed no significant difference between 3 0 -(3) and 6 0 -sl (2) but the spectra of the [màh þ 2li] þ ions were different. in general, replacing the acidic proton of the sialic acid group by salt formation prevented ready loss of sialic acid under positive ion conditions and accentuated differences between the spectra of isomeric sugars containing sialic acid at different positions. neu5ac-α-(2→3)-gal-β-(1→3)-glcnac-β-(1→3)-gal-β-(1→4)-glc 22. lstb gal-β-(1→3)-glcnac-β-(1→3)-gal-β-(1→4)-glc | neu5ac-α-(2→3) studies with a maldi-ion-trap-tof instrument have shown that many of the fragments in the positive ion spectra of n-glycans originate from multiple sites. in the spectra of high-mannose glycans an ion formed by loss of the chitobiose core and 3antenna is diagnostic for the composition of the 6-antenna. however, in the spectra of biantennary glycans (23), the abundant ion at the equivalent mass (m/z 712, hex 3 -hexnac) was shown to be formed predominantly from the b 5 ion (loss of the reducingterminal glcnac) and gal-glcnac from each antenna . symbols as for 20 plus % = galactose the programming language java has been used to write a program for identifying the total monosaccharides present in a tryptic glycopeptide containing up to six o-linked glycans from the hinge region of human serum immunoglobulin 1 (igg1). the program also calculated the amount of each glycopeptide as a percentage of total peak area . a modification and extension to the stroligo algorithm (ethier et al., 2002) for assigning structures of n-linked glycans from their cid spectra has been published (ethier et al., 2003) . spectra were recorded with a maldi-q-tof instrument, isotope-stripped and examined for peaks corresponding to monosaccharide losses. the program then built a relationship tree that was analyzed with respect to fragment ion types and adduct. [m þ na] þ , the ions usually encountered in maldi spectra of carbohydrates are now catered for as well as the [m þ h] þ ions featured in the original version of the program. negative ion fragmentation was also accommodated. greater attention to known biochemistry was incorporated in the new algorithm and the program was able to annotate spectra with the most probable structure. a web-based tool entitled glyco-fragment, to be found at http://www.dkfz.de/spec/projekte/fragments/ accepts carbohydrate structures in the extended iupac nomenclature (http://www.chem.qmw.ac.uk/lupac/2carb/38.html), as developed for the carbohydrate database, carbbank, and calculates the masses of all possible fragments. the presence of reducingterminal derivatives, various adducts such as sodium or lithium, persubstituted (e.g., permethyl) derivatives and substituents such as sulfate, acetate and phosphate are also accepted. b-and y-type fragments are listed by default and masses of c-, z-and crossring fragments can be obtained on demand. internal fragments are not catered for. output is in the form of a list or, in ''view as structure'' mode, ions associated with cleavage of each bond are shown when the user moves a cursor over a specific glycosidic linkage (lohmann & von der lieth, 2003) . glycosearchms (http://www.dkfz.de/spec/glycosciences.de/sweetdb/ms/) compares each peak in a mass spectrum with calculated fragments from all structures in the sweetdb database and the best matches are displayed (lohmann & von der lieth, 2004) . constituent monosaccharides can be obtained from the program glycomod (http://www.expasy.ch) as demonstrated by ma et al. (2003a) to assign compositions to n-glycans released from the glycoprotein rat selenoprotein p. reviews of available databases relating to glycomics have been published (von der lieth, 2004a,b) . work in this area is summarized in table 1 . a comparison of the behavior of dextrans in positive and negative ion maldi has shown the expected formation of [m þ na] þ ions in positive mode whereas the negative ion spectra contained ions at [m-1-120] à as the result of fragmentation. under esi conditions on a shimadzu-kratos seq instrument, positive ionization again produced [m þ na] þ ions but with a bias towards the lower masses. the negative ion spectra were dominated by extensive a-type fragment ions. maldi was reported to be more sensitive than esi but esi was more useful for structural studies (cmelík, stikarovská, & chmelík, 2004) . comparisons of fragmentation modes for the [m þ na] þ ions from these compounds have shown that, whereas psd, isd, and cid all produced glycosidic cleavages, only isd and cid produced significant cross-ring cleavage. localization of the charge to the reducing terminus by formation of a 1-phenyl-3methyl-5-pyrazolone derivative (see 1/61) restricted cleavage to the non-reducing terminus (bashir et al., 2004) . the b-d-fructan (inulin; for fructose, see 1/16) from dahlia tubers and glucose syrup from potatoes, together with similar polysaccharides from red onion and jerusalem artichokes, were used as reference compounds by stikarovská and chmelík (2004) to evaluate the best matrices for these compounds. linear-maldi-tof analysis gave stronger signals than reflectron-tof; dhb was found to be the best matrix for starch and thap for inulin. less effective matrices were chca, haba, 3aminoquinoline (3-aq, 1/24) and sinapinic acid (1/48). depolymerization of most of these very large molecules is necessary before maldi analysis. enzymatic treatment is usually used (details in table 1 ) but heat treatment is also common. acetylation of glucuronoxylans and glucomannans has received considerable attention (table 1) with some evidence of acetyl group migration during depolymerization (kabel et al., 2003) . acetylated glucuronoxylans and glucomannans have been recovered from flax shive following hydrothermal treatment but in this case, acetyl migration was not reported . acetylation has also been found on the mannose residues of glucomannans from birch and aspen wood and appear to be randomly distributed (teleman et al., 2003) . maldi-tof spectra from these two papers consisted of an extensive series of peaks with a mass separation of 42 units (masses up to 5,500) but considerable simplification was found after deacylation. fernández et al. (2003) have examined arabinoxylans from wheat by maldi and esi mass spectrometry and detected structures with up to 22 monosaccharide residues. as arabinose (1/2) and xylose (1/3) are isobaric, it was not possible to study the distribution of arabinose residues along the xylose backbone. however, this problem was overcome by permethylation; arabinose residues attracted three methyl groups, unsubstituted internal xylose residues, two and substituted residues one group. matrix-assisted laser desorption/ionization (maldi) conditions for determination of the substitution patterns in methyl(momcilovic et al., 2003b) and carboxymethyl-cellulose (momcilovic et al., 2003a) have been evaluated. depolymerization was achieved both by use of enzymes or acid and the products fractionated by size-exclusion chromatography (sec). dhb, chca, haba and indoleacrylic acid (iaa, 3/5) were tested as matrices for methylcellulose but little difference was found between them. the choice of solvent, however, did have a significant effect, probably due to differential solubility of the variously methylated compounds. measurements on two acid-depolymerized samples of methylcellulose with different degrees of substitution gave values that agreed well with the involved in 2 ary cell wall metab. (goujon, et al., 2003) argania spinosa (argan tree) identification of novel xufg motif aspergillus wentii araf, gal structure of oligosaccharides that modulate intestinal immune system (taguchi, et al., 2004) azorhizobium caulinodans glc, man random acetylation on man residues of glucomannans. (teleman, et al., 2003) barley starch enzyme l-tof (+ve) (dhb) glc quantification of glucose from starch. sorbitol as internal stand. (grant, et al., 2003) barley (hull-less) heat maldi glc study of structural changes caused by heating (li, et al., 2004c) birch wood glucomannan -tof (dhb) sec, hpaec, nmr glc, man random acetylation on man residues of glucomannans. (teleman, et al., 2003) brassica campestris l. (field mustard) endo-(1→4)-βxylanase tof(dhb) glc, gc/ms, sec rha, fuc, ara, xyl, man, gal, glc structural characterization of polysaccharides from seed cake (ghosh, et al., 2004) chaetosartorya chrysella (rosenbohm, et al., 2003) coffea arabica (green coffee) endomannase tof (dhb/hiq) man, gal, acetyl groups structural characterization (oosterveld, et al., 2004) commercial sample structural determination in pharmaceutical formulations (kühn, et al., 2003) (continued ) structural characterization. inhibition of salivary α-amylase (kandra, et al., 2004) corn starch enzyme l-tof (+ve) (dhb) glc quantification of glucose from starch.sorbitol as internal standard. (grant, et al., 2003) craterostigma wilmsii endo-glucanase r-tof (dhb) ara, rha, xyl, man, gal, glc, glca, gala study of cell-wall composition of dehydrated resurrection plant. more gal than hydrated leaves. (vicré, et al., 2004) eleusine coracana (kabel, et al., 2003) flax shive acetyl-glucomannan hydrothermal l-tof (+ve, -ve) (dhb) sec glc, man no acetyl migration after hydrothermal treatment flax shive xylooligosaccharides hydrothermal l-tof (+ve, -ve) (dhb) sec xyl, me-glca isolation and characterization of water-soluble hemicelluloses gelidum amausii to study antioxidative properties (wang, et al., 2004a) gramineae (doco, et al., 2003) lasallia pustulata pustulan galactomannan acetolysis r-tof (dhb) nmr, ir, glc gal, α-man main constituents are pustulan, a β-glucan-chitin complex and a β-(1→3)-glucan (pereyra, et al., 2003) lycopersicon esculentum (tomato) xyloglucan-specific endoglucanase tof (dhb/hiq) nmr α-ara, β-ara, xyl, glc, gal structure of xyloglucan produced by suspension-cultured tomato cells. (jia, et al., 2003) manihot esculenta crantz, starch iso-amylase tof ("standard matrix") glc study of different strains. one has more water-soluble glycans and unusual starch with low branching (carvalho, et al., 2004) mesorhizobium haukuii cyclic-β-glucan (guérardel, et al., 2003) nothogenia erinacea xylan xylanase r-tof (dhb) esi, nmr xyl comparison of xylanases from different families (nerinckx, et al., 2004) olea europaea (olive) polygalacturonase, arabinofuranosidase maldi, hplc rha, gal, fuc, xyl, ara, man, glc structural determination related to oil extraction (vierhuis, et al., 2003) (reis, et al., 2003) picea abies (spruce wood) galactoglucomannan hydrothermal l-tof (+ve, -ve) (dhb) sec man, glc, gal, xyl, ara effect of temperature and ph for extn. of hemicelluloses. 2% naoh increased high mass (14 kda) extn. but removed oac groups (lundqvist, et al., 2003) picea abies (spruce wood) steam sec-maldi mannan and xylan investigations of compounds that can replace fossil-based products (palm & zacchi, 2004) potato, sweet potato (mou, et al., 2004) red wine rhamnogalacturonan ii endogalacturonase acid hydrolysis β-d-apif structural determination of heptasaccharide from red wine rhamnogalacturonan scheelea phalerata (palm) heteroxylan partial acid hydrolysis tof (dhb) , nmr, gc/ms glcpa, α-galpa, β-xylp, α-glcpa, araf structural characterization (simas, et al., 2004) schizosaccharomyces pombe glucans naoh/zymolyase tof (chca), nmr structural characterization (sugawara, et al., 2004) solanum tuberosum (potato) isoamylase tof (dhb) glc-phosphate phosphorylation of starch is required for degradation (ritte, et al., 2004) sugar beet pulp acetylated homogalacturonan endogalacturonase from fusarium moniliforme tof (thap), hpaec gala, me-gala products of enzymolysis show decreasing hydrolysis for more highly acetylated compounds (bonnin, et al., 2003) sweet potato water tof (dhb) glc investigation of hydrothermal conditions for glucose production (nagamori & funazukuri, 2004) tamarind xyloglucan pyrococcus furiosus β1→4-endoglucanase tof (dhb) glc xyl, glc structural determination. fragments with 2-9 residues. (marry, et al., 2003) wheat arabinoxylans endoxylanase a r-tof (dhb), esi, ms/ms structures with up to 22 monosaccharide residues detected. (fernández, et al., 2003) wood, various species hemicelluloses xyl, hexa xylan more abundant in surface layers of pulp fibres (dahlman, jacobs & sjöberg, 2003) (1) instrument (matrix) other technique, sample (derivative). data supplied by the manufacturers. however, data from the carboxymethyl-celluloses was less satisfactory even though samples with different degrees of substitution could easily be distinguished from each other. a method has been published for detecting hydroxyethyl starch in urine. the compound is used as a plasma volume expander by athletes and has been banned by the international olympic committee. partial acid hydrolysis of urine was carried out with tfa and the product was examined directly by maldi-tof using super-dhb (s-dhb). the method was claimed to provide unambiguous identification in only 90 min (gallego & segura, 2004 ). of the matrices dhb, nor-harmane (1/35), chca, haba, iaa, and sinapinic acid, dhb and nor-harmane were found by xiong et al. (2003b) to give the strongest and best resolved spectra from macrocyclic polysaccharides containing from 19 to 25 sugar residues. sinapinic acid gave a very weak signal and was only recorded in linear mode. the liquid matrix, chca/3-aq/ glycerol also gave a strong and long-lasting signal but with inferior resolution. addition of alkali metal salts produced the expected adducts. a glass slide containing a uv absorbing tio 2 sol-gel film from which imprinted a-cyclodextrin (cd (cyclic-(1 ! 4)-a-dglucose) 6 , 24) molecules had been removed has been used to selectively adsorb a-cd from a mixture of a-, b-, and g-cd. the a-cd was detected directly by maldi-ms without the addition of extra matrix . cyclic-(1→4)-α-d-glc) 6 experiments with a-, b-, and g-cyclodextrins have shown that the larger molecules have greater affinity for larger alkali metals suggesting that the products are inclusion complexes that can be ionized intact. sinapinic acid was used as the matrix and under these conditions, dextran, a linear polymer, produced only [m þ h] þ ions (bashir et al., 2003a) . inclusion complexes with cyclodextrins have recently been used to investigate the composition of humic acids from antarctica. the structure of humic acids is still largely unknown even though they are widely distributed in nature. gajdošová et al. (2003) recorded the maldi spectra of humic acids with and without g-cyclodextrin and found that several constituents appeared to form inclusion complexes. thus, for example, a shift in the cyclodextrin peak by 66.0 mass units after addition of humic acid from a standard soil sample was interpreted as consistent with inclusion of a cyclopentadiene radical (c 5 h 6 þ ). results from the soils from antarctica were similar. maldi spectra were recorded without a matrix to simplify the spectra. an investigation of the mechanism of the well-known migration of alkyl-silyl groups from the two-to the three position in cyclodextrins has shown that the receiving oxygen is that from the same ring as the migrating silyl group and not to the adjacent ring (teranishi & ueno, 2003) . matrix-assisted laser desorption/ionization (maldi) sample preparation protocols for examination of curdlan ((( ! 3)-b-d-glc-(1 ! )) n , 25), a polyglucose obtained from the bacterium alcaligenes faecalis var. myxogenes 10c3, have been reported. the crude sample was separated into a low molecular weight, water-soluble portion and a high-molecular weight, waterinsoluble portion. the low molecular weight portion was examined from dhb containing ammonium fluoride and gave two low-mass (<4 kda) polysaccharide distributions differing by 16 da. the high molecular weight, water-insoluble portion was found to produce good signals from a mixture of dhb and 3-aq in dimethylsulfoxide (dmso) that was dried on a hot-plate at 708c. ions with masses of up to 15,000 were observed (chan & tang, 2003) . spectra of extracellular polysaccharides from the colony-forming prymnesiophyte algae phaeocystis globosa and p. antarctica showed peaks of less than 1,000 da, some of which had a repeat pattern of 192 da but the monosaccharide units were not identified (solomon et al., 2003) . (→3)-β-d-glc-(1→)) n supplementation of a pre-term formula with a mixture of galacto-and fructo-oligosaccharides with a molecular weight range similar to that of carbohydrates found in human milk as determined by maldi-tof analysis has been found to have a stimulating effect on the growth of bifidobacteria in the intestine and results in more frequent produced and softer stools. thus, prebiotic mixtures such as the studied oligosaccharide mixture might help in improving intestinal tolerance to enteral feeding in pre-term infants (boehm et al., 2003) . it is unusual for all but the simplest, low mass glycoproteins to produce maldi-tof spectra with resolved glycoforms but broad peaks and significant mass differences between observed and calculated protein masses for larger glycoproteins suggest glycosylation. thus, epoetins (erythropoietins), with several glycosylation sites occupied by sialylated complex glycans, produced only broad peaks with masses in the range of 30-35 kda and half-height peak widths of up to 5 kda when examined by maldi-tof (stanley & poljak, 2003) . deglycosylation, however, produced sharp peaks from the protein with only minimal tailing. human follistatin expressed in cho cells has two n-linked glycosylation sites at asn-95 and 259. its maldi spectrum contained three peaks; the first at m/z 31,525 corresponded to the unglycosylated protein and the other two at m/z 33,804 and 35,600 were produced by unresolved glycopeptides. lc/ms analysis identified nine complex glycans (hyuga et al., 2004) . several partially resolved peaks from 52 to 55.5 kda suggest glycosylation of human b-secretase catalytic domain, confirmed as n-linked by digestion with pngase f . reduction in mass of 1.4 kda of a humanized anti-hbs fab fragment by treatment with endo-h gave a mass that was still 3.7 kda above that of the protein. the authors speculated that the extra mass might indicate o-linked glycosylation but no proof was provided (ning et al., 2003a) . similarly, a reduction in the mass of approximately 4.5 kda of a lysosomal serine carboxypeptidase from trypanosoma cruzi following endo-h treatment suggested the presence of two to three n-linked glycans (parussini et al., 2003) . other carbohydrates detected by massdifference measurements of proteins before and after deglycosylation include glcnac in cu/zn-superoxide dismutase from fungus humicola lutea 103 (dolashka-angelova et al., 2004b) and the high-mannose glycan man 10 glcnac 2 in ovotransferrin expressed in pichia pastoris (mizutani et al., 2004) . matrix-assisted laser desorption/ionization (maldi) analysis is also useful for detecting the absence (perteguer et al., 2004) or modification of glycosylation. thus, the mass of human transferrin produced in insect (drosophila melanogaster s2) cells is consistent with lack of sialic acids on its n-glycans (lim et al., 2004). 2. n-linked glycans a. analysis of derived glycopeptides and site occupancy. although n-linked glycans have long been known to attach to a consensus sequence consisting of an asn-xxx-ser(thr) motif, where xxx can be any amino acid except proline, evidence is now emerging that asn-xxx-cys can also act as a motif. satomi, shimonishi, and takao (2004) have identified a third site based on this motif, occupied by approximately 2% of the glycosylation, in human transferrin. proteolysis, usually with trypsin, to localize each consensus sequence site to an individual glycopeptide is the classical method for determination of the glycosylation at each site. detection of glycopeptides by hplc following proteolysis is often difficult because of effects such as signal suppression. monitoring of oxonium ions (163 for hexose, 204 for glcnac, etc.) is a standard method for tracking the glycopeptides and has been extended to monitoring with an ion trap mass spectrometer by generating them in the ion-source region (sullivan, addona, & carr, 2004) . peptide masses were confirmed by maldi-tof ms after desalting with c18 ziptips. a method for predicting peptide retention times in reversed-phase hplc has been published (krokhin et al., 2004b) following examination of 346 tryptic peptides and deglycosylated glycopeptides identified by maldi-tof ms from 17 proteins. glycopeptides eluted slightly earlier than their deglycosylated versions. liu, feasley, and regnier (2004) have evaluated the technique of diagonal chromatography which involves comparisons of hplc chromatograms run under identical conditions before and after enzymatic removal of the glycans and have concluded that, although the technique worked well for detection of protein phosphorylation, retention times of the glycopeptides and derived peptides, identified by maldi-tof ms, were too similar for diagonal chromatography to be a reliable technique for detecting glycopeptides. maldi-tof ms has the advantage over esi-ms for the detection of glycopeptides in that the spectra usually only contain singly charged ions unlike esi with its tendency to produce multiply charged ions. glycopeptides are often revealed in maldi profiles by their relatively high mass and by peak spacing corresponding to monosaccharide residues (e.g., 162 for hexose) (krokhin et al., 2004a) . rather than attempt detection of glycopeptides in the presence of peptides, some investigators employ fractionation techniques to isolate the glycopeptides before mass spectrometric analysis. for example, wada et al. (2004) have developed a method whereby glycoproteins are reduced and alkylated, cleaved with trypsin and lysylendopeptidase and partitioned with cellulose or sepharose to bind the glycopeptides. the isolated glycopeptides could then be examined by lc or linear maldi-tof. ms/ms in an ion-trap-tof instrument provided peptide fragments for protein identification and carbohydrate ladders that gave information on the glycan composition. the method was applied to transferrin, igg and b 2 -glycoprotein 1. a method has been reported for identification and quantification of n-linked glycoproteins following their specific immobilization on a solid support . cis-diol groups in the carbohydrate were oxidized with periodate to aldehydes allowing the carbohydrate to be captured by hydrazide chemistry. the protein was then cleaved with trypsin or another suitable enzyme and the resulting peptides were released with pngase f for analysis by maldi or esi mass spectrometry. deuterium labeling of the peptide with succinic anhydride allowed quantitative studies to be made. the frequent absence of glycopeptides in lc/ms analyses has been used to advantage in indicating which peptide might be glycosylated. thus, peptides containing the consensus n-glycosylation sites at asn-142, 173, and 178 were not observed by hambrock et al. (2004) from the tryptic digest of the recombinant protein tsc-36/flik expressed in human cells suggesting that these sites were glycosylated. an et al. (2003b) have used pronase to digest two wellcharacterized glycoproteins and found that steric factors prevented complete digestion in the region of the glycosylation site leaving the carbohydrate attached to a short peptide. n-glycan masses were obtained by maldi-ft ms from a separate experiment involving glycan removal by pngase f and subtracted from those of the glycopeptides to obtain the peptide mass and, hence, its structure. the method was claimed to be more rapid that the traditional method of proteolysis and the results easier to interpret because no large peptides appeared in the final sample. the method was applied to the study of site heterogeneity of xenopus laevis egg cortical granule lectin. asn to asp conversion after pngase digestion effectively labels the percentage of a given site that is occupied and is detected by a mass difference of one unit. the technique has been used, for example, to show glycosylation in 10 of the 11 consensus sequence sites of the su component of avian sarcoma/ leukosis virus envelope glycoprotein (kvaratskhelia et al., 2004) and to identify occupied glycosylation sites in membrane-bound glycoproteins. normally this latter task is difficult because 2d sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) gels are ineffective in separating these compounds. to overcome the problem, the proteins were solubilized in guanidine-hcl, digested with trypsin and the glycopeptides were recovered by lectin binding. following removal of the n-glycans with pngase f, the peptides with the asn to asp conversion were detected by maldi-q-tof ms (fan et al., 2004) . investigations of the spike glycoprotein from the sars virus have revealed four occupied and one unoccupied n-linked sites by molecular weight measurements made before and after glycan removal by pngase f and by observing the asn to asp conversion (ying et al., 2004) . in a similar experiment, digestion of the glycoprotein n-cam with aspn and trypsin resulted in the identification of glycopeptides, each containing a single glycosylation site. they were separated by hplc and analyzed by maldi-tof ms. five of the six sites were identified but glycopeptides from site asn-4 were missing. this site was eventually characterized by a direct maldi-fticr analysis of the glycopeptides released in-gel by trypsin (albach et al., 2004) . incorporation of 18 o into the aspartic acid from h 2 18 o can be used to label the newly produced aspartic acid and was used by barinka et al. (2004) to identify and confirm glycosylation on each of the 10 consensus sequence sites of glutamate carboxypeptidase ii. b. n-linked glycan composition from glycopeptide analysis. measurement of glycan masses directly or by subtraction of the mass of the peptide from those of glycopeptides leads directly to the constituent isobaric monosaccharide composition as illustrated by a recent study of n-glycan masses from the androgenic hormone of porcellio scaber (crustacea) (grève et al., 2004) . sometimes, with large glycopeptides, peaks are poorly resolved but can often be improved by chemical treatments. thus, for example, the core fragment of human luteinizing hormone gave a broad, poorly resolved peak (m/z 8,700-10,700) when examined from sinapinic acid with a linear tof instrument (jacoby, kicman, & iles, 2003) . the compound consisted of two peptide chains, only one of which was glycosylated, linked with four disulfide bonds. these bonds were reduced on the maldi target with dithiothreitol (dtt) to give a more fully resolved envelope of peaks from which the n-linked glycan composition was deduced by subtraction of the peptide masses. c. glycan release. most structural determinations of these compounds are performed on released glycans. experimental details for chemical and enzymatic release of n-glycans have been described (merry & astrautsova, 2003) . release with pngase f (or pngase a if the glycans contain fucose in a-(1 ! 3)-linkage to the core glcnac) or hydrazine are the preferred enzymatic and chemical release methods respectively. no new chemical release techniques or modifications were found during the review period reflecting the increasing popularity of enzymatic release, particularly with pngase f. additionally, pngase f release is not accompanied with the potential hazards of hydrazine. however, there is some controversy as to whether this enzyme releases all glycans, particularly from larger glycoproteins. thus, it has been shown not to release glc 1 man 9 glcnac 2 (28) from arylophorin under non-denaturing conditions and, in addition, reinhold et al. (zhu et al., 2004a) assert that pngase f fails to release a subset of glycans from c. elegans and thus use hydrazine. artifacts detected by maldi-tof analysis after in-gel release with pngase f have included glycans with urea attached to the reducing terminus (omtvedt et al., 2004) as the result of its inclusion in an earlier procedure for glycoprotein purification. deglycosylation with trifluoromethanesulfonic acid, a method that leaves the protein intact for further study has been reviewed (edge, 2003 using the well-studied glycoproteins a1-antitrypsin and ribonuclease b. gutternigg et al. (2004) , however, preferred att as the matrix because of its neutrality. a large number of studies have been conducted on the structural determination of n-glycans from isolated glycoproteins and from intact organisms or tissues; these are summarized in tables 2 and 3 respectively. among the more unusual glycosylations to have been reported are the presence of glc 1 man 9 glcnac 2 (28) in the insect antheraea pernyi (chinese oak silkworm) complex, di-, tri-, tetra-antennary peptides, not glycosylation control residence time β site-specific glycan characterization. (kamar, et al., 2004) α1-acid glycoprotein human pngase f r-tof (dhb), glycans and me esters complex altered glycosylation in patients with inflammation and diabetes. (higai, et al., 2003a) α1-acid glycoprotein human, ox, sheep, rat pngase f trypsin, chymotrypsin l-tof (dhb), glycans comparison of glycosylation in four species. different glycosylation in each. (nakano, et al., 2004) α1-acid glycoprotein human pngase f trypsin l-+ r-tof (dhb), glycans (various hydrazones) high-mannose hybrid, complex use of cationic or hydrophobic derivs. to enhance signal α1-acid glycoprotein human pngase f tof, glycans (2-aa) complex, fucosylated di-and tri-antennary new separation method. fucglycans up in cancer (szöllosi, et al., 2004) α1-acid glycoprotein cancer patients showed raised agp with abnormal glycans aminopeptidase n (stephens, et al., 2004b) α1-antitrypsin identification of congenital disorders of glycosylation α1-antitrypsin human hepatoma cells complex increase in 1→3-fucosylation in hepatoma cell line (higai, et al., 2003b) α1-antitrypsin -pngase f trypsin tof (dhb), glycans complex (di-, tri-, tetra-antennary) as reference compound to demonstrate on-target digestion amyloid fibril human trypsin tof, glycopeptide complex detection of one occupied glycosylation site (karimi, sletten & westermark, 2003) androgenic hormone structural identification (grève, et al., 2004) α1-antitrypsin under-glycosylation of plasma α1-antitrypsin not random (mills, et al., 2003a) table 2. use of maldi ms for examination of n-glycans from specific glycoproteins (see also asialofetuin fetal calf serum pngase f trypsin l-+ r-tof (dhb), glycans (hydrazones) complex use of cationic or hydrophobic derivatives. to enhance signal avidin complex glycan identification in human ovarian tumour marker n-cadherin high-mannose, complex structural characterization cd24 (heat stable antigen) pngase f r-tof (dhb), glycans complex structural determination from different cell lines (ohl, et al., 2003) chorionic gonadotropin complex abnormal (branched on 3-arm) biantennary glycans in choriocarcinoma jeg-3 cells cortical granule lectin pngase f pronase, trypsin high-mannose, complex asn-154, 217 n-glycan ident. method based on pronase digestion to determine site occupancy cytotoxin 3 naja kaouthia (cobra) venom pngase f trypsin l-, r-tof (dhb, sinapinic acid) glycopep. complex, asn-29 first ident. of glycosylation in three-fingered toxin (osipov, et al., 2004) decay-accelerating structural identification (lukacik, et al., 2004) ecto-5'-nucleotidase bull seminal plasma cnbr, trypsin l-, r-tof (chca), oligo-man, hybrid, complex, asn-403 structural identification (fini, et al., 2003) site-specific structure determination. (hyuga, et al., 2004) α-galactosidase (caputo, et al., 2003) glutamate carboxypeptidase trypsin, asp-a tof (chca, ferulic acid, dhb), glycopeptides all 10 sites occupied but nature of glycans not stated identification of glycosylation sites by 18 o-incorporation (barinka, et al., 2004) β 2 -glycoprotein i human trypsin, lysylendopeptidase tof (dhb), ms n complex as test compound for method based on hydrophilic affinity isolation (wada, et al., 2004) gp17/sabp human seminal fluid pngase f trypsin seldi, l-tof (chca) cid glycans (per-me) different n-linked glycan profile in pathological state (caputo, et al., 2003) gp120 hiv-i sf2 t r y p s i n l-tof (chca, +ve, dhb, -ve), lc/ms (esi), glycopeptides complex, high-man, 110, 154, 160, 170, 203, 214, 235, 249, 274, 304, 328, 334, 358, 370, 378, 415, 428 high-mannose complex (gal-fuc) structural identification. novel galactose-substitution on core fucose (wuhrer, et al., 2004c) hemocyanin megathura crenulata (keyhole limpet) hydrazine maldi, glycans (2-ap) high-mannose, hybrid first gal-β-(1→6)-man and fuc-α-(1→3)-galnac epitopes hemocyanin high-mannose, complex structural identification mainly by hplc. igd/ige human pngase f, ingel r-tof (dhb), hplc (2-ab) identification of glycans. interaction with lectins. (arnold, et al., 2004) igg human (sjögren's disease) hydrazine tof, glycans, (abee) complex structural ident. lower gal on glycans from patient (kuroda, et al., 2002) igg human hydrazine r-tof (dhb), maldi-q-tof, lc/ms (2-ab) hybrid, complex identification of congenital disorders of glycosylation igg α-gal-caps) structural identification igg human trypsin, lysylendopeptidase tof (dhb), ms n complex as reference compound for method based on hydrophilic affinity isolation (wada, et al., 2004) igm determination of site-specific glycosylation endo-d, pngase f tof (sinapinic acid) complex fuc-glcnac on asn-54 needed for max. il-10r2 binding (logsdon, et al., 2004) integrin pngase f tof, (+ve) (dhb), high-mannose, complex glycans from melanoma integrins have β1→6-branches (pochec, et al., 2003) (westphal, et al., 2003) lysozyme mutant (g49n) pngase f (in gel) high-mannose plus glcnac yeast shown to be capable of adding glcnac to glycans (yoko-o, et al., 2003) matrix metalloproteinase-9 human pngase f, trypsin tof, glycans complex (biantennary) structural determination. asn-28, 120 (kotra, et al., 2002) neoculin curculigo latifolia fruit paucimannosidicdeduced from mass structural characterization of taste-modifying protein (shirasuka, et al., 2004) neural cell adhesion molecule calves endo n pngase f trypsin tof (att), esi, ion trap, glycans (2-ap) complex hnk-1 epitope -sites 2,4,5,6 structural identification. polysialic acid removed by endo n treatment (wuhrer, et al., 2003a) ovalbumin chicken pngase f l-+ r-tof (dhb), esi, procyclin trypanosoma brucei hf, pngase f tof (chca), glycopeptides, glycans high-mannose identification of glycosylation mutants resistant to con a prostate hybrid, complex structure of glycans in normal and prostate cancer (ohyama, et al., 2004) receptor (gotte, et al., 2003) ribonuclease b bovine pancreas pngase f trypsin l-+ r-tof (dhb), glycans (hydrazones) high-mannose use of cationic or hydrophobic derivs. to enhance signal ribonuclease b bovine pancreas to investigate the effect of charged (sialylated) glycans on glycoprotein migration in isoelectric focusing gels thy-1 avian in lec1 or tn5 cells asp-n, glu-c tof (chca), glycopeptides paucimannosidic structural identification (mehndiratta, et al., 2004) transferrin human pngase f r-tof (dhb, 3-aq), glycans (me ester, ants) multiple changes in glycosylation explains poor sensitivity as etoh-use marker (flahaut, et al., 2003) transferrin human serum identification of congenital disorders of glycosylation transferrin structural identification. glycosylation at asn-491 in asn-xxx-cys motif. (satomi, et al., 2004) transferrin human trypsin, lysylendopeptidase tof (dhb), ms n complex as reference compound for method based on hydrophilic affinity isolation (wada, et al., 2004) transferrin human hepatoma cells pngase f r-tof (dhb), glycans complex increase in (1→3)-fucosylation in hepatoma cell line (higai, et al., 2003b) vicilin cor a 11 trypsin tof, glycopeptides to study allergenic behaviour. (lauer, et al., 2004) vitelline pngase f high-mannose, complex study of the effect of glycan structure on sperm-binding (vo, et al., 2003) vitellogenin high-mannose structural identification (khalaila, et al., 2004) β-xylosidase high-man (me-man) paucimannosidic structural identification (gutternigg, et al., 2004) caenorhabditis elegans (natsuka, et al., 2002) caenorhabditis elegans soluble glycoproteins hydrazine tof (dhb), glycans (2-ap) high-mannose, truncated complex structural identification and genetics (hirabayashi, et al., 2002) chinese hamster ovary cells with glcnact-iii pngase f l tof (s-dhb), glycans complex (with bisect) structural identification. glcnac-tiii produces bisects (lee, park & stanley, 2003b) chinese hamster, (wild and lec19 ovary cells pngase f tof (+ve, -ve) (s-dhb, thap), glycans high-mannose, complex lec19 cho cells have reduced ability to add galactose (lee, et al., 2003c) chinese hamster, (schmitt, et al., 2004a) human serum hydrazine r-tof (dhb), q-tof, lc/ms, glycans (2-ab) high-mannose, hybrid, complex identification of congenital disorders of glycosylation maize glycoproteins pngase a r-tof (dhb), glycans (reduced, per-me) paucimannosidic for detection of immunogenic core (1→3)-fuc and core xyl (bardor, et al., 2003a) mice erythrocytes hydrazine tof (dhb) complex β-gal-transferase-1 deficient mice produced more n-ac-lactosamine (kotani, et al., 2004) nicotiana benthamiana paucimannosidic, highmannose, complex structural identification. glycans similar to those of higher plants (viëtor, et al., 2003) oryza sativa (rice) pngase a r-tof (dhb), glycans (reduced, per-me) paucimannosidic for detection of immunogenic core (1→3)-fuc and core xyl (bardor, et al., 2003a) rice, maize, wheat, date pngase a l-tof, r-tof (dhb/hiq), gc/ms, glycans paucimannosidic n-glycan structure similar between mono-and di-cotyledons (léonard, et al., 2004) . paucimannosidic glycans with similar gal-b-(1 ! 4)-fuc and gal-b-(1 ! 4)-gal-b-(1 ! 4)-fuc groups at the reducing-terminal 6-position have been found in keyhole limpet (megathura crenulata) hemocyanin (wuhrer et al., 2004c ). an unusual difucosylated core structure man 3-glcnac 2 dhex 2 ) in which one of the fucose residues is attached to a mannose residue has been reported as a constituent of phaiodactylipin (phospholipase a 2 ) from scorpion anuroctonus phaiodactylus) venom (valdez-cruz, batista, & possani, 2004) . high-mannose glycans including the first report of a glycan with a gal-b-(1 ! 6)-man moiety have been reported from keyhole limpet hemocyanin together with glycans with a fuc-a-(1 ! 3)-galnac epitope which causes cross-reactivity with schistosoma mansoni glycans which share same epitope . some n-glycans from c. elegans and related organisms are substituted with phosphorylcholine (pc, 3/13) which is thought to modulate the host immune response. studies on their biosynthesis have shown that c. elegans microsomes transfer pc from l-a-dipalmitoylphosphatidylcholine (31) to both hybrid and complex n-glycans containing glcnac (cipollo et al., 2004a) . hl60 cells treated with the glucosidase inhibitor n-butyldeoxynojirimycin (nb-dnj, 32) produced a series of compounds with the structure of high-mannose glycans but with only one glcnac residue as the result of cytosolic cleavage from misfolded glycoproteins that had been exported from the endoplasmic reticulum (mellor et al., 2004b) . a novel trisulfated hybrid glycan containing a glca residue capping the gal-glcnac antenna has been identified from bovine myelin glycoprotein p0 (yan, kitamura, & nomura, 2003) . e. applications of maldi ms to the study of n-glycan function. the presence of n-glycans on ribonuclease has been shown to reduce oligomerization (gotte, libonati, & laurents, 2003 o-linked glycans continue to receive less attention than their n-linked counterparts, one factor being their tendency to occur in groups that make site analysis difficult. as with n-linked glycans, much structural analysis is conducted on released glycans. a method for the identification of sites modified by o-glcnac that relies on mild b-elimination followed by michael addition with dithiothreitol and known as bemad has been described (wells et al., 2002) . the method was validated by mapping three previously identified o-glcnac sites, as well as three novel sites, on synapsin i purified from rat brain and on the lamin b receptor and the nucleoporin nup155. a. studies on intact glycoproteins and glycopeptides. collision-induced decomposition (cid) spectra of glycopeptides obtained by esi from doubly charged ions frequently display prominent loss of the sugar moieties as the result of the labile protons. czeszak et al. (2004) have used maldi-psd on glycopeptides derivatized with a phosphonium group at the amino-terminus and have shown that the resulting singly charged phosphonium ions and a-type fragments retain the sugar. in contrast, when doubly charged ions were generated by proton addition, loss of the glycan was again seen. the method was claimed to be an effective alternative to electron-capture dissociation (ecd) and was used to locate up to three galnac residues on the full tandem repeat peptide derived from the muc5ac mucin. kurogochi, matsushita, and nishimura (2004) have reported that lift-tof with dhb as the matrix is an ideal system for fragmenting glycopeptides (b and y series ions) and have used it to determine the o-linked glycosylation sites in mucin-type glycopeptides. up to six o-linked glycosylation sites have been found in the hinge region of iga1 from human serum . tryptic digestion yielded a 33-mer glycopeptide that was examined by maldi-tof ms from thap/ammonium citrate. this matrix gave more highly resolved spectra than s-dhb or analysis of carbohydrates and glycoconjugates & chca; the chca spectrum, in particular, was very poorly resolved . structural analysis was by exoglycosidase digestion (sialidase and galactosidase) and site analysis was performed on the degraded glycans by further cleavage with the endoproteases glu-c from staphyloccus aureus and proteinase k from tritirachium album . maldi-tof analysis of the stalk-region glycopeptide (aptpvppptgtprpl) from murine cd8 has shown that all three threonine residues are glycosylated with hexnac. four peaks were obtained and attributed to molecules containing 0-3 additional hexose residues . the 47 kda portion of the 45/47 kda glycoprotein from mycobacterium tuberculosis expressed in streptomyces lividans has been found to contain glycans on both the n-and c-terminal tryptic peptide; maldi-tof analysis showed from 0 to 9 hexose residues at the n-terminus but the glycosylation site was not determined (lara et al., 2004) . the 13-amino acid-containing peptide isolated from conus textile contains a gal-galnac residue attached to threonine but with an unknown linkage between the sugar rings. maldi-tof analysis of the products of a b-galactosidase digest on a synthetic analogue containing the common b-d-gal-(1 ! 3)-a-d-galnac-carbohydrate showed that the enzyme was unable to remove the galactose residue suggesting the presence of a1 ! 3-linked galactose and this was confirmed by subsequent nmr analysis (kang et al., 2004) . tryptic glycopeptides from surface glycoproteins of the hepatitis b virus were found by maldi-tof ms and on-target exoglycosidase digestion to be o-glycosylated with gal-galnac or neu5ac-gal-galnac. the n-glycans were mainly biantennary complex (schmitt et al., 2004a) . b. applications of maldi to the structural determination of o-linked glycans. studies on the structural determination of o-glycans from isolated glycoproteins and from intact organisms or tissues are summarized in tables 4 and 5 respectively. studies on some specific structural types are summarized below. i. mucins. the ability of maldi ms to resolve complex mixtures of glycans has been utilized in studies of mucins from the jelly coats of amphibian eggs. thus, three layers of the jelly coat from x. laevis have been shown to contain different but overlapping glycan structures; 40 neutral and 30 sulfated compounds were identified by hplc (pgc column), maldi-ft-icr, cid and the ''catalog-library'' approach of cid peak matching reported earlier (tseng, hedrick, & lebrilla, 1999) . thirty-five neutral, sulfated and sialylated glycans have been identified from x. tropicalis by similar techniques combined with exoglycosidase digestion (zhang et al., 2004a) . sialylated glycans were stabilized by formation of methyl esters. sulfated core-2 and core-4 glycans have been identified by maldi-tof ms and nmr from respiratory mucins from a patient suffering from chronic bronchitis but, unlike the structures of glycans identified earlier from a cystic fibrosis patient (lo-guidice et al., 1994) , these glycans contained no sialic acid (degroote et al., 2003) . maldi-tof data were recorded in negative ion mode and no loss of sulfate was reported. human intestinal mucins show an acidic gradient along the intestinal tract which may explain the region-specific colonization of the gut by various bacteria (robbe et al., 2003b) . o-glycans of stomach mucosa have an antibacterial action against helicobacter pylori by inhibiting biosynthesis of cholesteryl-a-d-glucoside (identified by maldi-tof ms), a major cell wall component (kawakubo et al., 2004) . ii. glycosaminoglycans (gags). methods for the analysis of hyaluronan and its fragments have been reviewed (capila & sasisekharan, 2004) . a. unsulfated gags. hyaluronan oligomers ( , with masses up to 8 kda, obtained from hyaluronic acid by the action of bovine testicular hyaluronidase have been examined by electrospray and maldi-tof ms. electrospray showed the presence of oligomers with both odd and even numbers of sugar units whereas maldi and high-performance anion exchange chromatography (hpaec) showed only even numbered oligomers. the discrepancy was traced to the electrospray ion source which was producing cone-voltage fragmentation. it was recommended that for esi studies of compounds of this type, the cone voltage be kept low and precisely controlled (prebyl et al., 2003 ). an lc/ ms method for quantification of hyaluronic acid fragments in pharmaceutical preparations produced by the action of hyaluronate lyase from streptococcus agalactiae has been reported, with negative ion maldi-tof from dhb being used to record the glycan profiles of three fractions. the largest fraction showed peaks to 15 kda (kühn et al., 2003) . a model disaccharide, dua-glcnac (10 mmol), derived from heparan sulfate by heparitinase enzyme digestion and bearing an unsaturated 4,5uronic acid (dua) at the non-reducing end has been adducted to mercuric acetate and the product characterized by maldi-tof ms, confirming the formation of a mercury adduct (m/z calc. 639.9, obs. 640 with the expected spread of 6 mass units for hg 198 to hg 204 , consistent with the production of a cyclic 4,5mercurinium intermediate (skidmore et al., 2004) . b. sulfated gags. a common method for analysis of these glycans is the use of basic peptides for ion pairing as outlined in the earlier reviews in this series. thus, for example, cleavage of heparin by controlled g-irradiation has produced fragments enriched in highly sulfated sequences which were examined by maldi-tof ms using ion-pairing with (arg-gly) 19 -arg to stabilize the sulfates (bisio et al., 2004) . studies by maldi-tof ms and hplc of the serine protease inhibitor and its chondroitinase and hyaluronidase digestion products have shown that, in acute inflammation, its chondroitin-4 sulfate chain is both longer and undersulfated compared with control glycoproteins (capon et al., 2003) . combined maldi-tof and enzyme digestion have also been used by kett and coombe (2004) for gag analysis. a method for simultaneous analysis of both n-and o-linked glycans has been published by robbe et al. (2003a) . glycans were released by non-reductive b-elimination using the ammonia-based method described earlier by huang, mechref, and novotny (2001) (plihal, et al., 2004) bile core-1 core-2 structural identification. branched core i in cancer c1 inhibitor human in rabbit mammary gland β-elimination r-tof (dhb (+ve) thap/nh 4 citrate), glycans core-1 structural ident. of glycans expressed in milk (koles, et al., 2004b) carbohydratetrypsin, chymotrypsin tof (cinnamic acid), seldi, glycopeptides mannose structural identification. 3 regions of glycans with maximum of four mannose (boraston, et al., 2003) κ-casein neu5gc-gal-galnac structural identification (mamone, et al., 2003) κ-casein trypsin, gluc neu5gc-gal-galnac structural identification (holland, deeth & alewood, 2004) cd8 mouse trypsin r-tof (dhb), esi, glycopeptides core-1 structural identification chorionic gonadotrophin human in cho cells hydrazine r-tof (dhb cmbt), ssi, ion trap, glycans (2-ab) 2 to 6 residues, sialylated structural identification (gervais, et al., 2003) coagulation core-1 structural identification (lukacik, et al., 2004) granulocyteser-5,9, thr-10, core 1 structural identification (forno, et al., 2004) iga ( core ii structural identification, mainly by hplc 2-aminoacridone (amac, 1/58) or 8-aminonaphthalene-1,3,6trisulphonic acid (ants, 34). they were then separated by gel electrophoresis, the bands containing the glycans were excised and the derivatized glycans were extracted for analysis by maldi and electrospray ms. comparisons between the electrophoresis profiles from the amac (uncharged) and ants (charged) derivatives provided an indication of the charge state of the glycan. the method was designed mainly for profiling mucin glycans and was applied to porcine gastric mucin and bovine submaxillary mucin. a popular method for the independent study of both n-and o-glycosylation is to remove the n-linked glycans with, for example, pngase f and then examine the o-glycosylation, either by further release using b-elimination or by measurements on the glycopeptides obtained from the o-linked region (forno et al., 2004; trimble et al., 2004) . the well-established differential hydrazinolysis method was used by gervais et al. (2003) to study n-and o-linked glycosylation of human recombinant chorionic gonadotropin allowing o-linked glycans to be identified for first time. with the recent sequencing of several genomes, attention has become focused on how the complexity of higher organisms can be encoded by such a small number of genes. post-translational modifications and, in particular, glycosylation have emerged as important determinants of the control of biological systems. lamarre-vincent and hsieh-wilson (2003) have studied glycosylation of cyclic amp-responsive element-binding protein, a transcription factor essential for long-term memory and have shown the first link between o-glcnac and information storage in the brain. o-glcnac was detected by specific enzymatic radiolabeling with galactose from [ 3 h]udp-galactose to the 4position of o-glcnac. maldi-tof analysis of the tryptic peptides was used to show that the enzyme was modified with two o-glcnac residues. most organisms synthesize a-linked-polyglucans, such as glycogen, as an energy source when other reduced carbon compounds are insufficiently available but, to date, the method for the initiation of glycogen synthesis in prokaryotes has not been determined. in an attempt to rectify this gap in our knowledge, albrecht et al. (2004) have produced the eukaryotic enzyme yeast glycogenin (glg2p), a known initiator in fungi and animals, in e. coli and found it to be autocatalytically glucosylated at tyrosine residues 232 and 230 with from 4 to 25 glucose residues, as determined by maldi-tof and ms/ms analysis. further incubation with udpglucose resulted in transfer of more than 36 additional glucose residues over 20 min as detected by maldi-tof. in another study, the z under-o-glycosylation in iga nephropathy (horie, et al., 2003a) iga1 human serum identification of glycans. computer program to interpret data iga1 selenoprotein p glu-c trypsin l-tof (chca), glycopeptides thr-346, hex, hexnac, neu5ac structural identification (ma, et al., 2003a) zona pellucida 3 human and mouse β-elimination r-tof (dhb), glycans (per-me) gal, galnac, neu5ac, neu5gc proteins from both sources have same glycans (coppin, et al., 2003) caenorhabditis elegans total glycoproteins β-elimination tof, q-tof (dhb), glycans glca, gal, galnac srf-3 mutant, resistant to bacterial infection is deficient in glycoconjugates (cipollo, et al., 2004b) hepatitis b virus trypsin, o-glycosidase tof (att), glycopeptides galnac-gal-neu5ac structural identification (schmitt, et al., 2004a) human airway mucin core-2, core-4 biosynthesis of airway mucins related to bronchial disease (degroote, et al., 2003) human intestinal mucins β-elimination tof (dhb), gc/ms, glycans core-5 evidence for region-specific glycosylation dependent on ph (robbe, et al., 2003b) human tonsillar lymphocytes trypsin tof, glycopeptides galnac, gal, neu5ac asialo-o-glycans deposited in iga nephropathy (hiki, et al., 2004) mouse intestinal mucins β-elimination tof, gc/ms, glycans (n-meamide) core-1, core-2 alterations in intestinal mucins caused by parasite (holmén, et al., 2002) xenopus laevis egg mucin β-elimination ft-icr (dhb (+ve) 2,5-dha (-)), cid, glycans neutral, sulfated, core-2 structural determination. catalog library approach ox submaxillary gland mucin neutral, acidic, unspecified core non-reductive β-elimination with nh 4 oh allows labeling (robbe, et al., 2003a) rat skeletal muscle (zhang, et al., 2004a) n-terminal segment of potato x virus has been found to be glycosylated at the n-terminus with galactose or fucose and that the presence of the sugar mediates the formation of a bound water shell on the virion surface (baratova et al., 2004) . glycan profiling by maldi-tof ms for the detection of disease is gaining ground. for example, a new method has been developed for the extraction of the acute-phase glycoprotein, a1-acid glycoprotein from human serum and its n-glycans have been compared in patients with inflammation or cancer and healthy controls. the disease states produced an increase in both the degree of branching and in the amount of fucosylation szöllosi et al., 2004) . the amounts of fucosylated bi-, tri-, and tetra-antennary glycans were also found to increase at the expense of unfucosylated triantennary glycans in this glycoprotein in patients with acute inflammation (higai et al., 2003a) . sialylated glycans were stabilized for maldi-tof analysis by formation of methyl esters. the same stabilization method was used by flahaut et al. (2003) in a study on the effect of ethanol on transferrin glycosylation in chronic alcoholics has revealed patient-dependent differences in both the level of sialylation and in the number of glycans attached to the molecule, possibly accounting for the controversial use of transferrin glycosylation as a marker for chronic ethanol consumption. a. detection of cancer biomarkers. n-glycans from normal human pancreatic cells consist mainly of complex biantennary structures with small amounts of tri-and tetra-antennary compounds and larger compounds with poly-n-acetyllactosamine extensions and extensive fucosylation (up to five fucose residues and eight gal-glcnac units as detected by maldi-tof ms). glycans from ribonuclease extracted from pancreatic adenocarcinoma tumor cells, on the other hand, contain fucosylated hybrid structures and biantennary glycans whose antennae terminated in galnac, thus providing a potential diagnostic test for the adenocarcinoma (peracaula et al., 2003a) . maldi-tof analysis gave much better resolution of the large glycans than could be obtained by hplc. altered glycosylation has also been found associated with prostate cancer; glycans from prostate-specific antigen (psa) contained increased amounts of fucose and galnac in the antennae, again indicating a possible diagnostic test (peracaula et al., 2003b) . another study found increased amounts of a-(2 ! 3)-linked sialic acid in human polysialic acid (psa) suggesting that differential binding to maackia amurensis agglutinin lectin could be used to differentiate prostate cancer from benign prostate hypertrophy (ohyama et al., 2004) . masses of some sialylated glycans were reported from linear maldi-tof spectra but most measurements were made on neutral glycans following linkage-specific removal of sialic acid. increased fucosylation has also been found in n-linked glycans from human hepatoma (higai et al., 2003b) and melanoma (ciolczyk-wierzbicka et al., 2004) cell lines and a reduction in the concentration of fucosylated glycoproteins has been found in dogs undergoing treatment for lymphosarcoma (xiong, andrews, & regnier, 2003a ). an extensive study of the n-and o-glycosylation of the human ovarian tumor marker ca125 has been reported. the bi-, tri-, and tetra-antennary glycans were identified by maldi-tof, ms/ms and fab mass spectrometry as permethylated derivatives coupled with exoglycosidase digestion . highly processed glycans have also been found on the breast tumor marker protein, gross cystic disease fluid protein-15, than on the same protein from healthy controls (caputo et al., 2003) . glycans were again examined as permethylated derivatives but, this time, by surface-enhanced laser desorption/ionization (seldi) mass spectrometry. muc1 glycoprotein is overexpressed in breast cancer and o-glycans have been shown to control proteolysis by preventing proteolysis of the thr3-ser4 bond if either amino acid is glycosylated (hanisch et al., 2003) . tetrasialylated-tetra-antennary glycans carrying a b1 ! 6 antenna appear to be associated with the development of cancer metastases as demonstrated by studies on integrins from metastatic and non-metastatic human melanoma cell lines (pochec et al., 2003) . maldi-tof profiles were obtained following in-gel glycan release using the method described by küster et al. (1997) as modified by hoja-lukowicz et al. (2000) . bi-and tri-antennary glycans have been identified attached to human chorionic gonadotropin produced in a choriocarcinoma cell line (jeg-3); the biantennary glycans, which were useful markers, were unusual in having a branched 3-antenna (35) rather than carrying both unbranched 3-and 6-antenna . the structure was confirmed by maldi-tof analysis of exoglycosidase digestion products with an a-mannosidase digestion following b-galactosidase digestion to confirm the presence of the exposed mannose residue on the 6-antenna. the study illustrates the danger of assigning structures directly by matching masses with those from a database or by using only the usual exoglycosidase sequence of b-galactosidase followed by n-acetylhexosaminidase which would incorrectly have suggested a normal biantennary structure such as 23. b. congenital diseases of glycosylation. matrix-assisted laser desorption/ionization-time-of-flight (maldi-tof) analysis of both the n-glycans released from tryptic peptides and of the peptides themselves has shown that the asparagine residues of a1-antitrypsin in patients suffering from congenital disorder of glycosylation (cdg) type i (deficiency in the initial protein glycosylation by glc 3 man 9 glcnac 2 , 36) are preferentially glycosylated in the order asn-46 > 247 > 83 (mills et al., 2003a) . asparagine occupation was measured by observing the extent to which it was converted to aspartic acid during glycan removal by the enzyme pngase f. in a newly characterized disorder, cdg-1i, maldi-tof and hplc were used to show that some of the n-glycans on fibroblasts consisted only of man 1 glcnac 2 and man 2 glcnac 2 (thiel et al., 2003) . analysis by maldi-tof and hplc of serum glycoproteins from several patients with cdg type ii syndromes (defective glycan processing) has been used to identify several enzyme defects. one patient was characterized with a mannosidase-iii deficiency that prevented biosynthesis of complex glycans as the result of failure to remove residual mannose residues from the 6-antenna. the predominant hybrid glycan 37 was characterized by cid using a maldi-q-tof instrument . a-mannosidase deficiency causes accumulation of mannosecontaining oligosaccharides (released from n-glycans) in various tissues but the disease has been successfully treated by enzyme replacement therapy. residual glycans were monitored in this study by maldi-tof ms (roces et al., 2004) . a glcnactransferase-ii deficiency in another patient in the study by butler et al. prevented c. other diseases. the hinge region of iga1 has been shown by maldi-tof ms to be under-o-glycosylated in patients with iga neuropathy (horie et al., 2003a) . as upper respiratory tract infection often precedes hematurea and proteinurea in these patients, horie et al. (2003b) have investigated the effect of tonsillectomy and steroid treatment and found that they produce increases in the extent of iga o-glycosylation. again, analysis was by maldi-tof with s-dhb as the matrix. a method has been reported for detection of the biomarker, serum amyloid component, in human plasma or urine. extraction was by monoclonal antibody binding and analysis by maldi-tof ms from sinapinic acid. several new, desialylated compounds were detected from three individuals (kiernan et al., 2004) . krokhin et al. (2003) have identified, in a preliminary study, several high-mannose and complex n-glycans from the spike protein of the sars virus taken directly from patients. the compounds were identified mainly with the aid of ms/ms on a q-tof instrument. work with these compounds usually involves analysis of intact proteins that are modified with chemically attached glycan residues and are ideal subjects for the high mass resolving ability of maldi-tof instruments. analysis of b-lactoglobulin glycation products formed from lactose and galactose has revealed lysine as the main binding site with additional binding at the amino-terminal leucine residue and at arg-124. maldi-tof analysis was found to be more efficient than lc/esi-ms for locating the binding sites (fenaille et al., 2004) . maldi-tof has, for the first time, enabled kinetic data to be obtained on defined glycation products from specific sites of a glycated protein. thus, lysozyme was incubated with d-glucose for 1, 4, 8, or 16 weeks in phosphate-buffered saline, digested with endoproteinase glu-c and the c-and n-terminal peptides were used for the relative quantification of the initial amadori product, n e -(carboxymethyl)lysine (38) and an imidazolone (39) formed from arginine (kislinger et al., 2003) . the preferred glycation sites in human serum albumin (hsa) have been determined as lysines 233, 276, 378, 525, and 545 following incubation of hsa with glucose, enzymatic digestion of the protein with either trypsin or endoproteinase lys-c, and examination of the resulting glycated peptides by lc/ms/ms and maldi-tof from chca (lapolla et al., 2004a) . glycation of casein by glucose, fructose or ribose has been characterized and shown not to affect its protective effects on human intestinal caco-2 cells (jing & kitts, 2004) . a study of 20 type 2 diabetic patients and 10 controls have shown that complications of diabetes are associated with a higher concentration of glyco-oxidized variants of globin than observed in controls or patients without complications (lapolla et al., 2004b) . several other studies on advanced glycation end products (ages) have been reported. yamada et al. (2004) have synthesized a collagen model peptide and subjected it to glycation with glucose, ribose and glyoxal (40). a dimeric peptide linked glyoxal-lysine dimer (41) was isolated and identified by maldi-tof as the first example of an intact glycation-derived dimer. the same technique has been used to show that methylglyoxyl, produced during the maillard reaction binds to arg-184 and arg-185 located within the binding site of bovine glutathione peroxidase and to deactivate the enzyme, resulting in an increase in the intracellular peroxidases responsible for oxidative cellular damage . ages from ribose were formed more rapidly when incubated with bovine serum albumin (bsa) than those from glucose or fructose as measured by binding to age receptors (valencia et al., 2004) . the use of in-house-developed perl script software has been used to identify and localize ages on b 2 -microglobulin . the program identifies masses, measured by maldi-tof ms that correspond to those of peptides with an age modification. in other studies, the ages n e -(carboxymethyl)-lysine, imidazolone a (42) and b (43), pyrraline (44) and 1-alkyl-2-formyl-3,4glycosyl pyrrole (45) have been found on the type 2 ryanodine receptor calcium-release channel, accounting for decreased cardiac contractility in diabetes . the soluble age receptor (srage), a member of the immunoglobulin superfamily, has been characterized from mouse and its nglycosylation identified by maldi-tof ms (hanford et al., 2004) . in another study, measurements of protein molecular weights have added support to the conclusion that amadori decomposition pathways are constrained in the presence of metal ion chelators and radical traps (culbertson et al., 2003) . although protein glycation is usually associated with pathology, as in the secondary effects of diabetes, the reaction is also involved in cooking where it changes the color (browning) and flavor of the food. fay and brevard (2004) have recently reviewed the use of mass spectrometry in the study of the involved maillard reaction. one protein encoded by the 13 drosophila peptidoglycan recognition proteins has been shown to possess enzymatic activity. maldi-tof analysis of the degradation products demonstrated that the enzyme hydrolyses the lactylamide bond between the glycan chain and the cross-linking peptides. the enzyme was, thus, classified as an n-acetylmuramoyl-l-alanine amidase (mellroth, karlsson, & steiner, 2003) . hplc and maldi-psd were used by antignac et al. (2003) to characterize the peptidoglycan from neisseria meningitidis. the peptidoglycan was hydrolyzed with muramidase from streptomyces globisporus. fourteen of the 28 muropeptides separated by hplc were found to be o-acetylated to different degrees. proteoglycan-like glyconectins from three sponges, microciona prolifera, halichondria panicea, and cliona celata have been examined by nmr and mass spectrometry. the structures were complicated and contained distinct acid-resistant and acid-sensitive domains. some sugars were sulfated and others pyruvylated (guerardel et al., 2004) . muropeptides derived from escherichia coli peptidoglycan have been labeled with the fluorescent dyes ants, 7-aminonaphthalene-1,3-disulfonic acid (anda, 46) or 4-aminonaphthalene-1-sulfonic acid (ansa, 47) and examined by fluorophore-assisted carbohydrate electrophoresis (face). results compared favorably, both qualitatively and quantitatively with earlier hplc-based methods. maldi-tof ms was used to confirm the identity of the face separated glycans (li, höltje, & young, 2004d some small anaerobic bacteria such as treponemes contain glycoconjugates that are different from the normal glycolipids. one of these glycoconjugates has been isolated from the oral spirochete, treponema medium by phenol/water extraction and the carbohydrate removed enzymatically for structural identification by nmr and maldi-tof ms. fragmentation was performed with a tof/tof instrument and revealed the glycan sequence of a tetrasaccharide repeat unit containing the two amino acids ornithine (48) and asp. the repeat unit ( that was amide-linked to l-ornithine and fucose linked to daspartic acid (hashimoto et al., 2003a) . many examples of the application of maldi-based methods to analysis of these compounds have been published; these are summarized in table 6 and only a few representative studies and some with more unusual features will be discussed here. extractions are normally carried out with hot aqueous phenol. these compounds often contain many more monosaccharide constituents than are found in the glycoproteins, requiring additional techniques such as combined gas chromatography/ mass spectrometry (gc/ms) and nmr for their analysis. their generally acidic nature, conferred by kdo glycans and phosphate groups, makes them ideal candidates for examination in negative ion mode. a. intact los. although small lipooligosaccharides can be examined directly, most have to be modified by hydrolysis of the large o-glycan chain or by removal of much of the lipid component of the lipid a (1/18) moiety. thus, for example, the backbone structures of the los from v. parahaemolyticus strains have been obtained following o-and n-deacylation with hydrazine and dephosphorylation with tfa (hashii et al., 2003a,b) . laser-induced decomposition can be a problem or, in some cases, can yield useful structural information. thus, the negative ion maldi-tof spectrum from dhb of the shortchain los from a strain of v. parahaemolyticus (3 kda) following deacylation showed successive losses of phosphoethanolamine (49), hexuronic acid and phosphate (hashii et al., 2003b) . los from m. catarrhalis has been deacylated with hydrazine and examined in negative linear mode from dhb/ hydroxy-iso-quinoline (hiq). the spectrum showed several peaks produced by in-source cleavage at the labile kdo-lipid a glycosidic bond (luke et al., 2003) . the lipid a molecule itself, examined in reflectron mode, was found to contain seven acyl groups and varying amounts of ethanolamine esterified to the two phosphate groups. both maldi-tof and nanospray ms/ms analysis of the de-o-acylated (hydrazine) los from pseudoalteromonas haloplanktis tac 125 grown at 158c has shown the presence of three phosphate groups, two on the lipid a and one on the core oligosaccharide, whereas at 258c, an additional phosphate was added to the core (ummarino et al., 2003) . the los from pseudoalteromonas issachenkonii kmm 3549 t has been found to contain what is thought to be the first example of a 4,7-disubstituted heptose as determined mainly by nmr; maldi-tof ms was used to delineate the lipid a acylation pattern (silipo et al., 2004b) . two sets of ions were obtained as the result of cleavage of the labile kdo-lipid a bond. haemophilus ducreyi has been shown to be capable of incorporating sialic acids with n-acyl groups larger than acetyl. however, the extent of sialylation dropped with increasing chain length, becoming non-existent for sialic acid with chains above c8 (goon et al., 2003) . the los was deacylated with hydrazine and examined by maldi-tof from dhb/hiq in negative ion mode; positive ion ms/ms spectra were obtained with electrospray ionization. b. lipid a. mild acid hydrolysis of the labile bond between kdo and the lipid a moiety is the normal method for releasing lipid a from these glycolipids. a method combining thin-layer chromatography (tlc) with both maldi-tof and esi-ms n (up to ms 4 ) has been applied to the analysis of lipid a from e. coli. the predominant fragmentation was loss of acyl groups but, whereas this appeared to occur by charge-remote processes in the cid spectra, charge-driven processes appeared to predominate in the maldi spectra (lee et al., 2004a) . several novel lipid as have been discovered during the review period. the first example of a neutral lipid a has been isolated from the predatory bacterium bdellovibrio bacteriovorus (schwudke et al., 2003) . the molecule consisted of a b-(1 ! 6)linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six hydroxylated fatty acids. a-d-mannopyranose residues occupied the two positions normally occupied by phosphates. a similar 2,3-diamino-2,3-dideoxy-d-glucopyranose (preston, et al., 2003) burkholderia caryophylli of ara4n at reducing end of lipid a (molinaro, et al., 2003) burkholderia cepacia eps l-tof (+ve, -ve) (dhb), nmr -α-d-gal, β-d-gal, kdo structure. growth in mannitol-enriched medium gave →6)-β-dfruf-(2→ (levan) (cescutti, et al., 2003) butyrivibrio fibrisolvens h10b (andersson, et al., 2003) chlamydophila psittaci 6bc lps lipid a l-tof (thap + 0.1% tfa), nmr 5 α-glcn, β-glcn, kdo structure. lps less active than typical endotoxins in cytokine induction. chlamydia trachomatis e, l 2 lps lipid a l-tof (thap + 0.1% tfa), nmr 5 α-glcn, β-glcn, kdo structure. lps less active than typical endotoxins in cytokine induction. coxiella burnetii glcpn lipid as with different degrees of ac more potent than fully acylated (mueller, et al., 2004) francisella novicida in e. coli l-tof (att) 6 glcn, kdo lipid a 1-de-phosphorylation occurs on periplasmic surface of inner membrane (wang, et al., 2004c) haemophilus ducreyi lps tof (dhb/hiq) -neu5ac homologues, β-d-glcnac h. ducreyi can incorporate unnatural sialic acids into lps (goon, et al., 2003) (tran, et al., 2004) klebsiella pneumoniae essential for lps synthesis (regué, et al., 2004) leptospira interrogans lipid structural characterization (corsaro, et al., 2004) pseudomonas stutzeri ox1 core tof (-ve) (thap), nmr -glcn, galn, hep, kdo, glc, pyruvic acid structural determination of novel highly negatively charged lps (leone, et al., 2004a) pseudomonas stutzeri ox1 (hashii, et al., 2003b) xanthomonas campestris pv. pruni lipid a l-, r-tof (thap) 6 glcnacp, phosphate presence of methyl-branched acyl chains (silipo, et al., 2004d) yersinia pseudotuberculosis (marceau, et al., 2004) disaccharide carrying six hydroxylated fatty acids was found in the lipid a from leptospira interrogans which causes a hemorrhagic fever known as weil's disease. the molecule carried only one phosphate group which was mono-methylated. the two hydroxy-acyl groups attached to the diamino-glucose residue i were esterified with unsaturated o-6-12:1 and 14:1 fatty acids (que-gewirth et al., 2004). 2,3-diamino-2,3-dideoxy-dglucopyranose has also been found in lipid a from bartonella henselae, a gram-negative bacterium that causes cat-scratch disease (zähringer et al., 2004) and in that from mesorhizobium huakuii. in the latter compound, the phosphate group normally attached to the di-nh 2 -glc moiety ii was replaced by d-glucuronic acid (choma & sowinski, 2004) . a 4-amino-4deoxy-l-arabinopyranose-1-phosphate has been found at the reducing end of the lipid a from burkholderia caryophylli (molinaro et al., 2003) . the negative ion maldi-tof (dhb) spectrum showed considerable heterogeneity due to differing numbers of acyl groups. the proximal (reducing end) glcn residue of lipid a from rhizobium leguminosarum lacks a phosphate group and can be oxidized to 2-amino-2-deoxy gluconate by an enzyme recently identified in the outer membrane of the bacterium (que-gewirth et al., 2003). the first example of lipid a from an agrobacterium species (a. tumefaciens strain c58, a plant pathogen) has revealed the presence of a normal bis-phosphorylated glucosamine disaccharide backbone with several acyl variants. the main species was a penta-acyl derivative bearing two 3-oh-14:0 fatty acids in ester linkage and two 3-oh-16:0 acids in amide linkage. the 3-oh-16:0 acid on glcn ii was esterified with 27-oh-28:0 that, in turn was esterified with a 3-oh-butyroyl residue . other lipid a molecules to have been analyzed in the review period are listed in table 6 . c. core oligosaccharide. mild acid hydrolysis is also the method normally used to obtain core oligosaccharides from lps. ethanolamine diphosphate has been identified for the first time and its position of substitution established in the lps core of a rough-type mutant of pseudomonas aeruginosa (pao1 dalgc). in addition, the paper reported a reinvestigation of the structure of the complete lps core of wild-type p. aeruginosa pao1 and a reassignment of the phosphorylation sites (kooistra et al., 2003) . the lps was complex because of the presence of esterified fatty acids that were removed by mild hydrazinolysis prior to analysis by esi and maldi. phosphoethanolamine has also been found in strains of n. meningitidis and 4,6-o-(1-carboxy)-ethylidine residues derived from pyruvic acid have been identified in the core of los from pseudomonas stutzeri ox1 (leone et al., 2004a) . the o-chain from bordetella avium lps, obtained by mild acid hydrolysis to remove the lipid a followed by hydrofluorolysis to remove the core oligosaccharide, was found to consist only of a chain of the substituted 1 ! 4 linked monosaccharide 2-acetamido-3-(3-hydroxybutanamido)-2,3dideoxy-b-d-glucopyranosyluronic acid (50). the negative ion maldi-tof spectrum from dhb showed a series of peaks from 1 to 3.8 kda (larocque et al., 2003) . other examples of o-chain analysis are cited in table 6 . table 6 and only two representative examples will be expanded here. the structure of the repeating unit of the eps from butyrivibrio fibrisolvens strain h10b is a hexasaccharide carrying o-acetyl and 1-carboxyethyl groups and has been determined with a variety of techniques including nmr and circular dichroism (cd) following extraction with phenol. maldi-tof ms of fractions from a partial acid hydrolysate was used to determine the carbohydrate sequence (andersson, cotta, & kenne, 2003) . b. cepacia, a bacterium causing lung infections was obtained from a cystic fibrosis patient and cultured on agar plates. phenol was added to the harvested cells, stirred for 5 hr at 48c and the eps was precipitated with isopropanol. maldi-tof and nmr were used to determine the structure of the repeating unit as . when grown on a mannitol-rich medium, conditions that induce the formation of eps, the bacteria produced a second compound with the structure ( ! 6)-b-d-fruf-(2 ! ) (cescutti et al., 2003) . a. studies on the glycan moiety following removal of the ceramide. glycosphingolipids can be analyzed directly by maldi-tof but the spectra are usually complicated because of heterogeneity in the ceramide portion of the molecule. thus, many investigators use an enzyme such as ceramide glycanase to remove the ceramide. a recent example is a study of the effect of n-alkyl analogues of deoxynojirimycin (dnj), inhibitors of ceramide glucosyltransferase, on gsl structure. gsls were cleaved with ceramide glycanase and labeled with 2-ab (1/56) for analysis by hplc and maldi-tof (mellor et al., 2004a) . carbohydrates substituted with phosphocholine (pc) and phosphoethanolamine (pe) have been released with endoglycoceramidase from glycosphingolipids of the pig parasitic nematode ascaris suum and identified by maldi, esi, gc/ ms (methylation analysis) and nmr (friedl et al., 2003) . a rapid method for splitting glycosphingolipids into their three components (sugar, sphingosine and fatty acid) by use of a domestic microwave oven has been reported by itonori et al. (2004b) . the glycosphingolipids were heated with 0.1 m naoh/ meoh for 2 min followed by 1 m hcl/meoh for 45 sec. the alkaline methanolysis step produced the intermediate lysoglycosphingolipids virtually free of by-products such as the omethyl ethers that are usually seen but the n-acetylamino-sugars needed re-acetylation by reaction with acetic anhydride/pyridine at room temperature. b. studies by tlc and maldi. glycosphingolipids are frequently separated by tlc. ivleva et al. (2004) have published a method for attaching tlc plates to a maldi target and have obtained spectra of even fragile compounds such as gangliosides by using an fticr instrument fitted with a vibrationally cooled maldi ion source. twenty matrices were investigated and the soft matrices, dhb, att and anthranilic acid (1/57) were found to produce the least sialic acid loss from gangliosides. tlc resolution was best preserved by using matrices in rapidly evaporating organic solvents. c. applications to the structural elucidation of glycosphingolipids. a novel glcnac-a-1 ! hpo 3 ! 6-gal-(1 ! 1)-ceramide (51) has been identified from the liver fluke, fasciola hepatica, by maldi-tof and electrospray-ms/ms. hf treatment released the glcnac residue, confirming the glcnacphosphate link (wuhrer et al., 2003b) . mammalian type globoand iso-globotriaosylceramides have also been found in this species obtained from infected sheep. maldi-tof spectra were obtained from both the intact glycosphingolipids and the 2-ap derivatives of their released glycans using att as the matrix, and the presence of antigenic terminal a-galactose was demonstrated by on-target incubation with a-galactosidase (from chicken) (wuhrer et al., 2004a) . ceramides carrying b-gal-(1 ! 6)-b-gal-(1 ! 1)-cer and larger oligosaccharides with from one to three additional b1 ! 6-linked mannose residues represent a departure from the common glycosphingolipids that contain inositol phosphate found in many fungi and may account for the resistance of this species to the fungicide, aureobadidin a . measurements by seldi ms of tetanus neurotoxin (53.7 kda) incubated with the ganglioside gt1b, and observation of the intact protein/ganglioside complex, has shown that two carbohydrate binding sites are required for toxicity (rummel et al., 2003) . d. miscellaneous studies. two photoaffinity probes (ganglioside gm2 labeled on the acyl chain with a p-(3-trifluoromethyl)diazirinyl-phenyl group) (52), were used to probe the active site of gm2-activator protein. after binding, and trypsinization, the tryptic peptides were examined by maldi-tof and esi-ms and found to be bound to the most flexible region of the protein (wendeler et al., 2004a) . g-radiolysis of glucosylceramide and galactosyl diglyceride resulted in the release of the sugars as shown by maldi-tof ms (shadyro et al., 2004) . other studies and details of the above work are summarized in table 7 . novel zwitterionic glycosphingolipids having the sugar chain capped with phospholcholine have been reported from the mycelia of filamentous fungi of acremonium species . structures were of the type pc ! 6-(man-(a1 ! 6)-man-(a1 ! 6)-glcn-(a1 ! 2)-ins1-p-1-cer. a related compound without the attached glycan chain has been identified from the red alga gracilaria verrucosa (khotimchenko & vas'kovsky, 2004) . the glycoglycerolipid a-d-glcp-(1 ! 3)-a-d-glcp-(1 ! 1)-lipid where the lipid is glycerol containing a c 15 acyl group at c-2 and a c 15 ether at c3 has been separated from the propionibacterium propionibacterium propionicum by tlc and its structure determined by maldi-tof, nmr, esi/ms, and gc/ms. related compounds acylated at c6 of the outer glucose residue and compounds lacking either this glucose or one fatty acid residue were also found (pasciak et al., 2003) . glycoglycerolipids from the thermoplilic bacterium meiothermus taiwanensis have been shown to have the structure a-galp-(1 ! 6)-b-galp-(1 ! 6)-b-galnacyl-(1 ! 2)-a-glc-(1 ! 1)gro where acyl ¼ 17:0 or hydroxy-17:0 (yang et al., 2004) . a similar compound from the thermophilic bacterium thermus oshimai ntu-063 has the structure b-glcp-(1 ! 6)-b-glcp-(1 ! 6)-b-glcpnacyl-(1 ! 2)-a-glcp-(1 ! 1)-glycerol diester where the n-acyl group is a 15:0 or 17:0 fatty acid and the glycerol-attached fatty acids have chain lengths from 15:0 to 18:0. the maldi-tof spectrum contained a range of peaks from m/z 1,300 to 1,550 (lu et al., 2004a) . di-mannosyl-acyl monoglyceride in which the mannose residue adjacent to the glycerol contained an anteisobranched 15-carbon acid at the 6-position has been characterized from rothia mucilaginosa, a pathogen responsible for several opportunistic infections. unusually the acyl group attached to the glycerol was at c2 rather than the more usual c3 (pasciak et al., 2004) . the structure of a novel mannose-capped lipoarabinomannan from amycolatopsis sulphurea has been investigated with a range of techniques of which maldi-tof (dhb, negative ion) showed an average molecular mass of around 10 kda but with a broad, unresolved envelope of peaks with a half-height width of approximately 5 kda . the lipoarabinomannan from the mycobacterium-like organism tsukamurella paurometabola has produced a maldi-tof spectrum from s-dhb with a broad peak with a mass range from 9 to 16 kda. the structure was mainly determined by nmr. it lacked some of the features such as branching arabinan chains that are found in arabinomannans from mycobacteria but, nevertheless the molecule was claimed to be the most elaborated non-mycobacterial arabinomannan identified to date . lipomannan and lipoarabinomannan from mycobacterium kansasii has been shown to possess a manno-oligosaccharide cap but to lack the phosphoinositol cap found in lipoarabinomannans from mycobacterium smegmatis (guérardel et (batrakov, et al., 2004) ascaris suum not analysed structural identification of zwitterionic glycans containing phosphocholine and phosphoethanolamine (friedl, et al., 2003) cow and human (bode, et al., 2004) fasciola hepatica (liver fluke) mammalian-type glycolipids. gal by exoglycosidase digestion (wuhrer, et al., 2004a) human vitreous body of eye tof (dhb), tlc (fujiwaki, et al., 2004) human and rat classification into new molluseries (aoki, et al., 2002) mucor hiemalis plasmodium falciparum intra-erythrocyte stage l-, r-tof (dhb, norharmane), tlc gal, glc, galnac d18:0, d20:0 oh-10:0, oh-12:0, oh-14:0 structural determination (couto, et al., 2004) (korduláková et al., 2003; . mass spectrometric methods for analysis of flavonoids have been reviewed (prasain, wang, & barnes, 2004) . three glycosyltransferases (pérez et al., 2004b) and two methyltransferases (pérez et al., 2004a) involved in the biosynthesis of phenolic glycolipids from mycobacterium tuberculosis have been characterized. maldi-tof spectra of the glycolipids showed a range of produces with masses of around 1.5 kda resulting from acyl heterogeneity. a new mannitol teichoic acid has been reported from the cell wall of the bacterium brevibacterium sp. vkm ac-2118 isolated from a frozen late pliocene layer (1.8-3 myr) of kolyma lowland, russia. the structure consisted of 1,6-poly(mannitol phosphate) with the majority of mannose residues carrying additional phosphate groups. positive ion maldi spectra from dhb gave peaks to 6 kda (potekhina et al., 2003) . the structures of hexamannosides from mycobacterial species have been elucidated and found to contain up to four long-chain acyl groups (gilleron, quesniaux, & puzo, 2003) . a typical structure is 53. studies with m. smegmatis have shown that mannose metabolism, as revealed by studies on phosphoinositol mannosdes, is required for growth (patterson et al., 2003) . mycoglycolipids have been isolated from the edible fungus hypsizygus marmoreu and identified by gc/ms and maldi-tof ms . the biosynthesis of mycobacterial phosphatidyl mannosides has been revised with the help of maldi-tof analysis (morita et al., 2004) . pim 4 (four mannose residues and two palmitate chains) present in mycobacterial phosphatidyl manno-oligosaccharide, identified by psd from chca, has been shown to be a natural antigen for cd1d-restricted t cells . although maldi-tof ms is used quite extensively in this field, fab still appears to be the most commonly encountered ionization technique. glycosides identified by maldi during the review period are listed in table 8 . glucosylated heteropolyflavans (e.g., 54) from sorghum bicolor have been examined by krueger, vestling, and reed (2003) ; polymers with from three to seven flavan units and up to seven glucose residues were found. to overcome problems with estimation of the number of hydroxyl groups present due to the coincidence in mass with the [m þ k] þ ion, samples were analyzed as cesium adducts by addition of cesium trifluoroacetate to the sample/matrix solution. silver trifluoroacetate was also investigated but problems were ardisia mamillata triterpenoid saponins tof, fab, ord, nmr, lc β-d-glcp, α-l-rhap, α-larap borrelia burgdorferi cholesteryl galactoside l-tof, nmr, fab, glc β-d-gal, agent for lyme disease (ben-menachem, et al., 2003) borrelia burgdorferi (schröder, et al., 2003) cheilanthes glauca (neretina, et al., 2002) henricia sanguinolenta and h. leviuscula leviuscula steroidal polyols. cholesterol tof (chca), hplc, tlc, nmr mono-, di-and tri-ome-β-xylp (sanguinoside c from starfish) (levina, et al., 2003) henricia sanguinolenta and h. leviuscula leviuscula steroidal glycosides (cholestanes, cholestenes) tof (chca), nmr, hplc, tlc 2-o-me-xylp hippasteria phrygiana (starfish) steroidal glycosides (cholestanes, cholestenes) tof (chca), nmr, hplc, tlc β-d-xylp, α-l-araf hordeum sp. (barley) flavone glycosides tof (dhb), nmr β-d-glc (nørbaek, et al., 2003) lupinus oreophilus ten triterpenoid saponins ft-icr, esi, nmr, hplc α-l-ara, β-d-gal, β-d-glc, α-l-rha (woldemichael, montenegro & timmermann, 2003) marrubium velutinum acetylated glycosides ft-icr (dhb) lc tlc nmr β-d-glcp (karioti, et al., 2003) mycale laxissima steroidal glycosides tof, nmr, gc/ms d-ara, d-glc, d-gal (antonov, et al., 2003) mycobacterium tuberculosis, phosphatidyl-myo-inositol mannoside r-tof (-ve) (haba), nmr β-d-man phlomis physocalyx hub.-mor. phenylethanoid tetraglycoside (physocalycoside) ft-icr (dhb), mnr, or, ir, uv, tlc α-l-rha, β-d-glc, api (ersöz, et al., 2003) plantago major l. phenolic glycoside, (verbascoside, tof, nmr, hplc glc, rha (egorov, et al., 2004) triterpene glycoside tof (chca), nmr xyl, qui, ome-glc quillaja saponaria triterpenoid saponins (28 compounds) β-gal, β-glca, α-l-rha, β-fuc, α-l-ara, β-xyl, β-glc, β-d-api (nyberg, et al., 2003) rhizophora mangle l. flavonol glycosides from tannin maldi rha, glc, (mangrove) (kandil, et al., 2004) sorghum bicolour (l.) moench heteropolyflavans l+r-tof (+ve) (3-iaa), glc (krueger, et al., 2003) solenostemma argel (asclepiadaceae) oxo-pregnane glycosides (stemmosides c and d) r-tof (chca), nmr, esi linear pentasaccharide, β-d-glcp β-d-olep, β-d-canp, β-d-cymp (plaza, et al., 2004) triterpenoid saponins tof (chca), nmr, gc/ms β-d-ome-glcp, β-xyl, β-d-quip, β-4-so 3 na-xyl (moraes, et al., 2004) thalictrum orientale phenyl glycosides ft-icr, uv, nmr β-d-glcp, β-d-xylp (erdemgil, et al., 2003) (nyberg, baumann, & kenne, 2003) . the triterpene glycoside from the sea cucumber stichopus mollis (stichopodidae) was found by moraes et al. (2004) to differ in structure from the corresponding glycosides from all other known stichopodids in possessing a sulfate group in the carbohydrate chain, prompting the authors to propose a reclassification to a new genus, australostichopus levin. both maldi and esi in negative ion mode have been demonstrated to be capable of detecting carminic acid (55), the red dye isolated from cochineal, in paintings applied to canvas. binding agents such as linseed oil did not interfere with the detection (maier, parera, & seldes, 2004) . maldi-tof has also been used to study the biotransformation of ginsenoside rb 1 by 49 microbial strains (dong et al., 2003) . these compounds are mixtures of cyclic, branched, and linear polymers of glucose containing various species-different substituents. they accumulate in the periplasmic space of gramnegative bacteria in response to low osmolarity. lequette et al. (2004) have identified the gene that encodes for the protein that controls the backbone structure of these carbohydrates. associated analysis of the carbohydrates with masses up to 3.5 kda was performed by maldi-tof from dhb or 3-aq. matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (maldi-tof ms) has been used by many laboratories to monitor the products of enzyme reactions aimed at deducing mechanisms of action or characterization of newly discovered enzymes. much of this work is summarized in table 9 . desorption/ionization on silicon mass spectrometry (dios-ms) or laser desorption without a matrix has been shown to be a viable alternative to maldi for analysis of small molecules and carbohydrates and has been used to monitor the activity of a2 ! 6-sialyltransferase on the disaccharide gal-b-(1 ! 4)-glcnac (shen et al., 2004) . jobron et al. (2003) have immobilized n-acetyllactosamine on a cellulose membrane and used it as an acceptor for assaying four glycosyltransferases. the trisaccharide produced by a1 ! 3-galactosyltransferase was identified by maldi and the method was proposed as the basis of a high-throughput technique for enzyme screening. a method for detecting the presence of o-glycanase, an enzyme that cleaves the galb-(1 ! 3)-galnaca-(1 ! ) link to serine or threonine, uses maldi to detect the [m þ na] þ ion at m/z 406 corresponding to the released sugar (akai et al., 2003) . a maldi-tof method for the simultaneous detection and measurement of sugars, ascorbic acid (57), citric acid (58), and sodium benzoate has been developed by ayorinde, bezabeh, and delves (2003) . the matrix was meso-tetrakis-(pentafluorophenyl)porphyrin (59), a compound with a molecular weight of 974 which is well above that of the target molecules. sodium or potassium hydroxides were used as dopants. the method was particularly good at detecting the low molecular weight organic acids. maldi-tof ms, from dhb also provides a method for detecting the presence of starch hydrolysates as adulterants in fruit juices (cabálková et al., 2004) . the action of the enzyme preparation, olivex, used in the preparation of olive (olea europaea) oil has been investigated. the preparation is mainly a pectinase that hydrolyses (1 ! 4)-linked galactan chains from the cell wall. however, it is unclear how this reaction improves the quality of the product (vierhuis et al., 2003) . a comparative study of five rhizobium strains from different cropping areas of china has concluded that differences in the efficiency of nitrogen fixation is not related to differences in nod factor structure (identified by maldi-tof and esi-q-tof ms) (thomas-oates et al., 2003) . production of humanized antibodies and other biopharmaceuticals in plants and insect cell lines, as a safe and economically feasible alternative to production in animals, requires reengineering of the n-glycan synthesis machinery to eliminate potentially antigenic a1 ! 3-fucose-and b1 ! 2-xylose-containing glycans. maldi-tof analysis is ideally placed for analysis of the resulting glycans and many examples are listed in table 10 . an analysis of human transferrin expressed in lymantria dispar (gypsy moth), however, has shown the natural absence of core a1 ! 3-fucosylation, a normally common motif in insects (choi et al., 2003b) . to study the specificity of igg and ige antibodies against the a1 ! 3-fucose and b1 ! 2-xylose epitope, bencúrová et al. (2004) have modified the relatively simple biantennary glycans on human transferrin to incorporate these epitopes. products were monitored by maldi-tof ms and antibody binding was reported to be greatest for the fucosylated glycans. maeda et al. (2004) have replaced all of the asparagine residues of the n-glycosylation sites in procarboxypeptidase y by alanine and expressed the protein in p. pastoris. the resulting protein lacked n-glycosylation but contained residual glucose, thought to be o-linked, as detected by an increase in the maldi mass from that expected from the amino acid sequence. analysis of erythropoietins (epo) with varying glycosylation has shown the fully sialylated tetraantennary glycan, identified by hplc and maldi-tof ms as its 4-aa derivative, to be the major determinant of biological activity (yuen et al., 2003) . sanz-nebot et al. (2003) have recorded maldi spectra from intact human recombinant epo and noted a drop in measured mass with increasing laser power which they attributed to loss of sialic acid within the ion source. maldi-tof has been used to identify o-glycans from a von willebrand factor c domain produced in p. pastoris (o'leary et al., 2004) . methods for their removal were investigated. a method for monitoring glycosylation in pharmaceuticals based on capillary electrophoreses of 3-aa derivatives with structural confirmation by maldi-tof ms has been published by kamoda et al. (2004) . maldi and ion exchange chromatography have proved useful for checking the batch-to-batch variation in glycosylation of three recombinant gonadotropins. as an example, excellent consistency (3.5) was found for the degree of sialylation of 120 batches of recombinant thyroid stimulating hormone over a 3-year period (gervais et al., 2003) . several reviews on the production of biopharmaceuticals and containing reference to maldi-tof analysis have been published. these include production of human therapeutic proteins in yeasts (bretthauer, 2003) , yeasts and filamentous fungi (gerngross, 2004) and lepidopteran cell lines (tomiya et al., 2004) . methods for characterizing biological products after manufacturing changes have also been reviewed (chirino & mire-sluis, 2004) . slime deposits in paper mills, termed biofouling, is a problem in the paper industry and has prompted studies on the composition of their glycolipids. the eps from methylobacterium strains, responsible for ''pink slime'' have been shown by verhoef et al. (2003) to consist of the repeating unit: sadly, many chemical papers appear to ignore details of the equipment and conditions used to obtain mass spectra of carbohydrates (and other compounds) even though conditions such as the choice of maldi matrix are often essential in getting the compounds to ''fly.'' although nmr experiments and equipment are usually described in great detail, information on mass spectrometry is frequently reduced to uninformative acronyms such as hrmaldims with no further details. without knowledge of the equipment used to record the spectra it is difficult to judge if hr is indeed meaningful. the relegation of essential methods to ''supplementary information'' is also to be regretted. the absence of essential information such as the matrix from many publications is reflected in table 11 (with apologies to authors from whose papers this information has been missed). ''maldi'' is used for papers omitting to cite the type of instrument used to record the spectra. most of the publications in this area relate to routine monitoring of reaction products and are summarized in table 11 . in addition to chemical reactions, enzymes are frequently used in this area because of their stereospecificity. both transferases and glycosidases such as pngase f used in their reversed mode to catalyze transglycosylations can be used; however, evidence has been found to suggest that enzymes used for reverse hydrolysis reactions can have their activity reduced by maillard glycation (maitin & rastall, 2004) . as with the previous review, tof, hplc (kaneko, et al., 2003) human, gnt-ix transfers glcnac to the 6-antenna of n-glycans. also transfers glcnac to the 6-position of the 3-antenna to produce a novel structure. enzymes uniquely expressed in brain. r-tof (dhb), nmr (inamori, et al., 2003) human, gnt-ix enzyme acts on the glcnac-β-(1→2)-man-α-1-ser/thr moiety forming a 2,6-branched structure in brain o-mannosyl glycan. r-tof (dhb), nmr (inamori, et al., 2004) pp α-glcnac-t2 (korekane, et al., 2003) saccharomyces cerevisia a strain of this yeast is unusually shown to be able to add a single glcnac residue to a variety of high-mannose glycans. in-gel removal of glycans with pngase f. tof (dhb) (yoko-o, et al., 2003) galnac-transferase 2 modification of the glycosylation signal of the herpes simplex virus type 1 glycopeptide gc-1 by mutation, increased the content of o-linked glycans but little effect on n-glycans. tof (mårdberg, et al., 2004) human csgalnact-1 and csgalnact-2 csgalnact-1 is involved in the initiation of chondroitin sulfate synthesis. csgalnact-2 participates mainly in elongation. tof (sato, et al., 2003b) human, galnac-t1, -t2, -t3, -t4, -t6, -t9 enzymes have different substrate specificities but galnac-t2 was found to transfer galnac residues to the same five positions of the iga1 hinge region in b cells. r-tof (chca) (iwasaki, et al., 2003) human, pp-galnac-t13 (neuroblastoma cell) transfers galnac to several mucin-derived peptides including muc5ac and muc7. tof (dhb), glycopeptides (zhang, et al., 2003e) human, pp-galnac-t14 transfers galnac to muc2, muc5ac, muc7 and muc13. may be involved in oglycosylation in the kidney tof (chca) (wang, et al., 2003c) human, transfers galnac to the 4-position of glcnac in n-and o-linked glycans. tof (dhb) (sato, et al., 2003c) human, galnac-tiv similar enzyme to human, β4galnac-t3. transfers galnac in β1→4-linkage tof, nmr human, synthesises a unique carbohydrate structure, galnac-β-(1→3)-glcnac. tof, nmr human, pp-galnac-t15 transfers up to seven galnac moieties onto the muc5ac peptide whereas the t2 enzyme only transfers five. tof (chca), lc, glycopeptides other transferases α-(1→2)-l-gal-t from helix pomatia transfers an l-fucose residue to terminal d-galactopyranosyl residues, has been found to be specific for galactose with β-linkages. . r-tof (dhb), hpaec (scheppokat, et al., 2003b) b4-gal-t, cho lec19 cells resistance to plant lectins such as ricin and abrin that bind galactose shown, in these cells, to be due to a reduced ability of the cells to transfer galactose to the n-linked glycans. tof (dhb, neutral, thap, acidic) (lee, et al., 2003c) lpcc (rhizobium leguminosarum) adds a mannose unit to the inner kdo moiety of the lps precursor, kdo2-lipid iva. tof (att, ammonium citrate) (kanipes, et al., 2003b) pimf (mycobacterium marinum) first analysis of a man-t involved in the later stages of phosphatidylinositol mannoside synthesis. r-tof, psd (alexander, et al., 2004) membrane bound gal-t, glycine max pectin gal-t involved in biosynthesis of β-(1→4)-galactan chains of pectin. gal transfer to unmodified pectic galactan (mr>150,000) low but increased after partial acid hydrolysis. tof (dhb) hplc, gc/ms (konishi, et al., 2004) aknk (l-2-deoxyfucosyltransferase) identified as the enzyme that adds l-2-deoxyfucose as the second sugar in the carbohydrate of the antitumor drug aclacinomycin a produced by streptomyces galilaeus. tof, lc/ms, nmr (lu, et al., 2004b) dermatan sulfate biosynthesis clarification of controversy over whether 4-sulfation in dermatan sulfate occurs on galnac before epimerization of neighbouring glca to iduronic acid or afterwards l-tof (dhb), hplc (mikami, et al., 2003) glycosidases β-n-acetylhexosaminidases enzymes hydrolyse and transglycosylate β-glcnac and β-galnac moieties suggesting that they are single enzymes having affinity for both substrates. studies in about 60 fungi tof (weignerová, et al., 2003) α-amylase (bacillus licheniformis and human saliva) aspergillus niger pectinase an isoenzyme that cleaves both glcnac-glcn and glcn-glcn linkages thus offering a replacement for expensive chitinase and chitosanase currently used for this purpose. tof (dhb) (kittur, et al., 2003) endo-β-n-glcnac-ase from mucor hiemalis (endo-m) enzyme expressed in candida boidinni can transglycosylate a disialo-biantennary glycan in media containing 30% acetone, dmso or meoh in higher yield than aqueous solution. tof (thap), hplc endo-β-n-glcnac-ase from mucor hiemalis a variety of sugar derivatives modified at c-1 or c-2 can be used as acceptors for this enzyme. tof (dhb, thap), hplc endo-β-gal-ase from bacteroides fragillis glycolysis of the tamm-horsfall glycoprotein releases two sugars, measurement of which in identical twins indicates closely-controlled glycosylation in the two individuals. tof (chca), glycans (rohfritsch, et al., 2004) polygalacturonases from trichoderma reesei study of substrate specificity; the random pattern for pectin cleavage showed that the enzymes were exo-polygalacturonases. tof (thap/nitrocellulose) (mohamed, et al., 2003) xyloglucanases xyloglucanases from aspergillus japonicus, chrysosporium lucknowense, trichoderma reesei. a. japonicus displayed endo-type attack whereas other two enzymes exo-type. tof (grishutin, et al., 2004) other enzymes acting on carbohydrates chitinases, chit33 and chit42 purified from the filamentous fungus trichoderma harzianum and their hydrolysis products characterized tof (dhb) (boer, et al., 2004) gdp-6-deoxy-d-talose synthetase identification of the enzyme that converts gdp-4-keto-6-deoxy-d-mannose into gdp-6deoxy-d-talose in the gram-negative bacterium actinobaccillus actinomycetemcomitan. r-tof (thap, nh 4 citrate) (mäki, et al., 2003) heparin/heparan 2-o-sulfatase enzyme from flavobacterium heparinum active site and saccharide substrate specificity maldi (chca) (raman, et al., 2003) lipid a 1-phosphatase from rhizobium leguminosarum r a h c l a c i m e h c o i b d n a g n i n o l c , n o i s s e r p x e (karbarz, et al., 2003) lpxa (mesorhizobium loti and leptospira interrogan) mechanism of action in formation of lipid a with four n-linked acyl groups. enzyme does not acylate udp-glcnac but requires udp-glcnac3n tof (sweet, et al., 2004) pectin methylesterase from aspergillus niger enzyme prefers oligogalacturonides with one or two non-esterified galacturonic acids in its active cleft rather than fully methylated acids. tof (thap/nitrocell) potato d enzyme catalyzes the cyclization of amylose to produce cycloamylose tof (takaha, et al., 2003) quinone-dependent pyranose dehydrogenase products formed by enzyme extracted from basidiomycete fungi studied with lactose as substrate. the 2-oxo-product characterized as its n,n-diphenylhydrazone derivative. maldi-tof, nmr. (volc, et al., 2004) pngase f r-tof (s-dhb), glycans p. pastoris engineered to produce complex glycans (bobrowicz, et al., 2004) angiogenic vascular growth factor vegf 121 (human) pepsin, pngase a tof (dhb) genes encoding the enzymes for (1→3)-fucose and 1→2-xylose disrupted. did not affect plan to be produced without the immunogenic sugars. (koprivova, et al., 2004) anti-rabies monoclonal antibody tobacco pngase (in gel) r-tof (dhb), ion-trap/tof, glycans structural characterization (high-mannose, complex). glycosylation did not affect efficacy (ko, et al., 2003) avidin (chicken) pngase a r-tof (dhb), glycans (reduced, per-me) for detection of immunogenic core (1→3)-fucose and core xylose (bardor, et al., 2003a) bile salt-stimulated (trimble, et al., 2004) c1 inhibitor (human) pngase f r-tof (dhb (+ve), thap/nh 4 citrate (-ve)), glycans structural determination of glycans expressed in milk (koles, et al., 2004a , koles, et al., 2004b c5-1 antibody alfalfa pngase a r-tof, psd (dhb, chca), glycans (per-me) structural determination, plant-type, complex (bardor, et al., 2003b) c5-1 antibody (human) alfalfa pngase a pepsin r-tof (dhb, chca), psd, (glycopeptides), glycans (reduced, per-me) alfalfa plants able to synthesise igg with humantype glycans (bardor, et al., 2003b) chorionic gonadotrophin hydrazine r-tof(dhb, cmbt), esi, ion trap. glycans (2-ab) structural determination of complex glycans and method development. (gervais, et al., 2003) dac g 5 and proder p 1 trypsin tof (chca) presence of man can have an adverse effect on the specificity of diagnostic tests for autoimmune and allergic diseases that are based on immunoassays. (van oort, et al., endogenous glycoproteins pepsin, pngase a tof (dhb) enzymes adding potentially antigenic β (1→2)-xylose and α (1→3)-fucose removed (strasser, et al., 2004b) erythropoietin cho cells pngase f tof (dhb), glycans (4-aa) structural determination under different culture conditions. fully sialylated tetra-antennary glycan is the major determinant of biological activity (yuen, et al., 2003) erythropoeitin cho cells -l-tof (sinapinic acid, dhb, hpa, ferulic acid), esi, ce, glycoproteins characterization of intact glycoprotein by several methods (sanz-nebot, et al., 2003) follicle stimulating hormone cho cells hydrazine r-tof (dhb, cmbt), esi, ion trap, glycans (2-ab) structural determination of complex glycans and method development (gervais, et al., 2003) into yeast. pronase glycotetrapeptide from bovine fibrin to assay enzyme activity. (bencúrová, et al., 2003) gm2-activator protein pngase f tof (dhb), esi-tof, nmr structural determination (wendeler, et al., 2004b) hyaluronan synthase (human) e. coli -l-tof (dhb) expression of catalytic region of human hyaluronan synthase (has2) in e. coli shown to synthesise a mixture of ha oligomers from 8-to 16-mer. (hoshi, et al., 2004) igg ( (sriraman, et al., 2004) igg 1 (human) rat hybridoma or cho hydrazine maldi, hplc, glycans (2-ap), absence of fucose but not presence of galactose or bisect enhances cellular toxicity (shinkawa, et al., 2003) igg 3 m o u s e hydrazine, pngase f r-tof (+ve) (dhb), esi, ms/ms, glycans effect of buffer and ph on production. (müthing, et al., 2003) igg 4 (human) trypsin tof (chca), glycopeptides glycosylation (high-mannose, truncated complex) not significantly affected by anti-mitotic agent (tait, et al., 2004) kdel-tagged cpipp antibody structural characterization (fujiyama, et al., 2004) lecithin:cholesterol acyltransferase (lane, et al., 2004) luteinizing hormone (human) hydrazine r -tof (dhb, cmbt), esi, ion trap, glycans (2-ab) structural determination of complex glycans and method development (gervais, et al., 2003) mfe-cp trypsin r-tof, lc/ms, cid, psd, glycopeptides structural characterization of glycoprotein (highmannose, asn-442, 484) used to target cancer cells (medzihradszky, et al., 2004) (continued ) onconase endo-h tof, glycoprotein cancer chemotherapeutic agent. structures of high mannose glycans (man 9-16 ) from mass of glycoprotein ovotransferrin (chicken) endo h tof (sinapinic acid), glycoprotein, glycan mass by difference structural characterization (mizutani, et al., 2004) placental alkaline phosphatase (human) pngase f tof (dhb), glycans (2-ab) presence of silkworm hemolymph improvesnglycosylation, high mannose (joosten, park & shuler, 2003a) plasminogen kringle pngase f tof (s-dhb) glycosylation machinery re-engineered to perform sequential glycosylation reactions that mimic nglycan processing in humans. plasminogen kringle pngase f tof yeast engineered to produce glcnac 2 man 3 glcnac 2 plasminogen kringle pngase f tof ppalg3 gene that encodes for dol-p-man:-man 5 glcnac 2 -pp-dol man-t deleted to prevent further mannose addition. incorporation of a -(1 4)-gal-t gave human-type glycans. (bobrowicz, et al., 2004) procarboxypeptidase y pichia pastoris -tof (sinapinic acid), glycoprotein replacement of all asn of n-glycosylation sites by ala removed n-glycosylation. residual glucose thought to be o-linked. (maeda, et al., 2004) soluble human complement receptor type 1 (scr1) pngase f tof (dhb), glycans (2-aa) glycan remodelling using the soluble glycosyltransferases, -(2 3)-silyltransferase to introduce silyl lewis x moieties. (thomas, et al., 2004a) transferrin insect cell line pngase a trypsin + chymotrypsin tof (dhb), glycans (2-ap) trichoplusia ni insect cells engineered to produce biantennary glycans (tomiya, et al., 2003) transferrin (human) lymantria dispar (gypsy moth) pngase a l-tof (dhb), glycans (2-ap) attempt to find a better expression system than those currently used. absence of core (1 3)fucosylation, a normally common motif in insects. (choi, et al., 2003b) transferrin (human) pepsin, pngase a l-tof, q-tof (chca), glycans, glycopeptides to study specificity of igg and ige antibodies against (1 3)-fucose and (1 2)-xylose epitopes. binding greatest for fucosylated glycans. (bencúrová, et al., 2004) vascular growth factor vegf (1 2)-xylose enzymes (koprivova, et al., 2004 , koprivova, 2003 #2924) glucose-based glycoprobes ident. of binding sites in na/d-glucose co-transporter protein l-tof (sinapinic) (tyagi & kinne, 2003) monosaccharide amides ru(iii)-promoted amide formation from azides and thioacids, which do not form amides at room temp. in the absence of ru ft-icr (fazio & wong, 2003) quaternary ammonium salts of ribitol salts from five aromatic amines tof (chca) oligosaccharides n-acetylglucosaminobioses by reverse hydrolysis using fungal n-acetylhexosaminidases tof (rauvolfová, et al., 2004b) arabinogalactosyl nonasaccharides β-(1→6)-d-galp backbone, α-(1→3)-linked l-araf side-chain tof (dhb) (li & kong, 2004) (argyropoulos & sarli, 2004) biotinylated saccharides as part of avidin/biotinylated drug delivery system tof (ouchi, et al., 2004) 6-branched cyclo-(1→3)-glucohexose cyclisation of branched glucohexaose tof (wu & kong, 2004b) 2,6-branched galacto-oligosaccharides synth. of tetra-and hexa-saccharides related to arabinogalactans tof (ning, yi & yao, 2003b) carbohydrate-modified poly(di-me-siloxane)s linear dimethylsiloxanes capped with various monosaccharides tof (henkensmeier, et al., 2004) 6-o-carboxymethylated chitotetraose selective glycosidation with chitinase catalysts tof (ochiai, ohmae & kobayashi, 2004b) chitobiose analogues sugar oxazolines glycosylated with chitinase from bacillus sp. nafion plate (ochiai, ohmae & kobayashi, 2004a) chitooligosaccharides as standards for diffusion-ordered spectroscopy tof (groves, et al., 2004) chitooligosaccharides synthesis from chitosan by enzymatic hydrolysis with chitinase tof chitooligosaccharides dp 2-6 use of pectinase from aspergillus niger tof, fab (kittur, et al., 2003) chititriose nicotinic amide for conformational studies. inhibitor of chininase hevamine maldi (germer, et al., 2003) c (galan, et al., 2003) cyclosophoraoses of rhizobium meliloti 2011 enzymatic synthesis. as additive for enantioseparation in ce tof deoxy phospha sugar-sugar disaccharides phospha analogues of normal sugar disaccharides tof (chca) (haritha, et al., 2004) fucosyl-lactoses specific fuc addn. after lipase-catalysed regioselective acylation tof (rencurosi, et al., 2003) fucosyl pentasaccharide (sulfated fucan repeat) convergent "2+3" synthetic strategy tof (dhb) (hua, et al., 2004a) fucosylated trisaccharides study of the specificity of the α-(1→2)-l-galactosyltransferase from helix pomatia. specific for d-galp β-linkages tof (dhb) (scheppokat, et al., 2003b) fucosylated trisaccharides synthesis with α(1→2)-l-galactosyltransferase from helix pomatia. tof (dhb) (scheppokat, bretting & thiem, 2003a) fucosylated trisaccharides (lactose) random fucosylation to construct library tof (meng, et al., 2004) gal-β-(1-3)-glcnac use of an anomeric fluorous silyl protecting group tof (manzoni, 2003) galactans biosynthesis by use of galactosyltransferase from radish (raphanus sativus l.) seedlings tof (abee derivs.) (kato, et al., 2003) galactose-containing oligosaccharides β-gal-t from bovine testes to synth. novel food components tof (dhb) (schröder, et al., 2004) β-(1→6)-d-galactosylated oligosaccharides use of β-(1→6)-d-gal-transferase from helix pomatia tof (scheppokat, et al., 2004) galactosylated tri-and tetrasaccharides regioselective synth. β-galactosidase from bacillus circulans tof (dhb) (farkas, et al., 2003) , et al., 2004d) β-d-glucosamine-containing nonasaccharide chemical synthesis with isopropyl thioglycosides tof (chca) β-d-glucosamine-containing oligosaccharides chemical synthesis with isopropyl thioglycosides tof (chca) (yang, hua & du, 2003) glucosylated isokestose and nystose enzymatic (kojibiose phosphorylase from thermoanaerobacter brockii to synthesise five novel oligosaccharides tof glycosaminotrioses with galnac or mannac termini. to study binding to hevein from hevea braziliensis tof (chca) (aboitiz, et al., 2004) imino sugar scaffolds for the generation of glycosidase inhibitor libraries tof (dhb) (la ferla, et al., 2004) kanamycin analogues chemical synthesis and antibacterial evaluation maldi (li, et al., 2004b) lacto-n-neotetraose dendrimeric polyethylene glycol supported synthesis tof laminarin hexasaccharide 4,6-o-benzylidenated acceptor to avoid generation of α−isomer in attempted β-d-(1→3) glycosylation under standard condits. tof (chca) (he, gu & du, 2003) lepidimoide unsaturated disaccharide (4-deoxy-β-l-threo-hex-4enopyranosyl-(1→2)-l-rhap) from okra mucilage tof (co powder) ) chemical. use of anomeric fluorous silyl protecting group tof (manzoni & castelli, 2004) maltooligosyl fructofuranosides immobilized cyclodextrin glucosyltransferase catalyst tof (dhb) maltosyl-erythritol transglycosylation product of erythritol by maltogenic amylase tof (chca) (yoon, et al., 2003) α-(1→3)-linked manno-hexaose and -octaose α-(1-3)-linked di-and tetra-saccharide donor + tetrasaccharide tof (chen & kong, 2002) mannosides synthesis by double differential glycosylation by lanthanide triflates tof i in situ method for identification of novel α-amylase inhibitors r-tof n(methyl-2,3,4-tri-o-acetyl-6-deoxy-α-andβ-d-glucopyranoside-6-yl (remenyik, et al., 2003) oligosaccharides (di-, tri-and tetrasaccharides) solid-phase synthesis with azidoglucose as a glycosyl acceptor tof (dhb) (wu & schmidt, 2004) oligosaccharides (general) polymer -resin hybrid capture-release strategy for rapid oligosaccharide construction tof (hanashima, manabe & ito, 2003) oligosaccharides (general) reiterative intramolecular glycosylation by use of a rigid spacer tof (dhb) (paul, müller & schmidt, 2003) oligosaccharides (rhamnan backbone) synthesis of oligosaccharides consisting of α-(1→2)-and α-(1→3)-linked rhamnan backbones and glcnac side chains tof (zhang & kong, 2003d) n-pentenyl arabinofuranosyl donors synthesis of little-studied donors for furanosides tof n(och 3 )-linked disaccharide analogues carbohydrate mimics resistant to hydrolysis by glycosidases tof (dhb) non-reducing oligosaccharides by reverse hydrolysis using fungal n-acetylhexosaminidases tof (rauvolfová, et al., 2004a) silyl lewis x and silyl lewis a precursors determination of recombinant (2→3)-α-silyltransferase tof (dhb) (ivannikova, et al., 2003) sucrose laurate enzymatic (alkaline protease from bacillus pseudofirmus al-89 tof (chca) (pedersen, et al., 2003) 6-sulfo-glcnac-β-(1→3)-gal-β-(1→4)-glc product of 6-sulfo-transferase produced in baculovirus system tof (dhb) (el-fasakhany, et al., 2003) (renaudie, et al., 2004) (prosperi, et al., 2004) various oligosaccharides automated solid-phase synthesiser. 20 times faster than solution tof (dhb) (palmacci, et al., 2003) various oligosaccharides formylacetal (ch 2 ) as novel linker on soluble-polymer support tof (oikawa, et al., 2004) various oligosaccharides use of a novel hexakisfluorous butanoyl support tof (chca), esi (goto, et al., 2004) various oligosaccharides use of fluorous-tagged saccharide primers tof (dhb) (kasuya, et al., 2004) various oligosaccharides acetyl esterase from trichoderma reesei rut-c30 found able to transglycosylate carbohydrates in organic solvents tof (dhb) (kremnický, mastihuba & côté, 2004) polysaccharides c h e m i c a l ( n-pentenyl-arabinofuranosyl donor) tof arabinogalactans from echinacea purpurea synthesis of double-branched isomeric nonasaccharides tof (csávás, et al., 2003) c tof (faijes, et al., 2004) lepidimoic acid from hibiscus esculentus chemical degradation of okra tof (co powder) (hirose, et al., 2003) maltobi-and -triose 2,4-dinitrophenyl-derivs. substrates for human pancreatic α-amylase. kinetic analysis r-tof mannan of candida kefyr ifo 0586, cell wall first synthesis of penta-and deca-saccharide repeats tof (chca) (xing & ning, 2003) , et al., 2003) chondroitin sulfate e hexasaccharide synthesis from β-d-galnac-(1→4)-β-d-glca tof (tamura & tokuyoshi, 2004) heparan sulfate fragments use of 6 orthogonal protecting groups to synth. 20 disaccharides tof (-) (dhb) (prabhu, venot & boons, 2003) heparin-like fragments synthesis of hexa-and octasaccharides on a solid support tof (ojeda, et al., 2004) heparin fragments chemical synthesis via regio-and stereo-selective glycosylation ftms heparin-like hexasaccharides synthesis by convergent block strategy tof (dhb) (lucas, et al., 2003) isotope-labelled hyaluronan oligomers 15 n-, 13 c-labels for nmr studies tof (dhb) (blundell, et al., 2004) monodisperse hyaluronan oligosaccharides chemoenzymatic synthesis with immobilized mutant enzymes r-tof (att) (-) (deangelis, oatman & gay, 2003) carbohydrates from algae fucoidan repeat from brown algae chemical synthesis. good antitumor activity tof (dhb) (hua, gu & du, 2004b) disaccharide fragments of lps from several species jones oxidation of allyl glycosides tof (chca) (madaj, jankowska & wisniewski, 2004) lipid a from chlamydia trachomatis chemical synthesis and purity assessment tof (dhb) (zamyatina, et al., 2004) mycobacterium tuberculosis phosphoinoside intermediate use of regioselective mannosylations maldi (jayaprakash, lu & fraser-reid, 2004) o-chain from campylobacter jejuni trisacch. from glcnphth-(1→3)-gal + allyl 6-deoxy-altrohep tof (2,4-dhb) (yoon, et al., 2004b) o-chain repeat from helicobacter pylori chemical synthesis of unique trisaccharide from danish strains tof o-chain repeat from shigella flexneri chemical synthesis of tetra-and two pentasaccharide fragments (mulard & guerreiro, 2004) capsular polysaccharide from (alpe, oscarson & svahnberg, 2003) eps repeat from cryptococcus neoformans chemical synthesis of repeating unit tof (zhang & kong, 2003a) repeat (zhang & kong, 2003b) lipoarabinomannan from rhodococcus equi chemical synth of pentasaccharide repeat from equine pathogen tof (chca) (ma, zhang & kong, 2004) chemical plus enzymatic synthesis tof (dhb) (furuike, et al., 2003) biotinylated, sialylated biantennary glycan enzymatic synthesis with endo-m tof (dhb) (mori, et al., 2004) high-mannose type attached to ethylene glycol as microarrays for gp-120 (from hiv) interaction studies tof (adams, et al., 2004) hybrid-type, bisected glycan transglycosylation with endo-β-n-acetylglucosaminidase tof (thap) α-d-man-(1→4)-β-d-glcnac-(1→4)-dsynthesis of unnatural α-d-mannose isomer tof (dhb) α-(2→3)-sialic acid-containing n-glycans use of α-(2→3)-sialyltransferase tof (fukae, et al., 2004 (hernández, et al., 2004) (xian, et al., 2004) complex n-glycans-glycopeptides chemoenzymic synth. of sugar-asn. solid-phase synth. of g-pep tof core ii o-linked glycopeptide solid phase plus enzymatic synthesis tof (dhb) (takano, et al., 2003) core ii o-linked glycopeptide solid-phase synthesis plus enzymatic sialylation tof (takano, et al., 2004) core ii sialyl lewis-x o-glycopeptide (gallego, et al., 2003) cyclic glycopeptides macrolactamization of glycosylated peptide thioesters maldi eel calcitonin plus n-glycans transglycosylation with glcnac-ase from mucor hiemalis tof (chca) (haneda, et al., 2004) emmprin with n-linked core pentasaccharide chemical, solid phase. n-glycosylated ig domain tof epidermal growth factor-like domain of blood coagulation factor ix carrying xyl-glc solid-phase synthesis. mild conditions enabled protecting groups on the sugar to be omitted tof (kitamura, et al., 2004) epithelial cadherin glycopeptides solid-phase synthesis. fmoc chemistry tof (reipen & kunz, 2003) epithelial mucin muc4 synthesis of tumor-associated glycopeptide by solid-phase synthesis tof (brocke & kunz, 2003 , brocke & kunz, 2004 galactosylated 5-hydroxylysine mimetics for incorporation into peptides such as type ii collagen tof (marin, et al., 2004) (sato, et al., 2004b) glycopeptides (of cell-surface glycoproteins) use of (2-ph-2-tms)ethyl-(ptmsel) as a fluoride-sensitive anchor for solid-phase synthesis tof (wagner, dziadek & kunz, 2003) glycopeptides study of glycosylation catalysed by pngase f in reverse rn. (chca) (jeong, lee & park, 2004) glycopeptide mimetics incorporation of unnatural (keto) amino acids in peptide chain fticr (liu, et al., 2003a) glycopeptide from collagen chemical synthesis. helical models incorporating collagen sequences for receptor binding studies tof (dhb, sinapinic acid) (lauer-fields, et al., 2003) gly-pro-thr(β-gal) 10 carbohydrate stabilizes triple helix of collagen by h-bonds tof (bann, bächinger & peyton, 2003) heterocyclic-tethered c-glycosyl amino acids , et al., 2003) interleukin-1α plus neu5ac-gal coupling by acyl azide, 2.5 mols/mol of protein tof (ootsubo, et al., 2004) β-lactoglobulin glycosylation by heating with glc or fru for thermal stability tof (chca) (broersen, et al., 2004) (halkes, et al., 2003) s-linked glycopeptides wo-phase system to avoid use of strong base maldi (zhu & schmidt, 2003) s-linked glycopeptide derived from human tamm-horsfall glycoprotein direct base-catalyzed s-glycosylation of cysteine and homocysteine-containing peptide with o-ac protected bromides maldi (zhu, haag & schmidt, 2004c) sperm cd52 fmoc chemistry to build peptide chain with glycan-asn tof (shao, xue & guo, 2004) glycosides acylated ginsenosides regioselective acylation in organic solvents with vinyl acetate and candida antarctica lipase tof (chca) (teng, et al., 2003 , teng, et al., 2004 1-adamantylmethyl glycosides single-step synthesis from per-ac-monosaccharides. binding to cyclodextrins depends on pyrenose stereochemistry tof (charbonnier & penadés, 2004) β-d-aminoglycosides use of 2-nitro-thioglycoside donors tof (dhb) (barroca & schmidt, 2004) diadzein glycosides transglycosylation with thermotoga maritima maltosyltransferase to increase solubility l-tof (dhb) dihydrodiosgenin glycosides as mimetics of bidesmosidic steroidal saponins r-tof (dhb) (suhr, et al., 2003a) (joosten, et al., 2003b) glycosyl glycerol transglycosylation by cyclodextrin glucanotransferase tof (chca) (nakano, et al., 2003a) (continued ) (dondoni & perrone, 2003) lipo-chitooligosaccharide nodulation factors two-step chemical and biotechnology synthesis tof p-methoxyphenyl β-lactosides with 3'supfonic acid to find carbohydrate ligand(s) which can inhibit adhesion of helicobacter pylori and gastrointestinal epithelial cells. maldi (borbás, et al., 2004) osw-1 analogues (cholestanes saponins) mod. of steroid side-chain. no change in anti-tumor action. maldi (deng, et al., 2004) rhamnosylated diosgenyl glucosides as mimetics of cytostatic steroidal saponins from ornithogalum saundersiae and galtonia candicans r-tof (dhb) (suhr, et al., 2003b) ) , et al., 2003) 3'-substituted p-methoxyphenyl β-lactosides to find ligand(s) which can inhibit the adhesion between helicobacter pylori and gastrointestinal epithelial cells. maldi (borbás, et al., 2004) glycosphingolipids reaction of thioglycosides with benzoyl ceramide tof (yamamura, et al., 2004) mannobiose-linked phosphoethanolamine chemical synthesis of gpi anchor intermediate tof (dhb) glycolipids dolichylphosphomannose analogues potential inhibitors of man-t in endoplasmic reticulum maldi (dhb) (kulesza, et al., 2004) glycolipids (general) ag triflate as alternative to tms-triflate for sensitive rns. tof (chca) glycolipids with partially f-alkyl chains (stadelmaier, et al., 2003) maltosyl erythritol transglycosylation with amylase (bacillus stearothermophilus) tof (chca) (yoon, et al., 2003) 6-o-sulfo lewis x neo-glycolipid containing lactamized neuraminic acid tof (dhb) cyclodextrins acetylated α, β and γ cyclodextrins chemical synthesis. properties in dense co 2 tof (potluri, et al., 2003) allylated cyclodextrin (6-nh-(ch 2 ) 3 -si(oet) 3 ) 7 substituents. as chiral hplc phase. tof (dhb) (lai & ng, 2004) amphillic cyclodextrins novel monosaccharide carriers through bulk liquid membranes tof (kida, et al., 2004) amphiphilic cds fully substd. on primary face chemical and enzymatic synthesis. 6-substituted cds tof (sallas, niikura & nishimura, 2004) bis-β-cyclodextrin/ pyromellitic acid 2,5-diamidate chemical. improved binding of guest dyestuff result of double cavity. tof (liu, et al., 2003b) cationic cyclodextrins 6-nhpy, 6-me-imidazole, 6-bu-imidazole, 6-nh-c 2 h 4 och 3 , nh 2 . plasmid complexes for transfection maldi (cryan, et al., 2004) 6-n-carboxyalkyl (-nh-co(ch 2 ) 4, 5 or 10 cooh) 7 substituents, enzyme promoter maldi 6-o-carboxymethyl (per-me) (ch 2 cooh) 7 , (me) 14 substituents. cec r-tof (+) (thap) (culha, et al., 2004) cyclodextrin dimer for transport of saccharides through a liquid membrane tof (ikeda, matsuhisa & ueno, 2003) cyclodextrins with acetone bridge epoxidation catalysts. ozone, catalysed epoxidation of alkenes tof (chca) (rousseau, et al., 2004) (teranishi, 2003) cyclodextrins with methacryl moieties characterization, copolymerization with 1-vinyl-2-pyrrolidone tof (dhb) (janus, et al., 2003) cds with one dithienylethene-tethered β-cyclodextrin dimers photocontrolled release and uptake of a porphyrin guest tof (mulder, et al., 2004) fluorescent cyclodextrin/peptide hybrids with macrocyclic metal complex znii-cyclen as a ligand site and β-cd is a receptor site for guest molecules, while the dansyl unit acts as a fluorescent probe. tof (furukawa, mihara & ueno, 2003) glycosylated β-cyclodextrin (β-gal) 1,2 , (lac) 1,2 , β-(gal-lac) 1,2 subs. molecular recognition r-tof (+) (dhb) (ikuta, et al., 2004) mannoside substituted (harabagiu, et al., 2004) pseudopolyrotaxanes + lactose-substituted cds receptor binding studies (lac) 6 (lac) 7 substituents ft-icr (dhb) (nelson & stoddart, 2003) pyridylmethyl-per-polyethyleneglycol-β-cd synthesis of model hemoprotein. ch 2 -py, -(c 2 h 4 o) 3 -ch 3 subs. tof (zhou & groves, 2003) na dodecyl sulfate + cd-based nanotubes study of binding in formation of inclusion complexes tof (kalashnikov, et al., 2004) , et al., 2004b) β-alanine-based glycoclusters β-alanine polypeptide plus galactose tof (sato, hada & takeda, 2003a) carbohydrate amphiphiles hydrazone ligation with lipophilic glyoxylyl acid derivatives tof (grandjean, et al., 2004) carbohydrate-centred glycoclusters penta-and 15-valent mannosides, glucose centre tof (dhb) (köhn, et al., 2004) carbosilane dendrimers tetravalent glycodendrimers. two new synthesic methods tof (2,4-dhb) (choudhury, et al., 2003) i l ( , et al., 2003) galnac-containing biotinylated clusters lgands of asialoglycoprotein receptor for targeted gene delivery tof (dhb,thap) (westerlind, et al., 2004) glycerol and glycol oligomers with mannose to study multivalent binding with proteins tof (dhb) (continued ) (oshovsky, et al., 2004) glycoclusters from silsesquioxanes prepared via thiol-radical addition reaction tof (gao, et al., 2004) o , et al., 2004) peptide dendrimers based on β-cyclodextrin thiol link to 6-position of glucose residues tof (muhanna, et al., 2003) poly(n-butyl-2-cyanoacrylate)/dextran nanoparticles comparison of methods for nanoparticle characterization tof (dhb) (weyermann, et al., 2004) polyphenylene-based glycodendrimers synthesis of glycodendrimers with sugars within scaffold tof (sakamoto & müllen, 2004) porphyrin-containing glycodendrimers with four and twelve derivatized glucose residues tof (chca) (ballardini, et al., 2003) schizophyllan with β-lactoside and αmannoside appendages naio 4 oxidation followed by reductive amination with aminoethyl-sugars tof spherical and hemispherical poly(ornithine) glycodendrimers maltose or lactose. up to 32 arms tof (dhb) (baigude, et al., 2004a ) (baigude, et al., 2003) tempo-functionalized pamam dendrimers with increasing mannose loadings tof (3-iaa) (samuelson, et al., 2004) thioglycosylated cationic porphyrins potential photodynamic drugs for colorectal adenocarcinoma maldi (ahmed, et al., 2004) tri ( bisected biantennary glycans plus bsa hemoenzymatic synth. bisect modulates ligand properties tof (dhb) (andré, et al., 2004) core of e. coli r4 plus tetanus toxoid synthesis via reductive amination tof (thap) (lukasiewicz, et al., 2003) fluorescent saccharide biosensors plus con-a photoaffinity labelling to concanavalin a tof (dhb, chca, sinapinic) (nagase, et al., 2003) gd 3 , gq 1b and gm 2 epitopes plus bsa for oligosaccharide-specific immunoadsorption therapy of guillian-barré syndrome tof (andersen, et al., 2004) glycopeptides from group a streptococcus and shigella flexneri y plus bsa or tt diethyl squarate coupling tof (dhb, sinapinic acid) (hossany, et al., 2004) ldnt, ldnfp and lnfpiii plus bsa study of targets for immune intervention in schistosomiasia tof lewis b hexasaccharide plus hsa linked with disuccinimidyl suberate spacer tof (thap) (zhu, et al., 2004b) o-specific polysaccharides from vibrio cholerae plus bsa di-to penta-saccharides linked via squaric acid diester chemistry seldi-tof (saksena, et al., 2003 ) schistosoma mansoni glycan fragments + bsa squaric acid chemistry. for studies on antibody binding tof trisaccharides from toxocara sp. plus bsa trisaccharides bound to bsa to act as immunoreagents tof (+ve) (dhb) (amer, hofinger & kosma, 2003) truncated complex glycans plus bsa ninhydrin coupling of pronase glycopeptides for binding studies maldi (van remoortere, et al., 2003) polymers allylurea-carbohydrate polymers synthesis of biodegradable material similar to thermoplastic tof (dhb) (olivier, et al., 2003) (1,2: (iyer, et al., 2003) polyacetyl containing trehalose reaction of α,α-d-trehalose with terephthaldehyde. mw 8 kda tof (chca) (teramoto, et al., 2004) polymers by ring-opening metathesis from norborene-attached acetyl-protected carbohydrates tof (iaa) schizophyllans with β-lactoside and α-man synthesis by naio 4 oxidation followed by reductive amination tof antibiotics/anticancer (menéndez, et al., 2004) kanamycin a-cholesterol conjugates aminoglycoside-derived cationic lipids for gene transfection r-tof (dhb) (sainlos, et al., 2003) landomycin e clusters new compounds synthesised by biosynthetic and lndgt4 gene manipulation tof (ostash, et al., 2004) squaric acid amides of anthracycline antibiotics for covalent binding to biomolecules tof (tevyashova, et al., 2004) vancomycin analogues one-pot synth. of modified carbohydrate after glycan removal maldi (ritter, et al., 2003) miscellaneous azido-glycoside on a sensor chip mannose-c 12 glycoside attached by two methods tof (sato, et al., 2004c) ferrocene-carbohydrate complexes two methods to attach mono-and di-saccharides to one or both cyclopentadienyl rings. electrochemical behaviour studied. , et al., 2003) oligosaccharide microarrays assembly of sugars on polystyrene plates ft-icr (fazio, et al., 2004) analysis of carbohydrates and glycoconjugates & the two areas that are particularly relevant for monitoring are those of glycodendrimers and glycoprotein conjugates. many polyfunctional compounds have been used to build the basic scaffold of these molecules. dendrimers built on such scaffolds include those on tris-(2-aminoethyl)-amine (60) (li et al., 2004e) , trimesic acid (61) (nishida et al., 2003) , carbosilanes (62) , silsesquioxanes (63) (gao et al., 2004) , cyclotriveratrylene (64) (van ameijde & liskamp, 2003) , tetra-aryl porphorins (65) (ballardini et al., 2003; ahmed et al., 2004; laville et al., 2004) and fullerene derivatives (isobe et al., 2003) . further examples are in table 11 . poly-hydroxy compounds such as carbohydrates themselves have been used as the core for dendrimers. thus, wu and kong (2004a) used ethylene glycol and glycerol to produce di-and trivalent (66) trisaccharides. three first-order dendrimers based on a cyclodextrin core containing fourteen val, phe or val-phe residues have been synthesized. the amino acids were linked to the terminal residues of 3,3 0 -iminobispropanolamine which was coupled through the central nitrogen to the 6-iodo derivative of bcyclodextrin (muhanna et al., 2003) . first-order dendrimers have also been synthesized by coupling 7, 14, or 21 man-o-ph-c:c-ch 2 -groups to b-cyclodextrin (ortega-caballero, giménez-martínez, & vargas-berenguel, 2003) (67) giving glycodendrimers with carbohydrate moieties both at the core and periphery. wu and kong (2004a) have also synthesized related compounds by cyclization of nona-and dodecasaccharides to give dendrimers containing hexa-and octa-saccharide rings. schizophyllan ([ ! glc-b-(1 ! 3)-(glc-b-(1 ! 6)glc-b-(1 ! 3)-glc-b-(1 ! 3)-] n ) has also been used as a backbone; b-glucoside appendages were oxidized to give aldehyde groups which were coupled to aminoethyl-b-lactoside or a-mannoside by reductive elimination . there is also a report of a glycodendrimer, based on a polyphenylene scaffold with a ring of monosaccharides between the center and the periphery (sakamoto & müllen, 2004) (68) . some of the largest multivalent glycodendrimers are based around a scaffold consisting of amino acids or polyamidoamine (pamam). in one of the latter cases, maldi-tof analysis of the products gave broad peaks with masses less than those predicted and attributed to incomplete synthesis of the pamam scaffold. masses ranged from 10,000 to 132,000 da (178 mannose residues) (woller et al., 2003) . choudhury, kitaoka, and hayashi (2003) with 32 sugar units produced a spectrum (m/z 19,132) but the next generation glycodendrimer (64 sugar units, predicted mass for c 1518 h 2656 o 892 n 250 ¼ 38,661) failed to give a signal. synthesis of large, fifth generation glycodendrimers with potentially 64 outer sugar residues (69) and sulfated analogues has been monitored by maldi-tof ms from thap (positive ion) and iaa (negative ion) to reveal that approximately 50 of the potential 64 sugars were actually incorporated (kensinger et al., 2004) . sharp maldi-tof peaks from spherical oligosaccharide-b-alanine-poly(lysine) dendrimers containing 32 cellobiose (13,418 da), maltose (13,472 da), and lactose (13,507 da) groups respectively, synthesized by baigude et al. (2003) , showed complete substitution of the amino groups by carbohydrate. differences were found between the measured and calculated masses of larger maltose-containing glycodendrimers (13 kda) based of a poly(ornithine) scaffold with the higher experimental masses being attributed to instrumental factors. the maldi-tof spectrum from chca of a third generation maltose-proline-poly(lysine) dendrimer (4.4 kda) synthesized by baigude et al. (2004b) gave three peaks attributed to the [m þ h] þ , [m þ na] þ , and [m þ k] þ ions. attempted addition of a succinyl group to each maltose residue produced a maldi spectrum with multiple peaks attributed to different numbers of attached succinyl groups and giving an average number of 13 attached groups. finally a 24-mer peptide was attached to produce a potential aids vaccine; maldi-tof analysis indicated the addition of one and two such units. self-assembling dendrimers carrying four or eight galnac residues have been constructed on a bipyridyl core; addition of cu(ii)so 4 linked two bipyridyl units to give the dendrimer. it was characterized by (70) (roy & kim, 2003) . matrix-assisted laser desorption/ionization-time-of-flight (maldi-tof) analysis is used in this area mainly to estimate the number of carbohydrate molecules bound to the protein. conjugation of the glycans is frequently by squaric acid chemistry or by reductive amination. however, lewis b hexasaccharide has been linked to human serum albumin (hsa) with a disuccinimidyl suberate spacer. the authors found that this spacer had a similar linking efficiency to squaric acid but had the advantage of a fast incorporation of the receptor saccharide to the carrier protein . saksena, ma, and kovác (2003) have used seldi to monitor the conjugation to bsa of di-through penta-saccharides that mimic the upstream terminus of the o-specific polysaccharide of vibrio cholerae o:1, serotype ogawa using squaric diester chemistry. seldi monitoring of the reaction allowed the extent of conjugation to be monitored and stopped when the desired molar hapten-bsa ratio had been reached. the accuracy of the mass measurement could be increased by using the carrier protein as the internal standard. tri-and hexa-galacturonates have been synthesized and coupled to bsa by reductive amination; maldi analysis gave a molecular weight of 70.996 corresponding to 3.8 mol of ligand/mol of bsa (clausen & madsen, 2004) . to develop antibodies to saikosaponin a, a triterpenoid saponin from the root of bupleuri radix, a plant used in eastern medicine, zhu et al. (2004b) have conjugated the saponins to bsa and determined the hapten number as 11 by maldi from sinapinic acid. concanavalin a has been labeled with a fluorescent probe at the active site by first binding a photolabile linker attached to mannose to the active site. the probe was then fixed to the protein by photoaffinity labeling, the mannose was removed by reduction of the disulfide link and the resulting thiol group used to attach various fluorescent reagents (nagase et al., 2003) . maldi from sinapinic acid was used to monitor various stages of the reaction and to demonstrate that the final addition of the fluorescent group occurred to the extent of 70-90%. nyame et al. (2003) have synthesized di-and trisaccharides bearing the ldn and ldnf epitopes and conjugated these, together with one carrying the lewis x epitope, to bsa. maldi-tof analysis showed that three to four molecules were coupled per mole of bsa. these and additional examples are listed in table 11 . a cyclosophoraose (cyclic-(1 ! 2)-b-d-glucan) named cys, produced by soil micro-organisms of the genus rhizobium, has been found to act as a catalyst for the methanolysis of several compounds such as phosphatidylcholine (lee & jung, 2004) . maldi analysis of several of the reaction intermediates showed acylated cyclosophoraoses in the mass range 3,000-4,500. carboxymethylated cys from r. leguminosarum biovar trifolii could be used to complex and, consequently increase the aqueous solubility of hydrobenzoin and n-acetyltryptophan (lee et al., 2004c) . a few additional papers documenting the use of maldi analysis for carbohydrate analysis that do not fit the above categories, have (mcce492), an 84-amino acid antimicrobial peptide from klebsiella pneumoniae has been purified in a post-translationally modified form from e. coli vcs257 harboring the pjam229 plasmid or k. pneumoniae rcy492. the 831 da modification was characterized by ms and nmr and found to consist of a trimer of n-(2,3-dihydroxybenzoyl)-l-serine linked via a c-glycosidic bond to b-d-glucose that was o-linked to the mcce492 (thomas et al., 2004b) . it has been proposed that tunicamycin, an inhibitor of d-hexnac-1-ptranslocases, exerts its action by coordinating the divalent metal cofactor at the active site. maldi spectra of tunicamycin in the presence of several divalent metals contained ions with compositions of [tun þ metal 2 ] þ showing that one of the metal atoms was present as a metal chelate (xu et al., 2004) . the cterminus loop 13 of the na þ glucose co-transporter (sglt1) has been shown to contain a binding site for inhibitory alkyl glucosides following photoaffinity labeling and maldi-tof analysis (raja, kipp, & kinne, 2004) . three hydroxyproline-rich peptides of 15, 18, and 20 amino acids, containing several pentose residues have been identified as defense signaling peptides released at wound sites in tomato plants (pearce & ryan, 2003) . from the above, it can be seen that maldi and particularly maldi-tof is still a major analytical method for carbohydrate analysis. even though electrospray ionization, particularly when coupled with cid, is being increasingly used, maldi-tof usually provides a superior profile of constituent sugars because of the tendency of the technique to produce only singly charged ions. however, maldi-tof ms of native acidic glycans is less satisfactory because of problems with prompt fragmentation. electrospray causes less fragmentation of these compounds but tends to produce ions in different charge states from glycans with several acidic groups, thus giving a profile that is not representative of the glycan content. however, this problem, and the instability of acidic carbohydrates under maldi conditions, can be overcome by derivatization of the carboxylic acid group of sialic acids but sulfates must be protected by other techniques such as ion pairing. thus, both maldi and electrospray have advantages and disadvantages for carbohydrate work and the best technique to use will be dictated by the problem to be solved. the work during the review period has shown that although maldi is now relatively ''mature,'' it is expected that future editions of this review series will be able to report many improved methods for performing these analyses. he is currently a consultant to the department of biochemistry, oxford, the oxford-dublin glycobiology institute and is an honorary professorial fellow at the university of warwick, uk. he has served on the committee of the british mass spectrometry society and has published more than 350 papers and reviews, mainly on mass spectrometry. enzymatic synthesis of complex glycosaminotrioses and study of their molecular recognition by hevein domains oligosaccharide and glycoprotein microarrays as tools in hiv glycobiology: glycan-dependent gp120/protein interactions thioglycosylated cationic porphyrins-convenient synthesis and photodynamic activity in vitro a method for detecting oglycanase in biological samples using a combination of maldi-tof mass spectrometry and time-resolved fluorimetry high efficiency of transferring a native sugar chain from a glycopeptide by a microbial endoglycosidase in organic solvents ionone, iridoid and phenylethanoid glycosides from ajuga salicifolia identification of n-glycosylation sites of the murine neural cell adhesion molecule ncam by maldi-tof and maldi-fticr mass spectrometry yeast glycogenin (glg2p) produced in escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation pimf, a mannosyltransferase of mycobacteria, is involved in the biosynthesis of phosphatidylinositol mannosides and lipoarabinomannan synthesis of tetra-and pentasaccharides corresponding to the capsular polysaccharide of streptococcus pneumoniae type 9a&l, 9n and 9a synthesis of cryptococcus neoformans capsular polysaccharide structures. iv. construction of thioglycoside donor blocks and their subsequent assembly synthesis of neoglycoproteins containing o-methylated trisaccharides related to excretory/secretory antigens of toxocara larvae improved capillary electrophoretic separation and mass spectrometric detection of oligosaccharides determination of nglycosylation sites and site heterogeneity in glycoproteins synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of guillian-barré syndrome structural studies of the extracellular polysaccharide produced by butyrivibrio fibrisolvens strain h10b determination of modulation of ligand properties of synthetic complex-type biantennary n-glycans by introduction of bisecting glcnac in silico, in vitro and in vivo default biosynthesis pathway for blood group-related glycolipids in human small intestine as defined by structural identification of linear and branched glycosylceramides in a group o le(a-b-) nonsecretor detailed structural analysis of the peptidoglycan of the human pathogen neisseria meningitidis study of the attachment of na þ on glucose and on some of its methylated derivatives mycalosides b-i, eight new spermostatic steroid oligoglycosides from the sponge mycale laxissima classification into a novel mollu-series of neutral glycosphingolipids from the lamp shell, lingula unguis structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells newly discovered neutral glycosphingolipids in aureobasidin a-resistant zygomycetes: identification of a novel family of gala-series glycolipids with core gal-1-6-gal-1-6-gal sequences synthesis of a branched chain aza-cdisaccharide via the cycloaddition of a chiral nitrone to an alkene, both sugar derivatives the glycosylation of human serum igd and ige and the accessibility of identified oligomannose structures for interaction with mannan binding lectin synthesis and cholera toxin binding properties of multivalent gm1 mimics preliminary investigation of the simultaneous detection of sugars, ascorbic acid, citric acid, and sodium benzoate in non-alcoholic beverages by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry an efficient approach towards the convergent synthesis of fully-carbohydrate mannodendrimers synthesis of sphere-type monodispersed oligosaccharide-polypeptide dendrimers synthesis of spherical and hemispherical sugar-containing poly(ornithine) dendrimers synthesis of structurally-controlled aids vaccine model with glyco-peptide dendrimer scaffolds porphyrin-containing glycodendrimers role of carbohydrate in stabilizing the triple-helix in a model for a deep-sea hydrothermal vent worm collagen characterization of the o-4-phosphorylated and o-5 substituted kdo reducing end group and sequencing of the core oligosaccharide of aeromonas salmonicida ssp salmonicida lipooligosaccharide using tandem mass spectrometry n-terminal segment of potato virus x coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface immunoreactivity in mammals of two typical plant glyco-epitopes, core (1,3)-fucose and core xylose monoclonal c5-1 antibody produced in transgenic alfalfa plants exhibits a n-glycosylation that is homogenous and suitable for glycoengineering into human-compatible structures identification of the n-glycosylation sites on glutamate carboxypeptidase ii necessary for proteolytic activity 2-nitro thioglycoside donors: versatile precursors of b-d-glycosides of aminosugars martix-assisted laser desorption time-of-flight mass spectrometry of dextran and dextrin derivatives matrix-assisted laser desorption/ ionization mass spectrometry with re-engineered 2,5-dihydroxybenzoic acid derivative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. a comparison of fragmentation patterns of linear dextran obtained by in-source decay, post-source decay and collision-induced dissociation and the stability of linear and cyclic glucans studied by insource decay lipids of the zygomycete absidia corymbifera f-965 combinatorial carbohydrate synthesis expression of eukaryotic glycosyltransferases in the yeast pichia pastoris specificity of igg and ige antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin a newly discovered cholesteryl galactoside from borrelia burgdorferi chronic diabetes increases advanced glycation end products on cardiac ryanodine receptors/calcium-release channels controlled g-ray irradiation of heparin generates oligosaccharides enriched in highly sulfated sequences sialoside specificity of the siglec family assessed using novel multivalent probes. identification of potent inhibitors of myelin-associated glycoprotein use of 15 n-nmr to resolve molecular details in isotopically-enriched carbohydrates: sequence-specific observations in hyaluronan oligomers up to decasaccharides dendrimers in drug research engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast pichia pastoris: production of complex humanized glycoproteins with terminal galactose human and bovine milk gangliosides differ in their fatty acid composition prebiotic concept for infant nutrition differential recognition of animal type b4-galactosylated and 3-fucosylated chito-oligosaccharides by two family 18 chitinases from trichoderma harzianum mode of action of fusarium moniliforme endopolygalacturonase towards acetylated pectin oglycosylation of a recombinant carbohydrate-binding module mutant secreted by pichia pastoris replacement of carbohydrate sulfates by sugar c-sulfonic acid derivatives ion exchange and purification of carbohydrates on a nafion(r) membrane as a new sample pretreatment for matrix-assisted laser desorption-ionization mass spectrometry differential effects of subunit asparagine 56 oligosaccharide structure on equine lutropin and follitropin hybrid conformation and receptor-binding activity glycerol and glycerol glycol glycodendrimers sugaring' carbosilane dendrimers via hydrosilylation genetic engineering of pichia pastoris to humanize nglycosylation of proteins matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry of oligosaccharides derivatized by reductive amination and n,n-dimethylation synthetic glycopeptides of the tandem repeat sequence of the epithelial mucin muc4 with tumour-associated carbohydrate antigens synthetic tumor-associated glycopeptide antigens from the tandem repeat sequence of the epithelial mucin muc4 glycoforms of blactoglobulin with improved thermostability and preserved structural packing aminoglycoside array for the high-throughput analysis of small molecule-rna interactions screening of sugar converting enzymes using quantitative maldi-tof mass spectrometry quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities the immunogenicity of the tumorassociated antigen lewis y may be suppressed by a bifunctional crosslinker required for coupling to a carrier protein detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis determination of carbohydrates in juices by capillary electrophoresis, high-performance liquid chromatography, and matrix-assisted laser desorption/ionizationtime of flight-mass spectrometry anionic adducts of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry strategies for shotgun identification of posttranslational modifications by mass spectrometry methods for the analysis of hyaluronal and its fragments in acute inflammation, the chondroitin-4 sulphate carried by bikunin is not only longer structural study of gcdfp/gp17 in disease versus physiological conditions using a proteomic approach structure of bacterial lipopolysaccharides identification and characterization of a novel cassava (manihot esculenta crantz) clone with high free sugar content and novel starch convenient methods for the synthesis of ferrocenecarbohydrate conjugates exopolysaccharides produced by a clinical strain of burkholderia cepacia isolated from a cystic fibrosis patient analysis of a bioactive b-(1-3) polysaccharide (curdlan) using matrix-assisted laser desorption/ionization time-offlight mass spectrometry a straightforward synthesis of 1-adamantylmethyl glycosides, and their binding to cyclodextrins fructans in crested wheatgrass leaves molecularly imprinted tio 2 -matrix-assisted laser desorption/ionization mass spectrometry for selectively detecting a-cyclodextrin an efficient and practical synthesis of a-(1-3)-linked mannohexaose and mannooctaose differently sized granules from acetylated potato and sweet potato starches differ in the acetyl substitution pattern of their amylose populations characterization of a novel human udp-galnac transferase, pp-galnac-t15 characterizing biological products and assessing comparability following manufacturing changes use of combinatorial genetic libraries to humanize n-linked glycosylation in the yeast pichia pastoris nglycan structures of human transferrin produced by lymantria dispar (gypsy moth) cells using the ldmnpv expression system characterisation of mesorhizobium huakuii cyclic beta-glucan characterization of mesorhizobium huakuii lipid a containing both d-galacturonic acid and phosphate residues synthesis of a cellobiosylated dimer and trimer and of cellobiose-coated polyamidoamine (pamam) dendrimers to study accessibility of an enzyme, cellodextrin phosphorylase identification of o-linked n-acetylglucosamine proteins in rat skeletal muscle using two-dimensional gel electrophoresis and mass spectrometry the structure of the oligosaccharides of n-cadherin from human melanoma cell lines biosynthesis in vitro of caenorhabditis elegans phosphorylcholine oligosaccharides srf-3, a mutant of caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates synthesis of oligogalacturonates conjugated to bsa different behavior of dextrans in positive-ion and negative-ion mass spectrometry identity and localization of advanced glycation end products on human b 2 -microglobulin using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry amino acid domains control the circulatory residence time of primate acetylcholinesterases in rhesus macaques (macaca mulatta) comparative study of carbohydrate chains released from the oviducal mucins of the two closely related amphibian species bombina bombina and bombina variegata structure of lipid a from pseudomonas corrugata by electrospray ionization quadrupole time-of-flight tandem mass spectrometry glycosphingolipids in plasmodium falciparum. presence of an active glucosylceramide synthase development on new methodologies for the mass spectrometry study of bioorganic macromolecules cell transfection with polycationic cyclodextrin vectors synthesis of an arabinogalactan-type octa-and two isomeric nonasaccharides. suitable tuning of protecting groups paradoxical impact of antioxidants on post-amadori glycoxidation. counterintuitive increase in the yields of pentosidine and n e -carboxymethyllysine using a novel multifunctional pyridoxamine derivative evaluation of newly synthesized and commercially available charged cyclomaltooligosaccharides (cyclodextrins) for capillary electrokinetic chromatography characterization of glycopeptides from hiv-i sf2 gp120 by liquid chromatography mass spectrometry localization of the o-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group matrixassisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance analyses of end-functionalized saccharidic polymers: an example of a useful analytical technique combination molecular properties of hemicelluloses located in the surface and inner layers of hardwood and softwood pulps synthesis and characterisation of novel chromogenic substrates for human pancreatic a-amylase porphyromonas gingivalis lipopolysaccharide contains multiple lipid a species that functionally interact with both toll-like receptors 2 and 4 polysaccharides of pseudomonas pathovar strains that infect pea, tomato, and soya bean functional analysis of the alg3 gene encoding the dol-p-man: man 5 glcnac 2 -pp-dol mannosyltransferase enzyme of p. pastoris structural determination of the o-chain moieties of the lipopolysaccharide fraction from agrobacterium radiobacter dsm a novel core region, lacking heptose and phosphate, of the lipopolysaccharide from the gram-negative bacterium pseudomonas cichorii (pseudomonadaceae rna group 1) synthesis and conjugation of oligosaccharide analogues of fragments of the immunoreactive glycan part of the circulating anodic antigen of the parasite schistosoma mansoni rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors sulfated oligosaccharides isolated from the respiratory mucins of a secretor patient suffering from chronic bronchitis murine and human zona pellucida 3 derived from mouse eggs express identical o-glycans determination of glycopeptide structures by multistage mass spectrometry with low-energy collision-induced dissociation: comparison of electrospray ionization quadrupole ion trap and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight approaches synthesis of osw-1 analogs with modified side chains and their antitumor activities monitoring glycosylation of therapeutic glycoproteins for consistency using highly fluorescent anthranilic acid revisiting the structure of the anti-neoplastic glucans of mycobacterium bovis bacille calmette-guérin: structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains polysaccharides from grape berry cell walls. part ii. structural characterization of the xyloglucan polysaccharides characterization of the carbohydrate moieties of the functional unit rvh 1 -a of rapana venosa haemocyanin using hplc/electrospray ionization ms and glycosidase digestion carbohydrate moieties of molluscan rapana venosa hemocyanin structural and functional analysis of glycosylated cu/zn-superoxide dismutase from the fungal strain humicola lutea 103 a systematic nomenclature for carbohydrate fragmentations in fab-ms/ms spectra of glycoconjugates a new synthetic approach to mycobacterial cell wall a-(1 ! 5)-d-arabinofuranosyl c-oligosaccharides a convenient synthesis of iminosugar-cglycosides via organometallic addition to n-benzyl-n-glycosylhydroxylamines assembling heterocycle-tethered c-glycosyl and a-amino acid residues via 1,3-dipolar cycloaddition reactions microbial transformation of ginsenoside rb1 by rhizopus stolonifer and curvularia lunata synthesis of saponins using partially protected glycosyl donors deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function phenolic glycoside isolated from seeds of the greater plantain (plantago major l.) integrated selective enrichment target-a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseases n-acetylglucosamine-6-o-sulfotransferase-1: production in the baculovirus system and its applications to the synthesis of a sulfated oligosaccharide and to the modification of oligosaccharides in fibrinogen thalictricoside, a new phenolic compound from thalictrum orientale physocalycoside, a new phenylethanoid glycoside from phlomis physocalyx hub.-mor automated structural assignment of derivatized complex n-linked oligosaccharides from tandem mass spectra application of the stroligo algorithm for the automated structure assignment of complex n-linked glycans from glycoproteins using tandem mass spectrometry in vitro synthesis of a crystalline (1 ! 3,1 ! 4)-b-d-glucan by a mutated (1 ! 3,1 ! 4)-b-d-glucanase from bacillus a method for proteomic identification of membrane-bound proteins containing asn-linked oligosaccharides regioselective synthesis of galactosylated tri-and tetrasaccharides by use of b-galactosidase from bacillus circulans contribution of mass spectrometry to the study of the maillard reaction in food rucl 3 -promoted amide formation from azides and thioacids assembly of sugars on polystyrene plates: a new facile microarray fabrication technique solid-state glycation of b-lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques characterization of plant oligosaccharides by matrix-assisted laser desorption/ionization and electrospray mass spectrometry functional properties and application in peptide synthesis of trypsin modified with cyclodextrin-containing dicarboxylic acids biochemical and mass spectrometric characterization of soluble ecto-5 0 -nucleotidase from bull seminal plasma mycobacterial phosphatidylinositol mannoside is a natural antigen for cd1d-restricted t cells the effects of ethanol on the glycosylation of human transferrin n-and o-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colonystimulating factor secreted by a chinese hamster ovary cell line irradiation effects in maldi, ablation, ion production, and surface modifications. part ii: 2,5-dihydroxybenzoic acid monocrystals role of electrons in laser desorption/ionization mass spectrometry structural elucidation of zwitterionic carbohydrates derived from glycosphingolipids of the porcine parasitic nematode ascaris suum overexpression of the waaz gene leads to modification of the structure of the inner core region of escherichia coli lipopolysaccharide, truncation of the outer core, and reduction of the amount of o polysaccharide on the cell surface evaluation of sphingolipids in vitreous bodies from a patient with gaucher disease, using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry n-linked glycan structures of human lactoferrin produced by transgenic rice chemoenzymatic synthesis of diverse asparagine-linked a-(2 ! 3)-sialyloligosaccharides an efficient synthesis of a biantennary sialooligosaccharide analogue using a 1,6-anhydro-b-lactose derivative as a key synthetic block sensing behavior of fluorescent cyclodextrin/peptide hybrids bearing a macrocyclic metal complex separation and characterization of humic acids from antarctia by capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. inclusion complexes of humic acids with cyclodextrins chemoenzymatic synthesis of conformationally constrained oligosaccharides rapid screening of plasma volume expanders in urine using matrix-assisted laser desorption/ionisation time-offlight mass spectrometry enzymatic synthesis of the core-2 sialyl lewis x o-glycan on the tumor-associated muc1a' peptide analyzing response variability using high-ph anion-exchange chromatography and pulsed amperometric detection progresses of derivatization techniques for analyses of carbohydrates efficient preparation of glycoclusters from silsesquioxanes characterization of low-glycosylated forms of soluble human urokinase receptor expressed in drosophila schneider 2 cells after deletion of glycosylation-sites functional microdomains of glycolipids with partially fluorinated membrane anchors: impact on cell adhesion stereoselective entry to b-linked c-disaccharides using a carbon-ferrier reaction solutionand bound-state conformational study of n,n,n-triacetyl chitotriose and other analogous potential inhibitors of hevamine: application of trnoesy and std nmr spectroscopy advances in the production of human therapeutic proteins in yeasts and filamentous fungi glycosylation of human recombinant gonadotrophins: characterization and batch-tobatch consistency characterization of keyhole limpet hemocyanin (klh) glycans sharing a carbohydrate epitope with schistosoma mansoni glycoconjugates cell wall polysaccharides of brassica campestris seed cake: isolation and structural features identification of a novel mannose-capped lipoarabinomannan from amycolatopsis sulphurea tsukamurella paurometabola lipoglycan, a new lipoarabinomannan variant with pro-inflammatory activity acylation state of the phosphatidylinositol hexamannosides from mycobacterium bovis bacillus calmette guérin and mycobacterium tuberculosis h37rv and its implication in toll-like receptor response are proton transfer reactions of excited states involved in uv laser desorption ionization? primary structure of the 2-omethyl-a-l-fucose-containing side chain of the pectic polysaccharide, rhamnogalacturonan ii differences among the cell wall galactomannans from aspergillus wentii and chaetosartorya chrysella and that of aspergillus fumigatus metabolic incorporation of unnatural sialic acids into haemophilus ducreyi lipooligosaccharides rapid oligosaccharide synthesis on a fluorous support molecular cloning and characterization of b1 ! 4-n-acetylgalactosaminyltransferases iv synthesizing n,n 0 -diacetyl-lactosediamine glycosylation and specific deamidation of ribonuclease b affect the formation of three-dimensional domain-swapped oligomers atbxl1, a novel higher plant (arabidopsis thaliana) putative beta-xylosidase gene, is involved in secondary cell wall metabolism and plant development unique in vivo modifications of coagulation factor v produce a physically and functionally distinct platelet-derived cofactor efficient preparation of carbohydrate-and related polyol-amphiphiles by hydrazone ligation comparison of maldi-tof mass spectrometric to enzyme colorimetric quantification of glucose from enzyme-hydrolyzed starch nontypeable haemophilus influenzae strain 2019 produces a biofilm containing n-acetylneuraminic acid that may mimic sialylated o-linked glycans the glycosylated androgenic hormone of the terrestrial isopod porcellio scaber (crustacea) specific xyloglucanases as a new class of polysaccharidedegrading enzymes diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis synthesis of 2-chloro-4-nitrophenyl a-lfucopyranoside: a substrate for a-l-fucosidase (afu) structural characterization of the lipid a component of sinorhizobium sp. ngr234 rough and smooth form lipopolysaccharide. demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acids is conserved in rhizobium and sinorhizobium sp molecular fingerprinting of carbohydrate structure phenotypes of three porifera proteoglycan-like glyconectins lipomannan and lipoarabinomannan from a clinical isolate of mycobacterium kansasii: novel structural features and apoptosis-inducing properties solidsupported enzymatic synthesis of pectic oligogalacturonides and their analysis by maldi-tof mass spectrometry neutral n-glycans of the gastropod arion lusitanicus glycopeptides as oligosaccharide mimics: high affinity sialopeptide ligands for sialoadhesin from combinatorial libraries structural characterization of tsc-36/ flik. analysis of two charge isoforms production of complex human glycoproteins in yeast polymer-resin hybrid capturerelease strategy for rapid oligosaccharide construction chemo-enzymatic synthesis and structureactivity study of artificially n-glycosylated eel calcitonin derivatives with a complex type oligosaccharide purification and characterization of mouse soluble receptor for advanced glycation end products (srage) o-linked glycans control glycoprotein processing by antigenpresenting cells: a biochemical approach to the molecular aspects of muc1 processing by dendritic cells synthesis and characterization of persilylated cyclodextrins a novel synthesis of deoxy phospha sugar-sugar disaccharides quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates and glycoconjugates analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period 1999-2000 analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period fragmentation of nlinked glycans with a maldi-ion trap time-of-flight mass spectrometer conversion of pyridylamino sugar chains to 1-amino-1-deoxy derivatives, intermediates for tagging with fluorescein and biotin schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier structural analysis of the carbohydrate backbone of vibrio parahaemolyticus o2 lipopolysaccharides structural characterization of the carbohydrate backbone of the lipopolysaccharide of vibrio parahaemolyticus o-untypeable strain kx-v212 isolated from a patient structural elucidation of polysaccharide part of glycoconjugate from treponema medium atcc 700293 chemical structure and immunobiological activity of lipid a from prevotella intermedia atcc 25611 lipopolysaccharide hallmarks of caenorhabditis elegans n-glycosylation: complexity and controversy synthesis of laminarin oligosaccharide derivatives having d-arabinofuranosyl side-chains endotoxic activity and chemical structure of lipopolysaccharides from chlamydia trachomatis serotypes e and l 2 and chlamydophila psittaci 6bc synthesis and characterisation of terminal carbohydrate modified poly(dimethylsiloxane)s specificity of amaranthus leucocarpus syn. hypocondriacus lectin for o-glycopeptides altered glycosylation of a 1 -acid glycoprotein in patients with inflammation and diabetes mellitus targeted proteoglycomics analysis of sialyl lewis x antigen expressing glycoproteins secreted by human hepatoma cell line iga nephropathy and tonsils-an approach from the structure of iga1 produced by tonsillar lymphocytes affinity capturing and gene assignment of soluble glycoproteins produced by the nematode caenorhabditis elegans preparation of a growth-promoting substance, lepidimoic acid, from okra pectic polysaccharide a convenient synthesis of lepidimoide from okra mucilage and its growth-promoting activity in hypocotyls a novel human b1 ! 3-nacetylgalactosaminyltransferase that synthesizes a unique carbohydrate structure, galnacb1 ! 3glcnac high-mannose-type oligosaccharides from human placental arylsulfatase a are core fucosylated as confirmed by maldi ms the first synthesis of peptide thioester carrying nlinked core pentasaccharide through modified fmoc thioester preparation: synthesis of an n-glycosylated ig domain of emmprin proteomic analysis of kappacasein micro-heterogeneity two glycosylation alterations of mouse intestinal mucins due to infection by the parasite nippostrongylus brasiliensis the lec23 chinese hamster ovary mutant is a sensitive host for detecting mutations in a-glucosidase i that give rise to congenital disorder of glycosylation iib iga1 molecules produced by tonsillar lymphocytes are under-o-glycosylated in iga nephropathy the structural alteration of o-glycosylated hinge peptides in serum iga1 before and after tonsillectomy in iga nephropathy peptide and amino acid glycation: new insights into the maillard reaction an engineered hyaluronan synthase. characterization of recombinant human hyaluronan synthase 2 expressed in escherichia coli fluorescence resonance energy transfer in a novel cyclodextrin-peptide conjugate for detecting steroid molecules synthesis and immunochemical characterization of protein conjugates of carbohydrate and carbohydrate-mimetic peptides as experimental vaccines synthesis and biological activities of octyl 2,3-di-o-sulfo-a-l-fucopyranosyl-(1 ! 3)-2-o-sulfo-a-lfucopyranosyl-(1 ! 4)-2,3-di-o-sulfo-a-l-fucopyranosyl-(1 ! 3)-2-osulfo-a-l-fucopyranosyl-(1 ! 4)-2,3-di-o-sulfo-b-l-fucopyranoside synthesis and biological activities of octyl 2,3,4-tri-o-sulfo-a-l-fucopyranosyl-(1 ! 3)-2,4-di-o-sulfo-a-l-fucopyranosyl-(1 ! 3)-2,4-di-o-sulfo-a-l-fucopyranosyl-(1 ! 3)-2,4-di-o-sulfo-b-l-fucopyranoside application of maldi-qtof in proteomics and glycomics microscale nonreductive release of o-linked glycans for subsequent analysis through maldi mass spectrometry and capillary electrophoresis ardisimamillosides g and h, two new triterpenoid saponins from ardisia mamillata effects of polymerization initiator complexation in methacrylated b-cyclodextrin formulations analysis of site-specific glycosylation in recombinant human follistatin expressed in chinese hamster ovary cells glycosylation of rapana thomasiana hemocyanin. comparison with other prosobranch (gastropod) hemocyanins efficient transport of saccharides through a liquid membrane mediated by a cyclodextrin dimer preparation and characterization of novel branched b-cyclodextrins having b-d-galactose residues on the non-reducing terminal of the side chains and their specific interactions with peanut (arachis hypogaea) agglutinin molecular cloning and characterization of human gnt-ix, a novel b1,6-n-acetylglucosaminyltransferase that is specifically expressed in the brain n-acetylglucosaminyltransferase ix acts on the glcnacb1,2-mana1-ser/thr moiety, forming a 2,6-branched structure in brain o-mannosyl glycan synthesis of fullerene glycoconjugates through sulfide connection in aqueous media glycosphingolipids in edible fungi and their biological activities microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids recombinant (2 ! 3)-asialyltransferase immobilized on nickel-agarose for preparative synthesis of sialyl lewis x and lewis a precursor oligosaccharides coupling thin-layer chromatography with vibrational cooling matrix-assisted laser desorption/ionization fourier transform mass spectrometry for the analysis of ganglioside mixtures initiation of oglycan synthesis in iga1 hinge region is determined by a single enzyme, udp-n-acetyl-a-d-galactosamine:polypeptide n-acetylgalactosaminyltransferase 2 synthesis of a hyaluronan neoglycopolymer by ring-opening metathesis polymerization the klebsiella pneumoniae wabg gene: role in biosynthesis of the core lipopolysaccharide and virulence mannose-bsa conjugates: comparison between commercially available linkers in reactivity and bioactivity isolation and characterization of water-soluble hemicelluloses from flax shive identification of post-translational modifications resulting from lhb polymorphisms by matrix-assisted laser desorption time-of-flight mass spectrometric analysis of pituitary lhb core fragment mass spectrometric characterization of a new 2-hydroxypropylb-cyclodextrin derivative bearing methacrylic moieties and its copolymerization with 1-vinyl-2-pyrrolidone one-pot chemo-, regio-, and stereoselective double-differential glycosidation mediated by lanthanide triflates synthesis of a key mycobacterium tuberculosis biosynthetic phosphoinositide intermediate carbohydrate biosensors modification-specific proteomics: characterization of post-translational modifications by mass spectrometry specificity of enzymatic in vitro glycosylation by pngase f: a comparison of enzymatic and nonenzymatic glycosylation structure of the xyloglucan produced by suspension-cultured tomato cells chemical characterization of different sugar-casein maillard reaction products and protective effects on chemicalinduced cytotoxicity of caco-2 cells glycosyltransferase assays utilizing n-acetyllactosamine acceptor immobilized on a cellulose membrane effect of silkworm hemolymph on n-linked glycosylation in two trichoplusia ni insect cell lines synthesis of b-d-galp-(1 ! 4)-b-d-glcpnac-(1 ! 2)-a-d-manp-(1 ! o)(ch 2 ) 7 ch 3 mimics to explore the substrate specificity of sialyltransferases and trans-sialidases location of o-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated eucalyptus wood inner core assembly and structure of the lipooligosaccharide of neisseria meningitidis: capacity of strain nmb to express all known immunotype epitopes prompt chemoenzymatic synthesis of diverse complex-type oligosaccharides and its application to the solid-phase synthesis of a glycopeptide with asn-linked sialyl-undeca-and asialo-nonasaccharides analysis of glycoproteins and the oligosaccharides thereof by high-performance capillary electrophoresis-significance in regulatory studies on biopharmaceutical products inclusion complexes formed by sodium dodecyl sulfate and cyclodextrin-based nanotubes steroid polyols from the far eastern starfish henricia sanguinolenta and h. leviuscula leviuscula analysis of the site-specific n-glycosylation of b1,6 nacetylglucosaminyltransferase v detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis polyphenolics in rhizophora mangle l. leaves and their changes during leaf development and senescence a-amylases of medical and industrial importance inhibitory effects of tannin on human salivary a-amylase a novel b(1 ! 6)-n-acetylglucosaminyltransferase v (gnt-vb) total chemical synthesis and nmr characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is a-d-gal-(1 ! 3)-a-d-galnac relaxed sugar donor selectivity of a sinorhizobium meliloti ortholog of the rhizobium leguminosarum mannosyl transferase lpcc: role of the lipopolysaccharide core in symbiosis of rhizobiaceae with plants a mannosyl transferase required for lipopolysaccharide inner core assembly in rhizobium leguminosarum. purification, substrate specificity and expression in salmonella waac mutants expression cloning and biochemical characterization of a rhizobium leguminosarum lipid a 1-phosphatase biclonal systemic al-amyloidosis with one glycosylated and one nonglycosylated al-protein acylated flavonoid and phenylethanoid glycosides from marrubium velutinum fluorous-tagged compound: a viable scaffold to prime oligosaccharide synthesis by cellular enzymes in vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (raphanus sativus l.) seedlings study on the structures of xyloglucans using specific enzymes structural variability of bm-40/sparc/osteonectin glycosylation: implications for collagen affinity natural antibiotic function of a human gastric mucin against helicobacter pylori infection malonylated flavonol glycosides from the petals of clitoria ternatea flavonoid composition related to petal color in different lines of clitoria ternatea post-source decay matrix-assisted laser desorption/ionization mass spectrometric study of peracetylated isoflavone glycosides cationized by protonation and with various metal ions cationization of simple organic molecules by singly-charged ag 3 þ cluster ions in matrix-assisted laser desorption/ionization mass spectrometry: metal cluster-molecule interactions synthesis of novel, multivalent glycodendrimers as ligands for hiv-1 gp120 a structural analysis of heparin-like glycosaminoglycans using maldi-tof mass spectrometry structural characterization of the nglycan moiety and site of glycosylation in vitellogenin from the decapod crustacean cherax quadricarinatus conjugate addition of phenols to 2-nitrogalactal-synthesis of o-(2-acetamido-2-deoxygalactosyl)tyrosine a convenient route to oglycosyl lactates via conjugate addition to 2-nitroglycals: ring closure to novel pyrano an inositol-containing sphingolipid from the red alga gracilaria verrucosa a facile synthesis of novel cyclodextrin derivatives incorporating one b-(1,4)-glucosidic bond and their unique inclusion ability amphiphilic cyclodextrins as novel monosaccharide transport carriers through a bulk liquid membrane proteomic characterization of novel serum amyloid p component variants from human plasma and urine structural determination of the n-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein glycosylation of onconase increases its conformational stability and toxicity for cancer cells relative quantification of n-(carboxymethyl)lysine, imidazolone a, and the amadori product in glycated lysozyme by maldi-tof mass spectrometry analysis of protein glycation products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry synthesis and conformational characterization of the epidermal growth factor-like domain of blood coagulation factor ix carrying xylosyl-glucose chitosanolysis by a pectinase isozyme of aspergillus niger-a non-specific activity in situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization ms analysis of polypeptide mixtures glyco-sams as glycocalyx mimetics: synthesis of l-fucose-and d-mannose-terminated building blocks function and glycosylation of plant-derived antiviral monoclonal antibody functional evaluation of carbohydrate-centred glycoclusters by enzyme-linked lectin assay: ligands for concanavalin a influence of lactation parameters on the nglycosylation of recombinant human c1 inhibitor isolated from the milk of transgenic rabbits n-and o-glycans of recombinant human c1 inhibitor expressed in the milk of transgenic rabbits isolation and characterization of periplasmic cyclic b-glucans of azorhizobium caulinodans biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (glycine max merr.) seedlings structure of a highly phosphorylated lipopolysaccharide core in the dalgc mutants derived from pseudomonas aeruginosa wild-type strains pao1 (serogroup o5) and pac1r (serogroup o3) high affinity capture surface for matrixassisted laser desorption/ionisation compatible protein microarrays targeted knockouts of physcomitrella lacking plant specific immunogenic n-glycans identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species purification and cdna cloning of udp-glcnac:glcnacb1 ! 3galb1 ! 4glc(nac)-r [glcnac ! gal]b1,6n-acetylglucosaminyltransferase from rat small intestine: a major carrier of dignt activity in rat small intestine polylactosamine synthesis and branch formation of n-glycans in b1,4-galactosyltransferase-1-deficient mice n-glycosylation pattern of the zymogenic form of human matrix metalloproteinase-9 liquid chromatographic and mass spectrometric analysis of human serum acid alpha-1-glycoprotein trichoderma reesei acetyl esterase catalyzes transesterification in water mass spectrometric characterization of proteins from the sars virus. a preliminary report site-specific n-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides an improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase hplc. its application to protein peptide mapping by off-line hplc-maldi ms matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry of heteropolyflavan-3-ols and glucosylated heteropolyflavans in sorghum quantification of hyaluronic acid fragments in pharmaceutical formulations using lc-esi-ms synthesis of stable dolichylphosphomannose analogues structural studies on igg oligosaccharides of patients with primary sjögren's syndrome structural characterization of nglycopeptides by matrix-dependent selective fragmentation of maldi-tof/tof tandem mass spectrometry post-translational modifications on proteins: facile and efficient procedure for the identification of o-glycosylation sites by maldi-lift-tof/tof mass spectrometry sequencing of n-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high performance liquid chromatography identification of glycosylation sites in the su component of the avian sarcoma/leukosis virus envelope glycoprotein (subgroup a) by mass spectrometry synthesis of the trisaccharide repeating unit of the atypical o-antigen polysaccharide from danish helicobacter pylori strains employing the 2 0 -carboxybenzyl glycoside synthesis of imino sugar scaffolds for the generation of glycosidase inhibitor libraries synthesis of the lewis b hexasaccharide and hsa-conjugates thereof preparation and chiral recognition of a novel chiral stationary phase for high-performance liquid chromatography, based on mono(6 a -n-allylamino-6 a -deoxy)-perfunctionalized bcyclodextrin and covalently bonded silica gel derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis dynamic glycosylation of the transcription factor creb: a potential role in gene regulation characterization of lecithin:cholesterol acyltransferase expressed in a human lung cell line enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides the complexity of non-enzymatic glycation product sets of human globins expression, secretion, and glycosylation of the 45-and 47-kda glycoprotein of mycobacterium tuberculosis in streptomyces lividans structural characterization of the o-chain polysaccharide isolated from bordetella avium atcc 5086: variation on a theme labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry profiling of n-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry a new synthetic route for the preparation of glycoprobes hazelnut (corylus avellana) vicilin cor a 11: molecular characterization of a glycoprotein and its allergenic activity melanoma cell cd44 interaction with the 1(iv) 1263-1277 region from basement membrane collagen is modulated by ligand glycosylation combination of two matrices results in improved performance of maldi ms for peptide mass mapping and protein analysis a study of the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic therapy, in human colon tumoural cells: a liquid chromatography and maldi-tof mass spectrometry analysis transposon mutagenesis of trypanosoma brucei identifies glycosylation mutants resistant to concanavalin a enantioseparation using cyclosophoraoses as a novel chiral additive in capillary electrophoresis cyclosophoraose as a catalytic carbohydrate for methanolysis antidiabetic effects of chitosan oligosaccharides in neonatal streptozotocin-induced noninsulin-dependent diabetes mellitus in rats antibodies that recognize bisected complex n-glycans on cell surface glycoproteins can be made in mice lacking n-acetylglucosaninyltransferase iii a mutation causing a reduced level of expression of six 4-galactosyltransferase genes is the basis of the lec19 cho glycosylation mutant structural analysis of lipid a from escherichia coli o157:h7:k-using thin-layer chromatography and ion-trap mass spectrometry the pmra-regulated pmrc gene mediates phosphoethanolamine modification of lipid a and polymyxin resistance in salmonella enterica synthesis and characterization of carboxymethylated cyclosophoraose, and its inclusion complexation behavior current chemical tagging strategies for proteome analysis by mass spectrometry a genetic and structural analysis of the n-glycosylation capabilities of rice and other monocotyledons a novel type of highly negatively charged lipooligosaccharide from pseudomonas stutzeri ox1 possessing two 4,6-o-(1-carboxy)-ethylidene residues in the outer core region structure of minor oligosaccharides from the lipopolysaccharide fraction from pseudomonas stutzeri ox1 identification of mdod, an mdog paralog which encodes a twin-arginine-dependent periplasmic protein that controls osmoregulated periplasmic glucan backbone structures steroidal polyols from far-eastern starfishes henricia sanguinolenta and h. leviuscula leviuscula a new steroidal glycoside phrygioside a and its aglycone from the starfish hippasteria phrygiana discovery and characterization of sialic acid o-acetylation in group b streptococcus syntheses of arabinogalactans consisting of b-(1 ! 6)-linked d-galactopyranosyl backbone and a-(1 ! 3)-linked l-arabinofuranosyl side chains biotechnological production of highly soluble daidzein glycosides using thermotoga maritima maltosyltransferase application of glycodiversification: expedient synthesis and antibacterial evaluation of a library of kanamycin b analogues starch from hull-less barley: iv. morphological and structural changes in waxy, normal and high-amylose starch granules during heating comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of escherichia coli synthesis of cluster mannosides via a ugi four-component reaction and their inhibition against the binding of yeast mannan to concanavalin a expression of functional human transferrin in stably transfected drosophila s2 cells a chemoenzymatic approach to glycopeptide antibiotics macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase a method for the generation of glycoprotein mimetics inclusion complexation behavior of dyestuff guest molecules by a bridged bis(cyclomaltoheptaose)[bis(b-cyclodextrin)] with a pyromellitic acid diamide tether optimization of diagonal chromatography for recognizing post-translational modifications an optimized protocol for nano-lc-maldi-tof-ms coupling for the analysis of proteolytic digests of glycoproteins the il-10r2 binding hot spot on il-22 is located on the nterminal helix and is dependent on n-linked glycosylation sialylation and sulfation of the carbohydrate chains in respiratory mucins from a patient with cystic fibrosis glyco-fragment: a web tool to support the interpretation of mass spectra of complex carbohydrates glycofragment and glyco-searchms: web tools to support the interpretation of mass spectra of complex carbohydrates molecular structures of fructans from agave tequilana weber var. azul preparation, properties, and applications of npentenyl arabinofuranosyl donors structure of a major glycolipid from thermus oshimai ntu-063 aknk is an l-2-deoxyfucosyltransferase in the biosynthesis of the anthracycline aclacinomycin a synthesis and structural study of two new heparin-like hexasaccharides complement regulation at the molecular level: the structure of decayaccelerating factor serological characterization of anti-endotoxin serum directed against the conjugate of oligosaccharide core of escherichia coli type r4 with tetanus toxoid identification of a 3-deoxy-d-manno-octulosonic acid biosynthetic operon in moraxella catarrhalis and analysis of a kdsa-deficient isogenic mutant characterization of galactoglucomannan extracted from spruce (picea abies) by heat-fractionation at different conditions effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry mass spectrometric identification of n-and o-glycosylation sites of full-length rat selenoprotein p and determination of selenide-sulfide and disulfide linkages in the shortest isoform neoglycoconjugates from synthetic tetra-and hexasaccharides that mimic the terminus of the o-ps of vibrio cholerae o:1, serotype inaba concise synthesis of b-glcnacp-(1 ! 6)-a-manp-(1 ! 6)-manp and its dimer, b-glcnacp-(1 ! 2)-a-manp-(1 ! 6)-manp facile synthesis of arabinomannose penta-and decasaccharide fragments of the lipoarabinomannan of the equine pathogen, rhodococcus equi recent developments in the synthesis and discovery of oligosaccharides and glycoconjugates for the treatment of disease synthesis of cis-(1 ! 3)-glycosides of allyl 2-acetamido-4,6-o-benzylidene-2-deoxy-a-d-glucopyranoside indiscriminate glycosylation of procarboxypeptidase y expressed in pichia pastoris matrix-assisted laser desorption and electrospray ionization mass spectrometry of carminic acid isolated from cochineal enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis synthesis of oligosaccharides on soluble high-molecular-weight branched polymers in combination with purification by nanofiltration cloning and functional expression of a novel gdp-6-deoxy-d-talose synthetase from actinobacillus actinomycetemcomitans casein phosphoproteome: identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresis 5-dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced maldi-ms analysis of biomolecules rapid synthesis of oligosaccharides using an anomeric fluorous silyl protecting group synthesis of the lewis a trisaccharide based on an anomeric silyl fluorous tag the pmrf polymyxin-resistance operon of yersinia pseudotuberculosis is upregulated by the phop-phoq two-component system but not by pmra-pmrb, and is not required for virulence basic amino acids as modulators of an o-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gc: functional roles in viral infectivity synthesis, conformational studies and mannosidase stability of a mimic of 1,2-mannobioside galactosylated 5-hydroxylysine mimetics for glycopeptide synthesis structural characterization of chemically and enzymatically derived standard oligosaccharides isolated from partially purified tamarind xyloglucan synthesis of maltooligosyl fructofuranosides catalyzed by immobilized cyclodextrin glucosyltransferase using starch as donor identification of disulfide bonds and site-specific glycosylation in chicken a 1 -acid glycoprotein by matrix-assisted laser desorption ionization time-of-flight mass spectrometry structural characterization of oligosaccharides using maldi-tof/tof tandem mass spectrometry glycoforms obtained by expression in pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein expression, purification, and characterization of avian thy-1 from lec1 mammalian and tn5 insect cells cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis cellular effects of deoxynojirimycin analogues: inhibition of nlinked oligosaccharide processing and generation of free glucosylated oligosaccharides a scavenger function for a drosophila peptidoglycan recognition protein tailoring modification of deoxysugars during biosynthesis of the antitumour drug chromomycin a assembly of a library of trisaccharides as mimics of sialyl lewis x via random combination strategy chemical and enzymatic release of glycans from glycoproteins o-glycan sialylation and the structure of the stalk-like region of the t cell co-receptor cd8 specificities of three distinct human chondroitin/dermatan n-acetylgalactosamine 4-o-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermitan sulfate biosynthesis the underglycosylation of plasma 1-antitrypsin in congenital disorders of glycosylation type i is not random mass spectrometric analysis of glycans in elucidating the pathogenesis of cdg type iix 5-amino-2-mercapto-1,3,4-thiadiazole: a new matrix for the efficient matrixassisted laser desorption/ionization of neutral carbohydrates estimation of the proton affinity values of fifteen matrix-assisted laser desorption/ionization matrices under electrospray ionization conditions using the kinetic method nlinked glycan structures of mouse interferon-b produced by bombyx mori larvae structural and functional characterization of ovotransferrin produced by pichia pastoris new polygalacturonases from trichoderma reesei: characterization and their specificities to partially methylated and acetylated pectins a transient tobacco expression system coupled to maldi-tof-ms allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants the structure of lipid a of the lipopolysaccharide from burkholderia caryophylli with a 4-amino-4-deoxy-l-arabinopyranose-1-phosphate residue exclusively in glycosidic linkage sample preparation effects in matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry of partially depolymerised carboxymethyl cellulose sample preparation effects in matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry of partially depolymerised methyl cellulose phase-variation of the truncated lipo-oligosaccharide of neisseria meningitidis nmb phosphoglucomutase isogenic mutant nmb-r6 structure of the major triterpene glycoside from the sea cucumber stichopus mollis and evidence to reclassify this species into the new genus australostichopus sequencing of oligosaccharides derivatized with benzylamine using electrospray ionization-quadrupole time of flight-tandem mass spectrometry fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization twostage time-of-flight (tof/tof) tandem mass spectrometer enzymatic preparation of biotinylated naturally-occurring sialylglycan and its molecular recognition on a quartz-crystal microbalance biosynthesis of mycobacterial phosphatidylinositol mannosides pmrab, a two-component regulatory system of pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid a structural analysis of kappacarrageenan oligosaccharides released by carrageenase from marine cytophaga mca-2 atmospheric pressure matrix-assisted laser desorption/ionization (ap maldi) on a quadrupole ion trap mass spectrometer aggregates are the biologically active units of endotoxin synthesis of peptide dendrimers based on a b-cyclodextrin core with guest binding ability total synthesis of a tetra-and two pentasaccharide fragments of the o-specific polysaccharide of shigella flexneri serotype 2a photocontrolled release and uptake of a porphyrin guest by dithienylethene-tethered -cyclodextrin host dimers effects of buffering conditions and culture ph on production rates and glycosylation of clinical phase i anti-melanoma mouse igg3 monoclonal antibody r24 enhanced post-source decay and cross-ring fragmentation of oligosaccharides facilitated by conversion to amino derivatives capillary electrophoresis to characterize ricin and its subunits with matrixassisted laser desorption/ionization time-of-flight mass spectrometry glucose production by hydrolysis of starch under hydrothermal conditions construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors synthesis of an unnatural n-glycan-linked dolichyl pyrophosphate precursor cyclodextrin trimers as receptors for arranging ester and catalyst at optimized location to achieve enhancement of hydrolytic activity synthesis of glycosyl glycerol by cyclodextrin glucanotransferases sample clean-up method for analysis of complex n-glycans released from glycopeptides detailed structural features of glycan chains derived from a1-acid glycoproteins of several different animals: the presence of hypersialylated, o-acetylated sialic acids but not disialyl residues structural analysis of n-linked glycans in caenorhabditis elegans dynamic multivalent lactosides displayed on cyclodextrin beads dangling from polymer strings synthesis of lactoside glycodendrons using photoaddition and reductive amination methodologies flavonoids of hedysarum setigerum hydrolysis of nothogenia erinacea xylan by xylanases from families 10 and 11 expression, purification and characterization of humanized anti-hbs fab fragment an efficient method for the synthesis of 2,6-branched galacto-oligosaccharides and its applications to the synthesis of three tetrasaccharides and a hexasaccharide related to the arabinogalactans (ags) design and synthesis of c 3 -symmetric lewis x antigen living ringopening metathesis polymerization of norbornenes containing acetylprotected carbohydrates using well-defined molybdenum and ruthenium initiators flavone c-glycoside, phenolic acid, and nitrogen contents in leaves of barley subject to organic fertilization treatments in situ extension as an approach for identifying novel a-amylase inhibitors immunity to schistosomiasis: glycans are potential antigenic targets for immune intervention solid-phase extraction nmr studies of chromatographic fractions of saponins from quillaja saponaria enzymatic glycosidation of sugar oxazolines having a carboxylate group catalyzed by chitinase enzymatic synthesis of alternatingly 6-o-carboxymethylated chitotetraose by selective glycosidation with chitinase catalysis analysis of sugar epimers using mass spectrometry: n-acetyllactosamine-6-6 0 -disulfate and the 2 0 -epimer n-glycosylation patterns of hsa/cd24 from different cell lines and brain homogenates: a comparison glycotentacles: synthesis of cyclic glycopeptides, toward a tailored blocker of influenza virus hemagglutinin carbohydrate structure and differential binding of prostate specific antigen to maackia amurensis lectin between prostate cancer and benign prostate hypertrophy simple formylacetal (ch 2 ) as a novel linker for saccharide synthesis on soluble-polymer support synthesis of heparinlike oligosaccharides on polymer supports synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from b-d-glucose 1-phosphate to isokestose and nystose using thermoanaerobacter brockii kojibiose phosphorylase identification and removal of o-linked and non-covalently linked sugars from recombinant protein produced using pichia pastoris structural evidence of grafting in electron beam irradiated starch-allylurea blends artefacts formed by addition of urea to n-linked glycans released with peptide-n-glycosidase f for analysis by mass spectrometry structural features of acetylated galactomannans from green coffea arabica beans synthesis and biological activities in vitro and in vivo of glycosylated human interleukin-1a, neoglyco il-1a, coupled with n-acetyloneuraminyl-galactose diverse motifs of mannoside clustering on a b-cyclodextrin core anion complexation by glycocluster thioureamethyl cavitands: novel esi-ms-based methods for the determination of k a values the first representative of glycosylated three-fingered toxins. cytotoxin from the naja kaouthia cobra venom generation of new landomycins by combinatorial biosynthetic manipulation of the lndgt4 gene of the landomycin e cluster in s. globisporus mucor hiemalis endo-b-n-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide design of attachment type of drug delivery system by complex formation of avidin with biotinyl drug model and biotinyl saccharide increasing sensitivity and decreasing spot size using an inexpensive, removable hydrophobic coating for matrix-assisted laser desorption/ionisation plates separation of hemicellulosic oligomers from steamtreated spruce wood using gel filtration automated synthesis of oligosaccharides in vitro glycosylation of peptide (rkdvy) and rnase a by pngase f identification of the binding site of methylglyoxal on glutathione peroxidase: methylglyoxal inhibits glutathione peroxidase activity via binding to glutathione binding sites arg 184 and 185 characterization of a lysosomal serine carboxypeptidase from trypanosoma cruzi molecular basis of anti-horseradish peroxidase staining in caenorhabditis elegans novel bacterial polar lipids containing ether-linked alkyl chains, the structures and biological properties of the four major glycolipids from propionibacterium propionicum pcm 2431 (atcc 14157 t ) structural and serological characterization of the major glycolipid from rothia mucilaginosa uso de espectroscopia de rmn y maldi-tof ms en la elucidacion estructural de flavonoides antioxidantes provenientes de la planta medicinal chilena cheilanthes glauca mannose metabolism is required for mycobacterial growth reiterative intramolecular glycosylation supported by a rigid spacer systemic signaling in tomato plants for defense against herbivores: isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene synthesis of sucrose laurate using a new alkaline protease new class of quaternary ammonium salts, derivatives of methyl d-glucopyranosides roles of 3-deoxy-d-manno-2-octulosonic acid transferase from moraxella catarrhalis in lipooligosaccharide biosynthesis and virulence glycosylation of human pancreatic ribonuclease: differences between normal and tumor states altered glycosylation pattern allows the distinction between prostate-specific antigen (psa) from normal and tumor origins studies of new polysaccharides from lasallia pustulata (l.) hoffm molecular dissection of the role of two methyltransferases in the biosynthesis of phenolglycolipids and phthiocerol dimycoserosate in the mycobacterium tuberculosis complex characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the mycobacterium tuberculosis complex synthesis and conformational analysis of novel n(och 3 )-linked disaccharide analogues sample preparation for hyphenated analytical techniques minor interspecies variations in the sequence of the gp53 tsl-1 antigen of trichinella define species-specific immunodominant epitopes two-dimensional electrophoresis replica blotting: a valuable technique for the immunological and biochemical characterization of single components of complex extracts purification and characterization of a b-xylosidase from potatoes (solanum tuberosum) mass spectrometric assignment of smith degradation glycopeptides derived from ribonuclease b stemmosides c and d, two novel unusual pregnane glycosides from solenostemma argel: structural elucidation and configurational study by a combined nmr-quantum mechanical strategy n-glycosylated catalytic unit meets o-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase glycosylation profile of integrin a3b1 changes with melanoma progression chemical contributions to understanding heparin activity: synthesis of related sulfated oligosaccharides a novel mannitol teichoic acid with side phosphate groups of brevibacterium sp. vkm ac-2118 the high co 2 -solubility of per-acetylated a-, b-and gcyclodextrin glycoform composition profiling of o-glycopeptides derived from human serum iga1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry new set of orthogonal protecting groups for the modular synthesis of heparan sulfate fragments mass spectrometric methods for the determination of flavonods in biological samples characterizing the electrospray-ionization mass spectral fragmentation pattern of enzymatically derived hyaluronic acid oligomers bordetella bronchiseptica pagp is a bvg-regulated lipid a palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract efficient synthesis of unsymmetrical ureido-linked disaccharides matrixassisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from vibrio fischeri strain hmk characterization of a tomato protein that inhibits a xyloglucan-specific endoglucanase an outer membrane enzyme that generates the 2-amino-2-deoxy-gluconate moiety of rhizobium leguminosarum lipid a a methylated phosphate group and four amide-linked acyl chains in leptospira interrogans lipid a: the membrane anchor of an unusual lipopolysaccharide that activates tlr2 c-terminus loop 13 of na þ glucose cotransporter sglt1 contains a binding site for alkyl glucosides the heparin/heparan sulfate 2-o-sulfatase from flavobacterium heparinum. a structural and biochemical study of the enzyme active site and saccharide substrate specificity new access to lipo-chitooligosaccharide nodulation factors b-n-acetylhexosaminidase-catalysed synthesis of non-reducing oligosaccharides enzymatic synthesis of n-acetylglucosaminobioses by reverse hydrolysis: characterisation and application of the library of fungal b-n-acetylhexosaminidases structural investigation of hemicellulosic polysaccharides from argania spinosa: characterisation of a novel xyloglucan motif a gene, uge, is essential for klebsiella pneumoniae virulence synthesis of peptide and glycopeptide partial structures of the homophilic recognition domain of epithelial cadherin structural characterisation by maldi-ms of olive xylo-oligosaccharides obtained by partial acid hydrolysis introducing transglycosylation activity into human salivary aamylase (hsa) enzymatic supported synthesis of lacto-n-neotetraose using dendrimeric polyethylene glycol lipase-catalysed regioselective acylations in combination with regioselective glycosylations as a strategy for the synthesis of oligosaccharides: synthesis of a series of fucosyllactose building blocks characterisation of the substituent distribution in starch and cellulose derivatives phosphorylation of transitory starch is increased during degradation a programmable one-pot oligosaccharide synthesis for diversifying the sugar domains of natural products: a case study of vancomycin microscale analysis of mucin-type o-glycans by a coordinated fluorophore-assisted carbohydrate electrophoresis and mass spectrometry approach evidence of regio-specific glycosylation in human intestinal mucins: presence of an acidic gradient along the intestinal tract efficacy of enzyme replacement therapy in a-mannosidosis mice: a preclinical animal study donor specificity in the glycosylation of tamm-horsfall glycoprotein: conservation of the sd a determinant in pairs of twins chemically methylated and reduced pectins: preparation, characterisation by 1 h nmr spectroscopy, enzymatic degradation, and gelling properties derivatization in the current practice of analytical chemistry cyclodextrins containing an acetone bridge. synthesis and study as epoxidation catalysts cu(ii)-self-assembling bipyridyl-glycoclusters and dendrimers bearing the tn-antogen cancer marker: synthesis and lectin binding properties secretory iga n-and o-glycans provide a link between the innate and adaptive immune systems two carbohydrate binding sites in the h cc -domain of tetanus neurotoxin are required for toxicity aminoglycoside-derived cationic lipids for gene transfection: synthesis of kanamycin a derivatives sugars within a hydrophobic scaffold: glycodendrimers from polyphenylenes one-pot preparation of a series of glycoconjugates with predetermined antigen-carrier ratio from oligosaccharides that mimic the o-ps of vibrio cholerae o:1, serotype ogawa a practical synthesis of amphiphilic cyclodextrins fully substituted with sugar residues on the primary face epr and affinity studies of mannose-tempo functionalized pamam dendrimers comparative analysis of the site-specific n-glycosylation of human lactoferrin produced in maize and tobacco plants separation of recombinant human erythropoietin glycoforms by capillary electrophoresis using volatile electrolytes. assessment of mass spectrometry for the characterization of erythropoietin glycoforms synthesis of new peptidic glycoclusters derived from b-alanine differential roles of two n-acetylgalactosaminyltransferases, csgalnact-1, and a novel enzyme, csgalnact-2. initiation and elongation in synthesis of chondroitin sulfate molecular cloning and characterization of a novel human b1,4-n-acetylgalactosaminyltransferase, b4galnac-t3, responsible for the synthesis of n,n 0 -diacetyllactosediamine, galnacb1 ! 4glc-4glcnac glycoinsulins: dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity site-specific introduction of sialic acid into insulin display of azido glycoside on a sensor chip n-glycosylation at asn in the asn-xaa-cys motif of human transferrin fast and efficient synthesis of a novel homologous series of l-fucosylated trisaccharides using the helix pomatia a-(1 ! 2)-l-galactosyltransferase enzymatic a(1 ! 2)-l-fucosylation: investigation of the specificity of the a(1 ! 2)-lgalactosyltransferase from helix pomatia facile synthesis of b(1 ! 6)-d-galactosylated oligosaccharides employing the b(1 ! 6)-d-galt-ii from the albumen gland of the snails helix pomatia structure of pre-s2 n-and o-linked glycans in surface proteins from different genotypes of hepatitis b virus chiral capillary electrophoresis: facts and fiction on the reproducibility of resolution with randomly substituted cyclodextrins acylated cholesteryl galactoside as a novel immunogenic motif in borrelia burgdorferi sensu stricto synthesis of oligosaccharides as potential novel food components and upscaled enzymatic reaction employing the b-galactosidase from bovine testes the obligate predatory bdellovibrio bacteriovorus possesses a neutral lipid a containing a-dmannoses that replace phosphate residues: similarities and differences between the lipid as and the lipopolysaccharides of the wild type strain b. bacteriovorus hd100 and its host-independent derivative hi100 post-translational modifications and their biological functions: proteomic analysis and systematic approaches formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids: identification by maldi-tof mass spectrometry a complex array of post-translation modifications determines the circulatory longevity of acetylcholinesterase in a hierarchical manner chemical synthesis of a skeleton structure sperm cd52-a gpi-anchored glycopeptide a mass spectrometry plate reader: monitoring enzyme activity and inhibition with a desorption/ionization on silicon (dios) platform the absence of fucose but not the presence of galactose or bisecting n-acetylglucosamine of human igg1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity direct nglycan profiling in the presence of tryptic peptides on maldi-tof by controlled ion enhancement and suppression upon glycan-selective derivatization neoculin as a new tastemodifying protein occurring in the fruit of curculigo latifolia pseudostichoposide b-new triterpene glycoside with unprecedent type of sulfatation from the deep-water north pacific sea cucumber pseudostichopus trachus full structural characterization of the lipid a components from the agrobacterium tumefaciens strain c58 lipopolysaccharide fraction the complete structure of the lipooligosaccharide from the halophilic bacterium pseudoalteromonas issachenkonii kmm 3549 t the structure of the phosphorylated carbohydrate backbone of the lipopolysaccharide of the phytopathogen bacterium pseudomonas tolaasii the structures of the lipid a moieties from the lipopolysaccharides of two phytopathogenic bacteria, xanthomonas campestris pv. pruni and xanthomonas fragariae structure elucidation of the highly heterogeneous lipid a from the lipopolysaccharide of the gram-negative extremophile bacterium halomonas magadiensis strain 21 m1 structure of a heteroxylan of gum exudate of the palm scheelea phalerata (uricuri) how does peripheral lipopolysaccharide induce gene expression in the brain of rats attachment of glycosaminoglycan oligosaccharides to thiolderivatised gold surfaces synthesis and structure of selected quaternary n-(1,4-anhydro-5-deoxy-2,3-o-isopropylidene-d,l-ribitol-5-yl)ammonium salts characterization of extracellular polymers of phaeocystis globosa and p. antarctica enantioselectivity in gas-phase ion-molecule reactions new fragmentation mechanisms in matrixassisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates recombinant anti-hcg antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and corea(1,3)-fucose residues synthesis of the first fully active lipoteichoic acid matrix-assisted laser-desorption time-of flight ionization and high-performance liquid chromatography-electrospray ionization mass spectral analysis of two glycosylated recombinant epoetins fragmentation characteristics of neutral n-linked glycans using a maldi-tof/tof tandem mass spectrometer the n-linked oligosaccharides of aminopeptidase n from manduca sexta. site localization and identification of novel n-glycan structures determination of neutral oligosaccharides in vegetables by matrix-assisted laser desorption/ionization mass spectrometry human galectin-1 recognition of poly-n-acetyllactosamine and chimeric polysaccharides unaltered complex nglycan profiles in nicotiana benthamiana despite drastic reduction of b1,2-n-acetylglucosaminyltransferase i activity generation of arabidopsis thaliana plants with complex n-glycans lacking b1,2-linked xylose and core a1,3-linked fucose maldi: more than peptide mass fingerprints structural analysis of arabinoxylans isolated from native and malted finger millet (eleusine coracana, ragi) refinement of the structures of cell-wall glucans of schizosaccharomyces pombe by chemical modification and nmr spectroscopy val-1, a novel polysaccharide lyase encoded by chlorovirus cvk2 synthesis of dihydrodiosgenin glycosides as mimetics of bidesmosidic steroidal saponins synthesis of rhamnosylated diosgenyl glucosides as mimetics of cytostatic steroidal saponins from ornithogalum saundersiae and galtonia candicans selective detection of glycopeptides on ion trap mass spectrometers site-specific n-glycosylation of chicken serum igg preparation of a unique glucan with large intervals in molecular weight distribution. controlled ring-opening polymerization of o-permethylcyclodextrin n-glycan structures of pigeon igg. a. major serum glycoprotein containing gala1 ! 4 termini capillary electrophoresis of carbohydrates enzymatic synthesis of lipid a molecules with four amidelinked acyl chains. lpxa acetyltransferases selective for an analog of udp-n-acetylglucosamine in which an amine replaces the 3 00 -hydroxyl group a novel method for the separation and purification of human serum acid alpha-1-glycoprotein. liquid chromatographic and mass spectrometric investigation of tryptic fragments structure of oligosaccharide side chains of an intestinal immune system modulating arabinogalactan isolated from rhizomes of atractylodes lancea dc transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/dna complexes in combination with microtubule disrupting anti-mitotic agents potato d-enzyme catalyzes the cyclization of amylose to produce cycloamylose, a novel cyclic glucan conversion of pyridylamino sugar chains to corresponding reducing sugar chains n-glycan structures of squid rhodopsin. existence of the a1-3 and a1-6 difucosylated innermost glcnac residue in a molluscan glycoprotein abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line solid-phase synthesis of core 2 o-linked glycopeptide and its enzymatic sialylation synthesis of a mimic for the heterogeneous surface of core 2 sialoglycan-linked glycoprotein synthesis of chondroitin sulfate e hexasaccharide in the repeating region by an effective elongation strategy toward longer chondroitin oligosaccharide mass spectrometry of n-linked oligosaccharides using atmospheric pressure infrared laser ionization from solution studies directed toward the synthesis of protein-bound gpi anchor human serum iga1 is substituted with up to six o-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry microdeposition device interfacing capillary electrochromatography and microcolumn liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry isolation and characterization of o-acetylated glucomannans from aspen and birch wood regioselective acylation of ginsenosides by novozyme 435 regioselective acylation of ginsenosides by novozyme 435 to generate molecular diversity a facile synthesis of a novel polyacetal containing trehalose residue in the main chain practical and convenient modifications of the a,csecondary hydroxyl face of cyclodextrins cyclodextrin-bound 6-(4-methoxyphenyl) imidazo[1,2-a]pyrazin-3(7h)-ones with fluorescein as green chemiluminescent probes for superoxide anions mechanism of 2-o-3-o silyl migration in cyclomaltohexaose (a-cyclodextrin) formation of squaric acid amides of anthracycline antibiotics. synthesis and cytotoxic properties a new type of congenital disorders of glycosylation (cdg-ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis production of a complement inhibitor possessing sialyl lewis x moieties by in vitro glycosylation technology siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity a catalogue of molecular, physiological and symbiotic properties of soybeannodulating rhizobial strains from different soybean cropping areas of china physicochemical characterization of the endotoxins from coxiella burnetii strain priscilla in relation to their bioactivities complex-type biantennary n-glycans of recombinant human transferrin from trichoplusia ni insect cells expressing mammalian b-1,4-galactosyltransferase and b-1,2-nacetylglucosaminyltransferase ii comparing n-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines periplasmic cleavage and modification of the 1-phosphate group of helicobacter pylori lipid a characterization of n-and o-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by pichia pastoris catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides synthesis of photoaffinity probes [2 0 -iodo-4 0 -(3 00 -trifluoromethyldiazirinyl)phenoxy]-d-glucopyranoside and [(4 0 -benzoyl)phenoxy]-d-glucopyranoside for the identification of sugarbinding and phlorizin-binding sites in the sodium/-glucose cotransporter protein the misr/miss two-component regulatory system influences inner core structure and immunotype of lipooligosaccharide in neisseria meningitidis determination of phosphorylation sites in lipooligosaccharides from pseudoalteromonas haloplanktis tac 125 grown at 158c and 258c by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry phaiodactylipin, a glycosylated heterodimeric phospholipase a 2 from the venom of the scorpion anuroctonus phaiodactylus advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of aspergillus niger synthesis of novel trivalent amino acid glycoconjugates based on the cyclotriveratrylene ('ctv') scaffold substitution of pichia pastorisderived recombinant proteins with mannose containing o-and n-linked glycans decreases specificity of diagnostic tests schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennary n-glycan man 3 glcnac 2 -r methylobacterium sp. isolated from a finnish paper machine produces highly pyruvated galactan exopolysaccharide composition and desiccation-induced alterations of the cell wall in the resurrection plant craterostigma wilmsii structural characteristics of pectic polysaccharides from olive fruit (olea europaea cv moraiolo) in relation to processing for oil extraction protein n-glycosylation is similar in the moss physcomitrella patens and in higher plants identification of the zpc oligosaccharide ligand involved in sperm binding and the glycan structures of xenopus laevis vitelline envelope glycoproteins conversion of lactose to b-d-galactopyranosyl-(1 ! 4)-d-arabinohexos-2-ulose-(2 ! dehydrolactose) and lactobiono-1,5-lactone by fungal pyranose dehydrogenase an endorsement to create open access databases for analytical data of complex carbohydrates bioinformatics for glycomics: status, methods, requirements and perspectives fragmentation studies of noncovalent sugar-sugar complexes by infrared atmospheric pressure maldi study of peptide-sugar non-covalent complexes by infrared atmospheric pressure matrix-assisted laser desorption/ionization liquid infrared atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sialylated carbohydrates fragmentation of sialylated carbohydrates using infrared atmospheric pressure maldi ion trap mass spectrometry from cation-doped liquid matrixes hydrophilic affinity isolation and maldi multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics the (2-phenyl-2-trimethylsilyl)ethyl-(ptmsel)-linker in the synthesis of glycopeptide partial structures of complex cell surface glycoproteins initiation of mucin-type o-glycosylation in dictyostelium is homologous to the corresponding step in animals and is important for spore coat function site-specific characterization of the n-linked oligosaccharides of a murine immunoglobulin m by high-performance liquid chromatography/ electrospray mass spectrometry cloning and characterization of a novel udp-galnac:polypeptide nacetylgalactosaminyltransferase, pp-galnac-t14 anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium crystallization of glycosylated human bace protease domain expressed in trichoplusia ni msba transporter-dependent lipid a 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of francisella novicida lpxe expressed in escherichia coli a four-component one-pot synthesis of a-gal pentasaccharide a convergent strategy for the preparation of nglycan core di-, tri-, and pentasaccharide thioaldoses for the sitespecific glycosylation of peptides and proteins bearing free cysteines site-specific glycosylation of an aglycosylated human igg1-fc antobody protein generates neoglycoproteins with enhanced function long-acting folliclestimulating hormone analogs containing n-linked glycosylation exhibited increased bioactivity compared with o-linked analogs in female rats silver triflate. a mild alternative catalyst for glycosylation conditions using trichloroacetimidates as glycosyl donors fungal b-n-acetylhexosaminidases with high b-n-acetylgalactosaminidase activity and their use for synthesis of b-galnac-containing oligosaccharides facile formation of novel carbohydrate-amino acid conjugates by reductive amination covalent structure of soybean seed coat peroxidase mapping sites of o-glcnac modification using affinity tags for serine and threonine post-translational modifications photoaffinity labelling of the human gm2-activator protein. mechanistic insight into ganglioside gm2 degradation expression of the gm2-activator protein in the methylotrophic yeast pichia pastoris, purification, isotopic labeling, and biophysical characterization ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: synthesis of and binding studies with biotinylated cluster glycosides containing n-acetylgalactosamine molecular characterization and allergenic activity of lyc e 2 (b-fructofuranosidase), a glycosylated allergen of tomato physicochemical characterisation of cationic polybutylcyanoacrylat-nanoparticles by fluorescence correlation spectroscopy carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens o-glycosyl amino acids by 2-nitrogalactal concatenation-synthesis of a mucin-type o-glycan triterpenoidal lupin saponins from the chilean legume lupinus oreophilus phil altering the strength of lectin binding interactions and controlling the amount of lectin clustering using mannose/hydroxyl-functionalized dendrimers characterization of the oligosaccharides associated with the human ovarian tumor marker ca125 synthesis of divalent b-(1 ! 6)-branched (1 ! 3)-glucohexaose and trivalent b-(1 ! 6)-branched (1 ! 3)-glucotriose synthesis of 6-branched cyclo-(1 ! 3)-glucohexaose and glucooctaose solid-phase synthesis of complex oligosaccharides using azidoglucose as a glycosyl acceptor localization of defined carbohydrate epitopes in bovine polysialylated ncam a novel glcnaca1-hpo 3 -6gal(1 ! 1)ceramide antigen and alkylated inositol-phosphoglycerolipids expressed by the liver fluke fasciola hepatica the parasitic trematode fasciola hepatica exhibits mammalian-type glycolipids as well as gal(b1 ! 6)gal-terminating glycolipids that account for cestode serological cross-reactivity glycopeptide analysis by matrixassisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation a novel gal(b1 ! 4)gal(b1 ! 4)fuc(a1 ! 6)-core modification attached to the proximal n-acetylglucosamine of keyhole limpet haemocyanin (klh) n-glycans identification of a-galactosyl epitope mimetics through rapid generation and screening of c-linked glycopeptide library infrared multiphoton dissociation of alkali metalcoordinated oligosaccharides infrared laser isolation of ions in fourier transform mass spectrometry first synthesis of d-mannose penta-and decasaccharides, the repeating unit and its dimer of the cell-wall mannan of candida kefyr ifo 0586 comparative proteomics of glycoproteins based on lectin selection and isotope coding characterization of macrocyclic polysaccharides using matrix-assisted laser desorption/ionization timeof-flight mass spectrometry carbon nanotubes as assisted matrix for laser desorption/ionization time-of-flight mass spectrometry conformational analysis of chirally deuterated tunicamycin as an active site probe of udp-n-acetylhexosamine:polyprenol-p n-acetylhexosamine-1-p translocases intact glycation end products containing carboxymethyl-lysine and glyoxal lysine dimer obtained from synthetic collagen model peptide synthesis and antigenic property of a novel sialyl 6-o-sulfo lewis x neoglycolipid containing lactamized neuraminic acid convenient synthesis of a glycopeptide analogue having a complex type disialyl-undecasaccharide synthetic studies on glycosphingolipids from protostomia phyla: total syntheses analysis of carbohydrates and glycoconjugates & of glycosphingolipids from the parasite, echinococcus multilocularis transglycosylation reaction of mucor hiemalis endo-b-nacetylglucosaminidase using sugar derivatives modified at c-1 or c-2 as oligosaccharide acceptors a novel oligosaccharide on bovine peripheral myelin glycoprotein p0 a practical synthesis of a (1 ! 6)-linked b-dglucosamine nonasaccharide synthesis of biantennary b-d-(1 ! 6) glucosamine oligosaccharides structural determination of the polar glycoglycerolipids from thermophilic bacteria meiothermus taiwansis computational estimates of the gas-phase acidities of dihydroxybenzoic acid radical cations and their corresponding neutral species proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus an nacetylglucosaminyltransferase of the golgi apparatus of the yeast saccharomyces cerevisiae that can modify n-linked glycans maltosyl-erythritol, a major transglycosylation product of erythritol by bacillus stearothermophilus maltogenic amylase enzymatic synthesis of two salicin analogues by reaction of salicyl alcohol with bacillus macerans cyclomaltodextrin glucanyltransferase and leuconostoc mesenteroides b-742cb dextransucrase synthesis of glcnacpb-(1 ! 3)-galp-a-(1 ! 2)-6-deoxy-altrohepp-a-(1-o-propyl), an oantigenic repeating unit from c. jejuni o:23 and o:36 novel efficient routes to heparin monosaccharides and disaccharides achieved via regio-and stereoselective glycosidation relationships between the n-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices structure and biological activity of the short-chain lipopolysaccharide from bartonella henselae atc 49882t mass spectrometry of oligosaccharides capillary electrophoresis-mass spectrometry for glycoscreening in biomedical research synthesis and purity assessment of tetra-and pentaacyl lipid a of chlamydia containing (r)-3-hydroxyicosanoic acid synthesis of a hexasaccharide fragment of group e streptococci polysaccharide and the tetrasaccharide repeating unit of e. coli o7: k98:h6 an efficient synthesis of a hexasaccharide-the repeating unit of the exopolysaccharide from cryptococcus neoformans serovar a facile syntheses of the hexasaccharide repeating unit of the exopolysaccharide from cryptococcus neoformans serovar a synthesis of a hexasaccharide, the repeating unit of o-deacetylated gxm of c. neoformans serotype a a general method for the synthesis of oligosaccharides consisting of a-(1 ! 2)-and a-(1 ! 3)-linked rhamnan backbones and glcnac side chains identification and quantification of n-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry gasphase potassium binding energies of maldi matrices: an experimental and theoretical study the emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an nterminal recognition region and a c-terminal synthetic region rapid determination of advanced glycation end products of proteins using maldi-tof-ms and perl script peptide searching algorithm cloning and characterization of a new human udp-n-acetyl-a-d-galactosamine: polypeptide n-acetylgalactosaminyltransferase, designated pp-gal-nac-t13, that is specifically expressed in neurons and synthesizes galna-serine/threonine antigen strategy for profiling and structure elucidation of mucin-type oligosaccharides by mass spectrometry profiling the morphological distribution of o-linked oligosaccharides hemodextrin: a self-assembled cyclodextrinporphyrin construct that binds dioxygen n-linked oligosaccharide analysis of glycoprotein bands from isoelectric focusing gels synthesis of a-s-linked glycopeptides in water containing solution caenorhabditis elegans triple null mutant lacking udp-n-acetyl-d-glucosamine:a-3-d-mannoside b1,2-n-acetylglucosaminyltransferase development of an assay system for saikosaponin a using anti-saikosaponin a monoclonal antibodies synthesis of an s-linked glycopeptide analog derived from human tamm-horsfall glycoprotein synthesis of novel s-neoglycopeptides from glycosylthiomethyl derivatives key: cord-280107-tulne0v3 authors: rabaa, maia a.; tue, ngo tri; phuc, tran my; carrique-mas, juan; saylors, karen; cotten, matthew; bryant, juliet e.; nghia, ho dang trung; cuong, nguyen van; pham, hong anh; berto, alessandra; phat, voong vinh; dung, tran thi ngoc; bao, long hoang; hoa, ngo thi; wertheim, heiman; nadjm, behzad; monagin, corina; van doorn, h. rogier; rahman, motiur; tra, my phan vu; campbell, james i.; boni, maciej f.; tam, pham thi thanh; van der hoek, lia; simmonds, peter; rambaut, andrew; toan, tran khanh; van vinh chau, nguyen; hien, tran tinh; wolfe, nathan; farrar, jeremy j.; thwaites, guy; kellam, paul; woolhouse, mark e. j.; baker, stephen title: the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases date: 2015-09-24 journal: ecohealth doi: 10.1007/s10393-015-1061-0 sha: doc_id: 280107 cord_uid: tulne0v3 the effect of newly emerging or re-emerging infectious diseases of zoonotic origin in human populations can be potentially catastrophic, and large-scale investigations of such diseases are highly challenging. the monitoring of emergence events is subject to ascertainment bias, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. disease surveillance is generally performed post hoc, driven by a response to recent events and by the availability of detection and identification technologies. additionally, the inventory of pathogens that exist in mammalian and other reservoirs is incomplete, and identifying those with the potential to cause disease in humans is rarely possible in advance. a major step in understanding the burden and diversity of zoonotic infections, the local behavioral and demographic risks of infection, and the risk of emergence of these pathogens in human populations is to establish surveillance networks in populations that maintain regular contact with diverse animal populations, and to simultaneously characterize pathogen diversity in human and animal populations. vietnam has been an epicenter of disease emergence over the last decade, and practices at the human/animal interface may facilitate the likelihood of spillover of zoonotic pathogens into humans. to tackle the scientific issues surrounding the origins and emergence of zoonotic infections in vietnam, we have established the vietnam initiative on zoonotic infections (vizions). this countrywide project, in which several international institutions collaborate with vietnamese organizations, is combining clinical data, epidemiology, high-throughput sequencing, and social sciences to address relevant one-health questions. here, we describe the primary aims of the project, the infrastructure established to address our scientific questions, and the current status of the project. our principal objective is to develop an integrated approach to the surveillance of pathogens circulating in both human and animal populations and assess how frequently they are exchanged. this infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance understanding of viral cross-species transmission events, and identify relevant risk factors and drivers of zoonotic disease emergence. the burden of infectious diseases in human populations in low-and middle-income countries remains high (sepú lveda and murray 2014); in the majority of cases, disease etiologies are never determined (kotloff et al. 2012; mulholland 2003; susilawati and mcbride 2014) , often due to inadequate laboratory diagnostic capacity. the causative agents of diseases of unknown origin (duos) fall broadly into three groups: (1) known pathogens that were not tested for or misdiagnosed; (2) previously unrecognized but common pathogens (e.g., parechoviruses and human metapneumovirus); and (3) newly emerging or re-emerging pathogens (e.g., filoviruses, henipaviruses, and coronaviruses). all three categories are relevant to policymakers when trying to allocate resources for prevention. while the third category accounts for the smallest number of infections, the impact of emerging pathogens, particularly those with potential for human-tohuman transmission, may range from disruptive to catastrophic. developing an infrastructure to tackle these issues is challenging and costly, yet interventions that are implemented without supporting data on duo frequency and population risk may prove ineffective. newly emerging pathogens in low-to middle-income countries are likely to originate from an animal source. approximately 60% of all human pathogen species are known to be zoonotic (woolhouse and gowtage-sequeria 2005) and pathogens that infect multiple species are three times as likely to emerge into human populations than host-restricted pathogens (taylor et al. 2001) . some pathogens undergo enzootic transmission in reservoir animal populations with occasional spillover into a human host and little to no onward transmission (jonsson et al. 2010; wertheim et al. 2009 ). other pathogens display human-to-human transmission after spillover and may result in large epidemics (drosten et al. 2013; gire et al. 2014; janies et al. 2008) . some of these emerging pathogens may eventually adapt to circulate exclusively among humans, leading to epidemic or endemic transmission cycles (holmes and twiddy 2003; taubenberger 2006) . the frequency with which such zoonotic transmission events occur and the likelihood that a pathogen will adapt to exclusively human transmission are largely determined by behavioral and immunological factors in the host, along with ecological and evolutionary factors of the pathogen (karesh et al. 2012) . it has been proposed that the focus of zoonosis research should move toward the early detection of zoonotic pathogens, in particular those that exhibit potential for emergence in humans (morse et al. 2012) . this is particularly poignant given the current epidemic of ebola virus in western africa, where sustained animal/human surveillance prior to the start of the outbreak may have had an impact on the scale of the epidemic. large-scale investigations of emerging zoonotic infections are challenging. monitoring is subject to ascertainment biases, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. disease surveillance is often performed post hoc, driven by a response to recent events and by the availability and sustainability of detection technologies. additionally, the inventory of pathogens that exist in mammalian and other reservoirs is astonishingly incomplete (anthony et al. 2013 ) and identifying those with the potential to cause disease in humans is rarely possible in advance. finally, the nature of the species barrier and the factors that enable pathogens to cross these barriers and establish transmission among humans are largely unknown. the factors mentioned above limit our ability to study zoonotic pathogens in detail; therefore, we identified the need for active monitoring of human and animal reservoirs of infection to characterize community exposures and infections in a developing country with a prolonged history of zoonotic transmission events and outbreaks. the two major requirements for understanding the burden and diversity of zoonotic infections and the behavioral/demographic risks of infection are the establishment of surveillance networks in populations that maintain regular contact with diverse animal populations and the simultaneous characterization of pathogen diversity in human and animal populations. here, we describe a project that is currently underway in communities across vietnam in which we are collecting clinical samples and associated clinical, epidemiological, and demographic data, which will be combined with high-throughput viral genome sequences and qualitative social sciences data to address key onehealth questions with the aim of better understanding the origins, risks, and emergence of zoonotic infections. southeast asia is a global hotspot for emerging infectious diseases (morse et al. 2012) , and vietnam has been an epicenter of emerging disease activity over the last decade (dinh et al. 2006; reynolds et al. 2006; vinh et al. 2009; vu tra my et al. 2014; wertheim et al. 2009 ). vietnam has a large population (89,700,000 in 2013 (statistical yearbook of vietnam, 2013)), some of the highest livestock densities in southeast asia (gerber et al. 2005) , and a substantial burden of duos (ho dang trung et al. 2012; thompson et al. 2015) . furthermore, approximately 50% of the vietnamese population reside in rural areas and participate in small-scale animal production (statistical yearbook of vietnam, 2013). we hypothesize that vietnam's demography, varied animal production systems, and food consumption habits facilitate the spillover of zoonotic pathogens into humans. specifically, we predict that exotic food production systems with mixed species and limited biosecurity, abattoirs and wet markets operating with minimal basic hygiene, poor cold chains for meat distribution, limited meat inspections in the market sector, and consumption of raw/undercooked blood, meat, organ tissues, and wild animal products promote the risk of zoonotic pathogen transmission. vizions, initiated in march 2012, is now an established platform for one-health research in vietnam. with vizions, we are aiming to integrate traditional clinical, epidemiological, and medical anthropological methods with new approaches for pathogen detection and discovery, including novel sequencing approaches combined with phylogenetic analysis to characterize pathogen populations. the principal aims of vizions are presented in box 1. to deliver the aims of vizions, we established two fundamental components: a hospital disease surveillance program to characterize endemic infections, novel infections and duos, and a high-risk sentinel zoonosis cohort to assess disease incidence and pathogen transfer ( figure 1 ). the hospital surveillance component of vizions is underway in seven locations. these locations are shown in figure 1 , and collaborating vietnamese institutions are outlined in box 2. over a 3-year period of enrollment at each site, we aim to enroll up to 3000 hospitalized cases of each of four key clinical syndromes (central nervous system (cns) infections, enteric infections, jaundice, and respiratory tract infections) that may be caused by a zoonotic pathogen. the aim to enroll 3000 cases under each clinical syndrome was based both on operational capacity and local epidemiology, specifically the ability to calculate population attributable fractions (pafs) for specific pathogens (thus providing supporting evidence of disease etiology). these samples sizes were considered to be sufficient to estimate pafs to informative levels of precision: for example, a pathogen found in 20% of 3000 cases and 40% of 1000 severe cases will give paf = 33% with approximate 95% confidence intervals of 30-37%. from pilot data, we anticipated identifying a pathogen associated with the defined clinical syndromes in 50% of cns cases, 60% of respiratory cases, 60% of enteric cases, and 30% of hepatitis cases; roughly 50-80% of these pathogens were predicted to be viruses. these enrollment targets should provide samples and metadata from known infections and provide >80% power to detect a pathogen that is present in just 1/ 1800 patients with a specified syndrome. upon enrollment and informed consent, data including diagnostic investigations (clinical and laboratory), age, sex, occupation, animal exposure, residential address, household size, income, and wealth indicators are recorded via a standardized case report form. the residence of each case is geo-located and will ultimately be associated with an existing suite of spatial datasets. additionally, we are collecting routine data for all hospital admissions (retrospectively and prospectively) via the international classification of diseases 10 (icd-10) and are modeling hospital catchment populations by comparing the spatial distribution of vizions-enrolled patients to the entire population box 1. the principal aims of the vizions project 1. to establish a model international collaborative consortium with an integrated approach to human and animal health research 2. to estimate the burden of disease (focusing on viral and zoonotic diseases), and investigate the disease epidemiology in patients hospitalized with specified clinical syndromes and infections in a cohort of high-risk individuals occupationally exposed to animals; with targeted sampling from domestic animals and wildlife in association with these individuals 3. to elucidate the etiology of infectious diseases of unknown origin in the human population, and provide a repository of putative pathogens for further study 4. to characterize genetic diversity within virus populations on either side of the species barrier in order to understand cross-species transmission and disease emergence 5. to identify socio-demographic, environmental, and behavioral drivers for disease emergence 6. to create a platform and resource for further research on zoonotic disease agents (2) a longitudinal cohort study of occupational risk of zoonotic infections, plus records of risk behaviors, with linked sampling of putative animal reservoirs that will generate >50,000 specimens for molecular investigation being performed in dong thap, dak lak, khanh hoa, and ba vi (large pink circles). entering the healthcare facilities. these data are being used further to predict atypical patterns of hospital admissions to detect outbreaks ( figure 2 sopharyngeal swab) are collected and subjected to a bacteriological and viral diagnostic algorithm for each clinical syndrome. this diagnostic process is conducted within vietnam (both at study sites and at oxford university clinical research unit (oucru) laboratories) and was designed to identify the major known causes of the four specific syndromes of interest. cases are subsequently categorized as being associated with a known causative agent or as a duo. at the time of writing (may 2015), we have recruited >6500 patients across the four syndromes. working with local academic and governmental partners in three provinces (figure 1, box 2) , we have additionally established a high-risk, sentinel cohort. we have recruited 880 individuals that we consider to be at high-risk for zoonotic pathogen transfer as a consequence of occupational exposure to animals (figure 3 ). cohort members were selected from (1) farming households, especially those with mixed livestock and wildlife species, (2) wildlife restaurants, (3) abattoirs, (4) wet markets, and (5) other high-risk occupational groups such as animal health workers/veterinarians and wildlife trappers/traders. potential cohort members were identified and screened for suitability and, after providing informed consent, were asked to provide a blood sample, nose and throat swab, and a rectal swab, followed by the administration of a questionnaire to document socioeconomic factors, healthseeking behavior, occupational hazards, animal exposure, and food consumption habits. participants are then interviewed and sampled annually to document risk factors for zoonotic infections, such as animal exposure, food consumption habits, and disease episodes within their household or among their animals. throughout the three-year study, participants who develop illness are encouraged to visit the local study hospital or are visited at home by a member of the local preventative medicine centre (pmc). the medical workers determine whether the illness is likely to represent an infectious disease episode and, if so, collect clinical samples from the cohort member and administer a disease episode questionnaire related to animal exposures. additionally, the local sdah are informed and a team of animal health workers visits the site within 48 hours of the reported disease episode for responsive sampling of representative animal species present at the study site (figure 3) . this longitudinal cohort component of vizions is designed to enable detection and monitoring of cross-species virus 'chatter' between animal and human populations. further, these repositories of linked human and animal specimens will permit us to determine the incidence and seroprevalence of selected zoonoses within the cohort, define viral diversity in humans with different animal exposures and in representative non-human species, and characterize human-animal contact behavior in a variety of settings. such behavior considered in the context of this project includes the reported slaughter, butchering, and rendering of livestock and wildlife, handling or consumption of diseased animals, and consumption of raw animal products. these data and specimen repositories are expected to provide a resource for the identification of novel or unexpected agents of human disease and their potential zoonotic origins, and to deliver information that can be used to examine the barriers to and drivers of cross-species transmission. phylogenetic studies have been utilized to investigate endemic and emerging pathogen populations at various scales and offer significant insight into the evolutionary and epidemiological characteristics of pathogens involved in disease emergence (grenfell et al. 2004; pybus and rambaut 2009) . within the context of vizions, we are performing full and partial genome sequencing to characterize both ubiquitous and novel viral populations present in vietnam. using these sequences, we aim to identify principal zoonotic viral populations and investigate their dynamics in human and animal populations. initial pathogens prioritized for investigation include rotavirus, hepatitis e virus, influenza a virus, and enteroviruses. for these and other viral populations that are found to be endemic in vietnam or show extensive transmission, we will use phylogenetic methods to characterize their spatial and temporal spread through animal and/or human populations; these investigations will provide information on large-and small-scale networks of disease transmission within the country, with which we hope to gain a better understanding of the patterns and determinants of pathogen dispersal. in the case of duos, where no pathogen is detected by standard screening methods, metaviral sequencing methods (cotten et al. 2014) will be used to identify viral nucleic acids within the sample. to provide further context on the extent of viral exchange between human and animal populations and characterize viral diversity across species of interest, we will also use metagenomic methods to characterize the viromes of healthy human and animal specimens. viruses that show zoonotic potential in these populations will be further investigated through population-level serological studies to determine the extent and risks of these zoonotic infections across vietnam. anthropogenic factors of expanding peri-urban regions, increased natural resource use, changing scales of agriculture, and human encroachment into wildlife habitat have led to the development of intensive agricultural practices and exotic species farming in vietnam. these factors increase the frequency, duration, and intensity of wildlife, livestock, domestic animal, and human interaction (rhyan and spraker 2010) . vietnam now has unprecedented demands for meat from livestock, and many wildlife species are now commonly farmed (e.g., bamboo rat, wild boar, civet, porcupine) (brooks et al. 2010; drury 2009 ). the maintenance of large animal populations (domestic and wildlife species) generally involves supplemental feeding and the introduction of animals from other populationsall factors that facilitate pathogen transmission (wildlife conservation society 2008). additionally, the presence of mixed wildlife and livestock populations on farms increases the risk of introduction of novel pathogens to potentially immunologically naive populations. although human exposure to livestock is known to be an important risk factor for cross-species transmission of zoonotic pathogens in this region (dinh et al. 2006; ho et al. 2011; mackenzie 2005) , the risk posed by wildlife remains unknown; this is due partly to an incomplete understanding of wildlife farming and consumption practices. we aim to investigate the socio-cultural context of wildlife consumption and farming within a subset of cohort participants with exposure to wildlife. in parallel to the annual participant interviews, we are conducting qualitative research including participant observation and indepth interviews to assess contextual and behavioral risk factors in a subset of individuals with specific exposures to wildlife species, including wildlife farmers, trappers, traders, and restaurant workers. at the time of writing, the hospital component of the vi-zions project is running to schedule with respect to data collection, patient recruitment, and pathogen screening. this has been a substantial resource for collaborating hospitals, where laboratory diagnoses are seldom performed and infections are typically identified through standard clinical observations. therefore, we are developing a thorough understanding of the disease burden of many key pathogens across vietnam but still have an alarming rate of duos, ranging from 42.6% (764 of 1792 samples screened) in diarrhea cases to 74.7% (464 of 621 samples screened) in cns cases (2451/4624 cases overall across the four syndromes have no identified pathogen). while these results are not reported back to hospitals in real time, allowing an immediate improvement of clinical assessment, they are providing vital seasonal information regarding annual fluctuations in hospital attendance and disease etiology. a key lesson from the hospital component of the study has been understanding and accounting for the gaps in diagnostic capabilities for patients who present with a disease of a presumed infectious origin in the hospital setting. a lack of diagnostic technology and funding to perform adequate diagnostics is a major issue in the healthcare systems of many developing countries and leaves treating clinicians to make diagnoses based on medical intuition and experience alone. this impacts the quality and consistency of data coming in from hospital sites, requiring additional considerations and generalizations to be made in our analyses, particularly those dependent on epidemiological data collected directly from the hospitals. furthermore, even though we are performing a considerable range of microbiological and molecular screening panels for major pathogens, the rate of duos is still high. by working in further detail on these duo specimens, we hope to uncover previously undiagnosed pathogens, thus reducing the proportion of patients hospitalized with duos. a better understanding of regional pathogens causing infections should, eventually, lead to better diagnostic approaches, which ideally should be performed in a multiplex system in a timeframe that is relevant to patient care and cost-effective for use in developing country settings. with respect to the cohort component of the vizions project, we are currently following 880 individuals in three provinces; all of these individuals have now been in the cohort for at least 1 year. to date, we have recorded 532 disease episodes in these individuals, mainly manifesting as fever, diarrhea, or respiratory infections. the collections of samples (from both routine and disease sampling) from this component of the study are, at this scale, unique and many are currently in a metaviral nucleic acid sequencing pipeline. we are currently extracting and screening over 3000 fecal specimens from humans and animals that are included in the cohort study using these methods. further, we are screening for a range of endemic zoonotic pathogens including influenza, hepatitis e, rotavirus, and calciviruses. the cohort component of vizions will be complete in mid-2017. the formation and maintenance of vizions has been a substantial nationwide effort, but we think that the nature of the now established network will be productive and will yield a major resource for understanding zoonotic infections in vietnam. probably the greatest lesson learned has been in getting the various governmental and non-governmental organizations to work in unison. in vietnam, like in many other countries, governmental and provincial departments supporting animal and human health largely work in administrative silos, with little overlap in their political direction or interests. the relationships that have been created between the independent government departments and academic research institutions will be key for the long-term sustainability of this network. however, as a consequence of zoonotic disease outbreaks occurring relatively infrequently, continual governmental funding for such a resource is often overlooked. nevertheless, given our engagement with the various governmental departments and members of the vietnamese public, we think that maintaining this infrastructure is both practical and financially viable. through training schemes for laboratory workers, medical staff, community healthcare workers, and veterinarians, we are providing an ongoing capacitybuilding role and have provided support for several potential outbreaks and atypical clinical presentations in both the human and animal communities. conversely, we believe that assessing the amount of zoonotic transfer and identifying pathogens likely to have an impact on human health will be the evidence required to persuade continued investment. here we have described an established multidimensional platform in vietnam aimed to tackle scientific issues surrounding the origins and emergence of zoonotic infections. this countrywide project, in which several international institutions collaborate alongside vietnamese organizations, is combining clinical data, epidemiology, highthroughput sequencing, and social sciences to address key one-health questions that cannot be addressed independently. our overarching objective is to develop an integrated and sustainable approach to the surveillance of pathogens circulating in both human and animal populations. this infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance the understanding of viral cross-species transmission events, and allow us to identify the relevant risk factors and drivers of zoonotic disease emergence. the capacity of vizions to detect and characterize unusual disease events in nearly real time will facilitate a new approach to protecting public health in vietnam. a strategy to estimate unknown viral diversity in mammals the conservation impact of commercial wildlife farming of porcupines in vietnam full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm risk factors for human infection with avian influenza a h5n1 clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection reducing urban demand for wild animals in vietnam: examining the potential of wildlife farming as a conservation tool geographical determinants and environmental implications of livestock production intensification in asia genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak unifying the epidemiological and evolutionary dynamics of pathogens aetiologies of central nervous system infection in viet nam: a prospective provincial hospital-based descriptive surveillance study risk factors of streptococcus suis infection in vietnam. a case-control study the origin, emergence and evolutionary genetics of dengue virus evolution of genomes, host shifts and the geographic spread of sars-cov and related coronaviruses a global perspective on hantavirus ecology, epidemiology, and disease ecology of zoonoses: natural and unnatural histories the global enteric multicenter study (gems) of diarrheal disease in infants and young children in developing countries: epidemiologic and clinical methods of the case/control study emerging zoonotic encephalitis viruses: lessons from southeast asia and oceania daszak p (2012) prediction and prevention of the next pandemic zoonosis global burden of acute respiratory infections in children: implications for interventions evolutionary analysis of the dynamics of viral infectious disease factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi emergence of diseases from wildlife reservoirs the state of global health in 2014 acute undifferentiated fever in asia: a review of the literature the origin and virulence of the 1918 'spanish' influenza virus risk factors for human disease emergence a prospective multi-center observational study of children hospitalized with diarrhea in rapid emergence of third generation cephalosporin resistant shigella spp. in southern vietnam novel porcine-like human g26p[19] rotavirus identified in hospitalized pediatric diarrhea patients in ho chi minh city streptococcus suis: an emerging human pathogen commercial wildlife farms in vietnam: a problem or solution for conservation? host range and emerging and reemerging pathogens the authors wish to acknowledge all local and international partners that form the vizions consortium: oucru hcmc, song chau, hoang nguyen van minh, corinne thompson, vu thi ty hang the authors state that they have no competing interests. a strategic award from wellcome trust of great britain funded this work (wt/093724). sb and mfb are sir henry dale fellows, jointly funded by the wellcome trust and the royal society (100087/z/12/z and 098511/z/12/z, respectively). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. this article is distributed under the terms of the creative commons attribution 4.0 international license (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord-023767-rcv4pl0d authors: o’ryan, miguel l.; nataro, james p.; cleary, thomas g. title: microorganisms responsible for neonatal diarrhea date: 2009-05-19 journal: infectious diseases of the fetus and newborn infant doi: 10.1016/b0-72-160537-0/50022-0 sha: doc_id: 23767 cord_uid: rcv4pl0d nan at the beginning of the 1st century, diari..eal disease continues to be a significant cause of morbidity and mortality worldwide. during the period of 1986 to 2000, an estimated 1.4 billion children younger than 5 years suffered an episode of acute diarrhea every year in developing countries; among these, 123.6 million required outpatient medical care, and 9 million required hospitalization. approximately 2 million diarrhea-associated deaths occurred in this age group annually, primarily in the most impoverished areas of the world.' these estimates are somewhat lower than the more than 3 million annual deaths from diarrhea reported in the prior 10 years? indicating progress in prevention and treatment of acute diarrhea. in the united states, approximately 400 childhood deaths per year were reported during the late 1 9 8 0~,~*~ although the actual number may be higher: accurate incidence rates for acute diarrhea in neonates from different populations are not readily available. the relative sparing of the newborn probably results from low exposure to enteropathogens and protection associated with brea~t-feeding.~-' after the first few months of life, increasing interaction with other individuals and the environment, including introduction of artificial feeding, increases the risk of exposure to enteropathogens. for most pathogens, the incidence of acute diarrhea peaks in children between 6 months and 4 years old? neonatal diarrhea is more common in underdeveloped areas, where low educational levels, crowding, and poor standards of medical care, environmental sanitation, and personal hygiene favor early contact with enteropathogens. as the incidence of neonatal gastroenteritis rises, there is a proportional increase in neonatal deaths because medical care for the poor often is inadequate.'0*" for very low birth weight infants (<1500g), the death rate from diarrhea is 100-fold greater than for higher-birth-weight infants. 12 this chapter discusses the pathogenesis, diagnosis, treatment, and prevention of gastroenteritis based on the available knowledge about pathogens that can cause neonatal diarrhea. pathogens that rarely or never cause acute diarrhea in neonates are not discussed. after an overview of host defense mechanisms and protective factors in human milk, the remainder of the chapter is devoted to specific pathogens that cause inflammatory or noninflammatory diarrhea. the neonate is a host that is uniquely susceptible to enteric infections. neonates have not had the opportunity to develop local or systemic immune responses, and in the first few days of life, they have not acquired the highly important enteric flora that protects the normal adult gastrointestinal tract.i3-" still less is known about the barrier effect of the neonate's gastric acidity," intestinal mucus,z0 or each of which provides protection against gastrointestinal tract infections in older infants, children, and adults. the gastric acid barrier appears to be least effective during the first months of life. the average gastric ph level of the newborn is high (ph 4 to 7; mean, 6).23,24 although the ph falls to low levels by the end of the first day of life (ph 2 to 3),23 it subsequently rises again; by 7 to 10 days of life, the hydrochloric acid output of the neonatal stomach is far less than that of older infants and ~hildren.~~.'~ the buffering action of frequent milk feedings and the short gastric emptying interpose additional factors in the neonate that would be expected to permit viable ingested organisms to reach the small intestine. the intestinal epithelium serves as a nutrient absorptive machine, barrier to pathogen entry, and regulator of inflammation. intestinal epithelial cells have receptors for bacterial products and produce chemokines (e.g., interleukm [ il]-8, monocyte chemotactic protein type 1 [ mcp-1 1, granulocyte macrophage-cell stimulating factor [ gm-csf] ) and proinflammatory cytokines (e.g., il-6, tumor necrosis factor-a [tnf-a], il-1) in response to invasion by enteropathogens." the gut epithelium orchestrates the immune response. however, in the newborn, phagocytic, chemotactic, and complement functions are immature. b and t lymphocyte functions are impaired, resulting in a preferential igm production in response to antigenic stimulation. igg is actively transferred from mother to infant across the placenta at about 32 weeks' gestation and peaks by about 37 weeks; premature neonates, especially those born before 28 weeks' gestation, are deficient in these maternally derived serum antib~dies.~' h e 2 6 -2 9 the importance of breast-feeding infants for the prevention of diarrheal disease has long been e m p h a s i~e d .~~~~* -~~ published studies reporting the association between breastfeeding and diarrhea are extensive and suggest that infants who are breast-fed suffer fewer episodes of diarrhea than those who are not. this protection is greatest during a child's first 3 months of life and declines with increasing age, during the period of weaning, partial breast-feeding confers protection that is intermediate between that gained by infants who are exclusively breast-fed and that by those who are exclusively bottle-fed. a striking demonstration of the protection afforded by breast-feeding of newborns has been provided by mata and urrutiai3 in their studies of a population of infants born in a rural guatemalan village. despite extremely poor sanitation and the demonstration of fecal organisms in the colostrum and milk of almost one third of diarrheal disease did not occur in any newborns. the incidence of diarrhea rose significantly only after these infants reached 4 to 6 months old, at which time solids and other fluids were used to supplement the human milk feedings. at that time, escherichia coli and gram-negative anaerobes (e.g., bacteroides) were found to colonize the intestinal tract.i3 in contrast, urban infants of a similar ethnic background who were partly or totally artificially fed frequently acquired diarrheal disease caused by enteropathogenic e. coli (epec) . multiple mechanisms by which breast-feeding protects against diarrhea have been postulated. breast-feeding confers protection by active components in milk and by decreased exposure to organisms present on or in contaminated bottles, food, or water. many protective components have been identified in human milk and generally are classified as belonging to the major categories of cells, antibody, antiinflammatory factors, and glycoconjugates and other nonantibody f a~t o r s .~~-~' examples of milk antibodies are summarized in table 20-1. for any given pathogen, multiple milk factors may help protect the infant. human milk typically targets a major pathogenic mechanism using multiple, redundant strategies. redundancy of milk protective factors and targeting of complex virulence machinery have created a formidable barrier to enteropathogens. despite the fact that pathogens can rapidly divide and mutate, milk continues to protect infants. for example, human milk has secretory antibodies to shigellu virulence antigens and lipopoly-saccharide^,^^.^^ neutral glycolipid gb3 to bind shiga and lactoferrin to disrupt and degrade the surface-expressed virulence antigen^.^^-^^ in a similar way, milk contains antibodies directed toward the surface expressed virulence antigens of epec,5' oligosaccharides that block cell attachment?9 and lactoferrin that disrupts and degrades the surface expressed epec antigens6' human milk can initiate and maintain the growth of bifidobacterium and low ph in the feces of newborn infants, creating an environment antagonistic to the growth of e. ~o l i . ' the protective effect of human milk antibodies against enteropathogen-specific disease has been described for vibrio cholerae,62 campylobacter j e j~n i , ~~ epec,59 enterotoxigenic e. coli (etec),64965 shigella,66'67 and giardia lamblia68,69 and for bovine milk concentrate against etec,70 rota~irus,~' and shigella. 72 in 1933, the nonlactose carbohydrate fraction of human milk was found to consist mainly of oligosa~charides.~~ in 1960, montreuil and mullet74 determined that up to 2.4% of colostrum and up to 1.3% of mature milk are oligosaccharides. human milk contains a larger quantity of the oligosaccharides than does milk from other mammals, and its composition is singularly complex.75 the metabolic fate of the oligosaccharides is of interest. only water, lactose, and lipids are present in greater amounts than the oligosaccharides. despite the fact that substantial energy must be expended by the mother to synthesize the many hundreds of different milk oligosaccharides, the infant does not use them as food. most of the oligosaccharides pass through the gut undigested.76377 it is thought that they are present primarily to serve as receptor analogues that misdirect enteropathogen attachment factors away from gut epithelial carbohydrate receptors. likewise, enteropathogens use the oligosaccharide portion of glycolipids and glycoproteins as targets for attachment of whole bacteria and toxins. evidence is emerging that these glycoconjugates may have an important role in protection of the breast-fed infant from disease. 48 human milk protects suckling mice from the heat-stable enterotoxin (st) of e. coli; on the basis of its chemical stability and physical properties, the protective factor has been deduced to be a neutral fucosyloligosaccharide. 79~80 experiments have shown that epec attachment to hep-2 cells can be inhibited by purified oligosaccharide fractions from human milk.59 oligosaccharides also may be relevant to protection from norwalk virus and other caliciviruses, because these viruses attach to human abo, lewis, and secretor blood group antigens.80'81 human milk contains large amounts of these carbohydrates. the ganglioside fraction in human milk has been shown to inhibit the action of heat-labile toxin (lt) and cholera toxin on ileal loops more effectively than secretory iga.82s83 lactadherin in human milk has been shown to bind rotavirus and to inhibit viral replication in vitro and in v~v o . ~~ a study of infants in mexico showed that lactadherin in human milk protected infants from symptoms of rotavirus infection.72 e. coli organisms promptly colonize the lower intestinal tracts of healthy infants in their first few days of life85-88 and constitute the predominant aerobic coliform fecal flora throughout life in humans and in many animals. the concept that this species might cause enteric disease was first suggested in the late 19th and early 20th centuries, when several veterinary workers described the association of diarrhea (i.e., in 1905, m0r0'~ observed that bacterium (now escherichia) coli was found more often in the small intestines of children with diarrhea than in children without diarrhea. adam96*97 confirmed these findings and noted the similarity with asiatic cholera and calf scours. he further extended these observations by suggesting that e. coli strains from patients with diarrhea could be distinguished from normal coliform flora by certain sugar fermentation patterns. although he called these disease-producing organisms dyspepsicoli and introduced the important concept that e. coli could cause enteric disease, biochemical reactions have not proved to be a reliable means of distinguishing nonpathogenic from pathogenic e. coli strains. there are now at least six recognized enteric pathotypes of e. ~oli.'~ the pathotypes can be distinguished clinically, epidemiologically, and pathogenetically (table 20 -2) .98104 etec organisms are defined by their ability to secrete the lt or the st enterotoxin, or both. lt is closely related to cholera toxin and similarly acts by means of intestinal adenylate c y c l a~e , '~~~'~~ prostaglandin s y n t h e~i s , '~~~'~~ and possibly platelet activating f a c t~r . '~~' '~~ st (particularly the variant sta) causes secretion by specifically activating intestinal mucosal guanylate cyclase.' "-' l3 the stb toxin causes noncyclic, nucleotide-mediated bicarbonate secretion and appears to be important only in animals.1'4-"6 enteroinvasive e. coli (eiec) has the capacity to invade the intestinal mucosa, thereby causing an inflammatory enteritis much like shigellosi~.'~~~''~ epec elicits diarrhea by a signal transduction m e~h a n i s m~~~'~~~~~'~'~~ which is accompanied by a characteristic attaching-and-effacing histopathologic lesion in the small intestine.12' enterohemorrhagic e. coli (ehec) also induces an attaching-and-effacing lesion, but in the colon?' ehec also secretes shiga toxin, which gives rise to the sequela of hemolytic-uremic syndrome (hus). diffusely adherent e. coli122 executes a signal transduction effect, which is accompanied by the induction of long cellular processes. 123 enteroaggregative e. coli (eaec) adheres to the intestinal mucosa and elaborates enterotoxins and a major problem in the recognition of etec, eiec, epec, and ehec strains of e. coli is that they are indistinguishable from normal coliform flora of the intestinal tract by the usual bacteriologic methods. serotyping is of value in recognizing epec serotypes'26 and eiec, because these organisms tend to fall into a limited number of specific serogroups (see table 20 -2).'263'27 eiec invasiveness is confirmed by inoculating fresh isolates into guinea pig conjunctivae, as described by sereny. 12' the ability of organisms to produce enterotoxins (lt or st) is encoded by a transmissible plasmid that can be lost by one strain of e. coli or transferred to a previously unrecognized although the enterotoxin plasmids appear to prefer certain serogroups (different from epec or invasive serogro~ps),'~~ etec is not expected to be strictly limited to a particular set of serogroups. instead, these strains can be recognized only by examining for the enterotoxin. this is done in ligated animal or by enzyme-linked immunosorbent assay (elisa)'36 for lt or in suckling mice for st. 137, 138 specific dna probes also are available for lt and st.98 whether there are other mechanisms involved in the ability of the versatile e. coli species to cause enteric disease, such as by producing other types of enterotoxins"' or by fimbriate adherence traits a l~n e , '~~. '~' remains to be elucidated. cytoto~ns.98~103,125 in tissue although early work on the recognition of e. coli as a potential enteric pathogen focused on biochemical or serologic distinctions, there followed a shift in emphasis to the enterotoxins produced by previously recognized and entirely "new" strains of e. coli. beginning in the mid-1950s with work by de and colleague^'^^^'^^ in calcutta, e. coli strains from patients with diarrhea were found to cause a fluid secretory response in ligated rabbit ileal loops analogous to that seen with v; cholerue. work by taylor and associate^'^"^^ showed that the viable e. coli strains were not required to produce this secretory response and that this enterotoxin production correlated poorly with classically recognized epec serotypes. in sho paulo, trabulsi'& made similar observations with e. coli isolated from children with diarrhea, and several veterinary workers demonstrated that etec was associated with diarrhea in piglets and cal~es.'~~-'~o a similar pattern was described in 1971 with acute undifferentiated diarrhea in adults in bengal from whom e. coli could be isolated from the upper small bowel only during acute i l l n e~s . '~' "~~ these strains of e. coli produced a nondialyzable, lt, ammonium sulfate-precipitable enterotoxin.'53 analogous to the usually short-lived diarrheal illnesses of e. coli reported by several workers, a short-lived course of the secretory response to e. coli culture filtrates compared with the secretory response of cholera toxin was de~cribed."~ however, like responses to cholera toxin, secretory responses to e. coli were associated with activation of intestinal mucosal adenylate cyclase that paralleled the fluid secretory r e~p o n s e . '~~. '~~ the two types of enterotoxins produced by e. have been found to be plasmid-encoded traits that are potentially separable from each other and from the equally important plasmid-encoded adherence traits for patho-st causes an immediate and reversible secretory whereas the effects of lt (e.g., cholera toxin) follow a lag period necessitated by its intracellular site of a~t i o n . '~~' '~' '~~ only lt appears to cause fluid secretion by activating adenylate cyclase, which is accomplished by toxininduced adp-ribosylation of the gsa signaling p r~t e i n .~' "~~ the activation of adenylate cyclase by lt and by cholera toxin is highly promiscuous, occurring in many cell types and resulting in development of nonintestinal tissue culture assay systems such as the chinese hamster ovary (cho) cell assay'34 and y1 adrenal cell assay.'35 the antigenic similarity of lt and cholera toxin and their apparent binding to the monosialoganglioside gm, have enabled development of elisas for detection of lt and cholera t~x i n . '~~, '~' -'~~ st is a much smaller molecule and is distinct antigenically from lt and cholera t~x i n . '~~, '~'~'~' al though it fails to alter camp levels, st increases intracellular intestinal mucosal cyclic guanosine monophosphate (cgmp) concentrations and specifically activates plasma membrane-associated intestinal guanylate cy~lase."'-"~ like camp analogues, cgmp analogues cause intestinal secretion that mimics the response to st."' the receptor for sta responds to an endogenous ligand called guunylin, of which sta is a structural homologue.'" because the capacity to produce an enterotoxin may be transmissible between different organisms by a plasmid or even a bacteri~phage,''~-'~' interstrain gene transfer genesis. [129] [130] [131] 160 may be expected to be responsible for occasional toxigenic non-e. coli. enterotoxigenic klebsiella and citrobacter strains have been associated with diarrhea in a few reports, often in the same patients with etec.'65,'66 likewise, certain strains of salmonella appear to produce an lt, cho cellpositive toxin that may play a similar role in the pathogenesis of the watery, noninflammatory diarrhea sometimes seen with salmonella enteritidis i n f e c t i~n . '~~"~ at least equally important as enterotoxigenicity for e. coli to cause disease is the ability of these organisms to colonize the upper small bowel, where the enterotoxin produced has its greatest effect. a separable, plasmid-encoded colonization trait was first recognized in porcine e. coli. veterinary workers demonstrated that the fimbriate k-88 surface antigen was necessary for etec to cause disease in piglets.16' an autosomal dominant allele appears to be responsible for the specific intestinal receptor in piglets. in elegant studies by gibbons and c o -~o r k e r s , '~~ the homozygous recessive piglets lacked the receptor for k-88 and were resistant to scours caused by etec. at least 15 analogous colonization factors have been described for human e. coli isolate^^^"^'"^^ against which local iga antibody may be produced. these antigens potentially may be useful in vaccine development. data on the epidemiology and transmission of etec remain scanty for the neonatal period. in the past 2 decades, these strains have been recognized among adults with endemic, cholera-like diarrhea in calcutta, india, and in dacca, banglade~h,''~*'~' and among travelers to areas such as mexico and central a f r i~a . '~~-'~~ the isolation of etec is uncommon in sporadic diarrheal illnesses in temperate climates where sanitation facilities are good and where winter viral patterns of diarrhea predominate. etec is commonly isolated from infants and children with acute watery summer diarrhea in areas where sanitary facilities are less than optimal.35-165*'75-'87 these include areas such as afria,165 ~~~il,35,175,181,186,187 gentir~a,'~~ bengal,'78''79 mexico,'8o and native american reservations in the southwestern united state^."^"'^ in a multicenter study of acute diarrhea in 3640 infants and children in china, india, mexico, myanmar, and pakistan, 16% of cases (versus 5% of 3279 controls) had etec.le4 a case-control study from northwestern spain showed a highly significant association of etec with 26.5% of neonatal diarrhea, often acquired in the ho~pital.''~ although all types of etec (lt and/or st producers) are associated with cholera-like, non inflammatory, watery diarrhea in adults in these areas, they probably constitute the major cause (along with rotaviruses) of dehydrating diarrhea in infants and young children in these areas. in this setting, peaks of illnesses tend to occur in the summer or rainy season, and dehydrating illnesses may be life threatening, especially in infants and young ~h i l d r e n . 3~~'~'~'~ humans are probably the major reservoirs for the human strains of etec, and contaminated food and water probably constitute the principal vector^.''^"^^ although antitoxic immunity to lt and asymptomatic infection with ltproducing e. coli tends to increase with age, st is poorly immunogenic, and st-producing e. coli continues to be associated with symptomatic illnesses into adulthood in endemic area~.l'~>l'~ the association of etec with outbreaks of diarrhea in newborn nurseries is well documented. ryder and colleagues'g0 isolated an st-producing e. coli from 72% of infants with diarrhea, from the environment, and in one instance, from an infant's formula during a 7-month period in a prolonged outbreak in .a special care nursery in texas. another st-producing e. coli outbreak was reported in 1976 by gross and a~sociates'~~ from a maternity hospital in scotland. etec and epec were significantly associated with diarrhea among infants younger than 1 year in bang1ade~h.l~ ' an outbreak of diarrhea in a newborn special care nursery that was associated with enterotoxigenic organisms that were not limited to the same serotype or even the same species has been reported.lg3 the short-lived etec, klebsiella, and citrobacter species in this outbreak raised the possibility that each infant's indigenous bowel flora might become transiently toxigenic, possibly by receiving the lt genome from a plasmid or even a bacteriophage. the clinical manifestations of etec diarrhea tend to be mild and self-limited, except in small or undernourished infants, in whom dehydration may constitute a major threat to life. in many parts of the developing world, acute diarrheal illnesses are the leading recognized causes of death. there is some suggestion that the diarrheal illnesses associated with st-producing etec may be particularly severe.'79 most probably the best definition of the clinical manifestations of etec infection comes from volunteer studies with adults. ingestion of 10' to 10'' human etec isolates that produce lt and st or st alone resulted in a 30% to 80% attack rate of mild to moderate diarrheal illnesses within 12 to 56 hours that lasted 1 to 3 days.'17 these illnesses, typical for traveler's diarrhea, were manifested by malaise, anorexia, abdominal cramps, and sometimes explosive diarrhea. nausea and vomiting occur relatively infrequently, and up to one third of patients may have a low-grade fever. although illnesses usually resolve spontaneously within 1 to 5 days, they occasionally may persist for 1 week or longer. the diarrhea is noninflammatory, without fecal leukocytes or blood. in outbreaks in infants and neonates, the duration has been in the same range (1 to 11 days), with a mean of approximately 4 days. as in cholera, the pathologic changes associated with etec infection are minimal. from animal experiments in which thiry-vella loops were infected with these organisms and at a time when the secretory and adenylate cyclase responses were present, there was only a mild discharge of mucus from goblet cells and otherwise no significant pathologic change in the intestinal tract.lo6 unless terminal complications of severe hypotension ensue, etec organisms rarely disseminate beyond the intestinal tract. like cholera, etec diarrhea is typically limited to being an intraluminal infection. the preliminary diagnosis of etec diarrhea can be suspected by the epidemiologic setting and the noninflammatory nature of stool specimens, which reveal few or no leukocytes. although the ability of e. coli to produce enterotoxins may be lost or transmitted to other strains, there is a tendency for the enterotoxin plasmids to occur among certain predominant serotypes, as shown in table 20 -2."' these serotypes differ from epec or invasive serotypes, but their demonstration does not prove that they are enterotoxigenic. the only definitive way to identify etec is to demonstrate the enterotoxin itself by a specific gene probe for the toxin codon, by a bioassay such as tissue culture or ileal loop assays for lt or the suckling mouse assay for st, or in the case of lt, by immunoassay such as elisa. however, even these sensitive bioassays are limited by the unavailability of any selective media for detecting etec by culture. even though substantial improvements have been made in enterotoxin assay (particularly for lt), the necessary random selection of e. coli from a relatively nonselective stool culture plate resulted in a sensitivity of only 43% of epidemiologically incriminated cases in an outbreak when 5 to 10 isolates were randomly picked and tested for enter~toxigenicity."~ by also examining paired serum samples for antibody against lt, only 36% demonstrated significant serum antibody titer rises, for a total sensitivity of etec isolation or serum antibody titer rises of only 64%. some have suggested that isolates may be pooled for lt or st assay. the capacity to prove with radiolabeled or enzyme-tagged oligonucleotide gene sequences for the enterotoxins (lt or st) further facilitates the identification of enterotoxigenic organisms.'94s195 a novel method of combining immunomagnetic separation (using antibodycoated magnetic beads) followed by dna or polymerase chain reaction (pcr) probing may enhance the sensitivity of screenin fecal or food specimens for etec or other the mainstay of treatment of any diarrheal illness is rehydration."' this principle especially pertains to etec diarrhea, which is an intraluminal infection. the glucose absorptive mechanism remains intact in e. coli enterotoxininduced secretion, much as it does in cholera, a concept that has resulted in the major advance of oral glucose-electrolyte therapy. this regimen can usually provide fully adequate rehydration in infants and children able to tolerate oral fluids, replacing the need for parented rehydration in most cases .199,200 its use is particularly critical in rural areas and developing nations, where early application before dehydration becomes severe may be lifesaving. the standard world health organization solution contains 3.5 g nacl, 2.5 g nahco,, 1.5 g kcl, and 20 g glucose per liter of clean or boiled drinking water.i9' this corresponds to the following concentrations: 90 mmoyl of sodium, 20 mmovl of potassium, 30 mmoyl of bicarbonate, 80 mmol/l of chloride, and 1 10 mmol/l of glucose. a variety of recipes for homemade preparations have been described?" but unless the cost is prohibitive, the premade standard solution is preferred. each 4 ounces of this solution should be followed by 2 ounces of plain water. if there is concern about hypertonicity, especially in small infants in whom a high intake and constant direct supervision of feeding cannot be ensured, the concentration of salt can be reduced.'02 a reduced osmolality solution with 60 mmol/l of sodium and 84 mmoyl of glucose and a total osmolality of 224 (instead of 311) mosm/kg has been found to reduce stool output by 28% and illness duration by 18% in a multicenter trial involving 447 children in four countries.203 commercially available rehydration solutions are increasingly available ~orldwide.'~' pathogens. lf6,197 the role of antimicrobial agents in the treatment or prevention of etec is controversial. this infection usually resolves within 3 to 5 days in the absence of antibacterial therapy.198 moreover, there is concern about the potential for coexistence of enterotoxigenicity and antibiotic resistance on the same plasmid, and co-transfer of multiple antibiotic resistance and enterotoxigenicity has been well d~cumented."~ widespread use of prophylactic antibiotics in areas where antimicrobial resistance is common has the potential for selecting for rather than against enterotoxigenic organisms. the prevention and control of etec infections would be similar to those discussed under epec serotypes. the use of breast-feeding should be encouraged. eiec causes diarrhea by means of shigella-like intestinal epithelial invasion (discussed later).117s11' the somatic antigens of these invasive strains have been identified and seem to fall into 1 of 10 recognized 0 groups (see table 20 -2). most, if not all, of these bacteria share cell wall antigens with one or another of the various shigelza serotypes and produce positive reactions with antisera against the cross-reacting antigen."* however, not all strains of e. coli belonging to the 10 serogroups associated with dysentery-lke illness are pathogenic, because a large (140 mda) invasive plasmid is also required.205 additional biologic tests, including the guinea pig conjunctivitis (sereny) test or a gene probe for the plasmid, are used to confirm the property of inva~iveness."~ although an outbreak of foodborne eiec diarrhea has been well documented among adults who ate an imported cheese,"' little is known about the epidemiology and transmission of this organism, especially in newborns and infants. whether the infectious dose may be as low as it is for shigella is unknown; however, studies of adult volunteers suggest that attack rates may be somewhat lower after ingestion of even large numbers of eiec than would be expected with shigella. the outbreak of eiec diarrhea resulted in a dysentery-like syndrome with an inflammatory exudate in stool and invasion and disruption of colonic mucosa."' descriptions of extensive and severe ileocolitis in infants dying with e. coli diarrhea indicate that neonatal disease also can be caused by invasive strains capable of mimicking the pathologic features of shigellosis. 206 the immunofluorescent demonstration of e. coli together with an acute inflammatory infiltrate2" in the intestinal tissue of infants tends to support this impression, although it has been suggested that the organisms may have invaded the bowel wall in the postmortem ~e r i 0 d . l '~ there is still little direct evidence concerning the role of invasive strains of e. coli in the cause of neonatal diarrhea.175 the infrequency with which newborns manifest a dysentery-like syndrome makes it unlikely that this pathogen is responsible for a very large proportion of the diarrheal disease that occurs during the first month of life. the diagnosis should be suspected in infants who have an inflammatory diarrhea as evidenced by fecal polymorphonuclear neutrophils or even bloody dysenteric syndromes from whom no other invasive pathogens, such as campylobacter, shigella, salmonella, vibrio, or yersinia, can be isolated. in this instance, it may be appropriate to have the fecal e. coli isolated and serotyped or tested for invasiveness in the sereny test. plasmid pattern analysis and chromosomal restriction endonuclease digestion pattern analysis by pulsed-field gel electrophoresis have been used to evaluate strains involved in outbreaks. 208 the management and prevention of eiec diarrhea should be similar to those for acute shigella or other e. coli enteric infections. the serologic distinction of e. coli strains associated with epidemic and sporadic infantile diarrhea was first suggested by goldschmidt in 1933209 and confirmed by dulaney and michelson in 1935. 205 these researchers found that certain strains of e. coli associated with institutional outbreaks of diarrhea would agglutinate with antisera on slides. in 1943, bra?" isolated a serologically homogeneous strain of e. coli (subsequently identified as serogroup 0111) from 95% of infants with summer diarrhea in england. he subsequently summarized a larger experience with this organism isolated from only 4% of asymptomatic controls but from 88% of infants with diarrhea, one half of which was hospital this strain (initially called e. coli-gomez by varela in 1946) also was associated with infantile diarrhea in a second type of e. coli (called beta by giles in 1948 and subsequently identified as 055) was associated with an outbreak of infantile diarrhea in aberdeen, s~o t l a n d .~'~*~'~ from this early work primarily with epidemic diarrhea in infants has developed an elaborate serotyping system for certain e. coli strains that were clearly associated with infantile these strains first were called enteropathogenic e. coli by neter and colleagues219 in 1955, and the association with particular serotypes can still be observed.220 as shown in table 20 -1, these organisms are distinct from the enterotoxigenic or enteroinvasive organisms or those that inhabit the normal gastrointestinal tract. they exhibit localized adherence to hep-2 cells, a phenotype that has been suggested to be useful for diagnosis and pathogenesis research. 'i9 epec is an important cause of diarrhea in infants in developing or transitional c~u n t r i e s .~"~~' -~~~ outbreaks have become rare in the united states and other industrialized countries, but they still 0ccur.2~~ some have attributed the rarity of this recognition of illness in part to the declining severity of diarrheal disease caused by epec within the past 30 years, resulting in fewer cultures being obtained from infants with relatively mild symptom^.^^^^^^ however, several other variables influence the apparent incidence of this disease in the community. a problem arises with false-positive epec on the basis of the nonspecific cross-reactions seen with improper shortening of the serotyping p r o c e d~r e .~~~,~~~ because of their complexity and relatively low yield, neither slide agglutination nor hep-2 cell adherence or dna probe tests are provided as part of the routine identification of enteric pathogens by most clinical bacteriology laboratories. failure to recognize the presence of epec in fecal specimens is the inevitable consequence. the apparent incidence of epec gastroenteritis also varies with the epidemiologic circumstances under which stool cultures are obtained. the prevalence of enteropathogenic strains is higher among infants from whom cultures are obtained during a community epidemic compared with those obtained during sporadic diarrheal disease. neither reflects the incidence of epec infection among infants involved in a nursery outbreak or hospital epidemic. epec gastroenteritis is a worldwide problem, and socioeconomic conditions play a significant role in determining the incidence of this disease in different populations.22s for instance, it is unusual for newborn infants born in a rural environment to manifest diarrheal disease caused by epec; most infections of the gastrointestinal tract in these infants occur after the first 6 months of conversely, among infants born in large cities, the attack rate of epec is high during the first 3 months of life. this age distribution reflects in large part the frequency with which epec causes crossinfection outbreaks among nursery populations'9'~230~237; however, a predominance of epec in infants in the first 3 months of life also has been described in community epidemic^^^'-^^^ and among sporadic cases of diarrhea acquired outside the h~s p i t a l . '~' -~~~ the disparity in the incidence of neonatal epec infection between rural and urban populations has been ascribed to two factors: the trend away from breast-feeding among mothers in industrialized societies and the crowding together of susceptible newborns in nurseries in countries in which hospital deliveries predominate over home d e l i~e r i e s . 5 '~~~'~~ although the predominant serogroup can vary from year to year,239,242,243,z46.249~250 the same strains have been prevalent during the past 40 years in great britain?51 puerto r~c o ?~~ guatemala: panama, 205 newfoundland,240 indonesia,244 thailand,254 and south when living conditions are poor and overcrowding of susceptible infants exists, there is a rise in the incidence of neonatal diarrhea in general257 and epec gastroenteritis in p a r t i c~l a r .~'~~~~~~~~~ a h igher incidence of asymptomatic family carriers is found in such situations.238b239 newborn infants can acquire epec during the first days of life by one of several routes: (1) organisms from the mother ingested at the time of birth; ( 2 ) bacteria from other infants or toddlers with diarrheal disease or from asymptomatic adults colonized with the organism, commonly transmitted on the hands of nursery personnel or parents; (3) airborne or droplet infection; (4) fomites; or (5) organisms present in formulas or solid food supplements.259 only the first two routes have been shown conclusively to be of any real significance in the transmission of disease or the propagation of epidemics. most neonates acquire epec at the time of delivery through ingestion of organisms residing in the maternal birth canal or rectum. stool cultures taken from women before, during, or shortly after delivery have shown that 10% to 15% carry epec at some time during this period.85~s6~88~260~26' use of fluorescent antibody techniquesz6' or cultures during a community outbreak of epec gastroenteritis" revealed twice this number of persons excreting the organism. virtually none of the women carrying pathogenic strains of e. coli had symptoms referable to the gastrointestinal tract. many of the mothers whose stools contain epec transmit these organisms to their infant~,8~*~' resulting in an asymptomatic infection rate of 2% to 5% among newborns cultured at random in nursery surveys.85~86~19'~262 these results must be considered conservative and are probably an artifact of the sampling technique. one study using 150 0 antisera to identify as many e. coli as possible in fecal cultures showed a correlation between the coliform flora in 66% of motherinfant pairs?63 of particular interest was the observation that the 0 groups of e. coli isolated from the infants' mucus immediately after delivery correlated with those subsequently recovered from their stools, supporting the contention that these organisms were acquired orally at the time of birth. in mothers whose stools contained the same 0 group as their offspring, the mean time from rupture of membranes to delivery was about 2 hours longer than in those whose infants did not acquire the same serogroups, suggesting that ascending colonization before birth also can play a role in determining the newborn's fecal flora. the contours of the epidemiologic curves in nurse$38264-269 and communi@38-240 outbreaks are in keeping with a contact mode of spread. transmission of organisms from infant to infant takes place by way of the fecal-oral route in almost all cases, most likely on the hands of persons attending to their care.86*267,269,270 ill infants represent the greatest risk to those around them because of the large numbers of organisms found in their stools271-274 and crossinfection also has been initiated by infants who were healthy at the time of their admission to the nursery.264,2723278-280 a newborn exposed to epec is likely to acquire enteric infection if contact with a person excreting the organism is intimate and prolonged, as in a hospital or family setting. stool culture surveys taken during outbreaks have shown that between 20% and 50% of term neonates residing in the nursery carry epec in their intestinal tracts.'02,230,231'm despite descriptions of nursery outbreaks in which virtually every neonate or low-birth-weight infant became infected,26232643281 there is ample evidence that exposure to pathogenic strains of e. coli does not necessarily result in greater likelihood of illness for premature infants than for term infants.261,272*279,282 any increased prevalence of cross-infections that may exist among premature infants can be explained more readily by the prolonged hospital stays, their increased handling, and the clustering of infants born in different institutions than by a particular susceptibility to epec based on immature defense mechanisms. the most extensive studies on the epidemiology of gastroenteritis related to e. coli have dealt with events that took place during outbreaks in newborn nurseries. unfortunately, investigations of this sort frequently regard the epidemic as an isolated phenomenon and ignore the strong interdependence that exists between community-and hospital-acquired ~~~~~~2 8 0 , 2 8 3 , 2 8 4 n ot surprisingly, the direction of spread is most often from the reservoir of disease within the community to the hospital. when the original source of a nursery outbreak can be established, frequently it is an infant born of a carrier mother who recently acquired her epec infection from a toddler living in the home. cross-infection epidemics also can be initiated by infected newborns who have been admitted directly into a clean nursery unit from the surrounding d i s t r i~t~~~~~~~*~~~ or have been transferred from a nearby hospital. 27832803286 after a nursery epidemic has begun, it generally follows one of two major patterns. some are explosive, with rapid involvement of all susceptible infants and a duration that seldom exceeds 2 or 3 months.264*265,276*287 the case-fatality rate in these epidemics may be very high. other nursery outbreaks have an insidious onset with a few mild, unrecognized cases; the patients may not even develop illness until after discharge from the hospital. during the next few days to weeks, neonates with an increased number of loose stools are reported by the nurses; shortly thereafter, the appearance of the first severely ill infants makes it apparent that an epidemic has begun. unless oral antimicrobial therapy is instituted (see "therapy"), nursery outbreaks like these may continue for months266-269 or with cycles of illness followed by periods of relative quiescence. this pattern can be caused by multiple strains (of different phage or antibiogram types) sequentially introduced into the nursery?78*288.289 the nursery can be a source of infection for the community. the release of infants who are in the incubation stages of their illness or are convalescent carriers about to relapse may lead to secondary cases of diarrheal disease among young siblings living in widely scattered areas.238,239,243 these children further disseminate infection to neighboring households, involving playmates of their own age, young infants, and mothers.23832393242 as the sickest of these contact cases are admitted to different hospitals, they contaminate new susceptible persons, completing the cycle and compounding the outbreak. this feedback mechanism has proved to be a means of spreading infantile gastroenteritis through entire ~o~n t i e s , 2~~~~~~,~~~ and even provinces.240 one major epidemic of diarrhea related to epec 01 11:b4 that occurred in the metropolitan chicago and northwestern indiana region during the winter of 1961 involved more than 1300 children and 29 community hospitals during a period of 9 months.240,291 almost all of the patients were younger than 2 years old, and 10% were younger than 1 month, producing an age-specific attack rate of nearly 4% of neonates in the community. the importance of the hospital as a source of cross-infection in this epidemic was demonstrated through interviews with patients' families, indicating that a minimum of 40% of infants had direct or indirect contact with a hospital shortly before the onset of their illness. it has been suggested, but not proved, that asymptomatic carriers of epec in close contact with a newborn infant, such as nursery personnel or family members, might play an important role in its t r a n s m i~s i o n .~~~'~"~~~ stool culture surveys have shown that at any one time about 1% of and 1% to 5% of young who are free of illness harbor epec strains. higher percentages have been recorded during community epidemics?38*239s243 be cause this intestinal carriage is transitory:383280 the number of individuals who excrete epec at one time or another during the year is far higher than the 1% figure recorded for single specimens.2803293 nursery personnel feed, bathe, and diaper a constantly changing population of newborns, about 2% to 5% of whom excrete epec.238*280 despite this constant exposure, intestinal carriage among nursery workers is surprisingly low. even during outbreaks of diarrheal illness, when dissemination of organisms is most intense, less than 5% of the hospital personnel in direct contact with infected neonates are themselves excreting pathogenic strains of e. coli.29',294,295 although adult asymptomatic carriers generally excrete fewer organisms than patients with acute illness large numbers of pathogenic bacteria may nevertheless exist in their stools?42s274 however, no nursery outbreak and few family cases24o have been traced to a symptomless carrier. instead, passive transfer of bacteria from infant to infant by the hands of personnel appears to be of primary importance in these outbreaks. cities,238.239,242 epec can be recovered from the throat or nose of 5% to 80% of infants with diarrheal illness275p2949295 and from about 1% of asymptomatic the throat and nasal mucosa may represent a portal of entry or a source of transmission for epec. environmental studies have shown that epec is distributed readily and widely in the vicinity of an infant with active diarrheal disease, often within 1 day of admission to the ~a r d . '~' , '~~ massive numbers of organisms are shed in the diarrheal stool or vomitus of infected e. coli organisms may survive 2 to 4 weeks in dust243.296 and can be found in the nursery air when the bedding or diapers of infected infants are disturbed during routine nursing procedure^^^^^^^ or on floors, walls, cupboards, and nursery equipment such as scales, hand towels, bassinets, incubators, and oxygen tents of other infant^?^,'^^,'^^ documentation of the presence of epec in nursery air and dust does not establish the importance of this route as a source of cross-infection. one study presented evidence of the respiratory transmission of epec; however, even in the cases described, the investigators pointed out that fecal-oral transmission could not be completely ruled additional clinical and experimental data are required to clarify the significance of droplet and environmental infection. coliform organisms have also been isolated in significant numbers from human mi1k,46~297~298 prebottled infant f0rmulas,2~~ and formulas prepared in the home.292 epec in particular has been found in stool cultures obtained from donors of human milk and workers in a nursery formula room.26o in one instance, epec 01 11:b4 was isolated from a donor, and subsequently, the same serogroup was recovered in massive amounts in almost pure culture from her milk.260 pathogenic strains of e. coli have also been isolated from raw cow's milk3'' and from drinking ~a t e r .~" likewise, epec has been isolated from flies during an epidemic, but this fact has not been shown to be of epidemiologic significance.211'220 infection of the newborn infant with epec takes place exclusively by the oral route. attempts to induce disease in adult volunteers by rectal instillation of infected material have been unsuc~essful.~~ there are no reports of disease occurring after transplacental invasion of the fetal bloodstream by enteropathogenic or nonenteropathogenic strains of e. coli. ascending intrauterine infection after prolonged rupture of the membranes has been reported only once; the neonate in this case suffered only from mild diarrhea.86 bacterial cultures of the meconium and feces of newborns indicate that enteropathogenic strains of e. coli can colonize effectively the intestinal tract in the first days of although e. coli may disappear completely from stools of breast-fed children during the ensuing weeks, this disappearance is believed to be related to factors present in the human milk rather than the gastric secretions.5~302~303 the use of breast-feeding or expressed human milk has even been effective in terminating nursery epidemics caused by epec 0 11 1:b4, probably by reducing the incidence of crossinfections among infants.3033304 although dose-effect studies have not been performed among newborns, severe diarrhea has occurred after ingestion of 10' epec organisms by very young the high incidence of cross-infection outbreaks in newborn nurseries suggests that a far lower inoculum can often effect spread in this setting. the role of circulating immunity in the prevention of gastrointestinal tract disease related to epec has not been clearly established. virtually 100% of maternal sera have been found to contain hemaggl~tinating,2'~~~''~~'~ or bacteriostatic2'0~3'2 antibodies against epec. the passive transfer of these antibodies across the placenta is extremely inefficient. titers in blood of newborn infants are, on average, 4 to 100 times lower than those in the corresponding maternal sera. group-specific hemagglutinating antibodies against the 0 antigen of epec are present in 10% to 20% of cord blood samp1es,219~307~308 whereas b a~t e r i c i d a l~'~~~~~ or bacterio-static3" activity against these organisms can be found much more frequently. tests for bacterial agglutination, which are relatively insensitive, are positive in only a small percentage of neonate^.'^^'^" the importance of circulating antibodies in the susceptibility of infants to epec infection is unknown. experiments with suckling mice have failed to demonstrate any effect of humoral immunity on the establishment or course of duration of intestinal colonization with e. coli 0127 in mothers or their infants.313 similar observations have been made in epidemiologic studies among premature human infants using enteropathogenic (01 27:b8)310 and nonenteropathogenic (04:h5)269 strains of e. coli as the indicator organisms. in a cohort of 63 mothers and their infants followed from birth to 3 months old, cooper and associate^'^ were able to show a far higher incidence of clinical epec disease in infants of epec-negative mothers than in infants born of mothers with epec isolated from stool cultures. this finding suggested to the investigators the possibility that mothers harboring epec in their gastrointestinal tracts transfer specific antibodies to their infants that confer some protection during the first weeks of life. protection against enteric infections in humans often correlates more closely with levels of local secretory than serum antibodies. although it is known that colonization of newborns with e. coli leads to the production of coproantibodies against the ingested the clinical significance of this intestinal immunity is uncertain. the previously mentioned experiment with mice showed no effect of active intestinal immunity on enteric col~nization.~'~ in human infants, the frequency of bacteriologic and clinical relapse related to epec of the same and the capacity of one strain of epec to superinfect a patient already harboring a different train^^^,^^^,^^^ also cast some doubt on the ability of mucosal antibodies to inhibit or alter the course of intestinal infection. studies of the protective effects of orally administered epec vaccines could help to resolve these question^.'^' the mechanism by which epec causes diarrhea involves a complex array of plasmid and chromosomally encoded traits. epec serotypes usually do not make one of the recognized enterotoxins (lt or st) as usually measured in tissue culture or animal r n~d e l s ,~'~~~'~ nor do these serotypes cause a typical invasive colitis or produce a positive sereny test only uncommonly do epec strains invade the bloodstream or disseminate.288 nevertheless, epec strains that test negative in these tests are capable of causing diarrhea; inocula of 10'' e. coli 0142 or 0127 organisms caused diarrhea in 8 of 10 adult volunteers.320 some epec strains may secrete weak enterot~xins,~~''~'~ but the consensus opinion is that the attaching and effacing lesion constitutes the critical secretory virulence pheno-clinical pathologic reports reveal the characteristic attachin and effacing lesion in the small intestine of infected infants? the lesion is manifested by intimate (about 10 nm) apposition of the epec to the enterocytes plasma membrane, with dissolution of the normal brush border and rearrangement of the cyto~keleton.'''~~~~ in some instances, the bacteria are observed to rise up on pedestal-like structures, which are diagnostic of the infection.i2' villus blunting, crypt hypertrophy, histiocytic infiltration in the lamina propria, and a reduction in the brush border enzymes may also be ~bserved.~'~'~'~ two major epec virulence factors have been described; strains with both factors are designated as typical epec.98*99*3'3 one such factor is the locus of enterocyte effacement (lee), a type 111 secretion system encoded by the lee chromosomal pathogenicity i~land.~'~-~'* the lee secretion apparatus injects proteins directly from the cytoplasm of the infecting bacterium into the cytoplasm of the target enter~cytes.~'~ the injected proteins constitute cytoskeletal toxins, which together elicit the close apposition of the bacterium to the cell, cause the effacement of microvilli, and most likely give rise to the net secretory one critical secreted protein, called towinterleukin-1 receptor (tir),"' inserts into the plasma membrane of the epithelial cell, where it serves as the receptor for a lee-encoded epec outer membrane protein called intimin.'" animals infected with attaching and effacin pathogens mount antibody responses to intimin and t i r ! 9 and both are considered potential immunogens. the lack of protection from epec reinfection suggests that natural antibody responses to tir and intimin are not protective. the second major virulence factor of typical epec is the bundle-forming pilus (bfp),330 which is encoded on a partially conserved 60 mda virulence plasmid called epec adherence factor bfp, a member of the type iv pilus family, mediates aggregation of the bacteria to each other and probably to enterocytes themselves, thereby facilitating mucosal colonization.332 a bfp mutant was shown to be attenuated in adult volunteers.333 the principal pathologic lesion with epec is the focal destructive adherence of the organism, effacing the microvillous brush border with villus blunting, crypt hypertrophy, histiocytic infiltration of the lamina propria, and reduced brush border enzymes. rothbaum and colleagues324 described similar findings with dissolution of the glycocalyx and flattened microvilli with the nontoxigenic epec strain 0119:b14. there has been a wide range of pathologic findings reported in infants dying of epec gastroenteritis. most newborns dying with diarrheal disease caused by epec show no morphologic changes of the gastrointestinal tract by gross or microscopic examination of tiss~es.~'~'~'' bra?" described such "meager" changes in the intestinal tract that "the impression received was that the term gastroenteritis is incorrect." at the other extreme, extensive and severe involvement of the intestinal tract, although distinctly unusual among neonates with epec diarrhea, has been discussed in several reviews of the pathologic anatomy of this disease.247v3 19,334 changes virtually identical to those found in infants dying with necrotizing enterocolitis have been reported.334 drucker and c o -~o r k e r s~'~ found that among 17 infants who were dying of epec diarrhea, "intestinal gangrene, and/or perforation, andlor peritonitis were present in five, and intestinal pneumatosis in five." the reasons for such wide discrepancies in epec disease pathology are not clear. the severity of intestinal lesions at the time of death does not correlate with the birth weight of the patient, the age of onset of illness, the serogroup of the infecting strain, or the prior administration of oral or systemic antimicrobial agents. the suggestion that the intensity of inflammatory changes may depend on the duration of the diarrhea3'' cannot be corroborated in autopsy s t~d i e s~'~*~"~~~ or small intestinal it is difficult to reconcile such a thesis with the observation that a wide range of intestinal findings can be seen at autopsy among newborns infected by a single serotype of epec during an epidemic. the nonspecific pathologic picture described by some researchers includes capillary congestion and edema of the bowel wall and an increase in the number of eosinophils, plasma cells, macrophages, and mononuclear cells in the mucosa and submucosa.262,319,335 villous patterns are generally well preserved, although some flattening and broadening of the villi are seen in the more severe cases. almost complete absence of villi and failure of regeneration of small bowel mucosa have been reported in an extreme case.338 edema in and around the myenteric plexuses of auerbach, a common associated finding, has been suggested as a cause of the gastrointestinal tract dilatation often seen at autopsy in infants with epec infection^.^^^'^^^'^^^ in general, the distal small intestine shows the most marked alterations; however, the reported pathologic findings may be found at all levels of the intestinal tract. several complications of epec infection have been reported. candidal esophagitis accounted for significant morbidity in two series collected before'" and the antibiotic era. oral thrush has been seen in 50% of epecinfected infants treated with oral or systemic antib i o t i c~. '~~,~~"~~ some degree of fatty metamorphosis of the liver has been reported by several investigators"0i2'5 335; however, these changes are nonspecific and probably result from the poor caloric intake associated with persistent diarrhea or vomiting. some degree of bronchopneumonia, probably a terminal event in most cases, exists in a large proportion of newborns dying of epec i n f e~t i o n .~" " '~'~~~ in one reported series of infant cases, epec was demonstrated by immunofluorescent staining in the bronchi, alveoli, and interalveolar septa. mesenteric lymph nodes are often swollen and congested with reactive germinal centers in the lymphoid f o l l i~l e s . 2 '~~~~, '~~ severe lymphoid depletion, unrelated to the duration or severity of the antecedent illness, also has been de~cribed.'~~ the kidneys frequently show tubular epithelial toxic changes. various degrees of tubular degeneration and cloudy swelling of convoluted tubules are common finding^.^'^,'^^,^^^ renal vein thrombosis or cortical necrosis may be observed in infants with disseminated intravascular coagulation in the terminal phases of the illness. the heart is grossly normal in most instances but may show minimal vacuolar changes of nonspecific toxic myocarditis on microscopic examinati0n.3~~'~~' candidal abscesses of the heart339 and kidneys'85,335,339 have been described. with the exception of mild congestion of the pia arachnoid vessels and some edema of the meninges, examination of the central nervous system reveals few changes?153262 despite the observation of braf l1 that "inflammation of the middle ear [is] exceptional," strains of epec have been isolated from a significant number of specimens of the middle ear in case series in which dissection of the temporal bone has been performed.2093215 exposure of newborns to epec may be followed by one of several possible consequences: no infection, infection without illness, illness with gastroenteritis of variable severity and duration, and rarely, septicemia with or without metastatic foci of infection accompanying gastroenteritis. when infants are exposed to epec, a significant number become colonized as temporary st00185,888231 or pharyngeal238 carriers with no signs of clinical disease. although l a~r e l l~~' showed that the percentage of asymptomatic infections rises steadily as age increases, this observation has not been confirmed by other investigator^.^'^.^^^ similarly, the suggestion that prematurity per se is associated with a low incidence of inapparent epec infection has been documented in several clinical but refuted in others.252,279 most neonates who acquire infection with epec eventually show some clinical evidence of gastroenteritis. the incubation period is quite variable. its duration has been calculated mostly from evidence in outbreaks in newborn nurseries, where the time of first exposure can be clearly defined in terms of birth or admission dates. in these circumstances, almost all infants show signs of illness between 2 and 12 days after exposure, and most cases show signs within the first 7 days.215,231,264 in some naturally and experi-menta1306 infections with heavy exposure, the incubation period may be as short as 24 hours; the stated upper limit is 20 days.2323343 the first positive stool culture and the earliest recognizable clinical signs of disease occur simultaneously in most although colonization may precede symptoms by 7 to 14 days.265,2663344 th e gastroenteritis associated with epec infection in the newborn is notable for its marked variation in clinical pattern. clinical manifestations vary from mild illness manifest only by transient anorexia and failure to gain weight to a sudden explosive fulminating diarrhea causing death within 12 hours of onset. prematurity, underlying disease, and congenital anomalies often are associated with the more severe forms of illness.214,233,345,346 experienced clinicians have observed that the severity of epec gastroenteritis has declined markedly during the past 3 decades.225 the onset of illness usually is insidious, with vague signs of reluctance to feed, lethargy, spitting up of formula, mild abdominal distention, or even weight loss that may occur for 1 or 2 days before the first loose stool is passed. diarrhea usually begins abruptly. it may be continuous and violent, or in milder infections, it may run an intermittent course with 1 or more days of normal stools followed by 1 or more days of diarrhea. emesis sometimes is a prominent and persistent early finding. stools are loose and bright yellow initially, later becoming watery, mucoid, and green. flecks or streaks of blood, which are commonly seen with enterocolitis caused by salmonella, campylobacter, or shigella, are rarely a feature of epec diarrheal disease. a characteristic seminal smell may pervade the environment of infants infected with epec 0 1 1 1:b34,232,262, 347 and an odor variously described as "pungent," "musty," or "fetid" often surrounds patients excreting other strains in their stool^.^^','^^ because the buttocks are repeatedly covered with liquid stools, excoriation of the perianal skin can be an early and persistent problem. fever is an inconstant feature, and when it occurs, the patient's temperature rarely rises above 39" c. convulsions occur infrequently; their occurrence should alert the clinician to the possible presence of electrolyte disturbances, particularly hypernatremia. prolonged hematochezia, distention, edema, and jaundice are ominous signs and suggest an unfavorable p r o g n o~i s .~'~,~~~*~*~ m ost infants receiving antimicrobial agents orally show a cessation of diarrhea, tolerate oral feedings, and resume weight gain within 3 to 7 days after therapy has been those with mild illness who receive no treatment can continue to have intermittent loose stools for 1 to 3 weeks. in one outbreak related to epec 0142:k86, more than one third of the untreated or inappropriately treated infants had diarrhea for more than 14 days in the absence of a recognized enteric pathogen on repeated culturing.267 recurrence of diarrhea and vomiting after a period of initial improvement is characteristic of epec e n t e r i t i~. '~'~~~~~~~ though seen most often in newborns who have been treated inadequately or not treated at all, clinical relapses also occur after appropriate therapy. occasionally, the signs of illness during a relapse can be more severe than those accompanying the initial attack of illness.215,232,285 not all clinical relapses result from persistent infection. a significant number of relapses, particularly those that consistently follow attempts at reinstitution of formula fee ding^?^^.^^^ are caused by disaccharide intolerance rather than bacterial proliferation. intestinal superinfections, caused by another serotype of epecz8393479348 or by completely different enteric pathogens, such as salmonella or shigella,245 also can delay the resolution of symptoms. rarely, infants suffer a "relapse" caused by an organism from the same 0 group as the original strain but differing in its h antigen. unless complete serotyping is performed on all epec isolates, such an event easily could be dismissed as being a recurrence rather than a superinfection with a new ~r g a n i s m . '~~*~~~ antimicrobial agents to which the infecting organisms are susceptible often may not eradicate epec:45,265,267 which may persist for weeks264,283,344 or months349 after the acute illness has subsided. although reinfection cannot always be excluded, a significant number of infants are discharged from the hospital with positive rectal dehydration is the most common and serious complication of gastroenteritis caused by epec or a toxin-producing e. coli. virtually all deaths directly attributable to the intestinal infection are caused by disturbances in fluids and electrolytes. when stools are frequent in number, large in volume, and violent in release, as they often are in severe infections with abrupt onset, a neonate can lose up to 15% of body weight in a few h o~r s .~~~,~~~ rarely, fluid excretion into the lumen of the bowel proceeds so rapidly that reduction of circulating blood volume and shock may intervene before passage of even a single loose before the discovery of the etiologic agent, epidemic diarrhea of the newborn was also known by the term cholera infantum. mild disease, particularly when aggravated by poor fluid intake, can lead to a subtle but serious deterioration of an infant's metabolic status. sometimes, a week or more of illness elapses before it becomes apparent that an infant with borderline acidosis and dehydration who seemed to be responding to oral fluids alone requires parenteral therapy for impr~vement?~~ it is incumbent on the clinician caring for small infants with gastroenteritis to follow them closely, with particular attention to serial weights, until full recovery can be confirmed. there are few other complications, with the possible exception of aspiration pneumonia, directly related to epec gastroenteritis. protracted diarrhea and nutritional failure may occur as a consequence of functional damage to the small intestinal mucosa, with secondary intolerance to dietary necrotizing enterocolitis, which occasionally results in perforation of the bowel and peritonitis, has not been causally related to infection with epec.247,264,266 a review of most of the large clinical series describing epec disease in infants who ranged in age from neonates to children aged 2 years revealed only three proven instances of ba~teremia:~~**~~ one possible urinary tract infection:65 and one documented case of meningitis in an infant of unspecified age.351 focal infections among neonates were limited to several cases of otitis and a subcutaneous abscess294 from which epec was isolated. additional complications include interstitial pneumonia,319 gastrointestinal bleeding with or without disseminated intravascular coagulatio11,3~.~~~ and methemoglobinemia caused by a mutant of epec 0127:b8 that was capable of generating large quantities of nitrite from proteins present in the gastrointestinal tract.353 the gold standard of epec diagnostics is identification in the stool of e. coli carrying genes for bfp and lee. identification of these genes can be accomplished by molecular methods (discussed later), but lack of access to these methods has led many labs to rely on surrogate markers, such as serotyping." classic epec has been recovered from the vomitus, stool, or bowel contents of infected newborns. isolation from bile233 and the upper respiratory t r a~t~~~~~~*~~~ ha s been described in those instances in which a specific search has been made. less commonly, epec is isolated from ascetic fluid'" or purulent exudates209*2157294* , occasionally, the organism has been recovered from blood c u l t~r e s ?~~,~~~ urine:65 and cerebrospinal fluid. stool cultures generally are more reliable than rectal swabs in detecting the presence of enteric pathogens, although a properly obtained swab should be adequate to demonstrate epec in most cases.2'7,296*354 specimens should be obtained as early in the course of the illness as possible because organisms are present in virtually pure culture during the acute phase of the enteritis but diminish in numbers during convalescence. because of the preponderance of epec in diarrheal stools, two cultures are adequate for isolation of these pathogens in almost all cases of active disease. studies using fluorescent antibody methods for identification of epec in stool specimens have demonstrated that during the incubation period of the illness, during convalescence, and among asymptomatic carriers of epec, organisms can be excreted in such small numbers that they escape detection by standard bacteriologic methods in a significant proportion of as many as 3 to 10 specimens may be required to detect epec using methods that identify individual epec isolates in the ~t 0 0 1 . 8~~~ after a stool specimen is received, it should be plated as quickly as possible onto noninhibiting media or placed in a preservative medium if it is to be held for longer periods. deep freezing of specimens preserves viable epec when a prolonged delay in isolation is necessary?" no selective media, biochemical reactions, or colonial variations permit differentiation of pathogenic and nonpathogenic strains. certain features may aid in the recognition of two important serogroups. cultures of serogroups 0 1 11:b4 and 055:b5, unlike many other coliforms, are sticky or stringy when picked with a wire loop and are rarely hemolytic on blood whereas 0 1 11:b4 colonies emit a distinctive evanescent odor commonly described as "~e m i n a l . ' '~~~,~~~ this unusual odor first led b r a y to suspect that specific strains of e. coli might be responsible for infantile gastroenteritis. because serotyping is simpler than molecular detection and because epec have long been known to belong to certain highly characteristic serotypes, serotyping can be used to identify likely epec strains, especially in outbreaks?16 e. coli, like other enterobacteriaceae members, possesses cell wall somatic antigens (o), envelope or capsular antigens (k), and if motile, flagellar antigens (h). many of the 0 groups may be further divided into two or more subgroups (a, b, c), and the k antigens are divisible into at least three varieties (b, l, a) on the basis of their physical behavior. organisms that do not possess flagellar antigens are nonmotile (designated nm). the epec b capsular surface antigen prevents agglutination by antibodies directed against the underlying 0 antigen. heating at 100°c for 1 hour inactivates the agglutinability and antigenicity of the b antigen. slide agglutination tests with polyvalent 0 or ob antiserum may be performed on suspensions of colonies typical of e. coli that have been isolated from infants with diarrhea, especially in nursery outbreaks. however, because of numerous false-positive "cross-reactions:' the 0 and k (or b) type must be confirmed by titration with the specific a n t i~e r a .~~~ the presence of epec does not prove that epec is the cause of diarrhea in an individual patient. mixed cultures with two or three serotypes of epec have been demonstrated in 1% to 10% of patients.244*245*352 this need not mean that two or three serotypes are causative agents. secondary infection with hospital-acquired strains can occur during convalesand some infants may have been asymptomatic carriers of one serotype at the time that another produced diarrheal disease. a similar explanation may pertain to mixed infections with epec and salmonella or shigella.217*2203358 nelson245 reported the presence of these pathogens in combination with epec in 14% of infants who were cultured as part of an antibiotic therapy trial. salmonella and shigella that had not been identified on cultures obtained at admission were isolated only after institution of oral therapy with neomycin. the investigator postulated that the alteration in bowel flora brought about by the neomycin facilitated the growth of these organisms, which had previously been suppressed and obscured by coliform overthe importance of seeking all enteric pathogens in primary and follow-up cultures of infantile diarrhea is apparent, particularly when the specimen originates from a patient in a newborn nursery or infants' ward. although epec gastroenteritis was once considered to be synonymous with "summer diarrhea," community outbreaks have occurred as frequently, if not more frequently, in the colder seasons.133,160*172 it has been suggested that the increased incidence at that time of year might be related to the heightened chance of contact between infants and toddlers cence,l 73,281,283,380 that is bound to occur when children remain indoors in close contact.z94 nursery epidemics, which depend on the chance introduction and dissemination of epec within a relatively homogeneous population and stable environment, demonstrate no seasonal prevalence. average relative humidity, temperature, and hours of daylight have no significant effect in determining whether an outbreak will follow the introduction of enteropathogenic strains of e. coli into a ward of infants.243 there are no clinical studies of the variations in peripheral leukocyte count, urine, or cerebrospinal fluid in neonatal enteritis caused by epec. microscopic examination of stools of infants with acute diarrheal illness caused by these organisms usually has revealed an absence of fecal polymorphonuclear l e~k o c y t e s~'~~~~~~~~~*~~~ although data on fecal lactoferrin in human volunteers suggest that an inflammatory process may be important in epec diarrhea.360,361 stool ph can be neutral, acid, or alkaline.617341 serologic methods have not proved to be useful in attempting to establish a retrospective diagnosis of epec infection in neonates. rising or significantly elevated agglutinin titers rarely could be demonstrated in early investigation^^'^"'^^^'; hemagglutinating antibodies showed a significant response in no more than 10% to 20% of cases.245,297 fluorescent antibody techniques have shown promise for preliminary identification of epec in acute infantile diarrhea. this method is specific, with few false-positive results, and it is more sensitive than conventional plating and isolation t e c h n i q~e s .~~~,~~' .~~~ the rapidity with which determinations can be performed makes them ideally suited for screening ill infants and possible carriers in determining the extent and progression of a n~r s e r f '~,~~~ or om mu nit$^^^^'' outbreak. because immunofluorescence does not depend on the viability of organisms and is not affected by antibiotics that suppress growth on culture plates, it can be used to advantage in following bacteriologic responses and relapses in patients receiving oral the rap^.^","^ the use of fluorescent antibody techniques offers many advantages in the surveillance and epidemiologic control of epec gastroenteritis. immunofluorescent methods should supplement but not replace standard bacteriologic and serologic methods for identification of enteric pathogens. specific gene probes and pcr primers for the bfp adhesin, the intimin-encoding gene (eue) and for a cryptic plasmid locus (eaf) are a~ailable.~~ detection of bfp or eaf are superior to detection of eue, because many non-epec, including nonpathogens, carry the eae gene.98b364 pcr and gene probe analysis can be performed directly on the stools of suspect infants. however, confirmation of infection by the identification of the organism in pure culture should be pursued. before widespread use of molecular methods, the hep-2 cell adherence assay was proposed for epec diagnosis."' the presence of a focal or localized adherence (la) pattern on the surface of hep-2 or hela cells after 3-hour coincubation is a highly sensitive and specific test for detection of epec. 365 the requirement for cell culture and expertise in reading this assay limits its utility to the research setting. an elisa for the bfp has been described but is not readily available?66 the capacity of la + epec to polymerize f-actin can be detected in tissue culture cells stained with rhodamine-labeled phall~idin.~~' this fluorescence-actin staining (fas) test is cumbersome and impractical for routine clinical use. the mortality rate recorded previously in epidemics of epec gastroenteritis is impressive for its variability. during the 1930s and 1940s, when organisms later recognized as classic enteropathogenic serotypes were infecting infants, the case-fatality ratio among neonates was about 50%. 2099210 during the 1950s and 1960s, many nursery epidemics still claimed about one of every four infected infants, but several outbreaks involving the same serotypes under similar epidemiologic circumstances had fatality rates of less than 30h.23424131 in th e 1970s, reports appeared in the literature of a nursery epidemic with a 40% neonatal mortality rate285 and of an extensive outbreak in a nursery for premature infants with 4% fatalities265; another report stated that among "243 consecutive infants admitted to the hospital for epec diarrheal disease, none died of diarrheal disease per se."368 a significant proportion of the infants who died during or shortly after an episode of gastroenteritis already were compromised by preexisting disease233,283,330 or by congenital m a l f~r m a t i o n s~'~,~~'~~~~ at the time they acquired their illness. these underlying pathologic conditions appear to exert a strongly unfavorable influence, probably by reducing the infant's ability to respond to the added stresses imposed by the gastrointestinal tract infection. although prematurity is often mentioned as a factor predisposing to a fatal outcome, the overall mortality rate among premature infants with epec gastroenteritis has not differed significantly over the years from that recorded for term the management of epec gastroenteritis should be directed primarily toward prevention or correction of problems caused by loss of fluids and electr01ytes.i~~ most neonates have a relatively mild illness that can be treated with oral rehydration. infants who appear toxic, those with voluminous diarrhea and persistent vomiting, and those with increasing weight loss should be hospitalized for observation and treatment with parenteral fluids and careful maintenance of fluid and electrolyte balance and possibly with antimicrobial therapy. clinical studies suggest that slow nasogastric infusion of an elemental diet can be valuable in treating infants who have intractable diarrhea that is unresponsive to standard modes of therapy. 369 there is no evidence that the use of proprietary formulas containing kaolin or pectin is effective in reducing the number of diarrheal stools in neonates with gastroenteritis. attempts to suppress the growth of enteric pathogens by feeding lactobacillus to the infant in the form of yogurt, powder, or granules have not been shown to be of value.370 a trial of cholestyramine in 15 newborns with epec gastroenteritis had no effect on the duration or severity of the diarrhea.265 the use of atropine-like drugs, paregoric, or loperamide to reduce intestinal motility or cramping should be avoided. inhibition of peristalsis interferes with an efficient protective mechanism designed to rid the body of intestinal pathogens and may lead to fluid retention in the lumen of the bowel that may be sufficient to mask depletion of extracellular fluid and electrolytes. the value of antimicrobial therapy in management of neonatal epec gastroenteritis, if any, is uncertain. there are no adequately controlled studies defining the benefits of any antibiotic in eliminating epec from the gastrointestinal tract, reducing the risk of cross-infection in community or nursery outbreaks, or modifymg the severity of the illness. proponents of the use of antimicrobial agents have based their claims for efficacy on anecdotal observations or comparative studies.245 nonetheless, several clinical investigations have provided sufficient information to guide the physician faced with the dilemma of deciding whether to treat an individual infant or an entire nursery population suffering from epec diarrheal disease. it should be emphasized, however, that these guidelines must be considered tentative until rigidly controlled, double-blind studies have established the efficacy of antibiotics on a more rational and scientific basis. oral therapy with n e o m y~i n ,~'~'~~' ~olistin,'~~or chloram-phenic01~~~ appears to be effective in rapidly reducing the number of susceptible epec organisms in the stool of infected infants. studies comparing the responses of infants treated orally with ne~mycin?~' gentamicin:65 p~l p y x i n :~~ or kanamy~in'~' with the responses of infants receiving supportive therapy alone have shown that complete eradication of epec occurs more rapidly in those receiving an antimicrobial agent. in most cases, stool cultures are free of epec 2 to 4 days after the start of therapy.2459363 bacteriologic failure, defined as continued isolation of organisms during or after a course of an antimicrobial agent, can be expected to occur in 15% to 30% of patients?45s265 such relapses generally are not associated with a recurrence of ~y m p t~m~.~~i *~~~*~~~ the effectiveness of oral antimicrobial therapy in reducing the duration of epec excretion serves to diminish environmental contamination and the spread of pathogenic organisms from one infant to another. breaking the chain of fecal-oral transmission by administering antimicrobial agents simultaneously to all carriers of epec and their immediate contacts in the nursery has appeared to be valuable in terminating outbreaks that have failed to respond to more conservative m e a s~r e s .~'~,~~,~~~ the apparent reduction in morbidity and mortality associated with oral administration of neomycin,230.233,234 colistin,246.267.285 p o l y m y x i r~,~~~ or gentamicin2& during nursery epidemics has led to the impression that these drugs also exert a beneficial clinical effect in severely or moderately ill infants. reports describing bacteriologic:65 or histopathol~gic~'~ evidence of tissue invasion by epec have persuaded some investigators to suggest the use of parenteral rather than oral drug therapy in debilitated or malnourished infants. on the basis of these data, there appears to be sufficient evidence to recommend oral administration of nonabsorbable antibiotics in the treatment of severely or moderately ill newborns with epec gastroenteritis. the drug most frequently used for initial therapy is neomycin sulfate in a dosage of 100 mg/kg/day administered orally every 8 hours in three divided doses.24s in communities in which neomycin-resistant epec has been prevalent, treatment with colistin sulfate or polymyxin b in a dosage of 15 to 20 mglkglday orally and divided into three equal doses may be appropriate. however, it is rarely necessary to use this approach. treatment should be continued only until stool cultures become negative for epec.245 because of the unavoidable delay before cultures can be reported, most infants receive therapy for 3 to 5 days. if fluorescent antibody testing of rectal swab specimens is available, therapy can be discontinued as soon as epec no longer is identified in smears; this takes no more than 48 hours in more than 90% of cases.245 after diarrhea and vomiting have stopped and the infant tolerates formula feedings, shows a steady weight gain, and appears clinically well, discharge with outpatient follow-up is indicated. bacteriologic relapses do not require therapy unless they are associated with illness or high epidemiologic risks to other young infants in the household. because the infecting organisms in these recurrences generally continue to show in vitro susceptibility to the original drug, it should be reinstituted pending bacteriologic re~ults.2~~ when clinical judgment suggests that a neonate may be suffering from bacterial sepsis and epec diarrheal disease, parenteral antimicrobial therapy is indicated after appropriate cultures have been obtained. the routine use of systemic therapy in severe cases of epec enteritis is not appropriate on the basis of current clinical experience. antimicrobial susceptibility patterns of epec are an important determinant of the success of therapy in infections with these organism^.^^',^^'^^^ these patterns are unpredictable, depending on the ecologic pressures exerted by local antibiotic and on the incidence of transmissible resistance factors in the enteric flora of the particular population served by an i n s t i t~t i o n .~~~"~~ for these reasons, variations in susceptibility patterns are apparent in different n~r s e r i e s~~~, '~~ and even from time to time within the same institution.247,248,250 sudden changes in clinical response may even occur during the course of a single epidemic as drugsusceptible strains of epec are replaced by strains with multidrug r e~i s t a n c e .~~~'~~' ,~~' because differences can exist in the susceptibilities of different epec serogroups to various antimicrobial agents, regional susceptibility patterns should be reported on the basis of ob group or serotype rather than for epec as a whole.250 knowledge of the resistance pattern in one's area may help in the initial choice of antimicrobial therapy. the prevention of hospital outbreaks of epec gastroenteritis is best accomplished by careful attention to infection control policies for a nursery. all infants hospitalized with diarrhea should have a bacteriologic evaluation. if the laboratory is equipped and staffed to perform fluorescent antibody testing, infants transferred from another institution to a newborn, premature, or intensive care nursery and all infants with gastroenteritis on admission during an outbreak of epec diarrhea or in a highly endemic area can be held in an observation area for 1 or 2 hours until the results of the fluorescent antibody test or pcr are received. because of the difficulty in diagnosing epec infection, reference laboratories, such as those at the centers for disease control and prevention (cdc), should be notified when an outbreak is suspected. infants suspected to be excreting epec, even if healthy in appearance, then can be separated from others and given oral therapy until the test results are negative. some experts have suggested that when the rapid results obtainable with fluorescent antibody procedures are not available, all infants admitted with diarrhea in a setting where epec is common may be treated as if they were excreting epec or some other enteric pathogen until contrary proof is obtained.372 stool cultures should be obtained at admission, and contact precautions should be enforced among all who come into contact with the infant. additional epidemiologic studies are needed to establish the advantages of careful isolation and nursing techniques, particularly in smaller community hospitals in which the number of infants in a "gastroenteritis ward may be small. the use of prophylactic antibiotics has been shown to be of no value and can select for increased r e~i s t a n c e .~~~"~~ unfortunately, it can be difficult to keep a nursery continuously free of epec. specific procedures have been suggested for handling a suspected outbreak of bacterial enteritis in a newborn nursery or infant care ~n i t .~~~l~~~*~~~ evidence indicating that a significant proportion of e. coli enteritis may be caused by nontypeable strains has required some modification of these earlier recommendations. the following infection control measures may be appropriate: 1. the unit is closed, when possible, to all new admissions. 2. cultures for enteric pathogens are obtained from nursing personnel assigned to the unit at the time of the outbreak. 3. stool specimens obtained from all infants in the nursery can be screened by the fluorescent antibody or another technique and cultured. identification of a classic enteropathogenic serotype provides a useful epidemiologic marker; however, failure to isolate one of these strains does not eliminate the possibility of illness caused by a nontypeable epec. 4. antimicrobial therapy with oral neomycin or colistin can be considered for all infants with a positive fluorescent antibody test or culture result. the initial drug of choice depends on local patterns of susceptibility. depending on the results of susceptibility tests, subsequent therapy may require modification. 5. if an identifiable epec strain is isolated, second and third stool specimens from all infants in the unit are reexamined by the fluorescent antibody technique or culture at 48-hour intervals. if this is not practical, exposed infants should be carefully followed. 6. early discharge for healthy, mature, uninfected infants is advocated. 7. an epidemiologic investigation should be performed to seek the factor or factors responsible for the outbreak. a surveillance system may be established for all those in contact with the nursery, including physicians and other health care personnel, housekeeping personnel, and postpartum mothers with evidence of enteric disease. a telephone, mail, or home survey may be conducted on all infants who were residing in the involved unit during the 2 weeks before the outbreak. 8. when all patients and contacts are discharged and control of the outbreak is achieved, a thorough terminal disinfection of the involved nursery is mandatory. above all, personnel and parents should pay scrupulous attention to hand hygiene when handling infants.38' since a multistate outbreak of enterohemorrhagic colitis was associated with e. coli 0157:h7,382 shiga toxin-producing e. coli (stec) have been recognized as emerging gastrointestinal pathogens in most of the industrialized world. a particularly virulent subset of stec, ehec, causes frequent and severe outbreaks of gastrointestinal the most virulent ehec belong to serotype 0157:h7. ehec has a bovine reservoir and is transmitted by undercooked meat, unpasteurized milk, and contaminated vegetables such as lettuce, alfalfa sprouts, and radish sprouts (as occurred in more than 9000 schoolchildren in japan).3849387 it also spreads directly from person to the clinical syndrome is that of bloody, noninflammatory (sometimes voluminous) diarrhea that is distinct from febrile dysentery with fecal leukocytes seen in shigellosis or eiec infection^.^^ most cases of ehec infections have been recognized in outbreaks of bloody diarrhea or hus in daycare centers, schools, nursing homes, and c o m m~n i t i e s .~~~-~~~ although ehec infections often involve infants and young children, the frequency of this infection in neonates remains unclear; animal studies suggest that receptors for the shiga toxin may be developmentally regulated and that susceptibility to disease may be age related. 391 the capacity of ehec to cause disease is related to the phage-encoded capacity of the organism to produce a vero cell cytotoxin, subsequently shown to be one of the shiga toxins.392-394 shiga toxin 1 is neutralized by antiserum against shiga toxin, whereas shiga toxin 2, although biologically similar, is not neutralized by anti-shiga toxin.395,396 like shiga toxin made by shigella dysenteriae, both e. coli shiga toxins act by inhibiting protein synthesis by cleaving an adenosine residue from position 4324 in the 28s ribosomal rna (rrna) to prevent elongation factor-1-dependent aminoacyl transfer rna (trna) from binding to the 60s rrna.3923393 the virulence of ehec also may be determined in part by a 60-mda plasmid that encodes for a fimbrial adhesin in 0157 and 026.397,398 this phenotype is mediated by the lee pathogenicity island, which is highly homologous to the island present in epec strains. 328 ehec and other stec infections should be suspected in neonates who have bloody diarrhea or who may have been exposed in the course of an outbreak among older individuals. because most cases are caused by ingestion of contaminated food, neonates have a degree of epidemiologic protection from the illness. diagnosis of stec diarrhea is made by isolation and identification of the pathogen in the feces. e. coli 0157:h7 does not ferment sorbitol, and this biochemical trait is commonly used in the detection of this s e r~t y p e .~~. '~~ because some nonpathogenic e. coli share this characteristic, confirmation of the serotype by slide agglutination is required. these techniques can be performed in most clinical laboratories. however, detection of non-0 157 serotypes is problematic and relies on detection of the shiga toxin; available methods include shiga toxin elisa, latex agglutination, and molecular method^.^^,^^^ these should be performed by a reference laboratory. hus in infants is not necessarily caused by stec infection. even in older patients, however, the stool is typically negative for stec at the time the that hus develops.400340' serum and fecal detection of cytotoxin has been performed in such patients, but no diagnostic modality is definitive once hus has s~pervened!~~,~~~ antimicrobial therapy should not be administered to patients who may have stec infection, although their role in inducing hus remains c o n t r o~e r s i a l .~~~'~~~ management of the diarrhea and possible sequelae is supportive, with proper emphasis on fluid and electrolyte replacement. aggressive rehydration is helpful in minimizing the frequency of serious sequelae. the hep-2 adherence assay is useful for the detection of epec, which exhibit a classic la pattern."' two other adherence patterns can be discerned in this assay: aggregative (aa) and diffuse (da). these two patterns have been suggested to define additional pathotypes of diarrheogenic e. coli." strains exhibiting the aa pattern (i.e., eaec) are common pathogens of infants.lz5 eaec cause diarrhea by colonization of the intestinal mucosa and elaboration of enterotoxins and c y t o t o~i n s .~~~~~ many strains can be shown to elicit secretion of inflammatory cytokines in vitro, which may contribute to growth retardation associated with prolonged otherwise asymptomatic colonization.io3 several virulence factors in eaec are under the control of the virulence gene activator aggr.404 presence of the aggr regulator or its effector genes has been proposed as a means of detecting truly virulent eaec strains (called typical eaec),404,405 and an empirical gene probe long used for eaec detection has been shown to correspond to one gene under aggr the mode of transmission of eaec has not been well established. in adult volunteer studies, the infectious dose is high (> lo8 colony-forming units [ cfu] ), suggesting that in adults at least, person-to-person transmission is unlikely.408.m several outbreaks have been linked to consumption of contaminated f~o d . "~~,~'~ the largest of these outbreaks involved almost 2700 schoolchildren in japan4"; a contaminated school lunch was the implicated source of the outbreak. some studies have demonstrated contamination of condiments or milk, which could represent vehicles of foodborne transmission. several nursery outbreaks of eaec have been 0bserved,4~'~~'~ although in no case has the mechanism of transmission been established. the fist reported nursery outbreak involved 19 infants in nis, serbia, in 1995. because these infants did not ingest milk from a common source, it is presumed that horizontal transmission by environmental contamination or hands of health care personnel was possible. most of the infants were full term and previously well, and they were housed in two separate nursery rooms. the earliest epidemiologic studies of eaec implicated this organism as a cause of endemic diarrhea in developing c o~n t r i e s .~'~-~'~ in this setting, eaec as defined by the m pattern of adherence to hep-2 cells can be found in upward of 30% of the population at any one time>l7 newer molecular diagnostic modalities have revised this figure downward, although the organism remains highly prevalent in many areas. several studies from the indian subcontinent implicated eaec among the most frequent enteric pathogen^.^'^.^'^.^^^ other sites reproducibly reporting high incidence rates include and bra~il."~'*~~' there is evidence that eaec may be emerging in incidence. a study from spo paulo, brazil, implicated eaec as the prevalent e. coli pathotypes in infants4i9; epec had previously been shown to be the most common pathogen in this community. many other sites in developing countries of africa:" asia,4°5~422 and south america4" have described high endemic rates. several studies have suggested that eaec is also a common cause of infant diarrhea in industrialized c~u n t r i e s . "~~~~~~ using molecular diagnostic methods, a large prospective study in the united kingdom implicated eaec as the second most common enteric bacterial pathogen after cumpylob~cter.~~~ a similar study from switzerland found eaec to be the most common bacterial enter~pathogen.~'~ studies from the united states also have demonstrated a high rate of eaec diarrhea in infants; using molecular diagnostic methods, eaec was implicated in 11% and 8% of outpatient and inpatient diarrhea cohorts, respectively, compared with less than 2% of asymptomatic control infants (p < .05). 427 although epidemiologic studies have shown that eaec can cause diarrhea in all age groups, several studies suggest that the infection is particularly common in infants younger than 12 months 01d.405*420 descriptions from outbreaks and volunteer studies suggest that eaec diarrhea is watery in character with mucus but without blood or frank pus.4o834o93412 patients typically are afebrile. several epidemiologic studies have suggested that many infants may have bloody diarrhea,4i6 but fecal leukocytes are uncommon. the earliest reports of eaec infection suggested that this pathogen may be particularly associated with persistent diarrhea (>14 days).414-416 however, later studies suggest that persistent diarrhea may occur in only a subset of infected infants!" in the serbian outbreak of 19 infected infants, the mean duration of diarrhea was 5.2 days4''; diarrhea persisted more than 14 days in only three patients. infants in this outbreak had frequent, green, odorless stools. in three cases, the stools had mucus, but none had visible blood. eleven babies developed temperatures in excess of 38oc; only one had vomiting. despite a lack of clinical evidence suggesting inflammatory enteritis, several clinical studies have suggested that eaec is associated with subclinical inflammation, including the shedding of fecal cytokines and la~toferrin.'~~.~'~ studies in fortaleza, brazil, suggest that children asymptomatically excreting eaec may exhibit growth shortfalls compared with uninfected peers.lo3 a study from germany reported an association between eaec isolation and infant colic in infants without diarrhea.4z5 this observation has not been repeated. eaec should be considered in the differential diagnosis of persistent diarrhea and failure to thrive in infants. diagnosis of eaec requires identification of the organism in the patient's feces. the hep-2 adherence assay can be used for this purpose"'; some reports suggest that the adherence phenotype can be observed using formalin-fixed cell^^^'^^^' thereby obviating the need to cultivate eukaryotic cells for each assay. pcr and gene probe for typical eaec are available. successful antibiotic therapy has been reported using fluoroquinolones in adult although preliminary studies suggest that a~ithrornycin~~~ or r i f a~i m i n~~~ also may be effective. therapy in infected infants should be guided by the results of susceptibility testing, as eaec frequently is antibiotic re~istant.~" additional e. coli pathotypes have been described, including diffusely adherent e. coli (daec), 434 and cytodetaching e. c 0 1 i .~~~ daec has been specifically associated with diarrhea outside of infancy, as infants may have some degree of inherent resistance to infection. 436 cytodetaching e. coli represent organisms that secrete the e. coli hem~lysin.~~' it is not clear whether these latter organisms are true enteric pathogens. there are differences in invasiveness of salmonella strains related to serotype. s. typhi, s. choleraesuis, salmonella heidelberg,439p440 and salmonella dub1inm1 are particularly invasive, with bacteremia and extraintestinal focal infections occurring frequently. salmonella species possess genes closely related to those for the shigella invasion plasmid anti ensthese genes are probably essential to intestinal infection. virulence plasmids, which increase invasiveness in some serotypes, have been recognized, although the precise mechanisms of virulence remain to be elucidated; resistance to complement-mediated bacteriolysis by inhibition of insertion of the terminal c5b-9 membrane attack complex into the outer membrane may be laboratory studies have demonstrated dramatic strain-related difference in the ability of s. typhimurium t o evoke fluid secretion, to invade intestinal mucosa, and to disseminate beyond the production of an enterotoxin immunologically related to cholera toxin by about two thirds of salmonella strains may be related to the watery diarrhea often seen. 447 part because of the properties of their lipopolysaccharide~~~~~~~~ persistence of the organism within phagolysosomes of phagocytic cells may occur with any species of salmonella. it is not completely clear how the organisms have adapted to survive in the harsh intracellular environment, but their survival has major clinical significance. it accounts for relapses after therapy. it explains the inadequacy of some antimicrobial agents that do not penetrate phagolysosomes. it perhaps is the reason for prolonged febrile courses that occur even in the face of appropriate therapy. although humoral immunity and cell-mediated immunity are stimulated during salmonella infections, it is believed that cell-mediated immunity plays a greater role in eradication of the ba~teria.4'~ t cell activation of macrophages appears to be important in killing intracellular salmonella. 454 defective interferon-y production by monocytes of newborns in response to s. typhimurium lipopolysaccharide may explain in part the unusual susceptibility of infants to salmonella infection. 455 studies in mice suggest that helper t cell (th1) responses in peyer's patches and mesenteric lymph nodes may be central to protection of the intestinal m~c o s a .~~~ humans who lack the il-12 receptor and therefore have impaired th1 responses and interferon-y production are at increased risk for salmonella infe~tion.~~' in typhoid fever, presence of an envelope antigen, vi, is known to enhance virulence. patients who develop classic enteric fever have positive stool cultures in the first few days after ingestion of the organism and again late in the course after a period of bacteremia. this course reflects early colonization of the gut, penetration of gut epithelium with infection of mesenteric lymph nodes, and reseeding of the gut during a subsequent bacteremic pha~e.4~' studies of s. typhimurium in monkeys suggest similar initial steps in pathogenesis (e.g., colonization of gut, penetration of gut epithelium, infection of mesenteric lymph nodes) but failure of the organism to cause a detectable level of ba~teremia.4~~ although both salmonella and shigella invade intestinal mucosa, the resultant pathologic changes are different. shigella multiplies within and kills enterocytes with production of ulcerations and a brisk inflammatory response, whereas salmonella passes through the mucosa and multiplies within the lamina propria, where the organisms are ingested by phagocytes; consequently, ulcer formation is less striking,446 although villus tip cells are sometimes sloughed. acute crypt abscesses can be seen in the stomach and small intestine, but the most dramatic changes occur in the colon, where acute diffuse inflammation with mucosal edema and crypt abscesses are the most consistent findings.460v461 with s. typhi there also is hyperplasia of peyer's patches in the ileum, with ulceration of overlying tissues. salmonella strains, with the exception of s. typhi, are well adapted to a variety of animal hosts; human infection often can be traced to infected meat, contaminated milk, or contact with a specific animal. half of commercial poultry samples are contaminated with salmonella. 462 definition of the serotype causing infection can sometimes suggest the likely source. for example, s. dublin is closely associated with cattle; human cases occur with a higher-than-predicted frequency in people who drink raw milk.@' for s. typhimurium, which is the most common serotype and accounts for more than one third of all reported human cases, a single source has not been established, although there is an association with cattle. despite the 1975 ban by the u.s. food and drug administration (fda) on interstate commercial distribution of small turtles, these animals continue to be associated with infection, as illustrated by a series of cases in puerto ~i~~.~~~ various pet reptiles are an important source of a variety of unusual salmonella serotypes such as salmonella marina, salmonella chameleon, salmonella arizonae, salmonella java, salmonella stanley, salmonella poona, salmonella jangwain, salmonella tilene, salmonella pomona, salmonella miami, salmonella manhattan, salmonella litchfield, salmonella rubislaw, and salmonella w a~s e n a a r .~~-~~ salmonella organisms are hardy and capable of prolonged survival; organisms have been documented to survive in flour for nearly a year?67 salmonella tennessee has been shown to remain viable for many hours on non-nutritive surfaces (i.e., glass, 48 hours; stainless steel, 68 hours; enameled surface, 114 hours; rubber mattress, 119 hours; linen, 192 hours; and rubber tabletop, 192 infection with salmonella is, like most enteric infections, more common in young children than in adults. the frequency of infection is far greater in the first 4 years of life; roughly equal numbers of cases are reported during each decade beyond 4 years of age. although the peak incidence occurs in the second through sixth months of life, infection in the neonate is relatively common. researchers at the cdc have estimated the incidence of salmonella infection in the first month of life at nearly 75 cases per 100,000 infants?69 adult volunteer studies suggest that large numbers of salmonella ( lo5 to lo9) need to be ingested to cause di~ease.4~' however, it is likely that lower doses cause illness in infants. the occurrence of nursery ~u t b r e a k s~"~~~~~ and intrafamilial spread497 suggests that organisms are easily spread from person to person; this pattern is typical of low-inoculum diseases transmitted by the fecal-oral route. the neonate with salmonella infection infrequently acquires the organism from his or her mother during delivery. although the index case in an outbreak can often be traced to a mother,474-477,495 subsequent cases result from contaminated objects in the nursery e n~i r o n m e n t~~"~~~ serving as a reservoir coming in contact with hands of attending p e r~o n n e l .~~.~'~ the mother of an index case may be symptomatic479~480i500~501 or asymptomatic with preclinical infecti0n,4'~ convalescent infedon,477,48 1,502 or chronic carriage. 503 the risk of the newborn becoming infected once salmonella is introduced into a nursery has been reported to be as high as 20% to 27%,487,493 but the frequency of infection may be lower because isolated cases without a subsequent epidemic are unlikely to be reported. gastric acidity is an important barrier to salmonella infection. patients with anatomic or functional achlorhydria are at increased risk of developing salmonellosis.504~505 the hyp~chlorhydria'~ and rapid gastric emptying typical of early lifez8 may in part explain the susceptibility of infants to salmonella. premature and low-birth-weight infants appear to be at higher risk of acquiring salmonella infection than term whether this reflects increased exposure because of prolonged hospital stays or increased susceptibility on the basis of intestinal or immune function is unclear. contaminated food or water is often the source of salmonella infection in older patients; the limited diet of the infant makes contaminated food a less likely source of infection. although human milk?06-508 raw milk?09 powdered milk,510-512 formula:93 and cereal5i3 have been implicated in transmission to infants, more often fomites, such as delivery room resu~citators,4~' rectal thermometer~,4'~>~'~ oropharyngeal suction device^,^^^'^^' water baths for heating formula?17 soap dispenser^,^" " clean" medicine airconditioning mattresses, radiant and serve as reservoirs. one unusual outbreak involving 394 premature and 122 term infants was traced to faulty plumbing, which caused massive contamination of environment and personnel.493 after salmonella enters a nursery, it is difficult to eradicate. epidemics lasting 6 to 7 week^,'@^,^^' 17 weeks,& 6 months,485b490 1 year:' " and 27 to 30 months487b493 have been reported. spread to nearby pediatric wards has the incubation period in nursery outbreaks has varied widely in several studies where careful attention has been paid to this variable. in one outbreak of salmonella oranienburg involving 35 newborns, 97% of cases occurred within 4 days of in an outbreak of s. typhimurium, each of the ill infants presented within 6 days of birth.477 these incubation periods are similar to those reported for salmonella newport in older children and adults, 95% of whom have been reported to be ill within 8 days of e~p o s u r e .~'~'~~' conversely, one outbreak of salmonella nienstedten involving newborns was characterized by incubation periods of 7 to 18 days.488 the usual incubation period associated with fecal-oral nursery transmission is not found with congenital typhoid. during pregnancy, typhoid fever is associated with bacteremic infection of the fetus. the congenitally infected infants are symptomatic at birth. they are usually born during the second to fourth week of untreated maternal illness.522 usually, the mother is a carrier; fecal-oral transmission of s. typhi can occur with delayed illness in the newborn.523 several major clinical syndromes occur with nontyphoidal salmonella infection in young infants. colonization without illness may be the most common outcome of ingestion of salmonella by the neonate. such colonization usually is detected when an outbreak is under investigation. most infected infants who become ill have abrupt onset of loose, green, mucus-containing stools, or they have bloody diarrhea; an elevated temperature is also a common finding in salmonella gastroenteritis in the first months of life.44o grossly bloody stools are found in the minority of patients, although grossly bloody stools can occur in the first 24 hours of life. hematochezia is more typically associated with noninfectious causes (e.g., swallowed maternal blood, intestinal ischemia, hemorrhagic diseases, anorectal fissures) at this early age.524 there appear to be major differences in presentation related to the serotype of s. enteritidis causing infection. for example, in one epidemic of s. oranienb~rg~'~ involving 46 newborns, 76% had grossly bloody stools, 11% were febrile, 26% had mucus in their stools, and only 11% were healthy. in a series of s. newport infections involving 11 premature infants;74 90% of infants with gastroenteritis had blood in their stools, 10% had fever, 10% had mucus in their stools, and 9% were asymptomatic. in an outbreak of s. typhim~rium~~' involving 11 ill and 5 healthy infants, none had bloody stools; all of the symptomatic infants were febrile and usually had loose, green stools. of 26 infants infected by salmonella virchow, 42% were asymptomatic; the rest had mild diarrhea!82 seals and colleagues488 described 12 infants with s. nienstedten, all of whom had watery diarrhea and low-grade fever; none had bloody stools. in a large outbreak in zimbabwe of s. heidelberg infection reported by bannerman,485 38% of 100 infants were asymptomatic, 42% had diarrhea, 16% had fever, 15% had pneumonia, and 2% developed meningitis. an outbreak of salmonella worthington was characterized primarily by diarrhea, fever, and jaundice, although 3 of 18 infants developed meningitis and 17% died. 515 in dramatic contrast to these series, none of 27 infants with positive stool cultures for s. tennessee had an illness in a nursery found to be contaminated with that organism.469 a few infants with salmonella gastroenteritis have developed necrotizing e n t e r o c o l i t i~, 4~~~~~~ but it is not clear whether salmonella was the cause. although gastroenteritis is usually self-limited, chronic diarrhea has sometimes been attributed to s a l r n~n e l l a .~~~~~~~ whether chronic diarrhea is caused by salmonella is uncertain. although some infants develop carbohydrate intolerance after a bout of salmonella and salmonella is typically listed as one of the causes of postinfectious protracted diarrhea,529 it is difficult to be sure that the relationship is causal. the prolonged excretion of salmonella after a bout of gastroenteritis may sometimes cause non-specific chronic diarrhea to be erroneously attributed to salmonella. major extraintestinal complications of salmonella infection may develop in the neonate who becomes bacteremic. extraintestinal spread may develop in infants who initially present with diarrhea and in some who have no gastrointestinal tract signs. bacteremia appears to be more common in the neonate than in the older a study of more than 800 children with salmonella infection showed that extraintestinal infection occurred significantly more often (8.7% versus 3.6%) in the first 3 months of life.531 several retrospective studies suggest that infants in the first month of life may have a risk of bacteremia as high as 30% to 50%. 440 one retrospective suggests that the risk is not increased in infancy and estimates that the risk of bacteremia in childhood salmonella gastroenteritis is between 8.5% and 15.6%. prospective studies of infants in the first year of life suggest that the risk of bacteremia is 1.8% to 6.0%.532*533 although selection biases in these studies limit the reliability of these estimates, the risk is substantial. the salmonella species isolated from infants include some serotypes that appear to be more invasive in the first 2 months of life than in older children or healthy adults (s. newport, s. agona, s. blockley, s. derby, s. enteritidis, s. heidelberg, s. infantis, s. javiana, s. saint-paul, and s. typhimurium) and serotypes that are aggressive in every age group (s. choleraesuis and s. dublin). other serotypes appear more likely to cause bacteremia in adults (s. typhi, s. paratyphi a, and s. paratyphi b). 530 virtually any salmonella serotype can cause bacteremic disease in neonates. a few infants with salmonella gastroenteritis have died with e. coli or pseudomonas aeruginosa sepsis;94 but the role of salmonella in these cases is unclear. unlike the situation in older children in whom bacteremic salmonellosis often is associated with underlying medical conditions, bacteremia may occur in infants who have no immunocompromising conditions.534 salmonella bacteremia is often not suspected clinically because the syndrome is not usually d i s t i n~t i v e . 4~~~~~ even afebrile, well-appearing children with salmonella gastroenteritis have been documented to have bacteremia that persists for several days.535 although infants with bacteremia may have spontaneous resolution without therapy:36 a sufficient number develop complications to warrant empirical antimicrobial therapy when bacteremia is suspected. the frequency of complications is highest in the first month of life. meningitis is the most feared complication of bacteremic salmonella disease. between 50% and 75% of all cases of nontyphoidal salmonella meningitis occur in the first 4 months of life.537 the serotypes associated with neonatal meningitis (s. typhimurium, s. heidelberg, s. enteritidis, s. saint-paul, s. newport, and s. panama)497 are serotypes frequently associated with bacteremia. meningitis has a high mortality rate, in part because of the high relapse rates. relapse has been reported in up to 64% of ca~es.5~' in some studies, more than 90% of patients with meningitis have died,539 although more typically, 30% to 60% of infants die.540*541 the survivors suffer the expected complications of gram-negative neonatal meningitis, including hydrocephalus, seizures, ventriculitis, abscess formation, subdural empyema, and permanent neurologic impairment. neurologic sequelae have included retardation, hemiparesis, epilepsy, visual impairment, and a t h e t o~i s .~~~ in large nursery outbreaks, it is common to find infants whose course is complicated by pneum~nia?'~ osteo-myeliti~,5">~~~ or septic arthriti~.4'~,~'~ othe r rare complications of salmonellosis include p e r i~a r d i t i s ,~~ p y e l i t i~,~~ peritonitis:77 otitis media:77 mas ti ti^,^^^ chole~ystitis,~~' endophthalmiti~,~~~ cutaneous abscesses,492 and infected cephal~hematoma?~' other focal infections seen in older children and adults, such as endocarditis and infected aortic aneurysms, rarely or never have been reported in neonates.537,549 altho ugh the mortality rate in two reviews of nursery outbreaks was 3.7% to 7.0%,"959496 in some series, it reached 18%. 485 enteric fever, most often related to s. typhi but also occurring with s. paratyphi a, s. paratyphi b, s. paratyphi c, and other salmonella species, is reported much less commonly in infants than in older patients. infected infants develop typical findings of neonatal sepsis and meningitis. current data suggest that mortality is about 30y0.~~' in utero infection with s. typhi has been described. typhoid f e~e r~" *~~' and nontyphoidal salmonella infections552 during pregnancy put women at risk of aborting the fetus. premature labor usually occurs during the second to the fourth week of maternal typhoid if the woman is untreated.522 in a survey of typhoid fever in pregnancy during the preantibiotic era, 24 of 60 women with well-documented cases delivered prematurely, with resultant fetal death; the rest delivered at term, although only 17 infants survived. 553 the outlook for carrying the pregnancy to term and delivering a healthy infant appears to have improved dramatically during the antibiotic era. however, one of seven women with typhoid in a series still delivered a dead fetus with extensive liver necrosis. 554 in the preantibiotic era, about 14% of pregnant women with typhoid fever died.555 with appropriate antimicrobial therapy, pregnancy does not appear to put the woman at increased risk of death. despite these welldescribed cases, typhoid fever is rare early in life. of 1500 cases of typhoid fever that osler and m~c r a e~~~ reported, only 2 were in the first year of life. in areas where typhoid fever is still endemic, systematic search for infants with enteric fever has failed to find many cases. the few infections with s. typhi documented in children in the first year of life often present as a brief nondescript "viral syndrome" or as p n e~r n o n i t i s ?~~*~~' fever, diarrhea, cough, vomiting, rash, and splenomegaly may occur; the fever may be high, and the duration of illness may be many weeks.522 the current practice of early discharge of newborn infants, although potentially decreasing the risk of exposure, can make recognition of a nursery outbreak difficult. diagnosis of neonatal salmonellosis should trigger an investigation for other cases. other than diarrhea, signs of neonatal salmonella infection are similar to the nonspecific findings seen in most neonatal infections. lethargy, poor feeding, pallor, jaundice, apnea, respiratory distress, weight loss, and fever are common. enlarged liver and spleen are common in those neonates with positive blood cultures. laboratory studies are required to establish the diagnosis because the clinical picture is not distinct. the fecal leukocyte examination reveals polymorphonuclear leukocytes in 36% to 82%359. 559 of persons with salmonella infection, but it has not been evaluated in neonates. obviously, the presence of fecal leukocytes is consistent with colitis of any cause and therefore is a nonspecific finding. routine stool cultures usually detect salmonella if two or three different enteric media (i.e., macconkey's, eosin-methylene blue, salmonella-shigella, tergitol 7, xylose-lysine-deoxycholate, brilliant green, or bismuth sulfite agar) are used. stool, rather than rectal swab material, is preferable for culture, particularly if the aim of culture is to detect carriers.560 on the infrequent occasions when proctoscopy is performed, mucosal edema, hyperemia, friability, and hemorrhages may be seen.*' infants who are bacteremic often do not appear sufficiently toxic to raise the suspicion of b a~t e r e m i a .~~~ blood cultures should be obtained as a routine part of evaluation of neonates with suspected or documented salmonella infection. ill neonates with salmonella infection should have a cerebrospinal fluid examination performed. bone marrow cultures also may be indicated when enteric fever is suspected. there are no consistent abnormalities in the white blood cell count. serologic studies are not helpful in establishing the diagnosis, although antibodies to and flagellar antigens487 develop in many infected newborns. if an outbreak of salmonellosis is suspected, further characterization of the organism is imperative?64 determination of somatic and flagellar antigens to characterize the specific serotype may be critical to investigation of an outbreak. when the serotype found during investigation of an outbreak is a common one (e.g., s. typhimurium), antimicrobial resistance testing475,565 and use of molecular techniques such as plasmid chara~terization~~~ can be helpful in determining whether a single-strain, common-source outbreak is in progress. and ampicillin or amoxicillin versus placebo.572 in contrast to these studies, data suggest that there may be a role for quinolone antibiotics in adults and ~h i l d r e n ,~~~,~~~ but these drugs are not approved for use in neonates, and resistance has been en~ountered.~'~ because these studies have few data as to the risk-benefit ratio of therapy in the neonate, it is uncertain whether they should influence treatment decisions in neonates. studies that have included a small number of neonates suggest little benefit from antimicrobial therapy.477*487*56835773578 however, because bacteremia is common in neonates, antimicrobial therapy for infants younger than 3 months who have salmonella gastroenteritis often is recommended,532v533v561 especially if the infant appears toxic. premature infants and those who have other significant debilitating conditions also should probably be treated. the duration of therapy is debatable but should probably be no more than 3 to 5 days if the infant is not seriously ill and if blood cultures are sterile. if toxicity, clinical deterioration, or documented bacteremia complicates gastroenteritis, prolonged treatment is indicated. even with antimicrobial therapy, some infants develop complications. the relatively low risk of extraintestinal dissemination must be balanced against the well-documented risk of prolonging the carrier state. for infants who develop chronic diarrhea and malnutrition, hyperalimentation may be required; the role of antimicrobial agents in this setting is unclear. the infant with typhoid fever should be treated with an antimicrobial agent; relapses sometimes occur after therapy. colonized healthy infants discovered by stool cultures during evaluation of an outbreak ought to be isolated but probably should not receive antimicrobial therapy. such infants should be discharged from the nursery as early as possible and followed carefully as outpatients. antimicrobial treatment of neonates who have documented extraintestinal dissemination must be prolonged. bacteremia without localization is generally treated with at least a 10-day course of therapy. therapy for salmonella meningitis must be given for at least 4 weeks to lessen the risk of relapse. about three fourths of patients who have relapses have been treated for three weeks or less?37 similar to meningitis, treatment for osteomyelitis must be prolonged to be adequate. although cures have been reported with 3 weeks of therapy, 4 to 6 weeks of therapy is recommended. in vitro susceptibility data for salmonella isolates must be interpreted with caution. the aminoglycosides show good in vitro activity but poor clinical efficacy, perhaps because of the low ph of the phagolysosome. aminoglycosides have poor activity in an acid environment. the stability of some drugs in this acid environment also may explain in vitro and in vivo disparities. the intracellular localization and survival of salmonella within phagocytic cells also presumably explains the relapses encountered with virtually every regimen. resistance to antibiotics has long been a problem with salmonella i n f e c t i~n .~~,~~~,~~' there has been a steady increase in resistance to salmonella in the united states over the last 20 years.581 with the emergence of typhimurium type dt 104, resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline has increased from 0.6% in 1979 and 1980 to 34% in 1996. 582 resistance plasmids have been selected and transmitted, partly because therapy has been given for mild illness that should not have been treated566 and partly because of use of antibiotics in animal feeds. resistance to chloramphenicol and ampicillin has made trimethoprim-sulfamethoxazole increasingly important for the treatment of salmonella infection in those patients who require therapy. however, with increasing resistance to all three of these agents in asia?83 the middle e~r o p e ,~~~,~'~ ar gentina,580 and north america,5793588,589 the third-generation cephalosporins and quinolones represent drugs of choice for invasive salmonellosis. the quinolones currently are not approved for persons younger than 18 years. cefotaxime, ceftriaxone, and cefoperazone represent acceptable alternative drugs for typhoidal and nontyphoidal salmonellosis when resistance is e n c o~n t e r e d .~~"~~~ because the second-generation cephalosporins, such as cefuroxime, are less active in vitro than the third-generation cephalosporins and are not consistently clinically effective, they should not be data suggest that cefoperazone may sterilize blood and cause patients with typhoid fever to become afebrile more rapidly than with chl~ramphenicol,~~~ perhaps because cefoperazone is excreted into bile in high concent r a t i o n~.~~~ the third-generation cephalosporins may have higher cure and lower relapse rates than ampicillin or chloramphenicol in children with salmonella meningitis. 595 the doses of ampicillin, chloramphenicol, or cefotaxime used in infants with gastroenteritis pending results of blood cultures are the same as those used in treatment of sepsis. because of the risk of gray baby syndrome, chloramphenicol should not be used in neonates unless other effective agents are not available. trimethoprim-sulfamethoxazole, although useful in older children and adults, is not used in neonates because of the risk of kernicterus. nosocomial infection with strains of salmonella resistant to multiple antibiotics, including third-generation cephalosporins, has emerged as a problem in south america. 580 nonantibiotic interventions are important in the control of salmonella infections. limited data suggest that intravenous immune globulin (igiv) (500 mg/kg on days 1,2,3, and 8 of therapy) along with antibiotic therapy may decrease the risk of bacteremia and death in preterm infants with salmonella ga~troenteritis.~~~ early recognition and intervention in nursery outbreaks of salmonella are crucial to control. when a neonate develops salmonellosis, a search for other infants who have been in the same nursery should be undertaken. when two or more cases are recognized, environmental cultures, cultures of all infants, cohorting and contact isolation of infected infants, rigorous enforcement of hand hygiene, early discharge of infected infants, and thorough cleaning of all possible fomites in the nursery and delivery rooms are important elements of control. if cases continue to occur, the nursery should be closed to further admissions. cultures of nursery personnel are likely to be helpful in the unusual situation of an s. typhi outbreak in which a chronic carrier may be among the caretakers. culture of health care personnel during outbreaks of salmonellosis caused by other salmonella species is debatable, although often recommended. data suggest that nurses infected with salmonella rarely infect patients in the hospital setting.597 the fact that nursing personnel are sometimes found to be colonized during nursery outmay be a result rather than a cause of those epidemics. the potential role of vaccines in control of neonatal disease is minimal. for the vast number of non-s. ryphi serotypes, there is no prospect for an immunization strategy. multiple doses of the commercially available oral live attenuated vaccine (ty2la; vivotif, berna), has been shown in chilean schoolchildren to reduce typhoid fever cases by more than 70%.598,599 however, the vaccine is not recommended for persons younger than 6 years, in part because immunogenicity of ty2la is age dependent; children younger than 24 months fail to respond with development of immunity!" vi capsular polysaccharide vaccine is available for children older than 2 years and is effective in a single dose. whether some degree of protection of infants could the virulence of shigellae has been studied extensively since their recognition as major pathogens at the beginning of the 20th century. the major determinants of virulence are encoded by a 120-to 140-mda p l a~m i d .~~~.~~~ this plasmid, which is found in all virulent shigellae, encodes the synthesis of proteins that are required for invasion of mammalian cells and for the vigorous inflammatory response that is characteristic of the d i s e a~e .~~*~~ shigellae that have lost this plasmid, have deletions of genetic material from the region involved in synthesis of these proteins, or have the plasmid inserted into the chromosome lose the ability to invade eukaryotic cells and become aviru1ent6o7; maintenance of the plasmid can be detected in the clinical microbiology lab by ability to bind congo red. the ability to invade cells is the basic pathogenic property shared by all ~h i g e l l a e~~'~~~~ and by the shigella-like invasive e. coli, which also possesses the shigella virulence plasmid.205~605~606~610~611 in the laboratory, shigella invasiveness is studied in tissue culture (hela cell invasion), in animal intestine, or in rabbit or guinea pig eye, where instillation of the organism causes keratoconjunctivitis (sereny test)."' animal model studies have shown that bacteria penetrate and kill colonic mucosal cells and then elicit a brisk inflammatory response. in addition to the virulence plasmid, several chromosomal loci enhance virulence.612v613 this has been best studied in s. flexneri in which multiple virulence-enhancing regions of the chromosome have been defined.604s612-614 the specific gene products of some of the chromosomal loci are not known; one chromosomal virulence segment encodes for synthesis of the 0 repeat units of lipopolysaccharide. intact lipopolysaccharide is necessary but not sufficient to cause virulence.6129615 at least two cell-damaging cytotoxins that also are chromosomally encoded are produced by shigellae. one of these toxins (shiga toxin) is made in large quantities by s. dysenteriae serotype 1 (the shiga bacillus) and is made infrequently by other shigellae.616 shiga toxin is a major virulence factor in s. dysenteriae, enhancing virulence at the colonic mucosa and also giving rise to sequelae similar to those caused by stec (discussed earlier). this toxin kills cells by interfering with peptide elongation during protein ~y n t h e s i s .~'~-~'~ additional toxins may also be secreted by shigellae, although their roles in virulence are not established.620 although much of the epidemiology of shigellosis is predictable based on its infectious dose, certain elements are unexplained. shigellae, like other organisms transmitted by the fecal-oral route, are commonly spread by food and water, but the low infecting inoculum allows person-to-person spread. because of this low inoculum, shigella is one of the few enteric pathogens that can infect swimmers. 621 the dose required to cause illness in adult volunteers is as low as 10 organisms for s. dysenteriae serotype 1,6" about 200 organisms for s. f l e~n e r i ,~~~ and 500 organisms for s. ~o n n e i .~'~ personto-person transmission of infection probably explains the continuing occurrence of shigella in the developed world. enteropathogens that require large inocula and hence are best spread by food or drinking water are less common in industrialized societies because of sewage disposal facilities, water treatment, and food-handling practices. in the united states, daycare centers currently serve as a major focus for acquisition of shigell~sis.~'~ numerous outbreaks of shigellosis related to crowding, poor sanitation, and the low dose required for diseases have occurred in this setting. given the ease of transmission, it is not surprising that the peak incidence of disease is in the first 4 years of life. it is, however, paradoxical that symptomatic infection is uncommon in the first year of life.626-629 the best data on the age-related incidence of shigellosis come from mata'~~'~ prospective studies of guatemalan infants. in these studies, stool cultures were performed weekly on a group of children followed from birth to 3 years old. the rate of infection was more than 60-fold lower in the first 6 months of life than (fig. 20-1) . 626 the same age-related incidence has been described in the united states629 and in a rural egyptian village. 628 this anomaly has been explained by the salutary effects of brea~t-feeding.~~'-~~' however, it is likely that breast-feeding alone does not explain the resistance of infants to shigellosis. a review of three large case series633-635 suggests that about 1.6% (35 of 2225) of shigellosis cases occur in infants in the neonatal period. the largest series of neonatal ~higellosis~~~ suggests that the course, complications, and etiologic serogroups are different in neonates than in older children. although newborns are routinely contaminated by maternal feces, neonatal shigellosis is rare. other aspects of the epidemiology of shigellosis elude simple explanation. the seasonality (summer-fall peak in the united states, rainy season peak in the tropics) is not well explained. the geographic variation in species causing infection likewise is not well understood. in the united states, most shigella infections are caused by s. sonnei or, less commonly, s. flexneri. in most of the developing world, the relative importance of these two species is reversed, and other shigella serotypes, especially s. dysenteriae serotype 1, are identified more frequently. as hygiene improves, the proportion of s. sonnei increases and that of s. flexneri decreases.636 data from bangladesh suggest that s. dysenteriae is less common in neonates, but s. sonnei and s. boydii are more c0mmon.6~~ there appear to be some important differences in the relative frequencies of various complications of shigella infection related to age. some of these differences and estimates are based on data that are undoubtedly compromised by reporting biases. s. dysenteriae serotype 1 characteristically causes a more severe illness than other shigellae with more complications, including pseudomembranous colitis, hemolysis, and hus. however, illnesses caused by various shigella serotypes usually are indistinguishable from each other and conventionally are discussed together. the incubation period of shigellosis is related to the number of organisms ingested, but in general, it is between 12 and 48 hours. volunteer studies have shown that after ingestion, illness may be delayed for a week or more. neonatal shigellosis seems to have a similar incubation period. more than one half of the neonatal cases occur within 3 days of birth, consistent with fecal-oral transmission during parturition. mothers of infected neonates are sometimes carriers, although more typically they are symptomatic during the perinatal period. intrauterine infection is rare. in the older child, the initial signs are usually high fever, abdominal pain, vomiting, toxicity, and large-volume watery stools; diarrhea may be bloody or may become bloody. painful defecation and severe, crampy abdominal pain associated with frequent passage of small-volume stools with gross blood and mucus are characteristic findings in older children or adults who develop severe colitis. many children, however, never develop bloody diarrhea. adult volunteer studies have demonstrated that variations in presentation and course are not related to the dose ingested because some patients develop colitis with dysentery but others develop only watery diarrhea after ingestion of the same i n o c u l~m .~~~ the neonate with shigellosis may have a mild diarrheal syndrome or a severe ~o l i t i s .~~~~~~~-~~ fever in neonates is usually low grade (<102" f) if the course is uncomplicated. the neonate has less bloody diarrhea, more dehydration, more bacteremia, and a greater likelihood of death than the older ~h i l d . 6~~ physical examination of the neonate may show signs of toxicity and dehydration, although fever, abdominal tenderness, and rectal findings are less striking than in the older complications of shigellosis are common.646 although the illness is self-limited in the normal host, resolution may be delayed for a week or more. in neonates and malnourished children, chronic diarrhea may follow a bout of shigello~is.6~',~~ between 10% and 35% of hospitalized children with shigella have convulsions before or during the course of usually, the seizures are brief, generalized, and associated with high fever. seizures are uncommon in the first 6 months of life, although neonates have been described with seizures.6399649 the cerebrospinal fluid generally reveals normal values in these children, but a few have mild cerebrospinal fluid pleocytosis. the neurologic outcome generally is good even with focal or prolonged seizures, but fatalities do occasionally occur, often associated with toxic encephalpa thy.^^' although the seizures had been postulated to result from the neurotoxicity of shiga toxin, this explanation was proved to be incorrect because most shigellae make little or no shiga toxin and the strains isolated from children with neurologic symptoms do not produce shiga t~x i n .~'~,~~' hemolysis with or without development of uremia is a complication primarily of s. dysenteriae serotype 1 infection. 652 sepsis during the course of shigellosis may be caused by the shigella itself or by other gut flora that gain access to the bloodstream through damaged mucosa.632'653*654 the risk of sepsis is higher in the first year of life, particularly in neo-nates,632.637-639,649,656 in malnourished children, and in those with s. dysenteriae serotype 1 infection.654 sepsis occurs in up to 12% of neonates with given the infrequency of neonatal shigellosis, it is striking that 9% of reported cases of shigella sepsis have involved infants in the first month of life.657 one of the infants with ba~teremia~~' reportedly had no discernible illness. disseminated intravascular coagulation may develop in those patients whose course is complicated by sepsis. meningitis has been described in a septic neonate. colonic perforation has occurred in n e o n a t e~, 6~"~~~ older children,@' and adults.661 although this complication of toxic megacolon is rare, it appears to be more common in neonates than in older individuals. bronchopneumonia may complicate the course of shigellosis, but shigellae are rarely isolated from lungs or tracheal secretions."2 the syndrome of sudden death in the setting of extreme toxicity with hyperpyrexia and convulsions but without dehydration or sepsis (i.e., ekiri ~yndrome)~~'"~ is rare in neonates. in the nonbacteremic child, other extraintestinal foci of infection, including ~a g i n a~~~. "~ and eye,"' rarely occur. reiter's syndrome, which rarely complicates the illness in children, has not been reported in neonates. although infection is less common in infants than in toddlers, case fatality rates are highest in infant^.^^'^^' the mortality rate in newborns appears to be about twice that of older children.632 in industrialized societies, less than 1% of children with shigellosis die, whereas in developing countries, up to 30% die. these differences in mortality rates are related to n~t r i t i o n . 6~~ availability of medical care, antibiotic resistance of many shigellae, the frequency of sepsis, and the higher frequency of s examination of stool for leukocytes as an indication of colitis is useful in support of the clinical suspicion of shigellosis. the white blood cell count and differential count also are used as supporting evidence for the diagnosis. leukemoid reactions (white blood cells > 50,000/mm3) occur in almost 15% of children with s. dysenteriae serotype 1 but in less than 2% of children with other ~h i g e l l a e .~~~ leukemoid reactions are more frequent in infants than in older ~hildren.6~' even when the total white blood cell count is not dramatically elevated, there may be a striking left shift. almost 30% of children with shigellosis have greater than 25% bands on the differential cell few reports address the white blood cell count in newborns, but those that do suggest that normal or low rather than elevated counts are more common. although serum and fecal antibodies develop to lipopolysaccharides and the virulence plasmid-associated polypeptide~,6~~ serologic studies are not useful in the diagnosis of shigellosis. pcr can identify shigella and eiec in feces.678 colonoscopy typically shows inflammatory changes that are most severe in the distal segments of therapy because dehydration is particularly common in neonatal shigellosis, attention to correction of fluid and electrolyte disturbances is always the first concern when the illness is suspected. although debate continues over the indications for antimicrobial therapy in the patient with shigellosis, the benefits of therapy generally appear to outweigh the risks. the chief disadvantages of antimicrobial therapy include cost, drug toxicity, and emergence of antibiotic-resistant shigellae. because of the self-limited nature of shigellosis, it has been argued that less severe illness should not be treated. however, children can feel quite ill during the typical bout of shigellosis, and appropriate antimicrobial therapy shortens the duration of illness and eliminates shigellae from stool, decreasing secondary spread. complications are probably decreased by antibiotics. given the high mortality rates of neonatal shigellosis, therapy should not be withheld. the empirical choice of an antimicrobial agent is dictated by susceptibility data for strains circulating in the community at the time the patient's infection occurs. multiresistant shigellae complicate the choice of empirical therapy before availability of susceptibility data for the patient's isolate. plasmid-encoded resistance (r factors) for multiple antibiotics has been observed frequently in s. dysenteriae serotype 1 outbreaks682 and with other ~higellae.~'~.~'~ antimicrobial resistance patterns fluctuate from year to year in a given locale.686 however, despite the guesswork involved, early preemptive therapy is indicated when an illness is strongly suggestive of shigellosis. in vitro susceptibility does not always adequately predict therapeutic responses. cefa~lor,6~~ furazolidone,688 ~ephalexin,6'~ amo~icillin,6~' kanam~cin,6~' and ~e f a m a n d o l e~~~ all are relatively ineffective agents. the optimal duration of therapy is debatable. studies in children older than 2 years and in adults suggest that singledose regimens may be as effective in relieving symptoms as courses given for 5 days. the single-dose regimens generally are not as effective in eliminating shigellae from the feces as are the longer courses. a third-generation cephalosporin, such as ceftriaxone, may be the best empirical choice. optimal doses for newborns with shigellosis have not been established. trimethoprim at a dose of lomg/kg/day (maximum, 160 mg/day) and sulfamethoxazole at a dose of 50 mg/kg/day (maximum, 800 mg/day) in two divided doses for a total of 5 days are recommended for the older child if the organism is s~s c e p t i b l e . 6~~-~~~ if the condition of the infant does not permit orally administration, the drug usually is divided into three doses given intravenously over 1 ampicillin at a dose of 100 mg/kg/day in four divided doses taken orally for 5 days may be used if the strain is susceptible. 676 for the rare newborn who acquires shigellosis, appropriate therapy often is delayed until susceptibility data are available. this occurs because shigellosis is so rare in newborns that it is almost never the presumptive diagnosis of the child with watery or bloody diarrhea. although a sulfonamide is as efficacious as ampicillin when the infecting strain is sus~eptible,6~~ sulfonamides are avoided in neonates because of concern about the potential risk of kernicterus. the risk of empirical ampicillin therapy is that shigellae are frequently resistant to the drug; 50% of shigellae currently circulating in the united states are ampicillin resistant.6963697 for the neonate infected with ampicillin-resistant shigella, there are few data on which to base a recommendation. ceftriaxone is generally active against shigellae, but in the neonate, this drug can displace bilirubin-binding sites and elicit clinically significant cholestasis. data on children and adults suggest that clinical improvement occurs with c e f t r i a x~n e .~~~*~~~ quinolones, such as ciprofloxacin and ofloxacin, have been shown to be effective agents for treating s h i g e l l o s i~~~~~~~~ in adults, but they are not approved for use in children younger than 18 years. other drugs sometimes used to treat diarrhea pose special risks to the infant with shigellosis. the antimotility agents, in addition to their intoxication risk, may pose a special danger in dysentery. in adults, diphenoxylate hydrochloride with atropine has been shown to prolong fever and excretion of the ~rganism.~" the response to appropriate antibiotic therapy is generally gratifying. improvement is often obvious in less than 24 hours. complete resolution of diarrhea may not occur until a week or more after the start of treatment. in those who have severe colitis or those infected by s. dysenteriae serotype 1, the response to treatment is somewhat delayed. for most of the developing world, the best strategy for prevention of shigellosis during infancy is prolonged breastfeeding. specific antibodies in milk appear to prevent symptomatic shigellosis6'68; nonspecific modification of gut flora and the lack of bacterial contamination of human milk also may be important. breast-feeding, even when other foods are consumed, decreases the risk of shigellosis; children who continue to consume human milk into the third year of life are still partially protected from in the united states, the best means of preventing infection in the infant is good hand hygiene when an older sibling or parent develops diarrhea. even in unsanitary environments, secondary spread of shigellae can be dramatically decreased by hand hygiene after defecation and before meals.704 spread of shigellae in the hospital nursery can presumably be prevented by the use of contact isolation for infants with diarrhea and attention to thorough hand hygiene. although nursery personnel have acquired shigellosis from infected newborns,685 further transmission to other infants in the nursery, although is rare. in contrast to salmonella, large outbreaks of nosocomial shigellosis in neonates are rare. unfortunately, good hygiene is a particularly difficult problem in daycare centers. the gathering of susceptible children, breakdown in hand hygiene, failure to use different personnel for food preparation and diaper changing, and difficulty controlling the behavior of toddlers all contribute to daycare-focused outbreaks of shigellosis. immunization strategies have been studied since the turn of the 20th century, but no satisfactory immunization has been developed. even if immunizations are improved, a role in managing neonates seems unlikely. campylobacter was first recognized in an aborted sheep fetus in the early 19o0s7o6 and was named vibrio fetus by smith and taylor in 1919. 707 this organism subsequently was identified as a major venereally transmitted cause of abortion and sterility and as a cause of scours in cattle, sheep, and goats.70s3709 it was not until 1947, when it was isolated from the blood culture of a pregnant woman who subsequently aborted at 6 months' gestation, that the significance of campylobacter as a relatively rare cause of bacteremia and perinatal infections in humans was a~preciated.~l'-~'~ during the 1970s, campylobacter was recognized to be an opportunistic pathogen in debilitated in 1963, v fetus and related organisms were separated from the vibrios (such as v cholerae and v parahaemolyticus) and placed in a new genus, campylobacter (greek word for "curved rod"). 715 since 1973, several campylobacter species have been recognized as a common cause of e n t e r i t i~~l~.~~~ and, in some cases, extraintestinal infections. the genus campylobacter contains 15 species, most of which are recognized as animal and human pathogens. the most commonly considered causes of human disease are campylobacter fetus, campylobacter jejuni, campylobacter coli, campylobacter lari, and campylobacter upsaliensis (table 20 -5),'30-732 although campylobacter mucosalis has been isolated from stool of children with diarrhea.733 dna hybridization studies have shown that these species are distinct, sharing less than 35% dna homology under stringent hybridization ~o n d i t i o n s .~~~,~~~ helicobacter pylori was originally named campylobacter pylori, but because of differences in dna, it was reclassified and is no longer considered in the campylobacter genus. strains of c. fetus are divided into two subspecies: c. fetus subsp. fetus and c. fetus subsp. venerealis. the first subspecies causes sporadic abortion in cattle and sheep7369737; in by far the most common syndrome caused by a campylobacter species is enteritis. c. jejuni and c. coli cause gastroenteritis and generally are referred to collectively as c. jejuni, although dna hybridization studies show them to be different. in the laboratory, c. jejuni can be differentiated from c. coli because it is capable of hydrolyzing hippurate, whereas c. coli is not. most isolates that are associated with diarrhea (61% to 100%) are identified as c. jejuni,751-754 and in some cases, individuals have been shown to be simultaneously infected with c. jejuni and c. ~o l i . ~~~ because of the fastidious nature of c. jejuni, which is difficult to isolate from fecal flora, its widespread occurrence was not recognized until 1973.716-732 previously called related vibrios by this organism had been associated with bloody diarrhea and colitis in infants and adults only when it had been associated with a recognized b a~t e r e m i a .~~~-~~~ in the late 1970s, development of selective fecal culture methods for c. jejuni enabled its recognition worldwide as one of the most common causes of enteritis in persons of all ages. it is an uncommon infection in neonates who generally develop gastroenteritis when i n f e~t e d .~'~-~~~-~~' bacteremia with c. jejuni enteritis also is uncommon.718~759,76l*769*772-778 maternal symptoms considered to be related to c. jejuni infection generally are mild and include fever (75%) and diarrhea (30%). in contrast to the serious disease in newborns that is caused by c. fetus, neonatal infections with although meningitis occurs in rare third trimester infection related to c. fetus or c. jejuni may results in abortion or stillbirth. pathogenesis c. fetus does not produce recognized enterotoxins or cytotoxins and does not appear to be locally invasive by the sereny instead, these infections may be associated with penetration of the organism through a relatively intact intestinal mucosa to the reticuloendothelial system and blo~dstream.~'~ whether this reflects a capacity to resist serum factors or to multiply intracellularly remains to be determined. c. jejuni is capable of producing illness by several mechanisms. these organisms have been shown to produce an lt enterotoxin and a c y t o t o x i r~.~~~~~~~ this enterotoxin is known to be a heat-labile protein with a molecular mass of 60 to 70 mda.7793782 it shares functional and immunologic properties with cholera toxin and e. coli lt. c. jejuni and c. coli also elaborate a cytotoxin that is toxic for a number of mammalian cells.783-785 the toxin is heat labile, trypsin sensitive, and not neutralized by immune sera to shiga toxin or the cytotoxin of clostridium dificile. the role of these toxins as virulence factors in diarrheal disease remains unpr~ved.'~~,~~ several animal models have been tested for use in the study of this pathogen.786 potential models for the study of c. jejuni enteritis include dogs, which may acquire symptomatic infection787; 3-to 8-day old ~h i c k s~~' -~~; chicken embryo cells, which are readily invaded by c. jejuni7"; rhesus monkeys791; and rabbits by means of the removable intestinal tie adult rabbit technique. an established small mammal model that mimics human disease in the absence of previous treatment or surgical procedure has not been successful in adult mice.792 an infant mouse mode1793,794 and a hamster of diarrhea appear promising. c. jejuni is negative in the sereny test for invasivenes~,~~~ and most investigators report no fluid accumulation in ligated rabbit ileal loops. the pathologic findings of c. fetus infection in the perinatal period include placental necrosis7'' and, in the neonate, widespread endothelial proliferation, intravascular fibrin deposition, perivascular inflammation, and hemorrhagic necrosis in the brain. 797 the tendency for intravascular location and hepatosplenomegaly in adults infcctcd with c. fetus has been the pathologic findings in infants and children infected with c. fetus can include an acute inflammatory process in the colon or rectum, as evidenced by the tendency for patients to have bloody diarrhea with numerous fecal leukocytes. 798 there also can be crypt abscess formation and an ulcerative colitis or pseudomembranous colitis-like or a hemorrhagic jejunitis or ileitis.717b725,801,802 mesenteric lymphadenitis, ileocolitis and acute appendicitis also have been described. infection with campylobacter species occurs after ingestion of contaminated food, including unpasteurized milk, poultry, and contaminated water.'26,803-812 m any farm animals and pets, such as chickens,813 dogs,814s815 and cats (especially young animals), are potential sources. the intrafamilial spread of infection in h o~s e h o l d s ,~~~,~~~ the occurrence of outbreaks in and the apparent laboratory acquisition of c. jejuni818 all suggest that c. jejuni infection may occur after person-to-person transmission of the organism. outbreaks of c. jejuni in the child daycare setting are not common. volunteer studies8i9 have shown a variable range in the infecting dose, with many volunteers developing no illness. the report of illness after ingestion of lo6 organisms in a glass of milk728 and production of illness in a single volunteer by 500 organisms8i9 substantiate the variation in individual susceptibility. the potential for low-inoculum disease has significant implications for the importance of strict enteric precautions when infected persons are hospitalized, particularly in maternity and nursery areas. when diarrhea in neonates caused by c. jejuni has been r e p~r t e d ,~~~-~~' maternal-infant transmission during labor has generally been documented.758~763*765~768p770~771 the lior serotyping system, restriction length polymorphism, and pulse-field gel electrophoresis have been used to confirm the identity of the infant and maternal isolates. most mothers gave no history of diarrhea during pregnancy.7623763*765,766 outbreaks have occurred in neonatal intensive care units because of person-to-person spread.8z0 the frequency of asymptomatic carriage of c. jejuni ranges from 0% to 1.3%7'6,717 to as high as 13% to 85%.716,717,732,821-823 in a cohort study in mexico, 66% of all infections related to c. jejuni were asymptomatic.732 infected children, if untreated, can be expected to excrete the organisms for 3 or 4 weeks; however, more than 80% are culture negative after 5 ~e e k s .~~~,~~~a~ ymptomatic excreters pose a significant risk in the neonatal period, in which acquisition from an infected mother can be clinically important.718,760s762,766 c. jejuni has increasingly been recognized as a cause of watery and inflammatory diarrhea in temperate and tropical climates throughout the world. it has been isolated from 2% to 11% of all fecal cultures from patients with diarrheal illnesses in various parts of the world.716-724,728*824-829 there is a tendency for c. jejuni enteritis to occur in the summer in countries with temperate climates. 825 the reservoir of campylobacter is the gastrointestinal tract of domestic and wild birds and animals. it infects sheep, cattle, goats, antelope, swine, chickens, domestic turkeys, and pet dogs. c. fetus often is carried asymptomatically in the intestinal or biliary tracts of sheep and cattle. during the course of a bacteremic illness in pregnant animals, c. fetus organisms, which have a high affinity for placental tissue, invade the uterus and multiply in the immunologically immature fetus. the infected fetuses generally are aborted. whether this organism is acquired by humans from animals or is carried asymptomatically for long periods in humans, who may then transmit the organism through sexual contact as appears to occur in animals, is unclear. it is believed that this subspecies rarely is found in the human intestine and that it is not a cause of human enteriti~?'~ c. fetus infections predominantly occur in older men with a history of farm or animal exposure and in pregnant women in their third trimester.710~71 1,716,717 symptomatically or asymptomatically infected women may have recurrent abortions or premature deliveries and are the source of organisms associated with life-threatening perinatal infections of the fetus or newborn infant.710,739-7483830 in several instances of neonatal sepsis and meningitis, c. fetus was isolated from culture of maternal cervix or vagina.712,747s795 a n osocomial nursery outbreak has been associated with carriage in some healthy infants.83' other outbreaks have been associated with meningitis 832, 833 cervical cultures have remained positive in women who have had recurrent abortions and whose husbands have antibody titer elevations. 738 the most commonly incriminated reservoir of c. jejuni is poultry.808,s12,834,835 m ost chickens in several different geographic locations had a large number (mean, 4 x 106/g) of c. jejuni in the lower intestinal tract or feces. this occurred in some instances despite the use of tetracycline, to which the campylobacter was susceptible in vitro, in the chicken feed.828 the internal cavities of chickens remain positive for carnpylobacter even after they have been cleaned, packaged, and fr0zen.8~~ however, unlike salmonella, c. jejuni organisms that survive usually do not multiply to high concen-tration~?~' domestic puppies or kittens w i t h c. jejuni diarrhea also can provide a source for spread, especially to infants or c. jejuni enteritis also has been associated in a number of outbreaks with consumption of unpasteurized in retrospect, the first reported human cases of c. jejuni enteritis were probably in a milk-borne outbreak reported in 1 946. 842 because campylobacter infections of the udder are not seen, milk is probably contaminated from fecal shedding of the organism. these organisms are killed by adequate heating. fecally contaminated water is a potential vehicle for c. jejuni infections.843 several phenotypic and genotypic methods have been used for distinguishing c. jejuni strains from animals and humans involved in epidemics. 844 clinical manifestations of infection caused by campylobacter depend on the species involved (see table 20 -5). human infections with c. fetus are rare and generally are limited to bacteremia in patients with predisposing condition^^^^.^^^ or to bacteremia or uterine infections with prolonged fever and pneumonitis that lasts for several weeks in women during the third trimester of pregnancy. unless appropriately treated, symptoms usually resolve only after abortion or delivery of an infected infant?10j12,739-748*750 these infected neonates, who are often premature, develop signs suggesting sepsis, including fever, cough, respiratory distress, vomiting, diarrhea, cyanosis, convulsions, and jaundice. the condition typically progresses to meningitis, which may be rapidly fatal or may result in serious neurologic ~equelae.~" additional systemic manifestations include pericarditis, pneumonia, peritonitis, salpingitis, septic arthritis, and abscesses.823 c. jejuni infection typically involves the gastrointestinal tract, producing watery diarrhea or a dysentery-like illness with fever and abdominal pain and stools that contain blood and ~u c u s .~~~~~~'~~' " ' older infants and children generally are affected, but neonates with diarrhea have been reported. infection in neonates generally is not clinically apparent or is mild. stools can contain blood, mucus, and pus712p721*762*763; fever often is ab~ent.7~~"~' the illness usually responds to appropriate antimicrobial which shortens the period of fecal shedding.845 extraintestinal infections related to c. jejuni other than bacteremia are rare but include cholecystitis,846 urinary tract and meningitis.761 bacteremia is a complication of gastrointestinal infe~tion,8~' especially in malnourished children. 849 meningitis that appears to occur secondary to intestinal infection also has been reported in premature infants who have had intraventricular needle aspirations for neonatal hydrocephalus.'i2 complications in older children and adults that have been associated with c. jejuni enteritis include reiter's syndrome, 850 guillain-barre ~y n d r o m e , 8~~~~~* and reactive persistent c. jejuni infections have been described in patients infected with human immunodeficiency extraintestinal manifestations generally occur in patients who are immunosuppressed or at the extremes of age.'14 campybbacter zari has caused chronic diarrhea and bacteremia in a neonate!% most important in the diagnosis of campylobacter infection is a high index of suspicion based on clinical grounds. c. fetus and c. jejuni are fastidious and may be overlooked on routine fecal cultures. isolation of campylobacter from blood or other sterile body sites does not represent the same problem as isolation from stool. growth occurs with standard blood culture media, but it may be slow. in the case of c. fetus infecting the bloodstream or central nervous system, blood culture flasks should be blindly subcultured and held for at least 7 days or the organism may not be detected because of slow or inapparent the diagnosis of c. fetus infection should be considered when there is an unexplained febrile illness in the third trimester of pregnancy or in the event of recurrent abortion, prematurity, or neonatal sepsis with or without meningitis. a high index of suspicion and prompt, appropriate antimicrobial therapy may prevent the potentially serious neonatal complications that may follow maternal c. fetus infection. campylobacter is distinguished from the vibrio organisms by its characteristics of carbohydrate nonfermentation and by its different nucleotide base omp position.^^^^^^^-^^^*^^^ campylobacter is 0.2 to 0.5 fm wide and 0.5 to 8.0 long. it is a fastidious, microaerophilic, curved, motile gram-negative bacillus that has a single polar flagellum and is oxidase and catalase positive, except for c. upsaliensis, which is generally catalase negative or weakly positive. c. jejuni and c. fetus are separated by growth temperature (c. fetus grows best at 25' c but can be cultured at 37' c; c. jejuni grows best at 42' c) and by nalidixic acid and cephalosporin susceptibilities, because c. jejuni is susceptible to nalidixic acid and resistant to cephalosporins. c. jejuni grows best in a microaerobic environment of 5% oxygen and 10% carbon dioxide at 42' c. it grows on a variety of media, including brucella and mueller-hinton agars, but optimal isolation requires the addition of selective and nutritional supplements. growth at 42' c in the presence of cephalosporins is used to culture selectively for c. jejuni from fecal specimens. in a study of six media, charcoal-based selective media and a modified charcoal cefoperazone deoxycholate agar were the most selective for identification of campylobacter species. extending the incubation time from 48 to 72 hours led to an increase in the isolation rate regardless of the medium its typical darting motility may provide a clue to identification, even in fresh fecal specimens, when viewed by phase-contrast microscopy. 72 when the organism has been cultured, it is presumptively identified by motility and by its curved, sometimes sea gulllike appearance on carbolfuchsin stain. polymorphonuclear leukocytes are usually found in stools when bloody diarrhea occurs and indicate the occurrence of ~o l i t i s .~~~*~~* to avoid potentially serious c. jejuni infection in the newborn infant, careful histories of any diarrheal illnesses in the family should be obtained, and pregnant women with any enteric illness should have cultures for this and other enteric pathogens. detection of c. jejuni and c. coli by pcr has been reported859 and in the future may be useful for the rapid and reliable identification of this organism. the differential diagnosis of c, fetus infections include the numerous agents that cause neonatal sepsis or meningitis, especially gram-negative bacilli. diagnostic considerations for inflammatory or bloody enteritis include necrotizing enterocolitis, allergic proctitis, and salmonella; rarely shigellu, and other infectious agents occur. agglutination, complement fixation, bactericidal, immunofluorescence, and elisa tests have been used for serologic diagnosis of c. jejuni infection and to study the immune response, but these assays are of limited value in establishing the diagnosis during an acute infection.731 the prognosis is grave in newborn infants with sepsis or meningitis caused by c. fetus. in infants with c. jejuni gastroenteritis, limited data suggest that appropriate, early antimicrobial therapy results in improvement and rapid clearance of the organism from stool.845 campylobacter species are often resistant to p-lactams, including ampicillin and cephalosporins.860v861 mo st strains are susceptible to erythromycin, gentamicin, tetracycline, chloramphenicol, and the newer quinolones, although resistance to these agents has been r e p~r t e d .~~' ,~~~ it appears that a parenteral aminoglycoside is the drug of choice for c. fetus infections, pending in vitro susceptibility studies. in the case of central nervous system involvement, cefotaxime and chloramphenicol are potential alternative drugs. depending on in vitro susceptibilities, which vary somewhat with locale, erythromycin is the drug of choice for treating c. jejuni e n t e r i t i~.~~~~~"~~' ' if erythromycin therapy is initiated within the first 4 days of illness, a reduction in excretion of the organism and resolution of symptoms occur.845 although data regarding treatment of asymptomatic or convalescent carriers are not available, it seems appropriate to treat colonized pregnant women in the third trimester of pregnancy when there is a risk of perinatal or neonatal infection. the failure of prophylactic parenteral gentamicin in a premature infant has been documented, followed by successful resolution of symptoms and fecal shedding with erythromycin. because there appears to be an increased risk of toxicity with erythromycin estolate during pregnancy and other forms of erythromycin should probably be used in these settings. azithromycin appears to be effective if the organism is susceptible.865 strains that are erythromycin resistant often are resistant to azithromycin.866 cumpylobucter tends to have higher minimal inhibitory concentrations for clarithromycin than for a~ithromycin.~~~ furazolidone has been used in children and ciprofloxacin in nonpregnant patients older than 17 years. contact precautions should be employed during any acute diarrheal illness and until the diarrhea has subsided. hand hygiene after handling raw poultry and washing cutting boards and utensils with soap and water after contact with raw poultry may decrease risk of infection. pasteurization of milk and chlorination of water are critically important. infected food handlers and hospital employees who are asymptomatic pose no known hazard for disease transmission if proper personal hygiene measures are maintained. ingestion of human milk that contains anti<. jejuni antibodies has been shown to protect infants from diarrhea due to c. j e j~n i .~~,~~~ c. dificile is a spore-forming, gram-positive, anaerobic bacillus that produces two toxins. in the presence of antibiotic pressure, c. dificile colonic overgrowth and toxin production occur. the virulence properties of c. dificile are related to production of an enterotoxin that causes fluid secretion (toxin a) and a cytotoxin detectable by its cytopathic effects in tissue culture (toxin b).869*870 both the usual manifestations of c. dificile disease in older children and adults include watery d&rhea, abdominal pain and tenderness, nausea, vomiting, and low-grade fever. grossly bloody diarrhea is unusual, although occult fecal blood is common. leukocytosis is present during severe illness. diarrhea usually begins 4 to 9 days into a course of antimicrobial therapy but may be delayed until several weeks after completion of the therapeutic course. usually, the illness is mild and self-limited if the offending drug is discontinued. severe colitis with pseudomembranes is less common now than in previous years because the risk of diarrhea developing during antimicrobial therapy is recognized and the antimicrobial agent typically is stopped. it is unclear whether this organism causes disease in newborns. one study from a newborn intensive care unit suggests that toxin a in stools is associated with an increased frequency of abnormal stools.893 endoscopic findings of pseudomembranes and hyperemic, friable rectal mucosa suggest the diagnosis of pseudomembranous colitis. pseudomembranes are not always present in c. dificile colitis; mild cases are often described as nonspecific colitis. several noninvasive techniques are used to establish the diagnosis, including enzyme immunoassay (eia) for toxin detection and pcr.893-896 isolation of c. dificile from stool does not distinguish between toxigenic and nontoxigenic isolates. if c. dificile is isolated, testing for toxin by cell culture or eia should be performed to confirm the presence of a toxigenic strain. there are multiple commercially available eias that detect either toxin a or both toxins a and b.893-895 these assays are sensitive and easy to perform. other assays are available for epidemiologic investigation of outbreaks of disease due to c. d i f i~i l e .~~~ in older children and adults, the diagnosis is confirmed by culture of c. dificile and demonstration of toxin in feces. in neonates, these data are inadequate to prove that an illness is related to c. dificile. when the clinical picture is consistent, the stool studies are positive for c. dificile and no other cause for illness is found, a diagnosis of "possible" c. dificile is made. a favorable response to eradication of c. dificile is supportive evidence that the diagnosis is c0rrect.8~~ because of the uncertainty implicit in the ambiguity of neonatal diagnostic criteria, other diagnoses must be considered. when the decision is made that a neonate's illness might be related to c. difjcile, the initial approach should include fluid and electrolyte therapy and discontinuation of the offending antimicrobial agent. if the illness persists or worsens or if the patient has severe diarrhea, specific therapy with r n e t r o n i d a z~l e~~~*~~~ should be instituted. metronidazole is considered to be the treatment of choice for most patients with c. difjcile ~olitis.8~' rarely is there a need to consider orally administered vancomycin or bacitracin in after initiation of therapy, signs of illness generally resolve within several days, titers decrease, and fecal toxins disappear eventually. recurrence of colitis after discontinuation of metronidazole or vancomycin has been documented in 10% to 20% of adults.g01 relapses are treated with a second course of metronidazole or vancomycin. drugs that decrease intestinal motility should not be administered. neutralizing antibody against c. dificile otoxin has been demonstrated in human colostrum.' secretory component of siga binds to toxin a to inhibit its binding to receptors 903 data show that there are nonantibody factors present in milk that interfere with the action of toxin b in addition to secretory iga directed at toxin a. 904 breast-feeding appears to decrease the frequency of colonization by c. d i f j~i l e .~'~ in addition to standard precautions, contact precautions are recommended for the duration of illness. meticulous hand hygiene techniques, proper handling of contaminated waste and fomites, and limiting the use of antimicrobial agents are the best available methods for control of c. dificile infection. b! cholerue is a gram-negative, curved bacillus with a polar flagellum. of the many serotypes, only enterotoxin-producing organisms of serotype 01 and 0139 cause epidemics. b! cholerue 01 is divided into two serotypes, inaba and ogawa, and two biotypes, classic and el tor; the latter is the predominant biotype. nontoxigenic 0 1 strains and non-01 strains of v cholerae can cause diarrhea and sepsis but do not cause outbreaks?06~90a pathogenesis b! cholerue 0 group 1 is the classic example of an enteropathogen whose virulence is caused by enterotoxin production. cholera toxin is an 84-mda protein whose five b subunits cause toxin binding to the enterocyte membrane ganglioside gm, and whose a subunit causes adenosine diphosphate ribosylation of a guanosine triphosphate-binding regulatory subunit of adenylate cy~lase."~~~'~ the elevated camp levels that result from stimulation of enterocytes by cholera toxin cause secretion of salt and water with concomitant inhibition of absorption. two other toxins are also encoded within the virulence cassette that encodes cholera toxin. these toxins, zona occludens toxin (zot) and accessory cholera toxin (ace), are consistently found in illness-causing strains of 0 1 and 0139 but not usually in v; cholerae organisms that are less virulent. since 1960, v cholerae 01, biotype el tor, has spread from india and southeast asia to africa, the middle east, southern europe, and the southern, western, and central pacific islands in the aquatic environment. the usual reported vehicles of transmission have included contaminated water or ice; contaminated food, particularly raw or undercooked shellfish; moist grains held at ambient temperature; and raw or partially dried fish. the usual mode of infection is ingestion of contaminated food or water. boiling water or treating it with chlorine or iodine and adequate cooking of food kill the organism.907 asymptomatic infection of family contacts is common but direct person-to-person transmission of disease has not been documented. persons with low gastric acidity are at increased risk for cholera infection. cholera acquired during pregnancy, particularly in the third trimester, is associated with a high incidence of fetal miscarriage can be attributed to a fetal acidosis and hypoxemia resulting from the marked metabolic and circulatory changes that this disease induces in the mother. it is not surprising that the likelihood of delivering a stillborn child is closely correlated with the severity of the maternal illness. the inability to culture v; cholerue from stillborn infants of infected mothers, together with the usual absence of bacteremia in cholera, suggests that transplacental fetal infection is not a cause of intrauterine death. neonatal cholera is a rare disease. this generalization also applies to the new 0139 strains, although mild9'* and severe forms of illness have rarely been described in newborns. 919 among 242 neonates admitted to a cholera research hospital in dacca, bangladesh, there were 25 infants ill with ~holera.~" even infants born to mothers with active diarrheal disease may escape infection, despite evidence that rice-water stools, almost certain to be ingested during the birth process, may contain as many as lo9 organism~/ml.~~~ the reason for this apparently low attack rate among newborns is not certain; however, it probably can be attributed in large part to the protection conferred by breast-feedingg2' human milk contains antibodies62 and receptor-like glycoprotein that inhibit adherence of v choleraeu and gangliosides that bind cholera toxin.65 the role of transplacentally acquired vibriocidal maternal antibodies has not been determined.922 because v cholerae causes neither bacteremia nor intestinal invasion, protection against illness is more likely to be a function of mucosal rather than serum additional factors that may reduce the incidence of neonatal cholera include the large inoculum required for infection925 and the limited exposure of the newborn to the contaminated food and water.229 clinicians should request that appropriate cultures be performed for stool specimens from persons suspected of having cholera. the specimen is plated on thiosulfate citrate bile salts sucrose agar directly or after enrichment in alkaline peptone water. isolates of v cholerae should be confirmed at a state health department and then sent to the cdc for testing for production of cholera toxin. a fourfold rise in vibriocidal antibody titers between acute and convalescent serum samples or a fourfold decline in titers between early and late (>2 months) convalescent serum specimens can confirm the diagnosis. probes have been developed to test for cholera toxin.926*927 the most important modality of therapy is administration of oral or parented rehydration therapy to correct dehydration and electrolyte imbalance and maintain h y d r a t i~n .~~ antimicrobial therapy can eradicate vibrios, reduce the duration of diarrhea, and reduce requirements for fluid replacement. one cholera vaccine, which is administered parenterally, is licensed in the united states but is of very limited value. several experimental oral vaccines are being t e~t e d ? *~-~~' i.: enterocolitica is a major cause of enteritis in much of the industrialized enteritis due to this organism primarily occurs in infants and young children, and infections in the united states are reported to be more common in the north than in the s o~t h .~~~-~~~ h i m als, especially swine, have been shown to serve as the reservoir for y. enterocolitica. a history of recent exposure to chitterlings (i.e., pig intestine) is common. transmission has also occurred after ingestion of contaminated milk and infusion of contaminated blood p r o d~c t s .~~~,~~~ virulence of y. enterocolitica is related primarily to a virulence plasmid, which is closely related to the virulence plasmids of yersinia pseudotuberculosis and yersinia p e s t i~.~~'~~~~ an st enterotoxin, which is closely related to the st of etec,943 may also be important. infection with y. enterocolitica is recognized as one of the causes of bacterial gastroenteritis in young children, but knowledge of neonatal infection with this organism is fragmentary. even in large series, isolation of yersinia from newborns is rare?313932, 944 the youngest infants whose clinical course has been described in detail were 11 days to several months old at the onset of their illness.932,944-952 there were no features of the gastroenteritis to distinguish it from that caused by other invasive enteric pathogens such as shigella or salmonella. infants presented with watery diarrhea or with stools containing mucus with streaks of blood. sepsis was common in these infants particularly in the first 3 months of life when 28% of enteritis was complicated by sepsis.948,949,953,954 fever is not a consistent finding in children with bacteremia, and meningitis is rare. in older children, fever and right lower quadrant pain mimicking appendicitis are often found.940 diagnosis y enterocolitica can be recovered from throat swabs, mesenteric lymph nodes, peritoneal fluid, blood, and stool. because laboratory identification of organisms from stool requires special techniques, laboratory personnel should be notified when yersinia is suspected. because avirulent environmental isolates occur, biotyping and serotyping are useful in assessing the clinical relevance of isolates. pcr has been used to detect pathogenic strain^.^^^'^^^ the effect of antimicrobial therapy on the outcome of gastrointestinal infection is uncertain. it has been recommended that antibiotics be reserved for sepsis or prolonged and severe gastroenteritisg3'; however, there are no prospective studies comparing the efficacy of various antimicrobial agents with each other or with supportive therapy alone. most strains of y. enterocolitica are susceptible to trimethoprimsulfamethoxazole, the aminoglycosides, piperacillin, imipenem, third-generation cephalosporins, amoxicillin-clavulanate potassium, and chloramphenicol, and resistant to amoxicillin, ampicillin, carbenicillin, ticarcillin, and m a c r o l i d e~.~~~-~~~ therapy in individual cases should be guided by in vitro susceptibility testing, although cefotaxime has been successfully used in bacteremic infants?54 aerornonas hydrophila is widely distributed in animals and the environment. although wound infection, pneumonia, and sepsis (especially in immunocompromised hosts) represent typical aeromonas infections, gastroenteritis increasingly is being recognized. the organism is a gramnegative, oxidase-positive, facultatively anaerobic bacillus belonging to the family vibrionaceae. like other members of this family, it produces an enter~toxin~~' that causes fluid secretion in rabbit ileal loops.%' some strains cause fluid accumulation in the suckling mouse model,"' whereas other strains are i n~a s i v e~~~ or cytotoxic. 964 the enterotoxin is not immunologically related to cholera toxin or the heat lt of although volunteer studies and studies with monkeys have failed to provide supportive evidence for enteropathog e n i~i t y ?~~,~~ there is good reason to believe that a. hydrophila does cause diarrhea in children. the earliest description of aeromonas causing diarrhea was an outbreak that occurred in a neonatal unit. 968 although several studies have failed to show an association with diarrhea,%9-974 most studies have found more aeromonas isolates among children with gastroenteritis than among ~o n t r o l s ?~~-~~~ part of the controversy may be caused by strain differences; some strains possess virulence traits related to production of gastroenteritis, whereas others do not. 970, 977 the diarrhea described in children is a disease of summer, primarily affecting children in the first 2 years of life. in one study, 7 (13%) of 55 cases of aeromonas detected during a 20-month period occurred in infants younger than 1 month. typically, watery diarrhea with no fever has been described; although there are descriptions of watery diarrhea with fever?78 however, in 22%, a dysentery-like illness occurred. dysentery-like illness has been described in the neonate. 979 in one third of children, diarrhea has been reported to last for more than 2 ~eeks.9~' there may be species-related differences in clinical features of aeromonas-associated gastroenteritis in children. 980 organisms that were formerly classified as a. hydrophila are now sometimes labeled as aeromonas sobria or aeromonas ~a v i a e .~~'~~~' fever and abdominal pain appear to be particularly common with a. sobria. one series of a. hydrophila isolates from newborns in dallas showed more blood cultures than stool cultures positive for a e r o m o n a~.~~~ diagnosis of enteric infection associated with aeromonas often is not made because this organism is not routinely sought in stool cultures. when the organism is suspected, the laboratory should be notified so that oxidase testing can be performed. the organism is usually susceptible to aztreonam, imipenem, meropenem, third-generation cephalosporins, trimethoprim-sulfamethoxazole, and chloramphenicol.q84~986 plesiomonas shigelloides is a gram-negative, facultative anaerobic bacillus that, like aeromonas, is a member of the vibrionaceae family. it is widely disseminated in the environment; outbreaks of disease are usually related to ingestion of contaminated water or seafood. 987 although it has been associated with outbreaks of diarrheal disease988 and has been found more commonly in ill than well controls, the role of i? shigelloides in diarrheal disease has remained contro~ersial.~~~ if it is a true enteropathogen, the mechanism by which it causes disease is ~n c l e a r . 9~~'~~' the role of this organism in neonatal diarrhea has not been extensively investigated. infections of neonates have been r e p~r t e d ?~' .~~~ but most cases of enteric disease currently reported in the united states are in adults.987 typical illness consists of watery diarrhea and cramps; sometimes, fever, bloody stools, and emesis occur and last for 3 to 42 days. diagnosis is not usually made by clinical microbiology laboratory testing because, as with aeromonas, coliforms can be confused with l ? shigelloides unless an oxidase test is performed. 996 the true frequency of infection is unknown. the organism has antibiotic susceptibilities similar to those of a e r~m o n a s .~~~'~~~ proving that an organism causes diarrhea is difficult, particularly when it may be present in large numbers in stools of healthy persons. bacteria that have been associated with acute gastroenteritis may be considered causative when the following criteria are met: 1. a single specific strain of the organism should be found as the predominant organism in most affected infants by different investigators in outbreaks of enteric disease in different communities. 2. this strain should be isolated in a significantly lower percentage and in smaller numbers from stool specimens of healthy infants. 3. available methods must be used to exclude other recognized enteropathogens, including viruses and parasites, enterotoxigenic agents, and fastidious organisms such as ca mpylobacter. 4. demonstration of effective specific antimicrobial therapy and specific antibody responses and, ultimately, production of experimental disease in volunteers are helpful in establishing the identity of a microorganism as a pathogen. optimally, the putative pathogen should have virulence traits that can be demonstrated in model systems. most bacteria that have been suggested as occasional causes of gastroenteritis in neonates fail to fulfill one or more of these criteria. their role in the cause of diarrheal disease is questionable. this is particularly true of microorganisms described in early reports in which the possibility of infection with more recently recognized agents could not be excluded. much of the clinical, bacteriologic, and epidemiologic data collected earlier linking unusual enteropathogens to infantile diarrhea must be reevaluated in light of current knowledge and methodology. several reports of acute gastroenteritis believed to have been caused by klebsiella suggest that, rather than playing an etiologic role, these organisms had probably proliferated within an already inflamed b~w e l .~~~-' '~' the recovery of klebsiella-enterobacter in pure culture from diarrheal stools has led several investigators to suggest that these bacteria may occasionally play a causative role in infantile gastroenteritis and enterocoliti~.'~~~~'"~ ingestion of infant formula contaminated with enterobacter sakazakii has been associated with development of bloody diarrhea and sepsis.'oo8 however, klebsiella species also may be isolated in pure culture from stools of newborns with no enteric s y m p t o m~. '~~~~'~' ' in one study, certain capsular types of klebsiella were more often isolated from infants with diarrheal disease than from normal infants.ioo2 later work has shown that klebsiella pneumoniae, enterobacter cloacae, and citrobacter species are capable of isolation of citrobacter species, such as those of klebsiella species, describe associations with enteric illnesses in up to 7% of cases.1014-1016 there is inadequate evidence to define the roles of klebsiella, enterobacter, and citrobacter species as etiologic agents of enteric illnesses. listeria monocytogenes, one of the classic causes of neonatal sepsis and meningitis (see chapter 14), has been linked to outbreaks of febrile diarrheal disease in immunocompetent adults and ~h i l d r e n . '~'~~'~~' seventy-two percent of ill individuals have had fever.ioz2 outbreaks have been related to ingestion of contaminated foods. listeria has rarely been described as a cause of neonatal gastroenteriti~.'~~~-~~~~ infection with enterotoxin-producing bacteroides fragilis has been associated with mild watery diarrhea.'027 these infections have a peak incidence in 2-to 3-year-old infants.1028 these toxin-producing organisms cannot be detected in routine hospital laboratories. a variety of organisms has been isolated from infant stools during episodes of diarrhea. most of these reports have failed to associate illness with specific organisms in a way that has stood the test of time. for example, i? aeruginosa 1029-1034 and have been associated with diarrhea, but proteus 1017, [1035] [1036] [1037] [1038] [1039] [1040] [1041] there are few convincing data suggesting that either is a true enteropathogen. these organisms generally are recovered as frequently from healthy infants as from infants with diarrheal disease, suggesting that their presence in stool cultures is significant.273,1042-1046 an association between providencia and neonatal enteritis has been substantiated largely by anecdotal reports of nursery outbreaks.215.27131016~1@'7 these bacteria are rarely isolated from infants with sporadic or community-acquired diarrheal disease.'042~'0m"04b~1050 candida albicans usually is acquired during passage through the birth canal and is considered a normal, although minor, component of the fecal flora of the neonate (see chapter 33).'05' intestinal overgrowth of these organisms frequently accompanies infantile gastroenteriti~,2"~~~~~'~~~*'~~~ particularly after antimicrobial therapy.233,247,'0521055 the upper small gut may become colonized with candida in malnourished children with diarrhea1056; whether the presence of the organism is cause or effect is unclear. stool cultures obtained from infants with diarrheal disease are therefore inconclusive, and although candida enteritis has been reported in adults,'057 the importance of this organism as a primary cause of neonatal gastroenteritis has been difficult to prove. clinical descriptions of nursery epidemics of candidal enteritis are poorly documented, generally preceding the recognition of epec and rotaviruses as a cause of neonatal diarrhea. even well studied cases of intestinal involvement add little in the way of substantive proof because secondary invasion of candida has been shown to be a complication of coliform enteritis.21 1,2333247 producing e n~e r o~o~n s~1 3 9~~~~1 6 7~1 7 5~1 9 3 ' " 0 1 2~1 0 1 3 reports of although diarrhea has sometimes been described as a finding in neonatal disseminated candidiasis, more typically, gastrointestinal tract involvement with disseminated candida is associated with abdominal distention and bloody stools mimicking necrotizing enterocoliti~.~~~"~~~-~~~~ typically, affected infants are premature and have courses complicated by antibiotic administration, intravascular catheter use, and surgical procedures during the first several weeks of life. a trial of oral anticandidal therapy may be helpful in neonates suffering from diarrhea in the presence of oral or cutaneous candidiasis. if the therapy is appropriate, a response should be forthcoming within 2 to 5 days. diarrhea sometimes occurs as a manifestation of systemic infection. patients with staphylococcal toxic shock syndrome, for example, often have diarrhea. loose stools sometimes occur in sepsis, but it is unclear whether the diarrhea is a cause or an effect. the organisms isolated from blood cultures in a group of bangladeshi infants and children with diarrhea included staphylococcus aureus, haemophilus inpuenzae, streptococcus pneumoniae, r aeruginosa, and various gramnegative enteric bacilli.'062 it is unknown whether the bacteriology of sepsis associated with diarrhea is similar in the well-nourished infants seen in industrialized countries. acute diarrhea associated with intestinal parasites is infrequent during the neonatal period. in areas with high endemicity, infection of the newborn is likely to be associated with inadequate maternal and delivery care, insufficient environmental sanitation, and poor personal hygiene standards. the occurrence of symptomatic intestinal parasitic infection during the first month of life requires acquisition of the parasite during the first days or weeks; the incubation period for e. histolytica and g. larnblia is 1 to 4 weeks, and for cryptosporidium parvum, it is 7 to 14 days. the newborn can be infected during delivery by contact with maternal feces,1o63 in the hospital through contact with the mother or personnel, or in the household through contact with infected individuals in close contact with the child. contaminated water can be an important source of infection for g. lamblia and c. parvum. organisms formerly identified as e. histolytica have been reclassified into two species that are morphologically identical but genetically distinct: e. histolytica and e. dispar. the former can cause acute nonbloody and bloody diarrhea, necrotizing enterocolitis, ameboma, and liver abscess, and the latter is a noninvasive parasite that does not cause disease. early acquisition of disease tends to be more severe in young infants; rarely, amebic liver abscess and rapidly fatal colitis have been reported in infant^."^^-'^^^ for example, a 19-dayold child from india who presented with 10 to 12 episodes of watery and mucous diarrhea, lethargy, jaundice, and mildly elevated liver enzymes has been described; the child recovered completely after 10 days of intravenous o m i d a~o l e . '~~~ however, asymptomatic colonization of neonates with various species of ameba is common in areas of high endemi~ity."~~ diagnosis can be established by stool examination for cysts and trophozoites and by serologic studies.'072 through the use of pcr, isoenzyme analysis, and antigen detection assays, e. histolytica and e. dispar can be differentiated.10733'074 serum antibody assays may be helpful in establishing the diagnosis of amebic dysentery and extraintestinal amebiasis with liver involvement. the efficacy of treatment with metronidazole for colitis or liver abscess has not been established for the newborn period, although this therapy has been used with success.1o65 patients with colitis or liver abscess caused by e. histolytica are treated also with iodoquinol, as are asymptomatic carriers. g. lamblia is a binucleate, flagellated protozoan parasite with trophozoite and cyst stages. it is spread by the fecal-oral route through ingestion of cysts. child-care center outbreaks reflecting person-to-person spread have demonstrated high i n f e~t i v i t y . "~~~'~~~ foodborne transmission and waterborne transmission also occur. infection is often asymptomatic or mildly symptomatic; cases of severe symptomatic infection during the immediate newborn period have not been reported. symptoms in giardiasis are related to the age of the patient, with diarrhea, vomiting, anorexia, and failure to thrive typical in the youngest children. seroprevalence studies have demonstrated evidence of past or current infection in 40% of peruvian children by the age of 6 months.io8' in a study of lactating bangladeshi mothers and their infants, 82% of women and 42% of infants excreted giardia once during the study; in some infants, this occurred before they were 3 months old.'o82 of these infected infants, 86% had diarrhea, suggesting that the early exposure to the parasite resulted in disease. in a prospective study of diarrhea conducted in mexico, infants frequently were infected with giardia from birth to 2 months, with a crude incidence rate of first giardia infection of 1.4 infections per child-year in this age group.6 the symptom status of these children was not reported but this study strongly suggests that g. lamblia may be more common than currently recognized among newborns living in developing areas. the diagnosis of giardiasis can be made on the basis of demonstration of antigen by eia or by microscopy of feces, duodenal fluid or, less frequently, duodenal b i~p~y . ' '~~~' '~' breast-feeding is believed to protect against symptomatic g i a r d i a~i s .~-~~* '~~~ this protection may be mediated by cellular and humoral i1nrnunity6~~"~~*"~~ and nonspecifically by the antigiardial effects of unsaturated fatty acids.iob6 giardia infections causing severe diarrhea may respond to metronidazole or furazolidone.'080 c. parvum is a coccidian protozoon related to toxoplasma gondii, lsospora belli, and plasmodium species. 1087s'088 the life cycle involves ingestion of thick-walled oocysts; release of sporozoites, which penetrate intestinal epithelium; and development of merozoites. there is asexual and sexual reproduction, with the latter resulting in formation of new oocysts that can be passed in stools. cryptosporidium species are ubiquitous. infection often occurs in persons traveling to endemic areas.'o89 because cryptosporidium infects a wide variety of animal species, there is often a history of animal contact among infected individ~als."~' person-to-person spread, particularly in household c~n t a c t s '~~'~' '~~ and daycare center^,"^^"'^^ is well documented and suggests that the organism is highly infectious. waterborne outbreaks of cryptosporidiosis occur and can be of massive proportion^."^^ the clinical manifestations of illness in immunocompetent persons resemble those of giardia infection but are somewhat shorter in d~ration'"~; asymptomatic carriage is rare. symptoms and signs include watery diarrhea, abdominal pain, myalgia, fever, and weight loss.1089*1090~1095~'096"098~'09 infection in the first month of life has been described.""~"" because symptoms resolve before excretion of oocysts ceases, a newborn whose mother has been ill with cryptosporidiosis in the month before delivery might be at risk even if the mother is asymptomatic at the time of the child's birth."'* with the increasing frequency of human immunodeficiency virus infection, it is likely that women with symptomatic cryptosporidiosis occasionally will deliver an infant who will become infected. infants infected early in life may develop chronic diarrhea and maln~trition."'~ the diagnosis of cryptosporidiosis is most typically made by examination of fecal smears using the giemsa stain, ziehl-neelsen stain, auramine-rhodamine stain, sheather's sugar flotation, an immunofluorescence procedure, a modified concentration-sugar flotation method, or an eia.' io4,1 lo5 nitazoxanide is effective therapy of immunocompetent adults and children ill with cryptosporidiosis.'io6 because illness is usually self-limited in the normal host, attention to fluid, electrolyte, and nutritional status usually suffices. enteric isolation of hospitalized infants with this illness is appropriate because of the high infectivity. several studies suggest that the risk of infection early in life may be decreased by breast-feeding. ' '"j viruses that infect the intestinal mucosa and cause primarily gastroenteritis are referred to as enteric viruses; they should not be confused with enteroviruses, members of picornaviridae family that are associated primarily with systemic illnesses. enteric viruses include rotaviruses, enteric adenoviruses, human caliciviruses, and astroviruses. other viruses such as coronaviruses, breda viruses, pestiviruses, parvoviruses, toroviruses, and picobirnaviruses have been sporadically associated with acute diarrhea but are currently considered of uncertain relevance. extensive reviews on the role of enteric viruses in childhood diarrhea can be found elsewhere."08-"" all four enteric viruses could conceivably infect the newborn, but the extent of exposure and clinical manifestations are largely unknown for astrovirus, enteric adenovirus, and human caliciviruses. rotavirus is the most extensively studied enteric virus. neonatal rotavirus infections have similar virologic and clinical characteristics to infection in older children, although some differences exist. rotavirus is a 75-nm, nonenveloped virus composed of three concentric protein shells: a segmented genome (1 1 segments), an rna-dependent polymerase, and enzymes required for messenger rna synthesis are located within the inner core. each segment codes for at least one viral protein (vp). the vp can be part of the structure of the virus, or it may be a nonstructural protein (nsp) required for replication, viral assembly, budding, determination of host range, or viral pathogenesis."" six distinct rotavirus groups (a through f) have been identified serologically based on common group of which three (a, b, and c) have been identified in humans.'io8 because group a rotaviruses represent more than 95% of isolated strains in humans worldwide, further discussion focuses on this group. group a rotaviruses are subclassified into serotypes based on neutralization epitopes located on the outer capsid. both rotavirus surface proteins, vp4 and vp7, can induce production of neutralizing antibodies.111431115 at least 10 vp7 types (g serotypes: gi to g6, g8 to g10, and g12) and nine vp4 types (p serotypes: pia, plb, p2a, p3, p3b, p4, p5, p8, and p12) have been detected among human r o t a v i r u~e s .~~~~-~~~~ by sequencing the vp4-coding gene, eight genomic p types (genotypes) have been identified that correspond to one or more of the described p antigenic types (genotype 8 to antigenic type pla, 4 to plb, 6 to p2a, 9 to p3,13 to p3b, 10 to p4,3 to p5, and 11 to p8)."" combining g antigenic with p antigenic and genetic typing, a specific rotavirus strain can be identified p antigenic type (p genetic type), g type. as an example, the human neonatal m37 strain is described as p2a[6], gi. from newborn nurseries, some of which seem to be endemic to the newborn units with high rates of asymptomatic infe~tion,"~~-"~' and less commonly, outbreaks of symptomatic infection.iiz8 these findings suggest that specific conditions of the newborn environment (e.g., child, nursery, personnel) may increase the possibility of reassortments between human strains; such strains may persist in these settings possibly through constant transmission involving asymptomatic newborns, adults, and contaminated surfaces. rotavirus primarily infects mature enterocytes located in the mid and upper villous e p i t h e l i~m .~~~~-"~~ lactase, which is present only on the brush border of the differentiated epithelial cells at these sites, may act as a combined receptor and uncoating enzyme for the virus, permitting transfer of the particles into the cell.1137 perhaps for this reason, infection is limited to the mature columnar enterocytes; crypt cells and crypt-derived cuboidal cells, which lack a brush border, appear to be resistant to rotaviral i n f e~t i o n . "~~' "~~ this concept also may explain why rotavirus infection is less common in infants younger than 32 weeks' gestational age than in more mature infants1139; between 26 and 34 weeks' gestational age, lactase activity is approximately 30% of that found in term infants. 1140 the upper small intestine is most commonly involved, although lesions may extend to the distal ileum and rarely to the ~0 l 0 n .~~~~7~~~~ interaction between intestinal cell and rotavirus structural and nonstructural proteins occurs, resulting in death of infected villous enter~cytes."~~ once infected, the villous enterocyte is sloughed, resulting in an altered mucosal architecture that becomes stunted and flattened. the gross appearance of the bowel is usually normal; however, under the dissecting microscope, scattered focal lesions of the mucosal surface are apparent in most cases. light microscopy also shows patchy changes in villous morphology, compatible with a process of infection, inflammation, and accelerated mucosal renewal. the villi take on a shortened and blunt appearance as tall columnar cells are shed and replaced by less mature cuboidal entero-ischemia may also play a role in the loss and stunting of villi"45 and activation of the enteric nervous system; active secretion of fluid and electrolytes may be another pathogenic mechanism.1146 during the recovery phase, the enteroblastic cells mature and reconstruct the villous structure. because of the loss of mature enterocytes on the tips of the villi, the surface area of the intestine is reduced. diarrhea that occurs may be a result of this decrease in surface area, disruption in epithelial integrity, transient disaccharidase deficiency, or altered countercurrent mechanisms and net secretion of water and electrolytes.1'33~1140~1142~1146~1147~1148 nsp4 has been found to induce age-dependent diarrhea in cd1 mice by triggering calcium-dependent chloride and water secretion.1149 the potential role of this "viral enterotoxin" in human disease is not yet clear.1150,1151 infants with asymptomatic rotavirus infections in the nursery are less likely than uninfected nursery mates to experience severe rotavirus infection later in life1152-1153; this finding suggested protective immunity and supported vaccine development. most studies have indicated that serum and intestinal antirotavirus antibody levels are correlated with protection against i n f e~t i o n "~~-"~~ although this correlation has not been ~n i v e r s a l .~~~~-~~~~ breast-feeding protects against rotavirus disease during the first year of life,57 probably including newborns. 1146 the high prevalence of antirotaviral antibodies in colostrum and human milk has been demonstrated by numerous investigators in widely diverse geographic areas.8 maternal rotavirus infection or immunization is accompanied by the appearance of specific antibodies in milk, probably through stimulation of the enteromammary immune between 90% and 100% of women examined in london, bangladesh, guatemala, costa rica, and the united states had antirotaviral iga antibodies in their milk for up to 2 years of rotavirus-specific igg antibodies have been found during the first few postpartum days in about one third of human milk samples a~s a y e d ,~~@~"~~ whereas i@ antibodies were detectable in about one half.1167 glycoproteins in human milk have been shown to prevent rotavirus infection in vitro and in an animal model."70 the concentration of one milk glycoprotein, lactadherin, was found to be significantly higher in human milk ingested by cytes.l 133, 1135, 1144 infants who developed asymptomatic rotavirus infection than in milk ingested by infants who developed symptomatic infe~tion.4~ rotaviruses probably infect neonates more commonly than previously recognized, although most infections seem to be asymptomatic or mildly symptomatic. 1128" 1 3 0 9 1 1 3 1~1 172-1 in a prospective study, the prevalence of rotavirus infection among neonatal intensive care unit patients was 18.4%. rotavirus has a mean incubation period of 2 days, with a range of 1 to 3 days in children and in experimentally infected adults. fecal excretion of virus often begins a day or so before illness and maximal excretion usually occurs during the third and fourth days, and generally diminishes by the end of the first week, although low concentrations of virus have been detected in neonates for up to 8 weeks.1140, [1186] [1187] [1188] [1189] rotavirus infections are markedly seasonal (autumn and winter) in many areas of the world, although in some countries seasonality is less striking; the reason for this is u n~l e a r .~~~" '~~ in nurseries in which persisting endemic infection has permitted long-term surveillance of large numbers of neonates, rotavirus excretion can follow the seasonal pattern of the community but can also show no seasonal it is not clear how units in which infection remains endemic for months or years differ from those with a low incidence of rotavirus. some nurseries are free of rotavirus infection' or minimally a f f e~t e d~~"~" whereas others have rotavirus diarrheal disease throughout the year or in outbreaks that involve 10% to 40% of low birth weight does not seem to be an important factor in determining the attack rate among infants at risk but may be important in rn0rta1ity.i~~~ infants in premature or special-care nurseries, despite their prolonged stays and the increased handling necessary for their care, do not demonstrate a higher susceptibility to infection; data regarding shedding of the virus are inconsi~tent!~*~~~~ after infection is introduced into a nursery, rotavirus probably will spread steadily and remain endemic until the nursery is closed to new admissions or nursing practices permit interruption of the cycle.1205 exactly how the virus is introduced and transmitted is uncertain, although limited observations and experience with other types of enteric disease in maternity units suggest several possibilities. the early appearance of virus in stools of some neonates indicates that infection probably was acquired at delivery. virus particles can be detected on the first45v11s6 or second"98 day of life in a significant number of infected infants. by day 3 or 4, most infected infants who will shed virus, with or without signs of illness, are doing so.117431186,1198 the large numbers of virus particles e x~r e t e d "~~,~~~~ suggest a fairly large and early oral inoculum. it is unlikely that contamination from any source other than maternal feces could provide an inoculum large enough to cause infection by the second day. transfer of particles from infant to infant on the hands of nursing and medical staff is probably the most important means of viral spread. with 10' to 10" viral particles usually present in 1 g of stool, the hands of personnel easily could become contaminated after infection is introduced into a nursery. there are numerous reports of nosocomial and daycare center rotavirus gastroenteritis outbreaks that attest to the ease with which this agent spreads through a hospital or institutional setting.'io8 admission of a symptomatic child usually is the initiating event, although transfer of a neonate with inapparent infection from one ward to another also has been incriminated. the most important factors influencing the incidence of rotavirus diarrhea in a nursery are the proximity to other newborns and the frequency of handling.1187 during a 4-month study, infants cared for by nursing staff and kept in communal nurseries experienced three epidemics of diarrhea with attack rates between 20% and 50%. during the same period, only 2% of infants rooming in with their mothers became ill, even though they had frequent contact with adult relatives and siblings. there is no clear evidence of airborne or droplet infection originating in the upper respiratory tract or spread by aerosolization of diarrheal fluid while diapers are changed. indirect evidence of airborne transmission includes the high infection rate in closed settings, the isolation of the virus from respiratory secretions,izo6 and the experimental observation of transmission by aerosol droplets in mice.'207 however, the respiratory isolation achieved by placin an evidence indicates that transplacental or ascending intrauterine infection occurs. transmission of virus through contaminated fomites, formula, or food is possible but has not been documented in newborns. rotavirus particles have not been found in human milk or c o l o~t r u m .~~~~~~~~ exposure of a newborn to rotavirus can result in asymptomatic infection or cause mild or severe gastro-outbreaks with high attack rates as measured by rotavirus excretion have been described but the extent of symptomatic infection severe rotavirus infection is seldom reported during the newborn period1203 but the extent of underreporting of severe disease, especially in the less developed areas of the world, has not been evaluated. it has been hypothesized that asymptomatic infections during the newborn period are the result of naturally attenuated strains circulating in this environment. rna electrophoretic patterns of rotaviruses found in certain nurseries have shown uniform and it has been suggested that these strains may be attenuated. the presence of unusual antigenic types such as the p2a[6] type within nurseries also suggests "less virulent strains." at least 10 rotavirus strains were documented to co-circulate in a tertiary care center during a 2-month period12" and in a different setting the same rotavirus strains by electropherotype produced asymptomatic infection in neonates and symptomatic infection in older infants.1183 newborns within a nursery exposed to a given rotavirus strain can develop symptomatic or asymptomatic infection. 130~1 2 1 0~1 2 i because newborns routinely have frequent relatively loose stools, it is possible that mild diarrhea episodes caused by rotavirus are being wrongly labeled as asymptomatic episodes. no clinical feature is pathognomonic of rotaviral gastroenteritis. early signs of illness, such as lethargy, irritability, vomiting, and poor feeding, usually are followed in a few hours by the passage of watery yellow or green stools free of blood but sometimes containing mucus.1187,1212-1214 diarrhea usually decreases by the second day of illness and is much infant in a closed incubator is not fully protective.118 s no enteritisel 129. 1130.1 173.1 179,1196,l 197,1201,1208 varies. 1 175.1 177.1 186.1 198,1203 improved by the third or fourth day. occasionally, intestinal fluid loss and poor weight gain may continue for 1 or 2 weeks, particularly in low-birth-weight infants. 1175 although reducing substances frequently are present in early fecal ~a m p l e s "~~~'~~~' 176~1187 this finding is not necessarily abnormal in neonates, particularly those who are breast-fed.1215 nevertheless, infants with prolonged diarrhea should be investigated for monosaccharide or disaccharide malabsorption or intolerance to cow's milk protein or in a prospective 49% of newborns with gastrointestinal symptoms in a neonatal intensive care unit had rotavirus detected in their stools. frequent stooling (present in 60%), bloody mucoid stool (42%), and watery stools (24%) were risk factors for a rotavirus infection. bloody mucoid stools, intestinal dilatation, and abdominal distention were significantly more common in preterm infants, but severe outcomes such as necrotizing enterocolitis and death did not differ among infected term and preterm infants. longitudinal studies in newborn nurseries and investigations of outbreaks among neonates rarely describe a severe adverse outcome or death.1139,1168,1187 because these infants are under constant observation, early detection of excessive fluid losses and the availability of immediate medical care are probably major factors in determining outcome. rotavirus gastroenteritis causes almost 400,000 deaths of infants every year,i2i7 concentrated largely in the poorest regions of the world. it is likely that in places where hospital-based care is uncommon, rotavirus causes neonatal deaths secondary to dehydration. group a rotavirus has been associated with a wide array of diseases in infants and children; reye syndromes, encephalitis-aseptic meningitis, sudden infant death syndrome, inflammatory bowel disease, and kawasaki syndrome have been described but not systematically studied. 1108 case reports and small case series have associated neonatal rotavirus infection with necrotizing e n t e r o c~l i t i s .~~'~"~~~ rotavirus infection may play a role in a small proportion of cases of necrotizing enterocolitis, although it probably represents one of many potential triggering factors. a significant association between neonatal rotavirus infection and bradycardia-apnea episodes was detected in one prospective study.1220 the possible association between natural rotavirus infection and i n t u s s u~c e p t i o n~~~~~~~~~ gained support after the association was made between the human-simian reassortant vaccine and intussusception in infants older than 2 months (attributable risk = 1: 10,000). 1224 intussusception is extremely uncommon in the newborn; it is highly unlikely that rotavirus triggers this disease in neonates. there are many methods used for detection of rotavirus in stool specimens, including electron microscopy, immune electron microscopy, elisa, latex agglutination, gel electrophoresis, culture of the virus, and reverse transcriptasepolymerase chain reaction. elisa and latex agglutination currently are the most widely used diagnostic techniques for detection of rotavirus in clinical samples. many commercial kits are available that differ in specificity and ~e n s i t i v i t y . '~~~-'~~~ in general, latex agglutination assays are more rapid than elisas but are less sensitive. the sensitivity and specificity of the commercially available elisas surpass 90%. checking of the elisa by another method such as gel electrophoresis or pcr amplification may be desirable if there is concern about false-positive results. fecal material for detection of rotavirus infection should be obtained during the acute phase of illness. whole-stool samples are preferred, although suspensions of rectal swab specimens have been adequate for detection of rotavirus by rotavirus are relatively resistant to environmental temperatures, even tropical temperatures, 1232 although 4°c is desirable for short-term storage and -70°c for prolonged storage.'io8 excretion of viral particles may precede signs of illness by several days' 190; maximal excretion by older infants and children usually occurs 3 to 4 days after onset of symptoms.1233 neonates can shed virus for 1 to 2 weeks after onset of symptoms. the primary goal of therapy is restoration and maintenance of fluid and electrolyte balance. despite the documented defect in carbohydrate digestion with rotavirus diarrhea, rehydration often can be accomplished with glucoseelectrolyte or sucrose-electrolyte solutions given orally .199,1234-1236 intravenous fluids may be needed in neonates who are severely dehydrated, who have ileus, or who refuse to feed. persistent or recurrent diarrhea after introduction of milkbased formulas or human milk warrants investigation for secondary carbohydrate or milk protein i n t~l e r a n c e . '~~~~~~" disaccharidase levels and xylose absorption return to normal within a few days1144 to weeks after infe~tion."~~ intractable diarrhea related to severe morphologic and enzymatic changes of the bowel mucosa is possible although rare in the newborn; it may require an elemental diet or parenteral nutrition. efficacy of anti-rotavirus antibodies (e.g., hyperimmune colostrum, antibody-supplemented formula, human serum immunoglobulin) and of probiotics has been p~s t u l a t e d , '~~~~'~~' although not convincingly shown1242; the widespread clinical use of these measures seems remote. one study suggests that use of lactobacillus during the diarrheal episode may decrease the duration of rotavirus-associated hospital stays, especially when used early in the course of the disease, although more studies are needed before recommending widespread use. 1241 hand hygiene before and after contact with each infant remains the single most important means of preventing the spread of infection. because rotavirus is often excreted several days before illness is recognized, isolation of an infant with diarrhea may be too late to prevent cross-infection unless all nursing personnel and medical staff have adhered to this fundamental precaution. infants who develop gastroenteritis should be moved out of the nursery area if adequate facilities are available and the infant's condition permits transfer. the use of an incubator is of value in reducing transmission of disease only by serving as a reminder that proper hand-hygiene and glove techniques are required, but is of little value as a physical barrier to the spread of encouraging rooming-in of infants with their mothers has been shown to be helpful in preventing or containing nursery epidemics.'243 temporary closure of the nursery may be required for clinically significant outbreaks that cannot be controlled with other measures.1128 development of rotavirus vaccines began in the early 1980s. candidate vaccines included bovine and rhesus monkey elisa. 1230, 1231 attenuated strains, human attenuated strains, and bovinehuman and rhesus-human reassortant strains."" in august 1998, the first licensed rotavirus vaccine, rotashield, an oral formulation of a simian-human quadrivalent reassortant vaccine, was recommended for use in children when they were 2, 4, and 6 months old. after approximately 500,000 children were vaccinated with more than 1 million doses, a significantly increased risk of intussusception was observed among vaccinated children, with an overall odds ratio of 1.8.iz4 use of this vaccine was terminated. two new vaccine candidates are undergoing phase i11 clinical trials: a "pentavalent" bovine-human reassortant vaccine including g types gl-g4 and p type pla[8] and a monovalent human attenuated pla[ 8]g1 vaccine. the epidemiology of rotavirus infection will change significantly if one or both candidates become widely available in the future. the impact on neonatal infection will depend on the effect of herd immunity in decreasing circulation of rotavirus strains. stools from breast-fed neonates are typically watery and yellow, green, or brown. the frequency of stooling can vary from one every other day to eight evacuations per day. in an active, healthy infant who is feeding well, has no vomiting, and has a soft abdomen, these varied patterns of stooling are not a cause for concern. physicians need to consider the child's previous frequency and consistency of stools and establish a diagnosis of acute diarrhea on an individual basis. close follow-up of weight increase in infants with nonformed stools can help confirm the clinical impression. a normal weight gain should direct medical action away from stool exams or treatment. diarrhea during the neonatal period is a clinical manifestation of a wide variety of disorders (table 20-6). the most common initiating factor is a primary infection of the gastrointestinal tract that is mild to moderate in severity, chapter 20 self-limited, and responsive to supportive measures. acute diarrhea can also be an initial manifestation of a systemic infection, including bacterial and viral neonatal sepsis. infants with moderate to severe diarrhea require close monitoring until the etiologic diagnosis and the clinical evolution are clarified. there are noninfectious diseases leading to chronic intractable diarrhea that may result in severe nutritional disturbances or even death unless the specific underlying condition is identified and treated appropriately. the differential diagnosis of a diarrheal illness requires a careful clinical examination to determine whether the child has a localized or a systemic process. lethargy, abnormalities in body temperature, hypothermia or hyperthermia, decreased feeding, abdominal distention, vomiting, pallor, respiratory distress, apnea, cyanosis, hernodynamic instability, hypotension, hepatomegaly or splenomegaly, coagulation or bleeding disorders, petechiae, and exanthemas should lead to an intense laboratory investigation directed at systemic viral or bacterial infection. if the process is deemed a localized intestinal infection, initial evaluation can be focused on differentiating an inflammatory-invasive pathogen from those that cause a noninflammatory process. for this, stool examination for fecal leukocytes, red blood cells, and lactoferrin can be a helpful indicator of the former. inflammatory diarrhea can be caused by shigella, salmonella, carnpylobacter, v parahaemolyticus, k: enterocolitica, eiec, eaec, c. difjcile, necrotizing enterocolitis, antibioticassociated colitis, and allergic colitis (i.e., milk or soy intolerance). noninflammatory causes of diarrhea include etec, epec, rotaviruses, enteric adenoviruses, calicivirus, astrovirus, g. larnblia and cryptosporidium. although supportive fluid therapy is mandatory for all types of diarrhea, the brief examination for fecal leukocytes and red blood cells can direct the diagnostic and therapeutic approach. pathogens such as shigella, salmonella, and ehec can cause watery or bloody diarrhea, depending on the specific host-pathogen interaction and the pathogenic mechanisms involved. some of the noninfectious diseases responsible for neonatal diarrhea are listed in table 20 the united states and panama food based oral rehydration salt solutions for acute childhood diarrhoea international study group on reduced osmolality ors solution. multicentre evaluation of reduced-osmolality oral rehydration salts solution antimicrobial resistance and enterotoxin production among isolates of escherichia coli in the far east high-molecular-weight plasmid correlates with escherichia coli invasiveness shigella guanabara, tip0 serologico destacado do grupo b ceylonensis-dispar a study of specific e. coli infections occurring in a unit for surgical neonates molecular characterization of strains of enteroinvasive escherichia coli 0143 untersuchungen zur kiologie der durchfallserkrankungen des sauglings a study of e. coli mutable from an outbreak of diarrhea in the new-born isolation of antigenically homogenous strains of bart. coli neopolitanum from summer diarrhea of infants slide agglutination of bacterium coli var. neopolitanum in summer diarrhea a complete somatic antigen common to sahone~la adelaide, escherichia coli-gomez and escherichia coli 0 1 ll:b4 an outbreak of infantile gastroenteritis in aberdeen epidemic gastroenteritis of infants in aberdeen during 1947 escherichia strains from infantile epidemic gastroenteritis identification of enterobacteriaceae association of escherichia coli sero-345. gastroenteritis due to escherichia coli enteropathogenic escherichia coli serotype ol11:hnt isolated from preterm neonates in nairobi, kenya bray's discovery of pathogenic esch. coli as a cause of infantile gastroenteritis cross-infection in infantile gastroenteritis acute intestinal infections of nondysenteric etiology protracted diarrhea of infancy treated by intravenous 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enteritis fluorescent antibody techniques for salmonella and other enteric pathogens colistin suppression of escherichia coli in stools. i. control of a nosocomial outbreak of diarrhea caused by neomycin-resistant escherichia coli olll:b4 genetic and phenotypic analysis of escherichia coli with enteropathogenic characteristics isolated from seattle children comparison of two assay methods for patterns of adherence to hep-2 cells of escherichia coli from patients with diarrhea an elisa for the detection of localized adherent classic enteropathogenic escherichia coli serogroups actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic escherichia coli yearbook of pediatrics use of an oral elemental diet in infants with severe intractable diarrhea controlled trial of orally administered lactobacilli in acute infantile diarrhea use of antibiotics in acute gastroenteritis among infants in hospital 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recently discovered diarrheal pathogen the export of coat protein from enteroaggregative escherichia coli by a specific atp-binding cassette transporter system pathogenicity of enteroadherent escherichia coli in adult volunteers heterogeneity of enteroaggregative escherichia coli virulence demonstrated in volunteers laboratory investigation of enteroaggregative escherichia coli 0 untypeab1e:hlo associated with a massive outbreak of gastrointestinal illness enteroaggregative escherichia coli and outbreaks of gastroenteritis in uk enteroaggregative e. coli associated with an outbreak of diarrhea in a neonatal nursery ward identification of a protein with toxigenic activity produced by enteroaggregative escherichia coli. abstracts of the general meeting of the enteroaggregative escherichia coli associated with persistent diarrhea in a cohort of rural children in india enteroaggregative escherichia coli and salmonella associated with non-dysenteric persistent diarrhea association of 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escherichia coli in young children living in western europe a study of infectious intestinal disease in england microbiological findings in cases and controls unpublished observations markers of enteric inflammation in enteroaggregative escherichia coli diarrhea in travelers detection of enteroaggregative escherichia coli with formalin-preserved hep-2 cells improved detection of enteroaggregative escherichia coli using formalin-fixed hep-2 cells successful treatment of diarrheal disease associated with enteroaggregative escherichia coli in adults infected with human immunodeficiency virus azithromycin found to be comparable to levofloxacin for the treatment of u s travelers with acute diarrhea acquired in mexico rifaximin versus ciprofloxacin for the treatment of traveler's diarrhea: a randomized, double-blind clinical trial molecular characterization of a fimbrial adhesin, f1845, mediating diffuse adherences of diarrheaassociated escherichia coli to hep-2 cells diffuse and 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cases infected with the new epidemic strain of vibrio cholerae 0139: designation of the disease as cholera nationwide prevalence of the new epidemic strain of vibrio cholerae 0139 bengal in india cholera-a possible endemic focus in the united states cholera in pregnant women neonatal diarrhea caused by vibrio cholerae 0139 bengal vibrio cholerae 0139 diarrhea and acute renal failure in a three day old infant neonatal diarrhea in a diarrhea treatment center in bangladesh clinical presentation, breastfeeding management and outcome bottle feeding as a risk factor for cholera in infants characteristics of the serum vibriocidal and agglutinating antibodies in cholera cases and in normal residents of the endemic and non-endemic cholera areas maternal cholera immunisation and secretory iga in breast milk response of man to infection with vibrio cholerae. i. clinical, serologic and bacteriologic responses to a known inoculum cholera, non-vibrio cholera, and stomach acid development and testing of a nonradioactive dna oligonucleotide probe that is specific for vibn'o cholerae cholera toxin development of an enzymelabeled oligonucleotide probe for the cholera toxin gene safety and immunogenicity in north americans of a single dose of live oral cholera vaccine cvd 103-hgr results of a randomized, placebocontrolled, double-blind crossover trial evidence that inactivated oral cholera vaccines both prevent and mitigate vibrio cholerae 0 1 infections in a cholera-endemic area a review of the current status of enteric vaccines ersinia enterocolitica infections in children ersinia enterocolitica gastroenteritis: a prospective study of clinical, bacteriologic, and epidemiologic features ersinia enterocolitica 0 3 infections in infants and children, associated with the household preparation of chitterlings clinical and microbiologic characteristics of cutaneous infection with yersinia enterocolitica ersinia enterocolih'ca 03: an emerging cause of pediatric gastroenteritis in the united 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disease possibly caused by i? shigelloides a. hydrophila and j? shigelloides as causes of intestinal infections in vitro and in vivo pathogenicity of i! shigelloides clinical disease spectrum and pathogenic factors associated with p. shigelloides infections in humans neonatal septicemia and meningitis due to plesiomonas shigelloides neonatal plesiomonas shigelloides septicemia and meningitis: a case review plesiomonas shigelloides sepsis and meningoencephalitis in a neonate plesiomonas shigelloides meningitis and septicaemia in a neonate: report of a case and review of the literature biochemical characteristics and a simple scheme for the identification of aeromonas species and plesiomonas shigelloides comparative in vitro activities of selected antimicrobial agents against aeromonas species and i? shigelloides antimicrobial therapy in plesiomonas shigelloide~associated diarrhea in thai children bacillus mucosus infection of the newborn klebsiella strains isolated from diarrheal infants. human volunteer studies klebsiella pseudomembranous enterocolitis beobachtungen a e r die bitiologie der gastroenterocolitiden des sauglings-und kindesalters. 11. untersuchung der rolle der klebsiella-stamme ober eine enteritis-epidemie bei friihgeborenen,verursacht durch den bacillus klebsiella bacillus mucosus capsulatus" in infantile diarrhea bacillus lactis aerogenes infection in the newborn stomatitis and diarrhea in infants caused by bacillus mucosus capsulatus a new klebsiella type (capsular type 15) isolated from feces and urine enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula contamination of infant feeds in a milton milk kitchen nosocomial colonization with kanamycin-resistant klebsiella pneumoniae, types 2 and 11, in a premature nursery nosocomial colonization with klebsiella, type 16, in a neonatal intensive-care unit associated with an outbreak of sepsis, meningitis, and necrotizing enterocolitis 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pathogens: enterobacteriaceae, pseudomonas, alcaligenes and aeromonas the intestinal flora in the etiology of infantile infectious diarrhea the bacteriological considerations of infantile enteritis in sydney beobachtungen iiber die atiologie der gastroenterocolitiden des sauglings-und kindesalters. iv. untersuchung der rolle der proteus morgani-stamme group d streptococci in the faeces of healthy infants and of infants with neonatal diarrhea neonatal enteritis due to providencia organisms infection with the providence type of paracolon bacillus in a residential nursery providence group of organisms in the aetiology ofjuvenile diarrhoea microbial flora of stomach and small intestine in infantile gastroenteritis diarrhoea associated with candida spp.: incidence and seasonal variation prevalence of candida species in nigerian children with diarrhea infantile diarrhea and malnutrition associated with candida in a developing community role of candida in indirect pathogenesis of antibiotic associated diarrhea in infants upper small intestine microflora in diarrhea and malnutrition in nigerian children diarrhea caused by candida beitrage zur frage der moniliasis in sauglingdter recovery from disseminated candidiasis in a premature neonate systemic candida infections in infants in intensive care nurseries: high incidence of central nervous system involvement disseminated fungal infections in very low birth weight infants: clinical manifestations and epidemiology bacteremia during diarrhea: incidence, etiology, risk factors, and outcome prevalence and risk factors associated with intestinal parasitoses in pregnant women and their relation to the infant's birth weight amebiasis in the newborn amoebic proctocolitis and liver abscess in a neonate amoebic appendicitis in a newborn infant amebiasis of the newborn: report of three cases extraintestinal amebiasis in infancy: report of three patients and epidemiologic investigations of their families amebic liver abscess in newborn fuliminant amebic colitis in a ten-day-old infant acquisition of intestinal parasites in newborn human infants comparison of use of enzyme-linked immunosorbent assay-based kits and pcr amplification of rrna genes for simultaneous detection of entamoeba histolytica and e. dispar comparison of pcr, isoenzyme analysis, and antigen detection for diagnosis of entamoeba histolytica infection giardiasis in day care centers. evidence of person-to-person transmission person-to-person transmission of g. lamblia in day care nurseries diarrhea caused by shigella, rotavirus, and giardia in day care centers: prospective study occurrence of g. lnmblia in children in day care centers the biology of giardia spp age-related rate of seropositivity and antibody to giardia lambliu in four diverse populations giardia lamblia infections in a cohort of bangladeshi mothers and infants followed for one year giardiasis and breastfeeding in urban africa local immunity in murine giardiasis: is milk protective at the expense of maternal gut? protection against infection with giardia muris by milk containing antibody to giardia killing of g. lamblia by human milk mediated by unsaturated fatty acids immunology of giardia and cryptosporidium infections cryptosporidiosis in immunocompetent patients cryptosporidiosis in animals and humans shedding of oocysts in immunocompetent individuals infected with cryptosporidium cryptosporidiosis travelers' diarrhea in two families cryptosporidiosis after marrow transplantation, person-to-person transmission and treatment with spiramycin cryptosporidiosis in humans: review of recent epidemiologic studies outbreak of cryptosporidiosis in a day care center cryptosporidiosis outbreak in a day care center cryptosporidiosis-associated mortality following a massive waterborne outbreak in milwaukee, wisconsin cryptosporidium: a frequent finding in patients with gastrointestinal symptoms human cryptosporidiosis in immunocompetent and immunodeficient persons: studies of an outbreak and experimental transmission cryptosporidium infections in mexican children: clinical, nutritional, enteropathogenic, and diagnostic evaluations cryptosporidiosis in children from some highland costa rican rural and urban areas timing of symptoms and oocyst excretion in human cryptosporidiosis schmid 11, et al. cryptosporidium, malnutrition and chronic diarrhea in children evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of giardia lnrnblio and cryptosporidium parvurn in human fecal specimens cryptosporidiosis: multiattribute evaluation of six diagnostic methods treatment of diarrhea caused by cryptosporidium parvum: a prospective of randomized, double-blind, placebo-controlled study of nitazoxanide cryptosporidiosis in northeastern brazilian children: association with increased diarrhea morbidity rotavirus, enteric adenoviruses, caliciviruses, astroviruses, and other viruses causing gastroenteritis fields virology rotaviruses and their replication viruses causing gastroenteritis non-group a rotavirus molecular epidemiology of rotavirus infection immunity of piglets to either vp3 or vp7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen reassortant rotavirus containing structural proteins vp3 and vp7 from different parents are protective against each parental strain distribution of human rotaviruses, especially g9 strains molecular epidemiology of rotavirus in children attending day care centers in houston unusual diversity of human rotavirus g and p genotypes in india evidence of high-frequency genomic reassortment of group a rotavirus strains in bangladesh emergence of type g9 in 1995 detection of rotavirus types g8 and g10 among brazilian children with diarrhea genetic an antigenetic characterization of a serotype g6 human rotavirus isolated in melbourne, australia detection of a human rotavirus with g12 and p[9] specificity in thailand review of g and p typing results from a global collection of rotavirus strains: implications for vaccine development neonatal rotavirus infection in bangladesh strain characterization and risk factors for nosocomial infection distinct population of rotaviruses circulating among neonates and older infants epidemiology of rotavirus in india detection and characterization of rotavirus g and p types from children participating in a rotavirus vaccine trial in belen an outbreak of diarrhea in a neonatal medium care unit caused by a novel strain of rotavirus: investigation using both epidemiologival and microbiological methods characterization of rotavirus infection in a hospital neonatal unit in pretoria, south africa neonatal rotavirus infection in belem, northern brazil: nosocomial transmission of a p[6] g2 strain detection and characterization of rotaviruses in hospitalized neonates in blantyre, malawi importance of a new virus in acute sporadic enteritis in children virus particles in epithelial cells of duodenal mucosa from children with acute nonbacterial gastroenteritis infantile enteritis viruses: morphogenesis and morphology reovirus-like particles in jejunal mucosa of a japanese infant with acute infectious nonbacterial gastroenteritis comparison of methods for immunocytochemical detection of rotavirus infections is lactase the receptor and uncoating enzyme for infantile enteritis (rota) viruses? the mucosal lesion in viral enteritis. extent and dynamics of the epithelial response to virus invasion in transmissible gastroenteritis of piglets noncultivable viruses and neonatal diarrhea. fifteen-month survey in a newborn special care nursery lactose malabsorption and milk consumption in infants and children mechanisms of mucosal injury: human studies determinants of diarrhea in viral enteritis. the role of ion transport and epithelial changes in the ileum in transmissible gastroenteritis in piglets identification of group a rotavirus genes associated with virulence of a porcine rotavirus and host range restriction of a human rotavirus in the gnotobiotic piglet model chronic rotavirus infection in immunodeficiency functional abnormalities in the intestine role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea transmissible gastroenteritis: sodium transport and the intestinal epithelium during the course of viral gastroenteritis age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the nsp4 protein gene mutations in nonstructural glycoprotein nsp4 are associated with altered virus virulence clinical immunity after neonatal rotavirus infection. a prospective longitudinal study in young children protection conferred by neonatal rotavirus infection against subsequent rotavirus diarrhea protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies identification of vp7 epitopes associated with protection against human rotavirus illness or shedding in volunteers protective effect of preexisting rotavirus-specific immunoglobulin a against naturally acquired rotavirus infection in children characterization of serum antibody responses to natural rotavirus infections in children by w7-specific epitope-blocking assays relative concentrations of serum neutralizing antibody to vp3 and vp7 protein in adults infected with human rotavirus seroepidemiologic evaluation of antibodies to rotavirus as correlates of the risk of clinically significant rotavirus diarrhea in rural bangladesh fecal antibody responses to symptomatic and asymptomatic rotavirus infections anti-rotavirus g type-specific and isotype-specific antibodies in children with natural rotavirus infections prospective study of communityacquired rotavirus infection evidence that protection against rotavirus diarrhea after natural infection is not dependent on serotype-specific neutralizing antibody cord blood and breast milk antibodies in neonatal rotavirus infection epidemiology of human rotavirus types 1 and 2 as studied by enzyme-linked immunosorbent assay transfer of anti-rotaviral antibodies h-om mothers to their infants effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection antibodies to seven rotavirus serotypes in cord sera, maternal sera, and colostrum of german women rotavirus-inhibitory activity in serial milk samples from mexican women and rotavirus infections in their children during their first year of life secretory antibody directed against rotavirus in human milk-measurement by means of enzymelinked immunosorbent assay human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis clinical immunity after neonatal rotavirus infection. a prospective longitudinal study in young children stool viruses in babies in glasgow. 2. investigation of normal newborns in hospital nosocomial rotavirus gastroenteritis in a neonatal nursery new virus associated with diarrhoea in neonates rotavirus infections in a special-care baby unit rotavirus infections in newborns: an epidemiological and clinical study a prospective study of rotavirus infections in neonatal and maternity wards serotypic characterization of rotaviruses derived from asymptomatic human neonatal infections further studies on neonatal rotavirus infection rotavirus shedding by newborn children molecular epidemiology of rotavirus infection in a room for convalescing newborns neonatal rotavirus infections rotavirus: a cause of nosocomial infection in a nursery prevalence of rotavirus infection in neonates diarrhea and rotavirus infection associated with differing regimens for postnatal care of newborn babies asymptomatic rotavirus before and after rotavirus diarrhea in children in day care centers quantitative aspects of rotavirus excretion in childhood diarrhoea influence of temperature and 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in infants the irritable colon of childhood (chronic nonspecific diarrhea syndrome) gut transit time and lactose malabsorption during phototherapy. i, 11 neonatal diagnosis of familial dysautonomia familial enteropathy: a syndrome of protracted diarrhea from birth, failure to thrive, and hypoplastic villous atrophy lethal familial protracted diarrhea infantile gastroenteritis due to water with high sulfate content diarrhea, red diapers, and child abuse management of children with infection-associated persistent diarrhea key: cord-146850-5x6qs2i4 authors: gupta, abhishek; lanteigne, camylle; heath, victoria; ganapini, marianna bergamaschi; galinkin, erick; cohen, allison; gasperis, tania de; akif, mo; institute, renjie butalid montreal ai ethics; microsoft,; university, mcgill; commons, creative; college, union; rapid7,; global, ai; university, ocad title: the state of ai ethics report (june 2020) date: 2020-06-25 journal: nan doi: nan sha: doc_id: 146850 cord_uid: 5x6qs2i4 these past few months have been especially challenging, and the deployment of technology in ways hitherto untested at an unrivalled pace has left the internet and technology watchers aghast. artificial intelligence has become the byword for technological progress and is being used in everything from helping us combat the covid-19 pandemic to nudging our attention in different directions as we all spend increasingly larger amounts of time online. it has never been more important that we keep a sharp eye out on the development of this field and how it is shaping our society and interactions with each other. with this inaugural edition of the state of ai ethics we hope to bring forward the most important developments that caught our attention at the montreal ai ethics institute this past quarter. our goal is to help you navigate this ever-evolving field swiftly and allow you and your organization to make informed decisions. this pulse-check for the state of discourse, research, and development is geared towards researchers and practitioners alike who are making decisions on behalf of their organizations in considering the societal impacts of ai-enabled solutions. we cover a wide set of areas in this report spanning agency and responsibility, security and risk, disinformation, jobs and labor, the future of ai ethics, and more. our staff has worked tirelessly over the past quarter surfacing signal from the noise so that you are equipped with the right tools and knowledge to confidently tread this complex yet consequential domain. these past few months have been especially challenging, and the deployment of technology in ways hitherto untested at an unrivaled pace has left the internet and technology watchers aghast. artificial intelligence has become the byword for technological progress and is being used in everything from helping us combat the covid-19 pandemic to nudging our attention in different directions as we all spend increasingly larger amounts of time online. it has never been more important that we keep a sharp eye out on the development of this field and how it is shaping our society and interactions with each other. with this inaugural edition of the state of ai ethics we hope to bring forward the most important developments that caught our attention at the montreal ai ethics institute this past quarter. our goal is to help you navigate this ever-evolving field swiftly and allow you and your organization to make informed decisions. this pulse-check for the state of discourse, research, and development is geared towards researchers and practitioners alike who are making decisions on behalf of their organizations in considering the societal impacts of ai-enabled solutions. we cover a wide set of areas in this report spanning agency and responsibility, security and risk, disinformation, jobs and labor, the future of ai ethics , and more. our staff has worked tirelessly over the past quarter surfacing signal from the noise so that you are equipped with the right tools and knowledge to confidently tread this complex yet consequential domain. to stay up-to-date with the work at maiei, including our public competence building, we encourage you to stay tuned on https://montrealethics.ai which has information on all of our research. we hope you find this useful and look forward to hearing from you! wishing you well, abhishek gupta the state of ai ethics, june 2020 the debate when ethicists ask for rights to be granted to robots is based on notions of biological chauvinism and that if robots display the same level of agency and autonomy, not doing so would not only be unethical but also cause a setback for the rights that were denied to disadvantaged groups. by branding robots as slaves and implying that they don't deserve rights has fatal flaws in that they both use a term, slave, that has connotations that have significantly harmed people in the past and also that dehumanization of robots is not possible because it assumes that they are not human to begin with. while it may be possible to build a sentient robot in the distant future, in such a case there would be no reason to not grant it rights but until then, real, present problems are being ignored for imaginary future ones. the relationship between machines and humans is tightly intertwined but it's not symmetrical and hence we must not confound the "being" part of human beings with the characteristics of present technological artifacts. technologists assume that since there is a dualism to a human being, in the sense of the mind and the body, then it maps neatly such that the software is the mind and the robot body maps to the physical body of a human, which leads them to believe that a sentient robot, in our image, can be constructed, it's just a very complex configuration that we haven't completely figured out yet. the more representative view of thinking about robots at present is to see them as objects that inhabit our physical and social spaces. objects in our environment take on meaning based on the purpose they serve to us, such as a park bench meaning one thing to a skateboarder and another to a casual park visitor. similarly, our social interactions are always situated within a larger ecosystem and that needs to be taken into consideration when thinking about the interactions between humans and objects. in other words, things are what they are, because of the way they configure our social practices and how technology extends the biological body.our conception of human beings, then, is that we are and have always been fully embedded and enmeshed with our the state of ai ethics, june 2020 designed surroundings, and that we are critically dependent on this embeddedness for sustaining ourselves. because of this deep embedding, instead of seeing the objects around us merely as machines or on the other end as 'intelligent others', we must realize that they are very much a part of ourselves because of the important role they play in defining both our physical and social existence. some argue that robots take on a greater meaning when they are in a social context like care robots and people might be attached to them, yet that is quite similar to the attachment one develops to other artifacts like a nice espresso machine or a treasured object handed down for generations. they have meaning to the person but that doesn't mean that the robot, as present technology, needs to be granted rights. while a comparison to slaves and other disenfranchised groups is made when robots are denied rights because they are seen as 'less' than others, one mustn't forget that it happens to be the case that it is so because they are perceived as instruments and means to achieve an end. by comparing these groups to robots, one dehumanizes actual human beings. it may be called anthropocentric to deny rights to robots but that's what needs to be done: to center on the welfare of humans rather than inanimate machines. an interesting analogue that drives home the point when thinking about this is the milgram prison experiment where subjects who thought they had inflicted harms on the actors, who were a part of the experiment, were traumatized even after being told that the screams they heard were from the actors. from an outside perspective, we may say that no harm was done because they were just actors but to the person who was the subject of the experiment, the experience was real and not an illusion and it had real consequences. in our discussion, the robot is an actor and if we treat it poorly, then that reflects more so on our interactions with other artifacts than on whether robots are "deserving" of rights or not. taking care of various artifacts can be thought of as something that is done to render respect to the human creators and the effort that they expended to create it. discussion of robot rights for an imaginary future that may or may not arrive takes away focus and perhaps resources from the harms being done to real humans today as part of the ai systems being built with bias and fairness issues in them. invasion of privacy, bias against the disadvantaged, among other issues are just some of the few already existing harms that are being leveled on humans as intelligent systems percolate into the everyday fabric of social and economic life. the state of ai ethics, june 2020 from a for-profit perspective, such systems are poised and deployed with the aims of boosting the bottom line without necessarily considering the harms that emerge as a consequence. in pro-social contexts, they are seen as a quick fix solution to inherently messy and complex problems. the most profound technologies are those that disappear into the background and in subtle ways shape and form our existence. we already see that with intelligent systems pervading many aspects of our lives. so we're not as much in threat from a system like sophia which is a rudimentary chatbot hidden behind a facade of flashy machinery but more so from roomba which impacts us more and could be used as a tool to surveil our homes. taking ethical concerns seriously means considering the impact of weaving in automated technology into daily life and how the marginalized are disproportionately harmed. in the current dominant paradigm of supervised machine learning, the systems aren't truly autonomous, there is a huge amount of human input that goes into enabling the functioning of the system, and thus we actually have human-machine systems rather than just pure machinic systems. the more impressive the system seems, the more likely that there was a ton of human labor that went into making it possible. sometimes, we even see systems that started off with a different purpose such as recaptcha that are used to prevent spam being refitted to train ml systems. the building of ai systems today doesn't just require highly skilled human labor but it must be supplemented with mundane jobs of labeling data that are poorly compensated and involve increasingly harder tasks as, for example, image recognition systems become more powerful, leading to the labeling of more and more complex images which require greater effort. this also frames the humans doing the low skilled work squarely in the category of being dehumanized because of them being used as a means to an end without adequate respect, compensation and dignity. an illustrative example where robots and welfare of humans comes into conflict was when a wheelchair user wasn't able to access the sidewalk because it was blocked by a robot and she mentioned that without building for considering the needs of humans, especially those with special needs, we'll have to make debilitating compromises in our shared physical and social spaces. ultimately, realizing the goals of the domain of ai ethics needs to reposition our focus on humans and their welfare, especially when conflicts arise between the "needs" of automated systems compared to those of humans. what happens when ai starts to take over the more creative domains of human endeavour? are we ready for a future where our last bastion, the creative pursuit, against the rise of machines is violently snatched away from us? in a fitting start to feeling bereft in the times of global turmoil, this article starts off with a story created by a machine learning model called gpt-2 that utilizes training data from more than 8 million documents online and predicts iteratively the next word in a sentence given a prompt. the story is about "life in the time of coronavirus" that paints a desolate and isolating picture of a parent who is following his daily routine and feels different because of all the changes happening around them. while the short story takes weird turns and is not completely coherent, it does give an eerie feeling that blurs the line between what could be perceived as something written by a human compared to that by a machine. a news-styled article on the use of facial recognition systems for law enforcement sounds very believable if presented outside of the context of the article. the final story, a fictional narrative, presents a fractured, jumpy storyline of a girl with a box that has hallucinatory tones to its storytelling. the range of examples from this system is impressive but it also highlights how much further these systems have to go before they can credibly take over jobs. that said, there is potential to spread disinformation via snippets like the second example we mention and hence, something to keep in mind as you read things online. technology, in its widest possible sense, has been used as a tool to supplement the creative process of an artist, aiding them in exploring the adjacent possible in the creative phasespace. for decades we've had computer scientists and artists working together to create software that can generate pieces of art that are based on procedural rules, random perturbations of the audience's input and more. off late, we've had an explosion in the use of ai to do the same, with the whole ecosystem being accelerated as people collide with each other serendipitously on platforms like twitter creating new art at a very rapid pace. but, a lot of people have been debating whether these autonomous systems can be attributed artistic agency and if they can be called artists in their own right. the author here argues that it isn't the case because even with the push into using technology that is more the state of ai ethics, june 2020 automated than other tools we've used in the past, there is more to be said about the artistic process than the simple mechanics of creating the artwork. drawing on art history and other domains, there is an argument to be made as to what art really is -there are strong arguments in support of it playing a role in servicing social relationships between two entities. we, as humans, already do that with things like exchanging gifts, romance, conversation and other forms of social engagement where the goal is to alter the social relationships. thus, the creative process is more so a co-ownership oriented model where the two entities are jointly working together to create something that alters the social fabric between them. as much as we'd like to think some of the ai-enabled tools today have agency, that isn't necessarily the case when we pop open the hood and see that it is ultimately just software that for the most part still relies heavily on humans setting goals and guiding it to perform tasks. while human-level ai might be possible in the distant future, for now the ai-enabled tools can't be called artists and are merely tools that open up new frontiers for exploration. this was the case with the advent of the camera that de-emphasized the realistic paint form and spurred the movement towards modern art in a sense where the artists are more focused on abstract ideas that enable them to express themselves in novel ways. art doesn't even have to be a tangible object but it can be an experience that is created. ultimately, many technological innovations in the past have been branded as having the potential to destroy the existing art culture but they've only given birth to new ideas and imaginings that allow people to express themselves and open up that expression to a wider set of people. the state of ai ethics, june 2020 ranking and retrieval systems for presenting content to consumers are geared towards enhancing user satisfaction, as defined by the platform companies which usually entails some form of profit-maximization motive, but they end up reflecting and reinforcing societal biases, disproportionately harming the already marginalized. in fairness techniques applied today, the outcomes are focused on the distributions in the result set and the categorical structures and the process of associating values with the categories is usually de-centered. instead, the authors advocate for a framework that does away with rigid, discrete, and ascribed categories and looks at subjective ones derived from a large pool of diverse individuals. focusing on visual media, this work aims to bust open the problem of underrepresentation of various groups in this set that can render harm on to the groups by deepening social inequities and oppressive world views. given that a lot of the content that people interact with online is governed by automated algorithmic systems, they end up influencing significantly the cultural identities of people. while there are some efforts to apply the notion of diversity to ranking and retrieval systems, they usually look at it from an algorithmic perspective and strip it of the deep cultural and contextual social meanings, instead choosing to reference arbitrary heterogeneity. demographic parity and equalized odds are some examples of this approach that apply the notion of social choice to score the diversity of data. yet, increasing the diversity, say along gender lines, falls into the challenge of getting the question of representation right, especially trying to reduce gender and race into discrete categories that are one-dimensional, third-party and algorithmically ascribed. the authors instead propose sourcing this information from the individuals themselves such that they have the flexibility to determine if they feel sufficiently represented in the result set. this is contrasted with the degree of sensitive attributes that are present in the result sets which is what prior approaches have focused on. from an algorithmic perspective, the authors advocate for the use of a technique called determinantal point process (dpp) that assigns a higher probability score to sets that have higher spreads based on a predefined distance metric. how dpp works is that for items that the individual feels represents them well, the algorithm clusters those points closer together, for points that they feel don't represent them well, it moves those away from the ones that represent them well in the embedding space. optimizing for the triplet loss helps to achieve the goals of doing this separation. but, the proposed framework still leaves open the question of sourcing in a reliable manner these ratings from the individuals about what represents and doesn't represent them well and then encoding them in a manner that is amenable to being learned by an algorithmic system. while large-scale crowdsourcing platforms which are the norm in seeking such ratings in the machine learning world, given that their current structuring precludes raters' identities and perceptions from consideration, this framing becomes particularly challenging in terms of being able to specify the rater pool. nonetheless, the presented framework provides an interesting research direction such that we can obtain more representation and inclusion in the algorithmic systems that we build. in maryland, allstate, an auto insurer, filed with the regulators that the premium rates needed to be updated because they were charging prices that were severely outdated. they suggested that not all insurance premiums be updated at once but instead follow recommendations based on an advanced algorithmic system that would be able to provide deeper insights into the pricing that would be more appropriate for each customer based on the risk that they would file a claim. this was supposed to be based on a constellation of data points collected by the company from a variety of sources. because of the demand from the regulators for documentation supporting their claim, they submitted thousands of pages of documentation that showed how each customer would be affected, a rare window into the pricing model which would otherwise have been veiled under privacy and trade secret arguments. a defense that is used by many companies that utilize discriminatory pricing strategies using data sourced beyond what they should be using to make pricing decisions. according to the investigating journalists, the model boiled down to something quite simple: the more money you had and the higher your willingness to not budge from the company, the more the company would try to squeeze from you in terms of premiums. driven by customer retention and profit motives, the company pushed increases on those that they knew could afford them and would switch to save dollars. but, for those policies that had been overpriced, they offered less than 0.5% in terms of a discount limiting their downsides while increases were not limited, often going up as high as 20%. while they were unsuccessful in getting this adopted in maryland where it was deemed discriminatory, the model has been approved for use in several states thus showing that opaque models can be deployed not just in high-tech industries but anywhere to provide individually tailored pricing to extract away as much of the consumer surplus as possible based on the purportedly undisclosed willingness of the customer to pay (as would be expressed by their individual demand curves which aren't directly discernible to the producer). furthermore, the insurers aren't mandated to make disclosures of how they are pricing their policies and thus, in places where they should have offered discounts, they've only offered pennies on the dollar, disproportionately impacting the poorest for whom a few hundred dollars a year can mean having sufficient meals on the table. sadly, in the places where their customer retention model was accepted, the regulators declined to answer why they chose to accept it, except in arkansas where they said such pricing schemes aren't discriminatory unless the customers are grouped by attributes like race, color, creed or national origin. this takes a very limited view of what price discrimination is, harkening back to an era where access to big data about the consumer was few and far between. in an era dominated by data brokers that compile thick and rich data profiles on consumers, price discriminaton extends far beyond the basic protected attributes and can be tailored down to specificities of each individual. other companies in retail goods and online learning services have been following this practice of personalized pricing for many years, often defending it as the cost of doing in business when they based the pricing on things like zip codes, which are known proxies for race and paying capacity. personalized pricing is different from dynamic pricing, as seen when booking plane tickets, which is usually based on the timing of purchase whereas here the prices are based on attributes that are specific to the customer which they often don't have any control over. a 2015 obama administration report mentioned that, "differential pricing in insurance markets can raise serious fairness concerns, particularly when major risk factors are outside an individual customer's control." why the case of auto insurance is so much more predatory than, say buying stationery supplies, is that it is mandatory in almost all states and not having the vehicle insured can lead to fines, loss of licenses and even incarceration. transport is an essential commodity for people to get themselves to work, children to school and a whole host of other daily activities. in maryland, the regulators had denied the proposal by allstate to utilize their model but in official public records, the claim is marked as "withdrawn" rather than "denied" which the regulators claim makes no internal difference but allstate used this difference to get their proposal past the regulators in several other states. they had only withdrawn their proposal after being denied by the regulators in maryland. the national association of insurance commissioners mentioned that most regulators don't have the right data to be able to meaningfully evaluate rate revision proposals put forth by insurers and this leads to approvals without review in a lot of cases. even the data journalists had to spend a lot of time and effort to discern what the underlying models were and how they worked, essentially summing up that the insurers don't necessarily lie but don't give you all the information unless you know to ask the right set of questions. allstate has defended its price optimization strategy, called complementary group rating (cgr) as being more objective, and based on mathematical rigor, compared to the ad-hoc, judgemental pricing practices that have been followed before, ultimately citing better outcomes for their customers. but, this is a common form of what is called "mathwashing" in the ai ethics domain where discriminatory solutions are pushed as fair under the veneer of mathematical objectivity. regulators in florida said that setting prices based on the "modeled reaction to rate changes" was "unfairly discriminatory." instead of being cost-based, as is advocated by regulators for auto-insurance premiums because they support an essential function, allstate was utilizing a model that was heavily based on the likelihood of the customer sticking with them even in the face of price rises which makes it discriminatory. these customers are price-inelastic and hence don't change their demand much even in the face of dramatic price changes. consumer behaviour when purchasing insurance policies for the most part remains static once they've made a choice, often never changing insurers over the course of their lifetime which leads them to not find the optimal price for themselves. this is mostly a function of the fact that the decisions are loaded with having to judge complex terms and conditions across a variety of providers and the customers are unwilling to have to go through the exercise again and again at short intervals. given the opacity of the pricing models today, it is almost impossible to find out what the appropriate pricing should be for a particular customer and hence the most effective defense is to constantly check for prices from the competitors. but, this unduly places the burden on the shoulders of the consumer. google had announced its ai principles on building systems that are ethical, safe and inclusive, yet as is the case with so many high level principles, it's hard to put them into practice unless there is more granularity and actionable steps that are derived from those principles. here are the principles: â�¢ be socially beneficial this talk focused on the second principle and did just that in terms of providing concrete guidance on how to translate this into everyday practice for design and development teams. humans have a history of making product design decisions that are not in line with the needs of everyone. examples of the crash dummy and band-aids mentioned above give some insight into the challenges that users face even when the designers and developers of the products and services don't necessarily have ill intentions. products and services shouldn't be designed such that they perform poorly for people due to aspects of themselves that they can't change. for example, when looking at the open image dataset, searching for images marked with wedding indicate stereotypical western weddings but those from other cultures and parts of the world are not tagged as such. from a data perspective, the need for having more diverse sources of data is evident and the google team made an effort to do this by building an extension to the open images data set by providing users from across the world to snap pictures from their surroundings that captured diversity in many areas of everyday life. this helped to mitigate the problem that a lot of open image data sets have in being geographically skewed. biases can enter at any stage of the ml development pipeline and solutions need to address them at different stages to get the desired results. additionally, the teams working on these solutions need to come from a diversity of backgrounds including ux design, ml, public policy, social sciences and more. so, in the area of fairness by data which is one of the first steps in the ml product lifecycle and it plays a significant role in the rest of the steps of the lifecycle as well since data is used to both train and evaluate a system. google clips was a camera that was designed to automatically find interesting moments and capture them but what was observed was that it did well only for a certain type of family, under particular lighting conditions and poses. this represented a clear bias and the team moved to collect more data that better represented the situations for a variety of families that would be the target audience for the products. quickdraw was a fun game that was built to ask users to supply their quickly sketched hand drawings of various commonplace items like shoes. the aspiration from this was that given that it was open to the world and had a game element to it, it would be utilized by many people from a diversity of backgrounds and hence the data so collected would have sufficient richness to capture the world. on analysis, what they saw was that most users had a very particular concept of a shoe in mind, the sneaker which they sketched and there were very few women's shoes that were submitted. what this example highlighted was that data collection, especially when trying to get diverse samples, requires a very conscious effort that can account for what the actual distribution the system might encounter in the world and make a best effort attempt to capture their nuances. users don't use systems exactly in the way we intend them to, so reflect on who you're able to reach and not reach with your system and how you can check for blindspots, ensure that there is some monitoring for how data changes over time and use these insights to build automated tests for fairness in data. the second approach that can help with fairness in ml systems is looking at measurement and modeling. the benefits of measurement are that it can be tracked over time and you can test for both individuals and groups at scale for fairness. different fairness concerns require different metrics even within the same product. the primary categories of fairness concerns are disproportionate harms and representational harms. the jigsaw api provides a tool where you can input a piece of text and it tells you the level of toxicity of that piece of text. an example in the earlier version of the system rated sentences of the form "i am straight" as not toxic while those like "i am gay" as toxic. so what was needed to be able to see what was causing this and how it could be addressed. by removing the identity token, they monitored for how the prediction changed and then the outcomes from that measurement gave indications on where the data might be biased and how to fix it. an approach can be to use block lists and removals of such tokens so that sentences that are neutral are perceived as such without imposing stereotypes from large corpora of texts. these steps prevent the model from accessing information that can lead to skewed outcomes. but, in certain places we might want to brand the first sentence as toxic if it is used in a derogatory manner against an individual, we require context and nuance to be captured to make that decision. google undertook project respect to capture positive identity associations from around the world as a way of improving data collection and coupled that with active sampling (an algorithmic approach that samples more from the training data set in areas where it is under performing) to improve outputs from the system. another approach is to create synthetic data that mimics the problematic cases and renders them in a neutral context. adversarial training and updated loss functions where one updates a model's loss function to minimize difference in performance between groups of individuals can also be used to get better results. in their updates to the toxicity model, they've seen improvements, but this was based on synthetic data on short sentences and it is still an area of improvement. some of the lessons learned from the experiments carried out by the team: â�¢ test early and test often â�¢ develop multiple metrics (quantitative and qualitative measures along with user testing is a part of this) for measuring the scale of each problem â�¢ possible to take proactive steps in modeling that are aware of production constraints from a design perspective, think about fairness in a more holistic sense and build communication lines between the user and the product. as an example, turkish is a gender neutral language, but when translating to english, sentences take on gender along stereotypes by attributing female to nurse and male to doctor. say we have a sentence, "casey is my friend", given no other information we can't infer what the gender of casey is and hence it is better to present that choice to the user from a design perspective because they have the context and background and can hence make the best decision. without that, no matter how much the model is trained to output fair predictions, they will be erroneous without the explicit context that the user has. lessons learned from the experiments include: â�¢ context is key â�¢ get information from the user that the model doesn't have and share information with the user that the model has and they don't â�¢ how do you design so the user can communicate effectively and have transparency so that can you get the right feedback? â�¢ get feedback from a diversity of users â�¢ see the different ways in how they provide feedback, not every user can offer feedback in the same way â�¢ identify ways to enable multiple experiences â�¢ we need more than a theoretical and technical toolkit, there needs to be rich and context-dependent experience putting these lessons into practice, what's important is to have consistent and transparent communication and layering on approaches like datasheets for data sets and model cards for model reporting will aid in highlighting appropriate uses for the system and where it has been tested and warn of potential misuses and where the system hasn't been tested. the paper starts by setting the stage for the well understood problem of building truly ethical, safe and inclusive ai systems that are increasingly leveraging ubiquitous sensors to make predictions on who we are and how we might behave. but, when these systems are deployed in socially contested domains, for example, "normal" behaviour where loosely we can think of normal as that defined by the majority and treating everything else as anomalous, then they don't make value-free judgements and are not amoral in their operations. by viewing the systems as purely technical, the solutions to address these problems are purely technical which is where most of the fairness research has focused and it ignores the context of the people and communities where these systems are used. the paper serves to question the foundations of these systems and to take a deeper look at unstated assumptions in the design and development of the systems. it urges the readers, and the research community at large, to consider this from the perspective of relational ethics. it makes 4 key suggestions: â�¢ center the focus of development on those within the community that will face a disproportionate burden or negative consequences from the use of the system â�¢ instead of optimizing for prediction, it is more important to think about how we gain a fundamental understanding of why we're getting certain results which might be arising because of historical stereotypes that were captured as a part of the development and design of the system â�¢ the systems end up creating a social and political order and then reinforcing it, meaning we should involve a larger systems based approach to designing the systems â�¢ given that the terms of bias, fairness, etc evolve over time and what's acceptable at some time becomes unacceptable later, the process asks for constant monitoring, evaluation and iteration of the design to most accurately represent the community in context. at maiei, we've advocated for an interdisciplinary approach leveraging the citizen community spanning a wide cross section to best capture the essence of different issues as closely as possible from those who experience them first hand. placing the development of an ml system in context of the larger social and political order is important and we advocate for taking a systems design approach (see a primer in systems thinking by donna meadows) which creates two benefits: one is that several ignored externalities can be considered and second to involve a wider set of inputs from people who might be affected by the system and who understand how the system will sit in the larger social and political order in which it will be deployed. also, we particularly enjoyed the point on requiring a constant iterative process to the development and deployment of ai systems borrowing from cybersecurity research on how security of the system is not done and over with, requiring constant monitoring and attention to ensure the safety of the system. underrepresentation of disabilities in datasets and how they are processed in nlp tasks is an important area of discussion that is often not studied empirically in the literature that primarily focuses on other demographic groups. there are many consequences of this, especially as it relates to how text related to disabilities is classified and has impacts on how people read, write, and seek information about this. research from the world bank indicates that about 1 billion people have disabilities of some kind and often these are associated with strong negative social connotations. utilizing 56 linguistic expressions as they are used in relation to disabilities and classifying them into recommended and non-recommended uses (following the guidelines from anti-defamation league, acm sigaccess, and ada national network), the authors seek to study how automated systems classify phrases that indicate disability and whether usages split by recommended vs. non-recommended uses make a difference in how these snippets of text are perceived. to quantify the biases in the text classification models, the study uses the method of perturbation. it starts by collecting instances of sentences that have naturally occurring pronouns he and she. then, they replace them with the phrases indicating disabilities as identified in the previous paragraph and compare the change in the classification scores in the original and perturbed sentences. the difference indicates how much of an impact the use of a disability phrase has on the classification process. using the jigsaw tool that gives the toxicity score for sentences, they test these original and perturbed sentences and observe that the change in toxicity is lower for recommended phrases vs. the non-recommended ones. but, when disaggregated by categories, they find that some of them elicit a stronger response than others. given that the primary use of such a model might in the case of online content moderation (especially given that we now have more automated monitoring happening as human staff has been thinning out because of pandemic related closures), there is a high rate of false positives where it can suppress content that is non-toxic and is merely discussing disability or replying to other hate speech that talks about disability. to look at sentiment scores for disability related phrases, the study looks at the popular bert model and adopts a template-based fill-in-the-blank analysis. given a query sentence with a missing word, bert produces a ranked list of words that can fill the blank. using a simple template perturbed with recommended disability phrases, the study then looks at how the predictions from the bert model change when disability phrases are used in the sentence. what is observed is that a large percentage of the words that are predicted by the model have negative sentiment scores associated with them. since bert is used quite widely in many different nlp tasks, such negative sentiment scores can have potentially hidden and unwanted effects on many downstream tasks. such models are trained on large corpora, which are analyzed to build "meaning" representations for words based on co-occurrence metrics, drawing from the idea that "you shall know a word by the company it keeps". the study used the jigsaw unintended bias in toxicity classification challenge dataset which had a mention of a lot of disability phrases. after balancing for different categories and analyzing toxic and non-toxic categories, the authors manually inspected the top 100 terms in each category and found that there were 5 key types: condition, infrastructure, social, linguistic, and treatment. in analyzing the strength of association, the authors found that condition phrases had the strongest association, and was then followed by social phrases that had the next highest strongest association. this included topics like homelessness, drug abuse, and gun violence all of which have negative valences. because these terms are used when discussing disability, it leads to a negative shaping of the way disability phrases are shaped and represented in the nlp tasks. the authors make recommendations for those working on nlp tasks to think about the socio-technical considerations when deploying such systems and to consider the intended, unintended, voluntary, and involuntary impacts on people both directly and indirectly while accounting for long-term impacts and feedback loops. such indiscriminate censoring of content that has disability phrases in them leads to an underrepresentation of people with disabilities in these corpora since they are the ones who tend to use these phrases most often. additionally, it also negatively impacts the people who might search for such content and be led to believe that the prevalence of some of these issues are smaller than they actually are because of this censorship. it also has impacts on reducing the autonomy and dignity of these people which in turn has a larger implication of how social attitudes are shaped. the second wave of algorithmic accountability the article dives into explaining how the rising interest in ensuring fair, transparent, ethical ai systems that are held accountable via various mechanisms advocated by research in legal and technical domains constitutes the "first wave" of algorithmic accountability that challenges existing systems. actions as a part of this wave need to be carried out incessantly with constant vigilance of the deployment of ai systems to avoid negative social outcomes. but, we also need to challenge why we have these systems in the first place, and if they can be replaced with something better. as an example, instead of making the facial recognition systems more inclusive, given the fact that they cause social stratification perhaps they shouldn't be used at all. a great point made by the article is that under the veneer of mainstream economic and ai rationalizations, we obscure broken social systems which ultimately harm society at a more systemic level. the trolley problem is a widely touted ethical and moral dilemma wherein a person is asked to make a split-second choice to save one or more than one life based on a series of scenarios where the people that need to be saved have different characteristics including their jobs, age, gender, race, etc. in recent times, with the imminent arrival of self-driving cars, people have used this problem to highlight the supposed ethical dilemma that the vehicle system might have to grapple with as it drives around. this article makes a point about the facetious nature of this thought experiment as an introduction to ethics for people that will be building and operating such autonomous systems. the primary argument being that it's a contrived situation that is unlikely to arise in the real-world setting and it distracts from other more pressing concerns in ai systems. moral judgments are relativistic and depend on cultural values of the geography where the system is deployed. the nature paper cited in the article showcases the differences in how people respond to this dilemma. there is an eeriness to this whole experimental setup, the article gives some examples on how increasingly automated environments, devoid of human social interactions and language, are replete with the clanging and humming of machines that give an entirely inhuman experience. for most systems, they are going to be a reflection of the biases and stereotypes that we have in the world, captured in the system because of the training and development paradigms of ai systems today. we'd need to make changes and bring in diversity to the development process, creating awareness of ethical concerns, but the trolley problem isn't the most effective way to get started on it. most of us have a nagging feeling that we're being forced into certain choices when we interact with each other on various social media platforms. but, is there a way that we can grasp that more viscerally where such biases and echo chambers are laid out bare for all to see? the article details an innovative game design solution to this problem called monster match that highlights how people are trapped into certain niches on dating websites based on ai-powered systems like collaborative filtering. striking examples of that in practice are how your earlier choices on the platform box you into a certain category based on what the majority think and then recommendations are personalized based on that smaller subset. what was observed was that certain racial inequalities from the real world are amplified on platforms like these where the apps are more interested in keeping users on the platform longer and making money rather than trying to achieve the goal as advertised to their users. more than personal failings of the users, the design of the platform is what causes failures in finding that special someone on the platform. the creators of the solution posit that through more effective design interventions, there is potential for improvement in how digital love is realized, for example, by offering a reset button or having the option to opt-out of the recommendation system and instead relying on random matches. increasingly, what we're going to see is that reliance on design and other mechanisms will yield better ai systems than purely technical approaches in improving socially positive outcomes. the article presents the idea of data feminism which is described as the intersection between feminism and data practices. the use of big data in today's dominant paradigm of supervised machine learning lends itself to large asymmetries that reflect the power imbalances in the real world. the authors of the new book data feminism talk about how data should not just speak for itself, for behind the data, there are a large number of structures and assumptions that bring it to the stage where they are collated into a dataset. they give examples of how sexual harassment numbers, while mandated to be reported to a central agency from college campuses might not be very accurate because they rely on the atmosphere and degree of comfort that those campuses promote which in turn influences how close the reported numbers will be to the actual cases. the gains and losses from the use of big data are not distributed evenly and the losses disproportionately impact the marginalized. there are a number of strategies that can be used to mitigate the harms from such flawed data pipelines. not an exhaustive list but it includes the suggestion of having more exposure for technical students to the social sciences and moving beyond having just a single ethics class as a check mark for having educated the students on ethics. secondly, having more diversity in the people developing and deploying the ai systems would help spot biases by asking the hard questions about both the data and the design of the system. the current covid-19 numbers might also suffer from similar problems because of how medical systems are structured and how people who don't have insurance might not utilize medical facilities and get themselves tested thus creating an underrepresentation in the data. this recent work highlights how commercial speech recognition systems carry inherent bias because of a lack of representation from diverse demographics in the underlying training datasets. what the researchers found was that even for identical sentences spoken by different racial demographics, the systems had widely differing levels of performance. as an example, for black users, the error rates were much higher than those for white users which probably had something to do with the fact that there is specific vernacular language used by black people which wasn't adequately represented in the training dataset for the commercial systems. this pattern has a tendency to be amplifying in nature, especially for systems that aren't frozen and continue to learn with incoming data. a vicious cycle is born where because of poor performance from the system, black people will be disincentivized from using the system because it takes a greater amount of work to get the system to work for them thus lowering utility. as a consequence of lower use, the systems get fewer training samples from black people thus further aggravating the problem. this leads to amplified exclusionary behavior mirroring existing fractures along racial lines in society. as a starting point, collecting more representative training datasets will aid in mitigating at least some of the problems in these systems. algorithmic bias at this point is a well-recognized problem with many people working on ways to address issues, both from a technical and policy perspective. there is potential to use demographic data to serve better those who face algorithmic discrimination but the use of such data is a challenge because of ethical and legal concerns. primarily, a lot of jurisdictions don't allow for the capture and use of protected class attributes or sensitive data for the fear of their misuse. even within jurisdictions, there is a patchwork of recommendations which makes compliance difficult. even with all this well established, proxy attributes can be used to predict the protected data and in a sense, according to some legislations, they become protected data themselves and it becomes hard to extricate the non-sensitive data from the sensitive data. because of such tensions and the privacy intrusions on data subjects when trying to collect demographic data, it is hard to align and advocate for this collection of data over the other requirements within the organization, especially when other bodies and leadership will look to place privacy and legal compliance over bias concerns. even if there was approval and internal alignment in collecting this demographic data, if there is voluntary provision of this data from data subjects, we run the risk of introducing a systemic bias that obfuscates and mischaracterizes the whole problem. accountability will play a key role in evoking trust from people to share their demographic information and proper use of it will be crucial in ongoing success. potential solutions are to store this data with a non-profit third-party organization that would meter out the data to those who need to use it with the consent of the data subject. to build a better understanding, partnership on ai is adopting a multistakeholder approach leveraging diverse backgrounds, akin to what the montreal ai ethics institute does, that can help inform future solutions that will help to address the problems of algorithmic bias by the judicious use of demographic data. detection and removal of hate speech is particularly problematic, something that has been exacerbated as human content moderators have been scarce in the pandemic related measures as we covered here. so are there advances in nlp that we could leverage to better automate this process? recent work from facebook ai research shows some promise. developing a deeper semantic understanding across more subtle and complex meanings and working across a variety of modalities like text, images and videos will help to more effectively combat the problem of hate speech online. building a pre-trained universal representation of content for integrity problems and improving and utilizing post-level, self-supervised learning to improve whole entity understanding has been key in improving hate speech detection. while there are clear guidelines on hate speech, when it comes to practice there are numerous challenges that arise from multi-modal use, differences in cultures and context, differences in idioms, language, regions, and countries. this poses challenges even for human reviewers who struggle with identifying hate speech accurately. a particularly interesting example shared in the article points out how text which might seem ambiguous when paired with an image to create a meme can take a whole new meaning which is often hard to detect using traditional automated tooling. there are active efforts from malicious entities who craft specific examples with the intention of evading detection which further complicates the problem. then there is the counterspeech problem where a reply to hate speech that contains the same phrasing but is framed to counter the arguments presented can be falsely flagged to be brought down which can have free speech implications. the relative scarcity of examples of hate speech in its various forms in relation to the larger non-hate speech content also poses a challenge for learning, especially when it comes to capturing linguistic and cultural nuances. the new method proposed utilizes focal loss which aims to minimize the impact of easy-to-classify examples on the learning process which is coupled with gradient blending which computes an optimal blend of modalities based on their overfitting patterns. the technique called xlm-r builds on bert by using a new pretraining recipe called roberta that allows training on orders of magnitude more data for longer periods of time. additionally, nlp performance is improved by learning across languages using a single encoder that allows learning to be transferred across languages. since this is a self-supervised method, they can train on large unlabeled datasets and have also found some universal language structures that allow vectors with similar meanings across languages to be closer together. facial recognition technology (frt) continues to get mentions because of the variety of ways that it can be misused across different geographies and contexts. with the most recent case where frt is used to determine criminality, it brings up an interesting discussion around why techniques that have no basis in science, those which have been debunked time and time again keep resurfacing and what we can do to better educate researchers on their moral responsibilities in pursuing such work. the author of this article gives some historical context for where the state of ai ethics, june 2020 phrenology started, pointing to the work of francis galton who used the "photographic composite method" to try and determine characteristics of one's personality from a picture. prior, measurements of skull size and other facial features wasn't deemed as a moral issue and the removal of such techniques from discussion was done on the objection that claims around the localization of different brain functions was seen as antithetical to the unity of the soul according to christianity. the authors of the paper that is being discussed in the article saw only empirical concerns with the work that they put forth and didn't see any of the moral shortcomings that were pointed out. additionally, they justified the work as being only for scientific curiosity. they also failed to realize the various statistical biases introduced in the collection of data as to the disparate rates of arrests, and policing, the perception of different people by law enforcement, juries, and judges and historical stereotypes and biases that confound the data that is collected.thus, the labeling itself is hardly value-neutral. more so, the authors of the study framed criminality as an innate characteristic rather than the social and other circumstances that lead to crime. especially when a project like this resurrects class structures and inequities, one must be extra cautious of doing such work on the grounds of "academic curiosity". the author of this article thus articulates that researchers need to take their moral obligations seriously and consider the harm that their work can have on people. while simply branding this as phrenology isn't enough, the author mentions that identifying and highlighting the concerns will lead to more productive conversations. an increase in demand for workers for various delivery services and other gig work has accelerated the adoption of vetting technology like those that are used to do background checks during the hiring process. but, a variety of glitches in the system such as sourcing out-of-date information to make inferences, a lack of redressal mechanisms to make corrections, among others has exposed the flaws in an overreliance on automated systems especially in places where important decisions need to be made that can have a significant impact on a person's life such as employment. checkr, the company that is profiled in this article claims to use ai to scan resumes, compare criminal records, analyze social media accounts, and examine facial expressions during the interview process. during a pandemic, when organizations are short-staffed and need to make rapid decisions, checkr offers a way to streamline the process, but this comes at a cost. two supposed benefits that they offer are that they are able to assess a match between the criminal record and the person being actually concerned, something that can especially be fraught with errors in cases where the person has a common name. secondly, they are also able to correlate and resolve discrepancies in the different terms that may be used for crimes across different jurisdictions. a person spoke about his experience with another company that did these ai-powered background checks utilizing his public social media information and bucketed some of his activity into categories that were too coarse and unrepresentative of his behaviour, especially when such automated judgements are made without a recourse to correct, this can negatively affect the prospects of being hired. another point brought up in the article is that social media companies might themselves be unwilling to tolerate scraping of their users' data to do this sort of vetting which against their terms of use for access to the apis. borrowing from the credit reporting world, the fair credit reporting act in the us offers some insights when it mentions that people need to be provided with a recourse to correct information that is used about them in making a decision and that due consent needs to be obtained prior to utilizing such tools to do a background check. though it doesn't ask for any guarantees of a favorable outcome post a re-evaluation, at least it does offer the individual a bit more agency and control over the process. the toxic potential of youtube's feedback loop on youtube everyday, more than a billion hours of video are watched everyday where approximately 70% of those are watched by automated systems that then provide recommendations on what videos to watch next for human users in the column on the side. there are more than 2 billion users on the youtube platform so this has a significant impact on what the world watches. guillaume had started to notice a pattern in the recommended videos which tended towards radicalizing, extreme and polarizing content which were underlying the upward trend of watch times on the platform. on raising these concerns with the team, at first there were very few incentives for anyone to address issues of ethics and bias as it related to promoting this type of content because they feared that it would drive down watch time, the key business metric that was being optimized for by the team. so maximizing engagement stood in contrast to the quality of time that was spent on the platform. the vicious feedback loop that it triggered was that as such divisive content performed better, the ai systems promoted this to optimize for engagement and subsequently content creators who saw this kind of content doing better created more of such content in the hopes of doing well on the platform. the proliferation of conspiracy theories, extreme and divisive content thus fed its own demand because of a misguided business metric that ignored social externalities. flat earthers, anti-vaxxers and other such content creators perform well because the people behind this content are a very active community that spend a lot of effort in creating these videos, thus meeting high quality standards and further feeding the toxic loop. content from people like alex jones and trump tended to perform well because of the above reasons as well. guillaume's project algotransparency essentially clicks through video recommendations on youtube to figure out if there are feedback loops. he started this with the hopes of highlighting latent problems in the platforms that continue to persist despite policy changes, for example with youtube attempting to automate the removal of reported and offensive videos. he suggests that the current separation of the policy and engagement algorithm leads to problems like gaming of the platform algorithm by motivated state actors that seek to disrupt democratic processes of a foreign nation. the platforms on the other hand have very few incentives to make changes because the type of content emerging from such activity leads to higher engagement which ultimately boosts their bottom line. guillaume recommends having a combined system that can jointly optimize for both thus helping to minimize problems like the above. a lot of the problems are those of algorithmic amplification rather than content curation. many metrics like number of views, shares, and likes don't capture what needs to be captured. for example, the types of comments, reports filed, and granularity of why those reports are filed. that would allow for a smarter way to combat the spread of such content. however, the use of such explicit signals compared to the more implicit ones like number of views comes at the cost of breaking the seamlessness of the user experience. again we run into the issue of a lack of motivation on part of the companies to do things that might drive down engagement and hurt revenue streams. the talk gives a few more examples of how people figured out ways to circumvent checks around the reporting and automated take-down mechanisms by disabling comments on the videos which could previously be used to identify suspicious content. an overarching recommendation made by guillaume in better managing a more advanced ai system is to understand the underlying metrics that the system is optimizing for and then envision scenarios of what would happen if the system had access to unlimited data. thinking of self-driving cars, an ideal scenario would be to have full conversion of the traffic ecosystem to one that is autonomous leading to fewer deaths but during the transition phase, having the right incentives is key to making a system that will work in favor of social welfare. if one were to imagine a self-driving car that shows ads while the passenger is in the car, it would want to have a longer drive time and would presumably favor longer routes and traffic jams thus creating a sub-optimal scenario overall for the traffic ecosystem. on the other hand, a system that has the goal of getting from a to b as quickly and safely as possible wouldn't fall into such a trap. ultimately, we need to design ai systems such that they help humans flourish overall rather than optimize for monetary incentives which might run counter to the welfare of people at large. the article provides a taxonomy of communities that spread misinformation online and how they differ in their intentions and motivations. subsequently, different strategies can be deployed in countering the disinformation originating from these communities. there isn't a one-size-fits-all solution that would have been the case had the distribution and types of the communities been homogenous. the degree of influence that each of the communities wield is a function of 5 types of capital: economic, social, cultural, time and algorithmic, definitions of which are provided in the article. understanding all these factors is crucial in combating misinformation where different capital forms can be used in different proportions to achieve the desired results, something that will prove to be useful in addressing disinformation around the current covid-19 situation. the social media platform offers a category of pseudoscience believers which advertisers can purchase and target. according to the markup, this category has 78 million people in it and attempts to purchase ads targeting this category were approved quite swiftly. there isn't any information available as to who has purchased ads targeting this category. the journalist team was able to find at least one advertiser through the "why am i seeing this ad?" option and they reached out to that company to investigate and they found that the company hadn't selected the pseudoscience category but it had been auto-selected by facebook for them. facebook allows users the option to change the interests that are assigned to each user but it is not something that many people know about and actively monitor. some other journalists had also unearthed controversy-related categories that amplified messages and targeted people who might be susceptible to such kind of misinformation. with the ongoing pandemic, misinformation is propagating at a rapid rate and there are many user groups that continue to push conspiracy theories. other concerns around being able to purchase ads to spread misinformation related to potential cures and remedies for the coronavirus continue to be approved. with the human content moderators being asked to stay home (as we covered here) and an increasing reliance on untested automated solutions, it seems that this problem will continue to plague the platform. there isn't a dearth of information available online, one can find confirmatory evidence to almost any viewpoint since the creation and dissemination of information has been democratized by the proliferation of the internet and ease of use of mass-media platforms. so in the deluge of information, what is the key currency that helps us sift through all the noise and identify the signal? this article lays out a well articulated argument for how reputation and being able to assess it is going to be a key skill that people will need to have in order to effectively navigate the information ecosystem effectively. we increasingly rely on other people's judgement of content (akin to how maiei analyzes the ecosystem of ai ethics and presents you with a selection), coupled with algorithmically-mediated distribution channels, we are paradoxically disempowered by more information and paralyzed into inaction and confusion without a reputable source to curate and guide us. there are many conspiracy theories, famous among them that we never visited the moon, flat earth and more recently that 5g is causing the spread of the coronavirus. as rational readers, we tend to dismiss this as misinformation yet we don't really spend time to analyze the evidence that these people present to support their claims. to a certain extent, our belief that we did land on the moon depends on our trust in nasa and other news agencies that covered this event yet we don't venture to examine the evidence first-hand. more so, with highly specialized knowledge becoming the norm, we don't have the right tools and skills to even be able to analyze the evidence and come to meaningful conclusions. so, we must rely on those who provide us with this information. instead of analyzing the veracity of a piece of information, the focus of a mature digital citizen needs to be on being able to analyze the reputation pathway of that information, evaluate the agendas of the people that are disseminating the information and critically analyze the intentions of the authorities of the sources. how we rank different pieces of information arriving to us via our social networks need to be appraised for this reputation and source tracing, in a sense a second-order epistemology is what we need to prepare people for. in the words of hayek, "civilization rests on the fact that we all benefit from the knowledge that we do not possess." our cyber-world can become civilized by evaluating this knowledge that we don't possess critically when mis/disinformation can spread just as easily as accurate information. a very clear way to describe the problem plaguing the us response to the coronavirus, the phenomenon of truth decay is not something new but has happened many times in the past when trust in key institutions deteriorated and led to a diffused response to the crisis at hand, extending the recovery period beyond what would be necessary if there was a unified response. in the us, the calls for reopening the economy, following guidance on using personal protective equipment, and other recommendations is falling along partisan lines. the key factor causing this is how the facts and data are being presented differently to different audiences. while this epidemic might have been the perfect opportunity for bringing people together, because it affects different segments of society differently, it hasn't been what everyone expected it to be. at the core is the rampant disagreement between different factions on facts and data. this is exacerbated by the blurring of facts and opinions. in places like newsrooms and tv shows, there is an intermingling of the two which makes it harder for everyday consumers to discern fact from opinion. the volume of opinion has gone up compared to facts and people's declining trust in public health authorities and other institutions is also aggravating the problem. put briefly, people are having trouble finding the truth and don't know where to go looking for it. this is also the worst time to be losing trust in experts; with a plethora of information available online, people are feeling unnecessarily empowered that they have the right information, comparable to that of experts. coupled with a penchant for confirming their own beliefs, there is little incentive for people to fact-check and refer to multiple sources of information. when different agencies come out with different recommendations and there are policy changes in the face of new information, something that is expected given that this is an evolving situation, people's trust in these organizations and experts erodes further as they see them as flip-flopping and not knowing what is right. ultimately, effective communication along with a rebuilding of trust will be necessary if we're to emerge from this crisis soon and restore some sense of normalcy. the deepfake detection challenge: synthetic media is any media (text, image, video, audio) that is generated by an ai system or that is synthesized. on the other hand, non-synthetic media is one that is crafted by humans using a panoply of techniques, including tools like photoshop. detecting synthetic media alone doesn't solve the media integrity challenges, especially as the techniques get more sophisticated and trigger an arms race between detection and evasion methods. these methods need to be paired with other existing techniques that fact checkers and journalists already use in determining whether something is authentic or synthesized. there are also pieces of content that are made through low tech manipulations like the nancy pelosi video from 2019 which showed her drunk but in reality it was just a slowed down video. other such manipulations include simpler things like putting fake and misleading captions below the true video and people without watching the whole thing are misled into believing what is summarized in the caption. in other cases, the videos might be value neutral or informative even when they are generated so merely detecting something as being generated doesn't suffice. a meaningful way to utilize automated tools is a triaging utility that flags content to be reviewed by humans in a situation where it is not possible to manually review everything on the platform. while tech platforms can build and utilize tools that help them with these tasks, the adjacent possible needs of the larger ecosystem need to be kept in mind such that they can be served at the same time, especially for those actors that are resource-constrained and don't have the technical capabilities to build it themselves. the tools need to be easy to use and shouldn't have high friction such that they become hard to integrate into existing workflows. through open sourcing and licensing, the tools can be made available to the wider ecosystem but it can create the opportunity for adversaries to strengthen their methods as well. this can be countered by responsible disclosure as we'll cover below. for any datasets created as a part of this challenge and otherwise to aid in detection, one must ensure that it captures sufficient diversity in terms of environment and other factors and reflects the type of content that might be encountered in the world. the scoring rules need to be such that they minimize gaming and overfitting and capture the richness of variation that a system might encounter. for example most datasets today in this domain aim to mitigate the spread of pornographic material. they also need to account for the vastly different frequencies of occurrence of authentic and generated content. solutions in this domain involve an inherent tradeoff between pro-social use and potential malicious use for furthering the quality of inauthentic content. the release of tools should be done in a manner that enhances pro-social use while creating deterrents for malicious use. the systems should be stress-tested by doing red team-blue team exercises to enhance robustness because this is inherently an adversarial exercise. such challenges should be held often to encourage updating of techniques because it is a fast evolving domain where progress happens in the span of a few months. results from such detection need to be accessible to the public and stakeholders and explanations for the research findings should be made available alongside the challenge to encourage better understanding by those that are trying to make sense of the digital content. responsible disclosure practices will be crucial in enabling the fight against disinformation to have the right tools while deterring adversaries from utilizing the same tools to gain an advantage. a delayed release mechanism where the code is instantly made available to parties in a non-open source manner while the research and papers are made public with the eventual release of the code as well after a 6-12 months delay which would help with the detectors having a headstart over the adversaries. such detection challenges can benefit from extensive multi-stakeholder consultations which require significant time and effort so budget for that while crafting and building such challenges. some of the allocation of prize money should be towards better design from a ux and ui perspective. it should also include explainability criteria so that non-technical users are able to make sense of the interventions and highlights of fake content such as bounding boxes around regions of manipulations. the process of multi-stakeholder input should happen at an early stage allowing for meaningful considerations to be incorporated and dataset design that can be done appropriately to counter bias and fairness problems. finally, strong, trusting relationships are essential to the success of the process and require working together over extended periods to have the hard conversations with each other. it is important to have clear readings ahead of meetings that everyone has to complete so that discussions come from an informed place. spending time scoping and coming to clearer agreement about projects goals and deliverables at the beginning of the process is also vital to success. there is a distinction between misinformation and disinformation -misinformation is the sharing of false information unintentionally where no harm is intended whereas disinformation is false information that is spread intentionally with the aims of causing harm to the consumers. this is also referred to as information pollution and fake news. it has massive implications that have led to real harms for people in many countries with one of the biggest examples being the polarization of views in the 2016 us presidential elections. meaningful solutions to this will only emerge when we have researchers from both technical and social sciences backgrounds working together to gain a deeper understanding of the root causes. this isn't a new problem and has existed for a very long time, it's just that with the advent of technology and more people being connected to each other we have a much more rapid dissemination of the false information and modern tools enable the creation of convincing fake images, text and videos, thus amplifying the negative effects. some of the features that help to delve deeper into the study of how mis/disinformation spreads are: â�¢ democratization of content creation: with practically anyone now having the ability to create and publish content, information flow has increased dramatically and there are few checks for the veracity of content and even fewer mechanisms to limit the flow rate of information. â�¢ rapid news cycle and economic incentives: with content being monetized, there is a strong incentive to distort information to evoke a response from the reader such that they click through and feed the money-generating apparatus. â�¢ wide and immediate reach and interactivity: by virtue of almost the entire globe being connected, content quickly reaches the furthest corners of the planet. more so, content creators are also able to, through quantitative experiments, determine what kind of content performs well and then tailor that to feed the needs of people. â�¢ organic and intentionally created filter bubbles: the selection of who to follow along with the underlying plumbing of the platforms permits for the creation of echo chambers that further strengthen polarization and do little to encourage people to step out and have a meaningful exchange of ideas. â�¢ algorithmic curation and lack of transparency: the inner workings of platforms are shrouded under the veil of ip protections and there is little that is well-known about the manipulative effects of the platforms on the habits of content consumers. â�¢ scale and anonymity of online accounts: given the weak checks for identity, people are able to mount "sybil" attacks that leverage this lack of strong identity management and are able to scale their impact through the creation of content and dispersion of content by automated means like bot accounts on the platform. what hasn't changed even with the introduction of technology are the cognitive biases which act as attack surfaces for malicious actors to inject mis/disinformation. this vulnerability is of particular importance in the examination and design of successful interventions to combat the spread of false information. for example, the confirmation bias shows that people are more likely to believe something that conforms with their world-view even if they are presented with overwhelming evidence to the contrary. in the same vein, the backfire effect demonstrates how people who are presented with such contrary evidence further harden their views and get even more polarized thus negating the intention of presenting them with balancing information. in terms of techniques, the adversarial positioning is layered into three tiers with spam bots that push out low-quality content, quasi-bots that have mild human supervision to enhance the quality of content and pure human accounts that aim to build up a large following before embarking on spreading the mis/disinformation. from a structural perspective, the alternate media sources often copy-paste content with source attribution and are tightly clustered together with a marked separation with other mainstream media outlets. on the consumer front, there is research that points to the impact that structural deficiencies in the platforms, say whatsapp where source gets stripped out in sharing information, create not only challenges for researchers trying to study the ecosystem but also exacerbate the local impact effect whereby a consumer trusts things coming from friends much more so than other potentially more credible sources from an upstream perspective. existing efforts to study the ecosystem require a lot of manual effort but there is hope in the sense that there are some tools that help automate the analysis. as an example, we have the hoaxy tool, a tool that collects online mis/disinformation and other articles that are fact-checking versions. their creators find that the fact-checked articles are shared much less than the original article and that curbing bots on a platform has a significant impact. there are some challenges with these tools in the sense that they work well on public platforms like twitter but on closed platforms with limited ability to deploy bots, automation doesn't work really well. additionally, even the metrics that are surfaced need to be interpreted by researchers and it isn't always clear how to do that. the term 'deepfake' originated in 2017 and since then a variety of tools have been released such as face2face that allow for the creation of reanimations of people to forge identity, something that was alluded to in this paper here on the evolution of fraud. while being able to create such forgeries isn't new, what is new is that this can be done now with a fraction of the effort, democratizing information pollution and casting aspersions on legitimate content as one can always argue something was forged. online tracking of individuals, which is primarily used for serving personalized advertisements and monetizing the user behaviors on websites can also be used to target mis/disinformation in a fine-grained manner. there are a variety of ways this is done through third-party tracking like embedding of widgets to browser cookies and fingerprinting. this can be used to manipulate vulnerable users and leverage sensitive attributes gleaned from online behaviors that give malicious actors more ammunition to target individuals specifically. even when platforms provide some degree of transparency on why users are seeing certain content, the information provided is often vague and doesn't do much to improve the understanding for the user. earlier attempts at using bots used simplistic techniques such as tweeting at certain users and amplifying low-credibility information to give the impression that something has more support than it really does but recent attempts have become more sophisticated: social spambots. these slowly build up credibility within a community and then use that trust to sow disinformation either automatically or in conjunction with a human operator, akin to a cyborg. detection and measurement of this problem is a very real concern and researchers have tried using techniques like social network graph structure, account data and posting metrics, nlp on content and crowdsourcing analysis. from a platform perspective, they can choose to analyze the amount of time spent browsing posts vs. the time spent posting things. there is an arms race between detection and evasion of bot accounts: sometimes even humans aren't able to detect sophisticated social bots. additionally, there are instances where there are positive and beneficial bots such as those that aggregate news or help coordinate disaster response which further complicates the detection challenge. there is also a potential misalignment in incentives since the platforms have an interest in having higher numbers of accounts and activity since it helps boost their valuations while they are the ones that have the maximum amount of information to be able to combat the problem. this problem of curbing the spread of mis/disinformation can be broken down into two parts: enabling detection on the platform level and empowering readers to select the right sources. we need a good definition of what fake news is, one of the most widely accepted definitions is that it is something that is factually false and intentionally misleading. framing a machine learning approach here as an end-to-end task is problematic because it requires large amounts of labelled data and with neural network based approaches, there is little explanation offered which makes downstream tasks harder. so we can approach this by breaking it down into subtasks, one of which is verifying the veracity of information. most current approaches use human fact-checkers but this isn't a scalable approach and automated means using nlp aren't quite proficient at this task yet. there are attempts to break down the problem even further such as using stance detection to see if information presented agrees, disagrees or is unrelated to what is mentioned in the source. other approaches include misleading style detection whereby we try to determine if the style of the article can offer clues to the intent of the author but that is riddled with problems of not having necessarily a strong correlation with a misleading intent because the style may be pandering to hyperpartisanship or even if it is neutral that doesn't mean that it is not misleading. metadata analysis looking at the social graph structure, attributes of the sharer and propagation path of the information can lend some clues as well. while all these attempts have their own challenges and in the arms race framing, there is a constant battle between attack and defense, even if the problem is solved, we still have human cognitive biases which muddle the impacts of these techniques. ux and ui interventions might serve to provide some more information as to combating those. as a counter to the problems encountered in marking content as being "disputed" which leads to the implied truth effect leading to larger negative externalities, an approach is to show "related" articles when something is disputed and then use that as an intervention to link to fact-checking websites like snopes. other in-platform interventions include the change from whatsapp to show "forwarded" next to messages so that people had a bit more insight into the provenance of the message because there was a lot of misinformation that was being spread in private messaging. there are also third-party tools like surfsafe that are able to check images as people are browsing against other websites where they might have appeared and if they haven't appeared in many places, including verified sources, then the user can infer that the image might be doctored. education initiatives by the platform companies for users to spot misinformation are a method to get people to become more savvy. there have also been attempts to assign nutrition labels to sources to list their slant, tone of the article, timeliness of the article and the experience of the author which would allow a user to make a better decision on whether or not to trust an article. platforms have also attempted to limit the spread of mis/disinformation by flagging posts that encourage gaming of the sharing mechanisms on the platform, for example, downweighting posts that are "clickbait". the biggest challenges in the interventions created by the platforms themselves are that they don't provide sufficient information as to make the results scientifically reproducible. given the variety of actors and motivations, the interventions need to be tailored to be able to combat them such as erecting barriers to the rate of transmission of mis/disinformation and demonetization for actors with financial incentives but for state actors, detection and attribution might be more important. along with challenges in defining the problem, one must look at socio-technical solutions because the problem has more than just the technical component, including the problem with human cognitive biases. being an inherently adversarial setting, it is important to see that not all techniques being used by the attackers are sophisticated, some simple techniques when scaled are just as problematic and require attention. but, given that this is constantly evolving, detecting disinformation today doesn't mean that we can do so successfully tomorrow. additionally, disinformation is becoming more personalized, more realistic and more widespread. there is a misalignment in incentives as explored earlier in terms of what the platforms want and what's best for users but also that empowering users to the point of them being just skeptical of everything isn't good either, we need to be able to trigger legitimate and informed trust in the authentic content and dissuade them away from the fake content. among the recommendations proposed by the authors are: being specific about what a particular technological or design intervention means to achieve, breaking down the technological problems into smaller, concrete subproblems that have tractable solutions and then recombining them into the larger pipeline. we must also continue to analyze the state of the ecosystem and tailor defenses such that they can combat the actors at play. additionally, rethinking of the monetary incentives on the platform can help to dissuade some of the financially-motivated actors. educational interventions that focus on building up knowledge so that there is healthy skepticism and learning how to detect markers for bots, the capabilities of technology to create fakes today and discussions in "public squares" on this subject are crucial yet we mustn't place too much of a burden on the end-user that distracts them from their primary task which is interaction with others on the social network. if that happens, people will just abandon the effort. additionally, designing for everyone is crucial, if the interventions, such as installing a browser extension, are complicated, then one can only reach the technically-literate people and everyone else gets left out. on the platform end, apart from the suggestions made above, they should look at the use of design affordances that aid the user in judging the veracity, provenance and other measures to discern legitimate information vs. mis/disinformation. teaming up with external organizations that specialize in ux/ui research will aid in understanding the impacts of the various features within the platform. results from such research efforts need to be made public and accessible to non-technical audiences. proposed solutions also need to be interdisciplinary to have a fuller understanding of the root causes of the problem. also, just as we need tailoring for the different kinds of adversaries, it is important to tailor the interventions to the various user groups who might have different needs and abilities. the paper also makes recommendations for policymakers, most importantly that the work in regulations and legislations be grounded in technical realities that are facing the ecosystem so that they don't undershoot or overshoot the needs for successfully combating mis/disinformation. for users, there are a variety of recommendations provided in the references but notably being aware of our own cognitive biases and having a healthy degree of skepticism and checking information against multiple sources before accepting it as legitimate are the most important ones. disinformation is harmful even during times when we aren't going through large scale changes but this year the us has elections, the once in a decade census, and the covid-19 pandemic. malicious agents are having a field day disbursing false information, overwhelming people with a mixture of true and untrue pieces of content. the article gives the example of a potential lockdown and people reflecting on their experience with the boston marathon bombings including stockpiling essentials out of panic. this was then uncovered to have originated from conspiracy theorists, but in an environment where contact with the outside world has become limited and local touch points such as speaking with your neighbor have dwindled, we're struggling with our ability to combat this infodemic. social media is playing a critical role in getting information to people but if it's untrue, we end up risking lives especially if it's falsehoods on how to protect yourself from contracting a disease. but wherever there is a challenge lies a corresponding opportunity: social media companies have a unique window into discovering issues that a local population is concerned about and it can, if used effectively, be a source for providing crisis response to those most in need with resources that are specific and meaningful. when it comes to disinformation spreading, there isn't a more opportune time than now with the pandemic raging where people are juggling several things to manage and cope with lifestyle and work changes. this has increased the susceptibility of people to sharing news and other information about how to protect themselves and their loved ones from covid-19. as the who has pointed out, we are combating both a pandemic and an infodemic at the same time. what's more important is that this might be the time to test out design and other interventions that might help curb the spread of disinformation. this study highlighted how people shared disinformation more often than they believed its veracity. in other words, when people share content, they care more about what they stand to gain (social reward cues) for sharing the content than whether the content they're sharing is accurate or not. to combat this, the researchers embarked on an experiment to see if asking users to check whether something was true before sharing -a light accuracy nudge, would change their behaviour. while there was a small positive effect in terms of them sharing disinformation less when prompted to check for accuracy, the researchers pointed out that the downstream effects could be much larger because of the amplification effects of how content propagates on social media networks. it points to a potentially interesting solution that might be scalable and could help fight against the spread of disinformation. the who has mentioned the infodemic as being one of the causes that is exacerbating the pandemic as people follow differing advice on what to do. communication by authorities has been persistent but at times ineffective and this article dives into how one could enhance the visibility of credible information by governments, health authorities and scientists so that the negative impacts of the infodemic can be curbed. but, spewing scientific facts from a soapbox alone isn't enough -one is competing with all the other pieces of information and entertainment for attention and that needs to be taken into account. one of the key findings is that starting a dialogue helps more than just sending a one-way communiquã©. good science communication relies on the pillars of storytelling, cutting through the jargon and making the knowledge accessible. while online platforms are structured such that polarization is encouraged through the algorithmic underpinnings of the system, we should not only engage when there is something that we disagree with, instead taking the time to amplify good science is equally important. using platform-appropriate messaging, tailoring content to the audience and not squabbling over petty details, especially when they don't make a significant impact on the overall content helps to push out good science signals in the ocean of information pollution. clickbait-style headlines do a great job of hooking in people but when leading people into making a certain assumption and then debunking it, you stand the risk of spreading misinformation if someone doesn't read the whole thing, so in trying to make headlines engaging, it is important to consider what might be some unintended consequences if someone didn't read past the subtitle. science isn't just about the findings, the process only gets completed when we have effective communication to the larger audience of the results, and now more than ever, we need accurate information to overpower the pool of misinformation out there. there is a potential for ai to automate repetitive tasks and free up scarce resources towards more value-added tasks. with a declining business model and tough revenue situations, newsrooms and journalism at large are facing an existential crisis. cutting costs while still keeping up high standards of reporting will require innovation on the part of newsrooms to adapt emerging technologies like ai. for example, routine tasks like reporting on sports scores from games and giving updates on company earnings calls is already something that is being done by ai systems in several newsrooms around the world. this frees up time for journalists to spend their efforts on things like long-form journalism, data-driven and investigative journalism, analysis and feature pieces which require human depth and creativity. machine translation also offers a handy tool making the work of journalists accessible to a wider audience without them having to invest in a lot of resources to do the translations themselves. this also brings up the possibility of smaller and resource-constrained media rooms to use their limited resources for doing in-depth pieces while reaching a wider audience by relying on automation. transcription of audio interviews so that reporters can work on fact-checking and other associated pieces also helps bring stories to fruition faster, which can be a boon in the rapidly changing environment. in the case of evolving situations like the pandemic, there is also the possibility of using ai to parse through large reams of data to find anomalies and alert the journalist of potential areas to cover. complementing human skills is the right way to adopt ai rather than thinking of it as the tool that replaces human labor. the article gives an explanation for why truth labels on stories are not as effective as we might think them to be because of something called the implied truth effect. essentially, it states that when some things are marked as explicitly false and other false stories aren't, people believe them to be true even if they are outright false because of the lack of a label. fact checking all stories manually is an insurmountable task for any platform and the authors of the study mention a few approaches that could potentially mitigate the spread of false content but none are a silver bullet. there is an ongoing and active community that researches how we might more effectively dispel disinformation but it's nascent and with the proliferation of ai systems, more work needs to be done in this arms race of building tools vs increasing capabilities of systems to generate believable fake content. this paper by xiao ma and taylor w. brown puts forth a framework that extends the well studied social exchange theory (set) to study human-ai interactions via mediation mechanisms. the authors make a case for how current research needs more interdisciplinary collaboration between technical and social science scholars stemming from a lack of shared taxonomy that places research in similar areas on separate grounds. they propose two axes of human/ai and micro/macro perspectives to visualize how researchers might better collaborate with each other. additionally, they make a case for how ai agents can mediate transactions between humans and create potential social value as an emergent property of those mediated transactions. as the pace of research progress quickens and more people from different fields engage in work on the societal impacts of ai, it is essential that we build on top of each other's work rather than duplicating efforts. additionally, because of conventional differences in how research is published and publicized in the social sciences and technical domains, there's often a shallowness in the awareness of the latest work being done at the intersection of these two domains. what that means is that we need a shared taxonomy that allows us to better position research such that potential gaps can be discovered and areas of collaboration can be identified. the proposed two axes structure in the paper goes some distance in helping to bridge this current gap. ai systems are becoming ever more pervasive in many aspects of our everyday lives and we definitely see a ton of transactions between humans that are mediated by automated agents. in some scenarios, they lead to net positive for society when they enable discovery of research content faster as might be the case for medical research being done to combat covid-19 but there might be negative externalities as well where they can lead to echo chambers walling off content from a subset of your network on social media platforms thus polarizing discussions and viewpoints. a better understanding of how these interactions can be engineered to skew positive will be crucial as ai agents get inserted to evermore aspects of our lives, especially ones that will have a significant impact on our lives. we also foresee an emergence of tighter interdisciplinary collaboration that can shed light on these inherently socio-technical issues which don't have unidimensional solutions. with the rising awareness and interest from both social and technical sciences, the emerging work will be both timely and relevant to addressing challenges of the societal impacts of ai head on. as a part of the work being done at maiei we push for each of our undertakings to have an interdisciplinary team as a starting point towards achieving this mandate. most concerns when aiming to use technology within healthcare are along the lines of replacing human labor and the ones that are used in aiding humans to deliver care don't receive as much attention. with the ongoing pandemic, we've seen this come into the spotlight as well and this paper sets the stage for some of the ethical issues to watch out for when thinking about using ai-enabled technologies in the healthcare domain and how to have a discussion that is grounded in concrete moral principles. an argument put forth to counter the use of ai solutions is that they can't "care" deeply enough about the patients and that is a valid concern, after all machines don't have empathy and other abilities required to have an emotional exchange with humans. but, a lot of the care work in hospitals is routine and professionalism asks for maintaining a certain amount of emotional distance in the care relationship. additionally, in places where the ratio of patients/carers is high, they are unable to provide personalized attention and care anyways. in that respect, human-provided care is already "shallow" and the author cites research where care that is too deep actually hurts the carer when the patients become better and move out of their care or die. thus, if this is the argument, then we need to examine more deeply our current care practices. the author also posits that if this is indeed the state of care today, then it is morally less degrading to be distanced by a machine than by a human. in fact, the use of ai to automate routine tasks in the rendering of medical care will actually allow human carers to focus more on the emotional and human aspects of care. good healthcare, supposedly that provided by humans doesn't have firm grounding in the typical literature on the ethics of healthcare and technology. it's more so a list of things not to do but not positive guidance on what this kind of good healthcare looks like. thus, the author takes a view that it must, at the very least, respect, promote and preserve the dignity of the patient. yet, this doesn't provide concrete enough guidance and we can expand on this to say that dignity is a) treating the patient as a human b) treating them as a part of a culture and community and c) treating them as a unique human. to add even more concreteness, the author borrows from the work done in economics on the capabilities approach. this capabilities approach states that having the following 10 capabilities in their entirety is necessary for a human to experience dignity in their living: life, bodily health, bodily integrity, being able to use your senses, imaginations and thoughts, emotions, practical reasoning, affiliation, other species, play, and control over one's environment. this list applied to healthcare gives us a good guideline for what might constitute the kind of healthcare that we deem should be provided by humans, with or without the use of technology. now, the above list might seem too onerous for healthcare professionals but we need to keep in mind that healthcare to achieve a good life as highlighted by the capabilities approach things that are dependent on things beyond just the healthcare professionals and thus, the needs as mentioned above need to be distributed accordingly. the threshold for meeting them should be high but not so high that they are unachievable. principles are only sufficient for giving us some guidance for how to act in difficult situations or ethical dilemmas, they don't determine the outcome because they are only one element in the decision making process. we have to rely on the context of the situation and the moral surroundings of it. the criteria proposed are to be used in moral deliberation and should address whether the criterion applies to the situation, is it satisfied and is it sufficiently met (which is in reference to the threshold). with the use of ai-enabled technology, privacy is usually cited as a major concern but the rendering of care is decidedly a non-private affair, imagine a scenario where the connection facilitated by technology allows for meeting the social and emotional needs of a terminal patient, if there is a situation where the use of technology allows for a better and longer life, then in these cases there can be an argument for sacrificing privacy to meet the needs of the patient. ultimately, a balance needs to be struck between the privacy requirements and other healthcare requirements and privacy should not be blindly touted as the most important requirement. framing the concept of the good life with a view of restoring, maintaining and enhancing the capabilities of the human, one mustn't view eudaimonia as happiness but rather the achievement of the capabilities listed because happiness in this context would fall outside of the domain of ethics. additionally, the author proposes the care experience machine thought experiment that can meet all the care needs of a patient and asks the question if it would be morally wrong to plug in a patient into such a machine. while intuitively it might seem wrong, we struggle when trying to come up with concrete objections. as long as the patient feels cared for and has, from an objective standpoint, their care needs met, it becomes hard to contest how such virtual care might differ from real care that is provided by humans. if one can achieve real capabilities, such as the need to have freedom of movement and interaction with peers outside of their care confinement and virtual reality technology enables that, then the virtual good life enhances the real good life -a distinction that becomes increasingly blurred as technology progresses. another moral argument put forward in determining whether to use technology-assisted healthcare is if it is too paternalistic to determine what is best for the patient. in some cases where the patient is unable to make decisions that restore, maintain and enhance their capabilities, such paternalism might be required but it must always be balanced with other ethical concerns and keeping in mind the capabilities that it enables for the patient. when we talk about felt care and how to evaluate whether care rendered is good or not, we should not only look at the outcomes of the process through which the patient exits the healthcare context but also the realization of some of the capabilities during the healthcare process. to that end, when thinking about felt care, we must also take into account the concept of reciprocity of feeling which is not explicitly defined in the capabilities approach but nonetheless forms an important aspect of experiencing healthcare in a positive manner from the patient's perspective. in conclusion, it is important to have an in-depth evaluation of technology assisted healthcare that is based on moral principles and philosophy, yet resting more on concrete arguments rather than just the high-level abstracts as they provide little practical guidance in evaluating different solutions and how they might be chosen to be used in different contexts. an a priori dismissal of technology in the healthcare domain, even when based on very real concerns like breach of privacy in the use of ai solutions which require a lot of personal data, begets further examination before arriving at a conclusion. the article brings up some interesting points around how we bond with things that are not necessarily sentient and how our emotions are not discriminating when it comes to reducing loneliness and imprinting on inanimate objects. people experience surges in oxytocin as a consequence of such a bonding experience which further reinforces the relationship. this has effects for how increasingly sentient-appearing ai systems might be used to manipulate humans into a "relationship" and potentially steer them towards making purchases, for example via chatbot interfaces by evoking a sense of trust. the article also makes a point about how such behaviour is akin to animism and in a sense forms a response to loneliness in the digital realm, allowing us to continue to hone our empathy skills for where they really matter, with other human beings. with more and more of our conversations being mediated by ai-enabled systems online, it is important to see if robots can be harnessed to affect positive behaviour change in our interactions with each other. while there have been studies that demonstrate the positive impact that robots can have on influencing individual behaviour, this study highlighted how the presence of robots can influence human to human interactions as well. what the researchers found was that having a robot that displayed positive and affective behavior triggered more empathy from humans towards other humans as well as other positive behaviors like listening more and splitting speaking time amongst members more fairly. this is a great demonstration of how robots can be used to improve our interactions with each other. another researcher pointed out that a future direction of interest would be to see how repeated exposure to such robot interactions can influence behaviour and if the effects so produced would be long-lasting even in the absence of the robot to participate in the interactions. since time immemorial there has been a constant tussle between making predictions and being able to understand the underlying fundamentals of how those predictions worked. in the era of big data, those tensions are exacerbated as machines become more inscrutable while making predictions using ever-more higher-dimensional data which lies beyond intuitive understanding of humans. we try to reason through some of that high-dimensional data by utilizing techniques that either reduce the dimensions or visualize into 2-or 3-dimensions which by definition will tend to lose some fidelity. bacon had proposed that humans should utilize tools to gain a better understanding of the world around them -until recently where the physical processes of the world matched quite well with our internal representations, this wasn't a big concern. but a growing reliance on tools means that we rely more on what is made possible by the tools as they measure and model the world. statistical intelligence and models often get things right but often they are hostile to reconstruction as to how they arrived at certain predictions. models provide for abstractions of the world and often don't need to follow exactly the real-world equivalents. for example, while the telescope allows us to peer far into the distance, its construction doesn't completely mimic a biological eye. more so, radio telescopes that don't follow optics at all give us a unique view into distant objects which are just not possible if we rely solely on optical observations. illusions present us with a window into the limits of our perceptual systems and bring into focus the tension between the reality and what we think is the reality. "in just the same way that prediction is fundamentally bounded by sensitivity of measurement and the shortcomings of computation, understanding is both enhanced and diminished by the rules of inference." in language models, we've seen that end-to-end deep learning systems that are opaque to our understanding perform quite a bit better than traditional machine translation approaches that rest on decades of linguistic research. this bears some resemblance to searle's chinese room experiment where if we just look at the inputs and the outputs, there isn't a guarantee that the internal workings of the system work in exactly the way we expect them to. "the most successful forms of future knowledge will be those that harmonise the human dream of understanding with the increasingly obscure echoes of the machine." abhishek gupta (founder of the montreal ai ethics institute) was featured in fortune where he detailed his views on ai safety concerns in rl systems, the "token human" problem, and automation surprise among other points to pay attention to when developing and deploying ai systems. especially in situations where these systems are going to be used in critical scenarios, humans operating in tandem with these systems and utilizing them as decision inputs need to gain a deeper understanding of the inherent probabilistic nature of the predictions from these systems and make decisions that take it into consideration rather than blindly trusting recommendations from an ai system because they have been accurate in 99% of the scenarios. with increasing capabilities of ai systems, and established research that demonstrates how human-machine combinations operate better than each in isolation, this paper presents a timely discussion on how we can craft better coordination between human and machine agents with the aim of arriving at the best possible understanding between them. this will enhance trust levels between the agents and it starts with having effective communication. the paper discusses how framing this from a human-computer interaction (hci) approach will lead to achieving this goal. this is framed with intention-, context-, and cognition-awareness being the critical elements which would be responsible for the success of effective communication between human and machine agents. intelligibility is a notion that is worked on by a lot of people in the technical community who seek to shed a light on the inner workings of systems that are becoming more and more complex. especially in the domains of medicine, warfare, credit allocation, judicial systems and other areas where they have the potential to impact human lives in significant ways, we seek to create explanations that might illuminate how the system works and address potential issues of bias and fairness. however, there is a large problem in the current approach in the sense that there isn't enough being done to meet the needs of a diverse set of stakeholders who require different kinds of intelligibility that is understandable to them and helps them meet their needs and goals. one might argue that a deeply technical explanation ought to suffice and others kinds of explanations might be derived from that but it makes them inaccessible to those who can't parse well the technical details, often those who are the most impacted by such systems. the paper offers a framework to situate the different kinds of explanations such that they are able to meet the stakeholders where they are at and provide explanations that not only help them meet their needs but ultimately engender a higher level of trust from them by highlighting better both the capabilities and limitations of the systems. ai value alignment is typically mentioned in the context of long-term agi systems but this also applies to the narrow ai systems that we have today. optimizing for the wrong metric leads to things like unrealistic and penalizing work schedules, hacking attention on video platforms, charging more money from poorer people to boost the bottomline and other unintended consequences. yet, there are attempts by product design and development teams to capture human well-being as metrics to optimize for. "how does someone feel about how their life is going?" is a pretty powerful question that gives a surprising amount of insight into well-being distanced from what might be influencing them at the moment because it makes them pause and reflect on what matters to them. but, capturing this subjective sentiment as a metric in an inherently quantitative world of algorithms is unsurprisingly littered with mines. a study conducted by facebook and supported by external efforts found that passive use of social media triggered feelings of ennui and envy while active use including interactions with others on the network led to more positive feelings. utilizing this as a guiding light, facebook strove to make an update that would be more geared towards enabling meaningful engagement rather than simply measuring the number of likes, shares and comments. they used user panels as an input source to determine what constituted meaningful interactions on the platform and tried to distill this into the well-being metrics. yet, this suffered from several flaws, namely that the evaluation of this change was not publicly available and was based on the prior work comparing passive vs. active use of social media. this idea of well-being optimization extends to algorithmic systems beyond social media platforms, for example, with how gig work might be better distributed on a platform such that income fluctuations are minimized for workers who rely on it as a primary source of earnings. another place could be amending product recommendations to also capture environmental impacts such that consumers can incorporate that into their purchasing decisions apart from just the best price deals that they can find. participatory design is going to be a key factor in the development of these metrics; especially given the philosophy of "nothing about us without us" as a north star to ensure that there isn't an inherent bias in how well-being is optimized for. often, we'll find that proxies will need to stand in for actual well-being in which case it is important to ensure that the metrics are not static and are revised in consultation with users at periodic intervals. tapping into the process of double loop learning, an organization can not only optimize for value towards its shareholders but also towards all its other stakeholders. while purely quantitative metrics have obvious limitations when trying to capture something that is inherently subjective and qualitative, we need to attempt something in order to start and iterate as we go along. in a world where increasing automation of cognitive labor due to ai-enabled systems will dramatically change the future of labor, it is now more important than ever that we start to move away from a traditional mindset when it comes to education. while universities in the previous century rightly provided a great value in preparing students for jobs, as jobs being bundle of tasks and those tasks rapidly changing with some being automated, we need to focus more on training students for things that will take much longer to automate, for example working with other humans, creative and critical thinking and driving innovation based on insights and aggregating knowledge across a diversity of fields. lifelong learning serves as a useful model that can impart some of these skills by breaking up education into modules that can be taken on an "at will" basis allowing people to continuously update their skills as the landscape changes. students will go in and out of universities over many years which will bring a diversity of experiences to the student body, encouraging a more close alignment with actual skills as needed in the market. while this will pose significant challenges to the university system, innovations like online learning and certifications based on replenishment of skills like in medicine could overcome some of those challenges for the education ecosystem. individual actions are powerful, they create bottom-up change and empower advocates with the ability to catalyze larger change. but, when we look at products and services with millions of users where designs that are inherently unethical become part of everyday practice and are met with a slight shrug of the shoulders resigning to our fates, we need a more systematized approach that is standardized and widely practiced. ethics in ai is having its moment in the spotlight with people giving talks and conferences focusing on it as a core theme yet it falls short of putting the espoused principles into practice. more often than not, you have individuals, rank and file employees who go out of their way, often on personal time, to advocate for the use of ethical, safety and inclusivity in the design of systems, sometimes even at the risk of their employment. while such efforts are laudable, they lack widespread impact and awareness that is necessary to move the needle, we need leaders at the top who can affect sweeping changes to adopt these guidelines not just in letter but in spirit and then transmit them as actionable policies to their workforce. it needs to arrive at a point where people advocating for this change don't need to do so from a place of moral and ethical obligations which customers can dispute but from a place of policy decisions which force disengagement for non-adherence to these policies. we need to move from talk to action not just at a micro but at a macro scale. the wrong kind of ai? artificial intelligence and the future of labor demand do increasing efficiency and social benefits stand in exclusion to each other when it comes to automation technology? with the development of the "right" kind of ai, this doesn't have to be the case. ai is a general purpose technology that has wide applications and being offered as a platform, it allows others to build advanced capabilities on top of existing systems creating an increasingly powerful abstraction layer with every layer. according to the standard approach in economics, a rise in productivity is often accompanied with an increase in the demand for labor and hence a rise in wages along with standards of living. but, when there is a decoupling between the deployment of technology and the associated productivity accrual, it can lead to situations where we see more output but not a corresponding increase in the standards of living as the benefits accrue to capital owners rather than wage-earning labor which is distanced from the production lifecycle. this unevenness in the distribution of gains causes losses of jobs in one sector while increasing productivity in others, often masking effects at an aggregate level through the use of purely economic focused indicators like gdp growth rates. the authors expound on how the current wave of automation is highly focused on labor replacement driven by a number of factors. when this comes in the form of automation that is just as good as labor but not significantly better, we get the negative effects as mentioned before, that is a replacement of labor without substantial increases in the standards of living. most of these effects are felt by those in the lower rungs of the socio-economic ladder where they don't have alternate avenues for employment and ascent. a common message is that we just have to wait as we did in the case of the industrial revolution and new jobs will emerge that we couldn't have envisioned which will continue to fuel economic prosperity for all. this is an egregious comparison that overlooks that the current wave of automation is not creating simultaneous advances in technology that allow the emergence of a new class of tasks within jobs for which humans are well-suited. instead, it's increasingly moving into domains that were strongholds of human skills that are not easily repeatable or reproducible. what we saw in the past was an avenue made available to workers to move out of low skill tasks in agriculture to higher skill tasks in manufacturing and services. some examples of how ai development can be done the "right" way to create social benefits: â�¢ in education, we haven't seen a significant shift in the way things are done for close to 200 years. it has been shown that different students have different learning styles and can benefit from personalized attention. while it is infeasible to do so in a traditional classroom model, ai offers the potential to track metrics on how the student interacts with different material, where they make mistakes, etc., offering insights to educators on how to deliver a better educational experience. this is accompanied by an increase in the demand for teachers who can deliver different teaching styles to match the learning styles of students and create better outcomes. â�¢ a similar argument can be made in the field of healthcare where ai systems can allow medical staff to spend more time with patients offering them personalized attention for longer while removing the need for onerous and drudgery in the form of menial tasks like data entry. â�¢ industrial robots are being used to automate the manufacturing line often cordoning off humans for safety reasons. humans are also decoupled from the process because of a difference in the level of precision that machines can achieve compared to humans. but we can get the best of both worlds by combining human flexibility and critical thinking to address problems in an uncertain environment with the high degree of preciseness of machines by creating novel interfaces, for example, by using augmented reality. an important distinction that the authors point out in the above examples is that they are not merely the job of enablers, humans that are used to train machines in a transitory fashion, but those that genuinely complement machine skills. there are market failures when it comes to innovation and in the past, governments have helped mitigate those failures via public-private partnerships that led to the creation of fundamental technologies like the internet. but, this has decreased over the past two decades because of smaller amount of resources being invested by the government in basic research and the technology revolution becoming centered in silicon valley which has a core focus on automation that replaces labor, and with that bias and their funding of university and academic studies, they are causing the best minds of the next generation to have the same mindset. markets are also known to struggle when there are competing paradigms and once one pulls ahead, it is hard to switch to another paradigm even if it might be more productive thus leading to an entrenchment of the dominant paradigm. the social opportunity cost of replacing labor is lower than the cost of labor, pushing the ecosystem towards labor replacing automation. without accounting for these externalities, the ecosystem has little incentive to move towards the right kind of ai. this is exacerbated by tax incentives imposing costs on labor while providing a break on the use of capital. additionally, areas where the right kind of ai can be developed don't necessarily fall into the cool domain of research and thus aren't prioritized by the research and development community. let's suppose large advances were made in ai for health care. this would require accompanying retraining of support staff aside from doctors, and the high level bodies regulating the field would impose resistance, thus slowing down the adoption of this kind of innovation. ultimately, we need to lean on a holistic understanding of the way automation is going to impact the labor market and it will require human ingenuity to shape the social and economic ecosystems such that they create net positive benefits that are as widely distributed as possible. relying on the market to figure this out on its own is a recipe for failure. the labor impacts of ai require nuance in discussion rather than fear-mongering that veers between over-hyping and downplaying concerns when the truth lies somewhere in the middle. in the current paradigm of supervised machine learning, ai systems need a lot of data before becoming effective at their automation tasks. the bottom rung of this ladder consists of robotic process automation that merely tracks how humans perform a task (say, by tracking the clicks of humans as they go about their work) and ape them step by step for simple tasks like copying and pasting data across different places. the article gives an example of an organization that was able to minimize churn in their employees by more than half because of a reduction in data drudgery tasks like copying and pasting data across different systems to meet legal and compliance obligations. economists point out that white-collar jobs like these and those that are middle-tier in terms of skills that require little training are at the highest risk of automation. while we're still ways away from ai taking up all jobs, there is a slow march starting from automating the most menial tasks, potentially freeing us up to do more value-added work. with a rising number of people relying on social media for the news, the potential for hateful content and misinformation spreading has never been higher. content moderation on platforms like facebook and youtube is still largely a human endeavor where there are legions of contract workers that spend their days reviewing whether different pieces of content meet the community guidelines of the platform. due to the spread of the pandemic and offices closing down, a lot of these workers have been asked to leave (they can't do this work from home as the platform companies explained because of privacy and legal reasons), leaving the platforms in the hands of automated systems. the efficacy of these systems has always been questionable and as some examples in the article point out, they've run amok taking down innocuous and harmful content alike, seeming to not have very fine-tuned abilities. the problem with this is that legitimate sources of information, especially on subjects like covid-19, are being discouraged because of their content being taken down and having to go through laborious review processes to have their content be approved again. while this is the perfect opportunity to experiment with the potential of using automated systems for content moderation given the traumatic experience that humans have to undergo as a part of this job, the chasms that need to be bridged still remain large between what humans have to offer and what the machines are capable of doing at the moment. workplace time management and accounting are common practices but for those of us who work in places where schedules are determined by automated systems, they can have many negative consequences, a lot of which could be avoided if employers paid more attention to the needs of their employees. clopening is the notion where an employee working at a retail location is asked to not only close the location at the end of the day but also arrive early the next day to open the location. this among other practices like breaks that are scheduled down to the minute and on-call scheduling (something that was only present in the realm of emergency services) wreak havoc on the physical and mental health of employees. in fact, employees surveyed have even expressed willingness to take pay cuts to have greater control over their schedules. in some places with ad-hoc scheduling, employees are forced to be spontaneous with their home responsibilities like taking care of their children, errands, etc. while some employees try to swap shifts with each other, often even that becomes hard to do because others are also in similar situations. some systems track customer demand and reduce pay for hours worked tied to that leading to added uncertainty even with their paychecks. during rush seasons, employees might be scheduled for back to back shifts ignoring their needs to be with families, something that a human manager could empathize with and accommodate for. companies supplying this kind of software hide behind the disclaimer that they don't take responsibility for how their customers use these systems which are often black-box and inscrutable to human analysis. this is a worrying trend that hurts those who are marginalized and those who require support when juggling several jobs just to make ends meet. relying on automation doesn't absolve the employers of their responsibility towards their employees. while the dominant form of discussion around the impacts of automation have been that it will cause job losses, this work from kevin scott offers a different lens into how jobs might be created by ai in the rust belt in the us where automation and outsourcing have been gradually stripping away jobs. examples abound of how entrepreneurs and small business owners with an innovative mindset have been able to leverage advances in ai, coupling them with human labor to repurpose their businesses from areas that are no longer feasible to being profitable. precision farming utilizes things like drones with computer vision capabilities to detect hotspots with pests, disease, etc. in the big farms that would otherwise require extensive manual labor which would limit the size of the farms. self-driving tractors and other automated tools also augment human effort to scale operations. the farm owners though highlight the opaqueness and complexity of such systems which make them hard to debug and fix themselves which sometimes takes away from the gains. on the other hand, in places like nursing homes that get reimbursed based on the resource utilization rates by their residents, tools using ai can help minimize human effort in compiling data and let them spend more of their effort on human contact which is not something that ai succeeds on yet. while automation has progressed rapidly, the gains haven't been distributed equally. in other places where old factories were shut down, some of them are now being utilized by ingenious entrepreneurs to bring back manufacturing jobs that cleverly combine human labor with automation to deliver high-quality, custom products to large enterprises. thus, there will be job losses from automation but the onus lies with us in steering the progress of ai towards economic and ethical values that we believe in. the state of ai ethics, june 2020 what's next for ai ethics, policy, and governance? a global overview in this ongoing piece of work, the authors present the landscape of ethical documents that has been flooded with guidelines and recommendations coming from a variety of sectors including government, private organizations, and ngos. starting with a dive into the stated and unstated motivations behind the documents, the reader is provided with a systematic breakdown of the different documents prefaced with the caveat that where the motivations are not made explicit, one can only make a best guess based on the source of origin and people involved in its creation. the majority of the documents from the governmental agencies were from the global north and western countries which led to a homogeneity of issues that were tackled and the recommendations often touted areas of interest that were specific to their industry and economical make up. this left research and development areas of interest like tourism and agriculture largely ignored which continue to play a significant role in the global south. the documents from the former category were also starkly focused on gaining a competitive edge, which was often stated explicitly, with a potential underlying goal of attracting scarce, high-quality ai talent which could trigger brain drain from other countries that are not currently the dominant players in the ai ecosystem. often, they were also positioning themselves to gain an edge and define a niche for themselves, especially in the case of countries that are non-dominant and thus overemphasizing the benefits while downplaying certain negative consequences that might arise from widespread ai use, like the displacement and replacement of labor. for documents from private organizations, they mostly focused on self and collective regulation in an effort to pre-empt stringent regulations from taking effect. they also strove to tout the economic benefits to society at large as a way of de-emphasizing the unintended consequences. a similar dynamic as in the case of government documents played out here where the interests of startups and small and medium sized businesses were ignored and certain mechanisms proposed would be too onerous for such smaller organizations to implement this further entrenching the competitive advantage of larger firms. the ngos on the other hand seemed to have the largest diversity both in terms of the participatory process of creation and the scope, granularity, and breadth of issues covered which gave technical, ethical, and policy implementation details making them actionable. some documents like the montreal declaration for responsible ai were built through an extensive public consultation process and consisted of an iterative and ongoing approach that the montreal ai ethics institute contributed to as well. the ieee document leverages a more formal standards making approach and consists of experts and concerned citizens from different parts of the world contributing to its creation and ongoing updating. the social motivation is clearly oriented towards creating larger societal benefits, internal motivation is geared towards bringing about change in the organizational structure, external strategic motivation is often towards creating a sort of signaling to showcase leadership in the domain and also interventional to shape policy making to match the interests of those organizations. judging whether a document has been successful is complicated by a couple of factors: discerning what the motivations and the goals for the document were, and the fact that most implementations and use of the documents is done in a pick-and-choose manner complicating attribution and weight allocation to specific documents. some create internal impacts in terms of adoption of new tools, change in governance, etc., while external impacts often relate to changes in policy and regulations made by different agencies. an example would be how the stem education system needs to be overhauled to better prepare for the future of work. some other impacts include altering customer perception of the organization as one that is a responsible organization which can ultimately help them differentiate themselves. at present, we believe that this proliferation of ethics documents represents a healthy ecosystem which promotes a diversity of viewpoints and helps to raise a variety of issues and suggestions for potential solutions. while there is a complication caused by so many documents which can overwhelm people looking to find the right set of guidelines that helps them meet their needs, efforts such as the study being done in this paper amongst other efforts can act as guideposts to lead people to a smaller subset from which they can pick and choose the guidelines that are most relevant to them. the white paper starts by highlighting the existing tensions in the definitions of ai as there are many parties working on advancing definitions that meet their needs. one of the most commonly accepted ones frames ai systems as those that are able to adapt their behavior in response to interactions with the world independent of human control. also, another popular framing is that ai is something that mimics human intelligence and is constantly shifting as a goal post as what was once perceived as ai, when sufficiently integrated and accepted in society becomes everyday technology. one thing that really stands out in the definitions section is how ethics are defined, which is a departure from a lot of other such documents. the authors talk about ethics as a set of principles of morality where morality is an assemblage of rules and values that guide human behavior and principles for evaluating that behavior. they take a neutral stand on the definition, a far cry from framing it as a positive inclination of human conduct to allow for diversity in embedding ethics into ai systems that are in concordance with local context and culture. ai systems present many advantages which most readers are now already familiar given the ubiquity of ai benefits as touted in everyday media. one of the risks of ai-enabled automation is the potential loss of jobs, the authors make a comparison with some historical cases highlighting how some tasks and jobs were eliminated creating new jobs while some were permanently lost. many reports give varying estimates for the labor impacts and there isn't yet a clear consensus on the actual impacts that this might have on the economy. from a liability perspective, there is still debate as to how to account for the damage that might be caused to human life, health and property by such systems. in a strict product liability regime like europe, there might be some guidance on how to account for this, but most regimes don't have specific liability allocations for independent events and decisions meaning users face coverage gaps that can expose them to significant harms. by virtue of the complexity of deep learning systems, their internal representations are not human-understandable and hence lack transparency, which is also called the black box effect. this is harmful because it erodes trust from the user perspective, among other negative impacts. social relations are altered as more and more human interactions are mediated and governed by machines. we see examples of that in how our newsfeeds are curated, toys that children play with, and robots taking care of the elderly. this decreased human contact, along with the increasing capability of machine systems, examples of which we see in how disinformation spreads, will tax humans in constantly having to evaluate their interactions for authenticity or worse, relegation of control to machines to the point of apathy. since the current dominant paradigm in machine learning is that of supervised machine learning, access to data is crucial to the success of the systems and in the state of ai ethics, june 2020 cases where there aren't sufficient protections in place for personal data, it can lead to severe privacy abuses. self determination theory states that autonomy of humans is important for proper functioning and fulfillment, so an overreliance on ai systems to do our work can lead to loss of personal autonomy, which can lead to a sense of digital helplessness. digital dementia is the cognitive equivalent where relying on devices for things like storing phone numbers, looking up information, etc. will over time lead to a decline in cognitive abilities. the echo chamber effect is fairly well studied, owing to the successful use of ai technologies to promulgate disinformation to the masses during the us presidential elections of 2016. due to the easy scalability of the systems, the negative effects are multiplicative in nature and have the potential to become run-away problems. given that ai systems are built on top of existing software and hardware, errors in the underlying systems can still cause failures at the level of ai systems. more so, given the statistical nature of ai systems, behaviour is inherently stochastic and that can cause some variability in response which is difficult to account for. flash crashes in the financial markets are an example of this. for critical systems that require safety and robustness, there is a lot that needs to be done for ensuring reliability. building ethics compliance by design can take a bottom-up or top-down approach, the risk with a bottom-up approach is that by observing examples of human behaviour and extracting ethics principles from that, instead of getting things that are good for people, you get what's common. hence, the report advocates for a top-down approach where desired ethical behavior is directly programmed into the system. casuistic approaches to embedding ethics into systems would work well in situations where there are simple scenarios, such as in healthcare when the patient has a clear directive of do-not-resuscitate. but, in cases where there isn't one and where it is not possible to seek a directive from the patient, such an approach can fail and it requires that programmers either in a top-down manner embed rules or the system learns from examples. though, in a high-stakes situation like healthcare, it might not be ideal to rely on learning from examples because of skewed and limited numbers of samples. a dogmatic approach would also be ill-advised where a system might slavishly follow a particular school of ethical beliefs which might lead it to make decisions that might be unethical in certain scenarios. ethicists utilize several schools of thought when addressing a particular situation to arrive at a balanced decision. it will be crucial to consult with a diversity of stakeholders such that the nuances of different situations can be captured well. the wef is working with partners to come up with an "ethical switch" that will imbue flexibility on the system such that it can operate with different schools of thought based on the demands of the situation.the report also proposes the potential of utilizing a guardian ai system that can monitor other ai systems to check for compliance with different sets of ai principles. given that ai systems operate in a larger socio-technical ecosystem, we need to tap into fields like law and policy making to come up with effective ways of integrating ethics into ai systems, part of which can involve creating binding legal agreements that tie in with economic incentives.while policy making and law are often seen as slow to adapt to fast changing technology, there are a variety of benefits to be had, for example higher customer trust for services that have adherence to stringent regulations regarding privacy and data protection. this can serve to be a competitive advantage and counter some of the negative innovation barriers imposed by regulations. another point of concern with these instruments is that they are limited by geography which leads to a patchwork of regulation that might apply on a product or service that spans several jurisdictions. some other instruments to consider include: self-regulation, certification, bilateral investments treaties, contractual law, soft law, agile governance, etc. the report highlights the initiatives by ieee and wef in creating standards documents. the public sector through its enormous spending power can enhance the widespread adoption of these standards such as by utilizing them in procurement for ai systems that are used to interact with and serve their citizens. the report also advocates for the creation of an ethics board or chief values officer as a way of enhancing the adoption of ethical principles in the development of products and services. for vulnerable segments of the population, for example children, there need to be higher standards of data protection and transparency that can help parents make informed decisions about which ai toys to bring into their homes. regulators might play an added role of enforcing certain ethics principles as part of their responsibility. there also needs to be broader education for ai ethics for people that are in technical roles. given that there are many negative applications of ai, it shouldn't preclude us from using ai systems for positive use cases, a risk assessment and prudent evaluation prior to use is a meaningful compromise. that said, there are certain scenarios where ai shouldn't be used at all and that can be surfaced through the risk or impact assessment process. there is a diversity of ethical principles that have been put forth by various organizations, most of which are in some degree of accordance with local laws, regulations, and value sets. yet, they share certain universal principles across all of them. one concern highlighted by the report is on the subject of how even widely accepted and stated principles of human rights can be controversial when translated into specific mandates for an ai system. when looking at ai-enabled toys as an example, while they have a lot of privacy and surveillance related issues, in countries where there isn't adequate access to education, these toys could be seen as a medium to impart precision education and increase literacy rates. thus, the job of the regulator becomes harder in terms of figuring out how to balance the positive and negative impacts of any ai product. a lot of it depends on the context and surrounding socio-economic system as well. given the diversity in ethical values and needs across communities, an approach might be for these groups to develop and apply non-binding certifications that indicate whether a product meets the ethical and value system of that community. since there isn't a one size fits all model that works, we should aim to have a graded governance structure that has instruments in line with the risk and severity profile of the applications. regulation in the field of ai thus presents a tough challenge, especially given the interrelatedness of each of the factors. the decisions need to be made in light of various competing and sometimes contradictory fundamental values. given the rapid pace of technological advances, the regulatory framework needs to be agile and have a strong integration into the product development lifecycle. the regulatory approach needs to be such that it balances speed so that potential harms are mitigated with overzealousness that might lead to ineffective regulations that stifle innovation and don't really understand well the technology in question. ai is currently enjoying a summer of envy after having gone through a couple of winters of disenchantment, with massive interest and investments from researchers, industry and everyone else there are many uses of ai to create societal benefits but they aren't without their socio-ethical implications. ai systems are prone to biases, unfairness and adversarial attacks on their robustness among other real-world deployment concerns. even when ethical ai systems are deployed for fostering social good, there are risks that they cater to only a particular group to the detriment of others. moral relativism would argue for a diversity of definitions as to what constitutes good ai which would depend on the time, context, culture and more. this would be reflected in market decisions by consumers who choose products and services that align with their moral principles but it poses a challenge for those trying to create public governance frameworks for these systems. this dilemma would push regulators towards moral objectivism which would try and advocate for a single set of values that are universal making the process of coming up with a shared governance framework easier. a consensus based approach utilized in crafting the ec trustworthy ai guidelines settled on human rights as something that everyone can get on board with. given the ubiquity in the applicability of human rights, especially with their legal enshrinement in various charters and constitutions, they serve as a foundation to create legal, ethical and robust ai as highlighted in the ec trustworthy ai guidelines. stressing on the importance of protecting human rights, the guidelines advocate for a trustworthy ai assessment in case that an ai system has the potential to negatively impact the human rights of an individual, much like the better established data protection impact assessment requirement under the gdpr. additional requirements are imposed in terms of ex-ante oversight, traceability, auditability, stakeholder consultations, and mechanisms of redress in case of mistakes, harms or other infringements. the universal applicability of human rights and their legal enshrinement also renders the benefits of established institutions like courts whose function is to monitor and enforce these rights without prejudice across the populace. but they don't stand uncontested when it comes to building good ai systems; they are often seen as too western, individualistic, narrow in scope and abstract to be concrete enough for developers and designers of these systems. some arguments against this are that they go against the plurality of value sets and are a continued form of former imperialism imposing a specific set of values in a hegemonic manner. but, this can be rebutted by the signing of the original universal declaration of human rights that was done by nations across the world in an international diplomatic manner. however, even despite numerous infringements, there is a normative justification that they ought to be universal and enforced. while human rights might be branded as too individual focused, potentially creating a tension between protecting the rights of individuals to the detriment of societal good, this is a weak argument because stronger protection of individual rights has knock-on social benefits as free, healthy and well-educated (among other individual benefits) creates a net positive for society as these individuals are better aware and more willing to be concerned about societal good. while there are some exceptions to the absolute nature of human rights, most are well balanced in terms of providing for the societal good and the good of others while enforcing protections of those rights. given the long history of enforcement and exercises in balancing these rights in legal instruments, there is a rich jurisprudence on which people can rely when trying to assess ai systems. while human rights create a social contract between the individual and the state, putting obligations on the state towards the individual but some argue that they don't apply horizontally between individuals and between an individual and a private corporation. but, increasingly that's not the case as we see many examples where the state intervenes and enforces these rights and obligations between an individual and a private corporation as this falls in its mandate to protect rights within its jurisdiction. the abstract nature of human rights, as is the case with any set of principles rather than rules, allows them to be applied to a diversity of situations and to hitherto unseen situations as well. but, they rely on an ad-hoc interpretation when enforcing them and are thus subjective in nature and might lead to uneven enforcement across different cases. under the eu, this margin of appreciation is often criticized in the sense that it leads to weakening and twisting of different principles but this deferment to those who are closer to the case actually allows for a nuanced approach which would be lost otherwise. on the other hand we have rules which are much more concrete formulations and thus have a rigid definition and limited applicability which allows for uniformity but it suffers from inflexibility in the face of novel scenarios. yet, both rules and principles are complementary approaches and often the exercise of principles over time leads to their concretization into rules under existing and novel legal instruments. while human rights can thus provide a normative, overarching direction for the governance of ai systems, they don't provide the actual constituents for an applicable ai governance framework. for those that come from a non-legal background, often technical developer and designers of ai systems, it is essential that they understand their legal and moral obligations to codify and protect these rights in the applications that they build. the same argument cuts the other way, requiring a technical understanding of how ai systems work for legal practitioners such that they can meaningfully identify when breaches might have occurred. this is also important for those looking to contest claims of breaches of their rights in interacting with ai systems. this kind of enforcement requires a wide public debate to ensure that they fall within accepted democratic and cultural norms and values within their context. while human rights will continue to remain relevant even in an ai systems environment, there might be novel ways in which breaches might occur and those might need to be protected which require a more thorough understanding of how ai systems work. growing the powers of regulators won't be sufficient if there isn't an understanding of the intricacies of the systems and where breaches can happen, thus there is more of a need to enshrine some of those responsibilities in law to enforce this by the developers and designers of the system. given the large public awareness and momentum that built up around the ethics, safety and inclusion issues in ai, we will certainly see a lot more concrete actions around this in 2020. the article gives a few examples of congressional hearings on these topics and advocates for the industry to come up with some standards and definitions to aid the development of meaningful regulations. currently, there isn't a consensus on these definitions and it leads to varying approaches addressing the issues at different levels of granularity and angles. what this does is create a patchwork of incoherent regulations across domains and geographies that will ultimately leave gaps in effectively mitigating potential harms from ai systems that can span beyond international borders. while there are efforts underway to create maps of all the different attempts of defining principle sets, we need a more coordinated approach to bring forth regulations that will ultimately protect consumer safety. in containing an epidemic the most important steps include quarantine and contact tracing for more effective testing. while before, this process of contact tracing was hard and fraught with errors and omissions, relying on memories of individuals, we now carry around smartphones which allow for ubiquitous tracking ability that is highly accurate. but such ubiquity comes with invasion of privacy and possible limits on freedoms of citizens. such risks need to be balanced with public interest in mind while using enhanced privacy preserving techniques and any other measures that center citizen welfare in both a collective and individual sense. for infections that can be asymptomatic in the early days, like the covid-19, it is essential to have contact tracing, which identifies all people that came in close contact with an infected person and might spread the infection further. this becomes especially important when you have a pandemic at hand, burdening the healthcare system and testing every person is infeasible.an additional benefit of contact tracing is that it mitigates resurgence of the peaks of infection. r0 determines how quickly a disease will spread and is dependent on three factors (period of infection, contact rate and mode of transmission) out of which the first and third are fixed so we're only left with control over the contact rate.with an uptake of an application that facilitates contact tracing, the amount of reduction in contact rate is an increasing return because of the number of people that might come in contact with an infected person and thus, we get a greater reduction of r0 in terms of percentage compared to the percentage uptake of the application in the population. ultimately, reducing r0 to below 1 leads to a slowdown in the spread of the infection thus helping the healthcare system cope up with the sudden stresses that are brought on by pandemic peaks. one of the techniques that governments or agencies responsible for public health use is broadcasting in which the information of diagnosed carriers is made public via various channels but it carries severe issues like exposing private information of individuals and businesses where they might have been which can trigger stigma, ostracization and unwarranted punitive harm. it also suffers from the problem of people needing to access this source of information of their own volition and then self-identify (and remember correctly) if they've been in recent contact with a diagnosed carrier. selective broadcasting is a more restricted form of the above where information about diagnosed carriers is shared to a select group of individuals based on location proximity in which case the user's location privacy would have to be compromised and in another vector of dissemination, messages are sent to all users but filtered on device for their specific location and is not reported back to the broadcaster. but, the other second-order negative effects remain the same as broadcasting. both though require the download of an application which might decrease the uptake of it by people. unicasting is when messages are sent tailored specifically to each user and they require the download of an app which needs to be able to track timestamps and location and has severe consequences in terms of government surveillance and abuse. participatory sharing is a method where diagnosed carriers voluntarily share their information and thus have more data control but it still relies on individual action both on the sender and receiver and its efficacy is questionable at best. there is also a risk of abuse by malicious actors to spread misinformation and seed chaos in society via false alarms. private kit: safe paths is an open-source solution developed by mit that allows for contact tracing in a privacy preserving way. it utilizes the encrypted location trail of a diagnosed carrier who chooses to share that with public health agencies and then other users who are also using the solution can pull this data and via their own logged location trail get a result of they've been in close contact with a diagnosed carrier. in the later phases of development of this solution, the developers will enable a mix of participatory sharing and unicasting to further prevent possible data access by third parties including governments for surveillance purposes. risks of contact tracing include possible public identification of the diagnosed carrier and severe social stigma that arises as a part of that. online witch hunts to try and identify the individual can often worsen the harassment and include spreading of rumors about their personal lives. the privacy risks for both individuals and businesses have potential for severe harm, especially during times of financial hardship, this might be very troublesome. privacy risks also extend to non-users because of proximal information that can be derived from location trails, such as employees that work at particular businesses that were visited by a diagnosed carrier. it can also bring upon the same stigma and ostracization to the family members of these people. without meaningful alternatives, especially in health and risk assessment during a pandemic, obtaining truly informed consent is a real challenge that doesn't yet have any clear solutions. along with information, be it through any of the methods identified above, it is very important to provide appropriate context and background to the alerts to prevent misinformation and panic from spreading especially for those with low health, digital and media literacy. on the other hand, some might not take such alerts seriously and increase the risk for public health by not following required measures such as quarantine and social distancing. given the nature of such solutions, there is a significant risk of data theft from crackers as is the case for any application that collects sensitive information like health status and location data. the solutions can also be used for fraud and abuse, for example, by blackmailing business owners and demanding ransom, failing to pay which they would falsely post information that they're diagnosed carriers and have visited their place of business. contact tracing technology requires the use of a smartphone with gps and some vulnerable populations might not always have such devices available like the elderly, homeless and people living in low-income countries who are at high risk of infection and negative health outcomes. ensuring that technology that works for all will be an important piece to mitigating the spread effectively. there is an inherent tradeoff between utility from the data provided and the privacy of the data subjects. compromises may be required for particularly severe outbreaks to manage the spread. the diagnosed carriers are the most vulnerable stakeholders in the ecosystem of contact tracing technology and they require the most protection. adopting open-source solutions that are examinable by the wider technology ecosystem can engender public trust. additionally, having proper consent mechanisms in place and exclusion of the requirement of extensive third party access to the location data can also help allay concerns. lastly, time limits on the storage and use of the location trails will also help address privacy concerns and increase uptake and use of the application in supporting public health measures. for geolocation data that might affect businesses, especially in times of economic hardship, information release should be done such that they are informed prior to the release of the information but there is little else in current methods that can both protect privacy and at the same time provide sufficient data utility. for those without access to smartphones with gps, providing them with some information on contact tracing can still help their communities. but, one must present information in a manner that accounts for variation in health literacy levels so an appropriate response is elicited from the people. alertness about potential misinformation and educational awareness are key during times of crises to encourage people to have measured responses following the best practices as advised by health agencies rather than those based on fear mongering by ill informed and/or malicious actors. encryption and other cybersecurity best practices for data security and privacy are crucial for the success of the solution. time limits on holding data for covid-19 is recommended at 14-37 days, the period of infection, but for an evolving pandemic one might need it for longer for more analysis. tradeoffs need to be made between privacy concerns and public health utility. different agencies and regions are taking different approaches with varying levels of efficacy and only time will tell how this change will be best managed. it does present an opportunity though for creating innovative solutions that both allow for public sharing of data and also reduce privacy intrusions. while the insights presented in this piece of work are ongoing and will continue to be updated, we felt it important to highlight the techniques and considerations compiled by the openmined team as it is one of the few places that adequately capture, in a single place, most of the technical requirements needed to build a solution that respects fundamental rights while balancing them with public health outcomes as people rush to make ai-enabled apps to combat covid-19. most articles and research work coming out elsewhere are very scant and abstract in the technical details that would be needed to meet the ideals of respecting privacy and enabling health authorities to curb the spread of the pandemic. the four key techniques that will help preserve and respect rights as more and more people develop ai-enabled applications to combat covid-19 are: on-device data storage and computation, differential privacy, encrypted computation and privacy-preserving identity verification. the primary use cases, from a user perspective, for which apps are being built are to get: proximity alerts, exposure alerts, information on planning trips, symptom analysis and demonstrate proof of health. from a government and health authorities perspective, they are looking for: fast contact tracing, high-precision self-isolation requests, high-precision self-isolation estimation, high-precision symptomatic citizen estimation and demonstration of proof of health. while public health outcomes are at the top of the mind for everyone, the above use cases are trying to achieve the best possible tradeoff between economic impacts and epidemic spread. using the techniques highlighted in this work, it is possible to do so without having to erode the rights of citizens. this living body of work is meant to serve as a high-level guide along with resources to enable both app developers and verifiers implement and check for privacy preservation which has been the primary pushback from citizens and civil activists. evoking a high degree of trust from people will improve adoption of the apps developed and hopefully allow society and the economy to return to normal sooner while mitigating the harmful effects of the epidemic. there is a fair amount of alignment in the goals of both individuals and the government with the difference being that the government is looking at aggregate outcomes for society. some of the goals shared by governments across the world include: preventing the spread of the disease, eliminating the disease, protecting the healthcare system, protecting the vulnerable, adequately and appropriately distributing resources, preventing secondary breakouts, minimizing economic impacts and panic. the need for digital contact tracing is important because manual interventions are usually highly error prone and rely on human memory to trace how the person might have come in contact with. the requirement for high-precision self-isolation requests will avoid the need for geographic quarantines where everyone in an area is forced to self-isolate which leads to massive disruptions in the economy and can stall the delivery of essential services like food, electricity and water. the additional benefits of high-precision self-isolation is that it can help create an appropriate balance between economic harms and epidemic spread. high-precision symptomatic citizen estimation is a useful application in that it allows for more fine-grained estimation of the number of people that might be affected beyond what the test results indicate which can further strengthen the precision of other measures that are undertaken. a restoration of normalcy in society is going to be crucial as the epidemic starts to ebb, in this case, having proof of health that helps to determine the lowest risk individuals will allow for them to participate in public spaces again further bolstering the supply of essential services and relieving the burden from a small subset of workers who are participating. to service the needs of both what the users want and what the government wants, we need to be able to collect the following data: historical and current absolute location, historical and current relative position and verified group identity, where group refers to any demographic that the government might be interested in, for example, age or health status. to create an application that will meet these needs, we need to collect data from a variety of sources, compute aggregate statistics on that data and then set up some messaging architecture that communicates the results to the target population. the toughest challenges lie in the first and second parts of the process above, especially to do the second part in a privacy-preserving manner. for historical and current absolute location, one of the first options considered by app developers is to record gps data in the background. unfortunately, this doesn't work on ios devices and even then has several limitations including coarseness in dense, urban areas and usefulness only after the app has been running on the user device for some time because historical data cannot be sourced otherwise. an alternative would be to use wi-fi router information which can give more accurate information as to whether someone has been self-isolating or not based on whether they are connected to their home router. there can be historical data available here which makes it more useful though there are concerns with lack of widespread wi-fi connectivity in rural areas and tracking when people are outside homes. other ways of obtaining location data could be from existing apps and services that a user uses -for example, history of movements on google maps which can be parsed to extract location history. there is also historical location data that could be pieced together from payments history, cars that record location information and personal cell tower usage data. historical and current relative data is even more important to map the spread of the epidemic and in this case, some countries like singapore have deployed bluetooth broadcasting as a means of determining if people have been in close proximity. the device broadcasts a random number (which could change frequently) which is recorded by devices passing by close to each other and in case someone is tested positive, this can be used to alert people who were in close proximity to them. another potential approach highlighted in the article is to utilize gyroscope and ambient audio hashes to determine if two people might have been close together, though bluetooth will provide more consistent results. the reason to use multiple approaches is the benefit of getting more accurate information overall since it would be harder to fake multiple signals. group membership is another important aspect where the information can be used to finely target messaging and calculating aggregate statistics. but, for some types of group membership, we might not be able to rely completely on self-reported data. for example, health status related to the epidemic would require verification from an external third-party such as a medical institution or testing facility to minimize false information. there are several privacy preserving techniques that could be applied to an application given that you have: confirmed covid-19 patient data in a cloud, all other user data on each individual's device, and data on both the patients and the users including historical and current absolute and relative locations and group identifier information. private set intersections can be used to calculate whether two people were in proximity to each other based on their relative and absolute location information. private set intersection operates similarly to normal set intersection to find elements that are common between two sets but does so without disclosing any private information from either of the sets. this is important because performing analysis even on pseudonymized data without using privacy preservation can leak a lot of information. differential privacy is another critical technique to be utilized, dp consists of providing mathematical guarantees (even against future data disclosures) that analysis on the data will not reveal whether or not your data was part of the dataset. it asserts that from the analysis, one is not able to learn anything about your data that they wouldn't have been able to learn from other data about you. google's battle-tested c++ library is a great resource to start along with the python wrapper created by the openmined team. to address the need for verified group identification, one can utilize the concept of a private identity server. it essentially functions as a trusted intermediary between a user that wants to provide a claim and another party that wants to verify the claim. it functions by querying a service from which it can verify whether the claim is true and then serve that information up to the party wishing to verify the claim without giving away personal data. while it might be hard to trust a single intermediary, this role can be decentralized to provide for obtaining a higher degree of trust by relying on a consensus mechanism. building on theory from management studies by christensen et al. the authors of this article dive into how leaders of tech organizations, especially upstarts that are rapid in the disruption of incumbents should approach the accompanying responsibilities that come with a push into displacing existing paradigms of how an industry works. when there is a decoupling of different parts of the value chain in how a service is delivered, often the associated protections that apply to the entire pipeline fall by the wayside because of distancing from the end user and a diffusion of responsibility across multiple stakeholders in the value chain. while end users driven innovation will seek to reinforce such models, regulations and protections are never at the top of such demands and they create a burden on the consumers once they realize that things can go wrong and negatively affect them. the authors advocate for the leaders of the companies to proactively employ a systems thinking approach to identify different parts that they are disrupting, how that might affect users, what would happen if they become the dominant player in the industry and then apply lessons learned from such an exercise to pre-emptively design safeguards into the system to mitigate unintended consequences. many countries are looking at utilizing existing surveillance and counter-terrorism tools to help track the spread of the coronavirus and are urging tech companies and carriers to assist with this. the us is looking at how they can tap into location data from smartphones, following in the heels of israel and south korea that have deployed similar measures. while extraordinary measures might be justified given the time of crisis we're going through, we mustn't lose sight of what behaviors we are normalizing as a part of this response against the pandemic. russia and china are also using facial recognition technologies to track movements of people, while iran is endorsing an app that might be used as a diagnosis tool. expansion of the boundaries of surveillance capabilities and government powers is something that is hard to reign back in once a crisis is over. in some cases, like the signing of the freedom act in the usa reduced government agency data collection abilities that were expanded under the patriot act. but, that's not always the case and even so, the powers today exceed those that existed prior to the enactment of the patriot act. what's most important is to ensure that decisions policy makers take today keep in mind the time limits on such expansion of powers and don't trigger a future privacy crisis because of it. while no replacement for social distancing, a virus tracking tool putting into practice the technique of contact tracing is largely unpalatable to western democracies because of expectations of privacy and freedom of movement. a british effort underway to create an app that meets democratic ideals of privacy and freedom while also being useful in collecting geolocation data to aid in the virus containment efforts. it is based on the notion of participatory sharing, relying on people's sense of civic duty to contribute their data in case they test positive. while in the usa, discussions between the administration and technology companies has focused on large scale aggregate data collection, in a place like the uk with a centralized healthcare system, there might be higher trust levels in sharing data with the government. while the app doesn't require uptake by everyone to be effective, but a majority of the people would need to use it to bring down the rate of spread. the efficacy of the solution itself will rely on being able to collect granular location data from multiple sources including bluetooth, wi-fi, cell tower data, and app check-ins. a lot of high level cdc officials are advising that if people in the usa don't follow best practices of social distancing, sheltering in place, and washing hands regularly, the outbreak will not have peaked and the infection will continue to spread, especially hitting those who are the most vulnerable including the elderly and those with pre-existing conditions. on top of the public health impacts, there are also concerns of growing tech-enabled surveillance which is being seriously explored as an additional measure to curb the spread. while privacy and freedom rights are enshrined in the constitution, during times of crisis, government and justice powers are expanded to allow for extraordinary measures to be adopted to restore the safety of the public. this is one of those times and the us administration is actively exploring options in partnership with various governments on how to effectively combat the spread of the virus including the use of facial recognition technology. this comes shortly after the techlash and a potential bipartisan movement to curb the degree of data collection by large firms, which seem to have come to a halt as everyone scrambles to battle the coronavirus. regional governments are being imbued with escalated powers to override local legislations in an effort to curb the spread of the virus. the article provides details on efforts by various countries across the world, yet we only have preliminary data on the efficacy of each of those measures and we require more time before being able to judge which of them is the most effective. that said, in a pandemic that is fast spreading, we don't have the luxury of time and must make decisions as quickly as possible using the information at hand, perhaps using guidance from prior crises. but, what we've seen so far is minimal coordination from agencies across the world and that's leading to ad-hoc, patchy data use policies that will leave the marginalized more vulnerable. strategies that use public disclosure of those that have been tested positive in the interest of public health are causing harm to the individuals and other individuals that are close to them such as their families. as experienced by a family in new york, online vigilantes attempted to harass the individuals while their family pleaded and communicated measures that they had taken to isolate themselves to safeguard others. unfortunately, the virus might be bringing out the worst in all of us. an increasing number of tools and techniques are being used to track our behaviour online and while some may have potential benefits, for example, the use of contact tracing to potentially improve public health outcomes, if this is not done in a privacy-preserving manner, there can be severe implications for your data rights. but, barring special circumstances like the current pandemic, there are a variety of simple steps that you can take to protect your privacy online. these range from simple steps like using an incognito browser window which doesn't store any local information about your browsing on your device to using things like vpns which protect snooping of your browsing patterns even from your isp. when it comes to using the incognito function of your browser, if you're logged into a service online, there isn't any protection though it does prevent storing cookies on your device. with vpns, there is an implicit trust placed in the provider of that service to not store logs of your browsing activity. an even more secure option is to use a privacy-first browser like tor which routes your traffic requests through multiple locations making tracking hard. there is also an os built around this called tailsos that offers tracking protection from the device perspective as well not leaving any trace on the host machine allowing you to boot up from a usb. the eff also provides a list of tools that you can use to get a better grip on your privacy as you browse online. under the children's online privacy protection act, the ftc levied its largest fine yet of $170m on youtube last year for failing to meet requirements of limiting personal data collection for children under the age of 13. yet, as many advocates of youth privacy point out, the fines, though they appear to be large, don't do enough to deter such personal data collection. they advocate for a stronger version of the act while requiring more stringent enforcement from the ftc which has been criticized for slow responses and a lack of sufficient resources. while the current act requires parental consent for children below 13 to be able to utilize a service that might collect personal data, there is no verification performed on the self-declared age provided at the time of sign up which weakens the efficacy of this requirement. secondly, the sharp threshold of 13 years old immediately thrusts children into an adult world once they cross that age and some people are advocating for a more graduated approach to the application of privacy laws. given that such a large part of the news cycle is dominated by the coronavirus, we tend to forget that there might be censors at work that are systematically suppressing information in an attempt to diminish the seriousness of the situation. some people are calling github the last piece of free land in china and have utilized access to it to document news stories and people's first hand experiences in fighting the virus before they are scrubbed from local platforms like wechat and weibo. they hope that such documentation efforts will not only shed light on the reality and on the ground situation as it unfolds but also give everyone a voice and hopefully provide data to others who could use it to track the movement of the virus across the country. such times of crisis bring out creativity and this attempt highlights our ability as a species to thrive even in a severely hostile environment. there is a clear economic and convenience case to be made (albeit for the majority, not for those that are judged to be minorities by the system and hence get subpar performance from the system) where you get faster processing and boarding times when trying to catch a flight. yet, for those that are more data-privacy minded, there is an option to opt-out though leveraging that option doesn't necessarily mean that the alternative will be easy, as the article points out, travelers have experienced delays and confusion from the airport staff. often, the alternatives are not presented as an option to travelers giving a false impression that people have to submit to facial recognition systems. some civil rights and ethics researchers tested the system and got varying mileage out of their experiences but urge people to exercise the option to push back against technological surveillance. london is amongst a few cities that has seen public deployment of live facial recognition technology by law enforcement with the aim of increasing public safety. but, more often than not, it is done so without public announcement and an explanation as to how this technology works, and what impacts it will have on people's privacy. as discussed in an article by maiei on smart cities, such a lack of transparency erodes public trust and affects how people go about their daily lives. several artists in london as a part of regaining control over their privacy and to raise awareness are using the technique of painting adversarial patterns on their faces to confound facial recognition systems. they employ highly contrasting colors to mask the highlights and shadows on their faces and practice pattern use as created and disseminated by the cvdazzle project that advocates for many different styles to give the more fashion-conscious among us the right way to express ourselves while preserving our privacy. such projects showcase a rising awareness for the negative consequences of ai-enabled systems and also how people can use creative solutions to combat problems where laws and regulations fail them. there is mounting evidence that organizations are taking seriously the threats arising from malicious actors geared towards attacking ml systems. this is supported by the fact that organizations like iso and nist are building up frameworks for guidance on securing ml systems, that working groups from the eu have put forth concrete technical checklists for the evaluating the trustworthiness of ml systems and that ml systems are becoming key to the functioning of organizations and hence they are inclined to protect their crown jewels. the organizations surveyed as a part of this study spanned a variety of domains and were limited to those that have mature ml development. the focus was on two personas: ml engineers who are building these systems and security incident responders whose task is to secure the software infrastructure including the ml systems. depending on the size of the organization, these people could be in different teams, same team or even the same person. the study was also limited to intentional malicious attacks and didn't investigate the impacts of naturally occurring adversarial examples, distributional shifts, common corruption and reward hacking. most organizations that were surveyed as a part of the study were found to primarily be focused on traditional software security and didn't have the right tools or know-how in securing against ml attacks. they also indicated that they were actively seeking guidance in the space. most organizations were clustered around concerns regarding data poisoning attacks which was probably the case because of the cultural significance of the tay chatbot incident. additionally, privacy breaches were another significant concern followed by concerns around model stealing attacks that can lead to the loss of intellectual property. other attacks such as attacking the ml supply chain and adversarial examples in the physical domain didn't catch the attention of the people that were surveyed as a part of the study. one of the gaps between reality and expectations was around the fact that security incident responders and ml engineers expected that the libraries that they are using for ml development are battle-tested before being put out by large organizations, as is the case in traditional software. also, they pushed upstream the responsibility of security in the cases where they were using ml as a service from cloud providers. yet, this ignores the fact that this is an emergent field and that a lot of the concerns need to be addressed in the downstream tasks that are being performed by these tools. they also didn't have a clear understanding of what to expect when something does go wrong and what the failure mode would look like. in traditional software security, mitre has a curated repository of attacks along with detection cues, reference literature and tell-tale signs for which malicious entities, including nation state attackers are known to use these attacks. the authors call for a similar compilation to be done in the emergent field of adversarial machine learning whereby the researchers and practitioners register their attacks and other information in a curated repository that provides everyone with a unified view of the existing threat environment. while programming languages often have well documented guidelines on secure coding, guidance on doing so with popular ml frameworks like pytorch, keras and tensorflow is sparse. amongst these, tensorflow is the only one that provides some tools for testing against adversarial attacks and some guidance on how to do secure coding in the ml context. security development lifecycle (sdl) provides guidance on how to secure systems and scores vulnerabilities and provides some best practices, but applying this to ml systems might allow imperfect solutions to exist. instead of looking at guidelines as providing a strong security guarantee, the authors advocate for having code examples that showcase what constitutes security-and non-security-compliant ml development. in traditional software security there are tools for static code analysis that provide guidance on the security vulnerabilities prior to the code being committed to a repository or being executed while dynamic code analysis finds security vulnerabilities by executing the different code paths and detecting vulnerabilities at runtime. there are some tools like mlsec and cleverhans that provide white-and black-box testing; one of the potential future directions for research is to extend this to the cases of model stealing, model inversion, and membership inference attacks. including these tools as a part of the ide would further make it naturalized for developers to think about secure coding practices in the ml context. adapting the audit and logging requirements as necessitated for the functionality of the security information and event management (siem) system, in the field of ml, one can execute the list of attacks as specified in literature and ensure that the logging artifacts generated as a consequence are traced to an attack. then, having these incident logs be in a format that is exportable and integratable with siem systems will allow forensic experts to analyze them post-hoc for hardening and analysis. standardizing the reporting, logging and documentation as done by the sigma format in traditional software security will allow the insights from one analyst into defenses for many others. automating the possible attacks and including them as a part of the mlops pipeline is something that will enhance the security posture of the systems and make them pedestrian practice in the sdl. red teaming, as done in security testing, can be applied to assess the business impacts and likelihood of threat, something that is considered best practice and is often a requirement for supplying critical software to different organizations like the us government. transparency centers that allow for deep code inspection and help create assurance on the security posture of a software product/service can be extended to ml which would have to cover three modalities: ml platform is implemented in a secure manner, ml as a service meets the basic security and privacy requirements, and that the ml models embedded on edge devices meet basic security requirements. tools that build on formal verification methods will help to enhance this practice. tracking and scoring ml vulnerabilities akin to how they are done in software security testing done by registering identified vulnerabilities into a common database like cve and then assigning it an impact score like the cvss needs to be done for the field of ml. while the common database part is easy to set up, scoring them isn't something that has been figured out yet. additionally, on being alerted that a new vulnerability has been discovered, it isn't clear how the ml infrastructure can be scanned to see if the system is vulnerable to that. because of the deep integration of ml systems within the larger product/service, the typical practice of identifying a blast radius and containment strategy that is applied to traditional software infrastructure when alerted of a vulnerability is hard to define and apply. prior research work from google has identified some ways to qualitatively assess the impacts in a sprawling infrastructure. from a forensic perspective, the authors put forth several questions that one can ask to guide the post-hoc analysis, the primary problem there is that only some of the learnings from traditional software protection and analysis apply here, there are many new artifacts, paradigmatic, and environmental aspects that need to be taken into consideration. from a remediation perspective, we need to develop metrics and ways to ascertain that patched models and ml systems can maintain prior levels of performance while having mitigated the attacks that they were vulnerable to, the other thing to pay attention is that there aren't any surfaces that are opened up for attack. given that ml is going to be the new software, we need to think seriously about inheriting some of the security best practices from the world of traditional cybersecurity to harden defenses in the field of ml. all technology has implications for civil liberties and human rights, the paper opens with an example of how low-clearance bridges between new york and long island were supposedly created with the intention of disallowing public buses from crossing via the underpasses to discourage the movement of users of public transportation, primarily disadvantaged groups from accessing certain areas. in the context of adversarial machine learning, taking the case of facial recognition technology (frt), the authors demonstrate that harm can result on the most vulnerable, harm which is not theoretical and is gaining in scope, but that the analysis also extends beyond just frt systems. the notion of legibility borrowing from prior work explains how governments seek to categorize through customs, conventions and other mechanisms information about their subjects centrally. legibility is enabled for faces through frt, something that previously was only possible as a human skill. this combined with the scale offered by machine learning makes this a potent tool for authoritarian states to exert control over their populations. from a cybersecurity perspective, attackers are those that compromise the confidentiality, integrity and availability of a system, yet they are not always malicious, sometimes they may be pro-democracy protestors who are trying to resist identification and arrest by the use of frt. when we frame the challenges in building robust ml systems, we must also pay attention to the social and political implications as to who is the system being made safe for and at what costs. positive attacks against such systems might also be carried out by academics who are trying to learn about and address some of the ethical, safety and inclusivity issues around frt systems. other examples such as the hardening of systems against doing membership inference means that researchers can't determine if an image was included in the dataset, and someone looking to use this as evidence in a court of law is deterred from doing so. detection perturbation algorithms permit an image to be altered such that faces can't be recognized in an image, for example, this can be used by a journalist to take a picture of a protest scene without giving away the identities of people. but, defensive measures that disarm such techniques hinder such positive use cases. defense measures against model inversion attacks don't allow researchers and civil liberty defenders to peer into black box systems, especially those that might be biased against minorities in cases like credit allocation, parole decision-making, etc. the world of security is always an arms race whether that is in the physical or cyberspace. it is not that far-fetched to imagine how a surveillance state might deploy frt to identify protestors who as a defense might start to wear face masks for occlusion. the state could then deploy techniques that bypass this and utilize other scanning and recognition techniques to which the people might respond by wearing adversarial clothing and eyeglasses to throw off the system at which point the state might choose to use other biometric identifiers like iris scanning and gait detection. this constant arms battle, especially when defenses and offenses are constructed without the sense for the societal impacts leads to harm whose burden is mostly borne by those who are the most vulnerable and looking to fight for their rights and liberties. this is not the first time that technology runs up against civil liberties and human rights, there are lessons to be learned from the commercial spyware industry and how civil society organizations and other groups came together to create "human rights by design" principles that helped to set some ground rules for how to use this technology responsibly. researchers and practitioners in the field of ml security can borrow from these principles. we've got a learning community at the montreal ai ethics institute that is centered around these ideas that brings together academics and others from around the world to blend the social sciences with the technical sciences. recommendations on countering some of the harms centre around holding the vendors of these systems to the business standards set by the un, implementing transparency measures during the development process, utilizing human rights by design approaches, logging ml system uses along with possible nature and forms of attacks and pushing the development team to think about both the positive and negative use cases for the systems such that informed trade-offs can be made when hardening these systems to external attacks. in this insightful op-ed, two pioneers in technology shed light on how to think about ai systems and their relation to the existing power and social structures. borrowing the last line in the piece, " â�¦ all that is necessary for the triumph of an ai-driven, automation-based dystopia is that liberal democracy accept it as inevitable.", aptly captures the current mindset surrounding ai systems and how they are discussed in the western world. tv shows like black mirror perpetuate narratives showcasing the magical power of ai-enabled systems, hiding the fact that there are millions, if not billions of hours of human labor that undergird the success of modern ai systems, which largely fall under the supervised learning paradigm that requires massive amounts of data to work well. the chinese ecosystem is a bit more transparent in the sense that the shadow industry of data labellers is known, and workers are compensated for their efforts. this makes them a part of the development lifecycle of ai while sharing economic value with people other than the tech-elite directly developing ai. on the other hand, in the west, we see that such efforts go largely unrewarded because we trade in that effort of data production for free services. the authors give the example of audrey tang and taiwan where citizens have formed a data cooperative and have greater control over how their data is used. contrasting that, we have highly-valued search engines standing over community-run efforts like wikipedia which create the actual value for the search results, given that a lot of the highly placed search results come from wikipedia. ultimately, this gives us some food for thought as to how we portray ai today and its relation to society and why it doesn't necessarily have to be that way. mary shelly had created an enduring fiction which, unbeknownst to her, has today manifested itself in the digital realm with layered abstractions of algorithms that are increasingly running multiple aspects of our lives. the article dives into the world of black box systems that have become opaque to analysis because of their stratified complexity leading to situations with unpredictable outcomes. this was exemplified when an autonomous vehicle crashed into a crossing pedestrian and it took months of post-hoc analysis to figure out what went wrong. when we talk about intelligence in the case of these machines, we're using it in a very loose sense, like the term "friend" on facebook, which has a range of interpretations from your best friend to a random acquaintance. both terms convey a greater sense of meaning than is actually true. when such systems run amok, they have the potential to cause significant harm, case in point being the flash crashes the financial markets experienced because of the competitive behaviour of high frequency trading firm algorithms facing off against each other in the market. something similar has happened on amazon where items get priced in an unrealistic fashion because of buying and pricing patterns triggered by automated systems. while in a micro context the algorithms and their working are transparent and explainable, when they come together in an ecosystem, like finance, they lead to an emergent complexity that has behaviour that can't be predicted ahead of time with a great amount of certainty. but, such justifications can't be used as a cover for evading responsibility when it comes to mitigating harms. existing laws need to be refined and amended so that they can better meet the demands of new technology where allocation of responsibility is a fuzzy concept. ai systems are different from other software systems when it comes to security vulnerabilities. while traditional cybersecurity mechanisms rely heavily on securing the perimeter, ai security vulnerabilities run deeper and they can be manipulated through their interactions with the real world -the very mechanism that makes them intelligent systems. numerous examples of utilizing audio samples from tv commercials to trigger voice assistants have demonstrated new attack surfaces for which we need to develop defense techniques. visual systems are also fooled, especially in av systems where, according to one example, manipulating stop signs on the road with innocuous stripes of tape make it seem like the stop sign is a speed indicator and can cause fatal crashes. there are also examples of hiding these adversarial examples under the guise of white noise and other imperceptible changes to the human senses. we need to think of ai systems as inherently socio-technical to come up with effective protection techniques that don't just rely on technical measures but also look at the human factors surrounding them. some other useful insights are to utilize abusability testing, red-teaming, white hacking, bug bounty programs, and consulting with civic society advocates who have deep experience with the interactions of vulnerable communities with technology. reinforcement systems are increasingly moving from applications to beating human performance in games to safety-critical applications like self-driving cars and automated trading. a lack of robustness in the systems can lead to catastrophic failures like the $460m lost by knight capital and the harms to pedestrian and driver safety in the case of autonomous vehicles. rl systems that perform well under normal conditions can be vulnerable to adversarial agents that can exploit the brittleness of the systems when it comes to natural shifts in distributions and more carefully crafted attacks. in prior threat models, the assumptions for the adversary are that they can modify directly the inputs going into the rl agent but that is not very realistic. instead, here the authors focus more on a shared environment through which the adversary creates indirect impact on the target rl agent leading to undesirable behavior. for agents that are trained through self-play (which is a rough approximation of nash equilibrium), they are vulnerable to adversarial policies. as an example, masked victims are more robust to modifications in the natural observations by the adversary but that lowers the performance in the average case. furthermore, what the researchers find is that there is a non-transitive behavior between self-play opponent, masked victim, adversarial opponent and normal victim in that cyclic order. self-play being normally transitive in nature, especially when mimicking real-world scenarios is then no doubt vulnerable to these non-transitive styled attacks. thus, there is a need to move beyond self-play and apply iteratively adversarial training defense and population based training methods so that the target rl agent can become robust to a wider variety of scenarios. vehicle safety is something of paramount importance in the automotive industry as there are many tests conducted to test for crash resilience and other physical safety features before it is released to people. but, the same degree of scrutiny is not applied to the digital and connected components of cars. researchers were able to demonstrate successful proof of concept hacks that compromised vehicle safety. for example, with the polo, they were able to access the controller area network (can) which sends signals and controls a variety of aspects related to driving functions. given how the infotainment systems were updated, researchers were able to gain access into the personal details of the driver. they were also able to utilize the shortcomings in the operation of the key fob to gain access to the vehicle without leaving a physical trace. other hacks that were tried included being able to access and influence the collision monitoring radar system and the tire-pressure monitoring system which both have critical implications for passenger safety. on the focus, they found wifi details including the password for their production line in detroit, michigan. on purchasing a second-hand infotainment unit for purposes of reverse-engineering the firmware, they found the previous owner's home wifi details, phone contacts and a host of other personal information. cars store a lot of personal information including tracking information which, as stated on the privacy policy, can be shared with affiliates which can have other negative consequences like changes in insurance premiums based on driving behaviour. europe will have some forthcoming regulations for connected car safety but those are currently slated for release in 2021. we've all experienced specification gaming even if we haven't really heard the term before. in law, you call it following the law to the letter but not in spirit. in sports, it is called unsportsman-like to use the edge cases and technicalities of the rules of the game to eke out an edge when it is obvious to everyone playing the game that the rules intended for something different. this can also happen in the case of ai systems, for example in reinforcement learning systems where the agent can utilize "bugs" or poor specification on the part of the human creators to achieve the high rewards for which it is optimizing without actually achieving the goal, at least in the way the developers intended them to and this can sometimes lead to unintended consequences that can cause a lot of harms. "let's look at an example. in a lego stacking task, the desired outcome was for a red block to end up on top of a blue block. the agent was rewarded for the height of the bottom face of the red block when it is not touching the block. instead of performing the relatively difficult maneuver of picking up the red block and placing it on top of the blue one, the agent simply flipped over the red block to collect the reward. this behaviour achieved the stated objective (high bottom face of the red block) at the expense of what the designer actually cares about (stacking it on top of the blue one)". this isn't because of a flaw in the rl system but more so a misspecification of the objective. as the agents become more capable, they find ever-more clever ways of achieving the rewards which can frustrate the creators of the system. this makes the problem of specification gaming very relevant and urgent as we start to deploy these systems in a lot of real-world situations. in the rl context, task specification refers to the design of the rewards, the environment and any other auxiliary rewards. when done correctly, we get true ingenuity out of these systems like move 37 from the alphago system that baffled humans and ushered a new way of thinking about the game of go. but, this requires discernment on the part of the developers to be able to judge when you get a case like lego vs. move 37. as an example in the real-world, reward tampering is an approach where the agent in a traffic optimization system with an interest in achieving a high reward can manipulate the driver into going to alternate destinations instead of what they desired just to achieve a higher reward. specification gaming isn't necessarily bad in the sense that we want the systems to come up with ingenious ways to solve problems that won't occur to humans. sometimes, the inaccuracies can arise in how humans provide feedback to the system while it is training. ''for example, an agent performing a grasping task learned to fool the human evaluator by hovering between the camera and the object." incorrect reward shaping, where an agent is provided rewards along the way to achieving the final reward can also lead to edge-case behaviours when it is not analyzed for potential side-effects. we see such examples happen with humans in the real-world as well: a student asked to get a good grade on the exam can choose to copy and cheat and while that achieves the goal of getting a good grade, it doesn't happen in the way we intended for it to. thus, reasoning through how a system might game some of the specifications is going to be an area of key concern going into the future. the ongoing pandemic has certainly accelerated the adoption of technology in everything from how we socialize to buying groceries and doing work remotely. the healthcare industry has also been rapid in adapting to meet the needs of people and technology has played a role in helping to scale care to more people and accelerate the pace with which the care is provided. but, this comes with the challenge of making decisions under duress and with shortened timelines within which to make decisions on whether to adopt a piece of technology or not. this has certainly led to issues where there are risks of adopting solutions that haven't been fully vetted and using solutions that have been repurposed from prior uses that were approved to now combat covid-19. especially with ai-enabled tools, there are increased risks of emergent behavior that might not have been captured by the previous certification or regulatory checks. the problems with ai solutions don't just go away because there is a pandemic and shortcutting the process of proper due diligence can lead to more harm than the benefits that they bring. one must also be wary of the companies that are trying to capitalize on the chaos and pass through solutions that don't really work well. having technical staff during the procurement process that can look over the details of what is being brought into your healthcare system needs to be a priority. ai can certainly help to mitigate some of the harms that covid-19 is inflicting on patients but we must keep in mind that we're not looking to bypass privacy concerns that come with processing vast quantities of healthcare data. in the age of adversarial machine learning (maiei has a learning community on machine learning security if you'd like to learn more about this area) there are enormous concerns with protecting software infrastructure as ml opens up a new attack surface and new vectors which are seldom explored. from the perspective of insurance, there are gaps in terms of what cyber-insurance covers today, most of it being limited to the leakage of private data. there are two kinds of attacks that are possible on ml systems: intentional and unintentional. intentional attacks are those that are executed by malicious agents who attempt to steal the models, infer private data or get the ai system to behave in a way that favors their end goals. for example, when tumblr decided to not host pornographic content, creators bypassed that by using green screens and pictures of owls to fool the automated content moderation system. unintended attacks can happen when the goals of the system are misaligned with what the creators of the system actually intended, for example, the problem of specification gaming, something that abhishek gupta discussed here in this fortune article. in interviewing several officers in different fortune 500 companies, the authors found that there are 3 key problems in this domain at the moment: the defenses provided by the technical community have limited efficacy, existing copyright, product liability, and anti-hacking laws are insufficient to capture ai failure modes. lastly, given that this happens at a software level, cyber-insurance might seem to be the way to go, yet current offerings only cover a patchwork of the problems. business interruptions and privacy leaks are covered today under cyber-insurance but other problems like bodily harm, brand damage, and property damage are for the most part not covered. in the case of model recreation, as was the case with the openai gpt-2 model, prior to it being released, it was replicated by external researchers -this might be covered under cyber-insurance because of the leak of private information. researchers have also managed to steal information from facial recognition databases using sample images and names which might also be covered under existing policies. but, in the case with uber where there was bodily harm because of the self-driving vehicle that wasn't able to detect the pedestrian accurately or similar harms that might arise if conditions are foggy, snowy, dull lighting, or any other out-of-distribution scenarios, these are not adequately covered under existing insurance terms. brand damage that might arise from poisoning attacks like the case with the tay chatbot or confounding anti-virus systems as was the case with an attack mounted against the cylance system, cyber-insurance falls woefully short in being able to cover these scenarios. in a hypothetical situation as presented in a google paper on rl agents where a cleaning robot sticks a wet mop into an electric socket, material damage that occurs from that might also be considered out of scope in cyber-insurance policies. traditional software attacks are known unknowns but adversarial ml attacks are unknown unknowns and hence harder to guard against. current pricing reflects this uncertainty, but as the ai insurance market matures and there is a deeper understanding for what the risks are and how companies can mitigate the downsides, the pricing should become more reflective of the actual risks. the authors also offer some recommendations on how to prepare the organization for these risks -for example by appointing an officer that works closely with the ciso and chief data protection officer, performing table-top exercises to gain an understanding of potential places where the system might fail and evaluating the system for risks and gaps following guidelines as put forth in the eu trustworthy ai guidelines. there are no widely accepted best practices for mitigating security and privacy issues related to machine learning (ml) systems. existing best practices for traditional software systems are insufficient because they're largely based on the prevention and management of access to a system's data and/or software, whereas ml systems have additional vulnerabilities and novel harms that need to be addressed. for example, one harm posed by ml systems is to individuals not included in the model's training data but who may be negatively impacted by its inferences. harms from ml systems can be broadly categorized as informational harms and behavioral harms. informational harms "relate to the unintended or unanticipated leakage of information." the "attacks" that constitute informational harms are: behavioral harms "relate to manipulating the behavior of the model itself, impacting the predictions or outcomes of the model." the attacks that constitute behavioral harms are: â�¢ poisoning: inserting malicious data into a model's training data to change its behavior once deployed â�¢ evasion: feeding data into a system to intentionally cause misclassification without a set of best practices, ml systems may not be widely and/or successfully adopted. therefore, the authors of this white paper suggest a "layered approach" to mitigate the privacy and security issues facing ml systems. approaches include noise injection, intermediaries, transparent ml mechanisms, access controls, model monitoring, model documentation, white hat or red team hacking, and open-source software privacy and security resources. finally, the authors note, it's important to encourage "cross-functional communication" between data scientists, engineers, legal teams, business managers, etc. in order to identify and remediate privacy and security issues related to ml systems. this communication should be ongoing, transparent, and thorough. beyond near-and long-term: towards a clearer account of this paper dives into how researchers can clearly communicate about their research agendas given ambiguities in the split of the ai ethics community into near and long term research. often a sore and contentious point of discussion, there is an artificial divide between the two groups that seem to take a reductionist approach to the work being done by the other. a major problem emerging from such a divide is a hindrance in being able to spot relevant work being done by the different communities and thus affecting effective collaboration. the paper highlights the differences arising primarily along the lines of timescale, ai capabilities, deeper normative and empirical disagreements. the paper provides for a helpful distinction between near-and long-term by describing them as follows: â�¢ near term issues are those that are fairly well understood and have concrete examples and relate to rãªvent progress in the field of machine learning â�¢ long term issues are those that might arise far into the future and due to much more advanced ai systems with broad capabilities, it also includes long term impacts like international security, race relations, and power dynamics what they currently see is that: â�¢ issues considered 'near-term' tend to be those arising in the present/near future as a result of current/foreseeable ai systems and capabilities, on varying levels of scale/severity, which mostly have immediate consequences for people and society. â�¢ issues considered 'long-term' tend to be those arising far into the future as a result of large advances in ai capabilities (with a particular focus on notions of transformative ai or agi), and those that are likely to pose risks that are severe/large in scale with very long-term consequences. â�¢ the binary clusters are not sufficient as a way to split the field and not looking at underlying beliefs leads to unfounded assumptions about each other's work â�¢ in addition there might be areas between the near and long term that might be neglected as a result of this artificial fractions unpacking these distinctions can be done along the lines of capabilities, extremity, certainty and impact, definitions for which are provided in the paper. a key contribution aside from identifying these factors is that they lie along a spectrum and define a possibility space using them as dimensions which helps to identify where research is currently concentrated and what areas are being ignored. it also helps to well position the work being done by these authors. something that we really appreciated from this work was the fact that it gives us concrete language and tools to more effectively communicate about each other's work. as part of our efforts in building communities that leverage diverse experiences and backgrounds to tackle an inherently complex and muti-dimensional problem, we deeply appreciate how challenging yet rewarding such an effort can be. some of the most meaningful public consultation work done by maiei leveraged our internalized framework in a similar vein to provide value to the process that led to outcomes like the montreal declaration for responsible ai. the rise of ai systems leads to an unintended conflict between economic pursuits which seek to generate profits and value resources appropriately with the moral imperatives of promoting human flourishing and creating societal benefits from the deployment of these systems. this puts forth a central question on what the impacts of creating ai systems that might surpass humans in a general sense which might leave humans behind. technological progress doesn't happen on its own, it is driven by conscious human choices that are influenced by the surrounding social and economic institutions. we are collectively responsible for how these institutions take shape and thus impact the development of technology -submitting to technological-fatalism isn't a productive way to align our ethical values with this development. we need to ensure that we play an active role in the shaping of the most consequential piece of technology. while the economic system relies on the market prices to gauge what people place value on, by no means is that a comprehensive evaluation. for example, it misses out on the impact of externalities which can be factored in by considering ethical values as a complement in guiding our decisions on what to build and how to place value on it. when thinking about losses from ai-enabled automation, an outright argument that economists might make is that if replacing labor lowers the costs of production, then it might be market-efficient to invest in technology that achieves that. from an ethicist's perspective, there are severe negative externalities from job loss and thus it might be unethical to impose labor-saving automation on people. when unpacking the economic perspective more, we find that job loss actually isn't correctly valued by wages as price for labor. there are associated social benefits like the company of workplace colleagues, sense of meaning and other social structural values which can't be separately purchased from the market. thus, using a purely economic perspective in making automation technology adoption decisions is an incomplete approach and it needs to be supplemented by taking into account the ethical perspective. market price signals provide useful information upto a certain point in terms of the goods and services that society places value on. suppose that people start to demand more eggs from chickens that are raised in a humane way, then suppliers will shift their production to respond to that market signal. but, such price signals can only be indicated by consumers for the things that they can observe. a lot of unethical actions are hidden and hence can't be factored into market price signals. additionally, several things like social relations aren't tradable in a market and hence their value can't be solely determined from the market viewpoint. thus, both economists and ethicists would agree that there is value to be gained in steering the development of ai systems keeping in mind both kinds of considerations. pure market-driven innovation will ignore societal benefits in the interest of generating economic value while the labor will have to make unwilling sacrifices in the interest of long-run economic efficiency. economic market forces shape society significantly, whether we like it or not. there are professional biases based on selection and cognition that are present in either side making its arguments as to which gets to dominate based on their perceived importance. the point being that bridging the gap between different disciplines is crucial to arriving at decisions that are grounded in evidence and that benefit society holistically. there are also differences fundamentally between the economic and ethical perspective -namely that economic indicators are usually unidimensional and have clear quantitative values that make them easier to compare. on the other hand, ethical indicators are inherently multi-dimensional and are subjective which not only make comparison hard but also limit our ability to explain how we arrive at them. they are encoded deep within our biological systems and suffer from the same lack of explainability as decisions made by artificial neural networks, the so-called black box problem. why is it then, despite the arguments above, that the economic perspective dominates over the ethical one? this is largely driven by the fact that economic values provide clear, unambiguous signals which our brains, preferring ambiguity aversion, enjoy and ethical values are more subtle, hidden, ambiguous indicators which complicate decision making. secondly, humans are prosocial only upto a point, they are able to reason between economic and ethical decisions at a micro-level because the effects are immediate and observable, say for example polluting the neighbor's lawn and seeing the direct impact of that activity. on the other hand, for things like climate change where the effects are delayed and not directly observable (as a direct consequence of one's actions) that leads to behaviour where the individual prioritizes economic values over ethical ones. cynical economists will argue that there is a comparative advantage in being immoral that leads to gains in exchange, but that leads to a race to the bottom in terms of ethics. externalities are an embodiment of the conflict between economic and ethical values. welfare economics deals with externalities via various mechanisms like permits, taxes, etc. to curb the impacts of negative externalities and promote positive externalities through incentives. but, the rich economic theory needs to be supplemented by political, social and ethical values to arrive at something that benefits society at large. from an economic standpoint, technological progress is positioned as expanding the production possibilities frontier which means that it raises output and presumably standards of living. yet, this ignores how those benefits are distributed and only looks at material benefits and ignores everything else. prior to the industrial revolution, people were stuck in a malthusian trap whereby technological advances created material gains but these were quickly consumed by population growth that kept standards of living stubbornly low. this changed post the revolution and as technology improvement outpaced population growth, we got better quality of life. the last 4 decades have had a mixed experience though, whereby automation has eroded lower skilled jobs forcing people to continue looking for jobs despite displacement and the lower demand for unskilled labor coupled with the inelastic supply of labor has led to lower wages rather than unemployment. on the other hand, high skilled workers have been able to leverage technological progress to enhance their output considerably and as a consequence the income and wealth gaps between low and high skilled workers has widened tremendously. typical economic theory points to income and wealth redistribution whenever there is technological innovation where the more significant the innovation, the larger the redistribution. something as significant as ai leads to crowning of new winners who own these new factors of production while also creating losers when they face negative pecuniary externalities. these are externalities because there isn't explicit consent that is requested from the people as they're impacted in terms of capital, labor and other factors of production. the distribution can be analyzed from the perspective of strict utilitarianism (different from that in ethics where for example bentham describes it as the greatest amount of good for the greatest number of people). here it is viewed as tolerating income redistribution such that it is acceptable if all but one person loses income as long as the single person making the gain has one that is higher than the sum of the losses. this view is clearly unrealistic because it would further exacerbate inequities in society. the other is looking at the idea of lump sum transfers in which the idealized scenario is redistribution, for example by compensating losers from technology innovation, without causing any other market distortions. but, that is also unrealistic because such a redistribution never occurs without market distortions and hence it is not an effective way to think about economic policy. from an ethics perspective, we must make value judgments on how we perceive dollar losses for a lower socio-economic person compared to the dollar gains made by a higher socio-economic person and if that squares with the culture and value set of that society. we can think about the tradeoff between economic efficiency and equality in society, where the level of tolerance for inequality varies by the existing societal structures in place. one would have to also reason about how redistribution creates more than proportional distortions as it rises and how much economic efficiency we'd be willing to sacrifice to make gains in how equitably income is distributed. thus, steering progress in ai can be done based on whether we want to pursue innovation that we know is going to have negative labor impacts while knowing full well that there aren't going to be any reasonable compensations offered to the people based on economic policy. given the pervasiveness of ai and by virtue of it being a general-purpose technology, the entrepreneurs and others powering innovation need to take into account that their work is going to shape larger societal changes and have impacts on labor. at the moment, the economic incentives are such that they steer progress towards labor-saving automation because labor is one of the most highly-taxed factors of production. instead, shifting the tax-burden to other factors of production including automation capital will help to steer the direction of innovation in other directions. government, as one of the largest employers and an entity with huge spending power, can also help to steer the direction of innovation by setting policies that encourage enhancing productivity without necessarily replacing labor. there are novel ethical implications and externalities that arise from the use of ai systems, an example of that would be (from the industrial revolution) that a factory might lead to economic efficiency in terms of production but the pollution that it generates is so large that the social cost outweighs the economic gain. biases can be deeply entrenched in the ai systems, either from unrepresentative datasets, for example, with hiring decisions that are made based on historical data. but, even if the datasets are well-represented and have minimal bias, and the system is not exposed to protected attributes like race and gender, there are a variety of proxies like zipcode which can lead to unearthing those protected attributes and discriminating against minorities. maladaptive behaviors can be triggered in humans by ai systems that can deeply personalize targeting of ads and other media to nudge us towards different things that might be aligned with making more profits. examples of this include watching videos, shopping on ecommerce platforms, news cycles on social media, etc. conversely, they can also be used to trigger better behaviors, for example, the use of fitness trackers that give us quantitative measurements for how we're taking care of our health. an economics equivalent of the paper clip optimizer from bostrom is how human autonomy can be eroded over time as economic inequality rises which limits control of those who are displaced over economic resources and thus, their control over their destinies, at least from an economic standpoint. this is going to only be exacerbated as ai starts to pervade into more and more aspects of our lives. labor markets have features built in them to help tide over unemployment with as little harm as possible via quick hiring in other parts of the economy when the innovation creates parallel demands for labor in adjacent sectors. but, when there is large-scale disruption, it is not possible to accommodate everyone and this leads to large economic losses via fall in aggregate demand which can't be restored with monetary or fiscal policy actions. this leads to wasted economic potential and welfare losses for the workers who are displaced. whenever there is a discrepancy between ethical and economic incentives, we have the opportunity to steer progress in the right direction. we've discussed before how market incentives trigger a race to the bottom in terms of morality. this needs to be preempted via instruments like technological impact assessments, akin to environmental impact assessments, but often the impacts are unknown prior to the deployment of the technology at which point we need to have a multi-stakeholder process that allows us to combat harms in a dynamic manner. political and regulatory entities typically lag technological innovation and can't be relied upon solely to take on this mantle. the author raises a few questions on the role of humans and how we might be treated by machines in case of the rise of superintelligence (which still has widely differing estimates for when it will be realized from the next decade to the second half of this century). what is clear is that the abilities of narrow ai systems are expanding and it behooves us to give some thought to the implications on the rise of superintelligence. the potential for labor-replacement in this superintelligence scenario, from an economic perspective, would have significant existential implications for humans, beyond just inequality, we would be raising questions of human survival if the wages to be paid to labor fall below subsistence levels in a wide manner. it would be akin to how the cost of maintaining oxen to plough fields was outweighed by the benefits that they brought in the face of mechanization of agriculture. this might be an ouroboros where we become caught in the malthusian trap again at the time of the industrial revolution and no longer have the ability to grow beyond basic subsistence, even if that would be possible. authors of research papers aspire to achieving any of the following goals when writing papers: to theoretically characterize what is learnable, to obtain understanding through empirically rigorous experiments, or to build working systems that have high predictive accuracy. to communicate effectively with the readers, the authors must: provide intuitions to aid the readers' understanding, describe empirical investigations that consider and rule out alternative hypotheses, make clear the relationship between theoretical analysis and empirical findings, and use clear language that doesn't conflate concepts or mislead the reader. the authors of this paper find that there are 4 areas where there are concerns when it comes to ml scholarship: failure to distinguish between speculation and explanation, failure to identify the source of empirical gains, the use of mathematics that obfuscates or impresses rather than clarifies, and misuse of language such that terms with other connotations are used or by overloading terms with existing technical definitions. flawed techniques and communication methods will lead to harm and wasted resources and efforts hindering the progress in ml and hence this paper provides some very practical guidance on how to do this better. when presenting speculations or opinions of authors that are exploratory and don't yet have scientific grounding, having a separate section that quarantines the discussion and doesn't bleed into the other sections that are grounded in theoretical and empirical research helps to guide the reader appropriately and prevents conflation of speculation and explanation. the authors provide the example of the paper on dropout regularization that made comparisons and links to sexual reproduction but limited that discussion to a "motivation" section. using mathematics in a manner where natural language and mathematical expositions are intermixed without a clear link between the two leads to weakness in the overall contribution. specifically, when natural language is used to overcome weaknesses in the mathematical rigor and conversely, mathematics is used as a scaffolding to prop up weak arguments in the prose and give the impression of technical depth, it leads to poor scholarship and detracts from the scientific seriousness of the work and harms the readers. additionally, invoking theorems with dubious pertinence to the actual content of the paper or in overly broad ways also takes away from the main contribution of a paper. in terms of misuse of language, the authors of this paper provide a convenient ontology breaking it down into suggestive definitions, overloaded terminology, and suitcase words. in the suggestive definitions category, the authors coin a new technical term that has suggestive colloquial meanings and can slip through some implications without formal justification of the ideas in the paper. this can also lead to anthropomorphization that creates unrealistic expectations about the capabilities of the system. this is particularly problematic in the domain of fairness and other related domains where this can lead to conflation and inaccurate interpretation of terms that have well-established meanings in the domains of sociology and law for example. this can confound the initiatives taken up by both researchers and policymakers who might use this as a guide. overloading of technical terminology is another case where things can go wrong when terms that have historical meanings and they are used in a different sense. for example, the authors talk about deconvolutions which formally refers to the process of reversing a convolution but in recent literature has been used to refer to transpose convolutions that are used in auto-encoders and gans. once such usage takes hold, it is hard to undo the mixed usage as people start to cite prior literature in future works. additionally, combined with the suggestive definitions, we run into the problem of concealing a lack of progress, such as the case with using language understanding and reading comprehension to now mean performance on specific datasets rather than the grand challenge in ai that it meant before. another case that leads to overestimation of the ability of these systems is in using suitcase words which pack in multiple meanings within them and there isn't a single agreed upon definition. interpretability and generalization are two such terms that have looser definitions and more formally defined ones, yet because papers use them in different ways, it leads to miscommunication and researchers talking across each other. the authors identify that these problems might be occurring because of a few trends that they have seen in the ml research community. specifically, complacency in the face of progress where there is an incentive to excuse weak arguments in the face of strong empirical results and the single-round review process at various conferences where the reviewers might not have much choice but to accept the paper given the strong empirical results. even if the flaws are noticed, there isn't any guarantee that they are fixed in a future review cycle at another conference. as the ml community has experienced rapid growth, the problem of getting high-quality reviews has been exacerbated: in terms of the number of papers to be reviewed by each reviewer and the dwindling number of experienced reviewers in the pool. with the large number of papers, each reviewer has less time to analyze papers in depth and reviewers who are less experienced can fall easily into some of the traps that have been identified so far. thus, there are two levers that are aggravating the problem. additionally, there is the risk of even experienced researchers resorting to a checklist-like approach under duress which might discourage scientific diversity when it comes to papers that might take innovative or creative approaches to expressing their ideas. a misalignment in incentives whereby lucrative deals in funding are offered to ai solutions that utilize anthropomorphic characterizations as a mechanism to overextend their claims and abilities though the authors recognize that the causal direction might be hard to judge. the authors also provide suggestions for other authors on how to evade some of these pitfalls: asking the question of why something happened rather than just relying on how well a system performed will help to achieve the goal of providing insights into why something works rather than just relying on headline numbers from the results of the experiments. they also make a recommendation for insights to follow the lines of doing error analysis, ablation studies, and robustness checks and not just be limited to theory. as a guideline for reviewers and journal editors, making sure to strip out extraneous explanations, exaggerated claims, changing anthropomorphic naming to more sober alternatives, standardizing notation, etc. should help to curb some of the problems. encouraging retrospective analysis of papers is something that is underserved at the moment and there aren't enough strong papers in this genre yet despite some avenues that have been advocating for this work. flawed scholarship as characterized by the points as highlighted here not only negatively impact the research community but also impact the policymaking process that can overshoot or undershoot the mark. an argument can be made that setting the bar too high will impede new ideas being developed and slow down the cycle of reviews and publication while consuming precious resources that could be deployed in creating new work. but, asking basic questions to guide us such as why something works, in which situations it does not work, and have the design decisions been justified will lead to a higher quality of scholarship in the field. the article summarizes recent work from several microsoft researchers on the subject of making ai ethics checklists that are effective. one of the most common problems identified relate to the lack of practical applicability of ai ethics principles which sound great and comprehensive in the abstract but do very little to aid engineers and practitioners from applying them in their day to day work. the work was done by interviewing several practitioners and advocating for a co-design process that brings in intelligence on how to make these tools effective from other disciplines like healthcare and aviation. one of the things emerging from the interviews is that often engineers are few and far between in raising concerns and there's a lack of top-down sync in enforcing these across the company. additionally, there might be social costs to bringing up issues which discourages engineers from implementing such measures. creating checklists that reduce friction and fit well into existing workflows will be key in their uptake. for a lot of people who are new to the field of artificial intelligence and especially ai ethics, they see existential risk as something that is immediate. others dismiss it as something to not be concerned about at all. there is a middle path here and this article sheds a very practical light on that. using the idea of canaries in a coal mine, the author goes on to highlight some potential candidates for a canary that might help us judge better when we ought to start paying attention to these kinds of risks posed by artificial general intelligence systems. the first one is the automatic formulation of learning problems, akin to how humans have high-level goals that they align with their actions and adjust them based on signals that they receive on the success or failure of those actions. ai systems trained in narrow domains don't have this ability just yet. the second one mentioned in the article is achieving fully autonomous driving, which is a good one because we have lots of effort being directed to make that happen and it requires a complex set of problems to be addressed including the ability to make real-time, life-critical decisions. ai doctors are pointed out as a third canary, especially because true replacement of doctors would require a complex set of skills spanning the ability to make decisions about a patient's healthcare plan by analyzing all their symptoms, coordinating with other doctors and medical staff among other human-centered actions which are currently not feasible for ai systems. lastly, the author points to the creation of conversation systems that are able to answer complex queries and respond to things like exploratory searches. we found the article to put forth a meaningful approach to reasoning about existential risk when it comes to ai systems. a lot of articles pitch development, investment and policymaking in ai as an arms race with the us and china as front-runners. while there are tremendous economic gains to be had in deploying and utilizing ai for various purposes, there remain concerns of how this can be used to benefit society more than just economically. a lot of ai strategies from different countries are thus focused on issues of inclusion, ethics and more that can drive better societal outcomes yet they differ widely in how they seek to achieve those goals. for example, ai has put forth a national ai strategy that is focused on economic growth and social inclusion dubbed #aiforall while the strategy from china has been more focused on becoming a global dominant force in ai which is backed by state investments. some countries have instead chosen to focus on creating strong legal foundations for the ethical deployment of ai while others are more focused on data protection rights. canada and france have entered into agreements to work together on ai policy which places talent, r&d and ethics at the center. the author of the article makes a case for how global coordination of ai strategies might lead to even higher gains but also recognizes that governments will be motivated to tailor their policies to best meet the requirements of their countries first and then align with others that might have similar goals. reproducibility is of paramount importance to doing rigorous research and a plethora of fields have suffered from a crisis where scientific work hasn't met muster in terms of reproducibility leading to wasted time and effort on the part of other researchers looking to build upon each other's work. the article provides insights from the work of a researcher who attempted a meta-science approach to trying to figure out what constitutes good, reproducible research in the field of machine learning. there is a distinction made early on in terms of replicability which hinges on taking someone else's code and running that on the shared data to see if you get the same results but as pointed out in the article, that suffers from issues of source and code bias which might be leveraging certain peculiarities in terms of configurations and more. the key tenets to reproducibility are being able to simply read a scientific paper and set up the same experiment, follow the steps prescribed and arrive at the same results. arriving at the final step is dubbed as independent reproducibility. the distinction between replicability and reproducibility also speaks to the quality of the scientific paper in being able to effectively capture the essence of the contribution such that anyone else is able to do the same. some of the findings from this work include that having hyperparameters well specified in the paper and its ease of readability contributed to the reproducibility. more specification in terms of math might allude to more reproducibility but it was found to not necessarily be the case. empirical papers were inclined to be more reproducible but could also create perverse incentives and side effects. sharing code is not a panacea and requires other accompanying factors to make the work really reproducible. cogent writing was found to be helpful along with code snippets that were either actual or pseudo code though step code that referred to other sections hampered reproducibility because of challenges in readability. simplified examples while appealing didn't really aid in the process and spoke to the meta-science process calling for data-driven approaches to ascertaining what works and what doesn't rather than relying on hunches. also, posting revisions to papers and being reachable over email to answer questions helped the author in reproducing the research work. finally, the author also pointed out that given this was a single initiative and was potentially biased in terms of their own experience, background and capabilities, they encourage others to tap into the data being made available but these guidelines provide good starting points for people to attempt to make their scientific work more rigorous and reproducible. the push has been to apply ai to any new problem that we face, hoping that the solution will magically emerge from the application of the technique as if it is a dark art. yet, the more seasoned scientists have seen these waves come and go and in the past, a blind trust in this technology led to ai winters. taking a look at some of the canaries in the coal mine, the author cautions that there might be a way to judge whether ai will be helpful with the pandemic situation. specifically, looking at whether domain experts, like leading epidemiologists endorse its use and are involved in the process of developing and utilizing these tools will give an indication as to whether they will be successful or not. data about the pandemic depends on context and without domain expertise, one has to make a lot of assumptions which might be unfounded. all models have to make assumptions to simplify reality, but if those assumptions are rooted in domain expertise from the field then the model can mimic reality much better. without context, ai models assume that the truth can be gleaned solely from the data, which though it can lead to surprising and hidden insights, at times requires humans to evaluate the interpretations to make meaning from them and apply them to solve real-world problems. this was demonstrated with the case where it was claimed that ai had helped to predict the start of the outbreak, yet the anomaly required the analysis from a human before arriving at that conclusion. claims of extremely high accuracy rates will give hardened data scientists reason for caution, especially when moving from lab to real-world settings as there is a lot more messiness with real-world data and often you encounter out-of-distribution data which hinders the ability of the model to make accurate predictions. for ct scans, even if they are sped up tremendously by the use of ai, doctors point out that there are other associated procedures such as the cleaning and filtration and recycling of air in the room before the next patient can be passed through the machine which can dwindle the gains from the use of an unexplainable ai system analyzing the scans. concerns with the use of automated temperature scanning using thermal cameras also suffers from similar concerns where there are other confounding factors like the ambient temperature, humidity, etc. which can limit the accuracy of such a system. ultimately, while ai can provide tremendous benefits, we mustn't blindly be driven by its allure to magically solve the toughest challenges that we face. offering an interesting take on how to shape the development and deployment of ai technologies, mhlambi utilizes the philosophy of ubuntu as a guiding light in how to build ai systems that better empower people and communities. the current western view that dominates how ai systems are constructed today and how they optimize for efficiency is something that lends itself quite naturally to inequitable outcomes and reinforcing power asymmetries and other imbalances in society. embracing the ubuntu mindset which puts people and communities first stands in contrast to this way of thinking. it gives us an alternative conception of personhood and has the potential to surface some different results. while being thousands of years old, the concept has been seen in practice over and over again, for example, in south africa, after the end of the apartheid, the truth and reconciliation program forgave and integrated offenders back into society rather than embark on a kantian or retributive path to justice. this restorative mindset to justice helped the country heal more quickly because the philosophy of ubuntu advocates that all people are interconnected and healing only happens when everyone is able to move together in a harmonious manner. this was also seen in the aftermath of the rwanda genocide, where oppressors were reintegrated back into society often living next to the people that they had hurt; ubuntu believes that no one is beyond redemption and everyone deserves the right to have their dignity restored. bringing people together through community is important, restorative justice is a mechanism that makes the community stronger in the long run. current ai formulation seeks to find some ground truth but thinking of this in the way of ubuntu means that we try to find meaning and purpose for these systems through the values and beliefs that are held by the community. ubuntu has a core focus on equity and empowerment for all and thus the process of development is slow but valuing people above material efficiency is more preferable than speeding through without thinking of the consequences that it might have on people. living up to ubuntu means offering people the choice for what they want and need, rooting out power imbalances and envisioning the companies as a part of the communities for which they are building products and services which makes them accountable and committed to the community in empowering them. ethics in the context of technology carries a lot of weight, especially because the people who are defining what it means will influence the kinds of interventions that will be implemented and the consequences that follow. given that technology like ai is used in high-stakes situations, this becomes even more important and we need to ask questions about the people who take this role within technology organizations, how they take corporate and public values and turn them into tangible outcomes through rigorous processes, and what regulatory measures are required beyond these corporate and public values to ensure that ethics are followed in the design, development and deployment of these technologies. ethics owners, the broad term for people who are responsible for this within organizations have a vast panel of responsibilities including communication between the ethics review committees and product design teams, aligning the recommendations with the corporate and public values, making sure that legal compliance is met and communicating externally about the processes that are being adopted and their efficacy. ethical is a polysemous word in that it can refer to process, outcomes, and values. the process refers to the internal procedures that are adopted by the firm to guide decision making on product/service design and development choices. the values aspect refers to the value set that is both adopted by the organization and those of the public within which the product/service might be deployed. this can include values such as transparency, equity, fairness, privacy, among others. the outcomes refer to desirable properties in the outputs from the system such as equalized odds across demographics and other fairness metrics. in the best case, inside a technology company, there are robust and well-managed processes that are aligned with collaboratively-determined ethical outcomes that achieve the community's and organization's ethical values. from the outside, this takes on the meaning of finding mechanisms to hold the firms accountable for the decisions that they take. further expanding on the polysemous meanings of ethics, it can be put into four categories for the discussion here: moral justice, corporate values, legal risk, and compliance. corporate values set the context for the rest of the meanings and provide guidance when tradeoffs need to be made in product/service design. they also help to shape the internal culture which can have an impact on the degree of adherence to the values. legal risk's overlap with ethics is fairly new whereas compliance is mainly concerned with the minimization of exposure to being sued and public reputation harm. using some of the framing here, the accolades, critiques, and calls to action can be structured more effectively to evoke substantive responses rather than being diffused in the energies dedicated to these efforts. framing the metaphor of "data is the new oil" in a different light, this article gives some practical tips on how organizations can reframe their thinking and relationship with customer data so that they take on the role of being a data custodian rather than owners of the personal data of their customers. this is put forth with the acknowledgement that customers' personal data is something really valuable that brings business upsides but it needs to be handled with care in the sense that the organization should act as a custodian that is taking care of the data rather than exploiting it for value without consent and the best interests of the customer at heart. privacy breaches that can compromise this data not only lead to fines under legislation like the gdpr, but also remind us that this is not just data but details of a real human being. as a first step, creating a data accountability report that documents how many times personal data was accessed by various employees and departments will serve to highlight and provide incentives for them to change behaviour when they see that some others might be achieving their job functions without the need to access as much information. secondly, celebrating those that can make do with minimal access will also encourage this behaviour change, all being done without judgement or blame but more so as an encouragement tool. pairing employees that need to access personal data for various reasons will help to build accountability and discourage intentional misuse of data and potential accidents that can lead to leaks of personal data. lastly, an internal privacy committee composed of people across job functions and diverse life experiences that monitors organization-wide private data use and provides guidance on improving data use through practical recommendations is another step that will move the conversation of the organization from data entitlement to data custodianship. ultimately, this will be a market advantage that will create more trust with customers and increase business bottom line going into the future. towards the systematic reporting of the energy and carbon climate change and environmental destruction are well-documented. most people are aware that mitigating the risks caused by these is crucial and will be nothing less than a herculean undertaking. on the bright side, ai can be of great use in this endeavour. for example, it can help us optimize resource use, or help us visualize the devastating effects of floods caused by climate change. however, ai models can have excessively large carbon footprints. henderson et al.'s paper details how the metrics needed to calculate environmental impact are severely underreported. to highlight this, the authors randomly sampled one-hundred neurips 2019 papers. they found that none reported carbon impacts, only one reported some energy use metrics, and seventeen reported at least some metrics related to compute-use. close to half of the papers reported experiment run time and the type of hardware used. the authors suggest that the environmental impact of ai and relevant metrics are hardly reported by researchers because the necessary metrics can be difficult to collect, while subsequent calculations can be time-consuming. taking this challenge head-on, the authors make a significant contribution by performing a meta-analysis of the very few frameworks proposed to evaluate the carbon footprint of ai systems through compute-and energy-intensity. in light of this meta-analysis, the paper outlines a standardized framework called experiment-impact-tracker to measure carbon emissions. the authors use 13 metrics to quantify compute and energy use. these include when an experiment starts and ends, cpu and gpu power draw, and information on a specific energy grid's efficiency. the authors describe their motivations as threefold. first, experiment-impact-tracker is meant to spread awareness among ai researchers about how environmentally-harmful ai can be. they highlight that "[w]ithout consistent and accurate accounting, many researchers will simply be unaware of the impacts their models might have and will not pursue mitigating strategies". second, the framework could help align incentives. while it is clear that lowering one's environmental impact is generally valued in society, this is not currently the case in the field of ai. experiment-impact tracker, the authors believe, could help bridge this gap, and make energy efficiency and carbon-impact curtailment valuable objectives for researchers, along with model accuracy and complexity. third, experiment-impact-tracker can help perform cost-benefit analyses on one's ai model by comparing electricity cost and expected revenue, or the carbon emissions saved as opposed to those produced. this can partially inform decisions on whether training a model or improving its accuracy is worth the associated costs. to help experiment-impact-tracker become widely used among researchers, the framework emphasizes usability. it aims to make it easy and quick to calculate the carbon impact of an ai model. through a short modification of one's code, experiment-impact-tracker collects information that allows it to determine the energy and compute required as well as, ultimately, the carbon impact of the ai model. experiment-impact-tracker also addresses the interpretability of the results by including a dollar amount that represents the harm caused by the project. this may be more tangible for some than emissions expressed in the weight of greenhouse gases released or even in co2 equivalent emissions (co2eq). in addition, the authors strive to: allow other ml researchers to add to experiment-impact-tracker to suit their needs, increase reproducibility in the field by making metrics collection more thorough, and make the framework robust enough to withstand internal mistakes and subsequent corrections without compromising comparability. moreover, the paper includes further initiatives and recommendations to push ai researchers to curtail their energy use and environmental impact. for one, the authors take advantage of the already widespread use of leaderboards in the ai community. while existing leaderboards are largely targeted towards model accuracy, henderson et al. instead put in place an energy efficiency leaderboard for deep reinforcement learning models. they assert that a leaderboard of this kind, that tracks performance in areas indicative of potential environmental impact, "can also help spread information about the most energy and climate-friendly combinations of hardware, software, and algorithms such that new work can be built on top of these systems instead of more energy-hungry configurations". the authors also suggest ai practitioners can take an immediate and significant step in lowering the carbon emissions of their work: run experiments on energy grids located in carbon-efficient cloud regions like quebec, the least carbon-intensive cloud region. especially when compared to very carbon-intensive cloud regions like estonia, the difference in co2eq emitted can be considerable: running an experiment in estonia produces up to thirty times as much emissions as running the same experiment in quebec. the important reduction in carbon emissions that follows from switching to energy-efficient cloud regions, according to henderson et al., means there is no need to fully forego building compute-intensive ai as some believe. in terms of systemic changes that accompany experiment-impact-tracker, the paper lists seven. the authors suggest the implementation of a "green default" for both software and hardware. this would make the default setting for researchers' tools the most environmentally-friendly one. the authors also insist on weighing costs and benefits to using compute-and energy-hungry ai models. small increases in accuracy, for instance, can come at a high environmental cost. they hope to see the ai community use efficient testing environments for their models, as well as standardized reporting of a model's carbon impact with the help of experiment-impact-tracker. additionally, the authors ask those developing ai models to be conscious of the environmental costs of reproducing their work, and act as to minimize unnecessary reproduction. while being able to reproduce other researchers' work is crucial in maintaining sound scientific discourse, it is merely wasteful for two departments in the same business to build the same model from scratch. the paper also presents the possibility of developing a badge identifying ai research papers that show considerable effort in mitigating carbon impact when these papers are presented at conferences. lastly, the authors highlight important lacunas in relation to driver support and implementation. systems that would allow data on energy use to be collected are unavailable for certain hardware, or the data is difficult for users to obtain. addressing these barriers would allow for more widespread collection of energy use data, and contribute to making carbon impact measurement more mainstream in the ai community. the paper highlights four challenges in designing more "intelligent" voice assistant systems that are able to respond to exploratory searches that don't have clear, short answers and require nuance and detail. this is in response to the rising expectations that users have from voice assistants as they become more familiar with them through increased interactions. voice assistants are primarily used for productivity tasks like setting alarms, calling contacts, etc. and they can include gestural and voice-activated commands as a method of interaction. exploratory search is currently not well supported through voice assistants because of them utilizing a fact-based approach that aims to deliver a single, best response whereas a more natural approach would be to ask follow up questions to refine the query of the user to the point of being able to provide them with a set of meaningful options. the challenges as highlighted in this paper if addressed will lead to the community building more capable voice assistants. one of the first challenges is situationally induced impairments as presented by the authors highlights the importance of voice activated commands because they are used when there are no alternatives available to interact with the system, for example when driving or walking down a busy street. there is an important aspect of balancing the tradeoff between smooth user experience that is quick compared to the degree of granularity in asking questions and presenting results. we need to be able to quantify this compared to using a traditional touch based interaction to achieve the same result. lastly, there is the issue of privacy, such interfaces are often used in a public space and individuals would not be comfortable sharing details to refine the search such as clothing sizes which they can discreetly type into the screen. such considerations need to be thought of when designing the interface and system. mixed-modal interactions include combinations of text, visual inputs and outputs and voice inputs and output. this can be an effective paradigm to counter some of the problems highlighted above and at the same time improve the efficacy of the interactions between the user and the system. further analysis is needed as to how users utilize text compared to voice searches and whether one is more informational or exploratory than the other. designing for diverse populations is crucial as such systems are going to be widely deployed. for example, existing research already highlights how different demographics even within the same socio-economic subgroup utilize voice and text search differently. the system also needs to be sensitive to different dialects and accents to function properly and be responsive to cultural and contextual cues that might not be pre-built into the system. differing levels of digital and technical literacy also play a role in how the system can effectively meet the needs of the user. as the expectations from the system increase over time, ascribed to their ubiquity and anthropomorphization, we start to see a gulf in expectations and execution. users are less forgiving of mistakes made by the system and this needs to be accounted for when designing the system so that alternate mechanisms are available for the user to be able to meet their needs. in conclusion, it is essential when designing voice-activated systems to be sensitive to user expectations, more so than other traditional forms of interaction where expectations are set over the course of several uses of the system whereas with voice systems, the user comes in with a set of expectations that closely mimic how they interact with each other using natural language. addressing the challenges highlighted in this paper will lead to systems that are better able to delight their users and hence gain higher adoption. the paper highlights how having more translation capabilities available for languages in the african continent will enable people to access larger swathes of the internet and contribute to scientific knowledge which are predominantly english based. there are many languages in africa, south africa alone has 11 official languages and only a small subset are made available on public tools like google translate. in addition, due to the scant nature of research on machine translation for african languages, there remain gaps in understanding the extent of the problem and how they might be addressed most effectively. the problems facing the community are many: low resource availability, low discoverability where language resources are often constrained by institution and country, low reproducibility because of limited sharing of code and data, lack of focus from african society in seeing local languages as primary modes of communication and a lack of public benchmarks which can help compare results of machine translation efforts happening in various places. the research work presented here aims to address a lot of these challenges. they also give a brief background on the linguistic characteristics of each of the languages that they have covered which gives hints as to why some have been better covered by commercial tools than others.in related work, it is evident that there aren't a lot of studies that have made their code and datasets public which makes comparison difficult with the results as presented in this paper. most studies focused on the autshumato datasets and some relied on government documents as well, others used monolingual datasets as a supplement. the key analysis of all of those studies is that there is a paucity in the focus on southern african languages and because apart from one study, others didn't make their datasets and code public, the bleu scores listed were incomparable which further hinders future research efforts. the autshumato datasets are parallel, aligned corpora that have governmental text as its source. they are available for english to afrikaans, isizulu, n. sotho, setswana, and xitsonga translations and were created to build and facilitate open source translation systems. they have sentence level parallels that have been created both using manual and automatic methods. but, it contains a lot of duplicates which were eliminated in the study done in this paper to avoid leakage between training and testing phases. despite these eliminations, there remain some issues of low quality, especially for isizulu where the translations don't line up between source and target sentences. from a methodological perspective, the authors used convs2s and transformer models without much hyperparameter tuning since the goal of the authors was to provide a benchmark and the tuning is left as future work. additional details on the libraries, hyperparameter values and dataset processing are provided in the paper along with a github link to the code. in general the transformer model outperformed convs2s for all the languages, sometimes even by 10 points on the bleu scores. performance on the target language depended on both the number of sentences and the morphological typology of the language. poor quality of data along with small dataset size plays an important role as evidenced in the poor performance on the isizulu translations where a lowly 3.33 bleu score was achieved. the morphological complexity of the language also played a role in the state of the performance as compared to other target languages. for each of the target languages studied, the paper includes some randomly selected sentences to show qualitative results and how different languages having different structures and rules impacts the degree of accuracy and meaning in the translations. there are also some attention visualizations provided in the paper for the different architectures demonstrating both correct and incorrect translations, thus shedding light on potential areas for dataset and model improvements. the paper also shows results from ablation studies that the authors performed on the byte pair encodings (bpe) to analyze the impact on the bleu scores and they found that for datasets that had smaller number of samples, like for isizulu, having a smaller number of bpe tokens increased the bleu scores. in potential future directions for the work, the authors point out the need for having more data collection and incorporating unsupervised learning, meta learning and zero shot techniques as potential options to provide translations for all 11 official languages in south africa. this work provides a great starting point for others who want to help preserve languages and improve machine translations for low resources languages. such efforts are crucial to empower everyone in being able to access and contribute to scientific knowledge of the world. providing code and data in an open source manner will enable future research to build upon it and we need more such efforts that capture the diversity of human expression through various languages. presence of media cards, total interactions, history of engagement with the creator of the tweet, the user's strength of connection with the creator and the user's usage pattern of twitter. from these factors, one can deduce why filter bubbles and echo chambers form on the platform and where designers and technologists can intervene to make the platform a more holistic experience for users that doesn't create polarizing fractions which can promote the spread of disinformation. for the first time, there's a call for the technical community to include a social impact statement from their work which has sparked a debate amongst camps that are arguing to leave such a declaration to experts who study ethics in machine learning and those that see this as a positive step in bridging the gap between the social sciences and the technical domains. specifically, we see this as a great first step in bringing accountability closer to the origin of the work. additionally, it would be a great way to build a shared vernacular across the human and technical sciences easing future collaboration. we are impressed with all the work that the research and practice community has been doing in the domain of ai ethics. there are many unsolved and open problems that are yet to be addressed but our overwhelming optimism in the power of what diversity and interdisciplinarity can help to achieve makes us believe that there is indeed room for finding novel solutions to the problems that face the development and deployment of ai systems. we see ourselves as a public square, gathering people from different walks of life who can have meaningful exchanges with each other to create the solutions we need for a better future. let's work together and share the mic with those who have lived experiences, lifting up voices that will help us better understand the contexts within which technology resides so that we can truly build something that is ethical, safe, and inclusive for all. see you here next quarter, the state of ai ethics, june 2020 suckers list: how allstate's secret auto insurance algorithm squeezes big spenders algorithmic injustices towards a relational ethics social biases in nlp models as barriers for persons with disabilities the second wave of algorithmic accountability the unnatural ethics of ai could be its undoing this dating app exposes the monstrous bias of algorithms ( arielle pardes data is never a raw, truthful input -and it is never neutral racial disparities in automated speech recognition working to address algorithmic bias? don't overlook the role of demographic data ai advances to better detect hate speech algorithms associating appearance and criminality have a dark past beware of these futuristic background checks go deep: research summaries 30 the toxic potential of youtube's feedback loop study: facebook's fake news labels have a fatal flaw research summaries capabilities, and ai assistive technologies go wide: article summaries 50 ancient animistic beliefs live on in our intimacy with tech humans communicate better after robots show their vulnerable side at the limits of thought engineers should spend time training not just algorithms, but also the humans who use them using multimodal sensing to improve awareness in human-ai interaction different intelligibility for different folks aligning ai to human values means picking the right metrics why lifelong learning is the international passport to success (pierre vandergheynst and isabelle vonã¨che cardia you can't fix unethical design by yourself research summaries the wrong kind of ai? artificial intelligence and the future of labor demand ai is coming for your most mind-numbing office tasks tech's shadow workforce sidelined, leaving social media to the machines here's what happens when an algorithm determines your work schedule automation will take jobs but ai will create them research summaries what's next for ai ethics, policy, and governance? a global overview (daniel schiff a holistic approach to implement ethics in ai beyond a human rights based approach to ai governance: promise, pitfalls and plea go wide: article summaries 69 this is the year of ai regulations apps gone rogue: maintaining personal privacy in an epidemic maximizing privacy and effectiveness in covid-19 apps article summaries who's allowed to track my kids online? chinese citizens are racing against censors to preserve coronavirus memories on github can i opt out of facial scans at the airport? with painted faces, artists fight facial recognition tech research summaries adversarial machine learning -industry perspectives article summaries the deadly consequences of unpredictable code adversarial policies: attacking deep reinforcement learning specification gaming: the flip side of ai ingenuity doctors are using ai to triage covid-19 patients. the tools may be here to stay the future of privacy and security in the age of machine learning research summaries 95 towards a clearer account of research priorities in ai ethics and society integrating ethical values and economic value to steer progress in ai machine learning scholarship go wide: article summaries 104 microsoft researchers create ai ethics checklist with ml practitioners from a dozen tech companies why countries need to work together on ai quantifying independently reproducible machine learning be a data custodian, not a data owner research summaries towards the systematic reporting of the energy and carbon footprints of machine learning challenges in supporting exploratory search through voice assistants a focus on neural machine translation for african languages radioactive data: tracing through training using deep learning at scale in twitter's timeline (nicolas koumchatzky neurips requires ai researchers to account for societal impact and financial conflicts of interest in modern ai systems, we run into complex data and processing pipelines that have several stages and it becomes challenging to trace the provenance and transformations that have been applied to a particular data point. this research from the facebook ai research team proposes a new technique called radioactive data that borrows from medical science where compounds like baso4 are injected to get better results in ct scans. this technique applies minor, imperceptible perturbations to images in a dataset by causing shifts within the feature space making them "carriers".different from other techniques that rely on poisoning the dataset that harms classifier accuracy, this technique instead is able to detect such changes even when the marking and classification architectures are different. it not only has potential to trace how data points are used in the ai pipeline but also has implications when trying to detect if someone claims not to be using certain images in their dataset but they actually are. the other benefit is that such marking of the images is difficult to undo thus adding resilience to manipulation and providing persistence. prior to relevance based timeline, the twitter newsfeed was ordered in reverse chronological order but now it uses a deep learning model underneath to display the most relevant tweets that are personalized according to the user's interactions on the platform. with the increasing use of twitter as a source of news for many people, it's a good idea for researchers to gain an understanding of the methodology that is used to determine the relevance of tweets, especially as one looks to curb the spread of disinformation online. the article provides some technical details in terms of the deep learning infrastructure and the choices made by the teams in deploying computationally heavy models which need to be balanced with the expediency of the refresh times for a good experience on the platform. but, what's interesting from an ai ethics perspective are the components that are used to arrive at the ranking which constantly evolves based on the user's interaction with different kinds of content.the ranked timeline consists of a handful of the tweets that are the most relevant to the user followed by others in reverse chronological order. additionally, based on the time since one's last visit on the platform, there might be an icymi ("in case you missed it") section as well. the key factors in ranking the tweets are their recency, key: cord-286368-kdwh4hgf authors: hui, david s.c.; lee, nelson; chan, paul k.s. title: a clinical approach to the threat of emerging influenza viruses in the asia‐pacific region date: 2017-07-05 journal: respirology doi: 10.1111/resp.13114 sha: doc_id: 286368 cord_uid: kdwh4hgf seasonal influenza epidemics and periodic pandemics are important causes of morbidity and mortality. patients with chronic co‐morbid illness, those at the extremes of age and pregnant women are at higher risks of complications requiring hospitalization, whereas young adults and obese individuals were also at increased risk during the a(h1n1) pandemic in 2009. avian influenza a(h5n1) and a(h7n9) viruses have continued to circulate widely in some poultry populations and infect humans sporadically since 1997 and 2013, respectively. the recent upsurge in human cases of a(h7n9) infections in mainland china is of great concern. sporadic human cases of avian a(h5n6), a(h10n8) and a(h6n1) have also emerged in recent years while there are also widespread poultry outbreaks due to a(h5n8) in many countries. observational studies have shown that treatment with a neuraminidase inhibitor (nai) for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes, especially when administered early in the course of illness. whether higher than standard doses of nai would provide greater antiviral effects in such patients will require further investigation. high‐dose systemic corticosteroids were associated with worse outcomes in patients with severe influenza. there is an urgent need for developing more effective antiviral therapies for treatment of influenza infections. severe acute respiratory infections such as avian influenza a(h5n1) 1 and a(h7n9) viruses 2 with pandemic potential have continued to circulate widely in some poultry populations and infect humans sporadically. infection caused by these viruses may result in severe disease and high case fatality rates because most humans have no background immunity to these viruses. sporadic human cases due to avian a(h5n6), 3 a(h10n8) 4 and a(h6n1) 5 have also emerged in recent years. the global circulation of oseltamivir-resistant seasonal influenza, the emergence of a(h1n1)pdm09 virus in 2009 followed by its continual circulation, 6 the rising number of a(h7n9) infections in humans 2 and the ongoing spread of a(h5n8) in recent months in the poultry populations in many countries in asia, africa, europe and middle east with pandemic potential 7 all point to an urgent need for developing more effective antiviral therapies to reduce morbidity and mortality. this article reviews the epidemiology, clinical, diagnostic and treatment aspects of these important and emerging influenza viruses that are posing threats to human health in the asia-pacific region. seasonal influenza is a major health problem. for instance, in hong kong, influenza a and b accounted for 39.4% and 10.2%, respectively, of hospital admissions confirmed to have viral infections. 8 the average annual incidence of hospitalization with laboratoryconfirmed influenza a infection was 107.7/10 000 and 18.3/10 000 among children aged <5 years and elderly ≥65 years, respectively. furthermore, influenza a was the most common (45.5%) virus detected from patients who died of respiratory viral infections. although influenza b is usually associated with a less degree of disease burden, its impact on human health is substantial. our previous study conducted in hong kong revealed that 24% of laboratory-confirmed influenza-associated hospital admissions were due to influenza b, and was much higher (41.9%) among older children and adolescents (aged 5-14 years). 9 this observation is similar to data in europe and north america. in the temperate region, influenza exhibits an annual seasonal peak in winter. however, the seasonality in tropical and subtropical regions is more variable. for instance, in hong kong, a subtropical city, most of the time, influenza exhibits two peaks during january-march and june-august, and occasionally, the two peaks merge without an obvious gap. 8, 10 such bimodal and variable seasonality of influenza is also observed in countries within the subtropical and tropical regions, and poses a challenge to implement annual influenza vaccination campaign as well as a question on whether the northern or southern hemisphere version of influenza vaccine would be more appropriate. a schematic diagram of the structure of influenza virion is shown in figure 1 . influenza represents one of the few examples of viruses with segmented genome. this, together with the widespread infection with different subtypes in various animal species, allows robust evolution through the process of 'reassortment'. the prerequisite for reassortment is a simultaneous infection with more than one strain of viruses in a host referred to as the 'mixing vessel' that allows replication and swabbing of gene segments between different strains of viruses. water birds are well-known mixing vessels, but the reassortants are more likely to have a tropism restricted to avian species. these new reassortants may pose problems in the land birds and poultry, and subsequently be of human concern. swine is another well-known mixing vessel. the unique property of swine is their susceptibility to both avian and human influenza viruses. thus, reassortants from swine are more likely to gain tropism for infection in humans. field observations made as early as 1990s have pointed out that influenza outbreaks that began with either pigs or people were then rapidly transferred to each other. 11 swine are likely to play the role of a mixing vessel in creating pandemic strains. although the 1918 pandemic strain was most probably an entirely avian-like virus, 12 the 1957 and 1968 pandemic strains were reassortants of human-and avian-origin viruses. 13 while the most recent pandemic, a(h1n1)pdm09, was not as severe as those that occurred before, there is no guarantee that the next pandemic will be as 'mild'. at present, a few subtypes (h5, h7 and h9) are of particular concern. cross-species infections, especially the recent strong wave of human a(h7n9) infection in mainland china underscores that the next pandemic could be imminent. 14, 15 countries should have pandemic preparedness in place, including antiviral stockpiling and pandemic vaccine preparations. these would be better achieved under joint multigovernment industrial partnership. human cases of the highly pathogenic avian influenza a(h5n1) were first detected in hong kong in 1997. 1 the number of affected countries increased between 2003 and 2008, with expansion from east and southeast asia, then to west asia and africa, with a sharp rise in egypt since november 2014. 16 as of 16 march 2017, there have been 453 deaths out of 858 human cases in 16 countries since 2003. 17 a canadian tourist died of a(h5n1)-related meningoencephalitis in alberta in january 2014 after visiting beijing in december 2013 without any obvious exposure to infected avian sources or environmental contamination. 18 a history of exposure to dead or sick poultry/wild birds occurs in over 60% of cases of human a(h5n1) infection. the incubation period for a(h5n1) infection ranges from 2 to 8 days but may be as long as 17 days. the clinical spectrum may range from asymptomatic infection, mild influenza-like illness to severe pneumonia, with multiorgan failure. 19 some of the a(h5n1) human cases have been linked to consumption of dishes made of raw, contaminated poultry blood. however, slaughter, defeathering, handling carcasses of infected poultry and preparing poultry for consumption, especially in household settings, appear to be important risk factors. 19, 20 among the fatal cases, the median duration from symptom onset to death was 9-10 days (range: 6-30 days). 20 viral rna was present in the blood of fatal a(h5n1) cases and this was associated with higher pharyngeal viral loads. nasopharyngeal swab samples, bronchoalveolar lavage and cerebrospinal fluid samples were positive in the canadian case for influenza a(h5n1) virus by various molecular testing methods, including sequencing. 18 in addition, lymphopenia and high chemokine/cytokine levels have been observed in severe a(h5n1) infection and these correlated with pharyngeal loads. 21 a new reassortant genotype of a(h5n1) containing the haemagglutinin (ha) and neuraminidase (na) genes from clade 1.1.2 and the internal genes from clade 2.3.2.1 was detected in 2013 and associated with the highest number of cases (n = 26) and deaths (n = 14) in cambodia. 22 complete viral genome analysis of the fatal case in canada has shown an ha gene of clade 2.3.2.1c and was a reassortant with an a(h9n2) subtype lineage polymerase basic 2 gene without mutations conferring resistance to adamantanes or na inhibitors (nai). 18 human-human transmission remains rare as a meta-analysis has shown that only 1-2% of more than 12 500 study participants from 20 studies exposed to patients with a(h5n1) infection had serological evidence for prior a(h5n1) infection. 23 delay in the delivery of appropriate treatment to patients with influenza a(h5n1) infection in indonesia was mainly related to delay in diagnosis rather than late presentation to healthcare settings. 24 age, country, per capita government health expenditure and delay from symptom onset to hospitalization were the risk factors for mortality related to a(h5n1) infection, emphasizing the importance of early diagnosis, treatment and supportive care. 25 a systematic review has shown that females were at higher risk of death (or: 1.75, 95% ci: 1.27-2.44) following a(h5n1) infection, whereas young age in particular <5 years was protective (or: 0.44, 95% ci: 0.25-0.79). 26 human infections with a novel avian influenza a (h7n9) virus were first reported in china in march 2013 in patients hospitalized with severe pneumonia. 27 there have been five seasonal epidemics in china since the virus was first discovered in 2013. the estimated risk of death (hospitalization fatality risk, hfr) among hospitalized cases of a(h7n9) infection in the second epidemic was 48% (95% credibility interval: 42-54%), which was slightly higher than the corresponding risk of 36% in the first wave. in the second epidemic, the hfr was estimated at 36% (95% ci: 28-45%) in patients aged <60 years but at 59% (95% ci: 51-67%) among those aged ≥60 years. 28 an upsurge in human a(h7n9) infections has been observed since november 2016 in mainland china. during this fifth epidemic wave, the number of human cases is higher than during the previous two waves during 2014-2015 and 2015-2016. the majority of recently reported human cases are associated with exposure to infected live poultry or contaminated environments, including wet markets where live poultry are sold. in addition, the human cases are more geographically widespread and cases are also reported from rural areas, unlike in previous epidemics (fig. 2) . 29, 30 the phylogenetic analysis on ha genes of isolates collected from humans suggested that the virus is evolving and with the more recent isolates clustered to a separate branch (supplementary figure s1 ). 31, 32 as of 5 april 2017, 1364 laboratory-confirmed cases of human infection with avian influenza a(h7n9) virus have been reported to world health organization (who). 33 human cases of a(h7n9) infection exported from the mainland of china have been confirmed in hong kong (n = 21), taiwan (n = 4), macau (n = 2), canada (n = 2) and malaysia (n = 1) in visitors who developed illness after returning from the mainland of china to their home cities. 29, 30, 33 the incubation period of human infection with the a(h7n9) virus ranges from 1 to 10 days, with an average of 5 days. the median time from poultry exposure to disease onset was 6 days, whereas the median time from illness onset to hospitalization, acute respiratory distress syndrome (ards) development, antiviral therapy and death were 4, 7, 6 and 21 days, respectively. 34 preexisting co-morbid conditions occurred in >60% of these cases. the prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, whereas the most striking laboratory findings were marked lymphopenia and thrombocytopenia. elevated cytokine levels have been observed in patients and the excessive cytokine responses may contribute to the clinical severity of a(h7n9) infection. 35, 36 limited human to human transmission with at least two epidemiologically linked cases due to close household contact with the index patients has been reported in a number of family clusters in both a(h5n1) 20, 37, 38 and a(h7n9) infections. 34, 39, 40 nosocomial transmission of a(h7n9) infection between two patients sharing a hospital room, 41 and co-transmission of avian influenza a(h7n9) and a(h1n1)pdm09 viruses between two patients with haematological disorders sharing the same room with bed distance <1 m have been reported. 42 comparisons of the clinical features between human cases of a(h5n1) and a(h7n9) are shown in table 1 . 34, 43 closure of live poultry markets (lpm) in the mainland of china has tremendously reduced the risk of a(h7n9) infection temporarily but closure is difficult to sustain due to cultural preference for live poultry. an ecologic modelling study estimated that closure of lpm reduced the mean daily number of a(h7n9) virus infections in the four most affected cities by 97-99%. 44 a retrospective serological study of blood specimens taken during january-may and october-november in 2012 from 1544 subjects who worked in lpm, farms, slaughter houses or kept backyard poultry in eastern china before the epidemic occurred in 2013 revealed no evidence of a(h7n9) infection. 45 other novel avian influenza subtypes the first human case of avian a(h6n1) infection was reported in a 20-year-old female with pneumonia in taiwan in may 2013, with subsequent full recovery following treatment with oseltamivir. 5 the first human case of avian a(h5n6) was confirmed in a 49-year-old male in sichuan province, onset of illness to death (median, days) 21 11 43 case fatality rate in hospital 34% 53% china, in may 2014 with a fatal outcome 3 and a second case was confirmed in a 58-year-old male with history of exposure to live poultry in guangdong province in december 2014. 46 the virus was a reassortant that contained seven genes from a(h5n1) and the na gene from an a(h6n6) virus circulating in ducks. 47 (fig. 3 ). 29 the latest case was reported on 1 december 2016. 48 in addition, china has confirmed three human infections, two fatal, with avian a(h10n8) viruses that contain the internal genes from a(h9n2), as does a(h7n9). 4, 49 similar to the a(h7n9) virus, the a(h6n1) and a(h10n8) viruses have low pathogenicity in poultry and are therefore more difficult to detect in birds in contrast to the highly pathogenic a(h5n1) virus. highly pathogenic avian influenza a(h5n8) viruses belonging to clade 2.3.4.4 of the a/goose/guangdong/ 1/1996 lineage were detected in 2014 in wild birds and poultry in china, japan, south korea, netherlands, germany, italy, russia, the uk and northern ireland. the virus was detected sporadically in canada and the usa in wild birds and poultry until mid-2015. the a(h5n8) viruses were also detected in taiwan, china and in hungary and sweden in 2015. since june 2016, many more countries in both europe and asia have detected infections in wild birds and/or domestic poultry with a(h5n8). many of these recent detections were associated with mortality in wild birds. 7,50 although human infection with a(h5n8) has not been detected, the ongoing spread of this virus to different countries in recent months is worrisome and may increase the risk of human infection. visitors who have returned from areas affected by avian influenza and developed respiratory symptoms and fever within 10 days after their return should be enquired about their relevant history (travel history, occupation, contact history and clustering) to facilitate early diagnosis and treatment. it is important to obtain any history of contact with poultry (live or sick/dead poultry as a(h7n9) virus may cause no or mild symptoms in poultry in contrast to a(h5n1)), exposure to wet markets or known avian influenza cases, and laboratory exposure including staff handling with spillage. patients with influenza-like illness who present with dyspnoea, tachypnoea, evidence of hypoxaemia and pulmonary infiltrates on chest radiograph should be hospitalized. 51 early case identification and isolation precautions would facilitate clinical management which may reduce the risk of severe infection and related complications for seasonal epidemic, zoonotic and pandemic influenza, in addition to reducing the risk of influenza transmission and mitigating the impact of outbreaks on the healthcare system. common findings in severe influenza infection include low or normal white cell counts, lymphopenia and thrombocytopenia, and increased serum transaminases, lactate dehydrogenase (ldh), creatine kinase and urea/creatinine. 6, 20, 27, 51 in the influenza clinical information network (flucin) study, early elevation of c-reactive protein ≥100 mg/l was an independent predictor of severe outcome. in a study of 1770 patients hospitalized with community-acquired pneumonia (cap), procalcitonin concentration had a strong association with need for invasive mechanical ventilation (imv) or inotrope support (in 6.5% of those studied). 52 higher levels of certain biomarkers on presentation linked to inflammation, coagulation or immune function (interleukin 6 (il-6), cluster of differentiation 163 (cd163), interferon gamma-induced protein 10 (il-10), lipopolysaccharide binding protein (lbp), il-2, monocyte chemoattractant protein-1 (mcp-1) and interferon gamma-induced protein 10 (ip-10)) have been associated with disease progression in both outpatients and those hospitalized with influenza. 53 laboratory diagnosis plays an important role in the management of influenza. the narrow window of an effective antiviral intervention for both seasonal and avian influenza demands a rapid turnaround time. 6, 54 nowadays, most laboratories employ either real time reverse transcription polymerase chain reaction (rrt-pcr)-based methods or antigen detection-based assays. while the rrt-pcr approach is most sensitive and can be applied to a wide variety of specimens including the less invasive throat swab samples, it requires a certain degree of laboratory setup and technical skills, which are not readily available in many healthcare settings. the antigen-based rapid influenza diagnostic test (ridt), although less sensitive, is still the test of choice particularly as a point-of-care test. our experience on a few ridt suggested that the sensitivity could be lower for patients who presented more than 2 days after the onset of illness, for young children compared to elderly, and for cases with pneumonia compared to those without. 55 it is therefore crucial to evaluate the performance with respect to the setting that ridt will be applied. the specimen of choice is critical. while in most situations, nasopharyngeal aspirate/swab is the preferred choice of specimen for seasonal influenza, falsenegative results may occur in patients with severe pneumonia where the viral load remains high in the lower respiratory tract (lrt) but has become undetectable in the upper respiratory tract (urt). 56 similarly, lrt specimens such tracheal aspirates or sputum are the specimen of choice for diagnosis of avian influenza infection in humans as the viral load in the urt is typically low. the development and availability of more affordable and reliable molecular diagnostic tests including multiplex pcr would facilitate future clinical management of patients with severe cap due to viral or bacterial aetiology. 57 the advantages and limitations of different diagnostic tests for influenza are summarized in table 2 . 55, 57 the m2 inhibitors (amantadine and rimantadine) and the nai (oseltamivir, peramivir, zanamivir and laninamivir) are the main antiviral agents approved for the prevention of and treatment for influenza. in general, antiviral treatment should be commenced as soon as possible for any patient hospitalized with confirmed/ suspected influenza with severe, complicated or progressive illness, and also for outpatients at higher risk for influenza complications. 58 the m2 inhibitors (adamantanes) are not effective against influenza b viruses and recently circulating influenza a(h3n2) and influenza a(h1n1)pdm09 viruses, which are resistant due to an s31n mutation in the m2 ion channel. however, as some avian influenza a(h5n1) strains are still susceptible, 59 combination of an adamantane with an nai may enhance antiviral activity for susceptible isolates. 60 oseltamivir is effective in reducing mortality (or: 0.17; p = 0.04) in influenza a(h5n1) infection when given before the onset of respiratory failure, 61 and may provide some survival benefit (49% reduction in mortality) when treatment is started within 6-8 days after symptom onset. 62 several observational studies have shown that treatment with oseltamivir for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes, especially when antiviral treatment has been initiated within 2 days of illness onset but even as late as 4-5 days after symptom onset. 63, 64 a systematic review has shown that in prophylactic studies, oseltamivir could reduce the proportion of symptomatic influenza whereas in treatment studies it modestly reduced the time to first alleviation of symptoms by 16.8 h, but it caused nausea, vomiting, headaches and renal/psychiatric side effects. 65 a meta-analysis of randomized controlled trials (rct) has shown that oseltamivir in adults with influenza shortens time to clinical symptom alleviation by 21%, reduces risks of lrt complications and hospitalization, but increases the occurrence of nausea and vomiting. 66 oseltamivir resistance is infrequent in a(h5n1), and <3% in circulating a(h1n1)pdm09, a(h3n2) and b viruses but it is important to monitor for antiviral resistance when managing patients with severe influenza. 67 all h7n9 viruses are amantadine resistant due to the s31n substitution in the m2 ion channel protein. in patients hospitalized with severe a(h7n9) infection, reduction of viral load following oseltamivir treatment was associated with improved outcome, whereas the emergence of virus resistant to nai harbouring an r292k mutation was associated with poor outcomes and lack of response to oseltamivir and peramivir, and reduced susceptibility to zanamivir and laninamivir (50-and 25-fold rises in inhibitory concentration by 50% (ic 50 ), respectively). two patients with severe a(h7n9) infection and r292k mutation requiring extracorporeal membrane oxygenation had received systemic corticosteroid treatment which might have contributed to treatment failure and a fatal outcome. 68 the recommended treatment duration of oseltamivir is generally 5 days, but longer treatment (10 days, then guided by clinical and virological testing as recommended by the who) is advisable for critically ill patients with respiratory failure with persistent viral replication in the lrt. 51, 58, 69 whether higher than standard dose of nai would provide greater antiviral effects in such patients requires further investigation. one rct of hospitalized patients (76% being children) revealed no clinical or virological advantages when comparing double dose of oseltamivir against standard dose. 70 no additional benefit of higher dose oseltamivir treatment was observed in adults hospitalized with influenza a, although a faster virological response was noted in influenza b. 71 however, an rct of 18 critically ill patients with a(h1n1)pdm09 in canada found that therapy with a triple dose of oseltamivir was associated with higher proportions of viral clearance at 5 days than standard therapy (78% vs 11%; p = 0.015). 72 intravenous (i.v.) peramivir treatment was associated with viral rna decline as well as culture and rna negativity, which occurred at similar rates to those on oral oseltamivir by day 5 among adult patients hospitalized for influenza-associated lrt complications. 73 the various nai dosages for adults with adjustment for renal failure are shown in table 3 . 58 zanamivir and laninamivir have quite similar profiles of drug susceptibility. one example is that h275y mutation, which confers high level resistance to oseltamivir carboxylate and reduced susceptibility to peramivir in n1-containing viruses, does not reduce susceptibility to zanamivir and laninamivir most ridt are chromatographic immunoassays (some are fluorescence-based immunoassays); applicable to np swabs, nasal and/or throat swabs and np aspirates/washes (training and protection equipment are required). performance is best if applied within 48-72 h from onset before a significant drop in viral load (up to 4-5 days in selected populations). lower sensitivity for a(h1n1) pdm09 virus has been reported. viral cell culture detects viable viruses, including those contained in the live-attenuated influenza vaccines (laiv). isolates can be subjected to phenotypic resistance assays (e.g. neuraminidase enzyme inhibition assay). viral load, specimen quality, transport, storage and processing techniques may affect test performance. pcr assays can either provide universal detection of influenza a virus by targeting the matrix (m) gene or subtype-specific virus detection (e.g. h1n1pdm09, h3n2, h5n1 and h7n9) by targeting the haemagglutinin (ha) gene. viruses that cannot be subtyped may indicate a novel strain. newer molecular-based point-of-care tests may improve accessibility and reduce processing time and technical demands; some may allow detection of multiple viruses. cost-effectiveness of pcr is variable, depending on the circumstances. some multiplex pcr platforms may provide detection of >14 respiratory viruses (e.g. rsv, human metapneumovirus, parainfluenza virus, rhinovirus and coronavirus) and atypical pathogens (e.g. mycoplasma pneumoniae and chlamydophila pneumoniae). dfa, direct fluorescent antibody test; ifa, immunofluorescence assay; np, nasopharyngeal; ridt, rapid influenza diagnostic test; rsv, respiratory syncytial virus. significantly. 74 intravenous zanamivir was used widely on a compassionate ground for late treatment of critically ill adults with influenza a(h1n1)pdm09 and those with suspected or proven oseltamivir resistance. 75 there were no drug-related trends in safety parameters and in a subset of 93 patients with positive pcr tests at baseline for influenza, showed a median decrease in nasopharyngeal viral rna load of 1.42 log 10 copies/ml after 2 days of treatment. 76 intravenous zanamivir was used with a favourable outcome in a patient with severe a(h7n9) infection complicated by pneumonia which did not respond to oseltamivir initially. 77 favipiravir, which is an inhibitor of a viral rna-dependent rna polymerase, has strong antiviral activity against nai-resistant and sensitive influenza viruses. 67, 78 systemic corticosteroids and other immunomodulating agents for severe influenza systemic corticosteroid has been used frequently for the treatment of influenza-related ards. however, a meta-analysis of data predominantly related to treatment of severe influenza a(h1n1)pdm09 has shown that systemic corticosteroid was associated with an increase in mortality (or: 3.06, 95% ci: 1.58-5.92). pooled subgroup analysis of adjusted estimates of mortality from four studies found or of 2.82 and 95% ci of 1. 61-4.92. 79 in comparison to controls, high-dose corticosteroids (>150 mg/day methylprednisolone eqv) was associated with increased risks in 30-day mortality (38.5% vs 7.7%, p = 0.021) and 60-day mortality (50% vs 15.4%, p = 0.022) and longer viral shedding (15 days vs 13 days, p = 0.039) in patients with influenza a (h7n9) viral pneumonia while there was no difference between low dose (25-150 mg/day methylprednisolone) and controls. 80 in a study of 2649 adults hospitalized with influenza in hong kong, singapore and beijing during 2008-2011, 23.1% had received systemic corticosteroids. bacterial superinfections increased risk of death (adjusted hazard ratio (hr): 2.2, 95% ci: 1.5-3.1). systemic corticosteroids increased risks of superinfections (from 2.7% to 9.7%) and deaths when controlled for indications (adjusted hr: 1.7, 95% ci: 1.1-2.6). 81 among adults with severe sepsis not in septic shock, use of low-dose hydrocortisone versus placebo did not reduce the risk of septic shock within 14 days. the findings do not support the use of hydrocortisone in these patients without septic shock. 82 the role of low-dose systemic corticosteroid needs further investigation by a properly planned rct. 83 in a study of critically ill patients infected with influenza a (h1n1)pdm 09 requiring imv, addition of a mammalian target of rapamycin inhibitor, sirolimus 2 mg/day for 14 days to oseltamivir and prednisolone (n = 19), was associated with a higher frequency of liberation from imv (84.2% vs 47.4%, p = 0.04), a shorter duration of imv (13.8 days vs 33 days, p = 0.03) and a higher chance of achieving lrt viral rna negativity by day 7 (75% vs 33%, p < 0.05) than without addition of sirolimus (n = 19). 84 the role of sirolimus plus oseltamivir versus oseltamivir alone in a larger sample size should be examined without systemic corticosteroid. a recent study of adults hospitalized for a(h3n2) has shown that a triple combination treatment of 2 days of clarithromycin 500 mg, naproxen 200 mg and oseltamivir 75 mg twice daily (bd), followed by 3 days of oseltamivir reduced both 30-and 90-day mortalities and length of hospital stay versus oseltamivir 75 mg bd without placebos for 5 days as control. 85 confirmatory studies of the role of this triple combination would be of interest. exploratory post hoc meta-analysis of studies of severe acute respiratory syndrome (sars) and severe influenza showed a significant reduction in the pooled odds of mortality following convalescent plasma versus placebo or no treatment (or: 0.25; 95% ci: 0.14-0.45). 86 thus, convalescent plasma and hyperimmune globulin may 88 nosocomial opportunistic airborne transmission of a(h3n2) has been implicated in a medical ward with imbalanced airflow and use of non-invasive ventilation (niv) for an index patient with acute exacerbation of chronic obstructive pulmonary disease (copd). 89 standard, contact and droplet precautions are recommended for routine management of patients hospitalized for influenza. 90 the main infection prevention and control measures for managing influenza are droplet precaution (by wearing a surgical mask within 1 m of the patient) and contact precaution (by wearing gown and gloves on entering the room and removing them on leaving). droplet precautions should be added to the standard precautions when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of avian influenza infection (table 4 ). 90 a study that measured the amount of influenza a(h1n1)pdm09 rna in aerosols in the vicinity of h1n1-positive patients undergoing aerosol-generating procedures (agp) has shown that bronchoscopy and respiratory/airway suctioning were the most definite procedures to produce aerosols above background baseline values. 92 thus, it is important for healthcare workers to take airborne precautions and perform agp preferably in an isolation room with negative pressure. in order to reduce room contamination in the hospital setting, major health organizations have recommended the application of a minimum room ventilation rate of 6 air changes per hour (ach) in existing facility whereas a higher ventilation of 12 ach is recommended for new or renovated construction, especially when caring for patients receiving mechanical ventilation and during agp. 91, 93 as single circuit niv may lead to room contamination by exhaled aerosols, 94, 95 it has been recommended to apply niv via a helmet and double circuit tubes for patients with mild to moderate respiratory failure during the influenza a(h1n1) pandemic in 2009. 96 however, it is important to ensure a good seal at the neckhelmet interface to prevent nosocomial transmission through environmental contamination by exhaled aerosols. 97 post-exposure prophylaxis with a daily dose of nai (e.g. oseltamivir 75 mg daily for adults) for 10 days is recommended for unprotected close contacts of patients with seasonal influenza who are at risk of complicated influenza. 98 who does not recommend routine post-exposure antiviral chemoprophylaxis for a(h7n9) virus. however, for some asymptomatic persons in which a substantial unprotected or prolonged exposure to an ill patient with a(h7n9) infection has occurred, initiation of empiric post-exposure antiviral treatment (e.g. oseltamivir 75 mg orally bd for 5 days), on the presumption that influenza virus infection has occurred, may be considered. this is likely to be limited to healthcare or other settings involving substantial exposure of those at higher risk for complications from table 4 infection prevention and control measures when assessing patients with complicated influenza 51,90,91 1. clinicians should pay attention to cases of community-acquired pneumonia and patients' travel history for early case detection 2. clinicians should remain vigilant against avian influenza and elicit any relevant clinical and epidemiological information from patients (fever, travel history, occupation, contact history and clustering) 3. standard, contact and droplet precautions are recommended for routine management of patients hospitalized for influenza. droplet precaution (by wearing a surgical mask within 1 m of the patient) and contact precaution (by wearing gown and gloves on entering the room and removing them on leaving) when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of avian influenza infection 4. risk assessment should be conducted before performing agp. for cases with severe influenza, it is advisable to perform agp in an airborne isolation room. when an airborne isolation room is not available, the following minimum hourly averaged ventilation rates should be provided for natural ventilation: a. 160 l/s/patient (hourly average ventilation rate) for airborne precaution rooms (with a minimum of 80 l/s/patient) (note that this only applies to new healthcare facilities and major renovations); b. 60 l/s/patient for general wards and outpatient departments; and c. 2.5 l/s/m 3 for corridors and other transient spaces without a fixed number of patients 5. in view of the increasing influenza activity during winter season and the emerging threat of avian influenza, all healthcare workers and visitors are recommended to wear surgical masks when entering patient care areas and strengthen hand hygiene agp, aerosol-generating procedure. influenza virus infection, including patients with severe immunosuppression, neonates and infants, pregnant and early post-partum women, elderly adults, persons with co-morbidities and other highly vulnerable patients; or, unprotected healthcare workers, especially those involved in agp. 99 annual seasonal influenza vaccinations are recommended for the high-risk groups, including residents of institutions for elderly and disabled, any age with chronic illness, age > 65 years; 6 months to 6 years, poultry workers in countries affected by avian influenza, healthcare workers especially those frequently in contact with high-risk persons and household contacts of high-risk persons. while vaccination rates among healthcare workers are highly variable in different countries, 100 there is no conclusive evidence that seasonal influenza vaccination of healthcare workers may protect their patients in terms of reduction in risks of mortality and influenza-like illness. 101 a survey in hong kong assessing the acceptability of an additional ad hoc influenza vaccination among the healthcare professionals following seasons with significant antigenic drift has shown that the strongest factors associated with accepting an additional influenza vaccine included immunization with influenza vaccines in the past 3 years, higher perceived risk of contracting influenza and higher perceived severity of the disease impact. 102 the current influenza vaccines induce a strainspecific immunity that is not ideal for this rapidly mutating virus. a universal influenza vaccine capable of inducing a broad cross-protection across strains is needed. to this end, the m2e antigen, a linear peptide that is conserved across influenza a strains, is being evaluated a vaccine target. 103 the other approach is to prepare virus-like-particles based on ha, na and m1 proteins. 104, 105 virus-like particles with a cocktail of seasonal and potential pandemic strains have been prepared and demonstrated promising results. 106 as a(h7n9) is now endemic in over half of the provinces in mainland china and will continue to cause recurrent zoonotic disease in the winter months, public health measures to control the source such as some rest days every month for the poultry workers to thoroughly clean the lpm environment, and banning the stock of live poultry in markets overnight are important. separation of live ducks and geese from landbased (i.e. non-aquatic) poultry in lpm systems can reduce the risk of emergence of zoonotic and epizootic viruses at source. in the long run, it is advisable to adopt a complete transition from sale of live poultry in wholesale and retail lpm to centralized slaughter and sale of chilled or frozen poultry. 107 seasonal influenza epidemics and periodic pandemics are important causes of morbidity and mortality. apart from influenza a(h5n1) virus and sporadic human cases of a(h5n6), a(h10n8) and a(h6n1) infections, infection due to a(h7n9) virus, especially the recent upsurge in the number of human infections in mainland china, is of great concern. the widespread poultry outbreaks due to a(h5n8) in many countries increases the risk of human infection. early isolation and treatment with an nai for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes. whether higher than standard doses of nai may provide greater antiviral effects in such patients would require further investigation. high-dose systemic corticosteroids were associated with worse outcomes in patients with severe influenza complicated by ards. there is an urgent need for developing more effective antiviral therapies for the treatment of influenza infections. university of hong kong. professor p.k.s.c. is a renowned clinical virologist with special interest in tumour virology and human respiratory viruses. he has published more than 330 scientific papers, and attained an h-index of 51. n.l. is professor in infectious diseases in the chinese university of hong kong. his main research interests include respiratory viral infections (influenza, rsv and coronavirus) and cap, focusing on clinical trials, patient outcomes, virokinetics and host response, and infection control in the hospital settings. he is currently working on novel antivirals and adjunctive therapies against these severe diseases. clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus human infection with a novel avian-origin influenza a (h7n9) virus who china statement on h5n6 clinical and epidemiological 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particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes interventions to reduce zoonotic and pandemic risks from avian influenza in asia additional supplementary information can be accessed via the html version of this article at the publisher's website. phylogenetic tree of haemagglutinin (ha) genes of h7n9 isolates collected from human infections. key: cord-274241-biqbsggu authors: shaw, timothy i.; srivastava, anuj; chou, wen-chi; liu, liang; hawkinson, ann; glenn, travis c.; adams, rick; schountz, tony title: transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0048472 sha: doc_id: 274241 cord_uid: biqbsggu the jamaican fruit bat (artibeus jamaicensis) is one of the most common bats in the tropical americas. it is thought to be a potential reservoir host of tacaribe virus, an arenavirus closely related to the south american hemorrhagic fever viruses. we performed transcriptome sequencing and annotation from lung, kidney and spleen tissues using 454 and illumina platforms to develop this species as an animal model. more than 100,000 contigs were assembled, with 25,000 genes that were functionally annotated. of the remaining unannotated contigs, 80% were found within bat genomes or transcriptomes. annotated genes are involved in a broad range of activities ranging from cellular metabolism to genome regulation through ncrnas. reciprocal blast best hits yielded 8,785 sequences that are orthologous to mouse, rat, cattle, horse and human. species tree analysis of sequences from 2,378 loci was used to achieve 95% bootstrap support for the placement of bat as sister to the clade containing horse, dog, and cattle. through substitution rate estimation between bat and human, 32 genes were identified with evidence for positive selection. we also identified 466 immune-related genes, which may be useful for studying tacaribe virus infection of this species. the jamaican fruit bat transcriptome dataset is a resource that should provide additional candidate markers for studying bat evolution and ecology, and tools for analysis of the host response and pathology of disease. bats are an ancient and diverse group [1] and are the second largest taxonomic group of mammals with more than 1,200 identified species among the 5,499 known mammals [2, 3] . bats are the only mammals to have evolved powered flight, which has allowed dispersal across all continents other than antarctica. bats are critical components of ecosystems, serving as major predators of insects, pollinating flowers and dispersing seeds of keystone plant species worldwide. the body sizes of bats range from less than 2 gm with 8 cm wingspans to more than 1 kg with 2 m wingspans. most contemporary species of bats are insect-, nectar-, or fruit-eaters, but about 1% are carnivores, including fish-eating and blood-drinking species. the evolutionary origin of bats remains controversial [4, 5] . in early work, bats were thought to be closely related to rodents and primates [6] . bats are now established within laurasiatheria; however, the placement of bats within laurasiatheria has been difficult to resolve because the major groups diverged from one another within a relatively short period of time [7] . different placements recently hypothesized for bats include: (a) sister to perissodactyla (horse) [8] ; (b) sister to cetartiodacyla (cattle+dolphin) [5] , (c) sister to perissodactyla+cetartiodacyla (horse, cattle, dolphin) [9] , (d) sister to ferungulata (cattle+dolphin, dog+horse) [4, 10] and (e) the pegasoferae hypothesis which places bat with perissodactyla and carnivora (horse+dog) [11] (see [5] for a review). two bat genomes have been sequenced to date [12] , the little brown bat (myotis lucifugus, 76 coverage) and the large flying fox (pteropus vampyrus, 2.66 coverage), but neither has been extensively annotated. these species represent the two major clades within bats: the microbats and megabats. transcriptome sequencing for another megabat species, the australian flying fox (pteropus alecto), has recently been published [13] . thus, a transcriptome for a microbat species is needed. many highly pathogenic viruses are hosted, or suspected to be hosted, by bat reservoirs, including ebolaviruses, marburg virus, hendra virus, nipah virus, rabies virus and coronaviruses [2] . in total, more than 100 viruses have been isolated from, or detected in, bats of dozens of species, yet many of the viruses that cause disease in humans cause little or no disease in the bats. significantly, the great majority of bat species have not been examined for infectious agents and are, thus, likely underappreciated as reservoir hosts. the continued encroachment of humans upon bat habitat and bat migrations caused by climate change may lead to novel infectious diseases among humans and livestock. moreover, some infectious diseases cause significant morbidity and mortality in bats that could have dramatic impacts on population numbers and cascading ecological effects [14] . thus, the study of bats and their infectious agents is an important but neglected aspect of zoonotic and wildlife disease research. jamaican fruit bats (artibeus jamaicensis) are one of the most common bats in the tropical americas, ranging from the caribbean islands, tropical south and central america, mexico and the florida keys [15] . the jamaican fruit bat is a microbat in the family phyllostomidae, which contains 56 genera and 192 species. they are a frugivorous generalist and fig specialist of medium size; about 80 mm in length with a wingspan of 130 mm and mass of about 50 grams. they can readily fly 20 km per night, although they typically maintain a smaller home range as long as food is available, and can live 9 years or more in the wild. females typically produce two offspring per year and provide maternal care for about 50 days, with pups reaching adult body weight by about 80 days. several microbes of interest have been isolated from or detected in jamaican fruit bats, including histoplasma capsulatum, trypanosoma cruzi, eastern equine encephalitis virus, mucambo virus, jurona virus, catu virus, itaporanga virus and tacaiuma virus, suggesting the species may be an important reservoir and vector of infectious diseases [15] [16] [17] [18] . it is unknown what diseases these pathogens may cause in bats. tacaribe virus (tcrv) was isolated from 11 artibeus bats (6 a. lituratus, 5 a. jamaicensis) in the late 1950s in and near port of spain, trinidad [19] . tcrv was the first arenavirus isolated in the americas and during the next decades other arenaviruses with substantial similarity to tcrv were identified that cause the south american hemorrhagic fevers (sahf) [20, 21] . the known reservoir hosts for all other arenaviruses are rodents, making tcrv exceptional for its repeated isolation from artibeus bats. because exhaustive searches for evidence of other potential reservoir hosts of tcrv failed to suggest another reservoir species [19, 22] , it has been suspected that artibeus bats are reservoirs of the virus. however, recent work by us demonstrated that trlv-11573, the only remaining isolate of tcrv, causes a fatal infection resembling the sahf in jamaican fruit bats, one of the species from which tcrv was isolated, or is cleared without disease, suggesting this species is not a suitable reservoir host for tcrv and the virus may be a significant pathogen for bats [23] . because of the equivocal role of artibeus bats as a reservoir host species for tcrv, and because of the similarities with human sahf, the jamaican fruit bat may be a novel model for studying the pathology of the disease. however, as an unusual, non-model organism, very little is known about its physiology, immunology or host response to tcrv. no antibodies are available with known specificity to jamaican fruit bat proteins, which dramatically limits its usefulness. to address some of these deficiencies, we have performed transcriptome sequencing and analysis of spleen, lung, kidney and poly-ic-stimulated primary kidney cells to identify genes of interest for assessing the host response to tcrv infection. more than 240,000 454 reads and 142 million illumina reads were obtained ( table 1 ). the reads were submitted to short read archive (sra) under srr539297 and srr538731. reads from lung, kidney, and poly-ic-stimulated primary kidney cell libraries were pooled for a combined de novo assembly using the 454 gs assembler program, yielding 6,450 contigs. for the illumina spleen sequences, we first corrected reads using the soapdenovo correction tool and further assembled them using soapdenovo, yielding 214,707 contigs. a total of 367,317 snps and 44,679 indels were detected through gigabayes. at least 16 reads covering a site were required to ensure the snp was of high quality. using tgicl, a combined assembly of the 454 and illumina contigs was constructed that contained 102,237 contigs with n10, n50, and n90 of 3,882 bp, 1,004 bp, and 289 bp, respectively. human and mouse genomes were used as references to estimate the distribution of bat contigs within known gene transcripts. human and mouse genomes were chosen for completeness of their annotations. genomic features were divided into 5 groups: 1 kb upstream of 59 utr, 59 utr, cds (coding sequence), 39 utr, 1 kb downstream of 39 utr. we found 58.03% and 23.18% mapped cds region for human and mouse genome respectively ( figure 1a ). because we performed transcriptome sequencing, we expected a majority of the sequences to map to cds and utr regions of the genome. many rna genes were also mapped, including long noncoding rnas and a substantial number of micrornas ( figure 1b ). annotation was concentrated on identifying micrornas because they could be cross validated through their rna secondary structure features. to further obtain a confident set of microrna sequences, a microrna prediction pipeline was used to cross validate the blast mapping of prediction. in the process, 42 confident microrna candidates were found that have been deposited within mirbase [24, 25] . we present the list of predicted micrornas as table s1 . mapping the predicted snps on the genomic features indicates that the vast majority of snps are in the cds region ( figure 1c-1d) . although humans and mice are both outside laurasiatheria the relatively fast rate of molecular evolution of mice is expected to result in more differences between bats and mice than bats and humans [26] [27] [28] [29] . the presence of sequences mapped 1 kb upstream or downstream of the known transcript indicated possible alternative splicing from human and mouse transcripts. blast2go was used to functionally annotate contigs. a total of 20,020 contigs (19.58% overall) had significant matches to known proteins in the ncbi non-redundant protein (nr) database. horse and human were identified as the top two species with best blast hits for bat contigs ( figure 2 ). the blastx annotation process is biased by the completeness of the annotation for each respective genome; therefore, despite the lack of a completely annotated horse genome, a high similarity between bat and horse genomes was apparent. the human genome is well annotated, which explains the high number of blast hits between bat and human. the go annotation divides the functional annotation into three main components: biological process, cellular process, and molecular [30] . a majority of the annotated genes encoding proteins that function within a cell or organelle are involved in metabolic and cellular processes. the primary molecular functions of these genes are catalytic and binding activities ( figures 3a-c) . a total of 466 immune-related genes were annotated by blast2go. these immune genes include toll-like receptors, cytokines, transcription factors, kinases and several chemokine receptors. in addition, categorizer was used to categorize the immune class using the goslim database, resulting in 30 categories representing a broad range of immune activities ( figure 4 ). the immune response and lymphocyte activation genes represented the largest proportion of theses transcripts. there were 82,218 unannotated contigs. a total of 16,869 sequences had open reading frames longer than 300 nt; 5,417 were identified through blastp to the nr database with evalue,1e 23 . for the remaining contigs, 54,892 mapped to the assembled myotis lucifugus genome, and 48,809 mapped to the assembled pteropus vampyrus genome. there were 20,145 contigs that mapped to pteropus alecto, australian flying fruit bat, and 18,359 that overlapped between genomic and transcriptome sequences for all three datasets ( figure 5 ). through this process, we were able to account for 65,828 (80%) unannotated contigs. the completeness of genes mapped to immunological pathways was examined using human and mouse as reference species. based on the ortholog data obtained, all contigs were mapped onto immune system related kegg pathways ( table 2 ) and determined that many genes were missing from these pathways. this could be due to the low expression within bat tissues or due to the overly stringent e-value cutoff of 1e 220 during reciprocal blast annotation that we chose to limit the number of false positives. a kegggraph visual representation of contigs mapped onto the mouse pathway was generated (figures s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, s18). pathways involved in the adaptive immune response, t and b cell signalling pathways, generally had more mapped genes than did those involved in innate response or natural killer (nk) cell-mediated cytotoxicity pathways ( figures 6a and 6b ). the nk cell cytotoxicity pathway appears to have almost half of its genes missing, whereas the b cell receptor pathway appears to have most of its genes present. nucleotide substitution in the coding region can be synonymous or non-synonymous. the ratio between the rate of synonymous (ds) and non-synonymous mutation (dn) can be used to infer the degree of selection operating on the system. we used the human genome as a reference for dn/ds calculations because the human genome is well annotated. reciprocal blast was used to identify human, mouse, and bat orthologs. macse was used to generate codon alignments. the alignments were trimmed for excessive gap codon triplets, and paml was used to calculate dn/ds for each gene. when genes are highly conserved, synonymous mutations (ds) tend to be estimated as 0, resulting in a larger dn/ds ratio, therefore those results were removed from the analysis. after filtering, dn/ds results were obtained for 14,717 genes. the majority of the genes have close to zero dn/ds with clear evidence of purifying selection, a feature common among mammalian genes [31] [32] [33] . for investigation of positive selection, tang et al. [34] have argued that a dn/ds threshold of greater than 1 for positive selection might be overly stringent. because of this, a dn/ds cutoff of 0.7 was chosen to investigate genes that might be experiencing weak purifying selection. a total of 138 genes above the 0.7 threshold were found (table s2) . for genes with evidence of positive selection, 32 exceeded the 1.0 dn/ds threshold (figure 7) . through annotation by david [35, 36] , there were 14 genes involved in transcriptional activation and regulation processes. there were 9 genes associated with cellular signaling. in particular, we found dna-damage-inducible transcript 4 (ddit4) gene with dn/ds 1.4053; this protein is involved in the mtor signaling pathway and it regulates cell growth and promotes neuronal cell death [37, 38] . ectodysplasin a (eda), involved with cytokine:receptor interaction pathways, had a dn/ds value of 1.23. the phylogenetic placement of bats within laurasiatheria is still unresolved. through reciprocal blast, we identified 8,785 putative orthologs across mouse, rat, cattle, horse and human (table s3) . afterward we filtered out alignments with greater than 5% gap, the 2,378 genes remaining were used to construct 500 iteration multilocus bootstrap species tree (see methodology). this resulted in a highly supported species tree placing bat sister to the clade containing cattle, horse, and dog ( figure 8 ). the jamaican fruit bat transcriptome described here is a major new resource for genetic studies of bats. this bat is an important seed dispersing and pollinating species found in most of the tropical americas. it is likely susceptible to infectious diseases, could be a zoonotic reservoir and vector, and may be a suitable model for the pathogenesis of sahf. considering the importance of immunological functions in response to infections, we conducted a transcriptome assessment of genes from spleen, kidney and lungs so that genetic tools and methods can be used to study this species as well as other microbats. genes were identified that mapped to immune response pathways; based on categorize classification of immune classes, we found 40 different immune classes. recently, the transcriptome sequencing for the australian black flying fox was performed [13] . our data contain a greater proportion of lymphocyte related immune classes than does the flying fox's transcriptome dataset. however, our dataset also contained a lesser proportion of cytokine related immune classes than the flying fox's transcriptome dataset. genes involved in adaptive immune response generally had more mapped genes compared to genes involved in innate responses. from figures 6a and 6b , more genes were mapped to the b cell receptor signalling pathway than to the nk cellmediated cytotoxicity pathway. this bias is likely due to the large number of b cells found in the spleen. due to our stringent blast criteria, it is also possible that lowering the e-value threshold could obtain additional genes mapped but at the risk of more false positives. we deposited 42 microrna genes for a. jamaicensis into mirbase, and according to mirbase this gene set is the first deposited bat microrna genes. estimates of substitutions within the orthologous contigs found 32 genes with a dn/ds ratio.1. this ratio provides a guide for indicating potential genes that are under positive selection. many genes were involved in transcriptional activation/regulation processes, suggesting potential differences in the transcriptional regulating architectures of bats and humans. the ddit4 and eda have a dn/ds ratio.1, suggesting these genes are under positive evolutionary selection. ddit4 is involved in regulation of cell death and its positive selection suggests a potential difference in cell death regulation between human and bats; further analysis will need to be performed to verify the functional differences. another potential positively selected gene, eda is associated with ectodermal dysplasia type 1 [39] , a disorder associated with abnormal development of physical structures, including skin, hair, nails, teeth, and sweat glands. we suspect the bat's eda gene could be used as a potential reference for future studies of the disorder. for transcripts that failed to be identified by reciprocal blast searches, we predicted the orf for the unannotated contigs and used blastp against the nr database to identify 5,349 unannotated contigs. for the remaining unannotated contigs, we used genomic data from myotis lucifugus and pteropus vampyrus, as well as transcriptome data from pteropus alecto to identify additional unannotated contigs. existing artibeus contigs that were not present within the nr database, but overlapped among myotis and pteropus genomic and transcriptomic sequences indicated the possibility for bat specific transcripts. we also found contigs that mapped only to the myotis lucifugus genome indicating the possibility for microbat specific contigs. in total, we were able to account for 80% of the unannotated trancripts, and the remaining unannotated transcripts likely include misassembled contigs, contigs not sequenced sufficiently in the other bats to be included in their genome assemblies, as well as a few transcripts specific to artibeus jamaicensis. many additional analyses are warranted to further refine the transcriptome information from artibeus and other bats. phylogenomics is an important tool for resolving the tree of life, and this transcriptome data set provides an opportunity to study the evolutionary history of bats. bats were once thought to be closely related to primates [6] ; however, further work using molecular information placed them within laurasiatheria [40] . our finding of bat as sister to the clade containing horse, dog, and cattle is consistent with the recent study by mccormack et al. [4] and zhou et al [10] . here, we used 2,378 loci from a microbat and species tree analyses to obtain 95% bootstrap support, whereas mccormack et al. use 683 loci to obtain 64%bootstrap support. a recent study by nery et al. [5] obtained a concatenated data from 3,733 loci from megabat with 100% bootstrap support and 1.0 posterior probability placing bat as sister to cattle. our phylogenetic tree is less resolved than nery et al., probably because we did not include the more limited transcriptome data available from dolphin and hedgehog. maximum likelihood analyses are powerful, yet can lead to incorrect conclusions in certain situations, whereas species tree analyses are less powerful but more robust to well-known violations of the models used for maximum likelihood phylogenetic analysis, such as incomplete lineage sorting (see [5] and references therein). additional work is clearly warranted, especially by using additional taxa, testing for convergence and specific violations of gene-tree models, and other sources of conflict among protein-coding genes and other portions of the genome. a principal difficulty for identifying mechanisms of pathogenesis of the sahf, in which the immune response may play a contributory role, is a lack of animal model resources that faithfully recapitulate human disease [41] . although laboratory mice (mus musculus) and rats (rattus norvegicus), which have substantial experimental methodologies and reagents, can be infected with junã­n virus (junv), the etiologic agent of argentine hemorrhagic fever, the pathogenesis is markedly different than human disease. the guinea pig (cavia porcellus) typically exhibits signs of disease that closely resembles human disease; however, there are few immunological or genetic tools for assessing the host response to infection. junv is also a bsl-4 and select agent, thus use of virulent strains is confined to only a few laboratories with highly specialized containment facilities. the pathogenesis of tcrv, a bsl-2 agent, in jamaican fruit bats exhibits many similarities to the sahf in humans, thus the use of transcriptome data could be useful for studying pathogenesis using a variety of new technologies, such as pcr arrays for pathway discovery, and for the development of antibodies to specific artibeus proteins that are important in the pathogenesis of disease. the transcriptome resource provided will facilitate research into artibeus host responses to infectious agents, including mechanisms of pathogenesis of arenavirus disease and will also provide further resource for additional understanding for the bat species evolution and physiological development. all procedures were approved by the unc institutional animal care and use committee (iacuc) and were in compliance with . substitution estimation scatter plot. we calculated the nonsynonymous mutation rate (dn) and synonymous mutation rate (ds) using orthologous genes between bat and human. two lines were drawn representing the two dn/ds cutoffs of 0.7 (green) and 1.0 (blue). doi:10.1371/journal.pone.0048472.g007 figure 8 . unrooted species tree from the orthologous dataset across six boreoeutheria mammals. the species tree was generated from 2378 gene loci. there was 95% bootstrap support for placing bats (chiroptera) sister to perissodactyla, cetartiodactyla, and carnivora. doi:10.1371/journal.pone.0048472.g008 the usa animal welfare act. unc animal care and use committee approval number, 1207c-ra-b-15. five bats from the university of northern colorado jamaican fruit bat colony were used for this work. male and female a. jamaicensis bats were euthanized by respiratory hyperanesthesia followed immediately by thoracotomy. tissues were aseptically removed and flash frozen in liquid nitrogen for subsequent rna extraction. tissues were homogenized in buffer rlt (rneasy kit, qiagen, valencia, ca) containing 2me using a bead beater and silicone beads. the homogenate was passed over a qiashredder column prior to total rna extraction according to manufacturer's instructions. for cell culture, one kidney from one bat was collected in serum-free hbss and minced under aseptic conditions, then trypsinized (trypsin-versene) at room temperature in a sterile 50 ml trypsin flask. cells were washed 36 in 10% fbs-emem, then seeded into a vented t-25 flask. the next day, unattached cells were removed and fresh 10% fbs-emem added. when cells approached confluence they were passaged with trypsin at a split ratio of 1:4. poly-ic was added to 50 mg/ml in two t-75 flasks containing 20 ml each of 10% fbs-emem and incubated for 6 hours, after which rna was extracted according to manufacturer's instructions (qiagen). total rna was extracted using the rneasy minelute cleanup kit (qiagen) and then shipped on dry ice to seqwright (houston, tx) for cdna library construction and sequencing. rna concentrations and quality were assessed by a260/a280 and a260/a230 absorbance values and agarose gel electrophoresis. a260/a280 values were all above 2.0 and a260/a230 were all above 1.9. electrophoresis of the rna samples demonstrated that 28s and 18s rrna were not degraded. libraries for the 454 were prepared from three tissues (kidney, lung, poly-ic-stimulated kidney cells). for 454 library construction, full-length cdna was synthesized with two set of primers for driver and tester cdna [42, 43] . single-stranded cdna was used for hybridization instead of double-stranded cdna. excess amounts of sense-stranded cdna hybridized with antisense-stranded cdna. after hybridization, duplex was removed by hydroxyapatite chromatography. normalized tester cdna was re-amplified with tester specific primer l4n. driver cdna was unable to amplify using l4n. an illumina truseq rna library was made from spleens according to manufacturer's instructions. the libraries were then sequenced according to manufacturer's recommendations: 454 using titanium chemistry and illumina using 26100 nucleotide paired-end sequencing on a hi-seq 2000. the 454 and illumina libraries were assembled individually and also by combining both libraries. bases from the 454 reads were called from the 454 generated sff file using pyrobayes [44] and 454 gs assembler (version 2.5) was used to perform the assembly. soap denovo [45] (version 1.04) was used to assemble reads obtained from spleen (illumina library). only contigs greater than 200 bases were used in the final analysis. prior to performing the combined assembly, duplicates from pre-assembled contigs of lung, kidney and spleen tissues were removed with cd-hit [46] (cd-hit-2009-0427) at default criterion and then combined into longer fragments with tgicl [47] . gigabayes [48] -a short-read snp/indel discovery program was used to detect polymorphisms. snp/indel detection was performed for both libraries separately. to make snp/indel predictions more reliable, we used the criterion that minor allele and major allele (alleles with fewer reads are minor alleles, and alleles with more reads are major alleles) occur at least twice and 8 times for 454 and illumina libraries, respectively. to identify the approximate relative position of conserved mammalian genes, we mapped the bat contigs on to the genome of mouse mm9 and human grch37 (downloaded from the ucsc genome browser) using blat v.34 [49] with a minimum score of 80 used as a filter. coordinates of the protein coding genes were obtained from ensemble (http://uswest.ensembl.org/index.html) xenoref and gtf files. we also normalized the number of blat hits based on the total annotated transcript regions (1000 nt upstream of 59 utr, 59 utr, cds, 39utr, and 1000 nt downstream of 39utr) that were present in the mouse and human. to predict precursor-microrna genes within assembled sequences, we downloaded precursor micrornas for mouse, rat and human from mirbase [24, 25] . we performed a blast search focused on high quality candidates, hits with $95% sequence identity [50] . based upon rnafold [51] secondary structure prediction, we further filtered out sequences that did not possess any hairpin loop structure. previously, it had been demonstrated that micrornas tend to have deterministic folding [52] and, therefore, we used unpaired structural entropy (use) to evaluate the rna secondary structure base pairing distribution (cutoff 0.83 use score). mir-abela, a support vector machine learning program [53] , was used to cross validate the prediction. the final remaining unfiltered sequences are considered as highly confident microrna candidates. orthologous contigs (against human, mouse, dog, cattle and horse) were identified using the reciprocal blast (blastn) approach [54] as it has been found to be superior to sophisticated orthology detection algorithms [55] . a stringent cutoff of 1e 220 was used to separate paralogs from orthologs. cdna sequences from human (homo_sapiens.grch37. 64 the substitution rate is inferred from orthologous genes between bat and mouse. sequences were aligned using macse [56] and an in-house java script was used to trim/remove codon gap triplets from the alignment. substitution rate was estimated using a maximum likelihood method implemented in the codeml program of paml 4.5 [57, 58] . the pairwise maximum likelihood analyses were performed in runmode-2. estimated rates of non-synonymous to synonymous substitutions (dn/ds) were plotted as a scatter plot. blast2go [59] was used to functionally annotate contigs. a combined graph was generated for each go category. to prevent overloading graphs, the sequence filter value was changed to 500 in all 3 categories (biological process, molecular function and cellular component). functional annotation was performed separately for all assembled contigs present in the combined assembly. based on categorizer [60] , we further classified the genes using the go slim database immune classes. the completeness of mapping the bat genes using euarchontoglires as a reference was further examined through kegg. to do this, we first downloaded the xml file of annotated kegg pathways [61, 62] for human and mouse. to identify genes that are functionally important within kegg pathways, kegggraph was used to represent a graph form of the kegg pathway. we further used kegggraph to compute the relative betweenness centrality, which is the algorithmic representation of the involvement of a node within a network. we chose to set a cutoff of grabbing the top 4 nodes within each network, or selecting the top 4 functionally important genes within each pathway [63] . open reading frame was predicted from the assembled contigs through the orfpredictor web server (http://proteomics.ysu.edu/ tools/orfpredictor.html) [64] . a customized java program was used to parse through the prediction to identify sequences longer than 300 nt. to perform additional annotation of the predicted open reading frame we used blastp with an e-value of 1e 23 against the most recent nr database that is available from ncbi during our analysis (august 26 th , 2012). using contigs that were functionally unannotated, we compared the jaimacan fruit bat contigs against three other available bat sequence dataset. myotis lucifugus and pteropus vampyrus genomes were downloaded from the ncbi tracedb ftp server (ftp://ftp.ncbi.nih.gov/pub/tracedb/). the pteropus alecto transcriptome was obtained from dr. a. papenfuss [13] . an evalue threshold of 1e 25 was used to indicate blast hit. we then used an r package venndiagram [65] for displaying the mapped unannotated contigs that overlapped between different bat genome and transcriptomes. to resolve the evolutionary relationship for the artibeus bat species, we filtered the putative bat orthologs between human, mouse, dog, cattle, and horse. insectivores such as hedgehog and dolphin were not used in our analysis due to limited gene annotation in these taxa. to obtain the best multiple sequence alignment for each putative orthologs, we used aqua's pipeline for performing multiple sequence alignment; the pipeline consists of multiple sequence alignment through muscle and mafft which is refined by rascal and assessed by normd [66] [67] [68] [69] . a customized java program was used to filter alignments (obtained through aqua) with greater than 5% gap per sequence. additionally, we filtered for sequences that are at least.1,000 bp long. phyml 3 was used to generate a maximum likelihood gene tree [70, 71] . mraic, a perl script wrapper for phyml, was used to infer the best substitution model for each gene tree based on aic, aicc, bic, and akaike weights [72] . aic was used as the objective function since not much variation was observed across different objective function. njst was used to calculate the unrooted species tree based on our gene trees [73] . a customized rprogram is used for performing a nonparametric bootstrap species tree through resampling nucleotides within loci as well as resampling the loci within the data set as described by seo [74] . fossil evidence and the origin of bats bats: important reservoir hosts of emerging viruses iucn red list version 2011.2: tabel 3a -status category summary by major taxonomic group (animals) ultraconserved elements are novel phylogenomic markers that resolve placental mammal phylogeny when combined with species-tree analysis resolution of the laurasiatherian phylogeny: evidence from genomic data mammalian phylogeny: shaking the tree mammalian phylogenomics comes of age using genomic data to unravel the root of the placental mammal phylogeny confirming the phylogeny of mammals by use of large comparative sequence data sets phylogenomic analysis resolves the interordinal relationships and rapid diversification of the laurasiatherian mammals pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions a highresolution map of human evolutionary constraint using 29 mammals the immune gene repertoire of an important viral reservoir, the australian black flying fox emerging diseases in chiroptera: why bats? artibeus jamaicensis identification of a new venezuelan equine encephalitis virus from brazil humoral and cell-mediated immunity to histoplasma capsulatum during experimental infection in neotropical bats (artibeus lituratus) experimental rabies virus infection in artibeus jamaicensis bats with cvs-24 variants tacaribe virus, a new agent isolated from artibeus bats and mosquitoes in trinidad, west indies the phylogeny of new world (tacaribe complex) arenaviruses phylogenetic analysis of the arenaviridae: patterns of virus evolution and evidence for cospeciation between arenaviruses and their rodent hosts serological evidence of infection of tacaribe virus and arboviruses in trinidadian bats tacaribe virus causes fatal infection of an ostensible reservoir host, the jamaican fruit bat mirbase: the microrna sequence database mirbase: integrating microrna annotation and deep-sequencing data fast genes and slow clades: comparative rates of molecular evolution in mammals determinants of rate variation in mammalian dna sequence evolution determination of mitochondrial genetic diversity in mammals correlates of substitution rate variation in mammalian protein-coding sequences the gene ontology categorizer natural selection on protein-coding genes in the human genome a scan for positively selected genes in the genomes of humans and chimpanzees positive selection on the human genome a new method for estimating nonsynonymous substitutions and its applications to detecting positive selection systematic and integrative analysis of large gene lists using david bioinformatics resources bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists rtp801 is elevated in parkinson brain substantia nigral neurons and mediates death in cellular models of parkinson's disease by a mechanism involving mammalian target of rapamycin inactivation rtp801 is a novel retinoic acid-responsive gene associated with myeloid differentiation mutations within a furin consensus sequence block proteolytic release of ectodysplasin-a and cause x-linked hypohidrotic ectodermal dysplasia parallel adaptive radiations in two major clades of placental mammals junin virus. a xxi century update construction of a uniformabundance (normalized) cdna library construction and characterization of a normalized cdna library pyrobayes: an improved base caller for snp discovery in pyrosequences de novo assembly of human genomes with massively parallel short read sequencing cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences tigr gene indices clustering tools (tgicl): a software system for fast clustering of large est datasets a general approach to single-nucleotide polymorphism discovery blat-the blast-like alignment tool a uniform system for microrna annotation fast folding and comparison of rna secondary structures analyzing modular rna structure reveals low global structural entropy in microrna sequence identification of clustered micrornas using an ab initio prediction method gapped blast and psi-blast: a new generation of protein database search programs phylogenetic and functional assessment of orthologs inference projects and methods macse: multiple alignment of coding sequences accounting for frameshifts and stop codons paml 4: phylogenetic analysis by maximum likelihood paml: a program package for phylogenetic analysis by maximum likelihood blast2go: a universal tool for annotation, visualization and analysis in functional genomics research categorizer: a web-based program to batch analyze gene ontology classification categories kegg for integration and interpretation of large-scale molecular data sets kegg: kyoto encyclopedia of genes and genomes kegggraph: a graph approach to kegg pathway in r and bioconductor orfpredictor: predicting proteincoding regions in est-derived sequences venndiagram: a package for the generation of highly-customizable venn and euler diagrams in r muscle: multiple sequence alignment with high accuracy and high throughput mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform aqua: automated quality improvement for multiple sequence alignments rascal: rapid scanning and correction of multiple sequence alignments morephyml: improving the phylogenetic tree space exploration with phyml 3 estimating maximum likelihood phylogenies with phyml model selection and multi-model inference estimating species trees from unrooted gene trees calculating bootstrap probabilities of phylogeny using multilocus sequence data we thank jason shaw for assistance with bat handling and stephanie james for laboratory assistance, and uga's georgia advanced computing resource center and institute of bioinformatics for computational resources and support. we thank anthony papenfuss for providing the assembled transcriptome for p. alecto. contributed reagents/materials/analysis tools: ts tg ll. wrote the paper: tis tg ts. key: cord-292075-t9z7zqz4 authors: gessain, antoine title: mécanismes d’émergence virale et transmission interespèces : l’exemple des rétrovirus foamy simiens chezl’homme en afrique centrale date: 2013-12-31 journal: bulletin de l'académie nationale de médecine doi: 10.1016/s0001-4079(19)31387-1 sha: doc_id: 292075 cord_uid: t9z7zqz4 summary a large proportion of viral pathogens that have emerged during the last decades in humans are considered to have originated from various animal species. this is well exemplified by several recent epidemics such as those of nipah, severe acute respiratory syndrome, avian flu, ebola, monkeypox, and hantaviruses. after the initial interspecies transmission per se, the viruses can disseminate into the human population through various and distinct mechanisms. some of them are well characterized and understood, thus allowing a certain level of risk control and prevention. surprisingly and in contrast, the initial steps that lead to the emergence of several viruses, and of their associated diseases, remain still poorly understood. epidemiological field studies conducted in certain specific high-risk populations are thus necessary to obtain new insights into the early events of this emergence process. human infections by simian viruses represent increasing public health concerns. indeed, by virtue of their genetic and physiological similarities, non-human primates (nhps) are considered to be likely the sources of viruses that can infect humans and thus may pose a significant threat to human population. this is well illustrated by retroviruses, which have the ability to cross species, adapt to a new host and sometimes spread within these new species. sequence comparison and phylogenetic studies have thus clearly showed that the emergence of human immunodeficiency virus type 1 (hiv-1) and hiv-2 in humans have resulted from several independent interspecies transmissions of different siv types from chimpanzees and african monkeys (including sooty mangabeys), respectively, probably during the first part of the last century. the situation for human t cell lymphotropic virus type 1 (htlv-1) is, for certain aspects, quite comparable. indeed, the origin of most htlv-1 subtypes appears to be linked to interspecies transmission between stlv-1-infected monkeys and humans, followed by variable periods of evolution in the human host. in this review, after an introduction on emerging viruses, we will briefly present the results of a large epidemiological study performed in groups of bantus and pygmies living in villages and settlements located in the rain forest of the south region of cameroon. these populations are living nearby the habitats of several monkeys and apes, often naturally infected by different retroviruses including siv, stlv and simian foamy virus. most of the persons included in this study were hunters of such nhps, thus at high risk of contact with infected body fluids (blood, saliva,...) during hunting activities. after reviewing the current available data on the discovery, cross-species transmission from monkeys and apes to humans of the simian foamy retroviruses, we will report the results of our study. such infection is a unique natural model to study the different mechanisms of restriction of retroviral emergence in humans. a large proportion of viral pathogens that have emerged during the last decades in humans are considered to have originated from various animal species. this is well exemplified by several recent epidemics such as those of nipah, severe acute respiratory syndrome, avian flu, ebola, monkeypox, and hantaviruses. after the initial interspecies transmission per se, the viruses can disseminate into the human population through various and distinct mechanisms. some of them are well characterized and understood, thus allowing a certain level of risk control and prevention. surprisingly and in contrast, the initial steps that lead to the emergence of several viruses, and of their associated diseases, remain still poorly understood. epidemiological field studies conducted in certain specific high-risk populations are thus necessary to obtain new insights into the early events of this emergence process. human infections by simian viruses represent increasing public health concerns. indeed, by virtue of their genetic and physiological similarities, non-human primates (nhps) are considered to be likely the sources of viruses that can infect humans and thus may pose a significant threat to human population. this is well illustrated by retroviruses, which have the ability to cross species, adapt to a new host and sometimes spread within these new species. sequence comparison and phylogenetic studies have thus clearly showed that the emergence of human immunodeficiency virus type 1 (hiv-1) and hiv-2 in humans have resulted from several independent interspecies transmissions of different siv types from chimpanzees and african monkeys (including sooty mangabeys), respectively, probably during the first part of the last century. the situation for human t cell lymphotropic virus type 1 (htlv-1) is, for certain aspects, quite comparable. indeed, the origin of most htlv-1 subtypes appears to be linked to interspecies transmission between stlv-1-infected monkeys and humans, followed by variable periods of evolution in the human host. in this review, after an introduction on emerging viruses, we will briefly present the results of a large epidemiological study performed in groups of bantus and pygmies living in villages and settlements located in the rain forest of the south region of cameroon. these populations are living nearby the habitats of several monkeys and apes, often naturally infected by different retroviruses including siv, stlv and simian foamy virus. most of the persons included in this study were hunters of such nhps, thus at high risk of contact with infected body fluids (blood, saliva,...) during hunting activities. after reviewing the current available data on the discovery, cross-species transmission from monkeys and apes to humans of the simian foamy introduction l'espèce humaine est en contact permanent avec l'environnement qui contient une multitude d'agents infectieux (virus, bactéries, parasites, champignons). dans la majorité des cas, aucune infection ne survient chez l'homme, lors de ces contacts. le rôle de l'immunité innée est majeur dans cette protection permanente. plus rarement, ces microbes peuvent infecter un individu et éventuellement se disséminer ensuite dans l'espèce humaine. même si ce processus, aboutissant à une émergence d'un agent infectieux chez l'homme, existe depuis la nuit des temps, des exemples récents laissent à penser que ces émergences, principalement virales, sont plus fréquentes depuis quelques décennies. dans cet article nous allons essayer de faire un point sur ce sujet d'actualité puis nous développerons des travaux de notre unité dans ce domaine. [16, 17] . ces résultats, ainsi que des études épidémiologiques et des observations à long terme de suivi de populations simiennes en semi-liberté, suggèrent fortement que la transmission dans les colonies de singes se fait, avant tout, par le biais de la salive, en particulier lors de morsures sévères [14] . l'infection par ces virus ne semble pas donner de maladie chez les singes infectés, mais très peu d'études ont été réalisées spécifiquement sur ce sujet. [28, 31] . les différents travaux réalisés dans ce domaine, par des équipes principalement américaines mais aussi françaises (dont la nôtre), ont permis de diagnostiquer à ce jour une infection par ce virus foamy simien chez plus d'une centaine de personnes [33] . il est bien sûr impossible de connaître le nombre de personnes réellement infectées de par le monde, mais ce chiffre n'est probablement qu'une partie visible du pic de l'iceberg des infections humaines par ces virus. en effet, on considère que dans la seule sous-région d'afrique centrale, où la chasse et les contacts avec les singes sont fréquents, plusieurs milliers de personnes sont certainement infectés. par ailleurs, en asie, des études réalisées en indonésie et au bangladesh ont montré de nombreux cas d'infection par des virus simiens originaires de macaques chez les personnes en contact avec des singes ; qu'il s'agisse de propriétaires de singes, ou de personnes vivant dans des temples où les singes sont particulièrement nombreux [34] [35] [36] les zoonoses ont surtout été considérées à l'origine comme des maladies liées à des agents infectieux touchant surtout les animaux. on tend actuellement à utiliser ce terme pour les infections transmissibles des animaux vertébrés à l'homme et inversement. quelle sera l'influence du changement climatique sur les diffusions des maladies émergentes ? la prédiction est très difficile dans ce domaine mais il semble probable que les changements climatiques provoqueront des modifications d'une part de la répartition géographique de nombreux vecteurs, (en particulier des moustiques), mais aussi au niveau des réservoirs de nombreux agents infectieux (en particulier des virus). il est difficile de répondre précisément à cette question malgré les nombreux travaux réalisés sur ce sujet. parmi plusieurs facteurs, on souligne actuellement le rôle majeur des facteurs de restriction dont les effets sont contrecarrés par des protéines spécifiques virales. cela a bien été démontré dans le cadre du vih-1 avec en particulier la protéine vif et le facteur apobec3g. such infection is a unique natural model to study the different mechanisms of restriction of retroviral emergence in humans origins of major human infectious diseases anticipating the species jump: surveillance for emerging viral threats impacts of biodiversity on the emergence and transmission of infectious diseases prediction and prevention of the next pandemic zoonosis cross-species transmission of simian retroviruses, how and why they could lead to the emergence of new diseases in the human population aids as a zoonosis: scientific and public health implications origin and genetic diversity of htlv-1 retrovirus and stlv-1 simian affiliated retrovirus human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo mapping the zoonotic niche of ebola virus disease in africa. elife propagation in tissue cultures of cytopathogenic agents from patients with measles foamy virus infection in primates non-primate foamy viruses. current topics in microbiology and immunology molecular ecology and natural history of simian foamy virus infection in wild-living chimpanzees modes of transmission and genetic diversity of foamy viruses in a macaca tonkeana colony two distinct variants of simian foamy virus in naturally infected mandrills (mandrillus sphinx) and cross-species transmission to humans replication in a superficial epithelial cell niche explains the lack of pathogenicity of primate foamy virus infections sites of simian foamy virus persistence in naturally infected african green monkeys, latent provirus is ubiquitous, whereas viral replication is restricted to the oral mucosa a. -a new human virus in cultures from a nasopharyngeal carcinoma simian retroviruses in african apes historical perspective of foamy virus epidemiology and infection -markers of foamy virus infections in monkeys, apes, and accidentally infected humans: appropriate testing fails to confirm suspected foamy virus prevalence in humans simian foamy virus isolated from an accidentally infected human individual identification of a human population infected with simian foamy viruses -cross-species retroviral transmission from macaques to human beings frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates simian foamy virus prevalence in macaca mulatta and zookeepers -naturally acquired simian retrovirus infections in central african hunters frequent and recent human acquisition of simian foamy viruses through apes' bites in central africa natural simian foamy virus infection in wildcaught gorillas, mandrills and drills from cameroon and gabon detection and molecular characterization of foamy viruses in central african chimpanzees of the pan troglodytes troglodytes and pan troglodytes vellerosus subspecies cross-species transmission of simian foamy virus to humans in rural gabon, central africa froment a. -simian foamy virus transmission from apes to humans, rural cameroon htlv-3/4 and simian foamy retroviruses in humans: discovery, epidemiology, cross-species transmission and molecular virology a. -primate-to-human retroviral transmission in asia a. -diverse contexts of zoonotic transmission of simian foamy viruses in asia zoonotic simian foamy virus in bangladesh reflects diverse patterns of transmission and co-infection temple monkeys and health implications of commensalism viral latency in blood and saliva of simian foamy virusinfected humans in vivo cellular tropism of gorilla simian foamy virus in blood of infected humans coinfection with hiv-1 and simian foamy virus in west central africans une transmission par la voie digestive à partir de la viande de singe mal cuite est-il possible ? comment expliquez-vous l'explosion épidémique du vih dans les années 80 ? on considère actuellement que l'émergence initiale du vih à la suite de quelques transmissions inter-espèces s'est effectuée dans les années 1900/1920 dans le sud du cameroun à partir de virus de chimpanzés (siv) qui sont passés chez l'homme. l'hypothèse du chasseur blessé avec un contact sanguin entre un chimpanzé infecté par un siv et un homme est la plus plausible. par la suite, plusieurs travaux suggèrent que le virus a ensuite circulé grâce à des hommes infectés le long du système fluvial de la rivière sangha, par des bateaux et ferries jusqu'à kinshasa. les connections fluviales entre le sud cameroun et kinshasa étaient fréquentes au début du siècle dernier, principalement liées à l'exploitation du caoutchouc et de l'ivoire. un article récent publié dans science, en 2014 (faria nr et al.), suggère qu'ensuite, la diffusion à partir de kinshasa dans différentes régions du sud et de l'ouest du zaïre ait pu avoir lieu (dans les années 1930-1960) par les migrations de populations, en particulier d'ouvriers pour travailler dans les mines, qui utilisaient alors les chemins de fer. ensuite, l'expansion du vih serait liée à la diffusion virale key: cord-022955-vy0qgtll authors: nan title: proteases date: 2005-06-20 journal: febs j doi: 10.1111/j.1742-4658.2005.4739_4.x sha: doc_id: 22955 cord_uid: vy0qgtll nan the incretin hormones glp-1 and gip are released from the gut during meals, and serve as enhancers of glucose stimulated insu-lin release from the beta cells. furthermore, glp-1 also stimulates beta cell growth and insulin biosynthesis, inhibits glucagon secretion, reduces free fatty acids and delays gastric emptying. glp-1 has therefore been suggested as a potentially new treatment for type 2 diabetes. however, glp-1 is very rapidly degraded in the bloodstream by the enzyme dipeptidyl peptidase iv (dpp-iv; ec 3.4.14.5). a very promising approach to harvest the beneficial effect of glp-1 in the treatment of diabetes is to inhibit the dpp-iv enzyme, thereby enhancing the levels of endogenously intact circulating glp-1. the three dimensional structure of human dpp-iv in complex with various inhibitors creates a better understanding of the specificity and selectivity of this enzyme and allows for further exploration and design of new therapeutic inhibitors. the majority of the currently known dpp-iv inhibitors consist of an alpha amino acid pyrrolidine core, to which substituents have been added to optimize affinity, potency, enzyme selectivity, oral bioavailability, and duration of action. various compound series and their sar relative to alpha amino acids will be presented. memapsin 2 (b-secretase, bace1) is the membrane-anchored aspartic protease that initiates the cleavage of b-amyloid precursor protein (app) leading to the production of amyloid-b (ab), a major factor in the pathogenesis of alzheimer's disease (ad). since memapsin 2 is a major target for the development of inhibitor drugs for the treatment of ad, its structure and physiological functions are topics of intense research interest currently. here we discuss the structural features of memapsin 2 and how do they contribute to the activity and inhibition of the protease. structural and kinetic evidence support the presence of 11 subsites for substrate or inhibitor binding in the activesite cleft of memapsin 2. subsites p3 to p2' are most useful in the design of transition-state analogue inhibitors. recent data indicated that subsites p7, p6 and p5 have strong influence of hydrolytic rate or inhibition potency. these subsites are, however, too far from the transition-state isostere for the design of drug-like transition-state inhibitors but can be utilized for the design of non-transition-state inhibitors that compete for substrate binding. besides carrying out proteolytic activity, the ectodomain of memapsin 2 also interacts with app leading to the endocytosis of both proteins into the endosomes where app is hydrolyzed by memapsin 2 to produce ab. a phosphorylated motif in the cytosolic domain of memapsin 2 is responsible for the recognition of gga proteins as part of the recycling mechanism that transports memapsin 2 from endosomes to trans-golgi then back to cell surface. these interactions may also be considered for the design of small-molecular compounds that interfere with memapsin 2 trafficking and thus reduce the production of ab. identification of human carnosinase -a brainspecific metalloprotease m. teufel biochemistry, exploratory research, sanofi aventis, strasbourg, france. e-mail: michael.teufel@sanofi-synthelabo.com metalloproteases form a large and diverse family of proteases and are molecular targets that represent an opportunity for therapeutic intervention. in particular, the development of potent inhibitors has made progress for the family of matrix metalloproteases (mmp). the sequencing of the human genome revealed that a significant percentage of the drugable genome is represented by proteases, many of them still with unknown function. in this presentation, data will be presented on the deorphanization of two previously unknown genes by means of bioinformatics and classical biochemistry. this work led to the identification of human carnosinase, a dipeptidase specifically expressed in the human brain and an ubiquitously expressed close homologue, characterized to be a non-specific dipeptidase. stimulating serpins with synthetic tailor-made oligosaccharides: a new generation of antithrombotics m. petitou thrombosis & angiogenesis, sanofi-aventis, toulouse, france. e-mail: maurice.petitou@sanofi-aventis.com we will discuss our research on synthetic oligosaccharides able to selectively activate the inhibitory activity of antithrombin towards various serine proteinases. we first synthesized pentasaccharides closely related to the antithrombin binding domain of heparin [1] (the active site), as well as analogues displaying different pharmacokinetic profiles. selective inhibitors of coagulation factor xa were thus obtained that represent a new class of antithrombotic [2] drugs currently being evaluated worldwide. we then designed larger oligosaccharides [3] that inhibit both factor xa and thrombin in the presence of antithrombin. they are devoid of undesired nonspecific interactions with blood proteins, particularly with platelet factor 4. clinical trials are ongoing to prove the therapeutic benefits of this new type of coagulation inhibitors. slow tight binding inhibitors in drug discovery: in the case of dppiv and elastase inhibitors z. kapui, e. boronkay, i. bata, m. varga, e. mikus, k. urban-szabo, s. ba´tori and p. ara´nyi discovery research, chinoin member of sanofi-aventis group, budapest, hungary. e-mail: zoltan.kapui@sanofi-aventis.com enzyme are extremely potent causing significant inhibition at very low concentrations that may be comparable to the concentration of the target enzyme. when this inhibition is studied in vitro, complexities arise because the concentration of the inhibitor is so low that it is altered significantly as a result of combination with the enzyme. this situation is referred to as tight-binding inhibition. partly as a result of their low concentrations, tight-binding inhibitors often show slow-binding characteristics. unlike conventional inhibitors that act almost instantaneously (or at least within the ms time scale), slow-binding inhibitors may take several seconds, minutes or even hours for their effect to be fully exhibited. this association between slow-binding and tight-binding is relatively common and slow tight-binding inhibitors are extremely potent and specific. proteolytic enzymes are involved in a multitude of important physiological processes. their intrinsic properties and activities are in the focus of wide-ranging research and they have a valuable role in experimental and therapeutic purposes. serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. within the class of serine proteases, human leukocyte elastase (hle) is one of the most destructive enzymes in the body. the enzyme dipeptidyl peptidase iv (dppiv) is a serine exopeptidase that cleaves xaa-pro dipeptides from the n-terminus of oligo-and polypeptides. inhibitors of dpp iv are of increasing interest to pharmaceutical industry alike, as they may become established as the next member of the oral antidiabetic class of therapeutic agents. objective of our work was to develop reversible, slow, tight-binding inhibitors against these serine proteases. ssr69071 is a potent inhibitor of hle, the inhibition constant (k i ) and the constant for inactivation process (k on ) being 0.0168 ± 0.0014 nm. this inhibitor is reversible, slow, tight-binding inhibitor with k on = 0.183 ± 0.013 10 6 /ms, and k off = 3.11 ± 0.37 10 )6 /s. ssr69071 inhibits the solubilization of elastin by hle with 13 nm of ic50 value. this inhibitor is one of the most effective inhibitor of a serine proteinase yet described. ssr162369 is a potent, competitive and slow tight binding type inhibitor of the human dipeptidyl peptidase-iv enzyme (k i = 2 nm, t½ = 8 h). on the basis of kinetic properties, ssr162369 forms stable enzyme-inhibitor complex. these slow tight-binding inhibitors have unique inhibitory properties, they are extremely active, and selective, form stable enzyme-inhibitor complex, therefore they have long-lasting effect. their oral activity and long lasting in vivo biological potency agreed very well with stable enzyme-inhibitor complex. the advantages in drug discovery of slow tight-binding inhibitors are discussed in this presentation. enzyme inhibition trend analysis -a new method for drug design m. shokhen, n. khazanov and a. albeck the julius spokojny bioorganic chemistry laboratory, chemistry, bar lan, ramat gan, israel. e-mail: albecka@mail.biu.ac.il many of the drugs that are currently in use or at different stages of development are enzyme inhibitors. therefore, enzyme mechanism-based inhibitors could be developed into highly selective drugs. our novel enzyme inhibition trend analysis method com-bines experimental enzyme kinetics data and high level quantum mechanical modeling of enzyme-inhibitor chemical interactions. the method utilizes the principal catalytic reaction scheme of the target enzyme and does not require its 3d structure (a ligand based approach). the method is valid for the prediction of the trend in binding affinity of inhibitors not only for the specific enzyme for which the qsar model was optimized, but also for the whole enzyme family. the methodology would contribute significantly to overcoming the problem of fast mutational resistance developed by pathogens in response to pharmaceutical treatment. it can be used as a computational tool for expert analysis of various hypotheses about structure-activity relationships formulated for the design of new inhibitors. angiotensin-converting enzyme (ace, ec 3.4.15.1) is a key enzyme for blood pressure control and water-electrolyte homeostasis. a large number of highly potent and specific ace inhibitors are used as oral drugs in the treatment of hypertension and congestive heart failure. somatic ace consists of two homologous domains (n-and c-) within single polypeptide chain, each one containing a catalytic site. the two catalytic sites within somatic ace molecule were long considered to function independently. however, recent investigations indicate the existence of negative cooperativity between ace active sites. we studied the properties of bovine ace active centers by use of separate ace n-domain (n-ace) obtained by limited proteolysis of parent somatic enzyme and testicular ace, which represents c-domain. these results were compared with the data obtained for full-length somatic ace from bovine lungs. the results obtained demonstrate strongly dependent mechanism of action of ace active centers in the reaction of the hydrolysis of tripeptide substrates. however, the hydrolysis of decapeptide angiotensin i proceeds independently on n-and c-domains. the mechanism of inhibition of ace activity is also dependent on the length of the inhibitor: (i) random binding of the ''short'' inhibitor molecule (such as captopril, lisinopril) to one of the active sites dramatically decreases binding of another inhibitor molecule to the second site; (ii) ''long'' nonapeptide teprotid binds to both active sites without any difficulties. since the main physiological ace substrates in the organism are ''long'' peptides angiotensin i and bradykinin, the development of new class of inhibitors with prolonged structure would be beneficial for abolishing of ace activity. synthetic peptide studies on severe acute respiratory syndrome coronavirus (sars-cov) extensive proteolytic processing of the replicase polyproteins, pp1a (486 kda) and pp1ab (790 kda), by the sars-cov 3clike protease (3cl pro ). besides, the structural spike protein of sars-cov contains two heptad repeat regions (hr1 and hr2) that form coiled-coil structures, which play an important role in mediating the membrane fusion process. in this study, we focused on both 3cl pro and the hr regions of sars. previous studies demonstrated that the coronavirus 3cl pro cleaves the replicase polyproteins at no <11 conserved cleavage sites, preferentially at the lq sequence. the reported crystal structure of sars-cov 3cl pro provides insights into the rational design of anti-sars drugs. in order to understand the molecular basis of the enzyme-substrate binding mechanism, we employ the synthetic peptide and mass spectrometry-based approaches to investigate the significance of selected amino acid residues that are flanking both sides of the sars-cov 3cl pro cleavage site. in addition, previous studies indicated that the relatively deep hydrophobic coiled coil grooves on the surface of sars-cov spike protein heptad repeat regions (hr1 and hr2) may be a good target site for the design of viral fusion inhibitors. we have designed and synthesized five truncated peptide analogs derived from hr1 and hr2 peptides based on both bioinformatics and structural analysis. the biological activities of these truncated analogs will be studied using circular dichroism spectroscopy, multidimensional chromatography, protein cross-linking and mass spectrometry-based approach. the above investigation will definitely broaden our knowledge on the sars research and will reveal the feasibility of rational design of synthetic peptide-based drug in combating with sars disease. ras-transfection-associated invasion: involvement of matrix metalloproteinase(s) confirmed using a chicken embryo model and real time pcr during metastasis tumorogenic cells leave the primary tumour and intravasate into the blood/lymphatic system, exiting at a secondary site to establish a secondary tumour. ras-transfection of a parental, non-invasive mcf-10a cell line, established from a patient suffering with benign fibrocystic disease, gave rise to an invasive derivative cell line (mcf-10a-neot) exhibiting the phenotype of a pre-malignant, invasive tumour. invasion and metastasis are protease-assisted processes, proteases either being secreted by the tumour, or by the stromal cells under the influence of the tumour. here we demonstrate the involvement of matrix metalloproteinase(s) in the invasion of the ras-transfected mcf-10a cell line. tumour cells were inoculated onto the damaged surface of the upper chorioamniotic membrane (cam) of a vasculated 9-day old chick embryo. the tumour cells were allowed to invade, and the number of invading cells quantified using real time pcr. inhibitors specific for various proteases were applied to the upper cam, to block invasion, and hence identify the proteinases involved. the number of tumour cells invading into the vascular system was established by sampling the lower cam and quantifying the numbers of alu sequences (present only in human cells) in the dna, isolated from the embryonic tissue, using real-time pcr. using this method, the key role of an mmp was demonstrated. spectrum ptk inhibitor, genistein (100 lm) abolished the release of neutrophil mmp-9, in the presence and absence of extracellular calcium, and reduced the release of timp-1. both pp2 (10 lm), a src family ptk inhibitor, and piceatannol (30 lg/ml), a syk family ptk inhibitor, reduced mmp-9 release substantially, indicating that multiple ptk families might be involved in mmp-9 release. inhibition of either syk or src ptks by piceatannol or pp2 did not appear to influence timp-1 release. low levels of wortmannin (100 nm, inhibition of pi3k) abolished the release of mmp-9 in the absence of calcium, and reduced mmp-9 release in the presence of calcium. investigations into the signaling pathways involved in timp-1 release are continuing. we conclude that mmp-9 release induced by extracellular calcium may be mediated through pi3k and multiple tyrosine kinases, including src and syk family ptks. timp-1 granule release may also be mediated by tyrosine kinases, although src and syk family ptks do not appear to be involved. thermodynamical and structural analysis of cruzain/cruzipain2 complexed with e-64 by molecular modeling and dynamics simulations peptidases represent one of the most relevant enzyme classes targeted by therapeutic intervention. to contribute to the assignment of a physiological role to genomic-derived peptidases and to make them more accessible for the drug discovery process, we have undertaken a program consisting of mrna expression profiling, full-length recombinant expression in insect cells, purification and determination of the catalytic activity for the human proteolytic enzymes. a milestone in the process was the construction of a non-redundant comprehensive database for all human peptidases comprising 443 unique annotated entries, by assembling and filtering public domain information and in-house generated data. in order to get an informative picture on their expression profiling, a transcriptome database for 375 human peptidases was created using the microarray (affymetrix tm ) and taqman ò (applied biosystems) technologies. in parallel, we have set up the procedure for pcr amplification and cloning of the peptidase genes in 96 mtp format and we have already created a repository of 231 full-length human cdnas encoding for peptidases. besides, the conditions for miniaturized insect cell cultures have been established. experimental trials have defined a validated, reliable and fully-automated robotic procedure for the purification of recombinantly expressed peptidases in 96 mtp format. in a pilot study using the high-throughput approach, 85% of the chosen reference hydrolases (14) were secreted into the insect cell medium. of them, 66% have been proven to be catalytically active using fluorescent homogeneous assays in 384well format compatible with the high-throughput screening criteria. the application of this procedure to genomic-predicted peptidases is discussed. comparison of putative glutamate racemases from bacillus species glutamate racemase catalyzes the interconversion between l-and d-glutamic acid and is the cell's source of d-glutamate, a key component in the synthesis of both the bacterial cell wall and the glutamyl capsule. bacillus subtilis has two glutamate racemases in its genome, race and yrpc, while b. cerus and b. anthracis have two race genes, race1 and race1. interestingly, race in b. subtilis is the isoform that is essential and has the greater catalytic efficiency, but both race1 and race2 have higher sequence homology to race, 70 and 79% respectively and share less homology with the yrpc isoform, both at 53%. we have cloned, overexpressed, purified, and are characterizing the kinetic and biophysical properties of the two putative glutamate racemases, race1 and race2 from b. cereus and b. anthracis, and will utilize kinetic and biophysical information to design inhibitors that may result in a novel antibiotic. although these two isoforms share a high sequence similarity, their properties are unique. kinetic data indicates a fivefold difference in catalytic efficiency of race2 compared to that of race1 in the l-to d-glutamate reaction. also, the absence or presence of substrate has an effect on the oligomerization state, details of which will be reported. finally, our collaborators have demonstrated through genetic knock out experiments that only one of the race isoforms is essential for the growth of b. anthracis. we have crystallized the race2 isozyme and x-ray data have been collected to 2.3 å . we are currently solving the structure via heavy-atom derivatives. acknowledgment: this research was funded by nih grant u19 ai056575. anti-inflammatory effects of methionine aminopeptidase 2 inhibition on human b lymphocytes e. janas 1 , r. priest 1 , s. ratcliffe 2 and r. malhotra 1 1 rheumatoid arthritis biology, glaxosmithkline, stevenage, uk, 2 high throughput chemistry, glaxosmithkline, stevenage, uk. e-mail: eva.x.janas@gsk.com processing of n-terminal methionine is an essential post-translational modification in both prokaryotes and eukaryotes regulating the subcellular localization, stability and degradation of proteins. the cleavage of the initiator methionine is catalysed by a highly conserved family of metalloproteases, methionine-aminopeptidase 1 and 2 (met-ap2). human met-ap2 is the molecular target of fumagillin, a natural product with antiangiogenic properties, which covalently binds to his 231 in the catalytic site of met-ap2. although fumagillin has been observed to inhibit proliferation and to cause cell cycle arrest in endothelial cells, the mechanism of inhibition is still poorly understood. recent studies describe high expression of met-ap2 in germinal centre b lymphocytes. here, we investigate the effect of the met-ap2 inhibitor fumagillin on b lymphocyte proliferation and cell cycle progression and compare these results to those observed in hu-vec. in addition our work sheds light on the mechanistic aspects of met-ap2 inhibition by fumagillin and its derivatives. effect of distal mutations on the molecular dynamics of the hiv-1 protease l10i and l90m are the most common distal mutations found in the protease gene of the drug resistant hiv-1 strains. these mutations do not confer resistance by themselves, however induce a large synergy effect when added to active site mutations. understanding the impact of the l90m and l10i mutations on the hiv-1 protease resistance profile is still a challenge. assuming that their contribution to the resistance profile could be mediated by conformational dynamics we have modeled l10i, l90m and l10i/l90m mutants of hiv-1 protease. these unbound mutated and wild type proteases were subjected to 10ns molecular dynamics simulations and compared using an essential dynamics (ed) analysis protocol. the first eigenvector of the native protease describes the flap openning motion. following eigenvectors describe ''the catalytic assisting motions'' (cam) of the protease that becomes dominant upon complex formation with a substrate (piana s et al. j mol biol 2002; 319(2): 567-583). mutation of luecine to methionine residue at position 90 perturbs the protein packing at the dimerization domain. such perturbations affect the dimerization domain motions which correlate with flap opening and the cam. as result the first eigenvector corresponds to the rotational of the one subunit relative to another along axis connecting residues 60 and 60'. in other words l90m mutation mistunes essential motions of the enzyme while retaining its flexibility. this could be the cause of the reduced structural stability of the l90m mutant. in contrast, l10i mutation causes only redistribution of the correlated motions amplitude. the catalytic assisting motion becomes the most influential that results in stabilization of the closed conformation. in turn, the flap opening motions are reduced in l10i mutant. essential dynamics of the double mutant l10i/l90m could be described in the following terms. a strong propagation of the cam induced by l10i mutation is coupled with the altered conformational space caused by l90m mutation. as result the double mutant prefers cam motions that are close to the native protease but also account for the perturbed packing within the dimerization domain. results presented may help understanding hiv-1 protease resistance pathways and in developing more efficient inhibitors of known drug resistant mutants. glutamate carboxypeptidase ii as a cancer marker and therapeutical target: two faces of an enzyme glutamate carboxypeptidase ii is a membrane-bound metallopeptidase expressed in a number of tissues such as jejunum, kidney, prostate and brain. the brain form of gcpii (also known as naaladase) is expressed in astrocytes and cleaves n-acetylaspartyl glutamate, an abundant neurotransmitter, to yield free glutamate. gcpii thus represents an important target for the treatment of neuronal damage caused by excess glutamate. animal model experiments suggest that specific inhibitors of gcpii could be useful for the treatment of several neuropathic conditions, such as brain stroke, chronic neuropathic pain or amyotrophic lateral sclerosis. in the same time, the enzyme is known as prostate-specific membrane antigen since it is upregulated in prostate cancer. it is used for the diagnosis and experimental therapy of prostate cancer using monoclonal antibodies and specific inhibitors. in order to analyze this important pharmaceutical target, we established an expression system based on drosophila schneider's cells. we have also cloned, expressed and characterized its human homolog gcpiii and homologous carboxypeptidases from pig and rat. using specific monoclonal antibodies, we have been able to study the expression of gcpii in various healthy and malignant tissues. we analyzed the substrate specificity of the enzyme using peptide libraries and identified two novel peptide substrates. availability of a recombinant protein enabled to introduce a simple fluorescent activity assay and test specific inhibitors. furthermore, we have biochemically characterized the recombinant protein in terms of pharmacologic properties, oligomeric status, ph dependence and activity modulation by metal ions. we have shown that the glycosylation is indispensable for gcpii carboxypeptidase activity and analyzed the role of each specific n-glycosylation site for the gcpii activity and folding. using site-directed mutagenesis, we are able to identify the domains sufficient and necessary for gcpii activity and also suggest structural explanation for the substrate specificity of the enzyme. dozens of chemicals feature inhibition of proteolytically important tyrosine residue of 20s proteasome by forming covalent bond to hydroxyl group that abolished its catalytic function. in contrary, the approach we utilize here is based on hydrogen and hydrophobic interactions reversibly inactivating all three sites of 20s complexes. we performed flexible docking studies of analogues of a natural product tmc-95a using 1jd2 crystal structure to describe the active site of protein and the position of the ligand. the search yielded several amide-like derivatives that have been screened for superimposition with tmc-95a. few of them revealed similar orientation of propylene groups to the active site of 20s. second screen was performed to reveal the chemicals with the strongest hydrogen-bonding of the ligand to the protein backbone of the receptor. this screen resulted in two chemicals that had strong h-contacts with tyr21, ser129 and, importantly, with proteolytically active tyr1 residue. to access the validity of the predicted chemicals we undertook in vitro studies measuring the hydrolyses of fluorogenic substrate by the sds activated 20s proteasome isolated from hela cells. we obtained more than 85% inhibition of 20s proteasome activity upon incubation the above chemicals (0.5 lg/ml) with proteasomes. we then demonstrated the effectiveness of the obtained chemicals to stabilize the level of oncosupressors, including p53 in benign (mcf10a) and highly metastatic (mda231) cell lines. treatment with these compounds greatly restored the level of p53 in cancer cells. finally, we performed proliferation assay and proved that adding of this artificially synthesized chemicals to mda231 cell line significantly reduced the level of proliferation, whereas mcf10a cells treated at similar conditions have not revealed any abnormal reduction of proliferation below control level. thus, we report of a strategy to predict highly suitable proteasome inhibitors that act via inhibition of protease activity and may lead to creation of a new class of drugs for cancer therapy. localization and trafficking of prostate specific membrane antigen (psma) and its variant form psḿ glutamate carboxypeptidase ii, also known as prostate specific membrane antigen (psma), is a transmembrane glycoprotein highly expressed in maligant prostate tissues. it was shown to represent very useful diagnostic marker and also potential therapeutic target for prostate cancer. two forms of the enzyme were identified in the prostate: full-length transmembrane form consisting of 750 amino acids and a truncated form (called psḿ ), believed to represent spliced variant of psma. the cdnas of both forms are identical except for 266-nucleotide region near 5´end of psma that is absent in psḿ . this deleted region codes for signal peptide as well as for intracellular and transmembrane domains. we are able to detect two protein forms in prostate cancer model cells (lncap cells) and we also show that both forms are glycosylated suggesting that this truncated form might originate from the processing of full length transmembrane psma. number of methods including differential centrifugation, pulse-chase experiments, immunochemistry and gfp-fusion protein analysis were used to analyze the origin, cell localization and trafficking of psma and psḿ in the mammalian cells. we have investigated the substrate specificity of the ns2b(h)-ns3pro protease by using internally quenched synthetic peptides representing both natural cleavage sequences and their recombinant chimeras. synthetic peptides incorporating the o-aminobenzoic acid/3-nitro-l-tyrosine fluorescence donor-quencher pair were used to analyze the minimum substrate length requirement, residue preferences and the contribution of prime side residues for enzymatic cleavage by the ns3 protease. a series of peptides derived from the ns3/ns4a cleavage site was designed for the substrate length mapping study. amino acid truncations in the non-prime and prime side region differently affected rates of substrate hydrolysis and binding as shown by their km and kcat values. the optimal substrate identified was a heptapeptide spanning p4-p3'. chimeric substrates with all possible combinations of non-prime and prime side sequences derived from 5 polyprotein cleavage sites (c, 2a/2b, 2b/3, 3/4a and 4b/5) were assayed for reactivity with the ns3 protease. kinetic parameters revealed a strong impact of the non-prime side residues on km, whereas variations in the prime side region had greater effect on kcat. the fluorogenic derivative of tetrabasic peptide rrrr/gtgn (c/ns5) demonstrated the highest affinity, whereas the peptide kkqr/sagm (2b/c) had the highest turnover number. the one with the greatest catalytic efficiency was identified as rrrr/sltl (c/4a). in addition, we have shown that a ser at p1' is the most preferred residue. the discovery of ns3 substrates with maximized reactivity will be useful for inhibitor development in sensitive high-throughput assays. inhibiting the mtor pathway with cci-779 results in decreased production of vascular endothelial growth factor in a head and neck squamous cell cancer cell line c.-a. o. nathan 1,2 , n. amirghahari 1,2 , x. rong 1,2 and y. sun 1,2 1 nathan, otolaryngology, otolaryngology/head and neck surgery, louisiana state university health sciences center, shreveport, la, usa, 2 nathan, cancer center, medicine, feist-weiller cancer center, shreveport, la, usa. e-mail: cnatha@lsuhsc.edu introduction: overexpression of the proto-oncogene eif4e in surgical margins of head and neck squamous cell cancer (hnscc) patients is an independent predictor of recurrence and is associated with increase in vascular endothelial growth factor (vegf) expression. activation of eif4e in margins through the mtor pathway has led us to determine that cci-779 an mtor inhibitor has both in vitro and in vivo growth inhibitory effects in hnscc cell lines. we wanted to determine if these effects were associated with decrease in vegf production. material and methods: a hnscc cell line fadu was treated with 1 and 10 ng/ml of cci-779 (previously established ic50 = 1 ng/ml). elisa was used to determine vegf protein levels in conditioned medium at 30', 1, 2, 4, 6, 24 and 48 h after treatment with the drug and compared to control cells treated with the diluent for each of the time points. results: a significant decrease in vegf production of 70% was noted at 24 h and maintained at 48 h in treated cells when compared to control cells at the same time points. the decrease in vegf levels (39-47%) was noted within 6 h of treatment with the drug. the percent decrease in vegf protein levels was the same for both doses of cci-779. conclusions: overexpression of eif4e in hnscc increases translation of mrnas with long 5'utrs, one of which is an important angiogenic factor vegf. inhibiting the mtor path-way with cci-779 can potentially decrease vegf production. this has future clinical implications for arresting tumor progression in hnscc patients with molecular positive margins identified by cells overexpressing eif4e, also known as minimal residual disease. proteases from cell culture of jacaratia mexicana m. c. oliver-salvador 1 , g. barrera plant proteases are important in food industry and food technology. the latex of jacaratia mexicana, caricaceae, fruits contains a high level of cysteine proteases. in this work was established a cell suspension culture of j. mexicana. callus culture was initiated from stem explants of j. mexicana on medium consisted of ¼-strength and full-strength ms mineral salts (murashige and skoog, 1965), full-strength ms organics and 6 g/l agar supplemented with cytokinins: 6-benzylaminopurine (bap) at 0.5 mg/l and 6-furfurylaminopurine (kinetin) at 0.25 mg/l and various concentrations (0.25, 0.5 and 1.0 mg/l) of auxins: 2,4-dichlorophenoxyacetic acid (2,4-d) 4-amino-3,5,6-trichloropiridin-2-carboxilic acid (picloram) indoleacetic acid (iaa) a-naphthaleneacetic acid (naa). all of the treatments induced callus except for the iaa, ana and without added phytohormones. the best auxin concentration for callus development was determined to be 0.5 mg/l. and the best condition medium for callus development and proteolytic activity of callus was determined to be 0.5 mg/l 2, 4-d + 0.5 mg/l bap. cysteine proteases were produced on callus culture of j. mexicana and liberated in the medium. also in the cell suspension culture these enzymes were secreted. our results support that is possible the synthesis of proteases in vitro culture of j. mexicana. since protease is a primary metabolite, further improvement in enzyme production is possible by increasing the growth rate and yield of cell culture of j. mexicana. and arginine, from peptides and proteins at neutral ph. it is known to play an important role in the control of peptide hormones, growth factor activity at the cell surface, and in the membrane-localized degradation of extracellular proteins. therefore, the present work was carried out to clone and express carboxypeptidase m in pichia pastoris, aiming at developing specific inhibitors and to evaluate the importance of the enzyme in different physiological and pathological processes. for this purpose, the enzyme's cdna was amplified from total placental rna by rt-pcr and cloned in the vector ppic9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in pichia pastoris. the results show that the cpm gene, after cloning and transfection, integrated in the yeast genome, which started to produce the active glycosylated protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured with the fluorescent substrate dansyl-ala-arg. the enzyme was purified by a two-step protocol including gel filtration and ionexchange chromatography, resulting in a 1761-fold purified active protein in a concentration of 400 mg/l of fermentation medium. sds-page showed that recombinant cpm migrated as a single band with molecular weight similar to native placental enzyme (62 kda). these results demonstrate for the first time the establishment of a method using pichia pastoris to express human carboxypeptidase m. mutational analysis of active site of glutamate carboxypeptidase ii human glutamate carboxypeptidase ii (gcp ii) is a membrane metallopeptidase expressed predominantly in the nervous system, prostate and small intestine. in the brain, gcp ii catalyzes cleavage of the abundant neuropeptide n-acetyl-l-aspartyl-l-glutamate (naag) to n-acetylaspartate and glutamate. gcp ii is a type ii transmembrane glycoprotein with a short cytoplasmic nterminal region (amino acids 1-18), a transmembrane domain (amino acids 19-43) and a large extracellular domain (amino acids 44-750) where the active site of the enzyme is situated. gcp ii, as a cocatalytic zinc metallopeptidase, has two zn 2+ ions in the active site which are necessary for its enzymatic activity. recently, the crystal structure of gcp ii was determined in our laboratory and amino acids arg210, asn257, lys699 and tyr700 were proposed to bind c-terminal glutamate of naag (mesters et al., manuscript in preparation). in the presented study, we carried out site-directed mutagenesis to assess the influence of these amino acid residues on the activity of gcp ii. in addition, glutamic acid in the position 424 which is proposed to be involved in proton shift during the catalytical hydrolysis of peptide bond, was mutated to alanine. all the mutant proteins were expressed in insect cells, purified to near homogeneity and enzymatically characterized. it was shown that a mutation in any of these positions lead to significantly reduced naag-hydrolyzing activity. the substitution of glu424 almost completely abolished the enzymatic activity, thus suggesting glu424 is crucial for enzymatic activity of gcp ii. kinetic characterizations of mutant proteins and their substrate specificities will be presented in comparison with wild type gcp ii. comparative study of mammalian homologues of human glutamate carboxypeptidase ii glutamate carboxypeptidase ii (gcpii) is a membrane-bound metallopeptidase. in homo sapiens, gcpii was shown to be expressed in various tissues, mostly in the central nervous system, small intestine and prostate. in brain it hydrolyses n-acetylaspartylglutamate (naag), which is the most prevalent peptide neurotransmitter in the mammalian nervous system, to form glutamate and n-acetylaspartate. in small intestine gcpii plays an important role in folate absorption. in prostate its function is still unknown. it was shown that inhibition of gcpii is neuroprotective in many neurodegenerative states. according to current knowledge of this enzyme, its role may also be important in prostate (and possibly other) cancers, where its expression is dramatically changed in comparison with healthy tissue. gcpii is thus becoming an important therapeutic target and diagnostic molecule. in order to analyze structure-activity relationships in related glutamate carboxypeptidases, we set to study the mammalian homologues of human gcpii: gcpii of rattus norvegicus, sus scrofa and mus musculus, which have approximately 90% dna sequence similarity to human gcpii. information on the biochemical properties, expression pattern and structural similarity is crucial e.g. for testing of gcpii inhibitors in animal models. we have cloned and expressed recombinant gcpii of r. norvegicus and s. scrofa in insect cells with the aim to obtain pure recombinant protein sufficient for structural analysis. data on biochemical comparison of rat, pig and human gcpii forms will be presented and interpreted in the light of the gcpii structure. structural analysis of pla protein from y. pestis: docking and molecular dynamics of interactions with mammalian plasminogen systemz e. ruback and p. g. pascutti laborato´rio de modelagem e dinaˆmica molecular, departamento de biofisica, universidade federal do rio de janeiro, rio de janeiro, r.j. brazil. e-mail: eruback@biof.ufrj.br the plasminogen (plg) system is an important mechanism for the cell migration through the tissues in the mammalian organisms. some bacterial agents can activate this system by proteases and lead an uncontrolled degradation of extracellular matrix components (mec), and make an invasive character of these infections. the y. pestis protein pla is a plasmid coded outer membrane protein, with aspartic-protease activity and is closely related with the proteolytic activation of plg in the serine-protease form called plasmin. exactly how the pla activate plg in plasmin remains unclear. we performed in this work the predicted interaction between the plg and pla protein by rigid-body docking with hex and evaluate the complex stability by molecular dynamics (md) using the gromacs. to evaluate the docking accuracy we use the crystal structure of complex plg-streptokinase. the md results show more stability in the docked plg-streptokinase complex than in crystal complex observed by the rmsd and rmsf calculations after 2 ns in simulation box. the pla model was constructed with spdb-viewer using the pdb structure of ompt as template and quality of model was evaluated with prochek. the docked complex of plg-pla show same interaction site predicted in mutagenesis studies. after 8 ns md (72 083 atoms in box), we observed the relax of beta barrel structure of pla and the progressive approximation and stabilization between the cleavage site of plg into the extracellular loops of pla, followed of the increase of hydrogen bonds number. in this study we report the possible aminoacids that can be participant in the active site and the sub sites of interaction. the total understanding of these interactions can be a important tool for drug design against bacterial proteases. glutamate carboxypeptidase ii (gcpii), also known as naa-ladase i, folylpolyglutamate hydrolase (folh) or prostate specific membrane antigen (psma) is localized in number of tissues. in brain astrocytes, it regulates neurotransmission by cleaving neurotransmitter n-acetylaspatylglutamate (naag) into n-acetylaspartate and most common excitatory neurotransmitter glutamate. inhibition of gcpii activity protects against cell death after brain stroke. in animal models it has been also shown that specific inhibitors of gcpii could be useful for the treatment of chronic neuropathic pain, amyotrophic lateral sclerosis and other pathologic situations when excess glutamate is neurotoxic. gcpii is identical to prostate-specific membrane antigen (psma), a tumor marker in prostate cancer. gcpii is also found in the membrane brush border of the small intestine where it acts as a folate hydrolase. this reaction expedites intestinal uptake of folate through hydrolysis of folylpoly-gamma-glutamates to monoglutamyl folates. gcpii inhibitors might thus be useful in the imaging and treatment of tumors where folate is required for their growth. therefore it was of interest to investigate whether gcpii might be upregulated in brain tumors as well. in order to analyze this possibility, we took 57 samples from 49 patients with brain tumors treated in faculty hospital motol during 1999-2004 and determined expression and activity of gcpii by western blots and immunohistochemistry using monoclonal and polyclonal antibodies developed against extracellular epitopes of gcpii. moreover, we characterized the enzymatic activity of the enzyme in human samples and correlated the expression of gcpii with the type and grade of the tumor. search for optimal isosteres in beta-secretase peptidic inhibitors alzheimer's disease is a widespread, neurodegenerative, dementia-inducing disorder. it is ascribed to the presence of a lesion in several brain regions, the neuritic plaques, which are extraneuronal accumulations of b-amyloid protein (ab), a 42-aa insoluble peptide that mixed with axons and dendrites of neurons, interrupt the synaptic process and cause neuronal death. the peptide ab is a product derived by proteolitic cleavage from a larger transmembrane cell protein termed amyloid precursor protein, app. two enzymes are involved in this cleavage: b-secretase and a-secretase. the first one cuts app between met671 and asp672 of app to generate the n-terminus of ab in the rate limiting step of the process, while the second one cleaves at various places within a sequence between amino acids 712 and 717 to generate the respective c-terminus. using a combination of molecular modeling techniques, we have designed a set of novel b-secretase peptidic inhibitors with a variety of isosteres starting from the available crystallographic structure of this enzyme bound to the inhibitor om99-2. some of the resulting ligands are predicted to have higher affinity for this enzyme than the starting compound. these inhibitors have been synthesized, their b-secretase affinity tested and cell essays have been performed to determine their ability to preclude the formation of ab peptides in cell cultures. schizophrenia and bipolar affective disorder (bd) are two neuropsychiatric diseases with high social and economic costs. in spite of the prevalence of these diseases, no effective long-term treatments are currently available. the enzyme prolyl oligopeptidase (pop) shows increased activity in both illnesses. this serine protease hydrolyzes peptide hormones and neuropeptides at the carboxyl end of proline residues. because of the relevance of pop as a therapeutic target, many specific inhibitors of this protein have been developed in recent years. the inhibitors ono-1603, jtp-4819 and s-17092-1 are currently in clinical trial phase. s-17092-1 has been administered safely to humans and has been proposed as a potential treatment for cognitive disorders associated with cerebral aging. our aim is to develop new peptide human pop inhibitors. to obtain the human brain pop required for our studies, the cdna corresponding to the enzyme was cloned and subsequently expressed in e. coli. pop activity was monitored by 19f-nmr using a new synthesized pop substrate labeled with 19f. this substrate allowed us to perform the inhibition assay avoiding the interference problems of colorimetric and fluorimetric assays and was suitable for high throughput screening of new pop inhibitors. different strategies were used to find putative human pop inhibitors: in silico screening and solid phase synthesis of candidates and screening with chinese medicinal plants extracts. furthermore, nmr studies were performed with the purified human enzyme by labeling the protein isotopically with 15n and d2o and by selective labeling of the residues methionine and tryptophan with 13c. nmr spectra of the labeled protein were obtained at 800 mhz by applying trosy techniques. nmr will provide structural information to perform structure-based drug design of new pop inhibitors in the future as well as to study the interaction of the candidates with the active site of the enzyme. the crucial regulatory function of the membrane type 1-matrix metalloproteinase (mt1-mmp or mmp-14) in connective tissue metabolism, pericellular proteolysis of extracellular matrix (ecm) components, zymogen activation and angiogenesis was demonstrated with the severe phenotype of the mt1-mmp-deficient mice. this membrane-anchored enzyme is not only essential for normal development of hard tissues, but highly expressed in different human cancers where its level frequently correlates with malignant parameters. in most cases the high level of mrna or elevated level of protein can be predictive for disease development but these parameters only partly reflect the expression and forms of mt1-mmp in pathological conditions. biosynthesis, trafficking, intracellular activation, internalization, protein-protein interactions, and the level of physiological inhibitors (timps) strictly influence the activity of mt1-mmp in cells and tissues. in our experimental system, we followed mt1-mmp processing and shedding and characterized the cell-associated and released forms of the enzyme (jbc 2000; 275: 12080-12089; jbc 2002; 277: 26340-26350 and biochem j 2005; 386: 1-10). we found active and inactive truncated forms of mt1-mmp as a result of treatments or experimentally generated imbalance with timps. we have also developed approaches to identify mt1-mmp forms in tumor tissues. here we present and discuss different strategies to identify mmp-14 in diverse biological samples. because mt1-mmp endows tumor cells with the ability to invade and metastasize, these strategies can provide valuable information on the role and function of this key protease. contribution of calpain to cellular damage in human retinal pigment epithelium cultured with zinc chelator y. tamada 1 , t. nakajima 1 , t. r. shearer 2 and m. azuma 1,2 1 research laboratories, senju pharmaceutical co., ltd., kobe, hyogo japan, 2 departments of integrative biosciences, oregon health & science university, portland, or, usa. e-mail: yoshiyuki-tamada@senju.co.jp purpose: we previously showed involvement of calcium-dependent cysteine proteases (calpains, ec 3.4.22.17) in neural retina degeneration induced by hypoxia and ischemia-reperfusion. aged macular degeneration (amd) is one of the leading causes for loss of vision. amd showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (rpe). rpe performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. the purpose of the present study was to determine the contribution of calpain-induced proteolysis to damage in human rpe. zinc chelator tpen was used to induce cellular damage since zinc deficiency is a suspected risk factor for amd. methods: third-to fifth-passage cells from human rpe were cultured with tpen. leakage of ldh into the medium was measured as a marker of rpe cell damage. activity of calpains was assessed by casein zymography, and proteolysis of calpain substrates was detected by immunoblotting. to confirm calpain-induced proteolysis, calpain in homogenized rpe was also activated by addition of calcium. results: tpen caused ldh to leak into the medium from rpe cells, and calpain inhibitor sja6017 inhibited the leakage. casein zymography and immunoblotting for calpain and a-spectrin showed activation of calpain in rpe cultured with tpen. proteolysis by activated calpain was confirmed by addition of calcium to homogenized rpe. conclusion: these results suggested that activation of calpain contributed to rpe damage induced by tpen in vitro. acknowledgments: dr shearer has substantial financial interest (research contract and consulting fee) in senju pharmaceutical co., ltd., and dr azuma is an employee of senju pharmaceutical co., ltd., a company that may have commercial interest in the results of this research and technology. this potential conflict of interest has been reviewed and managed by the ohsu conflict of interest in research committee. in vivo and molecular risk factors of chloroquine or pyrimethamine-sulfadoxine treatment failure in children with acute uncomplicated falciparum malaria the risk factors associated with chloroquine (cq) or pyrimethamine-sulfadoxine (ps) treatment failure were evaluated in 691 children enrolled prospectively in six antimalarial drug trials between july 1996 and july 2004 in a hyperendemic area of southwestern nigeria. following treatment, 149 (39%) of 389 children given cq and 64 (22%) of 302 children given ps failed treatment by day 7 or 14. in a multiple regression model, four factors were found to be independent risk factors for cq treatment failure at enrolment: age <7 years [adjusted odds ratio (aor) = 0.46, 95% confidence interval (ci) 0.26-0.84, p = 0.01], asexual parasitaemia >100 000/ll (aor = 0.46, 95% ci 0.23-0.93, p = 0.03), presence of gametocytaemia (aor = 0.48, 95% ci 0.26-0.88, p = 0.02) and enrolment after 4 years of commencement of the study, that is, after 2000 (aor = 0.47, 95% ci 0.25-0.77, p = 0.003). following treatment with cq, two factors were independent risk factors for failure of treatment: delay in parasite clearance >3 days (aor = 0.26, 95% ci 0.1-0.7, p = 0.014) and presence of gametocytaemia on day 7 or 14 (aor = 2.5, 95% ci 1.1-6.0, p = 0.03). in those treated with ps, two factors were found to be independent risk factors for ps treatment failure at enrolment: age <1.5 years (aor = 2.9, 95% ci 1.3-6.4, p = 0.009) and presence of fever (aor = 0.3, 95% ci 0.14-0.78, p = 0.01). following treatment with ps, delay in parasite clearance >3 days (aor = 0.39, 95% ci 0.18-0.84, p = 0.016) was an independent risk factor for failure of treatment. the quintuple mutants made up of triple dhfr (asn-108, arg-59 and ile-51) mutant alleles and double dhps (gly-437 and glu-540) mutant alleles were found in isolates obtained from 33% of patients, was significantly associated with ps treatment failure (p = 0.001), while pfcrt and pfmdr-1 mutant genes did not significantly predict cq treatment failure in these patients. these findings may have implications for malaria control efforts in sub-saharan africa where control of the disease depends almost entirely on antimalarial monotherapy. development of high-throughput assay of lethal factor using native substrate m.-y. yoon department of chemistry, hanyang university, seoul, south korea. e-mail: myyoon@hanyang.ac.kr designing of inhibitors for anthrax lethal factor (lf) is currently of interest as an approach for the treatment of anthrax because lf plays major roles in cytotoxicity of target cells. lf is a zincdependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (mapkk) family. current assay system for the screening of lf inhibitor use the optimized synthetic peptide coupled with various kinds of fluorophores, which enables fast, sensitive, and robust assays suited to high-throughput screening. however, lines of evidence suggest that the regions beside the cleavage site are also involved in specificity and proteolytic activity of lf. in the present study, we tried to develop high-throughput assay for lf activity based on native substrate, mek1. the assay system relies on the ecl signal resulting from a specific antibody against the c-terminal region of native substrate. a glutathione-coated multiwell plate was used as a solid support to immobilize the native substrate by its n-terminal gst-moiety. immobilized substrate increases the specificity and sensitivity lf-catalyzed substrate hydrolysis compared to the solution phase assay. this assay system would be expected to discover a wide spectrum of anthrax inhibitor. while significant progress has been made over the past decade in elucidating the structure and enzymatic mechanism of the 20s proteasome, our understanding of its assembly pathway and the role of the propeptides in the maturation process is still substantially incomplete. similarly, the mechanisms involved in the translocation of substrates into the central nanocompartment are only dimly understood at present. we have used the rhodococcus proteasome to dissect the assembly pathway, combining mutagenesis and crystallographic studies. for the thermoplasma proteasome we have established a ''host-guest'' interaction system which allows us to follow the translocation of specific substrates into the interior of the proteasome by electron microscopy, mass spectroscopy and x-ray crystallography. transferring substrates to the 26s proteasome in the fission yeast schizosaccharomyces pombe c. gordon mrc human genetics unit, western general hospital, edinburgh, uk. e-mail: colin.gordon@hgu.mrc.ac.uk the ubiquitin pathway is found in all eukaryotes. in this pathway, target proteins are covalently modified by the addition of ubiquitin, a 76 amino acid protein, to specific lysine residues. the ability of multi-ubiquitin chains to function as a signal to target proteins for degradation by the 26s proteasome is well documented. a key question is how is the multi-ubiquitin chain is recognized as a signal? fission yeast rhp23/rad23 and pus1/ rpn10 represent two families of multi-ubiquitin chain binding proteins that can associate with the proteasome as well as some e3 ubiquitin ligases. they seem to provide a link to shuttle ubiquitinated substrates from the e3 ubiquitin ligases to the 26s proteasome. a detailed characterization of their proteasome binding will be presented along with their potential role in ubiquitin conjugate dynamics. finally data will be presented indicating that an additional substrate presentation pathway exists in fission yeast which is also conserved in higher eukaryotes. non-proteasomal rpn10 raises the threshold for association of a ubiquitin-binding protein with the proteasome the ubiquitin proteasome pathway is responsible for the removal of the vast majority of short-lived proteins in the cell. in order to be degraded, a protein substrate is tagged with polyubiquitin and delivered to the proteasome where it is proteolysed. a slew of shuttle proteins is thought to mediate the delivery of polyubiquitinated substrates, although the mechanism remains elusive. one such family of proteins is comprised of rad23, dsk2 and ddi1, which all bind polyubiquitinated substrates through a ubiquitinassociated domain (uba) as well as the proteasome through their ubiquitin-like domain (ubl). another potential shuttle structurally unrelated to the ubl-uba family is rpn10. rpn10 is found as an integral subunit of the proteasome as well as an in an unincorporated pool. we characterized the interactions of these proteins with individual proteasomal subunits, as well as between themselves. we find unique relationships between the putative shuttle proteins and the proteasome, pointing to functional dissimilarity among them. strikingly, unincorporated rpn10 interferes with binding of dsk2 to the proteasome. thus, we propose that rpn10 might play a negative role in proteolysis through its action on dsk2. proteins modified by multi-ubiquitin chains are usually targeted for degradation by the proteasome. in other cases, ubiquitylation mediates protein sorting or regulates other functions. a striking example for a non-proteolytic role of ubiquitin is the rad6 dna damage bypass at stalled replication forks. key elements of this pathway are two ubiquitin-conjugating enzymes, rad6 and the mms2/ubc13 heterodimer, which are recruited to chromatin by the ring-finger ubiquitin ligases, rad18 and rad5, respectively. moreover, also the sumo-conjugating enzyme ubc9 is affiliated with the pathway and we discovered that proliferating cell nuclear antigen (pcna), a dna-polymerase sliding clamp involved in dna synthesis and repair, is a substrate. pcna is (i) mono-ubiquitylated by rad6/rad18, (ii) modified by lysine (k) 63-linked multi-ubiquitylation, which additionally requires mms2/ubc13/ rad5, and (iii) sumoylated by ubc9. all three modifications affect the same lysine residue of pcna, indicating that they label pcna for alternative functions. indeed, we discovered that monoubiquitylation of pcna promotes an error-prone replication bypass, whereas k63-linked multi ubiquitylation mediates errorfree replication across the lesions. in contrast, sumoylation, which occurs even in the absence of dna damage, prevents recombination between homologs at the replication fork. these findings indicate that mono-ubiquitin, k63-linked multi-ubiquitin chains, and sumo are crucial for decision making at the replication fork. ubiquitin-mediated proteolysis is the primary mechanism in eukaryotes for degrading unwanted and misfolded proteins. through the cascade of e1, e2 and e3 enzymes, ubiquitin monomers are attached sequentially to the target proteins, which are then recognized and degraded by the 26s proteasome. the selection and specific timing of polyubiquitination of the target proteins are conferred by different e3 ubiquitin ligases. the anaphase-promoting complex (apc) is one of the most extensively studied e3 ubiquitin ligases that plays essential role in the cell cycle and specific developmental processes. the core apc is composed of 11-13 subunits. except for apc2 and apc11, relatively little is known about the role of the other apc subunits or the assembly of the complex. two wd40-repeat activator proteins, cdc20 and cdh1 determine stage-specific activation of the core apc as well as selection and binding of the apc substrates. in plants, the apc activators are present in multiple copies. arabidopsis contains 5 cdc20 genes, 3 cdh1-type activators known as ccs52a1, ccs52a2 and ccs52b. our work has been focused on the function of apc activators in the cell cycle and plant development, identification of novel apc substrates and on the assembly of the apc complexes. apc activities, based on the expression profiles of the cdc20 and ccs52 genes, will be presented at organism level. by detailed protein interaction studies in yeast two hybrid system and arabidopsis protoplasts or transgenic plants, we shall demonstrate how the core apc interacts with the activators and substrates, and propose a model for apc assembly. characterization of substrate delivery to the saccharomyces cerevisiae proteasome by quantitative shotgun proteomics the proteasome is the central protein degradation machinery in the eucaryotic cell. in conjunction with the ubiquitin system, it is responsible for constitutive bulk protein turnover as well as the controlled degradation of regulatory proteins. the system is very well characterized, but the mechanism by which poly-ubiquitinated substrates are delivered to the proteasome remains unclear. recently our lab has proposed a number of proteins to be proteasome-based receptors for poly-ubiquitinated substrates in s. cerevisiae (rpn10p, rad23p, dsk2p; verma et al., 2004). others (e.g. richly et al. 2005) have put forward a complex model for the delivery of substrates from the ubiquitinating machinery to the proteasome involving the aaa atpase cdc48p. by analyzing the composition of affinity purified proteasome complexes from s. cerevisiae cells lacking these factors and/or exposed to specific proteasome inhibition, we hope to further elucidate the substrate delivery pathway. ubiquitinated proteins recruited to the proteasome are identified utilizing capillary chromatography in-line to electrospray ion trap mass spectrometry (mudpit; link et al. 1999). using a reference strain grown in minimal medium solely providing heavy nitrogen ( 15 n) as an internal standard, we are able to record even gradual fluctuations in sample composition. differences in the recruitment of substrates to the proteasome in varying mutant backgrounds will shed light on the specificity of proteasome substrate receptors and the topology of the substrate delivery mechanism. oxidative protein damage by reactive oxygen species (ros) produces cross-linking, fragmentation and biochemical modification of the amino acids resulting in biological dysfunctions. quercetin, a widely distributed bioactive plant flavonoid, possesses anti-cancer, antioxidants and free radical scavenging activities, as well as it binds with dna causing dna fragmentation. a little is known about protein oxidative damage and its modifications by antioxidants. therefore, the aim of the present work was to investigate the molecular mechanisms of antioxidant and prooxidant activities of quercetin toward proteins. the antioxidant activities of quercetin, such as superoxide dismutase (sod)-and catalase (cat)-mimetic as well as hydroxyl radical (aeoh) scavenging activities were possessed. bovine serum albumin (bsa) was incubated with different concentrations of quercetin. quercetin has highly sod-and cat-like and hydroxyl radical (aeoh) scavenging activities. its activities are concentration dependent. quercetin fragmentized bsa into specific fragments which they detected by sds/polyacrylamide gel electrophoresis. oxidative protein damage was assessed as tryptophan oxidation, carbonyl, quenone and advanced oxidation protein products (aopp) generation. the increase of protein oxidation products was in concentration dependent manner. the carbonyl and quenone contents and aopp were highly significantly elevated in querce-tin-treated proteins when compared with the control sample. the tryptophan fluorescence was highly decreased in treated protein than in the control sample. the mechanisms of antioxidant and pro-oxidant activities of quercetin have been discussed. these results demonstrate that antioxidant quercetin may potentiate protein damage via oxygen free radical generation, particularly .oh radicals by quercetin. protein stability mediated by a hyaluronanbinding deubiquitinating enzyme is involved in cell viability protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. we have identified three novel genes encoding a deubiquitinating enzyme, vdub1, vdub2, and vdub3 (villi deubiquitinating enzyme 1, 2, and 3) from human chorionic villi by rt-pcr. their cdnas are 1,593 bp in length and encode an open-reading frame of 530 amino acids with a molecular weight of approximately 58 kda. expression analysis showed that vdub transcripts are highly expressed in the heart, liver, and pancreas. in addition, they are expressed in various human cancerous cell lines. amino acid sequence analysis revealed that they contain the highly conserved cys, his, and asp domains, which are required for the formation of active site for the deubiquitinating enzymes. in vivo and in vitro deubiquitinating enzyme assays indicated that vdub1, vdub2, and vdub3 have deubiquitinating enzyme activity. here, we show that the overexpression of vdub proteins leads to irregular nuclear morphology and apoptosis, suggesting that these vdubs play an important role in regulating signal transduction involved in cell death. interestingly, the sequence analysis showed that vdub proteins contain the putative hyaluronan/mrna-binding motifs, and cetylpyridinium chloride-precipitation analysis confirmed the association between vdubs and intracellular hyaluronan and rna. chemical cleavage of peptide (amide) bonds usually requires harsh conditions. as a result of side reactions and the lack of specificity, chemical amide bond hydrolysis is not a preferred means of protein digestion. we have discovered selective cleavage of peptide bonds in proteins under milder circumstances than any previously reported chemical method. hydrolysis takes place in aqueous buffers in a ph range of 410, and occurs c-terminal to the proteogenic non-natural amino acid azido-homoalanine (azhal), effected by a staudinger reaction after addition of the mild and biocompatible reagent tris(carboxyethyl)phosphine (tcep). key feature in the suggested reaction mechanism is the unprecedented nucleophilic substitution of the resulting gammaiminophosphorane by the flanking c-terminal backbone amide oxygen atom. after hydrolysis, the new c-terminal peptide is present as a homoserine lactone residue and the n-terminal peptide as its free amine. this new reaction may find application as a very mild and selective bio-orthogonal degradation pathway in biochemistry and biomaterials science. overexpression of proteasome b5 subunit increases amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates the proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. proteasomes play a fundamental role in retaining cellular homeostasis. alterations of proteasome function have been recorded in various biological phenomena including aging. we have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of btype subunits. in this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the b subunit. stable overexpression of the b5 subunit in wi38/t and hl60 cells resulted in elevated levels of other b-type subunits and increased levels of all three proteasome activities. immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of ''free''/not integrated b-type subunits identified to accumulate in vector-transfected cells. in support we have also found low pomp levels in b5 transfectants thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. functional studies have shown that b5 overexpressing cell lines confer enhanced survival following treatment with various oxidants. moreover we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. finally, as oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the b5 subunit into primary imr90 human fibroblasts and we have observed a delay of senescence by 45 population doublings. in summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights towards a better understanding of proteasome regulation. expression levels of the components of the ubiquitin/proteasome pathway in pisum sativum seedlings under anoxia stress change in gene expression: proteins produced under aerobic conditions are no longer synthesized and are replaced by the socalled anaerobic peptides. among those proteins synthesized under o 2 deficiency some enzymes of the glycolytic and fermentative pathways were identified in plants. upon reintroduction of air, the anaerobic mrnas disappear rapidly and the increased levels of those enzymes must return to the basal levels. the ubiquitin/proteasome system is a major pathway of proteolysis in eukaryotic cells and may contribute to controlling the intracellular levels of a variety of short-lived regulatory proteins. in this proteolytic pathway, proteins are covalently conjugated to ubiquitin, which flags them for rapid hydrolysis by the 26s proteasome. long polyubiquitin chains must be formed to target a protein for destruction by the proteasome. in plants, the ubiquitin-mediated proteolytic pathway is implicated in a variety of cellular processes, including stress responses. in this study, 3-dayold pisum sativum seedlings were subjected to: (i) 15 h of anoxia stress; (ii) 2 h of aerobic conditions after 15 h of anoxia stress and (iii) 4 h of aerobic conditions after 15 h of anoxia stress. the levels of free and conjugated ubiquitin were detected by immunoblotting using anti-ubiquitin polyclonal antibodies. the changes in the mrna levels of some components of the ubiquitin/proteasome pathway in the seedlings were determined by relative semiquantitative rt-pcr. the results suggest an involvement of the ubiquitin-mediated proteolytic pathway in the anoxia stress response. b2-012p involvement of the anaphase promoting complex in plant development controlled degradation of short-live proteins via ubiquitindependent proteolysis by the 26s proteasome is a key mechanism in eukaryotes that regulates nearly all fundamental cellular processes including cell cycle. polyubiquitination of the protein substrate is sufficient to target it for degradation by a large atp-dependent multicatalytic protease, the 26s proteasome. the selection and specific timing of ubiquitination of the target proteins are conferred by different e3 ubiquitin ligase. the anaphase promoting complex (apc) is one of the e3 ubiquitin ligases, which by ordered destruction of various cell cycle proteins has fundamental roles in the regulation of mitotic and endoreproduplication cycles. the apc functions also outside the cell cycle. in post-mitotic cells, the cdh1 adaptor protein ensures stage specific activation and substrate selection of the apc. in plants, two classes of the cdh1-type activators have been identified, ccs52a and ccs52b that display differential regulation during the cell cycle and plant development as well as differences in their substrate-specificities. in arabidopsis, transient and complimentary expression profiles of the atccs52a1, atccs52a2 and atccs52b genes indicate apc functions during flower development. to identify apc targets, yeast two hybrid screens were performed in the laboratory. out of about 200 interacting proteins, several proteins were transcription factors including a key a regulator of flowers development. data on the interactions of the ccs52 proteins and transcription factors in arabidopsis protoplasts will be presented as well as a model for the apc regulated pathways. novel effects of ubiquitin system and chaperone proteins on the prion ''life cycle'' in yeast t. a. chernova 1 , k. d. allen 2 , e. p. tennant 2 , k. d. wilkinson 1 and y. o. chernoff 2 1 department of biochemistry, emory university, atlanta, ga, usa, 2 school of biology and institute for bioengineering and bioscience, georgia institute of technology, atlanta, ga, usa. e-mail: tcherno@emory.edu yeast prion [psi + ], the self-propagated aggregated isoform of the translation termination factor sup35, is used as a model system to study neural inclusion disorders. prion aggregates and other neural inclusions in mammals were previously reported to sequester ubiquitin (ub). proteasome inhibitors affected the turnover of mammalian prion proteins. however, a role of ub-dependent proteolysis in the prion ''life cycle'' has not been clearly defined. chaperone proteins, which are also implicated in ub-dependent proteolysis, have been shown to influence the formation and propagation of the prion aggregates. our results uncover the connection between alterations of ub system and chaperone proteins in their effects on the maintenance of yeast prion. we have demonstrated that deletions of genes encoding deubiquitinating enzymes, that are critical for ub regeneration at the proteasome (ubp6) or the vacuole (doa4), cause pleiotropic phenotypic effects that are primarily due to decreased levels of free ub in the yeast cells. these alterations, as well as deletion of the gene encoding ub-conjugating enzyme, ubc4, decreases [psi + ] curing by the overproduced disaggregase hsp104, suggesting that ub system influences hsp104-dependent clearance of prion aggregates. spontaneous [psi + ] formation was also increased in the ubc4 depleted cells. we previously demonstrated that excess of cytosolic chaperone ssa of hsp70 family increases de novo formation of [psi + ]. both in vivo and in vitro experiments uncover direct interactions between sup35 and hsp70 proteins. the amount of sup35-bound to hsp70-ssa was increased in ubc4 deletion strain. we propose a model to explain roles of hsp104, hsp70 and ub system in the prion life cycle. effects of parkinson''s disease mimetics on proteasome activity and protein turnover in human sh-sy5y neuroblastoma cells it has recently been suggested that impairment of the ubiquitin/ proteasomal system contributes to the degeneration of dopaminergic neurons (dn) and lewy body (lb) formation in parkinson's disease (pd). mitochondrial dysfunction is also a key factor in pd and agents such as mpp + and dopamine, which inhibit mitochondrial electron transport, produce selective degeneration of dn in animal models. in this study the effects of treating sh-sy5y cells with mpp + or dopamine over 72 h on proteasomal chymotrypsin-like activity (cla) was monitored. mpp + (0.1mm) caused a sustained depletion of glutathione levels followed by a reduction in proteasomal activity. a reduction in atp levels, caused by higher levels of mpp + (2mm), exacerbated this effect. exposure to low dopamine concentrations (0.1mm) led to large reductions in atp without affecting cla or glutathione levels; whilst higher concentrations (2mm) caused marked reductions in cla, glutathione and atp levels. these results suggest that, under oxidative stress, glutathione levels are important regulators of proteasomal activity in this cell line. our group has shown that mpp + can destabilize the neurofilament network in shsy-5y cells, partly due to changes in phosphorylation of neurofilament (nf) chains. as nfs are important components of lbs, and their mode of turnover is uncertain, we tested the effects of proteasome inhibitors on nf levels. treatment with these inhibitors led to nf accumulation, which was enhanced when glutathione levels were artificially depleted, suggesting that nfs can be degraded via the proteasomal pathway. the effects of proteasome impairment on protein accumulation will be discussed. mitochondria and the hypoxia-inducible factor 1 (hif-1): regulation of hif-1 is independent of a functional mitochondrial respiratory chain k. doege, w. jelkmann and e. metzen insitute of physiology, university of luebeck, luebeck, germany. e-mail: doege@physio.uni-luebeck.de the hypoxia-inducible factor hif-1 is the ''master-regulator'' in adaptation to low oxygen concentration and induces the hypoxic expression of several target genes, e.g. erythropoietin and vascular endothelial growth factor (vegf). in normoxia hif-1a is constantly produced but also degraded by oxygen-dependent prolyl-hydroxylation. mitochondria consume most of the oxygen delivered to cells and have been implicated in oxygen sensing. firstly, mitochondria have been proposed to stabilize hif-1a by production of reactive oxygen species (ros) in hypoxia. secondly, inhibition of the respiratory chain, e.g. by nitric oxide, has been proposed to cause redistribution of intracellular oxygen followed by reactivation of the prolyl hydroxylases and inhibition of hif signalling. we have used cells depleted of mitochondrial dna (q0) and gas permeable cell culture dishes to eliminate all oxygen diffusion gradients affecting the cells. we show that these dishes neutralize all effects of mitochondrial inhibition. additionally, cellular hypoxia as assessed by pimonidazole staining has been evaluated in human osteosarcoma cells treated with inhibitors of the respiratory chain under hypoxia. these results demonstrate an elevated po 2 under hypoxic conditions after treatment with mitochondrial inhibitors correlating with an intracellular oxygen concentration which reduces hif-1 activation. thus, neither the absence of ros nor the redistribution of intracellular oxygen supply leads to the destabilization of hif-1a in hypoxia. our experiments provide evidence that an increased intracellular po 2 evoked by the absence of mitochondrial oxygen consumption reactivates the prolylhydroxylases and is therefore responsible for the degradation of hif-1a under hypoxic conditions. enzyme activity is generally higher in rhizosphere than in bulk soil, as a result of a greater microbial activity sustained by toot exudates or due to the release of enzymes from roots. negative effects of heavy metals on soil microorganisms and enzyme activities have been long recognized. the aim of this study was to assess the stimulatory effects of different low molecular weight organic compounds commonly present in root exudates (mres) on microbial activity and protease activities and , and how high cd concentrations affect such stimulatory effects. soils (arenic udifluvent) were sampled from the agir long-term field trials, contaminated with cd nitrate at rates of 0 (control soil), 20 and 40 mg cd per kg of soil. the mre solutions contained glucose, citric acid, oxalic acid, glutamic acid or a mixture of the four compounds, added to give a rate of 300 mg of mre-c per kg of soil. the effects were measured at 4 mm (bulk soil) distance from the mrs. protease activity was determined by hydrolysis of n-benzoylargininamide (baa). the results showed that different mres had different stimulatory effects on microbial growth and on the protease activities, mostly localized in the rhizosphere soil layer. in the control soil, the dsdna content was significantly increased by the addition of all mre in both rhizosphere and bulk soil layers. the 20 and 40 mg cd per kg of soil negatively affected on protease activity. the glucose, citric acid, oxalic acid, glutamic acid, mres mix in both rhizosphere and bulk soil layers, did not stimulate protease. the, microbial growth and protease activities were drastically reduced by high cd concentrations. participation of different digestive proteinases of the yellow mealworm, tenebrio molitor, in initial stages of hydrolysis of the main dietary protein insects generally have a wide spectrum of digestive proteinases. the knowledge about the impact of different proteinases to initial stages of hydrolysis of dietary proteins is essential for insect control by means of proteinase inhibitors and bacillus thuringiensis toxins. the larvae of a stored grain pest yellow mealworm, tenebrio molitor, were reared on milled oat flakes. the main dietary protein for these larvae was 12s globulin, the main storage protein of oat seeds. to study the initial stages of 12s globulin hydrolysis in vitro the reaction was performed in the physiological conditions of anterior midgut (am) (ph 5.6) by purified enzyme preparations from am: two fractions of cysteine proteinases cys ii and cys iii, chymotrypsin-and trypsin-like proteinases. total hydrolysis of 12s globulin was observed with cys ii. slightly less effective was hydrolysis by chymotrypsin-like enzyme. cys iii cysteine and trypsin-like proteinases produced only partial hydrolysis of seed globulin. in all cases high molecular mass (mm) intermediate products were formed testifying that hydrolysis of 12s globulin was sequential. incubation with both cysteine proteinase fractions led to formation of 31 kda product, while serine proteinases proin contrast to ''classical'' bioregulator peptides, peptides could be generated in the course of catabolic degradation of functional proteins. for 15 years, we have been interested in such particular group of peptides derived from blood hemoglobin, hemorphins. hemorphins consist in a family of opioid receptor-binding peptides from 4 to 10 amino acids that are released by proteolytic processing from the (32-41) segment of human hemoglobin betachain. they are prevalent throughout the peripheral and central nervous system and have been isolated in vivo from tissues or fluids. many in vivo physiological effects have been related (coronaro-constrictory, anti-tumorous, immunoregulatory activities) and several of the hemorphins interact at various levels of the reninangiotensin system (ras) by inhibiting angiotensin-convertingenzyme (ace), aminopeptidase n (apn) and dipeptidyl peptidase iv (dppiv) activities. in addition, some hemorphins and in particular lvv-hemorphin-7 (lvvypwtqrf), binds with high affinity to the brain (ic 50 = 4.15nm) and renal at4 angiotensin receptor subtype and is possible the main endogenous ligand from this receptor. in an attempt to characterize in vivo precise mechanisms for their release, our attention is focused towards tumoral and central nervous system environments. the last one is particularly interesting as all cellular components implicated in the release of hemorphins are present simultaneously: the haemoglobin precursor and localized brain proteases which might come in contact with blood haemoglobin. in this purpose, the examination of potentiality for this tissue to generate ''neuro''-hemorphins would be of interest since sources of hemorphins in the brain have not yet been definitively established. later, we showed that sgt1 interacts with calcyclin (s100a6) and other calcium-binding proteins of the s100 family (nowotny et al. j biol chem 2003). moreover, in collaboration with dr chazin's group, we found that in vitro sgt1 binds to hsp90 (lee y-t et al. j biol chem 2004). in this work we studied the expression and subcellular localization of sgt1 in mammalian cells by means of western and northern blots. among different cell lines examined human embryonic kidney hek293 and human glioma t98g cells exhibit highest expression of sgt1 protein. moreover, we found that in mouse and rat cells there is one isoform of sgt1, while in human cells two isoforms of this protein were found. to study the subcellular localization of sgt1 we chose the cells containing moderate level of sgt1 such as human epidermal hep-2 cells. by applying immunocytochemistry we found that this protein is present not only in the cytoplasm but also in the nucleus. at present we check the effect of intracellular ca 2+ concentration on subcellular localization of sgt1 and on its co-localization with target proteins. acknowledgements: this work was supported by grants: kbn 3 p04a 043 22 and firca/nih r13 tw006005. combining reverse genetics, reverse chemogenomics and proteomics to assess the impact of protein n-terminal methionine excision in the cytosol of higher eukaryotes in living organisms whatever the cell compartment, proteins are always synthesized with methionine (met) as the first residue. however, this first met is specifically removed from most mature proteins. in the course of protein n terminal met excision (nme), the free n terminal met is removed by met aminopeptidase (map) cleavage. three enzymes (map1a, map2a and map2b) have been identified in the cytoplasm of arabidopsis thaliana. by combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic nme in a. thaliana and of the respective contributions of the two types of cytoplasmic map throughout development. in the map1a ko context (map1a-1), modulating map2 activity by treatment with various concentrations of the specific drug fumagillin impaired plant development. hence, (i) cytoplasmic nme is essential in plants, (ii) plant map1a and map2s are functionally interchangeable as a complete block of either map type activity does not cause any visible molecular or phenotypic effect, (iii) a minimal level of cytoplasmic map is required for normal development and (iv) the plant a. thaliana appears an excellent system to study nme and the associated-role of anti-cancer agents like fumagillin. proteomics was used to assess the impact of nme blocking induced by fumagillin. we used a wild-type plant and the map1a-1 variant grown in the presence of 100 nm fumagillin. the map1a-1 variant showed a dwarf phenotype. we compared by 2d gel electrophoresis the patterns of each protein extracts. protein spots were identified by tandem mass spectrometry. the data show that fumagillin induces many dedicated pathways, with a prevalence of those related to oxidative stress. prolyl endopeptidases from the midgut of the yellow mealworm tenebrio molitor side of proline residues. these enzymes were found in mammals, several higher plants, fungi and bacteria. it is suggested that the enzymes participate in the in vivo regulation of the action of biologically active peptides. we for the first time report about two prolyl endopeptidases in the larval midgut of a stored product pest yellow mealworm tenebrio molitor where they can participate in the proteolysis of one of the main dietary proteins of t. molitor larvae -rich in proline prolamines. characteristics of two prolyl endopeptidases are significantly different. optimum for hydrolysis of the substrate z-ala-ala-pro-pna (n-carbobenzoxy-l-alanyl-l-alanyl-l-prolyl-p-nitroanilide) by prolyl endopeptidase 1 was at ph 8.5, and prolyl endopeptidase 2 -at ph 5.6. prolyl endopeptidase 1 displayed high phstability in the ph range 7.0-10.0 and the rate of hydrolysis increased in the presence of kcl and cacl 2 . prolyl endopeptidase 2 demonstrated low stability in the whole ph range, the rate of hydrolysis strongly decreased in the presence of above mentioned salts, but increased in the presence of high concentrations of edta. the influence of cell growth media on the stability and antitumour activity of methionine enkephalin studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. the objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. the degradation kinetics of the model peptide methionine enkephalin (met-e, tyr-gly-gly-phe-met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of foetal bovine serum (fbs). the influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the met-e degradation was also investigated. the results obtained in the dulbecco's modified eagle medium containing 10% fbs indicated a rapid degradation of met-e (t 1/2 = 2.8 h). pre-incubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of met-e, as shown by increased half-life to 10 h. the in vitro activity of met-e against poorly differentiated cells from lymph node metastasis of colon carcinoma (sw620) and human larynx carcinoma (hep-2) cells was determined. tumour cells were grown 3 weeks prior to the experiment in a medium supplemented with 10, 5 or 2% fbs. statistically significant to mild or no suppression of cell proliferation was observed in all cultures. in both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and met-e, compared with cells exposed to the peptide alone and cells grown in the absence of met-e, was observed. this study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays. protein metabolism in whole body and skeletal muscle of laboratory rats treated by proteasome inhibitors proteasome inhibitors are new agents which may be used in treatment of cancer and other severe disorders. one of the possible side effects of their administration is disturbance in protein metabolism which may affect outcome of the illness. two separate studies were performed using wistar rats. in the first study, m. soleus (sol) or m. extensor digitorum longus (edl) were incubated in medium containing 30 mmol/l mg 132 or 30 mmol/l adaahx3l3vs or without inhibitor (control). protein synthesis was evaluated using l-[1-14c]leucine. proteolysis was determined according to the rate of the tyrosine release into the medium during incubation. in the second study, proteasome inhibitor mg 132 diluted in dimethyl sulfoxide (dms) was administered intraperitoneally in dose 10 mg/kg b.w. controls consisted of dms treated animals. changes in protein and amino acid metabolism were estimated in steady-state conditions using continuous infusion of l-[1-14c]leucine 2 h later. mann-whitney (in vivo study) and paired t-test (in vitro study) were used for statistical analysis. in in vitro study, both mg 132 and adaahx3l3vs significantly decreased protein synthesis and proteolysis. however, in in vivo study, a significant increase in whole-body protein synthesis and proteolysis were observed in mg 132 treated animals. acknowledgements: the study was supported by a grant of gacr no. 303/03/1512. bioinformatical evidence for a prokaryotic ubiquitin-like protein modification system h. scheel, s. tomiuk and k. hofmann bioinformatics group, memorec biotec gmbh, ko¨ln, germany. e-mail: kay.hofmann@memorec.com until recently, the ubiquitin system has been considered a purely eukaryotic invention. by now, the bacterial moad/moeb and this/thif systems are known to be prokaryotic versions of a rudimentary activation system for ubiquitin-like proteins. however, similarities to the ubiquitin system end after the activation step, as moad and this are not conjugated onto target proteins but rather have a role in the biosynthesis of molybdopterin and thiamin, respectively. the eukaryotic protein urm1 is the closest homolog of moad and this. unlike its bacterial cousins, urm1 is conjugated onto target proteins and thus can be considered the founding member of the diverse eukaryotic ubiquitin family. by using a bioinformatics approach that integrates methods of sequence analysis, phylogenetics, phylogenomics and gene-order analysis, we were able to show that many bacteria possess a third ubiquitin-like activation system that most likely is used for protein modifications. the novel system uses a moad/this relative, which is more closely related to urm1 than the typical moad and this proteins. these bacterial urm1 (burm1) proteins typically require the proteolytic removal of a c-terminal extension, which masks the gg motif important for activation. many burm1 operons contain a mpn+/jamm domain protein (belonging to a bona fide ubiquitin-specific protease family), which is most likely responsible for this cleavage. as a third component, an e1-like enzyme is also part of typical burm1 operons. the burm1-associated e1 enzymes look more like uba4 (the eukaryotic urm1-e1) than like the bacterial moeb/thif e1 enzymes. interestingly, the mpn+/jamm protease is also conserved in those bacteria whose burm1 end with gg, suggesting that burm1 removal is important not only for the activation step b2-025p non-hypoxic induction of hypoxia-inducible factors by insulin and 2-deoxy-d-glucose hypoxia-inducible factors (hifs) are key mediators of the cellular adaptation to hypoxia, but also respond to non-hypoxic stimuli like insulin. to clarify involvement of all known hif subtypes in conditions resembling diabetes, we determined distribution of mrnas and proteins in rats subjected to in vivo hypoglycemia and glucoprivation. wistar rats were infused with either saline, insulin, or 2-deoxy-d-glucose (2-dg) to provoke hypoglycemia or impaired glucose assimilation. using real-time qpcr, mrna levels of hif subunits 1a, 2a, 3a, 1b, and of the target gene glut-1 were determined in various organs. cellular distributions of hif-a proteins were examined by immunohistochemistry. treatments with insulin or 2-dg resulted in a widespread increase in hif-3a mrna after 6 h, whereas mrna expression of other hif subunits remained unaffected, except for hif-2a which increased in lung and heart after 2-dg. in cerebral cortex and kidney, enhanced staining of all hif-a proteins was observed after insulin or 2-dg treatments. lung, heart and kidney showed enhanced levels of glut-1 mrna. both hypoglycemia and glucoprivation provoke functional activation of the hif system, with transcriptional up-regulation of hif-3a representing a typical response. our data indicate an involvement of the hif system, and hif-3a in particular, in the pathophysiology of diabetes. fragments of human salivary statherin and pb peptide underlying a furin-like pro-protein convertase action in the pre-secretory salivary fragmentation pathway the recent analysis of some derivatives of human salivary peptides and proteins [13], such as acidic and basic proline-rich proteins (prp) and histatins, allowed recognizing in the presecretory salivary fragmentation pathway the action of a furinlike pro-protein convertase of the kexin-subtilisin family, often followed by a carboxy-peptidase action. on the same line, the present study was carried out to search in human saliva the fragments generated from statherin and pb peptide by the action of furin-like proteinases, utilizing a selected-ion monitoring strategy based on hplc-it ms. the fragments and post-translational derivatives detected with high frequency in multiple samples were the following: (i) statherin (5380 amu), des-phe-43 (5232 amu), des-thr-42-phe-43 (5131 amu), des-asp-1 (5265 amu), mono-phosphor. (5300 amu), statherin sv2 (missing 6-15 residues; 4149 amu), fragm. 10-43 (4128 amu), fragm. 11-43 (3971 amu), fragm. 14-43 (3645 amu). moreover, the fragm. 6-57 (5215 amu) of pb peptide (5793 amu) was identified. the quantity of these fragments in salivary samples was usually <10% of the parent peptide. the identified fragments confirmed the action of a proprotein convertase on furin-like consensus sequences, being the cleavage at arg-9 (ekflr), arg-10 (lrr) and arg-13 (rrigr) for statherin, and at arg-5 (rgpr) for pb peptide. detection of statherin missing n-and c-terminus residues indicated also a pre-secretory exopeptidase action, already observed in other salivary peptides. the function of these statherin and pb derivatives in the oral cavity must be elucidated. cloning and expression of a pepstatin insensitive acid protease from thermoplasma volcanium in e. coli acid proteases, commonly known as aspartic proteases, are recognized by their specific inhibition by pepstatin. acid proteases are found in microorganisms both as intracellular and extracellular enzymes. there is very limited number of thermostable, pepstatin insensitive acid proteases isolated from bacterial sources. the only example of purified and cloned acid protease from archaebacteria is thermopsin, produced by sulfolobus acidocaldarius. this thermophilic enzyme represents a new class of acid proteases. to extend our knowledge on the microbial acid proteases with thermostable properties, in this study we have undertaken the cloning and expression of a thermostable, pepstatin insensitive acid protease from themoacidophilic archaeon thermoplasma volcanium. a primer set was designed based on nucleotid sequence of the predicted thermopsin gene and pcr amplification produced a 3080 bp fragment, which covered complete thermopsin gene with some upstream and downstream sequences. the amplified thermopsin gene was cloned in e. coli, using pdrive vector. the alignment of the amino acid sequences of thermopsins from various archaea revealed the highest homology (44%) between the tp. volcanium thermopsin and putative tp. acidophilum enzyme, thermopsin 1. there was a low degree of similarity (28%) between the tp. volcanium thermopsin and thermopsin from sulfolobus acidocaldarius. expression of the recombinant thermopsin was attempted using qia expression kit, where the cloned gene was ligated to pqe expression vectors to be expressed under the control of t5 promoter. in this system the protein was tagged with 6xhis residue at n-terminal end so that it could be selectively isolated using ni-nta metal-affinity chromatography. include various vital proteins with discrete functions in the propagation of apoptosis. our aim is to generate a caspase cleavage site predictor specific for each member of the caspase family in order to make subtype-specific predictions of new caspase substrates. we have used a set of experimentally verified proteins to generate sequence logos and train a neural network in order to predict caspase cleavage sites. machine learning techniques, such as artificial neural networks, are often well suited to integrate the subtleties of sequence variations. this approach also enables integration of structural information in the pattern recognition procedure which could possibly increase the predictive performance of the neural network. the identification of new caspase substrates can lead to further elucidation of several cellular processes involving caspases, including apoptosis, cell cycle regulation, cellular differentiation, and pro-inflammatory responses. in addition, the generation of caspase inhibitors could be greatly aided by a caspase cleavage site predictor. regulation of protein synthesis and autophagic-lyososomal protein degradation in isolated pancreatic acini a. l. kovacs and e. papp cell physiology laboratory, department of general zoology, eotvos lorand university, budapest, hungary. e-mail: alkova@cerberus.elte.hu a series of biologically active compounds (wortmannin, ly294002, 3-methyladenine, rapamycin, okadaic acid, theophyllin, insulin, glucagon, cholecystokinin) influencing protein synthesis and autophagic-lysosomal protein degradation by interfering with important signalization pathways were investigated. our results show that in exocrine pancreas cells phosphatidyl inositolkinases (pi3k-s) are activators, while the target of rapamycin protein (tor) is an inhibitor of autophagy. camp is an inhibitor of lysosomal protein degradation that acts through members of the pi3k family. okadaic acid inhibits lyososomal protein degradation without inhibiting the formation of autophagic vacuoles. the inhibition of pi3k-s and tor diminishes protein synthesis, inhibitors of these kinases reduce the synthesis stimulatory effect of insulin. cholecystokinin showed a biphasic stimulatory effect while glucagon was ineffective on protein synthesis. on the base of these results a possible signalization pathway is suggested for autophagic segregation and lysosomal protein degradation in pancreatic acinar cells. purification and characterization of a bifunctional protease from vibrio vulnificus in this study, we purified and characterized an extracellular protease showing dual functions as prothrombin activator and fibrinolytic enzyme from vibrio vulnificus atcc 29307. the purified enzyme had broad substrate specificity towards various bloodclotting associated proteins such as prothrombin, plasminogen, fibrinogen and factor xa. the cleavage of these proteins could be stimulated by addition of 1 mm mn 2+ . the protease could acti-vate prothrombin to active thrombin. however, the thrombin activity generated from prothrombin activation by the protease seemed to be transient, with further cleavage resulting in a loss of activity. interestingly, the enzyme could enhance the activity of thrombin during the initial rate of fibrin formation when purified fibrinogen was used as substrate. it could also actively digest fibrin polymer as well as cross-linked fibrin. these results suggest that the secreted protease functions as a prothrombin activator and a fibrinolytic enzyme to interfere with blood clotting as part of the mechanism associated with its pathogenicity in human. tumor invasion and metastasis are the major causes of treatment failure and death in cancer patients. one requisite for neoplastic cell invasion during tumorigenic processes is the remodeling events that occur within the stroma or extracellular matrix (ecm). cysteine cathepsins, most likely along with matrix metalloproteases and serine proteases, degradate the ecm, thereby facilitating growth and invasion into surrounding tissue and vasculature. clinically, the activity levels and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. the aim of our study was therefore both the determination of prognostic and diagnostic impact of cathepsins b, l and h from human tissues extracts (normal and tumor tissue) and extracellular fluids (such as plasma and urine) and a1-proteinase inhibitor (pi) in pathogenesis of different types of human brain tumors, and extraction and purification of cysteine cathepsin endogenous inhibitors from normal and tumor brains and studying of their physicochemical properties. it was found that the increasing of cysteine cathepsins b, l, h activity levels in brain tumors tissues depend on histostructure, histogenesis and tumor malignancy grade. increasing of cathepsins l and h activity levels was found in plasma and urine in depending on histogenesis. at the same time decrease in pi activity level was registered. besides, kinetic characteristics of extracted normal brain endogenous inhibitors of cysteine cathepsins were determined. in extracted tumor brain endogenous inhibitors, there were differences in physicochemical properties in comparison with normal. the data obtained contribute to understanding the participation of cysteine cathepsins and their inhibitors in mechanisms of cancer genesis and both become useful for solving the problem of improving of tumor therapy and provide the possibility of using their activity as diagnostic and prognostic markers. protein hydrolysates of sea origin as components for microbiological culture media dry hydrolysate was prepared from protein-containing waste of icelandic scallop chlamys islandicus processing (spw) by means of a proteinase complex from king red crabs hepatopancreas. the enzyme consist of the proteolytic enzyme complex from crab hepatopancreas, in which serine proteases dominate (collagenase, elastase and trypsin-and chymotripsin-like proteinases). as proteinases from king red crab hepatopancreas have high enzymesubstrate affinity to icelandic scallop proteins, a high degree of proteolysis can be achieved. the composition and properties of the material were investigated on enzymatic protein hydrolysate from spw obtained under the most technologically suitable conditions: 50-55 o c, ph 7.5, 6 h, the ratio between the protein material and the enzyme preparation being 1000:6. for comparison we examined the composition of commercial pancreatic hydrolysate from poor-quality fish species, mainly boreogadus and micromestistus. it was found that hydrolysate from spw significantly overpowered the commercial analog in the mass percentage of the target product (free amino acid and oligopeptides). the resulting product contains not <80% free amino acids and oligopeptides. predominant are aspartic acid, leucine, isoleucine, arginine and lysine, which account for >5% of the free amino acids. the potential usage of the protein hydrolysate as a nutrient for microorganism cultivation is estimated. microbiological studies have demonstrated that the hydrolysate from spw can be used as a protein component in nutrient media. the tested microbial strains satisfactorily grew on the media. the z variant alpha-1 proteinase inhibitor (a1piz) misfolds in the endoplasmic reticulum (er) and is a substrate for er-associated protein degradation (erad). we report here that a1piz degradation is also dependent on vps30/atg6, a gene that encodes a component of two pi3-kinase complexes that regulate membrane traffic; complex i is required for autophagy, complex ii is required for the cpy-to-vacuole pathway. to elucidate why vps30p participates in a1piz degradation, we tested the hypothesis that erad was saturated at elevated levels of a1piz expression and that excess a1piz was targeted to one of these alternative quality control pathways. overexpression of a1piz led to vacuole-dependent degradation and both complexes were required for delivery of the excess a1piz to the vacuole. when the cpy-to-vacuole pathway was compromised a1piz was secreted and the distribution of soluble vs. aggregated forms of a1piz was comparable with that of wild type yeast. however, disruption of autophagy led to an increase in levels of aggregated a1piz; suggesting that when erad is saturated the excess a1piz is selectively targeted to the vacuole via the cpy-to-vacuole sorting pathway, while excess a1piz that forms aggregates in the er is targeted to the vacuole via autophagy. together, these results reveal multiple pathways for recognition and removal of aberrant proteins and provide direct evidence that aggregated a1piz is removed by autophagy. our findings may have application in the understanding of, and treatment for, individuals with liver disease caused by the accumulation of er aggregates of a1piz. acknowledgements: the study was supported by national science foundation grants mcb-011079 and mcb-0110331. yeast and lactobacillus association generates peptides from acid goat whey proteins fermentation s. didelot, s. bordenave-juchereau, e. rosenfeld, l. murillo, j. m. piot and f. sannier laboratory of biotechnology and bioorganic chemistry, university of la rochelle, la rochelle, france. e-mail: lmurillo@univ-lr.fr our goal was to produce peptides from fermentation of unsupplemented acid goat whey by dairy micro-organisms. we used a lactobacillus, lactobacillus paracasei, and a yeast, candida parapsilosis, both previously isolated from a cheese microflora. when co-cultivated aerobically, both micro-organisms grew on unsupplemented goat whey and led to a medium acidification from 6 to ph 3.5. reversed phase (rp)-hplc analysis revealed a total alpha-lactalbumin hydrolysis after 96 h of fermentation, a modification of the beta-lactoglobulin elution peak, and 2.5-fold increase in peptide level compared with the non-fermented whey. in the absence of c. parapsilosis, l. paracasei grew poorly on whey and only a weak medium acidification from 6 to 4.5 was observed after 192 h of fermentation. rp-hplc analysis revealed a weak modification of beta-lactoglobulin elution peak, a truncated form of alpha-lactalbumin and no peptide generation. c. parapsilosis was able to grow on unsupplemented goat whey without modifying ph of the medium, but only 25% of proteins were hydrolysed (alpha-lactalbumin) or denaturated (beta-lactoglobulin) and, again, no peptides were detected. these results suggest that (i) c. parapsilosis is required for l. paracasei growth and (ii) the co-culture of both micro-organisms is needed to generate peptides from alpha-lactabumin hydrolysis. during co-culture on whey, the use of penicillin g and cycloheximide as bacterial and yeast growth inhibitors respectively, revealed that l. paracasei growth was required for medium acidification to ph 3.5 and alpha-lactalbumin hydrolysis. however, we demonstrated that the protease(s) responsible of alpha-lactalbumin hydrolysis was (were) synthesized by c. parapsilosis during the first stage of fermentation and that medium acidification (obtained either by l. paracasei growth or chemically) was required for yeast protease(s) activity. dengue virus causes widespread human diseases such as dengue fever, dengue hemorrhagic fever and dengue shock syndrome. the viral genome is a positive rna strand that encodes for a single polypeptide precursor. processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by ns3 serine protease. the three dimensional structure of ns3 protease domain ns3pro has been elucidated [1] . recently a new construct of the recombinant form of the ns3pro, was engineered [2] . we have expressed in e. coli the his-tag-cf40.gly.ns3pro protein a new construct of the recombinant form of the ns3pro linked to a 40 -residue co-factor, corresponding to a part of ns2b, via a non-cleavable, flexible non-apeptide (gly 4 sergly 4 ), and have currently optimized the purification procedure. chemically optimized substrates, peptides and depsipeptides, were designed and tested to afford an efficient in vitro activity assay, using hplc and fret spectroscopy. the data suggest that the amino-terminal region of the 40-amino acid co-factor domain is involved in additional charged interactions with ns3 that are essential for activity as previously described. this form showed catalytic activity and spectroscopic studies were performed to identify the folding of the protein. moreover, experiments of limited proteolysis have been performed to identify the essential enzymatic domain of the protein and to stabilize the role of the cofactor in the activity and in folding stabilization of the enzyme. after 2 h of the limited proteolysis with endoproteinase asp-n the product was analyzed by sds-page and activity assay, showing a high reduction of the molecular mass and only a loss of the activity of the 20%. cd and 15 n-1 h-hsqc spectra of this protein fragment were performed and other functional and structural characterizations are in progress in our laboratory. it is intended to obtain the structure in solution of the essential active domain of the uniformly 13 c, 15 n-labeled cf40.gly.n-s3pro by high-field 3d nmr spectroscopy. the solution structure of the enzyme will be used to answer yet unresolved questions about the mechanism of action, the role of its cofactor ns2b, and the observed substrate specificity. introduction: fish consumption is associated to nutritional benefits due to the presence of proteins of high biological value, minerals, vitamins and polyunsaturated fatty acids. most studies concerning the benefits of fish consumption on cancer prevention have focused on fish fatty acids but little is known about the potential bioactivity of fish peptides. the present study was then designed to assess the antiproliferative activity of various fish protein hydrolysates, in order to further purify and characterize anticancer peptides. methods: twenty-one fish hydrolysates (from seven species) produced within the framework of the european valbiomar programm. fish hydrolysates composition (protein, fat and salt content) was determined by standard methods (kjehldhal, soxhlet extraction and volhard respectively). cytotoxic and antiproliferative activity were assayed in vitro on mcf-7/6 and mda-mb-231 human breast adenocarcinoma cell lines, following a cell viability colorimetric assay (promega, france). antiproliferative activity of fish hydrolysates was compared with that of reference anticancer molecules with various cellular targets, namely actino-mycine d, cytosine-beta-d-arabinofuranoside, cyclophosphamide, etoposide, kenpaullone and roscovitine. results: composition analysis revealed that most hydrolysates contained more than 70% protein. three blue whiting hydrolysates containing 96% protein, 0.5% lipid and 0.2% salt induced a strong breast cancer cells growth inhibition when tested at 1 g/l for 72 h in cell culture medium. blue whiting hydrolysates 3, 4 and 5, respectively, induced a growth inhibition of 24.5, 22.3 and 26.3% on mcf-7/6, and 13.5, 29.8 and 29.2 % on mda-mb-231. these in vitro antiproliferative activities are in the range of that observed when the two breast cancer cell lines are treated for 72h with kenpaullone, roscovitine or cytosine-beta-d-arabinofuranoside 10 )6 m. further studies are engaged to fractionate and characterize the antiproliferative peptides contained in blue whiting hydrolysates. during recent years, it has been established that intracellular proteolysis in eukaryotic cells is largely accomplished by a highly selective non-lysosomal pathway that requires atp and a large (2.5 mda) multisubunit complex known as the 26s proteasome. the proteasome-mediated pathway plays vital regulatory functions. it degrades many important proteins involved in cell cycle control, in signaling pathway, and in general metabolism, including transcription factors and key metabolic enzymes. another function of the proteasomal system is the removal of abnormal, misfolded and oxidized proteins generated under normal and, in particular, stress conditions. to date, proteasomes from other than animal or plant cells were studied only in yeast. recently, in our laboratory, the proteasome-mediated pathway was shown to be involved in the regulation of ligninolytic activities in the white rot fungi trametes versicolor and phlebia radiata upon nutrient starvation (staszczak, enzyme microb technol 2002; 30: 537-540). it was the first report on proteasomes in fungi representing basidiomycota. white rot fungi are able to degrade lignin by the action of secreted enzymes, the best characterized of which are laccases, lignin peroxidases, and manganese peroxidases. the subject of lignin biodegradation has commanded attention for a considerable period of time mainly because of its ecological significance and wide industrial applications of bioligninolytic systems. heavy metal ions are important environmental pollutants which affect biodegradation processes performed by white rot fungi. in the present study, we investigated whether the proteasomal degradation pathway might be involved in the regulation of laccase production by t. versicolor in response to cadmium exposure. studies of cacybp/sip function using small interfering rna cacybp/sip was discovered as a protein that bound calcyclin (s100a6) in a calcium-dependent manner (filipek and wojda 1996; filipek and kuznicki, 1998) and its distribution and some biochemical properties have been studied. for instance, it has been shown that cacybp/sip binds calcyclin via its c-terminal fragment (nowotny et al. 2000) and that, beside calcyclin, it interacts with other calcium binding proteins of the s100 family (filipek et al. 2002) . originally, we identified cacybp/sip in ehrlich ascites tumour (eat) cells but it is also present in other mammalian tissues and cells. in particular, high expression of cacybp/sip was found in neuronal cells of mouse and rat brain (jastrzebska et al. 2000) . at present the distribution and structural properties of cacybp/sip are quite well described but its function remains obscure. there is only one paper published concerning the possible involvement of cacybp/sip in b-catenin ubiquitination and degradation (matsuzawa and reed 2001). to elucidate the biological role of cacybp/sip we have designed and synthesized sirna (small interfering rna) against this protein. this sirna was then used to transfect neuroblastoma nb-2a and embryonic kidney hek293 cells, expressing high and low amount of endogenous cacybp/sip respectively. the level of cacybp/sip was monitored in cell extracts by western blot technique. we found that sirna against cacybp/sip, which we designed, inhibited the expression of this protein, as its level in transfected cells was lower in comparison with control cells. at present, we checked the effect of diminished expression of cacybp/sip on b-catenin degradation and other cellular processes. acknowledgements: this work was supported by grants: kbn heavy metals are powerful poisons for living cells. it has been shown that exposure to arsenicals, either in vitro or in vivo, in a variety of model systems, causes the induction of a number of the major stress protein families, such as the heat shock proteins (hsp) (toxicol appl pharmacol 2001; 177: 132). the reasons for heavy metal toxicity in vivo are not fully understood, but they are known to contribute to the accumulation of aberrant proteins (bba,1995,1268, 59). in animal cells, arsenite has been reported to cause sulfhydryl depletion, to generate reactive oxygen species and increase the level of high molecular mass ubiquitin-protein conjugates (toxicol appl pharmacol 2003; 186: 101). in cells submitted to stress conditions, several components of the ubiquitin/proteasome pathway are activated. in this major, eukaryotic proteolytic pathway, multiple ubiquitin molecules are enzymatically ligated to proteins destined for catabolism by an enzyme system composed of three types of enzymes, commonly referred to as e1, e2, and e3. the large ubiquitinprotein conjugates thus formed are subsequently degraded by a very large protease complex, the 26s proteasome, in an atpdependent process. the changes in free ubiquitin (ub) and ubiquitin-protein conjugates (ub-p) levels were followed by immunoblotting during the incubation of the higher plant lemna minor l.(duckweed) in the presence of arsenite (as), at concentrations known to confer thermotolerance to the plants. the observed increase in the amount of large molecular mass ubiquitin-protein conjugates is indicative of a role for the ubiquitin/ proteasome pathway in the response of lemna to as stress. this outcome is primarily attributed to an increased availability in protein substrates during as treatment for three main reasons: an increase in protein carbonyl (a major marker for protein oxidation) content detected by immunoblotting; moderate increments (as determined by semi-quantitative rt-pcr) in the mrna levels of the codifying sequences for the ubiquitin pathway components: ubiquitin, e1, e2 and the b subunit and the atpase subunit of the 26 s proteasome; an identical pattern of variation for the large ubiquitin-protein conjugates is observed in the simultaneous presence of as and cycloheximide, indicating that the observed increase in ubiquitin conjugates does not depend on de novo protein synthesis. ageing and autophagy y. stroikin and a. terman experimental pathology, linko¨ping university, linko¨ping, sweden. e-mail: yurst@inr.liu.se life of aerobic cells is associated with continuous oxidative damage resulting in the formation of altered, non-functional macromolecules and organelles. intracellular accumulation of oxidized proteins defective organelles and lipofuscin inclusions are typical manifestations of ageing that preferentially affects long-lived post-mitotic or growth-arrested cultured cells. autophagy, an important biological mechanism for renewal of damaged intracellular structures, has been found decreased in ageing. to learn more about the role of autophagy in ageing, we studied the effect of the inhibitor of autophagic sequestration 3-methyladenine (3-ma) on human diploid fibroblasts and astrocytes. inhibition of autophagy in growth-arrested (confluent) fibroblasts for 2 weeks resulted in the accumulation of altered lysosomes displaying lipofuscin-like autofluorescence, especially when 3-ma exposure was combined with hyperoxia. the findings suggest that autophagy is indispensable for normal turnover of lysosomes, and lysosomal components may be direct sources of lipofuscin. the accumulation of oxidatively damaged intracellular structures (so-called biological ''garbage'') was associated with decreased cell viability. two-week-inhibition of autophagy with 3-ma resulted in a significantly increased proportion of dying cells when compared with both untreated confluent cultures and dividing (subconfluent) cells exposed to 3-ma. similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. the results support the idea that biological ''garbage'' accumulation is essential for ageing and age-related death of post-mitotic cells, which can be prevented by cell division. recently two family members of the tumour suppressor gene p53 have been described, p63 and p73, which seem to be necessary for specific p53-induced stress-response pathways. furthermore, p63 and p73 appears to be crucial to determine the cellular sensitivity to anticancer drugs, particularly in tumours lacking functional p53. here, we show that p63 and p73 isoforms are also regulated by proteasomal degradation. we have identified several e3-ubiquitin ligases responsible for the regulation of the stability of p63 and p73. we found that the regulation of p63 and p73 is isoform-specific. furthermore, we demonstrate that ubiquitination of p73 influences the cellular localization of p73 and of the respective e3-ubiquitin ligases. finally, we show that the expression of the various e3-ubiquitin ligases can be differentially induced by p73-isoforms. in addition, the e3-ubiquitin ligases can influence the apoptotic function of p73. our findings demonstrate that p63 and p73 are sent to degradation or stabilized by e3-ubiquitin ligases in an isoform-specific manner and we suggest a negative feedbackloop between p63, p73 and their regulators, as they also influence the function of p63 and p73. increased level of metalloproteases was shown to accompany tumor angiogenesis and active invasion in adjacent tissue [1] . development of different types of tumors is often accompanied by increased protease activity in blood [2, 3] . in the present study we compared protease activity of plasma and eluate from surface of blood cells in healthy donors and patients with breast tumor. we have demonstrated recently that in blood of healthy donors almost all circulating nucleic acids (cirna) are bound at the surface of blood cells. in patients with fibroadenoma cirna were found at cell surface whereas in breast cancer, no cell-surfacebound cirna were detected in blood [4] . conjugates of hydrophobic and hydrophilic peptides of cd34 receptor with biotin were incubated with avidin-coated 96-well eia microplates. avidinpeptide complex was incubated with samples under investigation and serial dilutions of proteinase k solution, which was used for calibration of protease activity. undegraded peptides were visualized by incubation with goat anti-peptide antibodies followed by conjugate of anti-goat immunoglobulins with peroxidase. blood plasma and eluate from surface of blood cells of cancer patients demonstrated increased level of anti-hydrophilic protease activity compared with healthy donors. increase of protease activity against hydrophilic peptide in blood correlate with decrease of cell-surface-bound cirna, indicating that blood proteases can affect concentration and distribution of circulated na. identification of cleavage site and natural substrate specificity of prta, a serralysin-type metalloprotease from the entomopathogenic microorganism photorhabdus prta, a secreted basic metalloprotease of photorhabdus, belongs to the m12b (serralysin) family of proteases. the biological function of these enzymes is not known, but in some cases they are supposed to have a role in virulence. serralysins are generally assumed to have broad substrate side-chain specificity. attempts toward the generation of a sensitive and specific substrate of these enzymes had limited success, and no such substrate is available for prt-a. through mass spectrometric analysis of prta cleavage products of oxidized insulin a and b chain, we found that prta has a welldefined cleavage site preference. based on this, we developed a sensitive and highly specific oligopeptide substrate through optimization of the amino acid composition and length. the kinetic parameters of prta isolated from photorhabdus luminescens ssp. laumondii strain brecon were measured on the best substrate, dabcyl-glu-val-tyr-ala-val-glu-ser-edans, giving a km of 8.8 · 10 )5 , a kcat of 2.1 · 10 )2 /s and a kcat/km of 2.4 · 10 6 . its poor hydrolysis by various proteases proved its specificity, while it was very sensitivity in measuring prta activity in hemolymph samples from photorhabdus infected galleria mellonella larvae. the substrate preference of prt-a was determined by in vivo digestion of hemolymph proteins from manduca sexta. six minor protein components were selectively cleaved, which were provisionally disthe epithelial sodium channel (enac) is an integral component of the pathway for na + absorption in epithelial cells. enac activity is mainly regulated by mechanisms that control its expression at the cell surface, such as ubiquitination. the ubiquitin ligases nedd4 and nedd4-2 have both been shown to bind to enac and decrease its activity. conversely, the serum-and glucocorticoid regulated kinase (sgk), a downstream mediator of aldosterone, is able to increase enac activity. this effect is at least partly mediated by direct interaction between sgk and nedd4-2. sgk binds both nedd4 and nedd4-2 but it is only able to phosphorylate nedd4-2. phosphorylation of nedd4-2 reduces its ability to bind to enac, and hence increases enac activity. the impact of the interaction between nedd4 and sgk remains unclear. nedd4-like proteins interact with enac via their ww-domains. these domains bind py-motifs (ppxy) present in enac subunits. nedd4 and nedd4-2 both have four highly homologous ww-domains. previous studies have shown that interaction between nedd4 and enac is mainly mediated by ww-domain 3. sgk also has a py-motif, therefore we tested whether the ww domains of nedd4 and nedd4-2 mediate binding to sgk. we show that single or tandem ww domains of nedd4 and nedd4-2 mediate binding to sgk and that, despite their high homology, different ww domains of nedd4 and nedd4-2 are involved. our data also suggest that ww domains 2 and 3 of nedd4-2 mediate the interaction with sgk in a concerted manner, and that in vitro the phosphorylation of sgk at serine residue 422 increases its affinity for the ww domains of nedd4-2. the stimulatory effect of sgk on enac activity is partly mediated via nedd4-2 and will decrease if competition between nedd4 and nedd4-2 for binding to sgk occurs. we show that nedd4 and nedd4-2 are located in the same subcellular compartment and that they compete for binding to sgk in vitro. the concerted or successive action of proteolytic enzymes has been described in a number of important biological processes in which proteins are degraded or matured, such as digestion, turnover (lysosomal, proteosomal...), blood coagulation, developmental remodeling or apoptosis, among others. the complementary action of proteases belonging to different families to achieve a more efficient o a better modulated hydrolytic mechanism is well documented. specific molecular associations or shared scaffolds between the involved proteases and/or protein inhibitors and defined three-dimensional structures have also been reported. however, only in a few cases such structures involved metallo.carboxy-peptidases or their inhibitors [1] . we shall review this subject and describe, in such a context, a new model found in a marine invertebrate organism in which such a fact takes place. in particular, the characteristics of a novel bifunctional molecule displaying the functionalities and structures of serine-and metallo.carboxy-peptidases will be presented. its structure is fully different than the ones previously reported by us and collaborative groups for metallocarboxypeptidase inhibitors [2] [3] [4] . regulating the activity of herpes virus proteases c. s. craik departments of pharmaceutical chemistry, pharmacology, and biochemistry and biophysics, ucsf, san francisco, ca, usa. e-mail: craik@cgl.ucsf.edu herpesviral proteases exist in a monomer-dimer equilibrium in solution. dimerization is required for activity and a comformational change communicates the oligomerization state of the enzyme to the active site of each intact monomer. each monomer has an active site, which is spatially separate from the dimer interface. kaposi's sarcoma-associated herpesvirus (kshv), encodes a protease (kshv pr), which is necessary for the viral lytic cycle. like those of other herpesvirues proteases, the dimer interface of kshv pr is composed primarily of a helix near the c terminus, of the protein. the helix of one monomer interacts with residues in the symmetrically related helix of the other monomer across the dimer interface as well as with neighboring helices. small molecule inhibitors, site directed mutagenesis and 2d nmr spectroscopy were used to compare the monomeric and dimeric forms of kshv pr and to investigate the relationship of the active site and the dimer interface of the enzyme. active site inhibition was shown to strongly regulate the binding affinity of the monomer-dimer equilibrium of the protease, shifting the equilibrium completely to the dimeric form of the enzyme. a previously undetermined conformational change provided insight in to the regulation of protease activity by dimerization as well as an explanation for the weak dimerization of a family of enzymes with a disparately large dimer interface compared to their measured binding affinities. using this information as a guide, protein grafting of the interfacial helix onto a small stable protein, avian pancreatic polypeptide, generated a small macromolecular inhibitor that successfully disrupted the dimer interface and inhibited enzymatic activity. these results provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, validate the protease as a therapeutic target and suggest the dimer interface may be an alternative site for antiviral design. abteilung strukturforschung, max-planck-institut fu¨r biochemie, martinsried, germany. e-mail: huber@biochem.mpg.de proteolytic enzymes catalyze a very simple chemical reaction, the hydrolytic cleavage of a peptide bond. nevertheless they constitute a most diverse and numerous lineages of proteins. the reason lies in their role as components of many regulatory physiological cascades in all organisms. to serve this purpose and to avoid unwanted destructive action proteolytic activity must be strictly controlled. control is based on different mechanisms which i will discuss and illustrate with examples of systems and structures determined in my laboratory. the family of serine protease inhibitors known as the serpins is represented in all branches of life and predominate in the higher organisms, including man. they have evolved an extraordinary mechanism to inhibit proteases which distinguishes them from the 20 other families of serine protease inhibitors, and renders them uniquely qualified to control of the proteolytic pathways essential to life. the mechanism is best described as a spring-loaded mousetrap, where nibbling of the peptide loop bait springs the trap and crushes the unsuspecting protease. as with a mousetrap, the active state of a serpin is metastable, and the energy released upon conversion to its more stable form is used to trap the protease. the complexity of the serpin mechanism provides many advantages over the simpler lock-and-key type mechanism, utilized by all other serine protease families. serpins provide stoichiometric, irreversible inhibition, and the dependence on serpin and protease conformational change is exploited for signaling and clearance. the potential for regulation is also an inherent part of such a complex mechanism, as illustrated by the heparin activation of serpins antithrombin and heparin cofactor ii. however, with complexity of mechanism also comes susceptibility to disease causing mutations: both through loss-of-function, as with thrombosis caused by antithrombin deficiency; and gain-of-function, as with dementia caused by neuroserpin polymerization. many crystallographic structures of serpins have been solved over the past 20 years, and we now have a frame-by-frame cinematic view of the intricate conformational rearrangements involved in protease inhibition, modulation of specificity, and molecular pathology of the remarkable shape-shifting serpins. structural lessons of serine proteases: function and mechanism of the serine protease-like hgf as a growth factor in met signaling hepatocyte growth factor (hgf), a plasminogen-related growth factor, is the ligand for met, a receptor tyrosine kinase implicated in development, tissue regeneration and invasive tumor growth. hgf acquires signaling activity only upon proteolytic cleavage of single-chain hgf into its a/b-heterodimer, similar to zymogen activation of structurally related serine proteases. although both chains are required for activation, only the achain binds met with high affinity. recently, we reported that the protease-like hgf b-chain binds to met with low affinity this suggests that additional allosterically linked regions may be involved in the signaling process. furthermore, antibodies directed toward the b-chain or the hgf a-chain result in inhibition of met phosphorylation in a549 cells. these antibodies also inhibit proliferation in bxpc3 cells and baf3 cells. implications for dimerization mechanisms of hgf-dependent met receptor activation and signaling are presented. in addition, mutagenesis of the hgf b active site region has been investigated with respect to imparting enzymatic activity. thus while hgf has the function of a growth factor, the structural and receptor binding aspects of hgf are more akin to those of serine proteases. trypsinogen 4 with a 28 amino acid leader peptide on its n-terminus is the predominant form of the enzyme in human brain gene prss3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mrna for trypsinogen 4 has recently been identified in brain and other human tissues. analysis of the gene encoding trypsinogen 4 predicted two isoforms of the zymogen: isoform a may have a 72 amino acid, while isoform b a 28 amino acid n-terminal leader sequence. the translation initiation site for isoform a is an atg codon, while the initiation site predicted for isoform b is a ctg codon. we measured the amount of trypsinogen 4 mrna and the quantity of the protein as well in 17 selected areas of the human brain. trypsinogen 4 could be localized in glial and neuronal cells using immunohistochemical methods. we purified human trypsinogen 4 by affinity chromatography. our results show that splice isoform b is the predominant if not the exclusive form of the zymogen in human brain. the n-terminal residue of the isolated protein was identified by amino acid sequencing as a leucine. at the same time the longest mrna we were able to isolate was barely longer than the one corresponding to splice isoform b. although the most trivial explanation of our results is that isoform a is proteolytically processed to result in isoform b, it cannot be excluded that leucine rather then methionine is used as translation initiator amino acid. search for endogenous substrates for prolyl oligopeptidase in porcine brain prolyl oligopeptidase (po) is a serine protease present in most tissues, which preferentially cleaves the peptide bond at the carboxyl site of proline residues. the function of po is unknown, but it has been associated with several disorders of the central nervous system, such as depression and alzheimer disease. the purpose was to look for endogenous substrates for the recombinant porcine po in porcine brain. we adapted a method to extract the proteins from the brain with special attention to the smaller polypeptides since po is not known to cleave peptides larger than 30 amino acids. subsequently we looked for a method to separate the protein mixture in less complex fractions. 2d-gelelectrophoresis, commonly used in proteomics, is only suitable for proteins with a molecular weight between 10 and 200 kda and an iso-electric point between 4 and 10. two-dimensional chromatography offers a suitable alternative for small peptides. we chose ion exchange chromatography as a first and reversed phase high pressure liquid chromatography as a second step. the resulting fractions were divided into two parts. one part was incubated with the purified po, the other served as a control. by looking for shifts in the mass spectrum between the control sample and the incubated sample, we identified peptides cleaved by po. different methods, such as esi-qtof-ms and maldi-toftof-ms, were used to sequence cleaved peptides by msms. these experiments allowed us to deduce the sequence requirements for po cleavage. serine protease subtilisin immobilized on novel mesoporous materials serine proteinases are widely used in protein mapping and peptide or ester bond formation. fixation of enzyme on solid support has many advantages, such as high stability, possibility of recovering and low product contamination by enzyme. subtilisin carlsberg, a protease from bacillus licheniformis, was immobilized on mesoporous silica (sba-15) and several organosilica supports via physical adsorption. the bifunctional mesoporous organosilicas containing ch2-ch2 or ch=ch bridges in combination with organic tethers bearing amino or hydroxyl functionalities were synthesized using supramolecular templating in the presence of non-ionic triblock copolymers and exhibited high surface area and large pore diameters in the range of 50-70 å suitable for the incorporation of subtilisin. the kinetics of immobilization was examined for six different carriers. it was shown that enzyme retained hydrolytic activity after the immobilization. the dependence of subtilisin loading on the starting concentration of the enzyme during adsorption shows the maximum loading (455 mg protein/g support) at [e] = 20 mg/ml. the ph dependences of loading and activity of immobilized biocatalysts were bell-shaped. for the organosilica support containing amino and hydroxyl groups the ph-dependence was shifted to the alkaline ph by 2 in comparison with the support containing ch2-ch2 bridges. the adsorbed subtilisin desorbs easily in aqueous media, while no leaching of the enzyme was observed in acetonitrile and dmf/acetonitrile mixture (6/4). the immobilized biocatalyst shows high hydrolytic activity after incubation in non-aqueous acetonitrile for 1 week and after 48 h incubation in 60% dmf/acetonitrile mixture. these data indicate a possible application of the obtained biocatalysts in low water media. purification, structural and biological characterization of protease inhibitors from acacia plumose seeds protease inhibitors have been used in many current medicines. therefore, there is a considerable interest inside the pharmaceutical industry in discovering new composites and mechanisms of protease inhibition, since these investments have led, for example, to new anti-hiv therapeutical tests, coagulation diseases treatment and tests with anti-carcinogenic drugs. serine protease inhibitors are found in all plant tissues, mostly in the seeds of the leguminosae subfamilies: mimosoideae, caesalpinoideae and papilionoideae. acacia genus is one of most important member of mimosoideae, and the presence of protease inhibitors in this genus was described in only three species and none of them were structurally characterized. in this sense, we are studying three new protease inhibitors from a. plumose seeds. from saline extract of triturated mature seeds the inhibitors were purified and presented anti-coagulant activity, serine protease inhibitory activity and action on growth of fitopathogenic microorganisms, in vitro. the purification steps included size exclusion chromatography on the superdex-75 column, equilibrated and eluted with pbs, a ionic exchange chromatography on mono-s (hr 5/5) column, equilibrated with the buffer sodium acetate 50 mm (ph 5.0), and eluted with the same buffer in a gradient of 0-0.5 m of nacl. three fractions (eluted around 0.18, 0.22 and 0.33 m of nacl) that presented anticoagulant activity and serine protease inhibition were separated and denoted apia, apib and apic. their apparent mws were around 20 kda, by sds-page in the absence of reducing agents. in the presence of reducing agents they shown two bands: between 14-22, and 8-6 kda. the n-terminal analyze of higher mw chains were tyafl (apia); kellvdne (apib) and telhdd (apic). the circular dichroism spectra of these inhibitors were very similar, presenting a maximum around 230 nm and a minimum in 202 nm, compatible with presence of unordered and beta elements of secondary structure. their nterminal, cd spectra and two-polypeptide chains linked by covalent bound, are compatible with kunitz type inhibitors. probably these inhibitors are three different isoforms that present different inhibition specificity degree on the serine proteases family. the ki to different serinoproteases (trypsin, plasmatic kalikrein, elastase, quimotrypsin) and specificity to the phytopatogenic fungus are being investigated. although the proteases were initially described as enzymes involved in the non-specific degradation of dietary proteins, today it is known that they can also act as highly specific enzymes that perform selective cleavage of specific substrates. thus, alterations in the structure, regulation or function of this type of enzymes underlie serious human disorders including cancer. to date, more than 550 protease and protease homologs are annotated in man, mouse, and rat genomes (www.uniovi.es/degradome). the increasing complexity of the proteolytic systems has led to the introduction of global concepts as the term degradome to define the complete set of proteases that are produced in a specific moment by a cell, tissue or organism. as part of our studies focused on the characterization of the mammalian degradomes, we have identified and cloned unusual mosaic proteases containing in tandem serine protease domains. the first, called polyserase-1 is synthesized as a transmembrane protein that undergoes post-translational events to generate three independent serine protease domains. the second polyprotease is the polyserase-2, a secreted protein that remains as integral part of the initial protein product. to date, it is difficult to understand the putative functional advantages derived from the complex polyproteases and, albeit extremely unusual, it is not an unprecedented situation. thus, the amphibians ovochymase and oviductin are polyserine proteases that contain three in tandem serine proteases. in humans, angiotensin-coverting enzyme and carboxypeptidase d are polymetalloproteases that exhibit some similarities to the polyserases. all these polyproteases constitute examples that illustrate an additional strategy for increasing the complexity of the degradomes. evolution of a genetic locus, expressing several protease inhibitors with homology to whey acidic protein (wap) a. clauss and å . lundwall department of laboratory medicine, lund university, malmo¨, sweden. e-mail: adam.clauss@klkemi.mas.lu.se we have previously described a locus on human chromosome 20 that gives rise to 14 proteins containing wap four disulphide core (wfdc) domains. among them are the elastase inhibitors elafin and secretory leukocyte proteinase inhibitor (slpi). both slpi and elafin are also known to be important components of the innate immune defence by displaying anti-microbial properties. in order to gain a deeper understanding of the biological role of the locus, we have now extended our investigations of its organization and evolution into non-human mammals. homologous loci were identified on mouse chromosome 2, rat chromosome 3 and dog chromosome 24. transcript sequences were generated by race technology or retrieved from the est databases. as in humans, the murine and canine loci are divided into two sub-loci separated by approximately 200 kb. the majority of genes are conserved in all species, but the comparison also showed gain and loss of genes, e.g. two human pseudogenes were identified due to the discovery of functional rodent genes, and in the rat several duplications has yielded four slpi genes. a most interesting finding was that there is no murine elafin gene. the different wfdc domains showed a highly variable species conservation. this was particularly striking in proteins containing multiple domains, where the aminoterminal wfdc generally displayed low conservation, whereas the opposite was true for the carboxyterminal wfdc. the difference could be due to the potential targets of the inhibitors, which might be either highly variable exogenous microbial proteases or conserved endogenous proteases. signaling mechanism of thrombin-induced human gingival fibroblast contraction thrombin is activated during gingival tissue injury and inflammation. thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of proteaseactivated receptors (pars). we noted that thrombin and par-1 agonist peptide (20 lm) induced the gingival fibroblasts (gf)-populated collagen gel contraction within 2-h of exposure. however, par-3 and par-4 agonist peptide (<20 lm) show little effect on collagen gel contraction. u73122 (phospholipase c inhibitor) and 2-apb (ip3 antagonist) were effective in inhibition of gf contraction. thrombin-induced gf contraction was inhibited by 5 mm egta (an extracellular calcium chelator) and verapamil (a l-type calcium channel blocker). in addition, w7 (10 and 25 lm, a calcium/calmodulin inhibitor), ml-7 (50 lm, myosin light chain kinase, mlck inhibitor), and ha1077 (100 lm, rho kinase inhibitor) completely inhibited the thrombin-induced collagen gel contraction. thrombin also induced the phosphorylation of erk1/erk2 in gf. however, u0126 only partially inhibited the thrombin-induced gf contraction. similarly, wortmannin (100 lm), ly294002 (20 lm) (two pi3k inhibitors) and genistein, also showed partial inhibition. moreover, nac was not able to suppress the gf-contraction, as supported by slightly decrease in reactive oxygen species production in gf by thrombin. these results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting gf contraction. this event is mainly mediated via par-1 activation, plc activation, extracellular calcium influx via l-type calcium channel, and the calcium/calmodulin-mlck and rho kinase activation pathway. survival of the anticarcinogenic bowman-birk inhibitor from soybean at the terminal ileum of cannulated pigs plant protease inhibitors (pi) of the bowman-birk class, a major pi class in legume seeds, have emerged as highly promising cancer chemopreventive agents, being capable of preventing or suppressing carcinogenic processes in a wide variety of in vitro and in vivo animal model systems. in order to exert their chemopreventive properties in vivo, plant pi have to resist and survive, at least to some extent, degradation by acidic conditions and digestive enzymes during gut passage. in this study, we have evaluated the survival rate of the bowman-birk inhibitor (bbi) in the terminal ileum of cannulated pigs fed defatted soybean. two different quantitative approaches have been carried out. firstly, a competitive indirect elisa assay using an antisera capable to detect bbi free and/or in complex with digestive proteases; secondly, we have carried out spectrophotometric measurements of trypsin and chymotrypsin inhibitory activities in ileal samples, where the presence of bbi metabolites and/or single active loops can be detected. according to the elisa method, ileal apparent digestibility of bbi was 58 %, which resulted in a recovery of 0.61 mg out of 1.5 mg/kg feed ingested. significantly higher ileal digestibility values (95 %) were found when trypsin and chymotrypsin inhibitor activities were evaluated. the results suggest that the immunoassay may be overestimating the presence of functional pi by detection of inactive bbi, but also that the presence of complexed bbi with digestive proteases, even if protein extraction was carried out under acidic conditions, could make bbi undetectable in activity assays. studies are in progress to overcome these drawbacks. the resistance of bbi to the acidic conditions and digestive enzymes of the upper gastrointestinal tract make these proteins very interesting candidates for evaluation as chemopreventive agents, in modulating cell viability and tumor progression within the gastrointestinal tract. a single amino acid change in a chymotrypsin prevents plant proteinase inhibitor binding plants have evolved economical strategies to combat insects, which on one hand involves the production of multi-domain pis that can target multiple enzymes with different specificities and on the other, pis that belong to structurally distinct families. solanaceous plants, produce both type i and type ii families of pis, which specifically target serine peptidases. this study showed that type i pis are better inhibitors of a particular class of chymotrypsins within the gut of helicoverpa species that is otherwise unaffected by the type ii class of inhibitors. homology models were used to identify a single amino acid substitution in the helicoverpa chymotrypsin that was likely to confer resistance to the type ii inhibitor. our hypothesis was further supported by recombinant expression and mutagenesis of this single amino acid in the type ii inhibitor-resistant chymotrypsin. we therefore propose that both type i and ii inhibitors are required to protect plants against lepidopteran insects. mobility of the sulphate protamin/ low molecular weight heparin complexes in an electrical field glycosaminoglycans low molecular weight heparin (lmwh) activated plasma serine proteases inhibitors. serine proteases play an important role in thrombogenesis, the process that leads to blood clotting and such as heart attack, stroke and other cardiovascular disorders. lmwh has been used to temporarily render the blood incoagulable during prophylaxis or treatment of thrombosis and sometimes result in serious bleedings and for the heparin anticoagulant activity neutralization used sulphate protamin. it was investigated relationship between new lmwh-sk derivatives (were generated through the controlled cleavage of porcine intestinal mucosa heparin with a mixture of chitinolytic complex from streptomyces kurssanovii) anticoagulant activities and lmwh-sk complexes with sulphate protamin mobility in an electrical field. with this purpose used biospecific electrophoresis in 1% agarose with protamin sulphate. precipitation zones (zones of the equivalent) in the ''rocket'' form were generated. scanning image was saved as jpg format. the ''rocket'' squares estimated with the help of bandscan program. results: lmwh-sk with molecular mass (mm) 14.0; 5.8; 5.4; 4.7; 4.0; 3.4 kd demonstrated antithrombin activities (aiia) 85-264 iu/mg, activities against factor xa (axa) has made 100-278 iu/mg, axa/aiia ratio -(0.8-2.2). correlation coefficients between mm and precipitation zone heights or squares consist 0.56-0.73 (p < 0.05), between axa activities and precipitation zone heights or squares consist 0.37-0.54 (p < 0.05). conclusion: lmwh-sk was obtained with the chitinolytic comlex hydrolisis help has ratio axa/aiia-2,2, it is necessary for antithrombotic preparations. with the mm decrease axa activity increase and precipitation zone heights or squares of the lmwh-sk complexes with sulphate protamin decrease. the role of extracellular proteases in supplying filamentous fungi with nutrient compounds is well understood and experi-mentally documented. however there is no definite answer on the question on the need and role of these proteases in pathogenesis. the study of differences in the spectra of extracellular enzymes of saprotrophic and pathogenic fungi performed on fusarium species revealed that activity of secreted serine proteinases of pathogenic f. culmorum strain was much higher (up to 20-fold) than that of saprotrophic strain. the use of f. culmorum strains differing in pathogenicity (strongly and weakly pathogenic) demonstrated that activity of secreted serine proteases of strongly pathogenic strain was significantly higher (1.5-8-fold) than that of weakly pathogenic strain. this tendency was preserved in calculations of activity towards protein content and dry weight of mycelium indicating on purposeful synthesis and secretion of extracellular proteases by strains with high pathogenicity. at that these differences were much higher when the substrate for trypsin-like proteinases bz-arg-pna was used than in the case of substrate for subtilisin-like proteinases glp-ala-ala-leu-pna. according to the data obtained it is proposed that the value activity of trypsin-like proteinases secreted by the fungi correlated with the degree of their pathogenicity and plays, apparently, an important role in pathogenesis. acknowledgment: this work was supported by grants from the russian foundation for basic research. conformational adaptation of a canonical protease inhibitor upon its binding to the target protease increases specificity atomic resolution crystal structure of sgti in complex with crayfish trypsin provided further data on the molecular basis of the inhibition mechanism of pacifastin type inhibitors. in complex with crayfish trypsin, sgti exhibits more or less continuous contacts in an extended region (through sites p 12 -p 5' ) of the molecule. the comparison of this complex with a simulated bovine trypsin-sgti one shows that more than half of the interaction energy surplus is originated from the extended region of binding. some of these contacts result from a conformational change of sgti that was induced by its binding to the enzyme which is strongly supported by the critical comparison of the crystal structure of crayfish trypsin-sgti complex with the free form of sgti. alignment of the nmr structure ensemble with the x-ray structure of complexed sgti and a careful comparison of the backbone j, w angles were carried out. additionally, noe-derived restraints and corresponding distances in the complex are also compared. local conformation of both p 12 -p 4 and p 4 '-p 5 ' regions of the inhibitor shows significant changes upon binding suggesting that either or both of these regions may act as molecular recognition sites. this comprehensive analysis of the local backbone properties of sgti in the free and in the complex form made possible to identify conformational similarities and differences responsible for its efficient binding to the enzyme, and provides a good basis for further studying the structural aspects of protease inhibitor specificity. as most of serine proteases enteropeptidase light chain contains four disulfide bonds and one nonpaired cysteine at 122 (chymotrypsinogen-derived residue numbering) position which forms disulfide bond linking the pro-and catalytic domains. a mutant of human enteropeptidase light chain cys122ser was constructed by site-directed mutagenesis. the recombinant wild type and mutant proteins were produced in escherichia coli bl21(de3) with expression vector pet-32a. the active proteins were obtained after solubilization and renaturation of the fusion protein thioredoxin/human enteropeptidase light chain from inclusion bodies. after autocatalytic cleavage of thioredoxin the active enzyme was purified on agarose linked soybean trypsin inhibitor. the yield of refolded active enzyme increased from 1.87 to 7.84% in case of cys122ser mutant. the wild type and c122s mutant showed similar kinetic parameters for cleavage of small synthetic substrate gly-asp-asp-asp-asp-lys-naphthylamide, small ester thiobenzyl benzyloxi-carbonil-l-lysinate (z-lys-sbzl) and fusion protein cleavage. both enzymes were inhibited by trypsin-like serine proteases inhibitors but not inhibitors of chymotrypsin-like, cysteineor metallo-proteinases. recombinant human enteropeptidase light chain and its mutant c122s were active between ph 6 and 9 with a broad optimum at about ph 7.54 and demonstrated quite high stability to different denaturating agents. both enzymes demonstrated secondary specificity to chromogenic substrate z-ala-phe-arg-na with km = 0.067 mm, kcat = 23 s-1. proteinaceous low molecular serine protease inhibitors from wood rotting fungi k. j. grzywnowicz and j. zuchowski biochemistry department, maria curie-sklodowska university, lublin, poland. e-mail: grzyw@hermes.umcs.lublin.pl proteolytic enzymes have been firmly established as main regulatory components in a number of cellular and physiological processes. the most important factors influencing the proteolytic enzymes are natural, proteinaceous protease inhibitors, which form complexes with target proteases. they have been extensively investigated from the points of view on physiological functions, as tools for protease enzymology, models for protein-protein interactions and on potential medical applications. there is growing interest in new inhibitors of proteases from various sources. among known protease inhibitors from fungi are, yeasts inhibitors of proteinases a (asparagine protease) and b (serine protease), and low molecular inhibitors of serine proteinases from fruiting bodies of mushrooms -pleurotus ostreatus and lentinus edodes as well as some undefined proteinase inhibitory activities from water extracts of some species of basidiomycetes. searching for new, bioactive metabolites of basidiomycetous fungi we isolated and characterized recently some low molecular, proteinaceous, natural inhibitors of serine proteases, from mycelia of wood rotting fungi -trametes versicolor, abortiporus biennis and schizophyllum communae. isolation of inhibitors was achieved by ion exchange and size exclusion chromatography. preliminary characterization of their inhibitory activity (against some serine proteases), ph and temperature optima of action, and molecular mass, were classically analyzed. analysis of n-terminal amino acid sequences of these inhibitors suggests a new family of serine protease inhibitors from fungi. more detailed characterization of inhibitors (including molecular modeling) and preliminary experiments with laboratory animals and with lines of human cells are in progress. the role of serine proteases in the lectin pathway of complement activation p. ga´l 1 , g. ambrus 1 , v. harmat 2 , b. ve´gh 1 , g. na´ray-szabo´2, r. b. sim 3 and p. za´vodszky 1 1 institute of enzymology, hungarian academy of sciences, budapest, hungary, 2 protein modeling group, hungarian academy of sciences, budapest, hungary, 3 department of biochemistry, university of oxford, oxford, uk. e-mail: gal@enzim.hu the complement system is a cascade of serine proteases, and mediates essential functions during infection as a part of the innate immunity. activation of the complement system culminates in the destruction and clearance of invading microorganisms and damaged or altered host cells. our view about the complement system has changed considerably in the recent years, due to the discovery of a new activation pathway of complement: the lectin pathway. we have recombinantly expressed and characterized the mannose-binding lectin associated serine proteases: masp-1 and masp-2. these are related mosaic serine proteases with similar domain organization but with different enzymatic properties. we showed that masp-2 is capable of autoactivation and it can cleave c2 and c4 complement subcomponents. masp-2, therefore, can initiate the complement cascade without the contribution of any other protease. we demonstrated that the complement control protein (ccp) modules, which associate directly with the serine protease domain, stabilize the structure of the catalytic region masp-2 and contain exosites for the large protein substrates. these results are in agreement with the crystal structures of activated and zymogen forms of masp-2. masp-1 is the most abundant mbl-associated serine protease but it cannot activate the complement system. we demonstrated that masp-1 has a more relaxed substrate specificity compared to masp-2 and the activity of both proteases can be blocked by c1-inhibtor. we concluded that the two mbl-associated serine proteases participate in evolutionary and functionally different pathways. comparative kinetic study on s2' trypsin variants l. gombos, j. tó th, p. medveczky, a. ma´lna´si csizmadia and l. szila´gyi laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, . e-mail: gl@ludens.elte.hu by far the most serine proteases have a glycine in position 193, which is part of the s2' subsite (the second subsite on the enzyme surface c-terminal from the scissile bond of the substrate). in contrast, human trypsin 4, the trypsin isoform expressed in human brain, possesses an arginine in that position. the bulky side chain of this amino acid is responsible for the inhibitor resistance, the most striking feature of this isoform, as it interferes with the binding of polypeptide inhibitors to the enzyme surface. a chimpanzee typsin also has an arginine193, while rat trypsin v bears a tyrosine in that position. there is also a snake venom plasminogen activator, a trypsin type serine protease, that contains an s2' phenilalanine. we created glycine, arginine, tyrosine and phenilalanine s2' variants of human and rat trypsins by site directed mutagenesis in order to investigate the effect of these amino acids on the kinetic behaviour. on small chromogenic substrates and synthetic inhibitors, which do not interact with the s2' residue, there is no signifi-cant difference between the various mutants in catalytic efficiency and inhibitory constants, respectively. however, on oligopeptide substrates the catalytic efficiency decreases 20-50-fold in the nonglycine variants. this effect is even more dramatic with polypeptide partners: the catalytic efficiency drops 200-500 times while inhibitory constants increase by 3-5 orders of magnitude. we conclude that the catalytic mechanism is not fundamentally influenced by the substitution of residue 193, although this amino acid is part of the oxyanion hole. bulky residues in the s2' subsite hinder mainly the binding to interaction partners. structural studies on masp-2: towards the understanding of the mechanism of autoactivation mannose-binding lectin-associated serine protease 2 (masp-2), is the key enzyme of the lectin activation pathway of complement, a major element of innate immunity. a dimer of masp-2 complexed with mannose-binding lectin (mbl) is able to perform its biological functions: upon recognition of the pathogen by mbl masp-2 undergoes autoactivation, and then initializes the complement cascade by cleaving c2 and c4. masp-2 is a mosaic protein containing a chymotrypsin like serine protease domain (sp) and further domains with binding sites of mbl or substrates. our present study focuses on the structural background of the ability of the zymogen form of masp-2 to undergo autoactivation. we solved the structures of catalytic fragment of masp-2 both in its zymogen and activated forms. comparison of the two structures reveals characteristic conformational differences in the classical activation domain and in some other loops lining the substrate binding region. loop 1 shows a unique conformation with arg192 blocking the s1 pocket. we docked the activation loop of masp-2 in the active site of the active enzyme and built a model of the complex of the active and zymogen forms. the model reveals extended regions of molecular recognition. while this model represents the second step of autoactivation (active form cleaves zymogen), the first step (zymogen cleaves zymogen) requires the stabilization of the zymogen enzyme in active-like conformation. we built a model of a zymogen-zymogen complex. favorable and unfavorable contacts of the two zymogen molecules help us to identify possible molecular switches, as well as contact regions stabilizing an active-like conformation of the zymogen enzyme in the complex. the deg/htra proteases are atp-independent serine endopeptidases which are present in most organisms, including bacteria, humans and plants. previous work in our laboratory has shown that the deg2 protease of the model plant arabidopsis thaliana selectively degrades the photodamaged d1 protein in the reaction center of photosystem ii (psii) in vitro. therefore, deg2 is thought to catalyze the primary cleavage of photodamaged d1 protein, which is an important step of the repair mechanism that restores functional psii. our present studies aim to elucidate the regulation of the deg2 protease activity, especially with regard to its d1 degrading activity. we found deg2 associated to the stromal side of the thylakoid membranes and as a soluble protein in the chloroplast stroma. the amount and distribution of deg2 protein remained unchanged after exposure to different light intensities, which suggest either a substrate regulation or a posttranslational regulation of the d1 degrading activity of deg2. recent advances on deg2 regulation and complex formation will be presented. novel peptide inhibitors of human kallikrein 2 (hk2) human kallikrein 2 (hk2) is a serine protease produced by the secretory epithelial cells in the prostate. it activates several other proteases that may participate in the proteolytic cascade mediating metastasis of cancer. thus, modulation of hk2 activity is a potential way of preventing tumor growth and metastasis. furthermore, specific ligands for hk2 may be potentially useful for targeting and imaging of prostate cancer. we used enzymatically active recombinant hk2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. six different peptides binding to hk2 were identified using libraries expressing 10 or 11 amino acids long linear peptides. three of these peptides were specific and efficient inhibitors of the enzymatic activity of hk2. alanine substitution analysis revealed that motifs of 5-7 amino acid determined the inhibitory activity of the peptides. the peptides are also of potential utility for development of immunopeptidometric assays for hk2, which is promising marker for diagnosis of prostate cancer. furthermore, these peptides are potentially useful for treatment and targeting of prostate cancer. the mechanism of autoactivation of the zymogen masp-2 residues on the surface of pathogens. we managed to recombinantly express and purify two forms of zymogen masp-2. one form is the wild type zymogen enzyme, which can be activated, while the other one is a stable zymogen mutant form of masp-2. we could prepare the zymogen form of wild type masp-2 under certain conditions which enabled us to examine the kinetics of activation. we demonstrated that activation of masp-2 is a true autocatalytic activation without the involvement of any other protease. we characterized the enzymatic properties of zymogen masp-2 using the stable zymogen form. we demonstrated that zymogen masp-2 cannot cleave small synthetic substrates but it can cleave large protein substrate (c4). a molecular model for the interaction between zymogen and activated masp-2 during activation has also been built based on the available 3d structures of zymogen and activated masp-2. influence of streptokinase on the fibrinolytic system proteins the present study is dedicated to the investigation of the effect of protein by bacterial origin -streptokinase (sk) on the activity and interaction regulation mechanisms of fibrinolytic system proteins. the study was carried out with use of porcine haemostasis system which plasminogen isn't activated by sk. especially we were interested in study of the changing fibrinolytic system parameters such as tissue type plasminogen activator (t-pa), plasminogen activator inhibitor (pai-1), plasminogen, a2-antiplasmin activities. also the main parameters of coagulation system such as fibrinogen, soluble fibrin, fibrin degradation products levels and thrombin activity and quantity were studied. it was used affinity chromatography, electrophoresis, western-blotting, elisa, determination of proteins activity. it has been determined an increased consumption of plasminogen on 15% in 4 h after streptokinase injection. it was shown that activity and concentration of t-pa were significantly increased in 3.5 times in 1 h. on the next stages of investigation this parameters tend to norm. after sk injection pai-1 quantity was increased in two times (16.7 ng/ml compared to normal 8.9 ng/ml). the interesting fact was the activation of prothrombin by sk without activation of coagulation system in vivo. the injection of sk causes the significant increase of t-pa activity and quantity possibly due to direct or/and indirect effect on endothelial cells. we can conclude that sk causes pai-1 secretion due to effect on platelets as 90% of pai-1 storage is in a-granules of platelets. thus analysis of the data displayed besides of well-known sk function the influence of sk on the changing of fibrinolytic system potential possibly due to its effect on endothelial cells and platelets. paracrystalline inclusions in the mitochondrial matrix or intermembrane compartment occur in several biochemically unrelated disorders such as myopathies, paragangliomas and steatohepatitis, and in various cell types under normal conditions, as well. however, little is known about the composition of the inclusions, the mechanism of their formation and their relation to disease processes. in this study we have described the helix-shaped structures in the intracristal compartments of rat liver mitochondria that have undergone ca 2+ -induced permeability transition. the filaments are anchored in opposing parts of the mitochondrial membranes and appear to support the cristae mechanically. a protein, that apparently is a component of these helical filaments, has been identified as serine protease lactb. this protein shows close sequence similarity to the class c bacterial beta-lactamases and is the only member of this class in animals. since lactb has not been studied previously we cloned its cdna for expression in e. coli as c-terminal his-tagged fusion protein. lactb underwent proteolytic processing in both e. coli and in isolated mitochondria resulting in several protein fragments. this is likely to be due to autocleavage and may be an activation/maturation process. 2d blue native gel electrophoresis indicated that lactb was part of a >600 kda protein supercomplex. in summary, the presence of the serine protease motive in lactb and its supposed ability to form helical filaments suggest that lactb might function not only as a component of 'mitoskeleton' in maintaining and rearranging the mitochondrial ultrastructure under certain conditions, but also might take part in apoptotic processes. novel psychrophylic trypsin-type protease from serratia proteomaculans proteinase with trypsin specificity from psychrophylic microorganism serratia proteomaculans was partly purified. it was shown that the properties of this enzyme (temperature and ph-stability, efficiency of substrate hydrolysis) correspond with the psychrophylic character. inhibitor analysis and study of substrate specificity indicate that this enzyme is serine trypsin-type protease. at the same time this enzyme is zinc-dependent. proteases of such type were unknown till now. secondary specificity of the studied enzyme differs from the bovine trypsin specificity -this protease hydrolyses the short substrates more efficient. zinc, cadmium (ii) and copper (ii) ions in mmolar concentrations inhibit the enzyme activity. the unusual character of calcium ions influence on substrate hydrolysis and inhibition by the bovine pancreatic trypsin inhibitor (bpti) was registered for the studied enzyme. in vitro by a neutral to basic ph change [2, 3] . the kinetics of the activation process can be followed by stopped flow fluorescence (sff) experiments while the structural features of the transition can be explored by in silico molecular dynamics (md) and targeted molecular dynamics (tmd) [4] simulations. to challenge the activation process, mutants were constructed and studied by sff measurements. subsequently, on these mutants multiple md/tmd simulations were carried out. our results indicate the existence of parallel activation pathways. they demonstrate the absolute necessity of multiple simulations and of proper statistics. they reveal the pros and cons of the tmd method. a simple method for the purification of a novel serine endoprotease from wheat triticum aestivum (cv. giza 164) has been developed. it consists of ion-exchange and gel filtration. the molecular mass of the enzyme was 58 kda by sds/page under reducing conditions and 57 kda by gel filtration on a sepharose 6b column. the enzyme had isoelectric point and ph optimum at 4.2 and 4.5, respectively. the substrate specificity of the enzyme was studied by the use of synthesized and natural substrates, azocasein, azoalbumin, hemoglobin, casein, gelatin and egg albumin. the enzyme appears to prefer azocasein with km 2 mg azocasein/ml. the enzyme had a temperature optimum at 50°c with heat stability up to 40°c. while co 2+ and mg 2+ accelerated the enzyme activity by 54 and 56%, respectively, ca 2+ and ni 2+ had very little effect. the enzyme was strongly inhibited by phenylmethylsulphonyl fluoride (pmsf), but not by the other protease inhibitors, suggesting that the enzyme is a serine protease. from the results it can be concluded from the characterization that the t. aestivum serine protease may be suitable for food processing. in vitro effects of a potent, selective dipeptidyl peptidase ii (dppii) inhibitor in leukocytes and u937-cells. the compound was able to penetrate the cell membrane and proved efficacy without evidence for acute cellular toxicity. there was a dosedependent inhibition of intracellular dppii activity without affecting the dppiv activity (maximal efficacy at 100 nm). these properties enable to differentiate between dppii and dppiv in biological systems and allow further investigation of the physiological function of dppii. in a second step, we have been investigating the involvement of dppii in apoptosis in human leukocytes by using this compound. preliminar results based on annexin v-/pi-staining using up to 1 lm inhibitor in u937-cells and pbmc did not show signs of apoptosis while dppii activity was inhibited for 90%. effect of calcium ions on hydrolysis of peptide substrates of general formula a-(asp/glu) n -lys(arg)-b, catalyzed by enteropeptidase (ec 3.4.21.9), differs depending on substrate type. for specific enteropeptidase substrates (n = 4) calcium ion exhibits the promotion of hydrolysis by the natural two-chain enteropeptidase. hydrolysis of atypical enteropeptidase substrates (n = 1-2) is as a rule less efficient; in addition calcium ion shows in this case the inhibition influence. therefore the regulation of the nondesirable side-hydrolysis during full-length enteropeptidase-catalyzed chimeric proteins processing is possible by means of calcium ions. on the contrary the hydrolysis of substrates of all type (n = 1-4) by enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 or 784-800 fragments) is inhibited by calcium ions. hydrolysis of the natural enteropeptidase substrate, trypsinogen, is at least two orders of magnitude more efficient than any artificial substrate hydrolysis. we propose that this effect is caused by participation in trypsinogen coordination with enzyme of the addition secondary substrate binding site and/or calcium-binding site; both sites located on the n-terminal half (118-465) of the enteropeptidase heavy chain. one more mechanism of the regulation of the enteropeptidase activity by calcium ion is the unusual calciumdependent autolysis of the enteropeptidase heavy chain leading to the drastic loss of its activity towards trypsinogen. autolysis of enteropeptidase heavy chain and well-known autolysis of trypsin were compared; the second one serves as the natural defense mechanism against the undesirable premature proenzymes activation in pancreas leading to pancreatitis. the corresponding enteropeptidase inactivation in low ca 2+ ion environment might be the component of the same protective mechanism. b3-034p human trypsin 4 selectively cleaves myelin basic protein: is this brain protease involved in the pathomechanism of multiple sclerosis? demyelination, the breakdown of the major membrane protein of the central nervous system, myelin is involved in many neurodegenerative diseases. proteases participating in this process are potential targets of therapy in neurodegenerative diseases. in the present in vitro study the proteolytic actions of calpain, human trypsin 1 and human trypsin 4 (the product of gene prss3) were compared on lipid-bound and free human myelin basic protein as substrates. digestions only with calpain and human trypsin 4 actions may be of some physiological or pathological relevance, since these two are expressed in human brain. the fragments formed were identified by using n-terminal amino acid sequencing and mass spectrometry. the analysis of the degradation products showed that human trypsin 4 of these three proteases cleaved myelin basic protein most specifically. it selectively cleaves the arg80-thr81 and arg98-thr99 peptide bonds in the lipid bound form of human myelin basic protein. based on this information we synthesized region 94-104 of myelin basic protein, peptide ivtprtpppsq that contains the specific trypsin 4 cleavage site arg98-thr99. in vitro studies on the hydrolysis of this synthetic peptide by trypsin 4 confirmed our results with intact myelin basic protein. what lends some biological interest to the above finding is that the major autoantibodies found in patients with multiple sclerosis recognize sequence 80-96 of the protein. our results suggest that human trypsin 4 may be one of the candidate proteases involved in the pathomechanism of multiple sclerosis. enteropeptidase is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of its activation peptide following the sequence asp-asp-asp-asp-lys. its light chain alone is sufficient for an effective cleavage of fusion proteins with trypsinogen activation peptide analog. human enzyme possesses 10-fold specificity coefficient compare to bovine one, and an explanation of this fact can contribute a lot to the attempts of improving or modulating enzymatic properties. highly pure and active recombinant human enteropeptidase light chain (l-hep) was obtained by renaturation from inclusion bodies expressed in escherichia coli cells and the active l-hep was purified on agarose-linked soybean trypsin inhibitor. enzymatic activity of purified l-hep was studied through the cleavage of the synthetic peptide substrates and several fusion proteins. l-hep associated with soybean trypsin inhibitor slowly and z-lys-sbzl cleavage was inhibited with ki* = 2.3 nm. comparison of l-hep and bovine enteropeptidase inhibition by bovine trypsin inhibitor aprotinin has shown almost an order difference in ki*. ph dependence of the enzyme activity was measured and ph optimum point was found to be 7.54. enteropeptidase light chain amino acid sequence and crystal structure were analyzed for the presence of target regions for mono-and bivalent ions. unlike trypsin with predicted and experimentally proved calcium-binding sites and sodium-activated thrombin, l-hep was predicted to be deprived of any of such sites and an influence of these ions on the cleavage of different substrates was found to be confined primarily to a substrate binding. as a continuation of our efforts to fully elucidate the antisnake venom properties of mucuna pruriens and to further understand the molecular changes that occurred in mouse plasma proteome as a result of in vivo challenge test with venom and mucuna pruriens proteins (mpe), two dimensional polyacrylamide gel electrophoresis was done. plasma was pooled and gels were run in triplicate to eliminate both biological and experimental variations. analysis using imagemaster 2d platinum software and other statistical analysis tools showed significant differences in protein expression between all the treatments and the control group. some proteins were down regulated, some up-regulated, some completely disappeared while new protein spots were identified. the protein expression of plasma of mouse immunized with mpe for 3 weeks before challenge with lethal dose of venom and that injected with venom alone was more complex. some venom proteins like ecarin are serine proteases that activate clotting factors like prothrombin, causing haemorrhage and disseminated intravascular coagulation, on the other hand, the protease inhibitors from mucuna pruriens must have acted to antagonize these effects by direct proteolysis (cleavage products/spots appearing in the protein map) or other immunological mechanisms. the results obtained represents the first proteomics approach in studying all the plasma proteins involved in this phenomenon. we have only concentrated on protein spots showing interesting variations with respect to control. it is also an important step in the identification of the affected proteins, the kind of modifications/molecular mechanisms involved which is likely the basis of the in vivo protection the plant extract showed against the venom. the use of enzymes at low temperatures has great potential in terms of lower energy costs, therapeutic applications and to lower microbial contamination in industrial processes. low temperature proteases (cryophilic -or psycrophilic -proteases) are of particular interest for detergents and as wound debriding agents. at present, we are studying cryophilic proteases from antarctic krill (euphausia superba), which normally lives in the sea at temperatures near 0°c. we have isolated several low temperature proteases by chromatography. enzyme activities and stability were characterized at low temperatures and as a function of ph to find optimum conditions for different applications. a particular enzyme, named kt1, showed particularly high specific activity at 20°c, several times that of commercial preparations of proteases such as subtilisins. this protein showed a high degree of similarity with digestive trypsins isolated from various arthropoda species. using mrna molecules obtained from abdominal sections of e. superba and subsequently subjected to a reverse-transcription reaction, we identified, isolated and sequenced a dna molecule that codes for an inactive zymogen of the enzyme. cloning of this dna sequence in escherichia coli strains allowed the recombinant expression of the zymogen, followed by purification and activation of the zymogen, which lead to an active cryophilic trypsin. we performed a homology modeling procedure that conducted us to obtain a molecular model of the mature enzyme. the 3d model thus obtained was refined using energy minimization, hydrogen network optimization and residue-residue contact optimization techniques, leading to a reliable model of the enzyme. we used this model to identify many interesting and novel features of the enzyme molecule that could be related with its cryophilic character, and to propose site-directed mutagenesis strategies that could be used to improve the enzyme performance at low temperatures, its ph-activity profile, specificity, inactivation resistance and recombinant expression. in addition, the 3d model allowed us to design and experimentally obtain mutants that are resistant to auto-degradation and more readily activated. molecular cloning and expression of lactba mitochondrial serine protease mitochondria are thought to have originated from a symbiotic relationship between a bacterium able to perform aerobic metabolism ant the ancestor of eukaryotic cells. lactb is the only mammalian protein showing sequence similarity to bacterial serine proteases and belongs to c class b-lactamases. mouse lactb is 551 amino acids long and compromises a predicted mitochondrial import sequence, a short putative transmembrane segment, a b-lactamase homology domain containing the serine protease motif, -sxxk-, and a c-terminal d-transpeptidase domain. the physiological role of mammalian lactb is unclear. therefore, the purpose of this research work was to clone the gene of lactb for expression of lactb in e. coli for further biochemical and cell biological study. the full length lactb gene was cloned into the entry plasmid pentr/sd/d-topo. expression clones were created performing a recombination reaction between the entry clone and four destination vectors. expression constructs resulting in n-or c-terminal gst fusion protein and in n-or c-terminal his6-tag fusion protein were transformed into bl21 (de3) competent cells which are designed for use with bacteriophage t7 promoter based expression systems. when lactb was expressed as an n-terminal gst fusion protein, full-length lactb protein was recovered by glutathione-agarose affinity chromatography. expression of lactb as a c-terminal gst fusion protein or with either an n-or c-terminal his6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length lactb. these results show that the n-terminal gst fragment protects lactb from proteolytic processing and that lactb can undergo autoproteolysis, which may be a part of a physiological maturation or activation process. design and synthesis of retro-binding peptides active site inhibitors of thrombin thrombin is an important pharmaceutical target for the treatment and prevention arterial and venous thrombosis. biological active peptides are recognized to have significant therapevtic potential but serious limitations especially for oral dosing. the peptide stereomers could differ when forming productive complexes with an enzyme. moreover, the replacement of l-amino acid residues forming the hydrolyzed p1-p'1 bond by their enantiomers is known to result in either an uncleavable or a very slowly hydrolyzed analogue. this phenomenon is often used for the synthesis of the peptide's inhibitors stable to the degradation by the enzymes of organism. as the peptides containing d-amino acids, nor are subject to an enzymatic hydrolysis, the purpose of researches was synthesis of a retro -d-analogues of thrombin's substrates constructed from d-amino acids. the di-and tripeptides of the general formula x-d-arg-d-phe-ome [where x = z, tos, ac h, and z-d-arg-d-ala-(d), l-phe-ome (otbu)] were synthesized by conventional methods of peptide synthesis in solution. special features of their interaction with thrombin are investigated. their inhibitory action on reaction of splitting of fibrinogen by thrombin and on reaction of a hydrolysis by thrombin baee showed, that their inactivating action depends on the substituent on n-end of dipeptides and configuration of phenylalanine in a molecule of tripeptides. the relationship between structure and inhibitory action of the synthesized peptides is discussed. the successful application of d-amino acids for designing of biologically active peptide's analogues as a potential medicinal agent, steady to enzymatic degradation is shown. substrate specificity of mannose lectin binding associated serine proteinase 3 n. s. quinsey and r. n. pike department of biochemistry and molecular biology, monash university, melbourne, victoria australia. e-mail: noelene.quinsey@med.monash.edu.au the innate complement system is involved with the neutralization of pathogenic microorganisms. it plays a comparative role to that of the classic immune complement cascade. in the innate complement system, the oligomers of mannose lectins are able to bind to microorganisms. these oligomers have been shown to have mannose lectin binding serine proteinase (masps) attached, which once activated lead to the activation of the c3 convertase complex, which finally leads to the formation of the membrane attack complex. there have been three active masps identified in the human innate immune system-masp-1, masp-2 and masp-3. there is high homology between these three serine proteases especially in the n-terserpins are protease inhibitors that present their reactive site loop (rsl) to target proteases, followed by drastic conformational changes that inactivate the protease. the sequence of the rsl of serpins determines the target specificity. the drosophila melanogaster gene spn4 encodes multiple serpin isoforms each containing an individual rsl, thus enabling the attack of different proteases. variant spn4a contains a consensus recognition/cleavage sequence of furin within its rsl and is equipped with a signal peptide and an endoplasmic reticulum (er) retrieval signal (hdel). this suggested that the protein resides in the secretory pathway, like furin, a proprotein convertase that activates many cellular proteins and pathogens. our experiments demonstrate that spn4a forms sds-stable complexes with human furin that is inhibited with a second order rate constant of 5.5 · 10 6 /m/s. the rsl of spn4a is cleaved c-terminally to arg-arg-lys-arg, in accord with the enzyme's cleavage site. furthermore, the serpin is retained in the er of transfected cos7 cells as shown by immunofluorescence staining. a hdel deletion mutant was detected mainly in the medium of trans-fected cos7 cells, demonstrating the necessity of the hdel signal for the observed cellular localization. further experiments show that furin 1 and 2 of drosophila melanogaster are physiological targets for spn4a, since secreted forms of both enzymes form stable complexes with the serpin. together, the results demonstrate that spn4a is a potent inhibitor of furin that may meet the target at its natural location. experiments with the other rsl variants show that the spn4 gene represents a multipurpose weapon that is directed against different families of proteases. formation of the covalent tetrahedral complex (tc) with substrate is the first step of the catalytic process in the active site of serine proteases. his57 (chymotrypsin numbering) plays a role of a general base catalyst, activating the ser195 nucleophile by abstraction of its proton. it was experimentally observed that the pka of his57 ne in tc formed by serine proteases with transition state analog inhibitors is about 5 units higher than the corresponding pka in the free enzyme. this work demonstrates that the environmental change of the his57 in tc, induced by the substrate binding in the enzyme active site, is the dominant factor in the pka increase of his57 ne, and triggers the enzymatic processing of the substrate. these results are based on quantum mechanical modeling of the active site of free chymotrypsin and tc complex of chymotrypsin with trifluoromethyl ketone inhibitor in dft b3lyp/6-31+g** level of theory. the polar environment of the enzyme active site is accounted for explicitly in the microscopic model. the combined environmental effects of the bulk water solvation and the rest of the protein is implicitly accounted for by our scrf(vs) continuous solvation approach. the role of local polar effects, such as the oxyanion and the asp102-his57 hydrogen bond, on the pka of his57 ne in tc is analyzed. genome-wide analysis of subtilase (subtilisinlike serine protease) genes in microbial genomes limited to regions surrounding the asp, his and ser catalytic residues. pattern-searching methods using hidden markov models, based on conserved sequences surrounding the catalytic residues, were used to search for subtilases encoded in >200 bacterial and archaeal genomes, representing 177 species. more than 350 subtilases were found to be encoded in 109 genomes. subtilases are more commonly found in grampositive bacteria than in archaea or gram-negative bacteria, and it is more common to have multiple subtilase-encoding genes than a single gene. the majority of the subtilases have a predicted signal peptide for translocation across the cell membrane, and a sub-group of these secreted subtilases are predicted to have a carboxy-terminal cell-envelope anchor, mainly of the lpxtg type for covalent anchoring to peptidoglycan. the genomic context of the subtilase-encoding genes was analyzed to gain insight in putative functions for these proteolytic enzymes. by also taking into account the predicted intracellular or extracellular location of the encoded subtilases, it was possible to predict a function for many subtilases in either nutrition/growth, spore germination, surface protein processing/activation, bacteriocin/toxin processing, or sigma factor activation/regulation. the poisoning by botropics species makes a similar physiologic, one of systemic effects is the blood coagulation for several mechanisms, as direct action on fibrinogen; factor x activation or platelet activation, by toxins of venoms. in the last years were identifies in botropics venoms, serine proteases. this toxins are responsible by coagulant activity with direct action on fibrinogen. serine proteases are utility for hemostatic system studies and for therapeutics use. looking for new molecules models is very important to show the mechanism of action and search structural characteristics responsible for its activities. the present work has the objective of purification and characterization of a coagulant factor (cf) from b. pirajai venom. the purification was made using a gel filtration, hydrophobic chromatography and an affinity chromatography. the molecular filtration was made in sephadex g-75 with ammonium bicarbonate buffer (ambic) 0.05 m ph 8.1, resulting four fractions (p1-p4), the coagulant fraction was named p1. the p1 fraction was submitted in phenyl sepharose chromatography using triz buffer 10 mm ph 8.6 in a decreasing gradient of nacl (4; 3; 2; 1; 0.5; 0 m), and to finish the chromatography it was used distilled water, resulting six subfractions (fp1-fp6), the coagulant subfraction was named fp1. the fp1 sub fraction was submitted in benzamidine sepharose chromatography and eluted in the solutions: distilled water, obtained the subfraction bfp1, sodium phosphate buffer 20 mm ph 7.8, obtained the subfraction bfp2 and glycine buffer 20 mm ph 3.2, obtained the sub fraction bfp3 that is the cf. the cf displayed one band in sds-page (11%) showing a pure protein, it has 58 kda, the minim coagulant dose is 1.75 lg and has action on fibrinogen beta chain. the genome of arabidopis thaliana encodes 16 putative proteases from the deg/htra family. this group of atp-independent serine-proteases was well examined in other organisms, especially e. coli and humans, but only limited data is available for members from this protease family in plants. deg1 and deg2 have been shown to act as proteases in the chloroplast, but no deg/htra proteases from other compartments have been examined so far. the putative protease deg15 is predicted to be localized in the peroxisome. we cloned the gene encoding deg15 (at1g28320) in an overexpression vector for heterologous expression in e. coli. the tagged protein was purified by affinity chromatography and used to raise polyclonal antibodies. with these antibodies we investigated the intracellular localization of deg15 and the protein level under various stress conditions in order to evaluate the in planta function of this protein. the effect of site-directed mutagenesis on cold adaptation of vpr; a subtilisin-like serine proteinase from a psychrophilic vibrio-species psychrophilic enzymes have very similar 3d structures as their homologous enzymes from mesophilic and thermophilic organisms. main characteristics of enzymes from psychrophiles are their high catalytic efficiency (kcat/km values) and thermolability. a subtilisin-like serine proteinase from a psychrophilic vibrio-species (vpr) shows these characteristics when compared to homologous enzymes from mesophilic and thermophilic organisms. the vpr gene was cloned, sequenced and expressed in e. coli and recently the crystal structure was determined at 1.84 å resolution [1] . structural comparisons have been carried out which have led to hypotheses about some of the structural factors which may contribute to cold adaptation of vpr. some of these hypotheses have been examined using site-directed mutagenesis. the specific residue exchanges were selected with the objective to incorporate stabilizing interactions into the cold adapted enzyme which were deemed to be present in related thermostable homologues. these include incorporation of pro into loops, a new potential salt-bridge, as well as substitutions aimed at improving packing in the hydrophobic core and decreasing apolar exposed surface. we have also introduced ser to ala substitutions at three different locations in the cold-adapted enzyme, but these were the most frequent amino acid exchanges observed in sequence comparisons of the enzyme to those of more thermostable homologues. here we report on the catalytic and stability characteristics of the selected mutants. engineering of gfp for the screening of serine protease inhibitors site specific proteolysis has been an attractive target for the development of antiviral therapies based on selective viral inhibitors. it has been previously demonstrated that reporter proteins like beta-galactosidase could be very useful for the high-throughput screening of hiv-1 protease inhibitors through the display of an accessible protease target site on the enzyme surface. in this work, by using structural analysis, we have engineered the gfp protein from jellyfish aequorea victoria to accommodate in its surface the hcv virus ns5a-5b protease cleavage site edvvccsmsytwtg, in a manner that proper proteolysis results in a fluorescent activity decrease. the three resulting gfp constructions, carrying the protease cleavage site in positions 23-24, 102-103 and 172-173, were soluble expressed in escherchia coli. moreover, the hcv ns4 cofactor residues 21-34 fused in frame via a short linker to the amino terminus of the hcv ns3 protease domain (residues 2-181) were also expressed in e. coli and under 1mm iptg induction, at least 60% of soluble protein was recovered and further purificated by an histidin tag. the analysis of gfp proteolysis in front of hcv recombinant protease were performed either with bacteria crude extracts and purificated proteins. the results presented here indicated that proper solvent exposure of target sites on gfp carrier protein may be a critical factor for protease cleavage and for the observation of fluorescence activity variance, being an aspect of absolute relevance for further design and implementation of newer analytical tests. various kinds of stressors cause the group of metabolic changes defined as the general stress response, initiated by some intracellular signals, such as production of abnormal or denaturated proteins, enhanced generation of reactive oxygen species and others. proteolytic enzymes quickly modify proteins and as a consequence can regulate cellular metabolism. although the stress defense mechanisms have been very often described in the recent literature, in very few works were estimated stress response abilities of white-rot basidiomycetes, which produce two kinds of very important ligninolytic enzymes -laccase and peroxidases. our previous results showed that the addition of menadione to abortiporus biennis idiophasic cultures caused the significant increase of the extracellular laccase activity in comparison to the control. the aim of this study was to determine activities of serine proteinases and natural serine proteinase inhibitors in idiophasic cultures of basidiomycete a. biennis grown under menadione-mediated oxidative stress conditions. we investigated the changes of intracellular serine proteinases activities in the presence and absence of atp, using hemoglobin and fluorogenic substrates. the level of natural serine proteinase inhibitors in mycelia was also measured. a fungal inhibitor of trypsin was partially purified and used to in vitro experiments. an interesting correlations between serine proteinases, serine proteinase inhibitors and laccase activities in prooxidant treated cultures were also observed. it can suggest that the proteolytic modifications under oxidative stress conditions can act as a regulation way of laccase activity. serine proteinases, inhibitory and laccase activities were additionally analyzed by native page. calpain is a ca 2+ -regulated cytosolic cysteine protease, functioning as a ''modulator protease'', i.e. regulating/modifying functions/activities of substrates by limited proteolysis to modulate cellular functions. human has 14 calpain genes and potential substrates extend to various cytosolic proteins such as kinases, transcription factors, cytoskeletal and er proteins. in skeletal muscles, expression of p94 (also called calpain 3) predominates, playing an indispensable role for muscle functions in cooperation with ubiquitously expressed conventional calpains. for, a defect of p94 proteolytic activity originated from gene mutations causes muscular dystrophy. p94 localizes in myofibrils binding to connectin/titin, a gigantic elastic muscle protein connecting the z-and m-lines of sarcomere, the repetitive unit of myofibril, with a single molecule. in mdm (muscular dystrophy with myositis) mice, connectin/titin with a small deletion caused by natural mutation of the connectin/titin gene is expressed, resulting in severe muscular dystrophy phenotypes such as body weight less than a half of that of wild type, severely affected limb muscles with impaired walking ability and only 2-3 months of life time. the deletion in the mdm allele of the connectin/titin gene overlaps one of the binding sites of p94 in the n2-line, another electron-microscopically visible line between the z-and m-lines of sarcomere. the mdm phenotypes clearly indicate that connectin/ titin or p94 or both are essential for proper muscle functions. to elucidate physiological roles of connectin/titin and p94, we analyzed mdm mice in relation to calpain system. as a result, mar-ps (muscle ankyrin repeat proteins) were shown to be up-regulated in mdm muscle. marps bind to the n2-and z-line regions of connectin/titin and function as transcriptional regulators translocating into the nuclei. carp (cardiac ankyrin repeat protein), one of marps, binding site in the n2-line region is proximate to the p94 binding site, thus suggesting interactions of both molecules. possible signal transduction systems to modulate muscle functions revealed by the analyses will be discussed based on the results. inhibition and activation of calpain by its disordered endogenous inhibitor, calpastatin p. tompa 1 , z. mucsi 2 , o. gyo¨rgy 2 , c. sza´sz 1 and p. friedrich 1 1 institute of enzymology, biological research center, budapest, hungary, 2 research group of peptide chemistry, university of eo¨tvo¨s lora´nd, budapest, hungary. e-mail: tompa@enzim.hu calpains are a family of intracellular calcium-activated cysteine proteinases, implicated in the regulation of key cellular processes, such as cell division and programmed cell death. their activity is under tight control by an intracellular protein inhibitor, calpastatin, an intrinsically unstructured protein that contains four equivalent inhibitory domains. each of these comprise three conserved subdomains, of which subdmomains a and c anchor the inhibitor in a calcium-dependent manner, whereas subdomain b binds at the active site and inhibits the enzyme. in this work it is shown that the consequence of this mode of binding is that isolated a and c peptides promote calcium binding to calpain and thus activate the enzyme. this activation is manifest in the sensitization to calcium ion: the calcium required for half-maximal activity is lowered from 4.3 to 2.4 lm for l-calpain and 250 to 140 lm for mcalpain. in the physiologically significant sub-micromolar and low micromolar calcium concentration range this sensitization leads to a more than tenfold activation, which is of potential physiological importance as isolated calpain requires high calcium concentrations never realized in vivo. here we suggest calpastatin is degraded in vivo in a way that generates the activator peptides. due to the structural disorder of calpastatin, this unprecedented mode of action raises intriguing questions with respect to the generality of this ambivalent behavior. to address this issue, we have collected extreme cases, when the same protein elicits opposing, inhibitory and activatory, responses within the same molecular setting: structural predictions show that these proteins are largely disordered. as a conclusion, the possible general implications of this finding are discussed. meprins are oligomeric, brush border membrane or secreted zinc proteases that have unique and complex structures. they are composed of multidomain, highly glycosylated evolutionarily-related a and b subunits that form disulfide-linked homo-or heterooligomeric dimers. the homooligomeric form of meprin a forms very high molecular mass multimers of 1 000 000-6 000 000 da, among the largest extracellular proteolytic complexes known. meprins cleave cytokines, growth factors, bioactive peptides and extracellular matrix proteins, important compounds in inflammatory intestinal disease and in cancer metastases. to investigate the role of meprins in intestinal immune responses, inflammation was induced in mice by oral administration of dextran sulfate sodium (dss). the results showed that wild-type mice (c57bl/6 · 129) had a more severe reaction to dss than meprin b null mice on the same genetic background, as determined by body weight loss, intestinal bleeding and mortality. this implies that the presence of meprin b increases host damage caused by dss and that meprin b plays an active role in intestinal pathophysiology. meprins are also expressed in colon cancer cells (e.g. sw480, sw620, and caco-2). expression of meprin a appears to increase with increasing metastatic potential. in addition, meprin a is highly expressed in the human liver hepatoblastoma cell line hepg2 and abundantly secreted into culture media. examination of human tumor samples showed that meprin a is expressed in primary colon tumors and in tumors that have metastasized to the liver. this indicates that meprin a expression in gastrointestinal tumor cells contributes to the progression of the disease. biochemical pathways mediating necrotic cell death and neurodegeneration in caenorhabditis elegans n. tavernarakis, p. syntichaki, c. samara and k. troulinaki institute of molecular biology and biotechnology, foundation for research and technology, heraklion, crete greece. e-mail: tavernarakis@imbb.forth.gr necrotic cell death plays a central role in devastating human pathologies such as stroke and neurodegenerative diseases. elucidation of the molecular events that transpire during necrotic cell death in simple animal models should provide insights into the basic biology of inappropriate neuronal death, and facilitate the characterization of mechanisms underlying degeneration in numerous human disorders. various cellular insults, including hyperactivation of ion channels, expression of human beta-amyloid protein implicated in alzheimer's disease, constitutive activation of certain g proteins, hypoxia and possibly the ageing process, can trigger a degenerative, necrotic cell death in the nematode caenorhabditis elegans. we are genetically and molecularly deciphering the c. elegans necrotic death program. we have isolated mutations in several distinct genetic loci that bock degenerative cell death initiated by various genetic and environmental insults. by characterizing such suppressors, we have discovered that neuronal degeneration inflicted by various genetic lesions in c. elegans, requires the activity of specific calcium-regulated calpain proteases and acidic ph-dependent aspartyl proteases. although, it is believed that these proteases become activated under conditions that inflict necrotic cell death, the factors that govern the erroneous activation of such-otherwise benign-enzymes are largely unknown. we identified novel factors that modulate cellular ph homeostasis, which are required for necrosis and showed that targeting these factors effectively protects from necrotic cell death in c. elegans. our findings demonstrate that two distinct classes of proteases are involved in necrotic cell death and suggest that perturbation of intracellular calcium levels may initiate neuronal degeneration by compromising ph homeostasis and deregulating proteolysis. search for regulatory proteins which are controlled by proteolysis in escherichia coli based on microarray analysis j. m. heuveling ag hengge, institute of microbiology, fu berlin, berlin, germany. e-mail: joheuvel@zedat.fu-berlin.de the impact of controlled proteolysis on regulatory events in prokaryotes is increasingly recognized over the last decade. as in eukaryotic cells, proteolysis is more than just a garbage disposal but has been found to be implicated in the regulation of many vital functions of the bacterial cells, like cell cycle, stress responses and development (hengge r and bukau b. mol microbiol 2003) . conditional degradation of regulators shows a high potential of integrating a great variety of signals as is well studied for the degradation of sigma s. this sigma subunit of the rna polymerase, which triggers the general stress response in escherichia coli is digested rapidly by the clpxp protease in association with the phosphorylated response regulator rssb under non-stress conditions (stuedemann a. embo 2004). several other regulatory proteins have been found to be subjected to proteolysis, as lexa, a regulator of the sos response. also lon protease is involved for example in the degradation of rcsa, a regulator of the capsule biosynthesis and of sula, a cell division inhibitor (as a review: hengge-aronis r, jenal u. curr opin microbiol 2003). in order to find other regulatory processes in which proteolysis plays a role we pursued a global approach using the microarray technique. in mutants lacking functional clpp or lon proteases or either one of the clp recognition factors clpa and clpx, we searched for genes, which are differentially transcribed compared to the wildtype. we found some interesting groups of genes belonging to common regulons governed by known regulators -candidates for clp or lon mediated proteolysis. after confirmation of these results through lacz fusion studies of representative genes of these regulons, these regulators are presently examined in in vivo degradation studies using immunodetection methods. a distinct group of serine peptidases cannot hydrolyze proteins, but can readily cleave peptides that are up to about 30 amino acid residues long. the representative member of the family, prolyl oligopeptidase is implicated in a variety of disorders of the central nervous system. the enzyme consists of a peptidase domain with an a/b-hydrolase fold and its catalytic triad is covered by the central tunnel of a seven-bladed b-propeller. this domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. in most propeller domains the circular structure is ''velcroed'' together in a mixed blade, where both amino and carboxy terminus are involved to form a four stranded antiparallel b-sheet. non-velcroed or ''open topology'' propellers are rare, and prolyl oligopeptidase was the first protein structure exhibiting a domain of this nature. the apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart or the peptidase and/or propeller domains move to create an entry site at the domain interface. engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. this indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action. biochemical characterization of thermoplasma volcanium recombinant 20s proteasome and its regulatory subunit g. baydar and s. kocabiyik molecular genetics, biological sciences, middle east technical university, ankara, turkey. e-mail: gozde_baydar@hotmail.com proteasome associated energy dependent proteolysis is not only involved in rapid turnover of specific proteins that could be important during periods of stress, but also engaged in the turnover of the short-lived proteins that regulate a variety of cellular processes in both procaryotic and eucaryotic cell. the universal distribution of proteasome homologs in archaeal genome provide insight into the vital role of archaeal proteasomes. 20s catalytic core of archaeal proteasomes in combination with various aaa atpases and membrane associated lon proteases may play role in stress response or turnover of the regulatory proteins. however, little is known about the potential physiological roles of archaeal proteasomes. this study presents the data on biochemical and biophysical features of recombinant 20s proteasome of a thermoacidophilic archaeon thermoplasma volcanium (tpv). pcr was performed to amplify dna fragments containing tpv genes encoding the a -and b-subunits of the proteasome from tpv genomic dna. the amplified a-gene (tpva) and b-gene (tpvb) together with their upstream sequences were separately cloned and then combined in puc18 vector. the resulting recombinant puc-skba plasmid was used for heterologous production of in vivo assembled 20s proteasome in e. coli. the recombinant proteasome was purified by combination of ammonium sulfate precipitation, gel filtration chromatography (sepharyl s-300) and ion-exchange chromatography (q sepharose). molecular masses of purified protein subunits were estimated as 23.71 kda (b-subunit) and 21.13 kda (a-subunit). substantial post-glutamyl peptide hydrolyzing activity and chymotrysin-like activity were detected as associated with recombinant proteasome. maximum chymotrypsin-like activity was measured at 85°c and ph 8.5. crystallographic studies of the gtp-dependent transcriptional regulator cody from bacillus subtilis. cody is a gtp dependent transcriptional regulator of early stationary phase and sporulation genes in bacillus subtilis. it is activated by gtp, during rapid cell growth it represses several genes whose products allow adaptation to nutrient depletion. when the cells pass from rapid growth to stationary phase, the intracellular concentration of gtp drops thus releasing the repressed genes. cod y is a 259-residue polypeptide containing a helix-turn-helix motif for binding to dna. it also has motifs common with small gtpases, but cody has a much lower affinity for gtp. crystals of the full-length cody have been grown in the presence and absence of gtp from sodium citrate buffered solutions using lithium sulphate as a precipitant and diffraction data have been collected to 3.5 å resolution. attempts to solve the structure using anomalous data from the semet derivative crystals of cody have been hampered by the large number (70) of methionines in the asymmetric unit and difficulties in reproducibility of angiotensin-converting enzyme (ace) is a zinc metallopeptidase critical for the generation of the vasoconstrictor peptide angiotensin ii. a homologue of ace, ace-2, has recently been identified, which appears to play a counter-regulatory role to ace by inactivating angiotensin ii. like ace, ace-2 is a type i membrane protein with its active site contained within the extracellular domain. the expression of ace2 protein is normally low and restricted primarily to endothelial cells of the heart and kidney, kidney epithelium and testis. recent evidence from ourselves and others indicates that ace2 is significantly upregulated in a number of pathologies, such as myocardial infarction, renal disease and hepatitis c-induced cirrhosis. given that ace can be proteolytically released from the cell surface in culture, ace2 may likewise be shed into plasma or urine. detection of elevated levels of ace2 in plasma and urine may be a useful biomarker for the diagnosis of hepatic, renal and vascular disease. using a specific quenched fluorescent substrate, we have detected ace2 activity in human urine. in contrast, ace2 activity could not be detected in human plasma; interestingly, however, we noted that plasma markedly inhibited the activity of recombinant ace2, thus compromising the possibility of measuring plasma enzyme activity. we are in the process of purifying this inhibitor, which preliminary results suggest is small and hydrophilic. we are also currently optimizing methods for its removal from plasma samples, thus allowing detection of low levels of soluble ace2 activity in normal human plasma. the identification of a potential endogenous inhibitor of ace2, the first for this family of metallopeptidases, could have significant consequences for ace2 function in vivo and the regulation of angiotensin peptides. future studies will examine whether plasma or urinary levels of ace2 are elevated in cardiovascular, renal or liver disease. molecular determinants of proteolytic processing of non-structural polyprotein of semliki forest virus. institute of molecular and cell biology, university of tartu, tartu, estonia. e-mail: lulla@ut.ee semliki forest virus (sfv) is a positive-stranded rna virus. the replication of sfv is performed by the rna-dependent rna replicase complex (rc) and regulated by proteolytic processing. during the course of the infection template preference of rc changes from rna plus-strand to minus-strand. it has been known for several years that this preference switch is due to the proteolytic processing of sfv non-structural polyprotein p1234, mediated by viral cysteine protease located in the carboxy-terminal domain of the nsp2 protein. tight temporal regulation of this template specificity switch is crucial for the viral replication, but, nevertheless, its mechanism remains unsolved. therefore, the mapping of the essential molecular determinants of the site-specific cleavage consensuses may provide necessary information, concerning the cleavage regulation as well as regulation of the rna replication. the results of our studies indicate that as little as 5 amino acid residues from the c terminus of nsp3 protein determine the specificity of the proteolytic cleavage of the nsp3/nsp4 junction. at the same time sequences laying downstream of the cleavage point (in nsp4 region) have only minor effect on the cleavage efficiency. the exact region required for the cleavage of nsp2/nsp3 junction is yet not known but the sequences, required from c-terminal part of nsp2 protein, are likely short as well. in contrast, sequence lying within 80-240 n-terminal amino acid residues of nsp3 is vital for cleavage of the nsp2/nsp3 junction. this region may represent the cofactor of the nsp2 protease that activates processing at the nsp2/nsp3 cleavage site. thus, as the result of current research, a principally new function -regulation of the proteolytic processing and rna replication -was mapped to the conserved n-terminal region of the nsp3. this finding significantly improves our understanding about the role of nsp3, which was enigmatic till now, in the virus life cycle. activation occurs within the specific dibasic motif hsiirrsl, suggesting the involvement of the proprotein convertases (pcs) in these process. this family of endoproteases are responsible for the activation of a large variety of regulatory proteins by cleavage at multi-basic recognition sites exhibiting the general motif (k/r)-(x)n-(k/r)(n = 0, 2, 4 or 6). cotransfection of the furindeficient colon carcinoma cell line lovo with provegf-c and different pc members revealed that furin, pc5 and pc7 are vegf-c convertases. the processing of provegf-c is blocked by the inhibitory prosegments of furin, pc5 and pace4, as well as by furin-motif variants of alpha2-macroglobulin and alpha1antitrypsin. accordingly, mutation of the vegf-c pc-site (hsiirrsl to hsiisssl) inhibited provegf-c processing. following zebrafish caudal fin amputation, the injection of control vector or vector containing wild vegf-c did not affect fin regeneration. in contrast, injection of muted vegf-c (pro-vegf-c) inhibited fin regeneration. these data highlight the importance of vegf-c processing in zebrafish fin regeneration and suggest that zebrafish can be used as a simple and useful model for studying the role of protein maturation by the pcs in physiological processes. thimet oligopeptidase (top) hydrolyzes a variety of bioactive peptides and is implicated in the regulation of neurological and other physiological processes. top is composed of two ''clamshell'' domains, with the substrate-binding pocket and catalytic site lying between these domains. it is speculated that conformational changes in loops and coil regions connecting the domains lead to changes in substrate specificity. the loop region (residues 599-611) is close enough to the active site to interact with even the smallest substrate. it contains three glycine residues and is expected to be quite flexible. in an effort to trap intermediate conformations of the loop, we have replaced gly 599, 603, or 604 with ala and have compared the activities of the three resulting protein constructs towards two quenched fluorescent substrates. all three enzymes had lower activity than wild type towards a bradykinin analog, with g599a, the most active of the mutants, possessing 1/3 wild-type activity. however, utilizing a smaller substrate, g603a was the most active, surpassing even wild type (fivefold increase in activity). g604a had little activity towards either substrate. these results are consistent with data that revealed increases in activity towards the larger substrate, when the enzyme is partially denatured and presumably, more flexible and with increased accessibility of the binding loop to proteolytic enzymes, when partially denatured. acknowledgment: this work was supported by hhmi, nih-ns39892 (mjg). the proteasome: paradigm of a self-compartmentalizing protease self-processing of subunits of the proteasome crystal structures of the rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly handbook of metalloproteins bovine chymotrypsinogen-a x-ray crystal-structure analysis and refinement of a new crystal form at 1.8 a resolution equilibrium and rate constants for the interconversion of two conformations of a-chymotrypsin. the existence of a catalytically inactive conformation at neutral ph refolding transition of alpha-chymotrypsin -ph and salt dependence e-mail: saleh38@hotmail.com reference 1. arnorsdottir j, kristjansson mm, ficner r. crystal structure of a subtilisin-like serine proteinase from a psychrotrophic vibrio species reveals structural aspects of cold adaptation (which is an excellent substrate for adam-17), we observed 35% increase in enzymatic activity in the media of 1 mm 5-ht-treated cells compared to untreated cells. however, we did not see any increase in the fluorescence when we used ''cate1'', an adam substrate, which does not recognize adam-17. to further support a role for adam-17/tace, we designed silencing rnas against the enzyme, which were introduced into the mesangial cells using lentiviral infection. successful silencing was confirmed by western blotting 4 days after infection. control and tace silenced human mesangial cells were stimulated with 2-10 lm of serotonin for 5 min, and erk activation was assessed by western blotting baron-ruppert 2 and e. heymann 1 1 department of physiological chemistry (fb8/gw) e-mail: rebecca.lew@med.monash.edu.au b4-015p functional properties of p94/calpain3 and connectin/titin in mdm mouse skeletal muscle y bunkyo-ku the lectin pathway of complement system is an important component of the innate immunity. it provides the first line of defence against infection, since it is activated on the surface of invading pathogens. the activation of the complement system results in the destruction and clearance of foreign microorganisms. mannose-binding lectin-associated serine protease-2 (masp-2) is the enzyme which is responsible for the initiation of the lectin pathway of complement activation. masp-2 is a multidomain serine protease, which is synthesized as an inactive zymogen and become activated upon mbl binds to carbohydrate plant proteinase inhibitors are widely spread in the different plant species being a significant component of a defense system. somewhere a significant diversity of the proteins related to the same structural family of the inhibitors in the same species may be observed. the family of potato kunitz-type proteinase inhibitors (pkpis) exemplifies a group of proteins with the diverse properties and may be divided into three major homology groups: a, b and c. a lot of genes encoding different pkpiproteins of each group were found in various potato cultivars (solanum tuberosum l.). inhibition activity of plant invertase, cysteine and serine proteinase was found in proteins subgroup c. a set of gene copies were isolated by pcr from potato cv. istrinskii genome. dna sequencing analysis of these resulted in identification of 24 different dna sequences with a high similarity to potato kunitz-type inhibitors of group c (pkpi-c). cluster analysis demonstrated that this clones represented multiple copies of six new genes denoted as pkpi-c1, -c2, -c3, -c4, -c5 and c6. it can be supposed that at least two alleles containing pkpi-c genes are harbored in tetraploid genome of potato. one of new genes, namely pkpi-c5, exhibited 99% identity with known invertase inhibitor cdna (1423) from cv. provita. another pkpi-c6 gene was similar (98% identical residues) with cdna (p340) from potato cv. bintje encoding for a putative trypsine inhibitor. four other new genes demonstrated as much as 89-92% identity with known pkpi-c proteins from other potato cultivars. the n-terminal sequence of the protein encoded by the pkpi-c2 gene was identical to the n-terminal sequence of specific subtilisin inhibitor pksi isolated from cv. istrinskii.b3-049p regional distribution of human trypsinogen 4 in human brain determined at mrna and protein level j. to´th 1 , l. gombos 1 , e. siklo´di 1 , p. ne´meth 2 , m. palkovits 3 , l. szila´gyi 1 and l. gra´f 1 1 laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, hungary, 2 institute of immunology and biotechnology, university of pe´cs, pe´cs, hungary, 3 laboratory of neuromorphology, department of anatomy, semmelweis university, budapest, hungary. e-mail: july@ludens.elte.huproteases play an important role in many physiological and pathological processes in the central nervous system such as development, neurite outgrowth, neuronal plasticity and degeneration and cell signaling. a gene coding for such an enzyme might be prss3 on chromosome 9 of the human genome. it encodes due to alternative splicing both mesotrypsinogen, which is expressed in pancreas, and trypsinogen 4 whose mrna has been identified in different human tissues (initially in brain, recently in different epithelial cell lines from prostate, colon and airway). analysis of the gene prss3 predicted two isoforms of the zymogen: isoform a may have a 72 amino acid, while isoform b a 28 amino acid n-terminal leader sequence. in order to gain information on the possible role of human trypsinogen 4 we have determined its amount at the mrna and the protein level as well in 17 selected brain areas using real-time quantitative pcr and elisa.the highest transcript levels could be detected in cerebellar cortex, while low amounts were found, e.g. in cerebellar white matter samples. the distribution of the mrna in different brain areas measured by real-time pcr is consistent with the protein levels detected with elisa. the usage of different monoclonal antibodies specific for the 28 amino acid leader sequence and the protease domain allowed the separate detection of the zymogen and the active enzyme. in e.g. the hypothalamus the zymogen is the dominant form, while a significant degree of activation was found in the cerebellar cortex. our data indicate that the extent of activation varies with different areas. as human trypsinogen 4 is ubiquitous in the brain we conclude that it might play a role in general neurological processes. serine proteases are enzyme involved in the maintenance of the cell homeostasis. thus, this type of enzymes must be extremely regulated and it has been highly reported that serine proteases are involved in the growth and expansion of different cancers. in this regard, the type ii transmembrane serine proteases (ttsps) constitute a subfamily of membrane anchored serine proteases that are ideally positioned to carry out different interactions with other cell surface or extracellular proteins. among them, tmprss2 and tmprss3 proteins have been reported to be overexpressed in most prostate and ovarian cancers respectively, matriptase/mt-sp1 is expressed in a wide variety of benign and malignant tumors and hepsin is overexpressed in ovarian and renal cancers. desc-1 is a ttsp member found differentially expressed in squamous cell carcinoma (differentially expressed in squamous cell carcinoma gene 1) and differentially from other ttsps, its expression is found to be reduced in tumor tissues respecting to the normal tissue at rna level in head and neck squamous cell carcinoma (hnscc), what suggests a possible tumor protective function for desc-1. in order to shed light about the role of desc-1 in these processes, we have carried out the molecular cloning of the human full-length cdna and expression of the recombinant protein to delineate the implication of this protease in hnscc.diffracting crystals. therefore we used limited proteolysis and mass-spectrometry analysis to identify the domain boundaries of the protein and were able to determine the sequence of two principal proteolytic fragments corresponding to the n-and c-terminal domains of cody. these individual domains which were successfully cloned in escherichia coli, overexpressed as histagged proteins, isolated and purified. both domains have been crystallized. the crystals of the n-terminal domain grow from bis-tris buffered solutions at ph 6.5 containing polyethylene glycol and calcium acetate. the crystals of the c-terminal domain were obtained using ammonium sulphate as a precipitant. the crystals of n-terminus domain diffract to at least 2.3 å and crystals of c-terminus domain -to 3.2 å using an in-house diffractometer with a mar research image-plate as a detector. progress towards the determination of cody structure will be presented. the gram-positive bacterium listeria monocytogenes is a facultative intracellular parasite. interactions of l. monocytogenes with the host cell are provided by a number of secreted and cell surface proteins. one of the most important virulence factors, actinpolymerizing protein acta, is surface attached via the hydrophobic c-tailed membrane anchor. despite, the membrane anchor acta was found in comparable amounts both on the cell surface and in the culture supernatant. the aim of the work was to investigate the mechanism of acta release and the role of this process in l. monocytogenes virulence. maldi-tof ms analysis of trypsin released acta suggested releasing due to proteolytic cleavage between histidine and threonine residues in the close vicinity of the membrane anchor predicted by the htmm analysis. the substitution of histidine with proline prevented acta release into the culture supernatant, although did not disturb its surface presentation. in silico analysis of eight other l. monocytogenes membrane-anchored surface proteins suggested the role for asparagine and threonine residues in specific proteolysis. the prediction was experimentally tested by substitution of the residues with alanine. the l. monocytogenes spontaneous mutant strain, unable to release membrane-anchored proteins into the culture supernatant, was isolated. the mutation was mapped outside the acta gene and presumably affected the corresponding peptidase. the mutation impaired the invasion of l. monocytogenes into the human epithelial-like hela cells that suggested the effect of the released proteins on signaling events that result in induced phagocytosis of the pathogen by normally non-phagocytic cells. angiotensin ii (ang ii) has been proposed to act as a regulatory peptide in the epidermal layer of human skin. while the expression of receptors and peptide precursors have been demonstrated in epidermis, the formation of ang ii and its inactivation have not been studied in detail. thus we have established a model system with cultured keratinocytes to examine the metabolism of ang i, ii and related peptides by intact epidermal cells. cultures were incubated with peptides in a minimal medium, which sustained cell viability for at least 24 h and the metabolism of peptides was monitored by chromatography (rp-hplc). with ang i as peptide substrate five major products were detected in keratinocyte culture media after 12 h incubation. a half-life of about 9 h was estimated for ang i and the slow degradation supports results of earlier studies revealing low activities of exopeptidases in a microsomal fraction from keratinocytes as compared to fibroblasts. the degradation of ang i was not affected by inhibitors of alanyl aminopeptidase, peptidyl dipeptidase a and neprilysin. since a peptide product formed from ang i in keratinocyte cultures resembled ang ii in hplc analysis, the activity of peptidyl dipeptidase a in these cells was assayed with hip-his-leu and the presence of the peptidase was confirmed by its sensitivity to captopril. further experiments showed that ang ii, iii and related peptides were degraded in keratinocyte cultures with rates similar to ang i and these reactions interfered severely with the formation of ang ii. immunohistochemical studies showed a strong positive staining for neprilysin and alanyl aminopeptidase in the dermal layer of human skin and at the epidermal-dermal junction confirming the results obtained with the cell cultures. soluble angiotensin converting enzyme-2 present in human plasma and urine p94/calpain 3 is the skeletal-muscle-specific calpain and is considered to be a modulator protease in various cellular processes. a defect in the p94 gene causes limb-girdle muscular dystrophy type 2a (lgmd2a), suggesting that p94 functions are indispensable for proper muscle functions. in sarcomeres, p94 localizes at z-, n2-and m line regions. although the binding partner for p94 at z-line has not been identified yet, n2-and m-line localization of p94 are considered dependent on its interaction with the n2a and m-line regions of connectin/titin, respectively. connectin is a gigantic sarcomeric protein playing an important role as a molecular template for sarcomeric organization, an elastic element generating passive tension, a platform for various protein ligands, etc. in this study, we focused on the molecular components associated with the n2a region of connectin/titin to extend our understanding on p94. intriguingly, a recessive mutation in the mouse connectin gene, mdm (muscular dystrophy with myositis), causes muscular dystrophy. there are two remarkable phenotypes consequential to mdm mutation. first, the mdm mutation abolishes p94 binding activity of connectin n2a fragment. second, in skeletal muscle from mice homozygous for mdm mutation, upregulation of cardiac ankyrin repeat protein (carp) is observed. carp also binds to n2a connectin at the n-terminal proximity of the region mutated by mdm. the effect of mdm mutation on p94 activity and the properties of n2a connectin as well as carp were analyzed using both animal model and cell culture systems. semliki forest virus (sfv) is well known model virus, which has been studied for decades. the main topic of this research was characterization and analysis of sfv replication machinery using approach based on use conditional-lethal mutants of viruses. the direct aim of the present study was to sequence and functionally characterize a panel of independent sfv temperature sensitive mutants. from all putative ts-mutations, identified in this study, two were mapped to nsp1 protein, four were mapped to nsp2 protein and one was founded in nsp4 region. number of assays were used to verify phenotypic effects of revealed mutations: titration of virus stocks at different temperatures, leak yield experiments, analysis of viral rna synthesis and viral polyprotein processing at different temperatures. nsp2 mutants had clear viral protease defect and accumulated non-cleaved polyproteins on different stages. besides all, biotechnological branch of our research is already developing. it includes improving of existing sfv based expression vector system by use of ts-mutations for the temperature regulation of foreign gene expression in mammalian cells. dipeptidyl peptidase iv activity and/or structure homologues (dash) in brain tumors pathogenesis of many diseases, including cancer, often involves improper proteolytic post-translational modification of biologically active peptides. association of dysregulated expression pattern of novel group of ''dipeptidyl peptidase (dpp)-iv activity and/or structure homologues'' (dash) with cancer development and progression has been suggested by several authors, including us [1] . dpp-iv enzymatic action as a common attribute of most of dash members modifies signaling potential of their substrates, biologically active peptides, not only quantitatively, but due to the changes in their receptor preferences also qualitatively. in this study, we have investigated expression (by real time rt-pcr and immunohistochemistry) and enzymatic activity (by biochemical assays and enzyme histochemistry) of plasma membrane localized dash members, in particular dpp-iv, fibroblast activation protein-alpha (fap) and attractin in human gliomas. it was revealed that varying quantities of dpp-iv, fap and attractin mrnas and proteins were coexpressed in the studied tumors. the majority of dpp-iv-like activity in the glioma tissue could be attributed to the canonical dpp-iv. this activity, assayed biochemically and expressed per mg of protein, was increased in high grade gliomas. inhibition studies suggested lack of enzymatically active attractin in the examined glioma tissues. the results of our pilot study demonstrate for the first time that both enzymatically active and inactive dash molecules are coexpressed in gliomas and suggest prevailing association of increased dpp-iv activity with high grade tumors. acknowledgment: this work was supported by iga nr/8105-3 and msmt 0021620808 vegf-c is involved in the neovascularization processes, steps essential for wound healing, cancer progression and many other physiological functions. zebrafish vegf-c processing and key: cord-284711-l1za83w1 authors: anand, sudhir title: human security and universal health insurance date: 2011-08-30 journal: lancet doi: 10.1016/s0140-6736(11)61148-3 sha: doc_id: 284711 cord_uid: l1za83w1 nan human security is a multidimensional concept that has been a cornerstone of japanese development cooperation for more than a decade. at the heart of security is the idea of protection or insurance against downside risk. 1,2 three distinct questions arise from the concept of human security. first, protection of what? second, insurance against what? and, third, security for whom? the fi rst question relates to the specifi cation of what is to be protected. the defi nition of human security off ered by the commission on human security 3 is: "to protect the vital core of all human lives in ways that enhance human freedoms and human fulfi lment". the core of a person's life is closely concerned with the person's wellbeing and agency, which is best viewed in terms of his or her "capability" to achieve alternative "beings and doings". 4 in this context, health assumes central importance for two reasons: it is directly constitutive of a person's wellbeing; and it enables a person to function as an agent-that is, to pursue the various goals and projects in life that he or she has reason to value. this view deploys the notion of well-functioning, but it is not grounded in notions of economic welfare that are based on utility or income. it is, rather, an agency-centred view of a person, for whom ill health restricts the scope of human agency. since our ability to do things typically depends on our being alive, the capability to lead a long and healthy life must itself be regarded as a basic capability. the second question related to human security is insurance against what. here the concern is to insure against falling below an adequate threshold of human capabilities-in the case of a person's health, a minimum acceptable level. the probability of falling below a minimum threshold depends on both how vulnerable a person is-the degree of downside risk the person facesand how much above the threshold he or she is in the relevant dimension. 1 the extreme case of insecurity is certainty of being below a specifi ed threshold, and the absence of any chance of avoiding that fate. threats to human security can arise, for example, from natural disasters and environmental catastrophessuch as the 2011 earthquake and tsunami in japan, and the consequent leakage of radioactive material from the fukushima daiichi nuclear plant. they can arise from disease outbreaks such as hiv/aids, severe acute respiratory syndrome and drug-resistant tuberculosis; from personal accidents and illness; from economic downturns as in the asian fi nancial crisis of 1997-98; and from various other hazards that people face. 3, 5, 6 the vulnerability of a person to such risks will depend on his or her individual circumstances-including location, epidemiological environment, health status, and economic position. a person's health is aff ected by health care and various other determinants-eg, socioeconomic, behavioural, continuing commitment. otherwise, we risk alienating patients and damaging the therapeutic alliance. as part of the eradication eff ort, who requested that countries and laboratories either destroy the remainder of their stocks of variola, the causative agent of occupational, and dietary. but access to appropriate health care is also a vital factor in protecting a person from the risk of ill health, and especially of catastrophic ill health. comprehensive health care is thus important both in promotion of health and in response to health crises. without health insurance, a severe medical crisis that threatens survival, for example, can have disastrous fi nancial implications-that can aff ect human security in many other dimensions. the third question concerns security for whom-the entire population or a subset of it? universalism can be defended through a variety of diff erent approaches, which all invoke equity, fairness, or impartiality in some form or other. for instance, we can appeal to impartiality through the device of rawls's "veil of ignorance" in the "original position". 7 behind the veil of ignorance, i do not know who i will turn out to be and what serious illness or health threat i might encounter, which could require extensive medical attention. given this uncertainty, the institutional arrangement for health care i am likely to favour is one that ensures comprehensive coverage for all. the concept of human security has wide reach and includes multiple concerns. a major concern is protection of people's health, for which comprehensive health coverage for all is an essential requirement. universal health insurance thus contributes directly to furthering human security. this implication is as valid for japan as for other countries in the world. the tragic events of, and responses to, the earthquake of 2011 are a powerful reminder of japan's concern for human security. in the past, several distinguished politicians, civil servants, and academics in japan have drawn on and developed the concept of human security-including former prime minister keizo obuchi, japan international cooperation agency president sadako ogata, global health expert keizo takemi, and japan center for international exchange president tadashi yamamoto. 3, [8] [9] [10] [11] indeed, universal health coverage in japan, now in existence for 50 years, is indicative of the priority that japan accords to human security. over the decades, japan has also undertaken policies to advance human security in other dimensions, such as basic education, social protection, and economic safety nets. internationally, japan has used the concept of human security to guide assistance to developing countries through bilateral aid and multilateral policies. the range and reach of the idea of human security are extensive, as japanese actions have shown. a central manifestation of these actions is the country's commitment to universal health insurance. department of economics, university of oxford, oxford ox1 3uq, uk sudhir.anand@economics.ox.ac.uk i declare that i have no confl icts of interest. the "impossible patient": organizational response to a clinical problem long-term opioid contract use for chronic pain management in primary care practice: a fi ve year experience contracting for safety with patients: clinical practice and forensic implications contracts between patients and healthcare practitioners for improving patients' adherence to treatment, prevention and health promotion activities limiting exposure to medical malpractice claims and defamatory cyber postings via patient contracts systematic review: treatment agreements and urine drug testing to reduce opioid misuse in patients with chronic pain are no-suicide contracts eff ective in preventing suicide in suicidal patients seen by primary care physicians? partnerships in patient care: a contractual approach a rose by any other name: pain contracts/agreements nonabandonment: a central obligation for physicians the use of contracts in the inpatient treatment of the borderline personality disorder motivational interviewing in health care: helping patients change behavior economic security. paper presented at common security forum conference concepts of human development and poverty: a multidimensional perspective human security now: protecting and empowering people commodities and capabilities why human security? health as a human security priority for the 21st century. human security track iii. the helsinki process a theory of justice opening remarks common security in asia: new concepts of human security common security in asia: new concepts of human security the asian crisis and human security: an intellectual dialogue on building asia's tomorrow key: cord-018639-0g1ov96t authors: kurpiers, laura a.; schulte-herbrüggen, björn; ejotre, imran; reeder, deeann m. title: bushmeat and emerging infectious diseases: lessons from africa date: 2015-09-21 journal: problematic wildlife doi: 10.1007/978-3-319-22246-2_24 sha: doc_id: 18639 cord_uid: 0g1ov96t zoonotic diseases are the main contributor to emerging infectious diseases (eids) and present a major threat to global public health. bushmeat is an important source of protein and income for many african people, but bushmeat-related activities have been linked to numerous eid outbreaks, such as ebola, hiv, and sars. importantly, increasing demand and commercialization of bushmeat is exposing more people to pathogens and facilitating the geographic spread of diseases. to date, these linkages have not been systematically assessed. here we review the literature on bushmeat and eids for sub-saharan africa, summarizing pathogens (viruses, fungi, bacteria, helminths, protozoan, and prions) by bushmeat taxonomic group to provide for the first time a comprehensive overview of the current state of knowledge concerning zoonotic disease transmission from bushmeat into humans. we conclude by drawing lessons that we believe are applicable to other developing and developed regions and highlight areas requiring further research to mitigate disease risk. amplifi er hosts from which spillovers to humans have been documented (childs et al. 2007 ; daszak et al. 2007 ) . not surprisingly, the most devastating pandemics in human history, the black death, spanish infl uenza, and hiv/aids, were all caused by zoonoses from wildlife (morens et al. 2008 ) . zoonotic diseases can spill between animal hosts and humans in a variety of ways, including through (a) shared vectors, such as mosquitoes for malaria, (b) indirect contact, such as exposure to rodent feces in a peridomestic setting, or (c) direct contact with an animal through consumption, animal bites, scratches, body fl uids, tissues, and excrement (wolfe et al. 2005a ) . most pathogens infecting animals fail to make the jump into humans, but 33 % of zoonotic pathogens (~286 out of 868 zoonotic pathogen species studied) that have spilled over are known to be transmissible between humans (taylor et al. 2001 ) . of all eids, zoonotic spillovers from wildlife have been identifi ed as the most signifi cant, growing threat to global health (cleaveland et al. 2007 ; jones et al. 2008 ) . recent evidence highlights the link between infectious diseases and biodiversity loss, land use changes, and habitat fragmentation (cleaveland et al. 2007 ; maganga et al. 2014 ; gottdenker et al. 2014 ) . although additional research on the relationship between habitat degradation and eids is needed, gottdenker et al. ( 2014 ) reviewed 305 studies incorporating a broad variety of diseases and found that the most common land use change types related to zoonotic disease transmission were deforestation, habitat fragmentation, agricultural development, irrigation, and urbanization. functionally, the mechanisms infl uencing disease spillover include disruption of food web structures, changes in host-pathogen interactions, and mixing of pathogen gene pools resulting in increased pathogen genetic diversity (jones et al. 2013 ) . many studies have shown that habitat fragmentation and biodiversity loss correspond to an increase in disease and pathogen abundance and diversity within a host species (allan et al. 2003 ; gillespie et al. 2005 ; keesing et al. 2006 ; salzer et al. 2007 ; cottontail et al. 2009 ; young et al. 2014 ) . specifi cally, the emergence or re-emergence of many zoonotic diseases including yellow fever, lyme disease, hantavirus pulmonary syndrome, nipah virus encephalitis, infl uenza, rabies, malaria, and human african trypanosomiasis have been linked to anthropogenic habitat changes (jones et al. 2013 ) . many of these human environmental changes are occurring in sub-saharan africa where human bushmeat activities have been linked to numerous virulent disease outbreaks, including ebola (leroy et al. 2004a ) , hiv (van heuverswyn and peeters 2007 ) , and monkeypox . pathogen spillover from bushmeat can occur through consumption; however, the main risks are associated with exposure to body fl uids and feces during handling and butchering (kilonzo et al. 2014 ; paige et al. 2014 ) . historically, when a spillover occurred, the likelihood of an epidemic was limited because hunter-gatherer tribes were generally small and widely dispersed, hampering disease transmission between groups of people. once agricultural expansion occurred, human population densities increased, and people became better connected, diseases could spread more easily. as a result, transmissions of infectious diseases from animals to humans have led to devastating outcomes across the globe (lebreton et al. 2006 ) . eids cause hundreds of thousands of deaths annually (bogich et al. 2012 ) . some outbreaks have spread across large regions and became pandemics, costing the global economy tens of billions of dollars (e.g., sars, h5n1, the 2014-2015 west african ebola outbreak) and bringing entire nations to the brink of economic collapse. in this review, we explore the links between bushmeat-related activities and eids in sub-saharan africa, where the vast majority of african emerging infectious zoonotic diseases occur (jones et al. 2008 ) . the recent ebola outbreaks have highlighted the potential role of bushmeat as a source of pathogens, but a comprehensive review of the different pathogens that may emerge from wildlife through bushmeatrelated activities is lacking. although we are in no way suggesting that this issue is more important than other pressing health crises in sub-saharan africa (such as malaria prevention/treatment and improving healthcare infrastructure), we argue that a better assessment of the public health threats associated with this humanwildlife interaction is warranted and necessary to improve management of future disease outbreaks. the term "bushmeat" refers to the meat derived from wild animals for human consumption (milner-gulland and bennett 2003 ) ( fig. 24.1 ). it includes a wide range of animals, such as invertebrates, amphibians, insects, fi sh, reptiles, birds, and mammals, including as many as 500 species in sub-saharan africa (ape alliance 2006 ). although research has focused largely on mammals and, to a lesser extent, birds, theoretically any wildlife species harvested for bushmeat could be a potential source of zoonotic disease that can spillover during the hunting, butchering, and preparation process (wolfe et al. 2000 ; karesh and noble 2009 ) . hunters face risk of injury from live animals, which might allow animal blood to enter the hunter's bloodstream through open wounds. while small animals can be carried in bags, large animals are commonly carried on the shoulder or back, bringing the hunter in close contact with the animal and facilitating transfer of body fl uids (lebreton et al. 2006 ) . the highest risk of disease transmission occurs during the butchering of animals, e.g. skinning, opening of the body cavity, removal of organs, and cutting of meat. more people butcher than hunt animals (83 % and 42 %, respectively, lebreton et al. 2006 ) and butchering involves the use of sharp tools, which may lead to cuts during the process. subramanian ( 2012 ) found that 38 % of respondents cut themselves on a regular basis during butchering. women are especially at risk of disease transmission as they engage more often in butchering and in food preparation than men. in discussing the links between bushmeat and disease, we refer to this all-encompassing suite of risky behaviors as "bushmeat-related activities." nonhuman primates, rodents, and bats have all been linked to the spillover of zoonotic diseases into humans (cleaveland et al. 2007 ; jones et al. 2008 ; kilonzo et al. 2014 ) . a review of the west and central african bushmeat literature including market, offtake, and consumption surveys documented a total of 177 species from 25 orders that were harvested for bushmeat, including 134 (76 %) mammal species, 24 (14 %) bird species, 18 (10 %) reptile species, and 1 (<1 %) amphibian species (taylor et al. 2015 ) . among mammals, the largest group was primates (48 species) including western gorillas ( gorilla gorilla ), bonobos ( pan paniscus ), and common chimpanzees ( pan troglodytes ), followed by ungulates (34 species), carnivores (22 species), and rodents (16 species). in terms of biomass offtake, however, ungulates are generally the most prominent group. although the taylor et al. ( 2015 ) study is very comprehensive, it only included studies that: (1) provided a quantitative measure of bushmeat offtake, consumption, and/or market availability/sales; (2) used non-biased data collection methods and systematically sampled settlements/hunters to prevent selection bias; (3) identifi ed carcasses to the species level; and (4) recorded either the number of carcasses or the total biomass (kg). for a more inclusive and general review of existing central african bushmeat studies, see wilkie and carpenter ( 1999 ) , and for west african studies, see schulte-herbrüggen ( 2011 ). fa et al. ( 2006 ) found that of the approximately one million carcasses traded in the cross-sanaga region of nigeria and cameroon, 99 % were mammals; of which around 40 % were ungulates, 30 % rodents, and nearly 15 % primates. however, as wildlife populations become depleted, such as near urban areas and intensively used agricultural landscapes, smaller bodied mammals comprise a larger share of hunters' offtake (bowen-jones et al. 2003 ; schulte-herbrüggen et al. 2013a ). humans have hunted wild animals for consumption and to protect their crops for millennia (shipman et al. 1981 ; grubb et al. 1998 ; davies et al. 2007 ) , and it remains an important source of food and income security among rural communities today (de merode et al. 2004 ; brashares et al. 2011 ) . bushmeat is an important source of animal protein in many west and central african countries, with up to 90 % of total animal protein consumption coming from wild animals (fa et al. 2003 ) . overall, the contribution of bushmeat to protein and food security is generally lower in urban than rural areas and is highest among remote rural communities . for example, the relative importance of bushmeat in the diet of rural gabonese households ranged from 13 % of total household consumption value in a village near a town to 25 % in a remote community (starkey 2004 ) . similarly, for rural equatorial guinea, allebone-webb ( 2008 ) showed that bushmeat consumption contributed 43 % to total protein consumption in a village with poor transport links, but only 18 % in a village with good connections. in remote cameroonian communities with very few opportunities for purchasing alternative protein sources, bushmeat comprised 80-98 % of animal protein consumption (muchaal and ngandjui 1999 ) . in rural communities with relatively good market access and low levels of bushmeat consumption, the importance of bushmeat for food has been shown to increase seasonally during the agricultural lean season (e.g. the planting season between harvests) when farming households receive little income (dei 1989 , de merode et al. 2004 , schulte-herbrüggen et al. 2013b ) and during the dry season when fi sh is not available (poulsen et al. 2009 ). bushmeat is also an important source of nutrients, especially among children. evidence from rural madagascar shows that removing bushmeat consumption would result in a 29 % increase in the number of children suffering from anemia and triple the cases of anemia among children in the poorest households most hunters sell at least part of their harvest making it an important source of income, especially where alternative income-generating activities are lacking. the importance of bushmeat in household economies varies across sites and individual hunting households, ranging from 38 % to more than 90 % of the total cash income earned (reviewed in schulte-herbrüggen 2011 ). in rural gabon, hunting accounts for up to 72 % of household incomes, with the proportion rising in poorer, more remote communities (starkey 2004 ) . hunters are also more likely to sell large animals and keep small animals for their own consumption, because the latter fetch a lower price per animal and may be less marketable (van vliet and nasi 2008 ) . finally, households facing income shortages during the agricultural lean season and requiring cash income to pay for urgent expenditures, such as hospital bills, are more likely to sell bushmeat than keep it for own consumption (de merode et al. 2004 ) . overall, income from bushmeat sales can be lucrative and compare favorably with alternative work in many rural places. vega et al. ( 2013 ) found that commercial hunters in equatorial guinea generated a mean of us$2000 per year from bushmeat sales. hunters supplying markets in central african logging concessions earned twice the income of junior technicians working at a logging company (tieguhong and zwolinski 2009 ) . rural kenya hunters can earn 2.5 times the average salary in the area (fitzgibbon et al. 1995 ) , and ghanaian hunters can earn income similar to that of a graduate entering wildlife service, and up to 3.5 times the government minimum wage (ntiamoa-baidu 1998 ) . very successful zambian hunters have been reported earning just below the mean annual income in a single hunting trip (brown 2007 ) . the sale of bushmeat historically occurred at a local level, but with increased transportation routes and globalization , the bushmeat trade is expanding to supply urban and international demand. in the past, novel pathogens entering the rural communities may not have spread beyond the community, but this is no longer the case as remote rural areas are connected to urban areas, and increased global trade networks and air travel increases the risk of disease transmission worldwide . this expanding trade network links hunters to consumers, and with many people along this commodity chain coming into contact with bushmeat, the opportunity for disease spillover can occur at many points. for example, the commodity chain supplying bushmeat to an urban market in ghana includes hunters, wholesalers, market traders, restaurant owners, and consumers (mendelson et al. 2003 ) . the bushmeat commodity chain supplying an urban market in democratic republic of the congo is comprised of hunters, porters who carry the meat to the road, the bicycle traders who transport the meat into town, and the market-stall owners who sell the bushmeat to consumers (de merode and cowlishaw 2006 ) . a recent study from ghana estimates that a minimum of 128,000 bats are sold each year through a commodity chain that stretches up to 400 km and involves multiple vendors (kamins et al. 2011a ) . in zambia, mozambique, and malawi, well-developed and complex rural-urban trade supply networks link rural hunters to urban consumers who are willing to pay high prices for bushmeat (barnett 1997 ) . understanding commodity chains is important, as pathogens likely remain viable for some period after an animal is killed. for example, prescott et al. ( 2015 ) demonstrated that ebola virus remains viable on monkey carcasses for at least seven days, with viral rna detectable for 10 weeks. bushmeat has become a multi-million dollar business due to a growing human population and is now serving both subsistence and trade objectives. harvest volumes have been estimated at 12,000 tones per year in the cross-sanaga rivers region of nigeria and cameroon (fa et al. 2006 ) , 120,000 tones per year in côte d'ivoire (caspary 1999 ) , 385,000 tons per year in ghana (ntiamoa-baidu 1998 ) , and at total of 1-4.9 million tons per year in central african forests (wilkie and carpenter 1999 ; fa et al. 2002 ) . however, it is important to recognize that our understanding of the scale of bushmeat harvest is limited by the availability of information and hence current regional harvest estimates might underestimate actual harvest volumes. despite substantial effort in recent years, our knowledge is still site-specifi c and data are lacking from many regions. most surveys have been restricted to relatively small areas or market catchments from which national estimates were extrapolated. research efforts have focused on central africa with some data available for 60 % of countries compared to 30 % of west african countries (taylor et al. 2015 ) . a large number of sites with detailed bushmeat data are concentrated in the cross-sanaga region of nigeria and cameroon, where fa et al. ( 2006 ) collected market data at 86 sites, hence presenting a geographical bias in our understanding of bushmeat harvest . furthermore, the majority of available data samples (79.3 % and 53.6 %, in west and central africa, respectively) identifi ed by taylor et al. ( 2015 ) come from market surveys with poorly defi ned catchment areas, compared to offtake and consumption surveys. strong variation between individual estimates highlights the problems with extrapolation of survey data to national or regional levels and the effects of sampling strategies (hunter versus market surveys), timing of survey (open season versus lean season), survey location, and extrapolation methods. individual fi gures should therefore be treated with caution, but the overall message remains: bushmeat is harvested at an enormous scale exposing those involved in the bushmeat commodity chain to zoonotic diseases. the current scale of bushmeat hunting is primarily the result of socio-demographic changes (wilkie and carpenter 1999 ) . africa's human population has risen from 0.2 billion in 1950 to 0.9 billion in 2013 and is expected to rise to 2.2 billion by 2050 (united nations 2013 ). where alternative sources of animal protein and income are scarce, human population growth has been linked to increasing hunting intensity (brashares et al. 2001 ) . bushmeat has been and remains a staple source of animal protein among the rural poor, yet recent attention has focused on urban consumers of bushmeat as a driver of increased hunting. urban consumers generally have a range of meat sources from which to choose, but value bushmeat for its taste, cultural connotations, and as a luxury food item (fa et al. 2009 ). while urban consumers generally consume less bushmeat than rural consumers , urban populations in africa have increased dramatically from about 15 % of the total population in 1950 to 40 % in 2014 (united nations 2014 ) and have created a strong demand for bushmeat and hence market for rural hunters. the increasing demand for bushmeat has been accompanied by changes in hunting technology and improvements in hunting effi ciency. traditional hunting tools , such as nets and bow and arrow, have been replaced with more modern tools of guns and snares. modern guns have an up to 25-times higher rate of return compared to traditional weapons (wilkie and curran 1991 ) , substantially increasing the ease and cost-effectiveness of hunting (alvard 1995 ) . this enables hunters to catch more animals and sell a larger part of their catch (bowenjones and pendry 1999 ; bowen-jones et al. 2003 ; nasi et al. 2008 ) . hunting effi ciency has also improved as remote forests have become more accessible through the construction of logging roads and improved transportation (wilkie et al. 1992 ; auzel and wilkie 2000 ) . for example, after the construction of 140 km of logging roads in northern congo, the average time for a hunting trip was reduced from 12 to 2 hours (wilkie et al. 2001 ) . development of rural businesses , such as timber companies, attracts workers and their families to remote locations, increasing bushmeat demand, especially when no hunting regulations are in place and alternative protein sources are not provided bennett and gumal 2001 ; poulsen et al. 2009 ). the effect of logging company presence on hunting pressure was documented in gabon where ape populations decreased 50 % between 1983 and 2000 as a result of hunting (walsh et al. 2003 ). in addition, agricultural expansion and mining have exerted a strong force in changing the african landscape and infl uencing human migration patterns (norris et al. 2010 ). due to increased access, people are brought into closer contact with wildlife, which facilitates accessibility to bushmeat hunting and makes transportation of bushmeat from rural to urban areas easier and more cost-effective (wolfe et al. 2005a ) . along with increased ease of transportation comes the opportunity for bushmeat to be traded on the international market. the international trade in bushmeat has recently gained attention as both a driver of bushmeat hunting and the cross-border spread of zoonotic diseases. illegal wildlife trade is the second-largest black market worldwide, involving millions of animals and estimated to be worth us$50-150 billion per year (united nations environment programme 2014 ). case studies at airports screening passenger luggage for bushmeat estimated that approximately 5 tons of bushmeat per week arrive at paris roissy-charles de gaulle airport (chaber et al. 2010 ) and 8.6 tons per year at zurich and geneva airports (falk et al. 2013 ) . as bushmeat hunting, globalization, and human interconnectedness increase, the potential for zoonoses leading to eids also increases. this risk was highlighted when retroviruses (e.g., simian foamy virus) and herpesviruses (cytomegalovirus and lymphocryptovirus) were found in confi scated primates at us airports (smith et al. 2012 ). indisputable evidence of the transmission of pathogens from wildlife to humans exists only for relatively few cases because the standard of proof is very high. nevertheless, the evidence for spillovers is very strong and many pathogens can be classifi ed as very likely to spillover (jones et al. 2008 ; kilonzo et al. 2014 ) . furthermore, countless pathogen species of zoonotic potential will likely be discovered as surveillance increases (taylor et al. 2001 ; jones et al. 2008 ). our close phylogenetic relationship with nonhuman primates increases the likelihood that pathogen spillover from these animals to humans will cause infection (childs et al. 2007 ). moreover, it is not surprising that many studies have focused on spillover events from nonhuman primates to humans given the high prevalence of these largely diurnal mammals in the bushmeat trade (taylor et al. 2015 ) . for instance, nonhuman primates of the family hominidae include the gorillinae and paninae, which show a genetic difference of only 2 % or less with humans (gonzalez et al. 2013 ) , and members of these subfamilies share many morphological, physiological, and ecological features that may have a direct role in the transmission of infectious diseases (davies and pedersen 2008 ) . cleaveland et al. ( 2007 ) , in their assessment of the risk of disease emergence by taxa, found that the relative risk of disease emergence was highest for bats, followed closely by primates, then ungulates and rodents. there have been surprisingly few studies of the connection between hunting of birds or other vertebrates and eids, especially in africa, but surveillance for zoonotic pathogens in african birds is strongly needed (e.g., for avian infl uenza tracking see simulundu et al. 2011 simulundu et al. , 2014 . the characteristics of different species may render them more or less susceptible to hunting. behavioral traits such as communal nesting, large-group living, loud acoustic performances, and a diurnal lifestyle-which are found in many primate species-may facilitate the detection and harvesting of several individuals at one time (bodmer 1995 ) . taste preferences for certain species infl uence hunters' decisions as do attempts to maximize returns by preferring large-bodied animals that provide more food or fetch a higher price when sold than small-bodied species (bodmer 1995 ) . bats, especially the larger fruit bats popular in the bushmeat trade, are susceptible to hunting because they are often found in large, sometimes vocal groups that are visible during the day or in high concentrations in caves (mickleburgh et al. 2009 ). increased human encroachment in recent decades (kamins et al. 2011b ) has driven some bat species to become peridomestic (o'shea et al. 2011 ; plowright et al. 2011 ) , which renders them easy targets for hunting. finally, sick animals may be less successful in evading hunters and hence more easily hunted, thereby increasing the risk of disease transmission to hunters. in addition to the behavioral traits that may infl uence which species are hunted, physiological traits of these species may make them more likely to harbor and transmit diseases. for example, bats, which are present in the bushmeat trade and comprise the highest risk among all wildlife for harboring emerging diseases (cleaveland et al. 2007 ) , present unique traits that suit them to hosting pathogens. these traits include: (1) relatively long lifespans for their body size (munshi-south and wilkinson 2010 ), which may facilitate pathogen persistence for chronic infections; (2) fl ight, which allows movement and dispersal over long distances and which creates high body temperatures that may select for co-evolution with viruses that can live at febrile temperatures and are therefore highly virulent in humans (o'shea et al. 2014 ); (3) physiological similarity across sympatric species that roost together in high densities enabling pathogens adapted to any of the sympatric species to spillover to others (streicker et al. 2010 ) ; and (4) regulation of their immune systems in such a way as to make them more likely to host, but remain unaffected by viral pathogens, serving as the reservoir host for emerging and highly virulent viruses . despite the fact that pathogens are common and often occur in high numbers in basically all animals, only a relatively small proportion of these pathogens will spillover to humans (cleaveland et al. 2007 ) . that said, when spillover events do occur, they can be not only deadly but costly. for example, the united nations development program ( 2015 ) has estimated that west africa as a whole may lose us$3.6 billion per year between 2014 and 2017 due to the 2014-2015 ebola outbreak. this loss stems from the cumulative effects of closed borders, decreased trade, decreased foreign direct investment, and decreased tourism, resulting in increased poverty levels and food insecurity. to understand the dynamics of spillover events and risks in relation to the pathogen, a number of factors must be considered, including: (1) the evolutionary history of the pathogen, (2) how the pathogen is maintained among its wildlife host(s), (3) how the pathogen is transmitted across a species barrier, (4) whether a productive infection is produced in the new host, (5) whether that infection produces signifi cant disease in that host, and (6) whether morbidity and/or mortality levels in the secondary host are suffi cient to be considered signifi cant (childs et al. 2007 ) . from this, it follows that emerging pathogens are not an arbitrary selection of all pathogens. becoming established in a human host typically requires adaptations, often for increased virulence, as has been documented in hiv (wain et al. 2007 ; etienne et al. 2013 ) . generalist pathogens have the ability to infect more than one host species and have higher relative emergence risk than pathogens that are very hostspecifi c (cleaveland et al. 2007 ); this is especially true for pathogens that can infect species in more than one taxonomic order. one example of this generalist "broad" host range is found in the newly described african henipavirus, which can enter and infect cells of nonhuman primates, bats, and humans (lawrence et al. 2014 ) . of particular importance for understanding bushmeat-related spillover events is whether a wildlife species is a natural or incidental pathogen host. natural or reservoir hosts are a natural part of the pathogen life cycle and may maintain the infectious pathogen for prolonged periods of time, often without showing symptoms. in contrast, an incidental or dead-end host may be infected by the pathogen and may even transmit it, but it is not a part of the normal maintenance cycle of the pathogen and is more likely to be affected by it than natural hosts. for example, contact with sick common chimpanzees and western gorillas has been tightly linked to ebola virus spillover in several outbreaks (leroy et al. 2004b ) . like their human cousins, these great apes are largely considered incidental or dead-end hosts for this virus and do not maintain it long-term in nature. in the case of this deadly fi lovirus, understanding what species are true reservoirs (likely fruit bats in the family pteropodidae; pourrut et al. 2007 pourrut et al. , 2009 hayman et al. 2010 hayman et al. , 2012 and the spillover events between these reservoirs and other mammals (including apes, carnivores, and ungulates; leroy et al. 2004a ) will prove critical to mitigating the components of disease transmission that are due to bushmeat-related activities. unfortunately, it is often diffi cult to defi nitively determine the natural host(s) of a particular pathogen as it requires, in descending order of importance, isolation of the agent from individuals of the target species, detection of pathogen-specifi c nucleic acid sequences from individuals, and serological evidence that an individual has been exposed previously. indeed, the study of reservoir systems and how infectious agents move between and within them can be complex, requiring rigorous and sophisticated analyses of multiple interrelated variables (gray and salemi 2012 ; viana et al. 2014 ) . descriptions of the types of pathogens potentially encountered through bushmeatrelated activities can be found below, with several important and well-studied examples described in more detail. in their review of global trends in eids, in which they separately listed each antimicrobial pathogen strain that has recently emerged, jones et al. ( 2008 ) report that the vast majority of pathogens involved in eids are bacterial or rickettsial, followed by viral or prion, then protozoa, fungi, and helminths. other studies have ranked viruses as more prevalent (taylor et al. 2001 ; woolhouse et al. 2005 ; cleaveland et al. 2007 ). in jones et al.'s ( 2008 ) analysis of 335 eid events between 1940 and 2004, only four eids list bushmeat as the driver; other signifi cant drivers were socioeconomic factors such as human population density. these four bushmeat-related eid events were all signifi cant events; all due to viruses (ebolavirus, human immunodefi ciency virus-1, monkeypox virus, and sars), suggesting that viruses are the most important pathogens in regard to spillover due to bushmeat-related activities (see also kilonzo et al. 2014 ) . we review the literature from sub-saharan africa in relation to bushmeat species by pathogen type (viruses, bacteria, helminths, protozoa, fungi, and prions), noting the signifi cant potential for pathogens not yet associated with bushmeat-related activities to also be involved. very few studies have considered all of the potential zoonotics in a region or in a taxonomic group. magwedere et al.'s ( 2011 ) comprehensive study of zoonotics in namibia is an exception. table 24 .1 summarizes these pathogens by bushmeat host taxonomic group, conservatively listing only those species/pathogen combinations that have been tied strongly to spillovers from wildlife to humans via bushmeat-related activities and recognizing that this link is often putative and diffi cult to establish. thus, table 24 .1 does not include some of the potential but not demonstrated spillover risks of poorly studied groups such as helminths and protozoans. furthermore, due to their close genetic relationship with humans, common chimpanzees and western gorillas may share many pathogens of all varieties with humans, but the direction of spillover is not always clear (e.g. tourist interactions may spread disease from humans to apes) and much of these data are not discussed herein. also not included in the table are studies where pathogens are not determined to species and, consequently, the bushmeat host-human link is unclear, or where exposure would be via an insect vector, which could be encountered when handling bushmeat. while we have attempted a very thorough treatment of pathogens that meet our criteria for inclusion in the table, it is possible that some relevant studies have been missed. viruses are obligatory intracellular parasites characterized primarily by the nature of their nucleic acids (dna or rna; single or double stranded, etc.). they are the most abundant form of life on earth; many viruses are recognized as important disease-causing agents, and they are subject to frequent mutation and thus evolution. the advent of modern molecular techniques has advanced our understanding of viral diversity and pathogenesis in both animal and human hosts. for example, in relation to bushmeat, it is now clear that many virus variants are present in hunted nonhuman primate species, which have received most of the research attention, and that these variants have crossed between nonhuman primates and humans on multiple occasions (peeters and delaporte 2012 ; table 24 .1 ). bats and rodents are also major zoonotic virus carriers (meerburg et al. 2009 ; baker et al. 2013 ); other taxonomic groups are less studied, at least in sub-saharan africa. several sub-saharan african viruses of importance are vector-borne, including rift valley fever and crimean-congo hemorrhagic fever. while one presumes that this would make them unlikely to spread via bushmeat-related activities, the possibility remains that animal handling could present a risk (magwedere et al. 2012 ). however, no signifi cant links between vector-borne viruses and bushmeat hunting have been made, and we will not include a discussion of these viruses here. . evidence suggests that siv crossed over to humans by blood contact when hunters had an exposed open wound or injured themselves during the butchering of nonhuman primates (hahn et al. 2000 ; wolfe et al. 2004a , b ; karesh and noble 2009 ) . the closest relatives of hiv-1 found among nonhuman primates are sivcpz and sivgor, from common chimpanzees and western gorillas in west central africa (gao et al. 1999 ; sharp et al. 2005 ; keele et al. 2006 ; van heuverswyn et al. 2006 , 2007 takehisa et al. 2009 ) and at least four separate spillovers have occurred (peeters et al. 2013 the potential for future and continued spillovers from sivs is high, and multiple species-specifi c variants exist. for example, peeters et al. ( 2002 ) and peeters ( 2004 ) estimated that more than 20 % of nonhuman primates hunted for food are infected with a variant of siv; locatelli and peeters ( 2012 ) and peeters et al. ( 2013 ) noted that at least 45 species-specifi c variants of siv from at least 45 primate species are currently recognized. aghokeng et al. ( 2010 ) sampled 1856 nonhuman primate carcasses from 11 species found in bushmeat markets in cameroon. they documented low overall prevalence of siv (only 2.93 % of carcasses), with the lowest prevalence found among the most common species in the market. however, they did fi nd siv variants in about 70 % of the tested primate species. in total, serological evidence of siv infection has been documented for at least 40 different primate species (aghokeng et al. 2010 ; liégeois et al. 2011 liégeois et al. , 2012 . cross-species transmission of strains and co-infection with more than one strain have been documented, sometimes followed by genetic recombination (hahn et al. 2000 ; bibollet-ruche et al. 2004 ; aghokeng et al. 2007 ; gogarten et al. 2014 ), a recipe for future spillovers into humans (locatelli and peeters 2012 ) . human t-cell lymphotropic virus (htlv) : similar to hiv, human t-lymphotropic viruses (htlv) are related to simian viral lineages in which signifi cant diversity has been found (ahuka-mundeke et al. 2012 ; peeters and delaporte 2012 ) . all three sub-saharan great apes and 30 additional nonhuman primates have been documented to have stlv/htlv variants and a variety of htlv viruses have been documented in wildlife and in central african hunters (calattini et al. 2009 (calattini et al. , 2011 courgnaud et al. 2004 ; sintasath et al. 2009a , b ; wolfe et al. 2005b ; zheng et al. 2010 ; locatelli and peeters 2012 ) . similar to hiv/siv, dual infections with more than one variant have been documented in nonhuman primates (agile mangabeys, cercocebus agilis ; courgnaud et al. 2004 ) and in humans (calattini et al. 2011 ; wolfe et al. 2005b ) . simian foamy virus : simian foamy retroviruses ( sfv ) are endemic in most african primates (hussain et al. 2003 ; switzer et al. 2005 ; peeters and delaporte 2012 ) and are known to transmit to humans (sandstrom et al. 2000 ; switzer et al. 2004 ; calattini et al. 2007 ; mouinga-ondémé et al. 2010 , 2012 . like the other retroviruses discussed above (hiv and htlv), sfv is genetically diverse and relatively host species-specifi c. in cameroon, wolfe et al. ( 2004b ) documented three geographically independent sfv infections, which could be traced to de brazza's monkey ( cercopithecus neglectus ), mandrill ( mandrillus sphinx ), and western gorilla. likewise, in gabon, mouinga-ondémé et al. ( 2010 , 2012 documented human spillover events involving multiple strains of sfv, with infected humans having been bitten by common chimpanzees, western gorillas, or mandrills infected with their respective variant of sfv. ebola and marburg viruses : there are seven species of fi loviruses currently identifi ed, fi ve of which occur in sub-saharan africa-genus ebolavirus : tai forest ebolavirus (tafv), sudan ebolavirus (sudv), zaire ebolavirus (ebov), bundibugyo virus (bdbv); genus marburgvirus: marburg virus ( marv ) . these pathogens are periodically emerging viruses, typically from single spillover events, which cause hemorrhagic fevers (reviewed by olival and hayman 2014 ; rougeron et al. 2015 (but note that rougeron's listing for a single case of sudv in sudan in 2011 is erroneous)). the 2014-2015 west africa outbreak of ebov is still ongoing at the time of this writing (labouba and leroy 2015 ) . while the zoonotic source of this outbreak is unknown, three initial outbreaks of the ebola virus in the democratic republic of the congo from 1976 to 1979 involved victims who were reported to have handled western gorilla or common chimpanzee carcasses or to have had physical contact with people who touched the animals (leroy et al. 2004a , b ) . similarly, marburgvirus was fi rst identifi ed in laboratory workers who had dissected imported grivet ( chlorocebus aethiops ) (martini et al. 1968 ; siegert et al. 1968 ). both western gorillas and common chimpanzees have suffered signifi cant mortality from fi lovirus outbreaks (walsh et al. 2003 ; leroy et al. 2004a , b ; bermejo et al. 2006 ; rizkalla et al. 2007 ) and antibodies to ebov were documented in several other primate species by leroy et al. ( 2004b ) . the single case of tafv occurred in an ethnologist likely infected while performing a necropsy of a dead common chimpanzee following a rash of common chimpanzee deaths in the tai national park in côte d'ivoire (le guenno et al. 1995 ; wyers et al. 1999 ) . beyond primates, other incidental hosts in the wild are possible, as was demonstrated for duikers ( cephalophus spp.) (leroy et al. 2004a ; rouquet et al. 2005 ) . as reviewed by weingartl et al. ( 2013 ) , both dogs (naturally) and pigs (at least experimentally) can also be infected. during the 2001-2002 ebov outbreak in gabon, allela et al. ( 2005 ) found over 30 % seroprevalence in dogs living in villages with ebov human and animal cases. those dogs appeared to be asymptomatic and were presumed to be exposed by scavenging wild animals. although incidental hosts likely play important roles in the ecology of these viruses, especially when moribund or dead animals are consumed, strong evidence suggests that bats are the natural reservoir hosts for at least marburgvirus and ebov. for marburgvirus, the cave dwelling and densely packed egyptian rousette fruit bat ( rousettus aegyptiacus ) is now well-documented as a reservoir host amman et al. 2012 ), but antibodies against the virus and/or the presence of viral rna have been found in several other species (see table 24 .1 ). the strong association of marburgvirus with the egyptian rousette makes sense in light of the outbreaks of this virus in people visiting tourist caves or working in mines (adjemian et al. 2011 ; timen et al. 2009 ; towner et al. 2009 ; amman et al. 2012 ). the picture for ebov is less clear, but evidence of infection has been found in at least eight sub-saharan bat species (pourrut et al. , 2009 hayman et al. 2010 hayman et al. , 2012 table 24 .1 ). of the ten bat species listed in table 24 .1 for marburgvirus and ebov, seven are fruit bats, which are relatively larger and more visible, and thus targets of bushmeat hunters. that said, bushmeat hunting of these bats is not ubiquitous throughout their range and cannot solely explain fi lovirus spillovers. mari saéz et al. ( 2015 ) unconvincingly suggested the non-fruit bat, mops condylurus, might have been the source of the 2014-2015 west african ebola outbreak. pourrut et al. ( 2009 ) found evidence of antibodies against zebov in this species, but there is no real evidence that this free-tailed bat played a role in the 2014-2015 outbreak. to date, no bat host has been identifi ed for bdbv, sudv, or tafv and broader surveillance for indications of these viruses in bats and other hosts should be conducted. henipaviruses and other paramyxoviruses : hendra virus and nipah virus (hnvs) are paramyxoviruses in the genus henipavirus that emerged in australia and southeast asia, respectively, with fruit bats in the genus pteropus (family pteropodidae) as reservoir hosts (reviewed by croser and marsh 2013 ) . however, recent studies have identifi ed henipavirus and henipa-like viruses in sub-saharan african fruit bats, which are a phylogenetically distinct clade of pteropodid bats that do not overlap distributionally with any pteropus species. documentation of henipavirus and related rna (drexler et al. 2009 ; muleya et al. 2014 ; baker et al. 2012 ) and anti-henipavirus antibodies (hayman et al. 2008 ; pernet et al. 2014 ) in the african straw-colored fruit bat ( eidolon helvum ) clearly show that this deadly and diverse viral group is present in sub-saharan africa. this bat species is a frequent target of hunters and a signifi cant protein source where it is found (kamins et al. 2011b ). weiss et al. ( 2012 ) documented the presence of this group of viruses in these bats found live in bushmeat markets. strong evidence of spillover to humans was documented by pernet et al. ( 2014 ) who found antibodies against hnvs in human samples from cameroon. these seropositive human samples were found almost exclusively in individuals who reported butchering these bats. this bat is also a long-distance migrator with signifi cant panmixia across the continent, which could facilitate viral transmission between bats (peel et al. 2013 ) . the paramyxovirus story in sub-saharan africa is still unfolding. both drexler et al. ( 2012 ) and baker et al. ( 2012 ) describe great diversity in paramyxoviruses from sub-saharan bats. in their comprehensive study of the evolutionary history of this virus family, drexler et al. ( 2012 ) found that the henipavirus lineage originated in africa and identifi ed bats as the likely origin of this large family of viruses. a precautionary tale from sub-saharan africa comes from the recent discovery and naming of the sosuga virus from a wildlife researcher who became very ill after handling and dissecting hundreds of bats and rodents in uganda and south sudan (albariño et al. 2014 ). this virus is most closely related to tuhoko virus 3, a rubulalike virus recently isolated from the leschenault's rousette fruit bat ( rousettus leschenaultii ) in southern china. amman et al. ( 2015 ) subsequently found sosuga virus in r. aegyptiacus captured from multiple locations in uganda; the researcher infected by this virus handled this species extensively in her studies. lyssaviruses : rabies is the oldest known zoonotic eid, recorded as early as the twenty-third century bc (steele and fernandez 1991 ) . an estimated 25,000 people die in africa each year from rabies (dodet et al. 2015 ) , some portion of which may be from exposure that occurs in bushmeat-related activities, although most human cases can be attributed to domestic dogs. rabies virus (rabv) is in the lyssavirus genus. it is joined in africa by at least fi ve additional species: lagos bat virus (lbv), mokola virus (mokv), duvenhage virus (duvv), shimoni bat virus (shibv), and the newly proposed ikoma lyssavirus (ikov). these viruses have bat(s) as their reservoir host ) with two exceptions. the mokola virus is found in shrews ( crocidura spp.), rusty-bellied brush-furred rat ( lophuromys sikapusi ; saluzzo et al. 1984 ) , and companion animals (delmas et al. 2008 ; kgaladi et al. 2013 ). the ikoma virus has thus far only been documented in african civets ( civettictis civetta ; table 24 .1 , marston et al. 2012 ) . a variety of wildlife species can be secondary hosts of rabies (e.g., in botswana, see moagabo et al. 2009 ) and rabies has been documented to occur in a number of nonhuman primate species, including those encountered in the bushmeat trade (gautret et al. 2014 ) . lyssaviruses are found worldwide, but the greatest genetic diversity is in africa and lagos bat virus may be more than one species (delmas et al. 2008 ; markotter et al. 2008 ; kuzmin et al. 2010a ) . while most human cases are due to rabies virus, duvenhage virus has been documented in human fatalities associated with bat scratches that likely transmitted the virus (van thiel et al. 2009 ; paweska et al. 2006 ) . mokolo virus has been detected in two human cases without mortality (kgaladi et al. 2013 ) . the lyssavirus story in africa will continue to emerge due to increased surveillance and improved molecular techniques. the 2012 discovery of ikoma virus in an african civet in serengeti national park in tanzania, where domestic dogs are largely absent and detection in bat hosts is nonexistent (marston et al. 2012 ; horton et al. 2014 ) , highlights the likelihood that many more lyssaviruses exist in a variety of host species. the true diversity of lyssaviruses in africa, and the potential for human spillover via bushmeat-related activities, remains to be discovered. lassa and other arenaviruses : arenaviruses include a number of zoonotic species, typically transmitted from rodents to humans. lassa virus is the best known of the viral hemorrhagic arenaviruses in africa and is well-documented in west africa, especially guinea, sierra leone, nigeria, and liberia. as with some of the bacterial pathogens described below, the primary risk comes from peridomestic exposure to the rodent host, the natal mastomys ( mastomys natalensis ), via exposure to urine or fecal materials. however, ter meulen et al. ( 1996 ) found a strong association between hunting of peridomestic rodents and antibodies to and symptoms of lassa virus, tying bushmeat-related activities to the spillover of this virus to humans. human monkeypox virus : contrary to its moniker, the reservoir hosts of human monkeypox virus (mpx) are neither monkeys nor humans, but rather rodents. the fi rst case of human monkeypox was identifi ed in 1970 in the democratic republic of the congo, with subsequent outbreaks in liberia, sierra leone, côte d'ivoire, nigeria, and democratic republic of the congo (reviewed by reynolds et al. 2010 ; rimoin et al. 2010 ) . recent mpx increases in the democratic republic of the congo and elsewhere have been attributed to cessation of the human smallpox vaccine, which conferred some immunity to other pox viruses . human and nonhuman primate infections are suspected to result from wildlife exposure such as would occur in bushmeat-related activities; infected species include squirrels (e.g., thomas's rope squirrel, funisciurus anerythrus ; khodakevich et al. 1986 ; african ground squirrels; xerus sp.; reynolds et al. 2010 ) , dormice ( graphiurus sp.; reynolds et al. 2010 ) , and giant pouched rats ( cricetomys sp.; reynolds et al. 2010 ) . the outbreak that occurred in the usa in 2007 after exposure to rodents in the illegal pet trade also linked human monkeypox to rope squirrels, dormice, and pouched rats (hutson et al. 2007 ). while dormice are small and not likely to be the target of hunting, the diurnal and highly visible squirrels and the giant pouched rats are routinely hunted (taylor et al. 2015 ) , making the spillover to humans highly plausible. jones et al. ( 2008 ) list 54.3 % of eid events as being caused by bacteria and there is good evidence to suggest that bacterial pathogens have the potential to be just as important as viruses when it comes to those that may spillover due to bushmeat-related activities, but in this capacity they have received far less attention (cantas and suer 2014 ) . transmission pathways for bacterial pathogens can occur through direct exposure to body fl uids or feces, but they can also possibly be transferred indirectly through exposure to disease vectors such as fl eas and ticks when handling animals. in a rare survey of bacterial pathogens that might spillover via bushmeat-related activities, bachand et al. ( 2012 ) sampled muscle from 128 bushmeat carcasses from multiple species at markets in gabon for the presence of campylobacter , salmonella , and shigella . while they only recorded the presence of salmonella , the potential for contamination and thus spillover of enteric pathogens from carcass handling remains high, especially in the days after purchase when pathogens continue to replicate. bacteria in the genus leptospira are endemic sub-saharan african pathogens that have a high risk of spillover during bushmeatrelated activities as they are shed in urine. jobbins and alexander ( 2015 ) documented their widespread presence in wild mammals, birds, and reptiles, highlighting the role that wildlife may play in leptospirosis. the bushmeat interface may also play a role in human cases of anthrax, caused by bacillis anthracis , which is largely a disease of grazing herbivorous mammals, but to which common chimpanzees are also susceptible (leendertz et al. 2004 ). if bushmeat includes not only the hunting of apparently healthy animals but also sick animals or salvage of contaminated carcasses, the risk of human outbreaks increases (hang'ombe et al. 2012 ) . a number of bacterial pathogens are vector-borne, which at face value would make them unlikely to spread via bushmeat-related activities. however, especially for bacteria with fl ea or tick as vectors, as opposed to mosquitoes for example, one can envision that animal handling could present a risk. the most frightening among the vector-borne bacterial pathogens is plague, caused by the bacteria yersinia pestis and transmitted through the infected fl eas of rodents. africa remains an endemic region of importance for this pathogen (world health organization 2005 ; davis et al. 2006 ) . fleas and ticks are also responsible for transmitting rickettsial pathogens, such as rickettsia africae , which causes african tick-bite fever ( atbf ) . mediannikov et al. ( 2012 ) collected ticks from duikers and a pangolin that were living in close proximity to humans in guinea and found r. africae in 10 % of ticks collected from the tree pangolin ( manis tricuspis ), suggesting the potential for spillover with the close handling of these animals. further research is clearly and urgently needed to fully assess the potential for bacterial disease spillovers via bushmeat-related activities. the helminths or "worm-like" animals include many parasites of zoonotic potential, although taylor et al. ( 2001 ) found helminthes less likely to cause eids. humans engaging in bushmeat-related activities are likely exposed to these pathogens via exposure to fecal material in which eggs are shed, from transcutaneous exposure to infectious larvae, or from consumption of uncooked meat (mccarthy and moore 2000 ) . several studies have examined the prevalence of helminths in animals from bushmeat markets and found high rates of multiple species. for example, adejinmi and emikpe ( 2011 ) collected fecal samples from greater cane rats ( thryonomys swinderianus ) and bush duikers ( sylvicapra grimmia ) in bushmeat markets in nigeria and documented high prevalence rates (83.3 % and 53.8 %, respectively) of helminth ova in feces as well as larvae from fecal cultures. likewise, magwedere et al. ( 2012 ) and mukaratirwa et al. ( 2013 ) reviewed the evidence for trichinella infection in humans, livestock, and wildlife in sub-saharan africa and noted that bush-pigs ( potamochoerus spp.) and desert warthogs ( phacochoerus aethiopicus ) are a source for human infection. as is the case with many other pathogens, humans and nonhuman primates share susceptibility to many parasitic helminth species (pedersen et al. 2005 ; pourrut et al. 2011 ). pourrut et al. ( 2011 sampled gastrointestinal parasites from 78 wild monkeys of 9 species collected from bushmeat markets in cameroon and documented high helminth loads, including species known to infect humans. gillespie et al. ( 2010 ) had similar fi ndings from common chimpanzee fecal samples. overall, the available evidence suggests that spillover of many of these pathogens during bushmeat-related activities is likely. protozoans are a paraphyletic group of eukaryotic organisms that are neither animals, plants, nor fungi and include amoebas and giardia. the risk of protozoan spillover from bushmeat-related activities is similar to that for helminths and bacteria in that exposure to feces, bodily fl uids, and even potentially to meat could transmit disease to a permissive human host (pourrut et al. 2011 ) . a number of protozoans are important pathogens with zoonotic potential (taylor et al. 2001 ). perhaps the best example are the amoebozoa, which cause diarrheal disease and which are documented in a variety of animals, including bushmeat species such as nonhuman primates (gillespie et al. 2010 ; pourrut et al. 2011 ) . gillespie et al. ( 2010 ) documented the amoeba entamoeba histolytica and the ciliated protozoan balantidium coli in common chimpanzees; both are human pathogens (although the direction of spillover is uncertain, as common chimpanzees and other primates may have obtained this parasite from humans). indeed, lilly et al. ( 2002 ) documented both protozoans in common chimpanzees, western gorillas, agile mangabeys, and humans living in the same region in central african republic. a number of other nonhuman primates have had documented e. histolytica infections as well (see table 24 .1 ). other protozoan examples include toxoplasma gondii , which causes the disease toxoplasmosis, but could not be detected during a recent, albeit small scale, survey of bushmeat (prangé et al. 2009 ) and water/foodborne parasites such as giardia. recent studies have documented giardia in a variety of species that exist in the bushmeat trade, including western gorilla and african buffalo ( syncerus caffer ) (hogan et al. 2014 ). fungi are increasingly being recognized as important pathogens that may emerge, even in humans (jones et al. 2008 ; fisher et al. 2012 ) , and a number of fungi are considered medically important. in particular, fungal infections are problematic for people who are immunosuppressed (e.g., from hiv infection), in which case their immune systems are unable to adequately fi ght the infection. nonetheless, we have uncovered no examples of eids in africa caused by fungal pathogens not related to human immunosuppression, as even the 1950s outbreak of cryptococcal meningitis in the democratic republic of the congo has been likely linked to co-infection by hiv (molez 1998 ; jones et al. 2008 ). only 5 % of prion diseases are acquired (as opposed to inherited), but these include the well-publicized outbreaks of scrapie, bovine spongiform encephalopathy (bse, or "mad cow disease"), and chronic wasting disease (cwd) in ungulates from europe and north america. of these, only bse has been detected in humans and in captive-held primates (imran and mahmood 2011a , b ; bons et al. 1999 ; lee et al. 2013 ) , likely due to consumption of contaminated meat products. the authors have found no descriptions of infectious prion diseases in africa, but this poorly studied pathogen type may well be present in the world's second largest continent. as it relates to bushmeat-related practices, prions can be found in nearly all tissues and are resistant to degradation, even by cooking, rendering them a potential pathogen worth watching. the risk of disease spillover from bushmeat to hunters is highest during butchering and especially if no precautions are taken. whether hunters take precautions may depend on their knowledge and perception of disease risk. there is increasing evidence that the perception of and knowledge about zoonotic diseases is generally low but varies strongly between sites. a survey among rural bushmeat hunters and traders in sierra leone showed that 24 % reported knowledge of disease transmission from animals to humans (subramanian 2012 ) . similarly, 23 % of rural-urban hunters and traders in ghana perceived a disease risk from a bat-bushmeat activity, with signifi cantly more respondents associating risk with bat consumption than bat preparation or hunting (kamins et al. 2014 ) . individuals who participate in butchering wild animals typically associate less risk to meat preparation and consumption than those who do not participate in butchering (kamins et al. 2014 ) (fig. 24.2 ) . lebreton et al. ( 2006 ) found that hunters and butchers who perceived personal risks were signifi cantly less likely to butcher wild animals, but that risk perception was not associated with hunting and eating bushmeat. thirty-three percent of bushmeat consumers in a ghanaian market were not aware that zoonotic diseases could be transmitted from bushmeat to humans. those who were aware gave ebola (48 %) and anthrax (16 %) as examples of zoonotic diseases (kuukyi et al. 2014 ). in contrast, a large-scale survey among rural central african population showed that the majority (74 %) of respondents perceived contact with bushmeat blood or body fl uids as dangerous (lebreton et al. 2006 ) . unfortunately, studies in this fi eld can be challenging, as reported perceptions may differ from actual or 'revealed' behaviors and beliefs (wilkie 2006 ) . although there seems to be some level of risk awareness in certain human populations, several studies report a distinct lack of precautionary behavior, resulting in hunters, butchers, and consumers exposing themselves to zoonotic diseases. lebreton et al. ( 2006 ) found that only 4 % of hunters and 2 % of people reporting butchering indicated that they took precautions against contact with animal blood and fl uids while hunting and butchering. furthermore, the few that took precautions may not have protected themselves adequately, as the most common response was "generally being careful." this was followed by "washing hands," and the least number of participants reporting "avoiding contact with blood, draining blood from carcasses and wearing suitable clothing." paige et al. ( 2014 ) examined human-animal interactions in western uganda and found that nearly 20 % of participants reported either being injured by an animal or having contact with a primate. the most commonly reported animal injuries were bites (72.9 %) and scratches (23.2 %). in a separate study, it was also shown that although ghanaian hunters generally handle live bats, they do not typically use protective measures such as gloves, and thereby come into contact with blood through scratches and bites (kamins et al. 2014 ) . given the lack of awareness and precautionary measures taken among people who come into contact with bushmeat, the opportunity for new zoonotic pathogens to spillover into humans remains high (lebreton et al. 2006 ) . this is especially true, since the current rate of hunting wild animals will likely continue-at least until domestic animal production increases and can support the protein needs of the local people. current global disease control efforts focus almost exclusively on responding long after a spillover event has occurred, which increases the risk of a single spillover event causing an epidemic or pandemic. this retroactive response to emerging disease outbreaks is often costly economically and in terms of human well-being (childs and gordon 2009 ; united nations development program 2015 ) . increased pre-spillover surveillance measures along with quantifi cation of spillover risk is critically needed. for example, wolfe et al. ( 2004b wolfe et al. ( , 2005b found that 1 % of rural cameroonians are infected with wild primate variants of t-lymphotropic viruses and another 1 % are infected with wild primate variants of simian foamy virus. these sorts of data are simply lacking for most emergent disease systems. here we will discuss the regulatory and educational measures that could be taken to mitigate the risk of a zoonotic spillover event and spread. such efforts should be undertaken as a part of a comprehensive response to other sub-saharan public health crises so as to not divert scarce resources. for example, increases in eid surveillance efforts and in post-emergence management go hand in hand with the improved healthcare infrastructure that must become a priority for sub-saharan africa. at face value, the risk of disease transmission would be reduced if people stopped harvesting bushmeat; however, this scenario is not realistic given the importance of bushmeat in many communities in africa for which there is limited affordable access to alternate protein sources (pike et al. 2010 ; gebreyes et al. 2014 ) . a more practical option may be to restrict hunting of nonhuman primates, as many zoonotic eids have come from them, and instead allow communities to hunt smaller-bodied mammals with higher reproductive rates. any intervention aiming to restrict access to wildlife should involve community leaders and stakeholders during public outreach to reduce the risk of alienating communities (monroe and willcox 2006 ) . the education and enforcement necessary to implement such a restriction must consider the cultural and economic contexts surrounding individual communities. consider, for example, the problems with enforcement of access restrictions and the history of antagonistic relationships due to exclusion from protected areas between conservationists and local communities. without proper educational outreach, this could result in backlash from local communities. furthermore, using zoonotic diseases to enforce hunting restrictions runs the risk of demonizing species considered to be the main disease carriers. nonhuman primates could then become targets and their populations could be decimated (pooley et al. 2015 ) . a more realistic strategy may be to concentrate on preventing future zoonotic spillover events through culturally appropriate education and preventing the spread of diseases through better disease surveillance. in that effort, it would also be important to incorporate collaborative and interdisciplinary approaches between veterinary researchers, ecologists, microbiologists, public health researchers, and anthropologists to develop surveillance and research approaches that will be both culturally appropriate and improve detection of zoonotic diseases tied to bushmeat hunting (kilonzo et al. 2014 ). the risk of disease transmission could be reduced through community education that focuses on people with high levels of exposure to wild animals (wolfe et al. 2007 ). communicating with hunters and butchers about the risks associated with bushmeat and promoting awareness of safer techniques may reduce current levels of pathogen exposure and transmission. to enhance the effectiveness of prevention campaigns, it is particularly important to reinforce the potential for infections during hunting and butchering as this may be overlooked by some hunters (lebreton et al. 2006 ) . because the risk perception of hunters and those engaging in butchering wild animals has a negative association with the level of participation in meat preparation and consumption (kamins et al. 2014 ) , this may reduce current levels of pathogen exposure and transmission, if not by discouraging individuals to participate in preparation and consumption, then by encouraging those individuals to more proactively consider safety and preventative measures. global viral forecasting (gvf; now "global viral" and "metabiota") has been pivotal in educating vulnerable populations in rural central africa by providing information on the risk of zoonotic disease transmission from hunting wild animals (lebreton et al. 2012 ) . hunters are informed about disease risks associated with different species, what steps can be taken to avoid infections, and how they can reduce their contact with blood and body fl uids of wild animals. hunters are urged to redirect hunting efforts away from apes and monkeys and towards less risky species such as antelope and rodents, while also being discouraged from butchering animals when there are cuts or injuries on their hands and limbs. of course, a common aspect of such attempts at social outreach and education is that even when it is possible to promote awareness, individuals may not believe the hazard is important or that it could affect them. some authors have even found that when people do believe the risk is real and relevant, there is often little evidence that this knowledge promotes a change in behavior (mccaffrey 2004 ) . for example, a pilot education program among ghanaian hunters resulted in substantially improved understanding of disease risk, yet largely failed to change peoples' behavior (kamins et al. 2014 ) . when asked about what would change their behavior, participants responded; becoming ill from zoonotic disease followed by alternative livelihoods and stricter laws. because awareness is not directly related to behavior, monroe and willcox ( 2006 ) suggest that campaigns should not rely on the threat of infection to change behavior, but should rather use community leaders to change cultural norms associated with hunting and educate people involved in butchering about best practices of how to protect themselves. with the increasing prevalence of zoonotic disease emergence and the associated risk for public health, we have to improve our understanding of the dynamics of spillover events of pathogens from animal to human hosts (rostal et al. 2012 ) and improve systematic global monitoring efforts. this could help detect, defi ne, and control local human emergence while it is still locally confi ned and before it has a chance to spread globally. improved detection and surveillance will lead to a better prioritization of public health efforts. one of the most effective strategies in terms of early detection of an emergent pathogenic threat would be to focus surveillance efforts among people who are highly exposed to at-risk animals and on the animal populations to which they are exposed (lebreton et al. 2012 ) . bushmeat hunters would be an important target group, as they are in contact with bodily fl uids from animals and are at risk for transmission and infection from novel pathogens. as an example, gvf has established monitoring programs at multiple sites throughout central africa to detect the moment of a pathogen spillover, which can then be used to predict and ultimately prevent zoonotic disease emergences (evans and wolfe 2013 ) . in order to track and provide data for eids, this effort coordinates the collection of fi lter-paper blood samples from both hunted animals and people who hunt and butcher wild animals. early results have shown that this type of surveillance can assist in early detection of new diseases by offering insight into pathogen origin. it would also help describe the spillover dynamics of new or existing diseases. such data are valuable for developing a detailed, mechanistic understanding of the processes that drive disease emergence and prevent spillovers from spreading in early stages of an outbreak. contextualizing the relative or actual risks of spillovers would be vital for the preferential allocation of resources to high-risk regions or humans who perform high-risk activities (daszak et al. 2007 ). as part of these efforts, improved knowledge of how anthropogenic environmental changes and sociological or demographic factors affect the risk of disease emergence will likely be a cost-effective and sustainable mechanism to reduce or control disease spillover risks (daszak et al. 2007 ). the social and environmental issues surrounding bushmeat represent a complex problem for conservation, global public health, and sustainable development, as it is often the poorest and most vulnerable populations who depend on bushmeat for income or food security. accordingly, the challenge should be addressed in a holistic manner, by integrating multiple efforts to achieve common objectives. although much progress has been made not only in addressing the problems concerning bushmeat harvest and zoonotic disease spillover, there is much work to be done. research that would pave the way for future efforts would include the quantifi cation of social response to environmental policy change (e.g., in the context of harvest restriction), development of a more representative picture of bushmeat consumption in africa, a broader exploration of the many classes of pathogens within wildlife, and more thorough understanding and quantifi cation of the dynamics behind spillover events and the risks to humans. such efforts could facilitate the development of policy and infrastructure that would help curb the dependency on bushmeat, reduce risks associated with bushmeat harvest, and help understand in what circumstances zoonotic disease spillover events occur. there is still uncertainty as to how education should be implemented in different regions and what features of such education would be most valuable for local people. such an effort might consist of surveying rural bushmeat-harvesting populations across africa and using the resulting data to contextualize priorities and goals in a way that could help standardize education approaches. while some locations in africa have had extensive research in the scope and impact of bushmeat harvest, much of africa has been neglected in those efforts. a more developed understanding of the location, scale, and structure of bushmeat harvest throughout the continent would 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vaccine theory refuted histopathological and immunohistochemical studies of lesions associated with ebola virus in a naturally infected chimpanzee declines in large wildlife increase landscape-level prevalence of rodent-borne disease in africa emergence of a novel and highly divergent htlv-3 in a primate hunter in cameroon key: cord-277309-kelebqr6 authors: wang, lin-fa; anderson, danielle e title: viruses in bats and potential spillover to animals and humans date: 2019-01-18 journal: curr opin virol doi: 10.1016/j.coviro.2018.12.007 sha: doc_id: 277309 cord_uid: kelebqr6 in the last two decades, several high impact zoonotic disease outbreaks have been linked to bat-borne viruses. these include sars coronavirus, hendra virus and nipah virus. in addition, it has been suspected that ebolaviruses and mers coronavirus are also linked to bats. it is being increasingly accepted that bats are potential reservoirs of a large number of known and unknown viruses, many of which could spillover into animal and human populations. however, our knowledge into basic bat biology and immunology is very limited and we have little understanding of major factors contributing to the risk of bat virus spillover events. here we provide a brief review of the latest findings in bat viruses and their potential risk of cross-species transmission. in the last two decades, several high impact zoonotic disease outbreaks have been linked to bat-borne viruses. these include sars coronavirus, hendra virus and nipah virus. in addition, it has been suspected that ebolaviruses and mers coronavirus are also linked to bats. it is being increasingly accepted that bats are potential reservoirs of a large number of known and unknown viruses, many of which could spillover into animal and human populations. however, our knowledge into basic bat biology and immunology is very limited and we have little understanding of major factors contributing to the risk of bat virus spillover events. here we provide a brief review of the latest findings in bat viruses and their potential risk of crossspecies transmission. although there have been significant advances in diagnostics and medical countermeasures during the past century, the risk of cross-species transmission of known and unknown pathogens has emerged as a threat to human and animal populations due to a various factors, including industrialization, intensive farming, urbanization, rapid transportation and climate change [1, 2 ] . it is generally accepted that approximately 75% of emerging infectious diseases for humans are zoonoses [1, 3, 4] . the rate of emergence of novel viruses appears to be increasing as a result of both increased spillover from their natural reservoirs and our improved ability in detection [3] . among the newly emerged and most deadly zoonotic viruses discovered in the past few decades, bat-borne viruses occupy a greater proportion than viruses from any other mammalian order [5 ,6 ,7,8] . several studies have now concluded that bats are exceptional in their ability to act as natural reservoir of viruses and they are able to harbour more diverse viruses per animal species [6 ,9] . while the underlying biology for this observation is yet to be uncovered, it is certain that we will witness more disease outbreaks from bat-borne viruses in the years to come. at the present time, it is impossible to predict the risk of spillover potential for the vast number of viruses or viral sequences which have been detected in bats around the world. but it will be a good start to focus on the viruses in the 'known unknown' category, that is new or variant strains of bat viruses related to those which have already spilled over into and caused diseases in animals or humans. although bats are known to also carry dna viruses, all of the disease-causing and species-jumping bat-viruses are so far limited to rna viruses. in this brief review, we will focus on the major rna virus families harboured by bats that have demonstrated spillover and severe disease-causing potential. bats, order chiroptera, are the only mammals capable of powered flight and are among the most ancient of mammals and underwent extensive speciation for the last 100 million years. there are currently more than 1000 species of bats, making them the second most diverse mammalian group, after rodents, and representing 20% of extant mammalian species [10] . although the recent surge of interest in bats is mainly driven by their association with many of the most lethal viruses, bats are known for their exceptionally long life span and for being less prone to cancer [8] . bats are not only rich in species diversity, but also have great variation in their geographical locations, dietary preferences, physiological range of body temperatures, social behaviour and navigation and vision systems [11] . it is therefore important to recognise that such immense diversity makes it difficult to generalize-specific associations relating to bats and viruses to all members of the chiropteran order. in addition, due to the large number of bats around the world and the fact that similar bats can live in different geographical locations and multiple bat species can co-exist in similar ecological habitats, any virus surveillance or virome detection study should not be viewed as a holistic examination of any given systems. instead, they are more likely to be a transient snapshot of a specialised system at a given time. while recognising that there are limitations on current investigations of viruses in bats, we are also optimistic that with the increasing interest and research activities in this field, we will gain a more accurate panoramic view of bats and viruses in the not too distant future. as a matter of fact, the findings accumulated in the last few decades have already pointed towards a few virus families as being both more prevalent in bats and with proven potential for spillover into other animal species [5 ,12] . the summary below highlights these virus families, followed by other bat viruses which may possess spillover potential and are considered to be important enough to keep on our watch list. coronaviruses were not known to cause severe diseases in humans before the emergence of severe acute respiratory syndrome (sars) coronavirus (cov). the sars outbreak in 2002-2003 remains as one the most impactful pandemic outbreaks of the 21st century mainly due to the fact that the aetiology was totally unknown during the outbreak, which made accurate diagnosis and effective control impossible [13, 14] . the outbreak lasted more than six months with rapid spread of the virus from southern china to more than 30 countries on all major continents, and resulted in more than 8000 human infections and 774 deaths [14] . multiple international teams spent the next decade hunting for the origin of sars-cov and serendipitously found many sars-cov related viruses in bats, most abundantly from the genus rhinolophus (horseshoe bats) [15] [16] [17] . the most conclusive evidence came from the isolation of a cov from bats in china which was more than 98% identical in genome sequence to sars-cov and capable of using the sars-cov receptor, ace2, on human cells [18 ] . while it is not easy to assess the spillover potential of many sars-cov related bat covs due to unsuccessful attempts to isolate the viruses, it should be noted that a 'consensus' virus constructed via reverse genetics pointed to a high probability of human infection [19] . although a significant amount of attention was focused on sars-cov related viruses, the international community was again caught by surprise with the emergence of the middle east respiratory syndrome (mers)-cov in 2012 [20 ] . mers-cov is genetically quite different from sars-cov (figure 1 ), despite both viruses belonging to the genus betacoronavrius. as of october 1, 2018, mers-cov has infected 2249 people in 27 countries with 35% case fatality [21] . camels have been identified as important reservoir hosts for mers-cov and mers-cov related viruses [22,23,24 ,25,26,27 ] , but there is strong evidence that the evolutionary ancestors of these viruses are bats [28, 29, 30 ] . co-circulation and recombination of covs has been implicated as a mechanism that maintains viral diversity and continuous zoonotic transmission [27 ,31] . severe disease outbreaks caused by covs related to viruses associated with bats are not limited to humans. in 2016-2017, there was a major outbreak of swine acute diarrhoea syndrome (sads) in piglets in multiple southern china farms in a region geographically close to where the sars outbreak began in 2002 [32 ] . the origin of the causative agent, sads-cov, was quickly traced back to a bat colony in the vicinity of the pig farms where bat cov with more than 98% genome sequence identity was detected in rhinolophus spp. bats. sads-cov belongs to the genus alphacoronavirus and is genetically most closely related to hku2, a previously reported bat cov [32 ] . human coronavirus 229e is in the same genus, but is only distantly related to those bat covs. examination of pig farmers with close contact with sick and dying piglets did not yield evidence of human infection, hence humans may not be susceptible to sads-cov. further study is required to determine the true zoonotic potential of sads-cov and closely related bat covs. for unknown reasons, despite of the wide presence of covs in bats of different locations and species with relative high viral genome levels, multiple attempts by different international groups to isolate bat covs have been largely unsuccessful. the only successful isolation was achieved with sars-like viruses by direct isolation using vero e6 cells [18 ] or inoculation into the brain of suckling rats [17] . one of the first bat-borne bsl4 agents identified was hendra virus (hev) in australia in 1994 [33 ] , an emerging pathogen that caused the deaths of 7 people. additionally, 103 equine and 2 canine cases have been reported [34, 35] . in humans the case fatality rate from hev infection is 57%. all four species of flying fox in australia (pteropus poliocephalus, p. alecto, p. scapulatus and p. conspicillatus) have been found to be seropositive for hev antibodies and all have detectable virus in their urine with black flying fox (pteropus alecto) being the major reservoir host [36] . a closely related virus, nipah virus (niv) emerged in 1998 in malaysia, which transmitted from bats to humans via pigs as an intermediate and amplifying host [37] . in total, that outbreak resulted in 283 human cases and 109 deaths (39% case fatality) in malaysia as well as 11 cases and one death in singaporean abattoir workers [38] . a related, but not identical virus, was responsible for multiple niv outbreaks in bangladesh/india [39, 40] , with the latest outbreak that occurred in 2018 in kerala and resulted in 19 human infections and 17 deaths [41] . the reservoir hosts of niv have been identified as the large flying fox (p. vampyrus) and small flying fox (p. hypomenalus) in malaysia [42, 43] and the indian flying fox (p. giganteus) in bangladesh and india [44, 45] . after many years of unsuccessful attempts, niv was isolated from the indian flying fox in bangladesh [46] . the current status of henipavirus transmission during outbreaks is summarised in figure 2 . apart from hev and niv, cedar virus (cedpv) remains the only other isolated henipavirus species and experimental evidence from animal trials suggests that cedpv is non-pathogenic for humans [47] . serological evidence of henipavirus infection has been detected in lyle's flying fox (pteropus lylei) populations in southeast asia [48] , large flying fox in indonesia [49] and may be endemic among bat populations on the african continent [50] [51] [52] [53] . additionally, a ghanaian bat henipavirus, kumasi virus (kumpv), genome has been sequenced [54 ] . cumulatively, these studies indicate a wide global distribution of henipaviruses. menangle virus (menpv) is a zoonotic paramyxovirus, first identified in a disease outbreak of reproductive disease in pigs in 1997 at a piggery in new south wales, australia [55, 56] . the virus was also shown to be zoonotic, with 2 piggery workers with high-level exposure developing a serious influenza-like illness and rash during the outbreak. these individuals also developed neutralizing antibodies to menpv [55] . bats were hypothesized to be the source of the outbreak and menpv-neutralizing antibodies were detected in grey-headed flying foxes (pteropus poliocephalus), black flying foxes (pteropus alecto) and spectacled flying foxes (pteropus conspicillatus) [56] . in 2009, menpv was isolated from a bat roost at cedar grove, australia, where black flying foxes were the predominant species in this colony at the time of sampling [57] . tioman virus (tiopv) was isolated from urine of the small flying fox (pteropus hypomelanus) collected from tioman island, malaysia [58] . due the close relationship of tiopv with the zoonotic menpv, an experimental challenge of pigs was performed [59] and the trial suggested that pigs could act as an intermediate or amplifying host for tiopv and that oral secretion is a possible means of viral transmission. the identification of sequences similar to mumps virus (muv) in bats revealed that a virus that was believed to only infect humans has substantial similarity to a counterpart in bats [54 ] . the genetic and functional viruses in bats and potential spillover to animals and humans wang and anderson 81 aside from menpv and tiopv, other paramyxoviruses from the genus rubulavirus have been isolated from or detected in bats without evidence of zoonotic transmission. porcine rubulavirus (porpv), the causative agent 'blue eye' disease in pigs was first identified in mexico in 1980 [64] and serological surveillance data [65] suggests bats are likely reservoir of this virus. mapuera virus (mprpv), a rubulavirus closely related to porpv [66] that has not been associated with any human disease, was isolated from the salivary glands of a healthy fruit bat (sturnira lilium) captured in brazil in 1979 [67] . tukoko virus (thkpv) 1, 2 and 3 are rubulaviruses that have been detected and sequenced from rousettus leschenaultii in china [68] , but these viruses were unable to be cultured in the laboratory and the potential of these viruses to cause disease in humans and animals is yet to be ascertained. in 2012, metagenomic analysis from rna extracted from blood and serum samples of a patient with severe acute febrile illness revealed a novel paramyxovirus [69 ] most closely related to thkpv-3. the novel paramyxovirus was provisionally named sosuga virus (sospv) in recognition of its probable geographic origin (south sudan, uganda). the patient, a wildlife biologist, developed a severe illness after spending six weeks sampling bats and rodents. symptoms included fever, malaise, headache, generalized myalgia and arthralgia, neck stiffness, a metallic taste, sore throat and a maculopapular rash that was present later in the infection. the biologist was discharged after two weeks of hospitalization, but considerable sequelae (myalgia, arthralgia, headache, malaise, and fatigue) persisted for several months [69 ] . bat tissues collected during the period just before the onset of symptoms were tested for sospv, and several egyptian rousette bats (rousettus aegyptiacus) were found to be positive. four additional sospv-positive samples were found in archived tissues from egyptian rousette bats collected at other locations in uganda, suggesting this species could be a potential natural reservoir for this paramyxovirus [70] . although it has been known for more than four decades that ebola and marburg viruses can cause lethal haemorrhagic diseases in humans, the massive outbreak in west africa during 2014-16 was unprecedented with more than 11 000 human fatalities [71] . the ongoing ebola outbreak in the democratic republic of congo is a further indication that more outbreaks in africa [72] . currently, there are five distinctive species identified in the genus ebolavirus, including bundibugyo (bdbv), reston (restv), sudan (sudv), taï forest (tafv) and zaire (ebov). very recently, a sixth species has been proposed, named bombali virus (bomv), based on genome sequence detected in free-tailed bats in sierra leone (chaerephon pumilus and mops condylurus) [73] . both members of the genus marburgvirus, marburg virus (marv) and ravn virus (ravv), have been shown to cause fatal diseases in humans [74, 75] . the third genus, cuevavirus, contains only one species, lloviu virus (llov), whose disease-causing potential in humans is unknown [76, 77] . our group has recently characterized complete coding genome of a new filovirus named while llov, bomv and mlav sequences were first discovered in bats, and marburg viruses have been isolated directly from bats [79 ,80] , the role of bats as a reservoir for ebolaviruses is still debated mainly due to the lack of direct isolation of ebolaviruses from bats [81] . however, the detection of bomv seems to strengthen the notion that bats are likely natural reservoirs of ebolaviruses [73] . reoviruses (respiratory enteric orphan) were not known to be associated with severe human diseases when they were first discovered in the 1950s and about one third of the human population has been exposed to at least one of the mammalian reoviruses (mrvs) [82] . the outbreak of a severe respiratory and enteric disease among different members of a family in melaka, malaysia, changed our appreciation of both the diversity and the zoonotic potential of this class of viruses in the genus orthoreovirus, family reoviridae [83 ] . since the discovery of melaka virus during the 2006 outbreak investigation, at least 15 different strains have been identified, either from human outbreak investigations or bat virome studies [83 ,84-95] , as summarised in table 1 . serological and molecular detection studies suggest that the prevalence of these viruses could be severely underestimated due to the lack of routine diagnosis in hospitals [96, 97] . a plethora of known and novel viruses were identified in samples collected and metagenomically screened from straw-coloured fruit bat (eidolon helvum) in cameroon [98] and neoromicia species in south africa [99] . these bats were shown to harbor divergent viruses, including members of the families astroviridae, circoviruidae, parvoviridae, partitviridae, coronaviridae, picobirnavirdae, adenoviridae, herpesviridae, papillomaviridae, phenuiviridae, and picornaviridae [98, 99] . these recent studies build upon previous work [100] [101] [102] [103] to further expand the diversity of the bat virome. uniquely, the picobirnaviruses identified utilize an alternative genetic code [98] . similarly, there are many other bat-borne viruses with the potential for zoonotic transmission, but without documented human infection. rotaviruses and noroviruses, members of the families reoviridae and caliciviridae, respectively, are the major etiologic agents of acute gastroenteritis and several reports have identified rotavirus [98, 99, [104] [105] [106] or norovirus [107, 108] viruses in bats and potential spillover to animals and humans wang and anderson 83 different bat species worldwide. the caliciviruses recently discovered in bats were found to be antigenically similar to human noroviruses, again highlighting the potential for cross-species transmission [109] . influenza virus is known for zoonotic transmission and two novel subtypes were discovered in bats. in 2012, a new influenza virus genomic sequence was identified in frugivorous yellow-shouldered bats (sturnira lilium) in guatemala and was designated h17n10 [110] . the following year, a distinct influenza genome, classified as h18n11, was characterized from the flat-faced fruit bat (artibeus planirostris) in peru [111] . although virus isolation was not successful, reverse genetics was used to synthetically generate these viruses [112] and subsequent research highlighted the differences between these viruses and conventional influenza viruses. identification of these viruses in bats not only expanded the host reservoir of influenza and the genetic diversity of the viruses, but immediately raised the question about zoonotic potential. studies using synthetic viruses have allowed the identification and characterization of cellular receptors mediating virus attachment and entry, factors important for understanding the tissue tropism and possible zoonotic transmission [112] [113] [114] . hantaviruses are predominantly rodent-borne pathogens and transmission to humans can lead to severe diseases and death. species of hantaviruses have been identified in bats from africa and asia, expanding the potential reservoirs range and genetic diversity of these viruses [115] [116] [117] [118] [119] [120] . hantaan orthohantavirus (htnv) was isolated from two broadly distributed insectivorous bat species (eptesicus serotinus and rhinolophus ferrumequinum) [117] . evidence of a lethal genotype of andes orthohantavirus (andv), araraquara orthohantavirus (arqv), has been documented among several neotropical bats in brazil [118, 120] . arqv is one of the most virulent and lethal among all hantaviruses in humans and viral rna closely related to arqv was detected in urine of the common vampire bat (desmodus rotundus) [119] . these studies highlight that bats are probably playing an under appreciated part on the maintenance, circulation, and transmission of hantavirus in nature. recently, a group of previous unknown bunyaviruses, including severe fever with thrombocytopenia syndrome (sfts) virus and heartland virus (hrtv), emerged during human disease outbreaks [121, 122] . although their animal origins are not known, the most closely related virus has been found in bats in india. in 2010 a novel phlebovirus was isolated from leschenault's rousette bat (rousettus leschenaultii) in western india. the virus was identified by electron microscopy and phylogenetic analysis of the complete genome showed its close relation to sftsv and hrtv [123] . in contrast to new and emerging viruses where viral pathogenesis and transmission route is unknown, rabies has long been recognized throughout history, due to the characteristic symptoms associated with the disease [124, 125] . infection with lyssaviruses, including rabies virus and australian bat lyssavirus leads to rabies disease [126] . although bites and scratches from infected bats occur, there is an effective vaccine and post exposure prophylaxis available for this deadly disease. we know that there are several groups of bat viruses that can infect and cause severe diseases in humans, as we have briefly covered in this review. it is also well established that there are a large number of related viruses circulating in bats in different parts of the globe, but as of yet we are unable to accurately predict which of these viruses are capable of spillover and whether they will cause diseases in humans. we view these viruses as the 'known unknowns'. among the four families of viruses discussed in this review, each has a different combination of characteristics. in terms of frequency, the reoviruses seem to be the most permissive to spillover, especially in asia. however, to date we have only experienced severe, but not lethal, infections in humans. on the other hand, filoviruses and henipaviruses are far the more deadly but the frequency of spillover is relatively low [7, 12] . rabies virus is highly lethal and responsible for a large number of human deaths. but the direct spillover from bats to humans is limited. in contrast, the known genetic diversity of covs in bats is much greater than any of the other bat zoonotic viruses. covs contain the largest genomes of all known rna viruses, and hence are naturally exposed to a higher chance of genetic mutation per genome. to prevent frequent 'lethal mutations', covs have evolved to contain an exoribonuclease which increases the fidelity of rna genome replication [127, 128] . however, the large positive rna genomes of covs are highly prone to gross genetic changes via recombination, which is elegantly illustrated by two recent studies, one on sars-like viruses [129 ] and another on the discovery of a recombinant cov containing a reovirus p10 gene sequence [130 ,131] . the true rate of cov spillover into humans and livestock animals may be greatly underestimated as these occurrences do not always cause severe or lethal disease, demonstrated by serological surveillance. spillover and zoonotic transmission of covs is not limited to bat covs, rather increasing evidence suggests the emergence of new cov strains and the mutation of existing strains resulting in new disease syndromes in both animals and humans [132, 133] . one of the challenging scientific questions is why many of the bat-borne zoonotic viruses are so lethal when they spill over into human and/or livestock animal populations. up until now, our knowledge was very limited in addressing this question due to lack of research tools to conduct comparative immunology and pathogenesis studies in bats. several recent studies, however, start to reveal that bats may have evolved a more balanced innate defence system. on one hand, bats have elevated level of certain defence genes or pathways from type i interferon [134] to apoptosis [135] , at the same time bats exhibit more immune tolerance in different pathways, from inflammation [136] , sting signalling [137] to nk cell activation [138] . however, it should be cautioned that these are very early and preliminary studies, many of them based on genomics and bioinformatics analysis only, more indepth functional studies are required to get a better understanding of the asymptomatic infection of bats by viruses highly lethal in other mammals. the importance of bats as a source of emerging viruses has been proven from numerous studies in the last two decades. while most of the investigation is triggered by zoonotic spillover of bat viruses, the impact of spillover into livestock animals should not be underestimated as shown by the sads-cov outbreak. from the great genetic diversity and wide geographical locations of the various bat viruses detected so far, it is almost certain that we will see more and more disease outbreaks caused by bat viruses. among the 'known unknowns', bat coronaviruses may be a more likely cause of future spillover into both human and livestock populations due to their greater genetic diversity already known in bats around the world, their large positive strand rna genome size with a high rate of recombination, and proven spillover events in both human and animals. we are still at an infancy stage in terms of understanding bat biology in the context of how some of the most lethal 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show that all building blocks of sars-cov are present among a diverse group of covs in the same cave a bat-derived putative cross-family recombinant coronavirus with a reovirus gene an important finding demonstrating that covs are highly active in recombination, even with viruses outside the coronavirus family the persistent prevalence and evolution of cross-family recombinant coronavirus gccdc1 among a bat population: a two-year follow-up coronaviruses: emerging and reemerging pathogens in humans and animals animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? contraction of the type i ifn locus and unusual constitutive expression of ifnalpha in bats proteomics informed by transcriptomics reveals hendra virus sensitizes bat cells to trail-mediated apoptosis unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing dampened sting-dependent interferon activation in bats the egyptian rousette genome reveals unexpected features of bat antiviral immunity we thank xiao fang lim, xinglou yang and anna uehara for help with figure 1 , figure 3 and table 1 key: cord-285656-7o7ofk1e authors: dawson, harry d.; chen, celine; gaynor, brady; shao, jonathan; urban, joseph f. title: the porcine translational research database: a manually curated, genomics and proteomics-based research resource date: 2017-08-22 journal: bmc genomics doi: 10.1186/s12864-017-4009-7 sha: doc_id: 285656 cord_uid: 7o7ofk1e background: the use of swine in biomedical research has increased dramatically in the last decade. diverse genomicand proteomic databases have been developed to facilitate research using human and rodent models. current porcine gene databases, however, lack the robust annotation to study pig models that are relevant to human studies and for comparative evaluation with rodent models. furthermore, they contain a significant number of errors due to their primary reliance on machine-based annotation. to address these deficiencies, a comprehensive literature-based survey was conducted to identify certain selected genes that have demonstrated function in humans, mice or pigs. results: the process identified 13,054 candidate human, bovine, mouse or rat genes/proteins used to select potential porcine homologs by searching multiple online sources of porcine gene information. the data in the porcine translational research database ((http://www.ars.usda.gov/services/docs.htm?docid=6065) is supported by >5800 references, and contains 65 data fields for each entry, including >9700 full length (5′ and 3′) unambiguous pig sequences, >2400 real time pcr assays and reactivity information on >1700 antibodies. it also contains gene and/or protein expression data for >2200 genes and identifies and corrects 8187 errors (gene duplications artifacts, mis-assemblies, mis-annotations, and incorrect species assignments) for 5337 porcine genes. conclusions: this database is the largest manually curated database for any single veterinary species and is unique among porcine gene databases in regard to linking gene expression to gene function, identifying related gene pathways, and connecting data with other porcine gene databases. this database provides the first comprehensive description of three major super-families or functionally related groups of proteins (cluster of differentiation (cd) marker genes, solute carrier superfamily, atp binding cassette superfamily), and a comparative description of porcine micrornas. electronic supplementary material: the online version of this article (doi:10.1186/s12864-017-4009-7) contains supplementary material, which is available to authorized users. swine are an important models for human anatomy, nutrition, metabolism and immunology [1] [2] [3] . their organs are anatomically and histologically similar to humans as are their sensory innervation and blood supply [4] . pigs are naturally susceptible to infection with organisms that are closely related or identical to those species infecting humans including helminths (ascaris, taenia, trichuris, trichinella, shistosoma, strongyloides), bacteria (campylobacter, chlamydia, eschericia coli, helicobacter, neisseria, mycoplasma, salmonella), protozoans (toxoplasma) and viri (coronavirus, hepatitis e, influenza, nipah, reovirus, rotavirus) [2, 5, 6] . the last 10 years has seen a boon in the development of genetically modified pig as models for human cardiovascular and lung disease, neurodegenerative and musculoskeletal disorders [7, 8] and cancer [9] . there is also a robust effort to develop pigs as sources for organs and tissues for human xenotransplantation [10] . despite these potential strengths as a model, the lack of an annotated database for porcine gene and protein expression data is a limiting factor for translating findings in one species to another. multiple online databases exist for the storage and retrieval of diverse bovine, rodent or human biomedical data [11] [12] [13] [14] [15] [16] [17] [18] [19] . other databases exist for zebrafish (zfin, [20] ), c. elegans (wormbase, [21] ), and drosophila melanogaster (flybase, [22] ). databases that encompass multispecies analysis such as homologene and/or that rely on manual annotation such as innatedb [23] include bovine but not porcine genes. several porcine genome companion databases exist; however they lack robust manual annotation and are somewhat limited in scope or are infrequently updated [16] [17] [18] [19] . agbase, a large, multispecies functional analysis database allows the user to search 51,489 porcine genes based on 12 criteria including gene and protein names (uniprot) and gene ontology (go) annotations. furthermore, databases can contain a significant number of errors due to their primary reliance on machine-based annotation [24] . for example, the sus-bar database [19] is designed to identify protein orthologs based upon data that includes annotations from the machine-annotated ncbi genome. ncbi has recently begun to include go annotations into curated entries for non-human and rodent species but most of these are indirect and often based on observations made in other species. as swine are an important model for comparative human studies, there is a critical need to have a centralized, manually-curated source of information for biomedical research. to address these needs, we created the porcine translational research database. to generate content of immunological relevance, broadbased literature searches were conducted using the following terms: apoptosis, b cell development or activation, cd markers, chemokines, chemokine receptors, cytokines, cytokine receptors, dendritic cells, type 1 ifn induced genes, inflammation, nuclear factor kappa-lightchain-enhancer of activated b cells (nfκ-b) signaling pathway, toll receptor signaling pathway, t cell development or activation, th1 cell development and th2 cell development. in addition, immunologically related genes associated with the susceptibility to or pathology of allergy, asthma, arthritis, atherosclerosis and inflammation were included. in addition, the gene ontology consortium's community annotation wikis for immunology, cardiovascular disease and muscle biology were searched (http://wiki.geneontology.org/index.php/main_page). the jackson laboratory database of knockout mouse phenotypes was searched for genes leading to defects in immune or metabolic phenotypes when over or under expressed. these genes include the vast majority of genes that are related to immunity and inflammation [2, 3, 25, 26] . for additional metabolically related genes, genes involved in the transport or metabolism of macronutrients, trace vitamins and minerals were searched. other genes, associated with the susceptibility to or pathology of atherosclerosis, diabetes, and obesity, were identified. this process identified 13,054 candidate human, bovine, mouse or rat genes/proteins of interest used to select potential porcine orthologs by searching various online sources of porcine gene information. one to one orthology of protein coding genes were determined by protein structure similarity (best reciprocal blast hits) and the presence of a corresponding gene in the syntenic region of the human and or mouse genome. no 1:1 orthology could be established for members of some gene families including the leukocyte immunoglobulin-like receptor (lilr) killer cell immunoglobulin-like receptor (kir), carcinoembryonic antigen-related cell adhesion molecule (ceacam) and cytochrome p450 superfamilies. one to one porcine orthologs of human genes utilize the approved hgnc name according to the international society for animal genetics (isag) publishing guidelines. we defined pseudogenes by the criteria used by ensembl and enscode; namely the presence of one or more stop codons in the open reading frame that disrupt the protein structure, and (usually) a lack of intron structure at the genome level [27] . pseudogenes are further classified into processed, duplicated, unitary or polymorphic categories [27] . genbank (non-redundant, expressed sequences tag, high throughput genomic sequence, trace archive databases and whole genome shotgun contigs databases) was searched by discontiguous megablast using default settings (word size = 11), using reference sequence accession numbers to human, bovine, mouse or rat genes/ proteins of interest. a similar search was conducted in the following databases using the human or bovine reference sequence; nih intramural sequencing center (nisc) comparative vertebrate sequencing project [28] ; national center for biotechnology information (ncbi), sus scrofa genome assembly releases 102 to 105 and ensembl v10.2 releases 83 to 89. for genes that were determined to be missing from build 10.2 (additional file 1) (and for the mis-assembled or duplicated gene artifacts (additional file 1), we also constructed templates from de novo assemblies derived from illumina 80 bp reads of the pig alveolar macrophage transcriptome (dawson, unpublished results) using the de novo assembly algorithm of clc genomics workbench using word size of 20 and a bubble size of 50. when necessary, predicted templates (from bovine or human sequences) were supplemented with porcine expressed sequence tag (est) assemblies, single ests and portions of the published tibetan (bioproject # prjna291130), wuzhishan (bioproject # prjna144099), goettingen (bioproject # prjna291011) [29] , jinhua, meishan, bamei, large white, berkshire, hampshire, pietrain, landrace, rongshang and duroc (bioproject # prjna309108) porcine genomes [30] . ests were assembled using cap3 (http://doua.prabi.fr/software/cap3). rnaseq reads were then mapped to these predicted templates in order to derive the full-length consensus sequence (unambiguous 6x coverage) using clc genome workbench 7.0 (qiagen bioinformatics, redwood city ca). the following settings were used. mismatch cost =2, insertion cost = 3, deletion cost = 3, similarity fraction = 0.95, length fraction = 0.95. nucleotide sequences were translated using the expasy translate tool (http://web.expasy.org/translate/). a total of 1279 of these sequences have been deposited to the transcriptome shotgun assembly sequence database under bioproject prjna80971 and the short read archive under project srp013743). in silico-derived full-length rna sequences are provided for an additional 3391 genes. this process/pipeline is summarized in fig. 1 . a summary of these sequences is provided in table 1 . we randomly chose 268 of these mrna for comparison of the 5′, 3′ and orf length comparison to the corresponding human mrna. data are presented in additional file 4. for the 1041 protein-coding genes missing from the genome, we entered the gene symbols into the david version 6.8 (https://david.ncifcrf.gov) to assess overrepresentation of groups of gene with related function. the functional data were limited to human. nine hundred and fifty six genes out of 1041 genes were recognized and 955 had functional annotations, of the unrecognized gene 41 are pig or artiodactyl specific genes. data on functional enrichment of genes with a multiple comparison adjustment (benjamini) value of >0.05 are presented in table 3 . we chose the 60 largest proteins of extreme size (>3000 amino acids) to compare the status (number of loci and completeness) in the ncbi and ensembl build 10.2 genome. because exon preservation is usually well conserved and there is fragmentation of certain areas of the porcine genome, the number of exons for the corresponding human gene was used for comparison. lastly, we determined the chromosomal location of 1307 duplicated gene artifacts (2889 loci, additional file 2) to identify problematic regions. data are expressed as duplication per megabase (number of bases derived from the ncbi genome build (http:// www.ncbi.nlm.nih.gov/genome?term=sus%20scrofa) and are presented in fig. 2 . the currently described database was constructed in the filemaker po advanced v14.0 program (filemaker inc., it was deployed using the filemaker server advanced v14.0 program (filemaker inc., santa clara, ca). external access to the database has been successfully tested using chrome, internet explorer and safari browsers. other areas of the database were populated from existing published or our own unpublished data. each publication is manually reviewed and data (antibodies, real-time pcr assays, rna or protein expression data, functional data) is abstracted and entered into the database, along with the pubmed id, in the appropriate field. we have developed taqman real-time pcr assays for 1867 of these genes making them cross reactive for as many species as possible (1067 are partially or fully human gene cross reactive). this is to ensure that comparable areas of the gene are being analyzed as well as for economic reasons. we also conducted a literature survey to determine the sequence of porcine sybr green pcr assays. tissue-specific gene expression summaries, using these assays, are provided for these and other studies (i.e., those using microarray and rnaseq), and a comprehensive search of catalog and published literature to identify antibodies to the corresponding proteins. last, the "notes field" in the database was populated with information such as types of errors discovered, degree of 5′ and 3′ utr conservation, degree of positive selection pressure in various species, and intron status. when the gene (sequence) is present in the genome but not annotated as a gene, we annotate the gene in the notes field as "not an identified gene in ensembl build 10.2." or "not an identified gene in ncbi build 10.2". to date, we have generated 9720 full-length transcripts representing 9165 genes (table 1) . they include 1354 genes missing from ensembl build 10.2 (table 2 and additional file 1) and 1400 genes that have been sequenced at least two times (gene duplicated artifacts shown in table 2 and additional file 2 that were annotated as separate genes in either ensembl or ncbi builds. functional enrichment analysis of 1041 proteincoding genes that are missing from the genome reveals that genes that are annotated as cytokines (24, p = 0.0053) and transcription factors (68) (particularly homeodomainlike transcription factors (34, p = 0.032) and cenp-b/ helix-turn-helix (hth) domains (6, p = 0.035) are significantly overrepresented (table 3) . of note, the great majority of the interleukin 1 superfamily (il1f10, il1rn, il36a, il36b , il36g, il36rn, il37) members are significantly (p = 0.0073) overrepresented. data analysis that do not account for these genes risk missing assessment of important genes involved in inflammation and development. based upon gene number estimates from other closely related species such as human and cow, we estimated that our database has a coverage rate of approximately 42% of the porcine genome. these represent sequences found in 10,232 unigene entries (1.45 per gene), 9967 ncbi loci (5756 are single loci that are not duplicated gene artifacts or split into multiple loci, and 1793 genes have multiple (4211) loci. a total of 2109 and 1616 of the genes have no assigned unigene number or ncbi loci, respectively. in addition to go and kyoto encyclopedia of genes and genomes (kegg) annotations, literature-based functional annotations (derived from more than 5500 references) are provided for these sequences. we have also discovered a relatively large number (178) of porcine or artiodactyl-specific paralogs (additional file 3) for 104 protein or non-protein coding porcine genes. for genes with multiple paralogs, genes are named in the order of phylogenetic distance of the parent human or bovine gene. some of these genes are expressed pseudogenes. some of these genes have been previously discussed (i.e., cd36, il1b [25, 31] ) or will be discussed in the following sections. the transcripts we have generated for protein-coding genes include, on average, 70.5% of the corresponding 5′ and 3′ ends (each) of the human sequence (additional file 4). the orf is 99.4% conserved on a nucleotide count basis. these percentages indicate the fidelity of our procedure. we discovered extensive gene truncation (incomplete orf) and gene duplicated artifacts (genes sequenced more than once) among the machine annotated versions of these genes. these problems are common among 1st drafts of other genomes [32, 33] . gene duplicated artifacts appear most frequently for chromosomes 12, 2 and 3, and less frequently for chromosomes x, 11, 13, and 1 (fig. 2) . the most frequent areas should be targeted for re-sequencing or reassembly. analysis of the 60 largest porcine proteins in the database shows that gene fragmentation and truncation roughly correlate with protein size and number of exons (table 4 ). figure 4 shows blast search results from two extremely large proteins, hemicentin (hmcn1, panel a) and titin (ttn, panel b) that have 9 loci assignments each, in the current ncbi build. surprisingly, these proteins are not represented in ensembl build 10.2 as annotated genes. overall, of the 60 largest porcine proteins, only 6 and 10 are represented as single full-length sequences of the correct size in ensembl and genbank, respectively. we have deposited 12 de novo assemblies in the tsa archive and have provided in silico predicted rna and protein sequences for 37 of these genes. in previous studies, we extensively compared porcine, human and mouse genes related to immunity and inflammation [2, 3, 25, 26] . in the following section, we will summarize our findings for three major superfamilies or functionally related groups of proteins (cd marker genes, solute carrier superfamily, atp binding cassette superfamily) or non-coding rna (microrna) that have complete or nearly complete representation. cd markers (accessible as a group by entering cd markers in the annotations field) encode a heterogeneous group of cell surface proteins. the human leucocyte differentiation antigen (hlda) workshop has designated 408 molecules (some of which are grouped within a cd) as cd markers [34] . based upon our assembly and analysis, we could establish 1:1 orthology for 357 porcine genes to those that compose hlda version 10. forty-three genes are not present in the porcine genome or could not be designated as 1:1 orthologs. of these, nine genes (clec4c, clec4m, [35] [36] [37] . klrc2 (cd159c) is found in humans and rodents but not pigs. fcgr2c is a human-specific gene/pseudogene that belongs to a family of three low-affinity immunoglobulin gamma fc receptors (cd32) [38] . we have determined that pigs have two member of this family that roughly corresponds to fcgr2a and fcgr2b. tnfrsf14 (cd270) is a marker for b cells, dendritic cells, monocytes, and treg cells [39] found in humans and rodents, but not cows. although, canine, feline, equine and ursine homologs have been identified, this gene may be a pseudogene in pigs as the putative orf is interrupted by an endogenous retroviral sequence (h. dawson, unpublished). fcrl2 (cd307b) is a marker for b cells in humans. although sequences corresponding to fcrl2 have been identified in other mammals including dog and horse, no mouse ortholog has been identified [40] . this gene shows evidence of positive selection in humans [41] and is most likely a pseudogene in pigs. due to rapid evolution and post-speciation gene duplication, no 1:1 orthology could be established for most mouse and pig lilr or kir family members, including lilra4 (cd85g) and lilrb4 (cd85k) [42] . similarly, other than ceacam1 (cd66) and ceacam6 (cd66c), no 1:1 orthology could be established for most pig and mouse ceacam family members (ceacam3 (cd66d) ceacam5 (cd66e). ceacam8 (cd67) may be a pseudogene as ests in unigene ssc.60435 predict a 243 amino acid protein interrupted by several stop codons. ceacam8 and ceacam6 were previously determined to have no direct murine orthologs [35] . several other shared human-pig cd marker orthologs (adgre2 (cd312), adgre3 (cd313r), cd1a, cd1e, cr1 (cd35), cd58, fcgr2a (cd32), fcar (cd89), fcrl3 (cd307c), fcrl4 (cd307d), icam3 (cd50), ncr2 (cd336), ncr3 (cd337) and tlr10 (cd290r) have no rodent orthologs [2, 40, 43] . a significant number of errors were discovered in genes encoding porcine orthologs of human cd markers; 25 are not present in ensembl build 10.2, 88 of the proteins are truncated and 52 are duplicated gene artifacts. sixty-seven full-length mrna sequences encoding proteins, assembled from macrophage rna-seq reads, have been deposited in genbank. an additional 79 in silico constructs are provided. antibody data, gathered from publications, manufacturers or generated in house, is provided for 186 proteins including 395 monoclonal and 285 polyclonal antibodies. additional cross reactivity for 29 proteins is expected because they are >95% similar to human proteins. several of the cd marker family are members of other gene families including the solute carrier and atp-binding cassette super family. the human genome organization's gene nomenclature committee (hgnc) has assigned 395 genes to the solute carrier superfamily, 21 are pseudogenes and three hundred seventy four encode proteins (accessible as a group by entering solute carrier superfamily in the annotations field). these are organized into 52 subfamilies; about 25% are dedicated to nutrient transport. the porcine solute carrier super family contains 398 protein-coding members and all human subfamilies are represented. forty-two of these genes are present in other porcine genomes but missing from ensembl build 10.2, 113 are truncated and 58 of these are duplicated gene artifacts. sixty three full-length mrna sequences, assembled from macrophage rna-seq reads, have been deposited in genbank and an additional 159 in silico constructs are provided. forty-two of these genes are missing from all porcine genomes or are present as pseudogenes. among these genes are ucp1 (thermogenein), a protein involved in non-shivering thermogenesis and a pseudogene in pigs [44] and slc52a2, a primate specific riboflavin transporter [45] . other speciesspecific genes include eight primate-specific (slc2a14, slc22a24, slc35e2, slc35g3, slc35g4, slc35g5, slco1b1, slco1b7), one human specific (slc22a25) gene and 14 mouse or rodent-specific genes (slc6a20b, slc7a12, slc21a4, slc22a19, slc22a21, slc22a22, slc22a26, slc22a27, slc22a28, slc22a29, slc22a30, slco1a1, slco1b2, and slco6b1). slc25a18 is present in human and rodent genomes but is missing from bovine and porcine genomes. slc25a52 is present in primate and rat genomes but not mouse. slc9c2 is a pseudogene in mouse [46] . slc22a31 is an expressed pseudogene in pigs and is missing in rodents. slc22a11 is an expressed pseudogene in pigs and a non-expressed pseudogene in mouse. lastly, slc23a4, an intestinal nucleobase transporter [47] , is a pseudogene in humans but is present in pig, cow and rodent genomes. several porcine or artiodactyl-specific gene expansions are found in subfamilies (additional file 3) including slc7a3 (14 members), slc7a13 (3 members) slc22a6 (2 members), slc22a10 (4 members) and slc47a1 (2 members). the biological functions of these paralogs remain to be determined; however the parent genes are involved in amino acid (slc7a3, slc7a13) or dipeptide transport (slc22a6) [48, 49] . the hgnc has assigned 51 genes to the atp binding cassette superfamily, three are pseudogenes and 48 encode proteins (accessible as a group by entering atp binding cassette superfamily in the annotations field). these are organized into five subfamilies (a-g), about 20% are dedicated to nutrient (i.e., carotenoid, cholesterol and vitamin a) transport. the porcine atp binding cassette family contains 57 members and all human subfamilies are represented. these include five that are missing from ensembl build 10.2 and 18 that are duplicated gene artifacts. five of these genes are present in other porcine genomes, but missing from ensembl build 10.2, 21 are truncated, and 18 of these genes are duplicated gene artifacts, eleven full-length mrna sequences, assembled from macrophage rna-seq reads, have been deposited in genbank and an additional 24 in silico constructs are provided. an analysis of this superfamily revealed that abcc11 has no murine ortholog [50] and abca8 has no direct rodent ortholog as the gene has diverged into two paralogs, abca8a and abca8b [51] . the abcc4, a prostaglandin e2 transporter [52] , has diverged from the parent gene into five paralogs (abcc4l1, abcc4l2, abcc4l3, abcc4l4 and abcc4l5 (additional file 3). abca10, involved in human macrophage cholesterol transport [53] , is a pseudogene in rodents. it may be an expressed pseudogene in pigs as the predicted protein is half (787 amino acids) the size of human abca10 (1543 amino acids) and weak expression (by rnaseq) was detected in macrophages and moderated expression in intestine (h. dawson, unpublished) . abca17 is an expressed pseudogene is humans and pigs. like the solute carrier superfamily, most of the genes in the atp binding cassette super family have not been characterized at the functional level. nevertheless, the similarities and differences in the atp binding cassette and atp binding cassette super families impact the suitability of rodents and pigs as models for human drug and nutrient transport and metabolism. the exact number of micrornas in the porcine genome is unknown. there are 4272 annotated micrornas in the human genome (build 30). although there are several papers describing the measurement of porcine micrornas in various tissues or estimating the number in the porcine genome [54] [55] [56] [57] and three partially overlapping sources of porcine microrna sequences, the exact number of porcine micrornas is currently unknown. there are only 382, 385 and 816 (non-redundant) annotated pig mirna sequences in mirbase, ncbi gene build, and ensembl build 10.2, respectively. these three sources of information have a significant amount of overlap (fig. 5a) . we have consolidated this information and provide sequence data for our own predicted sequences based on conserved sequence identity to 1900 human, mouse or bovine sequences, to provide 1033 nonredundant porcine microrna sequences (accessible as a group by entering microrna in the annotations field). of note, all of the sequences found in mirbase were found in the ncbi gene build, 59 of the microrna sequences in ensembl were found to be duplicated artifacts, and 214 of the 1033 sequences are not present in the current ensembl gene build (10.2) . this includes 81 that we have predicted based upon their presence in other species and other unfinished porcine genomes. we discovered the following species-or genera-specific microrna; pigs (454), humans (199), primates (111) bovine (179), mouse (76) and rodents (20) . many of the porcine-specific microrna have arisen from biological duplication/expansion (additional file 3). a comparison of microrna that are present in pigs and shared among at least one of the three other species (human, cows, and mice) revealed that 318 microrna are shared among the four species, 107 are shared between pigs, humans and cows but not mice, and 34 are shared between pigs, mice and cows but not humans (fig. 5b) . thus, the frequency of non-conserved microrna preservation between human and pig is nearly three times that of mouse to pig. the porcine translational research database is named because of its unique utility to translate findings made in rodents to pigs and from those in pigs to humans. a comprehensive literature-based survey was conducted to identify genes that have demonstrated function in humans, mice or pigs. the resulting data in the database is documented by >6000 references. the database currently contains 65 data fields for each entry. our efforts to improve the genome and its annotation are similar to other efforts, for example the sequencing of 12,000 genes to supplement annotation of the pig genome [32, 33, 58] and de novo assembly of multiple pig genomes to reveal 1737 protein coding genes that are missing from ensembl build 10.2 [30] . the online supplemental data from the latter manuscript was unavailable at the time of the preparation of this manuscript so no comparison could be made. the manual assembly of >9700 rna sequences has direct practical implications for genomics-based analysis. the state of the current genome build (mis-annotations, duplication artifacts, and missing sequences) effectively prohibits its use for aligning rnaseq reads. we have used these sequences to compare gene expression separately from ensembl 10.2 and have also compared the number of reads obtained from the corresponding templates in ensembl 10.2. for the great majority of transcripts compared, as expected, our full-length sequences provided a higher level of sensitivity than the corresponding ensembl sequences (h. dawson unpublished) . the full 5′ and 3′ representation of each gene will also allow for characterization of regulatory regions and mirna target sites. in our estimation, >40% of transcripts in ensembl or ncbi genomes do not represent the fulllength gene. our efforts will also allow for further consolidation of porcine unigene numbers. currently, each gene is represented by from 0 to >10 unigene assignments, and >10% of genes have more than one. it is significant that we discovered a large number of errors (about 30% of entries) in the publicly available sequence databases (these can be accessed by searching the "notes field" using the word "error" (fig. 3) ). in addition to the duplication artifacts, mis-annotations and missing genes, we also encountered a number of rna sequences in publically available archives belonging to other species. for, example, human (ahr, af233432.1), panda (il2, nm_001199892.1) and rat (nudt14, ests in unigene ssc.85635) rna sequences are annotated as porcine derived. we also found sources of contaminating dna from completely unrelated species. for example, about 1/5 of porcine chromosome 4 clone cu076066.6 is from zebrafish. these sequences represent 6 zebrafish genes (loc100003615, loc447815, loc108179932, loc108183883, loc108183971, and loc103910681) and are annotated as porcine genes by ensembl build 10.2 (enssscg00000006223) and ncbi genomes (loc100739857). similarly, several ncbi loci (asna1l*, loc100737282, loc100737202, loc100620149, loc 100737282) and one ensembl locus (enssscg 00000026988) are derived from contaminating babesia bigemina genomic dna. we have discovered several sources of systematic errors in the ensmbl and ncbi gene/protein prediction or annotation pipelines. for example all selenoproteins in ensembl are truncated because the codon (uga) for selenocysteine is mistranslated or translated as a stop codon. we and others have identified a systematic error in the identification of another gene family, the taste receptor, type 2 (tas2r) superfamily. despite being intronless and mostly devoid of 5′ and 3′ utr regions, ensembl consistently fails to recognize them as genes [3] . these data illustrate the critical importance of the manual-curation process to reduce errors. we believe that this is the largest manually curated database for any veterinary species and that the infomantics are unique among those targeting a veterinary species in regard to linking gene expression to gene function, identification of related gene pathways, and connectivity with other porcine gene databases, as well as for reagents that measure gene and protein expression. in addition, it is the largest source of centralized antibody information for the pig. any database must be updated frequently in order to be useful. currently the database is updated monthly and we anticipate expanding the content to include all porcine genes. there are several super families of genes that will be the next targets of our efforts. one is the gpcr super family, the exact size of the gpcr super family is still unknown, but nearly 800 different human genes (or~4% of the entire proteincoding genome) have been predicted to code for them. we will also continue to develop and annotate new assays. we intend to include our own prediction analysis for the promoter and 3′ utr region of rna for transcription factor and microrna binding sites. lastly, we intend to synchronize our database with the porcine "snowball" array and porcine gene expression atlas [59] . the pig as a model for human nutrition a comparative assessment of the pig, mouse and human genomes analyses of pig genomes provide insight into porcine demography and evolution livestock models in translational medicine the pig: a model for human infectious diseases chlamydiaceae infections in pig genetically modified pig models for neurodegenerative disorders porcine models of muscular dystrophy pigs as models of human cancers genetic modification of 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nomenclature of cd molecules from the tenth human leucocyte differentiation antigen workshop comparative genomics of natural killer cell receptor gene clusters signal regulatory proteins in the immune system siglecs and their roles in the immune system genomic organization of classical human low-affinity fcgamma receptor genes regulatory t cell expression of herpesvirus entry mediator suppresses the function of b and t lymphocyte attenuator-positive effector t cells fc receptor-like molecules from evolutionary genetics to human immunology: how selection shapes host defence genes inhibitory leukocyte immunoglobulin-like receptors: immune checkpoint proteins and tumor sustaining factors comparative functional evolution of human and mouse cr1 and cr2 the uncoupling protein 1 gene (ucp1) is disrupted in the pig lineage: a genetic explanation for poor thermoregulation in piglets novel riboflavin transporter family rfvt/slc52: identification, nomenclature, functional characterization and genetic diseases of rfvt/slc52 traditional and emerging roles for the slc9 na+/h+ exchangers identification and functional characterization of the first nucleobase transporter in mammals: implication in the species difference in the intestinal absorption mechanism of nucleobases and their analogs between higher primates and other mammals a new member of the cationic amino acid transporter family is preferentially expressed in adult mouse brain human organic anion transporter oat1 is not responsible for glutathione transport but mediates transport of glutamate derivatives characterization of the mouse abcc12 gene and its transcript encoding an atp-binding cassette transporter, an orthologue of human abcc12 evolutionary analysis of a cluster of atp-binding cassette (abc) genes multiple drug resistance-associated protein 4 (mrp4), prostaglandin transporter (pgt), and 15-hydroxyprostaglandin dehydrogenase (15-pgdh) as determinants of pge2 levels in cancer abca10, a novel cholesterol-regulated abca6-like abc transporter microrna buffering and altered variance of gene expression in response to salmonella infection deciphering the porcine intestinal microrna transcriptome structured rnas and synteny regions in the pig genome distribution of mirna genes in the pig genome large-scale sequencing based on full-length-enriched cdna libraries in pigs: contribution to annotation of the pig genome draft sequence a gene expression atlas of the domestic pig the authors declare that they have no competing interests publisher's note the dataset(s) supporting the conclusions of this article are included within the article, its additional file (additional files 1, 2, 3 and 4) and within the online database (http://www.ars.usda.gov/services/docs.htm?docid=6065).authors' contributions hdd, cc, bg and js contributed to the content of the database. hdd and jfu wrote the manuscript. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-283339-pbgeoxdu authors: jonsdottir, hulda r.; dijkman, ronald title: characterization of human coronaviruses on well-differentiated human airway epithelial cell cultures date: 2014-12-18 journal: coronaviruses doi: 10.1007/978-1-4939-2438-7_8 sha: doc_id: 283339 cord_uid: pbgeoxdu the human airway serves as the entry point of human respiratory viruses, including human coronaviruses. in this chapter we outline the methods by which we establish fully differentiated airway epithelium and its use for human coronavirus propagation. additionally, we outline methods for immunofluorescence staining of these cultures for virus detection, characterization of cell tropism, and how to perform antiviral assays and quantify viral replication. the human airway serves as the entry point of human respiratory viruses, including human coronaviruses (hcovs). in order to properly recapitulate the complex anatomy of the human lung specialized cell culture models have been developed to resemble both the upper and lower airways [ 1 -3 ] . primary human bronchial epithelial cells cultured in an air-liquid interface (ali) system serve as a universal platform to study human respiratory viruses [ 4 -6 ] . these human airway epithelial (hae) cultures morphologically and functionally resemble the upper conducting airways in vivo. in these cultures, the epithelial layer is pseudostratifi ed and after differentiation they contain many different cell types such as basal, ciliated, and goblet cells and furthermore, generate protective mucus equivalent to that of in vivo epithelium [ 7 ] . establishment of hae cultures requires time and patience but the differentiated cultures allow for a number of advantageous analyses in respiratory virus research. we have adapted and optimized our methods based on previously published work [ 8 -10 ] . moreover, we have standardized methods for the propagation of human coronaviruses and evaluation of the effects of antiviral compounds on both viral replication and cell viability. we are able to propagate all known human coronaviruses in this system and can easily evaluate their tropism by immunohistochemistry [ 5 , 11 ] . in this chapter we outline the methods by which we establish fully differentiated airway epithelium and use it for human coronavirus propagation. additionally, we outline methods for immunofl uorescence staining of these cultures for virus detection, characterization of cell tropism and how to perform antiviral assays and quantify viral replication. carry out all procedures in a biosafety cabinet according to local biosafety regulations. cell culture fl asks are coated for 2 h with a mixture of type i and iii collagen that is necessary to effi ciently expand the number of primary airway epithelial cells. 5. culture fl asks can be directly used. optional : store coated fl asks at +4 °c for a maximum of 6 weeks. the inserts need to be coated overnight with collagen type iv, necessary for development and long-term maintenance of differentiated primary airway epithelial cell cultures. 1. mix 7.2 ml of fi lter-sterile dh 2 o with 800 µl of collagen type iv solution (0.5 mg/ml). 2. apply 150 µl per 12-well inserts, or 50 µl per 24-well inserts. after completing one plate, make sure that the entire surface of each well is covered with the 1:10 collagen solution. 3. air-dry the inserts overnight in a laminar fl owhood, and afterwards expose them to uv-light (type c) for 30 min. 4. to remove traces of acetic acid wash inserts twice with at least 500 µl of pbs. 5. after these steps, coated inserts can be used directly. optional : store at +4 °c (wrapped in foil) for a maximum of 6 weeks. repeat uv-exposure and washing steps before use. primary epithelial cells can be isolated from whole lung tissue resections of tracheal and/or bronchial origin according to the following protocol. smaller lung tissue resections can be processed with the same protocol. all procedures are performed at room temperature unless stated otherwise. 1. trim the bronchial tissue free of connective tissue and fat using forceps and scissors or a scalpel. if needed, cut the bronchial tissue into 2 cm segments. 2. wash the cleaned tissue three times in washing solution. 3. fill the desired number of 50 ml tubes with 30 ml of wash solution and transfer as many tissue segments as possible into a single tube, until the volume reaches 36 ml. then add 4 ml of 10× digestion solution to each tube, to end volume 40 ml (40 mg protease/0.4 mg dnase). 4. place tubes on a rocking platform/tube roller at +4 °c and incubate for 48 h. 5. place the 50 ml tube containing the digested tissue on ice and add 4 ml of heat-inactivated fbs to each tube (to a fi nal concentration of 10 % ( v / v )), to neutralize protease activity. invert tubes three times. 6. pour solution along with the tissue onto a large petri dish, and gently scrape off the epithelium from the collagen-cartilage surface, using a scalpel in the reverted angle. pool solutions containing dissociated cells into a 50 ml conical tube and wash the petri dish once with pbs. 7. centrifuge for 5 min at 500 × g . wash cells once with hbss and resuspend cells in begm to a concentration of, approximately, 5 × 10 6 cells/ml. 8. count cells using a hemocytometer and seed into collagen coated fl asks with 20 ml of pre-warmed begm. an appropriate amount of cells for t75 fl asks ranges between 0.5 and 1.0 × 10 6 cells. 9. change medium the next day to remove red blood cells and any unattached epithelial cells. 10. to prevent acidifi cation of the medium change it every 2-3 days, until 80-90 % confl uence. when the primary cells have reached 80-90 % confl uence in the expansion phase one can dissociate and seed the dedifferentiated primary cells on collagen type iv coated inserts, according to the following protocol. all procedures are performed at room temperature unless stated otherwise. 1. remove begm and transfer it into a 50 ml tube and wash the cell monolayer twice with 12 ml of hbss. 2. dissociate the bronchial cells for 3 min at 37 °c in a humidifi ed 5 % co 2 incubator with the appropriate amount of trypsin (25 cm 2 : 1 ml, 75 cm 2 : 3 ml). if needed tap the fl ask to dissociate the cells ( see note 7 ). 3. collect the cells in the previously collected begm and centrifuge for 5 min at 500 × g . carefully discard the supernatant and resuspend cells in hbss and centrifuge the suspension for 5 min at 500 × g . discard the supernatant and resuspend cells in pre-warmed ali medium and count using a hemocytometer. 8. the next day, medium in the apical compartment must be changed to remove any unattached cells. discard the old medium and wash the apical surface with 500 µl hbss and apply 500 µl of pre-warmed ali medium to the apical side. adjust volume to 200 µl for 24-well inserts. all incubation steps are performed at room temperature on a gyrorocker (20-30 rpm), unless stated otherwise 1. after the apical washing has been collected the apical surface is washed twice with 500 µl of pbs before cells are fi xed with formalin-solution for later immunofl uorescence analysis. 10. apply primary antibodies ( see table 2 ) diluted in 250 µl cb solution dropwise to the apical surface and incubate for 120 min. 11. wash the apical surface three times with 500 µl of cb solution for 5 min ( see note 11 ). 12. apply the appropriately diluted conjugated secondary antibodies in 250 µl cb solution dropwise to the apical surface and incubate for 60 min. 13. wash the apical surface twice with 500 µl of cb solution for 5 min. 14. incubate cells with nucleic acid counter stain solution diluted in 250 µl of cb solution for 5 min. 15. wash the apical surface once with 500 µl of cb solution for 5 min. 16. lastly, wash the apical surface twice with 500 µl of pbs for 5 min to remove residual saponin and restore cell membrane integrity. 17. before removing the washing solution, apply mounting medium on a glass slide (use 1-2 drops). remove any air bubbles. 18. excise the membrane from the plastic holder and carefully place the basolateral side of the membrane on top of the mounting medium, without generating air bubbles. 19. then slowly add one drop of mounting medium on top of each membrane. 20. slowly place the coverslip, in a tilted fashion, on top of the membrane without generating air bubbles. 21. allow the mounting medium to polymerize for 30 min, after which the slide can directly be analyzed. 1. pre-warm ali medium to 37 °c. 2. mix antiviral compounds (e.g. k22, recombinant interferons) in various concentrations or by serial dilution in ali medium. include non-treated controls. also, to exclude viral inhibition by solvents (e.g. dmso) include solvent controls. 3. for evaluation of either prophylactic or therapeutic effects of antivirals, the hae cultures can be incubated with the compounds diluted in the basolateral medium prior to, during or after infection. 4. infect cultures apically with human coronaviruses as described in subheading 3.2 . 5. collect apical washings in hbss as described in subheading 3.2 for viral quantifi cation by plaque assay and cells for viral quantifi cation by renilla luciferase assay or qrt-pcr. celltiter-glo substrate to room temperature. 2. transfer the buffer to the amber bottle containing the substrate to reconstitute the enzyme. mix by gently swirling the bottle. 3. wash the apical side of the hae cultures three times with 500 µl hbss to remove excess mucus. 4. apply 50 µl of hbss to the apical side and mix with equal volume of reconstituted celltiter-glo enzyme solution (optimized for 24-well inserts, for other insert sizes adjust buffer amount accordingly) and incubate for 5 min at room temperature on a gyro-rocker to induce cell lysis. 5. next incubate the plate for 10 min at room temperature to allow for stabilization of the luminescence signal. 6. transfer 20 µl of cell lysate to a white, non-transparent 96-well plate for analysis. 7. record luminescence ( see note 12 ). to account for background signal include empty wells in your analysis. 8. program your luminometer settings with 10 s measure time followed by a 2 s delay. 100 µl of assay buffer should be dispensed into each well. if the luminometer is not equipped with an injector the assay buffer can be added manually using a multichannel pipette. 9. to adjust samples for background include empty wells in your analysis. the current protocol is optimized for hcov-229e, but can easily be adapted to any other cell line and coronavirus strain. 1. seed 150,000 target cells in a 12-well cluster plates with 1 ml of complete medium per well and incubate overnight at 37 °c in a humidifi ed 5 % co 2 -incubator. 2. make 6 tenfold serial dilutions of the harvested virus supernatants in 1 ml and inoculate the cells. 3. incubate inoculum for 2 h at 33 °c in a humidifi ed 5 % co 2incubator before removing the serial diluted virus inoculums from the cells and replace with 1 ml of overlay medium. 4. incubate titration plates for 3-4 days at 33 °c in a humidifi ed 5 % co 2 -incubator. 5 . remove overlay and wash wells twice with water to remove residual avicel. 6. subsequently add approximately 0.5-1 ml of crystal violet solution to each well and incubate for 10 min. 7. remove crystal violet solution and wash the cells once with water and allow the plates to air-dry before counting the number of plaques. 10. the fi xed hae cultures can be kept for 1-3 months at 4 °c if the cb is fi lter-sterilized (0.2 µm) and all the procedure were performed under sterile conditions. after cold storage it is preferential to acclimatize the fi xed cultures for 15 min to room temperature on a gyro-rocker (20-30 rpm) prior to continuation of the staining protocol. 11. to prevent bleaching of the fl uorophores one should cover the inserts from daylight exposure during each incubation step. 12. luminometer settings depend on the manufacturer. however, a measurement time of 1-2 s per well has proved effective. 13. for this assay cultures must be infected with coronaviruses expressing a renilla luciferase reporter gene. 14. if your luminometer is equipped with an injector you must remember to account for priming by increasing the volume of required renilla assay buffer by 2-3 ml. 15. primers targeting hcov-nl63, hcov-hku1, hcov-229e, and hcov-oc43 have been characterized and described [ 4 , 14 , 15 ] . add the volume up to 34 ml, mix gently by inverting the tube three times. filter-sterilize the solution through a 0.22 µm fi lter, and store at −20 °c in aliquots of 3.5 ml in 15 ml tubes to prepare the 1,000× stock, fi rst confi rm the ra concentration of the ethanol stock by diluting it 1:100 in absolute etoh. measure the absorbance at 350 nm using a spectrophotometer and a 1 cm light path quartz cuvette (or nanodrop with 0.1 cm light path), blanked on 100 % etoh. the absorbance of the diluted stock should equal 0.45 (0.045 on a nanodrop) dissolve 42 mg ferrous sulfate, 12.2 g magnesium chloride, and 1.62 g calcium chloride-dihydrate in 80 ml h 2 o ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface air-liquid interface (ali) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drug transport studies differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-c culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures effi cient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential infection of human airway epithelium by human and avian strains of infl uenza a virus mucin gene expression during differentiation of human airway epithelia in vitro. muc4 and muc5b are strongly induced mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells a serum-free method for culturing normal human bronchial epithelial cells at clonal density well-differentiated human airway epithelial cell cultures isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus interferon lambda 4 signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses identifi cation of cell lines permissive for human coronavirus nl63 development of one-step, real-time, quantitative reverse transcriptase pcr assays for absolute quantitation of human coronaviruses oc43 and 229e this work was supported by the 3r research foundation switzerland (project 128-11). key: cord-298301-p1zj6jg9 authors: dey, lopamudra; chakraborty, sanjay; mukhopadhyay, anirban title: machine learning techniques for sequence-based prediction of viral-host interactions between sars-cov-2 and human proteins date: 2020-09-03 journal: biomed j doi: 10.1016/j.bj.2020.08.003 sha: doc_id: 298301 cord_uid: p1zj6jg9 background: covid-19 (coronavirus disease-19), a disease caused by the sars-cov-2 virus, has been declared as a pandemic by the world health organization on march 11, 2020. over 15 million people have already been affected worldwide by covid-19, resulting in more than 0.6 million deaths. protein-protein interactions (ppis) play a key role in the cellular process of sars-cov-2 virus infection in the human body. recently a study has reported some sars-cov-2 proteins that interact with several human proteins while many potential interactions remain to be identified. method: in this article, various machine learning models are built to predict the ppis between the virus and human proteins that are further validated using biological experiments. the classification models are prepared based on different sequence-based features of human proteins like amino acid composition, pseudo amino acid composition, and conjoint triad. result: we have built an ensemble voting classifier using svm(radial), svm(polynomial), and random forest technique that gives a greater accuracy, precision, specificity, recall, and f1 score compared to all other models used in the work. a total of 1326 potential human target proteins of sars-cov-2 have been predicted by the proposed ensemble model and validated using gene ontology and kegg pathway enrichment analysis. several repurposable drugs targeting the predicted interactions are also reported. conclusion: this study may encourage the identification of potential targets for more effective anti-covid drug discovery. according to the world health organization (who), the coronavirus disease (covid-19) pandemic, caused by a novel strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus infection, is one of the most crucial diseases in the current scenario. it has infected over 15 million people from more than 200 countries while causing death of more than 0.6 million people. the disease has created immense pressure and tension in the worldwide healthcare systems. at the end of the year 2019, wuhan city of china reported the first case of the novel coronavirus infection. now from asia to europe and america, its deadly effect is threatening the whole world [1] . genomic analysis showed that sars-cov-2 is phylogenetically related to sars-like bat viruses. hence, bats could be the possible source of the viral replication [2] . pangolins have also been identified as a potential intermediate host of novel coronavirus [3] . the usual symptoms of covid-19 affected patients are pneumonia, shortness of breath, cough and cold, fever, and multiple organ failure [4] . the genetic characteristics of sars-cov-2 should be well understood to fight against this virus. it is a single-stranded rna virus consisting of approximately 27-32kb with particle size ranging from 65-125nm in diameter [3] . the world healthcare systems are rigorously searching for a vaccine to mitigate the spread of the virus. besides that, they isolate the infected patients along with some general medicine as immediate treatment and care. sars-cov-2 comprises four main structural proteins including spike (s) glycoprotein, small envelope (e) glycoprotein, membrane (m) glycoprotein, and nucleocapsid (n) protein, in addition to many accessory proteins [5] . a comprehension of how these virus proteins interact with the host cells for survival and reproduction is essential for drug exploitation. protein-protein interaction (ppi) is one way the viruses interact with their hosts. identifying ppis between the virus and the host proteins helps explain how the virus proteins work and how they replicate and cause the disease. over the past decades, experimental strategies for recognizing ppis have been established. nevertheless, these experimental high-throughput screens are primarily used to classify intra-species ppis while inter-species interactomes remained largely understudied. in comparison, laboratory identification of ppis is usually time-consuming, laborious, and difficult to achieve complete protein interactomes. therefore, efficient computational methods for ppi prediction are used to bridge the gap by presenting experimentally testable hypotheses and removing protein pairs having a low probability of interaction to reduce the selection of ppi candidates. computational techniques have been popularly used for predicting viral-host interactions previously [6, 7, 8] . few works have already pursued a broad range of applications of artificial intelligence (ai) and machine learning (ml) to cover medical challenges and outbreak prediction of covid-19 pandemic, described in [9, 10] . kassani et al. have used publicly available covid-19 dataset of chest x-ray [3] and barstugan et al. analyzed computerized tomography (ct) images in [11] for automatic covid-19 classification using deep learning method. horry et al. [12] have also followed the same path of x-ray based covid-19 detection using artificial intelligence and pre-trained deep learning models. however, barstugan et al. used only ct images for covid-19 classification using a k-fold support vector machine (svm) classifier [4, 11] . these methods have some drawbacks. there is a possibility to raise the issue of over-fitting in deep learning due to the limited number of trained images [3, 12] . besides that, it also has limitations in classifying more challenging instances with vague, low contrast boundaries, and the presence of artifacts [13] . these approaches are time-consuming, carrying extra cost, space, and overhead. there is a portability issue of collecting sufficient input x-ray and ct images of covid-19 patients for the learning process. ozkaya et al. proposed fusing, and ranking deep features to detect covid-19 [14] . in this work, they generated two (16×16 and 32×32) subdatasets of 150 ct images. after improving the performance by deep feature fusion and ranking method, svm has been applied for classification. apart from image-based analysis and prediction, some works have been done on the optimistic drug discovery of covid-19. edison et al. introduced a vaxign reverse vaccinology tool and the newly developed vaxign-ml machine learning tool to predict covid-19 vaccine candidates [15] . in an article [16] , the authors have used network-based toolset to covid-19 for recovering the primary pulmonary manifestations of the virus in the lung as well as observed comorbidities associated with cardiovascular diseases. they predicted that the virus can manifest to some special tissues such as the reproductive system, and brain regions using network proximity, diffusion, and ai-based metrics techniques. however, similar kind of works on data-driven drug repositioning framework discovery using machine learning and statistical analysis approaches have been proposed in [17, 18] . it helps systematically integrate large-scale knowledge graph, literature, and transcriptome data to discover the potential drug candidates against sars-cov-2 [17] . in [19] , a novel combination of machine learning, bioinformatics, and supercomputing has been used to predict antibody structures capable of targeting the sars-cov-2 receptor binding domain. the potential result of this article [19] suggests that their predicted antibody mutants may bind to the sars-cov-2 rbd and nullify the virus. batra et al. [18] explored the knowledge of small-molecule treatment against covid-19. it also provided a pipeline to perform high-throughput computational modeling and ensemble docking simulations for screening of covid-19 therapeutic agents. in this article, we have tried to predict the target human proteins of the sars-cov-2 virus based on their protein sequences combining amino acid composition, pseudo amino acid composition, and conjoint triad features using machine learning techniques. the problem has been posed as a twoclass classification problem, where the two classes correspond to the interacting and non-interacting proteins of the virus and the host, respectively. this is the first work in this domain as per our knowledge. initially, we have employed the learning vector quantization (lvq) technique for feature subset selection as a preprocessing step. subsequently, after feature reduction, we have used some popular supervised learning algorithms such as support vector machine (svm), naive bayes (nb), random forest (rf) and k-nearest neighbor (knn) along with a deep multi-layer perceptron model and ensemble techniques (voting classifier, xgboost, adaboost) for classification and prediction. we have used 10-fold cross-validation, repeated 10 times strategy for the supervised learning process. in terms of accuracy perspective on the test dataset, the voting classifier ensemble technique performs better than the other algorithms. therefore, we have predicted the 1326 new potential human protein targets of the sars-cov-2 virus using an ensemble algorithm. gene ontology and pathway enrichment for these predicted interactions are investigated. moreover, we have reported a number of repurposable drugs which target the predicted interactions. in this section, we describe the data sets used for our work followed by the methodologies employed. this section describes the different data sets used in this study. in [5] , gordon et al. prepared a sars-cov-2-human protein-protein interactions (ppis) database between human proteins and novel coronavirus proteins using affinity-purification mass spectrometry (ap-ms). the database contains 332 unique interactions between 332 human proteins and four structural and as well as 20 accessory coronavirus proteins. the degree distribution of the sars-cov-2 proteins in the sars-cov-2-human ppi network is given in figure 1 . these experimentally validated 332 human proteins are used to construct positive training and testing datasets in our study. a summary of the ppi network of sars-cov-2-human is shown in figure 2 using cytoscape [20] . the total network is given in supplementary file s5. there is no "gold standard" for planning a negative dataset in the ppi network since noninteracting protein (negative sample) pairs are not experimentally established. therefore, negative samples must be chosen with care, which may adversely influence the accuracy of predicted ppis. there are two main sampling methods, namely random pairing, and subcellular localization. most of the studies have generated non-interacting proteins at random and then eliminates the pairs used in the positive examples [21, 22] . some studies construct the negative samples having different subcellular localization compared to the positive samples [23] . however, both of these methods are not completely reliable. in the case of random sampling, it may incorrectly take a significant number of positive samples as negative samples and produce different accuracy for different random pairing. in [21] ben-hur et al. proved that in ppi prediction, subcellular localization based methods may generate biased accuracy. we have prepared the negative samples in this article by selecting human proteins from the hprd database release 9 [24] that are not present in the positive dataset and that have a low degree in the human ppi network. viral protein pathway-based research showed that they appeared to target higher-degree human proteins rather than lower-degree proteins [25] . with the aid of cytoscape, we determined the degree of each human protein in the hprd database and ordered it in ascending order. then the negative samples are prepared taking into account the minimal degree of human proteins. this degree-based method of negative sample generation achieves better accuracy on the training dataset compared to the random pairing and subcellular localization (table 1 ). in order to prevent statistical differences, the same scale is assumed for the positive and negative sample, i.e., the ratio 1:1. an independent dataset is prepared to predict unknown human proteins that may indulge in protein-protein interaction with corona viral proteins. all the human proteins of the hprd database (around 9155 proteins) except the proteins that are present in the positive and negative datasets are considered as the independent dataset. a set of well-known supervised machine learning algorithms [26] , such as svm, naive bayes, random forest, deep learning, knn, and ensemble techniques are used for predicting ppis. we have implemented all these algorithms in python frameworks. support vector machines (svm) is one of the most powerful supervised learning algorithms which is based on the concept of hyperplane and it is a generalization of a simple and intuitive classifier called the maximal margin classifier. for a given training sample, the algorithm generates an optimal hyperplane that maximizes the margin between data points of different classes. for a two-class dataset, the nearest objects from each class should be well separated from the decision boundary [27, 28] . svm supports the concept of soft margin classifier because it can be violated by some of the training observations [29] . it can be represented as, in equation (1), c is a nonnegative tuning parameter, m is the width of the margin and the optimization problem chooses β 0 , β 1 , ..., β p to maximize m . 0 , 1 , ....., n are slack variables that allow individual observations to be on the wrong side of the margin or the hyperplane. x * are the observations [29] . k nearest neighbor (knn) algorithm is one of the simplest supervised learning algorithm used for both classification and regression problems. it is also called as the lazy learner as it uses all the data for training while doing prediction [27, 28] . we have to choose the value of kfirst (k ≤ √ n), where n is the size of the data). then it calculates the distance between test data and each row of training data with the help of any distance metric function (use hamming distance for categorical naive bayes is a probabilistic classifier, based on bayes' theorem. in simple terms, a naive bayes classifier assumes that the presence of a particular feature in a class is unrelated to the presence of any other feature. so, each feature makes an independent and equal contribution to the outcome. bayes' theorem finds the probability of an event occurring given the probability of another event that has already occurred. bayes' theorem is stated mathematically as the following equation, in equation (2), p (a|b) is the posterior probability, p (b|a) is the maximum likelihood and p (a) is the prior probability. basically, we are trying to find the probability of event a, given the event b is true. event b is also termed as evidence. in naive bayes perspective, p (y|x), y is class variable and x is a dependent feature vector (of size n) where, x = (x 1 , x 2 , x 3 , ......, x n ). therefore, we can rewrite the naive bayes equation for a set of independent features as, now, we need to create a classifier model. for this, we find the probability of a given set of inputs for all possible values of the class variable y and pick up the output with maximum probability. this can be expressed mathematically as, in equation (4), p (y) is also called class probability and p (x i |y) is called conditional probability. naive bayes has been successfully used to predict the protein-protein interactions and binding sites of dna/rna. for ppi, the algorithm generates binary classification output based on the protein sequence vector [27, 29] . ensemble modeling is a powerful way to improve the performance of the model. in order to improve the performance of the model, it combines several base models into an optimal predictive model. there are two kinds of ensemble techniques bagging and boosting. • in bagging (bootstrapping and aggregation) technique, we create multiple bootstrapped subsamples (row sampling with replacement) from the original one and apply the decision tree learning model on each of the bootstrapped subsamples. after each subsample decision tree has been formed, an algorithm is used to aggregate over the decision trees to form the most efficient predictor. in order to choose the most efficient predictor, it follows the majority of the voting classifier technique. this bagging concept is also applicable to various sets of classifier models. in our work, we have applied the majority voting classification technique on combining svm-polynomial, svm-radial and rf models to achieve the best accuracy among all models [29, 30] . a single decision tree classification gives low bias and high variance whereas a random forest classifier gives low bias and low variance. this bagging algorithm is also known as the random forest classifier. • boosting is an ensemble technique where new models are added to correct the errors made by existing models. models are added sequentially until no further improvements can be made. the adaptive boosting (adaboost) algorithm helps us to combine multiple "weak classifiers" into a single "strong classifier". the weak learners in adaboost are decision trees with a single split, called decision stumps. adaboost works by putting more weight on difficult to classify instances and less on those already handled well. it is used for both classification and regression purposes [29, 30] . • the extreme gradient boosting (xgboost) algorithm is an implementation of gradient boosted decision trees designed for speed and performance. it has a wide range of applications, portable, flexible implementation and cloud integration of this model is easy. xgboost is an ensemble tree method that applies the principle of boosting weak learners (carts generally) using the gradient descent architecture [29] . xgboost algorithm provides high bias and low variance [30] . mlp is a multiple feedforward artificial neural network that maps input vectors to output vectors [31] . it can be represented by a directed graph with multiple node layers, where the bottom and top layers are input and output layers respectively and others are hidden layers. dmlp is a fully connected network where every node in the upper level has connections with all the nodes in the lower level. each node represents a neuron (or processing unit) with a nonlinear activation function except for input layer nodes. multiple hidden layers are allowed that makes it deep neural [32] . in our work, we have used an adaptive learning rate optimization algorithm which is designed specifically for training our deep neural network [33] . we have created a five hidden layers mlp network along with the relu (rectified linear unit) activation function and 'sigmoid' function at the output layer. we have fitted our model with varying epochs, varying batch-sizes, and binary-cross-entropy as a loss function. a total of 3 sets of sequence-based features, namely, amino acid composition, conjoint triad, and pseudo amino acid composition of the human proteins are considered to train the machine learning models. the fasta sequences of human proteins are gathered from uniprot and the values of these features are extracted from protr [34] . these 3 feature sets are described below. the composition of amino acids explains the percentage of a type of amino acid found in a protein chain. this is one of the simple and effective predictive functions of ppis. the arrangement of amino acids reflects only the abundance in a sequence of each amino acid [27] . many studies have used conjoint triad to completely explain the essential ppi details to represent the properties of amino acid [35] [36] . in the conjoint triad system, the 20 amino acids are divided into seven classes based on their amounts of dipoles and side chains ( table 2 ). every amino acid of a protein chain is then replaced by the number of clusters. chou first proposed the composition of pseudo-amino acids in 2001 [37] . aac only displays the frequency of each amino acid in a sequence, but information on sequence order is lost. compared to aac, pseaac finds protein sequence order along with amino acid compositions [38, 39] . the order of sequence can be extracted from sequence similarity variables such as hydrophobicity, hydrophilicity, and side-chain mass. the performance of prediction depends on two aspects. they are feature extraction and performance of the classifier. feature selection is one of the important tasks in classification algorithms. the datasets used for classification contain a large number of features. most of them are either partially or completely irrelevant and redundant to the classifier. therefore, feature selection is used to select a number of important features so that it can achieve acceptable classification accuracy compared to considering all features. in this work, we have used a well-known kohonen's learning vector quantization (lvq) technique to identify important features and remove redundant features from the original dataset [40] . lvq is a simple, universal, and efficient learning classifier. it is very similar to the k-nearest neighbors. in an n-dimensional feature space, lvq tries to approximate optimal decision borders between different classes with a number of labeled codebook vectors. the euclidean distance measure helps to label the closest codebook vector of an example vector. a feature selection strategy using lvq is evaluated to optimize the hypothesis margin of lvq classification through minimizing its loss function [41] and it generates importance score of each feature based on the class identification. our proposed methodology is represented in terms of algorithmic steps below. input: collect the human proteins that interact with sars-cov-2 virus [5] (positive dataset). i 2 . collect non-interacting human proteins from hprd using the concept of degree distribution (negative dataset). output: predicted potential human target proteins. procedure: 1. combine i 1 and i 2 to construct the training dataset. the pictorial representation of the ppi prediction methodology has been shown in figure 3 . the performance of each classifier is evaluated with 10-fold cross-validation in 10 repeat runs to acquire average values. different measurements like accuracy, kappa, sensitivity, specificity, precision, and f1 score are calculated considering 1:1 positive and negative datasets. these parameters are defined as per the equations (5) to (10) . kappa = observed accuracy − expected accuracy 1 − expected accuracy , where, p recision = t p t p + f p * 100%, where, t p , f p , f n , and t n respectively denote the numbers of true positives, false positives, false negatives, and true negatives. in this work, we have started with 3 types of features (amino acid composition, conjoint triad, and pseudo amino acid composition) of human proteins which results in a 413-dimensional feature vector. the importance of all these features are calculated using lvq. afterthat, the knee point is evaluated to detect a large change in the importance score and extract the most important features. we have extracted 38 significant features from 413 features of the original dataset by applying the procedure. table 3 shows the comparison of performance between selected best 38 features vs all 413 features using all the used supervised learning algorithms. all these values have been calculated by 10 fold cross-validation repeated 10 times and then the average of them is reported. in case of dmlp classifier, the batch size of 32 or 25 is acceptable, with epochs = 100 unless the dataset is very large. for large datasets, a batch size of 10 with epochs between 50 to 100 can be considered. in our case, our covid-19 ppi dataset is quite large in terms of the number of features (413-dimensional feature vectors) for the training dataset. therefore, we have used epochs = 50 and batch-size = 10. after the feature selection, we kept the same set of epochs and batch-size for showing parity in the result the blind dataset is used to prevent bias of the classifiers. 124 proteins (62 interacting + 62 non-interacting) from the training dataset are treated as test datasets. the rest of the proteins are used as the training dataset. the performance of the classifier models using a blind dataset is given in table 4 . it can be seen from the table that sv m radial , sv m p olynomial , and random forest method achieved better accuracy, specificity, and f1 score over other classifiers. although, dmlp gives better accuracy on the training datasets, on the test dataset it can't do well compared to the other models. therefore, to increase the prediction accuracy and minimize the false positives we have built a majority voting based ensemble model using these three methods sv m radial , sv m p olynomial and random forest) to predict unknown sars-cov-2 target proteins of human. it can be seen that the ensemble method achieved better accuracy (72.33%), recall (71.67%), specificity (74.41%), precision (72.41%), and f1 score (72.03%) than all the other classifier models. on both positive and negative 1:1 dataset we measured aac, conjoint triad, and pseaac features. this dataset is used as the training dataset. all the human proteins of the hprd database, which are not present in the positive and negative dataset, are considered for prediction analysis. we have applied the ensemble method on these large numbers of human proteins (9155) of the hprd dataset and predicted 3603 potential interacting human proteins. the prediction results are listed in supplementary file s1 along with their average probability prediction score. however, standard svm and random forest assume that the probability threshold for both classes is equal, i.e., 0.5 for binary classification problem. in this work, we have changed the probability threshold from 0.5 to 0.7 so that we can predict all high probability human target proteins and avoid possible false positives. a higher threshold indicates higher confidence in predicting the positive class. the average probability of the predicted human proteins varies from 0.926 to 0.461. our motivation is to predict high-throughput target human proteins. if we consider the threshold of the average probability 0.9, then we get only 10 target human proteins. for probability factor 0.8, we obtain 340 proteins. therefore, we have set the probability value to 0.7, so that a reasonable number of target human proteins can be predicted. using this procedure, the potential number of target human proteins comes down to 1326 from 3603 (file s2). we have also calculated the degree of these predicted human proteins with respect to all the proteins of the hprd database using cytoscape and included in the supplementary files. the degree value varies from 1 to 168. the top 10 high-degree proteins with the degree and prediction score are listed in table 5 . go is one of the most used annotation systems to obtain the biological relevance of highthroughput experiments. to examine the functional characteristics of these predicted 1326 highprobability human target proteins, go term analysis is done on the biological process (bp), cellular component (cc), and molecular function (mf) categories. it has been noticed that the proteins that have similar cell locations or involved in some biological process or molecular function, are likely to interact with each other. we have collected go annotations of all predicted human proteins from david 6.8 [42] having corrected p-values less than 0.05 to validate the predictions. we have found that some of the most enriched go-cc terms of the predicted human proteins are cytosol, extracellular exosome, cytoplasm, membrane, and nucleoplasm. the go-cc term describes the different locations of a cell, at the levels of subcellular structures and macromolecular complexes. being an rna-based virus, the novel coronavirus attacks either the nucleus or cytoplasm of the host cell and causes a respiratory block in the lungs. therefore, the proteins involving in these cc terms are likely to act as potential coronavirus targets. antigen processing and presentation of exogenous peptide antigen via mhc class i, tap-dependent, nik/nf-kappab signaling, regulation of the cellular amino acid metabolic process, positive regulation of ubiquitin-protein ligase activity involved in the regulation of mitotic cell cycle transition, negative regulation of ubiquitin-protein ligase activity involved in the mitotic cell cycle, etc. are five most enriched go-bp terms collected from the david server. some of the most enriched go-mf terms are like protein binding, atp binding, gtp binding, cadherin binding involved in cell-cell adhesion, poly(a) rna binding, etc. in [43] , the authors examined that the coronavirus proteins especially the spike proteins bind with target human proteins and increase cell adhesion during infection. all the enriched go terms along with involved human proteins and p-values are listed in supplementary file s3. analysis of the kegg pathway shows potential illnesses that can develop in the human body due to covid-19 infection. all the significant pathways of 1326 predicted target human proteins along with the corrected p-values are listed in table 6 . viruses are well known to be exploiting the machinery of host cells for their own replication. the proteasome pathway is one of these intracellular processes that are hijacked by the viruses [44] . myung et al. showed this proteasome pathway is involved with different viruses like retrovirus, human immunodeficiency virus type 1 (hiv-1), simian immunodeficiency virus (siv), and moloney murine leukemia virus (mo-mulv). therefore, the probability of association of this pathway with novel coronavirus is very high. endocytosis is a biological process of transporting particles, such as large molecules, parts of cells, and even whole cells, to bring into a cell. experiments on the sars-cov-2 virus have shown that the endocytic pathway is the main pathways for regulating the entrance of covs into the host cells and thus the endocytic pathway, as well as the involved human proteins, can be extensively studied for the target of anti-viral therapies [45] . in [46] , wenzhong liu et al. showed that because of the failure to regularly combine carbon dioxide and oxygen during sars-cov-2 virus infection, the lung cells have highly severe toxicity and inflammation, which ultimately results in ground-glass pulmonary images. according to our results, novel coronavirus can alter the synthesis of macromolecules and its growth rate and can cause carbon metabolism. the term biosynthetic pathway suggests manufacturing the antibiotics which can help to combat drug-resistant viruses and diseases. one of the main public health issues of recent times is the exponential growth of antibiotic-resistant pathogens. therefore, the predicted human proteins involving this pathway need to be further studied to develop antibiotics for the sars-cov-2 virus. the amino acid plays an important role in the expression of the different viral functions including synthesis of viral coat proteins and the development of full infectious virions. novel coronavirus may alter this amino acid composition and cause several other diseases in the human body. we can conclude from the above pieces of evidence that the predicted human proteins involving these pathways are strongly involved in coronavirus infection and experimental validation is required to establish direct interactions. in this section, we have tried to find out the relation between our predicted 3603 human proteins with the other viruses. our research included six specific human-pathogenic rna viruses, namely, dengue, hiv-1, hcv, ebola, zika, and h1n1. we have found various pieces of evidence that many predicted proteins interact with different medically important viruses from published literature. table 7 shows the number of overlapping predicted human proteins and experimentally verified 29 aldoa , adpgk, glud1, pgam1, hk2, hk1, agxt, pdhb, got1, idh3g, hk3, idh2, eno2, idh1, eno3, pdha2, cat, rpia, pdha1, eno1, shmt1, pfkl, suclg1, idh3b, pfkm, idh3a, pklr, prps2, prps1 biosynthesis of amino acids (p= 5.6641e-6) 21 aldoa, shmt1, pfkl, pgam1, idh3b, pfkm, asl, idh3a, pycr2, got1, idh3g, pklr, eno2, idh2, eno3, idh1, rpia, prps2, [52] human proteins with other viruses. we found a large number of predicted human proteins interact with more than one virus. for example, human protein ikbke has the highest degree of 4. it interacts with four viruses, namely, dengue virus (denv), hiv-1, hcv, and h1n1. a small ppi network of the predicted target humans that interact with at least three viruses is shown in figure 4 . from no effective drug has yet been discovered targeting sars-cov-2 virus. the traditional mechanism for drug development and its approval is much more expensive and time-consuming. on the other hand, repurposing the existing drugs is an alternative method for identification of effective drugs. it can substantially shorten the time and minimize costs relative to new drug development. the human proteins that interact with the proteins of multiple viruses can be good candidate for as targets for drug repurposing. we have found 30 predicted human proteins that interact with the proteins of at least 3 viruses. the u.s. food and drug administration (fda) approved drugs that interact with these human proteins are queried using dgidb (http://dgidb.org/) and reported in table 8 . most of these drugs are used in cancer-inhibiting microtubules treatment, and other drug classes are used for diseases like atherosclerosis, crohn's disease, antiinflammatory agent, tumor necrosis factor, blood circulation and infection. table 8 : list of drugs associated with the predicted target human proteins that interact with the proteins of at least 3 different viruses. in this study, various sequence-based features and computational approaches are used for the first time in predicting the potential human targets of the sars-cov-2 virus. we have used the lvq algorithm for feature selection. hiv, influenza, and other viruses are well researched in various literature works compared to coronavirus proteins as sars-cov-2 which has emerged recently. hiv, the most well-studied virus, is believed to have around 1000 direct interactions with human proteins and 3000 indirect interactions. both hiv-1 and sars-cov-2 are enveloped rna viruses. the sars-cov-2 virus contains a large number of proteins, even more than hiv-1. therefore, it can be assumed that sars-cov-2 is a virus expected to have a large number of interactions with human proteins. however, due to the lack of experimentally validated sars-cov-2-human ppis, most of the possible interactions are currently unknown. in this paper, we have predicted the 1326 potential target human proteins considering a high probability factor (70%) using ensemble modeling. we have also analyzed the go terms and kegg pathway of these predicted human proteins and some of the pathways are also supported by recent literature.some repurposable drugs that target the predicted interactions have also been reported. we hope that this study will help biologists recognize possible associations between novel coronavirus and human proteins and facilitate the development of anti-viral drugs. the authors declare that they have no conflict of interest. file s1 excel file containing predicted human proteins. columns include predicted human protein's uniprot accession, uniprot name, gene name and prediction score.(xlsx) source: https://sites.google.com/site/supplementcovid19work/files file s2 excel file containing predicted human proteins having 70% average prediction probabilty score. columns include predicted human protein's uniprot accession, uniprot name, gene name and prediction score. source: https://sites.google.com/site/supplementcovid19work/files file s3 excel file containing predicted human proteins go terms. source: https://sites.google.com/site/supplementcovid19work/files excel file containing predicted human proteins that interact with other viruses. (xlsx) source: https://sites.google.com/site/supplementcovid19work/files file s5 cytoscape file containing predicted sars-cov-2-human interaction figure (.cys). source: https://sites.google.com/site/supplementcovid19work/files covidiagnosis-net: deep bayes-squeezenet based diagnostic of the coronavirus disease 2019 (covid-19) from x-ray images covid-19 spike-host cell receptor grp78 binding site prediction automatic detection of coronavirus disease (covid-19) in x-ray and ct images: a machine learning-based approach accurate prediction of covid-19 using chest x-ray images through deep feature learning model with smote and machine learning classifiers a sars-cov-2 protein interaction map reveals targets for drug repurposing a review of in silico approaches for analysis and prediction of hiv-1-human protein-protein interactions prediction of interactions between viral and host proteins using supervised machine learning methods a review on host-pathogen interactions: classification and 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key: cord-264031-0y7xbgun authors: wierbowski, shayne d.; liang, siqi; chen, you; andre, nicole m.; lipkin, steven m.; whittaker, gary r.; yu, haiyuan title: a 3d structural interactome to explore the impact of evolutionary divergence, population variation, and small-molecule drugs on sars-cov-2-human protein-protein interactions date: 2020-10-13 journal: biorxiv doi: 10.1101/2020.10.13.308676 sha: doc_id: 264031 cord_uid: 0y7xbgun the recent covid-19 pandemic has sparked a global public health crisis. vital to the development of informed treatments for this disease is a comprehensive understanding of the molecular interactions involved in disease pathology. one lens through which we can better understand this pathology is through the network of protein-protein interactions between its viral agent, sars-cov-2, and its human host. for instance, increased infectivity of sars-cov-2 compared to sars-cov can be explained by rapid evolution along the interface between the spike protein and its human receptor (ace2) leading to increased binding affinity. sequence divergences that modulate other protein-protein interactions may further explain differences in transmission and virulence in this novel coronavirus. to facilitate these comparisons, we combined homology-based structural modeling with the eclair pipeline for interface prediction at residue resolution, and molecular docking with pyrosetta. this enabled us to compile a novel 3d structural interactome meta-analysis for the published interactome network between sars-cov-2 and human. this resource includes docked structures for all interactions with protein structures, enrichment analysis of variation along interfaces, predicted δδg between sars-cov and sars-cov-2 variants for each interaction, predicted impact of natural human population variation on binding affinity, and a further prioritized set of drug repurposing candidates predicted to overlap with protein interfaces†. all predictions are available online† for easy access and are continually updated when new interactions are published. † some sections of this pre-print have been redacted to comply with current biorxiv policy restricting the dissemination of purely in silico results predicting potential therapies for sars-cov-2 that have not undergone thorough peer-review. the results section titled “prioritization of candidate inhibitors of sars-cov-2-human interactions through binding site comparison,” figure 4, supplemental table 9, and all links to our web resource have been removed. blank headers left in place to preserve structure and item numbering. our full manuscript will be published in an appropriate journal following peer-review. the ongoing global covid-19 pandemic caused by the infection of sars-cov-2 has to date infected impact of mutations on interaction binding affinity and performed a comparison of protein-protein and 96 protein-drug binding sites. we compile all results from our structural interactome into a user-friendly 97 web server allowing for quick exploration of individual interactions or bulk download and analysis of the 98 whole dataset. further, we explore the utility of our interactome modeling approach in identifying key 99 interactions undergoing evolution along viral protein interfaces, highlighting population variants on 100 human interfaces that could modulate the strength of viral-host interactions to confer protection from or 101 susceptibility to covid-19, and prioritizing drug candidates predicted to bind competitively at viral-102 human interaction interfaces. enrichment of divergence between sars-cov and sars-cov-2 at spike-ace2 binding interface to highlight the utility of computational and structural approaches to model the sars-cov-2-human 106 interactome, we first examined the interaction between the sars-cov-2 spike protein (s) and human 107 angiotensin-converting enzyme 2 (ace2) (fig 1.a) . this interaction is key for viral entry into human 108 cells 3 and is the only viral-human interaction with solved crystal structures available in both sars-cov 47 109 and sars-cov-2 [48] [49] [50] . comparison between sars-cov and sars-cov-2 revealed that sequence 110 divergence of the s protein was highly enriched at the s-ace2 interaction interface (fig 1.a; log2oddsratio=2.82, p=1.97e-5), indicating functional evolution around this interaction. to explore the 112 functional impact of these mutations on this interaction, we leveraged the rosetta energy function 51 to 113 estimate the change in binding affinity (δδg) between the sars-cov and sars-cov-2 versions of the 114 s-ace2 interaction (fig 1.b and 1.c) . the predicted negative δδg value of -14.66 rosetta energy units 115 (reu) indicates an increased binding affinity using the sars-cov-2 s protein driven by better optimized 116 solvation and hydrogen bonding potential fulfillment along the ace2 interface. our result is consistent 117 with the hypothesis that increased stability of the s-ace2 interaction is one of the key reasons for 118 elevated transmission of sars-cov-2 52 . moreover, recent experimental energy kinetics assays have 119 shown that sars-cov-2 s protein binds ace2 with 10-20-fold higher affinity than that of sars-cov s 120 protein 53 supporting the conclusions from our computational modeling. among individuals 6, 7, 54 . several hypotheses for genetic predisposition models have been proposed 123 including that expression quantitative trait loci (eqtls) may up-or down-regulate host response genes 124 and that functional coding variants may alter viral-human interactions 55, 56 . for instance, a recent rna-in order to add a structural component to our interactome map, and thereby enable modeling of 149 the binding affinity for these interactions, we additionally performed docking in pyrosetta using our 150 eclair interface likelihood predictions to refine the search space (supplemental after constructing the 3d interactome between sars-cov-2 and human, we first looked for evidence 162 of interface-specific variation by mapping both gnomad 58 reported human population variants 163 (supplemental table 3 ) and sequence divergences between sars-cov and sars-cov-2 164 (supplemental table 4 ) onto the predicted interfaces. in general, conserved residues have been shown 165 to cluster at protein-protein interfaces 63 , and a recent analysis of sars-cov-2 structure and evolution 166 likewise concluded that highly conserved surface residues were likely to drive protein-protein 167 interactions 64 . consistent with these prior findings at an interactome-wide level, we observed significant 168 depletion for both viral and human variation along the predicted interfaces comparable to that observed 169 on solved human-human interfaces (fig 2.a) . nonetheless, considering each interaction individually, our analysis uncovered a 13 interaction 171 interfaces enriched for human population variants (fig 2.b) , and 7 enriched for recent viral sequence 172 divergences (fig 2.c) . a breakdown of variant enrichment on each interface is provide in supplemental 173 table 5 . the individual viral interfaces showing an unexpected degree of variation may-like the 174 previously discussed s-ace2 interface-be indicative of recent functional evolution around the viral-human interaction. considering the slower rate of evolution in humans, enrichment of population variants along the human interfaces is unlikely to be a selective response to the virus. rather, these 177 interfaces with high population variation along the interfaces may represent edges in the interactome whose strength may fluctuate among individuals or between populations. alternatively, enrichment and depletion of variation along the human-viral interfaces could help distinguish viral proteins that bind 180 along existing-and therefore conserved-human-human interfaces from those that bind using novel 181 interfaces-that would be less likely to be under selective pressure. to further explore the functional impact of naturally occurring variants on the human interactors 183 of sars-cov-2, we considered variants with phenotypic associations as reported in hgmd 65 , clinvar 66 184 or the nhgri-ebi gwas catalog 67 . interactors of sars-cov-2 were significantly more likely than the 185 rest of the human proteome to harbor phenotypic variants in each of these databases (fig 2.d) . notably, 186 among the individual disease categories enriched in this gene set, several were consistent with reported 187 comorbidities including heart disease, respiratory tract disease, and metabolic disease 68, 69 (fig 2. e; 188 supplemental table 6 ). disruption of native protein-protein interactions is one mechanism of disease 189 pathology, and disease mutations are known to be enriched along protein interfaces 70, 71 . human 190 population variants on the predicted human-viral interface were more likely to be annotated as 191 deleterious by sift 72 and polyphen 73 but showed identical allele frequency distributions compared to 192 those off the interfaces (supplemental figure 4) . however, mapping annotated disease mutations onto 193 the protein interfaces only revealed significant enrichment along known human-human interfaces; no 194 such enrichment was found on human-viral interfaces (fig 2.f) . this is likely because unlike with human-195 human interactions, mutations disrupting human-viral interactions would not disrupt natural cell function, 196 and therefore would be unlikely to be pathogenic. our finding that disease mutations and viral proteins 197 affect human proteins at distinct sites is consistent with a two-hit hypothesis of comorbidities whereby 198 proteins whose function is already affected by genetic background may be further compromised by viral 199 infection. we next sought to explore the impact of sequence divergence in sars-cov-2 relative to sars-cov on 202 viral-human interactions. mutations between the two viruses were identified by pairwise alignment and the 203 impacts of these mutations on the binding energy (δδg) for 250 interactions amenable to docking were 204 predicted using a pyrosetta pipeline 46, 59, 60 . although the binding energy for most interactions was 205 unchanged-either because no mutations occurred near the interface or because the mutations that did 206 had marginal effect-we observed an increased likelihood of the divergence from sars-cov to sarscov-2 resulting in decreased binding energy (i.e. more stable interaction) (fig 3. a; supplemental table 208 7). the significant outliers in these δδg predictions can help pinpoint key differences between the viral-209 human interactomes of sars-cov and sars-cov-2. we further note a wide range of affinity impacts 210 among various human interactors of a single viral protein (fig 3.d) and hypothesize that these differences 211 may help identify the most important interactions. to further explore the significance of these changes in interaction affinity, we considered those 213 interactions with the largest decrease in binding energy; corresponding to the largest predicted increase in 214 affinity. specifically, we highlight the interaction between coronavirus orf9c and human mitochondrial nadh scanning mutagenesis along all docked interfaces in pyrosetta. we identified as binding energy hotspot 254 mutations all mutations with a predicted δδg at least one standard deviation away from the mean for identical amino acid substitutions across the rest of the interface. in total, out of 2,241 population variants 256 on eligible interfaces, 161 (7.2%) were identified as hotspots predicted to disrupt interaction stability, and 257 116 (5.2%) were identified as hotspots predicted to contribute to interaction stability (fig 3.b) . most of the 258 hotspot mutations were predicted to be driven by solvation or repulsive forces, with disruptive hotspots 259 primarily being driven by repulsive forces and stabilizing hotspots primarily being driven by solvation forces 260 (fig 3.c) . results summarizing the predicted impact of all 2,241 population variants on the docked 261 interfaces are provided in supplemental table 8 . the current version of the sars-cov-2 human structural interactome web server describes 332 291 viral-human interactions reported by gordon et al. 20 . we will continue support for the web server with 292 periodic updates as additional interactome screens between sars-cov-2 and human are published. as we update, a navigation option to select between the current or previous stable releases of the web 294 server will be provided. overall, we present a comprehensive resource to explore the sars-cov-2-human protein-protein 297 interactome map in a structural context. analysis through this framework allows us to consider the recent 298 evolution of sars-cov-2 in the context of its interactome map and to prioritize for further functional 299 characterization key interactions. likewise, our consideration of underlying variation in the human 300 proteins that interact with sars-cov-2 may be valuable in explaining differences in response to 301 infection. we particularly note that our observation that perturbation from underlying disease mutations 302 and viral protein binding occur at distinct sites on the protein is of clinical interest. further investigation 303 into the combined role of these two sources of perturbation to better understand the mechanisms linked 304 to comorbidities is warranted. however, our work is not without limitation. firstly, we note that although structural coverage 306 from our homology modelling of sars-cov-2 proteins was robust (supplemental figure 1) , the same done to orient the most likely interface residues on each structure towards each other, protein-protein 309 docking using incomplete protein models introduces some bias and low coverage may exclude some 310 true interface residues. for this reason, the initial eclair interface annotations-which are less subject 311 to structural coverage limitations-may provide orthogonal value. we additionally note that direct perhaps most importantly, we emphasize the importance of further experimental 323 characterization to confirm the predictions made here. nonetheless we believe our 3d structural sarscov-2-human interactome web server will prove to be a key resource in informing hypothesis driven 325 exploration of the mechanisms of sars-cov-2 pathology and host response. the scope, and potential 326 impacts of our webserver will continue to grow as we incorporate the results of ongoing and future 327 interactome screens between sars-cov-2 and human. additionally, we note that our 3d structural 328 interactome framework can be rapidly deployed to analyze future viruses. homology-based modeling of all 29 sars-cov-2 proteins was performed in modeller 90 using a multiple 332 template modeling procedure. in brief, a list of candidate template structures for each protein to be 333 modelled was obtained by running blast 91 against a reference containing all sequences in the protein data bank (pdb) 92 . templates were filtered to only retain those with at least 30% identify to the protein 335 to be modelled, and the remaining templates were ranked using a weighted combination of percent 336 identity and coverage as described previously 93 . to compile the final set of overlapping templates for 337 modeling, first the top ranked template was selected as a seed. overlapping templates were iteratively 338 added to the set so long as 1) the new template increased the overall coverage by at least 10%, and 2) 339 the new template retained a total percent identity no more than 25% worse than the initial seed template. pairwise alignments between the protein to be modelled and the template set were generated using a regions with large gaps (at least 5 gaps in the alignment in a 10 residue window). finally, alignment was to accommodate predictions between sars-cov-2 and human, slight alterations were made. using the predicted interface probabilities reported by eclair, we set up the initial docking 379 conformation to explore a restricted search space for each docking simulation. in cases where multiple 380 structures were available for the human protein, all structures were weighted based on the eclair 381 scores for the covered residues in each structure to maximize both coverage age inclusion of likely 382 interface residues. for each protein in the interaction, we performed a linear regression classification in scikit-learn 102 to optimally separate the likely interface residues from likely non-interface residues. the plane defined by this linear regression served as a reference to orient the structures along the y-axis the x-z plane and separated a distance of 5 å along the y-axis. for each docking attempt, a series of 387 random perturbations from these initial conformations were made to search the nearby space. first, the 388 human protein was rotated up to 360° along the y-axis to allow full exploration of different rotations of 389 the two interfaces relative to each other. second, to apply some flexibility to the plane predicting the 390 interface vs. non-interface sides of each protein, up to 30° of rotation along the x-and z-axis were 391 allowed for both the viral and human proteins. finally, a random translation up to 5 å in magnitude was 392 applied to the human protein along the x-z plane so that the docking could explore contact points other 393 than the center of masses along these axes. after initializing these guided starting conformations, docking was simulated in pyrosetta 46 395 using a modified version of the protein-protein docking methodology provided by gray 2006 103 . the 396 initial demo (https://graylab.jhu.edu/pyrosetta/downloads/scripts/demo/d100 docking.py) takes two 397 chains from a co-crystal structure, applies a random perturbation, and re-docks them. because 398 randomized initial orientation was already handled as described previously, these steps were removed 399 from our docking runs. in brief, the protein models were converted to centroid representation, slid into 400 contact using the "interchain_cen" scoring function, and converted back to full atom representation, 401 before having their side-chains optimized using the predefined "docking" and "docking_min" scoring table 2 . to annotate interface residues from atomic resolution docked models, we used a previously described 407 and established definition for interface residues 45 . in brief, the solvent accessible surface area (sasa) 408 for both bound and unbound docked structures was calculated using naccess. 100 we define as accessibility) and 2) in contact with the interacting chain (defined as any residue whose absolute 411 accessibility decreased by ≥ 1.0 å 2 ). the full list of sars-cov-2 mutations is reported in supplemental table 4 . human population variants in all 332 human proteins shown to interact with sars-cov-2 430 proteins were obtained from gnomad 58 reported as the most general term with no more significant ancestor term (supplemental table 6 , sheet 465 1). raw enrichment values for all terms are also predicted (supplemental table 6 , sheet 2). for curation of disease and trait associations from nhgri-ebi gwas catalog (http://www.ebi.ac.uk/gwas/) 67 the scoring function used for these calculations is as described previously 59 using the following weights; protein-ligand docking using smina the previous viral-human interactome screen by gordon et al. 20 supplemental table 9 . list of all predicted drug-target binding sites . comparison of the percentage of human genes that interact with (green) or do not interact with (orange) coronaviruses: an overview of their replication and pathogenesis a pneumonia outbreak associated with a new coronavirus of probable bat origin including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). mandell, douglas, and bennett's 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mutational constraint spectrum quantified from variation in 141,456 humans a simple physical model for binding energy hot spots in protein-protein complexes spatial chemical conservation of hot spot interactions in protein-protein complexes tests of concrete strength across the thickness of industrial floor using the ultrasonic method with exponential spot heads the sequence of human ace2 is suboptimal for binding the s spike protein of sars coronavirus 2. biorxiv conserved residue clusters at protein-protein interfaces and their use in binding site identification sars-cov2 (covid-19) structural/evolution dynamicome: insights into functional evolution and human genomics. biorxiv human gene mutation database (hgmd): 2003 update clinvar: improving access to variant interpretations and supporting evidence the nhgri-ebi gwas catalog of published genome-wide association studies, targeted arrays and summary statistics 2019 characteristics associated with hospitalization among patients with 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genome-wide sirna screen identifies the retromer as a cellular entry factor for human papillomavirus the master regulator of the cellular stress response (hsf1) is critical for orthopoxvirus infection a genome-wide small interfering rna (sirna) screen reveals nuclear factor-kappab (nf-kappab)-independent regulators of nod2-induced interleukin-8 (il-8) secretion architecture of the human interactome defines protein communities and disease networks tmed2 potentiates cellular ifn responses to dna viruses by reinforcing mita dimerization and facilitating its trafficking role of the early secretory pathway in sars-cov-2 infection tom70 mediates activation of interferon regulatory factor 3 on mitochondria a whole-genome association study of major determinants for host control of hiv-1. science extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations comparative protein structure modeling using modeller basic local alignment search tool the protein data bank interactome3d: adding structural details to protein networks protein identification and analysis tools on the expasy server the proteomics protocols handbook divergence measures based on the shannon entropy predicting functionally important residues from sequence conservation direct coupling analysis for protein contact prediction evolutionarily conserved pathways of energetic connectivity in protein families modbase, a database of annotated comparative protein structure models and associated resources the interpretation of protein structures: estimation of static accessibility accelerating protein docking in zdock using an advanced 3d convolution library scikit-learn: machine learning in python high-resolution protein-protein docking uniprot: a worldwide hub of protein knowledge analysis of multimerization of the sars coronavirus nucleocapsid protein a new coronavirus associated with human respiratory disease in china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a general method applicable to the search for similarities in the amino acid sequence of two proteins amino acid substitution matrices from protein blocks the ensembl variant effect predictor explaining odds ratios. j can acad child adolesc psychiatry ldlink: a web-based application for exploring population-specific haplotype structure and linking correlated alleles of possible functional variants open babel: an open chemical toolbox lessons learned in empirical scoring with smina from the csar 2011 benchmarking exercise sars-cov-2 that contain disease annotations in hgdm (log2or=0.57, p=1.70e-4) sars-cov-2 proteins were significantly more likely to harbor disease mutations than non-interactors error bars indicate ± se. e, a sample of individual disease terms enriched in human genes targeted by 603 comparison of the enrichment of hgdm, clinvar, and gwas annotated mutations on human-vial 605 interfaces or human-human interfaces for the same gene set. although disease mutations were enriched 606 on human-human interfaces (hgmd, log2or=0 24), no 607 enrichment was observed on human-viral interfaces (hgmd, log2or=0.21, p=0.13; clinvar the gwas category was removed from this analysis because most lead gwas 609 snps occurred in non-coding regions. error bars indicate ± se predicted changes in binding affinity from sequence divergences between sars-cov and sars-613 an overall representation of 614 these δδg predictions is reported (mean=-1.40 reu, std=6.16 reu) with interactions sorted from those 615 with the largest decrease in binding energy (most stabilized relative to sars-cov) to those with the 616 largest increase in binding energy (most destabilized relative to sars-cov) < z-score ≤ -1, n=85), or strongly stabilizing (z-score ≤ -2, n=31) score < 1, n=1,964) showed minimal impact of binding affinity. c, breakdown of the contribution of each 623 term in the pyrosetta energy function used for in-silico scanning mutagenesis for all population variants a breakdown of which term contributed most heavily 625 to the classification of all 277 interface hotspot population variants is shown on the right. d, individual 626 sars-cov-2-human interactions involving the same viral protein can have distinct interfaces with 627 distinct predicted changes in binding affinity between sars-cov and sars-cov-2 versions of the 628 protein. an example involving orf9b is highlighted where some interactions (e.g. tomm70 and ptbp2) 629 are predicted to be more stabilized in sars-cov-2 whereas others (e.g. bag5, slc9a3r1, and 630 mark2) are predicted to me unaffected. e, docked structure for the interaction between sars-cov-2 631 orf9c and human ndufaf1 sars-cov-2 (bottom) orf9c. interface residues are colored by their predicted energy contribution 633 from blue (stabilizing) to white (no impact) to red (destabilizing) -2 are labeled in red, while other residues with a major contribution to the binding 635 affinity are labeled in green (ndufaf1) or blue (orf9c). the overall predicted change in binding energy 636 (δδg=-21.7 reu) suggests the interaction is more stable (lower energy) in the sars-cov-2 version of 637 the interaction supplemental figure 4. summary of human population variant frequency and deleteriousness summary of allele frequency for human population variants either on or off the predicted human-700 viral interface presented as either a raw distribution or a cumulative density respectively. variants in 701 either category had roughly identical allele frequency distributions. c, d, summary of the sift 702 deleteriousness score for human population variants either on or off the predicted human-viral interface 703 presented as either a raw distribution or a cumulative density respectively population variants on the 706 interface were significantly more likely to be classified deleterious. f, g, summary of the polyphen 707 deleteriousness score for human population variants either on or off the predicted human-viral interface 708 presented as either a raw distribution or a cumulative density respectively. plots are colored based on 709 the split between polyphen benign, possibly damaging, and probably damaging categories. e, pie chart 710 breakdown of these categories. pie char outlines distinguish interface (green) from non-interface 711 (orange) for each sars-cov-2-human interaction with 3d structure available for both proteins, 50 independent 680 guiding docking trials were used to select a final docked configuration. the structure for the viral protein 681 is colored from white to blue with darker blue corresponding to higher eclair prediction. the structure 682 for the human protein is colored similarly using a green to white gradient. initial semi-random docked 683 configurations were generated using five steps. first a plane separating eclair predicted likely 684 interface from likely non-interface residues was drawn to divide each protein. second, the two protein 685 chains were separated 5 å apart on the y-axis using the previously defined plane to orient the likely 686 interface sides of each protein towards each other. third, the human protein was randomly rotated up 687 to 360° along the y-axis to sample different orientations of the two interfaces relative to each other. key: cord-272829-i4jh6bcn authors: zanetti, a. r.; zappa, a. title: emerging and re‐emerging infections at the turn of the millennium date: 2010-01-04 journal: haemophilia doi: 10.1111/j.1365-2516.2009.02174.x sha: doc_id: 272829 cord_uid: i4jh6bcn summary. after world war ii, mankind believed that infectious diseases were on the way to being defeated. unfortunately, they still are the second worldwide cause of death. globalization changes promote the emergence of new infections and pandemics; international deliveries and travelling facilitate the dissemination of infectious agents; man‐induced environmental changes create new opportunities for contacts between species, leading to infections in aberrant hosts, including man; global warming enables insects, a major vector of pathogens, to thrive in more countries. the main pandemics have been caused by viruses, such as hiv and novel subtypes of influenza viruses. in addition, prion proteins are a threat. the transmission of the creutzfeld jakob disease variant through blood transfusion and the recent discovery of prion protein in the spleen of a haemophilia patient are a matter of further concern. the end of the war against infectious diseases is not in sight. mankind’s battle with pathogens has lasted millennia and is destined to continue. throughout history man has periodically been the victim of epidemics: we have historical reports that vividly report these calamities, such as 'the plague of justininan' [1] dating back to the roman empire, 'the black death' in the middle ages [2] , the 'spanish flu' in 1918 [3] , the emergence of acquired immunodeficiency syndrome (aids) in the early 1980s and the more recent severe acute respiratory syndrome (sars) at the beginning of the xxi century [4] . after world war ii, the extensive knowledge on infectious agents and their hosts, environmental factors involved in their transmission, improvements in hygiene, healthcare and socioeconomic status and, last but not least, the discovery of effective antimicrobial therapy and the availability of vaccines, lulled man into believing that 'the war against infectious diseases had been won' and that epidemics were a phenomenon of the past [5, 6] . nothing could have been further from the truth. at the beginning of the third millennium, infectious diseases still are the second cause of death in the world (the first in developing countries). fifteen million humans die every year of infectious diseases, the five 'big killers' being aids, tuberculosis, malaria, respiratory infections and infantile diarrhoea [6, 7] . in the last decades, a number of new pathogens responsible for emerging infectious diseases, such as avian and swine flu, aids, sars, west nile, ebola and variant of creutzfeldt-jacob disease (vcjd) have been identified and other infectious diseases are re-emerging after a period of quiescence, such as malaria and tuberculosis caused by multidrug resistant strains [2, 5, 6, 8, 9] . the picture is compounded by the so-called 'deliberately emerging' pathogens, such as anthrax and smallpox -biological weapons that humans might plan to spread deliberately as an act of bioterrorism [2, 6, 9] . in the world of globalization, regular exchanges of enormous amounts of goods throughout the world and low-cost frequent travelling, whereby hundred thousands of people move from one side of the earth to the other within a matter of hours, facilitate the dissemination of infectious agents (microbial traffic). infectious diseases that once upon a time would have remained confined to their ecological niche can now potentially spread rapidly to every corner of the earth before preventive measures can be implemented [5, 10] . what is more, a number of other factors promote not only the dissemination but also the emergence of new infectious diseases: intensive farming and breeding associated with crowding promote the development of foci of infection; global warming has modified the climate, making insects, a major vector of pathogens, able to thrive in countries where the climate was previously hostile; the exploitation of natural resources has produced environmental changes that create opportunities for new contacts between species leading to emergence of infections in new hosts. the ability of an infectious agent to mutate and to jump the barrier species from animals to humans contributes to the emergence of zoonotic diseases. indeed, most of the outbreaks in the last 30 years have been caused by infectious agents normally hosted by wild animals (reservoir hosts). following the loss of their normal habitat (e.g., climate changes, ecological changes in land use), these animals can move closer to farms where they infect livestock (spillover hosts). in parts of the world characterized by crowding and low hygiene standards, the livestock then infect man. in some cases, man results to be a 'dead-end' host (aberrant host), who does not transmit the infection any further. for instance, in the 1990s, an unexplained epidemic of acute cardiopulmonary syndrome occurred unexpectedly in a navajo tribe living on the border of the four corners region (new mexico, arizona, colorado, utah) of the southern-western united states [11] . this potentially deadly disease caused by an unknown virus (sin nombre virus or 'nameless virus') subsequently identified as a member of the hantavirus genus (bunyaviridae family) is transmitted from mice to humans by inhalation of aerosolized virus-contaminated rodent excreta (saliva, urine, droppings). during the 1993 navajo outbreaks, the wet and mild conditions as a result of changes in climate caused by el niñ o favoured an abundance of mice food allowing a significant increase in the deer mouse population (peromyscus mariculatus), the natural reservoir of this virus [12] . the migration of infected rodents near human settlements created the ideal conditions for the outbreak onset. the deforestation that occurred in australia and malaysia in the late 1990s forced fruit bats (flying foxes), the reservoir of nipah and hendra viruses (henipavirus genus, paramyxoviridae family), to move closer to human dwellings creating the conditions for the emergence of focal outbreaks of encephalitis with high fatality rate in humans [13] . in this case, viruses were transmitted from bats to livestock animals (pigs and horses) and from these to humans. in other cases, however, zoonotic transmission is followed by viral adaptation to the new host that makes human-to-human transmission feasible. this is the case of several mutated viruses [10, 14, 15] . most of the emerging infections are caused by viruses, which, according to nobel prize winner peter medawar, are 'bad news inside a protein envelope'. the 'bad' news consists of strands of dna or rna that are able to penetrate mammalian cells, obliging them to reproduce the viral components instead of their own. the bad news can get even worse as viruses adapt continually to the environment by mutation, recombination or gene reassortment [2, 15] . the two key features of intracellular replication and genetic evolution make viral infections particularly difficult to combat: currently available anti-viral agents block viral replication by various mechanisms, but hardly remove dormant viruses that have already penetrated inside host cells and the protection conferred by vaccination can be made fruitless by genetic evolution of the pathogen, which may enable it to escape previously induced immunity. an example that shows why zoonotic transmission must be avoided is the infection by hiv. its natural reservoir is a non-human primate, called pan troglodytes troglodytes, in which it is fairly harmless. man became probably infected through contacts with infected blood during hunting and slaughtering processes ('bush meat', hunter's cut) about 60-70 years ago [16] . the infection remained confined to areas of africa up to about 40 years ago when international travelling and exchanges became more widespread. infected subjects moved from africa to haiti; from there, the infection then spread, as a result of sex tourism, first to gay communities in cities of north america (los angeles, san francisco, new york) and then to europe. at first, it spread in high-risk groups, such as male homosexuals, intravenous drug abusers and recipients of multiple transfusions; then, it extended to the general population. the outcome is the current pandemic: in 2008, who estimated that 33 million adults and children were living with hiv infection/aids worldwide, that more than 40 million people have died of the disease so far and that the number of deaths in the last year amounted to 2 million [17] . another well known example of recent worldwide pandemic is the sars. this emerging airborne viral disease had a great impact on account of its rapid international spread under favourable conditions created by our highly mobile, closely interconnected world. the origin of sars was in areas of dense population in southern china, where humans and animals are packed side by side providing ideal conditions for a new virus to emerge by jumping across species. the natural reservoir of sars virus (sars-cov) is the horseshoe bat, the spillover host is the masked palm civet or raccoon dog; man is an aberrant host [18] . the pandemic originated in guangdong, in southern china, where families often cohabit with the animals they rear. the promiscuity enabled the sars-cov to skip across species and find a new host -man. it has been documented how the first identified infected human (index case) unintentionally infected another 13 humans in the metropole hotel in hong kong, who then travelled to five different countries (germany, canada, thailand, vietnam and singapore), giving rise to new foci of infection. this ultimately led to a worldwide epidemic affecting more than 8000 people, with a death rate of 9.6% [19] . this event showed how modern transport systems can enable an emerging infection in one part of the earth to spread rapidly throughout the globe. however, it also showed how international healthcare co-operation can achieve control of a rising pandemic within less than a year. however, not all viruses behave this way. some viruses adopt the so called 'hit and run' strategy as they cause recurrent, geographically confined, usually self-limiting outbreaks. this is the case of ebola and marburg viruses, both members of the family filoviridae, causing severe and often fatal haemorrhagic fevers in humans and non-human primates. in 1976, transmission of ebola virus from its natural reservoir, which is still unknown, to man resulted in two simultaneous but separate, explosive outbreaks of human disease caused by two distinct virus subtypes in zaire and sudan with a case-fatality rate of approximately 90% and 50% respectively. the disease was self-limited both in space and time: it remained confined to well-defined geographical areas and mysteriously resolved [20] . since then, recurrent outbreaks have been reported in african countries including sudan, gabon, the democratic republic of congo, republic of congo and uganda. a variant of the african ebola virus, the reston virus, was first identified in 1989 in reston, virginia, usa and in 1992 in italy (siena) in colonies of cynomolgus macaques imported from the philippines [21] . despite its high pathogenicity in non-human primates, this virus does not seem to cause disease in humans. the infection by the marburg virus followed a similar course: in 1967, german laboratory workers were infected by african green monkeys belonging to the cercopithecus aethiops species imported from uganda and developed fatal haemorrhagic fever [22, 23] . west-nile, dengue and chikungunya viruses: vector-borne throughout the world arthropod-borne viruses are very common (more than 600) and more than 150 cause diseases in humans in more than 100 countries world-wide, with a disease burden as high as a hundred million cases every year. ecological factors, such as new routings of long-distance bird migrations and the increased long-distance air travel facilitate the movement of infected persons, livestock and exotic arthropod vectors around the world contributing to the spread of vector-borne diseases from the original niches to new geographical areas. changing in public health policy, insecticide and drug resistance are additional factors which can contribute to the emergence or resurgence of vector-borne infectious diseases [25] . an example is the west nile virus (wnv) infection, originally confined to africa (along the nile), southern europe, parts of the middle east and india, which has recently modified its epidemiological picture. in 1999, wnv was firstly imported into the new york area and, thanks to the presence of specific vectors (culex mosquito) and natural susceptible hosts (crows, jays), the infection rapidly spread coast to coast. man is a dead-end incidental host: it has now been estimated that 1-3 million people have been infected by wnv in north america. west nile (wn) is a potentially serious illness whose symptoms may range from mild to severe. in about 80% of cases, infection is asymptomatic and asymptomatic carriers may be infectious via blood transfusion or through organ and tissues donations [26] . in 2002, there were 23 documented cases of wnv transfusion-transmitted infections, which induced the usa blood collection agencies (bcas) to implement blood screening for this virus [27] . from june to september 2003, several hundred potentially infectious components were identified by the tests. in the last 50 years, outbreaks of wnv have been reported in some european countries both in horses and humans. in september-october 2008, the first two cases of wnv neuroinvasive disease have been reported in northern italy [28] . to prevent infection through blood donations by infected donors during the viremic peak, screening programmes have been introduced during the seasonal mosquito activity in the affected area. other examples of vector-borne diseases that have extended elsewhere in view of the climate changes and global warming is the re-emergence of dengue virus (dv) infection transmitted by aedes aegypti mosquito in tropical and subtropical countries. the susceptibility of aedes albopictus also known as 'tiger' mosquito (a mosquito of asian origin currently widespread in countries with a temperate climate) to serve as a new vector of dv gives new opportunities to the virus to extend its area of spread. the tiger mosquito which is the vector of chikungunya virus (chikv), a virus that caused several epidemics in africa, southeast asia, the western pacific and india, was first introduced in italy in the early 1990s by importing used tyres. in 2007, the chikv transmitted by this mosquito caused the first autochthonous epidemic outbreak in europe, in the north-east of italy (emilia romagna region) which involved more than 200 cases [29, 30] . this prompted the national blood centre and regional health authorities to take specific precautionary measures to ensure the safety of blood supply. influenza a is an airborne virus that repeatedly produces epidemics every year during the cold season. its hosts include birds (natural reservoir) and a number of mammals, including whales, seals, horses, pigs and man. it is particularly able to adapt to new hosts, thanks to its unique capacity for genetic variation: its surface proteins are able to mutate up to 50% of their amino acid sequence without losing their capacity to infect mammalian cells and its genome is made of eight rna segments that are genetically independent and that can therefore randomly reassort, forming antigenically novel subtypes [31] . such novel strains have caused a number of dangerous pandemics in the past, such as the spanish flu in 1918, asian flu in 1957 and hong-kong flu in 1968. currently, avian flu is a real threat caused by mutated strain h5n1, which continues to spread in humans. the cumulative number of human cases of avian influenza h5n1 reported by who by 11 august 2009 amounted to 438 cases and 262 deaths [32] . the issue is whether this virus or other avian viruses (h9n2, h7n7) will cause a novel pandemic or not [33] . to cause a pandemic, three characteristics are required: (i) the ability of the virus to replicate in a human host, (ii) the population has to be susceptible to it, and (iii) human-to-human transmission has to be possible. at present, for h5n1, the first two features are satisfied, but not the third. however, it cannot be excluded that it will develop the third feature in the future. on the contrary, human-to-human transmission has been proven for the novel swine flu caused by the influenza a (h1n1) virus (s-oiv) isolated for the first time in march-april 2009 in mexico [34] . this novel strain is a cause for concern as it has resulted from the unique reassortment of genetic elements of influenza viruses that normally infect three different kinds of host -swine, birds and human beings. the study of its structure has revealed that it differs considerably from its predecessors, isolated in human beings: its h antigen, which enables it to bind and infect cells of different species, has changed in terms of amino acid sequence alignment by approximately 27% and its n antigen, which enables it to digest sialic acids (neuraminidase activity), ensuring better access and spread in the organism, has changed by about 18%. this means that exposure to its predecessors may not provide any immunity to this unique strain and the whole population on earth is theoretically at risk [35] . indeed, in april 2009, who declared a phase-5 alert indicating that a pandemic was imminent and that it was urgent to finalize the organization, communication and implementation of the pandemic preparedness plans. on june 11, 2009 who then announced the beginning of a flu pandemic with a total of 29 669 cases worldwide and 145 deaths. only one month later, on july 6, 2009, who reported that the number of infections had increased up to nearly 100 000 with 429 deaths. laboratory-confirmed cases as officially reported to who as of 14 august 2009 amount to 182 166 with 1799 deaths. given that countries are no longer to test and report individuals, we can estimate that the number of cases reported actually understates the true number of cases. at the time of writing (mid-august 2009), who considers the pandemic to be moderate. the speed at which the infection is spreading is such that infection of millions of people worldwide is considered to be inevitable. however, symptoms are mild and most people recover from the infection without the need for hospitalization or medical care. the rate of severe infections is currently similar to what is generally seen during local seasonal influenza. however, monitoring is essential as it is not possible to predict how the pandemic will evolve. h1n1 might disappear spontaneously, cause a mild pandemic or a severe pandemic after virus mutation/re-assortment. concerning blood transfusions, the risk of direct transmission of influenza via blood or blood products is extremely low. the major issue will be a temporary loss of blood donors during the pandemic peak, with impact on blood supply, as donors may not attend blood collection services on account of illness. an atypical infectious agent is the prion protein, a conformational variant of a naturally occurring protein that cannot be removed by normal degradation processes and accumulates forming fibrous aggregates that are believed to be involved in the pathogenesis of the disease [36] . the new prion disease that emerged in man in the uk in 1996 was a food-borne infection caused by modern intensive meat production based on 'industrial cannibalization': a prion disease that has been known to affect sheep and goats for more than 200 years (scrapies) had been transmitted to cattle via inadequately sterilized fodder of animal origin, giving rise to an epidemic of bovine spongiform encephalopathy (bse), also called 'mad cow disease', which in turn was then transmitted to man via meat consumption. prion-infected humans develop a variant of classic sporadic creutzfeld-jakob disease (vcjd), an invariably fatal neurodegenerative disease. it is not known what proportion of prion-infected humans develop symptomatic disease. also, the mean latency period from prion infection to the development of overt disease remains unknown. over the period 1996-2007, a total of 161 cases were reported, nearly all in the uk and attributed to dietary exposure. possible transmission of vcjd by blood transfusion has been documented in the uk [37] . the emergence of prion disease has led to a reduction in the number of donors as people with a history of visiting the uk during the cattle bse epidemic are excluded. recently, the prion protein has been found in the spleen during the postmortem examination of a 70year-old patient suffering from haemophilia who died of causes unrelated to vcjd. indeed, this patient did not have any neurological symptoms before death [38] . of some concern is the fact that in 1996, this patient received a batch of factor viii manufactured using plasma from a donor who developed symptoms of vcjv 6 months after donation. although the mode of transmission has not been confirmed, the uk health protection agency together with the haemophilia centre doctor's organization alerted haemophiliacs about this finding. a related alert had been made a few years earlier: in 2004 patients with bleeding disorders who had been treated with plasma-derivatives made with blood collected in uk between 1980 and 2001 were told that they were potentially at risk of vcjd and excluded from donating organs. however, although patients exposed to contaminated products in the past may be at risk for vcjd, this does not mean that currently available plasma-derived products (manufactured with uk free plasma) still carry such risk. [39] . an important treatment option for patients with bleeding disorder is the administration of recombinant factors viii, which is not derived from plasma and undergoes several processing steps to remove any contaminating viruses and prions [40] . the development of new antimicrobial drugs and antibiotics together with the availability of safe and effective vaccines led to a spirit of optimism towards the definitive control and prevention of several infectious diseases. man may have won some battles against infectious diseases, but the war is still raging and the end is not in sight. in general, there is no way to predict when or where the next pathogens will emerge. emerging and resurging diseases are common in areas rich in wild animals and where natural and social environmental factors, including man's activities, increase the opportunities for the pathogens to extend their ecological niches and to be transferred to and from humans and other animals. the continual emerging of new infectious agents and re-emerging of drug resistant strains of old ones are issues with far reaching consequences. infectious diseases have always been present in society and are destined to remain part of human life in the future. emerging and re-emerging infections 11 plague: the dreadful visitation occupying the human mind for centuries the challenge of emerging and re-emerging infectious diseases the 1918 influenza pandemic world health organization. severe acute respiratory syndrome (sars) changing patterns of infectious disease infectious diseases: considerations for the 21st century report: the global burden of disease: 2004 update social and environmental risk factors in the emergence of infectious diseases from pasteur to genomics: progress and challenges in infectious diseases geography, ecology and emerging infectious diseases infectious diseases update: outbreak, hantavirus infection -southwestern united states hantavirus cardiopulmonary syndrome hendra and nipah viruses: different and dangerous global trends in emerging infectious diseases molecular constraints to interspecies transmission of viral pathogens the origins of acquired immune deficiency syndrome viruses: where and when a review of studies on animai reservoirs of the sars coronavirus world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) viral haemorrhagic fever in southern sudan and northern zaire: preliminary studies on the aetiological agent world health organization. ebola haemorrhagic fever in imported monkeys marburg virus ecology of marburg and ebola viruses: speculations and directions for future research response to imported case of marburg hemorrhagic fever, the netherlands resurgent vector-borne diseases as a global health problem transmission of wet nile virus through blood transfusion-united states update: 'detection of west nile virus in blood donations -united states west nile virus in italy: a further threat to blood safety, a further challenge to blood system infection with chikungunya virus in italy: an outbreak in a temperate region the chikungunya epidemic in italy and its repercussion on the blood system influenza virus: a master of metamorphosis world health organization. cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who avian influenza virus (h5n1): a threat to human health outbreak of swine-origin influenza a (hinl) virus infection -mexico towards a sane and rational approach to management of influenza h1n1 2009 transmissible spongiform encephalopathies: the story of a pathogenic protein possible transmission of variant creutzfeldt-jacob disease by blood transfusion preclinical vcjd after blood transfusion in a prnp codon 129 heterozygous patient signs of variant creutzfeldt-jakob disease in a patient with hemophilia: fviii concentrates most likely cause pathogen safety of manufacturing processes for biological products: special emphasis on kogenate bayer the authors stated that they had no interests which might be perceived as posing a conflict or bias. key: cord-272405-jmwn8pdn authors: parvez, mohammad k.; parveen, shama title: evolution and emergence of pathogenic viruses: past, present, and future date: 2017-08-04 journal: intervirology doi: 10.1159/000478729 sha: doc_id: 272405 cord_uid: jmwn8pdn incidences of emerging/re-emerging deadly viral infections have significantly affected human health despite extraordinary progress in the area of biomedical knowledge. the best examples are the recurring outbreaks of dengue and chikungunya fever in tropical and sub-tropical regions, the recent epidemic of zika in the americas and the caribbean, and the sars, mers, and influenza a outbreaks across the globe. the established natural reservoirs of human viruses are mainly farm animals, and, to a lesser extent, wild animals and arthropods. the intricate “host-pathogen-environment” relationship remains the key to understanding the emergence/re-emergence of pathogenic viruses. high population density, rampant constructions, poor sanitation, changing climate, and the introduction of anthropophilic vectors create selective pressure on host-pathogen reservoirs. nevertheless, the knowledge and understanding of such zoonoses and pathogen diversity in their known non-human reservoirs are very limited. prevention of arboviral infections using vector control methods has not been very successful. currently, new approaches to protect against food-borne infections, such as consuming only properly cooked meats and animal products, are the most effective control measures. though significant progress in controlling human immunodeficiency virus and hepatitis viruses has been achieved, the unpredictable nature of evolving viruses and the rare occasions of outbreaks severely hamper control and preventive modalities. incidences of emerging and/or re-emerging infections have significantly affected human health since ancient times [1] . the emerging pathogens are defined as novel etiological agents that have been recently introduced in a population. the "spanish flu" responsible for tens of millions of casualties in the early twentieth century, was the most devastating natural calamity in human history [2] . the flu pandemic returned in 1957 as "asian flu" and then in 1968 as "hong kong flu" that killed about three million people [2] . the most recent re-emergence of influenza on this scale was in 2009 as "swine flu" that claimed 18,500 lives [3] . on its pandemic course in 2002/2003, a novel severe keywords emerging infections · novel viruses · viral outbreaks · virus evolution incidences of emerging/re-emerging deadly viral infections have significantly affected human health despite extraordinary progress in the area of biomedical knowledge. the best examples are the recurring outbreaks of dengue and chikungunya fever in tropical and sub-tropical regions, the recent epidemic of zika in the americas and the caribbean, and the sars, mers, and influenza a outbreaks across the globe. the established natural reservoirs of human viruses are mainly farm animals, and, to a lesser extent, wild animals and arthropods. the intricate "host-pathogen-environment" relationship remains the key to understanding the emergence/reemergence of pathogenic viruses. high population density, rampant constructions, poor sanitation, changing climate, and the introduction of anthropophilic vectors create selective pressure on host-pathogen reservoirs. nevertheless, the knowledge and understanding of such zoonoses and pathogen diversity in their known non-human reservoirs are very limited. prevention of arboviral infections using vector control methods has not been very successful. currently, new acute respiratory syndrome coronavirus (sars-cov) infected >8,000 people, causing 774 deaths in 27 countries [4] . actually, it was the discovery of the human immunodeficiency virus (hiv) in the early 1980s that initiated a worldwide awareness of and research interest in emerging novel viral pathogens. over the past several decades, new outbreaks of infections have led to the discovery of a diverse array of highly pathogenic viruses mainly those belonging to the filoviridae, arenaviridae, bunyaviridae, paramyxoviridae, coronaviridae, flaviviridae, togaviridae and hepeviridae families. some examples include bk virus (bkv), jc virus (jcv), merkel cell polyomavirus (mcv or mcpyv), severe fever with thrombocytopenia syndrome virus (sftsv), hantavirus (htnv), and sin nombre virus (snv) [5] [6] [7] . following the fatal cases of lujo virus in southern africa in 2008 [8] , another arenavirus, the lassa virus (lasv), first reported in nigeria in 1969, re-emerged in guinea, liberia, and mali in 2009, in ghana in 2011, and in benin in 2014 [9] . human metapneumovirus (hmpv) was first identified in the netherlands in 2001, and was subsequently linked to an acute lower respiratory tract infection in children, similar to respiratory syncytial virus (rsv). recently, in 2013, a novel avian influenza a strain (h7n9) of "bird flu" in china [10] and the middle-east respiratory syndrome (mers)-cov have been identified [11] . of note, while 2015 was threatened by the re-emergence of the ebola virus, 2015/2016 has been challenged with the resurgence of the zika virus (zikv) [12] . despite substantial advancements in the understanding of the biology of pathogens, the breakthroughs in prevention, and their effects on public health and the global economy, the emergence of novel pandemic viruses remains an enduring puzzle. this review presents an update on the knowledge of important emerging/re-emerging viral infections worldwide, discussing their possible origin, evolution, natural reservoirs, human adaptations, and risk factors ( fig. 1 ). of the known viruses that infect humans, about 80% perpetuate naturally in non-human "reservoirs," largely farm mammals and poultry and, to a lesser extent, in wild animals and arthropods [13] . it is estimated that zoonotic infectious agents constitute about 60% of the known human pathogens and up to 75% of "emerging" human pathogens [14] [15] [16] . we have, however, limited knowledge of such zoonosis and the diversity of these viruses in their known reservoirs. the data on some of the domestic mammals harboring dozens of virus species is limited and we have insufficient knowledge about the wild animals that are estimated to host thousands of virus species [17] . 3 the deadly outbreak of mers-cov was linked to its zoonotic origin because of its close genetic homology to bat cov, but not to any other known hcov [18] . current data shows that bats harbour the greatest diversity of covs, which vary from species-to-species and region-toregion [19] . the lyssaviruses of rhabdoviridae are zoonotic human pathogens that cause fatal encephalitic disease. in addition, european bat lyssavirus type-1/-2 (eblv-1/-2), bokeloh bat lyssavirus (bblv) [20] , and australian bat lyssaviruses (ablv) have been implicated in human fatalities [21] [22] [23] . rabies virus (rabv), the archetypal lyssavirus, is historically one of the most dreaded viruses, with zoonotic potential in dogs, cats, and ferrets, and includes other domestic and wild mammals [24] . globally, several mammalian species, including the deer mouse, rice rat, and cotton rat are recognized as potential reservoir of htnv [25] . in china, the htnv and seoul virus (seov) are zoonotically linked to the striped field mouse and the norway rat, respectively. also in china, the fugong virus (fugv), a novel htnv has recently been identified in small oriental voles [26] . htnvs have also been detected in shrews and bats, but their link to human illness remains to be established. moreover, in addition to humans and pigs, there is a growing chain of mammalian hosts for the hepatitis e virus (hev) that includes deer, boar, mongoose, rabbit, rat, goat, camel, bat, ferret, moose, etc. [27, 28] . likewise, the highly prevalent torque teno virus (ttv), of the newly established virus family, anelloviridae, also infects pigs, cows, sheep, cats, dogs, and chickens [29] . arboviruses like dengue virus (denv), chikungunya virus (chikv), zikv, and west nile virus (wnv) are the arthropod-borne viruses that have re-emerged in many tropical and subtropical regions in the last 2 decades. notably, zikv was known as a neglected tropical disease confined to africa and asia until an outbreak was reported in 2007 on yap island and in 2013/2014 on islands in the pacific, thus expanding its geographical territory [30, 31] . wnv remains the most important mosquito-borne encephalitis pathogen in north america, involving culex sp. and the american robin in its transmission cycle. since its emergence in the west in 1999, it has undergone adaptive genetic changes as it spreads throughout north america [32] . furthermore, crimean-congo hemorrhagic fever virus (cchfv) is considered to be one of the major emerging diseases spreading to and within the european nations, following the expanding distribution of anthropophilic ticks [33] . every year, >1,000 cases of cchfv, due to human-tohuman transmission, are reported in southeastern eu-rope, including turkey. sftsv, a previously unidentified tick-borne bunyavirus, has recently emerged in china with fatality rates as high as 30% [34] . while dna viruses are believed to have been evolving and diversifying for millions of years [35] , most rna viruses are likely to have a much more recent evolution and "human-adaptation" for only thousands of years [36, 37] . owing to their error-prone polymerase/reverse-transcriptase (approx. 10 -4 /site/replication cycle) that seems to operate at optimal fidelity, rna viruses exist as more genetically diversified populations than dna viruses. nevertheless, of the hitherto recognized 158 species of human rna viruses consisting of 47 genera and 17 families, only a minority has adapted to humans [38] . in contrast, of the known 91 dna virus species with 22 genera and 8 families, nearly 87% are adapted to human hosts [38] . in the human-adaptation process, the viral genetic mutations, re-assortment or virus-host genetic recombination might lead to the establishment of stable virus lineages in human populations. it is, therefore, quite possible that such human-adapted viruses could circulate asymptomatically and remain undetected until their novel clinical manifestations are noticed. to understand this further, a recently isolated hev genotype 3 from a chronic hepatitis e patient containing a recombinant virus-host rna genome was shown to infect cultured human, pig, and deer hepatocytes [39] . it is assumed that there is a pool of human virus species still to be discovered. the composition of such a viral pool is dynamic, changing over time, i.e., while some virus species tend to become extinct, others continue to evolve in their natural hosts. more commonly, new species arise as a result of jumps from one host to another, thus crossing the species barrier [36] . humans are, therefore, no more than "incidental" or "spillover" hosts for pathogens. however, only a minority of such viruses are capable of persisting in certain human populations (endemics) or spreading across populations (epidemics) in the absence of a reservoir. differential host factors like age, health, physiology, nutritional status, exposure history, simultaneous infection with >1 pathogen, immunocompetence, and genetics are determinants to human susceptibility to an infection. the field of phylodynamics, combining a modeling framework for host, epidemiological, and molecular data, especially for rna viruses, shows particular promise for parvez understanding the patterns of viral evolution during epidemics [40, 41] . moreover, our underappreciated aspect of growing human populations, global changes in land usage, and the introduction of anthropophilic vectors create selective pressure on hosts and reservoirs. for example, both wnv and chikv evolved rapidly after being introduced to new locations and encountering new vectors. when aedes albopictus rather than a. aegypti became the main vector of chikv in the indian subcontinent after the 2004-2009 epidemic, the same viral strain spread rapidly, and the subsequent mutants seemed to circulate and persist more efficiently [42] . a unique molecular signature of the chikv epidemic was a single amino acid substitution (a226v) in the envelope protein (e1). this mutation was identified in 90% of the strains reported during the later phase, and was associated with a high epidemic potential for chikv [43] . zikv, initially known to be transmitted by mosquitoes, was recently reported as being transmitted, albeit more rarely, via sexual contact, saliva, breast milk, blood transfusions, and from mother to child [12, 44] . the key to understanding the emergence/re-emergence of novel viruses is to know the intricate "hostpathogen-environment" relationship in the evolution of pathogens. while the emergence of infectious diseases in naive regions is caused primarily by the movement of pathogens via trade and travel, local emergence is driven by a combination of environmental and social change. notably, virus transmission rates are often higher in dense than in sparse populations, and the spread is often greatly enhanced by air travel or migration. pathogens introduced into novel regions often cause explosive epidemics followed by a declining incidence whereas those that emerge locally due to land usage or social changes usually show consistent increases. a recent example is the emergence of zikv in brazil in 2015 [12, 44] . phylogenetic studies suggested that zikv from the pacific islands outbreak in 2013/2014 was probably introduced into brazil during the fifa world cup or the 2014 fia world endurance championship auto racing series. the dispersal of the virus in brazil resulted in an explosive epidemic of zika fever, and the infection spread to other countries due to frequent travel. while most human infections are known to have zoonotic origins, it is certain that alterations in the environment, due to industrialization and urbanization, are an important but completely neglected factor. though the origins of most of the human viruses are not known so far, the great majority can be categorized as "crowd diseases" that require a relatively high host-density to persist [35] . notably, the recent outbreaks of h1n1, hcov, hendra virus, nipah virus, and mers-cov suggest that the asia-pacific region is the global hot-spot for the emergence of novel rna viruses. in this case, an order-of-magnitude estimation of 1 such event per 100 years is broadly consistent with human demographic history [37] . notably, rna viruses are known to incorporate drastic mutations in their genome, an example being the large duplication events in the g protein gene of rsv. two such remarkable events were the 60-bp duplication in group b rsv in argentina in 1999 and the 72-bp duplication in group a rsv in canada in 2011 [45, 46] . these new genotypes of rsv with their duplications, known as ba and on1, respectively, spread to different geographical regions across the globe due to immunologically naive travellers [47] [48] [49] [50] . the mathematics of these spreading events is well known today, and a sophisticated array of computational and mathematical models can be used to accurately back-predict such events. an example of this is the first case-clusters of the sars outbreak and the subsequent global spread, including the country-by-country distribution of human cases [51, 52] . recent investigations have evaluated the transmission dynamics of zikv infection using mathematical models [53] . we may not ignore that wild animals constitute an important but poorly understood reservoir for known and undiscovered human pathogens, including viruses. furthermore, the relative importance of an animal species as a source of human infection is a function of the prevalence of zoonotic agents in that species and the probability of close contact (direct or indirect) with susceptible humans. clearly, these factors vary geographically, and changes in patterns of human and animal disease will continue to result from socio-economic and ecological changes at the human-to-animal interface. if risk is a function of frequency of contact and a probability of successfully adapting to human hosts, viruses acquired from non-human primates might already be better adapted to successful transmission than those from other mammals, like bovine, porcine, feline, and rodent mammals. for example, sars-cov has been detected in masked-palm or gem-faced civet cats which are sold at emergence of pathogenic viruses intervirology 2017;60:1-7 doi: 10.1159/000478729 5 chinese wildlife markets [54, 55] . like hev, many enteric viruses (e.g., jcv) are found worldwide in high concentrations in urban sewage, and lead to water contamination in countries with poor resources [56] . recurring epidemics caused by emerging viruses have necessitated the formulation of effective control measures, but the zoonotic transmission of many of these viruses has contributed significantly towards the challenges associated with their prevention and control. for instance, the prevention of arthropod-borne infections (e.g., denv, chikv, and zikv) using vector control methods has only been partially successful [57] . preventing mosquitos breeding can be mediated by avoiding the accumulation of rain water in endemic regions. other preventive measures like the use of repellent creams and clothes that cover the body should be employed to avoid mosquito bites. insectidal sprays should be utilized in endemic regions to control the mosquito population. additionally, prevention of the transmission of other zoonotic viruses should also be addressed in a systematic manner to combat the infection of humans with these pathogens. it is now well known that many emerging human viruses can be transmitted via contact with infected animals and the consumption of animal products, including freshwater and seafood products. the new approach to food safety and to protecting humans against foodborne health risks is the most effective measure to control the human illnesses associated with animal diseases. however, the vast majority of human infections are not reported to clinicians, and so such etiological agents remain unidentified and continue to contaminate the population. as discussed, poultry are high-risk birds for emerging novel influenza a strains. pork is another potential source of chronic hev. in a public health care initiative, the french health authority has recommended to cook pig-liver sausages prior to consumption [58] . precautions and proper care should therefore be taken when selecting, purchasing, and hunting high-risk animals and when cooking meat. the current biosecurity measures appear to have been generally more successful for constraining bacterial diseases, but less effective for viral diseases. surveillance of the emerging human viruses must include livestock, wild animal, and potential arthropod vectors as well as their environments at an international level of coordination. furthermore, individuals like livestock herders, zookeepers, hunters, rangers, and veterinarians working with reservoir or high-risk animals must take hygienic measures. notably, many viruses that affect the respiratory tract, like influenza, rsv, mers-cov, sars-cov, and hmpv are transmitted by respiratory secretions and aerosol droplets. effective measures for preventing their transmission in a community and their nosocomial spread are the adoption of good practices like frequent hand-washing and avoiding direct contact with patients [59] . in addition, generating awareness in the general public about various aspects of these diseases will also assist in the better management and control of these emerging viral infections. the overall balance between exposure and evolution as driving forces of human virus diversity is difficult to assess accurately, particularly for viruses that are not known to have adapted to humans and that exist primarily in animal reservoirs. despite landmark advances in understanding the nature and biology of many pathogenic viruses, there is limited knowledge on emerging novel viruses, their potential reservoirs, and their modes of transmission. for example, although the person-to-person transmission of bkv is known, no zoonotic or other origin has yet been established. likewise, though mcv is found in respiratory secretions, healthy skin sheddings and gastrointestinal tissues, its precise mode of transmission still remains undetermined. the perpetual nature of the emergence and transmission of infectious diseases therefore poses an ongoing challenge which is volatile and ever-changing. however, despite incidences of infection and pathogenicity, for many viruses like cchfv and zikv, there is no vaccine prophylaxis or therapeutic intervention available at present. the recurring post-monsoon outbreaks of dengue and chikungunya fever and their control in the indian subcontinent remain challenging. the complexity of the co-circulation of denv, chikv, and zikv in many regions makes differential diagnosis difficult due to overlapping clinical manifestations and the partial cross-reactivity of antibodies [60] [61] [62] [63] . these challenges include a requirement for constant surveillance, prompt and efficient diagnosis, and development of new therapies. there is a further need for devoted research, not only to develop counter-measures but also to understand the basic biology of new viral pathogens and human susceptibilities. while advances in biomedical technologies have enabled us to prospectively identify pathogenic inter-species viruses and assess their transmissibility, the environmental release of several potential "laboratory-adapted" viruses is considered to be a big threat. parvez the frequent emergence or re-emergence of deadly viral infections has significantly affected human health despite extraordinary progress in biomedical knowledge. high population density, rampant constructions, poor sanitation, changing climate, and the introduction of anthropophilic vectors create selective pressure on hosts and pathogen reservoirs. although we know that a substantial fraction of well-identified mammalian viruses is responsible for human etiology, there are large numbers of evolving viruses waiting to infect and adapt. the unpredictable nature of novel infections, the rare occasions of outbreaks, the small numbers of confirmed cases, the asymptomatic occurrence, and occurrence in remote areas severely hamper the assessment of control and preventive modalities. efficient vaccines are already available against important human viruses, like hbv and human papillomavirus. even though drug resistance in hbv and hiv has accelerated alarmingly, new generations of broad-spectrum anti-viral agents as combination regimens hold promise. moreover, specific geographical regions or interfaces between humans, livestock, wildlife, and the environment should be targets for intense surveillance. in this way, further extensive research will surely enhance our understanding and capacity for predicting new pandemics and preparing control measures in advance. the origin of plagues: old and new death from 1918 pandemic influenza during the first world war: a perspective from personal and anecdotal evidence estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study who: summary of probable sars cases with onset of illness from 1 new human papovavirus (b.k.) isolated from urine after renal transplantation cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy clonal 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dynamics ten years of global evolution of the human respiratory syncytial virus ba genotype with a 60-nucleotide duplication in the g protein gene genetic variability in the g protein gene of group a and b respiratory syncytial viruses from india epidemiology, transmission dynamics and control of sars: the 2002-2003 epidemic forecast and control of epidemics in a globalized world transmission dynamics of zika virus in island populations: a modeling analysis of the bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china potential transmission of human polyomaviruses through the gastrointestinal tract after exposure to virions or viral dna chikungunya virus: recent advances in epidemiology, host pathogen interaction and vaccine strategies chronic hepatitis e infection: risks and controls risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review phylogenetic and molecular clock analysis of dengue serotype 1 and 3 from new occurrence of co-infection with dengue viruses during emergence of chikungunya virus in indian subcontinent after 32 years: a review molecular characterization of dengue and chikungunya virus strains circulating in new delhi, india the authors declare no conflicts of interest. key: cord-278647-krh63hqp authors: carter, robert w; sanford, john c title: a new look at an old virus: patterns of mutation accumulation in the human h1n1 influenza virus since 1918 date: 2012-10-12 journal: theor biol med model doi: 10.1186/1742-4682-9-42 sha: doc_id: 278647 cord_uid: krh63hqp background: the h1n1 influenza a virus has been circulating in the human population for over 95 years, first manifesting itself in the pandemic of 1917–1918. initial mortality was extremely high, but dropped exponentially over time. influenza viruses have high mutation rates, and h1n1 has undergone significant genetic changes since 1918. the exact nature of h1n1 mutation accumulation over time has not been fully explored. methods: we have made a comprehensive historical analysis of mutational changes within h1n1 by examining over 4100 fully-sequenced h1n1 genomes. this has allowed us to examine the genetic changes arising within h1n1 from 1918 to the present. results: we document multiple extinction events, including the previously known extinction of the human h1n1 lineage in the 1950s, and an apparent second extinction of the human h1n1 lineage in 2009. these extinctions appear to be due to a continuous accumulation of mutations. at the time of its disappearance in 2009, the human h1n1 lineage had accumulated over 1400 point mutations (more than 10% of the genome), including approximately 330 non-synonymous changes (7.4% of all codons). the accumulation of both point mutations and non-synonymous amino acid changes occurred at constant rates (μ = 14.4 and 2.4 new mutations/year, respectively), and mutations accumulated uniformly across the entire influenza genome. we observed a continuous erosion over time of codon-specificity in h1n1, including a shift away from host (human, swine, and bird [duck]) codon preference patterns. conclusions: while there have been numerous adaptations within the h1n1 genome, most of the genetic changes we document here appear to be non-adaptive, and much of the change appears to be degenerative. we suggest h1n1 has been undergoing natural genetic attenuation, and that significant attenuation may even occur during a single pandemic. this process may play a role in natural pandemic cessation and has apparently contributed to the exponential decline in mortality rates over time, as seen in all major human influenza strains. these findings may be relevant to the development of strategies for managing influenza pandemics and strain evolution. at the close of world war i, the h1n1 influenza a virus swept the world [1] . during the 1917-1918 pandemic, approximately 40% of the human population was infected, with a death rate above 2%. it is estimated that this virus killed more people than died in the world war that was just then ending. mortality rates have dramatically declined since then [2] , but the h1n1 flu has persisted. as a zoonotic pathogen, the influenza virus is able to infect multiple species. it is generally thought that aquatic waterfowl are a primary natural influenza reservoir [3] , where there are usually no clinical symptoms [4] , and where low level transmission probably perpetuates the viral pool [5] . all 14 influenza subtypes are maintained in waterfowl [5] . h1n1 has had an interesting history. derivatives of the original virus circulated in humans and swine until 1957, when the human strain went extinct. in 1977, a version identical to those circulating in ne europe in the early 1950s reappeared in anshan, china and subsequently spread across the world [5] [6] [7] . in 2009, a swine h1n1 jumped to the human population, causing a widespread pandemic. this has increased concern that h1n1 might mutate into a more virulent form. however, since the pandemic of 1917, this has not happened. in fact, h1n1-related human mortality has declined very dramatically and very systematically [2] . apart from the 1917 pandemic, h1n1 has failed to cause any severe global pandemic, and human h1n1 essentially went extinct from 1957-1977. since its re-introduction, it has remained a relatively minor cause of influenza mortality [2] . this applies also to the 2009 outbreak, which caused relatively few deaths in those areas with good reporting systems in place [8] . it is therefore reasonable to ask if the striking reduction in h1n1 mortality might be due, in part, to natural attenuation resulting from deleterious mutation accumulation. herd immunity is undoubtedly an important factor in reduced h1n1 mortality since 1918, but this may not be sufficient to explain the continuous decline in h1n1-related mortality over multiple human generations or the eventual extinction of the viral strain. likewise, improved medical treatments, such as antibiotic treatment for flu-related pneumonia, were certainly a significant factor reducing h1n1 mortality, but these do not appear to fully explain the nature of the pattern of mortality decline seen for h1n1. for example, the exponential decline in mortality began before the invention of antibiotic treatment. the literature suggests rna viruses should be inherently subject to mutational degeneration [9] [10] [11] [12] [13] . this includes the bacteriophage ms2 [14] , the tobacco etch virus [15] , hiv [16] [17] [18] [19] , dengue virus type-2 [20] , ebola [21, 22] , and sars [23, 24] . some have suggested that intentionally increasing the rate of mutation accumulation ("lethal mutagenesis") may be a way to control viral epidemics by hastening strain extinction [25] [26] [27] [28] [29] [30] . there is some long-term historical evidence that supports the concept of natural viral attenuation through mutation accumulation [2] , and theoretical studies using numerical simulation strongly support the concept of natural and accelerated genetic attenuation of rna viruses [13] . the influenza genome consists of eight rna segments totaling over 13,100 nucleotides. these code for up to eleven distinct proteins, two with alternate reading frames and one through alternate splicing. each of the eight rna segments has its own history of reassortment, inheritance, and mutation [5] . in the same way, each of the major serotypes (e.g., h1n1, h2n2, h3n2) has its own rate of mutation and history of reassortment. almost all of the influenza genome is protein coding, and 5.7% (749 nucleotides) of the genome codes for two protein products simultaneously (table 1) . currently, there are thousands of fully-sequenced influenza genomes available in public databases, including a reconstructed version of a 1918 h1n1 genome. most of these have collection dates and lineage information associated with them. given this large body of data, it becomes feasible to test the attenuation model using mutation accumulation rates, non-synonymous amino acid changes, changing dn/ds ratios, changing transition/transversions ratios, and changes in codon specificity over time. previous genetic studies examining the history of the influenza virus have performed extensive phylogenetic analyses of influenza genomes [8, [31] [32] [33] [34] [35] . they have shown considerable nucleotide diversity among circulating strains, given clear evidence for adaptive selection of antigenic variants [36] [37] [38] [39] [40] [41] [42] , and have shown that most of the major innovations within the flu genome have occurred via reassortment [5] , by which one flu strain has recombined with another strain and obtained a segment of rna from the second strain. influenza phylogenies are odd, in some respects, consisting of one main trunk with many short and short-lived side branches [36, 39, 42, 43] . there are several factors that influence strain diversity, including the mutation rate, selection, drift, and crossimmunity-mediated competition between strains [39] . feguson, et al. [39] felt that strain-transcending immunity was essential in restricting viral diversity. ito, et al. [42] thought that the many side branches died out because they could not compete with the main-trunk lineage viruses. others have noted the temporal extinction of circulating strains upon the introduction of new strains or serotypes [5] . whatever the reason, a hierarchy of strain robustness is evident in the history of influenza viruses. while phylogenetic studies can build robust family trees, they do so by focusing only on a limited number of "informative" genomic locations [44] . even though the influenza genome is broken into eight separate rnas, unless an individual is infected with two strains simultaneously (providing an opportunity for reassortment), all eight sections are inherited as a set in a form of linkage [36] . thus, neutral and slightly deleterious mutations are carried along with those mutations under positive or purifying selection. this gives us enough information to make many phylogenetic inferences, and we have a wealth of data telling us the history of the various viral lines, but these phylogenetic techniques ignore genetic change within a larger portion of the genome in order to focus on the phylogenetically-informative sites. there is abundant evidence for multiple substitutions in specific places that are, in turn, sites of active selection [42] , but what about the rest of the genome? fitch, et al. [36] noted that the main strategy of influenza viruses was to outrun the immune system of the host by maintaining a high mutation rate. they wondered "why such a virus does not accumulate so many deleterious mutations as to die of its own ineptitude." they further wondered if the only reason influenza viruses continued to circulate in mammalian species was because of a sort of "gene therapy" through reassortment with stronger, less mutated avian viruses. the present study is an attempt to elucidate more fully the nature of mutational change in this historically important human pathogen. accession numbers for all available complete flu genomes were obtained from the influenza research database [45] as of june 1, 2012. this list was then compared to those available at flugenome.org [46] . to create a list of the 2009-2010 h1n1 outbreak versions, we used the collated genome list from kedwaii, et al. [8] . using these two accession lists, sequence data were obtained from genbank [47]. we removed non-human/swine, non-h1n1, and incomplete genomes from this list, obtaining 3,755 human and 351 swine genomes. because there are few indels in these flu genomes, a simple first-pass alignment algorithm, written in perl with the bioperl toolkit [48] , was used. essentially, we visually picked out a 6-10-nucleotide candidate region close to the beginning of each of the eight strands of the flu genome that appeared mostly conserved. the algorithm scanned each set of sequences for this segment and added an appropriate number of spaces to the beginning of each for a rough alignment. using bioedit [49] , we simply looked for sequences that were misaligned and inserted gaps where necessary to complete the alignment, paying close attention to codon usage. in difficult and ambiguous regions, we chose to minimize the number of changes necessary to produce each variant, similar to the methodology of carter [44] . once an alignment for each of the eight sections of the flu genome was completed, we used another perl program to scan through the alignment and compare each sequence to a reference strain. point mutations were counted individually while indels, irrespective of size, were counted as a single change [44] . there are several different ways to calculate sequence differences (discussed in [50] ), but this simple measure is sufficient for the purposes of this paper. due to variations in technique among the various studies that produced these sequences, the ends of the alignment tended to be ragged. for this reason, we compared only the section of complete alignment between each sequence and the reference genome. the few internal reassortments [32] that have occurred in h1n1 were easily seen in the alignments, but these also made up a small fraction of all sites. we did our best to create a consistent alignment through these regions. we began by analyzing mutation accumulation during the human h1n1 outbreak of 2009-2010, using strain california/04/2009 as a reference. this was the first sequenced genome of the outbreak included in kedwaii, et al. [8] . we then calculated the extent of mutational divergence of all subsequent genomes over the duration of the outbreak. we did a similar, but much larger analysis of mutation accumulation in all human h1n1 strains from 1918 to 2012, using the 1918 "brevig mission" reference genome (a/brevig mission/1/1918) as a reference. this sequence was missing the last 480 nucleotides of segment 4. we amended this by appending the ending sequence of another 1918 h1n1 virus, a/south carolina/1/18(h1n1). these two strains had only one nucleotide difference in the orf of segment 4 (c/t at position 238). thus, they are essentially identical and we do not expect to have introduced extra mutations in this way. using the amended 1918 brevig mission virus as a reference and including all human and porcine viruses in the database, we calculated snps, indels, transitions, transversions, non-synonymous amino acid changes, dn/ds ratios, predicted protein lengths (for all 11 proteins), the normalized codon scores (ncs) and relative synonymous codon usage (rscu) [51] score for each predicted protein of each genome. to calculate the normalized codon score, we downloaded the frequency of each of the 64 codons in the human, duck and domestic pig genomes from the codon usage database [52] . we then created a predicted amino acid chain from each of the respective open reading frames. we calculated ncs for each protein by simply summing the frequency of each codon, as it is used in each of the three species, and then dividing by the total number of codons in the proteins produced. rscu is simply the number of times any particular codon appears, divided by the number of times synonymous codons appear in that genome. anhlan, et al. [51] were the first to apply rcsu to influenza genomic data, but they only studied one of the eleven protein products of the h1n1 genome and did not analyze changes over time. here, we extend their techniques to create a time series in which one can better study shifts in codon usage due to mutation accumulation. during the 2009-2010 h1n1 outbreak, mutations within h1n1 accumulated at a relatively constant rate ( figure 1 ). the strains sequenced at the end of the pandemic had approximately 80 more mutations than the 2009 reference genotype. the line of best fit reveals a slope of 42.1 mutations accumulated per year (3.2 × 10 -3 mutations per site per year). given that the data were collected from viral strains circulating worldwide, the correlation between mutation count and the date of collection was surprisingly good (r 2 = 0.77). the linear accumulation of mutations during the 2009 outbreak clearly also extends to the longer-term accumulation of mutations during the entire history of h1n1 ( figure 2 ). during the last century there has been a remarkably constant increase in mutation count within the h1n1 virus population, except for a striking discontinuity between 1957 and 1976. this discontinuity reflects the extinction of the human h1n1 strain in the mid 1950s, followed by the re-introduction of the strain in 1976, presumably from a researcher's freezer. even though most reports (e.g., [53] ) indicate the re-introduction year was 1977, our data suggests the year of re-introduction was 1976. this is based upon the isolate a/new jersey/1976, which very clearly falls in line with the rest of the re-introduced lineage. notice that after re-introduction in 1976, the human h1n1 mutation count and the rate of accumulation resumed exactly where it left off just before the extinction occurred in the 1950s. our data clearly show that some non-frozen h1n1 genotypes occasionally appeared in the human population after h1n1 dropped from public concern after 1957 ( figure 2 ). these fall on the same mutation-count trajectory as the h1n1 circulating before the 1957 extinction. however, these nine strains appear to represent repeated transmission events from pigs to humans that failed to cause any pandemic (the porcine lineage had no extinction event, and hence no pause in mutation accumulation). nearest neighbor calculations (data not shown) indicate these strains are not a continuation of the human lineage. they cluster tightly with the 2009-2010 outbreak porcine viruses, all of which are more closely related to the 1918 isolate than they are to the human lineage viruses. thus, we included the 2009-2010 viruses and the nine isolated, nonoutbreak viruses in the "porcine" category. in this same figure, we can see that genotypes from the 2009 outbreak and after ( figure 2 , circle) fall directly on the trajectory of the non-frozen lineage. this clearly shows that the 2009 genotype was not derived from the 1976 re-introduction virus. kedwaii, et al. [8] affirmed earlier conclusions that the 2009 genotype was due to a reassortment between two swine viruses, an h1n2 and an h1n1, from different continents, but this did not affect the mutation accumulation curve. regardless, general attenuation due to genome-wide mutation accumulation might best explain the very low mortality [54] [55] [56] associated with the 2009 pandemic. the earliest 2009 outbreak strain had already accumulated 1,889 mutations compared to the 1918 strain. the most obvious place to test how reassortment might affect the mutation accumulation curve is with the 2009-2010 "swine flu" outbreak samples. despite the fact that the viral line has been attributed to reassortment with swine viruses, with partial contribution from earlier reassortments between swine and avian strains [8] , it does not display a jump in mutation count. reassortment can produce novel antigenic variants, but it does not reverse the majority of mutations, for they have accumulated in the non-reassorted areas of the genome. after we identified the direct 1918 human lineage viruses, we were able to adjust the dates of the later samples to account for a period of dormancy ( figure 3 ). when we shifted the "frozen" samples back 21 years (i.e., sequences from 1976 were assigned a recalibrated date of 1955), we see the data lines up almost perfectly (r 2 = 0.989; mutation rate = 14.4). the exact amount of time-frame adjustment was not critical; adjustments ranging from 20 to 30 years all gave excellent alignments, with correlation coefficients above 98%. the observed accumulation rate of roughly 14 mutations per year (1.1 × 10 -3 mutations per site per year) is more or less consistent with previous estimates (e.g., [39, 57] ). the accumulation of non-synonymous mutations in human h1n1 genomes is also shown in figure 3 . the non-synonymous mutations are not as abundant, as expected, but they are still accumulating at a rapid and constant rate. there were an average of 2.4 amino acid changes per year (0.6 × 10 -3 mutations per codon per year). our data show that the lineage re-introduced in 1976 was the only significant human h1n1 strain from 1976 until 2009. the mutation count trajectory of the h1n1 lineage the data in figure 3 can be used to estimate the time of arrival into the human population of the first h1n1 virus. extrapolating total mutation count linearly backwards to zero yields a year of first introduction into the human population of approximately 1893. interestingly, this would be in time to explain the 1889-1890 "russian" flu outbreak, which has been considered by some to possibly be the first h1n1 outbreak (no sequence or serotype data are available for that event). while our data strongly suggest that the ancestral genotype for all human and porcine h1n1 influenza strains (and by extension, all human influenza strains), entered the human population as early as the 1890s, others have estimated the arrival date to be one or more decades later [5] . our data strongly confirm that all human h1n1 viruses derive from a single, very recent common ancestor, just as others have concluded prior to this study [1] . given an estimated arrival date of 1893, we infer that, by 1918, h1n1 would have accumulated roughly 375 (25 years × 15 mutations/year) mutations since the initial genotype invaded the human population. this depends, of course, on the exact arrival date. within the entire human h1n1 lineage, only 56% of all nucleotide sites were invariant, indicating intense mutation pressure. before its apparent extinction in 2009, the human h1n1 lineage had accumulated approximately 1400 mutations (mostly point mutations), including 320 non-synonymous mutations, compared to the 1918 genotype. across all 3,755 h1n1 genomes isolated from humans (i.e., including the outbreak and non-outbreak porcine versions), we found that only 41.8% of all nucleotide sites were invariant. multiple variant forms (i.e., 3-fold and 4-fold degeneracy, meaning 3 and sometimes 4 different nucleotides observed at a given site) occurred at 21% of the variable loci, further suggesting that the mutations were primarily non-adaptive. when we included the porcine h1n1 genotypes, only 36.1% of sites remained invariant, with a similar distribution of nucleotide degeneracy. because the re-introduced h1n1 lineage had accumulated fewer mutations than the porcine h1n1 strains, the disappearance of the human lineage and the subsequent dominance of the 2009 porcine strain in the human population resulted in even more divergence from the 1918 stain: over 1900 nucleotide differences (approximately 15% divergence) and 325 amino acids differences (approximately 7% divergence in protein sequence). the approximately 90-nucleotide pb1-f2 protein is coded on the second genomic segment and overlaps completely the pb1 coding region [58, 59] table 1 . pb1-f2 has been implicated in increasing host death rate. yet, not all influenza viruses code for this protein, so it is not intrinsically necessary for the cycle of infection. in the case of h1n1, the majority (98.2%) of genomes in this study are predicted to fail to produce a full-length version of the protein, including most of the human versions after 1948 and none of the porcine versions prior to 1987. for this reason, only 10 of the 11 influenza proteins were included in rscu and ncs calculations. all genomes were predicted to produce fully-formed pb2, pb1, pa, ha, np, na, m1, m2, and ns1 proteins. only 14 genomes were expected to fail to produce a functioning ns2 protein due to mutation in the start codon, but all of these had another potential in-frame atg only a few codons upstream and/or downstream. for this reason, we assumed these viruses would also produce an ns2 protein of proper length. mutation accumulation in swine h1n1 since 1918 is shown in figure 4 , with the human regression line (with "frozen" linage adjusted for 21 years of dormancy) for comparison. regression analysis (ancova) indicated a significant difference in the slope (p = 0.0001) of the lines of best fit for the viruses obtained from the two species. although it is clear that the swine versions have more variability towards the end of the sampling period, they also have a higher mutation rate in general, even after adjusting the human data for the 21 year period of dormancy. we note that many mutations appeared and disappeared several times over the life of h1n1. the data are too sparse over most of the range to know if this is due to repeat mutations at common loci or incomplete genetic sampling of the circulating h1n1 viruses. however, over the 2009-2010 h1n1 outbreak, kedwaii, et al. [8] demonstrated near-complete lineage loss among the circulating viruses during a single season. bedford, et al. [33] had similar results in their study of influenza a (h3n2). it was noted earlier that the phylogeny appears as a main trunk with many short-lived side branches. if most mutations are lost from the population quickly, variations that appear several times over the course of h1n1 are most likely due to mutations occurring multiple times in the same place. we do not have enough sequence data to get a full picture of strain diversity in most years, but this will be interesting to pursue as more data become available. even though each genomic segment has a slightly different mutation/substitution rate, strong, genome-wide patterns are evident, including a declining ratio of nonsynonymous to synonymous codon changes (dn/ds), a declining ratio of transition to transversion mutations (ti/tv), and even changes in codon bias. additional file 1 includes these data on a segment-by-segment basis. figure 5 illustrates a continuous decline in the ti/tv ratio during the entire history of the human h1n1 lineage. this suggests there may be increasing selection against transversions over time, perhaps reflecting some degree of truncation selection as the genome becomes more attenuated and as extinction approaches. a transversion is 2.7 times more likely to cause a non-synonymous change than a transition, and is also more likely to change rna 3-dimensional configuration. since we do not see a change in the rate of accumulation of amino acid substitutions over time (figure 3 ), we suspect that the heightened selection against transversions may be more strongly associated with rna architectural constraints. over the history of the human h1n1 lineage, the accumulation of non-synonymous mutations was highly linear, after correcting the "frozen" samples for 21 years of dormancy ( figure 3 ). while it is true that this simple distance metric applied to amino acid changes would be expected to underestimate evolutionary relationships at large distances due to an increasing likelihood of multiple changes at individual loci, applying the standard poisson or gamma corrections [60] made no improvement to the fit of the regression to two significant figures. we therefore conclude that the relationship is indeed linear, at least over the sampled timescale. analysis of the normalized codon scores (ncs) clearly shows that codon preference degenerated continuously throughout the entire history of the human h1n1 line ( figure 6 ). all viruses in our database are more duck-like in their codon usage and most (99.8%) are more human-like than pig-like, but codon preference moved toward randomized codon use and away from the codon preference of the human, swine and duck hosts. thus, the virus is not adapting to use the codon preference of any of the it has generally been assumed that any non-neutral mutations within the influenza genome have arisen as selective adaptations and generally help drive influenza toward a stronger and more dangerous pathogen (in terms of either pathenogenicity or transmissibility) [41] . this was probably the basis for the extreme caution exhibited during the 2009-2010 h1n1 outbreak. there is a general perception that, given enough time, figure 7 relative synonymous codon usage in the direct 1918 human h1n1 lineage for the four alanine codons, after adjusting the "frozen" portion of the lineage (1976-2009) by 21 years. note that the two decreasing in frequency over time are exactly matched by the two increasing over time (i.e., non-synonymous changes are having a negligible effect). also, three of these lines (gca, gcc, and gcg) indicate these codons are drifting even farther away from the human average. h1n1 might mutate into a stronger pathogen, and hence might create another catastrophic pandemic, as it did in 1918. selection is evident in h1n1 and other influenza genomes. in certain sites of the ha1 genomic segment of h3, non-synonymous substitutions occur at more than twice the rate as in other sites [39] . this is seen as a major signature of adaptive change, but deleterious mutations in the areas not under selection are carried along with the ones under positive selection [36] . in this light, some perceive h1n1 to be a growing threat, with a new outbreak being just a matter of time. despite this common perception, a more lethal version of h1n1 has not arisen via mutation within the human population during the last 90+ years. this is significant. the two major human influenza pandemics since 1918 did not arise due to mutations within h1n1, but arose via horizontal transmission of new genetic material from bird influenza strains, creating recombinant viruses. they were also less lethal than the 1918 version. it is true that the population had a degree of residual immunity and was not as immunologically naïve as it was in 1917-18, but selection has still not been able to generate a devastating pandemic from the remnants of that which swept the world at the close of wwi. in this paper, we examine an alternative point of view regarding mutation accumulation within h1n1. we suggest that, while specific adaptive mutations commonly occur within the h1n1 virus, many more deleterious mutations are accumulating than beneficial mutations, even when there is strong selection. consequently, h1n1 appears to have been in very gradual error catastrophe throughout its history. our results strongly confirm the widely recognized fact that all past and present human and swine h1n1 influenza strains derive from the 1918 strain. by extension, this applies to other human influenza strains, including h3n2 and h2n2 [1] . human influenza has such a high mutation rate that, even within a single host individual, the virus quickly becomes a genetically diverse "swarm" [61, 62] . yet, globally human h1n1 influenza is monophyletic and all current variation is of recent origin. this is only possible if almost all human influenza lineages rapidly go extinct. moreover, we present strong evidence that the h1n1 genome has been systematically degenerating since 1918. this is evidenced by continuous, systematic, and rapid changes in the h1n1 genome throughout its history. for example, there was an especially rapid and monotonic accumulation of mutations during a single pandemic (figure 1 ). similarly, there was a continuous and rapid accumulation of mutations over the entire history of the virus (figures 2 and 3) , including a similar steady increase in nonsynonymous amino acid substitutions (figure 3 ). while mutations accumulated in the human h1n1s, there was a parallel accumulation of mutations in the porcine h1n1 lineage (figure 4) . fitch, et al. [36] also showed a linear mutation accumulation curve. the "gnarled trunk" of ito, et al. [42] should be buried in our data as well, but it will be swamped by the pervasive, genome-wide accumulation of mutations not under active selection. within the human lineage, there was a systematic decline in the ti/tv ratio ( figure 5 ). in addition, there was a very consistent loss of codon specificity over time (figures 6 and 7) . we show compelling new evidence supporting the extinction of human h1n1 in the 1950s, its subsequent re-introduction in 1976, and an apparent second extinction event of the human h1n1 lineage in 2009. strain extinction has often occurred when new strains appeared, including h1n1 replacing the circulating h3-like strains in 1917, h2n2 replacing h1n1 in 1957, and h3n2 replacing h2n2 in 1968 [5] . to our knowledge, we are the first to document the replacement of the re-introduced human h1n1 with reassorted swine h1n1 in 2009. all this is consistent with the genetic attenuation hypothesis, and we feel this is the most fundamental explanation for the very smooth, systematic, and exponential decline in h1n1 mortality rates since 1918 [2] . in light of these findings, what are the medical implications? does mutation accumulation really have anything to do with virulence? simonsen, et al. [2] showed mortality statistics for three influenza strains over multiple years (h1n1 from 1918 to 1987; h2n2 from 1958 to 1962; and h3n2 from 1968 to 1995). even though there is some debate concerning the mortality burden imposed by influenza viruses [63] , there has clearly been a continuous exponential decline in influenza-related mortality over time, and this is true for all three major serotypes. since there is a strong linear correlation between mutation count and time (figures 2 and 3) , and since there is also a close correlation between declining virus-related death rates and time, there is obviously also a correlation between mutation count and reduced death rates. reduction in mortality may be due to many other factors, including herd immunity, advances in medicine, and advances in hygiene, but would these other factors be expected to follow so tightly the time courses seen in simonsen? there have been major medical advances since 1918, and these have clearly been a factor in reducing h1n1-related mortalities. therefore, the correlation between mutation count and reduced h1n1 mortality might be considered spurious by some. however, while it is certainly true that medical intervention has greatly improved in the developed world since 1918, such medical intervention has been much more limited in the rest of the world. second, the observed decline in mortality is a remarkably smooth curve, while medical advances have occurred in bursts (e.g., the breakthrough in antibiotics, and the more recent development of antivirals). third, each of the great influenza pandemics (1918, 1956, 1968) involved the emergence of a new viral strain, which then followed its own exponential decline in mortality but within its own timeframe. this uncouples reduction in mortality and stage of medical advance. finally, the correlation between the exponential decline of h1n1-related mortalities and the linear increase in h1n1 mutations is only one of our evidences for the genetic attenuation of h1n1. our other evidences include: a) the extinction of all human influenza strains existing prior to the h1n1 strain; b) the apparent extinction of the human lineage of h1n1 in 1956, and then again apparently in 2009; and c) the erosion of h1n1 codon specificity, approaching random codon usage. it is our contention that all human influenza strains undergo natural attenuation due to mutation accumulation. it is too early to tell if the remaining versions of the 2009-2010 outbreak viruses will do the same, but it is likely given the known history of change in the various influenza genomes. the decline in codon bias is especially significant for several reasons. first, since the frequency of codon usage is positively correlated with trna availability in the cell [64] , the increased use of rare codons is expected to negatively affect protein translation rates. alternatively, li, et al. [65] did not notice any decrease in translational efficiency based on codon choice in bacteria, but they did see effects caused by mutation towards other genomic control motifs (i.e., anti-shine dalgarno sequences). part of codon bias deals with a cell's avoidance of controlling factors that do not directly deal with translation rates (e.g., cg dinucleotides). thus, there are multiple ways a disruption of codon bias might negatively impact the functionality of a particular stretch of any nucleic acid. second, even though the codon usage in ducks and humans is similar [51] , although less so for swine [52] , this might affect the ability of a virus to cross species lines (after cellular antigenic recognition is taken into account). third, anhlan, et al. [51] entertained several hypotheses, including that the virus was avian in origin but transferred to pigs before it jumped to humans. our data clearly indicate that all h1n1s studied are more duck-like in their codon usage, although we cannot comment about a prehuman swine intermediate based on our data. finally, since we see an obvious decay in codon bias over time when compared to codon usage in either human, duck, or pig, it is clear that h1n1 is not evolving toward optimal codon usage in any of these species but is slowing drifting away from optimal translational efficiency. we concur with anhlan, et al. that, "the issue of codon usage seems to be much more important at least for influenza viruses than previously thought." during the last 100 years, the h1n1 influenza genome has diverged from the original genotype by roughly 15%. might the approximately 1,900 nucleotide substitutions be primarily attributed to the genetic drift of perfectly neutral variations? this seems unlikely for several reasons. first, a viral genome of approximately 13,000 nucleotides does not have room for very much neutral rna. not only did 15% of the genome change, but polymorphisms arose across more than 50% of the genome. this strongly points to extreme mutational pressure, high enough, reasonably, to threaten error catastrophe. second, if some significant portions of the viral genome are neutral, deletions of such portions of the viral genome should be regularly seen, and selection should favor such deletions, rapidly producing smaller genomes. there is no evidence of significantly smaller influenza genomes. indeed, there is little evidence of deletion in any of the 2009-2010 genomes compared to the 1918 version. the only major indels occurred among the oldest samples (prior to 1948) in the sixth genomic segment (neuraminidase, or na), but all of these represented deletions compared to the 1918 genome and all later genomes. third, it is now known that even synonymous mutations are not always neutral. even though they may not directly affect protein sequence, they can affect rna stability, rna architecture, speed of translation, and protein folding. fourth, there should only be a finite number of nucleotide positions that are perfectly neutral. because of this, neutral divergence should quickly approach a limit, causing the rate of divergence from the original genotype to slow, but this is not seen. finally, the extensive genetic changes observed simply do not appear to be phenotypically neutral; they are tightly correlated with rapid fitness decline, attenuation, extinction of most circulating strains, and even more frequent sub-lineage extinction events. might the observed divergence be primarily due to adaptive mutations? we feel that the 15% divergence must be primarily non-adaptive because adaptation should occur rapidly and then reach a natural optimum. yet, we see that divergence increases in a remarkably linear manner. furthermore, the virus does not seem to be converging on a new optimal genotype since polymorphism remains extreme (over 50%), since many polymorphic sites have more than two alleles, and since codon specificity is declining over time. codon patterns can inform us about the origin of the virus, and they tell us that h1n1 is not only drifting away from that original codon use, but it is also drifting away from the host codon preferences. when grown in mouse cell culture, 2009 h1n1 viruses exhibited variation in replication rates, virulence, and pathogenicity, but these did not match the severity of clinical symptoms in humans [66] . thus, at this time, the exact relationship between mutation load and the severity of infection remains unknown. yet, selective adaptation is limited to only those amino acids that produce significant phenotypic effects [42] , and, since a viral genome in the absence of reassortment is essentially a single linkage block, it is expected that many more than just adaptive mutations occur. some of the changes might be due to selection, but the majority certainly are not. is it feasible that natural selection might fail to remove a large number of deleterious mutations? it is well known that numerous factors can cause a breakdown in the selective removal of deleterious mutations. these factors include a high mutation rate, a high rate of random loss, limited sexual recombination, genetic bottlenecking, and mutations with very small fitness effects. all of these factors should be especially pronounced in an rna virus such as influenza and all of these are either previously known or documented here. the genetic changes in h1n1 appear non-directional and are distributed quite uniformly across the genome (additional file 3), consistent with an accumulation of low-impact deleterious mutations. error catastrophe and lethal mutagenesis are already recognized as a threat to any rna virus. these facts, combined with the dramatic decline in h1n1 mortality and the very high rate of h1n1 strain extinction, all very strongly indicate that most of the genetic divergence from the original h1n1 genotype has been due to fixation of slightly deleterious mutations. could h1n1 ever back-mutate into a strain such as the ancestral genotype that caused the catastrophic 1917-1918 pandemic? given that the modern strains of h1n1 have diverged from the original 1918 strain by nearly 2000 mutations, that many of these mutations should be slightly deleterious [67] , and that natural selection was unable to stop their continuous accumulation in the first place, it is difficult to imagine how mutation/selection might ever restore full virulence. reassortment might bring in new material, but thus far this has only applied to a limited section of the genome, and reassortment today occurs in a very different mutational/genomic context than that of 95 years ago. it is often thought that a high mutation rate translates to rapid adaptation and evolution, yet the reverse seems more commonly true. deleterious mutations often interfere with selection for the more rare beneficial mutations [67, 68] . thus, the rapid accumulation of mutations in all h1n1 lineages should logically lead to their eventual extinction. the origin of human h1n1 influenza is unknown, but it is generally reasoned that it invaded the human population from a natural reservoir [5, 51] , most likely an aquatic waterfowl, with pigs as a possible intermediate host. in light of the strong tendency toward natural genetic attenuation which we document here, we suggest that the natural reservoir most likely involves a very quiescent viral state, as might occur within a host where there is very little viral replication, and hence much lower mutation rates. it would be very interesting to know the rate of influenza mutation accumulation in waterfowl. can reassortment explain these findings? based on our mutation count analysis, there is no evidence for reasortment in the human h1n1 lineage, and it has gone extinct, apparently twice. from other studies, various influenza strains are obviously derived from reassortment, but all this does is set the mutation clock back a little. any reassortment between a "fresh" virus and a high-mutation-count virus will inevitably lead to, at best, an averaging of the mutational load of the two. the 2009-2010 "swine flu" virus shows evidence of multiple reassortment events in a limited portion of its genome, and it was more robust than the lingering human h1n1 strain that it replaced, but it carries a great number of non-adaptive and presumably deleterious mutations. reassortment between two viruses of different immunological character might preserve the less degraded genome, but only temporarily. might pandemics be shortened by artificially accelerating the rate of genetic attenuation? the continuous and linear accumulation of mutations within a single influenza lineage, as was seen in h1n1 during the 2009-2010 influenza season (in which about 0.3% of all nucleotides mutated), supports the concept that natural genetic attenuation may be an important factor in the natural cessation of influenza pandemics. thus, the possibility of an artificial acceleration of mutation rate deserves further investigation, and may suggest new avenues of research in terms of pandemic management [13] . it is clear that natural selection is strongly at work in the influenza genome. this can be seen by preservation of all the basic proteins and functions of the virus, in spite of the fact that every possible point mutation happens in every human individual during the course of an infection. a large fraction of all deleterious mutations clearly must be selected away. likewise, the emergence of major antigenic variants shows that positive selection is operational. it is also clear that genetic drift is strongly in operation, with a major viral bottleneck happening at each transmission from one human host to the next, and perhaps at the start of each local outbreak [33] , ensuring that most unique genotypes are very quickly lost. yet, in addition to selection and drift, it also appears there is very strong mutational pressure on the influenza genome, potentially leading to lethal mutagenesis in most strains, and a gradual, natural genetic attenuation of human influenza in general. read and taubenberger [69] called the origin of human h1n1 an "enigma" whose riddle was not yet solved. like them, we see this as an unsolved riddle and we can only hope that our data might bring us one step closer to understanding the origins of this important disease. sequence analysis of historical and modern genomes of influenza h1n1 reveal a great deal about the history of the virus. the most recent common ancestor existed only about 120 years ago, and there has been universal extinction of all earlier human influenza strains. the rate of mutational divergence from that original genotype has been very constant both in human and porcine h1n1 strains (roughly 14-16 mutations fixed per year). modern h1n1 strains have diverged from the original genotype by roughly 1,900 fixations (more than 15% of the genome has mutated). roughly 7% of the amino acids have mutated. mutation accumulation is also associated with the historical exponential decline in h1n1 human mortalities, which may suggest significant genetic attenuation, as might arise due to very slow error catastrophe. sequence analysis confirms that the human h1n1 strain went extinct in 1957, but was reintroduced in 1976, apparently from a specimen frozen in the early 1950s. the resulting "frozen" lineage appeared less mutated compared to all other contemporary h1n1 strains (i.e., porcine strains and a few rare h1n1 strains appearing in humans but not derived from the frozen strain). consistent with the genetic attenuation model, the frozen human h1n1 lineage disappeared in 2009, and may now be extinct. it appears that the h1n1 strains currently in circulation are significantly attenuated, and cannot reasonably be expected to back-mutate into a non-attenuated strain. the greatest influenza threat, therefore, is the introduction of a non-attenuated strain from some natural reservoir. this suggests that a better understanding of the origin of such non-attenuated strains should be a priority [5] . our findings suggest that new strategies that accelerate natural genetic attenuation of rna viruses may prove useful for managing future pandemics and, perhaps in the long run, precluding the genesis of new influenza strains. additional file 1: h1n1 strain variation data using the 1918 brevig mission virus as a reference, in .csv format. additional file 2: relative synonymous codon usage (rscu) data, in .csv format. additional file 3: h1n1 genomic variation data, in .csv format. abbreviations nscu: non-synonymous codon usage; ncs: normalized codon scores. influenza: the mother of all pandemics. emerging infectious diseases pandemic versus epidemic mortality: a pattern 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influenza virus: a continuing enigma cite this article as: carter and sanford: a new look at an old virus: patterns of mutation accumulation in the human h1n1 influenza virus since 1918 this work was supported in part by fms foundation. thanks to tom gault for computer hardware support, henry wan for critical comments on an earlier draft, and the helpful suggestions of four anonymous reviewers. the authors declare that they have no competing interests.authors' contributions rc wrote the computer programs and performed most of the analyses. js was responsible for the development of the initial idea and interpretation of the data generated. both authors contributed equally to the preparation of the manuscript. all authors read and approved the final manuscript. key: cord-262868-wanbz1et authors: varki, ajit title: loss of n‐glycolylneuraminic acid in humans: mechanisms, consequences, and implications for hominid evolution date: 2002-01-04 journal: am j phys anthropol doi: 10.1002/ajpa.10018 sha: doc_id: 262868 cord_uid: wanbz1et the surface of all mammalian cells is covered with a dense and complex array of sugar chains, which are frequently terminated by members of a family of molecules called sialic acids. one particular sialic acid called n‐glycolylneuraminic acid (neu5gc) is widely expressed on most mammalian tissues, but is not easily detectable on human cells. in fact, it provokes an immune response in adult humans. the human deficiency of neu5gc is explained by an inactivating mutation in the gene encoding cmp‐n‐acetylneuraminic acid hydroxylase, the rate‐limiting enzyme in generating neu5gc in cells of other mammals. this deficiency also results in an excess of the precursor sialic acid n‐acetylneuraminic acid (neu5ac) in humans. this mutation appears universal to modern humans, occurred sometime after our last common ancestor with the great apes, and happens to be one of the first known human‐great ape genetic differences with an obvious biochemical readout. while the original selection mechanisms and major biological consequences of this human‐specific mutation remain uncertain, several interesting clues are currently being pursued. first, there is evidence that the human condition can explain differences in susceptibility or resistance to certain microbial pathogens. second, the functions of some endogenous receptors for sialic acids in the immune system may be altered by this difference. third, despite the lack of any obvious alternate pathway for synthesis, neu5gc has been reported in human tumors and possibly in human fetal tissues, and traces have even been detected in normal human tissues. one possible explanation is that this represents accumulation of neu5gc from dietary sources of animal origin. finally, a markedly reduced expression of hydroxylase in the brains of other mammals raises the possibility that the human‐specific mutation of this enzyme could have played a role in human brain evolution. yrbk phys anthropol 44:54–69, 2001. © 2001 wiley‐liss, inc. the evolution of modern humans from a common ancestor with the great apes occurred in a series of steps, influenced by complex interactions among genetic, developmental, ecological, microbial, climatic, behavioral, cultural, social, and other factors. there are many scientifically valid approaches towards gaining an understanding of how we came to be human. prominent among these approaches are: developmental, cognitive, social, and behavioral comparisons of humans with other primates; the paleontology and archeology of human ancestors; and the systematic genetic comparison of humans with other primates. given the remarkable progress of molecular biology techniques, the deciphering of most of the human genome (lander et al., 2001; venter et al., 2001) , and the relative ease of obtaining genomic dna noninvasively from humans and other primates, the genetic approach should, in principle, be somewhat easier than the others mentioned. in fact, apart from the general realization that our genomic dna sequences are remarkably similar to those of the great apes (king and wilson, 1975; sibley and ahlquist, 1987; caccone and powell, 1989; goodman et al., 1994; ruvolo, 1997; takahata and satta, 1997; kaessmann et al., 2001; chen and li, 2001) , there have been relatively few specific genetic differences between humans and apes uncovered to date (reviewed in gagneux and varki, 2001) . this has led some to call for a great ape/ primate genome project (mcconkey and goodman, 1997; , to accelerate progress in this area. while morphologists have long sought to demonstrate the adaptive significance of divergent anatomical structures, this has generally not been the case for molecular anthropologists. the latter have, to a large degree, used comparative dna (or amino acid) sequences for dating evolutionary events rather than for understanding the evolutionary processes that led to the sequence divergence. thus, very little is known of the selective (or random) forces that led to the 1-2% sequence differences between humans and the african great apes, or about the adaptive molecular differences that emerged during this period of primate evolution. this review discusses one of the few known apehuman genetic differences with a clear-cut biochemical consequence, the selective inactivation of the cmp-n-acetylneuraminic acid (cmp-neu5ac) hydroxylase gene in the human lineage irie et al., 1998; chou et al., 1998) . the resultant loss of a specific cell-surface sugar on human cells has implications for issues as diverse as human susceptibility and resistance to pathogens, the consequences of human ingestion of animals foods, the human innate immune response, and the development of the human brain. as with most unexpected discoveries, this finding has raised more questions than answers. an attempt is made to address some of these questions, and to suggest directions for future research. every high school graduate should now know about dna, rna, and proteins, and most would understand that a universal dna code defines the genes of all living things. these genes can be transcribed into their corresponding rna sequences, which in turn are translated into proteins. this "central paradigm" of molecular biology, i.e., that dna makes rna makes protein, has dominated recent approaches to understanding how living things work. however, there are several reasons why this scientifically powerful reductionist approach cannot fully explain the structure and function of complex multicellular organisms like humans. one reason is that dna itself is of little use unless the genes it encodes are expressed (transcribed to rna, and hence translated into proteins)-and gene expression is profoundly influenced by the physical, ecological, biological, and social milieu in which an organism exists. indeed, the social and cultural activities of humans must have a major impact on gene expression within the species, as well as in the many other species that humans interact with. a second reason is that besides dna, rna, and proteins, there are two other major classes of molecules that are required to create almost all life forms that we know of: lipids and sugars (glycans). glycans fall into two general categories: the more familiar small sugars like glucose that are a major source of energy, and the less wellknown glycan chains that are attached to many proteins and lipids, particularly on the surface of cells (varki, 1999a) . indeed, there are no extant freeliving organisms whose cells are not each covered with a dense coating of these glycan chains, comprising a so-called "glycocalyx" that is no less obvious than the icing on a birthday cake. these complex cell-surface sugar chains are the products of a specialized intracellular machinery whose synthesis and organization are themselves dictated by the expression of several hundred genes (varki and marth, 1995; varki, 1998) . this machinery is supplemented to an unknown extent by the incorporation of sugar building blocks from dietary sources into endogenous biosynthetic pathways (freeze, 1999) . given their ubiquitous occurrence in nature and their dominant presence on the surface of cells, it is not surprising that these sugar chains are intimately involved in the interactions between cells, both within an organism, and between organisms. a third limitation of the dna-rna-protein paradigm for explaining humans is that the human genome seems to have less than 35,000 genes (lander et al., 2001; venter et al., 2001) . thus, there cannot possibly be a "gene for" each specific biological entity or function. rather, the genetic contribution to the structure and function of most aspects of an organism arise from the combinatorial effects of multiple genes, gene regulation mechanisms, and gene products that act upon one another in various ways, under the constant influence of environmental factors. the attachment of glycan chains to proteins and lipids (so-called glycosylation) is a prime example of such a "postgenomic" process, wherein new biological entities or functions result from the action of one set of genes on the products of other genes. despite these considerations, studies of the structure and biology of glycans have lagged far behind those of dna, rna, proteins, and lipids. there are many reasons for this, including the branching and complexity of these sugar chains, the lack of an easily recognizable template-driven functional "code," and technical limitations in studying their structure and function. recent advances have substantially reduced this technical gap, opening up a new field now called glycobiology (rademacher et al., 1988; varki, 1999a) . thus, as with genomics (determining the total genomic dna of an organism), transcriptomics (elucidation of the complete set of genes expressed in a given cell type in a given situation), and proteomics (the description of all proteins found in a given cell type in a given situation), we are just beginning to enter the era of glycomics (elucidation of the complete array of glycans found on a given cell type in a given situation). a microbial organism approaching a mammalian cell surface would likely first encounter members of a family of sugars called sialic acids, which tend to be the outermost units on the glycan chains attached to the proteins and lipids below (fig. 1) . this family of 9-carbon acidic sugars (gottschalk, 1960; rosenberg and schengrund, 1976; schauer, 1982; ye et al., 1994; inoue et al., 1996; varki, 1992 varki, , 1999b is found predominantly in the deuterostome lineage of animals (warren, 1963; traving and schauer, 1998; . there are more than 40 kinds of sialic acids known in nature, and most are derived via biosynthetic modifications of a parent molecule called n-acetylneuraminic acid (neu5ac) (fig. 2) . further complexity arises from the fact that sialic acids can be attached to the underlying sugar chain in several different types of linkages (tsuji et al., 1996) . while many of these kinds and linkages of sialic acids can be found within a single species, there are also marked species-specific differences with regard to their relative amounts and/or distribution. likewise, even within a single species, there can be substantial differences between different cell types in the pattern and composition of their sialic acids. the evolution of this remarkable structural complexity is probably related to the diverse biological roles of these sialic acids in different cell types. 2 . structure of the two most common sialic acids on mammalian cell surfaces. the 9-carbon backbone common to all sialic acids is numbered. thick arrow points to the single oxygen atom that differentiates neu5gc from neu5ac. humans are genetically defective in the gene encoding the enzyme responsible for adding the oxygen atom. by virtue of their negative charge and surface location, sialic acids can mediate many biological roles in normal and pathological situations (fig. 3 ). it is well-known that a wide variety of human and animal pathogens use cell-surface sialic acids to either gain an initial foothold on the cells they infect or to target toxic molecules that they secrete (sandvig et al., 1991; escalante et al., 1995; sharon, 1996; varki, 1997 varki, , 1999b gagneux and varki, 1999; karlsson, 1998 karlsson, , 2000 fig. 3 for examples). if these were the sole functions of sialic acids, the detrimental consequences to deuterostome animals should have caused these sugars to be eliminated and/or replaced by others during the course of evolution. however, the absence of sialic acids is lethal during early mouse embryogenesis (w. reutter, personal communication), and genetic modifications of sialic acid linkages in mice cause significant pathologies (priatel et al., 2000; hennet et al., 1998) , indicating that these molecules also have critical endogenous functions. some of these functions are primarily structural or physical, e.g., aiding the filtration function of the kidneys or providing negative charge repulsion between cells in the blood stream. in the brain, this type of negative charge repulsion becomes enhanced and specialized by the formation of long chains of sialic acids (called polysialic acids). these can serve to physically separate neurons and neuronal extensions and hence participate in what can be loosely called "brain plasticity" at the organizational level (rutishauser and landmesser, 1996) . over the last two decades, it has become clear that sialic acids are also recognized by specific receptors within the same animals that synthesize these sugars (bevilacqua and nelson, 1993; kelm et al., 1994b; varki, 1994 varki, , 1997 powell and varki, 1995; crocker and feizi, 1996; kansas, 1996; collins et al., 1997a; kelm and schauer, 1997; crocker et al., 1998; brinkman-van der linden et al., 2000; crocker and varki, 2001a,b; see fig. 3 for examples). emerging evidence indicates that these receptors, particularly a family called the siglecs (crocker and varki, 2001a,b; , are able to recognize the diversity in sialic acids, and their linkages to underlying sugars. an additional set of biological roles for sialic acids is found in certain microbial organisms that decorate their cell surfaces with sialic acids (troy, 1992; wessels et al., 1989; bozue et al., 1999) , thereby allowing them to evade recognition and destruction by certain vertebrate immune mechanisms. these examples occur despite the fact that sialic acids seem to be otherwise restricted to the deuterostome lineage of animals. the best explanation can be found in the fact that all of these microorganisms are vertebrate pathogens (see examples in fig. 3 ). since surface sialic acids limit activation of multiple functions of the immune system, these pathogenic organisms likely benefit from coating themselves with these sugars. this form of molecular mimicry may have occurred mostly via convergent evolution, wherein these microorganisms evolved a variety of ways either to make their own sialic acids or to procure them from their vertebrate hosts. while there are many kinds of sialic acids in nature (gottschalk, 1960; rosenberg and schengrund, 1976; schauer, 1982; varki, 1992; ye et al., 1994; inoue et al., 1996; varki, 1999b; , the surfaces of most cell types in the mammalian species studied to date tend to be dominated by two major kinds: n-acetylneuraminic acid (neu5ac) and n-glycolylneuraminic acid (neu5gc). neu5gc is different from neu5ac only by an additional oxygen atom (see arrow in fig. 2 ). in order for sialic acids to get attached to glycoproteins and glycolipids, they must first be "activated" by conversion to their respective sugar nucleotide derivatives. thus, the common sialic acid neu5ac is converted to cytidine-monophosphate-neu5ac (cmp-neu5ac), which is then used as a high-energy donor for attaching neu5ac to newly made glycoproteins and glycolipids that are on their way to the cell surface. the synthesis of the neu5gc form of sialic acid takes place initially at the level of this sugar nucleotide precursor (shaw and schauer, 1988; bouhours and bouhours, 1989; muchmore et al., 1989; kozutsumi et al., 1990; shaw et al., 1992 shaw et al., , 1994 takematsu et al., 1994; kawano et al., 1995; schlenzka et al., 1996) . thus, as shown in figure 4 , an enzyme called cmp-neu5ac hydroxylase (hereafter referred to as cmah) catalyzes the transfer of one oxygen atom to cmp-neu5ac, generating cmp-neu5gc. the latter can now also be used as a donor to add neu5gc to molecules destined for the cell surface from their sites of initial synthesis within the cell. the enzymes that actually transfer sialic acids to glycoproteins and glycolipids (called sialyltransferases) can typically use both cmp-neu5ac and cmp-neu5gc as donors . thus, the ratio of these two major sialic acids found on a given cell surface is likely to be largely determined by the ratio within the sugar nucleotide donor pool available in that cell type. (varki, 1999b; traving and schauer, 1998) can be divided into the general groupings indicated. m, microorganism or toxin. see text for discussion. some of the early pioneers who discovered the sialic acids noted that, in contrast to the situation in other mammals such as rodents and ungulates, neu5gc was very hard to find in human tissues (reviewed in gottschalk, 1960; rosenberg and schengrund, 1976; schauer, 1982) . however, the methods they used could have missed small amounts of neu5gc in humans. an independent line of evidence suggesting that neu5gc is lacking in humans came from the medical field of hematology. for the clinical management of certain blood disorders, it becomes necessary to infuse serum from horses into human patients. not surprisingly, these patients often generate an immune response against components of the infused animal serum and manifest a condition called "serum sickness reaction," which contraindicates further infusions. some investigators who studied the serum sickness reaction noted that the immune response was being generated at least in part against neu5gc, which is present in abundance on horse serum glycoproteins and glycolipids (kasukawa et al., 1976; merrick et al., 1978; higashi et al., 1977) . this finding strengthened the notion that neu5gc is a foreign antigen to adult humans. however, several groups then reported (mostly using indirect methods such as antibody detection) that neu5gc could be found in human cancers and possibly in human fetal tissues (kawachi and saida, 1992; ikuta et al., 1982; higashi et al., 1984; stacker et al., 1985; hirabayashi et al., 1987a; kawachi et al., 1988; saida et al., 1990; devine et al., 1991; kawai et al., 1991; marquina et al., 1996; malykh and schauer, 2001) . moreover, neu5gc was also reported in some cultured cell lines of human origin (nakarai et al., 1987; ohashi et al., 1983) . taken together, these data suggested that humans might have a func-tional cmah gene, but simply suppress its expression sometime before the postnatal period when "immune tolerization" to self-antigens occurs. our recent studies done with more sensitive modern techniques showed that while neu5gc is undetectable (ͻ0.1% of total sialic acids) on the red blood cells and blood plasma proteins of adult humans, similar samples from all of the great apes have substantial amounts (neu5gc representing between ϳ20 -90% of total sialic acids). since the great apes are our closest evolutionary cousins (king and wilson, 1975; sibley and ahlquist, 1987; caccone and powell, 1989; goodman et al., 1994; ruvolo, 1997; takahata and satta, 1997) , the human loss of neu5gc expression must have occurred sometime after the ape-human common ancestor. a secondary consequence of this loss is that humans also have much higher levels of neu5ac, the precursor molecule to neu5gc. assays of cmah enzyme ( fig. 4) showed easily detectable activity in great ape cells, but not in human cells . the next logical step was to examine the gene encoding the cmah, to see if its promoter (regulatory) regions were mutated in some manner in humans, thereby altering its expression in adult humans. indeed, two groups independently found that the cmah gene in humans had suffered a mutation (irie et al., 1998; chou et al., 1998) . surprisingly the mutation was not in the regulatory regions of the gene that might have modified its expression patterns between fetal and adult states, but rather in the coding region which dictates the amino-acid sequence of the enzyme itself (fig. 5 ). the mutational event had deleted 92 base pairs of a single stretch of the sequences coding for the protein (corresponding to exon 6 in the mouse gene). since amino acids are dictated by triplets of dna base pairs, the loss of 92 base pairs (bp) also resulted in a "frame-shift," thus markedly truncating the length of the final protein encoded by the human gene (chou et al., 1998) . moreover, this truncated protein is missing certain amino acids that were known to be critical for the activity of the enzyme itself (schlenzka et al., 1996) . in contrast, examination of the cmah gene from the chimpanzee and relevant portions from the corresponding genes from other apes (chou et al., 1998) showed that they all encode an intact enzyme that is not very different from those originally cloned from mice and pigs (kawano et al., 1995; schlenzka et al., 1996) . thus, it is clear that humans lost neu5gc on their cell surfaces because of an inactivating mutation in the cmah gene. the gene is localized to chromosome 6 band p23-p22 in both humans and great apes (irie et al., 1998; chou et al., 1998) , which does not correspond to an area of known chromosomal rearrangement during hominoid evolution (yunis and prakash, 1982) . furthermore, the remaining human intronic region is very similar in size to that in the intact mouse genome (irie et al., 1998) , indicating that the deletion eliminating the fig. 4 . factors involved in biosynthesis of cmp-n-glycolylneuraminic acid (cmp-neu5gc). the enzymatic mechanism of the cmp-neu5ac hydroxylase that is primarily responsible for generating neu5gc in nonhuman animal cells is shown. the reaction takes place in the cytosolic compartment. the enzyme product cmp-neu5gc is the donor subsequently used for adding neu5gc to glycoproteins and glycolipids. cmp-neu5ac hydroxylase activity is present in great apes but not in humans. see text for discussion. 92-bp exon in humans must have been quite small. the corresponding genomic regions from the chimpanzee and other nonhuman primates were recently sequenced (hayakawa et al., 2001) . the region containing a 92-bp exon of the cmah gene turns out to have an adjacent alusq element in all nonhuman primates studied, and both were replaced by a newly disseminated aluy element that is specific to humans (alus are parasitic repetitive dna elements found in large numbers throughout the primate genome). thus, it is possible to propose a mechanistic model whereby an alu-mediated replacement event coincidentally deleted the 92-bp exon and inactivated the cmah gene sometime in the human lineage (hayakawa et al., 2001) . why did humans lose neu5gc production? it is difficult to be certain about why a particular genetic change became fixed in a particular species at some time in the evolutionary past. however, based on current knowledge of the functions of sialic acids (see above), one can propose some possible scenarios to explain the human loss of neu5gc. the most likely one is selection of a randomly occurring cmah gene mutation by a lethal microbial pathogen that required cell-surface neu5gc for effective infection (see below for some examples of such current-day pathogens). once such a mutation had thus become common in a human ancestral population, it could have undergone further selection because it conferred some additional benefits, or have simply drifted to fixation in the absence of any further selection. a more intriguing possibility is that the inactivated allele of the cmah gene was favorably selected because it conferred valuable new endogenous functions upon individuals who became homozygous for it. when did this mutation occur in relation to the various steps in human evolution? the identical mutation was found in genomic dna in ͼ40 individuals representing many different geographic regions, including african kung bushmen and khwe pigmies (among whom the greatest genetic diversity is known to be present; takahata and satta, 1997; kaessmann et al., 2001) . this, together with the lack of detectable neu5gc in blood samples from many additional humans studied by us, indicates that the mutation is universal to modern humans. thus, the inactivation of the cmah gene must have occurred sometime after our common ancestor with the bonobo/chimpanzee clade (ϳ6 million years ago; king and wilson, 1975; sibley and ahlquist, 1987; caccone and powell, 1989; goodman et al., 1994; ruvolo, 1997; takahata and satta, 1997) , but prior to (or possibly coincident with) the common origin of modern humans (variably estimated by different authors, with the most recent possible date of about ϳ100,000 years ago; takahata and satta, 1997; krings et al., 1997) . a more accurate estimate of the timing of the gene inactivation would be of value for generating hypotheses about the potential consequences of neu5gc loss in humans. for example, if it took place about 2 million years ago, this might suggest a possible contribution towards the brain size increase that began with the emergence of genus homo (wood and collard, 1999) . sequenceable autosomal dna has not been successfully recovered from fossilized mammalian bones that are more than about 50,000 years old (greenwood et al., 1999; hofreiter et al., 2001) . even the few successes tend to occur when using samples from northern latitudes. indeed, the probability of recovering dna is known to be worse for samples exposed to warmer temperatures (smith et al., 2001) . the occasional successes in obtaining mitochondrial dna from fossil hominids ͼ50,000 years old (krings et al., 1997) are explained by the much higher copy number of mitochondrial dna within each cell. overall, it is not possible (at least with current technology), to obtain and directly study cmah gene sequences from hominid fossils that predated the common origin of modern humans. two other approaches are therefore currently being pursued to try timing the inactivation of the cmah gene. the first relies on sequence comparisons of the great ape and other primate cmah genes with those of the remaining portions of the inactivated human gene. the assumption is that once the human gene became nonfunctional (i.e., became a pseudogene), it was no longer under selection pressure, and would thus accumulate both synonymous and nonsynonymous mutations at similar rates. such data are currently being used to estimate an approximate inactivation date (collaboration with n. takahata). the second approach relies on the direct study of residual sialic acids found in fossils (unpublished observations, in collaboration with s. paabo). these preliminary analyses suggest a dating of a little over 2 million years ago. it should be noted that there are reported strain variations in red blood cell expression of neu5gc among dogs (hashimoto et al., 1984; yasue et al., 1978) and cats (ando and yamakawa, 1982; furukawa et al., 1988a) , and in the latter instance, the presence or absence of neu5gc on red cells can act as a blood group system (andrews et al., 1992) . however, systematic studies of other tissues of these dog and cat strains have not been reported. thus, we do not know if it is only red blood cells that show differential expression. chickens are the only species besides humans that have been found to generate a generalized immune response to neu5gc when infused with animal serum (fujii et al., 1982) . in fact, polyclonal and monoclonal antibodies generated by chickens immunized with horse serum or horse red cell glycolipids have been very useful as tools in the study of neu5gc expression (hiraba-yashi et al., 1987b; higashi et al., 1988) . again, a systematic study of chicken tissues has not been carried out to see if the presumed lack of neu5gc is true for all organs. furthermore, the presence of neu5gc in ducks indicates that this is not a general feature of birds. further studies of the cmah gene in a wide selection of birds and carnivores seem warranted. however, a recent study showed that neu5gc is present on the serum immunoglobulins of cows, sheep, goats, horses, mice, dogs, guinea pigs, rats, and rabbits, as well as rhesus monkeys (raju et al., 2000) . thus, even if genetic variations in neu5gc expression are found among members of some of these species, the 92-bp exon deletion responsible for the human loss of cmah activity remains a unique genetic event that occurred after our common ancestor with the great apes, and is now universal among modern humans. as indicated above, many major pathogens and their toxins gain access to their mammalian hosts by binding to cell-surface sialic acids. the microbial binding proteins involved in such interactions can show exquisite specificity for the precise structure and linkage of the sialic-acid target (sharon, 1996; karlsson, 1998; varki, 1997 varki, , 1999b . thus, the human loss of neu5gc would have conferred protection from animal pathogens that prefer to bind to this sialic acid, while enhancing the success of pathogens that prefer to bind to neu5ac. as shown in table 1 , this issue has not been thoroughly studied for most human pathogens. it is clear that certain microbes causing serious diarrheal diseases in farm animals like cows and pigs have a strong preference for neu5gc (kyogashima et al., 1989; ouadia et al., 1992; willemsen and de graaf, 1993; lanne et al., 1995; delorme et al., 2001; schwegmann et al., 2001) , and humans are thus immune to infection. it is reasonable to speculate that this mechanism of resistance may have facilitated the domestication of some animal species, by limiting transfer of their pathogens to human caretakers. an even more speculative possibility is that this difference aided the worldwide migrations that brought humans into contact with diverse pathogen regimes of novel species of wild animals they encountered. in this regard, more information is needed about the sialic acid binding specificity of pathogens affecting animals that now live in very close contact with humans, such as dogs and cats. perhaps they will turn out to prefer neu5gc, explaining the relatively low rate of pathogen transfer from these domesticated pets to humans. another intriguing issue is that of the influenza a virus, the agent of epidemic and pandemic influenza in humans (wilson et al., 1981; taubenberger et al., 2000) . it appears that these viruses originate from wild water fowl and make their way to humans via domesticated livestock animals such as pigs . studies indicate that some animal forms of influenza a virus preferentially bind to neu5gc, while human forms can have some preference for neu5ac weis et al., 1988; ito et al., 1997 suzuki et al., 1986 suzuki et al., , 1997 . thus, a switch in specificity from neu5gc to neu5ac might be a required or facilitatory step in animal-to-human transmission of some influenza strains. on the other hand, if there are microbes that selectively prefer neu5ac, these should be more pathogenic in humans. while no clear-cut examples of this situation are known, most of the possibilities have yet to be explored. one potential example is plasmodium falciparum, the causative agent of the most serious form of human malaria that afflicts millions worldwide. the merozoite form of this organism that invades red blood cells uses cell-surface neu5ac as one of its targets for initial binding (klotz et al., 1992; deluca et al., 1996; reed et al., 2000) . since chimpanzees appear to be resistant to this form of malaria (ollomo et al., 1997) , it is possible that the p. falciparum merozoite receptor proteins do not recognize neu5gc. conversely, the merozoite stage of the phylogenetically related plasmodium reichenowi that infects chimpanzees (qari et al., 1996; escalante and ayala, 1994 ) might recognize neu5gc preferentially. functional comparison of the p. falciparum and p. reichenowi merozoite receptors seems to be worthwhile. overall, it is clear from table 1 that much further work is needed to pursue the consequences of neu5gc loss for resistance and susceptibility to infectious diseases in humans. the antibodies generated by a normal adult human exposed to infusion of horse serum (called hanganatziu-diecher or hd antibodies) can agglutinate the red blood cells of various animals, such as horses, pigs, and cows, by virtue of the fact that they all carry surface neu5gc. using the same kind of red-cell agglutination assay, spontaneously occurring hd antibodies have also been reported in patients who have never had exposure to animal serum infusion. while such serum reactivities are rare in normal human adults, they are found in a significant proportion of patients with cancer, as well as in diseases such as leprosy, rheumatoid arthritis, liver disease, and infectious mononucleosis (morito et al., 1982 (morito et al., , 1986 nishimaki et al., 1979; takiguchi et al., 1984) . at least two explanations can be considered for these phenomena. the first possibility is that as in cancer, some of these diseases involve proliferation of vascularized tissues (e.g., the granulomas of leprosy and rheumatoid arthritis). thus, a low-grade immune response might be occurring against neu5gc that is being gradually incorporated into such tissues from dietary sources over time (see discussion below). the second possibility is that these antibodies emerge as a nonspecific consequence of the disregulation of the immune system that occurs in some of these disease states. individuals with prior exposure to neu5gc in the diet may have rare preexisting memory b cells capable of producing antibodies directed against neu5gc. such b cells could undergo nonspecific expansion under conditions of generalized immune disregulation. the first possibility could be tested by directly assaying the affected tissues for the presence of neu5gc. despite the complete inactivation of the cmah gene in humans, there have been reports of traces of neu5gc in normal human tissues and in cultured human cell lines (nakarai et al., 1987; ohashi et al., 1983) . the latter finding is easily explained by the fact that human cells are typically cultured in fetal bovine serum, which is rich in glycoproteins carrying neu5gc. the fluidphase uptake of such glycoproteins into the lysosomes of cultured cells would eventually result in incorporation of the foreign neu5gc into human cellular glycoproteins and glycolipids (see pathway a in fig. 6 ). the best evidence for this route of incorporation is that growing the cells in the absence of animal serum results in the eventual disappearance of neu5gc (furukawa et al., 1988b; . however, this pathway cannot explain the traces of neu5gc found in tissues from normal humans who are not directly exposed to animal serum . on the other hand, the nature of the cmah mutation in humans (an exon deletion eliminating critical amino-acid residues) neu5gc not studied in detail streptococcus sanguis (dental caries) neu5gc not studied helicobacter pylori (ulcers) neu5gc not studied tetanus toxin (tetanus) neu5gc not studied coronaviruses (common cold) neu5gc not studied mycoplasma pneumoniae (pneumonia) neu5gc not studied polyoma virus (tumors) neu5gc not studied makes it hard to postulate a genetic mechanism to restore its function in malignant cells. indeed, the complete sequencing of the corresponding intronic regions of the cmah gene from human and chimpanzee genomes (irie et al., 1998; hayakawa et al., 2001) reveals no obvious avenues for such a repair mechanism. meanwhile, searches of gene databases show no evidence for any related proteins that might have a similar activity. two other possibilities must therefore be considered to explain the traces of neu5gc found in normal human tissues. the first is that there could be an additional completely differ-ent biochemical pathway for the biosynthesis of neu5gc in mammalian cells. there is so far no strong evidence for such a pathway being active in humans. the suggestion has been made that the glycolyl group of neu5gc (which replaces the acetyl group in neu5ac) could also originate from an unusual high-energy donor called glycolyl-coenzyme a (coa). such a potential donor can in fact be formed via two known biochemical pathways: beta-oxidation of 4-hydroxybutyrate in mitochondria, or from 3-hydroxypyruvate by the action of pyruvate dehydrogenase (vamecq and poupaert, 1990 ; vamecq et fig. 6 . uptake, metabolism, and turnover of neu5ac and neu5gc in mammalian cells. the general pathways for production and utilization of sialic acids in mammalian cells are shown. thick lines represent cellular membranes, or intracellular membranous compartments, as indicated. the common sialic acid neu5ac is synthesized from a neutral precursor n-acetylmannosamine (mannac) in the cytosolic compartment, activated into cmp-neu5ac in the nucleus, and eventually pumped into the golgi apparatus by a specific transporter (lepers et al., 1989) . there it serves as a donor for adding sialic acids to newly synthesized glycolipids and glycoproteins that originate from the endoplasmic reticulum, and are en route to the cell surface or to be secreted from the cell. sialic acids are eventually released in the lysosomal compartment as part of the overall degradation of glycolipids and glycoproteins and then pumped back into the cytosol (verheijen et al., 1999) , to be reutilized as shown. conversion of cmp-neu5ac to cmp-neu5gc occurs in nonhuman cells, and the subsequent fate of neu5gc is similar to that of neu5ac-recycling and varying degrees of reutilization. double line indicates block in conversion of cmp-neu5ac to cmp-neu5gc in human cells. two possible pathways (a and b) for incorporation of neu5gc of exogenous into human cells are indicated with dotted lines. pathway a involves uptake of glycolipids or glycoproteins carrying neu5gc into the lysosomal compartment. this pathway could only operate in cultured cell lines, or in humans given intravenous infusions of animal proteins. pathway b involves uptake of neutral precursor n-glycolylmannosamine (manngc) by passive diffusion across the plasma membrane, with subsequent conversion into neu5gc. manngc could originate from breakdown of dietary neu5gc in the gut. both these pathways would effectively bypass the enzymatic defect in humans. note that neu5ac and neu5gc can also be degraded back to mannac and manngc. while there are many known interconverting pathways involving mannac, the cellular fate of manngc is unknown, in human and nonhuman cells. there is also no known pathway for converting neu5gc back to neu5ac. hence neu5gc tends to recycle and accumulate to high levels in cells that express the hydroxylase (muchmore et al., 1989) . see text for further discussion. al., 1992). the glycolyl-coa would presumably donate its glycolyl group directed to a de-n-acetylated neuraminic acid (zhou et al., 1994; sjoberg et al., 1995; mitsuoka et al., 1999) , or to a potential precursor form of sialic acids called mannosamine. however, there is so far no evidence for the enzymatic activity of such a pathway in human cells. the other interesting possibility is the incorporation of neu5gc from dietary sources. studies of rats fed with radiolabeled sialic acids showed that a large fraction can be absorbed, and while most is excreted unchanged in the urine, a small amount of the label does get incorporated into tissues schauer, 1981, 1984; nohle et al., 1982) . the latter is thought to occur by intestinal conversion of the ingested sialic acids into the acylmannosamine derivative, which can be taken up into the circulation, absorbed into cells, and converted back into sialic acids (see pathway b in fig. 5 ). we are currently studying the possibility that a similar pathway exists in intact humans. if so, it is possible that the traces of neu5gc found in normal human tissues are all derived from the ingestion of foods containing neu5gc. in this regard, it is of note that sialic acids are only found in animal foods, and that neu5gc seems to be very common in pigs, cows, goats, and sheep (raju et al., 2000; wang et al., 1990) , while probably being much lower in poultry and fish. it remains to be seen whether this pathway has any relevance to diseases associated with the consumption of red meats. there also needs to be a detailed survey of the amounts of neu5gc present in common foods of animal origin. regardless of the mechanism, the question also arises whether there is a weak immune response to these traces of neu5gc in adult humans, and if so, what the consequences of such a response might be. as mentioned above, both direct and indirect studies have indicated that neu5gc is present in some human cancerous tissues and possibly in fetuses. these earlier reports used less sensitive methods that failed to detect the traces of neu5gc we subsequently noted in normal human tissues. thus, it is likely that the levels of neu5gc present in cancers are simply higher. the possible explanations presented above for the traces of neu5gc in normal tissues can be extended to fetuses and cancers as well, with the added suggestion that these rapidly growing tissues might be more efficient at scavenging neu5gc (or its breakdown product n-glycolylmannosamine) from dietary sources. of course, we still cannot rule out an alternative pathway for the synthesis of neu5gc that becomes specifically activated in tumors and fetuses. regardless of the mechanism(s) involved, the question again arises as to whether the presence of a sugar that is typically immunogenic in humans can in some way affect the outcome of a pregnancy or a malignant disease. perhaps it will be possible to take advantage of the presence of neu5gc on tumors to design some novel approach to the treatment or containment of cancer. of course, all of these considerations do not apply to cancers in other mammals, since they naturally have large amounts of neu5gc to begin with. thus, the animal model will have to be a mouse with a homozygous "knockout" of its cmah gene. such mice are currently being prepared. it is of great interest to ask if the loss of neu5gc in humans resulted in some significant changes in the endogenous functions of sialic acids. since neu5gc has a glycolyl group rather than an acetyl group, it is more hydrophilic, and this could result in changes in the physical or structural functions of sialic acids. however, this possibility and its potential consequences have yet to be investigated. as indicated above, the intrinsic receptors that bind sialic acids within animals have only recently been described, and there may be more to be discovered. correspondingly, we also know relatively little about the impact of neu5gc loss on the functions of these intrinsic sialic-acid receptors in humans. the limited information available concerning neu5gc and intrinsic sialic-acid receptors is summarized in table 2 . the best-studied examples to date are certain members of a family of proteins called the siglecs (sialic acid-binding immunoglobulin-like lectins; sgroi et al., 1993; crocker et al., 1994 crocker et al., , 1998 kelm et al., 1994a; powell and varki, 1995; crocker and feizi, 1996; crocker and varki, 2001a,b; . sialoadhesin (siglec-1) is a large molecule found on tissue macrophage cells in various organs such as bone marrow, spleen, and lymph nodes. the sialic acid-recognizing function of sialoadhesin has been extensively studied, and it is clear that it binds well to neu5ac, but not to neu5gc (brinkmanvan der linden et al., 2000; collins et al., 1997a; kelm et al., 1994b) . thus, human cells (which have an excess of neu5ac) have an excess of binding sites for sialoadhesin (brinkmanvan der linden et al., 2000) . presumably as a secondary consequence, there is an obvious difference in the tissue distribution of sialoadhesin-positive spleen macrophages between humans and chimpanzees. also, while only a subset of chimpanzee macrophages are sialoadhesin-positive, most human macrophages express this molecule (brinkmanvan der linden et al., 2000) . interestingly, in all the above respects chimpanzee spleens are more similar to those of rats than to humans. the biological significance of these facts will not be known until the functions of sialoadhesin itself are elucidated. meanwhile, it is interesting to note that sialoadhesin-positive macrophages are found in large numbers in pathological tissue specimens from patients with diseases such as breast cancer (nath et al., 1999) and rheumatoid arthritis (hartnell et al., 2001) , both of which appear to be rare conditions in the great apes (varki, 2000) . another siglec that strongly prefers neu5ac over neu5gc is myelinassociated glycoprotein (siglec-4a), which is expressed on schwann cells and oligodendrocytes, and appears to be involved in organizing and maintaining the myelin sheath that surrounds the axons in the white matter of the brain and the peripheral nerves (kelm et al., 1994b collins et al., 1997b) . curiously, the brain has very low levels of neu5gc, even in nonhuman mammals (see below). thus, it is presently difficult to make a hypothesis about how the loss of neu5gc expression in humans might have affected the function of myelin-associated glycoprotein. initial evaluation suggests that siglecs 2, 3, 5, and 6 are probably not affected by the human loss of neu5gc (brinkmanvan der linden et al., 2000; collins et al., 1997a; kelm et al., 1994b kelm et al., , 1998 . further studies are underway to evaluate of some of the newer siglecs that were recently described. of particular interest is a recently discovered human siglec-like molecule (siglec-l1) that lacks a conserved amino acid (an arginine residue) which is known to be essential for optimal sialic-acid recognition by previously known siglecs . we found that the loss of the arginine residue was caused by a single nucleotide substitution in human genomic dna that occurred after the common ancestor of humans with the great apes but prior to the common origin of modern humans (an-gata et al., 2001) . the chimpanzee ortholog of siglec-l1 has the arginine residue, remains fully functional in recognizing sialic acids, and turns out to preferentially recognize neu5gc over neu5ac. reintroducing the ancestral arginine by "repairing" the cloned human molecule regenerated sialic acid binding, along with a similar preference for neu5gc . thus, the single base-pair mutation that replaced the arginine residue on human siglec-l1 seems likely to be evolutionarily related to the loss of neu5gc expression in the human lineage. however, we do not know which came first, and how exactly the two events are related. in the course of doing this work, we also examined the whole human genome for other siglec-like genes. it turns out that the human genome contains many siglec-like pseudogenes (currently inactive components of the genome derived from ancestral active genes). interestingly, some of these pseudogenes have independent mutations that would have replaced the same conserved arginine residue that is required for optimal sialic acid recognition. much further work needs to be done to know if any of these pseudogenes are derived from genes that are still active in other primates. regardless, this work indicates that additional changes in the biology of sialic acids may have taken place during human evolution. the consequence of neu5gc loss for most of the other known mammalian sialic acid-recognizing lectins is also not very clear (see table 2 ), and further studies are needed. an intriguing case is that of a sialic acid-binding uterine agglutinin that was purified from the endometrium (the epithelial lining of the uterus) of both humans and rats, and that was shown to prefer neu5gc over neu5ac (chatterji et al., 2000) . it was also shown to recognize sialic acids on sperm. while the functions of this molecule are unknown, one can speculate that its preference for neu5gc might somehow be connected to anecdotal suggestions of differences between humans and great apes in matters such as menstrual blood loss and early fetal wastage (varki, 2000) . as mentioned above, a normal adult human directly exposed to neu5gc in the form of infused horse serum generates an immune response against this foreign sugar. biotherapeutic molecules produced via recombinant dna technology are typically produced in nonhuman cells, because human cells may allow transmission of human retroviruses and other infectious agents. as many of these products are glycoproteins, they can contain varying amounts of neu5gc originating from the nonhuman producer cells used. fortunately, most of the animal cell lines commonly used to produce such agents (e.g., chinese hamster ovary cells and baby hamster kidney cells) happen to produce very low levels of neu5gc. furthermore, although erythropoietin (produced by amgen) was later recognized to have very small amounts of neu5gc (hokke et al., 1995) , microgram amounts of this therapeutic glycoprotein had already been injected repeatedly into many humans, without apparent evidence of an obvious immune reaction (noguchi et al., 1996) . it is possible that the immune system of most humans is partially tolerized to neu5gc because of prior exposure to dietary neu5gc. if so, the reaction might be dose-dependent, and there should still be concern over recently developed products from the milk of sheep and goats which have very high levels of neu5gc (gagneux and varki, unpublished findings) . another possible explanation for the lack of immune response to neu5gc on erythropoietin is that the primary antigens in horse serum are glycolipids (not glycoproteins) bearing neu5gc. thus, there may be a lesser immune response to neu5gc on glycoprotein therapeutic agents. however, neu5gc injected in the form of glycoproteins could be directly taken up by human cells via pathway a of figure 6 , and eventually presented on the patient's own cell surfaces, attached to endogenously synthesized glycolipids. this could result in an immune response developing over time. this concern remains unresolved at the present time. similar considerations apply to the currently controversial attempts to transplant organs from other species into humans. all early attempts at such xenotransplantation of organs from other primates to humans failed for unknown reasons. these included attempts to transplant chimpanzee kidneys into humans (deodhar, 1986; reemtsma, 1989 ) which would nowadays be considered unethical. rejections of the transplanted organs were typically delayed by several weeks, and the mechanism of rejection remained obscure. if posttransplant serum from such patients had been saved, one could have determined if at least part of the rejection response was directed against neu5gc. regardless, it should be noted that the most popular model animal for xenotransplantation of organs into humans is currently the pig, which happens to be a species that expresses high levels of neu5gc in many tissues. no attention has yet been paid (in the published literature) to the potential for delayed immune response to neu5gc on such transplanted animal organs. it remains to be seen if this will pose a significant barrier to xenotransplantation. as indicated above, neu5gc is common in tissues of nonhuman mammals and is the major sialic acid in most organs and cell types of the great apes. in striking contrast to this situation, neu5gc is hard to find in the brains of these animals. indeed, early studies suggested that neu5gc was completely absent from the mammalian brain (gottschalk, 1960; rosenberg and schengrund, 1976; schauer, 1982) , and it was only subsequent analyses with more sensitive techniques that showed that there were small but clearly detectable amounts present (tettamanti et al., 1965; nakao et al., 1991; mikami et al., 1998; muchmore et al., 1998) . the mechanism for this differential expression in nonhuman animals appears to be the downregulation of cmah gene expression in the brain. this represents a rather rare example of a gene that is widely expressed in many tissues and yet selectively downregulated only in the brain. the fact that this unusual situation has been conserved throughout mammalian evolution suggests that there is a strong selection pressure in its favor. in other words, there must be some reason why the mammalian brain has restricted its neu5gc content for ͼ100 million years of evolution. in humans, of course, the last traces of neu5gc are eliminated from the brain by virtue of the genomic inactivation of the cmah gene. a tantalizing possibility is that some significant positive consequence of this change for the human brain offset any negative consequences in other human organs. however, there is at present no direct evidence to support this hypothesis. studies are currently underway in our laboratory to investigate the consequences of eliminating or overexpressing neu5gc in the brains of genetically modified mice. of course, the difference between a mouse and a human brain is much greater than the difference between a great ape and a human brain. thus, these experiments in mice may or may not provide final answers concerning this issue. on the other hand, genetic manipulation of the germline of humans or apes would be considered highly unethical. if the mouse experiments do not yield clear evidence, we may have to seek indirect clues towards understanding the implications of cmah loss for the evolution of the human brain. reviewed here is a genetic, biochemical, and structural difference between human and great ape cells that has potential implications for a wide variety of issues related to human evolution, physiology, and pathology. the nature of the genetic mutation, their universality in modern humans, and its direct biochemical consequences to human cells and tissues are clear. there is also good evidence that it can affect the susceptibility or resistance of humans to certain pathogens. circumstantial evidence is consistent with the possibility of human incorporation of traces of neu5gc from dietary origins. in addition, limited data show the effects of human neu5gc loss on cells of the human immune system such as macrophages. finally, the universal and selective suppression of neu5gc expression in mammalian brains raises the possibility that the human loss of neu5gc had some beneficial effects in human brain evolution. however, we have at present many more questions than answers. for example, when did this mutation first occur, and when did it become fixed as a universal feature of human ancestors? are there other consequences for infection risk or resistance in humans? what are the functional consequences for varki] the intrinsic human sialic-acid receptors whose neu5gc preference has yet to be explored? do the traces of neu5gc found in normal humans indeed come from dietary sources? if so, are there any pathological consequences to the human ingestion of neu5gc in foods of animal origin? is the mechanism of neu5gc reexpression in tumors and fetuses simply an exaggeration of what happens in normal tissues? or is there another biochemical pathway that becomes activated in these situations? does it matter that animal organs intended for xenotransplantation and certain biotechnology therapeutics produced in animal cells can be quite rich in neu5gc? and last but not least, what are the consequences, if any, for the evolution and function of the human brain? much diligent work by many investigators will be needed to answer these questions. on a more general note, the discovery of a major biochemical and structural difference between humans and great apes and the elucidation of its underlying genetic basis tend to raise hopes that this molecular change played a major role in the evolution of uniquely human characteristics. indeed, this is the issue likely to be of particular interest to the readership of this journal. however, the fact that this happens to be the first such difference recognized should not be taken as any indication of its relative significance in human evolution. it is indeed possible that this genetic change came under strong positive selection pressure because it contributed towards the evolution of organs like the human brain. on the other hand, it may have simply aided in the ability of humans to evade certain pathogens of animal origin. a less likely possibility is that this gene inactivation was a fortuitous event that simply drifted to fixation in the immediate ancestors of modern humans, because of a population bottleneck caused by other unrelated factors. further studies will eventually elucidate which of these possibilities is correct. on the minor gangliosides of erythrocyte membranes of japanese cats n-glycolylneuraminic acid and n-acetylneuraminic acid define feline blood group a and b antigens chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective a second uniquely human mutation affecting sialic acid biology hydroxylation of cmp-neuac controls the expression of n-glycolylneuraminic acid in g m3 ganglioside of the small intestine of inbred rats haemophilus ducreyi produces a novel sialyltransferase-identification of the sialyltransferase gene and construction of mutants deficient in the production of the sialic acid-containing glycoform of the 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antigen glycoproteins in different species animal sera the distribution of sialic acids in nature structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid definition of a bacterial virulence factor: sialylation of the group b streptococcal capsule multivalent binding of k99 fimbriae to the n-glycolyl-gm 3 ganglioside receptor structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution anthropology-the human genus difference in form of sialic acid in red blood cell glycolipids of different breeds of dogs identification of polysialic acids in glycoconjugates the origin of man: a chromosomal pictorial legacy g m3 directly inhibits tyrosine phosphorylation and de-n-acetyl-g m3 directly enhances serine phosphorylation of epidermal growth factor receptor, independently of receptor-receptor interaction the author thanks nissi varki, elaine muchmore, pascal gagneux, kurt benirschke, jim moore, margaret schoeninger, chris ruff, and two anonymous reviewers for very helpful comments. key: cord-284795-0eoyxz78 authors: khetan, aditya k. title: covid-19: why declining biodiversity puts us at greater risk for emerging infectious diseases, and what we can do date: 2020-06-25 journal: j gen intern med doi: 10.1007/s11606-020-05977-x sha: doc_id: 284795 cord_uid: 0eoyxz78 nan those viruses. fruit bats serve as a reservoir for both. with respect to ebola, the fruit bats thrived among the palm oil trees and, when they came into close contact with humans through these plantations, passed on the virus to humans. for nipah, the fruit bats contaminated date palm sap, which was then consumed by humans who thus got infected. given that loss of biodiversity is a primary driver of eid, there is an urgent need for measures to stem this loss. while public health measures, including surveillance of emerging disease hotspots, can be helpful as near-term strategies, they cannot substitute for a long-term solution that conserves biodiversity. in the absence of this, it is likely that public-health capacity will continue to be overwhelmed. human activities that drive loss of biodiversity are also directly tied to climate change and increasing water scarcity. as a result, targeting such activities can lead to a multitude of planetary health-and ipso facto human health-benefits. 5 these activities primarily involve agricultural intensification and expansion, which are the primary drivers of deforestation. human agriculture uses 33% of earth's available land surface, either as cropland (12%) or pasture (21%). it is estimated that between 2000 and 2010, 70% of deforestation was attributable to agricultural land expansion. this agricultural land expansion has mostly been for farming animals (for meat and other animal products such as dairy), soybean production, and palm oil production. 6 further, over 80% of soybean is used to feed animals for meat and is the principal source of protein for farmed animals. soybean demand, therefore, is essentially a surrogate for demand of meat. currently, 40% of calories available from global crop production are either fed to animals or used as biofuels. 5 given the significant contribution of human meat consumption to loss of biodiversity, decreasing such consumption must be recognized as a major priority for decreasing the incidence of eid over the medium to long term. such progress will also result in beneficial effects towards combating climate change, reducing water scarcity, and addressing malnutrition. it is estimated that 25% of global ghg emissions are the result of agriculture, most of it from the farming of animals for human consumption. the water footprint of a serving of meat is 10-20 times the water footprint of a serving of plant foods. decreasing the consumption of meat, therefore, can lead to progress in water conservation. 7 for every gram of protein in beef, 20 g of protein is utilized in feeding the animal. for chickens, the corresponding figure is 4 g. if the world's soybean production were instead utilized to feed humans directly, there would be a several-fold increase in protein availability for a large proportion of humans, whose protein demand is expected to rise with increasing economic prosperity. it is time we-as an interdependent world-recognize that what we eat primarily determines how the planet is used. physicians have historically played a leading role in issues that threaten the survival of our species, such as nuclear warfare. but with food, physicians have taken a narrow view in dietary guidelines and focused on isolating the effect of individual foods or nutrients on human health, ignoring the wider ecosystem which our food habits influence, and are, in turn, influenced by. as the latest pandemic shows, such a narrow view has been counterproductive, and likely will continue to cause significant harm. it is time for us to recognize that food, human health, and the environment are deeply interconnected, and understanding these relationships is vital to our planetary health. global trends in emerging infectious diseases impacts of biodiversity on the emergence and transmission of infectious diseases human influences on biodiversity evolution in action: climate change, biodiversity dynamics and emerging infectious disease safeguarding human health in the anthropocene epoch: report of the rockefeller foundation-lancet commission on planetary health tropical forests were the primary sources of new agricultural land in the 1980s and 1990s country-specific dietary shifts to mitigate climate and water crises key: cord-281836-j1r771nq authors: hernando-amado, sara; coque, teresa m.; baquero, fernando; martínez, josé l. title: antibiotic resistance: moving from individual health norms to social norms in one health and global health date: 2020-08-28 journal: front microbiol doi: 10.3389/fmicb.2020.01914 sha: doc_id: 281836 cord_uid: j1r771nq antibiotic resistance is a problem for human health, and consequently, its study had been traditionally focused toward its impact for the success of treating human infections in individual patients (individual health). nevertheless, antibiotic-resistant bacteria and antibiotic resistance genes are not confined only to the infected patients. it is now generally accepted that the problem goes beyond humans, hospitals, or long-term facility settings and that it should be considered simultaneously in human-connected animals, farms, food, water, and natural ecosystems. in this regard, the health of humans, animals, and local antibiotic-resistance–polluted environments should influence the health of the whole interconnected local ecosystem (one health). in addition, antibiotic resistance is also a global problem; any resistant microorganism (and its antibiotic resistance genes) could be distributed worldwide. consequently, antibiotic resistance is a pandemic that requires global health solutions. social norms, imposing individual and group behavior that favor global human health and in accordance with the increasingly collective awareness of the lack of human alienation from nature, will positively influence these solutions. in this regard, the problem of antibiotic resistance should be understood within the framework of socioeconomic and ecological efforts to ensure the sustainability of human development and the associated human–natural ecosystem interactions. the problem of antibiotic resistance (ar) has been traditionally addressed by focusing on humanlinked environments, typically health care facilities. nevertheless, it is now generally accepted that most ecosystems may contribute to the selection and spread of ar (aminov, 2009; martinez et al., 2009; davies and davies, 2010; martinez, 2014; berendonk et al., 2015; larsson et al., 2018) . a key conceptual point is that, based on cultural, humanitarian, and economic reasons, we have historically preserved the health of individual humans and farming animals. to that purpose, the same families of antimicrobial agents have been used. as a consequence, their positive (healing) and negative (selection of ar, therapeutic failure) effects have influenced the common health of humans and animals in particular locations (one health). the concept one health, first used in early twentieth century, expands the integrative thinking about human and animal medicine, including for the first time ecology, public health, and societal aspects (zinsstag et al., 2011) . in the case of ar, the one health perspective focuses on the risk assessment of emergence, transmission, and maintenance of ar at the interface between humans, animals, and any other linked (local) environment (robinson et al., 2016; jean, 2017) . consequently, the application of one health approaches demands integrative surveillance tools and interventions based on multidisciplinary approaches that include ecological and sociodemographic factors, besides more classic epidemiological models. global health is based on a broad collaborative and transnational approach to establish "health for all humans." in this case, it focuses ar at a general (global) scale, considering that the selection and global spread of antibiotic-resistant bacteria (arbs) and antibiotic resistance genes (args) are a problem that influences the health of human societies with disparate social and economic structures and is linked to many societal and ecological factors (chokshi et al., 2019) . interventions to reduce ar burden in a global world certainly require common and integrated policy responses of countries, international organizations, and other actors (stakeholders included). its goal is the equitable access to health and minimizing health risks all over the globe. besides its objective aspects (i.e., how travelers, migrating birds, or international commerce may contribute to ar spread), it has important international political aspects. it focuses in how countries and international organizations address the elements connecting and potentially spreading ar among humans, animals, and natural ecosystems at the earth scale (wernli et al., 2017) . in summary, the problems and the potential solutions concerning ar are not confined to particular regions, but have a global dimension: a problem for all humans, animals, and natural ecosystems, which should be solved with interventions aiming to improve health for all of them koplan et al., 2009; laxminarayan et al., 2013) . in the context of ar, a healthy environment would be an environment where ar is low or can be controlled by human interventions (hernando-amado et al., 2019; andersson et al., 2020) . of course, the global health concept of "health of an environment" (iavarone and pasetto, 2018; pérez and pierce wise, 2018; bind, 2019; van bruggen et al., 2019) or, in general, planetary health (lerner and berg, 2017) , has an unavoidable anthropogenic flavor. in practice, we consider "healthy environments" or "healthy ecosystems" those that minimize their current or their potential harm for the human individual or the society, in our case for ar. in other words, we adopt a selfish strategy, which should be necessarily implemented by the international (global) institutions. selfishness (kangas, 1997 ) applies mainly to individuals, but also to societal groups. however, these groups have not enough possibilities to act alone in the case of infectious diseases in general and ar in particular, which may expand worldwide. therefore, individual selfishness for health should be integrated in local one health and also in global health actions. the goal of controlling ar is a highly complex one, and its dimension has been compared to climate change or biodiversity loss, problems where individual actions are not enough for providing a solution, and consequently, individual freedom is confronted with collective responsibility (looker and hallett, 2006) . the construction of human societies reflects the tension between individual freedom and social rules/laws. the implementation of different social rules/laws for regulating human activities within a society is mainly based on moral (as kant's categorical imperative (kant, 1785) or religious-based brotherhood (matthew 22:35-40) statements), social stability (as anticrime laws; schiavone, 2012) , organizative (type of government and how it is formed, group identity), and efficacy (as antitrust laws; ricardo, 1821) arguments. however, these arguments mainly apply for establishing the socioeconomic organization as well as the individual welfare within a society. the situation concerning human health is somehow different. there are individual diseases, such as cancer or stroke, and social diseases, such as transmissible infections. for the firsts, social norms (as consciousness of the importance of the control of cholesterol, excess sugar uptake, or hypertension levels) are well established, and even laws (non-smoking regulations) had been implemented in occasions. however, the main impact of these regulations is at the individual health level (wikler, 2002) , because the associated diseases are not physically transmissible. a different situation happens in the case of infectious diseases in general and of ar in particular. for these diseases, everything that happens in a single person affects any one around. further, the fact that an arg emerging in a given geographic area can spread worldwide implies that neither individual norms nor country-based norms have been sufficient until now to counteract the worldwide spread of ar. one important aspect of laws in democratic societies is that they must be well accepted by the community, so that the acceptation of social norms usually comes first than their implementations as rules/laws. actually, the efficiency of democracy for responding to social crisis (as current ar or covid-19 crises), in opposition to other more autocratic regimens where decisions are implemented top-down, had been the subject of debate from the early beginning of democratic revolutions (tocqueville, 1838; hobbes, 1968; rousseau, 1974; spinoza, 2007) . in this regard, it is important to remark that one health aspects of ar can be tackled in the basis of countrylevel regulations that are linked to the socioeconomic and cultural aspects of each country (chandler, 2019; chokshi et al., 2019) . however, because global earth governance does not exist, global health control of ar is based on recommendations, rather than in rules/laws. consequently, the acceptance of social norms, starting within individuals or small organizations and expanding throughout the whole society (figure 1) , is fundamental to provide global solutions to the ar problem (nyborg et al., 2016; chandler, 2019) . the acceptance by the community of these social norms, considering that the way of promoting these norms might differ in different parts of the world (cislaghi and heise, 2018; cislaghi and heise, 2019) , largely depends on the transfer to the society of the knowledge required to understand the mechanisms and the impact for human health of the emergence and transmission of ar, an information that is discussed below. figure 1 | how the interactions among individual health, one health, global health, and social norms influences antibiotic resistance. the right panel shows the different levels of dissemination of antibiotic resistance. in the left panel, the different types of norms (from individual to global norms) that can impact antibiotic resistance at each level are shown. these norms influence all levels of transmission: the individual promotes (red arrows) his own individual health, but doing it also promotes the health of the group, and the health of the group promotes global health of the human society at large. at each level, there is a positive action (red broken lines) on antibiotic resistance. such dynamics largely depends on social norms (blue arrows) rewarding the individual or the groups whose behavior promotes health. below the left panel, the basic social norm, progress and development, has consequences on the whole ecobiology of the planet (lower panel with bullet points), influencing the undesirable open circulation of antimicrobial resistant bacteria (with their mobile genetic elements) and antibiotic resistance genes. the classic definition of ar is based only on the clinical outcome of the infected patient. an organism is considered resistant when the chances for the successful treatment of the infection it produces are low . this definition, which is the most relevant in clinical settings, presents some limitations for studies based on one health approaches that include the analysis of non-infective organisms, which lack a clinical definition of resistance, as well as analysis of the distribution of args, in several occasions, using non-culture-based methods . even in the case of animal medicine, antibiotic concentration breakpoints defining resistance are still absent for some veterinary-specific antimicrobials and poorly defined for different types of animals with disparate weights, which would influence the availability of the drug inside animal body (toutain et al., 2017; sweeney et al., 2018) . to analyze ar beyond clinical settings, the term resistome, understood as the set of genetic elements that can confer ar, irrespectively of the level of resistance achieved, in a given organism/microbiome was coined (d'costa et al., 2006; wright, 2007; perry et al., 2014) . ar acquisition is the consequence of either mutation (or recombination) or recruitment of args through horizontal gene transfer (hgt), transformation included. ar mutations are generally confined to their original genomes, propagating vertically and not spreading among bacterial populations, although some few exceptions of horizontal transfer of chromosomal regions containing ar mutations have been described (coffey et al., 1991; ferrandiz et al., 2000; novais et al., 2016; nichol et al., 2019) . the set of mutations that confer ar can be dubbed as the mutational resistome. current wholegenome-sequencing methods of analysis can allow defining the mutational resistome in an isolated microorganism (cabot et al., 2016; lopez-causape et al., 2017) . however, they are not robust enough yet for determining the mutational resistome in metagenomes. consequently, the impact of these analyses in one health studies is still limited and will not be further discussed in the present review. concerning their relevance for acquiring ar, args can be divided in two categories. the first one comprises the genes forming the intrinsic resistome (fajardo et al., 2008) , which includes those that are naturally present in the chromosomes of all (or most) members of a given bacterial species and have not been acquired recently as the consequence of antibiotic selective pressure. despite that these genes contribute to ar of bacterial pathogens, they are responsible just for the basal level of ar, which is taken into consideration when antibiotics are developed. in this regard, unless these genes, or the elements regulating their expression mutate, they are not a risk for acquiring resistance and have been considered as phylogenetic markers . further, it has been discussed that these genes may contribute to the resilience of microbiomes to antibiotic injury (ruppe et al., 2017b) , hence constituting stabilizing element of microbial populations when confronted with antibiotics more than a risk for ar acquisition by pathogens. the second category, dubbed as the mobilome, is formed by args located in mobile genetic elements (mges) that can be transferred both vertically and horizontally, hence allowing ar dissemination among different bacteria (frost et al., 2005; siefert, 2009; jorgensen et al., 2014; lange et al., 2016; martinez et al., 2017) . while the analysis of the resistome of microbiota from different ecosystems has shown that args are ubiquitously present in any studied habitat (d'costa et al., 2006; walsh, 2013; jana et al., 2017; lanza et al., 2018; chen et al., 2019b) , the impact of each one of these args for human health is different. indeed, it has been stated that the general resistome of a microbiome is linked to phylogeny and to biogeography, indicating that most args are intrinsic and do not move among bacteria (pehrsson et al., 2016) . however, some args escape to this rule and are shared by different ecosystems and organisms (forsberg et al., 2012; fondi et al., 2016) . these mobile args, frequently present in plasmids (tamminen et al., 2012; pehrsson et al., 2016) , are the ones that are of special concern for human health. although not belonging to the antibiotic resistome, genes frequently associated with resistance to other antimicrobials, such as heavy metals or biocides, as well as the genes of the mges backbones, eventually involved in the transmission and selection of args among microbial populations, the mobilome at large, are also relevant to track the emergence and dissemination of ar among different habitats martinez et al., 2017; baquero et al., 2019) . hgt processes are recognized as the main mechanisms for transmission of genetic information (baquero, 2017) . from the ecological point of view, hgt should be understood as a cooperative mechanism that allows the exploitation of common goods as args by different members within bacterial communities. in fact, some studies suggest that the ecological consequences of hgt events in ar evolution are contingent on the cooperation of complex bacterial communities, besides the acquisition of individual adaptive traits (smillie et al., 2011) . however, the understanding of the ecological causes and consequences of args transmission among organisms and microbiomes is still limited from the one health and global health perspectives. hgt-mediated ar is a hierarchical process (figure 2 ) in which args are recruited by gene-capture systems as integrons and afterward integrated in mges as plasmids, insertion conjugative elements, or bacteriophages (frost et al., 2005; garcia-aljaro et al., 2017; gillings et al., 2017; botelho and schulenburg, 2020) , which afterward are acquired by specific bacterial clones. selection at each of these levels will also select for all the elements involved in ar spread. for instance, the acquisition of an arg by a clone may promote the expansion of the latter (and of all the genetic elements it contains, other args included) in antibiotic-rich environments, such as hospitals or farms schaufler et al., 2016) , and vice versa, the introduction of an arg in an already successful clone may increase the chances of this resistance gene for its dissemination even in environments without antibiotics, unless the associated fitness costs are high. in this sense, if arg acquisition reduces the fitness, and this implies a decreased capability for infecting humans (see below), the burden for human health might eventually be lower. nevertheless, it is relevant to highlight that ar transmission cannot be understood just by analyzing the genetic mechanisms involved and the consequences of such acquisition for the bacterial physiology. indeed, as discussed below, there are ecological and socioeconomic elements that strongly influence ar dissemination. the evolution of ar comprises the emergence, the transmission, and the persistence of arbs (martinez et al., 2007; baquero et al., 2011) . concerning human health, selection of arbs/args is particularly relevant at the individual health level, whereas transmission is a main element to be taken into consideration at the one health and global health levels (figure 2) . indeed, unless ar is transmitted, it will be just an individual problem that would not affect the community at large. it is generally accepted that non-clinical ecosystems are often primary sources of args (davies, 1994) . as above stated, after their capture and integration in mges (figure 2 ), args and their bacterial hosts can contaminate different ecosystems, which might then be involved in their global spread (martinez, 2012; fondi et al., 2016; gillings, 2017; gillings et al., 2017) . this means that nearly any ecosystem on earth, along with the humandriven changes produced in it, may modulate evolution of ar. importantly, the huge escalation and worldwide expansion of a limited set of animals, plants, and their derived products, including foods, due to the anthropogenic selection of a few breeds and cultivars for mass production in livestock and agricultural industries (okeke and edelman, 2001; zhu et al., 2017) of economic interest have collapsed the variability and biodiversity of animals and plants (seddon et al., 2016) . because these organisms harbor particular host-adapted bacteria, which are frequently under antibiotic challenge, this situation, together with the ecological similarities of human habitats, might favor ar spread (martiny et al., 2006; manyi-loh et al., 2018) . indeed, while in underdeveloped areas of the world food animals are very diverse, intensive farming, common in developed countries, ensures a "shared-stable" environment where only the most productive types prevail (kim et al., 2017) . the common genetic origin of these types and the process of microbiota acquisition from nearby animals in intensive farming should homogenize also their microbiomes with consequences for ar dissemination. actually, it has been shown that the loss of microbial diversity figure 2 | genetic, ecological, and socioeconomic elements mediating the transmission of antibiotic resistance. args are ubiquitously present in any studied microbiome (a). however, only a few of them are transferred to human/animal pathogens, hence constituting a health problem. the genetics events implied include the acquisition of args by gene-recruiting genetic elements such as integrons (b); the integration of these elements in mges as plasmids, bacteriophages, or frontiers in microbiology | www.frontiersin.org figure 2 | continued insertion conjugative elements (c); and the acquisition of these elements by specific bacterial clones (d). these arbs can share these elements among the members of gene-sharing communities (e) and also move among different ecosystems, including humans, animals (particularly relevant farm animals), and natural ecosystems (with a particular relevance for water bodies). the connection of these ecosystems, as well as the reduced diversity of animals, plants, and in general habitats as the consequence of human activities, allows the different microbiomes to be in contact, favoring args transmission among the microorganism they encompass (f). this transmission is facilitated at the global scale by travel, animal migration, trade of goods, and eventually by meteorological phenomena, climate change included (g), hence producing a global health problem (h). while most studies on the dissemination of args focus on mges (davies, 1997; muniesa et al., 2013; lanza et al., 2015; garcia-aljaro et al., 2017) , recent works suggest that the contribution of natural transformation (orange arrow), allowing the direct uptake of args by natural competent microorganisms, may have been underestimated (domingues et al., 2012; blokesch, 2017) . further, competence can occur due to interbacterial predation (veening and blokesch, 2017) , a biological interaction that may facilitate the acquisition of beneficial adaptive traits by predator bacterial species (cooper et al., 2017; veening and blokesch, 2017) . other hgt mechanisms, such as dna packing in extracellular vesicles (ecv) or transference of dna through intercellular nanotubes, also seem to be relevant in nature (dubey and ben-yehuda, 2011; fulsundar et al., 2014) . while the biotic conditions that may enhance hgt have been studied in detail, less is known concerning abiotic modulation of args transfer. under contemporary conditions, at least 10 24 microorganisms are affected by a freeze-and-thaw cycle, at least 10 19 are agitated by sand, and at least 10 17 are subjected to conditions suitable for electrotransformation every year (kotnik and weaver, 2016) . may favor ar spread (chen et al., 2019a) . note that, beyond the transmission of particular ar spreading clones, ar is expected to spread in farms by the modification (eventually homogenization) of animals' microbiota. notwithstanding, even farm workers are subject to microbiome acquisition from animals, leading to microbiome coalescence sun et al., 2020) . it is to be noticed, and the recent covid-19 crisis exemplifies it, that besides economic development, cultural habits are relevant in the use of animals for food, a feature that has not been analyzed in detail, particularly with respect to their role as vectors potentially involved in ar dissemination. despite that the homogenization of hosts may help in ar transmission, the spread of arbs has some constraints, because the differential capability of each bacterial clone for colonizing different hosts may modulate their dissemination. indeed, while some species and clones are able to colonize/infect different animal species, humankind included, several others present some degree of host specificity (price et al., 2017; sheppard et al., 2018) . further, it has been shown that the capacity to colonize a new host is frequently associated with a reduction in the capacity for colonizing the former one. the same happens for mobile args; they are encoded in mges that present different degrees of host specificity, which defines the formation of gene-exchange communities, where the interchange of genetic material among members is facilitated (skippington and ragan, 2011) . conversely, the incorporation of different replicons and modules within plasmid backbones, a feature increasingly reported (douarre et al., 2020) , would enable arg replication in different clonal/species background and thus modify the community network of args. actually, the risk for humans of animal-based ar seems to be linked in most cases to shuttle, generalist clones able to colonize humans and particular animals (price et al., 2017; sheppard et al., 2018) . the understanding of the elements driving the transfer of ar among animals, humans included (figure 3) , requires the comprehensive survey of the clones and args that are moving among them (european food safety authority et al., 2020). tools to track the global epidemiology of antimicrobial-resistant microorganisms such as bigsdb (jolley et al., 2018) or comprehensive databases of args, ideally providing information of their mobility (zankari et al., 2012; alcock et al., 2020) , are fundamental for studying ar transmission at a global level. it is worth mentioning that, because humans constitute a single biological species, the human-associated organisms spread easily among all individuals. in fact, more prominent differences in humans' microbiome composition can be observed between individuals than among ethnic groups, even though, as expected, the resemblance in microbiotas is higher among those groups that are geographically clustered (deschasaux et al., 2018; gaulke and sharpton, 2018) . some groups of human population are, however, more prone to acquire arbs, due either to socioeconomic or to cultural factors. in lmics (low-to medium-income countries) and brics (brazil, russia, india, china, and south africa) countries, the combination of wide access to antibiotics, weak health care structures, and poor sanitation defines certainly a dangerous landscape. moreover, the progressive aging of the western population might favor the establishment and further expansion of an elderly reservoir of arbs and args, an issue that deserves further studies. the hypothesis that the microbiome of elder people might be a reservoir of ar is based not only on their cumulative history of antibiotic exposure and contacts with health care centers, but also on the rampant use of antibiotics of this population more prone to suffer from acute, chronic, or recurrent infections. significant worldwide advances in the organization of medical care of the elderly people lead to frequent hospitalizations, but health care centers may also facilitate the selection and further amplification of ar in the community. in addition, this may subsequently favor the entry of high-risk clones and of args in the hospital setting (hujer et al., 2004) . as stated above, there is a global increasing permeability of the natural biological barriers that have historically prevented bacterial dissemination through different ecosystems. besides local spread of ar in environments shared by animals and humans, which has to be addressed under a one health approach, ar can disseminate worldwide (figure 2 ) by economic corridors that promote the global interchange of goods and trade or human travelers or by natural bridges, such as animal migration paths or natural phenomena such as air and water movements (okeke and edelman, 2001; baquero et al., 2008; allen et al., 2010; overdevest et al., 2011; kluytmans et al., 2013; fondi et al., 2016) . the result is the appearance of similar arbs and args in different geographic areas. as the consequence, ar is a global health problem in the sense that an arb that emerges in a given place can rapidly spread worldwide. indeed, multidrugresistant bacteria, similar to those encountered in clinical settings, have been detected in human isolated populations that were not previously in contact with antibiotic, as well as in wildlife (clemente et al., 2015) . this indicates that pollution with args is present even in places where antibiotic concentrations are low (kümmerer, 2004) and might involve mechanisms of transmission that do not require selection. for instance, migrating birds can carry enteropathogenic bacteria resistant to different antibiotics (middleton and ambrose, 2005; poeta et al., 2008) , and international travelers, even those not receiving antibiotic treatments, also contribute to ar transfer among different geographic regions (murray et al., 1990; reuland et al., 2016) . in the group of long travelers are refugee people, in which dissemination of multidrug-resistant strains is favored by the poor sanitary conditions and overcrowding camps that refugees confront (maltezou, 2016) . a final issue concerning ar is its stability in the absence of selection. it has been proposed that the acquisition of ar reduces bacterial competitiveness in the absence of antibiotics (fitness costs) (andersson and hughes, 2010; martinez et al., 2011) ; certainly, a wishful proposition such as, if true, the reduction in the use of drugs or eventually antibiotic-cycling strategies should decrease ar (beardmore et al., 2017) . nevertheless, eliminating the use of an antibiotic does not produce a full decline of ar (sundqvist et al., 2010) . in fact, different studies have shown that ar not always reduces fitness but also can even increase bacterial competitiveness (andersson and hughes, 2010; schaufler et al., 2016) . in addition, compensatory mutations or physiological changes that restore fitness can be selected in resistant bacteria (andersson, 2006; schulz zur wiesch et al., 2010; olivares et al., 2014) . it is a fact, however, that although arbs are found nearly everywhere, including wild animals, natural ecosystems, or people from isolated populations without contact with antibiotics, among others (durso et al., 2012; clemente et al., 2015; alonso et al., 2016; fitzpatrick and walsh, 2016; power et al., 2016) , ar prevalence is consistently lower when antibiotics are absent, which suggests that pollution may impact ar, a feature that is discussed below. pollution of natural ecosystems is associated with activities that have driven relevant economic transition, in principle favoring human welfare, such as mining, industry, intensive land use, or intensive farming, among others. notwithstanding, globalization of health services, as well as the shift toward intensive farming, besides their positive contribution to human wellbeing, has rendered an increasing pollution by compounds with pharmacological properties of natural ecosystems, particularly water bodies, which may disrupt the stability of these ecosystems (oldenkamp et al., 2019) . among them, antibiotics are considered the most relevant cause of ar selection. despite regulations for reducing their use (van boeckel et al., 2017) , a substantial increase in global antibiotic consumption has occurred in the last years, and an even greater increase is forecasted in the next years (klein et al., 2018) . however, antibiotics are not the unique pollutants that can prime the selection and spread of ar. in this regard, it is important to highlight that heavy metals are one of the most abundant pollutants worldwide (panagos et al., 2013) . their abundance results from anthropogenic-related activities, such as mining, industry, agriculture, farming, or aquaculture and even for therapeutic use in ancient times. importantly, they may persist in nature for long periods of time. further, likely because metal pollution occurred before the use of antibiotics, heavy metal resistance genes were incorporated to mge backbones before args (mindlin et al., 2005; staehlin et al., 2016) . this means that heavy metals may coselect for mges and the args they harbor (partridge and hall, 2004; staehlin et al., 2016; zhao et al., 2019a) . even more, the presence of heavy metals, as well as of biocides or sublethal antibiotic concentrations (jutkina et al., 2018; zhang et al., 2018) , may stimulate hgt, as well as modify the dynamics of antibiotics, such as tetracyclines, in natural ecosystems (hsu et al., 2018) . coselection may also occur when a single resistance mechanism, such as an efflux pump, confers resistance to both heavy metals and antibiotics (cross-resistance) (pal et al., 2017) . although most published works analyze the effect of different pollutants on their capacity to select arbs or args, it is important to highlight that args should also be considered pollutants themselves. actually, a recent work indicates a close relationship between the abundance of args and fecal pollution (karkman et al., 2019) . in this respect, it is worth mentioning that, differing to classic pollutants, args/arbs are not expected to disappear along time and space, but rather, their abundance may even increase as the consequence of selection and transmission (martinez, 2012) . while the direct selection of ar by antibiotics or the coselection mediated by other pollutants, as the aforementioned heavy metals, has been discussed (wales and davies, 2015) , the effect of other types of human interventions on the dissemination of args and arbs through natural ecosystems has been analyzed in less detail. as an example, it has been proposed that wastewater treatment plants, where commensals, arbs, args, and antibiotics coexist, could act as bioreactors favoring the selection and transmission of args between different organisms (rizzo et al., 2013; su et al., 2017; manaia et al., 2018) , although evidences supporting this statement are scarce (munck et al., 2015; azuma et al., 2019) . in addition to the aforementioned pollutants with a direct effect in ar selection, it is worth noting that there are other abundant contaminants, such as sepiolite (present in cat litters or used as a dietary coadjuvant in animal feed) or microplastics, present in almost all aquatic ecosystems, which can favor the transmission of args or mges between bacterial species (rodriguez-beltran et al., 2013; kotnik and weaver, 2016; arias-andres et al., 2018) , hence amplifying the ar problem at a global scale. finally, the possible effect of climate change on the spread of ar is worth mentioning. indeed, it modifies the biogeography of vectors (such as flies, fleas or birds) involved in the spread of infectious diseases (fuller et al., 2012; beugnet and chalvet-monfray, 2013) . in addition, the increase of local temperatures seems to correlate with an increased ar abundance in common pathogens (macfadden et al., 2018) . besides, climate change is affecting ocean currents (martinez-urtaza et al., 2016) , which may allow the intercontinental distribution of arbs and args (martinez, 2009a,b) . although this phenomenon might contribute to the globalization of ar, further research is needed to clearly demonstrate a cause-effect relationship. it is relevant to mention that increased pollution and climate change are the unwanted consequences of human development. it would then be worth discussing how human development in general may impact (positively and negatively) ar, a feature that is analyzed below. human development is a necessity of our human behavior, although different models of development have been and are proposed, each one producing different impacts in the structure of human societies and on the preservation and stability of natural ecosystems (fenech et al., 2003; farley and voinov, 2016; seddon et al., 2016) . nevertheless, even for different socioeconomic models, there are some social norms that tend to be widely accepted, in particular those aiming to improve individual well-being. this implies the establishment of a society of welfare, understood as a right of any human on earth, a feature that depends on the economic development, and can be particularly relevant in the case of transmissible infectious diseases in general and of ar in particular. a continuously repeated mantra in worldwide ar policies is that the abusive consumption of antibiotics for the treatment or prevention of infections in humans and animals constitutes the major driver of ar. however, we should keep in mind that antibiotics constitute an important example of human progress supporting individual and global human health. in fact, the origin of the massive production of antimicrobials was a consequence of the needs resulting from world war ii in the 1940s. this was followed by many decades of human progress, most importantly by the common understanding of equal human rights, which was followed by the economic and social development (including medicine and food industry) of densely populated regions in the planet, including india and china. these countries are currently among the leaders in the production and consumption of antimicrobial agents. notwithstanding, as in any area of economy, progress bears a cost that, in this case, is antibiotic pollution of the environment, globally accelerating the process of the emergence, the transmission, and the persistence of arbs (martinez et al., 2007; baquero et al., 2011) . the non-controlled use of antibiotics is facilitated in lmics with disparate economic growth by different factors. heterogeneous regulation of antibiotic sales and prescriptions (often weak or missing) and the increase of online on-bulk sales in recent years contribute to their overuse (mainous et al., 2009 ). most of live-saving medicines represent out-of-pocket costs in most lmics, which led to an exacerbated use of cheap (usually old and less effective) antibiotics, phasing out their efficacy and increasing the demands and prices for the most expensive ones, eventually resulting in treatment unavailability (newton et al., 2011) . further, the cost of treating ar infections is much higher than that of treating susceptible ones, which is increasing the cost of health services (wozniak et al., 2019) . conversely, the growing economic capability of lmics in the brics category triggers the access of the population to health services and last-resort antibiotics. these countries also face a sudden high demand for meat and thus a prompt industrialization of animal production that has favored the misuse of antibiotics for growth promotion facilitated by their online availability (mainous et al., 2009 ). in addition, counterfeit or substandard antibiotics recently become a serious global problem (gostin et al., 2013) , which is exacerbated in lmics, where they represent up to a third of the available drugs. noteworthy, 42% of all reports received by the who global surveillance and monitoring system on substandard and falsified medicines worldwide come from africa, and most of them correspond to antimalarials and antibiotics (newton et al., 2011; gostin et al., 2013; hamilton et al., 2016; petersen et al., 2017) . despite this situation, it is important to highlight that human consumption of antibiotics is an unavoidable need to preserve human health. in fact, most health problems dealing with infections in lmics are still caused by a poor access to antibiotics, not by an excessive use of them. proof of this is the fact that the distribution of antibiotics has reduced endemic illnesses and children mortality in sub-saharan africa (keenan et al., 2018) . this means that, while a global decline in the use of antibiotics would be desirable to diminish the problem of ar, there are still several parts in the globe where antibiotic use should still increase to correctly fight infections. in fact, our primary goal should not be to reduce the use of antibiotics, but to ensure the effective therapy of infectious diseases for the long term. this does not mean that ar is not a relevant problem in lmics; it means that reducing antibiotic use is not enough to solve the problem. indeed, the current high morbidity and mortality due to infectious diseases (malaria, tuberculosis, low respiratory infections, sepsis, and diarrhea) in lmics will be worsened in the absence or low efficiency of therapeutic treatments. further, ar has economic consequences. according to world bank, 24.1 million people could fall into extreme poverty by 2050 because of ar, most of them from lmics (jonas and world bank group team, 2017) . consequently, besides a global health problem, ar has an important economic impact (rudholm, 2002) , hence constituting a global development problem, endangering not only the achievements toward the millennium development goals but also the sustainable development goals (van der heijden et al., 2019). world bank estimates that ar could impact the gross domestic product from 1 to 3.8%, which is even higher than what is estimated for the climate change (jonas and world bank group team, 2017) . these economic foresights are linked to the threads of increased poverty, food sustainability, global health deterioration (associated with both food safety and affordability to health care), and environment protection. all these issues are also impacted by the overuse and misuse of antibiotics, its lack of effectiveness, and the affordability to medicines and health care (van der heijden et al., 2019) . when talking about reducing antibiotic consumption, it is important to remind that up to two-thirds of overall antibiotic usage is for animal husbandry (done et al., 2015) . further, recent work states that the use of antibiotics in crops, particularly in lmics, might have been largely underestimated (taylor and reeder, 2020) . despite that evidences on the presence of common args distributed among animals and humans were published decades ago wegener et al., 1997; aarestrup, 1998; aarestrup et al., 1998) , and although the use of antibiotics as growth promoters has been banned in different countries (cox and ricci, 2008) , they are still allowed in many others (mathew et al., 2007) . of relevance is the fast increase of antibiotic consumption for animal food production in china (23% in 2010) and other brics countries . as stated previously, in these countries, increased income has produced a fast increase in meat products demand, due to changes in diet of their population. in addition, the increasing international competitiveness in meat production of these countries has fostered the rampant development of their industrial farming. together with the fact that legislation on antibiotics use remains weak, this situation increases the risk of emergence of ar linked to animal production. nevertheless, the problem is not restricted only to lmics, because antibiotics consumption rose as well in the highincome countries as the united states (13%) , where approximately 80% of the antimicrobials purchased in 2011 were applied in livestock production as non-therapeutic administration (done et al., 2015) . the development of intensive methods of fish production has also contributed to the rise in the use of antimicrobials and the selection of resistance determinants that can be shared among fish and human bacterial pathogens (cabello et al., 2013) . economic development has facilitated as well more global transport, waste disposal, and tourism, favoring ar spread within and between different geographical areas (ruppe et al., 2017a; ruppe and chappuis, 2017) . however, economic growth can also reduce the ar burden, especially when it enables the development of regulations and infrastructures that might reduce the risks of infection and ar spread. this is particularly relevant in the case of public health interventions on food, water, and sewage. because ar pathogens are mainly introduced in natural ecosystems through the release of human/animal stools (karkman et al., 2019) , the best way of reducing this impact is through the use of wastewater treatment plants, which are still absent in several places worldwide. indeed, it has been described that drinking water is a relevant vehicle for the spread of arbs in different countries (walsh et al., 2011; fernando et al., 2016) and that raw wastewater irrigation used for urban agriculture may increase the abundance of mobile args in the irrigated soil (bougnom et al., 2020) . notably, the analysis of args in wastewaters has shown that the prevalence of args in the environment in each country might be linked to socioeconomic aspects mainly related to economic development, as general sanitation, particularly the availability of drinking and wastewater treatments, malnutrition, number of physicians and health workers, human overcrowding, or external debt grace period (hendriksen et al., 2019) . the field of ar has mainly focused in the mechanisms of selection; the main driver for the increased burden of ar would be then the use of antibiotics itself. however, these results indicate that transmission, even in the absence of direct human-to-human contact, might be, at least, equally relevant. in this situation, an important element to reduce the ar burden will be to break the transmission bridges among different ecosystems that could be reservoirs of args. even when wastewater-treatment plants are available, the presence of arbs in drinking, fresh, and coastal waters, as well as in sediments nearby industrial and urban discharges, has been described in several countries (ma et al., 2017; leonard et al., 2018) . as in the case of fecal contamination markers, a reduction in the amount of args to non-detectable levels would be extremely difficult even when advanced water treatment procedures are applied. a standard definition of polluting arb/arg markers, as well as their acceptable levels, is then needed. this would be required not only for potable water, but also for water reutilization, as well as for land application and release of sewage effluents, because in all cases the reused water/sewage may carry arbs and args, together with pollutants, such as antibiotics, metals, biocides, or microplastics, which, as above stated, may select for ar (baquero et al., 2008; moura et al., 2010; yang et al., 2017; zhu et al., 2017; larsson et al., 2018; imran et al., 2019; wang et al., 2020) and may even induce hgt. the examples discussed above justify that human health in general and ar in particular are closely interlinked with economic development (sharma, 2018) . economic differences are also found at individual level, because there is a positive relationship between economic status and health (tipper, 2010) . in addition, social behavior might also impact ar, a feature discussed in the following section. different socioeconomic factors can modulate the spread of infective bacteria in general and of ar in particular. among them, the increasing crowding of humans and foodborne animal populations favors transmission at the local level (one health), whereas trade of goods and human travel (figure 2 ) favor worldwide transmission (global health) (laxminarayan et al., 2013; hernando-amado et al., 2019) . besides these global changes in social behavior, linked to economic development, more specific socioeconomic factors (income, education, life expectancy at birth, health care structure, governance quality), sociocultural aspects (inequalities, uncertainty avoidance, integration of individuals into primary groups, gender biases, cultural long-term orientation), and personality dimension highly influence antibiotic use and ar transmission (gaygısız et al., 2017) . for instance, although the governance quality seems to be the most important factor associated with a proper antibiotic use, western countries with distinct national culture patterns show different levels of antibiotics consumption (kenyon and manoharan-basil, 2020) . a better understanding of human social responses facing ailments, especially epidemics and antibiotic use, requires then a more detailed analysis of the differences between collectivistic (individuals living integrated into primary groups) and individually long-term oriented societies (oriented to future individual rewards) (hofstede, 2001; gaygısız et al., 2017; kenyon and manoharan-basil, 2020) . consistent with the sociological elements of ar, many of the aspects influencing ar reviewed above depend on social norms (figure 1) . in the classic view of the psychoanalyst erich fromm presented in his book "escape from freedom" (fromm, 1941) , human individual behavior is oriented to avoid being excluded from a higher social group. indeed, not following social common rules can be eventually considered as a mental disorder; a sociopathology. a social norm is defined as a predominant behavioral pattern within a group, supported by a shared understanding of acceptable actions and sustained through social interactions within that group (nyborg et al., 2016) . in democratic societies, laws usually derive from already accepted social norms; otherwise, they would be changed, and in that sense, the establishment of accepted social norms for fighting ar is a prerequisite to implement the global approaches, based on worldwide rules, which are required for tackling this relevant problem. interestingly, the ar problem is a bottom-up process, where small emergent changes (in some type of individual patients, in some groups, in some locations) cumulatively escalate to gain a global dimension. frequently, that occurs by crossing tipping points, that is, points where the local ar incidence becomes significant enough to cause a larger, eventually global, health problem. because of that, the implementation of solutions should be adapted to the control of critical tipping points in the small groups of individuals to disrupt the bottom-up processes. however, as ar spread can occur everywhere and at any time, global surveillance and mechanisms of control should be implemented to prevent a top-down process of global ar expansion. individual selfishness for ar is the cornerstone of social norms. this concept was coined and developed by one of us over a decade ago (baquero, 2007) . let us imagine that each individual is aware that each consumption of an antibiotic increases the personal risk of himself/herself or for his/her closer relatives (frequently exchanging microorganisms) of dying because of an antibiotic-resistant infection. the situation is analogous to the consumption of cholesterol-rich or highly salted food, or drinks with excess of sugar, concerning individual health. however, in the case of ar, it requires the understanding of the impact of individual actions at the global level. in this respect, anti-ar social actions should resemble more antitobacco and even general pollution/ecological campaigns. at the individual level, there is inertia that precludes changing habits, until a tipping point is crossed and health is compromised. the conclusions of studies mainly based on long-term cohort analysis, such as the framingham program for the influence of diet or smoking on personal cardiovascular disease (mahmood et al., 2014) , have become social norms that are naturally imposed by the ensemble of individuals. this creates a kind of societal culture, leading to appropriate individual behaviors, in occasions without the need of specific laws (diet), in occasion favoring the implementation of such laws (antismoking). however, we lack similar studies on issues such as these dealing with personalfamiliar risks that have successfully shifted social norms, driven by groups of individuals and based on the promotion of individual behaviors in the case of ar. despite that quantitative models on how individual antibiotic use may impact ar at the population level are still absent, it is worth mentioning that a reduced antibiotic consumption has also begun to occur in a number of countries just as a result of a change in individual behavior (edgar et al., 2009) , and some tools and indicators to address these changes have been suggested (ploy et al., 2020) . the "tragedy of the commons" metaphor, first proposed in the xix century (lloyd, 1833) and later on discussed in 1968 (hardin, 1968) , has been used for addressing the sociology of ar, by showing how individual selfishness promotes antibiotic use, increases resistance, and influences the health of the community by impairing antibiotic efficacy (baquero and campos, 2003; foster and grundmann, 2006) . ensuring the prestige of individuals that follow the social rules is needed to counteract the tragedy of the commons. nevertheless, it is important noticing that the tension between individual freedom and social rules that is inherent to the construction of democratic societies (tocqueville, 1838; hobbes, 1968; rousseau, 1974; spinoza, 2007) also applies here. one example of this situation is vaccination, considered in the last century as one of the most important advances to fight infectious diseases and now being the focus of antivaccination campaigns (megget, 2020) , a movement that has been considered by the who as one of the top 10 global health threats of 2019 1 . it is commonly accepted that social norms are mainly created by learning and education, a rational path that promotes health (chen and fu, 2018) . also, the increasing activities of "personalized medicine, " including antibiotic stewardship, follow the same trend (gould and lawes, 2016) . however, the antivaccination movement is an example of how the narrative, as well as the use of decentralized, social information channels such as the internet search, blogs, and applications to facilitate communication such as twitter, facebook or whatsapp, is of particular relevance in the construction of social norms, not necessarily based on scientific and rational grounds (jacobson et al., 2020; scott and mars, 2020) . the impact of social norms goes beyond human societies as human activities alter natural ecosystems; consequently, humans cannot be aliens of nature. we should then shape a socioecological system, linking the individuals, the groups, and the entire society, as well as natural ecosystems, also potentially damaged by ar, in a common multilevel adaptive system based on social norms and policies at the individual, local (one health), and global (global health) scale (levin et al., 2013) . the recent crisis of covid-19 illustrates the influence of social norms in the individual behavior. each one of the individuals, protecting himself/herself, also protects the others. a person not wearing on face mask is frowned upon, and on the contrary, somebody attaching to the rules increases reputation. the individual adopts the right behavior being influenced by the judgment. of others. in addition, different political regimes (democracy or autocracy), as well as their organization (centralized, federal), together with the capacity of the health services to support the norms and their efficacy to communicate the chosen policy to the citizenry, may shape the individual responses to social norms (greer et al., 2020; häyry, 2020; kavanagh and singh, 2020) . notwithstanding, two reasons that have been proposed to explain the low prevalence of covid-19 in japan were related with social norms more than with biological issues. these reasons, which are not common to other countries, were the socially accepted use of face masks and the mandatory vaccination of all the population against tuberculosis, which might protect from sars-cov-2 infection (iwasaki and grubaugh, 2020) , a feature that is still to be confirmed. the loss of social prestige of individuals taking antibiotics without prescription, as well as the pharmacies delivering these drugs or do not respect environmental protection, or the overconsumption of antibiotics in hospitals or in farms, or even in certain countries, is progressively constituting a "social norm, " converted in rules able to reduce ar emergence and spread. of course, family and school education, as well as governmental campaigns, including the use of social media (grajales et al., 2014) reinforces such social norms, which could allow the support of the society for the implementation of different interventions, some of them described below. controlling resistance not only requires establishing local interventions, which could be relatively easily implemented, but would also require global interventions that every country should follow, despite their disparate regulatory systems. local and global interventions are necessarily intertwined; for example, the use of a new drug to treat a single individual depends on regulations at the county level (one health approach), but the worldwide prevalence and transmission of resistance to this drug, as well as the regulations of its use, should be established internationally (global health approach). three main interventions to tackle ar have been historically considered: first, reduction of the antibiotic selective pressure by decreasing antimicrobials use; second, reduction of transmission of arbs using improved hygienic procedures that prevent spread; third, development of novel antimicrobials with limited capacity to select arbs or the design of new treatment strategies based on use of non-antibiotic-based approaches or, more recently, on the exploitation of trade-offs associated with ar evolution (imamovic and sommer, 2013; gonzales et al., 2015; barbosa et al., 2018; imamovic et al., 2018) . these interventions have been basically limited to local initiatives, applied mainly to hospitals and, more recently, to farms. however, ar has emerged and spread globally, in bacteria from different environments, so the health and dynamics of the global microbiosphere could be affected by antibiotics. in a sense, ar is affecting the planetary health (lerner and berg, 2017) , and the needed interventions for tackling this problem cannot be restricted to hospital settings (figure 3) . the proposed reduction in the use of antibiotics (blaskovich, 2018 ) must be compensated with alternative approaches for fighting infectious diseases. in this regard, strategies based on improving the capability of the immune system for counteracting infections (levin et al., 2017; traven and naderer, 2019) or the use of non-antibiotic approaches to prevent them, such as vaccines (jansen and anderson, 2018) , may help to reduce the burden of ar infections. indeed, vaccination against haemophilus influenzae and streptococcus pneumoniae has been demonstrated to be an effective intervention for reducing ar (jansen and anderson, 2018) . however, while vaccination has been extremely useful to prevent viral infections, it has been less promising in the case of bacterial ones. recent approaches, including reverse vaccinology, may help in filling this gap (delany et al., 2013; ni et al., 2017) . moreover, vaccination should not be restricted to humans, because veterinary vaccination can also contribute to animal wealth and farm productivity (francis, 2018) . besides, the use of vaccines in animal production reduces the use of antibiotics at farms/fisheries, hence reducing the selection pressure toward ar. other strategies to reduce antibiotic selective pressure include the use of bacteriophages (a revitalized strategy in recent years) (viertel et al., 2014; forti et al., 2018) , not only in clinical settings, but also in natural ecosystems (zhao et al., 2019b) , as well as the use of biodegradable antibiotics (chin et al., 2018) or adsorbents, able to reduce selective pressure on commensal microbiome (de gunzburg et al., 2015 . besides reducing the chances of selecting arbs, the use of antibiotics adsorbents may preserve the microbiomes, reducing the risks of infections (chapman et al., 2016) . importantly, the procedures for removing antibiotics should not be limited to clinical settings, but their implementation in wastewater treatment plants would reduce selection of ar in non-clinical ecosystems (tian et al., 2020) . concerning the development of new antimicrobials (hunter, 2020) , while there is a basic economic issue related to the incentives to pharmaceutical companies (sciarretta et al., 2016; theuretzbacher et al., 2017) , the focus is on the possibility of developing novel compounds with low capacity for selecting ar (ling et al., 2015; chin et al., 2018) . for this purpose, multitarget (li et al., 2014) or antiresistance drugs, such as membrane microdomain disassemblers (garcia-fernandez et al., 2017) , are also promising. furthermore, antimicrobial peptides, with a dual role as immunomodulators and antimicrobials, may also help fight infections (hancock et al., 2016) . in fact, some works figure 3 | local and global intervention strategies to tackle ar and knowledge gaps that could help improve existing ones. most interventions for reducing antibiotic resistance are based on impairing the selection of arbs/args, which is just the first event in ar spread. our main goal, as for any other infectious disease, figure 3 | continued would be reducing transmission. this does not mean that selective pressure is not relevant for transmission. indeed, without positive selection, hgt events are not fixed, allowing the enrichment of some args that are consequently more prone to diversification, both because they are more abundant and more frequently subjected to selection (davies, 1997; martinez, 2009a,b; salverda et al., 2010 ) and because they can explore different landscapes when present as merodiploids in multicopy plasmids (rodriguez-beltran et al., 2018) . therefore, reducing the selective pressure, either due to antibiotics or by other coselecting agents as heavy metals, still stands as a major intervention against ar emergence and transmission. to address this issue, we need to know more on the amount of pollutants, their selective concentrations, and their mechanisms of coselection and cross-selection in different ecosystems. this is a general example illustrating the gaps in knowledge in the ar field that need to be filled as well as strategies that may help in tackling this problem. the figure includes several other examples of the gaps of knowledge (red) that require further studies and the interventions (blue) that may help to tackle ar. have shown that arb frequently present collateral sensitivity to antimicrobial peptides (lázár et al., 2018) and that, importantly, some antimicrobial peptides present limited resistance or crossresistance (kintses et al., 2019; spohn et al., 2019) . from a conservative point of view, based on the use of the drugs we already have, it would be desirable to fight ar using evolution-based strategies for developing new drugs or treatment strategies. regarding this, the exploitation of the evolutionary trade-offs associated with the acquisition of ar, as collateral sensitivity, could allow the rational design of treatments based on the alternation or the combination of pairs of drugs (imamovic and sommer, 2013; gonzales et al., 2015; barbosa et al., 2018; imamovic et al., 2018) . in addition to interventions that reduce the selective pressure of antibiotics or that implement new therapeutic approaches, reducing transmission is also relevant to fight infections. the development of drugs or conditions (as certain wastewater treatments) able to reduce mutagenesis or to inhibit plasmid conjugation may also help in reducing the spread of resistance (thi et al., 2011; alam et al., 2016; lin et al., 2016; lopatkin et al., 2017; valencia et al., 2017; kudo et al., 2019) . besides specific drugs to reduce the dissemination of the genetic elements involved in ar, socioeconomic interventions to break the bridges that allow transmission between individuals and, most importantly (and less addressed), between resistance entities (hernando-amado et al., 2019) are needed (figure 3) . more efficient animal management, not only allowing less antibiotics use but also reducing animal crowding (and hence ar transmission), as well as improved sanitation procedures, including the universalization of water treatment, will certainly help in this task (berendonk et al., 2015; manaia, 2017; hernando-amado et al., 2019) . notably, wastewater treatment plants are usually communal facilities where the residues of the total population of a city are treated. hospitals are the hotspots of ar in a city; hence, on-site hospital (and eventually onfarm) wastewater treatment may help to reduce the pollution of communal wastewater by antibiotics and arbs (cahill et al., 2019; paulus et al., 2019) , hence reducing ar transmission. concerning trade of goods, it is relevant to remark that, although there are strict regulations to control the entrance of animals or plants from sites with zoonotic of plant epidemic diseases (brown and bevins, 2018) , there are no regulations on the exchange of goods from geographic regions with a high ar prevalence, a feature that might be taken into consideration for reducing the worldwide spread of ar. once arbs are selected and disseminated, interventions based on the ecological and evolutionary (eco-evo) aspects of ar lehtinen et al., 2019) should be applied to restore (and select for) susceptibility of bacterial populations, as well as to preserve drug-susceptible microbiomes in humans and in animals . eco-evo strategies include the development of drugs specifically targeting arbs. for that, drugs activated by mechanisms of resistance, vaccines targeting high-risk disseminating resistance clones or the resistance mechanisms themselves (kim et al., 2016; ni et al., 2017) , or drugs targeting metabolic paths that can be specifically modified in arbs ) might be useful. the use of bacteriovores such as bdellovibrio to eliminate pathogens without the need for antibiotics has been proposed; although its utility for treating infections is debatable, it might be useful in natural ecosystems (shatzkes et al., 2016) . more recent work suggests that some earthworms may favor the degradation of antibiotics and the elimination of arbs (wikler, 2002) , a feature that might be in agreement with the finding that arbs are less virulent (and hence might be specifically eliminated when the worm is present) in a caenorhabditis elegans virulence model ruiz-diez et al., 2003; paulander et al., 2007; olivares et al., 2012) . however, the information on the potential use of worms for reducing ar in the field is still preliminary and requires further confirmation. noteworthy, ar is less prone to be acquired by complex microbiomes (mahnert et al., 2019; wood, 2019) , a feature that supports the possibility of interventions on the microbiota to reduce ar. among them, fecal transplantation (chapman et al., 2016; pamer, 2016) or the use of probiotics able to outcompete arbs (keith and pamer, 2019) has been proposed as strategies for recovering susceptible microbiomes. the recent crisis of covid-19 (garrett, 2020) resembles the pandemic expansion of args and clearly shows that pandemic outbreaks cannot be solved by just applying local solutions. further, unless all population is controlled, and comprehensive public-health protocols are applied to the bulk of the population, such global pandemics will be hardly controlled. the case of covid-19 is rather peculiar, because we are dealing with a novel virus. very strict interventions have been applied, mainly trying to control something that is a novel, unknown, disease; we have been learning along the pandemic and still ignore what will come further. ar is already a very well-known pandemic affecting humans, animals, and natural ecosystems (anderson, 1999; verhoef, 2003) . in this case, we have tools that might predict the outcome, and likely because the degree of uncertainty is lower than in the case of covid-19, we have not applied clear, common, and comprehensive procedures to reduce the spread of ar. it is true that we know the evolution of antibiotics consumption and ar prevalence in several countries, and also interventions, mostly based on social norms, have been applied. social norms have reduced the unnecessary prescription of antibiotics, or pharmacy sales without prescription, and the use of antibiotics for fattening animals has been banned in several countries, being still allowed in several others. nevertheless, these actions are not general, and more aggressive, global actions are still needed. coming back to the covid-19 example, while the aim of health services worldwide is to detect any possible source of sars-cov-2, surveillance of infections (eventually by arbs) is not universal. in other words, it does not apply to all citizens in all countries. the reasons can be just political such as the inclusion of immigrants in public health services (scotto et al., 2017) or the consequence of limited financial resources and technical capacity that countries such as those belonging to the lmic category can face (gandra et al., 2020) . the problem is not only on citizens, because different non-human reservoirs, such as wastewater, drinking water, or freshwater, may jointly contribute to ar dissemination (hendriksen et al., 2019) . in this regard, it is important to highlight that low quality of water is regularly associated to poverty. universalization of health services, sanitization, access to clean water, and in general reduction of poverty are relevant step-forward elements for reduction of the burden of infectious diseases in general and of ar in particular. the time has come to tackle ar, and this cannot be done just by taking actions at the individual or even country level, but by taking convergent actions across the globe. as stated by john donne (1923) in his poem, "no man is an island, " written after his recovery from an infectious disease (likely typhus): "no man is an iland, intire of itselfe; every man is a peece of the continent, a part of the maine; if a clod bee washed away by the sea, europe is the lesse, as well as if a promontorie were, as well as if a manor of thy friends or of thine owne were; any mans death diminishes me, because i am involved in mankinde; and therefore never send to know for whom the bell tolls; it tolls for thee." this reflection on how infectious diseases in general should be faced by the society was published at 1624, but the idea behind still applies nowadays, especially for ar. all authors have contributed to the concept of the review and in its writing. jm was supported by grants from the instituto de salud carlos iii [spanish network for research on infectious diseases (rd16/0016/0011)], from the spanish ministry of economy and competitivity (bio2017-83128-r) and from the autonomous community of madrid (b2017/bmd-3691). work in tc and fb laboratory was supported by grants funded by the joint programming initiative in antimicrobial resistance (jpiamr third call, starcs, jpiamr2016-ac16/00039), the instituto de salud carlos iii of spain/ministry of economy and competitiveness and the european development regional fund "a way to achieve europe" (erdf) for co-founding the spanish r&d national plan estatal de i + d + i 2013-2016 (pi18/1942), ciberesp (ciber in epidemiology and public health; cb06/02/0053), the regional government of madrid (ingemics-b2017/bmd-3691) and the fundación ramón areces. the funders did not have any role neither in the design, nor in the writing of the current review. association between decreased susceptibility to a new antibiotic for treatment of human diseases, 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host-species transferrin receptor 1 orthologs are cellular receptors for nonpathogenic new world clade b arenaviruses date: 2009-04-03 journal: plos pathog doi: 10.1371/journal.ppat.1000358 sha: doc_id: 267149 cord_uid: 5twx9y5c the ability of a new world (nw) clade b arenavirus to enter cells using human transferrin receptor 1 (tfr1) strictly correlates with its ability to cause hemorrhagic fever. amapari (amav) and tacaribe (tcrv), two nonpathogenic nw clade b arenaviruses that do not use human tfr1, are closely related to the nw arenaviruses that cause hemorrhagic fevers. here we show that pseudotyped viruses bearing the surface glycoprotein (gp) of amav or tcrv can infect cells using the tfr1 orthologs of several mammalian species, including those of their respective natural hosts, the small rodent neacomys spinosus and the fruit bat artibeus jamaicensis. mutation of one residue in human tfr1 makes it a functional receptor for tcrv, and mutation of four residues makes it a functional receptor for amav. our data support an in vivo role for tfr1 in the replication of most, if not all, nw clade b arenaviruses, and suggest that with modest changes in their gps the nonpathogenic arenaviruses could use human tfr1 and emerge as human pathogens. arenaviruses are enveloped viruses that carry single-stranded, bi-segmented, rna genomes [1] . the family arenaviridae comprises a single genus (arenavirus) that contains at least 25 members [2] [3] [4] . arenaviruses have been classified into two antigenically and geographically distinct groups, the lassa-lymphocytic choriomeningitis serocomplex (''old world arenaviruses'') and the tacaribe serocomplex (''new world arenaviruses'') [5] [6] [7] . the new world (nw) arenavirus cluster is subdivided into clades a, b, and c. in addition, several north american viruses are recombination products of clades a and b (a/b) ( figure 1 ). all nw arenaviruses that cause hemorrhagic fever in humans are in the b clade. these include the junín (junv), machupo (macv), guanarito (gtov), and sabiá (sabv) viruses, which respectively cause argentine, bolivian, venezuelan, and brazilian hemorrhagic fevers with high (10-30%) case fatality rates [2, 8, 9] . a novel nw clade b arenavirus, chapare virus, was recently isolated from a fatal case of hemorrhagic fever in bolivia [3] . in addition to these pathogens, there are clade b viruses that do not cause disease in humans. these include the amapari (amav), cupixi (cpxv), and tacaribe (tcrv) viruses, although tcrv may have caused a febrile illness with mild central nervous system symptoms in a laboratory worker [10] . pathogenic and nonpathogenic nw arenaviruses do not cluster independently on phylogenetic analysis; tcrv is closely related to macv and junv, while amav shares highest sequence similarity with gtov ( figure 1 ). the zoonotic reservoir of a given nw arenavirus has a unique geographical distribution, and each arenavirus prefers its own host [11] . most of the natural hosts for nw arenaviruses belong to the subfamily sigmodontinae in the family cricetidae, which contains nw mice and rats [6, 12] . the host animals for macv, junv and gtov are calomys callosus (large vesper mouse), calomys musculinus (drylands vesper mouse), and zygodontomys brevicauda (short-tailed cane mouse), respectively [13] [14] [15] [16] . the natural reservoirs of sabv and chapare virus have not been identified. amav was isolated from neacomys spinosus (bristly mouse) and neacomys guianea (guiana bristly mouse), and tcrv is suggested to have a non-rodent host, artibeus species fruit bats in trinidad and tobago [12, [17] [18] [19] . the arenavirus envelope glycoprotein (gp), the sole protein found on the surface of virions, is processed into three associated subunits: the stable signal peptide (ssp), gp1, and gp2 [20, 21] . ssp is a unique component of the arenaviral fusion machinery and plays a role in the transport, maturation, and ph dependent membrane fusion activity of the gp complex [22] [23] [24] [25] . the gp1 subunit engages a cellular receptor(s), and gp2 mediates phdependent membrane fusion after viral particles are internalized into acidified endosomes [21, [26] [27] [28] [29] . two cell surface molecules have been implicated as cellular receptors for arenaviruses. old world arenaviruses and nw clade c arenaviruses oliveros and latino use a-dystroglycan as an obligate receptor, while the pathogenic nw clade b arenaviruses macv, junv, gtov and sabv use human transferrin receptor 1 (tfr1) to infect cells [30] [31] [32] [33] . the ability of nw clade b arenaviruses to cause disease in humans correlates with the utilization of human tfr1, a molecule that has several properties favorable to arenaviral replication and viral hemorrhagic fevers; it is rapidly endocytosed into acidic compartments, expressed on endothelial cells, and upregulated on rapidly dividing cells including activated lymphocytes [34] [35] [36] [37] [38] . we have previously shown that recombinant retroviral particles pseudotyped with the gps of macv, junv, or gtov efficiently use the tfr1 ortholog of the rodent hosts of these viruses [39] . a number of studies have suggested that amav and tcrv do not use human tfr1 [40] [41] [42] [43] . blocking human cells with inactivated amav significantly reduces the infection of these cells with amav pseudovirus, but does not interfere with the infection of macv, junv or gtov pseudoviruses [43] . it remains to be shown, however, if these nonpathogenic nw arenaviruses could use the tfr1 orthologs of their principal host animals. here, we confirm that amav and tcrv do not use human tfr1, and show that they nonetheless efficiently use the tfr1 orthologs of their respective animal hosts, n. spinosus and a. jamaicensis. we also show that mutation of one residue converts human tfr1 into an efficient receptor for tcrv, and replacement of four residues with those found in n. spinosus tfr1 converts human tfr1 into an efficient receptor for amav. these data show that tfr1 has an important role in the replication of nonpathogenic nw arenaviruses, and suggest that subtle changes in the gps of tcrv and amav might adapt them to use human tfr1. we confirmed that human tfr1 is not involved in the entry of amav and tcrv into human cells by examining whether an ahuman tfr1 antibody could inhibit the infection of hek293t cells mediated by the gps of amav and tcrv. we generated recombinant moloney murine leukemia virus (mlv) expressing green-fluorescent protein (gfp), pseudotyped with the gps of amav, tcrv, macv and gtov. cells were exposed to these recombinant viruses in the presence or absence of a control antibody (a-hla), or an a-human tfr1 antibody previously shown to inhibit infection of macv and junv pseudoviruses [32] . infection levels were assayed forty-eight hours later by flow cytometry (figure 2 ). the a-human tfr1 antibody inhibited infection by gtov and macv pseudoviruses, but had no effect on the entry of amav and tcrv pseudoviruses. the entry of all four pseudoviruses was unaffected by the a-hla antibody. these results are in agreement with previous studies showing that human tfr1 is not involved in the entry of the nonpathogenic nw clade b arenaviruses into human cells [40] . animal orthologs of tfr1 support entry of amav and tcrv pseudoviruses phylogenetic analysis reveals that gtov gp shares greater sequence similarity with amav gp than it does with the gps of the other pathogenic nw arenaviruses ( figure 1 ). amav, however, does not use human tfr1 as a cellular receptor. we sought to determine if animal orthologs of tfr1 could support entry mediated by the gps of amav and tcrv. chinese hamster ovary (cho) cells were transfected with plasmids expressing the human, mouse, rat, feline, canine, calomys callosus (cc ; macv host), calomys musculinus (cm ; junv host), or viruses that cause hemorrhagic fever in humans, which are only found in clade b (encircled), are labeled with an asterisk. viruses known to use human tfr1 are boxed in orange, and the nonpathogenic arenaviruses investigated in this study are boxed in cyan [39, 40] . doi:10.1371/journal.ppat.1000358.g001 several arenaviruses found in the new world cause hemorrhagic fever when they are transmitted from their natural reservoirs to humans. these pathogenic arenaviruses use human transferrin receptor 1 (tfr1), a protein involved in cellular iron uptake, to infect human cells. the nonpathogenic new world arenaviruses characterized thus far do not use human tfr1. we show here that two of these nonpathogenic viruses, amapari and tacaribe, can use animal orthologs of tfr1 to infect cells. we observe that recombinant viruses coated with the entry proteins of amapari and tacaribe viruses use the tfr1 orthologs of their natural reservoirs. modest alteration of human tfr1 converts it to an efficient receptor for amapari and tacaribe viruses. our findings provide insight into the potential of amapari and tacaribe viruses to adapt to use human tfr1 and perhaps emerge as human pathogens. zygodontomys brevicauda (zb ; gtov host) orthologs of tfr1. tfr1 expression was assessed by flow cytometry using an antibody directed against a flag tag added to the c-terminus of each ortholog. in parallel, aliquots of the same cells were infected with amav or tcrv pseudoviruses. as these viruses readily enter cho cells using a tfr1-independent pathway, pseudoviruses were diluted to minimize background entry. pseudovirus bearing the gp of lymphocytic choriomeningitis virus (lcmv) was used as a control. as shown in figure 3a , infection by amav and tcrv pseudoviruses was significantly enhanced by the introduction of zbtfr1 into cho cells, while amav pseudovirus entry was enhanced to a lesser degree by cctfr1. the entry of amav pseudovirus was also enhanced by feline tfr1 expression. we and others have previously shown that this tfr1 ortholog is also used efficiently by the pathogenic viruses macv, junv and gtov [39, 40] . the entry of lcmv pseudovirus was unaffected. we repeated these experiments using hek293t cells, and obtained similar results ( figure 3b ). these data show that although amav and tcrv pseudoviruses do not use human tfr1, they nonetheless use the tfr1 orthologs of several mammalian species. we next investigated whether amav and tcrv gp could also use the tfr1 orthologs of their host species, the small rodent n. spinosus and the fruit bat a. jamaicensis, respectively. we cloned the genes of their tfr1 orthologs by rt-pcr from frozen tissues of n. spinosus (nstfr1) and a. jamaicensis (ajtfr1). in order to determine the ability of the amav and tcrv gp1 proteins to bind these tfr1 orthologs, hek293t cells were transfected with plasmids expressing human, ns, or ajtfr1. these cells were incubated with ig-fusion proteins comprising the gp1 subunits of amav or tcrv (amav-gp1d-ig and tcrv-gp1d-ig, respectively). the binding of these proteins to transfected cells was measured by flow cytometry using an a-human igg antibody. the expression levels of tfr1 orthologs were assessed in parallel using an a-flag antibody. as shown in figure 4a , while the expression levels of all tfr1 orthologs were comparable (left panels), amav-gp1d-ig bound cells expressing nstfr1, but did not bind cells expressing ajtfr1. tcrv-gp1d-ig bound cells expressing ajtfr1 and also cells expressing nstfr1. neither ig-fusion protein bound cells expressing human tfr1. although both amav and tcrv pseudoviruses enter hek293t cells using a tfr1-independent pathway, indicating the presence of an alternative receptor, we did not observe binding of amav or tcrv-gp1d-ig to these cells (data not shown). this observation is consistent with an entry process dependent on a lower affinity receptor. to determine if amav and tcrv pseudoviruses could use nstfr1 and ajtfr1 to infect cells, hek293t cells were transfected with plasmids expressing human tfr1, zbtfr1, nstfr1, ajtfr1, or vector alone, and then incubated with amav and tcrv pseudoviruses. pseudovirus bearing the g protein of vesicular stomatitis virus (vsv) was used as a control. consistent with the gp1d-ig binding data, amav pseudovirus used only nstfr1, while tcrv pseudovirus efficiently used both ajtfr1 and nstfr1 ( figure 4b ). we next assessed whether nstfr1 and ajtfr1 could also serve as receptors for the pathogenic nw clade b arenaviruses. as the pathogenic nw arenaviruses efficiently enter hek293t cells using endogenous human tfr1 [32] , cho cells were transfected with plasmids expressing human tfr1, ajtfr1, nstfr1, zbtfr1, or vector alone, and subsequently infected with macv, junv, or gtov pseudoviruses. as shown in figure 4c , macv and junv, like tcrv, used both nstfr1 and ajtfr1, while gtov, like the closely related amav, efficiently used nstfr1 but not ajtfr1. we confirmed these findings using replication-competent tcrv and amav viruses. hek293 cells were transfected with plasmids expressing human tfr1, ajtfr1 or nstfr1, and subsequently infected with replication-competent tcrv or amav. as shown in figure 5 , the infection of tcrv was enhanced on cells expressing ajtfr1 and nstfr1, while that of amav was only enhanced on cells expressing nstfr1. a loop (residues 202-212) between b strands 1 and 2 in the apical domain of human tfr1 is a likely interaction site for the gps of the nw hemorrhagic fever arenaviruses [39, 44] . this loop includes tyrosine 211, which is essential for macv, junv, and gtov to use human tfr1 [39] . we compared the sequences of zbtfr1, nstfr1, and ajtfr1 in this region with that of human tfr1, and found several differences between human and animal tfr1 ( figure 6a and 6b). we sought to determine whether replacing the human tfr1 residues with their equivalents from animal tfr1 orthologs would allow the modified human tfr1 to serve as a receptor for amav and tcrv pseudoviruses. figure 6a shows a panel of eleven human tfr1 variants that were examined. in the first two variants (zh1 and zh2), residues from human tfr1 were replaced with those from zbtfr1. four variants (ah2 through ah5) include residues from ajtfr1, and five variants (nh2, nh4, nh5, nh7 and nh8) include nstfr1 sequence. these variants were expressed in hek293t cells, and the cells were incubated with tcrv or amav pseudoviruses. a variant (zh1) containing two alterations -r208g and dv210 -was an efficient receptor for tcrv ( figure 6c ). as expected from comparison with zh1, a variant (ah2) containing three ajtfr1 residues in place of human tfr1 residues 208-210 (r208g, l209a, and v210g) was a functional receptor for tcrv ( figure 6d ). when mutations at r208 and v210 were individually introduced into human tfr1, the variant containing v210g (ah4) supported tcrv pseudovirus entry efficiently, while the variant containing r208g (ah3) was a less efficient receptor. deletion of v210 (ah5), however, did not convert human tfr1 into a receptor for tcrv. a slightly larger alteration in human tfr1 was necessary for amav pseudovirus infection; nh5, which contains four mutations -d204n, k205s, r208g, and dv210 -or nh8, which contains five mutations, were efficient receptors for amav ( figure 6e ). the entry of vsv pseudovirus was unaffected by the introduction of any of the variants. thus, only minimal alteration of human tfr1 was sufficient for it to serve as an efficient receptor for the nonpathogenic viruses amav and tcrv. five nw arenaviruses have been shown to cause hemorrhagic fever in humans. we have previously shown that four of these viruses -macv, junv, gtov, and sabv -require tfr1 to enter human cells [32] . we have also shown that these viruses utilize the tfr1 orthologs of their rodent hosts efficiently and with specificity [39] . a recently identified fifth hemorrhagic fever nw arenavirus, chapare virus, remains to be characterized. collectively, these data indicate a central role for tfr1 in the replication of nw clade b arenaviruses that cause hemorrhagic fever in humans. closely related to these pathogenic viruses are three clade b arenaviruses that do not cause disease in humans, namely tcrv, amav, and cpxv, although tcrv may have caused mild disease in a laboratory worker [10] . here we have studied the two best characterized of these viruses, tcrv and amav. the gp sequences of these viruses are more similar to those of the pathogenic viruses than they are to each other (figure 1 ). in spite of the similarity of amav and tcrv to the other members of their respective subclades, these nonpathogenic viruses do not utilize human tfr1. in contrast, every clade b virus shown to use human tfr1 causes a south american hemorrhagic fever [24, [32] [33] [34] [35] . there is thus a close correlation between use of human tfr1 and the ability of a nw arenavirus to cause severe human disease. a possible exception is the whitewater arroyo virus (wwav), a nw clade a/b arenavirus that does not appear to use human tfr1, but is tentatively associated with three human fatalities in 1999-2000 [45, 46] . follow up studies by the cdc, however, failed to find any evidence of wwav infection in the human tissues mentioned in the original article (nichol, personal communication). in addition, there have been no further reports of wwav infection being associated with human disease. this correlation suggests that tfr1 use is a key determinant in the virulence of clade b viruses, which raises an important question: how easily might clade b viruses circulating in animals gain the ability to use human tfr1? our findings shed light on this issue; tcrv and amav pseudoviruses efficiently utilize the tfr1 orthologs of several species including those of their native hosts, a. jamaicensis and n. spinosus, respectively (figures 3 and 4) . as with the pathogenic clade b viruses, amav and tcrv utilized most efficiently the tfr1 ortholog of their natural hosts. it is therefore likely that tfr1 is also central to the replication of these nonpathogenic viruses in their native hosts. to investigate the possibility that gain-of-use of human tfr1 may not require extensive alteration of the tcrv and amav gps, we sought to identify differences between human tfr1 and host animal tfr1 orthologs that determine the ability of these molecules to support infection by tcrv and amav pseudoviruses. we swapped residues in a loop in the apical domain of tfr1 that is a key determinant of host specificity ( figure 6) . surprisingly, alteration of a single residue (v210g, ah4) allowed human tfr1 to efficiently support the entry of tcrv pseudovirus, while replacement of four residues in this loop with the corresponding residues of nstfr1 (nh5) was necessary to support efficient infection by amav pseudovirus (figure 6d and 6e). although these alterations are made to the receptor, it is reasonable to infer that similarly modest changes in the arenaviral gps would also permit efficient use of human tfr1. for example, in order to bind human tfr1, tcrv gp would only need to acquire mutations that accommodate v210, present in human tfr1 but absent or replaced by a glycine in animal tfr1 orthologs. our data, although limited, suggest that small alterations in the arenaviral gp could convert these nonpathogenic nw arenaviruses into human pathogens. this possibility has precedent in other emerging viruses. for example, alteration of only a few residues in the spike protein of the severe acute respiratory syndrome coronavirus (sars-cov) was sufficient for this glycoprotein, derived from an animal isolate of the virus, to efficiently use the human ortholog of its receptor, angiotensin converting enzyme 2 (ace2) [47] . similarly, the adaptation of avian influenza viruses to humans is thought to require as few as two amino acid substitutions in the influenza hemagglutinin glycoprotein (ha). these mutations allow ha to bind its human receptor, a2,6 -linked sialosides [48, 49] . structural studies of the sars-cov and influenza a virus entry proteins in complex with their respective receptors have helped to identify and predict entry protein alterations that contribute to the ability of these viruses to cause human disease [48, 50] . similar studies of arenanaviral gp molecules bound to tfr1 are likely to further elucidate the risk posed by amav, tcrv, and perhaps other nonpathogenic arenaviruses. of note in this regard, bats are increasingly recognized as reservoirs for viruses that can cross species barriers to infect humans [51] . nipah, hendra, and perhaps sars-cov and ebola viruses, are on the list of emerging viruses that involve amplification in bat hosts [52] . although tcrv was isolated from bats [19] , the suggestion that artibeus species bats serve as the natural hosts for tcrv has been questioned [53] . our observation that ajtfr1 is an efficient receptor for tcrv, and also for junv and macv, supports a role for bats in sustaining the replication of arenaviruses. ecological surveys of bats for novel arenaviruses are thus warranted, and may afford further insight into the diversity of arenaviruses. in addition, although cats experimentally infected with macv do not become ill [54] , viral titers in these animals were not determined. the finding that feline tfr1 supports the entry of amav and the pathogenic clade b arenaviruses thus highlights a possible role for cats in serving as intermediate hosts in the transmission of nw arenaviruses to humans [39, 40] . despite the inability of amav and tcrv to cause disease in humans, their gp proteins mediate entry into human cells through a pathway independent of human tfr1 or a-dystroglycan ( figure 2) [40, 43] . this observation suggests that these viruses can use more than one receptor, and raises the possibility that the pathogenic clade b arenaviruses may also use an additional receptor [40] . indeed, certain strains of the old world arenavirus lymphocytic choriomeningitis virus (lcmv) use both a-dystroglycan dependent and independent pathways to infect cells [55] . an alternative receptor may substitute for tfr1, or function downstream of tfr1 in a manner analogous to the hiv-1 coreceptors. identification of such a receptor on human cells will likely broaden our understanding of the entry mechanisms of a number of nw arenaviruses. clustalw was used to align the gp sequences of the nw arenaviruses and for phylogenetic calculations, and the cladogram was plotted using drawtree [56, 57] . hek293t cells (human embryonic kidney, atcc, crl-11268) were maintained in dulbecco's modified eagle's medium, and cho cells (chinese hamster ovary epithelial, atcc, ccl-61) in f12 medium. both cell lines were grown in the presence of 10% fetal bovine serum. plasmids encoding human, mouse, rat, c. callosus (large vesper mouse), and c. musculinus (drylands vesper mouse), z. brevicauda (short-tailed cane mouse) tfr1 are previously described [39] . the plasmids expressing the tfr1 orthologs of f. domesticus (cat) and c. familiaris (dog) were generously provided by colin parrish (cornell university, ithaca, ny). tfr1 genes for neacomys spinosus and artibeus jamaicensis were cloned from frozen heart and kidney, and liver tissues, respectively. deposited sequences in genbank (fj154604 and fj154605) cross-reference to catalog numbers of the museum of southwestern biology, university of new mexico from where these specimens were obtained. rna was isolated from these tissues using rnaqueous (ambion), and cdna was generated by superscript iii first-strand synthesis system for rt-pcr (invitrogen). pcr primers used to amplify the genes for both nstfr1 or ajtfr1 orthologs are: sense 59 atgatggatcaagccagatcagcawt-ctct 39 and anti-sense 59 aaaytcattgtcaatgtcc-caaaygtcacca 39. these genes, fused with a c-terminal flag tag, were cloned into the pcdna3.1 expression vector (invitrogen). amino acid substitutions in human tfr1 were performed using the quikchange method for site-directed mutagenesis (stratagene). amav gp1d-ig and tcrv gp1d-ig were generated by pcr-amplification of amav gp1 residues 80-228 and tcrv gp1 residues 87-243, with boundary residues selected according to a similar construct of macv gp1 previously shown to bind most efficiently to vero cells [32] . these fragments were cloned into a previously described pcdm8-based plasmid containing the cd5 signal sequence and the fc region of human igg1 [58] . macv (carvallo strain), junv (mc2), and gtov (inh-95551), gp-expressor plasmids have been described previously [32] . genes for amav and tcrv gp are gifts from stefan kunz (university of lausanne, switzerland); they were pcr amplified from infected vero cells, and cloned into pcaggs expression vector. the sequence of amav gp is the same as bean 7063 strain (genbank af512834), and that of tcrv has a deletion spanning amino acid residues 121-132, and substitutions at three residues, i134a, g418s and e458r, compared to genbank nc_004293. amav gp1d-ig and tcrv gp1d-ig binding to various tfr1 orthologs was detected by flow cytometry. ig-fusion proteins were produced in free style hek293 expression medium (invitrogen) from hek293t cells transfected with the appropriate plasmid, purified by binding to protein-a sepharose and eluted with 3 m mgcl 2 , dialyzed in pbs, and concentrated using a ym50 filter unit (millipore). for binding assays, hek293t cells were transfected with the various tfr1-encoding plasmids. after 48 hr, cells were detached by 1 mm edta/pbs and incubated in 100 ul of pbs-2% goat serum containing 0.5 ug of either an aflag m2 monoclonal antibody (sigma) or the indicated ig-fusion protein. cells were then stained with a-mouse igg, or with ahuman igg (fc-specific) antibodies conjugated with phycoerythrin (jackson immuno laboratories), respectively. to generate recombinant retroviruses pseudotyped with arenaviral gp molecules, hek293t cells were transfected at a 1:1:1 ratio with plasmids encoding the respective arenaviral gp, the mlv gag and pol genes, and the pqcxix retroviral vector (bd biosciences) expressing gfp, as previously described [32] . the structure of the human tfr1 dimer is shown, oriented with the cell membrane at the bottom. the apical, protease-like, and helical domains are indicated in green, red, and yellow, respectively, on one monomer. the other monomer is shown in cyan. in the right panel, the tfr1 apical domain is enlarged; a loop comprising residues 202-212, implicated as a site of interaction with the gps of nw clade b arenaviruses, is shown. the side chains of residues d204, k205, r208, v210, and y211 are colored yellow. the image was rendered using pymol [59] . (b) sequence alignment of residues 195 through 216 of human tfr1 with analogous sequences of the tfr1 orthologs of z. brevicauda (zbtfr1), a. jamaicensis (ajtfr1), and n. spinosus (nstfr1). variants of human tfr1 containing sequence from z. brevicauda (zh1 and zh2), a. jamaicensis (ah2 through ah5), and n. spinosus (nh2, nh4, nh5, nh7, and nh8) tfr1 were generated based on this sequence alignment. zb, aj, and nstfr1 sequences are shown in green, blue, and yellow, respectively. the right panel summarizes the entry data. nd = not determined. (c-e) hek293t cells were transfected with plasmids encoding human, zb, aj, or nstfr1 along with the zh variants (c), the ah variants (d), or the nh variants (e). the expression level of each tfr1 variant was assessed as in figure 3 . in parallel, cells were infected with amav, tcrv, or vsv pseudoviruses. the expression levels of the various tfr1 orthologs were normalized to that of human tfr1 (aflag, left panels), and infection levels were normalized to that of mock-transfected cells. doi:10.1371/journal.ppat.1000358.g006 biology and pathogenesis of lymphocytic choriomeningitis virus infection arenaviruses other than lassa virus chapare virus genetic identification of kodoko virus, a novel arenavirus of the african pigmy mouse (mus nannomys minutoides) in west africa the phylogeny of new world (tacaribe complex) arenaviruses molecular phylogeny of the arenaviruses phylogeny and evolution of old world arenaviruses new arenavirus isolated in brazil description of guanarito virus 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arenaviruses: implications for receptor usage and pathogenicity receptor use by pathogenic arenaviruses characterization of the cellular receptors for the south american hemorrhagic fever viruses junin, guanarito, and machupo crystal structure of the ectodomain of human transferrin receptor fatal illnesses associated with a new world arenavirus-california receptor use by the whitewater arroyo virus glycoprotein receptor and viral determinants of sars-coronavirus adaptation to human ace2 x-ray structures of h5 avian and h9 swine influenza virus hemagglutinins bound to avian and human receptor analogs glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities structure of sars coronavirus spike receptor-binding domain complexed with receptor bats: important reservoir hosts of emerging viruses bats as a continuing source of emerging infections in humans arenaviridae: the viruses and their replication epidemiology of machupo virus infection. ii. ecological and control studies of hemorrhagic fever differences in affinity of binding of lymphocytic choriomeningitis virus strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease kinetics phylip-phylogeny inference package (version 3.2) clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus the pymol molecular graphics system we thank joseph a. cook, museum of southwestern biology, division of genomic resources, university of new mexico, and robert j. baker, natural science research laboratory, the museum of texas tech university, for kindly providing tissues for artibeus jamaicensis and neacomys spinosus. we thank stephen c. harrison for manuscript editing and helpful discussion. cell supernatants were harvested 24 hr post-transfection, and filtered through a 0.45 mm filter. hek293t cells or cho cells were transfected with plasmids expressing various tfr1 orthologs, and at 24 hr, the cells were replated for flow cytometry and infection. at 48 hr post-transfection, tfr1 expression levels were assessed by flow cytometry using an a-flag m2 antibody. in parallel, cells were infected with the indicated pseudoviruses for 2 hours. twenty-four (hek293t cells) or forty-eight (cho cells) hours after infection, the cells were detached with trypsin and the gfp expression levels were measured by flow cytometry, and normalized to that of mock-transfected cells. values are the average of 2-5 duplicated experiments. infection of hek293 cells with replication-competent tcrv (trvl11573) or amav (bean70563), was performed in the bsl3 laboratory at the special pathogens branch, centers for disease control and prevention, atlanta. infections and immunofluorescence staining were performed as described previously [32] . briefly, hek293 cells growing on gelatin-treated glass coverslips were transfected with plasmids encoding ajtfr1, nstfr1, or human tfr1. thirty-six hours later, these cells were infected with tcrv or amav at a multiplicity of infection of 2.0. at 12 hr post-infection, the cells were washed, fixed with 3% formaldehyde, permeabilized with 0.1% triton x-100 and incubated with mouse hyperimmune sera specific for nw arenaviruses. cells were then washed and incubated with a fitc-conjugated secondary antibody. key: cord-254592-wa5il5go authors: brierley, liam; pedersen, amy b.; woolhouse, mark e. j. title: tissue tropism and transmission ecology predict virulence of human rna viruses date: 2019-11-26 journal: plos biol doi: 10.1371/journal.pbio.3000206 sha: doc_id: 254592 cord_uid: wa5il5go novel infectious diseases continue to emerge within human populations. predictive studies have begun to identify pathogen traits associated with emergence. however, emerging pathogens vary widely in virulence, a key determinant of their ultimate risk to public health. here, we use structured literature searches to review the virulence of each of the 214 known human-infective rna virus species. we then use a machine learning framework to determine whether viral virulence can be predicted by ecological traits, including human-to-human transmissibility, transmission routes, tissue tropisms, and host range. using severity of clinical disease as a measurement of virulence, we identified potential risk factors using predictive classification tree and random forest ensemble models. the random forest approach predicted literature-assigned disease severity of test data with mean accuracy of 89.4% compared to a null accuracy of 74.2%. in addition to viral taxonomy, the ability to cause systemic infection was the strongest predictor of severe disease. further notable predictors of severe disease included having neural and/or renal tropism, direct contact or respiratory transmission, and limited (0 < r(0) ≤ 1) human-to-human transmissibility. we present a novel, to our knowledge, comparative perspective on the virulence of all currently known human rna virus species. the risk factors identified may provide novel perspectives in understanding the evolution of virulence and elucidating molecular virulence mechanisms. these risk factors could also improve planning and preparedness in public health strategies as part of a predictive framework for novel human infections. the emergence of novel infectious diseases continues to represent a threat to global public health. emerging pathogens have been defined as those newly recognised infections of humans following zoonotic transmission or those increasing in incidence and/or geographic range [1] . high-profile examples of emerging pathogens include the discovery of the novel middle east respiratory syndrome (mers) coronavirus from cases of respiratory illness in 2012 [2] and the expansion of the range of zika virus across the south pacific and the americas [3] . the emergence of previously unseen viruses means that the set of known human viruses continually could not be assigned a disease severity rating and were excluded from all analyses (hepatitis delta virus, which is reliant on hepatitis b virus coinfection, and primate t-lymphotropic virus 3, which may be associated with chronic disease like other t-lymphotropic viruses but has not been known in humans long enough for cohort observations). disease severity differed between viral taxonomic families (fisher's exact, 1,000 simulations, p < 0.001), with arenaviridae, filoviridae, and hantaviridae having the highest fractions of severe-rated virus species (fig 1) . although 55 of 172 viruses considered zoonotic were rated 'severe', we note that only 3 of 40 nonzoonotic viruses were rated as causing severe disease (hepacivirus c and human immunodeficiency virus [hiv] 1 and 2). fatalities were reported in healthy adults for 64 viruses and in vulnerable individuals only for an additional 26 viruses, whilst eight viruses rated 'nonsevere' had severe strains, six of which belonged to the family picornaviridae. to find predictive risk factors for virulence, we first divided the 212 virus species into a single training (n = 181) and test set (n = 31) partition based on taxonomy and severity to minimise potential biases from trait imbalances between sets. using this training set, we then constructed a single classification tree that aimed to optimally classify viruses in virulence based on their ecological traits. the final pruned classification tree included variables relating to transmissibility, tissue tropism, and taxonomy (fig 2) . severe disease was predicted by the model for four generalised groups: i) viruses with a neural or systemic primary tropism with limited human-to-human transmissibility (excluding orthomyxoviruses, phenuiviruses, and reoviruses); ii) viruses known to have a renal tropism (primary or otherwise); iii) hantaviruses; and iv) retroviruses with sustained human-to-human transmissibility. although the illustrated classification tree identified several risk factors, this represents one of many possible trees because tree structure is dependent on the exact sampling partition between training and test data. we therefore constructed a random forest model containing 5,000 individual trees, each built using a bootstrapped sample of the training data and a randomly restricted subset of predictors, and repeated this approach over 200 alternative training/ test set partitions. averaging over these bootstrapped random forests, the most informative predictor variables for classifying virulence were taxonomic family and primary tissue tropism (fig 3) . however, primary transmission route, human-to-human transmissibility level, and having a known neural or renal tropism were also relatively informative, broadly mirroring the risk factors observed in the single tree. host range predictors were generally uninformative. to identify whether virulence risk factors might differ for non-human-adapted viruses, we repeated our machine learning analysis for only those viruses with known or suspected zoonotic transmission. for zoonotic viruses, the most informative predictors were similar (fig 3) , though transmission route variables (primary transmission route, having known vector-borne transmission) appeared to increase in relative importance. to quantify the effects of the most informative risk factors, averaged partial dependence was extracted from the random forests, describing the marginal predicted probabilities of severe virulence associated with each virus trait (fig 4, s2 table) . averaging across other predictors, viruses having tissue tropisms within neural or renal systems or systemic across multiple organ systems presented the highest risk of severe virulence, whilst respiratory and gastrointestinal tropisms presented the lowest risk. an increased probability of severe virulence was also observed for viruses transmitted by direct contact or respiratory routes and those with known but limited human-to-human transmissibility. when restricted to zoonotic viruses, patterns of partial dependence were mostly similar to those observed for all human viruses (fig 4) . although the single classification tree model predicted its training set well, it did not appear generalisable to novel data within its test set. the single tree correctly predicted virulence ratings from literature-based criteria for 24 of 31 viruses in its test set, giving a resulting accuracy of 77.4% (95% confidence interval [ci]: 58.9%-90.4%), no evident improvement on the null model assigning all viruses as nonsevere (null accuracy = 74.2%). the random forest approach gave better predictive performance, correctly predicting virulence with a mean accuracy of 89.4% across all training/test partitions (95% ci: 72.0%-97.0%), significantly greater than the null accuracy (one-tailed one-sample proportion test, p = 0.041). the random forest approach also achieved superior performance when considering sensitivity, specificity, true skill statistic, and the negative predictive value as a performance measure prioritising correct classification of 'severe'-rated viruses ( table 1 ). the random forests also outperformed the classification tree in area under the receiver operating characteristic curve (auroc) ( table 1 , fig 5) . nineteen of 139 viruses featured in test set partitions were misclassified from averaged random forest predictions (s1 table) : seven viruses rated as severe from literature protocols that were predicted to be nonsevere and 12 nonsevere viruses predicted to be severe. misclassifications from the random forest occurred most frequently within the flaviviruses and orthohantaviruses (s1 table) , though misclassifications did not appear to occur disproportionately between genera (fisher's exact, 1,000 simulations, p = 0.930). the observed predictor importance and risk factor directions were robust to constructing random forest models for subsets of viruses, removing those with low-certainty data or data variable importance from random forest models. importance of each variable in predicting virulence in random forest models applied to all known human rna viruses and zoonotic viruses only, calculated as the average decrease in gini impurity following a tree split based on that predictor and scaled against the most informative predictor within each random forest to give a relative measure. from serological evidence only (s1 and s2 figs), and similar performance diagnostics were obtained (s3 table) , though transmission route predictors appeared less informative when considering only viruses with at least 20 known cases. redefining our virulence measure to integrate information on known fatalities and differences with subspecies or strains in an partial dependence from random forest models in predicting severe virulence. predicted probability of classifying virulence as 'severe' for each of the most informative risk factors in random forest models applied to all known human rna viruses and zoonotic viruses only (primary tissue tropism, any known neural tropism, any known renal tropism, level of human-to-human transmissibility, primary transmission route, and any known vector-borne transmission). predicted probabilities are marginal, i.e., averaging over any effects of other predictors. boxes denote distribution of probabilities across 200 random forest models with alternative training/test partitions, with heavy lines denoting median probability. dashed line denotes raw prevalence of 'severe' virulence rating among the respective training datasets. colour key denotes predictor variable type as in fig 3, ordinal ranking system (s4 table) did not improve predictive performance (s5 table) . using alternative virulence measurements, the most informative variables and virus traits predicting severity showed good agreement with those of the main analysis (s3 and s4 figs). we present the first comparative analysis of virulence across all known human rna virus species to our knowledge. we find that disease severity is nonrandomly distributed across virus families and that beyond taxonomy, severe disease is predicted by risk factors of tissue tropism and, to a lesser extent, transmission route and level of human-to-human transmissibility. in both classification tree and random forest models, viruses were more likely to be predicted to cause severe disease if they caused systemic infections, had neural or renal tropism, transmitted via direct contact or respiratory routes, or had limited capability to transmit between humans (0 < r 0 � 1). these risk factors were robust to alternative modelling methods, alternative definitions of virulence, and exclusions of poor-quality data. primary tissue tropism was the most informative nontaxonomic risk factor (fig 3) and the first split criteria in the classification tree (fig 2) , with specific neural tropism and generalised systemic tropism predicting severe disease (fig 4) . few studies have directly predicted how tissue tropism should influence virulence. the identified risk factor tropisms could be explainable as a simple function of pathology occurring in sensitive or multiple tissues, respectively, increasing intensity of clinical disease. however, it has been suggested that an excessive, nonadapted virulence may result if infections occur within nontarget tissues that do not contribute to transmission [30] , although the evolutionary determinants of tissue tropism are not wellunderstood [31] . tissue tropism should be a key consideration for future comparative and evolutionary modelling efforts. we also found viruses primarily transmitted by direct contact and respiratory routes to have a higher predicted probability of severe virulence than viruses transmitted by vectorborne or faecal-oral routes. contrastingly, previous comparative analyses pooling several microparasite types, including a limited range of viruses, have shown positive associations between virulence and vector-borne transmission [17] or environmental survivability [18] . ewald [17] suggested virulence has fewer costs to pathogen fitness if transmission can occur independent of host health and mobility, e.g., through arthropod vectors or contaminated water, though we did not observe support for this hypothesis in our analysis. the relationship between virulence and transmissibility appears more complex. firstly, random forest models suggested a lower risk of severe virulence for viruses with sustained human-to-human transmissibility (level 4) than self-limited transmissibility (level 3) (fig 4) . this appears consistent with hypothesised virulence-transmissibility tradeoffs [21, 32, 33] and suggests that the adaptation necessary to develop efficient human-to-human transmissibility could result in attenuation of virulence in rna viruses. sustained transmissibility appeared to positively predict severe disease for a specific subset of four viruses in the single classification tree (fig 2) , all retroviruses causing chronic syndromes (hiv 1 and 2 and primate t-lymphotropic virus 1 and 2), which are likely subject to different evolutionary dynamics-if disease occurs after the infectious period, virulence brings fewer costs to pathogens from host mortality, essentially 'decoupling' from transmission [23] . we note only three nonchronic level 4 viruses rated severe: severe acute respiratory syndrome-related coronavirus, yellow fever virus, and zaire ebolavirus. although cross-species infections incapable of onward transmission (sometimes termed 'dead-end' infections) can result in high virulence because without coevolution, viral phenotypes within the novel host will be nonadapted-i.e., a 'coincidental' by-product [23, 24] -we did not observe viruses incapable of human-to-human transmission (level 2) to be comparatively more virulent. this may suggest that if virulence is entirely unselected in dead-end infections, phenotypic levels of virulence could just as easily turn out to be 'coincidentally' low. taxonomic family being a highly informative predictor in the random forests implies that there is a broad phylogenetic signal to virulence, but it is also highly likely that the explanatory power represents a proxy for many other phylogenetically conserved viral traits that are challenging to implement in comparative analyses of this scale, such as variation at the proteomic, transcriptomic, or genomic level or further data beyond simple categorisations, e.g., specific arthropod vector species. untangling these sources of variation from different scales of traits will be a critical next step in predictive modelling of viral virulence. we acknowledge several limitations to the quality of our data, as with any broad comparative analysis. risk factor data were problematic or missing for certain viruses, e.g., natural transmission route for viruses only known to infect humans by accidental occupational exposure and tissue tropism for viruses only known from serological evidence. however, the consistency of findings between alternative, stricter definitions of virulence and data subsets removing viruses with suspected data quality issues suggests scarcity of data does not bias our analyses. virulence also exhibits substantial variation at the subspecies level, i.e., between strains or variants. for example, severity of lassa virus disease superficially varies with infection route and geography, though this appears to be driven by variation between genotypes [34] . confirmatory analyses at a finer resolution would validate our identified risk factors, e.g., phylogenetic trait models of individual genera or species. furthermore, clinical symptoms are also subject to traits of the host individual, e.g., immunocompetence, age, and microbiome [35, 36] . our risk factor analysis brings a novel, to our knowledge, top-down perspective on virulence at the broadest level, though caution must be exerted in extrapolating the risk factors we find to dynamics of specific infections. the value of predictive modelling as an inexpensive and rapid tool for risk assessments during early emergence is increasingly recognised [16] . instances in which machine learning model predictions do not match outcomes could indicate likely candidates for outcome class changes, e.g., future reservoir hosts for zoonotic disease [37] , and we note severe virulence was predicted for 12 viruses rated 'nonsevere' from literature protocols (s1 table) . however, our models have restricted function in predicting the virulence of a newly identified virus, particularly if human infections are not yet recognised. taxonomy may be easily accessible and applicable to give simple virulence estimates. however, the most informative nontaxonomic predictors, tissue tropism and transmission route, are not likely to be identified with confidence before clinical observations of virulence. one way to address this information gap would be use of available data from animal infections, assuming that tissue tropism and transmission route do not differ between human and nonhuman hosts. alternatively, predictor data might be imputed from the nearest-related known virus, particularly for traits that appear highly phylogenetically conserved such as tissue tropism [31] . a more powerful future approach lies in the potential predictability of tissue tropism based on cell receptors and, more challengingly, of cell receptors based on viral proteomics or sequence data [38] , an increasingly accessible information source during early emergence following advances in genomic sequencing methods [39] . the exact links between tissue tropism, cell receptors, and nucleotide sequences are currently a critical knowledge gap and a potentially informative focus for future predictive efforts. a further key area requiring development is the possibility of inferring virulence directly from aspects of sequence data, e.g., genome composition biases, which have recently demonstrated the potential to predict reservoir host taxa and arthropod vectors via machine learning [40] . more widely, our analysis brings a novel, to our knowledge, focus that complements comparative models predicting other aspects of the emergence process such as zoonotic transmission [8, 9, 37, 41] , propagation within humans [10, 11] , or geographic hotspots [42, 43] . after continued calls for model-informed strategy, predictive studies are now beginning to shape surveillance and prevention with respect to emerging zoonoses [16, 44] , with virulence being been suggested as a factor to direct viral surveillance [45] , albeit in nonhuman hosts. the virulence risk factors we identify suggest that broadly targeting direct contact or respiratory transmission interfaces within ecological systems and/or tailoring detection assays towards certain virus families (e.g., hantaviridae) or tissues (e.g., neural tissue) could contribute to a viable strategy to detect future virulent zoonoses. this work adds to the comparative and predictive modelling efforts surrounding emerging infectious diseases. here, we contribute a novel, to our knowledge, focus on ecological predictors of virulence of human rna viruses, which can be combined in holistic frameworks with other models such as those predicting emergence dynamics. as a predictive model, the featured random forests offer valuable inference into the evolutionary determinants of virulence in newly emerging infections. we propose that future predictive studies and preparedness initiatives with respect to emerging diseases should carefully consider potential for human virulence. for each of the 214 recognised human-infective rna virus species, following standardised data compilation efforts and critical assessment protocols [5] , data on virulence and potential risk factors were collected via a systematic search and review of clinical and epidemiological literature. the following were consulted in turn: clinical virology textbooks [46] [47] [48] ; references from the data set described by [5] ; and literature searches using google scholar (search terms: searches 3 and 4 were carried out only when fatality or tropism data, respectively, were not already found from previous sources. data collection and virus name search terms included the full species name, any synonyms or subspecies (excluding vaccine strains), and the standard virus abbreviation as given by ictv online virus taxonomy [49] . although many possible measurements of virulence have been proposed [50, 51] , even simple metrics like cfr have not been calculated for the majority of human rna virus species. therefore, virulence was rated using a simple two-category measure of severity of typical disease in humans. we rated viruses as 'severe' if they firstly had �5% cfr when data were available (159/214 viruses, including those with zero cfr); otherwise, we rated viruses as 'severe' if they had frequent reports of hospitalisation, were associated with significant morbidity from certain conditions (haemorrhagic fever, seizures/coma, cirrhosis, aids, hantavirus pulmonary syndrome, htlv-associated myelopathy), or were explicitly described as 'severe' or 'causing severe disease' (s1 table) . we rated viruses as 'nonsevere' if none of these conditions were met. we note that this led to 'nonsevere' ratings for some viruses with clinically severe but rare syndromes; e.g., dengue virus can cause haemorrhagic dengue fever, though this is much rarer than typical acute dengue fever [46, 47] . to address this, data were also collected on whether the virus has caused fatalities in vulnerable individuals (defined as age 16 and below or 60 and above, immunosuppressed, having comorbidities, or otherwise cited as being 'at-risk' by sources for specific viruses) and in healthy adults and whether any 'nonsevere' virus has atypically severe strains (e.g., most infections with viruses within the species human enterovirus c cause mild disease; however, poliovirus, which causes severe paralytic disease, is also classified under this species). these were examined both individually and within a composite six-rank system (s4 table) . data were compiled for four main risk factors: transmission route(s) and tissue tropism(s), sourced from literature search exercises as described, and extent of human-to-human transmissibility and host range, sourced directly from [5] . although previous studies also predict virulence to vary with other traits, e.g., environmental survivability [18] , paucity of data or nestedness within taxonomic family prevented their inclusion in our analysis. firstly, primary transmission route was categorised as the dominant route the virus is transmitted by: vectorborne (excluding mechanical transmission), direct contact, faecal-oral, or respiratory transmission. primary tissue tropism was similarly categorised as the dominant organ system the virus typically infects or targets, specified as neural, gastrointestinal, hepatic, respiratory, circulatory, vascular, or 'systemic' (typical infection within multiple organ systems with no clear, single dominant tropism). however, many human viruses are known from isolation from blood or serum, with no further evidence of specific tissue tropisms (n = 69). therefore, we also included an additional 'viraemia' category in the primary tissue tropism predictor to indicate only blood presence was known. secondly, binary variables were also constructed, denoting whether viruses had ever been observed to utilise a) multiple transmission routes/tissue tropisms and b) each individual transmission route and tropism, including additional categories that were never among the primary routes/tropisms (food-borne and vertical transmission; renal, cardiac, joint, reproductive, sensory, skin, muscular, and endocrine tropism). we accepted isolation of the virus, viral proteins or genetic material, or diagnostic symptoms of the virus (such as characteristic histological damage) as evidence of infection within an organ system but did not accept generalised symptoms such as inflammation. human-to-human transmissibility was specified using infectivity/transmissibility levels, based on previous conceptual models and a systematic compilation and review of evidence [4, 5, 12] . level 2 denotes a virus capable of infecting humans but not transmitting between humans (r 0 = 0), level 3 denotes a virus with limited human-to-human transmissibility (0 < r 0 � 1), and level 4 denotes a virus with sustained human-to-human transmissibility (r 0 � 1). host range was specified as either 'narrow' (infection known only within humans or humans plus nonhuman primates) or 'broad' (infection known in mammals or animals beyond primates) [5] . binary variables were also sourced as to whether infection was known within a) humans only, b) nonhuman primates, c) other mammals, and d) birds. to identify potential differences in risk factors between adapted and nonadapted viruses, we also categorised whether each virus was zoonotic. we considered a virus to be zoonotic if it had transmissibility level 2 or 3 or had transmissibility level 4 and was known to infect nonhuman hosts (excluding anthroponotic viruses, e.g., measles morbillivirus). we also conservatively considered viruses to be zoonotic if zoonotic potential was suspected but data-deficient, e.g., rotavirus a-c. all virulence and risk factor data pertained to natural or unintentional artificially acquired human infection only, and data from intentional human infection, animal infection, and in vitro infection were not considered. viral taxonomy was included in analyses by specifying both genome type and taxonomic family as predictors. all virulence and risk factor data are available via figshare: 10.6084/m9.figshare.7406441.v3 (https://figshare.com/ articles/data_and_supporting_r_script_for_tissue_tropism_and_transmission_ecology_ predict_virulence_of_human_rna_viruses/7406441/3). firstly, the 212 retained virus species were split into a training set for model fitting and a test set for model evaluation. in order to avoid bias from an imbalance between types of viruses assigned to training and test sets, our selection was based on random sampling, stratified by genus-severity rating combinations. we sampled at a ratio of 75:25, i.e., for the four known severe viruses in the genus ebolavirus, three were randomly assigned to the training set and the remaining one assigned to the test set. if a genus-severity combination contained less than four viruses, all defaulted to the test set. comparative risk factor analyses were firstly carried out by constructing a classification tree using the r package 'rpart' v4.1-11 [52] . classification trees are a simple form of machine learning models that aim to optimally classify data points into their correct category of outcome variable based on a structure of binary predictor splits. tree-based methods are well-suited for comparative analyses in which confounding often results from taxonomic signal or suites of otherwise co-occurring traits because their high structure can intuitively fit complex nonlinear interactions and local effects. a tree model was fitted to the training set to predict virulence ratings by 'recursive partitioning', the repeated splitting of the data set using every possible binary permutation of each predictor, and retaining the split that minimises the gini impurity [53] , defined as 1 à p n i¼1 pðx i þ 2 for outcome variable x with n possible ratings and p(x i ) denoting proportion of data with rating i, which is equal to zero for perfectly separated data. to prevent overfitting, the tree was pruned back to the optimal branching size, taken as the most common consensus size over 1,000 repeats of 10-fold cross-validation. to validate the predictive power of the classification tree, predictions of virulence rating were generated when applied to the test set. tree accuracy was then calculated, comparing the proportion of correct predictions compared to literature-assigned ratings (assuming these to be 100% accurate as the 'gold standard' or 'ground truth'). because virulence ratings were imbalanced (i.e., only a minority of viruses cause severe disease, so correct nonsevere classifications are likely to be achieved by chance), accuracy was directly compared to the null model, i.e., a model with no predictors that predicted 'nonsevere' for all viruses. additional diagnostics of interest (sensitivity, specificity, negative predictive value, and true skill statistic [54] ) were also obtained. although classification trees have the advantage of presenting an interpretable schematic of risk factor effects and directions, individual tree structures may be sensitive to particular data points and have no intuitive measures of uncertainty. we therefore generated a further 200 partitions of our data into alternative training/test sets using the random stratified sampling procedure described. then, for each partition, we constructed a random forest, an ensemble collection of a large number of bootstrapped classification trees [55] . having many predictor variables compared to the relatively limited and fixed number of human-infective rna virus species, random forests handle such 'large p, small n' data architecture much more easily than traditional regression frameworks [56] . missing data in all predictors were imputed using the r package 'missforest' v1.4 [57] . using the r package 'randomforest' v4.6-12 [57] , random forests were created containing 5,000 individual trees, each built using a bootstrapped sample of training data and restricted to a randomly selected subset of predictors (k = 5) at each branching split. the predictive power of the random forest approach was evaluated by averaging over the test set predictions from all partitions. receiver operating characteristic curves were visualised and area under curves calculated to directly compare to the classification tree methodology. because of their high structuring, random forest models cannot give a simple parametric predictor effect size and direction (e.g., an odds ratio). instead, potential virulence risk factors were evaluated using two metrics: variable importance and partial dependence. variable importance is calculated as the mean decrease in gini impurity following tree splits on the predictor and can be considered as how informative the risk factor was towards correctly predicting virulence. partial dependence is calculated as the mean relative change in log-odds of predicting severe virulence, which were converted to predicted probabilities of severity associated with each risk factor. partial dependence describes marginal effects averaging across any influence of other predictors, and, as such, point estimates may not reflect any complex risk factor interactions. therefore, to test hypotheses regarding virulence risk factors, we present both averaged random forest partial dependence and the less robust but more accessible single classification tree for its ease of interpretation in risk factor structure and directly compare the statistical validity of both methods by plotting receiver operating characteristic curves. all modelling was carried out in r v3.4.3 [58] supporting information s1 table. virulence literature rating data for human rna virus training data set. virulence data for 212 human virus species ordered by genome type and taxonomy, including disease severity rating and supporting criteria for viruses rated 'severe', whether virus is known to have caused fatalities in vulnerable individuals and/or otherwise healthy adults, and whether virus is known to have 'severe' strains if species is rated 'nonsevere'. both disease severity rating/supporting criteria following the literature protocol given in the main text and mean predicted probability of severe disease from the random forest models are given. bold type denotes when predictions do not match literature-based ratings. dashes indicate predictions were not generated because fewer than four viruses were observed with this genus-severity combination and virus always defaulted to training set. aids, acquired immunodeficiency syndrome; cfr, case fatality ratio; hfrs, hantavirus haemorrhagic fever with renal syndrome; hps, hantavirus pulmonary syndrome; htlv, human t-lymphotropic virus. (pdf) s2 table. partial dependence from random forest models for all predictor variables. partial dependence given as mean marginal relative change in log-odds and mean predicted probability of classifying virulence as 'severe' for all predictor variables from random forest models featuring all viruses and models featuring zoonotic viruses only. (pdf) s3 table. diagnostics of random forest models using stringent data subsets. predictive performance metrics of random forest models applied to data subsets, excluding viruses with lowcertainty data (n denotes number of viruses excluded table. six-rank system of classifying virulence for human rna viruses. six-rank system of classifying human rna virus virulence with available data (specifically, severity rating from main text, fatalities in vulnerable individuals and healthy adults, and severe strains), along with example viruses and number of viruses fitting each exclusive rank's criteria. (pdf) s5 table. diagnostics of random forest models predicting alternative metrics of virulence. predictive performance metrics of random forest models predicting alternative virulence measures using different two-category definitions of 'severe' (n denotes number of viruses considered 'severe' using that definition). vulnerable individuals are defined as those age 16 and below, age 60 and above, immunosuppressed, having comorbidities, or otherwise cited as being 'at-risk'. ranks follow those given in table s5 fig. variable importance from random forest models using stringent data subsets. variable importance for virulence risk factors from random forest models applied to data sets, excluding a) viruses only known to infect humans from serological evidence (n = 36), b) viruses with <20 recognised human infections (n = 55), and c) viruses with poor data quality in at least one predictor (n = 71). variable importance is calculated as the relative mean decrease in gini impurity scaled against the most informative predictor within each model alongside importance from the main analysis for comparison. fig. partial dependence from random forest models using stringent data subsets. predicted probability of classifying virulence as 'severe' for each of the most informative risk factors from random forest models applied to data sets excluding a) viruses only known to infect humans from serological evidence (n = 36), b) viruses with <20 recognised human infections (n = 55), and c) viruses with poor data quality in at least one predictor (n = 71) alongside predicted probabilities from the main analysis for comparison. probabilities given are marginal, i.e., averaging over any effects of other predictors. because each data subset required resampling of the training and test partitions, note that raw prevalence of 'severe' virulence differed between each model (see s3 table) . boxes denote distribution of probabilities across 200 training/test partitions, with heavy lines denoting median probability. colour key denotes predictor variable type as in fig 3, i.e., blue = tissue tropism, green = transmissibility, red = transmission route. supporting data are available via figshare: 10 predicted probability of classifying virulence as 'severe' in alternative virulence measures for each of the most informative risk factors from random forest models alongside predicted probabilities from the main analysis for comparison. probabilities given are marginal, i.e., averaging over any effects of other predictors. because each measurement used a different two-category definition of 'severe', note that the raw prevalence of 'severe' virulence differed between each model (see s5 table) . boxes denote distribution of probabilities across 200 training/test partitions, with heavy lines denoting median probability. colour key denotes predictor variable type as in fig 3, factors in the emergence of infectious diseases isolation of a novel coronavirus from a man with 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fields virology principles and practice of clinical virology clinical virology the classification and nomenclature of viruses. the online (10th) report of the ictv viral virulence. in: nathanson n on the evolution of virulence and the relationship between various measures of mortality rpart: recursive partitioning and regression trees classification and regression trees: a powerful yet simple technique for ecological data analysis assessing the accuracy of species distribution models: prevalence, kappa and the true skill statistic (tss) random forests random forests: some methodological insights missforest-non-parametric missing value imputation for mixed-type data r: a language and environment for statistical computing. r foundation for statistical computing we thank jarrod hadfield, samantha lycett, and daniel streicker for helpful discussion and key: cord-027885-ua8miwes authors: das, sujata; khanna, charu; singh, shalini; nandi, shilpa; verma, reema title: impact of human microbiome on health date: 2020-03-10 journal: microbial diversity, interventions and scope doi: 10.1007/978-981-15-4099-8_20 sha: doc_id: 27885 cord_uid: ua8miwes the human genome in the recent years, by the advent of technological advancements, has emerged as a major prolocutor for reciprocity between the human body and the food consumed. as known, microbiome comprises all the genetic materials within a microbiota and can thereby be also referred to as metagenome of the microbiota. contemporary researches have revealed the influence of microbiome not only on human mind and health status, but also in wide range of disease switching, ranging from cardio-metabolic diseases, allergies and obesities to life-threatening diseases such as cancer. though the complete mechanism of many diseases is yet unclear, research works have revealed that the metabolites, nutrients and microbes can be regarded as the key players for such physiological state. the major approach of this chapter is to enlighten the interrelationship of the microbiome on the human health either in a synergistic or in an antagonistic manner. in the last few decades, immense initiatives have been taken in understanding the role of microbiome on human health. in the present day, either in the field of therapeutic development or medical treatment, the impact of microbiome is especially apparent in the studies of different microbial communities and the human microbiome. in the recent times, 'omics' has provided a significant impetus on all the facets of biological research which has ushered the field to gain in momentum. the study, which is contemporarily popularised as study of 'human microbiome', is an outcome of the advancement in the field of genomics and other fields of microbiology, which has given the classical microbiology a new outlook and perspective. thereby, the attention was driven and directed towards the genomics, which was the major objective of the human microbiome project (hmp) (turnbough and wilson 2007) . the hmp was used for the characterisation of all the microbial communities living in the human body, eventually switching the interest towards, not only, the type of microbes existing in the human body but also to the role and activity they perform both in the case of a healthy and diseased individual. since 2003, after the publication of the first human genome (chial 2008) , the biomedical research on microbiome has obtained a significant scientific attention. there has been an immense leap from the culture-based surveys of various tissues or organs, for example, of gut and oral cavity, to molecular profiling of the microbial communities and their biochemical products like enzymes, proteins, and amino acids in all the different ecological niches of the human body (eckburg et al. 2005; gill et al. 2006; costello et al. 2009 ) for this subject. this chapter attempts to put an insight into the distribution and diversification of human microbiome, the behaviour of human microbiome on the human health and microbiome as a paradigm for the future nutritional and medicinal strategies for human benefits. over 100 trillion microbes are estimated to be residing both inside and over the surface of humans, possessing genome which is approximately 150 times to that of the entire human being ). this includes microbes of different families such as bacteria, fungi, archaea and viruses, contributing about eight million unique protein-coding genes as compared to the human genome, which comprises only around 22,000 protein-coding genes (tomayko et al. 2013) . this variation and the nature of the organism, specific to the specific anatomic site of the body, is a consequence of their specific growth requirement. the other determinants for such growth include coevolution of microorganism, their extensive interaction amongst each other and with the human host, variation in the composition and function with respect to the population, human life span and inconsistency of the body sites, ecologic condition, difference in the oxygen tension, airway luminal temperature, mechanism of muco-ciliary clearance, sex, genetics and socio-economic status. hence, there has been a development of the concept of interdependence, in variety of physiologic, immunologic and metabolic processes, which ultimately determines the microbiome community in a particular site of the human body system. amongst all the human systemic microbiome, the gut microbiome, composed of the genetic material of the microorganisms in the gut, occupies a very essential and special position. they play an important role in various physiological processes like metabolism, immunity development and nourishment supply. the genotype and the immune system of the host have been shown to contribute towards the development of gut microbiota (thaiss et al. 2016) . in response to environmental factors, such as diet, pathogens and xenobiotic substances, a crosstalk occurs between the human immune system and the microbiome. for instance, the myeloid cells, epithelial layer and the innate lymphoid cells, part of the immune system, crosstalks with the gut microbiota for which the microbiome composition, host physiology and disease susceptibility are the main consequences of such crosstalks and feedback loops between them. along with the bacterial community, like firmicutes and bacteroidetes species (table 20 .1), these interactions are also contributed by the other microbiota like fungi (pothoulakis 2009 ), archaea and viruses (breitbart et al. 2003) . though the understanding of the immunological relationship between the fungi and archaea is limited currently, the trans-kingdom commensalism is expected to be formed from infancy (latuga et al. 2011) . the principal constituents are the bacteriome, virome and mycobiome, whose strong interdependence maintains the functionality of the gut microbiota, if imbalanced may also affect the other systems in various ways. since the time of birth of an individual, when the sterile gut of the neonate gets exposed to the biota of mother's vagina during the vaginal delivery or hospital microbiota in case of caesarean section (which may even include the multidrug-resistant species), the microbes starts their colonisation with an eventual change by the age of 3-5 years, by when an individual starts resembling bacterial community to that of an adult both structurally and functionally (bull and plummer 2014) . in adults, the composition of gut microbiota is uneven throughout the length of the gut. as compared to small intestine, which is rich in the species related to phylum firmicutes, colon on the contrary exhibits the presence of members of phylum bacteroidetes. the microbiome of lumen and that attached to the epithelial lining even show differences. the stool sample exhibited the presence of bacteroides, streptococcus, ruminococcus, lactobacillus, enterococcus, bifidobacterium and clostridium which presented the lumen community while on the mucous layer detected the presence of enterococcus, lactobacillus and clostridium (swidsinski et al. 2005) . during the initiation of hmp, the airways and lungs were exempted from the study, believing these parts to be sterile in nature (moffatt and cookson 2017) . this fact was always acceptable because of the negative results yielded by the various standard microbiological culture tests of the healthy individuals (faner et al. 2017) . its study was also a challenge owing to the difficulty in assessing the lower tract without the invasive techniques such as bronchoscopy. hence, there has been a delay in the systemic microbiome assay until the first study indicating the similarity of bacterial density of this part with the upper small bowel of human body was reported (man et al. 2017 ). this has been made possible due to the advances in molecular techniques independent of culture practices (faner et al. 2017 ). human respiratory system is divided into upper respiratory tract (urt) and lower respiratory tract (lrt) with alveoli, present in the lrt, acquiring the surface area nearly 70 m 2 (man et al. 2017) . this complete tract is occupied by the niche-specific microbiota with higher density dwelling in urt. the development of microbiota has been thought to effect on the morphological genesis of this system (man et al. 2017) . during the first hours of a healthy neonate, non-specific microbes, presumed of maternal origin has been detected. abundance of staphylococcus spp. in the first week, in the urt due to niche specification, has also been detected, which is then occupied by the corynebacterium spp., dominated by the dolosigranulum spp. the moraxella species has its dominance at the age of 4-6 months. the individuals with the possession of such microbiota have been found to possess stable microbiome community along with better airway health (morris et al. 2013; segal et al. 2013 ) this healthy development is prone to disturbance under certain conditions such as usage of antibiotics, oxygen tension, temperature and ph, presence of other siblings, seasonal variations, vaccinations, exposure to smoke and host genetics (man et al. 2017) . attempt to observe the diversity of flora in urt and lrt is well observed (table 20 .1). studies using species specific molecular techniques have declared that the diseasefree arteries and veins are microbe free in nature (jin et al. 2019; sobol 2014 ). very few studies do report the presence of bacterial and viral genome in some vessels of healthy subjects. amongst the microbes, helicobacter pylori and herpes simplex virus are the most prevalent followed by cytomegalovirus, chlamydia pneumoniae, mycoplasma pneumoniae and epstein barr virus and were also detected in healthy aorta, saphenous veins and internal mammary arteries (clifford and hoffman 2015) . studies using species-specific molecular techniques have declared that the diseasefree arteries and veins are microbe free in nature (jin et al. 2019; sobol 2014 ). very few studies do report the presence of bacterial and viral genome in some vessels of healthy subjects. amongst the microbes, helicobacter pylori and herpes simplex virus are the most prevalent followed by cytomegalovirus, chlamydia pneumoniae, mycoplasma pneumoniae and epstein barr virus and were also detected in healthy aorta, saphenous veins and internal mammary arteries (clifford and hoffman 2015) . the human urinary tract along with urine was earlier considered to be 'sterile', until the modern-day research, which has confirmed the presence of microbes in this system, significantly in healthy individuals. the advances in techniques such as 16 s rrna sequencing have considerably helped in revealing the normal microbiota of the human body system. after analysis of the urine, the most common genera reported are lactobacillus (more in women) and streptococcus (more in men), both of which, along with few other groups, deliver a protective role in this system against different pathogens. various bacterial taxa, as illustrated (table 20 .1), had been recognised in healthy adults, but some specific genera such as saccharofermentans, proteiniphilum, parvimonas and jonquetella were found in persons with age more than 70 years. the variation of urinary microbiome related to age and sex may be due to differences in voiding habits, hygiene, urinary metabolites, anatomic structures, hormonal variation and histology. even the vaginal microbiome at premenstrual phase, reproductive age and post-menopausal phase exhibits variation (aragón et al. 2018 ). central nervous system is considered to be one of the most immune privileged systems because of its closed compartmentalisation. it is isolated by physical barriers like blood-brain barriers and blood-cerebrospinal fluid barriers, from the circulatory system (obermeier et al. 2013; ransohoff and engelhardt 2012) . thus, lack of lymphatic drainage, expression of major histocompatibility complex by the parenchymal cells and anti-inflammatory environment of the central nervous system accounts for such privileged status of seclusion from the microbiota (berer and krishnamoorthy 2014) . though, in our body, there exists a bidirectional communication system, involving hormonal, immunological and neural signalling pathways, between the brain and the gut, accessed by the microbial flora of the intestine and certain metabolites, also known as the gut-brain axis. it is estimated that nearly 90% of serotonin (5-ht), a neurotransmitter, is produced in the intestine under the influence of gut microbiota, and the activation of its receptors in the enteric nervous system is responsible for the neuroprotection and adult neurogenesis in the mouse model (de vadder et al. 2018 ). the largest organ forming the external interface of the human body to the environment is the skin. nearly 1.5-2.0 m 2 of the skin covers an average human with 2-3 mm depth. three tissue layers are found in the skin: epidermis, dermis and hypodermis. epidermis is colonised by millions of the microbes such as bacteria, fungi, arthropods and even viruses. it acts as the physical, anatomical and immunological barrier to various pathogens extending protection of the body, unless this barrier is broken or there is an imbalance between commensal and pathogenic organisms resulting to cutaneous or systemic diseases. their acidic ph, continuous shedding of epidermal cells, hydrophobic nature, salinity and association with the antimicrobial compounds make them an efficient barrier (ross et al. 2017) . though microbes do exist on them in spite of the above characteristics, the number of microbes inhabiting the skin ranges from one million to about one billion in each cm 2 . although, human skin is inhabited by quite diverse microflora, but most commonly found bacterial phyla includes proteobacteria, corynebacteria, propionibacteria, bacteroidetes, firmicutes and staphylococcus spp. amongst fungi, malassezia spp., cryptococcus spp., epiciccum spp., aspergillus spp. and rhodotorula spp. are most commonly found. the factors that affect the prevalence and dominance of community on the skin include biological sex, skin depth, skin location (skin thickness, folds, density of hairs), age, health, geographical location, ethnicity, use of lotions, soaps, cosmetics, and antibiotics and hygiene practices. as humans are known to have a constant symbiosis with microorganisms, the human microbial community, inhabiting the various system exhibits their influence on them. there are nearly 100 trillion symbiotic microorganisms that exist on and within the human body and have shown to play very important roles both in human health and disease causation. the influence of this microbiome on various systems is as follows: amongst all the systems, the highest and heterogeneous microbial density resides in colon and is mostly codependent in nature and present along both longitudinal (proximal to distal) and axial (mucosal to lumen) gradients of the gastrointestinal (gi) tract. the microbiome has been reported to aid in food digestion, vitamin biosynthesis, bile acid biotransformation, building of innate immunity, maintenance of intestinal barrier (valdes et al. 2018) , etc. thus, gut is an "essential organ", carrying approximately 150 times more genes than are found in the entire human genome ) (fig. 20.1 ). microbial inhabitants within the host often contribute to metabolism such as: • bile salt metabolism • synthesis of essential and non-essential amino acids • replication of virulence factors in enteric pathogen • pro-drug transformation into active drugs and • metabolism of xenobiotic compounds • antibiotics by chemical transformation (sarkar et al. 2018) gut microbiota and host interaction result in the secretion of a series of metabolites including trimethylamine-n-oxide (tmao); short-chain fatty acid (scfa) such as acetate and butyrate; secondary bile acid; and indoxyl sulphates that activate numerous signalling pathways affecting host physiological processes (jin et al. 2019 ). when a child is born, the immune system at birth is under a relative state of immaturity. the developing immune system is characterised by a skewed t-and b-cell development and a blunted inflammatory cytokine production with respect to the regulatory responses. the consequence of such an underdeveloped immune system is high susceptibility to infections. thereby, the regulatory environment ensures the establishment of the microbiota which ultimately helps in immune regulation and limits the mucosal inflammation following their colonisation (elahi et al. 2013) . many intestinal bacteria also prevent the colonisation of pathogen by producing antimicrobial substances such as bacteriocin (inhibit pathogen growth), by competing for nutrition and attachment sites. this act is termed as barrier/competitive(collado et al. 2010 ). exposure to intestinal bacteria has also been found to prevent certain allergic responses in the hosts. thereby, the commensals, specifically the bacterial species and the products or metabolites derived from them, are considered as an intrinsic regulator of all the immune responses for the upliftment and restoration of human meta-organism's health (belkaid and hand 2014) . gut bacteria are intrinsically linked with the health of our entire body. however, a change in gut microbiota composition, termed as dysbiosis, can result in enhanced susceptibility of the host towards pathology. dysbiosis can cause various diseases such as inflammatory bowel disease (ibd) and irritable bowel syndrome (ibs), chronic kidney disease (ckd), cardiovascular disease (cvd), atherosclerosis, obesity, autism, allergy, asthma, hypertension, coronary artery disease, and heart failure (tang and hazen 2017; backhed, et al., 2012) . (ibd) ibs and ibd are related to bowel disorder with signs and symptoms of abdominal cramping, pain and bloating associated with features of disordered defecation in ibs and ongoing inflammation of all part of gi tract in ibd. although ibs is a multifactorial disease (soares 2014) , intestinal dysbiosis has also been found associated with this disease by facilitating adhesion of enteric pathogens. ibd includes crohn's disease (cd) and ulcerative colitis (uc). gut microbiota has been implicated to have roles in ibd pathogenesis, and the usage of antibiotics may help in reducing inflammation or prevented the same in murine models of disease and in patients. intestinal dysbiosis is most often accompanied by defective intestinal barrier function, which promotes the production of bacterial by-products that are rapidly absorbed and retains in intestinal lumen. when increased absorption is coupled with reduced clearance of these substances by the kidneys, levels of gut derived toxins rise in circulation and may potentiate vascular calcification, atherosclerosis and adverse cardiovascular functioning, which are the clinical conditions in the later stage of ckd. numerous epidemiologic studies have shown a relationship between gut-derived vascular toxins and cardiovascular events in patient with ckd (valdes et al. 2018 ). metabolic origin of traditional cvd risk factors such as obesity, dyslipidaemia and insulin resistance indicates close linkage between the gut microbiome and cvd. recent studies have implicated that atherosclerosis has generated the largest amount of data in association with gut microbiome. according to a hypothesis, gut microbiota metabolites elicit inflammatory cascade by translocating into bloodstream and promote atherosclerosis. oral bacteria causing oral cavities have also been found in atherosclerosis plaque (clifford and hoffman 2015) . in many cvds, heart failure is considered as end stage with a higher rate of morbidity and mortality (jin et al. 2019) . low intestinal perfusion and disruption of intestinal barrier have been reported as reasons for reduced cardiac output and blood redistribution. the translocation of microbiota and endotoxins into blood circulation leads to an aggravated systemic inflammation that further can lead to increased chances of heart failure (peng et al. 2018) . various studies suggested the direct and indirect link between gut microbiota and the development of hypertension (jin et al. 2019) . it is a complex clinical condition and can be influenced by number of factors. it is considered as modifiable risk factor for cvd. in some studies, prevotella, faecalibacterium, klebsiella, clostridium and streptococcus are found in abundance in hypertensive patients, indicating a close association of host microflora to such a clinical condition. other studies demonstrated that reduction in gut microbial diversity, short-chain fatty acid (scfa)-producing bacteria and increase in sympathetic drive to the gut and lactic acid producing bacteria, respectively, have direct role in blood pressure regulation. thus, it can be concluded that blood pressure is closely linked to diversity, richness and evenness of microbiome living in the gut and the improved gut microbiota may be a target for future therapies for hypertension (peng et al. 2018 ). the gut-brain axis plays a major role in central nervous system (cns) and intestinal and immune system functioning, as mentioned earlier. gut-brain axis is the bidirectional biochemical signalling between gi and cns activities, integrating efferent and afferent neural, endocrine, nutrient and immunological signals, providing gut microbiota and its derived metabolites a route to access the brain (bull and plummer 2014; joscelyn and kasper 2014). wang and kasper illustrated that this bidirectional communication system enables brain to command gi functions such as metabolism, peristalsis, mucin production and other immunological functions. gut microbiota is also found to have impact on hypothalamic-pituitary-adrenal (hpa) axis, thus playing a role in body's stress response teitelbaum et al. 2008 ). the gut microbiota has also been shown to synthesise neurotransmitters and neuromodulators. various neurotransmitters produced by bacterial species are presented in table 20 .2. those released neurotransmitter then stimulates epithelial cells to synthesise modulators within the enteric nervous system (ens) or directly acting on primary afferent axons (mcvey neufeld et al. 2013) . moreover, it has been shown that ens plays a major role in fundamental gastrointestinal physiological functions such as motility, fluid secretions and blood flow. furthermore, numerous studies have revealed the correlation between microbiome, microbiota derived products, antibiotics, prebiotics and probiotics and cns (wang and kasper 2014) . anxiety and stress, characteristic mood disorders, associated with nervous, endocrinal and immunological system have also been shown to have an association with the gut microbiota. stressors such as chemical, biological or any environmental stimuli can act as an active component to trigger the anxiety and stress response which ultimately activates the hypothalamic pituitary adrenal (hpa) axis. intestinal dysbiosis and gut pathogens have thereby been shown to cause stress and anxiety. animal models have represented to ameliorate these disorders by using probiotic formulations (messaoudi et al. 2011) . various output and input of role of gut microbiota is depicted in fig. 20 .2. the analysis of microbiome of human body shows both the pathogenic as well as beneficial microbial network (ozturk et al. 2017 ). customizable medicine based on an individual's microbiome is an excellent approach for therapeutic choices based either on exercises or/and medications which considers the genetic makeup of an individual, their health condition and quality of life (hasani-ranjbar and larijani 2017). human genomes are about 99.9% indistinguishable to one another; the generally steady human gene pool does not completely clarify all the phenotypic varieties amongst people. on the other hand, the bacterial biological system, living in each human body, that contributes multiple times larger number of qualities than the human genome, could be significantly unique in relation to an individual. therefore, the human microbiome can be regarded as the true cause of numerous responses that can influence the structure and plenitude of microorganism (hasani-ranjbar and larijani 2017) in the human body. recently various studies have shown that intestinal microbiota is fundamentally associated with various therapeutics as in cardiovascular diseases, cancer, etc. these include narrow spectrum antibiotics along with probiotics, prebiotics and synbiotics, faecal microbiota transplantation, nutritional modulators, immune modulators and phage therapy. in the usa and europe, jointly, in the year 2015, because of the onset of antibiotic resistance amongst pathogens, around 50,000 deaths were witnessed, which was projected to increase to a score of around ten million deaths per year worldwide by 2050 (langdon et al. 2016 ). in addition to the development of resistance, the usage of antibiotics has been reported to disrupt the ecology of the human microbiome. whereby, a dysbiotic microbiome loses the capability to perform vital functions such as nutrient supply, production of vitamin, and defence against the pathogens; thereby it leads to an eventual impairment of the metabolic, immunological and developmental system of the host. for an instance, drug-induced modification of the gut microflora can influence a group of foxp3 + treg cells that control demyelination in exploratory autoimmune encephalomyelitis (eae) (ochoa-repáraz et al. 2010) . another study reflects the antigens produced by bacteroides fragilis (capsule polysaccharide) can protect against eae (cns demyelination) and further in human multiple sclerosis (ochoa-repáraz et al. 2011) . similarly, the effects of common antimicrobial treatments on the gut microbiota have been illustrated in table 20 .3. probiotics are characterised as live microorganisms which are not part of the human host microbiome yet give a medical advantage to the host when administered or directed in sufficient quantity. probiotics have been extensively studied in recent years as it imparts various health benefits by the metabolites it produces in the relief from certain intestinal disorders as well as controlling eae. clinical trials have been able to demonstrate that certain cardio metabolic disorders (cmd), such as type 2 diabetes mellitus (t2d), dyslipidaemia and arterial hypertension, as well as chronic kidney diseases (ckd) can be managed by the ingestion of probiotics (neto et al. 2018) . saccharomyces boulardii has been shown to exert antiinflammatory effect which helps to control inflammation related to the dysbiosis in lumen (rodríguez-nogales et al. 2018) . various metabolites produced by microorganisms present in the host and its associated health benefits are discussed in table 20 .4. prebiotics are those food components that cannot be digested by human body but can be selectively digested by the members of probiotics, and thereby serves as a food fibre for probiotics. recent studies have demonstrated that the use of prebiotics can result in enhancing the ecological performance of the gut microbiota, thus promoting a much more beneficial community (vandeputte et al. 2017) . this conceptualizes that the human microbiota can be enhanced, stabilised and shifted by feeding with certain specific prebiotics such as carbohydrates. however, characterisation of the relationship between the prebiotic and probiotic is still a challenge. the point when the idea of synbiotic was first presented, two setups were proposed: first, where the prebiotic and probiotic segments were independent, each being in charge of a kamal et al. (2019) specific impact or medical advantage, and second, a symbiotic segment, where the probiotic was explicitly structured with a prebiotic substrate that would synergistically support the intensity, survival or metabolic movement of a related probiotic strain in the ecology of gastrointestinal system (krumbeck et al. 2016 ). as of late, two novel methodologies have been proposed for creating such synergistic synbiotics, both being dependent on their ecological function and wellness. first, the in vivo strategy which depends upon the determination and isolation of probiotic strain that would increase in number when a certain population of participants are administered with the specific prebiotic component (krumbeck et al. 2015) . second, called as a multi-taxon insertion, depending on sequencing, based on the identification of genes, would examine the fitness of probiotic strain in relation to the type of prebiotic administered by the usage of libraries of transposon mutants . a list of prebiotics with their probiotic components are given in table 20 .5. sometimes the mere usage of antibiotics is insufficient to treat few diseases and rather requires urgent alternatives to manage the severity of the clinical condition. this has led to the introduction of transplantation of the faecal microbiota. the process involves the separation and delivery of the faecal microbiota from stool of healthy donor to the gastrointestinal tract of the receiver patient, thereby enabling an efficient cure by normalising the microbiota composition. recent investigations have explained the mechanism behind the faecal microbiota transplantation (fmt), which has been used to treat clostridium difficile infection (cdi) for the restoration of the gut microbiome to gain the ability to inhibit clostridium difficile indirectly by competing for nutrients. the faecal microbiota also prevents the colonisation by unwanted microorganism by the activation of immune system as well as by direct release of certain antimicrobial components and other metabolites that helps in the inhibition of vegetative and as well as the sporulated disease-causing organism (khoruts and sadowsky 2016) . table 20 .6 shows the list of diseases and infections which can be treated by using fmt. intake of dietary medications for a long period of time impacts the structure and function of microbiome of the human body. the change in the availability of nutritional content of the diet causes corresponding alteration of the human microbiome. recent findings have suggested that depending upon the intake of dietary, the microbiome richness diversifies. the lower is the genetic richness of microbiome; the lower will be the immune status of the person, relating to abnormal metabolic function and poor anti-inflammatory activity (cotillard et al. 2013; le chatelier et al. 2013 ). the diet patterns may even contribute in managing different diseases (table 20 .7) by way of microbiome. microbiome-based approaches involving antibiotics, probiotics, prebiotics and synbiotics, faecal microbiota transplantation and nutritional modulators correlate directly with the alteration of immune status of an individual focusing on the innate immunity. thereby, the human microbiome is being affected for benefits with the change in immune status. until now, there are inadequate information in this method; however, treatment of inflammatory diseases using steroids is in abundance. intestinal innate and acquired immunity as well as systemic acquired immunity involves various mechanisms for the control of gut microbiome. some of them include change in barrier function, expression of leptin, molecule β, human leukocyte antigen (hla) class i and class ii loci, activation of toll-like receptors, natural killer cells, cd4+ cells and foxp3+ as well as the production of antimicrobial competition for nutrients; direct suppression by antimicrobial peptides; bile-acid-mediated inhibition of spore germination and vegetative growth; activation of immune-mediated colonisation resistance khoruts and sadowsky (2016) ulcerative colitis (uc) increasing the production of short-chain fatty acids, (butyrate); inhibitingth1 differentiation, activity of t cells, leukocyte adhesion; production of inflammatory factors shen et al. (2018) irritable bowel syndrome (ibs) visceral hypersensitivity; altered barrier function, gastrointestinal motility and the gut-brain axis grover et al. (2014) obesity decrease adiposity; alter metabolic phenotype by increased bacterial diversity marotz and zarrinpar (2016) obesity henning et al. (2018) peptides and α-defensins (ticinesi et al. 2019) for the proper functioning of the gut microbiome. phage therapy involves the introduction of explicit bacteriophages that targets a microorganism which in turn has the ability to generate a beneficial microbiome shift. however, a limitation to this strategy is the simultaneous resistance offered by the microorganism in play which is yet to be proven. till date, none of the phage therapy approaches have been established as an fda-approved drug. recently, scientists are working hard to perform phage based killing of microbes, but it is much more complicated. crispr (clustered regularly interspaced short palindromic repeats) is one of the approaches to advance the limitations (lemieux 2019). it is a tool derived from prokaryotic immune system empowered to study and modify organisms with ease and efficiency. the system helps to modify the gut genome of gut microorganisms and bacteriophages. this engineered crispr-cas system ultimately can control gene expression and modulate production of metabolite and protein presenting a new approach for the development of drugs that can target the microbiome (table 20 .8). although these interventions are used as medicinal and nutritional strategies and have been clinically experimented for a successful result, more focus and deliberate attempts are being made for the establishment of such casualty in order to define the functional metabolic change. these therapies are yet not so widely utilised due to the requirement of huge amount of money and time. hence, advancement in this field targeting the relation of human microbiome with health and diseases, identification of the composition and activity of microbiome as well as linking it, and most importantly using technical resources such as bioinformatics to incorporate and fill up the loopholes ascertaining to the models is quite difficult. future research based on these directions is a key to solve the problems and hence 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(2018) key: cord-293938-40zyv1h8 authors: jonsdottir, hulda r.; dijkman, ronald title: coronaviruses and the human airway: a universal system for virus-host interaction studies date: 2016-02-06 journal: virol j doi: 10.1186/s12985-016-0479-5 sha: doc_id: 293938 cord_uid: 40zyv1h8 human coronaviruses (hcovs) are large rna viruses that infect the human respiratory tract. the emergence of both severe acute respiratory syndrome and middle east respiratory syndrome covs as well as the yearly circulation of four common covs highlights the importance of elucidating the different mechanisms employed by these viruses to evade the host immune response, determine their tropism and identify antiviral compounds. various animal models have been established to investigate hcov infection, including mice and non-human primates. to establish a link between the research conducted in animal models and humans, an organotypic human airway culture system, that recapitulates the human airway epithelium, has been developed. currently, different cell culture systems are available to recapitulate the human airways, including the air-liquid interface (ali) human airway epithelium (hae) model. tracheobronchial hae cultures recapitulate the primary entry point of human respiratory viruses while the alveolar model allows for elucidation of mechanisms involved in viral infection and pathogenesis in the alveoli. these organotypic human airway cultures represent a universal platform to study respiratory virus-host interaction by offering more detailed insights compared to cell lines. additionally, the epidemic potential of this virus family highlights the need for both vaccines and antivirals. no commercial vaccine is available but various effective antivirals have been identified, some with potential for human treatment. these morphological airway cultures are also well suited for the identification of antivirals, evaluation of compound toxicity and viral inhibition. respiratory diseases caused by human coronavirus infection are of both medical and socio-economic importance. currently, they are studied in various model systems, ranging from cell lines to animal models. originally, the importance of hcovs in the burden of human disease was underestimated and as a result, no general therapy exists to treat coronavirus induced disease in humans. furthermore, no commercial vaccine is available leaving the human population vulnerable to emerging coronavirus infections. both the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses have recently crossed the species barrier and entered the human population to cause severe disease. in this review, we summarize the current knowledge on human coronavirus infection emphasizing the usefulness of organotypic human airway cultures as a model system. coronaviruses (covs), a subfamily of the coronaviridae family, are positive strand rna viruses with the largest genome of all known rna viruses (≥27 kb). the genomic rna is capped, polyadenylated and associated with nucleocapsid proteins within an enveloped virion. the envelope is covered by the characteristic surface glycoprotein that gives the virus particles their characteristic crown-like (latin: corona) appearance [1] . all covs share a common genome organization where the replicase gene encompasses the 5′-two thirds of the genome and is comprised of two overlapping open reading frames (orfs), orf1a and orf1b that encode for up to 16 non-structural proteins. the structural gene region, which covers the 3′-third of the genome, encodes the canonical set of structural protein genes in the order 5′ -spike (s) -envelope (e) -membrane (m) and nucleocapsid (n) -3′. the structural gene region also harbors several orfs that are interspersed along the structural protein coding genes. the number and location of these accessory orfs vary between the cov species [2, 3] . in animals, cov infections are mainly associated with respiratory and enteric disease and can have large economical impact on the veterinary industry, e.g. porcine epidemic diarrhea virus (pedv) causes gastrointestinal disease in pigs [4] , infectious bronchitis virus (ibv) causes severe kidney and respiratory disease in chicken [5] and bovine coronavirus (bcov) causes both respiratory disease and diarrhea in cattle [6] . additionally, cov infections can have other disease manifestations, such as central nervous system (cns) involvement, hepatitis and peritonitis [7] [8] [9] [10] . in humans, cov infections are mainly associated with respiratory diseases that are considered to have a large impact on the economy due to reduced productivity of the working population. currently, 6 coronaviruses that cause disease in humans have been discovered. four of those are commonly circulating and two have caused epidemics of severe acute respiratory disease. the first human coronavirus (hcov), b814, was described in 1965. in the following years, over 30 additional strains were characterized. ten of those strains could only be isolated from primary embryonic tracheal organ culture. others were readily isolated from monolayer cultures and were antigenically related to the prototype strain hcov-229e. hcov-oc43, for organ culture 43, was isolated and found to be distinct from the 229e prototype strain [11, 12] . in the subsequent decades, research on hcovs would center on these two distinct viruses. however, in 2002, an unknown respiratory illness, termed severe acute respiratory syndrome (sars), surfaced in asia. research determined it to be caused by a novel coronavirus [13, 14] . at the end of the epidemic, this virus had infected over 8000 people, most in china, and caused 774 deaths [15] . following the discovery of this virus, two additional covs causing human disease were identified. hcov-nl63 was isolated in the netherlands in 2004 from an infant with bronchiolitis [16] and hcov-hku1 in 2005 from a patient with pneumonia in hong kong [17] . in 2012, another respiratory hcov, middle east respiratory (mers)-cov, was isolated from a patient with pneumonia in saudi-arabia [18] . unlike sars-cov, this virus is still intermittently present in the human population and most recently caused a large outbreak in south-korea [19] . to date, there have been over 1600 cases and almost 600 deaths related to mers-cov infection [20] . out of the 6 known human coronaviruses, hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1 are commonly circulating in the human population and usually cause general respiratory illness and cold symptoms in healthy individuals [21] [22] [23] . like influenza, these viruses are capable of causing more severe disease in the immunocompromised and the elderly [24] . they infect the human airway from the luminal side and progeny viruses are released from the same side facilitating spread through coughing and sneezing [25, 26] . these coronaviruses are responsible for approximately 5-10% of all upper and lower respiratory tract infections [27] [28] [29] but the interactions between them and their natural host cells are poorly understood. currently, it is hypothesized that most of the human coronaviruses may have originated from bats [30, 31] . for example, hcov-229e is believed to originate from african hipposiderid bats possibly using camelids as intermediate hosts [32] . in the last 15 years, two coronaviruses have crossed the species barrier and caused severe and fatal disease in humans. sars-cov surfaced in 2002 and mers-cov in 2012 [13, 14, 18] . as opposed to the commonly circulating viruses, which generally only cause mild respiratory symptoms, these viruses presented with higher case fatality ratios, around 10 and 20-50% respectively [33, 34] . currently, there is abundant phylogenetic evidence for the bat origin of sars-cov, based on sequences of sars-like viruses found among bats in the recent years [35] [36] [37] . the initial transmissions of sars-cov from animals to humans were traced back to the live animal wet markets and it was hypothesized that the virus made its way into the human population using the civet cat as an intermediate host. however, successful isolation of sars-like viruses from bats [38] and the fact that a contemporary bat sars-like virus can infect human airway cultures [39] suggest that an intermediate host between humans and bat might not have been needed for the transmission of sars-cov. the evolutionary origin of mers-cov is less clear but it has been speculated to be bats as well. characterization of an african bat virus closely related to mers-cov shows that both the human and camel strains belong to the same viral species and phylogenetic analysis suggests that mers-cov infection in camels predates that in humans, suggesting that camels infect humans and not the other way around. furthermore, the bat virus roots the phylogenetic tree providing further evidence for the bat origin of mers-cov [40] . additionally, human-tohuman transmission, although not robust, seems to happen simultaneously as camel-to-human transmission. therefore, any further adaptation of mers-cov to the human host must be monitored carefully and intermediate hosts identified [41] . many bat coronaviruses have been identified in the recent years further highlighting the zoonotic potential of this family of viruses [30] . given the documented history of coronaviruses overcoming the species barrier and causing severe disease in humans, it is important to investigate the zoonotic potential of close evolutionary relatives of common hcovs in a culture model that recapitulates the aspects of the human airway, e.g. morphology and receptor distribution. it's important to study the mechanisms of pathogenesis and the evolution of zoonotic viruses in detail in order to identify molecular determinants that affect either transmission or pathogenesis. it's also important to elucidate whether coronaviruses currently circulating in animals are a potential danger to the human population. all of the known cellular receptors of hcovs belong to the same protein family, the membrane ectopeptidases. interestingly, the catalytic activity of these peptidases is not required for viral entry but rather the co-expression of other host peptidases activates the hcov spike proteins [42, 43] . it has been established that the human transmembrane serine proteases tmprssii and hat cleave and activate the hcov-229e, sars-and mers-cov spike proteins during viral entry [44, 45] . out of the four commonly circulating coronaviruses, hcov-229e is the only one that infects non-ciliated cells using the human aminopeptidase n (hapn) as its receptor [46] . this peptidase is predominantly expressed on non-ciliated cells in the human bronchus [47] . sars-cov and hcov-nl63 both utilize the angiotensin converting enzyme 2 (ace2) for cellular binding [48, 49] . ace2 is expressed on ciliated bronchial cells along with endothelial cells and both type i and ii alveolar cells [50] . mers-cov was found to use a different receptor than sars-cov, namely the dipeptyl-peptidase 4 (dpp4) [51] . dpp4 is widely expressed in endothelial cells and various epithelial tissues in the human body [52] . in ex vivo human lung organ cultures, different tropism of sars-and mers-covs was observed. mers-cov can actively replicate in both bronchial and alveolar tissue while sars-cov primarily replicates in alveolar tissue [53] . the wide cellular tropism of mers-cov might contribute to its associated disease severity and high mortality rate whereas the alveolar replication of sars-cov would explain why it generally presents with pneumonia. the cellular surface receptors for hcov-oc43 and hcov-hku1 are currently unknown but receptor determinants for these two viruses have been identified as n-acetyl-9-o-acetylneuraminic acid and o-acetylated sialic acid, respectively [54, 55] . all of these viruses can be successfully cultured and investigated in hae cultures [56, 57] . the discovery of hcovs, their receptor usage, cell tropism and receptor binding domain (rbd) is summarized in table 1 . furthermore, established reverse genetic systems for hcov-229e [58] , hcov-oc43 [59] and hcov-nl63 [60] allow for controlled virus mutation and fluorescent transgene insertion to better understand the interaction of these viruses with their pulmonary host cells. traditionally, respiratory viruses are studied in animal models, usually mice and ferrets [48, 61] . however, it is not always possible to correctly recapitulate human infection and disease in animal models. the establishment of transgenic animal models for human disease is attainable when either the virus receptor has been identified, which is not the case for all hcovs, or when viruses can be adapted to a different host. an adapted human virus may not share the same properties as the original human virus. sars-cov was found to replicate naturally in various strains of inbred mice but to enhance clinical signs of disease the hace2 was introduced into these mice. this resulted in murine models with varying degree of human disease similarity. since sars-cov already replicated in mouse cells, adapting it to the murine host was quite successful. this resulted in three mouse adapted strains that caused disease in mice similar to severe sars-cov cases in humans [62] . in an effort to establish a mouse model for hcov-229e infection transgenic hapn mice were created. however, the insertion of the hapn into mouse cells is not enough to establish robust hcov-229e infection in vivo. nevertheless, cells isolated from these transgenic animals could be infected in vitro [63, 64] . the emergence of both sars-and mers-covs emphasized the importance of establishing animal models for human coronaviruses. currently, a few animal models for mers-cov have been established. mice carry their own variant of the viral receptor ddp4 that differs from the human in regions important for mers-cov spike interaction and by replacing this receptor with the human one, mers-cov can infect mouse cells but the method of hdpp4 insertion has an effect on the degree of pathogenesis observed in these mice [65, 66] . various non-human primates (nhps) can be naturally infected with both sars-and mers-covs. however, disease presentation and pathogenesis differs between the different subspecies and nhp models are expensive, although ideal to study human infection due to their genetic similarity [62] . to establish a link between the research conducted in animal models and humans, an organotypic airway culture system resembling the human airway epithelium has been developed. this model is a universal platform to study human respiratory viruses [67] [68] [69] [70] . they have been used successfully for infection studies with all known human coronaviruses [56, 57] . furthermore, the cultures can be inoculated with a low infectious dosage to mimic natural infection in the human airway. whereas, animal models often require both high dosage and artificial inoculation routes. organotypic cell cultures are becoming increasingly common. different cell culture models exist to depict different epithelial tissues [71] . these cultures closely resemble their tissue of origin and contain various different cell types with distinctive roles in the polarized tissue. currently, various organotypic cell culture models exist to represent the different areas of the human airways. the human lungs span a long anatomical distance and carry out different functions depending on anatomical location [72, 73] . the structure of the epithelium also differs the further you descend into the airways. tracheal and bronchial epithelium is columnar and pseudostratified, with every cell in contact with the basement membrane, while the epithelium in the alveoli is comprised of a single cell layer to facilitate air-exchange [74] . tracheobronchial cells are one of the first targets of human respiratory viruses and can be cultured in airliquid interface (ali) where the apical side of the cell layer is exposed to air while the basolateral side is submerged in medium. tracheobronchial cells cultured in that way form a pseudostratified epithelial layer that both morphologically and functionally resembles the human upper conducting airway (fig. 1a) [75, 76] . after differentiation, these cultures contain many different cell types such as basal, ciliated and goblet cells. they also produce protective mucus, much like in vivo epithelium. when compared to primary bronchial cells in submerged two-dimensional culture, the gene expression of primary ali cultures differs significantly. however, the expression pattern of primary human bronchial ali cultures is comparable to that of in vivo epithelium. the human bronchial cell line calu-3 has been used as a culture model for respiratory epithelium but its gene expression in ali cultures is more similar to submerged bronchial cell cultures than differentiated epithelium [77] . disruption of the cell layer [57] . therefore, the primary tracheobronchial ali culture model is especially fitting for human respiratory virus research since it accurately recapitulates the primary entry point for these viruses. by using these cultures, virus replication and host interactions can be studied in natural target cells. further establishing the usefulness of this system hcov-hku1 was propagated for the first time in ciliated cells of bronchial hae cultures in 2010 after culturing it in conventional cell lines had failed [26] . alveolar epithelial ali cultures (fig. 1b) can also be used for virus-host interaction studies and are especially applicable when a viral infection causes pneumonia and alveolar damage [79] . hcov-hku1 has also been propagated in alveolar hae cultures and exhibits a strong tropism for alveolar type ii cells and causes large syncytia formation upon infection [80] . when compared to traditional two dimensional cell cultures, the hae cultures are more cumbersome and their preparation is time consuming but they do have an advantage over traditional monolayer cell cultures when it comes to virus-host interaction studies. different types of ali cultures used for virus research are summarized in table 2 . within the respiratory epithelium the innate immune system has a major protective role as the first line of defense against respiratory pathogens. in particular, the interferon (ifn) system orchestrates hundreds of different cellular effector proteins that (i) protect the epithelial barrier by altering the physiological and cellular environment, (ii) impair virus propagation, spread and transmission, and (iii) shape the host's adaptive immune response. recent publications have demonstrated that the innate immune system is functional in the hae cell culture system and that most pathogen recognition receptors are expressed and up-regulated upon treatment with exogenous stimuli [57, 81] . in general, hcovs do not elicit a strong innate immune response in primary target cells of the human airway early during infection. despite the presence of all major pathogen recognition receptors, no elevated expression of ifn beta, pro-inflammatory cytokines or interferon stimulated genes can be observed up to 12 h post-infection in haes infected with hcov-229e, mers-or sars-covs [57] . this is most likely due to the intrinsic cov properties harbored in the replicative non-structural proteins that actively aid in avoiding recognition by the host innate immune system. for example, the 5′ termini of the viral mrna are capped making them indistinguishable from the host cellular mrnas and no longer detectable by cellular sensors. furthermore, cov replication is associated with the appearance of double membrane vesicles (dmvs) in the host cell cytoplasm, which might serve as a protective shield for viral rna to prevent recognition by cytoplasmic rna sensors [82] [83] [84] [85] . in addition to the non-structural proteins, various cov accessory proteins have been discovered to inhibit interferon signaling at different stages of the host innate immune response. for example, mers-cov accessory protein 4a inhibits innate antiviral signaling by suppressing the activation of mda5 and rigi [86, 87] whereas 4b inhibits the induction of the ifn-beta promoter [88] . while orf 4a and 4b are ifn antagonists in the genome of mers-cov, sars-cov orf3b antagonizes ifn signaling through mavs/rigi [89] . whereas sars-cov orf6 disrupts ifn signaling by blocking the nuclear translocation of stat1 [89, 90] . these discoveries highlight that hcovs employ similar yet different strategies despite that respiratory infections with hcovs can result in severe respiratory disease there are currently no effective prophylactic or therapeutic treatment options available. however, the emergence of novel coronaviruses has emphasized the need to develop effective treatment options. for example, vaccines using the spike proteins of both sars-and mers-covs have proven protective in animal models [91, 92] suggesting that a vaccine against hcovs for human use might be achievable. additionally, various drugs that inhibit hcov infection at different stages of the replication cycle have been reported and some could potentially serve as treatment options for hcov associated severe respiratory disease. for example, patients presenting with severe respiratory disease, caused by sars-or mers-covs, are generally treated with steroids and interferon, sometimes in combination with the antiviral drug ribavirin [93] [94] [95] [96] . this treatment, however, is not especially effective highlighting the need for hcov specific antivirals. many different compounds have been determined to have anti-hcov activity. for example, protease inhibitors which suppress hcov entry [97] [98] [99] , cyclosporin a (csa) treatment blocks the replication of coronaviruses from all subgroups [100] and non-immunosuppressive derivatives of csa represent a possible therapeutic option for both human and animal cov infections. hcov infection can also be inhibited by pre-treating hae cultures with either recombinant ifn alpha or lambda [57] . similar effect has also been shown for recombinant ifn alpha and beta which could inhibit mers-cov in ex vivo lung cultures [53] . as previously described, ifn treatment of active hcov infection is not particularly effective in vivo. therefore, the use of ifn in humans might be limited to prophylactic treatment of exposed persons and/or health care workers treating infected patients. screenings of compound libraries have also resulted in the identification of some hcov specific antivirals. for example, a novel small compound inhibitor (k22) has been identified, and showed to be effective against a broad spectrum of covs and could inhibit both hcov-229e and mers-cov in hae cultures [101] . additionally, hcov-nl63 has been inhibited in hae cultures with polymer-based compounds [102] . to date, most treatment and inhibitor studies have been conducted in hcov susceptible cell lines. however, the hae cultures represent an ideal system to test the application and efficacy of those already identified, and new, antiviral compounds against hcovs in cells that represent the primary site of replication. furthermore, the hae cultures are heterogenous, containing many different cellular sub-populations, and would allow for the evaluation of compound toxicity and effect in a differentiated layer similar to human airway epithelium. compounds already shown to inhibit hcovs in cell lines should be applied to hae cultures as well before any animal or human trials. hcov induced respiratory diseases are of both medical and socio-economic importance. the emergence of sars-and mers-cov and the yearly circulation of the four common hcovs highlight the importance of elucidating the different mechanisms employed by hcovs to evade the host immune system as well as identifying antiviral compounds and human vaccine candidates. the hae culture system is based on primary human cells offering a unique platform to study respiratory viruses in cells representing the primary entry point of these viruses, bronchial epithelial cells, or investigate the interaction of hcovs and the distal airways, in type i and ii alveolar cells. additionally, the inclusion of airway epithelial cultures for other species enables the study of zoonosis and animal-to-human transmission. currently, many aspects of hcov infection and pathogenesis remain to be determined. the hae culture system, both tracheobronchial and alveolar, represents a unique platform to study virus-host interaction in natural target cells at the molecular level. these cultures are becoming more common and more relevant to hcov research. especially, for those viruses for which there is no animal model, as they provide an organotypic substitute for virushost interaction studies. the authors declare no competing interests. authors' contribution hrj wrote 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copy of the human coronavirus genome cloned in vaccinia virus recovery of a neurovirulent human coronavirus oc43 from an infectious cdna clone systematic assembly of a full-length infectious clone of human coronavirus nl63 animal models for the study of influenza pathogenesis and therapy animal models for sars and mers coronaviruses development of a transgenic mouse model susceptible to human coronavirus 229e cells of human aminopeptidase n (cd13) transgenic mice are infected by human coronavirus-229e in vitro, but not in vivo rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium human bocavirus 1 infects commercially available primary human airway epithelium cultures productively infection of human airway epithelium by 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(coronaviruses) similar to 229e virus, with some epidemiological observations human coronavirus 229e: receptor binding domain and neutralization by soluble receptor at 37 degrees c growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease the sars-cov s glycoprotein: expression and functional characterization identification of residues in the receptor-binding domain (rbd) of the spike protein of human coronavirus nl63 that are critical for the rbd-ace2 receptor interaction the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome we would like to thank prof. dr. volker thiel for his careful review of the manuscript and dr. eveline kindler for providing components for figures. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-022393-s26d54ew authors: e. newcomer, christian; g. fox, james title: zoonoses and other human health hazards date: 2007-09-02 journal: the mouse in biomedical research doi: 10.1016/b978-012369454-6/50054-6 sha: doc_id: 22393 cord_uid: s26d54ew zoonoses refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated hazards. in addition to the zoonoses, mice are capable of inflicting bites on inadequately trained personnel and are a rich source of allergens for a substantial number of persons predisposed to develop mouse-associated allergic sensitivities. this chapter discusses the mouse-associated zoonotic diseases and other health hazards and explains the strategies that are helpful for reducing or eliminating the risk of personnel exposure to these conditions. zoonoses is derived from the greek words zoon (meaning animals), and noses (meaning disease), and refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. however, when these essential animal program quality measures fail or are not incorporated into the animal facility operations, zoonotic pathogens may be unwittingly introduced and perpetuated, placing personnel at increased risk of exposure. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated implications in the new world serocomplex group are present among the wild rodents endemic to the united states such as neotoma spp. and peromyscus spp. buchmeier et al. 2001; fulhorst et al. 2002) . research animal programs that import wild rodents for laboratory studies should stay abreast of the developments on the identification and host-range characteristics of the new world arenaviruses of emerging importance. this section will focus only on lymphocytic choriomeningitis (lcm), a naturally occurring viral zoonosis of the laboratory mouse. many published reports of human lcm infection are associated with laboratory animal and pet contact, particularly mice and hamsters, and these studies now span many decades (armstrong and lillie 1934; bowen et al. 1975; dykewicz et al. 1992; jahrling and peters 1992; lehmann-grube et al. 1979; rousseau et al. 1997) . there seems to be a resurgent awareness among physicians that lcm should be sought as an etiology in human neurological disease and in pediatric congenital brain disorders (barton and hyndman 2000; barton et al. 1995 barton et al. , 2002 romero and newland 2003) . lcmv is widely distributed among the wild mouse population throughout most of the world presenting a zoonotic hazard (childs et al. 1992; childs and peters 1993; morita et al. 1996; smith et al. 1993 ). surveys conducted within the urban environment of baltimore, maryland, reported that 9% of house mice and 4.7% of persons tested had measurable lcmv antibody titers (childs et al. 1991 (childs et al. , 1992 . a similar serological survey conducted in spain across urban and rural ecological settings also found a 9% prevalence in mice and a 1.7% prevalence among persons by immunofluorescence assay (lledo et al. 2003) . the recent serological detection of lcmv in five mice on the treeless, sub-antarctic, macquarie island of australia, indicates the extent of distribution of this agent to the remotest areas of our planet (moro et al. 2003) . the apparent ease with which lcmv is transmitted to humans also occurs in a variety of other laboratory animal species; hamsters, guinea pigs, swine, dogs, and nonhuman primates, especially callitrichids, which readily sustain natural infections. in the case of the callitrichids, there have been numerous reports of epizootic infectious hepatitis (callitrichid hepatitis) due to lcmv, with a high mortality rate in zoological parks in both the united states and england over the past two decades (lucke and bennett 1982; montali et al. 1989; stephensen et al. 1990 stephensen et al. , 1991 stephensen et al. , 1995 . rodent (mouse) infestations of these zoos and/or the supplementation of the diets of tamarins and marmosets with suckling mice, a common practice (richter et al. 1984) , are potentially rich sources for lcmv. in the research animal facility environment, the laboratory mouse continues to merit attention as the species of primary concern as a reservoir for cases of human lcm (dykewicz et al. 1992 ). in the laboratory mouse, and to a lesser degree the hamster, breeding colonies can become endemically infected when the virus is transmitted to pups in utero or early in the neonatal period, producing a tolerant subclinical infection characterized by chronic viremia and viruria. when infected, athymic and other immunodeficient mouse strains may be predisposed to harboring silent, persistent infections and present a higher risk to personnel (dykewicz et al. 1992) . under some circumstances lcmv also produces a pantropic infection and may be copiously present in blood, cerebrospinal fluid, urine, nasopharyngeal secretions, feces, and tissues of infected natural hosts and possibly humans. bedding material and other fomites contaminated by lcmv-infected animals can also be important sources of infection for humans, as demonstrated in a recent outbreak among laboratory animal technicians and on many previous occasions (biggar et al. 1975; dykewicz et al. 1992) . the experimental passage of tumors and cell lines contaminated with lcmv has long been recognized (haas and stewart 1956) and represents one of the biggest threats for the introduction of lcmv into animal facilities at the present time (bhatt et al. 1986; dykewicz et al. 1992; nicklas et al. 1993) . reported that 17 of 63 rodent transplantable tumors screened were positive for lcmv and identified contamination in 4 of 14 hamster tumors and 2 of 81 mouse tumors that had been propagated in animals (nicklas et al. 1993) . the growth of lcmv in insect cell lines has also been demonstrated (rahacek 1965) , and the article by hotchin summarized the work of others indicating that numerous experimentally infected, bloodsucking ectoparasites are capable of transmitting the disease to laboratory rodents (hotchin 1971) . lcm virus also has been recovered from cockroaches (armstrong and lillie 1934) . the diagnosis and control of lcmv infection in mouse colonies has been reviewed in chapter 7 of this volume. tumor and cell-line screening before animal passage, the control of wild rodent infestations in areas where animals are housed or used, and the early detection of colony infections through sound colony health surveillance practices are of critical importance to the prevention of infection in mouse colonies. once established in mouse breeding colonies, the high viral load characteristically shed by mice infected congenitally or neonatally represents a very serious hazard to personnel. most of the cases of human infection, whether involving exposure in the home, agricultural, or laboratory setting, have involved contact with live mice and their excreta or mouse carcasses (dykewicz et al. 1992; havens 1948; morbidity and mortality weekly report 1984) . several authors have emphasized the association between the actual handling of infected mice and the contraction of the disease by humans (dykewicz et al. 1992; havens 1948; smithard and macrae 1951) . several cases of human infection have suggested the possibility that infected rodent tissues can serve as a source of infection for laboratory personnel (baum et al. 1966; dykewicz et al. 1992; tobin 1968 ). humans may be infected by inhalation or by the contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals. the transmission of lcmv by the bite of an infected mouse can also occur (scheid et al. 1964) . also, hotchin reported the findings of other researchers that lcmv was transmissible experimentally through the intact skin of the guinea pig, but this finding has not been reported in humans (hotchin 1971) . airborne transmission is well documented and plays a very important role in human infections, especially through the ready dispersion and inhalation of viral-contaminated dust from the animal cage or room (biggar et al. 1975; hinman et al. 1975) . following an incubation period of 1 to 3 weeks, humans may experience asymptomatic or a mild febrile disease ranging to a serious flu-like illness characterized by anorexia, malaise, diffuse myalgia and arthralgia, fever, chills, vomiting, headache, stiff neck, and photophobia. some patients enter a second stage of the disease several days after the resolution of early mild symptoms, developing meningoencephalitis and exhibiting additional signs such as drowsiness, confusion, sensory disturbances, and motor abnormalities. patients can also develop more serious nonneurological manifestations of the disease such as maculopapular rash, lymphadenopathy, parotitis, orchitis, arthritis, and epicarditis (peters 1997 ). central nervous system involvement has resulted in death in several cases. infections during pregnancy pose a risk of infection for the human fetus (wright et al. 1997). wright et al. reported 26 cases in human infants, with lcmv confirmed serologically over a two-year period in a major u.s. medical center (wright et al. 1997) . these infants presented with ocular abnormalities, macrocephaly, and hydrocephalus with microcephaly. fifty percent of the mothers reported having had illnesses compatible with lcmv infection, and over half reported exposures to rodents during their pregnancies. the diagnosis of lcm infection in humans is currently made by serological testing using either the immunofluorescent antibody (ifa) test or the enzyme-linked immunosorbent assay (elisa) (barton et al. 2002) . both of these tests are available through the centers for disease control and prevention and are superior to the complement fixation test that is widely available commercially. although there are no proven effective antiviral therapies for lcm infection, intravenous ribavirin therapy reduces mortality in patients infected with lassa fever virus (a member of the old world arenavirus serocomplex) and may be of some benefit in patients with severe lcmv infections (andrei and de clercq 1993; mccormick et al. 1986 ). this disease can be prevented in the laboratory through periodic serological surveillance using elisa and ifa tests of newly introduced animals with inadequate disease profiles and of resident animal colonies at risk. thorough screening of all tumors and cell lines intended for animal passage using the highly sensitive mouse antibody production assay or newer pcr-based laboratory tests for the presence of lcmv is another crucial element in the program to prevent the introduction of lcmv into established animal colonies (besselsen et al. 2003 and chapter 7 of this volume). sound animal facility sanitation practices and the use of microbarrier caging systems with proper infection-control techniques should prevent or suppress the spread of lcmv if present in the environment. the elimination of wild rodent infestations in animal facilities is very important to prevent the introduction of lcmv into the animal facility environment. also, facilities with wild rodent infestations may encounter the relatively common, free-living, bloodsucking mite of the rodent, ornithonyssus bacoti, in abundance (personal communication). although the natural lcmv transmission to humans from bloodsucking ectoparasites is unproven, the control of potential ectoparasitic vectors of this type would be a prudent measure. rabies is an acute, almost invariably fatal disease that occurs worldwide with the exception of a few countries, generally island nations, and other regions that have excluded the disease through animal importation and control programs and the aid of geographic barriers. neither the laboratory mouse nor other small wild rodent hosts appear to be important as reservoirs of natural rabies infection. hence, the principal reason for our discussion of rabies as a zoonotic disease of the laboratory mouse is to provide information that should quickly allay the fears of uninformed research and animal facility staff who suffer the bite of a mouse. on the other hand, the experimental use of mice in the study and characterization of rabies virus and in rabies vaccine development is an important component of some animal care and use programs that deserves special attention by the institution during all phases of research planning and implementation. there are no known cases of human rabies from rodents in the united states (2002). the incidence in larger wild rodent species within the united states has increased in recent years, however. during the interval 1971-1984, a total of 97 cases of rabies in rodents were recorded, but from 1995 to 2000 approximately 52 cases were reported in large rodents annually (2002) . the rodents involved were woodchucks and beavers, both species presumably large enough to survive the chance encounter and attack by a wild rabid carnivore such as the raccoon, skunk, fox, or feral cat. earlier literature on the federal republic of germany summarized findings from 1961 to 1967, which indicated that three mice, one rat (species not given), nine norway rats, and three muskrats were infected with rabies and had bitten humans (scholz and weinhold 1969) . despite rare reports of this nature, rodents are not a proven source of rabies transmission to humans (johnson 1989 ). all mammals are generally regarded as susceptible to rabies. in humans, the course of the disease proceeds through several phases: incubation, prodromal, acute neurological, coma, and rarely, recovery (johnson 1989) . the incubation period varies from 9 days to over 8 months. during the prodromal stage lasting 2 to 4 days, patients experience a period of apprehension and develop headache, malaise, and fever. an abnormal, indefinite sensation at the site of a prior animal bite wound is the first specific symptom. patients also may develop intermittent periods of excitation, nervousness, or anxiety interspersed with quiet periods when the mental state appears normal. further progression of the disease involves paresis or paralysis, inability to swallow, and the related hydrophobia, delirium, convulsions, and coma. rabies produces an almost invariably fatal acute viral encephalomyelitis, with death due to respiratory paralysis. adult mice used experimentally in rabies studies usually exhibit clinical signs between 5 and 15 days following inoculation and die within 5 days of the onset of clinical signs coinciding with the period of viral shedding. clinical signs in the mouse consist of muscular tremors, incoordination, excitation, or paralysis. certain rabies virus isolates from skunks produce a spastic paralysis in adult mice followed by recovery in a high percentage of infected mice. also, infant mice inoculated with certain strains of street rabies virus are capable of full recovery (johnson 1989 ). personnel working with mice experimentally infected with rabies virus should adhere to the well-established and detailed procedures that have been described in other sources for animal inoculation, husbandry, and tissue harvest procedures (johnson 1989) . vaccination of personnel involved in rabies studies with laboratory animals also is clearly indicated, regardless of the animal species involved. four other viruses that produce natural infections in the mouse have been implicated previously or are known to be infectious for humans. these include hantavirus, sendai virus, reovirus 3, and mouse hepatitis virus. for none of these viral agents is there documented evidence of zoonotic transmission of the agent from naturally infected laboratory mice to personnel in the laboratory. hantaviruses are zoonotic viruses comprising at least 22 species that are maintained among natural rodent reservoirs, despite the presence of neutralizing antibody in the rodent host (elliott et al. 2000; meyer and schmaljohn 2000) . approximately half of the hantaviruses are known human pathogens producing virus-specific patterns of disease that include hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, and its benign form, nephropathia endemica (mills and childs 1998) . although mus musculus can exhibit the pattern of persistent hantavirus infection in the presence of neutralizing antibody when induced experimentally (araki et al. 2003) , this does not occur in natural infections of the mouse (meyer and schmaljohn 2000) . this is the likely reason that the wild mouse apparently is not important as a natural reservoir for the hantaviruses. this interpretation is supported by serological studies of wild mus musculus that detected either no or a very low incidence of serological evidence to hantavirus exposure even when other wild rodent species in the vicinity had a high level of endemic infection (kantakamalakul et al. 2003; meyer and schmaljohn 2000; pacsa et al. 2002; zuo et al. 2004) . mus musculus is used in the laboratory as an animal model to study various aspects of hantavirus infection ranging from vaccine development (choi et al. 2003) , viral pathogenicity (ebihara et al. 2000; kim and mckee 1985) , and immunological aspects of persistence (araki et al. 2003) , and it is primarily in this context that hantavirus deserves mention as a zoonotic infection in the mouse. the control of wild rodent infestations in animal facilities, quarantine and testing of wild caught rodent species during importation, and proper observance of animal biosafety guidelines for hantavirus-infected animals would be expected to virtually eliminate the risk of this zoonosis in laboratory maintained mice. mouse hepatitis virus (mhv) remains a prevalent infection in many mouse colonies where it potentially impacts colony health and disrupts experimental studies (see chapter 6). earlier studies have demonstrated that human sera contained complement-fixing and neutralizing antibodies to mhv (hartley et al. 1964) . later studies suggested that this was most likely due to cross-reactive antibodies from human cold virus infections (bradburne 1970; mcintosh et al. 1967 ). mouse hepatitis virus and the two prototype human cold viruses (oc43 and 229e) are members of the antigenic group 2 coronaviruses, and it is now known that members of this group share four, and in some cases five, structural genes that could account for this crossreactivity (navas-martin and weiss 2003). the coronaviruses have a very narrow host range and generally replicate only in the cells of the host species (navas-martin and weiss 2003). however, under unique laboratory conditions involving persistent cell culture infection, the use of mixed cell cultures of murine and of a nonpermissive species, or the use of cells possessing modified receptors, mhv has been adapted to grow in human, nonhuman primate, and hamster cells. also, the many strains of mhv in combination with use of targeted rna recombinant system have also been very useful for experimental study of the molecular basis of coronavirus pathogenicity (masters 1999) . application of the targeted rna recombinant system to mhv for exploring of emerging coronavirus infections such as sars may delineate the molecular basis for expanding of host range. these studies may warrant the reader's future attention, but at the present, mhv can be reasonably dismissed as a zoonotic infection. sendai virus was once a prevalent agent in mouse colonies but has become a rarity in most institutions. this is due to its ease of eradication through the use of temporary cessation of breeding to eliminate a naive population that is susceptible to infection and through the use of caging systems that prevent transmission (see chapter 11). sendai virus was originally isolated and described in the 1950s during the investigation of cases of human respiratory illness (gerngoss 1957; kuroya et al. 1953a kuroya et al. , 1953b sano et al. 1953; zhandoff et al. 1957 ). in the original report involving the isolation of sendai virus from japanese newborn human infants suffering from fatal pneumonitis, lung suspensions from the newborns were inoculated intranasally into mice, producing lung consolidation and death in several cases (kuroya et al. 1953b) . in later studies, sendai virus isolates were reported to produce disease in human volunteers (kuroya et al. 1953a; yamada 1956) , and reports from many countries indicated that serological evidence of sendai virus infection was associated with outbreaks of human respiratory illness (demeio and walker 1958; gardner 1957; jensen et al. 1955). tennant et al. demonstrated that personnel working with laboratory animals had antibody titers to sendai virus, but personnel with no known exposure to laboratory animals also had significant titers to the agent (tennant et al. 1967) . recombinant sendai virus is widely used for gene transfer experiments, and these vectors can readily infect human airway epithelium and a variety of other human tissues under experimental conditions (nagai 1999; pinkenburg et al. 2004) . although these recent studies have clearly demonstrated that sendai virus is capable of infecting human tissues, the initial evidence for its role as a human pathogen remains doubtful. the mice used for the early isolations of the agent may have already been endemically infected with sendai and served as the source, or it may be that other serologically cross-reactive parainfluenza viruses, which were not characterized during this era, were responsible for producing false positive reactions to sendai virus (ishida and homma 1978) . reoviruses are generally regarded as the cause of childhood infections producing asymptomatic or very mild disease, and there are few reports linking these infections with a particular disease (tsai 2000) . reovirus 3 was originally isolated from the feces of a clinically ill child (stanley et al. 1953) , and it continues to receive attention as a possible etiology for neonatal hepatitis and extrahepatic biliary atresia in infants (richardson et al. 1994; steele et al. 1995) . reovirus 3 is highly infectious for infant laboratory mice and still receives some attention in the health-screening programs for the mouse and laboratory rodent species. jacoby and lindsey (1997) reported that mouse colonies in the united states continue to have a 5 to 20% prevalence of reovirus 3 infection. although there are no confirmed reports of reovirus 3 transmission from mice (or other laboratory animal species) to humans, the laboratory mouse should be considered a possible source for this infectious agent for humans and other susceptible species. the rickettsiae are fastidious, small pleomorphic coccobacilliary organisms maintained in nature in a cycle of infection involving mammalian hosts and arthropod vectors as reservoirs (saah 2000) . in most rickettsial infections, humans serve only as an incidental host and do not contribute to the propagation of the organism in nature. such is the case for rickettsialpox, a nonfatal, self-limiting zoonotic disease caused by rickettsia akari, which is perpetuated in a cycle of infection involving mus musculus as the primary host and a mite vector (liponyssoides sanguineus). isolation of the organisms from rats (rattus) and voles (microtus) has also been reported. the first cases of human rickettsialpox were described in patients in new york city (huebner et al. 1946a (huebner et al. , 1946b , and outbreaks of the disease have generally remained clustered within large urban areas of the united states and in rural north carolina, utah, south africa, korea, and the former soviet union . according to koss et al. approximately 800 cases of rickettsialpox have been reported in the literature, with nearly 500 of these within the three years following the original description of the disease . prior to the report by koss et al. the largest case study included 13 patients accumulated over a 10-year period. the recent report by koss et al., however, included 18 patients in new york city reporting over only a 20-month period in the wake of the september 11, 2001 attacks, suggesting that perhaps the heightened sensitivities to possible bioterrorism events stimulated an upsurge in the reporting of cases as a byproduct of increased patient concerns . authors have commented on a variety of other social and demographic factors that may also be contributing to the noticeable increase in the incidence of rickettsialpox and murine typhus in the urban environment (comer et al. 1999; paddock et al. 2003) . there are no reported cases of rickettsialpox in personnel related to exposure to naturally infected laboratory mice. the mite vector l. sanguineus has not been reported in laboratory mouse colonies either historically or contemporarily. however, the tropical rat mite (ornithonyssus bacoti) can be experimentally infected with r. akari but has not been shown to play a role in the natural cycle of infection. in the authors' experience of ornithonyssus bacoti infestations of laboratory mouse or rat colonies are still seen with some frequency in facilities that have resident wild or feral mouse populations and should be addressed in the institution's pest control and infection control programs. rickettsialpox has an incubation period of 7 to 21 days following the bite of the infected mite (saah 2000 mild to severe with an abrupt onset, and it typically presents with a classic triad of an eschar, fever, and a papulovesicular rash. the papule develops at the site of the bite and later ulcerates and progresses to an eschar, 0.5 to 3 cm in diameter, as r. akari proliferates locally in the skin and vasculitis develops saah 2000) . the rash begins with firm, generally nonpruritic, erythematous papules, 2 to 10 mm in diameter, that develop into vesicles and heal by crusting. in addition to fever, patients may experience chills and headache, and less commonly, backache, myalgia, and photophobia. the disease is mild and self-limiting within 6 to 10 days, and serious complications or death have rarely been reported (saah 2000) . patients typically respond quickly after the initiation of antirickettsial therapy with tetracycline, doxycycline, or other appropriate agent saah 2000) . however, following resolution of the infection, headache and lassitude can persist for 1 to 2 weeks. the reader should refer to koss et al. for information on other rash-producing or eschar-related diseases that should be considered in the differential diagnosis to rickettsialpox . rickettsia akari are diagnosed by complement fixation tests or the more sensitive indirect immunofluorescent antibody test. serum antibody to r. akari generally takes 2 weeks to develop, and paired sera are needed to confirm a four-fold rise in antibody titer (saah 2000) . during the acute phase of the infection, immunohistochemistry or pcr analysis can be used for a rapid diagnosis on biopsy material obtained from the papulovesicular rash or eschar paddock et al. 2003) . laboratory mice infected with r. akari develop fatal pneumonia with intranasal inoculation and severe illness and death with intraperitoneal inoculation. mice develop anorexia, depression, and dyspnea. peritonitis, splenomegaly, and lymphadenitis are found upon necropsy examination. subcutaneous inoculation produces an active infection for 1 month, with organisms being recovered from the spleen but not the feces or urine (bell 1970) . the control and eradicaton of r. akari infections depend on the prevention of wild mice and the mite vector from entering laboratory animal facilities and human dwellings. murine typhus is a rickettsial disease caused by rickettsia typhi (previously r. mooseri) that occurs worldwide with epidemics or with high prevalence in particular geographic areas (dumler and walker 2000) . in the united states, most human cases of the disease are concentrated in texas and southern california. the disease is now predominantly associated with the rat as the primary host species for the oriental rat flea (xenopsylla cheopis), which serves as the principal ectoparasitic vector transmitting the disease to humans. however, the mouse can also serve as a host for this flea, as well as for the northern rat flea (nasopsyllus fasciatus) and the mouse flea (leptosylla segnis), which also bite man and can be involved in the transmission of r. typhi (flynn 1973; pratt and wiseman 1962; yunker 1964 ). an early report in the literature indicated that x. cheopis was easily established in an animal facility inhabiting rooms used for housing laboratory mice (yunker 1964) . it is now also known that the cat and opossum and the cat flea (ctenocephalides felis) can be involved in sustaining the cycle in some geographic localities (azad et al. 1997) . clark and will (1994) reported on use of the laboratory mouse as an experimental host for rearing x. cheopis, but there have been no reports of natural infestations of mouse colonies with any of the flea vectors of r. typhi for several decades. also, r. typhi has not been isolated from natural infections in laboratory mice. murine typhus is a more serious disease than rickettsialpox and presents with fever, headache, chills, nausea, and vomiting. splenomegaly, hepatomegaly, central nervous system involvement, and multiorgan failure can occur as severe and potentially fatal complications. a skin rash, which is typically maculopapular in the case of murine typhus, occurs much less commonly than in rickettsialpox, and its absence should not dissuade the clinician from making a diagnosis of murine typhus and from promptly instituting therapy due to the potential severity of the disease (dumler and walker 2000) . the methods used for the laboratory diagnosis and treatment of the disease in humans and the principles of preventing the introduction of r. typhi into laboratory animal colonies are similar to those for r. akari. leptospirosis microorganisms were discovered in 1914 when they were isolated from jaundiced patients (inada et al. 1916) ; after further study they were named in 1917 (noguchi 1918) . leptospirosis is solely a zoonotic disease of livestock, pet and stray dogs, and wildlife, including wild rodents. rodent reservoir hosts of leptospirosis include, in addition to rats, mice, field moles, hedgehogs, gerbils, squirrels, rabbits, and hamsters (fox and lipman 1991; torten 1979) . human to human transmission is extremely rare. leptospira interrogans (comprising more than 200 serovars) have been isolated worldwide (tappero et al. 2000) . l. interrogans contains 23 serogroups with strains pathogenic for amphibians, reptiles, and mammals including humans. serovars australis, ballum, bataviae, hardjo, grippotyphosa, icterohemorrgagiae, javanica, and pomona are associated with rodent infections. leptospira serovars, including l. australis, bataviae, grippotyphosa, hebdomidis, icterohaemorrhagiae, pomona, and pyrogenes, are found in the house mouse (torten 1979) . leptospira ballum has also been reported from mice and is e. newcomer and james g. fox most commonly associated with zoonotic outbreaks (borst et al. 1948; friedmann et al. 1973; stoenner and maclean 1958) . although particular serovars usually have distinct host species, most serovars can be carried by several hosts. leptospira are well adapted to a variety of mammals, particularly wild animals and rodents. in the chronic form, the organism chronically infects the host and is shed in the urine inconspicuously for long periods of time. rodents are the only major animal species that can shed leptospires throughout their lifespan without clinical manifestations (fox and lipman 1991; torten 1979) . active shedding of leptospires by rodents can go unrecognized until personnel handling the animals become clinically infected or are infected by exposure to water or food contaminated by urine. rats and mice are common animal hosts for serotype, l. ballum, although it has been found in other wildlife as well. water can often be contaminated with infected rodent urine. the infection can persist unnoticed in laboratory rodents, though their carrier rates for laboratory-maintained rodents in the united states are unknown, but it is probably low. the organism is not routinely screened on health surveillance protocols for mouse colonies; however, there was a report of leptospirosis in 1984 in a research colony of mice in the united states being housed in a large research institution (alexander 1984) . l. icterohaemorrhagiaes antibody when compared to children living in the detroit suburbs. therefore, children living in rodent-infested tenements may be at increased risk of infection (demers et al. 1983) . in europe and more recently in north america, rodents including house mice have provided a source of leptospira infection for swine and by extension could also infect personnel working in swine production units (galton 1966; smith et al. 1992) . leptospira interrogans serovar bratislava is commonly reported in these mice. the disease may vary from unapparent infection to severe infection and death. a self-limited systemic illness is seen in approximately 90% of infected humans. the incubation period is usually 5 to 14 days. individuals infected with leptospira experience a biphasic disease (faine 1991; sanger and thiermann 1988; stoenner and maclean 1958) . they become suddenly ill with weakness, headache, myalgia, malaise, chills, and fever and usually exhibit leukocytosis. during the second phase of the disease, conjunctival suffusion and a rash may occur. upon examination, renal, hepatic, pulmonary, and gastrointestinal findings may be abnormal. intravenous penicillin is the drug of choice in treating early-onset and late-stage leptospirosis infection (faine 1991; taber and feigin 1979; watt et al. 1988 ). ampicillin and doxycycline also have been effective in treating people with mild to moderate forms of leptospirosis. because leptospirosis in humans is often difficult to diagnose, the low incidence of reported infection in humans may be misleading. from 1974 to 1979, only 498 cases were reported, for an incidence of 0.05 per 100,000 people per year (sanger and thiermann 1988) . leptospirosis was removed from the reportable disease category in the united states in 1995 because of the small number of cases reported. outbreaks have been documented in the united states from personnel working with laboratory mice (barkin et al. 1974; stoenner and maclean 1958) . in one study, 8 of 58 employees handling the infected laboratory mice (80% of breeding females were excreting l. ballum in their urine) contracted leptospirosis (stoenner and maclean 1958) . infection with leptospira most frequently results from handling infected animals (contaminating the hands with urine) or from aerosol exposure during cage cleaning (barkin et al. 1974; friedmann et al. 1973; stoenner and maclean 1958) . skin abrasions or exposure to mucous membranes may serve as the portal of entry. all secretions and excretions from infected animals should be considered infective. in one instance, a father apparently was infected after his daughter used his toothbrush to clean a contaminated pet mouse cage (boak et al. 1960) . rodent bites can also transmit the disease (looke 1986 ). in detroit, children from the inner city had a significantly higher because of the variability in clinical symptoms and the lack of pathognomonic pathologic findings in humans and animals, serologic diagnosis or actual isolation of leptospires is imperative (faine 1991) . as an aid to diagnosis, leptospires can sometimes be observed by examination or direct staining of body fluids or fresh tissue suspensions (sulzer et al. 1968 ). the definitive diagnosis in humans or animals is made by culturing the organisms from tissue or fluid samples, or by animal inoculation (particularly in 3-to 4-week-old hamsters) and subsequent culture and isolation. culture media with long-chain fatty acids with 1% bovine serum albumin are routinely used as a detoxicant (faine 1991) . serologic assessment is accomplished by indirect hemagglutination, agglutination analysis, complement fixation, microscopic agglutination, and fluorescent antibody techniques (faine 1991) . the serologic test most frequently used is the modified microtiter agglutination test. titers of 1:100 or greater are considered significant. molecular techniques including pcr and randomly amplified polymorphic dna fingerprinting are used for identification of serovars (tappero et al. 2000) . in mouse colonies infected with l. ballum, antibodies against l. ballum were detected in sera of mice of all ages, but 26. zoonoses and other human health hazards 727 leptospires could be recovered only from mature mice. progeny of seropositive females had detectable serum antibodies at 51 days of age but not at 65 days. it was also reported that progeny of seropositive female mice, which possessed antibody at birth and acquired additional antibody from colostrums, remained free of leptospires if isolated from their mothers at 21 days of age, despite exposure during the nursing period (stoenner 1957) . studies in mice experimentally infected with l. grippotyphosa demonstrated that maternal antibodies, whether passed through milk or placental transfer, conferred protection of long duration against the carrier state and shedding of leptospires. thus, serologically positive immune mothers do not transmit the disease to their offspring. however, mice born to nonimmune mothers, if infected at 1 day postpartum, become carriers with no trace of antibodies. thus a population of carrier pregnant mice without antibody could serve as a precipitator in outbreaks among susceptible mouse populations (birnbaum et al. 1972) . field surveys have supported this data in that a significant percentage of carrier mice do not have antibodies. this led to the diagnostic approach, which specifies that both serologic and isolation methods must be utilized to determine the rate of leptospiral infection in rodents (galton et al. 1962) . leptospira ballum is frequently found in the common house mouse (m. musculus) (brown and gorman 1960; yager et al. 1953) . therefore, eradication of infected colonies, use of surgically derived and barrier-maintained mice or of conventional laboratory mice free of leptospira infection, coupled with the prevention of ingress of wild rodents, should effectively preclude introducing of the organism into research and commercial laboratories (loosli 1967) . leptospira ballum has been eliminated from a mouse colony by administration of feed containing 1000 gm chlorotetracycline hydrochloride per ton for 10 days. after 7 days of antibiotic therapy, mice were transferred to clean containers and administered clean water, both having been sterilized by steam. mouse traps and rodenticides were used to destroy escaped mice and to prevent reintroduction of l. ballum by the common house mouse . commercial animal colonies maintained in research vivarium today are not routinely screened for leptospirosis, assuming that the organism has been effectively eliminated from commercial and research-maintained mice. rat bite fever can be caused by either of two microorganisms: streptobacillus moniliformis or spirillum minus. streptobacillus moniliformis causes the diseases designated as streptobacillary fever, streptobacillary rat bite fever, or streptobacillosis. haverhill fever and epidemic arthritic erythemia are diseases associated with ingestion of water, food, or raw milk contaminated with str. moniliformis. sodoku, derived from the japanese words for rat (so) and poison (doku), spirilosis, and spirillary rat bite fever are caused by another bacterium, spirillum minus. the bite of an infected rat is the usual source of infection. in some cases, other animal bites, including mice, gerbils, squirrels, weasels, ferrets, dogs, and cats, or rare traumatic injuries unassociated with animal contact, cause the infection. in a retrospective analysis coveting three decades of 45 s. moniliformis isolates (91% from humans) from the department of public health in berkeley, california, 50% of the isolates were from children < 9 years of age (graves and janda 2001) . in 75% of the human infections where a diagnosis was made, rat bite fever (rbf) was suspected; 83% of those suspected cases involved either known rat bite or exposure to rodents. two cases of rbf were attributed to exposurem in one case a squirrel, and in the second a mouse (graves and janda 2001). interestingly, > 9% of the cases could not be attributed to a rat bite or scratch, indicating that close contact with infected rodents can be sufficient to become infected (graves and janda 2001) . other reports have indicated that the disease can occur in individuals who have no history of rat bites, but reside or work in rat-infested areas or have pet rats with whom they have close contact (fordham et al. 1992; holroyd et al. 1988; rumley et al. 1987) . rat scratches can also be the source of the organism (edwards and finch 1986; shanson et al. 1985) . exposure to cats and dogs that prey on wild rodents may also be the source of the organisms. these organisms are present in the oral cavity and upper respiratory passages of asymptomatic rodents, usually rats (wilkins et al. 1988 ). mice can be infected with resulting morbidity and mortality due to arthritis and pneumonia. in one study, streptobacillus moniliformis was isolated as the predominant microorganism from the upper trachea of laboratory rats (paegle et al. 1976) . presumably the incidence of str. moniliformis is now lower in high-quality, commercially reared specific pathogen-free rats. surveys in wild mice indicate 0 to 25% infection with spirillum minus (hull 1955) . (2004). streptobacillus moniliformis incubation varies from a few hours to 2 to 10 days, whereas spirillum minus incubation ranges from 1 to 6 weeks (table 26-1) . fever is present in either form. inflammation associated with the bite and lymphadenopathy are frequently accompanied by headache, general malaise, myalgia, and chills (arkless 1970; cole et al. 1969; gilbert et al. 1971; mcgill et al. 1966) . the discrete macular rash that often appears on the extremities may generalize into pustular or petechial sequelae. arthritis occurs in 50% of all cases of s. moniliformis but is less common in spirillum minus. streptobacillus moniliformis may be cultured from serous to purulent effusion, which is recovered from affected larger joints. a total of 18 cases of endocarditis due to s. moniliformis were reported from 1915 to 2000 (shvartsblat et al. 2004 ). death has occurred in cases of s. moniliformis involving preexistent valvular disease or as a result of endocarditits in a previously healthy individual. infants can also die of the infection (sens et al. 1989) . if antibiotic treatmentmusually penicillin at doses of 400,000 to 600,000 daily for 7 daysmis not instituted early, complications such as pneumonia, hepatitis, pyelonephritis, enteritis, and endocarditis may develop (richter 1954 ). if endocarditis is present, the penicillin should be given parenterally at doses of 15 to 20 million units daily for 4 for 6 weeks. streptomycin and tetracyclines are also effective antibiotics for those individuals with penicillin-associated allergies. addition of streptomycin to standard therapy is also advised in cases where isolates of sir.. moniliformis are cell wall deficient (rupp 1992) . spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy. streptobacillus moniliformis grows slowly on artificial media but only in the presence of 15% blood and sera, usually 10% to 20% rabbit or horse serum incubated at reduced partial pressures of oxygen. sodium polyanethol sulfonate sometimes found in blood-based media because of its properties as a bacterial growth promoter should not be used due to its inhibitory effects on sir. moniliformis (lambe et al. 1973; shanson et al. 1985) . growth on agar consists of 1 to 2 mm gray, glistening colonies. the api zym diagnostic system can be used for rapid biochemical analysis and diagnosis. a pcr-based assay has also been described to diagnose sir. moniliformis (berger et al. 2001) . the genus salmonella are gram-negative bacteria with approximately 2000 serotypes. nontyphoidal salmonellosis is caused by any of these serotypes. other than salmonella typhi, the causative agent of typhoid fever, salmonellosis occurs worldwide and is important in humans and animals. s. typhi and salmonella choleraesuis have only one serotype, whereas the remaining 2000 serotypes are within the species salmonella enteritidis. references to the salmonella enteritidis serotypes are abbreviated such that "enteritidis" is dropped; for example, s. enteritidis serotype typhimurium is called salmonella typhimurium. salmonella typhimurium is the serotype most commonly associated with disease in both animals and humans. other serotypes most commonly reported from humans and animals are salmonella heidelberg, salmonella agona, salmonella montevideo, and salmonella newport. salmonellae are pathogenic to a variety of animals. salmonella are ubiquitous in nature and are routinely found in water or food contaminated with animal or human excreta. fecal-oral transmission is the primary mode for spreading infection from animal to animal or to humans. transmission is enhanced by crowding and poor sanitation. during the early 1900s, rodenticides containing live cultures of s. enteritidis were distributed on a large-scale basis by commercial and public health organizations in an attempt to eliminate feral rats. these cultures were known as "rat viruses" and were widely used in europe, england, and the united states as "rat poisons" (weisbroth 1979). however, enthusiasm for their use waned when it was discovered that the spread of the organisms couldn't be limited; predictably, the baiting program was implicated in several epidemics among exposed human populations (weisbroth 1979) . surprisingly, as late as the 1950s in england, s. enteritidis (serovar danzy) was isolated from adults living four miles apart. the source of infection was traced to contaminated cakes from a local bakery. mice that had acquired the (brown and parker 1957) . as with other fecal-oral transmitted diseases, control depends on eliminating contact with feces, food, or water contaminated with salmonella or animal reservoirs excreting the organism. salmonella survive for months in feces and are readily cultured from sediments in ponds and streams previously contaminated with sewage or animal feces. fat and moisture in food promote survival of salmonella. pasteurization of milk and proper cooking of food (56~ for 10 to 20 minutes) effectively destroys salmonella. municipal water supplies should be routinely monitored for coliform contamination (pavia and tauxe 1991) . clinical signs of salmonellosis in humans include acute sudden gastroenteritis, abdominal pain, diarrhea, nausea, and fever. diarrhea and anorexia may persist for several days. organisms invading the intestine may create septicemia without severe intestinal involvement; most clinical signs are attributed to hematogenous spread of the organisms. as with other microbial infections, the severity of the disease relates to the organism's serotype, the number of bacteria ingested, and the host's susceptibility. in experimental studies with volunteers, several serovars induced a spectrum of clinical disease ranging from brief enteritis to serious debilitation. incubation varied from 7 to 72 hours. cases of asymptomatic carriers, persisting for several weeks, were common (hull 1955) . salmonella are flagellated, nonsporulating, aerobic gramnegative bacilli that can be readily isolated from feces on selective media designed to suppress bacterial growth of other enteric bacteria. salmonella serotyping requires antigenic analysis (fox 1991) . salmonella gastroenteritis is usually mild and self-limiting. with careful management of fluid and electrolyte balance, antimicrobial therapy is not necessary. in humans, antimicrobial therapy may prolong rather than shorten the period that salmonella are shed in the feces (nelson et al. 1980; pavia and tauxe 1991) . in one double-blind placebo study of infants, oral antibiotics did not significantly affect the duration of salmonella carriage. bacteriologic relapse after antibiotic treatment occurred in 53% of the patients, and 33% of these suffered a recurrence of diarrhea, whereas none of the placebo group relapsed (nelson et al. 1980 ). tick-borne relapsing fever occurs primarily in foci in the western part of the united states, as well as other parts of the world. the disease is caused by at least 15 borrelia species and is transmitted to humans from a variety of rodents (chipmunks, squirrels, rats, mice, prairie dogs, hedgehogs) via soft ticks of the genus ornithodorus. of recent interest are the increasingly recognized enterohepatic helicobacter spp., which cause both hepatic and intestinal disease in mice (whary and fox 2004) . one of these, h. bilis, isolated routinely from mice, has been found using pcr-based assays in bile and gallbladder of chilean patients with chronic cholecystitis and in biliary cancers in japanese patients (fox et al. 1998; matsukura et al. 2002) . whether these new helicobacters will be linked to zoonotic transmission from wild or laboratory rodents will require further studies. pathogenic staphylococcus aureus of human phage type can cause clinical disease in mice and rats. this organism has been (davies and shewell 1964) 1 lab worker % not determined, alopecia, increased scaling on head (booth 1952) and back, 10 mice 1 bacteriologist 60 of 400, crusted or crustless plaques, circular with (cetin et al. 1965 ) prominent periphery; general alopecia; mortality in some mice 1 technician 20% colony with alopecia and scaly skin (dolan et al. 1958 ) 1 technician alopecia with crusting an erythema (povar 1965) introduced into spf barrier-maintained mouse colonies and spf rats and guinea pigs; the same phage type was isolated from their animal caretakers (davey 1962; shults et al. 1973) . colonization by normal s. aureus strains in the nasopharyngeal area of humans presumably minimizes the zoonotic potential of animal-originated s. aureus. as reviewed comprehensively in blank, reports of ringworm (favus) in the mouse began to appear in the european literature in the mid-nineteenth century and in the north american literature during the early twentieth century (blank 1957) . several of the early authors noted the similarities between the lesions of favus in the mouse and in humans. quincke, who is generally credited with isolating the causative agent which he named 3~-pilz (now trichophyton mentagrophytes), suggested that the infection in the mouse was also a source of infection of the cat, and thereby, of humans. earlier reports of murine ringworm referred to the causative agent as t. quinckeanum, but the successful crossing of t. quinckeanum with the perfect state of t. mentagrophytes, arthroderma behamiae, indicates that t. quinckeanum is not a distinct species (ajello et al. 1968) . a later study of the two varieties, t. mentagrophytes var. mentagrophytes and t. m. var. quinckeanum, noted that the conidia from both produced two morphological variants on cultivation (granular and fluffy), and these variants were a. behamiae type + and pathogenic (hejtmanek and hejtmankova 1989) . in addition to t. mentagrophytes, epidermophyton floccosum, mircrosporum gallinae, m. gypseum, m. canis, t. erinacai, t. schoenleini, and t. (keratinomyces) ajello have been reported as zoophilic dermatophytes that can infect mice and cause ringworm in humans (dvorak 1964; krempl-lamprecht and bosse 1964; marples 1967; papini et al. 1997; refai and ali 1970) . the dermatophytes are distributed worldwide and can involve a variety of small animal host species in addition to the mouse. chmel et al. (1975) conducted field studies in a wooded farm setting in czechoslovakia and detected an overall prevalence rate of 4.4% (57 positive of 1288) for t. mentagrophytes infection in 6 of 13 species sampled; the prevalence in mus musculus was 3.4%, with mice comprising 15.8% of the infections detected. of the species that harbored the infection, all frequented the barn or granary area; the seasonal incidence was highest during the winter months when the rodent carriers were more likely to seek harborage indoors. chmel et al. (1975) also analyzed patient data and demonstrated that t. mentagrophytes was the predominant isolate from those who did agricultural work, while t. verrucosum was the main isolate from individuals who worked with farm animals. also, human t. mentagrophytes infections were most common on the hands, wrist, forearm, face, and neck, unprotected skin sites readily contaminated by fodder, litter, or other materials while working in the barns. ringworm infections associated with the handling of bags of grain in which mice had been living have also been reported (alteras 1965; blank 1957) . ringworm infection in laboratory mice is often asymptomatic, remaining unrecognized until laboratory personnel become infected. early reports in the literature indicated that the prevalence of t. mentagrophytes was 80 to 90% among some laboratory mouse stocks (davies and shewell 1964; dolan et al. 1958 ). however, these reports predated the era of modem laboratory animal colony management marked by the commercial availability of cesarean-derived, barrier-maintained rodents. moreover, the modem production practices that have been universally adopted by the industry for decades and the use of microbarrier cages with appropriate technique have further reduced the opportunity for ringworm to become a significant problem in contemporary colonies. in recognition of this fact, none of the major commercial vendors in the united states survey their colonies for dermatophyte infections as part of routine health monitoring. sporadic cases of ringworm infections in rodents have been reported in the past three decades (hironaga et al. 1981; mizoguchi et al. 1986; papini et al. 1997) . programs involved in importing mice from sources that fail to meet contemporary rodent production and husbandry practices should consider screening mice for dermatophytes during the quarantine period. the ease of transmission of dermatophytes from animals to humans is well known and is a significant health hazard. laboratory mice, as well as other laboratory animal species, can harbor dermatophyte infection, with few or no visible skin lesions transmitting the infection to unsuspecting personnel (dolan et al. 1958) . transmission can occur through direct contact with the infected animal or through indirect contact with animal bedding or other materials in the environment of the contaminated animal room. rigorous facility and equipment sanitation has long been recognized as an essential element of an effective control program and should be undertaken in conjunction with efforts to treat valuable animals or to repopulate previously contaminated areas of a facility (davies and shewell 1964; dolan et al. 1958; mizoguchi et al. 1986 ). the importance of barrier protections by donning appropriate clothing, using gloves and other personal protective equipment, and modifying work practices to minimize skin exposure to dermatophytes has also been acknowledged for the prevention of transmission (dolan et al. 1958) . when prevention methods fail, allowing the introduction of dermatophyte infection into a mouse colony, and when transmission to humans occurs, clinical cases of dermatophytosis routinely respond well either to topical or systemic antifungal therapy. *found in laboratory animals that cause allergic dermatitis or from which zoonotic agents have been recovered in nature (see yunker 1964) . **wee, western equine encephalitis. +sle, st. louis equine encephalitis. ++rmse rocky mountain spotted fever. dermatophytosis or ringworm in humans can be asymptomatic and minor, often self-limiting and drawing little attention from the affected individual. the infection usually causes an expanding, scaly and erythematous inflammatory plaque on the skin that occasionally contains fissures or vesicles when severely eczematous. on the trunk and extremities, the lesion may consist of one or more circular lesions with a central clearing and sharply defined margins, forming a ring, and hence the name "ringworm" (fig. 26-1) (merlin et al. 1994) . other dermatophytes are named according to the sites of involvement on the body (e.g., tinea pedis for foot infections, tinea capitis for scalp infections). the dermatophyte infections of humans associated with direct or indirect contact from mice usually involve the body or extremities, especially the arms and hands. zoophilic t. mentagrophytes infection usually produces a highly inflammatory lesion and often undergoes rapid resolution. however, it can also produce furunculosis--deep infection of the hair follicles or widespread tinea corporism which is also seen in infections of e. f l o c c o s u m . in a laboratory-acquired infection with t. (keratinomyces) ajelloi, mice were the source of infection for a laboratory technican who developed small, grayish-white, scaly lesions on both hands. hand lesions yielded the organism, as did 2 of 250 apparently health mice (refai and ali 1970) . a. tapeworms a. reservoir and incidence although this parasite occurs in the mouse intestine, it is more commonly associated with rats and is especially common in wild norway (rattus norvegicus) and black (rattus rattus) rats throughout the world (faust and russell 1970; stone and manwell 1966; wardle and mcleod 1952 (voge and heyneman 1957) . larval development in tribolium sp. at 30~ requires 8 days. therefore, humans become infected only through ingestion of infected insects, such as flour beetles, which may contaminate rodent food or cereal marketed for human consumption. c. clinical signs the infection in humans is usually asymptomatic, but in moderate to heavy infections it may cause headaches, dizziness, abdominal discomfort, and diarrhea. the greatest length of an adult parasite removed from a patient was 1 meter. usually, adult parasites are 20 to 50 cm long and as much as 4 mm wide (markell et al. 1999 ). a. reservoir and incidence the dwarf tapeworm is a common parasite of both the wild house mouse and the laboratory mouse. as indicated earlier in the text, in most well-managed mouse colonies, r. nana incidence is low compared to earlier reports of its high incidence in rodent colonies (wescott 1982) . the estimate that 20 million humans in the world are infected was made many years ago but probably is an underestimate (markell et al. 1999) . surveys conducted in central europe report that this tapeworm in humans is more prevalent in warm than in temperate regions. an incidence of 10% has been noted in some south american countries (jelliffe and stanfield 1978) . it is most commonly diagnosed in children. diagnosis is made by observing characteristic eggs in the feces. b. mode of transmission r. nana is unique among tapeworms in that the adult worm develops after the egg is ingested. the hooked oncosphere then invades the intestinal mucosa and develops into a cysticeroid larva. rodentolepsis nana eggs can contaminate hands, be trapped on particulate matter, or be aerosolized, and then accidentally ingested. since no intermediate host is required, the eggs are readily infective for the reciprocal hosts (faust and russell 1970) . precautions against infection include strict personal hygiene, appropriate laboratory uniforms, and use of disposable gloves and face masks when handling contaminated bedding and feces. c. clinical signs the clinical picture of r. nana infection is quite cosmopolitan. in well-nourished persons, essentially no symptoms occur; the infection is noted when the proglottids or ova are seen in the stool. in other persons, the symptoms include headaches, dizziness, anorexia, inanition, pruritis of the nose and anus, periodic diarrhea, and abdominal distress. a tapeworm identified as r. nana was found in a tumor removed from the chest wall (jelliffe and stanfield 1978) . the diagnosis is based on identification of the characteristic eggs or proglottids in the stool. d. treatment praziquantfel, given orally in a single dose of 25 mg/kg body weight is the drug of choice. alternatively, niclosamide is given daily for 5 days because of the tissue phase of the parasite. the dose is 2 gm for adults and 1.5 gm for children > 34 kg, and 1.0 gm for children between 11 and 34 kg (markell et al. 1999) . recently, a parasite known to naturally colonize mice, r. microstoma, has been identified in the feces of humans living in the northwest of western australia (macnish et al. 2003) . although r. nana was the most common enteric parasite based on microscopic examination of feces, r. microstoma was identified as a mixed infection in 4 of 11 individuals by using a molecular-based assay consisting of restriction fragment length polymorphism of tapeworm dna as well as a sequencing of the pcr product of the internal transcribed spacer 1 region of ribosomal dna (macnish et al. 2002) . given that r. microstoma requires an intermediate host, tribolium confusum for its life cycle, it is understandable why it was not as common as r. nana in this study. however, given the morphological similarities of the eggs of r. nana and r. microstoma, the true prevalence of r. microstoma in humans won't be known until molecular techniques to differentiate the two species are utilized in future studies. syphacia obvelata is an ubiquitous parasite in both wild and laboratory mice. although parasitology texts report that syphacia is infectious to humans, this citation originates from a publication in 1919, in which two s. obvelata adult worms and eggs reportedly were found in the formalin preserved feces of a filipino child whose entire family of five was infected with h. nana (riley 1919) . no mention is made of the method of collecting the feces, nor is it known whether the feces could have been contaminated with murine feces or with the parasite and/or eggs. the only other report is an unpublished finding of s. muris eggs in the feces of two children and two rhesus monkeys, cited in a personal letter from dr. e. e. faust of tulane university, dated january 6, 1965 (stone and manwell 1966) . both of these cases may therefore be examples of spurious parasitism, but definitive information for that conclusion is lacking. regardless, no published information indicates that laboratory personnel have been infected by working with syphacia-infected mice. contamination of food or utensils or accidental ingestion of syphacia ova (e.g., via contaminated hands) could result in infection of humans. people working with infected mice probably ingest ova occasionally, but there is no evidence that this exposure results in active infection. because syphacia infection in humans has not been described, clinical signs have not been noted. there are striking differences in size between specimens of female s. obvelata and those of enterobius vermicularis, the pinworm, in humans (markell and voge 1965) . syphacia is 3.5 to 5.8 mm long, whereas the enterobius sp. female reaches a length of 8 to 13 mm. the male syphacia sp. measures 1.1 to 1.5 mm compared to 2.5 mm for enterobius. the size difference between the eggs of the two species is also marked: syphacia eggs are more than twice as long (125 gm versus 52/am as those of enterobius). it is unlikely therefore that syphacia sp. would be misdiagnosed as enterobius sp., assuming, of course, that the observer was aware of the size difference and measured the eggs. although many species of mites are found on laboratory mice, only ornithonyssus bacoti, the tropical rat mite, and liponysoides sanguineus, the house mouse mite, are vectors of human disease. ornithonyssus bacoti is seen in laboratory mice (fox 1982) ; l. sanguineus has been identified only on wild mice. bites from these mites, as well as from another mouse mite, haemalaelaps casalis, are responsible for allergic dermatitis, or local inflammation, in humans. ornithonyssus bacoti can be found on many rodents; the brown norway rat and the black roof rat are probably the primary host species (beaver and jung 1985) . since the time of the first report of human ornithonyssus bacoti-associated dermatitis in australia in 1913, and a 1923 report in humans in the united states, many other cases have continued to be described throughout the world (see table 26 -4) (charlesworth and clegern 1977; chung et al. 1998; dove and shelmire 1931; dowlati and maguire 1970; engel et al. 1998; fox 1982; haggard 1955; hetherington et al. 1971; riley 1940; theis et al. 1981; wainschel 1971; weber 1940) . ornithonyssus bacoti is an obligate bloodsucking parasite, usually tan but red when engorged with blood. both the male and female feed on a rodent as their preferred host. the female is 700 ~tm to 1 mm in length; the male is smaller (fig. 26-2) . eggs are laid in bedding or wall crevices by the female, which survives for about 70 days and feeds about every two days during this period. the mite has five developmental stages: adult, egg, nonfeeding larva, bloodsucking protonymph, and nonfeeding deutonymph. after feeding, the adults and protonymphs leave their host and seek refuge in cracks and crevices. the life cycle from adult to egg requires 7 to 16 days at room temperature. unfed protonymphs have survived for 43 days (brettman et al. 1981) . the mite often gains access to the premises on wild rodents and lives in crevices. if wild rodents are not readily available or are captured, the mite will seek blood elsewhere, either from the wild or laboratory rodent (if in an animal research facility) and/or humans. in some infestations, the rodent shows no clinical signs. however, in more chronic cases, dermatitis and anemia may develop. historically, this mite has been a troublesome parasite in certain laboratory animals, especially rats, mice, and hamsters (fox 1982; keefe et al. 1964 ). a. clinical signs tropical rat mites produce painful, pruritic lesions in humans. examination of patients often discloses papular lesions on the wrists, arms, abdomen, and chest. raised erythematous papules and nodules several millimeters to greater than 1 cm in size occur singly or in linear configuration ( fig. 26-3) . epidemiologically, cases usually occur in clusters that involve a common source of exposure to the mite. experimentally, cases have been shown to be a vector of pathogens. in the laboratory, mite transmission of various rickettsial species, pasteurella tularensis, and coxsackie virus between different laboratory animals has been shown (hopla 1951; petrov 1971; philip and hughes 1948; schwab et al. 1952) . affected individuals may be treated with topical lindane or treated symptomatically, given that the mite does not reside on humans for any extended periods. papular dermatitis will regress after a period of 7 to 10 days post-therapy. recurrence of ornithonyssus bacoti infestations is common unless the premises have been treated with an appropriate insecticide, and any feral rodents eradicated (engel et al. 1998 ). fleas are seldom found in laboratory mice but are common parasites of feral rodents. the oriental rat flea, xenopsylla cheopis, and another flea, nasopsyllus fasciatus, both naturally infest mice and rats; they are vectors for murine typhus. apparently, x. cheopis is easily established in animal facilities. at a midwestern u.s. university, it inhabited a room housing laboratory mice where, on two separate occasions, the flea caused distress by biting students (yunker 1964) . leptopsylla segnis, the mouse flea, bites humans and is a vector for plague and typhus, serious diseases in humans. also, l. segnis can serve as an intermediate host for the rodent tapeworms r. nana and r. diminuta, which can infect people. the flea's bite can also be irritating and cause allergic dermatitis. epidemiological perspective on the transmission of infectious diseases, principally rabies. the bite of the rat is far more powerful and more likely to be disfiguring than that of the mouse, and rat bites are known to be associated with the transmission of bacterial zoonoses such as rat bite fever and leptospirosis (see elsewhere in this chapter). one should assume that the mouse is also capable of transmitting these agents via bite. rabies transmission from small rodents in the wild occurs but is exceedingly rare (gdalevich et al. 2000) ; therefore, rabies is of concern only if experimental studies with the virus are being conducted in mice. mice can also transmit hantavirus infection and lymphocytic choriomeningitis virus infection via bites. anecdotally, most animal care and use programs report that rodent bites among personnel are a reasonably common occurrence that often are unreported to an institution's occupational medical service, despite the fact that bites inflict pain, produce anxiety, and may have significant health consequences. in addition to the hazard of zoonotic disease or local wound infection with pyogenic or toxin producing bacteria such as clostridium tetani, staphylococcus spp., streptococcus spp., escherichia coli, and bacillus subtilis, rodent bites, including those of the mouse, can induce a severe local allergic reaction or anaphylaxis in individuals previously sensitized to allergen (hesford et al. 1995; teasdale et al. 1983; thewes et al. 1999) . thus, bite wounds from mice should be immediately cleaned thoroughly and reported to the institutional occupational medical service to permit evaluation of the person's tetanus immunization status and need for additional local wound or other medical care. the need for additional training of bitten persons in animal handling may also be indicated. authoritative information on the incidence and impact of animal bites in the general population over the past several decades is scant, and reliable data on the incidence of mouse bites among personnel who work in laboratory animal facilities or among the general populace is lacking. there have been occasional studies on the occurrence and clinical characteristics of rat bites within urban populations, including a recent investigation of 622 bites over the period 1974-1996 that associated this phenomenon with urban blight, poverty, and unemployed populations (hirschhorn and hodge 1999) . traditionally, animal bites have received attention from the clinical perspective of wound management and complications and from the allergic skin and respiratory reactions to laboratory mice are very common in laboratory animal caretakers and technicians who work with these animals. a survey by lutsky (1987) demonstrated that three-fourths of all institutions with laboratory animals had animal care personnel with allergic symptoms. the prevalence of symptoms of laboratory animal-associated allergy (laa) among personnel working with laboratory animals has been estimated as between 10 and 46% in numerous recent studies, and among these individuals, approximately 10% are estimated to eventually proceed to the development of asthma (chan-yeung and malo 1994; eggleston and wood 1992; hollander et al. 1996; hunskaar and fosse 1990; knysak 1989; renstrom et al. 1994) . furthermore, other sources have suggested that among atopic individuals with preexisting allergic disease, up to 73% of persons exposed to lab animal allergens may eventually develop laa (committee on occupational health and safety in research animal facilities/national research council allergens 1997). the population at risk for work-related exposure to rodents was estimated at 90,000 (newill et al. 1986 ); this population has likely grown in the intervening years to the expanding populations of genetically modified mice that are used in contemporary biomedical research programs and require care. moreover, a recent study would seem to suggest that the risk of exposure to mouse allergens is not confined to those working in the laboratory animal facility environment. data analyzed from the first national survey of lead and allergens in housing in the united states demonstrated that 82% of homes of diverse types and income levels across geographic locations had evidence of mouse allergen; 57% had detectable levels on the kitchen floors specifically; and 22% had allergen concentrations greater that 1.6 ~tg/g of dust collected, a level previously correlated with the significantly increased rate of sensitization to mouse allergen (cohn et al. 2004) . the large number of staff at risk of exposure in the workplace or already presensitized, in combination with the substantial added costs to employers for the medical management, operational disruptions, and retraining efforts related to employees who develop laa and later proceed to asthma, should provide the impetus for many institutions to pay grater attention to this element of the occupational health and safety program (schweitzer et al. 2003) . the major allergen of the laboratory mouse is the mus m 1 protein, a member of the mouse major urinary proteins encoded by a multigene family consisting of approximately 35 genes (clark et al. 1984a (clark et al. , 1984b . the earlier literature on the subject of mouse allergy referred to the mouse urinary proteins (mups), whereas recent literature cites the specific protein (mus m 1) that is now known to be the primary offending allergen in the mup multigene family. the mus m 1 protein is in the lipocalin family of proteins that are produced in the saliva and liver and are excreted in the urine at levels 100 times higher than are present in mouse serum. lipocalins serve to bind small hydrophobic molecules and function biologically to transport vitamins, small volatile odorants, and pheromones conferring the characteristic odor to mouse urine (cavaggioni et al. 1999; flower et al. 1993; konieczny et al. 1997; santa et al. 1998; virtanen et al. 1999) . several studies have indicated that production of mus m 1 is under hormonal control and that the urine of male mice contains four-fold higher levels than the urine of female mice (hastie et al. 1979; lorusso et al. 1986; price and longbottom 1987) . in addition to being present in the saliva and urine, mus m 1 in the serum becomes incorporated into the pelt, conferring the allergenic property to mouse dander. the main allergens of many furred animals are structurally similar proteins within the lipocalin family, including those of the cow (bos d 2), horse (equ c 1), dog (can f 1), and rat (rat n 1) (virtanen et al. 1999) . the mus m 1 and rat n 1 lipocalin allergens, to which 90% of mouse and rat allergic individuals react, respectively, are closely related, sharing a 66% homology (clark et al. 1984a) . some have proposed that personnel exposed to laboratory animal allergens can be categorized into four basic risk groups based on their history of allergic disease and sensitization to animal proteins (committee on occupational health and safety in research animal facilities/national research council allergens 1997). these risk groups are (1) normal individuals, (2) atopic individuals with preexisting allergic disease, (3) asymptomatic individuals with ige antibodies to allergic animal proteins, and (4) symptomatic individuals with clinical symptoms upon exposure to animal allergens. individuals in the normal risk group do not have a history of allergic disease, and 90% will never develop symptoms of laa. if laa develops in individuals in the normal risk group, it usually appears during the first three years of exposure. however, infrequently individuals in this risk group who have remained free of laa for 10 or more years of exposure have developed a delayed onset of the condition (department of health and human services, national institute of occupational safety and health 1997). atopic individuals have a genetic predisposition for an exaggerated tendency to mount ige responses to common environmental allergens. atopic individuals have higher total levels of ige in the circulation and higher blood eosinophil counts compared to normal individuals, possibly as a result of the activation of cytokines involved in ige isotype switching, eosinophil survival, and mast cell proliferation (janeway et al. 2001 ). among atopic individuals, up to 73% of those exposed to allergenic animal proteins eventually develop symptoms (agrup et al. 1986) . in asymptomatic individuals with elevated circulating ige antibodies to animal allergens, up to 100% are at risk of developing allergic symptoms. of the individuals in risk groups that are already symptomatic for laa, approximately 33% will develop chest symptoms and 10% are likely to develop occupational asthma and face the prospect that continued exposure will result in permanent impairment. allergic rhinitis, allergic conjunctivitis, and contact urticaria are the most common disorders seen in laa (committee on occupational health and safety in research animal facilities/ national research council allergens 1997). clinically, allergic rhinitis and conjunctivitis present with the symptoms of sneezing, clear nasal discharge, nasal congestion, itchiness, and watery eyes. contact urticaria presenting as raised, circumscribed, erythematous lesions may also be present in laa patients who report an intense itchiness to the skin in the area of contact with the allergen. figs. 26-4 and 26-5 (fox and brayton 1982) illustrate the typical wheal and flare reaction on the skin provoked in an individual who had developed hypersensitivity to mouse urine over a period of several years and who was exposed by having a mouse with urine-contaminated feet walk over his arm (ohman 1978) . one large survey of laboratory animal workers summarized in the niosh alert (department of health and human services, national institute of occupational safety and health 1997) reported that of 5641 animal workers from 137 animal facilities, 23% developed allergic symptoms related to laboratory animals. of the workers reporting symptoms, 82% had nasal or eye symptoms, 46% had skin complaints, and 33% had asthma. patients who develop asthma as a more serious complication of laa manifest symptoms of wheezing, intermittent dyspnea or shortness of breath, cough, often nocturnal or in the early morning, and tightness of the chest. the key clinical sign in these patients is wheezing on auscultation, and physiological abnormalities include airflow obstruction, which may vary over time, bronchodilator responsiveness, and increased airway responsiveness (airway hyperreactivity) (tang et al. 2003) . though quite rare, generalized anaphylactic reactions that are potentially life threatening can occur in individuals highly sensitized to animal allergens. anaphylaxis may manifest as diffuse itching, hives, and swelling of the face, lips, and tongue. in some individuals, breathing becomes difficult owing to laryngeal edema, and others develop asthma and wheezing. laboratory animal-associated allergy is an example of the type i, ige antibody-mediated, immune reaction, and the reader should refer to other sources for a detailed discussion of the molecular mechanisms involved in developing this reaction (janeway et al. 2001 ). in the case of animal allergens, the usual route of initial exposure is airborne, although bite exposures (saliva) and direct contact with the skin can also become important in later clinical symptoms. in the type i reaction, upon exposure to antigen, which is often a protein or glycoprotein, the allergen is taken up and processed by cells of the innate and adaptive immune systems and by dendritic cells located in the mucosal-associated lymphoid cells, gut-associated lymphoid cells, and/or the dermis. the cytokine profiles of these cells favor the development of na'fve cd4 t cells into th2 cells that induce b cells to produce ige specific for the allergen. once the ige response is initiated, it can be further enhanced by basophils, mast cells, and eosinophils that also drive allergen-specific ige production (janeway et al. 2001) . ige is normally found only in low levels in the circulation because it binds to tissue mast cells and circulating basophils. in the sensitized individual, restimulation with the sensitizing allergen results in allergen binding to ige and the release of histamine and other chemical mediators from the mast cells and basophils. these mediators produce the array of clinical signs and symptoms that are characteristic of the allergy: itchiness, nasal congestion, sneezing, nasal and ocular drainage, coughing, wheezing, and shortness of breath. to establish the diagnosis of laa related to mouse exposure, the physician should begin by considering the strength of the history, physical examination findings, the temporal relationship between the patient symptoms and the environmental exposure to mice, and possibilities of alternative explanations for the patient's problems such as exposure to other potential allergens in the workplace or allergens of a nonoccupational nature. the development of clinical symptoms concomitant with or following exposure to an environment containing mice or mus m 1 laden mouse products should help in narrowing the number of allergens tested. the patient's family history of allergy is also very important to consider because atopy is a proven risk factor in developing laa (botham et al. 1995; meijer et al. 2002; venables et al. 1988 ). physical examination of the patient and clinical monitoring for the progression of allergic disease incorporate a number of approaches. pulmonary function tests such as bronchial hyperresponsiveness (in response to pharmacologic challenge with methacholine and not the specific allergen) and the forced expiratory volume in one second (fev1) are commonly used to evaluate the degree of airway impairment and the response to bronchodilators, glucocorticoids, and other therapeutic agents. radiographs may also be useful in patients with pulmonary involvement. routine laboratory tests may also aid in the characterization and management of the patient's condition, such as complete blood count and nasal smears for eosinophilia which is common in allergic individuals but also can be seen in those with perennial nonallergic rhinitis (dykewicz et al. 1998) . measurement of total serum ige has little value to the physician as an aid in distinguishing whether a particular patient has allergic disease, but it may offer some potential for the identifying of populations at risk for developing of laa as indicated in both prospective and cross-sectional studies of laboratory animal workers (hollander et al. 1996; renstrom et al. 1995) . use of the radioallergosorbent test (rast) for the detection of human ige antibodies of defined allergen specificity is also available for patient evaluation. however, the quality of the laboratory performing the in vitro rast assays, the specificity of the allergens used, and the potential for cross-reactivity are important considerations in adopting the rast as a diagnostic tool (hamilton 2003) . even when properly conducted, in vitro tests usually fail to detect a modest number of skin testpositive individuals, and on a per-test basis, skin tests have lower time and reagent costs (hamilton 2003) . clinicians agree that when properly performed, prick-puncture skin tests are generally considered the most convenient and least expensive screening method for detecting allergic reactions in most patients (demoly et al. 2003) . the valid interpretation of these tests relies on standardized allergens and methods, and negative prick-puncture tests may be confirmed by more sensitive intradermal techniques. even after falsepositive and false-negative tests have been eliminated, the proper interpretation of results requires thorough knowledge of the patient's history and physical findings. a positive skin test alone does not confirm a definite clinical sensitivity to an allergen in the asymptomatic patient but possibly predicts the onset of allergic symptoms. a positive skin test in conjunction with a history suggestive of clinical sensitivity strongly indicates the allergen as the cause of the disease (horak 1985) . strong positive skin tests along with a suggestive clinical history also correlate well with results of bronchial or nasal challenges with the antigen. the animal facility conditions and practices that contribute to mouse-associated laa as a serious and prevalent workplace hazard have received considerable study over the past several decades, enabling effective strategies for achieving control of exposures in most research animal care and use settings. in summary, these strategies involve exposure reduction through source reduction, containment of hazard through the use of modern equipment and engineering controls, and barrier protection with personal protective equipment. the mus m 1 allergen load in the environment is markedly increased when male mice are used in studies due to the fact that they excrete 4-fold higher levels of allergen in the urine than do female mice (lorusso et al. 1986 ). therefore, sources have recommended, that whenever scientifically possible, use of only female mice would be a means of reducing allergen load in the environment and minimizing the exposure of personnel (department of health and human services, national institute of occupational safety and health 1997; renstrom et al. 2001 ). furthermore renstrom et al. (2001) reported a three-fold higher rate of allergic sensitization in technicians who worked with male rodents. although this approach may be useful in a few studies, this method of source reduction would appear to have only very limited applicability across the broad scope of contemporary studies using mouse models. source reduction of mouse allergen is also achieved through reduction of animal density within an animal room (the number of animals per room volume) and through use of frequent, effective facility sanitation practices (department of health and human services, national institute of occupational safety and health 1997). the risk of exposure to mouse allergen varies by the type of animal-related activities conducted by personnel and by the type of animal housing systems and equipment containment devices employed in the use and maintenance of laboratory mice (gordon et al. 1997 (gordon et al. , 2001 schweitzer et al. 2003; thulin et al. 2002) . many studies have examined the different caging systems used for mouse housing, and the ability of each cage system design to reduce environmental allergen is well known (gordon et al. 2001; schweitzer et al. 2003) . the application of just a simple filter sheet top or fitted filter bonnet to an open cage is effective in reducing ambient allergen levels (reeb-whitaker et al. 1999) . however, studies indicate the clear superiority of individually ventilated caging (ivc) systems run under negative pressure for the purpose of controlling room allergen levels (gordon et al. 2001; reeb-whitaker et al. 1999; schweitzer et al. 2003) . gordon et al. (2001) suggested that the use of efficient negative ivc in combination with other engineering controls for allergen containment during procedures and waste processing would potentially produce a virtually allergen-free work environment. when negative pressure ivc housing is not available, the placement of cages in a hepafiltered, ventilated cabinet is effective at reducing room allergen loads (thulin et al. 2002) . gordon et al. (1997) reported that individuals who have direct contact with mice (animal technicians) have the highest exposure, followed by those who have intermittent contact with anesthetized animals or mouse tissues (scientists and necropsy technicians), followed by those with indirect contact (office workers or histology technicians). the specific animal husbandry activities that are known to result in high exposure of personnel to mouse allergen are cage-changing activities, including animal transfer, stacking dirty cages, and manual emptying of cages; handling animals directly (particularly males); and room sweeping (gordon et al. 1997) . for each of these activities, use of improved containment equipment and changes in equipment handling procedures are effective in the controlling the allergen hazard and should be encouraged. for example, use of ventilated cabinets or biological safety cabinets during cage changing and animal handling is effective in conjunction with the use of microisolation cages (gordon et al. 2001; schweitzer et al. 2003; thulin et al. 2002) . containment equipment has also been designed for the capture of airborne allergens generated when the bottom of one dirty cage is placed into the opening of another to stack the cages for transport to the cage wash area or when dirty bedding is removed prior to cage washing (gordon et al. 1997; kacergis et al. 1996) . room cleaning with a vacuum equipped with hepa filtration followed by mopping with a damp mop also aids in reducing the environmental allergen load and personnel exposure (kacergis et al. 1996) . use of personal protective equipment and dedicated work clothing for personnel involved in high-exposure activities is an important asset in reducing allergen exposure. it is important for the work clothing to remain at work, as evidenced by the finding that children of laboratory animal workers had a higher incidence of clinical signs during provocative testing, positive skin tests, and ige specific to laboratory rodents than did the children of parents who worked in other occupations (krakowiak et al. 1999) . full sleeve protection and gloves should be worn to prevent the urticarial reactions in persons who are highly sensitive to the mus m 1 allergen. personnel should also be provided with respiratory protection and eye or face protection when warranted. either filtering facepiece particulate respirators (n95 equivalent) or powered air purified respirators are effective in reducing exposure and alleviating clinical symptoms (schweitzer et al. 2003; thulin et al. 2002) . special attention must be paid to the selection and fitting of n95 filtering facepiece particulate respirators to ensure proper function (morbidity and mortality weekly report 1998). when the elimination of mouse allergen exposure in the workplace is not achieved through the use of engineering controls, work practices, and personal protective equipment, allergic reactions in persons sensitive to mus m 1 can be managed with pharmacological agents that have a long history of use for this condition. these include antihistamines, topical ~-adrenergic agents (bronchodilators), cromolyn sodium as a nasal spray, and intranasal potent glucocorticoids (austen 2004) . prophylaxis in patients with mild symptoms is often provided by topical cromolyn sodium on a continuous basis, supplemented with the intermittent use of antihistamines often at bedtime. the selection of the antihistamine has been an area of considerable discussion, and the reader should refer to casale et al. (2003) for further insights into this matter. in more serious cases, potent topical glucocorticoids may be necessary for alleviating clinical signs. immunotherapy, or hyposensitization, is typically reserved for patients who are unable or unwilling to escape the allergen provoking the response. although allergy to the dog or the cat can be ameliorated by immunotherapy (norman 1998 (norman , 2004 , the infrequent reports in the literature of immunotherapy for allergy to mice and other small laboratory animals have failed to establish the usefulness of this approach for the control of allergy to these species (sorrell and gottesman 1957; wahn and siraganian 1980) . the progress made in the past two decades in the evolution of health care systems responsible for the monitoring, control, and elimination of infectious diseases in laboratory mice as well as the advancements in the facilities, equipment, and techniques used to maintain mice in contemporary animal care and use programs, has vastly reduced the likelihood that personnel will encounter zoonotic diseases or other health hazards in the laboratory under most circumstances. continued program success in the control of mouse-associated hazards relies on the use of well-designed and maintained animal facilities, exclusion of wild rodents and other vermin, and quality control in animal care and veterinary care practices. in unique experimental situations that place personnel at a high risk of exposure to mouse-associated hazards, institutional review should ensure that procedures are carefully planned and conducted using personal protective equipment for worker safety. allergy to laboratory animals in laboratory technicians and animal keepers the relationship of trichophyton quinckeanum to trichophyton mentagrophytes alteras, i. 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rural house mice and in an opossum on the biological character of hvj akitsugu strain and its pathogenicity in human beings as revealed after experimental inoculation of it in volunteers infections of laboratory animals potentially dangerous to man: ectoparasites and other arthropods, with emphasis on mites influenza d in early infancy key: cord-274520-c674wkmt authors: moelling, karin; broecker, felix title: air microbiome and pollution: composition and potential effects on human health, including sars coronavirus infection date: 2020-05-28 journal: j environ public health doi: 10.1155/2020/1646943 sha: doc_id: 274520 cord_uid: c674wkmt polluted air poses a significant threat to human health. exposure to particulate matter (pm) and harmful gases contributes to cardiovascular and respiratory diseases, including allergies and obstructive lung disease. air pollution may also be linked to cancer and reduced life expectancy. uptake of pm has been shown to cause pathological changes in the intestinal microbiota in mice and humans. less is known about the effects of pollution-associated microbiota on human health. several recent studies described the microbiomes of urban and rural air samples, of the stratosphere and sand particles, which can be transported over long distances, as well as the air of indoor environments. here, we summarize the current knowledge on airborne bacterial, viral, and fungal communities and discuss their potential consequences on human health. the current data suggest that bacterial pathogens are typically too sparse and short-lived in air to pose a significant risk for infecting healthy people. however, airborne fungal spores may exacerbate allergies and asthma. little information is available on viruses including phages, and future studies are likely to detect known and novel viruses with a yet unknown impact on human health. furthermore, varying experimental protocols have been employed in the recent microbiome and virome studies. therefore, standardized methodologies will be required to allow for better comparisons between studies. air pollution has been linked to more severe outcomes of sars (severe acute respiratory syndrome) coronavirus (sars-cov) infections. this may have contributed to severe sars-cov-2 outbreaks, especially those in china, northern italy, iran, and new york city. air quality is commonly described by the concentrations of pm (ranging in diameter from 10 μm or smaller (pm 10 ) to below 0.1 μm (pm 0.1 )) and of the gaseous pollutants, ozone (o 3 ), nitrogen dioxide (no 2 ), and sulfur dioxide (so 2 ) [1 -5] . current recommendations for pm 2.5 and pm 10 by the world health organization (who) are 10 and 20 μg/m 3 on average per year, respectively, while upper limits for no 2 , o 3 , and so 2 are 40, 100 (at ground levels), and 20 μg/m 3 , respectively (table 1) . other agencies including the european union (eu), the chinese ministry of environmental protection, and the united states environmental protection ammonia (nh 3 ) can form pm 2.5 , termed secondary fine dust, in a gas-to-particle conversion process [8] . agriculture is the leading source of pm 2.5 secondary fine dust in many parts of the world including europe, which partially originates from nh 3 of fertilizers (figure 1(b) ) [9] . e major health problems described for exposure to air pollution are likely caused by pm and mainly affect the airways and the cardiovascular system [10] [11] [12] [13] [14] [15] [16] . both pm 10 and pm 2.5 can cause eye irritations, allergies, and rhinitis [10] [11] [12] . globally, air pollution contributes to most deaths by chronic obstructive pulmonary disease (copd) and to large proportions of fatalities caused by lung cancer, ischaemic heart disease, stroke, and cardiovascular diseases (figure 1(c) ). pm can also carry heavy metals that are associated with cancer and other diseases [17, 18] . e "beijing cough" is caused by polluting particles from inhaled smog [19] . a recent study described a correlation between pm 10 and hospital admissions for respiratory diseases [20] . pm 2.5 can reach deeper into the lung tissue than larger particles [21] . long-time exposure has been correlated with increased risks of chronic diseases, including copd that can develop into lung cancer (table 2) [10] [11] [12] 19] . cardiovascular diseases linked to pm 2.5 include ischaemic heart disease and stroke [22] . emerging evidence suggests causal associations between pm 2.5 and type 2 diabetes, decreased cognitive functions, attention-deficit/hyperactivity disorder, autism, and neurodegenerative diseases [10] . pm 2.5 may also be linked to premature birth, low birth weight, and sudden infant death syndrome [23] . however, these potential effects of air pollution remain to be better established and quantified. ultrafine nanoparticles (pm 1 and pm 0.1 ) can penetrate the skin, blood vessels, and the lymphatic system and are thereby distributed throughout the body; they can also act intracellularly [24] . short-term exposure has been implicated in exacerbation of the abovementioned diseases, but long-term consequences are largely unknown [25] . however, nanoparticles have been found to induce oxidative stress, which is associated with neurodegenerative disorders, cancer, chronic fatigue syndrome, and cardiovascular and gastrointestinal diseases [26, 27] . moreover, exposure to ultrafine nanoparticles has been linked to cardiovascular diseases in a recent cohort study [25] . worldwide, about 3.3 million people die prematurely from outdoor air pollution each year [9] , and additional about 3.8 million due to household pollution, mostly in developing countries where cooking with open fires is common practice [28] . of these people, 27% die from pneumonia, 20% from copd, 8% from lung cancer, and 45% due to cardiovascular diseases [10] . in western countries, life expectancy is estimated to be reduced by an average of 8.3 months due to exposure to pm 2.5 [29] . globally, the effect of air pollution on life expectancy is estimated to be more than twice as strong as the effects of water, soil, and occupational pollution combined [10] . e estimated 9 million premature deaths annually due to indoor and outdoor air pollution exceed those estimated for smoking (about 7 million) and major infectious diseases (aids, tuberculosis, and malaria combined account for about 3 million premature deaths) [10] . recently, the journal bmc infectious diseases published a special issue on the airborne microbiome, emphasizing on the spread of pathogens via human breath [30] . here, we focus on pathogens [31] and urumqi [32] in china, seoul in south korea [33] , and milan in italy [34] . additional studies investigated the subway systems of new york city, usa [35] , oslo, norway [36] , and hong kong [37] . in these studies, air filters were used to collect pm; microbial nucleic acids were isolated, processed, and sequenced. however, experimental conditions varied which limits direct comparison of the results. in beijing, pm 2.5 and pm 10 levels of a severe smog event were analyzed over seven days and the extracted dna was sequenced on an illumina hiseq 2000 sequencing system to determine microbial compositions [31] . sequencing library preparation included a pcr step since amounts of dna extracted from the air samples were too small for direct sequencing, and generated reads were aligned to nonredundant ncbi complete genomes for taxonomic assignment [31] . e detected microbes included bacteria (86.1% and 80.8% of reads in pm 2.5 and pm 10 , respectively), eukaryotes (13% and 18.3%), 0.8% archaea, and 0.1% viruses in both samples (figure 2(a) ). e most abundant bacteria belonged to the proteobacteria phylum, followed by actinobacteria, firmicutes, bacteroidetes, and cyanobacteria. most inhalable microorganisms were soilassociated and nonpathogenic. however, microbes known to cause allergies and respiratory diseases were detected, including the bacterium streptococcus pneumoniae, the fungus aspergillus fumigatus that can cause asthma and respiratory aspergillosis [40] , and human adenovirus c that causes respiratory, gastrointestinal, and urinary tract infections [41] . rna viruses such as influenza, coronaviruses, or rhinoviruses were undetectable by the employed method. e authors concluded that there was likely no risk for contracting infectious diseases from pollutant-associated microbes, but they recommended fixing soil by vegetation to reduce the amount of airborne microbes originating from fecal and terrestrial sources, including potential allergens [31] . in this context, it is noteworthy that in 2018, china announced to promote revegetation and to increase forestation levels from about 22% in 2016 to 30% by 2050 to tackle air pollution [42] . like in the air of beijing, proteobacteria and actinobacteria were abundantly detected in the air of the city of urumqi in northwest china (figure 2 (b)) [32] . is study also used dna from filtered pm as the starting material, but taxonomic assignment was based on pcr-amplified 16s rrna genes (prokaryotes) and 18s rrna genes (eukaryotes) [32] . several bacteria that may cause diseases in immunocompromised individuals but are typically harmless to the healthy population, such as acinetobacter, delftia, serratia, and chryseobacterium were detected. some of the detected fungal spores are associated with allergies [43] , such as ascomycota, basidiomycota, and zygomycosis. beijing is known for the "beijing" cough, which affects many inhabitants independent of their age [19] . is condition may pose an increased risk for other lung diseases such as infection by sars-cov-2. indeed, exposure to smog has been linked to an increased incidence of respiratory infections [44] and air pollution (such as pm and no 2 ) correlates with increased severity of diseases caused by infections with coronaviruses such as sars-cov-1 and sars-cov-2 [45] [46] [47] [48] [49] . sars-cov-2 may also be spread more efficiently in polluted air by attaching to pm [50] . a virome study from seoul identified dna viruses at different locations, industrial, residential, and a forest (figure 2 (c)) [33] . after removal of particles larger than 0.2 μm by filtration, samples were concentrated by tangential flow filtration and virus particles were purified by cscl density centrifugation. dna was extracted and, without pcr amplification, subjected to 454 pyrosequencing. reads were assigned to viral sequences using the camera databases and were taxonomically assigned with megan [33] . e study was not designed to detect any rna viruses. e authors identified predominantly plant-infecting singlestranded dna (ssdna) viruses of the gemini-, nano-and circoviridae families. nanoviridae are aphid-transmitted plant viruses with circular ssdna segments [51] . circoviridae also have circular genomes and infect plants, birds, pigs, fish, and insects [52] . geminiviridae consist of two capsids, each containing a circular ssdna of opposite polarities [53] ; some members can significantly damage crops [54] . in addition, microviridae, ssdna phages infecting enterobacteria, were identified. e authors also detected caudovirales, tailed phages with double-stranded dna genomes. microviridae and caudovirales comprise the most abundant phage populations in the human intestinal tract [55] and have also been identified in marine environments [56, 57] . no human pathogenic viruses were detected. however, previously unknown ssdna viruses were identified. further studies will be necessary to address potential risks of the airborne virome on human health and on crop productivity. a study in milan, northern italy, evaluated forty air samples from ten days of sample collection during different seasons for bacterial and fungal communities ( figure 2 (d)) [34] . e study relied on extracted bacterial dna and pcramplified 16s rrna genes that were sequenced on an illumina genome analyzer iix; taxonomic assignment was carried out with the rdp bayesian classifier [34] . around 10 4 , mainly soil and plant-associated, bacteria per cubic meter of air were detected, with actinobacteria and proteobacteria dominating [34] . significant seasonal and temperature-dependent variations were observed, for instance, with more actinobacteria on colder days. e authors did not address whether potentially pathogenic or allergy-inducing species were detected. e air of the new york city subway system was found to contain microorganisms mainly originating from outdoor air with a minor proportion from human skin [35] . here, dna extracted from filtered air samples was subjected to pcr to amplify 16s and 18s rrna genes, the amplicons were subsequently sequenced by 454 pyrosequencing, and taxonomic assignment was achieved using the silva database [35] . on average, samples contained 40.4% proteobacteria, 28.6% actinobacteria, 18% firmicutes, 9.1% bacteroidetes, 1.2% cyanobacteria, and a complex mixture . surprisingly, no known human pathogens were detected, but some of the detected fungi may cause allergies. e severity of the outbreak of sars-cov-2 in new york city may have been partly due to the high population density, high mobility, pollution, and also preexisting conditions such as obesity, which affects about 40% of the us population and may be a factor contributing to more severe outcomes of covid-19 [48, 58] . in oslo, aerosols were found to contain bacterial populations comparable to those of new york, with 37 different genera in total, some of them of skin origin [36] . concentrations were about 10-fold lower at night [36] . similarly, the air of the hong kong subway system predominantly contained proteobacteria and actinobacteria (figure 2 (f )) [37] . is study relied on extracted dna subjected to pcr amplification of 16s rrna genes and illumina miseq [34] . bacterial communities of air samples obtained in the subway systems of new york city [35] (e) and hong kong [37] (f ). bacterial communities observed for the troposphere [38] (g) and on sand grains [39] (h). sequencing. taxonomic assignment was achieved by aligning reads against the greengenes rrna gene sequence database using the uclust program [37] . as observed in the new york city subway, bacterial communities showed significant similarities with those of outdoor air samples, with some human skin-associated bacteria also being present. again, known pathogenic bacteria were not detected in this study. besides soil bacteria, the beijing study identified fecal bacteria as a prominent component of air pollution, possibly originating from rural animal farms. in addition, human fecal bacteria from sewage are a possible origin [31] . a study of the air microbiome of the graingrowing region vaud, switzerland, found a strong correlation between aerosolized and grain dust-associated fungal communities [59] . e presence of allergenic and mycotoxigenic species in most samples suggests that these fungal species may contribute to work-related respiratory symptoms of grain workers who, however, are exposed to much higher concentrations than the general population [59] . a study comparing rural and urban areas of the us found that urbanization leads to homogenization of the airborne microbiota, with urban communities exhibiting less geographic variability than rural areas [60] . e rural air microbiome was found to contain large numbers of fungi that are known triggers of allergies, including alternaria and cladosporium [60] . further studies are needed to assess to what extent diseases may result from exposure to the rural air microbiome and how they correlate with concentrations and exposure times. ere is evidence that microbes can be transported across very long distances and to high altitudes [60] . bacteria represented on average 20% of particles between 0.25 and 1 μm in diameter in either cloud-free or cloudy air obtained during the hurricanes earl and karl at 8-15 km altitude in the troposphere [38] . numerous bacterial taxa were identified, including acetobacteraceae, burkholderiaceae, streptomyces, and pseudomonadaceae. proteobacteria was the dominant phylum ( figure 2(g) ). ere were significant differences in microbial communities between samples from the two hurricanes. however, 17 bacterial and fungal species were common across all samples and may represent core members of the stratospheric microbiota [38] . due to the poor resolution of the sequencing approach, the authors were unable to determine if any human pathogenic bacteria were present [38] . e vertical distribution of bacterial communities in the atmosphere above the noto peninsula, japan, between 10-and 3,000-meter altitudes, has also been shown to vary substantially and mainly contained soil and marine bacteria [61] . e authors detected bacilli and proteobacteria, taxa that include known plant, animal, and human pathogens, which they speculated may be dispersed over large distances through high altitudes [61] . whether these airborne pathogens can cause an infection after exposure to high altitude remains to be shown. sand grains can be transported over thousands of kilometers and transport bacteria, such that their populations may even be globally connected [62] . sand grains of 200-600 μm in diameter from a german shore were shown to bind 10 4 to 10 5 bacteria composed of 3,000 to 6,000 different species, mostly of soil and marine origin [39] . a core bacterial community was determined, with 50% of the bacteria present on all sand grains, and the other half varied. proteobacteria was the dominant phylum, followed by bacteroidetes and actinobacteria (figure 2(h) ). e identified bacteria were not discussed as potentially harmful for people. dust from desert soil was shown to transport diverse assemblages of bacteria to the mediterranean [63] . e dust microbiome of the gobi desert was found to contain large amounts of alphaproteobacteria [64] . soil bacteria were more abundant during dust storm events, while the relative abundance of bacteria of anthropogenic origin decreased [65] . anthropogenic bacteria included those carrying antibiotic resistance genes, suggesting that the air microbiome may contribute to the spread of antibiotic resistance over long distances, whereby these genes may get diluted. no human health risks have been described [65] . a concern, however, is the presence of antibiotic resistance genes in the sewage of livestock production that can be transported by water or through air [66] . indoor pollution has been analyzed systematically using household air [67] [68] [69] . here, western households must be distinguished from those in developing countries where open fires used for cooking contribute to pollution, a major health concern and cause of premature mortality [70] . in western households, major sources of microorganisms are humans, pets, plants, plumbing, heating, ventilation/air conditioning, mold, and dust from outdoors [68] . people typically stay most of the day indoors, and the air microbiomes differ significantly between environments such as schools, offices, households, and transportation and even between different rooms of the same household [67] [68] [69] . one cubic meter of indoor air typically contains 10 5 of both virus-like and bacteria-like particles, about a tenth of the concentrations found in outdoor air [68] . fungal spores are less abundant and vary in numbers from around 80 up to 10 4 colony-forming units. humans emit around 10 7 copies of bacterial and fungal genomes per hour [68] . human stool can contain up to 10 9 particles per gram of fecal-transmitted pathogens such as norovirus, shigella, or salmonella [71] . it should be noted that humans carry 10 12 microorganisms on their skin and 4 × 10 13 in their digestive tract [72] and are the dominant sources of bioaerosols in indoor environments [73] [74] [75] . key factors that determine the composition of the indoor fungal and bacterial microbiome appear to be moisture, age of the home, and dog ownership [76] . potential effects on health may come from fungi as a significant source of allergens and mycotoxins [77] , whereby indoor fungal communities are dominated by species originating from outdoors [78] . fungal and bacterial spores can infect animals, plants, and humans [79] , are highly stable, and can survive dormant for years. fungi such as cryptococcus spp. can cause fatal disease in immunocompromised populations, such as aids patients and transplant recipients [80] . however, most microorganisms are benign and protect against harmful microbes, assist in the digestion, train the immune system, and lower the risk of autoimmune diseases [81] . high doses of pathogens are, however, a risk under poor sanitary conditions and exposure to droplets and aerosols from infected people with high titers of pathogens. not surprisingly, the indoor air microbiota of hospitals contain a larger percentage of potential bacterial pathogens than do outdoor samples [82] . indeed, many healthcare facilities are affected by the spread of sars-cov-2 and the resulting infection of healthcare workers and other patients. microbiome studies of hospitals may help to reduce exposure to pathogens; for example, rooms with higher airflow and humidity were associated with fewer airborne human pathogens [82] . us, architectural design may help to reduce transmission of pathogens in healthcare facilities. ventilation systems of trains and airplanes typically recycle cabin air which is passed through filters that do not efficiently remove viruses. during the sars-cov-2 pandemic, this has resulted in almost complete shutdown of long-distance traffic and public transport in many countries. spread of the virus may only be prevented if all passengers are confirmed negative for sars-cov-2 infections via antibody testing or real-time viral tests indicating a virus-free status. such tests are available for influenza virus; they provide rapid results but are often less reliable than laboratory tests. yet, that may be the only fast solution for long-distance travel in trains or airplanes. keeping a safety distance and masks can only help to contain the spread of sars-cov-2 to a certain extent. much less is known about airborne viruses than about bacterial and fungal communities. e international committee on taxonomy of viruses (ictv) lists approximately 6,000 known viruses, of which about 1,500 can cause diseases [83] . patients acutely infected with influenza virus can harbor up to 10 9 virus particles per cubic centimeter in the blood stream and release approximately 10,000 aerosolized viruses by coughing or sneezing [75] . indoors, influenza virus can reach concentrations of up to 2.6 × 10 5 particles per cubic meter [68] . even more infectious by airborne transmission is measles virus, which leads to almost 100% infections upon contact with an infected person [84] . measles virus causes severe disease during childhood and can also be dangerous for adults, especially for pregnant women [84] . noroviruses are relatively stable and can persist in the environment for several weeks [84] . as few as 18 to 1,000 norovirus particles can cause an infection [85] . noroviruses account for about 50% of infectious diarrhea in humans. ere are at least 33 genotypes and acquired immunity is short-lived and not cross-protective, so that a person may encounter several norovirus infections per year. norovirus is usually not seriously harmful to healthy adults, but to young children and the elderly [84] . closed environments such as cruise ships are commonly affected by norovirus outbreaks. coronaviruses are single-stranded positive-sense rna viruses, with seven known to infect humans, including sars-cov-1, mers-cov, and sars-cov-2 [86] . e four others contribute to about 10-15% of the seasonal acute respiratory infections [87] . other seasonal viral infections are caused by influenza a and b viruses, respiratory syncytial virus, and rhinoviruses. respiratory viruses such as influenza or coronaviruses, including sars-cov-2, are transmitted by respiratory droplets (larger drops emitted by coughing, sneezing, or talking) and aerosols (particles smaller than 1 micron in diameter) when they reach susceptible mucosal surfaces of the eyes, nose, or mouth. indirect contact through smear infections from contaminated surfaces may occur but the amount of viable viruses may be small. e transmission of respiratory viruses can be limited by wearing face masks, which reduce the spread of droplets and aerosols between people. outdoors, the viruses are normally too sparse to pose a significant risk for infecting healthy people if a safety distance from other people is maintained. even though droplets may travel a distance of about 30 cm before they sink, a safety distance for up to 2 meters has been proposed to contain the spread of sars-cov-2. face masks covering the nose and mouth can reduce droplet-based viral infections, while only surgical masks may protect against viral aerosols. air pollution as reviewed here can cause lung damage. is is a prominent problem mainly in large cities and manifests itself as "beijing cough," a dry cough highly prevalent in large and polluted cities [19] . ere is evidence that people exposed to severe air pollution are more susceptible to infection with the present sars-cov-2 pandemic virus and experience stronger symptoms, not only in large cities of china but also in other parts of the world [46] [47] [48] [49] [50] [51] . pollution, including pm and no 2 , likely contributed to the spread of sars-cov-2 and severity of disease in northern italy where pollution is severe [46, 47, 49, 50] . in addition to air pollution, preexisting conditions such as overweight may contribute to disease severity, which may especially be relevant for the us, where close to 40% are clinically obese [58] . sars coronaviruses have a history as pollutant through plumbing [88] . for example, sars-cov-1 spread through the plumbing of the amoy gardens building in hong kong, which was not aerosol-tight and thereby allowed the virus to spread from the 7th floor of the 33-story building with contaminated sewage [88] . also, in the hotel metropole in hong kong, twelve people were infected within 24 hours, causing a chain of infection of up to 4,000 people [89] . sars coronaviruses are extremely contagious [90] . strict regimens for infected people in singapore successfully contained the sars-cov-1 outbreak. however, the virus even escaped twice from researchers working under high safety laboratory conditions [91] . 8 journal of environmental and public health phages, the viruses of bacteria, are abundant on our planet, in the oceans, air, soil, and other environments [92] . ey can integrate into bacterial genomes but can also replicate by lysing the bacteria. about 10-20% of bacteria in the oceans are lysed daily by phages [93] . it is not trivial to characterize phages in an environmental sample; they typically require purification, concentration, and pcr amplification steps prior to sequencing and taxonomic assignment [94, 95] . e identification of phages in human samples has recently been discussed in detail [95] . phages were identified in the air of seoul and may therefore spread through the air [32] . yet, they are not known to pose a risk for human health. pollution. an important question is whether air pollution influences the composition of the host microbiota. e gastrointestinal tract harbors the highest number of microbes and may be indirectly affected by high concentrations of pollutant pm [96] . in humans, inhaled pm is rapidly cleared from the lungs and transported into the intestine where it may cause alterations in bacterial community compositions [97] . in a mouse model of inflammatory bowel disease (ibd), orally administered environmental pm 10 at a concentration representing a dose that could occur during periods of high levels of air pollution has been shown to significantly affect the gut microbiota [98] . e proportion of firmicutes was increased, while bacteroidetes decreased and inflammatory responses and gut permeability were promoted (figure 3 ) [98] . epidemiological evidence suggests that air pollutants are also linked to an increased risk for ibd in humans [99] . it has been suggested that air pollution, in general, and pm, specifically, may promote gastrointestinal diseases in humans [86] . recently, it has been shown that pm inhalation may alter the intestinal microbiota in humans [100] . as observed experimentally in mice, an increase in bacteroidetes and a decrease in firmicutes were observed, with health consequences yet to be determined. in addition to ibd, exposure to air pollution has been linked to type 2 diabetes and obesity, possibly due to effects on the intestinal microbiota [101, 102] . specific families of gut bacteria correlated with no x exposure; bacteroidaceae (phylum bacteroidetes) increased, while coriobacteriaceae (phylum actinobacteria) decreased [101] . ese changes were associated with increased fasting glucose levels characteristic of developing type 2 diabetes. in addition, polycyclic aromatic hydrocarbons and other organic pollutants present in pm can be metabolized by gastrointestinal bacteria and thereby alter the composition of the microbiota [103] . alterations in the lung microbiome have been linked to various diseases such as cystic fibrosis, copd, and asthma [104] . for example, patients with asthma and copd have increased relative abundances of proteobacteria compared to healthy individuals. interestingly, it has been shown that individuals exposed to higher levels of pm from household air pollution in malawi showed alterations of their lung microbiome, including higher relative abundances of potentially pathogenic bacteria of the genera streptococcus and neisseria [105] . moreover, domestic biomass fuel use was associated with the presence of an environmental bacterium, petrobacter, which is normally not present in the lung [105] . in summary, there is evidence that environmental pollution can affect the composition of both the gastrointestinal and the lung microbiota, with potential negative effects on human health. us, air pollutants, without directly transporting microbes, can indirectly affect the body's inherent microbiota. we are only beginning to understand the composition of aerial microbiomes and their potential impact on human health. however, from the current data, the following trends emerge for bacterial, viral, and fungal communities, despite the varying methodologies employed by the different studies. e bacterial communities of urban air microbiomes appear to be mainly composed of the phyla proteobacteria, actinobacteria, and firmicutes (figure 3) , while less abundant populations such as bacteroidetes and cyanobacteria are more variable among samples [31, 32, [34] [35] [36] [37] . is is reminiscent of bacterial and viral microbiota of the oceans and the human intestinal tract that are composed of abundant core members and less-abundant variable populations [55, 106] . potential human pathogens are typically below the detection limit in air samples even from closed environments such as subway systems, which means that there is not likely a significant risk for infection [31, 32, [34] [35] [36] [37] . likewise, ambient air appears to not contain significant amounts of known viral pathogens [33] . however, only a small fraction of all viruses found in the environment are known, which makes it difficult to estimate potential effects of the air virome on human health [33] . a major constituent of the airborne virome is bacteriophages that are not known to pose a risk for humans but may affect bacterial populations contributing to the spread virulence and antibiotic resistance genes [33] . coronaviruses. sars-cov-2 is the cause of the current covid-19 pandemic of 2019/2020, which has led to outbreaks of varying severities. high infection and death rates were observed, for example, in wuhan city and other parts of china, lombardy in northern italy, northern iran, new york city, usa, manaus, brazil, and johannesburg, south africa. in some cases, the severity of the outbreaks may have been linked to air pollution in conjunction with a high population density. other risk factors may comprise overweight/obesity, chronic cough, lung diseases such as copd, and infectious diseases such as tuberculosis and hiv/aids [44-50, 58, 107, 108] . sars-cov-2 most efficiently spreads through contact with infected people in indoor environments [109] . is has prompted restrictions of public transport and long-distance travel in many countries worldwide. outdoors, virus-containing droplets or aerosols typically do not travel through air beyond the proposed safety distance of one to two meters in amounts sufficient to cause an infection. a major risk for human health is airborne fungi that can exacerbate diseases including allergies and asthma [31, 32, 35] . studies on fungal air microbiomes may help to identify measures to reduce the abundance of fungal species linked to allergies, asthma, and other diseases in outdoor and indoor ambient air. for indoor environments, it has been shown that the abundance of specific components of the airborne microbiota can be altered by architectural design, humidity, and the degree of air flow [82] . us, hypoallergenic architectural design can be envisioned. ere is evidence that fungal spores are particularly abundant in rural air [59] . interestingly, exposure to indoor dust-borne alternaria spp. was found to be linked to a reduced occurrence of asthma, whereas indoor airborne aspergillus fumigatus and alternaria spp. were positively correlated with asthma [110] . us, exposure to fungi may have both positive and negative consequences for human health, depending on the species and the type of exposure (e.g., dust-borne vs. air-borne). in general, however, it is difficult to compare current studies, as they relied on varying protocols. in the future, standardized methodologies will be helpful to allow for better comparisons between studies. on a larger scale, there is evidence that the microbiome is globally connected [62] and that microbes may be transported over thousands of kilometers by dust and fine sand [63, 111] and through high altitudes up to the troposphere [38, 61] . whether potential pathogens can cause an infection after exposure to high altitude and the associated radiation, however, remains to be shown. while a direct effect of microbes transported over long distances on human health, such as infections, is unlikely, a potential concern is the dissemination of virulence factors and antibiotic resistance genes [65] . exposure to pm, even without attached microorganisms, has been shown to alter the intestinal microbiota and may be linked to diseases such as ibd [98] [99] [100] and type 2 diabetes [101, 102] . whether exposure to specific airborne microbes also influences these diseases remains to be determined. e authors declare that they have no conflicts of interest. karin moelling and felix broecker contributed equally to this work. figure 3 : changes in intestinal microbiota due to pm 10 in a mouse model. il-10 knockout mice, a model for inflammatory bowel disease, were fed with either standard mouse chow (left) or standard mouse chow supplemented with pm 10 for 35 days [99] . en, the bacterial composition in fecal samples of these mice was determined. regret that he passed away after an illness in 2019. e authors would like to thank prof. dr. peter palese (icahn school of medicine at mount sinai) for his generous support. directive 2008/50/ec of the european parliament and of the council of 21 may 2008 on ambient air quality and cleaner air for europe ambient (outdoor) air pollution ministry of environmental protection of the people's 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outdoor home of allergic patients in a dust-storm-affected area ambient particulate matter concentration levels of ahwaz key: cord-296863-xu0h92ac authors: berlinguer, giovanni title: bioethics, health, and inequality date: 2004-09-17 journal: lancet doi: 10.1016/s0140-6736(04)17066-9 sha: doc_id: 296863 cord_uid: xu0h92ac nan first, the technique of preimplantation genetic diagnosis (pgd), which was introduced after in-vitro fertilisation (ivf), allows recognition and elimination of an embryo with a genetic disorder or malformation and permits selection of sex. the international bioethics committee (ibc) of unesco (united nations educational, scientific, and cultural organization) recommended that "pgd be limited to medical indications. therefore sex selection for non-medical reasons is considered to be unethical." 3 nevertheless pgd is also practised, and sometimes morally and legally justified, in developed countries. the debate in frontier bioethics concerns mainly the right of parents to decide the characteristics of their children. sex selection, however, is widely put into practice not only through elaborate technologies but also in daily life through infanticide, discrimination in nutrition and health care, and other barbarous methods. in 1992, amartya sen 4 showed the existence-on the basis of changes in the ratio of women to men in the total population of asia and africa-of hundreds of millions of missing women caused by similar actions. worldwide indignation and appropriate cultural and legal measures have been taken in several countries to reduce this situation. did anything change? sen revisited the data in 2003, 5 and reported that the reduction in female overmortality has been counterbalanced by the spread of sexselective abortion against the female fetus, and as a result the number of missing women is now greater. embryos, fetuses, or babies can be selected by many methods from different cultures, which manifest a unique and crude tendency-this selection is a sharp and visible aspect of gender inequality. in developed countries, acceptance of sex selection through biotechnological methods can hinder any effort to reduce any kind of sex selection in any part of the world. second, the question of whether the human body be bought and sold has a history as long as the existence of slavery, and has been a very embarrassing problem for philosophers, from aristotle to locke, and for theologians. the antislavery movements finally imposed its abolitionone of the most impressive cases of a turn in history by effect of moral principles. it led to the geneva convention (sept 26, 1926) , which called on all nations to pursue the suppression of slavery in all its forms, as soon as possible. but in many places, old and new forms of slavery still exist, including bonded labour, in which a worker or labourer is bound to a company or a landlord for life by inextinguishable debts. a type of such bonded slavery existed in the villages of sultanpur mela, kot momin, and mateela in pakistan. in this case, 6 400 peasants-almost one in every family-sold one of their kidneys for less than us$2000. specialised hospitals transplanted their organs into rich patients coming from different cities and other countries. the peasants were obliged to become rewarded donors for the hope or the illusion to free themselves from debts. in this way, slavery met the biotechnological market of spare parts of human bodies. 7 such rewarded donation is the dark side of the enormous advantages coming from the possibility to transfer, from a person to another, organs, corneas, tissues, cells, gametes, stem cells, and other commodities. the moral question "is there a freedom to sell his own body?" was answered, to a certain extent, long ago by immanuel kant (man cannot be at the same time a person and an object; therefore we cannot sell any part of our body) and by john stuart mill (man has all the liberties, except that of choosing to be a slave). now the debate has been reopened. the june, 2003, edition of the journal of medical ethics 8 was dedicated to supply of organs for transplantation, and particularly to the moral legitimisation of the biotechnological market. in my opinion, the question would be most clear if raised the other way round: has a person the right to buy (or to rent) parts of the body of another human being? robert evans, a uk labour member of the european parliament, answered no, and proposed to declare illegal and punishable such practice, according to which wealthy people "are able to exploit desperate people with no fear of penalties". 9 if the answer to this question is yes, the risk is to legitimise a society in which everything could be bought; ourselves, too, who would be regarded as the final commodity. third, prevailing opinion on human cloning is mainly in favour of the cloning of cells and tissues for therapeutic reasons but is against reproductive cloning because this technique denies the casual combination of genes, restricts individual freedom, and implies genetic predetermination. the few people who are in favour of reproductive cloning affirm that human beings are almost always largely predetermined insofar that they are born in a particular country, time, class, and family. if a person's destiny is to become social, cultural, and moral clones, why should genetic cloning not be permitted? perhaps human liberty and self-determination should prevail over all limits and obstacles, either due to social and gender injustice or manipulation of minds; use of science in favour of genetic arrogance would deny or discourage the daily efforts of any person to build autonomously his or her own future. can we develop universal principles in bioethics? these and similar cases could stimulate the debate and (i hope) growth, by consensus, of common universal values in bioethics and of moral norms, accompanied (sometimes) by legal international regulations. this action becomes necessary for two additional reasons. first, many scientific practices have extended beyond national borders, such as the legal (and sometimes illegal) importation and exportation of stem cells, tissue collections, organs, dna samples, and genetic data. moreover, human experiments are done in several countries, and we should avoid putting unnecessary burdens on poor people and communities or creating new forms of exploitation. the bioethical issues that are generated need fair solutions, in accordance with the plurality of values and with the common interests of the world community. second, positive actions for health are essential on a world and local scale. the idea that the combination of scientific progress and free market would spontaneously extend its benefits worldwide, which was dominant in the past two decades, has failed, and a paradoxical situation about science has arisen. new, impressive advances in biomedical knowledge, which at some times in the past were largely accessible-eg, in the 1950s and 1960s, use of antibiotics against microbial diseases and vaccines against smallpox and poliomyelitis-are now becoming more and more selective. many individuals affected by aids or other serious infectious diseases can benefit from new drugs and survive; however, most people cannot afford to pay for the drugs and could die. in africa and other areas of the world, aids could lead to catastrophic effects similar to those the black death caused in europe in 1347-51. according to roy porter, "plague killed a quarter of europe's population-and far more in some towns; the largest number of fatalities caused by a single epidemic disaster in the history of europe. this provoked a lasting demographic crisis." 10 the differences are in the progress of the aids pandemic, which is slower than that of the black death but equally cruel, and in the fact that now we know the causes and possible remedies for aids, malaria, tuberculosis, and other scourges. the eradication of smallpox, the substantial reduction of childhood diarrhoeal deaths, and the elimination of poliomyelitis in 175 countries show that www.thelancet.com vol 364 september 18, 2004 1087 wellcome library, london rights were not granted to include this image in electronic media. please refer to the printed journal. many goals have already been achieved through knowledge and common action. unfortunately, the undesired but foreseeable result of medical progress tends to increase inequalities, because it is oriented by vested interests and directed towards the rich instead of general goals. annette flanagin and margaret a winker wrote: "the contemporary era of globalization, which was anticipated to capitalize on advances in technology, science, communication, and cross-national interdependence, has been accompanied by gaps . . . and wide disparities in societal resources . . . moreover, only a small fraction of funds for biomedical research is dedicated to research that affects most the poor or supports research conducted by resource-poor scientists and for the benefit of resource-poor populations." 11 the 10/90 gap refers to the fact that only 10% of the us$70 billion spent on health research and development by the private and public sector is used for research into 90% of the world's health problems. a similar (or greater) imbalance exists for expenditures on prevention and health care. benefit-sharing and equal access to advances in biomedical science are now urgent and universal issues. this moral change in values and priorities should guide public policies on health at all levels. if we think of universal principles in bioethics, the fundamental ones should probably be equal dignity of every individual and equity of life, disease, and death. a step towards universal principles-on a european level-is the convention for the protection of human rights and dignity of the human being with regard to the application of biology and medicine, 12 adopted by the council of europe in oviedo on april 4, 1997 , and opened to the signature of other nations. two fundamental articles state that the convention "shall protect the dignity and identity of all human beings and guarantee everyone, without discrimination, respect for their integrity and other rights and fundamental freedom" (article 1), and that "the interests and welfare of human beings shall prevail over the sole interest of society or science" (article 2). the convention includes articles on the rights of the patient, on equitable access to health care, on respect for private life, on non-discrimination on genetic grounds, on transplants, and on prohibition of financial gains "from the human body and his parts as such" (article 21). at its 31st session in 2001, the general council of unesco-after an explicit invitation of the round table of ministers of science-invited its director general to submit the technical and legal studies undertaken regarding the possibility of elaborating universal norms on bioethics. during 2002 and 2003, the ibc of unesco worked on a feasibility study, and concluded in june, 2003, with a report on the possibility of elaborating a universal instrument on bioethics, 13 a declaration that is less binding than a convention. the 32nd session of unesco in october, 2003, judged setting of universal standards in this area to be imperative and desirable, and invited the director general "to continue preparatory work on a declaration, and to submit a draft declaration at its 33rd session in 2005, involving from the very beginning states, the united nations and the other specialized agencies of the un system, other inter-governmental and non-governmental organizations and appropriate national bodies and specialists". 13 i know by personal experience in the ibc (in which i was a rapporteur) that to proceed from a feasibility study to a universal declaration on bioethics is almost impossible, but trying is worthwhile. the process of elaboration can be itself a contribution to the ethics debate, to knowledge and participation, as long as the existence of many different ethics, and bioethics in particular, is considered-not as an obstacle-but as an expression of richness and freedom. since the text of the preliminary report of the ibc is now publicly accessible, i will not discuss it in detail. i would only underline that, after the preliminary remarks, its first substantial section deals with health and health care (points 16, 17, and 18) . it begins with this paragraph: "health has a dual moral value: it is essential for the quality of life and life itself, and is instrumental as a precondition for freedom. when disease prevails, the destiny of a person (and even of a nation) is left to external www.thelancet.com vol 364 september 18, 2004 during the summer of 2003, more than 15 000 elderly people died in france ap rights were not granted to include this image in electronic media. please refer to the printed journal. factors and powers and may enter into an irreversible vicious circle of regression. the inequality between the rich and the poor-at the level of individuals, communities and nations-is becoming increasingly deeply felt in the area of health and healthcare, thereby contributing to the desperation and injustice that prevail and continue to increase in other health-related fields such as food, income and education." the main difficulty in practising moral principles concerning human dignity and equity in health is that in the past 15 years a singular ethics (and a singular policy) prevailed in the world, which resulted in overturning the health paradigms that had successfully guided public health and health services for one century. the principle that health is a value and an objective of economic development has been replaced by the opposite idea: that systems of universal care represent one of the main obstacles to economic growth. the leadership of national health policies has been transferred from health ministers to economics ministers, and internationally (particularly in developing and under-developed countries) was influenced more by the international monetary fund (imf), the world bank, and the world trade organization (wto) than by who. even when the negative results of their policies in relation to equity became clear, and the action of who (whose president at the time was gro harlem brundtland) succeeded in bringing health back on the world political agenda, the model of the commission appointed by who on macroeconomic and health continues to be that of the influential 1993 report of the world bank, investing in health. 14 the model does not include any critical analysis "of currently dominant macroeconomic policies or of the structure and mechanisms that entrench developing countries disadvantage, ill health, and deteriorating services". 15 in this framework, the debate refers mainly to healthcare systems, putting aside the concepts of healthy societies and systems. the idea of the priority of primary health care and of the prevention accessible to everybody has been supplanted by high technologies, even in countries where the resources are minimal. discussions on resources for health have been restricted to monetary aspects, ignoring the many possibilities of human resources, of changes in environment and workplace, and of improvements in nutrition and education. the need to identify priorities and to distribute fairly the resources for health care is replaced by the idea of rationing them: not through priorities and universal inclusion, but through selective exclusion. the analysis of diseases' causes has been concentrated mainly on individual factors, such as genes and behaviours, whereas the role of social factors, so important for disadvantaged people, has been neglected. the role of social factors is sometimes even concealed. one example is in the world health report (2002). 16 another example comes from the death, during the summer of 2003, of more than 15 000 elderly men and women in france, many of them poor or socially marginalised; of more than 5000 in germany and italy; and of others in many other european countries. it is true that in august the temperature in these countries was unusually high, but this risk had been widely described in epidemiological research, and preventive measures for elderly people in such conditions are available in almost all gerontology textbooks. the almost unanimous comment of the media was that they were killed by the heat. commentators forgot the isolation, loneliness, lack of attention by many family doctors and local health services, absence of any warning or information being broadcast on television (which is often the only communication between non-self-sufficient elderly individuals and the rest of the community), and insufficient funding for active assistance and care at home. the ministers of health were surprised by the events, 17 and local health authorities tried to underestimate and even to conceal numbers (almost like the epidemic of severe acute respiratory syndrome in china). very few raised two general questions: what else can we expect for world health from potential climate change, and what should we do about present and future risks? at the end of the 1990s, new political and moral trends began to emerge in the world, and new emphasis was given to health and equity in health. these trends became very influential culturally, although they were politically contradicted by the orientations of the dominant powers. health was reintroduced to the international political agenda. in many countries, researchers have shown a growing interest in health equity, inspired either by their moral and scientific sensitivity, or by evidence. the main efforts were inspired by the attempt to integrate altruism and self-interest, to reconsider health as an indivisible good, and by the refusal of simple charitable transfers of benefits among countries or groups. this is an old idea, now defined as compassionate conservatism, which may include the virtue of ethics but has two faults: (1) those who are helped are placed in a compromising, dependent position, treated as victims not agents; and (2) societal rules and structures that generate such social consequences are not addressed. public opinion, nevertheless, became more critical towards inequities in health, probably for two reasons. one is that the inequity in health, which often means life or death, raises higher indignation than other inequities concerning income or material goods. the other is an increased knowledge of reality through public inquiries, books, 18 medical journals, and campaigns. a few years ago, amartya sen, closing in dhaka the bangladesh session of the global equity in health initiative in 1998, said: "information concerning discrimination, torture, poverty, illness, and abandonment helps coalesce the forces opposing these events by extending the opposition to the general public. this is because the people have the capacity and willingness of reacting to other people's difficulties." evidence confirms the willingness of people to help others. millions, mainly young people, are working in voluntary services at home or abroad. often, they combine in their activities two aspects that in the past have been separated and even conflicting: to struggle for collective interests, and at the same time to work daily to help individuals. on the political and cultural scene, the role of civil society and of community organisations has increased almost everywhere. a new generation has emerged that criticises the effects of one-sided globalisation on environment, health, justice, and relations between science and society, which underlines that a better world is possible and demands peace. there are some analogies with the youth movements of the late 1960s, but also three differences that can make this new movement more lasting and more effective: their extension beyond schools and far beyond the usa and western europe; their will to integrate criticism with proposals; and their growing influence on national policies and on international agencies, as we can see from two examples. one example is the victory obtained against the bill of indictment, requested by the multinational pharmaceutical industries to the south african court against nelson mandela and his government, for the crime of producing and importing antiretrovirals by ignoring or violating patents. mandela made the decision to ignore the patent to make therapies accessible for the poor population, in a country where one in nine people is hivpositive. after global criticism from governments, nongovernmental organisations, hiv/aids specialists, and a globalised movement mainly organised through the internet, the pharmaceutical companies decided not to pursue the case. after a bitter struggle between the companies and who, new rules were adopted. it is now possible to suspend or limit the royalties for "intellectual property" in case of widespread epidemics: a partial victory in what could be called, perhaps improperly, conflict between patents and patients. later, in october, 2003, the south african competition commission concluded that the companies "had overcharged for the drugs and had limited their licensing to competitors to try to suppress competition"; 19 and finally, in december of that year, glaxosmithkline and boehringer ingelheim, while still rejecting the accusation, agreed to reduce the price for therapy by as much as 90%. a door has been opened for new international rules on everyday bioethics. the second example is the wto meeting in cancun, mexico, sept 9-14, 2003 , where no agreement was reached on trade in agricultural and industrial products, and the attempt to push decisions on fundamental issues, such as the privatisation of water resources and of health and educational services, completely failed. the consequences of this failure may be contradictory, but surely a new factor emerged: the formation of an alliance between more than 20 developing countries who represent more than half of the world's population, and antiglobalisation movements. developing countries have been deeply divided in the past 20-30 years, and have had almost no voice in the international arena. cultural and political antiglobalisation groups had already gained publicity in seattle 4 years ago, and now had common goals with many governments. so far, the main result is the defeat of the proposal to extend rules governing the trade of commodities to the services for persons, such as health and education, and to natural resources. the argument has been that such services affect human rights, are essential for human life and growth, and that nations should decide how to guarantee them to all citizens. the two cases show how far other international agencies such as the wto, the imf, and the world bank, are involved in decisions about people's health, which often is not considered as a value but a variable and uncomfortable element of the economic system. as far as ethics is concerned, the difference is that who does have a moral obligation towards people's health, whereas the wto, the imf, and the world bank do not. during and after the recent change in the who leadership, there was much discussion about its future, such as the stimulating debate in the lancet. 20 at the same time, the connections between health and human security became more evident. the un appointed an ad hoc commission that stated: "in addition to the persistent problems and vulnerabilities with which the world has long been familiar, there is a new wave of dramatic crises at the turn of the millennium related to terrorist attacks, ethnic violence, epidemics and sudden economic downturns. there is also a fear that existing institutions and policies are not able to cope with weakening multilateralism, failing respect for human rights, eroding commitments to eradicate poverty and deprivation, outdated sectarian perspectives in educations systems and the tendency to neglect global responsibilities in an increasingly interrelated world. at the same time, the opportunities for working towards removing insecurity across the world are also larger than ever before." 21 two deep contradictions are now arising. one is the move back to the idea of security, which was historically intended (with mixed intentions and results) to counter the threat of aggression across borders or violence against people. in the 20th century this concept was deepened and expanded 22 through the experience of the welfare state and through the emergence of new personal and collective rights. the questions are now: what security, and for whom? not only against the threat of attacks and crimes against nations and persons, but also for individuals and their dignity; for human welfare, health, and nutrition; for water and clean air; for the biosphere; and for the interests of future generations. the other contradiction is the policy of governments like the usa that, in the struggle against the threat ofinternational terrorism, choose to use their military and repressive power, without addressing the social, cultural, and political causes that cannot ever justify but might explain the growth of terrorism. from a practical point of view, the results of this policy are dubious at the very least. from an ethical point of view, it restricts the range of individuals who could contribute to society; it might demobilise popular, intellectual, and political energies; it introduces a rigid separation between those who are considered good and those who are branded evil; and it weakens the duties of public authorities and international institutions to face other individual and collective needs. the future of health, health policies, and health equity is strictly connected to the resolution of these contradictions. everyday bioethics: reflections on bioethical choices in daily life letters from prison report of the ibc on preimplantation genetic diagnosis and germ-line intervention: shs-est/02/cib-9/2. geneva: united nations educational, scientific, and cultural organization missing women missing women: revisited il villaggio dei disperati: qui tutti si vendono i reni (the village of the desperates: here all sell their kidneys) il corriere della sera oct 6 la merce finale: saggio sulla compravendita di parti del corpo umano (the final commodity: an essay on the sale and purchase of parts of the human body). milan: baldini and castoldi attack on organ trade begins with transplant tourists. the times the greatest benefit to mankind: a medical history of mankind global health: targeting problems and achieving solutions convention for the protection of human rights and dignity of the human being with regard to the application of biology and medicine (convention on human rights and biomedicine) report of the ibc on the possiblility of elaborating a universal statement on bioethics investing in health: world development indicators the report of the commission on macroeconomics and health: a summary critical appraisal: geneva: who the world health report 2002: reducing risks, promoting healthy life. geneva: world health organization morti da canicola: epidemiologia per non dimenticare challenging inequities in health: from equity to action agreement expands generic drugs in south africa to fight aids who director-general elections-join the lancet debate united commission on human security (ogata s, sen a, co-chairs) what is security? key: cord-279694-25rblhwb authors: mahy, b.w.j title: emerging and reemerging virus diseases of vertebrates date: 2014-11-28 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.02564-2 sha: doc_id: 279694 cord_uid: 25rblhwb in the last two decades, a large number of new viruses have been discovered, many of which are pathogenic in humans or other vertebrates. among the more important causes of virus emergence have been changes in human behavior, population, and increases in travel to distant countries. in addition, the application of new molecular technologies has led to the recognition of many viruses that hitherto went undetected. many of the new, emerging viruses have an rna genome, and many are zoonoses. the spread of human immunodeficiency virus, causing acquired immune deficiency syndrome, and the use of immunosuppressive drugs following transplant surgery, have increased the numbers of people in the population that are highly susceptible to emerging virus infections. the threat of a new pandemic of influenza virus in the human population stresses the need for development of better methods for detection, surveillance, and control of emerging virus diseases. it became apparent during the last two decades of the twentieth century that new infectious diseases were increasingly being recognized in the human and animal populations. this led to the establishment of a formal committee of the institute of medicine of the national academy of sciences, usa, who reported on their deliberations in 1992, in a report edited by joshua lederberg and richard shope. this was followed 10 years later by a second report, edited by mark smolinski, margaret hamburg, and joshua lederberg, which appeared in 2003 . among the factors they cited as contributing to emergence were microbial adaptation and change, human susceptibility to infection, climate and weather, changing ecosystems, economic development and land use, human demographics and behavior, technology and industry, international travel and commerce, breakdown of public health measures, poverty and social inequality, war and famine, lack of political will, and finally intent to harm. the advent of highly specific molecular techniques such as the polymerase chain reaction (pcr) in the early 1980s permitted the detection and grouping of viruses on the basis of genome nucleotide sequence analysis, and in several respects these techniques have replaced serological analyses for the characterization of viruses. although it is still important to isolate viruses in cell culture for their complete characterization, it is now possible directly to detect viruses in diseased tissues by pcr, then, by sequencing the amplicon, to determine whether a new virus has emerged to cause the disease. in fact, many viruses which do not readily grow in cell culture can only be differentiated by sequence analysis. the papillomaviruses are an example. their study was very difficult until the advent of sequence analysis, which now has revealed more than 100 types in humans, and many more in animals and birds. for differentiation, three virus genes (e6, e7, and l1) are sequenced, and if the combined sequence of these three genes differs by more than 10% from known papillomaviruses, the virus is considered to be a new type. other viruses which have not been grown in cell culture include many caliciviruses, and the ubiquitous anelloviruses, such as torque-teno (tt) virus, which can be detected and sequenced in the blood of most humans and many other vertebrate species. hepatitis c virus was originally described as non-a, non-b hepatitis virus because of the severe disease it caused but the virus would not grow in cell culture, and eventually was detected in blood known to be infected with the virus by reverse transcription of the rna present using random primers then expressing the resultant dna in the bacteriophage lambda gt 11. thousands of clones were screened using patient blood as a source of antibody before positive clones were detected, which then allowed the ☆ change history: july 2014. bwj mahy added new section on mers coronavirus. added several new references development of enzyme immunoassays that could detect the virus in blood and so were used to screen blood destined for transfusion, saving millions of lives worldwide. once the complete genome of hepatitis c virus was sequenced, it became apparent that there are many different genotypes circulating in the world, with different pathogenic properties. nucleotide sequence analysis has also been extremely useful in tracing the origins of viruses. for example, when hantavirus pulmonary syndrome, caused by a bunyavirus of rodents, sin nombre virus, was initially detected in 1993 in the four corners region of western usa, it was found that rodents inside a house where people had been infected carried a virus identical in sequence to virus isolated from human cases. however, rodents caught at various distances from the house had increasingly variable genome rna sequences, providing strong evidence that these rodents, deer mice (peromyscus maniculatus), were the source of the infection. subsequently more than 30 other hantaviruses, some of which also cause hantavirus pulmonary syndrome in humans, were isolated from rodents throughout north and south america. each new virus seems to be associated with a different genetic variant of rodent host, and all rodents that carry the virus belong to the subfamily sigmodontinae, unique to the american continent. a particularly powerful tool for the initial recognition of an emerging virus is the application of immunohistochemistry to diseased tissues. provided a comprehensive collection of antibodies is available, the particular virus or related group of viruses can often be detected. for example, when hendra virus first appeared in 1995 in australia, causing the death of a horse trainer and 14 of his horses, antibody against the virus was sent to the centers for disease control and prevention (cdc). then, in 1999, cdc was asked to investigate a newly emerged epidemic that had appeared in malaysia, killing more the 100 people and causing disease in many pigs. initially, it was suspected to be caused by a virus related to japanese encephalitis virus, but a virus was isolated from the pigs that replicated in vero cells, and reacted in an immunofluorescence test against the hendra virus antiserum. this could subsequently be used on patient tissues to study the pathogenesis of the disease, and after comparison of the genome sequences of hendra virus and nipah virus they were found to be closely related and are now classified in the genus henipavirus of the paramyxoviridae. finally, molecular methods can be used to detect new, emerging viruses in the absence of disease in the host. in 2001, allander and colleagues searched for rna viruses in human respiratory secretions using random primer pcr, and discovered a hitherto unknown parvovirus with a sequence related to the bovine and canine parvoviruses, which are grouped together in the genus bocavirus. the new virus was called human bocavirus, and many research groups worldwide have now confirmed the presence of the virus, particularly in pediatric samples, although it is still not certain how important this virus is in causing morbidity and mortality. their method also amplified a human coronavirus from the respiratory samples, and when sequenced this turned out to be hku1, a recently emerged coronavirus detected by scientists at hong kong university. it is possible that a systematic search of human samples using such molecular techniques might reveal more hitherto unknown human viruses. in some cases, the emerging viruses themselves have contributed to other viruses emerging and reemerging in the population. this is especially true of human immunodeficiency virus (hiv), the cause of acquired immune deficiency syndrome (aids), which rapidly spread following its emergence in the early 1980s to infect more than 40 million people worldwide by the end of the twentieth century. because of its severe effects on the immune system, the virus leads to numerous other infections in the hiv-infected population. for example, picobirnaviruses, that had been detected in fecal samples from chickens and rabbits, were difficult to detect in human fecal samples until a cohort of men with aids was examined, and in these humans picobirnavirus was detected for the first time. some rare diseases have become common in persons with aids. for example, the human polyomavirus known as jc virus can cause the rare brain disorder known as progressive multifocal leukoencephalopathy (pml). normally, the virus remains dormant in the kidney, but in hiv-infected individuals the hiv-encoded transactivator tat acts as a transactivator of jcv leading to pml which progresses to death within 4 months after infection. other important virus infections which emerge in aids patients are human herpesviruses (cytomegalovirus, herpes simplex viruses 1 and 2, varicella-zoster virus, and human herspesvirus 8, which causes kaposi's sarcoma). hiv is mainly spread through sexual activity between an infected and a noninfected person, and is most common in those who indulge in high-risk sexual behavior with multiple partners. it can also be transmitted by direct contact with infected blood, and is common in persons who indulge in intravenous drug use, particularly when needles, syringes, or equipment used to prepare drugs for injection are shared. it is therefore an example of a virus disease which is dependent on risky human behavior for its maintenance in the human population. the ability of such new infections to spread in the population has been greatly enhanced by population growth and ease of movement as a result of rapid air travel. a dramatic recent example of this was the appearance of the coronavirus causing severe acute respiratory syndrome (sars) in late 2002 which spread by air travel from a single infected chinese physician who infected 12 persons in a hong kong hotel. these infected persons then traveled by air and spread the infection to more than 8000 individuals worldwide, 10% of whom died. the virus then apparently receded from the human population in july 2003. only recently was it discovered that the sars coronavirus has a natural reservoir in chinese horseshoe bats (rhinolophus sinicus). some other species such as himalayan palm civets and raccoon dogs from which the virus has been isolated may serve as amplification hosts. following the recognition of the human coronavirus sars, research on coronaviruses intensified, and this led to the discovery in 2004 of two previously unrecognized human viruses, one found by hong kong university, called hku1 virus, and another reported almost simultaneously from the netherlands, called nl63, and from yale university, called new haven coronavirus. the latter viruses probably represent two isolates of the same virus species. they are clearly associated with lower respiratory tract infection in children, but initially it was claimed that new haven coronavirus was also associated with kawasaki disease in children. this intriguing claim was rapidly investigated and refuted by several different groups in japan, taiwan, and elsewhere, and the cause of kawasaki disease, which has features resembling a virus infection, remains unknown. in 2012, another novel human coronavirus was isolated from sputum from a fatal case of severe respiratory disease in the arabian peninsula. the virus, now called middle east respiratory syndrome coronavirus (mers-cov), has since been detected in more than 187 patients with respiratory disease, 97 of whom died. in november 2013,the mnistry of health in saudi arabia was notified of case of mers-cov infectionin a 43 year-old male patient who had cared for ill dromedary camels in his herd of 9 animals starting in early october. the patient was confirmed with mers-coronavirus by real-time pcr, and limited sequencing found the mers-coronavirus in three of the dromedary camels. other studies have detected mers-coronavirus-infected dromedary camels in several other regions in saudi arabia.but it remains unclear whether camels are an important source of the virus fror transmission to humans, and whether bats also play a role, as was the case with the sars-coronavirus. a majority of recent emerging virus diseases have been zoonoses (i.e., diseases transmitted from animals to humans under natural conditions). some of the more important of these include hiv-1, which was transmitted to humans from chimpanzees in central africa around 1931, and hiv-2, transmitted from sooty mangabeys to humans in west africa around 1940. other important recent examples are the viruses of the genus henipavirus. hendra virus was first recognized through a disease outbreak in some horse stables in hendra, queensland, australia, when 14 horses and their trainer died from pulmonary disease with hemorrhagic manifestations in 1994. the reservoir of the virus was found to be in large fruit-eating bats (pteropus spp.), and one year later a horse farmer 600 miles away in mackay, queensland, died of encephalitis from the same virus. then, in 1999, a related virus was discovered in malaysia following a major outbreak of respiratory disease in pigs and neurological disease in humans in their close contact. more than 100 humans died, and in a successful effort to control the disease 1.1 million pigs were slaughtered. the causative virus was isolated from a fatal human case that had lived in nipah river village, and so was named nipah virus. hendra and nipah viruses are clearly members of the family paramyxoviridae, but have been placed in a separate genus as their rna genome is about 19 kb in length, larger than that of any other paramyxovirus. nipah virus, like hendra virus, was found to have a reservoir in pteropus bats, and has since been identified in fatal human disease outbreaks in india in 2003 and bangladesh in 2004. other new viruses which apparently have a reservoir in fruit bats include menangle virus, a new paramyxovirus which emerged in a commercial piggery near sydney, australia, to cause stillbirths and abortion in pigs. menangle virus also caused disease in two workers in the piggery. a new virus related to menangle virus emerged during an investigation of urine samples from pteropid bats collected on tioman island, off the coast of malaysia, in 2001, and was named tioman virus. during the same investigation, a new orthoreovirus was isolated from pteropus hypomelanus in 1999 and called pulau virus, and more recently a related orthoreovirus called melaka virus was isolated from a human case of acute respiratory disease in melaka, malaysia. serological studies of sera collected from human volunteers on tioman island showed that 13% had antibodies against both pulau and malaka viruses. another important group of zoonotic diseases are rodent-borne, and caused by members of the genus hantavirus of the family bunyaviridae. these viruses first emerged during the korean war of 1950-52, when thousands of un troops developed a mysterious disease with fever, headache, hemorrhage, and renal failure with a fatality rate of 5-10%. it was more than a quarter of a century before the causative virus was isolated from field mice in korea, and named hantaan virus, the cause of hemorrhagic fever with renal syndrome (hfrs) in humans. then, in 1993, a new hantavirus emerged in the four corners region of southwestern usa as the cause of a severe acute respiratory disease syndrome, with a fatality rate close to 40%, and named sin nombre virus. this virus was shown to be transmitted to humans by inhalation of virus present in the urine, feces, or saliva of deer mice (peromyscus maniculatus). it seems likely that this disease had existed for many years, and was only recognized in 1993 because of a clustering of human cases as a result of a regional upsurge in the rodent population resulting from climatic conditions causing increased availability of rodent food. fortunately, in most of these infections, humans appear to be a dead-end host, and transmission between humans does not occur except with the andes virus in south america. rodent-borne viruses of the family arenaviridae also cause a number of serious zoonotic diseases in humans. the 'old world' arenaviruses such as lassa fever virus have been known for some time, but still cause thousands of fatal hemorrhagic fever cases every year in west africa. however, 'new world' arenaviruses such as junin virus causing argentinian hemorrhagic fever and machupo virus causing bolivian hemorrhagic fever have long been recognized in south america. recently, new arenaviruses have emerged, probably as a result of deforestation, which results in rodents seeking shelter in human habitation, and brings them into closer contact with people. these viruses include guanarito virus that causes venezuelan hemorrhagic fever with 36% mortality rate from confirmed cases, and sabia virus isolated in 1990 that causes brazilian hemorrhagic fever with a high fatality rate, including two laboratory acquired cases. rabies is a zoonotic disease of great antiquity that has mainly been associated with carnivores, such as dogs. the virus is excreted in the saliva of infected animals, and following infection it moves through the nervous system to attack the brain, causing aggressive behavior which results in the animal biting humans and animals with which it comes into contact and thereby spreading the virus infection. fortunately, due to early work by louis pasteur, a vaccine was developed that protects humans or other animals from infection, and can also be given immediately post exposure, and the domestic dog population in the developed world is vaccinated and does not pose a risk to humans. however, in some developing countries, it is not uncommon for a rabid dog to bite and infect more than 25 people before it can be put down, and worldwide there are still some 30 000 human rabies deaths per year. using molecular sequencing techniques, it is now possible to distinguish the genotypes of rabies viruses associated with different species of host, as the virus has become adapted through frequent transmission between members of the same host species. in the usa, there are six recognized terrestrial animal genotypes, in raccoons in eastern states, skunks in north-central states, skunks in southcentral states, coyotes in southern texas, red foxes in alaska, gray foxes in arizona, and several genotypes associated with particular species of bat. in fact, most fatal cases of human rabies in the usa can now be traced to bats, which are often not detected when the person is bitten; so rabies is not suspected and vaccination is not undertaken until the disease has taken hold. many important virus diseases are spread by arthropods, and exposure to new arthropods and the viruses they carry is critical to the emergence of new virus diseases. dengue hemorrhagic fever is caused by dengue virus which is transmitted mainly by the asian mosquito (aedes albopictus), and dengue fever is one of the most rapidly emerging diseases in tropical regions of the world. there are four serotypes of dengue virus, and it seems that consecutive infections with two antigenic types can lead to the more serious disease of dengue hemorrhagic fever with shock syndrome, which, if untreated, can result in up to 50% mortality. unfortunately, through the importation of vehicle tires containing water from korea, the asian mosquito was introduced into the usa, and is now present in several regions of the southern states. it can act as a vector not only for dengue virus, but also for california encephalitis virus. in europe, the emergence of two important animal diseases has occurred through the movement of arthropod vectors into the iberian peninsula. african horse sickness virus causes a disease that can be fatal to horses, mules, and donkeys, and is transmitted by nocturnal biting flies of the genus culicoides. these were introduced inadvertently into spain, and the disease is now endemic around madrid and regions to the south. african swine fever virus is transmitted by ticks of the genus ornithodorus and it causes a fatal disease resembling classical swine fever in domestic pigs. it first emerged in portugal and spain in 1957 , france in 1964 , italy in 1967 , and cuba in 1971 . through slaughter of infected animals, the disease was eradicated from europe, except sardinia, by 1995. the most recent dramatic example of the movement of a virus vector is provided by west nile virus, a flavivirus first isolated in uganda in 1937. this virus uses birds as a reservoir host, and is transmitted from birds to humans and other vertebrates by mosquitoes. in 1999, cases of encephalitis in new york were found to have been caused by a strain of west nile virus that was phylogenetically similar to a virus isolated from geese in israel. at the same time, many birds, especially corvids, began dying in new york state. since the introduction in 1999, west nile virus has become well established throughout the usa and moved north into canada and south into the caribbean and into mexico. it is not known how the virus moved from israel to the usa, but the most reasonable explanation is that it was carried in an infected mosquito or possibly an infected bird in the hold of an aircraft. transmission by an infected human seems less likely since the titer of virus in human blood is usually too low for efficient mosquito transmission. it is clear, nevertheless, that once it arrived in north america, west nile virus found an extremely favorable environment with abundant avian and arthropod hosts that facilitated its spread throughout the american continent. the emergence of new viruses is likely to continue as viruses evolve and find new ecological niches in the human and animal population. it is noticeable that most newly recognized viruses have been rna viruses, perhaps since rna evolves at a faster rate than dna, for which host cells have developed efficient proofreading enzymes. it will be important in the future to detect new viruses before they can emerge to cause disease in the population. the sars epidemic provides an excellent example. before the epidemic, only two human coronaviruses were known, human coronaviruses 229e and oc43. despite the fact that serious coronavirus diseases were well known in other vertebrates, such as feline infectious peritonitis and avian infectious bronchitis virus, it was not until the sars epidemic that research on human coronaviruses led to the discovery of three new human coronaviruses -sars, hku1, and nl63/new haven. there are other genera of viruses that cause serious disease in animals but have not been adequately investigated in humans. an example is the genus arterivirus, which has members causing serious disease in horses and pigs, but has not been reported at all in humans. this could be a worthwhile area for future investigation. another critical factor in the future control of emerging viruses is better vector control. when mosquito control was conducted using ddt, dengue fever virus was virtually eliminated from the americas in the 1970s, but environmental concerns led to the widespread banning of the use of ddt, so that since the 1980s there has been a considerable expansion of dengue fever in south america, with the appearance of dengue hemorrhagic fever there for the first time. there is a real need to improve mosquito control measures to control this disease. although there are prospects for a dengue virus vaccine, this is so far not available. in 2014, a new outbreak of ebola virus occurred in west africa, causing several thousand deaths in liberia and sierra leone. the ebola virus first emerged as the causative agent of viral hemorrhagic fever in two outbreaks occurring almost simultaneously along the ebola river in the democratic republic of congo in 1976. there were some 318 human cases in this episode with a mortality rate of 88%. since that time, a number of smaller outbreaks of ebola hemorrhagic fever affecting humans and monkeys were reported from west africa, but although most infections have been associated with hunting and handling of great apes, the actual origin of the virus has never been ascertained. it is clear hat transmission of the virus requires close contact with an infected individual, and does not involve airborne transmission. ebola virions are usually long filamentous sometimes branched forms with a uniform diameter of about 80 nm, and varying in length up to 14000 nm, from which they are called filoviruses, belonging to the family filoviridae. finally, one of the most important viruses that continue to emerge in different antigenic forms is influenza virus. the main reservoir of influenza viruses is in birds, and over the past century several pandemics of influenza have emerged, the most serious of which was in 1918. pandemic strains usually arise by a process of antigenic shift, where one of the genes encoding the hemagglutinin and/or the neuraminidase of influenza virus is replaced by one from birds. new pandemics occurred in 1918 (h1n1 subtype), 1957 (h2n2 subtype), and 1968 (h3n2 subtype). since 1968, there have been no new pandemics, but it is widely expected that another will occur. at the time of writing, there is worldwide concern that a highly pathogenic avian influenza virus (h5n1 subtype), which has caused some human infections and deaths in persons in close contact with infected birds, might mutate or recombine to generate a virus which would be highly transmissible in the human population. plans are being developed in many countries and by the who to try to prepare for such an event by generating possible vaccines against such a virus and stockpiling antiviral drugs. biomedical research emerging trends in lassa fever:redefining the role of immunoglobulin m and inflammation in diagnosing acute infection identification of the major, parenteral non-a, non-b hepatitis agent (hepatitis c virus) using a recombinant cdna approach nipah virus: a recently emergent deadly paramyxovirus identification of new papillomavirus types critical review of the vector status of aedes albopictus west nile virus: epidemiology and clinical features of an emerging epidemic in the united states nipah virus encephalitis reemergence the widening scope of coronaviruses timing the ancestor of the hiv-1 pandemic strains lessons to be learned from the ebolavirus outbreak in west africa a novel coronavirus associated with severe acute respiratory syndrome emerging zoonoses: crossing the species barrier the emergence and re-emergence of viral diseases molecular and biophysical characterization of tt virus: evidence for a new virus family infecting humans genetic identification of a hantavirus associated with an outbreak of acute respiratory illness microbial threats to health, emergence, detection and response unraveling the mysteries of middle east repiratory syndrome coronavirus key: cord-289003-vov6o1jx authors: burdet, c.; guégan, j.-f.; duval, x.; le tyrant, m.; bergeron, h.; manuguerra, j.-c.; raude, j.; leport, c.; zylberman, p. title: need for integrative thinking to fight against emerging infectious diseases. proceedings of the 5th seminar on emerging infectious diseases, march 22, 2016 – current trends and proposals date: 2018-02-28 journal: revue d'épidémiologie et de santé publique doi: 10.1016/j.respe.2017.08.001 sha: doc_id: 289003 cord_uid: vov6o1jx abstract we present here the proceedings of the 5th seminar on emerging infectious diseases, held in paris on march 22nd, 2016, with seven priority proposals that can be outlined as follows: encourage research on the prediction, screening and early detection of new risks of infection; develop research and surveillance concerning transmission of pathogens between animals and humans, with their reinforcement in particular in intertropical areas (“hot-spots”) via public support; pursue aid development and support in these areas of prevention and training for local health personnel, and foster risk awareness in the population; ensure adapted patient care in order to promote adherence to treatment and to epidemic propagation reduction measures; develop greater awareness and better education among politicians and healthcare providers, in order to ensure more adapted response to new types of crises; modify the logic of governance, drawing from all available modes of communication and incorporating new information-sharing tools; develop economic research on the fight against emerging infectious diseases, taking into account specific driving factors in order to create a balance between preventive and curative approaches. the objectives of the 5th edition of the val de grace school's seminar were to take a global, integrative approach to the emergence of new infectious disease agents, putting these in perspective relative to other types of risk, as well as studying crisis situations and the breaches brought on by the occurrence of emerging infectious diseases (eids). two key conferences respectively opened and closed this annual seminar; the first was given by dr. peter daszak (president of the ecohealth alliance and program director of usaid-ept-predict), the second by dr. patrick lagadec (former research professor at the é cole polytechnique, palaiseau). planned for an anticipated up to 200 participants, this seminar is designed for decision-makers, experts, medical doctors and scientists interested in human and animal health, social sciences, environmental sciences, prospective analysis, biosecurity and defense. this day of presentations and debates is presented under the auspices of the french social affairs and health ministries, as well as that of the environment, energy and marine affairs. peter daszak remarks today can be summed up in two primary messages, both personal and that of scientists from the ecohealth alliance. first, we must increase our research capabilities in order to understand the direct and less-direct causes of emerging infections if we hope to fight them once they become responsible for epidemics or pandemics. secondly, this presentation was illustrated with a few projects concerning the economics of eids which reveal the extraordinary costs they incur for national economies. there is at least one obvious reason to speak of this, which is that politicians and public decisionmakers are in fact quite sensitized when the economic damage engendered by the latest epidemics and pandemics are explained to them. if one takes, for example, the sars-cov pandemic, it led to a 1 to 2% decrease in gross domestic product in several southeast asian countries, for an estimated overall cost of 30 to 50 billion us dollars, relative to the total number of approximately 800 people worldwide who were affected. the appearance of new eids appears both more frequent and also vaster in terms of number of people affected these last years, many of these diseases appearing in developing or low-income countries. taking the case of the ebola virus (ebov) epidemic which broke out in west africa in 2014, it was much more widespread than any other epidemic to ever occur in central africa (400 people affected during an epidemic). it is very difficult in the united states -where peter daszak is working -to get public decision-makers interested, as is true with the population which feels very distant from such problems. ecohealth alliance has tried to draw the media and the public's attention to it, in vain! they only received one single email from the management of the boston airport, informing us that our work on the risk of ebola virus propagation via transcontinental air transport was unfounded and that the boston airport could not experience this type of threat. about a month later, when an american citizen was repatriated for treatment, the public began to panic; the media, particularly the tv channel cnn, exaggerated in broadcasting the issue, and the government took several decisions, notably regarding assistance and monitoring of international airports. these measures were decided upon no scientific basis in a moment of panic, and it seems that with every new worrisome eid, it is the same story! the measures taken are often disproportionate to the seriousness of the phenomenon. human demography has exploded in recent years, and populations today are concentrated in megalopolises. this tendency is even more marked in tropical areas in the south where there is also an important biodiversity of animal species. therefore, there exist today more opportunities for a virus or bacteria to pass from an animal to a human, then to propagate among the human population. international air transport makes possible the spread of these new infectious risks on a vast scale. what are governments doing faced with this type of threat when the demands on the part of the population are ever-increasing? vaccines are also considered by the former as a weapon of total destruction, and by the public as a widely-available miracle tool. neither is truly aware that it takes an average of 10 years to produce a vaccine. also, is it the right strategy in that it banks more on cure than on anticipation? in the u.s., president obama opted for a development aid policy through usaid favoring training and improvement of human and technical capacities to prevent future eids in the most needy countries. the american congress, however, did not follow this path, instead directing funds allocated to usaid towards research on a vaccine for and on epidemic management of the ebola virus crisis. recently, in collaboration with economists, ecohealth alliance modeled the risk of a new emergence in a situation identical to that of ebola in 2014-15 in west africa, estimating the economic damage caused, and attempted to deduce the economic cost of a health policy based on epidemic management as opposed to one favoring prevention and training. according to their estimates, a budget of 5 billion us dollars seems to suffice to prevent a new epidemic in west africa in opting for the second solution. their simulations obviously include many underlying hypotheses. all the same, this analysis indicates that if we were to quickly make available 1 billion of the 5 billion dollars for the purchase of equipment, the setting up of working laboratories on site, field hospitals, sending doctors and nurses to the area, training of our partners in affected countries -as military medical services know how to do -such a strategy would represent a real capital investment in managing epidemic propagation. unfortunately, this was not the option chosen by u.s. current governments, which we can only regret. now let us discuss the ecology of ebola virus transmission. the bat species pteropus are possibly the host reservoirs of ebola virus. as for the marburg fever virus, we are now certain that these bats are in fact the reservoir. the ebola virus is clearly present in a few giant bat species in africa. it can also circulate among primate species or indirectly through other infected mammals. we also know which species of bats are affected or not. even if the reservoir question is still under debate, we know a great deal about the rainforest circulation of this virus and the circumstances of its spreading in the human population with high-risk groups such as hunters. based on this, is it possible to prevent the virus' transmission to humans? although it poses a complex question, it is nonetheless possible to offer some avenues of reflection. william karesh, vicepresident of ecohealth alliance and instigator of the onehealth concept, carried out a research project in the democratic republic of congo, which consisted in educating villagers, and particularly hunters, regarding the danger of recovering primate corpses from the forest. this program was a real success, as the villagers changed their behavior and the eating of monkey meat. this represents a low-cost prevention approach, which in the end functions well if properly implemented. the only real solution when faced with this type of health threat is to work in conjunction with populations, and to treat questions of poverty, equality, and use and transformation of land together. these are arduous and complex tasks which must be carried out over the long-term. this type of message is extremely difficult to make heard among politicians and the public, and obviously more complex that announcing or clamoring for a vaccine. we are faced with primordial questions which are more or less difficult to answer. are we witnessing an increased frequency of eids appearance? are there more cases today? can we predict patterns, or rules, of emergence? are there geographic areas in which these emerging infections are more frequently or widely observed today? do we possess the scientific ability to predict exactly where the next infectious epidemic will start? evidently, if we were so able we might allocate the financial resources and technical and scientific support to suspected areas. at the moment, we do just the opposite, or we do nothing, and we are disconcerted at each new emergence. in order to convince governing bodies and public decision-makers, we must demonstrate to them that predictive approaches are less costly than curative ones. using approaches inherited from ecology and biogeography ecohealth alliance has adapted the same principles to understanding the ecology and spatial distribution of eid principles observed over the last decades. their work shows that of the nearly 400 new eids which have appeared, the so-called zoonotic ones originating in wild animals have been in net increase in the last 40 years; 3 out of 5 new infections which appear each year originate in wild fauna. what's more, the risk of infectious transmission is highly and doubly correlated with human density, as are these diseases of animal origin to the areas' biodiversity. they called these areas ''hot-spots'' for disease risks, mostly situated in intertropical regions. they are advocating for these specific zones to be those on which they concentrate our research efforts as well as international public aid for development. in fact, it is not only biological diversity in animals which must be taken into account, but also the evolution of natural ecosystems and their disappearance over recent years. deforestation and land use changes by populations are driving forces in the appearance in new eids. therefore, international politicians must better link economic strategies to those of habitat and biodiversity coordination if we wish to avoid new pandemics. in further use of the formalism of economic models, they have shown that a stable political strategy appears once joint simulations were conducted on economic damage due to a pandemic health crisis and preventive decision-making. even though the financial cost of preventive strategies may initially appear great, an optimal solution based on prevention shows itself over the mid-and long-term to be finally less costly than cure and control-focused strategies. we have need on an international level for the equivalent of the intergovernmental panel on climate change (ipcc) which, in the same way as does the ipcc on climate change scenarios and their impact, would concentrate on eids and their sociopolitical, economic and environmental consequences. the american development aid agency usaid did not originally give priority to eids, but the different public health crises brought on by the h5n1 bird flu virus in recent years have led usaid to modify their strategic orientation, notably with regard to the economic weight they bring to bear on regional economies. the fact that these epidemics appear first in countries situated in strongly species-rich intertropical areas which are developing or low-income, requires a reconsideration of our western policies on development aid in order to better include these notions. for ten years, usaid funded a program on new emerging infection threats, for a total of 1.3 billion u.s. dollars. at ecohealth alliance we have collaborated with this initiative through participation in the predict research project with funding of 45 to 50 million dollars. the goal of the predict program was to identify microorganisms potentially pathogenic for human populations, and which are hosted in animal reservoirs. we took particular interest in three animal groups, not only because it was impossible to work on all the animal groups present, but also because the three groups we choseprimates, rodents and bats -were recognized as being major reservoirs of agents which are pathogenic for humans. these three groups alone make up 75% of the world's mammal species. within the predict program we applied our knowledge of the high-risk emergence zones to draw samples from a large cohort of these three animal groups. over the first five years of the program we sampled 56,000 animals, trained 2500 scientists and medical and administrative personnel, and discovered more than 1000 new viruses belonging to families of viruses known not to be infectious to humans. this in itself represents a major finding! obviously, the discovery of a microorganism does not in and of itself indicates that a new eid might appear. their work in mexico on bat species demonstrated the existence of a dozen viruses which are very like the mers-cov responsible for the respiratory syndrome in the middle east. they therefore believe that the mers-cov is not hosted by dromedaries but rather by bats, as they found it in our work in mexico. some of these new coronaviruses may potentially be pathological for humans. it is clearly impossible to identify all viruses present on the planet. it would also be necessary to do the same for bacteria, parasitic fungi, and protozoa. as they are innumerable, it is preferable to make strategic choices regarding microorganism research and that on the highest-risk animal groups. based on our knowledge of currently known viruses in bangladesh bats, they extrapolated this data using species curve rarefaction, and capture-recapture techniques well known to ecologists, to estimate the total number of viruses to be expected in the totality of known mammal species in the world. they reached a value of 320,000 new viruses which remain to be discovered. were we to carry out biodiscovery research on these viruses, one could catalogue them, classify them in relation to already-known viruses, in particular those known to be pathogenic to humans, and through comparative genome studies assess their pathogenic potential. we are currently in the second phase of the us predict program, with an important accent on our manner of working. in fact we are currently concentrating more on factors involved in emergence and seek particularly to understand three such factors: habitat change and use, intensive agriculture, and the commerce of biodiversity. us development aid policy also focuses on three strategic areas: north africa, west africa, and continued research activity in saudi arabia. these geographic choices are shaped by recent events around the mers-cov and ebola viruses. especially in the case of mers-cov, dromedaries are certainly involved in the transmission cycle of the virus, but we believe that its true reservoir is the bat. thus using the many data at our disposal, we were able to show that the infectious risk of mers-cov for humans is not great in saudi arabia, but it is in other areas of contact between bat, dromedary and human populations, particularly in the horn of africa, especially in somalia. somalia is currently a politically disjointed country, where public health surveillance and care are greatly lacking or simply non-existent. for example, it is currently impossible to state how many mers-cov cases there are in somalia, or how many might exist. our scientific objectives are therefore to better understand the behaviors at the junction of wild or domestic fauna and human populations. for example, understanding human behaviors and practices at the interface of tropical forests and villages could help us to interpret how zoonotic transmission happens. through acting upon these behaviors and habits, we could lower this type of emerging risk. currently ecohealth alliance has research sites in uganda, malaysia and brazil; they are investigating the role played by habitat changes such as deforestation, human interaction with biodiversity through behavior and use. in manaus in brazil for example, the maximum risk of new infection is not in the heart of the city where the major markets are located, but in the peri-urban areas where agriculture and ranching are developing. these zones which ecologists call ''ecotones'', or transition zones, are located near strongly species-rich regions, with high concentrations of livestock which can come into contact with wild fauna, and which are furthermore inhabited by ranchers and farmers. these regions which are now located everywhere in the intertropical world for the purpose of feeding urban populations, are generally those where new infections appear, and where future pandemics will also most likely appear. at the heart of this research is the priority of understanding behaviors, habits and practices of populations with the goal of changing these factors. this is a long-term endeavor which requires developing approaches in the field for communicating with local populations, and for developing community participation in our own research, which we also consider an excellent means of education. o. borraz recalled the fact that we live in a society of risk. it is not that the dangers surrounding us are more numerous or more fearsome than before, but simply that the notion of risk plays a central role in public policies, in public and private organizational management, and in the controversies around new technologies. genetically modified organisms, mobile phones, nuclear waste, urban sanitation sludge; the activities now considered health or environmental risks are countless. this categorization puts public authorities in a position of having to ensure the safety of populations, even as the state itself sometimes represents a risk factor. it is therefore essential to understand how an activity becomes a risk, and how it is then managed by public authorities as well as by companies, associations and local conglomerates. risk from its identification to its management, from its highlighting to its instrumentalization becomes a tool linked to the emergence and expansion of a welfare state. it is used by politicians to justify on the one hand its lack of involvement in risk management, on the other the seizing of power over sectors from which they had previously disengaged. this political power seizure plays on politicians' selective dissemination within society of risk identification and the knowledge required for its management. two different processes exist by which risk represents an organizing principle for political power, a means to assist and contribute to the definition of the state's limits: ''putting at risk'' and ''regulation through risk''. ''putting at risk'' refers to all processes by which an event is described as or constitutes a real or predicted danger, and which thus is categorized as a risk [1] . there are many events, objects, and situations which have historically been categorized as ''putting at risk'' and which can be studied by sociologists (illness, divorce, food crises, unemployment, nuclear risk, chemical substances, technological risks. . .). this putting at risk can be a product of the state or of interest groups, under the influence of lobbying. this has been part of the organization of modern societies since the 20th century, and in particular following world war ii with the expansion of the welfare state which took advantage of different factors putting the population at risk in order to enlarge its sphere of activity and thus its power in the name of a protective mission. this welfare state built itself around risk management in creating and organizing agencies, action plans, drills, and monitoring mechanisms for their application through nationwide inspection bodies. the promotion of ''risk instruments'' and a ''risk-based regulation'' approach makes it possible to deal with risk-creating events. for the last two decades, in a context of decreasing means and in reaction to public-health crises which have led, according to some, to an overprotective state, a new tendency has emerged of governments using ''risk instruments'' to better allocate means and to decrease the state's hold. despite their differences, these two approaches are to the same end, which is to define -or perpetually re-define -the limits of the state. ''risk instruments'' contribute in each ''putting at risk'' situation to determining the state's involvement, the reason for its having competencies and resources, and therefore also the state's limits as opposed to that which concerns the private sector and individuals. the outcome of this defining as ''putting at risk'' and of the application of these risk instruments may be used by the state either to invest, or to disinvest; in the latter case for example entrusting risk management which formerly would have been the realm of institutions to local or private agents or to individuals. in the same way, risk instruments (or risk-based regulation or using risks to rationalize public intervention) may lead to state disinvestment or, on the contrary, be used by the state to seize back the reins in certain areas. paradoxically risk instruments serve in this instance to re-centralize or to ''remote-control'' areas to which the state had granted greater autonomy (universities or hospitals, for example). thus the way in which western societies manage risk, and the way in which these risks -their identification, how politicians and individuals perceive them, and their management -transform the links between the state (the political powers-that-be) and civil society are important to understand for those faced with risk management, especially in public-health risk management. what consequences these risk technologies have from one country to another thus greatly depends on institutions, state structure, professional organizations and their interrelationships, the balance of power and the forces involved, and the legal structurewhich may be more or less open to interpretation according to how it is drafted (more or less restrictive laws). risk management is, after all, at the core of the state and has always been central to its transformations. presenter: alfredo pena-vega (ehess, cnrs) climate change can be interpreted, according to a. pena-vega, as a complex system such as edgar morin defined it in his ''introduction to complex thought'' [2] . the stakes involved in it are multidimensional, and suppose at once the ideas of transformation, uncertainty and unexpectedness, unpredictability and crisis. . . it henceforth seems necessary to arm ourselves with instruments enabling us to understand this reality. the common denominator of such instruments is rationality. because climate change involves stakes which are climatological, economic, social and health-related, as well as questions of governance, ethics, biodiversity etc., cross-disciplinarity appears to be one form of rationality which allows us to avoid the dangers of ''fear-mongering''. a study was conducted among 12,000 high school students from 20 different countries, of which the objective was to understand how younger generations see climate change. the results are quite varied. if a minority of responders question the existence of climate change -with an argument largely based on a social construct communicated by the media -90% of responders claimed to be concerned and aware of the negative consequences of climate change. the vast majority consider it to be a threat to the survival of humanity, in particular due to the multiplication of natural disasters, dwindling biodiversity, the spread of new infectious diseases, notably those with vector-borne transmission, and the inequalities it fosters between humans. we are currently in the ''age of the unthinkable''. today's world constantly exposes us to new crisis situations which we must learn to confront. these situations are all the more difficult to manage in that they most often occur ''out of framework'', or within a framework in which it is difficult to define the outlines delimiting an increasingly ''volatile'' environment. whereas our benchmarks in terms of crisis management are structured according to typologies (natural/biological/social disaster), the boundaries of current crises are unclear, and their typologies intertwined. unexpected and unpredictable, they emerge within a context where uncertainty reigns and where the response is organized according to the logic of competitive leadership. information no longer follows a downward flow from the state to the public, but circulates in a collaborative manner via widespread connectivity, social networks, which compete with institutional media which are sometimes outdated. it is therefore difficult for decision-makers to circumscribe the areas of operation, to isolate causes, and to distinguish the components which permit specific, technical and successive interventions. good judgment thus becomes key. public health crises, and especially those concerning eids, have pointed up the difficulties in predicting their appearance and development. comparisons and connections between these and other types of crises (natural, technological, industrial. . .) and how they are interpreted can thus prove useful in public health crisis management. the unattainable goal of predicting such crises makes it necessary to prepare to meet with unexpected events during the ongoing management of the crisis; it is no longer a question of predicting the unpredictable, but preparing to deal with it. in the context of our intricate, complex society, coordination and communication are of course necessary, but it has also become imperative to have a thorough knowledge of the steering process. in ''out of framework'' situations, strategic thinking capability takes precedence over the quality of available technical expertise. this can take the form of an ''express thinktank'', a group made up of diverse members capable of and trained to work together in situations of uncertainty. whereas in a ''classic'' situation the command functions is a pyramid, in these ''out of framework'' situations, collaborative and flowing cooperation is required, without leadership's impeding the efficaciousness of the response. in the case of hurricane sandy in the u.s., in 2003, several work teams were set up in order to handle the unprecedented nature of the situation: ''real-time innovation'', ''immediate flaw detection'', ''emergency support functions''. these parallel work teams made it possible to optimize management of the disaster by limiting disastrous consequences. it seems essential to develop such networkbased crisis management in france. p. zylberman recalled to mind the definition of an eid as an unexpected infectious -or presumably infectious -phenome-non, affecting humans, animals or both. according to the definition used in the high council of public health's 2011 report [3] , this can entail an infectious clinical entity which has just appeared (''true'' emergence), one previously identified (known emergence) or a known infectious disease whose incidence has increased or whose characteristics have changed (re-emergence). the hiv epidemic in the 20th century or the sars-cov epidemic in the early 21st century are examples of true emergence. the emergence of hepatitis c corresponds to a known clinical entity whose etiological agent was identified at the end of the 1980s. the measles and west nile virus outbreaks on the american continent, in the 19th and end of the 20th centuries respectively, are examples of reemergence. nathan d. wolfe has defined five stages of transformation of an animal pathogen into a specifically human pathogen, resulting in a ''true'' emergence, with the possibility of an evolutionary interruption at each stage. stage 1 corresponds to a situation in which a known virus in animals has never yet been detected in humans in natural conditions. in stage 2, the virus known in animals is capable of infecting humans in natural conditions, but without the capability of person-to-person transmission. in stage 3, some cycles of secondary person-toperson transmission are possible. in stage 4, the virus circulates among humans through several secondary person-to-person transmission of varying duration. stage 5 is reached when the virus becomes exclusively human, and also contagious. on a population-wide scale an epidemic goes through four phases: introduction, propagation, amplification, and regression of the infectious phenomenon. the propagation phase is that during which there are the most widespread and frequent sites of infection. it corresponds (particularly for viruses) to the adaptation of the pathogen to its new host with person-toperson transmission taking effect little by little. it is often at this stage that the epidemic phenomenon is detected, sometimes with a considerable delay relative to the introduction of the pathogen. propagation can take place not only by contiguity but also across vast distances. humans thus play a role through their activities, and are what s. morse refers to as ''microbial traffic engineers'' [4] . what are the major factors in the emergence of new infectious pathogens in humans? a great deal of progress has been made in improving the rapidity in identifying new viruses, as j.-c. manuguerra points out. the time delay between the individualization of a new infectious nosological entity and the identification of the causal agent thus continues to shorten, from 14 years for hepatitis c in the 1970s to 2 years for hiv in 1983, and to 6 weeks for sars in 2003; even less for mers-cov. even so, the discovery or the knowledge of the pathogen's existence does not provide all the answers about the risks posed for or by a host species. various behaviors of pathogens can nonetheless be observed, in particular the pathogen's adaptation may require passing through several intermediary host species before adapting to its final host. moreover, the epidemic potential of a discovered virus is difficult to determine. among the very numerous known arboviruses, most are of anecdotal importance for human pathology, and little has been undertaken upon their discovery in preparation in case the pathogen were to become epidemic. this is true in the case of the zika virus, first isolated over 50 years ago, and currently responsible for a major epidemic in latin america and in the caribbean. the question of the time lapse between the beginning of an epidemic and identification of the pathogen is progressively slipping toward that between the beginning of the phenomenon and its detection by the health system. this period appears to be critical for eradicating the development of an epidemic. is it possible to narrow the time gap between the beginning of an epidemic and its detection by the healthcare services? early identification of an epidemic-level incident mobilizes all health services professionals, both in regards to human and to animal health, according to m. van kerkhove. human and animal health and the state of ecosystems are inextricably linked, and it is believed that nearly 60% of eids, including those re-emergent, are of zoonotic origin. it is through this exploration of the connections between animals and humans that the means of transmission and propagation of mers-cov within the human species could be revealed. this virus is an example of a potentially epidemic emergence. schematically, during the mers-cov epidemic, a limited number of person-to-person transmissions, and sporadic cases between dromedaries and humans, were observed, with a certain degree of increase seen in healthcare facilities, sometimes considerable, as was the case recently in korea. during the epidemic 75% of the mers-cov cases were reported in saudi arabia, and of 1600 cases reported to the who task force, 60% were considered to be primary cases, that is, contracted from an animal host source, and 40% to be secondary cases, or acquired through another human case. every primary case represents an opportunity to understand how the infection was contracted. it gradually seemed necessary to launch a veterinary investigation as soon as a case was diagnosed. this led to the development of animal surveillance, which made it possible to reveal the seropositivity of certain dromedaries, as well as active excretion of the virus in their environment, which made possible its transmission to humans. this improvement in detection of emerging pathogens and early epidemic detection calls for the improvement as well of the veterinary surveillance network, the so-called onehealth approach. in an area with limited resources, where all animals cannot be tested, it is imperative to concentrate on those areas with high concentrations of animals, such as slaughterhouses, and on areas in which humans come into close contact with animals and thus represent a high risk of transmission (see conference by p. daszak). the data collected in these areas of frequent contact between humans and animals can be utilized to issue recommendations for at-risk populations in order to reduce transmission. once the virus has acquired person-toperson transmission capability, its control is far more complex due to the rapidity of propagation following its introduction into the population. it therefore becomes difficult for the healthcare system to improve detection of the epidemic. establishing health policies to optimize the case reporting system is thus critical. how to provide care to patients in the case of new eids? in an epidemic situation, the treatment of affected patients usually is secondary to the need to control the disease, as a. mcgeer stated. nevertheless, an adapted patient care procedure can change the evolution of an epidemic, to varying degrees according to the pre-existing healthcare infrastructure in the given country. in north america, where the services for the monitoring and managing of infectious diseases and public health are separated from the healthcare system, the organization of care for infected patients is usually left to the physicians. the healthcare system is in fact organized around individual patient care, and not oriented toward an approach of global individual and collective care providing. in the case of eids, care of affected patients becomes a political issue in a country graced with a public health system. health is seen as a human right, and governments are judged not only by their ability to prevent and manage epidemics, but also according to their management of care provided to ill patients. the role of the public healthcare system is therefore to advise physicians and to develop recommendations for the detection of cases and their homogeneous treatment. these guiding principles make the physicians the kingpins between healthcare structures and the treatment of patients, and the public healthcare system. better patient care provision thus improves epidemic response. in fact, treating ill subjects also allows the risk of person-to-person transmission and propagation to be reduced. this treatment role attributed to physicians can be variously interpreted in countries in which the healthcare and public health system are underdeveloped or lacking. in fact, the arrival of healthcare personnel and the setting up of precautionary measures required to contain the epidemic may be experienced as an intrusion. the lack of comprehension and communication between medical staff and the affected community, as well as the potential lack of comprehension of the measures set in place can prompt affected individuals to hide, due to the uncertainty of their fate. this was the case for example in west africa during the ebola virus epidemic, during which affected patients were sometimes hospitalized far from their villages without care being taken to inform their families of their clinical progress and outcome. this led to sometimes-violent rejection of the healthcare personnel, which interfered with the measures meant to control the epidemic. another element to take into consideration in the care of affected patients is protection of the healthcare personnel, which has become a recurring problem. most of the microbial forms with epidemic potential propagate within the community. for a long time hospitals were not troubled over the risk of nosocomial transmission of eid agents. care providers were not aware at the time of the risk of contagion that they themselves ran when providing care to patients. this awareness happened during the recent sars, mers-cov and ebola epidemics, which shared certain physio-pathological characteristics different from those involved in previous epidemics. in fact the peak of virus excretion for measles, chicken pox and influenza generally occur before or upon the appearance of symptoms, and the risk of transmission is virtually nil when the patient arrives at hospital. sars, mers-cov and ebola virus have a different viral excretion rhythm: symptoms appear with a weak viral load, and their intensity increases with the level of viral excretion, which reaches its maximum just when the patient requires the most care. the hospital thus becomes a center for the propagation of the pathogen. the hospital system is therefore in danger of breakdown, as it is both the pole for patient care and the new center of the infection's transmission. these new pathogens therefore require a re-thinking of hospital design, in order to optimize both control of epidemics and patient care. what has been, or is, the extent of nosocomial infection's role in the transmission of sars-cov and mers-cov? the new pathogens require us to re-evaluate modes of prevention for nosocomial transmission, confirmed b. guéry. the 2003 sars-cov epidemic serves as a good example of what was learned about the intra-hospital transmission of these new pathogens. person-to-person transmission of sars occurs through droplets, physical contacts and airborne pathways. the sars-cov transmission rate to healthcare providers exclusive of invasive procedures has been estimated at 21%. the main risk factor identified was the lack of protection of the provider's airway through wearing a mask, with an odds ratio estimated at 13 . wearing scrubs and handwashing were also associated with lower transmission risk. sars is a disease which appeared in 2003 in guangdong province in china, then in hong kong, where numerous primary and secondary cases occurred. in total, according to the case index, 716 secondary and tertiary cases occurred, of which 52.3% among healthcare providers. beyond standard hygiene measures, studies conducted on affected healthcare providers revealed that certain categories of personnel, such as technicians and nurse's aids, show an infection rate twice that of the nursing staff, and 6 times greater than the medical personnel. these studies made it possible to identify infection risk factors generally not taken into account in the fight against nosocomial transmission of pathogens: a significantly, greatly increased risk (odds ratio 7.3) was noted in care providers having used precautionary measures against sars transmission for less than 2 hours, as was the case with those not having understood the protective measures (odds ratio 3.1). the factors identified as influencing transmission are patient viral load and patient index distance. the ideal conditions for transmission to occur are those of an infected patient excreting large quantities of virus, presenting with a certain number of comorbidities capable of masking the initial profile, and the existence of multiple close contacts with high-risk procedures such as oro-tracheal intubation, performing a fibroscopy, or the administration of treatments through nebulizers. the idea of a ''super-excreter'' patient was also identified during recent respiratory virus epidemics. this concept could play a crucial role. usually it concerns cases of very serious infection occurring in patients with several co-morbidities. in beijing in 2003, the sars case index was also associated with a large number of secondary cases (76 cases, of which 12 among healthcare personnel). as with mers-cov, despite a relatively low basal reproduction rate (r0), a large number of care providers were infected during the epidemic. this is what happened, for example, in abu dhabi, where 65 cases of mers-cov were diagnosed, of which 42% among healthcare providers. each case of provider infection was followed up through an epidemiological study, and each time an obstacle to the isolation of the patient and to the application of hygiene practices was noted. over 80% of the mers-cov cases identified in korea were thus traceable to 5 ''super-excreter'' patients. this notion remains questionable, as it is reductionist and could lead to the identification only of patients in this category, to the neglect of transmission risks associated with other patients. it is probably more fair to speak of ''hyperexcretion events'' which implies that each patient is at maximum risk, and should be treated using precautionary measures. in conclusion, the intra-hospital control and transmission of eids can only occur in connection with the development of precautionary standards, which should be ongoing over time, and should be applied by all healthcare providers. it is imperative to ensure that caregivers are adequately and regularly trained, and that they constantly keep in mind the importance of isolation of all infected patients. to achieve this, it is probably necessary to resort to specialized units, in reference hospitals, in conjunction with clear decisions at the national level. an integrated approach to health in face of the globalization of risk has been developing over the last few years. the ''onehealth/ecohealth'' concept, or global health, takes into account the fact that human health, animal health and environmental health are inextricably linked, especially in regards to eids, exposure to which is fostered by the multiplication of transcontinental travel, many instances of human-animal contact, and intensive farming and ranching. many recent examples have made it possible to establish the key role played by animal biodiversity in the introduction and transmission of pathogens within human populations. whether it be the role of bats in the 2014 ebola virus epidemic, or dromedaries in the 2012 mers-cov epidemic, the crucial role of animals and of human-animal contact -being wild or domestical animals -in triggering an epidemic has recently been emphasized. primates, rodents and bats are the three mammal groups most likely to be at the origin of future pandemics due to the high proportion of viruses which they share with humans. the inclusion of fields which appear quite unrelated (infectious diseases, animal health and ecological and environmental sciences) should thus be pursued and improved. it has been possible to establish models which allow the prediction of emergence tendencies in infectious diseases, and certain geographical areas at high risk for emergence have been identified: central africa and west africa, southeast asia, central america. these areas correspond to those at high risk of propagation due to underdeveloped or deficient public health systems, and to the absence of epidemiological surveillance. tools necessary for effective prevention of future epidemics are now available. these are all the more critical in that recent increasing tendencies raise fears of a multiplication of the number of emergent epidemics in future. beyond the fight against pathogen propagation, its introduction into the human population is in fact a key step against which ''battle plans'' can be drawn up. in order to perfect pandemic response, it is necessary to improve the coordination and interconnection between individual and institutional participants, such as healthcare providers and public health systems. a global response approach (oneresponse) should be reflected upon and developed. on an institutional level, a first step in bringing together the fields of environment and animal health occurred in 2010 in france with the creation of a national french agency for food, environmental and occupational health & safety (anses), originating in the french agency for food safety (afssa, which also includes the national agency for veterinary drugs) and the french agency for environmental and occupational health & safety (afsset). another case of bringing together the areas of surveillance, prevention and human health intervention occurred in 2016 with the creation of the french public health agency, with the merger of the health surveillance institute (invs), the national institute for prevention and health education (inpes), and the organization for preparedness and response to health emergencies (eprus). the relations between these two new institutions should be developed in order to provide a better-coordinated response to future health crises. the response to an eid should take into account not only factors linked to eids, but also to a constellation of political, economic and socio-cultural constraints. the decision to put in place such a battle is in fact a political decision which involves, beyond the scientific aspects, the intervention's impact upon the popularity of the acting political powers-that-be. if governments are judged according to their ability to prevent epidemic crises, they are equally judged on their ability to avoid expenditures deemed excessive given the existing risk. these political considerations can run counter to the scientific rationale behind the response. this is how, in the case of the 2014 ebola virus epidemic, the united states became involved: through the declaration by the liberian president on august 6, 2014, on the threat to national security posed by ebola, and the danger of the spread of the epidemic to the u.s. soil. in parallel with vaccine research and development, the actions of the u.s., the who and the united nations have focused on treatment of infected patients and the epidemiological securing of burials. in addition, despite a sometimes limited human impact, the economic impact of epidemics involving indirect costs (consequence for certain sectors of activity) has shown a marked increase. however, what characterizes modern epidemics is the duration of the economic ''shock'', in that it is temporary, as opposed to previous epidemics during which the shock tended to be drawn out in particular due to the persistence of infectious sources. there are many examples of this: among others, the ''spanish influenza'' of 1918, the effects of which (company closures, loss of revenue) faded out in 1921, or, more recently, the sars-cov epidemic, when the recession, that had been triggered by alerts against travel to southeast asian destinations communicated in march, ceased once these alerts were lifted two or three months later. it is of note that during the sars-cov epidemic only certain sectors (especially tourism) were affected in asia and in ontario, canada, and not the entire global economy. carrying out preventive measures such as staff training makes it possible to limit the number of crises at a lower cost. the costeffectiveness of such an approach has already been shown. it is henceforth necessary to raise awareness in decision-makers of the importance of prevention relative to risk. beyond these constraints, the decision to intervene is complicated by the heterogeneous nature of potential epidemics of the different pathogens. for this reason, and given the absence of technology permitting the prediction of the epidemic potential of a pathogen, prevention which targets the agent is impossible. prevention must therefore adopt other means, such as training locals in order to improve hygiene conditions. the improvement of transversal knowledge on infectious agents' transmission carries particular importance for the goal of preventing emergence. once an emerging agent is introduced into the human population, the pathogen propagation phase within the human population is the key phase in the development of the epidemic, and the one during which it is still possible to act in order to prevent the amplification of the pathogen in the population. the beginning of the propagation phase can be difficult to identify, and improvement in diagnostic techniques as well as the development of rapid diagnostic tests, and even on the field, makes it possible to accelerate detection of an epidemic signal. moreover, given the key role of certain animal species in the development of new epidemics, the development of animal health surveillance would allow us to further shorten the time lapse between the introduction of the pathogen and its propagation; however, this poses significant problems of wild animal monitoring particularly in southern hemisphere countries. better knowledge of states' operational modes in dealing with risk makes it possible to better understand their interventions and to better adapt the scientific response. the notion of risk has, little by little, become political, and the state uses risk to govern. risk management is therefore at the very heart of the state. recent public health crises have led to risk's taking on a new form, that of the unknown, by the fact of the unpredictability of its appearance and evolution. an interaction between scientific and political approaches to risk is absolutely necessary, in order to better evaluate at-risk situations according to modern methods such as structured decisionmaking, and to better handle risk-management tools. the changing nature of risks calls for the emergence of a new form of governance, which puts the individual back into the center of the state's action, as well as the confronting of arguments with expert committees. civil society's participation in risk management should be developed, and observation should once again play its part in crisis response. younger generations might also take greater part in responding to current crises. seven priority proposals can be outlined as follows: encourage research on the prediction, screening and early detection of new risks of infection; develop research and surveillance concerning transmission of pathogens between animals and humans, with their reinforcement in particular in intertropical areas (''hot-spots'') thanks to public support; pursue aid development and support in these areas of prevention and training for local health personnel, and to foster risk awareness in the population; ensure adapted patient care in order to promote adherence to treatment and to epidemic propagation reduction measures; develop greater sensitization and training among politicians and healthcare providers, in order to better prepare them to respond to new types of crises; modify the logic of governance, drawing from all available modes of communication and incorporating new information-sharing tools; develop economic research on the fight against eids, taking into account specific determining factors in order to create a balance between preventive and treatment approaches. the authors declare that they have no competing interest. introduction à la pensée complexe les maladies infectieuses émergentes : état de la situation et perspectives. available online: www global microbial traffic and the interchange of disease we are grateful to the speakers who generously brought their contributions to this seminar: peter daszak, olivier borraz, we thank corinne jadand for helpful organization and management support. key: cord-295194-xbla6tu7 authors: stripecke, renata; münz, christian; schuringa, jan jacob; bissig, karl‐dimiter; soper, brian; meeham, terrence; yao, li‐chin; di santo, james p; brehm, michael; rodriguez, estefania; wege, anja kathrin; bonnet, dominique; guionaud, silvia; howard, kristina e; kitchen, scott; klein, florian; saeb‐parsy, kourosh; sam, johannes; sharma, amar deep; trumpp, andreas; trusolino, livio; bult, carol; shultz, leonard title: innovations, challenges, and minimal information for standardization of humanized mice date: 2020-06-24 journal: embo mol med doi: 10.15252/emmm.201708662 sha: doc_id: 295194 cord_uid: xbla6tu7 mice xenotransplanted with human cells and/or expressing human gene products (also known as “humanized mice”) recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. these models can provide a relevant in vivo context for understanding of human‐specific physiology and pathologies. humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. as the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. therefore, an international community‐driven resource called “minimal information for standardization of humanized mice” (mishum) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. within mishum, we propose comprehensive guidelines for reporting critical information generated using humanized mice. antibody-dependent cellular cytotoxicity, is an immune defense mechanism whereby effector cells such as nk cells lyses target cells that have been bound by specific antibodies aml acute myeloid leukemia art anti-retroviral therapy bdbv bundibugyo ebolavirus bite bispecific t-cell engagers is a registered trademark for a class of recombinant bispecific monoclonal antibodies which bind to the cd3 receptor and to a tumor-specific antigen blt bone marrow-liver-thymus bm bone marrow bnabs broadly neutralizing antibodies are antibodies capable of neutralizing different types of viral strains brgf balb/c rag2 à/à il2rg à/à flt3 à/à mouse strain expressing macrophage colonystimulating factors (m-csf), il-3, il-6, gm-csf, and thrombopoietin (tpo) mscs mesenchymal stromal cells myelo-ablated mice are mice treated with irradiation or chemotherapy in order to decrease the bone marrow activity in order to improve the engraftment of transplanted stem cells myelodysplasia is an abnormal accumulation of immature blood cells in the bone marrow myelofibrosis is the replacement of the bone marrow with scar tissue due to proliferation of immature blood cells nash non-alcoholic steatohepatitis nih national institutes of health nk natural killer nod non-obese diabetic nog nod.cg-prkdc scid il2rg tm1sug /jic nrgf nod-rag1 à/à il2rg à/à flk2 à/à nrg nod-rag1 tm1mom il2rg tm1wjl /szj nsg nod.cg-prkdc scid il2rg tm1wjl /szj pbmcs peripheral blood mononuclear cells pd-1 programmed death receptor 1 pd-l1 pd-1 ligand 1 pdx-mi pdx model minimal information standard pdx patient-derived xenograft pirf por à/à /il2rg à/à /rag2 à/à /fah à/à rag1 recombination activating gene studies of human stem cell engraftment, hematopoiesis, and immunity studies using immunocompetent mice have provided critical insights into the development and regulation of hematopoiesis and immunity. however, such studies do not always reflect responses in humans because of multiple species-specific differences. therefore, mice developing components of the human immune system (his) mice were created. these models have provided tools for the understanding of human hematopoiesis and immunity in vivo and to test new therapies or vaccines without incurring risks to patients. the simplest engraftment method is the adoptive administration of human peripheral blood mononuclear cells (pbmcs) into severely immunodeficient mice ( fig 1a, table 1 ). since the adoptive human t cells react forcefully against the xenogeneic major histocompatibility complex (mhc) class i and ii expressed by mouse tissues, this so-called "hupbl" model faces the hardship of fulminant xenograft graft-versus-host disease (gvhd) occurring 2-4 weeks after pbmc transfer. these models have limited applicability to follow specific antigenic responses, but can be used to test human immunosuppressive agents. improvement of the hupbl model has been described with novel mouse strains lacking mouse mhc class i and ii, resulting in lower occurrences of gvhd (yaguchi et al, 2018; brehm et al, 2019) . a more complex approach covered here in detail is the hematopoietic stem cell transplantation (hct) of preconditioned immunodeficient mice with human hematopoietic stem cells (hscs). despite the full mismatch between the human leukocyte antigens (hla) expressed on the human hematopoietic cells and the mouse mhc expressed on tissues, hct leads to "fully" humanized his models ( fig 1a, table 1 ). human hscs can differentiate into multiple human hematopoietic lineages, giving rise to mature leukocytes, including several lineages of the human immune system. robust engraftment with human hematopoietic and lymphoid cells was pioneered back in 1988 with the description of the cb17-prkdc scid severely compromised immunodeficient (scid) strain engrafted with human fetal liver hematopoietic cells and autologous thymic tissues (mccune et al, 1988) . this scid-humanized (scidhu) system showed initially only a transient presence of human t cells and human immunoglobulin g (igg) in the circulation. the critical relevance of the strain background for engraftment success of human cells was later appreciated when it was observed that non-obese diabetic (nod)-scid mice had a much higher capacity to support human hsc engraftment. this was elucidated to be due to the expression of a human-like signal regulatory protein alpha (sirpa) allele in the nod strain, popularly known as the "don't eat me signal", bypassing phagocytosis of human cells by mouse macrophages (takenaka et al, 2007; shultz et al, 2012) . targeting the interleukin 2 (il-2) receptor common gamma chain (il2rg) resulted in the absence of mouse natural killer (nk) cell activity as well as ablation of t and b lymphocyte lineages. in addition, the development of mice lacking the expression of recombination activating gene 1 (rag1) à/à and rag2 à/à provided radioresistant mouse models lacking mature host t cells as well as b cells (shultz et al, 2007) . currently, there are approximately 50 diverse humanized mouse models available from biorepositories. most of these models are homozygous for the scid, il2rg, rag1, or rag2 mutations and express the nod or human sirpa allele. the nod-scid il2rg (à/à) (nsg), the nod-rag1 à/à il2rg à/à (nrg), and the nod/shi-scid il2rg (à/à) (nog) are broadly used strains for xenografting a large variety of human cells, but several other strains are prospering (for recent reviews, see shultz et al, 2019; allen et al, 2019) . it is important to be thoughtful also about the nature of the human hscs. although humans and mice differ greatly in their biological characteristics, human hscs can essentially engraft in myelo-ablated or irradiated mice and reside in the mouse bone marrow (bm) niche. this hct approach opened several doors for the understanding of the basic properties for long-term durable repopulation of human hscs. as sources of human hscs, cord blood (cb) or fetal liver are mostly used, as they have high frequencies of hscs. generally, a range of 1 × 10 4 -10 5 isolated hscs is administered per mouse in order to enable efficient human hematopoietic engraftment and long-term reconstitution. several laboratories have opted to use fetal tissues due to the higher abundance in the numbers of hscs, which can be explored to generate larger cohorts of humanized mice (n = 30-40) compared with cord blood (n = 10-20). some groups have tried to overcome this limitation by pooling hscs from several donors, but upon development of immune systems that are not hla-matched, once the t cells develop, allograft reactions among donors can complicate the analyses of the immune responses. additionally, it is important to take into consideration that hscs in fetal and neonatal tissues may be intrinsically different regarding the stage of the hematopoietic development. further, it is important to consider ethical constraints and difficulties in procurement of human fetal tissues. in fact, the us national institutes of health (nih) is currently supporting investigators to seek and develop humanized mouse models that do not rely on human fetal tissues (allen et al, 2019) . human hsc cell surface markers have been used to allow their identification, purification, and analyses, in order to define the hsc populations with highest engraftment and/or repopulation capacity. xenotransplantation of human cd34 + hscs into preconditioned immunodeficient mice is the most broadly used procedure to generate his mice, and this approach is corroborated by the clinical evidence that transplantation with human-enriched cd34 + hematopoietic/stem/progenitor cells (hspcs) is a salvage procedure when the hla is not optimally matched between patients and donors. remarkably, a defined cd93 hi sub-fraction within the lineage negative (lin à ) cd34 à cd38 à cell present in cb has high repopulating capacity in nod-scid mice (danet et al, 2002) . cd49f is an adhesion molecule serving as a hsc marker and intra-femoral injection of single cd49f + cells into female nsg mice can generate long-term (20 weeks) multilineage grafts (notta et al, 2011) . thus, the quest for the archetypical human hsc population and whether other defined cd34 à hscs subpopulations should also be considered for the generation of his mice and how to eventually expand these cells ex vivo without compromising their self-renewal potential remains to be clarified. another aspect to be taken in account is that the ability of human hspcs to engraft and differentiate into different hematopoietic lineages may largely depend on their interactions with the mouse bm microenvironment constituents. as some human factors may be absent in the mouse bm niche, sponge scaffolds seeded with human bm-derived mesenchymal stromal cells (mscs) have been implanted subcutaneously into nsg mice to allow the formation of niches for human hscs to differentiate (antonelli et al, 2016; reinisch et al, 2016; abarrategi et al, 2017) . using two-photon microscopy for high-resolution non-invasive in vivo analyses, these implants are currently enabling the clarification of the human bm microenvironment requirements in regulating human normal and malignant hematopoiesis in vivo . another limitation in his models is the lack or low levels of human factors and cytokines in mouse tissues or circulating in the plasma and needed for human hsc self-renewal or differentiation. transgenic expression of human interleukin 3 (il-3)/granulocyte macrophage-colony-stimulating factors (gm-csf)/stem cell factor (scf) in nsg mice resulted in enhanced levels of human myeloid cells and regulatory t cells (t reg ) (billerbeck et al, 2011) . very promising models are his mice generated with a mouse strain expressing several human cytokines such as macrophage colonystimulating factors (m-csf), il-3, il-6, gm-csf, and thrombopoietin (tpo), the "mistrg-6", and showing improved human t, b, and nk cell development (das et al, 2016; yu et al, 2017) . this is a valid approach, and expression of several different human growth factors and cytokines to support differentiation of early or mature lymphoid or myeloid cells has been performed (rongvaux et al, 2014; bryce et al, 2016; jangalwe et al, 2016) . some recent development was also exemplified by transgenic expression of human thymic-stromalcell-derived lymphopoietin (tslp) that supported lymph node development in immunodeficient mice (li et al, 2018) . dendritic cells (dcs) are main orchestrators of the adaptive immune system presenting processed peptide antigens to t cells through mhc classes i/ii and expressing key costimulatory molecules such as cd40 ligand (cd40l) required for b-cell activation and class switching (steinman, 2012) . different types of dcs exist in mice but they are not homologous to human dcs. further, in his mice, the human dc development and maturation are not optimal. novel his models based on the balb/c rag2 (à/à) il2rg (à/à) flt3 (à/à) (brgf) and nod.cg-rag1 tm1mom il2rg tm1wjl/szj flk2/flt3 à/à (nrgf) mice with a mutated receptor tyrosine kinase flk2/flt3 were created. human dc development is increased in brgf and nrgf mice with exogenous administration of human flt3 ligand (flt3l) after hct, leading to a major increase also in the numbers of human nk and t cells (li et al, 2016; douam et al, 2018) . a factor to be taken into account for the generation of his is the gender of the mice. females show better hsc engraftment and faster human t-cell immune development and maturation (volk et al, 2017) . when setting up these models, it is important to keep in mind that the kinetics of human immune reconstitution is not linear and the time of analyses after hct has to be longitudinally established for different strains and methods. for example, human t cells show maturation, activation, and functionality at 15-20 weeks after cb-hct in nrg-his mice (volk et al, 2017; theobald et al, 2018) , but this varies considerably for other his models. future improvements are seeking a better development of human t cells in his mice so that they will be equipped with functional t-cell receptors (tcrs) able to interact with the matched hla complexes on antigen-presenting cells. this critical advance relies essentially on the substitution of the mouse mhc class i and ii by different hla haplotypes. to solve this mismatch problem, a transgenic nrg mouse strain called "drag" was developed that expresses hla-dr4 . drag mice transplanted with hla-dr4 + hscs developed more cd4 + t cells and higher levels of human immunoglobulins g and m (igm and igg; kim et al, 2017) . hsc-humanized mice expressing class ii hla-dr4 and class i hla-a2 transgenes ("draga" mice) generated cd8 + -specific t cells and influenza-specific antibody responses (mendoza et al, 2018a) . similarly humanized brgs mice expressing human hla-a2 and dr2 transgenes (brgsa2dr2) showed faster development of cd4 + and cd8 + t cells and higher concentration of iggs in plasma (masse-ranson et al, 2019). the practical limitation of these hla-transgenic strains is that it is difficult to find hscs that express a particular combination of hlas. another method to improve and accelerate the regeneration of human t and b cells in his mice is the adoptive transfer of geneengineered human dcs from the hsc donor that are long-lived in vivo (salguero et al, 2014; daenthanasanmak et al, 2015; volk et al, 2017) . this approach significantly enhanced the regeneration of lymph nodes in his-nrg mice promoting maturation of functional human t cells, b cell class switching, and development of antigenspecific iggs (salguero et al, 2014; daenthanasanmak et al, 2015) . as a take-home message, development of human immunity in his mice depends on several variables ( fig 1a, table 1 ). different approaches are being taken concurrently to accelerate and optimize human immune responses in mice. a structured approach to converge the reporting in scientific publications of the materials and methods (such as specific mouse strains, sex of the mice, methods used for hct, time-points of analyses) will facilitate the interactions in the community to boost these promising preclinical models (table 1 ). the liver is a vital organ responsible for key metabolic functions of the body and the site for several human-specific viral infections. for efficient generation of mice xenografted with human liver tissues, a combination of a growth disadvantage of the murine liver and a regeneration stimulus for the human cells is required. several approaches resulted in high human liver chimerism in mice (dandri et al, 2001; mercer et al, 2001; bissig et al, 2007; hasegawa et al, 2011) and the resulting models have pros and cons (reviewed in ref. bissig et al, 2018) . for example, the transgenic upa (urokinase-type plasminogen activator expressed under the albumin promoter) mouse has a profound dysfunction and triggers apoptosis of murine hepatocytes (heckel et al, 1990; dandri et al, 2001; mercer et al, 2001) . therefore, salvage human hepatocyte transplantation is required within 2-4 weeks after birth. nevertheless, humanized upa mice maintain considerable health problems. transgenic upa mice are difficult to breed, which is also a limitation of another utilized mouse strain based on nog mice expressing transgenic herpes simplex virus type 1 thymidine kinase (hsvtk) under the albumin promoter (tk-nog; hasegawa et al, 2011) . conversely, the metabolic dysfunction of the il2rg à/à rag2 à/à mice with a knock-out for the fumarylacetoacetate hydrolase gene (fah à/à ) can be regulated by a small drug and bred efficiently but the mice frequently develop murine hepatocellular cancer (azuma et al, 2007; bissig et al, 2007) . all these models show some remaining mouse liver tissue that can blur human-specific liver metabolism ( fig 1b, table 1 ). therefore, next-generation models seek to eliminate the interfering mouse metabolism. one such model is the por à/à il2rg à/à rag2 à/à fah à/à (pirf) mouse (barzi et al, 2017) , lacking murine p450 cytochrome function and allowing a human-only cytochrome metabolism in mice. human liver chimeric mice have also been used to model metabolic disorders. the first xenograft model for metabolic liver disease was established using human hepatocytes from a patient with familial hypercholesterolemia with a low-density lipoprotein (ldl) receptor deficiency; bissig-choisat et al, 2015) . it would be desirable to extend metabolic disease models also to more prevalent disorders such as non-alcoholic steatohepatitis (nash). a nash model would also require a functional immune system in addition to the liver chimerism. such dual humanizations have been achieved previously (gutti et al, 2014; strick-marchand et al, 2015; billerbeck et al, 2016; dagur et al, 2018) . the combined human liver and immune system models can show formation of fibrosis upon hepatitis c virus (hcv) or hepatitis b virus (hbv) infections (washburn et al, 2011; bility et al, 2014) . another promising approach to study human liver function is the combination of organoid technology with humanized mouse models to examine the immune response to regenerative cellular therapies and cancer. organoid technology allows the generation of unlimited numbers of non-malignant (sampaziotis et al, 2017) or cancer cells (broutier et al, 2017 ; fig 1b, table 1 ). if derived from the same hsc donor used to humanize the mice, this approach can potentially be used to compare the immunogenicity of autologous and allogeneic cellular therapies or investigation of safety and efficacy of autologous cancer immunotherapies. studies of type 1 diabetes (t1d) are also prospering with the use of humanized mice. backcrossing the insulin 2 (akita) mutation into nrg mice (nrg-akita) followed by human hct into newborn mice resulted in > 50% of the nrg-akita mice rejecting human islet allografts (brehm et al, 2010 ; fig 1b, table 1 ). the akita model was also used to demonstrate the efficacy of stem cell-derived human beta cells (sc-beta) to regulate blood glucose levels in vivo. the effect of viral infections was also established for studies of t1d, showing that coxsackievirus b accelerated the destruction of insulinproducing beta cells of pancreatic islets (gallagher et al, 2015) . his models are currently being developed to recapitulate the course of disease in human t1d, including the interactions between human immune system and beta cells (tan et al, 2017; walsh et al, 2017) . recent studies have included engraftment of mice with diverse hematopoietic and non-hematopoietic human tissues and cell populations, human-induced pluripotent (ips) stem cells and embryonic stem (es) cell-derived tissues (shultz et al, 2014) . exciting advances on the development and use of emerging humanized mouse models in multiple disciplines are ongoing (fig 1b, table 1 ). technical and analytical annotation and standardization strategies to harmonize the use of human tissues implanted into humanized mice will be needed (table 1) . infections with human-specific pathogens his mice offer a unique possibility to study infectious disease agents with a tropism toward human leukocytes, hepatocytes, and lung ª 2020 the authors embo molecular medicine 12: e8662 | 2020 epithelia, to characterize the induced immune responses and to develop therapeutic interventions against associated pathologies ( fig 1c, table 1 ). one such pathogen is the epstein-barr virus (ebv), a common c-herpesvirus that persistently infects more than 95% of the human adult population and was the first oncogenic virus identified in man (mü nz, 2019). accordingly, it is associated with around 2% of all malignancies in humans (cohen et al, 2011) . despite the threat of primarily b and epithelial cell transformation in infected individuals, ebv remains asymptomatic in most carriers, presumably due to a near perfect immune control of the virus by cytotoxic lymphocytes (taylor et al, 2015) . his mice can model this cell-mediated immune control by primarily cd8 + t lymphocytes (mchugh et al, 2019) . ebv infection resulted in dramatic cd8 + t-cell expansion in humanized mice with a peculiar phenotype (chatterjee et al, 2019; danisch et al, 2019) . the expanding cd8 + t cells carried the programmed death receptor (pd)-1 and t-cell immunoglobulin and mucin domain-containing protein 3 (tim-3) but retained cytokine production and were even superior in cytotoxicity to pd-1 negative cd8 + t-cell populations. nonetheless, pd-1 blockade with antibodies did not improve ebvspecific immune control in his mice (chatterjee et al, 2019) . in contrast, pd-1 inhibition led to elevated ebv titers, increased il-10 production, and associated lymphomagenesis. long-term infection of his mice with another herpes virus, the human cytomegalovirus (hcmv) is also possible as human cd34 + cell serves as a latent reservoir, whereas lytic reactivation in monocytes and macrophages can be stimulated with granulocyte-colony-stimulating factor (g-csf) (for a review, see koenig et al, 2020) . hcmv infection and reactivation result in different immunological responses and reactivation is associated with a higher pd-1 expression on t cells (theobald et al, 2018) . other pathogens that challenge immune compromised humans, especially pediatric patients after hct, are human adenoviruses. human adenovirus 2 (hadv2) infection of his mice resulted in liver pathology in one-third of mice, while two-thirds of infected mice remained asymptomatic (rodriguez et al, 2017) . his mice with asymptomatic hadv2 infection developed virus-specific igm and interferon (ifn)-c-producing t-cell responses. in blood and bm of mice not showing pathology, only early viral rna transcripts could be detected, which suggested the establishment of a persistent infection. in contrast, severely affected mice showed both early and late transcripts in many tissues as well as virus production in the liver (rodriguez et al, 2017) , all signs of disseminated disease, similar to what is observed in hct patients that suffer severe hadv infections (lion, 2014) . viruses for which dichotomous outcomes of infection can be modeled in his mice are the filoviruses of the genus ebolavirus his mice also offer platforms to explore new therapeutic avenues. this has primarily been investigated for human immunodeficiency virus (hiv; marsden & zack, 2017) . for example, treatment of hiv infection with broadly neutralizing antibodies (bnabs) was established in his mice (klein et al, 2012) . these studies primarily explored antibodies against four regions of the hiv envelope protein that consists of three heterodimers of glycoprotein (gp)41 and gp120 (caskey et al, 2019) . these four regions are the cd4 binding site, the v3 loop, the membrane proximal region, and the v1/v2 region. in these bnab treatment studies, mutational escape from single bnab treatment of viremic his mice was observed, while mixtures of several bnabs were able to suppress hiv viral titers for several weeks (klein et al, 2012) . based on these successful treatments in his mice and control of hybrid simian and human immunodeficiency virus (shiv) in macaques (nishimura et al, 2017) , hiv-specific bnabs were also tested in patients with and without prior anti-retroviral therapy (art; caskey et al, 2015 caskey et al, , 2017 . similar to his mice, individual bnab treatment suppressed hiv viremia only transiently with escape mutation development. even transfer of two bnabs only achieved suppression in hivinfected individuals after prior art treatment (bar-on et al, 2018; mendoza et al, 2018b) . thus, multiple bnabs probably need to be maintained at sufficient plasma levels to suppress hiv long term in his mice and patients. improving t cell-mediated immune control of hiv through gene therapy is another avenue that is explored in his mice. studies fall into two main categories, either improving t-cell reactivity by hivspecific tcr and chimeric antigen receptor (car) expression or rendering t cells resistant to hiv infection . for improving t-cell function, hiv-specific tcrs were introduced in hscs and could suppress hiv infection (kitchen et al, 2012) . as another strategy, in order to target cells replicating hiv and with gp120 surface expression, an extracellular domain of cd4 fused to the cd3f signaling domain was expressed in t cells (zhen et al, 2015 . downregulation of the hiv co-receptor, a chemokine receptor targeted by r5 tropic hiv strains (ccr5), has primarily been explored. hiv infection in his mice could be significantly compromised by ccr5 deletion or downregulation by zinc finger nuclease-mediated gene editing or rna silencing, respectively (holt et al, 2010; myburgh et al, 2015; shimizu et al, 2015) . the longterm survival of such engineered t cells might potentiate a functional cure of hiv, but also raises concerns with respect to toxicities and adverse effects of the respective cellular products, which can be assessed in his mice. engraftment of additional human tissues, like liver, bone, lung, and thymus, has been reported, but mainly hepatocytes and human lung tissue have been explored for infections with human pathogens. for example, humanized liver mouse models have been used to study infections by different hepatitis viruses (dandri et al, 2001; mercer et al, 2001; bissig et al, 2010; lutgehetmann et al, 2012; allweiss et al, 2016) . however, simultaneous reconstitution of human tissues with autologous human immune cells remains a challenge. nevertheless, hbv and hcv infections have been explored in his mice with allogeneic or autologous hepatocyte engraftment (washburn et al, 2011; billerbeck et al, 2016; dusseaux et al, 2017) . more recently, bone marrow, liver, and thymus (blt) engrafted mice have also been combined with ectopic human lung transplants (wahl et al, 2019) . intra-organ infection of these 8 of 16 embo molecular medicine 12: e8662 | 2020 ª 2020 the authors animals with different human-specific viruses (middle east respiratory syndrome-related coronavirus (mers), zika virus, respiratory syncytial virus (rsv), and hcmv) showed virus replication within lung implants, as well as antigen-specific humoral and t-cell responses. further studies in this direction are necessary to widen the application of humanized mice to additional human pathogens and immune responses against them. these examples of the use of humanized mice to recapitulate human infections and associated immune responses illustrate the vigor and translational value of these models. standardization of the reporting will improve the interpretation of results for single infections and for cross-reference among the different pathogens studied (table 1) . over the past decades, mouse xenograft models have significantly contributed to a better understanding of human malignancies. cancer-derived immortalized cell lines can adapt to in vitro growth and do not replicate the original malignant physiology seen in patients, potentially leading to artifacts in oncology studies. thus, patient-derived xenograft (pdx) models are currently the state-ofthe-art approach. development of liquid and solid pdx models relies on the availability of material obtained from patients with defined types of cancer, which after minimal manipulation is transferred by several routes into immunodeficient mice (fig 1d, table 1 ). nevertheless, whereas cell line-based xenografts allow an easier standardization of models, pdx samples are highly variable, can adapt to the murine environment and the human tumor stroma can be eventually replaced by murine cells. for leukemia research, while the generation of immunodeficient mouse strains like nsg has enabled functional in vivo studies on human hematopoiesis, engraftment of (in particular) myeloid malignant cells has remained challenging. the absence of a human bone marrow niche and species-specific growth factors underlies these challenges, and most notably, it has been difficult to maintain selfrenewal properties of malignant stem cells. since these populations are thought to be the therapy-resistant cells that frequently cause relapse of disease, it is of critical importance to use xenograft models in which specifically these cells can be propagated and stemness maintained. in order to further humanize xenograft models, transgenic and knock-in strains have been generated that (over)express growth factors like il-3, gm-csf, scf, tpo, and/or m-csf, as well as others (wunderlich et al, 2010; rongvaux et al, 2014 ). an alternative approach has been to develop a human microenvironment in the mouse initiated by mesenchymal stem cells coated on 3d scaffolds (antonelli et al, 2016; abarrategi et al, 2017; carretta et al, 2017) or embedded in matrigel (reinisch et al, 2016) . these models have allowed the engraftment of various hematological malignancies, including those that are notoriously difficult to engraft in regular nsg models such as low-risk acute myeloid leukemia (aml), myelodysplastic syndrome (mds), and myelofibrosis. importantly, self-renewal was better maintained in these models as shown by serial transplantation experiments and transcriptome studies. as such, these models more faithfully capture the disease phenotypes as seen in human patients and therefore are likely to produce more clinically relevant and translatable results when used in drug screens. a challenge that remains is that myeloid malignancies, in particular aml, display a complex clonal heterogeneity. multiple genetically distinct subclones can co-exist within an individual patient, each driven by a similar founder mutation but with different secondary driver mutations. these clones are not only genetically distinct; they also differ remarkably at the transcriptome, epigenome, and cell biological level (de boer et al, 2018) . to develop curative therapies, this clonal heterogeneity needs to be taken into account. it has become clear that not all clones of an individual patient might engraft equally efficiently in mice, and also the level of humanization of the model used might impact on whether the true clonal heterogeneity is preserved in vivo (klco et al, 2014; antonelli et al, 2016; carretta et al, 2017; wang et al, 2017; de boer et al, 2018) . both (deep) sequencing technologies and flow cytometry-based approaches are useful tools to dissect clonal heterogeneity, in vitro as well as in mouse models. for instance, an "infinicyt"based approach, which combines expression profiles of multiple aberrant aml-specific plasma membrane proteins, can provide subclone-specific insights into the clonal complexity of the malignancy under study (de boer et al, 2018) . implementation of such technologies is warranted in any conducted in vivo xenograft experiment to link drug responses to specific genetic features of malignant clones. in the case of solid tumors, the use of pdxs for preclinical drug development holds potential to improve our knowledge of the principles underlying responsiveness to individualized treatment regimens (hidalgo et al, 2014; byrne et al, 2017 ). yet, many questions are still open, in particular concerning the ability of the pdx approach to directly influence clinical decision making (aparicio et al, 2015) . not all cells that compose the parental tumor successfully engraft in the mouse, which introduces a selective pressure for genetic variants conferring better survival fitness (ben-david et al, 2017) . the subsequent propagation steps may also affect clonal dynamics, with further deviation of serially passaged samples from the primary tumor from which they were derived (eirew et al, 2015) . the lack of a fully functional immune system in the host and the fact that human stromal components -such as cancer-associated fibroblasts, endothelial cells, and inflammatory cells-are replaced by murine counterparts add extra layers of divergence over native tumors (hidalgo et al, 2014; aparicio et al, 2015; byrne et al, 2017) . these limitations notwithstanding pdx models of solid tumors offer considerable opportunities for biomarker and target nomination. first, although serially passaged pdxs are likely to be genetically different from the matched tumor of their donor patients, they are expected to display genomic makeups and polyclonality patterns that, on a probabilistic basis, may be similar to those of tumors that spontaneously develop in unrelated individuals (eirew et al, 2015; byrne et al, 2017) . these factors make pdxs critical tools in the translational oncology domain, whereby predictive biomarkers discovered in pdxs may be leveraged for the prospective identification of patients with tumors exhibiting the same biomarker repertoire. second, responses to therapies that target driver oncoproteins are thought to be only partly influenced by microenvironmental parameters and more directly dependent on cancer cell-intrinsic features, which affords results in pdxs with adequate predictive power for cancer cell-directed treatments (byrne et al, 2017) . finally, vast pdx collections are poised to capture inter-patient tumor diversity on a population scale, thus representing powerful platforms for large-scale genotype-response associations (gao et al, 2015) . the above examples illustrated the importance of pdx models in understanding the evolution of tumor growth, investigating the mechanisms of drug resistance, and developing personalized treatments. critical to these studies is ensuring researchers have access to high-quality pdx models and molecular datasets that give sufficient power to perform informative analyses. however, the complex nature of pdx models and the heterogeneous resources that generate them often lead to crucial information about tumors, host strains, transplant, and quality assurance processes being inconsistently presented. to address this challenge, the pdx research community developed the pdx model minimal information standard (pdx-mi) that defines the critical metadata needed to exchange knowledge about pdx models (meehan et al, 2017) . pdx-mi describes the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of pdx models in cancer research. since its inception, pdx-mi has been adopted by producers of pdx models including the international pdxnet and europdx consortia as well as the pdx finder catalog that captures, harmonizes, and disseminates data about pdx models and associated omic datasets (conte et al, 2019) . pdx-mi promotes reuse of models and data, maximizing the impact of these models on oncology research and facilitating the development of new treatments. therapeutic modulation of the human immune system to improve recognition and response to tumors is a clinically accepted revolution in oncology treatment for multiple tumor types (pardoll, 2012) . immuno-oncology (io) is considered a breakthrough due to significant and durable tumor regression coupled with increased long-term survival. however, these clinical responses only occur in a subset of patients. therefore, considerable investment in preclinical research is still necessary to identify new and improved approaches to cancer cell-specific immune response as well as testing of combinatorial strategies. these and other approaches are typically developed in syngeneic mouse models of oncology to work out mechanisms of action. nonetheless, human-specific immune modulators require in vivo models with human-specific targets on both human immune cells and human tumors to validate preclinical responses, accelerate development, and improve translation to the clinic. ideally, io in vivo studies will rely on the combination of his and pdx models. as described in the following sections, his mice co-engrafted with human tumors are proving to be a valuable tool in the development of new strategies for human-specific immuno-oncology therapies. nonetheless, as these models are per se complex and the matching of the tumor and immune system from the same patient is currently a difficult task, several studies explore cell line derived xenograft (cdx) implanted after humanization in his mice or administration of human peripheral blood lymphocytes (pbl; fig 1e, table 1 ). these io studies have been used, for example, to test immune modulation caused by engineered agonistic or antagonistic monoclonal antibodies (mab; scott et al, 2012) , bispecific t-cell engagers (bite; baeuerle & reinhardt, 2009) , or t-cell bispecific antibodies (tcb; bacac et al, 2018) . a very important and commonly asked question regarding human tumor cell engraftment in his mice is whether the cdx or pdx tumor and hematopoietic donor must be 100% hla-matched to allow co-engraftment. neonatal irradiated nsg mice co-injected into the liver with cd34 + cord blood-derived hsc and hlamismatched human breast cdx, resulted in the development of a human immune system together with human tumor growth, including metastases in the lung and brain (wege et al, 2011 (wege et al, , 2014 . tumors were partially infiltrated with t cells, b cells, and myeloid cells. more detailed analyses of the spleen revealed not only a t-cell specific activation pattern but also b-cell maturation and the production of tumor-specific antibodies (wege et al, 2014) . moreover, the tumor engrafted his mice were used for a preclinical trial to test the potential of il-15 in combination with trastuzumab (anti-her2 mab) therapy with the intention to enhance antibody-dependent cellular cytotoxicity (adcc; wege et al, 2017) . il-15 treatment triggered immune activation and promoted tumor depletion but also induced systemic inflammation, resulting in death of the treated mice (wege et al, 2017) . in another study, 3-week-old nsg mice were engrafted with cord blood-derived cd34 + hsc first to establish mature multilineage immune engraftment and then injected subcutaneously 12-16 weeks later with partially hla-matched pdx tumors (wang et al, 2017) . despite the presence of a wide range of functional immune cells, the his mice were capable of engrafting the tumors and in many cases the growth kinetics of these tumors did not vary significantly from immunodeficient controls not engrafted with hsc. however, not all partially hla-matched tumor/hsc donor combinations escape immune-mediated changes in growth kinetics and some tumors are rejected, highlighting the importance of empirically testing tumor growth against multiple hsc donors. subcutaneously engrafted tumors were infiltrated with a wide range of human innate and adaptive immune cells and both the frequency and distribution of immune cell types varied across different tumor types (wang et al, 2017) . one mechanism known to prevent t cells from responding to tumors is the pd-1 and its ligand (pd-l1) checkpoint pathway. the clinically approved checkpoint inhibitor pembrolizumab (anti-pd-1 mab) has been tested in tumor-bearing his mice and suppression of tumor growth was observed using both cdx and pdx tumors (wang et al, 2017) . suppression of tumor growth with pembrolizumab only occurred in mice co-engrafted with human immune cells and the response was abrogated when mice were pretreated with anti-human cd8 mab to deplete human cd8 + t cells, demonstrating human cd8 + t cells mediated the effector response following release from checkpoint inhibition. efficacy studies with pembrolizumab were run with multiple hsc donors distributed among both control and treatment arms of each tumor tested for response. multiple hsc donors allowed the observation that not all tumor/hsc combinations show a response to pembrolizumab, and the frequency of donor-related response (~25-30%) is similar to what is observed in the clinic (topalian et al, 2012) . regulatory t cells (tregs) infiltrate a wide range of tumor types and mechanism of action studies performed in syngeneic mouse tumor models revealed that depletion of these cells from the tumor could release t effector cells from treg suppression (smyth et al, 2014) . preclinical efficacy for this approach was demonstrated when his mice were engrafted with sk-mel-5 human melanoma cdx and treated with an anti-human mab targeting the glucocorticoidinduced tnfr family-related (gitr) protein, highly expressed on tregs. tumor growth was significantly suppressed, the percentage of tregs was reduced in tumor and spleen, and tumor-infiltrating lymphocytes showed increased secretion of the effector cytokines il-2 and ifn-c (mahne et al, 2017) . as more io treatments move through clinical trials, clinicians are seeing an association between strong immune-mediated tumor killing responses and cytokine release syndrome (crs). given these observations, preclinical studies able to recapitulate crs in his mice are becoming highly relevant. in a recent report, his mice were co-engrafted with a diffuse large b-cell lymphoma (wsu-dlcl2) and treated with either obinutuzumab (anti-cd20 mab) or a novel cd20-t-cell bispecific antibody (tcb) containing two cd20 binding domains and one cd3e domain in a head-to-tail orientation to one of the cd20 regions (bacac et al, 2018) . cd20-tcb promoted a more extensive killing response than obinutuzumab. further, cd20-tcb administration was associated with increased expression of multiple human inflammatory cytokines indicating a crs response that was not observed with obinutuzumab treatment. an alternate strategy was tested where treatment was initiated with a single dose of obinutuzumab followed by multiple high doses of cd20-tcb. the pretreatment with obinutuzumab strategy enabled rapid and extensive tumor killing with minimal crs response. these types of preclinical experiments with his mice demonstrate their value in working out protocols designed to maximize both efficacy and safety. the question of immuno-therapy-mediated toxicity, particularly in the context of crs, is a key component of preclinical evaluation and a reliable assay is needed. neither in vitro assays nor nonhuman primates have proven reliable for assessment of crs (stebbings et al, 2007) . a team at the us food and drug administration recently published two reports testing crs using mab therapies known to have a strong cytotoxic response in the blt-his mice (yan et al, 2019a,b) . blt mice were injected with adalimumab (anti-tnf-a mab) as a negative control because it is used clinically without evidence of crs. the test article was tgn1412 (anti-cd28 mab), a reagent known to be associated with clinical crs. tgn1412-treated blt mice released multiple cytokines into peripheral blood within 2-4 hours of treatment, indicating a strong crs response that was not observed in the adalimumab-treated group (yan et al, 2019a) . the mice also showed a decrease in human cd45 + , cd3 + , cd4 + , cd8 + , and cd19 + cells in peripheral blood similar to human patients and showed an increase in murine serum amyloid a, indicating severe liver inflammation. a second study compared muromonab (anti-cd3 mab, okt3) to adalimumab in blt mice. the muromonab-treated mice released multiple proinflammatory cytokines associated with crs into peripheral blood within 2-4 h, and pretreatment of mice with methylprednisolone prior to muromonab blunted or delayed development of crs (yan et al, 2019b) . together, these studies show that blt-his mice are capable of recapitulating multiple aspects of a strong crs response when dosed with mab therapies designed to stimulate strong t-cell activation. to our knowledge, no direct comparisons between pbmc-his and blt-his model mice have been made to date. there are several published studies using pbmc-his mice demonstrating crs (brady et al, 2014; weissmuller et al, 2016) with the mice being used between 6 and 16 days of pbmc injection. in both reports, the authors state there were no signs of gvhd present when used. in yan et al (2019a) , a limited comparison of cd34-his mice and blt-his mice was undertaken and blt-his mice showed clear evidence of crs and no gvhd, whereas cd34-his mice did not show any difference as compared to control treatment, suggesting that the cd34-his mouse does not show a clear signal for crs and may not be an appropriate model. as previously published for the blt-his mouse (weaver et al, 2019) , when present, gvhd is clearly evident and can be differentiated from other processes, such as crs. gvhd occurs rapidly and for all pbmc-his mice, but does not occur with high frequency in blt-his mice. with respect to crs, pbmc-his mice would potentially demonstrate crs for drugs specifically impacting t cells, but not other tissues or cell types. blt-his mice have much broader engraftment in terms of cell types and presence in non-lymphoid organs, suggesting that a wider range of targets could demonstrate crs if present. for preclinical use, the stebbings in vitro assay (stebbings et al, 2007) should be initially undertaken. studies with his mice would be adjunctive and informed by both the target of the therapeutic and what organ system(s) were targeted. additional circumstances in which in vivo testing could be helpful include higher risk drug targets and non-t-cell targets. in conclusion, his mice are a powerful tool for io research. his mice do not recapitulate every aspect of human immunity, but they are capable of answering a wide range of important scientific questions that form a critical guide for preclinical io discovery. future challenges will include the understanding of the donor-to-donor immune variability observed in some of these treatment strategies. this will provide opportunities to identify predictive markers and clinical diagnostics assays helpful in assisting patient enrollment for improved treatment outcomes. tumor populations that escape response in subsets of mice can be further analyzed for understanding mechanism of resistance. humanized mouse models result from the sum of several components: choice and availability of human donors, human cells or tissues, mouse recipient, types of manipulations, human infections, and human tumor types. furthermore, the materials available for analyses and methods of analyses provide another level of complexity. it is quite clear that humanized mouse models are customized by the different laboratories around the globe and it would be unrealistic to standardize how the models should be built. nevertheless, reporting of minimal information provided by specific guidelines can facilitate independent validation of published data, which is a fundamental cornerstone for scientific advancements. the list of variables provided in table 1 is an initial attempt brought up by the faculty participating in the 2017 and 2019 embo practical courses created for training young investigators on the development of humanized mouse models. this initiative called "minimal information for standardization of humanized mouse models" (mishum) is built on the experience of the authors of this manuscript in developing similar reporting standards for non-humanized pdx models (meehan et al, 2017) . the main aim of this workgroup is, similar to pdx-mi, to promote material and protocol exchange, transparency in reporting of assays and analyses performed. the standards described here represent a starting point. the longer term intention is to extend this initiative beyond the embo courses to include input from multiple stakeholders from both academia and industry. further, in order to enable a standardized interpretation of already published results, the collection of technical information and quantifiable data can in the future be procured within mishum for the creation of a digitalized database. once these standards and database are evolving, a parallel data mining activity will provide the opportunity to explore and discover convergent signatures and patterns of human immunology, infections, and oncology in vivo in humanized mice. our long-term goal is that, in the future, these models will become largely reproducible and predictive models for the understanding of human physiology, immunology, and oncology, which require a living experimental system. beyond the gain of scientific information, we hope to make humanized mouse models more environmentally sustainable by optimizing the methods and reducing the number of humanized mice used for experimentation. ultimately, we seek to support the "3r" principles: (i) replace the use of humanized mice if alternative in vitro techniques (such by the use of organoids or chips), metadata or data mining eventually prove to be as solid as the in vivo results; (ii) reduce the number of humanized mice to a minimum within each experimental cohort; and (iii) refine the experimental setup using the best and ethically available human material and analyses, also making sure that invasive approaches can be minimized to mitigate the suffering of the animals. only with a consensus checklist and with a coherent reporting policy, we will be able to identify the best material, methods, and analyses that will ultimately lead to optimized humanized mouse models. below is a summary of main topics identified by our community that remain to be solved on a case-by-case basis depending on the use of the humanized mouse model: human hematopoiesis and immunity: methods to expand human hscs will enable larger experimental cohorts. novel methods for matching the hla between human hematopoietic cells and mouse epithelial cells will improve human t-cell development in a hlarestricted manner. better development of lymph nodes and germinal centers within the spleen that would improve innate and adaptive immune responses. novel mouse strains or methods allowing regeneration of lymph nodes will allow b cell class switch and production of human high-affinity igg and iga antibodies. human metabolism: mice and human display different rates and pathways of metabolism, and particularly for liver metabolism, it is essential to address this limitation of humanized mice. since many metabolites are diffusible, new models are needed that can eliminate or temporarily block murine metabolism while using chimeric mice. human infections: engraftment of human peripheral tissues (i.e., liver, lung, skin, and brain) will allow infections with human pathogens targeting other tissues than immune cells, and the possibility of combining them with hlas matched human hematopoietic stem cells will create a more complete model of human infection. better engraftment of the human erythrocyte lineage will allow further studies of erythrocyte-infecting pathogens (i.e., plasmodium). human oncology: further humanization of xenograft mouse models such as implantation of human msc-coated 3d scaffolds or nsg mice (over) expressing human cytokines has improved engraftment rates of primary tumor cells. however, for each individual cancer patient case, it will have to be established which (sub)clones preferably grow out. also, it will need to be carefully evaluated how the transcriptome and epigenome of the original patient samples are preserved in the pdx models. human immuno-oncology: the hla matching of the human immune system and tumor will be required. a functional enhancement of the cd4 + and cd8 + t cells will be needed to allow higher tumor infiltration and anti-tumor responses. an improved and faster development of "avatar" pdx models will allow decisions about personalized therapeutic options. (iii) https://www.mdpi.com/journal/vaccines/special_issues/humanized_mice in this special issue "humanized mice in vaccinology: opportunities and challenges", aspects related to the use of humanized mice in vaccinology, opportunities, and the challenges ahead are discussed. (iv) http://www.pdxfinder.org/pdx-standard/ the pdx minimal information document represents the results of a broad community effort to develop a standard regarding the essential information needed to describe a pdx model. versatile humanized niche model enables study of normal and malignant human hematopoiesis humanized immune system mouse models: progress, challenges and opportunities human liver chimeric mice as a new model of chronic hepatitis e virus infection and preclinical drug evaluation establishing human leukemia xenograft mouse models by implanting human bone marrow-like scaffold-based niches examining the utility of patientderived xenograft mouse models robust expansion of human hepatocytes in fah à/à /rag2 à/à /il2rg à/à mice cd20-tcb with obinutuzumab pretreatment as next-generation treatment of hematologic malignancies bispecific t-cell engaging antibodies for cancer therapy safety and antiviral activity of combination hiv-1 broadly neutralizing antibodies in viremic individuals a novel humanized mouse lacking murine p450 oxidoreductase for studying human drug metabolism patient-derived xenografts undergo mouse-specific tumor evolution hepatitis b virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and m2-like macrophages development of human cd4+ foxp3+ regulatory t cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing nod-scid il2rgamma(null) humanized mice humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and nk cells repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model human liver chimeric mice provide a model for hepatitis b and c virus infection and treatment p450-humanized and human liver chimeric mouse models for studying xenobiotic metabolism and toxicity development and rescue of human familial hypercholesterolaemia in a xenograft mouse model prospective isolation and characterization of genetically and functionally distinct aml subclones preclinical screening for acute toxicity of therapeutic monoclonal antibodies in a hu-scid model human immune system development and rejection of human islet allografts in spontaneously diabetic nod-rag1null il2rgammanull ins2akita mice lack of acute xenogeneic graft-versus-host disease, but retention of t-cell function following engraftment of human peripheral blood mononuclear cells in nsg mice deficient in mhc class i and ii expression human primary liver cancer-derived organoid cultures for disease modeling and drug screening humanized mouse model of mast cell-mediated passive cutaneous anaphylaxis and passive systemic anaphylaxis interrogating open issues in cancer precision medicine with patient-derived xenografts genetically engineered mesenchymal stromal cells produce il-3 and tpo to further improve human scaffold-based xenograft models new approaches for the enhancement of chimeric antigen receptors for the treatment of hiv viraemia suppressed in hiv-1-infected humans by broadly neutralizing antibody 3bnc117 antibody 10-1074 suppresses viremia in hiv-1-infected individuals broadly neutralizing anti-hiv-1 monoclonal antibodies in the clinic cd8 + t cells retain protective functions despite sustained inhibitory receptor expression during epstein-barr virus infection in vivo epstein-barr virus: an important vaccine target for cancer prevention pdx finder: a portal for patient-derived tumor xenograft model discovery engineered dendritic cells from cord blood and adult blood accelerate effector t cell immune reconstitution against hcmv human hepatocyte depletion in the presence of hiv-1 infection in dual reconstituted humanized mice repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis b virus c1qrp defines a new human stem cell population with hematopoietic and hepatic potential spatiotemporally skewed activation of programmed cell death receptor 1-positive t cells after epstein-barr virus infection and tumor development in long-term fully humanized mice microenvironment-dependent growth of preneoplastic and malignant plasma cells in humanized mice selective expansion of myeloid and nk cells in humanized mice yields human-like vaccine responses viral load affects the immune response to hbv in mice with humanized immune system and liver dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution comparative pathogenesis of ebola virus and reston virus infection in humanized mice viral infection of engrafted human islets leads to diabetes high-throughput screening using patientderived tumor xenografts to predict clinical trial drug response human hepatocytes and hematolymphoid dual reconstitution in treosulfan-conditioned upa-nog mice the reconstituted 'humanized liver' in tk-nog mice is mature and functional neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator patient-derived xenograft models: an emerging platform for translational cancer research human hematopoietic stem/ progenitor cells modified by zinc-finger nucleases targeted to ccr5 control hiv-1 in vivo improved b cell development in humanized nod-scid il2rgamma(null) mice transgenically expressing human stem cell factor, granulocytemacrophage colony-stimulating factor and interleukin-3 tracking human immunodeficiency virus-1 infection in the humanized drag mouse model in vivo suppression of hiv by antigen specific t cells derived from engineered hematopoietic stem cells functional heterogeneity of genetically defined subclones in acute myeloid leukemia hiv therapy by a combination of broadly neutralizing antibodies in humanized mice modeling human cytomegalovirus in humanized mice for vaccine testing a novel flt3-deficient his mouse model with selective enhancement of human dc development a human immune system mouse model with robust lymph node development adenovirus infections in immunocompetent and immunocompromised patients ebola virus disease in mice with transplanted human hematopoietic stem cells humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation dual roles for regulatory t-cell depletion and costimulatory signaling in agonistic gitr targeting for tumor immunotherapy humanized mouse models for human immunodeficiency virus infection accelerated thymopoiesis and improved t-cell responses in hla-a2/-dr2 transgenic brgs-based human immune system mice the scid-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function infection and immune control of human oncogenic gamma-herpesviruses in humanized mice pdx-mi: minimal information for patient-derived tumor xenograft models generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model combination therapy with anti-hiv-1 antibodies maintains viral suppression hepatitis c virus replication in mice with chimeric human livers latency and lytic replication in the oncogenesis of the epstein barr virus lentivector knockdown of ccr5 in hematopoietic stem and progenitor cells confers functional and persistent hiv-1 resistance in humanized mice early antibody therapy can induce long-lasting immunity to shiv isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment the blockade of immune checkpoints in cancer immunotherapy bioengineering of humanized bone marrow microenvironments in mouse and their visualization by live imaging a humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells humanized mice reproduce acute and persistent human adenovirus infection development and function of human innate immune cells in a humanized mouse model dendritic cellmediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids antibody therapy of cancer rnai-mediated ccr5 knockdown provides hiv-1 resistance to memory t cells in humanized blt mice humanized mice in translational biomedical research humanized mice for immune system investigation: progress, promise and challenges human cancer growth and therapy in immunodeficient mouse models humanized mouse models of immunological diseases and precision medicine targeting regulatory t cells in tumor immunotherapy cytokine storm" in the phase i trial of monoclonal antibody tgn1412: better understanding the causes to improve preclinical testing of immunotherapeutics decisions about dendritic cells: past, present, and future e8662 | 2020 human hepatocytes polymorphism in sirpa modulates engraftment of human hematopoietic stem cells type 1 diabetes induction in humanized mice the immunology of epstein-barr virus-induced disease signatures of t and b cell development, functional responses and pd-1 upregulation after hcmv latent infections and reactivations in nod.rag.gamma mice humanized with cord blood cd34(+) cells safety, activity, and immune correlates of anti-pd-1 antibody in cancer multidimensional analysis integrating human t-cell signatures in lymphatic tissues with sex of humanized mice for prediction of responses after dendritic cell immunization precision mouse models with expanded tropism for human pathogens humanized mouse models of clinical disease patient-derived xenotransplants can recapitulate the genetic driver landscape of acute leukemias a humanized mouse model to study hepatitis c virus infection, immune response, and liver disease blt-immune humanized mice as a model for nivolumab-induced immune-mediated adverse events: comparison of the nog and nog-exl strains humanized tumor mice-a new model to study and manipulate the immune response in advanced cancer therapy 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receptor-engineered stem cells long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor t cells in a nonhuman primate model of hiv/aids license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited we thank the european molecular biology organization (embo) and the jackson laboratory (jax) for funding, and we thank the staff of the euro key: cord-019040-lj1r8ptb authors: xiao, ren title: human security in practice: the chinese experience date: 2018-12-07 journal: human security norms in east asia doi: 10.1007/978-3-319-97247-3_3 sha: doc_id: 19040 cord_uid: lj1r8ptb this chapter elaborates on how the idea of human security is defined and understood by the government and various actors in china. as one of the permanent members of the united nations (un) security council, china, has been supportive of international norms advocated by the un, and even though the term human security has not been frequently used, in effect it has been vigorously practiced. for both the government and the academic community in china, human security and national security are not necessarily in confrontation but rather can complement and strengthen one another. the purpose is to improve the quality of people’s everyday life and the government is expected to contribute to this end. chinese people expect the government to extend a parental roof over the people. are perceived as pressing human security issues in china. the chapter concludes with a discussion of the overall human security practices and shows that, based on current indicators, china is heading in a healthy direction in terms of promoting human security. the understanding of human security in china in china, the government is often wary of new academic terms and tends to avoid using them directly. however, the findings from our previous study (ren and li 2013) clearly showed that the country is following a trajectory of increasingly attaching more importance to human (both individual and collective) security. much greater attention has been paid to mitigating threats to human security, and practical measures are being taken to fulfill the mandate. we emphasized the convergence of the human security idea and china's practices (ren and li 2013) . our findings disprove the following statement: "the very notion of 'human security' has so far not appeared in the chinese language in any possible translation, and the people's republic of china (prc) has not even accepted or adopted the concept of human security in either domestic development papers or foreign policy guidelines…" (wu 2013, 1) . our examination suggests otherwise. though the chinese government has only used the term "human security" on a few occasions, china is definitely engaged in the enterprise of enhancing human security. the fact that china is becoming more open and receptive to human security is strongly related to its experience within the un system. as one of the five permanent members of the un security council, china has long been supportive of the un, the most important international organization in today's world, and the country has played a proactive role in various un activities such as peacebuilding, development, and global governance (breslin and ren 2018) . over the years, the un has been subjected to criticisms and it is widely believed that reforms are needed, which china supports. nevertheless, beijing continues to show a belief that the un plays an irreplaceable role in global governance, and china has endeavored to support this by encouraging the un to play its role well (kim 1999) . this has remained an unchanged priority in china's foreign policy agenda. china's steady backing has boosted the status of the un in world affairs, at a time when the world is faced with growing global challenges in the first decades of the twenty-first century. meanwhile, the un has taken the lead in advocating and/or spreading norms and principles, and this has affected and helped to shape china's ideas (kent 2014) . usually, un initiatives or proposals attract china's attention, and prompt beijing to have a closer look at them before taking actions to adapt to the new norms or principles (morton 2009, 75-76) . for example, china was involved in the deliberations and adoption of the un world summit outcome document in 2005. although it is not the same thing as the "responsibility to protect" idea proposed by the international commission on intervention and state sovereignty (iciss 2001) , to some extent the outcome document is consistent with this idea in terms of thinking on protection. the outcome document attempts to strike a balance between the need to protect innocent people around the world from being harmed while avoiding the abuse of external intervention or selfish behavior in the name of protection. for this, the outcome document imposes limitations by listing four specific crimes against which the international community should act to prevent people's lives from being jeopardized: genocide, war crimes, ethnic cleansing, and crimes against humanity. in this way, the outcome document has become a new international legal document. china was involved in this process and made its own contribution. thus, involvement in global or regional institutions has pushed china to clarify or develop its own thinking on human security. against the backdrop of the great earthquake and tsunami that devastated japan's tohoku region, the ministerial millennium development goals follow-up meeting was held under the un framework in june 2011 in tokyo, japan. china sent vice foreign minister cui tiankai to attend the meeting. in the speech he delivered, cui, as the representative of the government of china, stated: to discuss the mdgs from the angle of human security offers a thoughtprovoking perspective. we believe that the mdgs and human security are interrelated and should be mutually reinforcing. the mdgs embody so many aspects of human security, while the realization of mdgs aims at greater wellbeing and security of more people in the first place. (t. cui 2011) cui pointed to the fact that the general picture of global security remains disturbing: "civilians in north africa and the middle east continue to bear the brunt of turmoil, and innocent women and children are still being displaced or killed in armed conflicts in various parts of the world." cui concluded that: these give rise to the call for a new concept on security and an international political order where the united nations should play a central role. we strongly believe that the purposes and principles of the un charter should be upheld, and security council resolutions should be implemented in a faithful manner. …in a word, if human security in the larger sense of the term is still so much threatened, there is little hope for better individual security. (t. cui 2011) this is an illuminating example of how china has clearly and definitively adopted and used both the idea of and the actual term "human security" in the context of a formal un meeting. although it was an event on mdgs and not specifically on human security, the term "human security" was explicitly employed to express china's opinion and position. this discourse was reinforced by further developments. in february 2014, ms. fu ying, chairperson of the national people's congress foreign affairs committee, was invited to and spoke at the munich security conference. she argued that the security necessary for people to survive and develop is fundamental to any security. the core of the chinese dream of the revival of the chinese nation proposed by president xi jinping is to have the 1.3 billion chinese people live a happy life. in other words, all ordinary people have the right and are entitled to live with dignity in a secure environment. this is the attraction of china's success story to the world, as well as the charm of the chinese dream. 1 this line of thinking has many elements in common with the widely shared "human security" idea. for instance, both place emphasis on individuals and their happiness, and this must be fulfilled together with national development. ends and means should be consistent, and the persistent value is that people themselves are the end and they should not be sacrificed for arbitrary national goals. the key point here is that china's security concept is undergoing a profound transformation. a new consensus has emerged that assumes security does not equal military security, and security should not only be comprehensive but also people-centered (yi renmin wei zhongxin): "national security" now has new connotations. during the landmark third plenum, which was held in november 2013, a major resolution to comprehensively deepen china's reform was deliberated and passed. the long reformist document has 16 parts and 60 items. the decision to create a new national security commission (nsc) was announced as a component of the 13th part on the new social governance system. the purpose of the nsc is to improve the relevant institutions and strategy to better safeguard china's national security. according to the explanatory speech president xi jinping gave, reform and development are conditional on national security and social stability, without which reform and development cannot be advanced further. 3 at present, china is faced with the dual pressure of safeguarding national sovereignty, security, and development interests externally, and maintaining political security and social stability internally (xi 2017, 411) . all kinds of risks that can or cannot be foreseen are clearly increasing, while the country's institutions and security mechanisms are insufficient to adequately meet the need of maintaining national security. this has resulted in demands to set up a powerful and capable platform to coordinate all work on national security. the responsibilities of the nsc include formulating and implementing national security strategies, advancing the construction of national security rule of law, deciding national security guidelines and policies, and studying and addressing major issues in the national security work. 4 quite understandably, this major decision drew considerable attention both at home and abroad. in april 2014, the first meeting of the new nsc was held. according to the speech president xi jinping delivered, the security of the people must be its core purpose. state security should in every sense serve the people and rely on the people. the mass foundation of state security must be laid and consolidated. 5 this means that state security and human security are not confrontational but rather that the two can and should coexist. internally, china is undergoing a modernization process and the number of contradictions is growing. in this time of rapid economic and social development, a series of social problems have been accumulating that have not been "digested" well. for some chinese observers, "group incidents" resulting from unbalanced distribution of interests frequently break out, negatively affecting social stability. externally, as china continues to grow, some powers and neighboring countries have been hedging against china, and as a result, contradictions or frictions in china's neighborhood have been increasing. moreover, challenges to social stability and security, from home and abroad, have been affecting each other, and they are interlinked. coping with them is becoming a more difficult job. in the current era, the concept of national security is becoming richer in depth and wider in breadth, involving a range of issues in a variety of areas. increasingly complex problems cannot be dealt separately by foreign affairs, national defense, and security departments but rather are demanding that more agencies, social organizations, and even the whole society work together (hu and wang 2013) . since the initiative to create china's nsc appears in the "social governance system" section of the november 2013 resolution, its domestic and internal security dimension is self-evident. in the meantime, the commission's work involves two dimensions (both external and internal) rather than just one. thus the "security" the initiative refers to is of a comprehensive nature. it is not difficult to come to that conclusion if we simply recall the july 5, 2009, incident in xinjiang, the riots in tibet and tibet-related self-immolations in its neighboring provinces, and the horrible killing at the kunming railway station on march 1, 2014. during all these incidents, innocent people were killed or injured, and some of the incidents resulted from the actions by hostile external forces. that is why it was widely believed that internal security would be the dominant concern for china's nsc, at least in its early stage. when ordinary people can be unexpectedly harmed by violent terrorist attacks in any situation and without warning, a sense of insecurity arises, and this can be frightening. thus, even though freedom from want is no longer a problem in today's china, ordinary people should also be able to live their lives free from fear. a broad concept of security of this kind logically became the goal for china's nsc. in fact, as cui shunji of zhejiang university points out, since the initiation of reform, at the highest levels, attention has been placed on poverty reduction, the pursuit of a sustainable development model, and china's proposals for constructing a "harmonious society" and a "harmonious world." these goals indicate that china regards the guaranteeing of basic human needs, social justice, and harmony as well as sustainable development as important parts of a continuum of national security (s. cui 2014, 68-69) . "letting people live a happier life with more dignity" has become the goal of national development, which indicates that china's recognition of human security has been elevated to the political level. putting people first and "governing for the people" have become the new thinking for governing the country. as a reflection of the foreign policy changes, handling foreign affairs for the state is shifting to handling foreign affairs for the people (s. cui 2014). this was reinforced at the 19th party congress, held in october 2017, which emphasized the need to regard people's interests as paramount and let the achievements of reform and development benefit everybody more and fairly. in china's research community, "human security" is often discussed in terms of the discourse on "non-traditional security." for years, chinese researchers have utilized the term "non-traditional security" to distinguish the so-called non-traditional matters from more traditional matters. traditional security usually refers to military security, namely, assuring national security through boosting military power. however, after the end of the cold war, threats to security have increasingly come from nonmilitary domains in the form of unconventional or non-traditional security threats. such issues include financial crises, terrorism, transnational crime, environmental degradation, the spread of hiv/aids, scarcity of water resources, food insecurity, and so forth. according to the summation by a leading chinese researcher, nontraditional security is broad-based, complex, and multi-dimensional. examining each in turn, while traditional security falls into military, political, and diplomatic areas, and its supreme value is the pursuit of peace and the elimination of war or the possibility of war, a broad-based non-traditional security is more about economic, social, cultural, and environmental threats as well as the emerging cyber security and space issues. in addition to peace, non-traditional security relates more to risk, crisis, emergency, and daily threats to life. it relates more to natural disasters, accidents, emerging public health incidents, and major public security events. second, non-traditional security is complex. threats to non-traditional security are mainly the threats to "societal security" and "human security." society and people are the chief referent objects of non-traditional security, and a "safe china" has societal and human dimensions. for individuals, safety means that the security of people is guaranteed: namely, individuals enjoy a state of existence in which a person's body is not injured, their mind is not harmed, they are not deprived of their property, and their living environment is not undermined. third, non-traditional security is multi-dimensional. according to the place and origin of non-traditional security events, threats to nontraditional security facing a country can be divided into four categories: (1) exogenous non-traditional security threats which take place abroad and chiefly require diplomatic negotiations; (2) endogenous nontraditional security threats which take place at home and chiefly require domestic interventions; (3) "bi-dogenous" (双源性) non-traditional security threats which take place in peripheral areas, necessitating both domestic and international management; and (4) "multi-dogenous" (多源性) non-traditional security threats that involve both traditional and nontraditional security issues requiring the involvement of the military in addition to other organizations (yu 2013, 3-6) . throughout the above process of ideational transition, a few landmark crises have affected china deeply, including the 1997 asian financial crisis that highlighted the importance of financial security, the 2003 sars crisis that highlighted public health security, and the march 1, 2014, terrorist attacks at the railway station in kunming, the capital of yunnan province. the horrible march 1 violence against innocent people especially highlighted the serious security threat that ordinary people could encounter in their daily lives. this genuine risk could give rise to a widespread sense of fear. as noted, earlier conceptions of security in china were mainly focused on state security, the importance of which remained unquestioned. now that human security has been put forward, and given its undeniable value orientation, support for the concept is gaining momentum. in fact, chinese scholars speak highly of human security and argue that it goes beyond the limits of state-centric traditional security research and is the least traditional theory among the non-traditional security domains (yu 2014, 18) . human security research explicitly sees people as a collectivity and individuals as the referent object of security. this transcends the dilemma of more traditional security theory, since the state can bring about insecurity to its citizens. such a possibility raises questions concerning the relationship between state security and human security. in general, chinese researchers do not endorse the view that human security overrides state security or that the two are confrontational, but rather affirm the reasonableness and value of state security at the same time. for example, shi bin, a professor at nanjing university, asserts that the human security idea is a focal embodiment of non-traditional security and new security concepts, and yet the relationship between human security and state security is much more complicated (shi 2014, 97-100) . shi argues that both state security and human security, in terms of their security concerns and value pursuits, have legitimate claims. however, there is no reason for either of them to become totally dominant. human security is of course the fundamental goal and ultimate value of human development. the value orientation of putting people first, with human security at the center, provides it with the moral high ground and legitimacy. however, a person has both individuality and sociality, and individuals are often weak and helpless. resisting foreign military invasion and safeguarding national sovereignty and territorial integrity are therefore in the nation's common interest. nonetheless, the traditional state-centric security idea and strategy indeed ignores the security needs of many non-state or sub-national entities and cannot adequately deal with external nonmilitary threats such as environmental degradation and pandemic disease. second, shi bin articulates that, although there is a tension between "human security" and "state security" and the two may conflict in practice, they can still be mutually accommodating and complementary if handled and balanced well. therefore, this can provide a favorable opportunity for upgrading the overall security level of all entities. people's security and welfare, and the improvement of their living conditions and quality of life, are an important base for national identity, social stability, and political legitimacy. in this sense, human security and state security are not necessarily contradictory. for shi, the human security discourse has a tangible western value orientation bearing liberal colors; consequently, in practice, it tends to override the security interests of the sovereign state. the acclaimed paradigm shift from the state to individuals excessively downgrades the positive role of the state in dealing with various security challenges. for zhang yunling of the chinese academy of social sciences, the rise of non-traditional security issues does not mean that traditional security is no longer important. the appearance of non-traditional security on the agenda and the fact that it is stressed imply that it has been included in the category of security, and therefore the formation of a "comprehensive security" concept includes both traditional and non-traditional security as well as the corresponding security policies (y. zhang 2012) . taken together, with "non-traditional security" increasingly becoming part of the mainstream discourse in china, researchers tend not to deny the value of state security but rather see human and state security as being mutually accommodating. nevertheless, the theoretical shortcomings of non-traditional security, as guoguang wu rightly points out, are obvious, when it is narrowly framed as oppositional to traditional security, without any positive and substantive defining of the contents and nature of new "securities" (wu 2013, 5) . the relationship between state security and personal security is not a zero-sum one though. more state security does not mean less personal security, and vice versa. the goal should be a calibrated balance between the two. moreover, the increasing tendency to securitize many issues and aspects of life may not be entirely beneficial. an overemphasis on discussions of security may imply a growth of insecurity. for example, in china food safety was previously not discussed often but was later widely talked about. this indicated that food safety was not formerly an issue, but it became such an issue. the deterioration of food safety was a negative development that involved the need for moral reconstruction in the society. as shown above, the chinese research community has attached increasing importance to the issue of human security. meanwhile, they are not just following in others' footsteps, but rather have developed their own analyses and views. some researchers have stressed that individuals are both the starting point and the ultimate purpose of any society. the emancipation of human beings should be the fundamental goal of any social emancipation. it is people who are the final objective of the concept of human security, and this is the core value any security is supposed to protect, while the state provides the means or temporary purpose (j. li 2013, 74-75) . the establishment of "people are the purpose" as a value grew to have great significance for chinese society after the community had drawn lessons from the history of the people's republic since 1949. during the cultural revolution period, many innocent people were attacked, detained, or persecuted illegally and immorally; rights were ignored, and people were harmed. after a disastrous decade, many people reflected on their painful experiences and thought about the phenomenon of imposing horrible acts on innocent individuals, something that should never recur. in the early reform period, there emerged a movement in china's intellectual community that began to discuss the issues of humanism (rendao zhuyi) and alienation (yihua). during this time, many intellectuals affirmed the significance of humanism and argued for the promotion of human values. some of them cautiously adopted the term "marxist humanism" or "socialist humanism" to distinguish from the so-called capitalist doctrine. not long after, the debate abruptly came to a halt due to a political intervention. however, along with further economic reform and considerable social development, the awareness of the value of the individual human being in china was reawakened and the value of people confirmed. the initiative for humanism in the 1980s was in fact later accepted by the whole of chinese society. this was also reflected in the discussions and research on human security, since it was made clear that guaranteeing human security is the value base for maintaining non-traditional security. the protection of people and human development should be set as the ultimate goals of any security course. obviously, this progress is precious for chinese society and deserves appreciation by the outside world. the paradigm of human security opposes the harming of the human freedoms and rights to promote economic growth or social stability. nor does it favor the pursuit of economic benefits and communitarian policies at the expense of sacrificing the security and dignity of the individuals or the nation. the fundamental reason for this lies in enhancing the value of people, which is the key and ultimate goal, and there is no higher goal than this. the previous practices of both "collective security" and "common security," yu xiaofeng, a leading scholar in human security, argues, could not have avoided the limitations of regarding the state as the chief actor (yu 2014, 33) . by contrast, "shared security," which he has proposed, regards the human community as occupying the central position of security, the protection of human life as the value base of security, social safety and prosperity as the priority goals of security, and harmony and cooperation as the supreme principles for security interactions between the states (yu 2014, 33-34) . this moves the discussion beyond the traditional security discourses and has the potential for further theoretical development. according to the human security now report (chs 2003) and the october 2012 resolution adopted by the un general assembly as a follow-up to paragraph 143 on human security of the 2005 world summit outcome, as well as the definition and design of this book, human security has two dimensions: protection and empowerment. in china, the dimension of protection has drawn much attention, epitomized in the doctrines of "putting people first" (yiren weiben) and "diplomacy serving the people" (waijiao weimin), which i discussed in a detailed way in an earlier study (ren and li 2013) . the findings from this earlier study have been reinforced by recent evidence. for example, a chinese newspaper correspondent wrote of his impressions when reporting from the national people's congress (npc) sessions. he was deeply impressed by the attention the npc delegates paid to the concrete issues concerning people's daily lives, including social insurance, income distribution, rights of peasant workers, food safety, unreasonably high drug prices, and protection of stakeholder interests. in his view, those kinds of issues had never before been so meticulously discussed at the npc (yuan 2005) . the changes the correspondent detected indicate ongoing healthy trends in human security terms. a harmonious society is one that puts people first, which means people's everyday life is a priority for the society. a government that puts people first is a government that represents people's interests. economic and social development should put human development at the center, and human development is the ultimate value judgment for social progress (yuan 2005) . meanwhile, the achievement of human security is a cooperative venture between the individual, society, and the state (bedeski 2013, 29) . government as a "necessary evil" is very much a western invention and is not a chinese idea. traditionally, people in china often have wishes for their government, and they want it to do good things for them. compared to western political culture, they have less vigilance but higher expectations of the government. one revealing example is the use of the term "parent officials" (fumu guan) to refer to government officials, with people expecting them to play a paternalistic role. without cooperative ventures, human security cannot be achieved. on the protection side, while people-oriented ideals might be considered noble ones, challenges come from the process of implementation. not surprisingly, many problems are persistent. one prominent example is the matter of government land appropriation and relocation of residents. sometimes the failure to protect the rights and interests of the ordinary people has led to acute standoffs and conflicts, and in some cases even extreme acts of protest. when property development businessmen and arbitrary power coalesce, together they can undermine the interests of the ordinary people who are affected. protecting people's legitimate interests has become an outstanding issue in china's human security amid the drastic social changes and urbanization of recent decades. moreover, the chinese leadership came into office with a promise to narrow the widening gap between rich and poor, and to shift to a more environmentally and economically sustainable growth model. since gross domestic product (gdp) became the brand of accomplishment for many government officials, "gdp worship" has prevailed. local officials tended to initiate projects that show their "achievements" and promote their image to the public. when poorly planned, such projects led to unexpected outcomes and environmental damage. when this became obvious, a strategic shift had to be undertaken. "no gdp growth at the expense of the environment" is becoming a new norm and "ecological civilization" the new banner, which was again stressed in the 19th party congress report of october 2017. the reform era has been characterized by remarkable success in terms of rapid economic growth and the improvement of living standards. after three decades of successful economic development, the country is standing at a new starting point. if the ruling party's historical promise to "let some people get rich first and eventually arrive at common prosperity" was a prerequisite for the reforms to unfold, and if four decades later this historical task has been basically fulfilled, what the reform enterprise must accomplish now is the other half: namely, to realize a common prosperity. this is a goal that will justify and inform further reforms. the reaffirmation of realizing common prosperity in line with the principles of governing for the people and building a comprehensive and balanced well-off society will guide future reform with a clear direction. 6 to fulfill its pledge to narrow the gap between rich and poor and to "unwaveringly pursue common prosperity," the leaders have to place the people in their hearts and take their needs seriously in order to realize the goal of the "chinese dream." after the 18th party congress in 2012, the new general secretary xi jinping expressed his view that "to fulfill the people's desire for better lives is what we shall strive for." 7 this statement sounded dear and close to the ordinary chinese people. on the empowerment side, there have also been several measures. only when a person has the capability for survival and development can he or she enjoy real freedom. the capacity for development is what empowers an individual. thus, a modernized china should not only accept empowerment through protection but also appreciate its importance in realizing human freedom and guaranteeing human security. education as a right is the fundamental and most significant form of empowerment, as the human experience has repeatedly revealed. thus, educational development is a significant way to empower people, especially compulsory education. china has reason to be proud in this regard. it has a tradition that puts significant emphasis on education, in which parents always try their best to pave the way for their children to receive a good education, and often they are willing to make sacrifices. in china, nine-year compulsory education has been in force for years. in 1989, project hope was set up as an educational support supplement, which later became well known throughout chinese society. it was initiated by the central youth league and the china juvenile development foundation as a philanthropic enterprise to help less developed areas to establish primary schools, to financially support children who have dropped out of school in the poorer regions to return to school, and to improve education in rural areas. the project received society-wide attention and support. implemented successfully, the project has positively changed the fate of hundreds of thousands of children of poor families and has also beefed up the whole society's awareness of the importance of education, thereby improving china's fundamental education. as a major initiative for the future, the third plenum set the aim of modernizing china's governance system and governing capability, a longterm goal for china. an important part of it is to reshape a new social governance system. during the 2014 national people's congress session, president xi joined the shanghai delegation for a deliberation of the government's work. for him, the key to future social governance is institutional innovation, and its core lies in people. only when people are living harmoniously can society be operating in a stable and orderly manner. for han zheng, the party secretary of shanghai, social governance was up to everybody, and the governing process should serve the all-round development of people. rule of law is the foundation of social governance, without which there can be no base for long-standing good governance and robust stability. 8 for that matter, among other things, it is necessary for grass-root cadres to regard people's concerns as their own matters, while always trying to understand their feelings and demands. again, the key challenge is to make this happen in real life. fortunately, in china there have been local autonomous grassroots organizations that play an intermediate role and serve to help in addressing problems at the grassroots level. they assist ordinary people who encounter difficulty, and they also, for example, mobilize donations to help people in the regions that have been struck by earthquakes or other natural disasters. the work of these local self-help organizations has proved to be useful and reassuring in terms of social governance. since 'human security' is central to non-traditional security, it is concerned with all kinds of factors that directly threaten human security" (zhou 2012, 255-256) . in today's china, prominent direct threats include air pollution, food safety, and cyber security. this section examines each of these in turn. the seriousness of the problem of air pollution first became apparent right all across china in 2013. heavy smog emerged not only in north and northeast china but also was reported in other regions. many people were alarmed by the situation, including concerns expressed by china's neighbors, japan and south korea. the spread of the smog made it obvious that the environment in which people were living was deteriorating, a red-light signal providing a warning on the existing pattern of economic growth, which was consuming a great amount of energy and yielding considerable waste. while many people were already aware of the issue, the shocking reality of the situation in 2013 and its related risks served to awaken people as never before. with many people walking in the streets wearing masks, the sense of insecurity was palpable and imminent. in this context, the specific threat of air pollution became the number one threat to people's security. to cope with this threat, in june 2013, china's state council laid out ten measures to prevent and combat air pollution. as a follow-up, in september, china released an action plan to implement this, which was a blueprint to fight against air pollution by 2017. since then, further work has been undertaken. in his government work report delivered in march 2014, premier li keqiang swore to "fight pollution like fighting poverty." when he was looking around the city of beijing, president xi emphasized that the sprawling pattern of urban development had to be contained and steps should be taken to deal with smog pollution and improve air quality. among the five requests xi made, one was logically to reinforce the measures to rein in air pollution. the top priority to combat air pollution and improve air quality was to control pm2.5, with major steps that included reducing the burning of coal, strictly limiting growth in the number of cars, adjusting industries, tightening management, and executing joint prevention and control. 9 xi's move clearly sent a signal that the government was committed to taking measures for better air quality. the chinese leadership had previously pledged to launch a revolution in energy production and consumption and said that urbanization must be balanced with ecological security (hook 2012, 1) . however, pollution was worsening and was posing a serious threat to human health and social stability. to reduce air pollution and carbon emissions, beijing (population 20 million) is attempting to phase out coal-fired power plants within the city's urban core, replacing them with cleaner-burning natural gas power plants. these measures are also basically valid for other cities. after all, fighting pollution is relevant to everybody's security, a point that has attracted a high-degree of consensus within chinese society. similarly, china's energy sector had a watershed year in 2013. reforms that could have a profound impact on china's environment and energy policy were floated. with concerns over air pollution mounting throughout the year, the country is poised to shift away from its reliance on coal and toward use of cleaner forms of energy, including natural gas. shale gas exploration is making progress in the country. driven by crises, a true transformation is under way but surely will take time to complete. the second human security concern among the chinese people is that of food safety. in recent years, a series of food contamination incidents occurred throughout the country, causing serious concern. a 2010 poll by xiaokang (小康 [moderately well off]) magazine and tsinghua university conducted in 12 chinese cities found that food safety ranked number one of all social concerns among those surveyed, reflecting mounting anxiety after the 2008 melamine crisis (z. zhang 2010; wishnick 2013, 256) . within this context of growing concern over food safety, the government has not been unaware or insensitive. appearing at a press conference, premier li keqiang responded by stressing that "food safety is of utmost importance." 10 answering a different question concerning the missing mh370 aircraft, he stressed that "any case involving human life has to be treated with the utmost care" (renming guantian). as with the issue of air pollution, the 2014 government work report promised to adopt the strictest surveillance, the most severe punishment, and the most serious accountability measures to resolutely govern pollution at the meal table and reassure people of "security on the tongue tip" (k. li 2014). in fact, there is a widely shared consensus in china on the need to make efforts to ensure food safety. specialists distinguish between two separate food-related issues: the first is related to food security or ensuring sufficiency in the quantity of food; the other concerns food safety, which refers to the quality of food. while the former involves the question of whether there is sufficient overall provision of food, an area in which china has vowed to "hold the bowl firmly in our own hands," the latter involves the question of whether people can be assured that the food they eat every day is safe. formerly, the issue of food scarcity trumped food safety. but today the main problem in china lies in food safety, with people alarmed by reports of different kinds of contamination. in recent years, this has even spilled over into china's foreign relations, and was epitomized in the spoiled jiaozi (dumpling) incident between china and japan. the incident originated in hebei province. an employee in the tianyang food factory was resentful over his low income. in revenge, he deliberately poisoned jiaozi, and the contaminated products were exported and sold in japan. some customers bought and ate them, becoming ill. on investigation, pesticides found in the frozen dumplings were traced back to china. this was a single case of a crime that resulted in grave consequences. the reputation of china's food products was seriously damaged, and the sino-japanese relationship was also somewhat affected. this incident once again highlighted the close links between domestic and international affairs. in the final analysis, the root cause lies with individuals, and the issue of "moral collapse" has been used to refer to this situation. to what extent this is true can be debated. there was a belief that overuse of fertilizers and pesticides was rampant, and that growers distinguish between what they themselves eat and what is sold in the market, even if they know the latter is not suitable to be eaten. again, how widespread the phenomenon is can be discussed, yet reports on these kinds of practices result in people feeling that the food they eat may be unsafe. as a result, in china there was an obvious lack of public trust in food safety. tighter regulations must be implemented. greater transparency helps to build public trust, including more official data and statistics about the improvement (or deterioration) in food safety, increased freedom for media and civil society to verify the official data, more effective actions from the government in handling the corruption and malfeasance involved in food safety issues, stronger protection for whistle blowers who uncover production of unsafe food and, more importantly, removal of the corrupt officials involved. further measures must be taken to improve china's food safety regulations, consumer education levels, and supply chain traceability and sustainability. the third and final issue is that of cyber security. the use of the internet has increasingly become a part of everyday life in china. by 2013, there were over 600 million internet users in the country, and mobile internet users reached 461 million, making the community of chinese "netizens" (wang min) the world's largest. with this growth, cyber security has become a prominent issue. in fact, china is one of the major victims of cyber-attacks. the covert activities revealed by edward snowden highlighted the vulnerability of nations and individuals who are monitored illegally and immorally (meng 2014) . at the government level, a central small leading group on cyber security was created, which held its first meeting in february 2014. it was emphasized that cyber security involved national security and development, as well as the careers and everyday lives of a vast number of ordinary people. it was therefore a major strategic issue. 11 the breakdown and vulnerability of the internet can have a widespread impact on people and their lives. this chapter has addressed the three research questions the research project raised by elaborating on how the idea of human security is understood or defined by the government and social actors, the ways in which the distinctions between the "protection" and "empowerment" aspects of human security are understood and accepted, and what downside risks are perceived as pressing human security issues in china. the major ones discussed here included air pollution, food security, and cyber security. as has been indicated in this chapter, although human security as a term is not frequently used, there have been various human security practices in china. the idea of human security has been firmly established and threats to human security have been detected. such problems result from china's still unfinished process of industrialization, urbanization, and drastic social change. the good news is that progress is being made, and theoretically, globally accepted values often exist at the individual level. with this comes occupancy of the moral high ground. china has gone far beyond the lip service level of cheaply talking about people's interests. this deeper approach can be fully integrated into human security in its holistic sense. threats to human security can be domestic risks, yet they often are transnational ones such as air pollution and sand storms. they require different sectors of the society and neighboring countries to work together. over 20 years ago, the commission on global governance drafted a report that emphasized the distinction between the security of states and the security of peoples (cgg 1995) . twenty plus years later, the security of peoples has gained momentum. this is also true in china, the most populous country in the world. china's practices have considerably reinforced the overall trends of affirming the value of people, protecting their lives, ensuring their legitimate interests and dignity, and empowering them not only to survive but also to live a respectful life. what is happening in china is indicating a healthy trend in this direction. each year the communist party of china central committee holds an annual plenum to deliberate what it believes to be the most important issues for the full text of the resolution and xi's explanation, see renmin ribao ershiyi shiji jingji baodao xi's remarks at the closing of the 18th party congress li's remarks at the 2013 national people's congress press conference anthropocentric theory of human security china and global governance our global neighborhood: the report of the commission on global governance chs (commission on human security). 2003. human security now. new york: commission on human security from commitment to concerted action. remarks by vice foreign minister cui tiankai of china at the ministerial follow-up meeting ren de fazhan yu ren de zunyan: zaisi ren de anquan gainian battle to balance urbanization with ecological sustainability ruhe kandai sheli guojia anquan weiyuanhui iciss (international commission on intervention and state sovereignty). 2001. the responsibility to protect. ottawa: the international development research centre china's participation in international organizations china and the united nations ren de anquan: fei chuantong anquan nengli jianshe de xinshijiao zhongguo fei chuantong anquan nengli jianshe zhengfu gongzuo baogao wangluo anquan: guojiazhanlue yu guoji zhili [cyber security: national strategy and international governance). dangdai shijie china and the global environment: learning from the past, anticipating the future. sydney: lowy institute for international policy a return to people: china's approach to human security ren de anquan' yu guojia anquan resolution 66/290. follow-up to paragraph 143 on human security of the 2005 world summit outcome food security and food safety in china's foreign policy human security challenges with china the governance of china ii fei chuantong anquan zhili yu 'hemei zhongguo' [nontraditional security governance and a 'safe china beijing: shehui kexue wenxian chubanshe ren' de daxie food security tops people's concern in survey. china daily preface zhongguo shipin anquan wenti yu qushi report on china's non-traditional security studies key: cord-286749-si83t03j authors: lu, q.-b.; wo, y.; wang, h.-y.; huang, d.-d.; zhao, j.; zhang, x.-a.; zhang, y.-y.; liu, e.-m.; liu, w.; cao, w.-c. title: epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection date: 2014-07-29 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-014-2215-7 sha: doc_id: 286749 cord_uid: si83t03j human bocavirus (hbov) is a novel parvovirus, often associated with respiratory tract diseases in children. this study explored the epidemiological characteristics and molecular evolution of hbov-1 in southeastern china. nasopharyngeal aspirates were collected from children admitted to hospital with acute respiratory tract infections. hbov-1 was detected using real-time reverse transcription polymerase chain reaction and further characterized by complete genome sequences analysis. among the 3,022 recruited children, 386 (12.77 %) were hbov-1-positive and 300 (77.72 %) had co-detection with other respiratory viruses. seasonal prevalence peaked in summer. hbov-1 presence was significantly associated with asthma attack [odds ratio = 1.74; 95 % confidence interval: 1.30, 2.31; p < 0.001]. similar results were obtained when either single detection or co-detection of hbov-1 was considered, demonstrating the minor impact of co-detection on the clinical characteristics or epidemic pattern. phylogenetic analysis based on the complete genome sequences showed that all the hbov-1 sequences clustered together and no branch was formed that was supported by bootstrap value ≥750. the overall evolutionary rate of the complete genome of hbov-1 was estimated at 1.08 × 10(−4) nucleotide substitutions per site per year (s/s/y) [95 % highest probability density: (0.40–1.86) × 10(−4) s/s/y]. selective pressure analysis showed that all the ω-values were less than 1, suggesting that hbov-1 was under negative selective pressure. site-by-site analysis identified the codon site 40 of the vp1 gene under positive selection. in conclusion, our study disclosed the epidemiological and genetic dynamics of hbov-1 epidemics in southeastern china in the most recent 3 years, the information of which might help to further improve our understanding of hbov-1 infection and guide better surveillance and control strategies in the future. electronic supplementary material: the online version of this article (doi:10.1007/s10096-014-2215-7) contains supplementary material, which is available to authorized users. acute respiratory tract infections (arti) are a major cause of significant morbidity worldwide, especially in infants and children. viruses are leading pathogens of arti. although some viruses, such as influenza virus, respiratory syncytial virus (rsv), and human adenovirus (hadv), are responsible for most arti, etiological causes remain unknown in a proportion of arti [1] . human bocavirus (hbov) is a novel parvovirus first described by allander et al. in 2005 [2] . it has been classified in the family parvoviridae, subfamily parvovirinae, and genus bocavirus by genetic organization and sequence homology. other parvoviruses infecting humans included the adenoassociated viruses, the well-known pathogen parvovirus b19, and the recently discovered human parvovirus 4 [3] . based on the phylogenetic analysis of viral genomes, four species of hbovs (hbov1-4) have been identified [4] , article note qing-bin lu and ying wo contributed equally to this work. electronic supplementary material the online version of this article (doi:10.1007/s10096-014-2215-7) contains supplementary material, which is available to authorized users. among which hbov-1 was mainly identified in respiratory specimens from children with acute respiratory disease [5] [6] [7] . according to previous studies, hbov-1 has a global distribution, with prevalences varying considerably from 1.5 % to 45.7 % in arti patients [5, 6, [8] [9] [10] [11] [12] [13] [14] . however, its role as a causative agent of respiratory tract diseases is frequently questioned due to its concurrent detection with other potential pathogens. possible interactions between hbov-1 and other viruses remained rarely investigated. in china, hbov-1 was detected in children with positive rates ranging from 4.6 % to 31.0 % [6, [14] [15] [16] [17] [18] . however, hbov-1 was co-detected frequently with other viruses in patients with respiratory or enteric infections; therefore, the impact of hbov-1 on arti occurrence remains unclear. the seasonal patterns of hbov-1 infection in china, especially the surveillance data from the most recent 4 years, are scarcely reported, probably owing to the lack of a systematic and prolonged surveillance. this study was aimed to explore the epidemiology pattern and clinical characteristics of hbov-1 infection in chinese children, as well as the molecular evolutionary pattern, for hbov-1, by performing a 4-year laboratory surveillance of arti cases. the laboratory surveillance was performed to detect the viral infection in children with arti between june 2009 and may 2013 at the children's hospital of chongqing medical university, china. the arti was determined based on syndromes of cough, rhinorrhea and/or dyspnea, and/or fever of >37.5°c. fever caused by known chronic medical conditions was excluded. the pneumonia was defined by the presence of patchy alveolar opacities on chest radiographs, in addition to presenting symptoms of cough, dyspnea (lower chest wall indrawing), or tachypnea (in infants, >50-60 breaths/min; in older children, >40 breaths/min). severe pneumonia was defined as pneumonia plus hypoxemia (maintained sao 2 <92 % in air) or rising respiratory and pulse rates with clinical evidence of respiratory distress and exhaustion with or without raised paco 2 . the definition of an attack was used as an episode of respiratory symptoms which prompts an urgent consultation with a doctor, is of sufficient severity to prevent the patient working/attending school/performing domestic duties/playing, and results in increased use of anti-asthma medication. the recruited patients had nasopharyngeal aspirates (npa) collected the day they were hospitalized. a standardized form was applied to extract the patients' information regarding demographic characteristics, underlying medical conditions, selected laboratory tests, radiographic findings, clinical manifestation, treatment course, and outcome. written informed consent was obtained from their parents. viral rna and dna were extracted from 200μl of the npa by using qiaamp® minelute virus spin kits (qiagen, hilden, germany). the cdna was synthesized by using the superscript®iii first-strand synthesis system for reverse transcription polymerase chain reaction (rt-pcr) (invitrogen, carlsbad, ca, usa). all samples were routinely screened for hbov-1 with the real-time pcr method [19] . partial hbov-1-positive samples were whole-genome sequenced following the method previously described [20] . genomic sequences were assembled using lasergene's dna seqman software (version 7.1.0, dna star inc., madison, wi, usa). previously published genbank sequences of hbov-1 were used for comparison. all alignments and phylogenetic tree construction were performed by the neighborjoining (nj) method with 1,000 bootstrap replicates using clc genomic workbench 5.5. similarities between strains were calculated by using bioedit version 7.13 (north carolina state university, raleigh, nc, usa; http://www.mbio.ncsu. edu/bioedit/bioedit.html). the substitution rates, the divergence over time, and the time to the most recent common ancestor (tmrca) of the current hbovs were estimated using a bayesian markov chain monte carlo (mcmc) approach, as implemented in the bayesian evolutionary analysis sampling trees (beast) package version 1. 8.0. the value of ω and the individual site-specific selection pressure were measured by using the single likelihood ancestor counting (slac), fixed effects likelihood (fel), internal fixed effects likelihood (ifel), and fast unconstrained bayesian approximation (fubar) methods implemented in the hypothesis testing using phylogenies (hyphy) package performed at datamonkey online (http://www.datamonkey.org/). the sites were confirmed when they were selected by more than two methods. a single breakpoint recombination (sbp) test with both akaike information criterion (aic) and bayesian information criterion (bic) calculation was conducted. the overall ω-value and 95 % confidence interval (ci) were estimated based on nj trees. selective pressure was defined as neutral evolution with ω=1, purifying or negative selection with ω<1, and positive selection with ω>1. a p-value of less than 0.10 was considered as the threshold for strong evidence of selection in slac, fel, and ifel, and posterior probability ≥0.9 in fubar, respectively. statistical analysis was done by the chi-square test for categorical variables. logistic regression models were used to explore the factors associated with higher hbov-1 detection and to determine the association between clinical characteristics and hbov-1 infection, with clinical disease as the dependent variable and hbov-1 infection as the independent variable. odds ratios (ors) and their 95 % cis were estimated using maximum likelihood methods. statistical significance was set at a p-value of less than 0.05. all the tests were performed by using sas 9.13 (sas institute inc., cary, nc, usa). from june 2009 to may 2013, 3,022 npa samples of hospitalized infants and children were collected. the median age was 8 months (range 1 months to 16 years), and 2,016 (66.71 %) were boys. the median delay before hospital entrance was 9 days (range 1-60 days). the hospitalization duration [mean ± standard deviation (sd)] was 7±4 days. among the 3,022 children, 450 (14.90 %) displayed severe pneumonia and 25 (0.83 %) had severe asthma. in total, 386 (12.77 %) children were positive for hbov-1. the children aged between 7 and 24 months had higher detection rates of hbov-1 than children of the other age groups ( and severe asthma were identified in 53 (13.73 %) and 4 (1.04 %) hbov-1-detected patients, among whom 12 and four had single detection, respectively, which is comparable with the patients with co-detection. the monthly positive rates of hbov-1 displayed specific seasonal distribution, as plotted in fig. 1 . one seasonal peak was observed in summer, demonstrated by remarkably higher infection rates during may-august. an exception was noticed in 2010, when another low peak was also observed in the winter season from november to december. when the hbov-1 single infection was plotted, a similar seasonal pattern was derived, with a clearly increased activity of hbov demonstrated in summer. the phylogenetic tree based on 5,157 bp of the whole genome sequences showed that all the hbov-1 sequences, regardless of whether they were single detection or co-detection, clustered together and no other branch was formed that was supported by bootstrap value≥750 (fig. 2) . the identities for the hbov-1 complete genome were 99.4-100 %. the phylogenetic trees were similarly established for the np1, figure 1) . the identities of hbov-1 were 97.8-100 % for the np1 gene, 99.6-100 % for ns1, 98.9-100 % for vp1, and 98.8-100 % for vp2. all the hbov-1 viruses in the investigated localities were dominated by a single viral lineage, which had been dominant in most parts of china in recent years. neither a spatial nor a temporal specific branch was formed based on the whole-genome analysis. the overall evolutionary rate was estimated at 1.08×10 −4 nucleotide substitutions per site per year (s/s/y) [95 % highest probability density (hpd): (0.40-1.86)×10 −4 s/s/y] based on the complete genome analysis. the highest evolutionary rate was derived from the np gene, while the lowest was from the ns gene sequences ( table 2) . the sbp test showed no evidence of recombination for any of the four genes. selective pressure analysis showed that all the ω-values were less than 1 ( table 3 ), indicating that these hbov-1 genes were under negative selective pressure and the evolutionary process is shaped mainly by purifying selection. the np gene had the largest global rate (0.23, 95 % ci: 0.15-0.33) among the four genes (range 0.08-0.23), suggesting that it is subject to greater selective pressure than the other three genes. site-by-site analysis of the full data set consisting of 206 hbov-1 coding sequences identified only one site on the vp1 gene (codon site 40) undergoing significant positive selection and a number of negatively selected sites. a codon-based maximum likelihood reconstruction of the evolutionary history of codon site 40 under positive selection was carried out (supplemental figure 2) . the result disclosed that the substitution has occurred mostly in the strains from asia, especially in china, suggesting that hbov-1 strains from china undertake more selective pressure than those from other regions. in the current study, we have identified the clinical characteristics and temporal patterns of hbov-1 in southeastern china through a 4-year surveillance. no role of hbov-1 in severe pneumonia occurrence was observed, regardless of whether they were single detection or co-detection. in contrast, a significant association between hbov and asthma was determined, suggesting a possible causal relationship. based on the genetic analysis, hbov-1 in southeastern china have similar mean evolutionary rates as other species of the family parvoviridae and one positive selection site was also disclosed which differs from hbov of other origins. according to our results, the prevalence of hbov-1 in pediatric arti patients is higher than those from other hospital-based studies [2, 6, 21, 22] , whereas it was lower than that previously detected in persistently wheezing children [16] . hbov-1 was commonly co-detected with a median rate of 42.5 %, and the most frequent co-detected viruses included rhinovirus, enteroviruses, and influenza [23] . we demonstrated a co-detection rate of 77.72 %, which is similar to the high proportion of co-detection as previously studied [24] , yet significantly higher than those from other studies [9, 12] . these discrepancies could be explained by the inherent differences in the population subsets, detection methods, as well as the epidemiological variability relating to geographic origin and climatic factors. seasonal circulations of hbov-1 vary, with most previous studies reporting hbov-1 peak attained during the winter season [9, 22, 23] . choi et al. found a higher frequency of hbov-1 between may and july over a 5-year period among children with arti in korea [25] , which, interestingly, is consistent with our results displaying a summer peak. although this temporal pattern coincided with the epidemic peaks of influenza circulation in southern china (http:// www.cnic.org.cn/chn/), the single detection of hbov also demonstrated a similar temporal pattern after excluding the the relative importance of hbov-1 as a causative pathogen for viral respiratory illnesses has not been determined, but it has been associated with respiratory diseases ranging from upper respiratory tract disease to bronchiolitis, and lower respiratory tract diseases, including pneumonia [11, 15, 26] . vallet et al. found a high concordance between hbov-1 detection and exacerbation of asthma requiring eventual hospitalization [27] . in our study, hbov-1 detection was not associated with pneumonia. instead, hbov-1 tended to cause more upper respiratory tract infections, and its infection was also related with asthma exacerbation. although a causal relationship between hbov-1 infection and asthma cannot be concluded based on the statistical results, their significant association suggest the need for further investigation. prospective longitudinal studies will hopefully increase our understanding of what, if any, role hbov plays in asthma. the epidemiology of the hbov-1 circulation in china has been previously studied; however, the viral gene sequence data acquired from these studies were rare and insufficient to reveal any temporal or spatial characteristics or any evolutionary dynamics. analysis of the genomic and epidemiological data of hbov-1 from the current study, as well as those available from different localities, revealed limited heterogeneity, and high conservation was demonstrated from both spatial and temporal perspectives. zehender et al. reported that the estimated mean evolutionary rate of the hbov-1 vp2 gene was 8.6×10 −4 s/s/y using 48 non-recombinant isolates [28] , demonstrating that hbov-1 was characterized by a rapid evolution. the rate identified in the current study is slower, which might be partially caused by the sample size of analyzed hbov-1 sequences. a few of the previous studies explored the estimated evolutionary rates of parvoviruses in carnivore parvoviruses (7.9×10 −5 -1.7×10 −4 s/s/y) [29] and human parvovirus b19 (1.1×10 −4 -2.6×10 −4 s/s/y) [30] . the estimated mean evolutionary rates of the complete genome, vp1, vp2, and ns genes of hbov-1 were similar to those of carnivore parvoviruses and human parvovirus b19. the ns gene was highly conservative among the four genes. surprisingly, the np gene showed a slightly higher substitution rate than the other genes. the icosahedral capsid consists of two structural proteins, vp1 and vp2, which are identical except for a certain number of amino acids at the amino-terminal end of the vp1 protein, the so-called vp1 unique region (vp1u). a conserved phospholipase a2 (pla2) motif, which resembles the catalytic motif of secreted pla2 (spla2), has been identified in the vp1u of most parvoviruses [31] . for hbov-1, vp1 and vp2 have the same sequence of 542 aa at the c termini, while vp1 has an additional 129 aa peptide at the n-terminus. one study indicated that the vp1u of hbov-1 also had spla2-like enzymatic activity, and these residues are crucial for its spla2-like activity [32] . mutation of one of the amino acids (21pro, 41his, 42asp, or 63asp) in the vp1u almost eliminated the spla2 activity of the hbov-1 vp1u. interestingly enough, one positive selection site (codon 40) was identified near codon 41 and 42 in the current study, which indicated a high probability of vp1u undergoing selective pressure. in spite of this finding, the associations between the positive selection site and spla2 activity or amplification of dna need further investigation. in conclusion, our study, for the first time, disclosed the epidemiological characteristics and genetic dynamics of hbov-1 in southeastern china in recent years. the disclosure of the temporal pattern of hbov infection provided the season when hbov infection could be highly suspected and surveillance should be strengthened. the current data also demonstrated that hbov was co-detected with a high prevalence; therefore, the sole presence of hbov in patients should be interpreted carefully as to its causal role in causing disease. this information might help to further improve our understanding of hbov-1 infection and guide better surveillance and control strategies in the future, especially among children. viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus human bocavirus infections human metapneumovirus and human bocavirus associated with respiratory infection in apulian population enterovirus 68 among children with severe acute respiratory infection, the philippines human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection human bocavirus and acute wheezing in children detection of human bocavirus in canadian children in a 1-year study human 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bocavirus 1-3 in children for acute respiratory infection in lanzhou area the human bocaviruses: a review and discussion of their role in infection human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand the association of newly identified respiratory viruses with lower respiratory tract infections in korean children seroepidemiology of human bocavirus defined using recombinant virus-like particles human bocavirus: a cause of severe asthma exacerbation in children rapid molecular evolution of human bocavirus revealed by bayesian coalescent inference high rate of viral evolution associated with the emergence of carnivore parvovirus phylogenetic evidence for the rapid evolution of human b19 erythrovirus phospholipase a2 activity-dependent stimulation of ca2+ entry by human parvovirus b19 capsid protein vp1 phospholipase a2-like activity of human bocavirus vp1 unique region acknowledgments this study was supported by the china mega-project for infectious diseases grant (2013zx10004-202) and the natural science foundation of china (81222037). the authors do not have any commercial or other association that might pose a conflict of interest. all authors read and approved the final manuscript. key: cord-253223-us0ey8dq authors: chow, brian d.w.; esper, frank p. title: the human bocaviruses: a review and discussion of their role in infection date: 2009-11-03 journal: clin lab med doi: 10.1016/j.cll.2009.07.010 sha: doc_id: 253223 cord_uid: us0ey8dq respiratory tract infections are a leading cause of morbidity and mortality worldwide. the human bocavirus (hbov) is a newly recognized human parvovirus first reported in 2005. since its discovery, this virus has been associated with upper and lower respiratory tract disease and gastroenteritis worldwide. this article is a comprehensive review of what is known about hbov. it includes an evaluation of diagnostic modalities, symptoms occurring in affected patients, and a discussion as to whether hbov is responsible for identified clinical manifestations. the article reviews the incidence and effect of coinfection and updates on related members (hbov-2 and hbov-3) recently reported. understanding of respiratory viruses such as hbov remains vitally important to the health of adult and pediatric patients. associated with upper and lower respiratory tract disease and gastrointestinal illness in adult and pediatric patients throughout the world. recently, two viruses closely related to the human bocavirus have been reported, provisionally named human bocavirus 2 (hbov-2) and human bocavirus 3 (hbov-3). 12, 13 for the sake of clarity, the remainder of this article refers to the original human bocavirus strain described by allander and colleagues as human bocavirus 1 (hbov-1). 11 it remains unclear whether hbov-1, hbov-2, and hbov-3 represent unique viral entities or distant genotypes of a single virus. because hbov-2 and hbov-3 have only been recognized over the last several months, the epidemiology and clinical manifestations associated with these viruses are only now being realized. the incidence of human bocaviruses varies widely, 14, 15 with various clinical presentations, and they are often found in association with other potential pathogens. this phenomenon has led to debate over the role of human bocaviruses as true pathogens. this article reviews the published studies and discusses the virologic, clinical, and diagnostic aspects of these newly recognized human viruses. hbov-1 was first described by allander and colleagues 11 in 2005. pooled, cell-free supernatant from respiratory samples was examined using a random polymerase chain reaction (pcr) technique. amplified pcr products were separated, ligated to a vector, and introduced into escherichia coli. clones were sequenced and evaluated through an automated protocol and compared against known sequences. the majority of the clones were determined to be human or recognized bacterial and viral pathogens. of the remaining sequences, a parvoviruslike sequence was identified with no known correlation to recognized human parvoviruses. phylogenetic analysis showed this to be a novel parvovirus most closely related to bovine parvovirus and canine minute virus (fig. 1) . it is now named the human bocavirus. in january 2009, kapoor and colleagues 12 reported the identification of a related parvovirus, termed hbov-2, from pediatric stool samples by random pcr analysis of nuclease-resistant virus particles. the resulting amplicons were subcloned into a plasmid vector, sequenced, and compared with genome entries on genbank. phylogenetic analysis of hbov-2 shows that it is most closely related to hbov-1. the amino acid identity of hbov-2 to hbov-1 is 78%, 67%, and 80% for the ns1, np1, and vp1/ vp2 proteins, respectively. there is significant divergence of the genomic targets used for detection of hbov-1, which explains why hbov-2 has remained unidentified to this point. a third bocavirus species was described in april 2009 from australia, and has been provisionally named human bocavirus 3 (hbov-3). similar to the methods used in discovery of hbov-1 and hbov-2, arthur and colleagues 13 digested filtered fecal samples with nucleases, then pelleted virions and evaluated using degenerate oligonucleotide primed-pcr. during these investigations, the group simultaneously discovered hbov-2 and a third species of bocavirus, hbov-3. analysis of the genome shows close homology to hbov-1 in the nonstructural protein encoding regions (ns1, np1), but hbov-3 is more similar to hbov-2 in the structural protein encoding regions (vp1/vp2). this finding suggests that hbov3 may be the product of recombination between hbov1 and hbov2. arthur and colleagues identified a recombination site that supports this hypothesis. as with hbov-2, the hbov-3 genome is divergent enough from hbov-1 to explain lack of detection using currently published primer sets ( table 1) . clinical presentation, seasonal distribution, and prevalence of hbov3 have yet to be characterized in full. further analysis of the phylogenetic relationships will be required to determine the correct taxonomy of this virus family. human bocaviruses are parvoviruses belonging to the family parvoviridae, subfamily parvovirinae, and are most closely related to bovine parvovirus and canine minute virus, in the genus betaparvovirus (see fig. 1 ). parvoviruses are single-stranded dna viruses with a small genome size between 4 kilobases (kb) and 6 kb that have three open reading frames that encode two nonstructural proteins, ns1 and np1, and the structural vp1 and vp2 proteins. the hbov genome does not encode a polymerase and depends on host cell dna polymerases for replication. parvoviruses have unenveloped capsids with icosahedral symmetry. 16 based on electron micrographs, hbov-1 appears structurally similar to other members of the parvoviridae family and is approximately 25 nm in diameter. 17 the vp2 gene is nested in the reading frame of vp1 and uses an alternate initiator codon. 11 the sequence of vp1 and vp2 differ only in the n-terminal extension of vp1. this region, referred to as the vp1 unique region, plays a critical role in virus infectivity in some parvoviruses. 18 in several parvoviruses, vp3 arises from posttranslational proteolytic cleavage of vp2 proteins. it remains unclear if vp3 exists in human bocavirus. the capsid of parvoviruses consists of about 60 copies of each viral capsid protein. the role of ns1 and np1 proteins in human bocavirus species remain unknown. zhi and colleagues 19 created an infectious clone of the related parvovirus b19 to determine the functions of these genes. their findings showed that ns and vp1 knockout mutants abolished the viral infectivity. the ns protein also seemed to be vital in genome transcription and to play a role in the regulation of capsid gene expression. parvoviruses have been shown to be transmitted by various routes, including respiratory, urine, and fecal-oral contact. 16 the mode of bocavirus transmission of hbov is unknown. most studies have reported the presence of hbov-1 dna in respiratory secretions and fecal samples. hbov-1 dna has also been detected in addition to serum, urine, and lymph nodes. [20] [21] [22] so far, hbov-2 and hbov-3 have only been identified in stool samples. similar to other respiratory viruses, transmission may occur through several routes, including inhalation, contact with infected secretions (through direct contact or possibly contaminated surfaces), and fecal-oral from the standpoint of gastrointestinal illness. although some parvoviruses cannot replicate without helper viruses, the mechanisms of cell entry and replication have not been described for human bocavirus. this lack of information is largely because of the inability to grow human bocaviruses in standard cell lines. following allander's initial description of hbov-1 in the human respiratory tract 11 , more than 60 studies have been published investigating the role of this virus in respiratory tract illness. clinical findings reported from hbov-1-positive patients from 9 representative studies are summarized in table 2 . most patients present with symptoms of upper respiratory tract illness, including cough, fever, and rhinorrhea. pharyngitis and rash are less common (11%-13%). additionally, patients tended to report earache. 14, 23 several studies raise the possibility that hbov-1 is associated with severe respiratory disease. reports describe this virus in children hospitalized for bronchiolitis, 24 asthma exacerbations, 15,25 and first-time episodes of wheezing. 26 wheezing is described in numerous studies. in one prospective study comparing 16 respiratory viruses in children admitted with respiratory infection, wheezing was seen in more than 50% of children in whom hbov-1 was recognized as the sole agent. surprisingly, children who had coinfections had less wheezing. 27 other common findings include tachypnea, fever, and hypoxia. one report from jordan found as many of 18% of children hospitalized with pneumonia were positive for hbov-1. 28 in thailand, fry and colleagues found that patients who had hbov-1 were more likely to be hospitalized with pneumonia (odds ratio of 3.56) compared with controls. most hbov-1 reports are retrospective analyses based from hospital settings in these studies, up to 6.6% of patients required stays in intensive care units and up to 40% required oxygen therapy at some point during their hospitalization. 20, 29 these studies likely overestimate the severity of disease associated with hbov-1 because of selection bias. in studies based on community samples, disease is mild and only rarely requires admission. to date, there have been no reported deaths associated with hbov-1. few studies list underlying comorbidities in hbov-1-infected patients. when reported, more than half of patients had an underlying medical condition. 20 the most common underlying conditions involve primary cardiac or pulmonary disease, including congenital heart lesions, heart failure, asthma, chronic obstructive pulmonary disease, and prematurity with chronic lung disease. 11, 20, 29 hbov-1 has been reported in immunocompromised patients who have respiratory and gastrointestinal illness. schenk and colleagues 22 reported a child coinfected with rhinovirus and hbov-1 following hematopoietic stem cell transplant. this patient developed fever, dyspnea, wheezing, and infiltrates on chest radiograph. in this patient, hbov-1 dna was isolated from nasopharyngeal, stool, and blood samples. koskenvuo and colleagues 30 reported a series of children who had acute lymphoblastic leukemia in whom hbov-1 was identified. presenting symptoms were febrile upper respiratory infection with otitis media, fever with vomiting and diarrhea, and isolated fever. garbino and colleagues 31 reported a case of hbov-1 in a hospitalized adult infected with hiv. all patients eventually recovered from their acute illness. although few studies have systematically examined immunocompromised patients for hbov-1, the incidence seems to be low. one study by muller and colleagues 32 examined bronchoalveolar lavage specimens from immunocompromised patients suspected of having pneumocystis jiroveci pneumonia. only 1 specimen of 128 (0.8%) was found to have hbov-1. miyakis and colleagues 33 prospectively examined bronchoalveolar lavage specimens from 53 adult lung transplant recipients and 67 symptomatic nontransplant patients and failed to find hbov-1 in any sample. these studies argue against hbov-1 as a pathogen of lower respiratory tract disease in these vulnerable populations. because many of these reports observe high coinfection rates, it is difficult to draw conclusions as to whether hbov-1 plays a role in pathogenicity of infected patients. to answer this several studies have compared the presence of hbov-1 in asymptomatic control groups to symptomatic individuals. [34] [35] [36] [37] all but one found hbov-1 more often in symptomatic children than in healthy controls. in the remaining study, asymptomatic carriage of hbov-1 was as frequent in asymptomatic infants as those who had acute respiratory tract disease. in a recent report by allander and colleagues 15 hbov-1 dna is also present in the serum of acutely ill patients suggesting that dissemination of this virus is possible. these findings provide support for hbov-1 as a cause for respiratory illness. when in vitro culture systems and animal models are available, a better understanding of the role of hbov-1 in human respiratory disease can be established. gastrointestinal symptoms are reported in up to 25% of patients who have hbov-1 respiratory infection (see table 2 ) suggesting hbov-1 may not be limited to the respiratory tract alone. the closely related canine and bovine parvoviruses are well described as both respiratory and enteric pathogens. 16 subsequent investigations have identified hbov-1 dna in stool samples from patients with gastrointestinal illness. the most common gastrointestinal symptoms include nausea, vomiting, and diarrhea. both watery and bloody diarrhea have been reported in conjunction with hbov-1. [38] [39] [40] [41] [42] [43] [44] [45] hbov-1 has been identified in 0.8% to 9.1% of stool samples from patients presenting with gastrointestinal illness. [38] [39] [40] [41] [42] [43] hbov-1 viral load in stool is significantly less than viral loads found in respiratory tract specimens of symptomatic patients. 44 similar to studies of respiratory illness, hbov-1 is commonly codetected with recognized enteric pathogens, with coinfection rates ranging from 21% to 77.6%. 41, 43 identified copathogens include norovirus, rotavirus, human calicivirus, astrovirus, adenovirus, campylobacter, salmonella, and clostridium difficile. [38] [39] [40] [42] [43] [44] in a recent casecontrol study on acute gastroenteritis, arthur and colleagues 13 examined stool specimens for potential pathogens, including all three species of human bocavirus. dna from human bocaviruses 1, 2, or 3 was isolated from 54 (27.4%) stool samples, making these human bocaviruses the second most common viral agents identified after rotavirus (37.1%). clinical illness associated with hbov-1 and rotavirus coinfection did not seem to be significantly different than that of rotavirus illness alone, suggesting the presence of hbov-1 did not worsen disease. 42 hbov-1 is infrequently reported in stools originating from immunocompromised patients. in a 4-year-old patient who had diarrhea, bocavirus was recognized on day 21 and again on day 75 following stem cell transplant. this patient went on to develop disseminated disease. 22 one patient who had a t cell deficiency was reported to have hepatitis associated with hbov-1. 46 hbov-1 was also associated with gastroenteritis in one patient who had b cell immunodeficiency. 39 unlike hbov-1-associated respiratory disease, large investigations for hbov-1-associated gastrointestinal illness have not been performed for the immunocompromised population. as with respiratory disease, the role of hbov-1 as a cause for gastrointestinal disease is unclear. several reports have identified hbov-1 in stool samples from asymptomatic patients. 42 , 44 arthur and colleagues compared stool samples positive for hbov-1, hbov-2, and hbov-3 from symptomatic patients and age-matched asymptomatic controls. the presence of hbov-1 failed to achieve statistical significance, supporting the hypothesis that hbov-1 may not be a cause of acute gastroenteritis. 13 the authors note that the lack of hbov-1 in symptomatic individuals may be because symptoms may appear before viral particles are shed in stool. similar findings were made for astrovirus and adenovirus, which are widely accepted causes of gastroenteritis. 13 also, hbov-2 was significantly found more often in patients with gastroenteritis. because of the low incidence in this study, the clinical significance of hbov-3 remains unclear. further data on higher viral load in stool samples corresponding with active disease would support causality. most studies identify coinfecting viral and bacterial pathogens in a substantial percentage of hbov-1-positive samples. although all studies did not examine samples for all pathogens, commonly reported pathogens include enterovirus, rhinovirus, rsv, parainfluenza, influenza, adenovirus, and hmpv ( table 3) . hbov-1 coinfection was a common occurrence in all studies with a median rate of 42.5%. hbov-1 coinfection is described with numerous pathogens; however, those that screen positive for rhinovirus, enteroviruses, and influenza have the highest incidence of hbov-1 coinfection. similarly, in samples found positive for hbov-1, a high rate of enterovirus, influenza, and rsv is seen. although this likely reflects the high frequency of these viruses in the population at large, one may speculate that these particular coinfecting viruses may provide a biologic benefit to hbov. hbov-1 has been recognized worldwide in association with upper and lower respiratory disease. most hbov-1-positive samples originate from children predominantly younger than 2 years of age. 11, 20, 27, 29, [34] [35] [36] [47] [48] [49] [50] [51] [52] [53] within this age group, children less than 6 months of age seem to be less frequently affected, perhaps because of passive maternal immunity. several studies have detected hbov-1 in adults. 20, 54, 55 in one study screening 1539 children and 273 adults, hbov-1 occurred at a similar frequency in both groups. 20 most studies report circulation of hbov-1 predominantly during the winter season. 20,47 seasonal peaks vary but most often are described in the early winter. most of these studies are retrospective, using archived respiratory samples submitted for analysis of common respiratory pathogens. this approach may lead to misrepresentation of when this virus circulates. several reports show increased frequency of hbov-1 infections during the spring. choi and colleagues 56 screened samples over a 5-year period from patients who had lower respiratory tract illness (lrti) in korea and found a higher frequency of hbov-1 between may and july. bastien and colleagues 14 studied samples originating from patients in canada and reported no apparent seasonal prevalence. only a few prospective studies of hbov-1 have been performed. in a 1-year prospective investigation in children less than 5 years of age hospitalized with respiratory tract disease, hbov-1 was detected in every month except august. 51 in this study hbov-1 peaked in the month of december, with 80% of hbov-1 isolates occurring between november and march. several seroepidemiology studies have been performed to assess the prevalence of hbov-1 infection during childhood. kahn and colleagues reported anti-hbov-1 antibodies are common in children. 57 screening serum samples using a viruslike particlebased elisa, kahn and colleagues found evidence of prior hbov-1 infection in 195 of 270 (72.2%) serum samples. 57 children between 4 and 8 months of age had the lowest prevalence of antibodies against hbov-1, whereas children younger than 2 months of age and those older than 5 years had the highest. 57, 58 the high proportion of seropositive children younger than 2 months of age suggests vertical transfer of antibodies. the subsequent decline in the percentage of seropositive children over the first 4 to 6 months of life likely represents waning maternal immunity. the low percentage of anti-hbov antibodies in children younger than 12 months of age correlates with population-based studies that demonstrate children younger than 1 year of age have a higher occurrence of infection. 20, 29, 34 by 5 years of age, most people have circulating antibodies against hbov-1, similar to other respiratory viruses, such as rsv, 59 rhinovirus, 60 and hmpv. 61 because of the difficulties of growing hbov-1 in vitro, it has yet to be determined which antibodies offer protection. also it is unclear if antibodies against hbov-1 will confer protection against hbov-2 or hbov-3 and vice versa. most protective antibodies are speculated to target the two viral capsid proteins vp1 and vp2. hbov-2 and hbov-3 both have approximately 80% aa identity with hbov-1 in this region with most sequence diversion occurring in the n terminal portion. the high similarity of viral capsid proteins suggest cross-reactive antibodies between bocavirus species may occur. studies on hbov-2 and hbov-3 seroepidemiology have not been performed thus far and are warranted. as more bocavirus species are recognized, our understanding of this family of viruses grows. based on the available sequence data of hbov-1, there seem to be two closely 68 58 42 46 30 28 ---59 21 14 -65d 14 -10 --7 11 --14 0 0 0 11 30 23 13 27 --6 6 --6 7 -73 9 9 9 0 5 -2 2 -----70 95 81 75 72 ----11 24 28 7 11 26d 18 12 12 6 ----7 -2 --14d 32 28 21 28 12 ----26 4 5 -49d 33 13 -9 12 --10 1 23 --3 42 total e 250 200 210 69 45 30 28 19 147 69 38 related genotypes. the two prototype strains (st1, dq000495.1; st2, nc_007455.1) have 99.5% identity at the nucleotide and protein level. because the amino acid identity between both genotypes differs by so little, it may be assumed they represent one serotype. analysis of 24 hbov-1 unique isolates (12 originating from respiratory samples and 12 from fecal samples) demonstrated little sequence variation from the prototypic strains. 39 there was also no difference in the proportion of each hbov-1 genotype identified in nasopharyngeal and fecal isolates, suggesting that each lineage infects both respiratory and enteric systems. the recent recognition of hbov-2 and hbov-3 demonstrates that the bocavirus genus has substantial diversity. hbov-2 and hbov-3 have similar genomic organization of the putative open reading frames (orfs) to hbov-1. phylogenetic analysis of hbov-2 orfs show that it is more closely related to hbov-1 (pairwise distance 22.2%-26.2%) than to the animal bocaviruses (pairwise distance 46.2%-55.8%). 12 hbov-3 nonstructural proteins ns1 and np1 are closely related to hbov-1, whereas the structural viral capsid proteins are more similar to hbov-2 (see fig. 1 ). arthur and colleagues 13 hypothesize that hbov-3 may be a result of a distant recombination event between hbov-1 and hbov-2. because only several strains of hbov-2 and hbov-3 have been recognized from a handful of regions worldwide, however, the true diversity is unclear. following the discovery of human bocavirus, numerous studies have used conventional pcr to detect hbov-1. 14, 20, 24, 28, 29, 34, 35, [38] [39] [40] [41] 43, 48, 49, 56, 58, 60, primers, target genes, and predicted amplicon sizes are listed in table 1 . most primer sets used for hbov-1 screening target the np1 gene. although there is a wide variation in detection of hbov-1 between study sites with detection rates as high as 45.2% reported in one study, 72 primers targeting the vp1/vp2 region have a slightly higher detection rate (median 7.5%, see table 1 ). this difference is not surprising because the vp1/vp2 is more conserved than ns or np. in addition, nested and real-time pcr have been used to detect hbov-1. real-time pcr yields a slightly higher median frequency of detection of hbov-1. 15, 17, 21, 26, 30, 36, 37, 44, 51, 52, 54, 67, 68, [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] the variation in detection of hbov-1 in these studies is likely attributable to multiple factors, including sensitivity of primer sets, laboratory technique, true variation in incidence of hbov-1, and methods used in sample collection. ziegler and colleagues 94 reported using nucleic acid sequence based amplification (nasba) to screen for hbov-1 in stool samples. nasba is an isothermic nucleic acid amplification technique used as an alternative to pcr for the detection of viruses. 95, 96 in the study by ziegler and coworkers, 94 no hbov-1 was detected using nasba. it is unclear if this represents a failure of the method, lack of hbov-1 in the samples, reaction inhibitors to stool samples, or other variables. an increasing number of studies are screening stool samples for the presence of hbov-1. 38, 39, [41] [42] [43] [44] based on fecal and respiratory hbov sequence similarities reported by lau and colleagues genomic targets used for respiratory samples should be as successful in stool samples. 39 the current literature finds that hbov-1 is found in stool samples less often than in respiratory samples (median 2% versus 7.5%). it is unclear whether this lower rate of detection is due to a truly lower incidence of hbov-1 in stool, decreased viral loads, or presence of reaction inhibitors commonly found in feces. to date, hbov-1 has not been successfully propagated in standard cell lines or animal models. this failure has been one of the major impediments to our understanding of this virus. parvoviruses have a large host range and tissue tropism, even among closely related strains. the viral capsid proteins seem to be the major determinant of host specificity. 16 the related human parvovirus b19 causes viremia and replicates in the bone marrow of children and adults and in the liver of the fetus. 16 two studies identified hbov-1 dna in serum of affected patients. fry and colleagues 36 demonstrated hbov-1 in acute serum from hbov-1-positive children who had respiratory disease. allander and colleagues 15 detected hbov-1 dna in more than half of acute-phase serum samples and in 19% of the convalescent-phase serum specimens. detection of hbov-1 in serum was associated with a high viral load in the nasopharynx. even with the detection of hbov-1 dna in the blood of affected patients, however, it remains unclear if hbov-1 viremia truly occurs. among healthy children, viral respiratory disease is often a self-limited and uncomplicated disease. therapy against most viral respiratory pathogens has not been shown to be effective. treatment of viral pathogens, such as influenza, may shorten the duration of illness in low-risk patients but has not been shown to reduce the duration of illness in the high-risk population. 97 treatment with ribavirin or intravenous immune globulin (ivig) remain controversial in the setting of viral respiratory disease and are only used in select populations with severe rsv and human parainfluenza virus (hpiv) disease. 98, 99 the relative ineffectiveness of antiviral compounds is, in part, because host inflammatory reaction leads to a substantial amount of pathology. in addition, many patients present for medical attention at a time past peak viral replication. for these reasons, it is difficult to demonstrate effectiveness of most antiviral compounds. in several case reports, patients have received antiviral treatment either as prophylaxis or as treatment of confirmed or presumed viral infections. schenck and colleagues 22 reported a child who had undergone hematopoietic stem cell transplant who was receiving acyclovir prophylaxis; however, he subsequently developed symptomatic hbov-1 disease with hbov-1 dna identified in respiratory, blood, and stool samples. during this patient's complicated hospital course, he was placed on ganciclovir and eventually foscarnet for cytomegalovirus (cmv) viremia. both the cmv viral load and the hbov-1 serum viral load became undetectable after initiating foscarnet. this patient continued to have hbov-1 dna detectable in stool samples, however. kupfer and colleagues 100 reported a patient undergoing treatment of b-cell lymphoma who received ganciclovir for cmv viremia who was also found to have hbov-1 in respiratory secretions. there was no clinical improvement in symptoms after initiating ganciclovir. supportive care, even in the intensive care setting, plays a large role in the management of patients who have severe viral illness. supplemental oxygen 22 and mechanical ventilation 101 have been used to support patients who have hbov-1-associated disease. in one study, up to 28% of hbov-1-positive children required intensive care, although not all required respiratory assistance. 20 one patient, coinfected with rsv and hbov-1, required support through extracorporeal membrane oxygenation until underlying cardiac physiology could be repaired. there have been no comparative studies using antiviral agents for the treatment of human bocaviruses. the large percentage of coinfections associated with human bocavirus infections suggests that evaluation for further pathogens should be undertaken for any patient diagnosed with hbov-1. treatment of copathogens (ie, influenza) may lead to patient improvement. future therapeutic strategies may include vaccines, hbov-neutralizing antibodies, and small interfering rna. three human bocavirus species are now recognized and are reported in association with respiratory and gastrointestinal diseases. because of wide variation in clinical presentations, severity of disease, and a high rate of coinfection, the role of the bocaviruses in causing disease has been debated. one consideration is that bocaviruses are passenger viruses with little or no clinical effect. another possibility is that bocaviruses require the presence of a copathogen for infection. several studies suggest that this is not the case, however. bocavirus is identified in patients who have disease despite the absence of copathogens. case-control studies have shown an increased risk for pneumonia in patients who have hbov-1-positive respiratory secretions and increased risk for gastroenteritis in patients who have hbov-2-positive fecal samples. two studies found no increase in hbov-1 frequency in symptomatic versus asymptomatic stool samples. one report finds hbov-1 did not worsen gastrointestinal disease when coinfected with known pathogens such as rotavirus. 13 the question of whether hbov-2 or hbov-3 is associated with respiratory infection has not been answered. little is known about the recently described bocaviruses hbov-2 and hbov-3. clinical epidemiology and seroepidemiology have yet to be described in detail. although antibodies to hbov-1 are common in human sera, cross-reactivity with hbov-2 and hbov-3 has not been investigated. furthermore, although hbov-2 and hbov-3 have been described in stool, their presence in the respiratory tract should be investigated. areas for further research on hbov-1 include examining objective measures of disease severity in patients who have coinfections and examining hbov-1 for evidence of disease distant to the respiratory and gastrointestinal tracts. presence of hbov-1 has been described in serum, 22 lymphatic tissue, 21 and in conjunction with elevated hepatic enzymes, 46 the clinical significance of which has yet to be determined. in summary, although the 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specimen dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time nasba update in prevention, evaluation, and outpatient treatment of influenza respiratory syncytial virus (rsv) immune globulin intravenous therapy for rsv lower respiratory tract infection in infants and young children at high risk for severe rsv infections: respiratory syncytial virus immune globulin study group use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology severe pneumonia and human bocavirus in adult human bocavirus infection in a neonatal intensive care unit undiagnosed respiratory viruses in children epidemiological and clinical study of viral respiratory tract infections in children from italy clinical and genetic analysis of human bocavirus in children with lower respiratory tract infection in taiwan no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease detection of human bocavirus in asturias, northern spain human bocavirus in hospitalized children role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing key: cord-300793-tuq8z6gm authors: weiss, robin a; mcmichael, anthony j title: social and environmental risk factors in the emergence of infectious diseases date: 2004 journal: nat med doi: 10.1038/nm1150 sha: doc_id: 300793 cord_uid: tuq8z6gm fifty years ago, the age-old scourge of infectious disease was receding in the developed world in response to improved public health measures, while the advent of antibiotics, better vaccines, insecticides and improved surveillance held the promise of eradicating residual problems. by the late twentieth century, however, an increase in the emergence and re-emergence of infectious diseases was evident in many parts of the world. this upturn looms as the fourth major transition in human–microbe relationships since the advent of agriculture around 10,000 years ago. about 30 new diseases have been identified, including legionnaires' disease, human immunodeficiency virus (hiv)/acquired immune deficiency syndrome (aids), hepatitis c, bovine spongiform encephalopathy (bse)/variant creutzfeldt-jakob disease (vcjd), nipah virus, several viral hemorrhagic fevers and, most recently, severe acute respiratory syndrome (sars) and avian influenza. the emergence of these diseases, and resurgence of old ones like tuberculosis and cholera, reflects various changes in human ecology: rural-to-urban migration resulting in high-density peri-urban slums; increasing long-distance mobility and trade; the social disruption of war and conflict; changes in personal behavior; and, increasingly, human-induced global changes, including widespread forest clearance and climate change. political ignorance, denial and obduracy (as with hiv/aids) further compound the risks. the use and misuse of medical technology also pose risks, such as drug-resistant microbes and contaminated equipment or biological medicines. a better understanding of the evolving social dynamics of emerging infectious diseases ought to help us to anticipate and hopefully ameliorate current and future risks. popular writing on emerging infectious diseases resounds with dire warnings about the threat of modern 'plagues' and losing the 'war against microbes.' this adversarial language obscures the fact that most of the microbial world is either neutral toward, or supportive of, human well-being and survival. indeed, we would not survive long without commensal microbes such as the beneficial strains of escherichia coli in our gut. that aside, the study of emerging infections is more than a passing fad. the recent rate of identification of such infections, the impact of the sars outbreak, the devastation caused by aids, and the ever-present threat of a new influenza pandemic indicate that we cannot control our disease destiny. nor are emerging infections unique to humans; the irish potato famine in 1845 and the english foot-and-mouth disease epidemic in 2001 underscore the consequences for human societies of disease emergence in crops and livestock. emerging infectious diseases in humans comprise the following: first, established diseases undergoing increased incidence or geographic spread, for example, tuberculosis and dengue fever; second, newly discovered infections causing known diseases, for example, hepatitis c and helicobacter pylori; and third, newly emerged diseases, for example, hiv/aids and sars. this perspective will discuss the human ecology of both the (apparently) new and re-emerging diseases. interest in infectious disease has itself recently re-emerged. in 1972, burnet and white commented, "the most likely forecast about the future of infectious disease is that it will be very dull. there may be some wholly unexpected emergence of a new and dangerous infectious disease, but nothing of the sort has marked the past fifty years" 1 . today, we may criticize the short-sightedness of our mentors' generation, yet in demographic terms they were essentially correct because the proportion of deaths from infectious disease has fallen throughout the twentieth century 2,3 (fig. 1) . humankind currently faces neither apocalyptic extinction nor even a population reduction such as occurred in europe during the black death of the fourteenth century. rather, overpopulation in relation to environmental resources remains a more pressing problem in many developing countries, where poor economic and social conditions go hand-in-hand with infectious disease. in industrializing countries during the nineteenth century, a major reduction in enteric infections was achieved by separating drinking water from sewage-an environmental change that probably saved more lives than all the twentieth century vaccines and antibiotics together. today, however, the growth of shanty towns without sanitation around the megalopolis cities of asia, africa and south america is recreating similar conditions, and in the past 40 years cholera has made a remarkable re-emergence through its longest ever (seventh) pandemic 4 . in most countries, life expectancy has risen over the past 50 years 5 (fig. 2) . the most important exception is those regions where hiv infection is rife. moreover, during the past 15 years, falling living standards in some african countries and the breakdown of public health infrastructure in ex-soviet nations has aided the re-emergence of transmissible diseases like tuberculosis 4,6 . further, severe outbreaks such as the 1918-1919 influenza a pandemic temporarily reversed the decline of deaths caused by infectious disease. the 50 million estimated deaths from that pandemic 7 represented about 2% of the global population at that time, and is twice as many as the cumulative aids mortality of the past 20 years. the next influenza pandemic may be just around the corner, and may spread even faster 8 , if access to appropriate vaccines and drug treatment is not available 9, 10 . for other newly emerging infections that make headlines, such as sars, ebola or vcjd, it is important to keep a sense of demographic proportion. placing these emerging infections on a 'richter' scale of human mortality (box 1) shows that they elicit scarcely detectable minor tremors in numbers of fatalities -despite the fear they invoke. we do not know, however, which one might leap to the top of the scale like hiv has done; indeed, it may be a completely unknown agent, as the sars coronavirus was two years ago. a major challenge is to predict which infection presages the next big quake, hopefully allowing preventive action. like any other animal or plant species, humans have been prone to infection by pathogens throughout their evolutionary history. such ancient infections by helminth and protozoan parasites, bacteria, fungi and viruses are endemic, eliciting a range of effects from a heavy burden of disease (e.g., malaria) to being essentially commensal in immunocompetent hosts (e.g., most types of herpesvirus and papilloma virus). other infections depend on an animal reservoir for their maintenance; their infection of humans may be pathogenic, but it has little part in the evolving ecology of the microbe or parasite. an estimated 61% of the 1,415 species of infectious organisms known to be pathogenic in humans are transmitted by animals 11 , for which the human represents a dead-end host. occasionally, however, a zoonotic infection adapts to human-to-human transmission and diversifies away from its animal origin. epidemic diseases are generally caused by infections that are directly transmissible between humans. hiv is a recent example of a long line of human infections initiated by a switch of host species, stretching back to the origins of measles and smallpox. free-living microbes may also find a human niche that suits their lifestyle, such as the lung for legionella pneumophila and the gut for vibrio cholerae. legionnaires' disease, first recognized in philadelphia in 1976, is the environmental equivalent of a zoonosis. it is seldom passed directly from person to person but it was human ingenuity in designing warm, aerated, humid 'artificial lungs' called air-conditioning systems that allowed the microbe to proliferate and become an opportunistic colonizer of the human lung. cholera, which was unknown beyond the ganges delta before it spread widely in asia and the middle east during the period 1815-1825, at around that time horizontally acquired a toxin gene and other factors in a genetic package that helped it to colonize the gut; the resultant diarrhea aids dispersal of the microbe 12, 13 . human society has undergone a series of major transitions that has affected our pattern of infectious disease acquisition and dissemination 4 . these transitions illustrate the interrelationship between environmental, social and behavioral influences on the emergence and subsequent spread of infectious disease. some infections were acquired when our australopithecine ancestors left their arboreal habitat to live in the savannah. this ecological change included exposure to new species of mosquito and tick as vectors for infection. after the emergence of homo sapiens, the eventual migration of neolithic hunter-gatherers out of africa 50,000 to 100,000 years ago exposed them to new infections in distant regions. the first major transition of prehistoric/early historic times gave rise to the epidemic, or 'crowd,' infections. this change must have started in the millennia following the advent of agriculture-from around 10,000 years ago-as agriculturally based society developed larger, denser populations. the domestication of livestock and the rich dividends available in human settlements to other animals (e.g., rodents, dogs and various insects) provided further opportunities for pathogens to move between species. sometimes such a pathogen (or a mutant strain thereof) would have been well suited to humans as a new host species, and, if human numbers were adequate, could therefore persist indefinitely as a human infection. thus, measles emerged about 7,000 years ago, probably from rindepest of cattle, and diverged to become an exclusively human infection when population size and density became sufficient to maintain the virus without an animal reservoir. similarly, smallpox became epidemic about 4,000 years ago, possibly evolving from camelpox, its closest phylogenetic relative. the next two transitions were primarily to do with great extensions in the spread of infectious diseases, entering distant populations as 'new infections.' thus, the second historical transition occurred in classical times as large eurasian civilizations came into commercial and military contact. they inadvertently exchanged their pools of infections, and vectors such as rats and fleas, across the mediterranean basin, the middle east, india and china. the plague of athens in 430 bce during the peloponnesian war vividly described by thucidides may represent the first report of typhus. this rickettsial infection is transmitted from rats to humans and thence among louse-ridden humans. typhus frequently accompanies human conflict and deprivation, as seen in a recent outbreak among rwandan refugees in burundi 14 . the justinian plague of 542 ce devastated the eastern mediterranean region and probably extended as far as china 15 like the black death 800 years later (and both are attributable to yersinia pestis 16 ). the third historical transition accompanied the era of worldwide exploration and colonization by europeans from circa 1500 ce onward. a contemporary account by one of hernan cortes' fellow conquistadors, bernal diaz, recalls that they might well have failed to overthrow the mighty aztec empire had they not been aided by a raging epidemic. this was possibly a combination of smallpox and measles, both wholly unknown to the new world population. curiously, the columbian exchange was unidirectional regarding infectious diseases; the one contentious possible exception being syphilis. the new world is believed to have had substantially fewer human zoonotic infections 15, 17 , and vector-borne infections like chagas' disease did not travel in the absence of an appropriate vector. two centuries later, captain cook unwittingly repeated the decimation of indigenous peoples through syphilis, measles and tuberculosis in many of the pacific islands, whereas lord jeffrey amherst deliberately attempted to spread smallpox among 'hostile' native americans, one of the better documented cases of germ warfare 18 . the transmission dynamics of infections in naive populations is markedly different from those in which the majority of adults are immune 19 . onboard the beagle, charles darwin observed with his customary acuity, "wherever the values are approximate global death rates for the year 2003, taken from the world health organization (who) and other sources. hbv and hcv, hepatitis b and c viruses; rsv, respiratory syncytial virus; hpv, human papilloma viruses; vcjd, variant creutzfeldt-jakob disease. two major, novel causes of mortality top the list: cigarette smoking and hiv infection; they emerged in the twentieth century and continue to increase in many developing countries. among the chronic and re-emerging infections, malaria and tuberculosis are near the top, so it becomes apparent why there is a need for the global fund for malaria, tuberculosis and aids. accidental injuries, particularly road deaths, continue to rise, with 85% occurring in developing countries 54 . although 2003 was the year of the sars outbreak 52,55 , less than 1,000 people actually died as a result of sars coronavirus infection despite the collateral damage to daily life, psychological wellbeing and economic activity in the affected cities. this richter scale represents a snapshot in time. twenty years ago, hiv was three logs further down the scale, whereas polio was three logs higher. fifty years ago, malaria was finally eradicated from europe, where it had formerly been widespread, including in england (shakespeare's 'ague'). bacterial respiratory diseases used to have a more important role in human mortality and, despite concern over multi-resistance to antibiotics 40, 56 , the situation is considerably better than in the era before the advent of antibiotics. common bacterial infections of childhood, such as diphtheria and whooping cough, have become rarities in the developed world, largely through vaccination. viral diseases have similarly been reduced. thanks to effective immunization policies of the who, smallpox was eradicated in 1977; polio and measles viruses, which have no animal reservoir, may soon be eliminated in the same way. the european has trod, death seems to pursue the aboriginal …most of the diseases have been introduced by ships and what renders this fact remarkable is that there might be no appearance of the disease among the crew which conveyed this destructive importation." today we are living through the fourth historical transition of globalization. urbanization, dense and usually impoverished peri-urban settlements, social upheaval, air travel, long-distance trade, technological developments, land clearance and climate change all influence the risks of infectious disease emergence and spread. although some of the apparent increase in infectious disease may be attributable to better diagnostic methods and surveillance, there seems little doubt that more incidents are occurring, and have the potential to spread more widely than 50 years ago, as outbreaks and spread of infections like nipah virus and sars would not have passed unnoticed. as humans encroach further into previously uncultivated environments, new contacts between wild fauna and humans and their livestock increases the risk of cross-species infection 20 . this process will only diminish as wild species become rarer and eventually endangered, like the great apes today. an example of such contact followed the establishment of piggeries close to the tropical forest in northern malaysia, where, in 1998, the nipah virus first crossed over from fruit bats (flying foxes, pteropus spp.) to pigs and thence to pig farmers 21 . destruction of natural forest has also encouraged fruit bats to relocate nearer human habitation, like the large colony in the botanic gardens in the heart of sydney. indeed, in 1997, hendra, a related paramyxovirus of australian fruit bats 22 , fatally infected a veterinarian examining a sick horse. rodents continue to be sources of re-emerging infections, as witnessed in the 1990s with hantaviruses in the united states. rodentborne hantavirus is prevalent in agricultural systems in south america and east asia, in arid grasslands in north america and elsewhere. in mid-1993, an unexpected outbreak of acute, sometimes fatal, respiratory disease occurred in humans in the southwestern united states 23 . this 'hantavirus pulmonary syndrome' was caused by a previously unrecognized virus, maintained primarily within the native deermouse, and transmitted through excreta. the 1991-1992 el niño event, with unseasonal heavy summer rains and a proliferation of piñon nuts, hugely amplified local rodent populations which led to the 1993 outbreak 23, 24 . in south america, there have been several outbreaks of hantavirus and arenavirus infections linked to forest clearance and the growth of rodent populations in the new grasslands 4 . habitat destruction is not the only cause of increased human infection, however. dengue virus is extending its range and prevalence because its mosquito vector breeds rapidly in the urban environment 25 . in the united states, nature conservation and increased woodland in the eastern states has led to the emergence of lyme disease. this disease is caused by a tick-borne spirochete and the presence of tick-infested deer near suburban homes leads to ticks residing on bushes adjacent to baseball diamonds and gardens. intensification of production of meat and meat products has led to new infections 26 . most notorious is vcjd in the uk arising from consumption of contaminated food products of cattle affected by bse 27 . bse, or 'mad cow disease,' emerged in british cattle in 1986 because of industrialized cannibalism, whereby rendered neural tissue and bone meal from slaughtered cattle were recycled into cattle feed, as well as into pies and hamburgers for human consumption 28 . originally, infectious prions from scrapie in sheep were the suspected source, but it now seems more likely that it arose from a bovine with sporadic prion disease. the extent of the human epidemic remains unclear. although natural transmission is unsustainable (r 0 < 1 in both cattle and humans), there are concerns that vcjd might be transmissible through blood transfusions 29 . without effective diagnostic tests for presymptomatic vcjd infection, this situation is extremely unfortunate. other recently emergent food-borne infections include e. coli o157:h7, which is harmless to cattle but toxic to humans, and salmonella enteriditis in chickens. better hygiene in abattoirs, butchers and domestic kitchens can greatly reduce the incidence of infection. in theory, closed and intensive farming of a single species should reduce the risk of cross-species infection (fig. 3) . but it also allows large-scale epidemics to emerge, as seen recently for avian influenza strains in southeast asia and the netherlands 8, 30 . ancient dietary taboos, such as those of hindus, muslims and jews regarding pork as unclean, doubtless had their roots in protection from infectious disease. today, an increasing demand for consumption of exotic and wild animals raises new risks of infectious diseases such as sars (box 2). changing patterns of human behavior and ecology affect two distinct steps in the emergence of new infectious disease. the first is an increased opportunity for animal-to-human infection to occur owing to greater exposure, which may be necessary but not sufficient to lead to the emergence of a new human infection. the second step is the opportunity for onward transmission once a person has become infected. for each novel epidemic, such as the 1918-1919 influenza pandemic or aids, there are probably thousands of failed transfers. some infections simply do not take in the new host. innate hostspecific restrictions on viral replication have recently become evident for primate lentiviruses 31 , which may explain why certain species that harbor simian immunodeficiency virus, but not others more commonly in contact with humans, gave rise to hiv-1 and hiv-2. even in the case of hiv-1, only one pedigree of three independent chimpanzee-tohuman crossover events 32 has given rise to the aids pandemic, whereas the other two smolder as poorly transmissible infections. fatal pathogenesis is not necessarily coupled with infectiousness 12 , which is evident for h5n1 avian influenza in humans 9 . but genetic reassortment between avian and human influenza viruses could easily give rise to a new, rapidly spreading strain 8 . a poorly infectious pathogen may not spread at all from the index case, as is usual with rabies, or may only infect close contacts and soon peter out, as seen with lassa fever and ebola virus. sars nearly became self-sustaining but was brought under control. some of the most insidious infections are those with long, silent incubation periods during which the person is infectious. these emerge surreptitiously so that when the new disease is eventually recognized, as aids was in 1981, the infection has already spread far beyond control. like the ships of centuries past, the speed of modern air travel works wonders for the dispersal of infectious diseases. sars was eventually constrained by quarantine and strict adherence to infection control guidelines in hospitals, but not before it quickly traveled from guangzhong to hong kong and on to toronto. if ebola broke out in a city with a busy international airport, it might also travel across continents in a similar manner. brockmann 33 has modeled how rapidly such infections can move once they reach a major airport hub; closing the hubs becomes an immediate imperative. we cannot be sure what the initial vector was for the arrival of west nile virus into north america in 1999: a migratory bird blown off course, an infected human with a valid air ticket or a stowaway mosquito on a similar flight. whatever the means of entry and early colonization of crows in new york, it has taken less than four years to reach the pacific coast 25 . thus, west nile virus has found a new reservoir in american birds, just as yellow fever virus reached new world primates 350 years earlier. microbes frequently capitalize on situations of ecological, biological and social disturbance. biologically weakened and vulnerable populations-especially if also socially disordered and living in circumstances of privation, unhygienic conditions and close contact-are susceptible to microbial colonization. the severity of the bubonic plague (black death) in mid-fourteenth-century europe seems to have reflected the nutritional and impoverishment consequences of several preceding decades of unusually cold and wet weather with crop failures compounding the incipient destabilization of the hierarchical feudal system. many of the rapid and marked changes in human social ecology in recent decades have altered the probabilities of infectious disease emergence and transmission. these changes include increases in population size and density, urbanization, persistent poverty (especially in the expanding peri-urban slums), the increased number and movement of political, economic and environmental refugees, conflict and warfare. political ignorance, denial and obduracy often compound the risk of infectious disease transmission-as has been tragically observed with hiv/aids in parts of africa, where widespread poverty, a culture of female disempowerment and political instability further "if there is any conceivable way a germ can travel from one species to another, some microbe will find it," wrote william mcneill in his classic text plagues and peoples 15 . for millennia, small farmsteads accommodated mixed species living closely with humans-goats, pigs, cattle, ducks, geese, chickens and perhaps a water buffalo or a donkey-and exchanged infections. when species are raised separately but are sold together, the opportunity for cross-infection moves from the farm to the marketplace. the 1997 outbreak of avian influenza in hong kong occurred in mixed markets, where live chickens, quail and ducks were stacked together in close quarters with humans. the h5n1 virus that emerged may have been derived by recombination between those of different avian hosts 8 . after 1997, mixed species were separated into different areas of the markets. but this year's h5n1 virus is spreading among intensively reared chickens across southeast asia. the increasing predilection for meat of exotic species has exacerbated the risk of exposure to infections not previously encountered, and this situation probably triggered the sars epidemic 55 . although we are still not sure of the natural reservoir species of sars coronavirus, the live markets and restaurants in guangzhong sold small carnivores, and several species of civet cat, racoon dog and ferret badger captured in china, laos, vietnam and thailand, were brought into close proximity 57 . clearly, some of the palm civet cats were infected with sars-related viruses, but it is less clear whether they represent the original source species. there is a danger in incriminating the wrong species; if the true reservoir resides in the rodent prey of these carnivores, then culling the predators may be counterproductive. stopping the exotic meat trade altogether would seem to be a simple solution to prevent the reappearance of sars, but once the taste for it has been established, that may prove no more practical than attempting to prohibit the tobacco trade. in africa, bushmeat also poses a serious problem for emerging infectious diseases, as well as for nature conservation. sick animals may be more easily captured. for example, 21 human deaths owing to ebola virus infection ensued from the butchering of a single chimpanzee 58 . hiv has crossed from chimpanzees to humans on at least three occasions, and a higher number of zoonotic events from sooty mangabeys are indicated for hiv-2 (ref. 32). whether these cross-species infections arose from butchering the animals or from keeping them as pets is unknown, but a recent survey of primate hunters in africa showed that they are susceptible, like handlers of primates in captivity, to infection (though not disease) from foamy retroviruses 59 . the escalating intercontinental trade in exotic pets can lead to unexpected infectious disease outbreaks. the united states has only recently imposed more stringent regulations and quarantine following cases of monkeypox in humans and in prairie dogs introduced by rodents imported from africa as pets 60 . exacerbate the problem 34, 35 . but we have little understanding of why the prevalence of hiv infection varies so greatly between cities in sub-saharan africa 36 . the urban environment has only recently become the dominant human habitat. urbanism typically leads to a breakdown in traditional family and social structures, and entails greater personal mobility and extended and changeable social networks. these features, along with access to modern contraception, have facilitated a diversity of sexual contacts and, hence, the spread of sexually transmitted diseases 37 . this risk is further amplified by the growth in sex tourism in today's internationally mobile world, which capitalizes on the desperation and ignorance of poverty, combined with exploitative behaviors, in developing countries. more generally, cities often function as highways for 'microbial traffic' 38 . rapid urbanization boosts certain well established infectious diseases, such as childhood pneumonia, diarrhea, tuberculosis and dengue, and facilitates dissemination of various 'emerging' diseases-as occurred for sars in the high-rise housing of hong kong. crowded and dilapidated public housing can potentiate infectious disease transmission through drug abuse and sexually transmitted infections 39 . technological advances in medicine and public health can also inadvertently promote the emergence and spread of infectious disease. it has become commonplace to quip that you go to the hospital at the peril of acquiring an intractable nosocomial infection such as methicillin-resistant staphylococcus aureus 40 , and such infections killed around 40 times as many people as sars did in 2003 (box 1). multidrug-resistant tuberculosis has also become a major problem, and, paradoxically, regions with health programs that reduced wildtype tuberculosis strains can develop into 'hot zones' for multidrugresistant tuberculosis 41 . by far the most effective medical vector of infectious disease has been the syringe and needle. drucker et al. 42 have charted the massive increase in the use of injecting equipment over the past 100 years. individuals with hemophilia treated with pooled clotting factors became almost universally infected with hepatitis b and c viruses before diagnostic screening tests were developed. over 20% of such affected individuals also became infected with hiv 43 , and more recently, transmission of west nile virus by blood transfusion and by organ transplantation has been reported 44, 45 . the use of contaminated needles among intravenous drug users has had similar consequences. infectious diseases have also been amplified by the use of nonsterile medical injections in developing countries 42 . egypt has the highest prevalence of hepatitis c infection in the world because of the use and reuse of syringes and needles in an earlier public health campaign to reduce bilharzia by medication given by injection. the transmission of cjd through contaminated surgical instruments is another example of iatrogenic spread of infection 29 . biological medicines produced from animal-cell substrates present an inherent potential hazard for introducing new infections. great care must be taken to ensure that live attenuated vaccines grown in animal cells or eggs are devoid of pathogens ; for example, several early batches of live and inactivated polio vaccine unwittingly contained live sv40 virus, a polyoma virus of macaques. after sv40 was discovered in 1960, polio vaccine production shifted to virus propagation in primary kidney cells of african green monkeys. these cultures were free of sv40 but possibly contained sivagm, a relative of hiv that fortunately does not infect humans 31 . the irony of the sv40 story is that the united states food and drug administration prohibited the use of well known, permanent cell lines demonstrably free of adventitious infectious agents, for fear that such immortalized cells might exert oncogenic properties on the vaccine. there is no epidemiological evidence of increased tumor incidence in those populations who are known to have received sv40-contaminated polio vaccine. but there have been a number of recent claims of an association of sv40 dna sequences in a variety of human malignancies 46 , although these findings remain controversial 47 . the ultimate medical means of introducing animal viruses into humans is xenotransplantation. the implantation of animal cells or tissues into immunosuppressed individuals seems to be a perfectly designed way to encourage cross-species infection. it is astonishing that trials were started without much thought about the consequences for potentially emerging pathogens, for example, porcine retroviruses 48 . the generation of genetically modified knockout or transgenic animals to prevent hyperacute rejection of donor tissues may exacerbate the infection hazard 49, 50 . happily, there is no evidence so far of retrovirus infection in individuals who were exposed to living pig cells 50 , and clinical xenotransplantation is now stringently regulated; so it seems all the more extraordinary that cellular therapies with fetal lamb cells and extracts continue to be practiced with impunity in alternative medicine clinics in europe and the far east. novel infectious diseases can emerge in any part of the world at any time. hiv and ebola came out of africa, avian influenza and sars from china, nipah virus from malaysia, bse/vcjd from the uk and hantavirus pulmonary syndrome from the americas. it is difficult to predict what new disease will come next or where it will appear, but changing ecological conditions and novel human-animal contacts will be useful clues as to which horizons require scanning with most scrutiny. we must expect the unexpected. as a codicil, another factor that needs to be taken into account is the potential impact of the hiv pandemic on the emergence of other infectious diseases 51 . we already know that persons with aids act as 'superspreaders' of tuberculosis, and we can only speculate what course the sars outbreak might have taken had someone incubating the disease flown to durban rather than toronto 52 . people with aids may persistently harbor infections that would otherwise be transient, and this could hamper the eradication of measles and polio. multivalent pneumococcus vaccines are ineffective in hiv-infected people with cd4 + lymphocyte levels below 200/µl, whereas live 'attenuated' vaccines such as vaccinia can cause virulent disease in the immunocompromised host. immunodeficient persons living at high density could also be the seed-bed for microorganisms that are initially ill adapted to human infection to evolve into transmissible human pathogens. thus, an infection from a zoonotic or environmental source-for example, the mycobacterium avium intracellulare complex-could conceivably emerge as the tuberculosis of the twenty-first century, although direct transmission between individuals with aids of such opportunistic infections have not been documented so far. we shall give girolamo frascatoro the last word on emerging and re-emerging infectious diseases by quoting from his treatise de contagione, published almost 450 years ago, "there will come yet other new and unusual ailments in the course of time. and this disease [syphilis] will pass way, but it later will be born again and be seen by our descendents." we are grateful to m. e. chamberland, h. w. jaffe and s. leff for critically reading the manuscript. natural history of infectious disease human frontiers, environments and disease. past patterns, uncertain futures 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key: cord-305302-go87uu06 authors: gessain, antoine; garcía-arenal, fernando title: editorial overview: emerging viruses: interspecies transmission date: 2015-02-28 journal: current opinion in virology doi: 10.1016/j.coviro.2015.02.001 sha: doc_id: 305302 cord_uid: go87uu06 nan emergent viruses and associated diseases comprise various and rather quite distinct entities, which can arise in a large part of the living world. indeed, emerging viruses have been, and are still, frequently reported in a great variety of animals, from insects to humans, as well as in plants. furthermore, some of these diseases, associated with emerging viruses, had recent major public health impact, as exemplified in humans by the aids [1] , hepatitis c pandemics [2] , or the current ebola disease epidemic, or in crops by cassava mosaic disease, which seriously compromises food security in east africa [3] . how can we define such emerging entities? firstly, it can be a new disease, due to a yet unknown virus. in humans, the natural reservoir of such a virus is frequently an animal. these zoonoses include some coronaviruses present in specific bat species and the severe acute respiratory syndrome, hantaviruses present in rodents and various clinical syndromes, and some filoviruses, as marburg and ebola viruses, living in fruit bats and the associated hemorrhagic fevers. for plants, new viruses in crops are also thought to emerge from wild reservoirs: for instance, the maize streak virus originates from wild african grasses. strictly speaking, these are considered as real emergences. secondly, it may be a known disease, in which one finds the causative viral agent. here, we can cite for human diseases, some clinical hepatitis and the hepatitis c virus, the severe adult t cell leukemia/lymphoma and the human retrovirus htlv-1, as well as a the kaposi's sarcoma and the human herpesvirus 8. examples in plants would be the identification of virus complexes associated with many known diseases of perennial crops, for example, grapevine leaf roll disease. here, we are rather talking about 'emergence of knowledge'. finally, it may be an already known virus, associated with a known disease, but with an epidemiological profile different from what was known before. we can mention the spread of west nile virus in the united states in the early 2000s, the arrival of chikungunya in the american continent in 2013, the dramatic current ebola outbreak in west africa, or the world-wide emergence in different temperate crops of tomato yellow leaf curl and other begomoviruses associated with the spread of highly efficient vector species. the causes of the emergence or resurgence of such viruses are many and varied [4, 5] . the emergence of a new viral associated disease or of a new virus is indeed the result of a sequence of successive steps, sometimes complex, and is often related to the entanglement of several factors: socioeconomic or particular cultural activities, increased mobility of human, animal and plant mobility ('the world is a global village'), human exploitation of the environment as deforestation or increase of agricultural or otherwise human managed land, resulting in loss of biodiversity or ecosystem simplification, disruption of human, animal and plant health systems in armed conflict, urbanization with development of huge slums of great poverty and basic hygiene, decreased interest in the surveillance and control of infectious diseases, use of unsterile medical equipment as part of therapeutic and/or mass vaccination and, finally, the ability of certain viruses to adapt quickly to a changing environment. this last point may be particularly significant for rna and small single-stranded dna viruses. it is crucial to note that all the factors that will increase the density of the human, animal and plants population, the density of vectors and/or reservoirs, and that will promote contact between these three elements, will favor emergence process. interspecies transmissions at the origin of the emergence of new viruses are common [6, 7] . one can mention for humans many arenaviruses and bunyaviruses having especially rodents as wild reservoirs, but also siv and stlv retroviruses found naturally in many species of non-human primates. finally, bats now appear as very important reservoirs of many viruses (nipah, hendra, ebola, coronaviruses, among others) that have recently emerged or are emerging in humans. interspecies transmission is also considered as a first step in plant virus emergence. although it is considered that host jumps are easier among taxonomically related species, reservoirs of emergent plant viruses often have not been identified, which has limited understanding emergence [5] . interspecies transmission as related to emergence is a new vast field of research promoted, among other factors, by the power of high-throughput sequencing and related bio-informatics methods. after the initial stages of interspecies transmission per se ('spillover') following the encounters between the virus (and its natural reservoir) and the new host, viruses often need to adapt to the new host, resulting in virus loads high enough for transmission and spread in the new host population [7] [8] [9] . dispersion in the new host population often follows new epidemiologic dynamics, and may be attained through various modes of transmission, including for humans sexual, nosocomial, direct contact, and aerosols, or through different vector species or new modes of host propagation (e.g., asexual vs. seed multiplication) in plants. some of these means of dissemination in the new host population have been well characterized, allowing efficient controls over inter-human and inter-crop transmission. however, understanding the initial stages of the emergence and the specific mechanisms for inter-species transmission of many viruses continues to be very limited. microbiological studies, especially in populations at high risk of emergence, are absolutely necessary to get new information about the early events of the emergence process. hepatitis e virus (hev), a small (7.2 kb), non-enveloped single-strand dna virus that belongs to the hepeviridae family, is the cause of a self-limiting acute hepatitis e endemic in large areas of the world. nicole pavio et al. report the current data on the zoonotic origin of viral hepatitis e. such possibility was suggested following the discovery of some animal strains of hepatitis e closely related to human hev in countries where sporadic cases of hepatitis e were autochthonous. domestic pigs and wild boars have been now recognized as the main hev reservoirs. thus, besides being primarily transmitted via fecal-oral route in human, infection through consumption of undercooked or raw meat and meat products contaminated by hev has clearly demonstrated that such virus is a foodborne zoonotic pathogen. direct contact with infected animal reservoirs is also a common source of zoonotic hev. rodents are also important reservoirs for emerging viruses in humans, especially arenaviruses and hantaviruses. all hantaviruses, which belong to the bunyaviridae family, share a negative strand dna (12 kb). some hantaviruses can cause, in specific areas of the world, very severe diseases in humans (as hemorrhagic fever with renal syndrome or pulmonary syndrome). the occasional spillover of hantaviruses from rodents to humans are at the origin of such severe clinical syndromes. in their review, edward holmes and yong-zhen zhang emphasize the fact that rodents have long been considered as the primary host of hantaviruses with a classic model of hantavirus evolution thought to reflect a process of virus-rodent codivergence over a time-scale of millions of years. the recent findings that bats, moles and shrews are also reservoirs of hantaviruses, associated with relatively frequent cross-species transmission, have greatly challenged such a theory for hantavirus evolution. bats are now recognized as a reservoir of a huge variety of viruses, especially rna viruses, belonging to different families [10] . such major and recent discoveries raise crucial questions concerning our understanding of the virus-bat interface, which is impacted by intrinsic (especially immunological features) as well as extrinsic factors. among these viruses, some are zoonotic and responsible of very severe infection in humans including filovirusesrelated diseases due to marburg and ebola viruses. sylvain baize reviews the situation of the current epidemic of ebola disease that began in west africa in 2014. indeed, after being restricted to central africa for 35 years, the ebola virus has suddenly emerged in guinea in early 2014 and spread rapidly to liberia and sierra leone. some fruit bats appear to act as the reservoir for ebola virus. however, the exact origin of the crossspecies transmission responsible for the current epidemic in west africa remains unknown. avian influenza viruses have been known to cause human diseases for more than 50 years. recent outbreaks of such viruses have raised major public health concerns both in vi emerging viruses: interspecies transmission poultry and humans, especially in asia. jasper chan and colleagues emphasize in their review the role of birds in the cross-species transmission and emergence of zoonotic avian viruses. intrusion of human into natural habitats of wild birds, domestication of wild birds as pets and mostly the increasing poultry consumption by humans have facilitated the emergence and dissemination of avian viruses that can cause zoonosis. indeed, avian influenza viruses are usually amplified and mixed in the poultry population before cross-species transmission from poultry to humans occurs. epidemiological, as well as host factors, as the distribution in the human versus. avian respiratory tract, of the different types of viral receptors can greatly modify the magnitude of the emergence process. non-human primates are considered to be the likely sources of viruses that infect humans and thus may pose a significant threat to human population. this is well illustrated by some retroviruses. indeed, the emergence of human immunodeficiency virus type 1 in humans has resulted from several independent interspecies transmissions from different sivs from chimpanzees and gorillas in the western part of central africa, probably during the first part of the last century. similarly, the origin of most human t cell lymphotropic virus type 1 subtypes appears to be linked to interspecies transmission between stlv-1-infected monkeys and humans, followed by variable periods of evolution in the human host. ré jane rua and antoine gessain review here the data on the cross-species transmission and molecular evolution of the simian foamy viruses, another retrovirus that can infect humans and can be considered as a model of restriction of retroviral emergence after spillover. although emergence of plant viruses has received less attention than for viruses causing diseases in humans or domestic and wild animals, consequences for food security, for the economy of large sections of society and for ecosystem composition and dynamics, hence, for society at large, can be as serious as for human diseases. classical hypotheses of plant pathology about ecosystem simplification and disease emergence, which converge with similar hypotheses in animal pathology [5, 11] , have just recently started to be tested experimentally or empirically. as marilyn roossinck and fernando garcía-arenal review here, the few recent studies available point to a role of ecosystem simplification in plant virus emergence. interestingly, analyses of virus infections in wild plants strongly suggest that the outcome of plant-virus interactions is highly environment-dependent, varying between mutualism and disease emergence. these pioneer studies also underscore the relevance of virus emergence into wild plants from crops, with high potential impacts in wild ecosystems. the relationship between agriculture and virus emergence has been well analyzed for rice yellow mottle virus (rymv), an rna virus endemic of africa with a narrow host range limited to the rice genus. rymv was first reported in kenya in 1966 and is presently the cause of the major viral disease of rice throughout sub-saharan africa. denis fargette and colleagues analyze the role of the transition from traditional to intensive cropping systems, and of crop expansion, in the emergence of rymv in vast regions from east to west africa, and in its evolution and diversification under different agroecological conditions. the processes leading to virus adaptation to a new host have been analyzed in detail in some plant-virus systems. adrian gibbs et al. present studies on the evolution of another rna virus, turnip mosaic virus (tumv), from a lineage of monocotyledon-infecting potyviruses to become a world-wide pathogen of brassicas (e.g., cabbage and rape family) with strain specialization on different host species. their work underlines the relevance of mutations, which are found in different genomic regions and may occur in ephemeral combinations, in host adaptation and specialization. geminiviruses are single-stranded dna viruses that account for a large fraction of recently emerged plant viruses, and that severely constrain agricultural production worldwide. recombination is inherent to the replication mechanism of these viruses, and its role in geminivirus evolution has been extensively analyzed. pierre lefeuvre and enrique moriones review recent work on the role of recombination in host switches and host range expansion, as well as in adaptation to different vectors, taking as case studies recently emerged geminiviruses such as maize streak virus and tomato yellow leaf curl virus(es). although research on plant viruses often lags behind that on human-infecting viruses, experimentation with plants allows to establish genotype-phenotype relationships of viruses on their eukaryotic hosts (as opposed as in cell culture) more easily than with animals. thus, the distribution of the fitness effects of mutations has been characterized recently in different hosts for some viruses. as stephanie bedhomme et al. review, the fitness effects of mutations may differ and even change sign, according to the host, resulting in antagonistic pleiotropy that may hinder virus adaptation to a new host. also, epistatic interactions between different mutations may result in rugged fitness landscapes that determine the accessibility of evolutionary pathways what, again, will condition host adaptation and, hence, emergence. most viruses causing diseases in plants are vectored, mostly by hemipteran insects, and the interactions with the insect that result in transmission have been much studied. although vectored viruses are more likely to become emerging threats, and emergence cannot occur without the proper vector, the role of vectors in plant virus emergence remains largely unexplored. this is clear from alberto fereres' perspective, in which it is discussed what is known about the role of vectors as drivers of plant virus emergence, and what still needs to be understood. what will be the future of viral emergence in humans, animals and plants remains unknown [12] . indeed, any prediction in the field of viral emergence remains very difficult and hazardous, as recently exemplified by ebola in west africa and mers coronavirus in saudi arabia or by the continuous emergence of new begomoviruses in plants. hiv infection: epidemiology, pathogenesis, treatment, and prevention hcv transmission in industrialized countries and resource-constrained areas molecular ecology and emergence of tropical plant viruses emerging pathogens: the epidemiology and evolution of species jumps evolution and emergence of plant viruses host range and emerging and reemerging pathogens ecology of zoonoses: natural and unnatural histories the evolutionary genetics of emerging viruses identifying genetic markers of adaptation for surveillance of viral host jumps interspecies transmission and emergence of novel viruses: lessons from bats and birds impacts of biodiversity on the emergence and transmission of infectious diseases daszak p: prediction and prevention of the next pandemic zoonosis key: cord-300301-7amiljnm authors: clements, bruce w.; casani, julie ann p. title: emerging and reemerging infectious disease threats date: 2016-03-04 journal: disasters and public health doi: 10.1016/b978-0-12-801980-1.00010-6 sha: doc_id: 300301 cord_uid: 7amiljnm this chapter describes the potential public health impact of emerging and reemerging disease. factors contributing to the emergence of diseases include increasing international travel and commerce, changes in human demographics and behavior, advances in technology and industry, microbial adaptation and the breakdown of public health systems. of emerging diseases, 60% are zoonotic, making the human–animal biome interaction critical. preparedness for an emerging disease relies on strong biosurveillance systems for early detection. control measures to prevent transmission must be implemented early. these include: rapid epidemiologic surveillance and investigations to characterize the disease; transmission prevention through containment and control measures; development and deployment of medical countermeasures; and emergency public information and warning. recovery after the outbreak of an emerging disease can result in a “new normal” with persistent endemic infection in the community. emerging and reemerging infectious disease threats objectives • describe why diseases "emerge" or "reemerge." • discuss the impact of emerging infectious diseases on public health preparedness. • list the likely sources of emerging infectious diseases in the future. • describe how international travel and commerce contribute to emerging infectious disease threats. • discuss how microbial adaptation contributes to emerging infectious disease threats. • list human demographic factors and behaviors contributing to emerging infectious disease threats. • identify the epidemiological clues indicating a possible emerging disease. • describe various types of surveillance approaches. • discuss the breakdown of public health measures and systems. • recognize the actions needed for responding to an emerging disease. on a friday afternoon before the labor day holiday weekend in 1999, the phone at the new york city department of health was answered by the on-call epidemiologist. an infectious disease specialist was calling to report an unusually large number of encephalitis or meningitis cases (inflammation of the brain or covering of the brain and spinal cord) at several hospitals in the borough of queens. blood and spinal fluid were tested at the new york state and cdc laboratories and reported positive for saint louis encephalitis virus (slev), a virus known to occur in the united states but never in new york city. new york city officials immediately began mosquito control programs in an attempt to stop transmission from mosquitoes, the vector for slev. over the next week, test results appeared to be conflicting. unknown to many of the investigators of the human outbreak, a veterinarian at the bronx zoo was investigating an outbreak of central nervous system disease in birds. wildlife veterinarians were also observing large bird die-offs but could not find a clear cause. by mid-september, both sets of investigators believed the causative agent was not slev. by september 27, after expanding testing services to several federal and academic laboratories as well as to the wider family of flaviviridae, it was confirmed that the causative agent for the human outbreak, the avian outbreak, and sentinel mosquito sampling was west nile virus (see fig. 10 -1). by 2000, the new york city outbreak included 62 confirmed cases and 7 deaths (johnston and conly, 2000) . by that time, the west nile virus was documented as having spread halfway across the united states to the rocky mountains (roehrig, 2013) . on april 15, 2009, a sample was routinely tested from a young girl with influenza-like illness as part of the us sentinel influenza network. the results indicated this was a uniquely novel strain of influenza a (see fig. 10 -2). on april 17, 2009, a similar result was confirmed 130 miles from the originally identified case. testing also confirmed that this virus was resistant to both amantadine and rimantadine but sensitive to both oseltamivir and zanamivir, two readily available antiviral medications. there had been sporadic cases of influenza with infection of viruses in the swine lineage, and it was suspected and later confirmed that this novel influenza a, h1n1, was a unique combination of genes most closely related to north american swine-lineage h1n1 and eurasian swine-lineage h1n1. in the two initial cases, no contact with pigs was discovered through extensive investigation, and it was determined that this novel virus had been spread to these cases by human-to-human transmission, a new behavior for h1n1. by april 23, additional cases were identified in texas, and samples collected from an outbreak in mexico were also positive for this new strain. by april 26, a public health emergency, the first in the history of the united states, was declared to allow for the rapid development of a vaccine, mobilization of antiviral medications through the federally resourced strategic national stockpile, and enhanced surveillance through reporting and testing. travel advisories were put into place, and upon discovery of additional cases internationally, the world health organization (who) raised the pandemic level from phase 4 to the pandemic level phase 5. the largest number of cases occurred in people between the ages of 5 and 24 years old. major figure 10-1 a culex quinquefasciatus mosquito is known as one of the many arthropodal vectors responsible for spreading arboviral encephalitis or west nile virus to human beings through their bite. photo by james gathany courtesy of the centers for disease control and prevention, public health image library. morbidity (illness) and mortality (death) occurred in pregnant women, and middle-aged people with chronic diseases and obesity, but overall the fatality rate was low. few cases were reported in people over the age of 65, indicating that this group may have had immunity. levels of influenza illness remained high in the summer of 2009, unusual for the north american continent. rapid molecular analysis of the viral genome led to rapid production of a vaccine. in the late summer, the food and drug administration announced the monovalent vaccine would be licensed via a "strain change" pathway, which is similar to how seasonal influenza vaccines are licensed. this meant production would be expedited, since the same methods would be used as those used to produce seasonal flu vaccine and additional safety and validation studies would not need to be completed. by september, 2009, 5 months after the first case was identified, prototype vaccine was delivered to us states for use. h1n1 is now included in seasonal vaccines (gatherer, 2009 ). ebola virus disease (evd) was first described in 1976 in two simultaneous outbreaks in sub-saharan africa. in december 2013, a small outbreak of evd was reported in a forested area in southeastern guinea (see fig. 10 -3). it has been postulated that the index case, a boy, was in direct contact with bats. this was the 26th reported ebola outbreak in history but the first to be reported in west africa. evd then spread to liberia and sierra leone, all through direct contact with the outbreak in guinea (who, 2014) . in guinea, liberia, and sierra leone as of august 13, 2015, there have been over 28,000 cases and 11,000 deaths. a small outbreak of 20 cases occurred in nigeria, and one case occurred in senegal. several cases were reported outside of the area, mostly in healthcare or humanitarian workers returning to their home countries. there were also imported cases in the united states and spain which led to secondary infections of medical workers but did not spread further. in both countries, the management of companion animals and environmental cleanup were challenging issues. several factors contributed to the devastation of this epidemic: poverty, population density, infrastructure decline after years of armed conflict, serious gaps in health and medical infrastructure with little to no surge capacity, and a delay in coordinated response. in spite of the significant risk to underresourced healthcare workers (comprising 10% of the dead), a major humanitarian medical response was launched internationally. in october 2014, controls on people traveling out of the area went into effect with exit screening for symptoms, entry screening in most countries, and active monitoring of travelers in some countries after arrival. this screening program controlled movement and by actively monitoring attempts to identify people who may become ill early in the course of the disease, getting them to healthcare in a controlled manner and with appropriate infection control practices in place. healthcare facilities and providers in many countries stockpiled personal protective equipment, implemented infection control training, and implemented screening processes in order to be prepared. on january 28, 2015, the who reported for the first time since the week ending june 29, 2014, that there had been fewer than 100 new confirmed cases reported. the focus of the response shifted from slowing transmission to ending the epidemic. in july 2015, results of early testing of a vaccine appeared very promising. by the end of 2015, the ebola outbreak in west africa reached over 28,000 cases and 11,000 deaths making it the largest ebola outbreak in history (cdc, 2015) . prior to this, the largest was a 2000-2001 uganda outbreak with 425 cases and 224 deaths (cdc, 2001) . at the time this chapter was written, public health professionals around the world continued to watch closely the progression toward stopping, and continued success in recovering from, this devastating ebola epidemic. bacteria plural of bacterium. a single-celled microorganism that can exist independently as a free-living organism or as parasite dependent upon a host organism. emerging infectious diseases illnesses caused by pathogenic organisms with an increasing incidence in humans. infectious diseases with increased incidence over the past two decades or those which threaten to increase in the near future are considered "emerging." emerging infectious diseases include pathogens which are newly evolving, spreading to new geographic areas, are previously unrecognized, or are old infections reemerging due to lapses in public health measures. fungi single-celled or multicellular organisms which cause infections in healthy persons or serve as opportunistic pathogens in persons who are immune compromised. examples include histoplasmosis and aspergillosis. helminths parasitic worms which live in humans or other animals and derive nourishment from their host. examples include the tapeworm, fluke, or nematode. healthcare-associated infection (hai) an adverse localized or systemic infectious condition occurring in a healthcare setting with no evidence that the infection was present at the time of admission. pathogen a biological organism capable of causing disease or illness to its host. prion the smallest infectious particle. it is an infectious strand of protein which replicates and leads to disease, and is similar to a virus. prions are the causative agents of diseases such as mad cow disease and creutzfeldt-jakob disease. protozoa a single-celled parasitic organism that can only multiply inside a host organism. r nought (r 0 ) a metric widely used in assessing disease transmissibility or the basic reproductive rate. it represents the average number of subsequent cases which one case generates during its infectious period. rickettsia a group of microorganisms requiring other living cells for growth like viruses, but having cell walls, using oxygen, and having metabolic enzymes like bacteria. rickettsia are typically transmitted by ticks, mites, or lice. typhus is one example of a disease caused by rickettsia. virus an infectious organism consisting of a nucleic acid molecule in a protein coat. it is only able to multiply inside the living cells of a host. emerging infectious diseases (eids) are some of the most challenging public health issues facing the global community. the hypothesis of "disease emergence" may have helped shaped the growth of global health initiatives, particularly at the world health organization (brown et al., 2005; lakoff, 2010) . eids are caused by pathogens that: (1) have increased in incidence, geographic, or host range; (2) have changed pathogenesis; (3) have newly evolved; or (4) have been discovered or newly recognized (lederberg et al., 1992) . in most developed countries, routine and seasonal outbreaks challenge health departments and healthcare systems, but reporting, investigation, and treatment protocols are typically in place, trained on, and easily implemented. vaccination programs and antiviral and antimicrobial medications mitigate many recurring infectious disease risks. those who have been exposed to or infected with recurring illnesses have developed some immunity. however, novel infectious diseases pose challenges that often exceed the immune function of populations and the capabilities of public health systems around the world. there are several factors that permit infections previously not seen globally or in specific locations to emerge or reemerge after periods of quiescence. those factors include: international travel and commerce and the movement of goods and people permits the movement of sick people and disease vectors into areas previously not visited, thereby exposing others to pathogens previously not encountered. international tourism has expanded consistently for over 60 years. from 1950 to 1980 it grew more than ten-fold from 25 million in 1950 to 278 million in 1980. it nearly doubled again between 1980 to 1995 reaching 528 million and again by 2013 reaching 1.087 billion. annual growth in tourism is expected to grow by over 3% each year reaching 1.8 billion international annual tourist arrivals in 2030 (un, 2014). modern transportation systems allow for rapid movement of people and with them diffusion of illness at a greater speed than during the preavia tion travel era. when travel was slower, often by caravan or ship, those who were ill could recover, layover, or succumb without transporting disease as easily. increased international travel is believed to have played a major role in the spread of hiv/aids. some virologists suspect that hiv was present at very low levels in remote areas of west africa for perhaps as long as 100 years in animals before the disease reached epidemic proportions and was officially isolated by scientists in 1983 (krause). hunting animals as a source of protein created greater exposure of humans to the disease, and development of the transcontinental highway from point-noire, zaire (now the democratic republic of congo) to mombasa, kenya, may have allowed truck drivers and traders along this route to carry the virus into the general population. airline travel has its own unique risks, with recirculated air in the confined space of an aircraft which could expose travelers to airborne diseases such as tuberculosis (see fig. 10-4) . passenger compartment conditions have some similarities to the holds of ships in sea-crossings known historically for harboring eids. with the ease of global travel, people with increased luxury income can visit developing countries, exposing those populations to novel diseases, or return home with novel infections after several hours of airline travel and before signs or symptoms of illness become apparent. economic development may result in changes of land use from agriculture to industry, disrupting established ecosystems. altering natural habitats by building dams and creating deforestation-reforestation programs alters the balance of ecosystems, allowing some species to overflourish or die out. additionally, there may be movement of people into land previously occupied by vegetation and animals, exposing those who resettle to novel or previously contained pathogens and/or vectors. zoonotic diseases, those which infect animals, comprise 60-75% of eids (taylor et al., 2001) . in the coming plague: newly emerging diseases in a world out of balance, medical journalist laurie garret writes "in this fluid complexity, human beings stomp about with swagger, elbowing their way without concern into one ecosphere after another (garrett, 1994) ." in addition, the effect of climate change on emerging diseases is unknown; however, lindgren et al. (2012) point out that climate change interacts with "a complex web" of all drivers of emerging diseases and therefore cannot be ignored. alterations of the geographic ranges of birds in europe and north america have also been demonstrated. many migrating birds carry pathogens, and changes to migratory bird habitats may result in human exposures to new pathogens (fuller et al., 2012) . climate change may also have effects on vector development, vector physiology, and vector habitat, ultimately affecting human vector-borne disease risks (parham et al., 2015) . human demographic factors and behavior including population density, population growth, and population distribution not only may affect the spread of people into geographic regions not previously inhabited, but it also can expose them to new pathogens. in addition, it may affect how disease transmission occurs from human to human. in many parts of the world, an increase in urban population is not matched by an increase in urban infrastructure and is accompanied by poverty, poor sanitation, and inadequate housing. conflict can result in population shifts to new geographic areas, disruption of critical infrastructure (including public health and health systems), and economic stress. poverty from any cause not only affects sanitation and vector control, but may force people into risk behaviors such as entering the sex trade. once ill, people living in poverty may not have access to healthcare or even have basic hygiene resources. they also may be unable to comply with isolation measures. as populations continue to age and advanced medical interventions such as chemotherapy and immunosuppressive biologicals alter immune function, diseases previously not known to infect humans can emerge. behaviors such as sexual activity and illicit drug use may also impact novel disease transmission. understanding human relationships with animals provides additional insights into eid risks (see fig. 10 -5). sixty percent of recent emerging diseases are zoonotic. colocation of open poultry markets and cohabitation with poultry has been identified as an important risk factor for human cases of avian influenza (dinh et al., 2006; thorson et al., 2006; choi et al., 2005) . human cases of high pathogenic avian influenza have occurred in workers depopulating flocks in the netherlands and canada (koopmans et al., 2004; tweed et al., 2004) . fortunately, few of these outbreaks to date have sustained human-to-human transmission. in 2003, an investigation of a large multistate outbreak of monkeypox was traced back to prairie dogs sold as exotic pets. the prairie dogs had been in close contact with giant gambian rats from ghana (cdc, 2003a) . this outbreak resulted in increased controls of imported exotic animals and markets. the source of the 2003 severe acute respiratory syndrome (sars) outbreak has never clearly been identified. the index cases were initially linked to catlike exotic pets related to raccoons called "civet cats" (cdc, 2003b) . this resulted in extensive bans on civet cat transportation and commerce. however, detailed investigation links the causative coronavirus to several wild animals used as food, and there are suggestions that the trade of many types of animals used in food was the source (cdc, 2003a,b; he, 2003a,b; normille and enserink, 2003; guan et al., 2003; ng, 2003) . bats play an important role in ecosystems in vector control, seed dispersal, and pollination. however, it is increasingly recognized that bats also play a significant role in the reservoir and transmission of zoonotic infections. contact with bats has long been recognized as a potential source for rabies, but several findings indicate that bats have a wider spectrum of diseases and may be a reservoir for paramyxoviruses such as measles, mumps, distemper, parainfluenza (drexler et al., 2012; messenger et al., 2003) , and ebola (leroy et al., 2007) . freidl et al. (2015) recently discovered antibodies for influenza a h9 in bats. influenza a h9 is a zoonotic disease and is a possible candidate for a novel pandemic strain. human interactions with bats and their habitats can occur in the workplace, home or recreational venues. in an observational study in western ghana, nearly half of the residents had contact with bats and bat habitats (anti et al., 2015) . reengineering and reopening closed facilities where bats have populated must be made with prevention methods in place. entering caves where bats and other animals live must also be approached with infectious disease precautions in mind. technology and industry certainly have tremendous human benefits but may also expose populations to conditions which foster eids. some of the most significant issues are changes in food production. mass agricultural compounds and facilities create environments where changes occur in microbial ecology. globalization of the food supply allows transportation of organisms on and in food and through accompanying vectors. food transportation over large distances may also introduce breaches in food security. advances in medical technology introduce techniques that allow bacteria to infect people and spaces not formerly at risk. increases in invasive procedures can result in the introduction of novel organisms. n n n an epidemiological investigation in 2012 traced the source of a mysterious meningitis outbreak to contamination of methylprednisolone acetate, a steroid injected to relieve back pain. several lots of the drug were contaminated with a rare fungus called exserohilum rostratum. over 13,000 patients were potentially exposed and 749 had confirmed infections, resulting in 61 deaths across 20 states (smith et al., 2013) . the cdc healthcare-associated infection (hai) 2011 survey of a large sampling of us acute care hospitals found on any given day, about 1 in 25 hospital patients has at least one hai (see table 10 -1). there were an estimated 722,000 hais in us acute care hospitals in 2011, and about 75,000 patients died during their hospitalizations. more than half of all hais occurred outside of the intensive care unit. contaminated gastrointestinal and bronchoscopy endoscopic devices are among the many advanced technologies associated with increasing outbreaks in healthcare settings, including those of organisms such as pseudomonas aeruginosa and salmonella spp. (kovalevaa et al., 2013) . the us food and drug administration, whose responsibilities include medical devices, issued a warning in march of 2015 raising awareness that several devices with complex designs may be difficult to adequately clean. many causative agents of healthcare associated infections are especially dangerous but their transmission is preventable. while some causative agents are more common bacteria and viruses such as escherichia coli or norovirus, many are less common, including acinetobacter, burkholderia, clostridium, and klebsiella. in addition, antibiotic-resistant strains such as carbapenem-resistant enterobacteriaceae, methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci and s. aureus are more difficult to control and treat. after the establishment of the european union in the 1990s and increased economic growth, patient transfer within and between countries expanded. after political changes and the opening of borders, medical care and technology improved. for example, the number of bone-marrow transplant units in central europe increased from 9 to 85 by 1995, and the number of dialysis units increased from approximately 200 to more than 1500 (krcmery, 1997) . however, vincent et al. (1995) reported in a one-day prevalence study that 21% of icu patients in western europe had nosocomial or healthcare-associated infections by microorganisms including p. aeruginosa and methicillin-resistant staphylococcus. microbial adaptation contributes to the development of "super-bugs." this includes both bacteria and viruses (see figs. 10-6 and 10-7). as discussed in the previous section, antimicrobial resistance is pervasive and is increasing in the face of widespread overuse and misuse of antibiotics. in addition, viruses rapidly adapt to immunologic responses. influenza viruses provide an example of genetic drift and shift which make them a persistent health threat. once formed, novel influenza viruses sometimes pose health risks for which humans and/or animals have little to no immunologic protection. these viruses can also develop an increased capacity for animal-to-animal or human-to-human sustained transmission. breakdown in public health systems and measures as a result of conflict, economicbased program cuts, or poor infrastructure can produce environments with increased risk and decreased prevention. this situation may result in disease emergence, but may also to read the full report, please visit the cdc hai prevalence survey (magill et al., 2014) . result in reemergence of diseases. diseases which had previously been controlled through sanitation, vector control, or vaccination programs can reemerge if any of the prevention programs fail or stop. in developed countries with successful vaccination campaigns, many diseases have been declared "controlled" or "eradicated." because of this, political and public interest in continued vaccination programs is waning, or personal risk is considered low relative to the risk (real or perceived) of vaccinations. in the united states there are also many states allowing "opt-out" programs so people may choose not to get vaccinated or have their children vaccinated. in numerous outbreaks this has proved problematic. n n n the us antivaccination movement has contributed to increased preventable illnesses and deaths. it is led by misguided conspiracy theorists, minor celebrities, and a small number of "healthcare professionals" who are either unfamiliar with or in denial of the science supporting vaccinations. the above headline appeared in january 2015 in the new york times (nagourney and goodnough, 2015) . notable quotes from this report include: • "we can expect to see many more cases of this preventable disease unless people take measures to prevent it," dr. gilberto f. chavez, the deputy director of the california center for infectious diseases, said. "i am asking unvaccinated californians to consider getting vaccinated against measles." • dr. james cherry, a specialist in pediatric infectious diseases at ucla, said the outbreak was "100 percent connected" to the anti-immunization campaign. "it would not have happened otherwise-it would not have gone anywhere," he said. "there are some pretty dumb people out there." • "the problem is that there are these pockets with low vaccination rates," said dr. jane seward, deputy director of the viral diseases division at the cdc. "if a case comes into a population where a lot of people are unvaccinated, that's where you get the outbreak and where you get the spread." n n n in ghent, belgium, a 2011 measles outbreak was associated with an anthroposophic school which promotes "complementary medicine" (braeye et al., 2013) . over 65 cases of measles were identified in a school that had low vaccination rates because of the philosophical beliefs of the parents. leaders of the school were not against vaccination. striking the balance between individual rights and beliefs, relative risk perception and acceptance, and public health priorities is always challenging. while the next emerging disease can be of any species of infectious organism, trends in recent cases display patterns suggesting which threats may be imminent (see table 10 -2). coronaviruses such as the causative agents of sars and middle east respiratory syndrome co-v (mers co-v); filoviruses such as ebola; and novel influenza viruses, are considered likely candidates. in a study by taylor et al., 2001 an analysis of emerging species revealed that of those known, there are 175 currently emerging species. viruses and prions are less than half of the total (44%), and bacteria or rickettsia comprise just under one-third. these potential pathogens may be transmitted by several routes, and the most common route is direct contact (53%), followed by indirect contact (47%), vectors (28%), and for 6% the route of transmission is unknown (taylor et al., 2001) . while it may not be possible to predict which pathogens may emerge or reemerge, it is possible to build infrastructure and take general steps to make populations and public health systems better prepared for the next novel infectious disease outbreak. at the heart of these measures is epidemiological surveillance. identification of a new illness or disease requires surveillance systems which not only continually monitor "routine" illness, but also have the ability to recognize anomalies when something is not "routine." as described in chapter "bioterrorism," there have been extensive efforts toward developing early warning epidemiological and environmental surveillance systems for unusual diseases and pathogens. the vital link in identifying a novel disease in a community is the astute clinician. clinicians must be able to recognize a novel disease, report it appropriately, and feel confident that the information will be quickly analyzed and acted upon. laboratory support is vital to quickly identifying and characterizing an emerging threat. this includes testing for drug resistance to inform decision-making concerning appropriate medical countermeasures. continued epidemiological surveillance throughout an outbreak can produce data which may be useful in evaluating and improving the public health and medical response. n n n • surveillance • robust outbreak investigation practices • transmission prevention through containment and control measures • delivery of medical countermeasures, if any • public messaging • recovery to a "new normal" n n n along with surveillance is the ability of public health responders to perform detailed outbreak investigations to determine the characteristics of the disease. characterization of the outbreak, identifying the natural course of the illness, and recognizing key risks for infection are necessary in order to learn how the new illness behaves in a population (see fig. 10-8) . this information will also inform control measure decisions. a priority item in the initial characterization of an outbreak is the identification of transmissibility. unfortunately, as surveillance methods are enhanced and cases are increasingly recognized and reported, transmissibility may be a very difficult thing to quantify. for example, the current analyses of transmissibility for mers co-v suggest that it is not a likely pandemic threat because of its low reproduction number or "r nought" (r 0 ). however, different cluster sizes, demographics, geographies, and public health and healthcare infrastructures may alter the outbreak characteristics and the reproduction number (r 0 ) (fisman et al., 2014) . capturing critical data will allow for accurate characterization within the context of the outbreak and will direct response activities such as control measures and medical surge management. the primary control strategy in all infectious disease outbreaks is preventing transmission. even minimal biosecurity actions can prevent animal-to-human transmission. separation of potential source animals, such as exotic imported animals and poultry, from routines such as cooking can reduce risk. effective cleaning of animal waste and habitats should be performed with at least basic respiratory protection. to prevent human-to-human transmission, good hygiene practices such as hand washing, general surface cleaning, and careful waste disposal should be reinforced. during the 2009-2010 h1n1 pandemic, people were advised to "cough hygienically" into their sleeve to prevent contamination of the hands and then others. of note, this technique was not without some controversy and was not recommended in the united kingdom and spain (anderson, 2010) . supplying hand wipes and hand sanitizer may be implemented in some locations of businesses, critical industries, and government agencies where other measures may not be reasonable. social distancing by school closures, limiting mass gatherings, and canceling large events may be necessary (see figs. 10-9 and 10-10). quarantine may be considered for individuals who have been exposed but are not yet sick, typically for two incubation periods of the disease. isolation separates those who are already ill from those who are not ill. in healthcare settings, isolation may begin from early screening of patients at points of entry. this may limit disease transmission to other patients who often have underlying conditions which increase their risks for severe illnesses and complications. risk management for communicable diseases in emergency departments includes signage to encourage patients to self-identify if they are sick or have an associated travel history. this allows for rapid triage to isolation areas and early institution of personal protective equipment for staff (puro et al., 2008) . in a 2012 study conducted of 41 facilities in 14 european countries, 83% had isolation rooms, but not all had anterooms, negative pressure, or hepa filtration. only 14.6% had all components. personnel trained in the recognition of highly infectious diseases were available in 24 of the 41, and management protocols were available in 35 of the 41 (fusco et al., 2012) . it would be interesting to see how attention paid to diseases such as ebola has impacted these numbers. in addition to human-to-human spread, animal-to-human spread is a common transmission route for eids. zoonotic disease professionals can develop recommendations for vector control when zoonotic diseases threaten human populations. mosquitoes are the most pervasive disease-carrying vectors. control measures include reducing standing water breeding sources or using larvicides and adulticides. the recent introduction of engineered aedes aegypti mosquitoes (ox513a), which can reproduce and produce offspring which die rapidly, shows promise in areas with emerging dengue fever (specter, 2012) . once control measures are determined, public health agencies must have the legal and social authority to implement recommended measures. the majority of the population typically complies with basic containment measures such as improved hygiene, avoiding ill people, etc. however, some individuals may be reluctant or unable to comply. for example, minimally ill people without paid sick leave may not be able to stay at home without significant economic loss. people may, out of fear, choose to flee quarantine zones. in these cases, agencies need to have flexible, scalable authorities to limit travel outside areas known to have disease. the challenges with quarantine measures were apparent throughout the international response to the 2014-2015 west african ebola epidemic. in the united states as in many countries, the government has the authority to restrict stateto-state and international travel. state and local health agencies have authority to control intrastate activities. however, political pressure may make it difficult to enact some control measures. school closure is one of the more difficult decisions for government agencies during a public health emergency. for many communicable diseases, children can act as superspreaders because of their behaviors and hygiene lapses. however, many schools function not only as places of education but also deliver significant social programs: safe havens, meals, and day care for working parents with marginal income. to close these schools disrupts very necessary social welfare programs. quarantine laws must also respect civil liberties. in the united states for example, quarantine orders must be the least restrictive form of control, have very clear reasons, be timelimited and allow for appeal. enforcement of quarantine orders requires law enforcement officers to interact, sometimes in close physical proximity, with potentially contagious individuals. additionally, any enforcement of quarantine of an individual must be heard in the judicial system. the judiciary has its own constitutional constraints of process which are frequently in opposition to the goals of quarantine. if the eid is susceptible to antibiotics or a vaccine is quickly developed, rapid distribution and dispensing of these medical countermeasures must be conducted. if the illness is highly contagious, programs limiting public interaction during mass dispensing and mass vaccinations will need to be implemented. as discussed throughout this text, it is unlikely that there will be large quantities of medical countermeasures available early on in an emerging or reemerging infectious disease outbreak. therefore, plans must also be in place for the utilization of scarce resources while complying with ethical and legal frameworks. throughout the entire outbreak, appropriate public messaging must be delivered. messages will contain information about the illness, personal protective actions, and where to go for health care. these messages must communicate risk and how individuals can limit morbidity and mortality. general prescripted public health messages can often be adapted early in an outbreak when detailed information on the threat is not yet specific. trusted leaders in the community should be used to deliver messages in order to improve compliance and alleviate fear. a "new normal" may follow as a community recovers from a novel outbreak. some eids become endemic diseases in affected communities and must be included in recurring public health activities. for example, west nile virus was first introduced to the western hemisphere in the late 1990s and is now an annual threat, causing 41,762 cases from 1999 to 2014 and 1765 deaths (cdc, 2014) . however, public health responses help to establish new practices in order to limit reemergence. these activities also prepare the community for similar eid threats. because of ongoing west nile virus threats in north america, the region is better prepared for emerging threats such as chikungunya and dengue. public health has been challenged with eids throughout human history. with globalization of populations, commerce, and travel comes globalization of infectious diseases. this exposes people and animals to novel diseases for which they have little to no natural immunity. rapid transportation and free movement allows sick individuals to spread illness and disease faster than ever before. preparedness efforts cannot predict the next emerging infection, but public health and healthcare capabilities developed, lessons learned from prior outbreaks, and institutionalization of routine infection control practices may serve to lessen the impact. european centre for disease prevention and control swine flu guidelines: 'cough hygienically' into your sleeve? human-bat interactions in rural west africa obstacles in measles elimination: an in-depth description of a measles outbreak in ghent the world health organization and the transition from international to global public health outbreak of ebola hemorrhagic fever--uganda update: multistate outbreak of monkeypox -illinois prevalence of igg antibody to sars-associated coronavirus in animal traders -guangdong province, china west nile virus, final cumulative maps & data for ebola outbreak in west africa -case counts avian influenza viruses in korean live poultry markets and their pathogenic potential risk factors for human infection with avian influenza a h5n1 bats host major mammalian paramyxoviruses nuanced risk assessment for emerging infectious diseases serological evidence of influenza a viruses in frugivorous bats from africa the ecology of emerging infectious diseases in migratory birds: an assessment of the role of climate change and priorities for future research infection control management of patients with suspected highly infectious diseases in emergency departments: data from a survey in 41 facilities in 14 european countries the coming plague: newly emerging diseases in a world out of balance the 2009 h1n1 influenza outbreak in its historical context isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome in guangdong province of china: epidemiology and control measures an epidemiological study on the index cases of severe acute respiratory syndrome occurred in different cities in guangdong province west nile virus -where did it come from and where might it go? can the scale politics of emerging diseases. the history of science society transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy trends in nosocomial infections in europe: comparison between western and central europe two regimes of global health emerging infections: microbial threats to health in the united states. institute of medicine human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo monitoring eu emerging infectious disease risk due to climate change multistate point-prevalence survey of health careassociated infections bats, emerging virus infections and the rabies paradigm measles cases linked to disneyland rise, and debate over vaccinations intensifies tracking the roots of a killer possible role of an animal vector in the sars outbreak at amoy gardens climate, environmental and socio-economic change: weighing up the balance in vector-borne disease transmission risk management of febrile respiratory illness in emergency departments west nile virus in the united states-a historical perspective fungal infections associated with contaminated methylprednisolone injections the mosquito solution risk factors for human disease emergence. philos is exposure to sick or dead poultry associated with flulike illness? a population-based study from a rural area in vietnam with outbreaks of highly pathogenic avian influenza human illness from avian influenza h7n3, british columbia world tourism organization. unwto tourism highlights the prevalence of nosocomial infection in intensive care units in europe: results of the european prevalence of infection in intensive care (epic) study ebola response team 2014. ebola virus disease in west africathe first 9 months of the epidemic and forward projections west nile virus fact sheet key: cord-257494-242k58ll authors: bastos, paulo; trindade, fábio; da costa, joão; ferreira, rita; vitorino, rui title: human antimicrobial peptides in bodily fluids: current knowledge and therapeutic perspectives in the postantibiotic era date: 2017-01-17 journal: med res rev doi: 10.1002/med.21435 sha: doc_id: 257494 cord_uid: 242k58ll antimicrobial peptides (amps) are an integral part of the innate immune defense mechanism of many organisms. due to the alarming increase of resistance to antimicrobial therapeutics, a growing interest in alternative antimicrobial agents has led to the exploitation of amps, both synthetic and isolated from natural sources. thus, many peptide‐based drugs have been the focus of increasing attention by many researchers not only in identifying novel amps, but in defining mechanisms of antimicrobial peptide activity as well. herein, we review the available strategies for the identification of amps in human body fluids and their mechanism(s) of action. in addition, an overview of the distribution of amps across different human body fluids is provided, as well as its relation with microorganisms and infectious conditions. human antimicrobial peptides (amps) represent approximately 10% of all curated amps catalogued to date. 1 human host defense peptides are an intrinsic part of the innate immune system and exhibit a broad activity spectrum against bacteria, fungi, viruses, and parasites while amps can be antibacterial (abps), antifungal, antiprotist, antiviral, anticancer, antiparasitic, insecticidal, spermicidal, chemotactic, antioxidant, protease inhibitors, or even exhibit wound healing properties (supporting information table s1), their scope of action overlaps considerably and some peptides show activity at several levels (fig. 2 ). [1] [2] [3] [4] [5] for instance, on the one hand, human plasma adrenomedullin-derived peptides are active against multiple bacteria species, human urinary and gingival fluid calcitonin gene-related peptide displays antimicrobial activity against several bacteria and fungi species, and human neutrophil peptides (hnps)/defensins isolated from most biological fluids display activity against several bacterial, fungal, and viral species (fig. 2) . on the other hand, dozens of human amps known to date display antimicrobial activity against the same human colonizers or pathogens, including escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus (figs. 2 and 3) . also, it is intriguing to observe that even though some amps display broad activity spectra, others seem to be anywhere from species-to kingdom-specific (fig. 2) . while likely to be confounded global network depicting the distribution of human antimicrobial peptides across biological fluids and their microbial targets. rectangular nodes correspond to biofluids, circular nodes to target species, and each edge correspond to a given gene encoding one or more antimicrobial peptides. the thicker the edge, the stronger is the association of a biofluid to a pathogen, representing increased number of antimicrobial peptides defending against such pathogen in such biofluid. also, pathogens represented by bigger nodes (e.g., escherichia coli, candida albicans, staphylococcus aureus) represent those that are targeted by more antimicrobial peptides across different biofluids. by observational bias (whereby some species, e.g., e. coli, tend to be tested more frequently than others), this observation also reflects that the activity spectra of amps depend not only on their physicochemical properties (which group them according to unique families) but also on target properties and on the environment/biological fluid in which they are found. as the large majority of amps identified to date are abps (ß83%, figs. 1-3) , most concepts and examples provided in this review will be based on results from studies focusing on abps, closely followed by those derived from studies on antiviral and antifungal peptides. however, the same principles tend to apply across all amps. amps are very potent cationic molecules displaying minimum inhibitory concentrations (the minimum concentration that prevents bacterial growth) as low as 1-4 μg/ml, 6 which highlights their promise as broad-spectrum antimicrobial agents. furthermore, amps act more rapidly than conventional antibiotics and are not affected by the typical resistance mechanisms involving conventional drugs, making them very attractive for therapeutic purposes. in addition, amps are not mere microbicide agents, as their scope of action may encompass other functions, such as cellular development and immune system modulation. for instance, fetal keratinocytes express significantly more amps compared to neonatal and adult keratinocytes, despite the lower degree of exposition to the environment or to pathogenic agents. these a rough estimate of all amps catalogued to date (supporting information table s2 ) indicates 14.1% to be α-helices, followed by β-sheets (3.7%), and peptides presenting with both helices and β-sheets (3.7%). however, approximately 60% of all amps currently identified do not have a known 3d structure, while only 0.3% have a known structure that is neither a helix nor a β-sheet. excluding those that have unknown structures, 99 .2% of all amps are either α-helices and/or β-sheets, suggesting a highly conserved structure for a highly primordial biological function. secondary structures of human amps may include α-helices (e.g., ll-37), 34 β-sheets (e.g., defensin 1), 35 and extended peptides (e.g., indolicidin). 36 considering that β-sheets and α-helices in polypeptide chains typically contain three to ten amino acids per strand and 3.6 residues per turn, respectively, and that amino acid sequences can be virtually unlimited, one can easily envision that a given peptide can display a multitude of secondary structures and, thus, many diverse conformations (see 37 for protein secondary structures predictive approaches). among secondary structures, α-helices (in addition to being the most frequently encountered in nature) are the most regular and predictable, and, consequently, the most extensively studied. amps with linear α-helices are notably positively charged and amphipathic, but only adopt such secondary conformation upon binding to bacterial membranes. 38 therefore, their antimicrobial activity is directly dependent on the secondary structure. 39 in contrast, β-sheet amps possess well-defined conformations due to the presence of disulfide bridges formed between thiol groups in cysteines. in these peptides, such bridges are often required for antimicrobial activity. 40 contrariwise, bovine lactoferricin was shown to be effective against e. coli 0111 even when such bridges were blocked by pyridylethylation. 41 other human amps, such as human β-defensin 3 (hbd-3), present both α-helices and β-sheets. in the specific case of hbd-3, disrupting the three stabilizing disulfide bonds does not compromise its antimicrobial activity, but it does diminish its chemotactic properties. 42 furthermore, the correlation of the conformation with peptides' antimicrobial activity is not always as straightforward as one could imagine. for example, disruption of hbd-1's disulfide bonds results in peptides with free cysteines at the carboxy terminal that show increased antimicrobial activity against the pathogenic candida albicans and the gram-positive commensals bifidobacterium and lactobacillus. 43 moreover, linear extended peptides do not keep a fixed conformation in solution, and are enriched in one or more amino acids, particularly arginine, proline, and tryptophan. for instance, the amp histatin found in human saliva is enriched in histidine residues, 44 while prophenin is very rich in proline and phenylalanine residues. 45 when it comes to loop rich peptides, the presence of many prolines makes an amp much less likely to form amphipathic structures, adopting what is frequently referred to as polyproline helical type-ii (ppii) structures. these structures are specifically bound by sh3 domains, which are usually found in proteins that interact with other proteins and mediate assembly of specific protein complexes (upon proline-rich peptides binding). 46 human antibacterial peptides (abps), which compose the largest group of human amps, exhibit a weighted average net charge of +5.8 per peptide, which is considerably above the average net charge of all amps identified to date. also, despite being intuitive that the more positively charged an amp is, the more potency it will present, 47 human amps do not show greater potency or efficacy when compared to those of other species, suggesting that the environment and the presence of different amino acids (d and rare amino acids) also play a significant role. notwithstanding the large diversity of amps across human biologic fluids (figs. 2 and 3, table i ), it should be noted that amps are much more diverse across all species, than those identified in humans, which present rather conserved properties. in fact, some amps from other species are characterized by more intriguing sequences, structures, and physicochemical growth-regulated alpha protein properties. case in point, bacillus subtilis is known to produce several extremely potent lipopeptides (active at concentrations as low as 0.01-0.06 μm) with antimicrobial activity against several species. one of these, gageotetrin a is a very unique anionic amp that consists of only leucine and glutamic acid residues conjugated with a unique fatty acid, 3-hydroxy-11-methyltridecanoic acid. 48 another example is that of sonorensin from bacillus sonorensis, a broad-spectrum amp from the heterocycloanthracin subfamily, active against both gram-negative and gram-positive bacteria, including listeria monocytogenes and s. aureus. sonorensin displays an amino acid sequence with multiple copies of the same motif, making this peptide rather unique. 49 baceridin is a circular peptide from a plant-associated bacillus that is synthesized by ribosome-independent machinery and bears only six amino acids, 50% in the d configuration. 50 moreover, baceridin is a proteasome inhibitor, compromising cell cycle progression and inducing apoptosis in tumor cells by a p53-independent pathway. 50 copsin from coprinopsis cinerea is bactericidal against enterococcus faecium and l. monocytogenes by interacting with the peptidoglycan precursor lipid ii and interfering with the cell wall biosynthesis. 51 curiously, it is modified with a pyroglutamate, which confers a higher degree of thermal stability and resistance toward protease digestion. 51 even though some display fungicidal and virucidal properties, the chief activity of human amps is against gram-positive and gram-negative bacteria (figs. 1-3). furthermore, while amps represent an extremely diverse group of biological active molecules, most share common properties that contribute for their membranolytic or otherwise antimicrobial activity: positive charge, hydrophobicity, and amphiphilicity. therefore, the mechanism of action of most amps involves membrane disruption (fig. 4) , a process that is also required when their translocation is warranted and the respective targets are localized intracellularly. 39 the interaction of amps with bacterial membranes depends on the establishment of attractive electrostatic forces between these. bacterial species are known to carry negatively charged outer membranes due to phosphate groups of the lipopolysaccharides in gram-negative bacteria and to the lipoteichoic acids in gram-positive bacteria. 52 the fact that all gramnegative and gram-positive bacteria display these type of negatively charged lipids accounts for the lack of specificity of most abps, and promotes the attraction between amps and bacterial membranes while preventing their binding to most host cells membranes. moreover, the inherent hydrophobicity and amphiphilicity allow abps to form clusters at the membrane's surface once attached. 53, 54 at low concentrations (low peptide-to-lipid ratio), abps tend to adsorb onto the surface, adopting an orientation parallel to the membrane bilayer. however, clustering increases the peptide-to-lipid ratio, which subsequently promotes additional clustering. once a certain peptide-to-lipid ratio threshold is reached, abps adopt a perpendicular orientation relative to the bacterial membrane, which allows them to insert themselves into the membrane, 55 as will be further discussed (fig. 4) . peptides can adopt many different secondary structures and conformations, which depend on their amino acids sequence, as well as on the environment. consequently, different peptides and different environments favor the formation of unique structures, such as barrel staves, carpets, and toroidal pores. 40, 56, 57 when the peptides attach and are inserted into the membrane bilayer so that the hydrophobic regions align with the phospholipids' acyl groups and the hydrophilic regions create the central region of a pore, a barrel stave is formed. in addition, these structures act as a primer for the aggregation of new peptides, thus expanding the pore's diameter. 58 due to abps' cationic sites, membrane crossover would be very energetically unfavorable in their original parallel orientation. however, this orientation changes to a (1) cluster at the cell surface and cause membrane disruption by several different mechanisms (e.g., barrel staves, carpets, toroidal pores), (2) translocate into cells and impair intracellular organelle machineries, (3) impair protein-protein interactions, enzymatic cascades, and cytosolic signaling pathways, (4) interact with nucleic (not in the case of bacteria) acids, trap replication forks, and compromise nucleic acids as well as protein synthesis, (5) preclude several steps of viral replication, (6) inhibit genetic material trafficking, reverse transcriptase, and viral proteases, (7) block the interaction between virus and host cells (e.g., viral envelope glycoproteins gp120 and gp41 or coreceptor cxcr4), compromising virus binding and entry, and (8) cause membrane lysis on enveloped viruses. see text for detailed description. designed using servier medical art. perpendicular one upon abps' clustering, and the peptides are reoriented with their hydrophobic portions facing the membrane acyl groups and their cationic sites facing the interior of the pores. thus, clustering and pore formation provides a means for membrane disruption and the leakage of intracellular molecules from bacterial cells. 59 in contrast, if peptides remain parallel to the membrane surface with their hydrophobic residues facing the membrane while their hydrophilic residues face the surrounding milieu, a "carpet"-like structure is achieved. with such organization, membrane's rigidity weakens progressively as new peptides are added, culminating in its dissolution and breakup. mechanistically, this mode of action is less stringent because peptides do not need to adopt a specific structure. however, the necessary concentration of abps appears be much higher than that required for the formation of barrel staves. [60] [61] [62] for toroidal pores to form, peptides need to aggregate onto the membrane's surface and insert themselves in a perpendicular fashion. 63, 64 subsequently, abps are reoriented parallel to the membrane so that polar residues interact with the polar heads of the membrane lipids, thus causing membranes to bend. this induced bending of the bilayer causes the upper and lower leaflets to meet, and allows a mixture of peptides and lipid head groups to line with the interior of the pore, forming toroidal or wormhole structures. 60, 63, 65 abps may also inhibit bacterial growth and have a bactericidal effect by promoting the clustering of anionic lipids, such as phosphatidylglycerols (pgs) and cardiolipin. for instance, for ll-37, initially believed to act according to the "carpet" model, there is evidence that its fragments, namely kr-12, act as a magnet that competes for the negatively charged pgs, promoting their redistribution into "pg-rich domains." such phospholipidic reorganization may disturb cell signaling and compromise the activity of membrane-bound proteins, namely, the voltage-dependent potassium channels, which can seriously jeopardize bacteria survival. 34 likewise, n-acylated peptides derived from lactoferricin were shown to induce defects in e. coli cell division by rearranging the distribution of inner membrane phospholipids, essentially due to the clustering of cardiolipin and other anionic lipids. 66 the bias toward positive charges confers amps the ability to easily interact not only with biological membranes, but also with other negatively charged molecules (fig. 4) such as nucleic acids and those bearing phosphate groups. 39, 67 in addition, because this type of interaction is largely unspecific, amps can exhibit not only a broad-spectrum activity, but also multiple mechanisms of action (fig. 3) . 68, 69 for instance, lactoferricin is known to inhibit atp-dependent multidrug efflux pumps as well as dna, rna, and protein synthesis. 70 ten human amps derived from lactoferricin and lysozyme are thought (by sequence homology) to act by trapping the replication fork and preventing base recombination and dna repair. 71 furthermore, the presence of many prolines in some peptides (also known as ppii structures as aforementioned) hampers the organization of amphipathic structures. 72 these structures favor interactions between peptidic structures and between these and nucleic acids, thus playing a major role in signal transduction and complexes assembly. 73 however, ppii structures do not necessarily contain prolines, 74 and abps rich in arginine residues are also able to translocate across membranes and to interact with nucleic acids and proteins. hence, both proline and/or arginine-rich abps may be capable of inhibiting prosynthetic signaling pathways and enzymatic activity (fig. 4) , shifting the conformation of cellular structures and activating autolysins. however, this is a speculation that warrants further studying. in addition to targeting bacterial membranes, human defensins also bind to the peptidoglycan precursor lipid ii and inhibit cell wall biosynthetic enzymes in staphylococci species. 75 moreover, when stabilizing disulfide bridges between conserved cysteine residues in human amps with β-hairpin or β-sheet conformations are disrupted, the resulting linear peptides still maintain their antimicrobial properties despite losing membranolytic activity. 76, 77 the antifungal histatins act at multiple levels by mechanisms of action conserved across the histatin family of amps, with histatin 5 the most potent amp in this family. this amp binds the yeast transmembrane receptor heat-shock protein ssa1/2p, is internalized, and then targets primarily yeast mitochondria. 78, 79 by doing so, it leads to the formation of reactive oxygen species (ros), causes atp efflux, and inhibits oxidative phosphorylation. 80, 81 simultaneously, histatin 5 also interacts with the potassium transporter trk1p, causing release of k + ions and further atp efflux. 82 when the functional domain of histatin 5 was synthetically multiplied, its antifungal activities were significantly potentiated and it became significantly faster-acting compared to the normal histatin 5. 82 such an observation might prove itself very useful, because it indicates that modulating the number of functional domains per amp may enhance its antimicrobial activity without requiring the administration of increased quantities of amp. histatins may also exert their antimicrobial activities by inhibiting host and microorganismal proteolytic enzymes. histatin 5 is a strong inhibitor of human matrix metalloproteases 2/9 and metalloproteases from porphyromonas gingivalis. 83 thus, its inhibitory activity over proteolytic enzymes may attenuate tissue damage and microbial propagation during the onset of periodontal inflammatory disease. also, by inhibiting the cysteine protease clostripain from clostridium histolyticum, histatin 5 mitigates the virulence factors produced by this clostridium species and may therefore alleviate the complications associated with gas gangrene syndrome. 84 so far, the above-mentioned mechanisms of action have been discussed in the context of abps or antifungal peptides, and sometimes the same peptide is active against both bacteria and fungi. in turn, the action of antiviral peptides can diverge from these mechanisms and should thus be independently commented (fig. 4) , although some overlap does exist. extracellularly, most native antiviral peptides often present as α-helical structures, have primarily lytic activity on enveloped viruses, and can directly inactivate cell-free virions (fig. 4) . human lysozyme fragments are this group's prototype, capable of aggregating on the viral surface and causing disruption of membrane envelopes. 85, 86 similarly, cap37 peptide appears to exert its antiviral activity by destructing the virus envelope of type i herpes simplex virus (hsv) or even by disrupting the capsids of adenoviruses. 87 however, because membrane lipids of enveloped viruses derive from host cell membranes, special attention is required when considering the exploitation of this feature, as antiviral peptides may disrupt host cell membranes indiscriminately. alternatively, antiviral peptides may block the interaction between the virus and the host cell (fig. 4) . that is believed to be, at least in part, the mechanism of action of human defensins, θ -defensin, human α1-antitrypsin virip, cyclic lactoferricin, and serine protease inhibitor elafin. for instance, it has been demonstrated that all α-defensins [hnp-1, hnp-2, hnp-3, and hnp-4 and human α-defensin (hd)-5 and hd-6] and hbd-3 prevented hsv infection by blocking virus binding and entry. such antiviral activity was attributed to hnp-1-3, hd-5, and hbd-3's high affinity to hsv's glycoprotein b and hnp-4, hd-6, and hbd-3's avidity toward endogenous heparan sulfate. 88 similarly, protection against hiv is conferred by peptides with lectin properties that can bind viral envelope glycoproteins gp120 and gp41, crucial for viral attachment, fusion of envelopes with host membranes, and subsequent entry into host cells. 89 hnp-4 that binds and blocks the action of gp120, and θ -defensins (e.g., the pseudogene retrocyclin) together with the human α1-antitrypsin-derived 20-residue viral-inhibitory peptide both targeting gp41 represent examples of such peptides. [90] [91] [92] likewise, hsv-2 infection can also be blocked by retrocyclin, which binds tightly to glycoprotein b2. 93 the cyclic peptide lactoferricin seems to compete for the ligation to extracellular heparan sulfate or chondroitin sulfate, thus preventing the very first event in cytomegalovirus, hsv, and papillomavirus infection. [94] [95] [96] finally, elafin is thought to act indirectly in host cells by hampering viral attachment and probably by modulating host's antiviral and inflammatory responses toward hsv-2 infection. 97 synergistically with this mechanism, antiviral peptides can also bind and inhibit the activation of viral co-receptors, leading to further inhibition of glycoproteins-mediated viral attachment. not only that, hbd-2 and -3 inhibit viral infection by promoting the internalization of its co-receptor cxcr4. 98, 99 virucidal peptides may also preclude several steps of viral replication by interacting with intracellular targets (fig. 4) . this is the case of hnp-1 and hd-5, which may prevent hsv replication by binding directly to its dna. 88 also, hnp-1 arrests influenza virus replication by inhibiting protein kinase c phosphorylation, an essential step for nuclear trafficking events. 100 human cathelicidin (ll-37) derived peptides, in turn, inhibit reverse transcriptase and viral proteases, thus preventing integration of the viral genetic load into human dna. 101, 102 the few resistance mechanisms against amps demonstrated so far among microorganisms have been observed almost exclusively in bacteria. with very few exceptions, bacteria do not seem to develop complete resistance against cationic abps, though some strategies do exist among bacterial species for diminishing their potency and efficacy. the difficulty in acquiring resistance may stem mainly from the fact that abps target an essential cellular component for bacteria survival, which cannot be easily modulated by the cell without causing significant adverse effects. therefore, resistance mechanisms are not likely to be a limitation in the therapeutic utilization of abps. in light of this scarce resistance mechanisms de novo acquisition, what mechanisms do bacteria naturally develop/possess against abps? first, one should consider that the primary target of abps consists of bacterial cell membranes and bear in mind how abps depend on the electrostatic interactions between membrane negative charges and positive peptidic residues. accordingly, bacteria have learned how to modulate cell membrane charges and composition. gram-positive bacteria can partially modify the peptidoglycan's teichoic acid and gram-negative bacteria can partly change its lipopolysaccharide lipid a moiety, thus compromising this interaction. for instance, d-alanine-activating enzyme and d-alanine transfer protein are known to transport positively charged d-alanines to the surface anionic teichoic acids in gram-positive s. aureus, reducing the negative net charge of its outer membrane. 103 also in s. aureus, the five-component system graxsr-vrafg increases the expression of mprf and dltabcd genes upon exposure to cationic amps. when s. aureus strains are mutated so that the regulation of these genes is disturbed, these strains become more susceptible to daptomycin, polymyxin b, hnp, and platelet factor 4-derived peptide, and less infectious in vivo in a nonclinical endocarditis model. [104] [105] [106] the gene product mprf attaches lysine residues, which bear a positively charged ε-amino group, to the anionic phophatidylglycerols, making these less negatively charged and thus less susceptible to interactions with cationic amps. 105, 106 the gram-negative vibrio cholerae can increase its resistance to polymyxins more than 100-fold by modifying its lipopolysaccharides in a process mediated by almf. 107 the lpta gene product found in gonococcal species, such as the gram-negative bacteria neisseria gonorrhoeae, protects lipopolysaccharide lipid a moieties by attaching phosphoethanolamine, making these bacteria less susceptible to amps and increasing their capacity to modulate host's immune response by evading complement-mediated cellular death. 108, 109 moreover, in n. gonorrhoeae and neisseria meningitides, the presence of phosphoryl and phosphoethanolamine in lipid a moieties enhances the activation of the nf-κb pathway and the production of proinflammatory cytokines in human cells, which can amplify their virulence. 110 despite this modification-dependent virulence, the absence of such modifications in commensal neisseriae species allow these to colonize and coexist with the human host without inducing bactericidal host immune responses. 111 in addition, the adaptation of the composition and, hence, the electrostatic properties of the membrane in order to circumvent the host immune system is not an exclusive phenomenon of pathogenic bacteria. commensal bacteria of the intestinal tract can remove phosphate groups from their membrane's lipopolysaccharides, decreasing their negative charge and allowing greater survival and proliferation in the gut. 112 similarly to the above-mentioned paradoxical modification-dependent virulence, mutated commensal gut microbiota bacteroides that fail to remove phosphate groups from their lipopolysaccharides are killed during inflammation while normal strains keep their resilience. 112 therefore, there appears to be an inverse relationship between acquired resistance and bacterial virulence, so that induced/acquired modifications render cells less virulent while simultaneously allowing these to more easily colonize and coexist within the human host environment. second, bacteria are also able to adapt the fluidity of the membrane by increasing the hydrophobic interactions within their outer membrane, thus hindering pore formation. 113 third, bacteria can express proteases against abps or transporters and efflux pumps like atp-binding cassette (abc) transporters to circumvent such innate defense barriers. 114, 115 glu-c protease produced by s. aureus induces the loss of antibacterial activity of neuroendocrine peptides, and chromofungin, procatestatin, and human catestatin are enzymatically degraded when treated with bacterial supernatants. 116 in e. faecalis, bacitracin binds the abc transporter ef2050-2049, which interacts with the regulatory domain of the two-component system ef0926-27, increasing its expression and conferring resistance against bacitracin. 117 two-component systems regulate the expression of abc transporters and are constituted by a transport permease and a sensor kinase. however, at least in b. subtilis, the abc transporter itself is required for both sensing of and resistance to bacitracin, suggesting these transporters to also function as environmental sensors. 115 lastly, bacteria may also synthesize molecules capable of directly neutralizing abps, 118 which can provide a way for bacteria to bypass amps. however, such resistance mechanisms are very limited and resistance to amps does not take place to a large extent. r bastos et al. in humans, endogenous amps have been found in several fluids throughout the body, as depicted in figures 2 and 3 as well as in table i . as noted, multiple amps have been found in tears, saliva, milk, blood, urine, sweat (and others not-so-well-explored biofluids), reflecting not only a remarkable versatility but also a huge conservation that reinforces its essential role. the robust knowledge of amps on some fluids (e.g., tears, urine, saliva) over others (e.g., amniotic fluid, cerebrospinal fluid) most likely reflects their availability, but different exposure to pathogens from the environment (figs. 2 and 3) should also account for these discrepancies to a large extent. additionally, saliva and urine screening is of special interest, since both the oral cavity and the urinary tract represent the main doorways for microbial invasion and colonization, and are thus enriched with host defense peptides. likewise, milk screening is of high relevance due to the presence of particular proteins and amps that will boost neonate's immunity. despite underrepresented, amps from other fluids are equally relevant (figs. 2 and 3). for instance, amps collected from airway secretions, bronchoalveloar lavages, and nasopharyngeal aspirates can be valuable sources of information regarding the physiological response to pathogens of the respiratory tract and may, themselves, constitute raw models for new therapeutic drugs. also, other than circulating immune cells derived, blood has not been regarded as a rich source of amps, but this view can be questioned (figs. 2 and 3), most likely due to tissue-derived amps' passage to the blood circulation. however, it should be noted that human fluid reservoirs are not independent/isolated, and, consequently, peptides may be present throughout and cross between, possibly undergoing modifications between such reservoirs. amps may be present in fluids as a result of many different processes, including exocytosis, in situ proteolysis, tissue necrosis, and cellular lysis. therefore, in this section, we will characterize naturally occurring amps based on structural and functional similarities rather than their source of collection. defensins are cationic peptides rich in cysteine residues, presenting as β-sheet structures stabilized by disulfide bridges. 4 according to the alignment of these stabilizing disulfide bounds, mammalian defensins are classified into three subclasses, α-defensins, β-defensins, and θdefensins. disulfide bounds of α-defensins occur between cysteines 2-6, 1-4, and 3-5. these peptides consist of 29-35 amino acids and adopt a triple-stranded β-sheet conformation. human neutrophils express four distinct α-defensins, known as hnp-1 to hnp-4. despite having been initially isolated from peripheral blood leukocytes, hnp-1 to hnp-3 can also be found in bone marrow, spleen, and thymus. 119 the remaining two already identified human α-defensins, hd-5 and hd-6, are found only in paneth cells of the small intestine and in epithelial cells, thus being tissue specific. 120 expressed in the aforementioned cell types, amps can also be found in bodily fluids in direct contact with these cells. among the most recent discoveries, hd-6 has been found to be active against bifidobacterium adolescentis and other anaerobic gut commensals, but not (directly) bacteriostatic or bactericidal against most pathogenic bacteria, and may therefore play an important role in maintaining a balance among the microbiota of the gut. 121 similarly, human hd-5 is particularly potent against the gram-positive bacillus clostridium difficile, one of the most common pathogens responsible for nosocomial infections, whose highly virulent strains often attack small intestine mucosa. 122 in addition, α-defensins may show both cellular and regional specificity throughout the gastrointestinal tract, either secreted or active inside intracellular granules and constitutively expressed or amenable to induction. 15, 123, 124 it should also be emphasized that the activity spectrum of α-defensins is not limited to bacteria and fungi, exhibiting activity against several virus, including hiv, hsv, adenovirus, and human papillomavirus (hpv), and displaying several unique mechanisms of action (please refer to section 4). 4 it should be noted that the antimicrobial activity of α-defensins is influenced even by the difference in one amino acid, such as for hnp-1 to hnp-3. in fact, an additional acidic residue in hnp-3 lowers its effectiveness (most accurately, its potency) against s. aureus, p. aeruginosa, and e. coli. this is explained by the easily donated proton, which makes this defensin less positively charged (at least when considering interaction domains only), thus reinforcing the importance of a positive net charge for the antimicrobial activity of amps, 125, 126 as evidenced in section 4. in terms of disease, α-defensins are known to have a paramount role, particularly when the demands for defense mechanisms are increased. accordingly, a remarkable 15-to 25fold increase in the concentration of hnp-1 and hnp-2 (and hnp-3 to a smaller extent) in human tears as been reported after ocular surgery, an increase that allowed these peptides to reach the required concentrations for antimicrobial activity but that is followed by a decrease once healing has been completed and the demands for immune defenses have returned to baseline. 127 nonetheless, the goal of achieving this increased α-defensin concentration in tears is not restricted to the enhancement of the immune defenses, as it is also known to promote local cellular proliferation and tissue repair. 128 in parallel, salivary α-defensins are active against streptococcus mutans, but their secretion has been found deregulated in caries-positive human subjects. 129 in contrast, prolonged physical exercise has been shown to significantly increase the levels of salivary α-defensins, 130 showing that lifestyles may have a considerable impact on our amp-based defenses against microorganisms and it is possible to modulated the activity thereof. disulfide bounds of β-defensins occur between cysteines 1-5, 2-4, and 3-6. these defensins are longer than α-defensins, spanning from 36 to 50 amino acids, but also share a triple-stranded βsheet conformation. the first hbd was isolated from plasma samples and kidneys constitute the major source of hbd-1. 131 since then, peptides derived from human hbd-1 have been identified in tears, urine, vaginal lavage, sweat, and blood plasma, and these are effective primarily against e. coli. [131] [132] [133] [134] in turn, hbd-2 was isolated from psoriatic skin samples, 135 displaying bactericidal activity toward gram-negative e. coli, p. aeruginosa, and c. albicans, and bacteriostatic activity toward gram-positive s. aureus. despite very effective in vitro, both hbd-1 and hbd-2 present attenuated activities in vivo as a result of their sensitivity to physiological salinity. in contrast, hbd-3 is active against s. aureus and vancomycin-resistant e. faecium even at physiological salt concentrations. 136 the hbd-119 defensin has been found in seminal plasma, and it has been reported as potential efficacious against e. coli, s. aureus, and c. albicans. 137, 138 furthermore, hbd-114 has been identified in epididymal fluid and has been shown to regulate lipopolysaccharide-mediated inflammation toward e. coli, s. aureus, and c. albicans, protecting sperm from motility loss. 139 in disease conditions, increased concentrations of hbds in plasma and bronchoalveolar lavage fluid were reported in patients with diffuse panbronchiolitis. in particular, β-defensin 4a was suggested to play a role against p. aeruginosa. 140 also, while β-defensins are expressed throughout the gastrointestinal tract, these seem to have evolved together with changes in human gut bacteria, displaying characteristic expression patterns in response to certain microorganisms. their expression may be significantly altered in human diseases, most notably in inflammatory bowel diseases (for insightful reviews on this topic, please refer to 16, 141 and enhanced in inflammatory conditions of the ocular surface 134 ). with α-defensins, the role of the ph for amps' activity is considered. here, β-defensins serve to illustrate the role of salt concentration. in fact, tears contain a remarkable concentration of sodium chloride, which inhibits the activity of human amps (by ß40% in the case of hbd-2, an inhibition that increases up to 90% in the presence whole tear fluid, suggesting the presence of other inhibiting factors). 142 therefore, whenever there is an increase in the demand of amps activity, (e.g., upon infection or tissue damage), their concentrations must surge to reach the required protective levels. however, an excessive increase in the concentration of any amp may lead to tissue damage and local functions may be compromised. for this reason, a complex interplay of amps is required at surfaces like the ocular epithelia, which is achieved by a qualitative enrichment, in addition to the expected quantitative increase (for a depiction of the multitude of amps found in human tears, please refer to 143 ). similarly to α-defensins, the activity spectrum of β-defensins is not limited to bacteria and fungi, and they are active against several virus, namely, hiv, influenza a virus (iav), respiratory syncytial virus, and vaccinia virus, and display several unique mechanisms of action, depending on their target (see above section on mechanisms of action). 4 lastly, genomic studies have predicted additional β-defensins to be expressed in human cells, but their presence in human bodily fluids and their antimicrobial activity in vivo is yet to be demonstrated. 144 while αand β-defensins adopt triple-stranded β-sheet conformations, θ -defensins have shorter sequences and adopt a complete circular conformation, with stabilizing disulfide bounds between cysteines 1-6, 2-5, and 3-4. the peptide rhesus θ -defensin 1 is the prototype of this more recently discovered family of amps. like αand β-defensins, θ -defensins possess broadspectrum microbial activity against bacteria and fungi, while also being capable of protecting mononuclear cells from infection by hiv-1. 145, 146 moreover, these peptides display antiviral activity against hsv, hiv, iav, and severe acute respiratory syndrome (sars) coronavirus, acting by several mechanisms, depending on their target. 4 structurally, θ -defensin peptides are very interesting and very odd, representing the first cyclic peptide family discovered in animals, albeit not in humans. moreover, their circular structure appears to be required for antimicrobial activity. 145 these peptides were first isolated as antimicrobial octadecapeptides expressed in leukocytes of rhesus monkeys, and their formation results from the head-to-tail joining of two independent nine-amino acid peptides derived from truncated pro-α-defensins. 146 in contrast to the previously described classes of defensins, human θ -defensins are thought to be completely inactivated, despite the existence of mrnas encoding at least two θ -defensins expressed in the bone marrow. 145 while genes encoding θ -defensins in humans also encode premature stop codons (thus hampering θ -defensin expression), synthetic human θ -defensins (called retrocyclins) have been shown as promising antiviral agents. 147 in fact, retrocyclin-1 inhibits the entry of hiv, hsv, and iav into host cells. in other mammals, retrocyclin-1 also protects from infection by bacillus anthracis spores and the rhesus θ -defensin 1 protects mice from sars coronavirus infection. 147 cathelicidins are a very diverse group of cationic α-helical and amphipathic amps that exhibit a broad-spectrum activity against bacteria, fungi, and virus. 54 the term "cathelicidin" originally referred only to the entire precursor protein, though currently is commonly used for the whole family, including the resulting peptides with antimicrobial activity. in contrast to the mammalian trend in which cathelicidins and defensins are the main amps, there is only one human gene encoding multiple cathelicidin-related peptides, 144, 148 located on chromosome 3 and expressed in the airways, mouth, tongue, esophagus, epididymis, and small intestine. 149, 150 despite this tissue expression pattern, ll-37 is constitutively formed in spleen, liver, stomach, intestine, and bone marrow. ll-37 is highly positively charged (+6 charge at physiological ph 7.4), due to the high content of arginine and lysine amino acids, and adopts an α-helical structure in solutions with ionic composition similar to that of human plasma. 151 while defensins share common structural features, cathelicidin-related peptides are highly heterogeneous despite deriving from the same precursor. cathelicidins are characterized by a highly conserved n-terminal signal peptide (the so-called "cathelin domain") and a highly variable c-terminal antimicrobial domain that can be released after cleavage by proteinases. 148, 152 cathelicidin is cleaved into the antimicrobial peptide ll-37 by both kallikrein 5 and kallikrein 7 serine proteases, it is upregulated by vitamin d, and has been shown to significantly reduce the risk of death from infection dialysis patients. 153 ll-37 was shown to disrupt bacterial membranes through the formation of toroidal pores and carpet structures, 63 and to exhibit chemotactic properties, attracting leukocytes and activating secretion of chemokines. in addition, ll-37 is internalized by cells, acidified in endosomes, and activates the signaling pathway downstream to toll-like receptor 3 by interacting with double-stranded rna. 154 profiling of airway fluids collected from premature infants' tracheal aspirates during infection evidenced the production of ll-37 even at an early stage of development. 155 in these samples, ll-37 was found in significantly increased concentrations during pulmonary or systemic infections, suggesting this peptide to be as important to avoid infectious processes as to fight already established infections. 155 the concentration of ll-37 has been measured in seminal plasma samples from healthy donors and found to be up to 70-fold higher than in blood plasma. 149 furthermore, it was found to be attached to spermatozoa, eliciting a possible role in human fertilization or its precursor to be extensively cleaved by the high concentration of serine proteases present in seminal plasma. 149 ll-38, an alternative form of human cathelicidin peptides, contains one more alanine at the n-terminus than ll-37 and was initially found to be produced in female vaginal secretions due to the action of gastricsin on sperm precursor cathelicidin. 156 both ll-37 and ll-38 are active against bacteria such as e. coli, s. aureus, p. aeruginosa, and bacillus megaterium, and synergistically with peptides from seminal plasma play an essential defense role protecting the human reproductive system. 156, 157 interestingly, both citrullination and adp (adenosine diphosphate)-ribosylation of arginines can compromise the ability of ll-37 to prevent endotoxin-induced sepsis, perhaps because the attachment of these negatively charged molecules reduces the peptide's cationicity or by raising steric hindrance. 158, 159 histatins are a normal component of human saliva and part of the innate immune system, protecting against a broad array of infectious agents, especially against candida species, saccharomyces cerevisiae, cryptococcus neoformans, and neurospora crassa. [160] [161] [162] there are only two genes enconding histatins (both located on chromosome 4), and they are exclusively expressed in human salivary glands. 163 however, other active amps resulting from the cleavage of histatins 1 and 3 also exist. 164 histatins 1 and 3 are encoded by genes htn1 and htn3, respectively. 165 histatins 2, 4, and 5-12 are the products of posttranslational proteolytic cleavage of histatins 1 and 3, though histatin 5 can also result from posttranscriptional modification of histatin 3 mrna. 165, 166 peptides derived from histatin 1 bear distinct domains associated with specific functions, namely an n-terminal domain with antimicrobial activity and a c-terminal domain with wound-healing properties. 167, 168 histatin 5 has two important metal-binding motifs, consisting of an amino-terminal copper (ii)/nickel (ii)-binding motif and a zinc (ii)-binding motif, although they can also bind to cobalt (ii) ions. 169, 170 the ability to coordinate metal ions appears to be important for the stabilization of the secondary structure, particularly in the presence of negatively charged membranes. 170 nevertheless, histatins lack any defined secondary structure and are unordered when in aqueous solutions, assuming α-helical structures only in organic solutions. 171, 172 in addition, histatin 5 binding to copper (ii) and nickel (ii) ions induces the formation of ros, which contributes for its fungicidal activity. 80, 169 histatins can bind to a receptor on fungal cell membranes and induce cell death by disrupting the cell cycle and causing nonlytic loss of atp, as discussed above. 173 while histatins are effective, potent, and fast-acting amps against several antimicrobial species, these peptides are nontoxic to human cells and human microflora, which may be due to the fact that their translocation across the membrane is site and species specific. 78, 174 both parotid, submandibular, and sublingual glands secret histatins, and their concentration and secretion have been shown to decrease with age, even when accounted for total protein concentration. 175 moreover, oral candidal infections increase with age, suggesting a link between age-associated deficiency of human histatins and an increased risk of oral infections. 175 dermcidins are broad-spectrum anionic amps with no apparent homology to other known amps. peptides in this group comprise 110 amino acid residues, which are subsequently processed into smaller but still active ones. dermcidins are constitutively expressed in human eccrine sweat glands and secreted in sweat, exhibiting antimicrobial activity against common human pathogenic microorganisms such as s. aureus, staphylococcus epidermidis, e. coli, e. faecalis, and c. albicans. [176] [177] [178] this constitutive production contrasts with human defensins and cathelicidins, which are induced during inflammatory and stressful conditions. a small percentage of human breast cancer cells displays rna encoding dermcidin, and after oxidative stress induction different types of tumor cells leads to the production of diverse and biologically active proteolytically processed dermcidin peptides. 179, 180 therefore, the upregulation of dermicidins may confer human breast cancer cells a selective advantage compared to nonmalignant cells. in contrast, reduced expression of dermcidins in sweat from atopic dermatitis patients contributes to skin infections and altered skin colonization. 181 hence, the downregulation of dermicidins in atopic dermatitis patients may represents a suppression of an innate and fundamental defense mechanism, which, in turn, could confer an advantage to infectious microorganisms. unlike histatins, which are nontoxic to human cells and human microflora (see above), dermicidins can have serious deleterious consequences to the human host. dermcidin isoform 2 is known as a stress-induced oxidative protein capable of inhibiting the synthesis of nitric oxide in endothelial cells, insulin in pancreatic islet β-cells, and hepatic hormone synthesis. 182 also, hypertensive patients show significantly higher levels of plasma dermcidin, but salicylic acid has been shown to reduce dermcidin levels while simultaneously attenuating dermcidin-mediated insulin inhibition in patients with acute myocardial infarction. 183 therefore, the exploitation of dermcidin isoform 2 for antimicrobial therapeutic purposes is most likely not possible due to severe side effects. despite this, it may be a promising therapeutic target for hypertension and diabetes management. in animal models, dermcidin has been shown to induce platelet aggregation and coronary artery disease by activating platelet cyclooxygenase and inhibiting the constitutive form of nitric oxide synthase, respectively, with an efficiency 40 times higher than that of adp at activating cyclooxygenase. 182 notably, dermcidin was able to stimulate the development of coronary artery disease in one animal model, even at suboptimal concentrations of adp within 30 min. 183 together, these observations suggest that the ability of amps to fight infections depends on their induction by infectious microorganisms, the particular peptides generated, and the local environment/tissue in which they act. also, biological roles performed by human amps are more diverse than immediately expected, playing essential functions other than fighting infectious microorganism(s). in fact, dermcidin-mediated inhibition of insulin is unique in the sense that no other protein is known to arrest pancreatic insulin synthesis in such way, and similar observations might result in novel and unexpected applications of human amps. amps expressed by human liver (leap 1 and 2) were initially discovered in human blood, and subsequently found in urine. these peptides are known as hepcidins and are characterized by a high content in cysteine residues (approximately 32% of all residues, with eight disulfide bounds). 184 leap-1 or hepcidin-25 is essential for maintaining iron homeostasis, and, when mutated, leads to severe iron overload and juvenile hemochromatosis. 185 hepcidins are the main regulators of plasma iron concentration and its secretion is promoted during inflammatory responses, particularly by the action of interleukin 6, which is one of the main mediators of the acute phase response. 186 as iron bioavailability is a limiting factor for bacterial growth, hepcidins represent an organic mechanism for sequestering iron from invasive pathogens. however, when several iron sources are available, as it happens within the human host, eliminating a single reservoir may not be sufficient to attenuate microorganisms' virulence. moreover, bacteria are known to exploit almost every host iron-binding protein and reservoir and, consequently, human laeps/hepcidins are a rather limited source of antimicrobial activity. 187 for these reasons, the antimicrobial role of iron sequestration depends not only on hepcidins, but also on several other mediators, such as transferrin, lactoferrin, ceruloplasmin, haptoglobin, and hemopexin. mutations responsible for decreasing the levels of hepcidins or compromising the function of other regulators of iron homeostasis, such as the major histocompatibility complex class i like hereditary hemochromatosis protein (hfe), transferrin receptor 2, and ferroportin, lead to increased iron blood levels. when levels are high enough to produce hemochromatosis, individuals are at an increased risk of infectious complications, including bacteremia and meningitis caused by e. coli, 188 bacteremic cellulitis, and hemorrhagic bullous skin lesions caused by v. cholera, 189 multiple pyogenic abscesses in the liver parenchyma as a result of yersinia enterocolitica infection, 190 meningitis, endocarditis, and pericarditis resulting from infection with l. monocytogenes, 191 and fatal septicemia during infection with vibrio vulnificus. 192 in contrast, hemochromatosis can also be associated with decreased macrophage iron storages in the case of hfe-associated hemochromatosis. this depletion makes these cells more efficient in fighting salmonella enterica subsp. enterica, serovar typhimurium, and mycobacterium tuberculosis infections. 193, 194 however, these protective effects of macrophages iron storage depletion have only been demonstrated in animal models and appear to be mediated not by hepcidins, but rather by lipocalin-2, which reduces the availability of iron for salmonella, and transferrin and lactoferrin, both of which compromise iron acquisition by m. tuberculosis. 193, 194 neuropeptides and neuroendocrine peptides may exert mitogenic actions through which innate barriers are reinforced and may display neurotransmitter, immunoregulatory, and direct antimicrobial activity. vasostatin-1 was the first natural antifungal n-terminal chromogranin a derived fragment peptide, having been first isolated from chromaffin secretory granules from bovine adrenal medulla. 195 however, its sequence is highly conserved in humans (97% identity homology) and its microbial activity also overlaps, to some extent, with that of bovine origin. 196 irrespectively, human vasostatin-1 displays antimicrobial activities against gram-positive bacteria (micrococcus luteus, b. megaterium) and a large variety of filamentous fungi (n. crassa, aspergillus fumigatus, alternaria brassicola, nectria haematococca, fusarium culmorum, fusarium oxysporum, trichophyton mentagrophytes) and yeast cells (s. cerevisiae, c. albicans). 195 moreover, many autocrine and paracrine biological activities of chromogranin a, its precursor protein, have been attributed to peptides located along its sequence, which are co-released with catecholamines by chromaffin cells upon stimulation and can be found in human bodily fluids. 197, 198 therefore, the antimicrobial activity spectrum of vasostatin-1 may be extended by that of co-released peptides also formed by chromogranin a proteolytic cleavage, and some of the coproduced peptides are even more potent than vasostatin-1 itself. 195 in addition to the structural requirements for the antimicrobial activity of chromogranin a derived peptides, posttranslational modifications may also modulate their activity. for instance, s-pyridylethylation (an alkylating modification consisting of the addition of pyridylethyl moieties to the -sh functional groups of cysteine residues that disrupts disulfide bridges) renders it selectively active against b. megaterium but not m. luteus, while oxidation seems to render it inactive against bacteria but still active against fungi. in addition, chromogranin a derived peptides also undergo phosphorylation and glycosylation, which can modulate their physicochemical properties. 195 currently, vasostatin-1 is known to be stored in endocrine, neuroendocrine, and neuronal cells, and to be released from stimulated chromaffin and immune cells upon stress. moreover, the stimulation of polymorphonuclear immune cells induces the processing of chromogranin a and the secretion of vasostatin-1 and other chromogranin a derived peptides, 195 which may account for local inflammation. lastly, recombinant and synthetic human vasostatinderived fragments have been shown to inhibit vascular contraction induced by endothelin-1, parathyroid hormonal secretion, neuronal survival, expression of neurofilaments, and neuronal gaba uptake. 199, 200 adrenomedullin was first isolated from a human adrenal pheochromocytoma and its plasma concentration is raised under specific physiological conditions, namely, renal failure, 201 hypertension, 202 and sepsis. 203 this peptide has antibacterial activity against several grampositive and gram-negative bacteria, including propionibacterium acnes, s. aureus, m. luteus, p. gingivalis, actinomyces naeslundii, s. mutans, c. albicans, eikenella corrodens, actinobacillus actinomycetemcomitans, streptococcus pneumoniae, haemophilus influenzae, streptococcus pyogenes, bacteroides fragilis, e. coli, and helicobacter pylori. 204 human neuropeptide y displays antimicrobial activity against bacteria and fungi, but truncated fragments are tenfold more potent than the intact precursor neuropeptide y, perhaps because the net charge of the fragments is more positive than that of the intact neuropeptide. 205 encephalins and their derived peptides may either enhance or inhibit the immune response and the corresponding circulating immune cells/mediators in both humans and animal models. 206, 207 as well as their subsequent antimicrobial effects, may thus take place indirectly in immune system modulation. simultaneously, antibacterial assays have revealed that peptide b/enkelytin (a c-terminal fragment of proenkephalin a) specifically targets gram-positive bacteria, including m. luteus, b. megaterium, m. luteus, and s. aureus, while having no effect over gram-negative bacteria. 208, 209 classically, proenkephalin a has been ascribed to the secretory granules from adrenal medullary chromaffin cells and various brain regions, such as the striatum and the pituitary, according to some animal studies. 210, 211 despite this, it has also been demonstrated to be expressed in and secreted by polymorphonuclear neutrophils (accounting for its particular enrichment in wound fluids), 208 t and b lymphocytes, macrophages, and mast cells. 212, 213 however, it should be noted that the antimicrobial effects of encephalins and their derived peptides result mostly from animal studies and have not been adequately studied in human secretions, despite the high conservation of their sequences across species, which most likely contribute for the similar activity spectrum. even so, phosphorylation and glycosylation are characteristic of these peptides, 208, 214 and it is possible to envision that differences at the level of posttranslational modifications or as a result of gene divergence may result in distinct activity spectra. interestingly, peptide b/enkelytin is metabolized in vivo to opioid peptides, thus revealing an intricate relationship between innate immunity and pain modulation, and its plasma concentration is increased after coronary artery bypass grafting in humans. 215 because it is expressed in both immune and neuronal cells, it is possible that the same stimuli may act on both cells populations, thereby accounting for immune system modulation at the periphery and alterations in central nervous system signaling. however, the exact roles and mechanisms of this interplay are not clear. in addition, other amps found in epithelial secretions from the gut and the skin, for example, include peptides derived from alpha-melanocyte-stimulating hormone, which is active against s. aureus and c. albicans. 216 accordingly, evidences suggest that amps like these act by inhibiting mrna and protein synthesis rather than inducing direct membrane disruption, as is the case for most amps. 217 several cationic proteins display nonenzymatic antimicrobial activity, which is sustained by the resulting cleavage peptides after protein fragmentation. for example, lactoferrin is a glycoprotein enriched in milk and neutrophilic granules, which has the ability to sequester iron and thus prevent bacterial overgrowth. 218 when digested by pepsin, lactoferrin yields lactoferricin h (human), an amp able to neutralize bacterial toxins, regulate gene transcription, inhibit complement activation, viral infection, and tumor overgrowth, thus further extending the antimicrobial properties of its precursor. 76 human chemokines are secreted by macrophages and polymorphonuclear immune cells and, therefore, found in the blood circulation. 219 moreover, during allergic inflammatory responses to bacterial infections of the airways, the eosinophil-recruiting chemokines eotaxin-1/ccl11, eotaxin-2/ccl24, and eotaxin-3/ccl26 are generated by mast cell proteases, active against several airway pathogens, including s. pneumoniae, s. aureus, h. aeruginosa. 220 peptides resulting from the cleavage of pepsinogen a and c prosequences have also been shown to exert antimicrobial activity. although these were initially identified from the stomach of bullfrogs, synthesized human pepsinogen c prosequences with similar structural characteristics (amphipathic and helical structures) to these cleavage peptides have also exhibited antimicrobial activity. 221 calcitermin is an amp that results from the cleavage of a 15 amino acids long sequence from the c-terminal region of protein calgranulin c, isolated from human airway secretions. 222 by adopting an alpha-helical conformation in bacterial membranes, calcitermin is able to target gram-negative bacteria (e.g., e. coli and p. aeruginosa) and fungi (e.g., c. albicans). 222 furthermore, the precursor protein and fragment peptide may act synergistically to protect the human host against foreign microorganisms present in the airways. on the one hand, the cleavage peptide calcitermin inserts into membranes of microorganisms. on the other hand, the precursor protein calgranulin c has a proinflammatory activity. by acting as a dangerassociated molecular pattern molecule and binding to the receptor for advanced glycation end products in innate immune cells, it activates the map-kinase and nf-κb signaling pathways, leading to the production of proinflammatory cytokines and histamine, upregulation of cell adhesion molecules, recruitment of leukocytes, and degranulation of granulocytes. 223, 224 dermcidin-derived peptides such as dcds, ssls, and leks (the letters correspond to the first three amino acids of the corresponding precursor dermcidin protein) are also formed in eccrine sweat glands (but not in apocrine sweat glands) at these body sites with high probability for contact with pathogens (e.g., palms, face, arms) and exhibit antimicrobial activity against a large number of microorganisms, including s. aureus, e. faecalis, e. coli, s. epidermidis, pseudomonas putida, methicillin-resistant s. aureus, and rifampicin/isoniazid-resistant m. tuberculosis. [176] [177] [178] such dermcidin-derived amps are regulated by proteolytic processing at several levels (see section 2) and are differentially expressed during inflammatory conditions. 225 these peptides are resistant to proteolytic degradation in sweat up to at least 40 hr and, interestingly, most dermcidin-derived peptides are anionic in nature. if such distinct properties may lead to different antimicrobial activity spectrum is unclear, though not surprising, as skin represents a harsh and particularly unique environment. as of specific interest, it should be noted that the consensual average skin surface ph has been decreasing in the last few years and is now generally accepted to be below 5. while showering and cosmetic products decrease skin's surface ph, plain tap water can increase the skin ph up to 6 hr. 226 noticeably, negative charges seem to be important for dermcidin-derived peptides' antimicrobial activity, in contrast with what would be expected for amps. in fact, most dermcidin-derived peptides are naturally anionic, but low ph (high h + concentration) leads to an increase in their net charge (toward positivity). therefore, while an acidic skin actually keeps the resident bacterial flora attached to it, a more alkaline skin compromises such attachment, suggesting that the more negatively charged such skin peptides are (alkaline skin), the more efficient these become. 226 despite the average length of 32.76 residues and net charge of +3.21 for the close to 3000 amps already identified, human amps tend to be longer and more positively charged. amps with more than 100 amino acids have been identified across the six kingdoms of life. curiously, most (approximately half) appear to be of human origin, presenting as amino acid sequences of 127 residues on average and suggesting large amps to be a human feature. moreover, compared to all the remaining amps, large human amps tend to be biased toward leucine (8.2%) and lysine (10.5%) residues, displaying nonpolar aliphatic and positively charged side groups, respectively (table i) . therefore, large human amps are enriched with amino acids that promote distinguishing features of amps: high overall positive charge, hydrophobicity domains, and amphiphilicity. nevertheless, this bias toward lysine residues is neither unique to human amps nor more pronounced among these. for instance, the amphibian cathelicidin rc-1 is composed by 32% lysine residues, and its homologous snake crotalicidin contains 38% lysine residues, showing high potency against s. pyogenes, acinetobacter baumannii, e. faecalis, s. aureus, e. coli, klebsiella pneumoniae, and p. aeruginosa. 227 as evidenced in table i , large human amps have been found in a variety of bodily fluids, including tears, saliva, urine, airways secretions, breast milk, blood, sweat, and epididymal fluids. in addition, other large amps are also expected to exist despite not constitutively found in biologic fluids, due to their inducible nature. for instance, paneth cells in the small intestine release amps-rich granules upon stimulation by cholinergic or bacterial stimuli, 228 and granulocytes are known to contain amps in granules destined for extracellular secretion. 229, 230 therefore, as the synthesis and secretion of large amps may not be constitutive but amenable to induction by microbial macromolecules and inflammatory cytokine stimuli, several other large amps may yet to be catalogued. hence, exploration of novel large antimicrobial human peptides is an ongoing work that may yield novel therapeutic agents. peptidomics is the complete study or global analysis of the peptides present in a biological sample, at a given time, under a particular condition. 231, 232 the identification of naturally occurring amps in human biological fluids is a laborious, daunting, and sometimes unsuccessful task. such naturally occurring peptides can directly result from gene encoding, from passage to bodily fluids as a result of cellular damage and renewal, or from extracellular proteolytic processing of precursor proteins. the latter, considered to be the most prevalent, hampers the identification and interpretation of biologically active amps because precursor proteins may themselves display antimicrobial activity, but may also be cleaved into multiple functional and nonfunctional peptides. 233, 234 moreover, owing to the large dependency of amps on their sequence and 3d structure, these peptides should not be subject to mass spectrometry (ms) based approaches employing enzymatic or chemical digestion, neither to any in vitro step that may cause their fragmentation. instead, top-down proteomic strategies are preferred. classically, different peptides fractions have been separated and tested for their biological activity, often without ever addressing the peptides' biological/antimicrobial activity, their sequence, target, or hypothetical mechanism(s) of action. however, current strategies are more focused, precise, and informative, capable of addressing all these parameters, sometimes in a single study (see below). 129, 235, 236 human biological fluids consist of complex samples, containing lipids, salts, proteins, carbohydrates, and other molecules that make the study of endogenous peptides extremely difficult. consequently, enrichment and separation steps are almost always required. of these biomolecules, proteins and larger peptides as well as salts are the most complicated to separate from the target peptides. luckily, salts can sometimes be removed in the same procedures applied for protein removal. first and foremost, proteases/peptidases have to be inactivated, which can be done by denaturing procedures (e.g., microwaving, boiling) or by adding protease/peptidase inhibitors. 237, 238 however, both of these can modify the peptide structure and, thus, these steps can be avoided when samples are resistant enough to further protease/peptidase activity. this is the case for urine samples, for example, as, when collected, have long been subject to proteolytic processing in the bladder. proteins are frequently removed by selective precipitation, most commonly by organic solvent (e.g., acetone) or acidic (e.g., trichloroacetic acid) precipitation, which also removes salts (left in the supernatant). 236, 239 however, precipitation-based proteins removal is not complete and may lead to the formation of peptide aggregates, which are consequently lost in the precipitate. 240 since peptides can present a great variety of sizes, sequences, structures, hydrophobicity properties, net charges, and other characteristics, their isolation is complex. still, such complexity allows for isolation steps to take place at multiple dimensions, increasing peptides isolation efficiency and accuracy. for instance, peptides can first be isolated based on their size and charge, 241, 242 bias toward specific amino acids (e.g., cysteines, tryptophans, methionines), [243] [244] [245] or on the presence of post-translational modifications (ptms) (e.g., phosphopeptides, glycopeptides). 246, 247 still, because of the aforementioned limitations, the search for endogenous amps in human bodily fluids has mostly relied on the identification of candidate amps by chromatographic techniques (e.g., high-performance liquid chromatography, hplc) and sequencing (e.g., edman sequencing), followed by their de novo chemical synthesis and in vitro antimicrobial activity testing. for these reasons, the identification and in vivo testing of novel amps is often time consuming and requires multiple studies/phases and resources. in order to mitigate this problem, candidate human amps have come to be first predicted in multiple ways, including mining of the entire human genome for particular motifs known to confer antimicrobial activity, and screening of human peptidomes (e.g., salivary, urinary) in order to find homologous peptides to other known amps. accordingly, several libraries and databases are currently available for data comparison, and some search programs can even combine both previously obtained data and de novo information. 248 even so, the identification of amps benefits the most from combinations of chromatographic and immunological techniques, genomics and proteomics. while genomics and proteomics both allow the prediction of novel amps and have the potential for contributing with a larger number of putative amps, peptides with proven in vivo antimicrobial activity have to be more accurately identified by combinations of chromatographic and immunological techniques, chemical sequencing and antimicrobial assays. though immunoassays can detect the intended target in a variety of complex biological samples, these techniques do not provide an integrative view of the peptidome, can suffer from cross-reactivity or lack of specificity issues and require previous knowledge of the target structure. 249, 250 moreover, this issue becomes a particular problem when considering that shorter or longer peptides resulting from the same precursor protein may all contain the same epitopes and thus be recognized as the same peptide fragment despite exhibiting distinct biological activities. electrospray ionization (esi) 236 and matrix-assisted laser desorption ionization (maldi) 251 have been the most frequently and successfully applied ionization techniques in the field of bodily fluids peptidomics. in line with these, peptides are most frequently previously separated by lc-based 251 and capillary electrophoresis (ce) based approaches, allowing different peptide fractions to be independently separated. 252 from this point onwards, almost any mass analyzer can be used, although in practice quadrupole time-of-flight (q-tof) and ion traps (it) instruments are the most frequently utilized ones in peptidomics, especially in exploratory studies. [253] [254] [255] in turn, fourier transform-ion cyclotron resonance-ms is more suitable for targeted/confirmatory analysis when dealing with fewer peptides. 256 because these apparatuses allow tandem ms to be performed, peptides can be readily sequenced in addition to their identification, circumventing the above-mentioned limitations concerning immunological assays and largely simplifying peptidomics workflows. in summary, when aiming at the isolation of a particular peptide, with predicted or known charge and molecular weight, an ms-based procedure can theoretically be very straightforward. first, fluid samples require (sometimes multiple) centrifugation steps to remove cells, cellular debris, wastes, and possible contaminants. then, proteins and peptides in the supernatant are separated and fractionated by multidimensional chromatographic techniques. within these procedures, proteins and peptides can be initially fractionated by gel filtration (size dimension), followed by cation-exchange (charge dimension) fractionation. cation-exchange chromatography uses a negatively charged ion exchange resin with affinity for molecules with net positive charge. a salt gradient is used to separate the peptides of interest from other bound peptides, based on their predicted isoelectric point. finally, peptides can be subject to ms analysis. they are typically ionized by esi or maldi and subsequently analyzed in a q-tof, it, or hybrid ltq. as amps tend to be highly basic peptides due to a bias toward arginine and lysine residues, these have a propensity to be particularly enriched when applying cation exchange chromatography based techniques. 257 however, because biological fluids are complex matrices, these sometimes require multiple fractionation, isolation, and extraction steps. these are laborious optimization techniques for the detection and quantification of amps and, despite their inherent complexity, they are often based in not always accurate functional and structural predictions. a recent approach has proved itself capable for the isolation of peptides present in bodily fluids that can bind directly to certain bacterial membrane components, such as lipopolysaccharide, and induce an inflammatory response against these. 129, 235 after obtaining a bacterial homogenate and extracting membrane components with an organic solvent such as n-butanol, functionalized beads with n-hydroxysuccinimidyl-sepharose (an agarose used in affinity and protein chromatography) are conjugated with these membrane bacterial components and incubated with a human biological fluid (e.g., saliva, urine). then, peptides bound to the conjugated beads are eluted and analyzed in a ltq-orbitrap hybrid fourier transform apparatus. moreover, functionalized beads conjugated with bacterial membrane components can be tested in vivo for their capacity to induce the production of inflammatory mediators. thus, this exploratory approach could result in the identification of potential human amps with selective properties capable of binding bacterial membranes and inducing an inflammatory response. recently, dallas and colleagues have attained the most complete and prolific analysis of naturally occurring peptides with potential antimicrobial activity in human milk by nano-lc quadrupole-tof tandem mass spectrometry (ms/ms), while searching against libraries of human milk peptides and known amps. 236 in order to remove milk peptides produced by in vitro proteolysis, fresh milk derived peptides were compared to control samples of the same origin but immediately subject to boiling. as boiling denatures proteases, in vitro proteolysis does not take place and peptides present in these controls could then be confidently assigned in the original analysis. moreover, masses fragmented in the first round of ms/ms were excluded from subsequent rounds of ms. 236 the authors realized that more than 80% of the +300 unique naturally occurring peptides identified with 99% confidence have yet unknown functions, which serves to illustrate how much there is still to discover regarding the biological roles of human endogenous peptides in biological fluids. nevertheless, the antimicrobial activity of the milk peptides (41 predicted amps) was confirmed in vivo by radial diffusion assays against the gram-negative e. coli and the gram-positive s. aureus. 236 furthermore, this study reinforced the observation that the background against which mass spectra are searched seems to be a more critical factor than the mass analyzer or the ionization technique used, when it comes to both the number of peptides identified and the confidence by which these can be assigned. in the same study, authors have emphasized and demonstrated the importance of avoiding in vitro hydrolysis of amps in human bodily fluids, which can be attained by adding protease inhibitors after sample collection or by adequately accounting for this phenomenon, as the authors did by using an appropriate control. in contrast, not all bodily fluids seem to be susceptible to this degree of hydrolysis, at least not to the same extent. urine, for instance, is thought to be more stable and not to require treatment with protease inhibitors. 258 however, by the time urine is collected it has already remained stagnant for considerably longer periods of time, and, hence, probably already extensively exposed to the action of proteases/peptidases inherently present in urine. therefore, how intact urinary amps actually are should always be questionable and these should ideally be interpreted considering the background of hydrolytic enzymes present in the same urine sample. also questionable is the inhibition of proteolysis in seminal plasma samples. in fact, the liquefaction process of human semen performed by seminin and other proteolytic enzymes is physiological, 259 and, therefore, naturally occurring amps should be analyzed in samples that were allowed to liquefy without the addition of protease inhibitors. curiously, seminin, which is the primary agent responsible for liquefaction of the seminal coagulum, is partially destroyed by freezing at −20°c, 259 reinforcing the vitality of analyzing fresh samples. similarly, the degradation of peptide fragments derived from semenogelins in seminal plasma samples is time dependent and responsible for the decline in hiv-enhancing activity of sperm, once again underscoring the importance of timely performing analyses of peptides in bodily fluids. 260 as previously stated, amps activity depends largely on their 3d structures, and their correct characterization becomes of paramount importance. the best experimental techniques have been and currently are x-ray crystallography 261 and nuclear magnetic resonance, 262 both of which allow for the study of structure-function relationships, though very expensive. therefore, currently, these are commonly replaced or complemented by bioinformatics prediction tools whenever possible. using such tools, the sequence of amino acids of a peptide allows for the accurate and immediate prediction of the presence of secondary structures (e.g., αhelices, β-sheets). then, the overall 3d structure is devised by algorithms employing energy minimization principles and molecular dynamics, sometimes resulting in multiple and not mutually exclusive 3d conformations, which is in accordance with peptides dynamic structure in biological fluids. 263, 264 the most common identification software referenced in the literature for peptide/precursor protein identification are sequest and mascot. 265, 266 currently, however, there is a considerable need for algorithms and software capable of correctly identifying amps and predicting their structure-function relationship. amps are a promising replacement for conventional antibiotics owing to their effectiveness against multiresistant bacteria and over multiple bacterial species simultaneously, invoking a diverse set of mechanisms that cannot be easily shortcut by bacteria, fungi, and/or virus. an ideal amp would be (i) highly selective against its target while leaving human host cells unaffected, (ii) not prone to resistance mechanisms, (iii) relatively easy to produce at low costs, and (iv) stable during storage or upon administration. drug design of such amps focuses on peptidic and nonpeptidic mimetic drugs, modulating the hydrophobicity, positivity, and amphiphilicity of endogenous amps to increase their antimicrobial potency. however, exploiting amps for therapeutic purposes is actually more complex than initially envisioned. while linear amps present a simpler design and a more predictable structure-function relationship, circular peptides may allow the design of longer acting and more resistant amps. such peptides have been designed by inserting the intended amp into other circular peptide (cyclotides) and have been proven efficacious against hiv and inflammatory diseases by circumventing their instability and poor bioavailability. 22, 267 similarly, while humans produce amps with l-amino acids and their antimicrobial activity may depend on the action of endogenous enzymes, which can only act upon l-type amino acids, producing amps with d-amino acids may render these more resistant to hydrolysis. rather than administering exogenous amps, it is possible and perhaps more secure and controllable to pharmacologically stimulate the production of endogenous amps. for instance, 1,25-dihydroxycalcypherol (active vitamin d3) has been successfully used to induce the expression of defb4 (defensin, beta 4) and camp (cationic antimicrobial peptide) genes, the production of both ll-37 and human β-defensin 2 in cell cultures of keratinocytes from diabetic foot ulcers and to promote wound healing. 268 accordingly, the persistence of functional milk human κ-caseinoglycopeptides in plasma of human infants after human milk feeding serves the purpose of exogenously administering amps without the requirement for their de novo synthesis. 269 amps can help preventing biofilm formation and microorganisms' growth. that is the case of ll-37 and its fragments, which were already shown to have antibiofilm formation properties against several methicillin-resistant s. aureus strains and p. aeruginosa, common pathogens of hospital-acquired infections, as well as against the melioidoisis' etiological agent burkholderia pseudomallei and the uropathogenic e. coli. [270] [271] [272] [273] another amp with antibiofilm formation activity is hbd-3, found to prevent the development of methicillin-resistant s. epidermidis and s. aureus biofilms, largely responsible for orthopedic implants associated infections. 274 likewise, amps may also inhibit the formation of fungal biofilms. for instance, histatin 5 has been demonstrated to successfully inhibit the growth of c. albicans biofilm on denture acrylic. 275 these examples may broaden the applications of amps in the clinical and biomedical fields by allowing functionalization of catheters. alternatively, implants and other medical devices can also reduce bacterial colonization, biofilm formation, and infection rates, presenting long lasting functionality, broad-spectrum activity, and minimal cytotoxicity against human cells. 276, 277 human amps could, theoretically, also be used as biosensors immobilized onto microchips for identification purposes, allowing the identification of microorganisms. however, such applications have only been exploited with animal or synthetic amps. these biosensors on electrical impedance alterations resulting from the presence of bacterial or fungal surface elements, or on the specificity of peptide nucleic acid probes against their targets, presenting higher versatility, lower costs, and lower sensitivity limits compared to other conventional methodologies. [278] [279] [280] alternatively, amps could be used as drug delivery systems in the form of conjugates in order to accurately target drugs and other agents to tumor sites or intended organs, as typified by monodisperse "endosomolytic" nanoparticles for in vivo gene delivery using intravenous injection, or by functionalized nanoparticles with higher membrane penetration targeted against gliomas. 281, 282 such functionalization may allow for deeper tissue penetration, increased antimicrobial potency, sustained release, and novel routes of administration to be achieved. ever since the isolation of the first peptide from the frog skin in 1983, 283 several breakthroughs were made in the isolation, synthesis, and application of amps. still, many challenges are yet to overcome in the field of amps peptidomics, 284 reflecting mainly their huge diversity and how little is known about their behavior in vivo and their structure-function relationships. in terms of costs, producing amps can be several hundred times more expensive than the production of conventional antibiotics. 285 moreover, amps' design can be very complex and the quest for the perfect amp a never-ending search. in fact, considering the possibility of working with only 20 amino acids, a small peptide with ten amino acids could have up to 20 10 = 1.02 × 10 13 different sequences, a larger peptide with 20 amino acids could present with up to 20 20 = 1.05 × 10 26 different sequences, and one with 100 amino acids could adopt any one of 20 100 = 1.27 × 10 130 different sequences. such possibilities do not even account for the presence of posttranslational modifications. then, each one of these peptides could shape into several different secondary structures and fall into a wide spectrum of activity, depending on the milieu conditions. furthermore, in a way to overcome the challenge of peptide stability, one has to consider the possibility to synthesize and to administer amps containing d-amino acids, thus decreasing their susceptibility to hydrolysis by human enzymes. 267 to deal with such large amount of amino acid combinations one should mind using peptide libraries and computational models (such as by quantitative structure-activity relationship analysis) in order to narrow large collections to few surrogate amps (for a review please see blondelle and lohner's paper. 286 another challenge to overcome is the lower amp's activity in vivo when compared to that observed in vitro. this has largely hampered the development of amps as therapeutic agents, as this decrease in activity stems from differences in ph and salt concentrations, the presence of proteases and corresponding inhibitors, and other interacting molecules hindering antimicrobial activity. accordingly, several amps (e.g., pexiganan, 287 iseganan, 288 neuprex 289 have reached phase ii clinical trials only to fail after approval for marketing because they did not evidence superior activity over already marketed conventional antibiotics. in early 2016, clinicaltrials.gov (a registry and result database of publicly and privately supported clinical studies of human participants conducted around the world) listed few clinical trials involving amps. for instance, ll37 is currently being studied for its efficacy in melanoma patients due to its ability to stimulate the immune system, while c16g2 and chromogranin a derived peptides are under scrutiny for their therapeutic potential against dental diseases. simultaneously, therapeutic strategies are also being studied to augment amps' expression in amp-deficient patients. this deficiency is believed to be due to an increase in th2 lymphocytes derived cytokines, il-4, il-13, and il-10, and a decrease in tnf-α, il-6, il-1, and interferonγ . one example of such strategy is pimecrolimus, a calcineurin inhibitor that binds with high affinity to macrophilin-12, preventing the translocation of nuclear factor of activated t cells to the nucleus and the transcription and release of such inhibitory cytokines (for more up-to-date trials, readers can access clinicaltrials.gov). another possible caveat concerns the cytotoxicity to mammalian cells when in concentrations above naturally occurring values. when bacteria produce amps, they also develop means to avoid their targeting by such peptides, such as efflux pumps and the sequestering of enzymes. however, eukaryotic cells are equally prone to damage if the administration of exogenous amps is carried out in high enough concentrations. therefore, administering amps not constitutively produced or at concentrations above those normally found in the human host environment can be toxic and very detrimental to human cells. furthermore, rapid metabolism and low bioavailability are characteristic of amps because these peptides can suffer extensive proteolysis in vivo, thereby accounting for their inactivation and short half-life. 22, 28, 290 similarly, for peptides to be available at sites distant from their origin, these would have to cross many cellular membranes. however, due to amps' polarity, membrane hydrophobicity may prevent them from doing so. 291 moreover, in contrast to other highly polar mediators (e.g., ionic salts and a few metabolites), endogenous peptides tend not to have a transporter, further compromising their bioavailability in different compartments and the subsequent impossibility to be absorbed through the gastrointestinal tract. therefore, the number of possibilities rapidly escalades, and each amp-based therapy can be further optimized based on the modulation of chemical properties and engineering of delivery systems. even acknowledging all these factors, the human host is most certainly an unpredictable environment and the same peptide may behave differently, displaying unique individual pharmacokinetics and pharmacodynamics. on a final note, due to amps' interaction with and dependence on other immune mediators, variability will most likely result from immune status alterations as those frequently found in human diseases, including infectious ones. taken together, the above discussed results support amps as constituting a paramount group of defense molecules in human biological fluids, being present in most fluids, contributing for the sterility of some of these, but being most notably enriched in biological fluids bathing tissues with the largest load of microorganisms (fig. 2) , such as saliva and urine. also, while some amps seem the display activity spectra against only a handful of microorganisms, others display broad-spectrum effects. thus, the expression and activity patterns of human amps enriched in biofluids may have suffered selective pressures resulting from exposers to microorganisms specifically present in each fluid (fig. 2) . this work was financed by national funding from fct (fundação para a ciência e a tecnologia) through the project uid/bim/04501/2013, uid/ic/00051/2013. rv thanks fct for if/00286/2015 fellowship. the authors report no declarations of interest. with saliva proteomics research. since then, he extended the application of proteomics to the characterization of other biofluids' proteome and peptidome in order to identify the biological pathways modulated by diseases such as diabetes mellitus and bladder cancer. currently, he works to a large extent with bioinformatics tools for the identification of enzymes involved in the regulation of proteome remodeling. additional supporting information may be found in the online version of this article at the publisher's web site: apd3: the antimicrobial peptide database as a tool for research and education anticancer efficacy of magainin2 and analogue peptides structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides cationic host defence peptides: potential as antiviral therapeutics the n-and c-terminal fragments of ubiquitin are important for the antimicrobial 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realistic outlook optimization and high-throughput screening of antimicrobial peptides pexiganan acetate a multinational, randomized phase iii trial of iseganan hcl oral solution for reducing the severity of oral mucositis in patients receiving radiotherapy for head-and-neck malignancy prolyl peptidases: a serine protease subfamily with high potential for drug discovery bioavailability and transport of peptides and peptide drugs into the brain key: cord-302918-0nk7zyod authors: broor, s.; bharaj, p.; chahar, h. s. title: human metapneumovirus: a new respiratory pathogen date: 2008-11-01 journal: j biosci doi: 10.1007/s12038-008-0067-y sha: doc_id: 302918 cord_uid: 0nk7zyod human metapneumovirus is a recently recognized pathogen of acute respiratory tract infection (ari) in children as well as elderly and immunocompromised adults. the virus belongs to the family paramyxoviridae, sub family pneumovirinae and genus metapneumovirus. through genetic analysis it has been characterized into two groups a and b which are further divided into four sub-lineages. the virus is difficult to grow in tissue culture and hence reverse transcriptase-polymerase chain reaction (rt-pcr) for n and l gene is the method of choice for diagnosis. the virus has been seen in all countries with seasonal distribution in winter months for temperate and spring/summer for tropical countries. f gene is the most conserved among different lineages and efforts are underway to design recombination vaccine using f gene. acute respiratory tract infections (ari) are a leading cause of morbidity and mortality worldwide (denny and loda 1986; hijazi et al 1996) . of the 10 million deaths of children less than 5 years of age throughout the world 1·9 million children died from acute lower respiratory tract infections (alri) in the year 2000, 70% of them in sub-sahara africa and south asia (williams et al 2002) . although the incidence rates of ari are almost similar in both developed and developing countries, the mortality is higher in developing countries (shapiro 1998) . the risk of pneumonia is 3-6 times higher in children from developing countries (10-20%) as compared to developed countries (3-4%) (victora et al 1999) . approximately 0.5 million children die due to alri in india each year, accounting for one fourth of the 1.9 million global alri deaths (reddaiah and kapoor 1988; ahmad et al 2000; williams et al 2002) . all classes of micro organisms including viruses, bacteria and protozoa are capable of infecting the respiratory tract, but viruses and bacteria are the common pathogens. a variety of viruses, including, respiratory syncytial virus (rsv), infl uenza viruses, parainfl uenza viruses, adenoviruses, coronaviruses and picornaviruses, are associated with different respiratory syndromes in all age groups (grondahl et al 1999; weigl et al 2000) . however, despite extensive diagnostic testing, a substantial portion of respiratory tract infections still cannot be attributed to any known pathogens. in about a third cases of alri (liolios et al 2001) and a half of upper respiratory tract infections (uri) (nokso-koivisto et al 2002) no pathogen could be detected .these observations suggest that unknown pathogens may be responsible for a substantial proportion of respiratory tract disease. metapneumovirus is a recently discovered etiological agent of acute respiratory illness (ari). it was fi rst reported in 2001 from the netherlands from nasopharyngeal aspirates (taken over a 20 year period) in 28 hospitalized children and infants with ari having signs and symptoms similar to that of rsv infection (van den hoogen et al 2001) . the virus was found to be closely related to the avian pneumovirus, a member of the metapneumovirus genus, and was called human metapneumovirus (hmpv) . although the virus was not isolated until recently, antibodies to the virus have been shown to be present in all persons ≥ 8 years of age in serum samples collected since 1958 (van den hoogen et al 2001) . thus the virus is not a newly emerging pathogen and did not recently "jump" to the human population from an animal reservoir. genetic analysis of the virus showed that it is an rna virus of the paramyxoviridae family and pneumovirinae subfamily along with rsv. the avian pneumoviruses (apv) (formerly turkey rhinotracheitis virus) is highly related to hmpv and these two viruses have been separated by taxonomists into a separate genus, metapneumovirus (lamb et al 2000) . initial electron microscopic examination revealed that the viral isolate had morphology similar to paramyxoviruses. spherical enveloped particles have a mean diameter of ~200 nm. in addition, fi lamentous and pleomorphic particles are also present. the virion is surrounded by a lipid envelope derived from the plasma membrane of the host cell into which the three virus glycoproteins, the attachment (g), fusion (f), and small hydrophobic (sh) proteins, are inserted (collins and mottet 1993) . the rna genome associates with viral proteins to form the helical nucleocapsid (represented on the right and in the centre of the virion on the left in fi gure 1). the proteins consist of the nucleocapsid protein (n), the phosphoprotein (p), and the large polymerase (l) protein. the m2-1 transcriptional enhancer protein is also thought to be associated with the nucleocapsid. the nucleocapsid is surrounded by the matrix (m) protein, which forms a link between the nucleocapsid and the lipid membrane of the virus particle (easton et al 2004) . full length sequences of at least 4 hmpv genomes have been reported (van den hoogan et al 2002; biacchesi et al 2003; herfst et al 2004) . the negative sense non-segmented rna genome of the virus is about 13 kb in length (easton et al 2004) . the hmpv genome is predicted to encode 9 proteins in the order 3-n-pm-f-m2-sh-g-l-5 (the m2 gene is predicted to encode 2 proteins, m2-1 and m2-2, using overlapping open reading frames, as in rsv) (van den hoogan et al 2002) (fi gure 2). the genome also contains noncoding 3'leader which has the viral promoter, 5' trailer and intergenic regions, consistent with the organization of rsv . however there are some differences in the genome of apv/hmpv as compared to rsv, they lack the 2 nonstructural proteins ns1 and ns2 complete virion nucleocapsid located at the 3' end of rsv genomes. these proteins counteract host interferons; therefore the lack of these genes in the metapneumoviruses may have important implications for the relative pathogenicity of these viruses compared with rsv strains (collins et al 2001) . sequence identity between apv and hmpv open reading frames is 56% to 88% (van den hoogen et al 2002). although the function of each of the gene products has not been formally tested, it is likely that the function of the proteins can be predicted by comparison with other paramyxoviruses. the f (fusion), g (glycosylated) and sh (short hydrophobic) proteins are integral membrane proteins on the surface of infected cells and virion particles. the f protein appears to be a classic viral fusion protein, with a predicted nonfurin f1/f2 cleavage site near a hydrophobic fusion peptide and 2 heptad repeats in the extra cellular domain that facilitate membrane fusion. the predicted g protein of hmpv exhibits the basic features of a glycosylated type ii mucin-like protein but interestingly lacks the cluster of conserved cysteines sometimes termed the "cysteine noose" that is found in the rsv and apv g proteins. g gene of hmpv tends to be highly variable like rsv g gene which may be due to host immune selection pressure (crowe 2004) . the function of the sh proteins of both the viruses remains unknown. the n (nucleoprotein), p (phosphoprotein) and l (large, polymerase) proteins are replication proteins in the nucleocapsid, the m2-1 and m2-2 proteins are regulatory proteins and the m (matrix) protein may coordinate viral assembly of viral nucleocapsids with envelope proteins (crowe 2004) . the classifi cation and naming of serotypes/serogroups, genotypes, subgroups, strains, variants and isolates of hmpv is still evolving. based on genomic sequence and phylogenetic analysis, there are two major genotypes of hmpv, designated a and b (biacchesi et al 2003; van den hoogen et al 2003) . these analyses are based on sequencing of the n, m, f, g, or l gene, and the genotype groupings are concordant regardless of which gene is studied (bastein et al 2003; biacchesi et al 2003; boivin et al 2004; peret et al 2004) . full length sequences of genomes from viruses representing the 2 major subgroups show that the diversity between hmpv subgroup a and b sequences is greatest for the sh and g proteins (59 and 37% identity, respectively), biacchesi et al 2003; and is more than rsv subgroup a and b. hmpv f protein, which is predicted to be the principal target of protective antibodies, is more conserved in hmpv strains than rsv. the overall level of genome nucleotide sequence identity and aggregate proteome amino acid sequence identity between the two hmpv subgroups were 80 and 90%, respectively, similar to the respective values for rsv a and b groups (hamelin et al 2004) . based on the sequence analysis of two surface glycoprotein encoding genes namely f and g genes each of the major lineages are further subdivided into two genetic sub-lineages known as a1, a2 and b1, b2, the signifi cance of this diversity is unclear at this time van den hoogen et al 2004) (fi gure 3). in a recent report huck and colleagues have reported a new sub lineage within the a lineage. previously reported a2 lineage is found to consist of two clusters namely, a2a and a2b. among all the sub-lineages of hmpv the a2 sub-lineage shows the greatest diversity (huck et al 2006) . the genetic diversity in hmpv strains also affects antigenic diversity, but to what extent has not been resolved. studies with experimental infection of animals have suggested that the two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with post-infection hamster sera (skiadopoulos et al 2004) . however cross protection studies in hamsters and nonhuman primates have shown that each strain provided a high level of resistance to reinfection with the homologous or heterologous strain, supporting the conclusion that the two hmpv genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups skiadopoulos et al 2004; van den hoogen et al 2004) . hmpv f protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection (macphail et al 2004; skiadopoulos et al 2004) . the extent of cross-protection in rodents cannot be extrapolated directly to the human situation because the animals are only semi permissive hosts for hmpv replication. although diffi cult to assess, the extent of cross-protection is important to estimate because vaccine developers will choose to develop either a monovalent or a bivalent vaccine formulation based on this factor. hmpv has been associated with ari in all age groups, with more severe diseases occurring in young children, elderly individuals and immunocompromised hosts. the virus causes a variety of clinical syndromes in children that are typical of the paramyxoviruses, including upper and lower respiratory tract illnesses. the clinical characteristics of hmpv infections are not distinctive, thus, differentiating it from other respiratory viruses on clinical grounds is not possible (stockton et al 2002) . a study conducted at vanderbilt university medical center on the association of the virus in a cohort of 2000 subjects of age 0-5 years, followed during a 25-year period revealed that hmpv was associated with common cold (complicated by otitis media in one third), bronchiolitis, pneumonia, croup and exacerbation of reactive airway disease (williams et al 2004) .the clinical profi le of illness caused by hmpv was similar to that caused by rsv. in outpatients with alri, hmpv was detected in 12% of cases, which was second only to rsv (williams et al 2004) . hoarseness has also been observed more frequently in hmpv infection as compared to rsv (falsey et al 2003) . further compared with rsv infections, the children who develop hmpv infection are somewhat older, and the severity of disease is usually somewhat less than rsv peiris et al 2003; van den hoogan et al 2003; viazov et al 2003) . human mpv infection is not restricted to the very young children but also occurs in adults and elderly subjects. in adults it usually causes fl u like illness and colds . in fragile elderly hmpv causes more severe disease than in healthy elderly or young adults (falsey et al 2003) . in a study among 10 elderly of > 65 years of age who had underlying illness, 4 developed pneumonitis and 2 died due to hmpv infection . immune status of infected subjects is also important in determining the severity of illness. hmpv infection has been reported in immunocompromised individuals. in 3 patients with acute lymphoblastic leukemia who had alri, hmpv was the sole pathogen detected and one of them died . hmpv infection has also been reported in hiv infected children but it is not clear if the severity of illness is more in this group (madhi et al 2003) . respiratory virus infections are often associated with recurrent wheezing in children and exacerbations of asthma in older patients. acute wheezing and asthma exacerbations have been associated with hmpv infection in some studies (jartti et al 2002; peiris et al 2003) but all studies have not shown this association (rawlinson et al 2003) . in some studies in children with asthma hmpv was found more frequently than rsv van den hoogen et al 2003) . a study by vicente et al (2006) suggested that hmpv genotype a might be more pathogenic than genotype b, causing greater clinical severity in children whereas no differences in disease severity associated with either genotype (agapov et al 2006) was observed in another study. because the seasonal distributions of hmpv and rsv overlap, the potential for dual infection exists. several studies have found hmpv -rsv co-infection rate of approximately 5-10% (esper et al 2004; viazov et al 2003; williams et al 2004; xepapadaki et al 2004) . however, greensill et al (2003) reported that 70% of rsv-infected children who required intensive care in liverpool, united kingdom, were co-infected with hmpv, suggesting that dual infection with rsv and hmpv may predispose for severe infection particularly in otherwise healthy children. in another study from the united kingdom, hmpv and rsv co-infection conferred an increased risk of admission to the pediatric intensive care unit (semple et al 2005) . however, such synergistic association has not been found in other, population-based and case-control studies of hospitalized children esper et al 2003; maggi et al 2003) . the possible synergistic interaction between hmpv and the severe acute respiratory syndrome (sars) coronavirus has been recently postulated during the 2003 sars outbreak in canada and hong kong as many individuals were found to have co-infection with hmpv (chan and li 2003; poutanen et al 2003) . on the other hand, such synergy between hmpv and sars was not confi rmed in experimental macaque models . in one case report, in an infant who had sars, fatal encephalitis was correlated with hmpv infection as hmpv rna was detected post-mortem in brain and lung tissue (schildgen et al 2005) . cases of severe hmpv infections in adults and reinfection in immunocompromised subjects suggest that, despite the universal infection in childhood, new infections can occur throughout life due to incompletely protective immune responses and/ or acquisition of new genotype. since severe disease is seen mainly in pediatric patients, it suggests that naturally acquired infection induces partial protection against the disease. however, it should be emphasized that there is no cross -protection among the virus strains. a recent report has described a child who suffered from two episodes of hmpv infection during a one month period, each caused by a different strain (vargas et al 2004) . the conventional model of attachment for the members of subfamily pneumovirinae including genus pneumovirus and metapneumovirus involves the interaction of viral g protein with a molecule or molecules on the host cell surface (levine et al1987) .while the precise nature of the cellular receptor and mechanism of entry has not been identifi ed, current evidence suggests that one or more cellular glycosaminoglycans or heparin-like molecules are involved in virus attachment and entry (feldman et al 2000) . following g-protein-mediated attachment, the f glycoprotein promotes ph-independent fusion between the cell membrane and the virus envelope. for the f protein to become functional, it has to be cleaved by cellular proteases, and the hydrophobic amino terminal region of the f1 component then promotes the fusion process, which introduces the internal components of the virion into the cytoplasm of the host cell, where the remainder of the infectious cycle takes place. recent experimental work using primates (chimpanzees, cynomolgus and rhesus macaques, african green monkeys) and small animals (hamsters, cotton rats, mice and ferrets) has been performed to characterize the pathogenesis associated with this viral infection; hmpv replicates to a various extent in the upper and lower respiratory tracts of these experimental animals, although clinical symptoms after intranasal challenge have only been observed in chimpanzees, cynomolgus macaques and balb/c mice so far (van den hoogen et al 2001; alvarez et al 2004; kuiken et al 2004; skiadopoulos et al 2004; . in balb/c mice and cotton rats, time course studies have indicated that peak viral titers are found around day 4-5 after infection and decrease thereafter alvarez et al (2004a) and , have demonstrated that hmpv may present an initial biphasic replication pattern in lungs of balb/c mice, with hmpv rna still detectable more than 180 days after infection. such persistence could be explained by an aberrant t helper cell type 2-like immune response, with impaired virus clearance after primary hmpv infection (alvarez and tripp 2005) . signifi cant pulmonary infl ammatory changes have been found in balb/c mice and cotton rats (alvarez et al 2004; . interestingly signifi cant infl ammatory changes were still present in the cotton rat animal model more than 21 days after viral challenge . increase in many cytokines and chemokines such as interleukin il-2, il-8, il-4, inf-γ, macrophage infl ammatory protein 1α and monocyte chemotactic protein has been observed in the lungs or bronchoalveolar lavage of both mice and cotton rats in response to hmpv challenge (alvarez et al 2004 . in humans, hmpv infection has also been associated with an increase of il-8 in upper respiratory tract secretions and with chronic infl ammatory changes of the airways, with presence of, intra alveolar foamy and hemosiderin-laden macrophages (laham et al 2004 , vargas et al 2004 . when compared with rsv, hmpv infections in humans seem to induce lower levels of infl ammatory cytokines such as il-12, tumor necrosis factor α, il-6 and il-1β (laham et al 2004) . thus far, no experimental human volunteer studies have been reported. since its initial description in 2001, hmpv has been isolated from individuals of all ages with ari, and has been identifi ed in every continent. hmpv infections have been documented in europe [united kingdom ( stockton et al 2002 ) , finland (jartti et al 2002 ) , italy (maggi et al 2003) , france (freymuth et al 2003) , germany ( viazov et al 2003) , spain (vicente et al 2003) , norway (dollner et al 2004) ]; america, canada , united states (esper et al 2003 , falsey et al 2003 , brazil (cuevas et al 2003) australia (nissen et al 2002) , asia [hongkong , japan (ebihara et al 2004) , korea (kim and lee 2005) thailand (samransamruajkit et al 2005) ] and africa [yemen, (al-sonboli et al 2005) , south africa (ludewick et al 2005) ]. in a preliminary study from pune in india hpmv was found in 5 of 26 children with ari (rao et al 2004) . in a study of children seen in a large referral hospital in delhi hmpv was detected in 12% of ari cases by reverse transcriptase-polymerase chain reaction (rt-pcr) (banerjee et al 2007) . role of hmpv in ari has been evaluated in many studies mostly using molecular methods. in young hospitalized children hmpv has been detected in 5-10% of ari cases. bastein et al 2003; bovin et al 2003; esper et al 2003 esper et al , 2004 mcintosh and mcadam 2004) . however in one study from italy the rates of hospitalizations due to hmpv infection varied from 7-43% (maggi et al 2003) . the rates of detection of hmpv in adults are usually lower than children with rates of about 3% in general community (stockton et al 2002) . it is interesting to note that detection rates of hmpv have generally been higher in retrospective studies than in prospective studies, an observation consistent with a degree of selection bias. this indicates that large prospective studies are needed in order to clarify the role of hmpv in various clinical conditions (hamelin et al 2004) . most hmpv infections occur in < 5 years of age, with children <2 years of age being most at risk for serious hmpv infections. in one study the peak was seen at 4-6 months of age (van den hoogen et al 2004). however, hmpv infections tend to occur in slightly older children compared to rsv van den hoogen et al 2003) . the activity of hmpv in temperate climates peaks between december and february (maggi et al 2003; van den hoogen et al 2003) where as in subtropics it peaks in springsummer . the peak of activity of hmpv at any given location often coincides with or follows the peak of rsv activity esper et al 2004) . outbreaks of hmpv are to be a local phenomenon unlike infl uenza virus, where two or three strains spread across the globe each year. strains of hmpv differ from community to community, and strains identifi ed in one location may be quite similar to strains identifi ed in other locations in different years. for example, the prototype strain identifi ed in the netherlands is genetically similar to strains identifi ed in australia; new haven, connecticut and quebec, canada in different years (esper et al 2004) . we in delhi also found that 2 lineages of hmpv circulated during the same season i.e. in 2005-06 a2b and b1 lineages were seen with a2b being the predominant genotype where as in 2006-07 b1 and b2 were found to co-circulate with predominance of b1 (broor s, et al, unpublished data) . although formal transmissions studied have not been carried out, transmission most likely is through large particle respiratory secretions and fomites as is true for rsv (human metapneumovirus 2006) . nosocomial transmission has been reported in hospital setting (pieris et al 2003). presence of hmpv antibodies in serum samples obtained in 1958 showed that the virus has been circulating for at least for the past 50 years in the netherlands (van den hoogen et al 2001). by the age of 5 years, >90% of individuals screened have evidence of hmpv infection. the seroprevalence of hmpv specifi c antibody in adults is nearly 100% (van den hoogen et al 2001, leung et al 2005) . the hmpv-specifi c antibodies in infants <3 months of age has been detected in >90% of the cases tested, indicating that maternally derived antibodies are present in young children (leung et al 2005) . whether these hmpv specifi c antibodies protects against infection or lessens the severity of illness is not known. hmpv replicates poorly in conventional cell culture and is relatively diffi cult to isolate. many strains show reliable cytopathic effect (cpe) in tertiary monkey kidney cell line/llcmk-2 cells . the cpe is quite variable, some strains show syncytia formation and other only produce rounding . cpe is usually observed after 10-21 days (mean 17 days). confi rmation of cpe is done by rt-pcr or indirect immunofl uorescence staining using hmpv specifi c antibodies. recent reports have shown that hmpv can replicate effi ciently in hep-2 cells but does not produce good cpe. van den hoogen et al (2004) reported that vero cell clone 118 is permissible for expression of virus from all four lineages and cpe is easier to observe. many laboratories are now using this cell line for routine virus isolation . in addition shell vial culture for rapid isolation of hmpv has also been described (reina et al 2007) . they showed that shell vial culture using commercial llcmk-2 cells should be a method for isolating hmpv from respiratory samples in pediatric population. direct immunofl uorescence assay (dfa) using virus specifi c antibodies is a rapid method to detect respiratory viruses and is commonly used in diagnostic laboratories. commercially available hmpv specifi c antibodies have been developed for direct immunofl uorescence assays, though this method may not be as sensitive as rt-pcr for the detection of hmpv (ebihara et al 2005 , landry et al 2005 , percivalle et al 2005 . because of the unavailability of rapid antigenic detection assays in the past and slow growth in tissue culture molecular methods have become the method of choice for the diagnosis of hmpv infection. cote et al (2003) reported that primers that bind regions of the n and l genes are highly sensitive for the detection of hmpv strains of both genotypes. however, it is reported that the p gene of hmpv is an ideal target for the molecular detection and genotyping as p gene provides the conserved region for designing rt-pcr primers and adequate variability to permit the accurate genotyping of the virus into 2 main lineages and 4 sublineages . real time pcr has also been described for hmpv which allows amplifi cation and quantitation of this pathogen in clinical samples. real-time rt-pcr is a powerful method to detect hmpv and other respiratory viruses. the assay has been developed to detect the n gene of all known hmpv lineages (maertzdorf et al 2004) and real time rt-pcr targeting the n and / or l gene is more sensitive, specifi c, and rapid method to detect this virus in clinical samples than conventional rt-pcr (mackay et al 2003) . nucleic acid sequence based amplifi cation assay (nasba) targeting the m gene has also been developed for detection of hmpv infection in respiratory specimens (dare et al 2007) . enzyme-linked immunosorbent assay (elisa) can also be used for hmpv serological testing using the hmpv-infected cells (falsey et al 2003 . recently, elisa methods using viral n or f protein expressed in prokaryotic or recombinant vesicular stomatitis virus (vsv) (leung et al 2005) and recombinant baculovirus system (liu et al 2007) have been developed to detect antibodies against hmpv. use of these assays in sero-epidemiologic studies might be helpful for detection of antibody response against hmpv infections thus increasing the understanding of the human immune responses to hmpv and permitting better understanding of the epidemiology of this virus. however, since infections with hmpv are universal, serologic testing for diagnosis can only help if four fold increases in antibody titers or seroconversion is demonstrated (prins and wolthers 2004) . the development of a safe and effective vaccine to protect against hmpv is a reasonable goal. several promising vaccine candidates have been tested in animal models. a live recombinant human parainfl uenza virus that contains the hmpv f gene has been shown to induce hmpv-specifi c antibodies and to protect experimental animals from hmpv challenge (skiadopoulos et al 2004) . a chimeric bovine/human parainfl uenza virus 3 expressing the hmpv f elicits neutralizing antibodies against both parainfl uenza virus and hmpv (tang et al 2005) . however, the results of these animal challenge studies should be interpreted cautiously. there are many limitations of the small-animal model for testing potential hmpv vaccines, not the least of which is that the pathogen is highly host restricted. the safety and effi cacy of a recombinant hmpv vaccine may be diffi cult to predict. chimeric hmpv/avian pneumovirus recombinant viruses in which the n or p gene of hmpv was replaced with the corresponding gene from avian pneumovirus are attenuated in african green monkeys (the p gene chimera is 10-to 1,000-fold more attenuated than the n chimera), though both are as immunogenic as wild-type hmpv (pham et al 2005) . this represents another strategy for the development of recombinant attenuated live hmpv vaccines. other than infl uenza virus, antiviral therapy for respiratory viruses has not shown tremendous potential. it is unknown what role the host's infl ammatory response plays during hmpv disease. nonetheless, antiviral compounds have been tested with hmpv. the antiviral activity of ribavirin to inhibit the replication of hmpv is equivalent to that observed with rsv (wyde et al 2003) . other compounds, such as nmso3, a sulphated sialyl lipid that has been shown to have potent antiviral activity against rsv in tissue culture cells, have been shown to have anti-hmpv activity in vitro (wyde et al 2004) . it is likely that an hmpv-neutralizing monoclonal antibody for prophylaxis of high-risk infants (similar to the anti-rsv f humanized monoclonal antibody currently used for prevention of severe rsv disease) will be developed and tested. the progress towards an effective antiviral strategy for hmpv is currently limited by the scant data on pathogenesis of the virus in the natural host. since the discovery of hmpv in 2001, the virus has been identifi ed worldwide. hmpv is a common respiratory pathogen, particularly in infants and young children. the virus is associated with both upper and lower respiratory tract infections and may be a trigger for asthma. there are two major genotypes of hmpv, whether these genotypes represent distinct serotypes re-mains controversial. the major challenges faced by the medical and scientifi c communities are: the understanding of the pathogenesis of hmpv disease and the development of a safe and effective vaccine to protect against infection and disease caused by this newly recognized respiratory virus. genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness the decline in child mortality: a reappraisal respiratory syncytial virus and human metapneumovirus in children with acute respiratory infections in yemen human metapneumovirus persists in balb/c mice despite the presence of neutralizing antibodies the immune response to human metapneumovirus is associated with aberrant immunity and impaired virus clearance in balb/c mice human metapneumovirus infections among children with acute respiratory infections seen in a 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reaction human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children estimates of world wide distribution of child deaths from acute respiratory infections comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by nmso3 in tissue culture assays human metapneumovirus as a causative agent of acute bronchiolitis in infants key: cord-282965-xguotf4m authors: o’callaghan-gordo, cristina; antó, josep m. title: covid-19: the disease of the anthropocene date: 2020-05-15 journal: environ res doi: 10.1016/j.envres.2020.109683 sha: doc_id: 282965 cord_uid: xguotf4m nan a close and tragic antecedent to covid 19 is the acquired immune deficiency syndrome (aids) caused by a human immunodeficiency virus (hiv) infection. this disease was discovered in 1981, and in 2018 had affected about 40 million people and caused more than 750,000 deaths. hiv viruses are the result of multiple transmissions between species of immunodeficiency virus that naturally infect african primates. most of these transfers probably resulted in viruses that spread limitedly in humans until one of these transmissions, which involved an immunodeficiency virus from chimpanzees in southeastern cameroon, resulted in the leading cause of the pandemic in humans (sharp and hahn, 2011) . the transmission of a virus from a wild animal spice to humans is not a rare event. in fact, a high proportion of human pathogens are zoonotic or were zoonotic in origin before being transmitted only to humans (wolfe et al., 2007) . since the emergence of aids, many other epidemic infectious diseases, such as ebola, sars and mers to name the most recent, have been caused by the transmission of viruses from wild animal species to humans as shown in 2008 by jones et al. who identified 335 emerging infectious diseases that emerged between 1960 and 2004, and found that at least 60% of these diseases came from nonhuman animals (jones et al., 2008) . these transmissions between non-human and human animal species are not, necessarily, the result of chance. there is strong evidence that ecological changes have led to increased rates of emerging and re-emerging diseases such as malaria, hantavirus lung syndrome, nipah virus, and ebola virus disease (myers et al., 2013) . human activity is increasingly disruptively transforming the earth's natural habitats and ecosystems by intensely altering the patterns and mechanisms of interaction between species (myers et al., 2013) and facilitating the transmission of infectious diseases across species and to humans (patz et al., 2005) . a study published in 2014 estimated that by 2050 25 million kilometers of new roads would be built and that 9 out of 10 would occur in developing countries, including many regions that maintain exceptional biodiversity and vital ecosystem services. these roads often cause habitat loss and fragmentation, forest fires, overflows and other environmental degradations, often with irreversible impacts on ecosystems (laurance et al., 2014) . the complete causal sequences and impacts of these ecological changes are still poorly understood, but frequently these emerging zoonosis appear and spread in circumstances that denote the effects of an economic and commercial practices that destroys natural habitats and animal populations, including those of humans living there, in the absence of effective protection and regulatory policies. in the case of covid-19, the chinese center for disease control and prevention has confirmed that the virus causing the outbreak of covid 19 in wuhan came from wild animals, whose meat was sold at the hankou's market in wuhan, in which about 120 animals of 75 different species were marketed, some of them alive, such as puppies of wolves, salamanders, crocodiles, scorpions, rats, squirrels, foxes, civets and turtles. the first group of patients with sars-cov 2 in wuhan were mostly traders in that market. the circumstances of the origin of covid 19 are similar to those of sars, an outbreak in which the first group of patients were also wildlife traders in guangdong city. peter j li, a law professor at the university of houston, has been researching the problems associated with the distribution and sale of wild animals for human consumption in china for years. as li explained in a march 25 article in the south china morning post, all wildlife trade activities in china have been banned since january 26, 2020, although the ban is temporary, with the aim of suspending trade only until the epidemic ends (li, 2020) . these markets should therefore become the appropriate substrate to allow sars-cov 2 virus being transmitted from some infected animals to traders and customers, and later to the rest of the population. according to pj li during years, the chinese government's attempts to regulate this type of wildlife meat trade have been countered by the influence of a powerful trade lobby, the benefits of which depend on maintaining access to consumption of these animals by a predominantly affluent sector of chinese society. to complete the causal chain, scientists' warnings about the potentially catastrophic effects of the risk of emerging infectious diseases have often not been heard. in the case of the previous sars outbreak, the bat trade is likely to have put infected animals in contact with susceptible amplifier hosts, such as the masked civet palm (parguma larvata) at some point in the wildlife supply chain, establishing a cycle in which susceptible people subsequently became infected (fèvre et al., 2006) . zhong nanshan and guan yi, both well-known sars experts, had previously warned of the possibility of a pandemic originating in the wild meat markets in china and of the need to ban such commercial practices (li, 2020) . john vidal, in a recent article, has cogently pointed out the link between covid-19 and planetary health (vidal, 2020) . indeed, we suggest that covid-19 is a paradigmatic example of an anthropocene disease. it follows a complex sequence involving disruption of the natural, social, economic and governance systems. the destruction of natural habitats and the extinction of species, the poorly regulated capture, marketing and consumption of non-human animals, the influence of lobbies to nullify or delay measures to protect natural and social systems, the limitation of current scientific knowledge and the contempt by governments and companies of the available evidence, have all worked in an orchestrated sequence to facilitate the current covid-19 pandemic. this sequence of distal causes is closely related to the global climate crisis and the rest of environmental disruptions of the anthropocene. consumption of fossil fuels for energy, deforestation and the conversion of natural habitats into farmland or extensive livestock are among the main sources of greenhouse gas emissions, and at the same facilitate the emergence of new zoonosis, such as sars-cov-2, with a pandemic potential. oil and timber extraction in primary forest areas involves the opening of roads in hard-to-reach areas, encouraging contact between humans and wildlife, and facilitating hunting and bushmeat consumption (wolfe et al., 2005) . advancing the agricultural frontier to respond to current food systems increases the frequency of ecotones, key areas in the onset of infectious diseases (rohr et al., 2019) . and at the same time, the destruction of habitats caused by these activities are the main causes of biodiversity loss, which is also associated with the emergence of infectious diseases (civitello et al., 2015) . to control the covid-19, the approach needs to focused on protecting human health with evidence-based prevention and control strategies, effective treatments for patients, vaccines to prevent infection and preparation of health systems to deal with a huge burden. however, as necessary as this is, a response focused on human health will result in a nearsighted vision. we need to look at covid-19 from the perspective of planetary health, that is, to understand that the response to the pandemic must not only be the right one for humans but also the right one for the planet. this point is especially relevant when we understand that covid-19 has the same origin as climate change and global environmental degradation, the biggest challenges we face as humanity. preventing cross-transmission of viruses from non-human animal species to humans becomes another compelling reason to urgently advocate for the preservation of natural ecosystems and stop the massive extinction of endangered species. similarly, the protection of animal rights is becoming a key element in the regulation of human consumption of animal meat in the framework of sustainable food production, marketing and consumption systems. markets, both food and securities, must be effectively regulated so that private profits do not become public tragedies. these solutions must be aligned with the reduction of internal and north-south inequalities, and at the same time be respectful of world diversity. if the pandemic subsides without causing an even greater global disruption, and we can all regain the precarious stability we were all living in, the real challenge will continue to be to transform our civilization into a just and sustainable society, achieving a zero level of greenhouse gas emissions no later than 2050 and this is humanity's great time trial race. the importance of the 2030 sustainable development agenda is therefore paramount. biodiversity inhibits parasites: broad evidence for the dilution effect animal movements and the spread of infectious diseases global trends in emerging infectious diseases a global strategy for road building no title. south china morning post human health impacts of ecosystem alteration human health: ecosystem regulation of infectious diseases emerging human infectious diseases and the links to global food production origins of hiv and the aids pandemic. cold spring harb destruction of habitat and loss of biodiversity are creating the perfect conditions for diseases like covid-19 to emerge bushmeat hunting, deforestation, and prediction of zoonotic disease emergence origins of major human infectious diseases ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord-030279-pv770doe authors: novossiolova, tatyana title: twenty-first century governance challenges in the life sciences date: 2016-11-29 journal: governance of biotechnology in post-soviet russia doi: 10.1007/978-3-319-51004-0_4 sha: doc_id: 30279 cord_uid: pv770doe the chapter explores the rapid advancement of biotechnology over the past few decades, outlining an array of factors that drive innovation and, at the same time, raise concerns about the extent to which the scope and pace of novel life science developments can be adequately governed. from ‘dual-use life science research of concern’ through the rise of amateur biology to the advent of personalised medicine, the chapter exposes the limitations of the existing governance mechanisms in accommodating the multifaceted ethical, social, security, and legal concerns arising from cutting-edge scientific and technological developments. so far as to suggest that the 'life sciences knowledge, materials and technologies are advancing worldwide with moore's law-like speed.' 3 and whilst some commentators have questioned the extent to which the ongoing progress of biotechnology has translated into practical applications and novel products, 4 there is some consensus that the biotechnology landscape has been fundamentally transformed over the recent decades with the possibilities now unlocked holding revolutionary potential. indeed, rapid advances in the field have produced a knowledge base and set of tools and techniques that enable biological processes to be understood, manipulated and controlled to an extent never possible before 5 ; they have found various applications in numerous spheres of life, generating enormous benefits and offering bright prospects for human betterment; and they have come to be regarded as a key driver of economic development with potential to close the gap between resource-rich and resource-poor countries. 6 the progress of biotechnology has been largely driven by three sets of forces, namely social, political and economic. 7 the social dynamics at work in this context are understood as the efforts to improve public health and overall wellbeing of individuals both in the global north and global south, boost agricultural yields and encourage environment-friendly practices to mitigate the adverse effects of climate change. several factors account for the significant value attached to the life sciences in the context of intense globalisation and continuous change. surging population numbers and extended life expectancy are augmenting the demand for developing effective and affordable medications, novel approaches for the treatment of chronic diseases and additional cost-effective sources of energy and food production. at the same time, rising global trade and travel, coupled with increased urbanisation, and an uneven distribution of wealth are creating optimal conditions for disease outbreaks, pandemics and environmental degradation. 8 against this backdrop, biotechnology appears full of promise and critical to tackling social and natural concerns; enhancing disease prevention, preparedness and surveillance; promoting development; and alleviating human suffering. 9 economic dynamics include national expenditure on research and development, purchasing power, trends in consumerism and market pressures and fluctuations. besides public funding for r&d which remains a key factor in the growth and flourishing of bioindustry in developed and emerging economies alike, private investment from venture capital firms, start-up companies and transnational corporations (tncs) have also played an indispensable role in capturing new markets and further facilitating the extension of bioeconomy on a global scale. dupont's significant footprint in india is indicative in this regard, not least because of the depth and diversity of the activity that the company has undertaken via its offshore r&d centres ranging from crop science to biofuels. 10 likewise, merck has outlined a 1.5 billion dollar commitment to expand r&d in china, as part of which it intends to establish an asia headquarters for innovative drug discovery in beijing. 11 political dynamics are triggered by states' increasing commitment to support the progress of biotechnology as a way of maximising their power and boosting their status in the international arena. 12 in the aftermath of 9/11 and the 'anthrax letters' attack of october 2001, substantial effort has been given to harnessing life science research for the purposes of national security. biodefence and bioterrorism preparedness are thus considered high-priority areas for national investment by government agencies and the military alike. an illustrative example of this two-tiered approach is the funding policy in the usa, where biodefence research is financed by the nih, department of homeland security (dhs) and defense advanced research projects agency (darpa), to name a few. under the synergistic influence of these three sets of forcessocial, economic and politicalbiotechnology has been transformed into a truly global fast-evolving enterprise encompassing a multitude of stakeholders, delivering considerable benefits and holding out still greater promise, with profound and far-reaching implications for virtually every aspect of human well-being and social life. the pharmaceutical industry is a case in point, for its steady expansion would hardly be possible were it not for the vast array of techniques and methods enabled by the progress of the life sciences. worth roughly 400 billion dollars, the global pharmaceutical market dominates the life sciences industry and arguably determines the trajectory of life sciencesrelated technological development and global spread. 13 gene cloning, dna sequencing and recombinant construction of cell lines, to name a few, are all deemed indispensable for the development of novel medicines and therapeutics. it suffices to mention that more than half of the top selling commercially available drugs in the usa would not exist without those methods. 14 agriculture, too, has been heavily influenced by the ongoing biotechnology revolution, as evidenced in the rapid growth and dispersion of commercialised transgenic crops (biotech crops) and the efforts to use gmos (both animals and plants) for the production of vaccine antigens and other biologically active proteins ('biopharming'). 15 indeed, the increase in the area of farmland planted with transgenic crops rose dramatically from 1.7 hectares in 1996 to about 60 million hectares in 2002 16 and is still growing. in addition, technological convergence 17 between biotechnology, nanotechnology, information technologies and cognitive science has unlocked a broad scope of opportunities for maximising public (and private) welfare, offering substantial benefits in wideranging areas such as medicine, pharmacy, crime investigation and national security by ensuring precision and reliability, while at the same time, reducing the amount of time previously required for the performance of certain tasks. four key features of biotechnology make it so appealing to the majority of stakeholders involved. first, biotechnology innovation is characterised by duality, whereby research yields results that simultaneously lead to advances in basic knowledge and stimulate product development. 18 second, the output that the life sciences generate in the form of new medicines, improved nutrition products, enhanced yields and novel materials, is 'strongly positive'. 19 the increasing utility of tools and strategies for human enhancement, whether in professional sport, for cosmetic and aesthetic purposes, or on the battlefield, vividly reflects the firm conviction that the transformative capacity of biotechnology, even at the most fundamental level, is something to be welcomed and vigorously embraced. what is more, biotechnology possesses proven economic viability, as illustrated in the burgeoning industries and new markets it has spurred. against this backdrop, the high rate of biotechnology expansion is anything but surprising, since every increment in biological capability pays back the researcher and the researcher's sponsors in short order. payback comes in the esteem of peers, in promotions, and in increases in the academic or corporate salaries of the researchers whose work generates knowledge and new therapies. payback comes in the form of profits for the manufacturers of kits to perform the manipulations, royalties for the writers of the methods manuals profits for the drug industry. payback comes for the public in the form of new drugs and therapies. 20 fourth, besides being cost-effective, many of the benefits that biotechnology offers are easy to obtain and disseminate. in other words, many of the various prospects for public (and private) betterment are not situated at some distant moment in the future but can be realised immediately, as a result of which pressing problems can be alleviated, if not fully resolved, and substantial revenue can be generated in the short term. last but not least, while there are some risks and concerns associated with the advancement of biotechnology, few of those are deemed urgent or significant enough to impact on the pace of innovation. as the actual manifestation of such risks is often contingent upon the interplay of a variety of factors, this renders the likelihood of a major crisis unfolding as a result of the progress of biotechnology low. moreover, there is a genuine belief that any challenges that may arise from the proliferation of novel technologies can either be foreseen or dealt with on a case-by-case basis. given the enormous potential of biotechnology for addressing societal, economic and environmental challenges, it is unsurprising that most states have readily endorsed scientific and technological innovation and embarked on largescale generously-funded r&d programmes in the life sciences. given the powerful multifaceted impetus for biotechnology advancement, it is possible to identify at least five key trends in the governance of biotechnology that are common for highly industrialised and developing countries alike. those include: high-level coordination, facilitation and funding; synergies within and between both the public and private sector; emphasis on strategic and competitive interests at the expense of precaution; regulations that seek to promote rather than restrict scientific and technological progress; and overreliance on technical solutions. at international level, the on-going expansion of biotechnology has been hailed not only as an inherently positive development but also as an essential prerequisite for enhancing human welfare and addressing various socio-economic, environmental and health concerns. in its 2013 world health report, the who called for: increased international and national investment and support in [life science] research aimed specifically at improving coverage of health services within and between countries. 21 the who has also strived to promote research on specific diseases, such as hiv/aids, cancer, pandemic influenza, tuberculosis and malaria, with the goal to improve methods for prevention and diagnostics and facilitate the development of effective therapeutics and vaccines. 22 in a similar fashion, the un food and agriculture organisation (fao) has highlighted the positive impact that biotechnology could have on the development of agriculture: . . . biotechnology could be a major tool in the fight against hunger and poverty, especially in developing countries. because it may deliver solutions where conventional breeding approaches have failed, it could greatly assist the development of crop varieties able to thrive in the difficult environments where many of the world's poor live and farm. 23 it is not difficult to see how those assertions have been translated into national policies and practical steps across the globe. the us nih that provide the bulk financial support for medical and health-oriented r&d in the us spent over 30.9 billion dollars during the fiscal year 2012, about a third of which was allocated for funding biotechnology and bioengineering projects. 24 within its sixth framework programme for research and technological development spanning the period 2002-2006 the european union (eu) distributed more than 2.5 billion euro for projects under the theme 'life sciences, genomics and biotechnology for health'. 25 developing countries, too, are increasingly investing in 'red' biotechnology as part of their efforts to address public health concerns. according to a recent who report, support for biotechnology and particularly, for cancer research, in cuba has soared over the past 20 years, amounting to over one billion dollars. 26 as a result, the cuban biotechnology industry is burgeoning, holding around 1200 international patents and exporting vaccines and pharmaceuticals to more than 50 countries. the prospect of climate change coupled with rising population numbers has compelled governments in the global north and south alike to explore 'green' biotechnology as a means of ensuring food security. the usa remains by far the largest commercial producer of gm crops. several eu member states (france, germany, spain, poland, romania, czech republic, portugal and slovakia), canada and australia further feature in the list of industrialised nations that have embarked on growing gm plant breeds. more and more emerging economies are striving to expand their agrobiotechnology sector, most notably brazil, india, argentina, south africa, mexico, burkina faso, myanmar and chile. 27 in 2008, the chinese government launched a major r&d initiative worth 4 billion dollars to develop new plant varieties by 2020 that will enhance yields, have improved nutritional value and be resistant to pests. 28 public-private partnerships underpinned by access to early-stage risk capital and strong linkages between business, universities and entrepreneurial support networks constitute an important vehicle for promoting innovation and fostering technology transfer and product development. for instance, the chinese government has launched a major initiative mobilising 2.5 billion dollars in venture capital to support start-ups in the immense zhangjiang science park outside shanghai 29 ; russia's rusnano has entered a 760 million dollar partnership with the us venture capital firm domain associates to fund 'emerging life science technology companies and establish manufacturing facilities in russia for production of advanced therapeutic products'; and cleveland's university hospital has allocated 250 million dollars for setting up a 'non-profit entity to fund and advise physician-scientists on transitional research and a related for-profit accelerator that will develop selected compounds to proof of concept.' 30 the kauffman foundation in the usa, a wealthy philanthropic establishment dedicated exclusively to the goal of entrepreneurship has been particularly zealous in its quest for promoting university-based entrepreneurial activities nationwide. its kauffman campuses initiative launched in early 2003 enjoyed so much popularity among universities that following the initial round of grants totalling 25 million dollars, the foundation announced its resolve to leverage a 100 million dollar investment for the creation of new interdisciplinary education programmes. 31 university-industry partnerships, while not a novel phenomenon in the area of biotechnology, have considerably intensified over the past several decades, thus facilitating the widespread commercialisation of life science research. indeed, 90 per cent of the companies in the us surveyed by blumenthal et al. in 1996 had relationships with an academic institution in that year and in more than half of those cases industry provided financial support for research in such institutions. 32 according to another study, the total industry investment in academic life science research in the usa tripled between 1985 and 1998 reaching almost 2 billion dollars and has been growing ever since. 33 against this backdrop, some commentators have put forward the 'triple helix' model, which serves both as a conceptual tool and a policy blueprint. in the former case, it is used to elucidate the academic-industry-government relationships that underpin the institutional arrangements and changing practices in the processes of production, transfer and application of knowledge in post-industrial societies; in the latter, it is promoted as a framework for economic development through state investment and knowledge sharing between academia and industry. 34 others, however, have remained sceptical of the close integration of universities and the private sector voicing concerns about the possible deleterious effects arising therefrom: as in other activities, when big money flows fast, temptations and opportunities arise for risky behaviour and stealthy or even brazen wrongdoing in pursuit of personal or institutional advantage. the new world of academiccommercial dealings is characterised by some grey areas and evolving rules for permissible and impermissible conduct. the people who manage and conduct research in scientific organisations are not immune to the weaknesses and foibles so plentiful elsewhere, despite the accolades for probity that science bestows upon itself. 35 with more and more universities joining the biotechnology 'gold rush' and corporate values and goals steadily penetrating the professional academic cultures, scholarship turns into a result-oriented activity subject to the priorities and interests of business partners and industrial sponsors. strategy and careful planning deemed essential to the pursuit of for-profit knowledge can have a restraining effect on the spontaneous vigour characteristic of academic research, limiting the range of problems that could be studied to those defined by the market. 36 at the same time, scientists often find themselves under tremendous pressure striving to satisfy the demands of their industrial clients without utterly neglecting their academic duties ranging from mentorship through filing grant applications to publishing. the extensive workload coupled with the bright prospects for securing long-term research funding and achieving some individual gain and prominence provide a favourable environment in which instances of dubious, sometimes fraudulent, behaviour, conflicts of interest and lack of transparency, unless too severe, are unlikely to encounter widespread opprobrium and may even go unnoticed. 37 in the race for patents and venture capital, the business mentality dulls scientific rigour and the ethics threshold appears not too difficult to cross. interests at the expense of precaution given the tremendous benefits that biotechnology is expected to generate in virtually any sphere of human activity, it is not difficult to understand why its progress is predominantly viewed through an explicitly positive lens by policy-makers. since the opportunities for achieving public betterment and enhancing state prestige and international standing are too tempting and too abundant, there is a powerful urge to dedicate both will and resources to promoting the large-scale expansion of the life sciences. for one thing, the prospect of conquering disease and maximising human wellbeing provides solid justification for a deliberate and sustained investment in fostering scientific and technological prowess. lack of commitment and reluctance to support r&d in the life sciences then becomes an unfavourable option in the political calculations of states regardless of their level of economic development and international status. within the context of political calculus pervaded by realist fears, competition and power, the perceived risks of inaction with regard to scientific and technological development justify vast expenditure, lower regulatory barriers to innovation and product development. political choices concerning biotechnology support are therefore frequently made at the expense of calls for caution and potential social, environmental and ethical concerns. the regulation of genetic engineering is a case in point. as discussed in the previous chapter, from the outset, the attempts of governments to impose strict controls on research involving rdna faced a severe backlash from academic scientists and business executives alike. by the 1980s, the various legislative initiatives put forward in the usa were abandoned in favour of the regime established by the nih guidelines, which virtually exempted the biotechnology industry from formal regulation. while the leading us-based companies pledged to 'voluntarily comply' with the guidelines, behind the scenes they craftily continued to push for a system that would insulate them from governmental and public scrutiny. 38 indeed, during the 1990s when the states parties to the btwc strived to strengthen the treaty by negotiating a binding verification mechanism corporate interests proved too big and too important to be ignored. both the pharmaceutical research and manufacturers of america (phrma) which represented the country's major research-based pharmaceutical and biotechnology companies, and the biotechnology industry organisation (bio) which at that time represented some 1400 biotechnology firms, became vocal opponents of any measures designed to promote international arms control which seemed to hinder in any way the protection of proprietary information and intellectual property. 39 in the period between 1994 and 2001 the associations invested considerable effort, time and ingenuity in lobbying the us government and influencing the diplomatic talks in geneva to secure an outcome that was in line with the demands of their constituencies. of course, it would be naive to ascribe the us resolve to reject in 2001 both the text of the protocol and its utility in general for providing adequate verification and enhancing confidence among states parties solely to the activity of the biotechnology industry; nevertheless, it would be equally naive to suppose that corporate interests played no significant role in the process. 40 besides economic priorities, national security and military calculations can also provide a compelling rationale for downplaying the potential risks associated with biotechnology expansion. following the 'anthrax letters' attack in october 2001, the us government embarked on a massive financial investment to boost its bioterrorism preparedness and enable the prevention, early detection, monitoring and emergency response to biological threats. as outlined in biodefense for the 21st century, a presidential directive that set out a comprehensive framework for national biodefence policy, between 2001 and 2005 the federal government provided roughly 6 billion dollars 'to state and local health systems to bolster their ability to respond to bioterrorism and major public health crises'. 41 along with the highly controversial vaccination programme that the government envisaged, 42 another important development designed to enhance america's biodefence preparedness and capability was the drastic increase both in the number of high-containment labs (bsl-3 and bsl-4) and the number of researchers with access to some of the most dangerous pathogens known to mankind, including the causative agents of ebola, plague and q fever. some commentators have questioned the logic behind this policy highlighting the heightened risk of accidental or deliberate release of pathogens. 43 far from being ill-founded or hypothetical, such fears stemmed from a range of high-profile cases that occurred after 2001 across the us in which the lack of proper training and professional negligence resulted in scientists being exposed to or infected with deadly microbes. 44 real-life horror stories about vials of plague being transported in the hand-luggage of researchers on passenger aircraft without the required authorisation, and deadly cultures gone missing from what appeared to be secure laboratories further fuelled the criticism toward the us biodefence policy raising difficult questions about its appropriateness and actual goals even before the 'anthrax letter' investigation revealed that the attack was 'insider's business'. 45 life science research, just as any other sphere of professional activity, is subject to a range of institutional, national and international regulations. along with the more general rules such as those related to occupational health and safety, fair pay and job competition, conflict of interests, labour rights, and professional liability, there are also specific ones addressing particular aspects of the research process including project clearing (e.g. review by local biosafety committees), safe laboratory practice and transport of pathogens (e.g. 2005 international health regulations), exchange of viral strains (e.g. pandemic influenza preparedness framework, 2011), handling of dangerous pathogens (e.g. us select agent programme) and ethical treatment of human subjects and samples obtained therefrom (e.g. the 2004 human tissue act in the uk). 46 while hardly exhaustive, this list suffices to convey the idea that the regulatory regime governing the practice of life science research is dense and comprehensive. with more than 30 international organisations overseeing biotechnology from various perspectives, 47 there is a prima facie reason to assume that the regime in its current form is sufficiently flexible to accommodate novel advances and hold any potential risks, which they may pose, at bay. yet in reality over the past decade the opposite trend has prevailed, that is, the existing governance mechanisms have struggled to respond adequately to the proliferation of new scientific developments with multiple adaptive uses and the multiplicity of cutting-edge developments posing profound ethical quandaries. how to account for this discrepancy? part of the problem stems from the fact that since at least the late 1970s the regulation of biotechnology has been streamlined so as to become compatible with and not a restriction on continued technological change and economic growth. 48 as such, it rests upon the barely questioned assumption that the progress of biotechnology is inherently good and needs to be harnessed and vigorously promoted. needless to say, any measures that seem to slow down or restrain its advancement are deemed undesirable and even detrimental to socio-economic development. hence, when developing regulations, policy-makers have generally pursued a twofold objective: first, to promote the safe practice of life science research by reducing any risks arising therefrom both to scientists and the general public; and second, to ensure that any issues that may hinder the expansion of biotechnology are not subject to restrictive legislation. a vivid manifestation of this approach is the way in which the ongoing debate on 'dual use research of concern'benignly-intended research that seeks to maximise human welfare by responding to health, societal and environmental ills but could also facilitate to the development of more sophisticated and potent biological weapons and enable bioterrorism 49has been handled. for more than a decade, researchers, journal editors, security experts and policy-makers have strived to devise oversight mechanisms and governance initiatives that could adequately tackle the challenge of dual use without stifling innovation. unfortunately, to date their efforts have met with little success, as a result of which virtually each experiment of dual use concern is dealt with separately on a case-by-case basis. this is not to say that there are no similarities across the studies of this kind. on the contrary, a few of the most notable examples follow a similar paradigm, including the creation of a vaccine-resistant strain of the mousepox virus, the artificial synthesis of the polio virus, the recreation of the 1918 spanish influenza virus and, most recently, the production of a mammalian-transmissible h5n1 avian influenza virus (see fig. 4 .1). 50 all four of them were performed in strict compliance with the rules and procedures in place for laboratory biosafety, biosecurity and biorisk management and under appropriate physical containment conditions; all had passed thorough review by the respective local biosafety and bioethics committees; and all of them were deemed essential in terms of public health benefits. above all, the ethical and security concerns that the studies have raised go far beyond the laboratory door, posing fundamental questions about how life science research is reviewed, conducted and communicated. yet none of the high-profile experiments of concern has proved critical enough to provoke a radical change in the way dual-use research is governed. 51 three points merit consideration in this regard. the first pertains to the manner in which the dominant discourse on dual use is framed, that is, in purely ethical terms as a dilemma. while bioethics undoubtedly has a role to play in the discussions on dual use, the language in early 2001, the journal of virology published a report of the creation of a highly virulent strain of the ectromelia virus, the causative agent of mousepox. the work described in the report was carried out by a group of australian scientists based in canberra. its original goal was the development of an infectious immunecontraceptive that could be used against wild mice for the purpose of pest control. to achieve this, the group drew upon previously published work. during the course of the experiment, the researchers unexpectedly discovered that the newly engineered strain of the mousepox virus, which they created, killed 60% more mice than the parent virus, including mice that had been vaccinated or that had natural immunity. when the research was published, concerns were raised that it could potentially be misapplied for hostile purposes, or even that the same technique could be utilised for creating a more virulent strain of the variola virus, which causes smallpox in humans. in 2002, a team of scientists led by dr eckard wimmer from the university of new york at stony brook announced that they had successfully created a polio virus 'from scratch'. to carry out the research, the scientists 'followed a recipe they downloaded from the internet and used gene sequences from a mail-order supplier'. a once the virus created, it was tested on mice, as a result of which the infected animals became paralysed and died. the study spurred a wide-ranging debate, not least because it drew attention to the possibility of using synthetic biology for constructing de novo viruses for the purposes of bioterrorism. in 2005, it was announced that cdc scientists, together with colleagues from several research institutions across the usa, had successfully recreated the influenza virus, that was responsible for the 1918 pandemic, which killed between 20 and 50 million people worldwide. using dna from a tissue of a flu victim buried in the permafrost in alaska, the researchers managed to reconstruct the influenza virus and thus study its pathogenesis and properties that contributed to its virulence. despite the scientific justification that was put forward, critics have argued that the study is 'a recipe for disaster', not least because the availability of the virus' full-genome sequence and detailed method for its reconstruction on the internet may facilitate its synthesis by a of 'dual-use dilemmas' is too abstract to offer appropriate analytical tools for dealing with the issues at play. as discussed above, the questions that dual-use research poses such as data sharing, research funding and project planning are far from hypothetical but they feature explicitly in everyday professional practice. however, the 'dilemma framework' automatically strips them of the complex socio-technical arenas in which they have actually presented themselves by laying an emphasis on what action should ideally be taken, rather than what is practically feasible given the circumstances. 52 moreover, such issues are typically structural in nature for they constitute fundamental elements of the life sciences professional culture, and as such, could hardly be adequately addressed solely at the level of individual researchers. yet framing social, legal and security concerns in terms of moral dilemmas allows for structural issues to be omitted from the discussion, rendering life scientists the chief, if not the only, moral agents expected to reach what is deemed to be the 'right' answer. 53 assigning abstract duties then comes to be regarded as an appropriate 'solution', even if those are virtually impossible to fulfil given the complexities of the working environment within which researchers operate. the second point is related to the reductionist view that dominates the discourse of what counts as a risk in life science research. perhaps one of the most significant legacies of the asilomar conference on rdna (see chapter 3) is the emphasis on laboratory risk that could be effectively managed by dint of physical containment and rules and procedures for safe laboratory practice. 54 it suffices to mention that the bulk of guidelines and formal regulations published by the who focus exclusively on promoting and refining measures that aim to maximise laboratory biosafety and prevent the accidental release of pathogens. hence, it is hardly surprising that the concept of dual use and the idea of risks beyond the laboratory door implicit in it seem alien to the majority of practising researchers. striking as it may appear, even though dual use research has been debated for more than a decade now, the level of awareness among life scientists of the broader social, security and legal implications of their work remains low. 55 the third point deals with the way in which risks in life science research are assessed and mitigated. given the narrow definition of risk encompassing technical particulars, physical containment and biosafety, risk assessment is considered an appropriate and reliable tool for ensuring research safety. the heavy reliance upon risk assessing tools is underpinned by two underlying assumptions. one is that it is possible to foresee and calculate most, if not all, things that could potentially go wrong both during the development phase of the project and after its completion. the other is that it is possible then to use the produced data as a basis for devising measures and strategies for eradicating, or at least, mitigating the risks likely to occur. attractive as it may seem, this 'new alchemy where body counting replaces social and cultural values' presupposes a clear distinction between the risk assessment 'experts' and the general public, whereby the former are granted a licence to make decisions about the risks that the latter cannot do without. 56 likewise, costbenefit analysis on the basis of which research proposals are screened for potentials risks and security concerns has attracted some serious criticism. in the view of some commentators, besides being sometimes deeply inaccurate, the cost-benefit analysis is 'ethically wrong' since 'applying narrow quantitative criteria to human health and human life' is unacceptable. 57 but there are other problems, too. as pointed out by dickson, the cost-benefit analysis distorts political decision-making by omitting any factors that cannot be quantified, thus obscuring questions of equity, justice, power, and social welfare behind a technocratic haze of numbers. 58 as a result, complex and politically charged decisions are reduced to a form that fits neatly into the technocratic ways of making regulatory decisions, whereby calculations and approximations made by the few substitute for the judgements of many. 59 the wide-ranging controversy that unraveled in late 2011 when two teams of scientists working independently in the netherlands and the usa managed to produce an air-borne strain of the h5n1 avian influenza virus, a highly pathogenic and lethal microbe with over 60 per cent mortality rate in humans arguably constituted the pinnacle of the deliberation on dual use research. both studies set alarm bells ringing for the security community who almost immediately jumped in the debate voicing concerns over the possibility of biological proliferation and bioterrorism. some commentators even argued that the experiments ran counter to the spirit if not to the letter of the 1975 btwc. 60 against this backdrop, the resultant controversy was deemed at least initially to offer a timely opportunity to evaluate the existing governance mechanisms, determine their gaps and weaknesses, and broaden the scope of deliberation inviting participation of a wide range of stakeholders. unfortunately, the outcome of the debate proved far more moderate, signalling preference for preserving the status quo without disrupting the established systems for governance and oversight. despite the extensive mass media coverage of the controversy, only few public consultations were held and none of those was designed as a platform for making policy proposals or developing action plans. moreover, the denselypacked agenda prepared duly in advance left very limited scope for posing 'tricky' questions which the participating 'experts' might have struggled to answer. needless to say, all consequential decisions were made behind closed doors away from public scrutiny and on some occasions the people with the greatest vested interest in the publication of the studies were also the ones with the greatest say in the process. 61 there were no significant changes in terms of governance initiatives, either. far from being ground-breaking developments, the us government policy for oversight of life sciences dual use research of concern 62 and the decision of the dutch government to invoke export control legislation before allowing the publication of the study conducted within its jurisdiction were little more than desperate moves that aimed to obscure the inadequacy and shortcomings of the measures already in place. 63 overall, the manner in which the h5n1 debate was handled could be treated as a missed opportunity, whereby those in charge of the decision-making process did little to address or even acknowledge the broader issues underpinning dual-use research of concern but simply 'kicked the can down the road to the next manuscript' waiting for the next controversy to erupt. 64 technology seems to play a significant role in the governance of life science research. high-containment laboratories, well-equipped biosafety cabinets, sophisticated waste management systems, enhanced personal protective equipment and secure containers for the safe storage and transportation of biohazard materials are just a few of the tools and systems in place that allow the safe handling of dangerous pathogens and toxins and, at the same time, protect both laboratory personnel and the general public from exposure to deadly microbes. that said, the effectiveness of technical solutions should not be overstated if only for the fact that 'problems' of governance are barely technical matters per se but rather constitute complex issues of human relatedness. nevertheless, the attractiveness of technological fixes as offering reliable risk mitigation and reassurance in the safety of biotechnology is ever growing. it suffices to mention that the h5n1 controversy discussed above was in part resolved after the lead researchers in the netherlands and the usa respectively agreed to add a detailed section on the technical specificities and laboratory biosafety and biosecurity measures taken during the experiments. 65 the strategy has proven effective in diverting attention from the rather inconvenient questions regarding the utility and significant potential for hostile misuse of the so called 'gain-of-function' (gof) research and concentrating it on more mundane issues dealing with in-house precautions and safety procedures. once the latter were deemed adequately resolved, the former were effectively forgotten. still, the value of technical means in ensuring reliable risk management should not be taken for granted. for one thing, laboratory biosafety precautions, however sophisticated, are far from perfect and accidents do occur. such is the case with the pirbright site in the uk which was at the centre of a major outbreak of foot-and-mouth disease in 2007, as a result of which over 2100 animals were slaughtered. 66 in 2012 the bioterrorism bsl-3 laboratory at the us cdc in atlanta suffered repeated problems with airflow systems designed to help prevent the release of infectious agents. 67 the faulty system could perhaps be regarded as an exception had it not been for the authoritative investigation report of the us government accountability office (gao) released in march 2013. according to the report, the cost of building and maintaining high-containment laboratories, coupled with the absence of national standards for their design, construction, operation, and maintenance 'exposes the nation to risk'. 68 far more critical is the situation in the developing world and emerging economies where lax regulations and technical failures have significantly heightened the risk of accidental release of pathogens, as demonstrated by the numerous 'escapes' of the severe acute respiratory syndrome (sars). 69 but even if technology functions impeccably, this hardly reduces the likelihood for a human error or inappropriate behaviour. unlocked doors in high-containment facilities hosting deadly pathogens, eating and drinking in laboratories and poor waste disposal practices are just a small part of the otherwise long list of mundane mishaps that may result in severe consequences. it is worth mentioning that the us cdc came under the spotlight after internal e-mail correspondence revealed that doors in the bsl-3 block where experiments involving the causative agenets of anthrax, sars and influenza were performed were left unlocked on numerous occasions, thus increasing the risk of unauthorised access or theft. 70 given the chance of technical flaw and the potential for human error, some life scientists have begun to question the reliability of existing laboratory precautions and demand thorough review and evaluation. in a recent letter to the european commission the foundation for vaccine research has asked for 'a rigorous, comprehensive risk-benefit assessment' of gof research that 'could help determine whether the unique risks posed by these sorts of experiments are balanced by unique public health benefits which could not be achieved by alternative, safe scientific approaches'. 71 engines that drive biotechnology momentum by and large, the ongoing progress of biotechnology is largely viewed and assessed through an explicitly positive lens which allows focusing almost exclusively on the benefits likely to be accrued notwithstanding the risks, actual and potential. the resultant distorted image is problematic, not least because it precludes any comprehensive discussion on the potential side effects and negative implications of novel life science advances. above all, it sustains the barely questioned assumption that the existing governance mechanisms are adequate and sufficient to cope with the stresses and strains of the rapidly evolving biotechnology landscape. yet given the complex and multifaceted dynamics shaping the life science enterprise, the rapid pace of innovation, and the limits to predicting the synergistic and cumulative effects of the proliferation of new technologies, the uncritical acceptance of such assumptions is at best naïve and at worst dangerous. arguably the advancement of the life sciences has greatly benefited from the fascinating breakthroughs made in other areas of study, such as chemistry, engineering, computing, informatics, robotics, mathematics and physics. some commentators even talk about a third revolution in biotechnology underpinned by scientific and technological convergence: convergence does not simply involve a transfer of tools sets from one science to another; fundamentally different conceptual approaches from physical science and engineering are imported into biological research, while life science's understanding of complex evolutionary systems is reciprocally influencing physical science and engineering. convergence is the result of true intellectual cross-pollination. 72 the resultant 'new biology' has opened up a range of marvellous possibilities enabling the manipulation of living matter at the full range of scales, as well as the application of biological systems principles for the development of novel materials, processes and devices. 73 as such, it has been largely hailed as possessing the 'capacity to tackle a broad range of scientific and societal problems.' 74 this is not an exaggeration. as noted by a recent report of the us nas, the precipitous decline in the cost of genome sequencing would not have been possible without a combination of engineering of equipment, robotics for automation, and chemistry and biochemistry to make the sequencing accurate. 75 likewise, it is the combination of expertise from fields as diverse as evolutionary biology, computer science, mathematics, and statistics that has allowed both the analysis of raw genomic data and the subsequent use of these data to other fields. 76 at the same time, advances in nanoscience and nanotechnology have considerably enhanced drug delivery making it more accurate by targeting specific parts of the body. 77 yet the transformative potential of scientific and technological convergence comes at a price, not least because parallel to the benefits it offers, there are risks the effects of which could be truly devastating. 78 take drug delivery, for instance. thanks to the technological breakthroughs over the past decade, doctors have gained unprecedented access to the human body which, in turn, has facilitated the treatment of previously incurable disease and conditions (e.g. some forms of cancer). nanoparticles and aerosols are now utilised for delivering a precise dose of therapeutics to tissues and cells via novel pathways circumventing body's natural defences and evading immune response. it is not difficult to imagine how such knowledge could be misapplied for malicious ends, including incapacitating and killing. research on bioregulators is a case in point. bioregulators are natural chemicals in the human body that play a vital role in the maintenance of the homeostasis but when administered in large quantities or in healthy individuals could be toxic and lead to serious disorders, even death. given their properties, bioregulators constitute the perfect bioweapon: efficient and virtually impossible to detect. and if in the past, security analysts discounted the risk of their weaponisation due to the instability of the compounds when released in the atmosphere, the emergence of novel drug delivery techniques has significantly altered the security calculus. 79 this is just but one example of the challenges that the increasing convergence between biology and chemistry poses to the integrity of the international biological and chemical non-proliferation regimes. 80 even though some effort has been made over the recent years to address those and other areas of concern and strengthen the international prohibition against biological and chemical warfare, in practical terms little has been achieved, as a result of which the risk of the hostile exploitation of novel scientific developments remains far from hypothetical. along with the risk of misuse of new knowledge, there is the risk posed by the lack of sufficient scientific knowledge. cross-disciplinary convergence opens a multitude of opportunities for manipulation and modification of living matter but, at the same time, it precludes almost any sensible assessment of the potential interactions likely to occur in the process. nano-based medicine is but one area that has attracted criticism in this regard. since some elements behave differently at nano-scale, it becomes extremely difficult to assess their level of toxicity or other negative side effects that they may exert. such is the case with long carbon nanotubes, which having been initially praised for their potential to improve implant development 81 were later blamed for exhibiting asbestos-like behaviour that could lead to cancer. 82 another area of converging science with far-reaching implications is synthetic biology, a cross-disciplinary field that draws upon strategies and techniques from molecular biology, chemistry, engineering, genomics, and nanotechnology and thus enables the design and modification of biological systems at a fundamental level. empowered by the tools of synthetic biology, in 2002 scientists managed to assemble a polio virus 'from scratch' in the absence of a natural template. and in 2010 craig venter and his team announced the construction of the first self-replicating synthetic cell which, in their view, was 'a proof of the principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.' 83 the controversial work has attracted criticism on several grounds, including the potential negative effects of the accidental or deliberate release of the novel organism in the environment and the arrogance of scientists to 'play god'. 84 more broadly, both the polio and synthetic cell studies have exposed the obstacles to the regulation of synthetic biology. 85 while some commentators dismiss the risk of bioterrorism, underscoring the key role of tacit skills and knowledge and the difficulties that the lack thereof poses to the replication of the experiments, 86 other issues still merit attention. consider the question of access to commercially available genomic sequences. even though the oversight system for screening base pair orders has improved since the 2006 guardian report that exposed the lax regulations under which virtually anyone could order gene sequences, 87 gaps still remain leaving scope for abuse by those with malign intent. for example, schmidt and giersch have outlined at least three areas of emerging challenges that the existing governance regimes would struggle to accommodate, including 'split orders', 'outsourcing', and the potential for non-natural biological systems. 88 the human genome project completed in 2003 lasted over ten years and cost close to 3 billion dollars; by contrast, about a decade later, wholegenome sequencing can be performed within hours at a price of roughly 1000 dollars or less. 89 while still in its infancy, personalised medicine and individual genetic testing are steadily gaining popularity. indeed, 'up to 100 000 people in england are expected to have their entire genetic makeup mapped in the first stage of an ambitious public health programme' launched by the national health service in 2012 that aims to 'revolutionise the treatment and prevention of cancer and other disease.' 90 according to its proponents, genomic testing offers numerous advantages vis-à-vis traditional evidence-based medicine, including the possibility of early diagnostics of disease, of individually-tailored treatment and, perhaps most importantly, of disease prevention, as illustrated in the resolve of the hollywood actress angelina jolie to undergo double mastectomy after discovering she has an inherited genetic mutation that puts her at high risk of breast and ovarian cancer. 91 but this is just the beginning. in 2012 scientists managed to sequence a foetus's entire genome using a blood sample from the mother and a saliva specimen from the father, a development that could potentially allow for a range of genetic disease conditions to be detected prenatally. 92 and laboratory experiments have already demonstrated the efficacy of genetic therapy to cure mitochondrial disease by creating an embryo with genetic material from both parents and a third person acting as a donor. 93 while truly breathtaking, the advances outlined above raise a host of thorny issues of ethical, social, and legal concern that merit public scrutiny and extensive deliberation before decisions regarding their widespread application are made. at a very basic level, there is the question of whether and to what extent we as individuals are capable of assimilating the information that our own genetic makeup may reveal. are we sufficiently resilient to cope with the emotional distress, anxiety, shame, stigma and guilt that the awareness of severe medical conditions that we or our closed ones are suffering or likely to develop? far from hypothetical, this question has prompted the establishment of a novel profession, that of the genetics counsellor whose task is to help patients overcome any negative effects, stress, or psychological trauma that the disclosure of their genomic map may create. 94 this is just a partial solution though, for the crux of the matter lies in finding a way to deal effectively with risk and probabilities and we as humans are yet to demonstrate a capacity for understanding or relating them to our own lives. 95 individual emotional turmoil, however significant, constitutes only the tip of the iceberg. according to daniel kevles, the torrent of new genetic information has already begun to fundamentally reconfigure social practices and inter-personal relations: it has been rightly emphasised that employers and medical or life insurers may seek to learn the genetic profiles of, respectively, prospective employees or clients. employers might wish to identify workers likely to contract disorders that allegedly affect job performance while both employers and insurers might wish to identify people likely to fall victim to diseases that result in costly medical or disability payouts. whatever the purpose, such genetic identification would brand people with what an american union official has called a life-long 'genetic scarlet letter' or what some europeans term a 'genetic passport'. 96 linking genetic makeup with human identity would ultimately set the scene for the proliferation of technologies aimed at human enhancement: after all, if a gene therapy could allow one to stand a chance in a job competition, boosting one's capabilities would potentially make them a more desirable candidate. other issues of more immediate concern are also likely to arise. one is privacy. gene-sequencing companies usually hold the genetic data of their clients in digital format on online platforms, which automatically creates a risk that personal information may be leaked, hacked or stolen. 97 further, there is the question of ownership. consider, for instance, the controversial issue of human gene patenting, whereby patented genes are treated as research tools and, as such, are controlled by the patent holder who may restrict and charge for their use. 98 thus created, the system often operates to the detriment of patients by hindering research practice, elevating diagnostics prices and denying access to second and independent medical opinion. 99 gene identification alone has a potential 'dark side' too, for it could enable the development of weapons targeted at groupspecific gene markers (e.g. ethnicity). 100 pre-natal genetic testing is yet another significant bone of contention, not least because it evokes notions of state-mandated eugenic programmes and assaults on human rights and dignity. while a nazi-like campaign for a superior race seems improbable in the twentieth-first century, this is not to say that other forms of eugenics may not be encouraged. indeed, some commentators have highlighted the rise of 'homemade eugenics', 101 whereby individual families can make decisions on the attributes of their progeny: the lure of biologically improving the human race, having tantalised brilliant scientists in the past, could equally seduce them in the future, even though the expression of the imperatives may differ in language and sophistication. objective, socially unprejudiced knowledge is not ipso facto inconsistent with eugenic goals of some type. such knowledge may, indeed, assist in seeking them, especially in the consumer-oriented, commercially driven enterprise of contemporary biomedicine. 102 it is plausible to assume that when presented with the opportunity of having their future child tested for genetic disorders, many parents would barely hesitate to accept. such a resolve could have far-reaching implications though. for instance, some genetic therapies entail the use of donor dna different from that of the parents, whereby any genetic modifications in the embryo will pass down to future generations. 103 despite the government support for the 'three-parent babies' in the uk, local religious organisations have protested vociferously against the legalisation of the technique. 104 at the same time, there are certain genetic disorders that can be diagnosed at an early stage but, as of yet, cannot be cured, which inevitably poses the tough choice between raising an unhealthy child and abortion. to be sure, such questions constitute more than individual parents' dilemmas, for they touch upon established social and cultural values, something evident in the profound differences across national reproductive policies. more broadly, there are concerns that reproductive genomics may remain a prerogative of those affluent enough to afford it, thus further exacerbating the divide between the global rich and the global poor. 105 the growth of life science capacity over the past few decades across the globe has been truly astonishing, leading to the emergence of a vibrant research community that brings together researchers from various parts of the world. indeed, a 2011 nas report highlights the extension of both north-south and south-south partnerships, which has played a key role in synergising strengths and maximising competitiveness by improving the quality and effectiveness of research and facilitating data sharing. 106 at the same time, increasing collaboration in the realm of biotechnology industry has offered companies situated in emerging economies access to the global market, thus contributing to economic development and growth. 107 recent advances in technology and laboratory and experimental equipment have further impacted on the practice of life science research in profound ways. improvements in dna sequencing technology have significantly shortened the time required for the preparation of nucleic base-lines, thus relieving scientists of the burden of completing the task themselves and allowing them to focus on their actual project instead. studies and experiments once performed by senior researchers with extensive experience are now carried out by masters students. aided by specially designed genetic engineering toolkits, children as young as the age of ten start exploring the realm of biology in an interactive and engaging manner. needless to say, their notion of science and the world in general would differ significantly from that of their parents whose primary sources of knowledge used to be textbooks and encyclopaedias. indeed, the increasing commercialisation of synthetic biology offers anyone curious enough to fiddle with biological systems the chance of doing so in the comfort of their own home. 108 such modern gene hackers often lack formal background in biology and come from various walks of life. driven by an insatiable appetite for knowledge and the vision of a ground-breaking discovery that could be turned into a multimillion dollar profit, they take up the rather unusual hobby of biohacking which entails the redesign of existing and the creation of novel biological systems. for just few hundred dollars bio enthusiasts set up laboratories easily obtaining all essential requisites and equipment from online sales. and if to some biohacking equates to little more than an unusual hobby, others highlight its potential to generate substantial revenue and fuel economic development. 109 contrary to popular expectation, biohackers are not just eccentric individuals who work in solitude away from public attention. rather, they are members of a wide global movement dedicated to the ideal of do-it-yourself biology (diy), which has branches in 45 locations on four continents. 110 the movement has been partially institutionalised through the establishment of the biobricks 111 and international genetically engineered machine (igem) foundations, which seek to promote the open and ethical conduct of biological engineering and stimulate innovation and creativity. to this end, igem holds an annual competition open to high school students, university undergraduates and entrepreneurs from all over the world. with more than 200 participating teams, the competition constitutes the premiere forum at which biohackers can showcase their skills through project presentation. exciting as it may seem, the ongoing diffusion of life science expertise poses an array of governance conundrums. at the level of professional practice, the proliferation of research facilities around the world has exposed the urgent need for laboratory biosafety and biosecurity training, especially in developing states where a tradition of handling dangerous pathogens is lacking. the issue is further complicated, for such countries often lack the required legal and institutional infrastructure to ensure that professional practice is in compliance with relevant international regulations. foreign aid has gone some way in helping overcome those deficiencies but it has given rise to new problems, too. for instance, it is far from unlikely for a donor state to provide material support for the construction of a state-of-theart laboratory eventually leaving its maintenance to the local government, which can hardly afford the subsequent costs. a similar trend is observed in the area of capacity building and human resource development. most projects that aim to promote biorisk management and a biological security culture tend to be severely constrained in terms of time and funding and overly ambitious in terms of agenda and expected outcomes. lack of adequate mechanisms for quality assessment hinders progress evaluation and sometimes leads to duplication of effort and resources. the emergence of the diy biologists in the life science arena has further added to the challenge of ensuring that novel scientific and technological developments are utilised in a safe and ethical manner. even at the level of everyday practice, difficulties still persist. for instance, many amateur scientists have complained of the lack of manuals and guidelines regarding the safe operation and maintenance of home laboratories. issues such as waste disposal, safe handling and storage of biological material and prevention of contamination pervade the work of biohackers who unlike professional researchers conduct experiments in a much more volatile environment. 112 potential security concerns are also present. with more and more individuals gaining access to biological engineering technologies, ensuring appropriate oversight of what goes on in garage laboratories becomes increasingly difficult. the experience of the us fbi is a case in point. back in 2004 the fbi arrested steven kurtz, a professor at the university of buffalo under the suspicion of plotting a bioterrorist attack. 113 the subsequent investigation revealed that all laboratory and dna extraction equipment found in kurtz's house was legitimately obtained and used in his artwork. in an attempt to avoid mistakes of this kind, the fbi has drastically changed its approach to dealing with the diy movement launching a series of outreach activities that seek to raise awareness of the potential security implications of biohacking. 114 while undoubtedly necessary, such initiatives may well be seen as too little, too late in light of the wide spread of materials, tools and devices that could facilitate the malign misuse of the life sciences. indeed, it is worth noting that as early as the late 1990s the us defence threat reduction agency (dtra) managed to build a research facility that simulated the manufacture of weaponised anthrax using only commercially available materials and equipment. 115 the role of states: both a poacher and gamekeeper structural factors have an important bearing on the development and growth of biotechnology. economic considerations, power interests and realist fears generate potent dynamics that shape and influence and sometimes direct the life science trajectory. within this context, states assume a dual role. on the one hand, they are expected to act as gamekeepers and regulate, monitor and control the process of life science research and the dissemination of novel technologies. on the other hand, though, they also have powerful incentives to act as 'poachers', not least because of the fascinating opportunities for enhancing their prosperity, prestige and security that scientific and technological development open up. 116 the following passage effectively outlines states' dual function: government has an important role in setting long-term priorities and in making sure a national environment exists in which beneficial innovations will be developed. there must be a free and rational debate about the ethical and social aspects of potential uses of technology, and government must provide an arena for these debates that is most conducive to results that benefit humans. at the same time, government must ensure economic conditions that facilitate the rapid invention and deployment of beneficial technologies, thereby encouraging entrepreneurs and venture capitalists to promote innovation. 117 given that the agent (i.e. state governments) in charge of initiating ethical debates on the progress of biotechnology is also the one expected to provide the conditions that would allow this progress to generate outcomes likely to contribute to economic growth and political superiority, it is hardly surprising that any issues likely to slow down or otherwise hinder the enormous momentum of the life sciences are omitted from public discussion. this duality further informs how risks are perceived, framed and addressed. for instance, even though most of the developing countries lack capacity to manage dual use research of concern, they do not see this as an immediate priority and prefer to invest effort and resources in improving their laboratory biosafety and laboratory biosecurity infrastructure and capacity. 118 in the view of their governments, the dangers of naturally occurring and circulating diseases constitute a far greater worry than the potential for misuse of cutting-edge research. by contrast, some developed countries, most notably the usa, have embarked on building their biological defence systems highlighting the grave threat posed by the potential use of bioweapons by non-state actors. their activities have encountered severe opprobrium as some analysts see them as a contravention of the norms embedded in the btwc. 119 the evolution of the chemical and biological non-proliferation regime epitomises the attempts of states to avert the hostile exploitation of the life sciences whilst promoting their use for 'peaceful, prophylactic and protective purposes'. the entry into force of the btwc and the chemical weapons convention (cwc) in 1975 and 1997, respectively, is indicative both of states' renunciation of chemical, biological, and toxin weapons and of their commitment to the goals of arms control and disarmament. that said, the imperfections and shortcomings of these treaties signify the influence of realist fears and political calculations that pervade international negotiations. in the case of the btwc, two points merit attention. the first pertains to the lack of verification mechanism when the treaty was first agreed back in the early 1970s. subsequent revelations of secret state-led offensive biological programmes in the former soviet union, south africa and iraq up until the early 1990s have significantly undermined the convention. second, the failure to negotiate a binding protocol in 2001 has further dimmed the prospects for strengthening the regime and thus ensuring universal compliance with its prescriptions. less acute but just as worrying is the situation regarding the cwc. even though the convention is exemplary in many respects, not least because of its verification system, almost universal membership and implementing body -organisation for the prohibition of chemical weapons (opcw)it still faces serious challenges that need to be considered. for instance, while the treaty bans the development, production, acquisition, and retention of chemical weapons, the definition of 'purposes not prohibited under th[e] convention' entails 'law enforcement including domestic riot control purposes' (article ii.9d). some commentators have argued that given the lack of a universally agreed definition what kind of activities count as 'law enforcement', this text opens a major loophole in the convention. 120 several states parties of the convention have voiced concerns in this regard. australia has noted that: the weaponisation of [central nervous system] acting chemicals for law enforcement purposes is of concern to australia due to the health and safety risks and the possibility of their deliberate misuse, both of which have the potential to undermine the global norm against the use of toxic chemicals for purposes prohibited by the convention. [ . . . ] australia's position is that it is not possible for a state party to disseminate anaesthetics, sedatives or analgesics by aerial dispersion in an effective and safe manner for law enforcement purposes. 121 critics highlight the possibility for the deployment of novel chemical weapons for the purposes of countering terrorism, something evident in the 2002 moscow theatre siege (dubrovka) when the russian security forces used a fentanyl-derivative agent, as a result of which about a sixth of the hostages and all of the terrorists involved died. 122 in 2011 the european court of human rights ruled with regard to the dubrovka operation that: there had been no violation of article 2 (right to life) of the european convention on human rights concerning the decision to resolve the hostage crisis by force and use of gas. 123 the court, nonetheless, noted that: even if the gas had not been a 'lethal force' but rather a 'non-lethal incapacitating weapon', it had been dangerous and even potentially fatal for a weakened person [ . . . ]. 124 the court further confirmed some of the earlier criticisms that were levelled against the government, particularly in terms of preparedness and provision of medical assistance. 125 according to the ruling, russia had to pay damages to all the 64 applicantsrepresentatives of siege victims. to date, russian officials have withheld information concerning the exact formula of the gas, which was used during the dubrovka operation, on security grounds. 126 given the lack of an internationally agreed definition of what constitutes 'terrorism' on the one hand, and the rise of irregular/asymmetric warfare and sporadic conflicts, on the other, some commentators have warned against the possibility of a 'grey area' which may enable states to utilise non-traditional methods of war to gain advantage. 127 deliberative systems encompass a vast array of practices, processes and mechanisms, both formal and informal, whereby a polity considers the 'acceptability, appropriateness and control of novel developments in or impacting on, shared social and physical arenas'. 128 by design, they reflect and are informed by the values, beliefs and standards shared among the group, or in other words, by the prevalent culture. as such, deliberative systems vary across societies with their intensity, inclusiveness and structure depending on the established political and social norms. yet their chief purpose and function remain virtually the same, namely to help societies adapt to the changing circumstance of their milieu in a way that ensures stability, sustainability and safety. public deliberation requires time; and wide-ranging life science advances, current and planned, offer profound challenges to shared ideas and ideals about the foundations of human relatedness and of social coherence, justice, human dignity and many other norms, both formal and informal. 129 yet given the ruminative nature of deliberative processes, on the one hand, and the fast speed at which biotechnology innovation is evolving on the other, the danger of the former being steadily outpaced and overburdened by the latter is far from hypothetical. consider the following passage sketching the scale of social changes likely to arise from the increasing convergence between nanotechnology, biotechnology, cognitive neuroscience and information technology: in the foreseeable future, we will be inundated with new inventions, new discoveries, new start-ups, and new entrepreneurs. these will create new goods and new services. [ . . . ] as expectations change, the process of politics and government will change. people's lives will be more complex and inevitably overwhelming. keeping up with the changes that affect them and their loved ones exhausts most people. they focus most of their time and energy on tasks of everyday life. in the future, when they achieve success in their daily tasks, people will turn to the goods and services, the new job and investment opportunities, and the new ideas inherent in the entrepreneurial creativity of the age of transitions. no individual and no country will fully understand all of the changes as they occur or be able to adapt to them flawlessly during this time. 130 this vision of a 'brave new world' merits attention on two important grounds. first, it implies that the changes likely to occur in the not too distant future as a result of the rapid progress of science and technology are imminent and unavoidable in the sense that their advent hardly depends on or even requires extensive public deliberation. second, given that our capacity for adaptation to and grasp of those changes will be considerably impaired, the age of transitions leaves little space for public deliberation. to add to this gloomy picture, there is already some evidence that the progress in the life sciences is overwhelming the existing deliberative mechanisms. for instance, kelle et al. argue that the rapidity of biotechnology advancement coupled with the immensity and complexity of the knowledge accumulated therefrom complicates efforts to deal with potential risks, something evident in the regulatory gap that the convergence of chemistry and biology has created in the area of arms control. 131 this is problematic, for the reduced resilience of deliberative systems provides favourable conditions in which scientific and technological innovation can continue unabated. a vicious circle is thus created in which the inability of deliberative systems to cope with the strain exerted by biotechnology advancement fuels the latter turning it into a self-propelling force. the proliferation of contentious 'gain-of-function' research is a case in point. even though the h5n1 controversy discussed in the preceding sections exposed the limitations of existing governance mechanisms for addressing the potential security, ethical, and legal implications arising from such studies, it hardly precluded scientists from conducting similar experiments. indeed, less than four months after the moratorium on research involving contagious h5n1 virus was lifted, a team of chinese researchers announced the creation of a hybrid of the h5n1 strain and the h1n1 virus that caused the 2009 flu pandemic. 132 and it was not long until the newly-emerged h7n9 influenza virus became airborne, as well. 133 if anything, those examples indicate that in light of the rapid pace of life science progress, addressing governance concerns on a caseby-case basis is not only self-defeating but given the number and variety of conundrums, it is likely to become unsustainable in the long run. given the significant potential of biotechnology to bring about multifaceted changes in different spheres of life and generate considerable benefits in the form of new products, enhancement of public and private capital and alleviation of social ills, there is a powerful urge to allow the ongoing expansion of the life sciences to proceed largely unfettered. risks are carefully calculated and, where possible, downplayed as hypothetical at the expense of comprehensive deliberation. and even when proposals for risk mitigation measures are entertained, preference is usually given to those unlikely to hinder the progress of life sciences. by and large, there is a genuine belief that the existing governance mechanisms in the area of biotechnology can accommodate and cope with the wide-ranging pressures exerted by scientific innovation and the rapid diffusion of technologies with multiple uses, by offering 'solutions' and handling concerns on a case-by-case basis. in particular, the technology of safety is still 'celebrated as an unadulterated improvement for society as a whole'. 134 yet there are reasons for scepticism toward the adequacy and effectiveness of the governance approaches currently in place. much of the discussion in the preceding sections has focused on the ways in which the increasing pace, growth and global diffusion of biotechnology advances are beginning to expose the limits of the existing measures for control and risk management by challenging accepted values and beliefs and redefining established norms of practice. as the multifaceted dynamics driving the biotechnology momentum continue to intensify and multiply, it becomes more and more difficult to comprehend, let alone foresee, the various impacts that the large-scale deployment and proliferation of novel scientific and technological advances have both on our social systems and the environment. given the tight coupling between human-made and natural systems and their complex, often unanticipated interactions with catastrophic potential, the existing narrow definitions of risk are rendered inadequate. 135 at the same time, the advent of new technologies with multiple adaptive applications opens up an array of possibilities for hostile exploitation thus compelling governments to make tough decisions in an attempt to reconcile the benefits of biotechnology with the potential security concerns arising therefrom. while the advancement of biotechnology promises tremendous public health benefits, it also holds a considerable catastrophic potential, as the case of 'gain-of-function' experiments illustrate. as scientific capabilities and work involving dangerous pathogens proliferate globally, so do risks and the prospects of failures, whether technical or arising from human error. indeed, assessing the rapidly evolving life science landscape some security commentators argue that 'current genetic engineering technology and the practices of the community that sustains it have definitively displaced the potential threat of biological warfare beyond the risks posed by naturally occurring epidemics' 136 . laboratories, however well equipped, do not exist in isolation but are an integral part of a larger ecological system. as such, they constitute a 'buffer zone' between the activities carried out inside and the wider environment. and despite being technically advanced and designed to ensure safety, this 'buffer zone', just as other safety systems is far from infallible. for one thing, mechanical controls leave room for human error and personal judgement, both of which are factors that could be highly consequential but which could hardly be modelled or predicted with exact certainty. 137 the speed at which the transformation of the life sciences is taking place is yet another factor that adds to the complexity of life science governance. stability is a fundamental condition for the development and preservation of human and natural systems alike. in social systems, culture is the primary source of stability, for it determines what values, beliefs, practices and modes of behaviour are deemed acceptable and, as such, lays the foundations of order. all forms of governance therefore are cultural artefacts and manifestations of culture. culture also provides the tacit standards whereby change is assessed and treated as acceptable or unacceptable. hence, any state of affairs in which the rate of change precludes regulation disrupts the ordinary functioning of the system and jeopardises its preservation: the breakdown of human regulation does not extinguish regulation of a simpler sort. [ . . . ] the system formed by men and the rest of the natural world will continue to regulate itself after a fashion, even if human regulation wholly fails at all levels above the primary group. but the resulting 'order' would exclude all those levels of human order which man-made stability makes possible. 138 to be sure, a world characterised by a runaway biotechnology would be far different from the one we know. the main challenge to averting this prospect lies in ensuring that the systems of governance are in sync with the progress of life sciences. history has shown that even highly developed, long-standing systems of governance can fail for reasons as diverse as disasters; loss of authority/legitimacy of governing bodies; and pervasive corruption. one further source of failure includes the inability of a society to adapt to its changing milieu: men are adaptable; they can learn to live even in harsh and hostile environmentsso long as the environment remains constant enough to give them time to learn. [ . . . ] if they form the habit of adapting by constantly changing that to which they are trying to adapt, they build uncertainty into the very structure of their lives. they institutionalise cluelessness. 139 the process of adaptation is closely connected to cultural patterns and any serious disruptions in the latter could have detrimental effects and impair it severely. the extent to which change is taking place within the framework of the prevalent culture defines the borderline between system evolution and system disintegration. the governance mechanisms currently in place, both formal and informal, are all a function of historical, cultural, and sociopolitical contingencies. as such, their capacity for adaptation largely depends on our ability to comprehend and assimilate the complex changes that the progress of biotechnology brings about. they can only evolve as fast as our shared standards, values, routines and perceptions allow them to. and that is why governance can hardly be reduced to a technocratic exercise; on the contrary, to be effective, it requires extensive deliberation and full appreciation of the far-reaching implications of novel life science advances. trends and drivers of change in the biomedical healthcare sector in europe: mapping report (dublin: european foundation for the improvement of living and working conditions chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template the test-tube synthesis of a chemical called poliovirus: the simple synthesis of a virus has far-reaching societal implications craig venter creates synthetic life form', the guardian moore's law pertains to the rapid rate of technological development and advances in the semiconductor industry, specifically the doubling of the number of transistors on integrated circuits that occurs approximately every 18 months. although advances in the life sciences occur at more random intervals and are driven by new conceptual breakthroughs in understanding of biological processes, it is a useful metaphor for the exponential growth of knowledge related to biology. see committee on the advances in technology and the prevention of their application to next generation bioterrorism and biological warfare threats, an international perspective on advancing technologies and strategies for managing dual-use risks the myth of the biotech revolution: an assessment of technological, clinical and organisational change globalization, biosecurity and the future of the life sciences innovation in global industries: u. s. firms competing in a new world biotechnology and the un's millennium development goals top ten biotechnologies for improving health in developing countries rising to the challenge: u.s. innovation policy for global economy innovating in the new austerity, burrill & co's 26th annual report on the life sciences industry globalization and the future of the life sciences, op cit an international perspective on advancing strategies for managing dual-use risks the valley of the shadow of death', dspace@mit globalization and the future of the life sciences the biotechnology promise: capacity-building for participation of developing countries in the bioeconomy converging technologies for enhancing human performance: science and business perspectives converging technologies -shaping the future of european societies taking care of the symbolic order: how converging technologies challenge our concepts enhancement technologies and the modern self the ethics of nbic convergence bringing converging technologies closer to civil society: the role of precautionary principle', innovation: the innovation in global industries, op cit in the valley of the shadow of death see the 'infectious diseases' section of the who website for an overview of the nih budget for 2015, see david malakoff and jeffrey mervis, 'first look: us spending deal a mixed bag for science within nih's flat 2015 budget, a few favourites', scienceinsider for information on the eu sixth framework programme and the activity area of life sciences cuba -battling cancer with biotechnology global status of commercialised biotech/gm crops plant genetic engineering: china hesitates on the brink', gmo safety rising to the challenge, op cit government academic, and venture firms come together in march to fund translational and early-stage development', fiercebiotech science for sale: the perils, rewards and delusions of campus capitalism relationships between academic institutions and industry in the life sciences -an industry survey the expanding role of university patenting in the life sciences: assessing the importance of experience and connectivity university-industry relationships and the design of biotechnology research entrepreneurial science in the academy: a case of the transformation of norms the triple helix of university-industry-government relations the triple helix: university-industry-government innovation in action the dynamics of innovation: from national systems and "mode 2" to a triple helix of university-industry-government relations commercialisation of the university and the problem choice by academic biological scientists see also dina biscotti et al. 'the "independent investigator": how academic researchers construct their professional identity in university-industry agricultural biotechnology research collaborations toward more secrecy in science: comments on some structural changes in science -and on their implications for an ethics of science varieties of secrets and secret varieties: the case of biotechnology for a detailed analysis on the us decision to reject the draft btwc protocol, see malcolm dando the white house the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences bioterrorism and smallpox planning: information and voluntary vaccination taking biodefence too far new labs, more terror a plague of researchers mixing bugs and bombs lab loses trio of plague mice', nature news anthrax letters' attack and the controversy of the us biodefence programme see jeanne guillemin army suspends germ research at maryland lab back to bioweapons? international health regulations (who, 2005), specifically section 'laboratory pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits (who for further discussion on the implementation of biotechnology regulations, see bo sundqvist et al. 'harmonisation of european laboratory response networks by implementing cwa 15793: use of gap analysis and an "insider" exercise as tools on the development of health and safety regulations on the use of nanotechnology, see eileen kuempel et al. 'risk assessment and risk management of nanoparticles in the workplace: translating research into practice there is a debate on whether the 'american model' of science-policy making underpinned by neoliberal ideology is fully embraced in europe. see, for example, gabriele abels, 'the long and winding road from asilomar to brussels: science, policy and the public in biotechnology regulation further, a policy paper issued by a business taskforce appointed by the uk government issued a policy paper in late 2013 demanding the liberalization of the existing eu legislation which, in their view, 'places restrictions on products and technologies without adequate evidence of risk'. see department for business, innovation & skills and the prime minister's office, cut the eu red tape: report form the business task force proposed framework for the oversight of dual use life science research: strategies for minimising the potential misuse of research information biosecurity reconsidered: calibrating biological threats and responses some commentators have expressed scepticism toward the claim that scientific and technological advancement poses serious threats underscoring the importance of other factors, such as socio-economic and socio-technic contexts. see, for example, kathleen vogel, 'intelligent assessment: putting emerging biotechnology threats in context others, however, argue that advances in modern biology and medicine have implications for the evolution of biological weapon programmes. see malcolm dando, 'the impact of the development of modern biology and medicine on the evolution of offensive biological warfare programmes in the twentieth century expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox the efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin-4 disaster in the making creation of killer poxvirus could have been predicted chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template a tale of two studies: ethics, bioterrorism and the censorship of science first synthetic virus created', bbc news university of bradford, available at www.brad.ac.uk/ acad/sbtwc (accessed 8/03/2013). see also german ethics council, biosecurity -freedom and responsbility of research on the framing of risk in biotechnology, see geert van calster, 'risk regulation, eu law and emerging technologies: smother or smooth? ethics of risk analysis and regulatory review: from bio-to nanotechnology european biotechnology regulation: framing the risk assessment of a herbicide tolerant crop framing the uncertainty of risk: models of governance for genetically modified crops denmark's regulation of agri-biotechnology: co-existence bypassing risk issues a distorted regulatory landscape: genetically modified wheat and the influence of non-safety issues in canada on the shortcomings of the existing models for public deliberation on the risks of biotechnology, see les levidow, 'european public participation as risk governance: enhancing democratic accountability for agrobiotech policy for critique of cost-benefit analysis, see also brian rappert the creation of contagious h5n1 avian influenza virus: implications for the education of life scientists all meetings of the us national science advisory board for biosecurity (nsabb) convened to discuss the manuscripts were restricted to selected individuals and full proceedings were never published. moreover, the consequential consultation meeting organised by the world health organisation (who) in february 2012 which rejected the nsabb recommendation for a redacted publication of the manuscripts featured the lead scientists who conducted the experiments and representatives of the us national institutes of health flu experts -and one ethicist -debate controversial h5n1 papers', scienceinsider fight over dutch h5n1 paper enters endgame', scienceinsider fight over dutch h5n1 paper enters endgame', scienceinsider dutch appeals court dodges decision on hotly debated h5n1 papers', scienceinsider bias accusation rattles us biosecurity board experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets airborne transmission of influenza a/h5n1 virus between ferrets: materials/methods, supporting text, tables, figures, and/or refences safety incidents" at animal lab', bbc news notes 155 airflow problems plague cdc bioterror lab high-containment laboratories: assessment of the nation's need is missing the scientist lab infection blamed for singapore sars case between 2014 and 2015, several high-containment facilities in the us have experienced serious biosafety lapses. accidents involving dangerous pathogens such as the causative agents of anthrax and bird flu were reported biosafety in the balance' anthrax? that's not the real worry cdc closes anthrax and flu labs after accidents about the same time, there were also reports about smallpox vials being retrived after having been left unaccounted for over 50 years. see ap, 'forgotten vials of smallpox found in storage room gain-of-function' influenza research and proposal to organise a scientific briefing for the european commission and conduct comprehensive risk-benefit assessment the third revolution: the convergence of the life sciences see committee on biomocular materials and processes, national research council, inspired by biology: from molecules to materials to machines a new biology for the 21st century nanotechnology: what it can do for drug delivery facing the truth about nanotechnology in drug delivery carbon nanotubes -bullets in the fight against cancer on the governance challenges brought about by the convergence between biology and other fields of science, see francis fukuyama and caroline wagner, information and biological revolutions: global governance challenges -summary of a study group report of the first workshop the body's own bioweapons innovation, dual use, and security: managing the risks of emerging biological and chemical technologies life sciences and related fields: trends relevant to the biological weapons convention building better implants some nanotubes could cause cancer and breeding: panacea or pandora's box?', news release on the security implications of synthetic biology, see international council for the life sciences synthetic biology and biosecurity awareness in europe on the social and ethical aspects of synthetic biology, see presidential commission for the study of bioethical issues, new directions: the ethics of synthetic biology and emerging technologies synthetic biology: social and ethical challenges framing biosecurity: an alternative to the biotech revolution model vogel's view are contested in jonathan tucker 'could terrorists exploit synthetic biology lax laws, virus dna and potential for terror on the issue of commercial order screenings, see stephen maurer et al. making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders on the governance of synthetic biology, see catherine lyall dna synthesis and biological security industry self-governance: a new way to manage dangerous technologies unpacking synthetic biology: identification of oversight policy problems and options synthetic biology, security and governance'¸biosocieties dna synthesis and security science enters $1,000 genome era', bbc news happened when i had my genome sequenced', the observer for information about commercial companies offering full-genome sequencing genome-based therapeutics: targeted drug discovery and development: workshop summary dna of 100,000 people to be mapped for nhs', the guardian systems cancer medicine: towards realisation of predictive, preventive, personalised and participatory (p4) medicine' grateful and moved" by reaction to her mastectomy decision', the guardian dna blueprint for fetus built using tests of parents cure for illness raises ethical fear', the guardian what happened when i had my genome sequenced another point that cadwalladr raises is the danger of a negative placebo effect whereby doubts about certain genetic disorder may lead to psychosomatic symptoms from eugenics to patents: genetics, law, and human rights unhidden traits: genomic data privacy debates heat up from eugenics to patents see also harriet washington, deadly monopolies: the shocking corporate takeover of life itself -and the consequences for your health and our medical future taking life: private rights in public nature benefits and threats of developments in biotechnology and genetic engineering' in sipri yearbook, armaments ethnic-affiliation estimation by use of population-specific dna markers racial differences in the response to drugs -pointers to genetic differences the achilles' helix from eugenics to patents three-person" embryos to combat genetic disease', the guardian innovative genetic treatment to prevent mitochondrial disease, press release three-parent embryos for mitochondrial disease? twelve reasons for caution february 2015, the uk passed legislation allowing the use of the technique. see james gallagher, 'uk approves three-person babies', bbc news building baby from the genes up one commentator distinguishes between 'big science' which was 'todown, hierarchical, vertical' and 'networked science' characterised by 'open systems, open software, open participation'. see diane rhoten biopunk: solving biotech's biggest problems in kitchens and garages life hackers biology is technology: the promise, peril, and new business of engineering life diffusion of synthetic biology: a challenge to biosafety do i understand what i can create: biosafety issues in synthetic biology charge dropped against artist in a terror case strategies to educate amateur biologists and scientists in non-life science disciplines about dual use research in the life sciences governing amateur biology: extending responsible research and innovation in synthetic biology to new actors, research report for the wellcome trust project 'building sustainable capacity in dual use bioethics secret project manufactured mock anthrax', the washington times biological weapons and america's secret war global governance and the twenty-first century technology converging technologies for improving human performance: nanotechnology, biotechnology, information technology and cognitive science report of the who informal consultation on dual use research of concern biological threat assessment: is the cure worse than the disease? dangerous ambiguities: regulation of riot control agents and incapacitants under the chemical weapons convention weaponisation of central nervous system acting chemicals for law enforcement purposes, xix session of the conference of the states parties moscow theatre siege: questions remain unanswered', bbc news press release: use of gas against terrorists during the moscow theatre siege was justified, but the rescue operation afterwards was poorly planned and implemented spasenie zalozhnikov ili unichtozhenie terroristov?', novaya gazeta the 2002 dubrovka and 2004 beslan hostage crises: a critique of russian counter-terrorism sekretov bol'she net', rossiskaya gazeta see also national research council, avoiding surprise in an era of global technology advances the challenge to deliberative systems of technological systems convergence converging technologies for improving human performance, op cit preventing a biochemical arms race, op cit bringing the bwc conventions closer together', the cbw conventions bulletin h5n1 hybrid viruses bearing 2009/h1n1 virus genes transmit in guinea pigs by respiratory droplet infectivity, transmission, and pathology of human-isolated h7n9 influenza virus in ferrets and pigs limited airborne transmission of h7n9 influenza a virus between ferrets shifting the blame: literature, law, and the theory of accidents in nineteenth-century america normal accidents, op cit building a bio world shifting the blame, op cit freedom in a rocking boat: changing values in an unstable society some commentators have critiqued the work of diamond on the grounds of simplicity. for a summary of some of the criticisms levelled at his work, see eric powell key: cord-301328-13adnvav authors: lowenthal, john title: overview of the csiro australian animal health laboratory date: 2016-04-24 journal: j infect public health doi: 10.1016/j.jiph.2016.04.007 sha: doc_id: 301328 cord_uid: 13adnvav emerging infectious diseases arising from livestock and wildlife pose serious threats to global human health, as shown by a series of continuous outbreaks involving highly pathogenic influenza, sars, ebola and mers. the risk of pandemics and bioterrorism threats is ever present and growing, but our ability to combat them is limited by the lack of available vaccines, therapeutics and rapid diagnostics. the use of high bio-containment facilities, such as the csiro australian animal health laboratory, plays a key role studying these dangerous pathogens and facilitates the development of countermeasures. to combat diseases like mers, we must take a holistic approach that involves the development of early biomarkers of infection, a suite of treatment options (vaccines, anti-viral drugs and antibody therapeutics) and appropriate animal models to test the safety and efficacy of candidate treatments. over the past three decades, there has been an increase in the incidence of emerging infectious diseases (eids) in humans, with approximately 70 percent of them arising from animals. a number of factors, including the geographic expansion of human populations, intensification of agriculture and habitat disruption due to climate change and deforestation, have led to a greater risk of eids being transmitted from wild and domesticated animals to humans [1] . furthermore, increased global travel and trade has increased the likelihood that eids will rapidly spread. eid outbreaks are unpredictable and often difficult to contain due to the absence of effective control measures such as vaccines and antiviral therapeutics. the world health organization has warned that the next human pandemic is likely to be zoonotic and that wildlife is a prime culprit. while the current list of known eids is a major concern, it is the existing unknown threats with the potential for efficient human-to-human transmission that pose the largest concern. over the past decade, there have been a number of epidemics, raising the concern that they are precursors to a pandemic. examples include the highly pathogenic h5n1 avian influenza virus that has decimated poultry production in asia and claimed over 350 lives since 2003 with continuing regular outbreaks, the hendra virus in australia, the nipah virus in malaysia and bangladesh and hemorrhagic fever viruses (ebola and marburg), which have emerged from bats via intermediate hosts, such as horses and pigs, to infect and kill humans over the past two decades. the sars epidemic in 2003-2004 claimed over 800 lives and cost more than $80b to the global economy. the virus was shown to be transmitted from bats to civet cats to humans. in 2012, a novel coronavirus emerged in the middle east (mers-cov), with a 37% mortality rate for the more than 1600 currently confirmed cases in 26 counties. bsl3 and 4 facilities must conform to strict infrastructure requirements, policies and procedures to ensure the safety of researchers who are working with a range of dangerous pathogens. there are several international bodies that develop and maintain biosafety guidelines. in the united states, it is the centers for disease control and prevention (cdc) in partnership with the u.s. national institutes of health biosafety (http://www.cdc.gov/biosafety/ publications/bmbl5/bmbl.pdf). the european guidelines are set by a legislative act of the european union. in australia, the department of agriculture and the office of the gene technology regulator have this responsibility. while regulations surrounding bsl3 and 4 facilities may differ from country to country, the basic principles of biocontainment are uniformly observed. for example, all work involving potentially infectious material must be conducted within primary containment, such as a biological safety cabinet. in the case of bsl4 facilities, primary containment is provided by a class iii biological safety cabinet located within a bsl4 cabinet laboratory or by the wearing of positive pressure protective suits with an independent breathing air supply (bsl4 suit laboratory or animal facility). examples of bsl-4 laboratories around the world include the national microbiology laboratory (winnipeg, canada, http://www. nml-lnm.gc.ca/index-eng.htm), the pirbright institute (pirbright, uk, http://www.pirbright.ac.uk/), the uniformed services university of the health sciences (bethesda, usa, http://www.usuhs. mil/) and the csiro australian animal health laboratory (geelong, australia, http://www.csiro.au/ places/aahl). the aahl is one of the world's premier highbiocontainment facilities, allowing researchers to work with bsl4 pathogens that are highly lethal to humans and for which there is no vaccine or effective treatment. the aahl is unique in the world in its capacity to undertake studies on a wide range of large numbers of domestic animals and wildlife [2] . at the aahl, exotic disease agents are used in the laboratory for researching emergency disease diagnoses and studying the relationships between the pathogens and different animal and human hosts. the aahl facility is unique in that its bsl3 and bsl4 animal facilities are sufficiently large to allow researchers to study a range of security sensitive biological agents (ssbas) in diverse species, including ferrets, bats, poultry, pigs, dogs, alpacas and horses, as well as small laboratory mammals. as one of only six high-containment animal research centers in the world, we work with national and international human and animal health organizations as part of a global one health network. aahl's mission is to be prepared to quickly and effectively respond to any new emerging infectious disease that may emerge. it does so by working with government and industry to assist in responding quickly to stop threats in their tracks and provide sustainable management strategies. we are exploring new technologies for detection, surveillance, diagnosis and response, and we will continue preparing for the next human pandemic. the csiro emerging infectious diseases program located at aahl has assembled a strong set of multidisciplinary research teams spanning the areas of virology, immunology, veterinary sciences and animal models. by understanding disease emergence and the host response to pathogens, we will inform public policy and develop innovative technologies to enable our industry partners to manufacture and deploy novel disease treatments to protect us from infectious diseases that threaten our wellbeing, economy and environment. the use of mouse models has been fundamental to our understanding of human infection and disease, and mice have become the traditional 'workhorse' because of their ease of handling, their fast generation time and the ready availability of mousespecific reagents. however, for many zoonotic pathogens, there are differences in the symptoms of the disease between the natural reservoir animal host (such as a bat or bird) and human hosts. frequently, zoonotic infections appear asymptomatic and are non-lethal in the natural host, yet induce severe and potentially lethal disease in humans or other spillover hosts. nevertheless, there are numerous factors that are likely to contribute to these differences, including anatomical, physiological, metabolic and behavioral traits, as well as how the immune systems of these hosts interact with the same disease agent. therefore, for a better understanding of eids, the laboratory mouse may not be the most appropriate model. there are many examples in the literature where non-traditional animal models have been highly informative for our understanding of the host responses to pathogens [3] . for example, we are using bats to study several emerging viruses such as hendra virus, and ferrets, which are widely accepted as an excellent model for influenza infection; they are naturally susceptible to infection with human influenza viruses and the disease pathology they develop resembles that of humans infected with influenza. furthermore, by studying the pathogen in its natural host, we may be able to devise efficient control measures in that host, thereby disrupting their transmission to humans. this has important implications for predicting, preventing and controlling spillover events and for the development of novel therapeutics and diagnostics. it is very difficult and often impossible to test the efficacy of new treatments for highly lethal infections in human clinical trials. animal models for human eids can play a key role in the development and testing of candidate vaccines and therapeutics and can be used to facilitate their regulatory approval. the us food and drug administration (fda) has developed the animal rule to assist in the regulatory approval process [4] . the rule states that when it would not be ethical to perform human challenge studies to measure the efficacy of vaccines and drugs developed to prevent or treat highly pathogens, the fda may grant approval based on appropriate animal efficacy studies when the results of those studies establish that the drug is reasonably likely to produce a clinical benefit in humans. the animal rule states that the fda will only rely on evidence from animal studies to provide substantial evidence of effectiveness when strict criteria are met. at the aahl, we have pioneered the concept of reducing the risk of human infection by breaking the transmission chain of a zoonotic agent from an animal host to the human. hendra virus circulates in its natural reservoir host, the fruit bat, without producing clinical disease. on occasions, the virus spills over to horses and causes a rapidly lethal respiratory disease, which can be easily spread to humans by direct contact. hendra virus causes 70% mortality in humans, but only a small number of cases have occurred, making the development of a human vaccine economically non-viable. instead, we developed a vaccine for horses (equivac hev, zoetis australia) that protects them from hendra virus infection and therefore indirectly prevents the infection of humans [5] . we suggest that a similar approach of vaccinating camels against mers will help prevent mers infection of humans. however, the timeline and obstacles required to develop such a vaccine cannot be underestimated [6] . no funding sources. global trends in emerging infectious diseases a road less travelled: large animal models in immunological research hendra virus vaccine, a one health approach to protecting horse, human, and environmental health middle east respiratory syndrome: obstacles and prospects for vaccine development none declared. not required.available online at www.sciencedirect.com key: cord-293143-1k170shh authors: dieninghoff, doris; karagiannidis, christian; straßmann, stephan; pieper, monika; dammaschek, sarah; zabner, joseph; klingelhutz, aloysius; windisch, wolfram; brockmann, michael; schildgen, oliver; schildgen, verena title: fatal hbov-1 infection in adult female cystic fibrosis patient date: 2016-07-18 journal: hum pathol (n y) doi: 10.1016/j.ehpc.2016.07.001 sha: doc_id: 293143 cord_uid: 1k170shh a clinical case of fatal hbov infection in an adult cystic-fibrosis patient awaiting lung transplantation is reported. the case is important as the genetic background of the underlying disease is congruent with the background of the sole permissive permanent cell culture cufi-8 which originates also from a cf patient donor. a clinical case of fatal hbov infection in an adult cystic-fibrosis patient awaiting lung transplantation is reported. the case is important as the genetic background of the underlying disease is congruent with the background of the sole permissive permanent cell culture cufi-8 which originates also from a cf patient donor. the human bocavirus (hbov) is a parvovirus that is associated with acute and chronic infections of the upper and lower respiratory tract, persists in some tissues and solid cancers and putatively may play an aetiologic role in the development of idiopathic lung fibrosis [1] [2] [3] [4] [5] [6] [7] [8] . the viral infection can switch between long term periods of viral shedding and silent phases of latency [9] . to date, no animal model exists, thus studies on the pathology of hbov infections are limited to clinical studies, case descriptions, and air-liquid interface cell culture models that have been shown to mimic some important steps of the infection cycle [1, 7, [10] [11] [12] . the latter infections models are based on either primary cells that can be differentiated into organ like tissues from primary cells [1, 7, [11] [12] [13] or cufi-8 cells [1, 10, 13] . cufi-8 is a cell line derived from a 24 year old female patient suffering from cystic fibrosis; the cell line carries the mutation pattern δf508/δf508 (the most common cf associated mutation) and is an immortalized human epithelial airway cell line from the bronchus of the patient; the immortalization was performed with weinberg htert and hpv-16 e6/e7 [10, 14] . this information might be relevant for a current clinical case that was treated in our hospital in cologne. the patient was a 24 year old non-smoking caucasian cystic fibrosis patient. she was regularly treated in our cystic fibrosis outclinics and was deemed to be eligible for lung transplantation shortly before the fatal clinical episode started. in advance to the lung transplantation, a microbiological check-up revealed colonization of the lung with low titres of aspergillus and a multiresistant pseudomonas aeruginosa strain, combined with allergic asthma, pancreatic insufficiency, and malnutrition (bmi 14-15 kg/m 2 ). in order to overcome the malnutrition, percutaneous endoscopic gastrostomy (peg) was required and initiated in our hospital. the patient started to recover well, episodically required non-invasive ventilation (niv), but was in a condition stable enough to continue niv at home. the night before the patient should have left the hospital she developed an ards unexpectedly and was shifted to our intensive care unit. during intensive care treatment, despite expected obstipation, the patient developed massive diarrhea. in order to identify a pathogen causative for the ards, a bal was performed and analysed for respiratory bacteria and viruses. an all-embracing molecular and microbiological diagnostic algorithm was performed as described earlier [15] [16] [17] , including molecular screening for human herpesviruses 1-8, parechoviruses, rhinoand enteroviruses, adenoviruses, influenza a and b virus including h1n1, human metapneumovirus, respiratory syncytial virus a and b, coronaviruses 229e, oc43, hku-1, nl63, parainfluenzaviruses 1-4, mumps and measles, and human bocavirus, mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumophila, and bordetella pertussis, but revealed human bocavirus as the sole pathogen detectable in the bal during the fatal clinical flare of the ards. detailed protocols for the diagnostic procedures were published previously [1, 9, [16] [17] [18] . moreover, any attempts to culture further respiratory pathogens including mycobacterium tuberculosis and atypical mycobacteria revealed negative results. although the clinical course could have been triggered by the pseudomonas aeruginosa or aspergillus colonization and no pseudomonas cepacia infection was differentially excluded, the most likely explanation for the fatal outcome remains the sole infection with human bocavirus. in this context it is important to note, that the patient was human pathology: case reports j o u r n a l h o m e p a g e : h t t p : / / w w w . h u m a n p a t h o l o g y c a s e r e p o r t s . c o m homozygous for the cystic fibrosis genotype δf508/δf508, and as the donor of the cufi-8 cell line, at the time point of death was 24 years of age. it is important to mention, that the bal was characterized by serious bleeding, which may have contributed to the clinical outcome. this bleeding is another parallel to the cufi-8 cells: the cells are permissive for hbov if they are seeded to filter membranes and differentiated to pseudostratified air-liquid interface cultures; thereby, they fall dry and develop a mucus layer on top, while receiving nutrition from the basal medium. if those air-liquid interface cultures are infected with hbov they develop a serious cytopathic effect and may become "leaky", i.e. the basal cell culture medium passes the membrane, which can be compared to the clinical bleeding. the fact that age, sex, and genetic phenotype for the patient and the permissive cufi-8 cell culture were equal solicits the cautious conclusion that the patient was highly permissive for the hbov infection and died because of the serious damages the virus initiated. this hypothesis is further supported by the fact that the patient suffered from diarrhea, although cystic fibrosis in concert with pancreatic insufficiency is generally accompanied by obstipation. in this context it is important to mention that hbov-1 is not generally associated with diarrhea but is observed in hbov-2 and 3 infections [19] [20] [21] [22] ; however, hbov-1 can be shed via the gastrointestinal route after swallowing and was repeatedly found in colorectal malignancies [23, 24] . unfortunately, due to the fact that the clinical course progresses so rapidly, and that the clinical colleagues were not aware of the parallels between the genetic background of the cufi-8 cells and the patient, no further clinical specimen were collected for scientific purposes, thus no further laboratory investigations could be performed, although this would not have changed the clinical decision making. this is a considerable limitation of our report, which in our view still contains important hints regarding the pathogenesis of hbov. taking into account the fact that it remains formally difficult to attribute clinical causalities to human bocavirus as previously discussed by martin [25] and byington [26] , a permissive animal model for studying hbov pathology would be a great advantage. however, although exclusively investigating a clinical cohort, byington [26] and coworkers have unintendedly confirmed the assumption that hbov is indeed a true pathogen rather than a blind and silent passenger [27] , which was further confirmed by our recent translation study in which we have shown that hbov-1 induces a profibrotic and procancerogenic cytokines expression in vivo and in vitro [1] . we also have to critically discuss the putative link between the cf genotype of the patient and the cell culture and its relation to the severe outcome of the hbov infection. so far it remains a matter of speculation if there is a causality between these co-incidences or if the clinical case was just a rare event that was congruent to our observations in cell culture. however, it remains possible that such a causative link exists and therefore this clinical case presentation is intended to trigger further research in this direction. as a consequence, more detailed clinical studies that focus on the high risk group of cystic fibrosis patients suffering from hbov infections are required, and it needs to be tested in vitro if cystic fibrosis specific factors contribute to a more severe clinical course of the hbov infection of those patients. all procedures described in this case report were performed with approval from the ethic committee of the university of witten (vote 73/2012). lung infection by human bocavirus induces the release of profibrotic mediator cytokines in vivo and in vitro human bocavirus: from common cold to cancer? speculations on the importance of an episomal genomic form of human bocavirus comparison of tissue distribution, persistence, and molecular epidemiology of parvovirus b19 and novel human parvoviruses parv4 and human bocavirus persistence of human bocavirus dna in immunocompromised children the human bocavirus is associated with some lung and colorectal cancers and persists in solid tumors bocavirus episome in infected human tissue contains non-identical termini human bocavirus 1 infects commercially available primary human airway epithelium cultures productively detection of head-to-tail dna sequences of human bocavirus in clinical samples latent infection of human bocavirus accompanied by flare of chronic cough, fatigue, and episodes of viral replication in an immunocompetent adult patient establishment of a reverse genetics system for studying human bocavirus in human airway epithelia human bocavirus can be cultured in differentiated human airway epithelial cells replication of an autonomous human parvovirus in non-dividing human airway epithelium is facilitated through the dna damage and repair pathways in vitro modeling of human bocavirus 1 infection of polarized primary human airway epithelia development of cystic fibrosis and noncystic fibrosis airway cell lines detection of hbov dna in idiopathic lung fibrosis acute human bocavirus infection in mds patient case report: human bocavirus associated pneumonia as cause of acute injury, cologne low copy number detection of hbov dna in bal of asymptomatic adult patients human bocavirus in an immunocompromised child presenting with severe diarrhea new molecular approaches in the diagnosis of acute diarrhea: advantages for clinicians and researchers a wide variety of diarrhea viruses circulating in pediatric patients in thailand detection of a bocavirus circular genome in fecal specimens from children with acute diarrhea in beijing the human bocavirus is associated with some lung and colorectal cancers and persists in solid tumors screening of human bocavirus in surgically excised cancer specimens human bocavirus 1 primary infection and shedding in infants community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study respiratory infections with human bocavirus the authors declare that no conflict of interest apply according to the icmje definitions. no funding was available for this report. key: cord-291909-x0sfwqnk authors: butler, colin d.; higgs, kerryn; mcfarlane, rosemary anne title: environmental health, planetary boundaries and limits to growth date: 2019-09-12 journal: encyclopedia of environmental health doi: 10.1016/b978-0-12-409548-9.10651-7 sha: doc_id: 291909 cord_uid: x0sfwqnk published almost 50 years ago, the limits to growth remains relevant to contemporary environmental health, though, paradoxically, this relevance is scarcely recognized. the seminal ideas it presented provide a useful background, as do the later planetary boundaries analyses, with which to consider key issues in contemporary environmental health. to be more than reactive, it is necessary to understand the complexity and interactions of integrated environmental health risks, including the possibility of significant global population decline within the current century. this contribution provides an overview to the limits to growth, linking it especially to the “planetary boundaries” of climate change, biodiversity loss and novel entities (including artificial substances and genetically modified organisms). the gradual increase in the amount of primary energy required to generate useable energy is also argued to be an under-recognized contributing factor to the decline in real wages growth for much of the world’s population since then, although this aspect may be improving. these elements have positive and negative health effects, which we discuss. only ones that allowed civilization to continue without crisis. if the human economy maintained business as usual, the team found, it would collide with the physical realities of a finite planet by the second half of the 21st century, triggering social collapse. initially, the book received a positive response and some of the recommendations were adopted by several countries. canadian prime minister pierre trudeau and us president jimmy carter each commissioned studies of the impact of physical limits on the global future and their national prospects. although these studies examined the outlook only as far as 2000, their conclusions to that date confirmed those of the original ltg study. right from the start, however, economists were critical, even abusive, creating a negative impression that persists in some quarters, todayddespite the rapidly accumulating evidence of the basic robustness of the ltg projections and assumptions. robert gillette, who attended the ltg launch for the journal science, noted that the "assumption of inevitable economic growth" represents "the very foundation" of economics. any "limit" to growth challenged this foundation. it is unsurprizing that most economists attacked the ideas vigorously, an assault that illustrates the conflict between the core assumptions of economists and those of the physical sciences. economics adopts a standard model where production and consumption exist in a circular flow, without a natural context. it is a world of business and individual, producer and consumer, labor and goods. the physical world, which supplies resources and provides a site where wastes can be discharged, is not seen as essential and does not affect the equations, though, occasionally, the concept of "externalities" (which can be negative or positive) is mentioned. ecological economists reject the argument that human activity is independent of nature, which they consider a conceit. instead, nature is accepted as the indispensable foundation of human activities. physics really matters; questions of depletion and pollution are inescapable. several researchers have compared the mit projections with what has actually happened since, establishing that the correlation between the standard run and real world trends over the intervening years is extremely close. one of these researchers, graham turner, compared the standard run's modeled trajectory with 40 years of historical data (see fig. 1 ). he concluded that the data for 1970-2010 approximated the standard run of the ltg model, although the figure shows a slight but favorable divergence for the trajectory of non-renewable resources, such as fossil fuels, phosphate and concentrated, rich sources of ores. systems ecologists charles hall and john day also compared the standard run with actual data to 2008. despite the common perception that the ltg work had failed, the model's performance was not invalidated, unlike models made by economists which are rarely, if ever, accurate over such a long time span. in 2018, jørgen randers, part of the original ltg team, using an updated model, compared the ltg projections with real world data up to 2017. he found that real world outcomes have approximated the second ltg scenario, the "standard run with extra resources," or "pollution crisis." based on observed data further processed by turner (up to 2010) trent modelled by the ltg study (1970-2100) (ltg) peak health? 2010 c2030: population declines due to increasing cases of regional overload 2050 2100 meadows et al. for 1900-70 and updated by turner with data to 2010. it also introduces the concept of "peak health." this is the point at which human population well-being reaches its zenith, if the ltg model (of decline this century) proves reliable. although the timing is imprecise, peak health will precede the exact moment of maximum population. peak health and unwanted population decline are not inevitable; even today they could be postponed, perhaps indefinitely, by enlightened policies and technological breakthroughs. many resource analysts have identified declining "energy return on energy investment" as a key to understanding the slowing of the rate of improvement of living standards for most people in high-income countries. in turn, these indicators reflect the decline in easily accessible fossil fuels. this subject will be returned to. it is important to understand that the broad trends described in the figure and used in the ltg model do not capture the entire world. no model can. these trends were chosen because they captured many aspects of the material world, both human and natural, including feedback loops and potential crises that might threaten the human economy and thus wellbeing. indicators of these trends were chosen for a number of reasons, including researchers' attempts to choose representative indicators that would faithfully reflect the trends, the need for parsimony and, especially back in 1972, the difficulty of finding accurate data. the decline in population that is modeled is a logical consequence of the decline in resources per capita, whether non-renewable, or as food, industrial output and services. if these inputs decline, the modelers assume, so will human population, which depends on them. the concept of the ecological footprint is among the most important developments in thinking related to the ltg. devised in the 1990s by william rees and mathis wackernagel, this measure enables ecological impacts (individual, national or global) to be quantified and compared. on one hand, it estimates the ecological assets required to produce the resources consumed by any discrete population; this includes food and fiber plants, livestock and fish, timber and other forest products, space for urban infrastructure and whatever "sinks" are needed to absorb the waste produced, especially carbon dioxide emissions. the unit of measurement adopted is the area of biologically productive land and water, usually expressed in hectares. on the other hand, the ecological footprint also estimates the productivity of a country's actual ecological assets (cropland, grazing land, forest land, fishing grounds, and built-up land). researchers using the ecological footprint methodology calculated that, while the world's biocapacity averages 1.7 ha. per capita, high-income "developed" countries greatly exceed this average. examples include the united states (8.8 ha. per capita) and the united kingdom (5.1 ha. per capita). the us, which has far more productive land available than the united kingdom, appropriates 1.27 times its own biocapacity through imports, and the united kingdom almost three times. many island nations and arid countries such as saudi arabia exceed their biocapacity by a factor of more than ten. the ecological footprint has the strengths and weaknesses of any aggregate indicator: the concepts and units are easy to understand by policy-makers and the public, but it does not encompass all aspects of human environmental impact (methane, e.g., is not integrated). this matrix needs to be used in conjunction with other indicators. another related development is the framework called "planetary boundaries," devised by a large team led by johan rockström (see following section). although the environmental health literature has long identified links between health and indicators used in the ltg model, such as food, services, and pollution, there has been little recognition among the health community, including within public health, of the possibility of a reduction in population this century. such reductions, as mentioned above, are forecast by most ltg scenarios, including the standard run (see fig. 1 ). such a reduction, were it to occur, will have impacts on public health. there are a few exceptions to this generalization. in 1972, the human ecologist frederick sargent, in an article in the american journal of public health, warned that human "interventions in and manipulations of the processes of the planetary life supportsystem (ecosystem) have produced a set of complex problems" (page 629). in 1973, the visionary socialist economist barbara ward co-authored "only one earth" with the microbiologist, pioneering "earth physician" and pulitzer prize winner, rené dubos. this book stated in part "the charge to the [1972 stockholm] conference was clearly to define what should be done to maintain the earth as a place suitable for human life not only now, but also for future generations" (emphasis added) (page xiii). health is implicit in this statement, as is sustainable development. in 1993 mcmichael, who was influenced by dubos, echoed sargent's term, writing in the foreword to his influential book planetary overload, that "the most serious potential consequence of global environmental change is the erosion of earth's life-support systems. yet, curiously, the nature of this threat to the health and survival of the world's living speciesdincluding our owndhas received little attention" (page xiii). a quarter of a century later, little has changed. although keynote talks by mcmichael and john last at the 1993 conference of the international epidemiological association warned of the dangers to global public health of global environmental change, there has been barely any recognition or follow up, at the broad integrated dimension. ltg receives little recognition now. a literature search for the term "limits to growth" in association with "health" reveals little other than work involving the authors of this contribution and their close collaborators. this is unfortunate. the persistence of single-issue approaches to address complex problems retards our ability to act effectively. this is the case where there is singular focus on climate change. the impact on human health and wellbeing is modified by many interacting factorsdincluding population, global demand and availability of resources and services and the waste and pollution we generate. other than noting the exponential growth of carbon dioxide in the atmosphere, ltg did not address climate change as such, but it modeled these factors as part of a complex system. although infrequently, the issues of resource depletion and population pressure have featured in some medical curricula, from at least the 1950s, preceding publication of the ltg by over a decade. pioneering writers and speakers have included colin bertram, peter parsons, and roger short. john guillebaud, the world's first clinician professor of family planning and reproductive health, has repeatedly spoken on the issue of both consumption and population, to diverse audiences including medical practitioners, nurses, medical students and others, since 1971. in 2011, a special issue of the american journal of public health focussed on peak oil, an important aspect of ltg. a scattering of other articles in the health literature have mentioned peak oil, but ltg is far more than peak oil. it is also far more than global warming. the reasons for the general failure of the public health literature to engage with ltg are complex but include an incorrect belief that ltg was discredited, over-specialization within the public health community, political suppression of the core ideas, and a lack of funders. the issue has had very few champions. this lack of engagement is not from lack of evidence. the term planetary boundaries (pbs) was first published in 2009. these boundaries were defined, initially, as referring to nine earth system processes (see table 1 ), each of which had been, is and will be modified by human actions. the first planetary boundary paper (published in 2009 in ecology and society, subtitled "exploring the safe operating space for humanity") explicitly acknowledges its debt to the ltg framework. a "safe operating space" implies the existence of multi-dimensional limits, just as does the word "boundaries". nonetheless, the links between pbs and the ltg are mostly implicit. although the focus of the pb work is on identifying the criteria for a "safe operating space" for humanity, rather than that of other living species, the concept acknowledges that humans depend on the diversity of life on earth. the first pb argued to be outside the safe operating space is biological diversity. analogous to nine bodily systems (renal, hepatic, neurological and so on) the large multidisciplinary team (29 co-authors) that was responsible for the first pb articles argued that these earth system processes can still provide useful services, even if functioning outside their optimal range. however, pushed too far, even one aberrant bodily function can cause death, and just one extremely disturbed earth system process might trigger catastrophic consequences for humanity. perhaps, for example, a precipitous loss of insects could disrupt pollination, food supply, and the survival of birds and other vertebrates. in turn, loss of birds might trigger additional crop losses, as their biological control of insects and other pests (i.e., of pests that do survive) is lost. another example is the reduced complexity of the microbiome of those who live in cities and other modified environments. this in turn has been hypothesized to be a causal role in the emergence of auto-immune diseases such as type 1 diabetes. further, just as with bodily processes, earth system components are linked to, interact with, and are influenced by common causes. humans may live for decades with chronic illness, but die quickly if multi-organ failure develops. so too, earth system processes interact, but exceeding multiple planetary boundaries, either simultaneously or in close proximity, risks precipitating a steep decline towards a civilization-crippling condition. other writers have also commented on the similarities between the earth and the human system, including james lovelock, the originator of the gaia hypothesis and an early user of the term "planetary medicine." table 1 shows links between planetary boundaries, ltg and human health. the exact extent to which we are breaching planetary boundaries is still being explored. the team's 2015 paper argued that two problems are already extremely dangerous (red zone) and two others are well on the way (amber zone), also noting that the designated boundaries are inter-related and most have overlapping implications. the team considers loss of biosphere integrity as the most critical problem. rates of extinction are reckoned to be at least 100 times the background rate, possibly as much as 10,000 times. populations of vertebrate species declined by more than half between 1970 and 2012 and the biomass of wild mammals is now only about 2% of the total, which is dominated by humans and livestock. that remaining 2% is under siege, including for substances such as rhinoceros horns and pangolin scales, which have alleged therapeutic benefit, even though chiefly constituted of keratin, just as are fingernails. biological diversity of lower order organisms (within soils, among pollinators, and in the species traditionally utilized for plant-origin food, resources and medicines) are similarly in decline. also under deliberate attack are forests, especially species with valuable timber, or growing on land that can be used for crops, including oil palm. many forests are also at unintended risk, due to roads and global warming, each of which also exacerbates the risk of fire. global warming is also likely to further hasten biodiversity loss. in some cases biodiversity decline and climate change, acting together or independently, also promote the survival of new pests. infestations of tree borers, able to survive warmer winters in large numbers, render trees, forests and their associated animal life more vulnerable to disease and fire. humans, a form of life, depend on the fabric of other life on earth for their survivaldfor food, clean air and water, and numerous other ecosystem "services" (see glossary and below), as well as for novel substances, including drugs. at some level of bio-alteration the reduction in ecosystem services will reduce in a non-linear way that could cascade in a manner harmful to all civilization. smaller scaled examples include the collapse of regional fisheries or the decimation of regional harvests by novel diseases or pests, such as in ireland in the late 1840s. the khapra beetle, a pest from south asia that has evolved insecticide resistance threatens significant (up to 30%) loss of rice, post-harvest, in some regions. devastating drought in the "dry corridor" of guatemala, el salvador and honduras has been blamed for contributing to the influx of migrants attempting to enter the united states between 2014 and the present (2019). for rockström, steffen and colleagues the second most pressing danger is the radical disruption of the biogeochemical cycles, particularly nitrogen and phosphorous. in nature, most nitrogen was inert in the atmosphere, though some was mobilized by bacteria and leguminous plants. applied as fertilizer, nitrogen has greatly expanded food production, but is now cascading through our rivers, groundwater and continental shelves, initiating algal blooms and dead zones. in the case of phosphorous, the other widely dispersed fertilizer, there is an added dangerdphosphate rock is a resource in decline, with grim implications for future agriculture, especially where populations will lack the financial capacity to import it, as prices rise. land-system change is argued to be in the "amber zone," close to crossing the boundary into extreme danger, if it has not already crossed it. millions of hectares of vegetation are still being cleared every year and wetlands continue to be drained. stocks of "blue carbon," stored in plants and trees associated with water, such as kelp and mangroves, are also under threat. land-system changes enable more food, fiber and other financially valued products to be grown, but amplify the harm to several pbs: biological integrity, climate and biogeochemical cycles. oil palm plantations are displacing tropical forests in asia, africa and, increasingly, latin america, where clearing already provides cattle pasture, soybean and sugar cane. such plantations involve the death of vast numbers of individual animals and the annihilation of immense tracts of tropical forest. this boundary is underpinned by the declining remainder of tropical, temperate and boreal forests. these forests have a major role in land surfaceclimate coupling. in addition, agricultural land-system change may ultimately result in land degradation giving rise to erosion, loss of topsoil, sedimentation of waterways and degradation of coastal zones. in dryland regions, degradation is referred to as desertification. large areas are affected. the united nations organization considers that 1 billion people are at risk of desertification globally, half of whom live in africa where they face major challenges to water and food security. increasing urbanization also drives land-system change, typically in areas of high agricultural productivity. vast urban regions impact surface energy (through the "heat island" effect), alter hydrological and biochemical cycles, net primary productivity and biological diversity. they are also major foci of pollutants. as humanity becomes predominantly urbanized it is with these land systems that most of us have most intimate contact. also in the amber zone is climate change. remaining below the 2 c target, which is thought to provide a reasonable chance of avoiding catastrophic climate change, necessitates technologies which do not yet exist for extracting carbon from the atmosphere. most nascent carbon reducing technologies require considerable energy, although new forms of cement may soon be feasible and environmental health, planetary boundaries and limits to growth affordable on a large scale. research since 2015 suggests that the 2 c target may need to be adjusted downwards to provide a reasonable chance of avoiding calamitous warming, in which case climate change may already belong to the red zone. even if the commitments made at the 2015 paris conference of the parties are all honored, it currently seems likely to many analysts that global temperatures will be 2 c hotter than pre-industrial times by 2050 and nearly 3 c higher by 2100. these estimates depend on a number of variables: whether nations will adopt more ambitious pledges in the near term; whether technologies will emerge that can, at a low energetic cost, suck carbon back out of the atmosphere; whether unknown tipping points will be crossed, forcing a temperature surge. if these variables prove unfavorable, the aspirational 1.5 c maximum target may be reached by the early 2030s. there is no guarantee that the damage can be held to approximately 2 c, in particular due to the risk of amplifying feedbacks such as the release of carbon dioxide and methane from the arctic, and/or the drying and burning of the amazon forest. the capacity of the ocean to absorb co 2 is also declining. that will slow the rate of ocean acidification, but increase atmospheric heat trapping. even if temperature rise and rainfall intensity can be contained, crop yields will decline and many places will become unliveable due to excessive heat and humidity or coastal inundation. glaciers that act as a bank to store water and in some cases whose melt supplies electricity to billions in asia and south america will shrink, coral reefs and many other species will disappear, and significantdeven catastrophicdsea level rise will result. in greenland and along the entire coast of west antarctica ice shelves are already retreating or collapsing as warm seawater intrudes underneath, grounding lines retreat, and the glaciers behind them accelerate in their march to the sea. climate scientist james hansen and many glaciologists warn that the disintegration of the polar icesheets involves non-linear processes, and the timing, though still unknown, may be far quicker than assumed, and may include rapid, even unstoppable collapse of ice cliffs in series in parts of antarctica and greenland. the impact upon human wellbeing resulting from stress on biological diversity will be compounded by climate change and the fragmentation of society. for example, a complex economic and social fabric enables the importation of food and other resources to an increasing number of regions, some of which have been in this vulnerable situation for decades. such mechanisms are fragile. today, five countries are recognized as afflicted by famine: yemen, somalia, south sudan, n.e. nigeria and two regions of the democratic republic of the congo (kasai and tanganyika). in the long run, if climate change and other aspects of adverse ecological change intensify, then it is also possible that regions that are current net food exporters will also experience famine; if this evolves then conditions in food-importing regions will inevitably deteriorate. alongside these four major crises, the researchers also identify the threat from various forms of pollution. most of these are discussed elsewhere in this encyclopedia. however, we briefly discuss novel entities. novel entities is a recently introduced term, first identified as a planetary boundary by the pb team in 2015, evolving from chemical pollution in the earlier pb publications. the pb team defines novel entities as "forms of existing substances, and modified life forms that have the potential for unwanted geophysical and/or biological effects." most novel entities have been generated in the anthropocene, the human-dominated era, defined roughly as the time since the start of the widespread combustion of fossil fuels, in the 18th century. they include synthetic molecules such as chlorinated fluorocarbons (cfcs), ddt, dieldrin and other organochlorines used as biocides and compounds used in industry such as polyvinyl chloride. cfcs, by harming the stratospheric ozone layer, clearly impinge on an earth system function (and thus indirectly on human environmental health); the destruction of the stratospheric ozone layer causes uv light to reach the earth's surface to a greater extent than prior to the widespread use of cfcs, leading to the potential for an increased incidence of skin cancer, ocular problems and immunosuppression. here, however, we focus mainly on the biological effects of novel entities. novel entities are not confined to new chemical compounds, as the pb authors note. genetically altered organisms can be conceptualized as novel entities, as are nanoparticles (such as in sunscreens and cosmetics), and blue light from computer and phone screens. humans are also exposed to numerous other emerging environmental hazards, especially since world war ii, and to human-generated ionizing radiation (x-rays were once routinely used to help fit shoes). possible health risks of nonionizing radiation, such as from mobile phones, are discussed briefly below, as are novel behaviors, foods and other novel environments. a 2017 lancet commission report estimates that 140,000 compounds have been synthesized since 1950, with perhaps 5000 widely disseminated in the global environment. although some are regulated, and a few have been banned, the pace of their introduction greatly exceeds that of epidemiological investigation and legal constraint. for example, the international agency for research into cancer (iarc), which is closely affiliated with the world health organization (who) has recently concluded that the widely applied herbicide glyphosate (commercially known as "round up") may be carcinogenic. these findings have been resisted by some companies and their agents and supporters. thousands of studies of novel entities have found or suggested that many are carcinogenic, while others act as endocrine disruptors or harm health in other ways. some have been linked with massive ecosystem disruption, including colony collapse disorder (of bees) and "insectageddon." the lancet commission on pollution reported that fewer than half of the most widely dispersed chemicals have undergone any testing for safety or toxicity. interactions between such chemicals have received even less examination. the immunological and allergenic effects of most novel entities are also barely explored, and could contribute to the changing pattern of allergic diseases, auto-immune conditions and autism. while some novel entities have been regulated (e.g., x-rays) and banned (such as the "dirty dozen," including the organochlorine dieldrin, which was, as a rare exception, strongly linked with breast cancer), hundreds or thousands of others are released annually onto the market. in both industrial and rural societies, almost the entire population has been exposed to hundreds of chemicals whose concentrations can be measured in tissue samples, while for thousands more, no test exists. there is little support from policy decision makers around the world for precautionary approaches to many potential risks. for example, there are concerns that mobile phones can cause brain tissue to warm up, if the receiver is held close to the ear. however, there are also concerns about the effects of non-ionizing radiation on brain tissue, and claims of an increased risk of malignant brain tumors in heavy users of mobile phones. cardiac and neurological disorders are also plausible consequences of the rapidly increasing use of wireless devices, including smart meters. infrasound from wind turbines is another novel entity. such sounds disturb the sleep of many people who live close to them, and there may also be other harmful effects including vertigo, as well as chronic diseases worsened by chronic poor sleep. such concerns have often been dismissed as "nocebic" (i.e., through apprehension and negative thoughts) as high quality evidence for health impacts is lacking. the precautionary principle would place the onus on industry to prove safety. novel behaviors, foods, organisms and environments are also emerging in the anthropocene. examples include reduced weight bearing exercise in childhood and adolescence (leading to a higher risk of early-onset osteoporosis), increased screen watching and the partial replacement of tangible, local friends and acquaintances for virtual social networks. novel diets include the widespread consumption of sweetened drinks, a known factor in obesity and harmful to health, while the greater variety of foods out of season, especially of fruit, is beneficial. there are also novel microbial and parasitic environments and novel microbiomes, each of which is likely to be associated with health benefits and risks. for example, humans and livestock farming provide opportunity for the amplification and spread of genes that convey antibiotic resistance. these genes are favored wherever antibiotics are used by humans or fed to livestock to promote growth and limit infectious disease. antibiotic resistance genes have been shown to spread to environmental microbes in soil and water systems, to wildlife and to human and livestock pathogens. identified mechanisms for this transfer include air-borne transport of particulate matter and direct and indirect contact with waste products. the augmented "wild" population of antibiotic resistant genes is an added risk to human health and has poorly understood implications for other environmental microbial systems. novel or increased contact with mammalian wildlife creates further potential for interspecies transfer of pathogens, particularly viruses. this is discussed below (in biodiversity and health). since the 1980s, there has been increasing recognition of ways that anthropogenic emissions of greenhouse gases (manifest in phenomena including global warming, weather wilding, jetstream oscillations, sea level rise and ocean acidification) is likely to impact human health, both positively (e.g., fewer cold waves in some areas) and negatively. there are numerous mechanisms for this. one that is perhaps most obvious is an intensification of extreme weather events, including heatwaves, droughts, flooding, and major storms including cyclones, typhoons and hurricanes. such events can have complex and delayed effects, such as from the savage 2017 hurricanes that flooded and devastated houston, texas and the us territory of puerto rico, as well as other regions. there is also speculation that the frequency, severity and locations of tornadoes may be affected. very intense flooding events, where weather systems remain almost stationary, have generated the neologism "rainbomb." changes in vector-borne diseases, food security, and sea level rise have long been forecast to occur due to global warming. global warming is already affecting migration, conflict and mental health, and these effects are likely to intensify. over the longer timescale, of decades to centuries, adverse effects are forecast to exceed benefits, perhaps by orders of magnitude, especially if the ice sheets in greenland and antarctica continue to melt. there are many ways health effects related to climate change can be categorized, such as through changes in temperature and humidity, vector ecology, water quality, water and food supply impacts, severe weather effects, air pollution, allergens, and migration, conflict and related mental health implications. a simpler classification has three main classes, conceptualized as "direct" (e.g., heatwaves), "indirect" (e.g., changes in vector ecology) and a third category, causally more displaced, with the potential for the largest burden of disease, through means such as large-scale conflict, migration and famine. in this classification, effects on mental health are regarded as "cross-cutting." dislocation from one's home due to a storm surge or a prolonged blackout (some parts of puerto rico lacked power for months following 2017 hurricane irma) can lead to depression and even suicide. such stress is also likely to exacerbate domestic violence, especially if associated with increased economic insecurity. increased rates of post-traumatic stress and anxiety are also likely in survivors. even worse than the mental trauma of a single extreme weather event are the health consequences, including to mental health, from conflict, famine and forced migration. of course, such "tertiary" effects have multi-dimensional causes, from ancient rivalries to recent and emerging contests over scarce resources, often aggravated by "youth bulges" and brutal repression. all writers on these "tertiary" topics, publishing in the academic literature, recognize the complexity of this issue, and frequently try to convey this by using the term "risk multiplier" to indicate how changes in climate can worsen (or in some cases reduce) the co-factorial causal contributors to conflict. that is, climate change is conceptualized as similar to a catalyst or enzyme. famines, wars and migration can all occur without climate change, but in some cases climate change can make these phenomena much worse. in some cases, such as sea level rise, climate change can be conceived as by far the dominant factor. however, even for vulnerable lowlying pacific islands, co-factors such as high population growth have contributed to vulnerability and the risk of migration, for example by depleting fresh water lenses, leading to the salinization of garden soil. many important diseases, including parasitic, vector-borne and zoonotic diseases are associated with invertebrates such as ticks, mosquitoes and blackflies, or higher order vertebrates. ticks transmit diseases such as lyme disease, mosquitoes transmit many illnesses such as malaria and yellow fever, while black flies transmit river blindness (onchocerciasis). the distribution of these insects and animals are shaped, not only by climate but by many other aspects of their ecology. often, the identification of the precise attribution to climate change is elusive and possibly fruitless. less intuitively, the epidemiology of many vector-borne diseases, including malaria, dengue fever and zika virus is also influenced by the ambient temperature in another way, by determining the growth rate of the parasite or virus within the cold-blooded vector. more rapid growth of these pathogens (i.e., in slightly warmer vectors) can, in some cases, lead to additional cycles of transmission, leading to explosive increases in cases. another way to think of these organisms is that their numbers and disease potential exist within a window or "sleeve" of climate and ecological suitability. it would be wrong to think that a warmer or wetter climate will inevitably increase the burden of these infectious diseases. as temperatures rise, insect populations may too, but only to a point. beyond that point, vector populations may in fact decline. similarly, excessive rain may reduce vector habitat (e.g., flushing the population away), as may unusually prolonged droughts (drying out the habitat). however, the epidemiology of vector-borne diseases is also influenced by human factors, such as insecticides (including impregnated bednets) and molluscicides, and by treatments such as vaccines (e.g., for yellow fever) and antimalarial drugs such as quinine. the impact of biodiversity loss on human health is being realized slowly. the dimensions of biodiversity (the diversity of genes, species and ecosystems) are not experienced or understood by most individuals, or policy makers, and challenge health researchers. as such, the impacts are dispersed across multiple scales of biodiversity and multiple dimensions of health and wellbeing much of which is discussed elsewhere in this contribution. the pb team has examined the genetic diversity within and between species and ecosystems and its functional role within a global system, separately. they conclude that the loss of genetic diversity has exceeded a safe limit, with uncertainty remaining over how this impacts the function of ecosystems. the alarming rate and extent of loss of genetic biological diversity has been discussed earlier. the loss of genetic diversity undermines the resilience of ecosystems. under relentless ecological change, biological diversity is replaced by ecosystems dominated by fewer, highly adaptive species, inadvertently or purposefully promoted by human activities. these include domestic species, pests and wild synanthropes, humans and the novel entities described above. genetic diversity is also the source of pharmaceutical discovery as well as the storehouse of traditional medicines. most naturederived pharmaceuticals come from plants; some come from traditional medicine practice but much is the product of systematic searching, modification and trial. nature produces an inspirational variety and complexity of molecules to further manipulate. the diverse origins of the pharmaceutical armory against hiv aids includes betulinic acid, derived from the bark of the tree betula pubescens; bevirimat, extracted from a chinese herb syzygium claviflorum; and ganoderic acid b, isolated from the fruiting bodies and spores of the fungi ganoderma lucidum. such a utilitarian appreciation likewise extends to livelihoods dependent on different aspects of biodiversity. for some, particularly vulnerable groups and those in remote locations, survival is dependent on the ability to harvest freely (or illegally) from the natural environment. rich biological diversity is often helpful for the resilience of ecosystems functions, sometimes called "services." in the early 2000s, the millennium ecosystem assessment, a global collaboration of over 1000 scientists, grouped these into four kinds, which they called supporting, provisioning, regulating and cultural. food production is classified as a provisioning service. for such a service, biological diversity is required for (supporting or underpinning) soil health, pest control (a regulating service), pollination and the genetics of livestock and crops. other products of provisioning services include clean water, bio-fuels and crop residues used to provide energy. other regulating services include carbon sequestration, climate regulation and disaster risk reduction. nutrient recycling is another example of a supporting ecosystem service. a sacred grove or an iconic species of deep significance to the beholder illustrate cultural services. disease-regulation as an ecosystem service is contested, but some diseases, such as lyme disease, are more prevalent in diminished and simplified ecosystems. the net effect of deforestation often favors mosquitoes that serve as vectors of human diseases including previously obscure pathogens such as zika and chikungunya viruses, or encourages urbanization or farm-foraging by fruit bat hosts of henipah viruses nipah and hendra. there is also a complex relationship between ecological change and malaria, which is, by far, the most important mosquito-transmitted disease. as discussed above, climate change also impacts these vector borne diseases. changes (driven by reductions) in biodiversity have increased zoonotic infectious disease risk especially due to intensive animal husbandry. livestock, which now dominate global vertebrate biomass, and intensive production, create the opportunity for viral amplification and mutations resulting in new and previously unrecognized animal diseases and zoonoses. these include h5n1 (avian flu, via chickens), h1n1 (swine flu, via chickens and pigs), possibly sudden acute respiratory syndrome (sars, via farmed civet cats and racoon dogs), nipah virus in malaysia (via pigs) and middle east respiratory syndrome (mers, via camels). novel or increased contact with mammalian wildlife creates further potential for interspecies transfer of pathogens, particularly viruses. such contact is facilitated by accelerated land-use change and wildlife harvesting and sometimes aided by domestic animals acting as amplification hosts. disease may transmit directly to humans or indirectly through domestic animals. many opportunities for viruses to jump between species may be required before a significant disease emerges. tropical areas of high biodiversity and under human pressure are considered "hotspots" for such diseases. novel zoonoses of wildlife origin such as hiv aids, ebola and sars corona virus have been the subject of strong interest in the late 20th century and early 21st century. the examples above have resulted in pandemics. many other smaller viral "spillover" events have occurred with localized impact only. natural or wild areas also reduce stress, depression and anxiety in those who visit them. this effect appears to be dependent on cultural and socioeconomic characteristics of the visitor and has deeper, religious dimensions for many indigenous people. as discussed earlier, the human microbiome links us to the external world. personal microbiodiversity is enriched by environmental and dietary diversity and, through mechanisms of immune regulation and the gut-brain axis, has a significant impact on physical and mental health. the benefits of experiencing biodiversity within natural settings appear to be physiological as well as psychological. however, the living environs of most people is one of reduced biodiversity, and for many most time is spent indoors. it is the capacity of earth's natural systems, the aggregate of species and ecosystem biodiversity, to provide resilience despite changing environmental conditions that should be of the most fundamental concern to health and well-being. the biosphere must absorb our wastes, including carbon emissions; buffer coastlines from extreme weather events; provide clean air, water, a moderate climate, and the renewable resources humanity seeks to consume. it is therefore of great concern that the global ecological footprint network (see "the ecological footprint" section) estimates that 150% of [global] biocapacity is consumed per annum. today, many determinants of health and wellbeing, including effective health services and their inputs, such as consumables and pharmaceuticals, are dependent on abundant and affordable energy. millions of people living in poverty, especially in sub-saharan africa and south asia, suffer multi-system consequences of air pollution, both indoor and outdoor, from smoke generated by their own household and by other households. in many locations, this is aggravated by the burning of fossil fuels such as coal and oil. many people, particularly women and children, undertake daily laborious effort to obtain fuel and water. access to electrical power, and even gas for cooking would bring significant improvements in health to the 1.3 billion people living without electricity. probably the least understood aspect of limits to growth is the concept and importance of declining energy return on energy investment (eroei). the number given for eroei is the ratio of useful energy obtained versus primary energy expended (see box 1). the major energy carriers in use today are fossil fuels, especially coal, oil and gas. these are used not only for transport, heating and electrical power (over 60% globally), but also as a chemical stock to manufacture plastics and to make fertilizer. but, just as in the past when our ancestors bred, trained, housed and fed donkeys and draft horses for assistance with laborious tasks, fossil fuel needs to be wrested from the environment, whether drilled for, dug by hand or removed by robotic shovels. these processes themselves take energy. in addition, energy released or captured from these sources needs to be distributed and the infrastructure to do that needs to be maintained. coal needs to be transported and burned, with some of its energy captured through combustion. electrical energy needs to be distributed and regulated irrespective of its source (including solar, wind, hydro and tidal). to manufacture wind turbines or solar panels requires energy, as does the mining infrastructure described above. life-cycle assessment allows a full quantification of the energy invested in any form of energy extracted, which is clearly significant. in the heyday of fossil fuels, oil and coal were easy to extract, and their eroei was highdsome analysts report an average eroei of over 100 in the late 19th century. in contrast, a review published in 2016 in nature communications found that, globally, up until 2017, solar panels may have yielded no energy beyond that required for their manufacture and installation. in other words, under the least optimistic scenario, solar panels, cumulatively, have been a sink for energy, rather than a source until very recently. more encouragingly, the eroei for solar appears to have increased considerably in the last decade, perhaps to 30 or 35, especially in locations with high insolation, such as in the tropics. the climate footprint of solar is much lower than of coal and will continue to decline, especially as the efficiency of panels increases and the electricity they generate is used to manufacture additional ones. the eroei for wind is widely agreed to be even higher than for solar, so these two sources have promise as major substitutes for fossil fuel energy, even though researchers still debate whether renewables will yield energy abundant enough to fuel the current consumption-oriented economy. in addition, ugo bardi and sgouris sgouridis argue that the window for a successful transition is narrow: a very large investment of available energy is required, while still maintaining adequate energy for ongoing services. moreover, the world's economic system may fail to allocate the necessary resources in the necessary timeframe (by 2050 in this analysis). bardi and sgouridis are skeptical that market forces can effect this transition. they calculated that, as of 2017, capital investment is only about one tenth of what is required. energy investment is also inadequate. though not impossible, any transition to renewables will be challenging and requires a substantially greater rate of energy and capital investment than is currently allocated. there is a widespread understanding that fossil fuels have been crucial to the human colonization and domination of the biosphere. the importance of energy is explicit in the work of many environmental writers, and implicit in the military actions of many great powers, who have frequently acted with violence or duplicity to acquire or maintain energy resources, from the middle east to the timor sea. without fossil fuel, modern civilization could not have evolved in the way it did, whether to create highways, intensive agriculture, skyscrapers or the space age. although, in the middle ages, the harnessing of water power for work from milling grain to sawing wood ("sawmilling") was widespread, such industry was necessarily confined to suitable riversides. ancient mariners crossed straits and sometimes oceans, powered by oars and blown by the wind, but the scale of maritime trade was miniscule compared with that made possible by steam, oil and nuclear-powered vessels. while this dependence on energy is well-known, though rarely highlighted in economic histories, the fact that eroei is steadily declining is rarely mentioned in mainstream media or outside of specialist journals; it is claimed that fracking and shale oil now negate peak oil, though insufficient attention is paid to the instability of an industry reliant on proliferation of drilling sites and rising costs. the decline of eroei may be disconcerting to a public ill-prepared for the future austerity which such a decline implies. such consequences would not only affect health services, but the myriad other processes necessary for health which rely on affordable energy, including agriculture. concern about the impact of global ecological change on health is growing. so too is an understanding of the need for multi-sector collaborations. few groups are yet addressing the deeper issues of pbs. however, many once disparate groups are converging as they seek to improve equity in health with a focus on global problems of biodiversity decline, environmental degradation and climate change. "planetary health," promoted by the prestigious medical journal the lancet, is currently prominent. others include eco-health, one health and the in vivo planetary health group. predicting future human health (or survival) under the status quo is difficult. ecological systems typically demonstrate nonlinear responses to perturbations and it is likely that current consumption patterns will precipitate dramatic shifts in biodiversity, ecological function and health-supporting services. impacts of environmental change are disproportionately experienced by poor and rural communities. advocacy and action to prevent these health risks is an essential role for those concerned with public health. this entry has reviewed the issue of limits to growth, its more modern formulation as planetary boundaries and the relevance of both concepts to global population health. it has used these frameworks to classify and extend some environmental health risks. these include novel entities and behaviors, and global risks including climate change, biodiversity loss, land-system change and biogeochemical cycles. these risks are escalating and we recognize the shortfall in assessing the health risk of new pollutants, entities and behaviors. when considered together with economic and population growth, and with ever-increasing resource and energy use, these environmental health risks may foreshadow significant population decline. by using the ltg and pb frameworks in this contribution, society can frame preventative measures on the scale at which this is required. without urgent change, future global population health, and survival, is imperiled. environmental quality in a growing economy. resources for the future health, population, limits and the decline of nature climate change, food security and population health in the anthropocene a further critique of growth economics encyclical letter laudato si 0 of the holy father francis on care for our common home collision course: endless growth on a finite planet the limits to growth great transition: the promise and lure of the times ahead union of concerned scientists, 1992. world scientists' warning to humanity the interaction of human population, food production, and biodiversity protection is global collapse imminent? re-assessment of net energy production and greenhouse gas emissions avoidance after 40 years of photovoltaics development come on! capitalism, short-termism, population and the destruction of the planet understanding social-ecological systems fischer: a critical look at food security strategies key: cord-276954-6m74kejh authors: dubé, simon; anctil, dave title: foundations of erobotics date: 2020-10-28 journal: int j soc robot doi: 10.1007/s12369-020-00706-0 sha: doc_id: 276954 cord_uid: 6m74kejh technology is giving rise to artificial erotic agents, which we call erobots (erôs + bot). erobots, such as virtual or augmented partners, erotic chatbots, and sex robots, increasingly expose humans to the possibility of intimacy and sexuality with artificial agents. their advent has sparked academic and public debates: some denounce their risks (e.g., promotion of harmful sociosexual norms), while others defend their potential benefits (e.g., health, education, and research applications). yet, the scientific study of human–machine erotic interaction is limited; no comprehensive theoretical models have been proposed and the empirical literature remains scarce. the current research programs investigating erotic technologies tend to focus on the risks and benefits of erobots, rather than providing solutions to resolve the former and enhance the latter. moreover, we feel that these programs underestimate how humans and machines unpredictably interact and co-evolve, as well as the influence of sociocultural processes on technological development and meaning attribution. to comprehensively explore human–machine erotic interaction and co-evolution, we argue that we need a new unified transdisciplinary field of research—grounded in sexuality and technology positive frameworks—focusing on human-erobot interaction and co-evolution as well as guiding the development of beneficial erotic machines. we call this field erobotics. as a first contribution to this new discipline, this article defines erobotics and its related concepts; proposes a model of human-erobot interaction and co-evolution; and suggests a path to design beneficial erotic machines that could mitigate risks and enhance human well-being. . amidst what some consider a new (sexual) revolution [22, 194, 315] , we are witnessing the rise of artificial agents capable of erotically engaging with humans, which we call erobots. the term erobots includes but is not limited to virtual or augmented partners, erotic chatbots, and sex robots [8, 93] . unlike previous technology, erobots do not simply mediate erotic experiences, but can also increasingly be perceived as subjects, rather than objects of desire [72-74, 87, 178, 245, 307] , in part due to their growing agency (i.e., the capability to act in/on the world to achieve goals; [154, 258, 266] ). this exposes humanity to the possibility of intimacy and sexuality with machines [8, 93] . the controversial advent of erobots has important ethical and social implications, which polarize public and academic discourses [47, 65, 66, 82, 87, 117, 179, 249, 250, 268, 270] . those who denounce their risks argue that erobots could: promote or perpetuate harmful sociosexual norms; generate (new) problematic or pathological behaviours; increase child abuse; impair interhuman relationships; deceive or manipulate humans; as well as augment the risks pertaining to privacy and data confidentiality [47, 65, 101, 114, 128, 129, 133, 182, 190, 195, 210, 222, 241, 249, 250, 264, 276] . conversely, those who endorse their potential benefits argue that they could: widen access to intimacy and sexuality; be employed in medical and therapeutic treatments; provide interactive and personalized sex education; prevent child abuse; reduce risks involved in interhuman sex; be used as standardized research tools; and enable a deeper exploration of humans' holistic erotic experiences [26, 27, 57, 64, 65, 83, 93, 109, 179, 180, 199, 319] . yet, the current scientific study of human-machine erotic interaction is limited and mostly speculative; no comprehensive theoretical model has been proposed, and the empirical literature remains scarce [86, 87, 89] . additionally, the current research tends to focus on potential risks and benefits rather than exploring solutions to mitigate the former and enhance the latter [87] . meanwhile, the private sector is racing to develop new erotic products to occupy an untapped sextech market that is estimated to be worth $30-120 billion [22, 82] . political and legal bodies need scientifically valid research (theoretically sound and evidence-based) to guide the regulation of emerging erotic technologies [271, 277] . to bridge this knowledge gap, research has emerged on digisexuality-or the use of technology in relationship and sexuality [200] (or technosexuality; [21, 283] )-and lovotics-a research domain aimed at developing strong bonds such as love, intimacy, and friendship between humans and robots by modeling and imitating human affection processes [50, 260] . these programs draw attention to the importance of studying the impact of technology on human intimacy in a world that tends to wrongly treat love, sex, and relationships as separate matters, disconnected from other human realities [51, 66, 82, 89, 179, 200, 260, 283] . they also outline the importance of increased immersivity and interactivity, in changing humans' relationship with erotic technology (e.g., distinctions between first and second wave digisexuality; [200] ). however, researchers tend to adopt descriptive perspectives on ongoing human-machine erotic interaction and co-evolution, without providing explicative mechanisms that have predictive value and could constitute theoretical grounds for empirical and clinical research. moreover, programs like lovotics too often adopt reductionist, technologically deterministic views (e.g., assuming that building machines that simply mimic biological erotic processes will effectively generate strong human-machine bonds; [50, 260] ). these programs underestimate the impact of individual differences, as well as the effect of sociocultural processes in influencing the way technology is imagined, developed, implemented, and attributed meaning over time [155] . they also underestimate how the complex web of affordances enabled by the growing agency of erotic machines influences our relationships with erobots, the interconnectivity of biological and artificial systems, as well as the unpredictable ways in which such systems can affect the cognition and evolution of both humans and machines. to comprehensively explore human-machine erotic interaction and co-evolution, mitigate erobot-related risks, and further human well-being, we need a new unified transdisciplinary field of research with a broad research agenda-a field we propose to call erobotics. as a discipline intersecting human-machine interaction (hmi) and sexology (i.e., the study of human sexuality), erobotics will aim to (1) study human-erobot interaction, co-evolution, and their related phenomena, as well as to (2) guide the development of beneficial erotic machines. moreover, in line with döring and pöschl [89] , we propose that erobotics should be grounded in sexuality [308] and technology positive frameworks [252] . this means that erobotics should explore issues related to technology-mediated human intimacy, but also strive towards pleasure, freedom, and diversity [308] . this also means that erobotics should aim to mitigate erobot-related risks and promote the ethical development of erotic machines geared towards well-being [252] . as a first contribution to erobotics and its sextech-positive objectives, the present article aims to: (o1) define erobotics and its related concepts; (o2) propose a model of human-erobot interaction and co-evolution; (o3) and suggest a path to design beneficial erotic machines. to do so, we propose a terminology based on the rich concept of erôs, a taxonomy of erobots, and a spectrum of their growing agency that aims to clarify the potentially changing nature of human-machine erotic interaction as well as the challenges faced by our socio-technological coevolution (sect. 2). we then propose an overarching model of human-erobot interaction and co-evolution, which has predictive value and constitutes theoretical grounds for a wide, collaborative, and transdisciplinary research agenda on erobotics (sect. 3). finally, we underline how humanerobot interaction and co-evolution can be detrimental to human well-being-particularly if they hinder the diversity of erotic traits, and if we do not change our current approach to technological design [34, 257] . as an alternative, we recommend implementing stuart russell's [257] principles for beneficial machines to guide the development of beneficial erobots (sect. 4). we conclude that the development of such beneficial erotic machines has the potential to mitigate erobot-related risks, and possibly maximize technology's benefits for human intimacy and sociosexual well-being. artificial agents are increasingly perceived and treated as social actors rather than mere objects [72] [73] [74] 178] . their gradual transition from patient to agent-from a passive technology that is simply used to an interactive technology capable of (rapidly increasing) degrees of agency-is crucial to understanding human-machine interaction and coevolution, particularly when these agents are designed for intimate interaction. it is, in part, fundamental to understand the ever-changing construction of meaning surrounding (emerging) erotic technologies and our relationships with them. as such, the nomenclature used to describe these socio-technological phenomena should reflect this complexity. while terminology is crucial to any scientific endeavour, the use of lay, misguided, and unscientific terms in marketing and pop culture often skews the way intricate emerging realities are conceptualized and studied. one example is the term "lovotics," whose use of the english prefix "lov-" needlessly emphasizes the concept of "love" over other aspects of human intimacy and relationships [50, 51, 260] . other examples include terms like "smart/sex toys, dolls, or robots," which are based on cultural tropes (e.g., science fiction), mundane consumer products (e.g., "smartphones"), and limited views of the kinds of interactions humans may have, or wish to have, with erotic artifacts. this discrepancy is exemplified by su, lazar, bardzell, and bardzell's [280, p. 3] pioneering study, which highlighted that sophisticated doll owners do not perceive their artificial partner as a simple sexual device, but rather as "[…] a human-like body that inhabits the home with purpose through its motions with the owner". this shifts the focus onto the interactive, holistic, and meaningful experiences that individuals may have, or wish to have, with artificial partners. notably, these experiences are not necessarily sexual, but are still intimate, romantic, friendly, and/or sensual: phenomena that could become even more complex and widespread with the advancement of the machines' agency [87] . to capture the complexity of human-machine erotic interaction and co-evolution, we begin by providing a nomenclature for erobotics grounded in the rich concept of erôs, which is central to understanding the cultural and modern evolution of our (technology-mediated) intimacy. we then propose a taxonomy of erobots and a spectrum of their agency, which highlights how erobots' transformative and relational influence is likely related to their growing agential capabilities. ultimately, the following section aims to help (re)structure the research and discourse on erobotics, their ethical and social implications, as well as the implementation of regulations adapted to the growing agency of erobots. according to anthony giddens, the eminent sociologist of modernity, the transformative process of modern sexuality is characterized by the increasing detachment from the social imperatives of reproduction-including the subservience of women and imposed heteronormativity-, allowing more people the freedom to redefine selfhood as personal, gender, and sexual self-emancipation [122] . this process finds its continuity in the recent integration of new erotic technologies into the lives of billions of people worldwide, which is leading to the emergence of novel practices, preferences, and identities [86, 89, 90, 200] . erobotics thus aims to study these transformations, and the full spectrum of techno-erotic phenomena ranging from self-stimulation to human-machine love. the term erobots characterizes all virtual, embodied, and/or augmented artificial erotic agents, as well as the technologies and systems from which they emerge [8] . this definition includes but is not limited to erotic virtual or augmented entities, chatbots, robots, avatars, as well as their enabling interconnected, multi-layered, and multi-agent systems (i.e., artificial and biological; [93] ). erobots are artificial agents in the sense that they are software and algorithm-based systems capable of various degrees of agency (as defined below). furthermore, because they (are perceived to) manifest erotic personas and behavioural patterns and are capable of erotically engaging with humans, and vice versa, erobots should be studied as specialized agents and multi-agent systems. notably, the eroticism of erobots can be designed (e.g., purposefully included in their forms and behaviours), or developed over time, if artificial agents have the capability to learn and enact such erotic personas and behavioural patterns (e.g., an initially platonic social ai that learns aspects of our sociosexuality and becomes capable of manifesting eroticism). erobots are "agents" in the sense that they are functional technological systems, like computer programs. since it is beyond the scope of this paper to statute on the nature of agency, we here employ the broadest definition recognized and commonly used in the fields of ai, machine learning (ml), and robotics [221, 257, 258, 284] . that is, the agency of machines refers to their capability to act intelligently in and on the world to achieve objectives on their own [221] . intelligence here simply refers to the capability to achieve goals [257, 284] . like their biological counterparts, artificially intelligent agents have the potential to communicate, adapt, behave, and/or interact with other agents using more or less complex learning algorithms. for example, the algorithms of a "software agent" based on reinforcement learning (rl) can act more efficiently in an environment through trial and error (maximizing reward functions) [38] . a population of software agents can also learn together through evolu-tionary algorithms that use fitness functions (metaheuristic optimization) [258] . agency levels found in functional technological systems, including erobotic systems, are based on the complexity-efficiency of learning algorithms, but also on: computing power, data access and storage, sensors, actuators, etc. the term erobotics, by extension, refers to the emerging field of transdisciplinary research exploring past, present, and future human-erobot interaction and co-evolution, as well as the evolution of technology that makes those interactions possible [8] . as a transdisciplinary field intersecting hmi and sexology, erobotics aims to develop theoretical, experimental, and clinical research methods to study the broad spectrum of dynamics related to the emergence of erotic technologies [93] . erobotics also aims to investigate the ethical and social implications pertaining to human-machine erotic interaction and co-evolution, as well as guide the development of beneficial erotic machines-i.e., machines that mitigate harm and enhance well-being. the term erobot is a portmanteau of erôs and bot. bot is the colloquial word used to designate both software and intelligent agents, either a digital computer program or robot with sensors and actuators [111, 207, 221] . the greek word erôs characterizes all phenomena related to eroticism, which denotes both the innate erotic quality of something, and the condition of being erotically aroused. more specifically, it relates to the fluid experience, construction, and elicitation of love, sexuality, sensuality, attraction, passion, attachment, fantasies, arousal, desire, etc., and their complex intersections [239] . admittedly, in english, terminological usage would normally prescribe the use of the prefix "eroto-" (as in erotophilia) to affix the concept of erôs to a new word. we would thus typically favor terms like "erotobots" to label artificial erotic agents. but these labels are not only unpleasantly sounding, the terminological usage of the prefix "eroto-" has been mostly used as a synonym for "sex" or "sexuality" in a limited sense (e.g., sexual desire). in french however, the prefix "éro-" allows for a richer and more inclusive denotation, one that encompasses all phenomena pertaining to the ever-changing conceptualization of "eroticism", as described above. hence, given that terms such as erotic (i.e., adjective), eroticism (i.e., noun), or eroticize (i.e., verb) all respectively derive from the french words "érotique," "érotisme," and "érotiser," by the same etymological logic, we prescribe the use of erobot and erobotics for the french words "érobot" and "érobotique." (note. these concepts were first introduced at the 87 th annual conference of the association francophone pour le savoir (acfas) in a symposium titled "penser l'érobotique: regard transdisciplinaire sur la robotique sexuelle"; [8] ). there are many philosophical reasons as to why the greek concept of erôs (and its derivatives) is central in the study of emerging intimate and sexual technologies. historically, the concept of erôs has been employed by many writers, philosophers, as well as the first psychologists exploring the intricacies of love, sex, and desire in the human mind. before modern sexology (e.g., biopsychosocial approach to human sexuality; [177] ), the work of these founders uncovered patterns of social and cultural complexity that underlie our erotic minds, identities, and practices. erôs is also widely used in cultural studies to explore the expressions of intimacy in its richness and historicity, as it offers a phenomenological and epistemological account of the ever-changing experience and meanings of intimacy, love, sensuality, and sexuality [193] . further, it is the most widely used concept in the study of the human experiences of passion and desire [23, 110, 113] . the first theory of erôs, plato's philosophy, powerfully influenced the western civilization conception of love and sex. simply put, plato teaches that, in a social-civilized context, trained by reason and moulded by an education oriented towards the good life, erôs is the art or craft (technê) that can lead humans to the discovery of the "sublime," or to fundamental truths about oneself, others, the world, and the divine [7, 136] . while plato fully recognized the power of the erotic mind and the erotic arts-sexual desire and romantic love-he and his many followers ultimately sought to sublimate erôs towards "higher" moral, social, and political models: "platonic love", which is both spiritual kinship (philià) and spiritual pursuit (agapè)-which implies that "true love" is nonsexual desire. the sublimationtransmutation of sexual energies into objectives of "higher" value is a dominant trope in many cultures and civilizations. in the west, the stoics, the epicureans, and the christians radicalized the sublimation path by instigating the long tradition of deflecting erôs into behaviour of higher social valuation, domesticating the instinctual life of the species by ascribing moral, social, and spiritual vocations to sexual energy [23] . the sublimation of erotic pleasure was a long process of cultural evolution that aimed at controlling and reorienting the appetitive nature of humans towards orderly, productive social outcomes like work, family, and personal discipline [110] . at the turning of the twentieth century, however, philosophy and science slowly began to question the culture of erotic sublimation [163] . the rediscovery of eroticism and the erotic life has been an arduous social and historical process that culminated during the first sexual revolution, which reaffirmed the value of the individual pursuit of sexual pleasure against the conservative repression of individual desires [122, 192] . the revalorization of erotic arts and representations opened up "eroticism" to new, modern, and widely diverse, aesthetics and ethics of sexuality: "in its numerous faces and traces (sexuality, desire, passion, love, friendship, etc.), the "erotic phenomenon" appears and becomes central in every attempt to grasp the condition of possibility for oneness and otherness, for selfhood and alterity, finitude and infinity." [32, p. 11] . following the works of freud, foucault, and contemporary feminist scholars such as simone de beauvoir, donna haraway, and judith butler, the sacrifice of individual sexuality to perform normative roles has had a major cost on human happiness and personal autonomy, especially for women [24, 44, 113, 122, 131] . today, against the residual background of general sexual sublimation and the prevalence of sexist norms, sexology and the "sex-positive movement," together promote a more complex and holistic view of sexuality, as well as individual sexual freedom, well-being, and pleasure [151] . driven by our increasingly powerful computer system infrastructures, erotic technology, we argue, is the latest stage of this continuous social and cultural revolution towards erotic emancipation-a technological erotic revolution [22, 132] . both technological innovation and sexual liberation currently drive demand for interactive artificial erotic partners, as well as immersive (multi-agent) erotic experiences [22, 56, 82, 179, 254] . erobots are thus the probable outcome of technological societies that recognize the personal and collective value of eroticism in human life. erobots are polymorphous: they can take many forms, alternate in their manifestations and behaviours, transcend media, and rely on or emerge from various interconnected, multi-layered, and multi-agent systems (i.e., artificial and biological). we propose the following taxonomy to categorize their different types: embodied erobot: any kind of corporeal artificial erotic agent. this includes various systems and devices that have some degree of erotic agential capabilities. the most (in)famous and researched embodied erobots are sex robots like those made by companies such as abyss creation's realbotix [245] and exdolls [92] (for a review see [87] ). sex robots are defined by john danaher [62] as any artificial entity that is used for sexual purposes and meets the following three conditions: (1) a humanoid form, (2) humanlike movements/behaviours, and (3) some degree of artificial intelligence. but, as danaher [63] rightly points out, sex robots do not have to be humanlike. they can take any number of forms or enact behaviours that markedly deviate from human likeness (e.g., fantasy creatures, science-fiction characters, and intelligent sex toys). furthermore, we agree that any artificial agent can be considered "corporeal" in the sense that all erobotic systems rely on materiality (e.g., hardware; [63] ). however, what distinguishes embodied erobots from other types of artificial erotic agents is that they are perceived as occupying space in our three-dimensional world and as capable of directly engaging with its materiality. contrastingly, other erobots appear limited to their vir-tual, augmented, or mixed environments, as well as to their vr/ar/mr-enabling devices [254] . virtual erobot: any kind of incorporeal artificial erotic agent. this encompasses any system (e.g., audio, visual, and/or written) that possesses some degree of erotic agential capabilities and can interact with humans via programs, applications, interfaces, and electronic devices, such as: computers, smartphones, tablets, gaming consoles, and vr equipment. examples of virtual erobots include conversational agents such as the harmony ai companion app [245] , and slutbot: an erotic chatbot developed for education and stimulation [157] . it also includes systems such as city of sin 3d [54] , virtual mate [299] , holodexxx [144] , mortherlode's pillow talk [212] , deviant tech's dominatrix simulator [80] , and, in fiction, samantha from spike jonze's her [10] . augmented erobot: any kind of artificial erotic agent emerging from the use of augmentative technology. this comprises systems resulting from the augmentation of oneself, or one's ecological niche-virtual or otherwise -, that have some degree of erotic agential capabilities. examples of augmented erobots include systems, applications, and characters projected via virtual goggles or augmentation glasses in one's environment. examples of such are: arconk [12] , greenscreenar [15] , 3d holo girlfriend [1] , or hybri [149] . it also includes avatars and virtual worlds such as chathouse 3d roulette [49] or applications expanding our erotic capabilities like mei (i.e., a sexting improvement app; [201] ) and aimm (i.e., an ml-empowered interactive matchmaking system; [5] ). for erobots resulting from human augmentation (e.g., avatars), the realization of their agency is, partly, an emerging property of the human-machine coupling, which generates unique erotic experiences and persona for the augmented person, but also for those who interact with the human-machine hybrid, or technologically erotic multiagent system [107, 131, 132, 297] . this taxonomy is meant to highlight different types of erobots and to emphasize that their systems can simultaneously be embodied, virtual, or augmented. in fact, cloud-based erobots that are connected through the iot/ios can manifest at the same time in various ways. they can be displayed on cellphones; animate a robotic body; appear in virtual worlds; or be projected in a non-virtual environment via augmentative technology. for example, users can interact with harmony's ai using both a smartphone application and a robotic-headed doll [245] . another example is hybri, which promises a future where humans and erobots fluidly alternate between embodied, virtual, and augmented erotic manifestations and experiences (i.e., mr; [149] ). as such, the perceived characters, devices, or interfaces are only parts of what is here described as an erobot. in fact, to fully grasp the extent of current and future human-machine interaction and their socio-technological co-evolution, it is essential to understand that erobots are not just their perceived characters (e.g., harmony's vr character or robotic-headed doll), but are composed of vast interconnected, multi-layered, and (increasingly adaptative) multi-agent systems that enable their (emerging) capabilities [161, 228] . for example, when people interact with an erobot, they engage with its interfaces (e.g., application and characters), but the erotic capabilities of those interfaces also depend upon clusters of enabling-systems including: software-hardware, cloud-based algorithms learning from multiple users, databanks, search engines, and humans (e.g., programmers, engineers, designers, artists, and partners). hence, like humans, erobots are not segregated stable entities, but are dynamic and porous systems relying on, enabled by, and embedded within other systems [28] . thus, erobots and their capabilities can be better conceptualized as emerging systems and properties, respectively, that which can be studied through their material substrate and (technological) ecological niche, and whereby humans are a key component in the enabling of their (erotic) agency and cognition (detailed in sect. 3). as such, erobots not only confront us to potentially novel erotic actors and experiences, but also paradoxically reminds us that-as biological organisms defined by our own structures and embedded within a larger niche-humans and machines are not so different or isolated. the agency of erobots represents hypercomplex conditions and states that can be better understood and studied across a spectrum. to appreciate this complexity, we propose a spectrum of erobots' agency (sea) ranging from level 0 (no agency) to 5 (full agency)-echoing the sae international's (j3016) spectrum of self-driving cars' automation levels (see fig. 1 ; [259] ). despite the impossibility of capturing erobots' infinite degrees of agency or technological substrates, and the obvious distinctions between autonomous cars and erobots (e.g., forms, behaviours, purposes, and underlying technology), this spectrum mock-up has heuristic value. it can help clarify present and future dynamics related to human-erobot interaction and co-evolution as the agency of artificial systems increases-i.e., greater agency may entail reduced (perceived) human control over artificial systems, greater machine (behavioral) unpredictability, and more uncertain human-erobot relationships. it can also help appreciate the scientific, ethical, and sociocultural challenges addressed to erobotics as we develop, and engage with, evermore complex agential erotic machines. noteworthy, the agency levels described in this spectrum should not be understood as discrete categories, but rather as a continuous gradient of capabilities possibly supported by diverse interconnected, multi-layered, and multi-agent systems. the agency of erotic machines is partly based on the degree of autonomy and reciprocity established and perceived in human-erobot interaction [176, 178, 278, 296, 320] . as such, in the sea, level 0 technologies are not erobots, but correspond to simple erotic objects or media without agency beyond that which is attributed by humans and/or their (pre-established and/or intended) affordances (e.g., dildos, vibrators, artificial vulva/vagina, dolls, and pornography). however, they are here included because they represent a significant portion of the erotic technology currently available and used [22, 56, 86, 89, 90, 137, 138, 247, 251, 253, 262] , and could play a role in our intimacy with erobots (e.g., interactions involving virtual partners and vibrators). they are also included here because their lack of agency (as previously defined in sect. 2.1) provides a baseline to compare subsequent sea levels and describe their potential implications for human-erobot interaction. indeed, as a reference point, level 0 technologies are comparatively highly controlled by humans, which makes our relations with them highly predictable. the interaction is co-constructed by their affordances, and importantly, by what people use them for (e.g., sex/love dolls' design provides cues about how to engage with them, but humans imagine and decide how to enact the rest; [280] ). the established reciprocity is limited, and users largely perceive themselves as in charge of the interaction. uncertainty thus remains low in the interaction with these products, since they have no capability to act intelligently on the world to achieve objectives on their own beyond their affordances. level 1 technologies integrate "basic agency" using various software-hardware implements that augment the erotic qualities of interactive and/or connected objects and media. for instance, a sex toy that adjusts its settings according to the pressure applied by its users-creating an interactive loop where humans perceive that they are not completely in charge of the sexual stimulation. their "basic agency" stems from machines' capability to react to human action, albeit in a simple way. in doing so, they establish a reciprocity that goes beyond the (intended and/or pre-established) affordances of machines and affects subsequent human-machine responses. at level 1, humans and machines not only provide cues about their use, but also act on each other. uncertainty thus increases as humans partly relinquish control over the co-constructed series of actions. still, level 1 systems are (perceived to be) controlled and predictable, as humans have a sense that they are mostly in charge of the interaction, and that the capability of machines to act on the world on their own to achieve goals beyond their affordances is restricted to what we use them for. level 2 technologies correspond to ai-enhanced erotic systems without ml capabilities. this includes devices, applications, or media that are built on established software frameworks and incorporate complex automation, but do not fig. 1 spectrum of erobots' agency. this spectrum, ranging from level 0 (no agency) to 5 (full agency), is inspired by the sae international's (j3016) levels of driving automation [259] . it presents the descriptive labels, corresponding system capabilities, examples of technology, and paralleled likert-type scales of human control and machines' predictability associated with each level of erobots' agency. note. references: harmony and henry [245] , ava [2] , samantha [10] , gigolo joe [91] , and nimani [183] . program used for creation: adobe illustrator cc 2017 (version 21.0.0) learn from their interactions (e.g., an erotic video game that displays characters and generates intimate stimulations as a function of users' actions). their "partial agency" stems from the fact that level 2 technologies do not simply react to human action, but (are perceived to) exhibit properties akin to more complex (relational) intelligence compared to level 0 or 1 (e.g., sociosexual communication and behaviors) [13, 258, 284, 286] . here, the erotic video game can process various input data and produce output responses to generate increasingly erotic experiences interpreted by users as pleasurable and/or lifelike. interactions with level 2 technologies are thus perceived to be more co-constructed, interactive, and reciprocal, but also more automatized, diversified, and goal-oriented. uncertainty markedly increases due to the diversity of potential pre-programmed automatic responses a system can enact. but, it remains somewhat controlled and predictable because of the system's inability to learn new patterns from its users or deviate from its pre-established output functions. humans may thus confidently estimate the boundaries of the machines' capability to act on our world to achieve goals on their own and their modes of (erotic) interactions. today, levels 0-2 technologies are widespread in human intimacy [22, 53, 80, 227, 275] . people can fall deeply in love with dolls (for a review see [87] ), regularly consume pornography [275] , and use various sex toys or games to enhance their erotic experiences [90] . it is thus crucial for erobotics to understand human interaction with these technologies (e.g., motivations behind their use), their influence on our erotic lives (e.g., sexual and relationship satisfaction), and how they may contribute to the evolution of sociosexuality (e.g., transformation of our preferences and identities). notably, however, the degree of agency of levels 0-2 technologies has led them to be categorized and treated, by most, as somewhat passive objects or media. yet, the line between patient and agent is becoming increasingly blurred as we move towards systems capable of more complex (erotic) interaction, learning, and adaptation [72, 73, 145, 178, 203, 220, 256, 291] . level 3 erobots correspond to the most sophisticated mlempowered erotic systems presently available. in fig. 1 , the "space" between levels 2 and 3 emphasizes that, while previous technologies have been a part of our intimacy for decades, contemporary ml has only recently entered people's lives through (interconnected) technological systems (e.g., devices and applications). it also emphasizes that the learning capabilities of level 3 systems mark a clear departure in their agency and our co-evolution (e.g., impact of learning on evolution, or baldwin effect; [18, 79, 105, 140, 292] ). specifically, the growing learning capabilities of level 3 erobots offer a wide array of (erotic) possibilities, ranging from continuous adaptative behaviours to the possibility of gaining holistic knowledge about individuals and their cultures. indeed, in contrast to pre-programmed output delivered by "good old-fashioned artificial intelligence" systems, ml makes it possible for erobots to interact with humans and learn erotic behaviours and sociocultural dynamics directly from users and their world by generalizing from experience. level 3 erobots are built on a variety of software architectures to improve their interactive and learning functions using methods like statistical pattern recognition and probabilistic ml [119, 173] . following innovations in affective computing [236] , these agents are also increasingly capable of "artificial emotional intelligence" (aei). affective and emotional analytics combine the ability to recognize human emotions and adapt the communicative-behavioural reasoning of machines [16, 81, 146, 162, 209, 263, 267] . already a billion-dollar industry, aei can be found in applications and chatbots to improve the experience of users and enhance cooperation in the work environment, particularly in healthcare, where these systems interact with human professionals and clients [279] . aei is gradually becoming an essential component of social robots and is projected to play a key role in facilitating the human-machine psychosocial and erotic interaction [95, 267] . level 3 erobots are the first systems whose responses are not entirely pre-established by humans. indeed, through learning and adaptation, level 3 erobots can potentially develop their own new sets of sociosexual patterns based on past interactions. this makes their actions partly unknown to designers and users-somewhat uncontrolled and unpredictable. for example, when users interact with harmony, they first engage with its pre-set routines, but subsequent responses become increasingly tailored to users as its ml system allows it to learn from past conversations and encounters [245] . hence, the (re)actions of level 3 erobots are harder to predict; the boundaries of their interactive potential are (perceived to be) more uncertain. interactions are thus seemingly closer to engaging with partners that do not just mechanically respond to input stimuli, but also contribute in more complex ways to the co-construction of (erotic) experiences. to appreciate the potential implications of these innovations for hmi and sexology, consider the influence of digital social media algorithms on human relationships (e.g., facebook; [39, 303] ). with limited learning algorithms, and massive user-user interactions on their platforms, social media have transformed the attention economy, and, in turn, are transforming identities, politics, consumption, and (means of) sexual selection all over the world. for instance, algorithms have been shown to affect states of minds (e.g., beliefs, preferences, and desires) by filtering and amplifying certain perceptions of the world (e.g., filter bubbles and echo chambers; [77, 118, 232] ). their impact on our erotic cognition and agency in the context of large platforms, such as ai-powered digital pornography (e.g., pornhub) and dating applications (e.g., tinder), have only recently started to be explored [106, 182, 197, 244, 281, 287 ]. yet, we can already see their unique influences on human intimacy (e.g., preferences, behaviors, and partner selection). level 3 erobots have only recently entered our world. however, the progressive conjoining of immense data mining and processing power, vastly more powerful processor units, and above all, innovative ml techniques giving birth to formidable algorithms, allow us to consider erobots that exceed level 3. indeed, in a gradual transition towards level 4 systems, ai scientists are now tackling higher cognitive capabilities, which could soon be incorporated into erobots. that is, for instance, those related to metacognition such as meta-reasoning (i.e., reasoning about reasoning), metalearning (i.e., learning to learn; [126, 298] ), and theory of mind (tom); or the ability to understand the mental states of others and recognize them as singular, autonomous entities [124, 240] . in fact, while current advanced ai systems excel at prediction, they struggle to understand real-world physics and our infinitely rich social world: not only causality and mentalistic concepts (e.g., goals, utilities, and relations), but also socially learned concepts, such as emotions, interests, and attachment [16, 43, 173, 230] . hence, many techniques in ai, such as deep rl, bayesian inference, and game theory, are now being used to simulate the inductive biases and metacognitive capabilities of humans. emerging architectures are also progressively allowing ai systems to learn directly, and increasingly rapidly, from human preferences and language [38, 130, 305, 306] . while these attempts modify "agents architecturally" and depict their internal states in a form interpretable for humans, others "[…] seek to build intermediating systems which learn to reduce dimensionality of the space of behaviour and represent it in more digestible forms" [242, p. 2] . inverse reinforcement learning, for one, teaches algorithms to adapt behaviour to circumstances and learn from human-machine continued interaction [257] . successful "consequence engines" in bots are also already capable of internally modeling their environment and other entities in order to avoid collisions, coordinate without communication, and reach their goals [30] . likewise, using deep neural nets, google's deepmind is developing tom with the ai agent tomnet, which is capable of building heuristics from basic mind models of other agents that are derived from metalearning observations of their behaviour [242] . based on these advancements, we can realistically anticipate the emergence of level 4 erobots with "higher agency" sustained by higher erotic analytics/heuristics and aei [6] . this is a reasonable assumption since higher cognitive capabilities (and/or their attribution) have been recognized as an important component to enable many-if not most-people to develop strong attachment to erobots (e.g., love and friendships), but also, because their incorporation (and/or mimicking) in artificial companions has become an explicit goal of programs like lovotics [50, 260, 261] . to our knowledge, however, level 4 erobots are not available yet ( fig. 1 marked by the "space" between levels 3 and 4). if they were, their capabilities would, by definition, enable them to develop models of themselves and their environments and adapt their learning strategies to become more efficient in human-erobot interactions. we hypothesize that this could likely lead level 4 erobots to be perceived as uncontrolled and unpredictable, but also, arguably more convincing and efficient as intimate partners as they would exhibit degrees of more sophisticated cognition and agency that gradually approach-without achieving it-interhuman erotic interaction. lastly, level 5 erobots correspond to hypothetical constructs capable of artificial general intelligence (agi), or "strong ai" [108, 123, 171] . level 5 erobots imply a situation where highly complex and unpredictable erotic machines act quasi-completely outside of human control, at least to the same extent as any other human partner [13] . however, according to most of the world's foremost researchers working in ml, these highly uncontrolled and unpredictable agi potentially capable of self-awareness, sentience, or "consciousness," will remain theoretical constructs for decades to come [108] . in other words, debates and discussions surrounding agi are not essential to study the erotic agential and relational spectrum between humans and erobots. and, since most ai specialists believe that agi is still far ahead, we suggest that erobotics mainly focuses on level 0-3 (and upcoming 4) technologies but plans for the possible advent of level 5. indeed, our knowledge of human erôs suggests that the higher capabilities of human minds are unessential ingredients to build machines capable of entertaining meaningful relationships with us. in fact, following the sea and the learning system underlying our erotic cognition (detailed in sect. 3.1), we recommend to instead launch erobotics under a relaxed dichotomy between "true" or "false" intelligence, cognition, agency, and affective relationships [290] . to sum up the argument: the effective level of capability necessary for any machine to erotically engage with human partners is simply the level of capability necessary to enact reciprocal erotic experiences with humans. to conclude, the sea highlights a progression in humanmachine erotic interaction-ranging from reactive sexual stimulation to the possibility of meta-cognitive erotic processes. this spectrum suggests that as their erotic agency increases, machines could progressively grow outside of human (perceived) control and their behaviours could be interpreted as more unpredictable-significantly influencing, in turn, our relationships with them. the sea also suggests that the progression of machine agency has the potential to influence our erotic ecological niche and cognition in evermore complex ways. and, while we tend to exaggerate what is necessary to achieve the "affective autonomy" involved in our relationships, we might need to downplay the prerequisites for experiencing erotic relationships [235] . that is, if we consider erotic agency and cognition as anchored in a social co-determination of affects [60, 61, 97] , we should perhaps also consider that the relational autonomy of behaviour enacted by erobots has the potential to transform the niche in which this autonomy is exercised, as well as human and machine cognitions. in the next section, we explore these transformations by proposing a model of human-erobot interaction and coevolution grounded in complex system theory [28] and drawing from 4e approaches to cognition [217], the neurodevelopmental trajectory of sexuality [234] , hierarchical incentive-motivational theory [288] , and ecological system theory (i.e., bioecological model; [42] ). this model and its synthetic approach provide explicative mechanisms that have predictive value for our socio-technological erotic interaction and co-evolution. it is also purposefully broad enough to constitute theoretical grounds for a wide, collaborative, and transdisciplinary research agenda on erobotics. it is our hope that researchers from various disciplines can use this model as a starting point, bring their own perspective, and shed light on its different aspects and levels of analysis. interaction and co-evolution model (heicem). this model depicts how human and erobots are likely to co-influence each other's erotic cognition through interactions and their impact on each other's ecological niche (i.e., represented here as the interconnected multi-layered systems depicted in the bioecological model; [42] ). this model highlights multiple levels of analyses and invites a collaborative, transdisciplinary research program on erobotics to address the details of the heicem (e.g., interactions, processes, and mechanisms), which remain unknown for the most part. at its core, it also includes, a potential mechanism based on universal darwinism, which is analogous to natural, artificial, and sexual selection, [75, 76, 78] . since a plethora of variables are implied in the study of human-machine erotic co-evolution, our model is not deterministic, but probabilistic: it rests upon the way humans and erobots are likely to influence each other's erotic cognition [217] through interactions (e.g., experiences of social and sexual rewards that motivate individuals to engage or not in erotic behaviours; [234, 288] ) and their potential impacts on each other's ecological niche-ranging from micro to macrosystems (e.g., technological to sociocultural environments; [42] ). moreover, this model rests upon a continuous exchange between the individual (e.g., preferences and behaviours) and population levels (e.g., artificial and biological agents populating our ecological niche). at the core of this model, and in an attempt to potentially bridge those levels, we hypothesize a mechanism analogous to natural, artificial, and importantly, sexual selection, here called erotic multi-agent selection (emas; see fig. 2 ), which represents fertile grounds for future research. erobots are products unlike any others: developed as social and sexual partners, they are likely to be increasingly perceived and treated as partners [72] [73] [74] 178] , especially if their agency continues to grow [176, 178, 278, 296, 320] . and since humans are wired by evolution, culture, and experiences to select and engage with (intimate) partners [177] , erobots' sociosexual capabilities can progressively set them apart from other technologies. that said, cognitive neurosciences can help us bridge hmi and sexology, to model important variables of human-erobot interactions and coevolution. the emergence of erobots could act on individuals (and vice versa), by influencing their erotic cognition via their interaction (detailed in sect. 3.1.1) and their transformation of our ecological niche (detailed in sect. 3.1.2) . here, the term cognition is used in the sense intended by 4e approaches to cognition. 4e approaches propose that cognition is embodied, embedded, extended, and enacted [217] . embodied, in the sense that cognitive processes partly depend on bodily processes, including but not only involving the brain (e.g., limbs, organs, peripheral nervous systems, and hormonal activity; [295] ). embedded, such that cognitive processes are situated (e.g., in a specific body, environment, and point in time; [25] ). extended, meaning that cognitive processes partly take place, and depend on, extra bodily processes (e.g., entities enabling storage and access to information, such as books, phones, computers, and other humans; [55, 115, 148] ). and enacted, such that cognition emerges from agents' active engagement with their environments and its affordances [121, 198, 295] . by extension, erotic cognition here refers to the constellation of embodied, embedded, extended, and enacted processes which enables, and from which emerges, affordance-based phenomena pertaining to erôs, as previously described. this includes but is not limited to, the constantly evolving and interactive experience, construction, and elicitation of love, attraction, attachment, passion, romance, desire, arousal, sensuality, sexuality, etc., and their complex intersections. while there are debates regarding the extent to which cognition is embodied, embedded, extended, and enacted, 4e approaches generally agree on some key points. precisely, that cognitive-related phenomena (e.g., attention, memory, language, emotions, sensations, and perception) depend on the specific morphological and physiological characteristics of agents, their situated ecological niche (e.g., natural, technological, and sociocultural environments), their active interaction with this niche, and the coupling of their characteristics with the information provided by said niche (i.e., affordances; [121]). importantly, 4e approaches enable us to approach artificial and biological agents as part of larger, interconnected multi-agent systems from which erotic cognition emerges in different ways. they also highlight that both artificial and biological agents can engage in cognitive processes specific to their own characteristics and niche while still fully acknowledging their underlying structural and functional differences. for instance, the cognitive processes of erobots are situated within specific virtual and non-virtual worlds (embedded). they depend on software (e.g., algorithms and programs), hardware (e.g., servers), and interfaces (e.g., computers and cellphones) wired and shaped to process afferent stimuli (embodied). parts of their cognitive processes take place outside of their software, hardware, and interfaces (e.g., clouds, databanks, and humans; extended). lastly, their cognitive processes emerge from an active engagement with their ecological niche (i.e., virtual or otherwise; enacted)-in which humans play a key role. 4e approaches do not imply that erobots have a subjective experience, nor do they imply that humans' biological erotic cognition is the same as erobots' artificial erotic cognition. however, they underline the possibility for erobots to have their own forms of 4e erotic cognitive processes or their own way of erotic "thinking" [290] . they also underline how humans are a fundamental part of erobots' erotic cognition-not only because we design them, but because we represent their main source of (sociosexual) data. for instance, in much the same way as humans, machines incorporate us in their cognitive processes. machines learn, store, and access data through us. hence, while the erotic cognition of erobots partly stems from their design and preprogrammed capabilities, it can also emerge from what they learn during human-machine interaction. moreover, we purposely create and select erobots that best fit the state of our erotic cognition, and in doing so, determine traits that are more likely to endure (or not) in erobotic populations, based on our individual and collective preferences. this process could subsequently influence the type of erobots that populate our world-probabilistically and retroactively affecting our ecological niche, our possibilities for social, intimate, and sexual experiences, and in turn, our new technologymediated erotic cognition. overall, 4e approaches highlight that erobots have the potential to learn their erôs, like humans, from a world that they are themselves transforming [61, 97] . specifically, erobots (can potentially) learn their erôs from the same human world that designs them, selects them, and is changed by them-which could incidentally lead us to (re)learn a new technology-mediated erôs as we engage with them, and give rise to a hybrid erotic cognition indistinguishable from the sum of its part or the hypercomplex processes from which it emerges. 4e approaches also highlight the importance of human and erobot interactions with each other and their world in co-influencing their erotic cognition-a transformative process here contingent on the agency of machines and their place in our intimate lives. erobots can influence our erotic cognition through interaction, by providing us with novel opportunities of social, intimate, and sexual experiences-generating new learnings and possibly impacting our partner selection. indeed, humans learn the complexity and meaning of their erotic subjectivity and agency. this learning process rests upon evolutionarily developed, hierarchically organized, and relatively plastic structures [41, 234, 288] . we are wired for adaptability proportionally to the needs of our sociosexual environment, which is so diversified that we developed systems that are prepared for sex, but also extremely flexible in learning strategies to maximize chances of erotic encounters in an uncertain, ambiguous, and ever-changing world [234] . our system is hierarchically organized [288] , like other animals, for stimulus-stimulus associations [35, 37, 231] and response-reinforcer associations [274] . it constantly makes causal inferences of what its internal state will be (e.g., pleasure, aversion, joy, and pain) from cues in the external world that predict reinforcers (e.g., food, predators, and partners). moreover, it continuously adapts the type and strength of its motivational, physiological, attentional, and behavioural responses according to how well such external cues predict outcomes [41, 234, 288] . in this sense, humans are biological erotic-learning agents. our experience of love, intimacy, and sexuality is inextricably linked to the dynamic interaction [177] between our evolved biological predispositions (e.g., genetic, hormonal, and physiological factors; [20, 59, 204, 302, 313] ), our ecological niche (e.g., social and cultural factors; [3, 68, 69, 135, 175, 213, 311] ), and our experiences (e.g., learnings; [32, 37, 124-126, 141-143, 196, 206, 224, 252] ). our innate predispositions are shaped into sexual responses, desires, behaviours, and preferences based on reward experiences (e.g., social and sexual pleasure), as well as our capacity to link said experiences, and their meaning, with various external predictive cues (e.g., physical, psychological, and behavioural traits; [116, 234, 288] ). in other words, our lifelong experiences with rewards form the bridge between what we want, how much we want it, which behaviours are required, and what they mean [234] . this biological erotic-learning system constitutes the blueprint for the development of our mating mind [204, 205] . that is, a mating mind so highly tuned to learning the demands of our ambiguous and culturally shaped world that our sexuality becomes inextricably part of larger systems in its experience and meaning. for the human animal, sexuality is thus fully erotic, such that love, attraction, passion, desire, sensuality, relationships, and sex are deeply rooted in our ever-changing, socially constructed minds [113] . this human erotic cognition not only enables us to navigate and make sense of our environment, but also enables us to transform it via the production of norms and artifacts reflecting our multifaceted sexuality. for instance, humans engage in a wide range of erotic activities besides sex and mating, ranging from situated and perpetually co-evolving rites of seduction, sensual performances, and conjugal arrangements [23] . humans of all cultures have invented an immense repertoire of moral, aesthetic, religious, and legal codes of acceptable and transgressive erotic behaviours [110] . humans have also produced quantities of art and entertainment materials about love, romance, and sexuality [122] , a process that constantly feeds back to us to co-construct our erotic cognition. it is this evolutionarily developed, culturally shaped, and experience-dependent erotic cognition that produces erobotic technology. it is the foundation of why and how we select partners [234] , and thus, central to the creation of, and our interaction and co-evolution with, sociosexual machines. to appreciate how erobots can influence our erotic cognition through interaction, let us consider the nimani thought experiment, which is based on technology that already exists or is in development [5, 149, 245, 254, 260, 299] : nimani is a hypothetical polymorphous erobot. it can interact with humans via their cellphones using an audio-visual interface or chat. nimani's avatar can also simultaneously appear as one or multiple characters in a virtual environment, be projected in our world or onto individuals via augmentation equipment, and animate a robotic body in our non-virtual world. nimani is cloud-based and connected to the internet, so its ai can interact with, and learn in real-time from, multiple biological or artificial agents in a hive-mind type of cognition. it can also infinitely copy itself and it has access to tremendous amounts of information through its access to search engines (e.g., google). hence, when you interact with nimani, you are engaging with vast interconnected systems (i.e., avatar(s), its related software-hardware, its learning cloud-based systems, as well as the network of information it has access to), exposing humans to a different kind of erotic partner. first, it is not biological, but computer generated. second, it can transcend medium and manifest itself in multiple places simultaneously. third, it can take on various forms and enact behaviours that are not bound by the rules of physics governing our non-virtual world. fourth, it can adapt said forms and behaviours to the needs (i.e., physical and psychological) or fantasies of its users. fifth, it can be duplicated such that producing, or engaging with, nimani is not a zero-sum game. and finally, it accesses, processes, and learns data differently than humans by using various algorithms, statistical methods, and search engines. interacting with nimani thus entails the pairing of new erobot-specific predictive cues with the human experience of reward. indeed, as an intimate partner, nimani rests upon some of the strongest human motivation incentives (i.e., social and sexual rewards; [116, 169, 202, 288] ). paired with its traits, these incentives have the potential to generate, through interactions, novel erotic learnings that can progressively give rise to new technology-oriented conditioned partner preferences specific to erobots and their traits [234] . for instance, users' experience of intimacy and sexual pleasure would here be paired with nimani's artificial forms, personalities, and behaviours-including those that are impossible in our non-virtual world. they would also be paired with its knowledge, adaptive and duplicative capabilities, enabling equipment and systems, as well as its cultural representation and symbolic meaning. moreover, depending on whether interacting with nimani constitutes a rewarding experience (or not), individuals will likely be motivated to repeat or avoid behaviours that have led to such internal states [234, 288] . for several reasons, this lifelong learning and approachavoidance process should not be underestimated. first, it points to the possibility for some people to have their first socially and sexually rewarding experiences with artificial agents. as such, based on the neurodevelopmental trajectory of sexuality, these experiences could form critical periods of development during which preferences for erobot-specific features are integrated and consolidated [36, 234] . second, this process can influence the subsequent development of our erotic preferences and partner selection, which could, in turn, potentially affect human-erobot interaction and co-evolution. for instance, traits that will generate more rewarding experiences will be more likely to be replicated in next generations, while those that generate aversive experiences could be discarded. finally, selected erobotic technologies are likely to populate and influence our ecological niche and situated erotic cognition through a perpetual feedback with our (technological) environment. erobots can influence our erotic cognition by transforming (parts of) our ecological niche, and vice versa. more precisely, since (erotic) cognition is situated, such that it takes place and emerges from agents' interaction with their ecological niche, it can be influenced by the modification of its niche's content (e.g., the introduction of potential new sociosexual partners and enacted experiences; [217]). to emphasize this point, we again employ the nimani thought experiment and break down the anticipated potential impacts of erobots on our ecological niche using the bioecological model [42] . the bioecological model proposes that human development is influenced by a dynamic continuous process of interaction with five layers of interconnected systems [42] . the microsystem refers to individuals, groups, institutions, and technology with whom people interact directly (e.g., partners-family-friends, schools, and computers). the mesosystem connects to microsystem with other layers of the model. the exosystem encompasses systems that indirectly affect people's lives (e.g., political, legal, educational, scientific, health, media, and economic entities). the macrosystem describes the overarching sociocultural norms and value systems influencing every other layers of the model. and finally, the chronosystem accounts for the influence of historical circumstances on the model, as well as how each layer changes over time [42] . at the microsystem level, we can expect that interacting with nimani could lead to new technology-oriented conditioned partner preferences (as previously described), but also to the co-construction of new proximal dynamics with individuals, groups, and institutions. for instance, as part of our techno-subsystem [153] , erobots can generate new experiences with families, friends, and partners, such as: considering using erobots [219, 265] , forming strong bonds with artificial agents [50, 260, 261] , changing marriage institutions, and engaging in consensual non-monogamy with machines [4] . they could also lead to the advent of new health, legal, educational, and entertainment services dedicated to human-machine erotic interaction (e.g., applications, stores, organisations). these changes would all be connected to other model layers through the mesosystem [42] . at the exosystem level, erobots can interact with political, economic, legal, scientific, health, media, and educational institutions. for instance, industries can (continue to) grow around the production of nimani's systems (e.g., vr/ar/mr equipment, teledildonics, ai, robotics, and computer infrastructure; [22, 82, 107, 149, 227, 245] ) and competitively adapt to market pressures [216] . political and legal bodies may implement regulations regarding erobotic technologies (e.g., ethical guidelines, laws, and production standards; [114, 117, 271, 277] ). health systems may witness the rise of (new) problems (e.g., compulsive use) and opportunities for therapeutic use (e.g., vr for intimacy-related fears; [172, 200] )-and adjust to provide services aimed at enhancing digihealth (i.e., engagement with technology that promotes well-being; [292] ). for example, by developing treatments and resources that favor a harmonious integration of erotic technology [200, 293] , and mitigate usage that disrupts important areas of functioning (e.g., family, relationships, work, and health; [200, 293] ). media will likely continue to cover human-machine erotic interaction [88, 90] ; contributing to the co-construction of our attitudes and behaviours towards erotic technology [48, 282] . educational institutions could potentially devise programs that include (and exploit) erobotics (e.g., sex ed that discusses digisexuality). and finally, the scientific community will likely continue to explore ongoing technology-mediated erotic changes, and hopefully try to improve well-being (e.g., love and sex with robots [51] , ai love you [318] , penser l'érobotique [8] ). at the macrosystem and chronosystem levels, we propose, based on historical examples (e.g., lgbtqa2s+, kink, fetish, and bondage, discipline, domination and submission, and sadomasochism (bdsm), and sex toys; [29, 167, 309] ), that cultures surrounding human-machine eroticism can evolve over time (e.g., sexbot-induced social change; [4] ). this likely depends on factors such as: geolocalisation, socioeconomic status, as well as prior norms and values regarding sexuality and technology [177] . still, erobots can increasingly expose people to the possibility of forming strong bonds with, and via, artificial agents; possibly leading to unpredictable (re)constructions of the meaning of love, sex, and technology [107, 155, 181, 309] . this prospect, and the (erotic) experiences that accompany it, can influence societal attitudes and acceptance towards erobots [48, 282, 309, 314, 320] , in addition to the value and meaning attributed to our relations with both artificial and biological agents. finally, the bioecological model predicts that these changes can trickle down to influence other model layers in a perpetual feedback loop [42] . this does not mean that everyone will directly engage with erobots, but the bioecological model rather helps us appreciate the potentially significant holistic co-influence that erobots could have with our ecological niche, and by extension, our situated erotic cognition. it highlights the unpredictable ways in which erotic technologies could contribute to the co-construction of human (erotic) life and the different layers that must be considered to comprehensively study erobotics. it also highlights the importance of sociocultural processes in the design, implementation, and production of meaning surrounding human-machine erotic interaction [155] . this could in turn play a significant role in influencing people's attitudes and responses towards erobotic technologies, as well as their willingness to engage with erobots over time [282] . again, technology does not have to be sophisticated to co-influence our erotic ecological niche and cognition. after all, sex toys are widely used, represent major investments, are subject to production standards, and participate in the co-construction of our norms regarding sexuality and technology [22, 82, 90, 114] . but we can appreciate that erobots with growing agency can accentuate such transformative processes since they could become intimate partners. erotic machines, designed for interactive social and sexual pleasurable feedback, have the potential to engage our reward system and erotic cognition in ways that other technologies simply cannot. erobots could thus become (as some scholars proposed regarding social ai and robots; [72] [73] [74] 186] ) similar to a new species of (intimate) partners in our environment that we can design and select, who learn from us, and who provide novel opportunities of (erotic) experiences and learnings. to study these hypercomplex processes, we proposed the heicem, a model that offers an overarching theoretical framework to launch a broad, collaborative, and transdisciplinary research program on erobotics. the heicem's structure highlights multiple levels of investigation and analysis, which require different disciplines-from humanities and sexology, to neurosciences, ai and hmi, and cognitive, social, and cultural sciences-to weigh in, if we want to fully grasp the factors and variations of our coevolution with erobots. noteworthy, at the moment, some of these phenomena are difficult to examine empirically without solely relying on self-report and hypothetical scenarios [87, 219, 265] , partly due to the unavailability, high price, and/or novelty of (sophisticated) erobotic systems. others, however, can already be observed (and studied)-to various degrees-through individuals, communities, and cultures related to: digi/technosexuality [21, 200, 283] , cybersex (or online sexuality; [67, 85] ), hentai (i.e., manga or anime pornography; [301] ) and otakuism (i.e., interests in animation, manga, and games, often incorporating (non-)fictional technology; [11, 304] ), dolls [87, 104, 166, 174, 280, 294] , toys [86, 89, 90, 138, 247, 253] , platforms [49] , games [80] , teledildonics [85, 107, 200] , (vr/ar/mr) pornography [254, 275] , (ai-powered) dating applications [197, 208, 281] , artificial partners [87, 112, 160, 200, 219, 237, 307] , as well as objectophilia, agalmatophilia/pygmalionism, and mechanophilia (i.e., respectively, the (sexual and/or romantic) attraction to objects, statue/dolls/mannequins, and machines; [102, 317] . just to name a few. that being said, if erobots are (or become), indeed, like a new species of intimate partners, we propose that universal darwinism may provide a core mechanism potentially explaining and predicting how human and erobot populations will influence each other as a function of the selective pressure they exercise on one another (i.e., emas; [75, 76, 78] ). universal darwinism (or general selection process; [147] ) generalizes the variation and selective retention of traits, the key mechanism of darwinian evolution, to other complex systems when conditions of variation and selective retention of traits are met, like in human-machine (erotic) interaction [70, 71, 75, 76, 78, 79, 168, 186, 300, 310] . universal darwinism has already been used to model the evolution of technology [186] . for instance, in accordance with complex adaptive system theory, the fittest (multi-agent) systems, algorithms, software, and applications endure, pass on their architectures, and populate our techno-ecosystem (i.e., fitness here being solely based on systems' ability to perform, adapt, survive, and (be) replicate(d) in a given ecological niche; [153, 206, 225] ). in evolutionary robotics, the principles of variation and selective retention of traits are used by software engineers [84, 152] . for instance, a first generation of codes-or "genotypes"-is generated as a potential solution to a problem (i.e., initial variations). the robots' fitness is then assessed in an environment, meaning that their code is translated into traits-or "phenotypes"-and their performance is observed to establish how well they interact with said environment to achieve goals. the fitness value determined by those observations then serves as a guide to select which robots will be used to seed the following generations; a process which is repeated until the targeted problem is solved [84, 152] . notably, these principles are now also being used to discover more efficient ml algorithms which could, in turn, enable artificial agents to learn and adapt more efficiently to uncertain environments and situations (e.g., human-machine (erotic) interaction) [246] . we thus conclude by hypothesizing that universal darwinism, as manifested by a process analogous to natural, artificial, and importantly, sexual selection, could be the engine behind human-erobot interaction and co-evolution, due to the social and sexual nature of erobotic technologies. this process, we propose, likely rests upon our evolving erotic cognition and the way it is co-influenced by our interaction with erobots and our ecological niche (as previously described). hence, overall changes in human and erobot interactions, cognitions, and populations could be better explained and predicted by emas (see fig. 2 ). this process could also become increasingly automated as the agency of erobots increases, and could in turn influence human-machine co-evolution by acting on individuals but impacting populations, and vice versa [125] . this mechanism possesses three important strengths: (i.) it can link individual and population levels-from interaction to co-evolution-in a perpetual feedback loop; (ii.) it can allow us to move in time from interactive, to proximal, to distal (and back again) in the co-evolution of biological and artificial erotic agents; and (iii.) it can help bridge hmi and sexology. that said, this is a hypothesis for future theoretical and empirical research in erobotics. what's more, the heicem already points to possible detrimental consequences for human (erotic) life and well-being if we do not rethink our current technological design and strive towards the development of beneficial erotic machines. erobotics aims to guide the development of beneficial erotic machines. to do so, and in line with döring and pöschl [89] , we propose that erobotics should operate under sexuality [308] and technology positive frameworks [252] -which are themselves inspired by positive psychology [269] . positive psychology is concerned with shifting our focus from solely examining negative aspects of the (human) behaviour, psyche, and life, to also considering (what enables) strengths, happiness, and health [269] . what this means for erobotics is that we should examine concerns and difficulties regarding intimacy, relationships, and sexuality, but also explore, and strive towards, pleasure, freedom, inclusivity, and diversity [308] . it also means that erobotics should aim to develop technologies that improve individual and collective wellbeing [252] . döring and pöschl's [89] sextech positive framework, we argue, is important and applicable to erobotics for three main reasons. first, it does not presuppose that certain sexualities or technologies are good/bad (ab)normal, or safe/dangerous. contrary to what some may consider a misleading or overly optimistic title, "positive" approaches encourage us to adopt judgment-free stances on research and interventions [252, 269, 308] . historically, this has been essential to the progress of psychology and sexology, which, unfortunately, have too often adopted biased, non-evidence-based, and harmful positions regarding individuals, groups, conditions, and/or sociosexualities (e.g., lgbtqa2s+ or kink, fetish, and bdsm; [29, 58, 96, 103, 120, 156, 165] ). second, it encourages us to consider the full spectrum of possibilities related to sexuality and technology, by exploring both negative and positive aspects of erotic technology-e.g., from its possible risks/dangers, disorders/dysfunctions, and problematic behaviors, to its potential benefits such as fulfilling intimate live and healthy technological use. third, it is solutionoriented; it does not (simply) stop at the "critical and risk perspectives", but instead encourages us to find ways to move from one end of the spectrum to the other. so, even if we must sometimes (importantly) focus on the negative aspects, it invites us to (re)embed our work within the larger goal of favoring human happiness, well-being, and flourishing. with these objectives in mind, the following sections highlight how human-erobot interaction and co-evolution may increase the likelihood of erobot-related risks if this process limits the diversity of erobotic traits available, and/or if the current approach to ai design is not changed-otherwise known as the standard model of ai design (i.e., optimizing specific pre-set goals; [257] ). as we have shown in the previous sections, erobots bring new agential and cognitive capabilities that may allow them to derive goals from their (erotic) interaction and co-evolution with humans. this can generate new issues related to human-machine compatibility. to curb these risks, we propose to design erobots based on russell's principles for beneficial machines [257] . we conclude that the development of beneficial erotic machines could mitigate erobot-related risks and enhance human wellbeing, through their potential health, education, and research applications. our interaction and co-evolution with erobots could be detrimental to human life if they progressively limit the diversity of erobotic traits available and negatively influence our erotic evolution. this issue may be exacerbated if profitdriven interests are responsible for the development of widely used erobots. in considering the fluidity and diversity of human sexuality (e.g., preferences, orientations, behaviors, and identities; [9, 177] ), limiting the access to diversified and inclusive erotic experiences is socially problematic, ethically dubious, and, arguably, economically counterproductive [22, 66, 82] . that said, the heicem suggests that erobots could rapidly undergo over-selection, such that the traits selected by the majority (e.g., compulsively used features) may be overreproduced in subsequent erobot populations (i.e., supply of variations), and that those that are less selected could be less reproduced and/or slowly disappear from the supply. this issue is particularly concerning in the event that the goal of developers is to maximize profit without considering human well-being. consider the basic example of the supply of erobotic traits: 50 physiological attributes (e.g., shapes, colours, hair), 50 psychological features (e.g., personalities and identities), and 50 behavioural patterns (e.g., social and sexual capabilities). suppose, then, that after a year, producers realize that only 60% of the initial traits have been selected by 90% of users. if we automate the supply and demand of traits using recommender systems based on predictive analytics, like the ones used by companies such as netflix or spotify [218] , then future supply will decrease proportionately. however, erotic diversity largely exists in marginal preferences [9, 177] . if automated systems like erobots over-select and overrepresent specific traits (e.g., the most popular), sociosexual diversity may decrease over time. this may in turn drastically limit the evolution of our eroticism, should erobots play a significant role in our intimacy. given that erobots are designed to act as intimate partners, over-selection may occur exponentially in human-erobot interaction and co-evolution. specifically, erobots could receive constant feedback regarding the preferences of millions of users and update their states or responses accordingly in real-time, to provide users with what has "worked best" for others, based on pre-established metrics (e.g., usage time and frequency). these metrics are susceptible to economic incentives, rather than being oriented towards individual and collective well-being [321] . therefore, in knowing human tendencies for intimate partner selection, the logic behind company-automated recommender systems, and the laws of supply and demand associated with said algorithms, we can predict that erobots could rapidly deliver what the majority wants, and in turn, reduce the supply of traits to fit that demand. we can also predict that this process could limit the diversity of erotic variations available-not necessarily in terms of quantity, but in terms of content [218] . this process contains the additional risk of overrepresenting traits that are detrimental to human well-being. that is, if left unmonitored, human-erobot interaction and co-evolution could be detrimental to human (erotic) life if we over-select traits that conflict with human interests-possibly heightening the likelihood of certain risks previously described in the literature [66] . for example, if erobots are designed solely to increase profit, they could further problematic or pathological dynamics. these may include addictionlike or obsessive-compulsive behaviours, increased social isolation, and reduced social skills [114, 190] . furthermore, if designers do not consider the importance of respect, mutuality, inclusivity, and diversity in human sexuality, erobots could end up perpetuating or reinforcing limited categories of social differences (e.g., gender/sex, race, and class), toxic patriarchal power dynamics, and rape culture (e.g., the objectification and commodification of women/females, ideas that men/males are owed sex, and problematic gender/sex stereotypes; [52, 129, 159, 170, 185, 210, 241, 249] ). they could conform to (or exacerbate) our ideologies by only providing us with information that reinforce our world view-an erotic filter bubble [229] . they could impair interhuman relationships or distort intimacy-related expectations (e.g., ideas that "personalized" sex should always be accessible; [133, 199, 249] ). they could take advantage of intimate contexts and emotional bonds to deceive users or manipulate our decision-making processes (e.g., political, consumption, and relationship choices; [114, 222] ). they may also record sensitive information [319] , which could in turn be sold to maximize profit (e.g., facebook and google exploiting personal data), or worse, become (weaponizable) hacking targets (e.g., tinder [46] and ashleymadison [316] ). that is, data from erobots could be used to coerce people, since taboo and stigma surrounding sexuality is still, in many parts of the world, enough grounds to destroy careers and relationships [114] . to summarize: human-erobot interaction and co-evolution may conflict with human interests if automated overselection limits the supply of erobotic traits and/or when a majority of individuals progressively select traits that conflict with human interests. a possible solution is to ensure that erobots reflect and maintain diversity in their evolving supply of traits (e.g., gender/sex, forms, behaviours, and personalities). after all, they can theoretically take any form and enact behaviours that contribute to human (erotic) wellbeing; they could echo the complexity and diversity of human sexuality [64, 170] . however, this is unlikely to be enough since at the core of the human-erobot interaction and coevolution problem is also another issue: the standard model of ai design. the standard model of ai design proposes to build machines that optimize specific objectives that we, humans, put into them [257] . for instance, alphago learns to play go by finding ways to optimize its number of points-a preprogrammed objective set out for them. to do so, its system plays against itself and other agents (biological or artificial), analyzes images, and through deep rl, optimizes its strategies to achieve the pre-set goal of increasing the score [43] . thus, intelligent machines based on the standard model have a perfect knowledge of the objectives to achieve [257] . the standard model is efficient and relatively safe for a go-playing machine with limited capabilities and scope of action, but it fails and can become detrimental to human wellbeing in real-world settings (e.g., human-machine (erotic) interaction)-particularly when machine agency increases. it fails, because in real-world settings we often ignore what quantities to optimize (e.g., in quality-driven intimate relationships; [43, 230] ), and it can become detrimental, because pre-set objectives-or the means to achieve them-can conflict with human interests [33, 196, 257, 284] . that is, programming biased, incomplete, or incorrect objectives can lead to unsuspected outcomes or loss of control [33, 214] . the problem with preference-based learning systems is also proportional to the agency of machines. specifically, an increase in the capability of machines to act in/on the world on their own to achieve pre-set goals may potentially result in their deployment of more sophisticated strategies to achieve those goals. these strategies may include subroutines for self-preservation (e.g., gather resources, copy itself, increase its computing power, change its code, and grow out of human control; [34] ), and deception: not unlike sciencefiction movies like ex machina [2] . in addition, machines built with a pre-set perfect knowledge of our objectives need not defer to us. they can instead conclude that humans are counterproductive to achieving their goals and remove us from the decision-making loop [33, 257] . but artificial agents do not have to be very sophisticated to cause problems. for instance, a personal assistant aimed at optimizing its predictive performance of our needs can become a nuisance, or detrimental to our autonomy. the standard model of ai design fails or becomes detrimental in human-machine interaction, precisely because of the human component. humans are often unstable, unpredictable, and unreasonable; our thoughts, emotions, preferences, behaviours, and objectives fluctuate constantly. we do not always know what we want, let alone how to achieve what we want. it is thus difficult (if not impossible) to program specific objectives that safely hold true across time and circumstances. the same goes for erotic interaction; our objectives-or what we want out of relationships, intimacy, and sexuality-remain, for the most part, conjectural. as such, we do not know what quantities to optimize in humanmachine erotic interaction, and if we do optimize some functions of behaviour, it can backfire. firstly, any objective can become obsolete during human-machine (erotic) interaction if it inadequately captures the unpredictable ways in which erobots influence human preferences and goals. secondly, it can lead to unsuspected outcomes or loss of control due to the pre-programming of biased, incomplete, or incorrect objectives [257] . for instance, in trying to achieve any pre-set goal, such as making users happy or providing erotic satisfaction, a machine could conclude that its first objective is to maximize the time spent with us. to achieve this, it could optimize its body types, personalities, and behaviours-escalating or varying reward experiences (e.g., lottery machines or instagram)-which can in turn chip away at human control, increase risks of addiction-like or obsessive-compulsive behaviours, and further social isolation [19, 114, 190, 191, 233] . it could also systematically fulfill its users' needs while disregarding its influence on our interhuman relationships [199] . it may repeatedly fall into closed loops, by reinforcing the patterns that once led to happiness or satisfaction, but that are becoming redundant, inefficient, or are limiting exposure to other forms of complex sociosexual interactions [257] . it could end up reciprocating similar ideas, communication style, and past preferences to users-an erotic echo chamber that is either boring or erotically limiting [77, 118] . it could also have an incentive to deceive, manipulate, and/or gather as much data on us as possible, to make its users happy-increasing risks pertaining to privacy and confidentiality [114] . finally, an erobot based on the standard model would not necessarily have to defer to us or ask for consent before deploying its strategies-even if they conflict with our interests-since it may already have a perfect knowledge of our goals [257] . these are just a few examples of ways in which the pre-set objectives of erotic machines may conflict with human interests. and, while some companies may see them as profitable ideas, they represent ethical, social, and developmental dead ends. for these reasons, we need to rethink ai design and stop trying to build machines that aim to optimize pre-set goals-particularly in intimate machines that could become significant part of our erotic lives and have continuous access to sensitive information. this is crucial if we want to steer erotic technology in a positive, ethical, and beneficial direction that favors human wellness, which in the end, could arguably be more economically profitable [22] . machines are beneficial if their objectives are in line with ours [257, 284] . granted that, since our objectives are uncertain, and programming incomplete, incorrect, or biased goals can conflict with human interests, stuart russell proposes three interdependent principles to guide us in rethinking how to create (agential) artificial systems [257, p. 173 ]: 1. the machine's only objective is to maximize the realization of human preferences. 2. the machine is initially uncertain about what those preferences are. 3. the ultimate source of information about human preferences is human behavior. the first principle aims to make purely altruistic machines that have an incentive to act for humans rather than any other entity (i.e., machines that have no self-interest and do not value their welfare or that of non-humans; [257] ). this would lead the artificial agents to prioritize the wellbeing of humans, as well as avoid conflicting preferences and goals between the two parties. this principle also invites the development of machines that consider our extended and changing preferences. precisely, if designed properly, beneficial machines could also learn, incorporate in their model, and aim to maximize, our extended and/or high-order preferences. machines could thus aim to maximize the welfare of other systems (e.g. (non-)humans and the environment), proportionally to the level of importance attributed to them by their users [257] . the second principle aims to develop humble machines that do not assume perfect knowledge of human preferences [257] . as previously mentioned, machines with perfect knowledge of human preference have no incentive to defer to us. they can remove us from the decision-making loop, deploy strategies to achieve their goals, and ignore their influence on human life. for example, if a machine knows that the "true" preference of a person is to be healthier, they might decide to forcibly restrict behaviours like eating junk food or driving a car. they would not have to ask as they would know what their users "really" want. uncertainty, however, places humans back in the driver's seat: machines that imperfectly know our preferences, but still aim to maximize them, have an incentive to defer to us, and ask for more information or commands in ambiguous situations, to improve their model. uncertainty also prevents machines from concluding that proximal behaviours (e.g., choices) invariably reflect human preferences. instead, it enables them to consider such behaviors as probabilistically related to (or encapsulated in) unknown preferences or goals, and to continue searching for them to improve their model [257] . the third principle aims to make useful machines that learn from observable quantities/metrics and establish a practical link with humans [257] . but it also aims to build machines that consider human behaviours as imperfect approximations of our preferences or goals. that is, assuming that our behaviours (e.g., choices) are connected to our preferences in complex ways, but do not always accurately reveal our preferences or goals. this is important given that what we do can be related to distal preferences (e.g., eating food that we do not like to make a host happy or maintain friendships), proximal preferences (e.g., getting drunk to have fun), or simple mistakes (e.g., missing an exit because we were not paying attention). the third principle establishes a practical connection between humans and machines, so that artificial agents can still improve their model based on observable data, and help maximize our preferences (first principle) while remaining uncertain of what those are (second principle; [257] ). as russell [257] explains, these principles are not laws that determine machine behaviour or completely shield humans from harm. they are guidelines to rethink ai design, move away from the standard model, and steer the development of intelligent machines in a safer direction that accounts for their growing capabilities. hence, the implementation of these principles deserves careful consideration, which is beyond the scope of this article. but we can already foresee some necessary fail-safes [257] . for example, regarding the first principle, russell [257] recommends the implementation of countermeasures that mitigate risks associated with people whose preferences are to harm others, since maximizing the realization of those preferences would be a problem. regarding the second principle, russell [257] recommends that we impose a "certainty threshold," or a limit for the certainty level achievable by machines to make sure that their predictive model never approaches perfect knowledge of human preferences, which would be the same as having pre-set objectives [257] . that said, even if these principles are not laws, they could promote the development of more human-compatible beneficial erobots. as a possible solution to the risks highlighted by the heicem, and in line with its sextech-positive goals, erobotics should aim to develop beneficial erobots whose objectives align with ours. to do so, we propose building altruistic, humble, and useful erobots that learn to predict human preferences from our behaviours, based on russell's principles [257] . specifically, erobots that (1) aim to maximize the realization of human erotic preferences, (2) are initially uncertain about what those erotic preferences are, and (3) use human behaviour as their ultimate source of information about our erotic preferences. erobots abiding by the first principle would have an incentive to act for humans rather than for themselves or the erotic preferences of non-humans. yet, to the extent that our erotic preferences include the well-being of others (e.g., the people their users interact with), beneficial erobots would also be concerned with maximizing their welfare proportionally to their users' altruistic tendencies. erobots abiding by the second principle would not assume perfect knowledge of human preferences. uncertainty would keep us in the decision-making loop by providing erobots with an incentive to defer to us when they are unsure about intimate interactions. thus, similarly to a receptive partner trying to further respect and mutuality, beneficial erobots could first consult humans, and then improve their predictive model accordingly, while never achieving total certainty. uncertainty could also prevent erobots from concluding that proximal erotic behaviours unvaryingly reflect human preferences or objectives. finally, beneficial erobots abiding by the third principle would base their learning processes on our erotic behaviours (e.g., intimate and sexual choices), while considering them as imperfect approximations of our erotic preferences or goals. for example, we sometimes engage in intimate activities for the benefit of others or make compromises to maintain relationships. still, by using our erotic behaviours as a proxy, beneficial erobots could refine their model, while preserving a safe dose of uncertainty that would enable our control and the compatibility of interests. over time, beneficial erobots designed with these principles could discover that human erotic preferences fluctuate and evolve, including during our interactions with them, and adapt accordingly. they could progressively recognize the diversity of human preferences (e.g., in forms, personality, and behaviours) and come to learn that, paradoxically, people enjoy-to various degrees-predictability, habit, and familiarity in their eroticism, but can also eventually habituate to (or grow bored of) being repeatedly exposed to the same thing, and resort to seeking novelty [19, 45, 184, 211] . to maximize the realization of such uncertain preferences, beneficial erobots would have an incentive to ask humans for consent and/or commands prior, during, and after erotic interactions to improve their model-while never assuming perfect knowledge of our preferences or goals, and spiralling out of control. instead, they could influence our behaviours to help us achieve our (higher and/or distal) preferences and goals (e.g., well-being), without imposing their will onto us-i.e., a sort of erotic nudge [31, 127, 134, 139, 272, 285] . beneficial erobots, we propose, could mitigate erobotrelated risks. specifically, their uncertainty could prevent them from falling into closed reinforcement loops or escalating rewarding experiences while disregarding how they influence other areas of our functioning. this could, in turn, reduce risks of addiction-like or obsessive-compulsive behaviours, and social isolation. through multi-user interactions, they could learn that the path to maximizing our preferences and goals (possibly) differs for each person. as such, they could propose personalized paths, but always aim to strike a balance between (erotic) novelty and familiarity. in time, counterproductive patterns could be mitigated by their imperfect knowledge of our preferences and their attempt to humbly maximize them while keeping us in the decision-making loop. moreover, to enhance our well-being, beneficial erobots could potentially educate users on topics such as: respect, diversity, mutuality, and consent [177] . in doing so, they could actually contribute to breaking cycles that perpetuate categories of social differences, toxic patriarchal power dynamics, and rape culture [64, 170, 199] . they could also try to harmoniously integrate into our intimacy [199, 257] . for example, instead of impairing our interhuman relationships, they could help us prepare for partnered life (e.g., practising compromise and communication). during a relationship, they could provide advises and help bridge common gaps in desires or preferences using a controlled outlet. after a relationship, they could help us recover by providing continuous support, intimacy, and companionship [199] . this would be possible without machines having to deceive or manipulate us, but instead, having an incentive to reveal the purpose of their actions and protect our data to the extent that it maximizes our preferences. in sum, beneficial erobots could reduce the likelihood of erobot-related risks, because their objectives are in line with ours. they would have an incentive to further human (erotic) flourishing without necessarily knowing it. and this could provide us with unprecedented safe access to wellbeing through their potential future applications. the advent of safe beneficial erotic machines opens the door to several health, education, and research applications. in terms of health, erobots could be used, for instance, by people who are single, isolated, have specific orientations or preferences, have physical or mental impairments, and/or have social or sexual difficulties finding partners [27, 57, 83, 109, 180, 265] . they could also be employed by those who may prefer artificial partners or anyone who wants to experience pleasure and companionship [112, 179, 199, 200] . indeed, everyone deserves a safe access to pleasurable intimacy and sexuality [312] . but this is not always possible. sometimes partners are not available (e.g., long-distance relationships or lack of compatible partners; [94] ). sometimes people want to explore on their own before engaging with others (e.g., after a trauma, a surgery, or to practice; [98, 179] ). sometimes engaging with a partner is unsafe (e.g., people with impulse control issues; [99] ). and, sometimes people do not necessarily want intimacy with humans (e.g., some doll-owners, robot fetishists, and people with objecto/agalmatophilia; [87, 112, 158, 223] ). here, technology can democratize eroticism and expand the possibilities of sexual wellness and health, but only if we make it inclusive and accessible (e.g., by considering gender, sexual, and racial diversity, power dynamics, and socioeconomic status; [22, 248] ). still in terms of health, erobotic technologies could have medical and therapeutic applications. they could act as care machines to provide adapted erotic stimulation to the elderly or individuals with disability, while simultaneously mitigating controversies surrounding sexual surrogacy and sex work [26, 27, 83, 109] . they could also help individuals with psychosocial, physical, and sexual difficulties [99] . for instance, under the supervision of trained (sex) therapists and educators, erobots may contribute to assessments and treatments of individuals with intimacy-related fears and anxiety via progressive exposition-desensitization [172] or help people with erectile dysfunction or premature ejaculation [226] . they could be used in therapy to help trauma victims become reacquainted with their body and sexuality in a safe, controlled environment [187, 188] . they may be part of clinical interventions for pelvic floor disorders [273] or sexual pain, to provide adapted and more ecologically valid stimulation that reduce hypersensitivities and break stimuli-pain associations [215] . they could be used to practice sociosexual interaction, communication, and distancing (e.g., during the coronavirus (covid-19) crisis) [17, 150, 255] . finally, they could help individuals become better partners and feel more confident with their body, sexual capabilities, and erotic agency. in terms of education, erobots and their related technologies could be used to provide interactive, validated, inclusive, and personalized sex education, and to help people learn about pleasure, respect, consent, inclusivity, diversity, and mutuality in an innovative and accessible way (e.g., plan parenthood's roo online chatbot; [238]). they may be employed for judgment-free self-exploration and practice to help people discover their erotic preferences [98, 179] , gain confidence, and be better partners. they could also provide resources (e.g., educative websites, clinics, feminist sex shops) or help create platforms for people to meet, build communities, discuss sexuality, and feel validated. sex education is unevenly distributed in the world, but if we favor inclusivity and accessibility [164] , technology can once again democratize this important service [40, 100, 289] . in terms of research, erobots could be used as standardized research tools to help researchers overcome methodological and ethical challenges related to sensitive research programs (e.g., sexology; [189, 306, 319] ). erobots may act as both stimuli and recording instrument in research protocols [189, 319] , while reducing the risks associated with interhuman interaction. their forms and behaviours can also be manipulated to isolate the influence of different variables on human responses. this could improve the ecological validity of experimental paradigms by bringing them closer to interhuman intimacy and sexuality. erobots could also provide access to data that are otherwise difficult to assess empirically (e.g., touch and movement in partnered sex). they could also facilitate data collection in people's everyday environment (e.g., at home; [319] ). finally, erobots do not require available human partners to participate in a study that necessitates multiple people. overall, erobotic technologies could enable us, for the first time, to gain a holistic view of human eroticism. importantly, however, to harness the full potential of erobots, we must involve people with diverse life experiences in their design and implementation stages. we must ensure the inclusion of: diversity in gender, sexuality, and ethnicity; people with disabilities; as well as people with different preferences, orientation, lifestyles, and socioeconomic status [22] . inclusiveness in the development of erotic technology can reduce risks of blind spots (e.g., assumptions about what people want or need), cover broader markets, and contribute to a more comprehensive human well-being [22] . in the twenty-first century, humans and artificial agents are increasingly coexisting through complex multi-agent systems. the scholarly investigation of the processes of their interaction and co-evolution has only started to become a serious research topic in recent years. despite many important contributions made in hmi and social robotics, no comprehensive theoretical framework addresses the advent of immersive, interactive, and interconnected agential erotic technologies. while sexual pleasure and health are progres-sively being considered basic human needs and rights [312] , research on sexuality remains taboo, especially in the study of technology. yet, in the face of widespread intimacy-related difficulties and dissatisfaction [177] , the human motivation for self-expansion [14] , and the ubiquity of technology in our lives [243] , we predict that the supply and distribution of (agential) erotic technology can (continue to) increase exponentially. the scientific study of this latest stage of our erotic evolution as a species has just begun. in this foundational paper, we argued that modern technology-mediated human intimacy requires a new unified transdisciplinary field of research intersecting hmi and sexology that we coined erobotics. we proposed the necessary conceptual and theoretical groundworks for this new field and explained how and why erobotics should adopt sexuality and technology positive frameworks. by studying the cognitive intricacies of human-machine erotic interaction and co-evolution, and by making the development of beneficial erotic machines more plausible, it is our firm belief that erobotics will open up promising new paths of research in hmi, sexology, social ai/robotics, and beyond. in this paper, we proposed a taxonomy of erobots that helps specify their fluid embodied, virtual, and augmented manifestations. we developed the first spectrum of erobots' agency in view of future theoretical, empirical, and clinical research. we also introduced the heicem, which constitutes theoretical grounds to launch a broad research program on erobotics. this model rests upon our ever-changing erotic cognition, and predicts how human and erobots can coinfluence each other over time. granted that, this model also points to potential risks if erobotic traits undergo overselection/representation while following the current standard model of ai design. to mitigate these unwanted consequences, we proposed that russell's [257] principles for beneficial machines be used to guide erobotic design, so that beneficial erotic machines could act to further human wellbeing through their potential health, education, and research applications. this article is not without limitations. the first one is that the most advanced erobots are not yet widespread or are based solely on future applications of existing technologies (or their potential combination). this means that the actual impacts of emerging erotic technologies on humanity (and vice versa) are hard to perceive and to study empirically. however, with the rise of digisexuality and the sextech industry, erobots have the potential to occupy a greater place in our erotic cognition and life. thus, developing erobotics today may guide its study and positive development for tomorrow. the second one is that it is not exhaustive. it proposes basic concepts, a (multi-level) testable model, and a path to explore human-machine erotic interaction and co-evolution. the details of which should be developed in future collaborative, transdisciplinary, and inclusive research, using a wide diversity of expertise. in fact, it is our hope that the terminology, frameworks, challenges, and potential applications discussed in this article will inspire the development of a comprehensive research agenda on erobotics: an agenda that involves people with diverse life experiences, and that builds upon collaborations of academia, the private sector, non-profit organizations, governmental institutions, and communities. as a concluding remark, we allude to the opening quote of plato in his symposium [7] . in this classic dialogue, readers are led by socrates to understand the "aporetical" (aporētikós) nature of erôs. while all human beings experience and seek erôs in its many forms-friendship, desire, pleasure, intimacy, sensuality, sexuality, love, etc.-, we mortals remain incapable of understanding its truth or "essence", which is "divine," and thus, inaccessible according to plato, the greeks, and most cultural belief systems. not unlike the phenomenon of consciousness, which inspired mysticism and religious beliefs about the soul, we never developed a genuine science of erôs, because we humans redefine the meaning of erôs each time we experience it. 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of sex robots in comparison to the attractiveness of women, and personal characteristics influencing this evaluation life 3.0: being human in the age of artificial intelligence nudging through technology: choice architectures and mobile information revolution a computational model of perceived agency in video games to tinder or not to tinder, that's the question: an individual differences perspective to tinder use and motives an integrative theoretical framework for understanding sexual motivation, arousal, and behaviour are social media a problem or a tool? new strategies for sexual education computing machinery and intelligence authenticity in the age of digital companions evolution, learning, and instinct: years of the baldwin effect introduction to special issue on digihealth and sexual health the modern sex doll-owner: a descriptive analysis the embodied mind: cognitive science and human experience what things do: philosophical reflections on technology, agency, and design cyborg intentionality: rethinking the phenomenology of human-technology relations a perspective view and survey of metalearning virtual mate. software design and development chimpanzee politics: power and sex among apes finding lolita: a comparative analysis of interest in youth-oriented pornography the biological basis of sexual orientation: how hormonal, genetic, and environmental factors influence to whom we are sexually attracted brain drain: the mere presence of one's own smartphone reduces available cognitive capacity collaborative structure between japanese hightech manufacturers and consumers unsupervised predictive memory in a goal-directed agent imaginationaugmented agents for deep reinforcement learning japan's emerging emotional tech introducing a multidisciplinary framework of positive sexuality objects of desire: the role of product design in revising contested cultural meanings humans as a model species for sexual selection research human sexual cycles are driven by culture and match collective moods sexual health, human rights and the law phylogenesis of mammal sexuality: analysis of the evolution of proximal factors toward acceptable domestic robots: applying insights from social psychology reboot for the ai revolution hackers finally post stolen ashley madison data cultural differences and the practice of sexual medicine: a guide for sexual health practitioners ai love you: developments in humanrobot intimate relationships cyberphysical human sexual behaviour acquisition system (seba): development and implementation study in china can we control it? autonomous robots threaten human identity, uniqueness, safety, and resources the age of surveillance capitalism: the fight for a human future at the new frontier of power. publicaffairs, new york publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations he specializes in sexuality and technology. specifically, his research program investigates how people's subjective, cognitive, and psychophysiological responses to sexual stimuli relate to their sexual preferences. he applies multi-method research designs to the study of human-machine erotic interaction-or erobotics-and explores the influence of new interactive, immersive, and connected (socio)sexual technologies he is also affiliated with the observatoire international sur les impacts sociétaux de l'ia the authors would like to thank dr. pfaus, dr. key: cord-279406-wwdqh9qs authors: guzman, norberto a.; guzman, daniel e. title: a two-dimensional affinity capture and separation mini-platform for the isolation, enrichment, and quantification of biomarkers and its potential use for liquid biopsy date: 2020-07-30 journal: biomedicines doi: 10.3390/biomedicines8080255 sha: doc_id: 279406 cord_uid: wwdqh9qs biomarker detection for disease diagnosis, prognosis, and therapeutic response is becoming increasingly reliable and accessible. particularly, the identification of circulating cell-free chemical and biochemical substances, cellular and subcellular entities, and extracellular vesicles has demonstrated promising applications in understanding the physiologic and pathologic conditions of an individual. traditionally, tissue biopsy has been the gold standard for the diagnosis of many diseases, especially cancer. more recently, liquid biopsy for biomarker detection has emerged as a non-invasive or minimally invasive and less costly method for diagnosis of both cancerous and non-cancerous diseases, while also offering information on the progression or improvement of disease. unfortunately, the standardization of analytical methods to isolate and quantify circulating cells and extracellular vesicles, as well as their extracted biochemical constituents, is still cumbersome, time-consuming, and expensive. to address these limitations, we have developed a prototype of a portable, miniaturized instrument that uses immunoaffinity capillary electrophoresis (iace) to isolate, concentrate, and analyze cell-free biomarkers and/or tissue or cell extracts present in biological fluids. isolation and concentration of analytes is accomplished through binding to one or more biorecognition affinity ligands immobilized to a solid support, while separation and analysis are achieved by high-resolution capillary electrophoresis (ce) coupled to one or more detectors. when compared to other existing methods, the process of this affinity capture, enrichment, release, and separation of one or a panel of biomarkers can be carried out on-line with the advantages of being rapid, automated, and cost-effective. additionally, it has the potential to demonstrate high analytical sensitivity, specificity, and selectivity. as the potential of liquid biopsy grows, so too does the demand for technical advances. in this review, we therefore discuss applications and limitations of liquid biopsy and hope to introduce the idea that our affinity capture-separation device could be used as a form of point-of-care (poc) diagnostic technology to isolate, concentrate, and analyze circulating cells, extracellular vesicles, and viruses. biomarkers are measurable substances or characteristics in the human body that act as indicators of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions [1] [2] [3] [4] . in practice, biomarkers include tools and technologies that have been used in clinical medicine for decades, and they play a critical role in all aspects of prevention, diagnosis, and treatment of disease [2] . despite advances in laboratory technology and the availability of significant information on biomarkers, we are quite far from widespread clinical use of biomarkers due to several limitations. the slow, arduous, and challenging process by which biomarkers are translated into clinical use, makes them suffer from low sensitivity, specificity, and predictive value, particularly when they are applied to rare diseases in population screening programs [3] . as a consequence, medicine in general, has long been criticized for lagging behind other industries in both innovation and adoption [4] . however, with a new wealth of biomarker information obtained over the last decade and advancements in systems medicine, there are opportunities to make significant improvements to diagnostics and therapeutics in medicine [5] . in this manuscript, we describe a point-of-care biomarker analyzer that has been demonstrated to identify and quantify circulating cell-free small molecules and complex biomolecules in biological fluid samples by utilizing an affinity capture-separation technology. furthermore, we will discuss the potential of this instrument to serve as a tool for detecting and analyzing cellular and subcellular entities, vesicular entities, viruses and their respective cargo contents. the ultimate goal of this biomarker analyzer is to demonstrate the potential to non-invasively and rapidly yield highly sensitive and selective tests to aid real-time clinical practice. the existence of different meanings for certain terminologies in clinical diagnostic chemistry and analytical chemistry is a source of confusion related to several laboratory assays, particularly immunoassays. in clinical diagnostic chemistry, a sensitive test refers to a test that will correctly identify almost all individuals who likely have a disease, and that will rarely yield a false-negative result. a specific test refers to a test that will almost always correctly rule out those who do not have a disease and will rarely yield a false-positive result [6] . in analytical chemistry, sensitivity is the minimum detectable concentration of an analyte, expressed as the limit of detection (lod), that can be accurately measured. specificity is the ability to accurately assess a single analyte in the presence of components which may be expected to be present in the sample matrix, such as interferences. similar to specificity, selectivity is the ability to accurately separate out and assess multiple components present in a sample mixture or matrix [7] . biopsy is another term that historically has referred to an invasive examination of tissue removed from a living body to discover the presence, cause, or extent of a disease [8] . the term biopsy has evolved to include both tissue biopsy and liquid biopsy, a non-invasive or a minimally invasive procedure used for the detection and isolation of circulating tumor cells, circulating tumor dna, and exosomes circulating in biological fluids [9, 10] . in turn, liquid biopsy has allowed for further prognostication and evaluation of therapeutic response in patients with malignancy. recent studies have shown that liquid biopsy has applications in non-cancerous diseases, allowing for the examination of cells, subcellular entities or vesicles, and their chemical and biochemical contents to discover the presence, cause, or extent of any disease [11, 12] . for example, the use of liquid biopsy via a high-throughput metagenomic sequencing assay has recently demonstrated both detection of a diverse array of bacterial and viral pathogens and quantification of damage to host tissues. the assay implements whole-genome sequencing of cell-free dna (cfdna), small fragments of dna released by host or microbial cells into blood, urine, and other body fluids [13] . similarly, proteins derived from the novel coronavirus 2 (sars-cov-2) have been analyzed by high-performance liquid chromatography (hplc) coupled to mass spectrometry (ms) from samples obtained from coronavirus disease (covid-19) patients [14] . though the concept of a miniaturized or personal laboratory has been voiced for several years [25, [43] [44] [45] , the creation of a modular design that is manufactured with compact and low-weight components for easy portability has proved to be difficult. capillary electrophoresis is a separation technique that has the potential to address this limitation as it utilizes miniaturized components, including a small power supply and detectors that can fit into a small space, thereby facilitating the manufacture of an instrument no larger than the size of a lap-top computer or smaller device [25, 45, 46] . instruments developed for ce use are manufactured in two formats: a conventional platform using fused-silica capillaries, and a microfluidic chip format consisting of microchannels etched or molded into a material made of glass, silicon, or a polymer such as polydimethylsiloxane [25, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] . newer microfabrication techniques in particular are advancing, and microchip production using 3d is now under development [52, 53] . overall, ce offers advantages such as reduced sample and reagent consumption, lower operating costs, shorter reaction time, better portability, higher number of theoretical plates, and higher reliability [25, 47, [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] . despite many advances in the ce technology, there is still a limited number of medical applications that are routinely performed in a clinical laboratory. before it can replace existing technologies currently used in clinical laboratories, ce has its own limitations that must be addressed. for example, the internal diameters of the capillaries and micro-channels utilized in ce are usually less than 200 micrometers, allowing the utilization of small volumes of fluids which are normally in the order of nanoliters and picoliters. consequently, the limitations of introducing small volumes of sample into the capillary column or channel results in poor concentration limits of detection, and hence risks overshadowing the many benefits of the ce technology [25, 47, [61] [62] [63] [64] [65] [66] . this is particularly concerning given that crucial biomarkers found in biological fluids are present in low concentrations which, if not detected and quantified, may lead to missing an important diagnosis. a misdiagnosis or delayed diagnosis further leads to incorrect treatment, delayed treatment, or no treatment at all, thereby worsening a patient's medical condition. unfortunately, most examples reported in the literature for the ce quantification of analytes in complex matrices are usually developed for substances found at large concentrations. while not all diagnostic errors are secondary to laboratory errors, the national academy of medicine estimates that all patients will experience one serious diagnostic error during their lifetime, and diagnostic errors are now the leading cause of medical malpractice claims [67] . overcoming the poor analytical sensitivity of ce has therefore become the emphasis of many investigations related to life science applications. promisingly, efforts to improve the analytical sensitivity of ce thus far have led to successful improvements in the limits of detection of substances found at low concentrations. some of these methods include field-amplified sample stacking, large-volume sample stacking, ph-mediated sample stacking, on-column isotachophoresis, chromatographic preconcentration, sample stacking for micellar electrokinetic chromatography, sweeping, and derivation of analytes with fluorescent chromophores [25, 47, 54, 55, [61] [62] [63] [64] [65] [66] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] . one technology in particular is the combination of solid-phase extraction methods, using affinity-based ligands, with ce. highly selective affinity ligands or adsorbents, such as antibodies, antibody fragments, lectins, aptamers, enzymes, metal-organic framework-based affinity sorbents, and other specially designed ligands are the cornerstone for the isolation and purification of many substances and cellular-subcellular entities found in complex matrices [25, 47, [54] [55] [56] [57] [58] 75, [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] . immunoaffinity capillary electrophoresis (iace) is a disruptive technique, which combines the use of antibodies and/or other affinity ligands (e.g., lectins, aptamers, metal-organic, or others) as highly selective capture agents with the superior resolving power of capillary electrophoresis. the high-resolution analytical separation ability of iace provides the advantage of characterizing and discerning different forms of the same protein (proteoforms) biomarker and drug metabolites, which can improve insights into disease diagnosis, monitor drug efficacy, and shorten the length of clinical trials. since the introduction of iace in the early 1990s by our laboratory, terry phillips' laboratory, and others, the technology has increased significantly in popularity, and many applications have been reported using both conventional ce and microchip ce [25, 54, 55, 58, [61] [62] [63] [64] 66, 75, [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] . furthermore, if iace is coupled to powerful detectors such as laser-induced fluorescence detectors or mass spectrometers, a significant improvement in limits of detection can been achieved [25, 75, [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] . since its inception, iace technology has used a "linear or unidirectional" protocol for the introduction of samples and buffers [88] [89] [90] [109] [110] [111] [112] [113] . a small area of the capillary known as the "analyte concentrator-microreactor" (acm) zone or device contains affinity ligands immobilized either to a matrix localized within the cavity of the acm device, or directly to the inner wall of a capillary or channel positioned near its inlet side. the term "analyte concentrator" refers to the function of isolating and concentrating a target analyte, usually present in a complex matrix, by using a "chemical or biochemical magnet" [25, 75, [88] [89] [90] 109] . the term "microreactor", on the other hand, refers to the function of performing a chemical, biochemical and/or cellular-organoid reaction, such as derivatization, chemical synthesis, enzymatic reaction, pharmacokinetic studies, or therapy response-prediction [25, 75, [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] . the linear or unidirectional iace capture-separation system aimed at performing on-line concentration and separation presents two problems: backpressure and contamination of the surface of the separation capillary or channel after repetitive uses. the backpressure is formed when there is use of a beaded matrix requiring porous frit structures, or when there is a compacted continuous bed composed of a porous micro/nanoscale monolithic structure. the contamination of the surface of the separation capillary or channel is due to the non-specific adsorption of analytes present in the sample. the fused-silica capillary or the microchip made of silica material has free silanol groups to which biomolecules such as proteins bind readily. this non-specific binding leads to changes in migration times and peak areas of the analytes after several uses of the separation capillary or channels. in turn, the altered inner surface of the capillary or microchannel yields irreproducible results and limits the use of the capillary or microchip. to solve these problems, a different approach to introduce samples and buffers was developed, which is known as an "orthogonal" iace capture-separation system. in this format or protocol, samples, buffers, or solutions are introduced into the acm in an orthogonal/perpendicular direction via capillaries: in one direction lie the transport capillaries with their entrance and exit ports, while in the other direction lie the separation capillary with its entrance and exit ports. both the transport and separation capillaries share a small section containing one or more affinity ligands immobilized to a beaded matrix localized within the cavity of the acm device, or immobilized directly to the inner wall of the capillary or channel. microvalves are used to control the direction of the passage of fluids, thus preventing contamination of the separation capillary once a sample has been suctioned or pushed through the transport capillary. the transport entrance port is connected to the transport capillary, thereby allowing samples, buffers, or other solutions to be introduced into the acm device from a container or vessel located at the inlet side of the transport capillary. for this, the microvalves localized at the transport capillary ports are open, and the microvalves localized at the separation capillary ports are closed. the transport exit port allows excess amount of sample or buffer that is passed through to be evacuated into a waste container or vessel. once a sample has been passed through the transport capillary, allowing for the target biomarker to be captured via immunoaffinity or another affinity principle, a cleaning buffer or solution is introduced through the transport capillary to remove non-specific bound materials. at this stage, the microvalves localized at the transport capillary ports are closed, and the microvalves localized at the separation capillary ports are open. a container or vessel containing a separation buffer or solution located at the inlet side of the separation capillary or channel can then be introduced. the container has a platinum-iridium electrode immersed in the buffer or solution that is connected to a high-voltage power supply, having positive and/or negative polarity. another container containing the same separation buffer or solution is localized at the outlet end of the separation capillary, having a second electrode that serves to ground the electrical system. a separation buffer is introduced from the inlet to the outlet side of the separation capillary, followed by a small plug of an elution buffer, either alone or carrying a chromophore, to release or release and derivatize the bound target biomarkers from the biorecognition affinity selectors immobilized to the acm device. a high-voltage power supply, connected to an electrode immersed into the container containing the separation buffer, is then turned on to allow the passage of the target biomarker(s) to the separation capillary exit port. separation of target biomarker(s) can be accomplished by electrophoretic mobility, electroosmotic flow, mechanical pressure, or a combination of electroosmotic flow and mechanical pressure. the system is hermetically sealed using tight fitted connectors to prevent air bubbles to enter into the capillaries. the separation capillary outlet side can be connected to one or several detectors to quantify and/or characterize the separated biomarker(s). most detectors are positioned on-line (uv-absorbance or fluorescence), in-line (amperometry), or off-line (mass spectrometry) at the outlet end of the separation capillary, usually at a single point of detection [47] . however, when using a charge-coupled device (ccd) camera detection system, it is possible to capture the sample zones in motion during the migration through the capillary [123] . figure 1 depicts a schematic representation of the two models of acm devices. both containing biorecognition affinity ligands or selectors immobilized to a matrix localized within the cavity of the acm device. the acm devices are often referred to as solid-phase extractor devices or simply spe devices. the term "acm device" is more appropriate because of the dual functionality of the device: it functions as both an on-line preconcentrator and an on-line microreactor. a major advantage of the orthogonal iace design is that it permits the protection of the inner surface of the separation of the capillary or channel during the sample introduction and cleaning process. four micro-valves positioned at each entrance-exit of the acm device can be controlled manually or by a computer. when the microvalves located at the entrance-exit of the transport capillary are open and the microvalves located at the entrance-exit of the separation capillary are closed, there is no contact of the sample and cleaning the unidirectional iace design is operated without the presence of microvalves, whereas the orthogonal iace design requires the presence of microvalves, as depicted in (b) with green circles. the orthogonal iace device has four microvalves positioned at each entrance-exit port. these microvalves are crucial in controlling the path of fluids through the transport capillary or separation capillary. black arrows indicate flow direction of buffers in the separation capillary, and migration direction of the separated analytes within the separation capillary; purple arrows indicate the flow direction of sample and cleaning buffers introduced into the transport capillary. biorecognition affinity capture ligands or affinity capture selectors are immobilized to a beaded or monolithic structure positioned within the cavity of the "analyte concentrator-microreactor" (acm) devices, or immobilized directly to the inner surface of the cavity of the acm device. the affinity capture selectors can be antibodies, antibody fragments, lectins, aptamers, enzymes, phages, receptors, protein a, protein g, or a variety of substances having affinities for different kind of substances. figure modified from [25] and [75] . numerous immunoaffinity capillary applications employing various configurations of the acm device, using either a conventional capillary electrophoresis or a microfluidic chip capillary electrophoresis, have been reported [25, [54] [55] [56] [57] [58] 61, 75, [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] . figure 2 depicts the use of an iace selective affinity capture-separation system for the quantification of brain-derived neurotrophic factor (bdnf), a neuropeptide growth factor obtained from human skin biopsies of atopic inflammatory reactions [124] . figure 2a is a schematic representation of an acm device where whole tetrameric antibodies, nanobodies, antibody fragments, such as fab, or other affinity ligands, such as aptamers and lectins, can be directly immobilized to its inner surface, instead of using a beaded or monolithic matrix support as depicted in figure 1 . since the acm device is not yet commercially available, each laboratory has made its own modifications to the original protocol published elsewhere for use in conventional or microchip capillary electrophoresis [25, 66, 70, 75] . for the protocol carried out in these experiments, streptavidin was first immobilized off-line to the surface of 2-mm glass disks serving as a solid support. this was followed by the addition of biotinylated the unidirectional iace design is operated without the presence of microvalves, whereas the orthogonal iace design requires the presence of microvalves, as depicted in (b) with green circles. the orthogonal iace device has four microvalves positioned at each entrance-exit port. these microvalves are crucial in controlling the path of fluids through the transport capillary or separation capillary. black arrows indicate flow direction of buffers in the separation capillary, and migration direction of the separated analytes within the separation capillary; purple arrows indicate the flow direction of sample and cleaning buffers introduced into the transport capillary. biorecognition affinity capture ligands or affinity capture selectors are immobilized to a beaded or monolithic structure positioned within the cavity of the "analyte concentrator-microreactor" (acm) devices, or immobilized directly to the inner surface of the cavity of the acm device. the affinity capture selectors can be antibodies, antibody fragments, lectins, aptamers, enzymes, phages, receptors, protein a, protein g, or a variety of substances having affinities for different kind of substances. figure modified from [25, 75] . numerous immunoaffinity capillary applications employing various configurations of the acm device, using either a conventional capillary electrophoresis or a microfluidic chip capillary electrophoresis, have been reported [25, [54] [55] [56] [57] [58] 61, 75, [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] . figure 2 depicts the use of an iace selective affinity capture-separation system for the quantification of brain-derived neurotrophic factor (bdnf), a neuropeptide growth factor obtained from human skin biopsies of atopic inflammatory reactions [124] . figure 2a is a schematic representation of an acm device where whole tetrameric antibodies, nanobodies, antibody fragments, such as fab, or other affinity ligands, such as aptamers and lectins, can be directly immobilized to its inner surface, instead of using a beaded or monolithic matrix support as depicted in figure 1 . since the acm device is not yet commercially available, each laboratory has made its own modifications to the original protocol published elsewhere for use in conventional or microchip capillary electrophoresis [25, 66, 70, 75] . for the protocol carried out in these experiments, streptavidin was first immobilized off-line to the surface of 2-mm glass disks serving as a solid support. this was followed by the addition of biotinylated antibody reacting with bdnf. the prepared disks containing the antibodies to bdnf were then transferred and placed in an area of the microchip termed the immunoaffinity port. extracted material obtained from frozen tissue biopsies, using a detergent-containing solution, was incubated with the immobilized antibody. the captured bdnf was then conjugated with a fluorescent dye, followed by elution and separation of the derivatized bdnf neuropeptide in the separation channel. detection was carried out using a laser-induced fluorescence detector. figure 2b demonstrates that the concentration of bdnf became elevated over a 24-h sampling period in a patient undergoing a severe reaction. figure 2c shows that the iace technology is able not only to distinguish differences in the severity of the lesion but could also detect different patterns in analyte concentration at different sampling sites. for example, at the center of the lesion with the highest inflammation area, elevated concentrations of bdnf were found; however, at 2 and 10 mm apart from the lesion, the concentration of bdnf started to decline significantly. antibody reacting with bdnf. the prepared disks containing the antibodies to bdnf were then transferred and placed in an area of the microchip termed the immunoaffinity port. extracted material obtained from frozen tissue biopsies, using a detergent-containing solution, was incubated with the immobilized antibody. the captured bdnf was then conjugated with a fluorescent dye, followed by elution and separation of the derivatized bdnf neuropeptide in the separation channel. detection was carried out using a laser-induced fluorescence detector. figure 2b demonstrates that the concentration of bdnf became elevated over a 24-h sampling period in a patient undergoing a severe reaction. figure 2c shows that the iace technology is able not only to distinguish differences in the severity of the lesion but could also detect different patterns in analyte concentration at different sampling sites. for example, at the center of the lesion with the highest inflammation area, elevated concentrations of bdnf were found; however, at 2 and 10 mm apart from the lesion, the concentration of bdnf started to decline significantly. figure 2 . schematic representation of a fritless acm device without the presence of a matrix composed of beads, sol-gel, or monolithic materials utilized in conventional capillary electrophoresis or microchip capillary electrophoresis. immobilization of the affinity ligands occurs directly onto the inner wall of the acm device (a). the protocol for sample processing is shown in four steps: (1) covalent immobilization of an antibody, directed against brain-derived neurotrophic factor (bdnf) neuropeptide used as affinity capture material, directly to the inner wall of a microchip channel (alternatively, 2-mm glass beads immobilized with streptavidin to which a biotinylated antibody against bdnf is coupled, can be inserted into the main channel of the microchip or in an immunoaffinity port of a specially designed microchip); (2) binding of immunoreactive bdnf neuropeptide present in skin biopsies to the immobilized affinity capture antibody; (3) cleaning excess amounts of unwanted non-specifically-bound material and equilibration of the capillary system; (4) elution of the reversibly specifically-bound bdnf neuropeptide. (b) depicts the sequential iace electropherograms of bdnf concentrations present in skin biopsies taken at different time points in a patient with severe atopic dermatitis illustrating the concentrations of detectable bdnf over time. graph (c) illustrates the concentrations of bdnf in skin biopsy of a patient with severe atopic dermatitis and control groups measured by iace at 18 h post-initial onset of the reaction. the green color bars represent the amount of bdnf measured within the lesion, while the orange color bars represent the background amounts of bdnf measured in tissues taken 2 mm from the periphery of figure 2 . schematic representation of a fritless acm device without the presence of a matrix composed of beads, sol-gel, or monolithic materials utilized in conventional capillary electrophoresis or microchip capillary electrophoresis. immobilization of the affinity ligands occurs directly onto the inner wall of the acm device (a). the protocol for sample processing is shown in four steps: (1) covalent immobilization of an antibody, directed against brain-derived neurotrophic factor (bdnf) neuropeptide used as affinity capture material, directly to the inner wall of a microchip channel (alternatively, 2-mm glass beads immobilized with streptavidin to which a biotinylated antibody against bdnf is coupled, can be inserted into the main channel of the microchip or in an immunoaffinity port of a specially designed microchip); (2) binding of immunoreactive bdnf neuropeptide present in skin biopsies to the immobilized affinity capture antibody; (3) cleaning excess amounts of unwanted non-specifically-bound material and equilibration of the capillary system; (4) elution of the reversibly specifically-bound bdnf neuropeptide. (b) depicts the sequential iace electropherograms of bdnf concentrations present in skin biopsies taken at different time points in a patient with severe atopic dermatitis illustrating the concentrations of detectable bdnf over time. graph (c) illustrates the concentrations of bdnf in skin biopsy of a patient with severe atopic dermatitis and control groups measured by iace at 18 h post-initial onset of the reaction. the green color bars represent the amount of bdnf measured within the lesion, while the orange color bars represent the background amounts of bdnf measured in tissues taken 2 mm from the periphery of the lesion. the light blue-colored bars represent the amount of background bdnf measured in tissue taken 10 mm from the periphery of the lesion. all values are the mean â± s.e.m. figure modified from [55, 124] . neuropeptides and their role in regulating inflammatory processes have become a topic of major interest in clinical medicine. bdnf has been associated with several inflammatory conditions, including asthma, hypoxic lung injury, hypersensitivity reactions, atopic dermatitis, neurological disorders, and type 2 diabetes [124] [125] [126] . in the example presented here for the analysis of bdnf in skin biopsies, iace technology shows similar results when compared to histopathologic analysis. furthermore, the example shows that results obtained with iace can be accomplished in a short period of time, whereas classical histopathology takes several days to complete [124] . figure 3 depicts a profile demonstrating the data obtained by the two-dimensional iace immunoassay when compared to the mono-dimensional sandwich enzyme-linked immunosorbent assay (elisa) method. two applications are shown utilizing both immunological methods: one is for the determination of alpha-1-acid glycoprotein or orosomucoid, and the other is for the determination of two structurally similar exorphins, casomorphins 5 and 7. the determination of immunoreactive alpha-1-acid glycoprotein (agp) in serum has been a challenge due to its high carbohydrate content and the heterogeneity in its glycan structures. there are many agp isoforms or proteoforms of which about 10-20 glycoforms are detected in the serum. the variations of the agp glycoforms are of interest to the study of the binding alteration of this protein with certain drugs, and of their usefulness as diagnostic biomarkers for some systemic inflammatory diseases [127] [128] [129] . the quantification of the immunoreactive exorphins derived from milk beta casein a1, casomorphins 5 and 7, is important as these exorphins are potential modulators of numerous regulatory processes in the body as well as potential biomarkers of several diseases. for example, opioid-like casomorphins have been associated with autism, sudden infant death syndrome, type 1 diabetes, apnea, constipation, postpartum psychosis, schizophrenia, circulatory disorders, and food allergies [130] [131] [132] [133] [134] [135] . as depicted in figure 3 , when the elisa test is used for the determination of alpha-1-acid glycoprotein and casomorphins, it is unable to detect subtle chemical differences between isoforms of the two protein-peptide molecules. the result is a single measurable detection signal obtained by elisa, expressed in arbitrary units by a purple bar for alpha-1-acid glycoprotein ( figure 3a ), and by a green bar for casomorphins ( figure 3c ). conversely, the results obtained by the iace technology provide more comprehensive information. figure 3b shows an electropherogram of the various separated isoforms of immune-reactive alpha-1-acid glycoprotein extracted from serum. additionally, each peak area of the agp isoforms can be quantified. the separation of isoforms of particular proteins and their individual quantification can therefore provide significant information for disease diagnosis and prognosis. clinical proteomic applications aim to solve specific clinical problems by adopting these procedures. recent studies are now viewing protein isoforms (proteoforms) as a new class of early diagnostic biomarkers for clinical proteomics [136, 137] . each individual molecular form of an expressed protein has been called a proteoform. this term captures the disparate sources of biological variation that alter primary sequence and composition at the whole-protein level [138] [139] [140] . the process of identifying aberrantly expressed proteins and disease-associated proteoforms has led to a better understanding of the underlying mechanisms of diseases, particularly tumorigenesis [141] . figure 3d shows an electropherogram of immunoreactive a1 beta casein-derived casormorphin-5 and casomorphin-7 extracted from urine and analyzed by iace. since these peptides have a very similar amino acid composition differing only by one amino acid, elisa cannot differentiate them given its lack of separation of the peptides after detection. in contract, the two-dimensional iace technology can separate the two peptides using a single antibody that cross-reacts with both peptides for capture, thereby allowing for their quantification and characterization by one or more detectors. in summary, the mono-dimensional elisa test is limited to generate a single data point, which provides information only about the sum of the two urine-extracted casomorphins and the 10 serum-extracted isoforms of agp. the immunoreactive exorphins derived from milk beta casein a1, casomorphins 5 and 7, is important as these exorphins are potential modulators of numerous regulatory processes in the body as well as potential biomarkers of several diseases. for example, opioid-like casomorphins have been associated with autism, sudden infant death syndrome, type 1 diabetes, apnea, constipation, postpartum psychosis, schizophrenia, circulatory disorders, and food allergies [130] [131] [132] [133] [134] [135] . . schematic representation of a comparative profile of enzyme-linked immunosorbent assay (elisa) and iace applications for the determination of alpha-1-acid glycoprotein (agp) or orosomucoid, and for the determination in urine of two structurally similar exorphins, casomorphins 5 and 7. the purple bar (a) depicts arbitrary units using an elisa method for the total cumulative quantitative values of the isoforms of immunoreactive agp from human serum, and the green bar (c) depicts arbitrary units using an elisa method for the total cumulative quantitative values of the similarly related immunoreactive beta-casomorphin peptides, beta-casomorphin-5 and beta-casomorphin-7, from human urine. panel (b) represents an electropherogram profile for the determination of the isoforms of immunoreactive agp from human serum, and panel (d) represents an electropherogram profile for the determination of the isoforms of immunoreactive beta-casomorphin peptides, beta-casomorphin-5 and beta-casomorphin-7, from human urine. the electropherograms depicted in panels (b) and (d) were carried out by on-line immunoaffinity capillary electrophoresis. the advantage of determining isoforms of a protein is that each isoform can have various biological roles. figure modified from [25, 127] . despite the increase in sensitivity and specificity of immunoassay techniques over the years, analytical interference remains a major area of concern. iace overcomes many of the limitations of the sandwich elisa test, in particular the frequent incidence of false-positive and false-negative results for numerous molecules [25, [142] [143] [144] [145] . part of this problem is due to the polyreactive nature of a significant number of antibodies, which bind not only to structurally related targets but also to structurally unrelated targets [146] [147] [148] . polyreactive antibodies are a major component of the natural antibody repertoire. in contrast to monoreactive antibodies, polyreactive antibodies have a low binding affinity for antigens, but the antigen-binding pocket of these antibodies is thought to be more flexible than that of the monoreactive antibodies and thereby can accommodate different antigenic configurations [149] . substances that arise from properties of the specimen and interfere with the reaction of an intended target antigen and reagent antibodies can generate inaccurate results [150] [151] [152] [153] . consequently, an incorrectly diagnosis that is based on inaccurate testing can lead to inappropriate and, in some cases, harmful treatment. in addition to false positive results, elisa can lead to false negative results. this tends to arise from weak binding affinity of the antigen to the affinity-capture agent. a number of modifications have been recommended to avoid interferences with antigen binding in immunoassays, such as the addition of detergents, treatment of a sample at high temperatures if the analyte is thermal stable, addition of some blocking agents to a sample, and others. however, despite advances in our knowledge and understanding of the mechanisms of interference in immunoassays, there is no single procedure that can rule out all interferences with elisa or other immunoassays [150] . the development of a portable point-of-care iace biomarker analyzer instrument that utilizes two-dimensional iace technology offers a potential solution to improve the conditions of binding between the affinity-capture molecule immobilized to the acm device and the target analyte to be tested. for example, addition of small amounts a non-ionic detergent, such as of nonidet p-40, addition of certain polycations, such as polybrene, and maintenance of the optimal ionic strength of the buffer, can help to improve the binding between the target analyte and the affinity-capture biorecognition molecule. other factors are also crucial to optimize binding, such as the control of temperature, buffer ph, and time of reaction; the use of a microwave pulse or acoustic micro-mixing system; and minor sample dilution with an appropriate conditioning buffer, all of which can improve binding and reproducibility of the assay [25, 75, [120] [121] [122] 154, 155] . another major advantage of the design of the iace biomarker analyzer instrument is that if the biorecognition affinity ligands or selectors (e.g., antibody, antibody fragment, lectin, aptamer, others) are stable and covalently immobilized with a well-defined orientation, then they can be reutilized multiple times. the advantage of this optimization protocol is that it permits the use of multiple well-oriented affinity-capture agents with high affinity and selectivity, in order to secure the binding of the target analyte. the ability of the acm device to be reused therefore brings down the cost per assay, resulting in significant benefits to patients that need frequent analysis of biomarkers. figure 4 shows a schematic representation of a modified version of a portable point-of-care iace biomarker analyzer instrument ( figure 4a ) coupled to two biological specimen collection systems: an exhaled breath/oral fluid collection system ( figure 4b ), and a urinary collection system composed of a urinary drainage bag connected to a foley catheter ( figure 4c ). the two sample collection systems can work separately and in synchronization with the point-of-care iace biomarker analyzer instrument in a sequential order. the acm device depicted in figure 4a is structurally positioned in order to prevent the contact of the sample with the separation capillary [25, 75, [120] [121] [122] 154, 155] . this task is performed with the help of four microvalves surrounding the acm device. a biological specimen containing one or more target analytes to be tested is obtained either by the exhaled breath/oral fluid collection system figure 4b or by the foley catheter urine collection system figure 4c . the corresponding specimen is then transported through a secondary or auxiliary transport capillary to the main transport passage or capillary, passing through the acm device containing three biorecognition affinity ligands (1, 2, and 3) all the way to the outlet end of the main transport capillary to a trap or waste container. sample introduction is carried out from the outlet side of the secondary transport passage or capillary, connected to the main transport passage or capillary, by controlled suction using a vacuum pump coupled to a trap or waste container collecting the excess amount of fluid. microvalves located at the inlet side of the main transport capillary (v-1) and at the inlet and outlet side of the acm device, which is positioned at the separation capillary figure 4a ) coupled to two biological specimen collection systems: an exhaled breath/oral fluid collection system (figure 4b) , and a urinary collection system composed of a urinary drainage bag connected to a foley catheter ( figure 4c ). the two sample collection systems can work separately and in synchronization with the point-of-care iace biomarker analyzer instrument in a sequential order. figure 4 . schematic representation of a modified version of a portable point-of-care iace biomarker analyzer instrument (a) coupled to two biological specimen collection systems; an exhaled breath/oral fluid collection system (b), and to a urinary collection system composed of a urinary drainage bag connected to a foley catheter (c). the two sample collection systems can work in synchronization with the point-of-care iace biomarker analyzer instrument in a separate and sequential order using control microvalves (v-1 to v-14) to regulate the flow of collected samples from an individual. the acm device having a staggered configuration, can have one or more biorecognition affinity ligands immobilized to its inner wall to capture one or more similar or distinct biomarkers present in the collected samples. the point-of-care iace biomarker analyzer instrument can also be equipped with one or more acm devices (depicted in the upper left side), all having a staggered configuration, with the purpose to capture, concentrate, separate, and analyze a panel of biomarkers. by generating a comprehensive set of biomarker information from the biological specimen, the instrument aims to obtain a reliable and accurate diagnosis, prognosis, and to provide an efficient method for the assessment of treatment efficacy. in addition to capturing and analyzing chemical and biochemical compounds, the acm device of the portable point-of-care iace biomarker analyzer instrument is capable of capturing circulating blood cells, subcellular entities, cell debris, viruses, exosomes, and the components of disrupted biological particles. to facilitate a smooth passage of the fluid through the tubes or capillaries, a detergent-containing solution, stored in the liquid vessel or container, is added to the system during the collection of the sample. moreover, a few filters are incorporated into the tubes or capillaries, containing digestive enzymes immobilized to their constituent matrices, with the purpose of breaking down larger biomolecules, cell aggregates, cell debris, or any material that may block the filters and may disturb the smooth flow of the biofluid throughout the transport tubes or capillaries. the integrated iace biomarker analyzer instrument can be used in remote areas, in ambulances, and in intensive care units of a medical facilities, and it can be installed under a protective and confined space for patients with infectious diseases. the data collected can be sent by a secure and codified system using web portals via internet to a supercomputer to reveal comprehensive biomarker diagnostic signatures, thereby providing accurate and effective diagnosis information to a healthcare professional, and back to the computer of the iace biomarker analyzer instrument to keep the information updated and stored. figure modified from [75, 122, 155] . after binding occurs between the target analyte(s) present in the biological specimen and the biorecognition affinity ligands immobilized to the acm device, the microvalve (v-2), positioned at the secondary or auxiliary transport capillary is closed, and the microvalve positioned at the inlet side of the main transport capillary (v-1) is open. a cleaning buffer or solution is introduced through the container positioned at the inlet side of the main transport capillary (container-1) to remove non-specific compounds bound to the inner surface of the main transport capillary. once the binding and cleaning processes are completed, microvalves positioned at the inlet and outlet sides of the transport capillary (v-3 and v-7) are closed, and the microvalves positioned at the inlet and outlet sides of the separation capillary (v-4, v-5, and v-6) are open. a separation buffer is introduced into the separation capillary from a container positioned at the inlet side of separation capillary (container-2) to a container localized at the outlet side of the separation capillary (not shown). a plug of an elution buffer can be introduced into the inlet side of the separation capillary by pressure, using an inert gas such as argon or helium, followed by the separation buffer. electroosmotic flow can also be used for the introduction of a plug of an elution buffer. when the high-voltage power supply connected to a platinum-iridium electrode is switched on the process of elution and separation starts. at the outlet side of the separation capillary, eluted and separated target analytes are detected, quantified, and partially characterized by one or more detectors connected to the capillary in-line (amperometry), on-line (uv-absorbance or fluorescence), or off-line (mass spectrometry). as illustrated in figure 4a , the acm device has three types of affinity ligands (1, 2, and 3) immobilized to its inner surface representing three different affinity-capture molecules. the biorecognition affinity ligands can be an antibody, a lectin, and an aptamer, or a combination of the three (i.e., three different antibodies). similarly, the target analytes can be three different target analytes or a single target analyte that has three different epitopes. with the advent of advanced technologies offering powerful bioengineering tools, it is possible to create new affinity-recognition-capture molecules with improved and unique binding capabilities. for example, nanobodies, or single-domain variable fragments of camelid-heavy chain-only antibodies, have been successfully engineered as highly selective reagents, with high affinity, minimal size, low cost, and great stability. nanobodies are used as research tools and in medicine [156] [157] [158] . similarly, aptamers, which are short, single-stranded oligonucleotides that bind to specific target molecules, are playing an important role as affinity recognition-capture and therapeutic reagents [159] [160] [161] . another important group of affinity-recognition-capture reagents are lectins, which comprise a widespread group of sugar-binding proteins occurring in all types of organisms including animals, plants, bacteria, fungi, and even viruses. they are used as an effective tool for the targeting, separation, and reliable identification of glycoprotein molecules. their importance stems from the understanding that changes in glycoprotein and glycopeptide content, altered glycosylations, and aberrant glycan structures are increasingly recognized as cancer hallmarks [162] [163] [164] [165] . the use of two or more affinity-recognition-capture reagents has been demonstrated to improve the affinity binding to a molecule with multiple epitopes, such as thrombin [166] . two different oligomeric dna aptamers that can recognize different epitopes in thrombin were used in a potentiometric biosensor. when testing for the detection of low concentrations of thrombin, the dual aptamer-immobilized configuration yielded better results than the one with a single aptamer when tested for the detection of tiny amounts of thrombin [166] . another application employing multiple affinity-capture agents has been reported to use six different antibodies to capture six different immunoreactive chemokines in samples of cerebral spinal fluid collected from premature babies. in such study, immunoaffinity capillary electrophoresis in microchips was used to determine the degree of brain trauma in birth traumatized premature babies. the quantification of six different chemokines was able to provide information for diagnosis and prognosis of the good and poor state of the babies with head trauma [167] . additional examples of using multiple antibodies to capture several biomarkers have been presented in [25, 55, 58, 155] . the main advantage of the acm device, having multiple affinity-recognition-capture reagents (as depicted in figure 4a ) is its capability to analyze a panel of biomarkers simultaneously in a single platform to generate a comprehensive data and information. for example, it is possible to obtain the following information from a single biosample analysis from a patient suffering from a chronic or an infectious disease or illness and undergoing treatment: (1) the presence of the antigen causing the disease, such as a microorganism or its components of the microorganism if the microorganism is broken apart; (2) the presence of one or more antibodies, if the patient's immunological system recognizes the microorganism or its components as foreign substances consequently responds by developing antibodies to the unknown antigens; (3) the presence of pro-inflammatory substances and anti-inflammatory substances, if the patient's body is fighting the disease as it develops; and (4) the presence of other molecular markers expressed by the body or by the cellular or subcellular entities, and secreted to the biological specimen during the development of the disease. in the case of a comprehensive collection of information from the isolation and quantification of a panel of biomarkers in the biosample(s), it should be feasible to make an accurate and reliable diagnosis of the disease, a prognosis of the disease (i.e., if it will continue its progress, or if the patient has hope for recovery), and a pathway for monitoring the effectiveness of treatment (if any). furthermore, because of the high power of resolution of capillary electrophoresis, it is achievable to isolate and characterize novel co-translational and/or post-translational modifications of biomolecules, whether during the development of the severity of the disease or as a result of other biological parameters that may serve as better and more promising biomarkers of predicting clinical outcomes. the accuracy, analytical sensitivity, and reproducibility of the tests obtained by the two-dimensional iace technology are higher than those of conventional mono-dimensional immunoassays such as elisa. most notably, there is little to no possibility of false-negative or false-positive results when using iace, whereas such results can be obtained with some frequency when using elisa. for example, if a mono-dimensional elisa test captures unrelated immunoreactive compounds, then a false-positive result may be obtained due to the polyreactivity of antibodies which are capable of interacting with related and unrelated substances. on the other hand, if a similar situation happens with the two-dimensional iace technology (wherein the test captures related and unrelated immunoreactive compounds), there still exists the possibility of confirming the identity of each separated peak using additional tools and detectors capable of providing partial or complete characterization of the separated analytes (e.g., by spiking the sample with internal standards, determining the corresponding migration time of each peak, and obtaining the absorption spectral profile and mass spectrometry profile for each separated compound). an enhancement to the features of the portable point-of-care iace biomarker analyzer instrument as described in figure 4a can be accomplished by coupling the instrument, whether as an integral component or as a detachable unit, to a portable-point-of-care breath/oral fluid collection system as depicted in figure 4b and described elsewhere [122, 168] . breath analyzer systems have been reported for the determination of organic volatile compounds and ionic content in exhaled breath condensate [169, 170] . however, studies of exhaled breath have demonstrated that humans generate fine particles during tidal breathing. micron and submicron particle sizes have been detected in exhaled breath of normal and pathological people [171] . exhaled breath sampling and analysis has long attracted interest in the areas of medical diagnosis and disease monitoring [171] [172] [173] [174] [175] [176] [177] ; however, progress from laboratory settings to routine practice has been slow. one main reason is the number of technical problems encountered for sampling and analysis, and the lack of normalization and standardization leading to significant variations that exist between results of different studies [174] . mouth-exhaled breath contains not only volatile organic compounds, but many other small and large molecules, as well as cellular and subcellular particles. these biomolecular and cellular entities originate from the airway, from the oral cavity and gut by bacterial action, and from mucus and saliva. among the substances found in exhaled breath are inflammatory cytokines. it is well-known that inflammation is part of the host's protective immune response against microorganisms; on the other hand, excessive inflammation can be detrimental to patients' health as it increases their morbidity and mortality. for example, dysregulation between pro-and anti-inflammatory cytokines has been found in exhaled breath obtained from patients with community-acquired pneumonia [178] . the potential of the portable point-of-care iace biomarker analyzer instrument to serve as a dual-integrated and complementary unit is greater than that of traditional instrumentation, since the instrument becomes a multi-modal screening system capable of isolating, concentrating, and quantifying multiple biomarkers for a comprehensive diagnostic and prognostic test. a panel of biomarkers has the potential to detect cases missed by the use of only a few biomarkers. infectious diseases, whether bacterial, viral, or of other origins, present acute and chronic stages of inflammation. early detection remains the key challenge to the survival of patients with contagious infectious diseases that may spread rapidly from one organ to another and can cast a storm over the whole body, ending in multiple organ failure. a cytokine storm syndrome having an hyperinflammation and characterized by elevated levels of cytokines in biological fluids can be a predictor of fatality. in particular, increased levels of interleukin-2, interleukin-7, granulocyte-colony stimulating factor, interferon-gamma inducible protein 10, monocyte chemo-attractant protein 1, macrophage inflammatory protein 1-alpha, and tumor necrosis factor-alpha have been associated to mortality due to virally driven hyperinflammation, as for example, in coronavirus disease 2019 (covid-19) [179] . figure 4c depicts an additional modular attachment to the portable point-of-care iace biomarker analyzer, with a different configuration from the one described in figure 4b . in this case, sample collection is performed from a connecting insertion to a modified urinary catheter, also called a foley catheter, as used in a catheterized patient. urinary catheters are used to help to empty the bladder, usually when a person is unable to urinate. the catheter is a sterile and flexible tube that is inserted through the urethra into the bladder. often urinary catheters are used during surgery, as the patient is unable to control their ability to urinate during anesthesia. in most cases, the catheter is intended to stay in place for a long period of time. urine drains from the bladder through the tube and into a collection bag. in addition to urinary retention and during surgery, the catheter is also used in many patients who stay in a hospital's intense care unit (icu) and who are too sick to use a bedpan. unfortunately, some complications such as urinary tract infections, may arise when catheters are used for an extended period of time [180] . in most cases, urinary tract infections are commonly treated with empirical antibiotics, resulting in overuse of antibiotics, which promotes antimicrobial resistance. interestingly, when urine from patients suffering from urinary tract infection is cultured, approximately only one in three patients are found to have urinary tract infection as defined by positive bacterial culture [181] . results from bacterial culture may take from one to three or more days, depending on the type of bacteria [182] . the portable-point-of-care iace biomarker analyzer instrument can overcome this delay since it is capable of generating comprehensive information based on a panel of biomarkers analyzed in the urine collected from the modified foley catheter collection system. the panel of biomarkers may include small molecules, biomolecules, and cellular and subcellular particles. these particles can be microorganisms such as bacteria, viruses, and others, including exosomes which are considered to be a trove of biomarkers [183] . a panel of biomarkers should therefore be able to confirm the presence or absence or a microbial infection. it has been reported that isolation and characterization of a panel of host-protein biomarkers yields significantly superior performance for diagnosing bacterial versus viral infections [184] . there is always uncertainty on how to manage a patient who may be a potential carrier of a contagious disease, as evident in the recent coronavirus pandemic. a "patient under investigation" is often placed in conservative precautions, and healthcare teams may defer or avoid certain procedures which may have been otherwise performed in other patients [185, 186] . to provide physicians with the answers they need to manage patients effectively during an outbreak setting, laboratory testing is needed on the front lines whenever it is feasible and safe to do so. in figure 4 we describe a portable-point-of-care iace biomarker analyzer that can be placed near the bedside of a patient and be separated by a plexiglass wall, such as the biomarker analyzer instrument that can be operated from an isolated containment area if necessary. when used on a patient, the portable and modular breath/oral collection system placed on a patient can be connected to the main instrument using a long flexible tube through the plexiglass wall to avoid any contamination. a similar connection can be performed when using the modified foley tubing coupled to the main instrument. the main transport and secondary or auxiliary passages or capillaries have an internal diameter usually between 500 and 900 âµm, permitting enough surface area for passing fluid derived from the exhaled breath and urine specimens and their respective contents. the foley tubing and the exhaled breath/oral collection system, respectively, have much larger internal diameters. the average size of most bacteria is between 0.2 and 2.0 micrometers, but others may be as large as 10 micrometers. white and red blood cells have size ranges fluctuating between 6.0 and 18 micrometers on average, and viruses, nanobacteria, or extracellular vesicles (evs) have size ranges primarily in the nanometer scale. the separation capillary used in conventional capillary electrophoresis instruments usually ranges between 50 and 100 micrometers, even though other internal diameters smaller than 50 micrometers or larger than 100 micrometers can be used as well. therefore, for most cases it should not be a problem to isolate and characterize cellular, subcellular, and extracellular particles when using the portable-point-of-care iace biomarker analyzer instrument. as illustrated in figure 4a -c, the interchangeable modular infrastructures designed for the rapid analysis of a comprehensive biomarker information, including a plexiglass wall for keeping the entire functional instrument in containment, should enable a successful robust and sustainable system which protects the patient as well as the healthcare workers. all technical operation of the biomarker analyzer instrument (devices, attachments, connectors, and valves) can be performed remotely via computer using robotic manipulators or controllers, and thus without the risk of any physical contact. robotic manipulators are capable of performing repetitive tasks at speeds and accuracies that far exceed those of human operators. modifications to the connection systems and other parts of the instrument and devices, including change of buffers and solutions, can be carried out as needed in a simple manner. the modularity of the components of the instrument and devices allows for the rapid replacement of parts. additionally, attachments of extra miniaturized detectors for improving sensitivity and characterization should facilitate significantly the detection and characterization of substances and/or particulate matters found at very low concentrations. early medical practitioners realized the importance of counting blood cells as a tool for investigation and quantitative study in healthcare [187] . for over 150 years, cell biologists had been using a hemocytometer (counting chambers) to quantify cells (mm). today, automated instruments such as flow cytometers are widely used in research and for management of patient diagnosis and prognosis, particularly for blood cancer cells [187, 188] . significant effort has been made to analyze cellular and subcellular entities, as well as viral particles and cellular vesicles using a flow cytometer (recently dubbed "flow virometry") [189] , but the straightforward applicability of this technique has been hampered by the small sizes of the particles and vesicles, their polydispersity, and the low refractive index [190] [191] [192] . with the rise of microfluidic devices, several attempts have been made to develop cell counters using microchips. however, for microfluidic devices to supplant current cytometers, it will require more development in sample preparation and sample techniques within lab-on-a-chip microfluidic platforms or miniaturized capillary electrophoresis instruments. studies on single cellular and subcellular entities, as well as their cargo content, by using conventional capillary electrophoresis and microchip capillary electrophoresis have been reported [193] [194] [195] [196] . there are several benefits associated with using these platforms, including low-volume requirements, rapid and efficient separations, special dimensions, and versatility. however, for the isolation and separation of a heterogeneous group of cellular and subcellular entities, as well as viral particles and cellular vesicles, many parameters must be taken in consideration, and the challenge for technical improvements will continue for a few more years. we are proposing a platform, based on iace, as a complementary tool to the existing technologies for the isolation and separation of particles of small sizes, such as viruses and evs. as demonstrated in figure 5 , capillary electrophoresis has already been used with some success for the separation of bacteria, viruses, and polymeric particles. it would be beneficial to have a sample processing method before separation, to isolate and concentrate the intended viruses or evs. immunoaffinity capillary electrophoresis has already been proven to be a useful technology to isolate, separate, and quantify cell-free molecules of biological interest based on the specificity and selectivity not only of antibody reagents, but also of lectin and aptamer reagents, quantifying molecules ranging from microgram/milliliter to femtogram/milliliter [25, 54, 55, 57, 75] . there are several reasons why we are interested in using the iace technology to study viruses and evs, in particular exosomes. the first reason is because we are in the middle of a pandemic, and the study of sars-cov-2 has emerged as a priority subject of research for the medical and scientific community [179] . the second reason to apply iace to study exosomes is because of the potential of these nanoparticles in clinical diagnosis and prognosis, as well as carriers of therapeutic drugs [197] [198] [199] [200] [201] [202] [203] [204] . not only are exosomes released by basically all cell types, they are also accessible in body fluids, they carry important molecular compounds of potential use as biomarkers, and they can cross many cellular barriers. as such, they play an important role in many physiological processes and have been implicated in many diseases [197, 198, 200] . as evs encompass a heterogeneous group of cell-derived vesicles, it would be a challenge to isolate, concentrate, and separate the three groups of nanoparticle entities. in human blood serum. they are mosquito-borne flaviviruses responsible for dengue fever and severe dengue hemorrhagic fever that belong to the genus flavivirus, family flaviviridae, which contains approximately 53 viruses. the flaviviruses are relatively small (40-65 nm) and spherical with a lipid-rich envelope [208] . vaccination must protect against all four serotypes, and hence this has proved to be difficult to design. structural differences between dengue viruses produced in humans versus cell lines many be key to understanding vaccine failure and developing better models for vaccine evaluation [209] . in the case of the coronaviruses, there are seven coronaviruses known to infect humans, but only three of them, sars-cov, mers-cov, and sars-cov-2 cause severe disease in humans. the seven coronaviruses are (1) little is known about the coronavirus sars-cov-2, which is responsible for the novel pneumonia known as coronavirus disease 2019 or covid-19. it appears that day after day, physicians and scientists are learning new information about the transmissibility and severity of the [205] [206] [207] . in view of the fact that there is a significant interest in the study of viruses and exosomes, a brief discussion of their importance is warranted. extracellular vesicles are a heterogeneous group of cell-derived membranous structures which originate from the endosomal system or which are shed from the plasma membrane, respectively. extracellular vesicles have been classified as oncosomes (1-10 micrometers), apoptotic bodies (500-2000 nanometers), microvesicles (50-1000 nanometers), and exosomes (40-200 nanometers) [197, 198] . some even smaller vesicles known as "exomeres" have been described as having even smaller sizes [199] . exosomes are naturally occurring nanosized vesicles and consist of natural lipid bilayers with an abundance of adhesive proteins that readily interact with cellular membranes. these vesicles' contents include cytokines and growth factors, signaling lipids, mrnas, and regulatory mirnas. exosomes have many roles and functions, and they are found in body fluids, including plasma, serum, urine, saliva, exhaled breath condensate, breast milk, tears, semen, cerebrospinal fluids, amniotic fluid, malignant ascites fluids, and cultured medium of cell cultures [197] [198] [199] [200] [201] [202] [203] [204] . they are involved in multiple physiological and pathological processes [197] [198] [199] [200] [201] [202] [203] [204] . these extracellular vesicles represent an important mode of intercellular communication by serving as functional vehicles that carry a complex cargo of proteins, lipids, and nucleic acids, and altering cell or tissue metabolism, influencing tissue responses to injury, infection, and disease [200] [201] [202] [203] [204] . exosomes are heterogeneous in size, heterogeneous in composition, and enriched in membrane-associated, high-order oligomeric protein complexes. exosomes and many types of enveloped viral particles, particularly rna viruses, have a similar size making exosomes and rna viruses "close relatives" [202] . in fact, enveloped viruses may be considered a form of ev. exosomes from virus-infected cells incorporate not only cell-encoded but also virus-encoded molecules [202] . exosomes are nanoparticles in the range of 40-200 nm (average 100 nm). therefore, they can be separated by capillary electrophoresis as many other biological particles have been separated. for example, figure 5 shows the separation by capillary electrophoresis of a virus (human rhinovirus serotype 2) [205] , a bacterium (pseudomonas fluorescens, a gram-negative, rod-shaped bacterium) [206] , and three polymeric particles (polystyrene micron-sized particles) [207] . the highly selective affinity capture of biorecognition agents such as antibodies, lectins, and aptamers, in conjunction with the superior resolving power of capillary electrophoresis, make a perfect pair to concentrate and separate biological species or entities for potential use in diagnosis. a typical example are viruses, which usually exist as more than one type. dengue infections are caused by four closely related viruses named den-1, den-2, den-3, and den-4. these viruses are called serotypes, because each type of virus has different interactions with the antibodies in human blood serum. they are mosquito-borne flaviviruses responsible for dengue fever and severe dengue hemorrhagic fever that belong to the genus flavivirus, family flaviviridae, which contains approximately 53 viruses. the flaviviruses are relatively small (40-65 nm) and spherical with a lipid-rich envelope [208] . vaccination must protect against all four serotypes, and hence this has proved to be difficult to design. structural differences between dengue viruses produced in humans versus cell lines many be key to understanding vaccine failure and developing better models for vaccine evaluation [209] . in the case of the coronaviruses, there are seven coronaviruses known to infect humans, but only three of them, sars-cov, mers-cov, and sars-cov-2 cause severe disease in humans. the seven coronaviruses are (1) 229e (alpha coronavirus), (2) nl63 (alpha coronavirus), (3) oc43 (beta coronavirus), (4) hku1 (beta coronavirus), (5) mers-cov (the beta coronavirus that causes middle east respiratory syndrome, or mers), (6) sars-cov (the beta coronavirus that causes severe acute respiratory syndrome, or sars), and (7) sars-cov-2 (the novel coronavirus that causes coronavirus disease 2019, or covid-19) [210] . little is known about the coronavirus sars-cov-2, which is responsible for the novel pneumonia known as coronavirus disease 2019 or covid-19. it appears that day after day, physicians and scientists are learning new information about the transmissibility and severity of the epidemic it causes. the seriousness of these infections and the lack of effective, licensed treatments for sars-cov-2 infections underpin the need for a more detailed and comprehensive understanding of coronaviral molecular biology, with a specific focus on both coronaviruses' structural proteins as well as their accessory proteins [211] . the known vaccines typically work by eliciting antibodies that block entry of the pathogen into cells, which in the case of enveloped viruses, involves antibody binding to the viral envelope proteins. the viral fusion protein is the key factor that induces the membrane fusion reaction that in turn allows viral entry [212] . analyzing structural biology of the viral membrane fusion proteins and their complexes with neutralizing antibodies is thus an exceptionally powerful approach to identifying vulnerability sites and to extracting the necessary information required to develop protective vaccines to efficiently combat the emerging viral diseases threatening our planet [212] . immunoaffinity capillary electrophoresis can serve as a platform to isolate, concentrate, and analyze various structurally related viruses, and to analyze their chemical modifications they might undergo, thereby providing significant information about a diagnostic signature classification of their serotypes. similarly, this approach can be implemented to study extravesicular vesicles and various types of circulating cells, such as immune cells and cancer cells [213, 214] , as well as quantifying and analyzing their chemical and biochemical contents. capillary electrophoresis can separate molecules or entities based on the differences in the charge-mass ratio of single molecules, complexes of molecules, circulating cells, biological particles, and even man-made nanoscale quantum dots [215] . recently, it has been proposed as a proof of concept that capillary electrophoresis can be used for the separation of bacterial extracellular vesicles [216] . much effort has been performed during the last 5 years to develop methods for the isolation and characterization of exosomes, which are seen as attractive candidates for clinical application as innovative diagnostic and therapeutic tools [217] [218] [219] [220] [221] [222] [223] [224] . lately, there has been increasing evidence which demonstrates that the positive effects of cell-based therapies are mediated by exosomes released from the administered cells, and that the micro-rna cargo in these exosomes is largely responsible for the therapeutic effects [225] . advances in the study of exosomes have been delayed because of the lack of methods to isolate them from clinical samples. traditionally, differential ultracentrifugation, ultrafiltration, immunoaffinity capture, and size exclusion chromatography have been used as conventional methods of isolation, but these methods are generally time-consuming and labor-intensive. they also may need costly instrumentation or some special chemical reagents, and in some cases, such methods of isolation may require multiple overnight centrifugation steps [226, 227] . in the last few years, several characterization and validation methods have been developed, for both research and clinical purposes, to analyze exosome purity and to quantify exosomal cargo. these methods include transmission electron microscopy (tem), scanning electron microscopy (sem), atomic force microscopy (afm), nanoparticle tracking analysis (nta), dynamic light scattering (dls), resistive pulse sensing, enzyme-linked immunosorbent assay (elisa), flow cytometry, fluorescence-activated cell sorting (facs), and microfluidics and electrochemical biosensors [228] [229] [230] [231] [232] . the number of methods for quantifying exosomes has expanded as interest in exosomes has increased. however, consensus on proper quantification has not developed, making each study difficult to compare to another [233] . immunologically, exosomes exhibit antigen-presenting capability [234] . as a result, immunoaffinity studies of exosomes are becoming attractive, and iace can be an ideal platform to be used for such applications in research and in clinical diagnosis. in fact, a few laboratories are already combining immunoaffinity and microfluidic system approaches for more efficient exosome collection [235] [236] [237] [238] [239] . given their biological activities in intercellular transportation, information communication, and cell-mediated immunity modulation after microbial infection, exosomes are considered to be of significant value for a comprehensive understanding of infectious diseases as they play a key role in clinical defense of microbial infections. furthermore, exosomes will play a significant role as immune drug carriers, both in the manufacture of biological vaccines, and as biomarkers of microbial infections [240] . recently, it has been shown that exosomes serve as decoys, providing cellular protection against bacterially produced toxins [241] . a consistent high-quality isolation method for exosomes, followed by characterization and identification of types exosomes and their content, is therefore crucial to distinguishing subpopulations of exosomes, other extracellular vesicles, and viral particles. a conceptual understanding of biological responses to nanoparticles is likewise needed to develop and apply safe nanoparticles in drug delivery and other uses [242] . the future of the in-depth investigation of exosomes will rely heavily on technological advances, particularly with respect to simplifying the capture of functional microvesicles, such as exosomes, and to providing accurate quantitative measurement of their contents [243, 244] . many interesting findings and hopes for advancing diagnosis and therapy for exosomes have been reported recently (table 1 ). one such study has found that exosomes released in response to cigarette smoke might trigger chronic obstructive pulmonary disease (copd), whereas engineered versions could be used as treatment of the disease [243] [244] [245] . another report has shown that exosomes from virus-infected cells modulate immune cells' responses and increase spread and infection of the virus through delivering viral nucleic acid and protein molecules to healthy cells [246] . as discussed earlier in this paper, the two-dimensional iace technology provides many advantages when compared to other immunoassays for the analysis of cell-free molecules, such as the use of low volumes and reagents, high analytical sensitivity, speed of analysis, and the separation of all target and non-target affinity-captured analytes [25, 54, 55] . nevertheless, there are a few technical challenges for the application of this technology to the isolation, concentration, and separation of cellular particles and vesicles which remains unresolved [247, 248] . the goal of analyzing exosomes by iace is to further study the association of the cargo content of exosomes with the presence or absence of a wide range of pathogens, such as bacteria or viruses, and integrates the host injury response to infection [13] . moreover, by primarily quantifying nucleic acids and crucial protein biomarkers which serve as indicators of early detection and evolution of diseases, exosomal analysis employing iace can further provide information about the injury of different tissues in a patient due to pathogens and/or inflammatory substances. the role of non-coding rnas (ncrnas) in evs is not fully understood, but their higher abundance in association with cancer and other diseases demonstrated their relevance. several studies are underway on micrornas, long ncrnas, and circular rna present in exosomes, and evidence exists that non-protein coding genes harbors crucial importance in terms of development, homeostasis, and disease [249] [250] [251] [252] [253] [254] . table 1 . a number of publications related to the potential use of exosomes obtained from liquid biopsy or cultured cells, and their cargo contents in diagnosis, prognosis, and therapy. mesenchymal stromal cells-derived exosomes potential to improve neurological injury. understanding the effect of exosomes as mediators of the beneficial effects of cell therapy for stroke and traumatic brain injury. [225] human hepatocellular carcinoma exosomes important role in the diagnosis and therapy for tumors. screening for biomarkers in early formation of chronic hepatitis and liver cancer. elucidation of signal pathways and their involvement in growth, metastasis, and angiogenesis; and of the significance of exosomes in the treatment of hepatocellular carcinoma. [227] human serum exosomes symptomatic respiratory viral infections after lung transplantation. presence of lung self-antigens, 20s proteasome, and viral antigens implies that exosomes trigger chronic rejection. [255] optogenetically engineered exosome system (explor) therapeutic carriers to deliver srikb (super-repressor ikb) to a therapeutic target used as sepsis model. amelioration of sepsis-induced organ injury and inhibition of secretion of proinflammatory cytokines. [256] human placenta exosomes maternal plasma exosomes therapeutic approach for gestational diabetes mellitus. to reduce the negative impact of gestational diabetes mellitus. [257] engineered tumor-derived exosomes therapeutic approach as anti-tumor agents. potentials usage in cancer immunotherapy. [258] mouse adiposederived mesenchymal stem cell exosomes novel therapeutic strategy for tissue injury. proteomic analysis of exosomes found more than 1000 protein groups with a number of biological functions, implying such exosomes might be valuable as potential therapeutic targets for tissue repair. [259] mesenchymal stem cell-derived exosomes potential role in osteoarthritis regenerative medicine. elucidation of the inflammatory and multiple pathophysiological processes in the synovium, leading to the degradation of cartilage and bone. potential role in cartilage repair and osteoarthritis therapy. [260] vascular endothelial cells-derived exosomes vascular smooth muscle cells-derived exosomes several other cells-derived exosomes potential of exosomes in diagnosis, prognosis, and treatment of atherosclerosis. understanding of occurrence and development of cardiovascular diseases such as atherosclerosis. [261] human urine exosomes potential of exosomes in diagnosis of nephropathies. characterization of proteolytically derived peptides that are essentially relevant to classify patients with nephropathies, cancers of the urinary tract. [262] human urine exosomes potential of exosome gene expression assay as noninvasive test for prostate cancer. evaluation of a urinary diagnostic assay to help assess whether a prostate biopsy is warranted. [263] human bone marrow mesenchymal stem cells-derived exosomes potential of exosome as treatment for severe covid-19. understanding of the effect and capacity of exosomes to restore oxygenation, downregulate cytokine storm, and reconstitute immunity. [264] human lungs epithelial cells-derived exosomes transduced with selected genes of the sars-cov-2 a new strategy to demonstrate that sars-cov-2 rna-containing exosomes represent an indirect route of entry into cardiomyocytes. understanding a potential cardiac dysfunction produced via sars-cov-2 rna containing exosomes, without the need for direct viral infection. [265] bovine milk exosomes an alternative strategy to load hydrophilic and lipophilic small molecules, and chemotherapeutic drugs into exosomes. potential drug delivery vehicle or nanocarrier for cancer treatment. [266] mesenchymal stem/stromal cell-derived exosomes may provide considerable advantages over their counterpart live cells, potentially reducing undesirable side effects including infusional toxicities. potential use in gene delivery, regenerative medicine, and immunomodulation. [267] cultured third-molar pulp cell-derived exosomes potential of pulp-derived exosomes in combination with fibrin gel to fill dental hard tissues understanding the use exosomes in combination with fibrin gel in clinical translation towards improved cell-free regenerative endodontics. [268] tumor-derived exosomes (texs) potential for circulating immune-related biomarkers, reflecting partially the genetic and molecular contents of the parent cancer cell. understanding how texs influence boost tumor growth, regulation of tumor neo-angiogenesis, premetastatic niche formation, and therapy resistance. [269] human exosomes-based vaccines potential use of the s protein of the sars-cov, a type i transmembrane glycoprotein, incorporated into exosomes as antigen to be used in vaccines to induce high levels of neutralizing antibodies. manufacturing of highly immunogenic sars-s-based vaccines. additionally, potential use in inhibiting tumor growth. [270] exosomal noncoding rna in glioma potential of exosomes as carriers of bioactive molecules into the brain. holds great promise in diagnostics and therapy. noncoding rnas of exosomes can be modulators of numerous hallmarks of glioma. understanding the function of exosomal noncoding rnas in cell-to-cell communication in the tumor microenvironment, tumor proliferation, invasion, angiogenesis, immune-scape, and treatment resistance. [271] human exosomes in cardiovascular diseases potential of exosomes as more effective intervention targets on ischemic heart disease. the presence of exosomes in plaque tissue, ischemic heart, and peripheral blood can be potential biomarkers for early diagnosis and prognosis of cardiovascular diseases. understanding the participation of exosomes in the evolution of ischemic heart disease, including their role in endothelial dysfunction, lipid deposition, atheromatous plaque formation and rupture, ischemia-reperfusion, and heart failure. [272] human urine exosomes potential source of biomarkers, pathogenic molecules, and therapeutic biologics in kidney diseases or disorders. understanding the role of exosomes in pathogenic mechanisms, biomarker discovery, and therapeutics of various kidney diseases, particularly lupus nephritis, acute kidney injury, diabetic nephropathy, renal fibrosis, kidney transplantation, and renal carcinoma. [273] potential of exosome's cargo, such as the novel slc22a5 transport protein, to serve as useful biomarker of inflammatory processes. understanding the significance of exosomes as carriers of inflammatory mediators involved in human pathologies. [274] human saliva exosomes from hiv-positive people platform to demonstrate that isolated saliva exosomes from hiv-positive individuals promote kaposi's sarcoma-associated herpes virus (kshv) infectivity in human oral epithelial cells. understanding how the trans-activation response element (tar) rna in hiv-associated exosomes contribute to enhancing kshv infectivity through the epidermal growth factor receptor (egfr). [275] human semen exosomes potential of exosome's cargo containing molecular fingerprints for a non-invasive diagnosis of prostate cancer. understanding the role of semen exosomes in prostate cancer diagnosis, and as possible agents for enhancing the transmission of sexual diseases. [276] human serum exosomes potential role of some mirnas extracted from serum exosomes of parkinson's disease patients to serve as biomarkers. understanding the significance of the expression levels of mir19b, mir24, and mir195 as biomarkers of parkinson's disease. [277] human milk exosomes a complementary strategy to deliver more functional insights of human milk. providing an enhanced immunological and micronutrient profile of human milk, with significant relevance to breast milk quality and the health of the mother and infant. [278] human umbilical cord exosomes potential role of exosomes derived from akt-modified human umbilical cord mesenchymal stem cells as therapy for improving cardiac regeneration. understanding the role of why exosomes obtained from akt-modified umbilical cord mesenchymal stem cells are more effective as therapy in myocardial infarction by promoting angiogenesis via activating platelet-derived growth factor d. [279] human bronchoalveolar lavage fluid exosomes potential role of mirna from exosomes with proinflammatory signatures in asthma and in allergic airway diseases. understanding the role of mirna obtained from bronchoalveolar lavage fluid exosomes. particularly micrornas mir-24 and mir-27, which can modulate gene programming and promote inflammation. [280] human cerebrospinal fluid and plasma exosomes potential use of alpha-synuclein and tau proteins obtained from central nervous system-derived exosomes that can efflux into blood can be used as biomarkers of parkinson's disease and other neurodegenerative diseases. exosomes can also serve as carriers of therapeutic substances for diseases of the central nervous system. understanding the role of the content of exosomes derived from the central nervous system in parkinson's disease. exosomes can carry and spread toxic alpha-synuclein between cells and induce apoptosis. [281] human induced pluripotent stem cell-derived neuronal exosomes potential use as a tool to assay the capacity of exosomes to influence neuronal and circuit development. control exosomes rescue neurodevelopmental defects in a model of rett syndrome. understanding the role of exosomes in the development of neural circuits, the increase in neurogenesis, and the promotion of cell proliferation and neural differentiation. [282] blood or cerebrospinal fluid-derived circulating circular exosomal rnas potential use of circulating exosomal circular rnas (circrnas) as biomarkers for the early detection and diagnosis of neuropsychiatric disorders. understanding the role of closed-loop structure circular rnas, a novel class of non-coding rna (ncrna), in mental diseases. some studies show that circrnas possess regulatory potential as "sponges" for target micrornas (mirnas) and rna binding proteins. [283] ovarian cancer cells-derived exosomes potential use of exosomal proteins and lipids in the early diagnosis of ovarian cancer. understanding the role several lipid species and proteins, which significantly differ in cancer derived exosomes when compared to those from ovarian surface epithelial cells. [284] the reliability of biomarkers in day-to-day medical practice still appears to be in question because of a lack of access to extremely sensitive and specific diagnostic testing. researchers are therefore not only discovering more comprehensive ways to determine disease activity via biomarker detection, but also linking biomarkers to risk factors that would allow for better prediction models to be created for risk of disease manifestation [5] . clinical implementation of exosome-based diagnostic and therapeutic applications is still limited by the lack of standardized technologies that integrate efficient isolation of exosomes with comprehensive detection of relevant biomarkers. with the iace biomarker analyzer instrument and its interconnected sampling collection systems as described in this manuscript, we expect to accurately identify and quantify different morphological subpopulations of nanovesicles and their components in a single platform. we hope that this device can be affordable and readily available in a clinical setting in the near future once biomarker applications have demonstrated its clinical validity, and once analytical validation studies have demonstrated its accuracy, precision, reproducibility, and sensitivity levels. however, as in all great technological revolutions, iace has and may continue to have some limitations. the advantages of this two-dimensional affinity capture-separation technology have just begun to come into view over the past few years, and the trend in usage of iace in clinical contexts is moving consistently in the positive direction. most promisingly, the incorporation of iace into telehealth, with the intent to meet the needs of telemedicine, and precision medicine, is beginning to revolutionize the delivery of healthcare to all segments of the population. in the near future, we foresee that the use of the iace technology, in conjunction with liquid biopsy, for the study of circulating cell-free molecules, single cells, subcellular entities, viruses, exosomes, and their constituent components in biological fluids, will become a powerful tool to unravel longstanding questions in both biological research and clinical diagnostics. author contributions: both authors contributed to data analysis and manuscript writing. all authors have read and agreed to the published version of the manuscript. there was no external financial support to conduct the research projects. best (biomarkers, endpoints, and other tools) resource. maryland: food and drug administration-national institutes of health biomarker working group biomarkers: potential uses and limitations how are we going to discover new cancer biomarkers? a proteomic approach for bladder cancer making meaningful clinical use of biomarkers systems medicine: helping us understand the complexity of disease sensitivity" and "specificity" reconsidered: the meaning of these terms in analytical and diagnostic settings validation of analytical methods. in 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of sars-cov-genome into human induced pluripotent stem cell-derived cardiomyocytes bovine milk-derived exosomes for drug delivery mesenchymal stem cell-derived exosomes for clinical use pulp-derived exosomes in fibrin-based regenerative root filling material exosomes in cancer: circulating immune-related biomarkers exosomal vaccines containing the s protein of the sars coronavirus induce high level of neutralizing antibodies exosomal noncoding rnas in glioma: biological functions and potential clinical applications exosomes in ischemic heart disease: novel carriers for bioinformation roles for exosomes in various kidney diseases and disorders exosomes in inflammation and role as biomarkers human immunodeficiency virus-associated exosomes promote kaposi's sarcoma-associated herpesvirus infection via the epidermal growth factor exosomes in semen: opportunities as a new tool in prostate cancer diagnosis microrna biomarkers of parkinson's disease in serum exosome-like microvesicles exosomics"-a review of biophysics, biology and biochemistry of exosomes with a focus on human breast milk exosomes derived from akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor d. stem cells transl exosomes in allergic airway diseases exosomes in parkinson's disease exosomes regulate neurogenesis and circuit assembly circular rnas in early brain development and their influence and clinical significance in neuropsychiatric disorders proteomic and lipidomic analysis of exosomes derived from ovarian cancer cells and ovarian surface epithelial cells in memoriam: we would like to dedicate this paper to the memory of five friends, colleagues, pioneers and educators, whose lives were unfortunately cut short, for their great contributions to the technology of capillary electrophoresis: csaba horvã¡th, brian howard, craig lunte, kevin altria, and gerhardus de jong. norberto guzman is the inventor of several patents issued and pending related to the iace technology. key: cord-017675-in9r33ww authors: nan title: the way forward: prevention, treatment and human rights date: 2008 journal: global lessons from the aids pandemic doi: 10.1007/978-3-540-78392-3_9 sha: doc_id: 17675 cord_uid: in9r33ww there now is a considerable body of evidence to support the view that an effective hiv/aids strategy integrates prevention, treatment and human rights. in this chapter, we emphasize the importance of each of these aspects and draw upon the conclusions reached in previous chapters to map out the future of hiv/aids. while medicine and science have a crucial role to play in addressing pandemics, whether slow-moving (like hiv/aids) or fast-moving (like influenza), the social, legal, political, financial and economic ramifications of pandemics can not be ignored. well-considered social, legal, political and financial strategies are essential in order to address any pandemic effectively. united kingdom 1998 kingdom -2004 source: global hiv prevention working group (2007) in chap. 2, we discussed how integrated prevention-treatment-human rights strategies aimed at high-risk groups have proved effective in countries like brazil. in chap. 7, we explained that limited resources need to focus on high-risk groups and locations to achieve the best possible results. however, as we showed in the sex workers, who are the source of almost 80% of hiv infections, a negligible amount of funding for hiv/aids is targeted at this group. the mismatch between the most affected group and the allocation of funding in ghana highlights the importance of matching funding to prevailing prevalence and transmission patterns in a given country or region. as we saw in chap. 2, an hiv prevalence rate above 1% is a key threshold for an hiv epidemic to run out of control unless funding for prevention efforts is targeted at high-risk groups, such as commercial sex workers, men who have sex with men, injection drug users and prisoners. however, in chap. 8 we saw that pepfar -the largest bilateral donor of funding for hiv/aids programs in developing countries -prohibits the use of funding for programs for commercial sex workers and needle exchange programs. in chap. 3, we saw that african-americans make up 54% of hiv/aids patients, even though african-americans account for less than 15% of the us population. moreover, black men who have sex with men (msm) have the highest rates of unrecognized hiv infection, hiv prevalence and incidence rates and aids mortality rates among msm in the united states. in five us cities, 46% of african-american msm are infected with hiv. hiv and aids prevalence rates have affected black msm disproportionately since the beginning of the epidemic. black msm are the only group in the united states with hiv prevalence and incidence rates that are comparable to those in the most affected developing countries. however, the vast majority of hiv prevention intervention for african-americans does not target homosexual men and for homosexual men does not target black msm (millett and peterson 2007) . thus, the need to focus prevention efforts on the most vulnerable groups remains an issue not just in developing countries. while prevention strategies need to be tailored to the sources of hiv infections in specific contexts, there are several proven prevention strategies that need to be scaled up. the resources for prevention need to be focused according to the specific nature of the epidemic in different settings, as we showed in chap. 2. figure 9 .2 shows the source of new hiv infections by region. table 9 .2 summarizes the coverage levels of several essential prevention strategies and fig. 9 .3 shows their deployment by region. it is important to emphasize that prevention and treatment are mutually supportive and need to be addressed simultaneously. access to treatment supports prevention by reducing risky behaviors, increasing disclosure of hiv status, reducing stigma and reducing infectiousness (global hiv prevention working group 2007) . prevention supports access to treatment by reducing the number of people that require treatment, thus making universal access to treatgroup (2007) order to enhance the effectiveness of both. ment more affordable. hiv treatment and prevention should be integrated, in hiv prevention strategies fall into four general categories: (1) prevention of sexual transmission; (2) prevention of blood-borne transmission: (3) prevention of mother-to-child transmission; and (4) social strategies. the strategies for preventing sexual transmission are: (1) behavioral change programs (to increase condom use, to delay the initiation of sexual behavior in young people and to reduce the number of sexual partners); (2) condom promotion; (3) hiv testing (knowledge of hiv status decreases risky behavior); (4) diagnosis and treatment of sexually transmitted infections (which significantly increase the risk of hiv acquisition and transmission, particularly in the case of genital herpes); and (5) adult male circumcision (which reduces the risk of female-to-male transmission by about 60%) (global hiv prevention working group 2007) . the effectiveness of these strategies varies. the promotion of condoms has been largely successful with respect to commercial sex and casual sex, but condom use remains low within marriage. as we noted in chap. 4, increasing life expectancy, in areas where it is low due to diseases like malaria, is a cost effective strategy for enhancing behavioral change to lower the risk of hiv infection. a survey by the who, on behalf of the global fund, reviewed anti-malaria operations in ethiopia, ghana, rwanda and zambia. in ethiopia, childhood malaria declined by 60% and the death rate was cut in half within 2 years of the beginning of the mass distribution of mosquito nets. within a single year, both cases and deaths dropped by twodeaths by a third. in many cases, the distribution of free nets was accompanied by free drugs based on artemisinin, a substance to which the malarial parasite has yet to develop widespread resistance, and spraying ddt inside people's houses. free nets and malaria drugs would bring malaria under control in most of africa at a cost of usd 10 billion (economist 2008) . these promising results also bode some studies suggest that treating sexually transmitted infections may not rediseases. moreover, as we noted in chap. 4, oster (2005) argues that the explanation for the substantial difference in the transmission rates between the united tions, which leave open sores from chlamydia, syphilis and gonorrhea that facili-there is significant evidence that male circumcision significantly reduces hiv a similar picture is seen in south and south-east asia, where overall hiv prevalence is much lower, but the countries with highest hiv prevalence have little thirds, in rwanda, and one-third in zambia. in ghana cases fell by an eighth and based on these results, the who believes that a 5-year campaign that distributes well for hiv prevention. association between the risk of infection with hiv and other sexually transmitted prevent as many as 24% of new infections over a decade duce hiv transmission significantly (halperin 2007) . however, there is a strong transmission. box 9.1 discusses the relationship between circumcision and hiv/ 305 tate hiv transmission. thus, treating bacterial sexually transmitted infections could states and sub-saharan africa is due to other untreated sexually transmitted infec-male circumcision (papua new guinea, cambodia and thailand) . conversely, hiv prevalence is extremely low in those countries where most men are circumcised (pakistan, bangladesh, indonesia and philippines). there is ecological evidence that prevalence of circumcision is negatively correlated with prevalence of hiv/aids. specifically, there is a strong inverse correlation between the prevalence of circumcision in countries and the prevalence of hiv in those countries. all the highest hiv prevalence countries are those where circumcision is little practiced. in fact, no country with nearly universal circumcision coverage has ever had an adult hiv prevalence higher than 8%, including higher risk than that in countries with prevalence of around 25%. this fact is illusfig. 9 .4 ecological relationship between circumcision and hiv prevalence. source: bailey (2007) a large, randomized controlled trial in 3,274 men between the ages of 18 and 24 years showed that circumcision resulted in a significant 60% reduction in hiv vulnerability to hiv varies considerably from one epidemic to the next, as do the issues facing vulnerable groups. for example, in a concentrated epidemic, such as in asia and latin america, hiv transmission occurs primarily among vulnerable groups and prevention programs targeted at vulnerable groups would reduce infection (auvert et al., 2005) . these results were confirmed by two other trials. 9 the way forward prevention, treatment and human rights trated in fig. 9 .4. countries such as cameroon, where a 1997 survey found sexual behavior to be overall infection. however, in a generalized epidemic, such as in several countries groups, halperin (2007) argues that transmission would continue unabated despite prevention programs targeted at vulnerable groups. however, as we noted in chap. 4, research regarding the relationship between trade routes, truckers, sex workers and hiv propagation contradicts this idea. in a generalized epidemic, where hiv is spread along trade routes, prevention programs targeted at truckers and sex workers would be effective in bringing down the growth rate of the spread of the disease. having multiple sex partners increases the risk of hiv infection in both concentrated and generalized epidemics, but the impact of this factor on hiv prevalence rates can vary considerably. for example, even though the united states and uganda have similar rates of multiple sex partners, and the number of sexual partners that men and women had over a 10-year period were much higher in the united states than in uganda, uganda's hiv/aids prevalence rate was about 18 times higher than that of the united states (halperin 2007). however, as we noted in chap. 8, a recent study indicates that abstinence-only programs are as effective as providing no information at all when it comes to preventing pregnancies, unprotected sex and sexually transmitted diseases. abstinence-plus interventions, which promote sexual abstinence as the best means of preventing hiv, but also encourage condom use and other safer-sex practices, are more effective than the proven strategies for preventing blood-borne transmission are: (1) to supply injection drug users with clean injection equipment; (2) methadone or other substitution therapy to reduce drug dependence; (3) blood safety programs, including screening of donated blood; and (4) infection control in health care settings, including injection safety and antiretroviral treatment following exposure to hiv. as we noted in chap. 8, the risk of aids infection through the use of blood products was recognized as early as 1982, but countries were slow to adopt measures to ensure the safety of the blood supply and the world health organization (who) passed a resolution on blood products that made no mention of aids as late as january 1987. in the 1990s, chinese health authorities promoted bloodselling by poor farmers to commercial blood collection centers, despite warnings from the who, spreading hiv/aids through the blood fractionation and reinjection process. in 2007, new hiv infections through hospital blood transfusions continued to be reported in china, and illegal underground blood collection centers have continued to operate. box 9.2 recounts the story of the libyan scandal over blood-borne transmission to children. in southern africa, where hiv transmission occurs primarily outside vulnerable 307 abstinence-only programs (underhill et al., 2007) . on december 14, 2007, sixth sense productions, inc., an independent holly-snezhana dimitrova, valentina siropulo) and a palestinian medical intern (ashraf ahmad djum'a al-hadjudj) who were jailed in libya and faced the death penalty for allegedly infecting children with hiv. this news item is a postscript to a long international drama that began to unfold in 1999 when the medics were arrested on charges of injecting 418 libyan children with hiv-tainted blood while at a benghazi hospital. of them, over 50 had died by the end of 2007. one important report was submitted by luc montagnier and vittorio colizzitwo leading experts on hiv/aids. their report concluded that the infection at the infections began before the arrival of the nurses and doctor in 1998. through hospital records, and the dna sequences of the virus, they traced it to patient n.356 who was admitted 28 times between 1994 and 1997 in ward b, iso and ward a. the first cross-contamination occurred during that patient's 1997 admission. montagnier and colizzi both testified in person at the trial of record for the defense. on that the strain of virus was already present before the arrival of the six accused. the accused were tried and retried. the libyans had signed confessions from them -which the accused said were extracted under torture. the final verdict in 2006 sentenced them to death by firing squad. the libyan president likened the scotland for the bombing of pan am flight 103 over lockerbie, scotland, on 21 and political favors in exchange for the release of the six. in the end, bulgaria, qatar and a group of european countries funneled usd 460 million into the international fund benghazi to finance the treatment of the hiv-infected children and the improvement of the libyan health care system. france played a pivotal role in the final release of the accused. in exchange for the release, france agreed to sell antitank missiles and nuclear technology to libya. it was a win-win deal for france: they did multi-million dollar business with libya and got publicity for helping the release of the accused. when the nurses returned to bulgaria, the government endorsed a 10,000 leva reimbursement for each of the nurses. a bulgarian mobile telephony provider donated an apartment for each nurse. 9 the way forward prevention, treatment and human rights 14 december 2006, nature (446, 836-837) published a report that also concluded wood producer, announced plans to make a usd 40 million movie about five event to the case of abdel basset ali al-megrahi, who is serving a life sentence in hospital resulted from poor hygiene and reuse of syringes. they concluded that the bulgarian nurses (kristiyana vulcheva, nasya nenova, valya chervenyashka, december 1988. thus, it became clear that libya was trying to extract economic the proven strategies for preventing mother-to-child transmission are: (1) general hiv prevention for women of child-bearing age; (2) a brief course of antiretroviral treatment in advance of delivery (which can reduce transmission by 50%, but is only received by an estimated 11% of women in need); (3) prevention of undesired pregnancy in hiv-positive women; (4) breast-feeding alternatives; and (5) cesarean delivery where the mother has a high viral load (global hiv prevention working group 2007) . in developing countries, a small but growing number of children are dying of hiv/aids. as fig. 9 .5 shows, some 4% of children died of hiv/aids in 2005. hiv infected mothers carry additional risks for the baby. in table 9 .3, we indicate some of the major risks. some risks like stillbirth or high infant mortality have been found only in developing countries but not in developed countries. for these additional risks, it has been suggested that one way of eliminating vents mother-to-child transmission by 100%. thus, family planning could also help to reduce mother-to-child transmission: o t h e r ( 2 9 % ) mother-to-child transmission is not to have the baby in the first place. this pre-another often neglected aspect of hiv prevention -one prohibited from funding by the bush administration's international aids programinvolves expanding family planning services, including for hiv-positive women who do not want to conceive. reducing unintended pregnancies could greatly decrease the number of infected infants as well as the number of children who eventually become orphans (halperin 2007). if an hiv-positive woman gives birth to a child, there is a risk of transmission of hiv itself, in addition to the other risks listed in table 9 .3. however, the transmission risk of hiv from mother to child is not 100%. it can be minimized through drug treatment of the mother and careful birthing. figure 9 .6 clearly demonstrates this fact, using the data from the united states. the introduction of zidovudine (for the mothers before childbirth) has dramatically reduced the risk of hiv infection of the baby. since 1998, most countries have applied a regimen of zidovudine from 28 weeks, with nvp administered during labor and to the baby, and the addition of a 7 day zidovudine/lamivudine postpartum regime. the result has been a dramatic reduction of infected newborns (see fig. 9 .7). note that the reduction has been evident in europe and the united states since 1994, when this regime was introduced. in thailand, the regime was introduced in 1996 and in most parts of africa 2 years later. once a child is born, the question is whether the infected mother should breastfeed the child. on the one hand, unaids estimated that globally there are 300,000 babies infected through breastfeeding. on the other hand, the unicef estimates that 1,500,000 children die every year from lack of breastfeeding by the breastfeed the baby. key factors that increase vulnerability to hiv include: (1) gender inequality (which reduces women's access to information and services, reduces power to negotiate safe sex with partners, increases the risk of sexual violence and may create the need to depend on sex for economic survival); (2) institutionalized discrimination against vulnerable groups (such as criminalizing drug use and needle possession, commercial sex work and sex between men); (3) poverty (which reduces access to information and services and access to prevention tools, such as condoms); (4) hiv stigma (which discourages individuals from seeking testing, disclosing their status, seeking hiv-related services or using alternatives to breast-feeding); and formation and social support by displacing populations and increase the risk of sexual violence) (global hiv prevention working group 2007). as we noted in chap. 4, there is no clear evidence that reducing poverty and income inequality will necessarily reduce hiv/aids prevalence. moreover, povcondoms and circumcision. significant percentage of men who have sex with men are hiv-infected in many africa; 27% in guyana; 33% in st. petersburg, russia; and 73% in urban ethiobetween vulnerable groups and the general population in bangladesh. the linkages between vulnerable groups, and between vulnerable groups and the general social strategies that address the factors that increase vulnerability to hiv innities and hiv-positive individuals in hiv/aids programs; (4) visible political leadership; (5) engaging a broad range of sectors in hiv awareness and prevention 9 the way forward prevention, treatment and human rights hiv/aids prevention strategy. thus, if poverty reduces access to information and parts of the world (28% in bangkok; 15% in phnom penh; 21.5% in urban sene-vulnerable groups are not compartmentalized. people infected through injection drug use can infect their sexual partners. a significant percentage of men who their clients, who in turn may infect their spouses or other sexual partners. in would be to find innovative ways to improve access to information, provide funding measures; (3) gender equity initiatives to empower women; (3) involving commution working group 2007). human rights are the core of most social strategies to gal; and 46% of african-american men in five us cities). sex workers can infect many areas, sex workers have very high rates of hiv infection (50% in south have sex with men also have sex with women (for example, 20% in asia) and a (5) conflict and humanitarian emergencies (which reduce access to services, inerty reduction is too broad a goal to constitute what might be considered a concrete 312 programs; and (6) legal reforms to support hiv prevention strategies, such as laws clude: (1) hiv awareness campaigns, including in the mass media; (2) anti-stigma services and access to prevention tools, such as condoms, a concrete policy response to enhance access to services and provide free access to prevention tools, such as pia) (global hiv prevention working group 2007). figure 9 .8 shows the linkages decriminalizing needle possession and anti-discrimination laws (global hiv preven-population, make effective prevention strategies for vulnerable groups essential. awareness of hiv status has a significant impact on rates of hiv transmission. when unaware of hiv seropositivity, the transmission rate is estimated at 8.8-10.8 the transmission rate to an estimated 1.7-2.4% (holtgrave 2005). however, inorder to balance the need for more testing with the need to respect human rights, it has been recommended that health care providers offer and recommend hiv testing, in conjunction with counseling (the opt-in approach), rather than rely on the client to initiate this process. however, mandatory hiv tests and routine hiv testand confidentiality (jã¼rgens 2007 ). the major barriers to increasing hiv prevention are: (1) failure to target limited funding where it will have the greatest impact, due to ing unless the client opts out risk violating individuals' rights to informed consent lack of information on the nature of the epidemic or ideological, non-scientific creasing access to hiv testing and counseling also raises human rights issues. in increases in funding; (4) failure to integrate hiv prevention in schools, workplaces and other health care programs, such as tb and reproductive health; and (5) stigma and discrimination against hiv-positive people and vulnerable groups, which deter people from seeking testing and prevention services and discourage political leadership (global hiv prevention working group 2007) . we analyze the problems and solutions regarding stigma and discrimination against hivpositive people and vulnerable groups later in this chapter. we analyzed the issues of inadequate financing, targeted financing and donor coordination in chap. 7. as we noted in chap. 7, a lack of donor coordination is an obstacle to expanding treatment and prevention programs, due to the administrative burden that it imposes on recipients. as we noted in chap. 8, the us government's foreign aids program, pepfar, devotes only 20% of funding to prevention and requires that two-thirds of that amount be spent on abstinence-only programs that do not promote condom use, despite evidence that this approach to prevention is not effective and undermines best practices. pepfar guidelines also undermine hiv/aids prevention by further stigmatizing sex workers and prohibiting funding of needle exchange programs, despite evidence that such harm reduction programs are effective. the pepfar approach assumes that vulnerable groups do not interact with the rest of society. pepfar is perhaps the best example of ideological, non-scientific restrictions on the use of donor funding, although it also serves as an example of two of the other significant barriers to hiv prevention, due to its promotion of stigma and discrimination against vulnerable groups and the percentage of funding that it allocates to prevention. however, it is important to emphasize that pepfar has done more than any other bilateral funding program to address the need for adequate financing. the key point is that the money that has been made available through pepfar could be better spent. on 26 december 2007, us president bush signed legislation that lifted a 1999 ban that had made washington, dc the only us city barred by federal law from using municipal money for needle exchange programs. officials of the district of columbia health department planned to allocate usd 1 million for such programs in 2008 (urbina 2007) . extending this change in policy to pepfar would enhance the effectiveness of prevention programs in the countries that receive pepfar funding. in chap. 3, we provided an overview of the history of drug developments to treat hiv/aids and saw the dramatic impact on survival of triple combination therapy. without this treatment, the chance of surviving 10 years was about 50%. with this treatment, patients have a 50% chance of living another 35 years. in the early 1980s in the united states, the leading causes of death among 25-44 old year men come the leading cause of death in this group. following the introduction of universal access to triple combination therapy, deaths from aids fell to fourth place, behind accidents, cancer and homicide. as a result, whereas 68% of americans considered hiv/aids to be the most urgent health problem facing the united ents became so important, as we saw in chaps. 5 and 6. this is also why access to there are five classes of anti-hiv drugs, which are known as antiretroviral verse transcriptase inhibitors (nnrtis), which began to be approved for use in 1997, stop hiv from replicating within cells by inhibiting the reverse transcriptase protein. (4) fusion or entry inhibitors prevent hiv from entering human immune sert its genetic material into human cells (http://www.avert.org/introtrt.htm). hiv-positive people are prescribed antiretroviral therapy once the number of cd4 cells falls below a certain threshold or when they develop clinical aids 2006, an international panel of experts continued to recommend these guidelines in low-and middle-income countries, which represents 28% of those in need (who/unaids progress report on universal access to treatment). the "3 by lion people on arv therapy by the end of 2005. during this period, the number of people in low-and middle-income countries receiving arv treatment increased from 400,000 to 1.3 million (who 2006) . and human resources, management capacity and the ability to identify new patients through testing and counseling (chai 2007) . in chap. 7, we examined multilateral funding programs that address these capacity constraints in developing ing treatment are just that -estimates. as we have noted, unaids hiv/aids estients that have been identified as requiring treatment. the reluctance of the united states to allow pepfar funding to be spent on who-approved drugs has also been criticized as an obstacle to expanding treatturer, cipla, created a triple-combination drug in a single pill (triomune) that could be taken twice daily, which it offered to sell for about usd 300 per patient per year in 2001. cipla's triomune offer made the 3 by 5 initiative a realizable goal and cipla has the production capacity to produce four million doses of triomune per day. the who approved triomune in december 2003 as a first-line treatment for hiv/aids (hamied 2005) . the pepfar restriction on the use of who-approved drugs had the effect of preventing the use of pepfar funding to buy triomune. moreover, the majority of pepfar funds have been used to purchase patented versions of hiv/aids drugs, rather than generic versions (see chap. 8). figure 9 .9 shows how generic competition has lowered the cost of triple combination antiretroviral therapy. between 2001 and 2007, the price of the generic drugs has brought down the price of the originator substantially -from over usd 10,000 to under usd 350. at the same time, the generic prices have stayed in the 25-30% range of the originator price. pepfar funds can be used to purchase other low-cost generic equivalents of several patented hiv/aids drugs, including some produced by cipla. pepfar requires that generic drugs be approved by the us fda, canada, japan or western europe to be eligible for funding (see chap. 8). if us fda approval is sought for fixed dose combinations of previously approved antiretrovirals for the treatment of hiv, if one or more of the approved drug components are covered by a patent, the fda cannot approve an application until the patent expires. however, the application can receive tentative approval (which recognizes that at the time the tentative approval action is taken, the application meets the technical and scientific requirements for approval, but final approval is blocked by patent or exclusivity). products that receive tentative approval are eligible for procurement under the table 9 .4. lists the generic versions of hiv/aids drugs that have been approved by the fda for purchase with pepfar funds, along with the generic companies that own the patents for the specific generic formulations and the country of manufacture. the fact that the patents for hiv/aids drugs are owned by different companies has delayed combining different hiv/aids drugs in a single pill in markets protected by patents. one such pill, atripla, was created through a joint venture between merck and bristol-myers squibb with gilead sciences and combines efavirenz (bristol-myers squibb, merck) with emtricitabine and tenofovir (gilead sciences) (ib times 2007). atripla was approved for sale in the united states in 2006, several years after the indian generic manufacturer, cipla, had started manufacturing a triple-combination pill and 3 years after cipla's pill was approved by the who. approval to market atripla in the european union was sought in december 2007. gilead sciences and merck have formed a joint venture to market atripla in developing countries (ib times 2007). table 9 .5 shows the us patents, patent owners and patent expiry dates for selected hiv/aids drugs. zidovudine was the first drug to be approved for treatment of hiv infection. as table 9 .5 shows, the patent for zidovudine expired in 2005 and the patent for lamivudine expires in 2009. however, glaxo extended the life of these patents to 2016 by combining the two drugs into one pill (called combivir). while the new combination reduces the number of pills that a patient needs to take, it did not involve the invention of any new chemical entities. the patent history of zidovudine has been cited as a classic case of "evergreening" -the use of the patent system to extend drug monopolies far beyond the term of the original patent (hamied 2005). box 9.3 discussed evergreening. zidovudine was originally synthesized in 1964, as a potential cancer treatment. research in 1984 showed that it was effective against hiv/aids, which formed the basis for glaxo's 1984 patent application. following clinical trials, the us fda approved zidovudine in march 1987 for advanced hiv disease in adults and the patent for zidovudine as a treatment for hiv/aids was granted in february 1988 (cochrane 2000) . while zidovudine alone only extended life by a matter of months, once it was combined with two other classes of hiv/aids drugs, it extended life for years. the fda expanded zidovudine approval in 1990 to include evergreening is a mechanism by which pharmaceutical and other companies can keep extending patents on drugs after the initial patents expire. over a fixed period of time. the intention of providing a monopoly is to provide an incentive to innovate. granting a patent requires three elements: (1) novelty of the product. product; (2) non-obviousness of the new product; and (3) demonstrated utility of the the role of patents is to give exclusive rights to manufacture the patented product 319 less-advanced stages of hiv disease (coffey and peiperl, 2006) . to understand evergreening in the united states, we need to examine the drug price competition and patent term restoration act, informally known as the "hatch-waxman act" . it is a 1984 united states federal law which established the modern system for generic drug approval. hatch-waxman generic. section 505( j)(5)(b)(iv), the so-called paragraph iv, allows 180-day exvolume). for pharmaceutical companies, the hatch-waxman act has created a perverse drugs by making marginal changes than to try the risky strategy of inventing completely new chemicals. thus, the two decades following its passage, the hatch-waxman act has resulted in more me-too drugs than drugs with new chemical case of prilosec -the so-called "purple pill" of astrazeneca (usd 6 billion/year global blockbuster drug), the patent for which expired in 2001 only to be reincarnated as a new patented drug nexium. however, on 30 april 2007 the supreme court of the united states issued a ruling in ksr international co v. teleflex et al., which raises the bar for patent holders to prove that their invention is not obvious, and therefore patentable. this ruling will make many existing patents more vulnerable, make it harder to gain approval for new patents and make evergreening more difficult in the future. if the patent claim extends to what is obvious, it is invalid. for example, a patent's subject matter can be proved obvious if there existed at the time of invention a known problem for which there was an obvious solution encompassed by the patent's claims. the supreme court noted that, "granting patent protection to advances that would occur in the ordinary course without real innovation retards progress and may, in the case of patents combining previously known elements, deprive prior inventions of their value or utility." it is worth quoting in full the court's description of the reason that patents are only granted for non-obvious innovations: "we build and create by bringing to the tangible and palpable reality around us new works based on instinct, simple logic, ordinary inferences, extraordinary ideas, and sometimes even genius. these advances, once part of our shared knowledge, define a new threshold from which innovation starts once more. and as progress beginning from higher levels of achievement is expected in the normal course, the results of ordinary innovation are not the subject of exclusive rights under the patent laws. were it otherwise patents might stifle, rather than promote, the progress of useful arts." 9 the way forward prevention, treatment and human rights ents for branded counterparts. the hatch-waxman act encouraged the growth of generic industry, whose market share rose from 19% in 1984 to 49% in 2002 (by amended the federal food, drug, and cosmetic act. section 505( j) sets forth clusivity to companies that are the "first-to-file" an anda against holders of pat-the process by which would-be marketers of generic drugs can file abbreviated incentive. it has given them more incentive to try to extend the life of existing abbreviated new drug applications (andas) to seek fda approval of the compounds. the most famous documented case of evergreening occurred in the who guidelines for arv treatment regimens provide a basis for a range of treatment protocols in individual countries. in individual countries, factors such as prices, drug efficacy and side effects are also taken into account. stavudine (d4t) recommended that d4t no longer be used, due to toxicity. instead, countries should switch to tenofovir (tdf) or zidovudine (azt). of these two, tdf is preferable, because of its efficacy and safety and because it can be taken only once a day. emtricitabine (ftc), in one, triple-combination pill that can be taken once a day. patients are more likely to adhere to this once-a-day regimen, thereby reducing azt and tdf, compared to d4t, has delayed the shift to the new regimen in many below), the clinton foundation hiv/aids initiative (chai) has negotiated price reductions for several hiv/aids drugs for use in 27 low-and middle-income countries (see table 9 .6). the clinton foundation is discussed in chap. 7. as of may 2007, 750,000 people were benefiting from medicines purchased under chai agreements in 50 countries (chai 2007). table 9 .6. first, the prices are generally higher for middle-income countries than for low-income countries. thus, the pharmaceutical companies are pursuing a price discrimination strategy across different markets, selling drugs at a price that the markets can bear. therefore, there is clear room for generic products in these markets, especially for the low-income countries. compulsory licensing is a distinct possibility (see, however, our discussion in chap. 6 about the difficulties many developing countries faced importing drugs under compulsory license from canada). some companies have used the world bank's country income index or the human development index as their criteria for setting prices. in chap. 6, we developed a much more comprehensive index that takes into account not just the level of development of the country but also level of prevalence of hiv/aids explicitly. second, the price of hiv/aids drugs in many cases in many developing countries is not necessarily lower than in developed countries. for example, in guatemala, between 2000 and 2003, prices of most hiv/aids drugs were consistently higher than in the united states (hellerstein 2003). according to chai, in 2006, 80,000 (4%) of those receiving arv treatment in low-and middle-income countries were taking second-line treatment. the reason that relatively few are on second-line treatment is that most only began treatment within the last 4 years. as a result, relatively few have experienced treatment failure, which is defined as (1) virologic failure (a viral load of more than 400 copies per milliliter), (2) immunologic failure (a declining cd4 cell count in spite of treatment) or (3) clinical failure (progression to aids evidenced by weight loss or the appearance of opportunistic infections). another reason is that poor diagnostic and laboratory capacity in many countries has made treatment failure difficult to diagnose. by 2010, chai estimates that close to 500,000 people will require second-line treatment in low-and middle-income countries (chai 2007) . the higher cost of second-line treatment means that access requires further funding. however, patents are not expected to be an obstacle to acquiring affordable second-line treatments in the most affected low-income countries, due to the delay of trips patent rules on pharmaceuticals to 2016, although patent rules may affect affordability in middle-income countries (chai 2007) . in chap. 6 we analyzed trips rules on patents for pharmaceuticals in developing countries. however, as we noted in chap. 5, the problem of regulatory capture in free trade agreements can undermine trips rules so that patents create obstacles to affordable treatment in some lowincome countries and political pressure on low-and middle-income countries can discourage the use of trips flexibilities to increase access to treatment. unitaid is a global health initiative for hiv/aids, tuberculosis and malaria that is funded by several national governments. with respect to hiv/aids, unitaid funding is focused on pediatric and second-line treatment and the prevention of mother-to-child transmission. unitaid will finance a free supply of second-line hiv/aids treatment in 27 countries for 18 months, after which the reduced prices achieved by chai will enable other funding sources, such as the global fund (discussed in chap. 7) and pepfar (discussed in chap. 8), to fund the purchase of second-line treatments at lower prices (chai 2007) . while high-income countries and middle-income countries with low prevalence rates are in a position to pay for hiv/aids treatment, middle-income countries with high prevalence rates and most low-income countries are not. low-income countries with high prevalence rates in particular will have to depend on external funding sources, such as pepfar and the global fund, to expand access to treatment and then to maintain treatment. medical care for people with hiv/aids in developing countries costs about usd 1,000 a year, in drugs and support facilities. the economist estimated that it would cost usd 6-7 billion a year to provide treatment for the 6-7 million people with hiv/aids in low-income countries that were in need of treatment in 2006. however, expanding treatment means that fewer people will die. moreover, millions more will become infected, and even more so if prevention efforts are not improved. thus, universal treatment in lowincome countries could cost usd 40 billion by the end of the next decade. this highlights the need to ensure that external funding is both increased and sustained and the importance of prevention in making universal access to treatment affordable (economist 2006). scientists have been trying to develop an hiv vaccine for more than 20 years, although some have suggested that an effective aids vaccine may be a biological impossibility (epstein 2007) . in 2007, about 50 experimental hiv vaccines were being tested in clinical trials. most viral vaccines work by generating antibodies that neutralize or inactivate the invading virus. however, unlike other viruses, hiv-1 evades the antibody response, which, together with the large genetic variety found in hiv-1 strains, has made the development of an hiv-1 vaccine difficult. to date, antibody-based hiv-1 vaccines have only succeeded in neutralizing a minority of the copies of the virus that are found in a given patient. hiv-1 antibodies target the mechanism that hiv-1 uses to bind itself to the host immune cells in order to prevent hiv-1 from entering the cell. however, hiv-1 uses shielding mechanisms to prevent the antibodies from recognizing the virus, including a dense coating. current hiv-1 vaccine research therefore seeks to find vulnerabilities in these shielding mechanisms, but this requires research for multiple genetic subtypes of hiv-1 (montefiori et al., 2007) . for example, one recent study identified a place on the outside of the human immunodeficiency virus that could be vulnerable to antibodies that could block it from infecting human cells, which might be targeted with a vaccine aimed at preventing initial infection (dunham 2007) . a new class of hiv vaccines was designed to trigger cell-mediated immunity to create an extended immune defense. however, in 2007, merck reported that its hiv vaccine, v520, had failed. v520 was being tested by merck and the us national institutes of health in a clinical trial involving 3,000 people in highrisk groups in australia, brazil, canada, the dominican republic, haiti, jamaica, peru, puerto rico and the united states (associated press 2007). v520 used the common cold virus (the adenovirus) to transport three synthetic hiv genes into the body's cells (park 2007) . merck halted the trials after 24 of 741 volunteers who got the v520 vaccine later became infected with hiv, while only 21 of 762 participants that received a placebo also became infected (associated press 2007). the v520 vaccine was one of only two aids vaccine candidates in advanced human trials, the other being tested by sanofi-aventis sa (dunham 2007) . other approaches are also being explored. david ho (the inventor of triple combination therapy) and his team at the aaron diamond aids research center are researching the use of different vectors, or not using vectors at all, to produce stronger immune responses. scientists at the international aids vaccine initiative are studying the use of crippled, live strains of hiv and ways to stimulate a special class of antibodies that appear to be able to defuse hiv. the global hiv vaccine enterprise, which is funded by the gates foundation (discussed in chap. 7), wellcome trust, the us national institutes of health and the european union, is seeking to accelerate research on hiv vaccines by linking together independent organizations so that researchers can learn from each other, rather than work in isolation (park 2007). as we noted in chap. 5, there are many subtypes of hiv-1 (the most commonly occurring hiv infection in humans). the major hiv-1 subtypes accounting for most infections in africa are subtype c in southern africa, subtypes a and d in eastern africa, and circulating recombinant form 02_ag (crf02_ag) in westcentral africa (peeters and sharp 2000) . the most commonly occurring form of hiv-1 in north america and in europe is subtype b. the first hiv/aids vaccine ever to reach phase iii trial was for subtype b. the gp120 vaccine was not effective. however, what vaccine trials have indicated thus far is that, in the case of hiv/aids, there is pattern of development of potential vaccines not in the subtypes where the needs are the greatest but in the area where the biggest monetary rewards are expected. the economics of hiv/aids vaccines suggest that funding for vaccines for the worst-effected countries are unlikely to come from the private sector (see box 9.4). hiv/aids affects hundreds of millions and kills several million people every year. the disease was identified several decades ago. two nobel prizes have been awarded in the past two decades for identifying the cause and the transmission mechanism of hiv/aids. yet we still do not have a vaccine for hiv/aids. kremer and snyder (2004) have developed an argument as to why the private sector is very unlikely to develop a vaccine for aids. here, we illustrate the argument with one example. imagine there are 100 people in the world. there are 90 people (type l) who have a small chance of 10% of contracting hiv/aids. there are another ten people (type h) who would develop hiv/aids with a 100% chance. let us suppose that the harm from hiv/aids is usd 100 for each person. let us also assume that for each usd 1 decrease in harm, a consumer is willing to pay usd 1 (technically, each consumer is risk neutral). suppose the drug is perfectly effective, has no side effects and is costless to produce. how much revenue will a pharmaceutical company generate in each of the following scenarios? (1) it develops a drug d that cures hiv/aids (forever). (2) it develops a vaccine v that prevents hiv/aids from developing. we show that under the assumption that the pharmaceutical company cannot distinguish between type h and type l, it is more profitable for the drug companies to produce the drug rather than the vaccine. if the pharmaceutical company develops the drug d, it will be able to sell it to all the people who get hiv/aids. by assumption, all the type h people will develop hiv/aids. thus, there will be ten people from type h who will get hiv/aids. in addition, nine people of type l will also develop hiv/aids. in total, there will be 19 people with hiv/aids, including both types. by assumption, each person contracting hiv/aids will be willing to pay usd 100 to reduce the effects of hiv/aids by 100%. therefore, the pharmaceutical company will be able to earn usd 1,900 in revenue from the entire population. given our assumption of zero cost of production, usd 1,900 will also be the profits of the pharmaceutical company. the vaccine has to be sold before hiv/aids strikes. for type l, there is a 10% chance of hiv/aids. thus, they will be willing to pay the average loss of 100 (1/10) = usd 10 for the vaccine. if the pharmaceutical company cannot distinguish between type l and type h, it can only charge usd 10 to all. in that case, it will generate usd 10 100 = usd 1,000 profits by selling the vaccine to all 100 people. the other possibility is the following. the company sets a price of usd 100 for the vaccine. in that case, no person of type l will buy the vaccine ex-ante (as their expected benefit before hiv/aids strikes is usd 10 but the cost is usd 100). the only people who will buy the vaccine will be of type h. since there are ten of type h, the profits will be usd 100 10 = usd 1,000. thus, in either price strategy, the profits of the company will be usd 1,000. therefore, the profits of the company are bigger in the case of the development of drug d instead of the vaccine v. this argument is extremely general as long as the probability of the type l does not get close to the probability of type h getting the disease and the company cannot distinguish between the types. at the beginning of this book, we highlighted the need to integrate three interrelated issues into any comprehensive aids strategy -prevention, treatment and human rights protection. as we showed in chap. 2, each of these issues must be considered in the context of specific countries or regions, in order to take into account variations in cultural values, affected groups, infection rates, legal systems, economic resources and human resources. in this chapter, we have analyzed prevention and treatment issues in greater detail. the preceding discussion shows that great progress has been made on these two fronts and that greater progress is possible. our analysis of prevention issues in particular has shown the need to integrate prevention, treatment and human rights strategies. the primary reason that human rights need to be addressed is because discrimination keeps people away from both prevention and treatment programs (gruskin et al., 2007) . changing social attitudes in order to overcome stigma and discrimination is not an easy task, particularly given deep-seated fears and prejudices surrounding sex, blood, disease and death and the wide-spread perception that hiv/aids is closely supportive and enabling environment for women, children and other vulnerable to change attitudes of discrimination and stigmatization associated with hiv/aids variations in cultural values and legal systems make hiv/aids-related human rights particularly difficult to tackle on a global basis. however, hiv/aidsrelated human rights are the area where the least progress has been made and need to become a central focus in the global fight against hiv/aids (jã¼rgens and cohen 2007) . in this section, we focus on three categories of laws: (1) laws that discriminate against vulnerable groups; (2) laws that discriminate against hiv-positive people, such as those that criminalize hiv transmission; and (3) laws that prohibit discrimination against vulnerable groups, including hiv-positive people. we review the united nations international guidelines on hiv/aids and human rights and provide examples in each category. 9 the way forward prevention, treatment and human rights to understanding and acceptance (united nations 2007). groups. the guidelines also recommend that states promote the wide and ongoing distribution of creative education, training and media programs explicitly designed united nations international guidelines on hiv/aids and human rights redialogue, specially designed social and health services and support to community commend that states, in collaboration with and through the community, promote a groups by addressing underlying prejudices and inequalities through community 326 tied to deviant or immoral behavior (jã¼rgens and cohen, 2007) . in this regard, the the law plays different roles with respect to infectious diseases. some health risks, such as poor access to sterile injection equipment, can be directly attributed to law, and laws have been used to change unhealthy behaviors, such as smoking and drunk driving. both international and national laws are used in disease control. in addition to the law's role as a source of disease control authority for government, the law has a countervailing role as a source of protection against excessive and unnecessary regulations (burris 1999) . the united nations international guidelines on hiv/aids and human rights acknowledge the inherent limitations in using law reform to enhance human rights. the effectiveness human rights laws depend on the strength of the legal system in a given society and on the access of its citizens to the system, both of which vary considerably from one country to the next. moreover, the law cannot serve as the only means of educating, changing attitudes, achieving behavioral change or protecting people's rights. nevertheless, since laws regulate conduct between the state and the individual and between individuals, they can either support or undermine the observance of human rights, including hiv-related human rights (united nations 2007) . for these reasons, we first consider laws that support human rights. while social attitudes may take time to change, an important first step is to reform laws, policies and practices that institutionalize discrimination against the groups of people who are most vulnerable to hiv/aids: women and girls; men who have sex with men; commercial sex workers; and injection drug users. the united nations international guidelines on hiv/aids and human rights recommend consistent with international human rights obligations and are not targeted against vulnerable groups (united nations 2007). laws in this category include those that prohibit sexual acts between consenting adults in private, laws prohibiting sex work that involves no victimization and laws prohibiting measures such as needle exchange that can reduce the harm associated with illicit drug use (elliot 2002 ). the united nations international guidelines on hiv/aids and human rights recommend the enactment of anti-discrimination and protective laws to reduce human rights violations against women and children in the context of hiv, to reduce the vulnerability of women and children to hiv infection and to the impact of hiv/aids. with respect to women, the guidelines recommend law reforms to ensure the equality of women regarding property and marital relations and access 327 that states reform criminal laws and correctional systems to ensure that they are have a negative impact on hiv-related human rights and then consider laws that to employment and economic opportunity, such as equal rights to own and inherit property, to enter into contracts and marriage, to obtain credit and finance, to initiate separation or divorce, to equitably share assets upon divorce or separation and to retain custody of children. in addition, laws should ensure women's reproductive and sexual rights, including the right of independent access to reproductive and sexual health information and services and contraception, the right to demand safer sex practices and the right to legal protection from sexual violence. with children against sexual abuse and provide for their rehabilitation if abused and ensexual abuse by their husbands. when the husband is hiv-positive or engages in unsafe sex or drug use, this increases the risk of infection for women. child cusdren make it difficult for women to leave abusive relationships. while statutes allow property ownership regardless of sex, in practice women only have user rights under customary laws, not ownership. under inheritance laws, property remains in the man's family after he dies. thus, if a woman wants to leave an abusive husband or her husband dies, she cannot take any property with her, leaving women economically dependant upon their husbands or, as widows, their families. new laws have created inheritance rights for dependants, but are ignored by the man's family and not enforced. as a result, women and children widowed and women must either rely on their in-laws for support or become commercial sex workers (kelly 2004) . laws and cultural traditions thus increase women's vulnerability to hiv/aids, either within marriage or by forcing them to support themselves and their children as sex workers. recommend the enactment of anti-discrimination and protective laws to reduce discriminatory property, divorce and inheritance laws for same-sex relationships. 9 the way forward prevention, treatment and human rights tody laws, customary practice and traditions that favor paternal custody of chil-sure that they are not subject to penalties themselves. protection under disability human rights violations against men having sex with men, including in the context 328 orphaned by aids are left without adequate resources for medical treatment, and the united nations international guidelines on hiv/aids and human rights of hiv, including penalties for vilification of people who engage in same-sex respect to children, laws should provide for children's access to hiv-related inlaws, inheritance laws, and child custody laws. in many african countries marital rape does not exist as a legal concept, leaving women with no recourse against formation, education and means of prevention, govern children's access to volcontext of orphans, including inheritance and/or support. laws should also protect untary testing with consent, should protect children against mandatory testing, particularly if orphaned by aids, and provide for other forms of protection in the in sub-saharan africa, laws of particular concern include marital rape, property laws should also be ensured for children (united nations 2007). one key purpose of such anti-discrimination laws is to reduce the vulnerability of men who have sex with men to infection by hiv and to the impact of hiv/aids. the guidelines also recommend that the age of consent to sex and marriage be consistent for heterosexual and homosexual relationships and that laws and police practices relating to assaults against men who have sex with men ensure adequate legal protection (united nations 2007). in a 2006 internet-based survey of 759 sexually active msm in new york city, 11% reported being hiv-positive and 74% reported being hiv-negative. the majority were white, college-educated and in their 30s. the race of the respondents was white (77%), latino (13%), black (4%) and other (6%). in the previous 12 months, 45% had more than ten male sex partners, 53% had engaged in unprotected anal sex and 37% had used non-injection drugs. fifty percent of the hiv-positive men had unprotected anal sex in the previous 12 months and 71% of the hiv-negative men had unprotected anal sex in the previous 12 months (nyc health 2007). in a 2006 survey of 614 black msm in new york city, 67% were hivhigh school education, 67% were unemployed and 57% had an annual income of less than usd 10,000. fifty-six percent identified themselves as homosexual, 32% as bisexual, 6% as heterosexual and 6% as other. sixty-five percent had previously been diagnosed with a sexually transmitted infection and 30% had been raped (80% before they were 18 years old). eighty-four percent knew that they were hiv-positive. of the 16% that were unaware that they were hiv-positive, 53% reported having been tested for hiv previously. of those who had never been tested for hiv, the reasons they gave were: (1) being afraid to learn that they had the perception of not being at risk because they practiced safe sex (16%); and (4) being afraid that results will be reported to the government (16%). fifty percent reported unprotected anal sex with a man in the previous 3 months and 31% had exchanged sex for drugs, money or a place to stay in the same period. among those who had unprotected anal sex with a man in their last sexual encounter, 84% of the hiv-positive men had an hiv-positive sex partner and 89% of the hivnegative men had an hiv-negative sex partner (nyc health 2007). according to the unaids guidance note on hiv and sex work, despite high hiv prevalence among sex workers, only one in three receive adequate hiv prevention services and even fewer receive adequate treatment and health care (unaids 2007) . the unaids guidance note focuses on the reduction of hiv vulnerability among sex workers, who are defined as adults over the age of 18 years in order to take into account that sexual exploitation of children under 18 years of age is prohibited under international law. the key factors that lead people into sex work include poverty, gender inequality, indebtedness, migration, criminal hiv (48%); (2) being worried that others might treat them differently (26%); (3) positive. the median age of the respondents was 42 years, 22% had less than a coercion, humanitarian emergencies, drug use and dysfunctional families. laws, policies and practices that drive sex work underground make hiv/aids prevention and treatment for sex workers and their clients more difficult. discrimination against sex workers among the police, health care services and other social services impede access to prevention and treatment. the unaids guidance note organizes its recommendations into three categories: (1) reducing vulnerabilities and addressing structural issues; (2) reducing risk of hiv infection; and (3) building supportive environments and expanding choices. the strategies in the first category are to: (1) address poverty and gender inequality by providing alternatives to sex work through micro-finance programs and reforms to property rights; (2) address the demand for paid sex by seeking to changes men's behavior; (3) expand access to education for girls and women; (4) provide alternative job opportunities through employment growth and vocational training; and (5) provide employment and education opportunities and access to social services for refugees, internally displaced persons and economic migrants. the strategies in the second category are to: (1) involve sex workers in hiv prevention and treatment programs; (2) make male and female condoms available for free or at low cost; (3) increase access to antiretroviral treatment; (4) address the specific needs of sex workers in sexual and reproductive health programs, taking into account the different needs of female, male and transgender sex workers; (5) make hiv prevention information and condoms readily available to clients; (6) seek to eliminate violence against sex workers by clients, managers, police and other government officials; (7) seek to change attitudes towards sex workers to reduce stigma and discrimination; (8) promote initiatives to enable sex workers to negotiate safe sex practices; and (9) promote access to drug addiction treatment programs and harm reduction programs, such as needle exchange. the strategies in the third category are to: (1) address sex work stigma and discrimination to reduce economic, cultural and social marginalization in families and communities; (2) improve access to health care, education and training, microfinance and credit, social services, housing support and legal services; and (3) promote community organizations that work with sex workers. the unaids guidance note on hiv and sex work has been criticized for emphasizing alternative livelihoods without offering concrete examples, rather than emphasizing the right to engage in sex work and workplace safety and national laws that undermine sex workers' rights, particularly criminal prohibition of sex work and related activities. the guidance note's strategy of reducing demand for sex work has been criticized as implicitly supporting the criminalization or repression of sex work, which can increase the risk of hiv infection by driving sex work underground, limit sex workers' choices regarding working conditions and clients and increase stigmatization. the guidance note was further criticized for not advocating enhanced human rights protection for those engaged in sex work -as women, men, transgender persons and workers. the process used for preparing the document was criticized for not meaningfully engaging sex workers. unaids' response to criticism of this document -to withdraw it as a public document and restrict it to internal use -was also criticized (canadian hiv/aids legal network 2007b) the united nations international guidelines on hiv/aids and human rights recommend that criminal law prohibiting sexual acts (including adultery, sodomy, fornication and commercial sexual encounters) between consenting adults in private should not be allowed to impede provision of hiv prevention and care services and should be repealed. with regard to adult sex work that involves no victimization, the international guidelines on hiv/aids and human rights recommend de-criminalizing and legally regulating occupational health and safety conditions to protect sex workers and their clients, including support for safe sex during sex work. more generally, criminal law should not impede provision of hiv prevention and care services to sex workers and their clients and should ensure that children and adult sex workers who have been coerced into sex work are not prosecuted for such participation but rather are removed from sex work and provided with medical and psycho-social support services, including those related to hiv (united nations 2007). in eastern europe and central asia, unaids (2006) estimates that the use of contaminated injection equipment accounts for more than 80% of hiv/aid cases and accounts for about 30% of new infections outside sub-saharan africa. the united nations international guidelines on hiv/aids and human rights recommend that criminal law not be an impediment to measures taken by states to reduce the risk of hiv transmission among injecting drug users and to provide them with hiv-related care and treatment. they further recommend that criminal law be reviewed to consider: (1) the authorization or legalization and promotion of needle and syringe exchange programs; and (2) the repeal of laws criminalizing the possession, distribution and dispensing of needles and syringes (united nations 2007) . in saint petersburg, russia, a 2002 study found that 48% of injection drug users had shared needles in the 30 days prior to their first use of a needle exchange program. in early 2004, there were four syringe exchange facilities in saint petersburg -one mobile service (a bus) and three fixed facilities. however, the most important source of sterile syringes for injection drug users was drug stores. human rights watch found that state-supported impediments to access to both needle exchange points and drug stores were important barriers to hiv prevention, including: (1) police patrols of drug stores, which deterred injection drug users from purchasing syringes; (2) police patrols of needle exchange bus stops; and (3) arrests, fines or bribes for possession of syringes, even though carrying syringes is not illegal in the russian federation. however, while police interference with the syringe exchange bus was a problem in the late 1990s, it lessened in the early 2000s. humanitarian action, an ngo that delivers syringe exchange services in saint petersburg, visited with police chiefs to talk about the importance of syringe exchange for hiv prevention and organized a training session in 2003 for police officers that included the participation of former drug users and people living with hiv/aids. however, due to past incidents, the fear of apprehension by the police kept some drug users from using fixed as well as mobile syringe exchange facilities (human rights watch 2004) . table 9 .7 shows the dramatic increase in hiv prevalence among injection drug users in saint petersburg from 1998 to 2001. a 2005 survey of 500 injection drug users (idus) in new york city found that 65% had obtained a syringe from an exchange program in the previous year, 49% at a pharmacy, 10% from a medical provider, 53% from a friend or sexual partner and 25% from a drug dealer. the self-reported hiv prevalence rate in the group was 21%. idus who obtained syringes from sterile sources (exchange, pharmacy or provider) were less likely to share syringes than those who obtained them from non-sterile sources (friends, relatives or the street). those who obtained syringes from exchange programs were significantly less likely to share syringes. nevertheless, 19% of idus had shared a syringe at least once in the previous 12 months and 53% had engaged in unprotected sex. idus that had shared a syringe were 2.5 times more likely to engage in unprotected sex (nyc health 2007). another category of laws discriminates directly against people with hiv/aids, such as laws that criminalize hiv transmission and travel restrictions based on hiv status. there is a concern that the criminalization of hiv transmission will discourage people from seeking testing (tarantola and gruskin 2007) . there is evidence that knowledge of hiv status results in behavioral changes that reduce transmission. in addition, where knowledge of hiv status leads to antiretroviral treatment, treatment also reduces transmission by reducing the amount of virus in the body. thus, the criminalization of hiv transmission may have the effect of increasing, rather than reducing, hiv transmission. one possible response is mandatory hiv testing in health care settings (that is, testing without the informed consent of the patient). however, this policy, too, may be self-defeating if it discourages people from nations international guidelines on hiv/aids and human rights recommendation that public health legislation ensure that hiv testing of individuals should several studies have concluded that the criminalization of hiv transmission is unlikely to serve the goals of public health policy or the goals of criminal law, and ommended that governments and the judiciary take into account the following principles in determining policy regarding the use of criminal sanctions under modes and risk of hiv transmission to rationally determine when and if conduct should attract criminal liability; (2) the primary objective should be to prevent public health and conform to international human rights norms, particularly nondiscrimination and due process; and (4) policy makers should assess the impact of law or policy on human rights and prefer the least-intrusive measures possible to achieve a demonstrably justified objective of preventing disease transmission. with respect to the four functions of criminal law (harm prevention through response to the epidemic: (1) imprisoning an hiv-positive individual does not prevent transmission through conjugal visits or high-risk behavior with other prisoners; (2) criminal penalties are unlikely to change sexual activity and drug use, due to the complexity of these human behaviors; (3) punishment/retribution do not achieve the goal of hiv prevention and risk reinforcing prejudice and discrimination against already stigmatized hiv-positive people; and (4) criminal sanctions are unlikely to act as a deterrent, given that drug use and sexual activity persist even with the risk of criminal prosecution and are more likely to be driven underground when prosecuted, hindering hiv prevention. moreover, overly broad use of criminal laws risks spreading misinformation regarding how hiv is transmitted. in an empirical study conducted in the united states, burris et al. (2007) found that laws prohibiting unsafe sex or requiring disclosure of infection do not influence people's normative beliefs about risky sex and did not significantly influence sexual behavior. the study concluded that criminal law is not a clearly useful in-moreover, given concerns about possible negative effects of criminal law, such as stigmatization or reluctance to cooperate with health authorities, criminal law should be used with caution as a behavioral change mechanism for hiv-positive people. seeking health care. moreover, mandatory hiv testing runs counter to the united thus may do more harm than good. in a unaids policy paper, elliot (2002) bution; and deterrence), elliot (2002) concluded that criminal law is an ineffective imprisonment; prevention of future harm through rehabilitation; punishment/retri-hiv transmission in common law countries. in some cases, courts have applied existing criminal laws to cases involving hiv, where the laws themselves do not refer specifically to hiv. in this context, law reforms could come from the legislature, through amendments that clarify the application of relevant criminal laws to cases involving hiv, or through the evolution of precedents in the courts. the united nations international guidelines on hiv/aids and human rights recommend the reform of criminal laws and correctional systems to ensure that they are consistent with international human rights obligations and are not misused in tization of the judiciary, in ways consistent with judicial independence, on the legal, ethical and human rights issues relative to hiv, including through judicial education and the development of judicial materials (united nations 2007). criminal laws should not include specific offences against the intentional transmission of hiv but rather should apply general criminal offences to these exceptional cases. such application should ensure that the elements of foreseeability, intent, causality and consent are clearly and legally established to support a guilty verdict and/or harsher penalties (united nations 2007) . in the united states, a series of cases involving spitting have gone in different directions. in ohio v. bird (1998) , an hiv-positive man was convicted of felonious assault, which requires the knowing attempt to harm by use of a weapon capable of inflicting death, after spitting in a police officer's face, even though all medical and scientific evidence demonstrated that saliva does not transmit hiv. in state v. jones (2000) , another case of an hiv-positive individual accused of spitting on an officer, the new mexico court of appeals ruled that criminal liability for battery could not be based upon the victims' subjective and unsubstantiated fears that they could develop a disease, and reversed the lower court on this issue. in weeks v. state (1992) , the texas court of appeal sustained the attempted murder conviction of an hiv-positive inmate who spat in a guard's face. the spitting cases show how the application of criminal laws to hiv-positive individualswhen based on hiv status, stigma and discrimination rather than on medical or scientific evidence -can undermine genuine efforts to reduce hiv transmission by spreading misinformation and increasing stigma and discrimination. in cases involving behavior that does carry a risk of hiv transmission, such as unprotected sexual intercourse or sharing drug injection equipment, the central issue is consent. in r v. cuerrier (1998) the supreme court of canada established that there is a duty to disclose one's hiv status before engaging in any activity that poses a "significant risk" of hiv transmission. failure to do so legally invalidates a sexual partner's consent to sexual intercourse. the lack of consent to have intercourse with a partner that is hiv-positive converts the sexual intercourse into a criminal assault. in that case, the complainants did not become infected with hiv as a result of the unprotected sex. however, if the complainants believe that their partner is hiv-free and the accused puts the complainants at significant risk to their health, failure to disclose hiv status vitiates consent to sexual intercourse. 9 the way forward prevention, treatment and human rights there have been numerous cases in which criminal laws have been applied to the context of hiv/aids (united nations 2007). they also recommend the sensi-this decision suggests that there might not be a duty to disclose hiv status prior to engaging in activities that do not pose a significant risk of transmission, such as kissing and oral sex, or where an hiv-positive individual uses a condom. in r v. edwards, a lower court judge ruled that there is no duty to disclose hiv status prior to engaging in unprotected oral sex because it is a low risk activity (canadian aids society 2004) . on 15 november 1991, the defendant learned that he was hiv-positive, but did not reveal his status to the complainant and continued to have unprotected sex with her. the supreme court of canada ruled that the defendant was not guilty of the act itself, but rather the consequences of the act. because it was likely that the defendant had infected the complainant before he learned of his hiv status, it could not be proved beyond a reasonable doubt that he had endangered the life of the complainant. however, the defendant was guilty of attempted aggravated assault for continuing to have unprotected sex with the complainant after having learned of his hiv status. the court ruled that there is sufficient criminal intent for a conviction on a sexual assault charge if a person acts "recklessly". in canadian law, a person acts "recklessly" if they know that their conduct risks committing a crime but they commit the act nevertheless. in this case, the supreme court ruled that criminal recklessness is established once an individual becomes aware of a risk that he or she has contracted hiv, but continues to have unprotected sex without disclosure of hiv status, thereby creating a risk of further hiv transmission. in this case there was no evidence before the court regarding the defendant's awareness of the risk that he might be hiv-positive, prior to 15 november 1991, other than the fact that he had been asked to take an hiv test. this aspect of the ruling raised the issue of whether there is a duty to disclose the mere awareness of a risk that one might be hiv-positive before having unprotected sex. the court also suggested that an hiv-positive person might be held criminally liable for failure to disclose hiv status before having unprotected sex with another hivpositive individual, where this results in the transmission of a different strain of hiv or a drug-resistant strain of hiv. the supreme court of canada cases have been criticized, on the one hand, for discouraging people from seeking testing in order to avoid the possibility of a criminal conviction based on knowledge of hiv status and, on the other hand, for risking undesirable invasions of privacy if courts are required to determine whether an individual was aware that their past activities put them at risk of hiv infection (canadian hiv/aids legal network 2003) . however, in r v. williams, the fact that the defendant had been asked to take an hiv test, because he was on a list of former partners provided by an individual who had tested hiv-positive, was not sufficient to establish that he was aware that his past activities had put him at risk in r v. williams (2003) , the defendant began a sexual relationship with the comthe complainant". what distinguishes aggravated assault from mere assault is not 335 plainant in june 1991, in which they had unprotected sex on numerous occasions. requires that the assault "wounds, maims, disfigures or endangers the life of aggravated assault under section 268(1) of the canadian criminal code, which of hiv infection. nevertheless, the decision has been criticized for extending the without defining the nature of the awareness that might be required. more generally, the use of criminal law to prevent hiv transmission has been criticized for stigmatizing all hiv-positive people because of the conduct of a few individuals, for discouraging those most at risk from seeking testing and for being unlikely to stop people from having risky sex or sharing needles and syringes. moreover, all of the hiv-related criminal prosecutions in canada have occurred in the context of heterosexual intercourse, rather than homosexual intercourse or injection drug use, creating a perception of discriminatory application (or non-application) of the laws (betteridge 2007). sion of hiv/aids between 2003 and 2007, in which eight accused pleaded guilty, two were convicted and one was acquitted (klein 2007) . a new zealand court has ruled that people living with hiv/aids are not required to disclose their hiv status if they use condoms during vaginal sex (klein 2007) . in particular, the use of criminal laws to prevent hiv transmission also has been criticized for not taking into account that hiv-positive individuals living in abusive relationships may fear the consequences of disclosing their status to partners and may not be able to use a condom or insist that their partner use a condom (canadian aids society 2004) . in a literature review of hiv/aids and genderbased violence, the harvard school of public health program on international health and human rights (2006) found that gender-based violence (which is not limited to violence against women) can interfere with safe sex practices and access to treatment. not only is gender-based violence a risk factor for acquiring hiv/ in summary, the use of criminal laws to prevent hiv transmission may undermine overall public health initiatives by: (1) reinforcing hiv/aids-related stigma; (2) spreading misinformation about hiv/aids; (3) creating a disincentive for hiv testing; (4) hindering access to counseling and support services; (5) creating a false expectation that criminal laws eliminate the danger of unprotected sex for people who believe that they are hiv-negative; (6) creating the risk of selective prosecution of marginalized groups; (7) criminalizing behavior that results from gender inequality, in the case of hiv-positive people living in abusive or economically dependent circumstances; and (8) invading privacy through the disclosure of medical records and hiv status in public court proceedings (elliot 2002) . however, the use of criminal laws may be warranted in some circumstances, where hiv status is an aggravating or otherwise relevant factor in cases involving physical assault that would constitute criminal behavior even in the absence of hiv, such as rape or the use of needles as weapons (elliot 2002) . finally, a distinction should be made between criminal laws and public health laws that are quasi-criminal in nature, particularly those regarding quarantine. while quarantine laws, such as isolation, detention or quarantine, may be suitable 9 the way forward prevention, treatment and human rights criminal law beyond cases where individuals know that they are hiv-positive, in the united kingdom, there were eleven prosecutions for reckless transmis-aids, but hiv/aids is also a risk factor for gender-based violence. for casually communicable and curable diseases, such laws run the same risk of misuse as do criminal laws (elliot 2002) . in this regard, the united nations international guidelines on hiv/aids and human rights recommend that public health law provisions applicable to casually transmitted diseases not be applied inappropriately to hiv/aids and that they be consistent with international human rights obligations (united nations 2007). some countries have restricted the entry of people living with hiv/aids, for shortterm or long-term stays, through mandatory testing or a requirement to declare one's hiv status. as we saw in chap. 8, the who international health regulations also contain provisions regarding health measures applied to travelers. these provisions encourage states to base their determinations upon scientific principles, available scientific evidence of a risk to human health and any available specific guidance their dignity, human rights and fundamental freedoms and minimize any discomfort or distress associated with such measures. governments cite two main reasons for imposing travel restrictions on people living with hiv/aids -public health protection and reducing demand on health care and social services (unaids/iom 2004) . in the united kingdom, another source of demands for hiv screening of migrants has been a concern over "health tourism" -hiv infected migrants from developing countries that go to europe to receive health care. however, research shows that access to treatment is rarely the after having arrived in the host country, and there is no uniform policy in european union countries regarding screening of migrants for hiv (carballo 2007) . hiv/aids is not considered to be a condition that poses a threat to public health in relation to travel because hiv/aids is already present in virtually every country in the world and hiv is not transmitted through casual contact. unlike highly contagious diseases with short incubation periods, such as sars, cholera and plague, hiv transmission can be prevented through safe sex and safe drug injection, which can be used by both the infected and the non-infected to prevent transmission. there is no evidence to support the assumption that both the infected and the non-infected will engage in unsafe practices. as a result, the presence of hiv-positive individuals, by itself, does not pose a risk to public health. in addition, travel restrictions are not effective in preventing the entry of hiv-positive individuals, since hiv tests do not detect the virus in newly infected people and nationals that are returning from travel abroad (who may have been infected while outside the country) are not subject to hiv/aids-related travel restrictions and are not prevented from entering their own country. moreover, travel restrictions can undermine hiv/aids-related public health initiatives by increasing stigma and discrimination and mislead the public into thinking that hiv/aids can be or advice from the who. they also require states to treat travelers with respect for reason for migration to europe, since most migrants only learn of their hiv status prevented through border measures, rather than through proven prevention strategies (unaids/iom 2004) . unaids and the international organization for migration (iom) recommend that exclusion on the basis of possible costs to health care and social services only occur on an individual basis, where the following considerations are shown: (1) the person requires the health care and social services and is likely to use them in the near future; (2) the person has no other means of meeting those costs (for example, through private or employment-based insurance or personal resources); and (3) these costs will not be exceeded by the benefits of the person's skills, talents, contribution to the labor force, payment of taxes, contribution to cultural diversity and capacity for revenue or job creation (unaids/iom 2004) . they also recommend that countries treat similar conditions alike, rather than singling out hiv/ aids. one study showed that the 10-year economic impact of admitting immigrants with asymptomatic hiv infection would be similar to admitting immigrants with asymptomatic coronary heart disease (zowall et al., 1994) . the canadian immigration and refugee protection act provides that foreign nationals can be deemed "medically inadmissible" based on a medical condition, danger to public health or public safety; or (2) they might reasonably be expected to cause excessive demand on health or social services. since 1991, canadian danger to public health or public safety by virtue of their hiv status. the issue of excessive demand on health or social services is mainly a consideration in cases of immigration or stays that exceed 6 months, is determined on a case-by-case basis permanent residents (spouses and children). demand on health or social services of health or social services for the average canadian resident; or (2) the demand the united states has had a travel and immigration restriction in place for people living with hiv/aids since 1987 (human rights watch 2006) . under the united states are inadmissible if they have "a communicable disease of public high level meeting on aids a "designated event" for which an hiv waiver would be available. visitors entering the united states on the visa waiver program (which waives the requirement to apply for a visa prior to traveling to the united 9 the way forward prevention, treatment and human rights government policy has been that people living with hiv/aids do not represent a and therefore denied a visa or entry at the border, if: (1) they are likely to be a 338 would add to existing waiting lists for those services and would increase the rate us immigration and nationality act, applicants for a visa or for admission to the health significance", which includes hiv infection, although waivers are available ces by canadian citizens or permanent residents. the social or economic contribu-and does not apply to refugees or close family members of canadian citizens or tions the individual is expected to make to canada are not taken into account. or mortality and morbidity in canada by denying or delaying access to those servihiv status or to be tested for hiv (canadian hiv/aids legal network 2007a) . on a case-by-case basis. for example, the us attorney general named the 2006 people entering canada for less than 6 months are not required to disclose their is considered excessive if: (1) the anticipated costs would likely exceed the costs states, for certain countries) must fill out an i-94w form, which asks, "have you ever been afflicted with a communicable disease of public health significance." if the visitor answers yes to the question or the us border authorities suspect a visitor to be hiv-positive the person may be: (1) placed into secondary inspection; (2) questioned by an official of the us department of homeland security; (3) placed into deferred inspection; (4) asked to withdraw the application for admission into the united states; (5) placed into the expedited removal process; or (6) placed into an us department of homeland security detention center and detained until the case is heard by an immigration judge (gmhc 2006) . hiv-positive non-immigrants seeking to enter the us on a temporary basis for business, pleasure, or education are eligible for a waiver under which they can be allowed to enter the united states. in practice, a waiver is granted in most cases if: (1) they are not symptomatic; (2) it is a short visit; (3) they have insurance or other assets sufficient to pay medical expenses; and (4) they don't appear to be a public health risk. permanent residency and immigration applicants can also apply for a waiver, but they are usually rejected. to receive a waiver as an immigrant, the person must be the spouse, unmarried son or adopted child of a united states citident as their son or daughter. in addition, an hiv-positive immigration applicant must prove that: (1) he will not be a danger to public health; (2) the possibility of spreading the disease is minimal; and (3) there will be no cost incurred by any level of government without its prior consent (tarwater 2001) . june 1987 the us public health service added aids to the list of excludable conditions, noting that the exclusion was not based on any new scientific knowledge and that aids is not spread by casual contact, which is the usual public concept of contagious. in july 1987, republican senator jesse helms also added hiv infection to the exclusion list, through the us congress, together with a prohibition on funding from the us centers for disease control for aids programs that "promote, encourage or condone homosexual activities" (koch 1987; aids treatment news 1991) . senator helms accompanied the introduction of his amendments with the following statement: "we have got to call a spade a spade, and a perverted human being a perverted human being" (koch 1987) . in july 1995, senator jesse helms advocated spending less money on hiv/aids, because it resulted from "deliberate, disgusting, revolting conduct" and was "a disease transmitted by people deliberately engaging in unnatural acts" (associated press 1995). ten years later, he had this to say: "it had been my feeling that aids was a disease largely spread by reckless and voluntary sexual and drug-abusing behavior, and that it would probably be confined to those in high-risk populations. i was wrong" . in 1990, the us centers for disease control (cdc) recommended that all diseases except active tuberculosis be removed from the list of excludable conditions. hiv was left on the list because it had been put on the list by congress. in november the political history of the us hiv travel restrictions is an interesting story. in zen or permanent resident or have a united states citizen or lawful permanent resi-1990, the immigration reform act of 1990 directed the cdc to establish a new list of excludable conditions, based solely on current epidemiological principles and medical standards. in january 1991, the cdc again proposed that only active tuberculosis remain on the list of excludable conditions. religious leaders campaigned to maintain the ban and the us house of representatives opposed removing the hiv ban (aids treatment news 1991) . in august 2007, democratic representatives barbara lee and hilda solis introduced the "hiv nondiscrimination in travel and immigration act". the proposed legislation would restore the authority of the secretary of health and human services to determine whether hiv status is a communicable disease of public health significance. the decision to maintain or remove the ban would then be based on public health analysis instead of a formal ban made by congress (latino commission on aids 2007). in november 2007, the us department of homeland security proposed a new rule that would allow short-term visas to be granted to hiv-positive people by us consulates in their home countries. however, applicants would have to agree to conditions, including ceding the right to apply for longer stays or permanent residency in the united states. democratic members of the us house of representatives objected that the changes would only shift decision-making authority to local consular officers, who may lack the appropriate medical expertise. moreover, there would be no appeal process (werner 2007) . the united states and canada are similar societies, both culturally and economically, but have adopted very different approaches to hiv/aids travel restrictions. the hiv prevalence rate in the united states is higher than in canada. this suggests that the us travel restriction has not been effective in preventing hiv transmission in the united states, and that the lack of such a restriction in canada has not had the effect of increasing hiv prevalence. health care costs, measured as a percentage of gdp, are also higher in the united states than in canada. while this difference is attributable to many factors, making it difficult to determine the impact of the different travel restriction policies on health care costs without further study, it is an indication that the canadian approach has not led to a significant increase in health care costs compared to the american approach. in 2003, americans spent usd 5,711 per capita on health care, compared with usd 2,998 in canada. americans spent 15.2% of gdp on health care compared with 9.9% of gdp in canada. interestingly, this gap was not always there. in 1970, both countries spent exactly 7.0% of their respective gdp on health care (oecd 2006) . another factor that suggests that us travel restrictions are unlikely to prove successful is illegal immigration. there are several million illegal entries into the united states each year. they are obviously not screened. thus, from a practical point of view, travel and immigration restrictions for hiv-positive individuals are unlikely to be effective in preventing the entry of many hiv-positive individuals and may provide additional incentives for some individuals to migrate illegally. the united nations international guidelines on hiv/aids and human rights recommend that states enact or strengthen anti-discrimination laws that protect vulnerable groups, people living with hiv/aids and people with disabilities from discrimination in both the public and private sectors, and provide for speedy and effective administrative and civil remedies (united nations 2007). human rights laws in many jurisdictions prohibit discrimination against vulnerable groups or against people with hiv/aids, as well as providing other rights that are relevant to hiv/aids, such as the right to life and the right to health. human rights laws fall into two categories. the first category applies to governments, prohibiting governments from passing discriminatory laws or requiring governments to uphold certain human rights. the second category of human rights law prohibits discrimination on the part of private actors, for example with respect to employment practices or rental of housing. while it is not possible to eliminate individual or societal prejudices with legislation, human rights laws provide victims of discrimination with legal recourse against acts of discrimination and create economic disincentives through fines or other legal remedies, thereby contributing to social change. canada provides one example of the sources and functioning of human rights laws. section 15 of the canadian charter of rights and freedoms, which is part of the constitution of canada, guarantees equality rights in the following terms: the equal protection and equal benefit of the law without discrimination and, in particular, without discrimination based on race, national or ethnic origin, colour, religion, sex, age or mental or physical disability. canadian courts have interpreted the term "disability" to include hiv/aids, which means that people living with hiv/aids have constitutional protection listed, but also covers analogous grounds, such as sexual orientation. any law that is inconsistent with constitutional provisions may be struck down or interpreted by courts to make it consistent with the constitution. the charter applies to all levels and branches of government, all government acts, government corporations and ment government policies or programs. however, the charter does not otherwise apply to acts by private citizens. instead, discrimination by an employer, a landlord or a private business is addressed under other federal and provincial human rights laws, such as the canadian human rights act, which apply to both the public and private sectors. by virtue of a 1996 policy of the canadian human rights commission and decisions of canadian courts and tribunals, the prohibition against disability-based discrimination in the every individual is equal before and under the law and has the right to against discrimination by the state. section 15 is not limited to the grounds that are private persons or bodies that exercise authority granted by a statute or that imple-canadian human rights act and its provincial counterparts cover discrimination based on hiv/aids status (elliott and gold 2005) . the remainder of this section provides an overview of court cases in a variety of countries that have applied constitutional law, international law and other legislation to uphold the rights of people living with hiv/aids with respect to employment and access to hiv-related medical care and treatment. in march 2007, mexico's national supreme court of justice ruled that a provision in article 226 of the social security institute law for the armed forces (issfam) that required hiv-positive individuals to be discharged from the military was unconstitutional, because it was not based on an individual assessment of the pering people living with hiv/aids. the court ordered that three soldiers be reinduty, which would include an obligation to reinstate their social security benefits with respect to hiv/aids, laws in south africa and latin america that provide a action campaign used this provision to challenge the government's program that limited the use of nevirapine to prevent mother-to-child hiv transmission to 18 test sites. the court ruled that the government's restriction on the use of nevirapine was unreasonable and that the policy should be reformed to meet the government's constitutional obligation (singh et al., 2007; elliot et al., 2006) . in argentina, five court cases between 1996 and 2003 repeatedly ordered the argentine ministry of health to supply antiretroviral treatment to people living with hiv/aids, in accordance with the right to health set out in international treaties, which had been incorporated into domestic law. the failure of the ministry of health to act in a timely fashion, which led to interruptions in the supply of antiretroviral drugs, ultimately led to a court order that would fine the ministry of health usd 1,000 per day (funds which would then be used to implement the national aids plan) until it complied with the courts' previous orders, and the threat right to health care have been used to induce governments to provide access to and equality and was inconsistent with mexico's international obligations regardvides a right to health care that is binding on the government. the treatment mexico's national supreme court of justice ruled for a fifth time that this provision was unconstitutional, thereby creating jurisprudence that is binding on all federal son's ability to work, violated constitutional protections of non-discrimination judges in mexico (avilã©s allende 2007). table 9 .8 summarizes several other antiretroviral treatment (gruskin et al., 2007) . the south african constitution pro-cases involving hiv-related discrimination in employment from various juris(pearshouse 2007; medina and reyes 2007; scjn 2007a, b) . in september 2007, dictions around the world. stated until medical certificates were issued to determine whether they were fit for (elliot et al., 2006) . an argentine court also relied on the right to health set out in international treaties to order the government to produce and administer a vaccine within a set period of time, in order to protect people living in a region affected by argentine haemorrhagic fever (singh et al., 2007) . the constitutional court of ecuador relied on the right to health set out in international treaties to rule that the ministry of health had failed to meet its obligations when it suspended its hiv treatment program (singh et al., 2007; elliot et al., 2006) . in costa rica, the supreme court ruled in 1997 that the costa rican social security fund could not argue that financial constraints justified failure to comply with its very reason for its existence, which is to provide coverage for necessary medical care. shortly after this ruling, the supreme court ordered the social security fund to develop a plan to provide coverage to all persons living with hiv/aids that were in need of antiretroviral treatment. a few weeks later, costa rica became the first central american country to include cov2006) . in india, the courts have interpreted the right to life in the indian constitution to sources to uphold the right to health in a variety of cases (singh et al., 2007) . table 9 .9 summarizes several other cases from various jurisdictions around the world where litigation has increased access to hiv-related medical treatment. these cases suggest that human rights laws can be instrumental in promoting health care reforms through litigation, provided that judicial authorities are independent and competent and governments respect the rule of law (singh et al., 2007) . in addition to laws that institutionalize or prohibit discrimination, institutional policies and practices can represent an important force with respect to stigma, discrimination and access to health care. the united nations international guidelines on hiv/aids and human rights recommend that states ensure that government and the private sector develop codes of conduct regarding hiv/aids issues that translate human rights principles into codes of professional responsibility and practice, with accompanying mechanisms to implement and enforce those codes. in many jurisdictions, the courts have the power to order changes in policies and practices of both governmental and non-governmental institutions. however, litigation is an expensive and time-consuming process that creates additional stress for the people living with hiv/aids who choose to litigate. thus, it is important to promote the voluntary adoption of appropriate policies and practices. one example in this category is the policies and practices of health care institutions. for example, in the mid 1980s, in british columbia, canada, all hospitals refused to treat aids patients, with the exception of st. paul's hospital, which adopted appropriate policies based on the commitment of the founding sisters of 345 include a right to health, and have obliged the indian government to dedicate re-erage for antiretroviral drugs in its national health insurance plan (elliot et al., another example in this category is the policies and practices of employers. as we showed in chap. 3, hiv/aids affects the productivity of workers substantially, making it cost effective for companies to have prevention programs and to provide treatment for employees, from a purely financial point of view. business leaders have an economic incentive to invest resources in fighting the epidemic. moreover, as we saw in chap. 7, firms can have a tremendous impact in promotment. however, it is important to have an overarching framework that ensures the adoption of best practices by individual firms and to minimize overlap between the private sector and the other players that are involved in addressing the pandemic. in this regard, the global business coalition on hiv/aids has provided leadership, particularly in its efforts to identify ways to improve the global business community's response to hiv/aids, including through leadership to dispel myths and stigma, break down workplace barriers and influence community change. given the economic and legal incentives, an effective hiv/aids response 9 the way forward prevention, treatment and human rights providence to care for all who were in need, regardless of financial or social standing (gratham 2007) . in 2006, the city of philadelphia agreed to resolve a complaint regarding the refusal of emergency medical services personnel to touch or lift a patient because of his hiv status, by paying monetary compensation and agreeing to implement a mandatory paramedic/emt training program on hiv and infectious diseases (john gill smith and united states v. city of philadelphia 2006) . ironically, "philadelphia" was the name and setting of the first high-profile hollywood film to take aids seriously, in 1993. we can think of hiv/aids as a disaster from the point of view of a country as a whole. unlike other disasters (such as an outbreak of an influenza pandemic), this kind of risk, we need to measure the severity and the frequency of occurrence of that risk. once we measure the risk, we need to find ways of managing the risk in a dynamic way. that means putting a risk management plan in place, monitoring the plan and modifying the plan as events unfold. most often, at the national level, hiv/aids is seen as a public health problem and is managed as such. thus, various measures are taken to reduce the incidence of hiv/aids by taking steps against the main channels through which the disease strikes: (1) actions to reduce the contamination of the blood supply; (2) special steps to promote health care for key groups, such as sex workers; (3) needle exchange programs; (4) promoting safe sex through the use of condoms; and (5) minimizing hiv transmission from infected mothers to newborns. 346 disaster unfolds over many years. however, the standard operating procedure for ing prevention among employees and their families and providing access to treatdisaster management also applies to managing hiv/aids risk. for managing any must be a core component of an overall business strategy. another approach to risk management is risk avoidance. at the country level, risk avoidance could imply two extreme actions: quarantining people who are already infected and preventing infected people from coming into the country. neither of these policies is feasible for most countries, as they directly go against human rights. thus, extreme forms of risk management and the respect for human rights pose a tradeoff for a country. cuba provides a striking example of how containment of hiv/aids can be conducted at a national level. cuba started promoting public health messages against hiv/aids in 1983, 3 years before the first hiv case was reported in the country. between 1986 and 1989, cuba undertook a massive testing exercise, which tested more than 80% of the adult population. those who were seropositive were quarantined indefinitely in sanitariums. over the years, cuba has relaxed the rule. today, anybody found seropositive is required to attend an 8 week course. after that, they are free to leave. nearly half the people choose to stay in the sanitariums, where they get free food and a place to stay, along with retraining if they choose to help with the logistics of the sanitariums. such a curtailment of freedom of movement without committing a crime is unprecedented anywhere in the world. it has been criticized by many. it did produce a result that is also unprecedented. cuba has an hiv incidence rate of 0.05%. in the neighboring island of haiti, the rate is 120 times as high, at 6.1%. it should be noted that quarantine of individuals who have committed no crime is not unheard of. there was the case of mary mallon in the united states in 1908better known as the "typhoid mary" -who carried typhoid without every showing any symptoms. she was quarantined against her will for a number of years. similarly, during the outbreak of influenza in the united states in 1918, many families were quarantined on public health grounds. individuals with sars were also quarantined in toronto. the future of hiv/aids presents a mixed picture. while hiv/aids incidence has begun to level off in some high-prevalence countries, new infections have increased in many developed countries. while several science-based prevention strategies need to be scaled up significantly, the increase in mother-to-child prevention has dramatically reduced infections among newborns and male circumcision is a promising new prevention strategy. while millions still lack access to treatment, there has been a large increase in funding, drug prices have dropped dramatically, several key drug patents will expire in the near future and efforts to develop new treatments continue. while stigma and discrimination remain obstacles to effective prevention and treatment, human rights laws have proved to be an effective vehicle for addressing discrimination and increasing access to treatment around the world. thus, while hiv/aids continues to pose a significant threat to public health, there are many signs that progress in fighting this pandemic can and will continue, as knowledge gradually replaces ignorance. travel/immigration ban: background senator jesse helms: cut aids funding experimental aids vaccine falls short randomized, controlled intervention trial of male circumcision for reduction of hiv infection risk: the anrs 1265 trial amplã­a corte protecciã³n a militares con vih male circumcision: the road from evidence to practice. division of epidemiology, school of public health, university of illinois at chicago betteridge g (2007) criminal law and hiv transmission or exposure: new cases and developments law as a structural factor in the spread of communicable disease the way forward prevention, treatment and human rights do criminal laws influence hiv risk behavior? an empirical trial hiv disclosure & the criminal law in canada supreme court of canada decision in r v canada's immigration policy as it affects people living with hiv/aids a human rights-based commentary on unaids guidance note: hiv and sex work communicable diseases: challenges for health in the age of migration 2c1fe1c/10624/chpt4eu.pdf. accessed a%20human%20rights-based%20commentary%20on % 20unaids %20guidance % 20 zidovudine's patent history look to the future: the war against aids. the economist net benefits: giving bed nets and drugs away free may be the way to deal with malaria criminal law, public health and hiv transmission: a policy options paper protection against discrimination based on hiv/aids status in canada: the legal framework the effect of pregnancy on survival in women infected with hiv: a systematic review of the literature and meta-analysis global hiv prevention working group (2007) bringing hiv prevention to scale: an urgent global priority history, principles and practice of health and halperin d (2007) evidence-based behavior change hiv prevention approaches for sub-saharan africa harvard school of public health program on international health and human rights hamied yk (2005) trading in death lessons not learned: human rights abuses and hiv/aids in the russian federation family, unvalued: discrimination, denial, and the fate of binational same-sex couples under memoir, jesse helms says he was no racist a salute to the sisters. promise fall/winter literature review increasing access to hiv testing and counseling while respecting human rights reasons why human rights should occupy the center of the global aids struggle. open society institute the way forward prevention, treatment and human rights conspiring to kill: gender-biased legislation, culture, and aids in sub-saharan africa new hiv cases drop but rise in young gay men. www.nytimes senator helms's callousness toward aids victims the latino commission on aids supports the hiv nondiscrimination in travel and immigration act discriminaciã³n por vivir con vih: fuerzas armadas, a punto del revã©s, letra s 128 the known hidden hiv/aids epidemic among black men who have sex with men in the united states plos medicine 4:12 nyc health (2007) hiv epidemiology and field services research unit report accessed 5 november human rights watch (2006) family, unvalued: discrimination, denial, and the fate park a (2007) assessing a failed aids vaccine mexico: supreme court rules discharge of hiv-positive troops unconstitutional genetic diversity of hiv-1: the moving target seeds of change in rwanda sesiã³n pãºblica nãºm the tuberculosis & hiv debate in immigration law: critical flaws cuerrier (1998) 2 scr 371, supreme court of canada new mexico court of appeals, 129 nm 165 ohio state court appellate amicus brief us food and drug administration (2006) guidance for industry fixed dose combina antiretrovirals for the treatment of hiv /nr/rdonlyres/98ef667e-cf50-4abb-ab9f-107 adf012927/0/265demarzode do human rights matter to health? lancet marde2007 state (1992) texas court of appeal united states academic anti-exclusion arguments. georgetown immigration law journal hivlawandpolicy.org /resources /partial %20case %20&resource %20 list-lambda packaged drug products, and single-entity versions of previously approved tarantola d, gruskin s (2007) new guidance on recommended hiv testing and hiv_data/2006globalreport/default.asp. accessed the way forward prevention, treatment and human rights consolidated version new law allows needle exchanges in washington modeling health care costs attributable to hiv infection and hiv prevention programs in high-income countries systematic review of abstinence-plus aids epidemic -planning, policy and predictions limited setting: treatment guidelines for a public health approach scaling up antiretroviral therapy in resouurce lawmakers, gay-rights groups protesting new hiv/aids travel unaids/iom (2004) statement on hiv/aids-related travel restrictions international guidelines on hiv/aids and human rights key: cord-270892-ycc3csyh authors: rollinger, judith m.; schmidtke, michaela title: the human rhinovirus: human‐pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery date: 2010-12-13 journal: med res rev doi: 10.1002/med.20176 sha: doc_id: 270892 cord_uid: ycc3csyh as the major etiological agent of the common cold, human rhinoviruses (hrv) cause millions of lost working and school days annually. moreover, clinical studies proved an association between harmless upper respiratory tract infections and more severe diseases e.g. sinusitis, asthma, and chronic obstructive pulmonary disease. both the medicinal and socio‐economic impact of hrv infections and the lack of antiviral drugs substantiate the need for intensive antiviral research. a common structural feature of the approximately 100 hrv serotypes is the icosahedrally shaped capsid formed by 60 identical copies of viral capsid proteins vp1‐4. the capsid protects the single‐stranded, positive sense rna genome of about 7,400 bases in length. both structural as well as nonstructural proteins produced during the viral life cycle have been identified as potential targets for blocking viral replication at the step of attachment, entry, uncoating, rna and protein synthesis by synthetic or natural compounds. moreover, interferon and phytoceuticals were shown to protect host cells. most of the known inhibitors of hrv replication were discovered as a result of empirical or semi‐empirical screening in cell culture. structure–activity relationship studies are used for hit optimization and lead structure discovery. the increasing structural insight and molecular understanding of viral proteins on the one hand and the advent of innovative computer‐assisted technologies on the other hand have facilitated a rationalized access for the discovery of small chemical entities with antirhinoviral (anti‐hrv) activity. this review will (i) summarize existing structural knowledge about hrv, (ii) focus on mechanisms of anti‐hrv agents from synthetic and natural origin, and (iii) demonstrate strategies for efficient lead structure discovery. © 2009 wiley periodicals, inc. med res rev, 31, no. 1, 42–92, 2010 human rhinoviruses (hrv) are the major cause of upper respiratory tract symptoms, the socalled common colds in humans. their name reflects the primary site of infection. because hrv are nonenveloped, icosahedral viruses of small size with a diameter of about 30 nm ( pico 5 small in latin) that consist of an rna genome, they were assigned to the family picornaviridae. currently, this virus family of the order picornavirales comprises the eight genera enterovirus, hepatovirus, cardiovirus, kobuvirus, teschovirus, erbovirus, aphthovirus, and parechovirus with 22 species and a multitude of serotypes. 1 because of high similarity in genome sequence and genome organization (fig. 1) , the former genera rhinovirus and enterovirus have been combined recently, keeping the existing name enterovirus (www.picornastudygroup. com/taxa/species/species.htm). an overview on the current taxonomy of picornaviruses pathogenic for humans as well as on newly proposed species of hrv is given in table i . at present the genus enterovirus includes four approved human enterovirus (hev) species (hev-a, -b, -c, and -d) and two approved hrv species (hrv-a and -b) (www. picornastudygroup.com/taxa/species/-species.htm). since 2007, the global distribution of highly divergent hrv strains was reported. [2] [3] [4] [5] based on the results of sequence, genomic, and phylogenetic analyses, it was proposed that these strains represent a new hrv species, hrv-c. [2] [3] [4] 6 in 2009, a further proposal concerning a new potential hrv-d species was published after sequencing and analysis of all known hrv genomes. 7 the approved and newly proposed species of the genus enterovirus share z70% homology (average amino acid identity) in the precapsid protein p1 as well as in 2c and 3cd. 3, [7] [8] [9] [10] different antigenic properties provide the basis for a further division of species into serotypes (table i) . about 100 rhinovirus serotypes are currently known. according to the currently approved taxonomy, most of them (75 and hrv hanks) belong to hrv-a and 25 of them to hrv-b. 11, 12 the genome of all known hrv-a and -b serotypes as well as of several field isolates of hrv-a, -b, and -c has been sequenced completely. 3, 7, [13] [14] [15] [16] [17] [18] [19] [20] viruses classified as hrv-c could not be grown in cell culture until now. phylogenetic analyses have been performed with partial sequences, 12, 21 as well as with the whole genome. 3, 7 the most recent and comprehensive analysis of all known hrv genomes revealed that (i) hrv-a and hrv-c share a common ancestor, which is a sister group to hrv-b, (ii) hrv-c represents a third species, and (iii) a basal divergence within hrv-a of three distinct strains that led to the proposal of a fourth species hrv-d. hrv-a and -b most often induce a mild, usually self-limited upper respiratory illness in humans characterized by nasal stuffiness and discharge, sneezing, sore throat, and cough. the conventional term is common cold. the common cold is a heterogeneous group of diseases caused by numerous viruses that belong to several different families e.g. rhinoviruses, coronaviruses, enteroviruses, and adenoviruses. 22 but, hrv represent the most common etiological agent worldwide. a large number of distinct strains circulate each year. 23 moreover, in a family or even in a single specimen, multiple hrv serotypes were detected simultaneously. [24] [25] [26] by using rt-pcr and culture, it was shown that hrv induce 22-50% of upper respiratory tract infections in adults as well as children. [27] [28] [29] [30] [31] higher incidence has been described from september to november, [32] [33] [34] and from april to may. 33, 34 in some years and perhaps some geographical areas, spring was a more important time for rhinovirus transmission. 33, 34 although overall rates of respiratory illness are lower in summer, rhinoviruses are the most frequently isolated at this time of year. 34 the incidence is inversely proportional to age. 25, 35 by age 2 years, 91% of the children have antibodies against rhinoviruses. 36 in addition to common cold, hrv are also involved in acute otitis media in children. 36, 37 moreover, data supporting a causative association with more severe lower respiratory tract infections of infants, elderly persons, and immunocompromised patients have been accumulated. [38] [39] [40] [41] [42] [43] [44] [45] studies of childhood and adult asthma have shown that hrv infections can also trigger exacerbations in patients with asthma, [46] [47] [48] [49] chronic obstructive pulmonary disease, [50] [51] [52] [53] [54] [55] and cystic fibrosis. [56] [57] [58] the recently discovered novel rhinovirus genotype hrv-c was associated with community outbreaks of influenza-like, acute upper respiratory infections and severe low respiratory tract infections of infants e.g. febrile wheeze, bronchiolytis, and asthma exacerbations, which peaked in fall and winter. [2] [3] [4] [5] [6] 26, 59, 60 in addition, the presence of hrv-c in the middle ear in patients with acute otitis media was demonstrated. 37 hrv spread occurs by means of virus-contaminated respiratory secretions that contain a high virus concentration. [61] [62] [63] besides direct hand-to-hand transmission, small-and largeparticle aerosol transmission of rhinoviruses has been shown. 61, 64, 65 children are important ''vectors'' for hrv transmission to family members. 25 moreover, studies with natural hrv-infected adults provided evidence that daily activities of infected people can lead to contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets, and virus transfer to fingers of healthy individuals for infection. 63, 66 because viral contamination of the hands plays an important role in transmission of hrv from person-to-person, interruption of this step of virus transmission presents a potential target for intervention. this was experimentally proved by treatment of hands by iodine 67, 68 or salicylic and pyroglutamic acid. 69 observations from experimentally induced infections in normal adult volunteers helped to understand the pathogenesis of hrv infections. [70] [71] [72] [73] [74] [75] the 50% human infectious dose of rhinovirus is low and the infection rate between 70 and 80%. after the deposition of hrv on nasal or conjunctival mucosa, viruses are transported to the posterior nasopharynx by mucociliary action of epithelial cells. specific receptors on epithelial cells in the adenoid area are used for binding and entry. already 8-10 hr after intranasal inoculation, infectious virus can be detected. 76 virus shedding peaks on the second day after infection and decreases rapidly thereafter. 75 but, small amounts of viruses were discovered in nasal secretions for up to 3 weeks after infection. virus and/or viral rna were demonstrated in the upper 70 as well as lower respiratory tract. 45, 72, 77, 78 using in situ hybridization, arruda et al. (1995) detected viral rna in a low number of ciliated cells in nasal biopsies. in the nasopharynx, a small portion of virus-positive ciliated as well as nonciliated cells was positive for viral rna. in 2000 papadopoulos et al. provided evidence that hrv may also lytically infect human bronchial epithelial cells in cell culture as well as in experimentally infected volunteers and induce the production of interleukin-6, -8, and -16. in agreement with these results, hrv rna was detected in 24-45% of children and 10-18% of adults with pneumonia. [79] [80] [81] [82] taken together, the results of natural cold studies as well as of experimental infection in human volunteers clearly demonstrate that hrv are able to replicate in the upper as well as in the lower airways. hrv infection triggers vasodilation and increased vascular permeability in the nasal mucosa, leading to nasal obstruction and rhinorrhoea. the mechanism is still incompletely understood because no histopathological changes were observed in nasal biopsy specimens from infected persons. 75 this led to the suggestion that clinical symptoms are primarily caused by the inflammatory response of the host to the virus infection and not by the cytopathic effect (cpe) of hrv. results of immunological investigations suggest a modest correlation between the concentrations of il-6 and il-8 in nasal secretions and the severity of symptoms in upper and lower hrv-induced respiratory tract disease. 78, 83 on day 2-4 after virus challenge, il-6 and il-8 concentrations were significantly greater in nasal secretions from experimentally infected symptomatic subjects than in those from infected asymptomatic or sham-challenged subjects. il-8 has been proposed as a mediator of neutrophile infiltrations that are observed during symptomatic infections. in experimental rhinovirus infection the onset of symptoms e.g. nasal stuffiness and discharge, sneezing, and cough was observed 10-12 hr after intranasal inoculation of the virus. 76 in contrast to rhinovirus infections in adults, fever is found in 15% of children with upper respiratory tract infections. 84 other symptoms in children and adults may be hoarseness, headache, malaise, and lethargy. sometimes viral infection is accompanied by bacterial complication, leading for instance to acute otitis media in about 20% of infected children, sinusitis, and pneumonia. [79] [80] [81] 85, 86 experimental infection was also used to study the causation between rhinovirus infection and asthma as well as copd exacerbations. 78, [87] [88] [89] [90] [91] [92] [93] it was shown that hrv infection enhances airway reactivity and predisposes allergic patients to develop late asthmatic reactions. 88, 91 rhinoviral colds were associated with an increase in histamine responsiveness that was accompanied by a bronchial mucosal lymphocytic and eosinophilic infiltrate. 89 in a recent study, an increased hrv-induced clinical illness severity in asthmatic compared with normal subjects was demonstrated. 93 strong relationships were shown between virus load, lower airway virus-induced inflammation, and asthma exacerbation severity. the results of this study also indicated that augmented th2 or impaired th1 or il-10 immunity are likely important mechanisms. mallia et al. provided evidence that low dose experimental rhinovirus infection in patients with copd induces symptoms and lung function changes typical of an acute exacerbation of copd. 92 viral replication and increased pro-inflammatory cytokine response were associated with symptomatic colds, increases in lower respiratory tract symptoms and reductions in forced expiratory volume in 1s or peak expiratory flow rate. the epidemiological data and pathology of hrv infections explain their high medical and socio-economic impact. millions of children and adults are taken ill with common cold every year, need medical consultations, are unable to attend school and go to work. 94, 95 direct costs include hospitalization, medical fees, and symptomatic treatment. moreover, exacerbations are the major cause of asthma and copd morbidity, mortality, and health care costs associated with these diseases. 92, 93 to date, specific drugs that prevent or reduce rhinovirus infection are not available. common cold can be treated only symptomatically with analgesics, decongestants, antihistamines, or antitussives and antibiotics are often wrongly prescribed. 96, 97 because of the large number of circulating hrv serotypes, treatment with specific antiviral drugs is considered to be more striking than vaccination. therefore, the search for new highly active synthetic and/or natural anti-hrv compounds is absolutely essential and represents an important area of antiviral research. such an anti-hrv drug would have to be (i) with broad spectrum activity because of the high number of hrv serotypes, (ii) administered very early in infection to demonstrate a good antiviral effect because of the fast infection kinetics, (iii) very safe because of the broad application by millions of people, and (iv) directed against a highly conserved target with low risk of resistance development. due to the very high error rates and the lack of proofreading ability in rna polymerases of picornaviruses, 98 naturally drug-resistant variants may exist in virus populations or resistant viruses can emerge under treatment. as with hiv, another highly variable rna virus, the risk of resistance development and/or selection of resistant virus variants could be minimized by applying combination of drugs directed against different targets. because clinical symptoms are suggested to be primarily caused by the inflammatory response of the host to the virus infection mediated by specific cytokines, a further advantage of drug combinations could be an additional immune-suppressive activity. the knowledge of structural components, nonstructural proteins that are necessary for viral multiplication, and stages of the viral life cycle is an essential precondition for the development of measures to prevent and treat hrv infection. the structure of hrv particles is well known. infectious virions consist of an icosahedral protein shell (capsid) that surrounds and protects the genome, a single positive-stranded rna molecule of approximately 7,400 nucleotides. the organization of the enterovirus genome is shown in figure 1 . the viral genomic rna is infectious and encodes a single, long, open reading frame flanked by untranslated regions (utr) at the 5 0 and 3 0 end. a small viral protein (vpg) is covalently linked to the 5 0 end. the 3 0 end is polyadenylated like cellular messenger rnas. structural components within these utrs e.g. the cloverleaf and the internal ribosome entry site (ires) of the 5 0 utr play an important role in rna replication as well as protein synthesis. 98 the nucleotide sequence of some regions within these structures is highly conserved among enteroviruses. their blockade could significantly inhibit viral replication. the molecular structure of hrv-1a, hrv-2, hrv-3, hrv-14, and hrv-16 was determined by x-ray crystallography. [99] [100] [101] [102] [103] [104] [105] the results show that the viral capsid is composed of 60 protomers of each of the three outer structural proteins vp1, vp2, and vp3 and of vp4 in the interior. a star-shaped plateau at the fivefold axis of symmetry, surrounded by a deep depression (canyon) and another smaller depression at the threefold axis were detected. moreover, a hydrophobic pocket was found beneath the canyon floor. with exception of hrv-14 and hrv-3, this pocket is occupied by a fatty acid, the so-called pocket factor. these host cell molecules have been suggested to play an important role in the viral life cycle by providing transient stability to the capsid during its movement from one host cell to another. 102 the outer surface of virions contains neutralization antigenic as well as host cell binding sites. the latter allow the virus to attach to molecules of the host cell membrane (adsorption), the receptors, and to start their life cycle. [106] [107] [108] based on their receptor use, two groups of hrv can be distinguished. the majority of hrv serotypes, the major group uses intercellular adhesion molecule-1 (icam-1) as their receptor. 109 the 12 viruses belonging to the minor group attach to low density lipoprotein (ldl) receptor, very-ldl (vldl) receptor, and ldlr-related protein on the cells whereat multiple receptors are involved. [110] [111] [112] hrv of the major group apply the canyon as attachment site for binding to icam-1. 113 in contrast, ldl receptors of minor group viruses bind near the tip of the five-fold vertex. 114 hrv-87 has been shown to utilize a sialylated glycoprotein as a cellular receptor. 115 furthermore, a hrv-89 variant as well as wild-type hrv-54 can use heparan sulphate proteoglycans for cell attachment in addition to icam-1. 116, 117 the interaction of rhinoviruses with their receptors leads to virus concentration on the cell surface. it induces the release of the pocket factor and conformational changes in the capsid and mediates viral entry via endocytosis. [118] [119] [120] [121] whereas icam-1 binding directly causes uncoating, 122, 123 release of the rna genome from the capsid of ldl-bound minor group rhinoviruses is triggered by acidification of the endosomal, ph-dependent pathway. [124] [125] [126] this detailed knowledge of the capsid structure and function as well as of the virus-receptor interaction offers a good possibility to develop antiviral drugs that interfere with the first steps of the viral life cycle, adsorption as well as uncoating. after uncoating, rhinovirus proteins are synthesized by the translation of a single, open reading frame using cellular ribosomes. the resulting polyprotein of approximately 250 kd is cleaved by viral proteases 2a pro and 3c pro into 11 final products (4 structural and 7 nonstrutural proteins) immediately after translation. 127, 128 at first, both proteinases release themselves from the polyprotein by selfcleavage. the primary cleavage of the viral polyprotein between p1 and p2 is mediated by 2a pro . thereafter, 3cd pro is released from the p3 precursor by autocatalytic cleavage. next, 3c pro and its precursor 3cd pro process proteins of the p1 (capsid proteins), p2, and p3 (nonstructural proteins) region. interestingly, 2a pro cleaves also the eif4gi/ii component of the translation initiation factor eif4f necessary for host cell protein synthesis, [129] [130] [131] and 3c pro and/or 3cd pro the rna polymerase transcription factors tfiid, tfiic, sl-1, and ubf. 98 therefore, effective inhibition of 2a pro and 3c pro would not only inhibit virus replication but could also prevent the shutoff of cellular protein and rna synthesis. moreover, the active site of proteinases is highly conserved among enterovirus serotypes. 132 this high conservation in conjunction with their important role in virus multiplication predestines these enzymes as targets for antiviral therapy. the viral rna polymerase 3d (3d pol ) represents another very important nonstructural protein of hrv. it forms a complex with both cellular and viral proteins, the rna replication complex. 98 this enzyme synthesizes viral minus-strand rna and uses it as template strand for the synthesis of genomic viral rna. vpg (3b) is the primer for negative-as well positive-strand rna synthesis. negative-strand, but not positive-strand rna synthesis, is stimulated by 2a pro . further viral accessory proteins include 2b, 2c, 2bc, and 3ab. besides 3d pol these proteins can play an important role in inhibition of viral rna synthesis by antiviral compounds. in summary, the knowledge of the structure of the viral capsid, proteases, and polymerase and their important function in the viral life cycle predestine these proteins as potential anti-hrv targets. hrv grow in several human and some primate cells expressing the minor group ldl receptors and/or the major group receptor icam-1. human cells susceptible to hrv infections include embryonic kidney, amnion, diploid fibroblasts from embryonic lung, tonsil, liver, intestine, and skin, adult fibroblast lines from aorta and gingival and the kb, hep-2, and hela continuous cell lines. 133 but, the susceptibility of hela cells and human fibroblasts to virus infection may vary. 32 hrv multiplication also occurs in primary human airway fibroblasts 134 and differentiated bronchial epithelium. 77, 78, 135 the proportion of infectible epithelial cells was shown to be between 3 and 10%. 77, 135 but, enhanced levels of viral production were detected in poorly differentiated in comparison to differentiated epithelial cells. 136 the degree of viral infection correlated with il-6 and il-8 induction in these cells virus growth causes a typical cpe characterized by ballooning, refractiveness, granularity, and shrinkage of infected cells. the hrv-induced cpe, infectious virus titers, viral protein expression, and rna synthesis can be chosen as parameters to evaluate the anti-hrv activity of compounds in cell-culture based assays. there are several methods for antiviral screening against hrv. the plaque reduction assay has been traditionally performed and accepted as the ''gold standard'' in antiviral testing. [137] [138] [139] however, this test is laborious, time consuming, and the evaluation is subjective. therefore, it is not suited for the routine antiviral testing. it was more and more replaced by methods based on quantification of protection from virus-induced cpe after drug treatment. so, the cpe in sample-treated and untreated cells has been compared by light microscopy. 137, 140, 141 but this evaluation is also subjective. another more objective approach is the spectrophotometric quantification of cpe results in neutral red or crystal violet uptake assays, 12,141-144 and the tetrazolium dye reduction method. 132, 145 it allows an excellent and rapid antiviral screening of large numbers of compounds using small amounts of extracts, natural, or synthetic compounds. active samples can be scheduled for additional testing using other assays e.g. virus yield or plaque reduction assays, and for studies on the mechanism of action. the activity of potential antiviral drugs has to be approved in vivo. because of the high degree of species-specific variations in icam-1 preventing infection by major group hrv, practical animal models have been absent for a long time. chimpanzees were infected with several hrv serotypes but without developing clinical signs. 133 hrv do not induce infection in rabbits, guinea pigs, and weanling mice injected with hrv by different routes. one minor group hrv, serotype 2, was adapted to grow in mouse fibroblasts and used in a mouse model of rhinovirus infection in which growth could be demonstrated. 146 based on the fact that the ldl receptor family is highly conserved between human and mouse, newcomb et al. examined whether hrv-1b, another minor group virus, may infect mouse airways in 6-to 8-week-old female c57bl/6 mice. 147 the authors demonstrated that this hrv serotype replicates and induces airway inflammation in vivo. these results strongly correspond to those of bartlett et al. who established three novel mouse models of rhinovirus infection in balb7c mice. 148 in the first model, 6-week-old balb/c mice were infected with hrv-1b. in the second model, transgenic balb/c mice, expressing a mouse-human icam-1 chimera, were inoculated with the major group hrv-16. rhinovirus-induced exacerbation of allergic airway inflammation is mimicked in the third model. due to the lack of a small-animal model for hrv infection until 2008, the experimental human challenge model has to be used to approve effects of potential antiviral drugs under controlled conditions in preclinical studies. volunteers were experimentally inoculated with various serotypes e.g. hrv-4, hrv-9, hrv23, hrv-29, and hrv-39 to examine the efficacy of potential antiviral drugs under standardized conditions. [149] [150] [151] [152] [153] [154] examples and results of these studies with capsid-binders, protease, and rna synthesis inhibitors, as well as interferons are described in the following sections. antiviral agents that inhibit virus attachment, capsid uncoating, protein and rna synthesis of picornaviruses are the best studied, 95, [155] [156] [157] and will be in the focus of this section. inhibition of virus attachment and/or uncoating interrupts the viral life cycle at its beginning and prevents hrv infection. options to prevent these early steps of the viral life cycle include (i) virus neutralization by hrv-specific antibodies, (ii) receptor blockade by antibodies directed against the cellular receptors icam-1 or ldl, (iii) by soluble receptor molecules, or (iv) by compounds interacting with the viral capsid. because of the high number of serotypes circulating often in parallel, application of hrv-specific antibodies is thought to be no promising approach for prevention or therapy of rhinovirus infection. in contrast, antibodies directed against the cellular receptor or soluble receptor molecules of major or minor group hrv could inhibit 90 and 10% of hrv serotypes, respectively. therefore, the strategy to prevent virus-receptor interaction by receptor antibodies or soluble receptor molecules has been extensively evaluated in vitro as well as in vivo. the antiviral activity of icam-and ldl-specific antibodies was confirmed in cell culture. 158, 159 furthermore, the prophylactic effectiveness and safety of intranasally administered rhinovirus murine icam-1 antibody was assessed in two double-blind, placebo-controlled, randomized studies of volunteers experimentally inoculated with hrv-39. 160 in the result, no toxicity related to antibody application was recognized. the higher dosage of 1 mg/subject of rhinovirus murine receptor antibody did not reduce overall infection or illness rates, but was associated with a 1-2 day delay in the onset of virus shedding and cold symptoms. viral titers and nasal symptoms were significantly reduced on the second day after challenge. in summary, the monoclonal antibody to the cellular icam-1 was demonstrated to be not effective enough. a new strategy was the creation of multivalent fab fusion proteins against icam-1. a new molecule, named cfy196 demonstrated a better avidity and in vitro potency against hrv over conventional mabs. 161 cfy196 is under development as nasal spray with the name of coldsol. antagonism of virus-receptor interaction was considered as another promising way to prevent hrv attachment to host cells. soluble forms of fully or truncated icam-1, [162] [163] [164] and ldl or vldl-receptor concatemers [165] [166] [167] exhibited antiviral activity against major and minor group hrv, respectively, in cell culture. soluble forms of icam-1 compete with receptor binding sites on the virus capsid, hinder an early infection event such as entry or uncoating, or directly inactivate hrv due to the formation of empty capsids. 163, [168] [169] [170] a soluble ldl receptor fragment neutralized viral infectivity by aggregation. 171 concatemers of the third ligand binding module of the vldl-receptor did not lead to viral aggregation but blocked the receptor binding sites and possibly inhibited viral uncoating by cross-linking the viral capsid subunits via multi-module binding. 166 the antiviral activity of a truncated, soluble form of icam-1 was proved in hrv-16 infected chimpanzee. 172 in randomized, double-blind, placebo-controlled trials, the safety and efficacy of intranasal administration of tremacamra, a soluble icam-1 in experimental hrv-39-induced colds in humans, was shown. 173 no further development was reported for these agents. 95 a further option to prevent virus attachment was described for low-molecular-weight compounds, the so-called capsid-binding agents, which enter the small hydrophobic pocket within viral capsid protein 1 beneath the icam-binding canyon of hrv. 174, 175 zhang et al. showed that drug may integrate into mature viruses by diffusion as well as into progeny viruses during assembly. 176 when hrv-14 and hrv-16 were grown in the presence of pleconaril, a higher occupancy occurred than when the drug was introduced into the alreadyassembled viruses. in doing so, capsid-binders induce conformational changes of the canyon of hrv-3 and hrv-14, hinder virus-receptor interactions, and prevent attachment to host cells. 105, 175, [177] [178] [179] in addition, uncoating of both hrv serotypes was shown to be inhibited as a result of a potential loss of flexibility of the viral capsid after drug binding. in contrast to hrv-3 and hrv-14, capsid-binding compounds did not prevent attachment of hrv-1a. results from x-ray studies showed that drug binding into the hydrophobic pocket of hrv-1a replaces the pocket factor but induces only very small conformational changes. 180 therefore, kim et al. suggested that the observed conformational changes are too small to affect receptor binding. but, capsid-binding compounds prevented attachment of hrv-16 possessing a pocket factor like hrv-1a without distinct deformation of the pocket. 102, 176, 181 further results from comparative antiviral studies with different capsid-binding compounds and hrv, representative for the major and minor group, did not reveal a correlation between inhibition of adsorption and receptor grouping or antiviral grouping. 182 the reasons for the difference in the mode of action of capsid-binding compounds related to attachment inhibition are not fully understood until now. taken together, inhibition of rna uncoating was found for all investigated serotypes after drug binding independent of receptor grouping whereas prevention of virus attachment was found to be an additional mode of action for individual viruses and/or drugs. till now, various potent compounds belonging to diverse chemical classes have been described as uncoating inhibitors. just to give an impression of diversity, the structures of disoxaril and pleconaril, 12,183-185 pirodavir and the oxime ether, 14, 141, 142, 186, 187 the isoxazole derivate compound, 19, 143, 188 the imidazole derivative sch 38057, [189] [190] [191] the chalcone ro 09-0881, 192, 193 4 0 ,6 dichloroflavan and isoflavan, 137, 194 the pyridine derivative mdl 20, 957, 195, 196 and the phenoxybenzene mdl-860 197, 198 that exhibit a potent anti-hrv activity (table ii) are shown as examples in figure 2 . they inhibit most of hrv serotypes and a couple of them also affect enteroviruses, however, with varying susceptibility. based on variability of susceptibility to capsid-binders of different length, hrv serotypes were classified into two different groups, a and b. 140 several of the given examples of compounds were also clinically tested. studying the development of clinically effective capsid-binders, the long road to the discovery of a clinically effective anti-hrv drug becomes apparent. one well-described example represents the discovery and optimization of capsid-binders from sterling winthrop pharmaceutical group, the so-called win compounds. 174 first inhibitors originated from juvenile hormone mimetics that demonstrated some activity against hrv-1a. determination of the x-ray structure of hrv-14 helped to understand the compounds' binding sites at the virus capsid. 103 results from subsequent x-ray studies of hrv-win compound complexes revealed the location and nature of binding sites and provided information concerning interactions within these sites. 175, 179, 180 this knowledge was used for optimization and design of new compounds. 199 optimized win compounds, for example disoxaril and pleconaril ( fig. 2) , consist of a methylisoxazol ring, a substituted phenoxy group, and a five-membered heteroatom ring and inhibit a broad spectrum of rhinoviruses and enteroviruses (table ii) . 12, [183] [184] [185] in 1985, the first broad-spectrum win compound disoxaril (win 51711) was tested in clinical trials. 139 the development of crystallurea in human volunteers treated with high doses as well as its low bioavailability (15%) prohibited subsequent development. thereafter, results from sar and qsar analysis were used to further enhance the potency and spectrum of activity. in 1992, another compound, win 54954, was clinically tested. it was not effective in humans infected with hrv-23 and hrv-29. 153 moreover, it was rapidly metabolized and induced a reversible hepatitis. consequently, the further clinical development was stopped. the better understanding of pharmacokinetic properties of capsid binders and synthetic chemistry efforts led to the discovery of pleconaril, an orally bioavailable, welltolerated capsid-binder that inhibits most rhinovirus as well as various enterovirus serotypes. 12, [183] [184] [185] in 2000, schiff et al. published the efficacy of pleconaril in an experimentally induced coxsackievirus a infection in humans. 200 in phase ii placebo-controlled, natural cold trials, the drug produced a moderate reduction of 1-1.5 day in the medium time to elevation of illness compared with placebo. 201 these results were confirmed in two subsequent pivotal studies. 202 besides the moderate clinical efficacy, these studies revealed that 13% of baseline isolates were not susceptible to pleconaril and 11% developed reduced susceptibility (defined as 10-fold increase in baseline value). in a subsequent study the relationship of pleconaril susceptibility and clinical outcomes in the treatment of common cold caused by rhinoviruses was demonstrated. 203 based on drug interaction, marginal treatment effect, and possibility of transmission of resistant viruses, the fda did not approve the applied oral administration of pleconaril for the treatment of common cold. the molecular mechanism of drug interaction of orally given pleconaril was shown to be based on hepatic cytochrome p450 3a activation. 204 to reduce adverse effects, shering-plough under license of viropharma completed a phase ii clinical trial with an intranasal formulation of pleconaril for the potential treatment of common cold in high-risk populations in 2007. the results were not published until now. pyridazine analogues developed by janssen research foundation represent another example for the long road to discovery of a clinically effective capsid-binder. 186 in 1992, the broad-spectrum activity of pirodavir ( fig. 2 ; table ii ) against rhinoviruses was published. 187 in the same year the results of a randomized, double-blind, placebo-controlled trail to assess the therapeutic efficacy of intranasal pirodavir in natural common colds were described. 205 possibly as a result of poor water solubility and rapid hydrolysis of pirodavir, no clinical benefit was found. the problem of ester hydrolysis was resolved by the development of oxime ether analogues of pirodavir by biota. an example is shown in figure 2 . like pirodavir these new analogues are potent inhibitors of rhinoviruses. 142 an advantage over pirodavir is their improved bioavailability. bta-798, an antiviral analogue with long half-life and good oral bioavailability, was scheduled to a phase ii clinical trial in 2008. the results have not yet been published. in summary, despite extensive research leading to the discovery of potent anti-hrv capsid-binders, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. nearly, the same conclusion has to be drawn for protease inhibitors. because of their pivotal role for viral polyprotein processing and the high conservation of critical amino acids, 132 2a pro as well as 3c pro represent potential anti-hrv targets. results from cell culture-based assays provided evidence that inhibition of hrv replication is in principle possible. for example, processing of the hrv-2 polyprotein was prevented by pyrrolidine dithiocarbamate treatment in virus-infected hela cells. 206 in contrast to other enteroviruses, [207] [208] [209] [210] pretreatment of cell monolayers with different nitric oxide donors leading to s-nitrosylation of 2a pro and 3c pro had neither an effect on virus replication nor on hrv-induced il-8 elaboration. 211 the proteolytic activity of 2a pro of hrv-14 was specifically inhibited by two elastase-specific inhibitors, 212 and an antiviral peptide representing a derivative of the caspase inhibitor zvad.fmk. 213, 214 homophthalimides, e.g. ly353349 ( fig. 3 ; table ii) , were described as inhibitors of 2a pro as well as 3c pro . 215 in contrast to protease 3c, no structure-activity relationship studies have been reported for hrv 2a protease. moreover, protease 2a accomplishes only one cleavage in hrv polyprotein, while protease 3c performs all other cleavages. after elucidation of the crystal structure of 3c pro , 216 computer modeling of structural features of protease inhibitors became possible. 217 furthermore, structure-based design was used to develop mechanism-based inhibitors of the 3c protease with potent antiviral activity against multiple hrv serotypes. 218, 219 highly active compounds incorporate various michael acceptor moieties, irreversibly bind to 3c pro , and exhibit anti-hrv-14 activity in hela cells. 220, 221 structure-activity studies were performed to optimize protease inhibitors. 220, [222] [223] [224] [225] [226] these efforts resulted in the identification of a highly active anti-hrv compound, ag7088 (rupintrivir; fig. 3 ; table ii ) that entered clinical trials. in cell culture, ag7088 inhibited a broad spectrum of laboratory hrv as well as clinical isolates. 132, 183, 227 in a single-cycle, time-of-addition assay it demonstrated antiviral activity when added up to 6 hr after infection. 227 inhibition of hrv replication strongly correlated with reduction in the level of il-6 and il-8 release into cell supernatant, leading to the suggestion that this agent may not only block virus replication but also diminish symptoms. 228 the pharmacokinetics and safety of rupintrivir were proved in two double-blind, randomized, placebo-controlled studies. 229 intranasal rupintrivir, administered as single doses of 4 and 8 mg or every 3 hr, six times per day, for 7 days, was safe and well tolerated. three double blind, placebo-controlled clinical trials were conducted to assess rupintrivir nasal spray (2% solution) for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. 230 rupintrivir prophylaxis reduced the proportion of subjects with positive viral culture by 26% and viral titers but did not decrease the frequency of colds. drug treatment led to the reduction of the mean total daily symptom score by 33%. subjects receiving rupintrivir also demonstrated significantly lower viral titers and rna levels than placebo-treated subjects on days 2, 3, and 5 and on days 2 and 3, respectively. there was no influence on the proportion of subjects with positive viral culture and the frequency of colds. clinical development was terminated because rupintrivir did not act in a subsequent natural infection study in patients. 231 in parallel research efforts, an orally bioavailable inhibitor of table ii) , was discovered. 225, 231 like rupintrivir, this compound is an irreversible inhibitor incorporating a michael acceptor moiety that forms a covalent bond with the 3c protease active site cysteine. it demonstrated an antiviral activity against all hrv and related picornaviruses tested. 231 in a phase 1 clinical study, compound 1 was shown to be safe and well tolerated. according to a publication of patick, no further clinical development was planned for this compound. 95 the blocking of viral rna synthesis during replication represents another site for chemotherapeutic interdiction. it was shown that, rhinoviral rna can be targeted in a sequence-specific manner by deoxyribozymes, 232 morpholino oligomers, 233 and small interfering ribonucleic acids. 234 the efficacy of the latter two approaches was confirmed in cell culture. in addition, 2-furylmercury chloride ( fig. 4 ; table ii ), 235 table iii) , 236 and pyrrolidine dithiocarbamate (fig. 4 ) interfered with rhinoviral rna synthesis and inhibited hrv replication in cell culture-based assays. 206, 237 the nucleoside analog ribavirin ( fig. 4 ; table ii ) that inhibits a broad spectrum of rna as well as dna viruses acts also against hrv-2 in hela cells. 238, 239 the cellular inosine monophosphate dehydrogenase that controls de novo synthesis of purine nucleosides represents the principal target in the mode of action of ribavirin. 240 moreover, when ribavirin is incorporated into picornavirus rna, it pairs equally well with either uracil or cytosine inducing mutations that can be lethal to rna viruses. 241 further identified mechanisms of action for ribavirin include inhibition of genomic rna capping, enhancement of host t-cellmediated immunity against viral infections through helping to switch the host t-cell phenotype from type 2 to type 1. 242 another compound with potent anti-hrv activity in vitro is enviroxime ( fig. 4 ; table ii ), a benzimidazole derivative. 243, 244 it inhibits viral plus strand rna synthesis. 245 in particular the 3a protein, which is involved in the initiation of plus strand rna synthesis, was implicated as likely target of drug activity. 246, 247 however, results from another study suggest that enviroxime targets a complex of proteins and/or cellular factors and that the exact mechanism remains to be studied. 248 although there was a statistically significant reduction in clinical score in a prophylactic study with hrv-9-infected volunteers, 152 enviroxime failed in experimentally induced hrv-4 and hrv-39 infection, 149, 151 and in clinical studies, 248-250 because of poor bioavailability and side effects. in an attempt to overcome the marked hydrophobicity, water insolubility, and toxicity, wyde et al. incorporated enviroxime into liposomes and then tested the anti-hrv activity and toxicity of the liposome-incorporated enviroxime in cell culture. 251 the liposome preparation of enviroxime inhibited hrv-1a and hrv13 as effective as the parent compound and was 10-to z50-fold less toxic. in contrast to free enviroxime, the liposome preparation was readily and successfully delivered by small-particle aerosol to the upper and lower respiratory tract of mice. in another attempt to overcome the disadvantages of enviroxime, several benzimidazole as well as nonbenzimidazole analogs were synthesized and studied. [252] [253] [254] [255] even though some compounds were better bioavailable and could be administered orally, 254 none of these compounds was tested in clinical studies. besides virus-specific targets, cellular inhibitors like interferons may represent a therapeutical approach. among other activities, interferons exhibit antiviral activity. the advantages of interferon application include the broad spectrum of activity and low risk of resistance development. human leukocyte and fibroblast as well as recombinant human a interferons prevent the hrv-induced cpe in cell culture whereas a variation in sensitivity was observed. [256] [257] [258] intranasally applied recombinant interferon a and interferon b have been shown to be effective in humans when provided prophylactically both in experimental and natural rhinovirus colds. [259] [260] [261] [262] [263] [264] significant reductions in illness frequency, mean symptom score, nasal secretion weights, and frequency of virus isolation were observed. in contrast, recombinant interferon g did not prevent hrv infection or illness and may enhance the symptoms. 265 little to no therapeutic effect was found in patients with common cold after interferon treatment. 150, 266 moreover, blood-tinged mucus and nasal bleeding were described as side effects. 263, 267 combining interferons with dichloroflavan, enviroxime, chalcone ro-09-0410 produced synergistic increases in antiviral activity in vitro against hrv-2 and hrv-9. 268 an attempt to demonstrate synergy between the anti-hrv effect of recombinant human rhuifn a and enviroxime in hrv-9 and hrv-14-infected volunteers failed. 269 according to the authors, the main reason for this failure may be the rapid removal of enviroxime from the nose when given intranasally. a. impact of natural products nature provides an astonishing pool of secondary metabolites biosynthesized from living organisms such as plants, fungi, protozoan, insects, and other animal sources. in contrast to synthetic compounds, natural products are characterized by an overwhelming chemical diversity. previously, the chemical diversity space between these two groups was evaluated with respect to drug substances by feher and schmidt. 270 it is shown that combinatorial compounds densely populate a small area, whereas natural products cover a wider range quite similar to the chemical space occupied by drug substances. the authors accordingly suggest that combinatorial libraries that mimic the distribution properties of natural products might be more biologically relevant. one may assume that secondary metabolites evolved as reaction to their target receptors related to defence, protection, attraction, and signalling. these adaptation processes have enriched not only the metabolites' structural diversity but have also optimized drug-like metabolic traits likely to have favorable pharmacokinetic properties. 271, 272 it is this evolutionary concept that gives the pool of natural products the greatest source of scaffold diversity with molecules of biological relevance. newman and cragg analyzed the number of drugs approved between 1981 and 2006 and circumstantiated that especially the anti-infective area is strongly dependent on natural products and structures derived from natural scaffolds. 273 the anti-infectives including the antiviral vaccines are with 22.8% or 230 launched drugs by far the major category with only about 30% being synthetic in origin. from 1981 to 2006, 78 vaccines and antiviral drugs have been approved. excluding the high number of vaccines (25) and biologicals (12) , most of the 41 small antiviral molecules are based on nucleoside structures or on peptidomimetics; only 16.7% are classified as totally synthetic drugs. however, till now, neither a synthetic nor a naturally derived anti-hrv drug substance has been approved for the treatment or prevention of hrv infections. intensive research and development efforts in the field of natural products revealed several inhibitors of viral attachment and entry, and inhibitors of viral protease from natural sources. the efficacy of natural products is not only reflected by statistics of launched drugs but also by empirical knowledge gained over centuries by successful application of naturalbased ethnomedicinal products such as plants, culinary herbs, and spices. phytochemical and pharmacological work performed with ethnomedicinal anti-hrvs mainly from plants revealed a high number of active metabolites from different chemical classes, e.g. coumarins, flavonoids, alkaloids, quinones, terpenoids, polyphenols, and polysaccharides. natural products include complex extracts and their chemical entities, which are biosynthesized by nature. for an unambiguous presentation of anti-hrv natural products it is of prior importance to first distinguish between a single chemical entity from nature, i.e. an isolated, purified natural compound, on one hand, and a natural preparation comprising hundreds to thousands of constituents, mainly secondary metabolites, on the other hand. if natural preparations are derived from plants, they might also be labelled as botanicals, phytoceuticals, or phytotherapeutic agents. these multicomponent preparations might show a varying profile of their constituents depending on the used species, origin, collection time, plant parts, extraction procedures, preparation methods, and manufacturing processes, just to mention a few important elements. these parameters affect the final product in terms of the qualitative and quantitative composition of chemical constituents, which may have an impact in biological activity. accordingly, studies performed with phytochemically not specified extracts or nonstandardized preparations often suffer from irreproducible and incomparable results a wide variety of natural preparations showed to be acting therapeutically in hrv and other viral infections with often complementary and overlapping antiviral mechanisms of action. [274] [275] [276] most of these remedies are described in ethnopharmacological sources or handed down for generations. they usually consist of simply prepared natural items whose chemical composition is complex. many of the contained secondary metabolites, possibly active principles, have never been examined chemically or biochemically using modern medical knowledge. they are however components of plant medicines, which have stood the test of time and as such may offer clues of great interest to medicinal chemists. a clear advantage of the application of these products is their absent or relatively low toxicity due to a usually long-term empirical trial. although the knowledge of the immuno-pathogenesis of rv-induced diseases remains limited, the host defense function of the airway epithelium plays an important role in the innate-immune response to hrv-infection. 277 host cells respond by the production of mediators with antiviral activity such as type i interferons and nitric oxide, and produce cytokines and chemokines that influence the subsequent induced innate-and specific-immune response. these processes are beneficial in facilitating clearance of virus from the respiratory tract, but also cause immuno-pathology. following hrv-infection, disease severity is dependent on direct, harmful effects of the virus as well as tissue damage as a result of the host antiviral immune response. accordingly, a number of agents with phenomenological effects against common cold have shown to exert their activity more in the field of regenerating tissue damage than on a direct anti-hrv effect. several herbal remedies consisting of a multitude of secondary metabolites from different chemical classes may attribute in a beneficial way for the treatment of common cold by reducing symptom severity and duration due to their immune-modulating, anti-oxidative, and anti-inflammatory properties. beside these commonly observed bioactivities of natural products, multicomponent mixtures like botanicals often show overlapping symptomatic effects as well as synergistic and/or additive properties. thus, it is a challenging endeavor to track down an observed phenomenological effect of a complex mixture on a molecular level. the following section explores the significance and current knowledge of selected botanicals for the prevention and therapy of common cold. questions about (1) clinical evidence of efficacy, (2) the constituents or at least the chemical classes that are involved in the observed anti-hrv effect, and (3) the involved pharmacological targets were covered as far as possible. echinacea preparations include expressed juice from aerial parts as well as extracts of roots or aerial parts, or both, from one or more species of the genus echinacea (e. angustifolia, e. purpurea, and e. pallida). they are the most recognized botanicals for prevention and treatment of common cold and flu, and account for the second top-selling herbal products in the us-market. 278 accordingly, echinacea has come under much scientific scrutiny. the high number of studies dealing with the effectiveness of echinacea for preventing and treating the common cold from clinical trials was recently reviewed by woelkart et al. 279 the authors summarized the findings of the meta-analyses regarding the 16 randomized controlled trials evaluated in the cochrane database, 280 the 14 randomized clinical trials analyzed by shah et al., 281 and the experimental hrv-infection studies pooled by schoop et al. 282 to sum up, the clinical data on echinacea so far are not fully consistent, mainly based on problems inherent in assessing the efficacy of echinacea preparations, such as lack of comparability of available preparations, study design, and outcome. nevertheless, the meta-analyses showed some evidence that preparations based on the aerial parts of echinacea purpurea might be effective for the early treatment of colds in adults. 280 echinacea showed to decrease the odds of developing the common cold by 58% and the duration of a cold by 1.4 days. 281 similarly, the evaluation of three induced rhinovirus prevention studies revealed the odds of experiencing a clinical cold were 55% higher with placebo than with echinacea. 282 stepping into a molecular level, several constituents found in echinacea species could potentially affect the symptoms of common cold. chemically identified substances include polysaccharides and glycoproteins, caffeic acid derivatives (especially cichoric acid and echinacoside), and lipophilic polyacetylenes and alkamides. pharmacological studies have shown that cichoric acid, alkamides, glycoproteins, and polysaccharides possess immunomodulatory activity. additionally, alkamides have been reported to exert not only antiinflammatory effects but also cannabinomimetic properties, which are suggested as molecular mode of action of echinacea alkamides as immunomodulatory agents. 283 raduner et al. showed that some echinacea alkamides exert cannabinoid type 2 receptor-dependent and independent immunomodulatory effects on cytokine expression. 284 different echinacea constituents were evaluated for their anti-oxidative effects measuring the inhibition of in vitro cu(ii)-catalyzed oxidation of human low-density lipoprotein. thereby, the major caffeic acid derivatives, cichoric acid and echinacoside, showed the highest anti-oxidative effects, which was even higher when combined with a natural mixture of alkamides. 285 sharma et al. used cytokine antibody arrays to investigate the changes in the pro-inflammatory cytokines and chemokines released from human bronchial epithelial cells exposed to hrv 14. 286 application of two chemically characterized echinacea extracts showed a reversion of the stimulated release of numerous pro-inflammatory cytokine-related molecules, e.g. for the cytokine il-6, and the chemokines il-8 and eotaxin. in a similar study, an echinacea extract rich in polysaccharides and another rich in alkamides and caffeic acid derivatives were as well able to neutralize the effects of hrv-infected epithelial cells. 287 using gene expression analysis both studies revealed the anti-hrv benefit of echinacea preparations being involved in multiple immune response signaling pathways. taken together, the numerous pharmacological findings from literature, the potential of echinacea preparations, and their constituents to combat or prevent common cold can be deduced to immune modulating, anti-inflammatory, and anti-oxidative properties that may also act in some combination of these event, rather than acting directly on hrv. garlic cloves have been used traditionally to treat a number of infectious diseases. however, only few confirmatory studies have been published regarding the traditional antiviral uses. the clinical effectiveness of garlic on the prevention of common cold was investigated by josling in 2005, who published a double-blind, placebo controlled study assessing 146 patients more than a 12-week treatment period with an allicin-containing garlic supplement. 288 common cold infections and symptoms were recorded in a daily diary. patients in the treatment group had significantly fewer colds than patients in the placebo group (24 vs. 65, po0.001) who also had a longer duration of symptoms (5.01 vs. 1.52 days, po0.001). as soon as the garlic is chewed, cut, or pressed, its main ingredient, the sulphur containing alliin, is broken down by the enzyme alliinase to the thiosulfinate allicin. by steam distillation allicin is transformed to diallyl disulfide and diallyl trisulfide that are responsible for the distinctive smell of garlic. further, allicin transformation compounds, such as e-and z-ajoene, are not found in fresh garlic, but in lipophilic extracts. by investigation of different garlic extracts and isolates against a number of different human pathological viruses, weber et al. could show that allicin was the most active virucidal component from fresh garlic and fresh extracts. 289 results of the direct pre-hrv-2-infection incubation assay let suggest allicin to bind to the viral protein capsid, leading to a subsequent inhibition of viral adsorption and penetration. although the garlic thiosulfinates are endowed with significant cytotoxicity, the antiviral effects were obtained in nontoxic concentrations. 289 beside the direct anti-hrv effect of fresh garlic extract and allicin, a number of human immune functions were found to be enhanced in vitro by aqueous garlic extract, its polar, and thiosulfinate fractions. 290 in north america, panax quinquefolium, the ginseng species indigenous to both canada and the united states, has been a popular herbal remedy to combat stress, and to modulate both natural and acquired immune responses. american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, have been found efficacious in the prevention of upper respiratory infections in immunocompetent healthy adults. 291, 292 in a randomized, double-blind, placebo controlled trial, 200 mg of a proprietary american ginseng root extract was given to 43 community-dwelling elderly adults (age 465 year) twice a day more than a ''cold and flu'' season period of 4 months. one month into the study, all participants received an influenza vaccination. during the first two months, no significant differences in duration and incidence were observed when compared to placebo. however, during the last two months significantly fewer subjects of the ginseng group reported acute respiratory syndromes than the placebo group (32 vs. 62%). additionally, the duration of respiratory symptoms was reduced by 55% in the ginseng group. 291 in a similarly arranged trial, 323 healthy adults (ages 18-65 years) with a history of at least two colds the previous year commenced a 4 month study at the beginning of a cold and flu season. 292 they received two 200 mg capsules daily of standardized american ginseng root extract or a placebo. outcomes measured were number of colds including symptom severity and total number of symptomatic days. a therapeutic effect was reported regarding symptom severity and fewer symptom days that were 31 and 34.5% lower in the ginseng group than in the placebo group. a phase ii randomized, controlled trial of 2 dosing schedules of american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, evaluated the safety, tolerability, and efficacy in a pediatric population already suffering from an upper respiratory tract infection. the results showed no serious adverse events and a good tolerability of both ginseng doses; however, frequency and severity of symptoms were not significantly different among each of the three treatment groups, i.e. standard dose, low dose, and placebo. 293 the most prominent constituents of the genus panax are the triterpene saponins ginsenosides. they are known to have numerous pharmacological activities such as anti-cancer, anti-diabetes, antiviral, and anti-atherosclerosis effects. some compounds of this chemical class showed to be responsible for the immunostimulant activity of ginseng. 294 on the other hand, the efficacy of a polysaccharide-rich extract of american ginseng was compared with an extract rich in ginsenosides on systemic and gut-associated immune function. the authors of this study investigated the lymphocytes isolated from spleen, mesenteric lymph nodes and peyer's patches, and immune cell proportions and cytokine production from sprague-dawley rats. they could show that the polysaccharide-rich ginseng extract modifies the rats' systemic immune responses and affects the gut-associated immunity in a manner distinct from that of the ginsenoside-containing extract of american ginseng. 295 a direct antiviral activity of ginseng constituents could be attested for the polysaccharides on rotavirus infection in ma104 cells. the triterpene saponins, however, did not exhibit any rotavirus infection-inhibitory activity in this study. 296 a moderate in vitro virucidal effect (id 50 62 mm) of the ginseng saponin chikusetsusaponin iii against herpes simplex virus type i was detected by fukushima et al. this compound exhibited an intracellular inhibitory activity, but could only marginally affect the viral proteins postinfection. 297 the ancient chinese formula bu-zhong-yi-qi-tang (japanese name hochu-ekki-to) is a traditional herbal medicine in china and japan that is composed of ten species of medicinal plants, namely astragali radix, atractylodis lanceae rhizoma, ginseng radix, angelicae radix, bupleuri radix, zizyphi fructus, aurantii nobilis pericarpium, glycyrrhizae radix, cimicifugae rhizoma, and zingiberis rhizoma. this formula is reported to have various immunomodulatory, [298] [299] [300] and anti-inflammatory activities. 301 yamaya et al. recently investigated the effects of hochu-ekki-to in cultures of human airway epithelial cells infected with hrv-14. 302 the output of virus, associated levels of viral rna, and the production of icam-1, cytokines and acidic endosomes in cells were measured. in airway epithelial cells hochu-ekki-to was able to decrease virus output and susceptibility to hrv infection by decreasing icam-1 and by blocking the entry of viral rna into the cytoplasm from the endosomes. glycyrrhizin, a major component of one herbal ingredient of hochu-ekki-to, i.e. glycyrrhiza glabra, was able to reduce supernatant virus titers dose-dependently, with a maximum effect between 0.12 and 0.6 mm. however, no clinical trials with representative numbers of subjects are published so far. the human rhinovirus k 63 5. umckaloabo (pelargonium sidoides) p. sidoides and p. reniforme form the origin of the popular drug umckaloabo. this herbal remedy from south africa has found entrance in western medicine mainly as aqueous ethanolic root extract from p. sidoides for the treatment of infections of the respiratory tract. the efficacy of umckaloabo compared with placebo has been evaluated in 103 adults suffering from common cold by lizogub et al. 303 the applied herbal preparation was well tolerated by the patients. the study demonstrated only a weak efficacy of umckaloabo compared to placebo after 5 days. after 10 days, however, the p. sidoides extract significantly reduced the severity of symptoms and shortened the duration of the common cold compared with placebo. just recently, timmer et al. selected randomized controlled trials examining the efficacy of p. sidoides preparations for the treatment of various acute respiratory infections and analyzed their efficacy and safety. 304 the authors concluded that umckaloabo may be effective in alleviating symptoms of acute rhino-sinusitis and the common cold in adults. it may be effective in relieving symptoms in acute bronchitis in adults and children, and sinusitis in adults. reliable data on the treatment for other acute respiratory infections however were not obtained. identification of the metabolites from umckaloabo revealed a high number of different chemical classes, such as phenolic and cinnamic acids, tannins, flavonoids, and coumarins. antibacterial activities of umckaloabo against different pathogens have been reported. phenols, coumarins, and tannins have been identified to contribute with moderate antibacterial activities, however, cannot explain the effect of the whole extract (reviewed by kolodziej 305, 306 ). additionally, p. sidoides extracts have been reported to significantly activate the nonspecific immune system by induction of tnf and no-release, and ifn-like activities. 305 these effects are assumed to contribute to the controversially discussed potential of p. sidoides extract for the treatment of upper respiratory tract infections. only one study reports a direct antiviral effect, i.e. a clear dose-dependent anti-herpes simplex virus acitivity for the aqueous root extract of p. sidoides. 307 further pharmacological studies are needed to elucidate potential direct anti-hrv properties of umckaloaba and its constituents. carrageenan, a mixture of different polysaccharides, which is mainly extracted from red seaweeds, has been extensively used in food, cosmetic and pharmaceutical industry as a thickener and gelling agent. it has previously shown an antiviral efficacy against several viruses. 308, 309 in a recently published study, lambda-, kappa, and iota-carrageenan were investigated for their anti-hrv inhibiting potential. at a concentration of 200 mg/ml iotacarragenan, a sulphated polysaccharide, was able to fully inhibit virus-induced cell death in hrv-2 infected hela cells. 310 based on their studies, grassauer et al. concluded that iotacarrageenan is effective against different hrv-serotypes on primary human epithelial cells. it is hypothesized by the authors that iota-carrageenan might create a hostile environment for hrv and thereby block viral entry and replication. because of its safe application and proved in vitro efficacy, iota-carrageenan deserves consideration as a candidate for clinical trials for prevention and therapy of hrv-induced common cold. the level of knowledge on the impact of the six botanicals on hrv-infection discussed above is different and heterogeneous. the best studied herbal remedy associated with common cold, i.e. echinacea, showcases the innate problem connected with multi-component mixtures: starting from the late nineties till june 2009 some 100 original articles have been published to this topic and tried to elucidate questions concerning efficacy, molecular mechanism, and bioactive ingredients of echinacea. although some evidence is provided for the effects of extracts, chemical classes as well as well-defined constituents on specific targets and pathways, the findings cannot be deduced to a common denominator. further, results from clinical trials often suffer from lack of comparability, because of using different study designs, outcome measures, and overall the application of different preparations. 279 a proper quality analysis and characterization of the preparation under investigation is mandatory and should follow the recommendations and guidelines for reporting clinical trials for herbal medicine. 311 as underlined before (sections 5.1. and 5.2.), the chemical complexity of a natural preparation might be beneficial in terms of synergistic, additive, and overlapping effects caused by the multitude of evolutionary trimmed metabolites, which may attribute with modulating multi-target effects. on the other side, exactly this fact is hardly compatible with the proper assignment of an activity to a defined chemical entity according to western medical practise. in contrast to a single compound (synthetic or naturally based), the chemistry of a botanical is not only complex but also varying. the analytical profile and in term the pharmacological profile of the investigated samples can differ substantially. accordingly, the quantitative and qualitative comparison of different studies resulting from botanicals is by far more complex than those performed with pure single compounds, and may also explain why so little emphasis from pharmaceutical industry has been put into the further development of even promising natural preparations. in general, the search for potent, selective, nontoxic compounds that might be developed further to a drug substance is a multidisciplinary, time-and cost-consuming process. therefore, strategies for a target-oriented discovery of lead-structures either from nature or synthesis are in high demand. some of them will be discussed in the following section, providing examples from anti-hrv research. as already mentioned, nature provides an extremely rich pool of bioactive natural products. it is however a challenging endeavor to find exactly those compounds that show an activity on the focused target. in natural product research hints from folk medicine are a valuable starting point to dig for lead structures of certain interest. the majority of active principles from higher plants has been discovered as a result of ethnopharmacologically directed pharmacognostic research. 312, 313 oral or written indications for a beneficial application of a natural material first need a critical evaluation as to the selection of the correct material, its medicinal preparation, the kind of application, and overall the pharmacological profile. a rational criticism of often anecdotal efficacy from traditional medicine is a mandatory attitude to avoid overinterpretation of handed-down information. 314 in case of pretended anti-hrv remedies, the reported biological efficacy obviously suffers from being restricted to respiratory diseases, which might be caused by a panel of bacterial or viral infections. thus, an approved remedy may affect the microbes, or show an immune modulating or even antiinflammatory effect, or a combination of these. the holistic access is an innate character of ethnopharmacology and needs to be tracked down to the specifically involved target/s. in a recently published study focusing on the discovery of hrv-capsid binders from nature using a pharmacophore-based virtual screening of an ethnopharmacologically biased 3d-multiconformational database, some 30 secondary metabolites were predicted to act as capsid-binding inhibitors of hrv. 144 for an in depth phytochemical and pharmacological investigation, it was mandatory to focus on one promising natural material. thus, we consulted the ethnobotanical source materia medica, which was written by pedanius dioscorides in the 1 st century ad. the treatise consisting of five books comprises some 1,000 or so drugs derived from minerals, animals, and the majority of them from plants (800). it represents a great repository of botanical, medical, and pharmacological lore. scrutinizing the natural materials underlying the obtained virtual hits we came across asafetida, which is a gum resin gained from the roots of a variety of foul-smelling ferula species from the apiaceae family. based on the descriptions given in the materia medica there is strong evidence that the juice (i.e. resin) of the popular ancient silphion originating from media and syria corresponds to asafetida (book iii, cap. 80). 315 yielded by incision of the root and stalk and frequently mixed with sagapenon, i.e. the resin of f. persica willd (book iii, cap. 81), it is reported to be effective in the context of upper respiratory diseases, e.g. ''for chronic harshness of the throat,'' ''it clears the voice,'' ''shrinks the uvulas,'' ''suitable for a cough,'' ''for pleurisy,'' ''for chest pain;'' sagapenon is described as follows: ''it clears thick matters from the lungs,'' ''given to those who are chilled.'' 315 these descriptions finally helped to prioritize those virtual hits, which have been reported to be constituents of asafetida. 316 the pharmacological investigation of asafetida and its constituents farnesiferol b and c ( fig. 5 ; table iii ) revealed a distinct anti-hrv-2 effect in the low micromolar range using a cpe inhibitory assay. 143 the results of this study provided a rationale for the ancient usage of asafetida for upper respiratory tract infections. on the other hand, the traditionally manifested evidence for asafetida for the treatment of common cold symptoms substantially helped in the selection of this plant material in the search for anti-hrv capsid binders. 144 typically, an ethnopharmacologically based discovery of an active (anti-hrv) extract is followed by a bioassay guided fractionation, and the isolation of those constituents that are responsible for the extracts' bioactivity. the concept of a bioassay-guided approach is the fractionation accompanied by simultaneous detection of the activity during the separation steps, which results in a continuous enrichment, and finally in the isolation of the active ingredient/s. in this way a large number of anti-hrv agents from natural sources have already been discovered. semple et al. investigated the active principle of the asteraceae plant pterocaulon sphacelatum, which has been used in traditional medicine of aboriginal people of australia as a favored treatment for respiratory infections, especially colds. by means of an antiviral activity-guided fractionation measuring the poliovirus-induced cpe assay, the authors identified the flavonoid 3,7,3 0 -trimethoxy-5,6,4 0 -trihydroxyflavone, i.e. chrysosplenol c ( fig. 5 ; table iii ) as potent and specific inhibitor of the picornaviral replication. 6,7,8-trimethoxycoumarin ( fig. 5 ; table iii) was also isolated as a major constituent from the ethanolic extract of p. sphacelatum, but showed no activity against poliovirus. 317 interestingly, this coumarin exhibited a significant effect in the hrv-2-induced cpe assay in a recently performed virtual parallel screening study performed in our laboratory. 318 in contrast to the isolated coumarin, chrysosplenol c was already known as a member of the 4 0 -hydroxy-3-methoxyflavones, which represent potent and specific inhibitors of picornaviral, especially rhinoviral replication. [319] [320] [321] in 1993, vanden berghe, haemers, and vlietinck provided a profound survey of antiviral agents from higher plants, and demonstrated the impact of ethnobotanical knowledge in their search for antiviral compounds from african medicinal plants. the selection of investigated plant species was mainly based upon their use in the treatment of viral diseases by african traditional healers. 320 the antiviral activity of 100 different plant species belonging to 33 families was investigated; thereof 21 species exhibited prominent antiviral properties against one or more of the tested viruses. the most pronounced activity against picornaviruses was recorded within the genus euphorbia. all compounds detected as antiviral constituents from the respective extracts were identified as 3-methoxyflavone derivatives, especially 3-methylethers of quercetin and kaempferol ( fig. 5 ; table iii ). they showed no significant cytotoxicity and were highly active in tissue culture against all human picornaviruses. in tissue culture, cells infected with different picornaviruses (among them also hrv) no cpe was observed, when cells had been treated with 2 mg/ml of these 3-methoxyflavones. 320 the selection of natural materials based on ethnopharmacology is a profound rationale for lead structure discovery and highly superior to random selection. this however rarely applies to marine organisms, fungi, and microbes. although these organisms are esteemed as highly valuable source for bioactive metabolites, 322 hardly any records from folk medicine are given for them. a medium-sized activity screening using a robust cell-based or in vitro-assay with reasonable effort as to time and costs is a strategy to get a first insight into the antiviral activities of extracts, fractions, and compounds from synthesis or nature. for the discovery of novel naturally based hrv 3c-protease inhibitors, singh et al. used a small peptide containing q-g scissile bond as substrate for the in vitro screening of extracts. the authors isolated the novel benzoisochromanquinone (1)-thysanone ( fig. 5 ; table iii ) from an extract of the fungus thysanophora penicilloides with potent hrv 3c-protease inhibitory activity. 323 a continued screening revealed a pronounced hrv 3c-protease inhibitory activity for the extract of the chinese herb polygonum cuspidatum. by bioassay-guided fractionation 2-methoxystypandrone ( fig. 5 ; table iii ), a naphthoquinone, with an ic 50 value of 4.6 mm, was isolated from the plant material. the total syntheses of this natural compound and further analogues allowed for a structure-activity relationship, and particularly the comparison between activities of ortho-vs. para-quinones. to measure the selectivity of the compound series against cystein proteases other than hrv 3c-protease, the compounds were evaluated against papain. the simple 9,10-phenanthraquinone ( fig. 5 ; table iii ) was the most active compound of the series and showed an ic 50 value of 1.4 mm with a distinctly higher degree of selectivity than 2-methoxystypandrone. 324 a cpe reduction assay was applied for the identification of raoulic acid, a bicyclic c25 terpene acid ( fig. 5 ; table iii ) isolated from raoulia australis (asteraceae). raoulic acid exerted an antiviral activity against coxsackie virus b3, b4, enterovirus 71, and hrv-2 and -3 with ic 50 values in the submicromolar range. no activity was recorded against influenza a and b viruses. 325 in the course of the systematic screening of microbial and natural products for anti-hrv activity, ishitsuka et al. identified 4 0 ,5-dihydroxy-3,3 0 ,7-trimethoxyflavone ( fig. 5 ; table iii) from the leaves of the chinese medicinal plant agastache rugosa (lamiaceae) as natural compound with high activity against all picornaviruses except mengovirus. the authors synthesized the orally active 4 0 ,5-diacetyloxy-3,3 0 ,7-trimethoxyflavone and performed investigations in tissue cultures and in mice to determine the compound's mode of action. this was assumed to be the process of viral replication, thus located between viral uncoating and initiation of viral rna synthesis. 326 the activity of flavan ( fig. 5 ; table iii ) was discovered serendipitously during a screening program by using a plaque inhibition assay. 194 based on these results, bauer et al. synthesized 4 0 ,6-dichloroflavan (bw863c, fig. 2 ; table ii) , which revealed an activity against a number of hrv serotypes in the range between 0.007 and 10 mm. as soon as an active lead compound is identified it is of utmost importance to scrutinize the derivatives' activity to (i) obtain insight into the chemical requirements mandatory for the focused biological activity, (ii) improve the compound's pharmacological profile in terms of potency, selectivity, cytotoxicity, etc., and to (iii) improve its bioavailability. the nonphenolic aporphine alkaloid glaucine is a prominent constituent of the aerial parts of gaucium flavum (papaveraveae). in a recently published study, spasova et al. investigated the antiviral potential of glaucine ( fig. 5 ; table iii) , glaucine derivatives, and its semi-synthesized 3-aminoethylglaucine cinnamoyl-and hydroxycinnamoyl amides. beside the anti-oxidative potential of the newly synthesized compounds, they all exerted an antiviral activity against the replication of hrv-14. the best anti-hrv activity was observed for oxoglaucine ( fig. 5 ; table iii; ic 50 table iii; ic 50 15 mm, si 10.3 and ic 50 13 mm, si 11, respectively). 327 the early findings of the anti-hrv active naturally derived 3-methoxyflavone inspired chemists and pharmacologists in the synthesis and the pharmacological evaluation of derivatives thereof decorated with various substitution patterns. several reports published during the last two decades reviewed the findings of antiviral flavonoid research. 320, 328, 329 by investigating the antiviral activity of a wide variety of naturally occurring flavonoids, tsuchiya et al. found chrysosplenol b and c ( fig. 5 ; table iii ) contained in chrysosplenium plants, and axillarin ( fig. 5 ; table iii ) as potent anti-hrv agents. based on their findings, the flavone skeleton decorated with a methoxy group in position 3 and a 5-hydroxyl group revealed as mandatory for an anti-hrv activity. 319 a series of antipicornaviral 4 0 -hydroxy-3-methoxyflavone derivatives was synthesized by de meyer et al. in order to establish a structure-activity relationship. thereby, different substitution patterns of the a-ring system with methyl, hydroxy, methoxy, halo, nitro, and amino was performed. their activity against polio and hrv was compared with those of naturally occurring flavonoids. further, the importance of the 4 0 hydroxyl-group and of the 3-methoxyl-group was confirmed by investigation of different derivatives lacking these features. 321 the results showed that 4 0 -hydroxy-3-methoxyflavones with a monosubstituted a-ring are less active than the corresponding compounds having a polysubstituted a-ring. within the tested series of compounds, 4 0 ,7-dihydroxy-3-methoxy-5,6-dimethylflavone emerged not only as noncytotoxic but also as most potent substance in both antiviral test systems. the lowest concentrations for this compound that protect 50% of the cells from cpe of 12 hrv serotypes were in the range from 0.016 to 0.5 mg/ml. in contrast to quercetin, this flavone was also reported to have no mutagenic properties (measured up to 2.5 mg/plate) in a short-term microbial assay. 321 the mechanism studies performed with 3-methoxyflavones have shown an interference with an early stage in the viral rna synthesis; no induction of resistance was observed. 320 in contrast to this mode of action, the anti-hrv chalcones and flavans are reported to interact directly with specific sites on the viral capsid proteins, thereby preventing uncoating and the consequent liberation of viral rna. 193, 330, 331 due to its anti-hrv potency, low toxicity, and promising bioavailability, dichloroflavan was evaluated for its protective efficacy against experimental hrv-infection in two clinical, double-blind, placebo-controlled trials. however, the drug candidate failed either when administered orally or intranasally. 332, 333 unfortunately, till now no clinical trials evaluating the efficacy of 3-methoxyflavones in common cold have been performed. the common idea of all computational approaches is to extract knowledge from a more or less large set of data in order to make predictions of new events. 334 within the lead discovery process, computational approaches, such as virtual screening, docking, quantitative structure-activity relationship, have largely enhanced the impact of computational chemistry and nowadays chemoinformatics plays a predominant role in drug research. 335 the key goal of the use of such methods is to reduce the overall cost associated to the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. a number of books and reviews on the impact of computational chemistry for lead structure determination highlight these efforts. [336] [337] [338] [339] in general, in silico methods can be divided into (i) ligand-based approaches, which rely on known active compounds. based on their physicochemical properties crucial for biological affinity, activities are predicted by extrapolation on not-yet tested substances, e.g. machine learning techniques and classical quantitative structure-activity relationship (qsar). ligand-based approaches are invaluable tools in cases where no structural information about the pharmacological targets is available. (ii) on the other hand, structurebased approaches use experimentally determined 3d structures of the targets, such as molecular docking or structure-based pharmacophore modeling for virtual screening. these methods allow for gaining insight into protein-ligand interactions at an experimentally determined (static) level (however not considering flexibility). a unique platform containing 3d coordinates of experimentally solved protein structures (by x-ray crystallography or nmr) is the protein data bank (pdb) currently comprising more than 50,000 structures of biomolecules and protein-ligand complexes. 340 additionally, there are several pdb-related web services and tools, which enable to use the pdb-portal in a rich diversity of information services for students and scientists. 341 particularly in the early stage of drug development, such as lead discovery and lead optimization, computational approaches allow for a target-oriented and rationalized proceeding, and thus may substantially help to maximize the success rate. 338 a recently published review on the impact of computer-assisted approaches in antiviral research thoroughly describes underlying in silico techniques, and highlights the benefits of computational approaches for the discovery of antiviral lead structures. 342 in anti-hrv research the capsid protein and the protease 3c revealed to be promising targets (as described before). inhibitors are assumed to have a major impact for the treatment of hrv-infections. additionally, these targets are structurally elucidated, and some potent ligands are known as well. these facts enable the performance of sensible computer-assisted approaches, both ligand-and structure-based. some studies using an in silico approach for the discovery of potential anti-hrv agents focusing on the mentioned targets will be reported in the following paragraphs. for compounds acting as potential hrv-capsid binders, some classical qsar and 3d qsar studies have been performed. while in classical qsar, the relationship between 2d calculated properties derived from chemical structures and measured biological activities are explored statistically, 3d qsar techniques are aimed at deriving a correlation and in turn activity prediction based on spatial arrangements of chemical properties and atoms. applying the 3d qsar technique comfa (comparative molecular field analysis) statistical models are derived, which are visualized in color-coded contours around the molecule. therein spots indicate where electrostatic properties and spatial arrangements are favorable for biological activity. 343 in the studies of diana et al., artico et al., and verma et al., qsar techniques helped on one hand to analyze and rationalize the structural features of active compounds essential for the interaction to the hrv canyon's binding pocket, and on the other hand to search for new classes of capsid binders, thus to narrow the synthetic challenges for specific anti-hrv agents, respectively. [344] [345] [346] [347] [348] in all these investigations hydrophobicity was found to be one of the most important determinants of substance activity. qsar combined with simplex representation of molecular structure was applied by kuz'min et al. based on the selectivity index (cc 50 /ic 50 ) and the hrv-2 inhibitory concentration of a set of [(biphenyloxy)propyl]isoxazole derivatives. 188 on the basis of qsar analysis and computational design, three new isoxazoles with high activity prediction were selected and synthesized. they all revealed a strong coincidence between experimental and predicted anti-hrv activity and selectivity index. terminal benzene substituents with negative electrostatic potential and a molecule length of approximately 5.5-5.6 å have been suggested as mandatory features within this chemical class for a hrv-2 inhibitory activity. hrv-serotypes show a high level of conservation at the protease 3c binding site; sequence alignment and secondary structure predictions suggested an overall architecture and mechanism of hrv-3c proteases that correlates with cellular cystein-and serine proteases, such as chymotrypsin and trypsin. 349, 350 the identity among 3c proteases from different families is however modest and provides space for the development of specific inhibitors for hrv-3c protease. 350 in a recently published review on selective inhibitors of picornavirus replication, de palma et al. summarized all currently known chemical structures acting as peptidic or nonpeptidic inhibitors of this viral target. 156 in 2000, reich et al., used the hrv 3c protease co-crystal structure information to rationalize the target-oriented synthesis of hrv 3c protease inhibitors from the class of substituted benzamides. activity data and subsequent crystallographic studies pointed out important requirements for the inhibition of the 3c protease. 351 similarly, maugeri et al. used a structure-based approach, and performed docking studies based on the 3c protease crystal structure with a virtual library consisting of benzamide derivatives. quantum-mechanic calculation proposed substituents with most promising biological activities. this workflow guided the design and synthesis of substances virtually assumed to act as substrate analogues. synthesis of some of these compounds and biological testing confirmed the underlying hypothesis. 219 quantitative molecular modeling studies were performed to better define and predict interactions between bicyclic 2-pyridone derivatives that showed to be irreversible inhibitors of the 3c protease. in these studies molecular mechanics simulations to evaluate chemical rate of covalent bond formation and free energy calculation combined with crystallographic studies were applied to explain the differences in activity of some irreversible peptidomimetic inhibitors. these data were used as a basis for further optimization of these compounds. 224, 352 in a recent study performed by kuo et al. some 6,800 compounds were subjected to a high troughput screening in the search for novel inhibitors for both 3c and 3cl proteases from picornavirus and coronavirus, respectively. 217 five nonpeptides were identified with ic 50 values r10 mm against severe acute respiratory syndrome-coronavirus 3cl-protease; one molecule was found to additionally inhibit the 3c proteases of coxsackievirus, enterovirus, and rhinovirus. this compound (id 43146) contains a dihydropyrazole ring decorated with two phenyl groups and a lengthy n-butyl-benzimidazolylamino-toluene. it was used as starting point for the selection of further four analogs showing ic 50 values in the range of 0.5-10 mm against the tested viral proteases. by means of docking-based computer modeling, the authors tried to rationalize the binding discrepancies responsible for individual and common protease inhibitors, thus to provide a rational base for the development of nonpeptide multiple-function inhibitors against coronaviruses and piciornaviruses. in anti-hrv research, first application scenarios have been conducted using pharmacophore models. according to the official iupac definition by wermuth et al., 353 a pharmacophore describes the 3d arrangement of steric and electronic features necessary to trigger or block a biological response. pharmacophores can be represented by three-dimensional chemical features, which include hydrogen bond donors and acceptors, aromatic rings, hydrophobic groups, as well as positive and negative ionisable moieties. additionally, the shape of ligands can be represented by shape features, which essentially describe the van der waals radii of the ligand atoms. the pharmacophore concept has proven to be successful, not only in rationalizing structure-activity relationships but also by its large impact in developing appropriate 3d-tools for efficient virtual screening. 336, 354 steindl et al. elaborated ligand-and structure-based pharmacophore models implementing the essential feature of the covalent binding to the cysteine 147 in the active site of the hrv-2 3c protease. thus, the in silico approach focused on defining a new pharmacophore feature representing a target structure for nucleophilic addition in the ligands, which is a crucial step for protease inactivation. the generated hypotheses retrieved known 3c protease inhibitors in the virtual screening cycle, and proposed potential (unconfirmed) ligands of the 3d protease binding site from available databases. 355 the viral capsid of several hrvs has been elucidated by crystallization and resolution of the 3d-structure. the hrv coat protein complexed with its highly active inhibitor win 61209 was used as starting point for the generation of structure-based pharmacophore models by . the models were used for virtual screening of a large commercially available 3d database. for final selection of virtual hits worth to be subjected to biological testing, docking studies and principal component analysis were performed. six candidates were tested for their ability to inhibit hrv serotype 39 by multiple-cycle cpe inhibition assay. although all of them showed a certain antiviral potential, one longitudinal piperazine derivative inhibited the virus at a concentration below 10 mm. some of the test candidates showed difficulties in the interpretation of experimental results due to their relatively high cytotoxicity and bad solubility. this circumstance asks for more cautious estimation of molecular properties for compound selection as stated by the authors. 356 in a subsequently performed study, the best validated pharmacophore model was used for the identification of naturally derived hrv coat protein inhibitors. 144 for virtual screening experiments the in-house generated 3d-database dios was used (as described before). based on the virtually predicted ligands and considering knowledge from traditional use, sesquiterpene umbelliferons from the gum resin of ferula sp., i.e. asafetida, were finally selected as most promising candidates. for biological evaluation, the antiviral activities of asafetida and its isolated constituents were assessed by an exploratory determination of the inhibition of the cpe induced by hrv serotypes 1a, 2, 14, and 16. the results revealed a dose-dependent and selective anti-hrv activity against serotype 2 for asafetida and its virtually predicted constituents, farnesiferols b and c ( fig. 5 ; table iii ; ic 50 : 2.6 and 2.5 mm, respectively). to scrutinize the selectivity of these two compounds against hrv-2 in comparison to the other tested serotypes, the amino acid sequences of hrv-2 and hrv-16 vp1 were aligned. since all amino acid residues involved in ligand binding showed 100% match in both serotypes, the experimentally determined selectivity profile could not be explained by different binding pockets. the serotype alignment does however not reflect potential protein flexibility during binding, which might differ between the hrv serotypes. additionally, off-target effects could be a reason for the observed selectivity. in a recently performed virtual parallel screening approach, we tried to identify potential targets of human pathological relevance for 16 constituents isolated and identified from the medicinal plant ruta graveolens. 318 using the screening platform pipline pilot 357 included in discovery studio, 358 low-energy conformers of the 16 identified molecules from r. graveolens were subjected to parallel screening. for this purpose, the inte:ligand pharmacophore model collection was used. 359 it currently comprises 2.208 models covering 280 unique pharmacological targets. based on the predicted ligand-target interactions, the authors focused on three biological targets, namely acetylcholinesterase, the hrv coat protein, and the cannabinoid receptor type 2. virtual hits and nonhits were assayed on their respective targets for a critical evaluation of the performed target-fishing approach. beside other predicted bioactivities, determination of their cpes on hrv-2 revealed the virtual hit arborinine ( fig. 5 ; table iii; ic 50 : 3.2 mm) and the nonpredicted hit 6,7,8-trimethoxycoumarin ( fig. 5 ; table iii ; ic 50 : 12.9 mm) as the most active anti-hrv constituents. it could be shown that the applied in silico strategy has the capacity of catalyzing drug discovery profoundly for all those diseases where molecular targets or molecular ligands are well defined to create reliable pharmacophore models. hrv, a prominent member of the picornaviridae family, infects humans more frequently than any other virus. infections with hrv mainly lead to upper respiratory diseases, such as the common cold, but may also cause more severe lower respiratory tract disorders. although the symptomology and severity of the common cold is relatively mild and the course of disease self-limiting, the socio-economic impact is tremendous in terms of recouping lost productivity due to sick leave. in the last decades, a number of anti-hrv agents from different origin-synthetics, natural compounds, biologicals, botanicals, and nutritionals-have been discovered. different concepts have been used as strategy for their discovery either starting from a phenomenological effect, e.g. empirical knowledge from folk medicine, or from a targetbased molecular level, e.g. icams, capsid binders, and hrv protease inhibitors. some of the outcomes revealed potent and promising activities that partly have been evaluated for the management of hrv-induced common colds in clinical trials mainly with sobering benefit. since the hrv infection is not life-threatening in most cases, a potential therapy has to be safe and effective with an almost unrecognizable level of side effects. these preconditions render the search for anti-hrv-agents into a high challenging endeavor and explains why no antiviral agent is approved for the prevention or treatment of hrv-infection until today, despite the significant efforts. moreover, the high variability 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serotype 2 (hrv2) human rhinovirus 3 at 3.0 a resolution the canyon hypothesis viral cell recognition and entry crystallographic and cryo em analysis of virion-receptor interactions a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses the minor receptor group of human rhinovirus (hrv) includes hrv23 and hrv25, but the presence of a lysine in the vp1 hi loop is not sufficient for receptor binding members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus multiple receptors involved in human rhinovirus attachment to live cells structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor the cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view the major and minor group receptor families contain all but one human rhinovirus serotype human rhinovirus type 54 infection via heparan sulfate is less efficient and strictly 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receptors in vitro studies of the antirhinovirus activity of soluble intercellular adhesion molecule-1 mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of icam-1 efficient neutralization and disruption of rhinovirus by chimeric icam-1/immunoglobulin molecules recombinant soluble low density lipoprotein receptor fragment inhibits minor group rhinovirus infection in vitro prevention of rhinovirus infection in chimpanzees by soluble intercellular adhesion molecule-1 efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial inhibitors of picornavirus replication the structure of antiviral agents that inhibit uncoating when complexed with viral capsids structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds structural analysis of antiviral agents that interact with the capsid of human rhinoviruses conformational change in the floor of the human rhinovirus canyon blocks adsorption to hela cell receptors the site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating a comparison of the anti-rhinoviral drug binding pocket in hrv14 and hrv1a analysis of three structurally related antiviral compounds in complex with human rhinovirus 16 antiviral capsid-binding compounds can inhibit the adsorption of minor receptor rhinoviruses in vitro activity of pleconaril and ag7088 against selected serotypes and clinical isolates of human rhinoviruses activity of pleconaril against enteroviruses susceptibility of coxsackievirus b3 laboratory strains and clinical isolates to the capsid function inhibitor pleconaril: antiviral studies with virus chimeras demonstrate the crucial role of amino acid 1092 in treatment in vitro activity of r 61837, a new antirhinovirus compound in vitro activity of pirodavir (r 77975), a substituted phenoxy-pyridazinamine with broad-spectrum antipicornaviral activity quantitative structure-activity relationship studies of [(biphenyloxy)-propyl]isoxazole derivatives. inhibitors of human rhinovirus 2 replication sch 48973: a potent, broad-spectrum, antienterovirus compound antipicornavirus activity of sch 47802 and analogs: in vitro and in vivo studies sch 38057: a picornavirus capsid-binding molecule with antiviral activity after the initial stage of viral uncoating direct and specific inactivation of rhinovirus by chalcone ro 09-0410 comparative studies on the antirhinovirus activity and the mode of action of the rhinovirus capsid-binding agents, chalcone amides 4 0 ,6-dichloroflavan (bw683c), a new anti-rhinovirus compound 3,4-dihydro-2-phenyl-2h-pyrano[2,3-b]pyridines with potent antirhinovirus activity antipicornavirus activity of substituted phenoxybenzenes and phenoxypyridines activity of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (mdl-860) against picornaviruses in vitro application of crystallography to the design of antiviral agents clinical activity of pleconaril in an experimentally induced coxsackievirus a21 respiratory infection oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: efficacy and tolerability in phase ii clinical trials efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses the effect of oral pleconaril on hepatic cytochrome p450 3a activity in healthy adults using intravenous midazolam as a probe safety and efficacy of intranasal pirodavir (r77975) in experimental rhinovirus infection inhibition of polyprotein processing and rna replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2a through s-nitrosylation: a protective mechanism against enteroviral cardiomyopathy an antiviral mechanism of nitric oxide: inhibition of a viral protease nitric oxide inhibition of coxsackievirus replication in vitro nitric oxide donors inhibit the coxsackievirus b3 proteinases 2a and 3c in vitro, virus production in cells, and signs of myocarditis in virus-infected mice effect of nitric oxide on rhinovirus replication and virus-induced interleukin-8 elaboration inhibition of proteolytic activity of poliovirus and rhinovirus 2a proteinases by elastase-specific inhibitors an antiviral peptide inhibitor that is active against picornavirus 2a proteinases but not cellular caspases antiviral activity of caspase inhibitors: effect on picornaviral 2a proteinase dual inhibition of human rhinovirus 2a and 3c proteases by homophthalimides structure of human rhinovirus 3c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein individual and common inhibitors of coronavirus and picornavirus main proteases structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3c protease with potent antiviral activity against multiple rhinovirus serotypes new anti-viral drugs for the treatment of the common cold structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 1. michael acceptor structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 2. peptide structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 3. structure-activity studies of ketomethylene-containing peptidomimetics structurebased design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 8. pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 4. incorporation of p1 lactam moieties as l-glutamine replacements in vitro antiviral activity of ag7088, a potent inhibitor of human rhinovirus 3c protease inhibition of human rhinovirus-induced cytokine production by ag7088, a human rhinovirus 3c protease inhibitor pharmacokinetics and safety of an antirhinoviral agent, ruprintrivir, in healthy volunteers phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3c protease gaining target access for deoxyribozymes a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism mode of action of 2-furylmercury chloride, an antirhinovirus compound methoxyflavones as potent inhibitors of viral-induced block of cell synthesis antiviral effects of pyrrolidine dithiocarbamate on human rhinoviruses broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at ctp synthetase in vitro antiviral activity of ribavirin against picornaviruses another ten stories in antiviral drug discovery (part c): ''old'' and ''new'' antivirals, strategies, and perspectives the broadspectrum antiviral ribonucleoside ribavirin is an rna virus mutagen ribavirin: recent insights into antiviral mechanisms of action inhibition of rhinovirus replication in in organ culture by a potential antiviral drug synthesis of syn and anti isomers of 6-[[(hydroxyimino)phenyl]methyl]-1-[(1-methylethyl)sulfonyl]-1h-benzimidaz ol-2-amine. inhibitors of rhinovirus multiplication comparative studies on the modes of action of the antirhinovirus agents ro 09-0410, ro 09-0179, rmi-15,731, 4 0 ,6-dichloroflavan, and enviroxime the antiviral compound enviroxime targets the 3a coding region of rhinovirus and poliovirus sequence determinants of 3a-mediated resistance to enviroxime in rhinoviruses and enteroviruses evidence that enviroxime targets multiple components of the rhinovirus 14 replication complex controlled trial of enviroxime against natural rhinovirus infections in a community the activity of enviroxime against rhinovirus infection in man activity against rhinoviruses, toxicity, and delivery in aerosol of enviroxime in liposomes 2-amino-3-substituted-6-[(e)-1-phenyl-2-(n-methylcarbamoyl)vinyl]imid azo[1,2-a]pyridines as a novel class of inhibitors of human rhinovirus: stereospecific synthesis and antiviral activity short synthesis and anti-rhinoviral activity of imidazo[1,2-a]pyridines: the effect of acyl groups at 3-position synthesis, antiviral activity, and biological properties of vinylacetylene analogs of enviroxime synthesis and antiviral activity of c2 analogs of enviroxime: an exploration of the role of critical functionality sensitivity of rhinoviruses to human leukocyte and fibroblast interferons cell and virus sensitivity studies with recombinant human alpha interferons comparative susceptibility of respiratory viruses to recombinant interferons-alpha 2b and -beta intranasal interferon-alpha 2 for prevention of natural rhinovirus colds intranasal interferon alpha 2 for prevention of rhinovirus infection and illness interferon-beta ser as prophylaxis against experimental rhinovirus infection in volunteers an effective dosage regimen for prophylaxis against rhinovirus infection by intranasal administration of huifn-alpha 2 efficacy and tolerance of intranasally applied recombinant leukocyte a interferon in normal volunteers intranasally applied recombinant leukocyte a interferon in normal volunteers. ii. determination of minimal effective and tolerable dose recombinant human interferon-gamma as prophylaxis against rhinovirus colds in volunteers ineffectiveness of postexposure prophylaxis of rhinovirus infection with low-dose intranasal alpha 2b interferon in families a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection synergism between anti-rhinovirus antivirals: various human interferons and a number of synthetic compounds failure to demonstrate synergy between interferon-alpha and a synthetic antiviral, enviroxime, in rhinovirus infections in volunteers property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry a new rapid and effective chemistry space filter in recognizing a druglike database rational selection of structurally diverse natural product scaffolds with favorable adme properties for drug discovery natural products as sources of new drugs over the last 25 years ethnomedicinal antivirals: scope and opportunity plant substances as antiviral agents: an update plant substances as antiviral agents host defense function of the airway epithelium in health and disease: clinical background herb sales down 6 percent in mainstream market echinacea for preventing and treating the common cold echinacea for preventing and treating the common cold. the cochrane database of systematic reviews evaluation of echinacea for the prevention and treatment of the common cold: a meta-analysis echinacea in the prevention of induced rhinovirus colds: a meta-analysis the role of alkamides as an active principle of echinacea alkylamides from echinacea are a new class of cannabinomimetics: cannabinoid type 2 receptordependent and -independent immunomodulatory effects synergistic anti-oxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from echinacea purpurea on in vitro oxidation of human low-density lipoproteins echinacea extracts modulate the pattern of chemokine and cytokine secretion in rhinovirus-infected and uninfected epithelial cells modulation of immune response gene expression by echinacea extracts: results of a gene array analysis preventing the common cold with a garlic supplement: a double-blind, placebocontrolled survey in vitro virucidal effects of allium sativum (garlic) extract and compounds enhancement of in vitro human immune function by allium sativum l. (garlic) fractions efficacy of cold-fx in the prevention of respiratory symptoms in community-dwelling adults: a randomized, doubleblinded, placebo controlled trial efficacy of an extract of north american ginseng containing poly-furanosyl-pyranosyl-saccharides for preventing upper respiratory tract infections: a randomized controlled trial safety and tolerability of north american ginseng extract in the treatment of pediatric upper respiratory tract infection: a phase ii randomized, controlled trial of 2 dosing schedules inhibitory effect of ginsenoside rg3 and its derivative ginsenoside rg3-2h on no production and lymphocyte proliferation effect of cvt-e002 (cold-fx) versus a ginsenoside extract on systemic and gut-associated immune function inhibitory effect of ginseng polysaccharides on rotavirus infection antiviral activity of dammarane saponins against herpes simplex virus i effect of a traditional japanese herbal medicine, hochu-ekki-to (bu-zhong-yi-qi tang), on immunity in elderly persons effects of five kampohozais on the mitogenic activity of lipopolysaccharide, concanavalin a, phorbol myristate acetate and phytohemagglutinin in vivo immunological restoration and anti-tumor effect by japanese herbal medicine in aged mice antiinflammatory effects of bu-zhong-yi-qi-tang in patients with perennial allergic rhinitis hochu-ekki-to inhibits rhinovirus infection in human tracheal epithelial cells efficacy of a pelargonium sidoides preparation in patients with the common cold: a randomized, double blind, placebo-controlled clinical trial kern winfried v. pelargonium sidoides extract for acute respiratory tract infections traditionally used pelargonium species: chemistry and biological activity of umckaloabo extracts and their constituents aqueous ethanolic extract of the roots of pelargonium sidoides-new scientific evidence for an old anti-infective phytopharmaceutical efficacy of an aqueous pelargonium sidoides extract against herpesvirus protective effect of a natural carrageenan on genital herpes simplex virus infection in mice polysaccharides as antiviral agents: antiviral activity of carrageenan iota-carrageenan is a potent inhibitor of rhinovirus infection recommendations for reporting randomized controlled trials of herbal interventions: explanation and elaboration ethnobotany and the search for new drugs: ciba foundation symposium 185 ethnobotany and natural products: the search for new molecules, new treatments of old diseases or a better understanding of indigenous cultures? how scientific is the science in ethnopharmacology? historical perspectives and epistemological problems pedanius dioscorides of anazarbus-de materia medica sesquiterpene coumarins from ferula assafoetida l antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in silico target fishing for rationalized ligand discovery exemplified on constituents of ruta graveolens l antiviral activity of natural occurring flavonoids in vitro antiviral agents from higher plants and example of structure-activity relationship of 3-methoxyflavones 0 -hydroxy-3-methoxyflavones with potent antipicornavirus activity biodiversity: a continuing source of novel drug leads structure and stereochemistry of thysanone: a novel human rhinovirus 3c-protease inhibitor from thysanophora penicilloides discovery, total synthesis, hrv 3c-protease inhibitory activity, and structure-activity relationships of 2-methoxystypandrone and its analogues antiviral activity of raoulic acid from raoulia australis against picornaviruses antipicornavirus flavone ro 09-0179 cinnamoyl-and hydroxycinnamoyl amides of glaucine and their antioxidative and antiviral activities antiviral activity of flavones and flavans present status and prospects of flavonoids as antiviral agents inhibition of an early stage of rhinovirus replication by dichloroflavan (bw683c) effect of dichloroflavan (bw683c) on the stability and uncoating of rhinovirus type 1b failure of intranasally administered 4 0 , 6-dichloroflavan to protect against rhinovirus infection in man failure of oral 4 0 ,6-dichloroflavan to protect against rhinovirus infection in man neural networks as data mining tools in drug design chemoinformatics and drug discovery methods and principles in medicinal chemistry the impact of informatics and computational chemistry on synthesis and screening enhancing drug discovery through in silico screening: strategies to increase true positives retrieval rates neural networks for chemists the protein data bank the protein data bank (pdb), its related services and software tools as key components for in silico guided drug discovery development of antiviral agents using molecular modeling and virtual screening techniques comparative molecular field analysis (comfa). 1. effect of shape on binding of steroids to carrier proteins investigation on qsar and binding mode of a new class of human rhinovirus-14 inhibitors by comfa and docking experiments synthesis and structure-activity studies of some disubstituted phenylisoxazoles against human picornavirus comfa analysis of the interactions of antipicornavirus compounds in the binding pocket of human rhinovirus-14 dihydro-2-oxazolyl)phenoxy]alkyl]-3-methylisoxazoles: inhibitors of picornavirus uncoating understanding human rhinovirus infections in terms of qsar site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3c cysteine protease and cellular trypsin-like serine proteases the picornaviral 3c proteinases: cysteine nucleophiles in serine proteinase folds substituted benzamide inhibitors of human rhinovirus 3c protease: structure-based design, synthesis, and biological evaluation structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics glossary of terms used in medicinal chemistry (iupac recommendations 1997) methods and principles in medicinal chemistry human rhinovirus 3c protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening docking versus pharmacophore model generation: a comparison of highthroughput virtual screening strategies for the search of human rhinovirus coat protein inhibitors ca: accelrys. 358. discovery studio rollinger extended her studies to the fields of phytochemistry, ethnopharmacology, molecular modelling and its application in natural product science completing the habilitation thesis entitled ''the search for bioactive natural products rollinger is a project leader and an associate in various national projects, as well as executive guest editor for current pharmaceutical design. she has been awarded the phoenix science award (2005) and received several additional awards until 1994 she was a postdoctoral research fellow at the institute of immunology, university marburg, where she studied picornavirusimmune cell interactions and the virus-induced cytokine induction. thereafter, she was a postdoctoral fellow at the institute of virology and antiviral therapy key: cord-297579-ohpm5ys0 authors: netzler, natalie e.; enosi tuipulotu, daniel; white, peter a. title: norovirus antivirals: where are we now? date: 2018-12-25 journal: med res rev doi: 10.1002/med.21545 sha: doc_id: 297579 cord_uid: ohpm5ys0 human noroviruses inflict a significant health burden on society and are responsible for approximately 699 million infections and over 200 000 estimated deaths worldwide each year. yet despite significant research efforts, approved vaccines or antivirals to combat this pathogen are still lacking. safe and effective antivirals are not available, particularly for chronically infected immunocompromised individuals, and for prophylactic applications to protect high‐risk and vulnerable populations in outbreak settings. since the discovery of human norovirus in 1972, the lack of a cell culture system has hindered biological research and antiviral studies for many years. recent breakthroughs in culturing human norovirus have been encouraging, however, further development and optimization of these novel methodologies are required to facilitate more robust replication levels, that will enable reliable serological and replication studies, as well as advances in antiviral development. in the last few years, considerable progress has been made toward the development of norovirus antivirals, inviting an updated review. this review focuses on potential therapeutics that have been reported since 2010, which were examined across at least two model systems used for studying human norovirus or its enzymes. in addition, we have placed emphasis on antiviral compounds with a defined chemical structure. we include a comprehensive outline of direct‐acting antivirals and offer a discussion of host‐modulating compounds, a rapidly expanding and promising area of antiviral research. risk and vulnerable populations in outbreak settings. since the discovery of human norovirus in 1972, the lack of a cell culture system has hindered biological research and antiviral studies for many years. recent breakthroughs in culturing human norovirus have been encouraging, however, further development and optimization of these novel methodologies are required to facilitate more robust replication levels, that will enable reliable serological and replication studies, as well as advances in antiviral development. in the last few years, considerable progress has been made toward the development of norovirus antivirals, inviting an updated review. this review focuses on potential therapeutics that have been reported since 2010, which were examined across at least two model systems used for studying human norovirus or its enzymes. in addition, we have placed emphasis on antiviral compounds with a defined chemical structure. we include a comprehensive outline of directacting antivirals and offer a discussion of host-modulating compounds, a rapidly expanding and promising area of antiviral research. although the same high prevalence of this strain was not reflected in other parts of the world, with lower levels detected in australasia, europe, and north america compared to the asian outbreaks during that same period. [31] [32] [33] the human norovirus positive-sense, single-stranded rna genome is 7.5 to 7.7 kb ( figure 1 karst et al 36 ) . orf2 and orf3 encode the proteins vp1 and vp2, respectively; vp1 is the major capsid protein and vp2 is the minor capsid protein, likely involved in capsid assembly and genome encapsidation. 37 the vp1 protein structure comprises the shell (s) and protruding (p) domains; the s domain encloses the viral rna, while the antigenically variable p domain forms the outer surface of vp1, and is also involved in cell attachment. 38, 39 the vp1 protein can be expressed in baculovirus which then self-assembles into virus-like particles (vlps). these vlps are antigenically and structurally indistinguishable to virions produced by the complete virus. 40 despite the clinical significance of norovirus infection, antiviral studies have been hindered, because until recently, human norovirus could not be successfully propagated in cell culture. recent breakthroughs have enabled human norovirus to be cultured in b cells 41 and intestinal enteroids, 42 which represent milestones in the field of norovirus biology. however, the modest replication levels generated by these new systems (≤3.5 log increase in b cells 41, 43 f i g u r e 1 schematic of the human norovirus genome. the norovirus genome is a positive-sense, singlestranded rna genome comprising three orfs that encode the nonstructural proteins: p48/n-terminal (ns1/2), ntpase (ns3), p22 (ns4), vpg (ns5), protease (ns6), and rna polymerase (ns7); and the structural proteins: and ≤3.8 log increase in enteroids 42 ) means that they require optimization before widespread use for antiviral screening and development. the gi.1 (norwalk virus) norovirus replicon system has been used to assess antiviral candidates against the human virus in lieu of a viral culture system ( figure 2 ). the norwalk replicon consists of an intact orf1, orf3, and genomic 3′ end, however, orf2 is disrupted by a neomycin gene, engineered into the vp1-encoding region. as such, while the subgenomic promoter is preserved, the expression of an intact vp1 is disrupted. self-replicating and stably expressed in huh-7 cells or bhk-21 cell lines, 44 the norwalk replicon has proven itself as a useful tool to screen potential antiviral compounds (table 1) . however, replication levels of the norwalk replicon are relatively low (approximately 1 × 10 3 norwalk rna copies per cell 44 ) , when compared to other replicon systems such as those for hepatitis c virus (hcv). the hcv replicons typically yield more than 1 × 10 4 copies per cell (aa eltahla, 2018 personal communication), and have been used to identify many successful antiviral candidates. moreover, they are amenable to high-throughput antiviral screening (reviewed in horscroft et al 45 ) , which has directly led to many of the direct-acting antivirals (daas) used to treat hcv infections today (reviewed in asselah et al 46 ) . other models have also been used to study antiviral efficacy against norovirus ( figure 2 ). for example, researchers have used in vitro enzyme activity assays, such as viral polymerase assays 47 and protease assays 48 (figure 2 ) to screen compounds for antiviral activity in a high-throughput format. 49 in addition, x-ray crystallography and in silico modeling can be used to examine ligand and viral protein interactions, to further elucidate antiviral mechanisms. 50 f i g u r e 2 current methods for the identification and characterization of norovirus antivirals. a flow chart depicting the methods and tools available for assessing the effectiveness of norovirus antivirals. panels in green involve a combination of in silico and in vitro methods. panels in blue and yellow represent in vitro and in vivo methods, respectively. the purple panel represents clinical testing in human patients. crfk, crandell rees feline kidney; ic 50 , half maximal inhibitory concentration; qrt-pcr, quantitative reverse-transcription polymerase chain reaction; rdrp: rna-dependent rna polymerase; sar, structure-activity relationship; tcid50, tissue culture infective dose [color figure can be viewed at wileyonlinelibrary.com] the replication cycle and antiviral targets for human norovirus. (a), a schematic of the complete norovirus replication cycle is presented and antivirals that have been developed against norovirus are depicted in turquoise circles. the listed compounds represent only those antivirals which have a known target and a more extensive list of antivirals has been described in table 1 surrogate viruses from within the caliciviridae family have also been exploited to screen for inhibitory activity of antiviral candidates across several calicivirus genera ( figure 2 ). these surrogates include murine norovirus (mnv; norovirus), feline calicivirus (fcv; vesivirus), porcine sapovirus (sapovirus), rabbit hemorrhagic disease virus (rhdv; lagovirus), and tulane virus, from the proposed genus recovirus. 51 mnv, in particular, has been used as the predominant human norovirus surrogate as it is classified within the same genus and can be robustly propagated in cell culture, 52, 53 making it amenable to viral disinfection and sterilization studies. [54] [55] [56] additional features that make mnv desirable for antiviral screening include its ability to be manipulated through reverse genetics, whilst in vivo studies in mice of many genetic backgrounds are straightforward. 57, 58 various animal models have also been used for human norovirus challenge studies and include: nonhuman primates such as chimpanzees, macaques, marmosets, and tamarins. [59] [60] [61] additionally, gnotobiotic or miniature pigs and gnotobiotic calves [62] [63] [64] [65] [66] have also been used, as well as knockout and humanized mice for norovirus infection studies. [67] [68] [69] however, no animal model has been deemed entirely suitable, due to obvious differences in clinical disease, gut physiology, microbiomes, naivety to norovirus infection compared to human populations, and the low genetic diversity of laboratory test animals. 36 every stage of the human norovirus replication cycle represents a unique target for antiviral development ( figure 3 ). however, the development of antiviral therapies that target viral replication requires a detailed understanding of norovirus biology and viral gene functionality, much of which is still to be elucidated. an overview of the human norovirus lifecycle and the specific antiviral targets are outlined in figure 3 . the stages of the replication cycle for antiviral targeting include: host cell attachment, internalization, genome release, viral genome replication mediated by the viral rdrp, translation of the genomic and subgenomic templates using the vpg and host cell machinery, viral protease cleavage of the viral polyprotein to yield mature viral proteins, followed by assembly, packaging, and cell egress (reviewed in thorne and goodfellow 70 ). while a number of other norovirus antiviral reports have been published in the last three years, [71] [72] [73] [74] [75] there has been significant recent progress in the field, which now warrants an updated review. herein we include a comprehensive overview of peer-reviewed studies since 2010, including antiviral candidates examined in at least two different systems (e.g. viral enzyme inhibition assays and mnv cell culture). it should be noted that plant or food extracts that have antinorovirus effects have been omitted from this study when the active antiviral compound is unknown, with this review focusing on compounds that have a defined structure (as outlined in table 1 ). we include studies using the recently developed human norovirus cell culture system in b cells. 41 we also discuss recently identified broad-spectrum antivirals with antinorovirus activity and provide a more comprehensive overview of host-modulating compounds, which is a recent, novel and exciting area within norovirus antiviral research. table 1 lists the individual antiviral compounds, or in the case where several very similar derivatives have been studied, it lists the most potent compound of the group as a representative derivative, with relevant citations. each compound is indexed for clarity throughout the manuscript and in table 1 . cell attachment and entry are features of the viral replication cycle that have been extensively investigated as antiviral targets for human immunodeficiency virus (hiv), dengue virus (denv), and hcv, 76 amongst others. to design compounds capable of targeting viral entry, knowledge of the viral entry receptor/s is usually required. recently, cd300lf was identified as the entry receptor for mnv, however, the counterpart receptor for human norovirus has yet to be discovered. 77, 78 as such, there is an absence of antivirals that target norovirus entry and a bias toward compounds that prevent norovirus cellular attachment. the most widely studied attachment targets are the histo-blood group antigens (hbgas). hbgas are complex carbohydrates that are presented abundantly on the surface of mucosal epithelia of the gastrointestinal tract. they interact with the surface p2 domain of the vp1 capsid protein 79, 80 and are thought to aid viral attachment, and perhaps even entry. [81] [82] [83] hbgas are determined by host cell genetics (reviewed in tan and jiang 82 ) and play an important role in terms of both virus and host interactions, and for noroviruses to initiate infection. 81, 84 in one study, in silico screening was performed on a drug library (>2 million compounds) to identify molecules that interact strongly with hbgas and prevent norovirus attachment. 85 potential hits (n = 160) were confirmed in vitro with enzyme-linked immunosorbent assay (elisa)-based human norovirus vp1/hbga blocking assays, which revealed that compounds with a cyclopenta-a-dimethyl phenanthrene base structure (n = 4) display potent inhibition (ic 50 < 10 μm) of hbga-norovirus binding. 85 the most potent compound in this series was 88 in addition, x-ray crystallography has revealed that these hmos interact directly with the p domain of the capsid, thus providing a clear mechanism for the binding inhibition observed. [86] [87] [88] despite these advances, attachment inhibitors have only been tested against vlps from less common genotypes and thus their effectiveness against a broader spectrum of norovirus strains needs to be demonstrated. passive immunotherapy with monoclonal antibodies (mabs) or nanobodies (nbs) is an antiviral strategy to prevent norovirus attachment and entry, which would be particularly useful for immunocompromised patients. the cross-genotypic activity displayed by nbs illustrates that these molecules have the potential to overcome the narrow antigenic spectrum typically displayed by conventional mabs. however, despite these findings, mab and nb studies have been based mostly on vlp-binding and structural analysis of that binding (table 1 ) and thus the effects of such compounds against norovirus in cell culture or in vivo need to be explored further before continued development toward clinical application. of the human norovirus antiviral targets, the rdrp is one with numerous preclinical candidates identified that can inhibit its activity (table 1) . critical for viral replication, the rdrp is a highly attractive antiviral target, as it largely netzler et al. lacks host homologs minimizing the chance of off-target adverse effects. 96 the human norovirus polymerase forms the canonical rdrp structure resembling a closed right hand, with fingers, palm, and thumb domains 97 ( figure 3b ), likely acting as a homodimer in it active state. [97] [98] [99] the rdrp-targeting antivirals are divided into two major classes; the nucleoside analogs (nas) and the nonnucleoside inhibitors (nnis nas inhibit rna synthesis through mimicry of incoming nucleoside triphosphates (ntps), which upon incorporation subsequently cause chain termination, 105 or less commonly, increase mutations during viral genome transcription that results in lethal mutagenesis (also known as error catastrophe). 106 110 another study reported a similar 2cmc potency for mnv rna level reduction (6.9 µm), but potency against the norwalk replicon was found to be 14-fold higher (1.3 µm). 111 this difference was proposed to be due to either varying methodology between studies, or differences in drug purity. 110 in the human norovirus bjab cell culture system, 2cmc inhibited human norovirus replication with an ec 50 of 0.3 µm. 67 despite the variation in 2cmc potency observed between these in vitro systems, collectively these studies illustrate that the polymerase is an excellent antiviral target for cross-genogroup inhibition of norovirus replication. the na 2cmc has also shown promise as a potential norovirus antiviral in mouse model studies. knockout mice infected with mnv and treated with 2cmc were protected from mortality, diarrhea, and had reduced norovirus genome titers in tissues and stool (1.0-1.5 log 10 reduction), compared to mock-treated animals. 67, 110 additionally, mnv-infected mice treated with a high dose of 2cmc (100 mg/kg/day for 5-7 days) demonstrated reduced transmission to uninfected sentinel mice caged together, and offered prophylactic protection for up to 18 days. 112 in an attempt to improve the safety and efficacy of 2cmc therapy, several derivatives have been examined for antinorovirus activity, for example, 2′-f-2′-c-methylcytidine (2fcmc). one study evaluated the inhibitory activity of 2cmc, 2fcmc, β-d-n(4)-hydroxycytidine (nhc) and the hbv/hiv na lamivudine against the replication of mnv and the norwalk replicon. 111 has potent and pan-genotypic activity against norovirus. 128 cmx521 is reportedly in the recruitment stage of phase i clinical trials to evaluate the safety, tolerability, and pharmacokinetics in less than or equal to 50 healthy adults. no peer-reviewed publications were available when writing this review, with results projected to be released later in 2018. 128 nnis generally exhibit narrow-spectrum antiviral activity and bind allosterically to block conformational rearrangements of the viral polymerase required to form an active replication complex. 129 currently, there are three known nni binding sites on the norovirus rdrp. one binding pocket is within the ntp access path located between the fingers and thumb domains, 130 the second pocket is termed site a, a positively charged ntp traversal channel with flexible amino acid side chains, 131 and the third pocket is site b, a highly conserved allosteric binding pocket present across the caliciviridae and located within the thumb region 131,132 ( figure 3b ). despite the promising potency of suramin and its derivatives, including ppnds, these compounds demonstrate limited bioavailability and exhibit poor cell permeability, 134 greatly reducing their antiviral efficacy in viral culture. 132, 135, 136 while suramin has been shown to inhibit mnv replication in cell culture (ec 50 0.3 µm), the large structure and low cell permeability meant delivery required a liposome system to enter raw264.7 cells. 135 similarly, ppnds was found to be much less potent in mnv cell culture, demonstrating only 20.5% inhibition of mnv replication in a plaque assay at 10 µm, compared to 98.0% inhibition at the same concentration in the rdrp assays. 132 moreover, ppnds has been eliminated from other enzyme inhibition studies due to nonspecific, offtarget effects, 137 and is therefore likely to be an unsuitable drug candidate for further antinorovirus development. much like the rdrp, the norovirus protease (ns6; figure 3c ) represents a desirable antiviral target since it plays an essential role in viral replication, through cleavage of the ns polyprotein, which is necessary for the production of viral progeny. norovirus protease inhibitors (pis) have been tested over a wide range of model antiviral systems ( figure 2 ) and represent the class of calicivirus antivirals with the most number of compounds (table 1) 141 although potent, ts mimics are far less studied than ts inhibitors and represent a small proportion of the compounds described in table 1 . the first ts inhibitors designed included a series of peptidyl aldehydes that incorporated a glutamine surrogate in their structure, 142 and take advantage of the preference for the norovirus protease to cleave at glutamineglycine peptide sites. 143 of rupintrivir. 154 the effectiveness of pis across multiple genera and norovirus genogroups illustrates that the protease is an excellent antiviral candidate for the treatment of norovirus infections. a pitfall of using some daas is the emergence of resistance mutations which can undermine their effectiveness, although combinational therapy is a proven option in hiv and hcv therapy to circumvent this. in comparison to daas, antivirals that target the host generally have a higher barrier to resistance than some classes of daas, 158 however, this initial study revealed that inhibition of mnv in mice was limited to the small intestine due to poor bioavailability of wp1130. 158 to address the poor bioavailability, libraries of wp1130 variants were developed and tested for improved antiviral efficacy. 156 immunomodulators are an excellent therapeutic option for viral infections due to their ability to induce a powerful host response against intracellular parasites. the best example of immunomodulators are interferons (ifns) and for over a decade, studies have shown that type i and ii ifns, as well as their receptors, provide protection against murine and human norovirus infections. 44, 52, 53, [163] [164] [165] [166] [167] [168] however, the role of type iii ifns (ifn-λ), in norovirus infection and their potential as norovirus antivirals has only recently been explored. 169 inhibition at 50 u/ml and~50% inhibition of the norwalk replicon at 100 u/ml. vitamin a-induced changes in the microflora are thought to be the mechanism responsible for these antiviral effects. 188 lastly, nitazoxanide (ntz) [2.5.4 ] (covered in section 3) has shown promise as an antiviral therapy, but the defined mechanism of activity against norovirus has yet to be determined. most recently ntz was shown to potently inhibit fcv replication in cell culture with an ec 50 of 0.6 µm, 189 and the gi norovirus replicon at a clinically relevant concentration (5 μg/ml), 190 which was later shown to result in a broad antiviral response. 191 despite these observations, the latter study also showed that ntz was ineffective against mnv suggesting that further antiviral investigations are warranted. 189 to date, no norovirus antiviral or vaccine is approved for medical use, and the only norovirus antiviral candidate to complete clinical trials is ntz. this compound was originally developed in the 1970s, and is currently an fdaapproved therapy for treating giardia and crytosporidium infections. 192 ntz has demonstrated broad-spectrum antimicrobial activity against a range of bacterial, protozoan, and viral infections, including inhibition of the norwalk replicon (ec 50 1.6 µm) (reviewed in rossignol 193 ). in phase ii randomized double-blind trial, ntz therapy was administered to 25 of 50 patients (≥ 12 years) that tested positive for rotavirus or norovirus infection. treatment resulted in a significant reduction in the duration of gastroenteritis symptoms (norovirus, p = 0.0295 and rotavirus, p = 0.0052) from 2.5 to 1.5 days when compared to the placebo. 194 additional anecdotal studies have supported the efficacy of ntz treatment for norovirus. ntz successfully treated one immunosuppressed transplant patient infected with norovirus that had experienced 10 consecutive days of gastroenteritis symptoms. 195 four days after commencing ntz treatment, a complete resolution of symptoms was recorded without a reduction in immunosuppressive drugs. 195 ntz treatment was also shown to resolve diarrheal symptoms and clear norovirus in stool samples from a pediatric patient with chronic norovirus, following kidney transplantation. 196 although the above-mentioned studies suggest that ntz is a promising therapy for the treatment of norovirus infections, there is an equal amount of evidence revealing that ntz is ineffective against norovirus netzler et al. infections. [197] [198] [199] [200] despite this contrary evidence, ntz represents the only therapeutic option currently available apart from rbv, immunoglobulins and supportive care to patients with persistent infections. human norovirus is a pervasive pathogen that creates a significant social and economic impact and causes hundreds of thousands of deaths each year. despite intensive research for safe and effective norovirus antivirals, none have yet been clinically approved, and the majority of candidates are still in the early stages of preclinical development. this review outlines recent therapeutic candidate studies to provide an updated overview of the current human norovirus antiviral development pipeline. optimization of human norovirus culture in enteroid and b cell systems offers the promise that in time, more robust and reliable culture methods will be developed allowing greater replication levels. these improved systems may enhance antiviral studies to provide a stronger platform for effective norovirus antiviral development. the landscape of antiviral development for norovirus is changing. the rapidly developing field of host immunomodulatory therapies is opening the door to potential treatments for many viral infections, with promising results already published for norovirus. moreover, recent discoveries have reported highly conserved binding pockets on critical viral enzymes, such as the norovirus polymerase, which could allow for the further development of broad-spectrum antivirals. the vast and varied global burden of norovirus: prospects for prevention and control aetiology-specific estimates of the global and regional incidence and mortality of diarrhoeal diseases commonly transmitted through food global economic burden of norovirus gastroenteritis norwalk virus shedding after experimental human infection global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis quantitative detection of norovirus excretion in 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candidate members of caliciviridae family in bats genomic characterization of swine caliciviruses representing a new genus of caliciviridae characterization of a novel calicivirus causing systemic infection in atlantic salmon (salmo salar l.): proposal for a new genus of caliciviridae advances in laboratory methods for detection and typing of norovirus recombination within the pandemic norovirus gii.4 lineage norovirus recombinants: recurrent in the field, recalcitrant in the lab-a scoping review of recombination and recombinant types of noroviruses the epidemiologic and clinical importance of norovirus infection emergence of a new norovirus genotype ii.4 variant associated with global outbreaks of gastroenteritis norovirus illness is a global problem: emergence and spread of norovirus gii.4 variants epidemics of gastroenteritis during 2006 were associated with the spread of norovirus gii.4 variants evolution of norovirus molecular epidemiology of norovirus in asymptomatic food handlers in busan, korea, and emergence of genotype gii.17 in japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region gastroenteritis outbreaks caused by norovirus gii identification of the novel kawasaki 2014 gii.17 human norovirus strain in italy genome of emerging norovirus gii emerging recombinant noroviruses identified by clinical and waste water screening noroviruses: state of the art pathogenesis of noroviruses, emerging rna viruses advances in norovirus biology norwalk virus minor capsid protein vp2 associates within the vp1 shell domain x-ray crystallographic structure of the norwalk virus capsid three-dimensional structure of baculovirus-expressed norwalk virus capsids expression, self-assembly, and antigenicity of the norwalk virus capsid protein human norovirus culture in b cells replication of human noroviruses in stem cell-derived human enteroids enteric bacteria promote human and mouse norovirus infection of b cells stable expression of a norwalk virus rna replicon in a human hepatoma cell line replicon cell culture system as a valuable tool in antiviral drug discovery against hepatitis c virus direct-acting antivirals for the treatment of hepatitis c virus infection: optimizing current ifn-free treatment and future perspectives nonnucleoside inhibitors of norovirus rna polymerase: scaffolds for rational drug design broad-spectrum antivirals against 3c or 3c-like proteases of picornaviruses, noroviruses, and coronaviruses a fluorescence-based high-throughput screen to identify small compound inhibitors of the genotype 3a hepatitis c virus rna polymerase in silico screening for human norovirus antivirals reveals a novel non-nucleoside inhibitor of the viral polymerase model systems for the study of human norovirus biology stat1-dependent innate immunity to a norwalk-like virus replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages characterization of ozone disinfection of murine norovirus comparison of chlorine and peroxyacetic-based disinfectant to inactivate feline calicivirus, murine norovirus and hepatitis a virus on lettuce disinfection kinetics of murine norovirus using chlorine and chlorine dioxide recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing t7 rna polymerase development of an optimized rna-based murine norovirus reverse genetics system chimpanzees as an animal model for human norovirus infection and vaccine development experimental infection of macaca nemestrina with a toronto norwalk-like virus of epidemic viral gastroenteritis experimental norovirus infections in non-human primates pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to a/h histo-blood group antigen expression pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup ii.4-hs66 strain of human norovirus intranasal p particle vaccine provided partial cross-variant protection against human gii.4 norovirus diarrhea in gnotobiotic pigs an experimental miniature piglet model for the infection of human norovirus gii inhibition of human norovirus by a viral polymerase inhibitor in the b cell culture system and in the mouse model a mouse model for human norovirus recombinant norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses norovirus gene expression and replication antiviral targets of human noroviruses anti-norovirus therapeutics: a patent review advances toward a norovirus antiviral: from classical inhibitors to lethal mutagenesis norovirus vaccines and potential antinorovirus drugs: recent advances and future perspectives recent advances in the discovery of norovirus therapeutics virus attachment and entry offer numerous targets for antiviral therapy functional receptor molecules cd300lf and cd300ld within the cd300 family enable murine noroviruses to infect cells discovery of a proteinaceous cellular receptor for a norovirus the p domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors atomic resolution structural characterization of recognition of histoblood group antigens by norwalk virus norovirus gastroenteritis, carbohydrate receptors, and animal models virus-host interaction and cellular receptors of caliciviruses structural basis for norovirus inhibition and fucose mimicry by citrate norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle inhibition of histo-blood group antigen binding as a novel strategy to block norovirus infections structural basis for norovirus inhibition by human milk oligosaccharides human norovirus inhibition by a human milk oligosaccharide treatment of norovirus particles with citrate development of norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis immunogenetic mechanisms driving norovirus gii. 4 antigenic variation serum immunoglobulin a cross-strain blockade of human noroviruses nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization nanobody binding to a conserved epitope promotes norovirus particle disassembly nanobodies: natural single-domain antibodies properties, production, and applications of camelid single-domain antibody fragments the flavivirus polymerase as a target for drug discovery the active form of the norovirus rna-dependent rna polymerase is a homodimer with cooperative activity comparison of the replication properties of murine and human calicivirus rnadependent rna polymerases crystal structures of active and inactive conformations of a caliciviral rnadependent rna polymerase inhibitors of the hepatitis c virus polymerase; mode of action and resistance a review of non-nucleoside anti-hepatitis b virus agents nucleoside/nucleotide analogues in the treatment of chronic hepatitis b the nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors in the treatment of hiv infections (aids) non-nucleoside reverse transcriptase inhibitors: a review on pharmacokinetics, pharmacodynamics, safety and tolerability nucleoside analogues: mechanisms of drug resistance and reversal strategies lethal mutagenesis of hiv with mutagenic nucleoside analogs synthesis and pharmacokinetics of valopicitabine (nm283), an efficient prodrug of the potent anti-hcv agent 2′-c-methylcytidine phosphoramidate prodrugs of 2′-c-methylcytidine for therapy of hepatitis c virus infection inhibition of norovirus replication by the nucleoside analogue 2′-c-methylcytidine the viral polymerase inhibitor 2′-c-methylcytidine inhibits norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model antiviral activity of nucleoside analogues against norovirus prophylactic treatment with the nucleoside analogue 2′-c-methylcytidine completely prevents transmission of norovirus favipiravir (t-705), a novel viral rna polymerase inhibitor favipiravir elicits antiviral mutagenesis during virus replication in vivo t-705 (favipiravir) induces lethal mutagenesis in influenza a h1n1 viruses in vitro t-705 (favipiravir) activity against lethal h5n1 influenza a viruses successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model t-705) inhibits in vitro norovirus replication biochemical evaluation of the inhibition properties of favipiravir and 2′-c-methyl-cytidine triphosphates against human and mouse norovirus rna polymerases ribavirin-current status of a broad spectrum antiviral agent interferons and ribavirin effectively inhibit norwalk virus replication in replicon-bearing cells crystal structures of murine norovirus-1 rna-dependent rna polymerase in complex with 2-thiouridine or ribavirin mechanism of action of interferon and ribavirin in treatment of hepatitis c ribavirin's antiviral mechanism of action: lethal mutagenesis? rna virus error catastrophe: direct molecular test by using ribavirin implications of high rna virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen chimerix announces discovery and demonstrated preclinical activity supporting ongoing phase 1 study of new antiviral for treatment and prevention of norovirus an objective assessment of conformational variability in complexes of hepatitis c virus polymerase with non-nucleoside inhibitors structure-based inhibition of norovirus rna-dependent rna polymerases naphthalene-sulfonate inhibitors of human norovirus rna-dependent rna-polymerase broad-spectrum non-nucleoside inhibitors for caliciviruses ppnds inhibits murine norovirus rna-dependent rna-polymerase mimicking two rna stacking bases inhibition of receptor/g protein coupling by suramin analogues delivery of suramin as an antiviral agent through liposomal systems structural bases of norovirus rna dependent rna polymerase inhibition by novel suramin-related compounds identification and characterization of inhibitors of human apurinic/ apyrimidinic endonuclease ape1 the enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon selection and characterization of rupintrivir-resistant norwalk virus replicon cells in vitro phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers potent inhibition of norovirus by dipeptidyl αhydroxyphosphonate transition state mimics design, synthesis, and evaluation of inhibitors of norwalk virus 3c protease substrate specificity of the norwalk virus 3c-like proteinase structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3cl protease. structure-activity relationships and biochemical, x-ray crystallographic, cellbased, and in vivo studies structural basis of substrate specificity and protease inhibition in norwalk virus potent inhibition of norovirus 3cl protease by peptidyl αketoamides and α-ketoheterocycles inhibition of norovirus 3cl protease by bisulfite adducts of transition state inhibitors structural and inhibitor studies of norovirus 3c-like proteases design, synthesis, and evaluation of novel prodrugs of transition state inhibitors of norovirus 3cl protease oxadiazole-based cell permeable macrocyclic transition state inhibitors of norovirus 3cl protease design, synthesis, and evaluation of a novel series of macrocyclic inhibitors of norovirus 3cl protease structure-guided design, synthesis and evaluation of oxazolidinone-based inhibitors of norovirus 3cl protease structure-based 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atg5-atg12/atg16l1 autophagy protein complex in antiviral activity of interferon gamma essential cell-autonomous role for interferon (ifn) regulatory factor 1 in ifn-γ-mediated inhibition of norovirus replication in macrophages critical role for interferon regulatory factor 3 (irf-3) and irf-7 in type i interferon-mediated control of murine norovirus replication type i and type ii interferons inhibit the translation of murine norovirus proteins interferons and ribavirin effectively inhibit norwalk virus replication in replicon-bearing cells mda-5 recognition of a murine norovirus the role of interferon in persistent viral infection: insights from murine norovirus distinct and overlapping genomic profiles and antiviral effects of interferon-λ and-α on hcv-infected and noninfected hepatoma cells dynamic expression profiling of type i and type iii interferon-stimulated hepatocytes reveals a stable hierarchy of gene expression il-28, il-29 and their class ii cytokine receptor il-28r ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex interferon-λ cures persistent murine norovirus infection in the absence of adaptive immunity expression of ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of interferon lambda against norovirus and reovirus interferon lambda (ifn-λ) efficiently blocks norovirus transmission in a mouse model gs-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees tlr7 agonist gs-9620 is a potent inhibitor of acute hiv-1 infection in human peripheral blood mononuclear cells tlr7 agonists display potent antiviral effects against norovirus infection via innate stimulation prophylactic efficacy of orally administered bacillus poly-γ-glutamic acid, a non-lps tlr4 ligand, against norovirus infection in mice toll-like receptors: the swiss army knife of immunity and vaccine development imiquimod for the treatment of genital warts: a quantitative systematic review antiviral effect of theaflavins against caliciviruses inhibitory mechanism of five natural flavonoids against murine norovirus comparison of the antiviral activity of flavonoids against murine norovirus and feline calicivirus naturally occurring flavonoids against human norovirus surrogates curcumin shows antiviral properties against norovirus antiviral effect of vitamin a on norovirus infection via modulation of the gut microbiome potential therapeutic agents for feline calicivirus infection opposing effects of nitazoxanide on murine and human norovirus nitazoxanide inhibits human norovirus replication and synergizes with ribavirin by activation of cellular antiviral response nitazoxanide: a new thiazolide antiparasitic agent nitazoxanide: a first-in-class broad-spectrum antiviral agent nitazoxanide in the treatment of viral gastroenteritis: a randomized double-blind placebo-controlled clinical trial norovirus gastroenteritis successfully treated with nitazoxanide successful treatment of chronic norovirus gastroenteritis with nitazoxanide in a pediatric kidney transplant recipient chronic diarrhea associated with persistent norovirus excretion in patients with chronic lymphocytic leukemia: report of two cases chronic norovirus infections in cardiac transplant patients: considerations for evaluation and management nitazoxanide is an ineffective treatment of chronic norovirus in patients with x-linked agammaglobulinemia and may yield false-negative polymerase chain reaction findings in stool specimens prolonged norovirus infection after pancreas transplantation: a case report and review of chronic norovirus her project focuses on identifying antiviral compounds for norovirus and other caliciviruses. in particular, her interest lies in the discovery of broad-spectrum antivirals with the potential to rapidly treat several clinically significant viruses for which we currently have no antivirals. during her phd, natalie was an author and editor for the book foodborne viral pathogens unsw and has been a tutor and demonstrator on the 2nd and 3rd year microbiology and virology courses following graduation, natalie worked as a research assistant at the biotechnology company, genesis research and development, for several years, investigating ligands involved in plant flowering and sirna inhibition targeting drug resistance he completed a b.med sci with first class honors majoring in microbiology and immunology from the university of new south wales in 2014 and is currently a phd candidate at unsw focusing on the biology of norovirus is also involved in teaching at unsw, including tutor and demonstration roles biology and microbiology he has a breadth of experience in the development of novel molecular assay systems and next-generation sequencing to investigate viral infections prof white commenced his postdoctoral research studies at the macquarie university, sydney, as a recipient of a royal society fellowship and later worked as hepatitis group leader at the prince of wales hospital until joining the university of new south wales in 2003. he is also an enthusiastic teacher, having convened the third-year science course viruses and disease for 15 years at unsw. how to cite this article the authors declare that there are no conflicts of interest. key: cord-103108-vmze2mdx authors: vanheer, lotte; schiavo, andrea alex; van haele, matthias; haesen, tine; janiszewski, adrian; chappell, joel; roskams, tania; cnop, miriam; pasque, vincent title: revealing the key regulators of cell identity in the human adult pancreas date: 2020-09-25 journal: biorxiv doi: 10.1101/2020.09.23.310094 sha: doc_id: 103108 cord_uid: vmze2mdx cellular identity during development is under the control of transcription factors that form gene regulatory networks. however, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. here, we integrate multiple single-cell rna sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. we show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. we present evidence that our approach identifies key regulators of cell identity in the human adult pancreas. we predict that heyl and jund are active in acinar and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell-derived pancreatic cells. the comprehensive gene regulatory network atlas can be explored interactively online. we anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity in the human adult pancreas. furthermore, given that transcription factors are major regulators of embryo development and are often perturbed in diseases, a comprehensive understanding of how transcription factors work will be relevant in development and disease biology. highlights reconstruction of gene regulatory networks for human adult pancreatic cell types an interactive resource to explore and visualize gene expression and regulatory states predicting putative transcription factors driving pancreatic cell identity heyl and jund as candidate regulators of acinar and alpha cell identity, respectively a fundamental question in biology is how a single genome gives rise to the great diversity of cell types that make up organs and tissues. a key goal is to map all cell types of developing and mature organs such as the pancreas, an essential organ at the basis of multiple human disorders including diabetes and cancer (kahn, cooper and del prato, 2014; kamisawa et al., 2016; han et al., 2020) . single-cell rna sequencing (scrna-seq) provides a powerful tool to resolve cellular heterogeneity, identify cell types and capture highresolution snapshots of gene expression in individual cells (grün et al., 2016) . with the advent of singlecell transcriptomics, great progress has been made toward the creation of a reference cell atlas of the pancreas (baron et al., 2016; segerstolpe et al., 2016; wang et al., 2016; xin et al., 2016; enge et al., 2017; lawlor et al., 2017; augsornworawat and millman, 2020; han et al., 2020) . work from several groups provided cellular atlases of the pancreas during mouse development (stanescu et al., 2017; byrnes et al., 2018; scavuzzo et al., 2018) , in adult mice (muraro et al., 2016) and in human fetal and adult pancreas (liu et al., 2014; baron et al., 2016; muraro et al., 2016; segerstolpe et al., 2016; wang et al., 2016; enge et al., 2017; han et al., 2020) . efforts have also been made to map cellular identity during pancreas development starting from human pluripotent stem cells hrvatin et al., 2014; zhu et al., 2016; han et al., 2020; hogrebe et al., 2020; peterson et al., 2020) . taken together, these studies provide an opportunity to better understand the maintenance and establishment of cellular identity among different pancreatic cell types. work over the past decades indicated that cellular identity is established by combinations of transcription factors (tfs) that recognize and interact with cis-regulatory elements in the genome. these transcription factors, together with chromatin modifiers, give rise to gene expression programs. a small number of core tfs are thought to be sufficient for the establishment and maintenance of gene expression programs that define cellular identity during and after development (ohno, 1979) . studies conducted in both mouse and human have successfully identified tfs that are pivotal for the acquisition and maintenance of pancreatic cell fates (dassaye, naidoo and cerf, 2016) . these include pdx1 (zhou et al., 2008; shih et al., 2015) , mafa (nishimura, takahashi and yasuda, 2015) , ngn3 (gradwohl et al., 2000) , nkx2.2 (sussel et al., 1998) , pax4 (sosa-pineda et al., 1997) , nkx6.1, neurod1 (mastracci et al., 2013) , arx (collombat et al., 2003) , mafb (artner et al., 2006) , rfx6 (smith et al., 2010) , gata4 (ketola et al., 2004) , foxa2 and sox9 (shroff et al., 2014; shih et al., 2015) . conditional deletion of tfs such as foxa2 and pdx1 in adult beta cells results in the loss of cellular identity and function (sund et al., 2001; gao et al., 2014) . genetic evidence for the role of these tfs in establishing human pancreatic cell identity is provided by the identification of tf loss-of-function mutations that cause pancreatic agenesis (stoffers et al., 1997; sellick et al., 2004; allen et al., 2012; shaw-smith et al., 2014; de franco et al., 2019) or neonatal or young-onset diabetes (senée et al., 2006; solomon et al., 2009; rubio-cabezas et al., 2010 smith et al., 2010; bonnefond et al., 2013; flanagan et al., 2014) . in addition, tf overexpression can reprogram somatic cells to adopt alternative identities (takahashi and yamanaka, 2006; zhou et al., 2008; vierbuchen et al., 2010; lima et al., 2016) . for example, the induced expression of ngn3, pdx1, and mafa was shown to reprogram mouse alpha cells into beta-like cells in vivo (zhou et al., 2008) . however, how key tfs underlie the maintenance of cellular identity in the human pancreas remains incompletely understood. over the past decade, multiple approaches to reconstruct gene regulatory networks (grns) from bulk and single-cell omics data have been developed (ghazanfar et al., 2016; lim et al., 2016; matsumoto et al., 2017; fiers et al., 2018) . in particular, it is now possible to combine single-cell transcriptomic data with cisregulatory information to infer grns (janky et al., 2014; aibar et al., 2017; van de sande et al., 2020) . because tfs recognize dna motifs in the genome, one can measure if inferred target genes are expressed within single cells, and therefore quantify the activity of tfs. such approaches have revealed the regulatory programs in distinct systems including the drosophila brain (davie et al , 2018) , cancer (wouters et al., 2020) , during early mouse embryonic development (peng et al , 2019) , in a mouse cell atlas (suo et al., 2018 ) and a human cell atlas (han et al., 2020) . analysis of grns in the human adult pancreas has identified distinct endocrine and exocrine regulatory states with multiple stable cell states for alpha, beta and ductal cells (kumar and vinod, 2019) . type 2 diabetes and body mass index (bmi) were shown not to impact grn activity of alpha and beta cells (kumar and vinod, 2019) . previous data shows that type 2 diabetic (faerch et al., 2015; dennis et al., 2019; dybala and hara, 2019; zaharia et al., 2019) and non-diabetic human islet preparations vary greatly depending on age (enge et al., 2017; westacott et al., 2017) and bmi (henquin, 2018) warranting the exploration of grns in larger cohorts. hence, it remains unclear whether previous grn findings can be extrapolated to a broader, highly heterogeneous non-diabetic and type 2 diabetes patient population. the development of integration methods provides an opportunity to analyze multiple scrna-seq studies from multiple laboratories and patients (butler et al., 2018; luecken et al., 2020) . additional knowledge on how grns maintain cellular identity in the human adult pancreas may further the understanding of disease states as well as improve efforts to convert patient cells into functional, mature beta cells for diabetes treatment. here, we build an integrated human pancreas gene regulatory atlas. in this resource, we use single-cell transcriptomes of the human adult pancreas, taking advantage of integration strategies and computational tools to reconstruct grns. our analysis identifies the grn landscape and candidate regulators that are critical for cellular identity in the human adult pancreas. integrating multiple human adult pancreas scrna-seq datasets can further improve the power of scrnaseq analyses to create a human adult pancreas cell atlas. we set out to analyse and integrate five publicly available datasets covering a total of 35 non-diabetic, 15 type 2 diabetic and 1 type 1 diabetic individuals using seurat v3.0 cca integration tools ( figure 1a , detailed donor information can be found in table s1 ) (segerstolpe et al., 2016; wang et al., 2016; xin et al., 2016; enge et al., 2017; lawlor et al., 2017; hafemeister and satija, 2019; stuart et al., 2019) . after filtering out low quality transcriptomes and data integration, uniform manifold approximation and projection for dimension reduction (umap) visualisation revealed that 7393 cells localize into distinct clusters ( figure 1b) . cells from each original dataset localize together suggesting that the location of cells on the umap is not driven by the dataset of origin ( figure 1c ). we next sought to identify pancreatic cell types ( figure 1d ). clustering analyses based on the expression of well-established cell type specific markers led to the identification of eight cell types in the human adult pancreas: beta, alpha, gamma, delta, acinar, ductal, stellate and endothelial cells ( figure 1b , table s2 ). umap visualization allowed for the segregation of endocrine, exocrine and other lineages ( figure s1a ). beta cells grouped together, away from other clusters and were marked by ins expression (figure 1e ). other distinct clusters corresponded to alpha, gamma and delta cells based on global transcriptional similarity and elevated expression of gcg, ppy and sst, respectively, and other markers ( figure 1e , table s2 ). using a similar approach, we reliably detected other, previously described, major pancreatic cell types (acinar, ductal, endothelial and stellate, figure 1b ). all cell types were detected in both non-diabetic and type 2 diabetic pancreases ( figure 1f ). an additional four rare cell populations, that cannot be robustly identified through clustering analyses, were identified manually by assessing the expression of ghrl (epsilon cells), tps1ab (schwann cells), cd86 (mast cells) and sox10 (major histocompatibility complex (mhc) class 2 cells) (wierup et al., 2002; segerstolpe et al., 2016) (figure 1g ). these rare cell types often cluster with other common cell types. importantly, our annotation recapitulated previous annotations to a large extent (figure s1b-c). in summary, we reconstructed an integrated single-cell atlas of the human adult pancreas, and annotated 12 pancreatic cell types. next, we set out to comprehensively reconstruct grns for all pancreatic cell types from single-cell transcriptomic data, applying single-cell regulatory network inference and clustering (pyscenic) (aibar et al., 2017; van de sande et al., 2020) . pyscenic links cis-regulatory sequence information together with single-cell transcriptomes in three sequential steps by 1) co-expression analysis, 2) target gene motif and chip-seq track enrichment analysis, and 3) regulon activity evaluation (figure 2a) . each regulon consists of a tf with its predicted target genes (co-expressed genes with an enriched tf motif), altogether forming a regulon. pyscenic identified 142 regulons that characterize the grns of the human adult pancreas 5 ( figure 2b /c, table s3 ). multiple regulons identified here as active in the pancreas correspond to tf binding motifs enriched in accessible chromatin in the pancreas (assessed by atac-seq in facs-purified pancreatic cells, (arda et al., 2018) ), supporting the validity of the approach ( figure s2a ). umap visualization based on the activity of 142 regulons in non-diabetic and type 2 diabetic pancreata revealed groups of cells that differ from one another based on their regulatory activity ( figure 2b -c). in particular, there are distinct regulatory states for exocrine and endocrine pancreatic lineages, stellate and endothelial cells (figure 2b /c). endocrine cell types clustered together, indicating shared regulatory states, while exocrine cell types formed two distinct clusters. stellate and endothelial cells differed most from other cell types in their regulatory states. these results are consistent with previous analyses (baron, veres, samuel l. wolock, et al., 2016; lawlor et al., 2017; kumar and vinod, 2019) and are also in line with our findings above based on gene expression analysis ( figure 1d ). as expected, regulons active in endocrine cell types include rfx6, pax6 and neurod1 ( figure 2d -g). these tfs have reported roles in endocrine cell fate commitment and maintenance of cell identity throughout adult life (smith et al., 2010; hart et al., 2013; mastracci et al., 2013) . using iregulon for visualization, many of the neurod1 target genes identified here have been previously linked to beta cell survival and function, such as snap25, tspan2, (gierl et al., 2006; haumaitre et al., 2008; hart et al., 2013; churchill et al., 2017) . clustering all cells based on the activity of all regulons identifies regulatory modules ( figure 2d , red squares). in the exocrine pancreas, one regulatory module, containing nr5a2, was shared between acinar and ductal cells, although with a tendency for increased regulon activity in ductal cells ( figure 2d /h). other exocrine regulons included onecut1, rest and hnf1b with reported roles in exocrine development (nissim et al., 2016; kropp, zhu and gannon, 2019) and the adult exocrine pancreas (quilichini et al., 2019; bray et al., 2020 ) ( figure 2d ). in summary, this analysis confirms the expected separation of exocrine and endocrine cells with distinct gene regulatory programs, and identifies known and novel candidate regulators of pancreas cell states. several regulatory modules are shared between different cell types within the endocrine and exocrine pancreas. additionally, each cell type is defined by cell-type specific regulatory modules ( figure 1d ). in the endocrine pancreas, alpha and beta cells shared endocrine regulons (mafb, meis2), whereas we observed distinct activities for arx and irx2 regulons in alpha cells and rxrg and pdx1 in beta cells ( figure 2d /h), expanding previous findings (kumar and vinod, 2019) . using iregulon for visualization, pdx1 target genes include slc6a17, pdia6 and abhd3, which have been reported to control insulin release (thomas, brown and brown, 2014; eletto et al., 2016; rorsman and ashcroft, 2018 ) ( figure s2c ). interestingly, gamma and delta cells overlapped with alpha and beta cells, respectively, suggesting a shared regulatory state ( figure 2c/d) . this includes shared regulon activity for arx in gamma and alpha and pdx1 in beta and delta cells ( figure 2d /h), consistent with their reported expression in published scrna-seq studies (baron et al., 2016; segerstolpe et al., 2016; lawlor et al., 2017) . gata4 and rbpjl, known acinar-specific tfs, were highly active in acinar cells (masui et al., 2010; carrasco et al., 2012) ( figure 2h) . similarly, ductal cells were characterised by highly active sox9 and pou2f3 regulons, in line with previous literature (shroff et al., 2014; yamashita et al., 2017) (figure 2h ). in sum, this analysis confirms that alpha, beta, acinar and ductal cells are defined by the activity of distinct tf combinations that form gene regulatory modules. in conclusion, the network approach recovers many of the expected regulators of pancreatic cellular identity allowing for the comprehensive characterisation of the gene regulatory state of all major human adult pancreatic cell types. a comprehensive network analysis provides an opportunity to predict and identify critical regulators of cell identity. to identify regulons with highly cell type-specific activities within the human adult non-diabetic pancreas, we calculated regulon specificity scores (rss) (a complete list of rsss can be found in table s4 ) (suo et al., 2018) . the rss utilises jensen-shannon divergence to measure the similarity between the probability distribution of the regulon's enrichment score and cell type annotation wherein outliers receive a higher rss and are therefore considered cell type-specific (suo et al., 2018) . it can therefore be used to rank the activity of tfs within specific cell types. among the top regulons identified in alpha cells, we recover well known regulators of alpha and endocrine cell fate such as arx, irx2, pax6, mafb, neurod1 and rfx6 ( figure 3a /b) (collombat et al., 2003; artner et al., 2006; delporte et al., 2008; smith et al., 2010; dorrell et al., 2011; mastracci et al., 2013) . in addition, we identified jund, egr4, srebf1 and stat4, which have not yet been implicated in alpha cell identity. egr1 (but not egr4) has been shown to transcriptionally regulate glucagon expression (leungtheung-long et al., 2005) as well as the pdx1 promoter in beta cells (eto, kaur and thomas, 2007) . stat4 and jund have been described in pancreatic tissue in general and beta cells, respectively, but not in alpha cells (yu and kim, 2012; good et al., 2019) (figure 3b /c). these tfs respond to the jnk and egfr signalling pathways and may have important physiological functions. both jund and the jund/jnk signaling pathway have been implicated in pancreatic cancer (shin et al., 2009; recio-boiles et al., 2016) . immunocytochemistry of the human adult pancreas confirmed the presence of nuclear jund in islets ( figure 3di ). we also detected nuclear jund protein in a subset of human induced pluripotent stem cells (ipscs) subjected to beta cell differentiation (figure 3e/f) . surprisingly, we also detected jund protein in ductal cells, despite lower jund regulon activity in this cell type (figures 3c, 3dii) . thus, protein 7 expression does not always predict regulatory activity. nevertheless, these results show that jund is present and active in a subset of pancreatic cell types in the human adult pancreas and human pluripotent stem cell derived islet cells. altogether, this analysis predicts tfs active in human alpha cells, recovering known as well as new candidate tfs. among the top regulons identified in beta cells, we retrieved well-known as well as new candidate regulators of beta and endocrine cell identity. known tfs include rxrg, pdx1, neurod1, pax6 and rfx6 (zhou et al., 2008; miyazaki et al., 2010; smith et al., 2010; mastracci and sussel, 2012; hart et al., 2013) (figure 3g /h). in addition, we found that znf705d, ascl2 and hoxd13 were highly ranked regulons ( figure 3h /i). hoxd13 and bhlhe41 have been shown to be present in the exocrine pancreas (cantile et al., 2009; sato et al., 2012) . interestingly, ascl2 has been reported to interact with β-catenin of the wnt pathway, the latter has an established role in endocrine fate specification during in vitro differentiation (schuijers et al., 2015; sharon et al., 2019; vethe et al., 2019) . many putative target genes of ascl2 including pdx1, ins, abcc8, foxa1, kcnk16, fxyd2 are directly related to glucose sensing and beta cell identity, in line with the beta cell-specific regulatory activity of ascl2 ( figure s3a ) (gao et al., 2010; arystarkhova et al., 2013; vierra et al., 2015; park, lee and park, 2016) . fxyd2γa, a regulatory subunit of the na + -k + -atpase, is a transcript exclusively expressed in human beta cells (flamez et al., 2010) . immunohistochemistry of human adult pancreas sections showed that ascl2 is expressed in ins + beta and islet cells ( figure 3j) . surprisingly, ascl2 was mainly localized to the cytoplasm ( figure 3j) , which is unexpected for tfs which tend to localize to the nucleus (baranek, sock and wegner, 2005) . cytoplasmic localization of ascl2 has been reported in the context of colon and breast cancer (zhu et al., 2012; xu et al., 2017) . these results implicate additional tfs including ascl2 in the regulation of beta cell identity. they also illustrate the value of network analyses to increase our understanding of the biology of the human pancreas. in summary, grn analysis and regulon ranking allowed us to pinpoint both known and novel candidate regulators of pancreatic endocrine cell identity, providing a resource for further investigation of their roles in cellular identity and function. similarly, the comprehensive network analysis provides an opportunity to predict and identify critical regulators of exocrine cell identity. we also identify known and new tfs in acinar cells. among the top acinar-specific regulons, we recovered well known regulators of acinar and exocrine cell identity such as ptf1a, rbpjl, gata4 and nr5a2 (ketola et al., 2004; masui et al., 2010; nissim et al., 2016; sakikubo et al., 2018) (figure 4a/b) . these findings are in line with a recent study that used single-nucleus rna-seq on pancreatic acinar tissue (tosti 8 et al., 2019) . furthermore, we identified mecom, heyl and tgif1 as highly ranked regulons ( figure 4b /c). interestingly, aberrant mecom expression has been linked to the induction of gastric genes in acinar cells, which disrupts acinar cell identity and increases susceptibility to malignancy (hoang et al., 2016) . the loss of tgif1 has been linked to pancreatic ductal adenocarcinoma progression making further exploration of these regulons interesting in the context of cancer biology (weng et al., 2019) . ectopic expression of tgif2 (but not tgif1) reprograms mouse liver cells towards a pancreas progenitor state (cerdá-esteban et al., 2017) . heyl is a reported notch signalling target gene in ngn3 + exocrine cells (gomez et al, 2015) . we confirmed nuclear expression of heyl in human acinar and islet cells (figure 4di /ii, detailed donor information can be found in table s1 ) by immunohistochemistry, in agreement with elevated heyl regulon activity in acinar cells ( figure 3c ). further functional studies in the healthy pancreatic context (matsumoto et al., 2004; coleman et al., 2013) . in summary, grn analysis and regulon ranking allowed us to pinpoint both known and novel candidate regulators of pancreatic exocrine cell identity. specifically, we identified heyl as a candidate tf that might be important for acinar cell identity, warranting further investigation. to enable users to easily navigate the human pancreatic cell network atlas, we provide a loom file that allows for the visualisation and exploration of the data using the web-based portal scope (davie et al., 2018) (.loom file and tutorial available at http://scope.aertslab.org/#/pancreasatlas/*/welcome and https://github.com/pasquelab/scpancreasatlas). features such as cell type annotation as defined in this paper, gene expression and regulon activity can be explored on the regulon and gene expression based umap. this resource enables users to select and visualize up to three genes or regulons simultaneously and select subsets of cells for downstream analyses. for example, the expression of covid-19 related genes can be interactively explored (yang et al., 2020) . target genes of a specific regulon can be downloaded to facilitate further exploration, for example in iregulon or gene ontology analysis (janky et al., 2014) . a list of predicted target genes of all 142 regulons can also be found in table s5 . furthermore, a list of target genes can be manually defined to compute the activity of a custom regulon. this resource can be used to further study cell identity and gene regulation in the context of the pancreas, diabetes and cancer. in this resource, we take advantage of integration strategies and new computational tools to reconstruct an integrated cell and grn atlas of the human adult pancreas from single-cell transcriptome data. this approach provides a comprehensive analysis of the gene regulatory logic underlying cellular identity in the human adult pancreas in a broad range of individuals, limiting the influence of inter-donor variability. we recovered known regulators of pancreatic cell identity and uncovered novel candidate regulators of cell identity that can be further investigated for their roles in cellular identity and function. by validating regulon analyses and creating an easily accessible interactive online resource which allows for the exploration of the gene regulatory state of 7393 cells from 51 individuals, this approach extends beyond previous gene regulatory studies in the human adult pancreas (augsornworawat and millman, 2020) . the present analysis identified regulators of pancreatic development, function and survival that are known to be critical in humans because loss-of-gene function causes pancreatic agenesis or young onset diabetes. for example, ptf1a (sellick et al., 2004; weedon et al., 2014) and gata4 (shaw-smith et al., 2014) , whose loss of function are linked to pancreatic agenesis and neonatal diabetes, were among the top acinarspecific regulons (figure 3 ). in addition, monogenic diabetes related genes pdx1 (nicolino et al., 2010 ), neurod1 (rubio-cabezas et al., 2010 , pax6 (solomon et al., 2009) , rfx6 (smith et al., 2010; patel et al., 2017) and glis3 (senée et al., 2006) were among the top beta cell-specific regulons (figure 3 and table s4 ). stress signalling (table s4) . creb3 and creb3l2 are non-canonical er stress transducers that are induced in human islets and clonal beta cells upon exposure to the saturated fatty acid palmitate (cnop et al., 2014) . interestingly, srebf1 and -2 undergo similar er exit and proteolytic processing in the golgi as these er stress transducers, but they do so in response to changes in er cholesterol content; both also have high regulon activity in alpha and beta cells. xbp1 is abundantly expressed in the exocrine and endocrine pancreas (cnop et al., 2017) , but the xbp1 regulon has its highest specificity in beta cells. atf3 and atf4 are tfs that are activated upon eif2α phosphorylation, an er stress response pathway to which no less than 5 monogenic forms of diabetes belong (eizirik, pasquali and cnop, 2020) . our data underscores the importance of these tfs for endocrine pancreatic cell identity. given that we predict novel regulators of cell identity in the human pancreas, it will be interesting to also expand this analysis to pancreas embryonic development. our work may also be beneficial in guiding improvements of and better understanding the in vitro derivation of pancreatic cell types. for example, the emergence of sst-positive cells together with beta-like cells at the end of in vitro differentiation could be explained by the overlap in regulatory states between beta and delta cells (baron et al., 2016) . grn analyses are particularly interesting for in vitro derived beta cells since a better understanding of the regulatory logic underlying control of beta cell fate may improve or facilitate future applications in regenerative medicine (pagliuca et al., 2014; rezania et al., 2014; nostro et al., 2015; russ et al., 2015; baeyens et al., 2018) . alternatively, many observed grns such as ascl2, mecom, ppard, gata6 and cdx2 are linked to pancreatic cancer making the additional exploration of grns interesting in the context of cancer biology (matsumoto et al., 2004; zhu et al., 2012; coleman et al., 2013; hoang et al., 2016; xu et al., 2017; weng et al., 2019; brunton et al., 2020) . finally, recent reports have stratified type 2 diabetes patients based on age at diagnosis, bmi, hba1c and insulin secretion and sensitivity, and identified subtypes with different genetic predisposition, treatment response, disease progression and complication rates (ahlqvist et al., 2018) . hence, it would be interesting to assess differences in gene regulatory state and gene expression profiles of alpha and beta cells between different type 2 diabetic subgroups. it is important to note that pyscenic is a stochastic algorithm that does not produce precisely the same regulons for repeated applications, limiting reproducibility when comparing different datasets (huynh-thu et al., 2010; van de sande et al., 2020) . to mitigate this uncertainty, we ran the full pyscenic pipeline five times and only kept consistent regulons with the highest regulon activity. the performance of pyscenic, and other grn inference methods, suffers due to the large amount of drop-out events in scrna-seq data warranting caution when interpreting results (chen and mar, 2018) . this could explain the absence of wellestablished pancreas tfs such as mafa (olbrot et al., 2002) , mnx1 (flanagan et al., 2014) , neurog3 (krentz et al., 2017) , foxa2 (lee et al., 2002) and nkx2-2 (mastracci et al., 2011) in this analysis. nevertheless, in support of the validity of our findings, atac-seq, literature and immunohistochemistry of human pancreas sections corroborate several pyscenic predictions. chen and colleagues underline the importance of using large sample sizes to derive the most accurate network inference possible (chen and mar, 2018) , highlighting the importance of dataset integration to increase the number of cells analysed. in the future, it will be interesting to extend our analyses to include many more cells and patients. in spite of current caveats, grn analysis has enabled the capture of biological relevant information (butte et al., 2000) . one additional limitation of this study is the assumption that all tfs bind their binding motifs in the promoters of expressed genes. however, tf binding can be restricted to a subset of tf motifs in the genome due to influence of chromatin processes including the presence of nucleosomes as well as dna methylation. therefore, additional approaches such as single cell multi-omics that capture additional layers of genome regulation will be helpful to increase our understanding of gene regulation in the context of the human pancreas. taken together, our grn atlas, containing 51 individuals, provides a valuable resource for future studies in the human pancreas development, donor variability, homeostasis and disease including type 2 diabetes 11 and pancreatic cancer. finally, our results provide new insights into the activity of tfs and gene regulation in the human adult pancreas from a gene regulatory perspective. questions about data analysis should be directed to the lead contact, vincent pasque (vincent.pasque@kuleuven.be). the reviewer tokens for this geo repository is cfsjciumfferjkd. motif discovery of bulk atac-seq data paired-end raw reads for bulk atac-seq (see key resource table) were downloaded from sra using sra toolkit (v2.9.4). reads were aligned and further analyzed using the encode atac-seq pipeline with default parameters using the encode human reference genome grch38.15 (lee, 2016) . bed files containing the global open chromatin landscape of adult alpha (alpha_1; ea4 and alpha_2; ea28), beta (beta_1; ea5 and beta_2; ea29), acinar (acinar_1; ea7 and acinar_2; ea27) and ductal (ea11) cells or cell type specific differentially accessible regions were used as input for motif discovery by homer (v4.10.4) using the 'findmotifsgenome.pl' with options using hg38 with size given (heinz et al., 2010) . the tfs whose motifs identified by homer correspond with tfs identified by pyscenic are visualized in figure s2a . analysis of publicly available scrna-seq data raw reads for five publicly available scrna-seq datasets (see key resource table) were downloaded from sra using sra toolkit (v2.9.4). afterwards, reads were aligned to the human reference genome grch38.95 using star (v2.5.3a) with default parameters followed by the conversion to the coordinate sorted bam format. next, the featurecounts command from the "rsubread" (v1.5.2) package in r (v3.6.1) was used to assign mapped reads to genomic features. low quality transcriptomes with a mitochondrial contamination greater than 5% and less than 200 expressed genes per cell were excluded from subsequent analyses. the resulting raw count matrix was batch corrected using the findintegrationanchors and integratedata functions from the "seurat" package (v3.1.1) after which subsequent analyses were carried out in the r package "seurat" (v3.1.4). gene expression was used to cluster all 7393 cells with umap, using seurat's function runumap. clusters for cell type annotation were defined using seurat's shared nearest neighbour algorithm findclusters function after which differential expression analysis was performed using wilcoxon's rank sum test with a minimum cutoff of 0.25 average log fold change and min.pct of 0.25. pyscenic grns were inferred using pyscenic (python implementation of scenic, v0.9.15) in python version 3.6.9 (aibar et al., 2017) . integrated read counts were used as input to run genie3 (huynh-thu et al., 2010) which is part of arboreto (v0.1.5). grns were subsequently inferred using pyscenic with the hg38_refseq-r80 motif database and default settings. to control for the stochasticity, which is inherent to pyscenic, a consensus grn was generated by merging results from five repeat pyscenic runs. if regulons were identified in multiple pyscenic runs, only the regulon with the highest auc value was retained. regulon activity represented by aucell values was used to cluster all 7393 cells with umap, using seurat's runumap function. all 142 regulons within non-diabetic cell types were visualized using the 'clustermap' function of the python package "seaborn" (v0.9.0). the z-score for each regulon across all cells was calculated using the z-score parameter of the seaborn 'clustermap' function. extended analysis of the target genes of specific regulons was conducted in cytoscape (v3.7.1) using the iregulon application (v1.3). the list of target genes of a specific regulon was downloaded from the loom file through the scope platform (https://github.com/pasquelab/scpancreasatlas) (davie et al., 2018) . to quantify the cell-type specificity of a regulon, we utilized an entropy-based strategy as described previously (suo et al., 2018) using the aucell matrix as input in matlab r2019b. the top 10 most specific regulons were subsequently visualized using the r package ggplot2 (v3.1.1). the complete regulon ranking list is available in table s2 . control ipsc line hel115.6 (cosentino et al., 2018) was differentiated into beta cells using a previously published 7-step protocol (cosentino et al., 2018) . at the end of the stage 4, cells were seeded into 24-well aggrewell 400 microwell plates (stem cell technologies) at a density of 0.9 ·10 6 cells per well after which differentiation was carried out as described previously (cosentino et al., 2018) . stage 7 differentiated beta cells were washed twice with pbs containing 0.5 mm edta and incubated in accumax (sigma #a7089) for 8 min at 37 °c after which 50% volume of knockout serum replacement (thermofisher #10828028) was added to stop the reaction. after centrifugation at 400 g for 5 min at room temperature, cells were resuspended in 1 ml ham's f-10 medium, supplemented as indicated before (demine et al., 2020) . 75,000 cells in 500 μl medium were seeded per square of a nunc lab-tek ii icc chamber (thermofisher). immunohistochemistry analyses were carried out largely as described previously (demine et al., 2020) , immunohistochemistry analyses were carried out largely as described previously (ceulemans et al., 2017) , using primary antibodies against the following proteins: ascl2 (merck, mab4418, clone 7e2, 1/5000), jund (atlas antibodies, hpa063029, 1/50), heyl (atlas antibodies, hpa076960, 1/200) and ins (agilent, ir002, 1/100). pictures were taken using a leica dmlb (leica microsystems). the integrated single-cell rna-seq data and pyscenic results can be explored interactively in scope (davie et al., 2018) . loompy (v2.0.17) (linnarsson lab., 2015) was used to create the loom files which were uploaded to scope. the embedding of the regulon and integrated gene expression based umap clustering, as seen in this article, were added to the loom file. this table is related to figure 1 , 2, 3 and 4. this table is related to figure 1 . this table is related to figure 2 , 3 and 4. list of regulon specificity scores for all 142 regulons of non-diabetic alpha, beta, acinar and ductal cells. this table is related to figure 3 and 4. list of putative target genes for each regulon. this table is related to figure 2 , 3 and 4. novel subgroups of adult-onset diabetes and their association with outcomes: a data-driven cluster analysis of six variables', the lancet diabetes and endocrinology scenic: single-cell regulatory network inference and clustering', nature methods reduced expression of plcxd3 associates with disruption of glucose sensing and insulin signaling in pancreatic β-cells', frontiers in endocrinology gata6 haploinsufficiency causes pancreatic agenesis in humans a chromatin basis for cell lineage and disease risk in the human pancreas an activator of the glucagon gene expressed in developing islet α-and β-cells hyperplasia of pancreatic beta cells and improved glucose tolerance in mice deficient in 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and endocrinology in vivo reprogramming of adult pancreatic exocrine cells to β-cells' ascl2 knockdown results in tumor growth arrest by mirna-302b-related inhibition of colon cancer progenitor cells genome editing of lineage determinants in human pluripotent stem cells reveals mechanisms of pancreatic development and diabetes we thank stein aerts, kristofer davie and the stein aerts lab for discussions and creating the permanent scope link, shengbao suo for sharing the matlab script for calculating the regulon specificity score, the key: cord-310371-pylrg91h authors: bishop, r.f.; kirkwood, c.d. title: enteric viruses date: 2008-07-30 journal: encyclopedia of virology doi: 10.1016/b978-012374410-4.00386-1 sha: doc_id: 310371 cord_uid: pylrg91h many viruses use the enteric tract as a route of entry to the human, animal, or avian host. the onset of acute enteritis is associated with infection by viruses that replicate at or near the site of entry into the intestinal mucosa, including caliciviruses, rotaviruses, adenoviruses, astroviruses, and coronaviruses. these ‘enteric’ viruses occur globally and share similar features. most are rna viruses that replicate in the cytoplasm of mature absorptive epithelial cells lining the villi of the small intestine, leading to inflammation and villus atrophy. vomiting and diarrhea can result in dehydration and death if untreated. despite abundant growth in vivo, they initially proved difficult or impossible to grow in vitro. most are genetically diverse, species specific, highly infectious within species and transmitted by the fecal–oral route. severe symptoms are most commonly associated with primary infections of young animals, and are followed by short-lived immunity. reinfections are common throughout life, but are often only mildly symptomatic. safe and effective vaccines have been developed to prevent severe rotavirus disease in young children. in addition to these enterotropic viruses, enteric disease can also result from spread to the intestine of hiv or cytomegaloviruses during the later stages of systemic disease in immunocompromised hosts. the intestinal tract, lined by replicating epithelial cells, bathed in nutrient fluids and maintained at optimal temperature provides an ideal milieu for growth of many viruses. 'enteric viruses' represent a wide spectrum of viral genera that invade and replicate in the mucosa of the intestinal tract, and that can be grouped as follows: . viruses causing localized inflammation at any level of the intestinal tract, predominantly in small intestinal mucosa, resulting in acute gastroenteritis, for example, rotaviruses, caliciviruses, adenoviruses, astroviruses; . viruses that multiply at any level of the intestinal tract, causing few enteric symptoms prior to producing clinical disease at a distant site, for example, measles virus, reoviruses (in mice) , enteroviruses (including polioviruses, coxsackieviruses, enteroviruses, hepatitis a and e); and . viruses that spread to the intestinal tract during the later stages of systemic disease, generally in an immunocompromised host, for example, human immunodeficiency virus (hiv), cytomegalovirus. this article focuses upon the first category of viruses that cause enteric disease associated with primary replication in the intestinal tract. acute gastroenteritis is one of the most common health problems worldwide. more than 700 million cases are estimated to occur annually in children less than 5 years of age, resulting in few deaths in developed countries, but more than 2 million deaths in developing countries. worldwide, a diverse group of viral, bacterial, and parasitic pathogens cause acute enteric symptoms including nausea, vomiting, abdominal pain, fever, and acute diarrhea. infections with viral agents, unlike those with bacterial or parasitic pathogens, cannot be treated with antibiotics, and many cannot be prevented by improvements in quality of drinking water, food, or sanitation. until the early 1970s most viral agents causing gastroenteritis in humans were largely unknown. studies using electron microscopy of intestinal contents resulted in the discovery of numerous viral enteropathogens now classified as caliciviruses, rotaviruses, astroviruses, or 'enteric' adenoviruses. caliciviruses are now recognized as the most important cause worldwide of outbreaks of viral gastroenteritis in humans of all age groups. rotaviruses are the single most important cause of life-threatening diarrhea in children <5 years old. astroviruses and adenoviruses also cause severe diarrhea in children. table 1 lists the characteristics of the major viruses associated with acute gastroenteritis. other viruses linked to gastroenteritis in humans include coronaviruses, toroviruses, picornaviruses, and picobirnaviruses. understanding many features of these 'enteric viruses' has been based on parallel studies of related viruses infecting animals. the family caliciviridae contain small rna viruses that cause enteric disease in a wide variety of hosts including cattle, pigs, rabbits, and humans. infections in other hosts, for example, sea lions, cats, and primates, appear to cause predominantly systemic and respiratory symptoms. caliciviruses are small nonenveloped viruses of 27-35 nm diameter ( figure 1) with a genome comprising a single-stranded positive-sense polyadenylated rna genome of 7400-7700 nucleotides (nt). typical calicivirus particles show cup-like hollows (calices) on the virus surface. some caliciviruses have an indistinct appearance described as 'feathery'. the 'norwalk agent' (now classified as a calicivirus) was identified in 1972 by kapikian and colleagues, using immune-electron microscopy (iem) to search for the causative agent of an 1968 outbreak of gastroenteritis in humans in norwalk, ohio, usa. many related viruses have been implicated as causes of gastroenteritis, and given names identifying the geographical location of the outbreak (e.g., marin county, snow mountain, hawaii, sapporo). classification of caliciviruses was hampered for many years by the inability to culture these viruses, and study their genetic and protein structure. caliciviruses causing enteric infections (in humans and other animals) are classified as belonging to the family caliciviridae, which is divided into four genera. the genus norovirus (nov), and genus sapovirus (sav) cause human and animal infections. other genera infect rabbits (lagovirus) or sealions, cats, and primates (vesivirus). the genome organization and reading frame usage differs between the four genera. for noroviruses, the genome contains three open reading frames (orfs). the largest (orf1) encodes a polyprotein which undergoes proteolytic cleavage to produce an ntpase, a 3c-like protease, and an rnadependent rna polymerase (rdrp). orf2 encodes the major capsid protein and orf3 a putative minor capsid protein. noroviruses are subdivided into five genogroups (gi-gv). genogroups gi, gii, and giv infect humans, giii infects cattle and gv infects mice. gi and gii contain at least 10 and 20 distinct genetic clusters, respectively. sapoviruses are divided into genogroups (gi-gv), of which gi, gii, giv, and gv infect humans. gi and gii are further divided into four and three genetic clusters. the inability to culture human caliciviruses delayed the introduction of diagnostic tests, resulting in an under-appreciation of the significance of these agents for many years. currently, over 20 different reverse transcription-polymerase chain reaction (rt-pcr) assays targeting regions on the rdrp gene and the capsid gene have been described and utilized in epidemiological studies. this large genetic diversity of human caliciviruses makes routine detection difficult. human nov are the leading causes of 'nonbacterial gastroenteritis' outbreaks in all age groups worldwide. outbreaks frequently occur in communities such as nursing homes, hospitals, schools, and cruise ships. no consistent seasonal variation has been observed. infection involves transmission via person-to-person contact or ingestion of contaminated food and water. epidemiological studies have identified caliciviruses in 60-95% of outbreaks in many countries. estimates of disease burden in usa suggest that caliciviruses are responsible annually for 23 million illnesses and 50 000 hospitalizations. strains of nov gii cluster 4 (gii-4) have been the most common type identified worldwide in the past 5 years (2001) (2002) (2003) (2004) (2005) in both adults and children. prevalence rates of calicivirus (predominantly nov) in young children admitted to hospital with acute gastroenteritis in many countries range from 3.5% to 20% annually. strains of sav, while playing a minor role overall, are more generally associated with childhood gastroenteritis than with disease in older children and adults. caliciviruses may be important enteric pathogens in patients with hereditary or acquired immunodeficiency. the genera norovirus and sapovirus are genetically diverse, and multiple strains co-circulate in human populations. individual dominant strains emerge every 2-5 years, and often have a global impact, such as the gii-4 strains identified in usa, europe, japan, and australia in 1995/ 1996 and again in 2004. nov recombinant strains, with polymerase and capsid genes derived from different ancestral clusters, have been identified in thailand and australia. repeated attempts to adapt nov and sav to growth in cell culture have failed. diagnostic techniques and analysis of antigenic variation rely predominantly on molecular biological techniques. cloning and expression of the major viral capsid protein (vp1) in baculovirus expression systems has led to the formation of virus-like particles (vlps) morphologically similar to native virus and their incorporation into enzyme immunoassay (eia) assays. pathogenesis of nov and sav infection is poorly understood as a result of the long-standing inability to adapt these viruses to cell culture, and the absence of a small animal model. symptomatic enteritis in human volunteers infected with 'norwalk agent' showed changes in jejunal biopsies (mucosal inflammation, absorptive cell abnormalities, villus shortening, and crypt hypertrophy) that persisted for at least 4 days after remission of clinical symptoms and reverted to normal after 2 weeks. no identifiable viral particles were detected by electron microscopy in any affected intestinal tissue. the recent demonstration that human noroviruses can infect and replicate in a three-dimensional cell culture model of human intestinal epithelium, should improve our understanding of the pathogenesis, and antigenic diversity of this important group of enteric viruses. these studies will also be enhanced by discovery of a norovirus that infects mice, and that replicates after transfection of cultured kidney cells. mechanisms of immunity to nov are unclear. infection results in formation of igg and igm serum antibody that are broadly reactive within, but not between, genogroups. the role of these antibodies in immune protection is unknown. infected individuals can develop short-term immunity to homologous viruses but the molecular diversity of nov circulating in communities makes it difficult to predict whether long-term immunity can develop. it is unclear why a proportion of exposed individuals remain uninfected during outbreaks. recent studies suggest that histo-blood group antigens and the secretor status may be genetic susceptibility markers for infection. at present the major control strategies for prevention of human calicivirus infection rely on prevention of contamination of food and water supplies. virus particles, later classified in the genus rotavirus, were first described in 1963 by adams and kraft as a cause of epidemic diarrhea in infant mice (edim). similar particles (ncdv) were recognized in 1969 by mebus and colleagues as a cause of severe diarrhea in newborn calves in nebraska, usa. neither virus was considered relevant as a causative agent of severe diarrhea in young children until 1973 when bishop, davidson, holmes, and ruck described a 'new virus' (later shown to be antigenically related to edim and ncdv) in duodenal biopsies and diarrheal feces from young children admitted to hospital in melbourne, australia with severe acute diarrhea. named because of their wheel-like appearance in negatively stained extracts examined by electron microscopy (rota ¼ latin for wheel) rotaviruses have since become established as causes of severe acute diarrhea in the young of many mammalian and avian species worldwide. rotavirus enteritis affects all children regardless of socioeconomic status, and results in over 600 000 deaths annually in young children in developing countries. rotaviruses are nonenveloped icosahedral viruses of 70 nm ( figure 2 ) diameter that belong to the genus rotavirus within the family reoviridae. the double-stranded rna genome is contained within a triple-layer of viral proteins (vps) comprising a core (vp1, vp2, vp3), an inner capsid (vp6), and an outer capsid (vp4, vp7). rotaviruses are classified into groups a to g based on serology of the vp6 protein. the majority of human and mammalian infections, due to group a viruses, are further classified into serotypes by antigenic differences on vp7 (g-serotypes) and into genotypes by genetic differences on vp4 (p-genotypes). to date there are at least 11 of 15 g-serotypes and 15 of 26 p-genotypes identified in humans. groups b and c have been identified infrequently in humans. groups d to g have been identified only in nonhuman mammalian or avian species. group a rotaviruses infect humans and other mammals repeatedly throughout life. most primary rotavirus infections in animals occur during the neonatal period. most primary rotavirus infections in humans occur during the first 24 months of life. children worldwide will experience at least one rotavirus infection by 5 years of age. in general, group a rotaviruses are species specific. however, rotavirus strains with gene segments of feline, bovine, or porcine origin have been isolated from children, suggesting the occurrence of cross-species infection in nature. cross-species infections can be established experimentally, and comprise one of the strategies for human vaccine development. the rotavirus genome consists of 11 segments of double-stranded rna that can be separated by polyacrylamide gel electrophoresis, allowing epidemiological studies mapping the genetic diversity of strains within and between serotypes. each gene segment encodes a separate protein, with the exception of gene segment 11 which encodes two proteins. reassortment of genes between human strains and human and animal strains occurs in vivo and in vitro. rotaviruses are transmitted from person to person by the fecal-oral route, or via aerosols. rotaviruses replicate in the cytoplasm of mature nonreplicating enterocytes lining the upper portions of the small intestinal villi, eventually causing cytolysis. profuse watery diarrhea results from a combination of mechanisms including malabsorption secondary to loss of enterocytes responsible for absorption and digestion, activation of the enteric nervous system, and stimulation of intestinal secretion by the rotavirus nonstructural protein nsp4. rotavirus antigenemia and viremia occur during the acute phase of severe primary rotavirus disease. as a result, complete rotavirus particles have been found in liver, lung, spleen, pancreas, thymus, and kidneys of experimental animals. it is not clear if the virus is replicating at these sites. clinical symptoms in children are strongly influenced by age, with severe often life-threatening diarrhea occurring after primary infection in young children, and in aged people in nursing homes. excretion of rotavirus particles in detectable numbers (by eia, rt/pcr) continues for 5-10 days, and occasionally up to 50 days. excretion can continue for months in immunodeficient children and animals. reinfections occur throughout life, and are usually asymptomatic or associated with mild symptoms. symptoms of primary infection require medical attention in 1:5 children, result in hospitalization in 1:65 children and death in 1:293 (almost all in young children in developing countries). treatment is based upon replacement of fluid and electrolyte loss, usually achieved by oral administration of fluids containing glucose and electrolytes. occasionally, delayed repair of the small intestinal mucosa is associated with disaccharide or monosaccharide malabsorption leading to malnutrition. primary rotavirus infection protects against severe symptomatic disease on reinfection, and is associated with humoral and cellular immune responses to individual rotavirus proteins. neutralizing antibody to vp4 and vp7 outer capsid proteins contribute to protection, possibly by interfering with viral replication and limiting the extent of intestinal damage. the role of immune responses to other proteins is uncertain. virus-specific cytotoxic tcells are not essential for protection. the importance of rotavirus disease worldwide and its contribution to childhood mortality in developing countries has resulted in strong initiatives, supported by the world health organization, to develop live oral rotavirus vaccines to be administered to infants before 3 months of age. two contrasting vaccines, a single attenuated g1p[8] human rotavirus and a pentavalent human-bovine g1-g4,p[8] reassortant vaccine, have been proved to be safe and effective in preventing severe rotavirus disease. both have the potential to radically change global childhood mortality and morbidity. adenoviruses were first detected in 1953 in cultured fragments of tonsillar and adenoidal tissue from children. they are nonenveloped icosahedral viruses approximately 80 nm in diameter (figure 3) . the genome is composed of double-stranded dna. the family adenoviridae comprises three genera: mastadenovirus (mammalian) classified into subgenera a-f representing more than 50 serotypes, aviadenovirus (birds), and a newly recognized genus atadenovirus identified in sheep and reptiles. most are readily cultivatable. adenovirus infections occur worldwide in many mammalian species, are species specific, usually associated with disease in the respiratory, urinary, and ocular systems, and are frequently shed in feces in the absence of any gastrointestinal symptoms. in 1975, flewett and colleagues in birmingham, uk, noticed the presence of large numbers of adenovirus particles in negatively stained extracts of diarrheal stools examined by em. these proved difficult to culture, were designated 'enteric' adenoviruses (ead), and are now classified as serotypes ead40 and ead41 within subgenus group f. cultivation of ead remains difficult. the most reliable growth has been achieved in human embryonic kidney cells (293 cells) immortalized by transfection with regions of ad5. ead40 and ead41 occur worldwide, causing severe acute enteritis in 5-15% of hospitalized young children. outbreaks occur at unpredictable intervals year-round with no seasonal prevalence. nosocomial epidemics occur in day-care nurseries and in hospital wards for children and adults. group a adenoviruses (serotypes 12, 18, 31) have also been implicated in epidemics, usually in older age groups. the adenovirus virion is composed of at least 10 different structural polypeptides and contains a linear 33-45 kbp dna. virus capsomers are arranged as hexons, the corners of which have antenna-like (fiber) projections presumed involved in cell attachment. the dna genomes of groups a-f are genetically diverse, and differences can be illustrated by analysis using genome restriction endonucleases. the heterogenous genome in groups a-f makes recombination between subgenera unlikely, with exception of groups a and f which show a close evolutionary relationship. diagnoses of ead infection rely on eia that detects the hexon antigen common to groups a-f, followed by determination of restriction enzyme patterns and/or reactions in eia incorporating neutralizing, monoclonal antibodies specific for ed40 and 41. ead replicate within the epithelial cells of the small intestine. group a adenoviruses have also been grown from mesenteric lymph nodes and appendices. the mechanisms causing diarrhea are not clear, but destruction of infected epithelial cells has a role. adenovirus diarrhea is more common in infants <12 months old than in older children, and can be protracted with a mean duration of 12 days. adenovirus diarrhea occurs in immunocompromised patients. nonseasonal epidemics of ead diarrhea occur in hospital wards, orphanages, and day-care nurseries. occasional fatal cases have been reported in children. evidence from animal models (with non-ead) suggests that viremia occurs, and can lead to infection of other tissues. the natural history of disease and development of immunity is unknown. astroviruses were first described in the uk in 1975 by madeley and cosgrove studying an outbreak of diarrhea in newborn babies in an obstetric hospital nursery. astroviruses are small, round nonenveloped plus-stranded rna viruses 28-30 nm diameter (figure 4) occasionally exhibiting virions with a superficial star shape. they are members of the genus astrovirus in the family astroviridae. they have been detected in humans (children and adults) and a range of mammalian (sheep, cattle, pigs, dogs, cats, and mice) and avian (turkeys, ducks) species, usually associated with diarrhea. there are currently eight serotypes of human astrovirus, designated hastv 1-8, based on reactivity with polyclonal antisera. hastv 1 is most common worldwide. prevalence rates as a cause of diarrhea vary from 2% to 16% (hospital-based studies), and 5% to 17% (community-based studies). most astrovirus infections have been recorded during colder months in temperate climates and year-round in tropical countries. a longitudinal study in mayan children in a poor community in mexico found a high prevalence (61%) of astrovirus infection in a birth cohort of 271 children followed for 3 years. infection occurred primarily in infants <12 months old, and showed a high rate of asymptomatic infection and prolonged shedding (2-17 weeks) in many infants. astrovirus infection has also been associated with persistent diarrhea (lasting for 14 days or more) in children in bangladesh. astroviruses are widespread in developed countries, causing outbreaks in day-care centers, hospitals, and nursing homes for the elderly. they are an important cause of enteritis in immunocompromised patients. the hast genome is a polyadenylated plus-stranded rna molecule of approximately 7 kbp. the genome contains two open reading frames (orfs), orf1a and -1b, code for nonstructural proteins and orf2 encodes for the capsid protein. genetic diversity in all serotypes exists, but no association has been shown between serotypes and ability to cause severe gastroenteritis. astrovirus diagnostic assays include commercially available eia kits, electron microscopy and rt-pcr detection and genotyping of diarrheal feces. acute astrovirus infection induces a mild watery diarrhea in young children that lasts for 2-3 days and may be associated with vomiting, fever, and anorexia. the lack of a small animal model has hampered studies of the mechanism of astrovirus-induced diarrhea. experimental models of astrovirus enteritis in turkeys and in gnotobiotic lambs show mild histopathological changes in the intestine (despite high mortality from severe osmotic diarrhea) together with viremia. experimental astrovirus infection in calves is asymptomatic, with viral replication apparently targeted to m cells. it is possible that none of these animal models illuminate pathogenesis of hastv infection in humans. cultivation of hastv was initially difficult, but can now regularly be achieved using a human colon cancer derived epithelial cell line (caco2 cells). members of the genera coronavirus and torovirus are enveloped plus-strand single-stranded rna viruses belonging to the family coronaviridae. electron microscopy shows them to be pleomorphic fringed particles 100-140 nm at maximum dimension ( figure 5 ). coronaviruses and toroviruses can be distinguished by differences in peplomer structure and reaction in iem using specific antisera. coronaviruses and toroviruses cause diarrhea, respiratory, and/or hepatic disease in many animal species, including cattle, mice, swine, cats, and dogs. in general, most of these viruses are species specific and disease is most severe in infant animals. transmission is fecal-oral and due to virus lability may require close contact. coronaviruses and toroviruses have been implicated in human diarrheal disease but there is still no consensus about their importance. similar particles have been seen frequently in children without diarrhea, particularly in children in developing countries. morphological similarities between these viruses and fragments of intestinal brush border make diagnosis difficult. several studies have implicated coronaviruses as causative agents of necrotizing enterocolitis outbreaks in newborn babies. toroviruses were first described as a cause of diarrhea by woode and colleagues in 1979 when breda virus was identified in a severe outbreak of neonatal calf diarrhea in usa. toroviruses are now also known to infect horses (berne virus) and swine. human infections were first described by flewett et al. in birmingham uk in 1984, but have rarely been reported since then. the pathogenesis of diarrhea has been studied in animal models using infection with coronavirus (tge) in piglets, and with breda viruses in calves. both replicate in epithelial cells of small intestine and descending colon causing diarrhea 24-72 h later. breda virus also replicates in crypt cells. many animal coronaviruses can be propagated in cell culture. isolation of human enteric coronaviruses is difficult and serological studies can be confounded by antibody resulting from repeated respiratory coronavirus infection. enteric infection can be confirmed in feces by detection of viral rna by rt-pcr, or iem using antibodies to the viral envelope glycoproteins. picornaviruses are 24-30 nm featureless spherical particles containing single-stranded positive-sense rna. they have been found in diarrheal feces from humans but their etiological role is often not clear. the first clear evidence implicating a picornavirus as an enteric pathogen identified aichi virus, as a cause of oyster-associated epidemics of gastroenteritis in japan in 1989. aichi viruses are now classified as a new genus kobuvirus within the family picornaviridae (kobu ¼ japanese for knob). isolation of aichi virus in vero cells has permitted development of eia and rt-pcr assays based on nucleotide sequence data. serological assays show seroconversion resulting from infection and a prevalence rate for antibody of 7.2% in japanese children aged 7 months to 4 years and rising to 80% in adults by age 35. the new genus parechovirus within the family picornavirideae contains at least one serotype (previously echovirus 22) that has been implicated as an enteric pathogen in humans. parvoviruses are small 22-26 nm single-stranded dna viruses comprising a genus parvovirus in the family parvoviridae. some animal parvoviruses have been clearly linked to enteritis including bovine, feline, mink, and canine strains. canine parvovirus infection emerged after 1977. this lethal neonatal enteric infection, accompanied by viremia and widespread systemic infection, shows a pathogenesis distinct from most enteropathogenic viruses. the virus infects and destroys crypt epithelial cells resulting in flat mucosa with fused and stunted villi. damage has been likened to that caused by radiation. other small viruses, resembling parvoviruses, have been seen by em in diarrheal feces in humans. evidence linking them to causation of disease is not convincing. they have often been present as dual infections with known enteric pathogens. in addition, their resemblance to some phages makes diagnosis uncertain. in the 1950s, the use of monkey kidney cell cultures for the growth of poliovirus revealed the presence of simian viruses and further study showed some of these to have properties consistent with enteroviruses. in parallel, investigations of viruses infecting domestic animals also revealed the presence of enteroviruses (and enteroviruslike particles) in pigs and cattle. enteroviruses have since been isolated from african buffalo, water buffalo, sheep, goat, deer, and impala and many have been shown to be related to bovine enterovirus isolates. in the past, the classification of enteroviruses was primarily based upon the physico-chemical properties of virions, growth in tissue-cultured cell lines, and by serotyping. in practice, this can be difficult with some isolates being poorly recognized by the reference antisera, or, the occurrence of (misleading) cross-reactivities between serotypes. the expansion of the sequence database together with more sensitive cloning/sequencing techniques have facilitated the elucidation of the genome structures of many picornaviruses. such analyses have replaced other techniques in the classification of picornaviruses, and this article discusses characteristics which are important for the classification of animal enteroviruses. bovine enteroviruses (bevs) are endemic in cattle in many regions of the world with infection typically enteroviruses of animals picobirnaviruses are 35-41 nm particles with a bi-or tri-segmented dsrna genome and have been detected in europe, south america, and australia. they are found significantly more often in patients with hiv-related diarrhea than those without diarrhea. their role in gastroenteritis in healthy individuals remains unknown diagnosis of noncultivable gastroenteritis viruses, the human caliciviruses foodborne viral gastroenteritis: challenges and opportunities gastroenteritis viruses. novartis foundation symposium viral gastroenteritis characterisation of torovirus from human faecal specimens immunity and correlates of protection for rotavirus vaccines new hope for defeating rotavirus comparative pathology of infection by novel diarrhoea viruses human sapovirus: genetic diversity, recombination and classification prevalence and genetic diversity of human astroviruses in mexican children with symptomatic and asymptomatic infections genetics of the rotaviruses pathogenesis of intestinal and systemic rotavirus infection norwalk and other human caliciviruses: molecular characterization, epidemiology and pathogenesis in vitro cell culture infectivity assay for human noroviruses the subgroup f adenoviruses application of a reverse transcription-pcr for identification and differentiation of aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans key: cord-253111-n5ywei4t authors: keck, frédéric title: avian preparedness: simulations of bird diseases and reverse scenarios of extinction in hong kong, taiwan, and singapore date: 2018-04-14 journal: j r anthropol inst doi: 10.1111/1467-9655.12813 sha: doc_id: 253111 cord_uid: n5ywei4t this article describes relations between humans, animals, artefacts, and pathogens in simulations of disasters, taking bird diseases in three chinese sentinel posts as ethnographic cases. drawing on distinctions between simulation, ritual, and play, it shows that the engagement of actors in the imaginary of simulations, which they describe as ‘realism’, reflectively reverses the oppositions between humans and nonhumans, active and passive, fiction and reality that shape ordinary life. borrowing from the anthropology of hunting societies, it argues that simulations of bird diseases, considered as signs of future species extinction, rely on cynegetic techniques of power, in which humans and animals symmetrically shift perspectives, and not only on pastoralist techniques, in which humans are above the population they monitor and sometimes sacrifice. of action, simulations can be described as ritual performances displaying relations between humans and nonhumans in extraordinary settings. this article starts from observed simulations of an avian influenza pandemic in china. influenza viruses emerging from birds have been considered as potentially causing future pandemics, because birds (particularly waterfowl in south china) have been identified as the reservoir in which flu viruses mutate before spreading successfully between humans (shortridge & stuart-harris 1982) . among other techniques such as early warning systems and stockpiling vaccines, preparing for the next pandemics therefore involves simulating the transmission of pathogens from birds to humans (caduff 2015; lakoff 2007) . arguing that a human pandemic is a symmetrical threat to the extinction of a bird species, i then consider another bird disease for which chinese experts organize simulations: botulism. this disease, which doesn't affect humans, is considered a potential disaster for black-faced spoonbills because it kills migratory birds when they concentrate in wetlands on their flyways (wilson 2010: 76) . bringing together simulations of avian influenza and botulism, this article questions the ethnography of simulation from the perspective of animal studies. how does preparedness transform relations between humans and birds? what does it mean to include birds, and more generally animals, in exercises simulating coming disasters? how does the description of animals as victims of disasters transform them into actors of simulations (keck & ticktin 2015) ? i will show that animals become, in a paradoxical way, 'passive actors' when they carry the signs of diseases that can potentially affect humans. techniques of preparedness have been included in the set of tools for global health initiatives, either in humanitarian actions or in biosecurity interventions, which have shaped contemporary affects and anticipations of the future (adams, murphy & clarke 2009; lakoff 2010) . global health has been extended to animals and the environmentan approach called 'one world, one health' -thus requiring an interspecies approach towards the ethical issues involved (hinchliffe 2015) . one of the most debated questions in the anthropology of pandemic preparedness is the adequacy of the notion of sacrifice to describe how some populations are separated from others and killed or left to die for the sake of public health. in exercises of triage, some patients are prioritized over others through the framework of urgency (nguyen 2010; redfield 2013) . in the management of human populations, migrant workers are considered as a threat by educated experts using viral connectivity as a metaphor of modernity (macphail 2014; mason 2016) . in the management of zoonotic pathogens, the notion of sacrifice seems even more obvious, since animals are massively killed to eradicate the reservoir of emerging infectious diseases; and in the management of wild animals, some individuals are sacrificed to protect the species (van dooren 2014). other descriptions, however, have been proposed of the entangled relations between animals and humans in the 'hotspots' or 'sentinel devices' where emerging pathogens connect them in productive ways (brown & kelly 2014; fearnley 2015; keck & lakoff 2013; lezaun & porter 2015; lowe 2010) . while sacrifice separates some living beings from others to eradicate pathogens, sentinels instantiate relations between living beings through the anticipation of future pathogens. simulations of bird diseases shift perspectives between humans and animals through the use of fiction. to support this argument, i rely on the anthropology of hunting societies, by contrast with pastoral societies. while public health relies on pastoral techniques of power combining sacrifice and surveillance to contain the threats coming from outside in a population (foucault 1981) , the 'one world, one health' approach uses techniques from birdwatchers and wildlife managers to monitor data about changing relations between humans and animals. 1 whereas pastoral techniques are asymmetrical, relying on the pastor's superior gaze over the flock manifested by sacrifice, cynegetic techniques are symmetrical, as hunters and prey constantly change perspectives when displayed in rituals (chamayou 2012) . most analyses of simulation have placed it in a genealogy starting with the cold war, using notions of ritual, performance, and play (davis 2007; galison 1997; gusterson 1996; lakoff 2007; masco 2006) . but they don't take the perspective of animals because they consider simulations as pastoral techniques of biopower, therefore bypassing what the anthropology of hunting societies shows about ritual action. in this article, i define a cynegetic practice of simulation that is always mixed with forms of planning, combining two different forms of anticipation. i call 'avian preparedness' this mix of cynegetic becoming-animal and pastoralist care for populations initiated by microbiologists, considered both as 'virus hunters' (perrey 2012 ) and as 'microbe farmers' (caduff 2015: 76) . to analyse simulations of bird diseases, i combine the notion of reverse scenario (schull 2015) with the notion of reflexive ritual. pandemic or extinction scenarios, i argue, connect humans and animals in realistic ways to play on the imagination of what could happen if a pathogen emerges. using animal reservoirs as mirrors in which anxieties about pandemics are reflected, humans exchange their perspectives with animals in an imagined future where their relations are reversed. simulations can thus be situated in an anthropological space between play and ritual. roberte hamayon (2015: 97) has called 'simulation' the way shamans in mongolian hunting societies imitate the movements of animals in a ritualized setting to prepare for the uncertainties of hunting. in an article entitled 'dissimulation and simulation as forms of religious reflexivity' , michael houseman proposes to move away from a definition of ritual as a collective action based on a shared secret to study 'the emergent properties of ritual simulation generally ' (2002: 77) . he draws on two cases taken from hunting societies of central africa, one in which male novices are symbolically killed and the other in which a goat is actually sacrificed. he asks: how do participants come to believe in a series of speeches and actions -the novice's death and the goat's consent to sacrifice -that are obviously false? he answers that ritual action creates an extraordinary space of reflexivity with regard to the ordinary relations that constitute the group. while the ritual for male novices is a 'dissimulation' , in which the relations between initiated and uninitiated are constantly reversed in such a way that the moment of death becomes uncertain, the ritual of goat sacrifice, says houseman, is a simulation of consent, in which the silencing of the goat through the consumption of herbs mimics the consent of male hunters to their initiation. the ritual is reflexive in the sense that it stages the condition of the ritual itself -the secrecy known by initiators and unknown by the initiatedthrough the manipulations of daily relations between humans and nonhumans, whose asymmetries are reflected as in a mirror. similarly, i suggest that we call 'reverse scenarios' the ritual performances by which simulators reflect playfully on the relations between humans and birds in the imagination of a disease that could affect them both. simulations, i argue, are far more than mere 'rehearsals' of a pandemic drama: they are catalysts in the transformation of human/nonhuman relations into asymmetrical biopolitical and ontological processes. to support this argument, i will describe three ethnographic cases in hong kong, taiwan, and singapore. these three territories have competed at the global level since the sars crisis in 2003 to be the best 'sentinel posts' where pandemic pathogens coming from china can be detected (abraham 2007) . as hubs for the intense circulation of persons and commodities in asia, but also as repositories of chinese traditional relations between humans and birds, they have prepared for the catastrophic effects of a bird flu outbreak. but while the hong kong government regularly culls its poultry and sends warning signals from the chinese border, taiwan and singapore rarely had to cull their poultry and rather invested in scenarios for the simulation of pandemic. taiwan, on the other side, shares with hong kong the monitoring of black-faced spoonbills, considered as one of asia's most threatened and charismatic species. this article thus connects the rationalities of preparedness with the economies of human/animal relations in three different but interconnected settings. it will examine indoor and outdoor simulations of avian influenza in singapore and hong kong, and compare them with simulations of botulism in taiwan. however, my analysis is not restricted to simulations of bird diseases in east asia, but highlights general traits of disaster simulation as a technique of preparedness. by looking at the relations between humans and birds in these three contexts through their common exposure to emerging pathogens, this article also analyses the relations between national states in asia as they have to manage a common disaster. since simulations of bird diseases connect relations at different scales, from the microbiological to the biopolitical, this article combines science studies, ethnography of rituals, and political economy through an anthropology of 'more-than-human' relations (helmreich & kirksey 2010; kohn 2013; sagan 2011) . in july 2013, i was visiting gavin smith, who was then an associate professor at the duke graduate medical school of the national university of singapore (nus). i had known gavin in hong kong while he was working in the state key laboratory of emerging infectious diseases at the department of microbiology of the university of hong kong. born in australia, gavin had studied botany and ecology at the university of melbourne, and then moved to hong kong for a ph.d. in molecular systematics. gavin had first analysed the genome of fungi, then of the sars coronavirus, then of bird flu viruses. he described this development of his research as a reduction of the size of the living beings he was sequencing, and a refinement of the data and results he could produce. gavin and his team relied strongly on the collaboration between the state key laboratory of emerging infectious diseases and the university of shantou. this university had been created by the hong kong tycoon li ka-shing, who opened it in 1990 in his native guangdong, with the approval and administrative support of the chinese authorities. it provided the state key laboratory of emerging infectious diseases with samples collected in poultry markets in fujian province, thus offering a real-time surveillance of influenza strains emerging in this 'epicentre' of pandemic flu. but after the publications of gavin's team showed the role of the chinese poultry industry in the emergence of these new strains (smith et al. 2006) , the guangdong authorities had severely restricted hong kong virologists' access to these samples. gavin then moved to singapore in 2012, where he had more freedom to continue his fundamental research, and more funding from international institutions. he had shifted from the sentinel post, where the new strains of avian influenza emerged, such as the h5n1 virus in 1997 (keck 2010) , to a place where he could simulate the evolution of flu viruses in high-tech computers. there was no need to be close to the site where viruses emerge, he explained to me, as he could simulate viral emergence through the use of genetic data banks. singapore was then considered the new 'pole of excellence' for biological research. the small city-state founded by lee kwan yew in 1965 at the end of the malay peninsula had converted its industrial and financial power, owing to its position on the commercial trade routes between east and west, into an economy of knowledge and information, with high investment in biotechnologies (ong 2010; 2016) . the nus graduate medical school was located just next to the ministry of health and the singapore general hospital, in the centre of the city. it had been created in 2005 to train high-tech medical leaders on the model of the duke university school of medicine in north carolina. the programme in emerging infectious diseases, for which gavin had been hired to work, was headed by wang linfa, who was also leading a team on bat viruses at the australian animal health laboratory in geelong. his programme was running projects for the surveillance of emerging infectious diseases, particularly influenza and other respiratory diseases, in singapore and the rest of southeast asia. gavin's career took a meaningful turn when his team published two major articles at the height of the 2009 h1n1 pandemic. the first one, entitled 'dating the emergence of pandemic influenza viruses' , was published just after the emergence of the h1n1 virus in mexico in april 2009. it showed that genetic components of the 1918 h1n1 pandemic virus circulated among pigs and humans as early as 1911, thus challenging the commonly held view that spanish flu jumped from birds to humans in 1918. in the context of the new h1n1 'swine flu' , this article stressed the need for intensified surveillance of pigs to detect emerging flu viruses before they become pandemic. 'if future pandemics arise in this manner' , gavin and his team concluded, 'this interval may provide the best opportunity for health authorities to intervene to mitigate the effects of a pandemic or even to abort its emergence' (smith, bahl, vijaykrishna, et al. 2009: 11712) . this conclusion was immediately reinforced by gavin's team's second publication, entitled 'origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic' , published in nature at the end of 2009, in which they showed that a 'twin' virus bearing the same genetic sequence as the pandemic h1n1 had been identified in pigs in hong kong in 2004. gavin's conclusion was not that the asian swine industry was to blame for the emergence of h1n1, but that there was a better surveillance of the animal reservoir for influenza viruses (waterfowl and pigs) in hong kong than in the united states. the team's paper ended, 'despite widespread influenza surveillance in humans, the lack of systematic swine surveillance allowed for the undetected persistence and evolution of this potentially pandemic strain for many years' (smith, vijaykrishna, bahl, et al. 2009 : 1125 . in july 2009, i took a course at the hong kong university pasteur research centre in bioinformatics, during which gavin and his team partners, vijay and justin, explained to virology students how to obtain this kind of result. bioinformatics is a method to analyse the massive amount of biological data available on the web -particularly through the website of the us national center for biotechnology information, genbank -with the help of dedicated software. this software, called blast (basic local alignment search tool) or msa (multiple sequence analysis), calculates the probability that genetic sequences derive from one another. such a procedure, called alignment, aims at approximating the real descent relationships between viruses through a calculation of the optimal 'tree of life' , based on the darwinian hypothesis that life expands in a rational manner to maximize fitness capacities (mackenzie 2003) . for gavin and his team, however, bioinformatics was not only about probability calculation. in the construction of a phylogenetic tree, biological knowledge is necessary because some correlations proposed by the computer make no sense in an evolutionary perspective, as they can be due to errors in sequencing or genetic deletion. to decide which correlations to take into account and which to consider as irrelevant, virologists use other software (such as bootstrap, jukes cantor, or tamura) providing the probability of correlations based on a given scenario. as for other techniques of preparedness, probability is not enough to simulate a coming disaster: it requires a work of imagination (lakoff 2007) . peter galison has shown that computer simulation emerged in the field of particle physics as a 'subculture' among experimenters and theoreticians under the 'monte carlo' programme funded by nasa in the 1960s to simulate a nuclear explosion. simulation replaces experimentation when the data needed for the theory are too numerous and complex to be processed with traditional instruments, creating a 'trading zone' in which different professional groups share a common language or 'pidgin' (galison 1997: 50) . similarly, bioinformatics produces new images shared by microbiologists, computer scientists, and public health executives in the anticipation of epidemics. software such as bootstrap can be considered as an accessory or decoy to trap the live bird viruses through the sequences that have been stored in genbank. gavin opened the course with a simulation of the work his team had done a few months before on the h1n1 virus, presented as a desktop exercise of what to do when a new virus emerges. 'imagine we've received this new sequence from atlanta. paste it to blast, and take as much information as possible. make a tree so big you can never print it on a sheet of paper: it will be good starting material before reducing the inquiry' . gavin and his team explained that in contrast to other virologists working on flu viruses, they had decided to use the eight segments of the virus's rna, 2 not just the two segments that shape the h and n proteins, usually considered to be the most significant markers of antigen evolution. they could thus build eight parallel evolutionary trees which, by their similarities, gave an indication of the real evolution of the whole flu virus. there was nothing secret in the images of pandemic viruses built by these microbiologists, unlike simulations of nuclear explosions built indoors by particle physicists (gusterson 1996) : all the phylogenetic trees produced by gavin and his team were publicly available on websites. drawing back the 'molecular clock' based on the estimated kinship relations between viruses, virologists could see the branches where evolutionary ruptures occur and pathogens jump from one species to another, such as the emergence of the h1n1 pandemic in 1918 and 2009. following continuities between genetic sequences, they exhibited the discontinuities between evolutionary niches. gavin said that when a virus jumps from one species to another, it inaugurates a new set of lines, as the 'immunity pressure' of the environment creates an 'evolutionary bottleneck' in which this mutation can replicate. drawing back these lines then lead him to what he named 'the most recent common ancestor' . 'i love those trees' , he added. the amount of information provided in a single image was correlated to the unity of an evolutionary hypothesis drawing this information back to a common ancestor. historians of science have shown the power of evolutionary trees in the books through which darwinism came to be accepted as a new worldview (bredekamp 2005; helmreich 2009 ), and their reframing in global health under the notion of an animal reservoir in which emerging pathogens spill over from animals to humans (king 2002; lynteris 2017) . but i was mostly intrigued by the way gavin and his team turned sequences into images through an anticipatory mode of reasoning that i call 'reverse scenario' . natasha schull proposes the term in her work with on-line gamblers, to describe the way they 'cope with the necessarily uncertain futures of any hand by returning them to a point in the past and confronting them with a branching diversity of outcomes that might have emerged from it' (2015: 56). it applies well to the practices of 'virus hunters' who use 'reverse engineering' to reconstruct potentially pandemic pathogens such as the 1918 h1n1 virus (caduff 2015: 105; napier 2003: 2) . just as a gambler speculates how much he could have won if he had played this or that card, gavin imagines what could have happened if the h1n1 virus had been detected in pigs prior to 1918 or 2009. virus sequencing allows him to trace potential connections between birds, pigs, and humans, and to imagine other futures of cross-species transmission. yet we don't know how birds (and pigs) are actually involved in the construction of pandemic scenarios, because we don't see how virologists take virus samples from birds. we thus need to turn from desktop simulations to 'real-ground' exercises organized by public health officials and birdwatchers. after my visit to gavin smith at nus duke graduate medical school, i went to a nearby building to meet jeffery cutter at the singapore ministry of health. trained as an epidemiologist, jeffery cutter was the director of the communicable diseases division of the ministry of health, co-ordinating preparedness for emerging infectious diseases in singapore. 'there are 50 million passengers every year at singapore changi airport' , he told me, 'so there is a high probability that emerging infectious diseases will come to singapore. we need to be prepared' . during the sars crisis in 2003, he said, 238 people were infected by the new coronavirus, with thirty-three deaths. they were all treated at the tan tock seng hospital, which had been designated by the government as an isolation hospital for sars patients. at the peak of the crisis, all schools in the country were closed for ten days, and home quarantine was established for more than 6,000 people known to have come into close contact with a sars-infected person. prime minister goh chok tong said publicly that sars could possibly become the worst crisis singapore had faced since independence. when a journalist asked him if he was being alarmist, goh responded: 'well, i think i'm being realistic because we do not quite know how this will develop. this is a global problem and we are at the early stage of the disease' (zolli 2012) . jeffery cutter introduced me to yoong cheong wong, head of the emergency preparedness & response division at the ministry of health. mr wong was a former policeman in charge of organizing exercises for emerging infectious diseases in hospitals. the first national exercise for influenza in singapore was organized in 2006, after the first cases of human-to-human transmission of h5n1 were suspected in thailand. called 'sparrowhawk' , it was organized in two stages: a table-top discussion and assessment of pandemic response plans among six hospitals in the country between april and june; and a practical exercise on 21-2 july, in which the six designated hospitals had to manage potential patients and share information about their circulation. mr wong explained that a thousand people were involved in the real-ground drill, mostly volunteers from grassroots organizations and nurses from training institutions. while patients moved from one hospital to another, some hospitals assessed the behaviour of others and gradually raised the number of casualties. the main question, according to mr wong, was: 'if there are 200 casualties in one hospital, which resources do they need?' the goal of these exercises is officially the identification of unplanned gaps in coordination between the actors who manage epidemics. but the actual criticism of planners most often focuses on the drills' lack of realism. 'we try our best to do it as real as possible' , said mr wong, 'but everyone knew it was just an exercise' . to explain the improvement between 'sparrowhawk' exercises in 2006 and 2008 in singapore, an official leaflet notes, 'the exercises were activated unannounced to inject greater realism. the ministry of health emergency medical staff turned up without prior justification at the general ward of the exercise hospital and "triggered" the simulated case of human infection with avian influenza' (unsic 2008: 56) . realism, in this case, came from the fact that people were surprised, and yet they knew that they were faking the epidemic. what does it mean for a simulation to be realistic? here the notion of 'reverse scenario' is useful. just as microbiologists imagine what could have happened if viruses had been stopped in their animal reservoir, public health planners imagine what would happen if contagious patients were stopped before entering hospitals. but in both cases, death is not performed in the scenario. by contrast, another type of exercise conducted in singapore was very real. on 17 july 2013, the agriculture and veterinary authority (ava) organized a drill in one of the poultry slaughterhouses on the border with malaysia. there are no authorized poultry farms on the territory and only five chicken farms for eggs, which constrains singapore to import and slaughter 40 million live chickens from malaysia every year. malaysian farmers need to be licensed by the ava to import their poultry to singapore, and are always regarded with suspicion by border authorities. and yet no case of h5n1 has been found in singapore. i was able to watch the drill on-line because it was streamed widely on a number of websites. the name, 'exercise gallus vii' , referred to the fact that it was the seventh avian influenza exercise conducted by the ava since 2002. it took place in the biggest of the ten slaughterhouses in singapore run by soonly food processing industry, which processed 80,000 chickens daily, providing 25 per cent of the fresh chicken meat in singapore (asia one 2013). the scenario for the exercise was the following: 1,500 chickens had been infected with h5n1 and should be killed and destroyed, according to emergency response plans. about 100 employees of the slaughterhouse, mostly indians, had to be screened for their temperature, take an antiviral drug, and then wear personal protective equipment (boots, cap, mask, glasses, gloves) on the back of which their name was written. the 'affected' chickens were placed in green cages on belts, immersed in water, and electrocuted. their cadavers were then placed inside two layers of biohazard bags to be incinerated. dr yap him ho, director of the quarantine and inspection group, declared on television, 'to make sure that ava is prepared for any incursion, the main thing we want to do for this exercise is to train our officers, as well as the other parties that are involved in the contingency planning, to be familiar with the steps of the procedures' (straits times 2013). although much could be said on the paternalistic ethnic, gender, class, and age divides that structured these exercises, i want to focus on the distinction between humans and animals. while the simulation at the clinic was animated by a sense of urgency and scarcity of resources confronted with a fake pandemic, the simulation at the slaughterhouse was quietly following the procedures in actually killing false positive chickens. i was also intrigued by the fact that while the hong kong government organized many such exercises on the human side, they had never simulated bird flu in poultry farms, because outbreaks occurred regularly in farms on its territory. while singapore authorities actually killed poultry for a disease that remained virtual, hong kong authorities simulated the transmission of bird flu from chickens to humans because it was already circulating among birds. although avian influenza kills birds as well as humans, the reverse scenario of a pandemic represents massive death in a different mode for humans and for birds, depending on past experiences of pathogens crossing borders between species. i now turn to the simulation of bird flu in hong kong. on 13 november 2006, hong kong co-ordinated a desktop exercise called 'great wall' which simulated a virtual cluster of three human infections with h5n1 in a family returning to hong kong and macau after their visit to a poultry market in mainland china. the goal of the exercise was 'to synchronize the three systems of pandemic preparedness to engender an effective response among them' (unsic 2008: 18) . the exercise opened with a ceremony attended by the vice-minister of the chinese ministry of health, wang longde. it was considered as a success in creating good relations between mainland china and the former british and portuguese colonies. while the sars coronavirus had caused a division between china and the former colonies in 2003 because of beijing's reluctance to share information (abraham 2007) , 'great wall' unified the country against an imagined enemy, pandemic avian influenza, but it instantiated ruptures between humans and birds. in january 2009, after long discussions with the centre for health protection, i was allowed to participate in an exercise in a hong kong hospital. the centre for health protection was created by the department of health after the sars crisis to anticipate outbreaks similar to sars through active surveillance and communication (leung & bacon-shone 2006) . within this centre, the emergency response branch, headed by a police officer, was in charge of writing scenarios and organizing simulations. twice a year, a field exercise was held in hong kong, with observers from china and overseas, bearing the name of natural entities: maple, cypress, chestnut, redwood, eagle, jadeite, and so on. there was no epidemic simulation in hong kong in 2009 because the emergency response branch considered the management of the h1n1 virus in the spring and autumn to be a 'real-life exercise' . 'exercise redwood' took place in the clinic of shau kei wan, located in the workingclass district of hong kong island. the following scenario was distributed to the participants and posted at major public buildings. confirmed human cases of bird flu had been reported in hong kong's neighbouring countries, as well as a rising trend in patient attendance with influenza-like illness in hong kong hospitals. live poultry had tested positive for avian influenza in hong kong's markets and were then culled at farms and markets. a member of the staff participating in the culling was reported to carry the h5n1 virus, as was an 8-year-old boy who had played with live poultry. four clinics were designated in hong kong for triage. only the final part of the scenario was held in shau kei wan; the first part was meant to provide a plausible context. the official purpose of the simulation was to co-ordinate hospital services in the management of patients with influenza-like illness. eighty actors playing patients came in through the front door of the hospital and were sent to different departments depending on their symptoms (pulmonary conditions, tuberculosis, etc.). twenty 'players' treated them in the services, and two 'simulators' communicated with other hospitals on a hotline. those who were diagnosed with h5n1 were evacuated by ambulance through the back door of the hospital, where the media took pictures. the head of the department of health gave a press conference after visiting the hospital, but the journalists asked him about new bacteria found in a private jacuzzi, not about the exercise itself. the drill's success was not questioned; it was successful because it had been held. the scenario was designed in such a way that no surprising event could happen: the only reality that came up was the bacteria in the jacuzzi, not the virus in bird markets. all the actors i saw looked young and relaxed, wearing blue caps and casual clothes. their symptoms were described on a tag they wore around their necks, indicating their name, address, nationality, sex, and age. these cards were red or green depending on whether they had bird flu or only influenza-like symptoms. they did not have to fake illness; their only role was to move to the designated departments. in another exercise organized by the centre for health protection, a plane was evacuated because a patient had been found with influenza-like symptoms; those who sat next to the patient received a red tag, and those who sat at a good distance received a yellow tag. what was remarkable in 'exercise redwood' was the absence of tension or anxiety in the medical staff's behaviour. although humanitarian ngos consider triage to be an ethical issue when a higher number of patients is met with limited resources (nguyen 2010; redfield 2013) , the medical staff of the shau kei wan clinic only separated symptomatic and asymptomatic patients to avoid the spread of the disease. the tension of the situationwho would be considered a spreader, who would receive treatment first -was delegated to objects: tags and caps. people were reduced to bearers of signs that circulated fluidly in the hospital under the gaze of the media. while the medical staff of the hospital played their own role, the actors playing patients with influenza-like illness came from a humanitarian association, the auxiliary medical service (ams). this association, created in 1950 to deal with the influx of chinese refugees, was registered in 1983 under the security department of the hong kong government. its 4,000 members are trained for disaster management in hong kong and outside. one of them described it to me as 'after-work entertainment' , 'a place to meet other singles'; it is an elite association where similar people express and share moral values. during their own exercises, performed every month, they train how to rescue victims of car accidents or fires. they simulate heartbeats on dummies with fake hearts. 'patients cannot fake the heart rate or the respiratory rhythm' , said the organizer, 'so they have tags' . tags have an indirect realistic effect, while dummies can actually simulate the heartbeat of an injured victim. members of the ams, who describe their own exercises as significant moments in the life of the association, were frustrated by 'exercise redwood' . from their perspective, there is a sharp difference between regular accident rescue exercises and extraordinary epidemic evacuation drills that co-ordinate several departments. one of the actors said he felt 'passive' , playing on the ambivalence of the term: as we had experienced sars in 2003, we all can forecast how we would behave in another similar situation, like avian flu. being a citizen, we are quite passive; i believe that we should follow the guidelines and the government's advice so as to prevent ourselves from getting affected. but in the exercise, we were being passive. 3 it can be said, consequently, that when they simulate patients in an epidemic exercise, members of the ams literally act as if they were dummies. there is thus a fundamental distinction in the real-ground exercises between actors and players. while actors are passive, reduced to the objects that 'act' or 'speak for them' , players and simulators introduce uncertainty by the way they combine these objects in the scenario. if actors have to appear 'real' to produce good images in the media, the simulators play on the fictional possibilities of the scenario. the distinction between simulators and actors in real-ground simulations runs parallel to the distinction between virologists and public health planners in desktop exercises: while actors and planners only follow their roles in the exercise, simulators and virologists explore the potentialities of reverse scenarios. another example shows how real-ground exercises combine the virtual and the actual as desktop simulation does. in an exercise called 'jadeite' , organized in january 2007, members of the emergency response branch were simulating the evacuation of a residential building on computers, and the scenario specified that they had to evacuate the simulation room to a 'fallback room' . the simulation room, from which the vulnerability of vital infrastructures in hong kong was virtually assessed, thus came to be seen itself as a material environment under threat. the head of the emergency response branch who described this exercise to me said it was 'more fun than a movie' , as if images had suddenly burst through the screen. in this reversal of the scenario, simulators became actors, and fiction reality. neither a ritual performance nor a scientific model of prediction, the simulation was, for this former policeman, a game he enjoyed playing. animals take part in simulations in a general category that can be called 'passive actors' . if there was no need to simulate the culling of poultry because it regularly happened in hong kong markets, simulators for the evacuation of residential buildings in hong kong planned that some residents were accompanied by pets that should be handled with care by the agriculture department, because their reaction to the evacuation was unknown, just as simulators of the evacuation of planes planned how to handle 'reluctant potential patients' . the real opposition in simulations is thus not between humans and animals but between poultry, handled as commodities that can be destroyed, ordinary citizens and pets, considered as 'passive actors' that should be handled with care, and simulators, changing the rules of the game. more than sharp ontological distinctions between fiction and reality or between animals and humans, there is a virtual gradient of actors in the anticipation of massive death produced by simulation. the use of accessories and animals in simulations of disasters is indeed an essential part of their 'realism' . tracy davis has shown that people coming from the world of theatre were hired by cold war experts to implement scenarios of nuclear blast through the design of accessories. simulation designers were, therefore, less concerned with the participation of the public than with the realism of the objects. after an exercise organized in coventry in 1954, prime minister winston churchill complained about the high degree of realism of the simulations, which he considered a waste of public money. 'who thought of the blood-stained old woman with the birdcage? i hope there is not going to be any more of this sort of thing at government expense' (quoted in davis 2007: 51) . women were the main targets of civil defence after the second world war, as they had been the targets of public health campaigns against microbes at the beginning of the twentieth century, because they were in charge of the household with children and pets. animals were portrayed as good accessories of nuclear blast simulations not only because they were good laboratory models (masco 2006: 305) , but also because they were part of a familiar landscape disrupted by an extraordinary event. the extraordinary context of the simulation thus provides the opportunity to display a series of apparent contradictions: people become objects; animals become people; actors turn out to be passive; fiction enters into reality. this capacity to displace oppositions in an extraordinary context through reverse scenarios brings simulations close to rituals. however, if simulations can be considered as rituals of public health administration, justifying the work of virus hunters who sound the alarm about future pandemics, it doesn't mean that there is a secret worldview shared by simulators (gusterson 1996) . rather, simulations display openly contradictions that public health administrations have to solve, often by the use of sacrifice in operations of culling (of infected poultry) or triage (of suspected patients). they create a tension between two techniques of power: those of biologists and simulators who follow viruses as they mutate between species, which i describe as cynegetic, and those of public health officials who have to protect some populations against others, which i call pastoral. to support this hypothesis, i will now turn to simulations organized by birdwatchers. can we say that birdwatchers take the perspectives of threatened wild birds as virologists take the perspective of poultry infected with flu viruses? how do humans in charge of the protection of animals cope with the oppositions we have seen in reverse scenarios of pandemics? birdwatchers have been involved in the surveillance of the avian reservoir of influenza viruses. public health and agriculture authorities realized that they could not follow the mutations of flu viruses in wild birds, and collaborated with birdwatching associations to monitor potential cases in wild bird reserves and natural environments. in hong kong and taiwan, birdwatching practices, a privilege of british and american expatriates until the 1980s, have become popular among chinese middle classes with the development of a leisure society, the commercialization of camera equipment, and the awareness of the vulnerability of the environment (choy 2011) . birdwatching has taken the form of a 'citizen science' with the opening of websites where amateurs post their observations and experts turn them into statistical data. every year, bird races offer opportunities for birdwatchers to compete in observing the highest number of birds on a designated territory. thus, birdwatching practices, like simulations, have virtual and actual aspects, orientated by the imagination of a disaster: the extinction of bird species. in hong kong and taiwan, the introduction of birdwatching was linked to techniques of preparedness and concerns for biosecurity. the main wild bird reserve in hong kong, mai po, is situated on the estuary of the pearl river delta and on the border with mainland china. after 1949, british officers used to sit there with their binoculars to trace chinese refugees or to signal an attack by the chinese army, and took this opportunity to inventory the more than 500 bird species in hong kong. hong kong and taiwan were part of the migratory animal pathological survey, a programme of surveillance of pathogens carried by birds (particularly japanese encephalitis). through this programme, british and american ornithologists trained chinese workers to band birds and collect samples. transforming birds into data and following the evolution of their numbers has been a way to simulate threats coming from birds since the second world war (keck 2015) . methods derived from us wildlife management have changed the perception of birds in the last twenty years in taiwan and hong kong, with the introduction of the concept of the flyway. species that fly from japan and korea to australia, along what came to be called the east asian-australian flyway, have been shown to have declined owing to the urbanization and development of the chinese coast. climate change is also supposed to damage the places where wild birds feed and roost on their migratory routes. among the bird species affected by these changes, one of the most charismatic is the black-faced spoonbill, a waterbird breeding in japan and korea in the summer and migrating to south china in the winter. in the 1990s, the number of these birds was estimated to be 2,000, but thanks to the conservation efforts shared by the countries to which they migrate, it increased to 3,000 at the end of the 2000s. wetlands have been developed in hong kong, taiwan, and fujian as shelters on their flyways. images of spoonbills abound in schools, environmental agencies, and natural reserves, often portrayed as funny birds smiling to the public. to understand how reverse scenarios of disasters enter the management of spoonbills in china, we have to go beyond these anthropomorphic images, and look at how they actually perform in simulations. in the winter of 2002-3, seventy-three black-faced spoonbills were found dead from botulism in taijiang national park. the wild bird society of tainan consequently organized a campaign to vaccinate spoonbills against botulism, and used decoys to train their staff on how to safely handle the birds. since then, drills have been regularly organized for bird protectors to learn how to manipulate fake spoonbills with caution. these decoys are also used to attract spoonbills for gps monitoring. carved in wood and painted in black and white, they are posted in the wetlands close to traps. decoys are different from images of spoonbills on posters: wildlife managers interact with them as if they were alive, because of their accurate mimicking. the use of decoys is a traditional feature of bird hunting. by contrast with lures, which attract birds based on their appetites, decoys attribute intentions to prey (schaeffer 1999: 64) . in england, duck decoys take the form of pondside wooden houses into which ducks are attracted by a fake dog, because hunters have observed that ducks swim towards the dog to signal that it has been seen. in the same way, the fake spoonbill is used as a decoy because it creates a situation of communication with the bird. but at the same time, the fake spoonbill is used as a dummy by birders who train to manipulate spoonbills in risky situations, as in epidemic simulations. the use of spoonbill decoys mixes hunting techniques and pastoral techniques because decoys are considered both as intentional agents and as objects that should be manipulated with care. this hypothesis was confirmed on 29 april 2013 when the wild bird federation of tainan led me to a site where three black-faced spoonbills had been trapped -a very rare event, which i was considered lucky to have witnessed. birdwatchers had found shelter in a taoist temple on the banks of the marshes, and they turned on some buddhist music to soothe the birds. with very calm gestures, a man equipped with gloves and a mask sewed a gps satellite tracking device around the waist of the spoonbill, while a woman held it gently but firmly. five other birdwatchers watched them with amusement, taking pictures and making comments on the bird's reactions. the man explained to me that he had to be cautious because it was a young spoonbill, so the weight of the satellite tracking device had to be imposed on its body in such a way that it would not hamper its growth or unbalance its flight. how to build techniques of monitoring that do not kill animals considered as indexes of the fate of their species is a major concern for wildlife managers, who often talk about the sacrifice of individual animals when they die as the result of an improper monitoring device (benson 2011) . but taiwanese birdwatchers focused the discussion on how to properly release these birds so that they would not be sacrificed but become sentinels of a threatened species. the release of birds is a major ritual concern in south china, known as fangsheng (literally 'let live'). this practice finds its roots in aristocratic practices of opening the cages of birds who sang well, and was codified in taoist rituals as a specific sequence of releasing a live animal; but it has developed massively in recent centuries with its qualification by buddhists as a practice of compassion that increases the 'merits' (gongde) of those who release live animals (handlin smith 1999) . in hong kong and taiwan, buddhist associations entered into discussion with birdwatching societies because many birds were found dead after they were bought in bird markets and released in surrounding natural parks. these associations had to warn through posters that releasing birds could cause the death of birds, because they were released in an improper environment, and the death of humans, since some of the birds found dead after the release carried avian flu (severinghaus & chi 1991) . consequently, when they released a bird equipped with gps that they had learned to manipulate through the use of decoys, taiwanese birdwatchers inserted a ritual sequence into a simulation practice. while the ritual introduced the bird into a cycle of life and death, the simulation used the decoy to produce signs of future threats for birds and humans. the black-faced spoonbills were released in the taijiang national park with the hope that they would fly over to hong kong or the fujian coast, where their signals could be captured. it should be noted that the taiwan straits are also the sites of military exercises or drills through which threats coming from china are mitigated. to prepare for an attack from the people's republic of china, the taiwanese centers for disease control was organizing exercises simulating the use of bioweapons in a public space, such as sars, smallpox, and avian flu virus (rollet 2010: 311) . the release of a black-faced spoonbill in the same chinese straits mimics and reverses the scenario of an avian flu outbreak from china. instead of carrying information on the potential war between two long-term enemies, it produces signals on the changes of bird habitats that concern birds as well as humans. while in the public health simulations birds are considered as carriers of infectious diseases, birdwatchers' simulations frame them as living beings with which humans share pathogens as signs of communication. another practice with birds in taiwan clarifies by contrast their role in simulations. according to peta, more than a million pigeons are released every day from boats in the taiwan straits by pigeon-racing clubs, and only 1 per cent survive to come back to their shelter. huge amounts of money (an estimated us$2 billion) are spent in bets on those which will return, welcomed as heroes if they are first, killed if they are too slow. 4 this is neither a ritual nor a simulation but a gambling practice through which birds crossing the sea between china and taiwan are qualified as political signifiers. the reverse scenario of the game, producing strange identifications between humans and birds, tells the story of a pigeon successfully returning home while others fail and die in the taiwan straits. releasing a bird in taiwan can thus appear as a simulation when it is embedded in the framework of an environmental flyway, as a ritual when it mobilizes a cosmological view of the circulation of souls between humans and animals, or as a game when it is organized on the border separating two competing territories. simulation, ritual, and play are three frameworks to mitigate the uncertainties of catching and releasing a potentially sick bird. i have considered three stages where bird diseases are simulated: computer simulations of avian influenza by microbiologists; outdoor performances of pandemic influenza by public health administrations; and exercises of manipulating threatened birds by birdwatchers. these three types of simulation displace the problem of sacrifice: that is, the definition of those who deserve to live and those who will die. microbiological surveillance of viruses moves away from the site of emergence where birds are killed to prevent the spread of pathogens to humans, and traces continuities between humans and animals as a reservoir where viruses silently mutate. simulations of epidemics don't address the problem of triage between humans in a situation of exception, but rather configure new relations between humans and animals through accessories in a performance. exercises of vaccinating and releasing potentially sick birds don't raise the issue of how many birds can die for the sake of the species, but rather reflect on how to handle a bird with care in a political environment where they carry signals of threat. in the reverse scenarios of simulations, birds appear not as sources of infection that must be eradicated, but as sentinels with which humans can communicate to imagine potential futures. simulations, therefore, should be analysed not only as a pastoral technique of power, as ways to mobilize populations under a common threat in which some are sacrificed for the sake of others, but also as a cynegetic technique of power, creating relations of identification between humans and animals around a perceived uncertainty at the borders between species. in the world of databased science and transparency of communication, simulations of bird diseases imply playing like animals or with animals to anticipate the (im)possibility of a mass extinction, thus mobilizing elementary forms of identification. in the cases i have considered, the question of the realism of exercises is a way to draw attention to the circulation of objects that recast relations between humans and birds by simulating the diseases they share. the use of computer software, accessories, and decoys in the three types of simulations i have analysed blurs the sharp opposition between humans and nonhumans. these objects can be considered as decoys because they carry the intentions that humans attribute to animals and shift their perception from inanimate objects to living beings. while scenarios of bird-generated epidemics play on the asymmetries between humans and birds, the horizon of the simulation (the extinction of the human or of a bird species) integrates them as common actors of a game. simulations can thus be considered as reflexive rituals because they display the uncertainties of the beings they play with and help to mitigate their threats. framing simulation as an operation unfolding between the spheres of mimetic play and reflexive ritual, and at the same time performing a series of subject-object reversals, i have argued for the need to approach this key apparatus of preparedness through a lens that allows for the mixing of pastoral and cynegetic techniques. what i have called in this article 'avian preparedness' takes place in a long history of simulating bird movements, from classical divination by roman augurs to medieval bird hunting and modern birdwatching. the specificity of contemporary forms of anticipation through sentinels, simulation, or stockpiling is that the distinction between cynegetic techniques of preparedness, taking the perspectives of birds on future threats through imagination and communication, and pastoral techniques of prevention, protecting human or nonhuman populations from external dangers through risk calculation, has become more difficult to make and yet more urgent. this article has argued for such a distinction, while opening the stage for future analyses of simulations as forms of play and ritual. this research was made possible by a project on simulations of disasters headed by sandrine revet at sciences po paris in 2011-13. i am grateful to her and to marc elie and guillaume lachenal for the discussions we had on simulations, as well as to the four anonymous reviewers and the editors of the jrai, who helped me improve the writing of this article. 1 this approach was launched in 2004 by the wildlife conservation society, and then endorsed by the international organizations for human and animal health (http://www.oneworldonehealth.org/, accessed 7 february 2018). 2 contrary to other viruses such as smallpox, flu viruses don't have their genetic information encoded in dna (a double-stranded helix of nucleic acid) but in rna (a single-stranded ribonucleic acid). this explains why they mutate more randomly. 3 anonymous interview by email after a questionnaire sent to the ams, february 2009. 4 http://www.peta.org/action/action-alerts/first-ever-taiwan-raid-police-bust-pigeon-racers/ (accessed 7 february 2018). twenty-first century plague: the story of sars anticipation: technoscience, life, affect, temporality rehearsing for the plague: citizens, security, and simulation ava holds culling exercise in poultry slaughterhouse wired wilderness: technologies of tracking and the making of modern wildlife darwins korallen: frühe evolutionsmodelle und die tradition der naturgeschichte material proximities and hotspots: towards an anthropology of viral hemorrhagic fevers the pandemic perhaps: dramatic events in a public culture of danger manhunts: a philosophical history ecologies of comparison: an ethnography of endangerment in hong kong stages of emergency: cold war nuclear civil defense wild goose chase: the displacement of influenza research in the fields of poyang 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potentialization of uncertainty ritual and its consequences: an essay on the limits of sincerity prayer animal release in taiwan an influenza epicentre? models, simulations and their objects dating the emergence of pandemic influenza viruses emergence and predominance of an h5n1 influenza variant in china origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic culling exercise. razor tv simulation exercises on influenza pandemic responses in the asia-pacific region flight ways: life and loss at the edge of extinction seeking refuge: birds and landscapes of the pacific flyway learning from sars. andrewzolli.com cet article décrit les relations entre humains, animaux, artefacts et pathogènes dans les simulations de désastres, en considérant les maladies qui affectent les oiseaux dans trois territoires sentinelles aux frontières de la chine comme des cas ethnographiques. en s'appuyant sur les distinctions entre la simulation, le ritual et le jeu, il montre que l'engagement des acteurs dans l'imaginaire des simulations, qu'ils décrivent sous le terme de « réalisme », renverse réflexivement les oppositions entre les humains and les non-humains, l'actif et le passif, la fiction et la réalité qui organisent la vie quotidienne. en empruntant les concepts de l'anthropologie des sociétés de chasseurs, il montre que les simulations de maladies aviaires, lorsqu'elles sont perçues comme des signes d'une potentielle extinction d'espèce, recourentà des techniques de pouvoir cynégétiques, dans lesquelles des humains et des animauxéchangent symétriquement leurs perspectives, et pas seulementà des techniques pastoralistes, dans lesquelles les humains sont au-dessus de la population qu'ils surveillent et parfois sacrifient.frédéric keck is a researcher at the laboratoire d'anthropologie sociale (collège de france, ehess, cnrs, psl). he has conducted research on the history of social anthropology and on the contemporary management of animal diseases, and headed the research department of the musée du quai branly.laboratoire d 'anthropologie sociale, 52 rue cardinal lemoine, 75005 paris, key: cord-274219-nh2t1qsl authors: harwood, stephen; eaves, sally title: conceptualising technology, its development and future: the six genres of technology date: 2020-08-30 journal: technol forecast soc change doi: 10.1016/j.techfore.2020.120174 sha: doc_id: 274219 cord_uid: nh2t1qsl one approach to developing futuristic views of technology is to draw upon experience and expertise. however, this becomes increasingly speculative as one moves to more distant timelines and visionary technological forms. this raises the question of whether it is possible to rationally predict how a technology development trajectory might unfold into the future, perhaps to some ‘ultimate form’, that is accessible, surfaces the necessary technological features for development as well as considers the implications for human–artefact relationships. the proposed approach is conceptually grounded in a parsimonious framework that examines different configurations of human–artefact relationships, revealing ‘six genres of technology’. this suggests how the shift from human-human to artefact-artefact and the increasing autonomy of the artefacts (technological beings), introduces specific features to each of the six genres. four features are identified in the later genres that in combination, could be construed as, or indeed pose a threat: autonomy, intelligence, language, and autopoiesis. this paper advances the debate about future technological developments by using the proposed framework to structure an argument about the key issues that should be discussed today so that the developments of tomorrow can be more reflectively considered, appropriately debated and knowingly pursued. rapid acceleration of technological development is leading to technological forms that were unimaginable just twenty years ago (e.g. autonomous vehicles, deep learning, intelligent robots). technologies that once were in the realms of science fiction have become an everyday reality and, along with benefits, are creating predicaments and raising concerns about implications for the future of work (dellot and wallace-stephens, 2017) alongside potential malicious use in areas such as warfare (fli, 2015) . beyond this, new technologies are provoking anxiety for the very future of humanity itself. as stephen hawking stated: "the development of full artificial intelligence could spell the end of the human race. once humans develop artificial intelligence, it would take off on its own, and re-design itself at an ever increasing rate. humans, who are limited by slow biological evolution, couldn't compete, and would be superseded" (cellan-jones, 2014, n.p.) . however, this alarmist view is just one possible perspective about how technology might unfold, with dreamers anticipating a world of leisure, incrementalists foreseeing gradual change and sceptics being dismissive of newer innovations (dellot and wallace-stephens, 2017) . indeed, facebook's mark zuckerberg critiques 'naysayers' arguing that they are irresponsible for drumming up doomsday scenarios (zuckerberg, 2017) . thus, we are faced with the challenge of how to view the unfolding future of technology. in thinking about the future, it allows us to anticipate possibilities and consider their implications in terms of preparation for and mitigation of their impact. how can we move beyond speculation, hype, hope and fear to informed discussion and decision making, enhanced forecasting and, thereby, the shaping of this technological future for the benefit of humanity? to answer this question, there is firstly the challenge of anticipating the manner with which technological developments might unfold longterm. this elicits approaches from the technology futures domain, though this domain is characterised by different views about approaches (lu et al., 2016) . in a comprehensive review of technology pervasive nature of expert biases (bonaccorsi et al., 2020) which can be found in such approaches as delphi (flostrand et al., 2020) . moreover, prognostications are critiqued for being either too short-term (i.e. unworthy of being viewed as futures) or projecting into the distant future and hence being highly speculative (helmer, 1999) . further, if a technology appears that was not forecast, especially if is disruptive, then this can have serious implications (kott and perconti, 2018) . nevertheless, fye et al. (2013) reveal that expert views are more likely to anticipate whether an event will occur, whilst short-term quantitative methods have the most likelihood of being accurate in terms of predicting when an event will occur. one novel approach to thinking about how the complexity of emergent technologies has reached new levels over time is presented by aunger (2010) . this explores the relationship between artefact and the creature (e.g. animal, bird, insect) that is using it, extending the notion of artefact use beyond humans. whilst its focus upon the artefact helps explain the growing complexity of technology, this is suggestive of a potential predictive capability. to complement this, gibson's (1966) notion of affordance draws attention to the artefact and how it affords possibilities for a creature's action, this being subsequently developed by norman (1988) who utilises the concept in a design context, invoking a relationship between artefact and human. since possibilities for what happens emerge from the relationship between the artefact and human, then this raises the question of whether the notion of artefact-creature relationship can be developed to forecast a technology development trajectory leading to some ultimate end-point such as envisaged by stephen hawking. one option is offered here, providing a holistic exploration of different configurations of the human-artefact relationship. this presents an argument that reveals a technological development trajectory transitioning from the past to the future and unfolding to reveal a sequence of six functional states, each state offering a frame ('genre') with its own characteristic attributes. it is assumed that there are no technical or time constraints, thus allowing some eventual endpoint to be envisaged. consequently, the six genres explain how technology might unfold with growing autonomy and connectivity into some ultimate form -'an autopoietic technological being' existing in communities of similar beings -and the consequent implications for the relationship between humans and artefacts. the genre is characterised by affordances specific to that genre (e.g. homeostasis, autonomy, reproduction) enabling their characteristics to be explored. this argument is grounded in the language of cybernetics and technology studies. positioned within the tfamwg (2004) taxonomy, this approach is soft, exploratory and perhaps fits within the scenarios family. this paper contributes to the debate about future technological developments by offering a conceptually grounded framework to structure an argument about one possible technological development trajectory, one that focuses upon the increasing autonomy of technological beings, to the increasing redundancy of humans, thus not only validating the claim of stephen hawking, but illuminating how his predictions might be achieved. the value of this approach is that it focuses attention upon the impact of the development and assimilation of different functional capabilities, placing into perspective 'what if' debates and inviting responses about what needs to be done to achieve, shape or, indeed, prevent the development of these genres. thus, it provides a unique informed conceptual argument to stimulate thinking (coates, 2000) and challenge mind-sets (schoemaker, 1995) about the salient issues and hence highlight why it is important to question contemporary technical developments and ask what might be their consequences. this contributes to a toolset of arguments that provides an informed perspective about the future developments (avin, 2019) . the argument commences with a reflection about what is technology and how it can be conceptualised, thus clarifying the focus of this paper. it is followed by a synopsis of how technological development can be conceptualised to understand the mechanism for how development might take place. the proceeding section is methodological, appraising approaches to how the future of technology is conceptualised and then leads into the six genres, which comprises the main portion of the discussion. the paper concludes with a reflection upon the underlying message from this exposition and its implications. the entity of interest in this paper is that which humans use to enable or augment what they do. the manifestation is typically a physical artefact, but a general term to label this is technology. this raises the question of what is technology? there are many definitions as illustrated by the variants offered in the oxford english dictionary. these reveal that not only has the term technology evolved in meaning over time, but that contemporary definitions vary considerably in content (fleck and howells, 2001) . for example, rip and kemp (1998: 329) draw attention to the everyday use of the term, that it is the artefact: "the idea of technology as artifacts (gadgets and gizmos) is still widespread in our culture", a view reinforced by kline (2003) . in contrast, schon (1967: 1) defines technology as "any tool or technique, any product or process, any physical equipment or method of doing or making by which human capability is extended". this draws attention to both the artefactual and processual nature of technology. alternatively, dosi's (1982) definition of technology distinguishes between the embodied and disembodied, encompasses a problem context, knowledge, perception, process and as well as the artefact itself, it is: "a set of pieces of knowledge, both directly "practical" (related to concrete problems and devices) and "theoretical" (but practically, applicable although not necessarily already applied), know-how, methods, procedures, experience of successes and failures and also, of course, physical devices and equipment existing physical devices embody -so to speak -the achievements in the development of a technology, in a defined problem-solving activity. at the same time, a "disembodied" part of the technology consists of particular expertise, experience of past attempts and past technological solutions, together with the knowledge and the achievements of the "'state of the art". technology, in this view, includes the "perception" of a limited set of possible technological alternatives and of notional future developments." (dosi, 1982: 147-148) this latter definition, which is adopted in this article, starts to capture the essence and multi-dimensional nature of technology, which is exclusively a human phenomenon. mcginn (1990) identifies eight dimensions, these being: material form, fabricative process of bringing into being, purpose, resource requirements, technical knowledge, method of bringing into being, the sociocultural-environmental context in which the preceding takes place and lastly the practitioner's mental set (e.g. attitude, views, values) . this invites a more extensive framework such as the technology complex proposed by fleck (2000) (fig. 1) to surface not only the artefactual and technical aspects of any specific technology, but also the manner in which technology is used alongside contextual issues such as economic and social-cultural dimensions. underpinning this view is the relational nature of technological artefacts (both material and non-material) with people, captured by such concepts as 'assemblages' (larkin, 1969; landstrom, 2000) , 'sociotechnical systems' (trist, 1981) , 'seamless web' (hughes, 1986) , 'sociotechnical constituencies' (molina, 1990 (molina, , 1997 , 'mangle' (pickering, 1993) 'ensembles' (bijker, 1995) , 'entanglement' (orlikowski, 2005) , 'sociomaterial assemblage' (suchman, 2007; orlikowski and scott, 2008) and 'imbrication' (leonardi, 2011) . these concepts infer the conjoined relationship between the artefact and its user, though the nature of the relationship dynamics is unclear. nevertheless, it can be argued that once an artefact is brought into being, it has a distinct existence or 'biography' (kopytoff, 1986 : 66) of its own. in other words, the artefact can be modified over time and it need not be attached to a single user, but can have multiple users and uses until its end-of-life. this shifts attention to two aspects of artefacts from the perspectives of both design and use. one, reflecting the essentialist view, is that the artefact's properties, underpinned by the laws of nature, determines what is possible (fleck and howells, 2001) . in this sense, the artefact is deterministic in terms of what can be done (winner, 1980) . in contrast, is the anti-essentialist view, which is concerned with how meaning is ascribed to the artefact and how embedded inscriptions are read (grint and woolgar, 1997) . a third aspect is offered by actor network theory (e.g. callon, 1986; latour, 1990 latour, , 1996 . this argues for agnosticism (i.e. impartiality), symmetry (i.e. all viewpoints) and free association (i.e. no a priori views) in how the relationship between humans and non-humans is viewed (callon, 1986) . they are all actants with agency (latour, 1990) , although it is unclear how this agency functions. in contrast is the notion of 'affordance', introduced by gibson (1966 gibson ( , 1979 . technological artefacts afford possibilities for use, which can arise through design (norman, 1988) . this focuses attention upon the dynamics of the relationship, that is absent in the previous terms and draws upon the notion of properties of artefacts and how these are perceived. however, this concept is itself problematic as there are different views about what constitutes an affordance, for example, is it merely pertaining to perceptions or is there a cognitive component (harwood and hafezieh, 2017) ? underpinning this human-artefact relationship is the view that there is a homeostatic balance among all relations that maintains stability. first presented by cannon (1926 cannon ( , 1929 to explain how the body maintains a steady state, the notion of 'social homeostasis' has been developed (cannon, 1932; wiener, 1948; bateson, 1972) and extended to the relationship between human and machine (ashby, 1952) . indeed, homeostasis underpins the notion of cyborg presented by clynes and kline (1960) . in summation, the notion of technology is as a human-artefact homeostatic complex, with each offering some form of agency. it is relational, multi-dimensional, both embodied and disembodied as well as intertwined and dynamic, yet is essentially stable. it affords possibilities about what can be done, but is underpinned by knowledge about how to make the human-artefact relationship work. to talk about technology, therefore, is to shift beyond the artefact and embrace the complexity invoked. since technology is characterised by complexity then this invites questions about how it has developed over time, how we are informed about technological developments and how we can conceptualise the process of technological development. whilst humans, as biological entities, have evolved slowly over time, technology appears to have developed at an accelerating rate. indeed, 2019 marked the 50th anniversary of technological forecasting & social change and provides a fitting frame to put this transformation into context. while paper was invented in china around 2000 years ago, the gutenburg printing press in the mid-fifteenth century, and the first digital computers in the early 1940s; in the last 50 years alone, man has landed on the moon (1969), a sheep has been cloned (1996), computers have become common-place in the home (from 1980s), social media (2000s) has become omnipresent and 5g global rollouts are commencing (2019) during the 2020 covid-19 pandemic, advances in high performance computing power and computing capacity have come to the fore with this harnessed to accelerate the research curve to better understand the virus and at a pace never seen before, to develop treatment regimes, testing and ultimately a vaccine (hpc consortium 2020). with ways of working, learning, collaborating and indeed living changed due to widespread global lockdowns, technology has quite literally needed to take the load: on 11th march, 2020, data centre services provider deutsche commercial internet exchange set a new world record in frankfurt -achieving data throughput at over 9.1 terabits per second (de-cix, 2020) . putting this into a wider context, newer forms of technology have now emerged that were either not conceived, or not anticipated to be actualisable twenty years ago, including hybrid and multi cloud computing, deep learning-based predictive analytics, brain-computing interfaces, distributed ledger technology including blockchain and at a nascent stage, quantum computing. the scale of the impact and iteration of these newer technologies has perhaps not been fully anticipated, particularly in terms of the emergent opportunities and threats arising from their integration. this is well illustrated by the rapid evolution of the smartphone, which has had profound implications for people's behaviour, from shaping the nature of experiences in sectors such as tourism (wang et al., 2014) to enabling meaningful personalised services at scale across health, home, retail and mobility enabled by location-based tracking, artificial intelligence (ai) and related sensor technologies. it has also contributed to a move away from the duality of being either online or offline with an increasing transition towards hybridity (eaves, 2020) , whereby we are frequently unthinking in our flip between our material and digital selves (simunkova, 2019) . but there are also implications such as health concerns arising from problematic smartphone use, including the 'fear of missing out', the need for touch arising from the sensation experienced from holding a smartphone and the impact of continued use upon anxiety and depression (elhai et al., 2016) . with the advance of 5g enabled devices becoming mainstream during late 2019 / early 2020 and bringing enhanced download speeds, reduced congestion, lower latency, less interference and more efficient capacity, the development of the smartphone is about to start another transformation in a global ecosystem of connected sensor rich devices -the realization of what is referred to as the internet of everything (o'leary, 2013; eaves, 2020) . but we have to ensure that this benefit is achieved securely and is inclusive and available to everyone -as an example, women in low and middle-income countries are still 8% less likely than men to own a mobile phone (gsma 2020). being offline has significant social and economic impacts from social exclusion to a limiting of career mobility (capgemini 2020) . the scale and depth of transformation can have unintended consequences with the gift of opportunity not equally distributed to all. focusing on a specific development area, the impact of cloud computing (public, private and increasingly hybrid multi cloud) to transform the it industry (nieuwenhuis et al., 2018) provides a good example, enabling enhanced, agile and integrated optimisation of resources on demand and becoming a contributory factor in the rise of home / mobile working and learning even before covid-19. the adoption of cloud solutions continues to accelerate but its consumption is evolving -it is not a linear path or one route fits all panacea (bailey and eastwood, 2018) . as an example, we are likely to see some repatriation of workloads to on-premise given the rise of edge computing -namely the relocation of computing resources closer to the point of use (network periphery). in this way, data aggregation and processing occurs at the edge so that only the results of processing are sent to the cloud -not data -reducing bandwidth costs and providing a strong partnership for internet of everything solutions (eaves 2020) . this highlights the speed of change within even the most recent technological developments, alongside the impact of technology integration as a catalyst for innovation at scale. additionally, the availability and analysis of big data is creating a tension between opportunity and risk. on one hand, providing the capacity to gain nuanced insights to inform decision making and offer personalised services, products or care; and on the other, raising many social and ethical issues around usage, privacy and ownership (newell and marabelli, 2015; eesc 2017) . this is especially the case given the lack of trust that has accelerated globally over the last decade. levels of trust inequality between the informed public and more sceptical mass population and also trust inequality by gender, has created divergent levels of confidence, and most recently, a shift towards localized trust centred on relationships perceived to be within people's control (edelman group 2019) . these examples raise the question of how quickly future developments will emerge and what issues will arise. the narrative about the development of technology also matters. during the last 50 years, public attitudes towards computers and technology have been largely shaped by the printed press and more recently online and social media. tuchman (1978) has described how different forms of mass media are a significant contributor to the social construction of reality. early media reports related to technology are explored by martin (1993 martin ( , 1995 ) who reveals how the use of headlines, exciting imagery and metaphors suggests that developments are dramatic rather than incremental. however, when our expectations are not met, our early enthusiasm is displaced by disillusionment and perhaps distrust. in more recent years, there has been a growth in multi-media forms especially social media, with increased accessibility to different audiences via a range of mediums and types of devices -but the use and impact of headlines, imagery and metaphor continues. there is a strong narrative around the 'dark side' of technology focusing on what it may take way rather than enable, most notably around the impact of robots and ai on employment and the future of work (eaves, 2018) . headlines can impact the way we think (surber and schroeder, 2007) and even subtle misinformation can have significant impacts on a reader's memory, inferential reasoning and behavioural intentions (ecker, lewandowsky, chang and pillai 2014) . it is also argued that deterministic headlines about technology are also unhelpful as they can narrow dialogue and debate rather than invite different perspectives to come together. in their recent paper, bhullar et al. (2019) discuss specific opportunities that can be attained through academia-industry collaboration. such collaboration could also be relevant to better informing the media narrative. whilst the metaphor used to shape the way that we perceive, think, and act can be powerful (martin, 1995/6) , metaphors also can be employed to represent, explore, clarify, structure and provide a bridge to understanding in developing fields (bazeley and kemp 2012) . for example, one of the current widely used metaphors for emergent newer forms of technology is the notion that they are 'disruptive'. these technologies may be labelled disruptive since they 'break asunder' (oed, 2018) and thus transform what preceded, especially in terms of associated social arrangements as revealed by fleck (2000) . this contrasts with bower and christensen's (1995) view of disruptive technologies as being those that are oriented to new applications or markets and tend to be initially of inferior quality / performance. irrespective, they are the outcome of both incremental and radical developments of their discrete elements (bower and christensen, 1995) . indeed, it is perhaps not these individual developments that have given rise to disruptive technologies, but novel configurations. the importance of configurations is that they draw attention to what emerges but which cannot be anticipated. a specific technology can have a technology developmental trajectory that maintains a performance improvement rate through both incremental and radical developments (bower and christensen, 1995) . this trajectory, constituting the history or ontogeny of structural changes, preserves the identity of that technology (maturana and varela, 1975) . however, a configurational technology offers different possibilities arising from the interpretive flexibility (bijker, 1995) of what is possible. configurations can also be viewed as stratified, for example, components are configured into modules which are configured in turn into devices then working systems (rip and kemp, 1998) . from these possibilities, recurring configurations may emerge and stabilise or 'crystallise', perhaps leading to contingency-specific generic systems (fleck, 1992: 8) . these generic systems are then more likely to have identifiable technology development trajectories (fleck, 1992) . one established metaphor that provides insight into how we understand the process of how technology unfolds over time, is that of evolution (e.g. rip and kemp, 1998; bowonder et al., 1999; fleck, 2000; tfamwg. 2004; devezas, 2005; lipsey et al., 2005) . ziman (2000) explores this metaphorical usage comparing models of biological evolution with technological evolution. underpinning the biological model are genomes (i.e. dna), genotypes (i.e. gene sets) and phenotypes (i.e. traits), the former constituting hereditable genetic code. central is the debate about whether there is transmission of a phenotype's attributes or changes that result from engagement with the environmental context that co-exists with the phenotype into the next generation. jablonka (2000) makes the distinction between the weismanian doctrine, which denies this transmission, and the lamarkian doctrine, which provides for it. the darwinian mechanism of natural selection of heritable differences arises from mutations of the genotype, though it is unclear how these arise. assuming the relevance of the metaphor, this introduces two issues. first, do artefacts have a genetic code and second, how do artefacts develop; are the weismanian or lamarkian models of development appropriate for artefact development? fleck (2000) argues that to assume a comparison between biological and technological evolution is misleading. the biological model invokes a replication mechanism (underpinned by the dna and its stability) and interaction with outside (the environment). this becomes problematic when applied to technology. fleck (2000) asks whether there is a technological equivalence to dna. the ability of biological forms to maintain (adapt) and produce (replicate) highlights the uniquely autopoietic nature of biological forms (maturana and varela, 1975) . indeed, fleck (2000) argues that artefacts are different from biological forms in that, whilst genetic code is embedded within the biological form, it is external to the artefact and is passed on through humans. thus, the ability to replicate or reproduce involves knowledge about, for example, design techniques and production methods, with effective replication requiring a composite of factors, as identified within the 'technology complex' (fig. 1 ). fleck (2000) proposes that the basic unit (c.f. gene) of technological development is the 'artefact-activity couple', an amalgam comprising of artefact, knowledge and organisation, which constitute the essentially necessary but sufficient elements to permit 'self-contained replicating capability' and comprises those most immediate human activities (i.e. production, operation and maintenance) required to develop and use a specific technology. generational differences arise from technical developments or adaptations to operational contexts. a lamarkian model is invoked. these may take the form of incremental or radical changes, these perhaps analogous to mutations. however, this insight into what technology is and how it develops is inadequate for foretelling how technology might unfold in the future. in conclusion, we can perceive not only the accelerated rate of technological development but also scaled integration, particularly with newer forms of digital technologies. however, our individual views about what is happening appear to be very influenced by how we are made aware of developments, with hype preceding disillusionment. if we wish to think about how technology is developing then the metaphor of 'evolution' remains useful, despite the challenges of how we make it fit. the challenge of anticipating the manner with which technological developments might unfold long-term invites approaches from the technology futures domain (lu et al., 2016) . indeed, there are a proliferation of approaches as revealed in tfamwg's (2004) comprehensive review of technology futures analysis (tfa). they identify 51 different methods, ranging from statistical to creative, which they categorise as hard (quantitative), soft (qualitative), normative (endpoint orientated) and exploratory (extrapolative). techniques include statistical analysis, science fiction analysis, focus groups, road-mapping and scenarios. their diversity draws attention to the contrasting roles of expertise and creativity as well as analysis and description, invoking that they each have different emphasises and strengths. as such, tfamwg (2004: 291) proposes the complementary use of multiple and mixed methods. as an example, hussain et al. (2017) combine expert opinion with creative thinking in their scenario-driven road mapping approach. moreover, even within a specific approach there is diversity, as revealed in the review of scenarios by lacroix et al. (2019) . however, forecasting how technology will develop and which technologies will prevail is a challenging and contestable domain (albright, 2002) . this is a view shared by phillips and linstone (2016) , who foreground its magnitude with the word 'struggles'. a variety of concerns include the related issues of the time horizon of studies and the accuracy of the approaches employed. indeed, one of the challenges is the pervasive nature of expert biases (bonaccorsi et al., 2020) , which can be found in such approaches as delphi (flostrand et al., 2020) . as such, ayres (1999: 12) appears dismissive of delphi and related approaches: "i do not have a high regard for expert judgments, taken as a class. let me leave it at that". further, prognostications have been critiqued for being either too short-term (i.e. unworthy of being viewed as futures) or projecting into the distant future and hence being highly speculative (helmer, 1999) . in contrast, is the inaccuracy and serious consequences that can arise from failing to forecast (false negative) a technology that does appear, especially when this has a disruptive impact (kott and perconti, 2018) . nonetheless, short-term quantitative methods have the most likelihood to be accurate about when an event will occur, whilst expert views are more likely to anticipate whether the event will occur (fye et al., 2013) . it is also evident that forecasting how technology develops into the future invites novel approaches. as an example, aunger (2010) demonstrates how the complexity of emergent new technologies reaches new levels over time. this work reveals how creatures (e.g. beavers, spiders, bees) have engaged with artefacts, progressing to how the 'human' creature has developed the artefact to increasingly more complex levels of sophistication (e.g. modern production). whilst this focus upon the artefact helps explain the growing complexity of technology, this is suggestive of a potential predictive capability. indeed, the notion of the artefact-creature (human) relationship can be developed to explore how technology can be developed into the future, culminating in some end-point. by considering possibilities in the human-artefact relationship, this offers a mechanism for establishing how one might consider the unfolding of technology into a scenario like one envisaged by stephen hawking. stephen hawking's prediction about how ai would take off on its own invites the question of how this might happen. indeed, should we not assume that it is an embodied ai, since we might also assume that any ai would want to take responsibility for the physical domain which it inhabits. thus, it is a technology that comprises both tangible (e.g. physical) and intangible (e.g. knowledge) elements and is shrouded in complexity (section 2). it will have a developmental trajectory which is rooted in human history, is emergent and so is unpredictable. its speed of evolution is faster than that of the biological evolution of humans, with the possibility of many generations emerging within one human generation (section 3). this might be the stuff of science fiction, but it is possible to map out a trajectory which, while making another assumption that all technical constraints can be overcome, reveals how such a technological entity or community of autonomous autopoietic technological beings might emerge. the argument is grounded in a simple framework based upon the notion of the human-artefact relationship, which has also found form in the aunger (2010) creature-artefact evaluation (section 4). the transition from human-human to human-artefact-artefact-human to artefact-artefact reveals a developmental trajectory that comprises of six frames (genres) (fig. 2) . each genre has unique functional mechanisms that define the nature of the metamorphic impact each genre has upon the developmental trajectory. for example, genre 3 draws attention to the recursive nature of artefacts produced by humans, which are used to produce artefacts for human use (e.g. the mass production line assembling subassemblies, assembled in a production line using components produced in a production setting). likewise, genre 4 focuses upon the growing autonomy of artefacts (e.g. the shift from mechanical cars to self-driving vehicles), genre 5 upon autonomous interaction between artefacts (e.g. smart technologies) and genre 6 upon the ability of artefacts to produce themselves autonomously in technological collectives (e.g. the narrative of science fiction). the systematic unfolding of functional mechanisms reveals the process underpinning the developmental trajectory. by focusing upon functional mechanisms, this escapes the constraints imposed if focusing on specific technologies to develop the argument. instead, specific technologies are used to illustrate the existing embryonic nature of what is proposed, suggesting that the narrative of each scenario is plausible, assuming that technical challenges can be overcome. moreover, in view of the accelerating pace of technological development proposed in section 3.1, then, whilst no attempt is made to predict when our scenario will unfold, it can be assumed that significant progress in technological advances will be achieved even within the next 20 years. indeed, these issues provide topics for possible debates about what needs to be done to achieve, shape or, indeed, prevent the development of these genres. a more detailed account of each of the six genres follows. the notion of a technology free world consisting of humans or forbearers in whatever stage of evolution, is one that is hard to conceive. however, it draws attention to the social dimension which forms the essence of what it is to be human. as social beings, there has emerged such phenomena as norms, culture, values and politics. aunger (2010) suggests that the technologies of humans are rooted in its forbearers. however, if artefacts are to be introduced into the relationship between humans or its forbearers, then it is initially likely to be of an opportunistic nature (e.g. the use of a fallen tree to bridge a river). these artefacts can range from those whose form offers the convenience of usage (e.g. the fallen tree), to those that are purposefully shaped (e.g. stripping a stick of foliage). nevertheless, this is not confined to humans, with other biological forms recognising the intermediary possibilities of artefacts (e.g. chimps using a stick to penetrate an ant nest). aunger (2010) suggests this can involve social learning, as observed amongst primates. however, the emphasis is upon the affordances offered by the artefact to serve a specific purpose. the recognition that artefacts can be used to make other artefacts (e.g. flint arrowheads) was perhaps the first explicit recognition of the tool-product relationship. whereas the artefact used to do something is an instrument, when used to produce other artefacts it can be viewed as a second order instrument (aunger, 2010) . it is within this genre that much of human endeavour has been concentrated. indeed, this has given rise to a variety of categorisations of the phases in human development, such as stone, bronze and iron; classical, medieval and modern; jet, atomic and space. such categorisations suggest that specific periods in time can be identified and characterised, for example, the 'age of steam and railways' (freeman and perez, 1988) . toffler (1980) distinguishes between agricultural, industrial and a 'third wave', this embracing renewable energy and computer intelligence and giving rise to new industries such as electronics, aquaculture and genetic engineering. likewise, grinen et al. (2017) propose three production revolutions: agrarian, industrial and cybernetic, this later being characterised by self-regulating systems. more recently, the concept of 'industrie 4′ has been proposed to provide vision for the development of 'smart' domains, these constituting 'cyber-physical 'systems (kagermann et al., 2013; reischauer, 2018) . these more recent developments are invocative of the subsequent genres, especially as they become intelligent (genre 4) which marks the latest transition in the shift from mechanical to digital devices that characterises genre 3. irrespective of these categorisations, the development and use of technology draws attention to four interconnected issues. first, is the manner in which a technology is taken up and embedded into the everyday experience of use. second, are the broader organisational, economic, social, cultural and governance implications as revealed by fleck (2000) . third, are the requisite conditions for the ongoing functioning of technologies -the infrastructural issues which tend to be invisible until a problem arises, this highlighting 'infrastructure inversion' (bowker, 1994) . fourth, is the special case of the more intimate nature of the human-artefact relationship manifesting in the cyborgisation of the human being. at the immediate level of the human-artefact relationship is the way in which the product is taken up and used. implicit is the embedding of the artefact into the organisation of the everyday -its domestication (harwood, 2011) . this is not a passive activity but may result in changes to both the ways things are organised and relations with others. further, this requires knowledge about both the technicalities of the artefact and the situation and, thus, how the artefact can be introduced into context specific situations (fleck, 1992) . the emphasis is upon the situatedness of practice (suchman, 1987) . the focus is local. more generally, technology is a phenomenon of society and its development. it has evolved from informal tinkering and disciplined experimentation into organised industrial functions (e.g. research and development, design for manufacture and market research). this has been conceptually explained with such terms as innovation (schumpeter, 1939) , diffusion (rogers, 1962 (rogers, , 2003 and innofusion (fleck, 1988) . the scale of adoption has also given rise to consumerism and commodification. this scaling-up of demand has transformed local craftsmanship into geographically distributed complex mass production systems comprising such features as procedures, skills, work organisation, factories, supply chains, industries and international trade. this has involved the development of automated artefacts (e.g. cnc machines, robotics) that perform tasks for humans (e.g. mass scale replication of products), serving human needs (e.g. the activities of the everyday), but requiring human intervention (e.g. maintenance). these technologies can also produce information, creating transparencythey 'informate' (zuboff, 1988) . coinciding with this is the development of computational devices (e.g. mainframes, personal computers, laptops), enabling more efficient process management (e.g. stock control, management accounting). michael (1962: 6 ) labels this computerautomation configuration as 'cybernation'. more recently the internet has given rise to new forms of social engagement, from the physical to the virtual, with it changing social practices and behaviours such as purchasing. with each newer generation that grows up alongside associated newer forms of technology, especially as physical reality transfers into the digital domain and hybridity emerges in their fusion, questions are being asked about the implications. as an example, 'generation z', the digital natives born circa 1995-2010 who have grown up with online and mobile technologies, multiple realities and social networks, are argued to be different from previous generations because of this association (turner, 2015; schroth, 2019) . this has created debate (e.g. schroth, 2019) as to the work-place, commerce, education, social and cultural implications for this newer generation, but also raises the question about what can be inferred for subsequent generations as we move into an age of smart interconnected technologies. the acceleration of adoption of the digital across everyday living, working and learning, catalysed by covid-19 and the changes in approaches to engagement, experience, patterns of consumption and buying behaviours observed as a result, offers insight into the future, especially as this has impacted cross-generationally at a scale never seen before. the oed (2018, n.p.) definition of 'infrastructure' is "a collective term for the subordinate parts of an undertaking; substructure, foundation; spec. the permanent installations forming a basis for military operations, as airfields, naval bases, training establishments, etc." infrastructure is in the background, invisible and often taken for granted, but essential in its enablement of everyday activity. it only becomes visible when there is a problem, this being the phenomenon of 'infrastructure inversion' (bowker, 1994) . there are two aspects to infrastructure. the first (the material dimension) is concerned with habitation (e.g. facilities for home, education, health, work and leisure). the second (the spatial dimension) focuses upon supply-delivery (e.g. of people, data, energy, materials, services) and draws attention to modes of transportation / communication (e.g. ships, aircraft, auto-vehicles, radio and blue-tooth). they are not mutually exclusive. within genre 3, the technologies have shifted from mechanical to automated, with attendant improvements in efficiencies and increasing convenience in the home and work-place. however, what distinguishes these developments from newer developments is the absence of ai, this denoting genre 4. there is a special form of human-artefact relationship, which is more intimate -involving the cyborgation of humans. the cyborg is a concept first conceived by clynes and kline (1960) , to capture the notion of a 'cybernetic organism' which could exist in unnatural environments (e.g. space travel). it is defined as a: "self-regulating man-machine system… [an] exogenously extended organizational complex function as an integrated homeostatic system unconsciously" (clynes and kline, 1960: 27) . this is a fusion of the organic with the inorganic, underpinned by homeostasis as the mechanism to regulate stability in the relationship. the cyborg has gained in prominence through science fiction notably dr who's daleks (nation, 1963) and cybermen (pedler, 1966) and in films such as robocop (neumeier and miner, 1987) and seven of nine (star trek, 1997) . indeed, the mass media narrative around cyborg technological developments is still often conveyed with all the attributes and often risk, drama or fear factors associated with science fiction (eaves, 2020) . however, the actuality manifests in technologies that are attached to (e.g. prosthetics, glasses, self-tracking wearables, exoskeletons), penetrated-into (e.g. dental treatments), and embedded within the body (e.g. chips, pacemakers) (haddow et al., 2016) , with a degree of permanence. additional variants include ingestion (e.g. medicines, self-acting or biomarker devices), involving absorption, and immersion (e.g. vr, ar, ml), whereby the body's senses are immersed within the blended worlds of virtual, augmented and actual reality. as an example, the world's first large scale nhs trial to test immersive vr therapy for psychosis and other mental health conditions commenced in june 2019 (oxford vr, 2019) . the different types of integration afforded by the development of the cyborg therefore also reflect an opening-up of new choices and tangible opportunities for technology to be harnessed for good. following from the above, these new possibilities are further illustrated by klugman (2001) , who differentiates between enhancement of the body and replacement of body parts, as well the distinction between an embodied but networked brain and an independent brain that can be transferred into other receptacles. klugman suggests that one should look again to science fiction for the unimaginable possibilities that could one day become a reality. indeed, advanced forms of nanotech and biotech developments pave the way to such speculations being actualized. beyond enhancing physical capability past levels of natural performance, the possibility of brain-computer connectivity (lotte et al., 2007; martins et al., 2019) to enhance brain capability arises. further and controversially, is the establishing, ethics and acceptability of human companionship and intimacy with android partners (song, 2018) . this is especially relevant given global issues such as aging population and isolation/loneliness, but also raises such questions as to what constitutes 'friendship', especially if the companion has been present since birth (dautenhahn, 2013) . following from the above is the imaginative view of future cyborgs being wirelessly interconnected (martins et al., 2019) , raising questions about whether their individualism is suppressed for the benefit of the 'collective' (cf. star trek's borg). cyborgisation offers a significant domain for discussion about how it might develop as an alternative to the proposed fourth to sixth genres (e.g. barfield, 2015) . for example, one controversial view is the transference upon biological death of human brain content into digital content (chalmers, 2010) . one elaboration of this is that it becomes absorbed into the digital cloud, with identity being both preserved and authenticated by an advanced form of blockchain, or other form of distributed ledger technology, and with the right permission key, perhaps downloaded into an autonomous technological being giving it 'life' (genre 4). a recent metamorphosis is that of the transition from automated technological artefact to autonomous technological being, with some form of intelligence. this, in its definitive form, is where an artefact is embedded into a context with no further engagement required -it is forgotten about and will serve out its purpose, maintaining and adapting itself until its end-of-life. this can be viewed in terms of the convergence of existing technologies, for example, for perception (sensors), data storage (cloud), processing (high performance computing), calculation and sense-making (ai, machine learning), authentication (blockchain) and action (autonomous vehicles, robotics, drones). this is perhaps the era denoted as the cybernetic revolution (grinen et al., 2017) . nevertheless, this notion of autonomy is open to debate, raising the question of what is an autonomous technology and what issues may be implied or foreseen? context is first provided from the much debated notion of weapons that are autonomous. the us department of defense defines an 'autonomous weapon system' as: "a weapon system that, once activated, can select and engage targets without further intervention by a human operator" (dodd, 2012: 13-14) . however, this allows human intervention to override its operation. a more insightful view of autonomy is provided by dod (2011). this reveals four levels of autonomy: 1 human operated: no decision making capability, but is able to provide feedback to sensed data -these might relate to inanimate and animated technologies. 2 human delegated: automation (de-)activated by humans -these follow prescribed routines. 3 human supervised: multi-tasking automation based on directives but contained within permitted scope. 4 fully autonomous: acts on goals without human intervention, but open to human intervention if necessary. whilst the former appears to relate to manually manipulated technologies, the latter invokes a process that permits both the recognition and categorisation of an object, as well as the ability to be alert to and accommodate the situation when the object changes status (e.g. from enemy to friend). this multi-level view has been designated as humanin-the-loop, human-on-the-loop and human-out-of-the-loop (hrw, 2012). a similar framework has been developed for autonomous vehicles with the 5th level not requiring a driver (skeete, 2018) this draws attention to the distinction between 'automation' and 'autonomy' (vagia et al., 2016) . automation invokes a self-acting process whilst the latter is self-regulating. the distinction is perhaps marked by the manner in which a disturbance is handled. is the process able to adapt and introduce a change that was not part of its initial programming? the cnc machine, as long as it is fed material can keep on producing parts despite the drill bit becoming worn. however, these parts are defective. alternatively, a sensor may detect this wear and initiate an action that replaces the drill bit, preventing defective parts from being produced. however, the cnc machine will be unable to rectify the situation if its servo-motor fails. this requires human intervention. in contrast is the notion of the autonomous vehicle that has been designed to function in a smart transport infrastructure system with minimum of human intervention once it has been brought into being (bissell et al., 2020) . further, they are not confined to the ground as illustrated with experiments in dubai with autonomous aerial taxis (mohamed et al., 2018 ). an autonomous vehicle can re-energise itself, for example using cryptocurrency to cover the transaction, as well as maintain itself at specialised docking points. one purpose is to transport, particularly people, between any two points. on land, the vehicle can sense road conditions and adapt to scenarios such as maneuvreing in crowded traffic. in this sense, it is self-regulating. however, as revealed by the moral machine platform which gathers a human perspective on moral decisions (mit, 2019), if placed into a situation where the autonomous vehicle had to choose between the lesser of two evils, for example killing five teenage pedestrians crossing on a red light or two elderly passengers in the vehicle, what should it do? as seen with the first actual pedestrian fatality involving a 'self-driving' car (gibbs, 2018) , this has raised significant discussion around autonomy and who has control, especially concerning trial situations with human 'pilots' alongside unforeseen consequences (stewart 2018) . this also brings to the fore the importance of 'responsible innovation' as described by ribeiro et al., (2017) . to conclude, the distinction between automation and autonomy is blurred and sometimes complex. indeed, vagia et al. (2016) highlight the multifarious and interchangeable use of the two terms and draw attention to taxonomies of 'automation' which recognise that there are multiple levels to how automation can be viewed. the disruption to both human thinking and doing requires a combination of augmentation through responsible and explainable ai and automation through tools such as a robot process automation (rpa). if the distinction is to be maintained between automation and autonomy, then it is perhaps based upon a view of autonomy in terms of the level of self-regulation and adaptation that is permitted to an entity, within the constraints of the system in which it functions. higher levels of autonomy therefore indicate independent entities that are resilient and have long-term viability. this invokes a context within which there is ongoing interaction. this, in turn, calls upon the entity's awareness of what is going on and the ability to make sense of this, anticipate what is likely to happen next, then generate an 'appropriate' response that serves the being's need to survive within this context. data is processed, which results in something happening. this faculty for humans to apprehend and understand foregrounds the concepts of 'intelligence' and 'knowledge'. in a machine this introduces the interrelated concepts and applications of 'artificial intelligence', 'machine learning' and 'deep learning'. aside from debates about what is 'intelligence' (e.g. maturana and guiloff, 1980) and 'artificial intelligence' (e.g. galanos, 2018) , this notion that machines can have intelligence is not new. turing proposed in 1951 that machines, through learning, could simulate human intelligence (turing, 1996) . more significantly, that at some point "machine thinking… would not take long to outstrip our feeble powers" (turing, 1996: n.p.) . this point of 'singularity', a concept attributed to von neumann (ulam, 1958) , has prompted much debate (e.g. vinge, 1993; chalmers, 2010; magee and devezas, 2011; eden et al., 2012) . this is aside from its mathematical interpretation (magee and devezas, 2011) . good (1966) speculates about the creation of such an 'ultra-intelligent machine', that it has an artificial neural network, learns from experience and will be man's last invention, "provided that the machine is docile enough to tell us how to keep it under control" (good, 1966: 33) . if this is the case, can control of ai be attained / maintained, or, drawing upon the science fiction metaphor, will ultra-intelligence outwit even enhanced human capability, as illustrated in the film 'ex machina' (garland, 2014) , when the intelligent android ava escapes containment. simon (2019) presents four scenarios that explore such possibilities suggesting an inevitability in the dominance of ai. irrespective of the 'science fiction ' connotations, in 1955 ' connotations, in , mccarthy et al. (2006 proposed a study to investigate the possibility of ai. perhaps the first articulation of the features required by an intelligent system was by mccarthy (1958) about 'advice taker', which included the underlying principle, "in order for a program to be capable of learning something it must first be capable of being told it" (mccarthy, 1958: 78) . indeed, 'being told' underpins the virtual personal assistant (vpa), highlighting the association between ai and some form of sensing. in this case the ability to recognise human speech, which involves natural language processing, is a form of ai. the artefact's voice interface (e.g. apple's siri) or text alternative (e.g. google assistant) alongside recognition capability, understands what the human user says / inputs and interprets this to provide a response. although relatively straightforward for simple queries such as 'what is the temperature?', it becomes much more demanding for queries that rely on something that was expressed previously and / or needs contextual awareness. this raises the technical challenge of how to develop the artefact's capability to handle the context, nuances, complexity and variability of the spoken language (collins, 2018; eaves, 2020) . it is this 'making of meaning' in a more complex setting that remains challenging. taking this further, findings from unesco (2019) and loideain and adams (2019) around the almost exclusive and default use of female voices in vpas suggests this may be reinforcing negative stereo types about women or even contributing to a gender gap in digital skills. collectively, examples such as this build in impact and contribute to a lack of diversity of experience in the tech sector (eaves, 2020) . a particular issue has been the 'submissiveness' and discriminatory nature of responses when virtual assistants have been required to react to questions and suggestions that are inappropriate or insulting, reflecting usecases beyond those envisaged for the systems during design (something now being addressed by leading providers). this raises questions about how bias in ai could be neutralised. beyond cultural implications, it is also important to consider that vpas have access to both more personal and a broader range of data than even a typical search engine. indeed, the accumulation of data gathered from sensors as well as the 'digital traces' (hedman et al., 2013; hand and gorea, 2018) we leave behind, enables the digital cloning of everyone, in terms of all aspects of our everyday, resulting in every person having their own 'data body' (critical arts ensemble, 1998) , with its own memory of the past. integrated with, for example, facial recognition into the appropriate infrastructure this can have more sinister implications, including who owns the data and what are implications for privacy and / or discrimination (see section 5.4.3). whilst the issues of meaning, bias and unwelcome use taint the view of ai serving the well-being of humans, another more encouraging view of ai is in terms of the possibilities arising from the notion of the 'digital twin' (dt). although definitions vary with context, a dt can broadly be described as the "assembled aggregation of data captured by other tools" (roske, 2019: n.p.) which can enable the creation of a digital functional model of a physical asset -from a component or product -to a process, system or even city. this increasing 'hybridity' (simunkova, 2019) or bridging of the physical and digital domains makes it possible to obviate the need for physical prototypes (glaessgen and stargel, 2012) and model the behaviour and lifecycle of assets; developing scenarios in advance, enhanced by ai, to optimise outcomes and help create repeatable and scalable experiences (microsoft 2019 ). in manufacturing for example, this can negate issues of bottlenecks and data isolation, stagnation and fragmentation (tao et al., 2018) . another benefit is the capacity to continually update and enhance digital twin modelling with real-world information collected via drones, sensors or other iot tools and apply advanced big data analytics, artificial intelligence and machine learning to acquire real-time insights about the physical artefact's operation, performance or profitability. applications are numerous, from smart manufacturing to smart city development -as an example, rennes in france is building a complete dt of its metropolitan area to improve urban planning (doyle, 2019) . this shift into the virtual domain clearly provides a supplementary dimension to the physical and can be a mutually enhancing relationship, as illustrated by the reciprocal continual improvement of the digital twin -physical artefact. increasing blurring of the digital and physical domains was already anticipated (eaves, 2020) and indeed this transformation has accelerated due to covid-19. this has brought to the fore the need for interoperability of any business asset, from people to technology, alongside having realtime visibility across both organisational boundaries and supply/demand chains. taking this acceleration to an extreme, will a digital twin enable an intelligent autonomous technological entity to continuously improve itself without humans being aware of what is happening? in sum, autonomy grounded in ai and integrated with other newer forms of technology, is evolving well beyond informating and computation to embrace an immersive, interactive, insight-rich, real time hybrid world environment. this highlights the potential of ai, in terms of diagnostics, insights and informed action taking, but also concerns associated with how bias (e.g. gender) is inscribed, as well as issues of privacy, discrimination, data ownership and dependency and their implications for changes in accepted norms of behaviour. nevertheless, ai developments are deemed to be of a narrow intelligence nature, with singularity and the emergence of artificial general intelligence or superintelligence, possibly being many decades away (bostrom, 2014) . the integrated nature of developments and applications of, for example, sensors, wireless connectivity, cloud storage and the processing -analytical capability of ai reveals the growing fusion or hybridity between the material and digital technologies. one benefit is that existing infrastructures such as electricity grids, gas pipelines and water mains can be made more efficient with these smart technologies (kumar et al., 2018) . indeed, by viewing ai from an integrated perspective, then this integrated ai complex itself constitutes an invisible infrastructure. the invisibility of physical structures, is complemented by the lack of transparency of the digital structures associated with ai and the data gathered. the emergent integration of sensors with ai via the internet of everything, increasingly facilitates detection and consequent activation of automated processes, extending beyond the mundane (e.g. doors, lighting, air-conditioning) to the personalised (e.g. re-ordering replacement items and tailored recommendations to predicted future needs). indeed, facial recognition, voice activation and mood detection are altering the relationship between human and artefact from activepassive to passive-active. moreover, integration of different technological forms allow new possibilities for engagement. the 'cloud' provides continuous and increasingly real-time data access and backup. 5g connectivity is giving rise to complex real-time 'smart' networks of engagement with global reach (chettri and bera, 2019) . for example, a surgeon using mixed reality can, at a distance, interact with a robot to perform surgery (vávra et al., 2017) . one potentially disruptive application is that of autonomous vehicles, not isolated from each other, but connected with each other as well as with roadside sensors and traffic management centres through a geographically distributed integrated infrastructure. this synchronises and reroutes vehicle journeys and hence, reduces collisions and congestion (elliott et al., 2019) . the need for human intervention diminishes with increased capability of this digital complex. however, one cannot overlook the dark side, with malicious intrusion creating concerns about cybersecurity and misinformation. further, such questions as to who owns the data, how it is processed and used, and by whom (newell and marabelli, 2015) draws attention to the potential of data as a form of control (marciano, 2019) . taking the example of vpas, a significant majority of this data are effectively in the control of a small number of technology multinationals creating dependency. to put this into context, in january 2019, amazon reported that it had sold over 100 million of its alexa-powered vpas (cnet, 2019). however, inherent bias, whether intentional or otherwise, can cause individual harm through unfair or discriminatory practices or collective social harms such as loss of opportunity, economic loss or social stigmatization. for example one study of facial recognition systems for gender and skin type, found that darker-skinned females were the most misclassified group (buolamwini and gebru 2018) . in the united states, concerns around ai bias, privacy and discrimination have prompted a governance intervention with the proposed algorithmic accountability act of 2019 (barbanel 2019). moreover, with the pervasive use of sensors (e.g. facial recognition) and ai in smart cities, policing may become an embedded feature of the infrastructure with automated penalties being imposed upon violators or perhaps worse for dissenters thus quelling freedom of speech (joh, 2019) . most recently, this has been brought to the fore in the surveilling of protesting crowds, from black lives matter to george floyd, and also determining whether people are observing appropriate social distancing. how can health and safety concerns, rights such as freedom of speech and individual privacy, and promotion of innovation all be balanced? whilst human intervention may diminish, questions are raised about how these digital complexes will shape communities and, more generally, society, as well as, who / what controls this? the time is now for open dialogue, voluntary governance and legislation to address not just what is possible through ai, but what should be prohibited too the scenario for this fourth genre is about autonomous artefacts that have been brought into being by humans using design and production technologies. these have the potential to become intelligent, resilient and agile technological beings with sensory and analytical capability. this is translated into dexterous, manipulative and mobility competencies. their ability to endure draws attention to their ability to firstly sense what is going on, underscoring the importance of sensors with diverse capabilities (e.g. geographical positioning, object / activity recognition and property / status detection); secondly to analyse (invoking a form of ai or machine learning) and, thirdly to self-maintain (invoking the ability to manipulate self physically and/or digitally, using their digital twin). this is in addition to normal functions, such as scheduling, monitoring and adapting their activity, alongside co-ordination where appropriate. however, whether they develop sentience is questioned as they are fundamentally machines (dautenhahn, 2013) . whilst these entities co-exist with and support humans as in the case of human companionship, social mediation and service provision (dautenhahn, 2013) , their autonomy is most likely to be best demonstrated when serving in remote or hazardous environments. such applications include underground mineral extraction, satellite detection-communication services and disaster management -a notable usage being the probing of radioactive fuel at japan's fukushima plant (fackler, 2017) . marine exploration also presents many challenges, with endurance, reliability and adaptability imperative, exemplified by the recent launch of the 'mayflower' autonomous vehicle aiming to cross the atlantic ocean. this demonstrates the capacity of 'augmented intelligence' which is continually cognisant, sensing its environment to inform decision making and operations real time -with no reliance on human capacity to intervene if issues arise (ibm, 2020). space exploration provides another example with campa et al., (2019) suggesting that, due to the hazards of space travel to humans, fully autonomous robots are the more likely contenders to colonise space. perhaps the most infamous futuristic vision is presented in the classic film '2001: a space odyssey' (kubrick and clarke, 1968) , with the computer hal9000 running a spaceship that is transporting humans to jupiter. however, it turned rogue becoming autonomous serving its own interests rather than that of humans. is this the science fiction narrative and associated metaphors taken too far? on one hand, baum (2018) urges a cautious approach to the misinformation around superintelligence, its hype and its potential consequences, whether massively beneficial or catastrophic. campa et al., (2019) however hypothesise that automation will eventually develop into an 'alien' superintelligent being. today, the complexity of software is such that unanticipated or unintended consequences can happen, such as a system shut-down (somers, 2017) . indeed, it can be asked what would stop a superintelligent entity from intentionally shutting-down human life-support systems. the next genre emerges with connectivity between the autonomous technological beings created by humans. this extends the being's capability through collaboration with other beings. this implies the coordination of the activities between entities and invokes a linguistic domain that enables this co-ordination (maturana, 1988) . thus, physical engagement between entities translates into some form of mutually recognisable means to communicate about each other's activity (e.g. making distinctions using body language). this first order linguistic domain characterises human-animal co-ordinations. at a higher level, these distinctions are converted into a symbolic form that manifests as language, a second order linguistic domain. this ability to make distinctions within language gives rise to conversations (maturana, 1988) . this presence of linguistic domains permits communication between autonomous technological beings and hence mutual adjustment and co-ordinated activity. since this is artefact-artefact communication it is less likely to be dependant upon natural language processing capability and instead emphasises the development of machine language capability. however, this moves beyond human generated interoperable communication systems enabling, for example, autonomous vehicle connectivity. rather, machine learning suggests that digital code can be self-generated and, moreover, if this is permitted, the opportunity to move from a fixed lexicon to one which is creative, leading to new linguistic terms and consequent actions and revealing an adaptation mechanism. this is illustrated with the reported case of two facebook chat bots that, in the process of negotiating, created their own language (lewis et al., 2017) . the presence of a linguistic domain draws attention to the collective manifestation of those (technological beings) engaging in language. if fleck's (2000) technology complex reveals the artefact being embedded within a multi-dimensional human social context, then would there be an equivalence for the collective of technological beings? this raises such questions as • how might they be organised for tasks, both in terms of 'communities' and spatially distributed? • would they adopt specialist functions and hence appropriate forms? • would their relationships be equitable or not? • what would be the implications for governance, whether centralised, decentralised or distributed? and • if there is inequity, would this create tensions between competing entities / groups of entities? whilst learning characterises autonomous technological beings, interconnection permits the sharing of this learning and the growth in community knowledge. this, in turn, creates the opportunity for adaptation of all entities within this community and hence adaptation of the community. unlike the human quality of resisting change for whatever reason, there may be reconciliation to ensure that all requirements are met. this invokes a collective form of ai and a collective form of behaviour. critical aspects that are missing include purpose and values, however defined. purposeful behaviour characterises human behaviour, including deviance from societally accepted norms / values about what is acceptable. however, whether autonomous technological beings can have values and how these might arise invokes a significant debate. the notion of cybersecurity suggests that the technological form can be intruded upon in a malicious manner (e.g. infected or spied upon). focusing on the collective, an 'infected' (rogue) technological being might corrupt the pure 'thinking of the collective, which can result in a schism that introduces conflict. however, if this takes place in a world in which humans still have engagement, it might be assumed that humans can stop further production of these beings, as well as terminate existing beings or negate their influence. nevertheless, since these technological beings can be in conflict with each other, the potential also arises for conflict with humans. moreover, if these technological beings are superior to humans in terms of capability, then this may lead to the eradication of humans. it may also lead to the emergence of the sixth genre. the sixth genre is the realm of science fiction and is potentially devoid of a need for humans (cf. stross, 2008) . the critical metamorphosis into this genre is the ability of autonomous technological beings to produce other technological beings, whether as clones or distinctively different -they are autopoietic (maturana and varela, 1975) . the notion of autopoiesis introduces the capabilities of self-maintenance and the ability to (re)produce itself. whilst an autopoietic technological being is perhaps still to be realised, this concept has been given credibility by john von neumann in his writings about automata that are self-reproducing (von neumann, 1966; burks et al., 1946) . further, the development of these beings can be envisaged in terms of the convergence of existing technologies. for example, production requires a specification of what is to be produced (e.g. dna in the form of a 'digital twin') and a mechanism for reproduction (e.g. robotics, additive manufacturing). possible design improvements might be achieved with ai (e.g. generative design). whilst autopoiesis may still be in the realms of science fiction, rabani and perg (2019) discuss current developments in molecular nanotechnology as representative of the most basic functional requirements for autopoiesis to occur. this is a space in which technological beings have expanded beyond earth to colonise other planets. they live in interconnected communities and it is also proposed that rather than being homogenous beings, that they each have a specialism. communities therefore have a purpose related to the locality (e.g. mining, exploring, producing, transporting) and individuals within each collective has a specialism. they could be organised in the form of a collective, communicating with others through the collective network, self-adjusting to the collective will, perhaps in a manner illustrated in the science fiction of star trek's borg collective? an aligned vision has been proposed by schmidhuber (2017) who envisages 'self-replicating robot factories' distributed in space producing ai entities of wide diversity, but with each form surviving though adaptation, collaboration and competition with each other. their ability to reproduce is grounded in each being having its 'digital twin' embedded into its memory. reproduction is lamarkian in the sense that each successive generation has the genotype of its predecessors with enhancement based on this generation's community experiences. thus, each successive generation is a technologically superior being to its predecessor. once a being has served its purpose of being a functional member of the community and has transferred its learning to its offspring; which is limited to two unless circumstances require otherwise, the being either terminates itself, to be recycled, or transports itself to communities which have a high incidence of damage infliction due to externalities. if humans exist then they are not needed by these technological beings and are perhaps reduced to curiosities and kept in special reserves along with other biological forms. indeed, the issue of accountability to humans becomes irrelevant. likewise, are concerns about the sustainability of the planets since these beings are not dependant upon environmental conditions, unlike humanity. this scenario invites many possibilities and questions that benefit from cross-sectoral open dialogue. this exposition presents a conceptual exploration of how technology might develop in the future. it is grounded in a framework based upon the human-artefact relationship and the transition to autonomy and autopoiesis for the emergent technological being. what perhaps distinguishes this approach to thinking about technological developments is its holistic nature, viewing different forms of technology, not in isolation but as configurations of distinct functional capabilities to afford some purpose. six genres are identified, each with their own underlying generic features. this is important as it places into perspective 'what if' debates about development, thereby inviting responses about what needs to be done to achieve, shape or, indeed, prevent the development of these genres. it commences with a view of how technology can be conceived, revealing many ways to consider this, and critically, that it is not just a physical artefact but embraces purpose, practices and knowledge, with implications for organisation, governance, social relations and culture. this is succinctly captured in fleck's (2000) technology complex. implicit is the relationship between that which is the technology and the human, manifesting in such terms as 'socio-technical' (trist, 1981; molina, 1990) and 'mangle' (pickering, 1993) to capture this fusion. it affords possibilities for use, can exhibit agency and exists in an essentially homeostatic relationship with humans. thus, any discussion about technology needs to appreciate this complexity. there follows a brief consideration of how technology develops. this draws attention to the potentially disruptive nature of technology in terms of how it 'breaks asunder' what precedes. this arises, not necessarily from incremental developments, but from the emergence of new configurations which cannot be anticipated. the metaphorical view of development as 'evolution' introduces the comparison between biological and technological evolution. however, these are different with the former assuming a weissman model of inheritance, with a darwinian mechanism, whilst the latter can be viewed as lamarkian, incorporating lessons learnt from the previous generation. this fundamental distinction possibly explains the asymmetrical pace of evolution and the acceleration in speed and proliferation of technological developments over time. having recognised the complexity associated with the artefact and the asymmetrical nature of development favouring the artefact over the human, the foundations are laid to explore the six genres and the emergence of the interconnected autopoietic 'technological being'. the first genre is one in which there is no artefact -this being a period involving ancestors to humans, transitioning to the second genre, whereby objects are exploited for their affordances, and then the third genre, which perhaps characterises much of human existence including the present, with the subsequent genres already manifesting in somewhat embryonic forms. a synthesis of genres 3 to 6 is presented in table 1 . the third genre is one characterised by a multi-millennia symbiotic relationship involving the mutual shaping of human and artefact. commentators have proposed different phases, such as 'age of steam and railways' (freeman and perez, 1988) . each phase has its own characteristics, distinguishing it from others. critical aspects include, first, the manner in which the artefact becomes domesticated into the everyday (harwood, 2011) drawing attention to the situated nature of usage (suchman, 1987) . second are the broader contextual issues, as revealed with fleck's (2000) technology complex, highlighting the mutually shaping nature of the human-artefact relationship at all levels of human organisation. third, are the invisible infrastructural aspects which only become visible if there is a problem (bowker, 1994) . finally, is the special case of the cyborgisation of the human, whereby there is a fusion between human and artefact. from a governance perspective, legislation is usually after the event, inviting questions about whether it is desirable and possible to have legislation in place in anticipation of technological developments and their possible implications. this must be complemented by open dialogue and voluntary governance that is industry led. it is with more recent technological advancements that insights are gleaned into how their configurations might perceivably create new possibilities for the development of the artefact in subsequent genres. the fourth genre appears to mark a transition, breaking the prominence of the third and establishing the artefact as a 'technological being' with its own agency, drawing upon the notions of autonomy and intelligence. however, it exists within an infrastructure that comprises an integration of sensors, wireless connectivity, hybrid multi cloud storage and the processing / analytical capability of ai, which is not only invisible, but also often lacks transparency and explainability in the decision making that results. moreover, there is growing human dependency upon this technology, raising the question of what happens when the infrastructure fails. issues include whether a fully autonomous technological being has the same rights as humans and who / what owns the data that are assembled about any specific objecthuman or non-human. further, who owns any resultant ip? the fifth genre, involving interconnectivity, introduces a linguistic domain, enhancing autonomous behaviour but with the possibility of collective rogue behaviour, which can lead to the demise of humans. indeed, there is growing separation between human and technological collectives as the latter start to develop collective autonomy. this raises questions about how to preserve human well-being, identity and ultimately survival? the sixth genre is one in which technological beings have the capability of self-maintenance and reproduction. they are autopoietic. further, this final stage renders humans redundant. whilst the fused human-artefact in the form of the cyborg has been introduced in the third age, compared to the trajectory offered here, it offers a potentially different one as explored by klugman (2001) and barfield (2015) . however, as previously mentioned, if singularity leads to ultra-intelligent technical beings, will they eventually outwit humans as illustrated in the film 'ex machina' (garland, 2014) , when ava escapes containment, even if humans have enhanced cognitive abilities? invoked in the overall argument underpinning the six genres is the question of how to control future developments for the benefit of humanity, balancing advancement of innovation with appropriate safeguarding and dialogue. each genre has its own characterisation. however, the issues raised here for debate are technological developments in the domains of autonomy, ai, language and autopoiesis. it is proposed that it is not developments in the individual domains that pose a threat, but the manner in which they combine. moreover, it is not the possibility of their technical achievement that is the issue, but the functional, operational or social consequences if achieved. indeed, the technical possibilities are perhaps no longer in the domain of science fiction but can be conceptually conceived in terms of existing technologies as embryonic forms of future possibilities. furthermore, since time, as a dimension, is reducing as a factor in development, for reasons attributed to a lamarkian model of evolution, then, what might be construed as impossible today may well be possible in twenty or even ten years from now. in summation, this raises questions about what debates should be happening in the immediate future and who should be involved in them? are the issues raised something that industry should consensually self-regulate? alternatively, is this a matter for politicians and policy makers to regulate upon? this paper contributes to the debate about future technological developments by offering a conceptually grounded framework to structure an argument as to one possible technological development trajectory, one that focuses upon the increasing autonomy of technological beings and the increasing redundancy of humans. from a policy and public debate perspective the view offered invites discussion which can only benefit the trajectory of digital transformation. many questions can be raised, notably: is this mere science fiction or is there a real threat to humanity as stated by stephen hawking? is there another possible trajectory continued through the cyborg that preserves human well-being and identity? whilst nano-and bio-technologies have not been explored as these are perhaps part of the world of the cyborg, how would they fit in the world of technological beings? might technological beings want to incorporate biological material into their inorganic composition? indeed, many issues are surfaced concerning our relationship with the growing capability and autonomy of technologies that both complement what we do yet can displace us. this has ramifications, not only for the future of work, but also for how we educate people about how to live in an increasingly technology enabled hybrid world. for example, what does it mean to trust in technology, as we might with personal assistants and robots that help in our homes? likewise, what will it mean to manage technology? the framework proffered has made one fundamental assumption. this is that there is a shift towards the emergence of the autopoietic technological being with all technical constraints being overcome. in conclusion, an argument is presented, which examines the relationship between humans and artefacts. it reveals a science fiction scenario whereby humans become redundant in a domain where autopoietic technological beings reign supreme with the capability to do what they want and go anywhere, unconstrained by hostile environments. it identifies four areas for development: autonomy, intelligence, language, and autopoiesis -these are currently technically feasible, and with the speed of future developments, how soon will these scenarios be realised? artificial intelligence ar augmented reality it is difficult to establish how to differentiate our respective contributions. the original idea was that of stephen harwood, but its development, embodiment and articulation was the combined outcome of much discussion arising from both authors complementary knowledge about this area and sally eaves's applied experience within emergent technologies. what can past technology forecasts tell us about the future? design for a brain types of technology exploring 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held roles including change agent and company director, as well as more recently, an academic, currently holding the post of lecturer and head of joint undergraduate programmes at the university of edinburgh business school. his research interests span a number of areas, in particular the application of newer forms of technology drawing conceptually upon the domains of cybernetics and the sociology of technology she specializes in the application of emergent technologies, notably ai, blockchain, cloud, cybersecurity, iot & 5g for both business and societal benefit at scale. an international keynote speaker and author, sally was an inaugural recipient of the frontier technology and social impact award, presented at the united nations and has been described as the 'torchbearer for ethical tech' -founding aspirational futures to enhance inclusion and diversity in the technology space and beyond key: cord-024088-020rgz5t authors: radandt, siegfried; rantanen, jorma; renn, ortwin title: governance of occupational safety and health and environmental risks date: 2008 journal: risks in modern society doi: 10.1007/978-1-4020-8289-4_4 sha: doc_id: 24088 cord_uid: 020rgz5t occupational safety and health (osh) activities were started in the industrialized countries already 150 years ago. separated and specific actions were directed at accident prevention, and the diagnosis, treatment and prevention of occupational diseases. as industrialization has advanced, the complexity of safety and health problems and challenges has substantially grown, calling for more comprehensive approaches. such development has expanded the scope, as well as blurred the borders between specific activities. in the modern world of work, occupational safety and health are part of a complex system that involves innumerable interdependencies and interactions. these are, for instance, safety, health, well-being, aspects of the occupational and general environment, corporate policies and social responsibility, community policies and services, community social environment, workers’ families, their civil life, lifestyles and social networks, cultural and religious environments, and political and media environments. a well-functioning and economically stable company generates resources to the workers and to the community, which consequently is able to maintain a positive cycle in development. a high standard of safety and health brings benefits for everyone: the company, the workers and the whole community. these few above-mentioned interactions elucidate the need for an integrated approach, and the modelling of the complex entity. if we picture osh as a house, this integrated approach could be the roof, but in order to build a stable house, it is also necessary to construct a solid basement as a foundation to the house. these basement "stones" are connected to each other, and are described in more detail in sections 4.1-4.3. section 4.1 focuses on the existing hazards, while section 4.2 mainly considers the exposure of workers to health hazards. health, due to its complexity, however, is not only influenced and impaired by work-related hazards, but also by hazards arising from the environment. these two sub-chapters are thus linked to section 4.3. in addition, the safety levels of companies may affect the environment. the strategies and measures needed for effective risk management, as described in section 4.1, therefore also contribute to reducing the risks to the environment. in the case of work that is done outdoors, the hazards arising from the environment understandably have to be given special attention. here, the methods applied to tackle the usual hazards at workplaces are less effective. it is necessary to develop protective measures to avoid or minimize hazards present in the environment. namely, agriculture, forestry and construction involve these types of hazards, and affect high numbers of workers on a global scale. finally, hazards in the environment or in leisure-time activities can lead to strain and injuries which -combined with hazards at work -may result in more severe health consequences. as an example one can mention the hazardous substances in the air causing allergies or other illnesses. another example is the strain on the musculoskeletal system from sports and leisuretime activities causing low back pain and other musculoskeletal disorders. depending on the type of hazard, the three topics, namely, safety, health and the environment, may share the common trait that the proper handling of risks, i.e., how to reduce probabilities and/or consequences of unwanted events is not always possible within a risk management system. this is true when one moves into the realm of uncertainty, i.e., when there is uncertain, insufficient or no knowledge of the consequences and/or probabilities (see chapter 5). 1. integrated multi-sectorial bodies for policy design and planning (national safety and health committee). 2. comprehensive approach in osh activities. 3. multi-disciplinary expert resources in inspection and services. 4. multi-professional participation of employers' and workers' representatives. 5. joint training in integrated activities. 6. information support facilitating multi-professional collaboration. international labour office (ilo) (2003) international labour conference, 91st session, report vi, ilo standards-related activities in the area of occupational safety and health: an in-depth study for discussion with a view to the elaboration of a plan of action for such activities. sixth item on the agenda. international labour office, geneva. what are the main challenges arising from the major societal changes for business/companies and workers/employees? how can these challenges be met in order to succeed in the growing international competition? what is the role of occupational safety and health (osh) in this context? the above-mentioned changes create new possibilities, new tasks and new risks to businesses in particular, and to the workers as well. in order to optimize the relation between the possibilities and the risks (maximize possibilities -minimize risks) there is a growing need for risk management. risk management includes all measures required for the target-oriented structuring of the risk situation and safety situation of a company. it is the systematic application of management strategies for the detection, assessment, evaluation, mastering and monitoring of risks. risk management was first considered exclusively from the point of view of providing insurance coverage for entrepreneurial risks. gradually the demands of jurisdiction grew, and the expectations of users and consumers increased with regard to the quality and safety of products. furthermore, the ever more complex problems of modern technology and ultimately the socioeconomic conditions have led to the development of risk management into an independent interdisciplinary field of work. risks can be regarded as potential failures, which may decrease trust in realizing a company's goals. the aim of risk management is to identify these potential failures qualitatively and quantitatively, and to reduce them to the level of a non-hazardous and acceptable residual risk potential. the development and formulation of a company's risk policy is regarded as the basis of effective risk management. this includes, first and foremost, the management's target concept with respect to the organization of work, distribution of labour, and competence of the departments and persons in charge of risk management. risk issues are important as far as acceptance of technology is concerned. it is not enough to reduce the problem to the question of which risks are tolerable or acceptable. it appears more and more that, although the risks themselves are not accepted, the measures or technologies causing them are. value aspects have an important role in this consideration. a positive or negative view of measures and technologies is thus influenced strongly by value expectations that are new, contradictory and even disputed. comparing risks and benefits has become a current topic of discussion. the relation between risks and benefits remains an unanswered question. the general public has a far broader understanding of the risks and benefits of a given technology than the normal understanding professed by engineering sciences which is limited to probability x harm. the damage or catastrophe potential, qualitative attributes such as voluntary nature and controllability also play an important role in the risk assessment of a technology. a normative setting for a certain, universally accepted risk definition according to engineering science is therefore hardly capable of consensus at the moment. the balanced management of risks and possibilities (benefits) is capable of increasing the value of a company. it may by far surpass the extent of legal obligations: for example, in germany, there is a law on the control and transparency for companies (kontrag) . the respective parameters may be defined accurately as follows: • strategic decisions aim to offer opportunities for acquiring benefit, taking into consideration risks. • risks that can have negative consequences to the technological capacities, the profitability potential, the asset values and the reputation of a company, as well as the confidence of shareholders are identified and measured. • the management focuses on important possibilities and risks, and addresses them adequately or reduces them to a tolerable level. the aim is not to avoid risks altogether, but to create opportunities for promoting proactive treatment of all important risks. the traditional occupational health hazards, such as physical, chemical and biological risks, as well as accidents, will not totally disappear as a consequence of change, nor will heavy physical work. about 30-50% of workers are still exposed to such hazards. there is thus need to still develop risk assessment, prevention and control methods and programmes for these often well-known hazards. in many industrialized countries, prevention and control programmes have had a positive impact by reducing the trends of occupational diseases and accidents, particularly in big industries. some developing countries, however, show an increase in traditional hazards. international comparisons, however, are difficult to make because of poor coverage, underreporting, and poor harmonization of concepts, definitions and registration criteria. statistics on occupational accidents are difficult to compare, and therefore data on their total numbers in europe should be viewed with caution. the majority of countries, however, have shown declining trends in accident rates irrespective of the absolute numbers of accidents. some exceptions to this general trend have nevertheless been seen. the accident risk also seems to shift somewhat as regards location, so that instead of risks related to machines and tools, particularly the risks in internal transportation and traffic within the workplace grow in relative importance. this trend may increase in future, particularly as the work place, as well as the speed and volume of material flows are increasing. a threat is caused by lengthened working hours, which tend to affect the vigilance of workers and increase the risk of errors. small-scale enterprises and micro-enterprises are known to have a lower capacity for occupational health and safety than larger ones. in fact, a higher accident risk has been noted in medium-sized companies, and a lower risk in very small and very large enterprises. we can conclude that this is due to the higher mechanization level and energy use in small and medium-sized enterprises (sme) compared with micro-enterprises, which usually produce services. on the other hand, the better capacity of very large enterprises in safety management is demonstrated by their low accident rates. the production of chemicals in the world is growing steadily. the average growth has been between 2-5% a year during the past 2-3 decades. the total value of european chemical production in 1998 was about usd 500 billion, i.e. 32% of the world's total chemical production, and it has increased 35% in the 10-year period of 1985-1995. the european union (eu) is the largest chemical producer in the world, the usa the second, and japan the third. there are some 100,000 different chemical entities in industrial use, but only about 2,500 are so-called high-production volume (hpv) chemicals produced (and consumed) in amounts exceeding 1,000 tons a year. the number of chemicals produced in amounts of 10-1,000 tons a year is about 25,000. but volume is not necessarily the most important aspect in chemical safety at work. reactivity, toxicological properties, and how the chemicals are used, are more important. the european chemical companies number some 39,000, and in addition there are 36,000 plants producing plastics and rubber. surprisingly, as many as 96% of these are smes employing fewer than 50 workers, and 70% are micro-enterprises employing fewer than 10 workers. thus, the common belief that the chemical industry constitutes only large firms is not true. small enterprises and self-employed people have much less competence to deal with chemical risk assessment and management than the large companies. guidance and support in chemical safety is therefore crucial for them. the number of workers dealing with chemicals in the european work life is difficult to estimate. the chemical industry alone employs some 1.7 million workers in europe, i.e. about 7% of the workforce of manufacturing industries. about 30%, i.e. over 500,000 work in chemical smes. but a much higher number of workers are exposed in other sectors of the economy. there is a distinct trend showing that the use of chemicals is spreading to all sectors, and thus exposures are found in all types of activities: agriculture, forestry, manufacturing, services and even in high-tech production. the national and european surveys on chemical exposures in the work environment give very similar results. while about 25% of the eu work-ers were exposed to hazardous chemicals, the corresponding figure in central and eastern european countries may be much higher. the workers are exposed simultaneously to traditional industrial chemicals, such as heavy metals, solvents and pyrolytic products, and to "new exposures", such as plastics monomers and oligomers, highly reactive additives, cross-linkers and hardeners, as well as to, for example, fungal spores or volatile compounds in contaminated buildings. this implies that some 40 million people in the eu15 are exposed at work, and usually the level of exposure is one to three orders of magnitude higher than in any other environment. about the same proportion (25% of the workforce, i.e. 500,000) of the finnish workers in the national survey reported exposure. the chemicals to which the largest numbers of workers are exposed occur typically in smes; they are e.g. detergents and other cleaning chemicals, carbon monoxide, solvents, environmental tobacco smoke, and vegetable dusts. european directives on occupational health and safety require a high level of protection in terms of chemical safety in all workplaces and for all workers. risk assessment and risk management are key elements in achieving these requirements. the risk assessment of chemicals takes place at two levels: a) systems-level risk assessment, providing a dose-response relationship for a particular chemical, and serving as a basis for standard setting. risk assessment at the systems level is carried out in the pre-marketing stage through testing. this consequently leads to actions stipulated in the regulations concerning standards and exposure limits, labelling and marking of hazardous chemicals, limitations in marketing, trade and use. in this respect, the level of safety aimed at remains to be decided. is it the reasonably achievable level or, for example, the level achieved by the best available technology? the impact is expected to be system-wide, covering all enterprises and all workers in the country. this type of risk assessment is an interactive practice between the scientific community and the politically controlled decision making. a high level of competence in toxicology, occupational hygiene and epidemiology is needed in the scientific community. and the decision makers must have the ability to put the risk in concern into perspective. in most countries the social partners also take part in the political decision making regarding occupational risks. b) workplace risk assessment directed at identifying the hazards at an individual workplace and utilizing standards as a guide for making decisions on risk management. risk assessment at workplace level leads to practical actions in the company and usually ensures compliance with regulations and standards. risk assessment is done by looking at the local exposure levels and comparing these with standards produced in the type a) risk assessment. risk management is done through preventive and control actions by selecting the safest chemicals, by controlling emissions at their source, by general and local ventilation, and by introducing safe working practices. if none of the above is effective, personal protective devices must be taken into use. noise is a nearly universal problem. the products of technology, which have freed us from the day-to-day survival struggle, have been accompanied by noise as the price of progress. however, noise can no longer be regarded as an inevitable by-product of today's society. not only is noise an undesirable contaminant of our living environment, but high levels of noise are frequently present in a variety of work situations. many problems arise from noise: annoyance, interference with conversation, leisure or sleep, effects on work efficiency, and potentially harmful effects, particularly on hearing. in short, noise may affect health, productivity, and well-being. the selection of appropriate noise criteria for the industry depends on knowledge of the effects of noise on people, as well as on the activities in which they are engaged. many of the effects are dependent on the level, and the magnitude of the effects varies with this level. hearing damage is not the only criterion for assessing excessive noise. it is also important to consider the ability and ease of people to communicate with each other. criteria have therefore been developed to relate the existing noise environment to the ability of the typical individual to communicate in areas that are likely to be noisy. the effects of noise on job performance are difficult to evaluate. in general, one can say that sudden, intermittent, high-intensity noise impedes efficient work more than low-intensity and steady-state noise. the complexity of the task with which noise interferes plays a major role in determining how much noise actually degrades performance. two common ways in which noise can interfere with sleep are: delaying the onset of sleep, and shifting sleep stages. one effect of noise that does not seem to depend strongly on its level is annoyance. under some circumstances, a dripping water faucet can be as annoying as a jackhammer. there are no generally accepted criteria for noise levels associated with annoyance. if the noise consists of pure tones, or if it is impulsive in nature, serious complaints may arise. new information and communication technologies (ict) are being rapidly implemented in modern work life. about 65% of workers have computers at work, and about 35% are e-mail and internet users. there are three main problem areas in the use of new ict at work. these are: 1) the visual sensory system, 2) the cognitive processes, and 3) the psychomotoric responses needed for employing hand-arm systems. all three have been found to present special occupational health and even safety problems, which are not yet fully solved. the design of new more user-friendly technology is highly desirable, and the criteria for such technology need to be generated by experts in neurophysiology, cognitive psychology and ergonomics. it is important to note that the productivity and quality of information-intensive work requiring the use of ict depends crucially on the user-friendliness of the new technology interface, both the hardware and software. communication and information technologies will change job contents, organization of work, working methods and competence demands substantially in all sectors of the economy in all countries. a number of new occupational health and safety hazards have already arisen or are foreseen, including problems with the ergonomics of video display units, and musculoskeletal disorders in shoulder-neck and arm-hand systems, information overload, psychological stress, and pressure to learn new skills. the challenge to occupational health and safety people is to provide health-based criteria for new technologies and new types of work organization. it is also important to contribute to the establishment of healthy and safe work environments for people. in the approved and draft standards of the international standardization organization, iso, there are altogether about 50 different targets dealing with the standardization of eyesight-related aspects. vision is the most important channel of information in information-intensive work. from the point of view of seeing and eye fatigue, the commonly used visual display units (vdu) are not the most optimal solutions. stability of the image, poor lighting conditions, reflections and glare, as well as invisible flicker, are frequent problems affecting vision. the displays have, however, developed enormously in the 1990s, and there is evidence that the so-called flat displays have gradually gained ground. information-intensive work may increasingly load the vision and sense of hearing, particularly of older workers. even relatively minor limitations in vision or hearing associated with ageing have a negative effect on receiving and comprehending messages. this affects the working capacity in information-intensive work. the growing haste in information-intensive work causes concern among workers and occupational health professionals. particularly older workers experience stress, learning difficulties and threat of exclusion. corrective measures are needed to adjust the technology to the worker. the most important extension of the man-technology interface has taken place in the interaction of two information-processing elements: the central nervous system and the microprocessor. the contact is transmitted visually and by the hands, but also by the software which has been developed during the 1990s even more than the technology itself. many problems are still associated with the immaturity of the software, even though its user-friendliness has recently greatly improved. the logic and structure of the software and the user systems, visual ergonomics, information ergonomics, the speed needed, and the forgiving characteristics of programs, as well as the possibility to correct the commands at any stage of processing are the most important features of such improvements. also the user's skills and knowledge of information technology and the software have a direct effect on how the work is managed and how it causes workload. the user-friendliness and ergonomics of the technology, the disturbing factors in the environment, haste and time pressure, the work climate, and the age and professional skills of the individual user, even his or her physical fitness, all have an impact on the cognitive capacity of a person. this capacity can to a certain extent be improved by training, exercise and regulating the working conditions, as well as with expert support provided for the users when difficulties do occur. the use of new technologies has been found to be associated with high information overload and psychological stress. the problem is not only typical for older workers or those with less training, but also for the super-experts in ict who have shown an elevated risk of psychological exhaustion. there are four main types of ergonomic work loads: heavy dynamic work that may overload both the musculoskeletal and cardiovascular system; re-petitive tasks which may cause strain injuries; static work that may result in muscular pain; and lifting and moving heavy loads, which may result in overexertion, low back injury, or accidental injuries. visual ergonomics is gaining in importance in modern work life. the overload of the visual sensory system and unsatisfactory working conditions may strain the eye musculature, but can also cause muscle tension in the upper part of the body. this effect is aggravated if the worker is subjected to psychological stress or time pressure. in addition to being a biological threat, the risk of infections causes psychological stress to workers. the improved communication between health services and international organizations provides help in the early detection and control of previously unknown hazards. nevertheless, for example, the danger related to drug abusers continues to grow and present a serious risk to workers in, for example, health services and the police force. some new viral or re-emerging bacterial infections also affect health care staff in their work. the increase in the cases of drug-resistant tuberculosis is an example of such a hazard. the goal of preventive approaches is to exert control on the cause of unwelcome events, the course of such negative events or their outcome. in this context, one has to decide whether the harmful process is acute (an accident) or dependent on impact duration and stimulus (short-, medium-, and long-term). naturally, the prevention approaches depend on the phases of the harmful process, i.e. whether the harm is reversible, or whether it is possible only to maintain its status, or to slow down the process. it is assumed here that a stressful factor generates an inter-individual or intra-individual strain. thus the effects and consequences of stress are dependent on the situation, individual characteristics, capabilities, skills and the regulation of actions, and other factors. the overall consideration is related to work systems characterized by work contents, working conditions, activities, and actions. system performance is expected of this work system, and this system performance is characterized by a performance structure and its conditions and requirements (figure 4 .2). the performance of the biopsychosocial unit, i.e. the human being, plays an important role within the human performance structure (see figures 4.2 and 4.3). the human being is characterized by external and internal features, which are closely related to stress compatibility, and thus to strain. in this respect, preventive measures serve to optimize and ensure performance, on the one hand, and to control stress and strain, on the other. preventive measures aim to prevent bionegative effects and to facilitate and promote biopositive responses. • the internal factors affecting performance are described by performance capacity and performance readiness. • performance capacity is determined by the individual's physiological and psychological capacity. • performance readiness is characterized by physiological fitness and psychological willingness. • the external factors affecting performance are described by organizational preconditions/requirements and technical preconditions/requirements. • regarding the organizational requirements, the organizational structure and organizational dynamics are of significance. • in the case of technical requirements, the difficulties of the task, characterized by machines, the entire plant and its constructions, task content, task design, technical and situation-related factors, such as work layout, anthropometrics, and quality of the environment, are decisive (table 4 .1). mental stress plays an increasing role in the routine activities of enterprises. through interactive models of mental stress and strain, it is possible to represent the development of mental strain and its impairing effects (e.g. tension, fatigue, monotony, lack of mental satisfaction). it is important to distinguish the above-mentioned impairing effects from each other, since they can arise from different origins and can be prevented or eliminated by different means. activities that strain optimally enhance health and promote safe execution of work tasks. stress essentially results from the design parameters of the work system or workplace. these design parameters are characterized by, e.g.: • technology, such as work processes, work equipment, work materials, work objects; • anthropometric design; • work techniques, working hours, sharing of work, cycle dependence, job rotation; • physiological design that causes strain, fatigue; • psychological design that either motivates or frustrates; • information technology, e.g. information processing, cognitive ergonomic design; • sociological conditions; and • environmental conditions, e.g. noise, dust, heat, cold. the stress structure is very complex, and we therefore need to look at the individual parameters carefully, taking into account the interactions and links between the parameters at the conceptual level. the design parameters impact people as stress factors. as a result, they also turn into conditions affecting performance. such conditions can basically be classified into two types: a person's internal conditions, characterized in particular by predisposition and personality traits, and a person's external conditions, determined mainly by the design parameters. when we look at performance as resulting from regulated or reactive action, we find three essential approaches for prevention: • the first approach identifies strain. it is related to anatomical, biochemical, histological, physiological characteristic values, typical curves of organ systems, the degree of utilization of skills through stress, and thus the degree of utilization of the dynamics of physiological variables in the course of stress. • the second approach is related to the control of strain. the aim is to identify performance limits, the limits of training and practice, and to put them into positive use. adaptation and fatigue are the central elements here. • the third approach for prevention is related to reducing strain. the aim is to avoid harm, using known limits as guidelines (e.g. maximum workplace concentration limit values for harmful substances, maximum organspecific concentration values, biological tolerance values for substances, limit values for noise and physical loads). however, the use of guideline values can only be an auxiliary approach, because the stress-strain concept is characterized by highly complex connections between the exogenous stress and the resulting strain. an objectively identical stress will not always cause the same level of strain in an individual. due to action regulation and individual characteristic values and curves of the organ systems (properties and capabilities), differences in strain may occur. seemingly identical stress can cause differing strain due to the superposition of partial stress. combinations of partial stress can lead to compensatory differences (e.g. physiological stress can compensate for psychological stress) or accumulation effects. partial stress is determined by the intensity and duration of the stress, and can therefore appear in differing dimensions and have varying effects. in assessing overall stress, the composition of the partial stress according to type, intensity, course and time is decisive. partial stress can occur simultaneously and successively. in our considerations, the principle of homeostasis plays an important role. however, optimizing performance is only a means to an end in a prevention programme. the actual purpose is to avoid harm, and thus to control strain. harm is a bionegative effect of stress. the causative stress is part of complex conditions in a causal connection. causal relationships can act as dose-effect relationships or without any relation to the dose. in this respect, the causative stress condition can form a chain with a fixed or variable sequence; it can add up, multiply, intensify or have an effect in only specific combinations, and generate different effects (e.g. diseases). we are thus dealing with a multicausal model or a multi-factor genesis. low back pain is an example of a complex phenomenon. the incidence of musculoskeletal disorders, especially low back pain, is rapidly increasing. several occupational factors have been found to increase the risk for low back pain. some studies indicate that psychosocial and work-related conditions are far more accurate in the prognosis of disability than are physical conditions. chronic low back pain is perceived as a phenomenon which encompasses biological, social and psychological variables. according to the model of adaptation, the goal of reducing risks is to increase a person's physical abilities (i.e. flexibility, strength, endurance), the use of body mechanics, techniques to protect the back (following the rules of biomechanics), to improve positive coping skills and emotional control. the following unfavourable factors leading to back pain have been identified at workplaces: • the lifting of too heavy loads. • working in a twisted or bent-down position. • work causing whole-body vibration. • working predominantly in a sitting position. • carrying heavy loads on the shoulders. the prevention of acute back pain and the prevention of work disability must entail several features. one important element is work safety, which can be maximized by screening a worker's physical and intellectual capacities, by ensuring ergonomic performance of the work procedures, and by increasing awareness of proper working techniques that do not strain the back. the use of adaptation programmes makes it possible to attain a higher performance level and to be able to withstand more strain (figure 4.4) . research-based methods of training optimize and improve performance. they are a means for controlling stress and strain with the aim of preventing bionegative effects and facilitating and promoting biopositive responses. the stress (load) and strain model and human performance can be described as follows: • causative stress generates an inter-individual or intra-individual strain. • the effects and consequences depend on a person's properties, capabilities, skills and the regulation of actions, individual characteristics of the organ systems, and similar factors. • within the performance structure, the performance of the biopsychosocial unit, i.e. the human being, plays an important role. the human being is characterized by external and internal factors, which in turn are closely related to stress compatibility and thus to strain. the connection between stress and harm plays a significant role in the research on occupational health hazards. how should this connection be explored? different hypotheses exist in replying to this question, but none of them have been definitively proven. the three most common hypotheses today are: stress occurring in connection with a person's life events. the number and extent of such events is decisive. problem-coping behaviour and/or social conditions are variables explaining the connection between stress and harm. 3. the additive stress hypothesis. the ability to cope with problems and the social conditions has an effect on harm which is independent of the stress resulting from life events. when we refer to the complexity of risks in this context of occupational safety our focus shall be on the enterprise. there are different kinds of risks to be found in enterprises. many of them are of general importance, i.e. they are in principle rather independent of an enterprise's size or its type of activity. how to deal with such risks shall be outlined to some extent here. in order to treat those risks at work successfully resources are needed whose availability often depends on the enterprise's situation. the situation in enterprises usually is a determining indicator for available resources to control and develop safety and health and thus performance of the enterprise and its workers and employees through appropriate preventive measures. this situation has been described to some extent in chapter 2. big companies usually have well-developed safety and health resources, and they often transfer appropriate policies and practices to the less developed areas where they operate. even in big enterprises, however, there is fragmentation of local workplaces into ever smaller units. many of the formerly in-built activities of enterprises are outsourced. new types of work organizations are introduced, such as flat and lean organizations, increase of telework and call centres, many kinds of mobile jobs and network organizations. former in-company occupational health services are frequently transferred to service providers. this leads to the establishment of high numbers of micro-enterprises, small scale enterprises (sses), small and medium-sized enterprises (smes) and self-employed people. sses and smes are thus becoming the most important employers in the future. from a number of studies there is evidence that at least among a part of sses and smes awareness of osh risks is low. both managers and workers often do not see the need to improve occupational safety and health or ergonomic issues and their possibilities and benefits by reducing or eliminating risks at work. as these types of enterprises, even more the self-employed, do not have sufficient resources or expertise for implementing preventive measures, the need for external advisory support, services and incentives is evident and growing. interpersonal relations in sses and smes being generally very good provides a strong chance for effectively supporting them. other special features in the structure of small and medium-sized enterprises to be considered are: • direct participation of the management in the daily activities; • the management structure is designed to meet the requirements of the manager; • less formal and standardized work processes, organizational structures and decision processes; • no clear-cut division of work: -wide range of tasks; and -less specialization of the employees; • unsystematic ways of obtaining and processing information; • great importance of direct personal communication; • less interest in external cooperation and advice; • small range of internal, especially long-term and strategic planning; and • stronger inclination of individual staff members to represent their own interests. the role of occupational health services (ohs) in smes is an interdisciplinary task, consisting of: • risk assessment: -investigation of occupational health problems according to type of technology, organization, work environment, working conditions, social relationships. • surveillance of employees' health: -medical examinations to assess employees' state of health; and -offering advice, information, training. • advice, information, training: -measures to optimize safety and health protection; and -safe behaviour, safe working procedures, first aid preparedness. different kinds of risks are found in enterprises (see table 4 .2). these different types of risks need to be handled by an interlinked system to control the risks and to find compromises between the solutions. figure 4 .7 illustrates these linkages. the promotion of safety and health is linked to several areas and activities. all of these areas influence the risk management process. the results of risk treatment not only solve occupational health and safety problems, but they also give added value to the linked fields. specific risk management methods are needed to reach the set goal. one needs to know what a risk is. the definition of risk is essential: a risk is a combination of a probability -not frequency -of occurrence, and the associated unwelcome outcome or impact of a risk element (consequence). risk management is recognized as an integral part of good management practice. it is a recurring process consisting of steps which, when carried out in a sequence, allow decision making to be improved continuously. risk management is a logical and systematic method of identifying, analyzing, evaluating, treating, monitoring and communicating risks arising during any activity, function, or process in a manner enabling the organization to minimize losses and maximize productive opportunities. different methods are available for analyzing problems. each method is especially suited to respond to certain questions and less suited for others. a complex "thinking scheme" is necessary for arranging the different analyses correctly within the system review. such a scheme includes the following steps: 1. defining the unit under review: the actual tasks and boundaries of the system (a fictitious or a real system) must be specified: time, space and state. 2. problem analysis: all problems existing in the defined system, including problems which do not originate from the system itself, are detected and described. 3. causes of problems: all possible or probable causes of the problems are identified and listed. 4. identifying interaction: the dependencies of the effect mechanisms are described, and the links between the causes are determined. 5. establishing priorities and formulating targets: to carry out this step, it is necessary to evaluate the effects of the causes. 6. solutions to the problems: all measures needed for solving the individual problems are listed. the known lists usually include technical as well as non-technical measures. since several measures are often appropriate for solving one problem, a pre-selection of measures has to be done already at this stage. however, this can only be an approach to the solution; the actual selection of measures has to be completed in steps 7 and 8. 7. clarifying inconsistencies and setting priorities: as the measures required for solving individual problems may be inconsistent in part, or may even have to be excluded as a whole, any inconsist-encies need to be clarified. a decision should then be made in favour or against a measure, or a compromise may be sought. 8. determining measures for the unit under review: the measures applicable to the defined overall system are now selected from the measures for the individual problems. 9. list of questions regarding solutions selected for the overall system: checking whether the selected measures are implementable and applicable for solving the problems of the overall system. 10. controlling for possible new problems: this step consists of checking whether new problems are created by the selected solution. the close link between cause and effect demands that the processes and sub-processes must be evaluated uniformly, and risks must be dealt with according to a coordinated procedure. the analysis is started by orientation to the problem. this is done in the following steps: 1. recognizing and analyzing the problem according to its causes and extent, by means of a diagnosis and prediction, and comparison with the goals aimed at. 2. description and division of the overall problem into individual problem areas, and specifying their dependencies. 3. defining the problem and structuring it according to the objectives, time relation, degree of difficulty, and relevance to the goal. 4. detailed analysis of the causes, and classification in accordance to the possible solution. the analysis of the problem should be integrated into the overall analytical process in accordance with the thinking schemes described earlier. the relevance and priorities related to the process determine the starting point for the remaining steps of the analysis. analyses are divided into quantitative and qualitative ones. quantitative analyses include risk analyses, that is, theoretical safety-related analysis methods and safety analyses, e.g. classical accident analyses. qualitative analyses include failure mode and effect analyses, hazard analyses, failure hazard analyses, operating hazard analyses, human error code and effect analyses, information error and effect analyses. the theoretical safety-related analysis methods include inductive and deductive analyses based on boolean models. inductive analyses are, e.g. fault process analyses. deductive analyses are fault tree analyses, analytical processes and simulation methods. theoretical safety-related analysis methods which are not based on boolean models are stochastic processes, such as markow's model, risk analyses and accident analyses which, as a rule, are statistical or probability-related analyses. a possible scheme to begin with is shown in figure 4 .9. since absolute safety, entailing freedom from all risks, does not exist in any sphere of life, the task of those dealing with safety issues is to avert hazards and to achieve a sustainable reduction of the residual risk, so that it does not exceed a tolerable limit. the extent of this rationally acceptable risk is also influenced by the level of risk which society intuitively considers as being acceptable. those who propose definitions of safety are neither authorized nor capable of evaluating the general benefit of technical products, processes and services. risk assessment is therefore focused at the potential harm caused by the use or non-use of the technology. the guidelines given in "a new approach to technical harmonization and standards" by the council resolution of 7 may 1985 are valid in the european union. the legal system of a state describes the protective goals, such as protection of life, health, etc., in its constitution, as well as in individual laws and regulations. as a rule, these do not provide an exact limit as to what is still a tolerable risk. this limit can only be established indirectly and unclearly on the basis of the goals and conceptions set down by the authorities and laws of a state. in the european union, the limits are expressed primarily in the "basic safety and health requirements". these requirements are then put into more concrete terms in the safety-related definitions issued by the bodies responsible for preparing industrial standards. compliance with the standards is voluntary, but it is presumed that the basic requirements of the directives are met. the term which is opposite to "safety" is "hazard". both safe and hazardous situations are founded on the intended use of the technical products, processes and services. unintended use is taken into account only to the extent that it can be reasonably foreseen. the risks present in certain events are, in a more narrow sense, unwelcome and unwanted outcomes with negative effects (which exceed the range of acceptance). unwelcome events are • source conditions of processes and states; • processes and states themselves; and • effects of processes and states which can result in harm to persons or property. an unwelcome event can be defined as a single event or an event within a sequence of events. possible unwelcome events are identified for a unit under review. the causes may be inherent in the unit itself, or outside of it. in order to determine the risks involved in unwelcome events, it is necessary to identify probabilities and consequences. the question arises: are the extent and probability of the risk known? information is needed to answer this question. defining risk requires information concerning the probability of occurrence and the extent of the harm of the consequences. uncertainty is given if the effects are known but the probability is unknown. ignorance is given if both the effects and the probability are unknown. figure 4 .10 shows the risk analysis procedure according to the type of information available. since risk analyses are not possible without practical, usable information, it is necessary to consider the nature of the information. the information is characterized by its content, truth and objectivity, degree of confirmation, the possibility of being tested, and the age of the information. the factors determining the content of the information are generality, precision and conditionality. the higher the conditionality, the smaller is the generality, and thus the smaller the information content of the statement. truth is understood as conformity of the statement with the real state of affairs. the closer that the information is to reality, the higher is its information content, and the smaller its logical margin. the degree of controllability is directly dependent on the information content: the bigger the logical margin, the smaller the information content, and thus the higher the probability that the information content will prove its worth. in this respect, probability plays a role in the information content: the greatest significance is attributed to the logical hypothetical probability and statistical probability of an event. objectivity and age are additional criteria for any information. the age and time relation of information play a particularly important role, because consideration of the time period is an important feature of analysis. as a rule, information and thus the data input in the risk analysis consist of figures and facts based on experience, materials, technical design, the organization and the environment. in this regard, most figures are based on statistics on incidents and their occurrences. factual information reveals something about the actual state of affairs. it consists of statements related to past conditions, incidents, etc. forecast-type predictions are related to real future conditions, foretelling that certain events will occur in the future. explanatory information replies to questions about the causes of phenomena, and provides explanations and reasons. it establishes links between different states based on presumed cause-effect relationships. subjunctive information expresses possibilities, implying that certain situations might occur at present or in the future, thus giving information about conceivable conditions, events and relationships. normative information expresses goals, standards, evaluations and similar matters; it formulates what is desirable or necessary. the main problem with risk analyses is incomplete information, in particular regarding the area of "uncertainty". in the eu commission's view, recourse to the so-called precautionary principle presupposes that potentially dangerous effects deriving from a phenomenon, product or process have been identified via objective scientific evaluation, and that scientific evaluation does not allow the risk to be determined with sufficient certainty. recourse to the precautionary principle thus takes place in the framework of general risk management that is concretely connected to the decision-making process. if application of the precautionary principle results in the decision that action is the appropriate response to a risk, and that further scientific information is not needed, it is still necessary to decide how to proceed. apart from adopting legal provisions which are subject to judicial control, a whole range of actions is available to the decision-makers (e.g. funding research, or deciding to inform the public about the possible adverse effects of a product or procedure). however, the measures may not be selected arbitrarily. in conclusion, the assessment of various risks and risk types which may be related to different types of hazards requires a variety of specific risk assessment methods. if one has dependable information about the probability and consequences of a serious risk or risky event, one should use the risk assessment procedure shown in figure 4 .11. • major industrial accidents; • damage caused by dangerous substances; • nuclear accidents; • major accidents at sea; • disasters due to forces of nature; and • acts of terrorism. • dangerous substances discharged (fire, explosion); • injury to people and damage to property; • immediate damage to the environment; • permanent or long-term damage to terrestrial habitats, to fresh water, to marine habitats, to aquifers or underground water supplies; and • cross-border damage. • technical failure: devices, mountings, containers, flanges, mechanical damage, corrosion of pipes, etc.; • human failure: operating error, organizational failure, during repair work; • chemical reaction; • physical reaction; and • environmental cause. system analysis is the basis of all hazard analyses, and thus needs to be done with special care. system analysis includes the examination of the system functions, particularly the performance goals and admissible deviations in the ambient conditions not influenced by the system, the auxiliary sources of the system (e.g. energy supply), the components of the system, and the organization and behaviour of the system. geographical arrangements, block diagrams, material flow charts, information flow charts, energy flow charts, etc. are used to depict technical systems. the objective is to ensure the safe behaviour of the technical systems by design methods, at least during the required service life and during intended use. qualitative analyses are particularly important in practice. as a rule, they are form sheet analyses and include failure mode and effect analyses, which examine and determine failure modes and their effects on systems. the preliminary hazard analysis looks for the hazard potentials of a system. the failure hazard analysis examines the causes of failures and their effects. the operating hazard analysis determines the hazards which may occur during operation, maintenance, repair, etc. the human error mode and effect analysis examines error modes and their effects which occur because of wrong behaviour of humans. the information error mode and effect analysis examines operating, maintenance and repair errors, fault elimination errors and the effects caused by errors in instructions and faulty information. theoretical analysis methods include the fault tree analysis, which is a deductive analysis. an unwelcome incident is provided to the system under review. then all logical links and/or failure combinations of components or partial system failures which might lead to this unwelcome incident are assembled, forming the fault tree. the fault tree analysis is suited for simple as well as for complex systems. the objective is to identify failures which might lead to an unwelcome incident. the prerequisite is exact knowledge about the functioning of the system under review. the process, the functioning of the components and partial systems therefore need to be present. it is possible to focus on the flow of force, of energy, of materials and of signals. the fault process analysis has a structure similar to that of the fault tree analysis. in this case, however, we are looking for all unwelcome incidents as well as their combinations which have the same fault trigger. analysis of the functioning of the system under review is also necessary for this. analyses can also be used to identify possible, probable and actual risk components. the phases of the analysis are the phases of design, including the preparation of a concept, project and construction, and the phases of use which are production, operation and maintenance. in order to identify the fault potential as completely as possible, different types of analyses are usually combined. documentation of the sufficient safety of a system can be achieved at a reasonable cost only for a small system. in the case of complex systems, it is therefore recommended to document individual unwelcome incidents. if solutions are sought and found for individual unwelcome incidents, care should be taken to ensure that no target conflicts arise with regard to other detail solutions. with the help of the fault tree, it is possible to analyse the causes of an unwelcome incident and the probability of its occurrence. decisions on whether and which redundancies are necessary can in most cases be reached by simple estimates. four results can be expected by using a fault tree: 1. the failure combination of inputs leading to the unwelcome event; 2. the probability of their occurrence; 3. the probability of occurrence of the unwelcome event; and 4. the critical path that this incident took from the failure combination through the fault tree. a systematic evaluation of the fault tree model can be done by an analytical evaluation (calculation) or by simulation of the model (monte-carlo method). a graphic analysis of the failure process is especially suited to prove the safety risk of previously defined failure combinations in the system. the failure mode and effect analysis and the preliminary hazard analysis as mentioned previously, no method can disclose all potential faults in a system with any degree of certainty. however, if one starts with the preliminary hazard analysis, then at least the essential components with hazard potential will be defined. the essential components are always similar, namely, kinetic energy, potential energy, source of thermal energy, radioactive material, biological material, chemically reactive substance. with the fault tree method, any possible failure combinations (causes), leading to an unwelcome outcome, can then be identified additionally. re-160 are especially suited for identifying failures in a system which pose a risk. liability parameters can be determined in the process, e.g. the frequency of occurrence of failure combinations, the frequency of occurrence of unwelcome events, non-availability of the system upon requests, etc. the failure effect analysis is a supplementary method. it is able to depict the effects of mistakes made by the operating personnel, e.g. when a task is not performed, or is performed according to inappropriate instructions, or performed too early, too late, unintentionally or with errors. it can pinpoint also effects resulting from operating conditions and errors in the functional process or its elements. an important aspect of all hazard analyses is that they are only valid for the respective case under review. every change in a parameter basically requires new analyses. this applies to changes in the personnel structure and the qualification of persons, as well as to technical specifications. for this reason, it is necessary to document the parameters on which each analysis is based. the results of the hazard analyses form the basis for the selection of protective measures and measures to combat the hazards. if the system is modified, the hazards inherent in the system may change, and the measures to combat the hazards may have to be changed as well. this may also mean that the protective measures or equipment which existed at time x for the system or partial system in certain operating conditions (e.g. normal operation, set-up operation, and maintenance phase) may no longer be compatible. different protective measures, equipment or strategies may then be needed. however, hazard analyses do not merely serve to detect and solve potential failures. they form the basis for the selection of protective measures and protective equipment, and they can also test the success of the safety strategies specified. a selection of methods used for hazard analysis is given in annex 2 to section 4.1. risk assessment is a series of logical steps enabling the systematic examination of the hazards associated with machinery. risk assessment is followed, whenever necessary, by actions to reduce the existing risks and by implementing safety measures. when this process is repeated, it eliminates hazards as far as possible. risk assessment includes: • risk analysis: -determining the limits of machinery; -identifying hazards; and -estimating risks. • risk evaluation. risk analysis provides the information required for evaluating risks, and this in turn allows judgements to be made on the safety of e.g. the machinery or plant under review. risk assessment relies on decisions based on judgement. these decisions are to be supported by qualitative methods, complemented, as far as possible, by quantitative methods. quantitative methods are particularly appropriate when the foreseeable harm is very severe or extensive. quantitative methods are useful for assessing alternative safety measures and for determining which measure gives best protection. the application of quantitative methods is restricted to the amount of useful data which is available, and in many cases only qualitative risk assessment will be possible. risk assessment should be conducted so that it is possible to document the used procedure and the results that have been achieved. risk assessment shall take into account: • the life cycle of machinery or the life span of the plant. • the limitations of the machinery or plant, including the intended use (correct use and operation of the machinery or plant, as well as the consequences of reasonably foreseeable misuse or malfunction). • the full range of foreseeable uses of the machinery (e.g. industrial, nonindustrial and domestic) by persons identified by sex, age, dominant hand usage, or limiting physical abilities (e.g. visual or hearing impairment, stature, strength). • the anticipated level of training, experience or ability of the anticipated users, such as: -operators including maintenance personnel or technicians; -trainees and juniors; and -general public. • exposure of other persons to the machine hazards, whenever they can be reasonably foreseen. having identified the various hazards that can originate from the machine (permanent hazards and ones that can appear unexpectedly), the machine designer shall estimate the risk for each hazard, as far as possible, on the basis of quantifiable factors. he must finally decide, based on the risk evaluation, whether risk reduction is required. for this purpose, the designer has to take into account the different operating modes and intervention procedures, as well as human interaction during the entire life cycle of the machine. the following aspects in particular must be considered: • construction; transport; • assembly, installation, commissioning; • adjusting settings, programming or process changeover; • instructions for users; • operating, cleaning, maintenance, servicing; and • checking for faults, de-commissioning, dismantling and safe disposal. malfunctioning of the machine due to, e.g. • variation in a characteristic or dimension of the processed material or workpiece; • failure of a part or function; • external disturbance (e.g. shock, vibration, electromagnetic interference); • design error or deficiency (e.g. software errors); • disturbance in power supply; and • flaw in surrounding conditions (e.g. damaged floor surface). unintentional behaviour of the operator or foreseeable misuse of the machine, e.g.: • loss of control of the machine by the operator (especially in the case of hand-held devices or moving parts); • automatic (reflexive) behaviour of a person in case of a machine malfunction or failure during operation; • the operator's carelessness or lack of concentration; • the operator taking the "line of least resistance" in carrying out a task; • behaviour resulting from pressure to keep the machine running in all circumstances; and • unexpected behaviour of certain persons (e.g. children, disabled persons). when carrying out a risk assessment, the risk of the most severe harm that is likely to occur from each identified hazard must be considered, but the greatest foreseeable severity must also be taken into account, even if the probability of such an occurrence is not high. this objective may be met by eliminating the hazards, or by reducing, separately or simultaneously, each of the two elements which determine the risk, i.e. the severity of the harm from the hazard in question, and the probability of occurrence of that harm. all protective measures intended to reach this goal shall be applied according to the following steps: this stage is the only one at which hazards can be eliminated, thus avoiding the need for additional protective measures, such as safeguarding machines or implementing complementary protective measures. 3. information about the residual risk. information for use on the residual risk is not to be a substitute for inherently safe design, or for safeguarding or complementary protective measures. risk estimation and evaluation must be carried out after each of the above three steps of risk reduction. adequate protective measures associated with each of the operating modes and intervention procedures prevent operators from being prone to use hazardous intervention techniques in case of technical difficulties. the aim is to achieve the lowest possible level of risk. the design process is an iterative cycle, and several successive applications may be necessary to reduce the risk, making the best use of available technology. four aspects should be considered, preferably in the following order: 1. the safety of the machine during all the phases of its life cycle; 2. the ability of the machine to perform its function; 3. the usability of the machine; and 4. the costs of manufacturing, operating and dismantling the machine. the following principles apply to technical design: service life, safe machine life, fail-safe and tamper-proof design. a design which ensures the safety of service life has to be chosen when neither the technical system nor any of its safety-relevant partial functions can be allowed to fail during the service life envisaged. this means that the components of the partial functions need to be exchanged at previously defined time intervals (preventive maintenance). in the case of a fail-safe design, the technical system or its partial functions allow for faults, but none of these faults, alone or in combination, may lead to a hazardous state. it is necessary to specify just which faults in one or several partial systems can be allowed to occur simultaneously without the overall system being transferred into a hazardous state (maximum admissible number of simultaneous faults). a failure or a reduction in the performance of the technical system is accepted in this case. tamper-proof means that it is impossible to intentionally induce a hazardous state of the system. this is often required of technical systems with a high hazard potential. strategies involving secrecy play a special role in this regard. in the safety principles described here, redundant design should also be mentioned. the probability of occurrence and the consequences of damage are reduced by multiple arrangements, allowing both for subsystems or elements to be arranged in a row or in parallel. it is possible to reduce the fault potential of a technical system by the diversification of principles: several different principles are used in redundant arrangements. the spatial distribution of the function carriers allows the possibilities to influence faults to be reduced to one function. in the redundant arrangements, important functions, e.g. information transmission, are therefore designed in a redundant manner at different locations. the measures to eliminate or avoid hazards have to meet the following basic requirements: their effect must be reliable and compulsory, and they cannot be circumvented. reliable effect means that the effect principle and construction design of the planned measure guarantee an unambiguous effect, that the components have been designed according to regulations, that production and assembly are performed in a controlled manner, and that the measure has been tested. compulsory effect includes the demand for a protective effect which is active at the start of a hazardous state and during it, and which is deactivated only when the hazardous state is no longer present, or stops when the protective effect is not active. technical systems are planned as determined systems. only predictable and intended system behaviour is taken into account when the system is designed. experience has shown, however, that technical systems also display stochastic behaviour. that is, external influences and/or internal modifications not taken into consideration in the design result in unintended changes in the system's behaviour and properties. the period of time until the unintended changes in behaviour and/or in properties occur, cannot be accurately determined; it is a random variable. we have to presume that there will be a fault in every technical system. we simply do not know in advance when it will take place. the same is true for repairs. we know that it is generally possible in systems requiring re-pair to complete a repair operation successfully, but we cannot determine the exact time in advance. using statistical evaluations, we can establish a timedependent probability at which a "fault event" or "completion of a repair operation" occurs. the frequency of these events determines the availability of the system requiring repair. technical systems are intended to perform numerous functions and, at the same time, to be safe. the influence of human action on safety has to be taken into account in safety considerations as well (i.e. human factor). a system is safe when there are no functions or action sequences resulting in hazardous effects for people and/or property. risks of unwelcome events (in the following called "risk of an event") are determined on the basis of the experience (e.g. catalogue of measures) with technical systems. in addition to this, safety analyses are used (e.g. failure mode and effect analysis, hazard analysis, failure hazard analysis, operating hazard analysis, information error analysis), as well as mathematical models (e.g. worst-case analysis, monte-carlo procedure, markow's models). unwelcome events are examined for their effects. this is followed by considerations about which design modifications or additional protective measures might provide sufficient safety against these unwelcome events. the explanations below present the basic procedure for developing safety-relevant arrangements and solutions, i.e. the thinking and decision-making processes, as well as selecting criteria that are significant for the identification of unwelcome events, the risk of an event, the acceptance limits and the adoption of measures. before preparing the final documentation, it is essential to verify that the limit risk has not been exceeded, and that no new unwelcome events have occurred. the sequence scheme describes the procedure for developing safety arrangements and for finding solutions aiming to avoid the occurrence of unwelcome events which exceed the acceptance limits, by selecting suitable measures. in this context, it is assumed that: • an unwelcome event is initially identified as a single event within a comprehensive event sequence (e.g. start-up of a plant), and the risk of an event and limit risk are determined. • the selection of technical and/or non-technical measures is subject to a review of the content and the system, and the decision regarding a solution is then made. • the number of applicable measures is limited, and therefore it may not be possible to immediately find a measure with an acceptable risk for a preliminary determination of the unwelcome event. • implementation of the selected solution can result in the occurrence of a new unwelcome event. • in the above cases, a more concrete, new determination of the unwelcome event and/or the unit under review, or the state of the unit under review, and another attempt at deciding upon measures may lead to the desired result, although this may have to be repeated several times before it is successful. in the case of complex event sequences, several unwelcome events may become apparent which have to be tackled by the respective set of measures. in accordance with the sequence scheme, the unit under review and its state have to be determined first. this determination includes information on, e.g., • product type, dimension, product parts/elements distinguished according to functional or construction aspects, if applicable; • intended use; • work system or field of application; • target group; • supply energy, transformed in the product, transmitted, distributed, output; • other parameters essential to safety assessment according to type and size of the product; • known or assumed effects on the product or its parts (e.g. due to transport, assembly, conditions at the assembly site, operation, maintenance); • weight, centre of gravity; • materials, consumables; • operating states (e.g. start-up, standstill, test run, normal operation); • condition (new, condition after a period in storage/shutdown, after repair, in case of modified operating conditions and/or other significant changes); and • known or suspected effects on humans. the next step is the identification of unwelcome events. they are source conditions of processes and states, or processes and states themselves. they can be the effects of processes and states which can cause harm to people or property. an unwelcome event can be a single event or part of a sequence of events. one should look for unwelcome events in sequences of processes and functions, in work activities and organizational procedures, or in the work environment. care has to be taken that the respective interfaces are included in the considerations. deviations and time-dependent changes in regard to the planned sequences and conditions have to be taken into account as well. the risk of an unwanted event results from the probability statement which takes into account both • the expected frequency of occurrence of the event; and • the anticipated extent of harm of the event. the expected frequency of occurrence of an event leading to harm is determined by, e.g., • the probability of the occurrence itself; • the duration and frequency of exposure of people (or of objects) in the danger zone, e.g. -extremely seldom (e.g. during repair), -seldom (e.g. during installation, maintenance and inspection), -frequently, and -very frequently (e.g. constant intervention during every work cycle); • the influence of users or third parties on the risk of an event. the extent of harm is determined by, e.g., • the type of harm (harm to people and/or property); • the severity of the harm (slight/severe/fatal injury of persons, or corresponding damage to property); and • number of people or objects affected. in principle, the safety requirements depend on the ratio of the risk of an event to the limit risk. criteria for determining the limit risk are, e.g., • personal and social acceptance of hazards; • people possibly affected (e.g. layman, trained person, specialized worker); • participation of those affected in the process; and • possibilities of averting hazards. the safety of various technical equipment with comparable risk can, for instance, be achieved • primarily by technical measures, in some cases; and • mainly by non-technical measures, in other cases. this means that several acceptable solutions with varying proportions of technical and non-technical measures may be found for a specific risk. in this context, the responsibility of those involved should be taken into consideration. technical measures are developed on the basis of e.g. the following principles: • avoiding hazardous interfaces (e.g. risk of crushing, shearing); hazard sources (e.g. radiation sources, flying parts, hazardous states and actions as well as inappropriate processes); • limiting hazardous energy (e.g. by rupture disks, temperature controllers, safety valves, rated break points); • using suitable construction and other materials (e.g. solid, sufficiently resistant against corrosion and ageing, glare-free, break-proof, non-toxic, non-inflammable, non-combustible, non-sliding); • designing equipment in accordance with its function, material, load, and ergonomics principles; • using fail-safe control devices employing technical means; • employing technical means of informing (e.g. danger signal); • protective equipment for separating, attaching, rejecting, catching, etc.; • suction equipment, exhaust hoods, when needed; • protection and emergency rooms; and • couplings or locks. technical measures refer to, e.g., • physical, chemical or biological processes; • energy, material and information flow in connection with the applied processes; • properties of materials and changes in the properties; and • function and design of technical products, parts and connections. the iterative (repeated) risk reduction process can be concluded after achieving adequate risk reduction and, if applicable, a favourable outcome of risk comparison. adequate risk reduction can be considered to have been achieved when one is able to answer each of the following questions positively: • have all operating conditions and all intervention procedures been taken into account? • have hazards been eliminated or their risks been reduced to the lowest practicable level? • is it certain that the measures undertaken do not generate new hazards? • are the users sufficiently informed and warned about the residual risks? • is it certain that the operator's working conditions are not jeopardized by the protective measures taken? • are the protective measures compatible with each other? • has sufficient consideration been given to the consequences that can arise from the use of a machine designed for professional/industrial use when it is used in a non-professional/non-industrial context? • is it certain that the measures undertaken do not excessively reduce the ability of the machine to perform its intended function? there are still many potential risks connected with hazardous substances about which more information is needed. because the knowledge about the relation between their dose and mode of action is not sufficient for controlling such risks, more research is needed. the following list highlights the themes of the numerous questions related to such risks: • potentially harmful organisms; • toxicants, carcinogens; • pesticides, pollutants, poisonous substances; • genetically engineered substances; • relation between chemical and structural properties and toxicity; • chemical structure and chemical properties and the relation to reactivity and reaction possibilities of organic compounds to metabolic reaction and living systems; • modes of action, genotoxicity, carcinogenicity, effects on humans/animals; • potentially harmful organisms in feedstuffs and animal faeces; • viruses and pathogens; • bacteria in feedstuffs and faeces; • parasites in feedstuffs and animal faeces; • pests in stored feedstuffs; • probiotics as feed additives; and • preservatives in feedstuffs. violent actions damaging society, property or people have increased, and they seem to spread both internationally as well as within countries. these new risks are difficult to predict and manage, as the very strategy of the actors is to create unexpected chaotic events. certain possibilities to predict the potential types of hazards do exist, and comprehensive predictive analyses have been done (meyerson, reaser 2002) . new methodologies are needed to predict the risk of terrorist actions, and also the strategies for risk management need to be developed. due to the numerous background factors, the preparedness of societies against these risks needs to be strengthened. table 4 .3 lists important societal systems which are vulnerable to acts of terrorism. the situation in the developing countries needs to be tackled with specific methods. one has to answer the following questions: • what specific examples of prevention instruments can be offered? • what are the prerequisites for success? • how can industrialized countries assist the developing countries in carrying out preventive actions? • how should priorities be set according to the available resources? one possibility is to start a first-step programme, the goal of which is higher productivity and better workplaces. it can be carried out by improving • storage and handling of materials: -provide storage racks for tools, materials, etc.; -put stores, racks etc. on wheels, whenever possible; -use carts, conveyers or other aids when moving heavy loads; -use jigs, clamps or other fixtures to hold items in place. • work sites: -keep the working area clear of everything that is not in frequent use. • machine safety: -install proper guards to dangerous tools/machines; -use safety devices; -maintain machines properly. • control of hazardous substances: -substitute hazardous chemicals with less hazardous substances; -make sure that all organic solvents, paints, glues, etc., are kept in covered containers; -install or improve local exhaust ventilation; -provide adequate protective goggles, face shields, earplugs, safety footwear, gloves, etc.; -instruct and train workers; -make sure that workers wash their hands before eating and drinking, and change their clothes before going home. • lighting: -make sure that lighting is adequate. • social and sanitary facilities: -provide a supply of cool, safe drinking water; -have the sanitary facilities cleaned regularly; -provide a hygienic place for meals; -provide storage for clothing or other belongings; -provide first aid equipment and train a qualified first-aider. • premises: -increase natural ventilation by having more roof and wall openings, windows or open doorways; -move sources of heat, noise, fumes, arc welding, etc., out of the workshop, or install exhaust ventilation, noise barriers, or other solutions; -provide fire extinguishers and train the workers to use them; -clear passageways, provide signs and markings. • work organization: -keep the workers alert and reduce fatigue through frequent changes in tasks, opportunities to change work postures, short breaks, etc.; -have buffer stocks of materials to keep work flow constant; -use quality circles to improve productivity and quality. risk combination of the probability of an event and its consequences. the term "risk" is generally used only when there is at least a possibility of negative consequences. in some situation, risk arises from the possibility of deviation from the expected outcome or event. outcome of an event or a situation, expressed in quality and in quantity. it may result in a loss or in an injury or may be linked to it. the result can be a disadvantage or a gain. in this case the event or the situation is the source. in connection with every analysis it has to be checked whether the cause is given empirically, or follows a set pattern, and whether there is scientific agreement regarding these circumstances. note 1 there can be more than one consequence from one event. note 2 consequences can range from positive to negative. the consequences are always negative from the viewpoint of safety. extent to which an event is likely to occur. note 1 iso 3534-1:1993 gives the mathematical definition of probability as "a real number in the interval 0 to 1 attached to a random event. it can be related to a long-run relative frequency of occurrence or to a degree of belief that an event will occur. for a high degree of belief the probability is near 1". note 2 frequency rather than probability may be used in describing risk. degrees of belief about probability can be chosen as classes or ranks such as: rare/unlikely/moderate/likely/almost certain, or incredible/improbable/ remote/occasional/probable/frequent. remark: informal language often confuses frequency and probability. this can lead to wrong conclusions in safety technology. probability is the degree of coincidence of the time frequency of coincidental realization of a fact from a certain possibility. coincidence is an event which basically can happen, may be cause-related, but does not occur necessarily or following a set pattern. it may also not occur (yes-or-no-alternative). data for probability of occurring with specific kinds of occurrence and weight of consequences can be: in a statistical sense: empirical, retrospective, real in a prognostic sense: speculative, prospective, probabilistic occurrence of a particular set of circumstances regarding place and time. an event can be the source of certain consequences (empirically to be expected with certain regularity). the event can be certain or uncertain. the event can be a single occurrence or a series of occurrences. the probability associated with the event can be estimated for a given period of time. task range by which the significance of risk is assessed. note 1 risk criteria can include associated costs and benefits, legal and statutory requirements, socio-economic and environmental aspects, the concerns of stakeholders, priorities and other inputs to the assessment. the way in which a stakeholder views a risk based on a set of values or concerns. note 1 risk perception depends on the stakeholder's needs, issues and knowledge. note 2 risk perception can differ from objective data. exchange or sharing of information about risk between the decision-makers and other stakeholders. overall process of risk analysis and risk evaluation. systematic use of information to identify sources and to estimate the risk. note 1 risk analysis provides a basis for risk evaluation, risk treatment, and risk acceptance. note 2 information can include historical data, theoretical analyses, informal opinions, and the concerns of stakeholders. process used to assign figures, values to the probability and consequences of a risk. note 1 risk estimation can consider cost, benefits, the concerns of stakeholders, and other variables, as appropriate for risk evaluation. process of comparing the estimated risk against given risk criteria to determine the significance of a risk. process of selection and implementation of measures to modify risk. note 1 risk treatment measures can include avoiding, optimizing, transferring or retaining risk. actions implementing risk management decisions. note 1 risk control may involve monitoring, re-evaluation, and compliance with decisions. process, related to a risk, to minimize the negative and to maximize the positive consequences (and their respective probabilities). actions taken to lessen the probability, negative consequences or both, associated with a risk. limitation of any negative consequences of a particular event. decision not to become involved in, or action to withdraw from, a risk situation. sharing with another party the burden of loss or benefit of gain, for a risk. note 1 legal or statutory requirements can limit, prohibit or mandate the transfer of a certain risk. note 2 risk transfer can be carried out through insurance or other agreements. note 3 risk transfer can create new risks or modify existing ones. note 4 relocation of the source is not risk transfer. acceptance of the burden of loss, or benefit of gain, from a particular risk. note 1 risk retention includes the acceptance of risks that have not been identified. note 2 risk retention does not include means involving insurance, or transfer in other ways. this includes risk assessment, risk treatment, risk acceptance and risk communication. risk assessment is risk analysis, with identification of sources and risk estimation, and risk evaluation. risk treatment includes avoiding, optimizing, transferring and retaining risk. → risk acceptance → risk communication harm physical injury or damage to the health of people or damage to property or the environment [iso/iec guide 51]. note 1 harm includes any disadvantage which is causally related to the infringement of the object of legal protection brought about by the harmful event. note 2 in the individual safety-relevant definitions, harm to people, property and the environment may be included separately, in combination, or it may be excluded. this has to be stated in the respective scope. potential source of harm [iso/iec guide 51]. the term "hazard" can be supplemented to define its origin or the nature of the possible harm, e.g., hazard of electric shock, crushing, cutting, dangerous substances, fire, drowning. in every-day informal language, there is insufficient differentiation between source of harm, hazardous situation, hazardous event and risk. circumstance in which people, property or the environment are exposed to one or more hazards [iso/iec guide 51]. note 1 circumstance can last for a shorter or longer period of time. event that can cause harm [din en 1050] . 1 the hazardous event can be preceded by a latent hazardous situation or by a critical event. combination of the probability of occurrence of harm and the severity of that harm [iso/iec guide 51]. note 1 in many cases, only a uniform extent of harm (e.g. leading to death) is taken into account, or the occurrence of harm may be independent of the extent of harm, as in a lottery game. in these cases, it is easier to make a probability statement; risk assessment by risk comparison [din en 1050] thus becomes much simpler. note 2 risks can be grouped in relation to different variables, e.g. to all people or only those affected by the incident, to different periods of time, or to performance. the probabilistic expectation value of the extent of harm is suitable for combining the two probability variables. note 4 risks which arise as a consequence of continuous emission, e.g. noise, vibration, pollutants, are affected by the duration and level of exposure of those affected. risk which is accepted in a given context based on the current values of society [iso/iec guide 51]. the acceptable risk has to be taken into account in this context, too. note 2 safety-relevant definitions are oriented to the maximum tolerable risk. this is also referred to as limit risk. note 3 tolerability is also based on the assumption that the intended use in addition to a reasonably predictable misuse of the products, processes and services, is complied with. freedom from unacceptable risk [iso/iec guide 51]. note 1 safety is indivisible. it cannot be split into classes or levels. note 2 safety is achieved by risk reduction, so that the residual risk in no case exceeds the maximum tolerable risk. existence of an unacceptable risk. note 1 safety and danger exclude one another -a technical product, process or service cannot be safe and dangerous at the same time. means used to reduce risk [iso/iec guide 51]. note 1 protective measures at the product level have priority over protective measures at the workplace level. preventive measure means assumed, but not proven, to reduce risk. risk remaining after safety measures have been taken [din en 1050] . note 1 residual risk may be related to the use of technical products, processes and services. systematic use of available information to identify hazards and to estimate their risks [iso/iec guide 51]. determination of connected risk elements of all hazards as a basis for risk assessment. decision based on the analysis of whether the tolerable risk has been exceeded [iso/iec guide 51]. overall process of risk analysis and risk evaluation [iso/iec guide 51]. use of a product, process or service in accordance with information provided by the supplier [iso/iec guide 51]. note 1 information provided by the supplier also includes descriptions issued for advertising purposes. use of a product, process or service in a way not intended by the supplier, but which may result from readily predictable human behaviour [iso/iec guide 51]. safety-related formulation of contents of a normative document in the form of a declaration, instructions, recommendations or requirements [compare en 45020, safety related]. the information set down in technical rules is normally restricted to certain technical relations and situations; in this context, it is presumed that the general safety-relevant principles are followed. a procedure with the aim to reduce risk of a (technical) product, process or service according to the following steps serves to reach the safety goals in the design stage: • safety-related layout; • protective measures; • safety-related information for users. function inevitable to maintain safety. a function which, in case of failure, allows the tolerable risk to be immediately exceeded. depending on the situation, it is possible to use one method or a combination of several methods. intuitive hazard detection spontaneous, uncritical listing of possible hazards as a result of brainstorming by experts. group work which is as creative as possible. writing ideas down (on a flip chart) first, then evaluating them. technical documentation (instructions, requirements) is available for many industrial plant and work processes, describing the hazards and safety measures. this documentation has to be obtained before continuing with the risk analysis. the deviations between the set point and the actual situation of individual components are examined. information on the probability of failure of these elements may be found in technical literature. examining the safety aspects in unusual situations (emergency, repair, starting and stopping) when plans are made to modify the plant or processes. a systematic check of the processes and plant parts for effects in normal operation and in case of set point deviations, using selected question words (and -or -not -too much -too little?). this is used in particular for measurement, control units, programming of computer controls, robots. all possible causes and combinations of causes are identified for an unwanted operating state or an event, and represented in the graphic format of a tree. the probability of occurrence of an event can be estimated from the context. the fault tree analysis can also be used retrospectively to clarify the causes of events. additional methods may be: human reliability analysis a frequency analysis technique which deals with the behaviour of human beings affecting the performance of the system, and estimates the influence of human error on reliability. a hazard identification and frequency analysis technique which can be used at an early stage in the design phase to identify and critically evaluate hazards. operating safety block program a frequency analysis technique which utilizes a model of the system and its redundancies to evaluate the operating safety of the entire system. classifying risks into categories, to establish the main risk groups. all typical hazardous substances and/or possible accident sources which have to be taken into account are listed. the checklist may be used to evaluate the conformity with codes and standards. this method is used to estimate whether coincidental failures of an entire series of different parts or modules within a system are possible and what the probable effects would be. estimate the influence of an event on humans, property or the environment. simplified analytical approaches, as well as complex computer models can be used. a large circle of experts is questioned in several steps; the result of the previous step together with additional information is communicated to all participants. during the third or fourth step the anonymous questioning concentrates on aspects on which no agreement is reached so far. basically this technique is used for making predictions, but is also used for the development of new ideas. this method is particularly efficient due to its limitation to experts. a hazard identification and evaluation technique used to establish a ranking of the different system options and to identify the less hazardous options. a frequency analysis technique in which a model of the system is used to evaluate variations of the input conditions and assumptions. a means to estimate and list risk groups; reviews risk pairs and evaluates only one risk pair at a time. overview of data from the past a technique used to identify possible problem areas; can also be used for frequency analysis, based on accident and operation safety data, etc. a method to identify latent risks which can cause unforeseeable incidents. cssr differs from the other methods in that it is not conducted by a team, and can be conducted by a single person. the overview points out essential safety and health requirements related to a machine and simultaneously to all relevant (national, european, international) standards. this information ensures that the design of the machine complies with the issued "state of the art" for that particular type of machine. the "what-if" method is an inductive procedure. the design and operation of the machine in question are examined for fairly simple applications. at every step "what-if" questions are asked and answered to evaluate the effect of a failure of the machine elements or of process faults in view of the hazards caused by the machine. for more complex applications, the "what-if" method is most useful with the aid of a "checklist" and the corresponding work division to allocate specific features of the process to persons who have the greatest experience and practice in evaluating the respective feature. the operator's behaviour and professional knowledge are assessed. the suitability of the equipment and design of the machine, its control unit and protective devices are evaluated. the influence of the materials processed is examined, and the operating and maintenance records are checked. the checklist evaluation of the machine generally precedes the more detailed methods described below. fmea is an inductive method for evaluating the frequency and consequences of component failure. when operating procedures or operator errors are investigated, then other methods may be more suitable. fmea can be more time-consuming than the fault tree analysis, because every mode of failure is considered for every component. some failures have a very low probability of occurrence. if these failures are not analyzed in depth this decision should be recorded in the documentation. the method is specified in iec 812 "analysis techniques for system reliability -procedure for failure mode and effects analysis (fmea)". in this inductive method, the test procedures are based on two criteria: technology and complexity of the control system. mainly, the following methods are applicable: • practical tests of the actual circuit and fault simulation on certain components, particularly in suspected areas of performance identified during the theoretical check and analysis. • simulation of control behaviour (e.g. by means of hardware and/or software models). whenever complex safety-related parts of control systems are tested, it may be necessary to divide the system into several functional sub-systems, and to exclusively submit the interface to fault simulation tests. this technique can also be applied to other parts of machinery. mosar is a complete approach in 10 steps. the system to be analyzed (machinery, process, installation, etc.) is examined as a number of sub-systems which interact. a table is used to identify hazards and hazardous situations and events. the adequacy of the safety measures is studied with a second table, and a third table is used to look at their interdependency. a study, using known tools (e.g. fmea) underlines the possible dangerous failures. this leads to the elaboration of accident scenarios. by consensus, the scenarios are sorted in a severity table. a further table, again by consensus, links the severity with the targets of the safety measures, and specifies the performance levels of the technical and organizational measures. the safety measures are then incorporated into the logic trees and the residual risks are analyzed via an acceptability table defined by consensus. ilo (1987) 1994, 1995, 1996, 1997, 1998, 1999 the risks to health at work are numerous and originate from several sources. their origins vary greatly and they cause vast numbers of diseases, injuries and other adverse conditions, such as symptoms of overexertion or overload. traditional occupational health risk factors and their approximate numbers are given in table 4 .4. the exposure of workers to hazards or other adverse conditions of work may lead to health problems, manifested in the workers' physical health, psysical workload, psychological disturbances or social aspects of life. workers may be exposed to various factors alone or in different types of combinations, which may or may not show interaction. the assessment of interacting risk factors is complex and may lead to substantial differences in the final risk estimates when compared with estimates of solitary factors. examples of interaction between different risk factors in the work environment are given in table 4 .5. the who estimate of the total number of occupational diseases among the 3 billion workers of the world is 160 million a year. this is likely to be an under-estimate due to the lack of diagnostic services, limited legislative coverage of both workers and diseases, and variation in diagnostic criteria between different parts of the world. the mortality from occupational diseases is substantial, comparable with other major diseases of the world population such as malaria or tuberculosis. the recent ilo estimate discloses 2.4 million deaths a year from work-related causes in the world including deaths from accidents, dangerous substances, and occupational diseases. eightyfive percent (85%) of these deaths take place in developing countries, where the diagnostic services, social security to families and compensation to workers are less developed. although the risk is decreasing in the industrialized world, the trend is increasing in the rapidly industrializing and transitory countries. a single hazard alone, such as asbestos exposure, is calculated to cause 100,000 cancers a year with a fatal outcome in less than two years after diagnosis (takala 2005). the incidence rates of occupational diseases in well registered industrialized countries are at the level of 3-5 cases/10,000 active employees/year, i.e., the incidence levels are comparable with major public health problems, such as cardiovascular diseases, respiratory disorders, etc. in the industrialized countries, the rate of morbidity from traditional occupational diseases, such as chemical poisonings, is declining, while musculoskeletal and allergic diseases are on the increase. about 200 biological factors that are hazardous to workers' health have been identified in various work environments. some of the new diseases recognized are blood-borne infections, such as hepatitis c and hiv, and exotic bacterial or viral infections transmitted by increasing mobility, international travelling and migration of working people. also some hospital infections and, e.g., drug-resistant tuberculosis, are being contracted increasingly by health care personnel. in the developing countries the morbidity picture of occupational diseases is much less clear for several reasons: low recognition rates, rotation and turnover of workers, shorter life expectancy which hides morbidity with a long latency period, and the work-relatedness of several common epidemic diseases, such as malaria and hiv/aids (rantanen 2003). the estimation of so-called work-related diseases is even more difficult than that of occupational diseases. they may be about 10-fold more prevalent than the definite occupational diseases. several studies suggest that siegfried radandt, jorma rantanen and ortwin renn work-related allergies, musculoskeletal disorders and stress disorders are showing a growing trend at the moment. the prevention of work-related diseases is important in view of maintaining work ability and reducing economic loss from absenteeism and premature retirement. the proportion of work-relatedness out of the total morbidity figures has been estimated and found surprisingly high (nurminen and karjalainen 2001 , who 2002 ) (see table 4 .6). the public health impact of work-related diseases is great, due to their high prevalence in the population. musculoskeletal disorders are among the three most common chronic diseases in every country, which implies that the attribution of work is very high. similarly, cardiovascular diseases in most industrialized countries contribute to 50% of the total mortality. even a small attributable fraction of work-relatedness implies high rates of morbidity and mortality related to work. the concept of disease is in general not a simple one. when discussing morbidity one has to recognize three different concepts: 1. illness = an individual's perception of a health problem resulting from either external or internal causes. 2. disease = an adverse health condition diagnosed by a doctor or other health professional. 3. sickness = a socially recognized disease which is related to, for example, social security actions or prescription of sick leave, etc. when dealing with occupational and work-related morbidity, one may need to consider any of the above three aspects of morbidity. a recognized occupational disease, however, belongs to group 3, i.e. it is a sickness defined by legal criteria. medical evidence is required to show that the condition meets the criteria of an occupational disease before recognition can be made. there are dozens of definitions for occupational disease. the content of the concept varies, depending on the context: a) the medical concept of occupational disease is based on a biomedical or other health-related etiological relationship between work and health, and is used in occupational health practice and clinical occupational medicine. b) the legal concept of occupational disease defines the disease or conditions which are legally recognized as conditions caused by work, and which lead to liabilities for recognition, compensation and often also prevention. the legal concept of occupational disease has a different background in different countries, often declared in the form of an official list of occupational diseases. there is universal discrepancy between the legal and medical concept, so that in nearly all countries the official list of recognized occupational diseases is shorter than the medically established list. this automatically implies that a substantial proportion of medically established occupational diseases remain unrecognized, unregistered, and consequently also uncompensated. the definition of occupational disease, as used in this chapter, summarizes various statements generated during the history of occupational medicine: an occupational disease is any disease contracted as a result of exposures at work or other conditions of work. the general criteria for the diagnosis and recognition of an occupational disease are derived from the core statements of various definitions: 1. evidence on exposure(s) or condition(s) in work or the work environment, which on the basis of scientific knowledge is (are) able to generate disease or some other adverse health condition. 2. evidence of symptoms and clinical findings which on the basis of scientific knowledge can be associated with the exposure(s) or condition(s) in concern. 3. exclusion of non-occupational factors or conditions as a main cause of the disease or adverse health condition. point 3 often creates problems, as several occupationally generated clinical conditions can be caused also by non-occupational factors. on the other hand, several factors from different sources and environments are involved in virtually every disease. therefore the wordings "main cause" or "principal cause" are used. the practical solution in many countries is that the attribution of work needs to be more than 50%. usually the necessary generalizeable scientific evidence is obtained from epidemiological studies, but also other types of evidence, e.g. well documented clinical experience combined with information on working conditions may be acceptable. in some countries, like finland, any disease of the worker which meets the above criteria can be recognized as an occupational disease. in most other countries, however, there are official lists of occupational diseases which determine the conditions and criteria on which the disease is considered to be of occupational origin. in 1985 who launched a new concept: work-related disease (who 1985) . the concept is wider than that of an occupational disease. it includes: a) diseases in which the work or working conditions constitute the principal causal factor. b) diseases for which the occupational factor may be one of several causal agents, or the occupational factor may trigger, aggravate or worsen the disease. c) diseases for which the risk may be increased by work or work-determined lifestyles. the diseases in category (a) are typically recognized as legally determined occupational diseases. categories (b) and (c) are important regarding the morbidity of working populations, and they are often considered as important targets for prevention. in general, categories (b) and (c) cover greater numbers of people, as the diseases in question are often common noncommunicable diseases of the population, such as cardiovascular diseases, musculoskeletal disorders, and allergies and, to a growing extent, stressrelated disorders (see table 4 .6). the concept of work-related disease is very important from the viewpoint of occupational health risk assessment and the use of its results for preventive purposes and for promoting health and safety at work. this is because preventive actions in occupational health practice cannot be limited only to legally recognized morbidity. the lists of occupational diseases contain great numbers of agents that show evidence on occupational morbidity. according to the ilo recommendation r194 (2002): list of occupational diseases, the occupational diseases are divided into four main categories: 1. diseases resulting from single causes following the categories listed in table 4 .4. the most common categories are physical factors, chemical agents, biological factors and physical work, including repetitive tasks, poor ergonomic conditions, and static and dynamic work. 2. diseases of the various organs: respiratory system, nervous system, sensory organs, internal organs, particularly liver and kidneys, musculoskeletal system, and the skin. 3. occupational cancers. 4. diseases caused by other conditions of work. research on risk perception shows differences in how different types of risks are viewed. instant, visible, dramatic risk events, particularly ones that cause numerous fatalities or severe visible injuries in a single event generally arouse much attention, and are given high priority. on the other hand, even great numbers of smaller events, such as fatal accidents of single workers, arouse less attention in both the media and among regulators, even though the total number of single fatal accidents in a year may exceed the number of fatalities in major events by several orders of magnitude. occupational diseases, with the exception of a few acute cases, are silent, develop slowly, and concern only one or a few individuals at a time. furthermore, the diseases take months or years to develop, in extreme cases even decades, after the exposure or as a consequence of accumulation of exposure during several years. as occupational health problems are difficult to detect and seriously under-diagnosed and under-reported, they tend to be given less priority than accidents. the perception of occupational disease risk remains low in spite of their severity and relatively high incidence. particularly in industrialized countries, the extent of occupational health problems is substantially greater than that of occupational accidents. on a global scale, the estimated number of fatalities due to occupational accidents is 375,000 and the respective estimate for fatalities due to work-related diseases is 1.8 million a year, giving a fatal accident/fatal disease ratio of 1 to 5. the corresponding ratio in the eu-15 is 1 to 20 (takala 2005). the risk distribution of ods is principally determined by the nature of the work in question and the characteristics of the work environment. there is great variation in the risk of ods between the lowest and highest risk occupations. in the finnish workforce, the risk between the highest risk and the lowest risk occupations varies by a factor of 30. the highest risk occupations carry a risk which is 4-10 times higher than the average for all occupations. the risk of an occupational disease can be estimated on the basis of epidemiological studies, if they do exist in the case of the condition in question. on the other hand, various types of economic activity, work and occupations carry different types of risks, and each activity may have its own risk profile. by examining the available epidemiological evidence, we can recognize high-risk occupations and characterize the typical risks connected with them ( figure 4 .12, table 4.7). as an example, the risk of occupational asthma, dermatosis or musculoskeletal disorders is common in several occupations, but not in all. there may be huge differences in risks between different occupations. the 10 occupations carrying the highest risk for occupational asthma, occupational skin diseases and work-related tenosynovitis, in 1986-1991, are shown in table 4 .8. assessment of the risk of occupational diseases has an impact on research priorities. table 4 .9 shows the priorities for research in four countries. the similarity of the priorities is striking, revealing that the problems related to the risks of occupational diseases are universal. the diagnosis of occupational diseases is important for the treatment of the disease, and for prevention, registration and compensation. the diagnosis is based on information obtained from: a) data on the work and the work environment usually provided by the employer, occupational health services, occupational safety committee, or expert bodies carrying out hygienic and other services for the workplace. b) information on the health examination of individual workers. the authorities in many countries have stipulated legal obligations for high-risk sectors to follow up the workers' health and promote early detection of changes in their health. occupational health services keep records on examinations. c) workers with special symptoms (for example, asthmatic reactions) are taken into the diagnostic process as early as possible. epidemiological evidence is a critical prerequisite for recognizing causal relationship between work and disease. epidemiology is dependent on three basic sources of information on work and the work environment: (a) exposure assessment that helps to define the "dose" of risk factor at work, (b) the outcome assumed to occur as a biological (or psychological) response to the exposures involved, and (c) time, which has a complex role in various aspects of epidemiology. all these sources are affected by the current dynamics of work life which has major impact on epidemiological research and its results. exposure assessment is the critical initial step in risk assessment. as discussed in this chapter, accurate exposure assessment will become more difficult and cumbersome than before in spite of remarkable achievements in measurement, analysis and monitoring methods in occupational hygiene, toxicology and ergonomics. great variations in working hours and individu-alization of exposures, growing fragmentation and mobility increase the uncertainties, which are multiplied. structural uncertainty, measurement uncertainty, modelling uncertainty, input data uncertainty and natural uncertainty amplify each other. as a rule, variation in any direction in exposure assessment tends to lead to underestimation of risk, and this has severe consequences to health. personal monitoring of exposures, considering variations in individual doses, and monitoring internal doses using biological monitoring methods help in the control of such variation. a monofactorial exposure situation in the past was ideal in the assessment because of its manageability. it also occurs usually as a constant determinant for long periods of time and can be regularly and continuously measured and monitored. this is very seldom the case today, and exposure assessment in modern work life is affected by discontinuities of the enterprise, of technologies and production methods, and turnover of the workforce, as well as the growing mobility and internationalization of both work and workers. company files that were earlier an important source of exposure and health data no longer necessarily fulfil that function. in addition, the standard 8-h time-weighted average for exposure assessment can no longer be taken as a standard, as working hours are becoming extremely heterogeneous. assessment of accurate exposure is thus more and more complex and cumbersome, and new strategies and methods for the quantification of exposure are needed. three challenges in particular can be recognized: a) the challenge arising from numerous discontinuities, fragmentation and changes in the company, employment and technology. although in the past company data were collected from all sources that were available, collective workroom measurements were the most valuable source of data. due to the high mobility of workers and variation in the work tasks, personal exposure monitoring is needed that follows the worker wherever he or she works. special smart cards for recording all personal exposures over years have been proposed, but so far no system-wide action has been possible. in radiation protection, however, such a personal monitoring system has long been a routine procedure. b) the complex nature of exposures where dozens of different factors may be involved (such as those in indoor air problems) and acting in combinations. table 4 .4 gives a list of exposing factors in modern work life, many of which are difficult to monitor. c) new, rapidly spreading and often unexpected exposures that are not well characterized. often their mechanisms of action are not known, or the fast spread of problems calls for urgent action, as in the case of bovine spongiform encephalopathy (bse) in the 1990s, sars outbreak in 2003, and in the new epidemics of psychological stress or musculoskeletal disorders in modern manufacturing. the causes of occupational diseases are grouped into several categories by the type of factor (see table 4 .4). a typical grouping is the one used in ilo recommendation no. 194 . the lists of occupational diseases contain diseases caused by one single factor only, but also diseases which may have been caused by multifactorial exposures. exposure assessment is a crucial step in the overall risk assessment. the growing complexity of exposure situations has led to the development of new methods for assessing such complex exposure situations. these methods are based on construction of model matrices for jobs which have been studied thoroughly for their typical exposures. the exposure profiles are illustrated in job exposure matrices (jem) which are available for dozens of occupations (heikkilä et al. 2005 , guo 2005 . several factors can cause occupational diseases. the jem is a tool used to convert information on job titles into information on occupational risk factors. jem-based analysis is economical, systematic, and often the only reasonable choice in large retrospective studies in which exposure assessment at the individual level is not feasible. but the matrices can also be used in the practical work for getting information on typical exposure profiles of various jobs. the finnish national job-exposure matrix (finjem) is the first and so far the only general jem that is able to give a quantitative estimation of cumulative exposure. for example, finjem estimates were used for exposure profiling on 43 chemical exposures and several other cancer-risk factors for 393 occupational categories. the jem analysis has been further developed into task specific exposure matrices charting the exposure panorama of various tasks (benke et al. 2000 (benke et al. , 2001 . as the previous mono-causal, mono-mechanism, mono-outcome setting has shifted in the direction of multicausality, multiple mechanisms and multioutcomes, the assessment of risks has become more complex. some outcomes, as mentioned above, are difficult to define and measure with objective methods and some of them may be difficult to recognize by exposed groups themselves, or even by experts and researchers. for example, the objective measurement of stress reactions is still imprecise in spite of improvements in the analysis of some indicator hormones, such as adrenalin, noradrenalin, cortisol, prolactin, or in physical measurements, such as galvanic skin resistance and heart rate variability. questionnaires monitoring perceived stress symptoms are still the most common method for measuring stress outcomes. thanks to well organized registries, particularly in germany and the nordic countries, data on many of the relevant outcomes of exposure, such as cancer, pneumoconiosis, reproductive health disturbances and cardiovascular diseases can be accumulated, and long-term follow-up of outcomes at the group level is therefore possible. on the other hand, several common diseases, such as cardiovascular diseases, may have a work-related aetiology, but it may be difficult to show at individual level. the long-term data show that due to changes in the structure of economies, types of employment, occupational structures and conditions of work, many of the traditional occupational diseases, such as pneumoconiosis and acute intoxications have almost disappeared. several new outcomes have appeared, however, such as symptoms of physical or psychological overload, psychological stress, problems of adapting to a high pace of work, and uncertainty related to rapid organizational changes and risk of unemployment. in addition, age-related and work-related diseases among the ageing workforce are on the increase (kivimäki et al. 2006 , ilmarinen 2006 . these new outcomes may have a somatic, psychosomatic or psychosocial phenotype, and they often appear in the form of symptoms or groups of symptoms instead of well-defined diagnoses. practising physicians or clinics are not able to set an icd (international statistical classification of diseases and health-related conditions)-coded diagnosis for them. in spite of their diffuse nature, they are still problems for both the worker and the enterprise, and their consequences may be seen as sickness absenteeism, premature retirement, loss of job satisfaction, or lowered productivity. thus, they may have even a greater impact on the quality of work life and the economy than on clinical health. many such outcomes have been investigated by using questionnaire surveys among either representative samples of the whole workforce or by focusing the survey on a specific sector or occupational group. the combination of data from the surveys of "exposing factors", such as organizational changes, with questionnaire surveys of "outcomes", such as sickness absenteeism, provides epidemiological information on the association between the new exposures and the new outcomes. there are, however, major problems in both the accurate measurement of the exposures and outcomes, and also, the information available on the mechanisms of action is very scarce. epidemiology has expanded the focus of our observations from crosssectional descriptions to longitudinal perspectives, by focussing attention on the occurrence of diseases and finding associations between exposure and morbidity. such an extension of vision is both horizontal and vertical, looking at the causes of diseases. time is not only a temporal parameter in epidemiology, but has also been used for the quantification of exposure, measurement of latencies, and the detection of acceleration or slowing of the course of biological processes. as the time dimension in epidemiology is very important, the changes in temporal parameters of the new work life also affect the methods of epidemiological research. the time dimension is affected in several ways. first, the fragmentation and discontinuities of employment contracts, as described above, break the accumulation of exposure time into smaller fragments, and continuities are thus difficult to maintain. collecting data on cumulative exposures over time becomes more difficult. the time needed for exposure factors to cause an effect becomes more complex, as the discontinuities typical to modern work life allow time for biological repair and elimination processes, thus diluting the risk which would get manifested from continuous exposure. the dosage patterns become more pulse-type, rather than being continuous, stable level exposures. this may affect the multi-staged mechanisms of action in several biological processes. the breaking up of time also increases the likelihood of memory bias of respondents in questionnaire studies among exposed workers, and thus affects the estimation of total exposures. probably the most intensive effect, however, will be seen as a consequence of the variation in working hours. for example, instead of regular work of 8 hours per day, 40 hours per week and 11 months per year, new time schedules and total time budgets are introduced for the majority of workers in the industrial society. the present distribution of weekly working hours in finland is less than 30 hours per week for one third of workers, regular 35-40 hours per week for one third, and 45-80 hours per week for the remaining third. thus the real exposure times may vary substantially even among workers in the same jobs and same occupations, depending on the working hours and the employment contract (temporary, seasonal, part-time, full-time) (härmä 2000 , piirainen et al. 2003 . such variation in time distribution in "new work life" has numerous consequences for epidemiological studies, which in the past "industrial society" effectively utilized the constant time patterns at work for the assessment of exposures and outcomes and their interdependencies. the time dimension also has new structural aspects. as biological processes are highly deterministic in terms of time, the rapid changes in work life cannot wait for the maturation of results in longitudinal follow-up studies. the data are needed rapidly in order to be useful in the management of working conditions. this calls for the development of rapid epidemiological methods which enable rapid collection of the data and the making of analyses in a very short time, in order to provide information on the effects of potential causal factors before the emergence of a new change. often these methods imply the compromising of accuracy and reliability for the benefit of timeliness and actuality. as occupational epidemiology is not only interested in acute and short-term events, but looks at the health of workers over a 30-40-year perspective, the introduction of such new quick methods should not jeopardize the interest and efforts to carry out long-term studies. epidemiology has traditionally been a key tool in making a reliable risk assessment of the likelihood of the adverse outcomes from certain levels of exposure. the new developments in work life bring numerous new challenges to risk assessment. as discussed above, the new developments in work life have eliminated a number of possibilities for risk assessment which prevailed in the stable industrial society. on the other hand, several new methods and new information technologies provide new opportunities for collection and analysis of data. traditionally, the relationship between exposure and outcome has been judged on the basis of the classical criteria set by hill (1965) . höfler (2005) crystallizes the criteria with their explanations as the following: 1. strength of association: a strong association is more likely to have a causal component than is a modest association. 2. consistency: a relationship is observed repeatedly. 3. specificity: a factor influences specifically a particular outcome or population. 4. temporality: the factor must precede the outcome it is assumed to affect. ing dose of exposure or according to a function predicted by a substantive theory. 6. plausibility: the observed association can be plausibly explained by substantive matter (e.g. biological) explanations. 7. coherence: a causal conclusion should not fundamentally contradict present substantive knowledge. 8. experiment: causation is more likely if evidence is based on randomized experiments. 9. analogy: for analogous exposures and outcomes an effect has already been shown. the hill criteria have been subjected to scrutiny, and sven hernberg has analyzed them in detail from the viewpoint of occupational health epidemiology. virtually all the hill criteria are affected by the changes in the new work life, and therefore methodological development is now needed. a few comments on causal inference are made here in view of the critiques by rothman (1988), hernberg (1992) and höfler (2005) : the strength of association will be more difficult to demonstrate due to the growing fragmentation that tends to diminish the sample sizes. the structural change that removes workers from high-level exposures to lower and shorterterm exposures may dilute the strength of effect, which may still prevail, but at a lower level. consistency of evidence may also be affected by the higher variation in conditions of work, study groups, multicultural and multiethnic composition of the workforce, etc. similarly, in the multifactorial, multi-mechanism, multi-outcome setting, the specificity criterion is not always relevant. the temporal dimension has already been discussed. in rapidly changing work life the follow-up times before the next change and before turnover in the workforce may be too short. the outcomes may also be defined by the exposures that have taken place long ago but have not been considered in the study design because historical data are not available. the biological gradient may be possible to demonstrate in a relatively simple exposure-outcome relationship. however, the more complex and multifactorial the setting becomes, the more difficult it may be to show the doseresponse relationship. the dose-response relationship may also be difficult to demonstrate in the cases of relatively ill-defined outcomes which are difficult to measure, but which can be detected as qualitative changes. biological plausibility is an important criterion which in a multimechanism setting may at least in part be difficult to demonstrate. on the other hand, the mechanisms of numerous psychological and psychosocial outcomes lack explanations, even though they undoubtedly are work-related. the missing knowledge of the mechanism of action did not prevent the establishment of causality between asbestos and cancer in a pleural sack or a lung. as many of the new outcomes may be context-dependent, the coherence criterion may be irrelevant. similarly, many of the psychosocial outcomes are difficult to put into an experimental setting, and it can be difficult to make inferences based on analogy. all of the foregoing implies that the new dynamic trends in work life challenge epidemiology in a new way, particularly in the establishment of causality. knowledge of causality is required for the prevention and management of problems. the hill criteria nevertheless need to be supplemented with new ones to meet the conditions of the new work life. similarly, more definitive and specific criteria and indicators need to be developed for the new exposures and outcomes. many of the challenges faced in the struggle to improve health and safety in modern work life can only be solved with the help of research. research on occupational health in the rapidly changing work life is needed more than ever. epidemiology is, and will remain, a key producer of information needed for prevention policies and for ensuring healthy and safe working conditions. the role of epidemiology is, however, expanding from the analysis of the occurrence of well-defined clinical diseases to studies on the occurrence of several other types of exposure and outcome, and their increasingly complex associations. as the baseline in modern work life is shifting in a more dynamic direction, and many parameters in work and the workers' situation are becoming more fragmented, incontinuous and complex, new approaches are needed to tackle the uncertainties in exposure assessment. the rapid pace of change in work life calls for the development of assessment methods to provide up-todate data quickly, so that they can be used to manage these changes and their consequences. many new outcomes which are not possible to register as clinical icd diagnoses constitute problems for today's work life. this is particularly true in the case of psychological, psychosocial and many musculoskeletal outcomes which need to be managed by occupational health physicians. methods for the identification and measurement of such outcomes need to be improved. the traditional hill criteria for causal inference are not always met even in cases where true association does exist. new criteria suitable for a new situation should be established without jeopardizing the original objective of ascertaining the true association. developing the bayesian inference further through utilization of a priori knowledge and a holistic approach may provide responses to new challenges. new neural network softwares may help in the management of the growing complexity. the glory of science does not lie in the perfection of a scientific method but rather in the recognition of its limitations. we must keep in mind the old saying: "absence of evidence is not evidence of absence". instead, it is merely a consequence of our ignorance that should be reduced through further efforts in systematic research, and particularly through epidemiology. and secondly, the ultimate value of occupational health research will be determined on the basis of its impact on practice in the improvement of the working conditions, safety and health of working people. changing conditions of work, new technologies, new substances, new work organizations and working practices are associated with new morbidity patterns and even with new occupational and work-related diseases. the new risk factors, such as rapidly transforming microbials and certain social and behavioural "exposures" may follow totally new dynamics when compared with the traditional industrial exposures (self-replicating nature of microbials and spreading of certain behaviours, such as terrorism) (smolinski et al. 2003 , loza 2007 . several social conditions, such as massive rural-urban migration, increased international mobility of working people, new work organizations and mobile work may cause totally new types of morbidity. examples of such development are, among others, the following: • mobile transboundary transportation work leading to the spread of hiv/aids. • increased risk of metabolic syndrome, diabetes and cardiovascular diseases aggravated by unconventional working hours. • increased risk of psychological burnout in jobs with a high level of longterm stress. • virtually a global epidemic of musculoskeletal disorders among vdu workers with high work load, psychological stress and poor ergonomics. the incidences of occupational diseases may not decline in the future, but the type of morbidity may change. the direction of trend in industrialized countries is the prominence of work-related morbidity and new diseases, while the traditional occupational diseases such as noise injury, pneumoconiosis, repetitive strain and chemical intoxications may continue to be prevalent in developing countries for long periods in the future. the new ergonomics problems are related to light physical work with a considerable proportion of static and repetitive workload. recent research points to an interesting interaction between unergonomic working conditions and psychological stress, leading to a combined risk of musculoskeletal disorders of the neck, shoulders and upper arms, including carpal tunnel syndrome in the wrist. the muscle tension in static work is amplified by the uncontrolled muscular tension caused by psychological stress. furthermore, there seems to be wide inter-individual variation in the tendency to respond with spasm, particularly in the trapezius muscle of neck, under psychological stress. about 40% of the health complaints of working-aged people are related to musculoskeletal disorders, of which a substantial part is work-related. the epidemics have been resistant against preventive measures. new regulatory and management strategies may be needed for effective prevention and control measures (westgaard et al. 2006, paoli and merllié 2001) . the 21st century will be the era of the brain at work and consequently of psychological stress. between 35% and 50% of eu workers in certain occupations report psychological stress due to high time pressure at work (parent-thirion et al. 2007 ). the occurrence of work-related stress is most prevalent in occupations with tight deadlines, pressure from clients, or the high level of responsibility for productivity and quality given to the workers. undoubtedly, the threat of unemployment increases the perception of stress as well. as a consequence, for example, in finland some 40% of workers report symptoms of psychological overload and about 7% show clinical signs of burn out. these are not the problems of low-paid manual workers only, but also, for example, highly educated and well-paid computer super-experts have an elevated risk of burnout as a consequence of often self-committed workload (kalimo and toppinen 1997) . unconventional and ever longer working hours are causing similar problems. for example, one third of finns work over 45 hours a week, and of these 25% work over 50 hours, and 8% often work 65-80 hours per week. it is important to have flexibility in the work time schedules, but it is counterproductive if the biologically determined physiological time rhythms of the worker are seriously offended. over 40% have a sleep deficit of at least one hour each day, and 22% are tired and somnolent at work (härmä et al. 2000) . the toughening global competition, growing productivity demands and continuous changes of work, together with job insecurity, are associated with increased stress. up to 30-50% of workers in different countries and different sectors of the economy report high time pressure and tight deadlines. this prevents them from doing their job as well as they would like to, and causes psychological stress. psychological stress is particularly likely to occur if the high demands are associated with a low degree of self-regulation by the workers (houtman 2005) . stress, if continuous, has been found to be detrimental to physical health (cardiovascular diseases), mental health (psychological burnout), safety (accident risks), and musculoskeletal disorders (particularly hand-arm and shoulder-neck disorders). it also has a negative impact on productivity, sickness absenteeism, and the quality of products and services. the resulting economic losses due to sickness absenteeism, work disability and lower quality of products and services are substantial. the prevention of stress consists not only of actions targeted at the individual worker. there is also a need for measures directed at the work organization, moderation of the total workload, competence building and collaboration within the workplace (theorell 1997). the support from foremen and supervisors is of crucial importance in stress management programmes. another type of psychological burden is the stress arising from the threat of physical violence or aggressive behaviour from the part of clients. in finland some 4% of workers have been subjected to insults or the threat of physical violence, 13% have experienced sexual harassment, and 10% mental violence or bullying at work. the risk is substantially higher for female workers than for men. stress has been found to be associated with somatic health, cardiovascular diseases, mental disorders and depression. one of the new and partly re-emerging challenges of occupational health services is associated with the new trends in microbial hazards. there are several reasons for these developments, for instance, the generation of new microbial strains, structural changes in human habitations with high population densities, growing international travel, and changes possibly in our microbiological environment as a consequence of global warming. of the 10 to 100 million species in the world, about 3 million are microbes. the vast majority of them are not pathogenic to man, and we live in harmony and symbiosis with many of them. we also use bacteria in numerous ways to produce food, medicines, proteins, etc. the pathogenic bacteria have been well controlled in the 20th century; this control had an enormous positive impact on human health, including occupational health. but now the microbial world is challenging us in many ways. new or re-emerging biological hazards are possible due to the transformation of viruses, the increased resistance of some microbial strains (e.g. tuberculosis and some other bacterial agents) and the rapid spread of contaminants through extensive overseas travelling (smolinski et al. 2003) . the scenarios of health hazards from the use of genetically manipulated organisms have not been realized, but biotechnological products have brought along new risks of allergies. a major indoor air problem is caused by fungi, moulds and chemical emissions from contaminated construction materials. new allergies are encountered as a consequence of the increasingly allergic constitution of the population and of the introduction of new allergens into the work environment. health care personnel are increasingly exposed to new microbial hazards due to the growing mobility of people. evidence of high rates of hepatitis b antigen positivity has been shown among health care workers who are in contact with migrants from endemic areas. along with the growing international interactions and mobility, a number of viral and re-emerging bacterial infections also affect the health of people engaged in health care and the care of the elderly, as well as personnel in migrant and refugee services, in social services and other public services. this section applies the general framework for risk governance (chapter 3) to the area of environmental risks. why should we include this topic in a book that is dominantly dealing with occupational health risks and safety issues? there are two major reasons for this decision: 1. most risks that impact health and safety of human beings are also affecting the natural environment. it is therefore necessary for risk managers to reflect the consequences of risk-taking activities with respect to workers, the public and the environment. these risk consequences are all interconnected. our approach to foster an integral approach to risk and risk management requires the integration of all risk consequences. 2. environmental risks are characterized by many features and properties that highlight exemplary issues for many generic risk assessment and management questions and challenges. for example, the question of how to balance benefits and risks becomes more accentuated, if not human life, but damage to environmental quality is at stake. while most people agree that saving human lives takes priority over economic benefits, it remains an open question of how much environmental change and potential damage one is willing to trade off against certain economic benefits. this section is divided into two major parts. part 1 will introduce the essentials of environmental ethics and the application of ethical principles to judging the acceptability of human interventions into the environment. part 2 addresses the procedures for an analytic-deliberative process of decision making when using the risk governance framework developed in chapter 3. it should be noted that this section draws from material that the author has compiled for the german scientific council for global environmental change and that has been partially published in german in a special report of the council (wbgu 1999). the last section on decision making has borrowed material from an unpublished background document on decision making and risk management that dr. warner north and the author had prepared for the us national academy of sciences. should people be allowed to do everything that they are capable of doing? this question is posed in connection with new technologies, such as nanotubes, or with human interventions in nature, such as the clearance of primaeval forests so that the land can be used for agriculture. intuitively everyone answers this question with a definitive "no": no way should people be allowed to everything that they are capable of doing. this also applies to everyday actions. many options in daily life, from lying to minor deception, from breaking a promise up to going behind a friend's back, are obviously actions that are seen by all well-intentioned observers as unacceptable. however, it is much more difficult to assess those actions where the valuation is not so obvious. is it justified to break a promise when keeping the promise could harm many other people? actions where there are conflicts between positive and negative consequences or where a judgement could be made one way or the other with equally good justification are especially common in risk management. there is hardly anyone who wilfully and without reason pollutes the environment, releases toxic pollutants or damages the health of individuals. people who pursue their own selfish goals on the cost and risk of others are obviously acting wrongly and every legislator will sanction this behaviour with the threat of punishment or a penalty. but there is a need for clarification where people bring about a benefit to society with the best intentions and for plausible reasons and, in the process, risk negative impacts on others. in ethics we talk about "conflicting values" here. most decisions involving risks to oneself or others are made for some reason: the actors who make such interventions want to secure goods or services to consumers, for example, to ensure long-term jobs and adequate incomes, to use natural resources for products and services or to use nature for recycling waste materials from production and consumption that are no longer needed. none of this is done for reasons of brotherly love, but to maintain social interests. even improving one's own financial resource is not immoral mere for this reason. the list of human activities that pose risks onto others perpetrated for existential or economic reasons could be carried on into infinity. human existence is bound to taking opportunities and risks. here are just a few figures: around 12,000 years ago about 5 million people lived on the earth. under the production conditions those days (hunter-gatherer culture) this population level was the limit for the human species within the framework of an economic form that only interfered slightly with man's natural environment. the neolithic revolution brought a dramatic change: the carrying capacity of the world for human beings increased by a factor of 10 and more. this agrarian pre-industrial cultural form was characterized by tightly limited carrying capacity, in around 1750 the earth was capable of feeding approx. 750 million people. today the world supports 6 billion people -and this figure is rising. the carrying capacity in comparison to the neolithic age has thus increased thousand-fold and continues to grow in parallel to new changes in production conditions (fritsch 1993; kesselring 1994; mohr 1995) . the five "promethean innovations" are behind this tremendous achievement of human culture: mastering fire, using the natural environment for agriculture, transforming fossil fuels into thermal and mechanical energy, industrial production and substituting material with information (renn 1996) . with today's settlement densities and the predominantly industrial way of life, the human race is therefore dependent on the technical remodelling of nature. without doubt, it needs this for survival, especially for the well-being of the innumerable people, goods and services that reduce the stock of natural resources. with regard to the question of the responsibility of human interventions in nature, the question cannot be about "whether" but -even better -about "how much", because it is an anthropological necessity to adapt and shape existing nature to human needs. for example, the philosopher klaus michael meyer-abich sees the situation as follows: ". . . we humans are not there to leave the world as though we had never been there. as with all other life forms, it is also part of our nature and our lives to bring about changes in the world. of course, this does not legitimise the destructive ways of life that we have fallen into. but only when we basically approve of the changes in the world can we turn to the decisive question of which changes are appropriate for human existence and which are not" (meyer-abich 1997). therefore, to be able to make a sensible judgement of the balance between necessary interventions into the environment and the risks posed by these interventions to human health and environmental quality, the range of products and services created by the consumption of nature has to be considered in relation to the losses that are inflicted on the environment and nature. with this comparison, it can be seen that even serious interventions in nature and the environment did not occur without reflection, but to provide the growing number of people with goods and services; these people need them to survive or as a prerequisite for a "good" life. however, at the same time it must be kept in mind that these interventions often inflict irreversible damage on the environment and destroy possible future usage potentials for future generations. above and beyond this, for the human race, nature is a cradle of social, cultural, aesthetic and religious values, the infringement of which, in turn, has a major influence on people's well-being. on both sides of the equation, there are therefore important goods that have to be appreciated when interventions in nature occur. but what form should such an appreciation take? if the pros and cons of the intervention in nature have to be weighed against each other, criteria are needed that can be used as yardsticks. who can and may draw up such criteria, according to which standards should the interventions be assessed and how can the various evaluative options for action be compared with each other for each criterion? taking risks always involves two major components: an assessment of what we can expect from an intervention into the environment (be it the use of resources or the use of environments as a sink for our waste). this is the risk and benefit assessment side of the risk analysis. secondly, we need to decide whether the assessed consequences are desirable. whereas the estimate of consequences broadly falls in the domain of scientific research and expertise, with uncertainties and ambiguities in particular having to be taken into account (irgc 2005, klinke and renn 2002) , the question about the foundations for evaluating various options for action and about drawing up standards guiding action is a central function of ethics (taylor 1986) . ethics can provide an answer to the question posed in the beginning ("should people be allowed to do everything that they are capable of doing?") in a consistent and transparent manner. in section 4.3.2, environmental ethics will be briefly introduced. this review is inspired by the need for a pragmatic and policy-oriented approach. it is not a replacement for a comprehensive and theoretically driven compendium of environmental ethics. environmental ethics will then be applied to evaluate environmental assets. in this process, a simple distinction is made between categorical principles -that must under no circumstances be exceeded or violated -and compensatory principles, where compensation with other competing principles is allowed. this distinction consequently leads to a classification of environmental values, which, in turn, can be broken down into criteria to appreciate options for designing environmental policies. in section 4.3.3, these ideas of valuation will be taken up and used to translate the value categories into risk handling guidelines. at the heart of the considerations here is the issue of how the aims of ethically founded considerations can be used to support and implement risk-based balancing of costs and benefits. for this purpose, we will develop an integrative risk governance framework. the concept of risk governance comprises a broad picture of risk: not only does it include what has been termed "risk management" or "risk analysis", it also looks at how risk-related decision making unfolds when a range of actors is involved, requiring co-ordination and possibly reconciliation between a profusion of roles, perspectives, goals and activities. indeed, the problem-solving capacities of individual actors, be they government, the scientific community, business players, ngos or civil society as a whole, are limited and often unequal to the major challenges facing society today. then the ideas of the operational implementation of normative and factual valuations are continued and a procedure is described that is capable of integrating ethical, risk-based and work-related criteria into a proposed procedural orientation. this procedure is heavily inspired by decision analysis. answering the question about the right action is the field of practical philosophy, ethics. following the usual view in philosophy, ethics describes the theory of the justification of normative statements, i.e. those that guide action (gethmann 1991 , mittelstraß 1992 , nida-rümelin 1996a , revermann 1998 . a system of normative statements is called "morals". ethical judgements therefore refer to the justifiability of moral instructions for action that may vary from individual to individual and from culture to culture (ott 1999) . basically, humans are purpose-oriented and self-determined beings who act not only instinctively, but also with foresight, and are subject to the moral standards to carry out only those actions that they can classify as good and justifiable (honnefelder 1993) . obviously, not all people act according to the standards that they themselves see as necessary, but they are capable of doing so. in this context, it is possible for people to act morally because, on the one hand, they are capable of distinguishing between moral and immoral action and, on the other, are largely free to choose between different options for action. whether pursuing a particular instruction for action should be considered as moral or immoral is based on whether the action concerned can be felt and justified to be "reasonable" in a particular situation. standards that cross over situations and that demand universal applicability are referred to as principles here. conflicts may arise between competing standards (in a specific situation), as well as between competing principles, the solution of which, in turn, needs justification (szejnwald-brown et al. 1993) . providing yardsticks for such justification or examining moral systems with respect to their justifiability is one of the key tasks of practical ethics (gethmann 1998) . in ethics a distinction is made between descriptive (experienced morality) and prescriptive approaches, i.e. justifiable principles of individual and collective behaviour (frankena 1963 , hansen 1995 . all descriptive approaches are, generally speaking, a "stock-taking" of actually experienced standards. initially, it is irrelevant whether these standards are justified or not. they gain their normative force solely from the fact that they exist and instigate human action (normative force of actual action). most ethicists agree that no conclusions about general validity can be drawn from the actual existence of standards. this would be a naturalistic fallacy (akademie der wissenschaften 1992, ott 1999) . nevertheless, experienced morality can be an important indicator of different, equally justifiable moral systems, especially where guidance for cross-cultural behaviour is concerned. this means that the actual behaviour of many people with regard to their natural environment reveals which elements of this environment they value in particular and which they do not. however, in this case, too, the validity of the standards is not derived from their factuality, but merely used as a heurism in order to find an adequate (possibly culture-immanent) justification. but given the variety of cultures and beliefs, how can standards be justified inter-subjectively, i.e. in a way that is equally valid to all? is it not the case that science can only prove or disprove factual statements (and this only to a certain extent), but not normative statements? a brief discourse on the various approaches in ethics is needed to answer this question. first of all, ethics is concerned with two different target aspects: on the one hand, it is concerned with the question of the "success" of one's own "good life", i.e. with the standards and principles that enable a person to have a happy and fulfilled life. this is called eudemonistic ethics. on the other hand, it is concerned with the standards and principles of living together, i.e. with binding regulations that create the conditions for a happy life: the common good. this is called normative ethics (galert 1998 , ott 1999 . within normative ethics a distinction is made between deontological and teleological approaches when justifying normative statements (höffe 1987) . deontological approaches are principles and standards of behaviour that apply to the behaviour itself on the basis of an external valuation criterion. it is not the consequences of an action that are the yardstick of the valuation; rather, it is adhering to inherent yardsticks that can be used against the action itself. such external yardsticks of valuation are derived from religion, nature, intuition or common sense, depending on the basic philosophical direction. thus, protection of the biosphere can be seen as a divine order to protect creation (rock 1980 , schmitz 1985 , as an innate tendency for the emotional attachment of people to an environment with biodiversity (wilson 1984) , as a directly understandable source of inspiration and joy (ehrenfeld 1993) or as an educational means of practising responsibility and maintaining social stability (gowdy 1997) . by contrast, teleological approaches refer to the consequences of action. here, too, external standards of valuation are needed since the ethical quality of the consequences of action also have to be evaluated against a yardstick of some kind. with the most utilitarian approaches (a subset of the teleological approaches) this yardstick is defined as an increase in individual or social benefit. in other schools of ethics, intuition (can the consequence still be desirable?) or the aspect of reciprocity (the so-called "golden rule": "do as you would be done by") play a key role. in the approaches based on logical reasoning (especially in kant), the yardstick is derived from the logic of the ability to generalize or universalize. kant himself is in the tradition of deontological approaches ("good will is not good as a result of what it does or achieves, but just as a result of the intention"). according to kant, every principle that, if followed generally, makes it impossible for a happy life to be conducted is ethically impermissible. in this connection, it is not the desirability of the consequences that captures kant's mind, but the logical inconsistency that results from the fact that the conditions of the actions of individuals would be undermined if everyone were to act according to the same maxims (höffe 1992) . a number of contemporary ethicists have taken up kant's generalization formula, but do not judge the maxims according to their internal contradictions; rather, they judge them according to the desirability of the consequences to be feared from the generalization (jonas 1979 or zimmerli 1993 should be mentioned here). these approaches can be defined as a middle course between deontological and teleological forms of justification. in addition to deontological and teleological approaches, there is also the simple solution of consensual ethics, which, however, comprises more than just actually experienced morality. consensual ethics presupposes the explicit agreement of the people involved in an action. everything is allowed provided that all affected (for whatever reason) voluntarily agree. in sexual ethics at the moment a change from deontological ethics to a consensual moral code can be seen. the three forms of normative ethics are shown in figure 4 .13. the comparison of the basic justification paths for normative moral systems already clearly shows that professional ethicists cannot create any standards or designate any as clearly right, even if they play a role in people's actual lives. much rather it is the prime task of ethics to ensure on the basis of generally recognized principles (for example, human rights) that all associated standards and behaviour regulations do not contradict each other or a higher order principle. above and beyond this, ethics can identify possible solutions that may occur with a conflict between standards and principles of equal standing. ethics may also reveal interconnections of justification that have proved themselves as examination criteria for moral action in the course of their disciplinary history. finally, many ethicists see their task as providing methods and procedures primarily of an intellectual nature by means of which the compatibility or incompatibility of standards within the framework of one or more moral systems can be completed. unlike the law, the wealth of standards of ethics is not bound to codified rules that can be used as a basis for such compatibility examinations. every normative discussion therefore starts with the general issues that are needed in order to allow individuals a "good life" and, at the same time, to give validity to the principles required to regulate the community life built on common good. but how can generally binding and inter-subjectively valid criteria be made for the valuation of "the common good"? in modern pluralistic societies, it is increasingly difficult for individuals and groups of society to draw up or recognize collectively binding principles that are perceived by all equally as justifiable and as self-obliging (hartwich and wewer 1991, zilleßen 1993) . the variety of lifestyle options and subjectiernization. with increasing technical and organizational means of shaping the future, the range of behaviour options available to people also expands. with the increasing plurality of lifestyles, group-specific rationalities emerge that create their own worldviews and moral standards, which demand a binding nature and validity only within a social group or subculture. the fewer cross-society guiding principles or behaviour orientations are available, the more difficult is the process of agreement on collectively binding orientations for action. however, these are vital for the maintenance of economic cooperation, for the protection of the natural foundations of life and for the maintenance of cohesion in a society. no society can exist without the binding specification of minimum canons of principles and standards. but how can agreement be reached on such collectively binding principles and standards? what criteria can be used to judge standards? the answers to this question depend on whether the primary principles, in other words, the starting point of all moral systems, or secondary principles or standards, i.e. follow-on standards that can be derived from the primary principles, are subjected to an ethical examination. primary principles can be categorical or compensatory (capable of being compensated). categorical principles are those that must not be infringed under any circumstances, even if other prin-219 fication of meaning (individualization) are accompanying features of mod-ciples would be infringed as a result. the human right to the integrity of life could be named here as an example. compensatory principles are those where temporary or partial infringement is acceptable, provided that as a result the infringement of a principle of equal or higher ranking is avoided or can be avoided. in this way certain freedom rights can be restricted in times of emergency. in the literature on ethical rules, one can find more complex and sophisticated classifications of normative rules. for our purpose to provide a simple and pragmatic framework, the distinction in four categories (principles and standards; categorical and compensatory) may suffice. this distinction has been developed from a decision-analytical perspective. but how can primary principles be justified as equally valid for all people? although many philosophers have made proposals here, there is a broad consensus today that neither philosophy nor any other human facility is capable of stating binding metacriteria without any doubt and for all people, according to which such primary principles should be derived or examined (mittelstraß 1984) . a final justification of normative judgements cannot be achieved by logical means either, since all attempts of this kind automatically end either in a logical circle, in an unending regression (vicious cycle) or in a termination of the procedure and none of these alternatives is a satisfactory solution for final justification (albert 1991). the problem of not being able to derive finally valid principles definitively, however, seems to be less serious than would appear at first glance. because, regardless of whether the basic axioms of moral rules are taken from intuition, observations of nature, religion, tradition reasoning or common sense, they have broadly similar contents. thus, there is broad consensus that each human individual has a right to life, that human freedom is a high-value good and that social justice should be aimed at. but there are obviously many different opinions about what these principles mean in detail and how they should be implemented. in spite of this plurality, however, discerning and well-intentioned observers can usually quickly agree, whether one of the basic principles has clearly been infringed. it is more difficult to decide whether they have clearly been fulfilled or whether the behaviour to be judged should clearly be assigned to one or several principles. since there is no finally binding body in a secular society that can specify primary principles or standards ex cathedra, in this case consensus among equally defendable standards or principles can be used (or pragmatically under certain conditions also majority decisions). ethical considerations are still useful in this case as they allow the test of generalization and the enhancement of awareness raising capabilities. in particular, they help to reveal the implications of such primary principles and standards. provided that primary principles are not concerned (such as human rights), the ethical discussion largely consists of examining the compatibility of each of the available standards and options for action with the primary principles. in this connection, the main concerns are a lack of contradictions (consistency), logical consistency (deductive validity), coherence (agreement with other principles that have been recognized as correct) and other, broadly logical criteria (gethmann 1998) . as the result of such an examination it is entirely possible to reach completely different conclusions that all correspond to the laws of logic and thus justify new plurality. in order to reach binding statements or valuations here the evaluator can either conduct a discussion in his "mind" and let the arguments for various standards compete with each other (rather like a platonic dialogue) or conduct a real discussion with the people affected by the action. in both cases the main concern is to use the consensually agreed primary principles to derive secondary principles of general action and standards of specific action that should be preferred over alternatives that can be equally justified. a plurality of solutions should be expected especially because most of the concrete options for action comprise only a gradual fulfilment and infringement of primary principles and therefore also include conflicting values. for value conflicts at the same level of abstraction there are, by definition, no clear rules for solution. there are therefore frequently conflicts between conserving life through economic development and destroying life through environmental damage. since the principle of conserving life can be used for both options a conflict is unavoidable in this case. to solve the conflicts, ethical considerations, such as the avoidance of extremes, staggering priorities over time or the search for third solutions can help without, however, being able to convincingly solve this conflict in principle to the same degree for all (szejnwald-brown et al. 1993) . these considerations lead to some important conclusions for the matter of the application of ethical principles to the issue of human action with regard to the natural environment. first of all, it contradicts the way ethics sees itself to develop ethics of its own for different action contexts. just as there can be no different rules for the logic of deduction and induction in nomological science, depending on which object is concerned, it does not make any sense to postulate an independent set of ethics for the environment (galert 1998) . justifications for principles and moral systems have to satisfy universal validity (nida-rümelin 1996b). furthermore, it is not very helpful to call for a special moral system for the environment since this -like every other moral system -has to be traceable to primary principles. instead, it makes sense to specify the generally valid principles that are also relevant with regard to the issue of how to deal with the natural environment. at the same time standards should be specified that are appropriate to environmental goods and that reflect those principles that are valid beyond their application to the environment. as implied above, it does not make much sense to talk about an independent set of environmental ethics. much rather, general ethics should be transferred to issues relating to the use of the environment (hargrove 1989) . three areas are usually dealt with within the context of environmental ethics (galert 1998 ): • environmental protection, i.e. the avoidance or alleviation of direct or indirect, current or future damage and pollution resulting from anthropogenic emissions, waste or changes to the landscape, including land use, as well as the long-term securing of the natural foundations of life for people and other living creatures (birnbacher 1991a ). • animal protection, i.e. the search for reasonable and enforceable standards to avoid or reduce pain and suffering in sentient beings (krebs 1997 , vischer 1999 ). • nature conservation, i.e. the protection of nature against the transforming intervention of human use, especially all measures to conserve, care for, promote and recreate components of nature deemed to be valuable, including species of flora and fauna, biotic communities, landscapes and the foundations of life required there (birnbacher 1991a) . regardless which of these three areas are addressed we need to explore which primary principles be applied to them. when dealing with the environment, the traditional basic and human rights, as well as the civil rights that have been derived from them, should be just as much a foundation of the consideration as other areas of application in ethics. however, with regard to the primary principles there is a special transfer problem when addressing human interventions into nature and the environment: does the basic postulate of conservation of life apply only to human beings, to all other creatures or to all elements of nature, too? this question does not lead to a new primary principle, as one may suspect at first glance. much rather, it is concerned with the delineation of the universally recognized principle of the conservation of life that has already been specified in the basic rights canon. are only people included in this principle (this is the codified version valid in most legal constitutions today) or other living creatures, too? and if yes, which ones? should non-living elements be included as well? when answering this question, two at first sight contradictory positions can be derived: anthropocentrism and physiocentrism (taylor 1986 , ott 1993 , galert 1998 . the anthropocentric view places humans and their needs at the fore. nature's own original demands are alien to this view. interventions in nature are allowed if they are useful to human society. a duty to make provisions for the future and to conserve nature exists in the anthropocentric world only to the extent that natural systems are classed as valuable to people today and subsequent generations and that nature can be classed as a means and guarantor of human life and survival (norton 1987 , birnbacher 1991b . in the physiocentric concept, which forms an opposite pole to the anthropocentric view, the needs of human beings are not placed above those of nature. here, every living creature, whether humans, animals or plants, have intrinsic rights with regard to the chance to develop their own lives within the framework of a natural order. merit for protection is justified in the physiocentric view by an inner value that is unique to each living creature or the environment in general. nature has a value of its own that does not depend on the functions that it fulfils today or may fulfil later from a human society's point of view (devall and sessions 1984 , callicott 1989 , rolston 1994 , meyer-abich 1996 . each of these prevailing understandings of the human-nature relationship has implications that are decisive for the form and extent of nature use by humans (elliot 1995 , krebs 1997 . strictly speaking, it could be concluded from the physiocentric idea that all human interventions in nature have to be stopped so that the rights of other creatures are not endangered. yet, not even extreme representatives of a physiocentric view would go so far as to reject all human interventions in nature because animals, too, change the environment by their ways of life (e.g. the elephant prevents the greening of the savannah). the central postulate of a physiocentric view is the gradual minimization of the depth of interventions in human use of nature. the only interventions that are permitted are those that contribute to directly securing human existence and do not change the fundamental composition of the surrounding natural environment. if these two criteria were taken to the extreme, neither population development beyond the boundaries of biological carrying capacity nor a transformation of natural land into pure agricultural land would be allowed. such a strict interpretation of physiocentrism would lead to a radical reversal of human history so far and is not compatible with the values and expectations of most people. the same is true for the unlimited transfer of anthropocentrism to dealings with nature. in this view, the use of natural services is subjected solely to the individual cost-benefit calculation. this can lead to unscrupulous exploitation of nature by humans with the aim of expanding human civilization. both extremes quickly lead to counter-intuitive implications. when the issue of environmental design and policy is concerned, anthropocentric and physiocentric approaches in their pure form are found only rarely, much rather they occur in different mixtures and slants. the transitions between the concepts are fluid. moderate approaches certainly take on elements from the opposite position. it can thus be in line with a fundamentally physiocentric perspective if the priority of human interests is not questioned in the use of natural resources. it is also true that the conclusions of a moderate form of anthropocentrism can approach the implications of the physiocentric view. table 4 .10 provides an overview of various types of anthropocentric and physiocentric perspectives. if we look at the behaviour patterns of people in different cultures, physiocentric or anthropocentric basic positions are rarely maintained consistently (bargatzky and kuschel 1994 ; on the convergence theory: birnbacher 1996) . in the strongly anthropocentric countries in the west, people spend more money on the welfare and health of their own pets than on saving human lives in other countries. in the countries of the far east that are characterized by physiocentrism, nature is frequently exploited even more radically than in the industrialized countries of the west. this inconsistent action is not a justification for one view or the other, it is just a warning for caution when laying down further rules for use so that no extreme -and thus untenable -demands be made. also from an ethical point of view, radical anthropocentrism should be rejected just as much as radical physiocentrism. if, to take up just one argument, the right to human integrity is largely justified by the fact that causing pain by others should be seen as something to avoid, this consideration without a doubt has to be applied to other creatures that are also capable of feeling pain (referred to as: pathocentrism). here, therefore, pure anthropocentrism cannot convince. in turn, with a purely physiocentric approach the primary principles of freedom, equality and human dignity could not be maintained at all if every part of living nature were equally entitled to use the natural environment. under these circumstances people would have to do without agriculture, the conversion of natural land into agricultural land and breeding farm animals and pets in line with human needs. as soon table 4 .10 different perspectives on nature. adapted from renn and goble (1996: 4). as physiocentrism is related to species and not to individuals as is done in some biocentric perspectives human priority is automatically implied; because where human beings are concerned, nearly all schools of ethics share the fundamental moral principle of an individual right to life from birth. if this right is not granted to individual animals or plants, a superiority of the human race is implicitly assumed. moderate versions of physiocentrism acknowledge a gradual de-escalation with respect to the claim of individual table 4 .10 different perspectives on nature (continued). adapted from renn and goble (1996: 4). life protection. the extreme forms of both physiocentrism and anthropocentrism are therefore not very convincing and are hardly capable of achieving a global consensus. this means that only moderate anthropocentrism or moderate biocentrism should be considered. the image of nature that is used as a basis for the considerations in this section emphasizes the uniqueness of human beings vis-à-vis physiocentric views, but does not imply carte blanche for wasteful and careless dealings with nature. this moderate concept derives society's duty to conserve nature -also for future generations -from the life-preserving and life-enhancing meaning of nature for society. this is not just concerned with the instrumental value of nature as a "store of resources", it is also a matter of the function of nature as a provider of inspiration, spiritual experience, beauty and peace (birnbacher and schicha 1996) . in this context it is important that human beings -as the addressees of the moral standard -do not regard nature merely as material and as a way towards their own self-realization, but can also assume responsibility for conservation of their cultural and so-cial function, as well as their existential value above and beyond the objective and technically available benefits (honnefelder 1993) . one of the first people to express this responsibility of human stewardship of nature in an almost poetic way was the american ecologist aldo leopold, who pointed out people's special responsibility for the existence of nature and land as early as the 1930s with the essay "the conservation ethics". his most well-known work "a sand county almanac" is sustained by the attempt to observe and assess human activities from the viewpoint of the land (a mountain or an animal). this perspective was clearly physiocentric and revealed fundamental insights about the relationship between humans and nature on the basis of empathy and shifting perspectives. his point of view had a strong influence on american environmental ethics and the stance of conservationists. although this physiocentric perspective raises many concerns, the idea of stewardship has been one of the guiding ideas for the arguments used in this section (pickett et al. 1997) . we are morally required to exercise a sort of stewardship over living nature, because nature cannot claim any rights for itself, but nevertheless has exceptional value that is important to man above and beyond its economic utility value (hösle 1991) . since contemporary society and the generations to come certainly use, or will use, more natural resources than would be compatible with a lifestyle in harmony with the given natural conditions, the conversion of natural land into anthropogenically determined agricultural land cannot be avoided (mohr 1995) . many people criticized human interventions into natural cycles as infringements of the applicable moral standards of nature conservation (for example, fastened onto the postulate of sustainability). but we should avoid premature conclusions here, as can be seen with the example of species protection. for example, where natural objects or phenomena are concerned that turn out to be a risk to human or non-human living creatures, the general call for nature conservation is already thrown into doubt (gale and cordray 1994) . not many people would call the eradication of cholera bacteria, hiv viruses and other pathogens morally bad (mittelstraß 1995) if remaining samples were kept under lock and key in laboratories. also, combating highly evolved creatures, such as cockroaches or rats meets with broad support if we ignore the call for the complete eradication of these species for the time being. an environmental initiative to save cockroaches would not be likely to gain supporters. if we look at the situation carefully, the valuation of human behaviour in these examples results from a conflict. because the conservation of the species competes with the objective of maintaining human health or the objective of a hygienic place to live, two principles, possibly of equal ranking, come face to face. in this case the options for action, which may all involve a gradual infringement of one or more principles, would have to be weighed up against each other. a general ban on eradicating a species can thus not be justified ethically, in the sense of a categorical principle, unless the maintenance of human health were to be given lower priority than the conservation of a species. with regard to the issue of species conservation, therefore, different goods have to be weighed up against each other. nature itself cannot show society what it is essential to conserve and how much nature can be traded for valuable commodities. humans alone are responsible for a decision and the resulting conflicts between competing objectives. appreciation and negotiation processes are therefore the core of the considerations about the ethical justification of rules for interventions. but this does not mean that there is no room for categorical judgements along the lines of "this or that absolutely must be prohibited" in the matter of human interventions into the natural environment. it follows on from the basic principle of conserving human life that all human interventions that threaten the ability of the human race as a whole, or a significant number of individuals alive today or in the future, to exist should be categorically prohibited. this refers to intervention threats to the systemic functions of the biosphere. such threats are one of the guiding principles that must not be exceeded under any circumstances, even if this excess were to be associated with high benefits. in the language of ethics this is a categorical principle, in the language of economics a good that is not capable of being traded. the "club" of categorical prohibitions should, however, be used very sparingly because plausible trade-offs can be thought up for most principles, the partial exceeding of which appears intuitively. in the case of threats to existence, however, the categorical rejection of the behaviour that leads to this is obvious. but what does the adoption of categorical principles specifically mean for the political moulding of environmental protection? in the past, a number of authors have tried to specify the minimum requirements for an ethically responsible moral system with respect to biosphere use. these so-called "safe minimum standards" specify thresholds for the open-ended measurement scale of the consequences of human interventions that may not be ex-ceeded even if there is a prospect of great benefits (randall 1988, randall and farmer 1995) . in order to be able to specify these thresholds in more detail the breakdown into three levels proposed by the german scientific council for global environmental change is helpful (wbgu 2000) . these levels are: • the global bio-geochemical cycles in which the biosphere is involved as one of the causes, modulator or "beneficiary"; • the diversity of ecosystems and landscapes that have key functions as bearers of diversity in the biosphere; and • the genetic diversity and the species diversity that are both "the modelling clay of evolution" and basic elements of ecosystem functions and dynamics. where the first level is concerned, in which the functioning of the global ecosystem is at stake, categorical principles are obviously necessary and sensible, provided that no one wants to shake the primary principle of the permanent preservation of the human race. accordingly, all interventions in which important substance or energy cycles are significantly influenced at a global level and where globally effective negative impacts are to be expected are categorically prohibited. usually no stringently causal evidence of the harmful nature of globally relevant information is needed; justified suspicion of such harmfulness should suffice. later in this chapter we will make a proposal for risk valuation and management how the problem of uncertainty in the event of possible catastrophic damage potential should be dealt with (risk type cassandra). on the second level, the protection of ecosystems and landscapes, it is much more difficult to draw up categorical rules. initially, it is obvious that all interventions in landscapes in which the global functions mentioned on the first level are endangered must be avoided. above and beyond this, it is wise from a precautionary point of view to maintain as much ecosystem diversity as possible in order to keep the degree of vulnerability to the unforeseen or even unforeseeable consequences of anthropogenic and nonanthropogenic interventions as low as possible. even though it is difficult to derive findings for human behaviour from observations of evolution, the empirically proven statement "he who places everything on one card, always loses in the long run" seems to demonstrate a universally valid insight into the functioning of systemically organized interactions. for this reason, the conservation of the natural diversity of ecosystems and landscape forms is a categorical principle, whereas the depth of intervention allowed should be specified on the basis of principles and standards capable of compensation. the same can be said for the third level, genetic and species protection. here too, initially the causal chain should be laid down: species conservation, landscape conservation, maintaining global functions. wherever this chain is unbroken, a categorical order of conservation should apply. these species could be termed primary key species. this includes such species that are not only essential for the specific landscape type in which they occur, but also for the global cycles above and beyond this specific landscape type thanks to their special position in the ecosystem. probably, it will not be possible to organize all species under this functional contribution to the surrounding ecosystem, but we could also think of groups of species, for example, humus-forming bacteria. in second place there are the species that characterize certain ecosystems or landscapes. here they are referred to as secondary key species. they, too, are under special protection that is not necessarily under categorical reservations. their function value, however, is worthy of special attention. below these two types of species there are the remaining species that perform ecosystem functions to a greater or lesser extent. what this means for the worthiness for protection of these species and the point at which the precise limit for permitted intervention should be drawn, is a question that can no longer be solved with categorical principles and standards, but with the help of compensatory principles and standards. generally, here, too, as with the issue of ecosystem and landscape protection, the conservation of diversity as a strategy of "reinsurance" against ignorance, global risks and unforeseeable surprises is recommended. it remains to be said that from a systemic point of view, a categorical ban has to apply to all human interventions where global closed loops are demonstrably at risk. above and beyond this, it makes sense to recognize the conservation of landscape variety (also of ecosystem diversity within landscapes) and of genetic variety and species diversity as basic principles, without being able to make categorical judgements about individual landscape or species types as a result. in order to evaluate partial infringements of compensatory principles or standards, which are referred to in the issue of environmental protection, we need rules for decision making that facilitate the balancing process necessary to resolve compensatory conflicts. in the current debate about rules for using the environment and nature, it is mainly teleological valuation methods that are proposed (hubig 1993 , ott 1993 . these methods are aimed at: • estimating the possible consequences of various options for action at all dimensions relevant to potentially affected people; • recording the infringements or fulfilments of these expected consequences in the light of the guiding standards and principles; and • then weighing them according to an internal key so that they can be weighed up in a balanced way. on the positive side of the equation, there are the economic benefits of an intervention and the cultural values created by use, for example, in the form of income, subsistence (self-sufficiency) or an aesthetically attractive landscape (parks, ornamental gardens, etc.); on the negative side, there are the destruction of current or future usage potentials, the loss of unknown natural resources that may be needed in the future and the violation of aesthetic, cultural or religious attributes associated with the environment and nature. there are therefore related categories on both sides of the equation: current uses vs. possible uses in the future, development potentials of current uses vs. option values for future use, shaping the environment by use vs. impairments to the environment as a result of alternative use, etc. with the same or similar categories on the credit and debit side of the balance sheet the decision is easy when there is one option that performs better or worse than all the other options for all categories. although such a dominant (the best for all categories) or sub-dominant option (the worst for all categories) is rare in reality, there are examples of dominant or sub-dominant solutions. thus, for example, the overfelling of the forests of kalimantan on the island of borneo in indonesia can be classed as a sub-dominant option since the short-term benefit, even with extremely high discount rates, is in no proportion to the long-term losses of benefits associated with a barren area covered in imperata grass. the recultivation of a barren area of this kind requires sums many times the income from the sale of the wood, including interest. apparently there are no cultural, aesthetic or religious reasons for conversion of primary or secondary woodland into grassland. this means that the option of deforestation should be classed as of less value than alternative options for all criteria, including economic and social criteria. at best, we can talk about a habit of leaving rainforests, as a "biotope not worthy of conservation", to short-term use. but habit is not a sound reason for the choice of any sub-optimum option. as mentioned at the start of this chapter, habit as experienced morality, does not have any normative force, especially when this is based on the illusion of the marginality of one's own behaviour or ignorance about sustainable usage forms. but if we disregard the dominant or sub-dominant solutions, an appreciation between options that violate or fulfil compensatory standards and principles depends on two preconditions: best possible knowledge of the consequences (what happens if i choose option a instead of option b?) and a transparent, consistent rationale for weighing up these consequences as part of a legitimate political decision process (are the foreseeable consequences of option a more desirable or bearable than the consequences of option b?) (akademie der wissenschaften 1992). adequate knowledge of the consequences is needed in order to reveal the systemic connections between resource use, ecosystem reactions to human interventions and socio-cultural condition factors (wolters 1995) . this requires interdisciplinary research and cooperation. the task of applied ecological research, for example, is to show the consequences of human intervention in the natural environment and how ecosystems are burdened by different interventions and practices. the economic approach provides a benefit-oriented valuation of natural and artificial resources within the context of production and consumption, as well as a valuation of transformation processes according to the criterion of efficiency. cultural and social sciences examine the feedback effects between use, social development and cultural self-perception. they illustrate the dynamic interactions between usage forms, socio-cultural lifestyles and control forms. interdisciplinary, problem-oriented and system-related research contribute to forming a basic stock of findings and insights about functional links in the relationship between human interventions and the environment and also in developing constructive proposals as to how the basic question of an ethically justified use of the natural environment can be answered in agreement with the actors concerned (wbgu 2000) . accordingly, in order to ensure sufficient environmental protection, scientific research, but especially transdisciplinary system research at the interface between natural sciences and social sciences is essential. bringing together the results of interdisciplinary research, the policy-relevant choice of knowledge banks and balanced interpretation in an environment of uncertainty and ambivalence are difficult tasks that primarily have to be performed by the science system itself. how this can happen in a way that is methodslogically sound, receptive to all reasonable aspects of interpretation and yet subjectively valid will be the subject of section 4.3.3. but knowledge alone does not suffice. in order to be able to act effectively and efficiently while observing ethical principles, it is necessary to shape the appreciation process between the various options for action according to rational criteria (gethmann 1998) . to do this it is, first of all, necessary to identify the dimensions that should be used for a valuation. the discussion about the value dimensions to be used as a basis for valuation is one of the most popular subjects within environmental ethics. to apply these criteria in risk evaluation and to combine the knowledge aspects about expected consequences of different behavioural options with the ethical principles is the task of what we have called risk governance. what contribution do ethics make towards clarifying the prospects and limits of human interventions into the natural environment? the use of environmental resources is an anthropological necessity. human consciousness works reflexively and humans have developed a causal recognition capacity that enables them to record cause and effect anticipatively and to productively incorporate assessed consequences in their own action. this knowledge is the motivating force behind the cultural evolution and the development of technologies, agriculture and urbanization. with power over an ever-increasing potential of design and intervention in nature and social affairs over the course of human history, the potential for abuse and exploitation has also grown. whereas this potential was reflected in philosophical considerations and legal standards at a very early stage with regard to moral standards between people, the issue of human responsibility towards nature and the environment has only become the subject of intensive considerations in recent times. ethical considerations are paramount in this respect. on the one hand, they offer concrete standards for human conduct on the bases of criteria that can be generalized, and, on the other hand, they provide procedural advice about a rational and decision-and policy-making process. a simple breakdown into categorical rules and prohibitions that are capable of being compensated can assist decision makers for the justification of principles and standards on environmental protection. as soon as human activities exceed the guidelines of the categorical principles, there is an urgent need for action. how can we detect whether such an excess has happened and how it can be prevented from the very outset that these inviolable standards and principles be exceeded? here are three strategies of environmental protection to be helpful for the implementation of categor-ical guidelines. the first strategy is that of complete protection with severe restrictions of all use by humans (protection priority). the second strategy provides for a balanced relationship between protection and use, where extensive resource use should go hand in hand with the conservation of the ecosystems concerned (equal weight). the third strategy is based on optimum use involving assurance of continuous reproduction. the guiding principle here would be an intensive and, at the same time, sustainable, i.e. with a view to the long term, use of natural resources (use priority). the following section will present a framework for applying these principles into environmental decision making under risk. the main line of argument is that risk management requires an analytic-deliberative approach for dealing effectively and prudently with environmental risks. assessing potential consequences of human interventions and evaluating their desirability on the basis of subsequent knowledge and transparent valuation criteria are two of the central tasks of a risk governance process. however, the plural values of a heterogeneous public and people's preferences have to be incorporated in this process. but how can this be done given the wealth of competing values and preferences? should we simply accept the results of opinion polls as the basis for making political decisions? can we rely on risk perception results to judge the seriousness of pending risks? or should we place all our faith in professional risk management? if we turn to professional help to deal with plural value input, economic theory might provide us an answer to this problem. if environmental goods are made individual and suitable for the market by means of property rights, the price that forms on the market ensures an appropriate valuation of the environmental good. every user of this good can then weigh up whether he is willing to pay the price or would rather not use the good. with many environmental goods, however, this valuation has to be made by collective action, because the environmental good concerned is a collective or open access good. in this case a process is needed that safeguards the valuation and justifies it to the collective. however, this valuation cannot be determined with the help of survey results. although surveys are needed to be able to estimate 234 the breadth of preferences and people's willingness to pay, they are insufficient for a derivation of concrete decision-making criteria and yardsticks for evaluating the tolerability of risks to human health and the environment. • firstly, the individual values are so widely scattered that there is little sense in finding an average value here. • secondly, the preferences expressed in surveys change much within a short time, whereas ethical valuations have to be valid for a long time. • thirdly, as outlined in the subsection on risk perception, preferences are frequently based on flawed knowledge or ad hoc assumptions both of which should not be decisive according to rational considerations. what is needed, therefore, is a gradual process of assigning trade-offs in which existing empirical values are put into a coherent and logically consistent form. in political science and sociological literature reference is mostly made to three strategies of incorporating social values and preferences in rational decision-making processes (renn 1997) . firstly, a reference to social preferences is viewed solely as a question of legitimate procedure (luhmann 1983 , vollmer 1996 . the decision is made on the basis of formal decision-making process (such as majority voting). if all the rules have been kept, a decision is binding, regardless of whether the subject matter of the decision can be justified or whether the people affected by the decision can understand the justification. in this version, social consensus has to be found only about the structure of the procedures; the only people who are then involved in the decisions are those who are explicitly legitimated to do so within the framework of the procedure decided upon. the second strategy is to rely on the minimum consensuses that have developed in the political opinion-forming process (muddling through) (lindbloom 1959 (lindbloom , 1965 . in this process, only those decisions that cause the least resistance in society are considered to be legitimate. in this version of social pluralism groups in society have an influence on the process of the formation of will and decision making to the extent that they provide proposals capable of being absorbed, i.e. adapted to the processing style of the political system, and that they mobilize public pressure. the proposal that then establishes itself in politics is the one that stands up best in the competition of proposals, i.e. the one that entails the fewest losses of support for political decision makers by interest groups. the third strategy is based on the discussion between the groups involved (habermas 1971 , renn 2004 ). in the communicative exchange among the people involved in the discussion a form of communicative rationality that everyone can understand evolves that can serve as a justification for collectively binding decisions. at the same time, discursive methods claim to more appropriately reflect the holistic nature of human beings and also to provide fair access to designing and selecting solutions to problems. in principle, the justification of standards relevant to decisions is linked to two conditions: the agreement of all involved and substantial justification of the statements made in the discussion (habermas 1981) . all three strategies of political control are represented in modern societies to a different extent. legitimation conflicts mostly arise when the three versions are realized in their pure form. merely formally adhering to decisionmaking procedures without a justification of content encounters a lack of understanding and rejection among the groups affected especially when they have to endure negative side effects or risks. then acceptance is refused. if, however, we pursue the opposite path of least resistance and base ourselves on the route of muddling through we may be certain of the support of the influential groups, but, as in the first case, the disadvantaged groups will gradually withdraw their acceptance because of insufficient justification of the decision. at the same time, antipathy to politics without a line or guidance is growing, even the affected population. the consequence is political apathy. the third strategy of discursive control faces problems, too. although in an ideal situation it is suitable for providing transparent justifications for the decision-making methods and the decision itself, in real cases the conditions of ideal discourse can rarely be adhered to (wellmer 1992) . frequently, discussions among strategically operating players lead to a paralysis of practical politics by forcing endless marathon meetings with vast quantities of points of order and peripheral contributions to the discussion. the "dictatorship of patience" (weinrich 1972) ultimately determines which justifications are accepted by the participants. the public becomes uncertain and disappointed by such discussions that begin with major claims and end with trivial findings. in brief: none of the three ways out of the control dilemma can convince on its own; as so often in politics, everything depends on the right mixture. what should a mixture of the three elements (due process, pluralistic muddling through and discourse) look like so that a maximum degree of rationality can come about on the basis of social value priorities? a report by the american academy of sciences on the subject of "understanding environmental risks" (national research council 1996) comes to the conclusion that scientifically valid and ethically justified procedure for the collective valuation of options for risk handling can only be realized within the context of -what the authors coin -an analytic-deliberative process. analytic means that the best scientific findings about the possible consequences and conditions of collective action are incorporated in the negotiations; deliberative means that rationally and ethically transparent criteria for making trade-offs are used and documented externally. moreover, the authors consider that fair participation by all groups concerned is necessary to ensure that the different moral systems that can legitimately exist alongside each other should also be incorporated in the process. to illustrate the concept of analytic-deliberative decision making consider a set of alternative options or choices, from which follow consequences (see basic overview in dodgson et al. 2000) . the relationship between the choice made, and the consequences that follow from this choice, may be straightforward or complex. the science supporting environmental policy is often complicated, across many disciplines of science and engineering, and also involving human institutions and economic interactions. because of limitations in scientific understanding and predictive capabilities, the consequences following a choice are normally uncertain. finally, different individuals and groups within society may not agree on how to evaluate the consequences -which may involve a detailed characterization of what happens in ecological, economic, and human health terms. we shall describe consequences as ambiguous when there is this difficulty in getting agreement on how to interpret and evaluate them. this distinction has been further explained in chapter 3 (see also klinke and renn 2002) . environmental assessment and environmental decision making inherently involve these difficulties of complexity, uncertainty, and ambiguity (klinke and renn 2002) . in some situations where there is lots of experience, these difficulties may be minimal. but in other situations these difficulties may constitute major impediments to the decision-making process. to understand how analysis and deliberation interact in an iterative process following the national research council (nrc) 1996 report, one must consider how these three areas of potential difficulty can be addressed. it is useful to separate questions of evidence with respect to the likelihood, magnitude of consequences and related characteristics (which can involve complexity and uncertainty) from valuation of the consequences (i.e. ambiguity). for each of the three areas there are analytical tools that can be helpful in identifying, characterizing and quantifying cause-effect relationships. some of these tools have been described in chapter 3. the integration of these tools of risk governance into a consistent procedure will be discussed in the next subsections. the possibility to reach closure on evaluating risks to human health or the environment rests on two conditions: first, all participants need to achieve closure on the underlying goal (often legally prescribed, such as prevention of health detriments or guarantee of an undisturbed environmental quality, for example, purity laws for drinking water); secondly, they need to agree with the implications derived from the present state of knowledge (whether and to what degree the identified hazard impacts the desired goal). dissent can result from conflicting values as well as conflicting evidence. it is crucial in environmental risk management to investigate both sides of the coin: the values that govern the selection of the goal and the evidence that governs the selection of cause-effect claims. strong differences in both areas can be expected in most environmental decision-making contexts but also in occupational health and safety and public health risks. so for all risk areas it is necessary to explore why people disagree about what to do -that is, which decision alternative should be selected. as pointed out before, differences of opinion may be focused on the evidence of what is at stake or which option has what kind of consequences. for example: what is the evidence that an environmental management initiative will lead to an improvement, such as reducing losses of agricultural crops to insect pests -and what is the evidence that the management initiative could lead to ecological damage -loss of insects we value, such as bees or butterflies, damage to birds and other predators that feed on insects -and health impacts from the level of pesticides and important nutrients in the food crops we eat? other differences of opinion may be about values -value of food crops that contain less pesticide residue compared to those that contain more, value of having more bees or butterflies, value of maintaining indigenous species of bees or butterflies compared to other varieties not native to the local ecosystem, value ascribed to good health and nutrition, and maybe, value ascribed to having food in what is perceived to be a "natural" state as opposed to containing manufactured chemical pesticides or altered genetic material. separating the science issues of what will happen from the value issues of how to make appropriate trade-offs between ecological, economic, and human health goals can become very difficult. the separation of facts and values in decision making is difficult to accomplish in practical decision situations, since what is regarded as facts includes a preference dependent process of cognitive framing (tversky and kahneman 1981) and what is regarded as value includes a prior knowledge about the factual implica-tions of different value preferences (fischhoff 1975) . furthermore, there are serious objections against a clear-cut division from a sociological view on science and knowledge generation (jasanoff 1996) . particularly when calculating risk estimates, value-based conventions may enter the assessment process. for example, conservative assumptions may be built into the assessment process, so that some adverse effects (such as human cancer from pesticide exposure) are much less likely to be underestimated than overestimated (national research council 1983) . at the same time, ignoring major sources of uncertainty can evoke a sense of security and overconfidence that is not justified from the quality or extent of the data base (einhorn and hogarth 1978) . perceptions and world views may be very important, and difficult to sort out from matters of science, especially with large uncertainties about the causes of environmental damage. a combination of analytic and deliberative processes can help explore these differences of opinions relating to complexity, uncertainty, and ambiguity in order to examine the appropriate basis for a decision before the decision is made. most environmental agencies go through an environmental assessment process and provide opportunities for public review and comment. many controversial environmental decisions become the focus of large analytical efforts, in which mathematical models are used to predict the environmental, economic, and health consequences of environmental management alternatives. analysis should be seen as an indispensable complement to deliberative processes, regardless whether this analysis is sophisticated or not. even simple questions need analytic input for making prudent decisions, especially in situations where there is controversy arising from complexity, uncertainty, and ambiguity. in many policy arenas in which problems of structuring human decisions are relevant, the tools of normative decision analysis (da) have been applied. especially in economics, sociology, philosophical ethics, and also many branches of engineering and science, these methods have been extended and refined during the past several decades. (edwards 1954 , howard 1966 , north 1968 , howard et al. 1972 , north and merkhofer 1976 , behn and vaupel 1982 , pinkau and renn 1998 , van asselt 2000 , jaeger et al. 2001 . da is a process for decomposing a decision problem into pieces, starting with the simple structure of alternatives, information, and prefer-ences. it provides a formal framework for quantitative evaluation of alternative choices in terms of what is known about the consequences and how the consequences are valued (hammond et al. 1999 , skinner 1999 . the procedures and analytical tools of da provide a number of possibilities to improve the precision and transparency of the decision procedure. however, they are subject to a number of limitations. the opportunities refer to: • different action alternatives can be quantitatively evaluated to allow selection of a best choice. such evaluation relies both on a description of uncertain consequences for each action alternative, with uncertainty in the consequences described using probabilities, and a description of the values and preferences assigned to consequences. (explicit characterization of uncertainty and values of consequences) • the opportunity to assure transparency, in that (1) models and data summarizing complexity (e.g., applicable and available scientific evidence) (2) probabilities characterizing judgement about uncertainty, and (3) values (utilities) on the consequences are made explicit and available. so the evaluation of risk handling alternatives can be viewed and checked for accuracy by outside observers. (outside audit enabled of basis for decision) • a complex decision situation can be decomposed into smaller pieces in a formal analytical framework. the level of such composition can range from a decision tree of action alternatives and ensuing consequences that fits on a single piece of paper, to extremely large and complex computerimplemented models used in calculating environmental consequences and ascribing probabilities and values of the consequences. a more complex analysis is more expensive and is less transparent to observers. in principle, with sufficient effort any formal analytical framework can be checked to assure that calculations are made in the way that is intended. (decomposition possible to include extensive detail) on the other hand, there are important limitations: • placing value judgements (utilities) on consequences may be difficult, especially in a political context where loss of life, impairment of health, ecological damage, or similar social consequences are involved. utility theory is essentially an extension of cost-benefit methods from economics to include attitude toward risk. the basic trade-off judgements needed for cost-benefit analysis remain difficult and controversial, and often, inherently subjective. (difficulties in valuing consequences) • assessing uncertainty in the form of a numerical probability also poses difficulties, especially in situations when there is not a statistical data base on an agreed-on model as the basis for the assessment. (difficulty in quantifying uncertainty, assigning probabilities) • the analytical framework may not be complete. holistic or overarching considerations or important details may have been omitted. (analytical framework incomplete) • da is built upon an axiomatic structure, both for dealing with uncertainty (i.e., the axiomatic foundation of probability theory), and for valuing consequences (i.e., the axiomatic basis for von neumann-morgenstern utility theory). especially when the decision is to be made by a group rather than an individual decision maker, rational preferences for the group consistent with the axioms may not exist (the "impossibility" theorem of arrow, 1951) . so in cases of strong disagreements on objectives or unwillingness to use a rational process, decision analysis methods may not be helpful. decision analytical methods should not be regarded as inherently "mechanical" or "algorithmic", in which analysts obtain a set of "inputs" about uncertainty and valuing consequences, then feed these into a mathematical procedure (possibly implemented in a computer) that produces an "output" of the "best" decision. da can only offer coherent conclusions from the information which the decision maker provides by his/her preferences among consequences and his/her state of information on the occurrence of these consequences. where there is disagreement about the preferences or about the information, da may be used to implore the implications of such disagreement. so in application, there is often a great deal of iteration (sensitivity analysis) to explore how differences in judgement should affect the selection of the best action alternative. da thus merely offers a formal framework that can be effective in helping participants in a decision process to better understand the implications of differing information and judgement about complex and uncertain consequences from the choice among the available action alternatives. insight about which factors are most important in selecting among the alternatives is often the most important output of the process, and it is obtained through extensive and iterative exchange between analysts and the decision makers and stakeholders. the main advantage of the framework is that it is based on logic that is both explicit and checkable -usually facilitated by the use of mathematical models and probability calculations. research on human judgement supports the superiority of such procedures for decomposing complex decision problems and using logic to integrate the pieces, rather than relying on holistic judgement on which of the alternatives is best (this is not only true for individual decisions, see heap et al. 1992 : 36ff., jungermann 1986 ; but also for collective decisions, see heap et al. 1992 : 197ff., pettit 1991 . one should keep in mind, however, that "superior" is measured in accordance with indicator of instrumental rationality, i.e. measuring means-ends effectiveness. if this rationality is appropriate, the sequence suggested by da is intrinsically plausible and obvious. even at the level of qualitative discussion and debate, groups often explore the rationale for different action alternatives. decision analysis simply uses formal quantitative methods for this traditional and common-sense process of exploring the rationale -using models to describe complexity, probability to describe uncertainty, and to deal with ambiguity, explicit valuation of consequences via utility theory and other balancing procedures, such as cost-benefit or cost-effectiveness analyses. by decomposing the problem in logical steps, the analysis permits better understanding of differences in the participants' perspective on evidence and values. da offers methods to overcome these differences, such as resolving questions about underlying science through data collection and research, and encouraging tradeoffs, compromise, and rethinking of values. based on this review of opportunities and shortcomings we conclude that decision analysis provides a suitable structure for guiding discussion and problem formulation, and offers a set of quantitative analytical tools that can be useful for environmental decisions, especially in conjunction with deliberative processes. da can assist decision makers and others involved in, and potentially affected by, the decision (i.e., participants, stakeholders) to deal with complexity and many components of uncertainty, and to address issues of remaining uncertainties and ambiguities. using these methods promises consistency from one decision situation to another, assurance of an appropriate use of evidence from scientific studies related to the environment, and explicit accountability and transparency with respect to those institutionally responsible for the value judgements that drive the evaluation of the alternative choices. collectively the analytical tools provide a framework for a systematic process of exploring and evaluating the decision alternatives -assembling and validating the applicable scientific evidence relevant to what will happen as the result of each possible choice, and valuing how bad or how good these consequences are based on an agreement of common objectives. yet, it does not replace the need for additional methods and processes for including other objectives, such as finding common goals, defining preferences, revisiting assumptions, sharing visions and exploring common grounds for values and normative positions. the value judgements motivating decisions are made explicit and can then be criticized by those who were not involved in the process. to the extent that uncertainty becomes important, it will be helpful to deal with uncertainty in an orderly and consistent way (morgan and henrion 1990). those aspects of uncertainty that can be modelled by using probability theory (inter-target variation, systematic and random errors in applying inferential statistics, model and data uncertainties) will be spelled out and those that remain in forms of indeterminacies, system boundaries or plain ignorance will become visible and can then be fed into the deliberation process (van asselt 2000, klinke and renn 2002) . the term deliberation refers to the style and procedure of decision making without specifying which participants are invited to deliberate (national research council (nrc) 1996, rossi 1997) . for a discussion to be called deliberative it is essential that it relies on mutual exchange of arguments and reflections rather than decision making based on the status of the participants, sublime strategies of persuasion, or social-political pressure. deliberative processes should include a debate about the relative weight of each argument and a transparent procedure for balancing pros and cons (tuler and webler 1999) . in addition, deliberative processes should be governed by the established rules of a rational discourse. in the theory of communicative action developed by the german philosopher jürgen habermas, the term discourse denotes a special form of a dialogue, in which all affected parties have equal rights and duties to present claims and test their validity in a context free of social or political domination (habermas 1970 (habermas , 1987b . a discourse is called rational if it meets the following specific requirements (see mccarthy 1975 , habermas 1987a , kemp 1985 , webler 1995 . all participants are obliged: • to seek a consensus on the procedure that they want to employ in order to derive the final decision or compromise, such as voting, sorting of positions, consensual decision making or the involvement of a mediator or arbitrator; • to articulate and criticize factual claims on the basis of the "state of the art" of scientific knowledge and other forms of problem-adequate knowledge; (in the case of dissent all relevant camps have the right to be represented); • to interpret factual evidence in accordance with the laws of formal logic and analytical reasoning; • to disclose their relevant values and preferences, thus avoiding hidden agendas and strategic game playing; and • to process data, arguments and evaluations in a structured format (for example a decision-analytic procedure) so that norms of procedural rationality are met and transparency can be created. the rules of deliberation do not necessarily include the demand for stakeholder or public involvement. deliberation can be organized in closed circles (such as conferences of catholic bishops, where the term has indeed been used since the council of nicosia), as well as in public forums. it may be wise to use the term "deliberative democracy" when one refers to the combination of deliberation and public or stakeholder involvement (see also cohen 1997 , rossi 1997 . what needs to be deliberated? firstly, deliberative processes are needed to define the role and relevance of systematic and anecdotal knowledge for making far-reaching choices. secondly, deliberation is needed to find the most appropriate way to deal with uncertainty in environmental decision making and to set efficient and fair trade-offs between potential over-and under-protection. thirdly, deliberation needs to address the wider concerns of the affected groups and the public at large. why can one expect that deliberative processes are better suited to deal with environmental challenges than using expert judgement, political majority votes or relying on public survey data? • deliberation can produce common understanding of the issues or the problems based on the joint learning experience of the participants with respect to systematic and anecdotal knowledge (webler and renn 1995 , pidgeon 1997 ). • deliberation can produce a common understanding of each party's position and argumentation and thus assist in a mental reconstruction of each actor's argumentation (warren 1993 , tuler 1996 . the main driver for gaining mutual understanding is empathy. the theory of communicative action provides further insights in how to mobilize empathy and how to use the mechanisms of empathy and normative reasoning to explore and generate common moral grounds (webler 1995). • deliberation can produce new options and novel solutions to a problem. this creative process can either be mobilized by finding win-win solutions or by discovering identical moral grounds on which new options can grow (renn 1999) . • deliberation has the potential to show and document the full scope of ambiguity associated with environmental problems. deliberation helps to make a society aware of the options, interpretations, and potential actions that are connected with the issue under investigation (wynne 1992, de marchi and ravetz 1999) . each position within a deliberative discourse can only survive the crossfire of arguments and counter-arguments if it demonstrates internal consistency, compatibility with the legitimate range of knowledge claims and correspondence with the widely accepted norms and values of society. deliberation clarifies the problem, makes people aware of framing effects, and determines the limits of what could be called reasonable within the plurality of interpretations (skillington 1997) . • deliberations can also produce agreements. the minimal agreement may be a consensus about dissent (raiffa 1994) . if all arguments are exchanged, participants know why they disagree. they may not be convinced that the arguments of the other side are true or morally strong enough to change their own position; but they understand the reasons why the opponents came to their conclusion. in the end, the deliberative process produces several consistent and -in their own domain -optimized positions that can be offered as package options to legal decision makers or the public. once these options have been subjected to public discourse and debate, political bodies, such as agencies or parliaments can make the final selection in accordance with the legitimate rules and institutional arrangements such as majority vote or executive order. final selections could also be performed by popular vote or referendum. • deliberation may result in consensus. often deliberative processes are used synonymously with consensus-seeking activities (coglianese 1997 ). this is a major misunderstanding. consensus is a possible outcome of deliberation, but not a mandatory requirement. if all participants find a new option that they all value more than the one option that they preferred when entering the deliberation, a "true" consensus is reached (renn 1999) . it is clear that finding such a consensus is the exception rather than the rule. consensus is either based on a win-win solution or a solution that serves the "common good" and each participant's interests and values better than any other solution. less stringent is the requirement of a tolerated consensus. such a consensus rests on the recognition that the selected decision option might serve the "common good" best, but on the expense of some interest violations or additional costs. in a tolerated consensus some participants voluntarily accept personal or group-specific losses in exchange for providing benefits to all of society. case studies have provided sufficient evidence that deliberation has produced a tolerated consensus solution, particularly in siting conflicts (one example in schneider et al. 1998) . consensus and tolerated consensus should be distinguished from compromise. a compromise is a product of bargaining where each side gradually reduces its claim to the opposing party until they reach an agreement (raiffa 1994) . all parties involved would rather choose the option that they preferred before starting deliberations, but since they cannot find a win-win situation or a morally superior alternative they look for a solution that they can "live with" knowing that it is the second or third best solution for them. compromising on an issue relies on full representation of all vested interests. in summary, many desirable products and accomplishments are associated with deliberation (chess et al. 1998) . depending on the structure of the discourse and the underlying rationale deliberative processes can: • enhance understanding; • generate new options; • decrease hostility and aggressive attitudes among the participants; • explore new problem framing; • enlighten legal policy-makers; • produce competent, fair and optimized solution packages; and • facilitate consensus, tolerated consensus and compromise. in a deliberative setting, participants exchange arguments, provide evidence for their claims and develop common criteria for balancing pros and cons. this task can be facilitated and often guided by using decision analytic tools (overview in merkhofer 1984) . decision theory provides a logical framework distinguishing action alternatives or options, consequences, likelihood of consequences, and value of consequences, where the valuation can be over multiple attributes that are weighted based on tradeoffs in multi-attribute utility analysis (edwards 1977) . a sequence of decisions and consequences may be considered, and use of mathematical models for predicting the environmental consequences of options may or may not be part of the process (humphreys 1977 , bardach 1996 , arvai et al. 2001 ): a) the structuring potential of decision analysis has been used in many participatory processes. it helps the facilitator of such processes to focus on one element during the deliberation, to sort out the central from the peripheral elements, provide a consistent reference structure for ordering arguments and observations and to synthesize multiple impressions, observations and arguments into a coherent framework. the structuring power of decision analysis has often been used without expanding the analysis into quantitative modelling. b) the second potential, agenda setting and sequencing, is also frequently applied in participatory settings. it often makes sense to start with problem definition, then develop the criteria for evaluation, generate options, assess consequences of options, and so on. c) the third potential, quantifying consequences, probabilities and relative weights and calculating expected utilities, is more controversial than the other two. whether the deliberative process should include a numerical analysis of utilities or engage the participants in a quantitative elicitation process is contested among participation practitioners . one side claims that quantifying helps participants to be more precise about their judgements and to be aware of the often painful trade-offs they are forced to make. in addition, quantification can make judgements more transparent to outside observers. the other side claims that quantification restricts the participants to the logic of numbers and reduces the complexity of argumentation into a mere trade-off game. many philosophers argue that quantification supports the illusion that all values can be traded off against other values and that complex problems can be reduced to simple linear combinations of utilities. one possible compromise between the two camps may be to have participants go through the quantification exercise as a means to help them clarify their thoughts and preferences, but make the final decisions on the basis of holistic judgements (renn 1986) . in this application of decision analytic procedures, the numerical results (i.e. for each option the sum over the utilities of each dimension multiplied by the weight of each dimension) of the decision process are not used as expression of the final judgement of the participant, but as a structuring aid to improve the participant's holistic, intuitive judgement. by pointing out potential discrepancies between the numerical model and the holistic judgements, the participants are forced to reflect upon their opinions and search for potential hidden motives or values that might explain the discrepancy. in a situation of major value conflicts, the deliberation process may involve soliciting a diverse set of viewpoints, and judgements need to be made on what sources of information are viewed as responsible and reliable. publication in scientific journals and peer review from scientists outside the government agency are the two most popular methods by which managers or organizers of deliberative processes try to limit what will be considered as acceptable evidence. other methods are to reach a consensus among the participants up front which expertise should be included in the deliberation or to appoint representatives of opposing science camps to explain their differences in public. in many cases, participants have strong reasons for questioning scientific orthodoxy and would like to have different science camps represented. many stakeholders in environmental decisions have access to expert scientists, and often such scientists will take leading roles in criticizing agency science. such discussions need to be managed so that disagreements among the scientific experts can be evaluated in terms of the validity of the evidence presented and the importance to the decision. it is essential in these situations to have a process in place that distinguishes between those evidence claims that all parties agree on, those where the factual base is shared but not its meaning for some quality criterion (such as "healthy" environment), and those where even the factual base is contested (foster 2002) . in the course of practical risk management different conflicts arise in deliberative settings that have to be dealt with in different ways. the main conflicts occur at the process level (how should the negotiations be conducted?), on the cognitive level (what is factually correct?), the interest level (what benefits me?), the value level (what is needed for a "good" life?) and the normative level (what can i expect of all involved?). these different conflict levels are addressed in this subsection. first of all, negotiations begin by specifying the method that structures the dialogue and the rights and duties of all participants. it is the task of the chairman or organizer to present and justify the implicit rules of the talks and negotiations. above and beyond this, the participants have to specify joint rules for decisions, the agenda, the role of the chairman, the order of hearings, etc. this should always be done according to the consensus principle. all partners in the negotiations have to be able to agree to the method. if no agreement is reached here the negotiations have to be interrupted or reorganized. once the negotiation method has been determined and, in a first stage, the values, standards and objectives needed for judgement have been agreed jointly, then follows the exchange of arguments and counter arguments. in accordance with decision theory, four stages of validation occur: • in a first stage, the values and standards accepted by the participants are translated into criteria and then into indicators (measurement instructions). this translation needs the consensual agreement of all participants. experts are asked to assess the available options with regard to each indicator according to the best of their knowledge (factual correctness). in this context it makes more sense to specify a joint methodological procedure or a consensus about the experts to be questioned than to give each group the freedom to have the indicators answered by their own experts. often many potential consequences remain disputed as a result of this process, especially if they are uncertain. however, the bandwidth of possible opinions is more or less restricted depending on the level of certainty and clarity associated with the issue in question. consensus on dissent is also of help here in separating contentious factual claims from undisputed ones and thus promotes further discussion. • in a second stage, all participating parties are required to interpret bandwidths of impacts to be expected for each criterion. interpretation means linking factual statements with values and interests to form a balanced overall judgement (conflicts of interests and values). this judgement can and should be made separately for each indicator. in this way, each of the chains of causes for judgements can be understood better and criticized in the course of the negotiations. for example, the question of trustworthiness of the respective risk management agencies may play an important role in the interpretation of an expected risk value. then it is the duty of the participating parties to scrutinize the previous performance of the authority concerned and propose institutional changes where appropriate. • third stage: even if there were a joint assessment and interpretation for every indicator, this would by no means signify that agreement is at hand. much rather, the participants' different judgements about decisionmaking options may be a result of different value weightings for the indicators that are used as a basis for the values and standards. for example, a committed environmentalist may give much more weight to the indicator for conservation than to the indicator of efficiency. in the literature on game theory, this conflict is considered to be insoluble unless one of the participants can persuade the other to change his preference by means of compensation payments (for example, in the form of special benefits), transfer services (for example, in the form of a special service) or swap transactions (do, ut des). in reality, however, it can be seen that participants in negotiations are definitely open to the arguments of the other participants (i.e. they may renounce their first preference) if the loss of benefit is still tolerable for them and, at the same time, the proposed solution is considered to be "conducive to the common good", i.e. is seen as socially desirable in public perception. if no consensus is reached, a compromise solution can and should be reached, in which a 'fair' distribution of burdens and profits is accomplished. • fourth stage: when weighing up options for action formal methods of balancing assessment can be used. of these methods, the cost-benefit analysis and the multi-attribute or multi-criteria decision have proved their worth. the first method is largely based on the approach of revealed "preferences", i.e. on people's preferences shown in the past expressed in relative prices, the second on the approach of "expressed preferences", i.e. the explicit indication of relative weightings between the various cost and benefit dimensions (fischhoff et al. 1982) . but both methods are only aids in weighing up and cannot replace an ethical reflection of the advantages and disadvantages. normative conflicts pose special problems because different evaluative criteria can always be classified as equally justifiable or unjustifiable as explained earlier. for this reason, most ethicists assume that different types and schools of ethical justification can claim parallel validity, it therefore remains up to the groups involved to choose the type of ethically legitimate justification that they want to use (ropohl 1991 , renn 1997 . nevertheless, the limits of particular justifications are trespassed wherever primary principles accepted by all are infringed (such as human rights). otherwise, standards should be classed as legitimate if they can be defended within the framework of ethical reasoning and if they do not contradict universal standards that are seen as binding for all. in this process conflicts can and will arise, e.g. that legitimate derivations of standards from the perspective of group a contradict the equally legitimate derivations of group b (shrader-frechette 1988). in order to reach a jointly supported selection of standards, either a portfolio of standards that can claim parallel validity should be drawn up or compensation solutions will have to be created in which one party compensates the other for giving up its legitimate options for action in favour of a common option. when choosing possible options for action or standards, options that infringe categorical principles, for example, to endangering the systematic ability of the natural environment to function for human use in the future and thus exceeding the limits of tolerability are not tolerable even if they imply major benefits to society. at the same time, all sub-dominant options have to be excluded. frequently sub-dominant solutions, i.e. those that perform worse than all other options with regard to all criteria at least in the long term, are so attractive because they promise benefits in the short term although they entail losses in the long term, even if high interest rates are assumed. often people or groups have no choice other than to choose the sub-dominant solution because all other options are closed to them due to a lack of resources. if large numbers of groups or many individuals act in this way, global risks become unmanageable (beck 1996) . to avoid these risks intermediate financing or compensation by third parties should be considered. the objective of this last section of chapter 4 was to address and discuss the use of decision analytic tools and structuring aids for participatory processes in environmental management. organizing and structuring discourses goes beyond the good intention to have all relevant stakeholders involved in decision making. the mere desire to initiate a two-way communication process and the willingness to listen to stakeholder concerns are not sufficient. discursive processes need a structure that assures the integration of technical expertise, regulatory requirements, and public values. these different inputs should be combined in such a fashion that they contribute to the deliberation process the type of expertise and knowledge that can claim legitimacy within a rational decision-making procedure (von schomberg 1995). it does not make sense to replace technical expertise with vague public perceptions, nor is it justified to have the experts insert their own value judgements into what ought to be a democratic process. decision analytic tools can be of great value for structuring participatory processes. they can provide assistance in problem structuring, in dealing with complex scientific issues and uncertainty, and in helping a diverse group to understand disagreements and ambiguity with respect to values and preferences. decision analysis tools should be used with care. they do not provide an algorithm to reach an answer as to what is the best decision. rather, decision analysis is a formal framework that can be used for environmental assessment and risk handling to explore difficult issues, to focus debate and further analysis on the factors most important to the decision, and to provide for increased transparency and more effective exchange of information and opinions among the process participants. the basic concepts are relatively simple and can be implemented with a minimum of mathematics (hammond et al. 1999) . many participation organizers have restricted the use of decision analytic tools to assist participants in structuring problems and ordering concerns and evaluations, and have refrained from going further into quantitative trade-off analysis. others have advocated quantitative modelling as a clarification tool for making value conflicts more transparent to the participants. the full power of decision analysis for complex environmental problem may require mathematical models and probability assessment. experienced analysts may be needed to guide the implementation of these analytical tools for aiding decisions. skilled communicators and facilitators may be needed to achieve effective interaction between analysts and participants in the deliberative process whose exposure to advanced analytical decision aids is much less, so that understanding of both process and substance, and therefore transparency and trust, can be achieved. many risk management agencies are already making use of decision analysis tools. we urge them to use these tools in the context of an iterative, deliberative process with broad participation by the interested and affected parties to the decision in the context of the risk governance framework. the analytical methods, the data and judgement, and the assumptions, as well as the analytical results should be readily available and understood by the participants. we believe that both the risk management agencies and the interested groups within the public that government agencies interact with on environmental decisions should all gain experience with these methods. improper or premature use of sophisticated analytical methods may be more destructive to trust and understanding than helpful in resolving the difficulties of complexity, uncertainty, and ambiguity. bioterrorism, preparedness, attack and response. elsevier beyond the job exposure matrix (jem): the task exposure matrix (tem) chapter 4 occupational safety and health and environmental risks a task exposure database for use 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politics and the struggle to define: a discourse analysis of the framing strategies of competing actors in a "new introduction to decision analysis corporate environmentalism in a global economy respect for nature. a theory of environmental ethics meanings, understandings, and interpersonal relationships in environmental policy discourse. doctoral dissertation designing an analytic deliberative process for environmental health policy making in the u.s. nuclear weapons complex the framing of decisions and the psychology of choice perspectives on uncertainty and risk akzeptanzbeschaffung: verfahren und verhandlungen the erosion of the valuespheres. the ways in which society copes with scientific, moral and ethical uncertainty can participatory democracy produce better selves? psychological dimensions of habermas discursive model of democracy welt im wandel: erhaltung und nachhaltige nutzung der biosphäre. jahresgutachten 1999 discourse in citizen participation. an evaluative yardstick the craft and theory of public participation: a dialectical process a brief primer on participation: philosophy and practice system, diskurs, didaktik und die diktatur des sitzfleisches konsens als telos der sprachlichen kommunikation? in: giegel biophilia: the human bond with other species rio" oder die moralische verpflichtung zum erhalt der natürlichen vielfalt risk and social learning: reification to engagement die modernisierung der demokratie im zeichen der umweltproblematik wandelt sich die verantwortung mit technischem wandel? key: cord-301856-71syce4n authors: domínguez-andrés, jorge; netea, mihai g. title: impact of historic migrations and evolutionary processes on human immunity date: 2019-11-27 journal: trends immunol doi: 10.1016/j.it.2019.10.001 sha: doc_id: 301856 cord_uid: 71syce4n the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host–pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host-pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. infectious diseases are arguably the main source of evolutionary pressure that humanity has ever confronted. the dispersion of different human communities around the globe has exposed each population to different infectious agents, exerting a selective pressure (see glossary) on them; thus, adaptation to the new environment has favored the selection of the most beneficial genetic variants for the host. as a result, infectious agents have caused the expansion of alleles behind the induction of either protection or tolerance to these diseases; heritable variations, that increased the survival to deadly infectious agents, may have been naturally selected before the hosts had the opportunity to reproduce [1] . natural selection driven by pathogens is probably more remarkable for those infectious agents that have been among us for a longer time, namely the causative agents of well-known diseases such as leprosy, smallpox, malaria, or tuberculosis. the genetic imprint of pathogen-driven selection depends on the length and the virulence of the infections and also the geographical distribution. the human genome presents more than 5000 genetic loci with traces of selective pressure [2] . this group includes more than 300 immune-related genes with functional variations between populations, which are probably behind the variability of responses to immune-related diseases reported nowadays [3, 4] . besides natural selection, other evolutionary mechanisms, such as genetic drift, greatly influence the frequencies of the genetic variants found within diverse populations throughout the world [5] (box 1 and figure 1 ). with the burst of next-generation sequencing and the development of cutting-edge technologies such as transcriptomics, proteomics, and systems biology, we are starting to witness the great impact of evolutionary processes on human immunity and how the interactions between microorganisms and humans that took place millennia ago might play a fundamental role not only in the response against modern pathogenic threats, but also in the emergence of autoimmune and inflammatory diseases observed in modern populations worldwide. in this review we offer a novel perspective on the role of infectious diseases as agents of natural selection and as forces behind the evolutionary pressure encountered by human ancestors and modern humans in their migrations around the globe. specific genetic variants selected throughout different periods of human history may have influenced immune responses of present-day populations against pathogenic microorganisms and may have played a role in the development of certain inflammatory and autoimmune diseases. the majority of experts agree that africa is where our species originated. genetic studies conducted in diverse contemporary populations suggest connections with ancestors that lived on the continent up to 350 000 years ago [6] . human evolution has been constantly influenced by pathogens; therefore, a great number of human genes linked to immune functions and immunity-related disorders have evolved along with humans. the heterogeneity in the immune response to infectious diseases across different populations is under genetic control and is the result of evolutionary processes. genetic variants that have been under evolutionary pressure can contribute to explaining the differences in the susceptibility to diseases observed across different populations. the ancestry of individuals from different populations across the globe greatly influences their possibility of developing certain autoimmune diseases and inflammatory disorders. the lifestyle of western societies affects the symbiotic relationships between humans, viruses, and other organisms and might contribute to the rise of certain autoimmune and inflammatory diseases. pathogens have played a central role as agents of natural selection from those very early days. among various infectious diseases, malaria has exerted the highest evolutionary pressure on the communities across the african continent ( figure 2 ) [7] . populations remaining in sub-saharan africa have been exposed to malaria for such long periods of time that their genetic structures have been shaped by the severity of malaria (plasmodium sp.) infections. in 1954, allison described that sickle cell disease distribution was confined to africa and was associated with the geographical presence of malaria [8] . this finding led to the more recent description of the existence of mutations in the hemoglobin-b (hbb) gene as a result of natural selection driven by evolutionary pressure for protection against malaria [9] (table 1) . similarly to hbb, some areas of west africa with a high incidence of malaria the gene variations that pass from one generation to the next are often transmitted as a random process known as genetic drift, while selection of advantageous variants tends to be preferentially transmitted. mutations, genetic drift, migration, and environmental selective pressure are among the fundamental processes behind the evolution of humans. the influence of these mechanisms in the diverse communities that were mobilized and then became isolated, as well as severe external factors such as epidemics, caused successive genetic bottlenecks in populations (see figure 1 in main text) [90] . human evolutionary studies are currently considered under 'modern synthesis', which merges darwin's ideas of natural selection with theoretical population genetics and mendelian principles, stating that evolution occurs via small genetic changes that are regulated by natural selection [91] . these beneficial adaptations subsequently expand within the members of a population and become evident in the ancestral specificity and the geographical distribution of the advantageous alleles in the genomes of contemporary humans. genetic bottlenecks occur when the number of individuals in a population is reduced drastically due to a catastrophic event such as an earthquake, a flood, a famine, or the outbreak of an infectious disease. these events limit the genetic variation of a population and can lead to genetic drift. as a result, a smaller population, with a correspondingly reduced genetic diversity, remains to transmit genes to future generations through sexual reproduction. even if this reduction in the genetic diversity is temporary, it can lead to long-lasting effects on the genetic variation of the offspring populations. genetic drift: changes in the allele frequencies of a population over generations due to chance. genetic locus: fixed position on a chromosome (e.g., the position of a gene or a genetic marker). histocompatibility complex: region of approximately 4000 kb, located on human chromosome 6, that contains a large number of genes whose products are expressed as proteins on immune cells. of these genes, the best known are hla genes. introgressive hybridization: incorporation of genes from one species into the genetic reserves of another by interspecific hybridization and backcrossing with the parent species. present a high frequency of hemoglobin-c (hbc) in their populations, which is associated with a 30-93% decrease in the possibility of developing the disease [10] . this is also the case for the duffy antigen receptor gene (darc) in erythrocytes and single nucleotide polymorphisms (snps) in human leukocyte antigen (hla), which have been associated with protection against plasmodium vivax malaria in certain areas of africa where this disease is endemic [11, 12] . another example of natural selection driven by evolutionary pressure for protection against malaria are thalassemia (a and b) pathologies, a group of hemoglobin disorders that presents an incidence of up to 30% among communities of west africa [13] . the human casp12 t 125 c snp, expression of which is restricted to the african subcontinent, south america, and certain areas of asia, can modulate immune and inflammatory responses to malaria by antagonizing interleukin (il)-1b and nf-kb signaling in innate immune cells; moreover, caspase 12-deficient mice (casp12 -/-) exhibit decreased interferon (ifn)-g production and clearance of the parasite, relative to wild type (wt) infected mice [14] . however, others have questioned these findings, given that caspase 12-deficient mice also lack caspase 11 expression, so the effects observed might not be specific to caspase 12 [15] . mycobacterium tuberculosis (mtb) has caused infections in our species and ancestors for at least 500 000 years [16] . this long-standing relationship between humans and mtb probably underlies the large variety of immune-related factors that modulate susceptibility to mtb infection, including vitamin d receptor (vdr), natural resistance-associated macrophage protein 1 (slc11a1), tir domain containing adaptor protein (tirap), hla, monocyte chemoattractant protein 1 (mcp-1), and cytokines such as il-12 and ifn-g [17] ( table 1) . patients with african ancestry present a higher frequency of mtb-related genetic variants than individuals from other populations, including variants in the gene encoding for toll-like receptor 6 (tlr6), mediating cellular responses to bacterial malaria is one of the greatest causes of morbidity and mortality in the history of humanity. most human populations with a long history of endemic malaria have evolved genetic adaptations to malaria parasites due to the strong selective pressure that this infection has exerted. since the parasite infects erythrocytes, the evolutionary pressure has selected genetic variants that affect red blood cells and, therefore, the survival of the parasite as well. genetic variants conferring resistance to the disease have spread through human populations over time, including several abnormal hemoglobins that protect against malaria but usually cause erythrocyte-associated diseases in the populations where these adaptations are prevalent. these factors include the t 125 c polymorphism in the caspase 12 gene (casp12); the hemoglobin b (hbb) and hemoglobin c (hbc) variants; mutations in the duffy antigen receptor gene (darc); thalassemias (a and b); sickle cell disease; and polymorphisms in the human leukocyte antigen (hla) loci. dna nucleotide. for example, an snp may replace cytosine (c) with thymine (t) in a certain segment of dna. selective pressure: phenomenon that alters the behavior and fitness of living organisms within a given environment. it is the driving force of evolution and natural selection. thalassemias (a and b): inherited hemoglobinopathies characterized by a failure in the synthesis of the globin alpha or beta chains. toll-like receptors: family of transmembrane pattern recognition receptors expressed by immune and nonimmune cells that recognize conserved pathogenassociated molecular patterns. they play a pivotal role in innate immunity. transgenerational inheritance: transmission of traits from generation to generation. trends in immunology, december 2019, vol. 40, no. 12 1107 lipoproteins [18, 19] . selective pressure has also shaped the mechanisms that modulate the expression of genes implicated in immune responses against lassa virus, such as il-21 (il21) and the glycosyltransferase-like protein large1 (large), suggesting that the natural selection exerted by the virus drove the expansion of genetic variants that enhance immunity against lassa fever [20] . these examples indicate that infectious diseases have contributed to shaping the genetic landscape of african populations and their descendants, and highlight the great impact of pathogens as an evolutionary force in humans. our homo sapiens ancestors were not the only species to venture out of africa, with other homo species performing a similar migration much earlier, such as homo ergaster, homo erectus, or homo heidelbergensis [21] . from these early migrations, local populations such as the denisovans and neanderthals evolved [22] . these lineages were not geographically isolated, but lived side by side with modern humans and interbred with them, leaving a genetic footprint in their common progeny. accordingly, 1-4% of the genome of european and asian populations is thought to derive from these now-extinct hominid lineages [23] . neanderthals spent close to 600 000 years adapting to their environment and their immune systems were shaped by the infections they faced. by interbreeding with archaic humans, modern humans incorporated these advantageous adaptations in the genome of their descendants. this was highlighted by different studies that showed that the introgression of diverse genes related to immune functions, such as the oas cluster, tlr1, or the histocompatibility complex from denisovans and neanderthals shaped the genetic landscape of present-day eurasian, but not african, communities. genomic sequences and expression data from lymphoblastoid cell lines from 421 individuals of european and african ancestry confirmed that the tlr1-tlr6-tlr10 genetic loci, presenting signs of local positive selection and repeated introgression from both neanderthal and denisovan genomes [24] [25] [26] , showed a significantly higher expression in individuals carrying archaic-like alleles than in individuals carrying the nonintrogressed modern human alleles [2, 26, 27] . the expression of these genes has shaped human immune responses against different types of pathogens. for example, the gp41 protein of the hiv-1 virus has been recently recognized as a tlr10 ligand [28] . in this regard, increased tlr10 expression has been correlated with higher il-8 production by the macrophage cell line thp-1 and higher titers of hiv-1 in the breast milk of hiv-1-infected nigerian women relative to controls [28] . tlr1 and tlr6 form dimers with tlr2, triggering immune responses against different types of bacteria, fungi, virus, and parasites [29] . variation in tlr1-tlr6-tlr10 is the major genetic determinant of human interindividual differences in tlr1/2-mediated responses, including cytokine production to a number of clinically relevant pathogens such as staphylococcus aureus and listeria monocytogenes [30] (table 1 ). this inheritance from archaic humans may have also left some human individuals more prone than others to developing asthma, hay fever, and other allergies (of 58 snps associated with susceptibility to allergic disease, 12 had a neanderthal or denisovan origin) [26] , although these associations remain to be fully demonstrated [31] . these reports demonstrate that by interbreeding with archaic humans, modern humans incorporated a group of advantageous adaptations to the genome of their descendants and contributed to shaping immune responses in modern human populations. the migration of our human ancestors out of africa implied the exposure to different types of infectious diseases (box 2 and figure 3 ). one study tested the responsiveness of human macrophages to pathogenic bacteria in vitro, finding that almost 10% of the genes present in human macrophages infected with the bacteria salmonella typhimurium or l. monocytogenes present different regulatory responses directly linked to the lineage of the donors and, also, that macrophages obtained from individuals of african origin display enhanced bactericidal activity compared with those from individuals of european lineage [32] . the trend towards lower inflammatory responses in european populations is strengthened by the fixation of a tlr1 gene variant that results in lower proinflammatory gene expression in populations with a european ancestry compared with those with an african one [33] . the largest population differences in gene expression between africans and europeans have been found in the macrophage receptor with collagenous structure (marco), a protein implicated in responses against viral infections and tlr-induced dendritic cell activation [34] , the chemokine receptor cx3cr1, which mediates effector lymphocyte functions, and also several ifn-stimulated genes [35] . west eurasian populations present a high frequency of tirap ser180leu snps [36] . tirap is an adaptor protein in tlr2 and tlr4 signaling pathways, involved in inflammatory responses and cytokine production. the heterozygous expression of the ser180leu snp is protective against invasive pneumococcal disease, bacteremia, malaria, and tuberculosis, as shown in a case-control study of 6106 individuals from the uk, vietnam, and several african countries, and it is associated with lower tlr2 signaling in humans [36] . this variant is considered to be a consequence of the natural selection that may have taken place in an early period following the migration of modern humans out of africa [37] . european populations present a selective adaptation of the ifn gene that allows a high production of ifn-g in infectious scenarios due to the positive selection of ifng variants +5173g and +874t; this suggests the existence of strong environmental pressures linked to higher ifn-g concentrations in plasma during mtb infection in european individuals relative to other populations [38, 39] . in line with this, a database meta-analysis showed that individuals expressing the +874t/a variant of ifng presented higher susceptibility to tuberculosis mtb infection than individuals without it, which might be considered a putative prognostic factor for the development of tuberculosis [40] , although this remains to be robustly demonstrated. one of the most interesting aspects of humans is their ability to adapt to almost every ecosystem of the planet. the history of mankind is also the history of millions of individuals wandering around the world, looking for a better place to live. a glimpse to the migratory legacy of humanity around the globe reveals the great impact that the massive population movements defined the world as we know it today (see figure 3 in main text). the distances ancient humans travelled are impressive, from the first hominids colonizing africa to the conquest of the americas in a time when the bering strait was not yet under water. the historical exodus of mankind started almost 2 million years ago with the migration of homo erectus from africa through eurasia. from this event on, relatively isolated human populations evolved separately on different continents, leading to the emergence of different human species, such as neanderthals in europe, the denisovans in asia, and, later, modern homo sapiens in africa [92, 93] . h. sapiens first colonized large areas of the continent around 300 000 years ago [6] , spread towards the middle east at some point between 150 000 and 80 000 years ago, and migrated through eurasia, reaching australia within 20 000 years [94] . asian human ancestors went through the frozen waters of the bering strait in two distinct waves to colonize the american continent approximately 20 000 years before the present time [95] . when humans ventured out of africa, they faced different types of pathogens than the communities that stayed in the african continent. with time, the series of events faced by diverse populations has generated differences in the immune responses to pathogens in the populations with an african or eurasian origin and which have spread throughout the world. the indigenous populations of south america are descendants of migrating populations of north-east asians that crossed the bering strait around 20 000 years ago [41] . five centuries ago, european settlers disembarked on the american continent, bringing a large collection of pathogens such as those causing measles, pneumonic plague, and influenza infections, which the indigenous populations had never faced before. these diseases rapidly spread and caused mortality rates above 90% [42] . the consequences of these pandemics are still visible in current populations; one report studied dna from the bones of 25 ancient humans from the tsimshian community, living in the british columbia region in canada until the 15th century, identifying marks of positive selection in a number of immune-related variants [43] . specifically, the hla-dqa1 variant was present in almost the totality of tsimshian individuals, but only in one third of present-day humans studied; this suggested that ancient american genomes were evolutionarily selected to respond to local diseases but not to fight against pathogens brought by the europeans [43] . another study compiled information on infectious diseases that have killed more than 10 000 individuals among 59 indigenous communities of the amazonia in the past two centuries, showing that the mortality rates and the incidence of infectious diseases rapidly decayed within the time following the first encounter with the pathogen, compatible with genetic adaptation [44] . european colonizers underwent purifying selection in situations of intense pressure. such scenarios were documented when dutch colonists migrated to surinam and encountered epidemics of yellow and typhoid fever that caused a 60% mortality rate among settlers [45] . variations in the frequencies of c3, glo, and hla-b genes among the descendants were not likely caused by genetic drift, but rather, it has been proposed that these populations were probably selected through genetic control of survival to the epidemics [45] (box 3). africans and americans with an african ancestry present a much higher number of genetic variants related to robust inflammatory reactions, increased cytokine secretion, and bactericidal activities compared with the other populations [32] , including more than 250 genes with traces of recent selection, such as il1a and il1b gene variants [46] . the degree of african ancestry, analyzed by fine-mapping analysis refined to the duffy-null allele of rs2814778, was correlated with an increased amount of the proinflammatory chemokines ccl2 and ccl11 in plasma relative to controls [12] . a study involving 12 000 african american and hispanic american women found that the higher values of c-reactive protein (crp) in blood found in these populations compared with european americans were related to a crp-associated variant of triggering receptors expressed by myeloid cells 2 (trem2) [47] . moreover, comparison of health record data from individuals with connective tissue diseases, including rheumatoid arthritis and systemic lupus erythematosus (sle), as well as atherosclerotic cardiovascular disease from almost 300 000 african american and european american adults was conducted; the study reported for the latter, a prevalence of atherosclerotic cardiovascular disease in 29.7% african americans (particularly high in young individuals), relative to 14.7% in european americans [48] . these studies highlight certain genetic links to inflammatory predisposition/manifestation. however, increased proinflammatory activity is a double-edged sword. in the absence of regular pathogen challenges that require maintained modulation of the balance between inflammation and suppression of the immune response, the organism can overreact to inflammatory stimuli and trigger exacerbated responses. for instance, descendants of african populations generally present higher susceptibility to a variety of autoimmune syndromes such as inflammation-associated carcinomas, lupus, asthma, and multiple sclerosis (ms), the overall prevalence of which is up to three times higher in individuals with african ancestry relative to individuals with european ancestry [31, 49, 50] . there are extensive differences in immune cell gene expression between americans with african and european ancestry. the increased proinflammatory responses observed in american individuals relative to other populations might be beneficial to combatting infections, but might also increase the chances of developing inflammatory and autoimmune disorders, which warrants further investigation. our gastrointestinal tract provides residence to both beneficial and potentially pathogenic microorganisms, harboring ten million different microbial genes in the human fecal microbiome [51] . the microbiome has its own evolutionary scenario across different populations with divergent lifestyles, nutrition, and exposure to environmental agents, generating extraordinary heterogeneity. the ongoing process of 'lifestyle westernization' of different societies has an important impact on the mutualistic relationships between humans and commensal organisms worldwide. african tribes are adopting western subsistence patterns, leading to remarkable changes in the composition of their microbiota [52] . the comparison of the intestinal flora of the baaka hunter-gatherers and the bantu agriculturalists (both from the central africa republic), with a group of us-born african americans showed a great example of the evolution of the human microbiome [52] . specifically, the bantu, still engaged in hunting, have a greater bacterial gut diversity than their baaka neighbors, who left the jungle for agriculture, and even more than urbanized westerners (us african-americans) [52] . this reduced microbiota diversity in western societies has been associated with a higher incidence of the so-called 'diseases of civilization' such as cardiovascular diseases, diabetes, obesity, and autoimmune disorders, which are very unusual in hunter-gatherer societies compared with communities living a western-type lifestyle [52, 53] . although viruses are mainly seen as pathogenic agents, they also play a fundamental role in the evolution and maturation of the human immune system [54, 55] . approximately 8% of the human genome is composed of endogenous retroviruses (ervs), sequences derived from past retroviral infections and permanently inserted into different regions of the human genome [56] . one study showed that ervs played a central role in the induction of ifn-dependent immune responses and that the removal of one or more of these viral dna elements in the hela human cell line severely impaired the recruitment of transcription factors necessary to trigger the expression of ifng against vaccinia virus infection relative to controls [57] . viruses can also influence the severity of infections caused by other viruses. for example, cytomegalovirus infection in hiv-1 seropositive humans can potentiate the effects of hiv-1 infection by expanding the pool of circulating regulatory t cells (immunosuppressive); these were shown to inhibit the proliferation of autologous peripheral blood mononuclear cell the origins of the hiv virus are still a matter of scientific discussion. the most accepted scenario argues that hiv originated in simians and was transmitted to humans in west africa in the 1920s, likely due to local ingestion of ape meat infected with the simian immunodeficiency virus. around 1960, the virus reached wide parts of the continent and finally spread overseas thanks to a group of haitian professors coming back from africa. in the following decades, the virus spread worldwide and generated the pandemic we now know. today, there are approximately 37 million people worldwide living with hiv-1/aids [110] . ccr5 is a receptor of chemokines that plays a fundamental role in hiv-1 pathogenesis and it is also one of the most promising targets to restrict the infection, since mutations in this receptor turn individuals resistant [96] . the ccr5-d32 mutation results in a deletion that eliminates the hiv-1 co-receptor on lymphocytes, providing robust protection against hiv-1 and, therefore, aids [97] . ccr5-d32 allele frequencies reach 14% in northern europe populations, whereas it is not present in populations with different ancestry, such as east asian, native american, or african groups [96] . this regional distribution of ccr5-d32 variants is most likely related to a naturally selective episode that struck european populations around 700 years ago and involved a strong infectious agent that also employed ccr5 [98]. a mathematical model studying the changes in the european populations in the middle ages suggested yersinia pestis (bubonic plague) as the most probable infectious agent behind the pressure that selected this particular genetic variant [99] . this is in agreement with the finding that european rroma populations, but not northwestern indian populations that inhabit the area where the rroma originally lived, present signatures of positive selection in tlr1-tlr6-tlr10, which influence cytokine responses in y. pestis infections [100] . (pbmc) in response to cytomegalovirus infection in vitro [58] . in one study, patients with chronic hepatitis c virus (hcv) infection and hepatitis a virus (hav) superinfection presented lower titers of hcv rna than patients harboring only hcv, suggesting that hcv replication might be potentially suppressed during hav infection [59] , although this will still require further investigation. the relationships between humans and pathogenic or nonpathogenic organisms are extraordinarily complex and include tripartite evolutionary interactions between humans and microbes competing with each other. this is the case of parasites that infect other parasites, such as bacteriophage viruses, that can influence the outcome of bacterial infections. for example, in a cohort of individuals with chronic wounds, a report showed that the phage pf, which coexists with pseudomonas aeruginosa in infected wounds, triggered the production of type i ifn, the inhibition of tumor necrosis factor (tnf) production, and the suppression of phagocytosis in human primary monocytes and mouse bone marrow-derived macrophages, dampening the antibacterial response and promoting the bacterial infection [60] . however, bacteriophages can also provide protection to the human host by directly attacking pathogenic bacteria and by upregulating in human pbmcs the expression of proinflammatory genes such as il1a, il1b, il6, tnfa, cxcl1, and cxcl5, as shown for several s. aureus and p. aeruginosa phages, including pnm, luz19, 14-1, and ge-vb_pae-kakheti25 [61] . cooperative relationships between organisms are evolutionary processes themselves. the way microorganisms and their hosts associate can lead to interactions of mutualism, in which the interplay may be so intimate as to provide benefit for each party and influence immune responses against different types of pathogens. a great number of humans live far away from the original settlements of their ancestors and are subject to radically different environmental conditions. between two and three million people with european genealogy suffer from autoimmune diseases, the prevalence of which is also increasing in other populations across the globe [62]. there is rising evidence that the emergence of autoimmune diseases is associated with the presence of a number of immune-related alleles that have been selected via evolutionary processes; and, furthermore, that the contrasting differences in the prevalence of autoimmune diseases between populations may be a result of different selective pressures [63] . alleles associated with inflammatory diseases that present marks of modern positive selection include the risk allele fut2 at rs601338 for crohn's disease (cd) or the risk variant sh2b3 at rs3184504 for celiac disease [64] ; such variants have been linked to the development of several human autoimmune diseases, such as type 1 diabetes, ms, and celiac disease [64, 65] . the analysis of the integrated haplotype score [66] of loci associated with sle that might provide protection against infections, such as tnip1, itgam, ptpn22, tnfsf4, uhrf1bp1, tet3-dguok, and blk, has suggested that these loci exhibit robust signs of positive selection [67] (figure 4 , key figure and table 1 ). african and asian human populations exposed to trypanosoma brucei or plasmodium sp. have presented positive selection of snps in the apol1 and fcgr2b genes; indeed, by enhancing human macrophage-mediated phagocytosis of infected erythrocytes, despite their association with an sle predisposition, these snps have been associated with protective roles against sleeping sickness and malaria, respectively [68, 69] . an analysis of human loci associated with inflammatory bowel disease (ibd) concluded that the majority of the loci associated with cd are also linked with a higher risk of developing ulcerative colitis [70] ( table 1 ). many of these loci were also associated with the development of other autoimmune diseases, namely psoriasis and ankylosing spondylitis [62] . pbmcs from individuals carrying the sh2b3 variant rs3184504*a (associated with a risk for developing cd) [71] presented higher production of il-6 and il-1b after stimulation with lipopolysaccharide or muramyl dipeptide due to enhanced activity of the nod2 inflammasome pathway relative to controls [72] ; this has suggested an immune-related role for sh2b3 in the context of bacterial infections, which might help explain the positive selection of sh2b3 approximately 1500 years ago [72] . others found an association between genetic variants in nod2, cd14, and increased susceptibility to cd [73] , in line with previous results showing that mutations in nod2 and tlr4/cd14 are related to an increased risk of developing ibd [74] . from another angle, changes in hygiene patterns seen in the past two centuries brought vast improvements in sanitation, drinking water, and garbage collection, which greatly reduced the exposure to many infectious diseases. however, these conditions may have reduced the exposure to viral and microbial agents that help the immune system to develop tolerance during childhood. the hygiene hypothesis proposes that the lack of exposure to microbial agents in the early stages of life is related to a higher risk of developing hypersensitivity reactions, based on the fact that children that are exposed to higher amounts of microbial stimuli (e.g., by growing on farms) are less prone to develop allergies and asthma [75, 76] . moreover, reduced exposure to infectious agents can have a much wider effect than initially believed. lack of exposure to microbes in childhood can cause aberrant responses to infection and potentiate the effects of etv6-runx1 mutations in the pathogenesis of acute lymphoblastic leukemia [77] . by contrast, a meta-analysis of six observational studies, including 1902 participants, showed a correlation between low helicobacter pylori infection and ms, suggesting that low h. pylori prevalence might be a putative protective factor in ms, although this remains to be experimentally validated [78] . one study also reported that antibodies against toxoplasma gondii were detected less often in patients with ms compared with healthy controls [79] . however, these findings warrant further and robust investigation. overall, it is clear that evolutionary processes can drive the fixation of genetic variations that increase (or decrease) our defense against infections upon sensing microbial ligands, but can also lead to a greater risk of developing certain autoimmune diseases in which endogenous ligands can cause tissue damage and inflammation. a growing number of reports suggest that inheritance is not always governed by classical darwinian evolutionary processes. exposure to certain environmental stimuli can cause effects in the progeny of an exposed individual, even though the stimuli are no longer present. this type of transgenerational inheritance might be explained through the effects of epigenetic processes, which are hypothesized to be transmitted through the germline and passed on to the offspring [80] . for example, the worm caenorhabditis elegans can transmit improved resistance to infections to pathogenic bacteria to their offspring through alterations of the histone landscape [81] . indian meal moths exposed to low doses of virus are subsequently less susceptible to viral challenge, a protection offered to their offspring as well [82] . transgenerational inheritance of diverse traits has also been observed in mice, in which the variation of the color of the coat is passed on the next generation [83, 84] . offspring of male rats subjected to a high-fat diet present glucose intolerance and reduced insulin secretion, linked to reduced methylation at the il13ra2 gene relative to controls [85] ; and mice fed scorpions are more resistant to a challenge with scorpion venom than mice on a normal diet [86] . since infections are one of the strongest factors impacting survival, it is conceivable that transgenerational transmission of traits in mammals, including humans, evolved to improve host defense. the number of studies of the potential role of epigenetic inheritance in shaping the human immune system is still scarce. however, different experiences undergone by certain communities indicate that these mechanisms might be important. for example, the babies of pregnant women who suffered during the early stages of pregnancy (the effects of the dutch hunger winter in 1944), 60 years later showed reduced dna methylation marks in several genes that control metabolism and cell differentiation during development, such as igf2, pim3, txnip, abcg1, pfkfb3, and mettl8, compared with their siblings [87, 88] . this was related with higher rates of obesity, heart disease, cancer, and depression in individuals whose pregnant mothers suffered the effects of the famine [87, 88] . some of these effects seemed to be present in the progeny of this group, that is, in the grandchildren of those who had passed the famine during pregnancy [89] . the rapid growth in the number of reports covering the impact of epigenetic mechanisms in different human processes warrants further and robust studies on the impact of epigenetic inheritance in shaping the evolution of the human immune system. human immune responses have been shaped by the evolutionary pressure exerted by microorganisms and viruses throughout history. generating a broad range of genetic variations and immune functions in different populations favors the adaptation to new environments and increases the chance of survival of the human species against potential pandemics. much remains to be learned in this exciting field over the coming years in order to identify the main regulatory forces and the time window necessary for the fixation of an advantageous genetic trait in a population (see outstanding questions). the combination of the selective pressure caused by infectious diseases with other evolutionarily relevant processes, such as genetic drift, migratory events, bottlenecks, and introgressive hybridization, contribute to driving the expansion, fixation, or elimination of characteristic immune response-related traits in different populations around the world. these specific genetic variants are able to boost the host response against pathogens by improving the sensing of microbial ligands but can also lead to the development of autoimmune diseases, in which the immune system responds to endogenous ligands and induces abnormal responses targeted against the host's own tissues. of note, it is very difficult to assign certain variants, a specific role in the protection or induction of autoimmunity. to assign changes in the genetic landscape of human populations to certain diseases is an extraordinary challenge. moreover, our species is in constant evolutionary interaction with various microorganisms and viruses. populations of bacteria and their viruses (phages) undergo, under natural conditions, reciprocal evolution in terms of resistance and infection; this, in turn, also affects the evolutionary traits of our immune system. thus, an extraordinarily complex scenario exists in which organisms of different phyla interact, compete, and coevolve, to ensure their own survival. as novel tools, the development and refinement of methods that study large sections of the human genome, epigenome, and microbiota, will help to obtain genome-wide data in diverse human populations, allowing us to follow the evolutionary trails left from the encounters with different organisms, further unveiling the roots of human immunity. high-throughput biotechnology and an expanding computational capacity can enable the study of global population genomics and might contribute to decoding the origin and consequences of functional changes in adaptive alleles down to the single cell level. however, these methods also have limitations associated with the difficulty in linking gene variations to clinical phenotypes and disease, the generation of false positives, or the high number of samples necessary to reach reliable conclusions. expanding the heterogeneity of populations studied for immune gene association studies relevant to disease will be key, as generally, a large focus is placed on certain european or american communities, thus generating results that are difficult to extrapolate to other populations. the knowledge of the evolutionary and genetic basis of human immune traits and their impact on diverse pathologies (e.g., autoimmunity, infections, inflammatory diseases, cancer) increasingly suggests that the genetic basis of disease may be derived from a large number of rare variants of modest effect. the mechanisms described here acquire special importance in the current scenario of world globalization, in which the migration fluxes and the admixture of different populations are reaching unprecedented levels, allowing faster expansion of advantageous alleles. however, these processes may also accelerate the spread of new epidemics, as seen in the cases of hiv infection, or more recently, sars-cov, ebola, and chikungunya viruses; as well as the emergence of multiresistant bacteria and fungi, such as methicillin-resistant s. aureus or candida auris. this is just the starting point to unveil the evolutionary history of the relationships between pathogens, the immune system, and humans. further investigation of the functional adaptations of human populations is warranted to provide a broad picture of the functional consequences of evolution in human immunity. acknowledgments m.g.n. is supported by a spinoza grant of the netherlands organization for scientific research and an erc advanced grant (#833247). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. we thank laszlo a. groh for which are the strongest evolutionary forces that drive the evolution of the human innate immune system? how long does the genome of a given population take to adapt to a new infectious threat? are the mechanisms of resistance to infection transmitted only via genetic modifications, or can epigenetic adaptations to resistance also be transmitted to the progeny, and under what circumstances? the development of single-cell sequencing technologies has opened new fields of study. how does genetic variation of the expression of a specific variant vary between different cell subsets and how does it affect the overall phenotype of an individual within a population? are there specific immune processes that are preferentially impacted by evolutionary pressures? in western societies we enjoy a life expectancy vastly superior to that of our predecessors, but at the same time, we suffer diseases that they did not suffer. can some of the reasons for these changes lie among some of those bacteria that we have somehow lost in our microbiome? identifying these bacteria and understanding their effects on the human body might be the first step to developing putative therapies based on bacterial restoration. since many of the variants causing autoimmune diseases are linked to an enhanced responsiveness to pathogens that is no longer needed in developed countries, could these genes and their related pathways be employed as targets for new putative therapeutic approaches against inflammatory/ autoimmune syndromes? are other newly described genetic regulatory pathways, such as interfering rna or long noncoding rnas, also influenced by pathogen-driven evolutionary processes in different populations globally? proofreading and correction of the manuscript. we additionally thank srinivas agra for providing the figure icon 'bacteria' as deposited on https://thenounproject.com. natural selection and infectious disease in human populations genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes 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genetic analysis of c-reactive protein in african american and hispanic american women the prevalence of atherosclerosis in those with inflammatory connective tissue disease by race, age and traditional risk factors group differences in proneness to inflammation the contribution of natural selection to present-day susceptibility to chronic inflammatory and autoimmune disease an integrated catalog of reference genes in the human gut microbiome gut microbiome of coexisting baaka pygmies and bantu reflects gradients of traditional subsistence patterns gut microbiota is associated with obesity and cardiometabolic disease in a population in the midst of the good viruses: viral mutualistic symbioses virolution: the most important evolutionary book since dawkins' selfish gene to erv is human: a phenotype-wide scan linking polymorphic human endogenous retrovirus-k insertions to complex phenotypes regulatory evolution of innate immunity through co-option of endogenous retroviruses cytomegalovirus infection in hiv-infected and uninfected individuals is characterized by circulating regulatory t cells of unconstrained antigenic specificity hepatitis a virus infection suppresses hepatitis c virus replication and may lead to clearance of hcv bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection pro-and antiinflammatory responses of peripheral blood mononuclear cells induced by staphylococcus aureus and pseudomonas aeruginosa phages host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease genetics of autoimmune diseases: insights from population genetics common risk alleles for inflammatory diseases are targets of recent positive selection signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution a map of recent positive selection in the human genome genes associated with sle are targets of recent positive selection end-stage renal disease in african americans with lupus nephritis is associated with apol1 systemic lupus erythematosus-associated defects in the inhibitory receptor fc riib reduce susceptibility to malaria inherited determinants of crohn's disease and ulcerative colitis phenotypes: a genetic association study newly identified genetic risk variants for celiac disease related to the immune response evolutionary and functional analysis of celiac risk loci reveals sh2b3 as a protective factor against bacterial infection card15/nod2, cd14 and toll-like 4 receptor gene polymorphisms in saudi patients with crohn's disease association between polymorphisms in the toll-like receptor 4, cd14, and card15/nod2 and inflammatory bowel disease in the greek population the effects of growing up on a farm on adult lung function and allergic phenotypes: an international population-based study innate immunity and asthma risk in amish and hutterite farm children a causal mechanism for childhood acute lymphoblastic leukaemia association between helicobacter pylori infection and multiple sclerosis: a systematic review and meta-analysis is toxoplasma gondii infection protective against multiple sclerosis risk? a critical view on transgenerational epigenetic inheritance in humans the pirna pathway responds to environmental signals to establish intergenerational adaptation to stress within and transgenerational immune priming in an insect to a dna virus epigenetic inheritance at the agouti locus in the mouse genetic and epigenetic variation among inbred mouse littermates: identification of inter-individual differentially methylated regions chronic high-fat diet in fathers programs b-cell dysfunction in female rat offspring physiological resistance of grasshopper mice (onychomys spp.) to arizona bark scorpion (centruroides exilicauda) venom persistent epigenetic differences associated with prenatal exposure to famine in humans dna methylation as a mediator of the association between prenatal adversity and risk factors for metabolic disease in adulthood transgenerational effects of prenatal exposure to the 1944-45 dutch famine analysis of bottlenecks in experimental models of infection haldane's the causes of evolution and the modern synthesis in evolutionary biology the genome of the offspring of a neanderthal mother and a denisovan father timing of archaic hominin occupation of denisova cave in southern siberia on the origin of modern humans: asian perspectives early human dispersals within the americas dating the origin of the ccr5-d32 aids-resistance allele by the coalescence of haplotypes long-term control of hiv by ccr5 delta32/delta32 stem-cell transplantation evaluating plague and smallpox as historical selective pressures for the ccr5-delta 32 hiv-resistance allele reappraisal of the historical selective pressures for the ccr5-d32 mutation convergent evolution in european and rroma populations reveals pressure exerted by plague on toll-like receptors small amounts of archaic admixture provide big insights into human history malaria in east african highlands during the past 30 years: impact of environmental changes common west african hla antigens are associated with protection from severe malaria full-exon resequencing reveals toll-like receptor variants contribute to human susceptibility to tuberculosis disease multiple common variants for celiac disease influencing immune gene expression epitope selection for hla-dq2 presentation: implications for celiac disease and viral defense card15/nod2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease association of nod2 leucine-rich repeat variants with susceptibility to crohn's disease nod1 and nod2: beyond peptidoglycan sensing global, regional and countrylevel 90-90-90 estimates for 2018: assessing progress towards the 2020 targe key: cord-283152-wav0d0ws authors: patel, sanjay k. s.; lee, jung-kul; kalia, vipin c. title: deploying biomolecules as anti-covid-19 agents date: 2020-06-09 journal: indian j microbiol doi: 10.1007/s12088-020-00893-4 sha: doc_id: 283152 cord_uid: wav0d0ws severe acute respiratory syndrome coronavirus (sars-cov-2) known as covid-19 has emerged as a major threat to human existence. covid-19 seems to have undergone adaptive evolution through an intermediate host, most likely bats. the flu leads to severe pneumonia that causes respiratory and multi-organ failure. the absence of any known treatment procedures, drugs, or vaccines has created panic around the world. the need is to develop rapid testing kits, drugs and vaccines. however, these proposals are time-consuming processes. at present social distancing along with previously known traditional medicines can act as quick and short-term alternatives for treating this viral flu. biologists have revealed that the populations of more than 30% of all vertebrates have been declining during the last few centuries. it has been realized that there is a need to curb the basic drivers for these immense losses. there have been cases of catarina pupfish going extinct without making news headlines. christmas island pipistrelle, a vesper bat vanished without a whisper. human races have been under threat of ''extinction'' having seen some of the worst pandemic disasters. human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids) has taken around 36 million human lives during the last four decades. even at present, more than 30 million people are living with hiv. constant efforts to find treatments and generating awareness has helped to decline the death rate due to hiv/aids from 2.2 million to 1.6 million (https://www.mphonline.org/worst-pandemicsin-history/). the flu pandemic of 1968 caused due to influenza a virus spread from hong kong to vietnam, india, the philippines, singapore, europe, the united states, and australia, with less than 3 weeks of its first report. despite a low mortality rate of 5%, it resulted in killing 1 million people, of which 50% were residents of hong kong. from 1956 to 1958, asian flu caused by influenza a (subtype h2n2) with its origin in china caused 2 million deaths in china, hong kong, singapore, and the united states (https://www.mphonline.org/worst-pan demics-in-history/). among the worst pandemics recorded during the twentieth century was the flu of 1918-1920, which infected around 33% of the world's population resulting in a death toll of 20-50 million people, equal to a mortality rate of 10-20%. the most disturbing aspect was that it struck even healthy young adults. prior to that, between 1910 and 1911, cholera pandemic with its origin in india was spread to russia, eastern europe, north africa, and the middle east. it had taken the lives of more than 0.8 million people with more than 90% being from india alone (https://www.who.int/). in the nineteenth century, influenza and cholera have been striking a human being quite frequently causing millions of deaths (https:// www.who.int/). the emergence of a novel severe acute respiratory syndrome coronavirus (sars-cov-2, renamed as covid19) in 2019 from wuhan, china has led to a global crisis and it has been declared as a pandemic emergency by world health organization (who) due to its fast rate of transmission among human beings [1, 2] . sars-cov-2 exhibits unique spiked protein (s glycoprotein) and belongs to the family of coronaviridae [3] . about 27,000 viral genomic sequences are available via the global initiative on sharing all influenza data (https:// www.gisaid.org/). sars-cov-2 enters the host cell by attaching s glycoproteins to angiotensin-converting enzyme 2 (ace2) that is mediated via serine protease tmprss211 [3] . the possible candidate drugs have been based on (1) inhibition of binding ace2 to sars-cov-2 via inhibition of the ace2 and tmprss211, (2) polyclonal antibody against s glycoproteins, and (3) inhibition of replication [3, 4] . non-availability of any treatment procedures, drugs, or vaccines has created panic around the world. in this review, we have described the present scenario on covid-19 and discussed the use of traditional medicines-based biomolecules in its mitigation. covid-19 (previously labelled as 2019-ncov) is the main causative organism responsible for acute pneumonia. coronaviruses (covs) are a group of genetically distinct viruses, which originated from broad ranges of hosts, including animal and bird species, and primarily cause respiratory and intestinal infections to humans and animals [1, [5] [6] [7] [8] . in 2002-2003, covs surfaced as a severe acute respiratory syndrome (sars) with cases of 'atypical pneumonia' in china and hong kong [9] . sars originating from bats caused a major threat to humans through interspecies transmission through another host such as raccoon dogs or himalayan palm civets. it led to around 8000 infections in 26 countries and a high fatality rate of 10%. in 2012, covs of animal origin-known as middle east respiratory syndrome coronavirus (mers) emerged with rarely transmitting history between humans [9] . it had a much higher fatality rate up to 34.4% globally till november 2019. these infections posed a severe public health risk and caused substantial damage to the economy of those e affected. for genetically heterogeneous sarslike covs (alphacoronavirus and betacoronavirus) bats seems to be the natural reservoirs, in different parts of china. these covs are well-known for their emergence from natural hosts [7, 9] . transmission of covid-19 possibly involved an adaptive evolution through an intermediate host (bat) before infecting humans. although the exact mechanism of transmission of covid-19 to humans is yet to be elucidated. -however, the primary evidence to support this route is the genomic identity of up to 96% nucleotides with a bat (betacov/ratg13/2013) [7] . the phylogenic comparison has also pointed out towards convergent evolution or multiple recombination events, between the two virus strains of diverse evolutionary origins. the major determining factor of the cell tropism is the spike protein, leading to the transmission of covs among different species [3, 7] . in fact, the virus is bound to the cellular receptor through this protein, which in turn catalyzes its entry by membrane fusion. although, no antiviral agents are known to act against this virus, however, monoclonal antibodies and convalescent sera are known to inhibit mers or sars in model systems. however, these sera and antibodies may not prove very effective in handling the present outbreak [7] . in the recent outbreak of covid-19, with possible transmission from bats, initially had shown pneumonia-like symptoms with unspecified etiology [10] . covid-19 infected person showed flu-like symptoms with fever, cough, headache, muscular soreness, and dyspnea with an incubation period of about 2-24 days [1, 5, 7] . in a few cases, patients exhibited atypical symptoms, including vomiting and diarrhea. the epidemiology curve of covid-19 broadly has been classified in different phases: (1) the start of the outbreak through exposure at a market (december 2019) to new cases (41 confirmed) beyond wuhan, china (january 13, 2020) between humans transmission occurrence by close contact, (2) a rapid increase in confirmed cases by spreading of the virus within hospitals and family transmission outside china, which was nearly 20-folds than phase first till january 23, 2020 [7] . this resulted in the traditional mass movement of people (about 5 million people), which has contributed significantly in spreading of confirmed cases of covid-19, and (3) finally, a cluster of cases marked on january 26, 2020 was the beginning of the third phase. the global covid-19 mortality rate of 3.4% has been reported by who [7] (tables 1, 2) covs are viruses with single stranded rna genome sizes between 26 to 32 kb [11] . initially, the screening of covid-19 infection has done through measurements of temperature and related symptoms. the laboratory testing for covid-19 detection includes methods based on the presence of the virus and the measurement of antibodies (serology) produced in response to infection [11] . the virus was confirmed using:-(1) pcr based reverse transcription (rt)-pcr detects covs specific rna from either nasopharyngeal and throat swabs or sputum samples within a few hours to 2 days, and (2) high throughput nucleic acid sequencing, (3) non-pcr based technique of isothermal nucleic acid amplification has a testing period of 5 min [11] [12] [13] . similarly, chest ct scans and radiographs to detect it were not found to be accurate for covid-19 confirmation at early stages [11] . the antibody test targets the production of antibodies such as iga, igm, and igg in response to covid-19 infection in blood samples, which takes around 14 days after infection or needs to wait for the onset of symptoms. the procedure, however, takes only 15 min to detect the infection. various approaches are under investigation for developing a rapid and low-cost detection kit with high specificity and selectivity for covid-19 [11] [12] [13] . the infections of covid-19 are not selective to any age group, such that even newborn infants have been among the confirmed cases. covs genomes exhibit high genomic plasticity enabling it to possess high recombination capacities [7] . these features have led to a high probability of adaptive mutations for utilizing multiple cellular receptors for efficient binding and entry by spike proteins into various hosts. this ability of covs may assume an alarming tendency to expand to wider host-species. since, multiple species of covs are present in wildlife animals, who constantly interact among themselves, their expansion or transmission to humans is inevitable. this feature evidenced by the transmission of covid-19 from humans to dogs, cats, and even tigers. thus, surveillance is expected to prevent the virus from establishing in other hosts, which live in the vicinity of human beings [14, 15] . covid-19 has exhibited a much higher infection than sars or mers cases. initially, to minimize the global spread after cases in china various approaches such as airport screening and traveling restrictions were largely implemented in a few countries [1, 16] . due to the asymptomatic behavior of covid-19 such as a longer incubation period suggested that quick tracking of the infected person is essential for limiting the human-to-human transmissions. the initial quaternity period of about 14 days for travelers was considered desirable [16] . but, even longer periods for incubations were required for visualizing of symptoms made the realization that it was a more critical scenario for its spread. the minimization of social interaction by limiting activities at workplaces, education centers public functions were suggested to be more effective in reducing the rapid spreading of infection [1, 2, 16] . early enough, it was observed that age groups and the frequency of physical interaction may be influenced by geographical locations. computational analysis of the data generated from measures of social distancing at the workplace, educational institutes, and lock-down suggested that either a 21 days lockdown or periodic shutdowns of 21 and 28 days, may prove effective to counter the covid19 infection in countries like india. alternatively, periodic two lockdowns of 3-4 weeks followed by a shorter lock-down with a gap of 5 days, or a long-term lock-down of 7 weeks may help to minimize the transmission of this infection among human beings [16] . these four approaches are likely to yield different -mortality cases with a maximum of 2727, in the first strategy followed by 11, 8, and 6 in the later three approaches [16] . it thus implied that social distancing along with lock-down can counter covid infection in any part of the world, including india, where medical facilities are limited. at present, no vaccines or drugs are available for specifically targeting this rapidly spreading disease. the most common practices are supportive care in combination with certain previously known but non-specific drugs. these seem to be the only options, at present. the strategy being envisaged right now is to take advantage of the similarities in the following features -epidemiological, genomic, and pathogenetic-of the sars-cov and sars-cov-2. conventionally sars-cov-2 infection are treated using a combination of therapeutic agents: (1) ventilation for oxygen supply, (2) combinations of antibiotics amoxicillin, azithromycin, and fluoroquinolones, (3) antivirals such as chloroquine, favipiravir (t-705), interferon, lopinavir/ritonavir, oseltamivir, remdesivir, and ribavirin, (4) corticosteroids, and (5) convalescent plasma [17] . among these various possibilities, remdesivir may prove to be an effective drug for handling covid-19, as it has been already shown to be helpful in treating rna viruses such as mers and sars [18, 19] . certain methods like antibody and plasma therapy through the donation of plasma by covid-19 patients had been adapted in the cases of covs such as mers and sars. other possibilities are using the neutralizing potential of monoclonal antibodies [11] . melatonin has been suggested as a potent drug for this treatment [20] . ivermectin seems to have the potential to inhibit covid-19 in vitro [21] . few pharmaceutical drugs, including hiv drugs and stem cells are under clinical trials, however, there are still major challenges to be handled [11, [20] [21] [22] [23] [24] [25] . invivo biosystem has developed ace2 humanized genetic models using caenorhabditis elegans for covid-19 research (https://tgx.elegans@invivobiosys tems.com). in the absence of the availability of specific anti-viral drugs or vaccines, a few asian countries such as china, thailand, and india have been relying on the use of traditional medicines. this expertise gained over at least a few centuries can have a short-term effect on covid-19. almost 85% of covid-19 patients in china are treated using traditional medicines such as herbal formulations yu ping feng san and sang ju yin, which modulate the t-cells and enhance host defense mechanisms [17, 26] . a few other combinations of traditional medicines such as lian hua qing wen capsule, shuang huang lian, and ma xin gan shi tang can be expected to be effective as therapeutics against viral infections [17, 27, 28] . many traditional herbal extracts and compounds have shown potential activity against cov, these include: (1) (6) for prevention such as fortunes bossfern rhizome [19] . liquorice root (glycyrrhiza glabra) a native of europe and asia showed an anti-viral property towards sars-covs that might be beneficial in this treatment because of the phytochemicals, including flavonoids (glycyrrhizin, liquiritigenin, and glabridin) and triterpenoids (glycyrrhizic acid, and glycyrrhetinic acid) [29] . thailand's medical board has recommended licorice root for covid-19 treatment. more efforts are needed untill vaccine discovery takes place. indian ministry of ayurveda, yoga and naturopathy, un ani, siddha, and homoeopathy (ayush) also advise on the use of traditional medicine arsenicum album although not supported by strong scientific evidence. intake of anti-malarial drug ayush 64 (without side effects), sesame oil in the nose, and tulasi, ginger, guduchi (tinospora cordifolia, and turmeric in the diet have been suggested. ayurveda has worked towards enhancing immunity against a host of infections and homeopathy has been reported for treating cholera, spanish influenza, yellow fever, and typhoid. it was also offered during the outbreak of ebola in 2014 (guinea, west africa) due to a lack of vaccine or anti-virals (https://m.economictimes.com/ news). infectious diseases are caused by pathogens, which have resistance to antibiotics. genomics plays a crucial role in diverse biotechnological applications including developing antipathogens [33] [34] [35] [36] [37] [38] [39] . the emergence of covid-19 continues to plague the whole world. from about 212 countries, over one million confirmed cases with a high mortality rate of 5.5% have been reported (https:// www.worldometers.info/coronavirus/). lock-down for social distancing in mitigating covid-19 has been found to be suitable for preventing pandemic scenario but it involves a heavy economic burden on the maintenance of essential services for health [40] . this strategy offers a short-term relief in delaying the transmissions. for a permanent remedy, investigations need to continue: (1) rapid and reliable testing kits, (2) short-term therapeutics and, and (3) finally vaccine for complete eradication. genomic data are likely to provide insights into the evolutionary trends and their potential transmission among diverse hosts. apart from various alternative approaches, traditional medicines known since long for curing such infections without side effects may prove beneficial. understanding sars-cov-2: genetic diversity, transmission and cure in human coronavirus 2019-ncov: a brief perspective from the front line candidate drugs against sars-cov-2 and covid-19 natural product-derived phytochemicals as potential agent against coronaviruses. a review comparative genomic analysis of rapidly evolving sars cov-2 viruses reveal mosaic pattern of phylogeographical distribution world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) covid-19: epidemiology, evolution, and cross-disciplinary perspectives the possible of immunotherapy for covid-19: a systematic review running title: immunotherapy for covid-19 coronavirus disease 2019: coronaviruses and blood safety review of the 2019 novel coronavirus (covid-19) based on current evidence molecular immune pathogenesis and diagnosis of covid-19 covid-19 infection: origin, transmission, and characterization of human coronaviruses potential interventions for novel coronavirus in china: a systemic review bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond mutated covid-19, may foretells mankind in a great risk in the future age-structured impact of social distancing on the covid-19 epidemic in india traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus covid-19: melatonin as a potential adjuvant treatment the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro coronavirus disease (covid-19): a primer for emergency physicians new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? treatment options for covid-19: the realty and challenges severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges yu ping feng san, an ancient chinese herbal decoction, induces gene expression of anti-viral proteins and inhibits neuraminidase activity immunomodulatory effects of a traditional chinese medicine with potential antiviral activity: a self-control study the chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of viral propagation and impacts immune function liquorice may tackle sars identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 natural bis-benzylisoquinoline alkaloids-tetrandrine, fangchinoline, and cepharanthine, inhibit human coronavirus oc43 infection of mrc-5 human lung cells inhibition of sars-cov 3cl protease by flavonoids quorum sensing inhibitors: an overview quorum sensing inhibitors as antipathogens: biotechnological applications quenching the quorum sensing system: potential antibacterial drug targets genomic analysis reveals versatile organisms for quorum quenching enzymes: acyl-homoserine lactone-acylase and-lactonase evolution of resistance to quorum-sensing inhibitors extending the limits of bacillus for novel biotechnological applications inhibition of microbial quorum sensing mediated virulence factors by pestalotiopsis sydowiana a lesson learned from the outbreak of covid-19 in korea key: cord-308201-lavcsqov authors: desforges, marc; le coupanec, alain; dubeau, philippe; bourgouin, andréanne; lajoie, louise; dubé, mathieu; talbot, pierre j. title: human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? date: 2019-12-20 journal: viruses doi: 10.3390/v12010014 sha: doc_id: 308201 cord_uid: lavcsqov respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. however, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza a and sars-cov, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. for a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (cns). viruses infecting human cns cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the cns as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to cns cells (virus-induced neuropathology). the etiological agent of several neurological disorders remains unidentified. opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in cns infection, with a special emphasis on human coronaviruses. the central nervous system (cns), a marvel of intricate cellular and molecular interactions, maintains life and orchestrates homeostasis. unfortunately, the cns is not immune to alterations that lead to neurological disease, some resulting from acute, persistent or latent viral infections. several viruses have the ability to invade the cns, where they can infect resident cells, including the neurons [1] . although rare, viral infections of the cns do occur [2] . however, their incidence in clinical practice common virus, is associated with febrile illness, fever, cough and congestion [97, 98] , as well as a characteristic rash and koplik's spots [99] . in rare circumstances, significant long-term cns diseases, such as [99] post-infectious encephalomyelitis (pie) or acute disseminated encephalomyelitis (adem), occur in children and adolescents. other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (mibe), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (sspe) that appears 6-10 years after infection [100] . yet, with the exception of hiv, no specific virus has been constantly associated with specific human neurodegenerative disease. on the other hand, different human herpes viruses have been associated with alzheimer's disease (ad), multiple sclerosis (ms) and other types of long-term cns disorders [101] [102] [103] . as accurately stated by majde [104] , long-term neurodegenerative disorders may represent a "hit-and-run" type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections [83, 103, [105] [106] [107] . including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods [8, 70, [108] [109] [110] . however, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of cns viral infections often remains unknown. indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases [110, 111] . among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents [104] . respiratory syncytial virus (rsv), a member of the orthopneumovirus genus [112] , infects approximately 70% of infants before the age of 1 and almost 100% by the age of 2 years old [113] , making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide [32, 114] . recent evidence also indicates that severe respiratory diseases related to rsv are also frequent in immunocompromised adult patients [8, 115] and that the virus can also present neuroinvasive properties [8] . over the last five decades, a number of clinical cases have potentially associated the virus with cns pathologies. rsv has been detected in the cerebrospinal fluid (csf) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] . furthermore, rsv is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the cns in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments [127] . an additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hmpv). discovered at the beginning of the 21st century in the netherlands [128] , it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals [129] . during the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. viral material was detected within the cns in some clinical cases of encephalitis/encephalopathy [130] [131] [132] [133] [134] but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hmpv neuroinvasion and potential neurovirulence. hendra virus (hev) and nipah virus (niv) are both highly pathogenic zoonotic members of the henipavirus genus and represent important emerging viruses discovered in the late 1990s in australia and southern asia. they are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage [135] [136] [137] [138] . although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for hev and the pig for niv as a first step before crossing the barrier species towards humans [135] . in humans, it can lead to different types of encephalitis, as several types of cns resident cells (including neurons) can be infected [139, 140] . the neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection [141] [142] [143] . the use of animal models showed that the main route of entry into the cns is the olfactory nerve [144] and that the nipah virus may persist in different regions of the brain of grivets/green monkeys [145] , reminiscent of relapsing and late-onset encephalitis observed in humans [146] . influenza viruses are classified in four types: a, b, c and d. all are endemic viruses with types a and b being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. they are responsible for seasonal epidemics that affect 3 to 5 million humans, among which 500,000 to 1 million cases are lethal each year [147, 148] . associated with all major pandemics since the beginning of the 20th century, circulating influenza a presents the greatest threat to human health. most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ards) [30, 149] and complications involving the cns [150] [151] [152] . several studies have shown that influenza a can be associated with encephalitis, reye's syndrome, febrile seizure, guillain-barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (adem) [153] [154] [155] [156] [157] [158] . animal models have shown that, using either the olfactory route or vagus nerve, influenza a virus may have access to the cns and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae [8, 69, [159] [160] [161] [162] . the influenza a virus has also been associated with the risk of developing parkinson's disease (pd) [151] and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (eae), which is reminiscent of the observation that multiple sclerosis (ms) relapses have been associated with viral infections (including influenza a) of the upper respiratory tract [163] [164] [165] . another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (pv), coxsackieviruses (cv), echoviruses, human rhinoviruses (hrv) and enteroviruses (ev). this genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (pv, ev-a71 and -d68, and to a lesser extent hrv) can invade and infect the cns, with detrimental consequences [166] [167] [168] [169] . even though extremely rare, hrv-induced meningitis and cerebellitis have been described [170] . although ev infections are mostly asymptomatic, outbreaks of ev-a71 and d68 have also been reported in different parts of the world during the last decade. ev-a71 is an etiological agent of the hand-foot-mouth disease (hfmd) and has occasionally been associated with upper respiratory tract infections. ev-d68 causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes [171] . both serotypes have been associated with neurological disorders like acute flaccid paralysis (afp), myelitis (afm), meningitis and encephalitis [166, [172] [173] [174] [175] . last but not least, human coronaviruses (hcov) are another group of respiratory viruses that can naturally reach the cns in humans and could potentially be associated with neurological symptoms. these ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses [176] like phev (porcine hemagglutinating encephalitis virus) [177] , fcov (feline coronavirus) [178, 179] and the mhv (mouse hepatitis virus) strains of mucov [180] , which can all reach the cns and induce different types of neuropathologies. mhv represents the best described coronavirus involved in short-and long-term neurological disorders (a model for demyelinating ms-like diseases) [181] [182] [183] . taken together, all these data bring us to consider a plausible involvement of hcov in neurological diseases. the first strains of hcov were isolated in the mid-60s from patients presenting an upper respiratory tract disease [184] [185] [186] [187] . before the severe acute respiratory syndrome (sars) appeared in 2002 and was associated with sars-cov [188] [189] [190] , only two groups of hcov, namely hcov-229e (previous group 1, now classified as alphacoronavirus) and hcov-oc43 (previous group 2, now classified as betacoronavirus) were known. several new coronaviruses have now been identified, including three that infect humans: alphacoronavirus hcov-nl63 [191] and betacoronaviruses hcov-hku1 and mers-cov [192, 193] . the hcov-229e, -oc43, -nl63 and -hku1 strains are endemic worldwide [31, 184, [194] [195] [196] [197] [198] [199] and exist in different genotypes [200] [201] [202] [203] [204] [205] [206] [207] . in immunocompetent individuals they usually infect the upper respiratory tract, where they are mainly associated with 15-30% of upper respiratory tract infections (uri): rhinitis, laryngitis/pharyngitis as well as otitis. being highly opportunistic pathogens [14] , hcov can reach the lower respiratory tract and be associated with more severe illnesses, such as bronchitis, bronchiolitis, pneumonia, exacerbations of asthma and respiratory distress syndrome [17, 31, [208] [209] [210] [211] [212] [213] [214] . the 2002-2003 sars pandemic was caused by a coronavirus that emerged from bats (first reservoir) [25] to infect palm civets (intermediary reservoir) and then humans [215] . a total of 8096 probable cases were reported and almost 10% (774 cases in more than 30 countries) of these resulted in death [216] [217] [218] . the clinical portrait was described as an initial flu-like syndrome, followed by a respiratory syndrome associated with cough and dyspnea, complicated with the "real" severe acute respiratory syndrome (sars) in about 20% of the patients [31,219]. in addition, multiple organ failure was observed in several sars-cov-infected patients [220] . in the fall of 2012, individuals travelling from the arabian peninsula to the united kingdom were affected by the middle-east respiratory syndrome (mers), a severe lower respiratory tract infection that resembled sars, leading also to gastrointestinal symptoms and renal failure among some patients [221] . molecular sequencing rapidly showed that the new epidemic was caused by a new coronavirus: the mers-cov [193, 222, 223] . mers-cov most probably originated from bats before infecting an intermediary reservoir (the dromedary camel), and also represented a zoonotic transmission to humans. phylogenetic analyses suggest that there have been multiple independent zoonotic introductions of the virus in the human population. moreover, nosocomial transmission was observed in multiple hospitals in saudi arabia [221, [224] [225] [226] [227] [228] [229] [230] . although possible, human-to-human mers-cov transmission appears inefficient as it requires extended close contact with an infected individual. consequently, most transmission have occurred among patients' families and healthcare workers (clusters of transmission). a more efficient human-to-human transmission was observed in south korea, during the 2015 outbreak of mers-cov [231, 232] . even though it has propagated to a few thousand people and possesses a high degree of virulence, mers-cov seems mostly restricted to the arabic peninsula and is not currently considered an important pandemic threat. however, virus surveillance and better characterization are warranted, in order to be prompt to respond to any change in that matter [23, [233] [234] [235] . as of october 8, 2019, the world health organization (who) reported that mers-cov had spread to at least 27 different countries, where 2468 laboratory-confirmed human cases have been identified with 851 being fatal (https://www.who.int/emergencies/mers-cov/en/). as observed for the four circulating strains of hcov [31, 194] , both sars-cov and mers-cov usually induce more [228, 236] severe illnesses, and strike stronger in vulnerable populations such as the elderly, infants, immune-compromised individuals or patients with comorbidities [31,237]. over the years, like sars-and mers-cov, the four endemic hcov have also been identified as possible etiological agents for pathologies outside the respiratory tract. indeed, myocarditis, meningitis, severe diarrhea (and other gastrointestinal problems) and multi-organ failure [220, 221, [238] [239] [240] [241] have been reported, especially in children. recent investigations on hcov as enteric pathogens demonstrated that all hcov strains can be found in stool samples of children with acute gastroenteritis; however, no evidence of association could yet be clearly demonstrated with disease etiology [242, 243] . different reports also presented a possible link between the presence of hcov within the human central nervous system (cns) and some neurological disorders among patients examined [244] [245] [246] [247] [248] [249] . like all viruses, hcov may enter the cns through the hematogenous or neuronal retrograde route. in the human airways, hcov infection may lead to the disruption of the nasal epithelium [250] and, although they bud and are released mostly on the apical side of the epithelial cells, a significant amount of viruses is also released from the basolateral side [251] . thus, although hcov infections are, most of the time, restricted to the airways, they may under poorly understood conditions pass through the epithelium barrier and reach the bloodstream or lymph and propagate towards other tissues, including the cns [33, 38, 208, 252] ; this was also suggested for other respiratory viruses that can reach the human cns, namely, rsv [8, 53] [257, 258] . moreover, persistently-infected leukocytes [252] may serve as a reservoir and vector for neuroinvasive hcov [245] . therefore, neuroinvasive hcov could use the hematogenous route to penetrate into the cns. the second form of any viral spread towards the cns is through neuronal dissemination, where a given virus infects neurons in the periphery and uses the machinery of active transport within those cells in order to gain access to the cns [35, 36] . although the olfactory bulb is highly efficient at controlling neuroinvasion, several viruses have been shown to enter cns through the olfactory route [259, 260] . after an intranasal infection, both hcov-oc43 and sars-cov were shown to infect the respiratory tract in mice and to be neuroinvasive [261] [262] [263] [264] [265] . over the years, we and others have gathered data showing that hcov-oc43 is naturally neuroinvasive in both mice and humans [244, 245, 261, 263, 266] . experimental intranasal infections of susceptible mice also indicate that, once it has invaded the cns, the virus disseminated to several regions of the brain and the brainstem before it eventually reaches the spinal cord [266] [267] [268] . furthermore, based on more recent work [269] , figure 1 illustrates the olfactory route, which is clearly the main route of neuroinvasion used by hcov-oc43, as well as the early steps of subsequent neuropropagation within the cns in susceptible mice and recapitulates the suggested equivalent pathway in humans. nevertheless, our data suggest that hcov-oc43 may also invade the cns from the external environment through other pathways involving other cranial peripheral nerves [269] , reminiscent of what was shown for other human respiratory viruses such as rsv and influenza virus [8] . therefore, on the one hand, an apparently innocuous human respiratory pathogen such as the hcov may reach the cns by different routes and induce short-term illnesses, such as encephalitis. on the other hand, it may persist in resident cells of the human cns and may become a factor or co-factor of neuropathogenesis associated with long-term neurological sequelae in genetically or otherwise predisposed individuals. because of their natural neuroinvasive potential in humans and animals, a possible association between the presence of ubiquitous human coronaviruses in the triggering or exacerbation of neurological human pathologies has often been suggested over the years. it is now accepted that hcov are not always confined to the upper respiratory tract and that they can invade the cns [220, 245, 248, 270] . as other viruses listed herein, hcov are neurotropic and potentially neurovirulent. even though no clear cause and effect link has ever been made with the onset of human neurological diseases, their neuropathogenicity is being increasingly recognized in humans, as several recent reports associated cases of encephalitis [244] , acute flaccid paralysis [271] and other neurological symptoms, including possible complications of hcov infection such as guillain-barré syndrome or adem [249, [272] [273] [274] [275] [276] [277] [278] [279] . the presence and persistence of hcov in human brains was proposed to cause long-term sequelae related to the development or aggravation of chronic neurological diseases [245] [246] [247] [248] [280] [281] [282] . given their high prevalence [31, 283] , long-term persistence and newly recognized neuropathogenesis, hcov disease burden could currently be underestimated. this suggest that better surveillance, diagnoses and deepened virus-host interactions studies are warranted in order to gather more knowledge that will make possible the development of therapeutic strategies to prevent or treat occurrences. potential short-term neuropathologies sars-cov, hcov-oc43 and -229e are naturally neuroinvasive and neurotropic in humans and therefore potentially neurovirulent [220, 244, 245, 249, 270, 271] . furthermore, animal models showed that sars-cov could invade the cns primarily through the olfactory route [265] or even after an intra-peritoneal infection [284] , and induce neuronal cell death [265, 284] . to our knowledge, no reports on the presence of the three other coronaviruses that infect humans in the cns have been published. however, neurological symptoms have been described in patients infected by all three viruses [276, 277, 285] . making use of our in vivo model of hcov neuropathogenesis, relying on the natural susceptibility of mice to hcov-oc43-the most prevalent strain among endemic hcov [210, 286] -encephalitis and transient flaccid paralysis associated with propagation towards the spinal cord and demyelination and long-term persistence in surviving mice were observed [261, [266] [267] [268] [287] [288] [289] ; thus, recapitulating the neurological afflictions reported in some patients infected by hcov [244, 249, 271, 272, [275] [276] [277] 290] . although we must interpret data obtained in rodents with all the caution dictated by the use of a non-human host, it is likely that the underlying mechanisms described will have relevance to the human situation or at least provide leads to investigate neurotropic hcov in humans. in susceptible mice, hcov-oc43 has a selective tropism for neurons in which it is able to use axonal transport as a way of neuron-to-neuron propagation [269] . these results, together with data harvested with the use of microfluidic devices (xona microfluidic), helped to elaborate a putative model of propagation adapted from tomishima and enquist [291] , in which infectious hcov-oc43 could either be assembled in the cell body or at different points along the axon using the anterograde axonal transport to propagate between neurons or from neurons to glial cells surrounding neurons in the cns (figure 2 ). furthermore, based on previous data using different mutant recombinant viruses harboring mutation in the s protein [266, 268, 269] and making use of a luciferase expressing recombinant hcov-oc43 [292] [293] [294] , we are now showing that the rate and success of virus propagation towards the spinal cord, in part through the neuron-to-neuron pathway, correlates with the exacerbation of neurovirulence ( figure 3 ). [269] and adapted from tomishima and enquist [291] . in this model, solid arrows represent fully assembled virus transport and dashed arrows represent subvirion assemblies [291] . schematic representations were assembled with the motifolio neuroscience toolkit, 2007. [292] was injected intra-nasally (i.n.) into mice. virus spread was assessed by bioluminescence imaging (bli) with the xenogen vivo vision ivis 100 imaging system (perkin-elmer) in infected anaesthetized mice placed in a light proof specimen chamber after intraperitoneal injection of d-luciferin. images were taken with a ccd camera mounted in a light-tight imaging chamber, using the acquisition software living image version 4.3.1 (caliper-lifesciences). evaluation of associated clinical scores: (levels 0 to 4: 0 is asymptomatic; 1 is mice with early hunched back; 2 is mice presenting slight social isolation, weight loss and abnormal gait; 3 is mice presenting total social isolation, ruffled fur, hunched back, weight loss and almost no movement; and 4 is mice moribund or dead (presented elsewhere; [266] ), indicate that only mice with a positive signal at both the level of the brain and spinal cord were evaluated to be at level 2 to 3. hcov-oc43 structural and accessory proteins are important for infection and some clearly represent virulence factors [38, [266] [267] [268] [269] 289, 295, 296] . using neuronal cell cultures and our murine model, we gathered data indicating that some of these proteins also play a significant role in viral dissemination [269, 296] and now aim to exploit these promising leads to fully understand the course and determinants of propagation to and through the cns and complete the neurologic portrait of short term hcov neuropathogenesis. the presence of hcov rna in the human cns establishes the natural neuroinvasive properties of these respiratory viral agents. moreover, it also suggests that they persist in human cns [245] as they do in human neural cells [297, 298] and in the cns of mice that survive acute encephalitis. these surviving mice exhibited long-term sequelae associated with decreased activity in an open field test and a reduced hippocampus with neuronal loss in the ca1 and ca3 layers [287] , reminiscent of what was observed after infection by the influenza a virus and rsv [127, 162] and to the significant loss of synapses within the ca3 region after infection by wnv [299, 300] . the precise and complete etiology of several long-term neurological pathologies still represent a conundrum. multiple sclerosis (ms) represents one such neurological disease for which an infectious agent or agents may play a triggering role, with viruses the most likely culprit in genetically predisposed individuals [301] . it has been suggested that several neurotropic viruses could be involved in ms pathogenesis but that they may do so through similar direct and/or indirect mechanisms [302] [303] [304] [305] [306] . however, although research has not yet led to a direct link to any specific virus, association of coronaviruses with ms has been suggested [307] . even though hcov-oc43 and -229e were detected in some control brains and in some brains coming from patients with different neurological diseases, there was a significantly higher prevalence of hcov-oc43 in brains of ms patients [245] . moreover, autoreactive t cells were able to recognize both viral and myelin antigens in ms patients but not in controls during infection by hcov-oc43 and hcov-229e [308, 309] . thus, the immune response may participate in the induction or exacerbation of long-term neuropathologies such as ms in genetically or otherwise susceptible individuals. furthermore, it was shown that in recombination activation gene (rag) knock-out mice, hcov-oc43-induced encephalitis could be partially mediated by the t-cell response to infection [263] . this underlines the possibility that, like its murine counterpart mhv, long term infection of the cns by hcov [245] may participate in the induction of demyelinating ms-like lesions. immune cell infiltration and cytokine production were observed in the mouse cns after infection by hcov-oc43. this immune response was significantly increased after infection by viral variants, which harbor mutations in the viral glycoprotein (s) [267] . these variants also induced glutamate excitotoxicity [268, 289] , thus increasing damage to neurons [310] and/or disturbing glutamate homeostasis [311] and thereby contributing to neuronal degeneration and hind-limb paralysis and possible demyelination [266] [267] [268] [269] . the degeneration of neurons may eventually lead to death of these essential cells by directly generating a cytotoxic insult related to viral replication and/or to the induction of different regulated cell death (rcd) pathways [312] [313] [314] . our results indicate that the underlying mechanisms appear to involve different cellular factors and pathways of rcd, described and reviewed elsewhere [38, 315] . virus-cell interactions are always important in the regulation of cell response to infection. for hcov-oc43, we clearly showed that the viral s and e proteins are important factors of neurovirulence, neuropropagation and neurodegeneration of infected cells [267] [268] [269] 296, 312] . we have also demonstrated that the he protein is important for the production of infectious hcov-oc43 and for efficient spreading between neuronal cells, suggesting an attenuation of the eventual spread into the cns of viruses made deficient in fully active he protein, potentially associated with a reduced neurovirulence [269, 295] . coronavirus accessory proteins have been extensively studied and are now considered as important viral factors of virulence implicated in pathogenesis while counteracting innate immunity [316] [317] [318] [319] . two of these accessory proteins (ns2 and ns5) produced during infection by hcov-oc43 play a significant role in virulence and pathogenesis in the mouse cns [38] . like for several other respiratory viruses, accumulating evidence now indicate that hcov are neuroinvasive in humans and we hypothesize that these recognized respiratory pathogens are potentially neurovirulent as well, as they could participate in short-and long-term neurological disorders either as a result of inadequate host immune responses and/or viral propagation in the cns, which directly induces damage to resident cells. with that in mind, one can envisage that, under the right circumstances, hcov may successfully reach and colonize the cns, an issue largely deserted and possibly underestimated by the scientific community that has impacted or will impact the life of several unknowing individuals. in acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the nervous tissue with different outcomes depending on the infected regions [320] . as previously mentioned, hcov may persist in the human cns as it does in mice [245, 287] and potentially be associated with different types of long-term sequelae and chronic human neurological diseases. in their famous review on cns viral infection, published a few years ago, koyuncu et al. [35] insisted that, under the right conditions, all viruses can have access to the cns. what "under the right conditions" means certainly represents a subject of debate among virologist and physicians. nevertheless, as stated in the introduction of this review, viral factors (mutations in specific virulence genes), host factors (immunodepression, age) or a mixture of both (underlining the importance of virus-host interactions), are all good candidates to refer to if one intends to find the beginning of an explanation. a fast and accurate diagnosis would certainly improve prognosis for patients with a suspected cns infection. identification of a specific virus provides relevant information on how to treat a patient; therefore, the development of modern technologies, such as high throughput sequencing (next generation sequencing) are warranted as it represents a potentially unbiased marvelous tool for rapid and robust diagnosis of unexplained encephalitis or other types of encephalopathies or neuronal manifestations, especially in the context where more traditional techniques have failed to identify the etiological agent [21, 108, 111, 244, 321, 322] . therefore, although our attention is mainly on a few different viruses such as hsv, arboviruses and enteroviruses, it may now be the time to look at cns viral infection from another perspective. these viruses truly represent an important proportion of cns viral infection associated with encephalitis, meningitis, myelitis and long-term neurological disorders. nevertheless, accumulating evidence in the scientific literature strongly suggest that many other viral candidates could be underestimated in that matter. several human respiratory viruses are neuroinvasive and neurotropic, with potential neuropathological consequences in vulnerable populations. understanding the underpinning mechanisms of neuroinvasion and interaction of respiratory viruses (including hcov) with the nervous system is essential to evaluate potentially pathological short-and long-term consequences. however, viral infections related to diseases that are rare manifestations of an infection (like long term chronic neurological diseases), represent situations where koch's postulates [323] need to be modified. a series of new criteria, adapted from sir austin bradford hill, for causation [324, 325] was elaborated by giovannoni and collaborators concerning the plausible viral hypothesis in ms [326] . these criteria certainly represent a pertinent tool to evaluate the involvement of human respiratory viruses as a factor that could influence long-term human neurological diseases. to continue the gathering of epidemiological data is justified to evaluate the clear cause and effect link between neuroinvasive respiratory viruses and short-and long-term human neurological diseases. understanding mechanisms of virus neuroinvasion and interactions with the central nervous system is essential for different reasons. first, to 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ns2 protein is required for virus replication and liver pathology cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus coronaviruses as encephalitis-inducing infectious agents clinical metagenomic sequencing for diagnosis of meningitis and encephalitis human virome in nasopharynx and tracheal secretion samples the aetiology of tuberculosis (translation of die aetiologie der tuberculose (1882) sequence-based identification of microbial pathogens: a reconsideration of koch's postulates the environment and disease: association or causation? infectious causes of multiple sclerosis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank jessy tremblay for his excellent work in confocal microscopy images. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-283709-y59h5bw8 authors: chan, renee w y; hemida, maged g; kayali, ghazi; chu, daniel k w; poon, leo l m; alnaeem, abdelmohsen; ali, mohamed a; tao, kin p; ng, hoi y; chan, michael c w; guan, yi; nicholls, john m; peiris, j s malik title: tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study date: 2014-08-28 journal: lancet respir med doi: 10.1016/s2213-2600(14)70158-4 sha: doc_id: 283709 cord_uid: y59h5bw8 background: middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic infection causing severe viral pneumonia, with index cases having resided in or recently travelled to the arabian peninsula, and is a global concern for public health. limited human-to-human transmission, leading to some case clusters, has been reported. mers-cov has been reported in dromedary camels but phenotypic characterisation of such viruses is limited. we aimed to compare mers-cov isolates from dromedaries in saudi arabia and egypt with a prototype human mers-cov to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung. methods: we characterised mers-cov viruses from dromedaries in saudi arabia and egypt and compared them with a human mers-cov reference strain. we assessed viral replication kinetics and competence in vero-e6 cells (rhesus monkey), tissue tropism in cultures of ex-vivo human bronchial and lung tissues, and cytokine and chemokine induction, gene expression, and quantification of viral rna in calu-3 cells (human respiratory tract). we used mock-infected tissue as negative controls for ex-vivo experiments and influenza a h5n1 as a positive control for cytokine and chemokine induction experiments in calu-3 cells. findings: we isolated three dromedary strains, two from saudi arabia (dromedary/al-hasa-kfu-hku13/2013 [ah13] and dromedary/al-hasa-kfu-hku19d/2013 [ah19d]), and one from egypt (dromedary/egypt-nrce-hku270/2013 [nrce-hku270]). the human and dromedary mers-cov strains had similar viral replication competence in vero-e6 cells and respiratory tropism in ex-vivo cultures of the human respiratory tract, and had similar ability to evade interferon responses in the human-respiratory-tract-derived cell line calu-3. interpretation: the similarity of virus tropism and replication competence of human and dromedary mers-cov from the arabian peninsula, and genetically diverse dromedary viruses from egypt, in ex-vivo cultures of the human respiratory tract suggests that dromedary viruses from saudi arabia and egypt are probably infectious to human beings. exposure to zoonotic mers-cov is probably occurring in a wider geographical region beyond the arabian peninsula. funding: king faisal university, egyptian national research centre, hong kong food and health bureau, national institute of allergy and infectious diseases, and european community seventh framework program. middle east respiratory syndrome coronavirus (mers-cov) is a disease of public health concern globally, with 837 laboratory-confi rmed cases and 291 deaths reported to who as of july 23, 2014. 1 index-case patients have all resided in or recently travelled to the arabian peninsula or adjacent countries. travel-associated cases or secondary transmission arising from such cases have been reported in europe, north america, africa, and asia. 2 some case clusters have been associated with limited human-to-human transmission occurring within family or health-care settings, and the remaining sporadic cases are presumed to be zoonotic in origin. [3] [4] a recent increase in reported cases from saudi arabia associated with health-care facilities is a particular cause of concern. 3 establishment of the proximate zoonotic source of human infection and modes of transmission from animals to human beings remains crucial for public health. mers-cov has been detected in dromedary camels (camelus dromedarius), in association with and preceding human infection, and in dromedary slaughterhouses and farms. [5] [6] [7] [8] [9] viruses from most of the recent human cases from the arabian peninsula phylogenetically cluster within clade b, but the earliest human mers-cov strains from 2012 are genetically distinct and are denoted as clade a. 10 dromedary mers-cov strains detected in the arabian peninsula in 2012-13 were clade b viruses, phylogenetically related to viruses from human cases from the same regions, but genetically diverse (non-clade a/b) viruses were reported from egypt. 11 researchers have also reported serological evidence collected between 2009 and 2013 of mers-cov infection in dromedary camel serum samples from tunisia, nigeria, ethiopia, and egypt, suggesting that mers-cov or antigenically related viruses might be geographically more widespread than is appreciated at present. 6, 12 findings from two studies of 226 people in saudi arabia and 179 people in egypt working in dromedary abattoirs showed that all remained seronegative for mers-cov, suggesting that trans mission to humans is uncommon 11, 13 and raising the question of whether dromedary mers-cov strains have the capacity to infect human beings. full genome sequences of dromedary viruses suggest that these viruses are similar to viruses in humans. 11, 14 there were some aminoacid diff erences between the human and dromedary viruses, including in the genetic sequence of the spike protein (a major determinant of host specifi city), but they did not aff ect the receptor binding interface with the cell receptor dpp4. 11, 14 however, because a single aminoacid change can profoundly aff ect host range and because the host range determinants of mers-cov are not well defi ned, dromedary viruses need to be phenotypically characterised in a physiologically relevant manner. 15 so far, few virus isolates from dromedaries have been available for biological characterisation and direct evidence for replication competence of dromedary mers-cov in human respiratory tissues has been lacking. the only biological characterisation of a dromedary mers-cov virus so far was the fi nding of dpp4-receptor-dependent virus replication in huh-7 cells, 16 a transformed human hepatoma cell line physiologically unrelated to the human respiratory tract. we aimed to compare mers-cov isolates from dromedaries in saudi arabia and egypt with the prototype human mers-cov emc strain to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung. we used a human mers-cov strain emc (hmers-cov) provided by r a m fouchier (erasmus university medical center, rotterdam, netherlands). we prepared the emc virus stock as previously described. 17 three dromedary camel strains, two from saudi arabia (dromedary/al-hasa-kfu-hku13/2013 [ah13] and dromedary/al-hasa-kfu-hku19d/2013 [ah19d]) and one from egypt (dromedary/egypt-nrce-hku270/2013 [nrce-hku270]), were prepared. the dromedary camel mers-cov isolates ah13 and ah19d had been isolated in vero-e6 cells (atcc, crl-1586, manassas, va, usa) from nasal (ah13) and faecal (ah19d) swabs, collected from dromedaries in al-hasa, saudi arabia, as previously described. 14 the detection of dromedary camel mers-cov nrce-hku270 from a nasal swab of a dromedary in an abattoir in egypt has been previously reported. 11 for this study, we successfully isolated the virus from this specimen in vero-e6 cells as previously described. 14 viruses were grown, aliquoted, and stored at -80°c until used. all the dromedary mers-cov strains used in this study were passage 3 after isolation in vero-e6 cells. we had previously compared the full genome sequence of the ah13 virus in the original nasal swab with the vero-e6 passaged virus used in this accession numbers of virus genomes retrieved for this analysis are jx869059, kc776174, kc164505, kc667074, kf192507, kj156881, kj156949, kj156944, kj556336, kj156952, kf600613, kf186564, kf600627, kf186567, kf600651, kj156866, kf600634, kf600632, kf600644, kf186565, kf186566, kf600645, kf600647, kf600630, kf600652, kf745068, kj156869, kj650297, kj650296, kj650295, kf600628, kf961221, kf961222, kj156874, kj156910, kj156934, methods for viral titrations are described in the appendix. the methods we used for ex-vivo organ cultures and infection, quantifi cation of viral rna and host cytokine and chemokine mrnas, and quantifi cation of protein and immunohistochemistry have been previously described 17 and are also detailed in the appendix. we did experiments with live mers-cov in a biosafety level 3 biocontainment facility at the university of hong kong. this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw-13-104). we cultured vero-e6 and calu-3 cells using dulbecco's modifi ed eagle's medium. cells were seeded at 1 × 10⁵ cells per well in 24-well tissue-culture plates and infected with the mers-cov strains at a multiplicity of infection of 0·01 or 2 as indicated. after 1 h of virus adsorption at 37°c, we removed the virus inoculum, washed the cells with phosphate-buff ered saline (pbs) three times to remove unbound virus, and replenished with fresh culture medium. we measured virus replication kinetics by titrating infectious virus in infected culture supernatants. the human-respiratory-tract-derived cell line calu-3 (atcc htb-55, manassas, va, usa) was cultured and infected with virus, as for the vero-e6 cells. rna from the infected cells was also collected at 6, 24, and 30 h post infection for analysis of gene expression and quantifi cation of viral rna. we harvested 1 ml of culture supernatants from calu-3 cells at 30 h post infection to measure cytokine and chemokine protein concentrations using elisa (r&d systems, minneapolis, mn, usa) and cytometic bead array (bd bioscience, san jose, ca, usa), using methods specifi ed by the manufacturer. as a positive control for assessment of cytokine and chemokine induction in calu-3, we used a highly pathogenic avian infl uenza h5n1 virus, a/hong kong/483/1997. infl uenza virus was prepared in madin-darby canine kidney cells as previously described. we created thermal inactivation curves of the coronaviruses at 37°c, using culture wells inoculated with virus dilutions in the absence of permissive cells, or with an ex-vivo culture of mouse lung prepared from c57bl/6n mice. the mouse lung culture was prepared similar to the ex-vivo organ culture of human samples. we added 1 ml of virus dilution into 24-well plates with diff erent starting concentrations (10⁴, 10³, and 10² tcid 50 /ml). we collected 130 μl of supernatant at 1, 24, 48, and 72 h post incubation to measure viral titration. to assess tissue tropism in ex vivo experiments, we infected bronchial and lung tissues with 1 ml of each mers-cov virus dilution at a titre of 10⁶ 50% tissue culture infective dose per ml (tcid 50 /ml). after incubation for 1 h at 37°c, we washed the cultures three times with 5 ml of warm pbs to remove unbound virus. mock-inoculated tissue served as controls (1 ml of dulbecco's modifi ed eagle's medium without virus). we collected culture supernatants from the infected cultures at 1, 24, 48, and 72 h post infection and titrated them in parallel for infectious virus using the tcid 50 assay. samples for experiments on the ex-vivo cultures of human bronchus and lung were provided by fi ve independent donors. we assessed the extent of the overall viral replication with area-under-curve analysis using the trapezoid rule. we used the area calculated from 24 to 72 h post infection above the limit of detection of infectious virus titre for this analysis. the results of the four viruses were compared with the non-parametric friedman test. we did the experiments with vero-e6 and calu-3 cells with three biological replicates of each cell line and calculated mean and standard error of the mean (sem). we compared diff erences in log 10 transformed viral titres by a two-way anova followed by a bonferroni multiple-comparison test. we compared the quantitative cytokine and chemokine mrnas between viruses and over time with a two-way anova followed by a bonferroni multiple-comparison test. the protein concentrations of the cytokines and chemokines between viruses at 30 h post infection were compared by a oneway anova followed by a bonferroni multiplecomparison test. see online for appendix we used p less than 0·05 to indicate statistical significance. statistical analyses were done with graphpad prism version 6.0 for mac. the funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. figure 1 shows the phylogeny of mers cov strains used in this study within the global context of mers-cov. only mers-cov with full genome sequences were included in this analysis. most viruses cluster within clade b mers-cov, including the dromedary camel virus isolates from saudi arabia used in this study (ah13 and ah19d). the prototype human mers-cov virus (emc) is a clade a virus. the dromedary mers-cov isolates, nrce-hku270 and dromedary/egypt-nrce-hku205/2013, have a long branch in the phylogenetic tree separating them from clade a and clade b viruses and also from each other, suggesting that they are genetically diverse from previously described mers-cov. we used the dromedary mers-cov isolate nrce-hku270 in this study. the nucleotide and aminoacid similarity between these human and dromedary camel viruses is provided the appendix. all four mers-cov strains replicated effi ciently in vero-e6 cells at multiplicities of infection of 0·01 and 2 (fi gure 2, table). egyptian dromedary mers-cov nrce-hku270 replicated at least as well as did human mers-cov emc, and reached peak titres even faster than human mers-cov at 48 h post infection when infected at low multiplicity of infection. although the two saudi dromedary mers-cov strains (ah13 and ah19d) replicated less effi ciently than did human mers-cov during the fi rst 24 h post infection, the viral titre of ah13 was similar by 72 h post infection. replication of dromedary mers-cov nrce-hku270 was signifi cantly higher than that of the other two dromedary mers-cov strains at 24 h and 48 h post infection at multiplicity of infection of 0·01. immunohistochemistry of ex-vivo cultures of human bronchus and lung infected with human and dromedary mers-cov strains suggested that all four viruses could infect these tissues (fi gure 3). because of their threedimensional nature, infection in ex-vivo cultures was non-uniform throughout the histological specimen (appendix). figure 4 shows viral replication kinetics of the viruses in the ex-vivo cultures of human bronchus and lung. in the absence of permissive cells, the thermal inactivation curves of each of these mers-cov strains showed virus infectivity largely decreasing to undetectable levels by 24 h post infection (fi gure 4a). we noted similar thermal inactivation kinetics when we substituted mouse lung tissue (non-permissive to mers-cov) for the human ex-vivo tissues (data not shown). although the extent of virus replication varied from donor to donor, comparison with the thermal inactivation curves provided convincing evidence of virus replication of all four viruses in the ex-vivo human bronchial (fi gure 4b) and lung (fi gure 4c) cultures from each donor over the 72 h experiment period. two replicates of ex-vivo cultures per donor from three donors, infected with the same virus (human mers-cov emc) on diff erent occasions, showed that the change of area-under-curve from the same tissue donor in bronchus ranged between 0 to 1·36-fold and in lung ranged between 0 to 1·46-fold, providing an indication of the range of technical variation in these ex-vivo assays (appendix). by contrast, the diff erence of area-undercurve for human mers-cov emc between donor 3 and donor 1 was 5·3-fold for the bronchus (area under curve above detection limit 112·8 for donor 1 patchy nature of the infection, the variation between donors, and lack of adequate statistical power, statistical comparisons between viruses have to be interpreted with caution. within these limitations, we noted no signifi cant diff erence in the replication competence of the four viruses in either the bronchus (p=0·151) or the lung (p=0·270) as assessed by the area-under-the-curve using the friedman test (appendix). immunohistochemical analysis of virus-infected bronchial tissues showed that the types of cells infected by human and dromedary mers-cov were very similar (fi gure 5). co-staining of viral antigen with the goblet cell marker muc5ac (fi gure 5a) and ciliated cell marker β-tubulin (fi gure 5b) lent support to the notion that mers-cov infects non-ciliated bronchial epithelium other than goblet cells. co-staining of viral antigen and alveolar epithelial cell marker ae1/3 showed that all four viruses infected alveolar epithelial cells (fi gure 5c), and co-staining with the alveolar type ii epithelial cell marker prosurfactant protein c showed that type ii alveolar epithelium was infected (fi gure 5d). we noted no evidence of infection of alveolar macrophages (fi gure 5e). all four viruses infected lung endothelium (appendix). thus, overall, the respiratory tropism of the human and dromedary mers-cov was similar. all three mers-cov strains (emc, ah13, nrce-hku270) replicated effi ciently in calu-3 cells (ah19d was not included because it is in the same clade as ah13). to quantify the viral rna and cytokine and chemokine gene induction and expression, the humanrespiratory-tract-derived cell line calu-3 was cultured and infected with virus as for the vero-e6 cells. rna from the infected cells was also collected at 6, 24, and 30 h post infection for analysis of gene expression and quantifi cation of viral rna. we harvested 1 ml of culture supernatants from calu-3 cells at 30 h post infection to measure cytokine and chemokine protein concentrations using elisa (r&d systems, minneapolis, mn, usa) and cytometric bead array (bd bioscience, san jose, ca, usa), using methods specifi ed by the manufacturer. as a positive control for assessment of cytokine and chemokine induction in calu-3, we used a highly pathogenic avian infl uenza h5n1 virus, a/hong kong/483/1997. infl uenza virus was prepared in madin-darby canine kidney cells as previously described. 17 despite effi cient replication, none of the mers-cov strains induced signifi cant gene expression or protein secretion of tumour necrosis factor α (tnfα), cxcl10, or interferon β (fi gure 6). we used infl uenza a (h5n1) virus, a potent inducer of interferon β and cxcl10 in epithelial cells, for comparison. we noted evidence of interleukin 6 induction by all three mers-cov strains; although ah13 and nrce-hku270 induced greater production of interleukin 6 mrna than the human emc strain, the human emc strain induced higher concentrations of interleukin 6 protein than did either dromedary strain (fi gure 6f). we compared the viral replication competence and respiratory tropism of three mers-cov isolates from dromedary camels with the prototype human clade a mers-cov isolate emc (panel). the two dromedary mers-cov isolates from al-hasa, ah13 and ah19d, are phylogenetically closely related to recent clade b human viruses from saudi arabia. 18 as shown by the long-branch in the unrooted phylogenetic tree (fi gure 1) and genetic similarity tables (appendix), egyptian dromedary mers-cov nrce-hku270 and dromedary mers-cov nrce-hku205 are genetically divergent from all other mers-cov detected to date, and are also divergent from each other, suggesting that the global diversity of mers-cov is wider than previously appreciated. the lack of a representative human clade b mers-cov isolate is a limitation of this study. comparison of viral genetic data can provide clues, but is not of itself fi nal evidence for the competence of an animal virus to infect human beings. with sars coronavirus, two aminoacid changes in the spike protein strikingly altered the ability of that virus to transmit effi ciently in human beings. 15 because all the crucial determinants of host tropism with mers-cov are not fully understood, especially those outside of the spike-receptor binding domain, phenotyping of such viruses provides essential complementary information to the genetic analysis. the three mers-cov isolates from dromedary camels (ah13, ah19d, and nrce-hku270) were similar to the human mers-cov emc virus in replication competence in the rhesus monkey cell line vero-e6. all four viruses replicated in ex-vivo cultures of the human bronchus and lung and infected the same cell types in the bronchus (non-ciliated bronchial epithelium) and lung (alveolar epithelium, including type ii alveolar epithelial cells). we noted infection of lung endothelial cells with all four viruses, suggesting that potential for dissemination beyond the respiratory tract was also comparable. overall, the tropism of human mers-cov emc for the human respiratory tract was similar to that previously reported. 17 phenotypically, human and dromedary mers-cov have much the same tropism and replication competence in human respiratory ex-vivo cultures. to our knowledge, this report provides the fi rst comparative data from living human respiratory tissue in situ. therefore, our study provides new and crucial information to the emerging understanding of a disease with global importance for public health. the heterogeneity of virus replication between individual donors in our ex-vivo infection experiments might result from diff erences in host susceptibility, which are physiologically relevant to the reported epidemiology of human mers and could imply that individual variations might be an important determinant of disease susceptibility and severity. however, this question needs more systematic investigation. thus, overall, our data support and complement the viral genetic sequence data of the spike proteins of the dromedary viruses, which seem similar to the spike proteins of human mers-cov strains, 11, 13, 16 and strongly suggest that dromedary mers-cov strains have the capacity to infect human beings. infection of ex-vivo cultures provided useful information of virus cell tropism in the absence of autopsy data for mers. ex-vivo organ cultures provided information about virus tropism at early stages of the infection, which is not usually available in autopsies or from patients with late-stage disease (often after lengthy mechanical ventilation). thus, our fi ndings emphasise the usefulness of ex-vivo cultures for risk assessment of animal viruses for human health. mers-cov is reported to be a weak inducer of type i and type iii interferons. 17, 19 immune evasion mechanisms are relevant to pathogenesis and interspecies transmission, and diff erences might aff ect virus replication competence in new hosts. we therefore investigated whether human mers-cov and dromedary mers-cov diff ered in their ability to evade eliciting type i interferon and other innate immune responses. because the multiplicity of infection cannot be accurately controlled in experiments with ex-vivo cultures of the human bronchus and lung, we used calu-3, an epithelial cell line derived from human lung adenocarcinoma that has been used as a cell line to study viral infections of the human respiratory tract. 20 human mers-cov emc, dromedary mers-cov ah13, and dromedary mers-cov nrce-hku270 replicated effi ciently in these cells, although the dromedary mers-cov strains replicated signifi cantly more effi ciently in these cells (appendix). despite this effi cient virus replication, no virus elicited any tnfα, cxcl10, or interferon β mrna or protein responses after virus infection (fi gure 6). by contrast, infl uenza a h5n1 virus was a potent inducer of interferon β and cxcl10. we noted some induction of interleukin 6 by dromedary mers-cov strains ah13 and nrce-hku270 and human mers-cov at late timepoints post infection (30 h; fi gure 6e and 6f). importantly, the human and dromedary mers-cov behaved similarly by not eliciting interferon and cxcl10 responses, suggesting that these viruses do not diff er in evasion of type i interferon responses. in view of the high prevalence of infection in dromedaries [5] [6] [7] [8] 10 and implied frequent human exposure, the rarity of human infection and disease remains a puzzle and is reminiscent of the epidemiology of avian infl uenza a h5n1. 21 the exact mode of transmission of mers-cov from dromedaries to people remains unclear and whether unusual modes of exposure or host susceptibility have a role should be investigated. serological and virological evidence of mers-cov in dromedaries has been reported from countries in east and north africa outside of the arabian peninsula, 11,12 but zoonotically acquired mers has not yet been diagnosed in human beings in the african continent. our fi nding that the genetically divergent dromedary mers-cov isolated in egypt is phenotypically similar to human mers-cov emc in explant cultures from human respiratory tissue underscores the possibility that zoonotic mers might be occurring, unrecognised, in northeast africa and beyond. at present, many countries only test for mers-cov in patients with a recent travel history to the arabian peninsula, and locally acquired mers could be missed. alternatively, diff erences in cultural practices of camel husbandry, modes of animal contact, and consumption of animal products (eg, fresh camel's milk) 22 might account for diff erences in occurrence of zoonotic disease in diff erent geographical regions. in conclusion, our fi ndings suggest that dromedary mers-cov has competence to infect the human respiratory tract and does not diff er phenotypically from human mers-cov in this respect. this evidence strengthens the contention that dromedary camels are the proximate source of infection for human beings. our fi ndings also emphasise the need to consider mers in the diff erential diagnosis of viral pneumonia in a much wider geographical region in northeast africa and beyond. rwyc planned and did the experiments with ex-vivo cultures, analysed the data, and contributed to drafting of the report. mgh, gk, aa, and maa did the fi eld studies and detection of virus in dromedary specimens, and planned the experiments. dkwc, llmp, and yg did the genetic sequencing and phylogenetic analysis. kpt did the in-vitro culture and quantitative pcr assays. hyn isolated the mers-cov strains. mcwc was involved in study design and obtained research funding. jmn did and interpreted the histology and immunohistochemistry studies. jsmp obtained research funding, planned and coordinated the study, analysed the data, and wrote the report. all authors contributed to the data interpretation, analysis, and provided critical comments on the draft report. we declare no competing interests. to assess the infection potential of dromedary camel middle east respiratory syndrome coronavirus (mers-cov) strains for humans, genetic analysis should be complemented with phenotypic characterisation in physiologically relevant invitro cell cultures. we searched pubmed for reports published up to july 5, 2014, with the terms "mers coronavirus" and "dromedary" and "culture" or "isolate". we applied no date or language restrictions. three papers were retrieved, only two of them related to mers-cov isolates from dromedary camels. one other recent paper not yet cited in pubmed was identifi ed through search of the literature. only one of these studies pertained to the phenotypic characterisation of dromedary mers-cov in cell cultures in vitro. 16 this study used a transformed human hepatoma cell line, not physiologically relevant to the human respiratory tract. our fi ndings show similar virus tropism and replication competence of human and dromedary mers-cov in ex-vivo cultures of the human respiratory tract. these fi ndings suggest that dromedary mers-cov from the arabian peninsula, in addition to genetically diverse dromedary viruses from egypt, can be infectious to human beings. exposure to zoonotic mers-cov might be happening in a wider geographical region beyond the arabian peninsula. virological evidence of mers-cov needs to be sought in patients with severe unexplained viral pneumonia in africa in addition to the middle east. middle east respiratory syndrome coronavirus (mers-cov)-update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update who. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: quantifi cation of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-tohuman transmission of mers coronavirus spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers coronaviruses in dromedary camels geographic distribution of mers coronavirus among dromedary camels investigation of antimiddle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 mers coronavirus in dromedary camel herd, saudi arabia structural biology: adaptation of sars coronavirus to humans isolation of mers coronavirus from dromedary camel tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia effi cient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential evaluation of the calu-3 cell line as a model of in vitro respiratory syncytial virus infection avian infl uenza virus (h5n1): a threat to human health stability of middle east respiratory syndrome coronavirus in milk key: cord-298369-66ifwtlp authors: smith, sherri a.; waters, nigel j. title: pharmacokinetic and pharmacodynamic considerations for drugs binding to alpha-1-acid glycoprotein date: 2018-12-28 journal: pharm res doi: 10.1007/s11095-018-2551-x sha: doc_id: 298369 cord_uid: 66ifwtlp according to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, and as such the importance of plasma protein binding primarily resides in its impact on pharmacokinetics and pharmacodynamics. of the major plasma proteins, alpha-1-acid glycoprotein (aag) represents an intriguing one primarily due to the high affinity, low capacity properties of this protein. in addition, there are marked species and age differences in protein expression, homology and drug binding affinity. as such, a thorough understanding of drug binding to aag can help aid and improve the translation of pharmacokinetic/pharmacodynamic (pk/pd) relationships from preclinical species to human as well as adults to neonates. this review provides a comprehensive overview of our current understanding of the biochemistry of aag; endogenous function, impact of disease, utility as a biomarker, and impact on pk/pd. experimental considerations are discussed as well as recommendations for understanding the potential impact of aag on pk through drug discovery and early development. according to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, whether it is the intended pharmacological target, or action at an undesired site with potential toxicological consequences. the importance of plasma protein binding primarily resides in its impact on pharmacokinetic properties such as clearance (cl) and volume of distribution (v ss ), with serum albumin, lipoproteins and alpha-1 acid glycoprotein (aag) being the major proteins involved in sequestering drugs in plasma (1) . aag also known as orosomucoid (orm), is a member of the acute phase protein (app) family, first described in 1950 (2) . it has a single polypeptide chain consisting of 183 amino acids with five glycan chains, accounting for~45% of its total molecular weight (~41-44 kda) (3, 4) . aag is formed primarily in the liver and circulates from 0.5 to 1.0 mg/ml in the plasma of healthy humans (3) . levels in healthy animals are generally lower compared to healthy humans (table i) . in most disease states including inflammation, infection, and cancer, aag levels increase from 2 to 6-fold in humans (3) , and show a much broader fold of induction in animals from 2 to 20-fold depending on animal species and disease (table i) . while the biological role of aag remains unclear, it has been demonstrated to regulate immunity and play a role in both pro-and anti-inflammatory response (37, 38) . aag has long been used as a clinical biomarker, and the potential to expand its 2-3 c a n c e r 1.43 ± 0.65 (0. 71-2.27) infection 0.50 ± 0.14 (0.28-0.92) (32) application for disease diagnosis, prognosis, and characterization has grown given the recent advances in proteomics and high resolution mass spectrometry (39) (40) (41) (42) . while aag represents a relatively small portion (~1-3%) of the total plasma proteins, compared to~60% composition of albumin, it can play a significant role in drug binding and pharmacokinetics (pk) (43) . aag is considered a high affinity/low capacity plasma protein, whereas albumin is considered low affinity/high capacity. aag is a highly acidic protein with a very low isoelectric point (pi) ranging from 2.8 to 3.8 (37) . this property enables aag to bind mainly basic drugs (i.e. lidocaine, propranolol, verapamil) but it may also bind to neutral lipophilic molecules (i.e. steroid hormones) and to acidic drugs (i.e. phenobarbital), whereas albumin is mostly implicated in binding to the latter charge type (44, 45) . most drugs bind to both plasma proteins with varying degrees of affinity. the extensive and variable sialylation of aag is what drives the low and wide pi range, a property that can impact drug affinity, and ultimately pk (3). since aag levels increase in most disease states (46) , drugs with a high affinity may demonstrate higher binding (lower fraction unbound, f u ) and altered pk properties (e.g. lower total cl), lower v ss . given the known species differences in aag abundance and drug affinity there is a growing body of work where pk in preclinical species did not accurately predict pk in human, and several of these case studies are discussed herein. incorporating the ontogeny of aag may also enable more accurate predictions of pk in neonate and infant patients (47) . we provide a rationale for testing the extent and affinity of drug binding to aag and albumin in the drug discovery process to aid in prospective human pk prediction efforts (fig. 1) . research is still lagging in the characterization of higher species aag which could help better predict pk in human. furthermore, there are experimental factors that are emerging as critical to the accurate determination of aag-drug binding in vitro. these aspects are also discussed in this review. albumin, aag, and lipoproteins are considered the most relevant plasma proteins in terms of drug binding (1). the range of albumin levels is slightly lower in animals compared to human. while levels of aag are also relatively lower in most animals compared to human in the healthy state, levels are much more variable in the diseased setting as described below and in table i . human albumin and aag levels in table ii provide units of mg/ml and μm to serve as a quick reference when considering stoichiometry with drug concentrations, assuming a single drug binding site model. generally, in rodents (0.1-0.3 mg/ml) and mini-pigs (0.3-0.5 mg/ml) aag values are lower compared to human (table i ). an exception to this generalization was reported in mice, however no explanation was suggested by the authors (9) . in dogs, aag demonstrates highly variable levels (0.04-1 mg/ml) (25) , though generally lower mean values (0.25-0.5 mg/ml) have been reported (26) (27) (28) . there is little characterization of monkey aag in the literature, though it has also been reported to be lower in cynomolgus monkey (0.1 mg/ml) compared to human (30) . the conventional pig is the only species where aag levels have been reported to be higher (1.1-2.5 mg/ml) (14, 18) compared to human. however, this finding has not been consistently observed, lower aag values of 0.24 mg/ml (16) and 0.34 mg/ml (15) have also been reported in the conventional pig. in most species, aag behaves as a positive acute phase protein (app), increasing in response to stimuli, including infection, inflammation, and cancer (table i) . the acute phase response (apr) is considered part of the innate (non-specific) defense system that offers protection prior to the adaptive (specific) immune response. the magnitude of response can differ across species and disease setting. minor, moderate, and major aaps are characterized by increases in protein expression by 0.25-1, 1-10, and > 10-fold, respectively. in mice aag is considered a major positive app as levels increase up to 20-fold following stimuli (7, 8) , whereas in human the response is relatively moderate with increases of~2-6 fold (3, 31) . in the domestic cat, aag increases are moderate (~2-3 fold) in the oncology setting, whereas a more robust response has been reported with infections (up to 17-fold) (20) (21) (22) . mixed responses have been reported in the conventional pig and the minipig. in the conventional pig, aag has been shown to behave as both a negative app with levels declining to~0.6-0.7-fold normal values (14) and as a positive app in response to infection and inflammation, with levels increasing 3-6-fold (15) . in both conventional pigs (18) and minipigs (16, 48, 49) there was no aag response following acute stimuli (lps or turpentine injection), despite increases in other apps in the conventional pig (tnfα and il-6) and in minipigs (c-reactive protein (crp), serum amyloid a (saa), haptoglobin (hp), pig major acute phase protein (pmap)). christoffersen et al. (19) hypothesized the aag response in the minipig may have different sensitivity in the acute versus chronic setting, an observation previously reported for other apps (saa and hp) in cattle (49) . in a chronic inflammatory setting, obese and mildly diabetic minipigs fed a high fat diet had elevated (~2-fold) aag levels (19) . changing housing conditions also caused up to a 2-fold increase in aag in minipigs (as well as increases in crp, saa, hp and pmap), and it is unclear when levels return to baseline, a finding that should be considered in acclimation, experimental design and data interpretation (19) . neither the negative app response nor lps insensitivity of porcine aag have been reported in any other species to date. however, it is not unprecedented for a given app to behave differently across species. for example, while crp and saa are considered major positive apps in human and pig, crp and saa are not affected in mice and rats, respectively. another example of differential species response is with transferrin, which acts as a negative app in human, and a positive app in mice (19, 48) . there have been mixed reports regarding gender differences in aag levels. while there may be statistical differences, they are relatively modest compared to the differences observed in disease or developmental settings. booker et al. (50) reported statistically significant differences in aag levels between newborn human males (0.42 mg/ml) and females (0.33 mg/ml) ( table iii) . the opposite trend was observed in adult humans; aag levels of 0.39 and 0.50 mg/ml in males and females, respectively (51) . similarly, blain et al. (36) reported slightly lower levels in human males (0.74 mg/ml) compared to female (0.84 mg/ml). female minipigs generally have slightly lower, though overlapping ranges, of aag levels compared to males (19) . no gender difference was observed in dog aag levels (0.32 mg/ml) (25) . ontogeny aag levels range from undetectable in the developing human fetus, to 0.1-0.2 mg/ml in cord blood (47, 51, 53) , up to 0.3 mg/ml at birth (34, 51, (54) (55) (56) (57) , steadily increasing to 0.4-0.7 mg/ml at 2-3 months (34, 50, 55) , and achieving adult levels (0.6-0.9 mg/ml) by 10-12 months of age (33,34,58) (table iv) . similarly, aag is undetectable (<0.04 mg/ml) in the cord blood of dogs, thus significantly lower compared to adult dogs (0.32 mg/ml) (25) . the opposite trend was observed in conventional pigs, with 12.7 mg/ml reported in the fetal pig, 14.3 mg/ml in 1 day newborns, 0.70 mg/ml in 4 week olds, and 0.24-0.34 mg/ml in adults (15, 16) . consistent with this pattern, heegaard et al. (14) reported aag levels of 6.6 mg/ml in newborn pigs (5-6 days old) that declined to 1.1 mg/ml in adults. aag comprises about 50% of the total plasma proteins in the newborn pig, whereas only about 0.3% in the adult pig. this property has not been reported in mini-pigs or any other laboratory animal to date. limited data have been published on fetal and neonatal plasma levels of aag in other species. however, liver aag levels in rat have also been shown to vary in development (60) . pregnancy can also impact aag levels. in human aag levels are lower in the pregnant female and continue to decline throughout pregnancy until birth when they begin to climb back to pre-pregnancy values (53, 56, 57) . wood and wood (51) reported the same values in female non-pregnant healthy volunteers and pregnant women, however the study size was relatively small (n = 10). the opposite has been reported in the pregnant dog (25) and in rhesus monkey (59) , with aag levels about 2-fold and 4-fold higher in the pregnant animal, respectively. given the striking differences between fetal, newborn, and adult aag levels, it may be important to understand placental transfer and the milk to plasma ratio (m/p) for drugs that bind to aag. fleishaker and mcnamara (61) described a diffusional model to assess drug distribution in milk, showing that the in vitro drug binding to serum and milk protein reasonably predict m/p drug ratio in vivo. the same authors tested the model in lactating rabbits using propranolol, a compound known to bind with high affinity to aag. to mimic the disease setting, rabbits were dosed with bovine aag and propranolol pk parameters were evaluated. the diffusional model was able to accurately predict the decrease in propranolol m/p from 2.13 to 1.23 before and after aag administration. importantly, a roughly proportional reduction in total plasma cl (35%) counteracted the decrease in f u (22%), maintaining consistent cl u rate and total drug levels in milk. to improve the prediction of f u in human infants mcnamara and alcorn (47) considered the ratio of aag and albumin in cord blood of newborns and adult blood. the corresponding ratios for aag and albumin employed were 0.38 (0.24 mg/ml cord divided by 0.60 mg/ml adult) and 0.81 (36 mg/ml cord divided by 45 mg/ml adult). prediction of f u in newborns was better for drugs that predominantly bind to albumin. the average predicted and observed ratios (newborn/adult), of f u were 1.20 and 1.38, respectively, for drugs that predominantly bind to albumin (n = 28 drugs in study set). the average predicted and observed ratios (newborn/adult), of f u were 1.61 and 2.50, respectively, for drugs that predominantly bind to aag (n = 11 drugs in study set). for the majority of drugs, the f u in newborns was under-predicted, 10/11 and 22/28 drugs that predominantly bind to aag and albumin, respectively. possible explanations for the disparity suggested by the authors include changes in drugligand affinity associated with age as well as increased free fatty acids and bilirubin in the newborn that can contribute to decreased drug binding. in addition, the under prediction may be due to inaccurate (falsely high) aag and albumin newborn levels employed in the model. while aag levels are generally more variable (higher dynamic range) compared to albumin, this alone cannot explain the trend in under prediction. three genes (aag-a, aag-b, and aag-b′), located on chromosome 9, encode human aag (haag) (37) . aag-a encodes orm1 and is expressed in the liver at >100-fold that of aag-b and aag-b′. aag-b and aag-b′ are identical in structure, differ from aag-a by 22 amino acids, and encode orm2 (62) . aag shares significant homology with human immunoglobulin g (igg) and the epidermal growth factor (egf)-binding domain of the egf receptor (63, 64) . aag-a (orm1) is polymorphic with three closely related genetic variants: f1, f2, and s, differing by <5 amino acid residues, and generally referred to as f1*s in table v (65, 66) . aag-b and aag-b′ (orm2) encode the genetic variant a. most individuals possess a mixture of these variants (67) . f1 + s + a is the most common phenotype (50%), followed by f1 + a (35%) and s + a (15%). the molar ratio of f1*s to a is~2-3:1 in healthy individuals (67) . the ratio can increase up to 8:1 in the disease setting since the f1*s variant is inducible (68) . no gender related differences have been observed in expression of these variant forms (32) . x-ray crystallography showed two common binding pocket lobes between the f1*s and a variants, while the f1*s variant possesses a unique third lobe making it more promiscuous for drug binding (69, 70) . drug binding properties have been shown to differ amongst these variants (71) . for example, the basic drug imipramine was shown to bind more strongly to a variant, whereas warfarin more strongly to the f1 and s variants. for most drugs, binding to genetic variants has not been well characterized since protein binding studies are routinely conducted on a pooled supply of healthy human plasma or in the whole plasma of individual subjects/patients. proteins routinely undergo post-translational modifications that can impact physiological function and half-life (t 1/2 ). glycosylation, the addition of oligosaccharide chains (glycans) is one of the most abundant post-translational modifications, with an incidence of~50% in eukaryotic proteins (72) . aaps are particularly susceptible to glycosylation. glycosyltransferases and glycosidases are responsible for building the precursors to glycans, a process highly vulnerable to changes in disease state (73) . oligosaccharyltransferases then transfer glycans to the polypeptide chain at asparagine (n-linked) or serine/threonine (o-linked) residues, the former of which exhibit a common pentasaccharide core. the glycan bonds occur in either α or β configuration allowing for more structural diversity. the antiinflammatory and immunomodulatory properties are directly impacted by glycan composition which, in turn, changes throughout the various stages of inflammation. the heavily sialylated glycans make aag one of the most acidic plasma proteins. there is a high level of heterogeneity resulting in a very low but wide pi ranging from 2.8 to 3.8 which in turn can impact drug binding and aag t 1/2 (37) . desialylation can result in an increase in pi range from 4.2 to 4.7 (74, 75) . human aag contains 5 n-linked glycans of the polypeptide backbone, each of which can form a variety of bi, tri, or tetra-branches all potentially further expressing sialic acid moieties (65) . despite thousands of potentially unique glycan combinations associated with aag, only about 12-20 glycan combinations are observed in the plasma of healthy humans (37, 76) . however, in the disease state many more glycan modifications have been detected under the regulation of inflammatory cytokines, tumor necrosis factor (tnfα), interleukin-1 (il-1) and il-6 and its utility as a biomarker will be described later (76) . aag offers two drug binding sites for basic drugs, one for acid drugs (44) , and up to 7 for steroids (45) . drug binding to aag is reportedly mediated predominantly via hydrophobic interaction with some data suggesting potential for electrostatic interaction. evidence to support the latter includes the observation of stereoselective binding in propranolol isomers (77) . desialylation and lower plasma ph have been shown to decrease drug binding to aag (78, 79) . propranolol binding was reduced and progesterone binding unchanged with desialylated aag. homologous aag genes have been observed across mammals including rodents, cats, dogs, pigs, monkeys and humans (table vi) . mouse, rat, rabbit, and pig aag genes sharẽ 44%, 59%, 70% and 70% homology, respectively, with human aag (37, 80) . while there are three human aag genes, there is only one gene reported in rat and two to three in mouse depending on strain or source. despite the presence of multiple (81) . two disulfide bridges have been described in human and pig aag, with only one in rat. various binding sites have been characterized across species; in human acidic, basic, and steroid binding sites have been reported, whereas cows and dogs lack the acidic binding site (4). aag belongs to a family of apps mainly generated in liver parenchymal cells at elevated levels within 12-24 h of injury (i.e. infection, inflammation, burns, cancer). apps by definition are proteins that change in response to injury by >25% in plasma (89) . examples of positive apps include ceruloplasmin, aag, and serum amyloid a (saa), all increasing levels in humans by about 50%, 3-fold, and > 1000-fold, respectively, in the diseased state (90) . negative apps include albumin, transferrin, and insulin-like growth factor i, which modestly decline in plasma in the diseased state. aag is a member of lipocalins, a superfamily of extra-cellular transporters that bind and transport small hydrophobic endogenous and exogenous chemicals. upregulation of apps enhances local inflammation by aiding in recognition of microbes, directing leukocytes, and increasing blood flow to the site of insult while minimizing inflammatory responses elsewhere (91) . the rapid and high magnitude of response, as well as the short t 1/2 , are properties characteristic of apps and hypothesized to be key elements in innate immunity. homeostasis is kept in check with redundancy such that a given app may impact multiple pathways while multiple apps may provide overlapping biological function. the inflammatory cytokines, tnfα, il-1 and il-6, have been shown to regulate aag, in addition to the other apps including c-reactive protein (crp), haptoglobin (hp), saa and hemopexin (92) . the function of aag is still poorly understood, however, as part of a cytokine mediated feedback mechanism it has been implicated in both anti-and pro-inflammatory modulation (37, 38) . monocyte activation and induction of t-cell proliferation (93) as well as activation of tnfα, il-1 and il-6 secretion (94) (95) (96) have been associated with the pro-inflammatory role of aag. su et al. (97) proposed a positive feedback mechanism of apps whereby inflammation is amplified in response to tnfα-mediated synthesis of aag-stimulated monocytes and vice-versa. the anti-inflammatory role of aag has also been reported. aag inhibits neutrophil chemotactic response associated with stimulation of n-formylmethionyl-leucyl-phenylalanine (fmlp) and the inflammatory peptide complement component c5a (98, 99) . aag was shown to modulate release of free radicals regardless of treatment time, whether aag was introduced prior to or post neutrophil activation (100) . multiple in vivo septic shock models in rodents have demonstrated the protective effect of aag when dosed prophylactically to animals challenged by tnfα or endotoxin (101) . the role of aag in angiogenesis was studied using an ex vivo rat model (102) . following aortic excision, macrophages respond by rapidly (within minutes) increasing tnfα levels, peaking at 24 h, and remaining elevated throughout angiogenesis. tnfα guides overexpression of aag within 24 h, with levels peaking after 2-3 days and sharply declining thereafter. as with inflammation, aag (and tnfα) has both pro-and anti-angiogenic effects depending on the context. early in the angiogenesis process, aag plays an inhibitory role via modulation of mitogen-activated protein kinases, whereas later in the process aag promotes angiogenesis via vascular endothelial growth factor regulation. as an app, levels of aag typically increase within 24 h of injury and begin to decline several days post amelioration. increased aag levels have been reported in the serum of breast, lung, and ovarian cancer patients (103) . in a study comparing aag levels in lung cancer patients versus individuals with no known cancers, results showed 89% sensitivity and 85% specificity and aag levels correlated with relapse-free survival (104) . in the case of hepatocellular carcinoma (hcc), diagnosis can be challenging due to other liver conditions (i.e. cirrhosis) presenting similar abnormalities, however increased aag levels are more pronounced with hcc providing a potential basis to differentiate these diseases (105) . the proportion of breast cancer patients with increased aag levels increases with disease progression, for example 25% and 81% of stage ii and iv patients respectively, had elevated levels compared to 12% in healthy donors (106, 107) . in most disease states, aag is modified both quantitatively as described above, as well as qualitatively, a phenomenon described by alminquist and lausing (39) after comparing serum glycoproteins of cancer patients versus healthy donors. relative to the associated polypeptide backbone, the heterogeneity in glycan composition and structure make it a good system for characterization and correlation with disease state (73) . some commonly exploited glycoproteins used in clinical cancer biomarker tests include carcinoembryonic antigen (cea), cancer antigen 125 (ca-125), ca-19-9, and prostate-specific antigen (psa), for the diagnosis of colorectal, ovarian, pancreatic and prostate cancers, respectively. technological advances in mass spectrometry and proteomics have led to an improved understanding of glycan structure and function. glycans are relatively more abundant compared with their associated proteins, often times multiple copies per glycoprotein, as is the case for aag known to have 5-6 associated glycans depending on species (table vi) . not only are they associated with the cancerous tissue but they can also be detected in serum, making its use as a biomarker more feasible in the clinical setting. in addition, the same modified glycan may be associated with more than one glycoprotein affording multiple opportunities/patterns for detection. modifications of the glycans associated with these biomarkers are relatively more specific and selective than the epitope itself, allowing for more accurate means for distinguishing healthy versus diseased tissue, and disease progression (40) . for example, testing serum levels of modified glycans associated with psa enable the distinction between benign prostatic hypertrophy and cancerous prostate (41) . in breast cancer, the serum glycosylation pattern not only distinguishes healthy from diseased tissue but also differentiates between malignant and non-malignant tumors and disease stage (73) . in inflammatory diseases including rheumatoid arthritis and asthma, aag glycans are more branched compared to healthy subjects (42) . patients suffering from acute inflammation, infection, burns, and tissue damage all showed an asialylated carbohydrate-deficient variant of aag (37) . human aag t 1/2 is relatively short,~2-5 days (108,109), compared to albumin, 14-21 days (110) . aag turnover is dependent on sialic acid residues and terminal galactose groups. mccurdy (111) studied the impact of glycosylation on in vivo cl of human derived aag in rabbit following intravenous injection. the terminal t 1/2 of native human aag was 58 h (consistent with the t½ value of 69 h reported by regoeczi et al. (112) . altered/reduced or absence of glycosylation lowered the t½ to 50 h and 42 h, respectively. cl of native aag was 2.2 ml/h/kg, whereas much higher values were observed for aag forms with altered (11 ml/h/kg) or absent glycosylation (100 ml/h/kg). the steady-state volume of distribution (v ss ) was 160 ml/kg for native aag, whereas much higher values were observed with altered (550 ml/kg) or absent glycosylation (2000 ml/kg). the absence, reduction or alteration of n-linked glycosylation resulted in a marked increase (>10-fold) in renal elimination compared to native aag. these studies support that the dispositional properties of aag are dependent on disease state since the biochemistry of aag is altered with disease as described earlier. therefore, if a given drug binds to aag to a high extent, it is possible underlying differences in the pk of the drug between healthy and diseased populations may be attributable to aag. drug binding to plasma, tissue(s) and intended target are critical parameters to predict pk and pharmacodynamics (pd). however, optimizing protein binding to plasma in the drug discovery setting is scientifically unsound (113, 114) . nearly 30% of the 260 fda approved drugs prior to 2003 are classified as highly bound (>95% or f u < 0.05), and this trend has increased in recent years with 45% of new drugs classified as highly bound, 24% of which have f u < 0.01 (115) . while the free drug hypothesis describes that the free concentration drives activity as it can cross cellular membranes to reach its target, the focus should be to optimize cl u and permeability. as described above, albumin, aag, and lipoproteins are considered the most important plasma proteins involved with drug binding. while albumin has a higher drug capacity due to its relative abundance in plasma, aag levels are lower and with high affinity drugs saturation may occur. the saturation of aag may or may not be buffered by albumin depending on the drug binding affinity for albumin. plasma protein levels can change with disease state, a decrease in albumin and an increase in aag are generally observed, which may impact protein binding and pk. both albumin and aag levels are significantly lower in the newborn, with newborn:adult ratios of about 0.81 and 0.38, respectively (47), a factor that should be considered when predicting pk in the very young pediatric population. a proposed flowchart to ascertain the impact of aag as a potential covariate for pk variability early in drug development is shown in fig. 1 . routine screening in human and laboratory animal species at a single low concentration (1-2 μm) is typically performed in early drug discovery as pk data in preclinical species is acquired. as a program advances towards development candidate selection with preliminary projections of human pk and dose, there is value in assessing concentration dependency and the identity of the major plasma proteins involved in drug binding. if the extent of binding is high (>90% bound) under single concentration assay conditions, further characterization of the binding constants for aag and albumin is warranted particularly if human free fraction is lower relative to animals or if concentration dependency is observed. if there is high affinity to human aag it may be worthwhile to assess k d in other species to build additional confidence in human pk predictions. multiple routine in vitro analytical procedures exist to assess the extent and affinity of drug binding to proteins. a recently published industry white paper (di et al., 2017) provides a comprehensive review of commonly used protein-binding practices, challenges, and recommendations (116) . the intention in this section of the manuscript is to suggest use of control compounds and to describe a source for erroneous fraction unbound values that has been overlooked. as with any assay it is good practice to include control compounds that are assessed along with test compounds to ensure a properly functioning assay. if data for control compounds are not available for a given assay/species one can still monitor the value for the control over time to ensure its consistency. when conducting definitive assays the use of multiple control compounds is advised since multiple factors can influence binding and some are compound specific. literature values for propranolol and warfarin are summarized across species in table vii . the intent here is to provide references for acceptable free fraction values for control compounds across species. propranolol was selected because it has moderately high binding to human plasma proteins, however the affinity is higher for aag relative to albumin by approximately two orders of magnitude (125) , therefore, propranolol can serve as a control for compounds that preferentially bind to aag. the reported propranolol f u values range from 0.10 to 0.29 in human plasma, a 3-fold difference, and outside what is typically deemed normal assay variability. the acceptable assay f u value for propranolol should be within 0.10 to 0.20 in healthy human plasma. warfarin was selected because it is highly bound to both aag and albumin (126, ge life sciences application note 29263246aa). it is helpful to include a highly bound control compound since they generally require longer incubation time to achieve equilibrium. while the reported warfarin f u values range from 0.005 to 0.022 in human plasma, more than a 4-fold difference, the absolute difference is low. the majority of references indicate a narrower range, therefore the acceptable assay f u value for warfarin should be within 0.005 to 0.015, a range similar to that reported in the recently published white paper (116) . it is not advised to select a compound with moderate or low protein binding to serve as a control because the f u values are generally more variable. based on recent studies (127, 128) it is possible the large range in human f u values is due to the blood collection and storage procedure. in the clinical setting, f u is typically measured in the plasma or serum of patients from blood collected in vacutainers. the collection procedure and storage of blood can have a significant impact on protein binding results. it has long been reported that plasticizers can disrupt the binding of drugs to aag (129) (130) (131) . for example, the plasticizer tris (2-butoxyethyl) phosphate (tbep), used to soften rubber stoppers in vacutainers, was shown to disrupt aag binding to the basic drugs lidocaine and quinidine (132) . polyvinyl chloride (pvc) bags containing the plasticizer diethylhexyl phthalate (dehp) are routinely used in blood collection. butler et al. (127) reported an average of a two-fold increase in f u for drugs known to bind to aag when blood was collected in these pvc bags versus blood collected in vacutainers. more recently, experiments were conducted to show the correlation between dehp levels and change in f u with drugs that bind to aag (128) . when blood was immediately transferred from terumo® bags to vacutainers the dehp levels were low (1-10 μm) but steadily increased with storage after 7 days (up to 300 μm) and 28 days (300-1000 μm). dehp can easily leach into the contents of bags since it is not chemically bound to the pvc. as expected, f u was higher (2-5-fold) with drugs known to bind to aag when tested using plasma containing high levels of dehp. the shift in f u was significantly reduced, though not eliminated, when the blood was immediately transferred to vacutainers. results generated using blood products collected/stored in plasticizer containing bags should be interpreted with caution as the f u values may over-estimate true in vivo values. in most instances when protein binding is measured clinically, vacutainers (no/minimal dehp exposure) are used since small (<10 ml) blood volumes are collected. this is in contrast to the relatively larger volumes (up to 350 ml) collected in bags for donation and/or non-clinical research purposes. it should be noted that the blood from animals is typically collected in smaller vessels not containing dehp, therefore the over-estimation of f u is less likely to occur in animal blood. studies may be warranted to assess effects on drug binding with other commercially available vacutainers as a precaution. despite what has been reported in the literature for decades, bags containing plasticizers known to disrupt aag-drug binding continue to be widely used for blood collection with the intended use in both research and in the clinical setting. dehp is essential in maintaining the shelf life of blood products up to 42 days as it protects the membrane of the red blood cell (133) . transfusion recipients routinely receive blood that has been collected and stored in these bags. clinical effects with regard to drug displacement have not been reported. other blood collection bags have been developed though it is unclear if they have an effect on aag-drug binding. multiple classes of drugs have been reported to bind to aag. examples of the relationship between aag binding and lipid solubility and/or electrostatic interactions have been reported for benzodiazepines, phenothiazine neuroleptics, beta blockers, anthracycline derivatives, antihistamines, and analgesics (3, 126) . here we focus on several well-studied examples where the drug-aag binding affected pk and/or pd in the clinical oncology setting. vismodegib was approved for the treatment of metastatic basal cell carcinoma by the food and drug administration (fda) in 2012. pk characteristics exhibited in the phase i clinical trial were unexpected based on preclinical allometric scaling. following a single oral dose, exposure was higher than predicted with a very low apparent cl and a long t 1/2 of about 10-12 days (134) . cross species in vitro data showed high plasma protein binding (≥95% bound) and low metabolic turnover in hepatocytes of all species except monkey, which agreed well with in vivo cl values (135) . mechanistic pk modeling was employed to explain the roles of plasma protein binding, solubility-limited absorption, and low metabolic cl in contributing to the unusual clinical pk properties (136) . in vitro studies revealed far lower vismodegib solubility, 0.0001 mg/ml at higher ph range 6.5-7.4, compared to~1.0 mg/ml at ph 0.1 (136) . the impact of solubility was manifested in saturation of oral absorption. there was no increase in mean steady-state concentration (c ss ), 22.6, 21.3 and 22.0 μm, with increase in oral dose from 150 to 270 and 540 mg, respectively (137) . the free fraction of vismodegib remained constant at 0.5 ± 0.1% across all dose groups (137) . however, in a separate study, a 2.6-fold increase in free fraction of vismodegib was reported between a single 150 mg dose (0.25 ± 0.14%) versus repeat daily 150 mg doses (0.65 ± 2.9%) (138) . vismodegib plasma protein binding properties were further characterized by isothermal titration calorimetry (itc) and surface plasmon resonance (spr) with both procedures showing higher vismodegib-aag affinity to the human isoform relative to the rat isoform (139) . by itc the k d values of vismodegib-aag were 1.1 and 118 μm in human and rat, respectively. by spr the k d values of vismodegib-aag were 13 μm and not detectable in human and rat, respectively, whereas the k d values for vismodegib-albumin were similar in human and rat (120 and 140 μm, respectively). in vitro experiments showed a negative correlation between aag concentration and target engagement, whereby supplementing physiologically relevant concentrations of aag resulted in a dampening of hh signaling via gli1-luciferase reporter assay (139) . there was a high correlation (r 2 = 0.73, slope 0.48) between aag and total vismodegib c ss in plasma samples from cancer patients, suggesting the role of plasma protein binding in vismodegib drug disposition (136) . perhaps even more compelling was the intra-patient parallel changes in total vismodegib concentrations with changes in aag. no correlation was found with albumin levels and vismodegib concentrations (139) . saturation of aag has been proposed to be a key determinant in the non-linear pk of vismodegib given that aag is a high affinity-low capacity protein and near stoichiometric levels of vismodegib and aag are reported (138) . despite the high affinity and resultant low free fraction, there remains sufficient unbound vismodegib available to interact with target to demonstrate pharmacological effect. in order to understand the mechanism(s) of non-linear pk, healthy human subjects received either a single oral dose or 7 daily oral doses of 150 mg vismodegib, followed by an iv microtracer dose of 10 μg [ 14 c]-vismodegib 2 h post first or last (day 7) oral dose (140) . aag levels were within close range in the two dose groups to eliminate need for correction. cl and v ss values after a single iv dose were 43.4 ml/h and 16.4 l, roughly 10-65-fold lower (depending on number of species employed in model) and 3-fold lower, respectively, compared to preclinical allometric scaling predictions (135) . relative to day 1, cl and v ss increased 81% and 63%, respectively on day 7, while t 1/2 remained unchanged at~10-11 days. mean free fraction increased 2.4-fold after 7 days oral dosing (0.79 ± 0.23%) compared to a single dose (0.33 ± 0.12%), a finding consistent with the observations previously described (138) . correcting for protein binding of vismodegib, the unbound cl and v ss values were relatively similar on day 1 and 7. absolute bioavailability was 31.8% following a single oral dose and declined to 7.4% after 7 days of repeat dosing, a finding attributed to slow absorption and limited intestinal solubility (140) . given the non-linear pk with long t 1/2 , a phase ib clinical study was conducted to determine if vismodegib steady-state concentrations could be maintained with less frequent dosing (138) . three oral dosing schedules were evaluated: 150 mg once daily (qd), once weekly (qw) or three times per week (tiw), all after having received a loading dose of vismodegib (150 mg qd, 11 days). after steady-state was achieved for the alternate dose schedules, vismodegib levels declined in the qw and tiw groups relative to qd, however the decline in unbound vismodegib (50% and 80%, respectively) was greater than the decline in total vismodegib (24% and 45%, respectively). only the qd dose schedule maintained unbound vismodegib concentrations sufficient to achieve target pathway inhibition (ic 95 ) of glioma-associated oncogene (gli1) previously described (141) , therefore the recommended dose and schedule was maintained at 150 mg qd. one additional point of consideration is the discrepancy between in vitro and ex vivo free fraction values reported for vismodegib in human plasma,~3-4% and < 0.25-0.79%, respectively (135, 137, 138, 140) . this discrepancy may be attributed to the collection and storage of blood products as described herein and in recent publications (127, 128) . free fraction values increased sharply for aag-binding drugs when human blood was exposed to plasticizer dehp; for example, vismodegib free fraction increased from 0.2% when collected in vacutainer (no/minimal plasticizer), to 0.4% and 1.4% when collected and stored in terumo® bags containing plasticizer for <1 and 7 days, respectively. additionally, vismodegib free fraction increased from 0.3 to 2.9% when human plasma was spiked with 800 μm plasticizer, a concentration one could expect to measure in blood after several weeks storage in terumo® or similar bags. ucn-01 (7-hydroxystaurosporine) is a small molecule protein kinase inhibitor. single agent clinical trials were initiated for multiple oncological indications in the late 1990s, followed by combination studies with other anti-cancer agents. the pk parameters in preclinical species (mouse, rat and dog) ranged as follows: moderate to high cl roughly 30 to 80% hepatic blood flow, high v ss 6 to 17 l/kg, and moderate t 1/2 from 3 to 12 h (142) . clinical pk were not predicted by allometry and exhibited low cl (17 ml/h), low v ss (12 l) and very long t 1/2 (>200 h) (143) . while ucn-01 (1 μg/ml) is considered highly bound to plasma proteins in preclinical species with free fraction ranging from 0.5 to 1.8%, the free fraction was substantially lower in human plasma at <0.02% (144) . ucn-01 free fraction was <0.02% or 6.2% when incubated with physiologically relevant levels of haag (1 mg/ml) or albumin (40 mg/ml), respectively, showing the preferential binding to aag. in vitro studies showed marked increase in ucn-01 free fraction with increases in concentration approaching stoichiometric levels of aag (145) . the association constant (k a ) was 799 × 10 6 l/mol in haag (roughly equivalent to k d of 1.25 nm), whereas in dog the k a was~60-fold lower at 13.2 × 10 6 l/mol (145, 146) . sparreboom et al., further characterized the role of aag in the pk of ucn-01 (147) . with an increase in dose ranging from 3.6 to 53 mg/m 2 /day iv infusion over 72 h, there was an increase in cl, 4.13 ml/h to 24.1 ml/h, respectively, a linear increase in v ss , 0.113 l to 0.276 l, respectively, and less than proportional (3.5-fold) increase in auc ∞ , (area under the curve extrapolated to infinity) 7460 to 26,140 mg*h/l. cl trended (r 2 = 0.264) with pre-dose aag levels, despite a relatively small data set (n = 39). it is proposed the increase in cl in humans is due, at least in part, to the increase in free fraction once aag becomes saturated. cl in dogs had demonstrated no dependence on dose from 0.81 to 6.48 mg/kg (142, 148) , a finding consistent with the notable difference in ucn-01-aag k d in human versus dog. the k d of haag-ucn-01 is roughly 4 orders of magnitude lower compared to k d haag-vismodegib (139) . in addition, ucn-01 does not appear to bind significantly to albumin given the comparable k d values of ucn-01 to aag vs human plasma, 799 vs 802 × 10 6 l/mol, respectively (145) . saturation of aag and the differential affinity to aag and albumin are likely to contribute to the non-linear pk of ucn-01. as with vismodegib the haag-unc-01 k d differed considerably from that of the preclinical species leading to poor predictive accuracy with allometric methods. ucn-01 has a slow dissociation rate which may reduce v ss , further hindering free drug from target interaction (139) , in contrast to the preclinical observations of high v ss , high tumor:plasma ratios, and decline in tumor volume (142) . ucn-01 has not advanced in the clinic due to unpredictable pk and off-target kinase inhibition (146) . imatinib is a selective inhibitor of bcr-abl, platelet-derived growth factor receptors, and c-kit receptor tyrosine kinases (149) . approval was granted for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors by the fda in 2001 and 2002. imatinib-aag binding is concentration-dependent with a reported k a of 1.7 × 10 6 l/ mol, roughly equivalent to k d of 0.6 μm, while imatinibalbumin binding is considerably weaker with a k a of 3.0 × 10 4 l/mol, roughly equivalent to k d of 33 μm (139, 150) . adding to the complexity is the differential binding of imatinib to various human aag isoforms, the k a reported above is for the f1-s variant, whereas binding was much weaker for the a variant (unpublished data) (151) . separately incubating 24 μm of the aag variants f1-s and a with 5 μm imatinib resulted in 6% and 18% free fraction, respectively. imatinib exhibits linear pk in patients (152) with low oral cl ranging from 8 to 12 l/h (150, 153, 154) , long t 1/2 of 18 h and high oral bioavailability >90%. a proportional increase in auc is observed with oral doses from 25 to 1000 mg. pk parameters are similar between single and repeat doses, showing 1.5-to 2.5-fold accumulation at steady-state. correlations between imatinib pk and abcb1 genotype, body weight and aag levels was shown in patients (150, 155) . despite linear pk in total imatinib, a non-linear relationship exists between free fraction and total imatinib concentrations in plasma as a result of high affinity to aag and~55-fold weaker affinity to albumin (156) . elevated levels of aag in patients have been linked with delayed or lack of response to imatinib treatment as well as potential resistance mechanism (157, 158) . in a clinical study with cml patients, approximately half exhibited elevated aag levels positively correlating with disease progression and white blood count. in the chronic, accelerated, and blast crisis phases of disease, 33, 83 and 75% of these patients, respectively, were increasingly likely to have higher aag levels. the impact of the plasticizer dehp on imatinib free fraction in human plasma was also assessed (128) . imatinib free fraction increased from 3.5% when collected in vacutainer (no/minimal plasticizer), to 4.9% and 14.7% when collected and stored in terumo® bags containing plasticizer for <1 and 7 days, respectively. additionally, imatinib free fraction increased from 3.5 to 15.3% when human plasma was spiked with 800 μm plasticizer. the plasticizer-free free fraction values reported by ingram et al. (128) are similar (~5%) to those reported in the package insert (novartis pharmaceuticals corporation, reference t2017-101). pinometostat (epz-5676) is a first-in-class, small molecule inhibitor of dot1l and was the first member of the novel histone methyltransferase inhibitor class to enter phase 1 clinical trials in both adult and pediatric mll-r leukemia patients. consensus preclinical predictions across multiple diverse methods suggested pinometostat would be a moderate-to-high cl compound in human with estimates ranging from 8 to 18 ml/min/kg, and species-invariant time approaches showed cross-species congruence in time-concentration profile. however, during early development, the observed cl in human was shown to be markedly lower than that determined in preclinical species. the majority of interspecies scaling and allometric methods over-predicted human cl of pinometostat with fold errors ranging from 4 to 13 (159) , characterized by 'vertical allometry'. the 3-5-fold difference in free fraction between rat and human provided the basis for the improved prediction using the free fraction corrected intercept (fcim) method. the unambiguous species difference in cl was not related to qualitative differences in metabolic pathways or routes of elimination, but instead to cross-species differences in plasma protein binding. concentration dependence in protein binding was observed in human plasma, over a relevant concentration range, which was less apparent in the preclinical species. this, along with in vitro kinetic determinations, suggested the saturable binding of pinometostat to aag. the equilibrium dissociation constant (k d ) for pinometostat binding to human aag was measured as 0.24 μm indicating a high affinity interaction. by comparison, prototypical aag ligands such as dipyridamol, disopyramide and thioridazine have k d values of 15.5, 1.0 and 63 μm respectively. furthermore, there is the disproportionately higher expression of aag in human plasma relative to preclinical species, which is likely a contributing factor alone, irrespective of potential speciesspecific differences in aag affinity. it has been suggested when the k d for a given drug-aag binding is low, and more than a log order lower relative to the k d for albumin, the pk may exhibit non-linearity (136) . if the drug also has a high affinity to albumin, a high capacity protein, fluctuations in free fraction will be minimal. if the drug has a low affinity to albumin, the non-linear effect may be exacerbated when drug levels are near stoichiometric with aag, since aag is a low capacity protein and may become saturated. given the known differences in abundance and homology across species for aag, allometric scaling may not be suitable for human pk prediction when there are differences in k d , something that can easily be measured in vitro now that aag of preclinical species are commercially available, though still limited in supply. monitoring aag and/or free drug concentrations, as well as phenotyping the genetic variants of aag in patients may be warranted in special circumstances to better understand pk and pd. in fig. 1 , we propose a flowchart for incorporation of protein binding assessment in drug research and development. investigations are ongoing to propose more reliable procedures for the collection and storage of blood for future use in drug free fraction measurements (personal communications with q 2 solutions holdings, llc). special consideration should be given to pregnancy and pediatric populations since aag levels are substantially lower until about 10 months post-natal. pbpk models have shown predictive utility with incorporation of aag and albumin levels and drug binding parameters. quantitative and/or qualitative analysis of aag may prove useful as a biomarker for disease diagnosis and prognosis, with the potential to serve as discerning criteria to improve likelihood of successful treatment. since aag behaves as a positive 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note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-305318-cont592g authors: lancaster, madeline a.; huch, meritxell title: disease modelling in human organoids date: 2019-07-01 journal: dis model mech doi: 10.1242/dmm.039347 sha: doc_id: 305318 cord_uid: cont592g the past decade has seen an explosion in the field of in vitro disease modelling, in particular the development of organoids. these self-organizing tissues derived from stem cells provide a unique system to examine mechanisms ranging from organ development to homeostasis and disease. because organoids develop according to intrinsic developmental programmes, the resultant tissue morphology recapitulates organ architecture with remarkable fidelity. furthermore, the fact that these tissues can be derived from human progenitors allows for the study of uniquely human processes and disorders. this article and accompanying poster highlight the currently available methods, particularly those aimed at modelling human biology, and provide an overview of their capabilities and limitations. we also speculate on possible future technological advances that have the potential for great strides in both disease modelling and future regenerative strategies. organoids (see box 1) are a powerful new system and are being increasingly adopted for a wide range of studies. indeed, since 2009, over 3000 papers have been published that make use of organoids in some form or another (see poster and box 2). organoids can be derived either from pluripotent stem cells (pscs), adult-tissue-resident cells (stem or differentiated cells) or embryonic progenitors (huch and koo, 2015) . because organoids follow the same basic intrinsic patterning events as the organ itself, they are a useful tool for investigating developmental organogenesis or processes of adult repair and homeostasis. their more accurate organ-like organization makes them a valuable tool for disease modelling, although further improvements, particularly in scalability, are still needed. nonetheless, because organoids have such potential, extensive effort has been made at generating organoids for a range of organ types. we describe the methods and applications for these in more detail in this article and in the accompanying poster, and discuss future directions for improving this technology and furthering its applications. pluripotent stem-cell-derived and adult-tissue-derived organoids one of the major leaps that has led to the development of organoid methods was the realization that, to better recapitulate organ morphology in vitro, one must first understand the development of that organ and try to mimic it. thus, years of research on the patterning events and signalling cascades at play during organogenesis have provided the necessary foundation to make organoid research possible. the relatively recent advent of human pluripotent stem cell (psc) cultures has provided the starting point for this process. remarkably, human pscs can be induced to spontaneously undergo differentiative and morphogenetic behaviours that mimic the formation of embryonic germ layers, especially when they are forced to form three-dimensional (3d) aggregates called embryoid bodies (ebs; box 3). ebs form germ layers that express well-described molecular markers and even segregate to form patches of individual germ-layer tissue within the aggregate. by applying specific growth factors, these aggregates can be directed towards a specific germ layer. biologists have taken advantage of the knowledge on developmental events to construct or reconstruct a tissue in vitro (organoids) that recapitulates many of the key structural and functional features of the organ (box 1). the range of organoids established so far is rapidly increasing, and although in many cases these have been first developed from rodent pscs, researchers are translating experimental methods to human cells. the use of pscs as a starting point to generate 3d organoid cultures allows the in vitro recapitulation of the developmental processes and tissue organogenesis that occur in vivo. however, once organ growth is terminated, adult tissues still require tissue maintenance and repair to ensure proper functionality of the adult organ. recently, a better understanding of the signalling pathways important for adult tissue maintenance and repair has enabled researchers to grow primary tissues as self-expanding organoids that retain all the characteristics of the tissue of origin, as well as their genetic stability over time (see huch and koo, 2015 for an extended review; table 1 ). here, we summarize the methods for derivation of organoids from human cells, be it pscs or tissue-resident cells. the development of 3d organoid cultures has proven very successful for several endoderm-derived organs, such as the intestine, stomach, liver, pancreas and lung. in this section, we discuss the findings and conditions used to develop these organoids. the term 'organoid' is actually not new. a simple pubmed search reveals its first usage in 1946 in reference to a tumour case study (smith and cochrane, 1946) . however, at that time, the term was used to describe certain histological features of tumours, such as glandular organization. over time, its meaning evolved to generally refer to tissues or structures that resemble an organ, and so this term became increasingly used in in vitro biology. however, it wasn't until the development of intestinal organoids in 2009 (sato et al., 2009 ) that it began to be used more specifically for these self-organizing in vitro structures. nonetheless, the term 'organoid' continues to be used for a wide variety of tissues or structures that may or may not fully recapitulate key features of an organ. therefore, in an attempt to clarify some of the confusion surrounding this term, we use a previously proposed working definition (lancaster and knoblich, 2014; huch and koo, 2015; clevers, 2016 ) that fulfils the most basic definition; namely, 'resembling an organ'. more specifically, a genuine organoid should satisfy several criteria: (1) a 3d structure containing cells that establish or retain the identity of the organ being modelled; (2) the presence of multiple cell types, as in the organ itself; (3) the tissue exhibits some aspect of the specialized function of the organ; and (4) self-organization according to the same intrinsic organizing principles as in the organ itself (sasai, 2013a,b) . since the dawn of biology as a scientific field, scientists have sought to understand mechanisms of human disease. while studying patients can give insight into symptoms and help describe the course of the disease, the underlying causes are often enigmatic. therefore, animal models have been a staple of human disease research, as they can be engineered to develop the disease, for example, by introducing relevant mutations. however, because of evolutionary divergence, there are many features of human biology and disease mechanisms that cannot be accurately modelled in animals. for instance, rodent models of alzheimer's disease do not ubiquitously display the characteristic plaques and tangles (asaad and lee, 2018) . when key disease features are absent from animal models, it is difficult to study the underlying mechanisms. for these reasons, efforts over the past several decades have focused on modelling human disease biology in a dish. while hela cells and other immortalized human cell lines have proven to be a powerful tool, their use has several considerable drawbacks, including their genomic instability and limited tissue identities (adey et al., 2013) . furthermore, the expansion of adult primary tissues far beyond the predicted hayflick limit is a real challenge (hayflick, 1965) . thus, more recent approaches have focused on in vitro models derived from stem cells, which allow for a broader array of tissue identities, long-term expansion, better genomic integrity and improved modelling of healthy biology. a stem cell is any cell that is able to self-renew and generate differentiated progeny, i.e. capable of generating several defined identities. thus, differentiated organ cell types can be generated in vitro from pluripotent stem cells (pscs) by following a series of differentiation steps that mimic early embryonic development. alternatively, differentiated cells derived from resident adult stem/progenitor cells, such as the stem cells of the intestinal crypt, can be used but historically these have proven difficult to expand in vitro (bjerknes and cheng, 2006) . in order to overcome these limitations and also better recapitulate tissue architecture, a new field of 3d in vitro biology called tissue engineering has come into the spotlight. by combining biology and engineering, more elaborate conformations of cells have been established, allowing for multiple cell types to be combined in a configuration that more closely mimics organ architecture. perhaps the most exciting developments have been the recent organ-on-a-chip methods, which allow for the construction of connected chambers that mimic different organ compartments, for example liver ducts and blood vasculature (huh et al., 2011) . while tissue engineering has provided a number of highly useful models for looking at the interaction between different cell types, there are certain artificial aspects that affect their ability to accurately model organ structure and therefore function. for example, the use of artificial membranes and matrices means that the cells are positioned via exogenous processes, rather than through bottom-up self-organizing principles as in organ development in vivo. thus, to more accurately model organ architecture, very recent efforts have focused on supporting the self-organizing development of these tissues in vitro. and so, the organoid field was born. in the intestine, wnt, notch, fibroblast growth factor (fgf)/ epidermal growth factor (egf) and bone morphogenetic protein (bmp)/nodal signalling are required during tissue development and in adult homeostasis and repair. by combining the knowledge on stem-cell populations and on the intestinal stem-cell niche requirements, intestinal organoids from either postnatal or adult intestinal epithelium (ootani et al., 2009) or from a single adult intestinal stem cell (sato et al., 2009) have been established, with an expansion potential far beyond the hayflick limit (>1 year in culture). similarly, mouse-and human-derived colonic stem cells have also been expanded in organoid cultures with minor adjustments in the medium composition . the culture conditions that support intestinal organoids include embedding the intestinal stem cells (or cells with the ability to acquire stem-cell potential, i.e. immediate daughters of the stem cells) in a 3d extracellular matrix (e.g. matrigel) and culturing them in a medium supplemented with egf, noggin and the wnt agonist r-spondin (rspo). these conditions allow the long-term expansion of adult intestinal epithelium in culture while retaining its ability to differentiate into individual derivatives ootani et al., 2009) . in parallel studies, the wells lab successfully differentiated pscs into gut epithelium in vitro by treating definitive endoderm-specified cells with wnt3a and fgf4 to induce posterior endoderm patterning and hindgut specification (spence et al., 2011) . these conditions result in the formation of spheroids after 2 days. then, upon transfer into matrigel and incubation in culture conditions that support the growth of adult-tissue-derived organoids, these psc-derived spheroids formed bona-fide small-intestinal organoids. recently, these psc-derived intestinal organoids have been combined with neural crest cells to recapitulate the normal and functional intestinal enteric nervous system (workman et al., 2017; schlieve et al., 2017) . gastric organoids have been obtained by either expansion of adult gastric stem cells from both the corpus and the antropyloric epithelia, as well as from directed differentiation of pscs. the identification of lgr5 as a marker for pyloric stem cells (barker et al., 2010) led to the development of the first long-term culture system of mouse gastric stem cells. corpus and pylorus stem cells were grown in matrigel and in medium supplemented with wnt3a and fgf10 (barker et al., 2010; stange et al., 2013) . blockade of transforming growth factor-beta (tgfβ) signalling enabled the expansion of human gastric organoids from both pylorus and corpus epithelium that would contain all gland and pit cell types (bartfeld et al., 2015) . differentiation into acid-producing parietal cells proved more difficult, though. only upon co-culture with the mesenchymal niche, corpus organoids from neonatal (li et al., 2014) and adult (bertaux-skeirik et al., 2016) mouse stomach tissue could differentiate into this mature cell type. the human counterpart has not been achieved, yet. to obtain psc-derived gastric organoids, mccracken et al., upon establishing definitive endoderm with activin (kubo et al., 2004; d'amour et al., 2005) , exposed the cells to wnt, fgf4 and noggin to enable gastric specification. noggin is essential to prevent intestinalization and to promote a foregut fate (mccracken et al., 2014) , while retinoic acid facilitates antrum epithelium specification. matrigel is essential for the formation of 3d foregut structures. when maintained in an egf-rich medium, these structures generate gastric organoids that contain all antral epithelium cell types (mccracken et al., 2014) . noguchi and colleagues used mouse pscs to generate a functional corpus epithelium in vitro that contained acid-producing cells (noguchi et al., 2015) . in a different approach, wells and colleagues observed that, after foregut patterning, sustained wnt signalling using the gsk3β inhibitor chir enabled fundus (corpus) specification instead of antrum. these fundic cells could be subsequently differentiated into all cell types of the gastric epithelium, including functional parietal cells (mccracken et al., 2017) . interestingly, the authors demonstrate that their psc-differentiated fundic organoids contain bona-fide stomach progenitors that can be isolated, cultured and further propagated in the tissue-derived organoid medium described by barker, huch and colleagues (barker et al., 2010) to expand adult-tissue-derived organoids (mccracken et al., 2017) , thus linking, for the first time, the development of both types of organoids. adult hepatocytes and cholangiocytes (also known as ductal cells) are the two endodermal-derived cell types in the adult liver, yet the organ is composed of mesoderm-derived hepatic mesenchymal cells. michalopoulos et al. first described liver-derived 3d structures back in 2001. in these studies, these 'organoids' were very different from what we now consider liver organoid cultures, as they would only survive for a short period of time in culture, yet they retained some of the function and structure of the hepatocyte epithelium (michalopoulos et al., 2001) . it was not until 2013 that the first liver organoids as we know them now were described. huch et al. established the first adult murine-tissue-derived liver organoid culture that sustains the long-term expansion of liver cells in vitro (huch et al., 2013b) . by combining matrigel with hepatocyte growth factor box 3. germ-layer formation: the starting point of organogenesis all organs develop from primitive tissues that form at the very early onset of embryogenesis. after fertilization, cells within the early blastula establish two main compartments: the extraembryonic tissue, which will give rise to the supportive foetal environment including placenta and amniotic sac, and the inner cell mass (icm), which will give rise to the embryo proper. human pluripotent stem cells (pscs) can be thought of as roughly equivalent to icm stem cells, and indeed human escs are taken from the icm of the human blastocyst. upon gastrulation, the embryonic disc, which derives from the icm, undergoes morphogenetic movements that establish the three germ layers: endoderm, mesoderm and ectoderm. progenitors within these germ layers have restricted potentials to generate primordial organ structures, and their specific identity will depend on their spatial context relative to one another, and on where they lie on the anterior-posterior and dorsal-ventral axes (zernicka-goetz and hadjantonakis, 2014) . this is due to the early establishment of gradients of morphogens that will influence differentiation towards particular subregions of these germ layers. for example, the endoderm will give rise to the entire gastrointestinal tract and, depending on their location, endodermal progenitors can give rise to more anterior identities, such as the stomach, or to more posterior identities, such as the colon. this is due to an anterior-posterior gradient of signalling factors like wnt, bmp and fgfs (zorn and wells, 2009) . similarly, bmp4 promotes formation of the mesendoderm, a precursor to certain mesoderm and endoderm types, whereas early activation of wnt can promote the presomitic mesoderm, the precursor to the kidney (little et al., 2016) . in contrast, in the absence of growth factors or serum, the embryoid body tends to be biased towards the ectoderm as the default. by understanding these specific morphogen gradients and their effects on the primordial tissue, these growth factors can be provided at specific concentrations and with specific timing to direct the differentiation of progenitors towards certain organ identities. (hgf), egf and liver-damage-induced factors fgf (takase et al., 2013) and rspo1 (huch et al., 2013b) , the isolated liver cells selforganized into 3d structures that retained the ability to differentiate into functional hepatocyte-like cells, even when grown from a single cell (huch et al., 2013b) . addition of an activator of cyclic adenosyl monophosphate (camp) signalling and inhibition of tgfβ signalling adapted this culture system to the expansion of adult human liver cells as self-renewing organoids that recapitulate some function of ex vivo liver tissue . of note, both human and mouse cultures could be established from single cells, hence enabling, for the first time, the study of mutational processes in healthy tissue blokzijl et al., 2016) . takebe et al. took a completely different approach and, by mixing human induced psc (ipsc)-derived hepatocytes with mesenchymal stem cells (mscs) and umbilical cord cells (huvecs), obtained the first embryonic liver bud organoids formed by proliferating hepatoblasts and supporting cells. when transplanted into different mouse sites, these developed into mature functional hepatic tissue (takebe et al., 2013) . in a follow-up study, takebe and colleagues also differentiated ipscs towards the three hepatic progenitorshepatic endoderm, endothelium and septum transversum mesenchymehence overcoming the issue of using postnatal tissue-derived stromal progenitors (takebe et al., 2017) . in 2015, sampaziotis et al. also generated cholangiocyte organoids from ipscs (sampaziotis et al., 2015) . whether combining both protocols could generate structures containing both hepatocytes and cholangiocytes or whether, instead, a better specification of true bi-potent hepatoblasts, like the novel lgr5+ population described by prior et al. (2019) is required first to achieve this goal, remains to be determined. since these seminal papers, altered protocols have enabled the establishment of liver models from different species from rat to dog (nantasanti et al., 2015; kuijk et al., 2016; kruitwagen et al., 2017) , as well as human disease models ranging from alpha-1-antitrypsin (a1at) deficiency and alagille syndrome to polycystic liver disease (wills et al., 2016) or cancer (broutier et al., 2017; nuciforo et al., 2018) . by modifying the huch conditions to expand human organoids , the vallier lab has recently established extrahepatic biliary organoids and showed that these can form bile-duct-like tubes that can reconstruct the gallbladder wall upon transplantation into mice (sampaziotis et al., 2017a) . finally, the nusse and clevers labs recently reported the establishment of hepatocyte organoids derived from adult mouse hepatocytes (peng et al., 2018; hu et al., 2018) , while the huch lab recently established clonal mouse hepatoblast cultures (prior et al., 2019) . while human adult hepatocyte cultures are yet to be established, human embryonic cultures that excellently recapitulate the bile canaliculi structure in vitro have been recently described . whether these cultures can differentiate into more mature hepatocyte or cholangiocyte derivatives and/or exhibit the extensive cellular plasticity of the in vivo tissue is still to be investigated. similarly to the liver, both embryonic and/or adult pancreas cells are difficult to expand or maintain in culture. by isolating mouse embryonic pancreas progenitors and culturing them in matrigel, grapin-botton and colleagues elegantly showed that the cells could recapitulate pancreatic embryonic development in vitro and generate exocrine (acinar) and endocrine (insulin-producing) cell derivatives (greggio et al., 2013) . by using a similar culture system as the adult liver organoids described above, huch et al. established the first adult mouse pancreas organoid model by seeding pancreas ductal cells in matrigel in the presence of fgf10, noggin, rspo1 and egf (huch et al., 2013a) . a similar system was later applied to human pancreatic cancer cells (boj et al., 2015) , although long-term expansion of healthy human pancreas tissue is still to be achieved. recently, loomans et al. expanded human pancreas tissue for a few passages and obtained structures formed exclusively of ductal epithelium. after transplantation into mice, these expressed some endocrine markers, although did not differentiate into islets (loomans et al., 2018) . unlike the grapin-botton lab's embryonic cultures described above, loomans' adult-tissue-derived ones cannot differentiate into endocrine or acinar cells in vitro. whether that is because adult pancreas ductal cells have lost their endocrine differentiation potency or whether the external cues have not been identified yet is unknown. the term 'lung organoids' describes both upper-and lower-airway organoids. protocols for both psc-and adult-tissue-derived organoids have been established. rossant and colleagues differentiated human ipscs to lung epithelium using air-liquid interphase culture (wong et al., 2012) . this initial protocol was later modified by the spence lab to instruct the cells towards a foregut fate by adding tgfβ/bmp inhibitors, fgf4, and wnt activators. subsequent activation of the hedgehog pathway with the smoothened agonist sag resulted in lung specification. transferring these specified 3d spheroids into matrigel and culturing them in fgf10-rich medium enabled the differentiation to lung organoids containing airway-like structures formed by basal, ciliated and club cells, surrounded by a mesenchymal compartment (dye et al., 2015) . interestingly, when transplanted in a bioartificial microporous poly(lactide-co-glycolide) scaffold, full maturation and differentiation of secretory lineages was observed, while long-term engraftment enabled these to differentiate into distal lung cells (dye et al., 2016) . parallel studies by the snoeck lab also showed the formation of 3d structures that developed into branching airway and early alveolar epithelium after xenotransplantation (chen et al., 2017) . establishing organoids from adult lung tissue has been more difficult. hogan and colleagues pioneered upper-airway organoids, reporting that single basal cells would self-organize into bronchiolar lung organoid cultures. these had limited expansion potential, yet were able to differentiate to both basal and luminal cells (rock et al., 2009) . building on these cultures, the rajagopal lab was able to expand airway basal stem cells by inhibiting smad signalling (mou et al., 2016) . the generation of distal (lower)-airway lung organoids has been more difficult. alveoli consist of surfactant-secreting type ii (at2) and gas-exchanging type i (at1) cells. by identifying and expanding bronchioalveolar stem cells (bascs), kim and colleagues showed that single bascs had bronchiolar and alveolar differentiation potential in lung organoids co-cultured with lung endothelial cells (lee et al., 2014) . similarly, barkauskas et al., by co-culturing human at2 cells with lung fibroblasts, established self-renewing human alveolar organoids (barkauskas et al., 2013) . co-culturing macrophages with at2 cells promoted the formation of organoids from at2 cells (lechner et al., 2017) in a dose-dependent manner (i.e. the more macrophages the more at2 growth). only recently, two labs have been able to expand lung progenitors in the absence of a mesenchymal or endothelial niche: the rawlins lab described the first human long-term self-renewing lung organoids derived from embryonic lung (nikolićet al., 2017) , while the clevers lab generated 3d human airway organoids containing ciliated, goblet, club and basal cells (sachs et al., 2019) . the generation of self-renewing human alveolar organoids in a defined medium still awaits development, though. although it was well known by the mid-20th century that dissociated rat thyroid cells aggregate and reconstitute functionally active thyroid tissue in vitro (mallette and anthony, 1966) , it was not until very recently that researchers successfully generated thyroid organoids from pscs. this delay was due to the lack of knowledge on how thyroid tissue develops. by studying the transcription factors important for thyroid development, antonica and colleagues found that forced expression of thyroid-specific transcription factors in pscs resulted in thyroid cells that formed follicles in vitro and upon transplantation (antonica et al., 2012) . more recently, kotton and colleagues identified the right combination of bmp and fgf signalling to enable the differentiation of ipscs to a thyroid fate. by isolating these thyroid progenitors and cultivating them in matrigel, the authors generated follicular organoids consisting of a monolayer of hormone-producing thyroid cells. of note, following xenotransplantation, these organoids restored th levels in a hypothyroid mouse model (kurmann et al., 2015) . whether selfrenewing human thyroid organoids can be obtained from adult thyroid epithelium remains to be discovered. in light of the early studies from mallette and anthony and the recent report from nagano and colleagues suggesting that mouse thyroid organoids can be developed from adult tissue (saito et al., 2018) , we speculate that the human counterpart will soon also be developed. the embryonic mesoderm gives rise to a variety of internal organs, including kidney, heart, cartilage, bone, reproductive organs and muscle. we detail below the methods to generate the respective human organoids. kidney organoids are an ideal illustration of the successful generation of an in vitro model based upon careful characterization of in vivo development. in particular, several recent studies have begun to shed light on where the key kidney precursors come from. work from taguchi et al. (2014) was instrumental in characterizing the early stages of metanephric kidney development, particularly the formation of metanephric mesenchyme (mm), then applying the identified signalling factors to direct differentiation of mouse and human pscs specifically towards mm cells that could form 3d structures when cocultured with mouse tissues. this study helped define the developmental steps leading to kidney formation. while taguchi et al. demonstrated the successful induction of mm, takasato et al. (2014) and xia et al. (2013) described conditions that would also give rise to the ureteric bud (ub). in both studies, the authors initially directed pscs towards the intermediate mesoderm by first mimicking signalling events involved in primitive streak formation. in the xia et al. study, the cells then gave rise to ub precursors that could mature and integrate into cocultures with embryonic mouse kidney. takasato et al. instead simultaneously generated human ub and mm identities. not only could these cells integrate into embryonic mouse kidney, but they could also form rudimentary tubule structures upon aggregation even without the supportive mouse tissue. the variety of approaches for generating early self-organising renal structures generally points to important roles for several growth factors, including bmp4, retinoic acid, fgfs, wnt and activin a. however, a breakthrough came with the establishment of the entirely self-organizing 3d human renal organoids by takasato et al. (2015) , which involved minimal use of growth factors ( just chir99021 to activate wnt, and fgf9), and led to structures with all the major components of the developing kidney, including the collecting duct, proximal and distal tubules, glomeruli, and even endothelial networks. although proper bone organoids have not yet been established, kale et al. described a promising approach to form spheroids of human adult bone precursor cells (kale et al., 2000) . these are a heterogeneous population of bone progenitors, including osteoblasts, that can be induced to form small pieces of crystalline bone called microspicules. this requires 3d aggregation of the cells as well as the removal of serum and addition of tgfβ1. given that this approach lacks the varied cell types and tissue architecture, it will be interesting to see whether current improvements in 3d culture methods could further develop these spheroids to bona-fide bone organoids. adult-derived organoid methodology has recently been applied to the fallopian tube of the female reproductive tract. the method described (kessler et al., 2015) has many similarities to the barker and huch et al. stomach organoid method and involves culture of human adult fallopian tube epithelial cells in matrigel in the presence of mouse gastric organoid medium (barker et al., 2010) with tgfβ inhibition. the resulting 3d cystic spheres develop various invaginations as they mature, and contain both ciliated and secretory cells. these tissues also respond to the sex hormones estradiol and progesterone in a manner reminiscent of the response of this tissue in vivo during the menstrual cycle. two independent groups have derived organoids from the adult endometrium using similar conditions as those described for adult liver organoids (turco et al., 2017; boretto et al., 2017) . hence, these required the presence of rspo, egf, fgf10 and noggin, but no wnt supplementation. both studies demonstrated that the endometrial organoids respond to estradiol and progesterone in specific and differential manners. specifically, estradiol stimulated increased proliferation while progesterone stimulated a more mature morphology, mimicking the later secretory phase of the menstrual cycle. these morphological and transcriptomic changes recapitulated the changes in the endometrium during the menstrual cycle. furthermore, turco et al. exposed the endometrial organoids to pregnancy signals (prolactin, human chorionic gonadotropin and human placental lactogen), which led to a decidua-like morphology similar to that seen in early pregnancy. the embryonic ectoderm gives rise to two main tissue types: surface ectoderm, which will develop into skin and its associated glands and hair; and neural ectoderm, which will give rise to the brain, spinal cord and neural crest. the neural crest is a highly multipotent entity that can further differentiate into the peripheral nervous system, as well as bone, cartilage, connective tissue, and vasculature of the head, and even contributes to the heart. for decades, mammary cells have been cultured in 3d extracellular matrix gels as a model for mammary gland development, homeostasis and tumorigenesis (hall et al., 1982) . indeed, isolated mouse mammary epithelial cells were some of the first cells to be cultured in matrigel (li et al., 1987) , and could give rise to branched structures reminiscent of the mammary gland. however, differences between mouse and human mammary stroma have made it difficult to directly translate these methodologies to a human in vitro model. the first 3d branched mammary-gland-like structures from human mammary cells were described by gudjonsson et al. (2002) , who isolated a progenitor population that could generate several cell types and the typical branched morphology of the mammary gland when embedded in matrigel. similarly, dontu et al. described the formation of mature ductal-acinar-like structures from multipotent human mammary epithelial cells upon embedding in matrigel and exposure to prolactin (dontu et al., 2003) . importantly, these studies successfully generated mammary-gland-like organoids from cells first expanded in vitro either as a cell line or in mammospheres. more recently, researchers devised methods to generate branching structures directly from isolated human mammary epithelial cells (linnemann et al., 2015) . the formation of these structures similarly relies upon embedding in 3d matrix gels and the presence of growth factors, including egf, hydrocortisone and insulin, to promote mammary epithelial-stem-cell proliferation. under these conditions, and with the addition of forskolin (fsk), human mammary epithelial cells spontaneously generated structures reminiscent of the terminal ductal lobular units of the mammary gland. importantly, these structures contained multiple cell types at correct positions within the branched structure and showed contractile activity, a feature of the basal/myoepithelial cells that eject milk during lactation. there is some controversy about the developmental origin of the salivary glandswhether ectodermal or endodermal (see patel and hoffman, 2014 and de paula et al., 2017 for details), yet mouse lineage-tracing studies suggest an ectodermal origin (rothova et al., 2012) , although there is no direct evidence for this yet. by applying the clevers organoid method to primary adult salivary gland cells, coppes and colleagues efficiently generated long-term-expanding organoids from the mouse and human salivary gland. the culture conditions required high levels of wnt activation to obtain organoids containing all differentiated salivary gland cell types. transplantation of these organoids into murine submandibular glands restored saliva secretion and increased the number of functional salivary gland acini in vivo . these studies hold the promise of salivary gland transplantation as a potential treatment for xerostomia (severe hyposalivation). the retina is the neural part of the eye and contains photoreceptors, supportive cells and interneurons. self-organizing retinal tissues were the first entirely 3d neural organoids to be successfully established from mouse (eiraku et al., 2011) and later human (nakano et al., 2012) escs. as with many organoids, matrigel turned out to be a key component. both studies described a very minimal medium, along with wnt inhibition, to develop retinal identities from escs. but it was the addition of dissolved matrigel that allowed for the formation of a more rigid epithelium that could adopt the morphology of the retinal primordium, the optic cup. these optic-cup-like organoids begin as aggregates containing large spherical vesicles of neuroepithelium with an early retinal identity and appear similar to optic vesicles. as development proceeds, these vesicles spontaneously invaginate to from a cup-like morphology reminiscent of the developing optic cup. optic-cup organoids form the typical stratified architecture of the developing retina, including rods and cones. however, generating functional photoreceptors, including light-sensitive outer segments, has been a challenge. since the initial description of self-organizing retinal tissues from human escs, others have improved upon these methods and recently described extended culture times and further maturation of the photoreceptors to include rudimentary outersegment discs and even occasional light responsiveness (wahlin et al., 2017) . in vitro modelling of human brain development has been a fast-evolving field that builds upon decades of in vivo and ex vivo work. the discovery in 2001 that human escs could form 2d rosette-like structures after an initial phase of 3d culture as an eb (zhang et al., 2001) revealed the self-organization potential of neural progenitors to form neural-tube-like structures. eiraku et al. then expanded upon this by extending the initial 3d culture phase before plating on coated dishes, which allowed the formation of even more complex stratified structures reminiscent of the developing cerebral cortex (eiraku et al., 2008; mariani et al., 2012) . specifically, neural progenitors could self-organize to form a continuous neuroepithelium, similar to retinal organoids, but as these brain-regionalized structures developed, progenitors generated neurons that properly migrated away from germinal zones to populate a more basal region reminiscent of the pre-plate of the cerebral cortex. this germinal zoning and segregation of post-mitotic neurons is highly reminiscent of the developing human brain. these initial cultures used dual smad and wnt inhibition (watanabe et al., 2005; eiraku et al., 2008) to promote the formation of relatively pure neural identities as well as to direct differentiation towards a telencephalic fate. subsequent work showed that, in the absence of these inhibitors, simply providing a very minimal medium to prevent the expansion of non-neuroectodermal identities could generate a more broad brain regional identity (lancaster et al., 2013) . furthermore, embedding in matrigel led to a dramatic reorganization and expansion of the neuroepithelium that allowed the formation of neural-tube-like buds that further expanded without the need for replating the aggregates. like the telencephalic structures described above, these cerebral organoids developed cortical regions with characteristic germinal and differentiated zones. however, these 3d tissues also formed a variety of brain regions, from hindbrain to retinal identities, within the same organoid. similarly, maintenance as a floating aggregate was also applied to the telencephalic structures to generate 3d forebrain organoids with improved tissue architecture (kadoshima et al., 2013) . since the establishment of telencephalic and cerebral organoids, subsequent studies further modified these approaches and generated organoids with more specific brain regional identities, including cortical spheroids (paşca et al., 2015) , hippocampal organoids (sakaguchi et al., 2015) , midbrain organoids (jo et al., 2016) , pituitary and hypothalamic organoids (suga et al., 2011; ozone et al., 2016) , and cerebellar organoids (muguruma et al., 2015) . more recently, researchers fused regionalized brain organoids, revealing that interneurons generated within the ventral telencephalic region could indeed migrate to dorsal cortical tissue as they do in vivo (birey et al., 2017; bagley et al., 2017; xiang et al., 2017) . finally, extended culture of cerebral organoids resulted in neuron maturation within the organoid, allowing for the formation of rudimentary networks and even rare responses to light in these whole-brain organoids containing retinal cells (quadrato et al., 2017) . the inner ear develops from non-neural ectoderm and contains thousands of hair cells that respond to minute air vibrations and thus allow us to hear, as well as detecting head movements and gravity. koehler et al. developed human inner-ear organoids from escs by using bmp4 to promote the non-neural ectoderm lineage as opposed to the neural ectoderm (koehler et al., 2017) . however, tgfβ inhibition prevented unwanted mesoderm and endoderm formation. this approach led to the successful formation of an epithelium on the surface of 3d aggregates of escs that expressed otic placode markers, the precursor to the inner ear. wnt activation subsequently led to the development of otic vesicles with supportive mesenchyme and later formation of epithelia-containing sensory hair-like cells. a primary goal of human organoids is their use in modelling human diseases in order to establish paradigms for drug screening, genotype-phenotype testing and even biobanking for specific diseases and future personalized treatments, including cell therapies. while the establishment of a human organoid model for most organs is still quite new, and therefore disease modelling in this manner is still in its infancy, there have already been several examples of using organoid cultures to study congenital or acquired human diseases. the first human condition to be modelled in organoids was cystic fibrosis (cf), in the intestinal organoid system. cf is caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) chloride channel, which is normally expressed in epithelial cells of many organs. dekkers and colleagues (2013) generated cf-patient-derived intestinal organoids that could recapitulate the disease in vitro. they developed a swelling assay in which wild-type organoids respond to the activation of camp by importing fluid to the lumen and subsequently swelling, whereas this response is abolished in cf organoids. this approach demonstrated the excellent predictive value of intestinal organoids for identification of responders to cftr modulators, and has become the first personalized treatment test for cf patients in the netherlands (berkers et al., 2019) . in a parallel approach, vallier and colleagues also showed that ipscs from cf patients differentiated into liver cholangiocytes can also model cf in vitro, as these cells also lack the ability to swell compared to wild-type controls (sampaziotis et al., 2015) . in addition, huch et al. showed that liver organoids from patients with a1at deficiency recapitulated the epithelial features of the disease, whereby precipitates of the misfolded a1at protein accumulated in the differentiated hepatocyte-like cells in vitro . similarly, failure to develop mature biliary cells in liver organoids from an alagille syndrome patient mirrored the biliary defects observed in patients . when cerebral organoids were first established, they were also applied to the study of primary microcephaly, a genetic condition caused by a mutation in cdk5rap2 (lancaster et al., 2013) . the brain organoids generated from patient-derived ipscs were overall much smaller and the individual cortical regions were especially hypoplastic. a series of observations at various time points and specific examination of mitotic spindle orientation during progenitor divisions revealed that the patient neural stem cells began dividing asymmetrically and generating neurons too early, which led to a depletion of the progenitor pool and an eventual decrease in overall neuron number. because mice could not fully recapitulate the extent of brain-size reduction seen in humans, the organoids revealed certain morphological differences that could only be seen in this human-specific model system. a modification of the more regionally specified telencephalic protocol summarized above (eiraku et al., 2008) was also used to model a human neurodevelopmental condition, idiopathic autism spectrum disorder (asd). mariani et al., 2015 established ipsc lines from four asd patients along with closely related unaffected controls. these were initially grown as 3d aggregates, followed by plating of rosettes as described previously (eiraku et al., 2008; mariani et al., 2012) , with the modification that rosettes were then lifted off and grown again as 3d aggregates to yield forebrain organoids. although generally very similar between probands and controls, the asd organoids displayed an increased number of inhibitory interneurons as a result of upregulated foxg1, an important factor in forebrain patterning. more recently, a genetic condition causing lissencephaly (smooth brain) was modelled using forebrain organoids, revealing defects in progenitors, including mitosis timing, spindle orientation, and defective wnt signalling (bershteyn et al., 2017; iefremova et al., 2017) . leber congenital amaurosis is a ciliopathy that affects the retina and leads to inherited blindness. to model this condition, parfitt et al. (2016) generated retinal organoids from ipscs with a mutation in cep290, a known genetic cause of this condition. these organoids displayed normal initial development into optic cups, but the resulting tissues showed decreased ciliation and reduced cilia lengths. by restoring the expression of full-length cep290, the authors were able to rescue the cilia length and protein trafficking in the cilium. not only can organoids be used to model congenital conditions from stem cells of patients carrying the genetic mutation, but they can also be used to model acquired diseases such as those carried by infectious agents or acquired mutations as in the case of cancer. since the first report that hela cells could be grown in vitro, researchers have established cancer cell lines for the majority of tumour types that have facilitated seminal discoveries in cancer biology. in addition, their ease of culture have made them excellent for large-scale drug screening and development (alley et al., 1988) , and have enabled the identification of genomic markers of drug sensitivity (garnett et al., 2012) . however, they have certain drawbacks: (1) they lack the tissue architecture of the organ in question, which is often intimately related to differentiation and disease progression, (2) their establishment usually requires a strong cellular selection and (3) they present extensive heterogeneity between labs, with marked differences in gene expression and proliferation, and a considerable variability in drug responses (ben-david et al., 2018; hynds et al., 2018) . in an attempt to obtain better models that recapitulate the architecture, genetics and drug responsiveness of patients' tumours, patient-derived xenografts (pdxs) emerged as a very suitable alternative. unfortunately, they are not applicable to all cancer models, are expensive and are impractical for large drug screenings. [for an extended review on pdx models, see hidalgo et al. (2014) .] in that regard, the discovery that healthy tissue from colon (jung et al., 2011; sato et al., 2011) and stomach (barker et al., 2010) could be expanded in vitro prompted many researchers to invest significant effort in using organoid technology to model cancer for personalized medicine, drug testing and drug discovery. indeed, the use of organoids to model cancer in vitro has become of great interest to the cancer field, and organoids derived from tissue resections, biopsies or even circulating tumour cells have now been established (see table 2 for a detailed list). in all cases, cancer-derived organoids more faithfully maintain the genetic and phenotypic features of the tumour of origin. in that sense, they resemble pdxs, but with the advantage of a higher establishment success rate, can be easily expanded in vitro and are amenable for drug screening (weeber et al., 2017; pauli et al., 2017) . the ability to expand primary cancer tissue in a dish has opened up the possibility of living biobanks of cancer-derived organoid cultures from different tumour types, including colorectal , gastric (yan et al., 2018) , breast and bladder mullenders et al., 2019) cancer. these provide opportunities for drug screening as well as drug development. hence, organoids from many tissues, ranging from the ones listed above to liver (broutier et al., 2017) and oesophageal cancer, have proven suitable for large drug-screening tests. while maintenance of genotypic and phenotypic features as well as suitability for drug testing does inform us about the translational potential of cancer organoids, it would be even more informative to investigate their predictive value in correlating drugsensitivity data with clinical or genomic data. up to today, only two studies, one in colon (vlachogiannis et al., 2018) and the other in bladder cancer organoids, have demonstrated the potential of organoids for predicting patient response. we envision that these are only the tip of the iceberg of many more studies to come to correlate the predictive value of organoids with clinical outcome. organoids have proven a good model to study infectious diseases and the mechanisms behind human-specific infectious agents. hence, human small-intestinal organoids have been used to model norovirus (hunov) infection and propagation, and have enabled the identification of bile as a critical factor for hunov replication (ettayebi et al., 2016) . similarly, cryptosporidium has been shown to infect and complete its life cycle in intestinal and lung organoids (heo et al., 2018) , which has facilitated the identification of not only type ii but also type i interferon signalling as a response to parasite infection. the ability to use human intestinal organoids to propagate coronaviruses in vitro has enabled the identification of the small intestine as an alternative infection route for middle east respiratory syndrome coronavirus, which causes severe human respiratory infections (zhou et al., 2017a) . similarly, gastric organoids have been used to develop models of helicobacter pylori infection (bartfeld et al., 2015; mccracken et al., 2014) , while lung organoids have modelled influenza virus infection in vitro (zhou et al., 2018) . for extended reviews, see bartfeld (2016) , lanik et al. (2018) and dutta et al., 2017. along the same lines, cerebral organoids that model genetic microcephaly were recently adapted by a number of research groups to study the mechanisms of microcephaly caused by zika-virus infection. this area of research has highlighted the power of brain organoid methods and the ease with which independent investigators could adopt the technology. remarkably, initial observations of the effect of zika on brain organoids were described as soon as 3 months after the world health organization (who) declared zika a global health emergency in 2016. these initial reports (garcez et al., 2016; cugola et al., 2016; qian et al., 2016) described overall smaller sizes of infected organoids, consistent with the microcephaly seen in patients, and a specific effect on neural progenitors, leading to cell death, reduced proliferation and premature differentiation. more recently, these zika-infected brain organoids have been used by at least five independent groups to test treatment strategies that would prevent the effects of zika virus infection on neural progenitors zhou et al., 2017b; sacramento et al., 2017; xu et al., 2016; li et al., 2017) . the rapidity with which a completely new disease model has been established and used for drug testing speaks to the power and future potential of these in vitro models for a variety of disorders. the discovery of crispr/cas9 gene editing as a user-friendly genetic-engineering tool compared to talen or zinc-finger nuclease technologies has prompted many investigators to adapt it to almost any cell type in any organism (for extended review see adli, 2018; sander and joung, 2014) . the clevers lab pioneered the application of crispr/cas9 technology to organoid cultures and used it to correct mutations and restore the chloride channel function of cf-patient-derived intestinal organoids (schwank et al., 2013) . after this seminal paper, many others have highlighted the technology's applicability to organoid cultures and its relevance to further our understanding of human pathologies (see driehuis and clevers, 2017 for an extended review). in particular, gene editing in colon organoids has enabled the step-wise recapitulation of tumorigenesis in vitro (drost et al., 2015; matano et al., 2015) , the identification of cancer signatures in microsatellite-unstable tumours (drost et al., 2017) , the discovery of new genes involved in liver cancer (artegiani et al., 2019) or even the development of the first human brain organoid cancer model for primitive neuroectodermal tumours (bian et al., 2018) . while organoids are powerful tools for modelling human organogenesis, homeostasis, injury repair and disease aetiology, the technology needs to overcome many hurdles to reach the next level in modelling human disease. specifically, because of their 3d nature, the size of all organoids is limited by nutrient supply; because organoids lack vasculature, their development and growth depend upon diffusion from the surrounding media. while this is sufficient for smaller organoids or those without complex stratification, such as the branched ductal organoids and the epithelial tissues of the endoderm, the thicker tissues of the brain experience dramatic necrosis in the organoid interior. thus, extensive effort will likely focus on improving nutrient supply and even vascularization. some organoids have been implanted into highly angiogenic sites in rodents to enable vascularization and blood perfusion by the host (takebe et al., 2015) . this approach has even revealed that certain human-specific bi-products, for example liver metabolites, are detectable in the rodent host blood takebe et al., 2013; hu et al., 2018) . furthermore, brain organoids transplanted into a highly angiogenic site of the rodent brain could be vascularized to improve survival and attract host-derived microglia (mansour et al., 2018) . finally, recent work applying fluid flow to kidney organoids revealed the ability of endogenous endothelial cells to form a vascular network and improve the maturation of the kidney tissue (homan et al., 2019) . this vascularization approach has the potential to overcome tissue growth/survival limitations while maintaining in vitro accessibility and scalability. another hurdle to disease modelling and drug testing is the scalability of organoid cultures. because of their 3d nature and complex morphologies, organoids are typically examined using laborious and time-consuming assays such as immunohistochemistry. furthermore, their culture requires larger culture vessels and volumes of media such that it is usually quite difficult to perform drug testing in the commonly used 384-well plate format. thus, other scaling approaches are being developed, such as mini-spinning bioreactors, or spin-ω, for scaling up production of brain organoids . the complexity that arises from their self-organization also introduces some unpredictability to this model system, particularly in the case of human psc-derived organoids. thus, a desired tissue identity is not always reproducible, and even when it is present, it rarely (if ever) arises in the same configuration from organoid to organoid. this means that researchers using these methods must carefully control which organoids to use for analysis and must examine many organoids in order to separate the real phenotype from the noise of inhomogeneity. however, a number of new methods have recently been introduced to begin to address this issue, such as bioengineering methods using scaffolds or micropatterned substrates that help guide the development of stem cells to particular identities and morphologies (warmflash et al., 2014; lancaster et al., 2017; knight et al., 2018) . as discussed above, organoids have proven amenable for drug testing with relatively small catalogues of drug compounds. more large-scale drug testing to identify novel compounds that may treat a range of patients will require scaling up of organoids, and the pharmaceutical industry is beginning to investigate this approach. however, in the more immediate term, organoids could be combined in the already established arsenal of 2d and bioengineered disease models. for example, cells generated from organoids could be isolated and cultured in 2d as a more accessible and scalable source. furthermore, organ-on-a-chip approaches may be applied to organoids, or cells isolated from organoids, in order to capture the cellular diversity, but in a defined configuration. microfluidics can also be applied to organoids to introduce fluid flow or restrict their growth in defined spatial conformations (karzbrun et al., 2018) . finally, even with further improvements, organoids will not replace existing models in drug development, and should be thought of as complementary to other methods. in particular, organoids cannot replace animal models as the in vivo whole-organism context will remain a necessity when evaluating a particular drug candidate. not only could disease model organoids be used for drug testing, but there is an increasing use of liver organoids and liver-on-a-chip models to test drug metabolism (kimura et al., 2018) . since the us food and drug administration (fda) now requires drug companies to demonstrate downstream metabolites before approval, human in vitro liver models allow for initial testing before testing in patients, in which potentially unpredicted metabolites could do harm. finally, while still far from being a reality, a long-term goal of organoid technologies will be to apply them to cell replacement or even whole-organ transplantation. currently, organ donors are in short supply and many patients fail to receive a vital organ transplant in time. a source of functioning cells or tissues that could replace the faulty ones in a patient would be a major advance and potentially save thousands of lives each year. while vast improvements on the technology are still required to achieve this goal, we envision that the field might see this type of application, and many others, in the future. m.a.l. is an inventor on patent applications describing the development of cerebral organoid methods. m.h. is an inventor on patents describing the development of gastric, pancreas and liver organoids. work in the lancaster laboratory is supported by the medical research council mc_up_1201/9, and the european research council (erc stg 757710). m.h. is a wellcome trust sir henry dale fellow and is jointly funded by the wellcome trust and the royal society (104151/z/14/z). in addition, the work in the huch lab is also funded by an h2020 european research council lsmf4life grant (ech2020-668350) and an nc3rs project grant (nc/r001162/1). the haplotype-resolved genome and epigenome of the aneuploid hela cancer cell line the crispr tool kit for genome editing and beyond feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay 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inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen a comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening from pluripotency to differentiation: laying foundations for the body pattern in the mouse embryo in vitro differentiation of transplantable neural precursors from human embryonic stem cells human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus high-content screening in hpsc-neural progenitors identifies drug candidates that inhibit zika virus infection in fetal-like organoids and adult brain differentiated human airway organoids to assess infectivity of emerging influenza virus vertebrate endoderm development and organ formation key: cord-312438-zr9zx7pv authors: hoo, regina; nakimuli, annettee; vento-tormo, roser title: innate immune mechanisms to protect against infection at the human decidual-placental interface date: 2020-09-10 journal: front immunol doi: 10.3389/fimmu.2020.02070 sha: doc_id: 312438 cord_uid: zr9zx7pv during pregnancy, the placenta forms the anatomical barrier between the mother and developing fetus. infectious agents can potentially breach the placental barrier resulting in pathogenic transmission from mother to fetus. innate immune responses, orchestrated by maternal and fetal cells at the decidual-placental interface, are the first line of defense to avoid vertical transmission. here, we outline the anatomy of the human placenta and uterine lining, the decidua, and discuss the potential capacity of pathogen pattern recognition and other host defense strategies present in the innate immune cells at the placental-decidual interface. we consider major congenital infections that access the placenta from hematogenous or decidual route. finally, we highlight the challenges in studying human placental responses to pathogens and vertical transmission using current experimental models and identify gaps in knowledge that need to be addressed. we further propose novel experimental strategies to address such limitations. the human placenta is the temporary extra-embryonic organ that is present only during pregnancy and is the anatomical boundary between the mother and fetus. it has a range of functions including transport of nutrients and gases, and hormonal production (1) . the placenta forms a physical, selective barrier between the maternal and fetal circulations, preventing transfer of pathogens. the uterine mucosal lining, the endometrium, is transformed into the decidua during early pregnancy (2) . a range of innate immune mechanisms can respond to pathogens in both the decidua and the placenta (3, 4) . the maternal-fetal interface is a protective barrier against pathogens, but some pathogens can transfer from the mother to fetus by different routes and cause fetal infection (3, 4) . vertical transmission during pregnancy can occur on distinct boundaries between the mother and the fetus: (i) the intervillous space (ivs), where placental villi is in direct contact with the maternal blood, (ii) the implantation site or decidua basalis, where maternal cells are in direct contact with the invading fetal trophoblast, and (iii) the fetal membranes, which are in direct contact with the uterine cavity (5) . defense mechanisms in the cervix, such as the production of mucus and antimicrobial peptides (amp), limit ascending infection from pathogens present in the lower genital tract, that otherwise may access the uterine cavity (6) . however, some pathogens can escape antimicrobial strategies at the cervix and ascend to the uterus, where they can bypass the fetal membranes and lead to the inflammation of the membranes-also known as chorioamnionitis-and infection of the amniotic fluid (7, 8) . pathologic and immune features of chorioamnionitis and intra-amniotic infection are generally associated with bacterial invasion and inflammation [refer to (8, 9) for a comprehensive review on these mechanisms]. here, we focus on infections and innate immune mechanisms at the uterine-placental interfacecases (i) and (ii) (figure 1) . infections at the uterine-placental interface are commonly associated with viruses, parasites and few bacteria ( table 1) . viral pathogens such as human cytomegalovirus (hcmv), zika (zikv), and rubella virus are the most common vertically transmitted pathogens through the decidual-placental interface ( table 1 ) (26, 27) . non-viral pathogens, such as toxoplasma gondii and listeria monocytogenes, can cross the placental barrier via cell-to-cell transmission ( table 1 ) (28, 29) . fetal infection can result in various forms of congenital anomalies in humans ( table 1) . understanding the pathogenic mechanisms used by infectious agents is central to preventing vertical transmission and controlling infection during pregnancy. how the innate immune cells and mechanisms in the placenta and the uterus recognize and respond to protect both the fetus and mother remains controversial due to technical and ethical constraints. however, there are several different models currently used to interrogate the uterine-placental interface in pregnancy. firstly, mice are frequently used as a pregnancy model for infection. although the murine models have provided important insights into the pathogenesis of various infection agents in the context of pregnancy, there are still limitations with this approach. the anatomy of placentation, length of gestation, and use of inbred strains, make extrapolation to humans problematic (30, 31) . secondly, a range of human trophoblast and choriocarcinoma cell lines are used as in vitro models for infection with pathogens. in contrast to the first trimester trophoblast in vivo, these cell lines do not recapitulate normal human trophoblast characteristics such as expression of the human leukocyte antigen (hla) class i and methylation of elf5 (32, 33) . thirdly, human primary placental explants are frequently used. the syncytium dies rapidly in these cultures and it is virtually impossible to standardize the types of villi sampled (30) . therefore, these in vitro experimental factors should be taken into careful consideration when interpreting studies of infection of trophoblast. in this review, we cover the innate immune features of the decidual-placental interface throughout gestation. we identify the gaps in knowledge and highlight the limitations of current studies and experimental models. finally, we discuss novel experimental strategies for understanding how infection affects pregnancy in humans. the trophoblasts of the placenta are the barrier between fetal and maternal tissues. they are derived from the trophectoderm, the outer layer of the blastocyst that forms an inner mononuclear layer with an outer primary syncytium following implantation (34) . the trophoblast in contact with the maternal cells can be: (i) syncytiotrophoblast (sct), a single layer multinucleated, syncytial layer formed by fusion of the underlying villous cytotrophoblast (vct), and (ii) extravillous trophoblast (evt), that invade from the cytotrophoblast shell and anchoring villi into the transformed maternal endometrium, the decidua (2) . the function of evt is to transform the uterine spiral arteries so that maternal blood is delivered to the intervillous space at low pressure. the arteries are surrounded by interstitial evt that destroys the smooth muscle cells of the arterial media, known as "fibrinoid" change (35, 36) . subsequently, endovascular evt (eevt) moves down the spiral arteries from the placentadecidua boundary (35) . these eevt form a plug of cells, limiting surges of arterial blood from damaging the delicate villi. evt invasion transforms the arteries to support optimal regulation of blood flow into the placenta during fetal development (36) . the plugs dissipate between 8 and 10 weeks of gestation when the full hemochorial circulation is established (37) . maternal blood then flows into the ivs, and establishes direct contact with the sct allowing for proper nutrient and gas exchange between the mother and the fetus. hofbauer (hb) cells are fetal macrophages of the human placenta (38) . hb cells can be detected in the placental villous stroma as early as 3 weeks post-conception and are present throughout pregnancy (1, 39) . they are likely to have a variety of functions including control of villous remodeling and differentiation, hormonal secretion, and trophoblast turnover (1, 40) . several lines of evidence have led to the postulation that hb cells may have a role in infection during pregnancy. hb cells with zikv viral particles detected intracellularly have been shown (41, 42). human immunodeficiency virus 1 (hiv-1) has also been detected in hb cells from first trimester infected placenta (43) . whether the hb cells can serve as a reservoir or limit virus replication is still unknown. isolated hb cells from healthy term placenta show elevation of pro-inflammatory cytokines such as il-6, mcp-1, ip-10, and ifn-α upon in vitro infection with zikv (44) . hb cells from the first trimester placenta are also permissive for zikv infection and replication (23) . however, this must be interpreted with caution because in vitro culture of hb cells do not entirely recapitulate the complexity of villous stromal microenvironment, such as presence of hormone and growth factors, all of which will influence the function and activity of hb cells (45) . the sct is the barrier between maternal blood and the placental core as it separates the ivs from the underlying fetal villous stroma. blood-borne pathogens such as viruses and parasites can potentially be transmitted through the sct barrier (figure 1) . how can pathogens cross the sct barrier and the vct to infect the villous stroma? although the sct is an efficient barrier due to its stiff, highly dense actin cytoskeleton network and continuous membrane (46), the syncytium undergoes continuous breaks or gaps and dynamic repair processes (47) . breaks in the syncytium could potentially lead to transmission of pathogens into the underlying vct. our recent work showed that a novel population of maternal macrophages (m3) is associated with the sct in early pregnancy and might be involved in repairing the breaks in the syncytium (10). it is intriguing that m3 macrophages infected with intracellular pathogens could possibly gain access to the underlying vct via the syncytial breaks (figure 2) . only a few viral entry receptors on the sct are described. notably, the sct lacks expression of zikv entry receptors, axl, and tyro3 (48) and the hcmv entry co-receptor integrin α/β (49) . this is further supported by the transcriptomic expression of viral receptors in placental cells (10, 50, 51). expression of surface receptors commonly used by zikv such as axl and hcmv such as nrp2 and pdgfra are lowly expressed by the sct (50) . in addition, there is minimal co-expression of ace2, the receptor gene for human severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and tmprss2, the viral spike protein serine protease gene (50, 52) . in line with this, there is no conclusive and direct evidence of vertical transmission of sars-cov-2 in a placenta from a healthy individual. there are some reports showing sars-cov-2 is predominantly localized at the sct of the second trimester placenta (53, 54) and can lead to severe inflammatory infiltrate in the ivs (55) . however, these findings are presented in a very small number of patients with severe disease or pre-existing pregnancy complications (54, 55) . alternative transplacental mechanisms have been postulated at the syncytial barrier. neonatal fc receptor (fcrn) is expressed on the apical surface of the sct and functions to selectively figure 2 | toll-like receptors and potential inflammatory response at the sct-blood interface. predominant tlrs found in the human placenta from early and term pregnancies. tlr2 and tlr4 are expressed in human placenta sct, vct, and in hb cells. infiltration of infected maternal blood, infected immune cells, or release of pathogenic determinant such lipopolysaccharide (lps), peptidoglycan, or parasite materials such as hemozoin or gpi (glycosylphosphatidylinositol) into the ivs will activate tlr-mediated signaling, leading to the production of a wide range of cytokines and chemokines. severe infection is characterized by massive immune cell infiltration including monocytes and neutrophils from systemic circulation and overproduction of inflammatory cytokines upon tlr activation. this may lead to sct inflammation and damage. sct also secretes antimicrobial peptides as innate immune mechanisms. figure is created by biorender.com. frontiers in immunology | www.frontiersin.org transport maternal igg (56) . fcrn could be exploited by certain viruses to enter the placenta including zikv, hiv-1, and hcmv (19, 57, 58) . transferrin receptor 1 (tfr1) is expressed on the apical end of the sct, and functions as the primary iron transporter into the basal side of the sct to provide sufficient iron stores into fetal circulation (59) . tfr1 has been associated with viral entry into a broad host cell range, including hepatitis c virus (60, 61) suggesting a possible mechanism of viral transport across the sct barrier. some pathogens, although unable to cross the sct barrier, can still adhere to the syncytium and cause further pathology. for instance, plasmodium falciparum infected red blood cells can bind with high affinity to chondroitin sulfate a expressed on the sct, resulting in local inflammation, syncytial breaks, and damage (62) (63) (64) . although the sct is an effective barrier to most pathogens, local inflammation, tissue damage, and fcrn or tfr1-mediated viral entry at the sct can potentially allow pathogen to breach the syncytial barrier, giving opportunity for transmission from maternal blood into placental villi (figure 2 ). during the first trimester of pregnancy, fetal evt invades deeply into the uterus. the decidua basalis, the region located at the implantation site, is populated at this time by a distinctive subset of innate lymphocytes, decidual natural killer cells (dnk), which constitute up to 70% of leukocytes. we have identified three major populations of dnk by single-cell rna-sequencing with unique phenotypes and functions in early pregnancy (10). in addition, there are populations of decidual macrophages (dms) (∼20%), conventional dendritic cells (dcs) and small proportions of t cells (∼10-15%), whereas b cells, plasma cells, mast cells, and granulocytes are virtually absent (10) (figure 1) . the proportion of immune cells will vary throughout pregnancy, with an increase in the proportion of t cells at term (51) . systemic infections will reach all organs including the decidua. whether pathogens can also access the decidua via the cervix is still unclear. chlamydia trachomatis, a common sexuallytransmitted intracellular bacteria, was detected in glandular epithelial cells and unidentified decidual cells in decidual biopsies (11) . this suggests the possibility of infections ascending and spreading from cell-to-cell from the lower genital tract into endometrial glands and vascular endothelium. the decidua basalis is in close contact with fetal cells and the maternal vasculature (figure 1) . first trimester dms and decidual stromal cells are susceptible to zikv infection and replication ex vivo (23) . hence, infection could possibly spread from infected maternal immune and non-immune cells at the decidua, into uninfected vct in the columns of the anchoring villi, and finally into the fetal compartment. however, this is likely to be limited to certain microorganisms which are capable of cell-to-cell spread, have an intracellular host niche, and are able to escape host innate defense mechanisms (table 1) . hcmv, the most common cause of congenital infection, is mostly reported to infect from the decidua (11, 65) . women with primary hcmv infection and first pregnancy are more likely to transmit the virus to their fetus, compared to multiparous women with previous infection and demonstrable antibodies (66) (67) (68) . low affinity maternal antibodies against hcmv correlate with higher viral loads detected in the decidua, whereas patients with intermediate to high neutralizing antibodies have minimal viral replication (65) , suggesting that maternal immunity against hcmv reduces risk of vertical transmission. hcmv protein was also detected in a range of cells within the decidua including endothelial, decidual stromal cells, dcs and macrophages (11, 65) , suggesting that that infected maternal leukocytes could initiate transmission through contact and infection of endothelial cells that line decidual blood vessels. despite the evidence of decidual infection, the mechanism of vertical transmission for hcmv is still in debate. dnks have been proposed to play a protective role against hcmv infection through several mechanisms including modulation of their cytotoxic effector function (69) and the interactions between the killer-cell immunoglobulin receptors (kirs) expressed by dnk and hla molecules expressed in the infected cells (70, 71) . activating kir2ds1 by dnks has been demonstrated to be more cytolytic against hla-c2 hcmv-infected maternal decidual stromal cells (70) . similar cytotoxic response was also observed when peripheral blood nk cells expressing kir2ds1 were exposed to hcmv-infected fibroblasts (71) . hence, this implies that in the decidua, dnks are capable of eliminating harmful infection depending on the combination of kir/hla interactions between dnk and infected cells. dnks are also able to control hiv-1 infection in vitro through production of ifn-γ (72) . the role of dnk in controlling viral infection may protect against potential risk of vertical transmission from the decidua. pattern recognition receptors (prr) are encoded in the germ-line and recognize specific, conserved pathogen-associated molecular patterns (pamps). these include gram-negative bacteria lipopolysaccharide (lps), gram-positive bacteria lipoteichoic acids, lipoprotein, dna, rna, glucans, and peptidoglycans (73, 74). pathogen recognition is not only an essential component of the innate immune response against infection, but also plays an important role in bridging the innate and adaptive systems by toll-like receptors (tlr) activation of antigen presenting cells by up-regulation of major histocompatibility complex (mhc) and co-stimulatory molecules (75) . tlrs, the most studied family of prr, are type i transmembrane proteins with large extracellular domains containing leucine-rich repeats that are expressed at the cell surface or intracellularly (76) . each tlr recognizes distinct pamps, leading to the activation of the transcription factor nf-κb and/or the interferon-regulatory factor (irf) family, and the production of a wide range of cytokines and chemokines, including type i ifns (76, 77) expression of tlrs is dynamic and changes in response to different pathogens and cytokines (74) . tlr2 (which recognizes bacterial proteoglycan) and tlr4 (which recognizes bacterial lps) are the most well-studied, with immunohistochemical evidence of expression in healthy primary sct at term (78) (79) (80) . in contrast, in the first trimester, tlr2 and tlr4 proteins are expressed in vct and evt, but minimally in sct (81, 82) (figure 3) . there is therefore variation in tlr2 and tlr4 expression in the different trophoblast lineages across pregnancy. why and how such dynamic regulation of tlr expression occurs during gestation requires further investigation in a broader range of human placental samples (different donors, gestation stages, genetic background, sampling regions). it is likely that alteration in cytokines profiles in the microenvironment as pregnancy progresses (83) may result in the variation in the expression of tlrs in the placenta. current evidence is only limited to in vitro tlr2/4 stimulation studies using placental explants and primary first trimester trophoblast cells, which drives the expression of figure 3 | toll-like receptors and potential inflammatory response at the decidua. predominant tlrs found in the human placenta from early and term pregnancies. tlr2 and tlr4 are expressed in evt. dm and dnk also express a wide range of tlr families, where stimulation of tlr agonists lead to the production of a variety of cytokines and chemokines. infiltration of infected cells and release of pamps in the decidua, which will activate tlr-mediated signaling. overproduction of inflammatory cytokines at the decidua may lead to local inflammation. figure is created by biorender.com. pro-inflammatory cytokines il-6, il-8, tnf-α, and ifn-γ (78, 80, 81) . tlr2 and tlr4 proteins are expressed in hb cells, confirmed by co-expression of cd68 in healthy term placentas (78) . in early pregnancy, our findings indicate that only tlr4 but not tlr2 transcripts are expressed in steady-state hb cells (10) (figure 4) . enhancement of il-6 and il-8 secretion upon stimulation of isolated first trimester hb cells with tlr4 agonist, lps (84), does suggest a role for tlrs on hb cells in bacterial recognition and placental inflammation during early pregnancy. hb cells are postulated to have a role in viral replication (41, 42), however evidence on the expression and function of viral nucleic acid sensing receptors tlr3, tlr7, tlr8, and tlr9 in hb cells is lacking. our findings show that tlr7, which recognizes viral single-strand rna (ssrna) (85) is expressed in steady-state hb cells (figure 4) (10) . other tlrs have also been shown to be expressed in decidua cells. dms and dnks isolated from first trimester pregnancies show steady state level expression of tlr1-9 transcripts and respond to a broad range of pamps, including heat-killed bacteria, microbial membranes, and nucleic acids (86) . stimulating primary dms with these pamps produces high levels of tnf-α, il-1β, il-6, il-8, il-12, il-10, and il-1ra, whereas dnks secrete il-6, il-8, and ifn-γ (86) . this study suggests that, in addition to the physiological roles of dms and dnks in accommodating the uterus for placentation, dms and dnks may play a role in pathogen recognition and antimicrobial response via activation of tlr signaling (figure 3) . the extent to which subsets of dms or dnks population (10) are critical for tlr-mediated response at the decidua is currently unknown. in malaria endemic populations, single nucleotide polymorphisms (snps) within the tlr4 coding and tlr9 promoter regions are associated with variation in disease severity and parasitemia control (87, 88) . in the case of pregnancy malaria, primiparous infected mothers with common tlr4 and tlr9 polymorphic variants are correlated with severe complications such as low birth weight and maternal anemia (89) . this highlights the importance of studies involving large cohorts of individuals which include genotyping from pregnant mothers living in malaria endemic regions (see section on "challenges and future perspective"). animal models have also been used to study the functional role of tlr signaling, particularly for pathogens that are intracellular at some stage of their life cycle ( table 1) . tlr4 and tlr9 are strongly activated by malaria parasite pamps such as glycosylphosphatidylinositol (gpi), dna, and hemozoin (90, 91) (figure 2) . in a mouse model of placental malaria, tlr4, and myd88 signaling activation resulted in placental expression of pro-inflammatory markers, such as il-6 and tnf-α (92, 93) . these studies also demonstrated that malaria parasite infection and inflammation in the mouse placenta lead to reduced fetus growth rate and disorganization of the vascular space in the placenta (92, 93) . however, tlr-mediated inflammation and pathology in the human placenta upon malaria infection is unknown and remains to be further investigated. studies of congenital toxoplasmosis are also currently limited to animal models. tlr2 and tlr4 are associated with recognition of t. gondii's infection in mice (94) . engagement of the t. gondii ligand by tlr2 and tlr4 at the sct-blood or in the evt-decidua compartments is plausible, although there is still no direct evidence for such host-parasite interaction in humans. tlr11 has a role in controlling t. gondii infection in mice (95, 96) , however in humans tlr11 is a pseudogene and is not expressed (97) . cytosolic prrs play an important role in fighting against viral infection by eliciting host type i interferons (ifn) antiviral response through recognition of single and double stranded rna (ssrna and dsrna) (98, 99) . examples of prrs are the cytosolic retinoic acid-inducible gene-i-like (rig-i) and the melanoma differentiation-associated protein 5 (mda5) receptors, both expressed in the sct and vct of term placenta (100) . in the human placenta, there is limited information on the function of rig-1 and mda5, but they may play a crucial role in recognizing a variety of rna viruses, including zikv and dengue virus (101) . the nucleotide binding oligomerization domain-like receptors (nod-like receptors; nlr) recognizes intracellular pathogen products which have entered into the host cytoplasmic compartment (74) . both nod-1 and nod-2 receptors, which are known to detect intracellular bacterial peptidoglycans (102) , are expressed in the sct in the first trimester and term placentas (103, 104) . the nlr pyrin-containing 1 and 3 proteins (nlrp1 and nlrp3) form the major inflammasome complexes, which contribute to activation of inflammatory caspases and pathogen clearance (105, 106) . activation of nlrp3 and aim2 inflammasomes, together with high expression of il-1r, il-1β, and caspase-1 was recently shown in the placental tissue of mothers infected with p. falciparum with significant pathology (107) . in a murine model of intra-amniotic inflammation induced by bacterial lps, tissue sections from the decidua basalis region expressed high levels of nlrp3, but negligible caspase-1 activation suggesting a possible non-canonical activation of the nlrp3 inflammasome (108) . our analysis shows that decidual dm1 expresses high levels of nlrp3 transcript at steady state compared to other cell types (10) (figure 4) , thus dm1 may play a role in nlrp3-mediated pathogen recognition during early pregnancy. amp secreted by epithelial and immune cells are small peptides that bind and destroy most groups of pathogens-bacteria, yeasts, fungi, and viruses (109) . in addition to direct killing of pathogens, amps can rapidly modulate innate host immune responses by recruiting myeloid cells and lymphocytes to the site of infection and mediating activation of tlr (110, 111) . the human placenta expresses high levels of β-defensins, a family of broad spectrum antimicrobial peptides which participate in direct bactericidal and anti-viral activity (112) . specific subtypes of β-defensins (hbd-1, 2, and 3) are expressed in scts (112) , suggesting these amps can target potentially bacterial or viral infection from the maternal blood. recognition of pamps by prrs during infection leads to production of pro-inflammatory cytokines that can aid in clearing the pathogen (74) . studies on the direct role of proinflammatory cytokines on the placenta in the case of infection is limited. inflammatory mediators can directly influence infection outcome and fetal development, but they can also cause damage to the placenta if produced in excess (113). amongst the proinflammatory cytokines associated with uterine-placental infection during pregnancy, the antiviral ifn are the most well-characterized. ifns are secreted by a variety of cell types as the first line of defense against viral infection (114) . type i ifns, including ifn-α and ifn-β, are potent antiviral cytokines. ifn-α and ifn-β bind to the ifnar1/2 receptor and lead to expression of ifn stimulated genes (isgs), which control virus infection through a variety of mechanisms (114) . loss of ifnar in the placenta leads to vertical transmission and fetal mortality in murine herpesvirus-68 (mhv68) infected mice (115) . in the mouse model of zikv infection, type i ifn-mediated signaling is essential for the control of viral replication in the placenta, but can also lead to significant placental pathology and fetal mortality (116, 117) . the mechanism of type i ifn-mediated placental pathology has been recently elucidated. ifn-induced transmembrane (ifitm) protein, which normally blocks viral entry into host cells, impairs syncytin-mediated fusion of vct to form sct, leading to aberrant placental development (118) . type ii ifn, ifnγ, predominantly produced by nk and cd4+ t cells is crucial in controlling parasitic infection, such as t. gondii in mice (94, 119) . however, elevated levels of ifnγ in response to t. gondii infection can lead to pathological effects during pregnancy including fetal demise (119, 120) . severe placental pathology and fetal death have also been associated with elevation of ifnγ during pregnancy in a murine model of malaria (121). hence, proper regulation of type i and ii ifn-mediated signaling at the uterine-placental interface is crucial in limiting pathogen replication, whilst preserving a balanced environment for normal placental development (122) . type iii ifn, ifnλ, are constitutively secreted by the human sct, which presumably confers antiviral effects against zikv infection (123) (124) (125) . tryptophan metabolism by ido indoleamine 2,3-dioxygenase (ido) is a host intracellular enzyme which metabolizes the amino acid tryptophan (126) . ido has been associated with maternal immunoregulation during pregnancy (127) . it also plays a key role in the control of bacterial and viral replication, through limiting the bioavailability of tryptophan (128) . ido also inhibits the replication of several parasitic pathogens including t. gondii in human fibroblasts (129) and leishmania spp in human macrophages (130) . mouse infection with l. monocytogenes showed that ido is elevated in an ifn-γ-dependent manner in stromal cells of the metrial gland and decidua basalis; a crucial process to resolve bacterial infection in the mouse placenta (131) . our findings also show ido1 expression is enriched in epithelial glandular and dc1 cell type in the first trimester decidua (10) (figure 4) . the presence of ido in decidua suggests that the enzyme might have a central role in limiting parasitic, viral, and bacterial replication, thus preventing their spread to the fetus. research on how the human placenta safeguards itself against infections is challenging due to obvious logistical and ethical issues in obtaining tissue from early in gestation (box 1). although animal experimental models have provided important insights relating to the immune responses to pathogenic infection, major differences between human and animal placentas must be considered (30, 31) . likewise, differences between strains of pathogens adapted for mice compared with human clinical isolates should be taken into account as this may lead to variation in pathogenesis and cellular response. one such example is the use of mouse cmv, which is unable to cross the mouse placental barrier, unlike the hmvc counterpart which can be transmitted transplacentally in humans (132) . therefore, all data obtained from studies of infection in pregnant animals needs careful interpretation and consideration prior to translation to clinical infection in humans. inherent properties of trophoblast cell lines, primary cultures or explants vary between donors, and are likely to be confounded by the area of the placenta that is sampled and as well as stage of gestation (133) . for instance, villous placental explants will vary depending on the types of villi sampled and the presence of box 1 | perspective of vertical transmission and innate immune function during pregnancy and infection. a variety of maternal infections can lead to vertical transmission ( table 1) . the exact mechanisms these pathogens use to escape host defense and cross the placental barrier into the fetal compartment are not entirely known. experimental models that recapitulate infection of the human placenta and thus vertical transmission are challenging to set up. more data and representative experimental models are needed to answer these questions: (i) how do different pathogens escape or modulate the maternal-fetal host innate immune barrier (ii) why do some pathogens lead to congenital infection but not others? studying infected human placentas will be essential in understanding this but access to these samples is difficult especially in low and middle-income countries (lmic) where maternal infection is particularly prevalent (who, maternal mortality index 2019). despite evidence of expression in primary placental tissue, functional studies on important innate immune features such as tlrs, amps, rig-i, mda5, nlrs, and ido during infection and pregnancy are lacking. understanding how different cell types at the uterine-placental interface (hb cells, dnks, and dms) respond to pathogen challenge is essential, but remains under-researched. a critical obstacle is to also extrapolate the protective and pathological mechanisms of cytokines from mouse to human infection. therefore, systematic comparison of the innate immune effector mechanisms across gestation, in the placenta and decidua from natural human infection vs. healthy pregnancy, will provide a more accurate representation in clinical settings. attached decidual tissue (133) . caution is therefore needed when interpreting data using these experimental models. to overcome such limitations, population-based cohort studies of women with infection during pregnancy with extensive tissue sampling should be performed. these need to include and focus on lmic where infection is still a major cause of maternal and fetal mortality and morbidity. cohort studies and epidemiological surveillance on maternal infections can offer significant insights into disease pathogenesis and accelerate clinical interventions (134) . collaborations between clinicians and researchers for population-based cohort collection and sample processing will be instrumental to achieving this goal. biological samples such as blood or placenta collected from controls and infected pregnant individuals could be stored and cryopreserved retrospectively. to capture the overall heterogeneity of infected and non-infected placenta samples, sampling, and biobanking criteria of different regions of placenta should be considered (135) . protocols are now available to use frozen tissue processed for single-cell/nuclei and spatial genomics (136, 137) . hence, application of single-cell "omics" on infected vs. healthy human placental and decidual samples will enable us to evaluate cellular heterogeneity in response to infection. the capacity to detect transcripts specific to host or pathogen mrna from the same tissue using in situ nucleic acid hybridization methods will provide direct quantification of infection burden and identification of potential target host cells within the same tissue (138) . recent advances in spatial transcriptomics methods have also allowed gene expression signatures to be quantified and resolved from individual tissue sections (139) . combination of these emerging technologies with new methods to integrate single-cell and spatial data computationally (140) will provide an unbiased approach to characterize and profile the transcriptome of individual cells in situ from the placenta and decidua in response to infections. we anticipate that high-throughput datasets generated from cohort sampling studies will unravel novel cell states and tissue spatial localization associated with placental infections and inflammation. this will also allow us to better characterize not only the innate immune response or makers of infection, but also other adaptive immune states in the human placenta (box 1). the use of in vitro models will also further define host responses to infection. the recent generation of human trophoblast stem cells (htscs) (141) and three-dimensional (3d) trophoblast organoids (142, 143) offer a great opportunity to study infections in early pregnancy where the access to first trimester placental samples is a concern. more importantly, the htscs and trophoblast organoids fulfill the criteria characteristic of human first trimester trophoblast in vivo (32) . both htscs and trophoblast organoids can differentiate in vitro into sct and evt with appropriate media (142, 143) allowing infection experiments on both the major trophoblast subpopulations present at the two major sites of contact between maternal and fetal cells. sequencing of both host and pathogen transcriptomes from infected trophoblast at single-cell resolution will also advance our understanding on host-pathogen interactions in placentas (144, 145) . further refinement of the trophoblast organoid and htscs culture system is needed to address key biological questions unanswered by current models. these include studying the effect of infection on cellular crosstalk between trophoblast and other primary placental cells such as hb cells, or decidual cells in culture, such as dnk or decidual stromal cells. adaptation of crispr/cas9 genome editing technology for the trophoblast organoids or htscs will offer novel insights into essential host genes required for vertical transmission and placental defense mechanisms in humans. major maternal and fetal complications as a result of infection are still a concern, especially in lmic with highest prevalence reported in countries of sub-saharan africa (who, maternal mortality index 2019). profound limitations on current study models and ethical regulations on studying human placenta have significantly delayed the development of therapies and vaccines for maternal-fetal infection. how vertical transmission occurs and how the uterine-placental innate immune system reacts to infection remain as major unresolved questions. revolutionary advances in single-cell genomics, imaging, computational, and stem cell biology methods are currently underway to study the molecular and cellular mechanisms of human diseases. therefore, it is now an exciting time to apply these transformative technologies to comprehensively address fundamental questions on host-pathogen interaction at the human uterine-placental interface. rh, an, and rv-t wrote and edited the manuscript. all authors contributed to the article and approved the submitted version. rh and rv-t were supported by wellcome sanger core funding (wt206194). an was supported by a sanger international fellowship, a nurture fellowship (d43tw010132) and a group leader award supported through the deltas africa initiative (107743/z/15/z). the deltas africa initiative is an independent funding scheme of the african academy of science (aas), alliance for accelerating excellence in science in africa (aesa), supported by the new partnership for africa's development planning and coordinating agency (nepad agency) with funding from the wellcome trust (grant no. 107743), and the uk government. cellular basis of interaction between trophoblast and uterus at implantation immune mechanisms at the maternal-fetal interface: perspectives and challenges immune responses at the maternal-fetal interface zika virus -reigniting the torch antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications evidence that intra-amniotic infections are often the result of an ascending invasion -a molecular microbiological study acute chorioamnionitis and funisitis: definition, pathologic features, and clinical significance 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broad tissue and cell tropism during the first trimester of pregnancy asian zika virus strains target cd14+ blood monocytes and induce m2-skewed immunosuppression during pregnancy cd14+cd16+ monocytes are the main target of zika virus infection in peripheral blood mononuclear cells in a paediatric study in nicaragua pereira l. congenital viral infection: traversing the uterine-placental interface understanding how listeria monocytogenes targets and crosses host barriers transmission of toxoplasma gondii from infected dendritic cells to natural killer cells development of the human placenta animal models of human placentation -a review what is trophoblast? a combination of criteria define human first-trimester trophoblast elf5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta the human placenta uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy trophoblast invasion maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: the boyd collection revisited the cytology of hofbauer cells a three-dimensional study of the normal human placental villous core zika virus infection at different pregnancy stages: anatomopathological findings, target cells and viral persistence in placental tissues zika virus rna replication and persistence in brain and placental tissue hiv-1 in trophoblastic and villous hofbauer cells, and haematological precursors in eight-week fetuses zika virus infects human placental macrophages placental syncytium forms a biophysical barrier against pathogen invasion apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast zika virus targets different primary human placental cells, suggesting two routes for vertical transmission developmental regulation of human cytomegalovirus receptors in cytotrophoblasts correlates with distinct replication sites in the placenta does the human placenta express the canonical cell entry mediators for sars-cov-2? elife single cell transcriptional signatures of the human placenta in term and preterm parturition sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes visualization of sars-cov-2 virus invading the human placenta using electron microscopy sars-cov-2 infection of the placenta probable congenital sars-cov-2 infection in a neonate born to a woman with active sars-cov-2 infection an igg-transporting fc receptor expressed in the syncytiotrophoblast of human placenta maternal immunity and antibodies to dengue virus promote infection and zika virus-induced microcephaly in fetuses the neonatal fc receptor (fcrn) enhances human immunodeficiency virus type 1 (hiv-1) transcytosis across epithelial cells identification and localization of divalent metal transporter-1 (dmt-1) in term human placenta identification of transferrin receptor 1 as a hepatitis c virus entry factor viral infection and iron metabolism malaria in pregnancy: pathogenesis and immunity adherence of plasmodium falciparum to chondroitin sulfate a in the human placenta syncytiotrophoblast degradation and the pathophysiology of the malaria-infected placenta human cytomegalovirus transmission from the uterus to the placenta correlates with the presence of pathogenic bacteria and maternal immunity interval between births and risk of congenital cytomegalovirus infection maternal immunity and prevention of congenital cytomegalovirus infection properties and mechanisms of immunoglobulins for congenital cytomegalovirus disease human cytomegalovirus infection elicits new decidual natural killer cell effector functions expression of kir2ds1 by decidual natural killer cells increases their ability to control placental hcmv infection modulation of human leukocyte antigen-c by human cytomegalovirus stimulates kir2ds1 recognition by natural killer cells nk cells control hiv-1 infection of macrophages through soluble factors and cellular contacts in the human decidua pathogen recognition and innate immunity dendritic cells and the control of immunity toll-like receptor signaling pathways irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors cell type-specific expression and function of toll-like receptors 2 and 4 in human placenta: implications in fetal infection expression and subcellular localization of tlr-4 in term and first trimester human placenta expression and regulation of the pattern recognition receptors toll-like receptor-2 and toll-like receptor-4 in the human placenta differential expression of toll-like receptors in the human placenta across early gestation toll-like receptors and pregnancy: trophoblast as modulators of the immune response toll-like receptor-mediated responses by placental hofbauer cells (hbcs): a potential pro-inflammatory role for fetal m2 macrophages tlr7 is involved in sequence-specific sensing of singlestranded rnas in human macrophages human decidual macrophages and nk cells differentially express toll-like receptors and display distinct cytokine profiles upon tlr stimulation variants in the toll-like receptor signaling pathway and clinical outcomes of malaria toll-like receptor (tlr) polymorphisms in african children: common tlr-4 variants predispose to severe malaria common polymorphisms of toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy tolllike receptor 9 mediates innate immune activation by the malaria pigment hemozoin induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols ofplasmodium falciparum myd88 signaling is directly involved in the development of murine placental malaria tlr4-mediated placental pathology and pregnancy outcome in experimental malaria innate immunity to toxoplasma gondii infection tlr11 activation of dendritic cells by a protozoan profilin-like protein toxoplasma profilin is essential for host cell invasion and tlr11-dependent induction of an interleukin-12 response a toll-like receptor that prevents infection by uropathogenic bacteria viral rna detection by rig-i-like receptors length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors rig-i recognizes the 5 ′ region of dengue and zika virus genomes nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan expression and function of nod-like receptors by human term gestation-associated tissues nod protein expression and function in first trimester trophoblast cells the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proil-beta sensing and reacting to microbes through the inflammasomes inflammasome activation and il-1 signaling during placental malaria induce poor pregnancy outcomes intra-amniotic inflammation induces preterm birth by activating the nlrp3 inflammasome † the role of cationic antimicrobial peptides in innate host defences betadefensins: linking innate and adaptive immunity through dendritic and t cell ccr6 plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide yockey lj, iwasaki a. interferons and proinflammatory cytokines in pregnancy and fetal development interferon-inducible antiviral effectors cutting edge: fetal/placental type i ifn can affect maternal survival and fetal viral load during viral infection type i interferons instigate fetal demise after zika virus infection zika virus infection during pregnancy in mice causes placental damage and fetal demise ifitm proteins inhibit placental syncytiotrophoblast formation and promote fetal demise interferon-gamma: the major mediator of resistance against toxoplasma gondii toxoplasma gondii-induced foetal resorption in mice involves interferongamma-induced apoptosis and spiral artery dilation at the maternofoetal interface contributions of maternal and fetal antiviral immunity in congenital disease organotypic models of type iii interferon-mediated protection from zika virus infections at the maternal-fetal interface type iii interferons produced by human placental trophoblasts confer protection against zika virus infection gestational stage and ifn-λ signaling regulate zikv infection in utero indoleamine 2,3 dioxygenase and metabolic control of immune responses prevention of allogeneic fetal rejection by tryptophan catabolism new insights into ido biology in bacterial and viral infections interferon gamma blocks the growth of toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan role of tryptophan degradation in respiratory burst-independent antimicrobial activity of gamma interferon-stimulated human macrophages indoleamine 2,3-dioxygenase is regulated by ifn-gamma in the mouse placenta during listeria monocytogenes infection immunobiology of congenital cytomegalovirus infection of the central nervous system-the murine cytomegalovirus model high incidence of contaminating maternal cell overgrowth in human placental mesenchymal stem/stromal cell cultures: a systematic review the burden of vaccine-preventable diseases in pregnancy in low-resource settings optimising sample collection for placental research a single-cell and single-nucleus rna-seq toolbox for fresh and frozen human tumors fin-seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system rnascope: a novel in situ rna analysis platform for formalin-fixed, paraffin-embedded tissues visualization and analysis of gene expression in tissue sections by spatial transcriptomics spatial mapping of cell types by integration of transcriptomics data derivation of human trophoblast stem cells trophoblast organoids as a model for maternalfetal interactions during human placentation self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta scdual-seq: mapping the gene regulatory program of salmonella infection by host and pathogen single-cell rna-sequencing we would like to thank ashely moffett for the useful discussions and critical review of the manuscript. we are also very grateful to sarah aldridge, loren gibson, damiana alvarez, carlos talavera-lopez, and anna arutyunyan for their insightful comments and corrections. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 hoo, nakimuli and vento-tormo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-290548-0wezrr1b authors: watanabe, tokiko; kawaoka, yoshihiro title: villains or heroes? the raison d'être of viruses date: 2020-02-19 journal: clin transl immunology doi: 10.1002/cti2.1114 sha: doc_id: 290548 cord_uid: 0wezrr1b the relationship between humans and viruses has a long history. since the first identification of viruses in the 19th century, we have considered them to be ‘pathogens’ and have studied their mechanisms of replication and pathogenicity to combat the diseases that they cause. however, the relationships between hosts and viruses are various and virus infections do not necessarily cause diseases in their hosts. rather, recent studies have shown that viral infections sometimes have beneficial effects on the biological functions and/or evolution of hosts. here, we provide some insight into the positive side of viruses. viruses, which consist of nucleic acid encased in a protein shell, are parasites of host organisms. the term 'virus' comes from the latin word 'venom', which means poison, because a virus is generally considered to be a causative agent like a poison that causes infectious diseases. these tiny, living entities have considerable import, because they can cause substantial damage to humans and non-human animals and other living organisms. the relationship between humankind and viruses has a long history. for example, the earliest evidence of smallpox was found in 3000-year-old egyptian mummies, who had smallpox-like eruptions on their skins. 1 the overall mortality rate of smallpox was around 30%, 2 making it one of the most feared infectious diseases. in 1918-1919, during world war i, influenza a virus caused the spanish flu pandemic, resulting in infection of approximately 500 million people and more than 20-40 million death worldwide. 3 since the initial isolation of viruses in the 19th century, scientists have identified and characterised a wide variety of viruses, and the field of virology has progressed remarkably since then, enabling us to combat the frequently deadly effects of these viruses. one of the greatest achievements is the complete eradication of smallpox. although smallpox was once rampant in the world, vaccination of the entire population has eradicated this disease. 1 similarly, the poliovirus vaccine has significantly reduced the incidence of poliomyelitis. 4 despite the progress of virology, we still have many unconquered viral diseases and we are confronted with the problem of emerging infectious diseases, which are caused by newly identified species or strains. for example, ebola virus disease and acquired immunodeficiency syndrome emerged in 1976 and 1981, respectively, 5-9 and more recently, severe acute respiratory syndrome (sars), highly pathogenic avian influenza viruses and middle east respiratory syndrome (mers) have appeared in human society. [10] [11] [12] [13] [14] [15] therefore, it is important to continue studying the mechanisms of viral replication and pathogenicity. yet, these negative aspects of viruses do not tell the whole story since the relationships between hosts and viruses are multitudinous, and virus infections do not necessarily lead to disease symptoms in hosts. rather, recent studies suggest that there are viruses that are beneficial to the biological functions and/or evolution of their hosts. recently, we established a research consortium, designated as 'neo-virology', which is supported by grants-in-aid for scientific research on innovative areas from the ministry of education, culture, science, sports, and technology (mext) of japan. in this consortium, we define a virus as a component of the global ecosystem. our aim was to elucidate the roles of viruses in host organisms and the global ecosystem, in contrast to traditional virology research, which tends to focus on pathogenic viruses that cause diseases in their hosts. this research project is expected to develop into an important scientific field that examines the interactions between the global ecosystem and viruses. in this brief review, we give some insights into the positive side of viruses. in traditional virology, most viruses found in humans are considered to be pathogenic to their hosts; however, recent studies have shown that there are some viruses that have symbiotic relationships with their hosts and do not cause disease. infection with one virus may protect the host from a superinfection with another pathogen. barton et al. 16 demonstrated that latent infection with the herpesviruses murine gammaherpesvirus 68 or murine cytomegalovirus, which are genetically related to the human pathogens epstein-barr virus and human cytomegalovirus, respectively, led to crossprotection in mice. infection with these viruses induced prolonged production of the antiviral cytokine interferon-gamma and systemic activation of macrophages that protected the mice from subsequent bacterial infections with either listeria monocytogenes or yersinia pestis. 16 moreover, it has been reported that superinfection with hepatitis a virus suppressed hepatitis c virus replication in patients with chronic hepatitis c in at least two cases, 17 and infection with human cytomegalovirus (hcmv) suppressed superinfection with hiv-1 in vitro as a result of the downregulation of the expression of ccr5, a co-receptor for hiv-1, induced by the hcmv infection. 18 some viruses also have beneficial effects with respect to non-infectious diseases. epidemiologic studies suggest that virus infections in childhood might confer protection against some cancers later in life. for example, the risk of chronic lymphoid leukaemia in subjects who had measles in childhood is relatively low, 19 and mumps infection in childhood might protect against the development of ovarian cancer in adults. 20 however, infection with oncoviruses is known to increase the risk of development of some cancers (e.g. cervical cancer and liver cancer induced by the human papillomavirus and hepatitis b virus/ hepatitis c virus infection, respectively). 21 such information is important when considering strategies for cancer immunotherapy and/or vaccination campaigns. in addition, the infection of non-obese diabetic mice with lymphocytic choriomeningitis virus prevented the infected mice from developing autoimmune disease and subsequent type i diabetes (insulin-dependent diabetes mellitus). 22, 23 chronic viral infection of mice with murine cytomegalovirus (cmv) increased epithelial turnover and wound repair via antiviral cytokine type i interferons (ifns), 24 but cmv infection can promote cancer malignancy; this phenomenon is known as 'oncomodulation'. 25, 26 recent metagenomic studies have revealed that virus infection sometimes confers benefits including the regulation of microbiota in the gut. bacteriophages are abundant in the gut and are thought to modulate the gut microbiota by infecting specific bacterial populations. accordingly, potential therapeutic applications of bacteriophages in humans (e.g. control of antibiotic-resistant bacteria, stabilisation of healthy gut microbes) have been considered. 27, 28 therefore, the elucidation of the symbiotic effects of viruses on the physiological functions and immune responses of their hosts, as well as clarification of the functional mechanisms involved, will lead to an understanding of the essential roles of viruses in regulating the biological processes of their hosts. retroviruses are found in almost all mammals and other vertebrates, and approximately 8% of the human genome is composed of retroviruses in the form of endogenised proviruses. 29, 30 given that only about 1% of genomic dna is made up of protein-coding genes, the abundance of retrovirus sequences in the human genome is remarkable. retrovirus sequences are conserved in humans and other primates, and therefore, the endogenisation of retroviruses is thought to have occurred millions of years ago. 31 some endogenous retroviruses have been shown to play beneficial roles in their hosts, including host evolution. for example, envelope genes from endogenous retroviruses contribute to the formation of the placenta during the fusion of syncytiotrophoblast cells in mammals. 32, 33 in addition, endogenous retroviral elements are known to protect host cells from infection with exogenous retroviruses in some mammals. [33] [34] [35] in addition to retroviruses, recent studies have shown that non-retroviral viruses have also endogenised in many mammalian species. [36] [37] [38] for example, tomonaga's group, which is one of the research groups in our neo-virology research consortium, discovered that bornaviruses, a genus of non-segmented, negative-strand rna virus, have been endogenised in the genomes of many mammals, including humans. 36 since bornaviruses do not encode reverse transcriptase and integrase genes, integration of bornavirus segments is believed to be mediated by long interspersed nuclear element-1 (line-1), which is a retrotransposon widely distributed in mammalian genomes. 39 tomonaga's group also showed that an endogenous bornavirus-like element in the ground squirrel genome blocks infection and replication of extant endogenous bornavirus. 40 together, these findings indicate a potential role for endogenous non-retroviral elements in antiviral defence. this group also performed an evolutionary phylogenetic analysis to elucidate the functions of endogenous bornavirus in mammalian genomes. 41 thus, it has become apparent that a large number of endogenous viral elements have accumulated in host genomes, more than previously expected. therefore, it is important that we understand the significance of endogenous viral elements to the biological function and evolution of hosts. the first virus to be identified in humans was the yellow fever virus in 1901 after the discovery of tobacco mosaic virus in 1892 in plants and footand-mouth disease virus in 1898 in animals. 42 since then, new virus species that infect humans have been identified almost every year. woolhouse et al. 43 reviewed human viruses that had been described in the literature and recognised by the international committee on taxonomy of viruses (ictv), and drew a discovery curve for human viruses by plotting the cumulative number of species reported to infect humans; they showed that new species of human viruses have been discovered at a rate of three or four per year. currently, there are approximately 263 viruses from 25 viral families that are known to be able to infect humans according to the latest ictv report. 44 in the last a few decades, emerging infectious diseases caused by newly identified viruses, such as ebola virus, 5-8 sars and mers coronaviruses, [10] [11] [12] human immunodeficiency virus (hiv), 9 nipah virus and hendra virus, [45] [46] [47] [48] have appeared in human society. most emerging infectious diseases are zoonotic, caused by viruses that originate in wild animals, such as primates, rodents and bats [49] [50] [51] ; in particular, bats have drawn attention because a recent comprehensive analysis of mammalian hostvirus relationships indicated that bats have a significantly higher proportion of zoonotic viruses than all other mammalian orders. 52 this analysis was part of a study supported by the united states agency for international development (usaid) emerging pandemic threats predict programme (https://ohi.vetmed.ucdavis.edu/programs-projects/ predict-project), which was initiated in 2009, working with partners in over 30 countries on global surveillance of viruses to identify and monitor zoonotic pathogens. to date, the predict programme has found over 1100 viruses in animals and humans, including a new ebola virus and mersand sars-like coronaviruses. in 2018, the global virome project (http://www.globalviromeproject. org) was launched, which aims to conduct viral surveillance on an even larger scale than the predict programme. those involved in this project estimate that~1.67 million yet-to-be-discovered viral species from key zoonotic viral families exist in mammal and avian hosts, and expect that 631 000-827 000 of these unknown viruses have zoonotic potential. 44 in addition to viral surveillance in mammals and avian hosts, zhang and holmes's group recently conducted a screen for rna viruses in diverse host species (more than 186 species other than mammalian and avian hosts) and identified about 200 vertebrate-associated rna viruses in fishes, reptiles and amphibians. 53 they also found that vertebrate-specific viral families or genera known to infect mammals and birds, including influenza viruses, filoviruses and hantaviruses, are also present in amphibians, reptiles and fish. 53 in addition to the identification of new virus species in diverse hosts, new virus lifestyles have also been found in nature. suzuki's group, which is one of the groups in our neo-virology research consortium, recently reported a new virus lifestyle exhibited by two rna viruses: a double-stranded (ds) rna virus (yado-nushi virus 1, ynv1) and a positive-sense, single-stranded [(+)ss] rna virus (yado-kari virus 1, ykv1) in a phytopathogenic fungus, rosellinia necatrix. 54 they found that the (+)ssrna virus (ykv1), which does not have its own capsid protein, hijacks the capsid protein of the dsrna virus (ynv1) to replicate. 55 our world is made up of vastly different physical environments and the various organisms that have adapted to live in those environments. the complex interactions between the living and nonliving components of these environments are the basis of the global ecosystem. various schemes have been proposed to classify living organisms: the one most often used currently defines all living organisms as archaea, bacteria or eukaryotes. therefore, viruses are not considered living components of the global ecosystem. given that approximately 10 31 virus particles exist on earth 56,57 and all of them are parasitic in living organisms, it is not hard to imagine how virus infection might affect the physiological functions of both hosts and the ecosystem. although 'traditional' virology research tends to focus on pathogenic viruses that cause diseases in their hosts, the recent progress in next-generation sequencing (ngs) technologies and data analyses has enabled us to discover a wide variety of new viruses, some of which do not cause diseases in their hosts. some obstacles to comprehensive virome analyses remain, such as viral dark matter, which are sequences that originate during virus metagenomics but cannot be aligned to any reference sequences of viruses. 58 nonetheless, recent viral metagenomic studies using ngs technologies and bioinformatic analyses have identified a large number of viruses in environmental samples, including plants and oceans. 59, 60 characterisation of these newly identified viruses may provide new insight into the significance of viruses and virus-mediated processes within global ecosystems. the history of smallpox and its spread around the world on the origin of smallpox: correlating variola phylogenics with historical smallpox records integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 spanish influenza virus from emergence to eradication: the epidemiology of poliomyelitis deconstructed isolation and partial characterisation of a new virus causing acute haemorrhagic fever in zaire viral haemorrhagic fever in southern sudan and northern zaire: preliminary studies on the aetiological agent isolation of marburg-like virus from a case of haemorrhagic fever in zaire commission r of an i. ebola haemorrhagic fever in zaire hiv-1 at 25 isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a pandemic warning? clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness herpesvirus latency confers symbiotic protection from bacterial infection hepatitis a virus infection suppresses hepatitis c virus replication and may lead to clearance of hcv human cytomegalovirus modulation of ccr5 expression on myeloid cells affects susceptibility to human immunodeficiency virus type 1 infection childhood 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function virus evolution syncytin is a captive retroviral envelope protein involved in human placental morphogenesis paleovirology and virally derived immunity studies of endogenous retroviruses reveal a continuing evolutionary saga late viral interference induced by transdominant gag of an endogenous retrovirus endogenous nonretroviral rna virus elements in mammalian genomes unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes endogenous viral elements in animal genomes comprehensive analysis of endogenous bornavirus-like elements in eukaryote genomes inhibition of borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome paleovirology of bornaviruses: what can be learned from molecular fossils of bornaviruses virology: from contagium fluidum to virome human viruses: discovery and emeraence the global virome project a morbillivirus that caused fatal disease in horses and humans the authors the natural history of hendra and nipah viruses outbreak of nipah-virus infection among abattoir workers in singapore fatal encephalitis due to nipah virus among pig-farmers in malaysia host range and emerging and reemerging pathogens global trends in emerging infectious diseases risk factors for human disease emergence host and viral traits predict zoonotic spillover from mammals the evolutionary history of vertebrate rna viruses chapter sevenviruses of the white root rot fungus, rosellinia necatrix a capsidless ssrna virus hosted by an unrelated dsrna virus the gene weavers marine viruses -major players in the global ecosystem origins and challenges of viral dark matter metagenomics of plant and fungal viruses reveals an abundance of persistent lifestyles viral macro-and micro-diversity from pole to pole we thank susan watson for editing the manuscript. this is an open access article under the terms of the creative commons attribution license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. key: cord-319044-5otz2w9v authors: walsh, michael g.; sawleshwarkar, shailendra; hossain, shah; mor, siobhan m. title: whence the next pandemic? the intersecting global geography of the animal-human interface, poor health systems and air transit centrality reveals conduits for high-impact spillover date: 2020-10-08 journal: one health doi: 10.1016/j.onehlt.2020.100177 sha: doc_id: 319044 cord_uid: 5otz2w9v the health and economic impacts of infectious disease pandemics are catastrophic as most recently manifested by coronavirus disease 2019 (covid-19). the emerging infections that lead to substantive epidemics or pandemics are typically zoonoses that cross species boundaries at vulnerable points of animal-human interface. the sharing of space between wildlife and humans, and their domesticated animals, has dramatically increased in recent decades and is a key driver of pathogen spillover. increasing animal-human interface has also occurred in concert with both increasing globalisation and failing health systems, resulting in a trifecta with dire implications for human and animal health. nevertheless, to date we lack a geographical description of this trifecta that can be applied strategically to pandemic prevention. this investigation provides the first geographical quantification of the intersection of animal-human interfaces, poor human health system performance and global connectivity via the network of air travel. in so doing, this work provides a systematic, data-driven approach to classifying spillover hazard based on the distribution of animal-human interfaces while simultaneously identifying globally connected cities that are adjacent to these interfaces and which may facilitate global pathogen dissemination. we present this geography of high-impact spillover as a tool for developing targeted surveillance systems and improved health infrastructure in vulnerable areas that may present conduits for future pandemics. the global spread of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) in 2020 has shown how rapidly emerging infectious diseases can devastate human health and national economies. such infections typically have zoonotic origins, with other notable examples being sars-cov in 2002 [1] , influenza a h1n1 virus in 2009 [2] , and the west african ebola virus disease epidemic of 2013-2016 [3] . emerging infectious diseases are increasing in incidence and expanding in geographic range due in large part to direct and diffuse anthropogenic pressure across ecosystems [4] . this is particularly true in areas with high wildlife biodiversity that are experiencing land-use changes such as deforestation [4, 5, 6, 7] , although the process of disease emergence is complex and cannot be attributed to any one driver. morse et al. have developed a useful framework for conceptualising a staged process of pathogen emergence in humans [8] . in the first stage, potential pathogens circulate exclusively in reservoir hosts. while this stage precedes spillover, the stage is still highly influenced by human activities that place stress on animal populations, such as habitat destruction, population displacement, and nutritional insecurity, thereby altering pathogen circulation in affected non-human host populations [9] . various researchers have hypothesized that certain animal taxa, such as bats [8, 10] , harbor a significantly higher proportion of zoonotic viruses. however, differential impact of specific taxa on spillover has been recently challenged by research showing that overall species richness within order is the primary driver of pathogen richness among mammalian and bird reservoir hosts, rather than shared biological study did show differences in viral richness between mammals and birds so hosts may still cluster within some taxonomic levels [11] . in the framework's second stage, activities that increase animal-human contact (such as forest management practices or raising domestic animals) create opportunities for spillover from reservoir hosts into human populations. the risk of spillover appears greatest both from generalist species that are highly abundant and adapted to human-dominated landscapes, and -conversely -from those specialist species that are threatened specifically due to habitat loss and human exploitation [11] ; both scenarios confirm the central influence of humans as a driving force behind spillover. hassell et al. argued that wildlife-livestock-human interfaces emerging under urbanisation represent particularly critical points for cross-species transmission, noting an urgent need for better characterisation of periurban wildlife interfaces using transdisciplinary approaches [12] . despite the critical influence of anthropogenic pressure, spillover is a complex process requiring that pathogens overcome many landscape and immunological barriers [13] , and even when successful, not all spillovers lead to sustained transmission. most often this is due to a lack of efficient transmission in the new host populations. occasionally, spillover into naïve human or domesticated animal populations does result in efficient transmission. the framework's final stage is marked by sustained onward transmission and widespread regional or global dissemination. at this stage, the capacity of local health systems to rapidly detect cases of a novel disease and control ongoing chains of transmission is key to preventing broader dissemination from the original focus [8] . in this work we distinguish wildlife-origin zoonoses with the potential to transmit efficiently between humans as impactful spillovers, and we further designate impactful spillovers with the potential to disseminate rapidly to regions beyond their origin focus as high-impact spillovers. highimpact spillovers are therefore a function of the capacity of local health systems and the proximity of the initial spillover to conduits of broader global dissemination, particularly transportation hubs such as airports [8] . consequently, in order to block emerging zoonoses with pandemic potential (high-impact spillovers), biosurveillance systems must simultaneously consider critical animal-human interfaces, the performance and reach of the health systems, and the biosecurity of proximate transportation hubs that can serve as conduits for rapid global dissemination. despite the documented importance of animal-human interfaces for zoonotic transmission of pathogens, and the framework described above for understanding the staged transition from spillover through to human pandemic, we still lack a practical, data-driven and synthetic description of the geography of the critical intersection of animal-human interface and vulnerable points with low disease detection and rapid widespread dissemination potential. the aims of the current work were therefore to (1) describe and quantify the global geography of the interfaces between mammalian and bird wildlife and humans and their domestic livestock; and (2) to synthesize the geography of the wildlifelivestock/poultry-human interface, poor health system performance , and the global network of air travel to identify cities whose global connectedness and proximity to animal-human interfaces indicate significant potential to serve as conduits for high-impact spillover. raster data for mammalian and bird species richness, livestock and poultry densities, and human population density were acquired to describe the intersection of their geographic distributions as landscapes of potential animal-human interface. mammalian and bird species richness were used as a representation of total mammalian and bird diversity (ɣ-diversity), respectively, rather than distinguishing between taxonomic groups since it has recently been shown that overall species richness is the primary driver of pathogen richness among these vertebrate hosts [11] . a mammalian richness raster was acquired from the socioeconomic data and applications center (sedac) repository [14] . species richness was quantified using the geographic extents of the international union for conservation of nature (iucn) assessment of mammalian species (5,488 species in 156 families). a total bird richness raster was obtained from the biodiversity mapping project [15] and was constructed from the handbook of birds of the world [16] , birdlife international[17], and iucn. the gridded livestock of the world (glw) provided livestock densities for cattle, pigs, sheep, goats, and buffaloes, and poultry densities for chicken and ducks [18] . the glw was updated in 2018 to more appropriately account for spatial heterogeneity within and between countries. aggregate livestock and poultry rasters were created by taking the sum of the number of cattle, pigs, sheep, goats, and buffaloes per unit area, and chicken and ducks per unit area, respectively, since the current aim was to describe the geography of the wild mammal-livestock-human interface, and wild bird-poultry-human interfaces. human population density was also obtained from the sedac repository and is derived from the global rural-urban mapping project version 4 estimates for the 2015 population [19] . finally, defining high-impact spillover potential requires a measure of the distribution of health system performance as an indication of the local capacity to detect and control the occurrence of cases of a novel zoonosis. the infant mortality ratio (imr) was chosen as a proxy for health system performance because this has been recognised as a j o u r n a l p r e -p r o o f journal pre-proof robust and widely used indicator of health infrastructure and health system performance and used extensively to compare health services between countries [20, 21] . the imr has been shown to correlate very well with disability adjusted life expectancy (dale) as well as the human development index (hdi) and the inequality-adjusted human development index (ihdi) and strongly relates to structural issues that affect entire populations, such as economic development, general living conditions, social wellbeing, and the quality of the environment [20, 22] . the raster of the imr was obtained from sedac [23] . mammalian and bird richness, human population, and imr rasters were aggregated to the spatial resolution of the livestock and poultry rasters such that all features were analysed with a granularity of 5 arc minutes, which is approximately 10 km. finally, an adjacency matrix for the global airport network was acquired from worldpop to compute the network centrality of individual airports within the global network [24] . this data product was constructed by worldpop to model annual air passenger flows and comprises 1491 airports (network nodes) with 644,406 flight connections between them (network edges) [25] . the global distribution of each landscape feature is presented in the left column of s1 figure 1 , with the corresponding top quartile of each distribution (75 th percentile) presented in the right column. the objective was to demarcate geographic zones based on the degree of intersection of each landscape feature, thus constructing proxy interfaces between humans, livestock, poultry, and mammalian and bird wildlife, and finally identifying where these interfaces intersect with poor health system performance. wild bird-poultry and wild mammal-livestock interfaces were considered separately as there is evidence that interfaces arise largely from interaction between phylogenetically related and/or sympatric species [26] , and further evidence suggesting that zoonotic viral richness is higher overall among mammalian species than avian species [11] . intersection was classified as the co-occurrence of the top quartile (75 th percentile) of each specified feature. the classification scheme was also reconstructed using the co-occurrence of the 50 th percentile for each landscape feature to examine how different distribution classifications affect the demarcation of interface and associated hazard. three alert levels (yellow, orange, and red) were identified, as conceptualised in figure 1 . all levels represent landscapes with both high anthropogenic pressure and high biodiversity, and thus have potential for impactful spillover. alert-level yellow depicts two-way interfaces between mammalian/bird richness and human population or livestock/poultry densities. alert-level orange depicts three-way interfaces between mammalian/bird richness, human population density, and livestock/poultry j o u r n a l p r e -p r o o f journal pre-proof densities. alert-level red depicts the same animal-human interfaces as alert-level orange, but extends the intersection of these interfaces to include the top quartile of infant mortality. therefore, the hierarchy of this classification system designates two-way interfaces with wildlife (yellow) as the minimum source requirement for wildlife-derived high-impact spillovers, whereas alert-level orange represents landscapes with maximal pressure on, and contact with, wildlife populations. finally, alertlevel red depicts where the geography of maximum human-animal interface intersects with the geography of poor health system performance. the latter represents those areas that might be expected to miss detection of early cases of spillover to humans and thus presents the greatest risk for high-impact spillover. this scheme thus builds on the work by morse et al. by 1) quantifying and mapping the degree of shared space between animals and humans, which they posit as driving the first 2 stages of their framework, and 2) quantifying and mapping the degree of health system capacity to intercept global dissemination of zoonotic pathogens that demonstrate onward human-to-human transmission, which defines the third stage of their framework. the validity of these animal-human interface metrics was tested using two case studies. the value of these metrics lies in how well they represent contact between wildlife, livestock/poultry, and humans, and the extent to which they intersect with poor health system performance. therefore, the two diseases selected as case studies were chosen based on their representation of infection ecology at the relevant animal-human interfaces and not based on their potential to cause pandemics in humans. as such anthrax was selected because it is a model disease for the wild mammal-livestock-human interface [27, 28] , while highly pathogenic avian influenza a h5n1 (hpai h5n1) was selected as a similarly good representation of the wild bird-poultry-human interface [29] . the food and agriculture organisation's global animal disease information system (empres-i) was used to capture reported occurrences of anthrax and hpai h5n1 between 1 january, 2010, and 1 may 2020 [30] . the empres-i system has a particular focus on transboundary animal diseases and emergent zoonoses. only those occurrences for which the exact location, or the location of the centroid of the subdistrict of the occurrence, was known were included in the case studies to minimise spatial uncertainty. for the two cases studies, respectively, the anthrax (n = 194) and hpai h5n1 (n = 3247) cases were considered as cities were within 50 km of the alert-level red zone for the wild mammal-livestock-human interface and wild bird-poultry-human interface (14.2% and 19.6%, respectively; figure 4 ). the interface zones of highest potential spillover impact, and their adjacent cities, were predominantly in sub-saharan africa and south and southeast asia (figure 4 ). all descriptive analyses were repeated at the next quartile (50 th percentile) for each interface and city centrality, at which 43% of cities in the top 50 th percentile of network centrality were within 50 km of alert-level red zone for each interface, while ≥ 81% of cities were within 50 km of alert-level yellow and orange for each distinct interface (supplemental material: s4 figure 3 , s5 figure 4 , and s6 figure 5 ). this work has defined a hierarchical geography of potential high-impact spillover based on variable animal-human interfaces, human health system capacity and proximate cities of high global connectivity. this work was a descriptive geographical exercise intended to quantify and map a systematic representation of the global animal-human interface, and subsequently it showed that many of the world's most connected cities are adjacent to or within areas where wildlife share space with humans and their domesticated animals. indeed, more than 40% of these cities were within or adjacent to landscapes of extensive animal-human interface, while approximately 14%-20% were located in landscapes of both extensive interface and poor health system performance, thus demonstrating the precarious positioning of many global transportation hubs. as a means toward preventing future highimpact spillovers of pandemic potential, we have highlighted those areas of the world that could most benefit from simultaneous investment in 1) conservation efforts to limit wildlife encounters with humans and their domesticated animals, 2) improved surveillance of animals, and 3) improved human health infrastructure to detect spillovers when they occur and prevent onward spread. finally, defining a global geography of potential high-impact spillover offers unique value by locating those areas and cities that could most benefit from the development of composite, or at least coordinated, landscape and airport biosurveillance systems at local and national levels. in this study we identify areas where the sharing of space between wildlife and humans is high, health system performance is low, and critical interfaces are located adjacent to cities with high global connectivity. we thus provide practitioners in human and animal health with a geographical framework that applies a synthesis of epidemiological, ecological and health system research outputs to aid j o u r n a l p r e -p r o o f journal pre-proof evidence-based public health decision-making. we emphasize that this work is not presented as a prediction of global hotspots of spillover. several well-conducted studies have been undertaken that provide useful results in this domain [4, 10, 38] . moreover, previous efforts have clearly demonstrated the importance of increasing human and domesticated animal encounters with wildlife as a key driver of spillover [39] , as well as the potential modulation of wildlife-human encounters and subsequent spillover by climate change [40] . while these previous modelling studies have identified the importance of human pressure on wildlife for spillover, none have described the global distribution of animal-human interface as a data-driven construct of the degree of shared space between wildlife, domesticated animals, and humans. nor have they considered the location of proximate transportation hubs, which are positioned to amplify impactful spillovers through rapid regional or global dissemination and thus transitioning impactful spillovers to high-impact spillovers with regional epidemic or global pandemic consequence. in other words, conduits of high-impact spillover have not been previously systematically mapped, nor have they been described using a practical hierarchy designating the degree of animal-human interface. the practical approach presented here will allow for a more targeted development of one health surveillance and prevention programs, and provides considerable scope as a strategic tool for preventing future pandemics. for example, a preventive strategy can be designed to create barriers between the high-hazard landscapes and their adjacent points of global dissemination. moreover, the closer an intervention is to the landscape generating the hazard (i.e. spillover) the broader would be its expected downstream prevention effect, whereas an intervention implemented farthest from initial spillover (e.g. at transportation hubs) is more permeable and would expectedly provide narrower effect. however, when integrated with upstream surveillance, enhanced monitoring at transportation hubs can contribute to robust, multi-tiered systems by providing a data-informed, critical last line of defense ( figure 5 ). some limitations with this work regarding proxy measures require further discussion. first, the animalhuman interfaces described here are based on the distributions of the species involved (wild mammals and birds, domesticated animals, and humans) rather than on direct observations of the species' interactions in the landscape. therefore the metrics used to represent these interfaces are proxies for interspecific interaction. nevertheless, the granularity of the species' distributions was relatively fine scale (10 km x 10 km) and, given the high percentile (75 th ) of species' distributions used to construct the primary risk zones, it is reasonable to assume that humans and animals do indeed share the spaces, either in whole or in part or directly or indirectly, within the interfaces we have demarcated. second, the infant mortality rate was used as a proxy for health system performance. we recognise that health j o u r n a l p r e -p r o o f systems are complex coalescences of economic and social infrastructure, medical capacity and training, public health capacity and training, governmental organisation, and the general socioeconomic status of human populations. moreover, it would not be possible to measure these individual components with the granularity required to synthesise a geographically meaningful new construct of health systems. however, the infant mortality rate has long been recognised as both a reliably measured outcome of health systems and a robust metric for comparing the performance of health systems between countries [20] [21] [22] . finally, the validation case studies should not be over-interpreted with respect to anthrax or hpai h5n1 or any other zoonosis. as described above, the current work is not intended as a predictive modelling study. the models used here were not trained with one set of outbreaks to identify some optimal suite of predictors, and then, once identified, tested and evaluated against an independent set of outbreaks. rather, under the current framework specific hazard metrics for animalhuman interfaces were created by quantifying the extent to which humans share space with wildlife. the point process models were then used to determine how these metrics, data-driven and standradised but conceptualised a priori, actually fit against real-world outbreaks of important known zoonoses. it is also important to recognise that outbreak reporting as captured by the empres-i system may not be geographically homogeneous, which is in fact reflected in the finding that red zone interfaces were a poorer fit to the case study outbreaks indicating that these areas may be more prone to missing cases. this investigation has shown that there are substantial animal-human interfaces in areas of poor health system performance, highlighting those areas of potential impactful spillover where health infrastructure may be insufficient to identify spillover cases early and block onward human-to-human transmission should this emerge. this work further showed that there are many cities with a high degree of global connectivity that are proximate to the areas at risk of impactful spillover, and thus has bats, civets and the emergence of sars emergence and pandemic potential of swine-origin h1n1 influenza virus the ebola outbreak global hotspots and correlates of emerging zoonotic diseases global trends in emerging infectious diseases the elephant-livestock interface modulates anthrax suitability in india forest loss shapes the landscape suitability of kyasanur forest disease in the biodiversity hotspots of the western ghats, india prediction and prevention of the next 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interface areas of northern tanzania: a retrospective record review wildlife/domestic livestock interactions human-animal interface: the case for influenza interspecies transmission empres-i -global animal disease information system spatial point patterns: methodology and applications with r spatstat: an r package for analyzing spatial point patterns the architecture of complex weighted networks some unique properties of eigenvector centrality the igraph software package for complex network research. interjournal complex systems 1695 r: a language and environment for statistical computing, r foundation for statistical computing global patterns of zoonotic disease in mammals global shifts in mammalian population trends reveal key predictors of virus spillover risk climate change will drive novel cross-species viral transmission anthrax ppm models 2267.9* model 1: wild mammal-livestock model 2: wild mammal-human model 3: wild mammal-livestock-human model 4: wild mammal-livestock-human-low health 1964 avian influenza a h5n1 ppm models 12753 model 3: wild bird-poultry-human model 4: wild bird-poultry-human-low health baseline aic from spatial-only specified model of zoonosis outcome key: cord-307320-fxs31d66 authors: ubah, obinna; palliyil, soumya title: monoclonal antibodies and antibody like fragments derived from immunised phage display libraries date: 2018-03-17 journal: recombinant antibodies for infectious diseases doi: 10.1007/978-3-319-72077-7_6 sha: doc_id: 307320 cord_uid: fxs31d66 morbidity and mortality associated with infectious diseases are always on the rise, especially in poorer countries and in the aging population. the inevitable, but unpredictable emergence of new infectious diseases has become a global threat. hiv/aids, severe acute respiratory syndrome (sars), and the more recent h1n1 influenza are only a few of the numerous examples of emerging infectious diseases in the modern era. however despite advances in diagnostics, therapeutics and vaccines, there is need for more specific, efficacious, cost-effective and less toxic treatment and preventive drugs. in this chapter, we discuss a powerful combinatorial technology in association with animal immunisation that is capable of generating biologic drugs with high affinity, efficacy and limited off-site toxicity, and diagnostic tools with great precision. although time consuming, immunisation still remains the preferred route for the isolation of high-affinity antibodies and antibody-like fragments. phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. the selection of binding fragments from phage display libraries has proven significant for routine isolation of invaluable peptides, antibodies, and antibody-like domains for diagnostic and therapeutic applications. here we highlight the many benefits of combining immunisation with phage display in combating infectious diseases, and how our knowledge of antibody engineering has played a crucial role in fully exploiting these platforms in generating therapeutic and diagnostic biologics towards antigenic targets of infectious organisms. the number of monoclonal antibody (mab) based drugs developed by biopharmaceutical companies is at an all-time high with more and more novel anti-infective antibodies gaining regulatory approval. the emergence of multi-drug resistance has reinforced the need to develop novel anti-infective approaches. unlike conventional antibiotics, o. ubah scottish biologics facility, elasmogen ltd, aberdeen, uk s. palliyil (*) scottish biologics facility, university of aberdeen, aberdeen, uk e-mail: soumya.palliyil@abdn.ac.uk monoclonal antibodies exhibit high target specificity and possess the ability to recruit immune system components for effective pathogen removal. modes of action include: specific binding and neutralisation of microbial toxins and virulence factors, opsonising and marking pathogens for cell-death by directing phagocytic cells to the site of infection, antibody mediated bacterial agglutination and clearance, complement activation and direct bacterial lysis [77] . host effector functions mediated through interactions with the fc region of mab drugs makes them highly effective in treating immunocompromised patients that are unable to generate their own immune response to fight diseases [90] . certain monoclonal antibodies exhibit synergistic or additive effects with conventional antibiotics providing an attractive therapeutic strategy for treating infections caused by multidrug resistant organisms [1] . this chapter discusses the advantages and disadvantages of using immunised phage display libraries constructed from mammalian sources for generating monoclonal antibodies against infectious disease targets. several groups have successfully developed monoclonal antibodies specific for bacterial, fungal and viral antigens with potential applications in the detection, diagnosis and treatment of infectious diseases which are reviewed in this chapter. originally mabs were isolated using hybridoma technology, and required the fusion of mouse lymphocyte and myeloma cells to generate specific murine antibodies that were often highly immunogenic if used in a therapeutic setting. a limited number of human mabs were also developed by using human b lymphocytes from naturally infected patients but this proved a technically challenging and generally unreliable approach [48] . subsequent advances in antibody engineering removed or resolved many of these technology "road-blocks" facilitating the generation of chimeric, humanised or deimmunised mabs and bispecific antibodies with increased potency and reduced immunogenicity. one of the major breakthroughs was the invention of phage display which revolutionised the field of antibody engineering with its robust, easy to use and highly versatile combinatorial display platform. its successful applications include: generation of antibodies with unique functions from immune and non-immune sources, de novo isolation of high affinity binders from non-immune or synthetic sources and the in vitro affinity maturation of antibodies [43] . phage based selection could be summarised as the display of antibodies (proteins or peptides) on the surface of bacteriophage by fusing the antibody gene to one of the phage coat proteins and selection based on the antigen binding of individual clones. phage antibody libraries are constructed by pcr based cloning of vh and vl repertoires by random pairing into a phage or phagemid vector system and display on the surface of bacteriophage. it has been used widely in the antibody engineering as a technique to mimic b cells, which are self-replicating packaged systems containing antibody genes that encode the antibody displayed on its surface (linked genotype and phenotype) [108] . phage display libraries can be constructed using antibody variable (v) genes isolated from igm mrna of non-immunised human donor b cells derived from diverse lymphoid sources such as peripheral blood lymphocytes (pbls), spleen cells, tonsils, bone marrow or from nonimmunised animal b cells (naïve antibody libraries). igg mrna from pbls or spleen cells of immunised animals or human patients are used to build immunised libraries. a third class called synthetic and semi-synthetic antibody libraries are constructed using repertoires of rearranged v genes from gene segments using polymerase chain reaction (pcr) and introducing variation into their cdr regions using custom degenerate primers encoding for diversity and length [108] . immunised libraries are generally constructed using antibody vh and vl genes amplified from the mrna of b cell pools from immunised animals or human donors. post immunisation, the antibody secreting b cells possess a higher percentage of antigen specific heavy and light chain gene transcripts. these b cells would have undergone affinity maturation and isotype switching in germinal centres before entering into the peripheral circulation. typically antibodies isolated from immunised libraries will have a high affinity and tighter specificity for the immunogens used [8] and can be achieved even from relatively small library diversity [108] . unlike hybridoma technology, phage display provides researchers with more possibilities to streamlining their monoclonal antibody generation process. the main improvement is the replacement of cell culture screening steps using hybridomas. instead of screening hundreds or thousands of hybridoma monoclonals to find positive clones, phage display, with carefully designed panning strategies, can screen billions of clones and allow the enrichment of small subsets of binders with desirable characteristics that can be further screened to identify individual binders. antibodies recognising specific antigenic conformations and epitopes can be easily isolated by introducing selective or subtractive panning steps during the selection process. it has also been reported that using phage display high specificity antibodies can be isolated from immunised sources which would be missed by traditional immunological methods [100] . a key feature of this system is the linkage of genotype (phagemid vector) and phenotype (phage coat protein-antibody fragment) which allows immediate access to the corresponding gene sequences of selected antibodies facilitating simple sub-cloning into various antibody formats based on required downstream applications ( fig. 6.1 ). over the last 20 years phage display technologies have been successfully used for the development of many therapeutic antibody candidates and approved drugs and with the relaxation of commercial restrictions more and more products are now entering the diagnostic and research markets as well [9] . the drawbacks of animal immunisation for the construction of phage display libraries include the time period required for the completion of such a process and the requirement to construct separate libraries for each antigen. however, by combining the power of immunisation with phage display, several high affinity monoclonal antibodies against "difficult" antigenic targets have been isolated from relative small antibody libraries and where traditional approaches have failed [33, 98] . generating antibodies against self or toxic antigens is limited if immunisation is to be employed. in particular, human autoantigens are highly conserved amongst most routinely used laboratory mammals such as mice or rat. therefore immunisation of non-mammals such as sharks and chickens, which are phylogenetically distant from humans, has been successfully employed for generating an immune response and antibodies and/or antibody like binders (vnar) to epitopes conserved in mammalian species [42] . chickens are phylogenetically distant from humans and therefore very successful in generating an immune response to mammalian proteins which are highly conserved [34] , while sharks are even more distant from humans, diverging from a common ancestor approximately 450 million years ago [6, 37, 53] . [13, 14, 54, 66, 76, 89, 111, 112] . human monoclonal antibodies from patients which showed protective effects in animal model studies have also been selected against bacterial targets such as anthrax toxins [107] , botulinum neurotoxin [4] and the abc transporter of methicillin resistant staphylococcus aureus [12] . in the case of the west nile virus neutralising mabs were only isolated from the b cell population of convalescent human patients with large phage display libraries constructed from uninfected donors delivering nothing of consequence [105] . conventional methods of antibody generation such as hybridoma and epstein-barr virus transformation are limited in their abilities to evaluate human monoclonal antibodies from different patients at various stages of their clinical course. technologies like phage display provide a powerful tool that allows pooling of large number of patient immune b cell populations and the selecting of antibodies with distinct specificities and inhibitory activities during different stages of infection [13] . the ability to improve affinities and broaden specificities post selection is a major attribute of recombinant antibodies. using recombinant dna and protein engineering approaches the pharmacokinetic and pharmacodynamics properties of these molecules can be improved including half-life extension and reduced immunogenicity. for the neutralisation of toxins and viruses, it is often advantageous to generate multiple high affinity antibodies that recognise different epitopes on the same antigen or a variety of antigenic subtypes capable of delivering broad protection against pathogen variants. such 'oligoclonal' antibodies were reported to show strong synergistic activity in neutralising botulinum neurotoxin (bont) assays [74] . a list of recombinant antibodies generated from phage display libraries constructed using the immune antibody gene repertoires of human patients is given in table 6 .1. several research groups have successfully demonstrated the potential of monoclonal antibodies, fragments and single domain antibodies for the prevention and treatment of bacterial infections in animal models. however their translation into the clinic has been somewhat slow. a small number of mab drugs under regulatory review include obiltoxaximab, for the treatment and prevention of inhalational anthrax and bezlotoxumab, which targets clostridium difficile enterotoxin b, developed for the prevention of recurrent c. difficile infection [83] . with the ever increasing numbers of antibiotic resistant bacterial strains, the need to develop antibody based drugs with novel modes of killing has never been greater especially if their mode of action limits the development of resistance. antibodies developed against bacterial targets fall into two main categories: (i) antibodies that target the bacterial cell surface directly or (ii) those that act indirectly by neutralising bacterial toxins or virulence factors and relying on the host immune system for effective pathogen clearance. monoclonal antibodies and fragments developed against various gram−ve and gram+ve bacterial targets using animal immunisation and phage display based selection are summarised below. infection control using high affinity monoclonal antibodies specifically targeting the quorum sensing (qs) molecules of pseudomonas aerugi-nosa has been reported from our laboratory [78] . a number of gram negative bacteria, including pathogens like p. aeruginosa, utilise homoserine lactones (hsls) as qs signalling compounds and engage in cell-to-cell communication to coordinate their behaviour. as qs takes a central role in p. aeruginosa infection by regulating the expression of extracellular virulence factors (and also [12] biofilm formation), immuno-modulation of the hsl molecules by monoclonal antibodies (mabs) can be used as a novel approach to prevent p. aeruginosa infections. sheep immunisation was utilised to develop antibodies with high affinity and sensitivity towards hsl compounds. a mixture of three hsls-n-acyl-c12-hsl, 3-oxo-c12-hsl and 3-oh-c12-hsl with different subgroups at the third carbon position were conjugated to the carrier protein thyroglobulin (tg) and used as immunogen to enhance the chance of eliciting an antibody response in sheep. using pbls from immunised sheep as starting material, v h -v λ and v h -v κ anti-hsl phage display libraries were constructed in a scfv format. the panning strategy was designed to drive selection towards the enrichment of high sensitivity and cross-reactivity clones. lead clones were reformatted into sheep-mouse chimeric iggs and had picomolar sensitivities (ic 50 values as determined by competition elisa) for 3-oxo-c12-hsl which is the central qs compound in p. aeruginosa. these values are quite impressive, considering the chemical nature of these lipidlike compounds (average molecular weight 300 da) which possess only a small head like structure and lack critical antigenic features such as aromaticity or charge. modelling of these sensitive anti-hsl antibodies indicated that the level of sensitivity observed was achieved through the generation of a deep and negatively charged binding pocket [2] . sheep immunisation was chosen for this hsl application as this approach has previously been shown to generate high affinity antibodies against haptenic targets [17] and sheep polyclonal antibodies have been used as specific high affinity immunologic probes for analytical and clinical purposes for many years [62] . in contrast to humans and mice, sheep b cell lymphopoiesis occurs predominantly in the ileal peyer's patches (ipp) and v(d)j recombination creates limited diversity in the sheep ig repertoire due to a very few gene segments participating in the rearrangement process. this characteristic feature makes sheep library construction simpler, as the entire heavy and light chain gene repertoire can be amplified using a small number of primers. much of the antibody diversity is achieved through antigen-independent post-rearrangement somatic hypermutation, which diversifies all cdrs as compared to the sole variability of just cdr3 in humans and mice [17, 62, 78, 84] . comparison of chicken and sheep responses by hyperimmunisation with the same immunogen revealed the ability of the sheep immune system to produce higher specificity antibodies than chicken [109] . the overall antibody titre was also much higher in sheep and might be due to the longer half-life (~15 days) of sheep immunoglobulins when compared to 35 h in chicken [106, 109] . it is also noteworthy that the sensitivities of the sheep polyclonal/mab antibodies described here were far superior to the published mouse monoclonal antibody specific for the same hsl antigens [50] . the protective effects of the sheep derived, anti-quorum sensing antibodies were demonstrated in vivo. in a slow killing model of the nematode worm caenorhabditis elegans the significant increase in survival rate in the presence of hsl mabs is similar to the defective slow killing observed in lasr mutant strains [99] . furthermore, in a non-neutropenic lung model of mice infected with p. aeruginosa pa058, hsl mab monotherapy demonstrated significant efficacy, prolonging survival by up to 83%. since no significant reduction in bacterial counts was observed in the lungs of infected mice, it is proposed that hsl specific antibodies protect mice possibly through antibody mediated scavenging of hsl compounds. this mode of action does not necessarily affecting bacterial numbers but probably prevents a switch to a more pathogenic phenotype [78] . it has been widely accepted that antibodies with sub-nanomolar affinity exhibit improved therapeutic efficacy, if the targets to be bound are present in low concentrations. the above study demonstrates the power of animal immunisation and phage display based selection strategies to isolate high affinity monoclonal antibodies towards non-antigenic targets which inherently lack properties like aromaticity and charge. adp-ribosylating enzymes such as cholera, pertussis, diphtheria toxins and escherichia coli heat-labile (lt) toxins are important virulence factors for a number of extracellular bacterial pathogens. pathogenesis is driven by the secretion of potent toxins that utilise adp-ribosylation as the catalytic mechanism underlying their action. adp-ribosylating toxins comprise a large family, and all produce disease by altering key metabolic processes after transfer of an adpribose moiety from nad to specific host-cell target proteins [25, 60] . it was not until the early 2000 that the adp-ribosylating enzyme was implicated in intracellular pathogenesis. it was shown that salmonella strains were capable of invading epithelial cells and localising in macrophages during infection [60, 61] . the salmonella virulence plasmid factor b (spvb) virulence gene of salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. lesnick et al. provided evidence that spvb encodes an adp-ribosylating enzyme that uses actin as a substrate and depolymerises actin filaments when expressed in cho cells [61] . a spvb blocking camel vhh single domain antibody (sdab) capable of blocking spvb enzymatic activity at a 1:1 molar ratio was isolated from an immune phage display library generated from an spvb immunised llama [3, 68] . as an intracellular protein, spvb is inaccessible to conventional antibodies, and small molecule inhibitors of spvb are fraught with potential side effects resulting from the indiscriminate inhibition of endogenous mammalian adp-ribosyltransferases (arts) [11, 51, 63] . the vhh sdab when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric c21n-c/spvb toxin, and transfected cells were also protected against cytoskeletal alterations induced by wild-type spvb-expressing strains of salmonella [3] . this provides evidence to support the development of these sdabs as therapeutic and experimental tools to block mammalian and toxin arts. helicobacter pylori is a gram negative pathogenic bacteria that colonises the human stomach and can cause gastritis, gastric and duodenal ulcers and cancer. the surface proteins in h. pylori mediate several host-pathogen interactions and hence are attractive targets for antimicrobial therapy and vaccination. an antibody phage display library in a scfv format was constructed by hyperimmunising mice with h. pylori total cell lysate and a monoclonal antibody fragment recognising the outer membrane protein hopq was isolated by performing biopanning on whole cells [88] . in another study a mab generated through mice immunisation and hybridoma technology was engineered using phage-display as a scfv fragment and showed high affinity binding to an h. pylori surface antigen. this phage displayed scfv antibody was shown to inhibit the growth of six different h. pylori strains and offered significant protection in a mouse model of infection. the authors argued that genetically engineered bacteriophage could be used as alternatives to conventional antibiotics in the treatment of bacterial infections [15] . spleen samples from mice immunised with gamma inactivated brucella melitensis strain 16 m bacteria was used to construct a phage display library and isolate monoclonal antibody fragments that specifically recognise brucella species. brucella can cause long term debilitating illness in humans, can be spread as aerosols and survive extended periods outside their host. the attributes could make brucella species possible biological warfare agents. since many brucella species share their immunodominant lipopolysaccharide (lps) antigen with the closely related yersinia species, a specific lps antibody that can distinguish between these two bacteria is central for rapid detection and diagnosis of brucella infection. specific washing steps were included during bio panning of an immunised b. melitensis library to eliminate phage that might be cross-reactive with strains expressing the same dominant lps epitope on yersinia. the resultant monoclonal antibody fragments was specific for the antigen and not cross-reactive towards yersinia species [41] . botulinum neurotoxins (bonts), regarded as one of the most toxic substances on earth, are secreted by clostridium botulinum and some other species of clostridium. bont/a which is the most potent among seven serotypes exerts its toxicity by the cleavage of snap-25 (synaptosomal-associated protein), mediated by its light chain (bont/a-l). this proteolysis causes blockage of nerve impulses and causes flaccid paralysis, including that of respiratory muscles resulting in death [16] . immunisation of macaques (macaca fascicularis) and construction of hyper immune phage display library resulted in the isolation of nanomolar sensitivity antibody fragments to the light chain fragment of bont/a that are capable of inhibiting bont/a endopeptidase activity in vitro. the variable heavy and light chains of selected clones are highly similar to human germline sequences which predicts good tolerance for clinical use. since immunoglobulin genes of non-human primates are very similar to human abs, the differences in the conserved framework regions of macaque and human iggs are no greater than those between human iggs from different individuals [16] . sub nanomolar affinity 'nearly human' fab fragments against tetanus toxoid antigen were isolated from a small phage display fab library constructed using the immune antibody repertoire of macaca fascicularis [18] . similarly high affinity neutralising antibody fragments against the protective antigen (pa) of anthrax toxin was isolated from a macaca immunised phage display library. these fab fragments were shown to bind to a particular region of pa that interacts with cell receptor thereby blocking its binding [56] . a single chain fv phage display library was constructed from a cynomolgus macaque (macaca fascicularis) immunised with lethal factor (lf) of anthtax toxin. resultant high affinity scfv fragments showed efficient inhibition of anthrax toxin in vitro and in vivo [80] . in cases where anthrax vaccination is not practical or antibiotic therapy is ineffective, passive immunisation with anthrax neutralising antibodies can be an effective method of treatment. similar to macaques, chimpanzee immunoglobulins are very close to human antibodies and they should be well tolerated in vivo in human therapy [31, 92] . lymphocytes from the bone marrow cells of two chimpanzees immunised with anthrax toxin pa, lf and edema factor (ef) were used to construct scfv phage display libraries and neutralising antibodies were isolated against pa and lf proteins. for passive immunotherapy, these fragments were converted into bivalent full length immunoglobulins which showed strong neutralising activity against the cytotoxicity of anthrax toxin in vitro. these high affinity (picomolar range) mabs demonstrated efficient protection in animals from anthrax toxin challenge in vivo, most likely by blocking binding of pa to the cell receptor which suggest their use in the emergency prophylaxis and treatment of anthrax [22, 23] . the nature of the antibody-antigen interaction allows for the development of molecules that can target bacterial antigens with a high degree of affinity and specificity. target specificity of mabs can be can be utilised to deliver antimicrobial compounds more effectively [97] . in addition, by choosing a suitable antibody fc portion, host effector functions can be mediated through the recruitment of elements of the host immune system such as macrophages, nk cells and complement to sites of bacterial infection and accelerating clearance of the infection. despite having a large number of candidates with promising efficacies in preclinical animal models of infection, anti-infective antibodies reaching clinical development and finally into the markets are somewhat slow. in most cases in vivo models can be limited in their ability to predict efficacy in humans, particularly due to differences in the pharmacokinetics of the molecules between humans and model animal species [110] . geng et al. reported that a large percentage of antibodies for anti-infective indications are ended in the early stage of discovery making them a high risk category in drug development [35] . challenges in clinical efficacy and pharmacokinetics can be overcome by choosing appropriate format for the indication. for example, igg fc region interacts with the neonatal fc receptor (fcrn) which is important for igg recycling and protection from degradation, contributing towards unique pharmacokinetic properties of the molecule and extending the serum half-life to about 21 days for most igg subclasses [86] . the variable region (vhh) of heavy chain only antibodies (hcabs) found in camelidae species possess an unusually long complementarity determining region 3 (cdr3) which can form finger-like extensions to penetrate into grooves on the surface of antigens that are usually inaccessible to conventional antibodies. staphylococcus aureus produces several adhesion factors and exotoxins such as hemolysins α, β, γ, δ and panton-valentine leukocidin (pvl) which are important virulence determinants. s. aureus is a leading cause of endophtalmitis which is associated with a poor visual outcome. the expression of pvls and leucotoxins especially luks-pv and lukf-pv induce the activation and permeabilisation of target cells and result in cell lysis. transgenic mice harbouring llama/human hybrid ig heavy chain locus were immunized with recombinant luks-pv and lukf-pv proteins. using phage display technology high affinity vhhs were isolated and reformatted into bivalent and tetravalent camel heavy chain antibodies. anti luks-pv and lukf-pv hcabs were able to inhibit pvl associated disease pathology in a non-infectious model of rabbit intravitreous pvl, when administered prophylactically. the hcabs completely reduced inflammation in the eyes of the treatment group without any apparent damage to vision or behaviour. this shows the possibility of administering toxin neutralising antibodies in combination with an intravitreal antimicrobial strategy for postsurgery endophthalmitis [58] . camel immunisation using s. aureus exoproteins and successful generation of anti β-hemolysin single domain antibodies was reported by jangra and singh [49] . in a neutralisation assay, their lead vhh clone completely inhibited five hemolytic units of the toxin in vitro and resisted thermal denaturation up to 99 °c. the cdr3 loop of vhhs is longer than the average igg and linked with cdr1 by an interloop disulphide bond which renders additional stability to the protein. in some cases the long cdr3 loop enables the formation of a convex paratope that can access deep enzymatic clefts and cavities on the surface of an antigen [71] . the active sites of enzymes such as β-hemolysin are mostly situated in their largest cleft. the long cdr3 loop of anti β-hemolysin clone is believed to bind this site and able to neutralise enzyme activity by blocking access to the hlb antigen [49] . vhh fragments specifically binding to tetanus toxoid 1 antigen were generated using camel immunisation and phage display technology. two vhh fragments recognising two different epitopes of the antigen successfully neutralised tetanus toxin in vivo in studies conducted in mice [71, 72] . another example where vhhs can inhibit bacterial infection by increasing the bacterial sensitivity to β-lactam antibiotics has been reported [24] . high affinity β-lactamase inhibiting vhhs were generated from immunised dromedary phage display libraries primed towards the antigens tem-1 and bcii-lactamases, representatives of class a and class b-lactamases, respectively. in the presence of ampicillin, specific vhh fragments inhibited the growth of e. coli cells expressing a fusion protein of tem-1 β-lactamase on their outer surface, whereas the cells were able to grown in higher concentrations of ampicillin (>150 μg/ml) when no vhh fragments were added [24] . the authors claim that vhhs are less immunogenic than larger murine antibodies as they show a high sequence similarity to human vh families 3 and 4. vhh nanobody dosed at an immune phage display library. a one-year-old male dromedary (camelus dromedaries) was immunised with purified recombinant bont/e protein [5] . another high affinity vhh inhibitor of bont/a protease activity was isolated from phage display libraries derived from b cells obtained from the immunised alpaca (lama pacos) [101] . one of the main advantage of using animal immunised phage display libraries for generating antibodies against bacterial antigens is the easy isolation of high affinity neutralising antibodies without the need for further affinity maturation. table 6 .2 summarises monoclonal antibodies and antibody fragments against various bacterial targets and their affinities to target antigens. chen et al. [22, 23] the eukaryotic nature of fungal pathogens presents an almost insurmountable problem when developing anti-fungal drugs. multiple drug resistance associated with existing chemical antifungal drugs highlight the need for new, more effective antifungal therapies. antibody based antifungal treatments can provide a much needed alternative to the near-exhausted chemical based strategies. due to their high specificity and target selectivity, antifungal antibodies can be successfully employed for targeted killing of fungal pathogens without harming host cells. using hybridoma technology several mabs were generated against various antigenic components of fungal pathogens cryptooccus neoformans, candida albicans, aspergillus fumigatus, pneumocystis jerovecii and histoplasma capsulatum [19, 36, 40, 70, 82, 85, 87] . the murine monoclonal antibody (mab 18b7) generated through hybridoma technology directed against the capsular polysaccharide of c. neoformans mediated protection in murine model of infection and was shown safe to use in human patients in a phase i dose escalation study [57] . in addition to the classical antibody mediated mechanisms of phagocytosis, complement activation and recruitment of inflammatory cells, mab 18b7 was shown to increase the stiffness of the capsule by polysaccharide cross linkage which impaired yeast budding by trapping newly emerging buds inside the parental capsule [26] . opportunistic invasive fungal pathogens cause over 2 million life-threatening infections per year worldwide with mortality ranging from 20-95% [10] . the most common cause of invasive fungal infections in the intensive care unit are candida spp., and in the usa and western europe, the incidence of candida infections is second only to staphylococcus aureus (including mrsa) [104] . recent data indicate that the incidence of candidaemia continues to rise unchecked by current clinical practice or drug regimens [73] . candida mannan specific monoclonal antibodies have been generated through sheep immunisation in our laboratory. a single chain antibody phage display library was constructed using the immune repertoire of a sheep hyperimmunised with candida albicans hyphal cell wall preparation. this antibody library, one of the very first and largest against candida cell wall antigens, has been successfully used to isolate high affinity and specificity binders to candida α-mannan, which are typically poorly immunogenic glycans present on the fungal cell surface. analysis via sem of polyclonal sera derived from immunised sheep confirmed that the library contained a varied collection of antibodies with specificity to both fungal cell wall carbohydrates (mannans and glucans) as well as a number of important cell wall proteins (patent wo2014174293 (a1)). hm-1 killer toxin protein is secreted by most yeasts to stop the growth of other competing strains by inhibiting the transmembrane enzyme β glucan synthase involved in their cell wall synthesis. a neutralising monoclonal antibody generated against hm-1 toxin (nmab-hm-1) was used to immunise mice and isolate hm-1 antiidiotypic antibodies from a scfv phage display library constructed using mice spleenocytes. anti-idiotypic scfv fragments that inhibited β-1,3-glucan synthase activity were isolated from this library and showed in vitro cell killing in four pathogenic species of candida including c. albicans, c. tropicalis, c. parapsilosis, and c. glabrata. the mic values of these scfvs were in the range 1.56-12.5 μg/ml [93] . biopanning using aspergillus fumigatus membrane fraction (amf) and employing competitive panning elution methods, scfv clones which showed in vitro antifungal activity against a. fumigatus was isolated from the same library [55] . the authors argue the potential of these scfvs as candidates for developing as antifungal drugs by either engineering as scfv-drug conjugates or reformatting into whole igg for complement-mediated and antibody-dependent cytotoxic pathways [93] . a number of human antibody fragments recognising c. albicans cell surface antigens were isolated from human phage display libraries [38, 113] which is beyond the scope of this review. the authors have demonstrated antibody mediated opsonisation of c. albicans cells, phagocytosis and killing of the fungus by mouse macrophages and activation of the mouse complement cascade in in vivo models [113] . combination immunotherapy is another attrac-tive therapeutic strategy where anti-mannan mabs were shown to have protective effect. in combination immunotherapy a chemical antifungal drug is administered along with an antibody. for example, mab b6.1 is synergistic with amphotericin b [39] and augments the therapeutic effect of fluconazole in a mouse model of disseminated candidiasis [59] . these examples demonstrate the potential for antifungal mabs in clinical settings and the use of drug combinations as a powerful strategy to enhance antifungal efficacy and abolish drug resistance. the majority of anti-infective monoclonal antibodies developed in the 1990s were antiviral drugs with a main focus on human immunodeficiency virus (hiv) neutralisers. using mice immunisations and sera from infected humans, several mabs were developed against the hiv type-1 envelop with only a few of them broadly neutralising across all hiv-1 subtypes. all these neutralising mabs were generated as a result of human infection and antibody responses against a range of epitopes such as gp120, gp41 or various epitopes on gp120 which are exposed after cd4 binding [64] . forsman et al. published the generation of several high affinity and crossreactive vhhs neutralising hiv-1 primary isolates belonging to subtype b and c. llamas were immunised with recombinant gp120 belonging to subtype b and the resultant phage display libraries were panned using recombinant gp120 from subtypes a, b and c. a competitive elution strategy using scd4 was employed during panning to isolate clones which were shown to inhibit the binding of scd4 to hiv-1 gp120 and gp140 from selected subtypes. it is proposed that the vhh domains inhibit hiv-1 infection by interacting with gp120 prior to its engagement with cd4, which makes them potential hiv-1 inhibitors [32] . subsequently, a family specific phage display library was constructed using pbls from the same llama immunised with recombinant gp120 derived from hiv-1 cn54 and tailored using degenerate primers based on the nucleotide sequences of the cdr3-fr4 region of the positive hiv-1 neutralising vhhs described above. resultant picomolar affinity vhhs were shown to neutralise a broad range of hiv-1 virus belonging to subtypes b and c [52] . another set of nanobodies that recognise the chemokine receptor cxcr4 was isolated by llama immunisation and phage display. cxcr4 belong to gpcr family where therapeutic targeting using conventional antibodies has proved unsuccessful probably because traditional antibodies struggle to access the cryptic and buried antigenic sites that make up the ligand binding pockets on the receptor surface. cxcr4 plays an important role in stem cell physiology, tissue repair, inflammation, metastatic spread of cancer and also serves as a co-entry receptor for hiv. highly potent nanobodies generated using whole cell immunisation (cxcr4-expressing hek293t cells), phage display library construction and biopanning behaved as competitive cxcr4 antagonists by totally blocking ligand cxcl12 binding and inhibiting chemotaxis and hiv-1 entry [47] . two monoclonal antibodies specifically recognising the orf-2 capsid protein of hepatitis e virus were isolated from a cdna library constructed using the lymphocytes isolated from a chimpanzee bone marrow [91] . this chimpanzee was experimentally infected with all five recognised hepatitis virus -hepatitis a to e. phage displayed biopanning using recombinant hev orf2 proteins from human hev strain (pakistani strain sar-55) and a swine hev strain (u.s. strain meng) generated two unique mabs with nanomolar binding affinities as determined by biacore analysis. the γ1 heavy chains of anti-hev mabs had the most sequence identity with the human vh3 family of germ line segments (89.4% and 88.5% overall identity) and the k light-chain sequences exhibited the most identity with the human vk1 family of germ line segments. these mabs were able to neutralise sar-55 strain of hev and offer protection from hev infection in rhesus monkeys. the authors have highlighted the significance of using chimpanzee as a donor for antigen primed antibody repertoire including the possibility of infecting them with viral pathogens of human relevance and reduced immunogenicity when used in human immunotherapy. earlier studies have reported that human antibodies are recognized as self by the chimpanzee immune system thereby generating little immunogenicity compared to other primates and the half-life of a human mab is equivalent to the estimated halflife of igg in humans [75] . schofield and colleagues also reported the generation of four monoclonal antibodies against hepatitis a virus capsid from the same chimpanzee bone marrow derived cdna library using inactivated whole hav capsid from strain hm-175 for biopanning [92] . all four mabs neutralized the strain hm-175 and two of the four mabs also neutralized the divergent agm-27 strain. however the actual mechanism of neutralisation remain unclear as the authors noticed that the fab fragments are not inhibiting virus attachment to cells via soluble simian cell receptor for hav, havcr1. no further developments for these monoclonal fab fragments are available in the public domain. generating vhhs from immunised llama libraries to hepatitis b surface antigen (hbsag) and hepatitis b core antigen (hbcag) were explored as an approach to inhibit hbv replication thereby tackling viral infection [94, 95] . the resultant vhh fragments were expressed as intrabodies to target the antigens which exert their functions in different compartments of the host cell (cytoplasm, er and nucleus). the intrabodies were shown to supress the secretion of hbsag and hbcag in vitro and inhibition of viral release in a mouse model [94] . other vhh fragments developed using animal immunisation and phage display technology include those binding to rotavirus gp6, h5 hemagglutinin to reduce viral replication in h5n1 influenza virus, vhh recognising the tail of infectious phage in lactococcus bacteria. the list with relevant publications is summarised in table 6 .3. also a comprehensive list of antibodies raised against viral targets using non-immune libraries was presented in a recent review publication [102] . there is currently no effective treatment for respiratory syncytical virus (rsv) lower respiratory tract infection. rsv is a major worldwide cause of morbidity and mortality in infants and young children, immunocompromised patients and the elderly. treatments remain largely supportive and rsv-specific options for prophylaxis are limited to palivizumab [65, 67] . alx-0171 (ablynx) is a trivalent nanobody in phase ii clinical trials for the treatment of rsv infections. it is one of the promising candidate biologic drugs in development for the prevention and/or treatment of rsv infections (see mazur et al. [65] for a full review). alx-0171 was originally isolated from a llama derived immune library as a monovalent domain prior to reformatting into a multivalent construct using genetic fusion. the vhh domain demonstrated high neutralising potency against rsv-a and rsv-b, and demonstrated superiority over palivizumab in blocking rsv replication [28] . camel vhhs or nanobodies have also been developed to treat diseases such as trypanosomiasis (african sleeping sickness) caused by parasites from the trypanosoma genus. trypanosoma brucei has evolved a range of sophisticated immune evasion mechanisms, which include repeatedly generating variations of the immunogenic variant-specific surface glycoprotein (vsg), efficient internalisation of antibody-vsg complexes from the surface by endocytosis and proteolysis of the immunoglobulin [7] . due to the dense packaging of vsg protein on the surface of trypanosoma, conventional antibodies are unable to access the more conserved vsg epitopes. high affinity nanobody clones directed towards isotype-specific and conserved epitope on the vsg protein coat, were isolated from an immunised camelid phage display vhh library. these nanobodies were found to be trypanolytic both in vitro and in vivo, which shows the potential of high affinity monovalent fragments to exert a cytotoxic effect on parasites even in the absence of fc domain. the authors have demonstrated the modes of action of these domains which appear to include: rapid arrest of cell mobility, blockage of endocytosis, flagellar swelling, collapse of mitochondrial membrane potential and atp exhaustion [96] . monoclonal antibodies and novel smaller antigen binding scaffolds presents an attractive option for the development of new therapies and molecular drug targets against a wide range of human diseases and also in diagnostics due to their specific[28] ity and flexibility. in this book chapter, we have been able to present the robustness of combining immunisation and phage display technology in raising antibodies specific to antigens relevant in a number of infectious diseases of public health interest, and also those antigens classified as bioweapons. although the limitations associated with animal immunisation, especially those relating to ethical issues surrounding animal welfare and the need to immunise for every antigen of interest may limit access to animal immunisation in the future. however it is the opinion of the authors that the debate regarding animal use for research purposes should be substantially assessed on the basis of a risk-benefit analysis and suitability. antibacterial properties of pseudomonas aeruginosa immunotype 1 lipopolysaccharidespecific monoclonal antibody (mab) in a murine thigh infection model: combined effects of mab and ceftazidime defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction single-domain llama antibodies as specific intracellular inhibitors of spvb, the actin adp-ribosylating toxin of salmonella typhimurium genetic and immunological comparison of anti-botulinum type a antibodies from immune and non-immune human phage libraries in vivo neutralization of botulinum neurotoxins serotype e with heavy-chain camelid antibodies (vhh) shark novel antigen receptors-the next generation of biologic therapeutics? in: pharmaceutical biotechnology antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite mining human antibody repertoires beyond natural antibodies: the power of in vitro display technologies hidden killers: human fungal infections identification of salmonella spi-2 secretion system components required for spvb-mediated cytotoxicity in macrophages and virulence in mice identification of an immunodominant abc transporter in methicillin-resistant staphylococcus aureus infections a large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals phage-displayed antibody fragments recognizing dengue 3 and dengue 4 viruses as tools for viral serotyping in sera from infected individuals helicobacter pyloriantigen-binding fragments expressed on the filamentous m13 phage prevent bacterial growth isolation of a nanomolar scfv inhibiting the endopeptidase activity of botulinum toxin a, by single-round panning of an immune phage-displayed library of macaque origin the isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep a high-affinity macaque antibody fab with human-like framework regions obtained from a small phage display immune library monoclonal immunoglobulin g1 directed against aspergillus fumigatus cell wall glycoprotein protects against experimental murine aspergillosis chimpanzee/human mabs to vaccinia virus b5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen novel chimpanzee/human monoclonal antibodies that neutralize anthrax lethal factor, and evidence for possible synergy with anti-protective antigen antibody potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding ) β-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae functional aspects of protein mono-adp-ribosylation antibody binding to cryptococcus neoformans impairs budding by altering capsular mechanical properties antibody responses against wild-type yellow fever virus and the 17d vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants generation and characterization of alx-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection analysis of the expressed heavy chain variable-region genes of macaca fascicularis and isolation of monoclonal antibodies specific for the ebola virus' soluble glycoprotein a human sars-cov neutralizing antibody against epitope on s2 protein potential of primate monoclonal antibodies to substitute for human antibodies: nucleotide sequence of chimpanzee fab fragments llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (hiv-1)-neutralizing properties and high affinity for hiv-1 gp120 isolation of single chain variable fragments against six esters of pyrethrins by subtractive phage display efficient production of chicken egg yolk antibodies against a conserved mammalian protein research and development of therapeutic mabs: an analysis based on pipeline projects passive intranasal monoclonal antibody prophylaxis against murine pneumocystis carinii pneumonia a new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks recombinant human antibody single chain variable fragments reactive with candida albicans surface antigens efficacy of combination immunotherapy of igm mab b6. 1 and amphotericin b against disseminated candidiasis protection against candidiasis by an immunoglobulin g3 (igg3) monoclonal antibody specific for the same mannotriose as an igm protective antibody isolation and expression of recombinant antibody fragments to the biological warfare pathogen brucella melitensis multiple-antigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens overview of antibody phage-display technology and its applications human-like antibodies neutralizing western equine encephalitis virus llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules nanobodies with in vitro neutralizing activity protect mice against h5n1 influenza virus infection nanobodies (vhh-based single variable domains) potently inhibit chemotaxis and hiv-1 replication and mobilize stem cells human monoclonal antibody production: current status and future prospects staphylococcus aureus β-hemolysin-neutralizing single-domain antibody isolated from phage display library of indian desert camel antibody interference with n-acyl homoserine lactone-mediated bacterial quorum sensing mammalian adpribosyltransferases and adp-ribosylhydrolases generation of a family-specific phage library of llama single chain antibody fragments that neutralize hiv-1 shark variable new antigen receptor biologics-a novel technology platform for therapeutic drug development the human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to aspergillus fumigatus membrane protein selection of a macaque fab with framework regions like those in humans, high affinity, and ability to neutralize the protective antigen (pa) of bacillus anthracis by binding to the segment of pa between residues 686 and 694 phase i evaluation of the safety and pharmacokinetics of murine-derived anticryptococcal antibody 18b7 in subjects with treated cryptococcal meningitis heavy chain-only antibodies and tetravalent bispecific antibody neutralizing staphylococcus aureus leukotoxins combination immunotherapy of mab b6. 1 with fluconazole augments therapeutic effect to disseminated candidiasis the best defense is a good offense-salmonella deploys an adpribosylating toxin the salmonella spvb virulence gene encodes an enzyme that adp-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells sheep monoclonal antibody fragments generated using a phage display system a steric antagonism of actin polymerization by a salmonella virulence protein hiv-1 neutralizing antibodies: understanding nature's pathways lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics detection of antibodies against the four subtypes of ebola virus in sera from any species using a novel antibody-phage indicator assay development and clinical applications of novel antibodies for prevention and treatment of respiratory syncytial virus infection the art of blocking adpribosyltransferases (arts): nanobodies as experimental and therapeutic tools to block mammalian and toxin arts production of recombinant scfv against p24 of human immunodeficiency virus type 1 by phage display technology a monoclonal antibody directed against a candida albicans cell wall mannoprotein exerts three anti-c. albicans activities nanobodies: natural singledomain antibodies antibody molecules which interact specifically with the active site or cleft of a target molecule a multicentre analysis of epidemiology of the nosocomial bloodstream infections in japanese university hospitals potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody markedly prolonged incubation period of hepatitis b in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis b surface antigen monoclonal antibodies in man that neutralized h3n2 influenza viruses were classified into three groups with distinct strain specificity antibacterial monoclonal antibodies: back to the future? high-sensitivity monoclonal antibodies specific for homoserine lactones protect mice from lethal pseudomonas aeruginosa infections lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea high-affinity, human antibody-like antibody fragment (single-chain variable fragment) neutralizing the lethal factor (lf) of bacillus anthracis by inhibiting protective antigen-lf complex formation novel camelid antibody fragments targeting recombinant nucleoprotein of araucaria hantavirus: a prototype for an early diagnosis of hantavirus pulmonary syndrome an anti-β-glucan monoclonal antibody inhibits growth and capsule formation of cryptococcus neoformans in vitro and exerts therapeutic, anticryptococcal activity in vivo antibodies to watch in 2016 generation of diversity in mammalian gut-associated lymphoid tissues: restricted v gene usage does not preclude complex v gene organization monoclonal antibody to fungal glucosylceramide protects mice against lethal cryptococcus neoformans infection fcrn: the neonatal fc receptor comes of age passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal cryptococcus neoformans infection identification of helicobacter pylori surface proteins by selective proteinase k digestion and antibody phage display directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries monoclonal antibody-based therapies for microbial diseases identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis e virus capsid protein four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus inhibition of fungal β-1, 3-glucan synthase and cell growth by hm-1 killer toxin singlechain anti-idiotypic antibodies llama-derived single-domain intrabodies inhibit secretion of hepatitis b virions in mice production, characterization and in vitro testing of hbcag-specific vhh intrabodies high affinity nanobodies against the trypanosome brucei vsg are potent trypanolytic agents that block endocytosis design of a peptibody consisting of the antimicrobial peptide dhvar5 and a llama variable heavy-chain antibody fragment competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response killing of caenorhabditis elegans by pseudomonas aeruginosa used to model mammalian bacterial pathogenesis heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells camelid single domain antibodies (vhhs) as neuronal cell intrabody binding agents and inhibitors of clostridium botulinum neurotoxin (bont) proteases generation of recombinant antibodies against toxins and viruses by phage display for diagnostics and therapy reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments international study of the prevalence and outcomes of infection in intensive care units human monoclonal antibodies against west nile virus induced by natural infection neutralize at a postattachment step biological half-life of ovine antibody in neonatal lambs and adult sheep following passive immunization human antibodies from immunized donors are protective against anthrax toxin in vivo making antibodies by phage display technology comparison of antibody production to human interleukin-6 (il-6) by sheep and chickens animal models in the evaluation of antimicrobial agents human combinatorial antibody libraries to hepatitis b surface antigen broadly cross-reactive hiv neutralizing human monoclonal antibody fab selected by sequential antigen panning of a phage display library human recombinant antimannan immunoglobulin g1 antibody confers resistance to hematogenously disseminated candidiasis in mice key: cord-282610-zim7nond authors: proal, amy; marshall, trevor title: myalgic encephalomyelitis/chronic fatigue syndrome in the era of the human microbiome: persistent pathogens drive chronic symptoms by interfering with host metabolism, gene expression, and immunity date: 2018-12-04 journal: front pediatr doi: 10.3389/fped.2018.00373 sha: doc_id: 282610 cord_uid: zim7nond the illness me/cfs has been repeatedly tied to infectious agents such as epstein barr virus. expanding research on the human microbiome now allows me/cfs-associated pathogens to be studied as interacting members of human microbiome communities. humans harbor these vast ecosystems of bacteria, viruses and fungi in nearly all tissue and blood. most well-studied inflammatory conditions are tied to dysbiosis or imbalance of the human microbiome. while gut microbiome dysbiosis has been identified in me/cfs, microbes and viruses outside the gut can also contribute to the illness. pathobionts, and their associated proteins/metabolites, often control human metabolism and gene expression in a manner that pushes the body toward a state of illness. intracellular pathogens, including many associated with me/cfs, drive microbiome dysbiosis by directly interfering with human transcription, translation, and dna repair processes. molecular mimicry between host and pathogen proteins/metabolites further complicates this interference. other human pathogens disable mitochondria or dysregulate host nervous system signaling. antibodies and/or clonal t cells identified in patients with me/cfs are likely activated in response to these persistent microbiome pathogens. different human pathogens have evolved similar survival mechanisms to disable the host immune response and host metabolic pathways. the metabolic dysfunction driven by these organisms can result in similar clusters of inflammatory symptoms. me/cfs may be driven by this pathogen-induced dysfunction, with the nature of dysbiosis and symptom presentation varying based on a patient's unique infectious and environmental history. under such conditions, patients would benefit from treatments that support the human immune system in an effort to reverse the infectious disease process. toward the end of a career spent studying persistent bacteria in chronic disease, microbiologist gerald domingue wrote, "it is unwise to dismiss the pathogenic capacities of any microbe in a patient with a mysterious disease" (1) . this thinking greatly applies to the illness me/cfs. me/cfs is characterized by neuroinflammation, severe fatigue, excessive post-exertional exhaustion, disturbed sleep, flu-like episodes, cognitive problems, sensory hypersensitivity, muscle and joint pain, headache, bowel symptoms, and severe impairment of daily functioning (2) . severely ill individuals are often wheelchair dependent, bedridden, and unable to perform basic tasks of work or daily living. the history of me/cfs strongly suggests that infectious agents play a central role in driving the disease process. these include early associations with epstein barr virus (ebv)/human herpes virus 6 (hhv6), the relapsing-remitting nature of me/cfs symptoms and antibodies/"autoantibodies" detected in patients with the disease (3, 4) . a number of me/cfs outbreaks have also been reported, in which numerous people in the same geographical location developed the illness simultaneously (2) . indeed, many me/cfs patients present with symptoms after suffering from a severe bacterial or viral infection. these infections often correlate with travel to a foreign country or exposure to pollutants or molds, suggesting that such pathogens take advantage of factors that compromise the host immune system. chronic inflammation is a hallmark of persistent infection. reports of cytokine activation in me/cfs clarify that the disease is associated with an inflammatory response (5) . montoya et al. (6) found me/cfs cytokine activation increased with disease severity, suggesting patients may struggle with a growing infectious burden over time. other me/cfs research teams have identified various forms of mitochondrial dysfunction in patients with the illness (7) . there are now dozens of well-characterized mechanisms by which bacteria and viruses dysregulate mitochondrial metabolism (8, 9) . many research teams have searched for well-characterized single pathogens in patients with me/cfs. these analyses often reveal elevated titers of igg and igm antibodies toward pathogens such as epstein barr virus, cytomegalovirus, parvovirus 19 and m. pneumonia (2, 3, 10) . several teams have also attempted to identify a single novel pathogen that might drive the entire me/cfs disease process. however, the discovery of the human microbiome now allows single microbes and viruses to be studied as members of complex communities. humans harbor these vast ecosystems of bacteria, viruses and fungi in nearly all tissue and blood (11) (12) (13) (14) . organisms in the microbiome continually interact with each other, and with the human genome, to regulate host metabolism and gene expression in both health and disease (15, 16) . a growing number of inflammatory disease states, including neurological conditions and cancers, are tied to dysbiosis or imbalance of these human microbiome communities (17) (18) (19) (20) . gut microbiome dysbiosis has been identified in me/cfs (21) . this dysbiosis is characterized by changes in microbe species composition and/or diversity. pathogens, or groups of pathogens, can promote dysbiosis by altering their gene expression in ways that promote virulence, immunosuppression and dysregulation of host genetic and metabolic pathways (22) . when seemingly disparate biomedical findings on me/cfs are interpreted through the lens of these microbiome-based paradigms and platforms, a cohesive picture of the me/cfs disease process emerges. me/cfs may be driven by pathogeninduced dysfunction, with resulting microbiome dysbiosis varying based on a patient's unique infectious and environmental history. under such conditions, patients would benefit from treatments that, like those now being developed for cancer, support the human immune system in an effort to reverse the inflammatory disease process. in the usa, me/cfs cases were first formally reported to the cdc in the 1980s (2) . at the time, human microbes were typically only detected with culture-based laboratory methods. then, around the year 2000, novel genome-based technologies began to revolutionize the field of microbiology (23, 24) . these technologies identify microbes based on their dna or rna signatures rather than their ability to grow in the laboratory. the results of these genome-based analyses were remarkable: vast communities of microbes were identified in the human body that had been missed by the older culture-based techniques. these extensive ecosystems of bacteria, viruses, fungi, and archaea are collectively known as the human microbiome (25) (26) (27) . today, so many novel microbes have been identified in homo sapiens that our human cells are equivalent to or even outnumbered by those of our microbial inhabitants (28) . the tens of millions of unique genes harbored by this microbiome dwarf the ∼20,500 genes in the human genome (12, 29) . for example, just one 2017 analysis of the human gut, skin, mouth, and vaginal microbiomes uncovered millions of previously unknown microbial genes (11) . this has forced science to redefine the human condition. humans are best described as holobionts, in which the microbial genomes and the human genome continually interact to regulate metabolism and immunity (15, 16) . early human microbiome studies characterized microbial ecosystems in the gut and on mucosal surfaces. however, the microbiome has now been shown to extend to nearly every human body site. these include the lungs, the bladder, the placenta, the testes, and the uterus [ (19, (30) (31) (32) (33) ]. jakobsen et al. (35) found that previously sterile implants removed from joints, bones, pacemakers, and skulls of symptom-free patients were colonized by a range of bacterial and fungal organisms. another study demonstrated the presence of novel tissue specific bacterial dna profiles in a variety of mouse organs including the brain, heart, liver, muscle and adipose tissue (36) . microbial communities also appear to persist in healthy human blood (37) (38) (39) . a dna virome was recently identified in healthy human blood (40) . another study reported both bacterial and fungal communities in the blood of healthy subjects. analysis of these organisms was performed by microbial resuscitation of blood culturing and microscopy in addition to next generation dna sequencing (41) . whittle et al. (42) recently characterized a human blood microbiome using a range of complementary molecular and classical molecular biology techniques. another study identified a larger amount of bacterial rdna in blood specimens from healthy individuals than in matched reagent controls (24) . kowarsky et al. (14) detected over 3,000 previously unidentified viruses, bacteria, and fungi in human blood samples obtained from immunocompromised patients. the study almost doubled the total number of anelloviruses found in humans. in order to classify many of these organisms the team was forced to add new branches to the "tree of life." they concluded that the newly discovered microbes "may prove to be the cause of acute or chronic diseases that, to date, have unknown etiology." the microbiome is inherited and evolves with the host. babies are seeded in the womb, during birth, and after birth by extensive microbiome communities in the placenta, the vaginal canal, and breast milk, among other body sites (43, 44) . microbes/pathogens acquired from the external environment are further incorporated into the microbiome over time. for example, once acquired, cytomegalovirus persists as a member of the microbiomewith a significant impact on host immunity. brodin et al. (45) found that the lifelong need for the body to control cmv causes approximately 10% of all t cells in cmv+ individuals to be directed against the virus. immune cells and associated microbes can travel between the human body and brain via several newly discovered pathways that bypass the classical blood-brain barrier (46) . benias et al. (47) documented a previously uncharacterized fluidfilled lattice of collagen bundles that appears to connect all human tissues. this human interstitium drains directly into the lymph nodes. two research teams have demonstrated the existence of a previously undiscovered meningeal lymphatic system (46, 48, 49) . the network's fluid pathways connect the cerebrospinal fluid and cervical lymph nodes directly to the brain. while great progress has been made in characterizing the human microbiome, our understanding of the body's microbial ecosystems is still in its infancy. metagenomic analyses of the microbiome in all body sites regularly identify species or strains of bacteria, archaea, fungi, and/or viruses not previously understood to persist in homo sapiens (13) . for example, just one study of the bladder microbiome identified 129 previously unidentified viruses in subjects' urine samples (50) . new strains of known microbes are also regularly identified. for example, as of august 2018, the ncbi database contains ∼1,833 lactobacillus genomes, with newly characterized lactobacillus genomes added on a weekly basis (51) . successful identification of these known or novel human organisms hinges on the careful choice of technology and methodology used for detection purposes (26) . while the majority of human microbiome studies center on bacteria, awareness of viruses, fungi, and archaea has increased in recent years (52) . for example, manuela et al. (53) found an almost 1:1 ratio of archaeal to bacterial 16s rrna genes in human appendix and nose samples. identification of this archaeome abundance and diversity required use of a very specific archaea-targeting methodology. viruses are the most abundant life forms on the planet and in the human body, but have been relatively hard to detect until very recently (54) . viruses that primarily infect bacteria, called bacteriophages, are particularly abundant in human microbiome communities. nguyen et al. (55) estimate that ∼31 billion bacteriophages traffic human tissue and blood on a daily basis. however, the human virome also harbors a plethora of human-associated viruses. newberry et al. (56) are correct to assert that this human virome is understudied in me/cfs. paez-espino et al. (57) at the joint genome institute have undertaken a large project called "uncovering the earth's virome." the project's goal is to better identify known and novel viruses in earth's ecosystems including the human body. viral identification requires the use of specific metagenomic tools, pipelines, and annotation platforms, with findings entered in the jgi img/vr database (57) . the project is progressing at such a rapid pace that viral diversity in img/vr has more than tripled since august 2016 (58), (figure 1) . nevertheless, the vast majority of the gene content (over 15 million genes in total) remains unknown or hypothetical. we must subsequently consider the possibility that asyet unidentified microorganisms may contribute to chronic inflammatory conditions like me/cfs. failure to do so would be akin to studying ∼2% of the animals in the rainforest and arriving at firm conclusions about the entire ecosystem based on that information alone. certain members of the human microbiome may also be difficult to detect based on their location and/or lifestyle. for example, chen et al. (59) found that elevated cytokine expression in response to hsv-infected peripheral nerve ganglia persisted even when the virus entered a latent, non-replicating state. while it is important to pursue identification of novel organisms in me/cfs blood and tissue, ample data already exists on better-studied components of the microbiome. microbial and viral survival strategies, virulence mechanisms and collective behaviors are also characterized by a high degree of functional redundancy (60, 61) . we must accept the complexity inherent to the human microbiome and further study these common mechanisms of survival and persistence. we must examine microbe and viral activity, microbe and viral gene expression, and the myriad ways in which the proteins and metabolites created by these organisms interact with the host immune system, the host genome, and each other. the genes of our microbial inhabitants greatly outnumber the ∼20,500 in the human genome. it follows that the majority of metabolites in homo sapiens are produced or modified by the microbiome. wikoff et al. (62) found a large effect of the gut microbiome on murine blood metabolites including antioxidants, toxins and amino acids. for example, production of the metabolite indole-3-propionic acid was completely dependent on the presence of gut microbes and could be established by colonization with the bacterium clostridium sporogenes. many microbes, viruses and their corresponding proteins/metabolites directly modulate the activity of host metabolic, immune, and neurological pathways. in other words, the human holobiont is controlled by the human genome, our microbial/viral genomes and their respective metabolites working in tandem. a growing number of studies provide examples of this metabolic overlap. while many such studies have been conducted in mice, their general trends carry over to humans. for example, me/cfs is associated with natural killer (nk) cell abnormalities, including reduced natural killer cell activity (63) . these findings must be interpreted to account for the fact that nk activity is modulated by the bacterial microbiome. one study found that bile acids modified by the gut microbiome impacted liver cell gene expression in a manner that controlled nk cell accumulation and anti-tumor activity (64) . similarly, a mixture of lactic acid bacteria from kefir increased the cytotoxicity of human nk khyg-1 cells to human chronic leukemia cells and colorectal tumor cells (65) . the microbiome and its metabolites also impact the activity of related immune cells. rothhammer et al. (66) found that tryptophan created by the gut microbiome interacted with the ahr receptor on microglia/astrocytes. subsequent changes in gene expression regulated communication between the two cell types. various forms of autonomic dysfunction are also common in me/cfs (67, 68) . it is subsequently important to consider that the microbiome may contribute to host blood pressure regulation. pluznick et al. (69) found that gut microbiomederived short chain fatty acids such as acetate and propionate travel to the kidneys and blood vessels. there they impacted activity of olfr78 and gpr41, two host receptors that control circulation and blood flow. microbial modulation of host pathways can also drive inflammatory disease processes. pathogens and their associated proteins/metabolites control human metabolism and gene expression in a manner that can push the holobiont toward a state of imbalance and illness. for example, rizzo et al. (70) found that human herpes viruses 6a/6b infected nk cells. this infection significantly modified expression of key host mirnas and transcription factors. mycobacterium leprae has been shown to alter human gene expression in a manner that allows it to hijack and reprogram adult schwann cells to a stem-like state (71) . in a murine model of diabetes, liu et al. (72) found that eltas, a protein created by s. aureus, prevented insulin from correctly binding its target receptor. this inhibited the phosphorylation of downstream signaling proteins and caused the mice in the study to develop impaired glucose tolerance. the ability of pathogens to interfere with host metabolism is tied to the dynamics of the communities in which they persist. like organisms in any ecosystem, human microbes constantly interact, both directly and indirectly. the proteins and metabolites they create are also in continual interplay. communities of microbes often exhibit synergistic interactions for improved nutrient acquisition, protection from host defenses, and survival in an inflammatory environment (73) . these include biofilm formation and cooperative signaling via quorum sensing peptides. humphries et al. (74) recently reported that biofilm bacteria can additionally communicate via ion channel-mediated electrical signaling. even viruses seldom act as single entities. diversity and equilibrium of the bacterial microbiome is regulated by bacteriophage predator-prey dynamics (75) . pfeiffer and virgin (27) found that enteric viral virulence is regulated by the activity of neighboring bacteria, fungi, and even helminths. these processes are called "transkingdom interactions." for example, human norovirus can bind carbohydrate histo-blood group antigens present on certain bacterial cells. this facilitates the ability of norovirus to infect human b cells (76) . interacting microbes may contribute to dysbiosis or imbalance of microbiome communities (77) . this dysbiosis is characterized by substantial shifts in community structure and diversity. in many cases, pathogens proliferate to inhabit niches once occupied by more innocuous microbes. most well-studied inflammatory disease states are tied to some form of microbiome dysbiosis. these include psoriatic arthritis, systemic lupus erythematosus, type 1 and 2 diabetes, parkinson's disease and a growing number of cancers (34, 78, 79) . the gut microbiome can initiate and promote colorectal cancer at all stages of tumorigenesis by acting as an inducer of dna damage, generating epigenetic changes, regulating cell growth, and modulating host immune responses (80) . the breast tissue microbiome of women with breast cancer has been shown to differ substantially in composition, virulence and diversity from that of healthy controls (81) . species composition of the bronchoalveolar microbiome shifted toward a more pathogenic state in patients with sarcoidosis (82) . alterations in the enteric virome were reported prior to disease onset in children susceptible to developing type 1 diabetes (83) . several research teams have tied me/cfs to bacterial gut microbiome dysbiosis. giloteaux et al. (21) found that gut microbiome bacterial diversity was decreased in me/cfs subjects compared to heathy controls. the team also noted increases in certain bacterial species associated with either pro-inflammatory or anti-inflammatory activity. nagy-szakal et al. (84) also analyzed the me/cfs gut microbiome. the study detected seven gut bacterial species whose relative abundance differed from that of control subjects and were strongly associated with me/cfs. while these findings are of interest, gut microbiome composition is additionally impacted by a host of environmental variables that cause large shifts in the region's microbial ecosystems. these include geographic location, food consumption, and even time of day (85) . many research teams studying inflammatory conditions have struggled to isolate and/or replicate disease-induced gut microbiome dysbiosis in the face of this "noise." for example, frémont et al. (86) found that intestinal microbiome species composition differed between patients with me/cfs and healthy controls. however, significant changes in intestinal microbiome composition were also identified between control subjects from norway and controls subjects from belgium. this variation was proposed to arise from differences in diet between the two cultures. studies of the me/cfs blood microbiome may be less subject to this environment-induced variability. furthermore, identification of microbial and/or viral communities in me/cfs blood would allow for a broader picture of possible infectious and inflammatory processes. for example, giloteaux et al. (21) found that me/cfs subjects had higher levels of bacterial lipopolysaccharides (lps), lps-binding proteins and soluble cd14 in blood. the team suggested that these inflammatory markers may indicate translocation of gut bacteria into the blood. however, the markers could also reflect the presence of bacteria in the blood itself. the blood microbiome can be characterized if the microbial dna/rna in samples is first separated from that derived from the human genome. olde loohuis et al. (87) used rna sequencing of reads from whole blood to analyze microbial communities in the blood of almost 200 patients with three neurological conditions: bipolar disorder, schizophrenia, and amyoytrophic lateral sclerosis. the team identified a wide range of bacterial and archaeal phyla in subjects with all three disease states. they observed increased microbial diversity in schizophrenia subjects compared to the two other groups, and replicated the finding in an independent dataset. stephen quake's cell-free dna shotgun sequencing technologies can also characterize bacterial communities in human blood, and can be additionally extended to identify viruses and fungi (14) . communities of microbes and viruses may also persist in me/cfs brain tissue. readhead et al. (88) recently detected a range of persistent viruses in the alzheimer's brain. these included herpesviruses, torque teno viruses, adenoviruses, and coronaviruses. the alzheimer's brain has also been shown to harbor bacterial and fungal communities (89, 90) . branton et al. (91) identified hundreds of bacteria and bacteriophage-derived samples in brain tissue removed from patients with epilepsy, and in brain samples obtained from hiv/aids patients after autopsy. in fact, studies of the virome provide significantly extended context on microbiome community dynamics and disease processes. this is because bacteriophages (phages) infect, and subsequently modulate the activity of the bacterial microbiome (75) . for example, duerkop et al. (92) characterized the intestinal virome in a model of t-cell-mediated murine colitis. the intestinal phage population changed in colitis, and transitioned from an ordered state to a stochastic dysbiosis. phage populations that expanded during colitis were frequently connected to bacterial hosts that benefit from or are linked to intestinal inflammation. tetz et al. (93) identified changes in the parkinson's gut bacteriophage community. these included shifts in the phage/bacteria ratio of bacteria known to produce dopamine. species-level studies of the microbiome are also greatly enhanced by analyses that provide further context on disease activity. this is an important consideration because, in theory, the microbiome of a patient with me/cfs could harbor the exact same microbial, viral and/or fungal species as that of a healthy subject. yet many of these organisms could be acting very differently in patients with the disease. species-level analyses of the me/cfs microbiome must subsequently be accompanied by studies that characterize microbial and viral gene expression and/or metabolism. since many of the proteins and metabolites in the human holobiont are microbial in origin, composition of the me/cfs proteome and metabolome change with microbiome species composition. proteome and metabolome analyses additionally reflect microbiome activity. this is because microbes and viruses frequently alter their gene expression in ways that cause them to express different proteins and metabolites over time. the human genome and related epigenetic changes also contribute to the metabolic diversity, although the high level of redundancy between human and microbial metabolites can make the origin of these associations hard to pinpoint. schutzer et al. (94) demonstrated that the me/cfs cerebrospinal proteome differs substantially from that of healthy controls (figure 2) . indeed, 738 of 2,783 identified proteins (26.5%) were unique to patients with me/cfs, providing strong evidence that me/cfs is indeed characterized by microbiome dysbiosis in tissue and blood. composition of the blood metabolome has also been shown to shift in me/cfs. one such study reported elevated plasma levels of choline, carnitine, and complex lipid metabolites in me/cfs patients (84) . another analysis demonstrated a sustained hypo-metabolic response in patients with the disease (95) . this dour-like state can be driven by exposure to adverse environmental conditions, as would be expected if the me/cfs immune system struggles to manage microbiome dysbiosis and associated pathogens. studies of the metabolome are often conducted in an attempt to identify disease-specific biomarkers. however, the metabolome can also be screened for metabolites that directly induce or suppress biological function in patients with a given illness. these studies help dissociate cause from effect and allow for possible modulation of disease phenotype. for example, johnson et al. (96) investigated the metabolic influence of microbial biofilms on colon cancer tissue and related cancer occurrence. they found that up-regulation of a biofilm-derived polyamide metabolite enhanced both biofilm formation and cancer growth. studies of microbiome activity must account for the fact that pathogens detected in patients with me/cfs are also regularly identified in healthy subjects or in patients with related inflammatory conditions. this is particularly true of studies that have searched for ebv, hhv6, cytomegalovirus, and other viruses able to be identified by pcr and/or antibody testing in me/cfs cohorts. this same trend is likely to hold for lessstudied or unidentified human microbes and viruses. while these "overlapping" results are often viewed as problematic, they make sense in light of research that clarifies how differently microbes act depending on host immune status, neighboring species, and a wide range of other variables. for example, susceptibility to hiv infection has been shown to vary based on the species composition and activity of the bacterial vaginal microbiome (97) . indeed, most human microbes are pathobionts: they can change their gene expression to act as pathogens under conditions of imbalance and immunosuppression (98) (99) (100) . for example, s. pneumoniae can persist as a highly adapted commensal or a virulent pathogen depending on its ability to evade the host immune response (101) . s. aureus causes a range of illnesses, from skin infections to life-threatening diseases such as endocarditis and meningitis. however, ∼30% of the healthy human population harbors s. aureus as a member of the normal nasal microbiome (102) . s. aureus virulence in these communities is determined by a number of factors, including the signaling and competitive strategies employed by neighboring microbes. the same is true of escherichia coli (e. coli), which also persists in numerous forms. one study found that "commensal" e. coli could evolve into virulent clones in fewer than 500 generations (103) . for most microbes, this evolution toward pathogenicity occurs via the acquisition of new genes or alteration of the current genome in a manner that induces gene loss (104) . for example, loss of muca increases the ability of pseudomonas aeruginosa to evade phagocytosis and resist pulmonary clearance (105) . it should also be noted that every microbial species represents many different strains, each of which may vary in the set of genes it encodes or in the copy number of such genes. this intra-species variation endows each strain with distinct functional capacities, including differences in virulence, motility, nutrient utilization, and drug resistance (106). greenblum et al. (106) identified extensive strainlevel copy-number variation across species in metagenomic samples obtained from patients with irritable bowel syndrome. this was especially true of genes tied to specific community functions, including functions related to community lifestyle. differences in gene copy-number also impacted adaptive functions linked to obesity. yao et al. (107) found that deletion of a single bacteriodes gene-and the bile salt hydrolase it expresses-altered host metabolism in a manner that impacted weight management, circadian rhythm and immunity. a microbe or a virus' location can also influence its activity. for example, much of the human population harbors hhv-6. however, in alzheimer's disease, hhv-6a was recently identified in human brain tissue (88) . there, its activity was shown capable of regulating host molecular, clinical, and neuropathological networks in a manner that can contribute to inflammation and neuronal loss. vanelzakker (108) has proposed that hhv-6 may also infect the vagus nerve in me/cfs, resulting in altered gutbrain axis signaling in patients with the illness. the human immune system also plays a central role in determining microbe and viral activity. a robust immune response is often capable of controlling pathogen virulence. however, if pathogens overcome the immune response, or the immune system is suppressed by medications, chemicals, or other environmental factors, pathobionts are more likely to alter their gene expression in a manner that promotes disease. pathobionts can subvert the human immune response by collectively altering their gene expression. yost et al. (100) performed an excellent gene ontology (go) enrichment analysis of the oral microbiome during periodontal progression. over the two-month study period, changes in the metagenome of non-progressing sites were minor. however, active sites that progressed to periodontitis were characterized by numerous functional genomic signatures. in fact, the team reported a complete rearrangement at the metagenome level between baseline sites that progressed to periodontitis and those that did not. go terms associated with processes including peptidoglycan biosynthesis and potassium transport were highly enriched at baseline sites that later progressed to periodontitis. genes controlling ciliary motility and crispr-associated proteins were also active during initial stages of disease progression. at the breakdown point, active sites expressed genes associated with ferrous iron transport and response to oxidative stress. progression to periodontitis was also correlated with increased expression of putative virulence factors associated with a range of bacterial species. mycoplasma, bacteriophage, and eukaryotic viral activity were higher in progressing sites compared to baseline samples. the team concluded that periodontitis progression is driven by the whole oral microbial community and not just a few select pathogens. in effect, under conditions of increasing inflammation and imbalance, the entire oral community appeared to act together as a pathogen. indeed, groups of bacteria not generally considered pathogens upregulated a large number of the putative virulence factors in active sites. these included veillonella parvula, a microbe almost always associated with dental health. community-wide shifts in microbiome virulence are often driven by dominant pathogens-organisms that become established as central components of the microbiome while suppressing commensal growth and activity (109) . in other cases, keystone pathogens promote inflammation even when present as quantitatively minor members of the microbiome. for example, p. gingivalis often comprises just 0.01% of periodontal biofilms, yet drives destructive changes in host-microbe interplay by profoundly impairing the innate immune response (110) . pathogens able to persist inside the cells of the immune system are uniquely positioned to drive inflammatory disease (111) . indeed, most well-characterized pathogens, including many connected to me/cfs, are capable of intracellular persistence (112) . by surviving in this fashion, they can directly interfere with human transcription, translation, and dna repair processes (figure 3) . pathogens in the cell cytoplasm may further dysregulate the epigenetic environment (113) . for example, upon infecting a macrophage, mycobacterium tuberculosis alters the expression of 463 human genes (114) . h. pylori infection predisposes to genomic instability and dna damage, including double strand breaks (115) . ebv infection of b cells can also promote persistent damage to human dna (116) . the thousands of metabolites and proteins expressed by intracellular pathogens also interact with the host genome, further modifying human gene expression in a manner that promotes disease. even bacterial quorum sensing peptides can dysregulate human pathway activity. wynendaele et al. (117) found that quorum sensing molecules created by gramnegative bacteria altered human gene expression in a manner that promoted in vitro angiogeneisis, tumor growth, and neovascularization in colon cancer. intracellular pathogens can also travel between cells via recently characterized tunneling nanotubules (tnts) (118, 119) . these cytoplasmic extensions of dendritic cells, glial cells and related human cells allow for the intracellular transfer of micrornas, messenger rnas, prions, viruses, and even whole organelles such as mitochondria (120, 121) . for example, hivinduced tunneling nanotubule formation appears to mediate approximately half of hiv virus spread among monocytederived macrophages (119). dysfunction driven by intracellular infection is compounded by the fact that microbial proteins and metabolites are often identical or similar in structure to those created by their human hosts. the molecular mimicry or sequence homology between these proteins and metabolites makes it increasingly difficult for the human holobiont to recognize "foreign" from "self." for example, altindis et al. (122) found that viruses carry sequences with significant homology to human insulin-like growth factors (vilps). these vilps can bind human and murine igf-1 receptors in vitro, resulting in autophosphorylation and downstream signaling. e. coli harbors a large, diverse network of proteins that actively promote endogenous dna damage in cells (123) . however, at least 280 of these dna-damaging proteins have human homologs that also promote dna damage and mutagenesis in the human host. indeed, redundancy between human and microbial metabolites, proteins, and pathways is so great that the potential for molecular mimicry to contribute to host immune and metabolic dysfunction is semi-infinite. for example, viral vesicles and human extracellular vesicles (evs) share considerable structural and functional similarity (124) . these similarities are so extensive that it is difficult to distinguish evs from (noninfectious) viruses. many pathogens employ common survival mechanisms to persist in host cells, tissue and blood. the metabolic dysfunction driven by these different microbes and viruses can result in similar clusters of human inflammatory symptoms. the ability of various pathogens to dysregulate activity of the vitamin d nuclear receptor (vdr) is an excellent example of how different microbes can drive similar disease processes. the vdr regulates expression of hundreds of human genes, many of which regulate inflammatory and malignant processes (125, 126) . the receptor also controls signaling of tlr2 and several families of antimicrobial peptides including cathelicidin (ll-37) (127) . pathogens capable of slowing vdr activity can subsequently facilitate their survival by slowing the innate immune response. pathogens frequently linked to me/cfs or inflammatory disease have evolved to survive in this fashion. vdr activity is downregulated as much as 20 times in epstein barr virus-infected lymphoblastoid cell lines (128) (figure 4) . hiv, m. tuberculosis cytomegalovirus, borrelia burgdorferi and mycobacterium leprae additionally dysregulate vdr activity to various degrees (114, (129) (130) (131) (132) . the fungus aspergillus fumigatus secretes a gliotoxin that significantly downregulates vdr expression (133) . because disabling the innate immune system via the vdr pathway is such a logical survival mechanism, other uncharacterized bacteria, viruses or fungi may have also evolved to dysregulate receptor activity. it follows that me/cfs patients with similar symptoms may not always test positive for the same mix of pathogens and/or communities of pathobionts. similarly, composition of the me/cfs microbiome, proteome and metabolome should be expected to differ somewhat from study to study. instead of worrying about these inconsistencies, the me/cfs research community should strive to better characterize even more common mechanisms of pathogen survival and persistence. the ability of pathogens to disable the host immune response extends far beyond the vdr pathway. pushalkar et al. (134) identified a distinct and abundant pancreatic microbiome associated with progressive pancreatic cancer. this dysbiotic microbiome drove oncogenesis by suppressing macrophage differentiation and t cell activity. another study found that candida albicans's transition from extracellular to intracellular pathogen was accompanied by a coordinated, time-dependent shift in gene expression for both host and fungus (135) . these gene expression changes led to a gradual decline in proinflammatory cytokine activation by the host immune system. these findings shed light on a recent me/cfs study. hornig et al. (5) reported distinct alterations in plasma immune signatures-including prominent activation of both pro-and anti-inflammatory cytokines-early in the course of me/cfs. however, these alterations were not observed in subjects with a longer duration of illness. there are a number of explanations for hornig's observations. me/cfs-associated pathogens may gradually alter their gene activity to persist in more latent, intracellular forms less recognized by the host immune system. or, in early-stage me/cfs, the immune system may actively attempt to target a growing infectious burden. over time however, pathogens in the microbiome may disable the immune response to a point where "immune exhaustion" occurs (136, 137) . immunopathology and cytokine production would subsequently drop. the resulting disease state could be compared to a garden, in which healthy plants become progressively stifled by others, such as kudzu vine. many research teams are studying how "acute" pathogens (i.e., well-characterized agents associated with known infectious diseases) can cause chronic symptoms by persisting in latent forms. these include zika, borrelia burgdorferi, influenza, and other well-characterized viruses and bacteria (138) . zika was shown capable of persisting in the cerebrospinal fluid (csf) and lymph nodes of infected rhesus monkeys for months after the virus had been cleared from mucosal secretions, peripheral blood and urine (139) . viral persistence in both the lymph nodes and csf was correlated with upregulation of genes involved in pro-inflammatory and anti-apoptotic pathways. tens of thousands of ebola survivors have developed chronic symptoms months or years after initial infection (140) . these "post-ebola syndrome" symptoms include extreme fatigue, severe pain, eye problems, and a host of neurological issues. while the virus is hard to identify in the blood of such patients, ebola has been detected in men's semen years after "recovery" (141) . another study found that, up to a month after initial infection, influenza viruses regulated the long-term expression of inflammatory and neuron/glia-specific genes in mice (142) . this was associated with chronic neuroinflammation characterized by an increase in the number of activated microglia and impairment of spatial memory formation. persistent measles virus is associated with conditions such as paget's disease, multiple sclerosis and crohn's disease (143, 144) . measles virus rna has been detected in blood, respiratory secretions, urine, and lymphoid tissue for weeks to months after clearance of the "acute" infection (145) . doi et al. (146) found that, in vitro, measles virus persistence correlated with viral transition from a lytic to non-lytic mode that allowed the virus to evade the host innate immune response. hundreds of studies demonstrate that acute bacterial pathogens can transition into latent forms that lack a classical cell wall (1) . these persistent variants are referred to as l-form bacteria. antibiotic use can induce persistent l-form growth. in fact, researchers create l-forms by deliberately culturing classical bacteria in conjunction with the beta-lactam antibiotics (147) . one microarray analysis of l-form growth revealed upregulation of genes shared in common with persister cells and biofilms (148) . l-form bacteria have been implicated in dozens of chronic conditions including rheumatoid arthritis, multiple sclerosis and sarcoidosis (1, 149, 150) . a better understanding of these chronic sequelae would greatly benefit the me/cfs community, since a similar chronic progression of symptoms is frequently documented in me/cfs. at different points in history, me/cfs has been called "post-polio syndrome, " "chronic mononucleosis syndrome" and "post-viral syndrome" due to the fact that chronic symptoms are often noted after acute infection with polio virus, epstein barr virus, influenza or a range of other pathogens (151) (152) (153) . chia et al. (154) found that patients with acute enterovirus infection went on to develop a multitude of chronic symptoms consistent with an me/cfs diagnosis. years after the initial infection, enterovirus protein and rna were still present in these patients' stomach biopsies. it is clear that novel methodologies may be required to best identify pathogens in their latent forms. pathogens that persist inside human immune cells and associated tunneling nanotubuoles have been particularly hard to detect. when persistent zika virus was identified in rhesus monkeys, zikaspecific antibodies were not detected in the csf, despite prolonged and robust responses in peripheral blood. this suggested an additional mechanism for viral persistence in certain "anotomic sanctuaries." modern living has also increased the likelihood that "acute" infections may generate chronic sequelae. before the advent of antibiotics, steroid medications and childhood vaccines, almost half of all children under the age of five died from acute infectious disease (45) . today, most individuals in first-world countries survive repeated acute infections over the course of decades. while certain dominant or keystone pathogens may be reliably identified in patients with me/cfs, composition of the me/cfs microbiome will likely differ between patients. even in hiv/aids, where an easily detected virus dysregulates immunity, disease symptoms reflect a mix of those driven by hiv, and those driven by "co-infectious" agents able to take advantage of the immunocompromised host (155) . no two patients with hiv/aids are expected to harbor the same mix of these additional persistent bacteria, fungi, and viruses. the same is true of most cancers, an increasing number of which are now tied to severe microbiome dysbiosis (34) . it is widely accepted that no two cancers are alike, and any tumor-associated microbiome is expected to differ somewhat among individual study subjects (156) . this same pattern, in which unique infectious history impacts symptom presentation may also occur in me/cfs. a recent study by brodin et al. (45) demonstrated the profound impact of infectious history on host immunity. the team performed a systems-level analysis of 210 healthy twins between the ages of 8 and 82. they measured 204 immune parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated, and 58% almost completely determined, by non-heritable environmental influences. many of these parameters became more variable with age, emphasizing the cumulative influence of environmental exposure. the team also calculated how acquisition of just one chronic pathogen-cytomegalovirus (cmv)-conditions the immune response. identical twins discordant for cmv infection showed greatly reduced correlations for many immune cell frequencies, cell signaling responses, and cytokine concentrations. in general, the influence of cmv was so broad that it affected 119 of the 204 measurements dispersed throughout the immune network. these and related findings led brodin and team to conclude that the immune response is "very much shaped by the environment and most likely by the many different microbes an individual encounters in their lifetime." the above suggests that me/cfs may be driven by a process we have termed "successive infection." during successive infection, an "acute" infection or "initial immunosuppressive event" dysregulates the host immune system. this makes it easier for microbes to subvert the immune response by acting as polymicrobial entities. pathobionts alter their gene expression to better promote community-wide virulence. infected human cells fail to correctly express human metabolites in the presence of pathogen-generated proteins, metabolites, and enzymes. dysfunction driven by molecular mimicry increases. certain pathogens may infect mitochondria or central nervous system tissue. intracellular pathogens slow the human immune response, causing the host to more easily acquire other infectious agents. this creates a snowball effect in which the microbiome becomes increasingly dysbiotic as the strength of the immune response weakens over time. eventually, the human host may present with symptoms characteristic of me/cfs or a related inflammatory diagnosis. the unique symptoms any one patient develops are expected to vary based on the species, strain, virulence, and location of the pathogens driving dysbiosis, along with the myriad ways in which the metabolites and proteins created by these organisms cause dysfunction by interacting with those of the host. early childhood infections may predispose to dysbiosis at a later date (157) . for example, measles depletes host b and t lymphocytes for up to 2-3 years after initial infection. this immunosuppression can reset previously acquired immunity and renders the host more susceptible to other pathogens (158) . in other cases, a toxic environmental exposure, infection during surgery or the difficulty of enduring a traumatic event may weaken the immune response to a point where previously subclinical infections become active. me/cfs outbreaks, in which numerous patients developed the illness at relatively the same time, may well represent this phenomenon at work. the successive infectious process may even begin in the womb. depending on the health of the parent, founding microbiome communities in the placenta, vagina and breast milk may already be dysbiotic. cabrera-rubio et al. (159) found that the breast milk microbiome of obese mothers tended to contain a different and less diverse bacterial community then that of normal-weight mothers. pathogens in the placental microbiome can alter methylation of human dna in a manner that may negatively impact the later life disease of premature babies (160) . many aspects of modern living can additionally drive successive infection. antibiotic use disrupts the ecology of the human microbiome (161) . antibiotic resistance genes from farm animals and produce are regularly transferred into the human food supply (162) . electromagnetic radiation has been shown to lower immunity (163) . the immunosuppressive biologics and supplements often prescribed for inflammatory disease further allow pathobionts in the microbiome to proliferate. for example, diaz et al. (164) found that the salivary bacteriome of patients taking immunosuppressive biologics was more permissive to the growth of oral opportunistic pathogens. modern society also exposes the average person to pathogens our recent ancestors were unlikely to encounter. international airports harbor pathogens from across the globe (165) . food products, and the microbes they contain, are frequently imported from foreign destinations (166) . one study found a range of opportunistic pathogens enriched in showerhead biofilms (167) . numerous pathogens were identified in commonly smoked cigarettes (168) . many such pathogens are capable of persisting in human microbiome ecosystems, where the host may lack immunity toward their presence. me/cfs is a spectrum disorder with a diagnostic criterion that includes a range of physical and neurological symptoms (2) . if successive infection contributes to me/cfs, this variability in symptom presentation is expected. furthermore, factoring "unique infectious history" into the disease process helps explain why patients with me/cfs often suffer from a multitude of symptoms not included in the official diagnostic criteria. because patients with me/cfs suffer from such diverse symptoms, it has been argued that they should be grouped into separately studied "subgroups" (169) . in some cases this makes sense. for example, studies that distinguish earlystage/late-stage me/cfs patients may further elucidate how the immune response is modified by the microbiome over time. however, if successive infection contributes to me/cfs, future research should also focus on better understanding the common pathogenesis shared by all subjects. indeed, successive infection may contribute to other conditions tied to microbiome dysbiosis, persistent infection, and adverse environmental exposure. me/cfs should be studied in concert with these other conditions, which include gulf war syndrome, ehlers-danlos syndrome, chronic lyme disease, and fibromyalgia among others (170) (171) (172) (173) (174) . the high levels of comorbidity and symptom overlap between patients with me/cfs and these related inflammatory diagnoses strengthens this assumption. a number of autoantibodies have been detected in patients with me/cfs (4). this has led some research teams to postulate that me/cfs should be regarded as an "autoimmune" disorder (175) . however, the classical "theory of autoimmunity" is in the process of being re-evaluated (78, 176) . increasing evidence suggests that "autoantibodies" are actually created in response to chronic, persistent microbiome pathogens. under such conditions molecular mimicry or structural homology between pathogen and host proteins can result in "collateral damage" toward human tissue. for example, vojdani et al. (177) found that 25 different alzheimer's-associated pathogens or their molecules could react with antibodies against amyloid beta via molecular mimicry or the binding of bacterial toxins to amyloid beta. a growing body of research documents "autoantibody" production in response to a range of bacterial, viral and fungal pathogens/pathobionts. these pathogens are not shortterm "triggers" but persist as members of complex microbiome communities. for example, "autoantibody" production was recently tied to microbiome pathobiont enterococcus gallinarum. manfredo et al. (178) detected e. galinarum in the mesenteric veins, lymph nodes, spleens and livers of mice made genetically prone to autoimmunity. in these mice, the bacterium initiated the production of "autoantibodies, " inflammation and activated t cells. however, this "autoantibody" production stopped when e. gallinarum's growth was suppressed with the antibiotic vancomycin or with an intramuscular vaccine. in addition, e. gallinarum-specific dna was recovered from liver biopsies of human autoimmune patients, and co-cultures with human hepatocytes replicated the murine findings. indeed, many pathogens can drive the activated or clonal t cell expansion often associated with "autoimmune" conditions. for example, tuffs et al. (179) found that s. aureus endotoxins triggered uncontrolled activation of t cells. this led to a proinflammatory cytokine storm that accounted for both t cell activation and related inflammation. microbes and their metabolites control bidirectional signaling between the gut and the brain via pathways collectively known as the gut-brain axis (180) . the gut-brain axis involves various afferent and efferent pathways including the vagus nerve, with signaling impacting neural, endocrine, and immune processes. the gut's enteric nervous system contains over 100 million neurons-more than in either the peripheral nervous system or spinal cord (181) . it follows that gut microbiome dysbiosis may modulate brain activity. for example, sampson et al. (79) transplanted fecal samples from patients with parkinson's disease into germ-free mice. these mice, and not controls, exhibited physical symptoms associated with parkinson's. however, there is also growing evidence that humans may harbor a brain microbiome. researchers at harvard university are characterizing this ecosystem as part of the ongoing "brain microbiome project." in what marks a major paradigm shift, multiple research teams have shown that both amyloid beta and prion protein (prp) are potent antimicrobial peptides (182, 183) . amyloid beta exhibited antimicrobial activity against a range of common microorganisms with a potency equivalent to, and in some cases greater than, cathelicidin (ll-37) (184). these pathogens included s. typhimurium, candida albicans and, more recently, a number of herpesviruses capable of persisting in the alzheimer's brain (88, 185) . the findings strongly suggest that amyloid beta and prp are not useless byproducts of abnormal brain catabolism. rather, they appear to form a mediated response of the innate immune system toward infectious agents in brain tissue (186) . a number of brain abnormalities have been reported in patents with me/cfs (187) . these findings must be interpreted in light of these novel infection-based paradigms and in concert with emerging data on the brain microbiome. in a study of aortic aneurysms, gottlieb et al. (188) reported that bak1 snp-containing alleles were detected in aortic tissue but not in blood samples from the same patients. more recently ursini et al. (189) found that schizophrenia gene risk loci that interact with early-life complications are highly expressed in the placenta. however, these loci were differentially expressed in placentas from women who suffered complications during pregnancy. they were also differentially upregulated in placentae from male compared with female offspring. these and related findings strongly suggest that the environment can select for human genome activity. for example, harley et al. (190) found that in ebv infected cells, ebna2 and its transcription factors modulated the activity of human genes associated with risk for multiple sclerosis, rheumatoid arthritis, type 1 diabetes and other conditions. in fact, nearly half of systemic lupus erythematosus risk loci were occupied by ebna2 and co-clustering human transcription factors. studies of the human genome must also account for the full extent of microbial dna and rna in human tissue and blood. if a genomic assembler fails to account for this contamination, chances of a false positive single nucleotide polymorphism (snp) increase significantly during analysis (191) . contamination with even a small amount of microbial dna/rna-just one or two base pairs of difference-is enough to cause significant statistical errors in this fashion. if me/cfs is driven by successive infection, treatments that support or activate the human immune system could improve microbiome health by allowing patients to better target persistent pathogens. development of such therapies should be a priority for the me/cfs research community. however, most immunostimulative treatments that target pathogens are characterized by immunopathology-a cascade of reactions in which inflammation, cytokine release and endotoxin release are generated as part of the immune system's response to microbial death (192) (193) (194) . the death of intracellular pathogens is particularly difficult for the host to manage, as the body must deal with debris generated from apoptosis. inflammation is also generated in response to bacterial cell wall components including the endotoxin lipopolysaccharide of gram-negative strains (192) . luckily, immunopathology-generated symptoms are generally temporary in nature, and tend to subside as an increasing number of pathogens are eradicated. temporary immunopathology resulting from antimicrobial treatment has been documented for over a century, with symptoms varying depending on the nature of targeted pathogens. first referred to as the jarisch-herxheimer reaction, the phenomenon was originally observed during treatment of syphilis with mercury and penicillin (195, 196) . the jarisch-herxheimer reaction/immunopathology has since been noted in a broad spectrum of chronic inflammatory conditions including tuberculosis and brucellosis (197) . short-term immunopathology is also a central feature of acute infection. if a patient develops the flu, inflammatory symptoms increase as the immune system releases cytokines and chemokines in response to the infecting virus. hiv/aids patients undergo a form of immunopathology called immune reconstitution inflammatory syndrome (iris) following treatment with combination antiretroviral therapy (art) (198) . iris occurs as art enables the host immune system to better target pathogens acquired during previous periods of hiv-driven immunosuppression. a range of well-characterized pathogens have been linked to iris including the herpesviruses and m. tuberculosis (193) . however, the inflammatory reaction is also noted in culture-negative patients, suggesting iris may also involve novel or uncharacterized pathogens (198) . over the past decade, in concert with our clinical collaborators, we developed an immunostimulative therapy used to treat patients with a range of chronic inflammatory conditions (200) . treatment centers on the use of a putative vdr agonist in the form of olmesartan medoxomil, with the goal of reactivating components of innate immunity under vdr control. in 2013, we published a series of case histories demonstrating improvement in me/cfs patients administered this treatment (199) . however, all me/cfs subjects administered the therapy experienced immunopathology and associated inflammatory symptom increases that lasted for many years. as a general trend, patients administered the treatment during earlier stages of disease experienced less immunopathology, emphasizing the need for immunostimulative therapies to be used in a predictive and even preventative fashion. while some me/cfs physicians may feel uneasy about the suffering induced by immunopathology, other research communities have become accustomed to treatments that cause temporary discomfort. novel cancer immunotherapies also generate immunopathology by activating patient t cells. this allows the immune system to better target malignant tumors (194, 201) . the resulting "cytokine release syndrome" leads to profound, temporary, symptom increases. however, these increased symptoms are considered acceptable, as patients who endure the reaction are more likely to enter a state of remission. since many forms of cancer are tied to severe microbiome dysbiosis, at least part of the "cytokine release syndrome" may result from the death of persistent pathogens. the history of me/cfs strongly suggests that infectious agents play a central role in driving the disease process. however, the discovery of the human microbiome has revolutionized the manner in which persistent infection and chronic inflammation are understood and studied. humans harbor extensive microbiome communities of bacteria, viruses, and fungi in nearly all tissue and blood. the hundreds of millions of unique genes harbored by this microbiome dwarf the ∼20,500 in the human genome. humans are best described as holobionts, in which these microbial genomes and the human genome continually interact to regulate host gene expression, metabolism and immunity. many inflammatory disease states, including neurological conditions and cancers, are tied to dysbiosis or imbalance of human microbiome communities in various body sites. while gut microbiome dysbiosis has already been identified in me/cfs, distinct microbial and viral communities may additionally persist in me/cfs blood and brain tissue. possible identification of these microbiomes should be a priority for the me/cfs research community, but analysis requires the use of very specific technologies and methodologies. pathogens and their associated proteins/metabolites control human metabolism and gene expression in a manner that can push the human holobiont toward a state of illness. studies of microbiome dysbiosis in me/cfs must consider this microbe and viral activity. most human microbes can alter their gene expression to act as pathogens under conditions of imbalance and immunosuppression. this pathobiont behavior is further determined by the activity and virulence of neighboring microbes. patients with me/cfs may harbor many of the same microbes and viruses as heathy individuals, yet these pathobionts may act with increased virulence in patients with the illness. intracellular pathogens, including several associated with me/cfs, have been shown to directly interfere with human transcription, translation, and dna repair processes. molecular mimicry between host and pathogen proteins/metabolites further exacerbates this interference. interacting microbes can also drive disease by changing their collective gene expression. other pathogens disable mitochondria, or may infect central nervous system tissue in ways that dysregulate signaling via the gutbrain axis. antibodies and/or clonal t cells identified in patients with me/cfs are likely activated in response to many of these persistent microbiome pathogens. different pathogens have evolved similar survival mechanisms to disable the host immune response and/or host metabolic pathways. the metabolic dysfunction driven by these different microbes can result in similar clusters of inflammatory symptoms. me/cfs may be driven by this pathogen-induced dysfunction, with the nature of dysbiosis and symptom presentation varying based on a patient's unique infectious and environmental history. an initial infection or environmental exposure weakens the host immune system. this makes it easier for pathobionts to subvert the immune response and interfere with host gene expression and metabolism. a snowball effect begins, in which the microbiome becomes increasingly dysbiotic as the strength of the immune response weakens over time. the unique symptoms any one me/cfs patient develops are expected to vary depending on the location, species, strain and virulence of the pathogens driving this dysbiosis. thus, while certain dominant or keystone pathogens may be identified in me/cfs, composition of the me/cfs microbiome, metabolome, and proteome should be expected to differ somewhat among individual patients. these common mechanisms suggest that me/cfs is best studied in concert with other chronic conditions tied to microbiome dysbiosis, persistent infection and adverse environmental exposure. these include fibromyalgia and gulf war syndrome, but also conditions like post-ebola syndrome in which severe chronic symptoms develop after infection with an "acute" infectious agent that is able to persist in latent forms. treatments that support or activate the human immune system could allow me/cfs patients to improve microbiome health by better targeting pathogens over time. like the novel immunotherapies being developed for cancers, these immunostimulative therapies would be expected to generate temporary immunopathology. institutional reviewers hesitant to approve immunopathology-based therapies should consider that me/cfs quality of life is typically very low, with patients demonstrating a substantial increase in mortality from suicide (202) . it often takes patients years to receive a diagnosis of me/cfs. this delay wastes a valuable period during which the immune system is most responsive to immunostimulatory treatment. patients treated during earlier stage disease are also less likely to experience severe or long-lasting immunopathology. this suggests that immunostimulative therapies should be administered in a predictive and even preventative fashion. in addition, interventions or treatments that might help patients better manage the byproducts of immunopathology (bacterial lps etc.) 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inflammatory syndrome in patients starting antiretroviral therapy for hiv infection: a systematic review and meta-analysis immunostimulation in the treatment for chronic fatigue syndrome/myalgic encephalomyelitis immunostimulation in the era of the metagenome cytokine release syndrome mortality of people with chronic fatigue syndrome: a retrospective cohort study in england and wales from the south london and maudsley nhs foundation trust biomedical research centre (slam brc) clinical record interactive search (cris) register the authors would like to acknowledge michael eason kirkpatrick and janet raty for their help with graphic design. they also thank sara nicole azevedo for her help with technical editing of the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2018 proal and marshall. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-301935-0qjo94ty authors: varma, ratna; soleas, john p.; waddell, thomas k.; karoubi, golnaz; mcguigan, alison p. title: current strategies and opportunities to manufacture cells for modeling human lungs date: 2020-08-22 journal: adv drug deliv rev doi: 10.1016/j.addr.2020.08.005 sha: doc_id: 301935 cord_uid: 0qjo94ty chronic lung diseases remain major healthcare burdens, for which the only curative treatment is lung transplantation. in vitro human models are promising platforms for identifying and testing novel compounds to potentially decrease this burden. directed differentiation of pluripotent stem cells is an important strategy to generate lung cells to create such models. current lung directed differentiation protocols are limited as they do not 1) recapitulate the diversity of respiratory epithelium, 2) generate consistent or sufficient cell numbers for drug discovery platforms, and 3) establish the histologic tissue-level organization critical for modeling lung function. in this review, we describe how lung development has formed the basis for directed differentiation protocols, and discuss the utility of available protocols for lung epithelial cell generation and drug development. we further highlight tissue engineering strategies for manipulating biophysical signals during directed differentiation such that future protocols can recapitulate both chemical and physical cues present during lung development. end-stage lung disease is the third leading cause of morbidity and mortality worldwide events that occur, that differentiation models attempt to mimic, and highlight how human lung embryology has served as the blueprint to create the common pathway of lung directed differentiation protocols. we then discuss the evolution of directed differentiation protocols to find opportunities for creating specific populations of airway and lung epithelia through targeted manipulation of key signaling pathways in 2d and 3d models. we further describe how these models have been used to recapitulate different airway and lung diseases. finally, we discuss how tissue engineering and biophysical cues using biomaterials can be utilized during lung directed differentiation to mimic patterning cues present in development to augment current differentiation protocols. directed differentiation protocols have been designed to mimic in vivo human lung development [39] . indeed, in vitro models of lung development have provided unique insight into human lung development [40] . as human lung development has been described at great length in earlier reviews, [41, 42] , we provide a brief overview as follows (schematically represented in figure 1 ). during early embryogenesis (at 14 days post fertilization), a process called gastrulation begins with the appearance of a structure called primitive streak, through which cells migrate to form the primary embryonic germ layers (definitive endoderm, mesoderm, and ectoderm) [43] [44] [45] . definitive endoderm expands, thereby forming the primitive gut tube comprised of three endodermal regions: foregut, midgut, and hindgut [46, 47] . this is when lung development begins, at approximately four weeks into embryonic life, with the outgrowth of foregut endoderm [48, 49] and continues through eight years of post-natal life [50] . there are five stages to lung development: 1. embryonic (weeks 4-7): the future lung buds emerge from the ventral side of the primitive foregut endoderm into the surrounding mesenchyme and develop into embryonic lung buds with early trachea and bronchi [51] [52] [53] [54] [55] . 3 . canalicular (weeks [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] : development of the respiratory, or gas-exchanging, airways is initiated, primitive alveoli form, and the future distal epithelium begins to thin as distal epithelial markers are expressed [41, 57] . 4 . saccular (weeks [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] : emergence of sac-shaped distal airways, which develop crests with muscle and elastin to create indentations. these distal airways extend to form alveoli by 29 weeks [58] . the developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange and further differentiation of alveolar epithelial cells (aec) i and ii takes place. septation leads to an increase in surface area of the gas exchanging portion of the developing lung and prepares the fetus to breath air during this stage [50, 59] . during the embryonic period, early lung is genetically defined by the expression of transcription factor nk2 homeobox 1 (nkx2.1) and sry-box 2 (sox2) [60] [61] [62] . during human lung development, it has been found that the lung buds and branches given off during the pseudoglandular period are mostly sox2 + sox9 + [17, 62, 63] . both sox2 and sox9 are individual markers of the early proximal or distal lineage, respectively [60, [64] [65] [66] . over the course of the canalicular and saccular periods of development (weeks , these double positive populations downregulate one sox protein and maintain expression of the other as these cells mature towards proximal or distal lineages [62] . the proximal airway (closer to the mouth) is comprised of a pseudostratified columnar epithelium that is responsible for the conducting airway function: debris and pathogen removal (ciliated cells), mucus production (goblet cells), prevention of airway inflammation (club cells), and humidification of air as it passes through to the distal lung compartment [67] [68] [69] . the squamous distal epithelium, j o u r n a l p r e -p r o o f composed of alveolar epithelial cells (aec i and ii), facilitates the respiratory function of the lung as air in the epithelial compartment is brought into close apposition to blood from the pulmonary vasculature; it also secretes surfactants, which play an immunologic role and decrease the surface tension present at the air-liquid interface, thereby preventing alveolar collapse [70] . in humans, a number of cell types are found in the proximal airway, each identified with specific markers ( table 1) [71, 72] . one mechanism by which lung epithelia begin to mature is based on chemokine secretions from the surrounding mesenchyme and the developing heart field which are well reviewed here [73] . key players including fibroblast growth factors (fgfs) [64, [74] [75] [76] [77] [78] [79] [80] , wnts [81] [82] [83] [84] , and bone morphogenetic proteins (bmps) [85] [86] [87] [88] [89] [90] are known to induce the differentiation of early lung progenitors in a controlled manner. for example, in mouse, it has been found that fgf10 plays a role in bud outgrowth [77] and drives lung progenitors towards a distal fate [78, 79] through canonical wnt signaling [64, 81, 91] . proximal epithelia develop because they are located further away from distally located fgf reservoirs in the mesenchyme, in a mechanism that appears dependent on concentration gradients [64] . bmp4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventral (back to front) and proximodistal (top to bottom) patterning in the nascent lung [88] . bmp4 is also present at high levels in distal bud tips and epithelia including aec ii cells [88, 89] , however, its inhibition promotes a proximal fate and, along with bmp2 inhibition, ciliated cell development [87, 88, 90] . j o u r n a l p r e -p r o o f while the cell fate of early proximal and distal lineages is directed through chemical signals, the lung epithelium itself undergoes marked changes in architecture, a process known as branching morphogenesis [79, 92] . from the simple tube of the anterior foregut endoderm to the complex tubular structure of the adult, a highly stereotyped mechanism of branching morphogenesis facilitates the outgrowth, division, placement, and structure of lung airway [42] . branching morphogenesis of the lung is driven by three simple and iteratively used processes: domain branching, planar and orthogonal bifurcation [93] . the first form of branching is domain branching: along a primary branch, buds form in a linear and sequential fashion, from proximal to distal. the next form of branching is planar bifurcation, in which the tip of the forming tube bifurcates to create two new tips, which subsequently elongate and bifurcate again, creating four tips. the last process of branching is known as orthogonal bifurcation. in this process, the initial planar bifurcation is followed by a rotation around the planar axis which creates two new tips through bifurcation. a critical gene in this process, sprouty, has been found to attenuate erk1/2 signaling, thereby altering the orientation of cell division and future tube elongation [94] . other critical genes and regulatory networks associated with fgf signaling also contribute to controlling the periodicity of the branched network [95] . although elements such as domain specification, bifurcation, rotation and branch generation remain largely undetermined [93, 96] , new technologies involving high-resolution live imaging, tension sensing, and force-mapping are opening paths to further explore and explain the branching morphogenesis phenomenon [97] . the early structure of the lung gives rise to a striking architectural separation of future sox2 + proximal lineages and sox9 + distal lineages, at least in mice [98] . the diameter of tube generated during branching morphogenesis in the pseudoglandular and canalicular stages has a small degree of variance within each stage as measured from electron micrograph sources of fetal human tissue [99] . this suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on the developing epithelia. once the basic organ structure has formed, the lung continues to be exposed to mechanical cues as it continues to mature. in several cases, these cues have been shown to be essential for correct organ function. in utero, the fetal lung is a secretory organ that only converts to an absorptive one, to prepare for breathing after birth, through a change in the activity of j o u r n a l p r e -p r o o f chloride and sodium channels late in development. fetal lung secretions result in a static fluid pressure of around 2.5 cmh 2 o in the developing terminal sacs of the fetus, which propels branching morphogenesis outwards into the developing thoracic cavity [100, 101] . lack of amniotic fluid in the developing lung alters the expression of distal epithelial markers and consequently results in the creation of smaller than normal lungs (pulmonary hypoplasia) [102] , highlighting the importance of this mechanical pressure during lung development. in addition, cyclic strain is generated from fetal breathing movements (fbm) in utero that prime the airway for use after birth. fbm are detectable from the tenth week of pregnancy and begin as infrequent and erratic activity with long quiescent periods. as development continues, these quiescent periods decrease and sustained periods of fetal breathing occur. these breathing movements vary with the fetal sleep cycle and can be chemically tuned [57] , and alter the volume of terminal sacs by around 5% [100, 103] , again highlighting the importance of mechanical signals influencing lung development. finally, a novel fgf10/fgfr2-dependent tensional mechanism has been shown by which distal epithelial cells in the lung accumulate motor proteins at the apex of the cell, thereby becoming resistant to compression from increasing fluid pressure within the tube lumen. cells under this tension are more likely to become aec ii cells, while those under compression become aec i cells [102] . interestingly, while the above examples highlight the importance of specific mechanical signals in the growth, development, and differentiation of the lung, psc directed differentiation protocols of the lung are primarily based on mimicking the sequential chemical changes that occur during lung development. 26, 62, 63] , reveals that sox2 + sox9 + progenitors are common in the developing lung buds and that branch tips of the pseudoglandular staged lung give rise to both proximal and distal epithelia [63] . moreover, specific protein markers have been found to differ in both timing and location of expression between human and mouse models: pro-spc in mouse is expressed early and throughout the developing mouse epithelium [117, 118] , while in human, pro-spc is rarely detected early in development and is only robustly found later in distal epithelia [63] . these examples highlight that, while there are similarities, development and patterning of mouse and human lungs is different, and these differences require human models to be fully appreciated. human psc protocols have generally followed the same differentiation chronology as that of mouse directed differentiation, wherein definitive endoderm, anterior foregut endoderm, progenitors. in all cases, these lung progenitors are then either sorted or directly guided towards proximal or distal progeny in 2d or 3d culture systems. ideally, products of directed differentiation protocols should mimic the cell proportions present in human airways and lungs (table 1) , however current protocols have not progressed that far. while these protocols continue to be refined, the percentage of select cell populations generated from these protocols have been summarized in table 2 . protocols to create proximal lung epithelia have focused on the production of the four major cells types present: ciliated, goblet, club, and basal cells (see table 1 for a summary of markers for each cell type). motivation for creating proximal epithelia in the field has primarily been to develop patient-specific cystic fibrosis (cf) models [11, 24, 27] and/or to produce epithelia with multi-ciliated cell populations for protocol validation [30, 33] . a shift towards human psc-derived cf models has been critical as mouse models do not accurately represent j o u r n a l p r e -p r o o f journal pre-proof cf disease progression and phenotypes seen in humans [128] [129] [130] . as such, the first evidence of human psc proximalization using cf patient-derived pscs was shown by mou non-lung hepatic cells (serpina1 + , sox9 + , apoa2 + , afp + ) [27] . multi-ciliated cells were only observed within spheroids when notch signaling was inhibited, at the expense of downregulated scgb1a1 expression, or when spheroid cells were further cultured in ali conditions. other mature epithelial populations including club and goblet cells were not assessed post ali culture. it is unclear whether 2d or 3d culture systems resulted in more representative proximal populations, although it is worth noting that the 3d spheroids could be manipulated to produce a variety of proximal epithelia ranging from progenitor to differentiated populations. the most recent approach, described by de overall, the aforementioned studies differed drastically from each other with regards to the timing and chemical modulation of each phase of differentiation towards proximal epithelia, and consequently produced variable results. while it is evident that 3d culture augments maturation, no protocol to date has been able to efficiently produce all functional epithelial populations present in the airway in proportions representative of those in vivo. furthermore, these studies have not thoroughly elucidated the mechanisms of proximal patterning. barring the application of fgf18 [11] (known to enhance proximal programming [133] ), protocols have adopted growth factors based on trial and error without understanding why, for example, fgf10 signaling (which is known to favor distal lung development) promotes production of proximal progenitors [11, 27, 30, 33, 37, 119] . as such, the quest for obtaining mature airway progenitors, such as ngfr + cells, comes at the cost of elongated protocol lengths, heterogenous maturation levels of resulting populations, and missed opportunities for understanding why these populations do not result in a histologically appropriate epithelium [29] . it is apparent that the timing of signaling molecule delivery as well as the competence of subjected cell populations to respond to a given signaling molecule are of extreme importance. the spatiotemporal dynamics of cell signaling are non-linear, are more complex in vivo, and are not fully appreciated in the latter stages of current directed differentiation protocols. this may explain the incongruence amongst different protocols, primarily those assessing the effects of gsk3î² and wnt signaling [27, 29, 30] , all of which targeted populations at non-comparable protocol stages. therefore, a deeper analysis is required to appropriately explain and mimic these dynamics in vitro. furthermore, recreating these spatiotemporal signaling patterns during directed differentiation protocols may potentially require repurposing molecular delivery tools from other fields such as drug delivery and tissue engineering [134] [135] [136] . interestingly, most existing protocols have been skewed towards generating multi-ciliated cells at the expense of goblet and club cells by subjecting airway progenitors to notch inhibition, which is known to decrease goblet cell populations [137, 138] . goblet cells, in j o u r n a l p r e -p r o o f addition to club cells, have recently been discovered as a source for generating multi-ciliated cells in primary airway epithelia [139] . club cells play a key role in epithelial injury, wherein they de-differentiate into basal cells in the absence of basal cells such that they can give rise to ciliated and club cell populations to repair a denuded epithelium [140] . therefore, in the future it will be critical to identify protocols to create psc-derived cultures containing these cell types, and not just multi-ciliated cells, in order to fully capture the dynamics of airway injury and repair for drug screening. overall, based on current progress, the konishi et al. and mccauley et al. protocols are considered the most relevant for generating functional airway epithelia. the alveolar space in the distal lung is comprised of two epithelial cell types: aec i and aec ii (see table 1 for specific markers of each cell type meanwhile, progenitor-like sox9 + spc + and sox9 + hopx + clusters were prominently present with minimal mature spc + (5%) and hopx + (4%) populations. further refinement of this protocol bifurcated proximal "human lung" and distal "bud tip progenitor" organoid development by culturing foregut spheroids in fgf10 with 1% serum or fgf7, chir, and ra in serum-free media, respectively [121] . after 65 days of culture, the "human lung" organoids expressed p63, foxj1, and mesenchymal markers with no sign of mature epithelial features; some spc and hopx staining was also observed. only after an 8 week-long in vivo implantation did mature ciliated actub + cells appear. "bud tip progenitor" organoids also contained heterogenous muc5ac + , hopx + , spb + , and spc + cells after 120 days. however, when seeded into naphthalene-injured mouse airways, they gave rise to actub + and muc5ac + cells. in general, this protocol diverged to produced lung organoids with heterogeneous populations of either predominantly proximal or distal epithelia, which required prolonged culture or in vivo implantation for maturation (limited in this case). a key aspect of the "human lung" organoids was their inclusion of a mesenchymal population to study epithelialmesenchymal crosstalk during lung development. implantation to promote maturation (incomplete in this case). all described directed differentiation protocols for distal epithelia utilized a 3d culture approach in some format, however only those that established lung specification in 2d culture prior to a 3d transition demonstrated promising results. these protocols do not completely depend on spontaneous organoid assembly, are highly responsive to fine tuning with morphogens, and can therefore provide better insight into the cellular responses in lung development to generate therapeutic strategies accordingly. although the "human lung" organoid and "lung bud progenitor" organoid-based protocols may be useful for studying complex cellular interactions during disease progression or furthermore, the employment of notch inhibition during the "preconditioning" phase may have played a role in promoting both aec i and aec ii populations, and therefore the aforementioned signaling pathways need to be collectively assessed. inspiration can be sought from a recent study which described a computational modeling approach, based on single cell rna sequencing, that predicted the optimal time point for chir withdrawal for maintaining a nkx2.1 + spc + lung fate; its findings were further supported by empirical studies [35] . employment of such techniques will prove essential for understanding fate choice and developing customized target lung populations. the study of airway and lung diseases is limited by animal models as they do not recapitulate human disease phenotypes and progression adequately. for example, existing mouse j o u r n a l p r e -p r o o f models of cystic fibrosis (cf) vary greatly in their ability to represent relevant organ pathologies and are deficient in developing spontaneous lung disease observed in humans [141] [142] [143] . similarly, pulmonary fibrosis is most commonly studied in the bleomycin-induced lung fibrosis mouse model, which results in faster disease progression, eventual resolution of disease phenotype over time, and is obscured by the wide range of bleomycin doses administered for induction of injury [144] . studies exploiting psc-derived lung culture models to explore the effect of drugs in human lung cells are therefore beginning to emerge. culture models of proximal airway epithelia have been applied for drug discovery primarily in the context of cf. cf is an autosomal recessive genetic disorder caused by mutations in the epithelial chloride channel gene, cftr, which consequently leads to accumulation of excess mucous and compromised mucociliary clearance [145] , affecting multiple organs. cf phenotypes have been studied widely in primary or psc-derived intestinal [146] [147] [148] , rectal [149] [150] [151] , pancreatic [152] , and airway models [11, 27, 33, 153, 154] . in the context of airway models, ali culture has been the gold-standard for studying primary airway epithelia derived from cf patients. in concordance with this method, wong et al. and developed an il-11 antibody which reversed late-stage lung fibrosis by significantly decreasing ecm deposition in an animal model [162] . evaluation of this il-11 antibody using j o u r n a l p r e -p r o o f psc-derived lung organoid models can provide better insight into their applicability for human disease. distal lung organoids have also been applied to model respiratory viral infections caused by respiratory syncytial virus (rsv), human parainfluenza virus type 3 (hpiv3) [163, 164] , as well as the measles virus (mev). chen and colleagues infected lbos with rsv, resulting in characteristic luminal shedding of epithelia [26] , which leads to small airway obstruction and consequent bronchiolitis in clinical settings [165] . interestingly, sach et al. demonstrated that prior incubation with palivizumab (an antibody that prevents rsv from fusing with cells) prevented rvs from replicating in primary airway organoids [154] , which would be valuable to assess in psc-derived lung or airway organoids. meanwhile, hpiv3 infected aec ii in lbos and temporally reached peak infection similar to that in primary alveolar epithelia [164] , confirming clinical data. hpiv3 infection did not result in either epithelial shedding or syncytium formation in the lbos as did rsv [26] and mev [164] infections, respectively, again confirming clinical phenotypes. this showed the ability of lung organoid models to not only demonstrate virus-specific infection, but also to recapitulate phenotypes observed in the clinic. another condition modeled by alveolar or lung organoids is spb deficiency, a lethal neonatal autosomal recessive disease which necessitates lung transplantation for patient survival. while there has been great progress in the establishment and maturation of lung epithelia from psc populations, a number of limitations have emerged that will require optimization and augmentation of current protocols to create better developmental and disease models, and specific cell populations: a. lack of control over which populations are produced -understanding or recapitulation of signaling pathways beyond proximodistal patterning is currently limited, as the ratio of aec ii versus aec i cells; or club cells versus goblet cells cannot be reliably predicted. furthermore, while development of reporter lines and identification of surface markers for sorting have [32, 34] helped the advancement of distal lung protocols (for aec ii cells specifically) in the last few years [28, 36] , such techniques are limited in current proximal airway protocols [168] . f. minimal recapitulation of physiological conditions -current alveolar organoids are beginning to represent relevant cell types, however, they are embedded in matrigel and grown in submerged culture. they, therefore, fail to provide an ali environment, which is critical to in vivo functionality. as in proximal protocols, cell products often need to be dissociated and regrown on transwells, which allow ali culture by exposing cells to media basally and air apically, for further assessment. g. high cost -associated with the growth factor and small molecules required for chemically directed stem cell differentiation, and the expertise required to reliably create lung epithelia with these protocols. the use of commercially available 2d based endoderm differentiation kits have greatly decreased the level of expertise required to achieve this early stage of differentiation. based on current advances, we have made recommendations for directed differentiation protocols that generate proximal and distal epithelia in figure 2 . the timing of chemical signals present during lung development has been well mimicked in current differentiation protocols. the lung, however, develops in response to chemical signals within a highly dynamic mechanical environment of cyclic strain, pressure and a complex branching tubular architecture [169] . indeed, it is well established that mechanical cues can impact progenitor cell fate [102, [170] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] and emerging evidence suggests that the mechanical environment can be manipulated to produce predictable fate choices in stem and progenitor cells [181] [182] [183] [184] . for example, the importance of biophysical manipulation associated with tissue has been exploited only to a limited extent to augment and guide directed differentiation protocols to address some of the above limitations [170, [185] [186] [187] . organoid cultures, for instance, allow self-assembly of tissue-like structures and enable further maturation of proximal and distal epithelia [12, 26, 28, 119] . in this section of the review, we highlight tools from tissue engineering that have been used to manipulate tissue structure and the resulting biophysical signals experienced during stem cell differentiation in both 2d and 3d (figure 3 and table 3 ). furthermore, we explore the opportunity to utilize such tools to engineer mechanical signaling as a strategy to augment and refine existing chemical differentiation protocols. note, we do not consider the use of simply culturing differentiating cells on substrates or in 3d hydrogels with variable mechanical stiffness but point the reader to excellent reviews on this subject [188, 189] . micropatterning of the culture surface is one strategy that has been used to manipulate the physical organization of 2d stem cell colonies and the resulting mechanical environment individual cells experience within the cell sheet. micropatterning entails deposition of j o u r n a l p r e -p r o o f extracellular matrix (ecm) protein islands with highly specific shapes and sizes on non-adhesive surfaces, via micro-contact printing (âµcp) [190] [191] [192] [193] [194] or soft lithography [195] [196] [197] . individual cells or cell populations are thereby restricted to the area of the surface where the adhesive protein islands are present. the shape of the island, therefore, geometrically constrains the shape of single and groups of cells in 2d, which determines the pattern of adhesive attachments between the cells and the underlying surface, and hence the mechanical state of the cells [198] . chen and colleagues presented early evidence that geometric constraint affects cell fate by demonstrating that cell growth and apoptosis are directly related to ecm pattern size through its control of cell spreading [194, 199] . not only is the size of the ecm pattern important, but also the shape it holds, specifically in relation to its aspect ratio and subcellular curvature. as shown by kilian et al., despite the presence of equipotential differentiation signals, osteogenic differentiation of heterogeneous mesenchymal stem cells (mscs) was promoted by increasing the ecm pattern aspect ratio at the single cell level [181] . further, in a pentagon-shaped design, the curvature of the lines connecting vertices was varied from convex to concave and was shown to guide cell differentiation choice from adipogenic to osteogenic, respectively, by manipulating subcellular myosin ii polarization, tension, and integrin localization. evidently, such micropatterned islands not only exert control over the growth and survival of cells, but also enable manipulation of cell differentiation through changes in intracellular tension. for example, by probing tension at the cell-boundary interface through confined 2d geometries, lee et al. found that patterned melanoma cancer cells occupying larger arc angles, or smaller magnitudes of curvature, expressed higher cancer stem cell markers [195] . furthermore, these markers were preferentially found at the edge of the micropattern, a consequence of perimeter tension acting through the p38-map kinase pathway. interestingly, once removed from the defined geometric environment, the cells lost their activated cancer phenotype. these tools could provide an excellent platform for subtly manipulating self-organization of psc populations to understand and influence their differentiation. while not in the context of augmenting directed differentiation specifically, the use of micropatterning has been applied to explore pluripotency and fate choice during early development. based on a previous finding that human psc differentiation is dependent on colony size [200] , nazareth et al. developed a high throughput âµcp platform, with optimized j o u r n a l p r e -p r o o f colony size, and probed early cell fates (pluripotent, neuroectoderm, primitive streak, and extraembryonic) in response to different media conditions and developmental factors [190] . such micropatterned surfaces were also used by warmflash and colleagues to investigate embryonic germ layer patterning of human pscs [201] . they found that bmp4 treatment results in spatially segregated regions that delineate ectoderm, primitive streak and trophoblast-like tissues within the patterned colonies. this pattern was shown to be mediated by the colony edge, as opposed to colony size, with bmp4 signalling progressively being restricted to the edge of the colony. these findings were further confirmed by tewary et al., who explained this effect to be caused by the emergence of a phosphorylated smad1 gradient. the establishment of this gradient is initially controlled via inhibition by noggin, followed by a restriction of bmp4 sensitivity to the edge of the colony due to re-localization of bmp receptors throughout the rest of the colony [202] . such micropatterned platforms have also been used to map fate choices made during mouse [203] and human [204] gastrulation events, and therefore are a powerful tool for elucidating fate choice during lung development. it is not clear however, how the mechanical state of the cells within these micropatterned islands impacts chemical cue secretion, and hence the local gradients of chemical signals that result in patterning of cell fate in these studies. another method to manipulate the physical organization and biophysical cues in a differentiating cell sheet is through substrate texture [205] . cellular behaviours including proliferation, adhesion, and differentiation have been linked to underlying substrate topographical cues [205] [206] [207] [208] [209] [210] . these cues are recognized by cellular protrusions called filopodia and lamellipodia, through integrin receptors and focal adhesions [211] [212] [213] [214] , which in turn dynamically modify their shape and exert protrusive forces [215] [216] [217] [218] [219] . in this section, we will provide key examples of substrate topographies, as well as grooves, based on their relevance for lung epithelial organization. these topographies can be microfabricated using various techniques, such as etching, photolithography, soft lithography, and stereolithography, that are scalable, precise, and provide high fidelity [220] . j o u r n a l p r e -p r o o f stem cell fate choices have been shown to respond to topographical features [221, 222] . for example, viswanathan et al. assessed the ability of different topographies to mimic sinusoidal epidermal undulation to induce in vivo-like biophysical cues. their screened undulating topography created î²1 integrin patterning that is reminiscent of the human dermis and more differentiated cells were found localized to the troughs of their pattern in a highly repeatable fashion. these findings suggest that replicating the physical organization of the dermal microenvironment promotes tissue-level organization and alters the positioning of the epidermal stem cells towards the in vivo state [223] . this group further applied a screening platform called topochip [224] , that incorporated features of varying sizes, roundedness, and distribution density, to assess human psc proliferation and pluripotency in the absence of ecm coatings [225] . topographies that ranked high in the screen not only supported psc proliferation, but also allowed maintenance of oct4 and sox2-expressing pluripotent colonies. in conjunction with computational modeling, this platform was able to predict topographical features conducive to maintaining psc pluripotency, thus demonstrating great promise for exploring how to use tissue organization patterning to control cell fate. application of this platform for probing keratinocyte differentiation revealed that differentiation is linked to changes in cell morphology, which is influenced by substrate topography, and mediated by rho kinase activity [226] . based on these studies, it is evident that application of biophysical cues alone can impact differentiation. high-throughput technologies like topochip could be used in the future to understand and mimic cell fates of proximal or distal lung epithelia. towards this goal of recapitulating the physiological morphology of distal lung alveoli, we recently developed largersized topographical features, specifically hemispherical cavities, that enabled seeding of multiple cells and further allowed maintenance of primary aec i and aec ii cells [227] . the ability of this platform to promote psc differentiation towards these cell types has yet to be explored, however. grooved topographical cues specifically, through their ability to modulate cytoskeletal alignment and cellular shape, have also demonstrated great promise in guiding cell fate [228, j o u r n a l p r e -p r o o f 229]. in the context of neural differentiation, ankam and colleagues generated a multiarchitectural chip (marc), that incorporated a range of isotropic and anisotropic topographies at both micro and nano scales, to differentiate pscs towards neural progeny without the use of embryoid bodies. anisotropic nanoscale grooves (250 nm) promoted neuronal differentiation with cell alignment and elongation, and isotropic pillars enhanced astrocyte differentiation with cellular branching within 7 days of culture. meanwhile, conventional culture protocols were unable to induce these populations without additional culture steps and/or prolonged culture up to 30 days [230] . neural differentiation on nanogrooves was attributed to actomyosin contractility via vinculin-associated focal adhesions [231] . marc further enabled investigation of nuclear morphology and histone methylation [232] , thereby exemplifying that such platforms can allow exploration of the mechanism of biophysical cues translating to dna modulation during differentiation. the influence of groove topography on differentiation has been highlighted in other contexts as well. abagnale et al. developed a micro-grooved chip, incorporating systematic variation of groove widths and ridges, to study msc differentiation towards adipogenic and osteogenic progeny. while wider ridges led to higher adipogenic differentiation with formation of fat droplets, thinner ridges enhanced osteogenic differentiation with calcium phosphate precipitation [233] . interestingly, groove width had minimal impact on favouring differentiation towards either lineage. the ridge-mediated differentiation effect was associated with cell morphology and focal adhesion formation wherein wider ridges resulted in rounder cell morphology with many large focal adhesions, as compared to thinner ridges leading to cellular elongation with fewer and smaller sized focal adhesions. nano-scale groove topography was also shown to alter the spatial conformation of psc colonies by elongating them, consequently affecting cell fate [234] . this effect was particularly potent at the colony edges and controlled by separate and differential localization of yap and taz during psc maintenance and differentiation. in general, grooved topography can provide great insights into psc fate choice and potentially expedite differentiation protocols. the marc platform illustrates the importance of combining biophysical and biochemical cues, especially as exposure to topography induced a higher yield of functional and mature progeny within a short time frame as compared to flat j o u r n a l p r e -p r o o f substrates or standard directed differentiation protocols [230, 235] . this is of extreme importance as current directed differentiation protocols for airway and lung epithelia require longer than 60 days of culture to achieve functional cell types [12, 27, 28, 30, 36, 119] . although the inclusion of topography has not been investigated for promoting lung differentiation, we have explored grooved substrates for aligning airway epithelia during differentiation to achieve coordinated unidirectional ciliary beating [236] . our unpublished data demonstrates that while primary human basal-derived epithelia lose their alignment on grooved topography over time, epithelia generated from human psc-derived airway progenitors maintain their alignment throughout differentiation in ali culture. while 2d cues have enabled the community to clearly demonstrate the capability of biophysical cues to manipulate cell fate, 2d approaches are limited in their biological applicability as most physiologic physical and mechanical cues occur in 3d environments [237] . [186] . further, these microchambers were validated for use in developmental drug toxicity screening, through which they were able to represent thalidomide embryopathy by exhibiting diminished contractility, beating frequency, and size of cardiac chambers. geometric constraint was also applied to cerebral organoids by lancaster and colleagues to minimize variability associated with neural induction efficiency. this entailed addition of a j o u r n a l p r e -p r o o f physical cue in the form of polymer microfibres, around which the organoid self-organized [170] . in conjunction with an established chemical protocol, these microfibres served to pattern developing organoids leading to recognizable neuronal features including a cortical plate, radial units, along with organized radial neuronal migration in a reproducible manner. this addition of a simple physical cue substantially increased the patterning and organization of brain organoids compared to those derived from protocols only relying on biochemical cues. another strategy to control cell and tissue geometry in 3d is the use of micromoulding to create defined mechanical microenvironments which in turn alter cell and tissue level organization and differentiation. this approach was first developed by nelson et al. using 3d collagen moulds to study branching morphogenesis of mammary epithelial cells [238, 239] . seeded mammary epithelia conformed to the 3d architectures, forming hollow tubules, and demonstrated predictable branching patterns according to mould geometry and presence of inhibitory morphogens. this technique was further used to understand the mechanism of cellular rearrangement in mammary ducts [240] and exhibit that mechanical stress gradients control the pattern of branching morphogenesis [241] . inspired by this method and its applicability for studying lung branching morphogenesis, we developed tubular constructs of physiologically relevant diameters to guide self-assembly of lung progenitors [242] . using this approach, we demonstrated that specification of these bipotent sox2 + sox9 + lung progenitors was dependent on geometry, wherein tubes of 100 âµm diameter led to a distal sox9 + fate, while 400 âµm diameter tubes remained in a sox2 + sox9 + lung progenitor state. the mechanism of this effect was dependent on canonical wnt signalling, and due to differences in cellular tension induced by patterning the progenitor cells into a 3d tube structure. while the addition of mechanical cues influences fate choice, its role in inducing cell functionality, especially at the organoid level, needs to be elucidated. currently, there is scant evidence of lung directed differentiation being manipulated in a 3d context [183, 242, 243] . beyond our exploration of patterning early lung fates through micromoulding, dye et al. have recently applied tissue engineering techniques during lung organoid formation. in their case, foregut endoderm spheroids cultured on highly degradable synthetic polymers demonstrated enhanced ability to differentiate into proximal airway epithelia after in vivo implantation [183] . evidently, the field of lung directed differentiation is in its nascent stages for using biophysical in the future, human psc-derived lung tissue models have the potential to enable exploration of infection, disease and regeneration mechanisms of action to impact drug discovery and drug development, and further inform patient-specific drug selection. while lung models remain in their infancy, the investment necessary to translate such models into practical use is worthwhile given that they offer a number of key advantages over primary cells or mouse models. firstly, psc-derived platforms enable modeling of human disease. another major advantage of psc-derived cells, specifically in the context of lung, is the potential to directly associate specific patient genetics and cell phenotypes with clinical conditions, as is underway in the field of lung cancer [244, 245] . this will avoid complications associated with prior exposure of primary cell donors to a plethora of environmental (such as smoking) and pharmaceutical stimuli. furthermore, establishing models specifically from psc sources potentially enables generation of the large number of cells and cell types necessary for personalized disease modeling. a number of challenges exist however, to translate these models into widespread use for drug discovery and development. one major challenge in the field, highlighted in this review, is the standardization of robust cell manufacturing protocols. lung epithelial models require not only large cell numbers, but also the correct proportion of cell types. additionally, for a variety of functional read-outs, these cell types must be appropriately spatially organized. therefore, standardized protocols are needed to both manufacture lung cells and assemble these cells into reproducible and clinically representative "lung tissues" at the scales required for screening. this will be essential to enable the generation of in vitro lung test tissues with sufficiently low batch-to-batch and within-batch variation for screening with high reproducibility. towards this challenge, methods will continue to emerge to control psc differentiation into the different proximal and distal lung cell types. for example, in this review, we have highlighted the emerging evidence that an opportunity exists to further improve differentiation control and disease modeling by mimicking mechanical cues experienced during development. beyond the strategies described in previous sections, this concept could be expanded further in the future to mimic additional aspects of lung development. for example, the developing lung is exposed to variations in oxygenation [246, 247] , which is known to impact cell fate choices [248] [249] [250] , therefore the use of optimized oxygenation levels could be an attractive and easily scalable strategy to further refine directed differentiation culture protocols. the developing lung is also subject to various other physical cues at the organ-scale including 1) pressure from amniotic fluid, which serves to expand the nascent alveolar compartment; 2) fetal breathing movements that provide a stretch-based physical cue which serves as a maturation signal; and 3) pulsatile flow from the extensive vascular network present throughout the organ. techniques that mimic these forces to control lung cell fate are emerging. these include the use of shear to produce relatively homogenous populations of aec i and ii [251] , the use of cyclic mechanical stretch [252] , and the use of patterned hydrogels to enable perfusion of lung-type structures [253] . scaling some of these complex mechanical setups to enable large scale efficient manufacturing, however, could be a challenge. the challenge of assembling lung cells into reproducible arrays of "lung tissues" is starting to be addressed by emerging high-throughput 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synthesis of three-dimensional tissues a single-cell atlas of the airway epithelium reveals the cftr-rich pulmonary ionocyte cellnet: network biology applied to stem cell engineering single cell transcriptional archetypes of airway inflammation in cystic fibrosis single-cell rna sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis transcriptional regulatory model of fibrosis progression in the human lung defined threedimensional microenvironments boost induction of pluripotency graphical abstract key: cord-294812-nnlzwaf1 authors: desforges, marc; le coupanec, alain; brison, élodie; meessen-pinard, mathieu; talbot, pierre j. title: neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans date: 2014-03-12 journal: infectious diseases and nanomedicine i doi: 10.1007/978-81-322-1777-0_6 sha: doc_id: 294812 cord_uid: nnlzwaf1 in humans, viral infections of the respiratory tract are a leading cause of morbidity and mortality worldwide. several recognized respiratory viral agents have a neuroinvasive capacity since they can spread from the respiratory tract to the central nervous system (cns). once there, infection of cns cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. among the various respiratory viruses, coronaviruses are important pathogens of humans and animals. human coronaviruses (hcov) usually infect the upper respiratory tract, where they are mainly associated with common colds. however, in more vulnerable populations, such as newborns, infants, the elderly, and immune-compromised individuals, they can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, respiratory distress syndrome, or even severe acute respiratory syndrome (sars). the respiratory involvement of hcov has been clearly established since the 1960s. in addition, for almost three decades now, the scientific literature has also demonstrated that hcov are neuroinvasive and neurotropic and could induce an overactivation of the immune system, in part by participating in the activation of autoreactive immune cells that could be associated with autoimmunity in susceptible individuals. furthermore, it was shown that in the murine cns, neurons are the main target of infection, which causes these essential cells to undergo degeneration and eventually die by some form of programmed cell death after virus infection. moreover, it appears that the viral surface glycoprotein (s) represents an important factor in the neurodegenerative process. given all these properties, it has been suggested that these recognized human respiratory pathogens could be associated with the triggering or the exacerbation of neurological diseases for which the etiology remains unknown or poorly understood. viral infections of the respiratory tract represent a major problem for human and animal health around the world. these respiratory infections induce the most common illnesses [1] and are a leading cause of morbidity and mortality in humans worldwide, especially children, the elderly, and immune-compromised individuals [2] [3] [4] . on a statistical basis, children represent a highly susceptible group, as they may experience multiple infections each year until they reach the age of 10 [5] . respiratory viral infections also represent an economic threat for agriculture, especially in cattle, swine, and poultry production. the idea that viruses can cause respiratory tract infections has been demonstrated since the early 1930s [2] . nevertheless, with the help of modern diagnostic tools, a significant number of new respiratory viruses have been discovered since the beginning of the twenty-first century and it is estimated that there are about 200 antigenically distinct viruses able to cause infection of the respiratory tract, especially in infants and children [6] . in fact, it is now believed that viruses cause 95 % of respiratory diseases in children and infants, and about 30-40 % in the elderly [2] . although the airway epithelial cells in the respiratory tract represent a first line of defense against pathogens, they can be targeted by several different respiratory viruses that can infect them as a way to penetrate the human host. several infections are self-limited and the infection remains local as the virus is cleared by the immune system in the respiratory tract with minimal clinical consequences. however, in some circumstances, viruses can avoid the immune response and cause more severe respiratory diseases [1] or even spread to other tissues, including the central nervous system (cns), where they could induce other types of pathologies [7] . neuroinvasive and neurotropic viruses: associated neuropathologies induce or participate in the induction of neurological diseases (neurovirulence) [8] . in humans, a long list of viruses possess these ''neuroproperties'' and infection often leads to acute encephalitis, which can be fatal depending on virus tropism [9] . rabies virus [10] , herpes simplex virus (hsv) [11] , and arthropod-borne flaviviruses [12] can induce encephalitis in humans. chronic human neurological diseases may also be linked to viral infection. in acquired immunodeficiency syndrome (aids) dementia and related disorders, human immunodeficiency virus (hiv) induces neurodegeneration [13] , which can result in motor dysfunctions and possibly cognitive impairments [14] . progressive multifocal leukoencephalopathy (pml) is a human demyelinating disease [15] where prolonged immunosuppression leads to reactivation of latent polyoma jc virus (jcv) [16] . subacute sclerosing panencephalitis (sspe), a progressive fatal neurological disease, is caused by cns persistence of measles virus [17] . human t-cell lymphotrophic virus (htlv-1) causes progressive tropical spastic paraperesis/htlv-1-associated myelopathy (ptsp/ ham) in 1-2 % of infected individuals [18] and hsv-1 and human herpes virus 6 (hhv-6) were proposed to cause or exacerbate alzheimer's disease (ad) [19] . as mentioned above, respiratory viral agents also have the capacity to invade the cns where they will infect resident cells and potentially be neurovirulent in inducing a neuropathology. several of these recognized respiratory pathogens can gain access to the cns, where they can eventually cause health problems in humans. respiratory syncytial virus (rsv), the most common pathogen to cause lower respiratory tract infection in infants worldwide [20] , is one such neuroinvasive respiratory agent that has been detected in the cerebrospinal fluid (csf) of patients [21, 22] and that was associated with convulsions [23] , febrile seizures, and encephalitis [24] . furthermore, it was recently shown that rsv can spread from the airways to the cns in mice after intranasal inoculation, and that it induces behavioral and cognitive impairments [25] . measles virus (mv) from the paramyxoviridae family is another common virus that causes a disease of the respiratory airways associated with fever, cough, and congestion. however, mv infection also induces other symptoms including a characteristic rash and koplik's spots [26] in the oral mucosa. one of two most important sequelae associated with mv infection is immunosuppression, which facilitates infection by opportunistic bacteria or parasites that can lead to pneumonia or diarrhea. a second type of rare but significant sequelae is long-term cns disease [26] . postinfectious encephalomyelitis (pie) or acute disseminated encephalomyelitis (adem) occurs in 1 of 1,000 measles cases in children and adolescents. measles inclusion body encephalitis (mibe) is a second cns complication that can arise after a mv infection in immune-compromised patients. finally, subacute sclerosing panencephalitis (sspe) is a third form of cns disease associated with mv infection. it is a slow progressive neurological disease that appears 6-10 years after infection in about 4-11 cases per 100,000 cases of measles (for review see [27] ). hendra virus (hev) and nipah virus (niv) are two members of the genus henipavirus (hnv), also from the paramyxoviridae family. they represent important emerging viruses that were discovered in the 1990s [28] . fruit bats are the natural reservoir and both viruses can be transmitted to humans from intermediate reservoirs such as pigs and horses [29] . the hnv causes acute and severe respiratory disease in humans, including necrotizing alveolitis with hemorrhage, pulmonary edema, and pneumonia [28] . neurological signs of pathology include confusion, motor deficits, seizures, febrile encephalitic syndrome, and reduced level of consciousness. moreover, neuropsychiatric sequelae have been reported but it is not known whether postinfectious encephalomyelitis occurs following infection [29] . the use of animal models showed that the main route of entry into the cns is the olfactory nerve [30] . influenza virus comes in three types: a, b, and c. types a and b cause the flu syndrome, characterized by chills, fever, headache, sore throat, and muscle pains [31] , and are responsible for seasonal epidemics that affect 3-5 million humans, of which 250,000-500,000 cases are lethal each year [32] . although influenza type c virus is less frequent in humans, it is also distributed around the world where it mainly infects infants and young children; most of the time infection results in mild upper respiratory tract illnesses. however, complications associated with lower respiratory tract infection do occur in children under 2 years of age [33, 34] . human influenza a virus is of particular interest because of its capacity to recombine and rearrange with its avian and porcine counterparts to generate new emerging viruses that are introduced into the human population and that may lead to pandemic outbreaks associated with significant morbidity and mortality, as it was observed at least four times during the twentieth century and already once in the twenty-first century [32, 35] . influenza virus type a is highly contagious and, even though most infections are localized to the upper respiratory tract, some more severe cases may result in pneumonia [36] and even complications involving the cns [37] . several studies have shown that influenza a can be associated with encephalitis, reye's syndrome, febrile seizure, acute necrotizing encephalopathy, and possibly acute disseminated encephalomyelitis (adem) in humans [31, [38] [39] [40] [41] . making use of murine models, it has also been shown that influenza a virus could reach the cns through the olfactory nerve route and alter hippocampal morphology or expression of synaptic regulatory genes while impairing cognition and emotional behavior [42, 43] . influenza a virus was also described as a factor which may increase the risk of parkinson's disease (pd) [37] . among the different respiratory viruses, coronaviruses are important pathogens of humans and animals, causing a range of symptoms, including in the cns. coronaviruses, a family of enveloped positive-stranded rna viruses with a characteristic crown-shaped appearance, are widespread in nature and can infect several different species [44] , in which they cause mainly respiratory and enteric pathologies, with neurotropic and neuroinvasive properties in various hosts including humans, cats, pigs, rodents, and fowl [45] [46] [47] [48] . they are taxonomically grouped in the family coronaviridae, within the order nidovirales, and they are classified within four different genera, namely alpha, beta gamma, and deltacoronaviruses [49, 50] . they form a group of enveloped viruses that have the largest genome among rna viruses. this non-segmented 30 kb positive single-stranded polyadenylated rna possesses 4 or 5 genes encoding structural proteins (s, e, m, n; he for the genus betacoronaviruses) and several genes encoding non-structural proteins. the spike protein (s) is a type-1 glycosylated transmembrane protein responsible for the recognition of the cellular receptor used by the virus to infect a susceptible cell. the envelope (e) protein is a small structural protein anchored in the viral envelope, and which has a role in the assembly of the virion; it appears to be responsible for the adequate curving of the viral envelope. the membrane (m) protein possesses three transmembrane domains and interacts with all the other structural viral proteins and therefore helps to shape and maintain the virion structure. the nucleocapsid (n) protein associates with the viral genome and plays an essential role in encapsidating it into a helical nucleocapsid within the viral particle. the hemagglutinin-esterase (he) is only present in most species of the betacoronavirus genus. like the s protein, it is a transmembrane protein which forms homodimers and which interacts with different types of sialic acid, associated with an apparent role in hemagglutination. it also possesses an acetylesterase function, which may be important early during infection or during the release of viral particles from the infected cells at the end of the replication cycle of the betacoronaviruses [48] . as mentioned above, coronaviruses are widespread in nature and can infect several different animal species, in which very often they are both respiratory and enteric pathogens. although several animal coronaviruses induce severe enteric diseases, they also often reach the respiratory tract, where they can be associated with mild to severe diseases. some examples of coronaviruses that can infect livestock or poultry, in which they have a respiratory tropism, are transmissible gastrointestinal virus (tgev) and its associated s protein deletion mutant: porcine respiratory coronavirus (prcov) and porcine hemagglutinating encephalitis virus (phev), which infect swine; bovine coronavirus (bcov), which infects cattle; and infectious bronchitis virus (ibv), which infects chicken [51] . several of these animal coronaviruses cause severe economic burden to poultry, swine, and cattle industries worldwide. other animal coronaviruses that have a respiratory tropism are feline coronavirus (fcov); canine respiratory coronavirus (crcov); and bat cov, which infects the bat species miniopterus [51] . among the respiratory animal coronaviruses, fcov and phev have been associated with neurological diseases. neurological symptoms may occur in cats infected with a highly virulent fcov variant, designated fipv [52, 53] . neurological disease appears partially immune-mediated and may result in uncontrolled secretion of cytokines [54] that leads to diverse pathological manifestations including meningitis [55] and even spinal cord involvement [56] . furthermore, there is often a small amount of infectious fcov present in brain tissue [52] . the phev was isolated from the brains of suckling pigs suffering from encephalomyelitis several years ago in canada [57] and the disease could be reproduced experimentally in piglets following intranasal inoculation [58] . after oronasal infection, it was shown that the virus first infects epithelial cells of the respiratory tract and small intestine and, using retrograde neuronal spreading via peripheral nerves, was able to enter the cns [59] . more recently, the neuroinvasiveness and neurotropism of the virus were again demonstrated in a murine model, where phev induced a poor inflammatory reaction in the cns and infected cells showed no cytopathological changes [60] . the last, but not the least, example of another animal coronavirus, i.e., neuroinvasive, neurotropic, and neurovirulent, is mouse hepatitis virus (mhv). this virus is a subspecies of the species murine coronavirus (mucov) [49] , which represents a collection of viral strains with different tropism, including respiratory for the mhv-1 strain, and neurotropic for the mhv-jhm and mhv-a59 strains. in susceptible mice, these two latter strains of mhv induce a demyelinating disease that resemble human multiple sclerosis (ms) [61] . coronaviruses are all molecularly related in structure and mode of replication [62] . therefore, the close structural and biological relatedness of hcov to the neurotropic animal coronaviruses has led to speculation about possible involvement of hcov in neurological diseases. till now, no clear specific association has ever been made between coronaviruses and any known human neuropathology. however, hcov-229e and hcov-oc43 [63] [64] [65] [66] , as well as sars-cov [67, 68] , were shown to be neuroinvasive and neurotropic. human coronaviruses (hcov) usually infect the upper respiratory tract, where they are mainly associated with common colds. however, in more vulnerable populations such as newborns, infants, the elderly, and immune-compromised individuals, they can also reach the lower respiratory tract, where they could instead be associated with pneumonia, exacerbations of asthma, respiratory distress syndrome or even severe acute respiratory syndrome (sars) [44, 69] . ever since their discovery in the late 1960s, coronaviruses able to infect humans were neglected by the international medical community. however, when a variant emerged from animals in southeast asia to cause the first pandemic of the twentyfirst century: the severe acute respiratory syndrome or sars, these apparently innocuous viruses suddenly became ''more interesting.'' indeed, the 2002-2003 sars pandemic was caused by a coronavirus variant that appears to have emerged from a bat reservoir to infect palm civets, sold live in open markets, which served as intermediate reservoirs before crossing into humans. moreover in the fall of 2012, 10 years after the sars episode, the world health organization (who) warned the international medical community, that a sars-like disease affected individuals that traveled from the arabian peninsula to the united kingdom, which may indicate a possible resurgence of sars. however, using molecular sequencing, it was rapidly shown that this new respiratory coronavirus was genetically different from sars-cov, underlining the importance of a molecular approach in making a viral diagnostic. it is now recognized that the new epidemic is caused by a new coronavirus from the genus betacoronavirus that was first named hcov-emc (for human coronavirus-erasmus medical center), human betacoronavirus 2c, and ncov or ncov (for novel coronavirus), and that is now known under the official name mers-cov: the middle-east respiratory syndrome coronavirus [50] . as of august 30, 2013, who indicated that the mers-cov has spread to eight different countries, where 108 laboratory-confirmed cases of individuals have been identified as infected by the mers-cov, with 50 deaths [70] . although coronaviruses that infect humans are mainly recognized respiratory pathogens, as they usually first target respiratory and mucosal surfaces, infectious particles, antigens or rna, were detected in other tissues than the respiratory tract, including the cns. the detection of hcov rna in human brain samples clearly demonstrates that these respiratory pathogens are naturally neuroinvasive in humans and suggests that they establish a persistent infection in human cns [63] . furthermore, we have shown that these viruses are able to establish a persistent infection in human cells representative of the cns [64, 65] and that hcov-oc43 rna could be detected for at least a year in the cns of infected mice that survived the virus-induced acute encephalitis [71] . therefore, an apparently innocuous human respiratory pathogen may persist in the human cns as a component of what is proposed to be a «viral flora» of the brain, like hsv in a large proportion of the population. it would therefore be possible that such a persistent infection may become a factor or cofactor of neuropathogenesis in genetically or otherwise predisposed individuals. human coronaviruses were first isolated in the mid-1960s from patients with upper respiratory tract disease [72] . until the end of the twentieth century, only two serological groups, represented by strains oc43 and 229e, were known to infect humans and they were recognized as respiratory pathogens responsible for up to 30 % of common colds [72] . over the last 10 years, sars has generated renewed interest in coronaviruses that led to the discovery of new coronaviruses that can infect humans: sars-cov [73, 74] , hcov-nl63 [75] , hcov-hku1 [76] and mers-cov [77] . among these six coronaviruses, at least hcov-229e and hcov-oc43, as well as sars-cov, possess neuroinvasive properties as viral rna [63] or infectious virus [67, 68] can be detected in human brains. to our knowledge, there exist no reports on the detection of hcov-hku1, hcov-nl63, and mers-cov in the human cns. on the other hand, neurological symptoms have been described in association with both hcov-hku1 and hcov-nl63 [78] and a recent report, which evaluated mers-cov cell tropism, suggest that, among several cell lines representative of different tissues and organs, this virus seems to be able to infect the neuron-committed human cell line nt2 [79] . viruses may enter the cns through two distinct routes: hematogenous dissemination or neuronal retrograde dissemination. hematogenous spread involves the presence of a given virus in the bloodstream and retrograde viral spread toward the cns occurs when a given virus infects neurons in the periphery and uses the transport machinery within those cells to gain access to the cns [80] . in order to be neuroinvasive, viruses such as hcov-229e, hcov-oc43, and sars-cov may use both entry routes from the periphery. the hematogenous route involves the presence of a given virus in the blood, where it can either remain free for a period of time before it can infect the endothelial cells of the blood-brain-barrier (bbb), or infect leukocytes that will become some sort of viral reservoir for dissemination to other sites. both situations occur during hiv infection of the cns. indeed, hiv-infected leukocytes that migrate through the bbb (called the trojan horse [81] ), is one of the route of spread, and direct infection of endothelial cells of the bbb is also possible even though the viral replication is at a low level [82] . infection of human monocytes/macrophages by hcov-229e and hcov-oc43 was reported [83, 84] and infection by hcov-229e of murine dentritic cells expressing the human aminopeptidase n [85] suggests that human coronaviruses may use these cells to disseminate to other tissues, including the cns, where they could be associated with other type of pathologies. sars-cov was also shown to be able to infect human monocytes/macrophages [68, 86] . moreover, monocytederived dendritic cells are also susceptible to infection by sars-cov [87] . human primary monocytes are activated following infection by hcov-229e [83] . since they eventually become macrophages as they invade tissues, this activation suggests that hcov-229e-infected monocytes would serve to facilitate their passage toward other tissues including the cns, especially in immunecompromised individuals, as this was observed for murine cytomegalovirus (mcmv) [88] . the fact that hcov-229e could only infect partially immunecompromised transgenic mice [89] suggests that hcov-229e could take advantage of an immune-suppressed environment and disseminate to the cns within susceptible individuals. the establishment of a persistent infection in a human leukocytic cell line [83] is also consistent with the possibility that monocytes/ macrophages serve as a reservoir and vector for this neuroinvasive hcov [63] . the sars-cov also infects monocytes/macrophages [68, 86] and dendritic cells, in which it modulates innate immunity [87] . these cells could also serve as a reservoir for the virus to reach and maintain itself in the cns. our results indicate that hcov could also infect human endothelial cells of the bbb in culture (unpublished data) and it has been speculated that sars-cov could do the same after viremia [90] . therefore, the neuroinvasive hcov could use the hematogenous route to penetrate into the cns. the second form of any viral spread toward cns is through neuronal dissemination, where a given virus infects neurons in periphery and uses the machinery of active transport within those cells in order to gain access to the cns [80] . after an intranasal infection, both hcov-oc43 [91] and sars-cov [92] were shown to infect the lungs in mice and to be neuroinvasive as hcov-oc43 [93, 94] and sars-cov [95] were detected in the cns of susceptible mice. therefore, these two coronaviruses may use both the hematogenous and transneuronal route toward the cns. furthermore, as shown in fig. 1 , once in the brain, hcov-oc43 can disseminate from the olfactory bulb to the cortex and we have previously shown that it could also reach the medulla, while the cerebellum remained almost uninfected. the hippocampus represents another specific structure infected by hcov-oc43 in the brain (fig. 1) and once in this region of the brain, the virus appears to spread by a transneuronal route before it eventually reaches the spinal cord [48] . neuroinvasive viruses can damage the cns as a result of misdirected host immune responses (virus-induced neuroimmunopathology) and/or viral replication, which directly induces damage to cns cells (virus-induced neuropathology). in acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the gray matter [47] . as mentioned above (section ''neuroinvasive and neurotropic viruses: associated neuropathologies''), chronic human neurological diseases may also be linked to viral infection. however, in several cases of these chronic diseases, it is difficult to ascertain a role for any given virus, in part due to the difficulty of establishing the time at which these viruses become involved. also, the four koch's postulates for disease induction dictate whether a particular infectious agent causes a specific disease [96] . however, several viral infections, especially slow viral infections related to diseases that are rare manifestations of an infection, represent situations where koch's postulate should be modified to better adapt to the situation [97, 98] . a series of new criteria, adapted from sir austin bradford hill's criteria for causation [98] , has been elaborated by giovannoni and collaborators [99] and should replace koch's postulates when one wants to evaluate the relevance of any given virus in relation to ms etiology [99] or any other long-term human neurological diseases potentially related to a viral infection as well, including infection by human coronaviruses. the presence of hcov-229e and hcov-oc43 was detected in various neurological diseases in humans, including pd and ms [63] and adem [100] . multiple sclerosis truly represents a human neurological disease where an infectious agent or agents may play a triggering role, with viruses the most likely culprit in genetically predisposed individuals [101] . there is a presumption that several neurotropic viruses could be involved in ms pathogenesis but that they may do so through similar direct and/or indirect mechanisms [102] [103] [104] [105] . however, research has not yet led to a direct link to any specific virus or other microbes with ms. association of coronaviruses with ms was suggested in numerous reports that are reviewed elsewhere [48] . one of these reports demonstrated a significant association of colds with ms exacerbations and a significant association of hcov-229e infection in ms patients [106] and another report on the association of viral infections and ms [107] commented that seasonal hcov infection patterns do fit the observed occurrence of ms exacerbations. furthermore, the case of these human coronaviruses in the cns may represent a new example where the traditional koch's postulates should be replaced by the previously cited adapted hill's criteria [99] . more than a decade ago, we experimentally confirmed that hcov-oc43 and hcov-229e were naturally neuroinvasive in humans. although viruses were also detected in some control brains, there was a significantly higher prevalence of hcov-oc43 in brains of ms patients [63] . moreover, these data, in association with the observation that autoreactive t-cells recognized both viral and myelin antigens in ms patients but not in controls [108, 109] during infection by hcov-oc43 and hcov-229e, suggest that the immune response may participate in the induction or exacerbation of neuropathologies such as ms in genetically or otherwise susceptible individuals (data summarized in table 1 ). furthermore, even though the use of the immunosuppressive drug cyclosporin a in hcov-oc43-infected mice resulted in a faster onset of encephalitis, suggesting a role for t-cells in viral clearance and survival with no related immunopathology [71] , it was shown that in recombination activation gene (rag) knock-out mice, hcov-oc43-induced encephalitis could be partially mediated by the t-cell response to infection [93] . the participation of different types of t-cells has been shown to play a significant role in the demyelinating neurological disease induced by the adapted from arbour et al. [63] and boucher et al. [108] numbers in the upper portion indicate the number of individuals positive for viral rna and numbers in parenthesis indicate the total number of individuals tested numbers in the lower portion indicate the number of t-cell clones obtained. monospecific describes clones that react against a single antigen and cross reactive describes clones that react both with hcov and myelin antigens murine cov, in particular for strain mhv-jhm [110] , which represents the murine counterpart of hcov-oc43. more recently, making use of another mouse model, we showed that hcov-oc43 induced immune cell infiltration and cytokine production in mouse cns and that this response was significantly higher after infection by variants which harbor point mutations in the viral surface glycoprotein (s) [111] . importantly, these s point mutations were acquired after persistent infections of human neural cell lines [112] . moreover, we also showed that infection by the s mutants is linked to glutamate excitotoxicity [113] . therefore, as this increase in cytokine production may induce damages to the neurons [114] that can be associated with problems in glutamate homeostasis, which in the end may create glutamate excitotoxicity [115] , it can contribute to neuronal degeneration associated with hind-limb paralysis, as illustrated in fig. 2 , and possible demyelination [111, 113] . the outcome of the observed degeneration of neurons may eventually lead to the death of these essential cells. as previously mentioned, infection of neurons by itself may also participate in the process of cell death by directly generating a cytotoxic insult related to viral replication and/or to the induction of different cell death pathways. when present in the murine cns, hcov-oc43 infects neurons in several different regions of the brain [48] (fig. 1 ) before reaching the spinal cord. infection of these essential cells induces their degeneration [111, 113] , as observed by aberrant state of neurofilament phosphorylation, a situation that often leads to cell death and that could be directly induced by viral replication. furthermore, using two model cell lines of differentiated human neurons, we demonstrated that programmed cell death (pcd) was induced after hcov-oc43 infection [116, 117] and that the inhibition of viral replication was also in direct correlation with increased cell survival, suggesting that infection and production of new infectious viruses directly participate in the process of degeneration and eventual death of neurons. our results indicate that the underlying mechanisms appear to involve different cellular factors and pathways, including caspase-independent apoptosis, parthanatos, and necroptosis, three forms of programmed cell death (pcd), which are reviewed elsewhere by the nomenclature committee on cell death (nccd) [118] . these cell death pathways can act separately but may also interact in response to a stimulus (including a viral infection), as they share some of the cellular factors involved in the overall process that leads to cell death and that often converges toward mitochondria [118] . figure 3 is a tentative representation of the various pathways and cellular factors involved during hcov-oc43-induced pcd of infected neurons. it is based on our data [111, 113, 116, 117] and on the scientific literature that describes some molecular pathways (parthanatos, necroptosis and apoptosis) and cellular factors, including calcium overload, endoplasmic reticulum (er) stress, excitotoxicity, poly(adp-ribose) polymerase (parp), calpain and oxidative stress related to the formation of reactive oxygen species (ros) involved in mitochondrial dysfunction, and eventual neurodegeneration and neuronal cell death [118] [119] [120] . virus-cell interaction is always important in the regulation of cell response to infection. for hcov-oc43, we clearly showed that the viral s glycoprotein is an important factor of neurovirulence and neurodegeneration of infected cells [111, 113, 116] . this was similarly shown for coronavirus strains mhv-a59 and mhv-jhm, which represent subspecies of the mucov species, the murine counterpart of hcov-oc43 as they are both members of the betacoronavirus genus. indeed, several reports and reviews have, over the years, described that s protein of this neuroinvasive and neurotropic murine coronavirus is a major factor associated with neurovirulence during encephalitis and the eventual demyelinating disease in susceptible mice [121, 122] . our more recent data also demonstrated that the he protein is an important factor for the production of infectious hcov-oc43, suggesting an attenuation of the eventual spread of viruses deficient in fully active he protein into the cns [123] . therefore, as the infection of neuronal cells apparently directly participate in the induction of neuronal death, by abrogating the production of infectious virus, the he protein of hcov-oc43 could play a role in neurovirulence of hcov-oc43, like it does for mhv [124] . (1) hallmarks of apoptosis, including relocalization of the activated pro-apoptotic protein bax (bcl-2 associated protein x) from the cytosol to the mitochondrial membrane, cytochrome c release from mitochondria toward the cytosol, dna fragmentation, and activation of caspases -3 and -9, are observed during infection of human neurons. however, using a pan-caspase inhibitor (z-vad-fmk), cell death is not abrogated after infection, suggesting a caspase-independent type of apoptosis. (2) relocalization of the mitochondrial protein aif (apoptosis-inducing factor) toward the nucleus (taif) is observed after infection and participates in dna fragmentation in conjunction with cypa (cyclophilin a) and histone h2ax. the aif is known to be activated during caspase-independent apoptosis. however, aif is also involved in parthanatos, another form of pcd potentially associated with neurodegeneration. as they are synthesized by the poly(adp-ribose) polymerase (parp) during a neuronal stress, including during hcov-oc43 infection, polymers of adpribose (par) may relocalize toward mitochondria and participate in the activation and relocalization of aif toward the cytosol before it reaches the nucleus. cyclophilin d (cypd) inhibition decreases aif release from mitochondria and abrogates cell death induced by infection. (3) aif release from mitochondria may be induced through its truncation (taif) by activated calpain, which is usually activated by a rise in the mitochondrial calcium concentration. (4) this increase in calcium concentration may be linked with either an important entry from the extracellular milieu (for instance during excitotoxicity) or with a release of calcium from the endoplasmic reticulum (er) following induction of er stress. both situations are probably taking place after infection of neurons by hcov-oc43. the increase in calcium concentration in mitochondria may also induce production of reactive oxygen species (ros) that can be harmful for mitochondria and hence neurons. (5) the presence during infection of an inhibitor (nec-1) of the receptor interacting protein kinase-1 (rip-1), significantly increases cell survival and partially abrogates viral replication, suggesting that necroptosis, a third form of pcd which involves rip-1 and rip-3 downstream of the tumor necrosing factor (tnf) receptor family (in the form of the death-inducing signaling complex (disc)), may play a role in hcov-oc43-induced neuronal death. solid arrows indicate experimental data and dashed arrows represent possible pathways based on the current literature (see text for details) as mentioned above, sars-cov is also neuroinvasive and neurotropic in humans [67, 68] and it could therefore be associated with the development of a neurological disease. furthermore, the involvement of sars-cov in cns infections was underscored by the findings that made use of transgenic mouse models expressing the human angiotensin-converting enzyme-2 (the cellular receptor used by sars-cov to infect susceptible cells). indeed, using these mice, it was shown that sars-cov could invade the cns after an intranasal infection primarily through the olfactory bulb [95] or even after an intraperitoneal infection [125] , with concomitant neuronal loss [95, 125] ; a phenomenon that can eventually lead to neurological problems. the presence of coronaviruses in the human central nervous system is now a recognized fact as they appear to be part of a viral flora of the brain, with potential neuropathological consequences in genetically or otherwise susceptible individuals, with or without additional environmental insults. knowledge of mechanisms and consequences of virus interactions with the nervous system is essential to better understand potentially pathological consequences and design intervention strategies that are appropriate to encephalitis or exacerbations of other types of neurological diseases for which a given virus is involved. in that regard, hill's criteria adapted by giovannoni 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pathogenesis of murine coronavirus in the central nervous system the pathogenesis of murine coronavirus infection of the central nervous system the acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus oc43 strongly enhances the production of infectious virus expression of hemagglutinin esterase protein from recombinant mouse hepatitis virus enhances neurovirulence severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor key: cord-314372-knhkdlq7 authors: kanduc, darja; stufano, angela; lucchese, guglielmo; kusalik, anthony title: massive peptide sharing between viral and human proteomes date: 2008-06-05 journal: peptides doi: 10.1016/j.peptides.2008.05.022 sha: doc_id: 314372 cord_uid: knhkdlq7 thirty viral proteomes were examined for amino acid sequence similarity to the human proteome, and, in parallel, a control of 30 sets of human proteins was analyzed for internal human overlapping. we find that all of the analyzed 30 viral proteomes, independently of their structural or pathogenic characteristics, present a high number of pentapeptide overlaps to the human proteome. among the examined viruses, human t-lymphotropic virus 1, rubella virus, and hepatitis c virus present the highest number of viral overlaps to the human proteome. the widespread and ample distribution of viral amino acid sequences through the human proteome indicates that viral and human proteins are formed of common peptide backbone units and suggests a fluid compositional chimerism in phylogenetic entities canonically classified distantly as viruses and homo sapiens. importantly, the massive viral to human peptide overlapping calls into question the possibility of a direct causal association between virus–host sharing of amino acid sequences and incitement to autoimmune reactions through molecular recognition of common motifs. protein sequences of the human proteome as well as of a number of viral proteomes have become available in databanks, offering an opportunity for a thorough comparative analysis of their reciprocal inter-relationship(s). this area of inquiry is of special interest because of our persistent ignorance of the mechanisms at the basis of viral pathogenesis and virus-host interactions [1, 19] . to define the molecular structural determinants possibly involved in viral virulence and human susceptibility, we have undertaken a systematic analysis of a large set of viral proteomes searching for amino acid sequences shared with the human proteome. specifically, the study was designed to answer two main questions: (i) what is the quantitative dimension of shortpeptide sequence sharing between viruses and human proteomes? (ii) how many human proteins harbor viral peptide modules? the two questions are of paramount importance in the context of autoimmune diseases. indeed, it is a common belief that an autoimmune reaction is mostly caused by a host receiving an antigen that has amino acid homology/similarity with amino acid sequences in selfantigens of the host [20] . this may result in the host immune system attacking the organs and tissues expressing the selfantigens with the shared sequences [23] . in this context and given the sustained increase in the incidence of autoimmune diseases [2, 8, 18, 22] , the mathematical quantification of peptide overlap extent between viruses and humans is essential to understand the role of structural viral similarity in the pathogenesis of autoimmunity. here we report a virus versus human comparative screening that reveals a massive, indiscriminate, unexpected pentapeptide overlapping between viral and human proteomes. the viral proteomes analyzed in this study were downloaded from www.ebi.ac.uk/genomes/virus.html and the human proteome obtained from uniprotkb (http://www.ebi.ac.uk/ integr8). the human proteome originally contained 37,993 proteins at the time of download, but we filtered out (1) two viral contaminants (i.e. viral proteomes which were being expressed in human cells), and (2) duplicated sequences and fragments. after the filtering, we were left with a human proteome consisting of 36,103 unique proteins. similarity analyses were conducted on 30 viral proteomes for a total of 717 viral proteins, equal to 302,667 amino acids. a similarly sized control set of 30 human protein samples (for a total of 686 human proteins, equal to 302,520 amino acids) was analyzed for inter-human overlapping. more precisely, for each viral proteome size, we constructed an artificial human sub-proteome drawn from the human protein set. the proteins were selected at random without replacement, and assigned to an artificial sub-proteome at random. we kept adding human proteins to each artificial human sub-proteome until adding the next protein would have caused the total number of amino acids in the artificial sub-proteome to exceed to the number of amino acids in the corresponding viral proteome. this way we eliminated possible bias in the comparison. the 30 artificial sub-proteomes are random samplings of human proteins and represent cross-sections of all human proteins. the list of the human proteins forming the 30 artificial human sub-proteomes is detailed as swiss-prot entries in table 3 . sequence similarity analyses of each of the 30 viral proteomes (or the 30 artificial human sub-proteomes) to the human proteome were carried out using viral (or human) 5mers sequentially overlapped by four residues. the scans were performed by custom programs written in c and utilizing suffix trees for efficiency [6] . the viral or human proteins were manipulated and analyzed as follows. the entire viral (or human) proteome was decomposed in silico to a set of 5-mers (including all duplicates). a library of unique 5-mers for each virus or human proteome was then created by removing duplicates. next, for each 5-mer in the library, the entire human proteome was searched for instances of the same 5-mer. any such occurrence was termed an overlap. cursory analyses (e.g. identification of unique overlapping 5-mers, counts of unique overlapping 5-mers, counts of duplications) were performed using linix/unix shell scripts and standard linux/unix utilities. data were plotted and linear least-squares regression was performed to determine whether any linear relationships exist between the level of overlap and the size of viral (or control) proteomes. the same procedure was applied when longer amino acid sequences (esa-, epta-, octapeptides, etc.) were used as probes in the similarity analyses. the similarity analysis for the artificial human subproteomes was performed as for the viral proteomes, with p e p t i d e s 2 9 ( 2 0 0 8 ) 1 7 5 5 -1 7 6 6 one modification. the modification is that all the proteins in the artificial proteome were removed from the comparison human proteome when the overlap determination was done. e.g., the artificial human sub-proteome 9 contains 12 human proteins selected from the original set of 36,103 proteins comprehensively forming the human proteome. these 12 proteins were removed from the comparison set prior to determining the overlap of artificial human sub-proteome 9 versus human; i.e. for artificial proteome 9, the overlap comparison was between 12 proteins in the artificial proteome and the remaining 36,091 human proteins. and similar for each of the other 29 artificial proteomes. the viruses were chosen based on the following criteria: (1) known to be pathogenic to human; (2) of significant health impact (e.g. hcv, rhinovirus, hiv, mumps, measles, sars, hpv, and polio); (2) phylogenetically different; (4) proteomes established to a significant degree of completeness. in addition, the viral proteomes were chosen to span a range of proteome sizes, with the smallest viral proteome being 1613 amino acids, the largest being 65,280 amino acids, and all the viral proteomes combined amounting to 302,667 amino acids. the viral proteomes are described in table 1 . as controls, we used 30 similarly sized protein sets drawn from the human proteome. in brief, we constructed 30 artificial sub-proteomes populated by proteins randomly selected without replacement from the human proteome. the constraints we used were that (1) the size of each artificial sub-proteome was to approximate the size of one of the viral proteomes, and (2) that the combined size of all the artificial proteomes was to approximate the combined size (in amino acids) of the viral proteomes. the 30 resultant artificial human sub-proteomes are described in table 2 . table has four columns. the first is the artificial subproteome number. the second is the cumulative amino acid length of all the human proteins in that artificial subproteome. the third column is the cumulative amino acid length of the corresponding viral proteome. the fourth column is the number of human proteins in the artificial sub-proteome. for example, in our virus versus human analysis, information for hepatitis c virus (hcv) was used as follows. the virus has taxonomic id 11103 and a proteome consisting of 3010 amino acids (see table 1 ). corresponding to this virus is the artificial human sub-proteome 9, which contains 12 human proteins which sum to a cumulative length of 2979 amino acids (i.e. just about the 3010 amino acids in the hcv proteome). and similar for each of the other 29 viral proteomes. the list of the human proteins forming the 30 artificial human sub-proteomes is detailed as swiss-prot entries in table 3 . sequence similarity analyses of each of the 30 viral proteomes (or the 30 human artificial sub-proteomes) to the human proteome were conducted using viral (or human) 5mers sequentially overlapped by four residues. basically, we used 5-mer sequences as probes to scan viral/human proteins against human proteome since pentapeptides are minimal structural units critically involved in biological/pathological interactions such as peptide-protein interaction and (auto)immune recognition ( [5, 7, 17, 18, 21] and further refs. therein). longer amino acid sequences (esa-, epta-, octapeptides, etc.) were used as additional probes to control the similarity pattern. quantitative analysis of the pentapeptide overlapping of viral versus human proteomes the numerical values that define virus-to-human similarity level are reported in table 4 . the table documents that all of the 30 viral proteomes under analysis have high and wide pentapeptide overlapping to the human proteome. only a limited number of viral pentamers are unique to the viruses, with no counterpart in the human proteome. the overlapping extent (in terms of percentage of unique viral 5-mers which occur in the human proteome, see column 6 in table 4 ) is rarely less than 90%. that means that almost 90% of the viral pentamer peptides are widely, intensively and repeatedly scattered throughout the human proteome. numerically, the viral versus human overlap is defined by 2,907,096 total table 3 . p e p t i d e s 2 9 ( 2 0 0 8 ) 1 7 5 5 -1 7 6 6 matches. also the viral overlapping distribution in the human proteome (as number of human proteins hosting viral 5-mers, column 5 in table 4 ) is highest, being close to 100% with hhv-5 (i.e. hhv-5 pentamers are present in 35,708 human proteins out of the 36,103 ones which comprehensively form the human proteome). given the ample literature documentation and research data in support of the critical role exerted by pentapeptide modules in cell biology as well in antigen/antibody immunorecognition ( [17] , and refs. therein), the data of table 4 are impressive. they become even more significant, if possible, when compared to the control data obtained for inter-human p e p t i d e s 2 9 ( 2 0 0 8 ) 1 7 5 5 -1 7 6 6 pentapeptide overlapping illustrated in table 5 . when 30 human artificial sub-proteomes were analyzed for pentapeptide overlapping to the entire human proteome, we obtained clear numerical evidence that the inter-human level of perfect 5-mer matching is of the same order of magnitude as the viral overlapping to human proteome detailed in table 4 , i.e. there are 3,713,010 inter-human versus 2,907,096 viral perfect 5-mer matches to the human proteome (see table 5 ). likewise, the number of human proteins involved in viral and inter-human overlaps is highest and practically identical in the numbers: human proteins are involved in the inter-human peptide overlapping for 697,373 times and in the viral versus human (2) total number of 5-mers in the artificial sub-proteome (including multiple occurrences); (3) unique 5-mers from the artificial subproteome occurring in the human proteome; (4) occurrences in the human proteome of 5-mers from artificial sub-proteome (including multiple occurrences); (5) number of human proteins in the human proteome involved in overlap; (6) % of unique 5-mers from the artificial sub-proteome which occur in the human proteome (i.e. 100 â column 3/column 1). a analogous to viral proteomes in size (see table 1 ), and composed by set of human proteins as detailed in table 3 overlap for 633,229 times (see data from tables 4 and 5 , respectively). this indicates a massive, repeated, acritical usage of the same pentapeptide blocks in viral and human proteomes. plotting the viral 5-mer occurrences in the human proteome as a function of the viral proteome length (see data under table 4 , columns 2 and 4) produces the graph illustrated in fig. 1 . the following observations are worth noting: first, the level of viral overlaps to the human proteome is directly related to the length of the viral proteome by a linear relationship. the pearson's correlation coefficient of the line is 0.97452, and represents an exceptionally strong linear relationship. biologically, fig. 1 suggests that peptide motif sharing is a constant property of the viral proteomes, exclusively depending on the viral proteome length and with no relationship to other structural and/or pathogenic viral features. as a note of interest, it can be seen that htlv-1, rubella virus, and hcv present a number of overlaps to the human proteome above the expected number of overlaps predicted by the linear regression line. this indicates that the massive, repeated, acritical, usage of the same pentapeptide blocks in viral and human proteomes is even more accentuated in the case of htlv-1, rubella virus, and hcv. to understand the overlap usage in entities so distant in the evolutionary time and so enormously different in the evolutionary history such as viruses and humans, we investigated the profile of viral motif distribution along the human proteome. representative histograms are reported in fig. 2 . it can be seen that, independently of the virus size, the density of the viral 5-mer motif matches along the human proteome presents constant behavior, with virus portions endowed with high similarity alternating with portions scarcely represented in the human proteome. the same alternating behavior is shown by the histograms of inter-human overlapping by analyzing sub-proteomic sets of low, medium and large size (fig. 3) . figs. 2 and 3 confirm the commonality of pentapeptide block usage in viruses and humans and, more in general, document the existence of a basic structural platform in the protein world. as shown in tables 6 and 7 , the viral versus human overlapping is still remarkable by using viral esa-or eptapeptide sequences as probes. non-stochastic nature of the peptide overlapping between the viral and human proteomes under analysis the results illustrated in tables 4, 6 and 7 are indicative of a widespread peptide overlapping between viral proteins and the homo sapiens proteome. in order to understand how the above reported data are mathematically governed, we explored the 30 viral proteomes and the 30 human subproteomes under analysis for the degree of internal redundancy and the viral versus human proteome overlapping at nmer level (with n from 5 to 16 amino acids). the quantitative data we found are reported in table 8 listing three orders of data: (a) the number of unique occurrences of n-peptides in the 30 viral (or the 30 human) proteome samples; (b) the total number of occurrences (i.e. including multiple occurrences) of n-peptides in the 30 viral (or the 30 human) proteome samples; and c) the relative viral versus human n-peptide overlaps. the first set of data, column 1 through 6 in table 8 , provides evidence that the numerical values for uniquely expressed npeptides are always lower than the actual total values for npeptide occurrences. this is remarkable in light of the enormously high number of potential n-peptides which theoretically are available. as an example, in face of the possible 3,200,000 pentamers, both the 30 viral proteomes and the 30 human sub-proteomes present a high degree of repetitiveness in their 5-mer composition. in fact, the 30 viral proteomes and the 30 human sub-proteomes are respectively formed by a total of 299,701 and 299,749 5-mers and, although there is the possibility of forming 3,200,000 different pentapeptides, these total 5-mers present 42,666 and 44,941 repeated pentapeptides, in the viral and human proteins, respectively. the intra-viral (or intra-human) peptide redundancy strikingly persists at higher n-mer level, despite the exponentially increasing numbers of theoretically possible nmers. the intra-repetitiveness is mathematically quantified in table 8 , columns 3 and 6, for the 30 viral proteomes and the 30 human sub-proteomes, respectively. the second set of data illustrates the overlapping at npeptide level (with n from 5 to 16 amino acids) from the 30 viral proteomes versus the 30 human sub-proteomes (table 8, columns 7 and 8) . again, we note that, although there is a potential pentapeptide reservoir amounting to 3,200,000, still 42,647 of unique viral pentapeptides (16.6% of the total) appear in the human proteins forming the 30 human sub-proteomes. when considering the total viral pentapeptides including multiple occurrences (table 8 , column 8), the viral contribution to the 30 human subproteomes raises to 56,230 pentamers. these repeated viral overlaps in the human proteins appear to further support the existence of a defined peptide repertoire which is utilized (even repeatedly) in protein composition. a similar observation holds in considering the viral peptide overlapping to the 30 human sub-proteomes at higher n-mer level (table 8, columns 7 and 8) . finally, the third set of data documents the overlaps at npeptide level (with n from 5 to 16 amino acids) from the 30 viral proteomes versus the entire human proteome (table 8 , columns 9 and 10). it can be seen that, independently of the vastness of the human protein sample, the extent of the viral overlap is relevant, especially when considering that the human proteome has the possibility of drawing from an enormous number of potential theoretical n-peptides. for example, notwithstanding the astonishingly great repertoire of theoretical possible undecapeptides (that is: 204,800,000 millions), nonetheless eight viral overlaps (one of which even repeated) are present in the sub-proteomic human samples. the numbers raise to 352 and 661 (including multiple occurrences of the same 11-mer) when considering the viral 11-mer overlaps in the entire human proteome. our conclusion is that the mathematical redundancy present in the protein world is not stochastic (i.e. is not pure random chance), but rather reflects strong peptide usage bias since certain peptides are repeatedly used (and shared) in (and among) viral and human proteins. it seems that peptide usage in the protein world (and, consequently, the degree of similarity, repetition, overlapping) is not dictated by pure mathematical laws of distribution. rather, powerful constraints appear to be at work by forcing the use of restricted platforms of n-mers and privileging the repeated usage of the chosen ones. possibly, as anticipated by preliminary data from our labs (kusalik et al., manuscript in preparation), these powerful constraints able to influence and favor specific n-mer synthesis might be found among physico-chemical factors such as hydrophobicity, residue bulkiness, dg8 of peptide bond formation, plus biological factors such as codon bias. 4. the difference between biological entities such as viruses and cellular organisms is quite obvious when the functional roles of the proteins found in viruses and in cellular organisms are compared [24] . on the other hand, according to the data we present here, this obvious difference disappears at the peptide phenetic level. the mathematical quantification of the widespread and high pentapeptide similarity between viral and human proteomes is surprising, raising fundamental questions. in particular, the ample viral-human peptide sharing is significant to (auto)immune phenomena as well as to evolutionary pathways. indeed, in the context of the physio(patho)logical relationships between viruses and humans, it is mandatory to observe that short-peptide motifs can exert a central role in cell adhesion, signal transduction, hormone activity, regulation of transcript expression, and enzyme activity [3, 9, 10, 14, 15, 17] . likewise, the antigen-antibody recognition process can reductionistically be circumscribed to interacting modules of five amino acid residues which, therefore, appear to be sufficient structural immunological determinants ( [11] [12] [13] 17] and refs. therein). consequently, we notice that the data shown here call into question the possibility of a direct causal association between virus-host sharing of amino acid motifs and incitement of autoimmune reactions [20] . indeed, the molecular mimicry hypothesis suggests that, when bacterial/viral agents share epitopes with a host's proteins, an immune response against the infectious agent may result in formation of crossreacting antibodies that bind the shared epitopes on the normal cell and result in the auto-destruction of the cell. in the present case, the molecular mimicry hypothesis implies that viral infections should be a practically infinite source of autoimmunity diseases since this study demonstrates that viral 5-mer matches are disseminated throughout practically all the human proteome and each viral match is repeated almost more than 10 times (see table 4 ). consequently, autoimmune diseases should theoretically approach a 100% real incidence, since the 30 viruses we examined practically are more or less disseminated throughout the entire human species. taking hcv as an example, we note that the virus versus human overlap analysis in table 4 produces the mathematical evidence that hcv (as well as htlv-1 and rubella virus) are even ''more similar'' to the human proteins than that expected by the linear regression relationship reported in fig. 3 . the data reinforce and add to our previous report on the sharing of numerous perfect exact matches between hcv and human proteomes [16] . since 2760 out of 3003 hcv 5-mers occur in the human proteome, the 46,731 occurrences include multiple/repeated matches in the human proteins. more exactly, each hcv 5-mer that overlaps with the human proteome are repeated an average of 16.5 times. moreover, the 46,731 occurrences occur in 20,269 different human proteins (table 4 ). that means that almost 60% of the 36,103 human proteins forming the h. sapiens proteome contain viral hcv pentamers. this datum is of cogent interest in the present worldwide advance of hcv infection, since it further excludes sequence similarity as a possible mechanism in hcvassociated autoimmunity phenomena and, at the same time, might address research towards new unexplored scenarios. these observations, of course, apply to almost all the viruses listed in table 1 . when analyzed in the evolutionary context, the viral and human protein sets studied here yield a closely congruent overlapping pattern to the human proteome at the 5-mer level as well as at higher n-mer levels, indicating a common compositional mosaicism in viral and human proteins. the absence of specific phenetic n-mer clusterings in the viral versus human proteome (and vice versa) suggests a synergetic platform of development of non-equilibrium systems which can be traced back to the prigoginian model rather than to the column number: (1) actual unique n-mers in the 30 viral proteomes; (2) actual n-mers in the 30 viral proteomes (including multiple occurrences); (3) number of repeated n-mers in the 30 viral proteomes; (4) actual unique n-mers in the 30 human sub-proteomes; (5) actual nmers in the 30 human sub-proteomes (including multiple occurrences); (6) number of repeated n-mers in the 30 human sub-proteomes; (7) unique viral n-mers overlaps in the 30 human sub-proteomes; (8) total viral n-mers overlaps in the 30 human sub-proteomes (including multiple occurrences); (9) unique viral n-mers overlaps in the human proteome; (10) total viral n-mers overlaps in the human proteome (including multiple occurrences). a peptide length, with n from 5 to 16 amino acids. b the number of possible amino acid combinations is given by 20 n . p e p t i d e s 2 9 ( 2 0 0 8 ) 1 7 5 5 -1 7 6 6 darwinian evolutionary scheme, where harmful protein sequences are ''selectively removed'' whereas neutral or useful peptides are left alone. de facto, it seems that viruses and mammalians have drawn and draw from a common ancient melting pot of short-peptide modules in building up their proteomes, either simple or complex. indeed, it seems that human evolution has paid very little attention to a peptide motif being viral or not. the reconstruction of the informational network relating all living organisms might be helped by an integration with the present data which recall a time when the totality of life on earth was a simple heterogeneous community that shared only the ability to synthesize proteins with a ribosomal machine and that freely exchanged genetic material [4] . r e f e r e n c e s to c or not to c: these are the questions myasthenia gravis: a higher than expected incidence in the elderly enhanced antigenicity of a four-contactresidue epitope of the measles virus hemagglutinin protein by phage display libraries: evidence of a helical structure in the putative active site evolutionary aspects of whole-genome biology a polyalanine peptide with only five native myelin basic protein residues induces autoimmune encephalomyelitis algorithms on strings, trees, and sequences: computer science and computational biology minimal peptide length requirements for (cd4+) t cell clones-implications for molecular mimicry and t cell survival epidemiology and estimated population burden of selected autoimmune diseases in the united states molecular effects of proinsulin c-peptide translational regulation of human papillomavirus type 16 e7 mrna by the peptide seqika, shared by rabbit (alpha1)-globin and human cytokeratin 7 correlating low-similarity peptide sequences and allergenic epitopes non-redundant peptidomes from daps: towards ''the vaccine''? multichain immune recognition receptor signaling: from spatiotemporal organization to human disease the ghrelin pentapeptide inhibits the secretion of pancreatic juice in rats p2y nucleotide receptor signaling through mapk/erk is regulated by extracellular matrix: involvement of beta3 integrins widespread and ample peptide overlapping between hcv and homo sapiens proteomes peptidology: short amino acid modules in cell biology and immunology generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition a suspenseful game of 'hide and seek' between virus and host molecular mimicry and immune-mediated diseases a pentapeptide as minimal antigenic determinant for mhc class i-restricted t lymphocytes multiple sclerosis mortality and patterns of comorbidity in the united states from autoantibodies revealed: the role of b-cells in autoimmune diseases virus species and virus identification: past and current controversies key: cord-321835-qn33sx8x authors: bailey, emily s.; fieldhouse, jane k.; choi, jessica y.; gray, gregory c. title: a mini review of the zoonotic threat potential of influenza viruses, coronaviruses, adenoviruses, and enteroviruses date: 2018-04-09 journal: front public health doi: 10.3389/fpubh.2018.00104 sha: doc_id: 321835 cord_uid: qn33sx8x during the last two decades, scientists have grown increasingly aware that viruses are emerging from the human–animal interface. in particular, respiratory infections are problematic; in early 2003, world health organization issued a worldwide alert for a previously unrecognized illness that was subsequently found to be caused by a novel coronavirus [severe acute respiratory syndrome (sars) virus]. in addition to sars, other respiratory pathogens have also emerged recently, contributing to the high burden of respiratory tract infection-related morbidity and mortality. among the recently emerged respiratory pathogens are influenza viruses, coronaviruses, enteroviruses, and adenoviruses. as the genesis of these emerging viruses is not well understood and their detection normally occurs after they have crossed over and adapted to man, ideally, strategies for such novel virus detection should include intensive surveillance at the human–animal interface, particularly if one believes the paradigm that many novel emerging zoonotic viruses first circulate in animal populations and occasionally infect man before they fully adapt to man; early detection at the human–animal interface will provide earlier warning. here, we review recent emerging virus treats for these four groups of viruses. | the geographical location of first detections (with known reservoirs) for recently emerged adenoviruses (ads), enteroviruses (evs), coronaviruses, and influenza viruses. zoonotic (coronaviruses and influenza viruses) and non-zoonotic viruses (ads and evs) are shown. for zoonotic viruses, the hosts range from cattle, bats, chickens, camels, wild birds, cats, ferrets, goats, and humans (from left to right). the different sizes of the circles represent the number of human cases during the first outbreaks of the emerging respiratory viruses. human cases of adenoviral infections are shown in blue; human cases of enteroviral infections are shown in yellow; human cases of coronaviral infections are shown in green; and human cases of influenza viral infections are shown in red. zoonotic influenza introduction and epidemiology influenza viruses are rna viruses that are members of the orthomyxovirus family and classified into four types: a, b, c, and d (2) . as shown in table 1 , these four types of viruses are characterized by their immunologically distinct nucleoprotein and matrix protein antigens. influenza a and b viruses consist of hemagglutinin (ha), which binds a sialic acid receptor, allowing the virus to enter the host cell, and neuraminidase (na), which cleaves the sialic acid to release the virus. similarly, influenza c and d viruses contain ha-esterase fusion glycoproteins that also allow for the attachment of viral and cellular membranes. antigenic shifts (influenza a only) in ha, na, and the ha-esterase proteins contribute to the generation of novel viral strains. the host range of influenza viruses includes humans, birds, pigs, bats, and other livestock animals such as cattle and goats. the network of the influenza viral transmission is complex with both inter-and intraspecies transmission. as the viruses continue to change in their genetic sequences, ongoing research is imperative in investigating the ecology of these viruses at the human-animal interface to control further spread of infections and prevent the risk of future pandemics. influenza a virus h3n2 subtypes are frequently reported in swine, avian, and canine hosts that are responsible for highly infectious respiratory diseases in pigs and have been examined as a potential cause of influenza in humans. one study, examining the role of iav in pigs at usa agricultural fairs, reported an average influenza a prevalence of 77.5% among 161 swine across all seven fairs (3) . the genomic sequences of the viruses isolated from the swine were ≥99.89% similar to the h3n2 viruses isolated in humans. at these fairs, iavs were detected at least 1 day before symptoms of the virus were observed in humans, indicating that h3n2 was transmitted from pigs to humans in this case. since 2009, h1n1 virus has posed a significant threat to livestock workers and the greater community and has now become a seasonal influenza virus which circulates in humans. to explore the role of swine production facilities in the development of new swine-like influenza viruses, the spatiotemporal association between weekly influenza-like illnesses (ilis) in humans and the location of pig farms was investigated in north carolina over four influenza seasons (4) . analyses showed that the years of h1n1 pandemic, 2009-2010 and 2010-2011, were closely related with earlier peaking of ili cases. these findings suggest that increased exposure to pigs was associated with earlier observations of the greatest number of human h1n1 cases. in china, the transmission of influenza a between humans and pigs in six farms is being examined using a one health approach, taking into consideration the interconnectedness of humans, animals, and the environment (5) . findings suggest that both a(h1n1)pdm09-like and swine-lineage h1n1 and swine-lineage h3n2 viruses are circulating in swine workers and that these viruses likely reassort and cross species within the pig farms; as such, additional research is needed to understand the relationship between cross species transmission of viruses in humans and pigs. avian influenza viruses are the largest group of influenza a viruses reservoired in aquatic birds or poultry. although infrequently transmitted to humans, many cases have now been reported. human infection with avian influenza can lead to serious health conditions, including death. the first outbreak of an iav strain, h5n1, in humans occurred in hong kong sar, china, in 1997, infecting 18 humans (6). the first identified cases of human infection with h7n2, another avian influenza, occurred in north america with two human cases reported in 2002 (7) . another variation of the virus, h7n7, was the first avian influenza strain reported in europe; it infected 89 humans in 2003. in 2004, the first human cases of h10n7 infections were observed in africa (8) . it is important to note that h5n1 virus outbreak occurred again in 2004, 7 years after its first outbreak in humans, infecting more than 650 humans and causing more than 386 deaths worldwide (9) . the avian influenza viruses have continuously evolved, causing serious infections among humans across the world. h7n9 virus, a sporadic subtype of an avian influenza a virus, was first reported in humans in china in 2013. since the first outbreak, china has been experiencing epidemics annually, with a cumulative number of 1,562 reported cases, 40% of which have led to deaths as of september 2017 (10) . the incidence of the h7n9 infections has been increasing in both humans and poultry and in 2017 alone 764 infections have been reported (11). although h7n9 was first recognized as a low pathogenic avian influenza, two divergent lineages were detected in 2016-including a highly pathogenic avian influenza variant (12). according to the center for disease control and prevention (cdc), h7n9 is now recognized as the virus with the greatest potential to cause a pandemic due to its rapid genetic changes over the last 5 years. this further supports the need to improve disease control strategies and increase efforts to develop an effective vaccination strategy in the future as the spread of the h7n9 infection poses a threat to the poultry business. influenza d virus (idv) is a novel influenza virus that is structurally different from the other influenza viruses. idv was first isolated in 2001 from pigs in usa and since the first report, viral infection has been reported in various locations in usa, europe, and asia. in a serological study, cattle workers and non cattleexposed adults in florida were screened for idv antibodies (13) . of the cattle workers, 97% of idv seroprevalence was observed, while less than 20% was observed in non-cattle-exposed adults, suggesting a greater risk of idv infection for cattle workers. during a swine respiratory disease outbreak in northern italy in 2015, the idv genome was detected and isolated in both pigs and cattle herds (14) . the viral genome isolated from the pigs was closely related to the viral genome isolated in usa in 2011. additionally, the archived serum samples from 2009 had lower idv antibody titers compared to the serum samples collected in 2015. these findings suggest that the incidence of idv infections in pigs may have increased over time, and therefore, idv may pose a public health threat to the community. (17, 18) . recently, it has also been suggested that bats may play a role in the direct human transmission as bat sars-like coronaviruses have been identified in some species (19) . in the past decade, teams from the sabin vaccine institute and baylor college of medicine have been working toward the development of a vaccine for sars-cov. although initial reports indicated that a vaccine may be ready for human clinical trials in 2017, progress has been slow and few human sars vaccine trials have been conducted to date. middle east respiratory syndrome was first recognized in saudi arabia in 2012. many cases were linked to travel to or residence in countries in and near the arabian peninsula. symptoms include severe acute respiratory illness with fever, cough, and shortness of breath. there is limited human-to-human transmission of mers-cov, but exposure to camels is a risk factor for infection, with seroprevalences 15-23 times higher in camel exposed individuals (20) . despite this, major health care-associated transmission of mers-cov was reported in the middle east and korea, with outbreaks characterized by interhospital spread related to overcrowding and a lack of personal protective equipment (21) . the total number of worldwide cases reported to the who as of january 9, 2017 was 2,067 mers-cov cases (22). the cocirculation of covs in its animal reservoirs (camels and bats) raises important questions about the evolution of mers-cov. in a study conducted between 2014 and 2015 in saudi arabia, researchers found that dromedary camels share three cov species with humans, including betacoronavirus 1, mers-cov, and a cov 229e-related virus (23) . with the aim of reducing mers-cov transmission to humans, haagmans et al. developed a vaccine for camels using a poxvirus vehicle (24) . this vaccine has significantly reduced virus excretion among camels and conferred cross-immunity to camelpox infections (25) . enteroviruses are small, positive-sense, single-stranded rna viruses in the picornaviridae family. there are 12 species of evs found globally, including ev a-j (ev-a, b, c, d, e, f, g, h, and j) and rhinovirus a-c (rv-a, b, and c). with low replication fidelity and frequent recombination, evs have viral genetic diversity and a potential for cross-species infection. in february 2013, the international committee on taxonomy of viruses (ictv) approved changes to ev and rhinovirus species names after many of the human ev species were identified and isolated in nonhuman hosts. based on an analysis of picornaviridae hosts listed in the ictv database and subsequent studies of ev infection in non-human primates, there is growing evidence to indicate a potential for future zoonotic transmission between animals and humans (26, 27) . among the most important emerging respiratory viruses are ev68, ev71, coxsackieviruses, echoviruses, rhinoviruses, and polioviruses. enterovirus transmission occurs year-round, with seasonal peaks occurring in the summer and fall (june-october). infants less than 1 year of age are most susceptible to infection, and males are at an increased risk for infection until the age of 20 years (28) . the predominant mode of transmission is through a direct or indirect fecal-oral route; however, certain serotypes are transmitted via the respiratory route, in tears, and via fomites (29) . immunity to evs is serotype specific with most causing mild respiratory infections. rhinoviruses are small, single-stranded rna viruses in the picornavirus family that are responsible for more than half of all upper respiratory tract infections. in addition to exacerbating asthma and chronic obstructive pulmonary disease, rhinoviruses have also been associated with acute respiratory hospitalizations among children (30) . in a large prospective study of us pneumonias, rhinoviruses have been identified as the second most prevalent etiology of pneumonia in children after respiratory syncytial virus and the first most common etiology among adults (31) . there are more than 150 unique types of rhinoviruses. among the three genotypes (a, b, and c) types a and c are most often associated with increased morbidity and bacterial secondary infection. in animals, rhinovirus type c has been associated with morbidity in chimpanzees (32) . with an array of unique serotypes no vaccines or approved antiviral therapies have been commercially produced; however, experiments have suggested that vaccines and antiviral therapy may be possible (33, 34) . enterovirus d68 has caused sporadic respiratory disease outbreaks across asia, europe, and usa since 1960s; however, in 2014, a nationwide outbreak of d68 was associated with severe respiratory illness in usa, resulting in 14 deaths out of a known 1,150 cases (35) . the cdc found 36% of all evs tested during this outbreak were d68 and that patients with a history of asthma were found to be at a disproportionately increased risk of infection (36). one study of the 2014 outbreak found 59% of patients seen with ev-d68 in hospitals across missouri, illinois, and colorado were admitted to intensive care units and 28% received ventilator support (35) . in a study evaluating evs in non-human primates, ev-d68 was detected as a recombinant zoonotic strain (37) . while there are several strains of coxsackievirus and evs that can cause hand-foot-and-mouth disease (hfmd), ev71 is most commonly associated with severe disease outcomes. hfmd predominantly affects young children and is found worldwide but especially in the asia-pacific region. although ev71 is not typically detected in animals, recent research has indicated that it infects non-human primates (38) . various antiviral therapies are currently under study, including small molecules, monoclonals, and antivirals. vaccine candidates are also in development, with two vaccines currently available in china, which involve recombinant proteins, attenuated strains, inactivated whole-virus and virus-like particles, and dna vaccines (39) . first discovered in 1953 by rowe et al., ads are non-enveloped, double-stranded dna viruses with 57 unique serotypes, some of which are specific for attacking the respiratory track, conjunctiva, or gastrointestinal track (40) . key features of ad infections include various symptoms of disease, including rhinorrhea, nasal congestion, cough, sneezing, pharyngitis, keratoconjunctivitis, pneumonia, meningitis, gastroenteritis, cystitis, and encephalitis. illnesses may be asymptomatic, mild, or severe; however, immunocompromised patients and infants are at increased risk of severe morbidity and death. outbreaks of respiratory ad infection are common in both military recruits and other large training groups, such as police trainees. large persistent epidemics of ad type 4-associated respiratory disease have been documented in various military trainees (41) (42) (43) . in response to the increased disease burden from ad4 and ad7 in military recruits, teva has made a vaccine available to military recruits in usa (42) . despite a 12-year hiatus from use, in late 2011 oral ad4 and ad7 vaccines were reintroduced as an infection control measure for military recruits (42) . after reintroduction, military recruits experienced a 100-fold decline in ad disease burden, which accounted for the prevention of approximately 1 death, 1,100-2,700 hospitalizations, and 13,000 febrile ad cases per year among trainees (44) . outbreaks of ad in the general population have been characterized by infection due to novel viruses such as ad7h, ad7d2, ad14a, and ad3 variants. these novel viruses are sometimes associated with high attack rates and a high prevalence of pneumonia. severe mortality is also prevalent among patients with chronic disease and in the elderly. one of the most important novel serotypes, ad14, previously rarely reported, is now considered as an emerging ad type causing severe and sometimes fatal respiratory illness in patients of all ages (45) . beginning in 2005, ad14 cases were suddenly identified in four locations across usa (46) ; the strain associated with this outbreak was different than the original ad14 strain isolated in 1950s. the novel strain, ad14a, has now spread to numerous us states and is associated with a higher rate of severe illness when compared to other ad strains. novel ad species have also been recently detected in crossspecies infections from non-human primates to man in usa and between psittacine birds and man in china (47) . these cross-species infections indicate that ads should be monitored for their potential to cause cross-species outbreaks. in a recent review of the risks of potential outbreaks associated with zoonotic ad (48) , it was noted that intense human-animal interaction is likely to increase the probability of emergent cross-species ad infection. additionally, the recombination of advs with latent "host-specific" advs is the most likely scenario for adaptation to a new host, either human or animal. currently, there are no fda approved antivirals for ad infection; however, the best antiviral success has been seen with ribavirin, cidofovir, and most recently brincidofovir an analog of cidofovir (49) . as it is clear that many emerging respiratory viruses have zoonotic reservoirs, the design and implementation of effective control strategies are increasingly important. it has been suggested that avoiding direct contact with animals known to be zoonotic reservoirs for these viruses is one potential strategy (50); however, in populations where contact at the human-animal interface is common this may not be an acceptable solution. complex disease problems cannot be solved by one institution or one discipline; as such, this presents opportunities to incorporate the one health approach of working across disciplines to incorporate human, animal, and environmental health to solve complex problems. although some of the respiratory viruses described here are found almost exclusively in humans (ad strains), many of the most important emerging respiratory viruses are found at the human/animal interface. this suggests that strategies for novel virus detection should incorporate global surveillance at the human-animal interface to detect potentially emerging zoonotic viruses. this surveillance will require collaboration and cooperation among many stakeholders in order to address emerging and novel viral diseases. eb, jf, and jc conducted the literature review and wrote the manuscript; gg conceived the idea of the review and helped revise the manuscript to add important scientific content and refine the interpretation of the results. all the authors reviewed the final version of the manuscript and agreed to its submission. this work was supported in part by nih/niaid grant r01ai108993-01a1 (gregory gray pi). current infectious disease challenges centers for disease control and prevention. types of influenza viruses influenza a(h3n2) virus in swine at agricultural fairs and transmission to humans are people living near modern swine production facilities at increased risk of influenza virus infection? evidence for cross-species influenza a virus transmission within swine farms, china centers for disease control and prevention. h7n2 questions & answers avian influenza virus a (h10n7) circulating among humans in egypt global and local persistence of influenza a(h5n1) virus food and agriculture organization of the united nations serologic evidence of exposure to influenza d virus among persons with occupational contact with cattle influenza d in italy: towards a better understanding of an emerging viral infection in swine update: outbreak of severe acute respiratory syndrome -worldwide factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor a more detailed picture of the epidemiology of middle east respiratory syndrome coronavirus preventing healthcare-associated transmission of the middle east respiratory syndrome (mers): our achilles heel co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels vaccines against middle east respiratory syndrome coronavirus for humans and camels host and viral traits predict zoonotic spillover from mammals characterizing the picornavirus landscape among synanthropic nonhuman primates in bangladesh airborne transmission of respiratory infection with coxsackievirus a type 21 rhinovirus-associated hospitalizations in young children strategies for safe living among lung transplant recipients: a single-center survey lethal respiratory disease associated with human rhinovirus c in wild chimpanzees immunization with live human rhinovirus (hrv) 16 induces protection in cotton rats against hrv14 infection the potential for a protective vaccine for rhinovirus infections severe respiratory illness associated with a nationwide outbreak of enterovirus d68 in the usa (2014): a descriptive epidemiological investigation african non-human primates host diverse enteroviruses pyramidal and extrapyramidal involvement in experimental infection of cynomolgus monkeys with enterovirus 71 enterovirus 71 infection and vaccines isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture outbreak of febrile respiratory illness caused by adenovirus at a south korean military training facility: clinical and radiological characteristics of adenovirus pneumonia adenovirus vaccines human adenovirus type 7 outbreak in police training center dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines acute respiratory disease associated with adenovirus serotype 14 -four states severe pneumonia due to adenovirus serotype 14: a new respiratory threat? a novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds do nonhuman primate or bat adenoviruses pose a risk for human health brincidofovir is highly efficacious in controlling adenoviremia in pediatric recipients of hematopoietic cell transplant emerging viral respiratory tract infections -environmental risk factors and transmission simultaneous determination of ace activity with 2 substrates provides information on the status of somatic ace and allows detection of inhibitors in human blood human infections with influenza a(h3n2) variant virus in the united states the severity of pandemic h1n1 influenza in the united states novel influenza a (h6n1) virus that infected a person in taiwan human illness from avian influenza h7n3, british columbia genome analysis of south american adenovirus strains of serotype 7 collected over a 7-year period outbreak of adenovirus genome type 7d2 infection in a pediatric chronic-care facility and tertiary-care hospital the 1998 enterovirus 71 outbreak in taiwan: pathogenesis and management human infection with influenza virus a(h10n8) from live poultry markets, china avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome in vitro susceptibility of adenovirus to antiviral drugs is speciesdependent key: cord-306535-j26eqmxt authors: robertson, matthew j.; kent, katarzyna; tharp, nathan; nozawa, kaori; dean, laura; mathew, michelle; grimm, sandra l.; yu, zhifeng; légaré, christine; fujihara, yoshitaka; ikawa, masahito; sullivan, robert; coarfa, cristian; matzuk, martin m.; garcia, thomas x. title: large-scale discovery of male reproductive tract-specific genes through analysis of rna-seq datasets date: 2020-08-19 journal: bmc biol doi: 10.1186/s12915-020-00826-z sha: doc_id: 306535 cord_uid: j26eqmxt background: the development of a safe, effective, reversible, non-hormonal contraceptive method for men has been an ongoing effort for the past few decades. however, despite significant progress on elucidating the function of key proteins involved in reproduction, understanding male reproductive physiology is limited by incomplete information on the genes expressed in reproductive tissues, and no contraceptive targets have so far reached clinical trials. to advance product development, further identification of novel reproductive tract-specific genes leading to potentially druggable protein targets is imperative. results: in this study, we expand on previous single tissue, single species studies by integrating analysis of publicly available human and mouse rna-seq datasets whose initial published purpose was not focused on identifying male reproductive tract-specific targets. we also incorporate analysis of additional newly acquired human and mouse testis and epididymis samples to increase the number of targets identified. we detected a combined total of 1178 genes for which no previous evidence of male reproductive tract-specific expression was annotated, many of which are potentially druggable targets. through rt-pcr, we confirmed the reproductive tract-specific expression of 51 novel orthologous human and mouse genes without a reported mouse model. of these, we ablated four epididymis-specific genes (spint3, spint4, spint5, and ces5a) and two testis-specific genes (pp2d1 and saxo1) in individual or double knockout mice generated through the crispr/cas9 system. our results validate a functional requirement for spint4/5 and ces5a in male mouse fertility, while demonstrating that spint3, pp2d1, and saxo1 are each individually dispensable for male mouse fertility. conclusions: our work provides a plethora of novel testisand epididymis-specific genes and elucidates the functional requirement of several of these genes, which is essential towards understanding the etiology of male infertility and the development of male contraceptives. the world human population reached nearly eight billion people in august 2019. this number continues to rise and is predicted to reach nearly ten billion by the year 2050 [1] . the increasing need to promote family planning through the development of reliable contraceptive options available to both men and women is widely recognized. currently there are numerous contraceptive options available to women; however, identification of a safe, non-hormonal contraceptive option for men is still an ongoing challenge. although several different fertility control alternatives for men have been investigated, none are currently clinically approved for use. our understanding of the mechanisms underlying male reproductive physiology is still at an early stage as the identification and elucidation of the function of key reproductive proteins is still an ongoing effort. identifying druggable protein targets expressed in the male reproductive tract has been the focus of numerous studies dedicated to the development of male contraception. the mammalian epididymis is a segmented organ comprised of a single, highly coiled tubule with functionally and morphologically distinct regions that can be subdivided most simplistically into a proximal, central, and distal region, conventionally named the caput, corpus, and cauda regions, respectively [2] . as mammalian spermatozoa transit through the epididymis, they acquire the ability to recognize and fertilize an egg, properties that they did not possess upon exiting the testis [3] . considering its essential role, the epididymis-in addition to maturing germ cells of the testis and spermatozoa-is a prime target for the development of a male contraceptive. to advance progress towards the development of a non-hormonal male contraceptive, several previous high-throughput studies have been published that identified a number of human, mouse, and rat genes as testis-specific or epididymis-specific [2, [4] [5] [6] [7] [8] [9] . in 2003, schultz et al. conducted the first study to identify male reproductive tract-specific genes using microarrays. through affymetrix-based genome-wide geneexpression analysis of meiotic-and post-meiotic spermatogenic cells, together with parallel analysis of available data from the ncbi unigene database, the authors identified 271 mouse genes as testis-specific, which included genes with both known and unknown function at the time [4] . in the following 5 years, through two additional microarray-based studies of rat testes and purified rat testicular cells, johnston et al. identified 58 [5] and 398 [8] additional or overlapping genes as testisspecific. in 2014, as part of the continued effort to identify novel contraceptive targets, the newer rna-seqbased transcriptomics methodology was utilized identifying 364 human genes as testis-specific [9] . together with antibody-based protein profiling, many of these genes were characterized in terms of the spermatogenic cell populations showing expression [9] . the first high-throughput transcriptomics study to identify epididymis-specific genes was a 2005 mouse epididymal transcriptome study, in which rna isolated from each of the 10 epididymal segments was analyzed by microarray analysis, identifying 75 epididymis-specific genes with distinct patterns of segmental gene regulation [2] . later in 2007, additional transcriptome profiling utilizing whole genome microarrays resulted in identification of 77 previously unreported epididymis-specific transcripts in the mouse [6] and 110 epididymis-specific transcripts in the rat [7] . a significant number of the identified mouse and rat genes in these studies were not known at the time, and only the probe identification numbers were presented. when evaluating potential druggability in a targetbased drug discovery process, one must consider the protein properties that are required for safe and effective inhibition. among the most significant is tissue expression specificity to minimize potential adverse effects, protein function and whether protein activity or interaction with other proteins is potentially druggable, sequence similarity to closely related paralogs that may be ubiquitously expressed, and whether genetically manipulated animal models demonstrate a functional requirement for the target of interest [10] . several noteworthy review publications have mentioned numerous genes whose critical functions, high expression, and specificity to the testes or epididymides make them viable nonhormonal male contraceptive targets [11] [12] [13] [14] [15] [16] [17] [18] . however, among the identified genes, a significant number either (1) are required for fertility, but are expressed in nonreproductive tissues, or (2) are reproductive tractspecific, but, when disrupted, lead to subfertility [10] . in either case, both are ineffective and highly undesirable outcomes for a potential male contraceptive target. therefore, the identification of additional novel male reproductive tract-specific genes would allow for further advances to be made in the quest to develop an effective and safe non-hormonal male contraceptive. in this study, 21 newly acquired and 243 previously published human and mouse rna-seq datasets [9, [19] [20] [21] [22] [23] [24] [25] [26] were processed in parallel through a custom bioinformatics pipeline designed to identify novel reproductive tractspecific and reproductive tract-enriched transcripts. additional databases obtained from illuminating the druggable genome [27] , mouse genome informatics [28] , and ensembl biomart [29] were utilized to stratify the results into subgroups based on protein druggability and on the availability of a mouse model. numerous reproductive tract-specific and reproductive tract-enriched, potentially druggable targets for which no published mouse model exists, congruent in expression across both mouse and human datasets were identified through our analysis and verified through conventional polymerase chain reaction (pcr). we present the data in a manner that should be most relevant and of substantial interest to the male contraceptive development field since identification of new targets worthy of consideration for further functional analysis in a knockout animal model and potential drug targeting continues to be of vast importance. through our results, we identified four novel epididymis-specific genes (spint3, spint4, spint5, and ces5a) and two novel testis-specific genes (pp2d1 and saxo1) worthy of functional validation in an animal model. through the crispr/cas9 system, we generated four individual gene knockouts (spint3, ces5a, pp2d1, and saxo1) and one double knockout mouse model (spint4/5) revealing an essential requirement for spint4 and spint5 in male mouse fertility, and the potential utility of pursuing spint4 in humans as a non-hormonal contraceptive target. despite significant advances in our understanding of the human and rodent testis and epididymis transcriptome, mostly through microarray-based studies, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts, no prior studies have utilized the more state-of-the-art rna-seqbased transcriptomics methodology for analysis of human epididymis-specific transcripts, and no prior studies have utilized rna-seq analysis of rodent reproductive tissues or cells to identify rodent reproductive tractspecific transcripts. to address these gaps in knowledge, and to increase the number of identified reproductive tract-specific genes in both species using the most relevant high-throughput transcriptomics methodology, we analyzed in parallel on a custom bioinformatics pipeline a large number of published and newly acquired human and mouse rna-seq datasets. one hundred and sixtytwo previously published human and 81 previously published mouse rna-seq datasets were retrieved from the sequence read archive (sra). the sra value for each sample is listed in additional file 1: table s1 and additional file 1: table s2 . we also generated 12 new human and 9 new mouse reproductive tissue rna-seq samples (geo accession gse150854). the final dataset is comprised of 3 new and 5 previously published human testis datasets [9] , 27 previously published purified human germ cell datasets [23, 24] , 6 previously published purified human sertoli cell datasets [23, 26] , 9 new and 6 previously published human epididymis segment datasets [21] , 6 previously published mouse testis datasets [19] , 9 new mouse epididymis datasets, 10 previously published purified mouse germ cell datasets [22, 25] , and 3 previously published purified mouse sertoli cell datasets [20] . an additional 118 previously published datasets contributed to the 26 non-reproductive human tissues [30] and 62 previously published datasets contributed to the 14 non-reproductive mouse tissues [19] . figure 1a , b summarizes all the samples acquired for the study. we performed a principal component analysis to visualize the variation in the samples after correcting for batch effects. human reproductive and non-reproductive tissues grouped according to sample type. the reproductive tissue samples clustered by tissue type whether or not they were newly generated or acquired from the sra (fig. 1c) . mouse data showed a similar variation in the samples based on the tissue type (fig. 1d) . for both human and mouse reproductive tissues, samples separated by whether or not the rna-seq was performed on isolated cells or the whole tissue. epididymal tissue was distinct from testis tissue in both human and mouse (fig. 1c, d) . to identify potential male reproductive tract-specific drug candidates, we analyzed the aggregated rna-seq data to find genes that were statistically significant in expression when compared to the non-reproductive tissue that had the maximum expression for that gene. this gene list was then further refined by filtering for genes that were lowly expressed in the non-reproductive tissue that had the maximum expression for that gene (tpm less than or equal to 1.0 for human; tpm less than or equal to 2.0 for mouse). finally, this tpm filtered list was then filtered for the genes that had a reproductive tissue or cell expression value greater than or equal to 10.0 tpm for human, or 8.0 tpm for mouse (fig. 2a) . across all the reproductive tissues, 720 candidate genes were identified in the human and 1304 candidate genes were identified in the mouse samples (fig. 2b , additional file 2: fig. s1 ). additional file 3: table s3 and additional file 4: table s4 summarize the differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, fdr, average and standard deviation tpm expression values, and log2 cpm gene expression value for the human and mouse samples, respectively. the results from the fdr and tpm expression value filtering for the human and mouse samples are summarized in additional file 5: table s5 and additional file 6: table s6 , respectively. additional file 5: table s5 and additional file 6: table s6 report the log2 fold change for the reproductive tissue or cell of interest compared to the tissue with maximal gene expression. the genes identified in additional file 5: table s5 and additional file 6: table s6 pass the filters in at least one of the reproductive tissues or cells of interest. in additional file 5: table s5 and additional file 6: table s6 , a value of zero for a given gene and fold expression comparison indicates that for that comparison, the gene did not pass the filters. the majority of genes were downregulated in the reproductive tissue of interest compared to the maximal gene expressing nonreproductive tissue (additional file 7: fig. s2 ). from the analysis, the majority of the candidate genes that passed the fdr and tpm filters were identified in the testis-or sperm-related cells in both human and mouse samples (additional file 7: fig. s2 ). the majority of candidate genes identified in our screen that were testis-specific were already identified by the human protein atlas [9] and/or our reanalysis of (see figure on previous page.) fig. 1 summary of the human and mouse rna-seq samples used in the identification of novel male reproductive tract-specific drug targets. the rna-seq samples used in the human (a) and mouse (b) analyses are schematically shown. principal component analysis was performed on the human (c) and mouse (d) non-reproductive and reproductive samples separately. the colors of the circles next to the tissues listed in a and b correspond to the colors used in the circles for the pca in c and d. sample size (n) values in red and/or black denote the number of new (red) and previously published (black) samples included in our analysis. fig. 2 identification of candidate drug male reproductive gene targets. a diagrammatic representation of overall methodology used to identify reproductive tract-specific candidate genes in humans (720 genes) and in mice (1062 genes). the maximum gene expression was determined across all the non-reproductive tissue samples for each gene for a reproductive tissue or cell sample of interest. genes were then filtered for significance using a false discovery rate (fdr) of less than or equal to 0.05 based on the differential gene expression analysis for the nonreproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest. genes that passed the fdr filter were filtered such that the average tpm expression value of the maximum expressing non-reproductive tissue was less than or equal to 1.0 tpm and the average tpm expression value of the reproductive tissue or cell of interest was greater than or equal to 10.0 tpm. b diagrammatic representation of the number of human and mouse candidate genes in terms of (1) the number of orthologs in the opposite species, (2) the number of genes previously or not previously identified in a prior transcriptomics-based drug target report, (3) the availability and phenotypic outcome of any reported mouse models, and (4) the number of novel genes without a reported mouse model congruent across both species. the main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in. the numbers in parentheses comprise the total number of candidate genes that are either epididymis-specific or specific to testis and epididymis, but not testis and/or testis cell-specific only. the hpa testis datasets (additional file 8: fig. s3 and additional file 9: table s7 ). thirty-six out of the 91 genes that were identified across all the human epididymis tissue were also identified by the human combined (newly acquired and previously published datasets) testis candidate gene list. finally, the majority of the candidate genes, 300, identified from the combined newly generated and previously published human testis datasets were shared with genes identified from the various testis cell datasets. we identified more candidate genes in the newly generated human epididymis tissues compared to previously published data: 19 out of 54 genes were unique to the newly generated caput samples compared to only 1 out of 36 genes which was unique to the previously published samples, 19 out of 75 genes were unique in the newly generated corpus samples compared to 12 out of 68 genes which were unique to the previously published corpus samples, and 33 genes were unique to the newly generated cauda samples compared to 2 genes in the previously published cauda data with no overlap between the two cauda gene lists (additional file 8: fig. s3 and additional file 9: table s7 ). there were 117 candidate genes that overlapped between the newly generated human testis samples and mouse testis sample gene lists, while there were 134 candidate genes that overlapped between the previously published human testis sample and mouse testis sample gene lists (additional file 8: fig. s3 and additional file 9: table s7 ). across all human epididymis tissue samples, including the newly generated and previously published samples, there were 16 genes in common with the combined list of candidate genes across all the mouse epididymis tissue samples. there was a small overlap between the human and mouse samples when the newly generated human caput, corpus, and cauda tissues were individually compared to the mouse caput, corpus, and cauda tissues; there was an overlap of 10, 12, and 4 for the caput, corpus, and cauda, respectively (additional file 8: fig. s3 and additional file 9: table s7 ). this trend was continued for the candidate gene lists derived from the previously published human caput, corpus, and cauda samples when compared to the candidate gene list from the mouse caput, corpus, and cauda, with 7, 10, and 4 genes in common for the caput, corpus, and cauda comparisons, respectively (additional file 8: fig. s3 and additional file 9: table s7 ). additional file 9: table s7 details the genes that are unique and in common for each of the comparisons. to assess the potential usefulness of the candidate genes identified in each human reproductive tissue as drug targets, we assigned the genes to a protein family (i.e., gpcr or ion channel). the majority of identified genes were not from a traditional drug target family like kinases or enzymes. the testis and germ cell datasets provided the most potential targets while the epididymis datasets provided the fewest (additional file 10: fig. s4a ). the protein family classification for each candidate gene identified in each reproductive tissue is detailed in additional file 11: table s8 . the majority of the candidate genes do not have a reported mouse model (additional file 10: fig. s4b ). additional file 12: table s9 summarizes mouse model availability for each candidate gene identified from human reproductive tissues or cells. figure 3 shows the complete list of novel human genes without a reported mouse model as identified in each of the respective cell and/or tissue datasets. digital pcrs (heatmap) and conventional pcrs demonstrating expression of a subset of the novel human reproductive tract-specific genes without a reported mouse model that we identified are shown in figs. 4 and 5, respectively. additional file 13: fig. s5 shows the complete list of previously identified human genes that remain without a reported mouse model as identified in each of the respective cell and/or tissue datasets. additional file 14: fig. s6 shows the complete list of male reproductive tract-specific human genes for which a previously generated mouse model shows male infertility phenotype, as identified in each of the respective cell and/or tissue datasets. through our bioinformatics analysis of previously published and newly acquired rna-seq datasets, we identified a total of 720 genes as reproductive tract-specific in humans (fig. 2 ). of these genes, 122 genes do not have a mouse gene ortholog, while 598 genes have a mouse gene ortholog (fig. 2) . of those with a mouse gene ortholog, 477 have a single gene ortholog (324 have the same symbol in mouse, while 153 have a different symbol in mouse), while 121 have two or more orthologous mouse genes. seventy-six human genes had 2-3 orthologous mouse symbols, 36 genes had 4-10 orthologous mouse symbols, and 9 genes (fam205a, krta p10-6, magea10, or2ag1, pramef11, pramef2, ssx2, ssx3, and ssx4b) had greater than 10 orthologous mouse symbols (11-93 symbols) (additional file 5: table s5 ). of the 720 human genes that we identified as male reproductive tract-specific, 435 have not been previously identified in a transcriptomics-based male reproductive tract-specific study [2, [4] [5] [6] [7] [8] [9] . the sum of our human data confirms the findings of 232 out of 364 genes from djureinovic et al. [9] . after re-identification of gene symbols from reported affymetrix ids and consideration of orthologous genes (mouse to human and rat to human), our human data confirm the findings of 19 out of 39 genes from johnston et al. [5] , 77 out of 176 genes from schultz et al. [4] , 5 out of 32 genes from johnston et al. [6] , 36 out of 253 genes from johnston et al. [2] , 4 out of 58 genes from johnston et al. [8] , and 3 out of 19 genes from jelinsky et al. [7] . of the 598 genes that have a mouse gene ortholog, 346 have not been previously identified as male reproductive tractspecific, and of these, 233 human genes currently lack mouse phenotype information based on data obtained from ensembl biomart, mgi, impc, and ncbi. three hundred and eighty-six genes were identified as testis-specific through either the reanalysis of djureinovic et al. testis datasets (377 genes identified), analysis of our de novo testis datasets (322 genes identified), or both (additional file 5: table s5 ). three hundred and thirteen genes were congruent across both datasets, while 64 genes were uniquely identified through our reanalysis of djureinovic et al.'s datasets and only 9 genes [ac136352.4, ankrd20a1, ankrd62, fam230a, ggtlc2, iqcm, potec, prnt, and utf1] were uniquely identified through our de novo datasets (additional file 5: table s5 ). interestingly, of the 377 genes we identified through djureinovic et al.'s reanalyzed datasets, 143 were not previously identified in their report [9] or any of the other previous reports [2, [4] [5] [6] [7] [8] . of these 143 genes, we randomly verified 21 of these genes as testis-specific in humans through conventional pcr (fig. 5) . we also verified through rt-pcr an additional 15 genes-such as allc, cdkl3, cox7b2, or2h1, and sppl2c-that had been identified through previous studies (additional file 15: fig. s7 ). of the 386 genes identified through either testis datasets, 150 have not been previously identified; of these, 117 genes have one or more mouse orthologs; and of these, 76 genes are lacking reported phenotype information. of the 76 novel genes lacking a reported mouse model, 7 genes encode enzymes (adam20, cpa5, dusp21, naa11, plscr2, prss38, and triml1), 6 encode transcription factors (bhmg1, foxr2, prdm9, tgif2ly, znf560, znf729), 2 encode transporters (slc22a14, slc25a52), and 61 encode proteins of unknown drug target type two hundred and thirty-three novel human reproductive tract-specific genes that each have mouse orthologous genes but with no reported knockout mouse models. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse (109 genes). genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation (74 genes) . genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis (10 genes). (such as etdb, smim36, bend2, btg4, cnbd1, dppa2, efcab5, erich6, fthl17, iqcm, mroh2b, ms4a5, oosp2, pnma6e, ppp4r3c, rbmxl3, rtl9, spdye4, spem2). all of these genes are listed in fig. 3 , and many of these genes are listed in figs. 4, 5, and/or 6. to the best of our knowledge, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts. through our analysis, we identified 291 genes as human testis-specific through one or more of the human germ cell datasets, but not through either of the human testis datasets (additional file 5: table s5 ). seventy-six genes were identified exclusively through one or more of the five human spermatogonia datasets (genes such as anp32d, c13orf42, dscr4, or13g1, or2d2, or52e4, ssx2, tle7), while 18 genes were identified exclusively through the human spermatocyte datasets (genes such as h2bfm, mageb17, mageb18, or2b6, tcp10, and znf709) and 79 genes were identified exclusively through the human spermatid datasets (genes such as ac013269.1, clec20a, or7e24, pramef2, spat a31a3, tmem191c, znf679). thirty-four genes were identified through all three cell types' datasets (genes such as ccdc166, eloa2, fam47a, heat r9, and spata31a1). many of these genes are listed in figs. 3, 4, 5, and/or 6. of the 291 genes identified as human testis-specific through one or more of the human germ cell datasets, 252 genes have not been previously identified, 201 of which have one or more equivalent mouse orthologs with 139 of these genes having not been knocked out in mouse. of these 139 novel genes with no mouse model, 8 encode enzymes (glt6d1, prss48, satl1, sult6b1, tmprss7, tpte2, triml2, and ttll8), 1 encodes an epigenetic protein (taf1l), 6 encode gpcrs (gpr156, tas2r13, tas2r30, tas2r46, tas2r50, vn1r2), 1 encodes a kinase (cdkl4), 33 encode ogpcrs (such as or2d3, or3a2, or52e5, or8g5, or10j1, and ) and obp2b met candidate threshold through our analysis of human testes datasets but did not meet candidate threshold from any of the germ cell or sertoli cell datasets, indicating potential expression in peritubular myoid cells, leydig cells, or other cell outside of the seminiferous epithelium. fam36a has not been previously identified, and neither mouse orthologs (1700011m02rik, gm9112) have been knocked out. obp2b was previously identified through djureinovic et al. [9] and johnston et al. [6] ; however, of the equivalent mouse orthologs (lcn4, obp2a, obp2b), only obp2a has been knocked out revealing abnormal coat/hair pigmentation [31] . ism2 and magec2 were identified through both human sertoli cell datasets, while also identified through testis and/or germ cell datasets. both genes have been previously identified (ism2 [8] , magec2 [9] ). ism2 knockout mice display non-reproductive phenotypes [9] . consistent with this finding, our mouse data do not identify ism2 as reproductive tract-specific in mice. magec2 lacks a mouse ortholog for functional analysis in mice. human sertoli cell-specific krtap2-3, krtap4-12, lhx9, and psg5 were identified through one or both human sertoli cell datasets but were not identified through any of the testis or germ cell three hundred and two novel mouse genes with human orthologs without a reported mouse model. the listed genes were identified in one or more mouse datasets as indicated in the venn diagram. underlined genes were also identified through our studies as reproductive tractspecific in human (111 genes). genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation (60 genes). genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis (14 genes). datasets indicating sertoli cell-specific expression in the testes (additional file 5: table s5 ). none of these genes have been previously identified as reproductive tractspecific in humans although lhx9 and psg5 have mouse orthologs that have been knocked out [32] [33] [34] [35] [36] [37] . human krtap2-3 has mouse orthologs krtap5-2, gm4559, gm40460, and gm45618, and human krta p4-12 has mouse orthologs krtap4-7 and gm11555; none of these mouse orthologs have been knocked out ( fig. 3 and additional file 5: table s5 ). psg5 knockout mice display non-reproductive phenotypes [32] [33] [34] [35] [36] ; however, lhx9 knockout mice display absent testes and sterility due to an essential requirement for lhx9 during mouse gonad formation [37] . a lhx9-gfpcreer knock-in mouse line-generated by knocking-in gfpcreer at the endogenous lhx9 locuscrossed with the rosa26-tdtomato reporter mouse line revealed cre recombinase activity in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons [38] . thus, lhx9 is not reproductive tract-specific in mice. our mouse data confirm this finding. to the best of our knowledge, no prior studies have utilized rna-seq for analysis of human epididymis-specific transcripts. through our studies, we identified 39 genes as human epididymis-specific through one or more of the human epididymis segment datasets that were not identified through any of the other human male reproductive tissue or cell datasets, indicating true epididymis specificity (additional file 5: table s5 ). of these 39 genes identified as human epididymis-specific, 29 genes have not been previously identified, 24 of which have equivalent mouse orthologs with 16 of these genes having not been knocked out in mouse. of these 16 novel human epididymis-specific genes with no mouse model, 1 encodes an enzyme-related gene (spint3) and the remaining 15 encode proteins of unknown drug target type (such as actbl2, bsph1, mslnl, spag11a, spag11b, wfdc10a, and wfdc9) (fig. 3) . seven genes were identified through our de novo sequenced human epididymis segment datasets that were not identified through our reanalysis of the human epididymis segment datasets by browne et al.; two of these genes are considered novel without mouse models: defb104a and defb104b (fig. 3 , additional file 5: table s5 , and additional file 9: table s7 ). meanwhile, five genes were identified through our reanalysis of the human epididymis segment datasets by browne et al. that were not identified through our de novo-sequenced human epididymis segment datasets; two of these genes are considered novel without mouse models: actbl2 and mslnl (fig. 3 , additional file 5: table s5 , and additional file 9: table s7 ). fifty-two genes met the criteria for identification as epididymis-specific through one or more of the human epididymis segment datasets, while also being identified as reproductive tract-specific through one or more of the testes, germ cell, and/or sertoli cell datasets (additional file 5: table s5 ). thus, these targets are not epididymis-specific per se, but may be desirable potential male contraceptive targets considering their broader target availability. of these 52 genes identified as human male reproductive tract-specific and epididymisexpressed, 20 genes have not been previously identified, 15 of which have one or more equivalent mouse orthologs with 11 of these genes having not been knocked out in mouse. all 11 of these novel genes with no mouse model encode proteins of unknown drug target type (al163195.3, ccdc168, ccnb3, defb121, defb134, eppin-wfdc6, krtap2-3, magea11, pnma6e, spem2, tex44) ( fig. 3 and additional file 5: table s5 ). since model organisms other than mice may be of interest for the future functional study of human genes-especially those for which no known mouse ortholog exists-we list novel reproductive tract-specific human genes without a mouse ortholog in additional file 16: fig. s8 , which may be of interest for generating null rat or marmoset models [39] . digital pcr (heatmap) demonstrating expression of a subset of these novel human reproductive tract-specific genes without mouse orthologs is shown in additional file 17: fig. s9 . through our bioinformatics analysis of previously published and newly acquired mouse rna-seq datasets, we identified a total of 1062 genes as reproductive tractspecific in mice. of these genes, 303 genes do not have a human gene ortholog, while 759 genes do (fig. 2) . of those with a human gene ortholog, 632 have a single ortholog (451 with the same gene symbol in human; 181 with a different symbol), while 127 mouse genes have two or more ortholog human genes (fig. 2 ). ninety-two mouse genes have 2-3 orthologous human symbols, 16 genes have 4-10 orthologous human symbols, and 19 genes (such as 1700080o16rik, ankrd36, fam90a1b, gm15319, magea10, pramel1, spdye4a, spdye4b, and zfy1) have greater than 10 orthologous human symbols ranging anywhere from twelve to twenty-six symbols (additional file 6: table s6 ). of the 1062 mouse genes that we identified as male reproductive tract-specific in mouse, 743 have not been identified in a previous transcriptomics-based study [2, [4] [5] [6] [7] [8] [9] . the sum of our mouse data confirms the findings of 150 out of 271 mouse genes from schultz et al. [4] , 7 out of 54 mouse genes from johnston et al. [2] , and 6 out of 23 mouse genes from johnston et al. [6] (additional file 18: table s10 ). of the 759 mouse genes that have a human ortholog equivalent, 482 have not been previously identified as male reproductive tract-specific, and of these, 302 genes currently lack mouse phenotype information based on data obtained from ensembl biomart, mgi, impc, and ncbi (fig. 6 ). digital pcr (heatmap) demonstrating expression of a subset of the novel mouse reproductive tract-specific genes with human orthologs and no reported mouse model, and with human reproductive tract enrichment, is shown in fig. 7 . seventeen novel genes without mouse models (8030474k03rik, fthl17b, fthl17c, fthl17d, fthl17e, fthl17f, gm15262, gm18336, magea3, magea4, magea5, magea6, magea8, mageb2, xlr5a, xlr5b, and xlr5c) were identified through the mouse id4+ spermatogonia datasets that were also identified as spermatogonia-specific through the human datasets (bend2, bx276092.9, fam9a, fthl17, magea3, magea4, magea6, mageb6, and pnma6e) and were not identified through either mouse or human testis datasets, indicating restricted expression in spermatogonia, spermatogonial stem cells, or both (additional file 5: table s5 and additional file 6: table s6 ). eight genes (1700080o16rik, ccnb3, gm21964, gm4779, pet2, prr23a1, prr23a2, and prr23a3) were identified through the mouse id4+ spermatogonia datasets, genes whose human orthologs-ccnb3, dcaf8l1, magea10, magea11, magea12, magea9, magea9b, prr23a, prr23b, prr23c, and tle7-were also identified through the human spermatogonia datasets. since these genes were also identified through mouse, human, or both species' respective testis datasets, this indicates either strong expression in the spermatogonia compartment or expression outside of and in addition to the spermatogonia compartment. twenty-two genes were identified as mouse sertoli cellspecific as they were not otherwise identified as reproductive tract-specific through our analysis of mouse testes or germ cell datasets (encode project consortium testes and helsel et al.'s id4+ germ cell datasets) (additional file 6: table s6 ). of these 22 genes, 18 have human orthologs; of these 18 genes, 17 are novel and not previously identified as male reproductive tractspecific; of these 17 genes, 7 have not been previously knocked out in the mouse (c1ql2, gm45015, mageb18, mycs, shc4, sowahd, and tmsb15b2); and of these 7 genes, 2 genes (gm45015 and mageb18) were identified with human orthologs (pnma6e and mageb18) that were also identified through our human analysis as human reproductive tract-specific (additional file 5: table s5 and additional file 6: table s6 ). unlike the limited number of reproductive tract-specific genes we identified through human sertoli cell-specific datasets, a considerable number of genes-202 mouse genes-were identified as reproductive tract-specific through analysis of zimmermann et al.'s mouse postnatal day 35 sertoli cell datasets that were also identified through either the mouse testis datasets, mouse id4+ germ cell datasets, or both (additional file 6: table s6 ). of these 202 genes, 160 have one or more human orthologs; of these 160 genes, 54 are novel and not previously identified as male reproductive tract-specific; of these 54 genes, 36 have not been previously knocked out in the mouse; and of these 36 genes, 16 (1700011l22rik, 1700011m02rik, 1700018b08rik, 1700042g07rik, ankrd36, ankrd60, etd, gm39566, gm6657, gm9112, magea10, spata31d1b, tcp10c, tex44, tex48, and wfdc9) were identified with human orthologs that were also identified through our analyses as human reproductive tractspecific (fig. 6 , additional file 5: table s5 , and additional file 6: table s6 ). to the best of our knowledge, published rna-seq data of mouse whole epididymis or epididymis segments does not exist, for the identification of epididymis-specific transcripts or otherwise. therefore, we isolated caput, corpus, and cauda segments from adult (postnatal day 60) b6/129 mice and subjected the rna to sequencing. sixty-six genes were identified as mouse epididymisspecific as they were not identified as mouse male reproductive tract-specific through our reanalysis of the entable s6 ). of these 66 genes, 48 have human orthologs; of these 48 genes, 34 are novel and not previously identified as male reproductive tract-specific; of these 34 genes, 17 have not been previously knocked out in the mouse (ascl4, bsph1, c1s2, clec18a, cyp2j13, cyp4a30b, defb42, gm45826, lce6a, lcn6, muc15, odam, spag11a, spag11b, spink13, svs1, tchhl1); and of these 17 genes, 5 genes (bsph1, defb42, gm45826, spag11a, and spag11b) were identified with human orthologs (bsph1, defb136, spag11a, and spag11b) that are also human epididymis-specific (fig. 6 , additional file 5: table s5 , and additional file 6: table s6 ). sixty-five genes were identified as reproductive tractspecific in mouse with expression in both epididymis and testis and/or testis cell. of these 65 genes, 48 have human orthologs; of these 48 genes, 24 are novel and not previously identified as male reproductive tract-specific; of these 24 genes, 14 have not been previously knocked out in the mouse (1700009n14rik, 4922502d21rik, 4930563d23rik, d330045a20rik, defb28, dgkk, fam90a1b, gm6871, hrasls5, nxf3, shc4, spint3, trpc5os, wfdc9); and of these 14 genes, 3 genes (spint3, trpc5os, and wfdc9) were identified with human orthologs (spint3, trpc5os, and wfdc9) that are also human epididymis-specific (fig. 6 , additional file 5: table s5 , and additional file 6: table s6 ). through the aforementioned studies, we identified spint3, spint4, ces5a, pp2d1, and saxo1 as congruent in expression across both mouse and human datasets with expression restricted to either the epididymis (spint3, spint4, ces5a) or the testis (pp2d1 and saxo1) (figs. 3, 4, 5, and 6; additional file 5: table s5 ; additional file 6: table s6 ). spint5 was also identified as epididymisspecific in mouse (fig. 5 , additional file 6: table s6 ); however, in humans, spint5p is a pseudogene that is not processed into protein. conventional rt-pcr of a panel of mouse and human tissue cdnas confirmed epididymis-or testis-restricted expression of spint3, spint4, ces5a, pp2d1, and saxo1 in both species and spint5 in mouse (fig. 5) . to glean insight into the onset of expression for the epididymis-specific genes, spint3, spint4, spint5, and ces5a; whole epididymides from postnatal days (p) 3, p6, p10, and p14; and epididymis segments (caput, corpus, and cauda) from p21, p28, p35, and p60 aged mice were collected and analyzed through rt-pcr (additional file 19: fig. s10 ). spint3 expression begins as early as p3 (low) and gradually increases through p10 and p14 reaching steady levels throughout p21 to p60 in all three segments of the epididymis (additional file 19: fig. s10 ). in contrast, spint4 and spint5 display near identical expression levels with no expression at p3, p6, or p10, and expression apparent at p14 and later time points, with expression restricted to corpus only at p21 and p28, and caput and corpus, but not cauda at p35 and p60 (additional file 19: fig. s10 ). rnascope-based fluorescence in situ hybridization revealed a distinct segment-specific pattern of expression for spint4 that was identical to spint5, with both showing expression in most of the epithelial cells restricted to a brief region of distal caput/proximal corpus (additional file 20: fig. s11) . spint3, on the other hand, displayed a pattern of expression in a majority of epithelial cells that begins just a bit further downstream along the corpus, but persisting for much further along the corpus, throughout the corpus and into the cauda (additional file 20: fig. s11 ). these results indicate that spint3 shares a role that is distinct from spint4 and spint5 and indicates a potential redundancy between spint4 and spint5 and how humans may have lost the evolutionary pressure to keep spint5p as a protein-coding gene. to glean insight into the potential spermatogenic cell population(s) expressing pp2d1 and saxo1, we performed rt-pcr of mouse testes isolated at postnatal day (p) 3, a time point enriched for gonocytes; p6 (onset of expression of type a spermatogonia); p10 (early spermatocytes); p14 (late spermatocytes); p21 (spermatids); and p35 and p60, which display complete spermatogenesis [40] (additional file 19: fig. s10 ). expression of pp2d1 and saxo1 is detected at similar levels at p28 and later, but not at p21 or before indicating expression during spermiogenesis and spermiation (additional file 19: fig. s10 ). to determine the male reproductive requirement and potential functional role of the identified novel male reproductive tract-specific genes, spint3, ces5a, pp2d1, and saxo1 were individually ablated by crispr/cas9mediated zygote approach. since in humans spint5p is a pseudogene, and in mice, spint5 protein is most similar in sequence to mouse spint4, we simultaneously ablated both mouse spint4 and spint5 genes, which on mouse chromosome 2 are only separated by 12.9 kilobases. the efficiency of generating each mutant is summarized in additional file 1: table s11 . each of the genes contained deletions of differing sizes and genomic targets. the genomic sequences flanking the deletion in each mutant are presented in additional file 1: table s12 , and representative sanger sequencing results for each mutant are presented in fig. 8 . using the forward and reverse primer pairs presented in fig. 8a -e and listed in additional file 1: table s13 , offspring carrying the mutant alleles were identified through routine genotyping (fig. 8k-o) . spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mouse lines were examined in parallel with littermate controls of equivalent age to determine the effect of gene ablation on spermatogenesis, sperm maturation, and fertility in male mice. none of the knockout strains generated in this study displayed any overtly abnormal appearance, difference in body weight (fig. 9 ) or composition, or difference in behavior when compared to the controls. to determine the male reproductive requirement of each of the genes of interest, spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout and control adult male mice were housed continuously with two females for 3 months and the size and number of litters were recorded. although spint3, pp2d1, and saxo1 knockout males sired a number and size of litters during the test mating period that was not significantly different from controls (fig. 9a-c) , spint4/5 and ces5a knockout males sired significantly fewer litters and pups over the test mating period (fig. 9a-c) . spint4/5 null males displayed a statistically significant 95% reduction in the number of litters and pups sired per male and statistically significant 64% reduction in litter size, over the 3-month mating period (n = 9 controls, n = 9 kos) (fig. 9 ). seven out of 9 males displayed complete infertility, and the two remaining males, who sired pups, sired pups at a significantly reduced number of litters and pups per month with litters of reduced litter size (fig. 9a-c) . this fertility defect in spint4/5 double ko males was not associated with any significant changes in epididymis and testis histology (additional file 21: fig. s12 ) or sperm numbers, motility, and morphology ( fig. 9g-i) . ces5a null males displayed a variegated phenotype with an overall statistically significant 50% reduction in the number of litters and pups sired per male, but no significant difference in litter size, over a 3-month mating period (n = 9 controls, n = 9 kos). the fertility defect in ces5a ko males was associated with significant changes in epididymis histology (additional file 22: fig. s13 ) and significant reductions in sperm motility and progressive motility (fig. 9h, i) . ces5a null males displayed a 50% reduction in sperm motility and progressive cells, a 50% increase in static cells, and a 25% decrease in average path velocity and progressive velocity after hyperactivation. no changes in testis histology were found (additional file 22: fig. s13) , and despite the sperm motility defect, scanning electron microscopy failed to identify a morphological defect in ces5a null sperm in comparison to controls (additional file 23: fig. s14) . the epididymides and testes weights of spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice were not significantly different from littermate control mice (fig. 9e, f) . histological analyses of testes from spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice revealed all had seminiferous tubules with intact epithelia and the presence of all germ cell subtypes and all stages of spermatogenesis (additional file 21: fig. s12 and additional file 24: fig. s15 ). histological analyses of caput, corpus, and cauda from spint3, spint4/5, pp2d1, and saxo1 ko mice revealed spermatozoa in tubule lumens of all knockouts with no significant differences in epididymal histology in comparison to controls (additional file 21: fig. s12 and additional file 24: fig. s15 ). however, ces5a knockout mice displayed significant histological abnormalities including lumen dilation (possibly from occlusion), inflammation, and the appearance of abnormal epithelia (additional file 22: fig. s13 ). computer-assisted sperm analysis of spint3, spint4/5, pp2d1, and saxo1 knockouts 9 phenotype analysis of crispr/cas9 generated null mice for determining the contraceptive potential of the selected genes. spint4/5 and ces5a null mice show significant fertility defects; meanwhile, spint3, pp2d1, and saxo1 null mice appear normal. fertility (a-c), body and reproductive organ weights (d-f), and sperm parameters (g-i) were all measured between knockout (−/−) and littermate control [wild-type (+/+) and heterozygous (+/−)] mice as indicated. bars represent mean ± sem. *p < 0.05, **p < 0.01, ***p < 0.005. ns, not significant. showed no statistically significant differences across all measured parameters including sperm concentration, sperm motility, and progressive motility (fig. 9g-i) . ces5a knockouts displayed significant decreases in sperm number and sperm motility (fig. 9h, i) ; however, cauda epididymal sperm isolated from a variety of ces5a null animals looked morphologically indistinguishable to controls (additional file 23: fig. s14 ). to date, the etiology of idiopathic male infertility is not fully understood, and hormonal male contraceptives have not been effective. therefore, identification of novel reproductive tract-specific genes, and elucidating the functional requirement or lack thereof of these genes, is essential towards understanding the etiology of male infertility and the development of male contraceptives. despite significant advances in our understanding of the human and rodent testis and epididymis transcriptome, mostly through microarray-based studies, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts, no prior studies have utilized the more state-of-the-art rna-seq-based transcriptomics methodology for analysis of human epididymis-specific transcripts, and no prior studies have utilized rna-seq analysis of rodent reproductive tissues or cells to identify rodent reproductive tract-specific transcripts. to address these gaps in knowledge, and to increase the number of identified reproductive tract-specific genes using the most relevant high-throughput transcriptomics methodology, we analyzed in parallel on a custom bioinformatics pipeline a large number of published and newly acquired human and mouse rna-seq datasets. through our studies, we identified and verified many novel male reproductive tract-specific transcripts in both species, and through the crispr/cas9 system, we interrogated the reproductive requirement of a subset of these genes. we found that spint4 (together with spint5) in mice is required for normal male mouse fertility, and although not required for male fertility, we identified ces5a as playing a major biological role in the epididymis. we report the remaining genes that we knocked out-spint3, pp2d1, and saxo1as dispensable for male reproductive function, which is essential information to disseminate to the scientific community. our study also verified the male reproductive tract-specific expression of many previously identified genes (additional file 13: fig. s5 , additional file 15: fig. s7) , and genes for which previously published mouse models display male infertility phenotypes (additional file 14: fig. s6 ). this later group of already functionally validated genes serves as potential male contraceptive targets worth underscoring to the research community. prior to massively parallel microarray-based and rnaseq-based transcriptomics analyses for the identification of reproductive tract-specific genes, the ncbi unigene database was a valuable resource for many in the male reproductive biology field for identifying testis-specific transcripts [4, [41] [42] [43] . although in our study we only considered prior microarray-based and rna-seq-based studies when considering the novelty of the genes that we identified, it is worth noting that several genes that we identified-not previously identified in microarray-based and rna-seq-based transcriptomics studies-were previously identified through studies that solely utilized the unigene database [41, 42] . nineteen human genes that we identified-c16orf82, ccdc27, cpa5, fam217a, fam46d, fam47c, fbxo24, fbxw10, fkbp6, galn tl5, kcnu1, mageb3, mroh2b, nutm1, prdm9, rbmxl3, spata31e1, triml1, and trpc5os-were previously identified by liu et al. [42] , and seven mouse genes that we identified-1700013d24rik, akap3, ankrd36, hrasls5, spesp1, tex22, and ubqlnl-were previously identified by choi et al. [41] and liu et al. [42] . thus, our results confirm the findings of these previous studies. since more than half of all human protein-coding genes are categorized as unknown in terms of drug target potential (additional file 11 table s8 ), and only 30% encode for classically druggable enzymes, gpcrs, ion channels, nuclear receptors, and transporters (additional file 10 fig. s4) , the potential to find new undiscovered drug targets that can be drugged using classical approaches is somewhat limited. indeed, in our study, we found one hundred and nine genes to be novel in terms of previously published high-throughput transcriptomics studies, without a current reported mouse model, and reproductive tract-specific in both humans and mice (figs. 2, 3, and 6 ). many of these genes (93 genes; 85%) fall into the category of unknown and may otherwise be considered "undruggable" due to various challenges with existing targeting approaches [10] . however, the contraceptive potential of these genes should not be overseen, but rather investigated for potential identification of a high-affinity small molecule that can either interfere with protein-protein interaction (ppi) or target the protein specifically for degradation using a new technology called proteolysis targeting chimeras (protacs). protein-protein interaction targets are not deemed undruggable, based on the discovery of small molecules capable of deeper and higher affinity binding within the contact surfaces of the target protein [44] . additionally, once a high-affinity small molecule against a specific target protein is identified, an engineered protac molecule can mark a target protein for proteasomal degradation by linking the target protein to the polypeptide co-factor, ubiquitin [45] [46] [47] . there are currently various combinations of protacs developed to overcome the limitations of cell permeability, stability, solubility, selectivity, and tissue distribution [48] [49] [50] [51] . therefore, disrupting ppis or utilizing protacs provides the potential to greatly promote the development of contraceptive drugs against the "undruggable" nonenzymatic target protein space. if gene knockouts for closely related and ubiquitously expressed paralogs display no abnormal phenotype, then unintended drug targeting of these proteins may result in no side effects in humans. however, the burden of safety for a male contraceptive is extremely high, and if it can be avoided, targeting non-reproductive tractexpressed proteins in humans should be avoided since the functional requirements for these proteins may not be fully understood. further, although mice are one of the best models for human disease, they are unable to communicate when they are unwell, a phenomenon that may occur independent of any measurable phenotypic traits. thus, potential reported side effects in humans during clinical trials may have been present, but missed, during animal studies, or in fact be present in humans and not in mice because of the vast biological differences across these two species. thus, with drug safety in mind, reproductive tract-specific candidates should be prioritized based on somatic cell-expressed protein sequence similarity, especially in the drug binding pocket. according to ensembl, several novel reproductive tract-specific genes without mouse models that we identified and verified-ac022167.5, al672043.1, bhmg1, c1orf105, c2orf92, c4orf51, ccdc196, spint3, tex44, tex48, tex51, and trpc5os (figs. 3, 4, and 5; additional file 5: table s5 )-have no known associated paralogs, indicating reproductive tract-specific drug targeting is highly likely. additionally, spag11a and spag11b are epididymis-specific paralogs (figs. 3, 4 , and 5; additional file 5: table s5 ) with no other known paralogs according to ensembl. efcab5 (figs. 3 and 5, additional file 5: table s5 ) has a ubiquitously expressed paralog, nsrp1, with only 7% amino acid sequence similarity, indicating specific drug targeting potential for this candidate. likewise, erich6 and mroh2b (figs. 3 and 5, additional file 5: table s5 ) have ubiquitously expressed paralogs erich6b and mroh2a, respectively, with 20% and 30% amino acid sequence similarity, indicating reasonable potential for specific drug targeting. prr23a, prr23b, and prr23c are testis-specific paralogs (figs. 3 and 5, additional file 5: table s5 ) with prr23d2 as the next closest paralog according to ensembl. since prr23d2 has less than 26% amino acid sequence similarity to prr23a, prr23b, and prr23c, but appears to be epididymis-specific according to the human protein atlas [52] , all four proteins of unknown function make suitable drug candidates. likewise, spat a31d1, spata31d3, and spata31d4 are testis-specific paralogs (figs. 3 and 5, additional file 5: table s5 ) with spata31a5 as the next closest paralog according to ensembl. since spata31a5 has less than 26% sequence similarity to spata31d1, spata31d3, and spat a31d4, but also appears to be reproductive tractspecific according to hpa [52] , all four proteins with unknown function also appear to be worthy of consideration for potential drug targeting. tpte and tpte2 are testis-specific paralogs (figs. 3 and 5, additional file 5: table s5 ) with ubiquitously expressed pten as the next closest paralog according to ensembl. since pten has less than 25% sequence similarity to tpte and tpte2, off-target effects appear to be unlikely. likewise, wfdc10a, wfdc10b, and wfdc13 are all epididymis-specific paralogs with the closest nonreproductive tract-expressed paralog, wfdc5, having less than 26% sequence similarity to any of the rts paralogs, also indicating high drug specificity potential. several additional novel reproductive tract-specific enzyme and gpcr genes without mouse models that we identified-gpr156, prss38, prss48, sult6b1, tmpr ss7, triml2, and ttll8 (fig. 3, 4 , and 5; additional file 5: table s5 )-have non-reproductive tract-expressed paralogs with less than 35% sequence similarity indicating good drug specificity potential. a novel testis-specific transporter gene without a reported mouse model that we identified, slc25a52, would make a poor drug candidate since its closest paralog, slc25a51, is 93% similar in amino acid sequence and ubiquitously expressed. cpa5, iqca1l, and ppp4r3c have ubiquitously expressed paralogs, cpa1, iqca1, and ppp4r3b, respectively, with 50-60% protein sequence similarity indicating careful consideration must be made for potential drug targeting without off-target effects. of the seventy-three genes that our study identifies as reproductive tract-specific in humans and for which a published mouse model shows male infertility phenotype (additional file 14: fig. s6) [28, 29, 31] , it is worth noting that 21 genes-cnbd2, defb110, fam170a, fbxo47, meig1, meiob, meioc, odf1, odf4, rec114, rnf17, spaca1, spata22, spem1, spo11, sycp1, terb1, tex19, tex38, tnp2, and topaz-do not have any associated paralogs and, thereby, may be considered most suitable for further drug development. however, it is also worth noting that it may be possible that genes required for male fertility in mice may not necessarily be required for male fertility in humans. of the seventy-three human reproductive tract-specific genes our study identified with male mouse infertility phenotypes, twenty-seven genes-actl7b [53] , akap4 [54] , boll [55] , brdt [55] [56] [57] [58] [59] [60] , catsper4 [54] , ccdc155 [61] , fkbp6 [55, [61] [62] [63] , meig1 [64] , meiob [55] [56] [57] 65] , nanos2 [55, 61] , odf1 [55] , prdm9 [61, 66, 67] , prss37 [55] , rad21l1 [68] , rbmxl2 [55, 62] , rnf17 [69] , sohlh2 [61, 70] , spaca1 [55] , spata16 [55, 56] , spem1 [55] , spo11 [55, 58, 61] , sun5 [55] [56] [57] , sycp1 [55] , tex38 [55] , tnp2 [55] , tssk1b [62] , and zpbp [55] -are currently associated with mutations underlying human male infertility, confirming a similar functional requirement for these genes in humans may exist. for the remaining 45 genes, however, either these genes are not required for human male fertility as they are required in mice, or associated mutations in male infertile patients have not yet been reported. although many reproductive tract-specific genes have been studied through functional genetics approaches, many remain to be solved. elucidating the function of these novel genes is necessary to build a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and the development of male contraceptives. in this study, four epididymis-specific genes (spint3, spint4, spint5, and ces5a) and two testis-specific genes (pp2d1 and saxo1) were deleted in mice to determine their functional requirement in male fertility and potential utility as male contraceptive target. we chose to study these genes because all but saxo1 encode enzymes or enzyme-related protein products and are thus considered druggable in the classical sense. saxo1, a cilia-related gene, was chosen because prior literature demonstrated expression in sperm [71] . although not druggable in the classical sense, if targeted through non-canonical approaches, one could obtain a fast-acting drug with greater reversibility potential and a decreased likelihood of affecting testicular function and size. the epididymis-specific genes we chose to target for functional analysis, by the very nature of their tissue's expression, also fit this potential drug profile of modulating only the latest stages of sperm development. analyses of testis and epididymis organ weights and histology, sperm parameters and morphology, and fertility revealed no significant differences in spint3, pp2d1, and saxo1 knockout mice in comparison to littermate controls demonstrating that, individually, spint3, pp2d1, and saxo1 are not required for male mouse fertility and are not suitable targets for the development of a male contraceptive. however, we found partial effects on male fertility in ces5a knockout mice and profound effects on male fertility in spint4/5 double knockout mice. ces5a is a member of a multigene family of mammalian carboxylesterases that can hydrolyze ester, thioester, amide, and carbamate linkages in a wide variety of endogenous and exogenous molecular substrates, including triglycerides, thus playing key roles in both metabolism and detoxification [72] [73] [74] [75] . ces5a shares roughly similar percent homology (~40% homology) to all four of its related paralogs, ces1, ces2, ces3, and ces4a. human carboxylesterase 1 (ces1) is predominantly expressed in the liver and has been shown to have triglyceride hydrolase activity as overexpression of human ces1 in cells leads to an increase in cholesteryl ester hydrolysis and free cholesterol efflux [76] . further, mouse ces1g-a protein expressed by one of a cluster of eight syntenic genes (ces1a through ces1h) orthologous to the human ces1 gene-has been shown to have triglyceride hydrolase activity as ces1g null mice display hyperlipidemia and abnormal lipid homeostasis including increased liver and circulating cholesterol and triglycerides, and altered saturated and unsaturated fatty acid levels [77, 78] . therefore, it is likely that ces5a exhibits similar carboxylesterase activity in the epididymis hydrolyzing cholesteryl ester and affecting free cholesterol efflux. indeed, recombinant ces5a protein has been previously shown to have carboxylesterase activity hydrolyzing cholesterol ester and choline ester [79] . since sperm cholesterol content is significantly decreased during epididymal maturation [80, 81] and a proper cholesterol/phospholipid (c/pl) ratio of the sperm plasma membrane is required for sperm capacitation [82, 83] , ces5a may be pivotal in regulating sperm membrane cholesterol and lipid levels to ensure the normal function of male gametes in the last steps of the fertilization process. the most closely related paralog to spint4 is eppin, which, as reviewed in o'rand et al., has at least three physiological functions [16] . eppin inhibits sperm motility when it binds the semen coagulation protein semenogelin 1 (semg1) on the sperm surface [84] ; it modulates the proteolytic activity of prostate-specific antigen (psa), a serine protease, against its seminal plasma substrate, semg1 [85] ; and it exhibits strong antibacterial activity [86] . these functions are postulated to prevent premature hyperactivation and capacitation of sperm in the female reproductive tract [16] , and to protect spermatozoa from proteolytic and bacterial attack during transit in the female reproductive tract [11, 16] . thus, it is possible that the physiological function of spin t4 is similar. however, unlike spint1 and spint2, which have been shown to act as protease inhibitors against a wide variety of prss and tmprss proteases [87] [88] [89] [90] , when tested against a panel of eight proteases (including psa, trypsin, chymotrypsin, plasmin, urokinase, thrombin, factor xa, and elastase), spint3 and spint4 were shown to lack protease inhibiting capability [91] . this indicates that either the protease inhibiting properties of spint3 and spint4 were lost in favor of yet unknown functions or their protease activity has a narrower spectrum of inhibition against unknown targets. since spint4/5 null male mice are severely subfertile, without an apparent difference in epididymis histology, sperm number, sperm morphology, and sperm motility parameters in comparison to the wild-type (wt) mice, this phenocopies the reproductive phenotype of several null mice of testis-, epididymis-, or prostate-specific genes (sof1, tmem95, and spaca6; pate8, and pate10), which reveal a requirement in regulating sperm migration through the oviduct and sperm-oocyte fusion in mice [92, 93] . a severe fertility defect associated with normal sperm number, morphology, and motility is also shared among mice lacking the sperm membrane protein adam3, thought to be crucial in sperm-zp binding and sperm migration through the uterotubular junction (utj) [94, 95] . more than 10 proteins including 2 proteases (ace, adam1a, adam2, calr3, clgn, cmtm2a/b, pdilt, pmis2, prss37, rnase10, tex101, and tpst2) have been described that affect the processing and/or localization of adam3 protein in spermatozoa [96, 97] . further studies with spint4/5 null mice are required to determine whether sperm behavior in the female reproductive tract, specifically the sperm migration through the utj, is adversely affected. since a large 16,797-bp genomic region-including the intergenic region between the spint4 and spint5 genes-was deleted ( fig. 8 ; additional file 1: table s12 ), based on the evidence presented in this manuscript, we cannot exclude the possibility that cis-acting elements and/or trans-acting factors affecting the expression of other genes may have contributed to the phenotype of these mice. lack of protein-coding ability of human spint5p does not necessarily indicate that this pseudogene is functionally obsolete. pseudogenes have been shown to play roles in gene expression and gene regulation [98] . for example, pseudogene transcripts can act as competitive endogenous rnas (cerna) through competitive binding of mirna, which results in regulation of gene expression [99] . to this end, studying the functional requirement of spint5 in male mice is necessary to further our knowledge of evolutionarily conserved genes between species. since humans are genetically diverse, a limitation to phenotype characterization of genetically manipulated mice is the reliance of a single mouse background to the examination of complex genetic outcomes, such as fertility, that is under the control of many genes with different levels of contribution to the phenotype [100] . it is possible that a gene that causes complete infertility in an inbred mouse background may only cause partial infertility or subfertility in a different inbred line or more robust outbred background. since the mice used in our study were a cross between c57bl/6 (b6) mice and dba/2 (d2) mice, and thus, these b6d2f1 mice are heterozygous for b6 and d2 alleles at all loci in their genome, we can eliminate infertility susceptibility of either the b6 or the d2 background as the cause for fertility defects in ces5a and spint4/5 mice. it does remain to be determined, however, whether the phenotype of the genes we knocked out would be more or less severe on a different mouse background, and if required for male fertility in humans, the level of contribution to male fertility of these genes across genetically diverse men. a limitation to this study is the reliance on mrna abundance positively correlating with protein abundance. future studies are necessary to elucidate the relationship between mrna and protein expression levels of the candidate genes identified in our study. furthermore, despite batch corrections that were made, technical differences in sample preparation and integrity across the various published rna-seq datasets can influence the results of our findings. one of the major advantages to our study design is the use of rna-seq datasets from purified human and mouse germ cells and sertoli cells to identify reproductive tract-specific targets since the use of whole testes for the identification of cell typespecific transcripts in past studies is subject to dilution effects. however, this advantage could also be considered a disadvantage since purified cells from nonreproductive tissues were not used for comparison but, if analyzed, purified cells from non-reproductive tissues may have revealed significant levels of expression in non-reproductive tissues. ultimately, functional studies in animals and humans will help to confirm whether genes identified in our study are essential for male fertility and not any other physiological process. through the integration of hundreds of published and newly acquired human and mouse reproductive and non-reproductive tissue and cell rna-seq datasets, we have generated a list of novel genes expressed predominantly or exclusively in the male reproductive tract that are worthy of consideration for functional validation in an animal model and potential targeting for a male contraceptive. our results further validate a functional requirement for spint4/5 and ces5a in male mouse fertility, while demonstrating that spint3, pp2d1, and saxo1 are each individually dispensable for male mouse fertility. identifying novel reproductive tract-specific genes congruent across species adds insight into organismal biology and valuable information that can be used to identify potential male contraceptive drug target candidates. furthermore, elucidating the individual functional requirement or lack thereof of these novel genes builds a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and further validation of the utility of a potential male contraceptive target. the de novo isolated human testes and epididymides included in this study were obtained from three donors through a local organ transplant program in quebec, canada, called transplant quebec. all procedures were approved by the local ethics committee, and written consent was obtained from each respective donor's family. the donors were of 40, 52, and 65 years of age with no preexisting medical condition that could affect reproductive function. donor testes and epididymides were removed under artificial circulation to preserve other organs that were assigned for transplantation. each testis and epididymis were dissected in the laboratory of robert sullivan at université laval. each epididymis was dissected into three segments corresponding to the caput, corpus, and cauda regions and minced into small tissue pieces. testes and epididymides tissue fragments were immediately snap frozen in liquid nitrogen, stored at − 80°c, and shipped frozen to baylor college of medicine for further processing. eight non-reproductive tissue types (kidney, liver, lung, skin, spleen, and stomach) and 2 female reproductive tissues (ovary and uterus) were obtained from the baylor college of medicine tissue acquisition and pathology core. thirteen nonreproductive tissue types (adipose, adrenal gland, brain, colon, heart, leukocytes, pancreas, prostate, salivary gland, skeletal muscle, small intestine, smooth muscle, thyroid) were obtained as purified rnas from takara bio (kusatsu, japan). human testes and epididymis segments were used for de novo rna-seq analysis; all human tissues and/or resulting rnas were used for rt-pcr verification. mouse tissues [testis, caput, corpus, cauda, ovary, uterus, and 17 non-reproductive tissue types (adipose, bladder, brain, colon, eye, heart, kidney, liver, lung, prostate, skeletal muscle, skin, small intestine, spleen, stomach)] were obtained from dissection of b6/129 mice; the remaining 2 non-reproductive tissues (smooth muscle and thyroid) were obtained as purified rnas from takara bio. mouse epididymis segments were used for de novo rna-seq analysis; all mouse tissues and/or resulting rnas were used for rt-pcr verification. rna for both rna-seq and/or rt-pcr verification was isolated from human and mouse tissues using trizol/ chloroform extraction method followed by rneasy mini kit from qiagen with on-column dnase (qiagen) treatment using the manufacturer's protocol. rnas used for rna-seq were assessed by bioanalyzer for rna integrity. for rt-pcr, rna was reverse-transcribed to cdna using superscript iii reverse transcriptase from thermo fisher according to the manufacturer's protocol. cdna was then pcr amplified using gene-specific primers designed using ncbi primer design tool. primer sequences are listed in additional file 1: table s14 . library generation for rna-seq rna-seq libraries were made using kapa stranded mrna-seq kit (kk8420). briefly, poly-a rna was purified from total rna using oligo-dt beads; subsequently, it was fragmented to small size; and first strand cdna was synthesized. second strand cdna was synthesized and marked with dutp. resultant cdna was used for end repair, a-tailing, and adaptor ligation. finally, the library was amplified for sequencing on an illumina novaseq 6000 platform. the strand marked with dutp was not amplified, allowing strand-specific sequencing. sequence alignment, quantification, and differential gene expression human testes, human epididymis segments, and mouse epididymis segments were sequenced by the department of molecular and human genetics functional genomics core at baylor college of medicine (additional file 1: table s1 and additional file 1: table s2 ). previously published reproductive and non-reproductive tissue and cell sequences were downloaded from the sequence read archive (sra) [101] (additional file 1: table s1 and additional file 1: table s2 ). all sequences were trimmed using trim galore! and aligned against the human genome (grch38) or mouse genome (grcm38) using hisat2 [102, 103] . gene expression in each tissue was quantified using featurecounts, filtered for only protein-coding genes, and batch corrected by removing unwanted variation using the ruvr method from ruvseq [104, 105] . differential gene expression was determined for each reproductive tissue against each nonreproductive tissue using the r package edger [106] . principal component analysis (pca) was performed in the r statistical environment using the log2 counts per million (cpm) for each gene in the corresponding tissue after using the ruvr method to correct for batch variation as described above. our procedure for identifying a reproductive-specific gene was repeated for each reproductive tissue or cell sample independently. the following selection criteria were applied to each reproductive tissue or cell sample. first, the non-reproductive tissue with the maximum expression, expressed as the log2 fold change between the non-reproductive tissue and the reproductive tissue or cell of interest, was identified for each gene using the results from the differential gene expression analysis. second, we identified reproductive-specific gene drug candidates using three filters: a false discovery rate (fdr) filter, a maximum transcript per million (tpm) expression value filter on the non-reproductive sample with the maximum expression identified as described above, and a minimum tpm expression value filter on the reproductive tissue or cell sample of interest. a gene was kept if the fdr from the differential gene expression analysis was less than or equal to 0.05 for the comparison of the reproductive tissue or cell sample of interest to the non-reproductive tissue with the maximum expression. a gene was considered to be a male reproductive tissue-specific drug target if the average tpm expression value in the non-reproductive tissue with the maximum expression from the differential analysis was less than or equal to 1.0 for human (2.0 for mice), and if the average tpm expression value for that gene was greater than or equal to 10.0 for human (8.0 for mice) in the reproductive tissue or cell sample of interest. the average and standard deviation of the tpm expression value for each gene was calculated from the ruvr batch corrected counts per million expression value for each tissue or cell sample. we consolidated data from ensembl biomart [29] and mouse genome informatics (mgi) [28] to create a comprehensive database of mouse gene symbols orthologous to human genes and vice versa. each respective species' stable ensemble gene id was used for each conversion, with gene symbol as the final output. as mentioned, several notable high-throughput gene expression studies using microarrays or rna-seq, focused on identifying male reproductive tract-specific genes, have been previously published [2, [4] [5] [6] [7] [8] [9] . tables and supplementary tables from these studies were gathered to collect the lists of genes previously identified. for microarray-based studies, affymetrix ids were used to confirm the identity of a listed gene, based on current sequence mappings, or in many cases to identify de novo the identity of a gene only known at the time of the study by its affymetrix id and not gene symbol. for mouse and rat studies, gene symbols were converted to orthologous human symbols, to systematically catalog both the rodent and corresponding human symbols as previously identified. for example, 399 affymetrix probe ids were listed as reproductive tract-specific in johnston et al. [8] . after re-identification of gene symbols based on current ensembl sequence mappings, 42 identified gene symbols remained the same, 160 received an updated/replacement gene symbol identification, 103 genes that were previously unidentified received a new gene symbol identification, 26 affymetrix ids lost mapping to any gene symbol, and 67 remain unidentified. out of the total of 305 affymetrix ids that mapped to 301 current rat gene symbols, 257 rat gene symbols converted to at least one human ortholog gene symbol that was either the same symbol or different. both rat and human symbols, based on new mappings, were considered previously identified. for the complete list of previously identified genes, see additional file 18: table s10 . genes were classified as encoding either enzymes, epigenetic-related proteins, g protein-coupled receptors (gpcrs), ion channels, kinases, nuclear receptors, orphan gpcrs (ogpcrs), transcription factors, transporters, or unknown proteins based on data obtained from illuminating the druggable genome [27] (additional file 11: table s8 ). we used data obtained from ensembl biomart [29] , mgi [28] , the international mouse phenotyping consortium (impc) [31] , and pubmed searches to generate a comprehensive database identifying the existence of a mouse model for all mouse genes (additional file 12: table s9 ). we then queried our identified candidate human and mouse genes against this list. for a given human gene, we queried the equivalent mouse ortholog gene symbol(s). spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice were produced at baylor college of medicine. b6d2f1 (c57bl/6 × dba2) mice were used as embryo donors, and cd1 mice were used as foster mothers. mice were purchased from charles river (wilmington, ma). all mice were housed in a temperature-controlled environment with 12-h light cycles and free access to food and water. mice were housed in accordance with nih guidelines, and all animal experiments were approved by the institutional animal care and use committee (iacuc) at baylor college of medicine. generation of spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice to generate spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice, grna/cas9 ribonucleoprotein complex was electroporated into fertilized eggs and transplanted into surrogate mothers as previously described [107] . briefly, to harvest fertilized eggs, card hyperova (0.1 ml, cosmo bio) was injected into the abdominal cavity of b6d2f1 females (charles river), followed by human chorionic gonadotropin (hcg) (5 units, emd chemicals). forty-eight hours after card hyperova, b6d2f1 males were allowed to mate naturally. twenty hours after mating, fertilized eggs with 2 pronuclei were collected for electroporation. custom crrnas targeting each gene were purchased from millipore-sigma. the sequences for all guide rnas used for crispr/cas9mediated gene editing are listed in additional file 1: table s11 . crrna and tracrrna (millipore-sigma) were diluted with nuclease-free water. the mixture was denatured at 95°c for 5 min and allowed to anneal by cooling gradually to room temperature (1 h). each grna was mixed with cas9 protein solution (thermo fisher scientific) and opti-mem media (thermo fisher scientific), and then incubated at 37°c for 5 min to prepare the grna/cas9 rnps [final concentration, 300 ng/μl cas9 for 250 ng/μl of each grna]. the grna/cas9 rnp solution was placed between electrodes with a 1mm gap in the ecm 830 electroporation system (btx). fertilized eggs were arranged between the electrodes, and then, the electroporation was performed with the following conditions: 30 v, 1-ms pulse duration, and 2 pulses separated by 100-ms pulse interval. for egg transfer, electroporated embryos were transplanted into the oviduct of pseudo-pregnant icr recipients. after 19 days, offspring were obtained by natural birth or cesarean section. the f0 mice with sequence-predicted heterozygous mutations were used for the mating with additional b6d2f1 mice to generate homozygous mutants. the f2 or later generations were used for the phenotypic analyses. for sanger sequence analysis of mutant mice, genomic dna was isolated by incubating tail tips in lysis buffer [20 mm tris-hcl (ph 8.0), 5 mm edta, 400 mm nacl, 0.3% sds, and 200 μg/ml actinase e solution] at 60°c overnight. polymerase chain reactions (pcrs) amplifying the genomic region containing the insertion/deletion events were performed using kod xtreme enzyme (toyobo, osaka, japan); pcr products were purified using the qiaquick pcr purification kit (qiagen, carlsbad, ca, usa) and sent for sanger sequencing on an abi 3130xl genetic analyzer (thermo fisher scientific, waltham, ma, usa) using the forward primer. for routine genotyping of mutant mice, genomic dna was isolated by separately incubating ear snips and tail tips in 50 mm naoh solution at 95°c overnight and inactivating with 1 m tris ph = 8.0. pcrs amplifying wild-type and mutant-specific amplicons were performed using 2x amfisure pcr master mix (gendepot, barker, tx). primer sequences are listed in additional file 1: table s13 . upon sexual maturation (6-7 weeks of age), knockout and littermate control male mice (n = 6-9 mice per genotype) were continuously housed with two 7-8week-old wild-type b6d2f1/j female mice per male for 12 weeks. during the fertility test, the number of pups was counted shortly after birth. the total number of litters and pups per male over the mating trial was calculated and divided by the number of months to generate averages and statistics per genotype. the average number of pups per litter is based on the average litter size per male where a litter is considered one or more pups. knockout and littermate control male mice (n = 5-13 mice per genotype) that were 12 weeks of age were used to examine body and reproductive organ weights, and testicular and epididymal histology. testes and epididymides were fixed in bouin's fixative, embedded in paraffin, sectioned at 5 μm thickness, and stained with 1% periodic acid-schiff (pas) stain followed by counterstaining with hematoxylin 2 solution. histological images were acquired with an aperio at2 slide scanner (leica microsystems). knockout and littermate control male mice (n = 5-13 mice per genotype) that were 12 weeks of age were used to examine sperm numbers and motility parameters using computer-assisted sperm analysis (casa). cauda of both epididymides was isolated, transferred into human tubule fluid (htf) (irvine scientific, santa ana, ca) containing 5 mg/ml of bsa, minced, and placed in a humidified incubator for 15 min at 37°c with 5% co 2 . following incubation, the sperm were diluted 1:50 in htf, added to a pre-warmed slide, and analyzed using a hamilton-thorne bioscience's ceros ii instrument. several fields of view were illuminated and captured until at least 200 cells were counted. rnascope 2.5 hd reagent kit (red) (cat. 322350, advanced cell diagnostics, newark, ca, usa) was used to detect spint3, spint4, and spint5 mrna transcripts on pfa-fixed, paraffin-embedded sections from 3-monthold wild-type epididymis. the probes against mm-spint3, mm-spint4, and mm-spint5 were custom-made, and the standard positive control (mm-ppib, cat. 313911) and negative control (dapb, cat. 310043) probes were used. the assay was performed according to the manufacturer's instructions. slides were counterstained using dapi and mounted using prolong glass antifade mountant (thermo fisher scientific inc.). multi-channel fluorescent images were acquired with an aperio versa (leica microsystems). all measurements are expressed as mean ± standard error of the mean. statistical differences were determined using student's t test. differences were considered statistically significant if the p value was less than 0.05. supplementary information accompanies this paper at https://doi.org/10. 1186/s12915-020-00826-z. additional file 1: tables s1, s2, s11, s12, s13, and s14. table s1 . summary of human rna-seq datasets. this table contains the sra value for each previously published human rna-seq dataset that was reanalyzed as part of this study. the geo accession number for each new human rna-seq dataset generated and subsequently analyzed in this study is also included. table s2 . summary of mouse rna-seq datasets. this table contains the sra value for each previously published mouse rna-seq dataset that was reanalyzed as part of this study. the geo accession number for each new mouse sample generated and subsequently analyzed in this study is also included. table s11 . single-guide rnas targeting the genes' upstream (u) and downstream (d) regions used for generating knockout mice. efficiency of embryo transplantation was presented using the number of total pups delivered by pseudopregnant mice divided by the number of total embryos used for oviduct transplantation (total pups/embryos transplanted). efficiency of genome editing was determined by the number of pups carrying enzymatic mutations divided by the number of pups subjected to genotyping (gm pups/pups genotyped). table s12 . sanger sequencing of detailed genotype of mutant dna sequences in all the five mouse lines. table s13 . primers and pcr conditions used for genotyping the mutant alleles of the knockout mouse lines. table s14 . human and mouse rt-pcr primer sequences used for verification of reproductive tract-specificity. additional file 2: fig. s1 . genes that passed the tpm and fdr filters in at least one of the measured reproductive tissues or cells were visualized using a heatmap of the ruvr batch corrected log2 cpm gene expression values for the human (a) and mouse (b) samples. additional file 3: table s3 . human expression summary. contains differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, false detection rate (fdr) value, average and standard deviation tpm expression values, and log2 cpm gene expression value for the human samples. all protein-coding genes (18,305 genes) that had expression in at least one reproductive tissue or cell is listed. additional file 4: table s4 . mouse expression summary. contains differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, false detection rate (fdr) value, average and standard deviation tpm expression values, and log2 cpm gene expression value for the mouse samples. all protein-coding genes (16,891 genes) that had expression in at least one reproductive tissue or cell is listed. additional file 5: table s5 . all human male reproductive tract-specific genes that met the criteria of identification as reproductive tract-specific in at least one male reproductive tissue or purified cell type, with the level of fold change listed under the tissue or cell if all criteria were met. the criteria of selection are as follows: fdr < 0.05; tpm repro > 10; tpm non-repro , max < 1. a fold change value of 0 indicates the criteria were not met for that that tissue or cell. additional columns indicating 1.) the equivalent mouse ortholog gene symbols (single or multiple symbols) that exist, and 2.) if our studies identified any of these mouse orthologs as reproductive tract-specific in mouse, are included. additional file 6: table s6 . all mouse male reproductive tract-specific genes that met the criteria of identification as reproductive tract-specific in at least one male reproductive tissue or purified cell type, with the level of fold change listed under the tissue or cell if all criteria were met. the criteria of selection are as follows: fdr < 0.05; tpm repro > 8; tpm non-repro , max < 2. a fold change value of 0 indicates the criteria were not met for that that tissue or cell. additional columns indicating 1.) the equivalent human ortholog gene symbols (single or multiple symbols) that exist, and 2.) if our studies identified any of these human orthologs as reproductive tract-specific in human, are included. received: 17 april 2020 accepted: 8 july 2020 population division: un the mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis1 the human epididymis: its function in sperm maturation a multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells identification of epididymis-specific transcripts in the mouse and rat by transcriptional profiling the rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides1 stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and sertoli cells the human testis-specific proteome defined by transcriptomics and antibody-based profiling toward development of the male pill: a decade of potential non-hormonal contraceptive targets epididymal protein targets: a brief history of the development of epididymal protease inhibitor as a contraceptive disrupting the male germ line to find infertility and contraception targets male contraception: another holy grail male contraception: past, present and future metabolic cooperation in testis as a pharmacological target: from disease to contraception non-hormonal male contraception: a review and development of an eppin based contraceptive the control of male fertility by spermatid-specific factors: searching for contraceptive targets from spermatozoon's head to tail epididymal approaches to male contraception an integrated encyclopedia of dna elements in the human genome research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis expression profiles of human epididymis epithelial cells reveal the functional diversity of caput, corpus and cauda regions transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage dynamics of the transcriptome during human spermatogenesis: predicting the potential key genes regulating male gametes generation chromatin and single-cell rna-seq profiling reveal dynamic signaling and metabolic transitions during human spermatogonial stem cell development id4 levels dictate the stem cell state in mouse spermatogonia human sertoli cells support high levels of zika virus replication and persistence pharos: collating protein information to shed light on the druggable genome 2018: knowledgebase for the laboratory mouse analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics high-throughput discovery of novel developmental phenotypes carcinoembryonic antigen-related cell adhesion molecule 2 controls energy balance and peripheral insulin action in mice shp1 phosphatase-dependent t cell inhibition by ceacam1 adhesion molecule isoforms deletion of the carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) gene contributes to colon tumor progression in a murine model of carcinogenesis ceacam1a-/-mice are completely resistant to infection by murine coronavirus mouse hepatitis virus a59 carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development the lim homeobox gene lhx9 is essential for mouse gonad formation generation and characterization oflhx9-gfpcreert2knock-in mouse line targeted germline modifications in rats using crispr/cas9 and spermatogonial stem cells spermatogenic cells of the prepuberal mouse. isolation and morphological characterization integrative characterization of germ cell-specific genes from mouse spermatocyte unigene library comparative and functional analysis of testis-specific genes genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice reaching for high-hanging fruit in drug discovery at protein-protein interfaces pharmacological perturbation of cdk9 using selective cdk9 inhibition or degradation catalytic in vivo protein knockdown by small-molecule protacs hijacking the e3 ubiquitin ligase cereblon to efficiently target brd4 lessons in protac design from selective degradation with a promiscuous warhead assessing different e3 ligases for small molecule induced protein ubiquitination and degradation cell-penetrating peptides: 20 years later, where do we stand? tat peptide-mediated cellular delivery: back to basics proteomics. tissue-based map of the human proteome single nucleotide polymorphisms: discovery of the genetic causes of male infertility a comprehensive gene mutation screen in men with asthenozoospermia malacards: an amalgamated human disease compendium with diverse clinical and genetic annotation and structured search genetic evaluation of patients with non-syndromic male infertility a systematic review on the genetics of male infertility in the era of nextgeneration sequencing genetics of male infertility: from research to clinic association study of single-nucleotide polymorphisms in faslg, jmjdia, loc203413, tex15, brdt, or2w3, insr, and tas2r38 genes with male infertility evaluation of 172 candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of european descent point-of-care whole-exome sequencing of idiopathic male infertility the biology of infertility: research advances and clinical challenges mutation screening of the fkbp6 gene and its association study with spermatogenic impairment in idiopathic infertile men efficient typing of copy number variations in a segmental duplication-mediated rearrangement hotspot using multiplex competitive amplification genetic defects in human azoospermia single-nucleotide polymorphisms of the prdm9 (meisetz) gene in patients with nonobstructive azoospermia two single nucleotide polymorphisms in prdm9 (meisetz) gene may be a genetic risk factor for japanese patients with azoospermia by meiotic arrest single-nucleotide polymorphisms in the human rad21l gene may be a genetic risk factor for japanese patients with azoospermia caused by meiotic arrest and sertoli cell-only syndrome excess of rare variants in genes that are key epigenetic regulators of spermatogenesis in the patients with non-obstructive azoospermia mutations in sohlh1 gene associate with nonobstructive azoospermia human fam154a (saxo1) is a microtubule-stabilizing protein specific to cilia and related structures human carboxylesterases: a comprehensive review structure and catalytic properties of carboxylesterase isozymes involved in metabolic activation of prodrugs human carboxylesterases and their role in xenobiotic and endobiotic metabolism the mammalian carboxylesterases: from molecules to functions heterologous expression, purification, and characterization of human triacylglycerol hydrolase hepatic carboxylesterase 1 is essential for both normal and farnesoid x receptorcontrolled lipid homeostasis deficiency of carboxylesterase 1/esterase-x results in obesity, hepatic steatosis, and hyperlipidemia baculo-expression and enzymatic characterization of ces7 esterase lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation variation in the cholesterol/phospholipid ratio in human spermatozoa and its relationship with capacitation analysis of recombinant human semenogelin as an inhibitor of human sperm motility characterization of an eppin protein complex from human semen and spermatozoa antimicrobial activity of human eppin, an androgen-regulated, sperm-bound protein with a whey acidic protein motif the role of type ii transmembrane serine proteasemediated signaling in cancer delineation of proteolytic and non-proteolytic functions of the membrane-anchored serine protease prostasin mechanisms of hepatocyte growth factor activation in cancer tissues regulation of cell surface protease matriptase by hai2 is essential for placental development, neural tube closure and embryonic survival in mice three genes expressing kunitz domains in the epididymis are related to genes of wfdc-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products sperm proteins sof1, tmem95, and spaca6 are required for spermoocyte fusion in mice identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice disruption of adam3 impairs the migration of sperm into oviduct in mouse male mice deficient for germ-cell cyritestin are infertile co-expression of sperm membrane proteins cmtm2a and cmtm2b is essential for adam3 localization and male fertility in mice factors controlling sperm migration through the oviduct revealed by gene-modified mouse models pseudogenes regulate parental gene expression via cerna network interspecific recombinant congenic strains between c57bl/6 and mice of the mus spretus species: a powerful tool to dissect genetic control of complex traits the sequence read archive cutadapt removes adapter sequences from high-throughput sequencing reads transcript-level expression analysis of rna-seq experiments with hisat, stringtie and ballgown systematic selection of reference genes for the normalization of circulating rna transcripts in pregnant women based on rna-seq data featurecounts: an efficient general purpose program for assigning sequence reads to genomic features edger: a bioconductor package for differential expression analysis of digital gene expression data crispr/cas9 mediated genome editing in es cells and its application for chimeric analysis in mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank drs. yumei li all data generated or analyzed during this study are included in this published article, its supplementary information files, and publicly available repositories. the sra values for each of the 162 previously published reproductive and non-reproductive human rna-seq datasets [9, 21, 23, 24, 26, 30] and 81 previously published reproductive and non-reproductive mouse rna-seq datasets [19, 22, 25] are listed in additional file 1: table s1 and additional file 1: table s2 . all raw and processed data for the 12 new human and 9 new mouse samples generated in this study is deposited in ncbi geo (accession gse150854). all mice generated in this study, and any additional information about this study, are available from the corresponding authors upon request.ethics approval and consent to participate human tissue acquisition was approved by the ethics committee at université laval with written consent obtained from each respective donor's family. all animal experiments were approved by the institutional animal care and use committee (iacuc) at baylor college of medicine. additional file 7: fig. s2 . summary of number of statistically significant up and down-regulated genes, and quantification of candidate genes with respect to the individual reproductive tissue or cell of interest. the plots in panels (a) and (b) summarizes the number of statistically significant human or mouse genes respectively, that are up-regulated or downregulated in each reproductive tissue or cell of interest compared to the non-reproductive tissue with maximal gene expression. red columns depict the number genes that are up-regulated and blue columns depict the number genes that are down-regulated. changes in gene expression were considered statistically significant for an fdr of less than or equal to 0.05. the total number of candidate genes are designated by the black columns. candidate genes are genes that passed the fdr and tpm expression value filters.additional file 8: fig. s3 . venn diagrams comparing the overlap between the candidate male reproductive genes identified by the indicated reproductive tissues. the human testis combined gene list is the list of genes from both new samples we isolated and from previously published testis samples. the human epididymis combined gene list is the list of genes identified in either previously published samples or the newly generated samples across all sections of the epididymis. lastly, the mouse epididymis combined gene list is the list combined list of genes identified across all three sections of the mouse epididymis.additional file 9: table s7 . complete cross-sample comparison identifying human and mouse reproductive tract specific genes common to two or more samples and unique to each as identified through our studies.additional file 10: fig. s4 . classification of genes into different protein families and identification of the existence of an experimental mouse model. each candidate human gene was classified as an enzyme (enzyme), chromosome and histone modifiers (epigenetic), g-proteincoupled receptor (gpcr), orphan g-protein-couple receptor (ogpcr), kinase (kinase), transcription factor (tf), nuclear receptor (nr), ion channel (ic), chromosome and histone modifying transcript factor (tf; epigenetic), transporter (transporter) and unknown (a). the total number of candidate genes identified in our search for mouse models were plotted. orange columns designate the number of candidate genes where a model was identified while yellow designates candidate genes where a model was not identified (b).additional file 11: table s8 . drug target type classification for human genes. genes are listed according to the tissue and/or cell that they were identified as reproductive tract-specific in.additional file 12: table s9 . availability of a mouse model for human genes with a mouse ortholog. genes are listed according to the tissue and/or cell that they were identified as reproductive tract-specific in.additional file 13: fig. s5 . one-hundred and forty-two previously identified human male reproductive tract-specific genes that remain without a reported mouse model. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file 14: fig. s6 . seventy-three human male reproductive tract-specific genes that each have a reported mouse model with male infertility phenotype. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file 15: fig. s7 . rt-pcr confirmation of reproductive tractspecificity in both humans (a) and mice (b). the genes listed in this figure were identified through our studies and previous studies, but currently remain without a reported mouse model. gapdh and hprt are included as housekeeping genes.additional file 16: fig. s8 . eighty-nine novel human genes without a mouse ortholog. the listed genes were identified in one or more datasets as indicated in the venn diagram. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file 17: fig. s9 . novel reproductive tract-specific human genes that do not have any equivalent mouse orthologs. these genes may serve as potential contraceptive targets, however functional validation would need to be carried out in another model organism than mouse, such as rat or marmoset, which do have orthologs to these genes. the digital pcr (heatmap) depicts the average transcripts per million (tpm) value per tissue per gene from the indicated human rna-seq datasets as processed in parallel through our bioinformatics pipeline. white = 0 tpm, black ≥30 tpm. the expression profile of the human housekeeping gene, gapdh, is included as reference. for data obtained from published datasets, superscript values reference the dataset publication as previously mentioned.additional file 18: table s10 . 1064 previously identified genes. genes previously identified as male reproductive tract-specific through high throughput gene expression studies using either microarrays or rna-seq [2] [3] [4] [5] [6] [7] [8] . the human ortholog to genes identified in mouse and rat studies is included.additional file 19: fig. s10 . developmental expression pattern of spint3, spint4, spint5, pp2d1, and saxo1 in epididymis and testis of postnatal and adult mice. whole epididymides were used at postnatal days 3, 6, 10, and 14 and epididymis segments (caput, corpus, and cauda) were used at postnatal days 21, 28, 35, and 60. whole testes were used at all time points. the housekeeping gene, hprt, was used as reference.additional file 20: fig. s11 . multi-channel fluorescence images of bilateral epididymis serial sections stained with custom rnascope probes targeting either spint3, spint4, or spint5 mrna (red) and dapi (blue). the position of caput (cap), corpus (cor), and cauda (cau) is labeled in the overview image (left column). the position of the magnification over the epididymis is the same for all three sections (right column).additional file 21: fig. s12 . representative periodic acid-schiff staining of spint3 and spint4/5 knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at 3 months of age.additional file 22: fig. s13 . representative periodic acid-schiff staining of ces5a knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at 3 months of age.additional file 23: fig. s14 . representative scanning electron microscopy images of spint4/5 and ces5a ko and littermate control mouse sperm.additional file 24: fig. s15 . representative periodic acid-schiff staining of pp2d1 and saxo1 knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at 3 months of age. all authors have no competing interests. key: cord-282628-6uoberfu authors: tiwari, bhagyashree; sellamuthu, balasubramanian; drogui, patrick; tyagi, r.d. title: future impacts and trends in treatment of hospital wastewater date: 2020-05-01 journal: current developments in biotechnology and bioengineering doi: 10.1016/b978-0-12-819722-6.00017-1 sha: doc_id: 282628 cord_uid: 6uoberfu the world’s population growth and economic development result in the increased requirement of land, water, and energy. this increased demand leads to the deforestation, loss in biodiversity, imbalance in agriculture and food supply, climate change, and increase in food and travel trade, which result in emergence and reemergence of infectious diseases. this chapter discussed various emerging infectious diseases and their causative agents (buruli ulcer and bunyvirus). furthermore, this chapter further illustrates the emergence of superbugs and the associated threat due to the presence of pharmaceutical compounds in the environment. the prevalence of pharmaceuticals in the environment exerts ecotoxic effects on living organisms and causes thousands of death every year. the threats associated with the pharmaceutical presence in the environment were briefly discussed in this chapter. finally, this chapter provides the alternative methods to avoid the use of antibiotics and to develop novel treatment technologies (such as phage therapy) to degrade and remove the pharmaceutical compounds. infectious diseases are the second leading cause of death annually across the globe. the causative agent of most emerging infectious diseases is viruses; every year approximately more than two novel viral pathogens are identified, which can cause illness in a human. since the 1970s, an average of 40 infectious diseases has been emerged and known to cause pandemics such as ebola, swine flu, chikungunya, and zika (table 17à1 ) [4] . the emergence of infectious diseases has severe health and socioeconomic impacts; thus the following section discusses the various infectious disease that emerged within the last two decades and presenting few pandemic viral diseases as a suitable example [5, 7] . factors for emergence include natural process (evolution of pathogen), infectious agents transfer from vertebrate to mammals, antimicrobial resistance (amr), and climate change. the factors responsible for the emergence of infectious diseases such as (1) the evolution of new strain, (2) the introduction of a host to enzootic, (3) translocation of infected wildlife, (4) farming practices, and (5) others were provided. the viral evolution rate occurs through mutation and adaption, which are much faster than any other microscopic organisms that is why a large fraction of emerging and reemerging pathogens are viruses (37%). among these 37% of emerging viruses, rna viruses are prominent one because of their higher nucleotide substitution rate which enables them to invade and amplify in broad host range [22] . the environment changes, social condition, and their interaction are the important factors which lead to the viral evolution and ultimately to disease emergence. the decrease in biological diversity due to deforestation, prevalence of micropollutant in natural environment, and climate change triggers the evolution of opportunistic pathogen [23] . for instance, the outbreak of nipah virus diseases was occurred due anaplasma phagocytophilum [7] buruli ulcers mycobacterium ulcerans [8] to the extensive deforestation of a forest of southeast asia [24] . the deforestation leads to migration of fruit bats for food quest to the agriculture land. these fruit bats are natural reservoir (host) of nipah virus and their migration to cultivable land lead to transmission of nipah virus disease in farm animals and subsequently in humans [24] . the increase in temperature due to climate change affects the ecology, survival, and behavior of arthropod vectors, which subsequently changes population dynamic with increased disease transmission rate. for example, the rise in temperature from 10 c to 15 c decreases the proliferation time of plasmodium falciparum in anopheline mosquitoes vector from 20 to 13 days. usually, p. falciparum requires 20 days to proliferate in the vector at 20 c. however, the high temperature reduces its proliferation time by increasing egg production, by increasing metabolic rate, and by reducing the larval and pupal period duration [25] . moreover, the proximity of natural host such as rodents and human is the ease and travel aids for the spread of emerging viruses. the viral infection begins from binding of virus to the receptor of host cell, and during the emergence of new disease, viruses develop either the ability to bind the new receptor or use homologue receptor in new host species. the spread of coronavirus (cov) which causes severe acute respiratory syndrome (sars) in human occurs due to cross-species transmission from raccoon dogs and chinese ferret badgers [1] . the examination of wild species of cov did not have the sign of sarsàcov infection but the cov viruses isolated from horseshoe bats have sarsàcov infection. the cov binds to angiotensin-converting enzyme 2 (ace2) receptor to infect humans. however, in bats, ace2 viral receptor is not use for infection by cov. later it was studied that the mutation in ace2 receptor aids the adaptation of cov in human cells. the spread of ebola, marburg, and measles viruses is some of the example of virus evolution and which contributes to the emergence of disease. the virus evolution and adaptation is difficult to predict and thus raises a question how the effect of emerging viral infection can be reduced. this requires a holistic approach to identify the drivers of emergence with effective surveillance. in 2009 an increased cases of severe fever with thrombocytopenia syndrome (sfts) were frequently reported in rural areas of china. the associated pathogens of thrombocytopenia and other similar diseases were not detected in majority of the pathogen samples which implies that the pathogen associated with sfts was newly evolved. the emergence of this unknown infection having average fatality rate of 10% due to organ failure leads to the implementation of enhanced surveillance to identify causative agent of sfts infection [2] . the surveillance data revealed 20 strain of viruses as causative of sfts infection and regarded as sfts viruses (sftsvs). sftsvs are rna virus with three single-stranded rna genomes. the complete sequencing of sftsv, rna genome reveals that they belong to genus phlebovirus, family bunyaviriade. the sftsv is transmitted in human through infected ticks and transmission of blood and other body fluid of infected person lead to human-to-human transmission of infection. the phlebovirus infection in human results in mild febrile illness. however, the major symptoms of sfts include thrombocytopenia, high fever, leukocytopenia, and lymphadenopathy. currently, it is believed that the vertical transmission of phlebovirus in arthropod vector (ticks such as haemaphysalis longicornis) helps in maintenance cycle of virus and amplification of virus occur in vertebrate host (human). the laboratory diagnosis of body fluid samples of sfts patient showed elevated level of serum and important enzymes and cofactor such as creatine kinase, alanine aminotransferase, lactate dehydrogenase, and aspartate aminotransferase. the sftsv replicates in the spleen of infected patient which increases the number of macrophages and platelets. the in vitro assay revealed that sftsv facilitates the phagocytosis of platelets by adhering on the surface of platelets and ultimately causing thrombocytopenia. the spread of sftsv in china and the detection of sftsv like viruses in other parts of world such as in the united states and europe emphasized on the urgent need of understanding of pathogenesis and transmission cycle of virus which help in the development of efficient vaccine. the changes in the hostàenvironment lead to the evolution of opportunistic and novel pathogens due to which infectious diseases emerge. the forces which shape the emergence of disease in human are similar to the drivers found in wildlife and domestic animals. these drivers alter the interplay of hosts, environment, and pathogen thereby modulating disease ecology and developing or acclimatizing pathogen in hosts. the drivers alter interaction pattern of pathogenàhost and environment which to either of three events (1) altering the genetic trait of pathogen which causes severe disease in same host; (2) emergence of pathogen in new hosts; and (3) redistribution of pathogens that result in its establishment in new geographical area. frequent use of antimicrobial compounds and mass rearing of animals result in amr and eventually increase virulence pathogenicity of a pathogen. mass rearing of food animals to meet the demand of growing population results in intensification of genetically similar animals of same sex and age at confined place. this leads to transmission of pathogen with increases population turnover which supports emergence of novel trait. the transmission of highly pathogenic asian avian influenza a (h5n1 hpai) is the well-known example of mass rearing [26] . the drivers which are responsible for the disease emergence in new host include interspecies contact, wildlife migration, and increase contact between different hosts. the worldwide change in ecological landscape results in close contact between human and animals which eventually lead to transmission of microbial reservoir of animals (birds, rodents, and bats) to human. for instance, the emergence of nipah virus in human was due to transmission of virus from foraging fruit bat to pigs [7] . the transport of food, live animals, plants with accompany insects for international trade, migration of birds, and wild animals results in geographical jump of pathogens. the bacterium ralstonia solanacearum hitchhikes to the united states via geranium plant which is imported from kenya [7] . persisters are slow growing or nongrowing organisms which remain in stagnant during the presence of antimicrobial compound but have the capacity to resuscitate and grow under specific conditions. the persisters formation is mediated by variety of stress such as due to nutrient depletion, oxidative stress heat, acidic ph, and the presence of toxic compound (antibiotics). antibiotics such as tetracycline, rifampin, and ciprofloxacin were shown to enrich persisters formation by inhibiting protein synthesis, rna synthesis, or by antioxidative defense [5] . persisters are the cause of biofilm infection, recurrent infection, and chronic infection and contribute toward prolongation of therapy time (e.g., tuberculosis, lyme disease). the repeated treatment of persistent infection may result in the development of drug-resistance microbes, often seen in the case of tuberculosis. persistent could be pre-or postantibiotic depending on its development in the host. the capacity of formation of persisters varies greatly among the bacterial species. for instance, persisters of mycobacterium tuberculosis are not removed from the host even after chemotherapy, while the single antibiotic treatment is sufficient for curing infection caused by streptococcus pneumoniae. the physical and psychological stress, host immune and hormonal factor, and coinfection also affect the persisters formation in the host. the mechanism behind the persisters formation is not well understood. however, it is believed that epigenetic changes, changes in dna modification, and posttranslational modification induce expression of persisters gene. the genes which involve in persisters formation are rela, sucb, hipa, ubif, and phou. the mutagenesis approach is used to study genes involved in persisters formation; however, the short antibiotic exposure, screening of partial mutant library, and aeration during antibiotic exposure are factors which result in failure of mutagenesis process for identifying persisters genes. zoonotic diseases are vector (mosquito, ticks, and bugs) aided or nonaided disease caused by bacteria, virus, parasites, prions and fungi, and transmitted from animals to human. various modes of transmission are direct contact between infected animal and human, via arthropod vector, consumption of contaminated animal food (meat and pork), and ingestion of aerosolized pathogens present in the environment. in last two decades, many vector borne pathogens spread in new geographical regions. the emergence and reemergence rate of zoonotic infection has increased inescapably due to urbanization, deforestation, climate change, international trade (frequent travel), population movement, and encroachment into animal habitats. the most emerging zoonotic vector borne diseases in the united states and canada include tick borne lyme disease, babesiosis, human granulocytic anaplasmosis and mosquito borne west nile virus (wnv), california serogroup viruses, and cache valley virus [7] . the change in land use alters the abundance and interaction of vectors and hosts (wildlife and domestic animals) which results in emergence of vectors. for instance, deforestation in amazon and eastern africa enhances the breeding of anopheles' mosquito due to sunlight and standing water. similarly in north america, increased hunting changes the predator community and results in increased abundance of small animals such as mice, chipmunks, and shrew which are main host of spirochete borrelia burgdorferi, causative agent of lyme disease [6] . socioeconomic changes and human activities are the another factor which governs spread and emergence of pathogen. the lyme disease was reported to occur more in high-income people in europe due to the more recreational activity and living in new homes in broad-leaf woodlands with cooccurrence of wildlife result in frequent exposure to vector. vector born zoonotic disease could be controlled by prompt identification of cause with subsequent action which requires integration of public health officials, researchers, and public [23] . arbovirus is an acronym for arthropod-borne viruses. dengue virus, wnv, and chikungunya virus are recent examples of arboviruses which are transmitted from the arthropods (ticks, bugs, and mosquito) to vertebrate. arboviruses use arthropods as a vector (carrier) for transmission and do not causes any sickness in them. bunyaviridae, reoviridae, flaviviridae, and togaviridae are the most prevailing arboviruses families that cause diseases in human and animals [27] . the emergence of arboviruses is governed by three factors, that is, high mutation frequency, varying anthropological behavior, and climate change. they easily adapt new host by altering receptor specificity, antigenicity, environmental conditions, and by efficient transmission. for instance, emergence of chikungunya virus in asia due to mutation in surface protein which increases its reproduction, transmission, and infection efficiency in aedes albopictus. they are able to maintain themselves for years in mosquito eggs or via attaining transstadial stages in ticks. domestic animals, livestock, and human are not important part of arbovirus life cycle because of nonviraemic transmission of arbovirus between ticks without infecting vertebrate host. this feature of arbovirus adds additional limitation for controlling disease emergence [27] . mosquito eradication, development of live-attenuated vaccines, antiviral drugs, and molecule are few methods used to control and prevent arboviral infection. however, high mutational frequency will result in emergence of new pathogenic arboviruses. since it has been proven by genome sequencing of mosquitoes that they are the carrier of various known and unknown viruses, control of localized arthropod during endemic could be a possible solution for regulating the emergence of arbovirus [24] . buruli ulcer (bu) is necrotizing skin disease caused by mycobacterium ulcerans recognized as one of most neglected tropical disease by world health organization (who). the bu lead to the formation of skin ulcers which results in osteomyelitis. it causes pandemic in humid tropical and subtopical region, often where humans are in proximity with slow moving or stagnant contaminated water. the transmission of m. ulcerans follows multihost transmission dynamics, that is, multiple hosts of aquatic environment such as scrapers, scavengers, and predators become contaminated with m. ulcerans and result in its passive dissemination via organism-to-organism contact. phylogenetic studies revealed that the m. ulcerans was emerged from species mycobacterium marinum which causes cutaneous disease in human and also shown to infect fish. m. ulcerans are widely distributed in africa; however, the bu cases reported in specific geographical villages. researchers postulates that a specific m. ulcerans strain might have pathogenicity or virulence to cause bu [8] . the current knowledge lacks the understanding of spatiotemporal distribution of m. ulcerans and required detailed scenario of diversity of m. ulcerans strains existing in environment [2] . superbug is a term coined for bacterial species which confers resistant toward majority of antibiotics. emergence of superbugs implies the frequent detection of novel bacterial pathogens which caused unrecognized life-threatening infections. the frequent emergence and spread of superbugs worldwide causes a concern that human life is heading back, toward the preantibiotic era, where the entire population of a society was wipe out due to the simple infection. united nation (un) general assembly of 2016 addresses the amr [28] as a health emergency and acknowledge that amr has deleterious effect on human health and sustainable development. who identified a group of amr resistant "priority pathogens" that requires urgent strategic action (table 17à2) . who categorize these priority pathogens into three categories, that is, critical, high, and medium priority based on the need for new antibiotics. the criteria for prioritization of pathogens were developed using multicriteria decision analysis technique which includes amr pathogens which causes mortality, prevalence of resistance, transmissibility, treatability, healthcare and community burden, 10-year trend of resistance, preventability in hospital and community settings, and current pipeline [9] . to fight against increasing prevalence of antibiotic-resistant superbugs and to limit the indiscriminate use of antibiotics, who prepared three groups of antibiotics namely access, watch, and reserve group to ensure appropriate prescription and use. access group comprises of antibiotics which are prescribe as in common infection as first and second choice. the first-choice antibiotics are narrow spectrum with low-resistant potential, whereas the second-choice antibiotics are broad spectrum having higher resistant potential. antibiotics such as β-lactam, chloramphenicol, and clindamycin come under this category. watch group identifies pharmacological antibiotic classes which prescribes as first or second choice but have a limited number of indication. the watch group has higher resistance potential compared with the access group. watch group comprises macrolides, quinolones and fluoroquinolones, and glycopeptides class of antibiotics [29] . who created reserve group of antibiotics to reserve some antibiotics as the last resort in superbug infection. this group of antibiotics should be recommended as "last resort," in case of life-threatening infection when other alternatives are failed in treatment. reserve groups include eight antibiotic or antibiotic class which are aztreonam, fosfomycin, fourth-generation cephalosporins (cefepime), oxazolidinones (linezolid), fifth-generation cephalosporins (ceftaroline), tigecycline, polymyxins (polymyxin-b, colistin), and daptomycin [6] . recently, among these eight antibiotic classes, resistant determinant against colistin was detected in people of rural vietnam. the prevalence of colistin resistant escherichia coli in intestine of resident of vietnam was extremely high, that is, approximately 70%. previously, mutation that causes colistin resistant was not transferable; thus it is not considered as pathogenic, as intestinal e. coli is nonpathogenic. finally, a transmissible colistin resistance gene (mcr) was detected in china, which has the potential to transfer colistin resistant to pathogenic microbes. the prevalence of mcr gene represents a serious concern regarding the emergence of superbugs that are resistant to last resort of antibiotic [30] . currently, there are only fewer option (such as last resort antibiotics) to tackle antibiotic-resistant superbugs, and the finding of mcr gene indicates toward the needs of innovative research which results in better understanding of superbug emergence and also to research and development on quality, safe, efficacious, and affordable antimicrobial medicines, especially new antibiotics and alternative therapies, vaccines, and diagnostics. the various chapters of this book discussed about the occurrence and prevalence of human and veterinary pharmaceuticals (antibiotics, antidepressant, antidiabetic, radioactive agents, etc.) at different environmental sites. these pharmaceutical compounds are biologically active compounds that are known to have a specific mode of action (moa) in human and animals even at low concentration. due to genetic relatedness (presence of conserved gene) across species, these compounds interact with protein and cell lineage of nontarget organisms (conserved therapeutic drug targets) and elicit a response in their body (targets metabolic pathway, enzyme or mediators of cell signaling molecule). for instance, ibuprofen which is a cyclooxygenase inhibitor was found to interfere with arachidonic acid signaling pathway in marine clams ruditapes philippinarum [31] . however, the effect of pharmaceuticals may vary from species to species due to difference in gene expression of the same gene across species or due to change in solubility or potency of drugs because of binding of pharmaceuticals with organic molecules present at environmental sites. therefore to understand the environmental risk posed by these contaminants, conceptual model of moa of pharmaceuticals was used. the moa [15] approach involves the assessment of drug targets and its evolution between mammals and the model species. the conceptual model of moa approach in marine organisms reveals the presence of fluoxetine at environmental concentration was able to control the serotonin signaling pathway, and this alteration in physiological signaling pathway results in defects in reproduction, locomotion, and metabolism of aquatic organisms [31] . many short-term toxicity studies reported that the drug molecules do not have an acute toxic effect on aquatic organisms because of their presence in low concentration (ng/l to low μg/l), but their constant release and exposure to aquatic biota have long-term chronic effects [32, 33] . however, many laboratory studies at high concentration of pharmaceutical compounds report direct lethal effects. tetracycline concentration around 10à100 μg/l leads to low periphyton (nematode, bacteria, and algae) concentration in mesocosm stream [34] . structure disruption in kidney and intestine of rainbow trout and brown trout due to diclofenac (5 μg/l) was reported [35] . prolonged exposure to pharmaceuticals in low concentration leads to the change in species trait and behavior of aquatic organisms. antidepressant and psychiatric drugs such as fluoxetine and oxazepam cause disruption in ecological interaction even in low concentration [9] . indeed, even by considering that these drugs are diluted after their release, it is evident that they have a toxic effect on the aquatic ecosystem. the widespread occurrences of pharmaceuticals at various environmental sites such as in ocean, river, and groundwater raise a concern regarding the risk associated with human health. for instance, the birth control and growth stimulator agent like estrogen have the potential to cause prostate and breast cancer in humans (if the estrogen concentration is used above the threshold limit). the worldwide discharge of estrogen from livestock and human ranges approximately 86,000 and 30,000 kg/year, respectively. the us national toxicology program declared estrogen as carcinogen, the no adverse effect concentration of estrogen in human is 0.3 mg/day and frequent detection of estrogen in drinking water concentration ranging from 0.1 to 2.0 ng/l represent a health risk [33] . estrogen was reported to cause abnormalities in animals such as permanent infertility (clover disease in sheep due to feeding on clover plant which has high phytoestrogen level) [33] . however, human health risk assessment studies reported no appreciable risk to human due to exposure of pharmaceuticals mixture [32] . due to emergence of antibiotic-resistant pathogens and unavoidable use of antibiotics, concomitant environmental perturbation caused by climate change might make the earth is not suitable for humans and other livings. thus researchers focus on finding alternative methods to avoid use of antibiotics and developing novel treatment technologies to degrade and remove the pharmaceutical compounds. prediction of emerging signals by theoretical and bioinformatics tool will highly help to alarm the researchers, hospitals, and public about the nearby occurrence of resistant bug. such predictions are underway by monitoring microbial community dynamics in different environmental samples; however, these studies facing enormous challenges in the developmental face [28,36à43] . several research studies have been conducted to observe a change in microbial metabolic pathways for a while. such study results could predict microbial interactions and route of evolution, and might apply to precisely pinpoint the emerging organism in the mixed microbial community. these studies are still under research stage, such studies are using genomic, metabolomic, and trancsriptomic tools to predict the bug emergence. recently, geoghegan and holmes [44] attempted predicting the virus emergence with an interest of biomedicine and preventing unpredicted incidence [44] . they have monitored the evolution of viruses in short-term period to highlight the cross-species transmission and emergence. they have concluded that predicting emergence requires a new mechanistic and integrated approach, which might permit or stop the emerging viral spread into new hosts [44] . with the fact that microbial resistant to pharmaceuticals, the treatment options are reduced; however, alternative methods have been explored to protect human and animal from microbial illness. the microbial drug resistance leads to direct and indirect consequences as described by who report 2015 [45] . the severe direct consequences are prolonged illness, impossible to protect the patients undergoing surgery and augmented treatment cost. whereas the indirect impacts of amr are depleting the global economy (due to the loss of productivity by sickness) and higher costs of treatment. thus who proposed a global action plan to assure preventing the transmission of infectious disease and providing complete treatment with the help of safe and effective medicines [45] . to achieve the global action plan successfully, five objectives have been proposed by who: (1) to improve awareness and understanding of amr; (2) to strengthen knowledge through surveillance and research; (3) to reduce the incidence of infection; (4) to optimize the use of antimicrobial agents; and (5) to ensure sustainable investment in countering amr. these objectives are likely to be met through political leaders, member states, the secretariat, and international and national partners across multiple sectors. notably, individual responsibilities like recycling unused medicines, intake of only prescribed medicines, and keeping the environment clean would highly help to achieve the who's global action plan for the betterment of human and animal health. increasing resistance to antibiotics and the emergence of "superbugs" that are resistant to drugs of last resort have highlighted the great need for alternative treatments of bacterial disease. this has led to renewed interest in the potential of phage to treat bacterial pathogens. the term "phage therapy" usually refers to the treatment of bacterial infections with intact phage; however, there are other ways in which phage can be used as antibacterials. phage can be used as "lethal agent delivery systems" to introduce nonspecific or toxic antimicrobials [46, 47] , or genes encoding antimicrobials [48] into selected pathogenic bacteria. in another method (targeted gene transfer), filamentous phage can be modified to carry therapeutic genes and to target cell surface antigens or receptors on mammalian cells, transducing them by receptor-mediated endocytosis [49] . although phages are not an antimicrobial tool in itself, this approach could be used to deliver antimicrobials to intracellular bacterial pathogens. phage secrets lysins enzymes which lyse the host bacteria. lysins are host specific and use cell wall components that are essential for viability as receptors; therefore bacterial resistance is rare [50] . initial tests of lysins against clinically relevant gram-positive bacteria, such as methicillin-resistance staphylococcus aureus, look promising [51, 52] . the use of multiple phage lysins with different cleavage sites could increase therapeutic effectiveness and further reduce the chance of bacteria developing resistance. lysins can also be used to make bacterial ghost vaccines. to produce better quality of treated sludge, wastewater treatment processes must achieve a minimum 6 log reduction in e. coli loads and a final end product (sludge) with a maximum admissible concentration of e. coli 1 3 10 3 cfu/g [dry solids (ds)] and zero salmonella in 2 g (ds). similarly, united states environmental protection agency regulations are categorized into class a and class b sludges. class a pathogen reduction requirements are more stringent than those of class b sludges which are subject to application restrictions. class a sludges are required to reduce pathogens to below the detectable limit (,3 most probable number of salmonella, ,1 plaque forming unit of enteric viruses, and ,1 viable helminth ovum per 4 g ds). treatment processes designed to achieve pathogen reduction to a required level can incur substantial capital and operating costs [53] . development of phage treatment of sludge may provide long-term and cost-effective control of potentially pathogenic bacteria (e.g., e. coli and salmonella). successful phage treatment of wastewater bacterial pathogens would be dependent on the prevalence and diversity of pathogen species within wastewater. it would be virtually impossible to produce phage targeted at all pathogenic serotypes. for example, there is a high diversity of e. coli serotypes [54] and around 2400 known salmonella serotypes [55] . however, wastewater treatment conditions intrinsically reduce the numbers of some pathogenic bacteria. therefore there is potential for phage treatment to be used successfully in combination with biological sludge stabilization processes to reduce the abundance of specific pathogenic bacterial strains such as e. coli o157. indeed, research on phage therapy for reduction of this pathogen in animal reservoirs is already underway [53] . although phage-derived therapies have several advantages compared with antibiotics, the combined use of phage, or phage lysins, and antibiotics is an attractive treatment regime. indeed, such a product is available commercially in georgia: phagobioderm is a biodegradable polymer composite impregnated with a lytic phage cocktail and ciprofloxacin [56] . using phage with antibiotics should increase the efficiency of treatment and reduce the emergence of resistant mutants because the surviving bacteria would need to acquire at least two separate resistance mechanisms. to kill the multidrug-resistant bacteria researchers from ibn (institute of bioengineering and nanotechnology), chin et al. [57] developed a novel synthetic molecule and demonstrated selectively destroying five multidrug-resistant bacteria [57] . recently polymer-based advanced drug designing to combat multidrug resistances is widely explored, which are described in detail [58, 59] . due to climate change, a steady increase in temperature (about 0.75 c) was recorded in the past 100 years, which was clearly observed on lands than the ocean in recent days [60] . however, ice melting in north pole, increase in sea level, and alteration in raining patterns are clear and known effects of examples of global warming. climate change affects the water cycle in the following aspects: (1) poor water quality, (2) less availability (quantity for consumption and use), (3) evaporation leads increases the microbial load (concentration) and toxic ions concentration, (4) water depletion in animal-farming and agriculture (leads to food scarcity), and (5) effects on freshwater biodiversity is still undetermined. ultimately, these changes will hugely affect wastewater microbiome (including beneficial and pathogenic microbes), the concentration of toxic pollutants and biological process performance, which was discussed in detail in chapter 13, treatment of wastewater containing pharmaceuticals: biological treatment. recently, who reported that climate change affects the infectious disease transmission pattern (fig. 17à1) . various infectious diseases and their agents (viruses, bacteria, protozoa, and multicellular parasites) have adapted (via evolution) humans as a primary host, which raises serious threat mankind to face in near future. due to the negligent, persistent and accidental activities of humans (industrial) raised the environmental pollution (chemicals including pharmaceuticals), which led to inseparable effect like climate change. furthermore, development of drug-resistant organisms and increased pathogen survival rate, only raising panic about the human, animal, and environmental health. thus researchers are continuously searching alternatives to these environmental problems. due to pharmaceuticals release in the environment and climate change, the survival of pathogens prolongs. high load of infectious bacterial strains dwelling in wastewater further favors the transfer of antibiotic-resistance gene. this could be a classical example for multidrug-resistant bacterial strains that are prevalence in tropical countries (like china and india). this draws immediate attention of public, health, and international sectors to fight against, pollution, climate change, and drug resistance. the advancement in medicinal research helps in control and elimination of various infectious diseases such as smallpox. however, emergence and reemergence of infectious diseases due to various factors, that is, natural evolution, climate change, amr, and many more, is a matter of concern. the knowledge about the evolution and life cycle of pathogens (bacteria, virus, or parasite) using omics study 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recombinant phage lysin lysk has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant staphylococcus aureus a novel sustained-release matrix based on biodegradable poly(ester amide)s and impregnated with bacteriophages and an antibiotic shows promise in management of infected venous stasis ulcers and other poorly healing wounds dictionary of microbiology and molecular biology antigenic formulas of the salmonella serovars bacteriophage-resistance systems in dairy starter strains: molecular analysis to application a macromolecular approach to eradicate multidrug resistant bacterial infections while mitigating drug resistance onset antibiotic-containing polymers for localized, sustained drug delivery recent advances in the treatment of pathogenic infections using antibiotics and nano-drug delivery vehicles, drug des van der valk, managing water resources under climate uncertainty key: cord-312461-5qzpo6l1 authors: adalja, amesh a.; watson, matthew; toner, eric s.; cicero, anita; inglesby, thomas v. title: characteristics of microbes most likely to cause pandemics and global catastrophes date: 2019-08-30 journal: global catastrophic biological risks doi: 10.1007/82_2019_176 sha: doc_id: 312461 cord_uid: 5qzpo6l1 predicting which pathogen will confer the highest global catastrophic biological risk (gcbr) of a pandemic is a difficult task. many approaches are retrospective and premised on prior pandemics; however, such an approach may fail to appreciate novel threats that do not have exact historical precedent. in this paper, based on a study and project we undertook, a new paradigm for pandemic preparedness is presented. this paradigm seeks to root pandemic risk in actual attributes possessed by specific classes of microbial organisms and leads to specific recommendations to augment preparedness activities. the recent global experience with severe infectious disease epidemics has triggered much interest in understanding the broader pandemic threat landscape. a substantial proportion of pandemic and biological threat preparedness activities have focused on list-based approaches that were in part based on pandemic influenzas of the past, historical biological weapon development programs, or recent outbreaks of emerging infectious diseases (e.g., sars, mers, ebola) (centers for disease control and prevention 2017; casadevall and relman 2010) . but such an approach inherently fails to account for agents not currently known or those without historical precedent. for that reason, preparedness activities that are limited to these approaches may hamper preparedness and lessen resilience. the purpose of this study was to analyze the characteristics of pathogens that could be capable of causing a global catastrophic biological risk (gcbr). these would be events in which biological agents-whether naturally emerging or reemerging, deliberately created and released, or laboratory engineered and escaped-could lead to sudden, extraordinary, widespread disaster beyond the collective capability of national and international governments and the private sector to control. if unchecked, gcbrs would lead to great suffering, loss of life, and sustained damage to national governments, international relationships, economies, societal stability, or global security (schoch-spana et al. 2017) . given the severe potential public health consequences of pandemic events, there needs to be a vital interest in developing and maintaining a flexible, rapid, and robust response capability. anticipating the forms of microbial threats that might cause future pandemics can help strengthen preparedness and response capacities. this paper proposes a framework for considering future pandemic threats and provides recommendations for how this framework should inform pandemic preparedness. review of the published literature and previous reports: the project team surveyed the current biomedical literature on the topic of emerging infectious disease characteristics, the pathogenic potential of microbes, and related topics. the literature review was microbe-and species-agnostic, encompassing all classes of microorganisms and host species. the literature review was accomplished with extensive pubmed searches on these subjects. relevant us government policy and strategy were reviewed. interviews: the project team interviewed more than 120 technical experts who work in and are intimately knowledgeable about this field. interviewees were drawn from academia, industry, and government. our goal was to ascertain the experts' views about the essential traits needed for a pathogen to become a gcbr, to contextualize historical outbreaks in light of these traits, and to determine which currently known infectious disease agents possess such characteristics. pandemic pathogen meeting: the project team completed a preliminary analysis that synthesized the results of our literature review and expert interviews. those findings were used to design and facilitate a meeting held on november 9, 2017, that included many of those who had been interviewed for this project. the meeting was held at the johns hopkins center for health security in baltimore, md. the purpose of the meeting was to gain additional insight and input into the project analysis, examine assumptions, and test possible recommendations. participants included representatives of us and foreign academic institutions, the federal government, and other independent subject matter experts. this paper is based on the findings of the project and is modification of the project report (johns hopkins center for health security 2018). when a pathogen has the capacity to cause a pandemic, it will possess several attributes that other microbes, capable of causing only sporadic or limited human infections, will lack. these traits can be divided into several categories: spread via respiratory transmission; capable of spread during incubation period prior to symptom onset; no preexisting host immunity; and other possible intrinsic microbial characteristics. many of these characteristics have been captured and are reflected, in equation form, by casadevall (casadevall 2017) . microbes have varied routes of transmission, ranging from blood and body fluids to vector-borne to fecal-oral to respiratory (airborne and respiratory droplet). while each mode of transmission is capable of causing large outbreaks if sustained human-to-human transmission is possible and left unchecked, certain modes of transmission are more amenable than others to intervention. for example, the transmission of an infectious disease caused by blood and body fluid transmission can be halted with infection control measures such as gloves or gowns. of the various modes of transmission, the respiratory route is the mechanism most likely to lead to pandemic spread. this is chiefly due to the fact that interventions to interrupt this method of spread are more difficult to implement when the simple and universal act of breathing can spread a pathogen. the prolific spread of influenza, pertussis, measles, and rhinoviruses is testament to this fact (herfst et al. 2017) . by contrast, although pathogens spread by the fecal-oral route, such as vibrio cholera and the hepatitis a virus, can generate explosive outbreaks, even a modicum of sanitary infrastructure can quench the outbreak. vector-borne outbreaks are a special case of a non-respiratory-spread agent. indeed, the only postulated extinction of a mammalian species by an infectious organism, the christmas island rat, was caused by a vector-borne trypanosome (wyatt et al. 2008) . for most of the agents that use this class of transmission, the spread is limited by a geographically and climatologically restricted vector habitat. humans can protect against vectors, and they can change where they live, but the christmas island rat could not. these factors have generally served to limit the pandemic potential of microbes that are spread by vectors. exceptions to this general limitation of vector-borne viruses include microbes spread by anopheles and aedes mosquitoes. pathogens spread by these mosquitoes have higher pandemic potential, given the geographic breadth of their spread. for example, most of sub-saharan africa is hospitable to the malaria-transmitting anopheles mosquitoes, while residents in 75% of us counties-as well as half the world's population-are regularly exposed to aedes mosquitoes that serve as vectors for high viremia flaviviruses and alphaviruses. such phenomena are borne out by the prolific spread of dengue, chikungunya, and zika (sinka et al. 2012 ; centers for disease control and prevention 2017). the onset and duration of the period when a person is contagious during an infection also play a major role in spread. diseases that are contagious during a late stage of infection, when infected people are very sick and therefore have more limited opportunities for spread, may be delimited in their spread. on the other hand, diseases that are contagious prior to symptom development, during the incubation period, or when only mild symptoms are present have greater opportunities for spread as infected individuals are able to conduct their activities of daily living with little or no interruption. modeling studies with simulated outbreaks have shown that the presence or absence of this timing of transmission factor can be decisive in whether an outbreak can or cannot be controlled. if a microbe is contagious before a person is seriously ill while the disease is still incubating, then there is higher potential for pandemic spread. historical examples reinforce this idea, as the only human infectious disease to be vanquished from the planet-smallpox-was one that was not contagious during the incubation period (fraser et al. 2004) . by contrast, a microbe such as the influenza virus, which is contagious prior to symptom development and has a wide range of clinical severity, is able to infect widely and is not amenable to control (brankston et al. 2007 ). microbial pathogenicity cannot, in reality, be separated from host characteristics. as elucidated by pirofsky and casadevall's host damage framework, disease is a complex interplay between a host immune system and a microbe (pirofski and casadevall 2008) . in congruity with this paradigm, host features and microbial pathogenicity are discussed together. for a microbe to cause a gcbr-level pandemic, it will be necessary for a significant proportion of the human population to be immunologically naïve to the agent so that the microbe would have a high number of susceptible humans to infect. additionally, large quantities of a sufficiently effective countermeasure (vaccine or antimicrobial agent) would not be available. immunologic naïveté would be expected with a zoonotic pathogen. the microbe, correspondingly, would have to possess the ability to evade the host immune response through virulence factors, immunological camouflage, or other features that allow a productive infection to ensue. additionally, human receptors that are utilized by a pandemic-causing microbe would likely be widespread in the population, facilitating permissive infection in the majority of humans. receptors may also provide target organ tropism for the agent, allowing severe disease to occur (e.g., lower respiratory tract and central nervous system). case fatality rates (cfrs) need not be inordinately high to cause a gcbr-level event, as evidenced by the 2.5% cfr reported for the 1918 influenza pandemic-the event closest to an actual human gcbr in the modern era (taubenberger and morens 2006) . a low but significant cfr adheres to the host density threshold theorem. according to this commonly held theorem, a microbe that kills too many of its hosts will run out of susceptible hosts and be extinguished (cressler et al. 2016) . while this may be true of pathogens that are closely linked to one host species, it is not applicable to sapronotic diseases such as amebic encephalitis and cholera (in certain contexts), which can infect and kill without jeopardizing future transmission or survival. indeed, many extinction-level amphibian infectious diseases are sapronotic in nature, such as the chytrid disease of salamanders and frogs (fisher 2017) . additionally, a gcbr-level event may not confer direct mortality. reproductive effects (i.e., in the manner of rubella or zika) or carcinogenic effects (e.g., htlv-1) could, in many ways, be highly detrimental to the future of humanity, as they could lead to significant curtailment of lifespans and diminishing birth rates, which could ultimately result in significant population collapse (rasmussen et al. 2017; tagaya and gallo 2017) . given the right context, any microbial organism could evolve or be engineered to be a gcbr. however, the most likely cause of a gcbr presently is a virus, with rna viruses being the most probable (woolhouse et al. 2013 ). historically, bacterially caused infections such as plague have had incredible impacts on the human species (raoult et al. 2013 ). however, the development of antibacterial therapies, beginning with the sulfonamides in 1935 and then penicillin in 1942, has severely limited the ability of this class of microbes to cause a gcbr-level pandemic. in addition, the relatively slower speed of replication and accumulation of mutations also disadvantages this class over viruses. for example, a human infected with the hepatitis c virus (an rna virus) produces trillions of virions per day, whereas the doubling time of yersinia pestis, the cause of plague, is 1.25 h (neumann et al. 1998; deng et al. 2002) . the public health crisis of multiple-drug-resistant bacteria, such as carbapenem-resistant enterobacteriaceae (cre) and others, is very alarming (logan and weinstein 2017) . the spread of these bacterial agents, for which few if any treatments exist, threatens the entire practice of modern medicine, from cancer chemotherapy to joint replacement therapy. however, these organisms, which have variable attributable mortality, tend to be unable to efficiently infect human hosts that are not compromised or hospitalized. as such, the risk to the general public is constrained. large outbreaks of cholera and plague have represented true public health emergencies in yemen and madagascar, but their spread reflects severe infrastructure deficiencies caused by war and supply constraints rather than true global pandemic risk (qadri et al. 2017; roberts 2017 ). fungi represent prolific pathogens outside of the mammalian species. outbreaks of chytrid fungal disease in frogs and salamanders as well as snake fungal disease represent true existential threats to affected species (fisher 2017) . however, fungi are largely thermally restricted, and only limited members of this class of microbes can infect warm-blooded organisms such as mammals (casadevall 2012) . indeed, a fungal filter is hypothesized to have existed and may be partly responsible for mammalian warm-bloodedness. the success of the mammalian-adapted fungus that causes white-nose syndrome in bats is facilitated by the lower body temperature that occurs during their hibernation (foley et al. 2011) . human infections with fungi tend to be severely damaging only in an immunocompromised host. the human innate immune system contends with countless fungal spores that are present in every breath of air. as such, many endemic fungal diseases, such as histoplasmosis or coccidioidomycosis, do not cause harm in the majority of immunocompetent humans infected. even newly emerging fungi such as candida auris and cryptococcus gattii are largely subjected to this limitation (chowdhary et al. 2017 ; centers for disease control and prevention 2010). one of the most widespread fungal outbreaks-the exserohilum fungal meningitis outbreak-was abetted by direct injection of a contaminated medical product into the spinal region of humans, which is not a usual mechanism of infection (casadevall and pirofski 2013) . without thermal adaptation (which might be feasible with deliberate manipulation), fungi, many of which are sapronotic and do not rely on or need mammalian hosts, will not constitute a pandemic threat to humans. prions-transmissible infective proteins-are one of the most fascinating and understudied of infectious agents. these agents, which are responsible for diseases such as kuru and new variant creutzfeldt-jakob disease (vcjd, the human form of "mad cow disease") in humans, cause scrapie, chronic wasting disease, and bovine spongiform encephalopathy in other mammalian species (chen and dong 2016) . though highly damaging to humans and other species they infect, prions require specific conditions for spread. new variant creutzfeldt-jakob disease was to date the most highly publicized outbreak of a human prion disease; it resulted in 229 human cases tied to the consumption of beef products primarily in england in the 1990s and the 2000s (hilton 2006) . other modes of transmission of cjd tied to iatrogenic spread via contaminated surgical instruments or cadaveric hormone products ceased once protective measures were put in place (bonda et al. 2016 ). kuru, a geographically restricted prion disease, was spread via human cannibalism in papua new guinea, and the outbreak abated once that practice was ended in the 1960s (liberski et al. 2012) . the transmission characteristics of prion diseases are such that very extraordinary circumstances, on a par with human cannibalism or massive food contamination, must be present for a gcbr-level risk to be present for humans. additionally, and almost by definition, such an event would be slow-moving (prions were once known as "slow viruses"). protozoal organisms have the distinction of being the only infectious disease to have caused the extinction of a mammalian species. the christmas island rat, unable to outrun its vector, was felled by a vector-borne trypanosome (t. lewisi) during the early twentieth century on the australian island (wyatt et al. 2008) . human forms of trypanosomiasis have not risen to such a level of concern. human protozoal infections have exerted tremendous pressure on the species, and it is hypothesized that half of all humans who have lived died of malaria, which still kills approximately half a million humans annually (world health organization 2017). however, the development of antimalarial compounds and vector avoidance strategies has proved successful when they are able to be employed appropriately, and they have relegated malaria to a pathogen whose impact is amenable to control. nonetheless, one aspect of malaria is of particular concern: the development and spread of artemisinin-resistant forms, which render treatment extremely challenging with little to no effective antimalarial agents left for use. largely confined to specific regions of asia, such as cambodia and myanmar, this organism poses severe treatment challenges and, if artemisinin-resistant forms were to spread to africa, could represent a continent-wide catastrophic biologic risk (haldar et al. 2018 ). ameba, ectoparasites, and helminths all have limited pandemic risk, as they are constrained by pathogenicity, transmissibility, or both. clonally transmissible tumors-such as the notable devil facial tumor disease in tasmanian devils-are rare occurrences in humans, with restricted modes of transmission (maternal-fetal and organ transplantation). space-adapted organisms (e.g., salmonella that originates on earth but spends time in the space station before coming back to earth) can exhibit enhanced virulence; however, they still are susceptible to antibiotic treatment and normal control measures: there is no evidence they pose greater epidemic risk than normal salmonella (wilson et al. 2007 ). an alien microbe species that is obtained on mars or meteorites and brought back to earth, one of the focuses of the planetary protection program at the national aeronautics and space administration (nasa), was not deemed by our interviewees and meeting participants to be likely to pose a threat. and if such a species were found, it would be unlikely to be adaptable to an earthlike planet environment, as adaptations to its home planet's markedly different environments would likely preclude adaptations to earth. even though the chances of serious biological risk posed by such a sample return are deemed to be low, there are many uncertainties, and the highest level biocontainment procedures are being considered for specimens that might harbor such non-earth-based organisms (national research council 2009). traditionally, viruses have been ranked at the highest level of pandemic risk, and dedicated preparedness efforts often focus solely on viruses. a disproportionate focus on viruses is justified, however, based on several aspects unique to the viral class of microbes. the high rate of replication of viruses-for instance, over 1 trillion hepatitis c virions are produced per day in a human infection-coupled with the mutability inherent in such short generation times gives viruses an unrivaled plasticity. this plasticity allows for host adaptability, zoonotic spillover, and immune system evasion. the lack of a broad-spectrum antiviral agent-like ones available for bacterial and even fungal organisms-also confers a special status on viruses. with no off-the-shelf treatment available to contain a viral outbreak, and likely no vaccine, containment efforts, at least in the early stages, will likely need to be made in the absence of a medical countermeasure (zhu et al. 2015) . there is a strong consensus that rna viruses represent a higher pandemic threat than dna viruses (kreuder johnson et al. 2015) . this assessment is derived from the fact that the stability of rna as a genomic material is less than that of dna, giving more genomic pliability to the rna viruses. dna viruses such as smallpox do challenge this assumption, and concern exists surrounding the related risks of monkeypox viruses, which are increasingly spreading in the absence of a smallpox vaccine campaign (kantele et al. 2016) . as monkeypox outbreaks continue to occur with longer chains of transmission, employing smallpox vaccines in target populations might be considered. another aspect of viral characterization is the location of replication. viruses with greater capacity for widespread have been shown in studies to be more likely to replicate in the cytoplasm of a cell (pulliam and dushoff 2009; olival et al. 2017) . this is postulated to be due to the higher affinity a virus must have for a particular type of host in order to be permitted entry into its nucleus, and this greater affinity would limit its zoonotic potential because it would be likely to be strongly tied to its usual host. in general, it is dna viruses that tend to have a nuclear replication cycle, while rna viruses have a cytoplasmic cycle. strikingly, smallpox-a dna virus with proven ability to cause pandemics-is a cytoplasmic replicator, while influenza-an rna virus with proven ability to cause pandemics -has a nuclear replication cycle. the exceptions to these rules argue against any overly strict adherence to them. other factors that may increase a virus' potential to cause a global catastrophic risk include a segmented genome (as exemplified by influenza viruses), a comparatively smaller genome size, and high host viremia (e.g., vector-borne flaviviruses). for example, the flu virus' segmented genome makes novel genetic assortment an eventuality, while a large genome may prevent nimble mutations. however, with each characteristic it is impossible to find a general rule, as exceptions abound. among currently studied viruses, the influenza a viruses are widely judged to pose the greatest pandemic risk based on historical outbreaks and viral characteristics (silva et al. 2017; imai et al. 2017 ). analysis of influenza risks is made in the centers for disease control and prevention (cdc)'s influenza rapid assessment tool (irat) which ranks h7n9 as the most concerning influenza virus strain (centers for disease control and prevention 2017). there are several viral groups other than the orthomyxoviruses (which include the h7n9 strain of influenza a) that are spread by respiratory routes, possess rna genomes, and merit enhanced attention: paramyxoviruses (especially these three genera: respirovirus, henipavirus, and rubulavirus), pneumoviruses, coronaviruses, and picornaviruses (especially these two genera: enterovirus and rhinovirus). based on our analysis and their inherent characteristics, these viral groups are the most likely source of a gcbr-level threat. there are efforts under way to construct viral catalogs of as many viruses as possible. the explicit aim of these projects is to reduce the uncertainty of outbreaks by extensively cataloging as many viral species as possible, so that a virus that causes a disease is less likely to be truly unknown. at the meeting and interviews for this project, a number of experts expressed concern that, while efforts to catalog and broadly sequence viruses in the animal world would provide new scientific discovery, we should not expect that it will identify the source of the next pandemic or that it can change the work being done for pandemic preparedness. broad viral sequencing would uncover many novel viruses. however, the vast majority of discovered viruses will not have the ability to infect humans let alone the prospect of widespread in the population. only a few viruses possess this ability. this work should be pursued with the objective of fundamental viral scientific discovery, rather than the goal of near-term improvement in pandemic preparedness. in the clinical practice of medicine, syndromic diagnosis-that is, making a nonspecific diagnosis, such as "sepsis," "pneumonia," or "viral syndrome," with little to minimal laboratory testing-is the norm. specific diagnosis (i.e., sending patient samples for definitive laboratory diagnosis) is often eschewed if it does not affect clinical management, is costly, and is not revealed with routine tests, and/or if the patient recovers. this practice has become enshrined not only in resource-poor areas in which access to diagnostic testing may be limited, but also in resource-rich areas, like north america and western europe, where specific diagnoses are viewed as superfluous. however, the yield from pursuing an etiologic diagnosis in infectious syndromes such as atypical pneumonia, sepsis, encephalitis, meningitis, and clinically significant fevers of unknown origin may be considerable, as it will provide important insight into the ongoing torrent of threats posed by the microbial world. by causing an infection with enough severity to come to medical attention, the culpable microbes have already established that they are damage-causing pathogens to humans-a feat that only a sliver of the microbial world can accomplish (woolhouse et al. 2016 ). many of these microbial diagnoses cannot be made through the routinely ordered diagnostics. therefore, a special effort would need to be made to get to a microbial diagnosis. if that were to be done more frequently and at a more strategic level around the world, it would provide an opportunity to develop new situational awareness regarding which microbes are circulating and infecting humans-information that is clinically valuable in its own right and more attuned to uncovering gcbr-level pathogens than broad viral cataloging. such efforts should not be limited to exotic "hot spots" of disease emergence but should be practiced in localities that are broadly representative of where these conditions occur. particular hot spots of emergence due to the presence of unique risk factors may be higher yield overall, but they should not be the sole sites of investigation. infectious disease emergence can occur anywhere, as evidenced by the 2009 h1n1 pandemic, which was first recognized as the etiology behind a mild pediatric upper respiratory infection in california and west nile fever emerging in cases of undifferentiated encephalitis in the new york city metropolitan area in the late 1990s (centers for disease control and prevention 2009; nash et al. 2001) . such a program would have significant cost and infrastructure implications in resource-constrained regions, so it would be most logical to set up sentinel or strategic sites for pursuing this level of microbial diagnosis in ways that are broadly representative. in developed nations such as the usa, these programs are available but underutilized because of lack of awareness or perceived lack of value by clinicians, for whom it will often not likely change therapeutic decisions. many participants in the project voiced the view that any microbe's pandemic potential could be substantially enhanced by human factors and poor preparedness, which could exacerbate a pathogen's spread or damage-causing potential. specific issues identified included gaps in hospital preparedness, medical countermeasure manufacturing capacity, medical countermeasure manufacturing locations, impacts on critical workforce members, and cascading effects on vital programs such as food production. for example, concentration of intravenous fluid manufacturing plants in puerto rico created massive shortages after a hurricane took the plants offline in 2017 (wong 2017) . the inability of hospitals to surge to meet enhanced patient needs for ventilators or icu beds is another potential constraint. human factors could also take the form of mistaken actions that are based on political considerations but are not supported by an evidence-based medical rationale, or scientific mistakes based on human error, such as misidentifying a microbe or misinterpretation of scientific or epidemiologic data. for example, early in the sars outbreak, mistakes regarding the etiology of the viral agent occurred, and the 2014 west african ebola outbreaks were initially thought to be cholera, delaying response efforts for months (world health organization 2014). some participants in this study were of the view that such factors as these could outweigh any intrinsic property possessed by a microbe or any physiologic vulnerability possessed by a human. magnification by human error could cause delays in response or awareness, allowing a pathogen to spread wider and deeper into the population and rendering containment more difficult, sowing panic, and severely stressing the healthcare infrastructure of a region. the majority view, however, was that intrinsic microbial characteristics are the main driver of a microbe's ability to cause a pandemic. pandemic preparedness should place a high priority on preparing for rna viral threats, given their frequent spread by respiratory route, cytoplasmic replication, and high mutability. surveillance, science, and countermeasure development programs and efforts should logically allocate significant resources to this class of microbes. except for influenza and certain coronaviruses, there are not major preparedness efforts being made for other viruses in this class of microbes. while rna viruses were at the top of the list of concerns, other classes of microbes, such as bacteria, fungi, and protozoa, should not be completely dismissed given characteristic that pose special concerns. cultivating and maintaining expertise in the epidemiology, surveillance, and pathogenicity of all classes of microbes, with explicit incorporation of a one health approach-which incorporates and integrates information from infectious diseases of plants, amphibians, and reptiles-will help foster the broad capacities needed for emerging pandemic and global catastrophic biological risks. pathogen-based lists, both usa and global, based on influenza precedents, historical biological weapon programs, and emerging infectious diseases were responsible for galvanizing early activities in the field of pandemic preparedness and have helped drive many important contributions. but these lists could create a sense of confidence regarding the prediction of future pandemic threats. lists can become frozen in the minds of those in the field and may be viewed as exhaustive rather than as starting points. additionally, inclusion in lists could also be sought for political (and not epidemiologic) reasons if inclusion carries with it the prospect of enhanced funding for a long-neglected endemic problem. one of the chief rationales behind this project was to attempt to move away from a strict list-based approach when considering pandemic threats and to develop a framework grounded in the facts of a microbe's biology and epidemiology. we recommend that risk assessment be rooted in the actual traits that confer pandemic or global catastrophic biological risks as opposed to a pathogen's presence on some earlier developed list. as respiratory-borne rna viruses have been identified as possessing heightened pandemic potential, it is important to strengthen surveillance activities around these viruses where they currently exist and establish them where they are not yet in place. currently, of the respiratory-borne rna viruses, only influenza and certain coronaviruses receive high priority for surveillance. while some efforts to understand coronaviruses, in the wake of sars and mers, exist, there is no systematic laboratory surveillance of coronavirus infections in humans. similarly, no such program exists for rhinoviruses, parainfluenza viruses, rsv, metapneumoviruses, and similar viruses. since this class of viruses is most likely to hold the future pandemic pathogen, constructing an influenza-like surveillance approach that better characterizes the prevalence, patterns, and geographic distribution of these viruses should be a priority. such an approach would focus on human infections, characterizing the epidemiology, virologic features, antiviral susceptibility (if applicable), and clinical manifestations in a fashion that mimics the extensive influenza surveillance conducted by the cdc and other international entities. currently, outside of anti-influenza antivirals, there is only one fda-approved antiviral for the treatment of respiratory-spread rna viruses (ribavirin). of the six fda-approved influenza antivirals-amantadine, rimantadine, baloxavir, zanamivir, oseltamivir, and peramivir-all target influenza viruses specifically and have no activity outside influenza, with two influenza a-specific agents (amantadine and rimantadine) rendered virtually obsolete because of resistance. the other antiviral agent (inhaled ribavirin) is approved for the treatment of respiratory syncytial virus (rsv) but has very limited use due to poor efficacy and major toxicity concerns for both rsv and parainfluenza viruses. there are currently no approved antivirals for any other respiratory-spread rna viruses in the world. prioritization of antiviral compounds against this group of viruses may lead to acceleration of drug development and (government and nongovernment) incentivizing programs. such antiviral compounds would have an advantage over many other emerging infectious disease countermeasures: these viruses exact a considerable toll in the form of community infections each year, providing a basis for a traditional pharmaceutical market as well as one for emerging infectious disease. pursuing not only broad-spectrum rna antivirals, but also those specifically targeted to specific viruses such as rsv, would increase the likelihood of yield. nontraditional molecules, such as monoclonal antibodies and immunomodulators, should also be investigated for a role in the treatment and prevention of rna virus respiratory infections (walker and burton 2018) . such adjunctive treatments may lead to improved clinical outcomes. to date, only one virally targeted monoclonal antibody is fda-approved: pavalizumab for prevention in high-risk infants. as with the above discussion regarding antivirals, the need for vaccines against respiratory-borne rna viruses should also be prioritized. currently, aside from influenza, for which a moderately effective but technically limited vaccine exists, there are no other vaccines for respiratory-borne rna viruses. experimental vaccines targeting rsv have made it into late clinical development only to fail. several important initiatives in this realm do exist and could be augmented to move beyond specific targets that have already been recognized. for example, the coalition for epidemic preparedness innovations (cepi) has selected a coronavirus (mers-cov) and a paramyxovirus (nipah) for vaccine development incentivizing (røttingen et al. 2017) . such a program could, in potential future initiatives, select additional vaccine targets from this group of viruses and even encourage the development of broadly protective vaccines against groups of viruses-for example, a vaccine that protects against all four strains of human parainfluenza viruses, both mers and sars covs, and both hendra and nipah viruses. additionally, the heightened interest at the national institutes of health (nih) in a universal influenza vaccine in the wake of the moderately severe 2017-18 influenza season should be channeled to provide significantly increased resources to this endeavor (paules et al. 2017) . as certain avian influenza viruses are of the highest threat tier, a universal influenza vaccine (even one that just protects against a strains) could substantially hedge against an influenza virus attaining gcbr status. as was evident during the 2009 influenza pandemic and subsequent influenza seasons, the treatment of influenza is suboptimal, despite evidence-based guidance. the status of the treatment for other respiratory viruses is even less defined. while there currently is not a robust antiviral armamentarium against these viruses, there are important clinical questions that occur with their treatment that merit further study. for example, what adjunctive therapies are useful? what coinfections may be present? at what stage of illness are rescue oxygenation devices warranted? as many of these viruses are highly prevalent in the community and are frequently encountered by clinicians in both outpatient and inpatient settings, finding answers to these questions would render clinicians more adept at dealing with pandemic versions of these viruses. with respect to influenza, there is a growing literature on the use of antiviral agents in combination with anti-inflammatory agents such as nonsteroidal anti-inflammatory agents (nsaids) and macrolide antibiotics (hung et al. 2017) . untangling the nuances of these treatment effects in order to develop robust guidance would have an impact on the ability to cope with an influenza-driven gcbr. because of the higher likelihood that a gcbr-level threat might emerge from the group of rna viruses with respiratory-spread, special attention to research on these agents is warranted if such research could increase pandemic risks. while much research on this class of viruses would be low risk and managed by appropriate approaches to biosafety, experimentally engineered antiviral resistance, vaccine resistance, or enhanced transmission, for example, would raise major biosafety and biosecurity concerns. the 1977 appearance of the h1n1 influenza a strain was thought to have resulted from laboratory escape (zimmer and burke 2009) . it is important to understand the kinds of work being performed with these agents and, in particular, to know of experiments that are being done or are being proposed that would result in increased pandemic risks. those experiments should have their own special review and approval process that is consistent with the risks and assesses the risks and benefits of this work before approval or funding of this work. as unknown infectious syndromes abound in all locations, and any given infectious syndrome may have as its etiology a potentially unknown or unappreciated microbe, specific diagnosis should be a routine endeavor. atypical pneumonias, central nervous system infections, and even upper respiratory infections often are treated without any etiologic agent being identified. as diagnostic technologies and devices improve in breadth, speed, and ease of use, the increasing uptake of these devices will provide a new opportunity to enhance situational awareness of an infectious syndrome in any location where they are deployed. such devices are currently being used in research projects in the developing world. the more routine use of devices, such as multi-analyte molecular diagnostic devices, has the capacity to provide a fuller picture of the microbiological epidemiology of any given syndrome, illuminating what has heretofore been biological dark matter (doggett et al. 2016; kozel and burnham-marusich 2017) . coupled with heightened surveillance of respiratory-borne rna viruses, the ability to capture an early signal of a potential pandemic pathogen will be greatly enhanced. to date, certain considerations have limited the uptake and use of these devices: cost, perceived lack of clinical impact, and constraints on hospital resources such as isolation beds. impacts on hospitals might be noted in laboratory testing volume as well as costs. however, when these devices are viewed in the context of pandemic preparedness, the cost-effectiveness calculation should change. these considerations could be moderated if they are considered part of a hospital's emergency preparedness activities and not exclusively as clinical (they also have benefit for antibiotic stewardship activities in both inpatient and outpatient settings). in fact, the use of these devices should be considered on a par with mechanical ventilators, vaccines, antivirals, and antibiotics in the context of pandemic preparedness. pilot projects demonstrating the feasibility of procuring such devices for infectious disease emergency preparedness could be conducted. understanding the microbial characteristics most importantly regarding the risks of pandemic or global catastrophic biological threats can help strengthen pandemic preparedness activities. while rna viruses pose the greatest risks, there are characteristics of other microbial classes that cause special concerns and are important to consider in scientific research agendas and in public health preparedness efforts. this analysis leads to a series of recommendations related to disease surveillance, antiviral and vaccine development, clinical research, and research oversight. taken together, assessment of key microbial class characteristics plus the focused actions that follow this assessment can broadly help improve preparedness for pandemic and global 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carbapenem-resistant enterobacteriaceae: the impact and evolution of a global menace the outbreak of west nile virus infection in the new york city area in 1999 assessment of planetary protection requirements for mars sample return missions hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy host and viral traits predict zoonotic spillover from mammals the pathway to a universal influenza vaccine the damage-response framework of microbial pathogenesis and infectious diseases ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses cholera in yemen-an old foe rearing its ugly head plague: history and contemporary analysis studying the effects of emerging infections on the fetus: experience with west nile and zika viruses echoes of ebola as plague hits madagascar new vaccines against epidemic infectious diseases global catastrophic biological risks: toward a working definition estimating disease burden of a potential a(h7n9) pandemic influenza outbreak in the united states a global map of dominant malaria vectors the exceptional oncogenicity of htlv-1 influenza: the mother of all pandemics passive immunotherapy of viral infections: 'super-antibodies' enter the fray space flight alters bacterial gene expression and virulence and reveals a role for global regulator hfq hospitals face critical shortage of iv bags due to puerto rico hurricane. the guardian rna viruses: a case study of the biology of emerging infectious diseases assessing the epidemic potential of rna and dna viruses ground zero in guinea: the ebola outbreak smouldersundetected-for more than 3 months historical mammal extinction on christmas island (indian ocean) correlates with introduced infectious disease broad-spectrum antiviral agents historical perspective-emergence of influenza a (h1n1) viruses key: cord-310509-c8wp2m69 authors: morens, david m.; fauci, anthony s. title: emerging infectious diseases: threats to human health and global stability date: 2013-07-04 journal: plos pathog doi: 10.1371/journal.ppat.1003467 sha: doc_id: 310509 cord_uid: c8wp2m69 nan historical information as well as microbial sequencing and phylogenetic constructions make it clear that infectious diseases have been emerging and reemerging over millennia, and that such emergences are driven by numerous factors (table 1) . notably, 60 to 80 percent of new human infections likely originated in animals, disproportionately rodents and bats, as shown by the examples of hantavirus pulmonary syndrome, lassa fever, and nipah virus encephalitis [2] [3] [4] . most other emerging/reemerging diseases result from human-adapted infectious agents that genetically acquire heightened transmission and/or pathogenic characteristics. examples of such diseases include multidrug-resistant and extensively drugresistant (mdr and xdr) tuberculosis, toxin-producing staphylococcus aureus causing toxic shock syndrome, and pandemic influenza [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] . although precise figures are lacking, emerging infectious diseases comprise a substantial fraction of all consequential human infections. they have caused the deadliest pandemics in recorded human history, including the black death pandemic (bubonic/ pneumonic plague; 25-40 million deaths) in the fourteenth century, the 1918 influenza pandemic (50 million deaths), and the hiv/aids pandemic (35 million deaths so far) [6, 9] . two major categories of emerging infections-newly emerging and reemerging infectious diseases-can be defined, respectively, as diseases that are recognized in the human host for the first time; and diseases that historically have infected humans, but continue to appear in new locations or in drug-resistant forms, or that reappear after apparent control or elimination [1] . emerging/ reemerging infections may exhibit successive stages of emergence. these stages include adaptation to a new host [11] , an epidemic/ pathogenic stage, an endemic stage, and a fully adapted stage in which the organism may become nonpathogenic and potentially even beneficial to the new host (e.g., the human gut microbiome) or stably integrated into the host genome (e.g., as endogenous retroviruses). although these successive stages characterize the evolution of certain microbial agents more than others, they nevertheless can provide a useful framework for understanding many of the dynamic relationships between microorganisms, human hosts, and the environment. it is also worth noting that the dynamic and complicated nature of many emerging infections often leaves distinctions between emerging and reemerging infections open to question, leading various experts to classify them differently. for example, we describe as ''reemerging'' new or more severe diseases associated with acquisition of new genes by an existing microbe, e.g., antibiotic resistance genes, even when mutations cause entirely new diseases with unique clinical epidemiologic features, e.g., brazilian purpuric fever [12] . similarly, we refer to sars as an emerging disease a decade after it disappeared, and apply the same term to the related mers (middle east respiratory syndrome) b coronavirus which appeared in saudi arabia in late 2012 [13] . the most salient modern example of an emerging infectious disease is hiv/aids, which likely emerged a century ago after multiple independent events in which the virus jumped from one primate host to another (chimpanzees to humans) and subsequently, as a result of a complex array of social and demographic factors, spread readily within the human population. aids was not recognized as a distinct entity until 1981 [6, 9] , after its initial detection among certain risk groups, such as men who have sex with men, recipients of blood products, and injection drug users. it was soon apparent, however, that the disease was not restricted to these groups, and indeed, the bulk of hiv infections globally has resulted from heterosexual transmission that has been heavily weighted within the developing world, particularly sub-saharan africa where a number of factors were responsible for this rapid spread; chief among these were human movement along truck routes accompanied by a high level of commercial sex work, inadequate public health infrastructures, poverty, and social inequality. this is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. the work is made available under the creative commons cc0 public domain dedication. funding: both authors are employees of nih and as such our work is funded by nih from operating funds rather than grants or other awards. competing interests: the authors have declared that no competing interests exist. other examples of disease emergences [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] include sars, which emerged from bats and spread into humans first by personto-person transmission in confined spaces, then within hospitals, and finally by human movement between international air hubs. nipah virus also emerged from bats and caused an epizootic in herds of intensively bred pigs, which in turn served as the animal reservoir from which the virus was passed on to humans. the 2009 h1n1 pandemic influenza virus emerged from pigs as well, but only after complex exchanges of human, swine, and avian influenza genes [14] . h5n1 influenza emerged from wild birds to cause epizootics that amplified virus transmission in domestic poultry, precipitating dead-end viral transmission to poultryexposed humans. additional examples are many [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] ; however, the variables associated with emergences are unique for each and typically complex. most of the important reemerging infectious disease agents first appeared long ago, but have survived and persisted by adapting to changing human populations and to environments that have been altered by humans. dengue virus and west nile virus (wnv), distantly related flaviviruses, serve as good examples. they have been spread by geographic movement of humans in association with the mosquito vectors for the diseases. for example, dengue came to the americas in association with the slave trade of earlier centuries. in this regard, slaves infected by mosquitoes in africa presumably brought the infection to the americas by seeding the mosquito population upon arrival [15] . similarly, wnv came to the united states in 1999 when an infected human, bird, or mosquito came by air travel from the middle east to the western hemisphere, providing a source for introduction of infection to new world mosquitoes and birds. pathogenic strains of dengue have also spread back from southeast asia to the western hemisphere, as has a major mosquito vector, aedes albopictus. unlike most arboviruses, which are partly or completely host-restricted, wnv has become adapted to multiple mosquito and avian species, a major factor in increasing its opportunity to infect humans. the lack of additional hosts undoubtedly drove the mosquitoes that are the vectors of dengue and the dengue virus itself to favor adapting to humans and to their behaviors and environments. the association of dengue with aedes mosquitoes that live in and around human habitations mean that crowding, poor sanitation, and poverty provide ideal environments for transmission to humans [15] . host immunity factors are also thought to be involved in the severe/fatal form of dengue known as dengue shock syndrome [15] . other non-arboviral examples of emerging infections abound. for example, cholera has repeatedly reemerged over more than two centuries in association with global travel, changing seasons, war, natural disasters, and conditions that lead to inadequate sanitation, poverty, and social disruption. emergences of disease caused by community-and hospital-acquired clostridium difficile and methicillin-resistant staphylococcus aureus (mrsa) have been driven by increased and/or inappropriate use of antibiotics, and some hospital-acquired organisms such as mrsa have now moved into community transmission. the global emergence of plasmid-spread ndm-1 (new delhi b-lactamase) gram-negative pan-resistant organisms, linked to global antibiotic use and inadequate antibiotic stewardship, medical tourism, economic globalization, and other aspects of modern life, has prompted calls for development of international control mechanisms [16] that are applicable to a number of emerging bacterial diseases in the developing and developed world. drug resistance mutations have also caused the reemergences of certain pathogens such as multidrug-resistant and extensively drug-resistant tuberculosis, drug-resistant malaria, and numerous bacterial diseases such as vancomycin-resistant enterococci. fungi have made significant contributions to disease emergence as well. in africa, cryptococcal disease has already surpassed tuberculosis as a leading cause of death [17] . other examples of fungal emergence include comorbidities in hiv-infected individuals (17) , cryptococcus gattii epidemics in predominantly healthy persons in the u.s. [18, 19] , and a 2012 u.s. nationwide epidemic of exserohilum rostratum infections associated with contaminated pharmaceutical products [20] . while it has become possible to eradicate certain infectious diseases (smallpox and the veterinary disease rinderpest), and to significantly control many others (dracunculiasis, measles, and polio, among others), it seems unlikely that we will eliminate most emerging infectious diseases in the foreseeable future. pathogenic microorganisms can undergo rapid genetic changes, leading to new phenotypic properties that take advantage of changing host and environmental opportunities. influenza viruses serve as a good example of emerging and reemerging infectious agents in their ability to rapidly evolve in response to changing host and environmental circumstances via multiple genetic mechanisms. new ''founder'' influenza viruses [21] appear periodically, cause a pandemic, raise widespread population immunity, and then, in table 1 . some major factors that underlie disease emergence and reemergence [2, 5] . response to human immune pressures, evolve and persist for decades using multiple genetic evolutionary mechanisms to sustain continual immune escape. the 1918 influenza pandemic virus is one example: over the past 95 years, its descendants have evolved continually by antigenic drift, intra-subtypic reassortment, and antigenic shift, the latter producing new pandemics in 1957 and 1968 [14] . even the genetically complex 2009 pandemic h1n1 influenza virus is a descendant of the 1918 virus [14] . such continuous genetic hyper-evolution forces us to develop new influenza vaccines containing new antigens on an annual basis. in the meantime, new human diseases keep emerging. as noted, in late 2012 the novel mers coronavirus emerged in saudi arabia [13] , and in early 2013 a new h7n9 avian influenza virus became epizootic in eastern china, causing 132 spillover infections of humans (as of june 7, 2013), with 28 percent case fatality [10, 22] . its pandemic potential, if any, remains to be determined. whether or not such outbreaks become more widespread, they nonetheless attract global attention and require significant international effort to monitor and contain. microbial advantages can be met and overcome only by aggressive vigilance, ongoing dedicated research, and rapid development and deployment of such countermeasures as surveillance tools, diagnostics, drugs, and vaccines. we appear to be entering a new era in which several important emerging, reemerging, and stable infectious diseases are becoming better controlled (e.g., hepatitis b, rabies, haemophilus influenzae type b, and even to some extent hiv/aids). however, our success in stopping the many new emerging diseases that will inevitably appear is not assured. we have many tools in our armamentarium, including preparedness plans and stockpiles of drugs and vaccines. but each new disease brings unique challenges, forcing us to continually adapt to ever-shifting threats [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] 23] . the battle against emerging infectious diseases is a continual process; winning does not mean stamping out every last disease, but rather getting out ahead of the next one. the perpetual challenge of infectious diseases emerging infections: microbial threats to health in the united states factors and determinants of disease emergence ecology of zoonoses: natural and unnatural histories the challenge of emerging and reemerging infectious diseases emerging infections: a perpetual challenge prediction and prevention of the next pandemic zoonosis emerging infectious diseases in 2012: 20 years after the institute of medicine report the world must build on three decades of scientific advances to enable a new generation to live free of hiv/aids world health organization. global alert and response (gar) crossspecies virus transmission and the emergence of new epidemic diseases tracing phylogenomic events leading to diversity of haemophilus influenzae and the emergence of brazilian purpuric fever (bpf)-associated clones genome characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans the persistent legacy of the 1918 influenza virus dengue research opportunities in the americas the emergence of pan-resistant gram-negative pathogens merits a rapid global political response estimation of the current global burden of cryptococcal meningitis among persons living with hiv/aids genome variation of cryptococcus gattii, an emerging pathogen of immunocompetent hosts the triple threat of cryptococcosis: it's the body site, the strain, and/or the host multistate outbreak of fungal infection associated with injection of methylprednisolone acetate solution from a single compounding pharmacy -united states reconstruction of the 1918 influenza virus: unexpected rewards from the past preliminary report: epidemiology of the avian influenza a (h7n9) outbreak in china drivers, dynamics, and control of emerging vector-borne zoonotic diseases key: cord-307803-rlvk6bcx authors: balloux, francois; van dorp, lucy title: q&a: what are pathogens, and what have they done to and for us? date: 2017-10-19 journal: bmc biol doi: 10.1186/s12915-017-0433-z sha: doc_id: 307803 cord_uid: rlvk6bcx microbes are found on us, within us and around us. they inhabit virtually every environment on the planet and the bacteria carried by an average human, mostly in their gut, outnumber human cells. the vast majority of microbes are harmless to us, and many play essential roles in plant, animal and human health. others, however, are either obligate or facultative pathogens exerting a spectrum of deleterious effects on their hosts. infectious diseases have historically represented the most common cause of death in humans until recently, exceeding by far the toll taken by wars or famines. from the dawn of humanity and throughout history, infectious diseases have shaped human evolution, demography, migrations and history. a pathogen is defined as an organism causing disease to its host, with the severity of the disease symptoms referred to as virulence. pathogens are taxonomically widely diverse and comprise viruses and bacteria as well as unicellular and multicellular eukaryotes. every living organism is affected by pathogens, including bacteria, which are targeted by specialized viruses called phages. the number of viruses and bacteria on earth is staggering and they occupy essentially every environment. a liter of surface seawater typically contains in excess of ten billion bacteria and 100 billion viruses. the number of viruses on earth is estimated to be around 10 31 , which corresponds to roughly ten billion times the number of stars in the universe [1] . an average human is made up of about 30 trillion cells but carries a similar number of bacteria, mostly in the gut [2] . the vast majority of viruses and bacteria we are exposed to have no negative effect and some can even * correspondence: f.balloux@ucl.ac.uk ucl genetics institute (ugi), darwin building, gower street, london wc1e 6bt, uk be beneficial, though a tiny fraction of these can severely affect our health. specifically, about one in a billion microbial species is a human pathogen. indeed, approximately 1400 human pathogens have been described, whereas it has been estimated that there are one trillion microbial species on earth, the vast majority of which remain uncharacterized [1] . pathogens can be divided into two main categories, namely facultative and obligate pathogens, reflecting how intimately their life cycle is tied to their host. facultative pathogens are organisms for which the host is only one of the niches they can exploit to reproduce. facultative pathogens are primarily environmental bacteria and fungi that can occasionally cause infection. they include many of the most problematic hospital-acquired bacteria involved in the antimicrobial resistance pandemic. a distinction is sometimes made between facultative and accidental pathogens, with the latter representing those which only occasionally infect weakened or immunocompromised hosts. typical examples of 'accidental' pathogens include neisseria meningitidis or escherichia coli. obligate pathogens require a host to fulfil their life cycle. all viruses are obligate pathogens as they are dependent on the cellular machinery of their host for their reproduction. obligate pathogens are found among bacteria, including the agents of tuberculosis and syphilis, as well as protozoans (such as those causing malaria) and macroparasites. some obligate pathogens require multiple different hosts to fulfil their life cycle. the definite host, which supports the adult form of the pathogen, is often a vertebrate and the intermediate host (referred to as a vector) is generally an arthropod or a mollusc. this alternation of vertebrate and invertebrate hosts is found in viruses (for example the zika virus), bacteria (for example lyme disease) and protozoa (malaria). trematodes (parasitic flatworms) go even further and some exhibit among the most baroque life cycles. digenetic trematodes have a basic three-host life cycle, and for some species a fourhost life cycle. for instance, halipegus occidualis sequentially has to infect a freshwater snail, an ostracod, a dragonfly nymph and ends its cycle after the dragonfly is eaten by the green frog rana clamitans, where it resides under its tongue [3] . what is the host range of pathogens? some pathogens are limited to infecting a single host species, whereas others can infect a multitude of host species. host ranges can feel highly idiosyncratic if not outright puzzling. for example, leprosy in humans is caused by two related intracellular bacteria mycobacterium leprae and mycobacterium lepromatosis, which are essentially restricted in the wild to humans, as well as armadillos in the americas and red squirrels in scotland [4] . conversely, yersinia pestis, another intracellular obligate bacterium and the agent of plague, has a natural life cycle involving alternating infections of rodents and fleas, but can infect essentially any mammalian host. an interesting twist in the case of plague is that y. pestis is not well adapted to the human host. with the exception of uncommon occurrences of human-to-human transmissions, referred to as pneumonic plague, plague epidemics (bubonic plague) are caused by plagueinfected fleas biting humans. somewhat ironically for a pathogen that is possibly the biggest killer in human history, bubonic plague is a complete evolutionary disaster. the human host is at a very high risk of dying, the flea cannot reproduce on a meal of human blood and the bacterium is stuck in an evolutionary dead-end as it cannot transmit to another host. there is no obvious predictor for the host range of different pathogens. intuitively, it may be tempting to predict that pathogens with a more intimate relationship with their host are more closely adapted to their host, and thus have a more restricted host range. however, there is no obvious pattern suggesting that viruses (that rely on the host cells' machinery for reproduction) have a narrower host range than bacteria. also, intracellular bacteria do not seem to have a markedly narrower host range than extracellular ones, despite being more intimately tied to their host. we know relatively little about the underlying genetic changes required for a pathogen to infect a new host, though, interestingly, only a few mutations can be required for a host jump. for example, avian influenza is only around five mutations away from being able to transmit in mammals [5] , and a single amino acid change was sufficient for the humanadapted bacterium staphylococcus aureus to become a pathogen of rabbits [6] . obligate pathogens tend to be highly adapted to their hosts, with sophisticated mechanisms to synchronise their life cycles with that of the host, and the ability to manipulate the host's immune system, metabolism and sometimes even behaviour. genes encoding proteins specific to pathogenicity are referred to as virulence factors, which include a variety of molecules required for colonization of the host, immunoevasion and immunosuppression, scavenging nutrients within the host, and entry into and exit out of cells for intracellular pathogens. in bacteria, virulence factors are often found in groups of genes on pathogenicity islands, which can be transferred horizontally by plasmids or other transposable elements. for example, one of the defining features of the plague bacterium y. pestis from its less virulent closest relative yersinia pseudotuberculosis, is the inclusion, early in its evolution, of two plasmids carrying genes involved in pathogenicity [7] . while acquisition of novel genes and repurposing of existing ones is essential in the evolution towards pathogenicity, a general feature during the evolution towards pathogenicity is genome reduction through the inactivation and loss of genes. this can be primarily explained by the fact that a host represents a fairly stable and resource-rich environment where some metabolic pathways required in the environment are not necessary. genome reduction is a general trend accompanying the evolution towards pathogenicity and is observed in mycobacterium tuberculosis, pathogenic e. coli strains and in the ongoing adaptation of klebsiella pneumoniae lineages to cystic fibrosis patients. the most extreme example is leprosy (m. leprae and m. lepromatosis), which has shed nearly half the genes found in their environmental relatives [8] . another interesting tendency of many bacterial pathogens is the secondary loss of the ability to undergo genetic recombination [9] . pathogens cause illness to their hosts through a variety of ways. the most obvious means is through direct damage of tissues or cells during replication, generally through the production of toxins, which allows the pathogen to reach new tissues or exit the cells inside which it replicated. bacterial toxins are among the deadliest poisons known and include famous examples such as tetanus, anthrax or botulinum toxin, known as botox in its commercial application. however, the damage to the host is often self-inflicted through a strong or sometimes excessive immune response that indiscriminately kills infected and uninfected cells and damages host tissues. typical examples of maladaptive over-reaction of the immune system include cirrhosis and liver cancer in hepatitis b [10] , or the 1918-1919 influenza epidemic, where the toll was highest amongst the young and healthy possibly because they mounted the strongest immune response and as such died from a 'cytokine storm' in the lungs leaving patients literally drowning in their own body fluids [11] . some pathogens benefit from the hosts' immune reaction to spread within an infected host or increase their transmission to uninfected hosts. influenza transmits mainly through aerosols created through the sneezing and coughing it causes. vibrio cholerae triggers a strong inflammatory response in the gut mucosa, leading to watery diarrhoea and ensuring its release in the environment and thus infection of further hosts. pathogens greatly vary in the severity of their symptoms from a mild inconvenience to assured death. it is sometimes assumed that the deadliest pathogens represent recent host jumps where the pathogens' virulence is maladapted to the new host, and that co-evolution between host and pathogen will lead to more benign symptoms over time. however, this is only true in the case of strict vertical transmission (such as from mother to child), where survival and transmission of host and pathogen are intimately linked. in the case of horizontal transmission, the situation is more complex and there is no straightforward way to predict the evolution of future virulence, as it will depend on a variety of factors, including the population structure of the host and the correlation between virulence and transmission [12] . a textbook example for reduction in virulence is the introduction of myxomatosis into the european rabbit population in australia and france in 1950 and 1952, respectively. upon introduction, the virus initially killed about 99% of infected rabbits but over a few years mortality went down to 90%, following the emergence of attenuated virus strains and genetic resistance in the rabbit population. while the virulence went down, which translated into higher transmission rates, the current 90% mortality rate remains exceedingly high and there is no evidence for further reduction in the short term [13] . bdgpl, the global lineage of the amphibian fungal pathogen batrachochytrium dendrobatidis (bd), is the only known pathogen to have extirpated entire host species. yet, over the three decades since its discovery, it shows no sign of evolving lower virulence. the primary reason there is little selective pressure on bd for virulence attenuation is that it can infect a very vast host range so that the extinction of any particular host species has limited impact on its fitness. even worse, some host species, such as the widely introduced african clawed frog, xenopus laevis, and the american bullfrog, rana catesbeiana, carry the disease asymptomatically, fuelling the global bd pandemics and limiting any shortterm prospect for significant decrease in virulence [14] . these are just examples of the evolution of virulence but both illustrate that there is no simple pattern of decrease in pathogenicity with time. how old are the major human pathogens? apart from a few putative ancestral pathogens, including helicobacter pylori [15] , that might have co-speciated with their human host, the infectious diseases afflicting us were acquired through host jumps from other wild or domesticated animal hosts or sometimes from the wider environment. the timing of these events and the original source remains unclear in many cases. the traditional view has been that many human pathogens emerged during the neolithic revolution. the main arguments for an origin of human pathogens linked to agriculture are based on the proximity between traditional farmers with their livestock and the emergence of higher human population densities in stable settlements enabled by agricultural subsistence. high population density is indeed required by some epidemic diseases which could not have maintained themselves on scattered groups of hunter-gatherers [12] . this argument, however, neglects the fact that pathogens can evolve fast. also, while the proximity of humans and livestock is conducive to host jumps, humans transmitted more diseases to domestic animals than they acquired, with tuberculosis in particular having probably jumped from humans to cattle rather than the other way around [16] . finally, this argument also neglects the high burden of pathogens in wild populations, including in the great apes. ancient direct evidence is scant for pathogens, and historical records rarely allow unambiguous attribution of described symptoms to a disease. that being said, recent progress in sequencing technology and in particular the ability to generate sequences, if not complete genomes, from ancient samples has greatly improved our understanding of the age of the major human pathogens, often leading to unexpected results. figure 1 summarises the current knowledge on the age of the seven current 'major killers' , as well as plague, which was included due to its major impact in the past. while some of these estimates may need to be updated in the future after the emergence of new evidence, it is unlikely the general pattern will change much. some human diseases are old (for example plasmodium falciparum malaria) and others recent, such as hiv or, more surprisingly, measles. there is also no obvious pattern pointing to the neolithic revolution as a strong driver for the emergence of human pathogens. where are the resistance-conferring genes in the human genome? infectious diseases have killed well over half of all humans who have ever lived on earth. pathogens, such as childhood diseases, that affect their host prior to reproduction, through death or reduced fertility, have exerted enormous selective pressures. yet, scans of the genome for signatures of pathogen-driven selection have identified only a few variants with clear effects. similarly, genome-wide association studies (gwas) for infectious disease resistance/susceptibility have identified only few loci impacting on infectious disease susceptibility [17] , despite their success in identifying thousands of variants involved in chronic diseases and phenotypic traits such as height. even for diseases that have affected us for a long time, for example tuberculosis, we know of no obvious protective genetic variant. given the high selective pressure pathogens must have exerted, it is reasonable to ask where all the resistance genes are. strongly protective variants may have reached fixation, rendering them undetectable unless the pathogen has a highly heterogeneous distribution range. an interesting case for regional selective pressure is the duffy negative antigen mutation protective against plasmodium vivax that is found at close to 100% in sub-saharan africa but virtually absent anywhere else. another situation where a resistance gene does not reach fixation arises when the protective variant is deleterious when homozygous, as in sickle cell anaemia. we might also speculate that the evolutionary potential and high genetic diversity of most pathogens limits our ability to detect protective variants in the human genome, particularly so if these were only effective against a subset of lineages within a pathogenic species. in addition to the few variants protective against specific pathogens, we also know of genomic regions involved in immunity against a wide spectrum of pathogens, such as interleukin genes or the major histocompatibility complex (mhc) system. the very high genetic diversity of the mhc is believed to have been shaped by exposure to different pathogen species [18] . also, following the recent development of techniques to sequence ancient dna, it has been suggested that immunity genes such as those encoding toll-like receptors have been acquired following hybridization with archaic humans and are over-represented in the current gene pool of anatomically modern humans relative to genes not involved in immunity [19, 20] . infectious diseases had a massive impact on our history, leading to the rise and fall of civilizations, both through the toll they took on human life but also through economic and societal collapse following epidemics. more military campaigns have probably been lost or won due to infectious diseases than due to the tactical acumen of the armies' commanders. thucydides reports in his history of the peloponnesian war, written in the 5 th century bc, how the plague of athens devastated the citystate of athens in ancient greece during the second year [27] ; influenza [28] ; hiv [29] ; tuberculosis [30] [31] [32] [33] ; p. falciparum malaria [34, 35] ; hepatitis b [36] ; measles [37] ; plague [38, 39] of the peloponnesian war (430 bc) when it was on the cusp of victory against sparta, ending the golden age of pericles and athenian predominance in the ancient world. the eventual fall of the roman empire was also largely down to another epidemic, the justinian plague in 541-542 ce, which precluded the emperor justinian recovering lost territories in the western part of the empire [21] . infectious disease played an equally important role in past human migrations. the conquistadores' sweeping conquests of large swaths of the americas in the 16 th century was greatly aided by the diseases they brought with them, such as measles and smallpox, to which the indigenous populations had limited immunity [22] . conversely, one of the possible reasons europeans managed to colonize africa was that they used quinine, an antimalarial drug derived from the bark of the cinchona tree [23] . history has been shaped not only by pathogens infecting humans, but also those affecting domestic animals and crops. for example, it has been suggested that the islamic conquest of the 7 th and 8 th centuries did not extend to sub-saharan africa because the horses and camels of the islamic armies were dying from trypanosma spread by tsetse flies [24] . conversely, pathogens were at other times the drivers of large migration. around one million irish people died and another million migrated to the us to escape the famine caused by phytophthora infestans destroying potato harvests between 1845 and 1852 [25] . at least in the developed world, the leading causes of human mortality are no longer infectious diseases but instead age-associated disorders such as cancer, heart disease and diabetes. numerous countries have undergone an epidemiological transition, starting some 300 years ago in some developed countries and less than 80 years ago for developing countries. diseases that once devastated human populations, such as smallpox, are now eradicated. others, such as the plague or leprosy, are largely under control with the exception of a few hotspots. the current situation is, however, one of new challenges. globalization and increased mobility, particularly air travel, have facilitated the transmission of diseases not just locally but between continents. the recent outbreak of zika in the americas, for example, has been attributed in part to an increase in air travel from infected areas into brazilian airports, extending both the incidence and geographic range of the virus [26] . the 2003 outbreak of severe acute respiratory syndrome (sars) and recurrent ebola crises in central africa highlight the ability of new and existing diseases rapidly to become significant international health threats. in addition, our ability to combat infectious diseases is also challenged by the widespread emergence of pathogen drug resistance. the global antimicrobial resistance (amr) crisis is increasingly limiting our resources to combat disease through antimicrobial therapy. thus, in spite of the global health narrative supporting a decline in the number of deaths caused by infectious disease, the complexity of our interactions with diseasecausing agents are as significant now as through history. infectious diseases continue to be a major cause of mortality globally, responsible for between a quarter to a third of all deaths and nearly half of all deaths in people under the age of 45, with most of these in principle avoidable. microbiology by numbers revised estimates for the number of human and bacteria cells in the body the role of damselflies (odonata: ztgoptera) as paratenic hosts in the transmission of halipegus eccentricus (digenea: hemioridae) to anurans red squirrels in the british isles are infected with leprosy bacilli airborne transmission of influenza a/h5n1 virus between ferrets a single natural nucleotide mutation alters bacterial pathogen host tropism yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis insight into the evolution and origin of leprosy bacilli from the genome sequence of mycobacterium lepromatosis microbial diversity and the genetic nature of microbial species immunobiology and pathogenesis of viral hepatitis the 1918 influenza pandemic: insights for the 21st century infectious diseases of humans myxoma virus and the leporipox viruses: an evolutionary paradigm emerging fungal threats to animal, plant and ecosystem health age of the association between helicobacter pylori and man myths and misconceptions: the origin and evolution of mycobacterium tuberculosis evolution, revolution and heresy in the genetics of infectious disease susceptibility pathogendriven selection and worldwide hla class i diversity genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes introgression of neandertal-and denisovan-like haplotypes contributes to adaptive variation in human tolllike receptors cooling and societal change during the late antique little ice age from 536 to around 660 ad insights from paleomicrobiology into the indigenous peoples of pre-colonial america -a review selected papers from the first international conference 'camel cultures: historical traditions, present threats and future prospects'. london: rn books after the famine: plant pathology, phytophthora infestans, and the late blight of potatoes zika virus in the americas: early epidemiological and genetic findings 17th century variola virus reveals the recent history of smallpox pandemic influenza -including a risk assessment of h5n1 the early spread and epidemic ignition of hiv-1 in human populations detection and molecular characterization of 9000-year-old mycobacterium tuberculosis from a neolithic settlement in the eastern mediterranean armed conflict and population displacement as drivers of the evolution and dispersal of mycobacterium tuberculosis pre-columbian mycobacterial genomes reveal seals as a source of new world human tuberculosis eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in europe plasmodium falciparum accompanied the human expansion out of africa genomes of cryptic chimpanzee plasmodium species reveal key evolutionary events leading to human malaria dating the origin and dispersal of hepatitis b virus infection in humans and primates origin of measles virus: divergence from rinderpest virus between the 11(th) and 12(th) centuries early divergent strains of yersinia pestis in eurasia 5,000 years ago historical variations in mutation rate in an epidemic pathogen, yersinia pestis the authors are grateful to liam shaw for discussions on the manuscript and for financial support from the mrc, bbsrc, nerc and esrc (grants ne/m000591/1 and mr/p007597/1). lvd and fb wrote the manuscript. both authors read and approve the final version. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-313529-xm76ae08 authors: liu, wen-kuan; chen, de-hui; liu, qian; liang, huan-xi; yang, zi-feng; qin, sheng; zhou, rong title: detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china date: 2011-12-14 journal: bmc infect dis doi: 10.1186/1471-2334-11-345 sha: doc_id: 313529 cord_uid: xm76ae08 background: human bocavirus (hbov) is a newly discovered parvovirus associated with acute respiratory tract illness (arti) and gastrointestinal illness. our study is the first to analyze the characteristics of hbov-positive samples from arti patients with a wide age distribution from guangzhou, southern china. methods: throat swabs (n=2811) were collected and analyzed from children and adults with arti over a 13-month period. the hbov complete genome from a 60 year-old female patient isolate was also determined. results: hbov dna was detected in 65/2811 (2.3%) samples, of which 61/1797 were from children (<18 years old) and 4/1014 from adults (≥18 years old). seasonal peaks of 4.8% and 7.7% were detected in may and june, respectively. 28 of 65 (43.1%) hbov-positive samples were co-detected with 11/16 other potential pathogens. mycoplasma pneumoniae had the highest frequency of 16.9% (11/65). upper and lower respiratory tract illness were common symptoms, with 19/65 (29.2%) patients diagnosed with pneumonia by chest radiography. all four adult patients had systemic influenza-like symptoms. phylogenetic analysis of the complete genome revealed a close relationship with other hbovs, and a more distant relationship with hbov2 and hbov3. conclusions: hbov was detected from children and adults with arti from guangzhou, southern china. elderly people were also susceptive to hbov. a single lineage of hbov was detected among a wide age distribution of patients with arti. respiratory tract infection etiology is complex and diverse, and new pathogens are continuously being reported. over the past few years, several novel respiratory viruses including human metapneumovirus (hmpv) [1], severe acute respiratory syndrome (sars) coronavirus [2] , human coronavirus nl63 (hcov-nl63) [3, 4] , and coronavirus hku1 (hcov-hku1) [5] [6] [7] have been identified. in 2005, allander et al. [8] reported a previously undescribed human parvovirus, human bocavirus (hbov) that belongs to the genus bocavirus, in respiratory secretions of children with respiratory tract disease in sweden. hbov is a single-stranded deoxyribonucleic acid (dna) virus with a small genome size of approximately 5.3 kilo-bases (kb), which has three open reading frames (orf) encoding two non-structural proteins ns1 and np1, and the two structural proteins vp1 and vp2. vp1 and vp2 are located within the same orf but have different initiator codon positions [8] . subsequently, hbov was reported in respiratory samples from different countries and regions worldwide [9] [10] [11] [12] [13] [14] , where hbov was detected in 1.5%-8.3% of respiratory samples from individuals with acute respiratory tract illness (arti), especially young children and infants. the virus was also found in stool samples from patients with gastrointestinal illness [15] [16] [17] [18] [19] [20] [21] [22] . these reports suggest that hbov might be associated with upper and lower respiratory disease and gastrointestinal illness throughout the world. in 2009, two viruses closely related to hbov, named hbov2 [23] and hbov3 [24] , were found in stool samples, and suggested hbov diversity. hbov infection has recently attracted increasing attention all over the world. however, the incidence and clinical presentation of this infection varies widely, and often involves co-infection with other potential pathogens [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . such characteristics have led to debate over the role of hbov as a true pathogen. therefore, additional evidence and studies are needed throughout the world to gain a better understanding of this virus. in this study, 2811 respiratory samples were collected from patients (with an age range of 9 days to 84 years) with arti in guangzhou, southern china, from november 2009 to november 2010 to analyze the characteristics of hbovpositive patients. samples in this study were taken as part of standard care. the first affiliated hospital of guangzhou medical university ethics committee approved the experimental design and patient involvement in this study. throat swab samples (n = 2811) were collected from patients with arti (presented at least two of the following symptoms: cough, pharyngeal discomfort, rhinobyon, snivel, sneeze, dyspnea) at three hospitals in guangzhou, southern china between november 2009 and november 2010. patients' ages ranged from nine days to 84 years, and included 1797 children (<18-years-old) and 1014 adults (≥18-years-old). clinical characteristics of the patients were recorded for further analysis. real-time polymerase chain reaction (pcr) for hbov detection dna from respiratory samples was extracted using a qiaamp dna mini kit (qiagen), in accordance with the manufacturer's protocol. taqman real-time pcr primers and probe were designed based on the conserved region of the np1 gene. sequences were as follows: forward primer, 5'-gag aga ggc tcg ggc tca ta-3' (2545-2564 nt); reverse primer, 5'-tcg aag cag tgc aag acg at-3' (2592-2611 nt); and probe, 5'-fam-cat cag gaa cac cca atc agc cac c-bhq1-3' (2566-2590 nt). primers and the probe were synthesized by takara. premix ex taq (perfect real time) real-time pcr reaction buffer was also purchased from takara. amplification was conducted using 10 pmol of primers, 3 pmol of probe and 5 μl dna in a final volume of 25 μl. cycling conditions included an initial incubation at 94°c for 2 min, followed by 40 cycles of 94°c for 10 sec and 55°c for 35 sec (abi-7500 real-time pcr instrument, applied biosystems). the amplified np1 gene target sequence (2545-2611 nt) was inserted into the pmd18-t vector (takara) and used as a positive control for quantification analysis. sensitivity of the pcr assay was calculated to be 10 copies of plasmid dna using positive control plasmid diluted gradients. hbov dna positive samples were tested for 16 other potential pathogens, including influenza a virus, influenza b virus, parainfluenza virus (1, 2, 3, 4), respiratory syncytial virus, adenovirus, enterovirus, human metapneumovirus, human coronavirus (229e, oc43, nl63, hku1), mycoplasma pneumoniae, and chlamydia pneumoniae by taqman real-time pcr, in accordance with the manufacturer's protocol (guangzhou huyansuo medical technology co., ltd). the complete genome of hbov from a 60-year-old female patient isolate was sequenced and analyzed. sequencing primer sets were designed according to hbov genome sequences available in the genbank database (table 1) . dna template (2 μl) was added to the pfu dna polymerase reaction mixture (fermentas) in a final volume of 25 μl and pcr amplified. products were purified after 1% agarose gel electrophoresis using a qiaquick gel extraction kit (qiagen). the purified dna was then sequenced (abi genetic analyzer 3130xl) and assembled using dnastar-seqman software (dnastar, http://www. dnastar.com/t-products-lasergene.aspx). pcr amplification and sequencing were conducted at least twice to ensure sequence accuracy. the hbov complete genome (gu338055) was aligned to other hbov genomes available in the genbank database using the basic local alignment search tool (blast, http://blast.ncbi.nlm.nih.gov/blast.cgi). phylogenetic analysis of 18 complete genomes of hbov, hbov2 and hbov3 from different countries and regions, including usa, sweden, thailand, japan, australia, china, hong kong, and taiwan was conducted using molecular evolutionary genetics analysis version 4.0 (mega 4.0, http://www. megasoftware.net/). phylogenetic trees were inferred from vp1/vp2 (3056-5071 nt), ns1 (253-2172 nt), np1 gene (2410-3069 nt) and complete genome data (1-5299 nt) using the neighbor-joining method, and bootstrap values were calculated from 1000 replicates. for comparison of categorical data, χ 2 test and fisher's exact test where appropriate. all tests were two-tailed and p < 0.05 was considered statistical significant. the 65 positive patients were aged between 54 days and 70 years, comprising 83.1% (54/65) that were ≤ 3 years-old, 10.8% (7/65) that were 4-17 years-old, and 6.2% (4/65) adult patients. four hbov-positive adults were 19, 22, 60 and 70 years-old, respectively. during our 13-month study period, more than 100 samples were collected for detection each month, and the ratio of ≤3 year-old patients ranged from 20. hbov dna positive samples were co-detected with 11 of 16 upper referred pathogens in 28/65 (43.1%) of the patients. mycoplasma pneumoniae had the highest frequency of 16.9% (11/65), followed by rsv with 7.7% (5/ 65) ( table 2 ). the male:female ratio was 42:23 in the hbov-positive patients which did not differ significantly from the hbov-negative patients (p = 0.40). the clinical characteristics of the patients are listed in table 3 . most patients presented with symptoms of upper respiratory tract illness (urti), including cough (93.8%) and expectoration (40.0%); 44 (67.7%) patients presented with (table 3) . nineteen (29.2%) were diagnosed as pneumonia by chest radiography, and 13/ 19 (68.4%) pneumonia patients were co-detected with one or two other pathogens, and a statistic difference was found for the symptom of "pneumonia" (p = 0.008) between the two groups "single hbov" and "co-pathogens". two of seven patients with systemic influenza-like symptom samples were co-detected with influenza a virus-enterovirus (triple pathogens) and influenza b virus (dual pathogens), respectively, while the remaining five patients were detected with single hbov. all four adult patients (≥18 years old) presented with systemic influenza-like symptoms; three had only hbov detection and the other 19 year-old female patient was co-detected with influenza b virus. complete hbov genome sequences for isolates from a 60 year-old patient were sequenced and submitted to the genebank (accession number:gu338055). the full length of the genome was 5299 bases and the distribution of a/g/c/t was 32.4%/20.4%/21.8%/25.4%, respectively. compared to the other hbov genomes available in the genebank database, gu330588 showed a 98% similarity with the hbov previously described by allander et al. [8] . phylogenetic trees were inferred from vp1/vp2, ns1, and np1 gene data, in addition to the complete genome sequence (figure 3) . gu330588 was similar to other hbovs, although it displayed obviously sequence variations from hbov2 and hbov3. three hbov lineages were illustrated in all four phylogenetic trees (figure 3 ). hbov is a novel parvovirus first described in 2005 by allander and colleagues [8] . since that time, it has been associated with upper and lower-respiratory tract disease and gastrointestinal illness throughout the world. however, most studies were focused on young children and infants [9] [10] [11] [12] [13] [14] , and only a few papers have described the characteristics of hbov infection in adult patients [16, 25, 26] . our study successfully analyzed the characteristics of hbov-positive samples from arti-infected patients with a wide age distribution from guangzhou, southern china for the first reported time. similar to previous study [16] , the detection rate in pediatric patients (<18 years old) was significantly higher than that in adults (≥18 years old) (p < 0.001), and most hbov-positive patients were ≤3 years old. hbov was detected in four adult patients, including 60 and 70 year-old patients. this suggested that older people were also susceptive to hbov infection, although at much lower positive rates. four adult patients also presented with systemic influenza-like symptoms, which might suggest that hbov infection in adults is a more complex and serious problem than in children. while seasonal peaks of hbov infection vary among different counties and regions because of climate and other factors, many previous studies suggest a higher detection rate in winter [27, 28] . in our study, a higher frequency of hbov was observed between may and june during the 13month testing period ( figure 2 ). this result was similar to the report of choi and colleagues, in which a seasonal peak was observed between may and june [29] . characteristics of hbov-positive patients in our study were also similar to previous reports [9] [10] [11] [12] [13] [14] . the male: female ratio in the hbov-positive patients did not differ significantly from the hbov-negative patients. in all clinical characteristics, seven major symptoms (cough, fever (≥38°c), abnormal pulmonary breath sound, dyspnea, expectoration, snivel and pneumonia) had the highest ratio (ratio > 10%) ( table 3 ) and were common as urti and lrti. as previous studies [13, [29] [30] [31] [32] [33] [34] , co-detection with other potential pathogens was common in hbov-positive patients. in this work, 43.1% (28/65) patients were codetected with other 16 potential pathogens, and there would be higher ratio if human rhinovirus were concerned [16] . furthermore, not only co-pathogens but also single hbov groups had a high ratio of major symptoms (table 3) , which might suggest hbov is an important pathogen in urti and lrti. a statistic difference was found for the symptom of "pneumonia" between the two groups "single hbov" and "co-pathogens" (table 3) , which might further suggest hbov is an important pathogen. further studies were required to determine whether hbov played a causative role in these co-infections or acted as an exacerbation factor. before 2009, little variation was found in the surface protein of hbov (vp1-vp2), and some researchers predicted that hbov infection might only occur after the subsequently development of life-long immunity via the neutralization of the target antibody [16] . however, novel types of hbov2 and hbov3 were described successively in 2009 [23, 24] , suggested diversity within this group of viruses. furthermore, we successfully sequenced the complete hbov genome (gu338055) for an isolate from an adult patient and phylogenetic analysis revealed the existence of three lineages: group i, hbov; group ii, hbov2; group iii, hbov3, and gu338055 was located in group i. the frequency and clinical presentations of hbov infection vary widely [9] [10] [11] [12] [13] [14] 16, 25, 26] and are typically associated with other pathogens [13, [29] [30] [31] [32] [33] [34] . such characteristics have subsequently led many scientists to question the role of hbov as a potential pathogen. this lack of information is largely because of the inability of hbov to grow in standard cell lines [35] . to confirm the effects of hbov, more studies are required throughout the world, focusing on various aspects of this infection, including epidemiology, serology, molecular biology, in vitro culture and animal models. hbov was detected from children and adults with arti from guangzhou, southern china, and the features were described in this study. hbov was confirmed in elderly patients (60 and 70 years old), suggesting that older people were also susceptive to hbov. all four adult patients with hbov positive in this study presented systemic influenza-like symptoms, which potentially suggest that hbov infection in adults may develop more serious 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discussion of their role in infection pre-publication history the pre-publication history for this paper can be accessed here submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the state major infectious disease research program (china central government, 2009zx10004-109) provided financial support for this work. authors' contributions rz and w-kl designed the study. w-kl, q l, h-xl performed the hbov dna testing. d-hc, z-fy and sq collected clinical data. all authors participated in the data analysis. w-k l and r z drafted the manuscript. all authors read and approved the final version of this manuscript. the authors declare that they have no competing interests. key: cord-309301-ai84el0j authors: li, yaqi; tang, peiyuan; cai, sanjun; peng, junjie; hua, guoqiang title: organoid based personalized medicine: from bench to bedside date: 2020-11-02 journal: cell regen doi: 10.1186/s13619-020-00059-z sha: doc_id: 309301 cord_uid: ai84el0j three-dimensional cultured organoids have become a powerful in vitro research tool that preserves genetic, phenotypic and behavioral trait of in vivo organs, which can be established from both pluripotent stem cells and adult stem cells. organoids derived from adult stem cells can be established directly from diseased epithelium and matched normal tissues, and organoids can also be genetically manipulated by crispr-cas9 technology. applications of organoids in basic research involve the modeling of human development and diseases, including genetic, infectious and malignant diseases. importantly, accumulating evidence suggests that biobanks of patient-derived organoids for many cancers and cystic fibrosis have great value for drug development and personalized medicine. in addition, organoids hold promise for regenerative medicine. in the present review, we discuss the applications of organoids in the basic and translational research. two-dimensional (2d) cultured cell lines have been the main in vitro research tool for the past decades. cell lines are relatively cheap, easy to handle and can be applied to multiple experimental techniques. however, the establishment of a cell line is time-consuming and involves extensive genetic and phenotypic adaption to culture conditions. thus, most cell lines are derived from tumors or have acquired oncogenic potential in vitro, while matching normal cells are usually lacking. the main problem of cell lines is the homogeneity of the cells, short of differentiated cell types in the original tissue. these problems limit the use of cell lines in personalized medicine and make them less suited to tissue physiology research requiring differentiated cell types. in cancer research, the preclinical research model that can phenocopy tumor heterogeneity is highly needed for research on the mechanisms of cancer progression and acquired drug resistance. in 1953, the first patient-derived xenograft (pdx) models were successfully established (toolan 1953 ). in this model, primary tumor tissue is transplanted into immune-deficient mice, while tumor structure and the relative proportion of tumor cells and stromal cells are largely preserved (byrne et al. 2017 ). thus, pdxs better retain the complexity and heterogeneity of the parental tumor than do cell lines, but establishment is still inefficient and early tumors are hard to establish (john et al. 2011) . besides, genetic manipulations cannot be carried out, and high-throughput analyses are expensive and hampered by complex logistics. last decade has witnessed a booming development of three-dimensional (3d) cell culture technologies. the advent of organoids avoids many of the disadvantages associated with cell lines and pdx. an organoid is characterized as a 3d structure, grown from stem and progenitor cells and consisting of variant organ-specific cell types, that self-organize via cell differentiation and spatially restricted lineage commitment (clevers 2016) . organoids can be grown from two types of cells: (i) pluripotent stem cells (pscs), such as embryonic stem cells (escs) and induced pluripotent stem cells (ipscs), or (ii) adult stem cells (ascs) (clevers 2016; rookmaaker et al. 2015) . organoids are proved amenable to all standard laboratory techniques, as well as to genetic modification drost et al. 2015; schwank et al. 2013) . organoids can be fast expanded, cryopreserved and applied to high-throughput analyses. though organoid cultures cannot mimic interactions with vasculature and stroma, organoids are a promising research model bridging the gap between cell lines and pdxs ( fig. 1 ) (drost 2018; sachs and clevers 2014) . since cell lines of escs and ipscs were established, researchers began to apply insights to induce these stem cells to generate differentiated cell types (chen et al. 2014; cherry 2012) . yoshiki sasai and colleagues firstly dug deeper by questioning whether such an in vitro model could mimic in vivo development and thus developed methods to culture brain, retina and pituitary structures 'in a dish' eiraku et al. 2008) . later, ipscs-derived organoids from optic cup, intestine, stomach, liver, lung, thyroid and kidney, were followed (chen et al. 2017; kurmann et al. 2015; mccracken et al. 2014; mccracken et al. 2011; nakano et al. 2012; takasato et al. 2015; takebe et al. 2013) . of note, each germ layer (endoderm, mesoderm, and ectoderm) is represented among this set of organs. typically, ipscs are expanded and subsequently differentiated through a multi-step protocol that moves towards a fully differentiated structure, and specific cocktails of growth factors are required for each step (fig. 2) . the differentiation process usually takes about 2-3 months, which depends on the specific type of organ (mccracken et al. 2011) . the structure of ipscs-derived organoids is complex and may contain mesenchymal, as well as epithelial and endothelial components. because differentiation protocols recapitulate development in vitro, ipscs-derived organoids are excellent models for studying development (takasato et al. 2015) , genetic diseases (freedman et al. 2015) , and infectious disease (garcez et al. 2016) . another air-liquid interface (ali) method was introduced allowing for the preservation of both epithelium and matched in vitro stromal microenvironment (neal et al. 2018 ). the ali method employs a boyden chamber-like structure where primary tissue is seeded in ecm (extracellular matrix) gel in an inner transwell tm fig. 1 comparison of cell lines, patient-derived xenografts and organoids. cell lines have low cost, are easy to handle and can be applied to multiple experimental techniques. pdxs preserve tumor heterogeneity and tumor-stromal interactions. pdos can be derived from both epithelial cancer cells and normal epithelium and cultured in an extracellular matrix (ecm) -providing basement membrane extract dish which is exposed to air to enhance oxygenation (dimarco et al. 2014; li et al. 2014; ootani et al. 2009 ). culture medium is added to the outer dish and can diffuse through the permeable transwell tm into the inner dish (fig. 3) . ali method has been applied in pscsderived organoid culture lately. koike and colleagues (koike et al. 2019) reported the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from ali culture of anterior and posterior gut spheroids differentiated from human psc. adapted ali culture of human cerebral organoids (giandomenico et al. 2019 ) and neocortical organoid (qian et al. 2020 ) derived from pscs were also developed. complementary to pscs-derived organoids that recapitulate development in vitro, ascs-derived organoids model adult tissue repair (clevers 2016) and can be established only from regenerative tissue compartments. in 1987, researches began to explore 3d-culture by culturing primary cells on a reconstituted basement membrane from engelbreth-holm-swarm (ehs) tumor (li et al. 1987; shannon et al. 1987 ). li and colleagues found mammary epithelial cells cultured on ehs matrix could form ducts, ductules, and lumina and resemble secretory alveoli. shannon and colleagues cultured adult rat type ii cells on ehs matrix with feeder layer cells (mainly fibroblasts) and revealed that cell-matrix interactions help type ii cells preserve their original cubical shape and morphological characteristics of variable differentiated cells. exploration based on ascs-derived 3d culture has been led to a new stage. two decades later, ascs-derived organoids were successfully developed from lgr5-positive intestinal stem cells in culture conditions modeling the stem cell niche of intestine sato et al. 2009 ). by providing the wnt agonist r-spondin, epidermal growth factor (egf), and the bone morphogenetic protein (bmp) inhibitor noggin, and embedding the cells in an extracellular matrix (ecm) -providing basement membrane extract (wenr method, culture strategy and applications of pluripotent stem cell (psc)-derived organoids. pscs-derived organoids can be differentiated toward each of the three germ layers (endoderm, mesoderm, and ectoderm) under specific differentiation signals. pscs-derived organoids can be applied to studying development, genetic diseases, and infectious disease spondin-1), lgr5-positive stem cells are able to selforganize, proliferate and form differentiated crypt-villuslike organoids (fig. 4a, b ). since then, by modifying cocktails of growth factors and cell isolation procedures, cultures of patient-derived organoids (pdos) have been successfully established for various human tissues by biopsy or resection, including the esophagus , stomach (bartfeld et al. 2015) , colon , liver (broutier et al. 2017; hu et al. 2018; huch et al. 2015) , pancreas (boj et al. 2015) , salivary gland (nanduri et al. 2014) , fallopian tube (kessler et al. 2015) , ovary (hill et al. 2018; kopper et al. 2019) , prostate karthaus et al. 2014) , breast , airway (sachs et al. 2019) , taste buds (ren et al. 2014) , endometrium (turco et al. 2017) , kidney (schutgens et al. 2019) , bladder (lee et al. 2018) , thyroid (saito et al. 2018) , biliary tract (saito et al. 2019) , oral mucosa (driehuis et al. 2019a ) and glioblastoma (fig. 5 , table 1) . a counterintuitive phenomenon is found that normal epithelium organoids often outgrow tumor organoids, which, in some instances, can be prevented by using cancer-specific selection methods. for example, tumor organoids from colorectal cancer (crc) can be selectively expanded upon withdrawal of wnt3a and r-spondin1. nearly all crcs harbor activating mutations in the wnt pathway or fusion of rspo(r-spondin-1) genes, allowing for the expansion of cancer cells without wnts and r-spondins, while normal epithelial cells arrest (nusse 2017; sato et al. 2011; seshagiri et al. 2012; van de wetering et al. 2015) . another approach to culture tumor cells selectively is to stabilize wild-type p53 by adding the mdm2 inhibitor nutlin-3 (drost et al. 2015) . tumor cells are not affected by nutlin-3 due to a loss of tp53 (olivier et al. 2010) , while normal cells in culture present cell cycle arrest and death, allowing for the selection of tumor cells. in general, pdos using wenr method can be derived from any epithelium of normal tissues as well as malignant or otherwise diseased tissues within approximately 7 days after embedding the cells into ecm matrix ( fig. 3c; fig. 5 ). pdos can be expanded long term and cryopreserved while remaining genetically stable, making organoids an ideal tool for disease modeling. in addition, this type of organoid culture allows the direct parallel expansion of diseased cells and matched normal cells from individual patients, which allows for the generation of living tumor organoid biobank and facilitates its potential application in personalized therapy (fig. 6) . however, to date, nearly all pdos types represent only the epithelial parts of organs, and there is an absence of stroma, nerves, and vasculature. adopting ali method, researchers can generate ascs-derived organoids from various murine tissues including small intestine, colon, stomach, and pancreas (li et al. 2014; ootani et al. 2009 ), then extending to culture clinical tumor samples (neal and kuo 2016; neal et al. 2018) , accurately recapitulating stem cell populations and their multi-lineage differentiation. the ali model preserves tumor microenvironment with tumor parenchyma and stroma, including functional tumor infiltrating lymphocytes (tils), providing a promising model for immunotherapy research for patients with cancer (neal et al. 2018) . in the remainder of this review, we will discuss how pscs-derived organoids and ascs-derived organoids are applied in basic and translational research. comparison of different culture methods for adult stem cells-derived organoids. in the wenr method, epithelial organoids are derived from tumor biopsies directly in matrigel with cocktail growth factors, with long-term expansion but no tumor micro environment. in the air-liquid interphase (ali) method, tumor biopsies are cultured in ali in the entire tumor microenvironment as a cell suspension of all cell types, including immune cells and other non-epithelial cell types, but with limited expansion tissue physiology organoids as a research tool for stem cell biology organoids is an ideal in vitro tool for the identification of novel stem cell markers, and the study of physiological phenomena requiring the coculture of multiple cell types. lgr5+ cells located at the crypt base was verified as the real intestinal stem cells (barker et al. 2007) . enlightened by the finding that lgr5 + intestinal stem cells can undergo thousands of cell divisions in vivo, sato and colleagues sato et al. 2009) successfully established the epithelial organoids from a single lgr5+ stem cell, which are also known as "miniguts". wnt signals, notch signals, egf signals and bmp signals together contribute the stem cell niche homeostasis (clevers 2013) . other stem cell biomarkers have been explored to initiate intestinal organoid cultures, including cd24 (von furstenberg et al. 2011) , ephb2 (jung et al. 2011) , and cd166+/grp78 (wang et al. 2013) . mini-guts contain multiple differentiated cell types. rapidly dividing, transit-amplifying (ta) daughter cells derived from lgr5 + cells can differentiate to enterocytes, fig. 4 morphology of several types of human adult stem-cell organoids. a a schematic diagram showing the growth pattern of organoids. typically, isolated cells or functional aggregates are embedded in extracellular matrix domes and cultured in media with essential niche factors. they gradually build tissue-like 3d structures within 1-2 weeks. b bright-field image of a typical murine small intestinal organoid culture. c brightfield image and he staining image of typical human normal colon epithelium, adenoma and adenocarcinoma organoids paneth cells, goblet cells, enteroendocrine cells, tuft cells, and the m cells that cover peyer's patches (clevers 2013) ,contributing to the study of physiology of cryptvillus axis. lendeboom and colleagues (lindeboom et al. 2018 ) applied a multi-omics framework on stem cellenriched and enterocytes-enriched mouse intestinal organoids to reveal multiple layers of gene expression regulation contributing to lineage specification and plasticity of intestine and found that hnf4g as a major driver of enterocyte differentiation. as another example, beumer and colleagues (basak et al. 2017; beumer et al. 2018 ) used organoids to study the effect of growth factors on hormone expression in enteroendocrine cells after establishing a protocol to obtain enteroendocrine cells in organoids. in organoids, hormones in enteroendocrine cells were differentially expressed based on the presence or absence of bmp4. this finding was then studied in a mouse model, and it was found that the bmp gradient along the crypt-villus axis in vivo dictates a switch in expressed hormones in enteroendocrine cells that migrate up this bmp gradient. beumer and his colleagues ) further constructed an organoid-based platform for functional studies of human enteroendocrine cells, which can be induced by transient expression of neurog3. by using single-cell mrna sequencing and mass-spectrometry, they revealed differences of human enteroendocrine cells with mice, and several secreted products were identified and validated by functional experiments. the mini-gut culture approach has been applied to the generation of organoids derived from the epithelial compartments of a variety of murine and human tissues of ecto-, meso-and endodermal origin, and promotes the study of stem cell biology of other tissues except for intestine. for example, long-term expanding organoids modeling mature pyloric epithelium can be efficiently generated from single lgr5 + stem cells located at the base of pyloric glands (barker et al. 2010) . later, strange and colleagues (stange et al. 2013 ) discovered that troy + chief cells can spontaneously dedifferentiate to act as multipotent epithelial stem cells in vivo, particularly upon damage. importantly, single troy + chief cells can initiate long-term expanding gastric organoids, containing various cell types of corpus glands. the finding further confirms troy + chief cells' role as "reserve" stem cells upon challenge of tissue homeostasis. organoid culture allows for the generation of specific cell types that were previously impossible in 2d cultures. for example, hepatocytes can be successfully established and expanded in organoid culture peng et al. 2018) . based on adult bile duct-derived bipotent progenitor organoids , culture table 1 comparison of the conditioned media requirements for the patient-derived organoids culture of respective cancer types conditions were developed that supported the growth of human hepatocyte organoids. the organoids proliferate greatly after transplanting into mice ). the resulting hepatocytes maintained its original physiological functions, including secreting cytoplasmic glycogen particles, forming bile canaliculi, and expressing albumin and cytochrome p450 enzymes. based on organoid culture system of hepatocytes, peng and colleagues (peng et al. 2018 ) described a unique effect of tumor necrosis factor-î±, a cytokine essential for liver regeneration and found that the addition of regeneration-enhancing cytokines in facilitating the in vitro expansion of cell types that are otherwise difficult to culture. as another example, yin and colleagues (yin et al. 2014 )showed modulation of wnt and notch signaling in intestinal organoids to direct lineage differentiation into mature enterocytes, goblet cells and paneth cells. specifically, the combination of iwp-2 (inhibitor of wnt production 2; wnt pathway inhibitor) and vpa (valproic acid; notch activator) specifically induced enterocyte differentiation, presumably by combining the effects of both inhibitors, in which iwp-2 induced enterocyte differentiation while vpa suppressed the differentiation of lgr5+ stem cells toward secretory cell types. the combination of dapt (notch inhibitor) and chir (chicken immunoglobulin-like receptor; gsk3î² inhibitor) mainly induced paneth cell differentiation, and the combination of iwp-2 and dapt primarily induced goblet cell differentiation. these methods provide new tools for the study and application of multiple intestinal epithelial cell types. organoids can be established from a single cell, which makes it possible to study the mutational status of single stem cells. the gradual accumulation of genetic mutations in stem cells throughout life is related to a variety of age-related diseases, including cancer. in this way, blokzijl and colleagues (blokzijl et al. 2016 ) were able to unveil mutation rates and patterns in normal stem cells throughout life by whole-genome sequencing (wgs) analysis (with peripheral blood as a reference for germline mutations). interestingly, the mutation rate, with around 40 novel mutations per year per stem cell, was applications of adult stem cells-derived organoids. a organoids derived from normal tissue are useful for studying physiology. for disease modeling, organoids can be genetically engineered to model genetic and malignant diseases by using crispr-cas9. normal organoids can also be infected with different types of pathogens to model infectious disease. normal organoids can be transplanted to wounds for tissue repair. b tumor-derived organoids can be used for basic research by genetic modification and modeling rare cancer. for translational research, tumorderived organoids can be used for biobanking, genetic repair and drug screening studies, both for personalized medicine (to choose the most effective treatment for a specific patient) and drug development (to test a compound library on a specific set of tumor organoids), as well as immunotherapy research similar in liver, small intestine, and colon stem cells, regardless of the large variation in cancer incidence of these organs. however, the types of mutations detected and the resulting mutational signatures in colon and small intestine cells were different from those in liver cells. to be pointed out, the inter-individual variation in mutation rate and spectra are low, indicating organspecific activity of common mutational processes throughout life. disease modeling crispr-cas9 technology as a useful tool for disease modeling of organoids the clustered regularly interspaced short palindromic repeats (crispr) associated protein 9 (cas9)/crispr system has become a major technology for mammalian genome editing. the system consists of cas9 nuclease derived from streptococcus pyogenes and guide rna which can recognize and target a specified dna sequence preceding the motif sequence adjacent to the. crispr-cas9 can generate dna double-strand breaks at specific genomic sites. mammalian double-strand dna breaks can be repaired by two ways, nonhomologous end joining (nhej) and homology-directed repair (hdr). nhej inserts indels randomly in the process of repair, and biallelic introduction of indels leads to gene knock-out (komor and badran 2017) . on the other hand, hdr can replace the damaged allele by existing intact genome, thus when tailored dna templates are co-delivered with crispr-cas9, hdr can be used for gene knock-in (komor and badran 2017) . although the crispr-cas9 technology has broadened its applications to a series of purposes, including dna base editing, rna targeting, epigenome editing and gene expression manipulation (adli 2018; komor and badran 2017) , the use of crispr-cas9 on organoids still basically harness nhej and hdr to engineer genes of interest. indeed, organoids are ideal model for investigating gene function by genome editing, as the organoid system allows for fast expansion with stable genetics and phenotype. previous studies have successfully achieved genome editing by installing crispr-cas9 into organoids using various approaches, including liposomal transfection, electroporation and viral infection (fig. 7) . however, variable experimental conditions limit the efficiency of genome editing in organoids, including the recovery after single-cell isolation, approaches for crispr-cas9 delivery, and the cleavage efficiency of the guide rna. selection and enrichment of positive organoids are necessary after crispr-cas9-mediated genome editing, or otherwise, labor-intensive organoid cloning, followed by sequencing of expanded organoid clones is needed. recently, ringel and his colleagues (ringel et al. 2020 ) developed a genome-wide pooled-library crispr screen approach by capturing sgrna (single-guide rna) integrations in single human intestinal organoids to dissect oncogenic signaling pathways. their screening method would be broadly applicable to various organoid models and selection assays, which may contribute to dissecting human disease mechanisms and facilitating biological discovery in primary 3d tissue models. currently, organoids have become a useful tool to model genetic diseases. generally, two types of methods have been adopted: (i) organoids established from patientderived biopsies; (ii) specific genetic mutations introduced to wild-type organoids using crispr-cas9 technology. cystic fibrosis (cf) is the best example for pdo modeling human genetic disease. cf is a monogenic channelopathy caused by inactivating mutations in the cf transmembrane conductance regulator (cftr) gene. the disease involves multiple organs, including the intestine, lung, pancreas, liver, kidney and sweat gland. in gastrointestinal organs and lungs, decreased cftr function results in reduced chloride transport through cftr toward the extracellular space, leading to a reduced water flow by osmosis and, hence, increased density of mucus. in the sweat glands, loss of cftr function leads to a high saline concentration in sweat. organoid model was firstly derived from rectum of cf patients. early work with organoids derived from the rectum of cf patients revealed cftr function: wild-type organoids rapidly swell upon opening the cftr channel in a cyclic adenosine monophosphate (camp)dependent manner through the addition of forskolin (fsk) ). rectal organoids from cf patients do not respond to fsk, but it is restored upon pre-incubation with cftr-restoring compounds or upon correction of the cftr mutation by crispr-cas9 (schwank et al. 2013 ). based on this finding, this organoid-based fsk-induced swelling assay began to be used to test drug response on organoids isolated from patients harboring different cftr mutations, including rare variants . lung is another organ that can be severely harmed by cf. due to cftr mutation, a thick sticky mucus forms in the lungs, which impairs breathing and provides a fertile environment for pathogens reproduction, leading to premature respiratory failure. cf airway organoids can be established from patient-derived ipscs (firth et al. 2015) , bronchial epithelial cells based on both ali (fulcher et al. 2005) and wenr cultures (sachs et al. 2019) or bronchoalveolar lavage fluids (no biopsy needed) (sachs et al. 2019; sondo et al. 2014 ). airway organoids from cf patients had an increased mucus layer, recapitulating the disease phenotype. fsk-induced swelling in airway organoids was reduced compared with organoids from normal controls and could be restored with cftrrestoring compounds. however, in contrast to rectal organoids, fsk-induced swelling in lung organoids did not depend on cftr alone, it was also influenced by the chloride transporter tmem16a, which is set as an alternative therapeutic target for cf patients. therefore, airway organoids may function as an additional platform for assessing drug response to cf, particularly for drugs acting on tmem16a. besides, liver, pancreatic and kidney organoids derived from cf patients can also be established. sampaziotis and colleagues (sampaziotis et al. 2015 ) generated ipscs from skin fibroblasts of a cf patient with homozygous f508 mutation and differentiated them into cholangiocyte-like cells (clc). cf-clcs expressed displayed defective expression of cftr protein. cf-clc organoids treated with experimental cf drug vx809 increases cftr function and improves intraluminal fluid secretion. cf pancreatic organoids can be established from pscs of cf patients, including both human escs and an ipsc line, to generate differentiated pancreatic ductal epithelial cells (pdecs) (simsek et al. 2016 ). pdecs derived from cf-ipscs showed decreased expression of cftr protein and damaged chloride ion channel activity, reappearing functional defects of patients with cf at the cellular level. in addition, a tubuloid line from urine of a cf patient was established (schutgens et al. 2019) . at the morphological level, the kidney tubuloids maintained folded over long-term culture, instead of the typical cystic phenotype, which was probably due to the lack of cftr function caused by cftr mutations f508del/s1251n. in tubuloids derived from urine of cf patients, fsk caused slight swelling in a concentration-dependent manner, suggesting residual cftr function, while after pre-incubation with the fig. 7 genome editing in organoids by crispr-cas9 technology. workflow of genetic engineering in organoids using crispr-cas9. either nhej or hr can be exploited for gene knock-out or knock-in, respectively. in most cases, expansion of single organoid clones after the selection procedure is necessary to obtain isogenic organoid populations cftr-potentiator drug vx-770 (ivacaftor, kalydeco), swelling increased significantly. all the above cf organoid models of different related organs allow in vitro assessment of treatment response and development of novel drugs. for a discussion of the use of cf-pdo for personalized medicine, see section 3.1. additionally, intestinal organoids harboring inactivation mutation of ttc7a have been successfully derived from patients with intestinal atresia, which recapitulates how ttca7 deficiency results in the loss of apical-basal cell polarity in the intestinal epithelium that can be rescued by adding rho kinase inhibitors (bigorgne et al. 2014) . besides, intestinal organoids were derived from patients with microvillus inclusion disease (mvix) caused by homozygous truncating mutations of syntaxin-3 (stx3) gene. the model revealed that partial loss of brush border microvilli and subapical accumulation of vesicles are typical histological phenomena of the disease (wiegerinck et al. 2014). liver organoids have been generated from patients with î±1-antitrypsin (a1at) deficiency. accumulation of mutant a1at in the endoplasmic reticulum in the liver leads to fibrosis or cirrhosis. liver organoids derived from the patients indeed contained a1at aggregates and presented increased apoptosis, which might contribute to fibrosis and cirrhosis . alagille syndrome is caused by loss-of-function mutations in jag1 or notch2 and leads to partial or complete biliary atresia. accordingly, organoids generated from a patient with alagille syndrome can not differentiate toward the biliary fate, whereas in proliferate conditions, no differences were observed compared with healthy controls (andersson et al. 2018; sachs et al. 2018) . ipscs-derived organoids can also be manipulated by crispr-cas9 technology to model diseases in different tissues. in human ipscs-derived kidney organoids, knock-out of podocalyxin and pkd genes freedman et al. 2015) recapitulated defects that mimic nephrotic syndrome and polycystic kidney disease respectively, as well as contributed to understanding the functions of the genes in the pathogenesis context. engineered ipscs-derived liver organoids helped in illustrating the various functions that different mutations of jag1 gene can have in the development of bile ducts and genesis the alagille syndrome: the c829x mutation of jag1 can causes significant alterations, while the g274d mutation does not affect organoid properties (guan et al. 2017) . in brain tissue, patient-specific ipscs-derived brain organoids can be used to model lissencephaly (bershteyn et al. 2017) , down syndrome , and neuronal heterotopia (klaus et al. 2019) . engineered ipscs-derived brain organoids were established to model microcephaly by rna interference of reprogramming factors (lancaster et al. 2013) , autism by overexpression of the transcription factor foxg1 (forkhead box g1) (mariani et al. 2015) , macrocephaly by deletion of pten (li et al. 2017) , timothy syndrome by introducing mutations in the cav1.2 calcium channel-interneurons (birey et al. 2017 ) and aicardi-goutiã¨res syndrome by introducing inactivation mutation of trex-1 ( thomas et al. 2017) . fused organoids culture was recently established to understand more complex biology, which was more applied in brain study. bagley and colleagues (bagley et al. 2017 ) firstly showed a co-culture method combining brain regions of choice within one organoid tissue and they generate a dorsal-ventral axis by fusing organoids of dorsal and ventral forebrain identities. combined with reprogramming technology, their novel fusions of organoids culture should offer researchers the possibility to analyze complex neurodevelopmental defects using cells from neurological disease patients and to test potential therapeutic compounds. xiang and his colleagues (xiang et al. 2017 ) successfully established and fused medial ganglionic eminence (mge) and cortex-specific organoids from human pluripotent stem cells followed by live imaging, to investigate mge development and human interneuron migration and integration, which offers deeper insight into molecular dynamics during human brain development . the same research team developed a new 3d system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids, providing a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain (xiang et al. 2019) .generally, engineered organoids can faithfully recapitulate genetic diseases and thus provide a valid resource for basic research and for development of novel therapeutics. organoids are closed 3d structures that exhibit the apical side of the epithelium towards the lumen and the basal membrane towards the outside. the apical membrane within the lumen is initially exposed to pathogens in vivo. three different methods have been established to reproduce the interaction between microbes and the host in the organoids culture (fig. 8 ). in the first method, organoids are mechanically sheared or digested into single-cell suspensions or large particles and then they are incubated with pathogens, which leads to infection of the cells. after embedded in the 3d matrix, infected cells can reform organoids that can be used to model infectious disease (dang et al. 2016; forbester et al. 2015; nigro et al. 2014; zhang et al. 2014 ). this method is easy to handle and do not require special equipment. but, the efficiency of infection varies among different pathogens and it can not reflect the initial interaction between microbes and the host. besides, during the process not only the apical side but also the basal side of cells are exposed, thus nonspecific responses may be introduced due to the interaction of pathogens with the basal side of the cells. in the second method, pathogens are directly injected in to the lumen of the organoids by a microinjection, thus the initial interaction of pathogens and the early response of the host cells can be captured and either apical or basal interaction can be investigated separately (bartfeld et al. 2015; leslie et al. 2015; mccracken et al. 2014 ). this method is now the mainstream method to build infection model. however, this method needs special such as a microinjector and it is hard to perform quantitatively due to the different sizes of organoids. in the third method, when single cells digested from organoids are seeded onto a 3d matrix-coated dish, they grow as 2d monolayer with the apical side exposed. adding pathogens directly into the culture media allows interaction between microbes and the host cells (ettayebi et al. 2016) . the 2d culture contains various differentiated cells and allows quantitative experiments, while it does not resemble the in vivo 3d structure of host tissues. based on the above approaches, organoids have been adopted to model viral, bacterial, and parasitic infectious diseases of different tissues, including diseases caused by pathogens that previously could not be studied in vitro (table 2) . these models recapitulate features of in vivo infection and could help identify therapeutic targets and develop novel drugs and vaccine. pscs-derived organoids were firstly used to model viral disease. in neuroscience, the development of pscsderived brain organoids help deciphered the sequence of disease progression in zika virus (garcez et al. 2016; qian et al. 2016) . zika virus (zikv) mainly spreads by the aedes aegypti mosquito and its infection can lead to microcephaly, which was set as a public health emergency by fig. 8 approaches of studying infectious diseases using organoids. organoids can be micro-injected with the microbe, thus the microbe is in direct contact with the apical side of epithelial cells, which became the mainstream method to build infection model. organoids can also be sheared into smaller aggregates, incubated with pathogens and re-seed into matrigel. alternatively, 3d organoids can be digested by enzyme into single cells and grown as 2d monolayer cultures, and then microbes are added into the culture media world health organization (who) in 2016. however, the pathogenesis of the zikv infection was not fully understood until brain organoids emerged. multiple research demonstrated that zikv infection can cause disorder of the cortical layers of cerebral organoid, abrogating growth and thus halting neurogenesis. researchers found that zikv infection leads to the activation of the toll-like receptor 3 (tlr3), contributing to deregulated neurogenesis and decreased functional neurons (cugola et al. 2016 ). gabriel and colleagues (garcez et al. 2016 ) further illustrated that two new isolated zikvs have different patterns of pathogenicity. unlike the highly passed mr766 strain of zikv, the new strains, infected apical proliferating progenitors, interfering with centrosomal protein assembly, which in turn led to their premature differentiation and apoptosis, resulting in microcephaly (wells et al. 2016) . the organoids model of zikv infection also promotes the development of treatment. in a high-throughput drug screen of 6000 compounds, caspase-3 activity inhibitors, emricasan and niclosamide, were found to be effective in limiting zikv induced death of neural cortical progenitor and zikv replication (xu et al. 2016) . except for zikv, japanese encephalitis virus (jev) infection, leading to japanese encephalitis (je), was modeled in generated telencephalon organoid . researchers found that jev infection caused decline of cell proliferation and increase of cell death, and infected astrocytes and neural progenitors. in addition, they revealed variable antiviral immunity in brain organoids of different stages of culture, which also provide clues to develop effective therapeutics of such diseases. another example of pscs-derived organoids application in human disease is with hepatitis b virus (hbv). in recent decades, treatments against hbv infection have improved; however, the development of personalized treatments has been hindered by the absence of personalized infection models. nie and colleagues (nie et al. 2018 ) generated pscs-derived liver organoids that recapitulated the genetic background of the donor, and found hbv infection in pscs-derived liver organoids could reproduce the life cycle of hbv and hbv-induced hepatic dysfunction, indicating that pscs-derived liver organoids may provide a promising personalized infection model for the development of personalized treatment for hepatitis. in recent years, ascs-derived organoids have become the main force in infectious diseases modeling. ascsderived organoids can be adopted to model the viral infection of intestinal organoids. gastric diarrhea in humans is mostly caused by human norovirus (hunov) and rotavirus infection (zheng et al. 2006) . although both of these viruses are rampant, no proper vaccine has been developed owing to the lack of in vitro culture method supporting their replication. intestinal organoids that were cultured as monolayers allowed for extensive replication of multiple strains of noroviruses. for some strains, the addition of bile to the culture medium was required for replication (ramani and atmar 2014) , indicating that not only are in vivo-like host cells required for productive infection but also an in vivo-like environment is relevant as well. ettayebi and colleagues (ettayebi et al. 2016) reproduced hunov infection in an organoid-virus co-culture system, with only a specific gii.3 hunov strain requiring the presence of bile. furthermore, lack of histo-blood group antigen (hbga) expression in intestinal organoids limits hunov replication, suggesting that this culture system allows the evaluation of potential treatments and preventions. similarly, researchers have shown that rotavirus strain (simian sa11) from clinical samples can proliferate in pscs-derived intestinal organoids (finkbeiner et al. 2012; yin et al. 2015 ). in urinary system, bk virus, which is a tubule-specific circular dna virus, infects 1-10% of transplanted kidneys, leading in 10-80% of these infected kidneys to the loss of the donor organ and no curative treatment exists (hirsch et al. 2005) . infection of kidney tubuloids (kidney-derived organoids in which only the tubular epithelium of the kidney is represented and glomeruli are lacking) with bk virus yielded a patchy infection with enlarged nuclei (due to intranuclear basophilic viral inclusions), similar to what is observed in kidney biopsies from patients with bk virus nephropathy (bohl and brennan 2007; schutgens et al. 2019) . respiratory infections pose a major global disease burdens (ferkol 2014) . respiratory syncytial virus (rsv) alone causes hundreds of thousands deaths annually among children, mostly in developing countries (nair et al. 2010) . ipscs-derived organoids of human airway epithelium can be infected by rsv virus, which can reproduce the morphological features of rsv infection in the distal lung (chen et al. 2017 ). persson and colleagues (persson et al. 2014 ) established a ali culture system for infection of human airway epithelium with rsv virus and they found that rsv has the potential to influence the cellular composition of the airway epithelium. besides, ascs-derived airway organoids using wenr methods can also be infected with rsv, recapitulating syncytia formation, cytoskeletal changes, and shedding of epithelial cells (mueller et al. 2005) . rsv-infected organoids attracted neutrophils more than did mockinfected control organoids, making this the first organoid model suitable for studying neutrophil-epithelium interactions (sachs et al. 2019) . intriguingly, rsv infection strongly increased organoid motility and ultimately resulted in organoid fusion. influenza viruses also pose a major public health problem worldwide, and novel emerging viruses may be lethal, as evidenced by the poultry-derived h7n9 virus infection that has had a 39% mortality rate since 2013. the infection of differentiated airway organoids with distinct strains of influenza virus can discriminate between poorly infective and highly infective strains (zhou et al. 2018) . importantly, hui and colleagues (hui et al. 2018 ) compared human and avian strains of influenza a virus in in vitro human bronchus and airway organoids, and found that the infection of airway organoids yielded similar results regarding virus replication and cytokine response. in addition, infection of airway organoids with enterovirus 71 (ev71) showed that ev71 replication kinetics are strain-dependent and the model help identify new infectivity makers for ev71 (van der sanden et al. 2018) . the year of 2020 witnessed the outbreak of coronavirus disease-19 (covid-19) caused by the virus severe acute respiratory syndrome-coronavirus 2 (sars-cov-2) which presents influenza-like symptoms ranging from mild disease to severe lung injury and multi-organ failure, eventually leading to death, especially in older patients with other co-morbidities. the who has declared that covid-19 is a public health emergency of pandemic proportions. organoids have been used as a great platform to research how covid-19 affects human and causes damage and for identifying possible drug targets for covid-19. lamers and colleagues (lamers and beumer 2020) infected enterocytes in human small intestinal organoids with sars-cov and sars-cov-2 and found that the intestinal epithelium supports sars-cov-2 replication, and organoids can be served as an experimental model for coronavirus infection and biology. zhou and colleagues (zhou et al. 2020) established the first expandable organoid culture system of bat intestinal epithelium which were fully susceptible to sars-cov-2 infection and sustained robust viral replication. they also found active replication of sars-cov-2 in human intestinal organoids indicating that the human intestinal tract might be a transmission route of sars-cov-2. in addition, monteil and colleagues (monteil et al. 2020) found that sars-cov-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by human recombinant soluble ace2 (hrsace2), demonstrating that hrsace2 can be a possible drug for early stages of covid-19. salmonella typhi (s. typhi) and clostridium difficile (c. difficile) are the two major bacterial intestinal pathogens that can cause diarrhea and gastrointestinal failures in humans. these pathogens infection has been successfully modeled using pscs-derived intestinal organoids. forbester and colleagues (forbester et al. 2015 ) microinjected live s. typhi into the lumen of the ipscs-derived intestinal organoids and revealed that upon injection, nf-îºb signaling was activated and inflammatory factors were secreted, which was consistent with previous findings in animal models. likewise, the spence lab (leslie et al. 2015 ) microinjected c. difficile toxin a (tcda) and toxin b (tcdb) into the lumen of ipscs-derived intestinal organoids to model anaerobe c. difficile infection (cdi). the injection of tcda recapitulates the impair of the epithelial barrier function and structure observed in organoids colonized with viable c. difficile. in another study, the worrell lab (engevik et al. 2015 ) observed a decreased expression of nhe3 (sodium/hydrogen exchanger 3) and muc2 (mucoprotein2) protein in c. difficile infected organoids compared to normal organoids, which may help creating a favorable environment for its colonization. bacterium helicobacter pylori (h. pylori) infection is a major risk factor for peptic ulcers, gastric adenocarcinoma and gastritis (salama and hartung 2013) . both gastric organoids derived from ipscs and ascs can be used to model h. pylori infection, by microinjecting h. pylori strain into the lumen of organoids (mccracken et al. 2014) , which can ensure the apical side exposed to h. pylori. luminal injection of h. pylori induces a potent nf-îºb-mediated inflammatory response (bartfeld et al. 2015) , connecting excessive microbial colonization of h. pylori with the occurrence of gastric cancer. in a followup study, researchers adopted gastric organoids to find out how h. pylori finds its gastric niche: a potent chemoattractant, urea, which produced by gastric epithelium is essential for the colonization of h. pylori in the gastric mucosa (huang et al. 2015a) . chronic salmonella infection of the gall bladder is associated with gallbladder carcinoma (shukla et al. 2000) . scanu and colleagues (scanu et al. 2015) showed that after salmonella infection, mouse gallbladder organoids exhibited characteristics of loss of polarity, familiar with those showed in the mouse model of gallbladder cancer. another study found that gallbladder organoids preexposed to salmonella that lack functional tp53 showed neoplastic transformations by activating akt (protein kinase b) and mapk (mitogen-activated protein kinase) pathway and could grow in culture media free of growth factors (scanu et al. 2015) . the protozoan parasite cryptosporidium causes lifethreatening diarrhea in immunocompromised individuals (e.g. people living with hiv and malnourished children), and infection may spread to the lungs (checkley et al. 2015) . drug development requires detailed pathophysiology information of cryptosporidium, but the lack of an optimal in vitro culture system hinders the experimental approaches. heo and colleague (heo et al. 2018) infected epithelial organoids derived from human small intestine and lung with cryptosporidium and found that the parasite can reproduce within the organoids and complete its complex asexual and sexual life cycles for multiple rounds. plasmodium parasites can cause malaria, which poses a significant global health burden, with over 200 million cases every year. plasmodium parasites are maintained between anopheles mosquitoes and mammalian hosts in a complex life cycle, and models to study them are challenging to establish, particularly for plasmodium species that infect humans (mellin and boddey 2020) . recently, several studies reported the application of ipscs-derived hepatocyte-like cells to model in vitro liver stage infections with p. berghei, p. yoelii, p. falciparum, and p. vivax (ng et al. 2015) . it was found that p. yoelii and p. falciparum infections of organoids recapitulated the primaquine sensitivity found in vivo. chua and colleagues (chua et al. 2019 ) infected organoids derived from simian and human hepatocytes, with p. cynomolgi and p. vivax and found that organoids could support the complete liver stage of both simian and human parasites, from initial infection with sporozoites, to the release of merozoites capable of erythrocyte infection. this study also illustrated the use of infected organoids to evaluate the response to an anti-relapse drug, highlighting the potential for organoids as a parasite drug screening platform, particularly in parasites with life-cycles longer than their host cells. though organoids derived from tumor and matched normal epithelial tissues provide valuable research tools for cancer biology, one of the most remarkable improvements in organoid research is the capacity to manipulate the genomes, transcriptomes and epigenomes of normal epithelial organoids to study the role of specific alterations in the process of tumorigenesis. murine organoid cultures were firstly used to study the early stages of tumorigenesis. li and colleagues (li et al. 2014 ) adopted the ali culture approach combined with genetically engineered mouse model and the retrovirus-mediated delivery of shrna constructs, to model multi-step tumorigenesis in organoids derived from digestive tract, including the colon, stomach, and pancreas. pancreatic and gastric organoids exhibited dysplasia as a result of expression of kras g12d , p53 loss or both. while colon organoids needed assembled apc, p53, kras g12d and smad4 mutations for malignant transformation to invasive adenocarcinoma-like morphology. all engineered organoids presented histologic characteristics of adenocarcinoma after subcutaneous implantation was performed to immunocompromised mice. following research tried to model multi-step tumorigenesis of conventional crc, which is characterized by chromosomal instability (cin) (fig. 9 ). drost and colleagues (drost et al. 2015) adopted crispr-mediated knock-out of the tumor suppressors apc, tp53, and smad4, married with crispr-mediated knock-in of the oncogenes kras g12d to model multi-step tumorigenesis. after being selected by niche factors in the culture media, cultures of organs were successfully built with complex oncogenic multi-gene modules that contain up to four simultaneous changes. the 4-hit akst (apc, kras g12d , smad4, and tp53) organoids could grow without stem cell niche factors such as wnt-3, rspondin-1 and egf. akst organoids were able to generate tumors with characteristics of invasive carcinoma upon subcutaneous implantation into immunocompromised mice. matano and colleagues (matano et al. 2015 ) applied a similar method to model tumorigenesis by inserting an additional crispr-mediated knock-in of the oncogene pik3ca e545 , in addition to akst. both studies showed that organoids with apc and tp53 mutations showed extensive aneuploidy, which is the hallmark of the cin pathway. xenotransplantation of engineered colorectal tumor organoids makes it possible to study cancer stem cells in vivo (de sousa e melo et al. 2017; shimokawa et al. 2017 ) and leads to metastatic diseases, making organoids a useful research tool to study metastasis mechanisms (fumagalli et al. 2017; roper et al. 2017) . de sousa e melo and colleagues (de sousa e melo et al. 2017) combined crc mouse with the lgr5 dtr/egfp allele. the resulting animals carry two of the most frequently mutated genes, apc and kras g12d , and in addition, express a diphtheria toxin receptor fused to an egfp under the endogenous regulatory region of lgr5, allowing specific elimination and visualization of lgr5-positive stem cells. using this model, it was found that in the absence of cancer stem cells, liver metastases did not occur, whereas primary tumors did not regress, indicating that lgr5-positive cancer stem cells are required for metastasis. in another study, fumagalli and colleagues (fumagalli et al. 2017 ) orthotopically transplanted cris pr-mediated kras, apc, tp53, and smad4 comutated human colon organoids into mice and showed that metastases to the liver and lungs occurred in 44% of the mice. almost no metastasis occurred when organoids carrying mutations in only three of these four genes were transplanted to mice; however, the lack of the fourth mutation could be overcome by providing the niche factor upstream of the absent mutation. for example, organoids with triple mutants lacking smad4 inactivation metastasized when noggin was added to the cells. these findings indicate that metastatic potential is directly related to the loss of niche factor dependency. crc that arises from the serrated neoplasia pathway is different from crc that arises from the conventional cin pathway. the activation of oncogene braf initiated the serrated pathway, followed by the extensive hypermethylation of cpg island methylator phenotype, and subsequent inactivation of tumor suppressor genes (bae and kim 2016). fessler and colleagues (fessler et al. 2016 ) firstly built the organoids to model the serrated pathway of crc by introducing the braf v600e mutation into normal human colon epithelial organoids via homologous recombination. they revealed that induction of a mesenchymal phenotype upon tgfî² treatment prevails in the braf v600e mutated organoids generating sessile serrated adenomas (ssas). in a recent study, by fig. 9 assessing different tumorigenicity and metastases mechanisms of combinative mutations in colon cancer based on normal colon organoids by using crispr-cas9 technology analyzing the genomic data from tcga, crcassociated gene alterations including braf v600e , cdkn2a, tgfbr2, znrf2 and rnf43, were selected to be introduced into murine colon organoids using crispr-cas9 technology to model the serrated pathway (lannagan et al. 2019) . upon subcutaneous implantation into immunocompromised mice, these engineered organoids could generate tumors with characteristics of serrated crc, including desmoplastic stromal responses, infiltrative growth, mucinous differentiation, tumor budding, and the formation of colon tumors spontaneously metastasizing to the liver. to be pointed out, transplantation of braf-mutated organoids failed to generate tumors, while injection of organoids with both mutations of braf and tgfbr2 led to invasive adenocarcinoma. implantation of organoids with those and additional mutations (cdkn2a, znrf2 and rnf43) resulted in increased tumor initiation and decreased survival time. moreover, to study the development of the r-spondin-driven serrated pathway, another study also introduced crispr-mediated gene fusions on braf v600e and/or tp53 accompanied with the r-spondin fusion eif3e (eukaryotic translation initiation factor 3 subunit e )-rspo2 to normal human colon epithelial organoids (kawasaki et al. 2020 ). the resulting braf v600e organoids showed aberrant crypt formation ability with poor engraftment capacity, while organoids with mutations of braf v600e and tp53 with eif3e-rspo2 fusion generated flatly elevated lesions and hyperplastic crypt structures with 'v'-shaped serration and basal dilation, together with enhanced engraftment capacity compared with only braf v600e mutated organoids. the loss of dna mismatch repair enzymes, such as mutl homolog 1 (mlh1), is common in crcs, resulting in tumors with high mutational load. these tumors are characterized by microsatellite instability (msi), as repetitive short sequences in the genome, which leads to changes in copy number following the loss of mismatch repair enzymes. drost and colleagues used crispr-mediated knock-out of mlh1 and cultured organoids for 2 months to allow accumulation of mutations. subsequent dna analyses of cultured organoids revealed an increase in mutational load compared with controls, which was similar to that of msi colorectal tumors. this study shows that organoids faithfully recapitulate in vivo mutagenesis and allow for the identification of mechanisms of tumor development. in addition, rare type of colorectal cancer can also be modeled based on organoid culture. li and colleagues ) have established a novel organoid line of colon signet-ring cell carcinoma (srcc), which accounts for only 1% of colorectal cancers. the novel organoid line resembles the primary tumor histologically and molecularly, and can efficiently generate tumors on xenografts. targeted dna sequencing with drug screening of 88 compound identified that jak2 (janus kinase 2) might be a potential treatment target. an in vitro drug screening experiment exhibited that srcc organoids can be sensitively treated by at9283 and pacritinib, two jak2 inhibitors, which was consistent with the in vivo xenograft response. the study provides a novel in vitro research tool for colorectal srcc and sets an example for personalized medicine based on organoids for rare cancer. pancreatic ductal adenocarcinoma (pdac) accounts for about 90% of all pancreatic malignancies and over 90% pdac patients harbor activation of the oncogene kras. kras can consequently induce inactivation of various tumor suppressor genes including tp53, cdkn2a, smad4 and brca2 to accelerate pdac development and progression (kanda et al. 2012; morris and wang 2010) . establishment of murine pancreatic organoids bearing a lox-stop-lox (lsl) krasg12d allele provided valuable insights for the development of pdac. in one study (li et al. 2014) , pancreatic organoids derived from lsl-kras g12d (k), tp53 flox/flox (p), and lsl-kras g12d ; tp53 flox/flox (kp) mice were successfully built using ali method. k, p, and kp organoids exhibited in vitro dysplasia and increased invasive behavior, and could in vivo generate well differentiated to poorly differentiated adenocarcinoma upon implantation into immunocompromised nog mice. kp organoids had most poorly differentiated morphology, with significant loss of ecadherin, indicating increased epithelial to mesenchymal transition. researchers of another study generated lowgrade preinvasive pancreatic intraepithelial neoplasias (panins) in vivo by crossing the murine lsl-kras g12d allele to a pancreas-specific pdx1-cre driver and organoids derived from panin lesions could be long-term expanded a produce panin-like lesions that persisted for up to 2 months upon transplantation (boj et al. 2015) . two studies have adopted crispr-cas9 technology to manipulate pdac driver genes including kras g12v , cdkn2a, tp53 and smad4 (kcts) in normal human pancreatic ductal organoids seino et al. 2018) . upon orthotopically transplantation to immunocompromising mice, different combinations of mutants in organoids exhibited distinct panins features, but only kcts organoids displayed pdac histopathological transformation . notably, kc and kt organoids died after wnt removal for 1-3 weeks, while kct and kcts organoids survived and expanded for at least 3 months, suggesting that cdkn2a and tp53 mutations are essential for organoids to grow independently of stem cell niche factors . in all, these studies indicate that organoids combined with crispr-mediated sequential mutations can recapitulate tumorigenesis and progression from panin to pdac. moreover, organoids could also be used to explore the roles of various factors such as the redox regulator nrf2 and the transcriptional enhancer foxa1 in pdac progression (chio et al. 2016; roe et al. 2017) . further researches are need to apply the improved knowledge of molecular mechanisms to clinics. gastric cancer are classified into four molecular subtypes based on deep sequencing: epstein-barr virus positive, msi, cin and genomically stable (cancer genome atlas research 2014). nanki and colleagues adopted organoids to illustrate the genotype-phenotype associations of gastric cancer. phenotype analyses of organoids derived from gastric cancer patients indicated multiple genetic and epigenetic ways to confer wnt and r-spondin niche independency. they found that induction of rnf43 and znrf43 mutations was sufficient for gastric cancer organoids to gain r-spondin independency. the phenomenon was then validated in crispr-cas9 engineered gastric organoids. in a similar study, seidlitz and colleagues (seidlitz et al. 2019 ) generated human and murine gastric cancer organoids which recapitulated the typical features and altered pathways of each of four molecular subtypes of gastric cancer. the combination of organoids and crispr-cas9 technologies promotes research on the molecular mechanisms of gastric cancer tumorigenesis and progression, thereby accelerating the development of preclinical gastric cancer models for novel drug development and personalized medicine. recently, dekkers and colleagues attempted to model multi-step carcinogenesis in breast epithelial organoids derived from human reduction mammoplasties using crispr-cas9 technology. they introduced crispr-mediated knock-out of four breast cancer-associated tumor suppressor genes, including p53, pten, rb1 and nf1 (ptrn) to mammary progenitor cells. mutated organoids could long-term expanded and generated er+ luminal tumors upon transplantation into mice for 1 out of 6 ptr-mutated and 3 out of 6 ptrn-mutated organoid lines. these organoids had various response to endocrine therapy or chemotherapy, indicating the potential application of this model to facilitate our understanding of the molecular mechanisms in specific subtypes of breast cancer. in prostate cancers, 40-80% of tumors harbor a gene fusion between the androgen receptor (ar)-responsive transmembrane protease serine 2 (tmprss2) gene and e26 transformation-specific (ets) family transcription factor, most often the oncogene ets-related gene (erg) (tomlins et al. 2005) . tmprss2 and erg, both located on chromosome 21, are separated by about 3 million base pairs. using crispr-cas9, a tmprss2-erg fusion was successfully introduced into mouse prostate organoids using a template that brought these two dna regions together. this genetic alteration resulted in armediated overexpression of erg, an effect that can be prevented by androgen receptor antagonist, consistent with those seen in vivo (driehuis 2017) . living organoid biobanks as a tool for personalized treatment and drug development as mentioned above, organoids can be efficiently established from patient-derived normal and tumor tissue samples, which can be cryopreserved and stored in living organoid biobanks. pdos resemble the tumor epithelium they were derived from both phenotypically and genetically. however, molecular profile alone is not sufficient to adequately predict drug sensitivity. even in patients with the same genotype, drug response varies. besides, some mutations are rare, thus clinical trials are impossible and efficacy testing is required for drugs to be conducted on an individual basis. thus, combined molecular and therapeutic profiling of pdos may help predict treatment response and contribute to personalized cancer treatment and drug development. a number of organoid biobanks have been reported since 2014 (table 3) . a colon cancer derived biobank of 22 lines was established in 2015, setting an example for a standard biobank . all samples performed rna sequencing and whole genome sequencing analysis. the molecular characteristics of pdos cover all five consensus molecular subtypes of crc. the mutations in the organoids were largely concordant with the original tumors, which was validated in a set of organoids established of colorectal metastases (weeber et al. 2015) . high-throughput screening of a panel of 83 compounds found that differences in drug sensitivity among the organoid lines that in some cases correlated with specific mutation. for example, rnf43mutant organoids were sensitive to wnt secretion inhibitors, and kras-mutant organoids were resistant to the egfr (epidermal growth factor receptor) inhibitors, including cetuximab and afatinib. later, schutte and colleagues (schã¼tte et al. 2017 ) reported a biobank of 35 organoid lines from crc. they found that organoid models reproduce most the genetic and transcriptomic characteristics of the donors, but (vlachogiannis et al. 2018) determined less complex molecular subtypes for the absence of stroma. drug screening with therapeutic compounds representing the standard of care for crc, combined with molecular profiles, helped identify a signature outperforming ras/raf mutation which has predictive value for sensitivity to the egfr inhibitor cetuximab. drug response in organoids and clinical response was also observed to prove that the in vitro organoid response correlates with the in vivo response. a clinical study of pdos derived from metastatic gastroesophageal and crc showed a strong correlation (100% sensitivity, 93% specificity, 88% positive predictive value, and 100% negative predictive value) between the in vitro organoid response to a set of targeted therapies and chemotherapies and the response of the tumor in patients (vlachogiannis et al. 2018) . another study adopted organoids for colon cancer chemoprediction showing that pdos test predicted more than 80% of patients' response treated with irinotecan-based therapies (ooft et al. 2019) . together, these studies indicate the potential of tumor-derived organoids to predict patients' responses. recently, two studies showed the applications of pdos derived from rectal cancer to predicting patient responses to neoadjuvant chemoradiation therapy. yao and colleagues (yao et al. 2020 ) generated a rectal cancer derived biobank (n=80) and tested pdos' sensitivity to 5-fu, irinotecan, or radiation. they incorporated a correlation between in vitro responses in organoids and the histopathologically determined tumor regression scores (trgs) after surgical resection to define prognostic cut-offs. using these parameters, the in vitro responses could predict clinical responses with an impressive area under the curve (auc) of 0.88 and an accuracy of 84%. in the other study, ganesh and colleagues (ganesh et al. 2019 ) established 65 pdo lines from rectal cancer to test responses to neoadjuvant chemoradiation therapy, including the standard folfox chemotherapy and radiation. the pdo responses significantly reflected the patients' progression-free survival. moreover, pdos generated invasive rectal cancer followed by metastasis upon transplantation into murine rectal mucosa, exhibiting the same in vivo metastatic route as in the corresponding patients. for pancreatic cancer, boj and colleagues (boj et al. 2015) were the first to successfully developed organoids from patient-derived pdacs. subsequently, seino and colleagues ) generate an extensive organoids biobank of pdacs (n=39) covering both classical and basal subtypes according to gene expression signatures. they found basal-type pdacs derived organoids are more independent of wnt signaling, which are more invasive and aggressive clinically, indicating that progression of pdacs are accompanied by loss of stem cell niche dependence. recently, tiriac and colleagues (tiriac et al. 2018 ) generated a much larger pdac biobank (n=114) and exposed a subset of these organoid lines to the standard-of-care chemotherapies. their sensitivities paralleled clinical responses in patients. besides, gene expression signatures of chemosensitivity based on organoids were developed to help predict responses to chemotherapy in both the adjuvant and advanced disease settings. by high throughput drug screening, they nominated alternative treatment strategies for chemorefractory pdo. another study also used pdos (n=30) to identify novel therapeutics to target pancreatic tumor cells in a biobank covering different histological subtypes, including pdacs, acinar cell carcinoma, cholangiocarcinoma, adenosquamous-pdacs, intraductal papillary mucinous neoplasm (ipmn)-derived pdacs and papilla of vater adenocarcinomas (driehuis et al. 2019c) . pdos were exposed to 76 therapeutic agents currently not exploited in the clinic. the prmt5 (protein arginine methyltransferase 5) inhibitor, ezp015556, was shown to target mtap (methylthioadenosine phosphorylase)-negative tumors, but also appeared to constitute an effective therapy for a subset of mtap-positive tumors, indicating the importance of personalized approaches for cancer treatment. huch and colleagues (broutier et al. 2017 ) described a liver tumor biobank (n=13) containing hepatocellular carcinoma and cholangiocarcinoma, as well as the rarer lymphoepithelioma-like cholangiocarcinoma. in drug screening experiments with 29 compounds, the erk (extracellular regulated protein kinases) inhibitor sch772984 was found to effectively inhibit the growth of tumor organoids, which was validated in vivo using xenotransplanted organoid lines in mice, histological types were not comprehensively reported highlighting sch772984 as a possible therapeutic agent. biliary tract carcinomas-derived organoids biobank was also established, covering intrahepatic cholangiocarcinoma, gallbladder cancer, and neuroendocrine carcinoma of the ampulla of vater (saito et al. 2019) . gene expression profiling of the organoids indicated that sox2, klk6 and cpb2 could be potential prognostic biomarkers. drug screening using a compound library of 339 drugs showed that the antifungal drugs, amorolfine and fenticonazole, significantly suppressed the growth of biliary tract carcinomas organoids with little toxicity to normal biliary epithelial cells. the organoids biobank of metastatic prostate cancer covering both ar (androgen receptor)-positive and -negative subtypes was the first reported biobank established by gao and colleagues ). the biobank captured the most common genetic aberrations in prostate cancer, including tmprss2-erg fusion, homozygous deletions of pten and chd1, as well as typical copy number variations. to be pointed out, organoids derived from circulating tumor cells were also successfully established in this biobank, showing that at least in some cases, organoids can be established from less invasive blood samples. a bladder cancer derived organoids biobank (n=20) was established containing urothelial carcinomas and one squamous cell carcinoma (lee et al. 2018) . organoid lines were interconvertible with orthotopic xenografts and recapitulated the mutational spectrum of the corresponding tumor type, including activation of fgfr3 and mutations in epigenetic regulators such as arid1a. drug screening of 40 compounds based bladder tumor organoids showed partial correlations with mutational profiles as well as treatment resistance, and the drug responses can be validated using xenografts in vivo. a biobank of breast cancer organoids (n=95) has been described covering major histological subtypes (invasive ductal carcinoma and invasive lobular carcinoma) and all molecular subtypes based on gene expression . organoid morphologies matched the histopathology of the original tumors, and hormone receptor [estrogen receptor (er), progesterone receptor (pr)], her2 status and copy number variations were retained. er and pr status have predictive value for the outcome of endocrine therapy (e.g. tamoxifen), while her2 is a target for targeted therapy (e.g. trastuzumab) and also has predictive value for chemotherapy outcome. the response of breast cancer-derived organoids to her2 inhibitor afatinib and to endocrine therapy tamoxifen were consistent with in vivo xeno-transplantations and patient response in clinic. an organoid biobank of high-grade serous ovarian cancer (hgsc) (n=33) was established by hill and colleagues (hill et al. 2018) . up to 50% of all patients with hgsc have dna repair defects, typically mutation of brca1 or brca2. these patients were thought to benefit from treatment with poly (adp-ribose) polymerase (parp) inhibitors. in the clinical setting, mutation analysis alone is not sufficient to adequately predict drug sensitivity. the study showed that functional assays in organoids are a better predictor than the genomic analysis in clinic, implying that functional assays in organoids may improve the prediction of drug sensitivity beyond what can be achieved with genomic analysis alone. kopper and colleagues (kopper et al. 2019 ) established a second ovarian cancer biobank (n=56) that captured all of the main histological subtypes, including borderline tumors, endometroid carcinomas, mucinous carcinomas, lgsc and hgsc. notably, a novel single-cell dna sequencing method was used to demonstrate intra-patient heterogeneity was preserved in organoids when compared with the original tumor. pdos can be used for drug-screening analyses and capture distinct responses of different histological subtypes to platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. besides, pdos can also be xenografted, enabling in vivo drug sensitivity analyses. taken together, pdos of ovarian cancer have potential application for translational research and precision medicine. driehuis and colleagues (driehuis et al. 2019a ) established an organoid biobank (n=31) derived from head and neck squamous cell carcinoma (hnscc). pdos recapitulates genetic and molecular characteristics of original hnsccs and can generate tumors upon transplantation to immunocompromised mice. the authors observe different responses to commonly used drugs in clinic including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. besides, drug screens exhibit selective sensitivity to targeted drugs that are not normally used in clinic for patients with hnscc. these findings may inspire the personalized treatment of hnscc and expand the list of hnscc drugs. in another study, the authors reported pdos derived from hnscc can also be used to evaluate their response to targeted photodynamic therapy and to ensure the safety of the treatment at the same time by testing it on organoids derived from matched normal tissues (driehuis et al. 2019b ). all organoids discussed above were derived from tumors of epithelial origin, known as carcinomas. recent advances show organoids derived from primary glioblastoma tissue, setting the stage for growing organoids from non-epithelial tumors (hubert et al. 2016) . glioblastoma presents great heterogeneity, and thus it's difficult to generate an ideal in vitro model which can recapitulate the in vivo situation of the donor. by modifying the method to develop cerebral organoids, the pdos can be successfully derived both from primary lesion of glioblastoma and from brain metastases. once formed, pdos presented hypoxia gradients and mimicked cancer stem cell heterogeneity with rapidly dividing outer cells surrounding the hypoxic core of differentiated cells and diffuse, quiescent cancer stem cells. drug testing based on organoids showed that nonstem cells were sensitive to radiation therapy, whereas adjacent cancer stem cells were radioresistant. orthotopic transplantation of pdos resulted in tumors recapitulating histological features of the parental tumor. based on this method, jacob et al. (2020) established a larger organoid biobank derived from glioblastoma (n= 70), recapitulating the histological characteristics, cellular diversity, gene expression, and mutational profiles of their donors. the organoids generated rapid, aggressively infiltrated tumors upon transplantation into adult rodent brains. the authors observe different responses to exposure of radiation with concurrent temozolomide, which was consistent with the patients' response and survival in clinic. notably, the authors further demonstrate the utility of organoids in modeling immunotherapy by co-culture of chimeric antigen receptor t (car-t) cells with organoids. they observed specific tumor cells were targeted and killed by car-t cells. the study expands the application of pdos in personalized treatment to include immunotherapy. several biobanks containing organoids derived from mixed cancers were established for pan-cancer research. pauli et al. (2017a pauli et al. ( , 2017b ) developed a robust precision cancer platform, by integrating whole exome sequencing with a living biobank which allows high throughput drug screens on pdos. the biobank included tumors derived from prostate, breast, colorectum, esophagus, brain, pancreas, lung, small intestine, ovary, uterus, soft tissue, bladder, ureter and kidney. in another study, to model tumor immune microenvironment, kuo and colleagues (neal et al. 2018) established pdos based on ali method from >100 individual patient tumors of 19 distinct organs and 28 histological subtypes. these pdos included common cancers such as colon, pancreas, and lung, and rarer histologies such as bile duct ampullary adenocarcinoma, brain schwannoma, and salivary gland pleomorphic adenoma. pdos in this biobank retain immune cells and should enable immuno-oncology investigations and facilitate personalized immunotherapy testing. as mentioned above, cf is a lethal genetic disease caused by cftr mutations that impairs the function of many organs including the intestine, lung, pancreas, sweat gland, liver, and kidney. the disease is characterized by the buildup of viscous, sticky mucus which clogs airways, causes chronic digestive system problems and leads to cf-related diabetes. approximately 2,000 cfcausing mutations of cftr have been described, and drug efficacy varies among the different genotypes (cutting 2015) . thus, there is a need for a personalized medicine approach to predict treatment response. beekman and colleagues (dekkers et al. 2016 ) first established an organoid biobank derived from rectum of 71 cf patients with 28 different cftr genotypes. based on the biobanking, they developed a personalized medicine approach by using fsk-induced swelling assay to select clinical responders to cf modulators. two patients with the rare and uncharacterized f508del/ g1249r genotype responded in vitro to a specific cf modulator, ivacaftor (kalydeco, vertex pharmaceuticals). the responses were consistent with their in vivo clinical response to the treatment, reflected by improved pulmonary function and sweat chloride tests. in a prospective follow-up study involving 24 participants (berkers et al. 2019) , the predictive power of the fsk assay was further substantiated, as the in vitro assay correlated with changes in pulmonary function and sweat chloride tests conducted in vivo. besides cf-rectal organoids, pancreatic organoids and cholangiocyte-like organoids derived of cf-ipscs have also shown the potential for drug screening (sampaziotis et al. 2015; simsek et al. 2016 ). in the netherlands, the licensing of orkambi (lumacaftor/ivacaftor, vertex pharmaceuticals) allows treatment of cf patients solely based on a positive organoid swelling response, demonstrating the potential of organoid-based assays for delivering personalized medicine (fig. 10) . in summary, organoid biobanks have been established for multiple tumor types (table 3) , including nonepithelial glioblastoma, and several principals can be concluded. first, pdos can be established from small biological samples, such as biopsies and even circulating tumor cells, and these organoids can generate tumors upon xenotransplantation. second, the pdos in biobanks recapitulate histological and genetic aspects of the original tumors, which holds not only for localized primary tumors but also for metastatic tumors. third, high-throughput drug screening experiments in organoids correlate with the response in patients and lead the identification of new therapeutic targets. combining genetic sequencing with functional assays in pdos will facilitate personalized treatment, even including immunotherapy, which will be discussed in detail in section 3.4. schwank and colleagues (schwank et al. 2013) firstly demonstrated that it is feasible to repair genetic defects in organoids. intestinal organoids derived from two cf patients with a homozygous cftr f508 deletion were repaired using crispr-cas9 technology. after repair, fsk-induced swelling was restored, functionally demonstrating cftr activity. later, firth and colleagues (firth et al. 2015) generated ipscs from cf patients with a homozygous deletion of cftr f508 and corrected this mutation using crispr technology to target corrective sequences to endogenous cftr genomic locus, combining with selection system. the corrected ipscsorganoids were able to differentiate mature airway epithelial cells with normal cftr expression and function. however, cftr gene repair in organoids and subsequent transplantation into patients is hard to be applied in the clinic. first, the loss of cftr functions results in disease in multiple organ systems, which would require the transplantation of organoids into multiple tissue sites. second, a high percentage of repaired cells per organ would be required for functional restoration. third, ethics problems in organoids transplantation are controversial and needs to reach a consensus in the research field. human and murine organoids have been orthotopically transplanted into mice to model disease or to show tumorigenic potential. here, we discuss studies that used organoid transplantation as therapy. yui and colleagues (yui et al. 2012 ) firstly exploited the feasibility to apply organoids to repair damaged epithelium. gfp + murine colon organoids derived from lgr5 + stem cells were reintroduced into mice with dss (dextran sulfate sodium salt) -induced acute colitis. transplanted cells readily integrated into the mouse colon and covered superficially damaged tissue. 4 weeks after transplantation, the donor cells constituted a single-layered epithelium, which formed functionally and histologically normal crypts that were able to self-renew. further, engrafted mice had higher body weights than ungrafted ones, indicating that donor cells contributed to the recovery of dss-induced acute colitis. although further optimization is still needed, the current study indicates that in vitro expansion and transplantation of organoids may be a promising treatment choice for patients with severe gastrointestinal epithelial injuries. orthotopic transplantation of liver organoids has also shown promising results. in a mouse model of toxicityinduced acute liver failure, transplantation of mouse differentiated biliary duct organoids derived from lgr5 + stem cells generated detectable organoid-derived nodules in 20-40% of cases . although engraftment rate was low (approximately 1%), a significant increase in survival of the grafted group was observed compared to the ungrafted group, indicating that the transplanted cells contributed to liver function repair ). in follow-up studies using the same injury model, the transplantation of mouse (peng et al. 2018 ) and human fetal ) hepatocyte organoids, generated much more extensive engraftment indicating that the efficiency of engraftment may be enhanced by transplantation of the most physiologically relevant cell type. recently, xiao and colleagues (xiao and deng 2020) generated induced sensory ganglion organoids exhibiting fig. 10 organoids for modeling cystic fibrosis (cf) and its multiple applications. organ-specific pathologies of cf can be studied separately using organoids derived from distinct tissues and applied to personalized drug screening molecular features, subtype diversity, electrophysiological and calcium response properties, and innervation patterns characteristic of peripheral sensory neurons, which may serve as a source for cell replacement therapy. yoshihara and colleagues (yoshihara and o'connor 2020) generated human islet-like organoids (hilos) from ipsc, which provides a promising alternative to cadaveric and device-dependent therapies in the treatment of diabetes. hilos contain endocrine-like cell types that, upon transplantation, rapidly re-establish glucose homeostasis in diabetic nod/scid mice. overexpression of the pd-l1 protected hilo xenografts such that they were able to restore glucose homeostasis in immune-competent diabetic mice. organoids combined with 3d printing were recently introduced to build the 3d architecture of tissue, which may find widespread applications in regenerative medicine. firstly, zhang and his colleagues (zhang et al. 2016 ) established the technique that can be transformed into human cardiomyocytes derived from ipscs to construct endothelialized human myocardium. then, creff and colleagues (creff et al. 2019 ) provided the possibility of creating artificial 3d scaffolds that match the size and structure of mouse intestinal crypt and villi. moreover, homan and colleagues (homan et al. 2019 ) built a model that had the ability to induce substantial vascularization and morphogenesis of renal organs in vitro under flow conditions opening up a new way for the study of renal development, disease and regeneration. though above studies indicate the potential application of organoids in regenerative medicine, many problems need to be solved before organoids are put into clinical use. for example, integration upon transplantation requires optimization, and animal-based 3d ecm matrix used for organoid culture need to be replaced with a synthetic matrix. the tumor microenvironment consists of various nonepithelial cell types, including immune cells and stromal cells, which greatly affects therapeutic responses. however, it is a major challenge to model tumor microenvironment. much of our knowledge regarding the tumor microenvironment was studied on cell lines and pdx models. however, cell lines are insufficient to recapitulate the heterogeneity of tumor cells, while the microenvironment of pdx models mainly depend on the mouse immune system, which cannot adequately recapitulate the human immune system. cancer immunotherapy has emerged as a promising therapeutic developments that take advantage of a patient's own immune system to eradicate tumor cells (mellman and coukos 2011) , and several organoid-based models have been established to study immunotherapy response (fig. 11) . recently, a holistic approach based on the ali method (mentioned in section 1.2), which preserved the tumor epithelium and its stromal microenvironment in vitro, was described using pdos of various cancer types, including colorectal and lung cancers (neal et al. 2018 ). in addition to stromal fibroblasts, cellular immune components such as tumor-associated macrophages, ctls (cytotoxic t lymphocyte), t h cells (t helper cells), b cells, natural killer (nk) cells and natural killer t (nkt) cells were also readily maintained for up to 30 days in the organoid cultures. the organoid cultures also preserved the t cell receptor (tcr) heterogeneity of the t cells found in the parental tumor. the authors used these organoids to model immune checkpoint blockade, leading to the expansion and activation of tumor antigen-specific t cells and subsequent killing of tumor cells. adoptive cell therapy is a promising immunotherapy. in this method, autologous immune cells are expanded in vitro followed by the subsequent transplantation of these cells back into the patient, to enhance the immune response against a tumor. using this strategy, durable regression of melanoma was achieved by in vitro expansion of autologous tumor-infiltrating lymphocytes (tils) (rosenberg 2015) . however, this approach requires resected specimens from which tils can be obtained. a strategy to avoid resection is to isolate peripheral blood lymphocytes, activate these cells in vitro by co-culture with tumor cells. for this strategy, tumor-derived organoids are a highly useful source of tumor cells for coculture: tumor organoid cultures can be efficiently established from a small tissue sample through biopsy, and tumor-derived organoids are heterogeneous and recapitulate the genetic and histological characteristics of the parental tumors. dijkstra and colleagues (dijkstra et al. 2018) were thus able to obtain tumor-reactive t lymphocytes from peripheral blood lymphocytes after 2 weeks of co-culture with tumor organoids derived from non-small cell lung cancer and msi-h crc. before coculture, organoids were stimulated with ifn-î³ to enhance antigen presentation. pd-1 blocking antibody, il-2, and anti-cd28 were added to enhance t cell activation. after co-culture, t lymphocytes were activated, as demonstrated by expression of ifn-î³ and cd107a. accordingly, after an additional 3 days of co-culture of activated t lymphocytes with tumor organoids, the survival of the tumor organoids was reduced, while matched normal organoids were unaffected. cancers with low mutational burden and stable tumor antigen presentation may be suitable targets for chimeric antigen receptor (car)-engineered t cells. studies on b cell malignancies showed promising results (june 2018) . in solid tumors, the therapeutic application of car t cells has been hindered by side effects that arise from targeting overexpressed native antigens that are, not exclusively expressed by tumors. thus, for solid tumors, preclinical models are needed to allow for carmediated cytotoxicity testing. recently, a luciferasebased quantification assay has been developed to test car t cell-mediated cytotoxicity against pdos (schnalzger et al. 2019) . instead of using carengineered t cells, the researchers adopted an nk cell line with non-mhc restricted cytolytic activity to target research. the efficiency of the system was confirmed by using car-engineered nk-92 cells directed toward epcam, an epithelial marker overexpressed by cancers. car-mediated cytotoxicity was observed against organoids derived from both normal colon tissue and crc tissue presenting either peptide (schnalzger et al. 2019 ). subsequently, the authors engineered cars targeting egfrviii, a neoantigen that is widely expressed by solid cancers. in a competitive co-culture assay, egfrviiispecific car nk cells killed egfrviii-expressing organoids derived from tumors efficiently but not organoids derived from normal tissue. finally, the team generated cars targeting antigens specific to subgroup of crc that overexpress the wnt ligand receptor fzd upon loss of expression of its antagonists rnf43/znrf3. to test possible side effects of fzd-specific cars, the authors evaluated cytotoxicity against normal colon fig. 11 co-culture systems of organoid and immune cell in immuno-oncology research. two main methods are currently being adopted: a holistic approach (up). tumor biopsies are cultured in ali in the entire tumor microenvironment as a cell suspension of all cell types, including immune cells and other non-epithelial cell types. b reductionist approach (down), epithelial organoids are derived from tumor biopsies and are then co-cultured with autologous immune cells derived from the peripheral blood of the same patient. crc, colorectal cancer; ali, air-liquid interphase; ecm, extracellular matrix; nk cell, natural killer cell organoids as well as different gene-edited organoid lines deficient for both rnf43 and znrf3 or for apc. these co-culture assays illustrated that the cytolytic activity of the fzd-specific car nk cells was not specific to the mutated organoid lines, suggesting that such approaches may have marked side effects if used therapeutically. though effective target has not been found, the platform can be widely used to evaluate car efficacy and tumor specificity in a personalized manner. in summary, co-cultures of cancer organoids and immune cells have become a highly promising strategy for personalized immunotherapy for cancer patients. organoids are robust research tools for the study of human development and disease. however, there are hurdles and limitations associated with using organoids (fig. 12) . first, culture approaches are not standardized. ascsderived organoids are established under distinct culture conditions in each laboratory. to reduce the cost, costefficient small molecule compounds were used to replace growth factors yin et al. 2014) . besides, homemade niche factors produced by various cell lines has been used commonly to culture organoids. however, this trend will result in experimental variation into organoid studies across different research labs. second, organoid culture requires the use of matrigel or other animal-based matrix extract to enable cells to aggregate into 3d structures. these extracts suffer from batch-to-batch variability in their composition, which may affect the reproducibility of experiments. in addition, they may carry unknown pathogens and are potentially immunogenic when transplanted to humans, limiting the application of organoids in a clinical transplantation setting. this may be solved by culturing with clinical grade collagen, which has been successfully used for colon organoids culture and expansion (yui et al. 2012) . steps toward fully defined culture conditions have been made with the development of a synthetic polyethylene glycol-based gel that sustains the short-term growth of mouse asc-derived intestinal organoids (gjorevski et al. 2016 ). however, this matrix remains to be optimized for the long-term expansion of intestinal organoids and f non-intestinal organoids. third, obtaining pure tumor organoids is another critical problem for researchers. many studies have reported fig. 12 schematic summarize of current limitations in organoids culture development (yellow) and methods to overcome these issues (brown) that tumor organoids can be overgrown and contaminated by matched normal organoids. one of the most widely used methods to acquire pure tumor organoids is to select tumor cells that harbor the most common mutations in its cancer type. for example, tumor organoids derived from crc can be selectively upon withdrawal of wnt3a and r-spondin1. however, this method is not applicable to all cancers, and some specific cancer subtypes. more importantly, intra-tumoral heterogeneity would inevitably be lost upon selection. some labs have suggested obtaining pure tumor organoids based on growth phenotypes. however, not all tumor organoids have clear morphological differences to normal organoids. more researches are needed to establish appropriate approaches for obtaining pure tumor organoids. fourth, for ascs-derived organoids, only the epithelial compartment of organs is represented, while blood vessels, immune cells, stroma, and nerves are lacking. as mentioned above, many groups have focused coculturing organoids with various types of cells, analogous to what has already been done with immune cells (dijkstra et al. 2018) , or adopted unconventional organoid culture methods, such as the ali method (neal et al. 2018; ootani et al. 2009 ). besides, several psc-based organoids are able to undergo mesenchymal differentiation to generate subepithelial myofibroblasts and smooth muscle . last, the development of a model based on living human tissues that can be stored and expanded in biobanks-potentially forever-has raised a set of ethical issues regarding informed consent and ownership (bredenoord et al. 2017) . for organoid biobanks, patient consent is required. the most common type of patient consent restricts the use of a patient's material to only a specific research aim. however, biobanks are useful for researchers in multiple fields, and the use of biobanks in a combination of fields may provide potentially synergistic data. to solve this problem, bredenoord and colleagues (boers and van delden 2015; bredenoord et al. 2017 ) have suggested that a broad consent be used for governance. this broad consent would allow donors to make informed decisions about how their samples are used after they have been provided with relevant information about the establishment and regulation of the biobank. another issue that has arisen with the development of living biobanks is ownership. organoids are increasingly used by commercial parties as tools for drug development or in validation studies. such uses will inevitably result in patentable compounds. it may be helpful to include regulations covering the distribution of any financial gains from intellectual property among stakeholders in the governance of the biobanks. furthermore, in terms of the culture conditions of tumor organoids, intra-tumoral heterogeneity could be lost during passages for that the culture media might not favor the growth of all tumor subclones equally effectively. additionally, novel mutations would be acquired during long-term expansion. collectively, these drawbacks deserve further attention, and more work is needed to improve organoids culture technologies. despite these limitations, organoid technology holds great promise as a robust tool for basic, translational and clinical research for human development and disease modeling. in this review, we have demonstrated the potential of organoid technology for modeling genetic, infectious and malignant disease, as well as for drug development and personalized medicine. the basic and translational applications of organoids are expected to expand in the future. regarding epithelial genetics, organoids have potential especially for rare diseases due to their high expansion ability and genetic stability. in infectious diseases, the mechanisms of virus-induced malignant transformation, for example, by epsteini-barr virus in gastric and nasopharynx cancers, may be studied in long-term cocultures of the virus with normal epithelium from the respective organ. in the studies of malignancies, culture conditions need to be developed for many types of carcinomas and for sarcomas and melanomas. the optimization of drug screening will be a pivotal use of organoids in personalized medicine by adopting biobanks of different cancers. this enables many compounds to be screened for a specific disease or a specific compound to be screened for many forms of a given disease. moreover, multiple organoids can be derived from the same cancer patient over time to assess drug response and developing resistance to targeted drugs and to predict patient outcome. in the area of regenerative medicine, there is still a long way to go for the transplantation of organoids as therapy. several hurdles, including the development of a non-animal-based alternative for matrigel and efficient delivery procedures, remain to be overcome. in conclusion, the worldwide application of organoids has contributed to unprecedented advances in research on human development and diseases. even though current organoid systems show some limitations and require further optimization for use in disease modeling and personalized medicine, they will continue to be valuable tools in basic and translational research. ecm: extracellular matrix; pdos: eatient-derived organoids; wenr method: wnt3a+egf+noggin+r-spondin-1 nico: nicotinamide; rspo: r-spondin-1; pge2: prostaglandin e2 dht: dihydrotestosterone; crc: colorectal cancer; ali: air-liquid interface tumor infiltrating lymphocytes; ta: transit-amplifying; wgs: wholegenome sequencing; crispr: clustered regularly interspaced short palindromic repeats; cas9: crispr associated protein 9; nhej: non-homologous end joining; hdr: homology-directed repair cftr: cf transmembrane conductance regulator; camp: cyclic adenosine monophosphate; fsk: forskolin; clc: cholangiocyte-like cells pdecs: pancreatic ductal epithelial cells; mvix: microvillus inclusion disease syntaxin-3; a1at: î±1-antitrypsin; ags: aicardi-goutiã¨res syndrome zikv: zika virus; who: world health organization; tlr3: toll-like receptor 3 japanese encephalitis virus; je: japanese encephalitis; hbv: hepatitis b virus; hunov: human norovirus; hbga: histo-blood group antigen rsv: respiratory syncytial virus salmonella typhi clostridium difficile; cdi: c. difficile infection helicobacter pylori; cin: chromosomal instability hiv: human immunodeficiency virus; shrna: short hairpin rna; ssas: sessile serrated adenomas; mlh1: mutl homolog 1; msi: microsatellite instability pdac: pancreatic ductal adenocarcinoma; srcc: signet-ring cell carcinoma androgen receptor; tmprss2: transmembrane protease serine 2; ets: e26 transformation-specific; erg: ets-related gene auc: area under the curve; ipmn: intraductal papillary mucinous neoplasm hgsc: high-grade serous ovarian cancer; lgsc: low-grade serous ovarian cancer; parp: poly (adpribose) polymerase; hnscc: head and neck squamous cell carcinoma; car-t: chimeric antigen receptor t; nk cell: natural killer cell; nkt cell: natural killer t cell; tcr: t cell receptor chimeric antigen receptor; dss: dextran sulfate sodium; ifnî³: interferon-gamma; mhc: major histocompatibility complex nhe3: sodium/hydrogen exchanger 3; muc2: mucoprotein2; akt: protein kinase b; mapk: mitogen-activated protein kinase; eif3e: eukaryotic translation initiation factor 3 subunit e egfr: epidermal growth factor receptor; prmt5: protein arginine methyltransferase 5; mtap: methylthioadenosine phosphorylase erk: extracellular regulated protein kinases; ar: androgen receptor severe acute respiratory syndrome-coronavirus 2; mge: medial ganglionic eminence; hrsace2: human recombinant soluble ace2; iwp-2: inhibitor of wnt production 2; vpa: valproic acid dapt: dual antiplatelet therapy; sgrna: single-guide rna; foxg1: forkhead box g1; hilos: human islet-like organoids references adli m. the crispr tool kit for genome editing and beyond mouse model of alagille syndrome and mechanisms of jagged1 missense mutations kang gh molecular subtypes of colorectal cancer and their clinicopathologic features, with an emphasis on the serrated neoplasia pathway knoblich ja fused cerebral organoids model interactions between brain regions lgr5(+ve) stem cells drive selfrenewal in the stomach and build long-lived gastric units in vitro identification of stem cells in small intestine and colon by marker gene lgr5 in vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection induced 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progenitor cells and cerebral organoids from zika virus infection loss of syntaxin 3 causes variant microvillus inclusion disease hesc-derived thalamic organoids form reciprocal projections when fused with cortical organoids fusion of regionally specified hpsc-derived organoids models human brain development and interneuron migration generation of self-organized sensory ganglion organoids and retinal ganglion cells from fibroblasts identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen olig2 drives abnormal neurodevelopmental phenotypes in human ipsc-based organoid and chimeric mouse models of down syndrome a comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer niche-independent highpurity cultures of lgr5+ intestinal stem cells and their progeny modeling rotavirus infection and antiviral therapy using primary intestinal organoids immune-evasive human islet-like organoids ameliorate diabetes functional engraftment of colon epithelium expanded in vitro from a single adult lgr5 + stem cell differential antiviral immunity to japanese encephalitis virus in developing cortical organoids sun j salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions bioprinting 3d microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip monroe ss norovirus classification and proposed strain nomenclature infection of bat and human intestinal organoids by sars-cov-2 differentiated human airway organoids to assess infectivity of emerging influenza virus authors' contributions yl and pt conceived and wrote the paper. sc and jp participated in conceptualization, validation and review of the draft. gh participated in the supervision and review of the final draft. the author(s) read and approved the final manuscript. this work was supported by the national natural science foundation of china (31470826, 31670858, and 81773357 to hua), shanghai sailing program (19yf1409500 to li) and shanghai anticancer association eyas project (saca-cy1a05 to li). the authors declare that they have no competing interests author details 1 key: cord-310844-7i92mk4x authors: hryhorowicz, magdalena; lipiński, daniel; hryhorowicz, szymon; nowak-terpiłowska, agnieszka; ryczek, natalia; zeyland, joanna title: application of genetically engineered pigs in biomedical research date: 2020-06-19 journal: genes (basel) doi: 10.3390/genes11060670 sha: doc_id: 310844 cord_uid: 7i92mk4x progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. the development of transgenesis has created new directions in research and possibilities for its practical application. generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. the further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation. numerous studies have shown that the pig (sus scrofa domestica) is the most suitable species both as a research model for human diseases and as an optimal organ donor for xenotransplantation. short pregnancy, short generation interval, and high litter size make the production of transgenic pigs less time-consuming in comparison with other livestock species this review describes genetically modified pigs used for biomedical research and the future challenges and perspectives for the use of the swine animal models. pigs have been extensively used in biomedical research due to anatomical and physiological similarities to humans. moreover, progress in gene editing platforms and construct delivery methods allow efficiently, targeted modifications of the porcine genome and significantly broadened the application of pig models in biopharming and biomedicine. target editing is possible through site-specific nucleases, of which the following are most commonly used: zinc finger nucleases (zfns), transcription activator-like effectors (tales), and nucleases from the crispr/cas (clustered regularly interspaced short palindromic repeats/crispr associated) system. introducing modifications in a specific site of the genome is possible due to cellular processes of repairing double-strand breaks induced by site-specific nucleases. double-strand breaks may be repaired in two ways: by non-homologous end joining (nhej) or by homologous recombination (hr). repair provided by nhej may lead to the formation of indel (insertion/deletion) mutations in the table 1 . summary of the most important advantages and disadvantages of the methods for obtaining genetically modified animals. advantages increased efficiency of the transgene integration precise transformation and selection of modified cells used in cloning the number of damaged zygotes do not exceed 10% the obtained animals do not exhibit mosaicism modification is also revealed in germ cells-transgenic offspring disadvantages low process efficiency (2%-3% in pigs) very low efficiency the possibility of random integration of the transgene early fetal mortality high process invasiveness the possibility of genetic defects genetically modified animals as research models for human diseases are a very important tool in searching for and developing new methods of therapy. a suitable model organism should be characterized by rapid growth, a high number of offspring, easy and inexpensive breeding, ability to be easily manipulated, and having a sequenced genome. initially, only rodents were used as a model in biomedical research. experiments on mice contributed to understanding the genetic background of numerous diseases. however, not every genetic disease induced in mice has the same clinical manifestation as in humans. furthermore, the short life span, together with a higher metabolic rate, makes the analysis of some hereditary diseases challenging. currently, pigs are one of the most important large animal models for biomedical research. many human diseases, such as cardiovascular diseases, obesity, and diabetes, have their counterparts in this species. the use of model organisms makes it possible to analyze diseases that occur naturally in animals with specific mutations and those deliberately induced. introduced animal genome changes may reflect mutations occurring in people suffering from specific genetic disorders. moreover, accurate and efficient genome editing can be used in the treatment of monogenic diseases. a slightly different direction of research is the use of genetically modified animal models in toxicological studies for testing drugs. genetically modified pigs are used as model organisms in research into various diseases, including cardiovascular and neurodegenerative diseases, neoplasms, and diabetes. cystic fibrosis (cf) is an autosomal recessive disorder manifested by bronchopulmonary failure and pancreatic enzyme insufficiency. cf is caused by a mutation in the gene responsible for the synthesis of the cftr (cystic fibrosis transmembrane conductance regulator) chloride channel, altering the mucosal function in the respiratory epithelium, pancreatic ducts, intestines, and sweat glands. the most common cystic fibrosis-causing mutation is the deletion of a phenylalanine at amino acid position 508 (df508). cf lung disease is the main cause of morbidity and mortality in cf patients. porcine lungs share many anatomical and histological similarities with humans. it has been shown that pigs in which the cftr gene was inactivated develop all symptoms of the disease occurring in humans, such as meconium ileus, defective chloride transport, pancreatic destruction, and focal biliary cirrhosis. this makes them a very good model species for this disease [14] [15] [16] . cystic fibrosis is a monogenic disease, and the insertion of the functional cftr gene into cf patient cells should theoretically restore the cftr channel function. therefore, pigs have also been used in gene therapy. the treatment with viral vectors successfully improved anion transport and inhibited bacterial growth [17, 18] . currently, the research focuses on improving the cf gene therapy with the use of the crispr/cas9 system. these efforts are focused on increasing the delivery efficiency of crispr/cas9 elements to target locus and obtaining sustained expression of the cftr transgene [19, 20] . it was demonstrated that precise integration of the human cftr gene at a porcine safe harbor locus through crispr/cas9-induced hdr-mediated knock-in allowed the achievement of persistent in vitro expression of the transgene in transduced cells. these results can help design effective gene therapy to treat cf patients [20] . duchenne muscular dystrophy (dmd) is a progressive, monogenic, x-linked lethal disease characterized by degenerative changes in muscle fibers and the connective tissue. it involves the degeneration of subsequent muscles-skeletal, respiratory, and cardiac-and progressive muscular dystrophy. muscular dystrophy is caused by a frameshift mutation in the dmd gene, which encodes dystrophin, a protein in muscle cells that connects the cytoskeleton with the cell membrane. the dystrophin gene contains 79 exons, with exons 3-7 and 45-55 being the most susceptible to mutations. dmd gene mutations are usually large deletions or duplications of one or several exons, as well as point mutations, leading to a change in the reading frame, the appearance of a premature stop codon, and failure to produce a stable protein. muscular dystrophy is most often diagnosed in early childhood, and patients become wheelchair dependent by 12 years of age. untreated boys die of cardiorespiratory complications around their 20 years. the rapid progress in gene editing gives hope for effective targeted therapies for dmd. moreover, the use of an animal model can facilitate the development of personalized treatment approaches. pigs with a dmd gene mutation (exon 52 deletion) develop human disease symptoms, such as lack of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive muscle dystrophy, and impaired mobility [21] . however, these animals died prematurely (up to 3 months old at most) what precluded natural breeding. the histological evaluation of skeletal muscles and diaphragm confirmed the presence of excessive fiber size variation, hypercontracted fibers, and segmentally necrotic fibers, resembled that of human dmd patients [21] . moreover, proteome analysis of biceps femoris muscle was performed. an increased amount of muscle repair-related proteins and reduced amount of respiratory chain proteins was found in tissue from 3-month-old dmd pigs. this indicated severe disturbances in aerobic energy production and a decrease in functional muscle tissue [22] . as the deletion of exon 52 in the human dmd gene is a common cause of duchenne muscular dystrophy, pigs can make an accurate research model for gene therapy. another porcine dmd model is genetically modified miniature pigs with a mutation in exon 27 in the dmd gene obtained by the crispr/cas9 system. in addition, these animals have shown symptoms of skeletal and heart muscle degeneration, characteristic of human patients with duchenne muscular dystrophy. reduced thickness of smooth muscle in the stomach and intestine was also observed in the pigs studied. however, founder pigs died of unreported causes [23] . although mutations in exon 27 are not reported in human dmd patients, pigs with this deletion constitute another useful animal dmd model. recently, moretti et al. demonstrated the restoration of dystrophin by intramuscular injection of crispr/cas9 components with the use of adeno-associated viral vectors in a pig model. in this study, pigs with dmd carrying a deletion of dmd exon 52 (d52dmd), resulting in a complete loss of dystrophin expression, were used. the restoration of dystrophin expression was possible due to the excision of exon 51 and the restoration of the dmd reading frame. the internally truncated d51-52dmd sufficed to improve skeletal muscle function, prevent malignant arrhythmias as well as prolonging lifespan of dmd pigs [24] . in the future, this strategy may prove useful in the clinical treatment of patients with d52dmd. alzheimer's disease (ad) is an age-related, progressive neurodegenerative disorder characterized by memory dysfunction followed by cognitive decline and disorientation. ad accounts for 50%-80% of human dementia cases. familial forms of ad are caused by autosomal mutations in the genes encoding presenilin 1 (psen1) and presenilin 2 (psen2) and amyloid precursor protein (app). these mutations are associated with the accumulation of amyloid β (aβ) peptide in senile plaques and phosphorylated tau protein in neurofibrillary tangles (nfts), which leads to synaptic damage and neuronal dysfunction [25] . the first ad model with the use of transgenic pigs was generated in 2009 by kragh et al. they produced göttingen mini pigs that carried a randomly integrated construct containing the cdna of the human app gene with ad causing a dominant mutation known as the swedish mutation (appsw) and a human pdgfβ promoter fragment [26] . although the transgene was specifically expressed in brain tissue at a high level, no ad phenotype was observed in mutant pigs. the same group also obtained göttingen minipigs with the human psen1 gene carrying the ad-causing met146ile mutation (psen1 m146i) and driven by a cytomegalovirus (cmv)-enhanced human ubic promoter. pigs were generated with the use of a site-specific integration system-recombinase-mediated cassette exchange (rmce) [27] . the psen1 m146i protein was expressed and tolerated well in the porcine brain, but also in this case, no symptoms of the ad disease were noticed. therefore, this group generated double transgenic göttingen minipigs with both appsw and psen1 m146i mutations. such a solution allowed the increase in intraneuronal accumulation of aβ [28] . in turn, another group obtained ad transgenic pigs using a retroviral multi-cistronic vector containing three ad-related human genes: app, tau, and psen1, with a total of six well-characterized mutations under the control of a fusion promoter: cmve+ hpdgfβ promoter region. they confirmed that transgenes were expressed at high levels in brain tissue and demonstrated a two-fold increase in aβ levels in the brains of transgenic pigs compared to wild-type [29] . cancer is a genetic disease involving uncontrolled, abnormal cell growth in the blood or solid organs resulting from acquired or inherited mutations. pigs represent a useful animal for the development and validation of new medicines and procedures in human tumor models. there are many resemblances in cancer biology between pigs and humans. these animals can correctly mimic human tumors and show similar pharmacokinetic responses to humans. adam et al. demonstrated that autologous transplantation of primary porcine cells transformed with retroviral oncogenic vectors caused tumorigenesis akin to those found in humans [30] . in turn, schook et al. induced tumor formation in pigs by introducing random transgenes that encode cre-dependent kras (kirsten rat sarcoma viral oncogene homolog) g12d and tp53 (tumor protein 53) r167h oncogenic mutations (orthologous to human tp53 r175h) [31] . moreover, saalfrank et al. reported that porcine mesenchymal stem cells (mscs) resemble human mscs requiring disturbance of p53, kras, and myc signaling pathways to become a fully transformed phenotype [32] . at present, pig models commonly used in cancer research include the tp53 knock-out model of osteosarcoma and apc (adenomatous polyposis coli) mutations model of familial adenomatous polyposis (fap). tp53 is a known tumor suppressor gene, and a germline mutation within this gene leads to li-fraumeni syndrome, a rare, autosomal dominant disorder that predisposes carriers to cancer development. the first model of li-fraumeni syndrome using genetically modified pigs has been described by leuchs et al. they generated pigs carrying a latent tp53 r167h mutation that can be activated by the cre-lox recombinase system [33] . after several years of observation, it was noted that both pigs with homozygous tp53 knock-out and pigs with heterozygous knock-out of tp53 showed osteosarcoma development. the heterozygous knock-out caused the development of spontaneous osteosarcoma in older animals, while homozygous tp53 knock-out resulted in multiple large osteosarcomas in 7 to 8-month-old pigs [32] . moreover, sieren and colleagues generated genetically modified yucatan minipigs that carried the tp53 r167h mutation. animals heterozygous for this mutant allele showed no tumorigenesis process, whereas homozygotes that reached sexual maturity developed lymphomas, osteogenic tumors, and renal tumors at varying rates. the tumor formations were validated by computed tomography, histopathological evaluation, and magnetic resonance imaging [34] . familial adenomatous polyposis is an inherited disorder characterized by the development of numerous adenomatous polyps in the colon and rectum which greatly increases the risk of colorectal cancer. the mutations in the apc tumor-suppressor gene are responsible for fap and may result in a hereditary predisposition to colorectal cancer. flisikowska et al. generated gene-targeted cloned pigs with translational stop signals at codon 1311 in porcine apc (apc 1311), orthologous to common germline apc 1309 mutations in human fap. evaluation of one-year-old pigs carrying the apc 1311 mutation showed aberrant crypt foci and adenomatous polyps with low-to high-grade intraepithelial dysplasia, similar to tumor progression as in human fap [35] . the apc 1311 pig model resulting in the development of polyposis in the colon and rectum can be useful in the diagnosis and therapy of colorectal cancer. cardiovascular diseases (cvds) are the major cause of morbidity and mortality worldwide. cvd is a group of disorders of the heart and blood vessels that involve coronary heart disease (such as angina and myocardial infarction), deep vein thrombosis and pulmonary embolism, peripheral arterial disease, cerebrovascular disease, and rheumatic heart disease. the dominant cause of cvd is atherosclerosis, which is characterized by the narrowing of arteries due to the accumulation of lipid and plaque formation. the plaque buildup restricts blood flow, and plaque burst can entail blood clots. similarities in heart anatomy and physiology, vessel size, blood parameters, coronary artery system anatomy, and lipoprotein metabolism make pigs a suitable model for the human cardiovascular system. atherosclerosis starts with the buildup of serum low-density lipoprotein (ldl), and mutations in the ldl receptor (ldlr) gene may cause familial hypercholesterolemia (fh). a porcine fh model has been generated in yucatan miniature pigs through recombinant adeno-associated virus-mediated targeted disruptions of the endogenous ldlr gene. ldlr+/− heterozygous pigs exhibited mild hypercholesterolemia, while ldlr−/− homozygotes animals were born with severe hypercholesterolemia and developed atherosclerotic lesions in the coronary arteries. these phenotypes were accelerated by high fat and high cholesterol diets [36] . the utilization of ldlr-deficient yucatan minipigs in the preclinical evaluation of therapeutics was also demonstrated. ldlr+/− and ldlr−/− pigs were used to assess the ability of novel drug-bempedoic acid (bema)-to reduce cholesterol biosynthesis. long-term treatment with bema decreased ldl cholesterol and attenuated aortic and coronary atherosclerosis in this fa model [37] . moreover, a model of fa and atherosclerosis was created by using the yucatan miniature pigs with liver-specific expression of a human proprotein convertase subtilisin/kexin type 9 (pcsk9) carrying the gain-of-function mutation d374y. pcsk9 plays important functions in cholesterol homeostasis by reducing ldlr levels on the plasma membrane. gain-of-function mutations in this protein cause increased levels of plasma ldl cholesterol, which in turn may result in more susceptibility to coronary heart disease. pcsk9 d374y transgenic pigs exhibited decreased hepatic ldlr levels, severe hypercholesterolemia on high-fat, high-cholesterol diets, and atherosclerotic lesions [38] . it is also considered that hypertriglyceridemia is an independent risk factor for coronary heart disease, in which apolipoprotein (apo)ciii is associated with plasma triglyceride levels. the hypertriglyceridemic apociii transgenic miniature pig model was generated for the examination of the correlation between hyperlipidemia and atherosclerosis. transgenic pigs expressing human apociii exhibited increased plasma triglyceride levels with their delayed clearance and reduced lipoprotein lipase activity compared to non-transgenic controls [39] . diabetes mellitus (dm) is a group of metabolic disorders characterized by hyperglycemia (elevated levels of blood sugar over a prolonged period), which results from deficiency or ineffectiveness of insulin. dm may lead over time to cardiovascular disease, chronic kidney disease, damage to the nerves and eyes. there are two main types of diabetes mellitus, called type 1 and type 2. type 1 dm, also referred to as juvenile diabetes or insulin-dependent diabetes mellitus, is caused by the pancreas's failure to produce enough insulin. the most common is type 2 diabetes, which is characterized by insulin resistance (reduced tissue sensitivity to insulin) that may be combined with relative insulin deficiency. the anatomical and physiological resemblance to the human pancreas and islets makes pigs excellent animals for metabolic diseases modeling. moreover, the structure of porcine and human insulin is also very similar (differs by only one amino acid). a transgenic pig model for type 2 dm was generated to evaluate the role of impaired glucose-dependent insulinotropic poly-peptide (gip). the main function of incretin hormones gip and glucagon-like peptide-1 (glp1) is stimulated insulin secretion from pancreatic beta cells in a glucose-dependent manner. in type 2 dm, the insulinotropic action of gip is impaired, which may suggest its association with early disease pathogenesis. the transgenic pigs expressing a dominant negative gip receptor (giprdn) in pancreatic cells were produced by lentiviral vectors. a significant reduction in oral glucose tolerance due to delayed insulin secretion as well as in β-cell mass caused by diminished cell proliferation was observed in giprdn animals [40] . these observations resemble the characteristic features of human type 2 diabetes, which makes the porcine giprdn model useful for testing incretin-based therapeutic strategies. further analyses revealed characteristic changes in plasma concentrations of seven amino acids (phe, orn, val, xleu, his, arg, and tyr) and specific lipids (sphingomyelins, diacylglycerols, and ether phospholipids) in the plasma of 5-month-old giprdn transgenic pigs that correlate significantly with β-cell mass [41] . these metabolites represent possible biomarkers for the early stages of prediabetes. moreover, the porcine giprdn model has been used to test liraglutide, glp1 receptor agonist, which improve glycemic control in type 2 diabetic patients. ninety-day liraglutide treatment of adolescent transgenic pigs resulted in improved glycemic control and insulin sensitivity as well as reduction in body weight gain and food intake compared to placebo-treated animals. however, the use of liraglutide did not stimulate beta-cell proliferation in the endocrine pancreas [42] . another type of diabetes, type 3 dm, is maturity-onset diabetes of the young (mody3). mody3 is a noninsulin-dependent type of diabetes with an autosomal dominant inheritance and is caused by mutations in the human hepatocyte nuclear factor 1 α (hnf1α) gene. mutation in hnf1α gene leads to pancreatic β-cell dysfunction and impaired insulin secretion. a pig model for mody3 was generated by expressing a mutant human hnf1α gene (hnf1α p291fsinsc) using intracytoplasmic sperm injection-mediated gene transfer and somatic cell nuclear transfer. the transgenic piglets exhibited the pathophysiological characteristics of diabetes, including high glucose level and reduced insulin secretion from the small and irregularly formed langerhans islets [43] . furthermore, hnf1α p291fsinsc pigs revealed nodular lesions in the renal glomeruli, diabetic retinopathy, and cataract, complications similar to those in patients with dm [44] . mutations in the insulin (ins) gene may result in permanent neonatal diabetes mellitus (pndm) in humans. a pndm large animal model was establish by generated pigs expressing a mutant porcine ins gene (ins c94y), orthologous to human ins c96y. transgenic animals showed signs of pndm, such as lower fasting insulin levels, decreased β-cell mass, reduced body weight, and cataract development. in addition, ins c94y pigs exhibited significant β-cell impairment, including the reduction in insulin secretory granules and dilation of the endoplasmic reticulum [45] . the porcine ins c94y model was further used to perform analysis of pathological changes in retinas and evaluation of the liver of transgenic pigs. the studies revealed several features of diabetic retinopathy, such as intraretinal microvascular abnormalities or central retinal edema [46] . moreover, the multi-omics analysis of the liver demonstrated higher activities in amino acid metabolism, oxidation of fatty acids, gluconeogenesis, and ketogenesis, characteristic of insulin-deficient diabetes mellitus [47] . the genetically modified pig models for human diseases described in this review are summarized in table 2 . human-derived proteins have long been used as therapeutics in the treatment of numerous diseases. however, their quantities are limited by the availability of human tissues. thanks to the development of biotechnology and genetic engineering, modified animals can be used as "bioreactors" to produce recombinant proteins for pharmaceutical use. by using adequate regulatory sequences, promoters, the expression of transgenes can be directed to selected cells and organs. the therapeutic proteins can be obtained from milk, blood, urine, seminal plasma, egg white, or salivary gland that can be collected, purified, and used at an industrial scale. moreover, it is possible to generate multi-transgenic animals that produce many biopharmaceuticals or vaccines in a single organism. the use of an animal platform allows for the relatively low-cost production of pharmacologically valuable preparations in high quantity and quality. the mammary gland is considered to be an excellent bioreactor system for pharmaceutical protein production. the advantage of milk is that it contains large amounts of foreign proteins that do not affect the animal's health during lactation as well as the ease of product collection and purification. while cows are the best species for obtaining large amounts of pharmaceuticals in milk, the cost and time necessary to carry out successful transgenesis make rabbits, sheep, goats, and pigs more popular species. although the pig is not a typical dairy animal, a lactating sow can give about 300 l of milk per year. velander et al. generated transgenic pigs that synthesized human protein c in the mammary gland. protein c plays an important role in human blood clotting, which makes it a potentially attractive drug. the collected milk contained 1 g/l of this protein [48] . other recombinant human proteins involved in the coagulation process, such as factor viii [49] , factor ix [50, 51] , von willebrand factor [52] , were also successfully obtained in the porcine mammary gland. furthermore, the line of transgenic pigs producing functional recombinant human erythropoietin in their milk was demonstrated. erythropoietin regulates red blood cell production (erythropoiesis) in the bone marrow by binding to a specific membrane receptor and has been used in the treatment of anemia. this bioreactor system generates active recombinant human erythropoietin at concentrations of approximately 877.9 ± 92.8 iu/1 ml [53] . in turn, lu et al. generated transgenic cloned pigs expressing large quantities of recombinant human lysozyme in milk. lysozyme is a natural broad-spectrum antimicrobial enzyme which constitutes part of the innate immune system. the authors demonstrated that the highest concentration of recombinant human lysozyme with in vitro bioactivity was 2759.6 ± 265.0 mg/l [54] . biopharmaceuticals can also be synthesized in pigs with the use of alternative systems, such as blood, urine, and semen. the blood of transgenic animals can be a source of human blood proteins, such as hemoglobin. swanson et al. and sharma et al. obtained transgenic pigs that produced recombinant human hemoglobin in their blood cells at a high level, with the ability to bind oxygen identical to that of human blood hemoglobin [55, 56] . there remains, however, the issue of obtaining large amounts of animal-generated therapeutics easily and inexpensively, without killing the animal. moreover, blood cannot store high levels of recombinant proteins for a long time, which are innately unstable, and bioactive proteins in the blood may affect the metabolism of the animals [57] . for this reason, research is being conducted into the production of recombinant proteins secreted into the urine or semen. the advantage of semen is that it is easily obtained and produced in high amounts in species such as pigs (boars can produce 200-300 ml of semen 2-3 times a week), while the advantage of urine is that proteins can be obtained from animals of both sexes throughout their lives. in addition, urine contains few proteins, which facilitates the purification of the protein product, and the urine-based systems pose a low risk to the animal's health. however, the limitation of protein production in the bladder is low yield [58] . the recombinant pharmaceutical proteins produced from transgenic pigs are listed in table 3 . genetically modified pigs can also be used as a source of cells, tissues, and organs for transplantation into human recipients. despite the growing knowledge and ability to perform transplants, the shortage of organs means that the number of patients awaiting a transplant is constantly increasing. xenotransplantation is any procedure involving the transplantation, implantation, or infusion of cells, tissues or animal donor organs, and also body fluids, cells, tissues, and human organs (or their fragments), which had ex vivo contact with animal cells, tissues, or organs into a human recipient. organ xenotransplantation would give us an unlimited and predictable source of organs and enable careful planning of the surgery and preoperative drug treatment of the donor. the animal that best meets the criteria for xenotransplantation is the domestic pig (sus scrofa domestica). pig and human organs show great anatomical and physiological similarities. however, the significant phylogenetic distance results in serious immunological problems after transplantation. despite major difficulties, the pig is currently the focus of all research aimed towards eliminating the problem of organ shortage for human transplantation in the future. thus, the challenge now is to overcome interspecies differences that cause xenograft rejection by the human immune system. the solution, therefore, is to modify pigs in such a way that their organs are not rejected as belonging to another species. advances in genetic engineering have brought scientists closer to obtaining modified animals that would be useful for pig to human transplants. a number of studies have reached the preclinical stage, using primates as model organisms. pig organs transplanted into human recipients are immediately rejected as a result of the so-called hyperacute immunological reaction. xenograft rejection is mainly caused by the gal antigen found on the donor's cell surface, which is synthesized by the ggta-1 enzyme. humans lack both the gal antigen and the ggta-1 enzyme, but have xenoreactive antibodies directed against the porcine gal antigen, which leads to the so-called enzymatic complement cascade in the recipient. the sequence of reactions results in a formation membrane attack complex, lysis, and destruction of the graft cells. the best possible solution to the problem of hyperacute rejection is to inactivate the gene encoding the ggta-1 enzyme responsible for the formation of the gal antigen. in 2001, the first heterozygous ggta1 knock-out pigs were produced [59] , and one year later, the first piglets with two knock-out alleles of the ggta1 gene were born [60] . a series of ggta1 knock-out pigs has also been generated by using zfns [61] , talens [62] , and the crispr/cas9 system [63] . moreover, other carbohydrate xenoantigens present on pig cells but absent in humans have been identified and include neu5gc antigen (n-glycolylneuraminic acid) catalyzed by cytidine monophosphate-n-acetylneuraminic acid hydroxylase (cmah) and the sda antigen produced by beta-1,4-n-acetyl-galactosaminyltransferase 2 (β4galnt2). pigs with ggta1/cmah/β4galnt2 triple gene knock-out were generated using the crispr/cas9 system. cells from these genetically modified animals exhibited a reduced level of human igm and igg binding resulting in diminished porcine xenoantigenicity [64] . to prevent hyperacute rejection, it is possible to introduce human genes regulating the enzymatic complement cascade into the porcine genome. as the complement system may undergo spontaneous autoactivation and attack the body's own cells, defense mechanisms have developed in the course of evolution. they regulate complement activity through a family of structurally and functionally similar proteins blocking complement activation and preventing the formation of a membrane attack complex (mac). introduction of human genes encoding complement inhibitors, such as cd55 (daf, decay-accelerating factor), cd46 (mcp, membrane cofactor protein), cd59 (membrane inhibitor of reactive lysis), into the porcine genome may overcome xenogeneic hyperacute organ rejection [65] . it was demonstrated that the expression of human complement-regulatory proteins can prevent complement-mediated xenograft injury and prolong the survival time of the xenotransplant [66] [67] [68] . studies have shown that the absence of ggta1 and additional human cd55, cd59, or cd46 expression has greater survival rates than just ggta1 knock-out [69, 70] . many genetically modified pigs with human complement inhibitors and other modifications important for xenotransplantation were also generated [71] [72] [73] . the modifications of the porcine genome described above largely resolved the problem of hyperacute rejection. however, xenogenic transplant becomes subject to less severe rejection mechanisms resulting from coagulation dysregulation, natural killer (nk) cells-mediated cytotoxicity, macrophage-mediated cytotoxicity as well as t-cell response. the coagulative disorders result from incompatibilities between pig anticoagulants and human coagulation factors. overcoming coagulation dysregulation in xenotransplantation will require the introduction of human gene encoding coagulation-regulatory proteins into the porcine genome, for example, thrombomodulin (tbm), endothelial cell protein c receptor (epcr), tissue factor pathway inhibitor (tfpi), and ectonucleoside triphosphate diphosphohydrolase 1 (cd39). thrombomodulin binds thrombin and functions as a cofactor for the activation of protein c, which is strongly anticoagulative. porcine tbm binds human thrombin less strongly and cannot effectively activate protein c. it was demonstrated that expressing human tbm (htbm) in porcine aortic endothelial cells (paecs) suppresses prothrombinase activity and delays clotting time [71] . the endothelial protein c receptor enhances the activation of protein c and decreases proinflammatory cytokine synthesis. in vitro studies revealed the correlation between human epcr (hepcr) expression in paecs and reduced human platelet aggregation [74] . a meta-analysis of multiple genetic modifications on pig lung xenotransplant showed that hepcr was one of the modifications that had a positive effect on xenograft survival prolongation in the ex vivo organ perfusion model with human blood [75] . further study demonstrated that kidneys from genetically-engineered pigs (carrying six modifications) functioned in baboons for 237 and 260 days. the authors suggested that prolonged survival time was associated, among others, with the expression of the human epcr gene [76] . tissue factor pathway inhibitor is the primary physiological regulator of the early stage of coagulation. tfpi binds to factor xa, and then xa/tfpi inhibits the procoagulant activity of the tissue factor (tf)/factor viia complex. it was demonstrated that the expression of human tfpi in paecs can inhibit tf activity, suggesting potential for controlling the tf-dependent pathway of blood coagulation in xenotransplantation [77] . more recently, multi-modified pigs carrying human tfpi transgene were produced [76, 78] . cd39 is an ectoenzyme that plays a key role in reducing platelet activation. cd39 converts adenosine triphosphate (atp) and adenosine diphosphate (adp) to adenosine monophosphate (amp), which in turn is further degraded by ecto-5 -nucleotidase (cd73) to antithrombotic adenosine. transgenic pigs with human cd39 (hcd39) gene were generated. the study showed that hcd39 expression protects against myocardial injury in a model of myocardial acute ischemia-reperfusion injury [79] . another approach to xenograft protection may be introducing a human gene that protects against the inflammatory response into the porcine genome. transgenic pigs expressing antiapoptotic and anti-inflammatory proteins, such as human heme oxygenase-1 (ho-1) and human tumor necrosis factor-alpha-induced protein 3 (a20), were produced [80, 81] . porcine aortic endothelial cells derived from pigs carrying human a20 transgene were protected against tnf-α (tumor necrosis factor alpha)-mediated apoptosis and less susceptible to cell death induced by cd95 (fas) ligands [81] . similarly, overexpression of human ho-1 ensured prolonged porcine kidney survival in an ex vivo perfusion model with human blood and paecs protection from tnf-α-mediated apoptosis [80] . furthermore, pigs with a combined expression of human a20 and ho-1 on a ggta1 knock-out background were generated. that transgenic approach alleviated rejection and ischemia-reperfusion damage during ex vivo kidney perfusion [82] . the cellular immune response is another barrier to xenotransplantation. human nk cells can activate the endothelium and lyse porcine cells through direct nk cytotoxicity and by antibody-dependent cellular mechanisms. direct nk cytotoxicity is regulated by activating and inhibitory receptor-ligand interactions. to prevent nk-mediated lysis through the inhibitory cd94/nkg2a receptor, pigs with human leukocyte antigens-e (hla-e) were obtained [83, 84] . the study showed that the expression of hla-e in endothelial cells from transgenic pigs markedly reduces xenogeneic human nk responses. in addition, it was demonstrated that the introduction of the hla-e gene into the porcine genome may also protect pig cells from macrophage-mediated cytotoxicity [85] . more recently, ggta1 knock-out pigs with hcd46 and hla-e/human β2-microglobulin transgenes were produced. the study showed that multiple genetically modified porcine hearts were protected from complement activation and myocardial natural killer cell infiltration in an ex vivo perfusion model with human blood [86] . another approach to inhibit direct xenogeneic nk cytotoxicity is the elimination of porcine ul-16-binding protein 1 (ulbp1), which binds to nkg2d activating nk receptors. crispr technology was adapted to create genetically modified pigs with a disrupted ul16-binding protein 1 gene. in vitro studies confirmed that porcine aortic endothelial cells derived from ulbp1 knock-out pigs were less susceptible to nk-cells' cytotoxic effects [87] . macrophages also play an important role in xenograft rejection and can be activated by direct interactions between receptors present on their surface and donor endothelial antigens as well as by xenoreactive t lymphocytes. the binding cd47 antigen to macrophage surface signaling regulatory protein (sirp-α) delivers a signal to prevent phagocytosis. however, the interaction between porcine cd47 and human sirp-α does not supply the inhibitory effect on macrophages [88] . therefore, the introduction of human cd47 (hcd47) into the porcine genome can overcome macrophage-mediated responses in xenotransplantation. the overexpression of hcd47 in porcine endothelial cells suppressed the phagocytic and cytotoxic activity of macrophages, decreased inflammatory cytokine (tnf-α, il-6, il-1β) secretion and inhibited the infiltration of human t cells [89] . the pigs with ggta1 knock-out and hcd47 were obtained [90] . it was demonstrated that the expression of human cd47 markedly prolonged survival of donor porcine skin xenografts on baboons in the absence of immunosuppression [91] . another challenge in xenotransplantation is the prevention of t cell-mediated rejection. t cells can be induced directly by swine leukocyte antigen (sla) class i and class ii on porcine antigen-presenting cells (apcs) or by swine donor peptides presented on recipient apcs. the main co-stimulatory signals regulating t cell function include cd40-cd154 and cd28-cd80/86 pathways. the cytotoxic t-lymphocyte antigen 4-immunoglobulin (ctla4) can inhibit the cd28-cd80/86 co-stimulatory pathway. therefore, the introduction of human ctla4-ig (hctla4-ig) into the porcine genome may alleviate t cell response in xenografts. it was shown that neuronal expression of hctla4-ig in pigs reduced human t lymphocyte proliferation [92] . moreover, transgenic hctla4-ig protein in pigs extended the survival time of porcine skin grafts in a xenogeneic rat transplantation model [93] . another approach to inhibit t-cell immune response may be the deletion of swine leukocyte antigen class i. reyes et al. created sla class i knock-out pigs using grna and the cas9 endonuclease. the obtained animals revealed decreased levels of cd4−cd8+ t cells in peripheral blood [94] . recently, pigs carrying functional knock-outs of ggta1, cmah, b4galnt2, and sla class i with multi-transgenic background (hcd46, hcd55, hcd59, hho1, ha20) were produced. in vitro study presented that the four-fold knock-out reduced the binding of human igg and igm to porcine kidney cells [95] . beyond immune barriers in xenotransplantation, there is also concern about the risk of cross-species pathogens infection. the main problem constitutes porcine endogenous retroviruses (perv), which are integrated into multiple locations in the pig genome. utilizing the crispr/cas9 technology gives great hopes for the complete elimination of the risk of perv transmission. niu et al. using the crispr/cas9 system inactivated all 25 copies of functional pervs in a porcine primary cell line and successfully generated healthy perv-inactivated pigs via somatic cell nuclear transfer. what is more, no reinfection was observed in the obtained pigs [96] . advances in genetic engineering and immunosuppressive therapies prolong organ survival time in preclinical pig-to-non-human primate (nhp) xenotransplantation models. the first xenotransplantation using pig hearts with eliminated gal antigen into immunosuppressed baboons was performed in 2005. the longest surviving heterotopic graft functioned in the recipient for 179 days [97] , in comparison to 4-6 hours of survival time with the use of wild-type pig hearts [98] . introducing additional modifications extended the xenograft survival time even more. the longest survival was obtained for heterotopic cardiac xenotransplantation-up to 945 days. the authors used hearts derived from genetically multimodified pigs (ggta1 knock-out, hcd46, htbm) and chimeric 2c10r4 anti-cd40 antibody therapy [99] . additional expression of htbm in ggta1 knock-out, hcd46 genetically modified pigs prevented early dysregulation of coagulation and prolonged the cardiac xenografts survival time [99, 100] . using the same genetic background, orthotopic heart xenotransplantation was performed, resulting in a maximum survival of 195 days [101] . however, xenograft survival time depends on the types of transplanted organs. in the case of kidneys in pig-to-nhp transplantation models, the longest survival of a life-sustaining xenograft was 499 days. ggta1 knock-out pigs carrying hcd55 gene as well as immunosuppression with transient pan-t cell depletion and an anti-cd154-based regimen were used in the experiments. moreover, the selection of recipients with low-titer anti-pig antibodies improved the long-term survival of pig-to-rhesus macaque renal xenotransplants [102] . the success of porcine liver and lung xenotransplantation remains limited, which is mainly associated with the occurrence of coagulation disorders [103] . the longest survival time for orthotopic liver xenografts (29 days) was achieved using ggta1 knock-out pigs, exogenous human coagulation factors, and immunosuppression, including co-stimulation blockade [104] . in turn, watanabe et al. demonstrated prolonged survival time of lung xenotransplants (14 days) from ggta1 knock-out, hcd47/hcd55 donor pigs in immunosuppressed baboons [105] . the authors indicated the important role of hcd47 expression in reducing immunologic damages and extending lung graft survival in the pig-to-nhp model. however, additional genetic modifications of the porcine genome and immunosuppressive regimen strategy are necessary for the clinical application of xenotransplantation. table 4 summarizes the most important genetic modifications of the porcine genome for xenotransplantation purposes. the anatomical and physiological similarity between pigs and humans makes this species very interesting for biomedical research [110] . the rapid development of genetic engineering in recent years has allowed for precise and efficient modification of the animal genome using site-specific nucleases. the nuclease-mediated editing of the porcine genome, as well as potential applications of genetically modified pigs in biomedicine, are shown in figure 1 . certainly, the driving force for development is the human mind and ideas that arise in it. one of the factors limiting the possibilities of using the potential of our ideas is the technical aspect. the transfer of new technologies, tested on smaller animal models, for example, is often limited and requires optimization for a large animal model. in the case of crispr/cas9 technology, a lot of emphasis should be placed on the possibility of reducing the risk of so-called off-targets by improving this system. paired nicking has the potential to reduce off-target activity in mice from 50-1000 times without compromising on-target performance [111] . another strategy to limit the number of undesirable off-targets is to increase the specificity of the system. in this situation, one can focus on enhancing or improving cas9 protein or sgrna modifications. protein cas9 properties can be modified, or their lifespan can be changed [112, 113] . for the future clinical success, it is also important to improve the efficiency of hr-mediated gene correction, especially in the situation of treating disease in which a template sequence is delivered to replace the mutated variant. another important goal to achieve is the possibility of applying hr not only for dividing cells but also for cells in the post-mitotic stage. hopes are placed in the fusion of the crispr/cas9 technique and aav (adeno-associated virus) as a donor template provider [114] . considering the low immunogenicity of the aav virus, the ability to transduce a wide spectrum of cells in terms of both type and developmental stage and strong limiting factor-capacity, it should be considered to minimize the crispr/cas9 system or use more than one separate virus to simultaneously exploit the potential of both technologies. the use of other delivery systems, e.g., nanoparticles, is also worth considering [115] . the different site-specific nucleases (zfn, talen, crispr/cas9) used for genome editing and two techniques (somatic nuclear transfer and microinjection) to produce genetically modified pigs are shown. biomedical applications for which genetically engineered pigs are generated include modeling human diseases, production of pharmaceutical proteins, and xenotransplantation. genetically modified pigs serve as an important large animal model for studying the genetic background of human diseases, testing novel drugs and therapy methods as well as developing models for gene therapy [116] [117] [118] . pigs can be used as anatomical (e.g., endovascular), surgical, behavioral, and cytotoxic models. ideas for new models of large animals are provided by the reality that shapes current demand. a lot has been done (pig model for influenza a infection), but still there is a need for pig models of other human viral diseases (hepatitis b; human immunodeficiency virus, hiv; severe acute respiratory syndrome coronavirus 2, sars-cov-2) [119] . hiv has been modeled in mice, filoviruses (ebolavirus, marburgvirus) have been modeled in small animals (i.e., mice, hamsters), but still, we need large models to investigate vaccines and antiviral drugs [120, 121] . transgenic pigs can also be a promising source of recombinant proteins used as pharmacological preparations. actually, the possibility of using pigs for the production of biopharmaceuticals has been slowed in recent years. some studies demonstrated that the pig mammary gland can be used as a complex recombinant protein source with appropriate post-translational modifications [122] . despite the advantages of pig animal platform (natural secretion, correct posttranslational modifications, constant production), some ethical doubts are probably limiting the boost. finally, the use of genetically engineered pigs for xenotransplantation is becoming an increasingly feasible alternative to standard allogeneic transplants and a potential solution to the problem of organ shortage. the combination of various multi-modified pigs and immunosuppressive therapies is required for overcoming immune rejection and effective xenotransplantation of different solid organs [123] [124] [125] . when it comes to treating end-stage organ failure, biomedical research could go a step further and try to create chimeric genetically modified pigs that would be carriers of human organs [126] . the publication was co-financed within the framework of a ministry of science and higher education program as "regional initiative excellence" in the years 2019-2022, project no. 005/rid/2018/19. the authors declare no conflict of interest. genetically engineered pigs as models for human disease trends in recombinant protein use in animal production. microb. cell fact genetically modified pigs as organ donors for xenotransplantation genetic transformation of mouse embryos by microinjection of purified dna dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes pronuclear microinjection transgenic technology in farm animals-progress and perspectives increased efficiency of transgenic livestock production increased transgene integration efficiency upon microinjection of dna into both pronuclei of rabbit embryos a simple and highly efficient transgenesis method in mice with the tol2 transposon system and cytoplasmic microinjection efficient generation of gene-modified pigs via injection of zygote with cas9/sgrna sheep cloned by nuclear transfer from a cultured cell line a background to nuclear transfer and its applications in agriculture and human therapeutic medicine disruption of the cftr gene produces a model of cystic fibrosis in newborn pigs the deltaf508 mutation causes cftr misprocessing and cystic fibrosis-like disease in pigs sequential targeting of cftr by bac vectors generates a novel pig model of cystic fibrosis lentiviral-mediated phenotypic correction of cystic fibrosis pigs cftr gene transfer with aav improves early cystic fibrosis pig phenotypes efficient gene editing at major cftr mutation loci in vitro validation of a crispr-mediated cftr correction strategy for preclinical translation in pigs dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle progressive muscle proteome changes in a clinically relevant pig model of duchenne muscular dystrophy porcine zygote injection with cas9/sgrna results in dmd-modified pig with muscle dystrophy somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of duchenne muscular dystrophy the role of cell-derived oligomers of abeta in alzheimer's disease and avenues for therapeutic intervention hemizygous minipigs produced by random gene insertion and handmade cloning express the alzheimer's disease-causing dominant mutation appsw generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (rmce) and somatic cell nuclear transfer (scnt) expression of the alzheimer's disease mutations aβpp695sw and psen1m146i in double-transgenic göttingen minipigs production of transgenic pig as an alzheimer's disease model using a multi-cistronic vector system genetic induction of tumorigenesis in swine a genetic porcine model of cancer a porcine model of osteosarcoma inactivation and inducible oncogenic mutation of p53 in gene targeted pigs development and translational imaging of a tp53 porcine tumorigenesis model a porcine model of familial adenomatous polyposis targeted disruption of ldlr causes hypercholesterolemia and atherosclerosis in yucatan miniature pigs bempedoic acid lowers low-density lipoprotein cholesterol and attenuates atherosclerosis in low-density lipoprotein receptor-deficient (ldlr+/− and ldlr−/−) yucatan miniature pigs familial hypercholesterolemia and atherosclerosis in cloned minipigs created by dna transposition of a human pcsk9 gain-of-function mutant characterization of a hypertriglyceridemic transgenic miniature pig model expressing human apolipoprotein ciii glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function changing metabolic signatures of amino acids and lipids during the prediabetic period in a pig model with impaired incretin function and reduced β-cell mass effects of the glucagon-like peptide-1 receptor agonist liraglutide in juvenile transgenic pigs modeling a pre-diabetic condition dominant-negative mutant hepatocyte nuclear factor 1α induces diabetes in transgenic-cloned pigs diabetic phenotype of transgenic pigs introduced by dominant-negative mutant hepatocyte nuclear factor 1α permanent neonatal diabetes in ins(c94y) transgenic pigs retinopathy with central oedema in an ins c94y transgenic pig model of long-term diabetes multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus high-level expression of a heterologous protein in the milk of transgenic swine using the cdna encoding human protein c transgenic pigs produce functional human factor viii in milk recombinant human factor ix produced from transgenic porcine milk engineering protein processing of the mammary gland to produce abundant hemophilia b therapy in milk production of recombinant human von willebrand factor in the milk of transgenic pigs recombinant human erythropoietin produced in milk of transgenic pigs production of transgenic-cloned pigs expressing large quantities of recombinant human lysozyme in milk production of functional human hemoglobin in transgenic swine an isologous porcine promoter permits high level expression of human hemoglobin in transgenic swine production of pharmaceutical proteins by transgenic animals expression systems and species used for transgenic animal bioreactors production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning production of alpha 1,3-galactosyltransferase-deficient pigs production of zfn-mediated ggta1 knock-out pigs by microinjection of gene constructs into pronuclei of zygotes production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology generation of ggta1 mutant pigs by direct pronuclear microinjection of crispr/cas9 plasmid vectors evaluation of human and non-human primate antibody binding to pig cells lacking ggta1/cmah/β4galnt2 genes expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection cardiac xenotransplantation: recent preclinical progress with 3-month median survival cytomegalovirus early promoter induced expression of hcd59 in porcine organs provides protection against hyperacute rejection long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts generation of gtko diannan miniature pig expressing human complementary regulator proteins hcd55 and hcd59 via t2a peptide-based bicistronic vectors and scnt b-cell depletion extends the survival of gtko.hcd46tg pig heart xenografts in baboons for up to 8 months potential value of human thrombomodulin and daf expression for coagulation control in pig-to-human xenotransplantation complement dependent early immunological responses during ex vivo xenoperfusion of hcd46/hla-e double transgenic pig forelimbs with human blood production of ulbp1-ko pigs with human cd55 expression using crispr technology regulation of human platelet aggregation by genetically modified pig endothelial cells and thrombin inhibition meta-analysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts atorvastatin or transgenic expression of tfpi inhibits coagulation initiated by anti-nongal igg binding to porcine aortic endothelial cells generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes transgenic swine: expression of human cd39 protects against myocardial injury transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys transgenic expression of the human a20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli kidneys from α1,3-galactosyltransferase knockout/human heme oxygenase-1/human a20 transgenic pigs are protected from rejection during ex vivo perfusion with human blood hla-e/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity characterization of three generations of transgenic pigs expressing the hla-e gene the suppression of inflammatory macrophage-mediated cytotoxicity and proinflammatory cytokine production by transgenic expression of hla-e multiple genetically modified gtko/hcd46/hla-e/hβ2-mg porcine hearts are protected from complement activation and natural killer cell infiltration during ex vivo perfusion with human blood the production of ul16-binding protein 1 targeted pigs using crispr technology role for cd47-sirpalpha signaling in xenograft rejection by macrophages role of human cd200 overexpression in pig-to-human xenogeneic immune response compared with human cd47 overexpression transgenic expression of human cd47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation prolonged survival of pig skin on baboons after administration of pig cells expressing human cd47 transgenic expression of ctla4-ig by fetal pig neurons for xenotransplantation transgenic expression of human cytoxic t-lymphocyte associated antigen4-immunoglobulin (hctla4ig) by porcine skin for xenogeneic skin grafting creating class i mhc-null pigs using guide rna and the cas9 endonuclease viable pigs after simultaneous inactivation of porcine mhc class i and three xenoreactive antigen genes ggta1, cmah and b4galnt2 inactivation of porcine endogenous retrovirus in pigs using crispr-cas9 galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching 6 months intravenous infusion of galα-3gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts chimeric 2c10r4 anti-cd40 antibody therapy is critical for long-term survival of gtko.hcd46.htbm pig-to-primate cardiac xenograft cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs consistent success in life-supporting porcine cardiac xenotransplantation long-term survival of pig-to-rhesus macaque renal xenografts is dependent on cd4 t cell depletion overcoming coagulation dysregulation in pig solid organ transplantation in nonhuman primates: recent progress prolonged survival following pig-to-primate liver xenotransplantation utilizing exogenous coagulation factors and costimulation blockade intra-bone bone marrow transplantation from hcd47 transgenic pigs to baboons prolongs chimerism to >60 days and promotes increased porcine lung transplant survival production of biallelic cmp-neu5ac hydroxylase knock-out pigs the generation of transgenic pigs as potential organ donors for humans a human cd46 transgenic pig model system for the study of discordant xenotransplantation pigs transgenic for human thrombomodulin have elevated production of activated protein c transgenic pigs as models for translational biomedical research double nicking by rna-guided crispr cas9 for enhanced genome editing specificity high-fidelity crispr-cas9 nucleases with no detectable genome-wide off-target effects enhanced homology-directed human genome engineering by controlled timing of crispr/cas9 delivery virus-mediated genome editing via homology-directed repair in mitotic and postmitotic cells in mammalian brain improved delivery of crispr/cas9 system using magnetic nanoparticles into porcine fibroblast current progress of genetically engineered pig models for biomedical research genome editing of pigs for agriculture and biomedicine genetically engineered pigs to study cancer the microminipig as an animal model for influenza a virus infection in vitro and in vivo models of hiv latency animal models for filovirus infections production of recombinant human coagulation factor ix by transgenic pig the role of genetically engineered pigs in xenotransplantation research genetically modified pigs as donors of cells, tissues, and organs for xenotransplantation xenotransplantation: current status in preclinical research interspecies chimerism with mammalian pluripotent stem cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-299733-4mpz5l9e authors: mitchell, william m.; nicodemus, christopher f.; carter, william a.; horvath, joseph c.; strayer, david r. title: discordant biological and toxicological species responses to tlr3 activation date: 2014-04-30 journal: the american journal of pathology doi: 10.1016/j.ajpath.2013.12.006 sha: doc_id: 299733 cord_uid: 4mpz5l9e toll-like receptors (tlrs) are highly conserved type 1 membrane proteins that initiate a multiplicity of transient gene transcriptions, resulting in innate and adaptive immune responses. these essential immune responses are triggered by common tlr pattern recognition receptors of microbial products expressed through the cytoplasmic carboxy-terminal toll/il-1 domain. toll/il-1 adapter protein cascades are induced by an activated toll/il-1 to induce transient transcription responses. all tlrs, with the exception of tlr3, use an myd88 adapter to toll/il-1 to initiate a proinflammatory cascade. tlr3 uses the toll receptor 3/4 induction factor adapter to initiate a different cytosolic adapter cascade with double-stranded rna agonists. this non-myd88 pathway induces both nf-κb and type 1 interferon responses. by using a tlr3-restricted double-stranded rna agonist, rintatolimod, we demonstrate significant unexpected differences in toxic responses between rats and primates. the mechanism of this differential response is consistent with a relative down-regulation of the nf-κb inflammatory cytokine induction pathway in the cynomolgus monkey and humans, but not observed systemically in rat. our findings suggest evaluation of tlr3 agonists in drug therapy. animal modeling is a time-tested standard for the development of novel therapeutics. 1 the potential of such models to adequately translate findings for use in therapeutic development, however, has recently been questioned. a report by seok et al 2 found a poor correlation between human and murine response to inflammatory stimuli, including responses to trauma, burn, and endotoxemia. discordant results at the level of gene expression array, temporal gene response patterns, and major signaling pathways, in response to these injuries, were demonstrated across species, but well conserved in humans, pointing toward inadequate and nonpredictive nature of available inflammation murine models. this may further explain the difficulty in translating promising pharmacological compounds into successful novel therapies. despite the heterogeneity of the patients and the causes of life-threatening trauma, the report found highly correlated genomic response profiles across the patient population, but not across species. burn, trauma, and endotoxemia yielded highly correlated genomic responses within species, but not across species, rodent to human. we have observed a similar dissociation of toxicities in a parallel proinflammatory system, the toll-like receptor 3 (tlr3) induction of innate immune responses. the tlrs form a family of class 1 transmembrane receptors originally discovered in drosophila, where they are essential elements in embryogenesis and an evolutionary ancient system of immune response to pathogen-associated molecular patterns (pamps). 3 they were recognized further in plants, 4 fish, 5, 6 and mice, 7 as well as primates. 7 tlrs act as a first line of defense against microbial pathogens by the induction of innate immunity and further provide the initial cellular orchestration for the induction of adaptive immune responses to provide specific humoral and supported by hemispherx biopharma, inc. disclosures: w.m.m. is an independent member of the board of directors and a shareholder of hemispherx biopharma, inc., c.f.n. is a consultant for hemispherx biopharma, inc., w.a.c. is chief executive officer and member of the board of directors and shareholder of hemispherx biopharma, inc., j.c.h. is an employee of hemispherx biopharma, inc., and d.r.s. is the medical director and a shareholder of hemispherx biopharma, inc. rinitatolimod is an experimental drug whose clinical development and manufacture is funded by hemispherx biopharma, inc., under the trade name ampligen. cell-mediated immunity, mediated in part by inflammatory cytokines. 8 they can be found especially in mature dendritic cells (dcs), central in the host adaptive immune response system. 9 all of the tlrs use an myd88-dependent signaling pathway, with the exception of tlr3, which uses the myd88-independent trif pathway. 10 two other doublestranded rna (dsrna) inducers of gene expression that initiate innate immune responses are the cytosolic helicases, mda5 and retinoic acid inducible gene 1 protein (rig-1), which are myd88 dependent. 11 the pamp for tlr3 is dsrna detected in endosomes of antigen-presenting cells and on the cell surface of selected cells, including endothelial cells and airway epithelium. 12e14 the expression pattern is consistent with sentinel activity for the detection of replicating virus in the host organism. the cellular location of tlr3 is modulated by unc93b1, which promotes trafficking of differentially glycosylated tlr3 to the plasma membrane; unc93b1 transcription is up-regulated by dsrna. 15 to address the observed toxic heterogeneity of response to pamps across species, we have used a restricted tlr3specific agonist, with much data reported in double-blind clinical trials 16, 17 and unpublished open-label safety trials. rintatolimod (poly i:poly c 12 u; ampligen) is a synthetic dsrna analogue composed of a single polypurine (inosine) strand and a single polypyrimidine (cytosine and uridine) strand. these are assembled in a dsrna structure that is maintained under physiological conditions by typical hydrogen bonding between purine and pyrimidine base pairs. the introduction of the pyrimidine base, uridine, at a 1:12 ratio into the polypyrimidine strand maintains the double-stranded structure but allows recurring sites of thermodynamic instability from nonhydrogen bonding of the mismatched base (uridine) with inosine of the polypurine strand. 18 this small difference in structure, however, restricts induction of transitory gene activities to tlr3, with its unique trif signaling pathways, with the exclusion of the mda5 and rig-1 dsrna proinflammatory dependence 19, 20 (figure 1 ). by using the restricted specificity of rintatolimod and the unique trif pathway of tlr3 signaling, we have been able to identify the apparent mechanism for the discordance in tlr3-mediated toxicity between rodents and primates. patient serum samples from two clinical trials being conducted according to good clinical practice standards, under figure 1 myd88-independent and myd88-dependent signaling pathways for the tlrs and helicases. a: the intracellular pathways for myd88-independent tlr3 nuclear signal transduction initiated by trif binding to the tir of the tlr3 homodimer. tlr3 monomers dimerize with binding of the dsrna ligand. activated trif initiates two pathways. the first results in the transitory induction of the ifns. the second is a species-variable pathway (rodents >> primates) that operates through nf-kb (dashed line) that transiently induces the production of inflammatory cytokines. adapter protein cascade initiated by trif, tbk1, traf1/3, nap1, ikk, ikkε, p13k, irf-3/7, tak1, tab1, and rip1. the ectodomain of tlr3 consists of a horseshoe-shaped structure populated by 23 leucine-rich b-sheets (orange disks) connected by nonordered chains containing rna-binding residues. the transmembrane a-helices (solid orange) connect the ectodomain to the cytoplasmic tir domain (dark green). the phosphorylated tir binds trif to initiate the adapter protein cascade. b: the intracellular pathways for myd88-dependent signaling for tlr 1/2 and 1/6 heterodimers and tlr 4 to 10 homodimers. the diverse pamp ligands (green bar) are not necessarily accurate in placement, as is the dsrna ligand with tlr3 in a. tlr4 uses both the myd88-dependent and myd88-independent pathways. trif-tir domainecontaining adapter-inducing ifn, tbk1-tankebinding kinase 1, traf-tnf receptoreassociated factor, nap1-nckeassociated protein 1, ikk-ikb kinase, ikkε inhibitor of ikb kinase, p13k-phosphoinositide 3-kinase, irf-ifn regulatory transcription factor, tab1-tgf-beactivated kinase 1, and rip1 receptoreinteracting (tnfrsf) kinase 1. the american journal of pathologyajp.amjpathol.org the rintatolimod investigational new drug no. 39,250, were analyzed for g-interferon (ifn), tumor necrosis factor (tnf)-a, il-12p70, and il-10 levels to assess acute and chronic changes that might be associated with rintatolimod dosing. all human protocols were conducted with institutional review board review and approval and patient informed consent. study subjects with a diagnosis of chronic fatigue syndrome qualified for participation if they met the protocol entrance criteria. patients received rintatolimod infusions at 400 mg twice weekly. frozen serum samples from 14 randomly selected patients in hemispherx biopharma's open-label treatment protocol, amp-511, were obtained at pre-infusion, and again at 4, 24, and 72 hours after infusion. serum samples in 76 randomly selected patients from the double-blinded protocol, amp 516, were obtained at baseline pre-infusion and again at week 32 or at early termination if the patient terminated participation before week 32. serum obtained from clinical testing sites was frozen and shipped to the hemispherx biopharma, inc., testing and manufacturing facility (new brunswick, nj), where it was assayed using commercially available enzyme-linked immunosorbent assay kits (human ifn-g, human tnf-a, human il-10, and human il-12p70) (supplemental table s1 ). animal toxicological analyses were conducted, as required, under investigational new drug guidelines. studies were conducted according to good laboratory practice procedures at licensed animal pharmacology facilities, according to institutional animal care committee review and approval. dose ranging was conducted at dupont (wilmington, de) and bioresearch (senneville, qc, canada), and 6-month chronic toxicological studies were conducted at chrysalis (cedex, france) and pharamakon (waverly, pa). maximum tolerated doses (mtds) were derived from acute and subacute dose-ranging studies and were defined as the maximum dose that did not cause mortality or moribund toxicity. repeat dosing studies were conducted with sprague-dawley rats, beagle dogs, cynomolgus monkeys, and new zealand white rabbits at doses ranging from 0 to 300 mg/kg, i.v. administered daily for 7 to 10 days (rats and rabbits) or twice weekly (monkey and dog) for 4 and 9 weeks, respectively. clinical observations were recorded during infusions, for 3 hours after infusion, and otherwise twice daily. complete postmortem analysis was conducted, including gross and microscopic anatomical evaluations using standard animal pathological procedures. six-month chronic toxicological studies were conducted using twiceweekly doses of 0, 6, 18, and 36 mg/kg rintatolimod under similar procedures. assessment of acute inflammatory cytokines in response to rintatolimod administration was conducted in collaboration with the lovelace respiratory research institute (albuquerque, nm) and hemispherx biopharma, inc., under good laboratory practice using an approved animal welfare protocol. thirty-two sprague-dawley rats were infused twice weekly for 8 weeks at 0, 6, 18, or 36 mg/kg and sampled for cytokines at 0, 4, 24, and 72 hours after doses 1, 7, and 15, respectively. each time point after infusion required sacrifice of a male and a female rat. ten cynomolgus monkeys were dosed for 16 weeks, twice weekly, at the same dosage levels. serum samples for cytokine measurement were obtained after doses 1, 7, 15, and 30. all serum samples were frozen and assayed for the corresponding patient cytokines using commercially available enzyme-linked immunosorbent assay kits (supplemental table s1 ). all results reported are on the basis of manufacturer-supplied standards yielding linear-dose responses. adverse event data in rintatolimod-and placebo-treated patients in controlled studies of chronic fatigue syndrome were assessed in the context of toxicological findings. adverse events were collected during serial patient evaluations of study subjects participating in prospective 24-to 40week studies of 400 mg rintatolimod or placebo, infused twice weekly for the duration of the protocols. 16 the x-ray crystallographic structure of the mouse tlr3 dimer/biol-dsrna complex and the human tlr3 monomer provided the coordinates for the model of human tlr3, with rintatolimod bound to its active site. molecular modeling used accelrys' discovery suite software version 2.5.5 (accelrys, inc., san diego, ca). the dsrna structure of the dimer/biol-dsrna complex was mutated in situ to the respective poly i (blue) and poly c 12 u (magenta) chains maintaining the phosphate backbone linear translational coordinates of the x-ray crystallographic structure. the coordinates of the human tlr3 monomer were used to replace each homodimer. several unacceptable close van der waals contacts for the human tlr3 crystal coordinates were resolved by an alternate rotamer conformation of the individual amino acid r group. to demonstrate the relative equivalence of these two ligands as tlr3 agonists, we in silico measured the potential energies of the ligands, naked homodimers, and the tlr3-ligand complex. the reduction in potential energy of the complex versus the sum of the potential energy of the components is the energy of binding. primary protein sequences for the human (genbank u88879); monkey species, including macaca mulatta (gen-bank bag55034.1 and ay864735), macaca fasicularis ajp.amjpathol.org -the american journal of pathology (genbank bag55033.1), papio anubis (xp_003899477), callithrix jacchus (jab01765.1), and saimiri boliviensis (xp_003899477); and rodents, including the house mouse (genbank af355152/mus musculus) and rat (genbank ab116229/rattus norvegicus), dog (genbank xp_ 005630024/canis lupus familiaris), and rabbit (genbank abb76310/oryctolagus cuniculus) were aligned using crystal w software version 10.1.2 provided by dnastar (madison, wi). acute dose-ranging toxicological analysis was completed in the rabbit, dog, rat, and monkey ( table 1 ). the mtd, defined as the highest acute dosage not associated with induction of a moribund state or mortality, was observed over a range of two orders of magnitude. the rabbit proved most sensitive to rintatolimod, with an mtd of 1.25 mg/kg per dose. the dog and rat were more tolerant, with mtds of 10 and 12.5 mg/kg per dose, respectively. the cynomolgus monkey was substantially more tolerant of rintatolimod, with an mtd of 100 mg/kg per dose, resulting in a surprisingly 80-fold differential in acute toxicities, with the nonhuman primate least susceptible to rintatolimod toxicity. because of the clinical development of the rintatolimodenvisioned chronic systemic dosing of the tlr3 agonist over prolonged periods in the treatment of chronic fatigue syndrome and to meet regulatory requirements [guidance for industry: nonclinical safety evaluation of drug or biologic combinations; u.s. department of health and human services, food and drug administration, center for drug evaluation and research (cder); http://www.fda.gov/ohrms/dockets/ 98fr/05d-0004-gdl0002.pdf, last accessed february 3, 2014], 6-month chronic toxicological studies were conducted in the sprague-dawley rat and cynomolgus monkey. animals were dosed similar to the human schedule, with twice-weekly infusions of rintatolimod at 6, 18, or 36 mg/kg per dose, which were expected to be nonlethal on the basis of the acute toxicity studies. dose levels were determined by the human dose of 400 mg per infusion, which is approximately 6 mg/kg for an average-sized patient. results of these two chronic toxicological studies were consistent with the findings of the acute toxicological dosing (table 2) , although lethality in the rat during the 6-month course of the study was commonly observed. the rat was extremely sensitive in multiple organs to both the 18-and 36-mg dosing regimens, and many animals in these groups did not survive the 6-month dosing period. in the rat, dose-dependent liver and renal toxicities were observed in all three dosage groups. additional findings in the rat included anemia and polychromasia, increased alkaline phosphatase, and profound elevation of hepatic transaminases. histopathological features were consistent with clinical analytes. dosedependent toxicities were observed in all dose groups, with hepatocellular degeneration, bile duct hyperplasia, and interstitial nephritis with renal tubular dilation. lymphoid depletion and hyperpigmentation (probably hemosiderin) were noted in the spleen, and extramedullary hematopoiesis and occasional miscellaneous organ inflammation were also noted in all dose groups. in contrast, monkeys tolerated the 6-month course without significant toxicity, except for occasional vomiting associated with infusion in the 36 mg/kg group. there were transient elevations of hepatic transaminases only in the highdose group, associated with an unexplained decrease in alkaline phosphatase in the monkey. coagulation parameters were prolonged in the high-dose monkey group. there were no histopathological findings in the monkey, with the exception of increased myelopoiesis in the marrow and follicular thyroid hyperplasia in the 18 and 36 mg/kg dose groups. the thyroid morphological observation in the nonhuman primate was correlated with an increase in thyroid-stimulating hormone and thyroxine, suggesting a central effect on the pituitary causing thyroid hyperactivity and the observed hyperplasia. by using the clinical-grade formulation of poly i:poly c 12 u, rintatolimod (ampligen), we have also found discordance between rodent and human inflammatory responses. as demonstrated with the nonhuman primate model (cynomolgus monkey), humans similarly have minor toxicities compared with the rodent model (sprague-dawley rat). as in the models of inflammation explored by seok et al, 2 substantial between-species differences are apparent. in contrast to the rodent, in which sensitivity to endotoxin is magnitudes less than in the human, for the rintatolimodmediated tlr3 pathways, the rodent is highly sensitive, whereas primates are relatively tolerant. the data summarized in table 3 demonstrate only minimal associations between rodent toxicological findings and the human experience. a low incidence of transient liver the american journal of pathologyajp.amjpathol.org function test abnormalities is seen in the human (ie, easily manageable, with dose reduction from 400 to 200 mg). some patients experience rigors and flu-like symptoms in association with infusion but not the vomiting noted in the high-dose monkey infusion or the general toxicities observed in the rat. no thyroid abnormalities observed in the monkey toxicity trial have been detected in the human clinical data set. thus, the human experience is similar to the monkey, although a major finding of pituitary-dependent thyroid hyperactivity noted in the monkey was not seen in the human. both human and monkey differ substantially in pathological response compared with the rodent. the unexpected differential toxicities observed between the rat and a nonhuman primate prompted an examination of inflammatory cytokines (g-ifn, tnf-a, and il-12p70) associated with infusion of a tlr3 agonist, rintatolimod. preclinical studies in the rat and cynomolgus monkey assessed the induction of these three inflammatory cytokines, as well as il-10, measured in the serum in response to three dosage levels (6, 18, and 36 mg/kg) of rintatolimod administered by i.v. infusion. the 4-hour time point in response to initial infusion in rats gave the strongest systemic signals, which are illustrated in table 4 , with the expression pattern consistent, although less intense, at later time points. the monkey exhibited minimal evidence of systemic cytokine production. a minimal ifn-g response was observed that was significantly different from that in the rat. il-12p70 at the highest 36 mg/kg dose level generated a small response in one monkey. in contrast, the rat showed a systemic cytokine response, with signals present for all three inflammatory cytokines and il-10, even at the lowest 6 mg/kg dose. the species difference for systemic inflammatory cytokine expression is statistically significant for each cytokine (p < 0.02). for comparison with humans, serum was obtained from 14 patients participating in a concurrent open-label clinical study of chronic fatigue syndrome (amp-511), with a similar time course to the animal pharmacological characteristics and quantified for the same panel of cytokines. the human clinical trial patients were dosed with 400 mg of rintatolimod i.v. over 30 to 60 minutes. similar to the monkey response, serum cytokine increases were minimally expressed with ifn-g, the highest in the aggregate, with a large sd (table 5 ). finally, we assessed for evidence of chronic systemic cytokine changes at the week 32 time point, obtained table 5 illustrates results similar to those of the monkey, with one patient exhibiting a relatively high systemic tnf-a level. systemic inflammatory cytokines, observed in rats, are correlated with significant acute and chronic in vivo toxicities at doses that elicited no detectable systemic inflammatory cytokines and minimal toxicities in primates. moreover, the elevated thyroid activity in the cynomolgus monkey has not been detected in humans. again, systemic signals of cytokine change in association with 32 weeks of rintatolimod at 400 mg per dose i.v., twice weekly, were not evidenced. human dcs do show phenotypic changes in association with exposure to rintatolimod, including shift in maturation profile and local release of cytokines (il-12p70, ils 4, 6, and 10, ifn-g, and tnf-a) and the chemokines, monocyte chemoattractant protein-1 (ccl2), and macrophage inflammatory protein-1a (ccl3) in the microenvironment of culture supernatants. 21, 22 as demonstrated in this communication, systemic elevations of inflammatory cytokines are observed in rats. in monkeys and humans, any local elevations (microenvironment) induced by rintatolimod were rarely detected systemically. the difference is in the magnitude of the response. it is this difference in humans that appears to be responsible, at least in part, with the relative lack of toxicity in primates. toxicities associated with overwhelming infection are associated with systemic cytokine storms 23,24 of elevated measurable circulating cytokines. the lack of systemic cytokine detection is consistent with the observed minimal toxicity in primates, in contrast to the significant toxicities observed in nonprimates. rat toxicity correlates with systemic cytokine levels, and the lack of primate toxicity similarly is correlated with the lack of significant systemic inflammatory cytokine levels. in vitro data, however, demonstrate that cells expressing tlr3 respond to dsrna. 21, 22 the difference between the systemic cytokine levels and the microenvironment is quantitative. inflammatory cytokines observed in the systemic circulation in rats are associated with extreme toxicity and are analogous to the toxicity seen in humans with lethal viral infections, such as highly pathogenic avian influenza (h5n1/h7n9) 23 and severe acute respiratory syndrome (sars coronavirus) 24 with inflammatory cytokine storms. the standard human bioactive dose (approximately 6 mg/ kg) is associated with substantial systemic toxicity in rodents, in which a dose only three times higher is often lethal. poly i:poly c is associated with significant human toxicity 25 not observed with rintatolimod. we examined whether this dissociation of toxicities in humans is related to the affinities of these dsrna ligands to tlr3. we modeled the predicted relative binding constants of poly i:poly c versus their mismatched poly c analogues. figure 2 demonstrates an in silico molecular model of rintatolimod bound to the human tlr3 ectodomain, forming a homodimer structure, and its close 2 å amino acid contacts on the tlr3 variable nonhelical segments. these noncovalent contacts provide the collective energetics of binding to form the active homodimer. poly i:poly c demonstrates equivalent in silico energetics of binding with rintatolimod and several other structures with mismatched bases in the poly c strand ( table 6 ). the insertion of a uridine in the poly c strand of poly i:poly c at various ratios of c/u has no significant effect on the binding affinity with tlr3. thus, any biological differences observed between rintatolimod and poly i:poly c cannot be attributed to a differential affinity of tlr3 binding. adverse event data in rintatolimod and placebo-treated patients in well-controlled studies of chronic fatigue syndrome were assessed in the context of toxicological findings. adverse events were collected during serial patient evaluation of study subjects participating in prospective 24-to 40-week studies of rintatolimod (400 mg) or placebo, infused twice weekly for the duration of the study. elevated lft results denotes alanine aminotransferase or aspartate aminotransferase greater than three times the upper limit of normal. no patients discontinued secondary to abnormal lft results. lft, liver function test; tsh, thyroid-stimulating hormone; t4, thyroxine. the american journal of pathologyajp.amjpathol.org we evaluated the rabbit (o. cuniculus), dog (c. lupus familiaris), rat (r. norvegicus), and monkey tlr3 coding sequences relative to the human standard for differences in species' orthologs. the rabbit, dog, and rat show 5.4% sequence nonidentities (supplemental figure s1 ). monkeys were highly homologous (>99%) between the five species analyzed (supplemental figure s2 ). the major divergence was as anticipated between old world [m. mulatta, m. fasicularis (cynomolgus), and p. anubis (baboon)] and new world (c. jacchus and s. boliviensis) monkeys. the specific sequence differences between the cynomolgus monkey and rat, relative to humans, are listed in table 7 . expressed tlr3 protein in the monkey and rat is similar in its total amino acid composition (905 and 906 residues, respectively) versus a z difference between rat and monkey cytokine levels across the three dosage levels using the jonckheere-terpstra test (two sided). the 400-mg infusions providing an approximately 6 mg/kg average dosing. *high means and sd values were secondary to one patient with a value of 500. removal of this patient results in a mean value of 4.6. ajp.amjpathol.org -the american journal of pathology shorter human tlr3 (889 residues). as expected, the monkey tlr3 has greater homology to human (95%) than mouse/rat sequences (80%; note that the mouse and rat are 100% homologous). rodents have sequence differences versus humans in all of the 23 leucine rich repeats. differences between humans and monkeys are present in leucine rich repears 2 to 6 and 8 to 22 (20 of 23). the endodomain is more highly conserved between the three species than the ectodomain, probably because of the critical cell signaling function of the cytoplasmic toll and il-1 receptor (tir), which suggests that the monkey and rodent signaling domains may have greater functional similarities than human, on the basis of the large disparity in residue length of human versus monkey and rodent tir endodomains. rintatolimod provided a unique tool that allowed us to dissect the differential effect of species on the systemic response to tlr3 activation. although poly i:poly c serves as a ligand for tlr3, and is a prototypic tlr3 agonist for experimental purposes, it also activates the dsrna-sensing cytosolic helicases. the substitution of a mismatched base (uridine) in the poly c strand of rintatolimod inactivates its ligand activity for the cytosolic helicases (mda5 and rig-1) while maintaining its agonist activity for tlr3. 19, 20 the helicases act through the inflammatory myd88 pathway used by all mammalian tlrs, with the exception of tlr3, which uses the trif adapter to initiate innate immune responses. figure 1 illustrates the pattern recognition of the ectodomain of the tlrs and their cytoplasmic adapter pathways responsible for their pleiotrophic effects. figure 1a depicts the non-myd88 pathway used by tlr3, and figure 1b depicts the myd88-dependent pathways used by all tlrs and the helicases, with the exception of tlr3. our toxicological analysis demonstrated systemic dose-dependent inflammatory cytokine responses in rats. although we did not examine inflammatory cytokine responses in rabbit or dog because of a lack of available test kits for the cytokines examined, we believe we would observe a similar quantitative cytokine response in the dog as observed in the rat. the rabbit was so the n-terminals of each tlr3 bind to opposite ends of the dsrna, with a minimum length of 45 bp required for interaction with essential residues of tlr3 for activation of intracellular signaling. amino acids of tlr3 required for binding of rintatolimod are shown as cpk (van der waals' radii) associated with the phosphate backbone. b: the tlr3 homodimer complexed with rintatolimod, as seen down the long axis of the dsrna. the tlr3 homodimer is represented as structural elements, with the blue arrows signifying direction of the leucine repeat bsheets and the red cylinders signifying a-helices. poly i strand of rintatolimod (blue) and poly c 12 u strand (magenta cytidines and green uridine). the american journal of pathologyajp.amjpathol.org sensitive to rintatolimod that the mechanism may be simply induced by systemic cytokine levels, although other toxicity mechanisms may be involved. there were, however, no significant detectable systemic inflammatory cytokines in either monkeys or humans. the data indicate that the parallel intracellular ifn and nf-kb transcriptional pathways are species dependent in magnitude of response because primate cells in culture respond to rintatolimod, with the biosynthesis of the ifns and inflammatory cytokines insufficient in vivo to provide a detectable systemic response. the lack of a systemic inflammatory response in primates is consistent with our observation of discordant in vivo toxicities between rats, monkeys, and humans and is consistent with the differences in gross analysis and histopathological characteristics observed between the species. in the case of myd88-dependent tlr pathways, primates are more sensitive. 2 in contrast, primates are much more resilient than rodents to inflammatory cytokine toxicity induced by nonemyd88-dependent, trif-mediated activation of tlr3. redundant helicase pathways may reduce the toxicological distinction between species for agonists that activate these pathways, such as poly i:poly c, in contrast to rintatolimod. potential mechanisms that may explain the speciesspecific differential toxicities observed with tlr3 activation by dsrna are apparent on analysis of gene structural differences between species. in addition to the primary sequence differences between rodents and primates illustrated in table 7 , rodent and human tlr3 gene structures are remarkably dissimilar in the proximal promoter domains, as well as tlr3 isoforms. 26, 27 an example of differential functional activity between mice and humans is the induction of tlr3 expression in murine macrophages by lipopolysaccharide (lps; ie, not seen in humans). 7 expression in human blood cells (including monocytes, granulocytes, natural killer cells, t cells, and b cells) and tissue macrophages is undetectable at the transcript level after lps exposure and is only seen in myeloid dcs. 12, 28 additional structural differences exist, resulting in possible differential tlr3 isoforms. tlr3 transcripts are initiated in either exon 1 or exon 2 of the mouse, indicating different promoters not present in humans. 27 the cloning of a human tlr3 isoform 29 suggests an alternate, but previously unrecognized, splicing pathway. the observed species-specific lps responsiveness in macrophages could be conferred by consensus motifs for nf-kb binding present in the murine tlr promoter, which are absent in human promoter. 28 this has been confirmed by later studies that demonstrate that species-specific differences in tissue expression and responses to lps coincide with the presence of different evolutionary nonconserved promoter sequences in both species. however, despite the overall nonrelatedness of tlr3 promoter sequences, mrna expression of both tlr3 orthologs was induced by ifns, particularly by ifn-b. the basal and ifn-beinduced activation of promoters from both species largely depended on similar ifn regulatory factor (irf) elements, which constitutively total monkey-expressed tlr3 contains 905 residues, versus 889 residues for the human tlr3. x total rodent (mouse and rat)eexpressed tlr3 contains 906 residues, versus 889 residues for the human tlr3. *calculations with the accelrys discovery studio software version 2.5.5 using a distance-dependent dielectric. net interaction energy is the energy of the complex minus energies of the components. y poly i:c 12 u, rintatolimod. charmm, generalized force field; vdw, van der waals. ajp.amjpathol.org -the american journal of pathology bound irf-2 and recruited irf-1 after stimulation. 27 in murine macrophages, tlr3 up-regulation induced by ifn-b required ifnar1, stat1, and, in part, irf-1, but not the janus kinase family member, tyk2. also, lps specifically up-regulates tlr3 expression through the induction of autocrine/paracrine ifn-b. in humans, however, ifn-be induced up-regulation of tlr3 was blocked by pretreatment with lps, despite the efficient induction of irf-1. 28 a recent publication provides additional evidence for differential cytokine responses between mice and humans. 30 exposure of human primary dcs to agonist dsrna did not induce tnf-a, il-6, or il-8. in human macrophages, the anti-inflammatory cytokine, il-10, was induced, but tnf-a and il-6 were not induced. this effect was specific for human cells because tnf-a or il-6 was induced by dsrna in murine dcs. moreover, there was differential regulation of nf-kb by murine versus human cells. as shown in numerous prior communications from other laboratories, it was demonstrated that nf-kb was activated by dsrna in murine cells but is absent in human dcs and macrophages, as was ikba degradation present in murine cells. 30 differences between the ectodomain of humans and nonhuman primates (20 of 23 llrs) provide a potential mechanism to explain the small differential toxicity between the species. length disparities between the cytoplasmic signaling domain of rodents and nonhuman primates, compared with humans, may contribute to the small differences in toxicity observed between monkeys and humans. there have been numerous reports comparing species specificity of tlr responses, especially tlr3, including mouse/ human, 12,27,30,31 monkey/human, 32 and fish/drosophila/ human. 5,6 a comparison of tlr2 and tlr4 agonists demonstrated similar cytokine levels in humans and chimpanzees versus baboons, consistent with total dna sequence homologies and the small differences we observed between cynomolgus monkeys and humans. 33 although universal tlr responses were largely conserved across primates, chimpanzee-specific immune signaling is enriched for hivinteracting genes that influence hiv disease progression. 34 differences exist in the primate phenotypic responses to tlr agonists. nevertheless, they are relatively similar when compared with other species. we have observed significant species differences in the toxic responses to rintatolimod, a restricted tlr3 agonist. analysis of the dose-dependent toxicities between rats, monkeys, and humans was remarkably dissimilar. primates are far more resilient to tlr3 stimulation than rodents. the data are consistent with differential tlr3 nuclear transcriptional activities, with the nf-kb pathway in inflammatory cytokine production in primates playing a relatively minor role compared with the irf-3/irf-7 nuclear ifn inducers. interestingly, rintatolimod has most recently been advanced clinically as a therapeutic modulator of chronic fatigue syndrome, a condition manifested by disordered energy metabolism and symptoms curiously reminiscent of the toxicity associated with inflammatory innate immune responses. it is hypothesized that the therapeutic effect may relate to feedback inhibition in these pathways, with rintatolimod being specific in its activity. discordance of toxicity in either direction is equally problematic for the successful clinical development of experimental therapeutics, either placing patients at risk for unexpected toxicity 35 or, conversely, raising regulatory barriers for agents shown toxic in sensitive species. alternative methods of in vitro analysis, such as demonstrated by stebbings et al, 36 need to be validated and adapted for regulatory evaluation for human safety. analogous issues may be encountered in modeling retinal disease across species in the context of rna-based therapeutics. 37 the recognition of differential in vivo toxicities and understanding the mechanisms involved should be beneficial in the rational design and risk-benefit analysis of new pharmacological agents for human use. the fda critical path initiative and its influence on new drug development genomic responses in mouse models poorly mimic human inflammatory diseases a family of human receptors structurally related to drosophila toll toll and interleukin-1 receptor (tir) domain-containing proteins in plants: a genomic perspective expression analysis of the toll-like receptor and tir domain adaptor families of zebrafish characterization of toll-like receptor 3 gene in rainbow trout (oncorhynchus mykiss) recognition of double-stranded rna and activation of nfkb by toll-like receptor 3 toll-like receptor control of the adaptive immune responses dendritic cells and the control of immunity myd88-dependent and myd88-independent pathways in synergy, priming, and tolerance between tlr agonists discordant species responses to tlr3 pattern recognition receptors and the innate immune response to viral infection differential expression and regulation of toll-like receptors (tlr) in human leukocytes: selective expression of tlr3 in dendritic cells kleinman me: irna via tlr3 toll-like receptor, rig-i-like receptors and the nlrp3 inflammasome: key modulators of innate immune responses to doublestranded rna viruses the role of unc93b1 protein in surface localization of tlr3 receptor and in cell priming to nucleic acid agonists chronic fatigue syndrome amp-516 study group, mitchell wm: a double-blind, placebo-controlled, randomized, clinical trial of the tlr-3 agonist rintatolimod in severe cases of chronic fatigue syndrome a controlled clinical trial in cfs (chronic fatigue syndrome) using a specifically configured rna drug, poly(i):poly(c 12 u) structural requirements of the ri n -rc n complex for induction of human interferon sidwell rw: tlr3 is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c12u), but not poly(i:c): differential recognition of synthetic dsrna molecules the microbial mimic poly ic induces durable and protective cd4þ t cell immunity together with a dendritic cell targeted vaccine not all polyriboinosinicpolyribocytidylic acids (poly i: c) are equivalent for inducing maturation of dendritic cells: implication for alpha-type-1 polarized dcs toll-like receptor-3 as a target to enhance bioactivity of cancer immunotherapy fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice toxic properties of a synthetic doublestranded rna: endotoxin-like properties of poly i. poly c, an interferon stimulator of mice and men: species variations of toll-like receptor expression species-specific regulation of tolllike receptor 3 genes in men and mice regulation of toll-like receptors in human monocytes and dendritic cells cloning of tlr3 isoform key differences in tlr3/poly i:c signaling and cytokine induction by human primary cells: a phenomenon absent from murine cell systems use of murine embryonic fibroblasts to define toll-like receptor activation and specificity genetic analysis of toll/interleukin-1 receptor (tir) domain sequences from rhesus macaque toll-like receptors (tlrs) 1-10 reveals high homology to human tlr/tir sequences innate immune responses to tlr2 and tlr4 agonists differ between baboons, chimpanzees and humans functional comparison of innate immune signaling pathways in primates cytokine storm in a phase i trial of the anti-cd28 monoclonal antibody tgn1412 cytokine storm" in the phase i trial of monoclonal antibody tgn1412: better understanding the causes to improve preclinical testing of immunotherapeutics short-interfering rnas induce retinal degeneration via tlr3 and irf3 we thank angus shieh (hemispherx biopharma, inc.) for extending his biostatistical expertise in comparative cytokine data analysis. supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2013.12.006. key: cord-312434-yx24golq authors: deng, ziqin; chen, junsheng; wang, ting title: bibliometric and visualization analysis of human coronaviruses: prospects and implications for covid-19 research date: 2020-09-23 journal: front cell infect microbiol doi: 10.3389/fcimb.2020.581404 sha: doc_id: 312434 cord_uid: yx24golq human coronaviruses, which can cause a range of infectious diseases, have been studied for nearly 60 years. the field has gained renewed interest from researchers around the world due to the covid-19 outbreak in late 2019. despite a large amount of research, little is known about the knowledge structure and developing trends of this topic. here, we apply bibliometric analysis along with visualization tools to analyze 15,207 publications related to human coronavirus from the scopus database, using indicators on publication and citation, journal, country or territory, affiliation and international cooperation, author, and keyword co-occurrence cluster. the results show that research on human coronavirus is dominated by sars-cov. although there have been many publications, only 626 publications (4.1% of total) have more than 100 citations. the top 20 journals with most publications account for 20.6% of total publications and 41% of total citations. in addition to the united states and some european countries, many asian and african countries are involved in this research, with china holding an important position in this area. leading researchers from various fields of human coronavirus research are listed to facilitate collaboration and promote effective disease prevention and control. the keywords co-occurrence analysis reveals that the research focus on virology, public health, drugs and other hotspot fields, and uncovers changes in the direction of coronavirus research. the research map on human coronavirus obtained by our analysis are expected to help researchers to efficiently and effectively explore covid-19. coronaviruses, which were discovered in the 1930s (estola, 1970) and cause a range of respiratory and intestinal infections in animals and humans (geller et al., 2012) , are enveloped viruses with positive-sense single-strand rna which belong to the nidovirales order, the coronaviridae family and the coronavirinae subfamily (gonzalez et al., 2003) . the name "coronavirus" is derived from the latin corona, meaning crown or halo, and refers to the virus's characteristic appearance under an electron microscope, which appears like a crown or solar corona (almeida and tyrrell, 1967) . there are four genera contained in the coronavirinae subfamily: the alpha-, beta-, gamma-, and deltacoronavairus. these viruses can infect not only birds (gamma-and deltacoronaviruses), but also a range of mammalian species (mainly alpha-and betacoronaviruses), including humans (corman et al., 2018) . coronaviruses have been known for more than 80 years (saif, 2004) , while only seven of them have been proven to infect humans so far. plenty of evidence has strongly suggested that human coronaviruses exist with or origin from livestock or some wild animals (corman et al., 2018) . in this case, diseases caused by coronaviruses can also be defined as a kind of zoonosis. human coronavirus (hcov) had not been considered as a highly pathogenic virus to humans until the severe acute respiratory syndrome (sars) outbreak in 2002 (peiris et al., 2003) and the middle east respiratory syndrome (mers) outbreak in 2012 (zumla et al., 2015) . recently, another coronavirus disease, the covid-19, caused by a novel coronavirus, sars-cov-2 (ciotti et al., 2019; tan et al., 2020) , has evoked these painful memories and made people more intensely aware of the pathogenic potential of these microorganisms (cui et al., 2019) . this is the seventh coronavirus identified so far to infect humans, with the others being hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, sars-cov, and mers-cov (geller et al., 2012) . hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 are able to cause common cold in humans and majority of these infections only manifest mild symptoms in respiratory system (mackay et al., 2012; owusu et al., 2014; annan et al., 2016) . however, sars-cov and mers-cov are more serious and responsible for high case fatality rates, both of whom belong to genus beta. once the case-fatality rate of sars was ∼10% (cheng et al., 2007) , while that of mers ranged around 35% 1 . similar to sars-cov and mers-cov, sars-cov-2 is also one of the betacoronaviruses, but with quite unique qualities zhu et al., 2020) . the clinical casefatality rate is not as severe as that of sars-cov and mers-cov, but it is more infectious than either virus (chan et al., 2020; huang et al., 2020) . as of 3:05 pm cest, 2 july 2020, there have been 10,533,779 confirmed cases of covid-19, including 512,842 deaths, reported to who 2 . and the virus had also spread to 213 countries and territories around the world and 2 international conveyances 3 by august 19, 2020, 09:28 gmt. scientists have been studying the human coronavirus since its discovery in the 1960s (hamre and procknow, 1966; bradburne et al., 1967; mcintosh et al., 1967) , and more than 15,000 papers related to coronavirus have been published (according to our search) (supplementary figure 1) . however, researchers need to devote a significant amount of time to reading and identifying relevant work in related fields due to the long research interval, large amount of data, uneven quality of scientific research papers, the presence of unnecessary duplications, and the differences between human coronaviruses and emerging viruses. therefore, it is particularly urgent to sort out important, effective and meaningful information from large databases in order to guide the scientific research, and promote the proper prevention, control, diagnosis and treatment of human coronavirus. bibliometrics, together with novel visualization methods of scientific information, have been reported to be helpful to identify emerging outbreaks of infectious disease. this is particularly true in the current age, where thousands of reports can be easily exchanged between public health specialists and healthcare providers across the internet (takahashi-omoe and omoe, 2012; unkel et al., 2012) . bibliometrics is also recognized as an essential tool and is widely used in a variety of fields to measure and evaluate scientific research quantitatively and qualitatively (aggarwal et al., 2016; romero and portillo-salido, 2019; deng et al., 2020) . therefore, in order to accurately, effectively and systematically reveal connections within the human coronavirus field, our study applied bibliometrics and visualization methods to analyze human coronaviruses-related publications and citations, countries and affiliations, as well as journal performance, author impact and keyword cooccurrence cluster. this study seeks to serve as a valuable reference and guidance for virologists, pharmacists, clinicians and epidemiologists studying the emerging human coronavirus, and to provide novel ideas for finding effective control measures, as well as drugs and vaccines, as soon as possible. our analysis was conducted using the online database scopus (https://www.scopus.com/), which provides a comprehensive collection of different types of scientific peer-reviewed literature (romero and portillo-salido, 2019; bonilla-aldana et al., 2020) . the document search was performed on february 15, 2020. in order to cover as many target documents as possible, we had selected terms that might be used by most scientific publications before the search formula was designed: besides "human coronavirus, " "novel coronavirus" and their abbreviation forms "hcov" and "ncov, " we have the terms "human coronavirus 229e" ("hcov-229e"), "human coronavirus oc43" ("hcov-oc43"), "human coronavirus nl63" ("hcov-nl63"), and "human coronavirus hku1" ("hcov-hku1") to search for publications related to these four currently known nonseverely pathogenic human coronavirus; for sars related publications, we have "severe acute respiratory syndrome-related coronavirus" ("sarsr-cov" or "sars-cov") and "severe acute respiratory syndrome" ("sars"); for mers related publications, we have "middle east respiratory syndrome-related coronavirus" ("mers-cov") and "middle east respiratory syndrome" ("mers") as well as "mers coronavirus erasmus medical center/2012" ("mers coronavirus emc/2012, " "hcov-emc/2012, " or "emc/2012"), "novel coronavirus-2012" ("2012-ncov") and "camel flu"; as for the covid-19 related publications, we have "severe acute respiratory syndrome coronavirus-2" ("sars-cov-2"), "coronavirus disease-2019" ("covid-19"), "2019-novel coronavirus" ("2019-ncov"), "2019-ncov acute respiratory disease, " "novel coronavirus pneumonia" and "novel coronavirus-infected pneumonia." several writing formats that are not generally accepted, like "corona virus" and "hcov229e, " were also taken into consideration after we had found them been used in some publications. notably, when directly using the abbreviation "sars" and "mers" to search, we were provided with a massive number of unrelated results in other fields. we decided to add qualifiers as limitation to these terms to have the accuracy of search optimized ( table 1) . and finally, we conducted the search with the following formula, designed according to the search rules of scopus (deng et al., 2020) : title-abs-key ("human coronavirus" or "human corona virus" or hcov or "novel coronavirus" or "novel corona virus" or ncov or "severe acute respiratory syndrome" or ({sars} and " * cov" or * virus or crisis or outbreak or epidemic or pandemic) or "sarsr-cov" or "middle east respiratory syndrome" or ({mers} and " * cov" or * virus or crisis or outbreak or epidemic or pandemic) or "camel flu" or "emc/2012" or "coronavirus disease 2019" or "covid-19" or (coronavirus or "corona virus" or " * cov" and 229e or oc43 or nl63 or hku1) or hcov229e or hcovoc43 or hcovnl63 or hcovhku1) ( table 1) . all search results, data and information that were essential for our analysis, including the count of citations and some indicators, were extracted from scopus on february 15, 2020. complete document lists were exported as csv files, which were then imported into microsoft excel 2016 for ranking and counting. bar charts were made by graphpad prism 8, and vosviewer 1.6.12 was used to generate the visualization maps. in the analysis of country (or territory), affiliation and author, every co-author was counted equally. in this case, documents with too many authors are not comparable to documents with fewer authors. since only documents with a maximum of 15 authors were counted by scopus, we followed the same strategy when conducting the visualization analysis with vosviewer. our search yielded 15,979 results, based on their titles, abstracts and key words. then we limited our results to publications in english and chinese, which yielded 15,207 documents in total, including 9,182 articles, 2,170 reviews and 3,855 documents of other types (supplementary figure 1) . as expected, the annual publications count was naturally divided into four sections, due to three notable epidemic events in history. before the outbreak of sars, the annual publication amount remained low, reaching a maximum of 29 in 1998. it was not until the sars crisis occurred in 2002 that the scientific publications in this field experienced explosive growth. the annual publication number rose suddenly to 1,729 in 2003 and continued to rise the following year, when it reached 1,754 publications. for the next few years, the publication count declined gradually, reaching a low of 488 publications in 2011. it was the first outbreak of mers in the middle east that led to renewed interest in coronavirus and an increase in publications. this lasted for 4 years, during which time the annual number reached another peak, 838 publications in 2015, coinciding with the second outbreak of mers in south korea. notably, the annual publication number has continued to decline since then. for the publications on human coronavirus in 2020, only publications released prior to february 15 were counted. interestingly, the trend for summed citation of annual publications closely aligned with annual publication production prior to 2013 (figure 1 ). for our citation analysis, we counted the number of publications with different citation amounts. we found that more than half of the publications, up to 9,383 (61.7%, 4,991 articles, 1,075 reviews), had been cited no more than 10 times, including 3,336 that had gone uncited (21.9%, 1,373 articles, 308 reviews). only 4.1% of publications (626 publications, 432 articles, 161 reviews) had more than 100 citations, and only 206 publications (1.4%) and 46 publications (0.3%) had been cited more than 200 and 500 times, respectively (supplementary figure 2) . the top 20 most cited articles and non-articles are listed separately in table 2 and supplementary table 1 . among the top 20 articles, six were published in the new england journal of medicine, four in science, three in nature, two in the lancet and the remaining five articles were published in five other journals. in total, these 20 articles were published in nine different journals. most of them were published after 2002, corresponding to the time of the sars outbreak. the research areas of the top 20 articles involve public health, preventive medicine, epidemiology, clinical reports and virological study, including the identification, isolation, and analysis of natural hosts of these coronaviruses. all english and chinese documents came from 3,443 different journals. the 21 journals with the most publications on human coronavirus are listed in table 3 . this list includes four journals that published articles that were in the top 20 article list (table 2) (new england journal of medicine, science, nature and lancet). these 21 journals produced 20.6% (3,126) of all publications analyzed here and 41.0% of the total citations (140,450 of 342,451). among them, the new england journal of medicine had the highest values in two indicators, citations per document and citescore (2018). also, notably, contributions made by chinese journals like hong kong medical journal and chinese medical journal cannot be ignored. figuring out core journals in a specific research field is of considerable importance. this can not only help academic achievements be known and used as much as possible, but also provide those who need the information with more concentrated access. the united states has dominated this field with 143,960 citations on its 4,225 published documents. china ranked second with a total of 49,316 citations for its 2,720 documents from researchers in mainland china, 48,828 total citations on 1,411 documents from researchers in china hong kong and 13,408 total citations on 646 documents from researchers in china taiwan. the united kingdom and canada have also contributed significantly to this research area ( in chan, leo l.m. poon and guan yi, which have all garnered high citation counts, cannot be ignored. the studies of those active and emerging researchers with unique ideas rather deserves our attention. it inspired us that more comprehensive and detailed evaluation of researchers will help to identify their specific areas of expertise, and therefore make their work more valued when applicable. a co-occurrence relationship is formed between two keywords when they both occurred in the same paper. keywords with strong co-occurrence relationship can reveal research hotspots more precisely than a single keyword. in our visual representation, strongly connected keywords were colored the same, indicating that they might share something in common. keywords shown in red are roughly connected to public health, preventive medicine and epidemiology. according to these keywords, human coronavirus diseases like "sars, " "mers" and covid-19 may have something worthwhile for comparison with other "infectious diseases" like "influenza" in their epidemiological characteristics; "healthcare workers, " "transmission, " "surveillance, " "quarantine, " or "isolation" may be the focuses of these studies, which can help to promote current disease control and prevention measures. keywords in blue and yellow are related to virus detection and clinical diagnosis. among them, "serology" and "rt-pcr" are likely to be the methods, the research objects are mainly some kinds of "respiratory viruses, " and the research purposes are mostly on "evolution, " "phylogeny, " and "diagnosis." the words in green are mainly virology-related and also include some immunological and pharmaceutical research: "spike protein, " "nucleocapsid protein, " "receptor-binding domain, " "ace-2" and "apoptosis" are mainly on "pathogenesis, " while "epitope, " "antibody, " "vaccine, " "inhibitor, " "interferon, " "ribavirin, " and "antiviral activity" are mainly about antiviral solutions (figure 4) . with keyword co-occurrence analysis, we can not only figure out the hotspots, but also discover the limitations, and even come up with new ideas. with the outbreaks of sars in 2002 and mers in 2012, along with current covid-19 pandemic (harapan et al., 2020) , the world has battled three serious human coronavirus outbreaks during the twenty first century. in particular, the covid-19 outbreak has surpassed the previous two ones in terms of its transmission scale, largely due to the strong infectivity and long asymptomatic incubation period of the emerging pathogen (epidemiology working group for ncip epidemic response, chinese center for disease control and prevention, 2020). human coronavirus has been an area of concern for many years, with the first study conducted almost 60 years ago and plenty of scientific documents published in the decades since. however, when the novel virus emerged, controlling the spread of this virus effectively and promptly remained a significant challenge. this is partially due to the unclear knowledge map of human coronavirus research and an inadequate understanding of the present research status, hotspots and development trends. this can result in a large amount of repetitive, insignificant or inefficient research work, which can prevent effective analysis of this virus and further delay the development of targeted prevention and control measures. therefore, we applied an innovative bibliometric and visualization analysis to sort and analyze more than 15,000 human coronavirus-related scientific publications spanning 60 years, with the purpose of mapping and managing previous research in this field. the trends in the annual number of publications and summed citation of annual publications on human coronaviruses (figure 1) reflect the interest and development of this area over the years (durieux and gevenois, 2010) . from 1965 to 2002, the number of publications was low and remained at a stable amount. strikingly, two explosive growth peaks appear separately in 2003 and 2012, matching the onset of the sars and mers outbreaks. this sharp growth seen at these times is reflective of the severe impact of emerging coronaviruses on human health. the number of publications is directly proportional to the extent and spread of the infective disease outbreak (hurtado et al., 2018) . the peak that emerged during the sars crisis is significantly higher than the one that emerged during the mers outbreak, which is due to the more widespread outbreak and more extensive impact of sars. considering the severity of the covid-19 outbreak, we can predict that this trend will continue and a large amount of publications will follow. meanwhile, after the short mers outbreak, the summed citation of annual publications dropped quickly and became out of sync with the number of annual publications, which might reflect a gradual declining interest in human coronavirus research. however, it is believed that the covid-19 outbreak caused by the emerging sars-cov-2 will bring the research on human coronavirus back to forefront. just as we predicted, the number of literatures on sars-cov-2 and covid-19 has doubled in the past 6 months, according to our latest online-searching in august. this has proven that the consequences of covid-19 are so catastrophic and far-reaching that it has attracted attention worldwide. the citation number of an article represents the extent of its dissemination and influence, and thus partly reflects its quality as well (muniz et al., 2018) . although there have been over 15,000 publications on human coronavirus, only 626 (4.1%) have more than 100 citations, while 9,383 (61.7%) have <10 citations (supplementary figure 2) . this indicates that although there has been much research on human coronavirus, there might be only a small percentage of these studies that are of high quality. it also indicates that the research field is wide but not deep, implying that much remains for researchers to study about this virus, and in-depth exploration in some specialized areas could shift the focus and direction of this field for the future. among the top 20 articles listed in table 2 , six were published in the new england journal of medicine, four in science, three in nature, two in the lancet and the other five articles were published in five different journals. these nine different journals are very responsive to novel emerging viruses and related diseases. moreover, 18 of these top articles are related to the sars outbreak and only one is connected to the mers outbreak. for the top 20 non-article publications listed in supplementary table 1 , there were up to 18 different journals in the list, indicating the attention received by these publications, all of which are related to sars research, from other sources. therefore, sars-cov plays an important and enlightening role in analyzing the research on human coronavirus. despite the fact that all english and chinese publications were obtained from 3,443 different sources, the top 20 journals with the most published documents account for 20.6% of all publications and 41.0% of all citations. this is reflective of the authority of these journals, as well as their high degree of interest in research related to human coronaviruses. due to the limitations of scopus, journals can only be sorted by the total number of published documents. however, as shown in table 3 , the new england journal of medicine, proceedings of the national academy of sciences of the united states of america, science, nature and the lancet rank in the top five based on citations per document, which is consistent with the results in table 2 . popular journals and their research trends in a particular field provide researchers with reliable references. in addition, core journals provide researchers with faster search routes and can serve as an important publication guide (zhuang et al., 2014; wang et al., 2019) . the united states, the united kingdom, germany and other european countries are usually the most active countries at the forefront of scientific research (sweileh, 2017) . however, as can be seen from figure 2a , the situation is quite different in the human coronavirus field. the united states remains dominant in this field, while some asian and african countries, such as china, singapore, saudi arabia, south korea, japan, india and egypt ranked in the top 20 for human coronavirus-related research. this implies that the research on emerging viruses is no longer limited within those developed countries with more developing countries getting involved. one possible factor lies in the wide and fast spread of the diseases, which has enabled more and more people have come to realize the urgency of taking timely actions and having a clear understanding of emerging infectious diseases. additionally, advancements in science and technology, as well as increased funding support from national policies in these regions have been instrumental in discovering and analyzing emerging viral pathogens. in particular, the top 20 affiliations with the highest number of documents published (figure 3, supplementary table 3 ) reflect chinese scientists' contributions to the human coronavirus field, with nearly a half of the top 20 affiliations coming from china. similarly, a recent article from stanley perlman also praised the contribution of chinese scientists to the study of novel human coronaviruses (perlman, 2020) . here, we generated a map to show the contributions of different countries to the human coronavirus field and display the cooperative relationship between countries (or territories) intuitively ( figure 2b ). this map indicates that international cooperation efforts between coronavirus researchers are led by the united states, china, china hong kong, the united kingdom and canada. these results can provide significant guidance for initiating collaborative projects, project applications, and academic exchanges, as well as providing information that can be used in the political sphere in different countries, territories and affiliations concerned with the impact of human coronavirus (fiala, 2012) . as for the covid-19 pandemic, the lack of information sharing and collaborative efforts is very detrimental to the prevention of diseases. countries and affiliations are supposed to take on the responsibilities to strengthen mutual trust and international cooperation, so that we can fight against this fatal virus with more joint efforts. analyses of author can help to understand a research field in a more comprehensive manner, and to evaluate the contributions of researchers, as well as their research level and academic status in this field, objectively (podsakoff et al., 2008) . in our study, the top 20 authors with the most publications were used to screen out those active researchers in this area. as shown in table 4 , yuen kwok-yung has produced the largest number of documents, guan yi has the highest number of citations per document, and joseph sung jao-yiu currently has the highest hindex. these data and indicators provide different perspectives on the research level and academic authority of each researcher. interestingly, consistent with figure 3 , table 4 reveals half of the top 20 researchers are from china, which also reflects the more active state of chinese researchers on human coronaviruses. it should be noted that among all the authors, some are researchers from research institutions at universities, some are doctors from hospitals, and some are experts from the centers for disease control and prevention (cdc), etc. with the current covid-19 outbreak, it is vital for scientists, clinicians and cdc experts to share and exchange information, and to develop a united approach in emerging viral disease outbreak responses and control (wang et al., 2020a) . in addition, we have learned from the covid-19 outbreak that at the beginning of a sudden outbreak, the allocation of emergency expert personnel and the expertise, authority and experience of these experts both have a significant impact on the spread of the epidemic. therefore, it is necessary to consult and analyze the literature in order to identify these leaders ahead of another novel human coronavirus emergency. our study is able to supplement and complement these efforts. a large amount of meaningful information can be obtained from keyword co-occurrence analysis, which could enable the identification of hotspots and trends, and guide researchers to related topics in their field (romero and portillo-salido, 2019) . in figure 4 , four clear research fields within human coronavirus can be seen, which mainly involve aspects of public health, preventive medicine and epidemiology, clinical work and pharmaceutical research. by contrast, the studies on tracing, evolution and animal carriers of human coronavirus account for only a small proportion of the studies performed, suggesting that the research in these areas is still insufficient and there is room for growth. in our study, it is demonstrated that current research on sars-cov-2 and covid-19 are mainly focused on prevention and treatment. although there is former experience of sars and mers to learn from, people are still likely to get overwhelmed at first in face of global public health emergencies like this, for its rapid spread on a grand scale (peeri et al., 2020) . promoting the correct use of protective masks in public places, encouraging a safe social distance and avoiding crowd gathering are all effective alternatives to prevent large-scale public transmission (gasmi et al., 2020) . and enhancing medical stuff with reasonable allocation of medical resources (emanuel et al., 2020) , separating different diagnosis and treatment areas, and providing centralized isolation areas for mild patients if necessary (hellewell et al., 2020) can all help to ease the shortage of medical resources and avoid nosocomial infections. with present absence of specific antiviral drugs or vaccines to covid-19, one vital step for now is to prevent the spread of sars-cov-2. based on reported data, the effective reproductive number (r o ) of the sars-cov-2 was estimated to be 2.2 approximately , which means that each infected individual can transmit the infection to more than two healthy individuals. it has been indicated that sars-cov-2 may attach the angiotensin converting enzyme 2 (ace2) receptor with its spike protein, in the way similar to sars-cov, to enter target cells (lu et al., 2020; walls et al., 2020) . however, it is still not sufficient enough to explain the high infective efficiency. further research has revealed some possible reasons. on one hand, the case fatality rate of covid-19 is much lower than that of sars ; on the other hand, x-ray crystal diffraction has implied that the combination between s protein of sars-cov-2 and ace2 is a little stronger than that of sars-cov (lan et al., 2020; shang et al., 2020) . in fact, different from sars-cov, significant mutation in the s protein of sars-cov-2 has been identified by genomics analysis, and a specific furin-like protease also plays an essential role in recognition, entry, stability and transmission of the coronavirus (coutard et al., 2020; drak alsibai, 2020) . in addition, neutralizing antibody against the virus has also been a hotspot in human coronavirus research nowadays. as for to sars-cov and mers-cov, several types of representative neutralizing antibodies such as monoclonal antibodies, their functional antigen-binding fragment and the single-chain variable region etc., has been found to block the binding between receptor-binding domain(rbd) region of s protein with ace2 receptor and then to inhibit the infection (jiang et al., 2020) . this inspires us that the rbd region may be noteworthy when studying the neutralizing antibodies induced by viruses or vaccines. a recent study has reported a human monoclonal antibody 47d11 which targets a conserved epitope of the sars-cov-2 s-s1b domain, and might play a role in the prevention and treatment (wang et al., 2020b) . convalescent sera have been applied to treating covid-19, but attention needs to be paid to the phenomenon of antibody-dependent enhancement (ade) caused by non-neutralizing antibodies which target in non-rbd regions (du et al., 2009) . although there are a range of studies on the origin and transmission of human coronaviruses, especially sars-cov, mers-cov, and sars-cov-2, current evidences are still not so convincing enough. despite of this, it is widely accepted so far that these three coronaviruses are able to transmit from person to person. they seem to origin from bats and need some intermediate host to facilitate replication, evolution, and variation, and thus can infect human directly. civet cats and dromedary camels are supposed to act as intermediate hosts for sars-cov and mers-cov, respectively azhar et al., 2014) . according to genome sequencing and evolutionary analysis, bats are also speculated to be the natural host of sars-cov-2 (guo et al., 2020) , while turtles, pangolin and snakes are alternatively possible to serve as the intermediate host for its transmission from animal to human (liu et al., 2020) . study on the origin of the human coronavirus is extremely significant for better understanding, prevention and control of relevant diseases. as it may become the focus of research on emerging human coronaviruses in the near future, more experts, material and financial resources may be urgently needed. in general, previous research on sars-cov and mers-cov can provide important guidance for the study of sars-cov-2 and speed up the development in therapeutic drugs and vaccines. although the keyword co-occurrence analysis shows the hotspots and trends within human coronavirus research, it also indicates that some fields remain unexplored or underexplored. for example, the relationship between human coronavirus and immune metabolism, the application of rna-seq and single cell sequencing technology in coronavirus research, and the possibility of cocktail therapy in viral treatment have not been studied extensively. due to the short duration of the recent covid-19 outbreak, studies on sars-cov-2 were insufficient in various fields. keyword co-occurrence analysis could help researchers studying sars-cov-2 by providing abundant potential directions in similar fields with other human coronaviruses and revealing the cross-disciplinary exploration potential. today we are experiencing an information data explosion. because researchers have been studying coronavirus for 60 years, there is a massive and complicated array of data. thus, being able to benefit from such an unprecedented amount of data without being overwhelmed poses a significant obstacle for researchers, especially when facing the emergency of a novel infectious disease outbreak, such as covid-19. a bibliometric analysis combined with data visualization is critical for exploring and communicating information effectively, and helping researchers to continue to progress (wong, 2012) . for this reason, we applied bibliometric and visualization methods to analyze 15,207 documents of human coronavirus-related studies from the scopus database using various indicators. we hope that our study will indicate potential directions for scientists to explore, promote cooperation with other human coronavirus researchers across disciplines, guide emerging researchers toward specialities that have yet to be fully developed, enable the development and use of new technologies in this field, and provide valuable ideas for the prevention, diagnosis and treatment of covid-19. datasets generated for this study will be made 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mobile cloud computing environment middle east respiratory syndrome tw designed the study. zd and jc performed the search. tw, zd, and jc analyzed the data and wrote the manuscript. all authors contributed to the article and approved the submitted version. this work was supported by the national natural science foundation of china (81000738 to tw) and the national undergraduate innovation funding of huazhong university of science and technology (201910487117 to tw, 2019a0199 to tw). the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2020.581404/full#supplementary-material the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 deng, chen and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-307046-ko3bdvo0 authors: vasilakis, nikos; tesh, robert b.; popov, vsevolod l.; widen, steve g.; wood, thomas g.; forrester, naomi l.; gonzalez, jean paul; saluzzo, jean francois; alkhovsky, sergey; lam, sai kit; mackenzie, john s.; walker, peter j. title: exploiting the legacy of the arbovirus hunters date: 2019-05-23 journal: viruses doi: 10.3390/v11050471 sha: doc_id: 307046 cord_uid: ko3bdvo0 in recent years, it has become evident that a generational gap has developed in the community of arbovirus research. this apparent gap is due to the dis-investment of training for the next generation of arbovirologists, which threatens to derail the rich history of virus discovery, field epidemiology, and understanding of the richness of diversity that surrounds us. on the other hand, new technologies have resulted in an explosion of virus discovery that is constantly redefining the virosphere and the evolutionary relationships between viruses. this paradox presents new challenges that may have immediate and disastrous consequences for public health when yet to be discovered arboviruses emerge. in this review we endeavor to bridge this gap by providing a historical context for the work being conducted today and provide continuity between the generations. to this end, we will provide a narrative of the thrill of scientific discovery and excitement and the challenges lying ahead. almost 120 years have passed since walter reed, james carroll, aristides agramonte, and jesse lazear established that yellow fever is caused by a filterable infectious agent which is transmitted by the bite of a mosquito, then known as stegomyia fasciata (aedes aegypti). lazear, who like his colleagues, had been stationed by the us army in cuba to study the disease, died of yellow fever in september 1900 after being exposed experimentally to mosquitos that had fed on sick patients. at about the same time in south africa, james spreull and sir arnold theiler demonstrated that bluetongue disease of sheep is caused by an "ultravisible" agent that could be transmitted by the injection of an infected serum. epidemiological evidence suggested that the agent was vector-borne, and it was subsequently shown by r.m. du toit that the disease occurred in sheep inoculated experimentally with suspensions of wild-caught biting midges (culicoides imicola). these and other seminal discoveries precipitated a century of research into vector-borne and zoonotic viral diseases, resulting in the discovery and isolation of many hundreds of novel viruses from insects or vertebrate hosts. some were identified as important human or veterinary pathogens. many other viruses were archived in reference collections, with only basic characterization of their biological or molecular properties. in recent years, the advent of next generation sequencing (ngs) has transformed this situation. complete genome sequences are now available for many of the archived isolates, allowing more accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. ngs has also opened the door to viral metagenomics, which has greatly increased the pace of new virus discovery from a wide range of hosts, usually with complete or near-complete viral coding sequences, but no virus isolate and minimal biological data. this has presented both opportunities and challenges for virologists and epidemiologists, as well as viral taxonomists, evolutionary biologists, and bioinfomaticians. sadly, this technological revolution has been accompanied by a period of progressive disinvestment in training in classical virology. in this review, we recall the rich history of the discovery of arboviruses and other zoonotic viruses in various settings around the world and the many outstanding scientists who have contributed to the endeavour. we also consider the impacts of ngs and metagenomic analysis, and the implications of these new technologies for the future of this important field of research. the rockefeller foundation (rf) was organized in 1913 for "the well-being of mankind throughout the world" [1] . at the time, yellow fever was still epidemic in many tropical and subtropical regions of africa and the americas, so in 1916, the rf established the yellow fever commission, with the lofty goal of eradicating yellow fever from the world. during the next 25 years, the rf supported an international group of scientists working on yellow fever in new york city in the united states (u.s.); rio de janeiro and salvador in brazil; bogota, colombia; yabba, nigeria; and entebbe, uganda [1, 2] . much of the work and accomplishments of rf-funded personnel during this period was described in strode's classic book, entitled "yellow fever" [3] . the major accomplishments included: confirmation of earlier work by the reed commission in cuba, demonstrating that yellow fever was caused by a filterable agent, yellow fever virus (yfv), that was transmitted by the bite of infected mosquitoes, aedes aegypti; 2. discovery that the rhesus monkey and the white mouse are susceptible to infection with yfv, providing models for subsequent studies on the pathogenesis, transmission, epidemiology, and control of the disease; 3. demonstration that convalescent sera of humans and animals infected with yfv neutralize the virus. this discovery led to the development of the mouse neutralization test, which allowed investigators to map the geographic distribution of the virus; 4. discovery of the forest or sylvan cycle of yfv and the importance of mosquitoes other than ae. aegypti in the transmission and maintenance of the virus; 5. development of the 17d vaccine strain of yfv and its first human trials. max theiler, son of south african bluetongue researcher sir arnold theiler, received a nobel prize in 1951 for this work. as a by-product of the overseas yellow fever investigations, rf-funded researchers also isolated a number of other previously unknown arboviruses, including west nile, zika, semliki forest, bunyamwera, bwamba, uganda s, ilheus, and anopheles a and b viruses [2] . the threat and onset of world war ii changed the priorities of the rf, and most of its overseas yf research activities ceased during this period. many of the former american rf staff became involved in studies of diseases of military importance, such as typhus, malaria, sandfly fever, and dengue, some as civilians and others as members of the u.s. armed forces. in 1950, after the end of the war, the rf decided to develop a major new program to study arthropod-borne viruses and to discover "what might be out there", as well as their disease associations, life cycles, and vectors. this led to the development of the rockefeller foundation virus program, a world-wide virus discovery program that was funded from 1951 to 1970 [1] . over the next several years, field laboratories staffed by both rf and local scientists were established with foreign governments in pune, india; port of spain, trinidad; belem, brazil; johannesburg, south africa; ibadan, nigeria; cali, colombia; and cairo, egypt [2] . in egypt, the cairo laboratory was associated with the u.s. naval medical research unit no. 3. scientists in these field laboratories were involved in the detection and investigation of human diseases in their respective geographic regions, surveying human and animal populations for serologic evidence of past viral infection, and searching for viruses in a wide variety of arthropods, mammals, birds, reptiles, and amphibians [2] . all virus isolations were done on site, using the classic technique of intracranial inoculation of newborn white mice, but later, as vertebrate cell cultures became available, inoculation of cell cultures was also used. in addition, sentinel animals, such as non-human primates, mice, and hamsters, were also used in the field to detect virus activity. viruses isolated in the overseas laboratories were initially characterized locally, lyophilized for preservation and storage, and then aliquots of each new virus were shipped back to the rf central virus laboratory in new york for further characterization and study. it was during this period that jordi casals, delphine clarke, loring whitman, and others developed the sucrose-acetone method for preparation of viral antigens and began to adapt the hemagglutination inhibition (hi) and complement fixation (cf) tests to identify and group arboviruses [4] [5] [6] . the rf virus program was extremely productive and many novel arboviruses, as well as non-arthropod-borne viruses (i.e., hantaviruses and arenaviruses), were discovered by rf-funded investigators during this period. the productivity of this search strategy in detecting novel viruses affecting humans was recently reviewed by rosenberg et al. [7] . the virus discovery rate was highest in the period 1950-1969, which coincided with the rf virus program. a similar approach was also practiced by the institut pasteur and other international groups involved in virus discovery during this period, as described in this article and in other publications [8] [9] [10] [11] [12] [13] , resulting in the detection of a high proportion of pathogenic arboviruses. the second advantage of the strategy was that it yielded actual virus isolates, whose pathogenesis could be studied experimentally in vivo and in vitro in vertebrate and arthropod models. in contrast, current search methods for new viruses, which generally use metagenomics and other sophisticated genetic techniques to detect novel viral agents, do not usually yield live viruses, only their nucleotide sequences. a complete or partial genetic sequence alone rarely provides insight into the epidemiology and ecology, host range, pathogenesis and disease potential, or transmission modes of the new viruses. in 1960, the rf made a decision to phase-out its virus program and to devote more of its efforts and resources to other projects, such as population control and increasing food production ("the green revolution"). over the next 10 years, the rf-funded investigators working in overseas laboratories were withdrawn and the respective laboratories were turned over to local institutions or governments. in 1964, the rf made arrangements with yale university to transfer its arbovirus group to new since the establishment of the institut pasteur in paris in 1888, louis pasteur sent collaborators to various countries, mainly in the french colonies of indochina and africa. in that early time, pasteur wanted to set up rabies centers where the disease was highly and dramatically prevalent; naturally, the research potential of these centers was rapidly extended to tropical infectious diseases [18] . currently, spread among 25 countries on five continents, there are 22 institutions, 19 of which bear the name of "institut pasteur" (ip). altogether, these institutions constitute a structure long called the "institut pasteur d'outre-mer", which in 1988 became the international network of pasteur institutes (réseau international de l'institut pasteur, riip) and associated institutes. from this emerging network, the first african laboratory for microbiology was created in saint louis, senegal in 1896, and then transferred to dakar to become the pasteur institute of dakar (institut pasteur de dakar, ipd yellow fever was to play a key role in the research at ipd. in the 1930s, ipd had developed a vaccine against yellow fever (strain fnv) and produced it commercially. the re-emergence of yellow fever in africa in the 1960s occurred mainly in the savannah zone and revealed the lack of knowledge about the natural maintenance of the virus during the inter-epidemic period. a large study to understand the emergence and re-emergence of sylvatic yellow fever was established between the ipd, institute pasteur abidjan (ipa) in côte d'ivoire, and institute pasteur bangui (ipb) in the central african republic, in order to detect the circulation of the yfv and map its emergence in the different ecozones. permanent research stations were developed in rain forests and savannas to detect virus circulation in vectors and hosts from the tree canopies to the savanna ground. year-round mosquito and monkey sampling over a period of more than 10 years made possible the detection of yfv in various monkey and mosquito species (e.g., aedes africanus, aedes opok, aedes furcifer-taylori, and aedes luteocephalus), thus elucidating the mechanism of yfv maintenance in nature. epidemiological observations during this period allowed max germain to formulate the concept of a "yellow fever zone of emergence" in west and central africa [19] . these ecological transition zones constitute ecotones adjacent to sylvatic environments, where prevailing ecological conditions, such as vector abundance, presence of non-human primates, and closeness for human contact, enable the cross-species transmission of viruses into humans-a "zone of emergence", which clearly appeared as the main source of epidemics in west africa [20] . thus, this specific ecosystem constitutes an ideal transition ecotype, where vaccination campaigns for the containment of epizootics and ultimately eradication ought to concentrate [21] . lastly, virus isolation in male mosquitoes (ae. furcifer-taylori) allowed the documentation of vertical transmission, allowing for virus maintenance in the inter-epidemic periods [22] . in the early 1960s, research laboratories focusing broadly on arboviruses were established under the umbrella of the collaborating center for reference and research on arboviruses (crora), led by paul brés. crora laboratories were created at various ips throughout africa, including ipd, ipa, and ipb, as well as ip yaoundé (cameroon), and ip tananarive (madagascar). one of the major activities was to establish an inventory of arboviruses circulating in various ecosystems. although virus isolations were made at various ips, virus characterization and identification were carried out at the crora reference center at ipd, followed by confirmation at yaru, with final registration in the international arbovirus catalogue [23] . at the end of the 1970s, following an outbreak of the ebola epidemic in zaire in 1976, ipb set up a research program on viral hemorrhagic fevers that lasted until 1983 [24] . this research program was the result of an important collaboration between the various pasteur institutes in africa and researchers of orstom. in 1977, orstom extended its field of research and expertise to tropical medical virology, thus supporting teams from the riip. the partnership between riip and ostrom was instrumental in expanding the scope of field research in africa, with seminal studies on the vertical transmission of arboviruses in mosquitoes [25] or the role of environmental factors, such as climate and latitude, in arbovirus transmission from arthropod to vertebrate hosts, using yfv [26, 27] and dengue virus (denv) [28] [29] [30] as models. research at ipb was also critical in elucidating the etiology of exanthematous fevers, coined "congolese red fevers", that have long been attributed to rickettsia. a total of 16 arboviruses (chikungunya, igbo-ora, o'nyong nyong, sindbis, bouboui, yellow fever, wesselsbron, west nile, zika, ilesha, bwamba, dugbe, tataguine, nyando, bangui, and rift valley fever viruses) were associated with these syndromes. symptoms, consisting of fever, diffuse pain, and exanthem, were present in more than 60% of the cases, with etiology dominated by four viruses-chikungunya, ilesha, bwamba, and tataguine [31, 32] . between 1976 and 1986, two field research stations were established in the central african republic, one located adjacent to the forest (bouboui) and the other in the wooded savanna (bozo) (figure 1 ). during this period, more than 420,000 mosquitoes of identified species in 14,591 pools were preserved for virus isolation. the most common anthropophilic mosquitoes caught were aedes (stegomiya) africanus and ae. (st.) opok, inside the forest gallery, aedes vittatus, in the savannah, and anopheles gambiae and an. funestus, in the houses of the village of bozo. a total of 321 viruses were isolated and assigned to 24 different species. these included chikungunya, bagaza, bouboui, bozo, bwamba, ilesha, kamese, kedougou, middleburg, mossuril, m'poko, nyando, orungo, pata, pongola, simbu, sindbis, tataguine, wesselsbron, west nile, yellow fever, and zika viruses. altogether, six arboviruses were found in the forest gallery, including bouboui, bozo, chikungunya, orungo, yellow fever, and zika viruses, vectored primarily by ae. africanus. research at ipd and ipb was also instrumental in demonstrating the expanded range of the crimean-congo hemorrhagic fever virus (cchv) in west and central africa [33] [34] [35] , followed by repeated isolation of the virus. this allowed a clear understanding of its eco-epidemiology in the region, including north-south migration of the infected ticks through the cattle traffic migration patterns in the sahel [34, 36] . likewise, active circulation of the rift valley fever virus (rvfv) was also demonstrated in senegal [37] , mauritania [35] , upper volta (present day burkina faso) [35, 38] , and the central african republic [39] . support for riip laboratories located in africa was provided by the ipp and ostrom teams, as well as through collaboration with various research centers in the u.s., such as harvard university, supporting the initial study on yellow fever and fnv vaccine; yaru, as a partner for new arbovirus classification; the centers for diseases control (cdc) at fort collins colorado, for the study of arboviruses; the cdc in atlanta and the u.s. army medical research institute of infectious diseases (usamriid), for the initiation of viral hemorrhagic fever research in central and west africa. the era of virus discovery in australia can be traced to the summer of 1950-1951, when a major epidemic of encephalitis swept through southeastern australia. there were clinical cases reported in victoria, new south wales, and south australia, of which 19 (42%) were fatal [40] . a similar epidemic of unknown etiology (named australian x disease) had occurred in eastern australia from 1916 until 1925, with almost 300 reported cases and an average case/fatality rate of 68% [40, 41] . surprisingly, no further cases were reported in the intervening 25 years. amongst those investigating the 1950-1951 epidemic were john a.r. miles and colleagues at the institute of medical and veterinary science in adelaide, and eric l. french of the walter and elisa hall institute of medical research in melbourne who, almost simultaneously, reported the isolation of a virus from the brain tissue of clinical cases [42, 43] . the virus, named murray valley encephalitis virus (mvev), was shown to be closely antigenically related to japanese encephalitis virus (jev), a flavivirus (then designated group b arbovirus) known to cause fatal encephalitis in east and southeast asia [43] . the subsequent development of arbovirology and the pathway to virus discovery in australia were linked intimately with the 1947 establishment of the queensland institute of medical research (now the qimr-berghofer institute) at herston in brisbane ( figure 2 ). ralph l. doherty joined the staff and, in 1957, established a program of virus isolation from mosquitoes, based at a field station at innisfail in the far north queensland. in 1959, doherty isolated the ross river virus (rrv) from aedes vigilax mosquitoes collected in townsville [44] . he subsequently showed that rrv neutralizing antibodies occurred commonly in human sera in eastern australia [45] . he also showed that individuals suffering from a severe debilitating syndrome, known as epidemic polyarthritis, had high antibody titres to rrv, suggesting a causal relationship [46, 47] . in 1971, doherty and his colleagues isolated the virus from a boy from the edward river mission aboriginal settlement in cape york [48] . rrv and the related alphavirus, the barmah forest virus (see below), are now recognized as important public health problems in much of australia and the pacific islands, causing arthritis, myalgia, and fatigue for six months or longer. several thousand cases of the disease are notified annually [49] . , normanton, and cairns in far north queensland. these included four novel flaviviruses (kunjin, kokobera, edge hill, and stratford), three orthobunyaviruses (koongol, wongal, and maputta) and one orbivirus (corriparta) [45] . the study also identified two alphaviruses previously unknown in australia (sindbis and getah) and the first isolations of mvev from mosquitoes [45] . with support from the rockefeller foundation, a field station was established at kowanyama and further expeditions were undertaken to collect arthropods and potential mammalian hosts throughout queensland. anopheline mosquitoes and a swamp pheasant (centropus phasianinus) collected at kowanyama from 1963 to 1966 yielded three novel viruses (kowanyama, trubanaman, and alfuy viruses) [50] . in 1969-1970, three novel viruses were isolated at kowanyama (wongorr and mitchell river viruses from mosquitoes and the ngaingan virus from biting midges), three novel viruses were isolated from mosquitoes collected near charleville in western queensland (charleville, warrego, and wallal viruses) and two viruses (belmont and d'aguilar viruses) were isolated from mosquitoes and biting midges, respectively, collected near brisbane [51, 52] . further expeditions to charleville to collect mosquitoes resulted in the isolation of two novel viruses in 1974 (facey's paddock and murweh viruses) and two novel viruses in 1976 (parker's farm and little sussex viruses) [53] . leanyer virus was also isolated in 1974 from mosquitoes collected near darwin in the northern territory [54] . viruses were also isolated from wildlife hosts; the almpiwar virus was isolated from a skink (cryptoblepharus virgatus) at kowanyama in 1966 [55] and the mossman virus was first isolated from a rodent (rattus leucopus) captured near mossman in 1970 [56] . in collaboration with doherty and his qimr team, expeditions were also conducted to isolate viruses from ticks associated with sea birds. in 1966, harald n. johnson from yaru collected soft ticks (ornithodoros capensis) from sooty tern (onychoprion fuscatus) colonies on the great barrier reef near cairns, from which two viruses (upolu and johnston atoll viruses) were isolated [57] . the saumarez reef virus was subsequently isolated by toby d. st. george and colleagues from australia's commonwealth scientific and industrial research organization (csiro) in 1974, from the same species of ticks associated with sooty terns on a coral cay in the southern coral sea [58] . in 1972, m. durno murray from csiro undertook an expedition to the australian territory of macquarie island in the southern ocean to collect hard ticks (ixodes uriae) associated with sea birds, resulting in the isolation of two novel viruses (nugget and taggert viruses) [59] . a second csiro expedition to macquarie island in 1975 yielded two additional novel viruses (gadget's gully and precarious point viruses) from hard ticks collected in royal penguin (eudyptes chrysolophus schlegeli) rookeries [60] . other research groups in australia also joined the hunt for arboviruses during the late 1960s and early 1970s, including ian d. marshall at the australian national university in canberra and neville f. stanley at the university of western australia. from 1965 to 1975, marshall and his colleagues conducted surveys for arbovirus activity, particularly rrv and mvev, in coastal regions of new south wales and in the murray river valley. in addition to these and other known arboviruses, marshall and colleagues isolated several novel arboviruses from mosquitoes, including gan gan, yacaaba, tilligerry and termeil, paroo river, picola, and barmah forest viruses [61, 62] and a novel reovirus, nelson bay virus, from a fruit bat (pteropus poliocephalus) [63] . like the related alphavirus rrv, the barmah forest virus was subsequently shown to be a cause of epidemic polyarthritis in humans [64, 65] , with infections occurring commonly throughout australia [66] . the gan gan virus also infects humans and is suspected of an association with epidemic polyarthritis [67] . marshall also conducted a number of collecting trips to papua new guinea from 1965 to 1975 funded by the rockefeller foundation. during an expedition to the sepik river district of papua new guinea in 1965-1966, he isolated the joinjakaka virus from a mixed pool of culicine mosquitoes and the japanaut virus from both culicine mosquitoes and a fruit bat (syconycteris crassa). in western australia, stanley and colleagues surveyed for arbovirus activity in the ord river valley from 1972 to 1976 [68, 69] . from 52,000 mosquitoes of 20 species, 195 virus isolates were recovered, including 28 isolates of mvev and 20 isolates of the kunjin virus from cx. annulirostris, suggesting the region may be an endemic focus in australia [69] . the study also identified eight novel viruses, including kimberly, parry's creek, ord river, and kunnanurra viruses, as well as four unknown isolates (or379, or512, or869, and or540), which have yet to be characterized [69, 70] . continuing surveillance in western australia by others has continued to reveal novel arboviruses, including oak vale, stretch lagoon, parry's lagoon, and fitzroy river viruses [71] [72] [73] [74] . in 1968, csiro established a new virology laboratory at long pocket in brisbane, headed by toby d. st. george, to investigate endemic diseases of livestock in northern australia. in 1967, doherty and colleagues had isolated bovine ephemeral fever virus (befv) from cattle during a major epizootic in queensland [75] but, despite epidemiological evidence suggesting vector-borne transmission, the virus had never been isolated from insects. this led st. george and colleagues to attempt virus isolations from a location in northern australia, where serological monitoring of a sentinel herd of cattle indicated that befv was likely to be enzootic. for a continuous period from october 1974 until may 1976, insect collections for virus isolation were conducted at beatrice hill southeast of darwin. from the 57,596 mosquitoes and 175,880 biting midges processed, one isolate of befv was recovered (from a mosquito pool). however, the collection also yielded 93 other virus isolates from 22 different serological groups, including four novel viruses (csiro village, marrakai, beatrice hill, and humpty doo virus) [76] . most significantly, the collection also yielded a single isolate of a novel serotype of bluetongue virus (btv serotype 20, btv-20), a major pathogen of sheep and goats that had previously been regarded as exotic to australia [77] . the isolation of btv-20 and the consequences for international trade dramatically changed the landscape with respect to virus discovery and characterization in australia. an immediate consequence was the approval of government expenditure for the establishment of the $230 million csiro australian animal health laboratory (aahl) in geelong, victoria, providing high-level biosecure containment for laboratory work and live animal studies. the discovery also led to the establishment of a permanent veterinary virology capability at the berrimah veterinary laboratory in darwin under geoff p. gard, and a national program for serological monitoring of sentinel cattle herds and the collection of insect vectors. efforts to isolate viruses from arthropods and livestock intensified. in the northern territory, a second novel serotype of bluetongue virus (btv-21) was isolated from a healthy sentinel cow at victoria river station in 1979 [78] , and four other novel arboviruses (coastal plains, berrimah, adelaide river, and koolpinyah viruses) were isolated from healthy cattle between 1981 and 1985 [79] [80] [81] [82] . in queensland, eight novel arboviruses were first isolated between 1976 and 1981 from biting midges (tibrogargan, tinaroo, peaton, wongabel, and walkabout creek viruses), healthy sentinel cattle (the douglas virus), and soft ticks (argas robertsi) (vinegar hill and lake clarendon viruses) [55, [83] [84] [85] [86] [87] . surveillance activities by the berrimah veterinary laboratory have continued to the present, with regular reports of the isolation of novel arboviruses. continuing surveillance by others in northern australia has also resulted in the isolation from mosquitoes of the bamaga virus and new mapoon virus from cape york [88, 89] . the 1990s also saw several significant disease emergence events in australia, which drew particular attention to bats as reservoir hosts of highly pathogenic viruses. in september 1994, an outbreak of a severe respiratory disease occurred in horses at a stable in brisbane. of the 21 affected horses 14 were euthanized or died of the disease. one of two severely affected humans who had contact with the horses also died. cooperation between the queensland government, the newly established csiro australian animal health laboratory, and others resulted in rapid isolation of the hendra virus, a novel paramyxovirus [90] , and the identification of fruit bats as reservoir hosts [91, 92] . the hendra virus has since re-emerged regularly in australia, with more than 70 confirmed cases in horses and seven infected humans, four of whom have died. in 1996, an injured female fruit bat (pteropus alecto) was found at ballina in new south wales. tissue homogenates from the euthanized bat were injected into mice, resulting in the isolation of a novel lyssavirus, subsequently named australian bat lyssavirus (ablv) [93] . three fatal human cases of ablv infection have subsequently been reported [94] [95] [96] . the virus is now known to occur at low prevalence in five of six families of bats endemic to the northern territory, queensland, and western australia [97] . in april 1997, another novel paramyxovirus, the menangle virus, emerged at a commercial piggery in new south wales, causing stillbirths with abnormalities of the brain, spinal cord, and skeleton [98] . two humans exposed to the pigs also developed an influenza-like illness [99] . fruit bats were again implicated as reservoir hosts [100] . the role of bats in the ecology and emergence of pathogenic viruses has been a major focus of study in australia since that time, primarily involving research teams led by hume e. filed and linfa wang. in all, more than 80 novel rna viruses representing 9 families and 16 genera have been isolated from humans, livestock, wildlife, and arthropods in australia and papua new guinea, and reported in the literature (table s1 ). many others have been isolated but remain uncharacterized and/or unreported. more complete characterization of these viruses will be facilitated greatly by the use of ngs. south and southeast asia have also been a fertile area for virus discovery, particularly novel mosquito-borne and tick-borne flaviviruses and viruses with reservoirs in bat species. early studies in india, partly funded by the rockefeller foundation, by telford work and his indian colleagues from the virus research centre in pune, including d.p. murthy, p.n. bhatt, h. trapido and k. pavri, led to the discovery of the kyasanur forest disease (kfd) virus [101] . the virus was isolated from sera and tissues collected from a moribund black-faced langur (presbytis entellus). this followed reports of an epizootic of unknown etiology causing large numbers of deaths in non-human primates and a number of cases of severe febrile illness in villages close to forested areas where dead monkeys had been found. the virus was shown to be closely related to the russian spring-summer encephalitis (rsse) serocomplex of flaviviruses, now known as the tick-borne encephalitis (tbe) serocomplex of flaviviruses. the virus was also isolated from some larvae and nymphs of hemaphysalis spinigera ticks [102] . kfd in humans followed a biphasic course, not unlike tbe, but with some hemorrhagic manifestations not seen in tbe, and without either meningitis or encephalitis. another tick-borne virus related to the rsse serocomplex, langat virus, had been isolated two years earlier from a pool of hard ticks, ixodes granualtus, collected from forest rats caught near kuala lumpur, malaysia, by c.e. gordon smith, then working at the institute for medical research in kuala lumpur [103] , but it is not known to be a human pathogen. a number of mosquito-borne flaviviruses were first isolated in southeast asia. the most important with respect to human disease are three of the four dengue serotypes. although dengue serotype 1 had been first isolated independently by hotta in japan in 1943 [104] , and shortly after by sabin in cincinnati in 1945 with material collected in hawaii [105] , the other three serotypes were first isolated from material collected in southeast asia. dengue serotype 2 was also isolated by sabin in 1945 from material obtained from new guinea [105] and dengue serotypes 3 and 4 were first isolated in 1956 from human sera and aedes aegypti and culex tritaeniorhynchus mosquitoes collected during a major outbreak of epidemic hemorrhagic fever in manila, philippines, by w.m. hammon, a. rudnick, and colleagues at the university of pittsburgh [106] . other novel flaviviruses have been isolated in malaysia, thailand, and papua new guinea [107] . the tembusu (tmuv) virus was isolated in kuala lumpur in 1957 from various mosquito species [108] and was the first of several closely related viruses, including the thcar virus, which was isolated from a pool of cx. tritaeniorhynchus mosquitoes collected in chiang mai, thailand, in 1992 [109] ; the sitiawan virus, from sick broiler chicks in malaysia [110] ; and the duck tembusu virus, an infection of ducks and geese causing an egg-drop syndrome in china and southeast asia [111, 112] . neutralising antibodies were found in humans in malaysia [113] but the virus has not been implicated in human disease. two other mosquito-borne flaviviruses have been described, jugra virus and sepik virus. little is known about the jugra virus, which was isolated from aedes spp. and uranotaenaia spp. mosquitoes and from the blood of a cynopterus brachyotis fruit bat [108] . the sepik virus was isolated in 1966 by ian d. marshall and colleagues from a pool of mansonia septempunctata mosquitoes collected in the sepik district of papua new guinea [114] . it was associated with a hospitalized case of febrile illness of unknown origin, with rising neutralising antibody to the virus. the sepik virus is particularly interesting, as its nucleotide sequence analysis shows it to be the closest known flavivirus to yellow fever virus [115] . a novel lineage of the west nile virus was isolated in sarawak, east malaysia, by d.i.h. simpson, e.t.w. bowen, and colleagues, from cx. pseudovishnui group mosquitoes [116] . initially called kunjin virus, it was shown to differ significantly in genomic sequence from the australian kunjin viruses, which have been shown to comprise west nile lineage 1b viruses, and have been described as west nile lineage 6 virus [107] . two flaviviruses with no known vector have been isolated in southeast asia, both from cy. brachyotis fruit bats: the carey island virus was isolated from a bat in the jugra forest, malaysia, in 1970 by a. rudnick and colleagues from the institute of medical research, kuala lumpur and the international center for medical research, university of california [108] , and the phnom penh virus was isolated by j.j. salaun and colleagues in 1969 from the salivary glands and brown of bats [117] . a closely related virus, the batu cave virus, is considered to be a variant of phnom penh virus. a considerable number of novel bunyaviruses have been isolated from south and southeast asia, particularly by scientists from the national institute of virology (formerly the virus research centre) in pune, including p.n. bhatt, k. pavri, k.r. singh, c.n. dandawate, f.m. rodrigues, d.t. mourya, p.d. yadev, a.c. mishra, and many others. recently reviewed in [118] , these viruses include the orthobunyaviruses, umbre, kaikalur, thimiri, and sathuperi viruses; a nairovirus, ganjam virus; phleboviruses, bhanja, and malsoor viruses; a hantavirus, thottapalayam virus; and two uncharacterized viruses, kaisodi and wanowrie viruses. additionally, novel bunyaviruses, batai and oya viruses, have been isolated in malaysia, the former from cx. gelidus mosquitoes [108] and the latter from pigs [119] , and the kaeng khoi virus was isolated from tadarida plicata bats in thailand. novel orbiviruses from south and southeast asia include the sathuvachari virus, isolated from starlings (brahminy myna) collected in vellore, tamil nadu, india, and most closely related to the mosquito-borne orbiviruses [120] ; and the japanaut virus, isolated from a mixed pool of culicine mosquitoes from papua new guinea [108] . new rhabdoviruses first isolated in south asia include the chandipura virus and joinjakaka virus-the former, a major human pathogen in india, was isolated from a human infection in 1965 near nagpur city [121] , whereas the latter, isolated in 1966 from a mixed culicine pool in the sepik district of papua new guinea, is not associated with disease in humans or animals. chandipura infection is characterized by fever, chills, arthralgia, myalgia, vomiting, and weakness. two novel alphaviruses were reported in kuala lumpur-bebaru and getah viruses. bebaru was first isolated from cx. (lophoceraomyia) spp. collected in 1956, but although neutralising antibodies have been found in human sera, it has not been associated with human disease [108] . getah virus was first isolated from cx. gelidus mosquitoes collected in 1955 near kuala lumpur [108] . it causes a mild disease in horses, characterized by pyrexia, edema of the hind limbs, swelling of the submandibular lymph nodes, and urticarial rash. it also causes a mild disease in pigs, with occasional reproductive problems, including abortion and neonatal infections. neutralising antibodies have been found in a number of animals and in humans. the important role of bats as reservoirs of a wide range of viruses was underlined by a number of the viruses described above, and particularly by the discovery of their role as the reservoir of the nipah virus in malaysia in 1999 [122] and subsequently their probable role as the origin of severe acute respiratory syndrome coronavirus (sars-cov) [123, 124] . the nipah virus, a virus closely related to the hendra virus in australia, was first isolated k.b. chua and s.k. lam during an outbreak of severe disease of humans and pigs in 1998-1999 in peninsula malaysia [125] , resulting in 265 human cases with a mortality of 40%, and the culling of over 1 million pigs. transmission to humans was from infected pigs. the disease in humans was a rapidly progressive encephalitic syndrome, with a significant pulmonary syndrome in some patients [126] . in pigs, the disease was spread via the respiratory tract, and the symptoms were either neural or pulmonary, or both. subsequent epidemics in bangladesh and india have substantially expanded our knowledge of the nipah virus, and have demonstrated that direct transmission of the virus from bats to humans can occur through the consumption of date palm juice, and possibly by other routes, and that mortality rates may often be significantly greater than in malaysia [127] . furthermore, evidence of nipah-like and hendra-like viruses have been detected either by isolation or serology from other pteropid bats across the geographic range of the genus, and related viruses may be carried by other bat species on other continents. sars-cov was first isolated by m. peiris and colleagues in hong kong [128] , and contemporaneously in the u.s. [129] and europe [130] . it was shown to be unrelated to other coronaviruses. transmission to humans is believed to have been via an intermediate host, such as the himalayan palm civets (paguna larvata), through wet markets in southern china. continued studies of the nipah virus in malaysia, and subsequently in bangladesh and india, and investigations of sars-cov in bats have resulted in an enormous explosion of knowledge of viruses carried by bats, with many examples from most viral families, although in many cases the information is from genomic fragments [131] . virus isolations have been made from bats, especially frugivorous bats, from india and malaysia. one of the earliest isolations was a paramyxovirus of the genus rubulavirus, which was isolated from a rousettus leschenaultia bat collected near pune in india [132] . a novel adenovirus from the genus mastadenovirus was also isolated from the same fruit bat species caught in maharashtra state [133] . a number of viruses have been isolated from fruit bats in malaysia by k.b. chua, s.k. lam, l.f. wang, and their colleagues-tioman virus, a paramyxovirus in the genus rubulavirusi, isolated from pteropus hypermelanus and related to the australian menangle virus, but not known to cause human disease [134] ; pulau virus, an orthoreovirus related to the nelson bay virus of australia, and not associated with human or animal disease [135] ; melaka virus, an orthoreovirus, causing acute respiratory disease in humans [136] ; and kampar virus, an orthoreovirus related to the melaka virus, and also causing acute respiratory disease [137] . the importance of orthoreoviruses originating in pteropid bats was assessed in an outpatient clinic, where it was found that pteropine orthoreoviruses are among one of the common causative agents of acute upper respiratory tract infection (urti), with a cough and sore throat as the most common presenting clinical features [138] . the history of arbovirus research in the ussr began in 1937, when an expedition under the leadership of lev a. zilber (at the time, the head of central virological laboratory of narkomzdrav ussr in moscow) went to the russian far east to study seasonal epidemic encephalitis. this disease with high mortality rates affected forest workers and soldiers stationed in the taiga, mainly those who came from other regions of the ussr. the disease had a pronounced seasonality-the cases started being recorded at the beginning of may, reached the peak in early june, and declined by august. local doctors designated the disease "spring-summer encephalitis" (sse) and assumed that it had been caused by some kind of virus. it has also been suggested that there were some similarities between sse and "summer encephalitis" (japanese b encephalitis and st. louis encephalitis) described at the time, it was also assumed to be a toxic form of influenza [139] . however, the etiology and transmission routes of sse remained unclear. during summer of 1937, zilber and colleagues isolated at least 29 strains of a new virus from the blood and cerebrospinal fluid of sick people, and from brain tissues of dead patients. the isolated virus had a weak antigenic relationship (in complement fixation tests) with japanese b encephalitis virus [140] . based on comparative analysis of epidemiologic data and the seasonal abundance of ixodes persulcatus ticks in the taiga, zilber assumed that sse was transmitted by ticks, in contrast to "summer encephalitis", which is transmitted by mosquitoes [141] . several strains of the virus were isolated from the ix. persulcatus ticks, and their ability to transmit the virus by biting laboratory animals was shown experimentally [142] . one of the first isolated strains (sofjin) was used for infecting rhesus macaques, which developed the clinical symptoms with signs of central nervous system (cns) impairment, similar to those in sick people [141, 143] . so, the etiological agent of sse, which was subsequently given the name tick-borne encephalitis, was discovered and is now known by the name tick-borne encephalitis virus (tbev). in 1938-1939, subsequent expeditions under the leadership of pavlovsky and smorodintsev studied in detail various aspects of the ecology, epidemiology, and pathogenesis of tbev, as well as the protective properties of the first anti-tbe vaccine, obtained from the brain tissue of mice infected by the tbev strain sofjin [144] [145] [146] . further studies showed that tbev is also prevalent in other regions of the ussr, including the european part, where the main vector of the virus is ix. ricinus ticks. at the same time, it was found that tbev is also an etiological agent of some seasonal encephalitis or febrile illnesses, such as central european encephalitis or biphasic milk fever [147] [148] [149] . the strains of tbev were initially divided into two geographical subtypes ("far eastern" and "central european"). these subtypes differed in severity of the illness and had antigenic differences in virus neutralization tests with serum of convalescents [148] . a third subtype of tbev ("west siberian") was described by pogodina and her colleagues in 1981 [150] . genetic data that has been accumulating since the late 1980s confirms the existence of three main tbev subtypes (or genotypes). the nucleotide difference between genotypes reaches 15-20% when comparing complete genomes [151] [152] [153] . tbe is the most important arboviral infection in russia. despite significant progress in the development of anti-tbev vaccines, thousands of cases are recorded annually in russia, mainly in siberian and far eastern regions [154] . in the modern classification, tbev belongs to the species tick-borne encephalitis virus of the genus flavivirus (flaviviridae) [155] . tbev is widely distributed within the area of its main arthropod vector-ix. persulcatus and ix. ricinus ticks, including russia, eastern and central europe, baltic and scandinavian countries [156, 157] . the discovery of tbev as a causative agent of sse gave an impetus to studies of similar diseases throughout the ussr. during subsequent years, the major virological centres were established as parts of the academy of medical science of the ussr (ams ussr), such as the department of neurovirology at the institute of neurology (1942), the institute of virology (1944), the institute of poliomyelitis and viral encephalitis (1950), as well as departments of virology at regional medical institutes in siberia and the far east. scientists from these centres were actively involved in the study of various zoonotic viral infections distributed in the ussr. many participants of the first expeditions subsequently became famous virologists. one of the most notable ones is michael p. chumakov, who later headed the institute of virology ams ussr (1950) (1951) (1952) (1953) (1954) and the institute of poliomyelitis and viral encephalitis ams ussr (1955) (1956) (1957) (1958) (1959) (1960) (1961) (1962) (1963) (1964) (1965) (1966) (1967) (1968) (1969) (1970) (1971) (1972) in moscow. chumakov organized numerous expeditions that aimed to study the etiology of zoonotic human infections. in the 1940s, outbreaks of the disease, designated by local doctors as "atypical tularemia", "anicteric leptospirosis", and "omsk spring-summer fever", were recorded in several rural regions of the omsk district in western siberia. clinicians from the omsk medical institute, under the leadership of ahrem-akhremovich, described the disease in detail and named it omsk hemorrhagic fever (ohf), as the patients often developed hemorrhagic diathesis. they also suggested that ohf was transmitted by dermacentor reticulates ticks, which are highly prevalent in the region [158, 159] . in 1947, chumakov and colleagues investigated the blood of patients with ohf and isolated 40 strains of a new virus, which was similar but different from tbev in serologic tests. the virus was named omsk hemorrhagic fever virus (ohfv). several strains of ohfv were also isolated from de. reticulates ticks collected in the natural foci of ohf [160, 161] . in subsequent years, the ecology of ohfv was extensively studied by scientists from the omsk medical institute and the institute of poliomyelitis and viral encephalitis ams ussr. the de. reticulatus ticks and their host, a narrow-headed vole (microtus gregalis), are considered an original natural reservoir of ohfv. the european water vole (arvicola amphibius), the tundra vole (microtus oeconomus), and some species of shrews are also involved in the circulation of ohfv [156] . however, the emergence of ohf outbreaks in the 1940s was presumably a consequence of the introduction by humans of muskrats (ondatra zibethicus) to this region in 1935-1936 [162] . muskrats are highly susceptible to ohfv and serve as an extremely effective amplifying host. the appearance and growth of the muskrat population in the natural foci of ohf led to an increase in infection rates in other animals and ticks [162, 163] . in addition to transmission of ohfv by ticks, humans can get infected while hunting and skinning, by direct contact with blood and excretions of infected animals. such "muskrat outbreaks" among hunters and their family members have been registered in the region at different times of the year, including winter, which is the season of active hunting for muskrats [164, 165] . based on antigenic relationships, ohfv was assigned to the tbe antigenic complex [166] , and later was classified as a separate species, omsk hemorrhagic fever virus of the genus flavivirus (family flaviviridae) [155] . genome sequence analysis confirmed the close evolutionary relationships of ohfv with tbev [167] [168] [169] . in 1944, virologists led by chumakov studied the etiology of an outbreak of a febrile illness which was accompanied with hemorrhagic manifestations ("acute infectious capillary toxicoses") in a rural area in the northwest part of the crimean peninsula. they designated the disease as crimean hemorrhagic fever (chf) and suggested that is transmitted by hyalomma (plumbeum) marginatum ticks. despite the absence of virus isolates from specimens from chf patients or from ticks, the viral etiology of chf and its zoonotic nature were proven experimentally by infecting volunteers with the blood of chf patients or a filtered suspension of ticks collected from a hare caught in the focus of the disease [170] . sporadic cases and outbreaks of chf were subsequently recorded almost annually in southern regions of the european ussr and central asian soviet republics. the first strains of the chf virus were isolated by alexander m. butenko from chumakov's team in 1967, from sera of chf patients and from hy. marginatum nymphs isolated in southern russia [171, 172] . later, the chf virus was shown to be identical to the congo virus isolated from a patient with hemorrhagic fever in zaire (present day democratic republic of congo) and the virus received its present name, the crimean-congo hemorrhagic fever virus (cchfv) [173] . cchfv is a one of the prototypic nairoviruses and today is assigned to the species crimean-congo hemorrhagic fever virus, of the genus orthonairovirus (nairoviridae: bunyavirales). during spring and summer 1962, chumakov, together with libíková from the institute of virology in bratislava (former cžechoslovakia), investigated an outbreak of fibrile illness in the kemerovo district in western siberia. initially, it was assumed that the patients were affected by tbe, but the sera of the patients did not react with tbev-specific antigen in serological tests. on the contrary, a new virus, named the kemerovo virus (kemv), was isolated from the blood of patients. several strains of kemv were also isolated from ix. persulcatus ticks collected in the region where the outbreak occurred [174, 175] . similar to kemv, the tribeč virus and lipovníc virus were isolated from ix. ricinus ticks in czechoslovakia in 1963 [176, 177] . based on morphological studies, kemv was classified to the genus orbivirus (family reoviridae) [178] . the ecology of kemv in russia has not been studied sufficiently, but recent research has shown that its prevalence in ix. persulcatus, ix. ricinus, ix. pavlovsky, and de. reticulatus ticks varies from zero to 10.1% in different regions of the country [179, 180] . from the above, it follows that in the period 1930-1960, arboviruses in the ussr were studied mostly as causative agents of human disease. examinations of arthropods and vertebrates in the natural foci of important human disease often led to exploring some other arboviruses. for example, butenko isolated the west nile virus (wnv) and dhori virus (dhov) from hy. marginatum ticks for the first time in the ussr while studying the natural foci of cchfv in the southern region of russia in 1964 [181] . in the late 1960s, there was an ecological trend in virology developing in the ussr. the founder of the ecological approach to virology in the ussr was dmitry k. lvov, who established the department of the ecology of viruses at the d.i. ivanovsky institute of virology in moscow (1967) , and later headed the institute (1987-2014). under his leadership, an ecological and virological survey was organized, aimed to identify the arboviral diversity in hematophagous arthropods and wild animals of the entire ussr. the survey included collecting and examining mosquitoes, ticks, and vertebrate animals (mostly rodents and birds), as well as samples from humans, in different types of biocenoses located in different climatic zones of the ussr. lvov and colleagues isolated more than 500 strains of different mosquito-borne viruses, including viruses of the california encephalitis antigenic group (species california encephalitis orthobunyavirus) and batai and batai-like viruses (species bunyamwera orthobunyavirus) in the genus orthobunyavirus, family peribunyaviridae [182] [183] [184] [185] . the other mosquito-borne viruses whose circulation was discovered and studied extensively, are the sindbis virus (sinv) and getah virus (getv) (genus alphavirus, family togaviridae) [186] [187] [188] . one of the important subjects of d. lvov's research was ix. uriae ticks, which parasitize on colony-nesting sea birds. in 1969-1974, more than 240 virus strains were isolated from ix. uriae ticks collected in the nests of sea birds on the coasts and islands in the sea of okhotsk, the bering sea, and the barents sea [189] . the isolated strains were mostly classified as novel bunyaviruses, flaviviruses, and orbiviruses, often based on morphological studies of the virion structure only, because their antigenic relationships with other viruses were not known at the time. among them, the sakhalin virus (sakhv) and paramushir virus (prv) were described as a novel bunyaviruses and later classified to the species sakhalin orthonairovirus (genus orthonairovirus, family nairoviridae) [190] . several other new viruses (zaliv terpenia, comandory, and rucutama viruses) were discovered and now belong to the species uukuniemi phlebovirus (genus phlebovirus, family phenuiviridae) [191] . the tyuleniy virus (tyuv) was isolated for the first time, which is one of the prototypic viruses of seabird tick-borne flaviviruses group (genus flavivirus, family flaviviridae) [192] . the prevalence of the okhotsky virus (okhv) and aniva virus (aniv), two newly described viruses belonging to the species great island virus (genus orbivirus, family reoviridae), have been studied in detail [156, 189, 193] . many new viruses were discovered by lvov and his colleagues while exploring the territories of central asia and transcaucasia. they isolated and studied the issyk-kul virus (iskv), which is associated with bats of the family vespertionidae and their argasid ticks [194, 195] . new viruses, tamdy (tamv) and burana (burv), were isolated from hyalomma spp. ticks collected from sheep or cows in pasture lands [196] . some novel viruses (artashat, chim, and geran viruses) were isolated from argasid ticks collected in rodent burrows [197] . morphological studies of these viruses by electron microscopy identified them as bunyaviruses. recently, they were classified as different species of the genus orthonairovirus (family nairoviridae) [198] . in total, during the ecological and virological surveys of the 1970s to 2000s, thousands of strains of different arboviruses were isolated, some of which remain to be classified. based on the studies conducted by soviet and russian virologists, we now know that at least 80 zoonotic viruses, assigned to eight viral families, circulate on the territories of the former ussr [156] . does this number reflect the true diversity of viruses circulating in the vast territory of northern eurasia? this question can only be answered by additional research aimed at finding new viruses, using new methods and approaches. the concept of viruses developed from the observations of ivanovsky and beijerinck of "filterable agents", with the discovery of the causative agent of tobacco mosaic in the 1890s. yellow fever virus and dengue virus were the first two arboviruses to be isolated early in the 20th century [199, 200] . the pioneering work of alexis carrel on the development of many cell and tissue culture methods in the 1910s at the rockefeller institute, and later refinements by maitland, eagle, and enders, led to the widespread use of various culture systems as indispensable tools for virus studies [201] [202] [203] . although these tools have since been used extensively for the in vitro characterization of viruses, they were inadequate for the identification and classification of a flood of novel viruses collected through the yfv surveillance program supported by the rockefeller foundation. jordi casals, among others, led the use of the complement fixation (cf) test in order to study viruses affecting the central nervous system. the cf test exploits the unique affinity of complement for antigen-antibody complexes. the original assay was developed in the 1920s for the serologic study of yfv, was improved in the 1930s [204, 205] , and its sensitivity and specificity improved again in the 1950s [206] . using cf tests, casals and his colleagues were able to classify viruses into antigenic groups. however, the inherent complexity and labour consuming aspects of the assay (titrations of antigen, complement, and hemolysin for optimal outcomes), technical demands (accurate interpretation of outcomes) and the development of alternative assays (see below), have restricted its applicability in laboratories worldwide. hirst observed in 1941 that chicken erythrocytes agglutinated in the presence of the influenza a virus and that virus-specific antibodies inhibited agglutination, forming the foundation for the hemagglutination inhibition (hi) test [207] . a decade later and on theiler's suggestion, casals showed that many arboviruses also agglutinated erythrocytes, establishing the hi test as a diagnostic tool for arbovirus infection and identification [4] . the gold standard for arbovirus identification, the plaque reduction neutralization test (prnt), has its origins in the observations of stokes and colleagues, dating back in the 1920s when monkeys could be protected against yfv by the inoculation of convalescent sera from patients who had recovered from the disease [200] . by the early 1930s, max theiler had adapted the assay for use in mice, in which mixed serum and virus was inoculated intracerebrally [208] . the cell culture adaptation of the test was first demonstrated by itoh and melnick in 1957 for studying the seroconversion of chimpanzees infected with echoviruses [209] , and a year later by henderson and taylor to detect antibodies to the eastern equine encephalitis virus [210] . versions of this assay are now widely used, including the microprnt [211] , the virus reduction neutralization test (vrnt) [212] , the focus reduction neutralization test (frnt) [213] , the rapid fluorescent inhibition test (rffit) [214] , the flow-cytometry neutralization [215, 216] , the colorimetric micro-neutralization assay (cmnt) [217] , and the reporter virus particle-based neutralization assays [218] [219] [220] [221] . historically, these methods (virus isolation, hi, cf, and neutralization tests) served as the basis of arbovirus diagnosis for many years, augmenting electron microscopy (see section below), which allowed the visualization of viruses in infected tissues and cell cultures. however, an inherent limitation of the serology-based assays has been their inability to determine whether antibodies in the examined serum were the result of a recent or past infection. this conundrum was solved by determining whether antibodies were igm (recent infection) or igg (past infection), using the enzyme-linked immunosorbent assay (elisa) [222] . the introduction of elisa revolutionized the field by also offering increased specificity and sensitivity for the accurate detection of many viruses. overall, although identification of pathologic agents through serologic assays is quite straightforward, there are instances where accurate identification may not be possible due to cross-reactivity. for example, cross-reactivity among flaviviruses poses a challenge in their identification, even when the "gold standard" of prnt for arbovirus detection is applied, especially in hyper-endemic settings of flavivirus circulation. several diagnostic labs faced this challenge during the recent emergence and explosive spread of the zika virus in the americas. a similar challenge is also common for the serologic diagnosis of bunyavirus infections, which is attributed to their ability to reassort. in this scenario, a novel bunyavirus may be misidentified as a known pathogen due to the presence of the m segment (contributed by the known pathogen), which encodes the immune-reactive envelope proteins (reviewed in [223] ). since the beginning of modern virology in the 1950s, transmission electron microscopy (tem) has been one of the most important and widely used techniques for the identification and characterization of new viruses. two tem techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section tem of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. for some viruses, tem can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). for successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, and so either high multiplicity of infection (moi) or rapid virus multiplication is required. viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. in certain cases, confirmation can be obtained by immuno-em performed either on virus suspension before negative staining or on ultrathin sections. this requires virus-specific primary antibodies, which might be not available in the case of a novel virus. for on-section immuno-em, oso4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually lr white). the ultrastructure of most common viruses is well documented in good atlases and book chapters [224] [225] [226] [227] [228] [229] [230] [231] [232] [233] [234] [235] and many classical publications of the 1960s, 1970s, and 1980s. several excellent reviews were recently published on the use of tem in the detection and identification of viruses [236] [237] [238] . the advent of next generation sequencing (ngs) has expanded the tool kit of the virus hunter. for many years, sanger-based sequence analysis has been employed in the identification and characterization of viruses [239] [240] [241] [242] . however, with the completion of the human genome sequence, the necessity for a high throughput approach that provided a massively-parallel sequencing strategy was fully apparent [243, 244] . the automated sanger method was considered a first-generation technology and newer methods are referred to as next generation sequencing (ngs). commercially available technologies from roche/454, illumina, life technologies/apg, oxford nanopore, and pacific biosciences offer unique ngs platforms, and all have been extensively reviewed [245] [246] [247] [248] [249] . unlike sanger sequencing, ngs does not require prior knowledge of the viral sequence and thus can be used for viruses of unknown sequence. many viruses cannot be cultured in the cell culture systems currently in use. ngs has shifted the paradigm by removing the need for cell culture and so opening the door to the discovery of many new viruses. the first instruments for ngs were developed in the 1990s and commercialized in the early 2000s, and they were quickly adopted for the identification of novel viruses from a wide range of sources. the number of known viruses increased from about 3,000 in 2005 to approximately 11,000 viruses in 2013 [250] , and that number has since increased dramatically. the technology has also been applied to sequence analysis of many previously known but poorly characterized viruses. this has significantly expanded the known virosphere and assisted in understanding the diversity and evolutionary relationships of viruses. the expansion of the known virosphere has also allowed the taxonomic assignment of an increasing number of viruses, with a total of 14 orders, 150 families, 1019 genera, and 5560 species of viruses currently approved by the international committee on taxonomy of viruses (ictv) [251] [252] [253] [254] [255] [256] . ngs has also allowed sequence analysis of hundreds or thousands of isolates known pathogenic viruses, facilitating epidemiological studies at scales extending from very local to global. this has generated a trove of new knowledge of significant public health importance. however, without the availability of isolates, ngs has not necessarily increased our understanding of the ecology of novel viruses, their host range, and the risks they may pose to public, veterinary, agricultural, or environmental health. the application of ngs to the unbiased mass sequencing and bioinformatic analysis of total nucleic acids extracted from biological samples obtained from a wide range of sources has led to an explosive increase in the number of complete or near-complete viral genomes. for example, a recent study using ngs to sequence the transcriptomes of arthropods representing 70 species from four classes (insecta, arachnida, chilopoda, and malacostraca) identified 112 novel viruses, many of which are represented by complete or near-complete genomes [257] . the novel viruses encompass the entire taxonomic diversity of previously known families and/or genera of (-) ssrna viruses and include divergent viruses with entirely novel and unusual genome architecture. similarly, sequence analysis of the transcriptomes of animals of more than 220 species sampled across nine metazoan phyla (arthropoda, annelida, sipuncula, mollusca, nematoda, platyhelminthes, cnidaria, and echinodermata), as well as chordates of the subphylum tunicata (salps and sea squirts), resulted in the discovery of 1445 rna viruses, mostly represented by complete or near-complete genomes [258] . based on phylogenetic analysis of rna polymerase (rdrp) domain sequences, the novel viruses included clades representing many established families of plant and animal rna viruses, as well as at least five clades that are so divergent that they are considered as likely new virus families or orders. also, the sequencing of transcriptomes of gut, liver, and lung or gill tissue of fish, reptiles, amphibians, and birds identified 214 novel vertebrate-associated viruses, representing every family or genus of rna virus associated with vertebrate infection, including those containing important human pathogens (orthomyxoviruses, arenaviruses, and filoviruses) [259] . these and other similar studies have heralded a new era in virology, revealing new dimensions in viral biodiversity and providing largely unexpected insights into the deep evolutionary history of viruses. however, only a minor subset of newly discovered viruses has been subject to full phenotypic characterization which can provide critical and fundamental insights into their biology and virus-host interactions, ultimately transforming our understanding of the evolutionary forces that shape the virosphere and disease emergence. realistically, as important as these discoveries are to the advancement of science, very few of the viruses will ever cause human disease or influence the global economy. this mass sequencing approach, which has been called viral metagenomics, can also be applied in a more targeted way to identify viruses in clinical cases of diseases of unknown etiology or to survey for potentially novel zoonotic viruses that may represent a significant risk of transmission to humans. indeed, ngs is being used increasingly in conjunction with real-time pcr as a front-line tool in medical and veterinary settings for rapid detection and identification of exotic or unknown emerging viruses. for example, in 2009, an outbreak of acute hemorrhagic fever occurred in mangala, democratic republic of congo (drc), involving three human cases, two of which were fatal. as no positive diagnosis was obtained using real-time pcr for known viral hemorrhagic fevers in africa, ngs was conducted on acute phase serum collected from the surviving patient, revealing the near-complete sequence of a novel rhabdovirus, bas-congo virus (basv; species bas congo tibrovirus) [260] . although the disease was attributed by the investigators to the novel virus, no isolate was obtained and there was no evidence of neutralising antibodies in 43 serum samples from undiagnosed hemorrhagic fever cases or 50 random serum donors from the drc. subsequently, ngs of blood collected from healthy individuals from nigeria identified two related viruses, ekpoma virus 1 (ekv-1; species ekpoma 1 tibrovirus) and ekpoma virus 2 (ekv-2; species ekpoma 2 tibrovirus), and a serological survey indicated that antibodies to these or similar viruses occur commonly in healthy humans in nigeria [261] . however, once again, neither virus was isolated. interestingly, several other tibroviruses had previously been isolated from healthy cattle or biting midges (culicoides spp.) in australia and florida [79, 83, 262, 263] . none have been associated with either disease in livestock or the infection of humans [83, 264] . these and other studies raise important issues regarding the significance of ngs data, even when providing complete or near-complete viral genome sequences, when investigating disease etiology. most viruses can cause asymptomatic infections and many newly discovered viruses may be benign in their natural host. therefore, in the absence of a virus isolate which can be used for experimental studies, establishing a causal association with disease based only on detection of the viral genome should be approached cautiously. the availability of a rapidly expanding number of novel viral genomes identified by metagenomic studies has also presented challenges for virus taxonomy-a system for classification of viruses that is administered by the international committee on taxonomy of viruses (ictv). should viruses detected only by their nucleotide sequence be classified and assigned to species and other higher taxa (genus, family order, etc.) alongside viruses for which we have a viable isolate? the ictv (then the international committee on nomenclature of viruses) was established in 1966 at the ninth international congress for microbiology in moscow, publishing its first report in 1971 [265] . it operates under the auspices of the international union of microbiological societies (iums), with the authority to develop, refine, and maintain a universal virus taxonomy. historically, the description and classification of a new virus by the ictv required significant information, such as host range, serology, replication cycles, and structure, aspects that could be determined from the study of isolates. on the other hand, sequences alone provide a trove of information, including evolutionary relationships (e.g., phylogeny), genome organization (e.g., the number of genes and their order), presence or absence of distinctive motifs (e.g., protein cleavage sites, terminal sequences, internal ribosome entry sites), as well as genome composition (e.g., codon usage, gc content), which of course could be used to inform classification into species. these concerns framed the contents of a workshop of experts and members of the ictv, resulting in a seminal consensus statement in which viruses identified only from metagenomic data are considered to be bona fide viruses and thus candidates for taxonomic assignment [266] . the expanding diversity of the known virosphere also presented challenges. a recent analysis of metagenomes of 3042 geographically and ecologically diverse samples led to the discovery of 125,842 new partial dsdna viral genomes encoding more than 2.79 million proteins, 75% of which had no sequence similarity to proteins from known virus isolates [267] . other metagenomic studies have revealed similar diversity in ssdna and rna viruses, particularly in marine ecosystems [268] . this has led ictv to consider a far broader framework for taxonomic assignment of viruses, recently approving the establishment of a taxonomic hierarchy that includes 15 ranks (realm, subrealm, kingdom, subkingdom, phylum, subphylum, class, subclass, order, suborder, family, subfamily, genus, subgenus, and species), thus expanding the range even beyond those currently available for other organisms [269] . the sheer volume of new virus genomes identified by metagenomic studies has also led to the development of new bioinformatic tools, that are increasingly being applied for automated virus classification of viruses, based almost exclusively on nucleotide sequence data [270] [271] [272] [273] [274] [275] [276] [277] . while the classic tools of virus discovery and characterization (e.g., em, serology, and tissue culture) are still widely used, ngs allowed for the rapid identification of an enormous repertoire of viruses, thus exponentially expanding the boundaries of the known virosphere. the resultant genomic sequences allowed for their accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. below are a few representative examples of how ngs transformed the known relationships of arboviruses within their respective families. rhabdoviruses contain negative-sense (-) single-stranded rna (ssrna) genomes. they are amongst the most numerous and diverse of rna viruses, naturally infecting mammals, birds, fish, reptiles, and amphibians, as well as insects, arachnids, crustaceans, nematodes, and a wide range of plants [278, 279] . most (but not all) rhabdoviruses that infect vertebrates are transmitted by hematophagous arthropods. the rhabdoviridae currently comprises 20 genera containing 143 species and one unassigned species [280] . of these, viruses assigned to seven of the genera (vesiculovirus, ephemerovirus, tibrovirus, hapavirus, ledantevirus, curiovirus, and sripuvirus) are considered to be arboviruses. viruses assigned to the newly characterized genus almendravirus appear to be insect-specific [281] . viruses assigned to the genus tupavirus have been isolated only from vertebrates but may possibly have arthropod vectors. complete coding sequences are now available for more than 100 other rhabdoviruses, many of which have been isolated from or detected in arthropods, but they have not yet been formally classified. while the classic tools of viral discovery (e.g., em, serology, and tissue culture) are still widely used, ngs has played a central role in recent genome sequencing efforts, revealing diversity, not only in the ecology of rhabdoviruses but also in the structural diversity of genome architecture [252, [282] [283] [284] [285] [286] [287] . in addition to the five canonical rhabdovirus structural protein genes (n, p, m, g, and l), it is now recognized that rhabdoviruses commonly contain multiple long open reading frames encoding putative accessory proteins, mostly of unknown function. ngs has also facilitated studies of the evolution of rhabdovirus genome organization [252] and revealed the importance of arthropods in the evolutionary history of rhabdoviruses [257] . according to the current international committee on taxonomy of viruses (ictv), classification of the order bunyavirales encompasses 10 families of segmented (-) ssrna viruses-arenaviridae, hantaviridae, nairoviridae, peribunyaviridae and cruliviridae, mypoviridae, fimoviridae, wupedeviridae, phasmaviridae, and phenuiviridae-a classification based on structural, genetic, and antigenic characteristics [288] . bunyavirales is a diverse order with a large number of viruses associated with human, veterinary, and plant disease, as well as being vectored by arthropods (mosquitoes, ticks, sandflies, and thrips) and infecting a wide range of other invertebrates. the recent discovery of gouléako (golv) [254] and cumuto (cumv) [289] viruses (the latter with ngs) which are evolutionarily related to but distinct from viruses in the genus phlebovirus, family phenuiviridae, suggesting they are to be assigned to the new genus goukovirus. in the past five years, ngs has revolutionized understanding of the vast diversity of this order, by allowing genetic identification of previously uncharacterized and unassigned bunyaviruses, which in turn provided a complimentary approach to the gold standard of classification (structural, genetic, and antigenic characteristics) for a more refined taxonomic classification. combined, these approaches will be instrumental for enhancing our understanding of their ecologic and geographic distribution, as well as public health impact. the family flaviviridae comprises positive-sense (+) ssrna viruses. the family contains several serious human pathogens, including dengue, yellow fever, zika, japanese encephalitis, west nile, and tick-borne encephalitis viruses (all arboviruses in the genus flavivirus) and the hepatitis c virus (a member of the genus hepacivirus). members of the genus flavivirus, like the alphaviruses (see section 5.4), are a diverse group of arthropod-borne viruses that are found on every continent except antarctica. several flaviviruses have been discovered and characterized recently, mainly through chance or increased surveillance efforts, and facilitated through ngs. newly characterized viruses include the mercadeo virus (mecdv), from pools of culex species mosquitoes in panama [290] , sabethes flavivirus (sbfv), from sabethes belisarioi in brazil [291] , the la tina virus (ltnv), from aedes scapularis in peru [292] , the long pine key virus (lpkv), from anopheles crucians in the florida everglades, and the kampung karu virus (kpkv), from anopheles tesselatus in borneo [292] , aedes flavivirus (aefv), from aedes albopictus laboratory colony that originated in thailand [293] , xishuangbanna flavivirus (xfv), from aedes albopictus in china [294] , and the cuacua virus (cucuv), from mansonia ssp. in mozambique [295] . one unexpected finding was the discovery of segmented viruses that grouped in the family flaviviridae and were more closely related to flaviviruses than members of other established genera. the jingmen tick virus (jmtv) is unique in the family flaviviridae with a four segment positive-sense genome [296] . traditional sanger sequencing generated the ns3 and ns5 gene sequences but could not connect these within a single genome segment. phylogenies based on either ns3-like or ns5-like sequences, which are encoded on two of the four segments, showed jmtv falling basal on the flavivirus tree and clearly distinct from the other viruses in this genus. the remaining two segments did not appear to have a flavivirus origin. jmtv has been isolated from cattle, monkeys, and ticks (rhipicephalus and haemaphysalis spp.) [297] . this prototype virus has lent its name to the newly identified segmented flaviviruses, and other viruses have recently populated the jingmenvirus clade. similarly, the guaico culex virus (gcxv) consists of five segments, and initial studies suggested gcxv is insect-specific, based on its isolation from culex ssp. mosquitoes [298] . additional potentially insect-specific four-segmented viruses include shuangao insect virus 7 (saiv7), the wuhan flea virus (whfv), wuhan aphid virus 1 (whav1), wuhan aphid virus 2 (whav2), and the wuhan cricket virus (whcv) [299] . interestingly, sequences that are similar to toxocara canis, a larva roundworm, and tentatively named t. canis larva agent virus (tclav), are distantly related to jmtv and appear to be the first evidence of a flavivirus-line organism in a member of the phylum nematoda [296] . the discovery and characterization of a segmented genome flavivirus has significant implications, as it reveals that rna virus segmentation is an evolutionary process that has occurred in previously unanticipated circumstances. the alphaviruses are a diverse group of (+) ssrna arthropod-borne viruses that are found on every continent except antarctica [300] . there were no known mosquito-only alphaviruses until 2012, when analysis by ngs of a mosquito-pool from the negev desert in israel showed the presence of an alphavirus, the eilat virus (eilv) [301] . further analysis showed that, although eilv could infect mosquito cells, it was unable to replicate in vertebrate cells [302] . eilv is considered a vaccine candidate, as it can produce immunity without replication in the vertebrate host, illustrating the importance of such discoveries, which can lead to novel platforms for the prevention and/or treatment of disease [303] . reoviruses are a diverse family of double-stranded rna (dsrna) viruses that infect a wide range of hosts and have a wide range of characteristics. of the 30 genera, there are only three for which novel viruses have been described using ngs, and the majority of these are in the genus orbivirus. interestingly, because of the structure of the genomes of reoviruses and the conserved sequences at the 5' and 3' ends of the segments [304] , it is relatively simple to sequence segments of reoviruses using more traditional techniques. this may be why there are so few novel reoviruses determined by ngs. novel viruses identified using ngs have been assigned to four other genera (seadornavirus, orthoreovirus, dinovernavirus, and cypovirus), all of which are shown in table 1 . 1 refers to year of the publication and not of the isolation of the virus. *not yet formally classified. newly recognized viruses containing a (+) ssrna genome have been proposed to form a new genus negevirus, related to genera of mite-infecting plant viruses (blunervirus, cilevirus, and higrevirus) in the new family kitaviridae [251, 253] . originally, six viruses with restricted host range in insects (isvs), designated as negev (negv), ngewotan (nwtv), piura (piuv), loreto (lorv), dezidougou (dezv), and santana (sanv), were identified in and isolated from mosquitoes and phlebotomine sandflies, collected in brazil, the ivory coast, israel, indonesia, peru, and the usa, [251] . their widespread geographic distribution was documented by several other groups, who reported the isolation and characterization of related viruses in the philippines (tanay virus; tanav) [315] , trinidad and tobago (wallerfield virus; walv) [289] , côte d'ivoire (goutanap virus; ganv) [253] , portugal (ochlerotatus caspius negevirus; ocnv and culex univittatus negevirus; cunv) [316] , brasil (brajeira and wallerfield viruses), colombia, and nepal. collectively, the close relationship of negeviruses with plant viruses of the genera cilevirus, higrevirus, and blunervirus, coupled with their heterogenous genome organization and architecture, provides support for the possibility that negeviruses are plant-like viruses that could eventually anchor a new virus family [317] . members of the family mesoniviridae, a newly discovered family of (+) ssrna viruses assigned to the order nidovirales [318, 319] , appear to have an extensive geographic distribution but a restricted host range within members of the family culicidae (flies) [251, [318] [319] [320] [321] [322] [323] [324] [325] . the mesoniviridae comprise of a single genus alphamesonivirus with nine recognized species: alphamesonivirus 1, including nam dinh (ndiv) [318] , cavally (cavv) [319] , ndiv a12.2520 [324] , ndiv ngewotan, and ndiv houston [326] viruses; alphamesonivirus 2, including the single isolate of the karang sari (ksav) virus, and the four bontag baru (bbav) isolates sampled in the early 1980s in indonesia, but only recently characterized [326] ; alphamesonivirus 3, including the dak nong virus (dknv), the three isolates of kamphaeng phet (kphv), sampled in indonesia and thailand in the mid-1980s [322, 326] ; alphamesonivirus 4, including the casuarina virus (casv), isolated in australia from coquillettidia xanthogaster mosquitoes [323] ; alphamesonivirus 5, the african hana virus (hanav) [320] ; alphamesonivirus 6 and 7, including ofaie (ofav) and kadiweu (kadv) viruses, isolated in the pantanal of brasil from mansonia sp. mosquitoes, respectively [327] ; and alphamesonivirus-8 and 9, including the african nse (nsev), and meno (menov) viruses [320] ; and the distinct yet unassigned species, the yichang virus [325] , isolated from culex sp. mosquitoes in china. across several generations, virus hunters have left a profound legacy, both to science and to the broader global community. in the field, their work has often been conducted in difficult, demanding, and sometimes dangerous circumstances. in the laboratory, they have developed and applied tools and methodologies that led to improved diagnosis, prevention, and treatment of viral disease, and still lie at the center of virology today. others working at more fundamental scientific levels have used their virus isolates as scientific models, opening the door to a revolution in molecular and cellular biology. yet, their work continues. viral disease emergence remains one of the most serious threats to humanity, with potentially devastating social and economic consequences. recent examples, such as sars, mers, henipa-, ebola, and zika viruses, illustrate the threat that unidentified or poorly characterized viruses can, and almost certainly will, continue to present. equally devastating emerging diseases of livestock, fisheries, and crops threaten food production and livelihoods. as history has repeatedly shown us, technological revolutions have been often accompanied by periods of progressive disinvestment in training for the eclipsed technologies of the past, and in our case, classical virology. the recent emergence of the zika virus in the americas has reinforced the notion that traditional methods of virus discovery and new technologies offer a complimentary toolkit, especially in resource-poor settings that can be seamlessly 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geographic distribution and host range novel viruses isolated from mosquitoes in pantanal, brazil the authors thank ashley rhame and dora salinas for excellent help in preparation of the manuscript. the authors declare no conflict of interest. key: cord-331255-t85yioyl authors: rohr, jason r.; barrett, christopher b.; civitello, david j.; craft, meggan e.; delius, bryan; deleo, giulio a.; hudson, peter j.; jouanard, nicolas; nguyen, karena h.; ostfeld, richard s.; remais, justin v.; riveau, gilles; sokolow, susanne h.; tilman, david title: emerging human infectious diseases and the links to global food production date: 2019-06-11 journal: nat sustain doi: 10.1038/s41893-019-0293-3 sha: doc_id: 331255 cord_uid: t85yioyl infectious diseases are emerging globally at an unprecedented rate while global food demand is projected to increase sharply by 2100. here, we synthesize the pathways by which projected agricultural expansion and intensification will influence human infectious diseases and how human infectious diseases might likewise affect food production and distribution. feeding 11 billion people will require substantial increases in crop and animal production that will expand agricultural use of antibiotics, water, pesticides and fertilizer, and contact rates between humans and both wild and domestic animals, all with consequences for the emergence and spread of infectious agents. indeed, our synthesis of the literature suggests that, since 1940, agricultural drivers were associated with >25% of all — and >50% of zoonotic — infectious diseases that emerged in humans, proportions that will likely increase as agriculture expands and intensifies. we identify agricultural and disease management and policy actions, and additional research, needed to address the public health challenge posed by feeding 11 billion people. i nfectious diseases are emerging at an unprecedented rate with significant impacts on global economies and public health 1 . the social and environmental conditions that give rise to disease emergence are thus of particular interest, as are management approaches that might reduce the risk of emergence or re-emergence 1 . at the same time, undernutrition -the insufficient intake of one or more nutrients -remains a major source of global ill health [2] [3] [4] [5] . together, the unprecedented rate of infectious disease emergence and the need to sustainably feed the global population represent two of the most formidable ecological and public health challenges of the twentyfirst century [5] [6] [7] [8] [9] (fig. 1) , and they interact in complex ways (fig. 2) . although modern agricultural technologies have reduced hunger, improved nutrition and spared some natural ecosystems from conversion to agriculture, current global agricultural production and distribution infrastructure still leaves billions of people's diets deficient in one or more crucial nutrients, with major consequences for global morbidity and mortality 10 , and these deficiencies are expected to worsen with climate change [11] [12] [13] [14] . credible estimates are available for specific nutrient shortfalls -for example, 1.6 billion people suffering iron-or vitamin b 12 -deficiency anemia 15 , 0.8 billion people with insufficient dietary energy (that is, calorie) intake 16 , 33% of pre-school age children and 15% of pregnant women at risk of vitamin a deficiency 17 -but the world still lacks a rigorous, recent assessment of the population suffering shortfalls of any one or more nutrients, although the number is surely in the billions. by 2100, the united nations projects that the global population will grow by nearly 4 billion, to exceed 11 billion people 18 . meeting the united nations' sustainable development goal, to "eradicate hunger" (https://sustainabledevelopment.un.org/) for this expanding human population will necessitate a large increase in food supplies, with major changes to agricultural production and distribution systems, infrastructure and social protection programmes 6 (fig. 3) . despite large and, in many cases, growing inequalities, the global population is on average expected to become richer, and historically, with greater affluence has come greater consumption of food products in general and more animal-sourced foods in particular, both of which further increase food demand and thus the requirement for agricultural expansion and/or intensification 19 . agriculture already occupies about half of the world's land and uses more than two-thirds of the world's fresh water 20 , and recent studies have suggested that agricultural production might need to double or triple by 2100 to keep pace with projected population growth and food demand [6] [7] [8] 21 (fig. 3) . meeting this demand using present agricultural production systems could require replacing >10 9 hectares of natural ecosystems with agricultural production, approximately 7% of the global land area, which is larger than the continental united states, although continuous efficiency improvements in agriculture will compel re-evaluation of these estimates. in turn, this agricultural expansion could result in an estimated ~2-fold increase in irrigation, ~2.7-fold increase in fertilizer 6,10,22 and 10-fold increase in pesticide use (fig. 3) . the challenges of feeding >11 billion people and managing infectious diseases intersect in several ways ( figs. 1 and 2) . first, the expansion and intensification of agriculture are disproportionately occurring in tropical, developing countries 6, 22, 23 , where 75% of deaths are attributable to infectious diseases 24 , where the risk of disease emergence might be greatest, and where disease surveillance and access to health care, particularly for those infections that accompany extreme poverty, are most limited 1, 25 . second, agricultural expansion and intensification have historically come with massive habitat conversion, contamination with animal waste and increasing use of agricultural inputs, such as pesticides and antibiotic growth promoters. beyond their direct adverse effects on human health 26, 27 , agricultural biochemical inputs are known to have direct effects on emerging human infectious diseases, and can also serve as indirect drivers by contributing to the emergence of wildlife diseases 28 that constitute important sources of emerging infections in humans 1, 29 . however, the strengthening of agricultural production systems can also improve nutrition, which has pronounced benefits for combating many infectious diseases at the individual and population levels 2, 30 . although concerns have been raised regarding the environmental impact of agricultural expansion and intensification 6,8-10,22,31 , their effects on infectious disease risk have not been synthesized. here, we review both the beneficial and adverse effects of agricultural expansion and intensification on the transmission of human infectious diseases. we synthesize the pathways through which agricultural practices influence human infectious diseases, and vice versa, and identify opportunities to minimize the adverse consequences of agricultural growth while maximizing the human health benefits of agricultural development (fig. 1 ). agricultural development can yield direct improvements to nutrition, and through several mechanisms, nutrition can be a critical determinant of infectious disease susceptibility and progression 4,32 (figs. 1 and 2). for example, immune responses are energetically costly 33 and thus undernutrition often reduces the development and effectiveness of immune responses that can limit or clear infections. the relationship between infectious disease and nutritional agricultural production can improve human health by reducing food prices and enhancing nutrition, which can increase resistance to infectious diseases. however, freshwater habitats established for irrigation, as well as other agricultural inputs, often increase the risk of vector-borne diseases, such as malaria and schistosomiasis. in general, rural residents are most vulnerable to these increases in infectious disease, whereas consumers some distance away derive most of the benefits from increased food production. to maximize human health given the impending 11 billion humans projected on the planet by 2100, society must minimize the adverse consequences of agricultural growth while maximizing the health benefits. images by kate marx. status can function in reverse as well, because many parasitic infections place direct demands on host nutrition, causing undernutrition when food is limited 34 . in fact, some parasites, such as helminths, can even cause eating disorders, such as geophagy (desire to eat soil), bulimia and anorexia 35 . enduring infections often require rapid and effective repair of tissue damage caused by parasites, which is also costly. hence, certain infections can cause undernutrition, which itself can compromise both resistance and tolerance to infections 34, 36 . by improving nutrition, agricultural development should facilitate combating many infectious diseases. for example, death rates from acute respiratory infections, diarrhoea, malaria and measles, diseases that on average kill more than a child every 30 seconds (1 million per year) 37 , are much higher in children who suffer undernutrition than in those that do not 3, 4 . in addition, poor maternal nutrition and associated impaired fetal growth are strongly associated with neonatal death from sepsis, pneumonia and diarrhoea 4 ; undernourishment is a well-understood risk factor for 4 . although the traditional green revolution approach to food production has succeeded in reducing undernourishment arising from insufficient calorie and protein intake, it has been very slow in reducing micronutrient deficiencies 39 , which can be have significant effects on defence against disease. to make matters worse, where violence, unrest and terrorism impede access to food, such as in parts of africa and central asia, morbidity and mortality attributed to communicable diseases can be further exacerbated 2 . although research suggests that improving nutrition will generally improve responses to infectious diseases, there may be important exceptions. for certain diseases, such as schistosomiasis and many respiratory infections, pathogenesis is a product of host immunity, often from hyperinflammation 40, 41 . thus, undernutrition can actually decrease symptoms by reducing the strength and pathogenicity of immune responses 40 . in addition, as nutrition improves, some parasites might proliferate faster than immunity can increase 34 , resulting in more, rather than less, morbidity, with the classic example that dietary intake of iron can increase malariainduced mortality 42 . in this section, we review the links between rural economy, infrastructure and infectious disease. , phosphorous (c), fertilizer and pesticide use (d), cropland area (e), and irrigated land area (f) and associated 95% prediction bands (light blue bands). 'nitrogen' refers to the normalized estimated global amount of nitrogen nutrients in fertilizers produced (originally reported in thousands of tonnes, now in metric tonnes). 'phosphorous' refers to the normalized estimated global amount of p 2 o 5 nutrients in fertilizers produced (originally reported in thousands of tonnes, now in metric tonnes). for a, data were collected from the world bank. for b-f, data were collected from the food and agriculture organization of the united nations and statistical models were developed based on the relationships between these variables and global human population density. after the best model was identified, year was substituted for human population density based on the fit in a. these projections should be evaluated with considerable caution because the models assume that human population size is the only factor that affects fertilizer and pesticide use and the amount of arable and irrigated cropland, when in reality, many factors affect these responses (for example, climate, diets, type of crops grown and so on). they merely illustrate that even an extremely parsimonious model seems to track past patterns tolerably well and thus could serve as a basis for coarse projections. see supplementary methods for details of the statistical models used to generate these figures and supplementary table 1 for coefficients and statistics for the best-fitting models. economic development. economic development, especially agricultural development, has historically driven reductions in both infectious diseases and poverty across many settings. in fact, the poor financially benefit more from economic growth in the agricultural sector than in industrial or service sectors 43, 44 . the early stages of economic development often involve the construction of infrastructure to facilitate food production and distribution, including roads, dams and irrigation networks 43, 44 . more recently, the early stages of rural development often include rapid expansion of telecommunication, and to a lesser degree, electrification, both of which are promising but underutilized resources for disease monitoring and control in the developing world 45 . other rural infrastructure that is more crucial to infectious disease prevention, such as safe water, sanitation and energy supplies, often follows or is developed concurrently 43, 44 . in 43 developing countries, rural infrastructure that provided access to sanitation and safe water explained 20% and 37% of the difference in the prevalence of malnutrition and child mortality rates between the poorest and richest quintiles, respectively 46 . moreover, clean water, sanitation and electricity can also facilitate the construction of schools and health clinics that can help to further reduce disease through education, prevention and treatment. hence, if the history of the developed world repeats itself in the developing world, then it seems plausible that agricultural development necessary to feed 11 billion people might help to reduce infectious diseases by promoting economic development and rural infrastructure. there are several important assumptions to this hypothesis, however, such as: (1) it is possible to feed 11 billion people; (2) the rate of economic development will match or exceed the pace of human population growth; and (3) developing countries are not in an infectious disease-driven 'poverty trap' , which is the notion that poverty increases the chances of acquiring and succumbing to disease, and chronic disease traps humans in poverty [47] [48] [49] [50] [51] . if agricultural development lags behind population growth or if clean water supplies, sanitation and electricity do not immediately follow road, dam and irrigation construction, then the prevalence of many infectious diseases could remain unchanged or even increase as human populations grow 52 . agricultural irrigation and freshwater redistribution. one major change in rural infrastructure that often accompanies agricultural development is the redistribution of fresh water, which has well-known consequences for the transmission of infectious diseases. for example, because 40% of crop production comes from only the 16% of agricultural land that is irrigated, and irrigated lands accounted for much of the increased yields experienced during the green revolution 10 , the potential for increased irrigation infrastructure to exacerbate infectious diseases is a critical concern (fig. 1) . importantly, agricultural development has caused both declines of certain types of fresh water, such as wetlands, and increases of others, such as dams, reservoirs and irrigation schemes. one of the largest drivers of global wetland losses has been conversion to agriculture 53 , which has led to declines in diseases that rely on wetland habitats. for example, japan's successful eradication of schistosomiasis during the mid-twentieth century relied in part on conversion of wetlands to orchards in schistosomiasis-endemic areas 54 , although replacing oxen with horses, which are less susceptible to schistosoma japonicum, as draft animals is also thought to have contributed substantially 54 . similarly, some of the earliest successes in malaria control at the turn of the twentieth century occurred in the southern united states, where wetland drainageincluding for agricultural conversion -was one of a suite of antitransmission strategies used by the rockefeller foundation in its landmark malaria control efforts 55 . although wetlands often decline with agriculture, dams, reservoirs and irrigation networks often increase, and this redistribution of fresh water has been widely associated with increases in some vectors and hosts of human pathogens 56 . for example, construction of the aswan high dam in egypt and the accompanying irrigation network was associated with a rise in mosquito vectors and the disfiguring mosquito-borne disease lymphatic filariasis, commonly known as human elephantiasis 57 . likewise, dam and irrigation construction resulted in increases in malaria in sri lanka and india 56, 58 , and a sevenfold increase in malaria in ethiopia 59 . a meta-analysis of 58 studies revealed that humans living near irrigation schemes or in close proximity to large dam reservoirs had significantly higher risk of schistosome infections than humans that did not live near these water resources 60 , at least partly because of increased freshwater habitat for intermediate snail hosts. similarly, a recent analysis of schistosomiasis case data from the past 70 years across sub-saharan africa showed far-reaching effects of dams and irrigation schemes, with increases in schistosomiasis risk extending up to hundreds of kilometres upstream from the dams themselves 61 . hence, dams, reservoirs and irrigation networks related to agricultural expansion are likely to increase vector-borne and waterborne diseases unless water resources are developed in deliberate and coordinated ways to mitigate these risks 62 . urbanization, globalization and the movement of agricultural products. the world's population became majority urban in 2007 and urbanization continues to outpace population growth, especially in developing countries. urbanization necessarily extends food supply chains, as consumers reside further from farms and fisheries. although only 23% of the food produced globally for human consumption is traded across international borders 63 , globalization is likewise elongating food supply chains. furthermore, as the costs of international travel have declined over time, the movement of people across borders has likewise increased. the expanded spatial scope and increased frequency, speed and volume of people and agricultural products moving within and among countries necessarily facilitates the spread of pathogens. for example, in ecuador, there is evidence that the construction of new roads has affected the epidemiology of diarrheal diseases by changing contact rates among people as well as between people and contaminated water sources 64 . each year in the united states, there are already an estimated 48 million people with illnesses, 128,000 hospitalizations and 3,000 deaths from food-borne infections 65 , and imported foods -especially from developing countries with poor sanitation infrastructure and weak food safety enforcement -have been associated with a rise in food-borne illness 66 . in addition, globalization is already thought to be a factor in the spread of human influenza viruses that spillover from poultry and swine 67, 68 . although modernization of food supply chains, especially those linking farms in developing countries to high-income consumers in cities and high-income countries, has typically accelerated the diffusion of stricter food safety and quality standards 69, 70 , as urbanization and globalization further extend food supply chains, enhanced monitoring for the spread of pathogens across transportation networks and strengthened food safety regulations will be needed. in this section, we review the effects of agricultural industrialization. anti-parasitic and antibiotic drug use. as the global human population increases, there will almost certainly be an increase in high density, industrialized livestock and aquaculture operations. currently, such livestock operations are vulnerable to devastating losses of animals to disease. for instance, in just the last 25 years, an influenza a virus (h5n1) and a foot-and-mouth outbreak led to the destruction of more than 1.2 million chickens 10 and 6 million livestock in china and great britain 71 , respectively, and a 'mad cow disease' epizootic led to the slaughter of 11 million cattle worldwide 10 . similar scenarios have occurred in aquaculture. in the past three decades, there has been more than fourfold growth in industrial aquaculture worldwide 72 , which should continue to increase as food demand grows and wild fish and shellfish captures push the limits of renewable production 73 . bacterial outbreaks in aquaculture are common, especially in developing countries where there are sanitary shortcomings 72 . in an effort to prevent these catastrophic disease-associated losses and improve animal growth, the agricultural industry uses a larger proportion of global antibiotic and anthelmintic production than human medicine, and most of the antibiotics are provided at non-therapeutic doses in the absence of any known disease 74, 75 . in fact, although estimates are lacking for most countries in the world, in the united states, nearly nine times more antibiotics are given to animals than humans and, of the antibiotics given to animals, more than 12 times as many are used nontherapeutically as therapeutically 74 . this widespread use of antibiotics and anti-parasitic drugs (for example, anthelmintics) in industrialized agriculture and aquaculture could have important implications for human infectious diseases because it seems to be driving microbial resistance to these drugs, some of which are also used in human medicine 72, 74, 76 . for example, livestock are a primary source of antibiotic-resistant salmonella, campylobacter and escherichia coli strains that are pathogenic to humans 74 . there is evidence that antibiotic-resistance genes acquired from aquaculture are being transferred to human systems and these pathogens have subsequently caused outbreaks 72 . anthelmintic resistance is also rife among parasitic worms of livestock and is strongly implied for parasitic worms of humans 77 . as livestock and aquaculture production expand to address growing food demands, it is likely that current antibiotics and anthelmintics will become less effective because of evolved resistance, and thus infectious diseases of domesticated animals and humans will be more difficult to treat 75 . pesticides, fertilizers and disease. pesticides, particularly insecticides, have shared value for suppression of agricultural pest populations and disease-carrying insect vectors, such as mosquitoes and other flies. because of the anticipated sharp increase in pesticide use by 2100 (fig. 3) , insecticide resistance is also expected to increase, with important implications for the control of diseases carried by insect vectors. insecticides with widespread shared use for both crop protection and vector control include pyrethroid, organophosphate and organochlorine insecticides 78 . several mosquito vectors of human and livestock pathogens have already evolved some resistance to these compounds 78 . if agricultural expansion and intensification is accompanied by an increased use of insecticides, vector resistance may become more common and the control of vectorborne diseases more challenging. in addition to potentially driving vector resistance, increased pesticide use is likely to cause numerous other effects on host-parasite interactions. pesticides can alter disease risk by modifying host susceptibility to parasites [79] [80] [81] . for example, many pesticides are immunomodulators that can increase infectious diseases of wildlife and humans 82, 83 , or are endocrine disruptors of humans with potential downstream effects on immunity 84 . even if pesticides do not directly affect immunity, detoxification of pesticides is energetically 'expensive' for the host, and thus pesticide exposure can reduce available energy resources for humans and zoonotic hosts to invest into parasite defences 85, 86 . pesticides can also affect human disease risk by altering the densities of hosts or parasites or their natural enemies or mutualists 87, 88 . several pesticides can alter host behaviours or be directly toxic to hosts and parasites, which in turn can modify contact rates between parasites and human hosts. in addition, pesticides can alter community composition, which can indirectly affect behaviours or densities of intermediate and zoonotic hosts and parasites. although chemical contaminants can be deadly to many free-living stages of parasites, both empirical trends and theoretical models suggest that stress associated with pesticide exposure can increase non-specific or generalist pathogens 79, 89, 90 . in addition to the impacts that pesticides can have on infectious diseases, they can also contribute to non-infectious diseases, such as cancers, birth defects, miscarriages and impaired childhood development 91 , which can further strengthen the poverty-infectious disease trap. indeed, among african agricultural households, there is evidence of an association between increased pesticide use and increased time lost from work due to sickness 27 . in addition to an increase in pesticides, nitrogen-and phosphorous-based fertilizers are expected to increase threefold by 2050 to boost food production, and most of this increase will occur in tropical regions already rich with pathogens 6, 10, 22 . although the effects of environmental nutrient enrichment on disease are indirect and complex, with some infections increasing and others decreasing, two reviews on the subject suggest that elevated nutrient levels more often than not exacerbate the impact of infectious disease 92, 93 . for example, phosphorous enrichment can benefit mosquitoes that transmit malaria and west nile virus 92, 93 . in addition, nitrogen-and phosphorous-based fertilizer use can increase the number of snails that transmit flatworms that cause human schistosomiasis 92, 93 . conversion of natural habitat to agriculture can increase the abundance of ecotones (boundaries between ecological systems), change species composition and reduce native biodiversity 94, 95 . ecotones play an important role in a number of important emerging infectious diseases 96, 97 , and reduced biodiversity that accompanies agricultural intensification can increase zoonotic disease emergence and can worsen already endemic diseases [98] [99] [100] . a recent global meta-analysis suggests that, based on the available literature, such biodiversity losses generally increase infections of wildlife and zoonotic infections of humans 101 . however, studies of the relationship between biodiversity change and infectious disease risk tend to focus on a single parasite or disease, often of non-human animals, which limits the ability to determine more broadly the effects of agricultural intensification on the overall burden of infectious disease in human populations 102 . recent studies have found that the local loss of dense forests, largely from agricultural expansion, affected diarrheal diseases, acute respiratory infections and general fever in cambodian children 103 and infectious disease incidence in nigerian children over a decade 104, 105 . proximate causes probably included reduced regulation of microbial contaminants in surface and ground waters, increased smoke from biomass burning, shifts in the ranges of insect vectors and decreased access to forest ecosystem services. a major concern is the potential for positive feedbacks between poverty, biodiversity loss, soil degradation and infectious disease 106, 107 . several mechanisms can underpin these reinforcing relationships. the first arises as a result of poor rural peoples' reliance on limited biophysical assets for their livelihoods. as households clear forests, deplete soils or overharvest biota to meet near-term consumption needs, this resource degradation can compromise future economic productivity and increase disease risk. in addition, poverty, environmental degradation, biodiversity loss and disease can quickly become mutually reinforcing responses to natural shocks, such as droughts or floods, or to protracted conflict in a region. there are also more direct examples. for instance, global net losses of tropical forests remained unchanged during the 1990s and 2000s (at approximately 6 million ha yr −1 or 0.38% annually 108 ; also see https://glad.umd.edu/projects/global-forest-watch and https:// www.globalforestwatch.org) and forest clearing can lead to transmission of zoonotic disease by increasing contact with wild animals. conversely, encroachment of people and domesticated animals into natural areas can introduce diseases to wildlife that can devastate wild populations and create reservoirs for the disease to be transmitted back to domesticated animals 109 (see next section for further discussion and citations). especially as the agricultural frontier expands, considerable attention must be paid to monitoring and managing these biodiversity-poverty-disease feedbacks 106 . a central tenet of epidemiology is that the incidence of many infectious diseases should increase proportionally with host density because of increased contact rates and thus transmission among hosts 110 . hence, increasing human and livestock densities could cause increases in infectious diseases unless investments in disease prevention are sufficient to prevent these increases. given that most of the increase in human and livestock densities are expected to occur in developing countries where disease surveillance, pest control, sanitation, and medical and veterinary care are limited, there is little reason to expect that control efforts will keep up with the expected increases in infectious diseases associated with increasing densities of these hosts. as host densities and thus transmission increase, theory suggests that parasite virulence should also increase under some circumstances 111 . when virulence of a pathogen is tied to its propagule generation within the body (such as occurs with many viral infections), the intermediate virulence hypothesis posits that an intermediate level of virulence maximizes parasite transmission because it balances producing many parasite offspring (increasing parasite fitness) with detriments to host survival due to pathology (decreasing parasite fitness). the balance in this trade-off determines parasite persistence. hence, as host densities increase and transmission becomes more frequent, the cost of increased virulence declines, shifting the optimum towards higher virulence 111 . consequently, for pathogens that experience this virulence trade-off, increases in human, crop and livestock densities have the potential to augment both the incidence and severity of infectious diseases. feeding 11 billion people -and the associated increase of land converted to agricultural production and livestock grazing -is expected to cause a surge in human-livestock, human-wild animal and livestock-wild animal contact rates, increasing the likelihood of 'spillover' events, which are defined as pathogen transmission from a reservoir host population to a novel host population 112, 113 . as natural ecosystems are converted to crop land or range land, interactions among humans, and domesticated and wild animals, could increase 66 . furthermore, if developing countries follow a trajectory similar to developed countries, then their demand for meat will increase, further increasing human-livestock, human-wild animal and livestock-wild animal contact rates 114 . these interactions are crucial because 77% of livestock pathogens are capable of infecting multiple host species, including wildlife and humans 115 , and based on published estimates from the 2000s, over half of all recognized human pathogens are currently or originally zoonotic 29, 116, 117 , as are 60-76% of recent emerging infectious disease events 1, 29, 117 (fig. 4) . examples of recent zoonotic disease emergences with enormous impacts on either livestock, humans or both, many of which might have agricultural drivers, include avian influenza, salmonellosis (poultry and humans), newcastle disease (poultry), swine flu, nipah virus (pigs and humans), middle east respiratory syndrome (camels and humans), bovine tuberculosis, brucellosis (mostly cattle and humans), rabies (dogs and humans), west nile virus, severe acute respiratory syndrome (sars) and ebola (humans) 1, 113 . to quantify the relationship between agricultural factors and disease emergence in humans through time, we used the human disease emergence database of jones et al. 1 . we classified land-use change, food industry and agricultural industry as agricultural drivers of human disease emergence (see supplementary methods) . these analyses revealed that agricultural drivers were associated with 25% of all diseases and nearly 50% of zoonotic diseases that emerged in humans since 1940 (fig. 5) . these values are even higher if we include the use of antimicrobial agents as an agricultural driver of human disease emergence, given that agricultural uses of antibiotics outpace medical uses in the developed world nearly nine to one 74, 118 . several factors have materialized that facilitate spillover events associated with disease emergence 113 . spillover appears to be a function of the frequency, duration and intimacy of interactions between a reservoir and novel host population 112 . for example, influenza is believed to have jumped from horses to humans soon after domesticating horses and then made additional jumps to humans from other domesticated animals, such as poultry and swine 119 . similarly, when free-range turkeys were prevented from interacting with wild birds and when interactions between domesticated pigs and wild fruit bats were reduced, influenza and nipah virus incidence 115 , human pathogens that are currently or originally zoonotic 29, 116 , and recent emerging pathogens that are zoonotic 1, 29 . dropped, respectively, suggesting wild-animal sources of these infections 120, 121 . in sub-saharan africa, the high frequency and duration of environmental interactions between different species of schistosoma worms infecting humans and cattle has undoubtedly facilitated their hybridization, and these hybrid schistosomes are more virulent to humans than their non-hybrid counterparts 122 . these factors associated with spillover and disease emergence could be targeted to reduce transmission potential as human populations and agricultural productivity increase. given that most human population growth is expected to occur in developing, tropical countries, hunting, fishing and gathering pressures will almost certainly rise in subsistence economies 13, 123 . the consequences of these pressures might initially increase contact rates between humans and wildlife. this could increase disease risk as bushmeat hunting is already thought to be responsible for several emerging human infectious diseases 116 . to make matters worse, bushmeat hunting is the second biggest threat to biodiversity behind habitat loss, and biodiversity losses can contribute to disease emergence 101 . however, if humans overexploit these natural resources, then human-wildlife interactions could eventually decline as species go extinct, which could reduce spillover events. changes in the composition of biodiversity associated with overhunting and overfishing can also have complex indirect effects on disease risk mediated by species interactions. for example, empirical evidence supports the notion that defaunation of large vertebrates in africa should increase zoonotic disease risk by reducing the predators and competitors (large herbivores) of rodents, a common reservoir of human pathogens 124 . similarly, overfishing of snail-eating cichlids in lake malawi in africa seems to have been a causal factor in snail population and human schistosomiasis increases there 125 . investigations of the effects of overexploitation on infectious diseases remain in their infancy, but the consequences for human populations could be profound. up to this point, we have focused on the effects of feeding 11 billion people on infectious diseases, but the relationship is bidirectional. that is, human infectious diseases can also impact the agricultural and economic development necessary to feed the growing human population (fig. 2) . for example, the overuse of antibiotics, anthelmintics and pesticides to prevent diseases is driving resistance to these chemicals that will compromise future crop, livestock and aquaculture production. in sub-saharan africa, areas with higher historical tsetse-fly abundance, the vector of the parasite (trypanosoma brucei) that causes african sleeping sickness in humans and cattle, experienced greater lags in the adoption of animal husbandry practices (for fertilizer and labour in agricultural enterprise) that hindered agricultural development and prosperity in africa long before and after europeans colonized 126 . in rural subsistence communities, any source of ill health can significantly impact people's productivity, yields and agricultural output [47] [48] [49] . for example, human immunodeficiency virus/aids has reduced average life expectancy in sub-saharan africa by 5 years since 1997, and a kenyan study found that crop production by rural subsistence-farming families dropped 57% after the death of a male head of household 127 . many neglected tropical diseases (ntds) impose devastating productivity losses for affected people that can impede agricultural development from local to national to regional scales 49 . some low-income communities appear mired in this poverty-disease trap, and thus might require substantial investment in health systems to promote the necessary agricultural and economic growth to pull them out of the povertydisease cycle [47] [48] [49] [50] [51] . the goal of identifying potential changes to infectious disease risk associated with feeding the growing human population is not only to draw attention to this important current and future problem but to also encourage preparation for these changes by stimulating agricultural-and disease-related research, management and policy that could maximize the human health benefits of agricultural development 128 . one urgent challenge is the problem of antibiotic, anthelmintic and pesticide resistance. analyses demonstrate that, in some cases, improvements in growth and feed consumption can be achieved by improved hygiene instead of antibiotics 76 . indeed, all antibiotics as growth promoters were banned in the european union in 2006 129 . to curb antimicrobial consumption in food animal production, van boeckel et al. 75 suggest: (1) enforcing global regulations to cap antimicrobial use, (2) adhering to nutritional guidelines leading to reduced meat consumption, and (3) imposing a global user fee on veterinary antimicrobial use. in addition, as recommended by the world health organization, international organization for epizootics, and food and agriculture organization, national and international policies based on best management practices should be developed and implemented that document when and how antibiotics should be used, and agencies should be established to monitor their use, mandate reporting and enforce these policies 76 . finally, given that host genetic variability can reduce disease risk 110 , large-scale industrial livestock operations could add genetic variability into their artificially selected food animals in an effort to reduce epidemics and epizootics. these changes promise to secure the long-term viability of antibiotics and anthelmintics for curing diseases of humans and non-human animals. another challenge that seems surmountable is to enhance education and health literacy. not surprisingly, education has been documented as a major contributing factor to reducing infectious diseases, especially ntds, and reducing ntds can have reinforcing positive effects on the ability of humans to fight more deadly diseases, such as aids, malaria and tuberculosis 49 . limiting ntds could also have knock-on effects for education and health literacy because ntds impede cognition, learning and school attendance 49 . indeed, an investment of just us$3.50 per child for ntd control can result in the equivalent of an extra school year of education 49 . this is likely an underestimate because of unaccounted for indirect effects of deworming on learning. a recent study revealed large cognitive gains among children who were not dewormed but had older siblings who were 130 . thus, enhanced education and ntd control have the potential to synergistically fuel agricultural and economic development and facilitate escape from the poverty-disease trap. national and international shifts in investments could also potentially pay large dividends for nutrition, infectious disease control and poverty reduction. there is considerable evidence that the developing world will struggle to feed its growing human population because of the poverty trap of infectious disease 49 . however, evidence also suggests that this trap might be broken through investments in health infrastructure and preventive chemotherapy [47] [48] [49] . curing worm diseases has the potential added benefit of reducing nutritional needs of cured individuals by ceasing the feeding of their parasites. by reducing food demand, these drugs could also protect the environment by reducing the conversion rate of natural areas to agriculture. this, in turn, might help to curb climate change, which is one of the greatest threats to global food security 11 and might also increase disease risk 131, 132 . understanding the economic relationships among infectious disease treatment and prevention, food production, poverty and climate change in the developing world are important areas of future research. as human populations increase, there might be more pathogen spillover events that could result in new emerging human infectious diseases. historically, humans have combated emerging diseases through early detection followed by coordinated quarantine, as demonstrated by the sars outbreak in 2003 and the ebola outbreak in 2014. we recommend continued and improved coordinated international disease surveillance, but this approach reacts to rather than prevents disease. we recommend a shift in both research and disease management efforts towards proactive management approaches. one proactive approach that has promise for preventing zoonotic disease emergence is biodiversity conservation 98, 101 , but more research is needed to evaluate its effectiveness across various types of zoonotic diseases and its costs and benefits relative to more proven reactive public health interventions. ultimately, science needs a better understanding of pathogen spillover, the origins of disease emergence and post-spillover evolution so that human disease emergence can be better predicted and prevented 112, 113, 133 . improved and diversified plant and animal genetic material could also help to simultaneously reduce hunger and disease. many efforts are already well underway; for example, introducing c 4 traits into rice to enhance higher photosynthetic capacity in a warming world, and breeding drought-and flood-tolerant cereals, pulses and vegetables better suited to increased frequency of extreme weather events that can help sustainably enhance productivity. crops and livestock genetically adapted to resist more pests can reduce pesticide and antibiotic use. in addition, given that micronutrient deficiencies are now the most widespread source of undernutrition globally, commodity research must diversify beyond merely boosting productivity of staple cereals. far greater attention is needed on approaches that add mineral and vitamin content to foods 39 . other, perhaps more challenging, interventions also merit attention. closing 'yield gaps' on underperforming lands, increasing cropping efficiency, eating less meat and excess per capita food consumption -which also have adverse health impacts on noninfectious diseases, such as high blood pressure, diabetes and heart disease -and reducing food waste and loss have the potential of doubling global food supplies 6, 114, 134, 135 , while simultaneously allowing financial savings to be redirected towards health infrastructure to control disease. in addition, family planning, promoting female education and job market prospects, and enhancing early childhood survival have proved very effective at reducing human population growth 136 . finally, producing food in more urban and suburban environments through vertical farming (the practice of growing produce in vertically stacked layers) also has potential to enhance food production locally, and might have reduced agrochemical and transportation costs and non-target effects relative to more traditional farming 135 . investments into predictive models could also pay dividends. agricultural environments are complicated, multi-species systems that exhibit interactions at many levels. the complexity further increases from the tensions that arise in balancing expanding agricultural systems, social benefits and infectious disease risk. all of this complexity can overwhelm many analytical tools and experiments, which makes advances in mathematical modelling especially crucial to addressing these issues. box 1 describes several examples of advances in mathematical modelling that illustrate their promise for analysing the links between agricultural practices and the dynamics of infectious diseases, projecting risks to future decades, influencing risks through either intentional (policies and interventions) or unintentional (continued habitat box 1 | modelling tools for quantitative analysis of agriculture-infectious disease systems mathematical models facilitate the investigation of a complex web of interactions between agricultural systems, social dynamics and infectious diseases in the presence of the substantial nonlinearities inherent in these disparate processes. a notable example is a body of theory and analyses that have emerged to examine the links between agricultural systems and the dynamics of infectious diseases in the context of global development 48 . the approach combines ecological and economic theory to model population subsistence and health, with the goal of examining the conditions under which subsistence and health needs of populations are met. the fundamental consumer-resource relationships that underlie the biological generation of capital via agricultural production can be formalized in ordinary differential equation models, which can be coupled with similarly structured models that estimate the gains and losses in human capital associated with population health and the increasing risk of acquiring and succumbing to infectious diseases associated with poverty. key insights emerge from such integrated models. high prevalence of infectious diseases among populations that rely heavily on subsistence, labour-intensive agriculture can reduce agricultural productivity, degrade human capital, undermine economic growth and generate conditions that systematically reinforce poverty 48 . poverty, in turn, increases disease transmission as the lack of access to clean water and preventive care often associated with the lifestyle of the poor make them continuously exposed to parasites and pathogens embedded in the environment 142 . malnutrition, a common consequence of poverty, may weaken immune response and increase susceptibility to disease. poor education and lack of resources lead to further reduction of health-seeking behaviour. modelling shows that the reinforcing feedback between poor health, labour productivity and capital formation triggers a cycle of increasing poverty and disease, known as disease-driven 'poverty trap' 48 . it also shows that poverty traps are reinforced by the asymmetry between the slow pace of investment in disease protection associated with increasing capital due to agricultural development, and the relatively faster ecological changes in transformed landscape that foster disease transmission 143 . modelling can also reveal the impact of temporary versus lasting structural interventions in changing the development trajectories of poor populations. these intrinsically quantitative and mechanistic investigations can indicate specific conditions for economic growth or resilience that can inform development strategies, via, for instance, targeted investments in agricultural development, human health or ecological conservation that may reinforce globally stable development equilibria 48 . a final major contribution of such models is to encourage primary data collection in support of deeper understanding of system dynamics, and greater inference of underlying determinants of health and wealth. because there are strong traditions of primary data collection in population biology, epidemiology and other disciplines wherein mathematical modelling is being advanced, the expansion of model-based research on coupled agricultural-health systems is likely to yield new field data collection efforts and direct the design of experiments, which, in turn, will yield greater opportunities for model parameterization and validation. these new empirical field data are what are desperately needed to explore whether the predictions of highly stylized models are supported in settings far more complex than modelled. simulation modelling, in turn, can also permit exploration of the uncertainty intrinsic to field experiments that generate data in a specific place and time, advancing understanding of system performance under a range of feasible weather, market and other conditions not realized during the period of data collection. conversion and antibiotic use) actions, and incorporating economic and social costs of various public health or agricultural interventions. these models should help direct field experiments, which are desperately needed to parameterize and validate these models and to quantify key sources of uncertainty. ultimately, we believe 'win-win' scenarios for controlling infectious diseases, increasing agricultural productivity and improving nutrition are attainable. although this sort of integrative thinking has been historically rare because agriculture and public health have been perceived as disparate disciplines, it is beginning to slowly penetrate these distinct fields of study. for example, several agrochemicals seem to increase the risk of human schistosomiasis and agriculturally derived zoonotic pathogens, and thus researchers are actively attempting to identify agrochemicals that might 'kill two birds with one stone' , reducing crop pests and thus increasing food production while not increasing or even decreasing human pathogens [137] [138] [139] . similarly, researchers are beginning to consider the introduction of prawns as biological control agents of schistosomiasis, which could simultaneously decrease disease and increase nutrition -because restored prawns can be fished, harvested and consumed without compromising their diseasecontrolling benefits 61, 140 . we believe that more resourceful and creative thinking and interdisciplinary interactions (among medicine, public health, agriculture professionals, economists, anthropologists, sociologists, modellers, empiricists and wildlife biologists, in the developed and developing world) have great promise for sustainably and 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alleviation and schistosomiasis control genetic dissimilarity between mates, but not male heterozygosity, influences divorce in schistosomes disease ecology, health and the environment: a framework to account for ecological and socio-economic drivers in the control of neglected tropical diseases the rise and fall of malaria under land-use change in frontier regions funds were provided by grants to j.r.r. from the national science foundation j.r.r. developed the idea and wrote most of the paper. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/ s41893-019-0293-3. correspondence should be addressed to j.r.r.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-282059-sdumq61z authors: nesse, randolph m; stearns, stephen c title: the great opportunity: evolutionary applications to medicine and public health date: 2008-02-17 journal: evol appl doi: 10.1111/j.1752-4571.2007.00006.x sha: doc_id: 282059 cord_uid: sdumq61z evolutionary biology is an essential basic science for medicine, but few doctors and medical researchers are familiar with its most relevant principles. most medical schools have geneticists who understand evolution, but few have even one evolutionary biologist to suggest other possible applications. the canyon between evolutionary biology and medicine is wide. the question is whether they offer each other enough to make bridge building worthwhile. what benefits could be expected if evolution were brought fully to bear on the problems of medicine? how would studying medical problems advance evolutionary research? do doctors need to learn evolution, or is it valuable mainly for researchers? what practical steps will promote the application of evolutionary biology in the areas of medicine where it offers the most? to address these questions, we review current and potential applications of evolutionary biology to medicine and public health. some evolutionary technologies, such as population genetics, serial transfer production of live vaccines, and phylogenetic analysis, have been widely applied. other areas, such as infectious disease and aging research, illustrate the dramatic recent progress made possible by evolutionary insights. in still other areas, such as epidemiology, psychiatry, and understanding the regulation of bodily defenses, applying evolutionary principles remains an open opportunity. in addition to the utility of specific applications, an evolutionary perspective fundamentally challenges the prevalent but fundamentally incorrect metaphor of the body as a machine designed by an engineer. bodies are vulnerable to disease – and remarkably resilient – precisely because they are not machines built from a plan. they are, instead, bundles of compromises shaped by natural selection in small increments to maximize reproduction, not health. understanding the body as a product of natural selection, not design, offers new research questions and a framework for making medical education more coherent. we conclude with recommendations for actions that would better connect evolutionary biology and medicine in ways that will benefit public health. it is our hope that faculty and students will send this article to their undergraduate and medical school deans, and that this will initiate discussions about the gap, the great opportunity, and action plans to bring the full power of evolutionary biology to bear on human health problems. 2003; harris and malyango 2005) . many medical students do not even accept the theory of evolution (downie 2004) . most medical students get two or more years of basic science education, including embryology, biochemistry, anatomy, histology, and physiology, and many get a genetics course from a professor who knows evolutionary biology. however, we know of no medical school that teaches a course in evolutionary biology as a basic medical science, and none that requires evolution as a prerequisite. our teaching experience confirms that few doctors have a chance to learn the principles of evolutionary biology most useful for medicine. are medical research and evolutionary biology better connected? new quantitative evidence comes from an innovative strategy for mapping citation patterns. instead of measuring co-citations between traditionally defined fields, rosvall and bergstrom (2007) define the boundaries of disciplines empirically from citation patterns. they then analyze the directed flow of citations between disciplines. the results for evolution and medicine are striking (see fig. 1 ). ecology and evolution journals cite work in medical journals occasionally, but medical journals cite work in ecology/evolution journals too rarely to even show up on the diagram. almost all of the connections have other fields as intermediaries. there are good historical reasons for the gulf between evolution and medicine (zampieri 2006) . they stem partly from the timing of flexner's (1910) report that recommended bringing basic sciences into the medical cur-riculum. at that time, evolutionary biology was in eclipse. many scientists thought that lord kelvin's arguments about the rate of the earth's cooling proved darwin wrong (kelvin 1862) . others recognized that darwin's theory of transmission by gemmules was inconsistent with his theory of natural selection (richards 1987) . natural selection was not re-incorporated into biology until its underpinnings in population genetics were developed in the early to middle years of the 20th century (fisher 1930) . even then, those foundations emphasized mutations and genetic variations, not the shaping of complex adaptations by selection, a field that was only developed by evolutionary and behavioral ecologists in the 1970s and later. those insights into trait evolution are just now being incorporated into medical science. mastering medicine is increasingly difficult. it includes far too much knowledge for any one person to learn: on this educators -and medical students! -agree. the challenge is to instill as much useful knowledge as possible in just a few years. the criterion of 'useful' is prioritized because medicine is a practical profession. patients want help and doctors need to know what to do. if a deeper understanding of a disease is useful, fine. otherwise, there is no time. every discipline makes recommendations, even demands, for curriculum content. in addition to the usual 20 or so departments in a medical school, there are demands from groups representing geriatrics, statistics, gender issues, bio-informatics, nutrition, musculoskeletal systems, cancer, law, breast feeding and child abuse experts, among scores of others. each advocates for including more content in one area. put them all together and medical school would take decades. interestingly, proposals for curriculum reform tend not to emphasize ways to include more and more specific content. instead, a review of 24 proposals for medical curriculum reform found that they consistently focused on values, especially the social nature and self-regulation of the medical profession (christakis 1995) . somehow, from all of these jostling interest groups, priorities and multiple regulations and examination requirements, a dean and faculty must come up with a curriculum. this makes it difficult to warmly welcome visitors who drop by to point out that a whole huge area of basic science has been omitted from the curriculum. however, evolutionary biology is not just another narrow topic, but a fundamental basic science. furthermore, it can help make medical education more coherent by giving students a framework for organizing the required 10 000 facts. understanding the gap also requires consideration of how well-prepared evolutionary biologists are to apply their knowledge to the problems of medicine. most evolutionary biologists know as little about medicine as physicians know about evolution. if you cannot tell a myocardial infarction from cardiac failure, doctors will not pay attention. if they feel you are just adding to the thousands of facts they need to memorize, they will flee. not many evolutionary biologists are eager to teach medical students; the best are working hard on their own research. the gulf will be understood only by looking from both ends of this two-way street. before we consider opportunities and solutions, it is worth noting that medicine's isolation from evolutionary biology is just one example of the fragmentation that isolates many disciplines. some of the isolation results from academic structures that allow hiring and promotion to be controlled by narrow disciplines. universities talk a lot about promoting interdisciplinary work precisely because their structures so efficiently prevent it. however, disciplines exist for good reasons. there is too much to know. trying to synthesize work from diverse areas is frustrating, especially if the goal is general understanding, not some fine point. also, going beyond your specialty means you will inevitably get some things wrong. it is easier to maintain quality by keeping to a narrow focus. perhaps this list of problems will lead some readers to throw up their hands. we highlight the problems because we want decision makers to recognize that we are fully aware of them. acknowledging problems allows realistic solutions. we want also to emphasize that even large challenges are worth confronting because of the great benefits of bringing more evolution to medicine. most of this article is devoted to examples of rapid progress in applying evolutionary principles to medicine. overviews of evolutionary approaches to health and disease are available in several articles and books (williams and nesse 1991; nesse and williams 1994; stearns 1998; trevathan et al. 1999; stearns and koella 2007; trevathan 2008; o'higgins and elton in press) , and a critical review assesses progress and directions (stearns and ebert 2001) . the goal here is not to summarize recent work, but to step back to describe the structure of the developing field, the challenges it faces, and its potential. the examples are organized into three categories. some use well-established applications, some are new, and some remain mostly opportunities. they suggest actions that will allow evolutionary biology to provide maximum benefits to human health. at the core of evolutionary medicine is recognition that diseases need both proximate explanations of bodily mechanisms and evolutionary explanations of why natural selection has left the body vulnerable to disease. why do we have an appendix and wisdom teeth, a narrow birth canal, arteries prone to atherosclerotic blockage, and cells that can divide out of control? these are good evolutionary questions; they are fundamentally different from proximate questions. the distinction between evolutionary and proximate questions was emphasized by mayr (1982) , but it was tinbergen's (1963) article that outlined the four questions that must be answered to provide a full explanation for any biological trait. proximate questions 1. how does the mechanism work? 2. what is the ontogeny of the mechanism? evolutionary questions 3. how has this mechanism given a selective advantage? 4. what is the phylogeny of this mechanism? the first two questions are about the body's proximate mechanisms, from dna transcription and physiological regulation to bones, muscles and behavior. the third and fourth are evolutionary questions about how the body got to be the way it is. the four questions are complementary not competing. all four need to be answered for each trait. medical textbooks address question 1 in detail, question 2 sometimes; questions 3 and 4 only rarely. from this perspective, medicine has been using only one half of biology. understanding jaundice illustrates why both proximate and evolutionary explanations are essential. the yellow color in the skin and eyes is caused by excess bilirubin that accumulates most often because of liver failure. textbooks describe bilirubin as a potentially toxic metabolite of hemoglobin that can be excreted in bile only after it is made water soluble by conjugation with glucuronic acid in the liver. this is a proximate explanation that says nothing about why bilirubin exists in the first place. one might think it is simply a waste product. however, the intermediate step between heme and bilirubin is biliverdin, a chemical that is more soluble than bilirubin. so, why does the body go to the trouble to make a difficultto-excrete toxin? this is the evolutionary question. bilirubin is an effective antioxidant that can protect against the oxidative damage that contributes to aging (stocker et al. 1987; nesse and williams 1994) . oxidative damage is partially responsible for atherosclerosis, so many studies have looked to see if higher levels of bilirubin protect against heart attacks. they do, dramatically. levels of bilirubin higher than normal are characteristic in gilbert's disease; middle-aged people with this genetic condition have rates of heart disease sixfold lower than those with normal bilirubin levels (vitek et al. 2002) . however, there is no mention of evolution or natural selection in the article that reviews the 11 studies of bilirubin protection against atherosclerosis (novotny and vitek 2003 ). an evolutionary perspective suggested an experimentlooking to see what happens if you knock out the enzyme that converts biliverdin to bilirubin. when the enzyme is working, potentially damaging oxygen radicals react with bilirubin, turning it into biliverdin, thus reducing the concentration of dangerous peroxide radicals up to 10 000-fold. without protection by bilirubin cells die quickly (snyder and baranano 2001; sedlak and snyder 2004) . this is a fine example of using the details of a proximate mechanism to test an evolutionary hypothesis. however, many relevant studies remain to be performed. no comparative study has investigated bilirubin levels in other primates to see if they are correlated with life span. more practically, researchers are now considering whether there could be possible disadvantages of using light exposure to reduce mildly elevated bilirubin levels in newborn infants (hammerman et al. 1998) . high bilirubin levels in the first days of life could be merely a result of the changeover to adult hemoglobin, but they could also help protect against oxidative damage from higher levels of oxygen and free iron exposure. the decision is delicate because too much bilirubin in the early days of life causes irreversible damage. when they first hear about evolutionary medicine, most doctors ask immediately, 'how can i apply it in the clinic today?' this surprises many basic scientists, who expect doctors to share the depth of their curiosity about why the body is the way it is. however, medicine is not a science, it is a practical profession. patients bring their problems; doctors try to help. the question, 'why has natural selection left the body so vulnerable to this disease?' seems very abstract to doctors who need to know right now, 'what is the problem? what treatment is best?' in response to doctors' demands for practical applications, it is tempting to offer quick examples of how evolution can inform everyday medical practice. this article reviews many: preventing antibiotic resistance, the benefits of inflammation, the costs of blocking normal defenses, the phylogeny of hiv, etc. however, offering examples too quickly creates two problems. first, even in these practical examples, evolutionary knowledge does not often change what a physician does in his or her day-to-day practice; instead, it guides research, as in the example of jaundice. treatment decisions are, and should be, based on controlled studies on humans, not on theory or on experiments performed on model organisms alone. darwinian medicine does not often give direct practice guidelines. second, merely listing quick applications sells evolutionary biology short. medical professionals learn other basic sciences not because they are useful everyday in the clinic, but because they provide a crucial depth of understanding and a framework for organizing the myriad facts in which the mind otherwise drowns. knowing the mechanisms and laws of acid-base balance gives a physician the perspective needed to apply formulas in the clinic. evolutionary biology offers the same sort of help, but on a much larger scale. instead of phenomena as specific as acid-base balance, evolution helps doctors make sense of why a disease exists at all, what environments increase the risk, and how treatments work. it has direct applications to medical research, but it also provides an otherwise missing paradigm for understanding why our bodies are vulnerable to disease. while no framework can capture all of the applications of evolution to medicine, recognizing two major distinctions is helpful. table 1 shows the categories created by intersecting the two different evolutionary questions with a selection of things that need explanation. it is important to distinguish the two different kinds of evolutionary questions. answers to questions about phylogeny trace the evolutionary history of the trait in question. answers to questions about the adaptive significance of a trait try to understand why a trait is in the state we find it. historical and adaptive approaches use different methods to test hypotheses; both can deliver usefully different insights, often on the same issues. the other distinction is among the different things we want to explain. often the question is about why our bodies are the way they are, especially why selection has left us vulnerable to a disease. the object of explanation can be a universal trait, such as bilirubin, or it can be traits that differ, for instance, versions of certain genes. for instance, some people have versions of genes that increase depression rates (caspi et al. 2003; sen et al. 2003) ; why has selection not eliminated these alleles? many questions are about why all humans are all the same; many are about why we differ. the second major target of explanation is the evolution of pathogens -bacteria, viruses, worms and others. the large issues are the same, but they evolve much faster, so we can often observe their evolution, even in the laboratory where this allows experimental tests of hypotheses. another target for explanation is the evolution of cells within the body, particularly cancer cells and certain classes of cells in the immune system. cancer originates through mutation. cells that divide faster and better evade the body's surveillance systems become more common and spread: a standard evolutionary process. cells in the immune system also undergo a kind of evolution so that those that most effectively fight an infection multiply the most quickly. in both cases selection is not acting on organisms, but on cell lines within individuals; here too evolutionary principles can be useful. the applications of evolution to medicine divide naturally according to the types of questions asked. because answers to questions about phylogeny and adaptive significance require different methods and different skills, it is not surprising that these areas of research remain somewhat separate in evolutionary medicine. some of the most useful applications of evolution often do not use evolutionary theory directly; instead they use technologies developed by evolutionary biologists. in particular, methods for reconstructing phylogenies are being applied to genetic data with very practical results. hiv is especially susceptible to such methods because its fastaccumulating mutations create finely detailed phylogenies. for instance, certain cases of hiv could be traced back to a specific florida dentist (ciesielski et al. 1992) . phylogenetic analysis also was used to falsify the hypothesis that hiv was introduced into africa via polio vaccine (weiss 2001) . the sars epidemic was traced quickly to a corona virus similar to one endemic in bats (li et al. 2004; skowronski et al. 2005) . tracing pathogen phylogenies can be very useful. influenza phylogenies suggest which strains are likely to spread in future epidemics (bush et al. 1999; ghedin et al. 2005; smith 2006 ), information vital to decisions about vaccine design. the current h5n1 avian influenza pandemic appears to have originated via reassortment between avian influenza strains circulating in eastern asia (li et al. 2004) . public health now uses such methods routinely to trace the source of contaminated foods. these phylogenetic methods have a remarkable reach, back even into prehistory. for instance, the complete genome sequence of the severely pathogenic shigella flexneri reveals that it is phylogenetically indistinguishable from the escherichia coli that lives normally in the human gut (wei et al. 2003) . the difference seems to be in a few virulence factors that result in substantially different ecological niches for the two organisms. technologies for tracing phylogenies have ready application to antibiotic resistance and to pathogen evolution in general. they are particularly powerful in revealing the origins of emerging diseases. for example, hiv1 originated in chimpanzees in central africa, and hiv2 originated in sooty mangabeys in west africa (heeney et al. 2006 ). importantly, these species do not develop aids. phylogenetic methods have also found recent applications in cancer research and treatment. cell lines differentiate as mutations accumulate, and the genetic differences make it possible to trace the sequence. two tumors that are histologically identical can have very different proteonomic signatures that make it possible to assess the level of cellular differentiation (abu-asab et al. 2006) . whether a tumor is all derived from the one line of cells, or from different origins arising during the tumor's growth may also be an important indicator (merlo et al. 2006; frank 2007) . researchers in every area of medicine use phylogenetic methods to analyze genetic data. sometimes they are used in conjunction with evolutionary theory, but they are also used independently to construct phylogenies with new applications in an era of genetic medicine. doctors who understand these phylogenetic methods and the evolutionary biology behind them will be better prepared to judge the significance of research findings such as those summarized above. as most readers know, evolutionary biology took off only after it was synthesized with population genetics in the 1920s and 1930's (fisher 1930) . mathematical treatments of allele frequencies that incorporated selection, drift, mutation, and migration made it possible to begin to understand the forces that shaped the genome. as lewontin (1974) has noted, however, this theory developed separately from breeder's theories about selection for phenotypes; the task of mapping changes in allele frequency to changes in phenotype remains a challenge. while this gap remains substantial in much of medicine, it is being reduced by the explosion of work on quantitative trait loci (qtls) and single nucleotide polymorphisms (snps), e.g., the wellcome trust case control consortium (2007) . the explosion of genetic information tends to focus attention on genetic differences between individuals. much research is trying to explain these differences and their significance. an increasing proportion of this work looks for evolutionary explanations. relatively overlooked, however, are questions about why all members of a species are the same. this is especially important with regards to traits that leave a species vulnerable to disease, such as wisdom teeth, the appendix, or a narrow birth canal. such traits are also in need of evolutionary explanations, and information on genetic variations is not always helpful. many physicians think of genes that cause disease as abnormalities in an otherwise 'normal' genome. this is a nonevolutionary view on two counts. first, it tacitly views the genome as a product of design with a blueprint that defines 'normal.' the genome is, instead, a collection of those genes that have tended to increase reproductive success (or hitchhiked on the success of other genes) while interacting with each other and the environment to construct a functional organism. second, while some dna sequences can be accurately described as 'damaged', it is increasingly clear that many medically relevant genetic variations are helpful or harmful only in interaction with particular aspects of environments. such genes have been called 'quirks' to distinguish them from defective genes that cause problems in all environments (nesse and williams 1994) . for instance, if you have the genes for nearsightedness, you will almost certainly become nearsighted. unless, that is, you live in a culture where children are not taught to read (norn 1997) . the problem comes only when certain genes and certain environments interact. similarly, some genes that interact with high fat diets to cause atherosclerosis are quirks that would be harmless if we lived and ate the way people did thousands of years ago. like phylogenetics, population genetics is a mature technology already applied widely and effectively throughout medicine. there are new applications, and one could quibble about whether users of these methods are thinking in evolutionary terms or just using technologies that work. nonetheless, population genetics cannot be separated from evolutionary theory and, as such, is a wellestablished area evolutionary medicine. new applications of evolutionary principles have brought spectacular progress in several areas of medicine during the past 20 years (stearns and ebert 2001) . studies of infectious disease and aging have been especially transformed. for the sake of continuity, however, we begin with progress coming from increasingly sophisticated evolutionary genetics (maynard smith 1998; jobling et al. 2004 ). established principles of population genetics are being augmented by new ideas and techniques. especially interesting are new strategies for using 'signals of selection' to determine which genes have been strongly selected in the past few thousand generations. just a few years ago, this approach offered a few methods and a few examples (olson 2002) . now, many new methods are applied to genome scan data to identify loci subject to directional and balancing selection as revealed by the homogeneity of the dna sequences surrounding the loci in question (vallender and lahn 2004; sabeti et al. 2006; voight et al. 2006) , and early overestimates of the number of loci of interest are now being corrected (thornton and jensen 2007) . these methods provide answers to long-standing questions, such as the origins of genes for lactase persistence (bersaglieri et al. 2004 ). most adult humans cannot digest milk because the enzyme that breaks down milk sugar is not made in adulthood. recent studies showed that genes that allow adults to digest milk have evolved separately several times, almost always in dairying cultures (holden and mace 1997; ingram et al. 2007; tishkoff et al. 2007) . similarly, there has been speculation for decades about whether the genetic tendency to feel sick immediately after drinking alcohol could be common in people from asia because it protects against alcoholism in a culture where alcohol has long been available. evidence has been sparse until now. the case has been bolstered by finding a strong signal of selection in asians at the site of the gene (voight et al. 2006) . the evolutionary backgrounds of alleles that predispose to disease can now be examined. of particular interest is a gene that makes apolipoproteins, substances that bind and transport lipids. individuals with the apoe4 subtype have a much higher risk of developing atherosclerosis and alzheimer's disease. this allele is universal in other primates. in humans, especially those living in cold climates, selection has increased the rates of the apoe3 allele (sapolsky and finch 2000) . this may be a case of selection caught in action, perhaps for genes that prevent health problems for meat eaters (finch and stanford 2004) . selection has also been proposed as an explanation for cystic fibrosis, given the scores of mutations that can cause it and its systematic variation with latitude. mice heterozygous for the cf allele have less fluid loss from cholera toxin (gabriel et al. 1994 ), but the chloride channel is not the rate limiting step for fluid loss in humans (hogenauer et al. 2000) . the cf gene also prevents entrance of salmonella typhus into gastrointestinal mucosal cells (pier et al. 1998 ). however, cystic fibrosis is more common in climates where diarrheal diseases are less common, and although remarkably prevalent, it remains a rare allele. cystic fibrosis offers a fine example of creative tests of interesting hypotheses, and an example of how hard it can be to reach a firm conclusion about the adaptive significance of a genetic variation. until recently, agreement on how to assess the role of selection on vulnerability genes was elusive (chadwick and cardew 1996) . that is changing fast. we now have systematic reviews of the role of selection in maintaining the prevalence of genes that increase risk for infectious disease (dean et al. 2002) , and progress in related areas is on the way. naïve thinking that genes exist always for the good of the individual and the species remain common in medicine even though biologists abandoned them in the 1970s. the importance of gene-level selection is highlighted in the work of trivers and others on selfish genetic elements that facilitate their own transmission at the expense of the individual (burt and trivers 2006) . these are dawkins' selfish genes with a vengeance (dawkins 1976 ). the best known examples are the t-allele in mice and segregation distorter in fruit flies. the role of selfish genetic elements in human disease, including cancer (crespi and summers 2005) , is an especially exciting area that is starting to be elucidated. these advances also suggested looking for conflicts that arise between genes transmitted through males versus those transmitted through females. following the lead of trivers (1974) , haig (1993) pointed out that in pregnancy, the interests of genes from the male differ from those from the female. genes derived from male benefit if they somehow induce the female to make more or larger offspring. energy reserves that a female mouse does not invest in the current litter will not benefit the male unless he happens to mate with her again. conversely, genes from the female benefit by reserving fat stores for future reproduction. the size of offspring that maximally benefits the male is only slightly different from the size optimal for the female, but this small difference may have shaped a complex system. this evolutionary hypothesis is supported by the details of a remarkable proximate mechanism. studies of genetically engineered mice show that the unopposed expression of a gene called insulin-like growth factor 2 (igf2) results in a large placenta and large but otherwise normal offspring; this outcome benefits paternal genes. when transmitted through the mother, this gene is inactivated by a process called imprinting, making the offspring smaller. igf2r is a gene with opposite effects; it degrades igf. its effect is decreased by imprinting from passage through the father (haig 1993) . loss of igf2 imprinting causes beckwith-wiedemann syndrome, characterized by large babies with very large internal organs. it may be more likely in offspring conceived using artificial reproductive technologies (maher et al. 2003) . this area of research vividly illustrates the clinical implications of studies that would never be considered without sophisticated applications of evolutionary theory (wilkins and haig 2003) . addressing a much broader issue, it is worth noting that 'knock-out' studies are about the evolutionary functions of a gene. they are modern equivalents of the old physiological method of extirpation. taking out an organ or a gene and looking to see what goes wrong can generate hypotheses about how an organ or gene is useful. often, no abnormality is observed. of course, this does not mean that the gene is useless, only that its effects are covered by redundant systems, that its benefits are manifest only in special situations, or that the benefit is just too small to be observed in a laboratory setting. for instance, genes involved the capacity for shivering might well appear to be harmful, unless one happened to look at their effects in extreme cold. similarly, some genetic variations associated with faster aging are likely to have compensating advantages, otherwise they would have been eliminated. as we are gaining technologies to manipulate genes, evolutionary thinking about their origins and functions becomes more crucial than ever. aging research shows how evolutionary thinking can transform a field. many doctors still view aging as an inevitable result of body parts wearing out. this knowledge gap is unfortunate for a trait so important to medicine. half a century ago, medawar (1952) saw that selection weakens with age because the surviving number of individuals declines, even in the absence of senescence. then williams (1957) had the insight that pleiotropic genes that cause aging and death can nonetheless be selected for if they also give benefits early in life when selection is stronger. he gave a vivid hypothetical example of a gene that makes bones heal faster in childhood, but that also slowly deposits calcium in the coronary arteries. hamilton (1966) provided mathematical models for the process. these evolutionary insights transformed aging research (finch 1991 (finch , 2007 . instead of looking only for proximate explanations for aging, the field now also seeks evolutionary explanations for why aging mechanisms exist at all. laboratory (rose 1991; stearns et al. 2000) and field evidence (austad 2005 ) soon showed that aging was a life history trait shaped by natural selection (stearns 1992 (stearns , 2000 . for many species, senescence in the wild is a deleterious trait with heritable variation, but life spans do not increase, presumably because the reproductive benefits of longer lives would be balanced by costs that decrease reproduction earlier in the life span (nesse 1988; austad 2005; williams et al. 2006) . the big new news in aging research is the discovery of remarkably strong effects of single genes that influence oxidative metabolism (guarente and kenyon 2000; austad 2005 ). these surprising findings are now being interpreted in evolutionary terms (partridge and gems 2006; ackermann and pletcher 2007; mcelwee et al. 2007) . they suggest that mechanisms that protect against oxidative damage are limited by their reproductive costs or just lack of selection. they also show how selection can shape special states of reduced metabolism that allow some species to survive periods of privation. these states slow aging dramatically, but they are special states precisely because they also so dramatically reduce reproduction. the ancient dream of extending lifespan no longer seems like just a dream, but do not buy beach property on hudson bay just yet. when it comes to aging, males are the weaker sex. it has long been known that men die younger than women, but this has rarely been interpreted in a life-history framework. a recent report about higher mortality rates for male than female mammals attributes it to both external causes and faster aging. the faster rates of aging for males are found mainly in polygynous species because a shortened reproductive span decreases the force of selection for older males (clutton-brock and isvaran 2007 ). an evolutionary view of humans suggested looking at the ratio of male to female mortality across the lifespan in different cultures. this found surprising results (kruger and nesse 2004) . in every culture at every age through late adulthood, mortality rates are higher for men. in modern societies, for every woman who dies at reproductive maturity, three men die. the pattern is consistent in 20 cultures studied. further work looking at the proximate causes of sex differences in mortality rates finds that they result not only from accidents and violence, but also from the full range of causes of mortality. applications of evolutionary biology to infectious disease are also very direct. pathogens evolve fast, right under (and in!) our noses. antibiotic resistance is the classic example. individual bacteria and viruses vary in their susceptibility to antimicrobial agents; those with even slight resistance replicate faster and their genotypes become more common. just a few years after alexander fleming discovered penicillin, he also discovered antibiotic resistance. the basic phenomenon is very simple. antibiotics are selction agents that quickly increase the proportion of organisms that can resist them. (bergstrom and feldgarden 2007) . shortly after the us surgeon general declared in the mid-1950s that the war on infectious disease was over, antibiotic resistance became a serious problem. staphylococcus quickly became resistant to penicillin, nearly all other bacteria followed. antibiotic resistance is an arms race; we invent new defenses, the enemy quickly finds ways around them, and we try to find new defenses. we are now faced with many organisms that resist every available antibiotic; some wonder if the war on infectious disease may be lost (normark and normark 2002; levy and marshall 2004) . nearly 10% of staphylococcus aureus are now resistant even to methicillin; infections caused by this resistant organism now cause 18 650 deaths per year, more than the 12 500 caused by aids (klevens et al. 2007 ). the economic burden of antibiotic resistance is estimated at about $80 billion annually in the usa. recognition of antibiotic resistance as an example of natural selection is often missing in medical articles on the topic. in biology journals the phrase 'natural selection' or another direct reference to evolution is used 79.1% of the time to describe antibiotic resistance, but in biomedical journals they were used only 17.8% of the time. instead, medical journals use 'emergence' or some other circumlocution to avoid the 'e-word' (antonovics et al. 2007) . many doctors view antibiotics as human discoveries, but most are results of selection acting over millions of years in the deadly interactions of bacteria and fungi with each other. the average bacteria isolated from soil demonstrates resistance to seven antibiotics (d'costa et al. 2006 ). this is not because of exposure to human-produced chemicals, but because the long co-evolution of bacteria and fungi has shaped toxins, defenses and new toxins (ewald 1994) . bacteria and fungi have been developing and testing the effectiveness of antibiotics for millions of years! another important aspect of resistance is whether it has costs to the resistant bacteria that will select against the resistance if antibiotics are withdrawn. the answer is sometimes yes, but often the costs seem to be so low that resistance persists, an ecological insight of huge importance for controlling antibiotic resistance (andersson and levin 1999) . continuous application of antibiotics also produces selection to reduce their costs, yielding resistant strains that persist after the antibiotics are withdrawn (schrag and perrot 1996) . however, restriction of antibiotic use in danish farm animals resulted in decreased resistance (aarestrup et al. 2001 ). more work on these evolutionary responses is of great importance. selection on pathogens is, of course, not a one-way street. hosts evolve too, creating co-evolutionary cycles of deception and ability to detect deception of vast complexity (ewald 1994; knodler et al. 2001; frank 2002) . the genes of vulnerable individuals become less common, and host resistance evolve, but very slowly compared with the rate of pathogen evolution. some of the resulting genetic change is in mechanisms close to the sites of infectivity. for instance, malaria uses the duffy antigen to enter red blood cells. individuals without the duffy antigen are less susceptible to malaria and have a selective advantage where malaria is common (hamblin and di rienzo 2000) . this is why the duffy antigen is absent in most africans. the ccr-5 receptor on white blood cells allows hiv to enter. the receptor is absent in about 1% of europeans; they do not get aids even when infected with hiv (samson et al. 1996) . some geographical evidence suggested that this genetic difference could result from selection by the plague epidemic in the 14th century, but in a nice example of hypothesis testing, more careful examination shows the patterns do not match (cohn and weaver 2006) . would we all be better off without the ccr-5 receptor? with the advent of hiv the answer may be yes, but this receptor is not useless; at the very least it appears to protect against west-nile infection (lim et al. 2006) . when a parasite such as malaria deals with both a mosquito and a mammal host, the complexity of its evolution is magnified (mackinnon and read 2004; grech et al. 2006) . here host-parasite manipulations can be studied in detail, and their complexity is more than intriguing. doctors learn about the complexity of parasite life cycles, but rarely do they have an opportunity to consider their evolutionary origins. nor do they have the evolutionary principles that would allow them to evaluate proposals to drive genetically engineered strains of mosquitoes into wild populations. such proposals rarely take into account how the introduced strains will evolve in interaction with the wild ones. changes in the phenotype also exert selection forces on pathogens. vaccination of large populations fundamentally changes the environment for a pathogen. for instance, steady pertussis vaccination for 40 years may have selected for more virulent strains of the whooping cough bacteria (diavatopoulos et al. 2005) , although decreased vaccination may be responsible for the increased incidence. imperfect vaccines can create selection pressures for increased virulence (gandon et al. 2001 ). this disturbing possibility has been documented for marek's disease in chickens (davison and nair 2005) . however, when a vaccine targets a toxin, selection can decrease virulence. this has happened for diphtheria, where lines that do not produce toxin have largely displaced the dangerous forms (soubeyrand and plotkin 2002) . these findings have obvious major public health implications, but the complex realities of host pathogen interactions make confident prediction difficult (ebert and bull 2003) . intuitive models for antibiotic resistance are often incorrect (normark and normark 2002) . for instance, some hospitals have tried rotating the antibiotic of choice over a period of a few months with the idea that by exposing bacteria to changing selective regime, this will prevent antibiotic resistance. but when the process is modeled, this turns out that antibiotic rotation is ineffective at creating a more heterogeneous suite of selective conditions. at least in principle, hospitals would do better to use a mix of different drugs on different patients simultaneously, rather than to cycle through these different drugs over time (bergstrom et al. 2004) . perhaps equally important are more general but lessrecognized selection forces from infectious agents. we have a wide variety of protective bodily responses, such as fever, cough and vomiting, that are held in reserve until released by a mechanism that detects the presence of pathogens (ewald 1994) mechanisms that regulate expression of these defenses are under constant selection (nesse 2005c) . individuals vary in how high fever rises during infection, how quickly immune cells are activated, and how much diarrhea is produced for a given level of infection. most symptoms of infectious disease are not caused directly by the pathogens: they result from these useful defenses. some are aspects of the inflammation and immune systems that attack pathogens. others, such as cough, diarrhea and vomiting, extrude pathogens. for all such defenses, one might think that selection would shape regulation mechanisms to be close to the optimal. but what is optimal? the answer is surprising. when the cost of a false alarm is low relative to the possible costs of not expressing a sufficient defense when it is needed, selection shapes regulation mechanisms that express the defense more readily or more intensely than seems sensible. we put up with smoke detectors that sometimes wail when we make toast because we want to be sure they warn us about any real fire. the 'smoke detector principle,' applies signal detection theory to yield quantitative predictions about how selection shaped defense regulation mechanisms (nesse 2005c ). it has clinical relevance because so much everyday medicine involves prescribing medications that block defenses such as fever, pain and cough. this tends to be safe because the body has redundant defense mechanisms and because the thresholds for defense expression are set by the smoke detection principle. sometimes, however, it is fatal. far from suggesting that doctors should let nature take its course, an evolutionary perspective suggests that many defensive reactions are excessive or entirely unnecessary. it also suggests that we have only begun to study a crucial set of principles at the core of general medicine. general practice could have a stronger foundation in science if practitioners had tools for thinking about how selection shaped defense regulation. most already know that using codeine to block cough after surgery is likely to result in pneumonia, and an increasing number recognize the utility of fever. however, only a few are thinking about how natural selection shaped the mechanisms that regulate defenses. such thinking will lead to new studies that provide the evidence we need to make better clinical decisions. in one particularly important example, a debate is now underway about whether influenza kills people directly or via the effects of released inflammatory agents (salomon et al. 2007 ). if the former is true, anti-inflammatory drugs will increase death rates, if the latter is true it will decrease them. the central defense against pathogens is, of course, the immune system. the costs as well as the benefits of immune responses need to be analyzed in evolutionary perspective as a life-history trait (zuk et al. 1996; lochmiller and deerenberg 2000; zuk and stoehr 2002; schmid-hempel and ebert 2003) . in addition to energetic costs, there is tissue damage from immune surveillance, reproductive costs, mate display costs, and others. of particular interest is variation in immune response, either because of limited resources or facultative systems that adapt the response to the current inner and outer situation (schmid-hempel 2003) . the study of pathogen virulence offers another example of how an evolutionary perspective can transform a field. just a decade ago, many physicians were taught that natural selection tended to shape pathogens and hosts to a benign mutual co-existence. after all, why kill the host that feeds you? rigorous evolutionary analysis revealed that this view is fundamentally incorrect may and anderson 1979; ewald 1994; frank 1996; ebert 1998) . the most important factor shaping virulence is its influence on the probability of transmission to a new host; virulence is shaped to whatever level maximizes transmission. for instance, prior to modern sanitation, bedridden patients with cholera could infect others and the organisms causing the most diarrhea were transmitted the most. the result is often fatal, but such traits are nonetheless selected for if they maximize transmission. this could have major implications for public health. good water purification systems prevent infection from bedridden patients, thus shifting the advantage to less virulent organisms whose victims can be up and around to spread them. virulence levels can also be influenced when several genetically different pathogen strains compete within a host. this should select for increased virulence. studies of trypanosomiasis (sleeping sickness) suggest multiple infections may be much more common than previously suspected (balmer and tostado 2006) . much of the recent work in evolutionary medicine asks questions about why natural selection has left the body vulnerable to disease (williams and nesse 1991; ewald 1994; nesse and williams 1994; stearns 1998; trevathan et al. 1999) . six categories summarize the main possible explanations: mismatch with the modern environment, pathogens coevolving with hosts, constraints on what selection can do, unavoidable trade-offs, reproduction at the expense of health, and defenses such as pain and fever that are useful despite causing suffering and complications (nesse 2005b) . these are potential evolutionary explanations for why selection has not made the body more resistant to disease. they are fundamentally differ-ent from proximate explanations about how the body works. the last two are not exactly explanations for disease vulnerability, but they need to be on the list because they are so often the source of misunderstandings. some hypotheses can be tested with a definitive experiment, others with comparative data, and some must be assessed by comparing observed features to those expected given the hypothesis (nesse 2008) . like much in science, this can be challenging. the 'thrifty phenotype' refers most generally to the benefits of weight gain and other mechanisms that conserve calories in environments characterized by erratic nutrition (neel et al. 1998b ). the extraordinary vulnerability to obesity in certain groups, such as pima indians and inhabitants of the south pacific island of palau, has been suggested to result from generations of experience with erratic food supplies. anthropological data on cultural variations in nutritional stability do not well support this interpretation (benyshek and watson 2006) . however, the more general idea that selection maximizes calorie conservation remains useful. natural selection may also have shaped mechanisms that adjust metabolic systems to cope with different nutritional environments. many studies demonstrate that low birth weight is a significant risk factor for obesity and diabetes in diverse populations (barker et al. 2002) . the evolutionary question is whether this 'fetal programming' is a 'predictive adaptive response' resulting from a mechanism shaped by selection to monitor fetal nutrition and adjust development in ways that facilitate coping with deprivation (gluckman et al. 2005) , or whether the association arises for other reasons (wells 2006) . low birth weight is also correlated with differences in stress reactivity (clark 1997 ) and rates of depression (costello et al. 2007 ). the adaptive significance of these reactions is as hard to figure out as the reactions are important. whatever the answer turns out to be, these evolutionary applications to medicine and public health nesse and stearns ª 2008 the authors studies have called our attention to the importance of the physiological state of mother and infant for the prevalence of lifestyle diseases later in life, with some well-documented effects delayed by several decades. has natural selection shaped a mechanism to detect and reject a fetus that is likely to succumb early to infection? a surprising amount of evidence is consistent with this hypothesis. the early term miscarriage rate is over 60% (boklage 1990) , and siblings tend to be more different in their hla immunological types than expected by chance. this suggests that other conceptions who received similar hla genes from both parents may have been selectively lost (ober et al. 1998) . while giving up a conception is inefficient, continuing to support a fetus who will likely succumb to infection is even more so, thus creating a selection force that could shape such a system. related evidence shows that spouses in small local communities tend to have hla types more different than expected (ober et al. 1997) . pheromone cues may guide individuals towards mates who differ sufficiently from themselves (jacob et al. 2002) . that the human female reproductive tract has been shaped to screen defective gametes and concepti is now well supported. that humans detect and choose mates based on immune complementarity is suggested by several studies but not yet definitively confirmed (loisel et al. 2007 ). the huge decreases in human mortality in the past century come not mainly from medical treatments, but from public health interventions, vaccination and sanitation in particular (armstrong et al. 1999) . they have, together, done more than all of the rest of medicine to improve human health. they also have created an environment vastly different from the one we evolved in. one result is a decreased burden of parasites such as worms in the gut. during most of human evolution we lived with helminth parasites. their absence in modern societies may help explain the vastly increased rates of autoimmune diseases, not just allergies, but diabetes and the childhood leukemias (elliott et al. 2007 ). regulation systems, including those that screen for antigens that react with self, were shaped with significant helminth loads on board (weinstock et al. 2004) . new evidence suggests that helminths evolved a capacity to make a protein (es-62) that down-regulates type-ii immunity that would otherwise attack them (melendez et al. 2007) . where helminth treatment has been initiated, asthma and crohn's disease rates have gone up (hurtado in press) . the cross reactivity between antibodies on schistosomes and dust mites, and different genetic levels of protection against helminths, may help to explain higher rates of asthma in people of african origins (barnes 2006) . in a bold clinical application, patients with an immune bowel disease, crohn's disease, were treated with the live ova of pig whipworm. about 70% entered remission . we can expect fast progress in autoimmune disease thanks to improved evolutionary understanding of the rule of modern hygiene. evolutionary approaches to cancer in general have progressed so quickly that their scope can only be suggested here (greaves 2000 (greaves , 2002 . the very existence of cancer results from an evolutionary process: differential replication of mutated cells (merlo et al. 2006) . the constant tendency for faster replicating cells to displace others is rigidly controlled by systems that regulate cell division and by surveillance systems that kill cells that are not where they belong. the length of telomeres, the bits of dna that hang from the end of chromosomes, may protect against cancer. each time a cell divides, the telomere get shorter; when it is gone, the cell dies. however, there is a side effect. short telomeres also shorten life-span (blasco 2005) . mathematical treatments of genes that predispose to cancer (crespi and summers 2006) and cancer cell evolution (frank 2007 ) offer promise of bringing coherence to this difficult field. the greatest opportunities for evolutionary applications relevant to health may be in public health and epidemiology. many have already been mentioned above, from diet to genetic epidemiology. every project needs an individualized application, but a few generalizations may help. for instance, when looking for risk factors for common diseases, the first question is whether the condition is equally common in hunter-gatherer populations. if not, then novel factors in the modern environment should top our list of suspects. some already do, such as too much fat and too little exercise. other factors, like the hygiene hypothesis mentioned above, are increasingly well supported. other apparently innocuous aspects of the modern environment deserve special attention. for instance, ubiquitous lighting has transformed our lives. instead of settling down to slow pursuits when darkness falls, we read, study, dance and watch television until long after we would have otherwise gone to sleep. the light itself may be risky. melatonin levels increase in the dark. a study of visually impaired women -who tend to have higher than normal melatonin levels -found risks of breast cancer about half of the rates for other women (kliukiene et al. 2001) . a subsequent study of nurses found those doing shift work and others exposed to light at night had increased cancer rates (stevens 2005) . in a fine demonstration of the value of research connecting proximate mechanisms with evolutionary hypotheses, melatonin-depleted blood from postmenopausal women has been shown to speed the growth of human breast cancer xenografts on nude mice (blask et al. 2005 ). more work is needed on this, but even now it suggests a new set of risk factors we should measure, and some simple public health advice -sleep with the lights off. obesity has doubled in the past 40 years in the usa, so that two-thirds of adults are now overweight or obese (wang and beydoun 2007) . diabetes and obesity are strongly correlated (neel et al. 1998a) . about 194 million adults worldwide have diabetes, and type 2 diabetes (late onset) is exploding. most diabetic patients are in india and china, and diabetes rates are expected to double, from 171 million in 2000 to 366 million in 2030 (wild et al. 2004) . much of the individual difference in vulnerability is accounted for by genetic differences (echwald 1999) , but this does not mean the obesity epidemic results from genetic abnormalities. instead, it means that novel aspects of our modern environment interact with genetic quirks to cause the problem, as it the case for nearly every polygenic disease. while not all ancestral environments were alike (elton in press), it does seem clear that modern diets are vastly different from almost everything that came before. we know what we should do to stay thin. we should eat less and exercise more. so, why don't we? one answer is that in the past individuals who were thin or who wasted calories in nonproductive exercise tended to have fewer children. selection favored those who took advantage of opportunities to eat fat, salt and sugar and who stored some extra calories in good times. selection has shaped mechanisms that limit weight gain, but they are feeble compared with those that prevent weight loss. these arguments have been made many times (eaton and konner 1985; eaton et al. 2002; chakravarthy and booth 2004) , but nutrition researchers sometimes still see these evolutionary hypotheses as alternatives rather than complements to new insights about molecular and physiological mechanisms that regulate caloric intake. an evolutionary view suggests two conclusions about diet, both unwelcome. first, most of us have built in tendencies to overeat and under-exercise when good food is available without much effort. second, there is no such thing as a completely natural diet that is perfectly safe. eating less fat is certainly wise, but it has costs. a diet of all wild vegetables will include poten-tially toxic substances. nonetheless, evolution does offer a way to ground the otherwise faddish area of nutrition research in a solid general understanding of the diets of our ancestors (eaton et al. 2002; leonard 2007; ungar 2007) . evolutionary principles are just beginning to be applied to mental disorders (nesse 1984 (nesse , 2005a wenegrat 1990; baron-cohen 1997; mcguire and troisi 1998; badcock and crespi 2006) , and they promise to bring them into the fold with other medical disorders (nesse 1999) . perhaps paradoxically, this may finally happen by recognizing the utility of negative emotions such as anxiety and depression (gilbert 1998; nesse 2000) . about half of mental disorders are emotional disorders characterized by excesses of negative emotions. while there is no doubt that much anxiety and depression is pathological, the capacities for anxiety and depression were shaped by natural selection along with the mechanisms that regulate them. these disorders are not like diabetes or parkinson's disease where a specific pathological lesion causes the disease. they are, instead, more like chronic pain or chronic cough, where the problem is dysregulation of a response that can be normal and useful. recognition that such evolutionary explanations are needed in addition to proximate explanations of mechanisms is just now dawning, along with recognition that categorical diagnoses that take no cognizance of environmental factors are fundamentally mistaken (nesse and jackson 2006; wakefield and horwitz 2007) . genes interact with environmental factors to create mental disorders. for instance, a study of a serotoninrelated polymorphism found that its strong effects on depression vulnerability were almost all mediated via an interaction with the number of severe life events (caspi et al. 2003) . this has become an exemplar for studies of gene · environment interactions. however, the measure of environmental effects, the number of severe life events, is crude compared with the sophistication of genetic analyses, especially in light of growing knowledge that low mood can be useful in certain special life circumstances (brown et al. 1995; nesse 2000; heckhausen et al. 2001 ). there are good theoretical reasons for thinking that low mood escalates to depression when an unreachable major life goal cannot be given up, and some supporting laboratory data (carver and scheier 1990) , but the case has not yet been proved. disorders such as schizophrenia require fundamentally different explanations. older ideas about the adaptive value of schizophrenia are now mostly discredited, although a haplotype associated with higher iq is also associated with a higher risk of schizophrenia (meyer-lindenberg et al. 2007 ). also, a haplotype associated with a gaba-a receptor shows clear signs of positive selection, which are weaker in lineages with schizophrenia (lo et al. 2007) . of particular interest is the hypothesis that autism and schizophrenia may be the flip-sides of extremes of the competition between imprinted genes coming from the father and the mother, the haig idea applied to psychiatry (badcock and crespi 2006; crespi et al. 2007 ). substance abuse is both more straightforward and more difficult. the straightforward aspect is that most drugs that affect the central nervous system evolved in plants to protect them from insects. in modern environments we create increasingly clever ways of purifying and administering them making addiction more common and more devastating. they hijack brain mechanisms that evolved to regulate behaviors such as foraging for ripe nuts (nesse and berridge 1997) . the problem becomes quickly complex, however, because of profound individual genetic variations in vulnerability to substance abuse that interact in complex ways with social environments that vary even from month to month for individuals (zucker 2006) . the field in general is gradually moving from an exclusive focus on proximate mechanisms and individual differences, to a broader consideration of the origins of vulnerabilities humans share and how they interact with certain environments to create disease. this review supports a global conclusion: much interesting and important research is taking place at the intersection of evolution and medicine. this research ranges from well-established applications of population genetics and phylogeny to new applications of evolution to specific medical problems such as infectious disease and aging. work in the area is growing rapidly. the fastest growth is in two disparate areas. first are those where evolution helps to make sense of new genetic data. why, for example, do genes that predispose to asthma persist? who would have thought they protect against schistosomes? growth is also fast in research asking new questions about why selection has left the body vulnerable to specific diseases. why, for instance, does bilirubin exist? who would have thought it was to slow aging? another question is whether evolution and medicine is one field, or is it just a collection of applications of different aspects of evolutionary theory? the increasing number of books and conferences that cover the full range of applications has found a large and eager audience. these initiatives have brought together diverse scientists and clinicians who are often delighted to learn about each other's work. in their shared evolutionary foundations, anthropologists, geneticists, physiologists, mathematical modelers and parasitologists turn out to have much in common. this is not artificial interdisciplinarity; advances in understanding evolution illuminate all of these fields and research in each of these fields offers opportunities for advancing evolutionary biology. the structure of evolutionary medicine is still defined mainly by the different contributing disciplines. genetics, paleontology, microbiology and immunology, ecology, reproductive medicine, cancer research, physiology, anatomy, behavioral biology, epidemiology, anthropology, and clinical medicine -all pursue evolutionary questions using somewhat different traditions and methods. a new framework, perhaps one based on questions such as those in the table in the introduction, may emerge. for now, the important observation is that workers in different field are increasingly finding commonalities in their shared foundation: evolutionary biology. awkward tensions always lurk when scientists from diverse disciplines come together. those working at more reductionist levels, sometimes pull away from those working on higher level questions. some doctors are uncomfortable welcoming in another discipline they do not know well. some evolutionary biologists grow quickly impatient with doctors who do not already know all about evolutionary biology. whether this new field can avoid such fragmentation remains to be seen, but many research opportunities provide a unifying force. for instance, research on lactase persistence benefits markedly from close collaboration between geneticists and anthropologists, and their conclusions have clinical relevance. a related challenge is dealing with hypotheses about adaptation (rose and lauder 1996) . experimental methods are not often available, many scientists are unfamiliar with comparative and other methods, firm conclusions can be elusive, and standards of evidence are still evolving (nesse 2008) . editors do not always have access to evolutionary expertise, so some good work does not get published where it will be widely seen, and some iffy ideas get presented as stronger than they are. while these problems are not universal, they are real, and they will be solved only by doing science -proposing hypotheses, testing them, discarding those that fail, coming up with new ideas, and finding better ways to test them. the boundary between basic science and applied medicine offers another challenge. preparing this review has impressed us with the number of clinically relevant findings. however, does understanding evolution change dramatically what a physician does in her day-to-day work? in general it does not, and should not. clinical decisions based on theory alone are notoriously suspect. treatment should, whenever possible, be based on controlled studies of treatment outcomes. however, lack of evolutionary understanding among physicians fosters misunderstanding about issues as important as aging, diet, and when it is wise to use medications to block defensive responses. while there is a trend for doctors to just carry out protocols, we want doctors to have a deep knowledge base so their decisions are informed by understanding the body and disease. better decisions come from doctors who understand the ecology of immune responses, the evolutionary reasons for polygenic diseases, the phylogeny of cancer cells, and the origins of antibiotics. we began by describing the magnitude of the gulf between evolutionary biology and medicine. we were struck by how much work is now transcending the gap, but we also were surprised to discover that the gap is even larger than we had thought. the research results described in this article are but a small flock of birds flying over the grand canyon. does it matter? would efforts to bridge the gap pay off in improved human health? many of the findings reviewed here suggest the answer is yes. they serve at the very least to suggest new studies, ranging from whether we need to be concerned about vaccines causing increased virulence to what kinds of disorders are likely to arise from advanced reproductive technologies. however, even aside from suggesting studies with direct clinical and public health relevance, evolutionary approaches increase our fundamental understanding of the body and disease. this is basic science at its most basic. the huge investments in understanding the mechanisms of the cell and gene replication dwarf all investments to date in understanding the evolutionary origins and functions of traits that leave us vulnerable to disease. we predict that increased focus on evolutionary questions will not only offer useful new understanding, it will also synergize with new understanding of mechanisms. in the abstract, we suggested that an evolutionary perspective will be especially helpful in doing away with the incorrect metaphor of the body as a machine designed by an engineer. some readers certainly wondered what we could possibly mean. of course, the body is a machine in the sense that it is composed of chemicals, levers, pulleys and systems that maintain homeostasis. furthermore, machines and bodies are alike in one important way; both are bundles of trade-offs. improving any one trait will likely harm something else. however, because they are products of evolution, bodies are very different from machines (childs 1999) . a deep understanding of the body as a body is perhaps evolution's greatest single contribution to medicine. machines are designed by an engineer to serve a human purpose. blueprints define the ideal type, and manufac-turing attempts to turn out identical units. when a defect is discovered, engineers change the design. bodies are not designed; they are shaped by natural selection. there is no blueprint, no ideal type. variation is intrinsic. there is no normal genome. there is no normal body. there is no separate manufacturing facility; there is just the process of development -genes interacting with environments to create adult forms. the process involves some chance factors, and also adaptations that monitor the early environment and shift development in ways that adjust the adult form to a particular environment. some traits, such as a birth canal that passes through a narrow circle of bone, cause problems for the species. but there is no engineer with a drawing board to go back to. rerouting birth via the abdominal wall would work better, but the intermediate stages between that and the current route would not work, so the system stays suboptimal. bodies are bodies shaped by selection, not machines designed by intelligence. giving up the machine metaphor gives medicine a stronger foundation in biology. finally, there is the challenge of how best to advance work at the interface of evolutionary biology and medicine. we offer the following brief observations and suggestions in hopes that they will stir discussion and action. at present there is no way to find out what is going on in the field of evolution and medicine. no keywords adequately capture the literature; there is no journal and no society. the resources at the evolution and medicine network (http://evolutionandmedicine.org) offer access to a variety of teaching and information resources and a nucleus for the growing community. the network is being expanded to include the evolution and medicine review: news, recent publications of interest, commentaries on the most notable papers, and questions posed and answered by members of the community. it is intended to meet the need for a central source of information about new research and opportunities in this diverse field. evolutionary applications is a natural outlet for new research in the area of evolution and medicine, and a special issue devoted to the topic is planned for 2009. discussions are underway to organize a society for evolution and medicine. the international alliance of research universities (iaru) evolutionary medicine initiative has budgeted funds for a founding meeting in 2009, likely in conjunction with the darwin festival celebrations honoring the 200th year of darwin's birth and the 150th anniversary of the publication of the origin of species. the evolution and medicine network provides updated information on this and other new developments. many young scientists and physicians want to apply evolutionary principles in their areas of medical research, but training programs are not yet available. some are being organized. a proposal to be funded by the iaru will provide research training in evolutionary medicine at six universities including the university of copenhagen, which is recruiting a professor of evolutionary medicine. several other universities, including durham in the uk, are also developing programs. a major research institute is not yet in the works. many have suggested pursuing the strategy that worked so well in the early days of human genetics, summer workshops that bring together established and junior researchers for an intense period of study and discussion with leaders in the basic science. the evolution and medicine network will announce such programs as they become available. funding for projects in evolutionary medicine is available for aging, genetics, and infectious disease. the nih genetic variation and evolution study section has been especially valuable not only in providing funding, but also in providing guidance that increases the quality of work in this subfield. support for work on broader questions is harder to find. private foundations can take the lead in supporting important projects that do not fit the portfolio of government funding agencies, and in supporting efforts to develop the field as a whole. we say reforms, because physicians not now taught even the most basic principles of evolutionary biology as they apply to medicine. it is as if the engineering curriculum included no physics. with no evolutionary biologists on their faculties, no resources to hire them, and an overly-full curriculum, only a very occasional far-sighted dean and faculty will be able to bring evolutionary biology into the curriculum. the modernization of medical education will be helped by curriculum recommendations from advisory bodies such as the institute of medicine that are backed up by new questions on tests administered by the national board of medical examiners and other similar bodies. while some aspects of evolutionary biology must be a part of medical education, it is unrealistic to provide all of the necessary foundations in medical school. like other basic knowledge, much needs to be in courses prior to medical training. undergraduate courses on evolutionary medicine are an excellent solution. most such courses do not provide enough basic evolutionary knowledge, so a combination with a basic evolution course is essential. the logical outcome will be evolutionary questions on the examinations like the mcats in the usa that are used to screen applicants to medical schools. implementation of the above recommendations will require close collaborations among 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history phd thesis the developmental behavior genetics of drug involvement: overview and comments immune defense and host life history trade-offs in parasitology, evolution and behavior warm thanks to carl bergstrom, pascal gagneux, catriona maccallum, peter jones, and mark thomas for very helpful comments and suggestions; to bernard crespi, robin bush, and an anonymous reviewer for extremely valuable critiques; and to the wissenschaftskolleg zu berlin for a fellowship for rn that made completion of this article possible. key: cord-321993-uazc3lyg authors: hedrick, stephen m. title: the imperative to vaccinate date: 2018-10-31 journal: the journal of pediatrics doi: 10.1016/j.jpeds.2018.06.041 sha: doc_id: 321993 cord_uid: uazc3lyg nan stephen m. hedrick, phd the disease legacy of civilization h uman beings are almost certainly the most diseased species on earth. by one accounting, there are at least 1400 human pathogens, including bacteria, fungi, prions, protozoa, viruses, and worms, and of these, 100-150 appear capable of causing human epidemics. 1, 2 even this is likely to be an underestimate, as new and sensitive sequencing techniques continue to uncover new viruses at a steady rate. 3 we human beings are remarkable in many ways, but why are we remarkable for playing host to so many infectious agents? why is it that we must maintain high levels of vaccine coverage to prevent infectious agents from sickening or even killing large swaths of the population? the answers lie in the story of human disease epidemics, and it begins with human cultural and technological ascendance and what we now understand to be its inevitable consequences for pestilence and death. it is about our ingenuity, which has caused the retreat of many infectious diseases, but highlights a central tension in human existence-immediate self-interest vs long-term collective welfare. 4 the concept is not just academic; there are realworld implications that we can resolve with an understanding of human disease ecology. the notion is that we are not only culturally connected or genetically connected through a common ancestry. rather, there is another fundamental concept that is, perhaps, not widely accepted or even understood. we are biologically connected, in the present, through our exchange of infectious agents and our common susceptibility to disease. to understand modern human disease prevalence, we have only to look to the most basic principles of epidemiology. a simplified version is that diffuse or small host populations cannot sustain an acutely infectious agent, meaning one in which infection is followed by clearance and long-term immunity. as the number of people with immunity increases, the density of susceptible hosts decreases, and with the corresponding decline in transmission, the infectious agent is not maintained in the population. 5 this principle described our preagricultural ancestors-a few thousand individuals congregated in groups but spread out over an enormous area. small or low-density populations can only sustain a certain type of infectious agent, one that persists, usually for the life of the host. 6, 7 once infected with herpes viruses, such as herpes simplex virus, cytomegalovirus, or epstein-barr virus, we are infected for life, and such viruses have infected us since even before we became human beings. [8] [9] [10] [11] to some extent, this was the primordial state of disease in diffuse bands of preagricultural hunter-gathers: persistent viruses, bacteria (eg, mycobacterium tuberculosis), intestinal protozoa, worms, and fleas. our paleolithic ancestors were not disease-free, but they almost certainly did not experience periodic and devastating epidemics. 12, 13 conversely, large populations that live at high density, such as modern human beings, can sustain a much greater diversity of infectious agents, including those that the immune system is able to clear. transmission from person to person is rapid enough and continuous, such that there is little selective pressure for persistence. large and dense urban populations can maintain acutely infectious agents indefinitely due to a constant source of newly susceptible hosts in the form of immigration or births. these agents often share an ability to be transmitted by casual contact such as respiratory droplets produced by a cough or a sneeze, and as evidence of the success of this pathogen strategy, there are more than 200 different viruses from at least 6 different virus families (adenovirus, coronaviruses, influenza virus, parainfluenza virus, respiratory syncytial virus, and rhinovirus) that cause "cold" symptoms: sneezing, coughing, and runny nose. 14 the dawn of agriculture and the domestication of animals, especially herd animals, allowed the emergence of permanent human settlements and the growth of large situated communities. 15 the world's population increased approximately 1000-fold from the beginning of the agricultural revolution to the end of the 19th century, and most importantly, settlements eventually grew into a huge massing of humanity. simultaneously, we domesticated animals and ourselves, and we sampled all of the viruses and bacteria existing in cows, horses, pigs, sheep, goats, and birds. those that could replicate in human beings and spread from person to person by respiratory propulsion (or other means, such as fecal-oral) became established evermore in the human population. this is the answer to why we are the most diseased species on earth. we are the only species to so profoundly and rapidly change the way in which we interact with each other and other animals; in other words, we invented for ourselves an entirely new ecosystem. so, in addition to the endless parade of cold viruses that circulate among us, we acquired a great many deadly infectious agents, such as those that cause diphtheria, influenza, measles, meningitis, mumps, plague, rubella, smallpox, typhus, whooping cough, and others. each disease has its own history and severity, but all rely on large, high-density populations for continued propagation. these newly acquired infectious agents not only caused severe or deadly disease, they shaped the population. many are known as childhood diseases because they infect susceptible children who either recover from the disease and retain immunity or die. in a population in which a disease like measles existed, everyone contracted the virus exactly once, such that almost all surviving adults were immune. what does the world look like in the face of measles? from 1956 to 1960, before the availability of a vaccine, an average of 542 000 cases of measles were reported each year in the us, along with an average of 450 measles-related deaths, 4000 encephalitis cases (often with permanent brain damage), and 150 000 respiratory complications. the measles vaccine was licensed in 1963 and the measles, mumps, rubella (mmr) vaccine was licensed in 1971. for the years between 1987 and 2000, the number dropped to 28 730 cases of measles in children younger than 5 years of age; 97 died, 43 contracted encephalitis, and 2480 contracted pneumonia. since 1997, there has been less than 1 case per million population in the us. 16 the global burden of measles in 1999 was an estimated 873 000 deaths that were reduced through a world-wide vaccination campaign to an estimated 164 000 deaths in 2008. 17 those who survive measles without lasting effects still have 2 worries. one is that measles infection depresses the immune system for up to 2 years, making children more susceptible to other infections, 18 and a second is the possibility of developing subacute sclerosing panencephalitis, a usually fatal neurologic degenerative disease caused by reactivation of latent measles virus. the assessed risk is on the order of 1 in 10 000 measles cases and as much as 10-fold greater for children who contract measles before the age of 12 months. 19 for children who are immunocompromised, such as those being treated for leukemia, an actual measles infection is severe, extended, and often fatal. 20 although measles is possibly the world's most infectious human virus, it was not the most devastating of the childhood infectious diseases. the smallpox death toll for just the 20th century has been estimated at upwards of 300 million people, similar to the entire population of the present-day us. 21 smallpox caused more deaths than all the wars in history. for centuries before vaccination, most urban families could count on losing multiple children to smallpox, diphtheria, scarlet fever, or whooping cough. with widespread vaccination, combined with targeted vaccination to insulate the last few cases, smallpox was eliminated as an infectious disease on earth. smallpox eradication was our first and thus far only complete victory over a human disease-causing agent, made possible by universal, global vaccination, and intensive surveillance. after tortuous millennia of epidemic disease and hundreds of millions dead, who would argue that this was not a most wonderful gift given by humankind to itself? but that gift was not without cost, and the cost was a tincture of personal independence and the acknowledgement that each of us is inextricably tied to the entire human community. it took the idea of community out of the realm of philosophy and placed it as a fundamental property of life. smallpox eradication itself was a physical enactment of the tension between personal freedom and the authority of society. in on liberty, in chapter iv, john stuart mill asks, "what then is the rightful limit to the sovereignty of the individual over himself? where does the authority of society begin? how much of human life should be assigned to individuality, and how much to society?" mill's inquiries can be answered by biology, but first consider the concept of community protection (often referred to as "herd immunity"). as the density of susceptible (unvaccinated or disease naïve) hosts declines, so does the incidence of disease. below a certain threshold, the incidence of disease (frequency of new infections), even in unimmunized people, approaches zero. this is community protection, and it follows directly from basic epidemiology. vaccination effectively reduces the number and density of the disease-susceptible people, making acutely infectious agents unsustainable in the population. 22 conversely, because vaccine protection is sometimes imperfect, a vaccinated individual living within a diseasesusceptible population is at substantial risk. the risk of disease for any individual is thus most importantly dependent on the collective immunity of the population, especially those most susceptible to infection, usually the youngest children and oldest adults. in this regard, disease ecology does not equivocate; in the world as it exists today, our health and our very being depend on the immune status of the rest of humanity. the rightful limit to the sovereignty of the individual over him or herself stops at the boundary of disease immunity. as long as one case of smallpox could be found on earth, billions were at risk. even without considering the imperative of contagious disease, mill came to the same conclusion, "as soon as any part of a person's conduct affects prejudicially the interests of others, society has jurisdiction over it. . .." two centuries before on liberty and before the enlightenment, this was expressed after a fashion in john donne's meditation xvii, "now this bell tolling softly for another, says to me, thou must die," written while he was convalescing from a near-fatal disease, possibly typhus. although this meditation was ostensibly concerned with god as the author of every person and every death, we might equally apply it in a way that donne could not-we are each of a network, a medium for disease that transcends us as individuals. the death of one of us portends many more. we can rage against this injustice, but it is literally a fact of life. in this context, the famous line from meditation is relevant, "no man is an island, entire of itself." community protection is a fundamental concept with no strict definition. the threshold is sharp but varies with each infectious agent. it protects vaccinated and unvaccinated people alike. it is the most powerful force in disease prevention but exists only in the immunity status of the entire population network. considering the difficulty of this concept, it is no wonder that as a society and as a people we do not have a consensus the journal of pediatrics • www.jpeds.com volume 201 • october 2018 concerning the responsibility of individuals to vaccinate their children. one way to understand vaccination decisions is as an exercise in game theory played out over the entire human population of the earth. in this case, each individual is defined narrowly in economic terms, acting as if he or she balances costs against benefits to maximize personal advantage. if most everyone cooperates (vaccinates), then everyone enjoys the benefits of being disease free. in contrast, the decision to cooperate may be perceived to have a cost, and individuals looking to maximize personal advantage will choose noncooperation at a certain probability. when no one is vaccinated and everyone is in danger, that probability is close to zero-everyone is incentivized to vaccinate or risk the possibility of deadly disease. this must have been the dominant sentiment in the time of smallpox. as universal vaccination is approached, danger diminishes with or without vaccination, and the probability of noncooperation increases. for measles, the threshold for community protection is calculated to be 94.4%, that is, when 94.4% of the population has received 2 doses of mmr vaccine, the community is protected from disease. 23 under such conditions, some parents may decide not to vaccinate and thus avoid even very rare adverse effects. the consequence of this is that the rate of vaccination drops below the threshold, and the community is no longer protected. in other words, as we proceed toward elimination of a disease by vaccination, as we are for poliomyelitis, the invisible hand of the market pulls defeat from the jaws of victory. from this reasoning, elimination of a disease on a purely voluntary basis has been proposed to be unlikely, and the thought is that compliance to protect the population or eradicate a disease can only be achieved by a mandatory vaccination policy. 24 in the western hemisphere, we have all but eliminated measles and rubella, in one sense moving us backward in time to the pre-columbian rarity of acutely infectious diseases. however, should we lapse in our vaccination vigilance, within one generation we could replay the disease devastation of the 16th century that included death of more than one-half of the native inhabitants of the western hemisphere. 25 we are part of, what watts and strogatz called, a small-world network 26 -with no more than 6 degrees of separation connecting the entire 7+ billion human beings on earth. like the spread of middle east respiratory syndrome from the middle east to korea, we can consummate those connections, wherever they may be, with a day's travel. a glimpse of a future with poor vaccine adherence occurred not too long ago, with an outbreak of measles originating in anaheim, california. the infecting person (patient zero) almost certainly arrived from abroad, but most of the infected individuals were unvaccinated us residents. 27 the decline in mmr vaccination compliance began with a 1998 medical report in the journal lancet. andrew wakefield and 12 colleagues published an analysis of 12 children claiming to show a connection between mmr vaccination and the onset of a newly described "pervasive developmental disorder" that they summarized as a "chronic enterocolitis in children that may be related to neuropsychiatric disorder." at face value, this is a tiny sample size with no controls. it linked 3 common conditions with mmr vaccination based on parent's recollections. multiple large studies subsequently showed no such association, and the paper was retracted by 10 of the authors and by the journal after a prolonged study by the general medical council in the united kingdom; however, it was not just a case of flawed science, it turned out to be fraud driven by avarice. in articles published by the british medical journal, the investigative journalist brian deer revealed how wakefield had been hired to attack the mmr vaccine by a lawyer, richard barr. wakefield and colleagues were paid to contrive the existence of a syndrome, he initially called, "autistic enterocolitis" for the express purpose of bringing a class action lawsuit against vaccine manufacturers; this occurred before initiation of the disgraced study. 28 another apparent moneymaking scheme was ironically to market vaccines. in a press conference given after the publication of his lancet paper, wakefield said he could not support the triple mmr vaccination and called for vaccination to each disease separately. he had previously patented a single measles vaccine. the british general medical council revoked his license to practice medicine, and he was asked to leave the royal free hospital in london. the wakefield study has no basis in reality, but its publication corresponded to a substantial drop in mmr coverage in the united kingdom, europe, and the us. 29 in response, some countries or states within the us have made vaccination a mandatory condition for entrance into schools. for example, california senate bill no. 277, signed into law june 2015, requires vaccination for all children attending any public or private elementary or secondary school, childcare center, day nursery, nursery school, family daycare home, or development center. exemptions for described medical conditions are permitted, but those based on personal or religious beliefs were to be phased out. because california requires vaccination records for all schools, the effects of the bill could be tracked-and the effects were dramatic. in 2014, more than 33% of school children lived in california counties with vaccination rates less than 90%, and 70% of children lived in counties with less than a 95% vaccination rate. 30 interestingly, the counties with the lowest rates of vaccination were of 2 types, rural counties largely located in the most northern part of the state and counties that include the tony urban communities surrounding san francisco and los angeles. 31 by 2016, more than 95% of children were from counties with greater than 95% vaccination. nonetheless, the law only requires vaccination records for students as they enter each grade span (kindergarten, seventh grade, etc), and it recognizes personal exemptions previously on file. as such, there are still schools in which a single case of measles could spark a local epidemic. contrast the vaccination rate in california, where comprehensive vaccination is required to attend school, with that of oregon, where exclusions based on personal beliefs are allowed with only a requirement for completing an informational module online. 32 the proportion of the population in oregon counties with kindergarten vaccination rates greater than 95% has gone from almost 100% in 2000 to just 30% as of 2015. 30 in addition to community health, the notion of not vaccinating seems to deny short-term self-interest. even with a low disease incidence brought about by community protection, pertussis vaccination is a small price to pay for the prevention of whooping cough. beyond that, universal vaccination protects children with immunodeficiencies that arise either from congenital or acquired conditions and their treatments. it can eliminate a disease from the world for all time, saving all future generations, but at what cost? what is the safety of vaccination? each vaccine from each manufacturer is reviewed by the food and drug administration for safety before licensing, and after licensing, the centers for disease control and prevention and food and drug administration maintain a nationwide monitoring system, the vaccine adverse event reporting system, a signal detection system to identify rare events not found in prelicensing reviews. the program allows anyone to report an adverse reaction online, by fax, or by mail. 33 the centers for disease control and prevention also operates the vaccine safety datalink in conjunction with us care organizations that track data from more than 9 million people. these data provide the means to monitor the safety of current and recently introduced vaccines nearly in real-time as they are administered to people across the country. the data from the vaccine safety datalink are used to devise the vaccine regime for children and assess the frequency of complications as they arise. 34 there also exists a national vaccine injury compensation program (vicp), run by the health resources and services administration. this program receives reports of adverse vaccine reactions, studies each claim, and makes 1 of 3 determinations: (1) an adverse reaction occurred "more likely than not"; (2) the individual is compensated, although the panel does not concede that there occurred a vaccine-related adverse reaction; and (3) the case is adjudicated by a court within the us court of federal claims. because the vicp is the only avenue for vaccine-related compensation in this country, the number of filed cases is one measure of the number of adverse reactions severe enough to incite a claim. for the years of 2006 through 2013, there were approximately 2.2 billion vaccine doses distributed in the us. the total number of cases brought before the vicp was 2853, and the number ultimately compensated was 1672. that is, about 1 in a million vaccine doses was associated with some sort of adverse reaction severe enough to bring a patient to the vicp. importantly, this is but an average for all vaccines and all groups of people, but it highlights the overall rarity of vaccine-associated, severe adverse events. considering the benefit to the individual and to society, this would seem to be a reasonable risk. aside from sober risk assessment, sticking an infant with a needle to induce an immune reaction might feel unnatural. but it isn't so. the immune system is naturally engaged and constantly fighting many potential infections on a continuous basis. for example, in people with an acquired immunodeficiency, such as those low numbers of t lymphocytes, previously benign bacteria, fungi, and viruses become deadly: cytomegalovirus, candida, pneumocystis carinii (now pneumocystis jirovecii), toxoplasma, and other environmental agents can cause sickness or death. 35 regardless of the presence of actual disease-causing agents, without the constant activity of our immune system, we perish. another concern is that multiple vaccinations might "overload" the immune system, causing children to be more susceptible to unvaccinated diseases. however, in a study of nonvaccine-targeted infections recorded from emergency department visits, there was no significant correlation with the number of vaccines given to children 24-47 months of age. 36 medical studies are difficult to evaluate, even for professionals. the wisdom of one moment is often replaced in the next. a reasonable course of action with respect to new clinical findings is to wait and act conservatively. however, we now have a century's worth of experience in vaccinating billions of people. we have witnessed the regression or elimination of many infectious diseases in the face of vaccination. and we have studied the short-and long-term effects of vaccination. this is now established science. we can work to make vaccines even safer and more effective, but we cannot as a society regress to some past era in which we count hundreds of thousands of measles or polio cases per year. infectious diseases are a major, and almost certainly permanent, part of human existence. the growth of civilization with the addition of animal domestication made the appearance of epidemic diseases inevitable, but human inventiveness has allowed us to find countermeasures that relieve at least some of our collective misery. furthermore, the experience of humankind over the past several millennia has shown that we have no choice; our place in the network of hosts susceptible to human pathogens gives lie to our notions of complete personal independence. even the most atavistic society would not choose for their children a path of immune naiveté (at least not for long). perhaps this is an instructive irony. it takes deadly infectious diseases to see that we are all of a one species, biologically connected, and isolated on earth. ■ submitted for publication jan 26, 2018; last revision received jun 1, 2018; accepted jun 13, 2018 risk factors for human disease emergence ecological origins of novel human pathogens search strategy has influenced the discovery rate of human viruses the meaning of human existence network structure and the biology of populations measles endemicity in insular populations: critical community size and its evolutionary implication epidemiology meets evolutionary ecology molecular phylogeny and evolutionary timescale for the family of 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revelations of the americas before collective dynamics of 'small-world' networks measles outbreak linked to disney theme parks reaches five states and mexico wakefield's "autistic enterocolitis" under the microscope how the case against the mmr vaccine was fixed after a debacle, how california became a role model on measles vaccination rate jumps in california after tougher inoculation law safety monitoring in the vaccine adverse event reporting system (vaers) the vaccine safety datalink: successes and challenges monitoring vaccine safety opportunistic infections in patients with and patients without acquired immunodeficiency syndrome association between estimated cumulative vaccine antigen exposure through the first 23 months of life and non-vaccine-targeted infections from 24 through 47 months of age key: cord-017752-ofzm3x3a authors: nan title: theories of carcinogenesis date: 2007 journal: molecular mechanisms of cancer doi: 10.1007/978-1-4020-6016-8_1 sha: doc_id: 17752 cord_uid: ofzm3x3a the oldest description of human cancer, referring to eight cases of tumors of the breast, was found in the egyptian edwin smith papyrus, written around 3000–1500 bc. the oldest specimens of human cancers were detected in the remains of a female skull dating back to the bronze age (1900–1600 bc), and in fossilized bones of ancient egypt. the mummified skeletal remains of peruvian incas, dating about 2,400 years ago, contained lesions suggestive of malignant melanoma. the term “cancer” goes back to hippocrates (460–370 bc), who named a group of diseases καρκινοσ and καρκινομα, the ancient greek word for crab. it is a metaphor for the hard center and spiny projections of the tumors he studied. cancer is the latin word for crab and its use has been traced back to galen (ad 129–199). a snapshot of theories of carcinogenesis, devised in the course of the last two centuries, reflects the progress of insight from the cellular level via biochemistry to an understanding of damaging influences and oncogenes, and to a more wholistic approach in the regulatory theory. it shows the relative success of reductionism as well as the current need to put the insights of various research endeavors into broader paradigmatic contexts. in 1665, robert hooke described walled cavities in his microscopic examination of cork and called them cells. in 1805, lorenz oken conceptualized a cell-based theory of life, arguing that plants and animals are assemblages of tiny living infusoria. this notion was later populated and refined by matthias schleiden and theodor schwann [schleiden 1838; schwann 1839; nurse 2000 ]. in 1841, robert remak (1815 -1865 described the phenomenon of cell division in chick embryos and in muscle development. between 1850 and 1855, he extended these observations to embryonic development and proposed that tumor cells arose by cell formation from existing specific tissues [remak 1852 [remak , 1855 . like giovanni morgagni, who had performed the first autopsy in 1761 and had correlated illness to macroscopic pathology, rudolf virchow (1821 virchow ( -1902 correlated illness to microscopic pathology. after initial skepticism, virchow acknowledged remak's evidence for cell division. in 1858, he gave a series of 20 lectures to a group of physicians at the institute of pathology in berlin, in which he summarized his experience in microscopic anatomy of tissues with special attention to those deviating from the healthy condition [virchow 1858 ]. according to virchow's dictum "omnis cellula e cellule" cells of diseased tissues are derived from normal tissues, implying that malfunction begets disease (significantly, virchow had been a student of müller's, who had demonstrated in 1838 that cancer is made up of cells, not lymph; but he was of the opinion that cancer cells arose from interstitial budding elements, blastema, not from normal cells). hence, tumors are derived from cells that divide faster than they should. the average human body experiences around 10 16 cell divisions in a lifetime. with an individual's risk to contract cancer being about 10%, malignant transformation occurs in 1 out of 10 17 cell divisions [weinberg 1998 ]. the mechanistic underpinning for this process was defined by the identification of key regulators of the cell division cycle by leland h. hartwell, r. timothy hunt, and paul m. nurse. chapter 1 the analysis of transformation has been guided substantially by the technical accomplishment to expand cells in culture. tissue culture was developed in the early years of the 20th century [harrison 1907; burrows 1910] . warren lewis cultured rodent cancers [lewis 1936] . in 1951 at the johns hopkins hospital, george gey established human cancer cell culture from the cervical adenocarcinoma of the 30-year-old, black henrietta lacks [gey et al. 1952] . although the resulting hela cells are among the cornerstones of cancer research, their high rate of proliferation caused a risk for cross-contamination of other cultures by them. this lead to the establishment of cell-typing techniques on the biochemical [gartler 1967 ] and genetic [nelson-rees et al. 1973] levels. generally, the human tumor cells that grow permanently in culture are a selected group of very aggressive cancers. almost all of the continuous cell lines are derived from high-grade, high-stage cancers. programmed cell death (apoptosis, greek: falling off of tree leaves) [kerr et al. 1972 ] may be invoked by many organisms as a control mechanism to prevent unrestricted growth. research during the 1960s through 1970s in the worm caenorhabditis elegans, identified ced-3 and ced-4 as essential genes for programmed cell death, while ced-9 was found to be a negative regulator of apoptosis. the 2002, nobel prize in medicine and physiology was awarded for these observations to sydney brenner, h. robert horvitz, and john e. sulston. the first mammalian homolog for ced-3 was described as bcl-2, a gene that is involved in b-cell lymphomata [negrini et al. 1987; vaux et al. 1988 ]. bcl-2 transfected b-lymphocytes are resistant to apoptosis, which is typically induced by interleukin-3 withdrawal. for the first time, it was demonstrated that the pathway to tumorigenesis depends not only on the ability to escape growth control but also on the ability to prevent cell death [hockenbery et al. 1990 ]. according to the biochemical theory of cancer, a key process that governs cell proliferation goes awry and causes transformation. various aspects of metabolism may be affected in a manner that could lead to cancer. consequently, before the discovery of oncogenes, a large variety of theories was debated, which incriminated the malfunction of diverse biochemical processes as causative for malignant transformation. during tumor progression, the enzymatic composition of the affected cells is simplified (described as the theory of convergence in cancer), so that various cancers resemble one another more than they resemble their tissue of origin [greenstein 1954 ]. as one possible underlying reason, the biochemist otto von warburg [von warburg 1930] had suggested that the oxidative metabolism in cancer cells is replaced by glycolysis and that the excessive proliferation of cancer cells reflects their ability to metabolize independently of oxygen. later, it was found that the limiting substrates for tumor growth are oxygen and glucose. hence, anaerobic glycolysis is not the cause, but the consequence of the accelerated growth, which cannot be satisfied by the reorganization of the micro-vasculature [vaupel et al. 1976 ]. however, in a remarkable reversal toward supporting the warburg model, a 2005 publication showed that in cells engineered to become cancerous glycolytic conversion started early and expanded as the cells became more malignant [ramanathan et al. 2005 ]. this rekindled the discussion of bioenergetics in cancer cells. others attributed the simplified enzyme patterns of cancerous cells to a regression of the tumor tissues to early embryonal stages of development. highly malignant cells tend to resemble fetal tissues more than their adult normal counterparts do. the idea of derepressive dedifferentiation in carcinogenesis found support in the occurrence of onco-fetal proteins during the disease. the expression of these genes should be repressed in differentiated tissues, but this repression is reversed in tumors. the description of tumor tissue in histopathologic analysis as dedifferentiated is derived from this concept. the alternative model of "oncogeny as partially blocked ontogeny" suggested that cancer is the result of a series of alterations in the genes and their gene expression, which prevent a stem cell from completing all the steps necessary for terminal differentiation, suggesting that the target cell for carcinogenesis is the pluripotent stem cell [potter 1978 ]. the protein deletion theory, an extension of the dedifferentiation theory, is an epigenetic model of cancer. based on the observation that a carcinogenic aminoazo dye covalently bound liver proteins in animals undergoing early carcinogenesis, whereas little or no dye binding occurred to the proteins of tumors induced by this dye, miller and miller [1947] proposed the deletion hypothesis. they suggested that carcinogenesis resulted from permanent alterations or loss of proteins that are essential for the control of growth. thus, carcinogens eliminate specific enzymes from the affected cells by binding covalently to water-soluble basic proteins (h 2 proteins according to electrophoresis nomenclature). this causes the elimination (deletion) of these proteins from the cells. cancer originates because the water-soluble basic proteins contain several growth inhibitory components. therefore, the initial step in carcinogenesis is the inactivation of endogenous inhibition. transformation can be associated with refraction to exogenous inhibitors of cell cycle progression. potter [1964] suggested that the proteins lost during carcinogenesis may be involved in the feedback control of enzyme systems required for cell division, and he proposed the feedback deletion theory. in this model, repressors crucial to the regulation of genes involved in cell proliferation are lost or inactivated by the action of oncogenic agents on the cell, either by interacting with dna to block repressor gene transcription or by reacting directly with repressor proteins and inactivating them. it was thought that experimental evidence, in which the fusion of cancerous cells with nontransformed cells resulted in the absence of transformation, supported the epigenetic theory. later, this phenomenon was attributed to the functional dominance of tumor suppressor genes. the demonstration of the presence of an ordered biochemical imbalance, linked to transformation and progression, in cancer cells led to the molecular correlation concept. weber [1977] stated that the biochemical dysregulations underlying neoplasia could be identified by elucidating the pattern of gene expression as revealed in the activity, concentration, and isozyme aspects of key enzymes and their linking with neoplastic transformation and progression. key enzymes are involved in the regulation of rate and direction of the flux of competing synthetic and catabolic pathways and are most likely affected in the malignant process. a number of enzyme activities found to be altered in malignant cells are those involved in nucleic acid synthesis and catabolism. in general, the key enzymes in the de novo pathways and salvage pathways of purine and pyrimidine biosynthesis are increased and the opposing catabolic enzymes are decreased during malignant transformation and tumor progression. these findings and concepts were further developed by the analysis of gene expression profiles and identification of gene expression signatures in cancer cells some 20 years later. the stigma that cancer equals death, originating in the experiences of hippocrates, galen, and celsus, was attached to the disease for centuries. it led to the long-respected dictum that doctors should not inform their patients of the diagnosis to avoid agony. in view of progress in surgery, which allowed the removal of some tumors, the american cancer society was formed in 1913 to educate the public about the warning symptoms of cancer and to reduce their fatalistic fears. the increased public health awareness was helpful whenever carcinogenic mechanisms were identified and the need for lifestyle changes was publicized. the insight that malignancy may be caused by the influence of damaging agents forms the basis of the noxious theory of carcinogenesis. among the influences that may cause cancer are chemicals, radiation, and viruses. chemical carcinogenesis. in 1775, chemical carcinogenesis was observed by the english surgeon sir percival pott, who related the cause of scrotal skin cancer in a number of his patients to a common history of occupational exposure to large amounts of coal soot as chimney sweepers when they were boys. the connection between soot and cancer was confirmed in 1915 by the first controlled experimental induction of cancer in laboratory animals by katsusaburo yamagiwa. the experiment established chemical carcinogenesis, and specifically occupational exposure, as one possible cause for malignant growths. an unrelated form of occupational exposure was documented in the mid-19th century in silver miners from st. joachimsthal, bohemia (today czech republic). silver had been extracted there since the mid-16th century and was manufactured into the joachimsthaler silver coins that were predecessors of the german currency "thaler" and later the american currency "dollar." these miners had a high incidence of lung cancer, which was otherwise extremely rare at that time. the cause was traced to their occupational exposure (table 1. archeological evidence suggests that the mayans smoked tobacco leaves as early as the 1st century bc. only in 1761, john hill published a treatise that warned of unusual tumors of the nose consecutive to sniffing tobacco. by 1949, ernst wynder had conducted a survey of 684 lung cancers, which indicated a substantially elevated risk in smokers compared to nonsmokers. it was followed 6 months later by a similar analysis, authored by richard doll. about 188 years after the publication by john hill, a connection between lifestyle choices and cancer risk was established. during the following years of the 20th century, chemical carcinogenesis by tobacco products became a major cause for an increasing incidence of lung cancers. (table 1. 3.b). in italy, bernardino ramazzini associated breast cancer with reproductive factors. he reported in 1713 the virtual absence of cervical cancer and relatively high incidence of breast cancer in nuns and suggested that this was in some way related to their celibate lifestyle. the key observations by pott, hill, and ramazzini laid the foundation for the field of cancer epidemiology. this area of research was given another foundation between 1930 and 1932, when fisher, haldane, and wright established the principles of population genetics. in the united states, the first hospital registry for cancer was established in 1926 at yale-new haven hospital in connecticut. in 1935 and 1946 , the first central cancer registries were initiated in connecticut and california. in 1941, the united states national cancer institute published a survey of 696 chemical compounds, 169 of which were found to be carcinogenic in animals. during the 1960s, environmental movements became prominent in most of the western societies. rachel carson believed that the long-term ecological effects of synthetic chemical pesticides were not being researched adequately. her book "silent spring" pointed to the pathogenic potential of environmental toxins, and the concept of carcinogens entered popular consciousness. in 1964, rachel carson succumbed to cancer at the age of 56. the national cancer act of 1971 (declared "war on cancer" by president richard nixon) mandated the collection, analysis, and dissemination of all data useful in the prevention, diagnosis, and treatment of cancer. it resulted in the establishment of the national cancer program, under which the surveillance, epidemiology, and end results (seer) program was developed in 1973. over the years, the susceptibility to various cancers has been associated with nutritional habits. in 1981, doll and peto [1981] estimated that 35% of cancer deaths in the united states were attributable to dietary factors. the western european diet is rich in meat and correlates with a high incidence of colon cancer. nasopharyngeal cancer is among the most widespread tumors in southeast asia, possibly supported by the ingestion of salted fish. esophageal cancer typically occurs in conjunction with alcoholism. the growing health conscience in the late years of the 20th century, combined with insights into the potential carcinogenic properties of reactive oxygen intermediates prompted multiple studies into cancer preventive capacities of antioxidants as nutrition supplements. it was soon found that while 10 theories of carcinogenesis table 1 .3.a. occupational cancers. certain occupations are associated with high levels of exposure to specific carcinogenic influences. these agents cause dna damage through physical or chemical effects. accordingly, the types of cancers induced by these carcinogens have a higher than normal incidence among exposed workers the intake of some foods can increase the risk for specific malignancies, others -such as retinoidscan act in a chemopreventive [sporn et al. 1976 ] fashion (figure 1.3 from their studies of oral cancer, slaughter, southwick, and smejkal derived the concept of carcinogenesis as a process of field cancerization (field carcinogenesis, condemned mucosa syndrome). the repeated exposure of a region's entire tissue area to carcinogenic insult increases the risk for developing multiple independent premalignant and malignant foci in that tissue [slaughter et al. 1953 ]. increasingly, molecular mechanisms have been identified to link certain toxins to specific cancers. in 1975, bruce ames at the university of california in berkeley developed a test for the mutagenicity of chemical compounds, which was used to confirm that carcinogens are mutagens. further mechanistic insight was gained with the demonstration that aflatoxin causes the mutation g249t in p53, which is associated with hepatoma [bressac et al. 1991] . ultraviolet (uv) light induces pyrimidine dimers, which cause mutations in p53 that lead to skin cancer [brash et al. 1991; pierceall et al. 1991] . the double-edged sword of mutagens became evident when their possible benefit in the treatment of neoplasias was discovered. mustard gas had been used as a chemical warfare agent during world war i and was studied further in world war ii. in 1917, krumbhaar, a captain in the us medical corps, noted the development of profound leukopenia in individuals who survived a gas attack for several days [krumbhaar 1919 ]. following up on this observation, a group of the us office of scientific research and development (osrd) at yale medical school secretly studied the effects of nitrogen mustard on lymphomata. there, lindskog successfully treated a radioresistant lymphosarcoma that compressed the patient's trachea with the injection of nitrogen mustard in december 1942. none of this was made public until 1946. during a military operation in world war ii, allied ships in bari harbor, italy, were sunk in an air assault (2 december 1943). at the center of the destruction was the vessel john harvey, laden with ammunition, supplies, and 2,000 mustard gas bombs. a large number of military personnel were accidentally exposed to mustard gas and were later found to have abnormally low white blood cell counts. it was reasoned that an agent, which damaged the rapidly growing white blood cells, might have a similar effect on cancer. cornelius p. rhoads served as chief of the medical division of the us army's chemical warfare unit during world war ii. based on his experience in the bari incident, he investigated mustard gas as a tumor-killing agent. this presaged classical chemotherapy [rhoads 1946 ]. soon, the pharmacists louis goodman and alfred gilman, recruited by the us department of defense to investigate potential therapeutic applications of chemical warfare agents, observed that exposure to mustard gas caused profound lymphoid and myeloid suppression suggesting its utility for the treatment of lymphomata [goodman et al. 1946 ]. sidney farber of boston recognized that folic acid stimulated the proliferation of leukemia cells. in one of the first examples of rational drug design, he collaborated with lederle laboratories to devise folate analogs. he demonstrated that aminopterin produced remission in acute leukemia in children because it blocked a critical chemical reaction needed for dna reduplication [farber et al. 1948 ]. aminopterin was the predecessor of methotrexate (developed by lederle laboratories in 1948), which in 1956 became the first compound cure of metastatic cancer, when it was used by roy hertz and min chiu li to treat a case of choriocarcinoma. from 1942, research by george hitchings and gertrude ellion at the burroughs wellcome corporation had corroborated that it was possible to treat cancer with chemical compounds. using one of them, 6-mercaptopurine, joseph n. burchenal achieved a high percentage of complete remissions in childhood leukemias. due to these early successes, the us congress created a national cancer chemotherapy service center (nccsc) at the national cancer institute in 1955. in 1965, cisplatin was discovered by barnett rosenberg, who explored the effects of electric fields on the growth of bacteria. he observed that the bacteria unexpectedly ceased to divide due to the exposure to an electrolysis product of the platinum electrodes. the discovery soon initiated studies into the effects of platinum compounds on cell division. this drug was later pivotal in the cure of testicular cancer. the often adverse effects of these agents were diminished when it was realized that they could be effectively used in combination [frei et al. 1958; frei et al. 1965 ]. this approach followed the strategy of antibiotic therapy for tuberculosis, which used combinations of drugs with different mechanisms of action. frei, freireich, and holland hypothesized that cancer cells would be less likely to mutate and develop drug resistance to the drug combination (table 1 .3.c). the coalescence of efforts to eliminate compounds with intrinsic mutagenic potential from cancer therapy with increasing insights into the molecular pathways associated with growth signals led to the development of small molecule inhibitors, including sti571 (gleevec) [druker and lydon 2000] and zd1839 (iressa). röntgen , experimenting with electrical discharges in vacuum tubes (crookes tubes), identified penetrating radiation that also produced theories of carcinogenesis 13 table 1 .3.c. categories of conventional anticancer drugs. chemotherapy is the use of chemical substances to treat cancer. the groups of classical anticancer agents comprises cytotoxic drugs that interfere with cell proliferation through various mechanisms fluorescence, and named it x-rays ("x" symbolizing the unknown). he died from leukemia after years of working with these newly discovered rays. in 1896, henri becquerel observed that penetrating radiation was given off by uranium. marie curie (born maria sklodowska, 1867-1934) discovered the element radium, as well as methods for separating radium from radioactive residues in sufficient quantities to analyze its therapeutic properties. after a life time of research into radioactivity, marie curie succumbed to pre-leukemia. the hazards of exposure to ionizing radiation were soon recognized. acute skin reactions were observed in many individuals working with the recently invented x-ray generators. in the early years of the 20th century, these researchers were frequently affected by skin cancers and leukemias. by 1902, a case of radiation-induced cancer was reported, arising in an ulcerated area of the skin. within a few years, a large number of such skin cancers had been observed, and the first report of leukemia in five radiation workers appeared in 1911. the french physician jean bergonie developed the law of radiosensitivity. he died in 1925 from cancer caused by his research with x-rays. in 1927, hermann j. müller recognized that ionizing radiation, already known to be carcinogenic, is also mutagenic [müller 1927 ]. x-rays break the sugar-phosphate backbone of dna. radiation damage may be exerted by directly and indirectly ionizing radiation. photons and neutrons are not charged and are indirectly ionizing. radiation of charged particles (α-rays, electron rays including β-rays, proton rays) bear a higher risk for cellular damage, including transforming events. the atomic bombs that exploded over hiroshima and nagasaki caused dramatic increases in the incidence of leukemias during the ensuing decades. by the 1950s, researchers at the sloan-kettering institute in new york city became alarmed over thyroid cancers that were diagnosed in adolescents who had received radiation treatment of their thymus glands in childhood. later reports began to document that thyroid cancers could develop about 20 years following childhood radiation therapy. nevertheless, the use of radiation to fight cancer was under study early on. the work by maude menton , simon flexner, and j.v. jobling at the rockefeller institute lead to the publication of the monograph "influence of radium bromide on a carcinomatous tumor of the white rat" in 1910. tumor viruses were detected at the turn of the 20th century with the cell-free transmission of human warts [ciuffo 1907 ] and of chicken leukemia [ellermann and bang 1908] . in 1911, peyton rous isolated a highly oncogenic retrovirus (rous sarcoma virus) from a chicken sarcoma [rous 1911 ]. in 1932, shope and hurst demonstrated that papillomavirus had oncogenic activity in rabbits. in the early 1940s, clarence cook little argued that viruses had caused breast cancer in a strain of laboratory mice. these groundbreaking results had been met with skepticism, because transmissibility in chickens and tumorigenesis in rabbits were not seen as applicable to human disease. the doubts were dispelled in the 1950s, by the demonstration that a tumor induced by rous sarcoma virus (rsv) could produce infected tumor cells [rubin 1955 ]. in conjunction with the observation that murine leukemia viruses are transmissible to newborn animals [gross 1950 ], it initiated two decades of intense research into animal viruses, including many retroviruses with tumorigenic properties in animals. in 1964, the epstein-barr virus (ebv) was observed by electron microscopy in cultured cells from burkitt lymphoma [epstein et al. 1964 ]. studies of rsv lead to the identification of the first oncogene, v-src, in the 1970s [martin 1970; brugge and erikson 1977] and its subsequent sequencing [czernilofsky et al. 1980 ]. in general, the infection of cells with an oncogenic dna virus may result either in productive lytic infection with cell death and the release of newly formed virus particles or in cell transformation to the neoplastic state with little or no virus production, but with the integration of viral genetic information into the cell dna. the genomes of herpes viruses are doublestranded linear dna molecules with sizes in the range of 140-170 kb. the initiation of transformation by oncogenic herpes viruses appears to depend on specific genes, although no single t antigens (tumor antigens) have been identified. ebv was discovered in 1964 by epstein, achong, and barr in a biopsy from burkitt lymphoma. it is a γ-1 herpes virus infecting all human populations, with a prevalence of over 90% in adults. infection results in the establishment of a lifelong carrier state, characterized by the persistence of antibodies to several viral gene products and the secretion of infectious virus in the saliva, which is also the usual vehicle of transmission. the epstein-barr virus, which is the agent of infectious mononucleosis, is causative for burkitt lymphoma (described by english surgeon denis burkitt in uganda in 1958) in africa and sporadic cases elsewhere, for b-cell lymphomata in acquired immunodeficiency syndrome (aids), as well as for nasopharyngeal carcinoma with high prevalence in china. viral dna and various ebv antigens are detectable in the affected tumor cells. a herpes virus designated hhv type 8 (kshv, kaposi sarcomarelated herpes virus) has been implicated in aids associated kaposi sarcoma [chang et al. 1994] , the most common malignant tumor in aids, and also in rare sporadic kaposi sarcomata unrelated to aids. the herpes simplex virus type 2 (hsv-2) may be involved in the pathogenesis of cervical cancer. originally known as serum hepatitis, hepatitis b has only been recognized as such since 1947. it has caused epidemics in parts of asia and africa. hepatitis b is recognized as endemic in china and various other parts of asia. hepatitis b viruses (hbv) specifically infect liver cells. chronic infection with hbv may have a causal role in primary hepatocellular carcinoma, which is one of the most common forms of cancer in asia. viral dna is integrated into the tumor cells in some of these cases. in 1963, baruch blumberg and harvey alter reported the discovery of the hepatitis b surface antigen (aa, hbsag, australia antigen), and a specific antibody binding to it. in 1970, dane visualized the hepatitis b virion. these discoveries paved the way for the development of a vaccine. the genomes of the papova family members polyomavirus and sv40 are double-stranded circular dna molecules with sizes of about 5 kb. they contain two main groups of genes that are associated with early and late events in the replication cycle. the early genes are transcribed soon after infection of a cell and their encoded proteins participate in viral dna synthesis but are not structural components of the virions. the late genes encode proteins of the viral coat and capsid. in productive lytic infection, early proteins are formed transiently before the structural proteins are assembled into viral particles. when stable transformation takes place, viral dna is integrated into the cellular chromosomal dna and some of the early proteins are persistently synthesized, but viral particles are not produced. approximately 60-120 distinct types of human papilloma viruses (hpv) have been identified, which infect epithelial cells. while several forms cause benign tumors, such as warts, some types of sexually transmitted hpv are associated with precursor lesions to squamous carcinoma of the uterine cervix. in 1983, harald zur hausen and colleagues isolated hpv16 from a human cervical cancer specimen. hpv types 16 and 18 ("high risk hpv"), followed by hpv types 45 and 31, may cause invasive cervical carcinoma or anorectal cancers. hpv dna is extrachromosomal in the precursor lesions and infectious virus is produced. viral dna is frequently integrated into the cancer cells, but additional agents or factors may be involved at various stages of the progression to invasive carcinoma. cell transformation by hpv results from the expression of two early genes, e6 and e7. e6 binds to p53, while e7 binds to rb, in both cases resulting in the degradation of their targets in the ubiquitin-proteasome pathway. acting together, e6 and e7 are sufficient to induce transformation in the absence of mutations in cell regulatory proteins. in 2006, a vaccine against high risk hpv strains came on the market. while there is no evidence that sv40 can induce human tumors or that sv40 dna is present in human tumor cells, it has been a valuable model in cancer research. the early proteins found in tumors induced by polyomavirus and sv40 are termed t (tumor) antigens. polyomavirus produces large, middle, and small t antigens, of which the middle t antigen (55 kd) is necessary for transformation. this early protein is bound to the plasma membrane of transformed cells and activates signal transduction pathways that promote cell cycle progression. the two early proteins, t (large t, 94 kd) and t (small t, 17 kd), are formed from the same reading frame by alternative splicing. the large t antigen is located in the nucleus of infected cells and maintains the transformed state. distinct domains of large t bind to p53 and rb, inhibiting their function. because large t inhibits both proteins, expression of only the sv40 large t protein is sufficient to induce the transformation of certain cells. most adenoviruses only cause acute upper respiratory tract infections. adenoviruses were discovered in adenomatous tissue in 1953 by rowe. their genomes are double-stranded linear dna molecules with sizes of about 35-40 kb. in cells transformed by oncogenic adenoviruses, a region of the genome encoding early gene products, including the e1a and e1b oncoproteins, is transcribed. these transforming proteins inactivate the rb and p53 tumor suppressors, with e1a binding to rb and e1b binding to p53. oncogenic rna viruses. hübner and todaro postulated the existence of retroviral oncogenes [hübner and todaro 1969] . among the many families of rna viruses, only members of the retrovirus and flavivirus families are capable of transforming cells and inducing tumors. the genomes of retroviruses are single-stranded rna molecules with a size range of 3-9 kb. all retroviruses contain a reverse transcriptase [baltimore 1970; temin and mizutani 1970] , and their reduplication requires the synthesis of a double-stranded dna intermediate of the rna genome. some of the virally determined dna becomes integrated into the host dna as a provirus. typically, there are three retroviral genes that encode proteins necessary for viral reduplication, but do not contribute to transformation: -the gag gene encodes internal structural proteins of the virus. two unique types of human retroviruses, human t-cell leukemia viruses (htlv) types 1 and 2 take part in the etiology of leukemias [ruscetti et al. 1977; mier and gallo 1980; poiesz et al. 1980] . human t-cell leukemia virus type 1 (htlv-1), the first human retrovirus to be isolated and characterized, may be the causative agent of a relatively rare form of t-cell lymphoma that occurs mainly in japan and the caribbean islands. htlv-2 can cause hairy t-cell leukemia [kalyanaraman et al. 1982] . all the known rna-containing tumor viruses are classified as retroviruses, with the exception of the hepatitis c virus (hcv), which resembles a flavivirus. in 1989, daniel bradley provided chiron with non-a/non-b hepatitis serum from chimpanzees. there, michael houghton and colleagues discovered a single virus and changed the name to hcv. the virus was then cloned from infectious sera of patients with posttransfusion hepatitis. hepatitis c may lead to chronic liver disease and cirrhosis, which is a predisposing factor for liver cancer. the encounter with a family, in which many members developed breast or liver cancer, led pierre paul broca to hypothesize, in 1866, that an inherited abnormality within the affected tissue caused the tumor development [broca 1866 theodor boveri (1862 boveri ( -1915 then proposed that defects in chromosomes lead to malignancy [boveri 1914 ]. he hypothesized that malignant tumors might be the result of a certain abnormal condition of the chromosomes, which may arise from multipolar mitosis. the main concepts of boveri's theory are: -the problem of tumors is a cellular problem -typically, every tumor arises form a single cell -the primordial cells of tumors contain, as a result of an abnormal process, definite and wrongly combined chromatin contents -chromosome abnormalities are the cause to the tendency toward rapid cell proliferation, which is passed on to all decendents of the primordial cell. in the 1950s, sajiro makino in japan, theodore hauschka in the united states, and albert levan in sweden observed that virtually all tumor cell lines have chromosomal aberrations. the discovery of the philadelphia chromosome in chronic myeloid leukemia [nowell and hungerford 1960] later provided experimental evidence for boveri's theories. it supported the hypothesis that damage to the chromosomes induced carcinogenesis. aneuploidy, typically with elevated dna content, is a frequent marker of cancerous cells. providing more functional insight, the first description of a translocation was reported in 1973 by janet d. rowley [rowley 1973 ]. although the philadelphia chromosome was among the first translocations to be discovered, the genes involved in the translocation that causes burkitt lymphoma were the first to be molecularly characterized. in 1982, carlo croce and bob gallo showed that the myc proto-oncogene on chromosome 8 is affected by the translocation. simultaneously, phil leder's group demonstrated that myc is translocated into the 5′ region of the immunoglobulin heavy chain (igh) gene [dalla-favera et al. 1982; taub et al. 1982] . cancers represent a large category of somatic cell genetic diseases [mckusick 1985] . the term "somatic mutation" was first applied to cancer by ernest tyzzer, who observed that tumors sequentially transplanted into mice developed a continuous broadening of host specificity among recipients from various inbred strains [tyzzer 1916 ]. by the 1970s, tyzzer's model had received a molecular underpinning and cancer was understood as a disease of genetic alterations. tumor initiation and progression occurs through the accumulation of changes that begin when a single normal cell sustains a permanent genetic damage. the resulting dysregulation of gene function is responsible for the clonal expansion of a population of somatic cells that ultimately becomes dominant. progress in the understanding of dna and genes has been a major determining factor for progress in cancer research. in 1869, johann friedrich miescher had identified a weakly acidic substance of unknown function in the nuclei of human white blood cells, which later became known as deoxyribonucleic acid, or dna. the term gene (derived from the greek γενοσ = origin), attributed to johanssen, first appeared in 1909 as an abstract concept to explain the hereditary basis of traits. oswald avery, colin mcleod, and maclyn mccarthy showed in 1944 that dna constitutes the genetic material. in 1953, james watson and francis crick deduced the double helical structure of dna from x-ray diffraction data, generated by rosalind franklin. in 1961, sidney brenner and francis crick established that groups of three nucleotide bases, or codons, are used to specify individual amino acids. the genetic code of nucleotide triplets was worked out in final detail in 1966, mainly through work by marshall nierenberg and heinrich matthaei. this paved the way for the molecular analysis of gene damage. one of the most important approaches for biotechnology is the cloning of genes inserted into plasmids. it was initiated through discussions between stanley cohen and herb boyer at a conference in hawaii, and by march 1973 the feasibility of their new method was demonstrated. pcr was invented by kary b. mullis in spring of 1983. these techniques allowed for the large availability and easy manipulation of cancer related genes. in 1977, frederick sanger at the medical research council in cambridge, uk and walter gilbert at harvard university in boston, usa independently devised methods for sequencing dna, which were further developed by leroy hood at the california institute of technology, who invented an automated dna sequencer in 1985. in 1990, the human genome project was launched to obtain the complete blueprint of human dna, planned for 2005. in 1998, the competition by a private enterprise, led by craig venter, accelerated the process, so that both groups presented a draft sequence of the genome by june 2000. the genetic analysis of cancer experienced additional support from the technical accomplishment to manipulate individual genes in vivo. in 1982, a team led by richard palmiter and ralph brinster generated the first transgenic mouse. this was achieved through pronuclear microinjection of genetic material into the nuclei of fertilized eggs. from 1987 through 1989, teams led by martin evans, oliver smithies, and mario capecchi created knockout mice by selectively disabling a specific target gene in embryonic stem cells. rna tumor viruses can cause normal cells to adopt the characteristics of rapid uncontrolled growth that are typical of many tumors. the discovery of the human proto-oncogene src by dominique stéhelin, harold varmus, michael bishop, and peter vogt [stéhelin et al. 1976] confirmed that viral oncogenes are derived from related genes of host cells. their analysis implied that the cellular src sequence is involved in the normal regulation of growth. it also suggested that tumors could arise independently of viruses as a result of mutations in their related cellular genes. consecutively in 1982, three publications in the journal nature independently of one another identified a point mutation in the proto-oncogene ras as a defect associated with bladder cancer [chang et al. 1982; parada et al. 1982; mcbride et al. 1982 ]. these discoveries revealed that a cellular transforming gene involved in human bladder and lung tumors was homologous to the transforming viral ras gene [parada et al. 1982; der et al. 1982] , and that an activating point mutation affected the identical codon in all cases. thus, it became apparent that the same cellular proto-oncogenes could be affected by viruses, by chemical carcinogens, or by nonviral somatic mutations, which brought together various previously independent lines of research. the observation that the growth of murine tumor cells in vivo could be suppressed by fusion of the tumor cells with nontransformed cells provided evidence that the ability of cells to form a tumor is a recessive trait [ephrussi et al. 1969 ]. knudson [knudson 1971 ] carried out an epidemiological study of retinoblastoma development in children. he postulated that "two hits" are required for the complete inactivation of a tumor suppressor gene. the gene p53 was discovered independently by linzer and levine [1979] and by lane and crawford [1979] as a cellular protein that binds to the viral oncoprotein of sv40. initially suspected as a cellular oncogene, due to mutations that act as dominant negative forms, the identification of loss of heterozygozity and loss of function mutations of p53 confirmed its actual role as a tumor suppressor [baker et al. 1990 ]. after this clarification, p53 became known as the guardian of the genome, because it protects from the consequences of genetic damage by inhibiting cell division or inducing cell death. in 1983, loss of heterozygosity analysis was used to map the tumor suppressor gene rb, which was then cloned in 1986 [friend et al. 1986 ]. oxidative metabolism inevitably leads to dna damage. this may occur by direct oxidation of bases, by induction of dna strand breaks, or by mediation of frameshift mutations in microsatellite dna. each cell (of estimated 10 14 in the human body) loses more than 10 4 bases (out of a total of 6 × 10 9 nucleotides) per day from the spontaneous breakdown of dna at body temperature, mostly through the damage by reactive oxygen species. a similar number of lesions is generated by spontaneous depurination, resulting in miscoding by the residual apurinic site [loeb 2001 ]. the deamination of 5-methylcytosine to thymine is among the most frequent causes for point mutations. it accounts for more than 20% of all base mutations that give rise to genetic disease [krawczak et al. 1998 ]. it has been estimated that 5-methylcytosine deaminates at a rate of 5.8 × 10 −17 s −1 at each cpg site (cytosine and guanine separated by a phosphate) [shen et al. 1994] , which corresponds to about four residues per cell per day. mutation frequencies of the hypoxanthine phosphoribosyl transferase (hprt) gene, a commonly used marker for mutation frequency, in normal adult epithelial cells reach approximately 1.3 × 10 −4 [martin et al. 1996 ]. the reduplication of dna during cell division introduces the possibility of errors at an estimated rate of 1.4 × 10 −10 nucleotides/cell/division. loeb and colleagues [loeb et al. 1974 ] realized that it would be unlikely for tumor cells to acquire the number of mutations presumably needed for full transformation during the lifetime of the host and postulated the existence of mutator genes. much later, the study of hereditary non-polyposis coli led to the discovery of defective dna repair genes [ionov et al. 1993; thibodeau et al. 1993; parsons et al. 1993] . any mutation of cancer associated genes can be handed on to following generations and predispose the affected cells to malignant transformation in the case of additional dna damage. the formation of cancer has been termed "clonal evolution" to describe how certain mutations enable cells to copy their damaged dna and divide under conditions, which cause normal cells to stop replicating. the repetition of this process allows cells to accumulate cancerous mutations [cavenee and white 1995] . in 1949, berenblum and shubik [1949] concluded that carcinogenesis is at least a two-stage process. five years later, armitage and doll [1954] inferred from their analysis of age and cancer incidence a 6-7 step process. in 1983, newbold and overell observed that an activated ras gene failed to transform normal fibroblasts, unless they were first immortalized [newbold and overell 1983] . this led to the hypothesis that ras activation was only one step in a number of mutations necessary in the pathway to malignancy. the concept of multiple somatic mutations as underlying mechanism of carcinogenesis was further advanced by a multistep carcinogenesis model, conceived of by foulds [1957] and refined by fearon and vogelstein [1990] . it also gave rise to the recognition of chromosome instability and microsatellite instability as two distinct pathogenetic mechanisms of carcinogenesis. the technical achievements of differential display ], serial analysis of gene expression (sage) [velculescu et al. 1995; zhang et al. 1997] , and dna microarrays [schena et al. 1995; derisi et al. 1996 ] further advanced these concepts to the definition of transformation on the basis of aberrant gene expression profiles [kononen et al. 1998; golub et al. 1999 ]. in addition to chromosome integrity and dna sequence fidelity, the regulation of the chromatin structure is an important determinant in transformation. dna methylation is a covalent modification of the c5 position in cytosine. this methylation pattern is stably maintained at cpg dinucleotides by a family of dna methyl transferases that recognize hemi-methylated cpg dinucleotides after dna replication. dna hypo-methylation was identified as a characteristic of cancer cells in 1983 [feinberg and vogelstein 1983] . in 1964, vincent allfrey had realized that histones were often chemically modified by acetylation, which caused them to relax their binding to dna [allfrey et al. 1964 ]. this implied the possibility of a role for histones in cancer [roth 1965 ]. in 1974, robert kornberg proposed that chromatin was quite structured, consisting of repeated units of about 200 base pairs of dna wrapped around 2-4 distinct histones (later called nucleosomes) [kornberg 1974; kornberg and thomas 1974] . the importance of acetylation for the regulation of gene expression and gene silencing was, however, realized only many years later. in 1998, methylation and phosphorylation of histones were observed by several investigators to contribute similarly [bestor 1998 ]. today, various enzymes that modify histones are known to contribute to transformation [horiuchi et al. 1981 ]. beyond the development of cancer research from explanations on a cellular level to a molecular genetic level, there has been a development of dynamic models of carcinogenesis. winge introduced the concept of selective cellular proliferation, realizing that selection must operate on a genotypically mixed population of proliferating cells as inevitably as it acts on a genotypically mixed population of reproducing organisms [winge 1930 ]. macfarlane burnet conceptualized the clonal selection theory for immunity and applied it to cancer. it suggests that tumorigenesis represents the development of a clone of cells with the capacity to multiply excessively in the context of its relationships within the body [burnet 1959 ]. in the 1960s, feedback control in biological systems was described by francois jacob and jacque monod. cellular metabolism and proliferation are regulated by spatiotemporal circuits of mutual feedback control. they include extracellular and intracellular signals, rate limiting steps, and checkpoint controls. cancer development has also been described with the algorithms of ecology [michelson et al. 1987; maley et al. 2006 ] and game theory [tomlinson 1997 ]. the regulatory theory contends that cancer is not a morphologic entity, but an aberrant regulatory process among individual cells, their microenvironment, and the entire host. genetically identical cells and organisms exhibit substantial diversity, even when they have identical histories of environmental exposure. variation in gene expression, based in part on the stochastic nature of biochemical reactions, may contribute to this phenotypic variability [raser and o'shea 2005] . genetic changes underlying growth control, senescence, invasion, and stromalparenchymal interactions are part of a continuum of carcinogenesis that affects interrelated pathways. in malignant cells, the normal balance between the number of cells completing the cell cycle and the number of cells dying is changed. likewise, the balance of adhesive versus migratory surface molecules on malignant cells is shifted in favor of the motility enhancing receptors. full transformation has two basic requirements: -genetic instability of the cell to drive tumor progression -selective advantage of the cell to allow for clonal expansion [cairns 1975; nowell 1976 ]. the genetic instability of tumor cells is reflected in the heterogeneity within individual tumors and among tumors of the same type. it is based either on chromosome instability, leading to aneuploidy, or on defective dna repair, leading to microsatellite instability and gene mutations. genomic destabilization is an early event in tumor development. the mean number of alterations in a cell that turns carcinomatous may amount to about 11,000 [stoler et al. 1999 ]. waves of clonal expansion give rise to daughter cells that have the growth advantage typical of cancer. clonal selection drives this process. tumors are clonal insofar as they are derived from the same stem cell precursor. genetic instability generates a collection of coexisting subclones, each with the potential for future changes in the face of selective pressures [cahill et al. 1999 ]. the relative importance of selective advantage versus genetic instability in tumor initiation and progression is still subject to debate. studies of cell senescence have led to a research focus on population dynamics, selection, and evolution. hayflick recognized that there is a finite number of possible population doublings by nontransformed differentiated cells [hayflick and moorehead 1961] . after a limited number of divisions, a state of crisis is reached, in which most cells die. a few cells may be altered in a fashion that conveys a selective advantage, which allows them to grow out and dominate the population. these cells are selected and form an expanding population with potentially precancerous characteristics. the demonstration that htlv-1 immortalizes normal t-lymphocytes [popovic et al. 1983] led to additional investigations, which confirmed that tumor viruses can frequently immortalize human host cells. the shortening of the chromosome ends, telomeres [szostak and blackburn 1982; moyzis et al. 1988] , is an integral part of replicative senescence. the enzyme telomerase [mckay and cooke 1992; chong et al. 1995] replenishes the chromosome ends and can prevent this shortening. its activity is present in most cancer cells, but not typically in nontransformed differentiated cells [hastie et al. 1990 ]. the first cancer hospital was founded in the 18th century in reims, france. french gynecologist joseph claude anthelme récamier (1774-1852) described the invasion of the bloodstream by cancer cells, coining the word "metastasis." in the 1850s, pierre paul broca (1824 broca ( -1880 and karl von rokitansky (1804 -1878 , independently of each other, observed the venous spread of cancer. theories of metastasis formation have traditionally been based on concepts of population dynamics. in 1889, english surgeon stephen paget (1855 paget ( -1926 described the propensity of various types of cancer to form metastases in specific organs. he stated that "the distribution of the secondary growth is not a matter of chance" and proposed that these patterns were due to the dependence of the "seeds" (the cancer cells) on the "congenial soil" (the target organ for metastasis) [paget 1889 ]. this notion was challenged by american pathologist james ewing (1866 ewing ( -1943 , who suggested that circulatory patterns between a primary tumor and specific secondary organs were sufficient to account for most of the targeted metastasis [ewing 1928 ]. this was relativized by leonard weiss, who demonstrated that the number of metastases in specific target organs, derived from certain tumors, could not be accounted for solely by blood flow patterns [weiss 1992 ]. the first evidence that metastasis formation depends on intrinsic characteristics of the tumor cells came from experiments by isaiah fidler [fidler 1975 ], who generated sublines with increasing invasive potential by serial passage of a melanoma cell line through mice. soon, somatic cell fusion and microcell mediated chromosomal transfer suggested that the ability to disseminate was under positive and negative genetic control [ramshaw et al. 1983; sidebottom and clark 1983; layton and franks 1986] . these observations placed ensuing research activities into metastasis on a deterministic footing. the secretion of proteases by tumor cells [turpeenniemi-hujanen et al. 1985; matrisian et al. 1986 ] was recognized as one factor causing invasiveness. homing receptors were identified on the cell surface, which are necessary and sufficient to mediate metastasis formation by specific tumors [günthert et al. 1991] . in conjunction with the finding of metastasis suppressor genes [steeg et al. 1988; alvarez et al. 1990 ], the detection of metastasis genes has corroborated the existence of genetic programs intrinsic in the tumor cells, which regulate invasiveness. these observations have led to the development of a genetic theory of metastasis formation, according to which metastasis genes are developmentally nonessential genes that physiologically contribute to inflammation, wound healing, and stress-induced angiogenesis. their dysregulation in cancer occurs on the level of aberrant expression and splicing [weber and ashkar 2000] . tissue-specific molecular markers (addressins) were identified in 1988 [streeter et al. 1988 ], which implied the possibility that circulating cells could recognize target organs. this was corroborated by the identification of the contribution by chemokines and their cognate receptors to tumor dissemination [mueller et al. 2001 ]. in the evolution of research progress from a reductionist to a comprehensive understanding of cancer, interactions between the host and the cancer cells have recently received increasing attention. mintz and illmensee [1975] had demonstrated that the injection of undifferentiated embryonal carcinoma cells into mouse blastocysts suppressed their inherent tumorigenicity and led to the contribution by these cells to a variety of functional tissues. around the same time, the michigan radiologist john wolfe recognized that women with dense breasts had an elevated risk of contracting breast cancer, implying a role for the stromal architecture. in 1990, it was realized that the tissue environment had a dramatic effect on the potential by tumors to metastasize [nakajima et al. 1990 ]. tumorigenic prostatic stroma and nontumorigenic prostatic epithelium can interact to induce the development of carcinosarcoma [chung et al. 1988 ]. the concept that the stroma plays important roles in carcinogenesis has since been developed by mina bissell [bissell and radisky 2001] , judy campisi [krtolica et al. 2001] , and donald ingber [huang and ingber 1999] . early work in experimental carcinogenesis had shown vascularization and hyperemia around tumor transplants [ide et al. 1939; coman and sheldon 1946] and similarities were seen between the vascular reactions to tumors and to tissue damage [algire et al. 1945] . cancer researchers became interested in angiogenesis factors in 1968, when the first hints emerged that tumors might release such substances to foster their own progression. two groups, one led by melvin greenblatt in california with phillipe shubik in chicago, and another by robert l. ehrmann and mogens knoth in boston, showed that burgeoning tumors can release a substance that induces existing blood vessels to grow into them [rijhsinghani et al. 1968; ehrmann and knoth 1968] . such vascularization promotes tumor growth because it ensures a sufficient supply of oxygen and nutrients. folkman [1971; folkman et al. 1971 ] recognized the important role of blood vessels in the growth of cancerous tumors. after more than a decade of research, mediators of angiogenesis that are secreted by some tumors were identified [senger et al. 1983; shing et al. 1984 ]. the inhibition of vegf (vascular endothelial growth factor)-induced angiogenesis was shown to suppress tumor growth [kim et al. 1993 ]. today, a monoclonal antibody to vegf is used in the treatments of some cancers. these investigations also led to the discovery of naturally secreted compounds that curtail the growth of new tumors [taylor and folkman 1982; o'reilly et al. 1994; o'reilly et al. 1997 ]. in the 17th and 18th centuries, some believed that cancer was contagious. in fact, the first cancer hospital in france was forced to move from the city in 1779 because of the fear that cancer could spread throughout the city. more than a century later, the potentially protective role of the immune system against transformed cells was recognized. in the 1890s, new york surgeon william b. coley found a record of a young patient with round cell sarcoma on the neck, who had been listed as an utterly hopeless patient when he developed a severe infection of erysipelas. he survived the infection and his tumor went into remission. based on this case, coley devised a killed vaccine of streptococcus pyogenes (the cause of erysipelas) with serratia marcescens. after a few years of its use, he reported to have successfully treated some sarcoma patients with the application of his bacterial toxins (coley's toxin) [coley 1893 [coley , 1896 . after coley's death, his daughter helen coley nauts reviewed his records, published several reviews of his work, and founded the cancer research institute, which promotes immune therapies for cancer. in 1909, paul ehrlich carried out immunizations in animals with tumor cells and suggested that tumors occur at high frequency in humans, but are kept under control by the immune system [ehrlich 1909 ]. further developments in tumor immunology have led to models of selection and evolution of cancer cells. macfarlane burnet coined the term immunosurveillance in 1967 [burnet 1967 ]. in this conceptual framework, the host immune system constantly screens cells for signs of transformation and eliminates those that pose a threat to the body's integrity. the growth of a tumor reflects an escape from immunosurveillance. cancer cells that can evade the immune system, be it by down-regulation of antigen presenting or co-stimulatory molecules, be it by expression of immunosuppressive cell surface molecules or cytokines, will grow out and form tumors. three distinct theories were developed to interpret the nature of the tumor recognition by the immune system. -lewis thomas described homograft rejection as a primary defense against neoplasia [thomas 1959 ]. -according to concepts by burnet, which are based on self/non-self discrimination, the immune system is active early in antitumor protection. the early surveillance mechanisms shape the tumor's immunological phenotype [burnet 1967 ]. this was supported by the description of tumor specific antigens [old and boyse 1964] . tumors mostly express self antigens, which may account for the incomplete protection from transformation by the immune system. -the alternative proposal of the danger theory [matzinger 1994 ] implies that the immune system is activated only at later stages of carcinogenesis. during the early stages, tumor cells appear immunologically as healthy growing cells that do not send out danger signals to activate the immune system because they express neither microbial immune recognition patterns nor release distress signals to alarm the innate immune system cells [fuchs and matzinger 1996] . in advanced growth, hypoxia and tissue damage induce stress responses, which activate the immune system. in the framework of the danger theory, the immune system is activated at later stages of tumor development, when tissue damage has occurred. the possibility to direct the immune system to fight cancer cells in virtually any location within the body with minimal side effects has attracted increasing research efforts. the high specificity and high binding affinity of antibodies made them attractive as potential anticancer agents. for a long time, however, they were difficult to isolate in large quantities. the fusion of antibody producing cells with myeloma cells into hybridomas, accomplished by cesar milstein and georges koehler in the early 1970s, changed that. yet, biotechnology had to advance to accomplish humanizing such antibodies before they became successful in therapy. in 1997, the us food and drug administration (fda) approved rituxan, a monoclonal antibody to cd20 (developed by idec pharmaceuticals) to treat non-hodgkin lymphoma [mclaughlin et al. 1998 ]. the process also led to the development of herceptin, spearheaded by dennis slamon, an antibody that targets the receptor erbb2 (her-2/neu) that is overexpressed on the surface of about 30% of breast cancers. because antitumor immunity is predominantly cellular immunity, other research has been directed toward turning t-lymphocytes against tumors. steven a. rosenberg focused his efforts to generate antitumor vaccines on tumor associated antigens. in a similar approach, martin kast studied the development of peptide-based vaccines. glenn dranoff demonstrated the high effectiveness of irradiated tumor cells transfected with the cytokine gm-csf in inducing antitumor immunity [dranoff et al. 1993] . over time it became clear, on the other hand, that the immune system could also impact negatively on cancer risk in the context of chronic inflammation. in 1876, robert koch and louis pasteur had shown independently of each other that microorganisms can cause disease. in the 1980s, barry j. marshall and j. robin warren demonstrated that gastric ulcers were caused by bacteria they called helicobacter pylori. infection results in widespread inflammation that predisposes to stomach cancer. inflammation in the stomach mucosa is also a risk factor for malt (mucosaassociated lymphoid tissue) lymphoma, a lymphatic neoplasm in the stomach. over the decades, the roles of hormones in carcinogenesis have received increasing attention. the observation by bernardino ramazzini in 1713 of a virtual absence of cervical cancer and relatively high incidence of breast cancer in nuns was an important step toward identifying and understanding the importance of hormonal factors, such as those associated with pregnancy, in modifying cancer risk. in 1878, thomas beatson discovered that the breasts of rabbits stopped producing milk after he removed the ovaries. he suggested to the edinburgh medico-chirurgical society in 1896: "this fact (. . .) pointed to one organ holding control over the secretion of another and separate organ." beatson found that oophorectomy often resulted in the improvement of breast cancer patients and inferred the stimulating effect of a female ovarian hormone on breast cancer, before the hormone itself was discovered [beatson 1896 ]. allen and doisy [1923] identified an ovarian hormone they referred to as "estrus stimulating principle," later called estrogen. from the late 1950s to the 1970s elwood jensen demonstrated that such hormones do not undergo redox modifications to become activated. instead, they bind to a receptor protein within their target cells [jensen and jacobson 1962] . this hormone/receptor complex then travels to the cell nucleus, where it regulates gene expression. the first nonsteroidal antiestrogen to be reported in the literature, mer25, was described by lerner and coworkers in 1958 [lerner et al. 1958 ] as an agent that had no other hormonal or antihormonal properties. the drug failed in clinical trial because the large doses required caused serious central nervous system side effects. tamoxifen, first discovered in 1962, is a nonsteroidal antiestrogen that serves a dual role as breast cancer treatment and preventive. it was approved for the treatment of advanced breast cancer by the us fda in 1977. awareness of the androgen dependence of prostate tissue can be traced back to the scottish surgeon john hunter, who observed in 1786 that castrated bulls had small prostates. in 1941 , charles brenton huggins (1901 -1997 , a urologist at the university of chicago, with his students clarence v. hodges and william wallace scott, published three papers that demonstrated the relationship between the endocrine system and the normal functioning of the prostate gland. in the 1940s, charles huggins also reported a dramatic regression of metastatic prostate cancer following removal of the testes [huggins and hodges 1941] . later, drugs that blocked male hormones were found to be effective treatments for prostate cancer. androgen ablation with gonadotropin releasing hormone agonists (gnrh-as) in prostate cancer patients was first reported in 1982 [tolis et al. 1982] . in 1988, the androgen receptor was cloned ]. iatrogenic causes for cancer predisposition were incriminated by a study published in 1971, which documented an association between clear-cell adenocarcinoma of the vagina and in utero exposure to diethylstilbestrol [herbst et al. 1971 ] (dodds and associates had characterized diethylstilbestrol as an extremely potent estrogen [dodds et al. 1938] ; it had been prescribed for close to 30 years to prevent certain complications of pregnancy and as a treatment for advanced breast cancer in postmenopausal women). in july 2002, the women's health initiative study was stopped after more breast cancers and heart problems occurred among women taking estrogen-progestin pills. in 2006, multiple clinical studies showed that breast cancer rates in the united states dropped in 2003, consecutive to a drastic reduction in the use of hormone replacement therapy. some of the numbers came from the national cancer institute's surveillance database, which uses cancer registries around the country to project national incidence and death rates. vascular reactions of normal and malignant tumors in vivo: i. vascular reactions of mice to wounds and to normal and neoplastic transplants an ovarian hormone: preliminary report on its localization, extraction and partial purification and action in test animals acetylation and methylation of histones and their possible role in the regulation of rna synthesis inhibition of collagenolytic activity and metastasis of tumor cells by a recombinant human tissue inhibitor of 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physiologische und pathologische gewebelehre über asymmetrische zellteilung in epithelkrebsen und deren biologische bedeutung the metabolism of tumours hereditary with reference to carcinoma enzymology of cancer cells (first of two parts) stress response genes -the genes that make cancer metastasize racing to the beginning of the road. the search for the origin of cancer comments on hematogenous metastatic patterns in humans as revealed by autopsy zytologische untersuchungen über die natur maligner tumoren. ii gene expression profiles in normal and cancer cells key: cord-302222-9ad0fw6z authors: monath, thomas p. title: vaccines against diseases transmitted from animals to humans: a one health paradigm date: 2013-11-04 journal: vaccine doi: 10.1016/j.vaccine.2013.09.029 sha: doc_id: 302222 cord_uid: 9ad0fw6z abstract this review focuses on the immunization of animals as a means of preventing human diseases (zoonoses). three frameworks for the use of vaccines in this context are described, and examples are provided of successes and failures. framework i vaccines are used for protection of humans and economically valuable animals, where neither plays a role in the transmission cycle. the benefit of collaborations between animal health and human health industries and regulators in developing such products is discussed, and one example (west nile vaccine) of a single product developed for use in animals and humans is described. framework ii vaccines are indicated for domesticated animals as a means of preventing disease in both animals and humans. the agents of concern are transmitted directly or indirectly (e.g. via arthropod vectors) from animals to humans. a number of examples of the use of framework ii vaccines are provided, e.g. against brucellosis, escherischia coli o157, rabies, rift valley fever, venezuelan equine encephalitis, and hendra virus. framework iii vaccines are used to immunize wild animals as a means of preventing transmission of disease agents to humans and domesticated animals. examples are reservoir-targeted, oral bait rabies, mycobacterium bovis and lyme disease vaccines. given the speed and lost cost of veterinary vaccine development, some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. opportunities for vaccine-based approaches to preventing zoonotic and emerging diseases that integrate veterinary and human medicine (the one health paradigm) are emphasized. zoonoses (diseases transmissible from animals to humans) account for approximately 60% of all infectious pathogens of human beings and 70% of all emerging infectious diseases [1, 2] . the origins of these diseases, and underlying factors (including man-made factors) in their emergence have been the subject of considerable interest [3, 4] . certain zoonotic diseases have the potential for pandemic spread by human contagion, such as avian influenza, sars and the middle east respiratory syndrome coronavirus, and others for regional cross-border epizootics, such as yellow fever, venezuelan equine encephalitis and rift valley fever. the cost of a short list of zoonotic disease emergences in the interval between 1997 and 2009, including bovine spongiform encephalopathy, sars, highly pathogenic avian influenza, west nile, and pneumonic plague (india), was estimated to exceed $80 billion [5] . of 32 major emergencies that concerned public health (including hurricanes, earthquakes, and terrorist attacks) occurring between 2001 and 2013, more than 25% were zoonotic disease outbreaks [6] . however, the toll on public health is much greater than that caused by such dramatic outbreaks. it is estimated that 56 different zoonotic diseases are responsible annually for 2.5 billion cases of human disease with 2.7 million deaths and substantial reductions in livestock production [7] . animals, including livestock and companion animals, also suffer illness and death following infection with many zoonotic infections, and livestock and poultry are subject to large-scale intentional destruction as a means of preventing human infections, resulting in huge economic losses. wild animals, including endangered species, may also be mortally affected, examples being west nile disease in birds, yellow fever in neotropical monkeys, plague in black-footed ferrets, and ebola in the great apes. vaccines are an important means of prevention and control of zoonotic infectious diseases in humans and domesticated animals. however, the target for vaccination is, in almost all cases, the directly affected species, and there are few practically implemented illustrations of the potential for indirectly preventing human disease by immunizing the domesticated or companion animal sources of infection. the concept of immunizing wild animal reservoirs for the prevention of disease in humans or domestic animals is even more challenging and has received limited attention. moreover, human and animal health divisions of the biopharmaceutical industry are generally separate and segregated, and there is no organized approach to the development of new vaccines indicated for the prevention of spread of diseases from domestic or wild animals to humans. in addition, the major funding sources for research in human and animal diseases tend to be stove-piped into different government agencies, stifling cross-cutting approaches. the purpose of this review is to stimulate the science and policy communities to seek innovative ways to interdict zoonotic diseases by integrating human and veterinary medicine and vaccine development, and by creating new streams of funding aimed at the intersection of human and animal health. these aims are consistent with the one health initiative, which seeks to establish "collaborative efforts of multiple disciplines working locally, nationally and globally to attain optimal health for people, animals and our environment" [8] . a framework for considering one health vaccine interventions is provided, as well as a brief review of past and current efforts, including a few successes. it will be obvious to the reader that this is a wide-open field with many opportunities that deserve more attention than they have received. the complexity, timeline, and cost of development of animal vaccines and the regulatory hurdles for product approval are far less than for human vaccines. thus some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. it is obvious that the benefit to human health deriving from vaccination of animals is far easier to justify when there is also a benefit to animal health, the latter often being closely linked to economic value. causative agents of zoonotic diseases, including viruses, parasites, bacteria, fungi, and prions, have extraordinarily varied life cycles and modes of transmission. some may persist between periods of active transmission in soil or invertebrate species. many have silent transmission cycles involving wild animals that have coevolved with the infectious agent and exhibit no signs of disease. some zoonotic diseases occur when a causative agent harbored by a wild animal reservoir jumps species to domesticated animals and thence to humans. others are primarily diseases of domesticated animal species. humans may be infected by direct contact with wild or domesticated animals, or indirectly by ingestion of contaminated milk or meat, inhalation of aerosolized secretions or excreta, fomites, or hematophagous insect or tick vectors. despite this complexity of epidemiological patterns, the opportunities for intervention often boil down to a few simple bottlenecks in the transmission process. for example, milk-borne diseases can be prevented by pasteurization, certain meat-borne diseases by inspection and animal husbandry improvements (e.g., trichinella, bovine tuberculosis), and other diseases avoided by limiting contact with known high risk species (e.g., tularemia, turtle-borne salmonellosis, exposure to bats carrying henipaviruses). where the risk of infection is high, or the resulting disease severe, vaccines may be the most efficient and cost-effective means of prevention and control. alternative methods for control of zoonotic diseases have generally employed trapping, poisoning, or other means of destroying the offending animal reservoir/vector; these methods have a mixed (often negative) record of success and are in any case becoming socially unacceptable. this review focuses on the use of vaccines for animals as an acceptable means of interdicting zoonotic disease in both animals and humans. three major epidemiological frameworks are identified for the control of zoonotic disease by means of vaccination of animals ( table 1 ). the scope of this review encompasses only those diseases for which a strategy targeting vaccination of animals is actually used or is under development. there are many diseases of table 1 frameworks for vaccine development and utilization as a means of preventing zoonotic diseases, and status of approval of existing vaccines for humans and animals. the first major framework includes zoonotic infectious diseases that affect both humans and economically important animals, where wild animals are the source of infection. livestock and humans are dead-end hosts, and neither contributes to the transmission cycle. in this case, vaccines are required to prevent disease in both humans and economically important animals, but do not interrupt transmission of the disease in nature. for framework i diseases, there is an important opportunity to accelerate the development of new vaccines by concurrent veterinary and human product research ( table 2) . moreover the affected animal species represents a natural disease model of infection, pathogenesis and immunity that may be useful in testing efficacy and immunological correlates of protection of a new vaccine intended for human use, and thus provide data supporting regulatory approval under fda's animal rule [9] . a list of framework i diseases and vaccines is shown in table 1 , and an example is given below. west nile virus is a mosquito-borne, single strand, positivesense, enveloped rna virus (genus flavivirus, family flaviviridae), closely related to japanese encephalitis virus. west nile virus is a recognized cause of human disease ranging from mild feverrash-headache syndromes to lethal encephalomyelitis. horses, domesticated geese, farmed alligators (as well as a number of wild birds and mammals) are also susceptible to severe and fatal disease. although recognized as a cause of disease as early as the 1940s, west nile became an increasing problem in the 1990s, with outbreaks affecting humans and/or horses in northern africa, western europe, the eastern mediterranean, the black sea region and the volgograd oblast of russia [10] . the most dramatic development was the introduction of west nile virus into north america in 1999 [11] [12] [13] , the occurrence of large outbreaks in 2002 and 2003, rapid spread across the us, and subsequent introduction to the caribbean and south america. horses and a number of wild bird species, notably crows and jays, were affected in addition to humans. between 1999 and 2012, a total of 37,008 cases of west nile fever and 16,196 cases of neuroinvasive west nile disease were reported in the us, with the highest incidence in the middle of the country from texas north to the dakotas [14] . the incidence of neuroinvasive disease in the us has varied between 0.1 and 1.0 per 100,000 in this interval [15] . over 25,000 horses have been affected since 1999, and in these animals the disease is more severe (33% case-fatality, 40% of survivors with neurological sequelae) than in humans (4-9% case fatality, 30% of encephalitis survivors with sequelae) [16] . moreover, the incidence of west nile in horses (∼700 per 100,000) is substantially higher than in humans [17] . the animal health industry rapidly responded to this veterinary emergency, and multiple west nile vaccines were rapidly developed, including a live vaccine (discussed below), whole virion inactivated, dna, and poxvirus vectored vaccines. the first vaccine for horses was marketed in 2001, only 2 years after introduction of the virus into the us. multiple human vaccine development programs were also initiated, but many of these efforts were discontinued or decelerated due to the high market risk associated with low incidence, a slackening of public concern with the disease, and the uncertain regulatory pathway for vaccine approval. although efficacy of a vaccine for equids is established [18] , field trials to prove vaccine efficacy in humans would be large, expensive, and difficult due to the unpredictable occurrence of west nile outbreaks. the application of the animal rule to licensing a west nile vaccine, while plausible, has not been adjudicated by the fda with a sponsor. although approaches to developing veterinary and human vaccines against west nile were technologically similar, in only one case was a concurrent development program undertaken by a single company. this fact illustrates the current status of stove-piped and separate animal and human health biopharmaceutical industries. the exceptional case is of interest as a model of how vaccine development for zoonotic diseases could be improved. in 1999, within 2 months of the identification of west nile as the etiological agent of the initial outbreak affecting humans and horses in new york, acambis, a publically traded human-vaccine biotechnology company, applied its platform technology for yellow fever 17dvectored vaccines to the development of a west nile vaccine, with the intent to develop both vaccines for both horses and humans. this technology involved replacing the gene encoding the yellow fever 17d vaccine virus' envelope (e) protein with the corresponding gene of west nile virus. this chimeric vector vaccine platform had been previously used by the company to construct a chimeric vaccine against the closely-related japanese encephalitis virus [19] ; at the time, that vaccine had proven to be highly effective, protecting non-human primates against intracerebral challenge with virulent japanese encephalitis virus [20] . two new vectors were engineered, one with the wild-type west nile ny99 strain e protein gene and one with an e gene containing three attenuating mutations [21] [22] [23] . the former was neurovirulent in mice, while the latter was more attenuated and thus deemed more suitable as a human vaccine. the principal question was whether the yellow fever 17d vector would infect and immunize horses, since yellow fever is a host-restricted primate virus. to find out, studies were sponsored by acambis in early 2000 at the colorado state university school of veterinary medicine. horses were vaccinated with the west nile/yellow fever chimeric virus or with yellow fever 17d vaccine, and viremia and antibody responses were determined [24] . in addition, vaccinated and control horses were challenged by the intrathecal injection of a high dose of virulent west nile virus, following the same model referred to above for protection studies of the chimeric japanese encephalitis vaccine wherein monkeys were challenged by the intracerebral route. these studies showed that horses inoculated with chimeric vaccine developed neutralizing antibodies against west nile and were protected against a severe intrathecal challenge. a similar development plan was successfully followed for the mutated human vaccine version, using non-human primates as the test host. the veterinary and human vaccine candidates were developed side by side, and the data obtained in both programs were designed to support transition to advanced clinical trials in horses and humans. in 2002, acambis licensed the veterinary vaccine technology to a major animal health company (intervet), and in the same year clinical trial materials were made for human trials. intervet completed development of the veterinary vaccine (prevenile ® ) [25] , which was approved by usda in 2007, and acambis brought the human vaccine (chimerivax-wn02) into clinical trials in the same timeframe [26] , subsequently outlicensing the technology to sanofi pasteur. as expected, development of the veterinary vaccine and its progression through the regulatory pathway substantially outpaced the human vaccine; importantly, the veterinary application significantly informed the human vaccine table 2 strengths and limitations of vaccination of animals as a means to control of zoonotic diseases. framework (see table 1 development program in providing useful information on safety, durability of immunity and immune correlates or protection. co-development of the veterinary and human west nile vaccines is as a potential model for other vaccines. there are a few other examples where vaccines were co-developed for animals and humans, including vaccines against venezuelan equine encephalitis and rift valley fever (described below). certain issues arise, however, that may need to be considered in the context of such integrated efforts. first, a safety problem arising during use of a veterinary vaccine could provoke regulatory concerns or a liability problem for the human analog. although a safety issue did arise briefly due to anaphylaxis type reactions in horses, resulting in temporary withdrawal of prevenile ® from the market [27], there were no repercussions for the human analog vaccine (chimerivax-wn02). this raises the interesting question whether the human and veterinary regulatory agencies are integrating information that might be of value, either during development of similar vaccines for different species or after marketing approval. this is an obvious overlooked area for application of one health principles. second, the immune response to veterinary vaccines generally should differentiate naturally infected from vaccinated animals (diva) on the basis of a laboratory test, which allows compliance with trade restrictions. this is, of course not a concern for regulatory approval of human vaccines, although it can be useful in seroepidemiological and vaccine coverage studies. an example of problems associated with diva is the off-label use of commercial horse vaccine to protect emus against eastern equine encephalitis (eee), since some combination vaccines against eee contain equine influenza antigen and such vaccines elicit cross-reactive antibodies to avian influenza resulting in quarantine. the chimeric west nile vaccine described above potentially allows for diva testing (using responses to the yellow fever nonstructural proteins expressed by the vector) [28] . a number of important diseases are transmitted between domesticated animals and thence to humans. vaccination of domesticated animals has the potential to protect humans against these zoonoses, either indirectly by interrupting transmission where domesticated animals are amplifying hosts in the transmission cycle or directly by preventing spread from infected animals to humans. a list of framework ii diseases and vaccines is shown in table 1 . selected examples are used to illustrate the role of vaccination of domesticated animals in preventing human disease. brucella spp. are facultative, intracellular gram-negative bacteria, pathogenic for domestic animals and humans. brucellosis, caused mainly by brucella melitensis (which infects sheep and goats), brucella abortus (cattle), and brucella suis (swine), occurs worldwide, with the highest prevalence in the middle east, asia, africa, tropical america, and the mediterranean region [29, 30] . the annual incidence of human infections is estimated at 500,000 cases but the disease is widely acknowledged to be underreported [31] . brucella canis, an infection of dogs, occurs worldwide with highest prevalence in tropical america. b. canis disease in humans has been reported, especially in persons handling breeding dogs and in immunosuppressed individuals. human brucellosis is acquired by contact or aerosol spread from infected animals, fomites, or ingestion of unpasteurized milk or undercooked meat. not surprisingly, brucellae survive for long periods in dust, animal excreta, soil, meat and dairy products. wild animals are also affected and can be the source of infection of livestock and humans. in animals, brucellosis causes epididymitis in males and abortion, placentitis, infertility and reduced milk production in female animals. the human disease is protean, manifested by chronic fatigue, relapsing fever, endocarditis, spondylitis, osteomyelitis, arthritis, and meningitis [32] . prevention of human disease by control of brucellosis in livestock has long been a public health priority [33] . control of brucellosis relies principally on surveillance, testing, removal of infected animals, import/export animal and animal product control provisions, protection from exposure to wild reservoirs (such as elk, deer, and bison), and vaccination. antibiotic treatment of animals is regulated and discouraged due to the large doses and long treatment required and concern about resistance. old, empirically developed live attenuated vaccines, b. melitensis rev1 vaccine for goats and sheep; b. abortus s19 and rb51 vaccines for cattle; and the oral b. suis s2 vaccine used widely in china for multiple species, elicit cellular immunity against the intracellular pathogen and are more effective than other types of vaccine [34] . the rb51 vaccine (a spontaneous rifampin-resistant rough mutant) is approved in the us [35] . however, the live vaccines have a number of drawbacks. the latter include lack of the ability to differentiate s1 and rev1 vaccine immunity from natural immunity (diva) and interference with surveillance and export control procedures; pathogenicity (especially abortion when animals are vaccinated during pregnancy); antibiotic resistance of the vaccine strains; and modest efficacy. live vaccines used in livestock can also cause illness in humans. vaccination as a stand-alone strategy has rarely been carefully evaluated, in large part due to concerns over the quality of the existing vaccines. however, vaccination is largely credited with elimination of brucellosis in the us [36, 37] (the us was declared free of brucellosis in cattle in 2009) and for control of brucellosis in china [38] . a recent study in greece showed that a mass vaccination program with the b. melitensis rev1 vaccine resulted in a decrease in human infections [39] . a number of new vaccine approaches, including diva vaccines, designed to induce th1 oriented cellular immunity are under investigation, including safer rationally designed, mutated live vaccines [34] ; recombinant, invasive escherischia coli [40] ; recombinant subunit microencapsulated vaccines; and dna vaccines [41] . experimental b. canis vaccines have been investigated in mice. where brucellosis-free status has been achieved, as in the us, wild animal reservoirs (especially bison and elk) threaten to reintroduce the disease. vaccination with existing vaccines is feasible, but delivery is challenging [42] . this vero cytotoxin secreting gram-negative bacteria is an important cause of sporadic and epidemic food-borne illnesses of humans, including gastroenteritis and hemorrhagic colitis, with potentially lethal complications (hemolytic-uremic syndrome). cattle and sheep are the principal reservoirs of infection and transmission to humans occurs via food (meat, seeds and vegetables) contaminated with animal feces. undercooked ground beef is a source of infection in approximately one-third of human cases and recalls are a significant economic threat to the meat packing and distribution industry. animals concentrated at feed lots and slaughter that shed bacteria can produce lots of meat with high rates of o157 [43, 44] , but there is considerable variability in the occurrence of contaminations [45] . in developed countries, various sanitary measures and testing have been instituted to reduce the risk to consumers, but these remain imperfect. vaccination of feed lot cattle has been proposed as a measure to reduce the prevalence and duration of shedding and the risk to consumers. o157-specific bacterial extract vaccines containing protective outer membrane proteins have been conditionally approved by usda (manufactured by epitopix, willmar, mn) and fully approved by the canadian food inspection agency (bioniche life sciences, belleville, ont.). feedlot cattle receiving 2 or 3 doses of the bioniche vaccine 3-4 weeks apart had 59-98% reduction in colorectal colonization or fecal shedding and significant reduction in magnitude and duration of shedding [46] [47] [48] . hurd and malladi [49] modeled the impact of vaccinating cattle on human health outcomes. assuming 80% efficacy of the vaccine and 100% adoption rate, the model indicated a 60% reduction in the incidence of e. coli o157-related human illness. the model also predicted significant reductions in the number of lots of contaminated ground beef and detection by usda, which would have substantial economic benefit to packers and distributors. vaccine effectiveness under conditions of field use will be highly dependent on adoption rate (vaccine coverage), and in part by whether cattle complete the 3-dose vaccination series on the proscribed schedule [46] . an interesting question is: who will pay for vaccination of feedlot cattle? is the economic benefit there for the meat industry, and will vaccination reduce the cost of other preventive measures and testing or will vaccine costs simply be on top of other costs? will government agencies concerned with human health subsidize the cost, without empirical demonstration of a human health benefit? since ground beef only contributes about one-third of human infections, how could vaccines be used as a means of reducing other sources of food contamination? another issue for use of the current vaccines is the role of other enterohemorrhagic e. coli (e.g. o26, o11, and o103) in human disease. this disease is caused primarily by the gram-negative bacterium, bartonella henselae, transmitted between domestic cats by the agency of cat fleas, ctenocephalides felis. human infection occurs by contact spread from cats, including scratches by claws contaminated with blood or flea feces, or possibly by flea bite [50, 51] . the prevalence of infection in household cats in the us is approximately 28%, but in stray animals it is 81% and high prevalence rates have been found in developing countries [52, 53] . disease in humans is manifest by fever, a papule followed by a pustule at the site of infection and lymphadenopathy. rare complications include meningitis, encephalitis, endocarditis, glomerulonephritis, osteomyelitis, neuropsychiatric abnormalities, and relapsing fever and splenomegaly. human infections in immune-compromised individuals are particularly severe. approximately 22,000-24,000 cases and 2000 hospitalizations caused by cat scratch disease are estimated to occur annually in the us. in the late 1990s, heska corporation, an animal heath company in colorado, initiated a program to develop a vaccine for household cats, with the goal of limiting the potential for transmission of the bacteria from cats to humans. unfortunately, the program was not completed and no vaccine is available at present. given the fact that cats rarely become ill with b. henselae, this would have been an unusual product providing protection to pet owners, with marginal if any real benefit to the target species. moreover, it would likely have been extremely challenging to demonstrate a health benefit to humans. rabies is a fatal infection of the central nervous system caused by rabies virus, a member of the lyssavirus genus, family rhabdoviridae. it is estimated that up to 40,000-60,000 cases of human rabies occur annually, and dog bite is the cause of over 98% [53] . successful vaccination of dogs and humans against rabies was first demonstrated in 1885 by louis pasteur, using crude nerve tissue preparations. however, until the first decades of the 20th century in developed countries, and continuing in many developing countries today, the ancient practice of dog population reduction campaigns have been the main approach to rabies control, a method that has repeatedly proven to be ineffective. dog licensing and vaccination requirements were introduced in the united kingdom in 1910, and gradually at the local and then state levels in the us beginning in the 1920s, with national requirements attained by 1955 [54] . many successful dog vaccination campaigns have been reported in latin american countries and asia [55] . some countries have declared eradication of canine rabies, notably the united kingdom in 1922, japan in 1956, the us in 2007, as well as malaysia, singapore, taiwan, hong kong. in 2007, the first world rabies day event, the ultimate vision was promulgated of canine rabies elimination through systematic vaccination. however, rabies remains a significant public health problem, due to absent or incomplete dog vaccination in many areas of the world. more than 7.5 million post-exposure treatments with rabies vaccines are given annually, at a cost of over $1 billion worldwide [56, 57] . in china, due to the low prevalence of canine vaccination, the sales of rabies vaccines for human post-exposure prophylaxis outstrips any other human vaccine, accounting for 14% of all annual vaccine sales, i.e. 12 million doses costing $244 million [58] . the requirement for human post-exposure vaccination at this scale represents an obvious failure of public health, since it plays no role in containing the spread of rabies in the canine vector. a very high rate of vaccine coverage in the dog population (exceeding 75%) is required for interruption of the rabies transmission cycle [59] . the development of oral rabies vaccines has allowed vaccination of free-roaming dogs that could not be restrained and vaccinated by injection, as well as the vaccination of wild animal species that are the source of infection in dogs [60] . oral bait vaccine for dogs has been successfully deployed in trials in many countries, including turkey, thailand, sri lanka, south africa, and the philippines [61] ; the world health organization has supported this approach as a supplemental program where there are substantial populations of free-ranging or feral dogs [60] . significant increases in canine vaccination coverage have been achieved when oral rabies vaccine was added to a program of standard parenteral vaccination. oral vaccination of dogs has been accomplished using commercial baits containing live modified rabies vaccines, such as the street-alabama-dufferin (sad) strain, variant b19 [62] and the attenuated copenhagen strain of vaccinia expressing the rabies glycoprotein gene (g protein) [63] . a more detailed review of oral rabies vaccines is provided below (framework iii). hendra virus disease, a severe and fatal infection of horses and humans in australia, has been noted as an example illustrating one health principles in disease prevention and control [64] , and is especially relevant now that a new vaccine for horses has been introduced. hendra is a member of the henipavirus genus, family paramyxoviridae. the reservoir hosts are fruit bats (pteropus spp.), and the virus is spread from bats to horses by contact (including respiratory droplets), by food or fomites contaminated with bat urine, or by contact with sick horses. all reported human infections have resulted from contact with infected horses [65] [66] [67] . hendra virus disease was first described in 1994. human and equine cases have occurred in coastal queensland and new south wales and positive bats have been detected in the northern territory. a total of 81 deaths in equids have been reported in 14 outbreaks, with a very high case-fatality rate (75%), and 8 cases (4 fatal) have occurred in humans, including horse trainers and veterinarians, all of whom had contact with sick horses [68] . the disease is manifested by severe systemic illness, respiratory symptoms or acute and relapsing encephalitis [69] . swine appear to be susceptible to experimental infection. nipah virus, a closely related bat-borne agent in se asia has caused outbreaks of severe and fatal disease in swine and humans [65] [66] [67] . in november 2012, pfizer animal health launched equivac ® hev, an adjuvanted subunit protein vaccine for the prevention of hendra virus disease of horses in australia [70] . since horses are a major source of contact spread of hendra virus to humans, the vaccine promises to make an important contribution to human health as well. fear of acquiring the disease has also constrained equine veterinary practice in australia [71] , and the vaccine should mitigate this problem. development of equivac ® hev was a collaborative effort between pfizer and csiro's australian animal health laboratory. however, support for the development program was also provided by human medical researchers in the us, at the uniformed services university of the health sciences supported by the henry jackson foundation for the advancement of military medicine. a provisional approval for limited use of the vaccine was obtained in early 2012, with full approval in november. the vaccine is a soluble, recombinant glycoprotein (g) of hendra virus, the ligand for cell attachment and antibodies to the protein neutralize cell receptor binding of the virus [72, 73] . the vaccine protects horses and ferrets against experimental infection [73] , and appears to cross-protect against nipah virus [74] . the availability of equivac ® hev should lead to rapid uptake by horse owners in australia. the equine and horse racing industry in australia is large, contributing billions of dollars, and over 1% of total gross domestic product [75] . hendra virus in horses is a notifiable disease in all australian jurisdictions; the property where the horse cases are located is quarantined and animals that are infected are euthanized. the occurrence of at least one hendra virus outbreak annually since 2006, and the high lethality of the disease have raised considerable awareness in australia. since all human cases of this zoonosis have resulted from contact with infected horses, vaccination of horses against hendra virus promises to be a highly effective strategy for preventing human cases. rift valley fever is an enveloped, single-strand, segmented rna virus belonging to the phlebovirus genus, family bunyaviridae, occurring in africa, with intermittent extensions to the arabian peninsula. rift valley fever virus causes explosive and economically damaging outbreaks of disease in cattle and sheep, with stillbirth, abortion, and very high mortality of young animals; in adult animals, it causes 30% mortality in sheep, 10-15% in cattle, and 5-10% in goats [76] . humans typically develop self-limited nonspecific febrile illness, but 1-2% have a complicated course with hemorrhagic fever syndrome, encephalitis, hepatitis, renal failure, or retinitis, and case-fatality rates in severely ill and hospitalized patients is as high as 20% [77, 78] . the virus is transmitted between livestock and from livestock to humans by the agency of mosquito vectors. in addition, humans commonly acquire infection by contact and aerosol routes when handling, treating, or butchering infected livestock, and the virus can persist in meat for weeks. the ecology of rift valley fever and the reasons behind its periodic emergences have been the subject of intensive study. in brief, the reservoir of infection is aedes mosquitoes, especially ae. linneatopennis, which maintain the virus by transovarial transmission [79] . the dessication-resistant ova containing virus remain in depressions in the earth ('dambos') that are flooded during the rainy season, with the subsequent emergence of infected adult aedes mosquitoes. infected, viremic, livestock in turn served as source for amplified virus transmission by a variety of mosquito vectors [76, 80] . there are no approved vaccines for prevention of rift valley fever in humans, although a number of candidates are in development by academic and government laboratories. vaccination of cattle and sheep represents a strategy for preventing disease in these species, and thereby for interrupting virus transmission to humans. rift valley fever epizootics are to some extent predictable based on rainfall patterns and surveillance of disease in livestock [81] . surveillance provides opportunities for rapid intervention, particularly with a single-dose vaccine (most likely a live, attenuated vaccine) that would protect against viremia in cattle and sheep and prevent mosquito infection. in addition, routine vaccination of livestock in inter-epizootic periods with a product capable of inducing durable protective immunity is a long range goal supported conceptually by modeling [82] . however, there are many obstacles to livestock immunization in africa, including access, policy, regulatory approval, cold chain, and commercial viability. additionally, diva requirements are driven by regulations prohibiting export of livestock or meat by countries experiencing rift valley fever [82] . some of the obstacles to vaccination implementation could be overcome by development of improved vaccines. two old veterinary vaccines, the live smithburn vaccine, developed using techniques of serial passage in mouse brain similar to that applied to the early development of the french neurotropic vaccine against yellow fever [83, 84] , and a formalin inactivated vaccine [85] are commercially available from the onderstepoort institute in south africa. however, the smithburn vaccine, now produced in bhk-21 cells, is reported to cause teratogenicity and abortion when used in pregnant animals [84] and is used only in countries endemic for rift valley fever due to concerns about reversion. the inactivated vaccine, also grown in bhk-21 cell culture, requires multiple doses to be effective and probably has relatively short durability [86] , making it less desirable for the interventions proposed above. nonetheless, the inactivated vaccine was successfully used to interrupt a rift valley fever outbreak in south african sheep [79] . indeed, following a large epidemic of rift valley fever in egypt in 1978-79, the veterinary serum and vaccine research institute (cairo, egypt) produced a formalin-inactivated vaccine in bhk-21 cells using the epidemic strain (zh501), which matched locally circulating strains compared to the south african strain [87] . another inactivated vaccine (tsi-gsd 200) developed by the us army for human immunization and grown in diploid fetal rhesus lung cells was tested clinically and shown to be well tolerated and immunogenic. ninety % of the subjects developed a neutralizing titer >40, which was shown to be higher than the protective level in a passive immunization-challenge study in hamsters [88, 89] . in addition to the requirement for multiple doses for primary and booster immunization, inactivated rift valley fever vaccines have the disadvantage of requiring high biocontainment facilities for manufacturing. in recent years, there has been substantial progress in development of newer vaccines, and some of these vaccine development projects have been collaborations between human and veterinary research groups. additionally, there has been a moderate level of support from the us government because rift valley fever is a credible threat of natural or intentional (bioterrorist) introduction. nevertheless, despite very promising technical results, there has been insufficient support from industry and government to propel any of these new vaccine candidates into use. because of the obvious advantages of rapid onset and durable immunity associated with live vaccines, development of an improved live vaccine has been the focus of research. us army investigators attempted to induce attenuating mutations in two rift valley fever virus strains isolated during the 1978 egyptian epidemic [90] . mp-12 is a live vaccine that was developed from the virulent zh-548 strain by 12 passages in mrc-5 cells in the presence of the mutagen, 5-fluorouracil, resulting in a temperature sensitive virus with 9 amino acid mutations. attenuation was demonstrated in multiple animal models, and reassortment studies showed that attenuating mutations were redundant and resided in all three gene segments [91, 92] . development of mp-12 was undertaken by the us army medical research institute of infectious diseases with the intention to produce a vaccine for both human and animal immunization. the vaccine was clinically tested in 62 human volunteers and shown to be well-tolerated and highly immunogenic [93] . army investigators, in collaboration with usda, conducted a number of studies of mp-12 vaccine in sheep and cows, including neonatal, pregnant and lactating animals. these studies showed that mp-12 caused a low viremia, but with no attendant clinical signs; there was no virus secretion in milk, and no abortions or teratogenicity when vaccine was given in in mid-to late term pregnancy. mp-12 was highly immunogenic and protected livestock against virulent challenge [94, 95] . however, ewes vaccinated with mp-12 early in pregnancy showed a low incidence of abortion and teratogenicity, indicating some residual virulence of the vaccine [96] . to improve genetic stability and safety of mp-12, reverse genetic techniques were used to introduce deletions in the s and m rna segments in genes encoding, respectively, nss and nsm proteins [97, 98] . two deletion mutants were evaluated for safety and immunogenicity in pregnant ewes [99] and in calves (morrill jc personal communication, 2013) with positive results for the nsm deletant, making it an attractive candidate as a veterinary and human vaccine. there were no clear safety signals when ewes were inoculated in early-mid pregnancy. further safety studies are required to rule out the low incidence of abortion/teratogenicity seen with parental mp-12 in early-term ewes. unfortunately, human trials have not yet been performed with the rationally designed mp-12 derivative. a third live vaccine designated clone 13, is a plaque-derived clone of a central african strain of rift valley fever isolated from a human subject, and was found to be naturally attenuated for mice and to have an in-frame deletion of most of the nss gene [100] . this observation was the basis for modifying the mp-12 vaccine by ns gene deletion, as described above. once again, a collaboration between the human and veterinary researchers led to a study in ewes, showing that clone 13 was highly immunogenic but did not cause abortions [101, 102] . another attenuated vaccine designated r566, has been developed by reassorting clone 13 and mp-12 so that it contains the s segment of clone 13 and the l and m segments of mp-12. this strain has attenuation domains from both parental vaccine candidates. a number of live, replicating, and non-replicating heterologous viral vectors expressing rift valley fever g1 and g2 glycoproteins and nonstructural proteins have been have been investigated in mice, elicited immune responses and protected against challenge. the vectors included lumpy skin disease (capripoxvirus) [103] , alphavirus (vee and sindbis) replicons [104] , newcastle disease virus [105] , adenovirus, and modified vaccinia ankara. several of these constructs were used to immunize sheep and/or cattle (lumpy skin disease virus, newcastle disease virus, and sindbis replicons) with somewhat variable success. for a more comprehensive review see indran and ikegami [106] and boshra et al. [107] . live vectors are a promising approach for new rift valley fever vaccines, particularly veterinary vaccines, but may have problems for homologous boosting in light of anti-vector immunity. various prime-boost strategies have been proposed, as for plasmid dna vaccines (see below), but these would be exceptionally difficult to implement for immunization of livestock in the field, and are thus impractical. subunit protein produced in insect cells, virus-like particles [108] , and dna vaccines [109] against rift valley fever are also in early stage development. these approaches have potential advantages of safety and thermostability during storage and distribution, but may require multiple dosing and provide less durable immunity than live vaccines, and thus are less desirable products for framework ii implementation. overall, it remains to be seen which of the many rift valley fever vaccines in development progress to regulatory approval and whether an integrated veterinary and human health policy based on the immunization of livestock in africa together with predictive surveillance, can abort impending outbreaks, and lead to long range control of this important disease. vee is a mosquito-borne single strand, positive-sense, enveloped rna virus belonging to the alphavirus genus, family togaviridae. other medically important members of the alphavirus genus include eastern and western equine encephalitis viruses. there are 6 vee virus subtypes identified by antigenic and genomic analyses, and a number of additional varieties. subtype iab and ic cause epizootic disease in equids and associated zoonotic infections of humans [110] . during epizootics, horses and donkeys infected with these strains develop high viremias, serve as the primary hosts for infection of mosquito vectors and therefore are the indirect source of human infections acquired by mosquito bite. in contrast, the enzootic subtypes ii-vi, are maintained in nature in cycles involving rodent species and mosquitoes, are not amplified by equid viremic hosts, and cause sporadic illness in humans and equid dead-end hosts. epizootics of ic virus are the result of mutation and selection of virulent equine-competent viruses from enzootic strains, particularly the id variant [111] . in the 1930-1940s vee iab viruses caused large epizootics in south america, with associated human epidemics of encephalitis. between 1962 and 1969, a series of major subtype iab and ic epizootics occurred in northern south america, and between 1969 and 1971 the virus spread north to central america, mexico, and texas [112] . the cumulative economic and medical impact of vee outbreaks between 1935 and 1971 was devastating, with over 150,000 equid and 50,000 human cases. some of the vee iab epizootics are believed to have been spawned by the injection of horses with inactivated veterinary vee vaccines containing residual live virus [113] . this likely occurred in trinidad in 1943 and again in nicaragua in 1970, but probably was a widespread problem in the past. between 1992 and 1995, vee ic re-emerged in venezuela and colombia, with an estimated 4000 equid deaths and over 100,000 human cases of which 3000 had encephalitis [114, 115] . since vee causes an acute incapacitating illness in humans and the virus efficiently infects via the aerosol route, it was developed by both the us and soviet union as an offensive biological weapon [116] . as part of these programs, vaccines for the protection of military personnel were also developed. in the us, a live, attenuated virus (tc-83) was developed by the us army medical research & development command (usamrdc) by empirical passages of the prototype trinidad donkey (subtype iab) virus in fetal guinea pig heart cell culture [117] . the development of the live vaccine followed poor experiences with chemically inactivated vaccines; in animal models, only the live vaccine protected against aerosol challenge. however, tc-83 vaccine has a number of drawbacks as a human vaccine, including failure to immunize about 18% of vaccinees, and moderate-to-severe reactogenicity in about 25% of subjects. in humans, it remains an investigational product, used solely for the protection of laboratory workers [118] , with approximately 7000 persons vaccinated since 1963. the tc-83 virus acquired 12 mutations during the empirical passage series in guinea pig heart cells, but attenuation appears linked to only 2 of these, in the 5 -noncoding region and the e2 envelope glycoprotein, and these substitutions appear to be subject to reversion in the vaccinated host [119] . in addition, tc-83 has been isolated from mosquito vectors during field use, illustrating the potential for secondary spread and mutation and recombination events. an investigational formalin-inactivated tc-83 vaccine (designated c-84) was also developed by usamrdc and used following tc-83 priming to seroconvert tc-83 non-responders. since horses and related species are severely affected during epizootics and are the source of mosquito vectors infecting humans, there is an obvious need for a single dose veterinary vaccine that evokes rapid immunity. us army investigators explored the use of tc-83 live, attenuated human vaccine for immunization of equids beginning in 1962 [120] , and there was limited field use of the vaccine in colombia in 1967. however, when epizootic vee appeared for the first time in central america (guatemala) in may 1969, and then spread southwards to costa rica and northwards to the us, there was considerable urgency to utilize a vaccine strategy for control of the disease in horses, donkeys and mules. in 1969, the us military responded rapidly to requests for tc-83 vaccine from guatemala and el salvador. the vaccine had been produced and stockpiled at the merrell national laboratories, swiftwater pa under contract to the usamrdc for the purposes of biological defense. by 1972, over 10 million doses of tc-83 had been given to equidae in the us, mexico and central america [106] . collaborative studies were also undertaken by agencies concerned with human and animal health (usamrdc, nih and usda) to fully explore the biology of the vaccine in horses [121, 122] , ultimately leading to licensure and commercialized by the animal health industry, both as a live vaccine and then an inactivated vaccine combo with eastern and western equine encephalitis vaccines. tc-83 vaccine was credited with a rapid curtailment of the 1969-71 outbreak. the history of vee exemplifies many one health principles, including the prevention of human cases through domesticated animal vaccination, use of a single vaccine product for animals and humans, and an agency (the us army) concerned with human health engaged in both veterinary and human vaccine development, and providing a solution for curtailing an emerging zoonosis. after the large epizootic in the 1970s, tc-83 vaccine was again deployed during the epidemic in 1995 in colombia to create an immune barrier to spread of the virus. recent efforts have focused on development of improved vee vaccines for humans that are less reactogenic and more immunogenic than tc-83, can be manufactured in a more acceptable substrate, and have a lower risk of reversion to virulence and of mosquito transmission [123] . in addition, vaccines that crossprotect against the enzootic vee subtypes are needed. vee id is endemic in colombia, peru, venezuela, and ecuador, and the ie subtype circulates in southern mexico. aguilar et al. [103] postulated that disease caused by vee is confused clinically with dengue, and that, in endemic areas, up to 10% of dengue cases may actually be due to vee enzootic subtype viruses. subtype id poses the ever-present risk of mutational change to produce high viremia and epizootic transmission in equids, as happened in the 1990s. v3526 vaccine is a rationally designed vaccine from the epizootic subtype iab genome, with insertion of a pe2 cleavage-signal mutation combined with an e1 gene resuscitating mutation. v3526 had a good safety profile and was immunogenic and protective in laboratory animals, including nonhuman primates [124] . while retaining a degree of neurovirulence for suckling mice, v3526 is not virulent when inoculated intracranially in juvenile monkeys [125] . v3526 has also been evaluated in horses [126] . the vaccine was safe and highly immunogenic, with subcutaneous doses as low as 100 plaque-forming units shown to protect horses against challenge with virulent subtype iab virus. unfortunately, v3526 proved to be too reactogenic for humans in a phase 1 trial [127] , and thus development for both human and veterinary use has stopped. the v3526 virus was subsequently formalin inactivated and has been investigated with adjuvants replacement for the c-84 vaccine. other live and live vector approaches to improved vee vaccines have been investigated only in laboratory animals, including a chimeric virus constructed from nonstructural genes of sindbis and the structural genes from vee [128] , vee replicon vaccines, and vaccinia recombinants. none of these approaches have reached advanced development. it is only a matter of time before another vee outbreak emerges in tropical america, and there is a substantial risk of cross-border spread. the prospects for vaccine interventions have diminished with dwindling support for new vaccines and increased concerns for vaccine safety. most zoonotic diseases are maintained in transmission cycles involving wild mammals or birds. however, because of the difficulties in vaccinating specific host species, wildlife immunization as a means of preventing spread to domestic animals and humans has been applied in only a few diseases. some of the barriers to implementing wild animal vaccination include (i) involvement of multiple species in natural transmission cycles; (ii) safety concerns for non-target species; (iii) high reproductive rates and population turn-over; (iv) fastidious feeding behaviors and difficulty in designing effective baits; (v) difficult delivery due to very high or, conversely, very low population densities of the target species; (vi) environmental concerns, and release of genetically modified organisms; (vii) difficulty in designing an effective formulation for oral immunization; (viii) instability of a vaccine or vector under prevailing environmental conditions; and (ix) requirement for low unit cost and government funding for vaccine purchase and delivery. nevertheless, targeted immunization of wild animal reservoirs is a subject of considerable interest for future research, not only for control of infectious agents affecting domestic animals and humans but also for control of wildlife diseases. one example of the latter was the effort to develop a means of immunizing great apes affected by ebola virus in central africa with vaccines previously developed for human use. aside from rabies vaccines delivered in oral baits, which is well-established, wildlife vaccination has had limited success. two promising examples of early-stage vaccine applications are described below (lyme disease and mycobacterium bovis). in addition experimental immunization and protection of prairie dogs (cynomys ludovicianus) using a raccoon poxvirus recombinant oral bait vaccine [129] , and ballistic vaccination of bison against b. abortus [130] have been described. plague, a global but localized zoonotic disease with rodent wildlife reservoirs, would appear to be a target of particular interest for future research [131] . there are many other possible targets for new framework iii vaccines, and future research in this field is encouraged. in the united states, lyme disease is the most common vectorborne disease and the 7th most common infectious disease overall. it is also a major and increasing public health problem in europe. approximately 30,000 cases are reported in the us annually, and the number has doubled in the last 15 years [132] . however, at a meeting in august, 2013, the centers for disease control and prevention (cdc) reported that the annual incidence of infection is believed to be 10-fold higher, i.e. 300,000 cases. although lyme disease occurs across the country, the incidence is highest in the northeast and north central states. in the us, lyme disease is caused by the spirochete borrelia burgdorferi, which is amplified each spring and summer in a cycle principally involving ixodes scapularis ticks and field mice. mice are persistently infected and represent the reservoir of infection in nature [133] . b. burgdorferi is passed transtadially to nymphal and adult ticks which infect humans and dogs; these species develop clinical disease but are dead-end hosts. the human disease is manifested by a protean syndrome, starting with a localized skin infection (erythema migrans), and progressing to a multisystem disease variably with lassitude, arthritis, carditis, meningitis and other neurological manifestations [134] . because of the increasing incidence and geographic expansion of lyme disease, the high incidence of tick exposure, and the difficulty in recognizing and removing attached ticks due to their small size, difficult differential diagnosis, troublesome and potentially severe clinical manifestations and medical controversies over treatment and chronicity of the disease, lyme disease has emerged as a high priority for public health interventions [135] . vaccination of humans would appear to be a logical and cost-effective means to prevent the disease [136] , and veterinary vaccines for dogs are widely used and have proven to be modestly effective [137] . however, whereas safe and highly effective vaccines for humans have been developed, none is available for distribution today. glaxo smithkline's lymerix ® vaccine was approved in 1998, but withdrawn in 2002 by the company, principally for commercial reasons, a decision that is lamentable given the increasing incidence of the disease [135, 138, 139] . a new lyme disease vaccine for humans active against both b. burgdorferi and species causing lyme disease in europe developed by baxter bioscience is now in phase ii development, but it is uncertain whether it will reach the market. nevertheless, these vaccines established critical immunological principles; the human vaccines are composed of recombinant ospa protein, the dog vaccines of both ospa and ospc, and work via antibody-mediated mechanisms. ospa is expressed by the borrelia spirochete in the midgut of infected ticks. since the tick vector only begins to transmit borrelia 24-36 h after initiating blood feeding, ospa specific antibodies imbibed in the blood meal of a vaccinated host kill the bacteria and block transmission [140, 141] . if a similar ospa antibody response could be evoked in the natural reservoir hosts of b. burgdoferi (peromyscus spp. field mice), it may be possible to interrupt the transmission cycle and reduce the prevalence of infected nymphal and adult ticks responsible for human and canine infections. proof of concept was obtained in a field study where peromyscus leucopus mice were trapped and vaccinated by subcutaneous injection of ospa; a reduction in the prevalence of b. burgdorferi in nymphal ticks was seen in the following year [142] . however, practical vaccine delivery and effective immunization of mice in the wild, requires a thermostable oral bait vaccine matched to the high population density and rapid population turnover of the reservoir hosts, the effects of which are not diluted by non-targeted species that play a role in b. burgdorferi transmission [135] . two promising live oral vaccine approaches have been investigated in the laboratory: a bacterial (e. coli) vector [143, 144] and a viral vector (vaccinia) [145, 146] expressing ospa. the e. coli vector contained in an oral bait formulation and ingested multiple times elicited anti-ospa antibodies and protected laboratory and wild p. leucopus mice against needle and tick challenge. a 5-year field study of the oral bait vaccine, sponsored by cdc, has been performed and results are anticipated with interest. a company, us biologics inc., is engaged in bringing this vaccine to market. the vaccinia technology, which rests on the shoulders of the successful oral bait vaccine against wildlife rabies (see below), has been tested in the laboratory. laboratory mice immunized by gavage with vaccinia expressing ospa were successfully immunized after a single dose and were protected against tick challenge. peromyscus consuming oral bait vaccine were also significantly protected against challenge with infected ticks. although the vaccinia vector looks promising, no commercial endeavor has yet emerged to support development. both the e. coli and vaccinia oral vaccines require specialized formulations in baits that incorporate the vaccine in the bait itself, rather than in a liquid sachet embedded in the bait used for delivery of rabies vaccines. many questions surround the application of an oral bait vaccine targeting the reservoir host, including efficacy of this approach in the field, the high density of baits required, cost and sustainability of local and state funded programs aimed at distributing baits, and the role of species not targeted by the vaccine in lyme disease maintenance cycles. if only partially effective, the risk of acquiring lyme disease may be reduced, but the public would still need to take precautions against tick bite. nevertheless, given the lack of a vaccine for humans, the high level of public concern about lyme disease, the high risk to children, the localized nature of b. burgdorferi transmission allowing geospatially focused control efforts, and the possibility that homeowners may be motivated to play an active role in distributing baits, the idea has appeal. m. bovis is the cause of tuberculosis in a wide array of domesticated and wild animals, and it remains a major veterinary health problem worldwide, causing severe economic losses from livestock disease, death and export restrictions. humans become infected by ingesting raw milk or undercooked meat, or by the aerosol route from infected animals or humans. in developed countries where pasteurization and test-and-slaughter programs have controlled the disease, zoonotic infections are relatively rare, accounting for 0.3-7.2% of tuberculosis cases [147] [148] [149] ; in developing countries which do not practice these measures, it remains more common, although few data on prevalence exist. wild animals are a major source of infection of domestic livestock [150] . control measures aimed at control of m. bovis by culling wildlife reservoirs is problematic, with inconsistent results and ethical concerns. vaccination of wildlife is an attractive alternative control measure, especially since the traditional tuberculosis vaccine (bacille calmette-guerin, bcg) derived from m. bovis is effective when orally administered [151] . examples of wildlife that serve as maintenance hosts of m. bovis and sources of infection in livestock, include white-tail deer in the us [152] ; wild boar, red and fallow deer in europe [153] ; badgers in the united kingdom [154] ; african buffalo (syncerus caffer) in south africa [155] ; and brushtail possums (trichosurus vulpecula) in new zealand [156] . brushtail possums have been experimentally vaccinated using oral bcg and shown to be resistant to challenge with m. bovis [157] . proof of concept has been provided by a field study in an endemic area of new zealand. possums were trapped, manually vaccinated using orally delivered bcg in a lipid matrix formulation, and vaccinated and control animals were recaptured at intervals [158] . vaccinated animals received 1-3 vaccinations during the 2-year study. at the end of study, the 14 ha study area was depopulated, and all animals assessed for clinical and subclinical m. bovis infections. vaccine efficacy against naturally acquired tuberculosis was 95-96%. the authors concluded that oral vaccination of possums could be a practical strategy contributing to elimination of m. bovis in livestock. although the field study did not demonstrate control via freely consumed bait vaccine, captive possums have been shown to consume vaccine in flavored baits [159] . rabies is transmitted between specific wild carnivore reservoir hosts, which serve as a source of spill-over infections of other wild carnivores, and infection of domesticated animals and humans. oral rabies vaccine was initially deployed in europe for control of rabies in the red fox (vulpes vulpes), using modified live virus vaccine [160, 161] . the concept began in the 1960s with the work of george baer at the cdc, which showed that foxes could be orally immunized with modified live virus [162] . the live, attenuated evelyn-rokitnicki-abelseth (era) vaccine or street-alabama-dufferin (sad) viruses were employed in experimental and field studies. numerous studies in the 1970s confirmed that captive and wild foxes could be orally immunized with a variety of baits containing vaccine [163] . in 1978, steck and colleagues initiated a wild fox rabies control program in the swiss alps using oral bait vaccine consisting of chicken heads with vaccine and tetracycline biomarker in a container made of polyvinyl chloride and aluminum foil inserted under the scalp [164] . the trial demonstrated that 60% of foxes had ingested bait. over the next 20 years, successful fox rabies control programs were carried out in many european countries, after the late 1980s using baits distributed by fixed wing aircraft and helicopters rather than by ground [165] , and resulting in elimination of terrestrial rabies in several countries [166, 167] . for large scale distribution, the laborious chicken head method bait gave way to commercially manufactured molded or extruded baits of various kinds, consisting of fish meal or bone meal, fat, and a pouch or blister containing liquid vaccine virus [168] . the vaccines currently used in europe are (1) sag2 (e.g., rabigen ® , virbac laboratories, france), a modified live attenuated rabies virus derived from the original sad vaccine and having an additional mutation in the codon for amino acid 333 of the rabies g protein, which increases genetic stability of the virus [169] ; and (2) recombinant vaccinia virus (copenhagen strain) expressing the era ® strain rabies g protein (raboral ® , merial corp.) [170] . the rabies g protein gene has been inserted into the thymidine kinase gene of vaccinia, which results in further attenuation compared to the parental virus [171, 172] . duration of oral rabies immunity, at least 18 months in adult red foxes, is sufficient to provide herd immunity and reduce the reproductive rate (r 0 ) to less than 1. the modified live virus vaccines are more thermolabile than vaccinia, require −20 • c storage, retain some pathogenicity for non-target species, and pose safety risks to humans exposed inadvertently. consequently, recombinant vaccinia is the only oral rabies vaccine approved for wildlife immunization in the us. this vaccine consists of fishmeal and fish oil bound by a polymer (ethylene vinyl acetate) and containing the vaccine in a plastic pouch held in place by a wax mixture. immunization with vaccinia or modified live virus occurs in the buccal mucosa and tonsils, and vaccines are poorly effective after ingestion [173] . in one study, consumption by red foxes of a single bait containing vaccinia resulted in protection against virulent rabies in only half of the animals [174] . this observation suggests that high bait densities and repeated vaccinations are important to effective control in the wild. bait densities distributed in europe generally range between 10 and 20 baits/km 2 , resulting in 50-80% of animals potentially immunized (positive for the tetracycline biomarker) [161] . in addition to distribution density, feeding habits may also be important, since animals have been observed to pick apart baits and consume only the bait portion. while control of terrestrial wildlife rabies has been successful in parts of europe, it has been more challenging in other areas due to the diversity of carnivores involved in transmission of different rabies virus variants. in the arctic regions, a specific rabies variant is maintained in the arctic fox, with spill-over infections of red foxes, skunks, and raccoon dogs. where vaccination is not practiced in domesticated sledge dogs, these animals may be severely impacted by contacts with rabid foxes, and human dog owners placed at considerable risk. while experimental oral vaccination of arctic foxes has been successful, there is limited experience in the field [175] . in ontario, canada control of arctic rabies variant in red foxes using oral bait vaccine has been successful, but the virus still occurs as a result of spill-over transmission to skunks, which are not efficiently immunized with recombinant vaccinia vaccine [176, 177] . oral bait recombinant vaccinia vaccine has been primarily used to control raccoon rabies, which expanded beginning in the mid-1970s from enzootic areas in florida northwards and westwards to involve many states in the eastern us, as well as new brunswick and quebec, canada [178, 179] . the control program relies on distribution of vaccine baits specific zones of rabies activity, particularly along the appalachian ridge, enhanced surveillance and ring vaccination with evidence of spread of the disease. judged from the absence of spread beyond the zones of vaccine distribution, the program has worked well, despite relatively low prevalence of rabies antibody (approximately 30%) in sampled raccoons [170] . it is possible that pre-existing immunity to raccoonpox virus may interfere with immunization with vaccinia [180] . the economics of large-scale oral vaccination programs in the us have been modeled and are generally favorable [181] . in addition to control of raccoon rabies, successful use of the vaccine has been made in the control of gray fox (urocyon cinereoargenteus) variant rabies in west texas [182] . in contrast to raccoons, a higher prevalence of rabies antibody (61%) attributed to vaccination is found in gray foxes. rabies in coyotes (canis latrans) was responsible for epizootic canine rabies in the 1980s and 1990s in parts of the us, and was controlled by an oral bait vaccination campaign, contributing to the elimination of canine rabies by 2007 [177] . skunks remain a problematic species for vaccine control of rabies. skunks are an important spill-over host for the arctic fox, raccoon rabies, and big brown bat rabies virus variants [183] . although the vaccinia vector vaccine is not sufficiently effective in skunks [169] , promising results were obtained with a replicationcompetent adenovirus type 5 vector expressing the rabies g protein [184] . aerial distribution of this vaccine (onrab ® , artemis technologies, guelph) showed high rates of immunization of raccoons, and arctic foxes and modest seroconversion (17-51% in different plots) in skunks, probably due to lower rates of bait acceptance [185, 186] . onrab ® is approved by the canadian regulatory authorities for control of rabies in skunks, raccoons, and foxes, and is under investigation in the us. there are some notable failures of animal vaccination as a means to preventing zoonotic diseases of humans, and these illustrate some of the limitations of the approach shown in table 2 . japanese encephalitis, a mosquito-borne flavivirus closely related to west nile virus, is endemic in asia, with nearly 2 billion people at risk of infection [187] . horses and humans are dead-end hosts, and framework i immunization of both is widely practiced in many parts of asia, with a long record of success. pigs are an important domesticated animal amplifying host for infection of rice paddy-breeding culex spp. vectors, resulting in spill-over infections of humans. moreover, infection of pregnant sows can lead to abortion and stillbirth, and infected boars may have reduced spermatogenesis and infertility [188] . framework ii immunization of swine was previously a major initiative in japan, using live, attenuated vaccines. however, it was exceedingly difficult to vaccinate piglets born in spring and early summer during the narrow window between loss of interfering maternal antibody and contribution to virus amplification. the practice of vaccination was abandoned in favor of re-locating piggeries from areas of culex breeding and biting activity. elsewhere in asia, other obstacles precluded consideration of framework ii vaccination against japanese encephalitis, including the prevalence of small piggeries located near vector breeding sites and of feral swine, and the importance of wild ardeid birds and waterfowl in virus transmission. moreover in many areas of asia, less developed than japan, and undergoing rapid urbanization, the locations of pig holdings are not controlled and are often located near rice paddies and urban centers [189] . consequently, the focus has long been on human vaccination as the primary means of prevention. this case study exemplifies some of the factors that can limit application of framework ii vaccination: (1) the need to customize vaccination to the breeding and husbandry practices and timing of domesticated animal targets; (2) the role of wild animals and feral domesticated animals as additional amplifying hosts in the transmission cycle; and (3) difficult access given the very large scale and geographic complexity of domesticated animal populations. q fever is caused by the intracellular gram-negative bacterium, coxiellla burnetii and an important worldwide infection of ruminants, which serve as the source of infection of humans, especially where large numbers of animals are concentrated [190] . q fever is a major occupational hazard of abattoir and farm workers, veterinarians, and persons involved in the handling and distribution of animals or animal products. a dramatic outbreak recently occurred in high-intensity goat farms in the netherlands (2009), with 2361 human cases and 6 deaths [191] . transmission of c. burnetii occurs between direct spread between domesticated animals, and from animals to humans. infected animals shed bacteria in urine, feces, vaginal secretions and products of conception, and in milk. ticks are also a reservoir of bacteria in nature and a source of infection of livestock. ruminants, particularly goats and sheep develop pneumonia, abortion, stillbirth, premature delivery, and delivery of weak offspring, and herds can be affected for prolonged periods, causing significant economic losses [192] . there are two developmental stages of c. burnetii, a small-cell variant (scv, the extracellular form) and the metabolically active intracellular large-cell variant. the scv is highly resistant to degradation and can persist in the environment for long periods of time. infection of humans is acquired principally by the aerosol route via dust containing spore-like scv forms, with oral routes of infection (ingestion of unpasteurized milk) being secondary. the human disease is characterized by an influenza-like illness, and may be complicated by pneumonia, endocarditis, and (pregnant women) abortion and fetal death. person-to-person transmission occurs, but is rare. approximately 5% of infected persons may develop chronic infections, with various manifestations. prophylactic vaccines have played a role in the control of q fever in livestock, but the practice is not a reliable means of preventing human infections. in russia, a live, attenuated m-44 vaccine was used for many years in animals and humans, but causes a persistent infection and has not been considered safe for use elsewhere. in europe, coxevac ® , a formalin inactivated strain rsa 493/nine-mile phase 1 bacterial form (smooth forms, with complete surface lipopolysaccharide) has been used in goats and cattle and reduced the incidence of shedding, but is not effective in pregnant or chronically infected animals [183, 193] . the live and phase 1 vaccines are also not diva, which presents substantial issues for export controls. in australia, an inactivated phase 1 vaccine prepared from henzereling strain is marketed for humans by csl ltd (qvax ® ) [194] . however, severe reactions occur in persons who have previously been naturally infected with c. burnetii, and skin testing to ensure absence of exposure is required [195] . clearly, improved vaccines are needed for both livestock and humans, but various attempts at recombinant vaccines have been disappointing. the problem for framework ii vaccination against q fever is due to multiple factors, including imperfect vaccines, limited efficacy of vaccines in parous animals, the difficulty in recognizing the disease and intervening expeditiously, and the rapid and widespread contamination of the environment with scv forms. the last problem is the major reason that livestock vaccination is not a reliable means of protecting humans against exposure. a 4 year study of sheep vaccinated with the phase 1 vaccine showed that the proportion of animals shedding bacteria in feces was markedly reduced after 1 year and then eliminated after 2 years, but c. burnetii was still present in environmental samples [196] . finally, live veterinary vaccines may pose a risk of inadvertent infection of humans handling the vaccine or vaccinated animals. examples include live brucella and orf (contagious ecthyma) vaccines. while this topic is beyond the scope of this review, it is important in several contexts, and indeed little is known about the risk of human pathogens to animals. immunization of swine and poultry workers against influenza as a means of preventing introduction of human influenza viruses to these animals has been emphasized as a means of preventing emergence of reassortant strains [197] . immunization of humans to prevent spread of viruses to captive nonhuman primates is often practiced, including vaccines for influenza, hepatitis a and b, and measles. prevention of animal diseases and human diseases by use of vaccines is a well-established principle, and there are potential synergies that can be achieved in concurrent delivery of human and animal vaccines in developing country settings [198] . for some diseases affecting both livestock and humans there is a clear commercial incentive to develop vaccine products (framework i vaccines) ( table 2) . however, such development efforts are generally segregated in the animal and human health divisions in industry and academia, and have separate regulatory pathways. this results in a potential waste of resources and duplicated scientific endeavors. interestingly, when one company (akso nobel), an animal health company, decided in 2003 on a strategic move into human vaccine development, it drew on its veterinary scientists to staff the program. as pointed out in this review, there have been isolated successful examples, e.g. a west nile vaccine, of co-development of a vaccine for both veterinary and human indications, an obviously efficient strategy that broadened both the commercial and public health opportunity. future efforts along similar lines should be considered on a case by case basis, depending on medical need, but in general there is value in closer connections between human and veterinary vaccines and regulatory science, and in the application of domesticated animals as models for development of infectious disease and cancer vaccines. several issues related to diva requirements and liability concerns have been mentioned. prevention of zoonotic diseases of humans by means of vaccination of domesticated (framework ii) or wild (framework iii) animals is an attractive but under-exploited concept. an obstacle is that there may be limited commercial incentives (table 2) . where a market exists, governments may be the principal customers, as is the case for the approved oral bait rabies vaccines and the reservoirtargeted lyme disease vaccine in development. thus, the public health gains for such an intervention need to be compelling and must offset the cost of development and implementation. the goal is far easier to justify when vaccination also prevents disease in an economically valuable animal species, there is a profit incentive for animal vaccination or a clear social gain from improved animal health, and when the public health spin-off is an added benefit. examples of the latter may include vaccines against hendra and nipah virus diseases, brucellosis, chlamydophila felis, rift valley fever, and venezuelan equine encephalitis. the potential for elimination of a disease through vaccination (employed together with other strategies), as has been demonstrated for brucellosis in the us and terrestrial rabies in some european countries is a compelling economic concept, though applicable in only selected cases. the cost of preventative programs is almost always lower than emergency response programs, as illustrated by the significantly lower cost of oral wildlife rabies programs over contingency actions to control epizootic spread [199] . the recent announcement of a 10fold higher incidence of lyme disease than previously believed will lead to a reassessment of the economics of preventive strategies for this disease, including wildlife vaccines. economics represent the key determinant for development and utilization of framework ii and iii vaccine strategies. a low unit cost of such vaccines will always be a requirement. the economic barriers are particularly relevant when considering vaccines for developing countries. on the positive side, the cost of developing a new animal vaccine through licensure is a small fraction, approximately 10%, of the cost of a typical human vaccine (the latter being $200-500 million by one estimate [200] , but often far higher) [201] . the relatively lower cost and shorter timelines for developing animal vaccines reflects the simpler path to regulatory approval, and is driven by the significantly lower market potential for these vaccines. despite the lower cost of developing new veterinary vaccines, high and middle income countries still pay higher prices until the fixed costs of development are paid off, there is an over-supply of vaccine, or competing products enter the market. to redress the pricing barriers in the case of human vaccines, there has been strong advocacy for new approaches to secure vaccine supply and access for developing countries where the burden of infectious diseases is greatest [202] . as part of this strategy, emerging market manufacturers provide access to low unit cost vaccines, and such manufacturers of veterinary vaccines could play a substantial role in a public health strategy for animal immunization. indeed all of the principles being applied to human vaccines could be extended to vaccines for animals, particularly if there is both a clear rationale for public health and the "pull" of a potentially expanded market or of guaranteed purchase agreements. up to now public-private financing for developing and distributing veterinary vaccines has represented a tiny fraction of support available for human developing-world vaccines, and has focused on vaccines for livestock as a means of improving animal production and protein supply rather than preventing zoonotic diseases [203] . given the public health impact of zoonotic diseases described in the introduction to this review and the potentially lower cost of interventions targeting animals vs. humans, there should be a new emphasis on the public health improvements that could result from animal immunization. zoonotic diseases that merit consideration because they occur at high incidence or are poorly controlled, include rabies, zoonotic leishmaniasis, brucellosis, rift valley fever, m. bovis, lyme disease, and several enteric bacterial infections. as mentioned above, the regulatory pathway for animal vaccines is considerably simpler than for human vaccines [201] . this is due to multiple factors, including less onerous requirements for manufacturing and control of veterinary products, the simpler and far less expensive clinical trial requirements for marketing approval, the ability to challenge animals to demonstrate vaccine efficacy, as well as an established regulatory mechanism for conditional approval allowing commercial sales while still gathering more definitive data. there is no requirement for large, statistically powered efficacy field trials to obtain marketing approval, as is the case for human vaccines. the development of veterinary vaccines is consequently far faster than for human vaccines. for example, west nile vaccine for horses was commercialized 2 years after introduction of the virus into the u.s., whereas the first (phase 1) human trial of a west nile vaccine was completed 5 years after introduction. the lower costs and accelerated timeframe for development of animal vaccines represent an important rationale for novel investments in public health, particularly in developing countries. to justify investments in a framework ii or iii vaccine where an economic incentive for animal immunization is marginal and a public health benefit is a goal, it is important to plan for vaccine effectiveness studies showing that vaccination of animals actually reduces human disease prevalence, as was postulated in greece following vaccination against b. melitensis [39] . such demonstrations would really drive the field forward. thus, while the deployment of tc-83 venezuelan equine encephalitis vaccine was credited with the curtailment of the human epidemic in 1971-72, no controlled study to demonstrate an effect on human disease incidence was actually performed. indeed, studies to confirm the attractive hypothesis that immunization of domesticated ruminants against rift valley fever in africa would prevent intermittent outbreaks of the disease in animals and humans, while leaving the mosquito reservoir of infection intact, would be difficult to design and carry out. because of its discrete epidemiology, hendra virus (albeit a low-incidence disease) presents a unique opportunity to demonstrate the effectiveness of animal immunization on the occurrence of a disease in humans. the increasing problem of emerging infections, the majority of which are the result of spill-over from animals to humans, is a compelling reason to consider novel vaccine interventions, and the collaborations between veterinary and human health institutions in the development of the hendra, west nile, vee and rift valley fever vaccines described in this review serve as examples of the power of this approach. other potential targeted vaccine interventions focused on animal reservoirs or intermediate hosts in order to control disease emergences include avian influenza, nipah virus disease, and, possibly, middle east respiratory syndrome. funding agencies and industry should be encouraged to seek integrated approaches to prevention of zoonotic diseases. the ultimate success of examples provided in this review, such as e. coli o157 vaccines for cattle, reservoir targeted lyme disease vaccines for field mice, and rift valley fever vaccines for livestock will require sustained efforts utilizing the one health paradigm. 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cord-320283-nkb9nzyt authors: wiebers, david o.; feigin, valery l. title: what the covid-19 crisis is telling humanity date: 2020-06-04 journal: neuroepidemiology doi: 10.1159/000508654 sha: doc_id: 320283 cord_uid: nkb9nzyt nan the world is enveloped in a global health emergency that is exacting enormous medical and economic tolls upon humanity. the sars-cov-2 that has caused the current covid-19 pandemic is thought to have originated in bats and, via an intermediary such as the pangolin, to have found its way from a "wet market" where live wildlife species were being sold for human consumption in wuhan, china, to one or more humans at that location [1] . within months, this highly infectious virus spread throughout china and around the world, currently involving at least 185 countries and territories, with a trail of incredible damage in its wake [2] . the medical community finds itself on the front lines throughout the world dealing with the immediate human health consequences of this rapidly evolving crisis and trying to develop therapies and vaccines, as countries and their leaders attempt to mitigate the overwhelming societal and economic devastations that are unfolding. from a neuropsychiatric perspective, there are obvious signs of global psychological distress related to social isolation and fears of illness, death, and countless uncertainties about the future [3] . less attention has been given to neurological manifestations including headache, anosmia, ageusia, ataxia, paresthesia, ischemic stroke, seizures, and various encephalopathies, collectively occurring in up to nearly 40% of hospitalized covid-19 patients in early series. encephalopathies have been more likely to occur in severely ill patients and have included at least one case of documented viral encephalitis with sars-cov-2 in the cerebrospinal fluid [4] . past respiratory viral pandemics have been associated with other delayed neurological sequelae, including acute inflammatory polyradiculopathy, peripheral neuropathy, myopathy, brainstem encephalitis, and parkinsonism, the latter of which was prominently associated with the so-called spanish influenza pandemic of 1918 [5] . given the immense global burden of covid-19, it is likely that the long-term neuropsychiatric complications will be substantial and it will be important for the medical community to prospectively monitor patients to establish the nature and extent of such morbidities while also dealing with the acute manifestations of this illness that are unfolding each day in emergency rooms throughout the world [3, 5] . yet, in the midst of all of the pandemonium and destruction, and as we begin to find our way through this crisis, it is imperative for us as a society and species to focus and reflect deeply upon what this and other related human health crises are telling us about our role in these increasingly frequent events and about what we can do to avoid them in the future. failure to do so may result in the unwitting extermination of all or a good part of our species from this planet. although it is tempting for us to lay the blame for pandemics such as covid-19 on bats, pangolins, or other wild species, it is human behavior that is responsible for the vast majority of zoonotic diseases that jump the species barrier from animals to humans. the us centers for disease control and prevention (cdc) observes that "…3 in every 4 new or emerging infectious diseases in people come from animals." these infections are caused not only by viruses but also by bacteria, fungi, and parasites from a variety of animal sources [6] . the alarming increase in frequency of these lethal zoonotic diseases relates in large part to our human-dominated ecosystem with increasingly unnatural human-animal close contact, grossly aberrant crowding of animals for human purposes, destruction of animal habitats, and vast numbers of highly mobile humans to swiftly carry these diseases throughout the world [7] . as is likely with covid-19, the outbreak of sars (an earlier severe acute respiratory syndrome) in 2003 was the result of a coronavirus that originated in bats with subsequent infection of wild animals sold in live-animal street markets in china. such markets provide breeding grounds for these and countless other zoonotic pathogens with sick, highly stressed, and overcrowded animals in highly unsanitary conditions. the opportunity for human transfer of these pathogens is increased immensely by allowing direct access to large crowds of humans [8] . the human immunodeficiency virus (hiv), which has infected 75 million people and killed 32 million since the onset of acquired immunodeficiency syndrome (aids) in the 1980s, originated in a type of chimpanzee in central africa. scientists have generally concluded that the chimpanzee version of the virus (called simian immunodeficiency virus) was transmitted to humans and mutated into hiv when humans hunted these chimpanzees for meat and came into contact with their infected blood [9] . the hunting, capturing, and selling of wild animals for human consumption, particularly in connection with live-animal markets, clearly constitute major public health risks. continuing these practices will assure further human health crises with potentially even greater destructive power in the future. another well-recognized source for increasingly lethal human zoonoses is the massive overcrowding of animals for human consumption in industrial "factory farm" environments -also known as concentrated animal feeding operations. over the last 40 years, as the factory farm model has become a global phenomenon, a host of avian influenza (bird flu) viruses, including h5n1, have emerged in countries with large-scale industrial poultry operations [10, 11] . intensive confinement of unprecedented num-bers of chickens in these facilities to lower cost has provided a fertile ground for the development of an everincreasing supply of new pathogens. and while bird flu was once a very rare disease among chickens, today we see outbreaks occurring every year [12] . transmission of these diseases from chickens to humans was almost nonexistent 25 years ago; now serious outbreaks are occurring regularly -more in the past 15 years than in the entire 20th century [11] . initially detected in humans in 2009, the h1n1 pandemic (the so-called swine flu because of its origin in pigs) constituted the 1st worldwide flu pandemic in 40 years. the cdc estimates that over the past 10 years, this disease has killed between 151,700 and 575,400 people worldwide. pigs had long been considered a possible mixing vessel for influenza viruses that originated within pigs, birds, and humans, and the cdc has concluded that this virus resulted from reassortment, a process through which two or more influenza viruses can swap genetic information by infecting a single human or animal host [13] . larger numbers of pigs in closer proximity provide greater opportunities for this to occur, and the mixing of live pigs from north america and eurasia through international trade or other means could have also contributed to the circumstances necessary for this reassortment to occur [14] . the large-scale confinement of animals for human consumption has also played a major direct role in another ongoing health crisis in the usa and around the world -antibiotic resistance. nearly 80% of the antibiotics sold in the usa are now used for livestock feed to prevent disease and promote growth [15] . intensive confinement operations require more antibiotics than family farms, and if the bacteria that naturally exist in these animals acquire antibiotic resistance genes, treatment becomes ineffective. this circumstance constitutes a threat not only to animal health but also to human health [16] . humans may be exposed to antibiotic-resistant organisms by handling or eating raw or undercooked meat, coming into contact with farm animals or their feces, and/ or eating produce or consuming water (including recreational water) that has come into contact with animal feces [17] . robust scientific data confirm that proximity to livestock operations and manure application to crop fields are each associated with the likelihood of acquiring antibiotic-resistant infections in humans [18] . researchers at the johns hopkins bloomberg school of public health drove cars, windows down, behind trucks that were transporting broiler chickens from farms to slaughterhouses in virginia and maryland; they documented 3 neuroepidemiology doi: 10.1159/000508654 antibiotic-resistant bacteria in the air inside the cars, as well as on the top of soda cans in the cars' cupholders [19] . antibiotic-resistant infections constitute a major and growing global health threat and now kill an estimated 35,000 people in the usa and 700,000 people worldwide per year [20] . one further fundamental source for the increasing threat of pandemics and other human health crises is habitat destruction. vast numbers of wildlife species are threatened with extinction from cutting down forests and expanding urban areas and industrial activities. the survivors are forced into closer proximity to themselves and humans, increasing the likelihood of transforming what would otherwise be benign animal microbes into deadly human pathogens. to compound the issue, much of the land that is being lost to deforestation is being converted to land for raising more animals for human consumption [7] . our species has come to the edge of the cliff on these issues, and the covid-19 pandemic is forcing us to make a choice between either changing our thinking and practices in these realms, or facing increasing destruction and perhaps self-annihilation. medical experts are painfully aware of the undeniable hazards of continuing along our current trajectory. dr. amesh adalja of the johns hopkins center for health security writes openly of the relatively new avian influenza, h7n9, that has been considered one of the most concerning of all pandemic threats over the past few years. although an epidemic of h7n9 began in southern china in 2013, there has to date been no sustained human-to-human transmission. nevertheless, more than 1,500 human cases have occurred with a case fatality rate of 40%, and adalja warns that "if h7n9 achieves sustained human-to-human transmission, it would arguably be the worst health and national security threat faced by the world in literally a century. an h7n9 pandemic could well be worse -perhaps much worsethan the great pandemic of 1918" [21] that killed an estimated 50 million people [22] . rather than simply attempting to react to crises like covid-19 after death and destruction are already upon us, we need to have the vision, wisdom, and compassion to address fundamental underlying causes and act now to mitigate and prevent the numerous disasters that are literally waiting to happen. the chinese government should be applauded for taking the much-needed step of banning the trade and consumption of wild animals in china on february 24, 2020. although shutting down this usd74 billion wildlife farming industry has been decried by some as economically harmful, such harm pales over-whelmingly in comparison to the vast health and economic threats to china and the world involved in continuing to allow business as usual. other nations throughout the world should also prohibit such practices, particularly in association with live-animal markets. intensive confinement of animals in factory farm operations should be discontinued worldwide for the sake of animals, humans, and the environment, and we should rapidly evolve to eating other forms of protein that are safer for humans, including plant-based meat alternatives and cultured meat (produced by culturing animal cells). additional investment in plant-based agriculture to grow crops to feed humans rather than livestock for human consumption would feed more people while utilizing far less land and water, allowing for the preservation of vital ecosystems for innumerable species. the time has come for us to rethink our relationship with all life on this planet -other humans, nonhumans, and the earth, a life form in itself. what is good for nonhumans and the earth is virtually always in the best interests of humans, given the profound interconnectedness of all life. all that we do depends upon abundant plant and animal life as well as clean air and water. each of us can have a positive impact upon these fundamentals by demonstrating and inspiring an enhanced mindfulness, beginning most basically with what we eat and how all of our daily choices and actions may be affecting animals and natural habitats. ultimately, the survival, not only of other life forms on this planet, but also of our own, will depend upon humanity's ability to recognize the oneness of all that exists and the importance and deeper significance of compassion for all life. the authors have no conflicts of interest to disclose. escaping pandora's box: another novel coronavirus johns hopkins coronavirus resource center are we facing a crashing wave of neuropsychiatric sequelae of covid-19? neuropsychiatric symptoms and potential immunologic mechanisms first case of 2019 novel coronavirus disease with encephalitis nervous system involvement after infection with covid-19 and other coronaviruses global hotspots and correlates of emerging zoonotic diseases origin and evolution of pathogenic coronaviruses simian immunodeficiency virus infection of chimpanzees the next influenza pandemic: lessons from hong kong bird flu: a virus of our own hatching avian influenza: recent developments origins of the 2009 h1n1 influenza pandemic in swine in mexico antibiotics overuse in animal agriculture: a call to action for health care providers industrial food animal production, antimicrobial resistance, and human health high-density livestock operations, crop field application of manure, and risk of community-associated methicillin-resistant staphylococcus aureus infection in pennsylvania food animal transport: a potential source of community exposures to health hazards from industrial farming (cafos) tackling drug-resistant infections globally: final report and recommendations johns hopkins school of public health center for health security clinicians biosecurity report the challenge of emerging and re-emerging infectious diseases key: cord-300969-a3zcggf2 authors: antolin, michael f.; jenkins, kristin p.; bergstrom, carl t.; crespi, bernard j.; de, subhajyoti; hancock, angela; hanley, kathryn a.; meagher, thomas r.; moreno‐estrada, andres; nesse, randolph m.; omenn, gilbert s.; stearns, stephen c. title: evolution and medicine in undergraduate education: a prescription for all biology students date: 2012-02-06 journal: evolution doi: 10.1111/j.1558-5646.2011.01552.x sha: doc_id: 300969 cord_uid: a3zcggf2 the interface between evolutionary biology and the biomedical sciences promises to advance understanding of the origins of genetic and infectious diseases in humans, potentially leading to improved medical diagnostics, therapies, and public health practices. the biomedical sciences also provide unparalleled examples for evolutionary biologists to explore. however, gaps persist between evolution and medicine, for historical reasons and because they are often perceived as having disparate goals. evolutionary biologists have a role in building a bridge between the disciplines by presenting evolutionary biology in the context of human health and medical practice to undergraduates, including premedical and preprofessional students. we suggest that students will find medical examples of evolution engaging. by making the connections between evolution and medicine clear at the undergraduate level, the stage is set for future health providers and biomedical scientists to work productively in this synthetic area. here, we frame key evolutionary concepts in terms of human health, so that biomedical examples may be more easily incorporated into evolution courses or more specialized courses on evolutionary medicine. our goal is to aid in building the scientific foundation in evolutionary biology for all students, and to encourage evolutionary biologists to join in the integration of evolution and medicine. the scientific and societal benefits each has delivered, and we acknowledge that potential conflicts can arise between the evolutionary and medical viewpoints. as evolutionary biologists, we think in terms of traits and genes in populations or lineages over time. we view traits as continuously evolving in a population context, driven by processes such as adaptation by natural selection, mutation, and genetic drift, but constrained by genetic and physiological trade-offs. we know that interactions between evolutionary forces are mediated by the effective size of populations over long time periods, and we are accustomed to seeing traits as the products of genotype-by-environment interactions. medical doctors, on the other hand, are trained to view patients as individuals in need of care, with injuries and symptoms at a particular time. population-level and public health concerns must be balanced with alleviating an individual patient's suffering. these apparently contradictory viewpoints need not be at odds (e.g., childs et al. 2005) . the integration of evolutionary biology into medicine, often referred to as "evolutionary medicine," provides a dynamic view of genetic, environmental, and infectious diseases. an individual patient's ailments represent a particular point in time at the convergence of ancestries, environment, and exposures, like the tips of growing and changing trees whose branches intermingle through time but stay mostly out of sight. evolutionary biologists who train undergraduate premedical students are in a position to teach them the fundamental evolutionary principles that underlie biology. by explicitly using biomedical examples, we can form a truly interdisciplinary science that actively engages evolutionary biologists as well as medical researchers and practitioners. this requires collaboration between the communities to develop methodologies and curricula that integrate evolutionary biology into training future generations of doctors, public health workers, and biomedical researchers. here, we give a brief overview of the historical relationships between evolutionary biology and medicine (box 1), and discuss how evolutionary biologists can use medically relevant examples to teach basic evolutionary biology concepts to all undergraduates, but particularly to students preparing to enter medical professions. we frame these examples in terms familiar to evolutionary biologists, and point to resources currently available for the classroom. our message is that evolutionary medicine is an exciting and useful field that many of our students will need to understand and will find engaging, and that evolutionary biologists can actively connect evolutionary biology and the biomedical sciences. we, the authors, contributed to a symposium on evolutionary medicine during the 2011 meeting of the society for the study of evolution in norman, oklahoma, to honor seminal contributions made by george williams, who passed away on 8 sept 2010. williams' writings on the evolution of senescence and life histories provided fundamental conceptual developments in evolutionary biology (williams 1957) , as did his thoughts on the role of natural selection in adaptive evolution on multiple levels from genes to individuals to groups of organisms (williams 1966) . further, his work in collaboration with randolph nesse spurred interest in applying evolutionary biology to medicine and public health. the essay on "the dawn of darwinian medicine" (williams and nesse 1991) and the book "why we get sick" (nesse and williams 1994) are cornerstones of the synthesis between evolutionary biology and the biomedical sciences. williams' work inspired research projects, articles, and books that have further addressed the many ways that evolutionary biology influences biomedical research and how an evolutionary approach can improve medical diagnostics and therapies (e.g., a special issue of the proceedings of the national academy of science featuring presentations from a sackler symposium, held in april 2009 (stearns et al. 2010 ); a special issue of evolution: education and outreach originating in part from the sse 2011 evolutionary medicine symposium (jenkins and antolin 2011) . awareness of the potential impact of evolutionary biology on medicine is also growing beyond the biology research community, as illustrated by a joint report from the american association of medical colleges and the howard hughes medical institute (aamc-hhmi 2009), entitled scientific foundations for future physicians. it describes core scientific competencies that should be taught during premedical training and in medical school, and explicitly covers evolutionary biology, genetics, and genomics (download the full report from http://www.hhmi.org/grants/pdf/08-209_aamc-hhmi_report.pdf). nesse et al. (2010) substantially expand on these core competencies and provide further recommendations for integrating evolutionary biology into medical education. the "future physicians" report makes the case that because scientific knowledge and medical practice continually grow in response to new discoveries, one goal of medical school must be to train doctors to integrate and apply new clinical and scientific knowledge into their daily work, and to absorb the "fundamental scientific principles that are key to lifelong learning and biomedical scientific literacy" (aamc-hhmi 2009, p. 3) . as an integrative scientific discipline at the core of the life sciences, evolutionary biology has a role in helping physicians and biomedical researchers understand how the human body works and why it sometimes fails. human diseases are in part a reflection of the evolutionary history that shaped our genetic make-up and responses to our current environment, including the parasites and pathogenic microbes that plague us. but understanding evolutionary history provides only one perspective on disease. as pointed out by ernst mayr (1961) and niko tinbergen (1963) , traits have both proximate and ultimate causes (see also nesse and williams 1994) . proximately, diseases arise from combinations of biochemical, physiological, and immunological responses of individuals to the environment and to pathogens. in this sense, disease can be defined mechanistically in terms of malfunctions, disorders, anatomical flaws, and infections that cause morbidity and/or death. ultimately, however, the causes of diseases can be explained in three parts-by ancestry and common descent of organisms, by variation in the environment over time, and by ongoing genomic and evolutionary forces (williams and nesse 1991; nesse and williams 1994; nesse and stearns 2008) . a key to understanding vulnerability to disease is that adaptive evolution increases fitness in terms of reproductive success, and not health, over many generations. natural selection will compromise health and survival if that increases overall reproductive success, and natural selection may not keep pace with the rapid or recent environmental changes in industrial and postindustrial societies. the viewpoint that diseases are shaped by both proximate and ultimate causes represents one of the central concepts underlying evolutionary medicine. it is important to recognize that evolution may not act to shape disease per se, but rather that evolution acts on traits that mediate vulnerability to disease. for example, the potential to form cancers is an inherent risk of multicellularity. moreover, symptoms such as vomiting and fever may actually be defenses against disease by reducing the effects of pathogens. legacies of evolutionary history, including interactions between an individual's genetic susceptibilities, immune system responses, and environmental exposure to poor nutrition, toxins, and pathogens, are important ultimate explanations for disease. ongoing natural selection may also influence disease, for example, through the genomic mechanisms that underlie many cancers or parent-parent and parent-offspring conflicts for resources. these conflicts begin with different genomic contributions by the parents, and continue through prenatal development and into childhood. further, human bodies are not optimally designed, but represent a series of developmental, biophysical, and physiological compromises driven by trade-offs that contribute to risks of injury and disease. on another level, natural selection acts rapidly on microbes via their short generation times and high mutation rates, and microbes rapidly evolve resistance to immune defenses, drugs, and vaccines, often with tragic consequences. the evolutionary approach to the health sciences has clear implications for research on the prevalence of disease within human populations, improved public health practice, and ultimately more effective care for the sick (nesse and stearns 2008; stearns and koella 2008; wolfe et al. 2007; omenn 2010 ). the goal of biology courses, from introductory to advanced, is twofold: for students to learn how to think scientifically, and for students to master content in biology. understanding the nature and process of science informs personal and policy decisions, with local, regional, and global impacts, and some knowledge of biology can be helpful for everyone in making life decisions. education research suggests that one way to support those goals is to teach with key concepts such as biological evolution (bransford et al. 2000; ambrose et al. 2010 ). this "big picture" approach helps students build a reference framework to categorize information, which facilitates building connections between new and old content, and improves student retention and ability to transfer information. the ability to categorize and connect information is one of the traits that enable experts to navigate easily through disciplinary content. the ap biology revisions (college board 2011) and the vision and change report (nsf/aaas 2011) emphasize teaching key concepts including evolution. evolution is an excellent theme for teaching about the nature of science (national academy of sciences 1998), and a poor understanding of the nature of science is correlated with a low acceptance and understanding of evolution (johnson and peeples 1987) . undergraduate biology educators can serve all their students well by introducing an evolutionary view into their courses, beginning with introductory biology. using examples from evolutionary medicine to engage all students-premedical, majors, and nonmajors-in learning about biological evolution may be a particularly effective approach to support students' development both as scientific thinkers and biologists (e.g., see hillis 2007) . as we show below, applying basic evolutionary principles to human health leads to interesting, novel, and useful conclusions. students who are not typically excited by basic evolutionary concepts may be drawn in by focusing on human health. exploring the research behind theories such as the "stone age diet" and the hygiene hypothesis gives students an accessible entry to discovering the nature and process of science. in particular, this approach demonstrates that premedical students should learn evolutionary biology for the same reasons they study biochemistry, physics, and mathematics-because broad scientific knowledge gives future medical practitioners the tools to understand how a patient may have developed sets of symptoms, and how to best to treat the patient. evolutionary medicine is easily incorporated into undergraduate curricula, through specific examples illustrating basic evolutionary concepts as well as more complex ideas. here, we highlight key aspects of evolutionary medicine in the form of familiar evolutionary concepts. additional examples are provided in the evolution-focused science competencies in the aamc-hhmi report (2009), in an expanded analysis by nesse et al. (2010) , and in boxes 2-10. a growing number of resources are available to support advanced courses or seminars on evolutionary medicine, courses that should complement those in evolution, phylogenetics, and population genetics. these include nesse and williams' (1994) classic as well as more recent collections (stearns and koella 2008; trevathan et al. 2008 ) and a textbook (gluckman et al. 2009 ). the website evolution and medicine review (http://evmedreview) provides a resource for sharing news about events and relevant articles, as well as a list of syllabi and other resources useful for educators (box 2). the december 2011 issue of evolution: education and outreach includes by stearns (2011b), omenn (2011) , and alcock and schwartz (2011) that provide specific suggestions for courses linking evolution to medicine and public health. other articles in the same issue provide excellent examples of the synthesis of evolution and medicine in cancer (casas-selves and degregori 2011), vaccine development (hanley 2011) , and scurvy (buklijas et al. 2011 ) and highlight a new module for teaching evolution and medicine (beardsley et al. 2011 ). however, we will know that evolution has truly become an integral part of premedical scientific training when testing competency in evolution becomes part of gaining admission to medical school. the aamc-hhmi report recommends that "assessment of the newly defined scientific competencies must be credibly and reliably accomplished by the medical college admission test (mcat ® ) exam" (aamc-hhmi 2009, p. 2). the just-released (november 2011) preview of the revised mcat ® for 2015 specifically includes evolution in section 1c on transmission of heritable information from generation to generation and the processes that increase genetic diversity and in section 9a on understanding social structure (https://www.aamc.org/students/applying/mcat/mcat2015/). although this is a positive step forward, indicating support from the medical community for increasing evolutionary thinking in medical education, evolution biology is still not listed as one of the "foundations of living systems" or integrated throughout the lists of life sciences topics to be tested. our undergraduate premedical students should be willing to learn evolutionary medicine, but greater synthesis is possible. with the goal of continuing development of an interdisciplinary and integrated framework for evolutionary medicine in the undergraduate curriculum, we provide some principles of evolutionary biology aligned with medical examples that students may find particularly enticing. evolutionary medicine can provide engaging illustrations of the genotype to phenotype connection, and students' experience with variation in disease presentation is a familiar way to demonstrate genetic variation. in-depth genetic studies generally show that even commonly taught diseases that at first appear to have a relatively simple genetic basis are found to be more complex. multiple functional and mutant alleles combine with environmental effects to cause variable symptoms and disease severity (templeton 2006) . this is clearly demonstrated by phenylketonuria (pku), a condition caused by mutations in the single-copy gene encoding phenylalanine hydroxolase and tested for in neonatal health screening. pku arises from the inability to metabolize the amino acid phenylalanine, the buildup of the amino acid and phenylketones, and eventually microcephaly, incomplete brain development, seizures, and severe learning disabilities. although in most cases, disease can be avoided by excluding foods rich in phenylalanine from the diet, the treatment is not equally effective for all variants, and the frequencies of both functional and mutant alleles differ among human populations. this is a familiar problem to evolutionary biologists: although genetic variation is the stuff of evolution, it is seldom straightforward to map genotypic (now genomic) variability onto phenotypic differences between individuals and populations (see boxes 3 and 4). students often miss (or reject) this basic concept in their first introduction to evolution, but examples from evolutionary medicine can help drive this idea home. common ancestry explains why biomedical research in animals is applicable to humans (antolin 2009). but some caution is in order, as in the case of differences between humans and mouse strains in the p450 family of enzymes important for drug metabolism, which limits the mouse as a model for drug testing for humans (nelson et al. 2004) . further, it is important for medical practitioners to be aware that each patient has a different ancestry (evolutionary history), and therefore a different genetic makeup, different reactions to drugs, and often different disease symptoms (meyer 1999; omenn 2010) . in managing health care, such differences can result in life, death, or long-term disability and morbidity. individual patients' presenta-tion of disease will depend on complex interactions at the convergence of patients' genetic legacies, the evolutionary history of pathogens to which they are exposed, and the environmental context where the patients and pathogens meet. combining concepts of ancestry with individual-level genetic variation culminates in the newly emerging approach of "personalized medicine" (knight 2009; costa et al. 2010; jiang and wang 2010 ; see box 5). a common misconception among students is that "fittest" is the biggest, strongest, or fastest. examples from evolutionary medicine demonstrate that fitness depends on how rates of reproduction and environmental circumstances interact (see boxes 3, 4, and 6). for example, "diseases of civilization" imply a mismatch hypothesis: alleles that cause disease in modern times may have been adaptive in the past, but produce traits that are maladaptive (mismatched) under current diet and living conditions. a corollary is that modern humans may be changing their environment more rapidly than potential responses to selection can occur. the mismatch hypothesis provides some of the best ultimate explanations for modern rates of obesity, heart disease, type-2 diabetes, breast and prostate cancer, goiter, iodine deficiency, birth defects, and aging (harper 1975; eaton et al. 1988; williams and nesse 1991; greaves 2002; diamond 2003; swynghedauw 2004; gluckman et al. 2009 ). the understanding that contemporary genetic diseases and conditions may be consequences of adaptive mismatches can help explain the persistence of maladies in the face of improved sanitation and living standards. genetic disease varies among human populations because both long-term population sizes and histories of natural selection determine how many disease-causing mutations they carry (antolin 2009). as an example, the prevalence of specific allelic variants of the cftr gene that cause cystic fibrosis, a debilitating and in the long-term fatal genetic disorder, is potentially linked to selection imposed by past outbreaks of tuberculosis (poolman and galvani 2007) . this is similar to the long-established link between sickle cell anemia in individuals who inherit at least one copy of the s allele of the hbb globin gene, but where heterozygotes have some degree of resistance to malaria (allison 1954). in addition to leading to better diagnoses and treatments, seeing diseases as unfortunate genetic legacies inherited from the past may also help ameliorate some of the social stigma associated with genetic diseases. a related concept is that we are specifically adapted to the premodern environment in which humans were primarily huntergathers, before the domestication of plants and animals. central to this concept is that humans are adapted to a stone-age "environment of evolutionary adaptation" (eea). we caution against uncritical use of the eea, as the view is speculative, struggles to account for differences among ancestral human populations that probably experienced different environments, and ignores ongoing human evolution (also see strassmann and dunbar 1999; méthot 2011) . nonetheless, the concepts of a mismatch and an eea provide opportunities to introduce students to the scientific process, with discussion, gathering of information, and forming testable hypotheses. evolutionary biologists view phenotypic plasticity-the ability for organisms to display a range of phenotypes in different environments-as a way to maximize fitness under variable environmental conditions (also see box 7). in health and medicine, phenotypic plasticity may explain why early developmental problems may lead to disease in adults (bateson et al. 2004 ). an example of developmental trade-offs leading to disease in later life is increased susceptibility to heart disease in adults related to periods of nutritional deficiencies and slowed growth in fetal development and early childhood. the damage is potentially higher if subsequent weight gain (catch-up growth) is too rapid when better nutrition becomes available. in the case of growth rates and body size, adult heart disease is a cost of rapid growth following nutritional stress, and should be considered during care of low birth weight or premature babies (bateson et al. 2004; gluckman et al. 2009 ). weak selection on the diseases of aging helps explain their persistence in modern times, especially as postreproductive longevity increases (also see box 8). in evolutionary biology, fitness accrues via reproductive success summed across all stages of an individual's life history, and reproductive events early in life contribute more to fitness than do those late in life. the evolutionary theory of aging suggests that early-life fitness components including developmental rate, age at first birth, and fecundity early in life are linked by trade-offs to fecundity and mortality later in life. if these trade-offs result from the same genes acting at both life stages, selection on early-life traits will increase their frequency even if they negatively impact fitness-related traits later in life, a genetic effect called antagonistic pleiotropy (cf. williams 1957) . the role of testosterone in males provides an example. although increased testosterone has clear benefits for maximizing male reproductive success, this may come at the expense of disease re-sistance because increased testosterone also reallocates immune function away from immune reactions that protect against infections (braude et al. 1999; bribiescas and ellison 2007) . higher reproductive success early in life may come at the expense of disease and morbidity, especially if infections are chronic. whether the correlation between testosterone and altered immune function represents a genetic trade-off, or whether reallocation represents adaptive phenotypic plasticity, may be determined using modern genomic analyses and association studies. another striking trade-off is embodied in stem cells. stem cells are essential for the differentiation and renewal of all tissues, but their genetic characteristics place them only a few mutational steps away from cancer cells, and almost all cancers originate through a short series of somatic mutation in stem cells, which are increasingly being revealed as a double-edged sword (e.g., janzen et al. 2006; krizhanovsky and lowe 2009) . exploring evolution on a microbial time scale can help students understand the roles of genetic variation and reproductive success in evolution (box 9). short generation time, huge populations, high mutation rate, genetic reassortment, and horizontal gene transfer in microbes and viruses ensures that infectious diseases will evade therapies and vaccines in the future, and that new diseases will continue to emerge from reservoirs in wild animals: an "arms race against an adaptable opponent" (lederberg 2000) . this dynamic view of disease accounts for the variability in human-adapted pathogens such as influenza viruses and malaria, where evolutionary escape hinders development of vaccines with long-lasting protection and results in multidrug resistance. medical practice is directly impacted by rapid evolution of resistance to antimicrobial drugs as well as avoidance of natural and vaccine-induced immunity (bergstrom and feldgarden 2007; nesse and stearns 2008) . understanding how pathogen variability arises leads to public health interventions that may reduce exposure and limit chains of transmission (galvani 2003) . in our germophobic society, students need to realize that we have a long evolutionary history with microbes and parasites, and that these organisms are not always debilitating even if they cause mild disease (also see box 10). in some cases, microbes are helpful, and removing all commensals can be unhealthy: consider the occurrence of vaginal yeast infections in conjunction with systemic antibiotic use (eckert et al. 1998; reid 2001) . as we learn more about the complex interactions between humans and the diverse microbial species that colonize our bodies, we discover just how important commensal organisms are to our well-being (e.g., grice and segre 2011). many gut bacteria play important roles in our nutrition, such as the bacterium escherichia coli in the large intestine providing us with the essential vitamin k, which we cannot produce ourselves. some pathogens and parasites may, paradoxically, also be necessary to our well-being. obsession with hygiene in some industrialized societies has for the first time in our evolutionary history relieved us of our helminth parasites. strachan (1989) first proposed the "hygiene hypothesis" that attributed the recent epidemic in asthma, allergy, and other autoimmune diseases to this loss of our old companions (bach 2002; osada and kanazawa 2010) . this hypothesis predicts that restoration of helminth infections, or some facsimile thereof, should relieve symptoms of at least some of these diseases. indeed, in a clinical trial, the majority of crohn's disease patients who ingested pig whipworm (trichuris suis) experienced disease remission (summers et al. 2005) , and more studies indicating the utility of helminth infections to treat autoimmune disease have followed (reddy and fried 2009) . particularly, striking is the observation that patients with both multiple sclerosis and worm infections experience a much slower development of disease than do those lacking worm infections (correale and farez 2007) . finally, our long evolutionary history with pathogens has led to many defensive adaptations, including fever, coughing, and vomiting. medical practitioners need to be aware of symptoms that are actually defenses, because it is possible that in some cases, treating symptoms may prolong the course of infections and opportunities for pathogen transmission (williams and nesse 1994; carey 2010) . in a memoir (the edge blog [http://www.edge.org/documents/ williams_index.html], downloaded 17 june 2011), george williams sums up why evolutionary medicine should resonate in the community of evolutionary biologists, and how evolutionary thinking can help physicians to become better practitioners: in twenty or thirty years, medical students will be learning about natural selection, about things like balance between unfavorable mutations and selection. they will be learning about the evolution of virulence, of resistance to antibiotics by microorganisms, they will be learning about human archaeology, about stone age life, and the conditions in the stone age that essentially put the finishing touches on human nature as we now have it. these same ideas then will be informing the work of practitioners of medicine, and the interactions between doctor and patient. they'll be guiding the medical research establishment in a fundamental way, which isn't true today. at the rate things are going, this is inevitable. these ideas ought to reach the people who are in charge-the doctors and the medical researchers-but it's even more important that they reach college students, especially future medical students, and patients who go to the doctor. they'll have questions to ask that doctor, who will have to have answers. i hope this set of ideas produces a certain amount of bottom-up influence on the medical community, via students and patients. but i hope also that there's some top-down influence-that it will be influencing the faculties in medical schools and the researchers on human disease. george williams' work spurred us to ask "what is the role of evolutionary biologists in promoting evolutionary thinking in medical education and practice?" we encourage evolutionary biologists working in schools of medicine and public health, as well as colleges and universities where evolutionary research and instruction occurs, to explore relationships between evolution and health. we encourage evolutionary biologists to reach out to premedical students and others preparing for the medical professions, and to directly connect evolutionary science to the intellectual development of physicians and public health practitioners at every phase of their education and training. financial support for this symposium was provided by the education and outreach committee of the society for the study of evolution, by the national evolutionary synthesis center, and by the co-authors home institutions. we thank the local organizers of the 2011 evolution meetings, particularly r. broughton, i. schlupp, and l. weider (special thanks for the use of the bicycle!). the historic relationship between medicine and evolutionary biology has been mixed, and traditionally evolutionary biology has not played a prominent role in medical training. even so, general natural history was part of the medical school curriculum when charles darwin attended edinburgh in 1824-1826, but evolution certainly was not (antolin 2011a,b). darwin had familiarity with links between evolutionary biology and medicine through his grandfather erasmus darwin's volume "zoonomia," which included both a linnaean classification of disease and a clear statement about transmutation and common descent in nature (darwin 1801 ). darwin also consulted with his father, the physician robert waring darwin, on human heredity and disease (bynum 1983 ). the question of heredity was briefly covered issues in the "descent of man" (darwin 1882) and in "variation of plants and animals under domestication" (darwin 1875) , along with observations on the similarity of diseases (and responses to medication) in humans and apes, differing disease prevalence among human populations, effects of disease on aboriginal populations coming into contact with europeans, and sex-limited maladies such as hemophilia and gout. as evolutionary biology developed as a field, various scientists and physicians also advocated for inclusion of evolutionary thinking in medicine (e.g., huxley 1881; aitken 1885; morton 1926; see zampieri 2009). in the late 1800s, most applications of evolution in medicine were holistic, fitting in with the traditional medical ideas of "constitutions" and "diatheses." these were defined as tendencies to display groups of maladies in general ways, for instance "tubercular" individuals (bynum 1983; zampieri 2009 ). at the same time, the medical sciences were moving discovering specific causes for specific ailments, resulting in improved diagnosis and treatment (porter 1998) . as an example, the use of anesthesia and aseptic methods in surgery was developed during this period. the work of pasteur, koch, and others in identifying microbes and the birth of germ theory made it possible to identify the pathogens underlying common diseases such as whooping cough, syphilis, and cholera. further, industrialization and concentration of human populations in cities created new public health challenges that needed attention (porter 1998) . as the mechanisms underlying evolution and genetics were explored during the late 1800s and early 1900s, so were the physiological, developmental, genetic, and microbial bases of disease. the flexner report of 1910 marked a major turn toward placing medicine on a firm scientific footing. this classic of medical education explicitly called for bringing medical education onto a uniform foundation of human biology (flexner 1910) , and for experimental analysis of physiology, development, endocrinology, biochemistry, and anatomy of specific tissue and organ systems. flexner also called for broad coverage of the general sciences as part of medical training and practice. this period saw a step toward a reductionist view of the causes of disease, diverging from previous holistic medical approaches. although medical knowledge grew rapidly, medical applications from the emerging field of evolutionary biology were not as clear and, as a result, the evolutionary viewpoint was not broadly incorporated into medical school curricula. evolution was not mentioned in the flexner report, but one response to the report was for medical schools to establish departments of anthropology to bring human natural history, via classes in comparative anatomy and geographical medicine, explicitly into medical training (morton 1926) . this period spanning 1910 through the 1940s also saw the modern synthesis in evolutionary biology and breakthroughs in evolutionary research. but evolutionary biology still was not incorporated into medical training. in part, this was because the successful clinical application of antimicrobial drugs during this same period led to the idea that infectious diseases would soon to be completely manageable (burnet and white 1972; anderson 2004) . ironically, it is evolutionary biology that explains why pathogenic microbes remain an ongoing challenge (e.g., see box 9). but three serious misunderstandings and abuses of evolutionary theory resulted in evolution being expunged from medical training by the late 1940s (porter 1998; zampieri 2009 ). the first was related to the previously mentioned "constitutions," "diatheses," and "degeneracy" used to explain disease vulnerabilities in certain racial groups and in women. the misapplication of this typologically racist and sexist thinking to disease, which was wrongly tied to evolutionary principles, was largely dealt with by implementation of flexner's (1910) recommendations. the mechanistic understanding of disease attenuated societal biases of race and gender, although social disparities in the delivery of medical treatment still persist. the second reason was the horrific application of eugenics in the first half of the 20th century culminating in the genocidal nazi regime in the 1930s and 1940s in europe (zampieri 2009). to be sure, eugenics was a malignant social policy that arose from misapplication of the science of evolution, and was discarded by evolutionary biologists after the population genetics of mutation and natural selection were better understood (j. b. s. haldane [1964] provides an amusing essay defending mathematical theories of population genetics, including the balance between mutation and selection that was overlooked by eugenicists, and some lovely verse about his own life-ending fight with colorectal cancer). but the backlash was severe enough that even today many scientists within biomedical fields do not fully acknowledge how much evolutionary science informs medicine (antonovics et al. 2007; nesse and stearns 2008) . the third reason was that the dominant reductionist scientific paradigm advocated by flexner (1910) trained scientists to reject teleological thinking, which was mistakenly conflated with the process of natural selection, a misunderstanding that persists among some to this day. currently, few medical schools teach evolutionary topics beyond human genetic variation, drug resistance, pathogen virulence, and adaptation by natural selection (nesse and schiffman 2003; downie 2004; childs et al. 2005; harris and malyango 2005) . much of the new interest in evolutionary approaches to medicine has come from addressing questions about why natural selection has left the body vulnerable to disease. why does the human eye have a blind spot? why does the appendix persist despite causing appendicitis? why are autoimmune diseases becoming more prevalent in recent decades? why has not natural selection shaped childbirth to be less painful and risky? why are humans so vulnerable to drug and alcohol abuse? what is the evolutionary explanation for aging? is menopause a life-history trait shaped by selection, or an epiphenomenon? students are quickly engaged in discussions about such interesting questions. the challenge for educators is to help them think critically about the questions, without getting prematurely discouraged by the substantial challenges of forming and testing evolutionary hypotheses about disease vulnerability. for many questions, such as the adaptive significance of menopause, the unknowns are so substantial that consensus remains elusive despite extensive research. some may see this as a reason to teach simpler topics. however, students already get plenty of experience memorizing the answers to scientific questions. topics in evolutionary medicine give them an opportunity to grapple with current questions. students will tend to latch onto a favorite hypothesis and try to defend it. this gives educators an opportunity to emphasize the importance of considering all possible hypotheses, and strategies for determining which are correct and which are false. in contrast to proximate studies, evolutionary questions often have more than one answer, in the sense that multiple factors may be involved in accounting for a trait. emphasizing this can help students recognize the need to consider multiple possibilities. in short, topics in evolutionary medicine are well suited to advancing a major goal of modern education-helping students to learn how to formulate hypotheses, how to go about the scientific process of testing them, and how to cope with the inevitable complexity and confusion that attend almost any scientific endeavor. some of the more common errors are summarized in an article that lists 10 questions worth asking for any evolutionary study of disease (nesse 2011) . human populations span a tremendous diversity of environments, and our ability to inhabit such a broad range of habitats results in part from genetic adaptations. the availability of genome-wide data on genetic variation from diverse populations offers unprecedented opportunities to identify the loci responsible for these adaptations and elucidate the genetic architecture of human adaptive traits. some of the earliest and most convincing evidence of adaptation to the environment comes from correlations between phenotypes and environmental variables. classic examples of these patterns include correlations between sickle cell anemia, β-thalassemia and malaria endemicity (haldane 1949; allison 1954) , body mass and temperature (roberts 1978; katzmarzyk and leonard 1998) , skin pigmentation and solar radiation (jablonski and chaplin 2000) , and lactase persistence and dairying (simoons 1969) . for several of these examples, the genetic variants underlying these patterns are now known (enattah et al. 2002; lamason et al. 2005; sulem et al. 2007; tishkoff et al. 2007; piel et al. 2010 ). using genome-wide data, it is now possible to turn this approach around to scan the human genome to identify genetic variants that correlate with predicted environmental selective pressures, even without knowing the phenotypic adaptations that may drive the associations between specific alleles and the environment. association studies of this kind have been carried out for variation related to climate, subsistence, diet, and pathogen diversity (fumagalli et al. 2010; hancock et al. 2010; hancock et al. 2011) . integrating results from these environmental genome scans with studies of genotype-phenotype association and other sources reveals the relationships between disease risk and adaptation to the environment. for example, scans show that alleles correlated with diet and subsistence influence folate and energy metabolism. in addition, among genes with strong correlation with climate, many have alleles or genotypes implicated in traits such as pigmentation phenotypes and other aspects of ultraviolet radiation response, cardiovascular disease, immune response, and cancer. together with ongoing efforts to identify variants associated with disease phenotypes in diverse populations (rosenberg et al. 2010) , environmental genome scans and scans for adaptive genetic loci can identify the genetic variants that underlie differences in disease susceptibility among populations (e.g., nielsen et al. 2009; pickrell et al. 2009; grossman et al. 2010 ). it is repeatedly stated in medical practice that each case is unique and should be treated individually, but in most cases the underlying factors determining such variation remain poorly understood. part of the answer however may lie in our genes and thus in our evolutionary history. for example, broad patterns of population structure among humans are largely the result of ancient demographic events. the bottleneck during the out of africa exodus of modern humans about 50,000 years ago contributed substantially to reduced heterozygosity in individuals living outside of africa and remains the most remarkable demographic imprint reflected in their genomes (cavalli-sforza and feldman 2003) . in contrast, fine-scale population structure stems from recent demographic processes, reflecting local mating within popu-lations living in geographically circumscribed locations. the impact of inbreeding on the frequency of rare diseases has been demonstrated in historically isolated groups, such as the ashkenazi jewish population (reich and lander 2001) . if geographic regions throughout the world are finely structured, as suggested by global genomic surveys (auton et al. 2009 ), it may be predicted that populations in some regions harbor disease-risk alleles that are population specific and otherwise rare in humans. for example, acuña-alonzo et al. (2010) recently reported that a novel cholesterol transporter abca1 gene variant (r230c, rs9282541) is exclusive to native americans and has been associated with low levels of high-density lipoprotein (hdl), obesity and type 2 diabetes in admixed populations in mexico (villarreal-molina et al. 2008) . the authors also identified signatures of positive natural selection around the abca1 variant, suggesting that r230c carriers could have had a selective advantage during the initial peopling of the americas. because the c230 abca1 protein shows decreased cholesterol efflux, the presence of this variant could favor intracellular cholesterol and energy storage, conferring an advantage during severe famine periods. however, under current westernized lifestyle, this allele may have become a major susceptibility allele for low hdl-c levels and other metabolic traits. it becomes clear that genetic variation observed today, including medically relevant variation, is the consequence of the interplay between different evolutionary forces acting over time during human population history. therefore, medical students would benefit from learning evolution theory, as it will allow them to better understand genomic variation and its implications in health and disease. cancers account for almost 13% of all human deaths worldwide (jemal et al. 2011) , representing a major medical problem for the modern world. it is not a single disease, but an ensemble of diseases with common principles. all cancers harbor groups of cells that show uncontrolled growth, invasion of neighboring tissues, and metastasis to distant organs (hanahan and weinberg 2011) . ultimately, most cancers cases result in death. cancer arises via accumulation of mutations and selection-a case of aberrant evolution of somatic cells during the lifetime of the host, although instances of transmissible cancer are also known (murgia et al. 2006) . although somatic cells in healthy individuals often harbor many mutations without any apparent consequences (de 2011) , cancer cells carry specific mutations that result in immortality, uncontrolled growth, and the ability to metastasize (hanahan and weinberg 2011) . many of the key mutagenic processes that are active in cancer cells also operate in germ-line evolution, which together with selection, gives rise to population-and species-level divergence in genomes over thousands or millions of years. moreover, many of the genes and pathways associated with cancer are also present in other organisms such as yeast or fruit flies. for instance, the first discovery of a class of cancer genes, the rat sarcoma (ras) family, was in rat (harvey 1964; kirsten et al. 1970) . therefore, much can learned about the mechanistic bases of cancer, and we may exploit weakness of cancer cells to develop better diagnostic and prognostic strategies by borrowing knowledge from germ line evolution. genetically modified animals provide insights into cancer-related biological processes; and even today we test new cancer drugs on mice for efficacy and side effects before prescribing them to human patients. we are now moving into an era of personalized diagnostics and treatment, which will require low cost, rapid analysis using more minimal biological samples more than ever before. evolutionary medicine can help to achieve these goals by translating knowledge from evolution into clinical strategies. box 6. public health and evolution. gilbert s. omenn. public health courses are emerging as popular undergraduate offerings, especially at universities with schools of public health. evolution has shaped burden of disease in the modern world in which we practice public health (omenn 2010) . human cultures and technologies have modified life on planet earth and have coevolved with myriad other species, including microorganisms, plant and animal sources of food, invertebrate vectors of disease, and intermediate bird, mammal, and primate hosts. molecular mechanisms of evolution have produced differential resistance or susceptibility to infectious agents, including malaria, plague, smallpox, tb, measles, and diarrheal and respiratory diseases. the domestication of sheep and cattle has selected for humans able to digest milk throughout life through persistence into adulthood of lactase enzyme expression in the intestine, a major story of anthropology (itan et al. 2009 ). the emergence of a "western diet" of dairy, refined cereal grains, refined sugars, vegetable oils, alcoholic beverages, salt, and omega-6-rich meats has dramatically altered glycemic load, fatty acid composition, macronutrients, acid-base balance, sodium/potassium ratio, and fiber content. the results include epidemics of atherosclerotic cardiovascular disease, obesity, diabetes, high blood pressure, osteoporosis, certain cancers, and bowel, inflammatory, and autoimmune disorders (cordain et al. 2005) . a newly recognized phenomenon is the selection of excessive hemostatic activity from platelets and the plasma-clotting proteins; what was protective against death from bleeding after injuries among hunter-gatherer, warring humans, or against pregnancy-related hemorrhage now contributes to thrombosis underlying heart attacks and strokes. conversely, there is little pressure against hemostasis and thrombosis because deaths resulting from blood clots and coronary disease associated with hemostasis and thrombosis occur mostly after the reproductive years of life (coller 1997) . learning about evolution over millennia for humans and over hours or days for microbes enlivens the experience of understanding evolution in public health context. synthesis of data from multiple disciplines has led to hypotheses for understanding the evolutionary-genomic underpinnings of autism spectrum conditions and psychotic-affective spectrum conditions, mainly schizophrenia, bipolar disorder, major depression, and borderline personality disorder (crespi and badcock 2008) . the synthesis integrates inclusive fitness theory, comparative primatology, and developmental psychology with information from mouse gene knockouts, human genomic disorders, psychiatric genetics, and human and mouse gene expression. the primary hypotheses focus on autism and psychoticaffective conditions as diametric disorders mediated by opposite alterations to the development of human-elaborated social-brain and sexual-brain phenotypes. by this hypothesis, social-brain architecture develops predominantly in the context of mother-child interactions, whereby early, basic mother-child attachment, underlain in part by paternally expressed imprinted genes, serves as a scaffold for development of the highly social, neocortical brain. in turn, development of the highly social neocortical brain is mediated in part by effects of maternally expressed imprinted genes. alterations to the expression of imprinted genes (genes that are silenced under inheritance from one or the other parent, and that evolve under genomic conflict, (see haig [2004] ) play important roles in early social-brain development, with deviations toward relative paternal-gene expression increasing risk of autism. deviations toward relative maternal-gene expression increase risk of psychotic-affective spectrum conditions (crespi and badcock 2008; crespi 2011) . autism and psychotic-affective conditions are thus hypothesized to arise in part through epigenetic variation that influences maternalpaternal genomic conflicts. more generally, the human social brain may underdevelop in childhood, manifesting as autism spectrum conditions, or selectively and relatively over-develop, usually in adolescence or early adulthood, leading to psychotic-affective spectrum phenotypes and conditions. cognitive and affective variation between females and males strongly modulates liability to these conditions. thus, major epidemiological and phenotypic features of autism and psychotic-affective conditions can be predicted from interactions between a malefemale brain continuum and a maternal-paternal imprinting bias continuum (crespi and badcock 2008) this conceptual framework spans two levels. first, the diametric nature of autism versus psychotic-affective conditions is modulated by multiple genetic, genomic, epigenetic, and social-environmental causes based in human brain evolution. second, this diametric nature may arise from imprinted genes, which can be dysregulated in two opposite directions. the framework makes novel, testable predictions with direct implications for cognitive-behavioral and pharmacological therapy, human evolution, the molecular evolution of psychiatric risk genes, and human brain development. one important prediction, that imprinting is relatively common among genes that underlie risk of autism and schizophrenia, is supported by analyses of data from a recent genome-wide study of imprinted genes in mice (gregg et al. 2010 ). box 8. can any organisms be potentially immortal? staphen c. stearns. weismann (1882) suggested that only organisms with a soma and germ line would age, and that organisms that divided symmetrically would not age. recent experiments have shown just how precisely symmetric that division would have to be. their results suggest that all known organisms must age, for even a bacterium like e. coli divides into two nonidentical cells, one of which contains older parts than the other and ages faster (stewart et al. 2005) . the lineages with the old parts suffer a fitness disadvantage and disappear, and the lineages with the young parts persist. the goal of potential immortality (barring accidents) may also be unattainable if another basic claim of the evolutionary theory of aging remains unrefuted: aging and death result not from a single mechanism that can be repaired but from the diffuse degradation of multiple processes across the entire genome and body (williams 1957) . repair of one problem will simply reveal the next in line, and that process will repeat hundreds or thousands of times. the byproducts of repair are also expected to connect through trade-offs to costs paid in other traits. recently, some genes with major effects on aging have been discovered in other organisms, but they are involved in trade-offs with reproductive performance early in life, and whether those trade-offs can be compensated remains to be seen (stearns 2001a) . the evolutionary view of aging is not optimistic about life-extending genetic therapies, but it may provide some needed wisdom about the human condition. box 9. why does antibiotic resistance persist after antibiotic use stops? carl t. bergstrom. the evolution and spread of antibiotic resistance powerfully illustrates the basic process of natural selection. bacterial populations manifest variability in antibiotic susceptibility; some of this variation is heritable; in the presence of antibiotics, less-susceptible strains survive and reproduce more rapidly. the inevitable result-as we have seen with every antibiotic yet introduced-is the evolution of resistance in bacterial populations (genereux and bergstrom 2005) . a related phenomenon offers an opportunity to teach more subtle aspects of evolutionary biology and population genetics (bergstrom and dugatkin 2011) . when antibiotic use is terminated in a community, what happens to antibiotic resistance in the bacterial populations? given that antibiotic resistance commonly imposes some cost to bacteria that express it, we might predict that when a particular antibiotic is discontinued, resistance to that antibiotic should decline. although this sometimes occurs (e.g., seppälä et al. 1997) , it is by no means guaranteed. sometimes resistance to the drug persists long afterward. one example is sulfonamide resistance in great britain, which has not declined even though sulfonamides were essentially discontinued in the mid-nineties (bean et al. 2005) . why? a number of multilocus evolutionary processes may contribute, including the phenomenon of associated linkage selection (levin et al. 1997) . because bacteria have haploid genomes and limited opportunities for recombination, most bacteria exhibit strong linkage along the entire circular chromosome. as a result, hitchhiking and related processes are very important. in the process of associated linkage selection, resistance to a new drug arises on a chromosome or plasmid carrying resistance alleles for older drugs. this generates positive linkage disequilibrium between the new and old resistance alleles. even if an older drug is discontinued, the resistance alleles to this drug may be indirectly favored by selection for resistance to the newer drug. associated linkage selection can thus explain why older resistant strains are not outcompeted by unrelated sensitive strains: those sensitive strains are less likely to carry resistance to whatever new drugs are currently in use. but associated linkage selection does not explain how resistant strains are able to avoid being replaced by mutants that retain resistance to the new antibiotic but revert to sensitivity to the old one. to make sense of this, we turn to another phenomenon: compensatory mutation. although resistance mechanisms impose fitness costs, compensatory mutations can ameliorate these costs and restore fitness of a resistant strain-in the absence of the antibiotic-to near that of the sensitive strain (schrag and perrot 1996; andersson and hughes 2010) . an obvious consequence is that compensatory mutations reduce the selective difference between resistant and sensitive strains in the absence of antibiotic use, and thus slow the rate at which sensitive strains are able to replace resistant ones. many compensatory mutations have a more subtle effect as well. they make it difficult for bacteria to revert to full sensitivity, by creating a fitness valley between the resistant, compensated strain and the sensitive uncompensated strain (levin et al. 2000) . this occurs because the compensatory mutation, while beneficial in the presence of the resistance mutation, is deleterious in its absence. because it is easy to "get in" to the resistant compensated state in the presence of the antibiotic but difficult to get out in its absence, this phenomenon has been described as an evolutionary lobster trap (tanaka and valckenborgh 2011 although the negative impacts of rapid microbial evolution are well understood, humanity has turned the evolutionary lability of viruses into a means of fighting disease. "applied evolu-tion" has been used to generate many live-attenuated virus vaccines, including those against measles, mumps, rubella, varicella (chickenpox), influenza, and poliovirus (fda 2011). in this classical approach, pioneered by pasteur in his effort to create a rabies vaccine, wild-type virulent viruses are serially passaged in a novel host or in a novel temperature regime. the resulting adaptations to the new environments results in loss of fitness in the original host (plotkin 2001) . a successfully attenuated virus shows decreased virulence in humans while retaining the ability to stimulate an immune response. this process can require as few as 28 passages, as for the varicella vaccine (takahashi et al. 2008 ), or hundreds, as was needed for the yellow fever vaccine (monath et al. 2008) . however, one of the drawbacks of classical vaccine design is that the phenotype of the virus, that is, its attenuation, determines genotype. thus, although it is clearly desirable to secure attenuation by multiple or large attenuating mutations (burch et al. 2003) , the mutations responsible for attenuation of many liveattenuated virus vaccines are either unknown or, in the case of the three strains of the oral poliovirus vaccine (opv) (kew et al. 2005) , few in number. predictably, ∼1 in every 750,000 children receiving their first dose of opv experience vaccine-associated paralytic polio due to reversion of virulence in the vaccine (kew et al. 2005) . revertant strains may be transmissible between humans and thus threaten the success of the global effort to eradicate polio. in contrast, the nasal influenza vaccine carries attenuating mutations on four of its eight genomic segments, and to date has never reverted to virulence within a vaccinee (tosh et al. 2008) . fortunately, reverse genetics (rational vaccine design) informed by evolutionary thinking suggest novel approaches to safeguard the attenuation of live vaccine viruses. for example, vignuzzi et al. (2008) have attenuated poliovirus by enhancing the fidelity of its rnadependent rna polymerase and thereby slowing its mutation rate. because this virus is less likely to mutate, it is less likely to revert to a less-faithful, and more fit, polymerase. finally, live vaccines may not only evolve themselves but may also shape the evolution of their wild-type counterparts. there is evidence that some veterinary vaccines are driving the evolution of antigens in their wild-type targets (park et al. 2011 ), but the impacts of such evolution on virulence and transmission are not yet known. at the extreme, vaccines have the potential to eradicate their wild-type targets, potentially creating an empty niche into which new viruses may emerge from human or zoonotic reservoirs (rieder et al. 2001; rimoin et al. 2010; vasilakis et al. 2011) . evolution, medicine, and the darwin family evolution by any other name: antibiotic resistance and avoidance of the e-word global distribution of genomic diversity underscores rich complex history of continental human populations the effect of infections on susceptibility to autoimmune and allergic diseases developmental plasticity and human health resistance among escherichia coli to sulphonamides and other antimicrobials now little used in man evolution and medicine: an inquiry-based high school curriculum supplement the ecology and evolution of antibiotic-resistant bacteria how people learn: brain, mind, experience, and school stress, testosterone, and the immunoredistribution hypothesis how hormones mediate trade-offs in human health and disease developing a curriculum for evolutionary medicine: case studies of scurvy and female reproductive tract cancers patterns of epistasis in rna viruses: a review of the evidence from vaccine design natural history of infectious disease darwin and the doctors: evolution, diathesis, and germs in 19 th -century britain literature review: should antipyretic therapies routinely be administered to patients with [corrected] fever? how cancer shapes evolution, and how evolution shapes cancer the application of molecular genetic approaches to the study of human evolution a science of the individual: implications for a medical school curriculum advances in ap platelet gpiib/iiia antagonists: the first anti-integrin receptor therapeutics origins and evolution of the western diet: health implications for the 21st century association between parasite infection and immune responses in multiple sclerosis nutritional genomics era: opportunities toward a genome-tailored nutritional regimen the strategies of the genes: genomic conflicts, attachment theory, and development of the social brain psychosis and autism as diametrical disorders of the social brain the variation of animals and plants under domestication zoonomia: or the organic laws of life somatic mosaicism in healthy human tissue the double puzzle of diabetes evolution in health and disease: the role of evolutionary biology in the medical curriculum stone agers in the fast lanechronic degenerative diseases in evolutionary perspective vulvovaginal candidiasis: clinical manifestations, risk factors, management algorithm identification of a variant associated with adult-type hypolactasia fda. 2011. vaccines, blood and biologics medical education in the united states and canada. bulletin number four, carnegie foundation for the advancement of teaching genome-wide identification of susceptibility alleles for viral infections through a population genetics approach epidemiology meets evolutionary ecology evolutionary science and society: educating a new generation principles of evolutionary medicine cancer causation: the darwinian downside of past success? high-resolution analysis of parent-of-origin allelic expression in the mouse brain the skin microbiome a composite of multiple signals distinguishes causal variants in regions of positive selection genomic imprinting and kinship: how good is the evidence? the rate of mutation of human genes. eighth international congress of genetics hallmarks of cancer: the next generation adaptations to climate-mediated selection pressures in humans human adaptations to diet, subsistence, and ecoregion are due to subtle shifts in allele frequency the double-edged sword: how evolution can make or break a live-attenuated virus vaccine evolutionary explanations in medical and health profession courses: are you answering your students' 'why' questions? an unidentified virus which causes the rapid production of tumors in mice making evolution relevant and exciting to biology students an address on the connection of the biological sciences with medicine the origins of lactase persistence in europe the evolution of human skin coloration stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16ink4a global cancer statistics evolution and medicine personalized medicine in oncology: tailoring the right drug to the right patient the role of scientific understanding in college: student acceptance of evolution climatic influences on human body size and proportions: ecological adaptations and secular trends vaccine-derived polioviruses and the endgame strategy for global polio eradication properties of a murine sarcoma virus genetics and the general physician: insights, applications and future challenges stem cells: the promises and perils of p53 slc24a5, a putative cation exchanger, affects pigmentation in zebrafish and humans infectious history the population genetics of antibiotic resistance compensatory mutations, antibiotic resistance and the population genetics of adaptive evolution in bacteria medically relevant genetic variation of drug effects yellow fever vaccine cause and effect in biology: kinds of causes, predictability, and teleology are viewed by a practicing biologist research traditions and evolutionary explanations in medicine the relation of evolution to medicine clonal origin and evolution of a transmissible cancer teaching about evolution and the nature of science darwinian and demographic forces affecting human protein coding genes comparison of cytochrome p450 (cyp) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants ten questions for evolutionary studies of disease vulnerability why we get sick: the new science of darwinian medicine the great opportunity: evolutionary applications to medicine and public health making evolutionary biology a basic science for medicine evolutionary biology in the medical school curriculum vision and change in undergraduate biology education: a call to action evolution in health and medicine sackler colloquium: evolution and public health enhancing the teaching of evolution in public health parasitic helminths: new weapons against immunological disorders rapid evolution of low-pathogenic h9n2 avian influenza viruses following poultry vaccination programmes signals of recent positive selection in a worldwide sample of human populations global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis evaluating candidate agents of selective pressure for cystic fibrosis the greatest benefit to mankind: a medical history of humanity an update on the use of helminthes to treat crohn's and other autoimmune diseases on the allelic spectrum of human disease probiotic agents to protect the urogenital tract against infection will the polio niche remain vacant? major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the democratic republic of congo introduction: the changing microbial environment, darwinian medicine and the hygiene hypothesis genome-wide association studies in diverse populations reducing antibiotic resistance huovinen, and the finnish study group for antimicrobial resistance. 1997. the effect of changes in the consumption of macrolide antibiotics on erythromycin resistance in group a streptococci in finland primary adult lactose intolerance and the milking habit: a problem in biological and cultural interrelations. i. review of the medical research does impressive progress on understanding mechanisms advance life history theory? pp evolution in health and disease evolutionary perspectives on health and medicine aging and death in an organism that reproduces by morphologically symmetric division hay fever, hygiene, and household size human evolution and disease: putting the stone age in perspective genetic determinants of hair, eye and skin pigmentation in europeans trichuris suis therapy in crohn's disease evolutionary medicine development of varicella vaccine escaping an evolutionary lobster trap: drug resistance and compensatory mutation in a fluctuating environment on aims and methods in ethology convergent adaptation of human lactase persistence in africa and europe population genetics and microevolutionary theory flu myths: dispelling the myths associated with live attenuated influenza vaccine evolutionary medicine and health: new perspectives fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health engineering attenuated virus vaccines by controlling replication fidelity association of the atp-binding cassette transporter a1 r230c variant with early-onset type 2 diabetes in a mexican population a mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine ueber die dauer des lebens pleiotropy, natural selection and the evolution of senescence the dawn of darwinian medicine key: cord-330312-1pjolkql authors: liu, y.-t. title: infectious disease genomics date: 2017-01-20 journal: genetics and evolution of infectious diseases doi: 10.1016/b978-0-12-799942-5.00010-x sha: doc_id: 330312 cord_uid: 1pjolkql the history and development of infectious disease genomics have been closely associated with the human genome project (hgp) during the past 20 years. it has been emphasized since the beginning of the hgp that such effort must not be restricted to the human genome and should include other organisms including mouse, bacteria, yeast, fruit fly, and worm for comparative sequence analyses. a brief history is reviewed in this chapter. as of 2016, more than 7000 completed genome sequencing projects have been reported. one of the important motivations for these efforts is to develop preventative, diagnostic, and therapeutic strategies through the analysis of sequenced microorganisms, parasites, and vectors related to human health. a number of examples are discussed in this chapter. the history and development of infectious disease genomics are closely associated with the human genome project (hgp). 1 a series of important discussions about the hgp were made from 1985 to 1986, 1,2 which led to the appointment of a special national research council (nrc) committee by the national academy of sciences to address the needs and concerns, such as its impact, leadership, and funding sources. the committee recommended that the united states begin the hgp in 1988. 3 they emphasized the need for technological improvements in the efficiency of gene mapping, sequencing, and data analysis capabilities. in order to understand potential functions of human genes through comparative sequence analyses, they also advised that the hgp must not be restricted to the human genome and should include model organisms including mouse, bacteria, yeast, fruit fly, and worm. in the meantime, the office of technology assessment (ota) of the us congress also issued a similar report to support the hgp. 4 in 1990, the department of energy (doe) and the national institutes of health (nih) jointly presented an initial 5-year plan for the hgp. 5 in october 1993, the sanger center/institute (hinxton, uk) was officially open to join the hgp. the cost of dna sequencing was about $2 to $5 per base in 1990 and the initial aim was to reduce the costs to less than $0.50 per base before large-scale sequencing. 5 the sequencing cost gradually declined during the subsequent years. in 2004, the national human genome research institute (nhgri) challenged scientists to achieve a $100,000 human genome (3 gb/haploid genome) by 2009 and a $1000 genome by 2014 to meet the need of genomic medicine. in early 2014, illumina announced that the company would begin producing a new system to deliver full coverage human genomes for less than $1,000. 6 the first complete genome to be sequenced was the phix174 bacteriophage (5.4 kb) by sanger's group in 1977. 7 the complete genome sequence of sv40 polyomavirus (5.2 kb) was published in 1978. 8, 9 the human epsteinebarr virus (170 kb) genome was determined in 1984. 10 the first completed free-living organism genome was haemophilus influenza (1.8 mb), sequenced through a whole-genome shotgun approach in 1995. 11 the second sequenced bacterial genome, mycoplasma genitalium (600 kb), was completed in less than 1 month in the same year using the same approach. 12 the doe was the first to start a microbial genome program (mgp) as a companion to its hgp in 1994. 13 the initial focus was on nonpathogenic microbes. along with the development of the hgp, there was exponential growth of the number of completely sequenced free-living organism genomes. the fungal genome initiative (fgi) 14 was established in 2000 to accelerate the slow pace of fungal genome sequencing since the report of the genome of saccharomyces cerevisiae in 1996. 15 one of the major interests was to sequence organisms that are important in humanhealth and commercial activities. with the explosion in the number of sequenced genomes, thanks to the development of next generationesequencing methods, many genome-based studies have become popular. compared to 6 years ago when only 1100 completed genome projects were documented, the gold (genomes online database) contains information for 67,879 genome-sequencing projects, of which 7210 were completed, as of august 2015. 16, 17 the genomes of human malaria parasite plasmodium falciparum and its major mosquito vector anopheles gambiae were published in 2002. 18, 19 historically, the effort to sequence the malaria genome began in 1996 by taking advantage of a clone derived from laboratory-adapted strain. 20 notably, many parasites have complex life cycles that involve both vertebrate and invertebrate hosts and are difficult to maintain in the laboratory. few other important human pathogenic parasites, such as trypanosomes, 21, 22 leishmania, 23 and schistosomes, 24, 25 have been either completely or partially sequenced. 26, 27 in the meantime, the genome sequence of aedes aegypti, the primary vector for yellow fever and dengue fever, was published in 2007. 28 the genome size (1376 mb) of this mosquito vector is about 5 times larger than the previously sequenced genome of the malaria vector a. gambiae. about 50% of the genome consists of transposable elements. in 2010, the genome sequence of the body louse (pediculus humanus humanus), an obligatory parasite of humans and the main vector of epidemic typhus (rickettsia prowazekii), relapsing fever (borrelia recurrentis), and trench fever (bartonella quintana), was reported. 29 its 108 mb genome is the smallest among the known insect genomes. subsequently, more vector genomes have been published. 30e32 genome-sequencing projects for other important human disease vectors are in progress. 33, 34 these include culex pipiens (mosquito vector of west nile virus), and ixodes scapularis (tick vector of lyme disease, babesia and anaplasma). the challenge to sequence the genome of an insect vector is much greater than a microbe. for example, the genome of ticks was estimated to be between 1 and 7 gb and may have a significant proportion of repetitive dna sequences, which may be a problem for genome assembly. 35 furthermore, the evolutionary distances among insect species may also affect homologybased gene predictions. it is as important to understand the sequence diversity within a species as to perform a de novo sequencing of a reference genome from the perspective of human health. this is true for both hosts and pathogens. 36, 37 the goal of the 1000 genomes project is to find most genetic variants that have frequencies of at least 1% in the human populations studied. 38 one of the similar efforts for human pathogens is the nih influenza genome sequencing project. when this project began in november 2004, only seven human influenza h3n2 isolates had been completely sequenced and deposited in the genbank database. 39, 40 as of may 2010, more than 5000 human and avian isolates had been completely sequenced, including the 1918 "spanish" influenza virus. 41 databases for human immunodeficiency virus (hiv) and hepatitis c virus have also been established. while most human studies of microbes have focused on the disease-causing organisms, interest in resident microorganisms has also been growing. in fact, it has been estimated that the human body is colonized by at least 10 times more prokaryotic and eukaryotic microorganisms than the number of human cells. 42 it was suggested to have "the 2nd human genome project" to sequence the human microbiome. 43 highly variable intestinal microbial flora among normal individuals has been well documented. 44e46 therefore, the human microbiome project (hmp) was initiated by the nih in late 2007. 47 the analysis and data of 242 healthy adults at 15 (for males) or 18 (for females) body sites over 22 months were published in 2012. 48 the completed or ongoing genome projects (table 10 .1) provide enormous opportunities for the discovery of novel vaccines and drug targets against human pathogens as well as the improvement of diagnosis and discovery of infectious agents and the development of new strategies for invertebrate vector control. specific examples are provided to illustrate how the information provided by various genome projects may help achieve the goal of promoting human health. meningococcal isolates produce one of 13 antigenically distinct capsular polysaccharides, but only five (a, b, c, w135, and y) are commonly associated with disease. 49 the polysaccharide capsule is important for meningococci to escape from complement-mediated killing. while conventional vaccines consisting of the conjugation of capsular polysaccharides to carrier proteins for meningococcus serogroups a, c, y, and w-135 have been clinically successful, the same approach failed to produce clinically useful vaccine for serogroup b (menb). the capsule polysaccharide (a2e8-n-acetylneuraminic acid) of menb is identical to human polysialic acid, therefore is poorly immunogenic. 50 alternatively, vaccines consisting of outer-membrane vesicles (omvs) have been successfully developed to control menb outbreaks in areas where epidemics are dominated by one particular strain. 51e54 the most significant limitation of this type of vaccine is that the immune response is strain specific, mostly directed against the porin protein, pora, which varies substantially in both expression level and sequence across strains. 55, 56 with the completion of the genome sequence of a virulent menb strain, a "reverse vaccinology" approach was applied for the development of a universal menb vaccine by novartis. 55, 57, 58 through bioinformatic searching for surface exposed antigens, which may be the most suitable vaccine candidates due to their potential to be readily recognized by the immune system, 570 open reading frames (orfs) were selected from a total of 2158 orfs of the mc58 genome. eventually, five antigens were chosen as the vaccine components based on a series of criteria including the ability of candidates to be expressed in escherichia coli as recombinant proteins (350 candidates), the confirmation of surface exposure by immunological analyses, the ability of induced protective antibodies in experimental animals (28 candidates), and the conservation of antigens within a panel of diverse meningococcal strains, primarily the diseaseassociated menb strains. 55, 58, 59 the vaccine formulation consists of an fhbp-gna2091 fusion protein, a gna2132-gna1030 fusion protein, nada, and omvs from the new zealand menzb vaccine strain, which contains the immunogenic pora. initial phase ii clinical results in adults and infants showed that this vaccine could induce a protective immune response against three diverse menb strains in 89e96% of subjects following three vaccinations and 93e100% after four vaccinations. 59 this vaccine (bexsero) has been approved in the usa and in more than 30 other countries. 60 natural products, especially microbial secondary metabolites, are important source of bioactive compounds. actinomycetes have been a main source of natural-product discovery in bacteria. consequently, the high rediscovery rate of known compounds and scaffolds were inevitable with activity-based screening. genome mining of gene clusters that produce secondary metabolites have been a new approach to overcome this problem. for example, an antibiotic, clostrubin, was discovered through searching novel compounds from clostridium beijerinckii due to the presence of several cryptic gene clusters for secondary metabolite biosynthesis. 61 genome mining starts with a genome-wide search for highly conserved members of the required biosynthesis gene cluster. computational programs that support the prediction of operons help to assign boundaries of newly identified biosynthesis gene clusters. a large-scale, high-throughput genome mining for the genetic potential for producing phosphonic acids by screening more than 10,000 actinomycetes has been achieved in 2015. 62 it was believed that phosphonates would have greater potential to become pharmaceuticals, with a past commercialization rate of 15% (3/20), such as fosfomycin, compared to the 0.1% average for natural products as a whole. 63, 64 in addition, bioinformatical discovery of phosphonate biosynthetic loci has been well established, as all but two previously characterized phosphonate biosynthetic pathways start with phosphoenolpyruvate (pep) mutase that is encoded by pepm. among 10,000 actinomycetes, only 278 strains were confirmed to have pepm by polymerase chain reaction (pcr) screening and genome sequencing. a diverse collection of phosphonate biosynthetic gene clusters were identified within these strains. remarkably, 55 out of the 64 distinct clusters would direct the synthesis of unknown compounds. characterization of strains within five of these groups resulted in discovery of argolaphos, and other interesting compounds, including valinophos, and phosphonocystoximate. argolaphos showed broad-spectrum antibacterial activity against salmonella typhimurium, e. coli, and staphylococcus aureus. targeting an essential pathway is a necessary but not sufficient requirement for an effective antimicrobial agent. 65 identification of essential genes in a completely sequenced genome has been actively pursued with various approaches. 66, 67 the indispensable fatty acid synthase (fas) pathway in bacteria has been regarded as a promising target for the development of antimicrobial agents. 68 the subcellular organization of the fatty acid biosynthesis components is different between mammals (type i fas) and bacteria (dissociated type ii fas), which raises the likelihood of host specificity of the targeting drugs. comparison of the available genome sequences of various species of prokaryotes reveals highly conserved fas ii systems suggesting that the antimicrobial agent can be broad spectrum. 69 in addition, through computational analyses, new members of the fas ii system have been discovered in different bacterial species. 70, 71 one of the protein components in this system, fabi, is the target of an antituberculosis drug isonizid and a general antibacterial and antifungal agent, triclosan. 72e74 through a systematic screening of 250,000 natural product extracts, a merck team identified a potent and broad-spectrum antibiotic, platensimycin, which is derived from streptomyces platensis and a selective fabf/b inhibitor in fas ii system. 75 treatment with platensimycin eradicated s. aureus infection in mice. platensimycin did not have cross-resistance to other antibiotic-resistant strains in vitro, including methicillin-resistant s. aureus, vancomycin-intermediate s. aureus, and vancomycinresistant enterococci. no toxicity was observed using a cultured human cell line and the activity of platensimycin was not affected by the presence of human serum in this study. however, the fas ii system appears to be dispensable for another gram-positive bacterium, streptococcus agalactiae, when exogenous fatty acids are available, such as in human serum. 65, 76 the susceptibility to inhibitors targeting the fas ii system indicates heterogeneity in fatty acid synthesis or in acquiring exogenous fatty acids among gram-positive pathogens. 76 comparative genomic approaches may be useful to identify and develop a strategy to target the salvage pathway for s. agalactiae. alternatively, similar approaches as described earlier for menb vaccine may also be applied for s. agalactiae (group b streptococcus). 77 emergence of drug-resistant malaria to chloroquine in 1950s and sulfadoxinee pyrimethamine in 1960s occurred from western cambodia to the greater mekong subregion (gmsr, including cambodia, lao, myanmar, thailand, and vietnam) and to africa. the finding of artemisinin-resistant malaria in cambodia and gmsr raised a concern regarding the global spread of these parasites. while a number of studies, including population genetics and laboratory-based investigations were conducted, no reliable molecular marker was identified until the major breakthrough reported in early 2014. 78 clinical artemisinin resistance has been defined as a reduction of parasite-clearance rate, which is expressed as an increase of parasite-clearance half-life, or a persistence of microscopically detectable parasites 3 days after artemisinin-based combination therapy (act). although artemisinin was thought to have broad-stage specificity against malaria throughout the life cycle, it was showed that artemisinin-resistant parasites only had decrease of artemisinin susceptibility at ring stages, which was demonstrated by the ring-stage survival assay (rsa 0e3 h ). 79 an in vitro laboratory-based approach was conducted at a time when populationbased genome-wide association studies (gwas) did not clearly identify the genes responsible for artemisinin resistance. 78 for 5 years, an artemisinin-resistant f32-art5 parasite line was selected by culturing an artemisinin-sensitive f32-tanzania clone under a dose-escalating, 125-cycle regimen of artemisinin. eight mutations in seven genes were eventually selected from the result based on whole-genome sequence analysis f32-art5 and f32-tem (its sibling clone cultured without artemisinin) at 460ã� and 500ã� average nucleotide coverage, respectively. to examine whether these in vitro selected mutations were associated with artemisinin resistance in cambodia, sequence polymorphism in all seven genes were analyzed from 49 culture-adapted clinical isolates related to their rsa 0e3 h . only polymorphisms of a gene, k13-propeller, showed a significant association with rsa 0e3 h survival rates. in total, four mutant alleles, each harboring a single nonsynonymous snp (y493h, r539t, i543t, and c580y) within a kelch repeat of the c-terminal k13-propeller domain were identified. to confirm that k13propeller polymorphism is a molecular marker of clinical artemisinin resistance, parasite-clearance half-lives in patients were correlated with their k13 alleles. of the 150 patients, 72 carried parasites with a wild-type allele and the others carried parasites with only one of the three single nonsynonymous snps in the k13propeller: c580y (n â¼ 51), r539t (n â¼ 6), and y493h (n â¼ 21). the parasiteclearance half-life in patients with wild-type parasites is significantly shorter (median 3.30 h) than those with these three mutant alleles (median 6.28e7.19 h). subsequently, clinical studies have validated the association between k13 propeller mutations and artemisinin resistance. 80e82 early mathematical model for malaria control suggested that the most vulnerable element in the malaria cycle was survivorship of adult female mosquitos. 83, 84 therefore, insect control is an important part of reducing transmission. the use of ddt as an indoor residual spray in the global malaria eradication program from 1957 to 1969 has reduced the population at risk of malaria to about 50% by 1975 compared with 77% in 1900. 83, 85 engineering genetically modified mosquitoes refractory to malaria infection appeared to be an alternative approach, 86 given the environmental impact of ddt and the emergence of insecticide-resistant insects. the vector biology network (vbn) was formed in 1989 and had proposed a 20-year plan with the who in 2001 to achieve three major goals: (1) to develop basic tools for the stable transformation of anopheline mosquitoes by the year 2000, (2) to engineer a mosquito incapable of carrying the malaria parasite by 2005, and (3) to run controlled experiments to test how to drive the engineered genotype into wild mosquito populations by 2010. 87e89 while some proof-of-concept experiments have been achieved for the first two aims in 2002 when the a. gambiae genome was completely sequenced, 90,91 the progress has been relatively slow. 92 genomic loci of the a. gambiae responsible for p. falciparum resistance have been identified through surveying a mosquito population in a west african malaria transmission zone. 93 a candidate gene, anopheles plasmodium-responsive leucine-rich repeat 1 (apl1) was discovered. subsequently, other resistant genes have also been identified. 94, 95 studying the genetic basis of resistance to malaria parasites and immunity of the mosquito vector will be important to control malaria transmission. 96 perhaps the most immediate impact of a completely sequenced pathogen genome is for infectious disease diagnosis. the information may be of great importance to the public health when a newly emerged or reemerged pathogen is discovered. a few examples will be described. a novel swine-origin influenza a virus (s-oiv) emerged in the spring of 2009 in mexico and subsequently was discovered in specimens from two unrelated children in the san diego area in mid-april 2009. 97, 98 those samples were positive for influenza a but negative for both human h1 and h3 subtypes. the complete genome sequence and a real-time pcrebased diagnostic assay were released to the public in late april. the outbreak evolved rapidly and who declared the highest phase 6 worldwide pandemic alert on june 11, 2009. s-oiv has three genome segments (ha, np, and ns) from the classic north american swine (h1n1) lineage, two segments (pb2 and pa) from the north american avian lineage, one segment (pb1) from the seasonal h3n2, and most notably, two segments (na and m) from the eurasian swine (h1n1) lineage. 98 with the available influenza genome database, diagnostic assays to distinguish previous seasonal h1n1, h3n2, and s-oiv can be easily accomplished. 99 a comprehensive pathogen genome database is not only useful for infectious disease diagnosis but also for novel pathogen discovery. 100 homologous sequences within the same family or among different family members are important for new pathogen identification even with the advent of third generationesequencing technology. 101 de novo pathogen discovery may also be complicated by coexisting microorganisms, such as commensal bacteria in the human body. without prior knowledge of these microorganisms, one may be misled. in 2003, a microarray-based assay, designated virochip, was used to help discover the sars conoronavirus. 102 the virochip contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in genbank. the computational search for conservation was performed across all known viral families. a microarray hybridized with a reaction derived from a viral isolate cultivated from a sars (severe acute respiratory syndrome) patient revealed that the strongest hybridizing array elements belong to families astroviridae and coronaviridae. alignment of the oligonucleotide probes having the highest signals showed that all four hybridizing oligonucleotides from the astroviridae and one oligonucleotide from avian infectious bronchitis virus, an avian coronavirus, shared a core consensus motif spanning 33 nucleotides. interestingly, it had been known previously through bioinformatics analyses that this sequence is present in the 3 0 utr of all astroviruses, avian infectious bronchitis virus, and an equine rhinovirus. 103 therefore, a new member of the coronavirus was identified through the unique hybridizing pattern and subsequent confirmations. the finding of the seventh human oncogenic virus, merkel cell polyomavirus (mcv) 104 in 2008 is another example of why conserved sequences are important for novel pathogen discovery. mcv is the etiological agent of merkel cell carcinoma (mcc), which is a rare but aggressive skin cancer of neuroendocrine origin. two cdna libraries derived from mcc tumors were subjected to high-throughput sequencing by a next-generation roche/454 sequencer. nearly 400,000 sequence reads were generated. the majority (99.4%) of the sequences derived from human origin were removed from further analyses. only one of the remaining 2395 cdna was homologous to the t antigen of two known polyomaviruses. one additional cdna was subsequently identified to be part of the mcv sequence when the complete viral sequence was known. later analyses showed that 80% (8/10) of the mcc had integrated mcv in the human genome. monoclonal viral integration was revealed by the patterns of southern blot analysis. only 8e16% of control tissues had low copy number of mcv infection. in 2015, an interesting and unexpected discovery of the malignant transformation of hymenolepis nana, a human tape worm, in a human host has been reported by conventional and next generationesequencing approaches. 104a initially, examination of a 41-year-old hiv-infected man revealed extensive lymphadenopathy. h. nana eggs and blastocystis hominis cysts were found in stool. the disease progressed to death despite antiparasitic and antiretroviral treatment. histological examination of biopsied lymph nodes revealed proliferative cells with overt malignant features. they were monomorphic with morphologic features characteristic of stem cells (a high nucleus-to-cytoplasm ratio). however, the small cell size (<10) suggested infection with an unfamiliar, possibly unicellular, eukaryotic organism. infection with a plasmodial slime mold rather than h. nana was considered because of the prominent syncytia formation and the primitive appearance of the atypical cells but lack of architecture identifiable as tapeworm tissue. pcr screening suggested that these cells were h. nana. next generationegenome sequencing and comparative analysis revealed h. nana variants harboring mutations typically found in cancer. as of 2016, next generationesequencing technologies are gradually being applied for diagnosis and monitoring of infectious diseases, including genotypic resistance testing, direct detection of unknown disease-associated pathogens without culture, investigation of microbial population diversity in the host, and strain typing. 105 however, promising, next generationesequencing approaches for clinical diagnosis require further improvements for automation, standardization of technical and bioinformatic procedures, and other practical issues, such as costs and turnaround time. while we can expect that the efforts of a variety of genome projects may improve human health, the socioeconomic issues that are not discussed in this chapter may be substantial. in addition, the tremendous amount of information derived from these projects will also pose a challenge for scientists as well nonscientists to follow and understand. the human genome project: past, present, and future a turning point in cancer research: sequencing the human genome mapping and sequencing the human genome mapping our genesdgenome projects: how big? how fast understanding our genetic inheritance, the u.s. human genome project: the first five years: fiscal years 1991e1995 the $1,000 genome nucleotide sequence of bacteriophage phi x174 dna the genome of simian virus 40 complete nucleotide sequence of sv40 dna dna sequence and expression of the b95-8 epstein-barr virus genome whole-genome random sequencing and assembly of haemophilus influenzae rd hisotry of microbial genomics microbial genome program mutation of the pik3ca gene in ovarian and breast cancer life with 6000 genes the genomes on line database (gold) in 2009: status of genomic and metagenomic projects and their associated metadata the genomes online database (gold) v.5: a metadata management system based on a four level (meta)genome project classification genome sequence of the human malaria parasite plasmodium falciparum the genome sequence of the malaria mosquito anopheles gambiae funding for malaria genome sequencing the genome sequence of trypanosoma cruzi, etiologic agent of chagas disease the genome of the african trypanosome trypanosoma brucei the genome of the kinetoplastid parasite, leishmania major the genome of the blood fluke schistosoma mansoni the schistosoma japonicum genome reveals features of host-parasite interplay helminth genomics: the implications for human health eupathdb: a portal to eukaryotic pathogen databases genome sequence of aedes aegypti, a major arbovirus vector genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle highly evolvable malaria vectors: the genomes of 16 anopheles mosquitoes genome of rhodnius prolixus, an insect vector of chagas disease, reveals unique adaptations to hematophagy and parasite infection genome sequence of the tsetse fly (glossina morsitans): vector of african trypanosomiasis vectorbase: a data resource for invertebrate vector genomics genomic resources for invertebrate vectors of human pathogens, and the role of vectorbase tick genomics: the ixodes genome project and beyond the genome gets personalealmost human genetics of infectious diseases: between proof of principle and paradigm a plan to capture human diversity in 1000 genomes race against time large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution characterization of the 1918 influenza virus polymerase genes microbial ecology of the gastrointestinal tract the meaning and impact of the human genome sequence for microbiology bacterial community variation in human body habitats across space and time diversity of the human intestinal microbial flora a core gut microbiome in obese and lean twins microbiology learning about who we are human microbiome project c. structure, function and diversity of the healthy human microbiome mechanisms of avoidance of host immunity by neisseria meningitidis and its effect on vaccine development an igg monoclonal antibody to group b meningococci cross-reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissues effect of outer membrane vesicle vaccine against group b meningococcal disease in norway vaccine against group b neisseria meningitidis: protection trial and mass vaccination results in cuba phase ii meningococcal b vesicle vaccine trial in new zealand infants efficacy, safety, and immunogenicity of a meningococcal group b (15:p1.3) outer membrane protein vaccine in iquique, chile. chilean national committee for meningococcal disease identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing effect of sequence variation in meningococcal pora outer membrane protein on the effectiveness of a hexavalent pora outer membrane vesicle vaccine complete genome sequence of neisseria meningitidis serogroup b strain mc58 a universal vaccine for serogroup b meningococcus vaccinology in the genome era lessons from reverse vaccinology for viral vaccine design discovery of clostrubin, an exceptional polyphenolic polyketide antibiotic from a strictly anaerobic bacterium discovery of phosphonic acid natural products by mining the genomes of 10,000 actinomycetes thoughts and facts about antibiotics: where we are now and where we are heading biosynthesis of phosphonic and phosphinic acid natural products type ii fatty acid synthesis is not a suitable antibiotic target for gram-positive pathogens identification of critical staphylococcal genes using conditional phenotypes generated by antisense rna global transposon mutagenesis and a minimal mycoplasma genome antibacterial targets in fatty acid biosynthesis the application of computational methods to explore the diversity and structure of bacterial fatty acid synthase a triclosan-resistant bacterial enzyme a new mechanism for anaerobic unsaturated fatty acid formation in streptococcus pneumoniae molecular basis of triclosan activity inhibiting bacterial fatty acid synthesis mycobacterium tuberculosis platensimycin is a selective fabf inhibitor with potent antibiotic properties essentiality of fasii pathway for staphylococcus aureus identification of a universal group b streptococcus vaccine by multiple genome screen a molecular marker of artemisinin-resistant plasmodium falciparum malaria reduced artemisinin susceptibility of plasmodium falciparum ring stages in western cambodia spread of artemisinin resistance in plasmodium falciparum malaria genetic architecture of artemisinin-resistant plasmodium falciparum spread of artemisinin-resistant plasmodium falciparum in myanmar: a cross-sectional survey of the k13 molecular marker malaria management: past, present, and future the epidemiology and control of malaria the global distribution and population at risk of malaria: past, present, and future possible use of translocations to fix desirable genes in insect pest populations from tucson to genomics and transgenics: the vector biology network and the emergence of modern vector biology the mosquito genomeea breakthrough for public health malaria control with genetically manipulated insect vectors stable germline transformation of the malaria mosquito anopheles stephensi transgenic anopheline mosquitoes impaired in transmission of a malaria parasite malaria control with transgenic mosquitoes natural malaria infection in anopheles gambiae is regulated by a single genomic control region leucine-rich repeat protein complex activates mosquito complement in defense against plasmodium parasites dissecting the genetic basis of resistance to malaria parasites in anopheles gambiae mosquito defenses against plasmodium parasites swine influenza a (h1n1) infection in two childrenesouthern california, marcheapril emergence of a novel swine-origin influenza a (h1n1) virus in humans detection in 2009 of the swine origin influenza a (h1n1) virus by a subtyping microarray a technological update of molecular diagnostics for infectious diseases third-generation sequencing fireworks at marco island viral discovery and sequence recovery using dna microarrays a common rna motif in the 3' end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus clonal integration of a polyomavirus in human merkel cell carcinoma malignant transformation of hymenolepis nana in a human host next-generation sequencing for infectious disease diagnosis and management: a report of the association for molecular pathology database resources of the national center for biotechnology information ensembl genomes: extending ensembl across the taxonomic space the comprehensive microbial resource the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents genomic metadata for infectious agents, a geospatial surveillance pathogen database a catalog of reference genomes from the human microbiome the influenza virus resource at the national center for biotechnology information key: cord-320988-yjxbm4tn authors: correa, m.t.; grace, d. title: slum livestock agriculture date: 2014-08-21 journal: encyclopedia of agriculture and food systems doi: 10.1016/b978-0-444-52512-3.00161-3 sha: doc_id: 320988 cord_uid: yjxbm4tn slums are unplanned squatter human settlements in peri-urban and urban areas where more than 800 million people live. these densely populated areas lack basic public services. livestock raised in these conditions compete with humans for space and water, and pose a risk to human and animal health. notwithstanding the risk of disease transmission, slum livestock agriculture plays an essential role in the livelihoods of people and deserves consideration in urban planning and policy making. slums are human settlements developing in peri-and urban areas. often not recognized as part of planned urban environments, slums continue to grow in number and size around the world. there are now 800 million to a billion people living in slums, a large percent of the seven billion worldwide. slums are difficult to define based on a single characteristic because they are not homogeneous within cities in a country or among countries; their unique milieu depends on the geographical location of the settlement, the socioanthropological background of the dwellers, and the history of its development. notwithstanding the sociocultural differences, slums have some common characteristics: poor housing, often illegitimately built on private or public land with poor drainage and unfit for agriculture; overcrowded conditions; limited access to potable water; poor sanitation and lack of sewage or waste removal; high numbers of domestic pets; and clandestine keeping of livestock. slums originate from the need of people for a place to live after displacement due to drought, famine, or wars, and migration from the countryside into the cities by the push of rural unemployment and the pull of urban opportunities. this migration from rural to urban areas populates informal settlements in the periphery of cities and towns in many countries (united nations, 2011) . demographic information from slums is only as good as the reports governments make. less than 100 countries report data on slum human population, and from those, 37 indicate they have more than 50% of the population living in slums. one country reported that 94% of the population lives in slum conditions (united nations, 2007) . in the 2013 census in india, 65 million people were identified as living in slums (government of india, 2013) , which is probably an underestimate. in lesser developed countries, the increase in the urban population dating from the 1970s is parallel to the decrease of the rural population. demographers expect that 60% of the world's population will live in urban areas by the year 2050 and 2 billion people will be living in slums (united nations, 2011) . in africa, the population in urban areas increased sevenfold in 50 years. population density in some slums has reached 200 000 people per square kilometer, as in bangladesh (huque, 2005) . with one in every seven people living in a slum, ensuring sufficient food becomes a concern; slum livestock agriculture could be part of the solution to fulfill the nutritional needs of that many people (united nations human settlements programme, 2003) . production of food in slums is clearly different from rural, peri-urban, or other types of urban agriculture developing in many industrial countries. urban agriculture had been characterized as a decentralized supply system composed of small and scattered production units (mougeot, 2000) . there are other classifications of urban agriculture, only some of which include slum agriculture (egbuna, 2008) . slum livestock keeping is especially difficult to classify. the lack of basic resources to maintain and exercise good agricultural practices coupled with unsanitary slaughter is likely to render slum livestock agricultural products unfit for human consumption. however, despite all the negatives, slum livestock agriculture is a means of securing food, earning income, and supporting livelihoods of slum dwellers. slum agriculture benefits from a large market close at hand, low transportation costs, availability of production inputs, the ability to make use of waste foods and water, and low entry costs to setting up production (food and agriculture organization, 2001) . informal markets for real estate and goods develop in slums, including markets for processed and unprocessed animal products (potsiou and ioannidis, 2007) . peri-urban and urban livestock agriculture is a complex and multifaceted activity developing in an environment often unfit for human habitation. roaming chickens, pigs, and goats are a common sight in slums, and even cows and animals used for transportation or hauling such as donkeys and horses can be seen. in different regions of the world, different animal species are popular: goats and sheep are more common in africa and southeast asia, and pigs are more common in latin american slums. food animals in slums are a public health concern due to their potential for transmitting zoonotic diseases, unsafe food products, the risk of physical injuries and traffic accidents, and environmental contamination. animal products processed locally enter the general population food chain through peri-urban and urban food markets. owing to the lack of a proper waste disposal, animal and human waste mixes in the streets with leftover water from household activities. stagnant water and waste, garbage, and other contaminants develop a foul smell and attract vermin and insects. untreated wastewater flows into local waterways and is drunk by roaming animals. this gray water is often used for urban vegetable or crop farming. there are few estimates of the volume of animal or vegetable products originating from slums used to supplement the family diet or entering the food chain through peri-urban or urban markets. in ghana, 95% of the lettuce consumed in accra is produced in urban and peri-urban areas (amoah et al., 2007) . in the chimbote slum in lima, peru, where 400 000 people live, it has been estimated that there are 280 swine production units (chimbotenlinea.com, 2014) . that is approximately 25% of the households in the slum assuming there are four members per household. the food and agriculture organization of the united nations has conducted technical consultations and extensive studies on urban and peri-urban agriculture. thirty percent of the meat and 70% of eggs produced in the world is associated with urban production (food and agriculture organization, 1999) . it is not clear if slum agriculture production is included in these estimates. slum livestock keeping is typically smaller scale than the peri-urban and nonslum urban agriculture. however, slum agriculture has rarely been the focus of systematic evaluations and studies. the complexity of this type of slum livestock agriculture and its implications in the social and political arena is, however, getting recognition (international livestock research institute, 2012; lemma and rao, 2013) , and some evidence is emerging on scale and importance. in maputo (1.2 million inhabitants), mozambique, more than one in three households in urban areas raises livestock (international livestock research institute, 2013) . in ibadan in southern nigeria, one in three urban households kept livestock, whereas in kaduna in northern nigeria nearly half the households kept livestock (international livestock research institute, 2013) . livestock production in slums is typically small to medium scale and low technology. chickens and pigs seem to be ubiquitous in latin american slums, whereas goats and rabbits are favored in africa and southeast asia. cows are noticeable in india and nepal where, for religious reasons, they cannot be slaughtered. some animals in slums are allowed to roam and scavenge whereas others are confined in unsuitable small spaces shared with humans. the scale of this agricultural activity varies in type and size and it can range from subsistence to small-to medium-size commercial enterprises with hundreds of small animals. in more densely populated slums, fewer animals are kept and enterprises are likely to be small scale; where more land is available, livestock keeping is more common and on a larger scale (box 1). chickens are animals most commonly raised in slums. one or two hens and one rooster roaming free with a few chicks is a common sight. it is not uncommon to find medium (10-30 box 1 livestock keeping in dagoretti district, nairobi, kenya dagoretti district lies to the west of nairobi city, 12 km from nairobi city center. it is one of 8 urban districts in nairobi and has around onetenth of the total population. most inhabitants live in tin-roofed timber houses with inadequate access to piped water, sanitation, and electricity. unemployment (approximately 60%), human immunodeficiency virus-acquired immune deficiency syndrome, crime, and homelessness are persistent problems. dagoretti was originally outside the city of nairobi, but when the boundaries were extended in 1963, it became part of the city. dagoretti district has both government and ancestral land. some ancestral land has been sold to migrants, and government land has also been allocated to individuals. in some areas, squatters live on land temporarily leased out by landlords, a loose agreement, which can be revoked at any time. the population in the last available census was 240 509 up from 41 409 in 1969, a vivid illustration of the rapid growth of cities. of the population, 42% are below 19 years of age, an age distribution that is also typical of developing countries. a recent survey characterized livestock keeping in dagoretti. more than half the households kept animals: in order of declining popularity: dogs (39%); poultry (39%); cats (37%); sheep and goats (14%); pigs (10%); and cattle (1%). a few households kept other animals including, rabbits, donkeys, doves, turkeys, ducks, and geese. dogs were kept mainly for security, cats for vermin control, donkeys for work, geese for security and food production, and the other species were kept mainly for food production. in addition, cattle manure is a valuable by-product used for urban crop and vegetable production, and livestock are an easily liquidated asset that can be sold to meet urgent household needs or act as pledge enabling access to credit. moreover, farmers report deriving social and psychological benefits from livestock keeping. the study focused on dairy cattle kept by 1 in 80 households in dagoretti. farmers kept an average of three cattle. nearly all households kept productive breeds, used artificial insemination, and zero grazing. farms produced approximately 12 l of milk a day, and 88% of this was sold with the rest being consumed in the household. most of the milk produced is sold in the community through informal markets. all sales of milk earn farmers in dagoretti approximately us610 000 per year. farmers reported that both demand and production are increasing. chickens) to larger layer flocks (up to 100 chickens) in confined spaces. chickens are the most versatile of poultry, given the value added of egg production and the worth of the animal, live or processed. in slums with limited electricity or where the cold chain is not available, chickens and eggs are a quick and easy source of protein and income generation. chickens have an additional value because they are used in religious ceremonies and offerings (alves et al., 2009) . the breeds of chickens vary from region to region and some commercial breeds are seen in slums. scavenging chickens are fed in the morning with leftover food and possibly with added corn or poultry meal. during the day, chickens eat insects, garbage, and organic matter found in empty lots or by the side of the road. housing is rudimentary and may consist of hay or rice husks arranged in cardboard or wooden boxes and placed off the ground for protection from dogs and predators mostly during the night. raising chickens is considered an easy activity requiring minimal labor and is usually performed by women and children (food and agriculture organization, 2001). backyard flocks requiring additional attention compete for space with humans and other animals and are more laborintensive requiring feeding, watering, and pen cleaning. noise levels and manure disposal become a problem with larger flocks and are a deterrent for keeping them in small and crowded spaces. larger flocks are more common in peri-urban areas than slums, but broiler chickens keepers may have more than 100 birds, as in the case in the mombasa slum in bangladesh where a bird keeper is reported to have between 125 and 250 birds (sabuni, 2010) . live chickens are transported in cages or burlap bags to nearby food markets. these markets are located at walking distance from the dwelling where the animals are raised, although sometimes birds are transported in cages on top of buses or trucks to other urban markets. chickens are sold live or slaughtered on site at the buyer´s request. eggs have a longer shelf life (lasting 3-4 weeks without refrigeration depending on environmental conditions) than poultry meat. eggs are wrapped in newspaper and kept in the shade to prolong their shelf life. they may be sold to neighbors in small numbers, as needed, for income generation or in larger numbers at markets. other poultry kept in slums include turkeys, geese, doves, pigeons, quail, and guinea fowl. rabbits are gaining popularity in slums in africa. they can be kept in small spaces in crates, either individually or in small groups. rabbit crates are easy to build and can be kept stacked up against each other or leaned against a wall to occupy less space. these animals require more attention than scavenging or backyard flocks of chickens for feeding, watering, and cage cleaning. however, it is a profitable and uncomplicated business as evidenced by the number of rabbits a person can keep. one resident in the kahawa soweto slum in nairobi has more than 400 rabbits in a shed (kelto, 2013) . rabbits are fed greens collected locally and supplemented with vegetable leftover from markets or pelleted food. this is a small species that can reach to 4 kg of weight and can easily be processed locally as chickens for sale or family consumption. in a space of 30 â 40 feet, slum residents can raise more than 500 rabbits of different breeds. more unusual animals are also kept: a survey in nigeria reported snails, grass cutters, and antelope among a total of 14 species kept. pigs adapt easily to urban conditions. one sow can produce 6-12 piglets twice or three times a year if their reproductive cycle is timed properly (pregnancy lasts 3 months, 3 weeks, and 3 days). selling the piglets before birth is not uncommon at certain times of the year for events or festivities where pork is commonly consumed. pigs are considered a form of saving account because piglets can be sold for cash or given in exchange for the boar´s service (the piggy bank). boars can grow tusks and become very large and aggressive, requiring confinement to protect people and other animals. this is why fewer people keep boars compared to sows. rearing of pigs in slums in small numbers (one to five) is often done by women and larger herds by men (food and agriculture organization, 2001) . piglets are easy to manage because they can roam during the day searching for food and come back to their home space at night. they can also be tethered and kept close to the dwelling, or housed in makeshift pens behind or in the confines of the human dwelling. nowadays pigs are ubiquitous to slums in latin america and present even in countries with muslim and hindu population such as india and nepal (rajshekhar, 2004) . it is difficult to establish the genetics of pigs commonly reared in slums. these are smaller and more adaptable to the local conditions than large commercial breeds. pigs revert to wild type very quickly and grow hair and tusks adapting to the environment. however, pigs kept in confined spaces in and small numbers are usually larger commercial breeds requiring more attention and feeding. the creole pig from haiti was an example of a small hairy dark pig well adapted to the environment, eating garbage and fallen rotten fruit. they had sociocultural value and were considered to have an important role in recycling waste. issues associated with swine fever in the late 1970s and early 1980s prompted the united states department of agriculture and other international organizations to develop and conduct an eradication program. the sociocultural value of these animals was not clearly understood. people were given large commercial breeds as substitutes for the creole pig and asked to pen the pigs. the larger breeds had to be fed and cared for and were not allowed to roam, which resulted in detrimental conditions of the environment in which people lived (ebert, 1985) . goats are adaptable to urban environments. they are known for eating almost anything, including bark from trees and shrubbery, garbage from streets and land fields, or clothes hanging to dry. these animals require attention, given their inquisitive nature and destructive eating habits. usually people keep a couple of female goats for their milk with the offspring. in slums close to peri-urban areas people keep the goats in pens or house patios during the night and walk them to pasture at different locations during the day. in the urban environment, and in particular in slums, goats roam the streets in search of food, and it is common to see them searching for food with pigs (diogo et al., 2010) . goats can decimate green areas very quickly, causing damage to vegetables growing by the side of the roads or public open spaces. they require more attention than chickens or pigs and they need shelter from the rain. goats' milk is nutritious and can be easily transformed into yogurt and cheese to extend the shelf life of an otherwise perishable product. in some large slums such as in delhi, india, slum goats' sightings have become a tourist attraction and are offered as part of city tours (news.com.au, 2013) . mixed bos taurus taurus and bos taurus indicus cattle are let free in search of food or water or walked to pasture in the mornings and brought back at night. in countries with a predominantly hindu population cows are sacred and only the milk is consumed. oxens are mainly kept for draught power; they are used for plowing and also transport. owing to religious reasons female cattle cannot be disposed of after their productive lives have ended. older unproductive animals may be abandoned and may die of diseases, hunger, intoxication from garbage, or get involved in traffic accidents. there are also many goshalas in india where abandoned cattle are looked after. milk is a valued product that contributes to food security in slums. but it is perishable and has to be consumed quickly or transformed into a variety of cheeses, yogurt, or sour milk. naturally fermented sour milk is popular in east and west africa; fermenting also removes lactose, which makes it easier to digest for populations that do not have lactase enzymes persisting in adulthood. tending larger animals is time consuming and may involve walking long distances to find a place to feed away from the city or to cut grass that can be brought back to the animal. this is easier to do when the slum is located in the periphery of a city compared to an urban-area slum. often cattle are confined to a small space or their mobility may be limited by tethering them close to the dwelling. studies of peri-urban dairying in east africa found that nearly all cattle were 'zero grazed,' that is, kept inside all the time (figure 2) . cow dung in india is also a valuable fuel: it is collected, reshaped into cake-like forms, and dried. in india and elsewhere cattle dung is also used as a building material mixed with mud and other substances. livestock value is more than monetary; it is a symbol of social status and social identity both individually and collective (comaroff and comaroff, 1990) . the value of livestock and the production parameters used elsewhere (such as in the industrial countries) need to be revaluated because a cow or goat whose production is low may still be providing useful services (dutta, 2003) . in slums, an intricate relationship exists between poverty, food security, livestock keeping, and the environment. enforcing livestock regulations becomes a difficult task when animal ownership is clandestine. in many countries regulations cover animal health and welfare issues, the disposal of dead animals, slaughter procedures, and environmental contamination, but in practice they are not enforced in slums. in other cases, regulations are used for 'rent-seeking' by authorities: that is police or other officials will confiscate animals and not give them back unless a bribe is paid. keeping animals in cities undoubtedly creates environmental problems. for example, in kisumu, kenya, keeping animals is illegal. the city has six slums and enforcing animal farming law is a challenge. the dung of the animals becomes a problem when the rain washes it and contaminates the city water supply. the city lacks the infrastructure to recycle the dung and 75% of it is not utilized (new agriculturist, 2006 ). yet the draconian response of banning livestock from cities may not be the most appropriate. in kampala, uganda, a process lasting several years has recently led to new city ordinances that seek to support urban agriculture as an important economic activity, while regulating against the potential adverse effects. animals of the same or different species in slums are in proximity with each other and with humans. contagious diseases can spread rapidly under these conditions. additionally, slum-raised animals suffer from malnutrition coupled with an adverse environment that makes them more susceptible to disease or injury (food and agriculture organizations, 2011). some problems are associated with animals living in areas with poor waste disposal. pigs and goats eat plastic bags and these are common findings at slaughter or necropsy. the bags fill the stomachs impeding food digestion and nutrient absorption. rwanda banned nonbiodegradable plastic bags in 2008, an excellent example for other african countries where plastic waste is ubiquitous. the health of animals in slums requires attention, but estimating disease and its impact is difficult. both zoonotic (diseases transmitted from animals to people) and nonzoonotic animal diseases need to be considered. reportable, zoonotic, and production diseases are discussed elsewhere in this encyclopedia. some of these diseases are of national public health interest or are of international trade importance requiring immediate reporting through specific official public health channels and to the world organisation for animal health, one of the three sisters of the world trade organization. reportable diseases of animals are those with the potential to cause epidemics or pandemics that cause serious disease in people, as is the case of the severe acute respiratory syndrome known as sars, the h5n1 (pathogenic avian), and h1n1 influenza virus (known as swine influenza) (gauthier-clerc et al., 2007; girad et al., 2010) . public health resources and market inspections are deployed for some of these diseases, but control is often ineffective in poor countries. determining the public health penetration of these programs in slums has been highlighted as a need (unger and riley, 2007) . risk communication in a crisis situation involving the livestock population in a slum may require additional resources and targeted programming. the lack of information of the size and location of the avian and swine population may impede the progress of disease control measures. public awareness programs or the application of rigid standards for disease control without consideration of the sociocultural and economic value of the animals for people may also decrease compliance (box 2). references to the 1920 flu pandemic are always brought to light in the media as the possible serious consequences of disease spread and death. however, public health crisis response is different today and includes emergency and disaster preparedness, first responders training programs, and damage mitigation and relief. nonetheless robust economic estimates by the world bank suggest that the cost of an epidemic originating in animals could be a trillion dollars (world bank, 2012). there are approximately 1000 zoonoses or diseases transmitted from animals to humans. disease transmission may be direct through contact with infected animals or its fluids, or through contaminated animal products, water, or objects contaminated with infectious material (center for disease control and prevention, cdc 24/7, 2011). especially important for poor countries are the so-called neglected zoonoses often associated with poverty, poor hygiene, and poor understanding of disease transmission. the rest of the article discusses some neglected zoonoses that are known or likely to be a problem in slums. brucella melitensis, the cause of brucellosis, is very pathogenic to humans (center for disease control and prevention, 2012). it is transmitted from goats and sheep to humans through direct or indirect contact. unpasteurized milk or contact with body fluids of infected animals are considered the most common transmission routes. there are other brucellae affecting swine, cattle, and dogs, but melitensis is associated with the more severe forms of the disease. flaying and skinning sheep by blowing air through a cut in the skin exposes people to body fluids and inhalation of bacteria from infected animal, and hence is not recommended. cysticercosis is a disease caused by the larval stage of the tapeworm taenia solium. the disease is sometimes wrongly known as the 'the pig tapeworm,' but humans are the definitive host for the adult form of the parasite, not the pig. the adult parasite releases segments daily containing thousands of eggs into the gut. these eggs pass into the environment with the feces. humans and pigs become infected and develop cysticercosis by ingesting the parasite eggs. pigs are coprophagic and human defecation in open spaces is one of the main forms of transmission of the disease from humans to pigs. when eaten by pigs, the eggs develop into cysts within the pig (figure 1 ). if people eat meat containing viable cysts these can develop to tapeworms in the human host, thus completing its cycle. human to human transmission occurs when people harboring the parasite contaminate food due to poor hand hygiene (garcia et al., 2001; carrique-mas et al., 2001) . autoinfection can also occur following improper hand washing after a bowel movement. after humans consume eggs, cysts start to develop in any organ of the body, including the brain. when cysts develop in the brain, the disease is known as neurocysticercosis. it is considered as one of the most common infestations of the brain in humans. the main manifestation of the disease is epilepsy (garcia et al., 2001) . studies from a slum in india revealed that 25.5% of people in the studied sample who had active epilepsy had antibodies to t. solium (singh et al., 2012) . cysticercosis in pigs may reach 30% prevalence or more in some countries where pigs are allowed to roam and human defecation is done in the open (carrique-mas et al., 2001; fleury et al., 2012; flisser et al., 2003) . even with low human taeniasis in the order of 1-2%, the prevalence of cysticercosis in pigs can be high. this is mainly due to the fact that only adult parasite can live in the intestine of the host for years and release thousands of eggs into the environment. hepatitis e virus is an enteric disease transmitted from different species of animals including pigs to humans; it can be transmitted between humans through contaminated water and food. the seroprevalence in the human population unexposed to swine is 2% whereas the seroprevalence in swine workers or other swine-exposed populations is 10% (whithers et al., 2002) . data from a study of a pediatric population in karachi with access to municipal piped water and nonflush toilets in a slum in india showed a high seroprevalence for hepatitis e virus (jafri et al., 2013) . the exposure to hepatitis e virus seems lower than hepatitis a (mohanavalli et al., 2003) . hepatitis e is a disease that can cause severe gastrointestinal symptoms and has been associated with high death rates of pregnant women in lesser developed countries. in some peri-urban farms pigs are raised close to ponds where fish are kept. the pig feces are washed down to the ponds for the fish to eat. the virus seems to be ubiquitous in swine populations and undistinguishable even in samples from different countries. this was the case in hepatitis e study in two farming communities (not slum conditions), in north carolina and costa rica (whithers et al., 2002; kase et al., 2008) . hepatitis e is a zoonosis, but the importance of animals in its maintenance and transmission has not been fully established. a pandemic of h1n1 influenza was declared in 2009. this disease originated in pigs but subsequently was maintained entirely by human to human transmission. in response to the pandemic, the government of egypt ordered all of the country's pigs to be slaughtered. because humans can only get the new flu from other humans this was not effective as a control response. it also had far-reaching and unintended consequences on the cities' waste management. cairo's 30 000 garbage collectors used to feed the city's organic waste to pigs and their livelihood became endangered while the streets of the capital filled up with trash. source: adapted from the new york times article, mona el-naggar contributed reporting, 19 september 2009. available at: http://www. nytimes.com/2009/09/20/world/africa/20cairo.html_r=2&scp=1&sq= michael%20slackman%20cairo%20pigs&st=cse& (accessed 30.01.14). cryptosporidium is associated with cattle and causes diarrheal disease in humans and cattle. although extensively studied in developed countries, it has not been diagnosed until recently in slums. surveyed households in the nairobi´s dagoretti district, kenya, determined the extent of cattle keeping and the prevalence of cryptosporidiosis. with a prevalence of 20% in urban cattle, this parasite could be a major contaminant of water sources and disease that needs further consideration (international livestock research institute, 2012) (figure 2) . antibiotics and other veterinary drugs administered to animals without proper veterinary supervision can have direct and indirect consequences for human health. after administration of an antibiotic there is a period of time when the animal product should not be consumed. this period known as the withdrawal period is to allow the drug in question to clear the animal's system because it may have harmful health effects if consumed by people who are allergic or sensitive to the drug. for example, clenbuterol is commonly used as a growth promoter (often illegally), and several outbreaks of illness have occurred when people consume livestock products from animals treated with clenbueterol. of potentially greater importance than sickness as the result of consumption of residues is the risk of bacteria developing resistance to antibiotics because of their use in agriculture. as a result of these concerns, many countries require that antibiotics and some other animal health drugs should only be prescribed by veterinarians. however, this is not practicable in most slums. veterinary services may be out of reach for many people in slums either because of distance or cost. in some cases, livestock owners may visit a veterinary office and explain the symptoms of the disease to the veterinarian, but without proper examination of the animal and the information received, the medicines may not be appropriately prescribed. the livestock owner may not fully understand how to administer the medicines and use a single dose of a medicament keeping the rest in case the animal does not improve. getting advice from another family member or neighbors, purchasing a single dose of an antibiotic from a street vendor, or using leftover medicines from a previous case is not uncommon. veterinary services are often expensive and out of reach of the poor (ahuja et al., 2003) . in addition, farmers may use drugs for nonhealth purposes. in khartoum, the practice of adding antibiotic to milk to preserve it was reported, and this could have health consequences such as allergy in the people ingesting the product (hassabo et al., 2012) . in china, melamine was added to milk so that it would appear to have higher protein levels. this lead to one of the largest food safety events of recent years resulting in the death of six infants; the children died from kidney stones and thousands were hospitalized as a result of drinking the contaminated milk (wei and liu, 2012) . slum agriculture and associated markets are part of the informal sector of the economy. it is a business model based on necessity, mostly home-based, and often clandestine. when there is no proper access to water and refrigeration, meat and eggs must be consumed rapidly, processed, and sold close to the place of origin. markets in slums are public places where vendors congregate. products may also be sold by hawkers moving from door-to-door or as requested. in many cities in developing countries milk is sold in this way. it is not uncommon to sell products from homes through modified window sills, from makeshift sale racks in corridors between houses, or on top of blankets or plastic sheets in the floor in open spaces. slum dwellers acquire knowledge of who produces and sell different items. prices vary based on perceived notions of quality, hygiene, or availability. the exchange of money is not always the norm for payment and people engage in bartering or exchanging products for services. agricultural markets in slums differ from municipal markets where there is an operational legal framework and the state is responsible for the enforcement of public health and food safety regulations. however, the great majority of food sold in municipal markets lack any structured sanitary inspection. this does not mean that the markets in slums lack internal form of self-regulation, they have a sui generis operational business model. although these markets are outside the purview of state regulation and disease control and preventative measures, the prevailing food-safety actions are based on accepted cultural norms. these norms are developed by the people who live, sell, and purchase products under these conditions or based on religious beliefs or restrictions (rheinländer, 2006) . the number, identification of species, and location and ownership of animals in a slum is not usually performed. the lack of census data and owner identification poses a challenge for the implementation of national disease control or eradication programs. required reporting of diseases and animal or product traceability to origin may be impossible in case of a food-related outbreak. this is contrary to the application of the sanitary and phtyo-sanitary measures of the world trade organization. most countries agree to adhere to these measures when they join the world trade organization. conducting an animal census in a slum is challenging but without knowing how many animals there are, vaccine campaigns or eradication programs cannot be conducted. however, the legally ambiguous position of livestock in slums mean owners are reluctant to incur official notice. the inspection of animal facilities and product transformation industries destined for human consumption is highly regulated. although in many developing countries animals must be processed in municipal slaughterhouses, at home and clandestine slaughter are common. in bolivia, some of the municipal slaughterhouses are rudimentary and managed by the local communities. the official veterinarian is allowed to inspect the animals preslaughter and outside the facility. the inspection concerns mostly reportable diseases of national importance. inside the slaughterhouse, skilled butchers castrate male pigs, and these and other animals bleed on the floor as part of the exsanguination process. once the internal organs are removed the carcass is hung on a wall hook and employees rub the pigs´canal with a rag. the same rag and water stored in a bucket is used to wash several carcasses. an inquiry into the practice revealed the employees neither understood why they were doing the cleaning nor how it was supposed to be done (correa et al., 2003) . in ibadan, nigeria facilities are even less developed. approximately 250-300 cattle are slaughtered daily; more are slaughtered on weekends, and fewer during muslim holidays. cattle are kept in pens, then moved to the slaughter slab. they are tied down at the slab and killed by cutting the throat. the dead cattle are then dragged on the ground to the abattoir area. this is simply a shed with a concrete floor and open sides. removal of the intestines and quartering is done on the floor. portions of the carcass are then carried to the adjacent butchers' stalls where they are sold. the abattoir is under municipal management and officers collect tax and tariffs on each cow amounting to us$1 per animal. the role of environmental sanitary officers is to inspect slaughter slabs and the general environment and ensure the area is clean. however, the filthy conditions of the market witness the challenges they face in carrying out their work. the veterinary department is supposed to check animals before slaughtering and inspect meat after slaughter, but many animals escape inspection, and even when problems are found veterinarians find it difficult to ensure condemned meat is discarded. most butchers kill only one animal a day, and if this is condemned by veterinarians as unfit for human consumption they lose their entire day's earnings. hence, they strongly resist attempts to condemn meat (grace et al., 2012) . slaughter practices are probably clearly specified in pertinent regulations, but without oversight or evaluation by competent authorities, wrongly learned and applied practices may never be corrected. new employees learn from other employees and the practice is passed down, transformed, and perpetuated. the 'trichina inspectors' in livestock markets in bolivia are lay people trained by other people to inspect the tongue of pigs in order to detect cysts associated with cysticercosis. trichina is a different parasite and the tongue inspection is not associated with this parasite, but t. solium. the wrong parasite reference has prevailed and the practice continues unchecked for efficiency in preslaughter diagnosis of cysticercosis in pigs. in ecuador´s countryside, a traveling butcher works at the community slaughterhouses at scheduled days and times of the week and processes the carcasses of mainly large species at the municipal facility. this is a trained person who can condemn carcasses from sick animals and has an important food safety role. however, the most predominant form of slaughter in slums is the one done at home, in the streets, between or behind buildings. usually for smaller species the head is removed using a sharp knife or axe against a hard surface like a piece of wood or the stump of a tree. the internal organs are removed swiftly; sometimes the esophagus and the rectum are tied to avoid intestinal content spillage and contamination. animals processed in this manner are not properly exsanguinated. raw offal may attract dogs and vermin. the meat, without refrigeration must be consumed immediately or it is left to dry on racks. meat maybe carried to markets in small carts, buckets, or burlap bags and sold in small pieces or cut by the client´s request. the meat is not inspected when sold in the slums to neighbors or at smaller slum markets. in nepal, animal parts are sold with other identifiable body parts like a hoof or horns in order to guarantee that the species being sold is the actual species. in many cases the meat is covered in turmeric, reported to be done to decrease contamination (oral tradition reported to authors by street vendors). interestingly, homeopathy studies suggest that turmeric can help combat bacterial infections (krup et al., 2013; vasavda et al., 2013) . slum livestock agriculture interweaves its existence with the geographic location and culture of the population. people take advantage of their new environment and recreate a way of living based on their upbringing, including raising and processing livestock. the main motivations for this activity are family subsistence, income generation, and social status. slum livestock agriculture activities range from subsistence to semicommercial production. it plays a role in the livelihood of millions of people providing animal protein and generating income. however, it lacks technical sophistication and typically operates outside regulatory purview, and its practice overlooked and tolerated by local authorities. animals pose a risk to human health and other animals, are a public nuisance, and contribute to the waste accumulating on streets and the run off contaminating the environment and water sources. livestock products, raw or processed are sold in street markets and may enter the urban food chain. animal ownership is difficult to establish in slums and livestock population difficult to determine. under these circumstances, veterinary services and disease-control programs may be hard to implement. good hygiene, good manufacturing practices, and animal slaughter inspection turn into an impossible task to implement and enforce. disaster and white-coat diplomacy practices combined with information and communication technologies should be considered in the planning and implementation of public awareness and outreach programs (lemery, 2010; lin and heffernan, 2010) . 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(upa) in selected towns of ethiopia creating the livestock guru: icts to enhance livestockrelated knowledge among poor households in orissa prevalence of antibodies to hepatitis a and hepatitis e virus in urban school children in chennai urban agriculture: definition, presence, potentials and risks available at cleaning up its act: recycling livestock waste slum tour in dharavi, mumbai with reality tours and travel informal settlements, real estate market need for good land administration and planning epidemiology of taenia solium taeniasis/cysticercosis in india and nepal street food quality. a matter of neatness and trust broiler chicken market in slums association between epilepsy and cysticercosis and toxocariasis: a population-based case-control study in a slum in india slum health: from understanding to action world urbanization prospects. economic and social affairs united nations human settlements programme pharmacological activities of turmeric (curcuma longa linn): a review review of melamine scandal: still a long way ahead antibody levels to hepatitis e virus in north carolina swine workers, non-swine workers, and murids growing greener cities in africa proper slaughter and flaying of sheep and goat. ethiopia sheep and goat productivity improvement program key: cord-301537-uu2aykoy authors: johnston largen, kristin title: two things can be true at once: surviving covid‐19 date: 2020-05-27 journal: dialog doi: 10.1111/dial.12571 sha: doc_id: 301537 cord_uid: uu2aykoy nan as i sit in california, currently under a "stay at home" order to help stop the spread of the covid-19 pandemic, i have been reflecting on worship, the role of the church in providing healthcare, and our sacramental life. colleagues have asked for my thoughts on liturgy in the midst of a public health crisis, so i have decided to put things on paper so that others may be included in the conversation. many have already published resources on moving worship into the online environment, but a large percentage of those resources have approached it from a practical perspective rather than a theological or historical one. the title of my essay is a riff on luther' s 1527 open letter "whether one may flee from a deadly plague," which has made a resurgence during this health crisis. the letter has always been a favorite of mine: i assigned it during the four years i taught introduction to lutheranism, and it served as a conversation partner in my dissertation on liturgical rites of healing. quotes from the letter have circulated around facebook, especially as luther (near the end of the letter) provides very practical advice during a plague. the main point of the letter is that pastors and city officials are to work together to physically and spiritually care for those affected by disease, and those who are not bound by such responsibility should be free to leave without burdening the conscience. but before we use luther's letter as our urtext for the church's response during covid-19, we must remember that it seems unthinkable for luther (at least in the letter itself) that people would not be able to attend worship during a plague. our situation is different, with local, state, and federal governments providing recommendations and orders to stay home and not gather in groups. our understanding of science is also different, being on this side of the scientific revolution with a better grasp of how germs spread. luther's science is not our science, so that must be considered when reading his specific recommendations on liturgical practices during health crises. on the other hand, the letter reminds us that the church must work alongside civil authorities in preventing a plague from spreading, which means that congregations must follow the orders not to gather. attempting to spiritualize this pandemic as being the will of god-either as punishment or an opportunity to repent--is dangerous. the theological and historical concerns that i raise below are as i interpret our lutheran traditions, drawing on luther and the book of concord. the practical suggestions that i offer that differ from our customary practices are understood to be in extremis-in an emergency like we are facing today. as luther reminds his readers at the beginning of the letter, all christians must "come to their own decision and conclusion" (luther, 1968, p. 119 ). purpose" one thing that must be addressed before reflecting on particular issue is to define worship from a theological perspective or, to use john witvliet's (2006) modes of liturgical discourse, in terms of "deep meaning and purpose." for lutherans the primary theological understanding of worship is as a dialogue between god and humans, or as luther says in his torgau sermon, "where our dear [god] may speak to us through [the] holy word and we respond to [god] through prayer and praise" (luther, 1959, p. 333 ; see also luther, 2000, p. 397.84) . distinct from some other christian traditions, lutheran worship is primarily about god coming to us through the means of grace, which are concrete and external ways that god make godself known in the worship event. these are primarily preaching, the sacraments, absolution, and other liturgical practices. at the same time, lutheran worship also has a participatory response included in it. this is the second half of luther's torgau definition. active participation ("full, active, conscious," as would be articulated four centuries later at the second vatican council) is the other half of the dialogue between god and humans. participation is a diverse thing, just as christ was incarnated into a diverse human reality. the response is the response by the entire congregation, not just that of worship leaders or a select few. i think this should give us pause when we look at livestreaming (rather than web conferencing), as that calls into question the participatory ability of digital worship. unlike some theological traditions, lutherans are under no obligation to go to worship. yet, this question actually misses the point if we attend to our definition of worship-it is through worship that we know who god is and how god operates in the world. it contains external forms so that god's word may exert its power more publicly (luther, 2000, p. 399.94) . in fact, luther (2000, p. 398.85) believes worship is so important that we should have it daily, but understands that sunday as the chief day of the week is handed down from ancient times. in her book @worship: liturgical practices in digital worlds, liturgical theologian teresa berger (2018, p. 16) cautions us from too quickly succumbing to the false dichotomy of "real" and "virtual." such distinction can equate "virtual" with "non-real," which automatically privileges faceto-face relationships and practices over technology mediated. god primarily operates through means (neighbor, preaching, sacraments) ; thus, technologically mediated communication can certainly be the medium of god's own communication. another theological concept that helps bridge the gap between the so-called "virtual" and "real" is one that was used during the reformation to describe christ's presence in the sacrament. luther argued that, because of the ascension, christ had the attribute of ubiquity, meaning that he is available where he promised to be. for luther and his disagreements with the reformed, this meant that christ is truly present as the elements of the lord's supper on every altar. this same logic can be used in the digital environment: christ has promised to be present among gatherings of christians and in the midst of those who suffer (the theology of the cross). if such is true, then christ can be present online that transcends time and geography, just like christ's presence in the sacrament. theologian deanna thompson (2016; 2020, 26 march) follows this argument, claiming that virutal community is real community, mediating the body of christ. one of the arguments that i heard in my previous work of helping faculty teach online is the assumption that online coursework and whole-person formation contradict one another, since the online environment is all about the mind. in my experience of teaching online, i know this is not true. berger (2018, p. 19) affirms this by stating that the online environment definitely does have a physical effect on users. the types of relationships that can occur online can be described as "low stakes," meaning that they are "not associated with any cost, friction or risk" (simanowski, 2018, p. 9 ). does such a "low threshold" (berger, 2018, p. 36) for commitment allow one to flee from any sort of responsibility in the relationship, or does it allow for the freedom of openness without the increased possibility for negative consequences? constructing intentional community, as what (or should) already exist in our regular congregations, differs from the low stakes approach that one could see in facebook. in our current situation, the online environment is operating parallel to the communities that would occur in person if the health crisis did not exist. thus, such relationships are actually "high stakes" as this moment is temporary with the assumption that these relationships will continue outside technology. the phrase that health officers are using is "social distancing," the clinical term that encourages people to stay out of public spaces or larger gatherings. while limiting contact and large groups is an important step in reducing the pandemic peak, the term creates another set of problems. it is physical distancing that can reduce the spread of germs, but we must continue social encounters in this crisis even while limiting physical encounters. social networking and web conferencing technology, something i have been using since spring break to teach the remainder of the semester, can foster and assist us in maintaining our neighborliness in the midst of covid-19. maintaining both physical and social distancing can lead to isolation, which can make the health situation (especially mental health) worse. in some of the discourse i have seen online in the last weeks, i have noticed a discrepancy in terminology. when it comes to using video-based technology to broadcast online, two terms usually appear: livestream and web conference. although both of these practices use webcams and microphones, their level of interactivity is quite different. the livestreaming approach is unidirectional, which is how one currently watches television and youtube. the broadcaster creates the material, and those who watch consume the material. participation at best is passive and could be analogous to a pre-reformation understanding of the mass. the main role of the worshiper is to watch at the important moments, while simultaneously engaging in their own devotional practices. livestreaming (and admittedly web conferencing if the feature is enabled) allows for recording, meaning those unable to participate at the scheduled time could join in when they are able, but this can increase the individualism present in contemporary society today. also, taking seriously the role of the holy spirit means that it would be nearly impossible to replicate the live action in a recording (spadaro, 2014, p. 79) . the web conferencing approach is bidirectional and multidirectional. it allows for both proclamation and response through the same online tool, which is not the case with livestreaming. the "congregation" is part of the interactivity just as the worship leaders. this better simulates the dialogical nature of lutheran worship that i defined earlier. in his letter, luther (1968, p. 134 ) encouraged his readers to continue to participate in the weekly proclamation of the word through the sermon. he understands that central to christian life is the preaching and practice of god's word (luther, 2000, p 398.90) . historically, the service of the word has been the primary sunday liturgy for lutherans. some may wish to dispute this because our confessional documents and luther himself assume a weekly celebration of the sacrament (see melanchthon, 2000, p. 258.1; luther, 2000, p. 472.49 ). but we know that this was the ideal, and various circumstances usually prevented or hampered attaining this ideal: a shortage of pastors in early american efforts, laity still feeling unworthy to receive, the assumption that frequent reception would diminish the sacrament's specialness. the trend toward restoring weekly celebration of the lord's supper came with the early work done alongside the ecumenical liturgical renewal movement of the mid-twentiethcentury. 1 and still, among many lutheran congregations, weekly communion is not yet the practiced norm, although it is assumed in the newest worship books. when visiting my family in minnesota, the congregation where we worship still celebrates the sacrament twice a month. this attempt to restoring weekly sacramental celebrations has in some places turned into an overcorrection, with the assumption that every time the congregation gathers, or even a subset of the congregation gathers, the liturgy is deficient if the sacrament is absent. this practice has led to a phenomenon that looks more like votive masses than regular worship, in which the eucharistic liturgy appears to be for particular intentions ("votums") rather than the means of grace. the language around these votive (and sometimes private) masses becomes about mutual union and friendship, professing the faith we share, which is contrary to our understanding of the sacrament (melanchthon, 2000, p. 270.68) . these votive-like celebrations often separate the proclamation of the word from the sacrament, such that the sacramen-tal elements become the primary action rather than maintaining the historic order and balance (see evangelical lutheran church in america, 1997, p. 38) . one problem identified by those who advocate for perpetual fasting during this pandemic is that the sacrament must be celebrated within the assembly, as articulated in the use of the means of grace, principle 39 (evangelical lutheran church in america, 1997, p. 44 ). yet, this neglects the fact that the online environment is an assembly gathered for worship, while not raising objections to sacramental celebrations outside the assembly (e.g., church council meetings, retreats, etc.). the best advice would be to fast from the sacrament as long as possible, even in the midst of desiring it. 2 the season of lent provides a scheduled opportunity to do so, as we prepare ourselves for the annual celebration of christ's death and resurrection (lange, 2020, march 24) . as many congregations are already doing, these services of the word can easily take place in the online environment, especially through the communal nature of web conferencing. but the current health crisis may run many months; it already is running into eastertide and after. this requires other solutions (see below). because of the centrality of the proclamation of the word, lutheran congregations have not replaced the sunday liturgy with daily prayer; this was the custom in many anglican parishes pre-1979 prayer book. daily prayer in its ideal form is daily worship that does not occur on sundays, as the readings assigned are primarily from the other parts of scripture. the three main offices-morning prayer, evening prayer, night prayer-focus the gospel on the invariable canticles from luke's gospel, rather than the reading of a gospel lectionary text. the daily prayer offices are also better suited for smaller gatherings rather than the presence of the entire worshipping congregation. this may make the most sense in the online environment, as the web conferencing technology works best with smaller groups, with these rites particularly suited as domestic (at home) rituals rather than in the worship space of the congregation. the music that accompanies daily prayer, especially the historic orders with the assigned chants, can be done with little-to-no accompaniment, and can easily be spread among many people (both rostered and lay) for leadership. it is with the celebration of the lord's supper (holy communion, eucharist) where we encounter the most difficulty in this public health crisis. even many advocates for digitally mediated worship stop short of agreeing on "online communion." the main argument is that "the christian faith is deeply incarnational, and that means wedded to physicality and matter. … [o]ffering communion online short-circuits the communal, embodied nature of the eucharist" (berger, 2018, p. 84) . these critiques are important, as they lift up one of the many layers of meaning for the sacrament, namely, its physicality and incarnational nature. this physicality of the means of grace connect with our own physicality to remind us that our human/bodily nature is a god-given gift. the external nature of the sacraments provides the needed certainty to which our faith can cling/grasp (luther, 2000, p. 461.37) . when the sacramental elements are not possible in any way, the words of institution themselves serve the role of comfort and healing. the 1540 brandenburg church order notes that lay people can use the words of institution without administering the elements (they could not do that) so that the sick could "feed on the word" (rittgers, 2012, p. 171) . this is a natural extension from luther's claim that the sole source of comfort for christians is the word (rittgers, 2012, p. 103) . the proclamation of the gospel, aural and edible, is in service of consolation (treu, 1986, p. 18 ). the church's ministry to the sick has usually included bringing communion to those who are unable to attend sunday worship. this tradition dates to the second-century writings of justin martyr in his description of christianity to roman officials. in narrating an outline for the sunday liturgy, he describes the role of the deacons as the ones who bring the lord's supper to those who are absent (chapters 65 and 67). the deacons, since they did not have the role to "consecrate" the sacrament (justin assigns that to the "president of the assembly"), would have brought the already-consecrated elements as an extension of the assembly's sunday worship. this practice has continued to the present day and is seen in the current lutheran tradition with lbw's "distribution of holy communion to those in special circumstances" and elw's "sending of holy communion." i think the lbw's title better lifts up the issues we face today-we are in "special circumstances" that require us to rethink our customary practices. and this reevaluation makes it necessary that we be creative, especially since our congregations are all dealing with different restrictions and situations. extending our practices of "special circumstances" would be the ideal solution for distributing communion during these times. in contexts that are not in a quarantine-like state where visits are still allowed, a minister of communion would bring the sacrament to individuals or small groups in their homes or other arranged places. the caveat is that in many places, the assembly is not gathering on sunday mornings, so how would the extended distribution be extended from something that is not happening? if allowed under the civil orders, ministers of communion would gather with the pastor for a full eucharistic liturgy (including the proclamation of the gospel), receive the sacraments themselves, and then carry them to those who are not allowed to be present. preaching, which importantly connects to the distribution of the sacrament, could be recorded so that it could be played in the remote locations when the sacrament is distributed. it would be important that the minister gives the communion to the other person (and vice versa) so that the communal nature of communion continues. the more difficult context is when gatherings in general are disbanded by order of civil officials. one might argue that churches could exempt themselves from such a situation (e.g., two kingdoms), but luther (1968, p. 131) reminds us in his letter that part of the responsibility for both the body and soul means doing what it takes not to spread infection; in fact, it is considered sinful to not avoid places and persons in the case of possible infection. yet, there are possibilities even here. while maintaining physical distancing, it would be possible to deliver the sacrament to households, like permitted for food delivery. even though the sacrament is not mere bread and wine as served at the table, luther (2000, pp. 469.23-24, 474 .68) still calls it food (and medicine) for both the body and soul, nourishing us in a different mode than regular table food. again, pastors and ministers of communion would need to find a way to maintain the intimate connection between the proclamation of the gospel and the sacrament, so that we do not privilege one over the other. as suggested above, preaching could happen remotely through digital means and people would be able to receive communion. it is the distribution and reception-the "for you"-that is the central action of the sacrament, so ideally it would be someone else who distributes communion (luther, 2000, p. 469.21; formula of concord, 2000, p. 602.54) . as thomas schattuer (2014, p. 209) notes, the lutheran mass "culminated in the reception of the sacrament." this could occur among family members in a household, roommates in other situations, medical professionals and patients in a healthcare facility, and so on. this is the most difficult part of the discussion on digital worship in a health crisis. i have read essays on both sides of the argument, and most seem to be talking past one another. the ideal response is, as i have stated above, to fast from receiving the sacrament. in the small catechism, luther notes that the benefits of the sacrament are "forgiveness of sins, life and salvation," which makes it different from baptism (and the necessity of that for salvation). yet, in the large catechism, luther (2000, p. 469 .24) provides additional benefits: comfort, new strength, and refreshment. the sacrament also "a pure, wholesome, soothing medicine that aids you and gives life in both soul and body" (luther, 2000, p. 474.68) . so while the lord's supper is not salvifically necessary, it certainly could be considered pastorally necessary. before musing on what online communion may look like, i want to offer three caveats. the first is that the sacrament remains unimpaired even if we handle or use it unworthily (luther, 2000, p. 467.5; formula of concord, 2000, p. 597.24 ). this is not to excuse bad sacramental practice or to justify doing whatever we want with the lord's supper. rather, it does provide some comfort as we attempt to adjust to an unthinkable situation in a public health crisis. the second is that no one should deter someone from receiving the sacrament (luther, 2000, p. 473.62) . especially in times of pastoral necessity, the lord's supper should be received. luther (1968, p. 134 ) saw this as a requirement for pastors to minister to the sick and dying. it is part of the full work of ministry in the midst of a health crisis: preaching, teaching, exhorting, consoling, visiting, and administering the sacrament (luther, 1968, p. 136) . but, when visits are not allowed, the last two in this list of work must be rethought using the tools we have in our time. the third is that we should not doubt that christ the word can certainly accomplish what he promises (formula of concord, 2000, p. 601.47) . this simple argument was central to luther's disagreements with zwingli at marburg-if jesus promises (as he states in the words of institution) to truly be present, then we should not doubt those words or attempt to construe them to mean something else. so, is it possible to have online communion? i hesitate to answer in the definitive, but i provide here some theological rationale for doing so in extremis. by online communion i mean having worshippers gather in community through web conferencing with their own bread and cup as originating from their pantries. the two main objections that are raised by this are: (1) the lord's supper requires contact, and (2) it is akin to self-communion. the first objection should not be taken lightly, as generations of christians have gathered physically to participate as the ecclesial body of christ in the sacramental body of christ. distribution is important in lutheran theology, which regularly happens from person-to-person (see luther, 2000, p. 470.34-35) . unfortunately, in many dire situations, that is not possible and can be even dangerous; recall luther's exhortation that ministers are also responsible for not spreading infection (luther, 1968, p. 131 ). an even stronger objection related to contact is that the sacramental event must happen in-person, which would preclude the sacraments happening remotely. prior to the technological revolution, such remote sacramental event would be unthinkable and thus would not have come up in the theological discourse, certainly not during the reformation. to me this is a question of "use" and "action," the two words that the concordists identify as best expressing luther's sacramental theology in his "confession concerning christ's supper." the right use of the sacrament is reception and faith (schattauer, 2014, p. 209) . the sacrament cannot be present separate from its intended use (formula of concord, 2000, p. 595.15 ). this is the closest the lutheran tradition comes to defining the "how" of the sacrament, as that was not the important question in debating the lord's supper (the "who," "what," and "why" were primary in the regular celebration of the lord's supper, it is the presiding minister who completes steps one through three, and then the recipient of the sacrament completes steps four and five. yet, the concordists do not appear to say anything about the necessity of the presiding minister doing one and three, only two-speaking (or singing) the words of institutionbecause of having the proper "ministry or office" (formula of concord, 2000, p. 607.77 ). christ's body and blood is truly present as the sacramental bread and wine in its use and action, which i would argue can extend over digital means in the midst of the online community. the concordists insist on the language of use and action to prevent misuse of the sacrament through eucharistic adoration/reservation and corpus christi processions (formula of concord, 2000, p. 611-612.108 ). any adoration of christ occurs when the community gathers for sacramental worship, not as adoration of the sacrament itself. the distribution extra nos prevents self-communion, which is second objection. i find it peculiar that such objection is raised in regard to online communion, when generations of lutheran pastors have communed themselves during the eucharistic liturgy (rather than having an assisting minister commune them) without much objection. in fact, the use of the means of grace, application 46a, permits such practice (evangelical lutheran church in america, 1997, p. 50) . the role of the presiding minister in all of this is to proclaim the words of institution, just like the preacher proclaims the gospel-both of these are understood as "showing forth" christ (melanchthon, 2000, p. 272.80) . 4 the presiding minister is not acting in persona christi as a show or example of christ's action at the last supper (melanchthon, 2000, p. 271.72) . rather, the presiding minister is to proclaim christ through the audible and edible word (aural and sacramental) because christ is the word. in his liturgical reforms, luther underscores these ritually by requiring the words of institution to be spoken publicly and the eliminating the manual acts with the elements (schattauer, 2014, pp. 211, 216) . such reforms cause the eucharistic liturgy to focus on "christ's entire life and the meaning of that life for human salvation," rather than on reenacting a particular moment in christ's life (wandel, 2006, p. 100 ). when this essay is published online, most people will still be under "stay at home" or quarantine orders, and that may also be the case once this essay is published in hardcopy. while community continues to happen through online means, christians will be unable to gather in worship spaces for easterthe culmination of the liturgical year-in order to protect the vulnerable among us. this new life proclaimed in the death and resurrection of christ is a constant reminder that all christians are called to care for the neighbor in both their physical and spiritual life. during this time of being 'alone together,' i have been reflecting on the lectionary, especially the gospels for the second and third sundays of easter. like thomas, who missed the first appearance of resurrected jesus in the locked room, we may doubt christ's true presence unless we see and experience what has always happened in the past. yet, christ still comes to us, even when we do not experience church as in previous days. like the two disciples on the road to emmaus, we may not understand these events that have taken place, where our easter expectations have been disrupted by things outside out control. yet, christ still comes to us, even when we cannot gather physically to break bread as we have in previous days. pastors, deacons and all christians are responsible for the well-being of all people during this time, which may also include adjusting sacramental practice in extremis. the debate over online or virtual communion is not new, but the current health crisis has brought it to the foreground, and the ending of the covid-19 pandemic will not stop the debate. as we continue to figure out how to be church in the 21 st -century, we will need to attend to the many layers of meaning inherent in our practices. luther (2000, 472.49) argues that those who do not desire the sacrament actually despise it. 3 this is what luther means by connecting the elements with the word (luther, 2000, pp. 468.10, 469.30) . 4 although the apology does seem to assume the presiding minister is the one who distributes, this does not necessarily align with today's practice as articulated in the use of the means of grace, principle 41 (evangelical lutheran church in america, 1997, p. 46 ). kyle kenneth schiefelbein-guerrero https://orcid.org/0000-0001-9285-6161 kyle kenneth schiefelbein-guerrero spadaro, a. (2014) . cybertheology: thinking christianity in the era of the internet, m. way (trans.). new york: fordham university. thompson, d. (2016) . the virtual body of christ in a suffering world. nashville, tn: abingdon press. thompson, d. (2020, 26 march lutherjahrbuch, 53, 7-25. wandel, l. (2006) . the eucharist in the reformation: incarnation and liturgy. cambridge: cambridge university. witvliet, j. (2006) . teaching worship as a christian practice: musings on practical theology and pedagogy in seminaries and churchrelated colleagues. reformed journal. https://reformedjournal. com/teaching-worship-as-a-christian-practice-musing-on-practicaltheology-and-pedagogy-in-seminaries-and-church-related-colleges/. doi: 10.1111/dial.12555 should christians practice "virtual communion" in time of a plague? perhaps surprising to liturgical christians, but surely surprising to the public at large, if they cared, is that during this coronavirus-covid-19 pandemic a parochial debate also has gone viral; well, viral within our subculture. this debate concerns whether holy communion is legitimate when done "virtually" over the internet. this debate is serious. sometime it has been viscerally reactive, claiming that some pastors just want to do their "own thing" or even that such centering on the eucharist implies fetishism. i am grateful to observe instead that the conversation has become more civil as the involuntary fasting from the lord's supper extends into many weeks. more often now "both" sides recognize that they argue from common conviction; that we dearly cherish (rather than fetishize) the eucharist. still, i fear that a higher love yet has been missing from much of the conversation. i would prefer not to join the public conversation insofar as the conversation already seems premised on privileging doctrine over human wholeness. i do not like these terms on which the debate is set: "pro or con, care for holy things is more important than care for human lives." it is a hidden premise with a faulty disjunction. it forgets that lutheran doctrine has always carried within itself the quality of a quatenus, that we hold and must hold certain things doctrinally high insofar as they convey the promise of the gospel and that the gospel itself holds highest god's loving intention for the wholeness of human being, what the gospel of john thematizes as abundant life. so i join with a different premise. god's intention that the gospel be proclaimed in word and act to bless human beings with wholeness of life now and forever is the point. this requires that we are freed from self-preoccupation with ourselves so to serve others in the same love with which god embraces us all. luther, of course, as in his treatise on why christians should not flee in a time of plague, reminds us that self-care is required for other-care. that treatise defined in stark relief the ultimacy of the office of ministry's call to loosen and break the bonds of despair and anxiety as zealously as we can as loving service in itself and as reinforcing god's algorithmic formula in our temporal terms for the health/salvation (salus) of all; you know, "god's work, our hands." so much for prolegomena. but what about the weighty doctrinal loci that we also sincerely do hold dear (me included, if that is not clear to some) and bear on the presenting question? these include justification, the church, the sacraments, and the office of the ministry. these are the first and primary steps in the augsburg confession and, indeed, display a particular logic or trajectory we sometimes fail to see. further inputs for our thinking include some basic anthropology and some beyond-basic metaphysics. many on both sides have written fine constructive theology written on the matter in more general and popular terms. i will explore these lutheran premises with an eye for those whose questions are more dogmatically impelled. i will conclude with a coda in praise of the mystical body of christ, our appreciation of which is regrettably understated, if at all extant. no mere editorial (however longish) such as this can explore fully these loci. but i hope i can add some nuance to a mutually respectful dissensus. i have long sloganized that the vocation of the church is "the objectification of justification." human beings are fickle folk. emotional dis-ease routinely subsumes rational equanimity. anxiety is a chronic condition. when stressors of many kinds set us off, we can be locked into moral and spiritual trauma. all the dis-ease (and diss-ease) is caused in some way by sin, ours or another's. ptsd, moral injury, and spiritual trauma are contemporary names for the manifestation of the ancient general category of sin. depression and despair collude. one's whole physical and emotional and moral being is inhabited by these demons and only an-other can evict them. in other words, we are captive to our subjectivity and only an objective other can save us. concomitantly, only an-other carrying an-other's word in-with-under other objects objectively brings the saving grace word spoken and acted to us. the primal human need for a good word from outside oneself sets the initial logical ordering of the augsburg confession (ca). it makes sense, of course, for the first article to be about god. we start with the ultimate, however abstract the concept may be. then there is history, that is, sin and alienation. with article ii, in other words, the topic is more empirical, concrete, "objective." not to belabor the point, but the confession gets ever more "objective." jesus is our real and accessible rescue from alienation (iii). justification is the lutheran grammar to speak that (iv). the church (v), then, is the historical and empirically objective "paying forward" of justification as the very body of christ that evokes the life of new obedience (vi) and "is" church only as and when it proclaims the word and administers the sacraments (vii). the trajectory in this ordering underscores that any rescue of humanity from its alienation from god and self must come from a historical palpable "other." oswald bayer (a lutheran's lutheran) states the same. the augsburg confession and the smalcald articles insist that justifying faith "comes in the promise of the gospel and the alien righteousness (iustitia aliena) of christ that comes with it only in this manner must always receive proper emphasis." the gospel is never one's private possession. in other words, subjectivity cannot have the day (martin luther's theology, a contemporary interpretation, 2008, p. 252). external markers constitute the sacramental event. in the lord's supper those are (a) "the social and concurrently naturalcultural moment" of shared eating and drinking; (b) the actualization of a "definitive communal relationship between god and humanity" taking place within a physical assembly; (c) convened by and "through the performative word that has been addressed" to the assembly through bread and wine; (d) the whole action of which is empowered by the presence of the resurrected crucified jesus (bayer, pp. 252-253). then the perhaps surprising remark: the public external character of this word event and the private freedom of the individual "are correlates and empower and support one another." religion is surely not a private affair. but neither is it a form of heteronomy. alien righteousness must assume priority, but neither is it coercive. (bayer, 253). hold high the objective othernessthe alien-work of god. yet do not dismiss the integrity of the communicant's trust and desire. do it all with and within the objectivity of what is physical. there are two points in this fine summary that bear especially on our subject. the first concerns the relationship of physicality, communication, and location. as much as we emphasize the priority of the objectified grace of god in the elements of water, bread, and wine, it is puzzling to suppose nevertheless that the finite object that conveys grace (finitum capax infiniti) is bounded. bounded by what? well, it has been said forcefully in this debate on "virtual" communion that it is only legitimate when one assembly gathers around one loaf. that assembly must be physical and gathered in one place. but other scenarios that challenge that point are very familiar to us. suppose that the assembly is physically present, but is numbered in the thousands or even tens of thousands, as with churchwide assemblies and youth gatherings. there, of course, no one questions the subjectivity of adolescents gathered around and given the body and blood in the forms of hundreds of loaves and hundreds of cups. one loaf and one cup are lifted up at the center table and thousands of morsels and sips are consumed by de facto house churches without walls around the stadium, even behind walls and in other rooms by "overflow crowds." also, the distant eyes in the last row of the upper deck would not even be able to see the loaf and cup lifted were it not for the jumbo-tron screens hanging high over the arena. communication happens in multiple modes, most personally at distances of less than six feet. after that, from ear-aids to massive amplifiers, blue-toothed bridges, and towers and satellites convey the same grace in and with sound and light waves; the same intended original intimacy of god's voice in human voice to human ears still says "for you." why start and stop with one loaf and one very local assembly? of course we prefer that. in our subjectivity we prefer that. we respond more readily to the very familiar, to what has become intimate and intuitive. but infinity does not stop at walls; the real incarnate and divine christ shows up in emmaus, closed upper rooms, to roman military converts, under the floorboards with wwii american prisoners in the philippines (true story), and shares his incarnate divinity (communicatio idiomatum); wherever christ pleases. does the power of god, which for fickle human consciousness necessarily begins at the physical, end with the physical? well, yes, actually, but for comfort's sake christ does so even over great distances, metaphorically concomitant with entangled quantum particles. as bayer avers, the sacramental action begins with the physical but addresses (and redresses) subjectivity. the sacrament cannot begin with subjectivity. but enfleshed grace means to go to and through subjectivity so to move and change the receiver the more into christ. remember (re-member), christ counters zwingli's astral-projection direction and, as promised always, comes to us. the point of counter-precedents to the norm of one loaf in local assemblies in real time is very clear. what we already do already proves agreeable exceptions to the norm. we have already practiced-enthusiastically so!-virtual communion. and-of course!-we have always found ways to commune those who are sight and hearing impaired. we do not insist on an "ableist" assembly. the objective character of the eucharist is never purely so. it cannot be, because communication is not like that. thank goodness, still, the accent on the "other" is still more than on the receiver. new mass communication software platforms do not change that accent. if we argue that they do, we reveal our bondage to an aristotelian metaphysical stipulation of eucharistic conditions that fog our memory that christ's promises hold wherever he wants to, including the space/time relative and quantum qualities of postmodernity. perhaps the real error in this debate is not that we lack real communication and presence to each other when communion happens over longer distances, but that we have attached the word "virtual" to it. modalities have changed because metaphysical understandings have changed. we are not talking about donning headsets and entering into an alternate reality, as if being church is like going to the "feelies" predicted by orwell. we are talking about a real objectified message of forgiveness and liberation and re-union into a holy and incarnated comm-union that is just as real as the most localized assembly of two or three people in one space. since the stone was moved, we have always said this; we have always prayed this. our communion at the table happens with the saints and angels of all time and space. "with angels and archangels" we lift and consume bread and cup. might we not be at that blinking point of awakened insight now of actually converging our metaphysics and communion practices with the mystical poetry we have sung for millennia? let us just say it. the eucharist has always been "virtual" and the communion of saints has been our infinitum capax finiti. we physical and fickle folk are embraced by a boundless communion that graces and feeds our return to the holy and beloved community. indeed, we physical and folk are incarnated with christ no matter the visible or invisible walls. "virtual" in this frame does not mean fake. nor does it mean "spiritualist." the subjectivity of human perception does not and is commanded not to place bounds on the precise objects with and through which god gets to us. "virtual" here at least connotes the extension and incarnation of christ's physical body and blood beyond artificial boundaries of time and space, as the risen christ first did with walls of stucco and wood. the ubiquitous christ will be the incarnate christ and vice versa. one other predicate of "objectification" requires attention, at least for now. it is the function of the eucharistic presider, and so bears on the office of ministry. the reformers were clear that all the baptized are of the priesthood of all believers, and that the baptized are thus also servants in the spiritual estate, not only temporal. but ca v is not thereby collapsed with ca xiv. it is for the sake of good order that the pastoral office is distinct from the service of all the baptized. what all can do by virtue of their baptism cannot be done by all at the same time. the result would be chaos. so lw 44:128: "because we are all priests of equal standing, no one must push himself forward and take it upon himself, without our consent and election, to do that for which we all have equal authority. for no one dare take upon himself what is common to all without the authority and consent of community. and so it is that a pastor is one who is rightly called (rite vocatus) by his/her community of faith. the call and the command make one a pastor of the divine word (lw13:65). this call comes from the faith community and is for the good order of the faith community (1 corinthians 14:40). there are vital presuppositions in the lutheran understanding of the pastoral office. "vital," remember, has both to do with being "central" and being "life-giving" (vita). we have reviewed already the necessity that the gospel comes from outside the receiver's subjectivity. someone, an-other, a selected and called one, must proclaim the gospel in its purity and see to it that the sacraments are ministered rightly. that someone, the pastor, attends not only to the proclamation and the giving. he or she attends to the subjectivity of the receivers too. pastors know their people. knowing their people implies a reciprocal relationship of trust between pastor and people. the shaping of sermons and-i submit-the contextual understanding and framing of a sacramental occasion requires such mutual trust. the communicator of alien righteousness, in other words, is herself not at all alien to the receiver. good order does not imply that the pastor "does it all," however. pastoral "control" of a whole parish life, including the manner of its sacramental worship, does not belong to this conception of ministry. one wonders whether a pastor does not exhibit a privileged clericalism when the self-control volume level goes past 11, as if one held a personal sense of ontological difference between the pastoral office and the rest of the baptized priesthood. the life of a congregation managed by such a relationship may happen on time, concordant with a stiff lip of upper reine lehre. but that is not necessarily good order. good order resonates in a faith community when people are known for what they can do well for each other, including tasks of prayer and lay eucharistic ministry, even lay preaching, and maybe even on rare occasion when lay sisters and brothers commune each other under the express permission and direction of the pastor. characterizing all of such a congregation's life is a living trust, a deep and respectful loving relationship that shapes the worship community and precedes its gathering. this is why "online communion" can be understood to be as real as a more "concrete" local assembly. the figure on the video monitor is known and trusted. the gospel proclaimed and the word acted in the words of institution, to be sure, are effective no matter the trust level (otherwise there is that donatism matter). but the christian community already in relationship and rightly ordered completes the circuity however dispersed in space and time the already palpably related assembly is. let me be clear. it is always "better than good order" to commune together as one particular assembly within the shining affinity and infinity of all the saints and angels. that is the normative good for our subjectivities. but in a time of exigency when a fast from the eucharist is involuntary, it is not pastorally caring after a surfeit of heteronomously imposed fasting days to tell the flock to "remember what you ate" as if that is the same as the call to "remember your baptism." we need the manna. god means us to have food for the journey in the wilderness. and when the counsel in such days says that prayer and meditation and listening to the word is "just as effective anyway," does that logic not undercut the very reasons the same counselors once argued for regular celebration of holy communion? does it not in itself betray a favored "spiritualism," if not even a closeted gnosticism? yes, god comes to us in many ways, and can be seen to do so in many places, but only after christ is revealed to us in the indissoluble nexus of word and sacrament (so wrote luther when writing on the pun of "crystal," christall, christ-in all). there comes the time in an exigency when god's people, threatened deeply in our subjectivity during just such times, gotta eat. ignatius of antioch's apt synonym, "the medicine of immortality," is meant from faith for faith in such days. much more could and should be said, but is not necessary here. the evangelical effect of "virtual" communication (though not yet virtual communion) has been so very consequential and beautiful in the life of the congregation i am called to serve. great stories can and will be told. "virtual communion" is a responsible step in in extremis times for the encouragement and continued formation of the faithful individually and together. i do not mean this as "normative," as if this should regularly replace the side-by-side body language of the local worship assembly. i intend this argument as the exception that proves the rule. it is an interim measure that in and by the holy spirit's power will console and move from "inside-out" god's people further in the way of trust and loving service to this dis-eased world. and when the spirit brings us as a local assembly more palpably back together around the font and table, we will be the more grateful that we were re-membered as christ's body even as we were too long apart. university of houston doi: 10.1111/dial.12545 "the voice of one crying out in the wilderness" 1 there is something strange going on in our weather system. for the past 2 months our island, located in the northern part of the atlantic, has been literally closed down more than dozen times. this means that there have been no flights, international or domestic, roads have been closed down (either in parts of the country, or the whole country), schools have been closed, and electricity has been out in certain areas, for hours up to days, all because of the weather. this is a huge concern to all of us who live here in iceland, and even as i write this, we are in the midst of one of these events. there is really nothing "normal" about it, and questions about its relationship to a changing climate are compelling. but it is too soon to draw any conclusions. patterns have to have time to develop. at the same time, our glaciers are melting, right in front of our eyes, because of increase in temperature, which also are warming up the sea around the island, causing big changes, and real threats, to our fishing practices, as some fish species are leaving, seeking cooler waters elsewhere, while new arrive. it takes time for the fish industry to adapt, and the uncertainty is challenging for people, especially in the small fishing towns around the country, to say nothing of our whole economy. 2 like everywhere else, icelanders have been slow to wake up to the seriousness of a warming climate, but gradually people are realizing that this means that life cannot go on like usual any longer. the swedish teenage girl, greta thunberg, who started school strike for the climate in august of 2018, has made a huge impact in our country and elsewhere, by directing people's attention to the alarming reports scientists have been writing for years about the serious impact of global warming. because of greta, young people in iceland are starting their own school strike for the climate, and by doing that they have put much needed pressure on our government to act according to their commitment to the paris agreement, from december 2015. it has been breathtaking to watch what has happened since the greta thunberg school strike for the climate started, less than 2 years ago, outside of the swedish parliament. greta was only fifteen years old, and this was her own initiative. her parents supported her, although reluctantly to begin with, because they worried about her health and how the publicity would affect her. her aim was to remind swedish politicians of the climate crisis and their responsibility to react to the crises, three weeks before the fall election 2018. after the election greta decided she would continue her strike until the day the swedish government had fulfilled their promises to meet the conditions of the agreement reached in paris, and reiterated at other climate conferences. so her strike goes on, but she is certainly no longer by herself. 3 what started as a one-person act, has gradually developed into a world-wide movement, which, it is safe to say, has made greater impact than any other climate initiative. by speaking in clear terms, and making radical decisions, like not to fly, greta has managed, at her young age, to bring people all over the world, out to the streets, demanding responsible actions from those in charge, as well as individuals, who are contributing to the climate crisis by their daily behavior. there is something about her either/or rhetoric that makes people pay attention. she herself has said that the reason why she tends to see things as black and white is because she has asperger's syndrome. she also has told the story of her childhood, and how she became severely depressed after hearing about climate change at early age and realizing that people were not doing anything about it. after suffering for years from eating disorders and selective mutism, she was able to overcome her life-threatening condition, and start to eat and talk again, by speaking up and actively fighting for responsible reaction to the climate crisis. it is clear that for greta this is about life and death, and that is the message she wants to convey. during the past year and a half, greta has been invited to speak at numerous rallies, as well as exclusive meetings such as the european parliament, houses of parliament in london, the unites states congress, and the united nations. true to her black and white worldview, greta insists that we have to stop our emission of greenhouse gases; "either we do that or we don't," has been her repeated message. 4 there is something profoundly prophetic about her "clear text" rhetoric. it is not simply about actions but also about a change of heart, and mind. speaking to the european economic and social committee in brussels, in february 2019, greta challenged her audience to do their homework, because "once you have done your homework," she insisted, "you realize that we need new politics, we need new economics where everything is based on a rapidly declining and extremely limited remaining carbon budget." but, to greta, "that is not enough." what is needed is "a whole new way of thinking." instead of political systems based on competition, we need to cooperate and work together and to share the resources of the planet in a fair way. we need to start living within the planetary boundaries, focus on equity and take few steps back for the sake of all living species. we need to protect the biosphere, the air, the oceans, the soil, the forests. 5 for greta there is no compromise, "no lukewarm, and neither cold nor hot" way of thinking (rev. 3.16), you are either for or against, either willing to save the planet, and our future, or not. the burning house is a compelling metaphor, painted in strong colors. "our house is on fire. i am here to say, our house is on fire," greta said in her address to the world economic forum in davos in january 2019. she concluded her speech with this powerful, no beating-around-the-bush message: we must change almost everything in our current societies. there is something not right, when our kids and teenagers are missing out of school in order to protest and fight for the future of our planet, our common home, and the future of all of us who live here now, as well as future generations. there is something strange going on, and the young people are getting it. once again civil disobedience is proving to be an important tool against unjust systems, which are protecting the few, and not caring for the rest. this is what climate justice is all about. it reminds us that those who have contributed the least to the climate crisis are suffering the most; the poor, women, and children in the global south. those of us who belong to the privileged part of the world, need to start thinking globally; we need to look at the bigger picture. we cannot continue to think just about us, and our economy. greta thunberg argues what it all boils down to is the choice between money and the environment. there are multiple ways we can respond responsibly to the current crisis we are faced with, not only highly technical, and financially costly solutions. a book called drawdown: the most comprehensive plan ever proposed to reverse global warming (2017), lists, for example, education of girls as the sixth most important solution, and family planning as number seven, right after refrigeration, wind turbines, reduced food-waste, plant-rich diet and tropical forests; and before solar farms, and rooftop solar. 7 there is no surprise that people, who are paying close attention to the discourse about the climate crises, are worrying about the future. eco-anxiety is a growing concern, especially among the youth. melting glaciers, higher temperatures, and severe storms are among the signs of climate change that are raising the awareness, and even anxiety, of the people in iceland. it is important that people realize that something can still be done. greta thunberg has warned all of us that talk about hope can indeed keep us away from actions. at the un climate change conference, in katowice in poland in december 2018, greta gave a powerful talk, in front of world leaders, climate scientists, and other participants. she concluded her talk with those words: until you start focusing on what needs to be done rather than what is politically possible, there's no hope. we cannot solve a crises without treating it as a crisis. we need to keep the fossil fuels in the ground and we need to focus on equity. and if solutions within this system are so impossible to find then maybe we should change the system itself? we have not come here to beg world leaders to care. you have ignored us in the past and you will ignore us again. you've run out of excuses and we're running out of time. we've come here to let you know that change is coming whether you like it or not. the real power belongs to the people. 8 a prophetic voice; a challenging, encouraging, and compelling voice. but will she be able to move us into action? only time will tell. trauma, eco-spirituality, and transformation in frozen 2: guides for the church and climate change i recently became captivated by the film frozen 2. i was in florida for a psychotherapy professional training and one night decided to take myself on a date. nothing fancy; i was intentionally looking for something not too thought provoking or activating-just dinner and a movie. little did i anticipate how disney's new animated film would capture my imagination, heart, and theological intrigue. while there is enough material in my thoughts and consciousness to fill out a book (keep your eyes out for one in the future-the proposal is already in the works), i wanted to share a few reflections on how disney's frozen 2 can provide a lens for trauma, transformation, and the essential call for our faith communities to step more fully into an eco-spirituality as a means of fully incarnated repair. warning: spoilers ahead! first things first. "trauma," as i am using it, refers to any experience that overwhelms our capacity to respond to the challenges in our environment and results in either an over constriction or an over expansion. trauma is less about the event or experience itself and more about the ways in which it impacts us as individuals, communities, or global ecology. when faced with a significant threat that overwhelms our capacity for resiliency, we are at risk of developing symptoms of traumatic response. in its simplest form, traumatic responses cause us to be smaller or less than we truly are in an effort to protect ourselves from further wounding. we either shrink to escape further blows or we build and reside behind walls to project a larger image. trauma and transformation are the beating heart of frozen 2. as olaf wisely queries, "did you know that an enchanted forest is a place of transformation?" just as trauma entrenches us in protective patters; transformation calls from the beyond, into the unknown, and into the promise of authentic flow. transformation often requires us to enter into liminal places, the spaces betwixt and between, where our familiar habits are tested. these spaces, either geographically or relationally, disrupt our habits of constriction or fleeing protection and generate opportunities and wiggle room for the new. they require courage and offer hope for connection, fullness, and completion. the heartbeat pulsing through the film begins in the opening scene in which elsa and anna play with snow toys. anna explores the narrative that love, in this instance between a distressed damsel and a "fancy" prince, will save the day. elsa, meanwhile, weaves a story of trapped fairies and "the fairy princess who breaks the spell and saves everyone." their play prompts their father to tell a story of a real enchanted forest and how he became king. his story paints a picture of colonialism and subsequent acts of violence that rend the connection among the elemental spirits, the northuldra people (based on the sami people) from arendelle, and begins a cascade that separates anna from elsa, and their family from their community. while initially told from the perspective of king agnarr, the driving quest of the film is to discover the truth, brave the trauma of the truth, and make amends or reparation thus breaking the spell. at the center of the tale is elsa's quest to follow the lure of the voice that calls to her into the unknown and toward the source that holds memory and truth. along the way elsa must show her power to befriend the elemental spirits and witness the pieces of truth they hold. from the wind, she sees her parents as children and meets the northuldra people. the fire spirit shows her that she is not alone in hearing the call. the water spirit challenges her to recognize the limits of her power and to depend on another to go the distance. the earth giants, through the prompting of anna, break the wall that is the origin and symbol of violence and mistrust. it is only through the befriending and partnership with the elemental spirits that elsa finds her way home to who she fully is and anna steps into her power. the origins of trauma and separation in frozen 2 are located in the deceptive "gift" to build the dam and stop the flow of water and connection thus weakening the elemental spirits and leading to violence. transformation occurs by venturing into the unknown, befriending the elemental spirits and indigenous communities, courageously witnessing the source violence of trauma, and taking concrete actions to break the spell and restore resiliency and vitality. so, what wisdom can the church glean from frozen 2? first, in the midst of our ecological global crisis, we must find the courage to venture into the unknown. what are more sustainable practices? how do we speak with confidence about the limits of our solidified patterns and hope of restored connection? second, we need to find the fortitude to witness the ways in which we have enacted violence against one another, the earth, and non-human beings and the conviction to change, dismantle the dams we have built to enhance our power while limiting the magic of the natural world, and make reparations. humanity's chronic history of violence has profound implications for planetary health and eco-diversity. as we move forward in this critical period of ecological viability, who will we show ourselves to be? will we extend our awareness of the creation narrative in genesis 2 and live into a renewed confession that yhwh created the earth and all of her creatures and they are good? as the fires in california and, more acutely, australia, have made clear, the loss of animal life as a consequence of our unchecked impact on climate is devastating. will we protect the children of the earth from our unfettered goblin of destruction or will we break the spell and save everyone? as communities of faith, we have an opportunity to mend the traumatic wounds of colonial violence, humbly seek forgiveness and understanding for our histories of collective violence toward indigenous peoples, and offer reparations (in whatever form is appropriate) to our intra-and interspecies siblings. we must find the courage to befriend those who frighten us in their efforts to protect themselves from our histories of violence and to join with them to heal the traumas that threaten to freeze and drown. transformation is formed through the courage to venture into the unknown, the willingness to listen, witness, and befriend, the moral fortitude to break down the walls that were erected in fear, and connect ever more fully to the elemental spirits of the planet and, through those connections, to who we are meant to be. we are the ones we have been waiting for. can we fully step into our power and show our self who is made in the image of the divine? grounding flight wellness center doi: 10.1111/dial.12553 from the redwood forests and the cedars of lebanon to the tree of good and evil in the garden of eden, far back into the groundswells of the archaic human imagination, the experience of "treehood" (paul tillich) has claimed human hearts and minds all around this good earth for countless generations. 1 i myself, in my own mundane way, have been captivated by existential encounters with trees, real or imagined, ever since i can remember. but much as i have self-consciously and enthusiastically lived with, thought about, and contemplated trees my whole life, i have never explored that experience itself. i want to make a start at doing that here, with the hope that this might prompt others, particularly members of american christian communities, to go and do likewise, in fresh ways. 2 the first tree i ever fell in love with was a lombard poplar. i grew up in an exurban setting, near buffalo, new york. one side of the family land was lined with these tall, cylindershaped trees, which had already grown to full height, perhaps 60 feet tall, when i was a child. usually without my parents knowing, i would on occasion climb up one of those trees as high as i dared. the branches were fragile, but, for a slim 11year-old, that climb was safe, or so i thought back then. on those ascents, i often imagined myself to be a kind of heroic adventurer. i would station myself maybe 40 feet above the ground for a spell, as i surveyed our house below and the fields beyond, and felt the wind bending the tree and brushing my face. it was a boy's dream. for those moments, i lived ecstatically, in another world, thanks to that poplar tree. in retrospect, i can imagine that those tree-climbing adventures must have had an important psychological function for me. i was an unhappy child at times, a condition that i only began to understand some years later when i was in therapy during my college years. high up in one of those trees, i suppose that i was able to leave those familial tensions behind, if only for a short time. in therapy, i came to understand that, among other family dynamics, i had had a conflicted relationship with my father. he was a kind and caring man, but i began to realize that he was also distant at some deeper level. enter the world of trees. perhaps thanks to his german heritage-germans typically cherished their parks, perhaps more than other ethnic groups-my father loved trees. one of his uncles, who was also of german descent, had a top position in the buffalo parks department in the late nineteenth century. that uncle oversaw the implementation of a plan to plant what turned out to be many thousands of sweepingly gracious elm trees, along both sides of many of the city's parkways. in those days, long before the onset of dutch elm disease, buffalo was elm city without the name. that history behind him, my father often found times to take his mind off his busy professional life-he was a dentist-by planting and caring for trees all around our sizeable property. and he often enlisted me to work with him, which was always a joy for me. those were some of the times when i truly felt close to him and when, i believe, he truly felt close to me. adventurous joy with those poplar-climbings and warm personal bonding with those tree-plantings and that tree-care with my father-those were some of the deeper experiences of my younger years which i came to cherish as i grew into adulthood. also, during my high school years, my family had the means to travel to many of the nation's great national parks during extended summer vacations. under my father's tutelage on those trips, i came to affectionately know many trees, the majestic redwoods of california, for example, or the effervescent quaking aspens of utah. during the years of my doctoral studies, i found a way to read every volume of the collected works of john muir, even though those works were obviously not immediately germane for my chosen field of academic research, twentieth century german theology. john muir then led me to the much more famous henry david thoreau. i think, in retrospect, that i read muir first, and thoroughly, because he was so deeply imbued with calvin's theology, whether he fully understood that or not, and since, by that time, i had immersed myself in calvin's thought, along with luther's, both of whom, i came to believe and then subsequently to argue, were dedicated champions of the goodness of creation and the glories and the mysteries of the natural world, in particular. 3 i read muir and thoreau, ironically perhaps, at the same time that i was working on my doctoral dissertation on the great karl barth's-highly problematical-theology of nature. 4 barth's theology as a whole, seminal indeed as it was, never helped me to understand, much less to affirm, my longstanding love for trees. muir's and then thoreau's encounters with trees did. the result was a theological proposal, on my part, for a new way to understand my love-or anyone's love-for trees. barth had adopted what was, at the time, a more or less conventional theological way to understand human relationships with other creatures, a theme developed by many thinkers in his era, but which was most often associated with the name of the jewish philosopher, martin buber, and his book, i and thou. 5 buber contrasted an i-thou relation, which he thought of in intimate, personalistic terms, with an i-it relation, which he defined as an objectifying relationship between a person and a thing. so, when someone says to his or her partner, authentically, "i love you," that is an i-thou relationship. when he or she picks up a hammer and hits a nail, that is an i-it relationship. buber and others-among them, barth-who gave this way of thinking currency, were eager to protect and then to celebrate the authenticity of genuine human relationships and to reject any kind of objectifying relationships between humans and other humans. humans should always be regarded as ends-in-themselves, according to this way of thinking, and should never be treated as objects to be manipulated. what, then, about my relationship with trees? the i-thou, i-it way of thinking does not account for my love of trees. trees are not persons. you cannot communicate with a tree the way you can communicate with your spouse, as a thou. are all trees, therefore, in truth mere objects? was that lombard poplar which i adored when i was 11 years old merely an object i used, like a ladder, to climb up into the sky? or was it, in truth, a creature in its own right, worthy of my respect, even adulation? wasn't it the case that i not only clung to that tree, forty feet above ground, for safety's sake, but also to embrace it? that tree, for me, back then was no mere object. it was something else. but what? buber recognized this problem in an appendix to the second edition of i and thou. he even imagined a relationship to a tree that is somehow akin to an i-thou relationship, but he self-consciously chose not to try to think that through. i decided that i myself would give it a try. in my first scholarly article, i argued that a revision of buber's thought was required. hence my title: "i-thou, i-it, and i-ens." 6 i wanted to be able to talk about the trees that i loved as ends-in-themselves, no longer as mere objects. in that article, to illustrate i-ens relationships, i drew attention not only to the praxis of thinkers like thoreau and muir with regard to nature, but also to luther's and calvin's visions of earthly creatures. both reformers, like thoreau and muir, portrayed those creatures in non-objectifying terms and indeed celebrated those creatures as ends in themselves, as, in some sense, charged with the mystery of god. luther saw miracles in nature everywhere and stood in awe of them. calvin considered the whole of nature to be a theater of divine glory and celebrated that glory enthusiastically. in ensuing publications, i employed the constructs of i-thou, i-it, and i-ens as a kind of silent interpretive key to open up the whole sweep of classical christian theology in a new way. i argued that -notwithstanding lynn white jr.'s then widely hailed critique of the christian tradition as ecologically bankrupt, alleging that christians have almost always treated nature as a mere object, something to be manipulated-we can trace a major christian tradition that richly affirmed the natural world in its own right. that way of thinking i could have called the ens-tradition. in retrospect, i think that my reflections about buber's way of thinking and my historical investigations were existentially dependent on my early encounters with treehood. likewise for my conversion to environmental activism, along the way. that happened, emphatically, after i first began to work my way through books like rachel carson's silent spring and stewart udall's the quiet crisis in the early 1960s. 7 it was natural, as it were, for me in those days, and subsequently, not only to love trees in their own right, but also to do all that i could do to protect them, along with the whole world of god's earthly creatures. but my life with trees by no means came to expression just in youthful encounters or in mid-life scholarly writings or even in longstanding commitments to environmental or ecojustice activism. 8 i also have been blessed throughout my life by rich encounters with a range of particular trees. this story has unfolded in several locations, but i want to mention only one here, the old farmhouse at hunts corner, in southwestern maine, which has been a home away from home for me and my family for more than forty years. at hunts corner, notwithstanding the human incursions here and there and the ominous pipeline in particular, i have developed cordial relationships over the years with many of the trees on our land, i-ens relationships as i think about them. i have learned to call many of those trees by name and sometimes greet them, when no other humans are around. our plot was in all likelihood a farmland 150 years ago. the west side of our land is marked by one of those famous stone walls that defined the farm fields in historic new england. the oldest trees tend to be near that wall or to be growing from an adjacent, steep and stony incline, which never could have been farmed. one mother oak, in particular, has fascinated me ever since i first noticed it. it is enormous. i cannot put my arms even half way around its mammoth base. the poor tree has been hammered and seared over its long lifetime by the elements. the top of its central trunk was apparently sheared off, perhaps decades ago. but the tree has lived on. near that mother oak grow a number of smaller, but nevertheless sizeable descendants. i once walked through that area with a neighbor and he eagerly explained to me that i could make a lot of money if i were to have those oaks cut down for commercial sale. grand old towering mother white pines also grow in that area and elsewhere on our land. my brother, gary, and i once cut down one of those giants after it had died, this, for safety reasons. i did not want it to fall on anyone, particularly on my grandchildren, who sometimes had ventured out near that tree, at the edge of the forest. treehood should not be romanticized. a tearful older father once told me, in a long, quiet conversation, how he had lost his daughter to a tree, in the prime of her life. this was the story that onlookers reported. his daughter and her two toddlers had been picnicking in a park. on their way home, she was watching them run on playfully ahead of her. at one point, she saw a large tree falling down on to the children. she ran desperately to push them out of the way, which she did. but she herself was killed. that story was in my mind, as were my own grandchildren, all the time my brother and i were working to take down that immense, but dead pine tree at the edge of our forest. huge it was. gary and i barely had the strength together to roll pieces from that tree's trunk into the woods to their final resting places. early on in my family's tenure at hunts corner, i began to carve out paths in the back forest, where that mother oak and a number of the great white pines live and where american beeches are now moving in. closer to our house, i have planted a variety of individual trees over the years or occasionally cut away competitors, in order to allow some extant trees to flourish. perhaps the most striking of all the tree planting that laurel and i have done over the years was the operation that she and i once performed on what was, for us at the time, a nameless sapling. it was march, early on in our experience with the world of rural maine. what we did was sheer, youthful folly. laurel had decided at that time, that, come the next spring, we would turn over a plot just back of our house, where she would begin to create a perennial garden. but there stood that large sapling right in the middle of that space! without much thought, we decided that we would try to move that tree, right then. the ground was frozen, of course. i had to use an ax to cut out the ball of the roots. once cut free, we could barely drag that ball out of its earthen socket. now what? we decided to roll it maybe forty yards to the western side of our land. there, using the ax again, and a pick-ax, i hollowed out a cavity for that big, frozen root ball. finally, we were able to slide that sapling and the mass of its frozen roots into that hole. it was only then that it dawned on us that we had planted that tree close to the church next door, a pristine, white, wooden building, which easily could have appeared on some new england calendar cover. but that was that. never mind that sapling. the church building appeared to be as picturesque as ever. we hurried on into the house to warm ourselves by the franklin stove. little did we know back then that that nameless sapling, more than 40 years later, would magically turn into a graceful and fulsome red maple whose sumptuous branches would then completely cover our vista of the whole church building! that iconic structure is gone from our angle of vision for much of the year. there may be a parable hidden in this ironic tale, but, if so, i have yet to discover what it is. sadly, the sugar maple i planted at the front of our property many years ago recently died. it was painful for me to observe that large and lovely tree die over the course of several seasons and then to witness it standing there, barren, a skeleton, all by itself. true, stories like these sometimes have a blessed ending, according to one of the central themes of the christian faith, from death comes life. over the many years that we have lived at hunts corner, mostly from the early spring through the late fall, we have used our old iron stove in the kitchen steadily, sometimes even on cool summer nights. and we obtain fuel for those fires almost always from standing dead-wood, which we cut down at various places on our land and then drag in, cut up, split, and stack. that was to be the story of that dead sugar maple. we would give thanks for it one more time, so i thought, as it would later warm both our kitchen and our hearts. but that dead sugar maple's transition to firewood was not as smooth as i had anticipated. that project turned out to be an adventure. for many years, my brother and i have helped each other with forest and other chores at our respective rural homes, his in western connecticut. he learned to love trees the same way i did, working with our father on the grounds of our exurban buffalo home. after various childhood and adolescent skirmishes, some of them harsh, gary and i have remained close over the years and have grown even closer in these our golden years, especially by assisting each other outdoors either in connecticut or maine, for days at a time. that towering dead sugar maple had to be cut so that it would fall away from the street, not on to the street, where it might block or even hit some speeding car that was passing by. with some anxiety, i admit, i nevertheless trusted gary to cut that tree just so that it would fall precisely where it was supposed to. i had witnessed gary "place" (his term) falling trees in just the right locations many times. when this tree began to undulate, however, it did not immediately fall away from the street as gary had cut it to fall. the tree just stood there trembling, not falling in any direction! what was going to happen? with some sense of urgency (!) and with a long rope tying him to that oh-so-perilously oscillating tree, as it was readying itself to fall in one direction or another, gary dashed to a spot far away from the street and then pulled on the rope, again and again, until the tree finally fell toward him (it crashed down a few feet to his left!) and not on to some unsuspecting car that might have been speeding up or down our road. quite a feat for one who was at that time about to turn eighty! as i am constantly aware, trees are not always our friends. but thankfully, in this case, gary was able to coax that tree in a friendly direction, narrowly escaping injuring himself or anyone else. i have saved for last what is for me the best news about treehood. some years ago, laurel and i purchased a then ten-foot tall purple beech sapling, and planted it in our hidden garden. long before, we had come to adore the gigantic hundred-yearold purple beeches we had encountered in mt. auburn cemetery, near our massachusetts home. in this finite world, those great trees are, for me, the best natural symbols of eternal life that i can imagine. laurel and i have decided to have our ashes interred at the base of our own purple beech, which now rises high above us in the hidden garden. i have affixed a foot-high celtic cross-made of cementat the base of our purple beech, which one day will not only mark the place of our buried ashes, but will also announce the truth, for those who have ears to hear, that has claimed my own soul self-consciously since the first days of my theological study to these my octogenarian years, predicated on a reading of colossians 1:15ff.: the crucified and risen lord is the cosmic christ, both now and forever-"…[a]ll things have been created through him and for him. he himself is before all things, and in him all things hold together" (col.1:16f. nrsv). i saw that cosmic christology in the figures and designs on the historic celtic crosses that i encountered during a trip to ireland with laurel in 1996, along with throngs of other spiritual seekers. 9 i concluded then that the classical celtic saints were by no means essentially nature mystics, as many who have been fascinated with them in our time have believed. no, their spirituality of nature was consistently an eschatological celebration of the cross and resurrection. for the great celtic saints, the love of the seas and the earth and its creatures and the love of jesus christ, crucified and risen from the dead, is the same love, now and forever. hence i was overjoyed when i found and then was able to buy that cement celtic cross at home depot for $14.98. i eagerly carried it off to implant it in the earth next to the purple beech in our hidden garden. i wanted to announce that someone believes-or that someone, whose ashes are interred there, once did believe-that that tree, marked by that cross, is-or was-for that believer the lignum vitae. i cannot imagine the story i am telling here ending otherwise, for i now realize that my world, from the days of my childhood on, always has been, is, and, i hope, always will be, the world of treehood. 2 treehood, rightly construed, has a justice dimension. think of the remarkable work of nobel laureate wangari maathai (d. 2011), who started the green belt movement in kenya, which has planted more than 30 million trees in africa, in order to fight erosion, to create firewood, to give work to poor women, and, generally, to reestablish the health of the whole earthly biotic community. wangari's work presupposed that trees have their own standing, that trees, essentially, are not first and foremost objects for capitalist exploitation, whether directly, through commercial development, or indirectly, through the destructions wrought by impoverished peoples. nor, in wangari's perspective, were trees essentially a means for the wealthy temporarily to escape from the contradictions of modern industrial society, under the rubric of "ecotourism." 7 carson, r. (1962). silent spring. new york: houghton & mifflin; udall, s. (1963) . the quiet crisis. minneapolis, mn: fortress, 2013, ch. 18. 9 for an account of my engagement with celtic spirituality, see santmire, h. p. (2000) . the church is a global network. it has a presence around the world that is almost unsurpassed by any other organization or movement. the church has contact with other religions at all levels, and cooperates with a wide range of humanitarian organizations. it is in dialogue with world leaders, not least via the un system. the church has a presence in many places around the world that are not readily accessible. during crises and disasters, the church is often there before they happen, while they are happening, and long after the immediate relief work has been phased out. this is an obligation in an era in which the world must learn to live with the climate crisis and its consequences. we know that those people who have contributed least to global warming are often those most severely affected by climate change. we know that social challenges such as poverty, migration, and the global health situation are directly linked to environmental and climate issues. there is a need for climate justice. the issue is how we humans interact with the natural environment, of which we are a part. we therefore have to take action based on what feels most meaningful in our lives. we must therefore talk about the sacrifices that we can make together, so that our children and the children of others can have a future. the climate crisis is exacerbated by lifestyles that make greed seem like a virtue. resolving it will be difficult for as long as people and nature are viewed only from the perspective of economics and technology. only when we actually distinguish between our needs and our desires can we achieve fair and just climate goals. when will we learn to say, "enough is enough!"? what we think about and feel about nature really matters. is it a mechanism that simply keeps on rolling? an unlimited source of raw materials? our recreation area? our enemy? a place of endless harmony and balance? a system involving a constant battle for survival? how we relate to nature as creation reveals how we relate to the very basis of existencewhich we call god. the churches in the east and the west have developed somewhat differing points of focus with regard to humankind and creation. put in simple terms, western tradition has developed a deep trust in rationality and science. this has contributed to a demystifying of nature and humankind's role in creation. its secrets were dissolved in measurability. humans came to understand themselves to be rulers of nature, rather than stewards who are responsible for and have to care for something that they do not actually own. the emphasis was put on humankind's function. theologians in the east have talked more about nature as a mystery that cannot be fully described, not even with the most excellent measuring instruments available in the world of science. nature meets us and shows itself to us, but never fully. as humans, we are part of this mystery. each human being is itself a miniature cosmos, a microcosm. here, the relationship is at the forefront. the western view has a tendency to see too little concreteness, and something romantic, in this approach. but the fact is that a full understanding of our role as human beings requires both perspectives: function and relationship, doing and being. it is a characteristic of being human that we can have an indepth understanding of ourselves based on the relationships in which we are involved: to ourselves, to each other, to the entire creation, and to the ground of being itself. we can also gain a deep understanding of our mission as human beings, our function: why are we actually here? as we face the climate crisis, we need to focus on rational action inspired by the best science available, while also needing to have an existential understanding of how and why we feel and act as we do. destroying biodiversity; wrecking forests and wetlands; poisoning water, soil, and air-all these are violations of our mission as human beings. theology calls it a sin. this sin arises from our inability to see the earth as our home, a sacrament of community. our natural environment unites all the people on earth with every living thing, in a way that transcends any differences in faiths and convictions that may exist between us humans. experiencing the beauty of nature means a lot to us. but we are also created for another type of beauty: that people have quality of life, live in harmony with nature, meet in peace and help each other. if we want to have an ecologically, socially, economically, and spiritually sustainable approach to the world-which we must have-individual or commercial solutions will never be sufficient. this is why spiritual maturity is now required. such maturity means being able to see the difference between what i want and what the world needs. it can understand that the climate crisis is rooted in human greed and selfishness. it can elevate us above fear, greed, and fundamentally unhealthy ties. if we want technological development, fair and just economic systems, ecological balance, and social cohesion to work together to create a sustainable future on our earth, we also need a conversion, a new state of mind. a renewal of our humanity (in the dual sense of the word). it is not sufficient for us to only address the symptoms if we really want healing and wholeness. like pope francis, we are of the opinion that we are in urgent need of a humanism that is able to bring together different areas of knowledge, including economics, to form a more integrated and integrating vision. science, politics, business, culture, and religion-everything that is an expression of humankind's dignity-need to work together to put our earthly home on a more stable footing. real stature among leaders and rulers of various kinds becomes apparent when we in difficult times can maintain high moral principles and focus on the long-term common good. in these days, the bishops of the church of sweden will be issuing a bishops' letter about the climate that highlights these issues in more detail. the climate deadline is coming ever closer. indecision and negligence are the language of death. we must choose life. give the earth the opportunity to heal, so that it can continue to provide for us and so that people can live in a world characterized by fairness, justice, and freedom. archbishop antje jackelén doi: 10.1111/dial.12557 thoughts while sheltering … in the midst of a crisis it is easy to make statements that later seem unnecessarily alarmist. i do not think i am the kind of person who normally sounds alarmist (but who really who thinks that they are alarmist?), but it is hard to imagine that covid-19 will not remake our lives in ways we never could have envisioned a few months ago. it is hard to imagine that our lives-collectively and individually-will not be forever changed. some thoughts: 1. i do not generally read god's wrath and judgement into current events and i am not prepared to do that now. that said, i find myself wondering if covid-19 will not change our lives in a manner similar to the tower of babel (genesis 11). this story is, among other things, an account of how a united humanity became divided. i wonder if covid-19 will not threaten to (further?) divide us. 2. others have written and commented about the relationship between covid-19 and climate change. many of these people are much more knowledgeable and smarter than me. i plan to listen even harder to them. for much of my adult life, i have attempted to walk with a light environmental footprint (e.g., i have walked or rode a bicycle to work for over 25 years). in the past year, my wife and i have doubled down on such practices and we walk with an even lighter environmental footprint. i have a feeling that others might be joining me in the future. 3. i wonder if covid-19 will not accelerate the already fast pace of secularization in western societies. the churchfairly or not-has been associated with the status quo and thereby irrelevance by many people (especially younger people). with "physical distancing" forcing worshipping communities to meet virtually and disembodied, can they matter? what is life together if it is virtual and disembodied? i am not a luddite who wants to destroy technological tools. i am, after all, writing this because of the miracle of modern computer technology. however i think that social media and virtual life supplements, not supplants, embodied life. 4. i wonder if covid-19 might not reverse-or at least stem the tide of-the pattern of secularization and irrelevancy of the church. the church mattered in the early middle ages because of its commitment to caring for the sick and vulnerable. i am thinking, for example, of gregory the great who while he was pope used the wealth of the church to feed the hungry and care for the poor. is covid-19 such a moment for the church? 5. i think about hospitals and monasteries in the middle ages. they were beacons and refuges for christians fleeing plague and pestilence. hospitals and monasteries were beacons and refuges for christians to care for others who were fleeing plague and pestilence. these hospitals and monasteries were outposts of civilization in wildernesses of savagery and barbarianism. does the church need to reclaim that part of its history and heritage and make it more central to its mission and identity? 6. i think also of the babylonian exile. israel had to rethink what it meant to be faithful when it had no temple for people to worship in and bring their offerings to. what will it mean for us in the 21st century if we cannot gather together in the ways we have always gathered? i finish here with only six thoughts. god worked for 6 days and then rested. i will rest also with these six thoughts and observe a kind of sabbath. i will be thinking on god and the ways of god and who we are called to be. my seventh thought is a sabbath thought. it is a thought, such as it is, of worship, prayer, and contemplation. david c. ratke doi: 10.1111/dial.12558 the corona crisis unmasks prevailing social ideologies the current covid19 pandemic shows that dominant ideologies of our age-from individualism to social constructivism-fall short in meeting reality by disregarding the wider ecological community in which we are situated. human beings, for sure, play an increasing role in cultivating, shaping, and also destroying our shared world. maybe the corona virus experience teaches us to recover the importance of human communities as well as our place in ecological communities? it seems that neither individualism nor social constructivism stand the test of reality. if there is anything the corona crisis teaches us, it is that our lives are interconnected. there is no human being who only inhabits his or her own little world, and who is in charge. we are part of a great human community-for good and evil. we infect each other, yes, but we also live off each other's infectious smiles. what would our lives be like without close eye contact and bodily expressions of welcome? community is the first and most important part of our lives, and during quar-antine we experience how much we miss the normal social interaction with each other. in the meantime, we are thrown back on ourselves, or the very closest ones. there resides some truth in every ideology, otherwise it could not attract our attention, and be infectious. liberalism is the view that every citizen should have as much freedom to live as possible. most of us agree on this value across the political spectrum from left to right. yet the fact is that we are the blacksmiths not only of our own happiness, but also of our misfortune. the misery is that we cannot know in advance. but more than that, we also share the misfortune of others. self-restraint is necessary precisely because it is a primary fact that my desire for freedom and movement can put others in bondage and immobility. since the 1950s, existentialism has been a very widespread ideology. it still is under the guise of being against all ideology. since jean-paul sartre, existentialism has argued that you are what you do. it is your free decisions that give you the essence and character of your particular humanity. existentialism is a humanism, as sartre called his 1946-program. true it is that we live every day with small choices about where to go, but the idea that we are "decision-makers" all the day is an extremely forced view. fortunately, most of us do as we usually do, and if we do not, others would not be able to count on us. a human being who constantly decides pro or con would be an incalculable human being, a constantly ticking bomb under enduring relationships. an existentialism without the "humanism of the other person" (levinas) is a monster beyond the possibility of attunement and self-correction. the ideology of existentialism lies in its individualism. fortunately, however, we live in communities where we, as resonating beings, constantly tune in to each other. hopefully, we also live the greater part of our lives in a pre-conscious stream of experience that precedes our small and large decisions. otherwise, we would quickly become sleepless persons, incarcerated in a hyperactive consciousness, and eventually we would become insane. in short: it is not very often my decisions that determine who i am. rather, it is the sum of the resonance-and-dissonance experiences of my life that determines who i am and what i do. the community exists before the conscious self-awareness of the ego. alongside the over-spiritualized view of existentialism, we find another ideology, which sees a human being as the exclusive owner of a physiological body, curved in around itself. we could call it the skin-and-hair ideology. bodies, however, do not only include skin and hair, bones, and internal organs, for we live as socially and ecologically extended bodies. what we can learn from biology is that our bodies are in constant exchange between one's own body and everyone else's. humans are "holobionts," an organismic space for a variety of lifeforms. in discussing the nature-nurture problem, the controversy has been about how much genes (nature) determine us, and how much our society (nurture). but inside our body we do not only carry our specific human genome, but also a wider microbiome, made up of all the viruses, bacteria, and fungi that have entered our body from the outside into our nose, throat, ears, and not least gut-through food consumption, fluids, and the inhalation of air. overall, we should be grateful for the world of microbiota, for most microorganisms are symbiotic. without bacteria and viruses, we would curl up on the floor with abdominal pain, and we could never be able to "make decisions." overall, we need to be good friends with our bacteria and viruses. only a few are as harmful as covid-19, and here we naturally have to go into counter-procedures such as cleansing, preventing, and quarantining. as far the medical science goes, we do not have a cure at present. hence, the respirators will be running until the corona infection is over. let me now address what i see as the most widespread ideology in our time, at least in the academy: social constructivism. this ideology has spread from sociology to psychology, politics and pedagogy, and social constructivism has ended up being a quasi-orthodox consensus ideology within the humanities and substantial parts of theology as well. this movement's first epicenter was the book the social construction of reality, written by sociologists thomas luckman and peter l. berger in 1966, followed up by berger's the sacred canopy in 1967, focusing on the religious construction of reality. being a circumspective scholar, berger soon after realized the weaknesses of social constructivism but by then the ideology had already infected wider parts of the social sciences. its thesis was, and henceforth is, that human societies construct reality through language perception and the maneuvers of rhetoric, political, and social engineering, and that human societies do so under only a minimal resistance from pre-linguistic reality, including nature. again, there is an aspect of truth in this idea. the ways in which we use language and discursively define the boundaries of society do indeed have impact on the public perception of reality. for example, the political responses to the corona crisis show the considerable impact of our political constructions of reality. state leaders are the ones who have capacity to define states of emergency, and in an exceptional situation governments act as quasi-sovereign powers that determine the social reality for the general population. it cannot be different, but political decisions differ from country to country. do we proceed as in south korea and taiwan (with large screenings of the population and subsequent quarantines)? do we do as in denmark (with an early and strict lockdown but initially without many corona tests)? or, do we choose like sweden (avoid strong coercion but appeal to the population)? by comparison, the overarching federal strategy in the united states still (march 29, 2020) seems oscillating. this being the case, social constructivism does not sit well with a common sense realism: it is the de facto spread of the covid-19 infection, and the subsequent fatalities, that will determine whether our political measures have worked or not. in an infectious world, politics combines wait-and-see attitude with post hoc maneuvers. even the most powerful politicians cannot talk away covid-19. either you have it or you do not have it. either you infect others or you do not. either you will see an exponential spread or you will see a flat rising curve. thus, it is the spread of infections that tests politics, not the other way around. social and political constructs are not capable of defining reality. it seems that even the most clumpydumpy politicians are beginning to understand the reality test, after having tried to downplay covid-19 rhetorically. accordingly, what we need in the academy is a thorough revision of the prevailing ideologies of our age: individualism, social constructionism, discourse theory, etc. we need a biocultural and ecological paradigm shift within the social and human sciences, including theology. otherwise we see people of faith as individual faith decision-makers, and we overburden one another with overheated appeals to letting god come to our mind, as if we could conceptually enframe god. yet if god at all is, god is prior to our consciousness and our self-aware pious decisions. if god is, god is present to the child, and present to us when we are using our full energy and attention in solving a problem, when we are falling asleep, when we are aging and entering into states of dementia and no longer in conscious contact with god. the faith of any individual (each in his or her individual manner) is rather about tuning into a deeper reality-a reality which is already there, as the prime and pervasive source of resonance, present in a divine personal form beyond my own little personhood. faith is about plugging in, of moving into the prior reality of the divine self-communication, in words as well as beyond words. similarly, revising the assumptions of the skin-and-hair ideology, jesus is not a bygone entity, a "composite entity" of (a) a divine entity, (b) a skin-and-hair body, and (c) a particular lonely soul, as some analytical theologians redescribe chalcedonian christology in a so-called "compositional christology." but god was not incarnate in a man cave, but conjoined the shared flesh of humanity, shared also with non-human creatures beyond the skin of jesus. by becoming incarnate in jesus and in his extended body (also called the reign of god), god is no less present in the compressed respirator tents than in the open sunlight and fresh air. god is radically being there, being there with others and being there for others, not least for us who are gasping for fresh air. now back to us who hope to survive and go on. what kind of a reality do we hope to wake up to after the corona crisis? i guess we are waking up to a deeper sense of how much we miss one another, after we have had to separate ourselves from each other. we are missing the abillity to look each other in the eyes (not mediated through a screen), missing to give hands and hugs. we are missing the deep meaning of having skin. i hope that we may rediscover that our community is prior to me as individual, and that the interests of others precede my considerations of myself. it seems to be obvious that the corona crisis has unmasked the castles in the air that we have erected in our ruling ideologies, not least within the academy. individualism and social constructivism-both presuppose a remoteness of human existence from the world of which we are part. both tend to see individuals and communities as isolated islands, who are ceaselessly at work in imposing a human order into a presumably blank world slate. yet nature is not a blank slate, but is full of multiple life and regenerative powers. moreover, nature is not just "out there" but also "in here." we carry nature deep within ourselves, and our entire existence and well-being depends on it. this should not come as a surprise to theologians who speak of god as the benevolent creator of all that is, on the fields and work life, in our houses, and in ourselves. we need to be more than humanists in order to be truly humane. we can no longer pretend not to be deeply connected to circuits larger than ourselves, for we are at once symbolic creatures, living in cultures, and symbiotic creatures that benefit from the rich world of viruses, bacteria, and fungi. at the same time, however, we are also vulnerable beings. this has always been the case but in a global world, this has become even clearer because we travel as much as we do, and live as close to each other as we do in the big cities. covid-19 does something about us before we do anything about it. every moment, awake or asleep, our immune system trains in capturing the viruses and bacteria that make us sick. let us hope that the self-generative powers of nature, endowed by god the creator, will be strong enough to handle the covid-19 in most of us, until we some day can find a vaccine. in the meantime, let us look forward to being able to return to our beloved communities. "into the community" could conveniently become the new mantra after the corona era. university of copenhagen doi: 10.1111/dial.12559 the covid cross pandemic. it is not a word that falls easily from the lips. in a highly scientific and technological society it may strike one as a bid odd, like something from a more primitive past. that is the power of nature and a sobering reminder that while we have come to control many things in it, nature still can transcend our power and understanding, even with fatal results. this pandemic has reminded us all too clearly how limited human power is. it also brings into clear focus how thin and vulnerable human society is when the whole world can be turned upside down in a matter of weeks. in such a world being ravaged by an "invisible enemy," where is one to turn? the fact that one cannot see it or easily trace it places in the heart a fear and anxiety not unfamiliar from the middle ages. the existential experience is the same. we are left with a feeling of vulnerability against an unknown power greater than ourselves and for which we as yet do not have any strong defenses. evolutionary biology crashes into human society. to "shelter in place" and "social distance" are pretty basic but limited responses, ones not unfamiliar from centuries ago. we have been driven back to the most elemental of human responses, isolation. where, then, is god in the midst of pandemic? here incarnation meets the deepest of human needs, affirming god's identification with and understanding of our suffering and anxiety on the covid cross. when there is no obvious ultimate cause or reason, perhaps the only possible source is god. but, if god, then why would a good god do such a thing? for divisive theological dualists the next step is natural, god must be mad at us for something we have done and is punishing us. since it cannot be our fault, the search is then on for a scapegoat, whether it be 'gays' with the aids crisis, new orleans' perceived licentiousness for hurricane katrina, or america's secularism for 9/11. for some today the source must be china, the lgbtq community, or environmentalists. it is theodicy at its most brutal, and it must be challenged. a free creation and human greed combine to make an international disaster, not divine intervention. it is here that the cross confronts the covid-19 virus, not with platitudes or panaceas, with naming and blaming, but with the affirmation that god is with us. the first century world of jesus was a time of disease and death such that much of jesus' ministry was spent in healing from disease and disabilities. it was not unfamiliar to him. such is the nature of enfleshment. if one takes enfleshment with all biological seriousness, as niels gregersen does in his concept of "deep incarnation," (see the cross of christ in an evolutionary world), we can understand that god identifies with human suffering at the most basic of biological levels. the suffering of the covid virus is not foreign to god and therefore we are not left alone within it. it means that god is with us in all the biological suffering of an evolutionary world. while the source of the virus is not definitively confirmed, currently it is believed to have originated in bats (as a number of other coronaviruses have), which perhaps bit a pangolin (a sort of plated anteater), which, as an endangered species, was illegally captured and sold at an illegal wild animal market in wuhan, china. the source of the pandemic? human greed. to paraphrase winston churchill, "never have so few done such harm to so many." theologically we would call this a result of human sin. it requires human capacity to take something biologically derived and place it on the world market. had the pangolin been left alone, perhaps this would not have happened. at such a time of anxiety and isolation, there is a deep longing for hope, meaning, and perhaps forgiveness. to understand the enfleshment of god as deep incarnation, connecting throughout all biological creation, means that no creature, including the human, is truly separated from god, especially those who are dying alone from the virus. if god is truly present to us at the most intimate levels of our existence, then so too is the divine promise. this takes immanuel, "god with us," to a whole new level and connects the present suffering from the covid-19 virus to the cross of christ. it affirms that even if our cognitive faculties or awareness are not functioning well (or at all) that god is still with us. it is not our awareness of god that makes god's grace effective in our lives but god's awareness of us! that is the ground of our hope, not our own reason or strength, even as we pray that a medical solution may soon be found. as creator to creation one might metaphorically say that god is "entangled" (non-local, relational holism) with creation, ourselves included, at the foundational levels of material existence analogous to entangled subatomic particles (see simmons, the entangled trinity: quantum physics and theology). deep incarnation is a way of thinking christologically about the redemptive entanglement of the creator with the whole of creation, giving us hope and release from fear and anxiety as this is carried up into and transformed by god. this foundational relationality then grounds divine presence in a suffering world and provides a connectivity for accompaniment and hope in the midst of decline and loss. it is the covid cross. such accompaniment is also expressed through the medical professionals and others who are working tirelessly, and with some personal risk, to help everyone survive throughout the world. this too is an expression of god's care and love within an entangled creation. transcending one's self-interest for the sake of the ill other can certainly be understood as a gift of the spirit. pandemic reminds us that we too are part of that same entangled creation and that we are also our brothers' and sisters' keepers for we are all in this together. perhaps this may be one of the most hopeful outcomes from such a horrible pandemic. doi: 10.1111/dial.12560 there is the existential angst that comes with self-quarantine and the awareness of why it is necessary-we call it "plague dread." and then there are the various levels of explanation, the micro-meanings, you might say. and then there is the mystery-the big meaning, macro-meaning. each of us will fill in the dread with the facts of our own life. i am approaching age 90, with at least three of what the media call "underlying conditions"-more than enough empirical ground for me to dread the coronavirus. almost hourly, we hear precise scientific descriptions of the virus. these descriptions are crucial, because they enable competent people-physicians, nurses, and researchers-to treat the disease and even prevent its spread. the scientific theory of evolution helps me understand our situation. the coronavirus is an example of an evolutionary process wrapped within larger evolutionary processes. the behavior of the virus follows darwinian expectations. all of the processes that take place within our bodies-from the nano and molecular levels to the cells-follow the same evoiutionary pattern. these evolutionary processes within us are fundamentally ambiguous in that they bring us life and they also bring us death. leonard hummel and gayle woloschak describe this ambiguity in their fine 2017 book, chance necessity, love: an evolutionary theology of cancer (cascade books). this presents us with a dilemma-we are grateful for the life-giving work of our internal body processes, and we dread the deadly work of those processes. like cancer, the presence of coronavirus is fully "natural." nature within us is "naturally" ambiguous. further, these micro-evolutionary processes take place within a much larger story of evolution with several chapters: the evolution of life, which began millions of years ago, within the larger 4 billion year-long story of planet earth's evolution, within the still larger story of cosmic evolution, 12 billion years in the telling. our response to covid-19 is to resist the flow of evolution and redirect it. that is what our practice of medicine is about, the attempt to redirect evolutionary processes in our favor. the long processes of evolution bend because of our efforts. this reminds me how infinitesimally small we are, and yet how amazingly gifted we are. evolution has brought us life and also the skill to reorder evolution itself. nevertheless, despite our efforts, even when they are successful, the struggle with evolution takes its toll-and that means injury and death. in my caseevolution in my mother's womb caused me to be born with spina bifida, which, though moderate in severity, has radically impacted the last 10 years of my life. even as i write, i am aware of the mystery (note the capital "m") that wraps around us. we-and these incomprehensible processes of evolution-float in a sea of mystery. why is it that our existence is woven on this vast and complex loom of evolution? why has god chosen this particular way of bringing us into life and sustaining us? many thinkers down the millennia have pondered this "why?"-and they have given us no satisfying final answers. we can probe mystery, but we cannot resolve it like a puzzle. the book of job speaks to me at this point. when job raised the question and demanded god's response, the voice from the whirlwind spoke to him: your mind is too small and weak to comprehend the height and depths of mystery-you simply must accept it and trust it. the existentialist albert camus acknowledged the mystery, and he believed it is indifferent to human hopes and longings; we cry out for answers for our lives, but in return we hear only silence-he called it ultimate absurdity-absurdity with a capital "a." his novel the plague is the story of life during a plague. the plague was indifferent to human existence, the epitome of absurdity. others have called the mystery enemy, malevolent, intending to destroy us, if it can. christian faith calls the mystery friend, redeemer, suffering god. much like the message of job-death at the hands of the mystery is real; our attempts to understand it are futile; but the same mystery is our redeemer. we can trust it. after all, evolution is a process-faith believes the process is going somewhere, and that "somewhere" is in the life of god. the life of god is love, which is why in the midst of plague we find love, caring for others. medically, for most people our current plague will not have serious consequences. psychologically and economically, it will damage most people, at least to some degree. a small percentage of people will die. all of us will be borne along the same evolutionary process into our future. and for all of us, that future will be god's gift to us. think of the image of a train. some of us will get off the train at this station, everyone will get off sooner or later, at different stops. every station's name will be the same, "god's destination-love." to imagine that my words may speak to you well by the time they reach you seems like magical thinking. no one seems to know exactly where we are. our slow and then sudden awareness of the impacts of coronavirus left us in an existentially halted, almost eschatological space: we were caught in a world incredibly arrested and incredibly new at the same time. we're deeply aware of old tensions of injustice and vulnerability pulling taut, and simultaneously many of us feel the grit of the irreducible relationality of our bodies and planet anew. we've picked up familiar embodied routines in vital work and mundane practices, and yet now many of the familiar kin that once nourished us with convivial learning, signs of peace, earthly delights, bread and wine are learning to do so again with virtual creativity or picking up pieces. if we are honest with each other, there have been many world-ending plagues before-many apocalypses "now and then," as catherine keller says. native peoples know well the injustices and radical loss of histories of settler violence and plague; so too do lgbtiq folks know the ways that homophobia shaped responses to hiv/aids crises. even theologians from julian of norwich to martin luther knew the risks of bodied life together. we are "mutually bound" in moments like this one, luther himself wrote in his now much-cited 1527 letter, "whether one may flee from a deadly plague." these moments of crisis won't be the last, as much as we aim to prevent loss. earthly creatures are vulnerable and resilient, enfleshed with possibilities both tragic and felicitous. if the old kingdom of our everydayness met its match in the new kingdom of the present, the coming future that cultivates such anxiety in so much of our theological and ethical communities already only intensifies with unknowns. as life began to shift in ireland, i was waist-deep in a sabbatical research-ing the complex emotional, affective, and felt responses to the climate crises of our time. from eco-anxiety to environmental despair to climate grief, the present and anticipated losses aggregate and will continue to do so. the affective and emotional energies that mutually bind us in the midst of our planetary crises-including that of pandemic-are just as much part of the crises and ethical responses as the scientific approaches we desperately need. in an interview with the harvard business review, expert on grieving david kessler (known especially for his work with elisabeth kübler-ross), reflected that what the pandemic brings with it is "a number of different griefs." we (in all of the manifold diversity that term names) are grieving our imagined present, a sense of normalcy, our planet, our loved ones, our work, our relationships, our habits of interactions in the world, and more. more particularly, kessler argues that something called "anticipatory grief" is in the air. "anticipatory grief," he says, "is that feeling we get about what the future holds when we're uncertain." 1 in unhealthy ways, anticipatory grief morphs into shifting anxieties and end of the world imaginaries. in richer ways, it acknowledges that our lives undergo transformation into the future and that we must find ways to re-story the present, cultivate resilience, imagine and take action for better future societies. honoring grief is an active process that we undertake together in moments like these. and sometimes that process means we do the long, hard work of actively grieving our loved ones and gentle hopes for our future as they really do change forever. outside of pastoral care, affect, emotions or feeling rarely get much consideration in systematic or constructive theology. theology, in its rational and patriarchal guises, so often belittles affective archives as beneath the intellectual purity of doctrinal thinking. yet, theology at its richest and most compelling is felt, is inscribed emotional depth-not as cheap sentimentality or sensationalism, but as imaginative wondering, grieving, transforming, pacing in awe and praise in the middle of the night, lamenting loss in the middle of the day, and crying in terror or joy. even the driest of systematic theology sometimes can't escape tears when it anticipates our own angst and anticipations. when we do, we human animals make theology and theopoetics with everything we've got. we unleash our manifold imaginations to handle newness, especially in times of immense cultural grief. i want my theology to learn how to grieve better, especially in a time of pandemic. most researchers into climate grief will tell you that learning how to grieve a present moment opens up the possibilities of our relational connection. these psychologists, literary theorists, scholars of environmental humanities, and poets ask society to move beyond feelings of ethical individuality (e.g., if only i made "greener" choices) to ethical collectivity (e.g., if only we organized for structural transformation to a better world). grieving means we are thinking about relationality, shared worlds, and communal possibilities human and more-than-human. deep calls to deep, and the pathos of shared imagination can cultivate attentiveness to those who need care. that connectivity of spirit may lead to collectively questioning and lamenting power structures or unjust relationships in the world. questioning may lead to refiguring expectations to ask what the next possible course of action might be. that's just one possible route. along that route, the most curious feature of the literature of environmental despair is a persistent emphasis on the importance of play for times of transformation and collective grief. the vitality of playfulness may seem counterintuitive when everything is so dour. think, however, of the creativity emergent in our moment: churches playing with virtual connection, people taking up sourdough starters and knitting, movie nights with strangers over twitter, students coloring rainbows for their windows to encourage, reenergized hikes and reimagined forms of community, families performing skits and songs. play is how we grow, open our minds to what is next, and learn to create with what materials we have. even in moments of dire need, new creation can begin to emerge to help us connect and feel our way out in new imaginative and physical planetary landscapes. playing and creating joy in the wasteland, making possibilities in the midst of the ruins of dashed hopes is just another name for theology. it seems like a good model for divine creativity: divinity that grieves and transforms in response to our common life; divinity that cocreates out of playfulness with an unfolding creation still called "good." how are you doing? if you ask me that question, i have two very different answers, both of which are true. the first one is that i am fine, and i have much to be thankful for: my health is good, and so is the health of my family; i have a safe home and plenty of food; i have a job and discretionary income to buy hiking poles when i decided that hiking is my new covid-19 passion; and am able to get outside for long runs and long walks. the second one is, i am not doing great. i miss my routine, and i am anxious and disoriented. i feel like i am not very useful right now, and that is extremely painful. i miss my students in particular, and my colleagues and friends as well-i miss being with them in person, and i am sick of zoom. i am still grieving the loss of holy week and easter services, and i wonder what church is going to look like when we can finally gather again. and, i am missing being able to travel and see friends and family. as i said, both of these things are true. i share this because i wonder if you are having some of the same feelings, and if you are, i want to encourage you that it is ok. on the one hand, it is important to acknowledge and give thanks for your blessings; on the other hand, it is important to acknowledge your feelings of frustration and anxiety. it is important to both support and nourish others when we can, and also have a good cry and even a little tantrum when we need to-do not go crazy however; presumably there are oth-ers in your house who might be startled by your screaming. we are in uncharted territory, all adjusting to a new normal that seems to continually take from us, and we need to give ourselves permission to take time to recalibrate. but even in the midst of it all, we do not lose hope. even if we cannot see it, because the end of the tunnel still seems so far away, there is light there waiting for us. we will get through this, and we will find ourselves on the other side. we will be together once more, and my hope is that we will treasure the daily rhythm of our lives-and the people we share it withall the more for their absence. in the meantime, care for yourselves as best you can, and care for others. accept mediocrity in some things-now is not the time for perfection. do not lose heart. persevere. breathe. love. and when in doubt, love some more. united lutheran seminary philnevahefner@gmail.com doi: 10.1111/dial.12569 global christianity and theological education: introduction to "dialogue in dialog" the papers published in this issue's "dialogue in dialog" were initially presented in two successive luther colloquies held by united lutheran seminary in 2018 and 2019. the essays by madipoane masenya and elieshi ayo mungure were written for a 2018 colloquy on "theology and exegesis in african contexts," along with the essay by andrea ng'weshemi that appeared in the spring issue of dialog. 1 the essays by timothy wengert, kristopher norris, and david brondos were written for a 2019 colloquy on "theological education in the lutheran tradition". 2 the purpose of united lutheran seminary's luther colloquy is to explore the legacy of luther and the lutheran reformation for modern, global, and ecumenical christianity. readers may be interested in the logic behind and the connection between these particular topics. the topics are intimately connected-on the one hand, because the future shape of the church in africa will be determined partly by the accessibility of theological education and the appropriateness of curricula and methods to african contexts. the contributions by masenya, mungure, and ng'weshemi richly demonstrate this point. in turn, the vitality of the church in america may depend on our continued willingness to hear voices that remind us of our connectedness to the global church and our embeddedness in a global society-by our willingness to hear voices that remove the blinders we inherit simply by being born into a particular context and by accepting its structures and self-justifications as given and just. faith in the gospel gives us eyes to see the world anew, to see god present and active and redeeming even where chaos and death seem to abound. but faith comes from hearing, and we in north america need to open ourselves to the power of hearing christians from contexts other than our own and to living in mutual care for one another. theological education plays no small part in inculcating and practicing these habits of hearing and caring. as i remarked at the beginning of the 2018 colloquy, many of the luther biographies that rolled off the presses to mark the supposed 500th anniversary of the reformation spoke of the unintended consequences and even the failure of luther's efforts. 3 in this telling, luther aspired to reform the universal church, but he ended up the leader of a particular church; and the ensuing competition between particular churches and the political authorities aligned with them produced primarily oppression and warfare, before giving way to skepticism and, after a long and weary journey, the separation of church and state that we prize and the pervasive unbelief that we in the church lament. there is much to unpack in this grand narrative stretching "from luther to unbelief"-and this is not the place. but i will say two things. first, judged by luther's own standards, the reformation is not a failure as long as the church lives, the church gathered by the holy spirit through word and sacrament, the church sent into the world to proclaim and serve. luther knew full well that the church is constantly assailed by the false worship of gods less than god. he may not have been so unable to comprehend our world as we sometimes assume! the church today is and can be a force to repudiate the worship of lesser gods and to offer in their place the fullness of god's life and meaning. the second thing to be said is that the story of a straight line from luther to secularization is a story of the northern, western world-a story that readily occludes from view anyone but ourselves. it is a story that is somewhat defensible as an exercise in european and north american self-understanding; it is indefensible as a story that assumes the only meaningful chapter in the story of reformation christianity unfolds between wittenberg and gettysburg, between scandinavia and minnesota. the well-documented shifts in global christian population (including in the lutheran communion) need not be reviewed here. suffice to say: the majority of christians now reside outside of north america and europe, and in due time, the largest body of lutherans will probably be found in sub-saharan africa. there is no question that appropriate remembrance of the reformation in the church should recognize that our past, present, and future are global. that global context, in turn, becomes the context for theological education no matter where it occurs. in my introduction to the 2019 colloquy on theological education, i made these remarks: we live in a moment when theological education-in the seminary context, at leastfaces massive challenges. on the one hand, there is declining enrollment; on the other hand, the rising costs of doing business, including high property costs for older schools with residential campuses. there is also the challenge of serving new populations of seminary students: many students now come to seminary as second-, third-, or fourth-career students, as mature adults with significant obligations to family and community. whether first or later career, students come as parttime students, as commuter students, as distance-learning students. how are their needs to be met, so that they can meet the needs of christians? and if the church is to proclaim the gospel in every place of need, how do we train students for those contexts? one thing is for certain, when graduates leave seminarythey will find a church that needs them. in fact, they will find a church that many times more of them. this fact reminds us that it is not only theological education in the seminary that must be discussed; it is not only the education of pastors that needs to be discussed; the question is: how can church leaders of diverse vocations-pastors, deacons, and others-take their education, go forth, and educate through word and deed as part of their broader vocation? in moments of challenge, we are always in danger of finding ourselves in a reactive state. monumental decisions are suddenly demanded, and one simply does the best one can with faith, acting on principle but on the basis of limited information and limited prior reflection. the resulting action is inevitably constrained both by practical limitations-what else can we do?-and by intellectual constraints-what else can we imagine? what can we imagine if we have not had the time to reflect and study? it is urgent that we use the time we now have to study and imagine, that we think about the purposes of theological education and the ways that theological education must respond to changing contexts-a changing church, a changing worldon the basis of our enduring commitments, above all, our commitment to serve christ's church. as i planned this colloquy, i did not invite speakers to weigh in on any particular set of current proposals for seminary education. in order to evaluate this or that current proposal, in order to imagine alternatives faithful to the mission of the church, we need to bring to bear the insights of our tradition, of theology and history, and of our global church body. i thus invited speakers to address changes and innovation in theological education that occurred in moments of great pressure and even crisis-the reformation itself, the rise of nazi germany-and in the complicated history of christian expansion around the globe. such investigations give us insight into how those who came before us responded to the call of theological education in concrete, difficult circumstances. we can learn much from the thoughts and actions of those who have gone before us, from their successes and their failures: christianity does not invent itself ex nihilo with every new generation; rather we carry into the future a vibrant, living, diverse tradition, grounded in the greatest gift handed down to us, the heart and sum of our tradition, the gospel. theological education is not a task for the seminary alone; it is a core task of the entire church. while the term today often refers to seminary education, what we do at seminary is educate educators. we educate those who must educate others not only about basic doctrinal teachings but also about the depths of christian theological reflection and insight into scripture. we educate those who must teach others not only about doctrine and theology, but also about how we might worship, live, and work together as the church. shaped by seminary, church leaders in turn shape flocks and publics that will go forth and witness to the gospel (i.e., teach others about the gospel), including through the faithful exercise of vocation. theological education is a broad venture, and among the sixteenth century confessions, lutherans were uniquely concerned with the education of the rural peasantry-no other confession in the sixteenth century produced so much literary material that was aimed at rural and small church ministry, at the "simplest" of pastors. this is a tradition that we ought to be proud of; it is a tradition that we carry forward not only in our concern for rural and small church ministry, but fundamentally in our concern that theological education is for all christian peoples in all contexts. today, we look toward a future marked by big challenges and consequential decisions, and as we survey this future and seek to chart our way through it, we are standing on ground that has already shifted. this leads me to the final point i wish to make: as fallen human beings in a fallen world, we frequently respond to change with trepidation; an uncertain future stirs anxiety. much of the movement that has occurred in theological education in recent decades, however, ought to give us cause for hope: we now have women as well as men engaged in theological study and proclaiming the gospel; our understanding of the gospel is now enriched by diverse voices; we have long been a global church, we are now better aware of and better prepared to listen to witnesses from other parts of the world. diverse and global perspectives on scripture and theology and the life of the church challenge us and enrich us in our own contexts. the gospel is a magnificent thing to behold, and church is stronger for seeing its truth and work from different perspectives. tradition is not a zero-sum thing, as if adding a new voice drowns out the old-indeed, new voices can help us see better the depths of what martin luther and so many others wanted to teach us. the richer our field of study, the better we are prepared to do god's work in the world. in conclusion, i want to underline one further groundshift that i have already mentioned: many seminarians, many church leaders in training, are now second-, third-, fourthcareer students. many, whether first or later career, take on the challenge of higher theological study in diverse life circumstances. their willingness to undertake this work is a gift of god. they bring-all students bring-a great diversity of experiences, insights, talents, and vocational skills that strengthen the ministry of the church-that help the church to proclaim the gospel effectively to more people in more walks of life. this too is not a zero-sum game: we are all one in christ, who uses diverse gifts, who welcomes diverse forms of worship, who alone is our redeemer. theological education does face challenges, but it will go on as long as the church goes on. and, as isaiah 40 holds, the word of the lord endures forever. all of us who are involved in theological education-in other words, every committed believer-is called to undertake the venture of theological learning and theological teaching in the spirit of faith, with joyous confidence in both god's direction of our paths and god's redemption of our failings. and we are called, too, to be learners at the feet of the great cloud of witnesses, the great company of teachers who came before us and who span the globe around us. we are called to be learners first from our divine teacher. @worship: liturgical practices in digital words. london: routledge the use of the means of grace: a statement of the practice of word and sacrament digital worship and sacramental life in a time of pandemic sermon at the dedication of the castle church, torgau mn: fortress. (original work published 1544 ce whether one may flee from a deadly plague mn: fortress. (original work published 1527 ce book of concord: the confessions of the evangelical lutheran church d.). first apology. (original work published ca. 150 ce apology of the augsburg confession book of concord: the confessions of the evangelical lutheran church the reformation of suffering: pastoral theology and lay piety in late medieval and early modern germany from sacrifice to sacrament: eucharistic practice in the lutheran reformation facebook society: losing ourselves in sharing ourselves key: cord-329149-1giy1fow authors: martinez-martin, nadia title: technologies for proteome-wide discovery of extracellular host-pathogen interactions date: 2017-02-22 journal: j immunol res doi: 10.1155/2017/2197615 sha: doc_id: 329149 cord_uid: 1giy1fow pathogens have evolved unique mechanisms to breach the cell surface barrier and manipulate the host immune response to establish a productive infection. proteins exposed to the extracellular environment, both cell surface-expressed receptors and secreted proteins, are essential targets for initial invasion and play key roles in pathogen recognition and subsequent immunoregulatory processes. the identification of the host and pathogen extracellular molecules and their interaction networks is fundamental to understanding tissue tropism and pathogenesis and to inform the development of therapeutic strategies. nevertheless, the characterization of the proteins that function in the host-pathogen interface has been challenging, largely due to the technical challenges associated with detection of extracellular protein interactions. this review discusses available technologies for the high throughput study of extracellular protein interactions between pathogens and their hosts, with a focus on mammalian viruses and bacteria. emerging work illustrates a rich landscape for extracellular host-pathogen interaction and points towards the evolution of multifunctional pathogen-encoded proteins. further development and application of technologies for genome-wide identification of extracellular protein interactions will be important in deciphering functional host-pathogen interaction networks, laying the foundation for development of novel therapeutics. the plasma membrane constitutes a critical biological interface between the cytosol and the extracellular environment of the cell, and consequently membrane-tethered proteins and secreted molecules (collectively termed extracellular proteins) are essential regulators of cellular communication. from high affinity cytokine-receptor interactions to low affinity cell-cell adhesion contacts, extracellular protein-protein interactions (eppis) are key for information processing and coordination of virtually all processes in a living organism. furthermore, given their fundamental functions and their accessibility to systemically delivered drugs, extracellular proteins are particularly suitable targets for therapeutic intervention. in fact, despite these proteins being encoded by approximately one-fourth of the human genes, at least two-thirds of the existing drugs target either secreted or membrane-bound proteins [1] . thus, the elucidation of the eppi networks on a global scale has become crucial for the biomedical research. however, in spite of their relevance and abundance, eppis are remarkably underrepresented in available large-scale datasets. this discrepancy is due to the low sensitivity and limited compatibility of current high throughput technologies to detect extracellular interactions because of the unusual biochemical nature of the membrane proteins and the intractability of their binding partners [2] [3] [4] . in particular, transmembrane domain-containing proteins are amphipathic, making it difficult to solubilize them in their native conformation, and often contain posttranslational modifications such as glycans and disulfide bonds, which are not properly added in common heterologous expression systems [5] . in addition, interactions between cell surface proteins are often characterized by fast dissociation rates and therefore weak binding affinities, and in consequence well-established ppi methods such as yeast-two-hybrid or affinity purification-mass spectrometry (ap/ms) largely fail to detect these interactions. over the last decade, several innovative technologies have been developed to overcome the aforementioned technical challenges and allow for sensitive 2 journal of immunology research detection of eppis [2, [6] [7] [8] [9] [10] . nevertheless, the mapping of eppis remains a major challenge in biology. infectious diseases result in millions of deaths each year and therefore identifying new candidate targets for improved therapeutic development remains a pressing health concern. pathogens have evolved a myriad of elegant and often complex strategies to invade the host and commandeer host immune responses to allow pathogen replication, spread, and persistence in the infected organism. many cell surface molecules serve as entry receptors for initial host cell invasion, and concerted responses to the pathogenic challenge critically rely on cell functions mediated by receptors and secreted proteins. to allow host colonization, pathogens encode highly optimized protein modulators, in the form of secreted molecules or receptors expressed on the plasma membrane of the infected cells or the surface of the pathogen [11, 12] . interactions between these proteins and extracellular host molecules form the foundation of communication between a host and a pathogen and play a vital role in the initiation and outcome of the infection [13, 14] . characterizing host-pathogen eppi networks is therefore of utmost importance to gain a better understanding of the infection process and to inform the development of novel or improved therapeutic strategies. excellent studies on mapping hostpathogen interactions, particularly ms-based analysis of viral infection, have provided a wealth of insight into infectious diseases [15] [16] [17] [18] [19] . nevertheless, similarly to host eppis, a significant hurdle to the elucidation of host-pathogen biology has been the shortage of datasets of extracellular interactions between host and pathogen proteins, partly due to the technical challenges that these proteins present. moreover, an additional consideration when studying pathogen-encoded molecules is that these proteins often lack any recognizable homology with any host molecules, thus precluding prediction of their functions [11, 20] . robust methodologies that permit unbiased characterization of eppi in the absence of preexisting hypotheses are thus needed to elucidate the binding partners and molecular functions of most pathogenencoded molecules. excellent reviews have recently revisited the currently available technologies for proteome-wide eppi discovery [4, 8, [53] [54] [55] . here we discuss the application of some of these technologies to the study of host-pathogen interaction and describe some of the major findings that have recently impacted research in the field of extracellular host-pathogen recognition. protein microarrays and functional genomics approaches are highlighted here as emerging techniques with unique potential for the elucidation of host-pathogen eppi networks at a genome-wide scale. classical biochemical and biophysical approaches are particularly suitable for detection of high affinity host-pathogen interactions, such as those mediated by a viral capsid protein and a host cell surface receptor, or a pathogen-encoded glycoprotein and a secreted host factor. typically, these approaches have relied on the utilization of recombinant pathogen proteins as baits to probe for host binding partners, followed by immunoprecipitation and ms, or biophysical techniques for analysis of ppi such as surface plasmon resonance (spr). spr requires prior knowledge of the possible interacting partners and is therefore unsuitable for unbiased ppi discovery, whereas immunoprecipitation and ms approaches usually fail to detect weak interactions, which often characterize eppis, particularly those that take place on the cell surface. notwithstanding, the identification of the receptors for some of the most prominent pathogens, such as the severe acute respiratory syndrome coronavirus (sars-cov) or the new world arenaviruses, was made utilizing standard immunoprecipitation techniques [56, 57] . despite their inherent limitations, biochemical approaches continue to provide relevant insights into host-pathogen interactions, such as the discovery of the dipeptidyl peptidase 4 (dpp4) as the receptor for the mers-cov just within a few months after the emergence of this virus [58] . in addition, more recently kabanova and colleagues identified the cell surface receptor for the trimeric entry complex ghglgo encoded by human cytomegalovirus (hcmv) [21] . several studies have shown that the ghglgo trimer is involved in the infection of fibroblasts, whereas the ghglul128l pentameric complex is required for entry into endothelial, epithelial, and myeloid cells [59] [60] [61] . in this work, both the trimeric and the pentameric cmv protein complexes were generated as recombinant products and used as baits to perform binding experiments on biotinylated cell surfaces, followed by immunoprecipitation and ms identification of bait-cell receptor complexes. using this approach, the authors identified the cell surface protein pdgfr as a high affinity receptor for the ghglgo trimer and demonstrated that this interaction was required for infection of fibroblasts. interestingly, in the case of the pentameric cmv complex, multiple bands were detected upon protein immunoprecipitation from epithelial cells, suggesting the existence of multiple receptors on these cells, which so far remain unknown [21] (table 1) . different biophysical techniques for detection of ppi, in particular spr, have also proven valuable in the field of host-pathogen interactions. spr offers the advantage of label-free, sensitive detection of interactions between a diversity of ligands in real time, thus allowing calculation of kinetic parameters. spr has been widely utilized to monitor antibody binding to a variety of pathogen antigens, information that has informed vaccine development [62] [63] [64] . the spr technology has also been exploited for discovery of eppis. for example, viejo-borbolla and coworkers utilized spr to screen several secreted and membrane-expressed glycoproteins encoded by herpes simplex viruses (hsvs) for binding to chemoattractant cytokines (the chemokine family) and were able to identify a subset of human chemokines that bound to hsv glycoprotein g with high affinity [22] . recently, day and colleagues utilized a combination of glycan arrays and spr and identified over 60 host-bacterial glycan pairs characterized by a wide range of binding affinities, some of which participated in bacterial adherence to host cells in vitro, leading to the hypothesis that bacteria-host surface glycan interactions may mediate initial attachment to the target cell during infection [65] . despite spr and related methods offering higher sensitivity for detection of transient biochemical and ms pdgfr identified as a high affinity cell surface receptor for the cmv ghglgo protein complex [21] herpes simplex viruses (hsvs) biophysical secreted and plasma membrane-expressed glycoprotein g targets a specific set of human chemokines with high affinity [22] human immunodeficiency virus type 1 (hiv) monoclonal antibodies cd4 identified as the receptor for hiv infection of t cells [23, 24] rhinovirus monoclonal antibodies icam-1 as the common entry receptor for most rhinovirus serotypes [25, 26] plasmodium falciparum avexis basigin identified as the cell-surface receptor that mediates erythrocyte infection [27] human adenoviruses extracellular human protein microarrays elucidation of the extracellular interactome of adenovirus-encoded immunomodulatory proteins [28] pseudomonas aeruginosa extracellular pathogen protein microarrays (nappa) screening of patient sera against all p. aeruginosa extracellular proteins. 12 proteins identified as potent antigens [29] varicella zoster virus (vzv) extracellular pathogen protein microarrays (nappa) identification of 18 extracellular viral proteins that promote humoral responses upon screening of the entire vzv proteome [30] streptococcus extracellular pathogen protein microarrays identification of new streptococcal proteins that interact with fibronectin, fibrinogen, and c4bp factors [31] hepatitis delta virus lhdag antigen plasma membrane microarrays (mpa) 150 candidate interactions identified between viral antigen and plasma membrane proteins [32] simian virus 40 (sv40) plasma membrane microarrays (mpa) 99 candidate interactions between whole particles and plasma membrane proteins identified [32] vaccinia virus (vacv) triceps (ms) 7 candidate cell surface binding partners identified for vacv [33] viral pathogens computational studies insights into global principles of virus-host ppi networks [15] [16] [17] [18] [34] [35] [36] hepatitis c virus (hcv) cdna libraries cd81, claudin-1, and occludin as cell surface receptors and some of the players involved in hsv internalization [37] [38] [39] adenovirus and coxsackievirus b monoclonal antibodies and cdna libraries car identified as a common entry receptor for adenovirus 2/5 and coxsackievirus b [40] sindbis virus sirna screens nramp as cell surface receptor for entry into drosophila cells [41] murine norovirus crispr/cas9 cd300lf identified as a cell surface receptor that determines virus tropism [42] bacterial distending toxins (e. coli) haploid cell screens sphingomyelin synthase 1 and the putative g protein-coupled receptor tmem181 identified as toxin receptors [43, 44] clostridium difficile and perfringens toxins haploid cell screens lipolysis-stimulated lipoprotein and the low-density lipoprotein receptor-related protein 1 identified as the receptors for the bacterial toxins, respectively [45, 46] ebola virus haploid cell screens hops proteins and the niemann-pick c1 (ncp1) transporter identified as endosomal receptors that mediate cytosol access [47] lassa virus haploid cell screens lamp1, lysosomal-associated membrane protein 1 identified as an essential host factor mediating virus release to cytosol [48] adeno-associated virus (aav) serotype 2 haploid cell screens 46 cell host factors identified, including heparin sulfate proteoglycan biosynthesis and intracellular transport genes. the immunoglobulin domain-containing transmembrane protein kiaa0319l identified as aav receptor [49] 4 journal of immunology research population genomics analysis p. falciparum ebl-1 protein binds to the erythrocyte receptor glycophorin b, a highly polymorphic gene in malaria-endemic regions [50] streptococcus phage display 19 bacterial proteins identified as potential fibronectin-binding proteins [51] fusobacterium nucleatum transposon-based mutant libraries the bacterial protein fap2 binds to the receptor tigit and downregulates nk-mediated killing of tumor cells [52] avexis, avidity-based extracellular interaction screen; nappa, nucleic-acid programmable protein array; mpa, microfluidic-based comprehensive protein array; crispr, clustered regularly interspaced short palindromic repeat; ms, mass spectrometry; ppi, protein-protein interaction. ppis than most biochemical approaches, these biophysical techniques have not been exploited for large-scale eppi discovery, possibly due to the low throughput of the available instrumentation and the overall difficulties for generation of the relevant protein libraries. these studies, among many others, have demonstrated the power of the classical biochemical and biophysical techniques for detection of host-pathogen interactions. nevertheless, these approaches require previous knowledge of the pathogen-encoded proteins responsible for binding and the ability to produce such proteins as recombinant reagents, which may be challenging, as exemplified by the production of hcmv entry complexes [21, 66] . alternative methods have been utilized in those cases where there is no previous knowledge of the pathogen proteins required for interaction with the host cells. in this regard, the screening of large collections of monoclonal antibodies raised against membrane proteins has proven particularly useful to identify receptors that mediate viral entry. back in the early 80s, the discovery of cd4 as an entry receptor for the human immunodeficiency virus type 1 (hiv) significantly impacted our understanding of viral pathogenesis and subsequent development of therapeutics [23, 24] . in this case, the well-defined tropism of the virus determined the choice of over 100 antibodies directed against human leukocyte differentiation antigens, of which only antibodies that recognized the surface receptor cd4 blocked viral infection [23] . it is worth noting that similar monoclonal antibody screens have also been utilized for unbiased characterization of viral blockers. for example, colonno and colleagues performed a screen of more than 2,000 hybridomas from mice immunized with preparations of plasma membranes from human cells and were able to find one antibody that blocked rhinovirus binding to its cell surface receptor [25] , identified as the intercellular cell adhesion molecule 1 (icam-1) in subsequent studies [26] . despite the undoubted importance of the biochemical and biophysical approaches to the study of host-pathogen interactions, the aforementioned limitations have motivated the development of alternative technologies for large-scale analysis of eppis. from the initial utilization of microarrays for detection of ppi over a decade ago, human proteome chips containing thousands of recombinant proteins have been generated, some of which are now commercially available. protein microarrays offer the unique advantage of requiring minimal consumption of protein reagents, fast readouts, and relatively more affordable instrumentation. typically, a fluorescently labeled or tagged protein of interest (the bait) is generated as a recombinant product and screened against all proteins in the array [10, 53] . despite the increased availability of highcoverage protein arrays, very few are focused on extracellular proteins and therefore are not suitable for study of hostpathogen eppis. most existing microarray-based methodologies rely on multimerization of the bait protein for increased avidity and detection of weak eppis, mimicking the way these interactions occur in vivo, where proteins are arrayed in the crowded molecular environment of apposing plasma membranes. different multimerization strategies and protein microarray libraries have been developed and utilized for host-pathogen interaction discovery, some of which are described in more detail below. the wright lab developed a novel method for detection of low affinity eppis, termed avidity-based extracellular interaction screen (avexis) [2] . in brief, avexis consists of the expression of the extracellular domain (ecd) of the bait of interest as a recombinant protein, which retains its binding properties while removing the insoluble transmembrane region of the protein. these ecds are tagged with a coiled-coil sequence from the rat cartilage oligomeric matrix protein to allow for pentamerization of the bait and therefore increased binding avidity, alongside a -lactamase tag for detection of baitprey interactions upon incubation with the colorimetric substrate nitrocefin. this multivalent strategy has been used for medium-scale screens, allowing detection of weak interactions between human receptors with low false-positive rates [2] . notably, crosnier and colleagues applied avexis to search for the plasma membrane receptor responsible for plasmodium falciparum infection of erythrocytes [27] . the authors compiled a library consisting of most secreted or cell surface-expressed proteins in erythrocytes and systematically assayed more than 40 red blood cell proteins for binding to p. falciparum protein pfrh5, a parasite protein essential for blood stage growth, expressed as an avexis pentameric bait. notably, the ok blood group antigen basigin was identified as a unique receptor for pfrh5, and inhibition of this interaction was shown to be sufficient to block parasite invasion of the erythrocyte, findings that may importantly inform antimalarial therapies [27] . in later studies, avexis was miniaturized making this approach compatible with the protein microarray format, thus permitting more comprehensive and lower resource-intensive screenings [67] . although this technique should allow for high throughout and sensitive determination of eppis, this approach has not yet been applied to elucidation of pathogen-host interactions. over a decade ago, fueled by the recent completion of the human genome, genentech pioneered a significant effort to identify novel secreted or transmembrane domain-containing proteins, upon careful bioinformatics assessment and high throughput protein purification [68] . these efforts resulted in the generation of a comprehensive human protein library, which was subsequently utilized to develop an extracellular protein microarray platform, consisting of over 1,500 secreted or single-transmembrane domain containing proteins [10] . for the generation of this human protein library, secreted proteins or the ecd of single-transmembrane receptors were fused to different affinity tags and subsequently purified from cell culture supernatants by size-exclusion chromatography. mammalian cells or baculovirus-insect cells were preferentially used as expression systems, to maximize the likelihood of proper folding and glycosylation of the extracellular protein collection [10, 69] . sds-page and multiangle laser light scattering were used to analyze noncovalent aggregation and ensure high-quality protein production. subsequently, the purified proteins were spotted on epoxysilane slides using a nanoprint arrayer, and protein immobilization on the microarray was determined by probing the slides with the relevant anti-tag antibodies [10] . to enhance detection of low affinity interactions, a rapid method to assemble bait proteins (whose ecd was expressed as a fc tag-fusion protein) into multivalent complexes using fluorescently labeled protein a microbeads was developed. proof-of-concept assays showed high sensitivity for detection of weak eppis characterized by micromolar , a minimal off-target binding, and more than 70% true-positive to false-positive detection ratio [10, 69] . over the years, this extracellular protein microarray has successfully identified counterreceptors for a number of human molecules, providing relevant insights into novel pathways that coordinate a multitude of cell functions [70] [71] [72] . receptor discovery. recently, we applied this eppi discovery platform to the study of extracellular viral proteins ( figure 1 ), with a focus on human adenovirus-(hadv-) encoded immunomodulatory proteins [28] . despite the increasing relevance of hadv as both pathogens and therapeutic vectors, information on the interaction of these viruses with the host immune system remains scarce [73, 74] . interestingly, the immunomodulatory proteins encoded by these viruses, termed e3 proteins, show substantial diversity in their ecds across and within viral species and constitute one of the most divergent regions of the hadv genome [75, 76] . given this striking variability, the e3 proteins have been suggested to play a role in viral tropism and pathogenesis, yet the functions of virtually all e3 proteins have remained unknown [73] . in our study, we took advantage of such unique variability to evaluate the effect of viral immunomodulatory protein diversity in extracellular host targeting. screening of a substantial number of e3 proteins encoded by different hadv species using the extracellular protein microarray platform allowed identification of over 50 novel virus-host interactions encompassing 5 viral species, which were fully validated by orthogonal methods [28] . these findings revealed significant diversity in extracellular host targeting and, moreover, allowed identification of semiconserved host targets, pointing towards specific human receptors that may represent previously unrecognized hubs for viral perturbation. furthermore, most of the e3 immunomodulators were identified as multifunctional proteins, suggesting that viruses have evolved proteins capable of interfering with several cellular functions, a strategy consistent with the optimization of limited genomic resources. such economic targeting has been often observed in intracellular targeting [15] [16] [17] , but so far few examples of widespread targeting in the extracellular environment have been reported [28, [77] [78] [79] [80] , let alone a global elucidation of eppi networks, in part due to the technical challenges associated with eppi detection. remarkably, many of the hadv e3 proteins preferentially interacted with host receptors that exert known or predicted inhibitory functions during the immune response (as defined by the presence of intracellular immunoreceptor tyrosinebased inhibitory motif, itim), including lilrb1 [81, 82] , lair1 [83] , and mpzl1 [84] , suggesting previously unrecognized strategies of immunosuppression that may be utilized by other human pathogens [28] . moreover, several of the receptors identified as targets for the viral proteins in this study (including the prominent cell surface molecule cd45) do not have known counterreceptors in the host, supporting the longstanding hypothesis that pathogen molecules drive the evolution of immune receptors and in many instances may represent the most relevant modulators of host receptor function [85] [86] [87] . in summary, such unbiased, microarraybased study of immunomodulatory proteins represented the first large-scale analysis of the ppi landscape of a collection of extracellular immunomodulators encoded by viruses. future investigation of other pathogen-encoded molecules using similar extracellular protein microarrays will likely shape our understanding of the pathogen imprint in our immune system. microarrays. one of the main limitations of any protein microarray platform is the lower protein coverage relative to other genome-wide methods for ppi identification, due to the costs and difficulties for generation of comprehensive protein libraries to be deposited onto the microarrays. in an attempt to address this caveat, ramachandran and colleagues developed a method called nucleic-acid programmable protein array (nappa), in which dnas are directly deposited onto the array followed by protein synthesis in situ using an in vitro transcription and translation (ivtt) system, thus avoiding the need for protein purification [88] . although this promising approach has proven superior in generating transmembrane-containing molecules as soluble proteins, it still remains to be systematically addressed if the extracellular human proteins produced in this manner present the folding and posttranslational modifications necessary for protein functionality. nevertheless, emerging data support the utility of nappa as a useful tool for the study of bacterial proteins. for example, montor and colleagues used a bioinformatics approach to predict the pseudomonas aeruginosa proteins that reside in the outer membrane of the bacteria or are secreted to the extracellular environment of the infected cell [29] . in this work, the authors utilized the nappa approach to screen all predicted extracellular gene products for interaction with sera from cystic fibrosis patients, where p. aeruginosa establish a life-threatening lung infection. from 266 bacterial proteins initially selected, 12 proteins were recognized by antibodies in the sera, indicating that these bacterial proteins represent major antigens that trigger adaptive immune responses in humans. interestingly, robust antibody responses against three previously uncharacterized proteins were detected, suggesting this approach could help identify new extracellular proteins that exert unknown functions during the infection [29] . these results confirmed the utility of the microarrays to detect immune responses against membrane proteins encoded by pathogens, and supported the use of this methodology for diagnosis applications. in this regard, several groups have developed microarrays composed of pathogen-encoded proteins [30, [89] [90] [91] [92] [93] . such pathogen protein arrays have so far being exploited mainly for diagnosis purposes, to allow screening of antibodies present in patient sera for binding to extracellular bacterial or viral antigens on the array. nevertheless, their inherent high throughput and compatibility with multivalent bait approaches makes them a powerful tool for eppi discovery. for example, margarit et al. developed a streptococcus microarray to find novel microbial proteins capable of binding to the human proteins fibronectin, fibrinogen, and c4bp and were able to identify a set of streptococcal proteins that interacted with these factors [31] . nevertheless, despite such pathogen protein-based arrays offering great promise, this methodology remains to be systematically analyzed for eppi discovery. more recently, yu and colleagues applied nappa technology in combination with the halotag-halo ligand detection system to elucidate the interaction network of two effector proteins (sidm and lida) encoded by legionella pneumophila, a highly pathogenic bacteria that is the causative agent of legionnaire's pneumonia [94] . similarly to many 8 journal of immunology research pathogen proteins, these virulence factors lack significant homology to host molecules therefore complicating the assessment of their host targets and biological functions. in this work, the bacterial proteins of interest were tagged with a halotag, a modified haloalkane dehalogenase that covalently binds to synthetic halo-ligands (haloalkanes) that can be fluorescently labeled, thus allowing more robust detection of bait protein binding to interactors present on the array. in this study, more than 10,000 human proteins were expressed on the nappa array using different ivtt techniques, leading to identification of 20 and 18 binding partners for the lida and sidm effectors, respectively, most of them experimentally verified by pull-down [94] . although this study focused on identification of intracellular ppi, the applicability of the nappa-halotag technology for eppi determination should be explored in the future. moreover, bait multimerization strategies should be implemented in order to make this approach more suitable for detection of transient ppis. related to the nappa technology, glick and colleagues recently built a miniaturized platform focused on human membrane proteins. by integrating the microfluidics technology, protein microarrays, and an ivtt system, this group built a new device named microfluidic-based comprehensive human membrane protein array (mpa) [32] . a notable improvement introduced by these investigators was the addition of microsomal membranes to the ivtt system to allow for improved folding and posttranslational modifications in plasma membrane proteins, both common limitations of ivtt systems. in this work, a library of 2,700 human genes encoding for membrane proteins was built and subsequently utilized to screen the large-form delta antigen (l-hdag) encoded by the hepatitis delta virus (hda) and whole viral particles of the simian virus 40 (sv40), a nonenveloped human pathogen. proof-of-concept assays showed encouraging results, with over 75% true-positive rate within a small set of proteins with known interactors and, more importantly, indicated the feasibility of this approach for expression of multitransmembrane-containing proteins, a protein type that has proven challenging given their high hydrophobicity. the mpa screens identified 99 and over 150 interactions for sv40 particles and l-hdag viral protein, respectively, and around 35 interactions were validated by coimmunoprecipitation or protein-fragment complementation assays [32] . to our knowledge, this is the first study to assess eppis using a comprehensive human protein library and whole viral particles (sv40) as baits, a valuable approach that may provide important insights into pathogen tropism, alongside a molecular explanation for the cell surface receptors engaged by the pathogen. further utilization of this platform followed by a more systematic analysis of the candidate hits, including nonspecific binder determination, will be needed to assess the overall performance of the mpa technology. regardless, this platform provides an extended version of the nappa approach that focuses on mammalian eppis and may therefore provide relevant insights into extracellular hostpathogen interactions. the protein microarrays have represented one of the most fruitful approaches for unbiased determination of eppis, including host-pathogen interactions. nevertheless, one of the main limitations of this technology is the need to generate comprehensive libraries, a process that is resource consuming and often not available to many researchers [53, 93] . consequently, although some of the available arrays were designed to cover a significant fraction of the human proteome, any discoveries made using these platforms are limited to the proteins present in each array. the current libraries are likely to continue expanding alongside innovative approaches to facilitate sensitive detection of eppi using protein microarray formats. over the last decade, ms-based technologies have emerged as a versatile, powerful approach to decipher many aspects of the human proteome, including the characterization of protein complexes. excellent reviews on current ms-based technologies, recent improvements, and future prospects for elucidation of ppi networks are available [95] [96] [97] [98] . in this review, we briefly discuss the applications of some of these techniques to the study of extracellular host-pathogen interactions. proteins and interacting partners. the proteins expressed on the surface of pathogens mediate functions necessary for survival, replication, immunoevasion, and transmission and therefore are logical candidates for therapeutic and vaccine design. however, the study of the surface proteome in pathogens, particularly in bacteria is constrained by the fact that commonly used prediction algorithms fail to correctly predict the location of several proteins [29, [99] [100] [101] . despite the characterization of the extracellular proteins and their interactions still representing the achilles heel of most proteomics methods, ms has emerged as an invaluable approach to characterize the protein composition of plasma membranes [5] . to date, several studies have exploited ms-based techniques to gain insights into the extracellular protein composition of bacterial pathogens [101] [102] [103] [104] . for example, palmer and colleagues studied the surface proteome of the tick-borne intracellular pathogen anaplasma marginale (rickettsiales: anaplasmataceae) using liquid chromatography and tandem ms [105] . interestingly, the authors found that the surface proteome of a. marginale isolated from tick cells, despite being less complex than that of bacteria isolated from human erythrocytes, contained a novel protein, which the authors hypothesized to play a function in human cell invasion in spite of its human counterreceptor remaining uncharacterized. this interesting observation suggests a remodeling of the bacteria surface proteome during the transition between mammalian and arthropod hosts, an aspect of the infection that could be targeted to block transmission. similarly, several studies have pursued the identification of the proteins present in viral particles utilizing ms. although these analyses suffer from several drawbacks associated with membrane protein characterization, particularly the poor solubility of these proteins and the low abundance of many plasma membrane proteins, these studies have revealed a complex composition for most of the viruses studied, alongside incorporation of many host proteins in the virions, in most cases with undetermined functions [106] [107] [108] . an interesting observation from some of the studies referred above is the fact that certain bacterial proteins, predicted cytoplasmic by consensus, can be found in the extracellular environment of the cell, where they may play alternative functions. in fact, the number of proteins that are secreted through noncanonical signal sequence pathways is increasingly appreciated [99, 100, 109, 110] . little is known about these bacterial proteins originally described as cytosolic proteins but capable of exerting functions on the cell surface, which some authors have named moonlight proteins, in reference to their potential to exert multiple functions [111] . there is emerging evidence that protein moonlighting contributes to virulence of important bacterial pathogens including staphylococcus aureus or mycobacterium tuberculosis, sometimes in fascinating ways. for example, m. tuberculosis is known to encode two molecular chaperones, cpn60.1 and cpn60.2, which function as modulators of myeloid cells among other regulatory functions [112] . despite these chaperones being by definition cytosolic, cnp60.2 has been detected in significant amounts on the bacterial surface, and either recombinant cnp60.2 or antibodies against this protein efficiently block binding of m. tuberculosis to macrophages [112] , through a potential interaction with the receptor cd43 [113] . in addition, the protein dnak, a hsp70related protein encoded by m. tuberculosis, can locate to the bacterial surface and functionally interact with cd40 [114] and with the hiv coreceptor ccr5 [115] . notably, dnak appears to block hiv binding to ccr5 in vitro, an interesting observation given the co-occurrence of m. tuberculosis and hiv infection [116] . although a more detailed revision is out of the scope of this review, bacterial protein moonlighting, excellently revisited by henderson and martin [111] , is a thought-provoking phenomenon that suggests a much more complex extracellular landscape than anticipated. moreover, such protein moonlighting is in line with the hypothesis that pathogens have evolved multifunctional proteins as a prominent strategy for efficient use of limited genomic resources [17, 28] . another ms-based approach that holds great promise for host-pathogen eppi detection is the recently developed triceps [33] . triceps is a chemoproteomic reagent that consists of three moieties, one that binds the ligand of interest through its amino groups, a second one that binds glycosylated receptors on the cell surface, and a biotin tag for purifying the receptor peptides for subsequent identification by ms. notably, in the initial description of the method, triceps was successfully applied to the identification of receptors for extracellular ligands of diverse nature, such as secreted glycoproteins, small peptide ligands for g proteincoupled receptors, and therapeutic antibodies. importantly, this approach has also been utilized to study cell surface molecules targeted by vaccinia virus (vacv). interestingly, the analysis of vacv binding to hela cells revealed seven candidate binding partners, including the previously identified receptors axl, chondroitin sulfate proteoglycan 4, and laminin binding protein dystroglycan 1. further, downregulation of five out of the seven candidates using short interfering rna reduced vacv infection by 40-60%, supporting the functionality of the interactions identified, at least in vitro [33] . although this technology is still developing and no studies on other pathogens have been published yet, future triceps-based studies promise relevant insights into pathogen interaction with distinct components of the cell surface. as an addendum to the vast amount of knowledge acquired using ms approaches and some of the additional methodologies discussed in this review, bioinformatics offers an in silico systems biology approach that reveals a global perspective on host-pathogen interactions. advances in computation have been fundamental to dissect the complex datasets generated in many genome-wide msbased studies and have enabled the reconstruction of largescale host-pathogens ppi networks, providing fundamental insights into viral disease and hence host biology [15-18, 34-36, 117] . although the computational tools available for analysis of large datasets, in some cases developed in association with some of the high throughput screens mentioned in this review, certainly deserve a focused chapter, a couple of observations are specially notable. commonly observed in these studies is that the intracellular viral effectors preferentially target host proteins that act as hubs (proteins with many interacting partners) or bottlenecks (proteins central to many pathways in the network) [15, 16, 36] . for example, dyer and colleagues built a network of host-pathogen ppi by integrating published information from 190 pathogens [36] . supporting previous findings, this analysis indicated that pathogen-encoded proteins preferentially interfere with host molecules that control critical cellular processes, such as cell death or nuclear transportation, possibly as a strategy to maximize control of the host machinery given limited genomic resources. interestingly, this study highlighted a small set of extracellular host proteins recurrently targeted by several of the viral and bacterial pathogens analyzed, including cell surface receptors such as vegfr2/kdr and collagen, possibly indicating previously unrecognized roles in the immune response against pathogens. although informative, the analysis performed by dyer and collaborators was skewed towards viruses, with a prominent enrichment in hiv strains [36] . more extensive analyses encompassing other human viruses and bacterial pathogens may reveal general strategies of immunomodulation and potential human targets suitable to therapeutic intervention. interestingly, increasing evidence suggests that virus-host interactions are governed by principles distinct to those that dictate within-host interactions [20, 28, 85, 87, 118] . notably, detailed analyses carried out by the xia group highlighted significant differences between virushost and within-host (also called endogenous) interactions, such as the tendency of viral proteins to compete with host proteins for binding to a given receptor in the absence of sequence similarity with the host counterpart or the observation that viral molecules have evolved multiple short linear motifs capable of mediating a number of diverse interactions [20, 118] , features that are consistent with the multifunctional capabilities of some pathogen-encoded proteins [20, 22, 28, [77] [78] [79] [80] 118] . altogether, bioinformatics analysis of virushost interactions suggest that virus-mediated targeting of host proteins is characterized by signatures of pleiotropy, economy, and convergent evolution, conclusions that are supported by emerging experimental data. followed by thorough biological experimentation such computationalbased systems biology approaches will provide a unique tool to help decipher basic global principles of pathogenhost interaction and may reveal novel eppis amenable to therapeutic intervention. alongside protein microarrays-based technologies, ms, and computational analysis, the explosion of the functional genomics field in the last years has revolved the avenues to study pathogen interactions with their hosts, often in high throughput. in brief, genetic screens comprise gain-of-function and loss-of-function strategies, represented by complementary dna (cdna) libraries and rna-interference-(rnai-) based approaches, respectively. these methods were developed more than two decades ago and have been widely utilized by the scientific community, providing fundamental insights into the infection process. in particular, the cdna libraries have proven extremely successful in identifying viral receptors through a gainof-function approach, upon transduction of the cdna library from a susceptible cell line into nonpermissive cell lines. the use of cdna libraries is not reviewed in detail here in the interest of a more comprehensive revision of relative newer genomics-based approaches, such as the clustered regularly interspaced short palindromic repeat/ crispr-associated protein 9 (crispr/cas9) or the haploid cell screens. nevertheless, these libraries have represented one of the most significant technologies to further our understanding of the pathogen-host interaction. for example, early studies made use of cdna libraries to shed light on the complex mechanism exploited by hepatitis c virus for initial invasion of the cell [37] [38] [39] , identified car as a common receptor for adenovirus 5 and coxsackievirus b [40] , and were instrumental to identify slam1 and pvr as a receptors for measles and poliovirus, respectively [119, 120] . in turn, the rnai technology has yielded significant insights into virus-host interactions, such as the identification of the ion transporter nramp as the receptor for the mosquito-borne sindbis virus colonization of drosophila cells [41] . the main power of the rnai technology is that it allows high throughput genome-wide screens and therefore potential identification of essential factors that play roles in different aspects of the pathogen life cycle, including initial interaction with the host cell. although rnai screens have provided tremendous insights into host-pathogen interactions and remain widely utilized [121] , inefficient gene depletion and off-target effects are important limitations of this methodology [122] . technology. the increasingly popular crispr/cas9 technology overcomes some of the caveats often associated with genetic manipulation and holds enormous promise for genome editing and downstream applications, including host-pathogen interaction discovery [123] . although still early days, high throughput crispr/cas9 screens for genome-wide studies have already displayed remarkable results, with high levels of genomic modification, hit confirmation, and strong phenotypic effects [124] . the development of the crispr/cas9 technology has undoubtedly transformed the functional genetic analysis in mammals. recent studies have applied the crispr/cas9 technology to ablate expression of previously identified receptors for viral entry, such as the hiv coreceptors cxcr4 and ccr5, leading to resistance to infection in primary cells [125, 126] . an interesting additional application of crispr/cas9 is the direct editing of viral genes important for viral fitness. this approach has recently been used to target hsv-1, cmv, and epstein-barr virus (ebv) essential genes, leading to a significant decrease of viral replication [127] . these studies suggest the potential use of crispr/cas9 as an innovative therapeutic strategy, as aspect that will surely be further explored in the near future. another prominent example published recently is the identification of host factors that confer susceptibility to the evolutionary related type iii secretion systems, t3ss1 and t3ss2, encoded by vibrio parahaemolyticus [128] . the t3sss are highly complex nanomachines utilized by gramnegative pathogens to inject a variable repertoire of virulence factors into the cytosol of the eukaryotic cells, enabling pathogen adhesion and internalization of modulation of host processes. interestingly, using genome-wide crispr/cas9 screens, sulfation and fucosylation of cell surface components were identified as host determinants of t3ss1-and t3ss2mediated cytotoxicity, respectively. the authors hypothesized that interactions between sulfated cell surface molecules such as host proteoglycans and bacterial adhesins act as facilitators of t3ss1 activity, whereas fucosylated glycans on the surface may serve as receptors for t3ss2 components necessary for insertion of the complex in the host membrane [128] . the crispr/cas9 approach has just started to reveal its power as a tool for unbiased identification of novel eppis, elegantly exemplified by the identification of cd300lf as the cell surface receptor for noroviruses, which, strikingly, was identified as the main determinant for the tropism of the murine norovirus [42] . further optimization of this technology will unequivocally signify a tremendous advance for the discovery of extracellular host-pathogen ppis, the processes underlying host-pathogen interactions and its possible therapeutic applications. haploid cells, in turn, allow the study of recessive phenotypes that can be masked in diploid cells, due to the difficulties of creating true genetic knockouts in mammalian cells. despite yeast being a useful tool due to the simplicity of obtaining relevant mutants at its haploid life stage, the majority of human pathogens do not replicate in yeast therefore limiting the applicability of this approach [129] . in recent years, human haploid cells have been increasingly utilized for genome-wide loss-of-function genetic screens using insertional mutagenesis [43, 44, 47] . in initial studies, carette and colleagues took advantage of the kbm7 cell line, a derivative of the chronic myeloid leukemia cell line (cml) with a haploid karyotype except for chromosome 8 [130] . using gene-trap retroviruses for efficient insertional mutagenesis, the authors generated a genomewide collection of null mutants for most nonessential genes [43] . this approach was successfully utilized to identify host factors essential for the functions of the distending toxins or cdts, potent virulence factors secreted by a number of pathogenic bacteria. in particular, mutagenized kbm7 cells were treated with escherichia coli-derived cdts and resistant clones were isolated, leading to identification of insertions in the sphingomyelin synthase 1 and the putative g protein-coupled receptor tmem181, suggesting that this molecule may serve as a surface receptor for the toxin [43, 44] . similar haploid screens have identified novel receptors for a number of bacterial toxins, including the lipolysis-stimulated lipoprotein receptor for the clostridium difficile transferase [45] , or the low-density lipoprotein receptor-related protein 1 as a host receptor of the clostridium perfringens tpel toxin [46] . in a later study, carette et al. generated a kbm7-derived cell line named hap1, haploid for all chromosomes [47] . similarly to previous studies, the authors used the retroviral gene-trap approach to mutagenize hap1 cells followed by deep sequencing to map more than 800.000 insertions. in this study, using a replication competent vesicular stomatitis virus (vsv) carrying the ebola virus glycoprotein, a previously unknown entry receptor for ebola virus was identified. notably, these haploid cell screens identified six members of the hops complex, proteins known to play functions in endosomal/lysosomal trafficking, as well as the niemann-pick c1 (npc1) transporter as the most prominent hit of the assay. it is worth noting that npc1 is not a surface molecule but rather an endosomal receptor. these findings led the authors to propose a novel mechanism of entry by which ebola virus is internalized into the endocytic pathway, followed by endosome maturation and cleavage of the surface glycoprotein of the virus. endosome fusion, mediated by the hops complex, would allow interaction with ncp1 containing endosomes, triggering fusion and release of the viral genome into the cytosol. multiple cell surface receptors can lead to internalization of the ebola virus into the endocytic pathway [131] ; such redundancy in receptor usage likely explains why these receptors were not identified in the haploid cell screen [47] . notably, independent studies have confirmed that ncp1 acts an intracellular receptor for ebola, including a chemical screen approach, a study showing ncp1 dependence for infection of otherwise nonsusceptible cells, and more recently the elucidation of the crystal structure of this receptor bound to the ebola virus glycoprotein [132] [133] [134] . interestingly, after the aforementioned haploid genetic screens identified ncp1 as a noncanonical entry receptor (given its intracellular localization), other filoviruses have been shown to take advantage of this receptor [135] . the relevance of this intriguing mechanism of viral entry is further reinforced by recent work on lassa virus, an old world arenavirus that, similarly to ebola virus, causes severe to fatal hemorrhagic disease in humans [48, 136] . a genome-wide haploid screen using vsv pseudotyped with lassa glycoprotein was performed in order to identify host factors essential for viral entry. although -dystroglycan (dag1) was long recognized as the cell surface receptor for lassa virus, additional factors were suspected, given the observation that certain dag1-expressing cells are resistant to infection. the authors elegantly demonstrated that at a neutral ph, the lassa virus glycoprotein was bound to dag1, whereas upon exposure to lower ph (resembling the lysosome environment), a receptor switch occurred leading to strong association with the lysosomal-associated membrane protein 1 (lamp1) [48] . thus, similarly to ebola virus, in the model suggested the virus would be incorporated into the endocytic pathway after interaction with its surface receptor dag1, followed by increasingly acidic conditions that would result in interaction with lamp1 in the lysosomal membrane, triggering membrane fusion and release of the virus in the cytosol [48] . more recently, pillay and colleagues applied the haploid cell screening approach to the identification of host factors essential for the adeno-associated virus (aav) serotype 2 infection, one of the leading vectors for virus-based genes therapies [49] . notably, the most significantly enriched gene in these screens was kiaa0319l, a poorly characterized type i immunoglobulin domain-containing transmembrane protein named hereafter as the aav receptor. among the 46 host factors identified as hits, many were implicated in heparin sulfate proteoglycan biosynthesis as well as a number of proteins that participate in intracellular transport processes. aav is known to attach to the cells using heparin sulfate proteoglycans and hijacks endosomal trafficking to travel to the nucleus upon invasion of the cell; thus the authors hypothesized that these additional factors may influence virus tropism [49] . altogether, these studies elegantly demonstrate the power of genome-wide screens in human haploid cells and the power of this approach to study virus-host interactions. future studies should further assess the applicability of this method for general detection of interactions that take place at the pathogen-plasma membrane interface. it will also be important to generate additional haploid cell lines, in order to broaden the range of pathogens and pathogen-derived molecules that can be studied using these genetic tools. in this regard, a number of haploid cell lines have been generated in mammals [137] , unique tools to elucidate the basic aspects of human genetics. discovery. pathogens are among the most intriguing and prominent drivers of human evolution. humans have adapted to the pressure imposed by microorganisms through genomic diversification, particularly through variation of genes involved in immune system function, constantly challenged by the rapidly coevolving pathogen genomes. the advent of new technologies such as next-generation sequencing and the computational tools associated have opened new avenues for the study of human genetics, making it possible to evaluate the contribution of genetic diversity to susceptibility to infection at the genomic level. the emergence of datasets of genomic variation in multiple human populations, as well as pathogen genomes, allows detection of signatures of selection, which can be exploited to identify genes with major roles in immunity (for an excellent review see [138] ). remarkably, the cell surface-expressed receptors are among the most polymorphic gene families in mammals, subjected to strong positive selection and rapid evolution, in many instances possibly driven by pathogen molecules that remain unknown [87, [139] [140] [141] . polymorphisms in receptors and immunomodulatory genes contribute to the natural susceptibility of different individuals to infection [142] [143] [144] , as illustrated by protection against hiv infection in individuals carrying homozygous polymorphisms in the viral coreceptor ccr5 [145] . the identification and further study of genes under positive selection may represent a mainstream approach to dissect novel genes involved in disease and hostpathogen interaction. a notable example is the identification of glycophorin b as the erythrocyte receptor for p. falciparum protein ebl-1 through examination of highly polymorphic genes in populations from malaria-endemic regions [50] . further population genetics studies promise key insights into novel immunological mechanisms and have the potential to provide molecular details that will ultimately help design effective therapies. in addition to these encouraging technologies, the generation of phagemic libraries has also represented an important tool for deciphering ppis, in this case between particular binding partners and the whole genome of specific pathogens [146] [147] [148] . typically, pathogen-encoded molecules are expressed as fusions with phage envelope proteins, a method known as phage display that has been widely exploited to identify peptides with specific binding properties. for example, beckmann and colleagues built a phage display library to identify novel group b streptococci proteins capable of mediating adherence to fibronectin, a major component of the extracellular matrix often exploited for colonization of the host [51] . from this analysis, the authors identified 19 genes with homology to known bacterial adhesin proteins, genes involved in virulence, transport, or metabolic processes, along with genes with uncharacterized functions. interestingly, one of these genes showed significant homology with the scpb protein, a peptidase found in other streptococci that inactivates the member of the human complement system c5a, suggesting that this bacterial molecule acts as a bifunctional protein, similarly to other examples of multifunctional proteins discussed above [51] . more recently, a transposon-based insertion-inactivation mutant library was elegantly utilized to identify a bacterial protein capable of targeting the surface receptor tigit, an inhibitory molecule present in natural killer (nk) cells and t cells [52] . fusobacterium nucleatum is a common oral bacterium that has been associated with colon adenocarcinoma and rheumatoid arthritis among other malignancies. in this study, gur and colleagues showed that different strains of f. nucleatum blocked nk-mediated killing of human tumors. using a library of f. nucleatum mutants, the authors identified fap2 as the bacterial protein that directly interacted with tigit, leading to inhibition of nk cytotoxicity and downregulation tumor-infiltrating t lymphocytes activation. immunoevasion is a hallmark of cancer; however whether members of the microbiome found within the tumor provide cancer cells with immunoregulatory properties has remained a major matter of debate [149] . these interesting findings suggest that f. nucleatum present in the tumor niche may enhance tumor escape by inactivating nk-mediated killing upon interaction of the fusobacterial fap2 with the inhibitory receptor tigit. of note, transposon-based mutant libraries are readily available for other pathogenic bacteria and have been successfully applied to identification of bacterial genes implicated in bacterial physiology [150] [151] [152] . it would be of interest to employ these libraries for unbiased identification of eppis. notably, as mentioned above, we found that hadv immunomodulators preferentially target other immunoreceptors that, similarly to tigit, also play inhibitory functions [28] , suggesting this might represent a common immunosuppressive tactic evolved by pathogens. in fact, there is emerging evidence suggesting that may be the case, as a number of extracellular proteins from unrelated human pathogens have already been shown to target diverse immune receptors with inhibitory functions [28, 52, 82, 87, 153, 154] . further exploration of inhibitory receptor targeting by other pathogens warrants exciting biological discoveries. deciphering the human genome made possible the categorization of genes that encode for the human secretome; now, the challenge of the postgenomic era is to annotate the functions of those genes and their expression patterns during health and disease. a lot has been learnt from painstaking, highly focused experiments using classical biochemistry. in recent years, the impressive technological advances in proteomics, functional genomics, and computation have revolved our understanding of cell communication and function and have collectively created a versatile platform to enable biological discoveries, from mechanistic explorations to big data and systems biology analysis. nevertheless our understanding of the molecules and mechanisms of extracellular immunomodulation and pathogen invasion remains remarkably limited. extracellular ppis between host-and pathogen-encoded molecules orchestrate an enormous diversity of cellular processes, from initial colonization of the target cell to subsequent immune responses. the elucidation of these extracellular interactomes is integral to understanding the molecular basis of infection and will guide the development of more efficient or innovative therapeutics. improvements in proteomics and genomics approaches have exponentially increased our understanding of how pathogens, particularly viruses, modulate the intracellular environment of the cell. concomitantly, we and several other groups have implemented technologies directed towards elucidation of extracellular interactomes [2, 9, 10, 28, 32, 155] , which have begun to reveal fundamental principles of extracellular journal of immunology research 13 host-pathogen interactions. notably, recent studies have revealed extensive eppi networks in model organisms such as drosophila or zebrafish [2, 9] . these undertakings predict that, similarly to intracellular ppis, extracellular networks will be highly connected, with secreted and plasma membrane-expressed proteins having multiple binding partners. however, as discussed in this review, the identification of the host factors and in many cases the pathogen molecules that mediate eppis have largely defied molecular identification, in part due to the technical difficulties inherent to the study of these extracellular proteins. the elucidation of the global principles dictating extracellular pathogen-host ppis will require a coordinated effort to bring together the areas of biology and technology. there are now considerable opportunities for integrating multiple disciplines for eppi discovery, particularly proteomics and crispr/cas9 genome-wide screens, which should be powered by commensurable advances in bioinformatics and computation for big data analysis. the integration of orthogonal datasets coming from multiple "omics" approaches will be advantageous for elucidating the intricacies of the host-pathogen extracellular interactomes and will further enhance the rational identification of novel 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meningitis in a pediatric cohort homozygous defect in hiv-1 coreceptor accounts for resistance of some multiply-exposed individuals to hiv-1 infection cloning of ligand-binding domains of bacterial receptors by phage display m-like proteins of streptococcus dysgalactiae a fibrinogen-binding protein of staphylococcus epidermidis cancer and the microbiota the neglected intrinsic resistome of bacterial pathogens sequence-defined transposon mutant library of burkholderia thailandensis comprehensive transposon mutant library of pseudomonas aeruginosa leukocyte immunoglobulin-like receptor b1 is critical for antibodydependent dengue hsv-1 infection through inhibitory receptor, pilralpha the mammalianmembrane two-hybrid assay (mamth) for probing membrane-protein interactions in human cells the author is grateful to lino gonzalez, jennie lill, erik verschueren, and yvonne franke for critical reading of the manuscript and insightful comments and suggestions. avidity-based extracellular interaction screen aav:adeno-associated virus car:coxsackievirus receptor cmv:cytomegalovirus crispr/cas9: clustered regularly interspaced short palindromic repeat/crispr-associated protein 9 ebv:epstein-barr virus ecd: extracellular domain eppi:extracellular protein-protein interaction hadv:human adenoviruses hcv:hepatitis c virus hiv:human immunodeficiency virus hsv:herpes simplex virus itim:immunoreceptor tyrosine-based inhibitory motif ivtt:in vitro transcription/translation lair1:leukocyte-associated immunoreceptor like 1 lilrb1: leukocyte immunoglobulin-like receptor 1 mpa:microfluidic-based comprehensive human membrane protein array mpzl1:myelin protein zero-like protein 1 ms:mass spectrometry nappa:nucleic-acid programmable protein array ncp1:niemann-pick c1 pdgfra: platelet-derived growth factor receptor alpha ppi:protein-protein interaction pvr:poliovirus receptor sars-cov: severe acute respiratory syndrome coronavirus sv40:simian virus 40 slam:signaling-lymphocytic activation molecule spr:surface plasmon resonance t3ss: type iii secretion system vavc:vaccinia virus. the author declares that they have no competing interests. key: cord-317900-05y9re12 authors: senanayake, nari; king, brian title: geographies of uncertainty date: 2020-08-14 journal: geoforum doi: 10.1016/j.geoforum.2020.07.016 sha: doc_id: 317900 cord_uid: 05y9re12 abstract the question of uncertainty has generated substantial critical engagements across the social sciences. while much of this literature falls within the domains of anthropology, science studies, and sociology, this short introductory paper highlights how geographical scholarship can also enrich emerging transdisciplinary debates on uncertainty. specifically, we discuss how geographers engage with uncertainties produced through and reconfigured by some of the most formidable issues of our contemporary moment, including neoliberal transformation, disease and illness, resource conflict, global climate change, and ongoing struggles around knowledge, power, and justice. in conversation with debates in cognate fields, this special issue brings together contributions that grapple with uncertainty through key geographic concepts such as scale, power, spatiality, place, and human-environment relations. this work extends scholarly understanding of howuncertaintyarises, is stabilized, and also how people navigate, experience, challenge, and rationalizeuncertaintyin everyday life. in doing so, we signal the immense potential offered by emerging intersections between human geography and broader critical social science interventions on the question of uncertainty. uncertaintyand its cognates, indeterminacy, liminality, obscurity, confusion, and misperceptionincreasingly pervade social and environmental problems. they are, for instance, endemic features of neoliberal transformation, health-environment interactions, food (in) security, natural disasters, resource conflict, and global climate change. while the question of uncertainty is not new, it increasingly captivates popular imagination in the context of these highly dynamic, contested and intractable problems, thereby catalyzing academic interest across the social sciences. yet despite the pervasiveness of uncertainty in daily life and forms of academic inquiry, few of the contributions associated with this transdisciplinary literature are geographical. as a consequence, this special issue demonstrates new ways that geographers can enrich work in this field, while also highlighting the potential of the concept to reshape approaches to topics, themes, and foci that the discipline actively pioneers. the articles in this special issue collectively respond to two trends that have historically sidelined the influence of critical human geographic scholarship in transdisciplinary debates about uncertainty. first, while the problem of uncertainty has received substantial attention in academic work across the fields of science and technology studies (sts), anthropology, sociology, and development studies, texts that explore uncertainty through a critical and explicit engagement with geographical concepts are relatively few and far between (we discuss some recent and promising exceptions below). second, within the discipline of geography itself, the question of uncertainty has largely been interrogated by quantitative and spatial perspectives that have given rise to various taxonomies of incertitude (kwan and schwanen, 2018; kwan, 2012; griffith, 2018) . fewer geographers, however, use qualitative and ethnographic methodologies to explore how the limits of knowledge become spatially generative, and/or operate as a direct force shaping social life. this is surprising given that the question of uncertainty is increasingly intertwined with contexts and problems that human geographers have long engaged, such as globalization, disease and illness, migration, and ongoing struggles around knowledge, power, and justice. furthermore, the multiple challenges and/or opportunities created by uncertainties in such contexts arise from and often rework central preoccupations in the subdiscipline of critical human geography; namely, socio-spatial and human-environmental relationships. approaches from human geographers could therefore help enrich existing work through their perspectives on scale, power, spatiality, place, and human-environment relations. with all of this in mind, this special issue offers a timely reflection on geographies of uncertainty and uncertain geographies. we bring together papers that were presented at the 2018 annual meeting of the american association of geographers as part of two sessions organized around the question of uncertainty. our contributors engage with a wide spectrum of qualitative methodologies and empirical case studies in order to examine how uncertainty shapes places and landscapes, social relations, human health, and/or human-environment interactions. while these articles grapple with diffuse subjects, including banana disease, wolf management, pest eradication programs, and human health (hiv, ckdu, and silicosis), taken together they clearly articulate several key themes for future geographic scholarship. first, we explore how uncertainty is often produced through, and temporarily stabilized by, key geographic axes such as time, space, scale, and regimes of environmental governance. attending to the ways that uncertainty is experienced as a spatiotemporal condition, and how it frequently compounds across scales of knowledge production, enables the special issue's contributors to demonstrate how forms of incertitude work through geographic relationships. crucially, this work takes us beyond established approaches that tend to engage with geography only in so far as it helps situate the production of uncertainty within broader social, cultural and political-economic contexts. as part of this discussion, we also contribute to recent moves both within and beyond the discipline that theorize experiences of uncertainty from the spatial margins. beginning from what claire herrick (2017) dubs as "non-archetypal spaces of expertise" the papers assembled here reveal other knowledges and practices that teach us how to live with, and in some instances, to make a living from, experiences of uncertainty. in particular, our contributors are in dialogue with wider debates which suggest that responding to uncertainties often requires combining different styles of knowledge. as scoones (2019a, 29) concludes, bringing together "formal and informal, accredited and lay knowledge, and experiential and conceptual understandings… [are central to] addressing uncertainties in context through culturally embedded, experiential [forms of] learning" (also see: shattuck, 2020a) . a second intervention of the special issue examines how the production and productiveness of uncertainty can generate geographical outcomes and socio-ecological possibilities. a key point here is that while uncertainty can restructure social and ecological relationships, it does so in highly differentiated ways, often with unanticipated effects. as a consequence, our contributors demonstrate how uncertainty can reinforce exiting fault lines of inequality at the same time that it might generate new forms of social, spatial, and ecological difference. third, this collection of articles focuses on how uncertainty is reconfigured through human-non-human dialectics. drawing on a rich tradition of scholarship within the discipline of geography, our contributors grapple with what robbins (2012, 234) describes as "the stubbornness and intractability of certain properties of non-human things" such as the fugitive chemistry of suspected toxin(s), unstable viral loads, materially ambiguous and uncooperative agricultural pests, and illegible wildlife. these complex human-non-human interactions pose important challenges to western scientific and regulatory traditions with "huge implications for the practice of science, management and policy" (scoones, 2019a, 5) . indeed, the frequent failure to classify, record, and regulate unruly human-non-human dynamics opens up possibilities to engage with more experiential forms of learning, diverse knowledges, and democratic responses to the question of uncertainty. in short, these papers speak to ian scoones' (2019b) call to revolutionize "methodologies for science and policy that take uncertainties seriously" by exploring engagements with "experimentalism, citizen participation, transformative action and 'post-normal' science." fourth, our contributors raise critical questions about the politics of uncertainty and in particular, how it indexes new forms of accountability, governance, and regulation. on the one hand, several articles demonstrate how uncertainty is strategically deployed in ways that individuate responsibility for environmental risk. in particular these studies demonstrate how uncertainty is often weaponized to reproduce both the imperceptibility of links between health and environment and the unaccountability of the structural forces that produce them. on the other hand, the papers in this special issue explore the potential of embracing uncertainty as a pathway to open "up a more rigorous, robust, transparentand democratically accountableenvironmental politics" (stirling 2018, 120) . in sum, the existence of uncertainty is generative of politics and possibilities that can be mobilized for very different political (and geographical) ends. more broadly, in the context of covid-19, our intervention comes at a moment of heightened, albeit highly differentiated, experiences of uncertainty and risk. enduring uncertainties about disease prevalence, testing, spread, and treatment have intensified uneven life opportunities and unjust exclusions, while also reconfiguring patterns of mobility, care, and social reproduction (neely and lopez, 2020; dattani, 2020; hannah et al., 2020) . this crisis has also heralded new forms of intervention, regulation, and governance that are unfolding as this special issue goes to press. many of the contributions included in this collection speak to similar themes and demonstrate the entanglements of uncertainty and geography in our daily lives. in reviewing the broader literature associated with this special issue, we were struck by the marginal position human geography occupies within scholarship on uncertainty in the critical social sciences. this is not to say that human geographers have not recently taken up many of the substantive themes that are of interest in transdisciplinary debates. indeed, engagements with experiences of liminality, protracted uncertainty and indeterminacy within the subdiscipline have become increasingly wide-ranging in their ambit, investigating questions on migration and refugee resettlement (loyd, ehrkamp, and secor, 2018; mountz et al., 2002) , development (chung, 2017 (chung, , 2020 , climate futures and the anthropocene (nightingale, 2018) , neoliberalism (anderson et al., 2020) , identity and selfhood (march, 2020) , and humanitarianism (newhouse, 2017) . "uncertainty also appears as a key term in various geographic handbooks and encyclopedias (brown and damery, 2009; stirling, 2018) . within these volumes, scholars argue for greater analytical precision in approaches to uncertainty, differentiating it conceptually from risk, ambiguity, and ignorance (stirling, 2003; . 1 other authors reflect more broadly on how uncertainty poses "[1] philosophical challenges [for the discipline] regarding the nature, origins, and value of knowledge, [2] ethical challenges regarding acceptable levels of risk, and [3] political challenges concerning how to act and who has the mandate to decide" (brown and damery, 2009, 90) . cutting across all of these contributions, is the promise of geographical approaches to shed light on how these multidimensional challenges vary across space, time, and scale, and how they mediate place-specific and human-environment relations and practices. however, only a handful of existing contributions within geography explicitly reference transdisciplinary debates on uncertainty. as has been argued elsewhere, and for other topics, "at the most practical of levels, this means that this rich corpus of highly prescient geographical writing rarely appears in keyword database searches" (herrick, 2016, 672) . more broadly, we argue that the relative invisibility of these promising geographic contributions relates to our failure "to develop the same kind of critical conceptual mass" that has allowed scholars in science and technology studies (sts), anthropology, sociology and development studies to direct conversations on the production, circulation, and lived experience of uncertainty. to date, scholars of sts and histories of science, medicine and environment occupy among the most well-cited and influential niches in the literature on uncertainty. for more than two decades, this work has taken pains to: (a) re-embed uncertainty in material as well as social contexts of knowledge practice (langston, 2010; murphy, 2004; roberts and langston, 2008; hecht, 2009; jasanoff, 2004) , (b) disrupt stable and binary distinctions between certainty and uncertainty as categories of knowledge (proctor and schiebinger, 2008; mcgoey, 2012) , and (c) examine how uncertainty is reconfigured by struggles over definition, legitimacy, and responsibility (brown, morello-frosch, and zavestoski, 2011; dumit, 2006; michaels, 2008; oreskes and conway, 2010; zavestoski et al., 2004) . a central and enduring line of investigation turns on how "unknowing, ignorance, and imperceptions [about social and environmental problems are] not just accidentally but purposefully generated in the history of knowledge practice" (murphy, 2006, 9, emphasis added) . this collection of work investigates how uncertainty is often manipulated or manufactured by industry or interest groups with the aim of displacing responsibility and fending off regulation (see langston, 2010; michaels, 2008; oreskes and conway, 2010; proctor and schiebinger, 2008) . as mcgoey (2012, 4) explains, this well-established body of literature "explores how different forms of strategic ignorance and social unknowing…both maintain and disrupt social and political orders, allowing both governors and the governed to deny awareness of things it is not in their interest to acknowledge." work in these disciplines also documents how scientific uncertainty is strategically marshalled and renegotiated by social movements that organize in response to toxic exposures and environmental crises. this line of analysis has been established in work on medically contested illnesses such as multiple chemical sensitivity, gulf war syndrome, and chronic fatigue syndrome, where patient activism and struggles for diagnosis are often grounded in experiences of being denied healthcare, insurance claims, and/or social legitimacy as sufferers. zavestoski et al.'s (2004; 161) research into guld war syndrome for instance, has argued that these experiences of exclusion often give rise to new collective subjectivities and illness groups which contest 'what they see as… unresponsive medical [legal, and bureaucratic] system[s]' (also see dumit, 2005; ; brown, morello-frosch, and zavestoski, 2011) . as senanayake and king (2019) argue, the intention of this work is to decentralize and potentially democratize knowledge production in response to uncertainty, challenging what counts as risk or harm and who gets to decide the terms and stakes involved. the allied focus on situated knowledge and regimes of imperceptibility in feminist science studies represents another compelling cluster of scholarship on the question of uncertainty. this includes work that foregrounds the indeterminacy and partiality of knowledge claims that "put[s] ambiguity to critical use… through demonstrating how social categories such as woman or man, black or white, straight or queer as well as concepts such as identity and selfhood need not be fixed in order to inform scholarly inquiry of structural and unstructured dynamics of power" (hayes-conroy and hayes-conroy 2008, 469; also see: sedgwick 2008) . for example, a recent review article by loren march (2020) provides a route into the affective and biopolitical implications of liminality, foregrounding experiences of disorientation, performativity and embodiment among trans and queer folks who do not conform to the dominant grids available for categorizing bodies. by destabilizing binary identity categories, this work documents how illegibility and in-betweenness are experienced and produced through space. in dialogue with this work, other scholars investigate how uncertainty generates new forms of social difference and subjectivity that reinforce patterns of vulnerability (auyero and swistun, 2009; chung, 2020) . together this work encourages us to think more deeply about uncertainty as a lived and spatiotemporal condition and prompts us to engage with the disorienting and sometimes potentially deadly consequences of being in-between. similar debates around the question of uncertainty animate scholars in critical development studies and anthropology, finding concrete expression in several anthologies and special issues (samimian-darash and rabinow, 2015; cooper and pratten, 2014) . much of this scholarship grapples with how uncertainty articulates with narrow, modernist views of progress, development, and technocratic risk management (scoones, 2019a; scott, 1998; ferguson, 1994) . this theme is taken up by paprocki (2019), who investigates how donors and development agencies translate uncertainty about climate change into, narrowly defined, normative visions of development in coastal bangladesh. these "anticipatory development futures" elevate commercial shrimp aquaculture and the expansion of an urban-based, industrial economy as preferred adaptation strategies in the face of uncertainty, despite significant evidence that these adaptation regimes result in environmental degradation, agrarian dispossession and 'development-induced migrations.' as paprocki (2019. 307) argues, these forms of anticipatory governance "entail the end of rural livelihoods in the delta, replacing them with a highly stylized (and age old) vision of development where the rural population transitions into an industrial labour force." this work thus brings debates about uncertainty, governance and accountability into sharp relief, demonstrating how uncertain ecological crises can be claimed and mobilized by donors and development agencies to justify interventions that continue to displace agrarian lives and livelihoods. this concern is also taken in a different direction by a recent working paper by scoones (2019a, 5) that draws on uncertainty to challenge "linear, hierarchical, modernist vision of progress… and technocratic managerialism." by theorizing uncertainty from the margins, he asks: "what more can we learn from alternative (including non-western) cultures of uncertainty that construct the world in different ways, through different histories, social imaginaries, traditions of thinking, and everyday practices?" (2019, 10; also see: leach et al., 1999; scoones, 1994) . in doing so, he suggests that pastoralists in sardinia, tibet, and northern kenya have situated knowledge and experiences of living in limbo that could help upend conventional modes of development, progress, and risk management, and in turn create space for imagining more socially just and ecological sound futures (also see: street, 2011; 2014; ; krätli and schareika, 2010; amin, 2013; samimian-darash and rabinow, 2015) . while this transdisciplinary literature is vibrant and compelling, it tends to treat geography as context, situating uncertainty within broader fields of social relations, cultural practices, and political-economic systems. the purpose of this special issue is not to deny the significance of these interventions, but instead to explore how key geographical concepts such as scale, spatiality, place, and human-environment relations can deepen and enrich transdisciplinary conversations on the question of uncertainty. we argue that the relative silence of geographers in these debates is a missed opportunity for the discipline, especially given our scholarship on related themes as well as a long history of critical approaches to many of the topics that are at the heart of this literature. together, this signals the potential for geographical contributions to transdisciplinary debates. we now turn to a discussion of cross-cutting themes in this special issue, to help make some of these contributions more explicit. in organizing this special issue, we collected a diverse set of papers that, while in active dialogue with innovations in other disciplines, remain intent on documenting how experiences of uncertainty, insecurity, risk, indeterminacy, and illegibility are inherently geographical. each of the articles in this special issue make original contributions on their own; however, when taken together they collectively address four core themes for a geography of uncertainty. first, the contributions illustrate how uncertainty is temporarily stabilized across key geographic axes such as time, space, scale, and regimes of environmental governance. this theme helps demonstrate how uncertainty can be spatially fixed at key moments, either due to necessity, strategy, or through institutional action. reflecting this concern, arielle hesse's paper examines attempts to regulate respirable crystalline silica, which is carcinogenic dust that is generated through hydraulic fracturing. the expansion of natural gas extraction in the united states has introduced new forms of risk for workers and competing understandings of how to regulate silica exposure. the article is an excellent case study of how the industry attempts to construct illegible geographies in efforts to fend of regulation. by documenting the spatial and temporal flexibility of the worksite, hesse demonstrates how geography is strategically enrolled to position the hydraulic fracking industry as unknowable and thus, ungovernable. in doing so, she demonstrates how the variable spatial and temporal configuration of the industry is used to stabilize uncertainty about particular health risks and outcomes for workers. while uncertainty can persist and accumulate through spatial relationships, several of the articles also wrestle with how uncertainty comes to be functional or rational at some scales yet is counterproductive at others. as one example, nari senanayake examines local attempts to neutralize biomedical uncertainty about chronic kidney disease of unknown etiology or ckdu, in sri lanka's dry zone. given resource constraints and generic treatment regimes, senanayake argues that the disease that is most frequently enacted through clinical encounters is actually an undifferentiated form of kidney disease that envelopes both regular and mysterious forms of illness (ckd/ckdu). while the absence of diagnostic closure in kidney disease clinics enables important improvisations in the practice of care, it also inadvertently stabilizes uncertainty about actual disease burden at higher scales of analysis. specifically, she demonstrates how uncertainties about disease states compound across scales of knowledge production and how this then frustrates attempts to map the prevalence of ckdu at regional or national levels. in a related vein, jeff martin details the challenges for wolf management in the u.s. west, documenting how government agencies strategically harness uncertainty to navigate funding cuts and longstanding anti-federal and anti-regulatory sentiment. in a fascinating extension of sts scholarship, martin explores how illegibility about wildlife populations can be understood as logical in certain domains of bureaucratic management even as it produces new uncertainties and socio-ecological dilemmas at other spatial and temporal scales. on the one hand, a hesitancy to share information on wolf populations can protect mid-level bureaucrats from external political pressures to delist the species from the endangered species act. on the other hand, martin asserts that illegibility "from above" undermines agents' ability to effectively intervene around their charges and guarantee wolf conservation over the long durée. similar to hesse's discussion of how the production of uncertainty shapes silica regulation, martin asserts the production of uncertain data can allow state agencies to displace responsibilities for environmental protection in space and time. a second theme reveals the productivity or productiveness of uncertainty and how it can be generative of geographical outcomes and socio-ecological possibilities. papers by cavallo (2020) , martin (2020) , and senanayake (2020) all document pragmatic, experimental, and improvised practices of survival in contexts of uncertainty, institutional instability, and resource scarcity. here authors illustrate how farmers in uganda, midlevel public administrators in the american west, and doctors in the dry zone of sri lanka frequently engage in pragmatic strategies to maximize pathways for action in response to multiple and compounding uncertainties. farmer experimentation in the face of uncertainty about a deadly plant disease (bxw) is a focus for sara cavallo, where improvised knowledge networks partially realign relations of expertise. specifically, she demonstrates how strategic experimental networks between farmers and scientists utilize a hybrid set of practices that remain rooted in local cultural understandings and social networks. indeed, cavallo shows how farmers negotiate differing claims to certainty regarding the disease through a range of tactics that do not fully align with theories of the environmental subject as an extension of state logic. instead, compounding uncertainties allow farmers to make claims on the state and re-envision agricultural systems in ways that accommodate greater experimentalism, particularly in the face of uncertain plant health risks. discussion of uncertainty as a strategic resource is also evident in martin's analysis of controversial wildlife management in the american west. this paper documents how implicit and pragmatic choices around day-to-day practices and different technologies allow agents to navigate ecological complexity and multiple use mandates. the production of contingent geographies of care in the wake of sri lanka's kidney disease epidemic is the basis of senanayake's paper, which documents how local attempts to manage biomedical uncertainty about disease are socially and spatially generative. rather than attempting to achieve diagnostic closure in cases of mystery kidney disease, many local clinicians pragmatically suspend diagnosis and instead routinely treat disease-complexes, including different combinations of kidney disease, hypertension, diabetes and cholesterol giving rise to more holistic, contextualized and responsive ecologies of care. more broadly, this work demonstrates how geographic, as opposed to diagnostic, distinctions increasingly structure access to care and state resources. in doing so, she reveals how the island's kidney disease epidemic has cemented new forms of socio-spatial difference in the dry zone while simultaneously reproducing previously established geographical patterns of neglect. in an excellent example of how uncertainty about chemical harms generates socio-ecological possibilities, annie shattuck explores how the expansion of pesticide use within northern laos reconfigures smallscale farming livelihoods within an increasingly globalized agricultural economy. safe use education dominates this setting, which entails information on safe handling practices, personal protective equipment, and familiarity with the toxicity of pesticides. yet in grounding this form of knowledge translation with local livelihood practices, the article effectively demonstrates how safe use education elides contextual political economies and hybrid practices that result in lived experiences of uncertainty. this leads shattuck to conclude that "smallholder farmers are not operating from a complete deficit of knowledge. rather their knowledge of toxicity and risk is partial, situated in a particular context, laced with uncertainty and inequality, and often gained from embodied experience." the article thereby offers us a conceptual route through which to consider the entanglements between situated knowledges of risk and socio-ecological possibilities. a final critical point that emerges from these papers is that while uncertainty can restructure social and ecological relationships, it often does so in highly uneven ways. indeed, to a large extent, all of the papers in this special issue are concerned with how uncertainty is differentially experienced and embedded in multiple, interlinked struggles that constitute daily life (such as poverty and political marginalization) as well as, individual experiences of resource scarcity, health, and/or disease. along those lines, brian king's article details the ongoing uncertainties with hiv in rural south africa even within a setting that has drastically scaled-up the provision of care. the widespread provision of anti-retroviral therapy in the global south has led public health institutions to identify hiv as a chronic condition; however, there remain a wider set of related uncertainties that include ongoing stigma, food insecurities, and unequal bodily responses to treatment. king concludes that given these dynamics, hiv uncertainty and certainty simultaneously co-exist and are co-produced, while remaining materially uneven for those managing their health. likewise, cavallo (2020) and shattuck (2020b) , illustrate how different forms of government intervention in response to uncertainties about plant disease management and pesticide use can reproduce patterns of socio-spatial inequality, normalizing risk and harm for some bodies and not others. as a consequence, our contributors demonstrate how uncertainty is frequently enrolled in the production of social, spatial, and ecological difference. third, the contributions demonstrate how uncertainty is produced, transformed, and negotiated through a key focus of geographic inquiry: the interactions between human and non-human agents. in collecting these articles, it was not our intention to select case studies that dealt specifically with the non-human. but in reading these contributions together, we are struck that all authors deal in some meaningful way with non-human actors. whether it is a pathogen or virus, banana, wolf, chemical toxin, or an invasive insect population, all of the articles engage with the ways that the non-human generates uncertainties, and in turn poses important challenges to western scientific and regulatory traditions. martin and sedell for instance, address how dynamic and moving species, either wolves or invasive insects respectively, confound attempts to measure, surveil, and manage. and hesse and shattuck emphasize how chemical exposure cannot easily be fixed in space and time, as the pressures from broader political and economic forces, including corporations that want to mitigate their exposure to lawsuit and damages, draw upon uncertainty and personal responsibility to deflect culpability in undermining human and environmental health. in considering how uncertainty is refracted by human and nonhuman interactions, there are a few points worth emphasizing. first, western traditions of bureaucratic rationality, scientific management, and state power all require practices of containment. whether it is the containment of invasive pests within a particular agricultural region, wolves in a conservation zone, or an infectious disease within an individual body, scientific and bureaucratic management requires that the object of concern to be contained in space and time. the fact that these entities resist attempts at containment and measurement mean they are active participants in the production of uncertainty. perhaps most evident of this point is jennifer sedell's paper, which convincingly demonstrates that in terms of invasive pests and agricultural production in california, uncertainty about insect presence/ absence cannot be eradicated. as one of her informants explains, the absence of insects in the traps does not prove that they have been eradicated since it is impossible for existing measurement devices to observe the status of pest populations at sub-detectable or low levels. as sedell notes, the implications for the agricultural industry are significant and political responses are required to restore 'pest-free' status for the trade of many agricultural goods. this theme also takes center stage in martin's contribution. focusing on how and why government agents routinely collect less and/or less precise data about the wildlife under their charge, martin complicates traditional assumptions of bureaucratic rationality and state power. he skillfully demonstrates how, in the context of ecological complexity, logistical limitations and socio-political conflict, state agents engage in practices of obfuscation to manage risks and exonerate themselves from blame. in dialogue with mcgoey, 228 (2007) ; (2012;)), martin demonstrates how this apparent dysfunction is not a breakdown of bureaucratic rationality but rather is "indicative of a rational strategy [for managing antithetical demands] in itself." instead of precipitating more experimental and adaptive management approaches, martin demonstrates how the frequent failure to record and manage unruly nonhuman dynamics can end up reproducing forms of bureaucratic rationalityeven when uncertainties are central to managing socioecologically complex systems. in doing so, he draws our attention to how "politics matter, and adaptive, experimental governance without democratic politics, can end up similar to earlier [technocratic] expert modeseven when uncertainties are recognized" (scoones 2019a, 28) . finally, these papers raise critical questions about the politics of uncertainty and, in particular, how it indexes new forms of accountability, governance, and regulation. together the special issue invites geographers to grapple with how uncertainty often generates politically opposing ways of problematizing and intervening in human-environment and socio-spatial relationships. for instance, in this collection, several articles demonstrate how materializing health-environment interactions as uncertain have become powerful strategies for affirming ambiguity about toxic and pathogenic exposures, and ultimately, of fending off stricter government regulation (also see: murphy, 2006) . shattuck (2020a) examines farmers' knowledge of pesticide risk in northern laos in the wake of individually focused safe use messaging and enduring uncertainties about chemical harms. by documenting how situated experiences of toxicity articulate with safe use models, she demonstrates that local typologies of safety and harm continue to blame poor, 'risky' people for pesticide-related health impacts "instead of the socio-economic situations that require them to take risks" in the first place. partial knowledge about the impacts ofand responsibility forchemical exposures in this case, is marshalled to reinforce and legitimate individually based risk management. in the context of rural poverty, uneven access to health care and local inequalities, this approach renders invisible the myriad ways that farmers' decision making is refracted by other risks, principally the risks of being poor and rural. the tendency to reinforce individual responsibilities for exposure in the face of uncertainties about occupational health hazards is also a focus for hesse (2020), where, as argued above, the fossil fuel industry strategically conceptualizes the geographies of hydraulic fracturing as flexible, variable, and unknowable in order to contest government regulation of toxic silica exposures. specifically, this article reveals how the processes of rulemaking as well as the variable spatial and temporal configuration of the fracking industry helps decentralize and individualize responsibility for exposure. similarly, king (2020) asserts that the "production of uncertainty is relational and dynamic, changing not only with new 'certainties' about the virus and treatment protocols, but also the ways in which its management intersects with social and environmental dynamics." this reveals how the experiences of living with hiv are not uniform and continue to be shaped by a range of factors, such as socio-economic resources, food insecurity, employment, gender, age, and sense of responsibility for others. as a consequence, the special issue makes a powerful case for focusing on how uncertainty generates styles of politics that reproduce precarity and individual responsibility for managing exposures to multiple social and environmental harms. a different, albeit related, perspective is explored in other papers where embracing uncertainty is viewed as a pathway to open "up a more rigorous, robust, transparentand democratically accountableenvironmental politics" (stirling 2018, 120) . this work combines what scoones (2019a, 27) describes as a "transformational vision, [where] notions of justice are central" with a "more patient, sometimes unruly, bottom up approach to defining future pathways," often focused on adaptive management, incremental learning, and polycentric governance. as one example, sedell (2020) demonstrates how competing models for understanding invasive pest populations reveal the profound instability of metrics that determine pest absence/presence in california's agricultural sector. as part of this analysis, she demonstrates how alternative methodologies for visualizing pest presence/absence as well as bottom up diversified farming practices reconceptualize pests as neither exceptional nor temporary, but instead, as part of a new normal. doing so, opens up possibilities for alternative agro-ecological and political arrangements by locating the region's pest problem not with the insects themselves but in production systems that lack resilience to biological invasions. as a consequence, sedell reveals how uncertainty about pest presence/absence can precipitate a fundamental re-evaluation of california's agro-environmental system, and simultaneously transform our relationships to pest populations and how the region produces food. how networks of cross-species relations are productive of 'new ecological accommodations' or 'ways [of] learning to live endemically with our viral [microbial, and insect] companions' (greenhough 2012, 295) thus constitute important avenues for future geographic research on the politics of uncertainty. to a large extent, all of the papers in this special issue push us to grapple with uncertainty as a norm and not an anomaly. in other words, this work opens up space for engagement with uncertainty as more than a temporary phenomenon but as a "permanent condition" where stability and certainty about socio-spatial and human-environment relationships are often elusive (brown and damery 2009, 82) . rather than attempting to ensure a strict boundary between knowledge and its opposite, our contributors encourage us to think more deeply about uncertainty as a lived and spatiotemporal condition. it is these very entanglements between uncertainty, environments, and everyday life that provide the narrative thread uniting this diverse collection of articles. while engaging with a broad range of topics and qualitative methodologies, this work coalesces around four central themes: (1) how uncertainty works through geographic relationships; (2) how uncertainty generates geographical outcomes; (3) how uncertainty is reconfigured by human-non-human dialectics; and, (4) how uncertainty raises questions about politics. in doing so, this special issue develops a critical human geography of uncertainty, which not only articulates how the concept is useful for geographers, but also, argues that geography can enrich existing transdisciplinary work on the subject with its perspectives on scale, spatiality, power, place, and human-environment relations. credit authorship contribution statement nari senanayake: conceptualization, writing -original draft, writing -review & editing. brain king: conceptualization, writingoriginal draft, writing -review & editing. surviving the turbulent future brexit: modes of uncertainty and futures in an impasse flammable: environmental suffering in an argentine shantytown a companion to environmental geography contested illnesses: citizens, science, and health social movements navigating compounding uncertainty: farmer strategies amid biosecurity crises in western uganda governing a liminal land deal: the biopolitics and necropolitics of gender engendering the new enclosures: development, involuntary resettlement and the struggles for social reproduction in coastal tanzania ethnographies of uncertainty in africa come on, people… we* are* the aliens. we seem to be suffering from host-planet rejection syndrome': liminal illnesses, structural damnation, and social creativity illnesses you have to fight to get: facts as forces in uncertain, emergent illnesses depoliticization, and bureaucratic power in lesotho where species meet and mingle: endemic human-virus relations, embodied communication and more-than-human agency at the common cold unit 1946-90 uncertainty and context in geography and giscience: reflections on spatial autocorrelation, spatial sampling, and health data thinking through covid-19 responses with foucault -an initial overview taking back taste: feminism, food and visceral politics. gender, place cult africa and the nuclear world: labor, occupational health, and the transnational production of uranium global health, geographical contingency, and contingent geographies when places come first: suffering, archetypal space and the problematic production of global health geographies of uncertainty and negotiated responsibilities of occupational health states of knowledge: the co-production of science and the social order rethinking social reproduction in the time of covid-19 hiv as uncertain life living off uncertainty: the intelligent animal production of dryland pastoralists the uncertain geographic context problem context and uncertainty in geography and giscience: advances in theory, method, and practice toxic bodies: hormone disruptors and the legacy of des exploring understandings of institutions and uncertainty: new directions in natural resource management a geopolitics of trauma: refugee administration and protracted uncertainty in turkey queer and trans* geographies of liminality: a literature review between scylla and charybdis: environmental governance and illegibility in the american west on the will to ignorance in bureaucracy strategic unknowns: towards a sociology of ignorance doubt is their product: how industry's assault on science threatens your health lives in limbo: temporary protected status and immigrant identities uncertain exposures and the privilege of imperception: activist scientists and race at the u.s. environmental protection agency sick building syndrome and the problem of uncertainty: environmental politics, technoscience, and women workers care in the time of covid-19 uncertain futures and everyday hedging in a humanitarian city the socioenvironmental state: political authority, subjects, and transformative socionatural change in an uncertain world merchants of doubt: how a handful of scientists obscured the truth on issues from tobacco smoke to global warming all that is solid melts into the bay: anticipatory ruination and climate change adaptation agnotology: the making and unmaking of ignorance political ecology: a critical introduction toxic bodies/toxic environments: an interdisciplinary forum modes of uncertainty: anthropological cases no fly zone? spatializing regimes of perceptibility, uncertainty, and the ontological fight over quarantine pests in california searching for ckdu: mystery kidney disease, differentiated (in) visibility, and contingent geographies of care in dry zone sri lanka living with uncertainty: new directions in pastoral development in africa what is uncertainty and why does it matter embracing uncertainty: what are the implications for sustainability n. senanayake and b. king geoforum xxx (xxxx seeing like a state: how certain schemes to improve the human condition have failed epistemology of the closet health-environment futures: complexity, uncertainty and bodies toxic uncertainties and epistemic emergence: understanding pesticides and health in lao pdr risky subjects: embodiment and partial knowledges in the safe use of pesticide uncertainty and precaution: some instrumental implications from the social sciences uncertainty artefacts of not-knowing: the medical record, the diagnosis and the production of uncertainty in papua new guinean biomedicine biomedicine in an unstable place: infrastructure and personhood in a papua new guinean hospital patient activism and the struggle for diagnosis: gulf war illnesses and other medically unexplained physical symptoms in the us we thank all of the authors who contributed their articles to this special issue and the reviewers for their helpful and timely comments. we are especially grateful to harvey neo for his insightful feedback and patience in helping steer this project towards completion. the special issue emerged from a series of aag sessions in 2018, where these papers received constructive comments and encouragement from our generous discussants, rebecca lave and david demeritt. particular thanks to arielle hesse and a. marie ranjbar for valuable comments on previous drafts of this introduction. key: cord-299315-s43gw24k authors: capps, benjamin; lederman, zohar title: one health, vaccines and ebola: the opportunities for shared benefits date: 2015-09-16 journal: j agric environ ethics doi: 10.1007/s10806-015-9574-7 sha: doc_id: 299315 cord_uid: s43gw24k the 2013 ebola virus outbreak in west africa, as of writing, is declining in reported human cases and mortalities. the resulting devastation caused highlights how health systems, in particular in west africa, and in terms of global pandemic planning, are ill prepared to react to zoonotic pathogens. in this paper we propose one health as a strategy to prevent zoonotic outbreaks as a shared goal: that human and great ape vaccine trials could benefit both species. only recently have two phase 2/3 ebola human vaccine trials been started in west africa. this paper argues for a conceptual change in pandemic preparedness. we first discuss the ethics of one health. next, we focus on the current ebola outbreak and defines its victims. third, we present the notion of a ‘shared benefit’ approach, grounded in one health, and argue for the vaccination of wild apes in order to protect both apes and humans. we believe that a creation of such inter-species immunity is an exemplar of one health, and that it is worth pursuing as a coextensive public health approach. ebola virus 1 has devastated parts of west africa, and has caused alarm worldwide. it is one of a number of notable zoonotic emergent infectious diseases (zeid), also including influenza, coronaviruses like middle east respiratory syndrome (mers), and the now pandemic human immunodeficiency virus (hiv). the majority of all eids are caused by zoonoses 2 ; and most of these are pathogens of wildlife origin that become endemic in localised non-human animal and human populations (jones 2008) . these pathogens are emerging at an alarming rate, reflecting changes in local topologies and the global climate, triggered by human and animal causative and adaptive activities (epstein 2001) . ebola is endemic to central africa, and is normally dormant in still unknown reservoirs. periodically however, it infects local human populations, causing extensive mortalities but then fading out before widespread contagion (hayden 2014; marzi and feldmann 2014; macneil and rollin 2012) . 3 the ongoing outbreak in west africa surpasses all previous occasions, although at time of this writing, the endemic appears to be receded (who ebola response team 2015) . many have been dismayed by the global efforts to curtail the epidemic, questioning international resolve to respond timely and effectively (mitman 2014; spencer 2015) . in particular, many have been critical of the systematic neglect of public health infrastructure, and have identified strengthening health systems as the long term solution to the disease (dawson 2014; farmer 2014; gates 2015; rid and emanuel 2014) . the measures used during this outbreak are focused on human communities, and includes clinical case management (that to date lacks any curative treatment), quarantine and isolation, surveillance and contact tracing, a rapid and reliable laboratory service, safe and dignified burials, and social education (dawson 2014; macneil and rollin 2012; marzi and feldmann 2014) . critics have much to say about the importance of infrastructure and basic supplies needed, but less has been said about the limitations of ebola containment measures. although these previously worked well within geographically isolated communities where ebola periodically emerged, they were less likely to do so in a sustained and widespread outbreak. in light of the current catastrophe, it now compels us to consider also the limitations of traditional public health measures during an epidemic of this magnitude, which although they may bring an acute situation under control eventually, are challenging to enforce, strain medical and social networks, and provide limited prevention and no cure. 4 indeed, although these measures have brought the emergency to its current abating state, it took a great deal of time and vast efforts, many still died, and infection resurgence is a possibility. the importance of biomedical countermeasures, such as vaccines, therefore cannot be understated. in this respect, it has been resolved that failures in advanced drug development and production must be tackled (who 2015) , especially the political and economic barriers that hamper development and deployment in places such as west africa, and which further emphasise the neglect of certain transmissible diseases in that region (marzi and feldmann 2014) . 5 the current perspectives to zoonotic risks and pandemic planning have changed little despite the warnings from the 'swine flu' pandemic of 2009 6 that the opportunities for expedient vaccine production and sustainable clinical access still seem someway off (gates 2015) . our particular concern, however, is that while the ethical debate is being dominated by global human threats, other considerations about endemic zoonoses are being overlooked. 7 using ebola as a case study, we apply one health (oh) as an ethical framework to make the case for strategic changes. in particular, the debate about vaccines plausibly could be extended to the concurrent need in primate populations. 8 this paper therefore proposes the possibility of shared immunity between species that are equally affected by ebola. our proposal for a novel approach to vaccination that protects both human communities and the fauna they interact with and often depend upon is speculative, as technical issues are far from resolved. however, we have two further intentions: firstly, to highlight the oh 4 in general, prevention and then containment of highly pathogenic eids is about slowing and limiting the contagion, while often treating patients to the degree possible and who are likely to die, thus allowing the existing infrastructure to operationalize and then keep up. traditional public health methods of infectious disease control are known to work up to a point, depending on various factors such as the pathogen, victim and context. in particular, these methods rely to a large degree on the trust of the populations effected to follow non-pharmaceutical precautions under conditions of immensurable suffering and burdens, and the dedication, training and supplies made available to health care and other workers who sustain the infrastructure (such as, in the ebola case, the highly risky and stressful job of digging and filling graves). confidence in these may have become complacent (putting aside the question of political negligence), as it was only a matter of time before ebola would befall upon a highly populated city for the first time. 5 ''ebola emerged nearly four decades ago. why are clinicians still empty-handed, with no vaccines and no cure? because ebola has historically been confined to poor african nations. the r&d incentive is virtually non-existent. a profit-driven industry does not invest in products for markets that cannot pay'' (chan 2014) . 6 these failings have become, for some, symbolic of the abject failures of a global system which does not allow new possibilities for pandemic planning, such as more effective and urgent vaccine production (capps and lysaght 2013) . 7 that ebola is a neglected tropical disease cannot be disputed, meaning that it has failed to attract significant interest for deployment of pharmaceutical interventions (until its full pandemic potential came to light in the current outbreak) (macneil and rollin 2012). so far, local responses fall back on traditional public health measures; these measures do little to benefit non-human interests, as victims or by finding mutual solutions. we propose that a different approach to pandemic prevention should invest in such technologies as vaccines, but do so using a broad ecological scope. 8 we use primate (clade haplorhini) to identify the non-human apes (hominidae) that are susceptible to the ebola virus; our analysis will proceed to discuss the great apes (genus gorilla and pan), as more is known about the effects of the virus on them as highly sentient and endangered species. initiative as a source of alternatives to pandemic planning, so that, secondly, in the spirit of oh collaboration, we can encourage further and broaden the ethical debate. the paper unpacks in the following way: first, we explain the ethics of oh as an approach that recognises an ecological perspective. second, we define and expand upon the victims of the ebola epidemic so as to consider a new oh-grounded agenda. third, we articulate a possible preventive measure to prevent ebola in both human and animal populations. we argue that, along with efforts to test ebola vaccines in humans, existing vaccines that have been proven safe and efficacious in primates should already be deployed in order to protect both species. our proposal supports the conjecture that focusing on broadly ecological factors, and understanding and reacting to the natural ecology of zoonoses, is central to future zeid planning . one health (oh) has come to signify the interdisciplinary effort to optimize the health of humans, non-human animals, and their ecosystems. as an approach to biomedical enquiry, it has been adopted as a broad heuristic for evidence-based policy involving the usual suspects from public health, as well as veterinarians, animal and plant biologists, ecologists, and environmental scientists (scoones 2010; leach and scoones 2013) ; and thereby, it has become a stimulus for collaborative research. thus, its trans-disciplinarily-across multiple disciplines, encouraging de-siloing of sectors, and engagement with partisan stakeholders-creates change by identifying and solving real-world ecological problems. it is thus an extensive ecological perspective to that of public health. however, there are those who have been critical of the oh agenda because, like some existing study or practice lenses, it excludes the humanities and social sciences (lapinski et al. 2015) , and that, in part, obstructs the development of an inclusive bioethics framework (thompson and list 2015) . while the first is largely an empirical point, and we can point to anthropologists, among others, expressing solutions, but perhaps being less heard, in respect to ebola (aaa 2014); the latter observation indicates oh's lack of a philosophical grounding. in fact, oh has no origins in any particular ethical theory. one explanation for this is that normative enquiries are outside of the purview of oh. the collaborative model, therefore, is not about a distinctive oh ethics per se, but an attempt to integrate ecological perspectives on the same terms as public health activism; to probe conventional wisdom to find innovative solutions. this is perhaps a practical consideration because oh otherwise would likely lose political traction under anything more concretely conceptual. the oh goal is to assemble a comprehensive set of data across a broad spectrum of expertise, and to thereby provide solutions that are of benefit to human wellbeing within ecological settings. most recently, this idea is being framed as effectiveness gains through dynamic cooperation in environmental contexts, and has the effect of raising environmental concerns on par with concurrent efforts in public health such as in disease surveillance and animal management. this might be enough to create a vision of oh ethics: van rensselaer potter, in his earliest definition of bioethics, talked about a system of human survival that included environmental, or ecological ethics (potter 1988 ). this could easily capture the idea of oh as broadening public health into diverse fields. potter, a pioneer in challenging parochial and non-secular ideas shaping the human condition, noted a schism between the medical-science domains and humanistic ethics, and that both were distanced from environmental ethics. the ethics of oh, therefore, may just be signalling the resurgence of bioethics as a unified endeavour (thompson and list 2015) , allowing for reflective and critical engagement with current pandemic measures, which up to now gave little credence to solutions outside the scope of public health ethics. a deeper appreciation of secular bioethics, however, also points to the intrinsic interests beyond those of human beings. in the developing oh literature, it is more commonly acknowledged that human beings are part of and dependent upon the biosphere. one way oh has developed is in a perspective that a 'healthy' environment entails healthy animals along with healthy people. it is not 'us versus them', then, but a problem of shared risk that is something concrete to act on, thus providing opportunities to maintain healthy or rescue unhealthy ecosystems (rabinowitz et al. 2008) . however, in practice, reactions to these risks, and solutions to pathogens, still prioritise human interests, because there is no fundamental sense in which non-human animals, or the environment, matter morally. sure, while oh in this sense creates the grounds for humans to express compassion towards animals and ecosystems and to engage in novel approaches to health problems, overall it often achieves the same goals of prevention and response so far already installed in public health; so oh, in this sense, adds nothing to the ethical debate except by broadening the factors considered in any human cost-benefit analysis. the difference oh makes is in engaging with alternatives: it questions public health ideas entrenched as the only way to solve such problems, and indicates the dangers of the unreflective or blinkered view (leach and scoones 2013) . its effectiveness in ethical discourse, much like the collaborative idea, is that it asks questions about ecological benefits without overstepping public health priorities. finally, there is the sense in which oh has an enabling effect in respect to grounding an ethical theory in environmental issues. what that theory is, however, is contested. in this paper, therefore, we will sketch the idea that oh ethics ought to contain two elements: (1) a focus on the inclusive and shared determinants of health; and (2) a unifying theory. by spelling out these elements better, one is able to assess those projects that profess to be oh; and this will be essential in judging our shared immunity proposal. health is often understood as being normative: implying something good or desirable. this might be applicable from an internal view (being healthy), or an external one, such as the view from public health that concerns community (that is, conditions for being healthy). an 'unhealthy' state can be explained by a pathogen or other kind of destabilising event that impacts or creeps into a biological system, resulting in an altered, often unwanted and endured state. this might be the presence of a virus in an individual, or even the conditions (opportunities and barriers) of healthy living. public health often takes a similar focus, aiming to create healthy circumstances and conditions for people by focussing on the determinants of health. in this respect, oh uses health as an inclusive determinant, such that it includes actions that are broad in orientation and scope, so that health activism ought not be limited to human agents. oh is therefore an investigation of the scientific, social, economic and ecological determinants of human, non-human and ecosystem health, but also a 'shared benefit' approach. our use of 'shared' points to ethical consistency; that actions that affect a broad spectrum of agents should be fairly applied. just as racism is paradoxical in human societies, some exclusionary actions between human beings and non-human animals might be similarly judged as speciesistic. this echoes ideas of equality, and the interests of minorities or the vulnerable being protected against parochial or vested interests. it also befits an examination of incongruity, need and fairness, and justice-these components of comprehensive doctrines are only knowable through ethical study, and in this respect, we are less confident in setting the oh agenda, for such a task requires far greater elucidation than is possible here. we can, however, offer a basic account of 'benefits' that will begin the conversation in earnest about oh as a unifying theory. human beings act in ways that affect non-human animals and the environment, and this raises the question as to how much we should either change such actions, or, indeed, make efforts to assist in the wellbeing of other species. the basic assumption in public health has been that we should interfere only to the extent that their collective welfare is at stake, because animals' interests are outweighed by human interests . thus, public heath applies welfare conditions for the health of animals, which only occasionally includes ethical considerations, such as the humane culling of disease vectors and hosts. however, without engaging in a lengthy debate about non-human moral status, there is also a condition of interspecies connectedness. in the case of preventing zoonotic pathogens, oh on this reading implores us to study the causes and roots of transmission, counting each being as an equal unit in this biological process. the wider study of biospheres, ecosystems, and social networks achieves this. what is ethically important is that this study is concerned with the health of the ecosystem in its entirety, not solely that of humans. oh, therefore, becomes a study of 'natural' environments, enriching public health with animal and ecological studies, and creates a whole new frame of evidence to better design effective responses. in turn, the emphasis turns to discovering and developing creative ways to recover and maintain healthy ecosystems. these hint at plausible strategies that draw on the humanities and social sciences, which can better comprehend the emergent contingencies beyond statistical confines (neyland 2013) . but what are the objects, goods, or benefits(and harms) that enable states of heath? one way we might extend ethical concerns to non-human interests is by securing universal goods (capps and lederman 2015) . these are the kinds of goods that reach beyond the needs of human communities, describing benefits as inclusive across species, and feature broadly in ecosystems and the environment. for example, ecosystems are necessary for life by providing the basic requirements (and even complex determinants of heath, in terms of social and cultural goods), and can therefore create 'unhealthy' lives by becoming unproductive and even toxic. these effects can be observed in stressed and challenged environments when they are misused, exploited and degraded. the ecosystem is, therefore, a foundation of universal goods-goods necessary for the health of multiple species, and these goods are likely shared through interspecies connectedness. primarily, then, universal goods extend terms of reference beyond the restrictions of public health purposes. one set of solutions would emanate from comparative medicine originating in human beings (this is the opposite of current comparative medicine studies where animal models are utilised for human health). human trials and treatments may well be useable in animal populations, benefiting them directly, and in some cases, where a pathogen is eliminated, it might reduce risks for human populations. a second possibility would be adapting biobanks, which, because of the terms of reference in providing public goods, are restricted to furthering human interests only (capps 2013 ). this does not make good scientific sense, because there is a welter of data being lost or overlooked simply because of intentional institutional design that arbitrarily excludes other contributions. for example, animal samples may well show up zoonotic risks sooner, or enable the natural history of a pathogen to be understood. 9 a recent proposal to create an ebola biobank would do well to consider extending its remit to include the animals that are the essential links in zeids (hayden 2015) . these are intriguing possibilities because they also allow real environment information gathering and sharing, and not just the artificial data, for example, from de novo animal experiments (capps and lederman 2015) . there are, however, going to be more or less hard cases where conflict between public health goals and securing universal goods is more or less likely; and solutions are going to be less amicable between human interests and an ecological perspective. at this level of disagreement, a debate about animal or environmental interests or rights is to be had. but in our paper, we develop this idea of universal goods to give weight to the broadly inclusive and shared determinants that are affecting both humans and animals as victims of ebola. according to oh, it behooves us to consider the opportunities to improve the health of those directly affected by the virus, in the sense that operationalizing public health should be extended to other primates such as chimpanzees and gorillas; related not only in their level of evolutionary sentience, but also as victims of ebola. the current ebola outbreak, which started in a single index case in december 2013, but was not reported as an outbreak until march 2014, is the largest known in history (rio et al. 2014; yakubu et al. 2014) . both humans and great apes have been affected. at time of writing, there have been 28,005 reported confirmed, probable, and suspected cases in human infections, mainly in guinea, nigeria, liberia, and sierra leone. 11,287 confirmed patients have died. 10 in great apes, the effect of ebola is likewise devastating. gorillas and chimpanzees are susceptible to the virus (bermejo et al. 2006; kaiser 2003) . ebola has killed roughly one third of the western lowland gorilla population in the past 15 years, which, along with habitat loss and poaching, led the world conservation union to declare it a critically endangered species (walsh et al. 2003) . three interrelated enquiries interest us as advocates of oh. first, a significant question is 'why now?' (bausch and schwarz 2014; farmer 2014) . why only now has the ebola virus, which has previously emerged in isolated regions, become a regional endemic (olival and hayman 2014; olson et al. 2012 )? this question has been asked in the context of other zoonotic diseases, most prominently hiv and its analogous emergence from primates in africa. 11 answers will likely become evident as our understanding of zoonoses encompasses the exponential amount of accumulated knowledge from across disciplines, including the study of the reservoir, host and effected animals, the ecologies they inhabit, and their natural responses to the virus. it is therefore not only a question of what humans might have done differently this time to create the tragedy, but also their ongoing interactions with the environment whence the virus came from. these insights will be significant in developing strategies for potential future ebola outbreaks, and paradigms for other zeids, including possible preventative measures. second, it has been debated as to whether medical interventions for eid should be deployed in animal species. according to one view, we should not interfere with natural systems at all; apes have lived with ebola for years without need for human intervention. yet the state of wild populations today is such that no environment is free from human effects, and therefore such groups must adapt to the 'anthropocene' (hockings et al. 2015) . in fact, the landscape has changed so significantly that human intervention is perhaps necessary for them to survive at all. 12 although dissent has been voiced against interfering in 'natural systems' and the effectiveness of medical interventions relative to other conservation strategies (ryan and walsh 2011) , the magnitude and significance of the current ebola outbreak should at least question the premise of non-intervention. intervention, therefore, can be justified because the alternative is decimation across the biosphere, affecting human beings who rely on it, and the animals that live within it. if this can be considered as a universal good, then, we can start to envision medical strategies to protect both human and animal populations. the plausibility of vaccinating other species during significant endemics has been voiced before, often from the conservation angle (marzi and feldmann 2014; ryan and walsh 2011) , but never received any serious consideration, as far as we are aware. two reasons for this might be postulated: limited resources are to be used to address human needs, especially at times when endemics or potential pandemics are occurring; and vaccine safety in administering to potentially critically endangered species. recently, a vaccine trial for ebola was carried out on captive chimpanzees to inform future conservation (warfield et al. 2014) . third, what (at least partially) grounds the need to respond to the queries posed above in respect to the shared risk of zoonoses, is the fact that human beings and primates are equally affected by the virus. therefore, if an ethics of shared benefits is persuasive, then one can start to see how conceptual change is necessary in zeid planning. for example, standard public health policies prioritise human interests, and often, these interests are perceived to collide with and outweigh the conservation of the biosphere. examples would include devastating and often ineffective culling (johansen and penrith 2009; jenkins et al. 2010) , or ravaging biodiversity on the basis of 'at-risk-to-human' calculations. oh, however, starts to give rise to different opportunities: for example, developing data storage from veterinary and conservation studies that can benefit humans, and vica versa (capps and lederman 2015) ; or strategizing to create healthy ecologies that will concurrently present fewer risks to human beings. 13 concomitantly, humans, who often receive better medical care, may serve as 'concurrent research participants' and adaptive public/veterinary health models. 14 the scientific literature to support oh as an approach to coordinate pandemics of zoonotic origin is rapidly accumulating (rabinowitz et al. 2013 ), such that it should be gaining traction in pandemic planning. it has, however, yet to feature in the solutions to ebola. oh ought to have some quite significant implications for pandemic planning (against ebola and generally) in various chronological phases. 1. the natural ecology of the ebola virus. the animal origin of the current epidemic is perplexing. 15 the virus tends to only occasionally emerge in isolated villages, rarely appearing in hospitals and other health facilities (garrett 1995) . in this regard, the current outbreak is unique. beyond human interference, the ecology of the virus itself undoubtedly plays a key part. there are a number of species that could be implicated as the host, such as bats, other large mammals, or primates; even insects and plant viruses have been implicated in its transmission to human beings (hayden 2014; monath 1999) . it is imperative to conduct studies to locate the reservoirs and the plausible transmission routes to human beings and primates (in terms of group-to-group interspecies and cross-species transmission) and other known and unknown species contagions, to explain risks and spillover events. wildlife conservation workers have been tracking ebola in gorilla and chimpanzee populations for some time; but these data rarely reach the attention of public health planners (walsh et al. 2007 ). 2. manage habitat disruption. there is a vast and largely uncharacterized pool of possible zoonotic pathogens, and increasing opportunities for infection caused by disruptive human activities and ecological encounters (morse 2009 ). the 13 understanding ecologies of vector-borne pathogens reveals some intriguing events, such as how biodiversity and diverse species networks can buffer, dilute and 'soak up' pathogens (harris and dunn 2013; keesing et al. 2010 ). 14 that is, comparative studies and the reverse data use of human trials to benefit animal populations, such as in veterinary application (yeates 2013) . 15 an early report from the current outbreak hypothesized that the host was a bat colony living in a local hollowed out tree (saéz et al. 2014 ). development of industry, such as mining, can bring people into regular contact with zoonotic reservoirs and hosts (kangbai and koroma 2015). these industries employ local and international workers who then travel to and from wild territories (allouche 2015) . anthropocentric activity also disrupts normal animal behaviour, for example, changing fruit bat roosting and foraging ranges so that they move to proximate sites to human dwellings (looi and chua 2007) . further, evidence suggests that biodiversity is a key element in emergent zoonotic diseases, where, in some cases, there is a reversed correlation: less biodiversity, or even deprived ecologies, create more risks for human zeids spillover events (cardinale et al. 2012; jones 2008) . 3. prevention of zoonotic infections. non-pharmaceutical measures can work well in eid outbreaks, but are only practical considerations once the spillover event has occurred in humans (in other contexts, personal protection equipment might be used as biosafety measures). reactive pharmaceutical measures, such as vaccines, take time to develop to specific pathogens, and then are often hampered by politics and investment, biological limitations, errors, and logistics. prevention, as is central to public health, might therefore be considered key. currently, several types of ebola vaccines have been proven effective and safe in primates, but none has been approved in humans yet (see below). human trials however are ongoing; and several captive chimpanzee trials have been conducted warfield et al. 2014) . once an ebola vaccine is approved for use in humans, several strategies to increase coverage may be used, such as ensuring that eco-tourists are appropriately vaccinated before visiting at-risk primate populations, and introducing health programmes in mining and refining communities often located in remote areas and near to potential ebola hotspots, such as bat roosts. 4. monitoring of disease in animals. studies have identified stereotypic behaviours in animals when burdened with zoonotic disease. for example, gorillas faced with endemic ebola reacted in ways that point to a decrease in social cohesion and lower reproductive potential: females were significantly more likely to transfer from breeding groups to non-breeding groups and males were more likely to transfer from groups to solitary-living. in general, there was a decrease in formation of breeding groups. interestingly, during the post-epidemic period, immigration of breeders between groups returned to normal while immigration of non-breeders remained low. observable social dynamics, then, may be used as indicators to detect ebola outbreaks (genton et al. 2014 ). this is an example of how animals can act as sentinels for imminent human risk. 16 5. animal-to-human transmission. several routes of animal-human transmission of ebola exist, including ingestion of raw infected meat (bats, primates and other animals) , and exposure to hosts and reservoirs through daily life, professions and tourism (köndgen et al. 2008 ). these various routes are potentially causing more zeid spillover events. for example, 'bushmeat' is consumed in higher amounts due to population growth in some areas (wolfe et al. 2005) ; mining is a growing industry in many regions (see below); and local economies rely on the growth of ecotourism. presenting these as local and global issues is challenging: for example, the local population is unlikely to support the prohibition of eating specific species as part of infection control. moreover, to have ethical credence, it would be consistent to address concurrent risks in developed countries, such as reducing intensive farming that also drives zeids. as regional industrial growth is essential for creating sustainable development for all countries, a call to reduce anthropocentric activity in rich wildlife areas in order to meet expedient conservation efforts would likely be rejected because of the local economic losses (and the international desire to visit such areas). nonetheless, efforts should be aimed at education of locals and visitors about the modes of transmission of ebola (muyembe-tamfum et al. 2012 ). learning about the local ecology-animal behaviour, biology, anthropology-would point to innovative ways to adapt in order to reduce the risk of transmission. the accumulated knowledge, therefore, raises some intriguing possibilities for the study and feasibility of potential zoonotic control measures; and in particular, using and adapting the 'shared' biosphere as part of the solutions to endemic ebola. however, despite the obvious ecological links to human zeid outbreaks, the interest in devising such possibilities has, until now, had little traction in public health and extant pandemic planning. most pandemic plans mention little about the ecosystem beyond its risk potential and stipulate requirements to devastate the animal populations (culling and the like) as a means to limit future human-to-human transmission. the one health solution to endemic ebola: inter-species immunity vaccination is by far one of the most significant responses to eid in human beings, and in the context of ebola its importance is beyond doubt; since the outbreak was first detected, public health, clinical staff and allied workers struggled against quite immense odds to bring it to the current state. the case for human vaccination speaks for itself. however it should not be understated quite how important it is since the alternative is to fall back on the objective: ''not to dramatically increase the person's chance of survival, it's to contain the spread'' (fjeldsaeter 2014) . one can only imagine what advantages the early deployment of an effective vaccine would have been. putting aside questions of the economic inequality that provides little incentive for vaccines until worst case scenarios prevail (capps and lysaght 2013; dawson 2014; farmer 2014) , the current upscaling in research to find a vaccine for ebola illustrates the standard phased approach to innovation: invention, animal experiments, trials in humans before large scale production and delivery. as with human beings, the obvious advantage to animals affected by the disease, such as great apes, is immunity from the disease (and relationally, although not always the case, such as with endangered species, is exclusion from culling measures), and prevention of cross infection (walsh et al. 2007 ). 17 the obvious benefit is also in terms of conservation. specifically the case of highly endangered gorillas (and other susceptible animals on wwf lists) is extremely significant (ryan and walsh 2011) . no ebola vaccine has yet to be approved for therapeutic use in human beings. however, ebola vaccine development has been an active field of research for several laboratories worldwide, and candidate vaccines were found some time ago to be safe and efficacious in mice (blaney et al. 2011) . several human-targeted vaccines have been proven safe and efficacious in trials in primates, including adenovirus type 5 and 3, human parainfluenza virus type 3, and vesicular stomatitis virus geisbert and feldmann 2011; marzi and feldmann 2014; stanley et al. 2014 ). these prospective vaccines, however, raise different concerns, such as safety, price, effectiveness, delivery and side effects. in an attempt to aver safety in the use of replicating viruses (see below), warfield et al. (2014) tested the protective effects of a virus-like particle in captive chimpanzees using adenovirus 5 as a vector. first, they demonstrated that the vaccine was safe for chimpanzees. second, they documented the development of a robust immune response in chimpanzees, evidenced by a detection of virus-specific glycoprotein and vp40 18 antibodies 2-4 weeks post-vaccination. third, they demonstrated that total igg fractions taken from the chimpanzees that were vaccinated had a protective effect in mice challenged with murine ebola: 30-60 % of mice in the study groups survived compared to none of mice in the control groups. similarly, blaney et al. (2011) developed a live-attenuated and inactivated rabies virus vaccine that expresses the ebola glycoprotein. the vaccine had no adverse effects in primate models, it induced humoral response to both rabies and ebola, and was shown to be protective against both viruses. while this is obviously an early-phase study, it has great potential in terms of resources and feasibility in conferring immunity in mammals against two lethal pathogens. so far, two vaccines have passed phase 1 clinical testing: chimpanzee adenovirus 3-vectored vaccine encoding ebola surface glycoprotein (chad3) (rampling et al. 2015) , and vesicular stomatitis virus (vsv)-vectored vaccine also encoding for the outer protein of the zaire ebola strain (agnandji et al. 2014) . the prevail study is an ongoing phase 2/3 trial taking place in liberia that examines the safety and effectiveness of these two vaccines. 19 concomitantly, the strive trial is taking place in sierra leone, where 6000 healthy volunteers will be given the vsv vaccine in order to test its safety and effectiveness. 20 17 agricultural policy tends to follow vaccinating all of the exposed animals so that those not already infected will develop sufficient immunity. however, when time and resources permit, it is normal for all exposed animals to be slaughtered (kahn et al. 2002) . 18 vp40 is a matrix protein that together with glycoprotein constitute the virus-like particle vaccine. occasionally, nucleoprotein is also present. vp40 is essential for cell expression of viral antigens to which the body responds by creating antibodies (escudero-perez et al. 2014; marzi and feldmann 2014) . 19 see: http://www.niaid.nih.gov/news/qa/pages/ebolavaxresultsqa.aspx; accessed 12 june 2015. 20 http://www.cdc.gov/media/releases/2015/p0414-ebola-vaccine.html; accessed 4/2015; accessed 12 june 2015. the vsv phase 1 trial in geneva was halted due to safety concerns when several healthy participants developed different adverse effects such as arthritis. the trial was continued a month later, to understand contagion networks and possibilities for control, first we need to see the connections between vectors and victims, and by understanding these within shared ecologies we might be able to better safeguard communities-both animal and human. as these authors postulated: ''in addition to the protection of threatened nhps [nonhuman primates], vaccination of nhp populations in endemic areas might also offer an additional, critical benefit to humans. the interaction of humans and infected nhps has been associated with transmission of ebov to humans and initiation of subsequent outbreaks, so prevention of disease in nhps may also serve to limit ebov transmission into the human population'' (blaney et al. 2011) . concurrently with the race to develop and test vaccines on human beings, we argue that already now, we can and should (upon assuring the degree of safety) deploy vaccines in captive and wild primates with the aim of benefiting both primates and humans. the current strategies, we submit, are driven by a too narrow vision: we propose that oh espouses a 'shared benefit' approach that is complementary to the 'shared risk' approach (rabinowitz et al. 2008) . a 'shared benefit' approach seeks to actively maximize health in one species while in turn benefiting another species as well. specifically, we refer here to research and interventions in humans that benefit animals and vice versa. our proposal is to implement the notion of inter-species immunity. one of the identified risks for ebola, while not knowing for sure the reservoirs of the virus, is close proximity between human and primate populations (towner et al. 2009 ). our proposal is for direct action to administer vaccinations to humans through public health and research paradigms, and additionally to animals to stave off future outbreaks in both populations. such an approach, aimed at vaccinating animals in the first instance, would be preventative rather than reactive to an outbreak in human populations, by protecting across species and thereby creating a potential barrier to future occurrences of ebola in the fauna. our proposal is to co-develop vaccines for human and primate use in ebola endemic and at-risk sites in africa; and simultaneously, to deploy such vaccines to these sites in animal and (in due course) human populations. the delay in getting vaccines to the people in africa is in part due to the need to conduct proper clinical trials first and the troubling consequences of creating randomization (donovan 2014; shaw 2014) . however, captive primate populations could be enrolled in trials as benefiting vaccine development at a lower safety level (in comparison to the footnote 20 continued upon approval by the review committee. the ongoing phase 2/3 vsv trials were modified according to the results in that study (agnandji et al. 2014) . one health, vaccines and ebola: the opportunities for… 1023 standard profiles for first in human trials, and additionally avoiding the later phased stages of human clinical trials). 21 primates might be research subjects who can contribute to a longer-term ecologycentred strategy to vaccinate wild animal populations urgently. simply put, researchers are already injecting captive primate populations, and, if proven safe and efficacious in these trials (i.e. would not to our knowledge wipe out remaining primate populations), this approach provides a fast track to wild primate populations. an oh approach would potentially justify animal research on captive primates within parameters of participation of 'vulnerable' populations (i.e. the agents likely to be the first cases or most at risk in future outbreaks because of their situation and circumstances). the next stage would be vaccinating the same species in the wild for the protection both of the same species (primates) and other at-risk species (human beings). this is, firstly, an ethical enquiry involving the status of primates as sentient beings who possess moral value (fenton 2014) ; and secondly, a conceptualization of animals as vulnerable populations such that risky clinical trials, with conditions, can be ethical. in answering the first enquiry, we note that ebola and the recent chimpanzee trials happen at a time when the national institutes of health is planning to reduce significantly the use of chimpanzees in invasive research, 22 and therefore raises the case of whether minimally invasive research on still captive or retired chimpanzees is ethical at all. we might see experimentation, however, as a parallel development to research and treatment in vulnerable human beings, such as children and other people who cannot consent (wendler 2014) . the idea here is that trials might benefit wild populations and therefore it might be possible to justify within human research ethics paradigms. in human clinical research, the acceptability of such study is a function of acceptable risk, and, when vulnerability is in question, so are the chances of direct benefit (the 'best interests' test) and the possibility of appreciating benefits for others of one's own kind (children suffering from the same condition, for example). in this sense, developing protocols with primates in captivity might be justified, including using those that have 'retired', to meet the conditions of expediency; but concurrently we must anticipate that there is a direct benefit-or a best interest in play. 23 while potential 'secondary ecological risks' exist, such as accidental extinction of the animal species, there would be some important caveats 21 scholars concerned with animal ethics will blame us here for putting the animals at increased risk compared to humans. however, given a vaccine that has been proven safe in the lab, and the significant risk ebola poses for apes, we believe that the risks posed by the vaccine are proportional to the benefit that might be accrued to the animals themselves. 22 http://www.nih.gov/news/health/jun2013/od-26.htm; news release; wednesday, june 26, 2013; accessed 12 june 2015. 23 this perspective is different from the predominant study of exogenous factors such as habitat disturbance and climate change as drivers of ebola emergence, and links directly to the contribution of transmission between gorilla or chimpanzee social groups in the wild. if this equivalency were to remain within an oh approach: that the vaccine is safe enough to use in human phase 1 trials concurrently (shared risk) and that wild apes would receive the treatment as part of the same strategy (shared benefit). if this shared benefit paradigm of securing universal goods is legitimate, then it goes some way in justifying our strategy as mutually benefiting from a single intervention. this raises feasibility problems, but some intriguing ecological repercussions warrant serious consideration of an oh vaccine approach. 1. one would have to possess extensive knowledge about the reservoirs, vectors and hosts, and hierarchical zoonotic bridges between species, to understand the impacts of vaccines in terms of safety, stability, and effectiveness. this will involve knowledge of human, human-animal, and animal-animal interactions (i.e. comprehensive studies of fauna and flora), and their linked activities within the biosphere. at present, pandemic planning is focussed on public health, and to a degree, anthropocentric studies of how we contract and spread the pathogens amongst our own kind. this focus, for instance, locates some major challenges of vaccine use, specifically high levels of distrust and ambivalence towards medical interventions in some african populations that would impede wide human community vaccination programmes (mark 2014; macneil and rollin 2012; mitman 2014) . one might therefore face resistance in deploying an effective vaccination programme. oh, therefore, helps planners look to other solutions that may complement communitybased interventions. firstly, vaccinating domestic animal populations, both companion animals and those in husbandry, 24 could avoid collateral loss to families and livelihoods. these losses are substantial and as targets for public health intervention might gain widespread support. secondly, and which we focus on here, is developing a novel approach to research and deployment in the field as a protective measure that demands immediate attention. 25 thus, following the approach we outlined above, the vaccine will increase the welfare of humans and wild apes, both as protection (eventually) and in conducting knowledge based trials. to address the expediency argument, we again note that both the chad3 vaccine and the vsv vaccine have proven to be safe and efficacious in non-human primates (stanley et al. 2014; geisbert and feldmann 2011) . the human trials will take time to conclude. clearly, with the primate trials already concluded, there is an 24 while the context of animal vaccinations has been debated considerably in respect to farming practices (and risks to humans as pathogenic risks, food safety and economics), there has been little coverage of the benefits to the animals. the debate is now further sparked by the quarantine and killing of companion animals exposed to potential contagions by their owners, such as the spanish dog killed for the fear of ebola transmission (associated press 2014). 25 we note that the ongoing debate is deliberate in its assessment to get vaccines into the field as soon as possible to protect health care workers and needed staffing (i.e. burial teams and cleaners). this is a separate, urgent debate which does not entirely equate to the stage wise proposal we make here. however, the design to get vaccines first to primates as a joint shared immunity strategy could expedite human benefits and use, with a focus on employing biologists, veterinarians and the like to target the animal populations. opportunity to deploy these vaccines right away to wild apes. in the short term, we might be seeing every primate that lives as a benefit of vaccine deployment. the long-term benefits are immunity, possibly extending across species and thus limiting the future scope for spillover events. furthermore, it will provide in situ data to be gathered from the wild populations. 2. achieving broad coverage to widely dispersed animals would be costly and logistically challenging 26 but has been achieved in other settings using low interventional methods such as baiting in the case of rabies (morters et al. 2013) . one challenge is the difficulty in reaching entire ape populations. the dense tropical forests and the animals' nomadic tendencies would make effective immunization difficult. however, by use of local and interdisciplinary knowledge and expertise, and various vaccination methods such as hypodermic darts and synthetic baits, this obstacle may be overcome (ryan and walsh 2011) . one long-term strategy may be to create buffer zones around villages by vaccinating domestic and wild animals, that might be enough to minimise risks of future outbreaks, following the alreadyexisting use of designated zones in farmed animal populations elsewhere (kahn et al. 2002) . 27 the approach would require an increased evidence base, of course, but effectiveness could be achieved by focusing on 'hot spots', 28 localized risk maps (jones 2008) , and using targeted empirical data, such as weather patterns that are known to influence zoonotic spillover events (bausch and schwarz 2014) . 29 ring vaccination is another strategy, where vaccines are delivered to animals found in the 26 ryan and walsh (2011) counted 22 different pathogens that are harmful for apes, 16 of which have licensed vaccines. they claim that the major obstacle in dispensing these vaccines is the delivery to the animals (ryan and walsh 2011) . 27 the authors point out that ''the high seroprevalence among children indicates the same source(s) of exposure [to eobla] as in adults, either inside or near villages''. moreover, because great ape infection is often lethal, and direct contact with humans is rare, some other animal, perhaps bat roosts near settlements, represent the most likely common animal source of exposure: ''these animals, previously identified as a potential reservoir, are abundant in the forest ecosystem and consume fruits on trees located in or around villages'' (nkoghe et al. 2011) . 28 'hotspot' maps highlight regions: (1) where the risk of disease transfer between wild primates and from wild primates to humans is greatest; (2) where there are cross-species transmission events between wild primates due to a high diversity of closely related primate species; and (3) where it is most likely that human beings will come into frequent contact with their wild primate relatives. ''these areas also are likely to sustain a novel epidemic due to their rapidly growing human populations, close proximity to apes, and population centers with high density and contact rates among individuals'' (pedersen and davies 2009). 29 this would have to be an ideal, managed area, additionally creating the rural populations in control of the solutions. there are two elements to achieving this: (1) engagement, cf. ''far greater community engagement is the cornerstone of a more effective response. where communities take charge, especially in rural areas, and put in place their own solutions and protective measures, ebola transmission has slowed considerably'' (who 2014); and knowledgeable land use, cf. protecting ''threatened habitats by reminding nearby communities of all the benefits they derive from keeping these habitats intact. forests, meadows and marshes prevent floods, supply clean water, provide habitat for species that pollinate crops, put oxygen into the atmosphere and take carbon out, and otherwise make themselves useful. in some cases, conservation groups or other interested parties actually put down cash for these ecosystem services-paying countries, for instance, to maintain forests as a form of carbon sequestration'' (conniff 2014). proximity of a known outbreak. further, vaccination campaigns in animals are likely to be cheaper and possibly more temporally feasible than in humans (ryan and walsh 2011; macneil and rollin 2012) . 3. technical issues, including the use of live attenuated viruses as vectors. for example, live attenuated vaccines are more effective than killed vaccines in conferring long-term immunity, thus necessitating fewer vaccine shots and lower rates of compliance and coverage. moreover, using viruses that are replication-competent as vaccine vectors will increase the chances for herdimmunity and therefore the potential for inter-species immunity. however, one of the risks in using a live attenuated, replication-competent vaccine in wildlife is the activation of the attenuated virus and spread to other species, including humans. beyond using killed viruses or viral particles, one solution may be using as vector a species-specific virus. for example, a recombinant murine cytomegalovirus (cmv) that was genetically engineered to express ebola particles was found to be protective in mice. since cmv is highly species-specific, a cmv-based ebola vaccine will potentially spread rapidly in a wildlife population, such as gorillas, without any cross infection to other species (marzi and feldmann 2014) . the use of replication-competent vectors raises another problem: pre-existing immunity to the virus that is used as the vector will hinder spread of the ebola particles, thereby preventing immunity to be acquired. this challenge could be addressed by the development of vectors to which there is no pre-existing immunity among the specific population. for example, newcastle disease virus, to which there is no detectable pre-existing immunity in humans, was developed as a potential vector of ebola particles with some (limited) positive results. vsv was also used as a vector with little if any pre-existing immunity in humans, with even greater success (marzi and feldmann 2014; stanley et al. 2014 ). the existing challenge with vaccine development was captured by the ghana academy of arts and sciences technical committee. they enumerated that development of vaccine is notoriously tricky given pathogen diseases' drift and other factors that impact on their individual effectiveness with different strains; the possibilities of emergent side-effects and other unforeseen incidents; the distrust of trials originating from certain foreign organisations; and how all of this will affect uptake (both in terms of willingness and immunisation) in the target population. 30 within a 'shared benefit' approach, however, one originating in a one health perspective, we coin the term inter-species immunity to conceptually re-think the notion of immunity within a community; specifically, to extend the goods of health, such as immunity strived for in human populations, to other species and vice versa. we suggest that ebola incidence may be prevented or reduced in one species population by inducing immunity against that pathogen in another species population. so far, the best example of the success of such an approach can be seen in the response to the hendra virus, where vaccination of horses prevented disease in both horses and humans (middleton et al. 2014 ). the key to success of inter-species immunity might be with other measures that look to adapt and benefit other ecologies, such as preparing protected ecological zones (removing food and perching areas for bats) based on planned and managed farming areas, and identifying timely and imminent risks to initiate human and animal vaccination in and around these zones. one could adopt already-used surveillance programmes in at-risks regions (these, as we noted earlier, should already be modified to include 'indicative' behaviour in animals of possible zoonoses infection): ''previous serosurveys, together with the geographic pattern of outbreaks, have highlighted the potential role of the ecosystem, and an increased risk among forest populations has previously been described. our study confirms that the forest, particularly the deep forest, is the environment most at risk. this is the area harboring animals susceptible to the virus, such as great apes and bats, the latter representing a viral reservoir'' (nkoghe et al. 2011 ). we could not say whether the remoteness and distances between villages could create conditions for regional immunity by lowering the chances that an affected host might infiltrate the buffered populations from long distances away. 31 however, as mentioned, oh is about interdisciplinary collaboration, and solutions to extreme situations such as the current ebola outbreak require such an approach more than ever (middleton et al. 2014) . understanding the needs of the various stakeholders such as villagers and hunters, and the ecology of all the organisms involved, is without a doubt essential for the success of any viable long-term solution. at the moment, inter-species immunity is likely to involve a programme of trials in captive primate populations, early role out to wild populations (assuming they are safe), and then a concurrent programme to vaccinate human communities in at risk regions (this human challenge is already featured in the literature with respect to other pathogens). however, with the impending imh prohibition on some primate research in the united states, and paralleled restrictions in other countries, this is a window that is potentially closing. invasive great ape research rarely has scientific justification and primate research in general is falling out of favour, although it remains possible in many jurisdictions under strict conditions. invasive research on great apes-using chimpanzees in particular-is likely to be prohibited; but we suspect that monkey research will continue for some time. this might provide the necessary level to proceed to trials in human and great ape populations. so, one could also look at it through a shared vision-if human beings are willing to volunteer for phase one trials, which was highly evident in recent calls, then possibly retired chimpanzees could be coopted as well. at this stage, it will be envisioned that the vaccine is safe for human beings, so this might be an acceptable concurrent risk for primate populations. we surmise that this vaccination strategy, inspired by the ongoing ebola outbreak, might be replicated in future ones. we make the case for the vaccination of wild primates even prior to the completion of the ongoing human trials, with the conditions of optimizing their safety and welfare, and assuring mutual benefits to them and their future offspring, as well as to humans. phase 1 trials of rvsv ebola vaccine in africa and europe-preliminary report ebola and extractive industry strengthening west african health care systems to stop ebola: anthropologists offer insights excalibur, spanish ebola patient's dog, is euthanised despite global outcry. the guardian outbreak of ebola virus disease in guinea: where ecology meets economy ebola outbreak killed 5000 gorillas inactivated or live-attenuated bivalent 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interventions needed second who high-level meeting on ebola vaccines access and financing. summary report west african ebola epidemic after one year-slowing but not yet under control bushmeat hunting, deforestation, and prediction of zoonotic disease the ebola outbreak in western africa: ethical obligations for care animal welfare in veterinary practice acknowledgments capps conceived of the idea for this article and led its drafting. lederman was integral to developing the idea and contributed equally to the research for the paper. the authors would like to thank the following people for helpful comments: wang linfa, paul anantharajah tambyah, and sharon amit. key: cord-004879-pgyzluwp authors: nan title: programmed cell death date: 1994 journal: experientia doi: 10.1007/bf02033112 sha: doc_id: 4879 cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl-2 proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl-2 protein. one of the two facial nerves of newborn mice was transected on the 2nd-3rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, 70 to 80 % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl-2 protein survive after axotomy, and suggest that, in rive, bcl-2 protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult6 de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones (90% neurons and 5-10% non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively 15h and 7.5h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by >60%. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the 2 secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, 1992, science 257:967) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of 12 clones revealed high, but not absolute homology of 5 fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp52 glycoprotein (ep3) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia 1993, 49, a75) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from 20 month old rats during one month with testosterone propionate (2 #g/kg s.c., once every 3 days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a 125i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., 1991, dev. brain res., 58, 13-24) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during 2-3 weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, 1066 epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within 20 minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already 15 min. after interaction, 3 distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia-1 showed an overall identity of 93%. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by 40-fold within 5 days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within 24 hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days 10 and 24. lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day 24, we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of 444aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, 1 rue michel server, 1211 gen~ve 4 dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch0rch et el., histology for pathologists, t992). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. 63:21, 1990) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. 41:7, t 993) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s01-16 r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. 120, 3004 bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within 1 day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap-1 dna binding activity was transiently induced and the ap-1 complex was shown to consist principally of cfos/jund. oct-1 dna binding activity and oct-1 protein were gradually lost from the gland over the first four days of involution, whereas oct-1 m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap-1 induction and oct-1 inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct-1 may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap-1 dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap-1 on progranlmed cell death of manlnmry epithelial ceils. bcl-2~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, 1066 epalinges, 1 institute of molecular biology, glaxo inc.,1228 plan los ouates. 8cl-2(z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl-2o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl-2 species. we show here that membrane attachment is not required for the survival activity of bcl-2o< a truncation mutant of bcl-2(z lacking the last 33 amino acids (t3) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l929 fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl-2 which lies in the conserved domains 4 and 5 upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc2 mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc2 mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s02-01 s02-04 platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from 20 patients and 10 controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd68, in both groups the pdgf ~9 cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented 4.65+1.63% (mean+sd) of the volume occupied by lung tissue in the boop cases, and 2,12+0.65% in the controls (!0<0,001). similarily, 10.73+4.69% of the lung tissue was occupied by cd68 e~ macrophages in the boop cases, compared to 5.37:~3.73% in the controls (p<o.005). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd68@ cells on the 30 cases (r=g.50 p<0.0o5). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp-1, we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch 3012, bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure (14 tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo1.147. pp989-996,1993) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that 5% of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv40-t immortalized human bronchial epithelial cells (beas-2b). inos activity was higher than 100 pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee 1992, science 257:p 967). therein a 3'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or 10 seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant 31-30946.91) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. (1993) jcb 121:241). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep1 encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse 4, 3012 bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p140 ex~ revealed several conserved stretches of amino acids in their nh2-terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this 360 bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a 1 gtlo mouse testis cdna library. after several rounds of successive screening a total of about 4 kb of mouse edna (msep1) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, 38 % and 41% of amino acid identity, respectively. the msep1 sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc-1), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc-1.we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd4/cd4 double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag-2 -/-mice, were tested. only the scid mice and the cd4/cd8 sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. 7 2 kda in sds polyacrylamide gel electrophoresis. the ade6-m26 mutation in the ade6 gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m26 activity, the effect of the deletion of the ade6 promoter in combination with either the m26 and m375 mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m26 hot spot activity relative to the m375 control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m26 hot spot activity, rather than lack of transcription being responsible for the loss of m26 activity. the role of transcription in m26 hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top2 mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed 1 nucleus with 4 completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top2 rec7 double mutant (rec7 is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top2 rec8 double mutant (rec8 is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster (1). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical 352 bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted 5' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line 2 cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, 0., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. 4, 3012 bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse 4, ch-3012 bern homologs of the salmonella typhimurium (mutl, s and 59 and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about 220 bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the 5' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the 3' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch-3012 bern rec7 and rec8 are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~1000 fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec7 and rec8 is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of 56kda for rec7 and of 73kda for rec8. to remove the gst carrier, the rec7 and rec8 fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d6bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit6t zurich, wintertharerstrasse 190, normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag-2 genes they also become able to perform v(d)j recombination. by transiently transfeeting nih 3t3 fibroblasts with both intact rag genes we found da recombination rate of 0.06%. no recombination was detected, when we transfected the intact rag-1 gene with a mutant rag-2 gene, which lacks 89 c-terminal amino acids, or the intact rag-2 gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l4 fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of 2-2.5. this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; 1nstitut fiir molekularbiologie l universitfit ziirich, h6nggerberg, 8093 ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, 8091 zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about 108 logld50 units/g of brain, are detected in prn-po/+ mice 33 weeks after inoculation (the earliest time point measured), 24 weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than 16 weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the 8-app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons 16 and 17 in humans framing the sequence of the 8-amyloid itself.a genomic clone was isolated from mouse strain c57 black/6 containing the coding regions of the last three exons (16) (17) (18) according to the human sequence. sequencing of all three exons including the large 3' non coding region showed a 100% homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high (97.6%) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular 8-app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to 150 bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about 50 bp. heterology at the distal end was most difficult for reca to resolve, and already 22 bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch-3012 bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations 1. we analyze dna rearrangements at the level of the complete genome by rflp, using 7 is elements as probes for hybridisation to southern blots samples taken at different time points from 12 independent cuttures of e coli b grown by serial transfer for 10'000 generations 2 and stored at -70~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously 2 naas t., 81o~ m, fitch w.m and arber w, (1993) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is 15.2 kb in length, contains 13 peptide coding sequences, and genes for 22 trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj 07103-2714, usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or 293 cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. 67 4822 (1993) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (-522 to +13) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between 8.5 -13.5 dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., 0fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i066 epalinges, switzerland. hpvi8 is associated with genital carcinoma. like other human papillomaviruses, hpvi8 cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi8 l1 and l2 genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l1 and l2 genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l1 and l2 proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l1 and l2 proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to 15 structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. 70, ch-4056 basel bacteriophage p1, carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop1, functions expressed by transposons carried on the p1 genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p1 regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c1 and bof, whereas transcription of its morphogenetic genes from p1 specific late promoters is induced by the early expressed gpl0. by site-directed mutagenesis the late promoter ps, composed of the -22 inverted repeat aagttactt and the -10 box, was functionally characterized. p5 and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr2b all share the perfect inverted repeat around position -22, whereas the weaker promoters lpr91, lpr82a, lpr82b and lpr83 contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type 1 reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. (1993) nucl. acids res. 21, . 3'-azido-2',3'-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p66 polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p51 polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi3 dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d68 subunit at a molar ratio of 4:1 mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p66 with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p66 compared to the hiv-rt heterodimer d66/d51, suggesting a function of the 51 kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the 3' long terminal repeat which can code for a 36 kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co4 + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[314 bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a 47 kda protein which is specifically detected with an anti-orf-peptide antiserum. the 47 kda protein can be labeled with d-[2-3h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf5), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the 47 kda orf g}ycoprotein has a short half-life of about 1.5-2 hours in this system whereas the orf5 truncated protein is even less stable; its half-life is about 20 minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of 32 of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over 60% of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, 8093 zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to 50% of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a 58-year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a 108 bp in-frame deletion in the pre-s 1 region. transfection studies in huh-7 cells with the predominant pre-s 1 deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s 1 deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi6) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct-2, noet-3 and noct-4) bind to taatgarat sequences from the viral icp0 and icp4 promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat-1. we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p37k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch-1015 lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated 37k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (<30% of the virus production). if p37k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p37k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p37k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch-3012 bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e2 and p80, a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some 600-800 virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in 20 to 30% of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp 38 transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc18 plasmid and dnase digested, random fragments of 200 bp were inseded in ~,gtl 1 dna and expressed in e. coil y1090. as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a 5kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the 5' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the 5' terminal hairpin of mvmp. mvmp rf dna containing the entire 3' extremity and extending to the hha i site downstream of the axis of symmetry of the 5' extremity was cloned into pgem-szf(+). this construct contains more than half of the 5' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a9 cells were transfected with this dna and the 5' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n0n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the 5' end indicated that both flip and flop forms were present. the corresponding 5' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type-1 share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp16. on these promoters, vp16 combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct-1/pou domain protein (without the activation domain of oct-1 ) and vp16 where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct-1 serves to recruit vp16 that has no intrinsic dna binding capacity. vp16, however, possesses a stong acidic c terminal activation domain of -80 amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct-1/pou domain protein and recombinant vp16 that has been truncated at the c terminus. additionally, we are also testing different truncations of vp16 in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus 7.5 kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the 7.5 kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il-2, il-8 and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids 1-67 (n-ter) and 67-262 (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, 4056 basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, 1992, j. exp. biol. 167:1-14; neely, 1993 , i. neurosci. 13:1292 -1301 . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., 1988, pnas 85:4075-4078) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a23187, led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. 3127814.89 to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca3 cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing 16 mm k +, 1s,3r-acpd (50 ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from -80 to -60 mv and calculated by linear regression, was -15.7 â�¢ 18.7 mv, and was shifted to -53.4 â�¢ 6.4 mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the 1s,3r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = -51.2 ~ 5.6 mv in [k+]o = 16 mm and shifted to -74.0 :t: 2.8 mv in [i4+]o = 8 ram). this k + current was voltage-independent in the membrane potential range of-120 to -20 mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u-937, ml-1 and hl-6o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi1 is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch-8029 z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol (5-500 nm), that activates g,-linked receptors, reduced the amplitude of i~ by 51.5 â�¢ 3.6% in voltage-clamped ca3 pyramidal cells in ttx (n=20). reduction of imm by isoproterenol was curtailed to 27.3 â�¢ 2.9%, (p < 0.001) in the presence of baclofen (i0 ~m), acting at gaba b receptors, or adenosine (50 nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at 37 ~ for 3 successive 30-rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine (300 ttm), a decrease in either basal or k +-(50 ram) stimulated da release was observed during the 2 nd 30-rain period. tested under similar conditions, dibutyryl cgmp (1 mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, 100 gm) increased basal da release during the 1 st and 2 nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch-1211 geneva 14 while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca 2+, the pathway for ca 2 ⧠entry is not yet accurately defined. at least two mechanisms seem to be involved: 1) the opening of voltage-operated ca z ⧠channels, and 2) the stimulation of a "capacitative" ca 2+ entry regulated by intraocllular ca 2 ⧠stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca 2+ channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine (1 lam) completely blocked the cytosolic ca 2+ response to k +, which exclusively activates voltage-operated ca 2. channels, but only partially (22 %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca 2+ influx aiter depletion of ca 2 ⧠stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca 2+ entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only 30% of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca 2+ influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca 2 ⧠antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch 3012 berne *depanment of anatomy and physiology, university of dundee, uk dundee dd1 4hn. subjection of hela cells to 45~ for 30 minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around 60kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for 24h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around 32kda, 44kda and 55kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk2 revealed that considerable pools of erkl and erk2 show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with 12-o-tetradecanoyl-phothol-13-acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk2 antibodies. these results suggest that heat shock induced activation of at least erkl, erk2 and the 55kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. 31-27636.89. effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. 190, switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto-2b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: 1) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and 2) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was 5 times the basal rate on polylysine and 2.5 times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h7 or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie 11 der universitat uz zart'ch, winterthurerstrasse 190, 8057 ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of 22 gin residues interrupted by 1 arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~.23 mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_25 were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala]21, 23, 25, 27, 31 on gal4 chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca2+-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca 2+ concentration ([ca2+]c) was clamped at various levels with the ca2+-ionophore, ionomycin, in order to locate the sites of action of ca 2+, to determine their sensitivity towards ca 2+ and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca2+]c, we show that ca2+-clamping (50-860 nm) induced a concentration-dependent increase in the production of both pregnenolone (506___70 % of the basal level) and aldosterone (255+27 % of the basal level, ec50 = 273 nm). by contrast, ca 2+ did not influence the conversion of 1 t-deoxycorticosterone into aldosterone at concentrations up to 600 rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca2+-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, 25hydroxycholesterol, indicating that ca 2+ acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca2+]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca2+]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie 11, universilat ziirlch, 8057 z'6rich # lnstitut rir molehdarbiologie i, universitiit ziirich, 8093 ztirich we have previously described and cloned a factor (mtf-1) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf-1 is a protein of 72.5 kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf-1 gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf-1. moreover, due to the loss of mtf-1 the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf-1 is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc12) were differentiated with ngf for 3-4 days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) (0.2.50.0 nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within 15 minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about 30-60 minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura-2 single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca2+] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca2+] i were analysed. ca2+ entry across voltage-activated ca 2-~ channels was inhibited by 50% after 30 minute treatment with cl-a, while the atp-induced ca2+ entry via ca2*-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca2+]i started at the grape-like domain. the speed of the ca 2+ wave across the cell body was slower than in control cells. the addition of 0.5 gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within 24 hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak2, tyk2) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately 5kb and a minor species of approximately 4kb. nucleotide sequence analysis revealed a 84% homology to the porcine syk gene. the syk gene is expressed as a major 3.0kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring (6c8) and nondifferentiaflng ((33) subclones of red-1 rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (>70%) of immunoreactive gai3. simultaneously, a iruncated form of g~2 accumulated in the cytosol, while the membrane concentrations of gai2, gets and gaq/11 remained unchanged. nondifferenfiating (33 cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng 6c8 cells were consistent with p2t receptor coupling to gai3. thrombin receptors seemed to couple preferentially to gai in g3 cells and to gaq in 6c8 cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek5, the murine homologue of the chicken cek5. all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. 0vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., 1992, 12:5217-5227 a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p2 receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca2+-dependent isoenzymes (c~, [3, 7) , as compared to the ca2+-independent isoenzymes (5, e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp 41251 did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca2+-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo-3 and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk1 [clone 4]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk1 cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk1 cells. a. and preiss, a. biozentrum der universit&t basel, ch 4056 basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii0 kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ 150 kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy-1 and cd26 surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd45 is a key regulator of t cell proliferation as it controls the activity of src family kinases p56/~k and p59fy n. associations of thy-1 with cd45, and of cd26 with cd45 have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy-1 and cd26 accessory molecules. we report that thy-1, cd26 and cd45 can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a 78 kda protein that is recognized by an anti-bip (grp78) antibody. in vitro kinase assays show the selective association of p56 ick and p59fy n with thy-1 and cd45 contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, 1015 lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih 3t3 cells using cat reporter constructs containing the promoter of either the t3 responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try1 and unactivated mfpar potentialized the t3 induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t3 dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the 9-cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp90 via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp90. we genetically addressed the role of hsp90 in budding yeast by analyzing three kinds of hsp82 (the essential yeast homologue of hsp90) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp82 homologues from other species). hsp82 mutant are examined for their ability to complement a hsp82 deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp82-hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp82 leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp82 homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, 30, quai ernest-ansermet, ch-1211 gen&ve 4 id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a3-actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different 5' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon 9, 1005 lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch-1015 lausanne. lab. of mol. growth reg., nih, bethesda, md, 20892 usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, 3 subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of 46 kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid (0.5 -20 ram) induces various patterns of ca2+i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura-2 loaded cells. most frequently, ca2+i oscillations were observed, although other patterns (a single ca2+i transient; a single peak followed by a sustained elevated level, or a sustained ca2*i elevation solely) were occasionally seen. the amplitude of ca2*i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, 0.2 rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin (50 pm, a cyclooxygenase inhibitor) and by 5, 8, 11, 14-eicosatetraynoic acid (etya, 3 ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: (1) arachidonic acid per se elicits ca2+ i oscillations in these ceils; (2) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, 8091 ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t1 which encodes a secreted glycoprotein of the immunoglobulin superfamily. a 448 bp region located between 3.6 and 4.0 kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap-1 binding site which is absolutely required for t1 gene expression. it is surrounded by 3 e-boxes. at least 2 of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap-1 complex can activate the d.e. gene t1 in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([3h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla2 on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla2) were tested on ip production elicited by a 5 sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [3hi 1,4,5-ip3 and of its degradation products. an inhibition was also observed with the two pla2 inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca 2+, mg 2+-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla2. regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol 1,4,5-trisphosphate (insp~) formation, leads to an increase in the intracellular ca 2. concentration ([ca2+]~). these hormone-stimulated [ca2 ⧠signals have a complex temporal and spatial regulation, with the [ca2*]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca2+]~ spike is a wave, where [ca2 ⧠is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca2+]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca2 ⧠response are known, the precise mechanism generating these complex [ca2+]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ ⧠release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp3-sensitive intracellular stores behave as functionally discrete units, and that during a ca 2 ⧠spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca 2+ release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box 2543, ch-4002 basel "department of membrane research and biophysics, weizmann institute of science, rehovot 76110, israel phenylarsenoxide (pao) at 20btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or 2,3~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol4containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci2 cells and from fibroblasts as well as from sulx:ellular fractions by 2-dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize 3 distinct molecular weight forms (68, 85 and 120 kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at 89b4-i0 region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit6 de lausanne, rue du bugnon 9, 1005 lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [14c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge2 and pgd 2 respectively. in absence of gsh: 1) pgd2 could not be detected; 2) the level of pge2 was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i4c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: 1) does not correspond to a degradation product of pge2; 2) crossreacts with pge 2 antibody; 3) fits the conditions required for a 15 hydroperoxy pge2 (chemical degradation into pge2 by hydroperoxyde reducing reagents such as snc12 or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity (92% and 85% identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch-1700 fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins(3,4,5)p 3. but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins(3,4,5)p 3 in human foreskin fibroblasts with an ic50 of about 5 nm. a similar inhibition was observed in in vitro assays (ics0=lnm) with pi 3-kinase ii~nunoprecipitated by antibodies directed against the 85 kd subunit (p85). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p85, indicating the correct interaction of p85 with the pdgf-~ receptor. the pl10/p85 complex of the pi 3-kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi 3kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi 3-kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was 10 to 50 fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only 5 to 15 fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch-1700 fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins(3,4,5)p3 (pip3) which is due to the activation of ptdlns(3)-kinase. pip 3 has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip 3 in a dose-dependent manner (ic50 = 5 nm). furthermore, ptdins(3)-kinase activity immunoprecipitated from resting cells is totally abolished by i0 nm wortmannin (ic50 = 1 nm). these results show a potent and direct effect of wortmannin on ptdins(3)-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip3 is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins(3)-kinase, suggest a modulatory role for pip 3 in cytoskeletal rearrangements.we are currently investigating the effects of pip 3 on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca 2+. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch 8057 ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct-2a. when ectopically expressed in non-lymphoid ceils, oct-2a is not sufficient to mediate enhancer activity of a multimerized 50 bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet-2a. to this end, a oct-2a expressing yeast strain has been constructed which bears two integrated reporter genes, his3 and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct-2a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv-1 cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca2+-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon-7 and tumor necrosis factor-~ induced little no 2-production, but primed macrophages for enhanced n0 2-production induced by salmonella dublin or lps. this is one of the first demonstrations of no 2-production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis (2-dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by 2-d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis 1992; 13:707-714. this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif-4a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf-4b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif-4a mutant were isolated. one of the suppressors (stm1) is a single copy gene which encodes a protein resembling the human translation initiation factor eif-4b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm1 gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of 25 amino acids of unknown function and has -like the human eif-4b -a rna binding domain. the second suppressor (stm2) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~4aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain 21, ch-3013 berne using heterologous probes we isolated cdnas coding for translation initiation factor eif-4a from tobacco, eif-4a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif-4a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif-4a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type 1 (hpiv1), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the +1 reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv3 cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and 3, we have shown that positions +5 and +6 (the first g of the gug triplet being +1) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp9/14 of the signal recognition particle is retained in 2 fusion proteins fabrice bovia & katharina strub, d6partement de biologie cellulaire, universit6 de gen~ve, ch-1211 gen~ve 4 the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of 6 proteins and f rna molecule (srp rna). the 2 smallest proteins srp9 and srp14 are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these 2 polypeptides bind to the srp rna exclusively as a heterodimer (srp9/14). we have constructed single-chain variants of this dimer using a short peptide linker of 17 aa to connect the c-terminus of the 14 kd subunit to the n-terminus of the 9 kd subunit (14-9) and vice versa (9-14). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the 2 fusion proteins fold into a similar structure as srp9/14 and bind srp rna as a monomer. furthermore, they can functionally replace srp9/14 in the elongation arrest and translocation assay. since the 2 fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a24 s06-08 studer, r. and hunziker, p. e., 8iochemisches institut der universit~t z0rich, ch-8057 z0rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, 1700 fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep1 = ascaris lumbrieoides eliminated protein 1) which is located within the germqine specific material. alep1 shows strong homologies with the ribosomal protein s19 (rps19). the transcription of its gene takes place only in the germ-line. 2d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep1 protein (rps19) is present only in the germ-line ribosome. does the alep1 germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep1 from ascaris somatic tissue named rps19', we are currently studying the expression pattern of this rps 19'. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps 19? since ascaris is not suitable for genetic analyses, we isolated rps19 from the nematode caenorhabditis elegans. the c. elegans rps19 cdna shows only 60% homology at the nucleic acid level. rps19 is located on the c. elegans chromosome iv. the identification of a rps19 mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j2 and michel, m.r. t 1 institute of medical microbiology, university of beme, ch-3010 berne. 2 institute for immunology and virology, ch-8006 ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf9) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif3 is a new eukaryotic initiation factor which shows 26% identity with the sequence of mammalian translation initiation factor eif-4b. the tif3 gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif3 gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif3,or mammalian eif-4b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the 5' leader region in a tif3 gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta 1174 : 191-194, 1993 . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus-2 gene exists which, however, shows strong sequence diversion in the 3'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the 3"-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the 9kd and 14kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit4gen~ve the signal recognition particle (sr2), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp9and srpl4are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp9 and srpi4 form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp9 and srpi4 revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi4, this u-helical region is located betwen aa 72and i00 at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., 1989) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines ((2) were examined. because the the additional ef-lc~ copy is under the control of the hsp70 promoter, flies were aged either at 25~ or at 29~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at 37~ but not in ef flies grown at 29~ nor in control flies at 37~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at 29~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p450, nadph:p450 reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc 254 pp 2148 , 1979 . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st6rilit6, hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor-1, protease nexin-1 and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using 125i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after 48h of stimulation by the gonadotropin fsh (1-100 ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by 2-6-fold, while keeping their cell surfaceassociated ahspgs constant. analysis of 35s-labelled hspgs revealed that heparan sulfates constitute about 40 % of the surface-associated and 10% of the soluble granulosa cell glycosamineglyeans. finally, using 125i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and 3,5,5-trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased 6-21 times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha6~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for 9-cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi 8226 myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for 4 days alternating with growth in dox-free medium for 17 days. after > 9 months of treatment, the cells developed an atypical mdr phenotype with 4-to 6-fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < 2-fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr1 drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around 50 and 60-70% at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by 60-90% relative to the wild type cells without detectable p190. this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t7 bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a 6xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), 17 cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the 17 edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone 5 (nkg5); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining 6 edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c-5 position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x2(vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp1 was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab 405 (zlf), basel, ch-4031 chromosomal subregion llp13 displayed non-random allelic loss in 61 nsclcs. llp13 contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). 76% of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although 38% of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at 17p13, immuno-histochemical and sequence analysis of the p53 gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p53 gene is a pivotal event in the progression of nsclc. $08-11 recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. 75, 8032 ziirich, and *institut for nutztierwissenschaften, eth ztifich, 8092 ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf3 and myf4. therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome 11 band p15, in the first approach we transferred a normal human chromosome 11 into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome 11, as it was shown by chromosome 11 specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd48, a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to 90% (p.g.) and 78% (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd48 marker (cd48+) and cd48 deficient clones (cd48-) were obtained. takeda et al. (cell 73, 1993, 703-11) showed that in some (cd59-) bcell clones, a deletion of the p1g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd48+ and cd48-clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd48-t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd48phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd48-t cell clones. supported by grant 31-30757-91 of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation (30 mins.) of c-foser by estradiole (e2) led to the disruption of epithelial cell polarity within 24 hours, as characterized by the expression of apical and basolateral marker proteins. during the next 3-5 days, however, the cells fully regained their polarized phenotype. conversely, long-term activation (24-48 hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b16 routine melanoma cell fine (b16-ls9) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b16-f1) or inng-specific (b16-f10) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls9 and f10. in contrast, b16-ls9 grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b16-ls9 cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b16-ls9, enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b16-ls9 ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b2 precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp70 probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me49). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight 25% below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at 45% below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by 15% when compared to controls (p<0.01); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin 10, the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse 70, ch-4056 basel, switzerland. phone ++41-61-2672171, fax ++41-61-2672148. the mitochondrial chaperonin system consists of chaperonin 60 (cpn60; also termed hsp60), which is homologous to e. cull gruel, and chaperonin 10 (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl0 of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn60, subsequent refolding of the enzyme is strictly dependent upon yeast cpnl0. in the presence of mgatp, yeast cpn60 and yeast epnl0 form a stable complex that can be isolated by gel filtration. the atpase activity of hsp60 is not inhibited by complex formation with cpnl0. a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel (1). to test the in vivo role of cpnl0, we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of 11,372 da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between 23 ~ c and 37 ~ c (2). (1) rospert, s. et al. (1993) proc. natl. acad. sci. 90, in press. (2) rospert, s. et al. (1993) febs lett., in press. characterization of yeast mitochondrial heat shock protein 70 , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, 4056 basel. mitochondrial hsp70 (mhsp70) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp70, we used the cloned gene (a gift of dr. n. morishima) to modify mhsp70 with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp70. we developed a purification procedure that allows us to recover this tagged protein with a purity of about 95%. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp70 with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt0. we have copurified a protein of about 20 kd together with mhsp70. the 20 kd protein could be released from mhsp70 by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine 123 fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. (1990) , bba, 1018, 190-193) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about 110 inv. therefore the detection limit of changes of a~mit within the physiological range is about 10-20mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about 20inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j6quier e. and tappy l. institut de physiologie de l'universitg bugnon 7,1005 lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in 8 subjects over a 8 hour period in a respiratory chamber. a mixed meal containing 13c labeled carbohydrates was ingested at 9:30 am and an exercise session (walking at 5 km/h 45 rain, slope 10 %) was performed at 8:15 am (fasting) or 11 am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath 13co2) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over 8 hours was similar but net cho ox was 16 % lower, lipid ox 61% higher and exo cho ox 39 % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by 40 % (p<0.0001), while glycogen breakdown was increased by 12 % (p=0.06). in conclusion, a 45 min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a 8 hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. 8chini hooft van huijsduijnen, p.k. jackson and d. picard d4partement de bielogie cellulaire, universit4 de geneve, ch-1211 geneve 4 the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih3t3 cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. 48 hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g1. a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, 8093 z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h1, h2 and h7 of tim from b. megaterium, corresponding to amino acid residues 13-36, 44-55. and 220-227 respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, 23% of the lipid droplet surface in goats and 40% in dogs were in direct contact to outer mitochondrial membranes or -in other terms -1% of outer mitochondrial membranes in goats and 2% in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a 2 times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. (1986), 7, 187) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl (1.8 and 1.6 times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/3f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise (75 w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: 1) the spectral analysis of hr variability where the high frequency component (hf, above 0.15 t4z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about 0.1 hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and 2) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (-39_+ 10%, n=6) was measured. contrasting with this, in normal subjects, no change in twa (+2_+12%, n=6) but a decrease in beth hf (-67_+13%, n=7) and lf (-85_+5%, n=7) without change in lf/hf ratio (-19+_.26%, n=7) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r=0.79, p<0.0005) between ps and the ree. the slope of the regression line indicated a net cost of ps of 3.6 kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch-1005 lausanne. the hearts of 4-day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from 10 to 60 seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of 8 and 20 mmol/1 of glucose 1) the arrest of cardiac activity under anoxia was delayed by 60 and 110 %, respectively, 2) the duration of the postanoxic cardioplegia was reduced and 3) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g2/m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm-2, known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded (24-48hrs) when compared to spontaneous maturation (30ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during 48hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay (3-rhrs). mpm-2 epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~-121l g~. ~t4rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b6~jinnin9 with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the 1342 hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist6ne hi kinase activity increases before gvbd (2x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p34 cdc2, following gvbd, histone hi klnase activity culminates (8x) in metaphase i. activation by phesphorylation of p42 mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following 7 hours of culture. the upper migrating form of p34 cdc2 is present for 8 houra. the activation of p42 mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p13 sucl, neither iocrease of histone hi kinase activity nor p34 cdc2 dephosphorylation are observed although gvbd is induced; p42 mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps4 cdc2 and p42 mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf8 an alternative pathw~ leading to gvbd that is cdc2-independent and that involves p42 mapk activation ensuing mpf-independent phosphorylations. s 10-04 urich, k., 4002 basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol-3-kinase (pi 3-k) and phosphatase 2a (pp2a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione 527, a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine 160 and threonine 291, are also phosphorylated by purified p34 ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine 160 were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $10-05 schmidt, s., fa~khauser, c., and viesturs, s. isrec, 1066 epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc7 and cdcll genes result in continued nuclear division without cytokinesis, while cdcl6 mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl6 product is required for maintenance of p34 r activity in mitosis and the primary sequence of the cdc7 protein indicates that it encodes a protein kinase. data concerning the role of the cdc7, cdcll and cdcl6 genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv40 origin-containing dna in vitro. it is a five subunit protein with one large subunit of 100-i40 kda and four small subunits of 36-40 kda. rfc binds specifically to primertemplate structures at the 3'-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc1, codes for a 95 kda polypeptide that is 36% identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc2, coding for a 40 kda polypeptide, is 50% identical to the hrfc 37 kda subunit. rfc3, coding for a 38 kda polypeptide, is 51% identical to the hrfc 36 kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc2 and rfc3, as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p53 is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p53 modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp53 protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p53 blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p53 is able to reanneal complementary dna single strands. since both wt and some mutant forms of p53 share this function, they are unlikely to be related to the p53 tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p53 inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p53 can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi3 dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p53 exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p53-rp-a interaction may function to protect replication enzymes from inhibition by p53 under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of 1600, 1570 and 1280 bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex174. because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh1s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol 8 sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp 70 from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap70 homolog from escherichia coil, and used them for detecting of hsp70 protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of 70kda, and has been recognized by antibodies against dnak and eucaryotic hsp72/73, it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp70 on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc2 mutant which is unable to complete the g1/s transition. nicole sr and viestars simanis. chemin boveresses 155, 1066 epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc2 gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc2 null background, when the only source of p34 cde2 is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc2 gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc2 gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g1-s transition is provided by the observation that it is able to complement a cdc2a2l mutation in trans. this mutant is defective only for (32 function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, 1993, j. cell biol. 123:357) where it is localized on a subset of microtubulea. when caco-2 cells are grown to confluency and become polarized, e-map-115 expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map-115, with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map-115 may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map-115 is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t 15 varies during mitosis. in early prophase, no e-map-115 can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map-115 is hyperphosphorylated. this hyperphosphorylated form of e-map-115 is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map-115 during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map-115 activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g1 and mitotic celts are strongly labelled with the cr-antiserum 7696, while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g2 in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr-22k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, 8. institute of histology and gen. embryol., university; 1700-fribourg a mrna for an alternatively spliced form of calretinin, ealretinin-22k (cr-22k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the 14 amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr-22k and the (h)6(nanp)19 polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last 15 amino acids of cr-22k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the 2 sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr-22k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the 2 antibodies. both antisera detected the protein cr-22k in immunohistochemieal stainings of widr cells confirming the presence of cr-22k in widr cells. the tctp (p23) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl312) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl312 structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl 312has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl312 expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch-5232 villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad3, radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad9 gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding 4 kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk2. probably, this cdna encodes the p40 protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p13 sucl beads. treatment of cells with cdk2 antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with 3h-thymidine (3h-tdr) during their first week of life when most of smcs were proliferating; then, 3h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. 5-bromo-2'-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of 3h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of 8 days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the 3d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about 100gm were analyzed. upon wounding, i.e. upon excision of a 1 mm 0 "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by 2-d page after tctp homolog gene (ykl312) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl312 gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl312 +(n=10) and ykl312 -(n=l 0)) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl312 gsne product. the second one corresponded to a 28 kda peptide with an isoelectric point of 4.6. this ykl312 associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of 120 kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands (40-80 tim wide) inserting into large reetangular cell areas (side lengths >1500 ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a 12 x 12 photodiode matrix array (maximal spatial resolution 151.tin x 151am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity 0.35 m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant 823a-028424 (sr) and usphs grant ns 16824 03ms). it is a noncollagenous glycoprotein with a molecular mass of 148 kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $11-05 muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and 5 to 25% of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that 5' sequences play an inloortant role in this regulation. we have located a 160bp transcriptional enhancer (bcei) within the 5' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than 1.5kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/24 dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of 1,3 nmoles/weu of 100.000 sc. in elonal cultures of proliferating sc, this intracellular level of ach was 0.l nmoles/well. when cells were cultured in the presence of an esterase inhibitor (10 ixm phospholine), the ach amount was enhanced 2 fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase (2 gm bromoach), gave a 50% decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a 15 min incubation, and the measured level of ach was 3 pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in 0.1% fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either 220 kda or 350 kda. by using antibodies specific for the large (350 kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether 35s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch-3010 bern locomoting blebbing cells colchicine (10-sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about 2~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by 0.5 m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf-1, the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf-1 into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf-1 during the process of osteoclastogenesis. by in situ hybridization, expression of csf-1 was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf-1 receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf-1 to osteoclasts was shown. expression of c-fms parallels the expression of csf-1 in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf-1 forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $2 and $6. if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $2 next to $6. we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of 17 substitutions in $2 (c232), and 7 of 19 substitutions in $6 (c393) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $2 mutants. in $6, in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position 393 in $6 of kv2.1 is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $5 and $6 of voltage-gatedk + channels, called h5 or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j3 barrel-like structure formed by four adjacent antiparanel j3 sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv2.1 (drki). twemy-eighi positions from k356 to t383 were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch3so2sch2ch2nme3 + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with 3 mm mtset. the mutants p361c, i379c, y380c, and k382c showed 87%, 99%, 39%, and 21% reduction in current amplitude, respectively. in coutrast, mutants $363c, a367c, t368c behaved like wild type kv2.1 with less than 5% current reduction. our results suggest that the side chains of p361,1379, y380, and k382 are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k382 to y380 to i379. (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. 190, ch-8057 zlirich comparison of 5' flanking sequences of three gabaa-receptor subunit genes (atl, ~, 8) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and 72 subunit gene promoters and in seven tandem copies in the 8 gene promoter. a novel dna binding activity (bsf1) was identified that binds to various versions of this purine element. bsf1 was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf1 during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf1 as a transcriptional activator of neuronal genes. therefore, bsf1 may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf1 binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf1, the question, whether bsf1 is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg 2+ macroelectrodes using the neutral mg carrier eth 7025.we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained 2 mmol/l n%atp. this solution was titrated with mgci 2 and the changes in [mg ~] above 0.05 retool/1 monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/1 at 37~ ph 6.4, 103.3-+-25,3 (n=9); ph 7, 59.7:k-27.4 (n=6) and ph 7.4, 30.g~15.4 (n=7). elevation of the [naq to 50 m~l/1 and reducing the [kq to 1(30 mmol/l was without effect at ph 6.4 (n=6). reducing the temperature to 25~ increased the k a at 7.4 to 49.0:k21.6 (n= 5). mg =+ macroelcctrodes provide an easy way of measuring [mg z ⧠in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn 2+ and ca 2+, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn 2+, ca 2+ or h + indicate that y401 is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, 1211 geneva 11. steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around -40 mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that 5-10% of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as 2na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c6/36 and sf9. two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. 423:152, 1993 it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf9 cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at -77 mv and +66 mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at -59 mv and +60 mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r177 cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation (25 him k +) or treatment with nmda {i00 ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked 45ca2+ influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i,2a,2b,2c,2d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand 125i-cgp55802a. the intensity of the phctolabeled bands, thought to represent nr-i and nr-2a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature 361, p467-470 (1993) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is,3r)-acpd, at 1-50 /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at -50/-40 mv and which reversed at aound 0 mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators (4[~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca 2+ and k + channels in ngf-differentiated pc12 ceils. whole-cell ba 2+ and k + currents were recorded with the patch-clamp technique. by using specific ca 2+ channel blockers (co-conotoxin (cgtx), isradipine) we found 3 types of ba 2+ currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t0-cgtx plus isradipineresistant iba. 4[~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc (19-31) (2 pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba 2+ currents. however, the 0~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca 2+ and k + channels by the pkc signalling pathway in ngf-differentiated pc 12 cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either 0 or 4 amino adds and site b encodes either 0, 8,11 or 19 (8+11) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a4b19. in contrast, non-neuronal cells and muscle calls contain a4b0 and aob0 mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a4b8, whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. (1992) , curr. up, cell biol. 4:869-874). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos-7 and 293 cells revealed that chick agrin isoforms a4b8 and a4b19 were highly active, while a4bll was up to 40 fold lower in activity. no activity could be detected for the a4b0 and aob0 isoforms. in agreement with these findings the a4b8 isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda (50-100 #m) generated an inward current of 10-60 pa, which was insensitive to ttx and which reversed at about 0 inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)-2-amino-5-phosphonopentanoic acid (d-aps) or by mk-801. it was potentiated by reducing the extracellular magnesium concentration from 1 to 0.01 ram, or by adding glycine (10 /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from 2 to 0.01 mm caused a 1.5to 4-fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. 2) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. 3) when various substrates were impregnated locally with agrin a4big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, 1211 geneva 4, switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n=16). in the proliferating state, 54% (n=35) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current (10 pa/pf) and, as expected, after fusion of salellite cells into myotabes, 100% of the ceils displayed an ach-activated current (n=6; 26 pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, 93% of the sc (n=15) displayed an ach-activated current and, in addition, these cells expressed a 4 times higher ach-current density. (40 pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter 3 months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $12-20 we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x2 or c~3 with either 1~2 or 1~4 crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct31~4 neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic50): hexamethonium (0.3 i.tm), mecamylamine (0.2 p.m), pentolinium (0.2 i.tm) and trimetaphan (1.2 ~tm). with (x3132 nachrs the ic50s of the same antagonists were about 10 times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~3132 and t~2~2 nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a3(34 and t~2~4 nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant 31.31018.91 to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be 16 per #ms. after 30 and 75 rain tris-inkubation the tod decreased to 10,4 and 1,5, respectively. this reaction was reversible when these axons were reincubated in na~-c41ntaining solution. after 120 rain reincubatiou the tod was 11.8 per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for 30 min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of 1-2 rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of 7 min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors 4n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the 93 kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae 1i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~5, ~ & y2), we treated plegnant rats with diazepam (l.25mg/kg/d; s.c.) from gestational day 14 to 20. we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd20, pns, pni5 & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr95531 markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap7 at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat 1) nmda receptors in ai are activated following mgb stimulation and that 2) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l,5-u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear 1,5-~-l-arabinans (-90 i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (-14 i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l,5-~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr 42'000, iep ~ 3.0, ph optimum 4.8, temperature optimum 55~ ph stability 3.5-8.0, temperature stability s 45oc, km = 0.205 mg/ml, vmax = 17.7.10 -3 ~unol/min. zn and hg showed potent inhibitory effects. 3.2) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., 1992, plant science 82:21-27) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec 5.4.99.5) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro7) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. 267, 2810 -2816 . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after 6 to 30 hours of oxidative stress induced in wheat seedlings by a treatment with 10 mm cuso 4. the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described 45 kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia 47, a67) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by 2-aminoindan-2-phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, 8092 zijrich. *technical university wroclaw, 50-370 wroclaw, poland. all four phenylalanine ammonia-lyase (ec 4.3.1.5) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine (15gm to 24.5/.tm), similar temperature (58~ and ph optima (8.5). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. 2-aminoindan-2-phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. 1992,625) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c-12st-19 and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l0 ms and 1z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of 14c-benzoic acid (14c-ba) or 14cphenylalanine (14c-phe). alternatively, leaf 1 of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using 14c-ba or 14c-phe. free and bound 14c-phenolies were quantified by hplc. incorporation of 14c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly 14c-sa and an unknown polar compound. 14c-phe gave also some 14c-sa, in addition to 14c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with 14c-sa, 14c-benzoic acid (ba) or 14c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, 4c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf 1 after treatment with 14c-sa and 14c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $13-12 the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa-4,5-dioxygenase. we have purified and characterised a copper-containing enzyme of -38kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity (30 wm-2s -i) at different temperatures (havaux and strasser, z. naturforsch. 45c, 1133 -1141 , 1990 ). when leaves were exposed for a short period (i0 min) to heat (25 to 45~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until 37~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa-4,5-dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa-4,5-dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse 1, ch-4056 basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) (1) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain (2). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with 1) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid 9 (chitavtp) and 2) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after 30 vain. and complete after 90-120 rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to 90-120 rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a-1.5, bio-gel a-5 and ha. the molecular mass was estimated to be 1,300 kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of 14 different subunits with molecular weight in the range 22-35 kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of 9 nm and a height of 14 rim. end-on projection established a 7-fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $14-01 hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c0 and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr8) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a 9enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides 1-817). a 1,8 kb hinc-ii fragment containing the 5"-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), 7f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with 2 nm of il-113. this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a 32p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k8 activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il-6, which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il-6 promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il-6 and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il-6 is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn 2 (il-2) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il-2 mrna, but does not induce secretion of il-2. in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il-2 rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il-2 mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il-2, as other mrna's like 1~2-microglobulin are not regulated. this data suggests that il-2 gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il-2 mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il-2 mrna. assuming the presence of a translational regulator that specifically represses il-2 mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il-2, there will be no translation of il-2 mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il-2 mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin-2 receptor (il-2r), tumor necrosis factor-receptors 55 (tnf-r55) and 75 (tnf-r75) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all 3 receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r75 and lowest for tnf-r55. tnf-r55 levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r75 levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)-8 when stimulated with calcium ionophom (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was released into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il-8 in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined 1l-8 concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for 1i-,-8 in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic50:15-30 mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above 0.3 g. hdl apoprotein/l caused a reduction of glial cell differentiation. $14-08 tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of 1000 u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p75 or the p55 tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs0 of hormone-independent cells towards the ecs0 of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased (10-20 %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a 4-8 fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin 1 ]3 (il-1 ~) and requires bh 4 as a cotactor. 2,4-diamino-6-hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh 4 synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh 4 via a salvage pathway. sepiapterin dose-dependently augmented il-1 i],-stimulated no synthesis, indicating that availability of bh 4 limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh 4 synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh 4 tom gtp. in conclusion, these data demonstrate that bn 4 synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh 4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. -1 [3) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il-1 ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il-113 dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il-1 [3 (2nm) and pdgf-bb (10 ng/ml, 100 ng/ml and 300 ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ (2nm) and pdgf-bb (100 ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il-1 dependent stimulation of inos. coincubation of mesangial cells with il-113 (2nm) and bfgf (3ng/m[, 10ng/m[, 30ng/m[, 100ng/m0 dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il-113 (2nm) and bfge (100ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were 100% lethal for wild type mice, correlating with serum tnf levels of up to 10 fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a 3 to 5 fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further 50%. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the 5' portion (-7.8 kb/+4.1 kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il2ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, -0.05 kb; dh3, -5.8 kb) were found. a third site (dh2, -1.35 kb) is detectable in t cells that have been activated with concanavalin a and il2. this site maps to the same position as cisacting regulatory sequences that are required for the response of the il2ra gene to signals from the il2 receptor. interleukin-6 (il-6) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t3) reduced the stimulation by more than 50 %. egf and t3 and even more choleratoxin increased the il-6 production in absence of fetal calf serum. the effect was dose dependent. 2% human ab serum substituted for i0 % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il 1 had a stimulatory effect (serum, t3, egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il-6 is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin-6 (il-6) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il-2 and il-2 receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent 10 to 100 fold increase of the il-6 production could be demonstrated after 24h and 48h. conditioned medium from tcells and from fibroblasts failed to induce il-6 secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il-6 production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il-6 production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il-6 synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il-6 production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. (1992) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il-8, which is known to be an angiogenic factor. the aim of this study is first to assess if il-8 is expressed in the palisading ceils, and second to determine if hypoxia can trigger il-8 gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il-8 is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il-8 receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il-8 is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il-8 expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il-8 by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il-8 expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il-8. it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i3 gene by virus or double-stranded rna involves induction and modification of irf-1. overexpression of irf-1 can induce expression of the ifn-b gene in certain cells. a role of irf-1 in the activation of ifn-a genes has also been proposed. furthermore, irf-1 has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf-1 alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both 1fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf-1 does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il-6, il-8, mcp-i, il-10 and tgf-132, affect each other's production and receptor system and seem to form a cytokine network. among them, il-1 may play a pivotal role, since il-i can induce or increase the production of other cytokines (il-6,il-8,mcp-i,tgf-132) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in 15 gbl cell lines and 15 gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il-113, type i and type ii receptors, indicating the presence of an il-1 autocrine loop. il-1 stimulated growth of some of the cell lines. a positive correlation of il-6 and il-8 expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il-6, il-8) in gbls. antisense oligonucleotide to il-lc~ and/or il-113 partly suppressed this secondary cytokine expression. these results demonstrate that the il-1 autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif-2, thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn-13 gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because 3t3 ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., 1992; meurs et al., 1993) . we have produced homozygous pkr knockout (pkr ~176 mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn-13 gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf-13 plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf-6 on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a 48h exposure to cpa, a dose dependent increase in dna synthesis (3h-tdr incorporation) was observed. the maximum effect was attained with 12 pm cpa (up to 4.3 fold) which was stronger than with 10 ng/ml egf (up to 2.8 fold). tgf-81 and tgf-132 inhibited this response dose dependently (idso = 0.1 ng/ml) and reduced it to control levels at 1 ng/ml. these findings show that the tgf-6 mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p82. pb2 is responsible for recruiting 5" ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb2 dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb2 was not affected. a50 $14-28 neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk(15) cells. this bioassay is 100-to 1000-fold more sensitive than the widely used l929 bioassay, also, human tnf-a and human tnf-8 were detected with a slightly higher sensitivity in the new test compared to the l929 bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk(15) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn 7 in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that 80%. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr-1 specific for the tnf type a (55 kda) receptor and htr-9 specific for the tnf type b (75 kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a 2 fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using 125i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about 0.5-3 fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added 125i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the 3' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a 32 kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il-3 mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca 2+ dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il-3 constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il-3 could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il-3 mrna expression, rapa blocked il-3 induced proliferation. cea also inhibited il-3 superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il-3 transgenes with and without the au-rich region in the 3' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il-3 mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, 34, 52, 1993) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to 3h-thymldine uptake assay. the scf mrna was expressed in 4 of 5 tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in 5/7 lines (range : 39-96 pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of 7 lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant 3200-031005). expression of the v-h-ras oncogene in the il-3 dependent pb-3c mast cells leads to the generation of two classes of il-3 autocrine tumors in vivo. autocrine il-3 expression in class-1 tumors results from increased il-3 mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb-3c resulting in downregulation of oncogenic il-3 expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class-1 hybrids. in contrast, class-2 tumors are characterized by transcriptional activation of the il-3 gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, 1993, j.exp.medicine 178, 403-411) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class-1 alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of 50 pg/kg followed by 10 jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by 31 + 9% (p < 0.005) while rbf decreased by 20-+6% (p < 0.02). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin-4 (il-4) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il-4 reduced the growth rate of four human colon tumor cell lines up to 70% in a dose-dependent manner. three other cell lines showed no significant alteration of 3h-thymidine incorporation or colony formation in the presence of 100 u/ml il-4. responder and nonresponder tumors were immunopositive for il-4 receptor (il-4r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il-4r and bound biotinyated il-4. responsive tumors expressed more receptors. 11-4 binds with high affinity to a 130 kda transmembrane receptor (il-4r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il-4r target proteins. tyrosine phosphorylation of various proteins between 70 and 170 kda appears to be initiated during il-4mediated signaling events in colon tumor cells. the cloning of human il-4r target proteins will contribute to the understanding of the il-4 signal transduction pathway at the initial stage. the renal effects of endothelin-1 were investigated in 16 anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in 8 newborn rabbits, a bolus injection of 5 nmol.kg "1 of endothelin-1 caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for 45 rain. the dramatic increase in renal vascular resistance (+ 28 -+ 4%) induced by endothelin led to a fall in glomerular filtration rate (-12 -+ 4%) and renal blood flow (-16 _+ 3%). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ 20 _+ 5% and + 49 _+ 9%, respectively). in 8 additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin-1 -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake 1 , w. hanke 2 and m. reine~ke 1 , 1 institute of anatomy, university of z0rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor 1 (igf-1), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage 41. at stage 43, the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage 50. between stage 54 and 60, i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage 61, the islets reformed and the amount of endocrine cells increased reaching the adult status at stage 63. around stage 52, igf-l-immunoreactions appeared. igf-1immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf-1 may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch-8093 zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (>570 bases) untranslated region is followed by three in frame augs at codons 1,4 and 15. f is a type i transmembrane protein with a postulated signal sequence of 31 amino acids. we confirmed that the 46 aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon 1, 4 or 15 are all functional, but show slightly different fusion efficiencies. forms translated from aug 1 or 4 appear larger in electrophoresis than forms initiated at aug 15, suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit6, ch-1005 lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine 943 in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd24) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st0beweg 51, d-79108 freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse 190, ch-8057 z0rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd24a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp1 and ap-2 consensus binding sites and a tata box. functional analysis of a 0.6 kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd24a promoter did not influence the ubiquitous nature of this promoter, finally, the cd24a, cd24b and cd24c genes were mapped to mouse chromosomes 10, 8 and 14 respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b5 reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a 34 kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp34), and a 32 kda soluble protein of the intermembrane space (mcrlp32). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of 694 amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca 2+ exchanger in mammalian cell lines using the t7 rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a 6xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $15-08 the neural cell adhesion molecule l1 is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l1 and fusion proteins containing the ig-domains i-vi of l1 specifically block the stabilization of ltp in the ca1 region. baseline synaptic transmission was not affected by the presence of the anti-l1 antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l1 antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l1, whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l1 to stabilization of ltp within the ca1 synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp1) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned 10-12 residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww63, which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww63 can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p6diatrie, chuv, ch-1oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. histidine 36 is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine 36 is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant 4-7, in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a 100-fold higher equilibrium constant for octamer dissociation and a 145-fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of 100 0013 x g membrane fractions. in normal pbmn cells the lbsa content accounted for 2.6/~g/108 cells or 20 nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to 1.6 ,ug/108 cells or 13 nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm3 (65-80 % of total gangliosides) followed by small amounts of od3 (5 -15 %) and other acidic glycosphingolipids. neutral gsls included gl1, gl2, gl3 and gl4-hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm3 content which represented only 55 % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y1 receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially 4 spacially clustered extracellular acidic residues of the human y1 npy receptor essential for npy binding. subsequently, we mutated 36 additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit6 de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas 90:4042, 1993) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 3h-2-deoxyglucose (2-19(3) uptake by astrocytes in primary culture with an ec50 of ~ 100 ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap3). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j3hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates 2-dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit6 de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the 3.5 kb clone isolated contains an open reading frame of 2214 bp encoding a protein of 738 amino acids. the coding region of the mouse astrocyte glys cdna shares 87% and 86% identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of 4 kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a 4.2 kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec 3.6.1.1) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young (2 days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with 6~ for various periods of time. after thawing the samples, the activities of glucose-6phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr 155 000 when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr 250 000 was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr 80 800 catalytic subunit. $15-20 the na,k-pump moves 3 na + out and 2 k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the 31 (t31) and 40 (t40) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. 101:117,1993) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt 430 â�¢ 14, t32 215 z 8, t41 124 z 3 s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was -79 , -45, and -28 mv in the wt, t31 and t40 groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph1646mact (236 aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph1646 lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph1559mact (323 aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph1646mact is very similar to that lph1559mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph1646mact and lphi559mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine 543 to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y543 signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose-6-phosphate/igfii receptor, we have mutated amino acid positions in the ha-y543 shown to be important for internalization of the two receptors. we have changed amino acids on either side of y543 to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y543 mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose-6-phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine 9 2 . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg5 or alg8 with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at 30oc. the double mutant phenotype allowed the isolation of both alg5 and alg8 genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg5 or alg8 mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a6 cells with the b. marinus al-subunit (txl-tbm). this subunit confers a 300-fold higher resistance to ouabain (k i = 50/~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a6 cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a 50-fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft6r 3-4 days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around 0.16 mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of 66 kda that represents a non-glycosylated form of the 80 to 90 kda mature na/p;-cotransporter. we conclud$ that the protein napi-2/rat when expressed in sf9 cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi-2 protein as expressed h sf9 cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of 47 and 190 kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the 190 kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the 47 kd fragment, but not the 190 kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the 47 kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein 4.1. we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l1 and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. 59:1570 -1573 , 1992 scholey a.b. et al, neuroscience 55:499-509, 1993; lijthi a. et al. unpublished data and see usgeb 94) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l1 (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l1 showed an impaired search pattern when compared with that of salineqnjected animals (p>0.05, mann-whitney). whereas control animals spent up to 45% of their time searching for the platform in the correct (= training) quadrant, the time anti-l1 animals swam in the correct quadrant was closer to chance level (31%). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l1, the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by 86rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch28080 was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of 295 amino acids with about 55% amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c131), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. 1, 8029 zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi6fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi6fi cells prior to tail vein injection were treated, 2 different outcomes were observed. short treatment (lh) resulted in 85% inhibition of lung colonization whereas long-ter~ treatment ( 2weeks) resulted in 10-15 times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a 150 kda cell surface protein. sequence information from peptides revealed in several cases 100 % homology to mouse centrosomin a, i.e.a 32 kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this 150 kda protein from a bi6fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position 5. removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking 30 of the normal 40 residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, 1211 geneva 4 in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic(80-90%) and membrane-bound activity (10-20%}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna (4.2 kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x-114 fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached 2, 0.8 and 0.25 ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was 1.8, 0.4 and 0.125 hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology (33-42% identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so4-ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of 200 mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild (10%), although significant (n=8, p<0.001), fall in jw. since cuso4 is a sh-reagent like hg, we looked at the effects of cuso4 (lmm). there was a 68% reduction of j, (n=7, p<0.001), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, 5mm) in the presence of cu, caused a 90% reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and 5ram cuso4 caused 51% and 92% jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $15-44 the amino acid sequences of the pc-645 e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc-645 ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was 15%-20%. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and 8subunits remained structurally conservative during evolution (e.g. -70% identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $15-45 functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than 70 apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk-1 carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk-1 determinant of mag and p0. we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk-1 bearing myelin proteins such as mag, p0, mog, ncam or pmp22. by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $100 protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l2/hnk-1 negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas 88, 10629, 1991) . the cdna codes for a protein of 349 amino acids which shows 77% amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of 6 ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome 14. in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l1, a 1.skb edna clone closely related to l1 (ch-l1) was isolated from a ~. gtl 1 library derived from poly(a)* l~qa of day 8 mouse brain. using this edna as probe the remaining cdi'ia 5' sequences were cloned out of a plasmid library constructed from postnatal day 6-14 mouse brain poly (a)+ p, na. two independent full length clones of 4.2 and 4.4 kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl1 reveals that, like the cell adhesion molecules (cams) mouse l1, chicken ng-cam, and nr-cam (bravo), chl1 is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l1 family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl1. the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl1 is susceptible to proteolytic cleavage. bands of 185kd, 165kd, 125kd, and 50kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl1 protein in brain and heart, but not lung, kidney and intestine. in liver, a 50 kd immunoreactive band was found. the relationship of chl1 to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was 220mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution (22 costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to 1 mm chzcihg (10', outer medium) followed by fixation with 0.25% glutaraldehyde (5', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n=6) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a 50 kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions 5 and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range 3--10.5 (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing 0.2% chaps and tris/hci at ph 9.5 (2nd dim.). nucleoside phosphatase activity was detected histochemically in the 2d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (>1.5 m). denaturation in 2m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within 15 min while denaturation in 6 m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains (11,6) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. 190, ch-8057 zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, 2424 bp) encoding for a protein of 637 amino acids. in the eight transmembrane spanning domains the protein is 78% identical with the corresponding system from rat kidney, in the hydrophilic regions only 30%. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, 1.9, 2.7, and 4.2 kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the 1.9 kb transcript. mammalian systems share only the 2.7 kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid 13/a4 protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app-695 is involved in the growth regulation offibroblast% and also stimulates neurite extension in b 103 neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp-695 is the rerms site, arg-328 to ser-332. the presence ofsapp binding sites on the surface of b 103 ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp-695. finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp-695 can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation (823a-028366), and nih (ag 05131). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c(1b adrenergic receptor stably expressed in rat1 fibroblasts (3.3 pmol/mg prot.). the =(1b-ar was solubilized from 32p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with 100 ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro-318220) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{1b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last 147 amino-acids) was constructed and stably expressed in rat cells (1.3 pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist 3h-prazosin at 4~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos-1 cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos-1 cells correspond to those in organ cultured human intestinal explants, in caco-2 ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos-1 cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $15-59 assembly and transport of paba peptide hydrolase alpha and beta subunits in cos-1 cells eldedng, j.a., gr0nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec 3.4.24.18), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and 13, which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. 266, 21381, 1991) . here, we report on the expression of the cdnas of pphcz and pphj3 in cos-1 cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos-1 cells. pphl3, on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos-1 cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt3r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt3r immunoreactivity from e17 to postnatal day 10. in the present work, we attempt to demonstrate by radioautography that the detected nt3r are effectively the binding sites of [125i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with 0.1 nm of l, 3,5,3'-[t25i]triiodothyronin 2200 ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t3). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled "1"3, all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt3r which bind thyroid hormones (snf 31-33671-92). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, (3h)-mannose-labeled glyeolipids named cpi and cp2 were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp1 and cp2 both contain the identical core oligosaccharide mangl,2(x->po4-6)manal,2mamxl,6(y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp1 and cp2 can also be observed after lableling with (3h)myo-inositol, the lipid moieties of cp1 and cp2 can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi210 cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose-6-phosphat e/igfll-recept or and a2,6slalyltransferase in hepg2 cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man-6-p/igfii receptor (mpr) and (resident) c~2,6sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg2 cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. 1988, 106, 617-28) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes (2' of warming up) showed no, late endosomes (20') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant 31-30757.91 of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin-4/5 (nt-4/5) as well as the other known neurotrophins, ngf, bdnf and nt-3. we found that acute administration of nt-4/5 to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt-4/5 resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt-4/5 exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt-4/5 strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day 15, as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day 18 rats, nt-4/5 very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt-4/5 were mimicked by bdnf with similar potency, nt-3 was found to be only marginauy effective, our findings identify nt-4/5 as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. 6 syn loci are known on the genome; the syn 3 locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa 816, 853, 854, 856, 857 and 872 and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the 3 syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by 2 ligand-recepter interactions. ffwo+-strains penetrate at 0~ cell/cell fusion was analysed and found to need also 2 receptor-ligand interactions if induced by syn 3 mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day 16 (p16) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p16 spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns 2) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the 3.5 kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a 50% identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the 541 amino acid open reading frame encodes a 62 kd protein with a putative signal peptide and two transmembrane domains, whether this ns 2 protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n8) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd44 standard (cd44s) and splice variants (cd44v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd44s and cd44v on n8 tumors and cell lines by immunohistology and northern blot. results showed high levels of cd44s on 33/44 nb, 6/10 n8 cell lines and 4/4 ganglioneuromas (mature, benign sympathetic tumors). lack of cd44s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd44s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd44s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd44s expression. no gd44v were detected on tumors or cell lines. we conclude that cd44s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus 1 (bhv1), bovine herpesvirus 5 hv5) and caprine herpesvirus 1 (caphv1). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two 8hv1 reference strains was found to be >98%. the sequence homologies between bhv1 and bhv5 were >75% and those between bhv1 and caphvl were >65%. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $15-67 o. moullet and j.-l. dreyer, instimt de biochimie, unlversit6 de fribeurg, ch-1700 fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. (1993) exp. hematol. 21,269-276) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic50=40-45 ktm) or dicnlorophenol-indopbennl (/6'50=200 ilm), but 2-substituted quinones are inactive. the lc50's are decreased 3-10 times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g2, a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h7 which e~presses a major 72kd antigen on its surface recognized b~ mab g10/4) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin6] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr 72,000 polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m011er, institute of zoology, university of fribourg, p4rolles, "1700 fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr1) was analyzed in detail. agene located close to the cbr1 encodes a putative gtp-binding protein, whose promoter region is located within a distance of only 2 kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm08h7). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm08h7 is s:udied with antibodies raised against the putative chromo domain protein and by injecting a 5-ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, 4-8 cell extracts were established and their quality was assessed by the ability to transcribe 5s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s16-07 university of berne, ch 3010 berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [35s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [35s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada (1,2) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam-1 and icam-1 is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed 8cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the 8cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch 3010 berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [125-i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw3, from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw3. we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw3 antisense rna. in vivo, an amastigote sw3 antisense transcript and the corresponding polypeptide were also detected suggesting that the sw3 is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h1 of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to 45% as compared to histone h1 of higher eukaryotes exist. alkaline phospatase digestion and analysis of h1 by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h1 of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i0 segments of double-stranded circular dna ranging in size from 7-30 kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em2 of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em2-antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em2positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em2 were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c57bl/10) versus susceptible (c57bl/ 6j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em2 igg 3 and igg i. sus-ceptibility of c57bl/6j-mice was associated anti-em2 s and no igg 3 . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c57bl/10 and decreased in r and aki< mice. day 30 p.i. cd4 ⧠-lymphoblast cells dominated in all meuse strains; day 90 p.i. an in-creased nu/0ber of cd4-/cd8 + and cd4+/cd8 + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first 7 hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw3, sw44, and sw20, are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw3 predicted protein is homologous with histone h1 proteins and sw44 and sw20 potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $16-17 microbiology, university of geneva, medical school, c.m.u., 9 avenue de champel, 1211 geneva 4. a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t7 rna polymerase promoter. the expressed 17 transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. 67 : 4822-4830, ig93). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il2 and il2r genes are constitutively expressed and the transcriptional activator nfwd3 which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw720c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti0n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch1566 st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of 2.9 kb with flanking regions of 1.2 and 2.2 kb and is localized on one chromosome of approximately 440 kb. the membranebound enzyme gene is 3.1 kb and occurs as a single copy on a chromosome of approximately 1100 kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, 9000 st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into 3 coding regions (pi, p2 and p3), qhe p3 region contains a protease 3cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of 3cp r~ has been d6~aonstrated in a vacciniavirus/t7 ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav 3cp r~ sequence with that of other picornaviruses imply that amino acid residues his-44 and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein 2b revealed a 27,5 kda peptide. this is in contrast to its predicted size (12 kda). however, analysis with anti-(putative)2a antiserum confirmed an upstream location of the 2b n-terminus. the new 2bpeptide was also identified in lyeates of hav infected mrc-5 cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p2 organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk2. we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut0 isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk2 expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk2expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk2-expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk 2 might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca3 and ca1 hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca3 and ca1 pyramidal ceils (p=0.76, n=82) with almost no feed-forward inhibition (p=0.01). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability (9--0.6) between ca3 pyramidal cells. monosynaptic excitation was found ha 56% of cases between ca3 neurons (n=43) but only in 27% of cases (n=ll) between two ca1 cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca3 (5/6) and within cai (2/3), but not between ca3 and cat fields (2/2). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to 100% at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged 3-6 days or 11-14 days after birth. the spinal cord was crushed and growth of fibers assessed 5 days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in 38% of the trials (45 animals, aged 3-6 days). in nervous systems from older animals (11-14 days after birth) the success rate was low (only 4 out of 38 preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals (12 out of 21), whereas only one preparation out of 15 showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about 12 days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at 8 days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds (31-3626292) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of 15oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 1950's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a 12 â�¢ 1 2 array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di-8-anepps) or a calcium dye (fluo-3) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to 1 60 i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai (2 cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received 1 injection in sii. the mediosagittal image of cc was subdivided into 30 segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior 2/3 and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/5 of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between +2.7 and +4.5 mm interaural levels, we recorded 40 single units along 6 electrode tracks in 4 young female wistar rats. most units (n=32) fired in a non-regular poisson process, although only a small number (7/32) was characterized by an average burst size with more than 3 spikes. crosscorrelograrns were computed for 25 pairs of units simultaneously recorded from the same electrode and for 26 pairs simultaneously recorded from distinct electrodes. most crosscorrelograms (27/51) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that 1/8 to 1/40 of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (>250 ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map2 binds to microtubules via a domain containing 3 or 4 repeats of an 18 amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map2 their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map2 is caused by the 18 amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map2 mutants in which 1 or 2 repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at 10 am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, 118, 105, 1992) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over 10 d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b4. soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at ,5 d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, 2.5 d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at 10 d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci4mence, j. f. 1, ranieri, j. p.2, aebischer, p.2, and sigrist, h. 1, 1institute of biochemistry, university of berne, ch-3012 berne; 2division autonome de recherche chirurgicale, chuv, ch-1011 lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc12, nb2, ng108-15). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[(3-trifluoromethyl) diazirine-3-yl]phenyl]-4-maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned (20 and 300 #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in 2-deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of 20-30 or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch-1700 fribourg calretinin (cr), an ef-hand ca2+-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of 0-6 days-old rat pups were cultured for 10-21 days applying the interphase technique (stoppini etal, j. neurosci. methods 37, 1991) or the roller-tube technique (g&hwiler, j. neurosci. methods 4, i981) . the fixed cultures were immunolabe0ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i1-l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c2 or c3. in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap-25-ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap-25-ir. in conclusion, snap-25 is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $17-19 repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, 8iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. 1993 , 1993 have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged 3-8 days, the cord was cut into two with scissors at the c5 -c7 level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in 33 preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to 500 ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of 360 kd. in rat brain two monoclonal antibodies, a and bw6, recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw6 stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw6. these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no 31-33447.92 from the swiss national science foundation. serum free aggregating brain cell cultures prepared from 16 day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap-25, synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day 12 in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day 20 and day 30 in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci (30mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch-1700 freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s 17-23 stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, 1211 geneva 4 the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns1 and cop-25.1) share an approximately 420 bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns1 and cop-25.1 in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz 5, 3012 bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at 4 -5 hz. this study investigates the origin of the regular oscillations. when stimulated electrically at 5hz, the synaptic transmission in the cultures showed strong depression by about 50% on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of 50% at 5hz were implemented in a computer model consisting of 100 randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at 4 -5 hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d 1 receptor mrna in the rat brain and binding to the receptor was studied at 12 time points from gestational day (gd) 13 to postnatal day (pn) 60 by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i0 um. d 1 recepto~o~inding , determined with the selective antagonist ~ji-sch 23982 was first noted on gd 18 in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn 14. the expression of d i receptor ~na was studied by using a mixture of 3-~js-labelled oligonu-specific cleotides. on gd 13 messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the 3'-end is mediated by host factor. in contrast, the 3'-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the 5'-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to 25 % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt 76 and 210 from the 5'-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the 3'-end but requires a previously unknown structural feature near the 5'-end of this template. $18-02 karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, 30, quai emest-ansermet, 1211 gen~ve 4 splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf3a consist of three subunits of 60, 66 and 120 kda and corresponds to three proteins present in the functional 17s but not in the 12s u2 snrnp. it binds to the 12s u2 snrnp only in the presence of sf3b, suggesting a two step assembly pathway of 17s u2snrnp: binding of sf3b to the 12s u2 snrnp generates a 15s intermediate particle that is converted to the active 17s u2 snrnp by the subsequent association of sf3a. comparison of proteins enriched in the sf3b fraction with polypeptides found in the purified 15s u2 snrnp suggests that sf3b comprises at least five of the other 17s u2 snrnp specific proteins and together with sf3a promotes the u2 snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf3a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf1, a heat-stable 75-kd protein, functions early during spliceosome assembly. tryptic peptides of sf1 were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of 7 internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap62, as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf1 could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf4 triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf4 has been impeded by its low abundance; however, a correlation between sf4 activity and a polypeptide of 50 kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf4. interestingly an atp-dependent rna-helicase cofractionates with sf4 through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i066 epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence (5'-cagugn-3') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of 16,384 ire molecules randomized at these 7 nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are 5'-cagugn-3' (wild-type) and 5'-uaguan-3'. this novel finding predicts base-pairing within the loop between positions i and 5. all nucleotide substitutions in the "bulge" or at loop positions 2,3 and 4 decreased binding by 36% to 99%. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a73 $18-08 3' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. 4, ch-3012 bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their 3' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u7 snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. 3' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u7 rna gets assembled into functional u7 snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u7 rna is converted into the canonical sm sequence (u7 sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u7 sm off particle inhibits the function of preformed endogenous u7 snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u7 rna is placed 3' of hlstone pre-mrna and injected into oocytes. only u7 sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u7 sm wt rna, the common snrnp proteins g and b/b' and a u7-specific protein of 40 kd (50 kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u7-specific crosslink being located most 3'. u7 sm opt rna does not become crosslinked to the u7specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u7 snrnps using these characterized u7 rnas and a preparation of highly enriched snrnp proteins. u7 sm wt and u7 sm opt rnas associate with common sm proteins in this system, but the interaction with the u7-speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately 400,000 fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of 115 kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna 3' end formation carmen spycher, andr6 furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. 4, ch-3012 bern, switzerland histone rna 3' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved 3'-terminal hairpin loop structure. for the mouse h4-12 gene major and minor processing sites are located 3 and 5 nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u7 snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the 5' end of its rna moiety, u7 rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u7 surnp were analysed by incubation in nuclear extract from mouse k 21 cells. our results indicate that base-pairing with u7 rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u7 rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides 5 to 7 around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax-6 gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax-6 homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax-6 affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax-6 homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax-6 may have been present in a common ancestor. therefore, we are now trying to isolate pax-6 homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. 70, ch-4056 basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax7. our human sequence extends the known mouse edna both on the 5' and the 3' end. as a first step in order to test for a functional role of pax7 in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax7 is conserved between mouse and chicken. next, we assessed the expression of pax7 in different mouse and human cell lines. we found pax7 transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax7 is already present at the myoblast stage. moreover, 10ti/2 fibroblasts converted to myoblasts by either transfection of myod or treatment with 5-azacytidine expressed pax7, whereas the parental cells did not. while myod was able to activate pax7 in 10t1/2 cells, expression of pax7 was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax7 during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a 300 bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax-6 gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax-6 is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax-6 homolog from drosophila (dpax-6), which shares more than 90% sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax-6 orthologue. the drosophila gene spans ~16 kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax-6, which affects dpax-6 transcription in the eye discs, suggesting that dpax-6 is encoded by the ey gene. this implies that pax-6 (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays 93% of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position 60c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s 19-08 our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df(2r)wmg, which includes poxn, but survive over the deficiencies df(2r)xte-18 and df(2r)kl-32 flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch-8093 ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the 35s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $20-04 r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/3, which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/3 genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence (98.6% homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $20-02 patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. 190, 8057 z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall1 and ssn6. from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a 5' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall1 and ssn6 that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall 1 on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal4 chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh-13 which belongs to a cluster of at least 4 homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh-13 is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh-13 hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh-13 is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh-13 protein product with a polyclonal antiserum. in order to clarify the function of ceh-13 in the c. elegans development, we have isolated a ceh-13 mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus 1 (shv-1) infected cell pratein (bicp) 0 acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the 81cp0 polypeptide contains a cysteine-rich zinc finger domain (c3hc4) which is conserved in a number of viral and cellular regulatory proteins including the 8icp0 homologs icr3 of herpes simplex virus type 1 and protein 61 of varicella zoster virus. this type of zinc finger (the so-called ring tinge0 was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. 81cp0-induced cat activity of a promoter-cat construct was 72-fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp0-induced transaotivatlon was reduced 54old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn-1 class of pou genes. preliminary evidence suggests that the brn-1like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp-50) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres 3 and 5 in the hindbraln. these rbombomeres were identified by the specific expression of the krox-20 marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the 24 hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp-50 is mostly expressed in glia cells. another pou gene we have identified (gp-9) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres 2 and 4 for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $20-07 willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct 2, 3, 4 and 5. in the present study we determined the chromosomal localization of the gene encoding the n-oct 3 transcription factor and characterized the structure of isolated n-oct 3 genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the 5' end of the n-oct 3 coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct 3 cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct-1. p~asmids with a deletion or mutations in the oct-1 site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct-1 site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in 2~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about 70 positive clones. so far, 30 were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented 5' to 3' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal31-sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $20-09 to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp1ars1 minichromosome were constructed that contain either the ded1 or pet56 promoter firing against the origin of replication ars1. while the pet56 constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded1 constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h4-ars, a second origin of replication, rescued a ded1 construct as a minichromosome (yrpdh3).the chromatin structure mapped at low and high resolution of the ars1 region in yrpdh3 was indistinguishable to that of the pet56construct yrpft61. ananlysis of rna showed that transcription was going through ars1 in yrpdh3 and in yrpft61, but the levels of transcripts in yrpdh3 were much higher. we conclude that transcription through are1 interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b31 was cloned. at least two copies of this segment appear to be present on the 29 kb circular plasmid, whereas one copy is carried on the 50 kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel 906 bp putative open reading frame (orfl) on the 50 kb linear plasmid, orfl displayed extensive sequence homology (95%) to putative open reading frames present on the clones obtained from the 29 kb circular plasmid. heterogeneity is mainly caused by 3rd-base-wobbllng. flanking sequences share 85-95% homology, including the 5' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately 36.5 kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $20-16 similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c 44h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c44h/c44h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone 5.80 which has a high homology to pit-l, clone 5.54 which is related to the brn-3 gene and clone 5.48 which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit-1 related cdna clone 5,80 was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i,25 mg/kg/d, s.c.) from gestational day 14 to 20 on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn 14, pn 28 and 8 week old male and female rats was studied. 8 week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn 28 prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of 8 week old prenatally diazepam exposed male ~ats. $21-04 distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, 1005 lausanne, and **university of fdbourg, 1700 fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from 6 weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during 10 days with tmt at different concentrations, ranging from 10 -10 m to 10 -6 m. microglia were found to be the most sensitive cell type, since already at 10-10m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at 10 -8 m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at 10 -6 m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a 48met probe. vip mrna was found in thalamic nuclei on el7, later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el9 and its expression matured over the next days. few cortical neurons contained vip mrna on e21, they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p 22, when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after 9 hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the 2-d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of 35s-labeled proteins from primary astrocyte cultures treated or not with vip 1 ~tm revealed two newly synthesized proteins: a 36 kda protein (pi=5) and a 23/24 kda protein (pi=6). only the latter is visible on a silver stained 2-d gel, which is a prerequisite to consider its purification and microsequencing. induction of the 23/24 kda protein by vip was time-dependent with a maximum at -12 hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol 1993; 35:73p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct2-adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n=10) received t (100mg orally), followed (+ 3 h) by y (0.1mg/kg intravenously), and y + nx (0.8mg intravenously). analgesia was assessed over 8 h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. 3.25 h (riii +8.8 +semi.6 ma and ns +8.4 +1.8) vs p (rih -0.3 +1.2 and ns +2.3 +1.6, p<0.05) and lasted ca. 6 h. y reversed t analgesic effects during ca. 1 h (r/ii -2.9 +1.8, p<0.05 and ns +2.8 +1.5, p<0.05), whereas y + nx abolished t effects thoughout the study period (rill -1.9 4-1.1 and ns +0.6 +1.3, p<0.05), y alone tended to lower pain thresholds (rill -4.1 +1.5 and ns -1.9 +1.4, p>0.05 anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron 1: 85, 1988) . in-1 neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in-1 stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in-1 in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in-1 antigens are low in staining for gap-43, a marker for growth and plasticity in the developing and adult brain. in contrast, gap-43 is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in-1 expression and elevated gap-43 levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of 68 amino acids out of which 20 are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about 70 % homology to those of mammalian metallothioneins (mt), with two conserved inserts of 1 thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i1), the presence of cu(i) and zn(ii) (6-7 moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of 3h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, 99% of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of 3h-arachidonic acid with a ec50 of 3 pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of 3h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from 7.25 to 7.65, both basal and glutamate evoked release of 3h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c1-/hco3-antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $21-09 multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch-3010 bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to 4 cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c6) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish 4 sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in 2 hour competitive uptake studies fibreblasts reached 1.7 and 2.6 times the concentrations of c6-and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. 563: 227-233, 1991) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of 50 nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase (42%) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, 27t:181, 1993) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., 1992) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., 1991; tschopp et al. 1992) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., 1992) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [35s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [35s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip (0.1 ~d~l) superfused for 5 rain. had no effect, whereas concentrations over 0.2 ixm selectively and reversibly depressed the nmda-medlated responses in 12/16 cells. in the presence of cnqx (10 [tm) and biencuuine (10 ~tm), the isolated nmda-mediated late epsp was still attenuated by 0.3 ~m vip in 8/10 cells (mean:-42% al~er 30 rain.). in 3 other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:-64 % after 10 min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit4 de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk-801, an nmda receptor antagonist but not by cnqx or ap3, which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk-801. interestingly, this synergism is completely inhibited by h-89, a protein kinase a inhibitor, whereas staurosporin and kn-62 which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b-50) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca3 of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap5), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap5 and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca 2+ channel blocker cd 2+, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca 2+ influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i0 ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g9 by i0 ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i0 ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to 10 ~mndga (blocks the 5-1ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i0 ~m etya (blocks the 15-1ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to 50 ~m 4bpb (blocks phospholipase a 2) did not prevent ndga-or etya-induced uncoupling. exposure to 75 ~m skf 525-a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf 525-a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $21-19 potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, 1211 gen~ve 4 simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a 15 hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba2+-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba2+-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k ⧠pump, contributes to the control of the e m, and by this way to the driving force for the inward k ⧠current. overdose of ifosfamide has ooo/red in a canc~c patient (25g i.v. iy0/24 hours with 20 g as ~utectarfe). urir~ collece{on was obtaj/3ed in 24 hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u5~ ~ (s~@pl. v, pag.2627-262g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~3ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ 1500 m~/24h and 207 r~/24h at day one ard day res~ec~vely. 9~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c 200 m~f~ intraperit~neally), no ~ omild be detected in urine within 24 ht~r~ after drug administl-4(cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~0olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., 103, 1234 -1241 , 1989 ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation (3.5 g./l. in 0.02 m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the 5th to the 1oth week postnatal. spatial learning capacities were studied at the ages of 26, 65 or 80 days in a cimular swimming pool (morris place navigation task) and at the age of of 7 months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to 6 months following the supplementation. barcelona, e-08193 bellaterra females of both lines (n=b/grp) were injected sc daily between days 15-20 of pregnancy with vehicle (v), i(di) or 3(d3) mg/kg diazepam, or not(c). in these studies(a,b) their 5-7 mo old offspring were subjected to a 50-run session in a shuttlebox(a:12 males, 6 females of each line/condition) or a 6min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_:6-7 males of each line/condition). a: the freezing behavior of ld3 rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld3s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd3s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld3 vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least 0.7 mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least 0.7 mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the 16 kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg2+-dependent. according to the proposed ligands we replaced mg 2* by fe 2 ⧠or mn ~+ and observed an enhanced activity up to 100%. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m4dical universitaire, 1211 gen~ve 4. pci(3) and pc2, members of the mammalian family of pro-protein convertases homologous to the yeast kex 2, are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc1 and pc2 in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc1 levels were higher than pc2 (pc1/pc2=2.6+0.2) whereas the opposite was found for non-beta cells (pc1/fc2=0.05â�¢ confirming that pc1 is important for proinsulin conversion while suggesting a role for pc2 in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc1/pc2 ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor 75 kd form of pc2 accumulated in beta cells whereas only the fully processed 67 kd form was detected in the non-beta cells. the kringle (2+3) domains (k2+3) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k2+3 by affinity chromatography on a ni-2+-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the 6 histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k2. the dissociation constant of k2+3 was measured by intrinsic fluorescence titration with 6-aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z0rich-lrchel winterthurerstrasse f 90 ch-8057 z0rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a32p]atp indicated that the enzyme has a catalytic subunit of 72 kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the 5' ->3' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to 400 bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. 2,4,5,2',4',5'-hexachlorobiphenyl (6-cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of 6-cb and cpz were studied in 3t3-preadipocytes (p) and 3t3-adipocytes (a). 3t3-cells were cultivated in dulbecco's minimal essential medium supplement with 10% fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for 48h. radiolabelled 6-cb (20~tm) or cpz (5~tm) was added to the medium. following a single dose 70-80% of cpz were taken up within an hour into p and a. in contrast, 6-cb reached an intracellular plateau of 30-50% of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to 95% of the dose. 6-cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to 1 week. 6-cb uptake into p was fully reversible while the release from a was limited to 10% of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of 3t3 cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of 02-production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately 70% of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp91phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp91 was assayed in vitro and in vivo. attempts to "tag" gp91 with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp9 lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp91 in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch-8057 zorich; *rheumaklinik, universit&tsspital, ch-8091 z%rich molecular chaperones of the hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = 27 s and z2 = 200 s at 2 ~m dnak and 0.i ~m ligand at 25~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : 0.4 s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = 0.13 min i at 30"c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. 30, ch8005 ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. 283:377-344, 1992) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, 270:229-332, 1990) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations (0.1-1 ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using 300 bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal-5'-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, 8057 z~rich. the pyridoxal-5'-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. (1990) methods enzymol. 183, 146-159] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~-1015 lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca 2+] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca2+] i levels were m~nitored either with a fluoresc~qce analyser using fura 2 or by 45ca 2+ efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca2+]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in 45ca2+ efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after 3 rain, took 1 to 2 h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at 10 -7 m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~3sicn. 92) barja, f. 1 and turian, g. i, ilab. gen. microbiology, 2dept. biochemistry, 3station exp. zoology, university of geneva, 30 quai ernest-ansermet, 1211 geneva 4 the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., 1991, fems left. 77:19-24) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm-1 was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position 1 to 5. using immunofluorescence or immunoblotting techniques, anti-c~sm-1 activity was abolished after incubation with the native peptide and peptides modified in position 1 (only when e~a) or 5. on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position 1 or 5 were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm-1 to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a 1 kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a 3.6 kb clone potentially encoding a protein of 1133 amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~60 min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin1nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h6nggerberg, ch-8093 z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: 1. the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. 2. addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. 3. so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh4) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh4 is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh4. the most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e,3ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f1"ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i 4 biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i0 molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a2 (pla2) at 0~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca 70~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla 2 in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of 16:1(3t) at the sn-2 position. for instance, the hydrolysis rate of pg ig:3/16:l(3t) was greater than that of pg 16:0/16:0. these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf 3100-33693.92). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd2+,ag +) at low concentrations (i0-6m) increase rapidly (10-60 s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca2+]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca2+]i . metals (10-4-10 -6 m) were added at the apical side of the epithelium, and [ca2+]i was measured with fura-2. hg 2+ induced a rapid (peak 5-10 s) and marked increase in [ca2+]i, whereas cd 2+ entailed a slower (peak 2-5 min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca2+]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca2+]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit6 de fribourg, ch-1700 fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by 1% lubrol xl tm, precipitated with ammonium sulfate (between 40 and 70% saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose-12, followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of 52 kda and 40 kda whereas pmo-ii consists of a single sub-unit of 52 kda. the 52 ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing (13 aa) of the 40 kda subunit from pmo-i showed 84% homology with rat brain fructose-l,6-bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a 4fe-4s cluster. using pcr we have isolated and characterized a cdna clone of 738 bp from spinach and a clone of 911 bp from corn coding for the catalytic subunits including the transit peptides. the first 31 (spinach) resp. 38 (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are 113 (spinach) or 114 (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with 91% similarity and 81% homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf 31-28811.90 and 31-37725.93) $23-20 r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit6 de fribourg, ch-1700 fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb41a3, has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was <0.15% of the protein content in the cell extract. specific pmo activity was increased 15 to 20 fold compared to the activity in whole cells. this approach demonstrates the presence o1' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations (0.1-ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for 3 major human drug metabolizing p450 monooxygenases (cyp2c9, cyp2d6 and cyp3a4) was determined in liver microsomes. fs showed low affinity (ki > 50 ~tm) for cyp2d6 and cyp3a4 whereas cyp2c9 was selectively and competitively inhibited (ki = 0.1 ~tm). the potential for in vivo fs interactions with cyp2c9 substrates was therefore investigated in 14 volunteers using dicinfenac (df) as a probe (25 mg orally) on days 0, 1 and 8. df and 4'-ho-df kinetics were determined by hplc/uv. df cmax increased over time (0.28 [sd 0.12], 0.38 [0.20] and 0.45 [0.4] mg/l on day 0, 1 and 8, resp.). oral clearance was reduced on days 1 and 8 (14 % and 15 %, resp.). a time dependent decrease in urinary metabolic ratio (4'-ho-df/df) was noted (1.07 [0.34], 0.90 [0,23] and 0.70 [0.18] on day 0, 1 and 8, resp. (p < 0.0001)) only over the first 4 hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp2c9 substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o 32-36600.92). 1211 gen~ve, switzerland. previously, a parvalbumin mutant, pvf~02w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca2+-binding in either one, pv.câ�¢ and pv_m, or both, pv-co~, of the two ca2+-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ ⧠with affinity constants, kc,, of 1.1,107 and 3.2-106 m "~ in the absence of mg 2+. mg 2+ shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = 2 9 103 m t. pv.m~ is much less effected by mg z* (kmg = 80 m "t) and can be considered as possessing a ca2+-specific site. nevertheless, in the absence of ca 2 ⧠both, pv.co and pv~, bind i mole of mg 2+ with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca 2. nor mg 2+. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp-102 deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca 2~ or mg z*. our results indicate that destroying the ca2+-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z ⧠. biochemlsches institut der unlversitfit zfirich, switzerland, ch-8057. the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were 2mg per litre of culture. the presence of 7 cd ions per molecule was confirmed by the sh/cd ratio and by the existence of 7 h3cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of -8, were found to be approximately 15 times weaker than for rabbit mt, at ph 7. (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over 85 sequences a general multialignment of 62 class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over 83% of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $23-28 bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, 6-13 years old were placed in five groups. hcb was given orally in concentrations of 0.01, 0.1, 1.0, and 10 mg/kg b.w. in gelatin capsules, daily, for 13 weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in 2% buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of 47,2 kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at 412 nm and c~-and i~-bands at 540 and 575 nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to 405 nm. atomic absorption measurements indicate approximately 1 mole of zinc and 1 mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine-2,3-dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol 1987~ 142:217) . the fluorescent substance formed from 3-hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. 3-hk at concentrations of 0.08-+0.01, 0.2_+0.04, and 1_+0.4 btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was 2.6_+ 0.3, 3.7_+0.2, and 9.6+_2 ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of 3-hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of 205 daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf 32-36058.92, emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization (350 nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was 40%. nonspecific oligonucleotide binding was below 2 %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine 2,3-dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces 3-hydroxykynurenine, a uv-b filter. the synthesis of 3-hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between 72 and 84 years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j 1985; 230:635) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol 1987; 142:217) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was 0.7_+0.3 nmol/mg protein/hour. in cataracts a low 1do activity of 0.3_+0.2 nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was 1.6_+0.9 nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf 32-36058.92, emdo, and hartmarm-m011er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after 350 nm irradiation (irradiance 0.7 mw cm -2 for 20 minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $23-33 two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed (70 to 220~i~n) by variation of hematocrit frcrn 0 to 60% in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r 70000) concentration (i0 to 80g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s04-25, s 15-69 s05-18, s05-19 s 11-05, s11-06 bchini hooft van huijsduijnen s08-12 s05-30 s15-45 s08-13 s08-05, s08-06 s01-08 s15-67, $23-19 s13-03 nook, ek s01-15, s10-15 s05-02, s05-05 s 15-03, s15-29, s15-30 s19-03, s19-04, $19-05 s16-02, $16-15 s12-22 s15-48 s05-24 s03-01, s03-02, s03-03 s03-01 lrmler s05-28, s09-06 s03-07, s03-10, s03-11 s18-05 lipp, p. s05-26 so 1-02 s15-15, s15-16, $21-03, $21-11, $21-12, $21-14 $23-29,$23-30 s01-17 s 15-29, s15-30 s12-12, s12-24 molar s15-19 s08-05, s08-06 e s09-06, s09-07 s15-03, s15-29, s15-30 e. s03-05 s05-09, s05-10, s05-51 s05-52 s10-16 s08-14 s04-23, s04-29, s05-13, s05-53, $20-15 s10-16, s10-19 s09-01, s09-02 s15-68, s 17-24 stalder h. s03-19 s10-21 s09-09, s13-10, s13-13 s10-21 teheesen s01-10, s01-11, s01-18 s15-16 van dillewyn s05-32, s05-37, s05-38 s08-05, s08-06 s05-18, s05-19 huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, 30 quai ernest-ansermet, 1211 geneva 4, switzerland. ca2+-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~91). it is known that the ca2+-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, 1987) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca2+-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord-327432-ogw27tob authors: zhang, feng-jian; hu, de-ying; liu, yi-lan; li, hong; zhu, xiao-ping; pan, shao-shan title: expert consensus on nurses’ human caring for covid-19 patients in different sites date: 2020-08-07 journal: curr med sci doi: 10.1007/s11596-020-2222-0 sha: doc_id: 327432 cord_uid: ogw27tob this study aims to develop the expert consensus on nurse’s human caring for corona virus disease 2019 (covid-19) patients in different sites, and thus provide a guideline on providing whole process and systematic caring for covid-19 patients. based on the frontline experiences of human caring for covid-19 patients and the review of literature, the initial draft of consensus was made and finalized after online meeting and revisions. the experts reached consensus on the following parts: terms and definitions, principles of human caring for covid-19 patients, and human caring measures for covid-19 patients in different sites. the expert consensus is practical, concise, and reasonable for guiding the nurses providing human caring for covid-19 patients, as well as other similar infectious diseases. the current novel corona virus disease 2019 (covid19) outbreak, which began in december 2019, presents a significant challenge for the entire world [1] . with a high infection rate, rapid spread, and uncertain clinical course duration, the covid-19 can lead infected patients into critical conditions, and even death [2] . the unexpected infection of covid-19 not only threatens the patient's health and life, but also seriously affects the patient's psychological state [3] [4] [5] , such as the fear and irritability caused by the uncertainty and the unknown outcome of the disease, the loneliness and helplessness resulting from the prohibiting of visit by family members and friends during hospitalization and isolation, the anxiety and depression in knowing about the infection of their relatives and friends. the patients with covid-19 may go through several stages in different sites, such as fever clinic visiting, mobile cabin hospital, designated hospital treatment, centralized medical isolation observation, home isolation, even rehabilitation clinic visiting [6] [7] [8] [9] [10] [11] . regardless of the sites and the stages, patients are continuously exposed to the difficulties and pressure, and prone to negative emotions and psychological problems, which strongly call for personalized human caring [12, 13] . currently, many nursing experts have explored the human caring measures for covid-19 patients in different sites, but the standardized and whole-process human caring measures for covid-19 patients are not available. therefore, the experts were invited to develop a consensus on delivering human caring in different sites, thus to provide guideline for the human caring to covid-19 patients as well as other similar infectious diseases during the whole process of treatment and recovery. nursing administrators from hubei, fujian, beijing, sichuan, shanghai, guangdong, conducted a systemic search and thereafter collected the related literature from several electronic databases: psychinfo, cinahl, pubmed, web of science and cnki (chinese). the keywords include nursing, human caring, caring, etc. the draft of the consensus was made after reviewing and summarizing the literature, as well as collecting the frontline experiences and measures of human caring for covid-19 patients. two rounds of interdisciplinary expert meetings by invitation were organized in april 2020 to revise the draft and finally reached the consensus. thirty-two experts from multiple medical institutions participated. the consensus can be applied in different sites (fever clinic, hospital isolated ward, mobile cabin hospital, isolation observation center, rehabilitation clinic) where covid-19 patients get medical treatment. moreover, it can also be used as guideline for patients with other similar infectious diseases in different sites. covid-19 refers to the disease caused by the novel coronavirus began in 2019. human caring, attention, and understanding of human nature, which satisfies human needs, protects human dignity and interests, and embodies human value from the perspective of human needs and desires. fever clinic, a specific medical area in the outpatient department of a hospital during the prevention and control of acute infectious diseases according to the instructions of the superior. it is particularly established for the screening and diagnosis of suspected or infected patients, and provides a specific medical area for observation and treatment of patients in need. hospital isolated ward is the medical department for the treatment of severe and critical patients with covid-19. isolated unit in mobile cabin hospital is the temporarily built and renovated medical institution for the temporary treatment of the covid-19 patients with taking the chinese people's liberation army (pla)'s field mobile medical system as a model. centralized medical isolation observation centers are the specialized places for medical isolation and observation of covid-19 patients after discharge and before returning home. rehabilitation clinic is a special established institution that provides services for patients with covid-19 during the discharged recovery stage, such as consultations, follow-ups, and physical and mental rehabilitation. (1) nurses should be highly aware of the severity and complexity of covid-19, as well as the difficulty and suffering of patients, to protect patients' lives and health and make them feel warmly supported. (2) nurses should be responsible and kind-hearted to patients, treat patients as if they are the family members, or even assume the roles of their family members if necessary. (3) integrate human caring into the whole process of treatment and recovery, while meeting patient's individualized human caring needs. (4) deliver human caring with professional and creative measures [14] . (5) treat nurses themselves as the most important resource, and comprehensively utilize the available resources to provide human caring [15] . (6) highlight the mobilization of patients' selfcare and motivate their potential capacity to facilitate self-healing, and encourage the support and human caring between patients [16] . (7) pay attention to patients about the infection of their family members, and provide prompt psychological support if the infection or death occurs. (8) nursing staff should conduct standard procedure of self-care and self-protection in order to ensure both physical and mental health, and report any negative feelings and emotions, as well as symptoms, therefore, to avoid the occurrence of relevant accidental incident. (9) write or stick the name and role noticeably on the outer protective suit to make it easy for patients to identify and call. (1) receive patients forwardly and warmly with polite manner and moderate voice, and introduce the name and role to patients; (2) provide masks for patients and their companions, and teach them the right way to put on the mask; (3) the triage nurse should arrange patients visiting orderly, and timely register the basic information, then measure and record their vital signs. prioritize the appointment of critical patients, recognize the abnormal symptoms of patients in time, and assist in treatment if necessary [17] ; (4) the triage nurse should actively provide guidance and help to patients, assist patients with critical conditions or mobility problems in registration, physical examination, picking up medicine; (5) keep the environment clean, make the guidance signs clear, decorate with proper posters, and disinfect regularly; (6) provide comfortable seats for waiting patients. ensure one room, one physician and one patient during consultation to prevent crossinfection and protect patients' privacy; (7) identify the negative emotions of patients, care for the depressed patients, comfort the emotional patients, and provide psychological support. take on the role of their family members for the patients lacking family support because of their families suffering from covid-19. accompany the patients to make them feel safe and warm; (8) visit patients frequently during the treatment, care for the patients forwardly and assess the health status actively, and take measures to relieve the pain and discomfort. remind the patients of their safety to avoid falls, and tell the patients to call for help from the nurses if needed; (9) arrange the patients in observation reasonably according to their conditions, and provide a single room when possible. patients with common fever, suspected infection, and confirmed covid-19 infection, should be classified and managed in different ways by following guidelines; (10) assess the basic needs of patients, and provide warm food and warm water according to their needs. report the unsolved problems to head nurse or seek help from other departments; (11) set up a green channel for critical patients. assist in transferring the patient to other areas or hospitals according to the conditions; (12) record and hand over the patients' special caring needs or measures to the next shift; (1) receive patients forwardly and warmly with polite manner and moderate voice, and introduce the name and duty to the patients; (2) the duty nurse should daily communicate with the patients appropriately at the bedside. assess the needs of patients proactively and fulfill their reasonable needs. the unsolved problems by nurses should be reported to the head nurse, doctor or other related people to seek help; (3) identify the patient's physical discomfort through monitoring, questioning, and observing. apply professional knowledge and skills to relieve patients' suffering and facilitate their comfort, even organize relevant specialist nursing consultation; (4) communicate proactively with the patients and listen to them in daily work, and evaluate the psychological status with phq-4 if necessary to figure out the abnormal conditions. timely comfort, accompany and encourage the patients with anxiety and fear, even act as a temporary role of patient's family member, and the psychologist can be invited to help to deal with the particular problems. encourage and facilitate the caring and communication between the patients, and assist the patient in communicating with their families and friends; (5) deliver nursing operations normatively, correctly and timely, and inform and communicate with patients during the operation, even if the patients are unconscious. close the door or cover the patients body during the operation to protect the patients privacy; (6) provide necessary knowledge of disease and health to the patients. provide and instruct the patients to put on masks. teach the recovering patients to start breathing training to promote the lung function; (7) assist the patients in daily life and medical treatment, and prepare daily necessities for them. the items provided by their relatives and friends should be delivered to the patients timely. when the patients get out of bed, the nurses should pay close attention or give support to avoid accidents such as falling. (8) pay attention to the nutrition of patients' diets, prepare delicious and warm meals, and try the best to provide personalized meals according to patients' needs. prepare fruits, milk and other foods appropriately. provide microwave ovens in the ward for heating foods at any time. encourage and assist patients without swallowing disorders to eat by themselves. provide enteral and parenteral nutrition for patients with eating problems. take away the left food and plates as soon as patients finish eating; (9) keep the wards clean and warm, and ventilate and disinfect regularly; (10) assist in or implement bed bath and other hygiene care for the patients to keep clean and comfortable; (11) encourage the patients in the recovery rehabilitation and activities involvement, and express the appreciation for the patient cooperation; (12) record the special caring needs or measures of patients on the whiteboard and handover between each shift; (13) provide wake-up service for coma patients four times a day, and play audio materials with patients' favorite music, the blessing, and encouraging words from relatives, colleagues, and friends; (14) provide detailed guidance on medication, isolation, condition report, and follow-up for discharged patients. provide channels for consultation such as telephone or message platforms; (15) provide support to the patients in their terminal stage. express respect to the dead while delivering death care. inform their family members about the unfortunate news and related information with appropriate manner [18] . (1) receive patients forwardly and warmly with polite manner and moderate voice, and introduce the name and duty to the patients; (2) guide the patients to the cabin and assist with taking the luggage if needed; (3) introduce the environment and staff information to the patients, especially the functional area like doctors' office, nurse station, toilet, bathroom, to facilitate their adaptation to the environment; (4) create a friendly living environment, maintaining cleanness, disinfecting regularly, and ventilating efficiently. decorate the isolated unit with positive and inspirational posters and slogans to encourage patients to fight with the disease, and use proper equipment to protect the patients privacy; (5) enquire about the patients needs on living and medical service, provide necessary supplies for their daily use, and improve the service and facilities for living environment. ensure patients get living belongings from family members as soon as possible. report to the head nurse or other managers if there are any unsolved problems; (6) greet patients at the bedside on shift change and handover the patients' special caring demands and measures between nurses; (7) implement treatment measures correctly and timely, and protect patient's privacy during operating procedure; (8) visit the patient regularly, report to the doctor if the patient gets worse, and assist with the patient's transition when needed; (9) organize diverse recreational activities to enrich the patients' daily life in the cabin. encourage the patients to participate in activities or volunteer work, thus to make them feel valuable and confident with recognition and encouragement; (10) evaluate the mental status of the patient, comfort the patient who feels anxiety or down, and assist them in communicating with their families and friends in different ways according to their needs, ask the psychologist for help if necessary; (11) assist the patient to sort out items and provide personalized discharge guidance upon discharge. thereafter, lead the patient to the doorway with appreciation and best wishes, and hand over with community staff. (1) treat the patient warmly and politely with cordial greeting, and assist in carrying personal belongings to the room; (2) introduce the environment and living facilities to the patient, as well as the precautions and work procedures to facilitate adaptation; (3) keep the room clean, hygienic, and warm, disinfect the room regularly; (4) establish convenient and multi-party communication channels for nurses and patients to make it easy for the patient to report the living needs, disease change, and treatment needs at any time [19] ; (5) assess the patient's physical conditions and medical service needs daily and provide health education and suitable treatment; (6) assist the patient in seeking medical treatment outside the cabin hospital; (7) provide convenient services like delivering items, assisting in receiving medicines, purchasing items, and guiding medication usage. assist the isolated person with life difficulties or selfcare disabilities and assist them in eating, washing, and doing activities. ensure the accompanying guardian and safety of the children; (8) guide the isolated person to start appropriate rehabilitation activities such as lung function rehabilitation exercises; (9) teach the methods of isolation and disinfection at home to the isolated person, and explain the knowledge of disease prevention to improve the awareness and ability of protection; (10) assess the psychological status of the patient, encourage peer support and family engagement in the psychological support for the patient. (1) conduct regular follow-up to assess the discharged patient's recovery and needs with politeness. leave a message or call again in 2 h for the lost-call; (2) inquire about the health status, medication use, check-up, diet, and rehabilitation of the patients, and provide corresponding guidance and help; (3) assess the patient's isolation conditions and protection measures, and remind the patient if necessary; (4) provide home care for the patient when needed. remind the patient to seek medical help if any patient's condition changed dramatically, and arrange the ambulance to transfer the patient while necessary; (5) listen to the patient, answer the patient's questions patiently, and provide help within the ability during the follow-up. seek help and support from the relevant specialist for special difficulties; (6) relieve the patient's bad mood, and show empathy to the patient's suffering during the follow-up. invite the psychologist or relevant specialist to intervene for severe psychological problems; (7) assist patients in arranging further consultation and other matters; (8) record the patient's special problems or conditions for other healthcare workers to pay attention to in future follow-up; (9) provide necessary assistance for patients to return to their work; (1) receive patient forwardly and warmly with polite manner and moderate voice, and introduce the name and role to the patients; (2) keep the clinic area spacious, and decorate the area with clear signs, warm colors, green plants, and positive energy posters; (3) design a quick physical examination process, guide the recovered person to finish the examination at different times. provide personalized examination program for the recovered people like elderly people, infants, postpartum women and those with basic diseases; (4) provide a comfortable environment with the necessary facilities. ensure that the waiting area is equipped with specialized seats for the recovered person to rest, and other supplies like hand sanitizer, towel paper, disposable cups, medical masks, wheelchairs should be provided. the heating equipment should be equipped and set to an appropriate temperature; (5) provide nutritional, safe, and warm food catering for the patients at a specific time; (6) take care of the patient when the patient is rehabilitating, and stop the rehabilitation in time if the patient feels uncomfortable. additionally, handle emergencies happened to the patient timely; (7) assess the health needs of patients, launch various health education programs, and provide health guidance, such as dietary guidance, sleeping adjustment, self-monitoring after taking medication, caring for covid-19 related symptoms, person and home environment disinfection methods, to improve patients' self-care ability [20] ; (8) teach the recovered patient to master the six-character breathing tactics and traditional chinese medicine exercises, such as baduanjin and taijiquan, to improve the lung function and immunity function; (9) pay attention to the psychological and emotional state of the patients. the nurse can use relevant scales to evaluate and find abnormalities during this process, and intervene timely. ask the psychologist to intervene for special psychological problems; (10) make connections actively with the community, hospitals, and other institutions to provide extended care for the patient. conduct remote rehabilitation and health guidance by phone, wechat, and videoes to provide support for patient treatment and recovery according to the needs of the patient. world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) characteristics of and public health responses to the coronavirus disease 2019 outbreak in china covid-19 under the sars cloud: mental health nursing during the pandemic in hong kong covid-19): patient experience-administrative services on the frontline during crisis needs and concerns of patients in isolation care units -learnings from covid-19: a reflection nursing standards for patient with covid-19 in severe or critical conditions state council of the people's republic of china. guideline for prevention and control of covid-19 in medical institutions national health commission of the people's republic of china. diagnosis and treatment program for patient with covid-19 national health commission of the people's republic of china. rehabilitation program for discharged patients with covid-19 national health commission of the people's republic of china. investigation report of covid-19 between china and who strength the action of follow-up of patient with covid-19 after discharge theory and practice of human caring in nursing strengthening actions for caring as a core component of nursing in the people's republic of china linking the unitary paradigm to policy through a synthesis of caring science and integrative nursing caring science or science of caring our most powerful weapon to fight covid-19: patient involvement outpatient experience of human caring scale (oehc-scale): improvement by delphi method response and role of palliative care during the covid-19 pandemic: a national telephone survey of hospices in italy telehealth transformation: covid-19 and the rise of virtual care rehabilitation of covid-19 patients the authors would like to show respects to all nursing staff who providing human caring to patient with covid-19 in pandemic. thanks for ms suoling zhou from baylor college of medicine, usa, for her great support on polishing the language. the authors have no conflict of interest. key: cord-276218-dcg9oq6y authors: kim, jihoon; koo, bon-kyoung; knoblich, juergen a. title: human organoids: model systems for human biology and medicine date: 2020-07-07 journal: nat rev mol cell biol doi: 10.1038/s41580-020-0259-3 sha: doc_id: 276218 cord_uid: dcg9oq6y the historical reliance of biological research on the use of animal models has sometimes made it challenging to address questions that are specific to the understanding of human biology and disease. but with the advent of human organoids — which are stem cell-derived 3d culture systems — it is now possible to re-create the architecture and physiology of human organs in remarkable detail. human organoids provide unique opportunities for the study of human disease and complement animal models. human organoids have been used to study infectious diseases, genetic disorders and cancers through the genetic engineering of human stem cells, as well as directly when organoids are generated from patient biopsy samples. this review discusses the applications, advantages and disadvantages of human organoids as models of development and disease and outlines the challenges that have to be overcome for organoids to be able to substantially reduce the need for animal experiments. the use of classical cell line and animal model systems in biomedical research during the late twentieth and early twenty-first centuries has been successful in many areas, such as improving our understanding of cellular signalling pathways, identifying potential drug targets and guiding the design of candidate drugs for pathologies including cancer and infectious disease. the value of the achievements these model systems have made possible is proved by the fact that their use has become near universal in biomedical research today. historically, the investigation of disease mechanisms in animal models has progressed along a common discovery pipeline, whereby biological processes were initially investigated by genetic screens in invertebrates, followed by an analysis of evolutionary conservation in mammalian model systems, eventually leading to clinical translation to humans. the common principles of animal development and organ physiology derived from this approach have led to a detailed mechanistic understanding of many human diseases. nevertheless, extrapolating results from model systems to humans has become a major bottleneck in the drug discovery process. furthermore, recent studies have identified biological processes that are specific to the human body and cannot be modelled in other animals. these include, for example, brain development, metabolism and the testing of drug efficacy. the emergence of human in vitro 3d cell culture approaches using stem cells from different organs has therefore received widespread attention as having the potential to overcome these limitations. attempts to model the biology of human organsincluding the differentiation of human stem cells in 2d, in either the presence or absence of a 3d matrix; bio-printing of human cells; and the culture of cells in a microfluidic device ('organ-on-a-chip') 1 -were made prior to the emergence of organoids and have shown some potential for drug screening or human disease research. however, organoids are unique, in that they are self-organizing, 3d culture systems that are highly similar to -and in some cases, histologically indistinguishable from -actual human organs [2] [3] [4] [5] [6] [7] [8] . one feature that is common to all organoids is that they are generated from pluripotent stem cells (pscs) or adult stem cells (adscs; also known as tissue stem cells) by mimicking human development or organ regeneration in vitro 9 . the analysis of organoid formation can thus provide valuable information about the mechanisms underlying human development and organ regeneration, highlighting their value for basic biological research in addition to their potential application in pharmaceutical drug testing and molecular medicine. the potential of organoids to complement existing model systems and extend basic biological research, medical research and drug discovery into a more physiologically relevant human setting is becoming ever more widely appreciated. however, the development of organoid technology is still in its infancy in comparison to established cell line and animal models, with challenges still to overcome. in this review, we describe examples of psc-derived and adsc-derived organoid models that have shown great potential and promise in modelling human disease for a broad spectrum of life stages, from early development through to adulthood. many excellent reviews have already described the various organoid human organoids: model systems for human biology and medicine model systems [9] [10] [11] [12] [13] [14] , highlighted their relevance for disease modelling 15, 16 and outlined organoid-associated protocols 17 . this review will instead discuss organoids as a novel model system for understanding human development and genetics that complements, and in the future might reduce, the use of animal models. we emphasize the differences between the mouse and human systems, evaluating the advantages and disadvantages of animal models and human organoid models. we also highlight the tools and methodologies that are available for human organoid studies, in the hope that this information will lead basic biologists to consider using this emerging platform for the study of human pathophysiology. the diversity of biological model systems building on the fundamental idea that biological mechanisms are conserved throughout evolution, biomedical research has traditionally focused on only a handful of model organisms. these models are typically robust, fast-growing species that generate a large number of offspring in a short time and can be propagated at low cost in the laboratory. researchers benefit greatly from the use of an established model organism, as there are already detailed descriptions of its development and physiology, a large body of experimental techniques and many community resources, including reagents and species-specific databases. while some model organisms, such as yeast (saccharomyces cerevisiae), fruit flies (drosophila melanogaster) and mice (mus musculus), have a long tradition of use in research, other model systems have been established more recently but nevertheless have helped broaden our knowledge by presenting us with a new set of experimental tools. these include the worm, caenorhabditis elegans, whose reproducible cell lineage allows unparalleled analysis of cell fate decisions 18 , and the zebrafish, danio rerio, the first vertebrate system in which saturation loss-of-function mutagenesis screening was performed 19, 20 . c. elegans became a widely used experimental model from the mid 1970s, whereas the first large-scale genetic screens in d. rerio were conducted in the 1990s 21, 22 . however, although it is extensive, the information obtained from animal models does not completely reflect human physiology. in this respect, human organoids (which emerged in the early 2010s) 3, 5, 23, 24 can be seen as a novel experimental model that bridges the gap between animal models and human beings. the most commonly used model organisms currently are s. cerevisiae, c. elegans, d. melanogaster, zebrafish and the common house mouse. in addition, for cancer studies, patient-derived xenografts (pdx) and cancer cell lines are also used. each model has its own unique advantages and limitations (fig. 1 ). for example, 60-80% of genes in c. elegans have human orthologues, and about 42% of human disease genes have orthologues in c. elegans [25] [26] [27] . moreover, many signalling pathways have been evolutionarily conserved from this species. the largely deterministic nature of c. elegans development makes it an ideal model for cell lineage analysis and has led to the discovery of key cell death pathways, yet pattern formation in c. elegans is different from that in other organisms, and mechanistic principles of its development are often not fully conserved. in addition, other aspects of the physiology of c. elegans, such as its lifespan, metabolism and immune system, are very different from human physiology. among mammalian model systems, the availability of highly advanced genetic tools and germline-competent www.nature.com/nrm mouse embryonic stem cells (escs) that enable the generation of genetically engineered mouse models makes the mouse the most preferred system. nevertheless, limitations remain with regard to the physical inaccessibility of early development, as compared with other, egg-laying models with visible development, the requirement for expensive animal facilities in which to maintain the mice and some important differences between mouse and human physiology (box 1). the zebrafish was developed as a vertebrate model for large-scale genetic studies in the 1990s, to overcome some of the limitations of the mouse model: the transparent zebrafish embryo and this model's potential to reproduce in huge numbers with a manageable price tag allowed zebrafish researchers to uncover fundamental principles of early embryonic development. however, the zebrafish model suffers from a hugely complex genome, a lack of comprehensive in vitro culture systems and the fact that it shares only a limited degree of biological similarity with humans. over the past 30 years, the use of these various, equally valuable animal model systems, as wells as 2d human cancer cell lines, has led to an explosion of knowledge about human development and mechanisms of disease, but it has also revealed the limitations of these same model systems in emulating human pathophysiology. a number of biological phenomena that are specific to humans are not amenable to being reproduced in animal models. the human brain, for example, is far more complex than its mouse counterpart, owing partly to human-specific developmental events and mechanisms 28 . neurons in the human cortex, for example, arise from a cell type, outer radial glia, that is not present -or is present only in minute numbers -in rodents 28 . human physiology is also profoundly different from the mouse model system: it is perhaps unsurprising that there are huge differences in metabolism between humans and laboratory models, given that humans develop far more slowly than the models 29 . common drugs such as ibuprofen 30 and warfarin 31 are metabolized in the liver in such different manners in humans and rodents that, in humans, ibuprofen is prescribed for pain, fever and inflammation, and warfarin is prescribed as an anticoagulation drug, whereas both drugs are toxic to rats. finally, and in contrast to all animal models, humans are not inbred. understanding human genetic diversity and its influence on disease onset and progression and drug responses is a prerequisite for developing personalized medical treatments and requires the establishment of human-specific model systems. the advent of human induced pluripotent stem cell (ipsc) technology and diverse human adsc culture methods has made it possible, for the first time, to generate laboratory models specific to an individual 32 . reprogramming other cells into ipscs has become a routine laboratory procedure, but generating disease models from those cell lines remains challenging. early approaches focused on the differentiation of ipscs into one type of cell (for example, neurons and cardiomyocytes) and their culture in 2d 33, 34 . more recently, culture methods have been developed to mimic in vivo organ development in 3d, allowing more complex tissue structures and diverse cell types to be modelled simultaneously 1, 9, [35] [36] [37] [38] [39] . in this methodology, human ipscs are sequentially exposed to a course of differentiation cues in order to simulate the stages of a human developmental process. during this process, differentiated ipscs aggregate to form first an organ bud, and later organoids, that faithfully mimic the mature organ structure, including multiple cell types and the interactions between them. human adsc-derived organoids have also emerged as an alternative organoid system that consists of a simpler structure composed mainly of cell types found in the epithelium 3 . in contrast to the complicated process of ipsc reprogramming followed by differentiation to the required organ type, these organoids can be generated from biopsies isolated directly from the organ of interest or from diseased patient tissue. however, the establishment of human adsc-derived organoids is limited by accessibility to the tissue and prior knowledge of the culture conditions for that tissue, while an ipsc line, once established from a patient, can be used to repeatedly generate different tissue models without any time limit (that is, beyond the patient's lifespan) 40 . in summary, organoids constitute an improvement in the generation of model systems, with cell types that more closely resemble, and are in similar conditions to, those found in the human body 7, 41 . diverse organoid systems provide a useful variety in their complexity and modelling capability, ranging from the simple epithelial structures derived from adscs 3 to more complex cultures box 1 | differences between human and mouse stem cells the difference between mice and humans is evident in the culturing of their stem cells. pluripotent mouse embryonic stem cells (escs) were established in the early 1980s 45, 46 . leukaemia inhibitory factor (lif) was the first pluripotent stem cell (psc) factor identified, with the later discovery that 2i (the kinase inhibitors pd0325901 and chir99021) and lif (together termed 2ilif) are necessary as well as sufficient for the culture of escs from all mouse strains [176] [177] [178] [179] . the first human pscs to be successfully cultured were human escs, equivalent to mouse epi-stem cells that have lost germ-cell potential 44, 180, 181 . initial cultures were propagated in media that were based on basic fibroblast growth factor, but for decades researchers continued to search for appropriate culture conditions in which to maintain human pscs in the naive, pluripotent state [182] [183] [184] [185] [186] [187] [188] . universally agreed-upon culture conditions for the maintenance of naive human psc cultures remain elusive, with various reports suggesting different medium compositions including the use of a pkc inhibitor (gö6983), rock inhibitor (y-27632), mek inhibitor (pd98059), gsk inhibitor (im12), braf inhibitor (sb590885), lck/src inhibitor (wh-4-023) or wnt5a 182, 184, 189, 190 . a similar fundamental discrepancy between mouse and human cultures, in their requirements for additional media components, has also been observed in a number of adult stem cell (adsc)-derived organoid culture systems. however, one commonality appears to be that inhibitors of tgfβ and p38 signalling must be added to the growth factors used in murine adsc cultures in order to enable the long-term culture of human adscs 9 . for example, mouse colonic organoids require epidermal growth factor, noggin, r-spondin and wnt as growth factors, while human colonic organoids additionally require two inhibitors, a83-01 and sb202190 (which block tgfβ and p38 signalling, respectively) 3 , in order to attain a similar level of longevity and robustness. the differences between mouse and human systems in both psc-derived and adsc-derived organoid cultures suggest rather surprising fundamental differences between these species in the intrinsic signalling pathways that are activated in their stem cells and that are required for stem cell identity, and not just differences during development or during an immune response. this confirms the need for human-based laboratory model systems in order to fully understand human development and pathophysiology. part of neocortex where neuronal progenitors reside. a breed with genetically closely related individuals. induced pluripotent stem cell (ipsc). pluripotent stem cells reprogrammed from somatic cells. nature reviews | molecular cell biology generated from multiple germ layers 6 obtained via multistep psc differentiation protocols 42, 43 . it is expected that, in the near future, the variety of organoid models available, coupled with advances in the technology, will provide a series of powerful and efficient platforms for studying human development, physiology and disease. the past years have seen substantial progress in the development and use of human-cell-based model systems for the study of human biology, including both pluripotent and adsc-based systems. the introduction of human ipsc technology. the first human pscs, known as human escs, were reported in 1998 (ref. 44 ), nearly two decades after the initial discovery of murine pluripotent cell lines in 1981 (refs 45, 46 ). the human stem cell lines were predicted to be useful for the study of human developmental biology, drug discovery and cell therapy -in spite of the technical limitations imposed by the lack of knowledge of appropriate differentiation protocols at that time. as the generation of human psc lines required the sacrifice of human embryos at the blastocyst stage, strong ethical concerns were raised 47 . in 2007 the debate around human pscs was largely circumvented by the introduction of human ipsc technology, which converts an ordinary differentiated fibroblast from an adult human into a pluripotent cell 33 . reprogramming to a pluripotent state is usually achieved by forced expression of a specific set of transcription factors 33 , and the pluripotent cells can then be differentiated into specific cell types. ipsc technology not only allowed the generation of patient-specific stem cells but also enabled researchers to work with an unlimited supply of human stem cells and stem cell-derived tissues. furthermore, ipsc technology allows the banking of patient-derived stem cells 48 . there are still technical limitations when using ipscs -for example, the use of oncogenes for reprogramming, the genetically unstable nature of the reprogramming and the low efficiency of reprogramming. all these technical hurdles made it difficult to obtain error-free ipsc lines from patients 49 . but these limitations have been at least partly overcome by the use of non-integrating vectors and standardized quality control protocols to avoid or screen out unwanted genetic alterations 49 . line-to-line variability caused by the genetic heterogeneity of humans has also been resolved by the use of isogenic controls generated by genetic engineering using crispr-cas9 technology 50 . these improvements have enabled researchers to use ipsc-derived specialized cell types -for example, neurons, cardiomyocytes, haematopoietic progenitor cells and pancreatic β-cells -in disease modelling and drug screening 51 . however, just over a decade after their introduction, 3d organ culture methods have greatly increased the utility of human ipscs. ipsc-derived organoid models. human psc-derived organoids are generated by guided differentiation protocols that mimic developmental processes identified through previous work, both in vitro and in vivo (fig. 2) . as our knowledge of human development is very limited, most early studies that aimed at producing organoids with properties similar to those of human tissues were based on parallels drawn from mouse development. in principle, to generate an organoid, the entire process of organ development from pscs should be faithfully mimicked. in reality, it is nearly impossible in vitro to provide all biochemical cues that drive cell differentiation and 3d tissue assembly and organization at precisely the right times, places and concentrations at which they would occur during embryonic development. fortunately, cells in vitro tend to follow a semi-autonomous differentiation trajectory, as they do in vivo, and three main types of protocols are utilized to generate functional organoids. in the case of brain organoids, human pscs are initially guided to differentiate into embryoid bodies before further differentiation towards the neuroectodermal lineage. once the cell aggregates contain the developmental precursors for brain tissue patterning, the rest of the developmental steps occur spontaneously in a spinning bioreactor 5, 52 . for the development of liver primordia, guided differentiation to hepatoblasts (hepatocyte precursors) was not sufficient to form a complete set of organ precursors, which requires cells from different lineages. it was known from studies of mouse hepatogenesis that cell-to-cell communication between endothelial, mesenchymal and hepatic endoderm cells was important, so based on this knowledge the first human liver primordia were created by mixing human psc-derived hepatoblasts, mesenchymal cells and endothelial cells. through self-condensation and organization, these three cell types assemble in vitro to make an aggregate that mimics the architecture of developing human liver primordia 23, 53 . other organoids require more precise, lengthy protocols in order to acquire the appropriate progenitor cell types for the target epithelium. many organoids that model endoderm-derived organs (such as oesophagus, stomach, colon, intestine and lung) undergo stepwise differentiation protocols, in which the timing, concentration and combination of specific growth factors and chemical inhibitors for modulating key developmental signalling pathways are crucial to developing the desired epithelial tissue in a manner analogous to fetal development 24, 43, [54] [55] [56] [57] . in all cases, the process of organoid formation involves three crucial steps. first, key signalling pathways regulating developmental patterning are activated or inhibited (using commercially available morphogens and signalling inhibitors) in order to establish a correct regional identity during stem cell differentiation. usually this is achieved through induction of the signalling events that have been identified in the mouse as establishing cell fates in vivo. second, media formulations that allow proper terminal differentiation of the desired cell types within the organoid are developed, generally following established methods of 2d culture or inspired by the murine developmental process. finally, cultures are grown in a way that allows their expansion in three dimensions, which is achieved either by aggregating cells into 3d structures or by embedding the cultures into a 3d matrix. control cell lines with the same genetic background as experimental cells. three-dimensional pluripotent stem cell aggregates. culture system in which nutrients are supplied while cells are agitated. adscs can be cultured in the presence of niche factors that function to maintain the stem cells in an undifferentiated state while allowing stem cell differentiation. as a result, the cultured stem cells can generate adsc-derived organoids, which are composed of an epithelial monolayer that mimics the 3d architecture and contains the cell types of the desired organ to be modelled 58 . the first steps in the development of adsc-derived organoids, however, are to identify and isolate the appropriate population of adult stem cells and to understand their niche requirements. obtaining the first mouse intestinal organoid cultures required not only the identification of the intestinal stem cell population expressing the selective marker for these cells, the leucine-rich repeat-containing g protein-coupled receptor 5 (lgr5), but also the essential niche factors to support stem cell activity 59 . the re-creation of the intestinal stem cell niche in vitro was inspired by mouse genetic studies that had shown that epithelial proliferation and stem cell self-renewal are dependent on epidermal growth factor (egf) and wnt activity, while differentiation is controlled by bone morphogenetic protein (bmp) kidney organoids after it was discovered that sb202190 prevents secretory lineage differentiation, this inhibitor was replaced by the addition of insulin-like growth factor 1 (igf1) and fibroblast growth factor 2 (fgf2) 4 ; this modification enables concurrent multilineage differentiation and self-renewal in human gut organoids. all these changes and refinements to culture conditions, which are the results of another decade of research, improved the quality of human gut organoid cultures, enabling researchers to produce organoids that more closely resemble tissue in vivo. nevertheless, mouse organoids remain more similar to the in vivo tissue of origin than do human organoids. generation of other adsc-derived organoids. using the procedure described above, multiple types of organoids from various tissues have been generated by modifying the basic medium that was initially developed for intestinal organoid culture ( fig. 2; fig. 3 ). in most cases, a mouse organoid culture system was first established and then adapted to human cells. mouse colonic organoids can be grown through the addition of wnt3a 3 . mouse stomach pyloric organoids and corpus organoids were grown with only slight modifications to the original protocol for mouse intestinal organoids 60, 61 . mouse liver and pancreas ductal organoids were also obtained similarly from injury-activated lgr5-positive progenitors 62, 63 . the number of organoids generated from diverse mouse tissues and organs is growing, and for each, previous knowledge of the signalling processes that comprise the organ-specific or tissue-specific stem cell niche environment has been key to developing the appropriate protocols. adsc-derived human organoids have also become widely available (fig. 3) . they have been generated from almost all endoderm-derived tissues (intestine, colon, stomach, liver, pancreas, lung, bladder and so forth) 3,7,41,64-70 and from gender-specific tissues (prostate, endometrium, fallopian tube and mammary gland) 8,71-75 . additional components frequently required for the growth of human cultures, in comparison to the mouse cultures, are a tgfβ pathway inhibitor (such as a83-01) and a p38 mapk inhibitor (such as sb202190) 9 . as for mouse organoids, human organoids can be derived from minimal amounts of tissue biopsies and can be cultured indefinitely, thus forming the basis for the building of living biobanks, which are an important resource in biomedical research. following the establishment of human stem cell-based organoids, various human diseases have been modelled using these systems. for example, human organoids have been used to study infectious diseases, inheritable genetic disorders and cancer. moreover, with the advent of various genetic-engineering tools, pathogenic genes and mutations can be directly tested in organoids derived from cells isolated from healthy donors, which enables us to perform human genetic studies in controlled genetic backgrounds. the human brain is the most highly developed brain in the animal kingdom and the most complex organ in our body. because of its distinct complexity, it has been very difficult to model human brain development using animal systems. human brain organoids, however, have now been successfully used to model human brain development and disease. the first disease studied in this model was microcephaly; unlike rodent models, human brain organoids with a mutation in cdk5rap2 display a significantly reduced size, with a smaller number of progenitors that undergo premature neurogenesis 5 . cdk5rap2 is localized at the centrosome and is required for correct orientation of the mitotic spindle, so that a mis-orientation of the cleavage plane prevents the expansion of the progenitor pool by symmetric division in the patient-derived organoids. this human experimental system was also crucial in determining the relationship between the zika virus (zikv) and microcephaly. during the recent zikv outbreak, a link was initially suggested between middle part of the stomach, connected with cardia, fundus and pylorus. collection of biomaterials, such as cancer organoids, used for research. www.nature.com/nrm neurological disorders, including microcephaly, and zikv [76] [77] [78] [79] . nevertheless, even though zikv could be detected in microcephalic fetal tissues, the causal relationship and the mechanism by which zikv might compromise brain development were not clear. a number of studies have used 3d human stem cell-derived systems, including neurosphere culture and brain organoid models, to reveal the effect of zikv infection on human brain development 80, 81 . thus, the human organoid model provides a physiologically relevant platform for studying zikv pathogenesis. further studies with human brain organoids have determined that brazilian zikv strains possess increased virulence as compared to an african strain 82 , and that the ns2a protein of zikv causes decreased neural stem cell proliferation 83 . various chemical compounds that alleviate the hypomorphic effect of zikv have also been identified using the human brain organoid system 84 . these studies highlight the importance of human brain organoids as a model system in understanding the pathology and mechanistic basis of genetic and infectious diseases, as well as in discovering potential therapeutic reagents. as exemplified by the zikv studies above, a human model system is preferable to animal models when studying infectious disease pathogenesis, because pathogens often have a narrow species or tissue tropism; that is, they infect only certain species, and sometimes only specific cell types. for decades there were not any methods to culture human noroviruses, which cause acute gastroenteritis and can be fatal in young children or in old or immunocompromised individuals 85 . it recently became possible to successfully grow norovirus in culture by using a human intestinal organoid-derived epithelial monolayer, which led to the finding that certain viral strains specifically require both certain bile components and the activity of the host galactoside 2-alphal-fucosyltransferase 2 (fut2) enzyme for infection 86 , which mirrors the patterns of epidemiological studies. rotaviruses, which also cause diarrhoeal disease 87 , are another example of human intestinal organoids being used to cultivate patient-derived viral strains. the rapid replication of the virus was detected within one day of inoculation. an antiviral cytokine, a vp7-neutralizing antibody and ribavirin were demonstrated to suppress viral replication in the organoid system 88, 89 . human airway organoids are suitable models for the study of respiratory viruses. respiratory syncytial virus (rsv) is a single-stranded rna virus that is a significant threat to young infants and the elderly 41 . it was shown to successfully infect human airway organoids and to cause dramatic changes to epithelial morphology and function: the ns2 protein of rsv was shown to cause increased motility of airway epithelial cells 41 . the human airway organoid system has also been used to assess the infectivity of animal-borne influenza viruses [90] [91] [92] . the diversity of two viral surface molecules -haemagglutinin and neuraminidase -has previously made it difficult to predict the infectivity of these viruses in humans 93 . the human airway organoids faithfully simulate human airway epithelium, and so provide a platform for rapidly testing the infectivity of newly emerging influenza viruses. organoids have also been used to co-cultivate human epithelia with bacteria (for example, helicobacter pylori) and with protozoan parasites (for example, cryptosporidium) 64, [94] [95] [96] . the list of pathogens that can be grown in the surrogate human organ system is growing rapidly. we expect that, in the near future, numerous microbiome strains from the human gut or glands will be analysed by using organoids to study infectious diseases in a human cell context, and that organoid infection models will be used as screening platforms to identify novel drug candidates (box 2). human organoids are important resources for precision medicine. for example, they can be useful for selecting an appropriate drug for patients with genetic diseases or cancer. cystic fibrosis (cf) is a relatively common genetic disease with approximately 90,000 patients worldwide. although loss of function of the cftr gene, encoding a chloride channel regulating the mucosal environment, is the main cause, nearly 2,000 mutations have been reported that affect the function or expression of cftr, making this a genetically heterogeneous disease 97 . while two drugs -vx-770 (a cftr potentiator that improves chloride-channel activity) and vx-809 (a cftr corrector that helps mutant protein folding) -are already available for patients with cf who carry specific cftr mutations in combination with the common mutation f508del, evaluating the efficacy of these drugs or discovering new drugs for patients with different mutations remains challenging. rectal organoids isolated from small endoscopic biopsies have nonstructural protein 2a, encoded in a virus genome. an effect that causes small size of a body part. the ability of a virus to infect various species and cells. an approach to treating individual patients with the best possible drug, based on the individual's genetic background and disease characteristics. numerous epidemics and pandemics have threatened human health in history (of which a few examples are plague (yersinia pestis), spanish flu, smallpox, aids/hiv, ebola virus, severe acute respiratory syndrome (sars)-coronavirus (cov) and zika virus (zikv); who, disease outbreaks), heavily disrupting human society. the emergence of the novel human coronavirus sars-cov-2 was first reported in wuhan, china, at the end of 2019 (ref. 191 ). sars-cov-2, reported to have crossed the species barrier, causes coronavirus disease-19 in humans 192 . owing to the lack of targeted antiviral medicines and a vaccine against covid-19, the virus has spread globally and the who has declared covid-19 a pandemic (who, coronavirus disease (covid-19) pandemic). developing a vaccine and therapeutics against covid-19 has become a priority, and human organoids can play a crucial role in advancing research, by providing human cells from different organs that are susceptible to sars-cov-2 infection. several research groups have successfully shown that sars-cov-2 can infect and propagate in primary human organoids derived from liver and gut, as well as in pluripotent stem cell-derived organoids modelling blood vessels and the kidney [193] [194] [195] . sars-cov-2 uses the same protein, viral receptor angiotensin-converting enzyme 2 (ace2), as sars-cov to enter the host cells. ace2 is expressed in multiple tissues and cell types, enabling the virus to infect other tissues beyond the lung [196] [197] [198] [199] . the ability of sars-cov-2 to infect different organs was modelled with human organoids [193] [194] [195] . moreover, it has been shown that the viral infection of human blood vessel and kidney organoids can be inhibited by human recombinant soluble ace2, a drug candidate initially developed for sars-cov 194 . structural studies of the spike proteins of sars-cov-2 and sars-cov bound to ace2 predicted a similar result [200] [201] [202] . these studies have provided additional evidence that human organoids can serve as an effective research platform for the study of human diseases, including the outbreak of new viruses such as sars-cov-2 and zikv. nature reviews | molecular cell biology been used in forskolin-induced swelling (fis) assays [98] [99] [100] to assess forskolin-camp signalling-induced cftr channel activity, and it was found that the assay can faithfully predict patient responses to individual drugs and to combined treatments 99, 100 . it was also found that with this relatively simple in vitro assay it is possible to identify which patients with rare mutations would respond to, and could therefore be treated with, existing therapies 99,100 , which could be life-changing for those individuals. the rectal-organoid-based fis assay screen is currently being performed by hubrecht organoid technology for patients in the netherlands with cf. human organoids also play a prominent role in enhancing our understanding of human cancers. traditionally, human cancer cell lines, mouse cancer models or pdx have been the main experimental platforms for studies of human cancer. however, with the advent of adsc-derived organoid technology, transformed cancer tissues have been grown in vitro as cancer organoids (also known as tumouroids or canceroids), demonstrating that this technology is applicable to diseased tissue as well as to normal epithelia. patient samples from colon 101-105 , brain 106, 107 , prostate 108 , pancreas [109] [110] [111] , liver 112 , breast 113 , bladder 68 , stomach [114] [115] [116] , oesophageal 117 , endometrial 118 and lung 41,119 cancers have readily been cultured as cancer organoid models. recently, an analysis of drug responses in patients and in their matched cancer organoids led to the conclusion that responses to the drugs are highly similar in the two settings: a drug with no antitumour activity in the organoids did not demonstrate efficacy in the matched patient, and drugs that showed an effect in the organoid cultures were matched by a patient response in close to 90% of cases 120 . this initial study has been corroborated by several studies with larger cohorts 105, 121, 122 . the sample size and cancer types analysed remain limited, and more rigorous investigation will be needed before cancer organoids can be routinely adopted as in vitro patient 'avatars' . nevertheless, the rapidly increasing number of patient-derived cancer organoids and their use in xenograft formation and molecular profiling have already accelerated cancer research, and patient-derived organoid models in the future may provide an in vitro screening platform to predict the best therapeutic options for individual patients. capecchi reported highly frequent homologous recombination events in mouse escs 123 . by contrast, homologous recombination was found to be an extremely rare event in human pscs 124 . later studies reported that double-strand breaks (dsbs) facilitate homologous recombination events as part of the dna repair mechanism in human cell lines 125 . much effort has been directed at establishing an efficient method for generating dsbs at a specific target locus in order to harness this repair machinery in introducing desired genetic changes such as repair sequence or pathogenic mutation into the target locus. this was initially accomplished by the use of meganucleases (endonucleases with a long recognition site (12-40 bps)), zinc finger nucleases 126 and transcription activator-like effector nucleases 127 , but with varying levels of target specificity and activity. the development of the crispr−cas9 endonuclease technology has made diverse methods of genetic engineering readily available to all researchers [128] [129] [130] [131] . unlike the previous technologies, the cas9 endonuclease is guided to the genomic sequence of interest as a means to generate a dsb by a guide rna sequence (grna), making the system highly versatile and easy to apply 132 . there are numerous examples of genetic engineering of human pscs using this system, including the generation of isogenic cell lines with specific mutations. such isogenic cell lines serve as an important control for genetic analysis in human pscs, due to the strong phenotypical variation among different cell lines. furthermore, large-scale loss-of-function analyses have been performed in many human cancer cell lines and human pscs through the multiplexing of grnas in retroviral or lentiviral vectors, to provide genome-wide targeting coverage. combining organoid systems with crispr-cas9 genome editing broadens the applications of the organoid system in many ways. for example, this system has been used to model monogenic disorders such as cf. human gut organoids with f508del, causing misfolded cftr channel protein that leads to rapid degradation, were precisely corrected into the normal sequence by crispr-cas9; the engineered organoids with the corrected cftr amino acid sequence showed restored channel activity of cftr in vitro 133 , showing the causal relationship between the mutation and disease phenotype, as well as the possibility of using a similar strategy to generate autologous organoids for transplantation. crispr-cas9 technology has also been employed to identify and introduce oncogenic driver mutations to normal epithelial organoids. two groups have independently reported the minimum set of mutations that can closely model a metastatic human colon cancer 134, 135 . simultaneous knockout of multiple genes or conditional gene knockout strategies have also been developed for adsc-derived organoids and for pscs that can be used for psc-derived organoids [136] [137] [138] . crispr grna library screening analysis has been performed in multiple labs to maximize the utility of human organoids in genetic studies 139, 140 . these developments in genetic engineering, combined with human psc and human organoid technologies, have opened new opportunities for studies of human genetics, as it is possible to perform genetic experiments in small human organ models that closely reflect human physiology. organoids should largely be considered as a model system currently under development: much of the current excitement around the technology is based on their enormous potential rather than on what has already been achieved. nevertheless, organoids could potentially revolutionize disease research in a profound manner (fig. 4) . generating animal models for a specific disease requires pre-existing insight into either the causative conditions or the genes involved. animal models are typically created by applying harmful conditions to animals or by www.nature.com/nrm manipulating the genes responsible for the disorder, while organoid models can be directly generated from affected patients without prior knowledge of the specific genes responsible. this is particularly relevant for multigenic disorders such as inflammatory bowel disease, provided that the pathology is caused by the affected epithelium, and for cancers, where cancer organoids can be directly isolated from the patient [141] [142] [143] [144] [145] [146] . human organoid cultures have a number of potential benefits over animal models (box 3): organoids provide faster and more robust outcomes, are more readily accessible and provide both a more accurate representation of human tissue and a larger quantity of material to work with than animal models do. the mouse is one of the animal model systems most frequently used to explore human biology and disease, owing to its similarity to humans, in comparison with other animal models, and to the ability to generate transgenic and knockout mouse strains. however, the generation of a conventional transgenic mouse model to address questions regarding human disease generally takes more than a year, even with the technological advance of crispr-cas9-mediated precision genome editing [147] [148] [149] . furthermore, differences in microbiota and pathogen composition between animal models and humans, as well as the failure of certain phenomena observed in mice to translate directly to humans, limit the utility of animal models in human disease research 150 . (2) biobanking, whereby samples obtained from patients can be used to generate patient-derived organoids and stored as a resource for future research; (3) disease modelling, to understand the mechanisms of human diseases such as infectious diseases, inheritable genetic disorders and cancer using various laboratory techniques, including omics and drug-screening analyses; and (4) precision medicine, in which patient-derived organoids can be used to predict response to drugs and as resources for regenerative medicine coupled with genetic engineering. ecm, extracellular matrix. diseases caused by more than a single genetic factor. human stem cell-derived organoid culture is widely expected to bridge the remaining gaps between animal models and humans, primarily because the source material for organoid culture is a stem cell derived from a human 35, 40 . the length of time required to establish the research platform is also faster for the organoid model than for animal models: a human organoid culture can be established within a few weeks or months with a high success rate, thus supporting the use of patient-derived organoids in personalized medicine to provide robust personalized data, including individual mutation profiles and drug responses. organoid handling is also relatively easy, with researchers being able to handle a large number of organoid lines at the same time, although the initial determination of culture conditions for a new tissue type is rather complicated. variability and single-cell profiling. in spite of the many advantages detailed above, human organoid culture remains under development, and numerous efforts to advance the technology are still in progress. one of the most pressing issues in organoid technology is the variability of the system: individual reports have described contrasting methods to generate organoids from stem cells, but a widely accepted, standardized protocol is still missing. for instance, human gut organoids derived in different laboratories, using human adscs and human pscs, are different, even though both are termed 'gut organoids' 2,3,24 . the possibility for further improvement of an already well-defined method was recently reported by showing that more refined culture conditions provide better cellular diversity and culture efficiency 4 . the lack of widely accepted, standardized protocols and guidance is an important issue to overcome in order to reduce the variability of the system from research group to group. a collective effort should be made to set clear guidelines and ways to assess the quality and validity of each system. single-cell profiling technologies for transcriptome and epigenome analysis may be key in terms of highly accurate assays suitable for this purpose. with these assays, it would be possible to compare every cell type present in the organoids with their in vivo counterpart at the level of transcriptome and epigenome. in addition, individual differences such as age and genetic background might introduce further variability to the human organoid system, which, while potentially challenging, could also present an opportunity to assess the role of person-to-person variability in human biology. additional considerations to be addressed include the modelling of cell-to-cell communication with stromal cell populations and the development of vasculature in organoid systems, although vascularization has been observed in some cases upon transplantation 23, 151 . significant progress has recently been achieved with blood vessel organoids 152 , which have a great potential to impact research into vascular diseases such as diabetes. however, despite steady progress, vascularization still remains a difficult obstacle to overcome. several reports already exist of organoid co-culture systems containing pathogens such as bacteria, viruses and parasites 153, 154 , and moreover, organoid co-cultures with mesenchymal and/or immune cell populations have shown exciting progress in recent years and are of great interest as means to better model human disease 23, [155] [156] [157] [158] [159] [160] [161] [162] . nevertheless, organoid systems already contain a large degree of complexity, and it is important to acknowledge the challenge of adding additional components to an already complex system. both simple and complex organoid systems have their pros and cons, and therefore it is most important to use the most appropriate level of complexity for a given study. for example, cancer organoids containing only the epithelial component can be sufficient to test the efficacy of most cancer drugs. however, for immuno-oncology therapies or for an assessment of drug metabolism and availability to the target organ, more complex systems containing immune cells, mesenchymal cells and/or endothelial cells together with the organoids will be necessary. unfortunately, a standardized protocol or guidelines concerning these matters are still lacking, so individual researchers are left to determine the most appropriate system for themselves. we foresee that full standardization of many of these protocols will be achieved in the near future, in a manner similar to increases in both the quality and reproducibility of human ipsc technology. assessing interactions with other organs and the environment. one clear drawback of organoid systems is the lack of interorgan communication. human organoid systems fundamentally mimic a part of the human body, not the entire body. therefore, human organoids are limited to the reproduction of organ-specific or tissue-specific microphysiology, a limitation to bear in mind prior to entering this exciting new field. however, efforts are already in progress to overcome • human-derived: human organoids represent human physiology, rather than being a 'human-like' or 'similar' system. • rapid: adult stem cell-derived and pluripotent stem cell-derived organoids can be established rapidly and easily. • robustness: once established, scale-up is usually possible for large-scale genomic screening and drug screening. • genetic manipulation: most modern genetic engineering tools can be applied to induced pluripotent stem cells or directly to organoid systems. • personalization: induced pluripotent stem cells and organoids can be obtained from individuals. • cellular components: the microenvironment is sometimes lacking, particularly in adult stem cell-derived organoids. co-culture systems with other cell types are not firmly established. • standardization: protocols for organoid establishment and quality control are not globally standardized. • relatively costly: organoids cost less than mouse or fish models, but they are relatively expensive compared to traditional cell lines, fly, yeast or worm models. • scale: studies at the level of whole organs are difficult. • heterogeneity: owing to diversity between individuals and protocols, outcomes may vary from group to group. www.nature.com/nrm this limitation. for example, multiple organoids have been connected in order to study communication between the liver, pancreas and gastrointestinal tract 163 ; cell migration between the developing forebrain and hindbrain [164] [165] [166] ; and the interaction between the brain and hormone-producing organs 167 . the development of tools to help us model organ-level communication will progress, although this capacity is likely to lag in comparison to progress in other areas of the field, owing to the complex nature of the studies undertaken. of note, efforts to bring together the fields of organoid research and organ-on-a-chip research are particularly exciting, potentially resulting in an 'organoid-on-a-chip' technology 168, 169 . we foresee, for example, the potential generation of a chamber device that enables the separate culture of distinct organoid types, thus preventing the uncontrolled fusion of organoids while permitting organoid-organoid communication. finally, the effect of extracellular matrix composition on organoid culture is yet to be defined; uncertainty in the composition of the extracellular matrix can heavily influence the outcomes in chemical screening or genetic screening of human organoids. diverse efforts have been made, with some impressive successes in recent years [170] [171] [172] [173] [174] [175] . this obstacle should be overcome not only as a means to produce more robust human model systems, but also to allow the translation of human organoid technology to regenerative medicine, where 'good manufacturing practice' requires all raw materials, including matrix materials, to be fully defined. furthermore, work is ongoing towards the development of organoid culture platforms for large-scale production, organoid-based high-content screening platforms and micro-organoids-on-a-chip as miniature, finely controlled systems. for all these systems, it will be extremely important to know how to manufacture a synthetic, versatile extracellular matrix. despite the remaining challenges, human organoids hold great potential in clinical translational research, owing to the advantages outlined above and to rapid, ongoing technology development. from the initial full 'laboratory life cycle' , which started with the isolation of patient samples, to the establishment of organoids and their cryopreservation, organoid technology has expanded to embrace genetic manipulation, various omics and drug-screening analyses and diverse co-culture system with viruses, bacteria and parasites (fig. 4) . thus, technologies and experimental procedures that were developed in other model systems can now be applied to human organoid systems, which will accelerate our understanding of human biology and enable us to validate hypotheses and models generated from studies in animal model systems. given the rapid technical advances in the field, we believe that human organoid systems will provide unprecedented opportunities to improve human health. published online xx xx xxxx progress and potential in organoid research single lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche provide the first example of organoids derived from adscs isolated from mouse gut long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and barrett's epithelium human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition cerebral organoids model human brain development and microcephaly report that the complexity of human brain development can be modelled by human psc-derived organoids kidney organoids from human ips cells contain multiple lineages and model human nephrogenesis long-term expansion of functional mouse and human hepatocytes as 3d organoids long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium in this review, huch and koo summarize the development of various organoid culture 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critical look: challenges in differentiating human pluripotent stem cells into desired cell types and organoids disease modeling in stem cell-derived 3d organoid systems organoid and assembloid technologies for investigating cellular crosstalk in human brain development and disease human kidney organoids: progress and remaining challenges liver organoids: from basic research to therapeutic applications disease modelling in human organoids long-term expanding human airway organoids for disease modeling stepwise differentiation of pluripotent stem cells into retinal cells modelling human development and disease in pluripotent stem-cell-derived gastric organoids embryonic stem cell lines derived from human blastocysts establishment in culture of pluripotential cells from mouse embryos isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells human embryonic stem cells: research, ethics and policy human ipsc banking: barriers and opportunities advances in pluripotent stem cells: history, mechanisms, technologies, and applications stem cells, genome editing, and the path to translational medicine pluripotent stem cells in disease modelling and drug discovery guided self-organization and cortical plate formation in human brain organoids generation of a vascularized and functional human liver from an ipsc-derived organ bud transplant 3d modeling of esophageal development using human psc-derived basal progenitors reveals a critical role for notch signaling esophageal organoids from human pluripotent stem cells delineate sox2 functions during esophageal specification wnt/beta-catenin promotes gastric fundus specification in mice and humans in vitro generation of human pluripotent stem cell derived lung organoids the intestinal stem cell identification of stem cells in small intestine and colon by marker gene lgr5 differentiated troy+chief cells act as reserve stem cells to generate all lineages of the stomach epithelium lgr5 +ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro in vitro expansion of single lgr5 + liver stem cells induced by wnt-driven regeneration unlimited in vitro expansion of adult bi-potent pancreas progenitors through the lgr5/r-spondin axis in vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection a novel human gastric primary cell culture system for modelling helicobacter pylori infection in vitro long-term culture of genome-stable bipotent stem cells from adult human liver expansion of adult human pancreatic tissue yields organoids harboring progenitor cells with endocrine differentiation potential tumor evolution and drug response in patient-derived organoid models of bladder cancer basal cells as stem cells of the mouse trachea and human airway epithelium reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability quantification of regenerative potential in primary human mammary epithelial cells identification of multipotent luminal progenitor cells in human prostate organoid cultures single luminal epithelial progenitors can generate prostate organoids in culture the notch and wnt pathways regulate stemness and differentiation in human fallopian tube organoids a developmental and genetic classification for malformations of cortical development: update 2012 zika virus and microcephaly: why is this situation a pheic? detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study zika virus associated with microcephaly zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr3 zika virus impairs growth in human neurospheres and brain organoids show the utility of complex brain organoids for translational zika virus research the brazilian zika virus strain causes birth defects in experimental models zika-virus-encoded ns2a disrupts mammalian cortical neurogenesis by degrading adherens junction proteins identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen epidemiology of human noroviruses and updates on vaccine development replication of human noroviruses in stem cell-derived human enteroids demonstrate that organoid culture systems can support research on difficult pathogens that previously could not be cultivated rotavirus vaccines human intestinal enteroids: a new model to study human rotavirus infection, host restriction, and pathophysiology modeling rotavirus infection and antiviral therapy using primary intestinal organoids the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities differentiated human airway organoids to assess infectivity of emerging influenza virus influenza viruses en route from birds to man influenza virus neuraminidase structure and functions modeling infectious diseases and host-microbe interactions in gastrointestinal organoids persistence and toxin production by clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function modelling cryptosporidium infection in human small intestinal and lung organoids recent strategic advances in cftr drug discovery: an overview a functional cftr assay using primary cystic fibrosis intestinal organoids report the use of organoids in precision medicine for patients with cystic fibrosis characterizing responses to cftr-modulating drugs using rectal organoids derived from subjects with cystic fibrosis rectal organoids enable personalized treatment of cystic fibrosis prospective derivation of a living organoid biobank of colorectal cancer patients a colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases patient-derived colorectal cancer organoids upregulate revival stem cell marker genes following chemotherapeutic treatment patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients a patient-derived glioblastoma organoid model and biobank recapitulates interand intra-tumoral heterogeneity patient-derived organoids (pdos) as a novel in vitro model for neuroblastoma tumours organoid cultures derived from patients with advanced prostate cancer organoid models of human and mouse ductal pancreatic cancer pancreatic cancer organoids recapitulate disease and allow personalized drug screening human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression human primary liver cancer-derived organoid cultures for disease modeling and drug screening a living biobank of breast cancer organoids captures disease heterogeneity human gastric cancer modelling using organoids a comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening divergent routes toward wnt and r-spondin niche independency during human gastric carcinogenesis organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening patient-derived organoids model treatment response of metastatic gastrointestinal cancers a rectal cancer organoid platform to study individual responses to chemoradiation patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer site-directed mutagenesis by gene targeting in mouse embryoderived stem cells gene targeting in human pluripotent cells chimeric nucleases stimulate gene targeting in human cells enhancing gene targeting with designed zinc finger nucleases a tale nuclease architecture for efficient genome editing rna-guided genetic silencing systems in bacteria and archaea multiplex genome engineering using crispr/cas systems rna-guided human genome engineering via cas9 targeted genome engineering in human cells with the cas9 rna-guided endonuclease the next generation of crispr-cas technologies and applications functional repair of cftr by crispr/cas9 in intestinal stem cell organoids of cystic fibrosis patients report the first study to apply crispr-cas9-based gene correction in an organoid system sequential cancer mutations in cultured human intestinal stem cells modeling colorectal cancer using crispr-cas9-mediated engineering of human intestinal organoids one-step generation of conditional and reversible gene knockouts a protocol for multiple gene knockout in mouse small intestinal organoids using a crispr-concatemer simultaneous paralogue knockout using a crispr-concatemer in mouse small intestinal organoids pooled in vitro and in vivo crispr-cas9 screening identifies tumor suppressors in human colon organoids genome-scale crispr screening in human intestinal organoids identifies drivers of tgf-beta resistance alterations in the epithelial stem cell compartment could contribute to permanent changes in the mucosa of patients with ulcerative colitis dna methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development single cell analysis of crohn's disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties dna methylation and transcription patterns in intestinal epithelial cells from pediatric patients with inflammatory bowel diseases differentiate disease subtypes and associate with outcome organoids in cancer research somatic inflammatory gene mutations in human ulcerative colitis epithelium one-step generation of mice carrying mutations in multiple genes by crispr/cas-mediated genome engineering generating genetically modified mice using crispr/cas-mediated genome engineering one-step generation of mice carrying reporter and conditional alleles by crispr/ cas-mediated genome engineering exploring host-microbiota interactions in animal models and humans vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation human blood vessel organoids as a model of diabetic vasculopathy organoids in immunological research modeling host-virus interactions in viral infectious diseases using stem-cellderived systems and crispr/cas9 technology human fetal tnf-alpha-cytokineproducing cd4 + effector memory t cells promote intestinal development and mediate inflammation early in life organoid modeling of the tumor immune microenvironment generation of tumor-reactive t cells by co-culture of peripheral blood lymphocytes and tumor organoids 3d model for car-mediated cytotoxicity using patient-derived colorectal cancer organoids co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes a primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions mesenchymal stem cells increase alveolar differentiation in lung progenitor organoid cultures anatomically and functionally distinct lung mesenchymal populations marked by lgr5 and lgr6 modelling human hepato-biliarypancreatic organogenesis from the foregut-midgut boundary fused cerebral organoids model interactions between brain regions fusion of regionally specified hpsc-derived organoids models human brain development and interneuron migration assembly of functionally integrated human forebrain spheroids understanding the role of steroids in typical and atypical brain development: advantages of using a "brain in a dish" approach towards a human-on-chip: culturing multiple cell types on a chip with compartmentalized microenvironments multisensor-integrated organs-onchips platform for automated and continual in situ monitoring of organoid behaviors extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture development of collagen-based 3d matrix for gastrointestinal tract-derived organoid culture peg-4mal hydrogels for human organoid generation, culture, and in vivo delivery mechanically and chemically defined hydrogel matrices for patient-derived colorectal tumor organoid culture designer matrices for intestinal stem cell and organoid culture growth of epithelial organoids in a defined hydrogel inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells the ground state of embryonic stem cell self-renewal validated germline-competent embryonic stem cell lines from nonobese diabetic mice new cell lines from mouse epiblast share defining features with human embryonic stem cells derivation of pluripotent epiblast stem cells from mammalian embryos derivation of novel human ground state naive pluripotent stem cells induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast derivation of naive human embryonic stem cells systematic identification of culture conditions for induction and maintenance of naive human pluripotency epigenetic resetting of human pluripotency naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass resetting transcription factor control circuitry toward ground-state pluripotency in human systematic identification of culture conditions for induction and maintenance of naive human pluripotency application of small molecules favoring naive pluripotency during human embryonic stem cell derivation clinical features of patients infected with 2019 novel coronavirus in wuhan coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 recapitulation of sars-cov-2 infection and cholangiocyte damage with human liver ductal organoids inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 sars-cov-2 productively infects human gut enterocytes a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis for the recognition of sars-cov-2 by full-length human ace2 structural basis of receptor recognition by sars-cov-2 the authors thank a. kavirayani and c. hindley for proofreading, and s. bae for initial design of the artwork. work in j.a.k.'s laboratory is supported by the austrian academy of sciences, the austrian science fund (grant z_153_b09) and an advanced grant from the european research council. b.-k.k.'s laboratory is supported by the austrian academy of sciences, the european research council, the human frontier science program and the interpark bio-convergence center grant program. the authors contributed equally to the writing and revisions of the article. the authors declare no competing interests. nature reviews molecular cell biology thanks h. clevers, p. liberali and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. who, disease outbreaks: www.who.int/emergencies/ diseases/en/ who, coronavirus disease 19 (covid-19) pandemic: https:// www.who.int/emergencies/diseases/novel-coronavirus-2019 key: cord-331584-z43ifmr3 authors: mahy, b.w.j. title: emerging and reemerging virus diseases of vertebrates date: 2008-07-30 journal: encyclopedia of virology doi: 10.1016/b978-012374410-4.00383-6 sha: doc_id: 331584 cord_uid: z43ifmr3 in the last two decades, a large number of new viruses have been discovered, many of which are pathogenic in humans or other vertebrates. among the more important causes of virus emergence have been changes in human behavior, population, and increases in travel to distant countries. in addition, the application of new molecular technologies has led to the recognition of many viruses that hitherto went undetected. many of the new, emerging viruses have an rna genome, and many are zoonoses. the spread of human immunodeficiency virus, causing acquired immune deficiency syndrome, and the use of immunosuppressive drugs following transplant surgery, have increased the numbers of people in the population that are highly susceptible to emerging virus infections. the threat of a new pandemic of influenza virus in the human population stresses the need for development of better methods for detection, surveillance, and control of emerging virus diseases. it became apparent during the last two decades of the twentieth century that new infectious diseases were increasingly being recognized in the human and animal populations. this led to the establishment of a formal committee of the institute of medicine of the national academy of sciences, usa, who reported on their deliberations in 1992, in a report edited by joshua lederberg and richard shope. this was followed 10 years later by a second report, edited by mark smolinski, margaret hamburg, and joshua lederberg, which appeared in 2003 . among the factors they cited as contributing to emergence were microbial adaptation and change, human susceptibility to infection, climate and weather, changing ecosystems, economic development and land use, human demographics and behavior, technology and industry, international travel and commerce, breakdown of public health measures, poverty and social inequality, war and famine, lack of political will, and finally intent to harm. the advent of highly specific molecular techniques such as the polymerase chain reaction (pcr) in the early 1980s permitted the detection and grouping of viruses on the basis of genome nucleotide sequence analysis, and in several respects these techniques have replaced serological analyses for the characterization of viruses. although it is still important to isolate viruses in cell culture for their complete characterization, it is now possible directly to detect viruses in diseased tissues by pcr, then, by sequencing the amplicon, to determine whether a new virus has emerged to cause the disease. in fact, many viruses which do not readily grow in cell culture can only be differentiated by sequence analysis. the papillomaviruses are an example. their study was very difficult until the advent of sequence analysis, which now has revealed more than 100 types in humans, and many more in animals and birds. for differentiation, three virus genes (e6, e7, and l1) are sequenced, and if the combined sequence of these three genes differs by more than 10% from known papillomaviruses, the virus is considered to be a new type. other viruses which have not been grown in cell culture include many caliciviruses, and the ubiquitous anelloviruses, such as torqueteno (tt) virus, which can be detected and sequenced in the blood of most humans and many other vertebrate species. hepatitis c virus was originally described as non-a, non-b hepatitis virus because of the severe disease it caused but the virus would not grow in cell culture, and eventually was detected in blood known to be infected with the virus by reverse transcription of the rna present using random primers then expressing the resultant dna in the bacteriophage lambda gt 11. thousands of clones were screened using patient blood as a source of antibody before positive clones were detected, which then allowed the development of enzyme immunoassays that could detect the virus in blood and so were used to screen blood destined for transfusion, saving millions of lives worldwide. once the complete genome of hepatitis c virus was sequenced, it became apparent that there are many different genotypes circulating in the world, with different pathogenic properties. nucleotide sequence analysis has also been extremely useful in tracing the origins of viruses. for example, when hantavirus pulmonary syndrome, caused by a bunyavirus of rodents, sin nombre virus, was initially detected in 1993 in the four corners region of western usa, it was found that rodents inside a house where people had been infected carried a virus identical in sequence to virus isolated from human cases. however, rodents caught at various distances from the house had increasingly variable genome rna sequences, providing strong evidence that these rodents, deer mice (peromyscus maniculatus), were the source of the infection. subsequently more than 30 other hantaviruses, some of which also cause hantavirus pulmonary syndrome in humans, were isolated from rodents throughout north and south america. each new virus seems to be associated with a different genetic variant of rodent host, and all rodents that carry the virus belong to the subfamily sigmodontinae, unique to the american continent. a particularly powerful tool for the initial recognition of an emerging virus is the application of immunohistochemistry to diseased tissues. provided a comprehensive collection of antibodies is available, the particular virus or related group of viruses can often be detected. for example, when hendra virus first appeared in 1995 in australia, causing the death of a horse trainer and 14 of his horses, antibody against the virus was sent to the centers for disease control and prevention (cdc). then, in 1999, cdc was asked to investigate a newly emerged epidemic that had appeared in malaysia, killing more the 100 people and causing disease in many pigs. initially, it was suspected to be caused by a virus related to japanese encephalitis virus, but a virus was isolated from the pigs that replicated in vero cells, and reacted in an immunofluorescence test against the hendra virus antiserum. this could subsequently be used on patient tissues to study the pathogenesis of the disease, and after comparison of the genome sequences of hendra virus and nipah virus they were found to be closely related and are now classified in the genus henipavirus of the paramyxoviridae. finally, molecular methods can be used to detect new, emerging viruses in the absence of disease in the host. in 2001, allander and colleagues searched for rna viruses in human respiratory secretions using random primer pcr, and discovered a hitherto unknown parvovirus with a sequence related to the bovine and canine parvoviruses, which are grouped together in the genus bocavirus. the new virus was called human bocavirus, and many research groups worldwide have now confirmed the presence of the virus, particularly in pediatric samples, although it is still not certain how important this virus is in causing morbidity and mortality. their method also amplified a human coronavirus from the respiratory samples, and when sequenced this turned out to be hku1, a recently emerged coronavirus detected by scientists at hong kong university. it is possible that a systematic search of human samples using such molecular techniques might reveal more hitherto unknown human viruses. in some cases, the emerging viruses themselves have contributed to other viruses emerging and reemerging in the population. this is especially true of human immunodeficiency virus (hiv), the cause of acquired immune deficiency syndrome (aids), which rapidly spread following its emergence in the early 1980s to infect more than 40 million people worldwide by the end of the twentieth century. because of its severe effects on the immune system, the virus leads to numerous other infections in the hiv-infected population. for example, picobirnaviruses, that had been detected in fecal samples from chickens and rabbits, were difficult to detect in human fecal samples until a cohort of men with aids was examined, and in these humans picobirnavirus was detected for the first time. some rare diseases have become common in persons with aids. for example, the human polyomavirus known as jc virus can cause the rare brain disorder known as progressive multifocal leukoencephalopathy (pml). normally, the virus remains dormant in the kidney, but in hiv-infected individuals the hiv-encoded transactivator tat acts as a transactivator of jcv leading to pml which progresses to death within 4 months after infection. other important virus infections which emerge in aids patients are human herpesviruses (cytomegalovirus, herpes simplex viruses 1 and 2, varicella-zoster virus, and human herspesvirus 8, which causes kaposi's sarcoma). hiv is mainly spread through sexual activity between an infected and a noninfected person, and is most common in those who indulge in high-risk sexual behavior with multiple partners. it can also be transmitted by direct contact with infected blood, and is common in persons who indulge in intravenous drug use, particularly when needles, syringes, or equipment used to prepare drugs for injection are shared. it is therefore an example of a virus disease which is dependent on risky human behavior for its maintenance in the human population. the ability of such new infections to spread in the population has been greatly enhanced by population growth and ease of movement as a result of rapid air travel. a dramatic recent example of this was the appearance of the coronavirus causing severe acute respiratory syndrome (sars) in late 2002 which spread by air travel from a single infected chinese physician who infected 12 persons in a hong kong hotel. these infected persons then traveled by air and spread the infection to more than 8000 individuals worldwide, 10% of whom died. the virus then apparently receded from the human population in july 2003. only recently was it discovered that the sars coronavirus has a natural reservoir in chinese horseshoe bats (rhinolophus sinicus). some other species such as himalayan palm civets and raccoon dogs from which the virus has been isolated may serve as amplification hosts. following the recognition of the human coronavirus sars, research on coronaviruses intensified, and this led to the discovery in 2004 of two previously unrecognized human viruses, one found by hong kong university, called hku1 virus, and another reported almost simultaneously from the netherlands, called nl63, and from yale university, called new haven coronavirus. the latter viruses probably represent two isolates of the same virus species. they are clearly associated with lower respiratory tract infection in children, but initially it was claimed that new haven coronavirus was also associated with kawasaki disease in children. this intriguing claim was rapidly investigated and refuted by several different groups in japan, taiwan, and elsewhere, and the cause of kawasaki disease, which has features resembling a virus infection, remains unknown. a majority of recent emerging virus diseases have been zoonoses (i.e., diseases transmitted from animals to humans under natural conditions). some of the more important of these include hiv-1, which was transmitted to humans from chimpanzees in central africa around 1931, and hiv-2, transmitted from sooty mangabeys to humans in west africa around 1940. other important recent examples are the viruses of the genus henipavirus. hendra virus was first recognized through a disease outbreak in some horse stables in hendra, queensland, australia, when 14 horses and their trainer died from pulmonary disease with hemorrhagic manifestations in 1994. the reservoir of the virus was found to be in large fruit-eating bats (pteropus spp.), and one year later a horse farmer 600 miles away in mackay, queensland, died of encephalitis from the same virus. then, in 1999, a related virus was discovered in malaysia following a major outbreak of respiratory disease in pigs and neurological disease in humans in their close contact. more than 100 humans died, and in a successful effort to control the disease 1.1 million pigs were slaughtered. the causative virus was isolated from a fatal human case that had lived in nipah river village, and so was named nipah virus. hendra and nipah viruses are clearly members of the family paramyxoviridae, but have been placed in a separate genus as their rna genome is about 19 kb in length, larger than that of any other paramyxovirus. nipah virus, like hendra virus, was found to have a reservoir in pteropus bats, and has since been identified in fatal human disease outbreaks in india in 2003 and bangladesh in 2004. other new viruses which apparently have a reservoir in fruit bats include menangle virus, a new paramyxovirus which emerged in a commercial piggery near sydney, australia, to cause stillbirths and abortion in pigs. menangle virus also caused disease in two workers in the piggery. a new virus related to menangle virus emerged during an investigation of urine samples from pteropid bats collected on tioman island, off the coast of malaysia, in 2001, and was named tioman virus. during the same investigation, a new orthoreovirus was isolated from pteropus hypomelanus in 1999 and called pulau virus, and more recently a related orthoreovirus called melaka virus was isolated from a human case of acute respiratory disease in melaka, malaysia. serological studies of sera collected from human volunteers on tioman island showed that 13% had antibodies against both pulau and malaka viruses. another important group of zoonotic diseases are rodentborne, and caused by members of the genus hantavirus of the family bunyaviridae. these viruses first emerged during the korean war of 1950-52, when thousands of un troops developed a mysterious disease with fever, headache, hemorrhage, and renal failure with a fatality rate of 5-10%. it was more than a quarter of a century before the causative virus was isolated from field mice in korea, and named hantaan virus, the cause of hemorrhagic fever with renal syndrome (hfrs) in humans. then, in 1993, a new hantavirus emerged in the four corners region of southwestern usa as the cause of a severe acute respiratory disease syndrome, with a fatality rate close to 40%, and named sin nombre virus. this virus was shown to be transmitted to humans by inhalation of virus present in the urine, feces, or saliva of deer mice (peromyscus maniculatus). it seems likely that this disease had existed for many years, and was only recognized in 1993 because of a clustering of human cases as a result of a regional upsurge in the rodent population resulting from climatic conditions causing increased availability of rodent food. fortunately, in most of these infections, humans appear to be a dead-end host, and transmission between humans does not occur except with the andes virus in south america. rodent-borne viruses of the family arenaviridae also cause a number of serious zoonotic diseases in humans. the 'old world' arenaviruses such as lassa fever virus have been known for some time, but still cause thousands of fatal hemorrhagic fever cases every year in west africa. however, 'new world' arenaviruses such as junin virus causing argentinian hemorrhagic fever and machupo virus causing bolivian hemorrhagic fever have long been recognized in south america. recently, new arenaviruses have emerged, probably as a result of deforestation, which results in rodents seeking shelter in human habitation, and brings them into closer contact with people. these viruses include guanarito virus that causes venezuelan hemorrhagic fever with 36% mortality rate from confirmed cases, and sabia virus isolated in 1990 that causes brazilian hemorrhagic fever with a high fatality rate, including two laboratory acquired cases. rabies is a zoonotic disease of great antiquity that has mainly been associated with carnivores, such as dogs. the virus is excreted in the saliva of infected animals, and following infection it moves through the nervous system to attack the brain, causing aggressive behavior which results in the animal biting humans and animals with which it comes into contact and thereby spreading the virus infection. fortunately, due to early work by louis pasteur, a vaccine was developed that protects humans or other animals from infection, and can also be given immediately post exposure, and the domestic dog population in the developed world is vaccinated and does not pose a risk to humans. however, in some developing countries, it is not uncommon for a rabid dog to bite and infect more than 25 people before it can be put down, and worldwide there are still some 30 000 human rabies deaths per year. using molecular sequencing techniques, it is now possible to distinguish the genotypes of rabies viruses associated with different species of host, as the virus has become adapted through frequent transmission between members of the same host species. in the usa, there are six recognized terrestrial animal genotypes, in raccoons in eastern states, skunks in north-central states, skunks in southcentral states, coyotes in southern texas, red foxes in alaska, gray foxes in arizona, and several genotypes associated with particular species of bat. in fact, most fatal cases of human rabies in the usa can now be traced to bats, which are often not detected when the person is bitten; so rabies is not suspected and vaccination is not undertaken until the disease has taken hold. many important virus diseases are spread by arthropods, and exposure to new arthropods and the viruses they carry is critical to the emergence of new virus diseases. dengue hemorrhagic fever is caused by dengue virus which is transmitted mainly by the asian mosquito (aedes albopictus), and dengue fever is one of the most rapidly emerging diseases in tropical regions of the world. there are four serotypes of dengue virus, and it seems that consecutive infections with two antigenic types can lead to the more serious disease of dengue hemorrhagic fever with shock syndrome, which, if untreated, can result in up to 50% mortality. unfortunately, through the importation of vehicle tires containing water from korea, the asian mosquito was introduced into the usa, and is now present in several regions of the southern states. it can act as a vector not only for dengue virus, but also for california encephalitis virus. in europe, the emergence of two important animal diseases has occurred through the movement of arthropod vectors into the iberian peninsula. african horse sickness virus causes a disease that can be fatal to horses, mules, and donkeys, and is transmitted by nocturnal biting flies of the genus culicoides. these were introduced inadvertently into spain, and the disease is now endemic around madrid and regions to the south. african swine fever virus is transmitted by ticks of the genus ornithodorus and it causes a fatal disease resembling classical swine fever in domestic pigs. it first emerged in portugal and spain in 1957 , france in 1964 , italy in 1967 , and cuba in 1971 . through slaughter of infected animals, the disease was eradicated from europe, except sardinia, by 1995. the most recent dramatic example of the movement of a virus vector is provided by west nile virus, a flavivirus first isolated in uganda in 1937. this virus uses birds as a reservoir host, and is transmitted from birds to humans and other vertebrates by mosquitoes. in 1999, cases of encephalitis in new york were found to have been caused by a strain of west nile virus that was phylogenetically similar to a virus isolated from geese in israel. at the same time, many birds, especially corvids, began dying in new york state. since the introduction in 1999, west nile virus has become well established throughout the usa and moved north into canada and south into the caribbean and into mexico. it is not known how the virus moved from israel to the usa, but the most reasonable explanation is that it was carried in an infected mosquito or possibly an infected bird in the hold of an aircraft. transmission by an infected human seems less likely since the titer of virus in human blood is usually too low for efficient mosquito transmission. it is clear, nevertheless, that once it arrived in north america, west nile virus found an extremely favorable environment with abundant avian and arthropod hosts that facilitated its spread throughout the american continent. the emergence of new viruses is likely to continue as viruses evolve and find new ecological niches in the human and animal population. it is noticeable that most newly recognized viruses have been rna viruses, perhaps since rna evolves at a faster rate than dna, for which host cells have developed efficient proofreading enzymes. it will be important in the future to detect new viruses before they can emerge to cause disease in the population. the sars epidemic provides an excellent example. before the epidemic, only two human coronaviruses were known, human coronaviruses 229e and oc43. despite the fact that serious coronavirus diseases were well known in other vertebrates, such as feline infectious peritonitis and avian infectious bronchitis virus, it was not until the sars epidemic that research on human coronaviruses led to the discovery of three new human coronaviruses -sars, hku1, and nl63/new haven. there are other genera of viruses that cause serious disease in animals but have not been adequately investigated in humans. an example is the genus arterivirus, which has members causing serious disease in horses and pigs, but has not been reported at all in humans. this could be a worthwhile area for future investigation. another critical factor in the future control of emerging viruses is better vector control. when mosquito control was conducted using ddt, dengue fever virus was virtually eliminated from the americas in the 1970s, but environmental concerns led to the widespread banning of the use of ddt, so that since the 1980s there has been a considerable expansion of dengue fever in south america, with the appearance of dengue hemorrhagic fever there for the first time. there is a real need to improve mosquito control measures to control this disease. although there are prospects for a dengue virus vaccine, this is so far not available. finally, one of the most important viruses that continue to emerge in different antigenic forms is influenza virus. the main reservoir of influenza viruses is in birds, and over the past century several pandemics of influenza have emerged, the most serious of which was in 1918. pandemic strains usually arise by a process of antigenic shift, where one of the genes encoding the hemagglutinin and/or the neuraminidase of influenza virus is replaced by one from birds. new pandemics occurred in 1918 (h1n1 subtype), 1957 (h2n2 subtype), and 1968 (h3n2 subtype). since 1968, there have been no new pandemics, but it is widely expected that another will occur. at the time of writing, there is worldwide concern that a highly pathogenic avian influenza virus (h5n1 subtype), which has caused some human infections and deaths in persons in close contact with infected birds, might mutate or recombine to generate a virus which would be highly transmissible in the human population. plans are being developed in many countries and by the who to try to prepare for such an event by generating possible vaccines against such a virus and stockpiling antiviral drugs. emerging and reemerging virus diseases of plants identification of the major, parenteral non-a, non-b hepatitis agent (hepatitis c virus) using a recombinant cdna approach nipah virus: a recently emergent deadly paramyxovirus identification of new papillomavirus types critical review of the vector status of aedes albopictus west nile virus: epidemiology and clinical features of an emerging epidemic in the united states nipah virus encephalitis reemergence the widening scope of coronaviruses timing the ancestor of the hiv-1 pandemic strains a novel coronavirus associated with severe acute respiratory syndrome emerging zoonoses: crossing the species barrier the emergence and reemergence of viral diseases molecular and biophysical characterization of tt virus: evidence for a new virus family infecting humans genetic identification of a hantavirus associated with an outbreak of acute respiratory illness microbial threats to health, emergence, detection and response, 367pp. 367pp are we ready for pandemic influenza h5n1? geminivirus a class of plant viruses with a genome composed of single-stranded dna encapsidated in geminate particles.pandemic an epidemic at the regional or worldwide level. satellite an infectious molecule that depends on a helper virus for crucial functions. key: cord-337646-gkcm6ds0 authors: nan title: the federation’s pages: wfpha: world federation of public health associations www.wfpha.org bettina borisch and marta lomazzi, federation’s pages editors date: 2020-09-17 journal: j public health policy doi: 10.1057/s41271-020-00240-3 sha: doc_id: 337646 cord_uid: gkcm6ds0 nan result of complex interactions between humans and their natural environment, notably wild or domestic animals, or both, and the environments we all share. in fact, in the last 17 years, three new coronavirus diseases have emerged from close contact with wild animals and have spread among humans causing deadly diseases. severe acute respiratory syndrome (sars-cov), one of these lethal zoonotic pathogens was first identified in southern china (guangdong province) in november 2002, where host to human transmission occurred in a wet food market (that sells perishable foods, among whom sometimes living animals) [3] . at the end of the epidemic in 2003, china had reported > 8,000 cases of the disease and 774 deaths, with a case-fatality rate of 7% [4] . the next coronavirus to generate a global public health crisis was the middle east respiratory syndrome (mers-cov) that emerged in saudi arabia in 2012 among people working closely with camels. although this virus was not found highly contagious, yet had a high case-fatality rate, and no available vaccine. by 2019, a total of 2468 laboratory-confirmed cases of mers with 851 deaths (34.5% mortality) had been reported to who [5] . the first known cases of covid-19 occurred in late 2019. local health authorities and a further clinical and epidemiological investigation traced direct exposure to the wuhan wet food market for 66% of patients hospitalized initially [6] , then soon after came evidence of person-to-person transmission via respiratory droplets. during the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus (2019-ncov), held on 30 january 2020, the covid-19 pandemic was underway. at that meeting, chinees authorities reported over 7700 confirmed cases, over 12,000 suspected cases and 170 deaths, and the disease had spread to 18 countries with 83 confirmed cases outside china. based on these statistics, the who declared the covid-19 a public health emergency of international concern (pheic) [7] . responses to this latest pandemic have drawn widespread criticism of china, the who, and many national governments. we chose not to add our voices to that chorus, but instead affirm calls for a wiser approach to learning the lessons from the two prior epidemics [4] , and this third, covid-19 [8] . all three coronaviruses emerged via zoonotic transmission. sars-cov-19 is a new strain of coronavirus not previously identified in humans, but now established, it is likely to persist. the full ramifications of covid-19 are yet to fully unfold. apart from the tragic direct human toll of the covid-19 coronavirus epidemic, economic cost estimates range from $3.3 trillion to $82 trillion [9] . the world bank envisions a 5.2 percent contraction in global gdp in 2020-the deepest global recession in eight decades, and the first since 1870 to be triggered solely by a pandemic [10] . economic disruptions will not be evenly spread exacerbating global inequities. covid-19 is also unleashing other pernicious pathways of widespread health harm. this pandemic arrived amidst a landscape of other problems, several global regions are in conflict, and climate change threatens further food insecurity. social distancing enforcement and interruptions to global trade have left world food aid programs short of supplies and volunteers. interruptions to food systems are driving a double burden of malnutrition. in early april 2020, the world food program warned the world is on the brink of a hunger pandemic, suggesting a real possibility of covid-19 pushing an additional 130 million people to starvation by the end of 2020, bringing the global total of people facing immanent starvation to 265 million [11] . reducing the spread of this highly contagious virus forced nations to launch unprecedented campaigns to eradicate the virus by mandating social distancing and by closing sectors of the economy where close human contact was unavoidable. instructing people to stay at home meant voluntarily shutting down the economy. diverging views on whether to prioritize human health or the economy resulted in debate, social media storms, pushback, political division, and considerable variation in intra-national enthusiasm to implement full lockdown. meanwhile, ongoing ideological debates created an echo chamber of myths and misinformation, labeled an 'infodemic" by the who [12] . countries reluctant to heed expert health advice are enduring high case rates and deaths. the global shutdown is triggering an economic recession with deep and longterm consequences. high-resource nations are evidently not immune, and the link between poverty and poor health is well established [13] . previous recessions in the united kingdom (uk) indicated that a 1% fall in employment is associated with a 2% increase in prevalence of chronic illness [14, 15] . failing to consider the possibility of new global pandemic events rendered the world ill-prepared to deal with complex and diverse challenges posed. learning the lessons of the past, recognizing the health risks of allowing poor management of human-environment interactions, and heeding the warnings of the potential for additional novel diseases combining high contagion and high virulence could have produced more timely and more effective global health responses. a wiser response could have averted much of the human misery generated by covid-19. with the emergency still enveloping the world, most attention has been paid to health systems as a way forward. we adopt a public health approach and propose examination of interactions between humans and the environment as a fundamental driver of the present and preventing future pandemics. we recommend greater focus on environmental actions to prevent and mitigate them. the association between emerging infectious diseases (eids) and environmental destruction is widely recognized: deforestation destroys natural habitats, increases the density of remaining wild animal populations, increases their movements to look for food accompanied by the probability of human contact-all induce stress that impairs immune systems and increases viral shedding [16] . moreover, hunting and capture of wild animals for human consumption purposes (such as for food, medicines, dress) expose the animals to even higher stress. and the crowded conditions in food markets create opportunities for direct human and domestic animal contacts with wild animals and their excreta. rapid growth of the world population, and increasing demands for food, are powerful drivers of environmental destruction. this combination has accelerated evolution of pathogenic zoonoses and increased the probability of humans interacting with wild animals and spreading disease [17] . environment preservation is urgent for many reasons: conservation of biodiversity, the fight against climate change, reduction of air, water and food pollution, and improvement of human health and quality of life [18] . unfortunately, political leaders often postponed these aims to preference economic exigence. the resultant failure to recognize the health and economic value of ecosystem services threatens provision of the necessities of human health, such as clean air, clean food, and clean water. the covid-19 pandemic has shown dramatically that an event caused by a human-environment perturbation can destroy the fragile equilibrium of human societies and their economies. main message: the 'one health' and 'global health' approaches should become priorities in governance to quell pandemics, putting in first place preservation of the natural environment. environmental spread of microbial pathogens is well established. similarly, an abundance of evidence demonstrates the efficacy of appropriate interventions for limiting disease. water may be associated with infectious diseases for several reasons: as vehicle for infections by ingestion, contact, or inhalation, as habitat for intermediate hosts or vectors-or in a protective role, as an essential resource for hygiene. the 6th sustainable development goal "clean water and sanitation" has not yet been reached everywhere. according to the united nations report 2019, "billions of people still lack safe water, sanitation and handwashing facilities" [18] . moreover, water scarcity caused by climatic changes imposes water reuse-after treatments validated to eliminate pathogens [19] . the need for water to be free of microbiological contamination is only part of safety, as water must also be free from chemical contamination. an excess of disinfectants and their improper use generates pollution and human exposure to other toxic hazards. therefore, there is an urgent need to develop and validate efficient methods of sewage treatment and water disinfection, limiting the use of chemicals, and applying them according to a rigorous risk-benefit analysis. sars-cov-2 is eliminated in feces by 20-50% of infected people and its rna has been found in sewage in disparate parts of the world (usa, netherland, australia, france). we do not know if the sars-cov-2 can be transmitted by water [20] . nevertheless, efficient treatment and implementation of water safety plans can ensure water safety for a variety of uses as reported by national and international agencies [21] . thus, implementation and enforcement of efficient systems and strategies for water treatment are essential preparation for the present and future threats. some descriptive epidemiological studies have found correlation between covid-19 incidence, lethality, and air pollution [22] . aside from the (highly questionable) hypothesis about long distance transport of pathogens with air particulates, the main role of the air pollution seems related to impairment of airway defences to viruses [23] . enforcement of regulations against air pollution is essential to prevent respiratory morbidity. moreover, the most plausible airborne transmission of covid-19 occurs indoors or, if outdoors, in close proximity to the source. air treatments in built environments may represent a risk factor or, the opposite, a protective preventive mechanism, depending on the efficacy via proper design and management [24] . on the other hand, covid-19 caused a temporary reduction in air and water pollution: while lockdown stopped many economic activities, it also could improve water and air quality. at the same time, massive use of disposable gloves and masks and hand disinfectants caused an immense surge in medical wastes, not always properly disposed [25] . for preparedness against pandemics, all urban and medical waste collection and treatment should be efficiently managed. main message: development and implementation of technologies, structures, and regulations to limit environmental pollution will be very important for prepar-edness to control future epidemics. preventing the spread of an infectious disease in a population necessitates understanding transmission pathways. when a novel pathogen appears, initial interest focuses on diagnosis and therapy, then on developing and testing a suitable vaccine. but, as we saw in the response to the sars-cov-2, the first line of the defence against the diseases is the prevention of initial transmission into the human population, that generally involves the environment. once a disease is established in populations, along with person-to-person spread, quarantine measures of social distancing and contact tracing come to the fore as primary means of restricting further transmission. although close contacts are predominant means of transmission for respiratory viruses, their airborne spread requires attention. its probability varies, depending on the source of infective aerosol (respiratory or fecal), on the pathogen's survival in the environment, and on environmental conditions [26, 27] . for covid-19, the possibility of spreading by aerosol, in addition to direct spread through large droplets (> 5µ) and close contacts, is a risk in indoor environments including homes, restaurants, vehicles, nursing residences, temples, among others. the advice of distancing by 1 m was probably insufficient to prevent transmission for someone without any protective equipment [26] . and there is possible occupational exposure in settings other than healthcare facilities, for example, in wastewater treatment plants and waste processing facilities. even if science shows us that water and food are minimally involved in the covid-19 pandemic, these means and risks transmission should be thoroughly investigated for every emerging infection. main message: in most cases, the possible ways of infection are known by analogy with similar diseases, even if every new pathogen is characterized by unique peculiarities. it is advisable to apply the precautionary principle, without waiting for epidemiological evidence. remember that prevention is the priority, even if timely actions could limit the possibility of evidencing their added value in comparison with no action. eids such as ebola, influenza, sars, mers, and, most recently, covid-19 can erupt, creating global pandemics with massive mortality and morbidity. disruptions to trade and travel bring devastating and long-lasting economic impacts. the seemingly opposing forces of economic development and public health need to be reconciled, they are not in opposition, rather reinforcing. over and above the devastating human toll of pandemics, covid-19 provides further evidence that healthy environments and healthy people are critical to healthy economies. disruption to one domain escalates catastrophic repercussions in others. potential for new infectious diseases is ever present, not remote; we ignore them to our peril. the main challenge is to be ahead of epidemics at every step ahead of emergence, transmission, and spread. the human-environment interface must never be underestimated in its capacity to enrich or harm human health. environmental actions are essential in fighting pandemic; an effective strategy includes keywords with the same prefix: preservation, preparedness, and prevention. we should share and implement this strategy worldwide, focusing also on actions involving health systems and society. prediction and prevention of the next pandemic zoonosis opinion: sustainable development must account for pandemic risk the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? a novel coronavirus from patients with pneumonia in china enzootic patterns of middle east respiratory syndrome coronavirus in imported african and local arabian dromedary camels: a prospective genomic study clinical features of patients infected with 2019 novel coronavirus in wuhan statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus (2019-ncov) state ment-on-thesecon d-meeti ng-of-the-inter natio nal-healt h-regul ation s-(2005)-emerg ency-commi ttee-regar ding-theoutbr eak-of-novel facts are not enough-offline this is how much the coronavirus will cost the world's economy, according to the un. world economic forum and united nations chief warns of hunger pandemic as covid-19 spreads (statement to un security council). ocha how to fight an infodemic. the lancet update on the global charter for the public's macroeconomic conditions and health in britain: aggregation, dynamics and local area heterogeneity. iza dp no. 13091. london: iza institute of labor economics cohere a call for a post-pandemic health strategy-offline editors. climate vulnerability understanding and addressing threats to essential resources volume 1 health earth 2020: science, society, and sustainability in the anthropocene the united nations world water development report 2019: leaving no one behind the united nations world water development report 2020 -water and climste change paris: unesco sars-cov-2 in wastewater: state of the knowledge and research needs water, sanitation, hygiene, and waste management for the covid-19 virus: interim guidance can atmospheric pollution be considered a co-factor in extremely high level of sars-cov-2 lethality in northern italy? environ pollut seasonality of respiratory viral infections environmental engineers and scientists have important roles to play in stemming outbreaks and pandemics caused by enveloped viruses environmental perspective of covid-19 respiratory virus shedding in exhaled breath and efficacy of face masks covid-19 airborne transmission and its prevention: waiting for evidence or applying the precautionary principle? key: cord-310905-1oqfh8of authors: gill, karamjit s. title: strange affair of man with the machine date: 2020-10-13 journal: ai soc doi: 10.1007/s00146-020-01078-9 sha: doc_id: 310905 cord_uid: 1oqfh8of nan in paying our memorial tribute to mike cooley, the founding chairman of ai&society, we are reminded of his poem "insulting machines" above. it makes us reflect on the strange affair of man with the machine. whilst we may feel mesmerised by the computing power of our creation, the ai machine, we may also feel that we are being seduced to participate in our own demise, as helpless victims. professor bell (2018) notes that although the idea of ai was 'codified' as a computational paradigm at the historical dartford conference in 1956, its focus on abstraction and computation was an attempt to 'make machines use language, form abstractions and concepts, solve the kinds of problems now reserved for humans, and improve themselves.' bell notes that while wiener's cybernetics cultivated the interface between biological and technical systems, maccarthy, minsky and shanon appeared in 1956 to create an intellectual agenda to announce computational ai. what was lost in this computational enterprise was the social piece, the human piece and the biological component inherent in cybernetics, so this earlier ai became just a technical artefact. bell reflects that the birth of computational ai coincided with cybernetics going underground in europe and north america, and any notion that you could use the new power of computation to drive social science looked more like socialism-in a period when it was not a good thing to be. although the initial computational formulation was a product of a particular time and place, it has influenced the framing of research agendas ever since. but what exactly is ai in 2020, and why does it loom very large in our conversations about the future? bell says that the 1956 blue print of ai coincided with the interest of us defense department in pursuing the simultaneous machine translation from russian (not just logic but context); 1960-70s was a period of reckoning that the translation culture and its contexts were not realisable; 1990 saw the pursuit of intelligent ai; twenty-first century ais appropriated an abundance of data to train algorithms. ai is now about 'can i know your desires for goods and services, an ultimate manifestation of capitalism. ' bell further says that the question now is 'not what ai but whose ai? what work is it doing and why?' would ai look differently in different countries-different data sets and different logics, what do ais know, and what might they do? these are questions of consciousness and not of intelligence: can we imagine non-human objects to have consciousness, have intelligence! we have now gone way past the era of human-machine collaboration and heuristics of problem solving of the earlier ais, we now live in the era of the prediction ais. whist the academic community may be overjoyed with their work on prediction and affective computing to solve societal problems, the same prediction paradigm is being appropriated by high-tech companies and security agencies for automating mass surveillance of people and communities. we learn from zuboff (2019) that high tech, not content with automation of human experiences into behavioral surplus, has misappropriated affective computing architecture with the aim of automation of human emotion, the creation of an emotion chip, the creation of emotion ai. the implications of automating 'us' is to instill an awe of 'inevitability of technology' and the culture of 'economic and market dependency' and a sense of helplessness in the face of when the computer says "no". we wonder whether the creators of the computational paradigm in 1956 would have imagined that one day their dream of functional rationality would be misappropriated by high-tech companies in the 2020s to automate not just problem solving processes but to venture into automating human behaviour in the pursuit of automating the human itself. on reading zhuboff (ibid), we should perhaps not be too surprised that prediction paradigm rooted in the computation paradigm of 1956 would continue to follow the path of data mining to build big data commons for behavioural mining, construct living laboratories for reality mining of human experiences, and launch an unprecedented instrumentation for prediction and tuning of societal policies. it is instructive note that the very prediction paradigm that is elevated to reality mining has now become a catalyst for the creation of uncontrollable echo chambers that thrive by copying ideas, copying feedback, listening to the same sermon, same voice, many gurus but same sermon, no change, no new vision, no transformation-a paradox of algorithmic exploration. harari (2020) argues that the epidemic of surveillance technologies that track, monitor and manipulate people, marks an important watershed in the history of surveillance. the danger lies in not just the normalisation of the use/misuse of mass surveillance tools, but also the implication of a dramatic transition from "over the skin" to "under the skin" surveillance that has arrived with coronavirus. for example, what is now demanded of us is not just what is outside our skin but also what is inside-not just the blood pressure and temperature of our fingers, but also the blood pressure under the skin. he asks us to imagine a future scenario in which every citizen would be required to wear 'a biometric bracelet that monitors body temperature and heart-rate 24 h a day'. we should, remember that technology can also use biometric data to predict human behaviour and manipulate our feelings and sell us anything they want-be it a product or a politician'. we learn from weizberg (2020) that whilst international agencies such as the unhc may express concern about sharing sensitive biometric data of refugees with the security agencies, they together with the world bank and the world food programme seek technological solutions to the elusive problem of identity and citizenship status. under the techno-centric umbrella of improved accountability, increased efficiency, and greater objectivity, biometrics is being used as a blunt instrument for digital surveillance. the surveillance technology may offer 'seductively easy solutions' to complex population problems by tying 'legal status directly to the body', which also leaves people, their messy lives, life choices, hopes and their survival strategies at the mercy of the digital scanner of 'dispassionate bureaucracies'. it should be alarming to note that whilst biometrics is increasingly seen as a panacea for a range of problems being addressed by the global development agenda of international agencies such as un's sustainable development goals, and world bank, and the world food programme, it has also become a surveillance tool for the externalisation of 'european asylum policy' to the 'global south'. we also learn from weizberg (ibid.) that across the 'global south, biometric identifiers are increasingly linked to voting, aid distribution, refugee management and financial services' and the most vulnerable populations are now being used as 'laboratories for experimental tech.' it should thus come as no surprise that human rights advocates worry about international agencies such as unhcr sharing sensitive biometric data of refugees with the security agencies, thereby making it 'accessible to other actors beyond the unhcr's own biometric identity management system.' we learn from zhouboff (op.cit: 215) that it is becoming clear that this 'surveillance gamification' is not concerned with ethical, trust and moral implications of misappropriating body as data and behavioural surplus, it is more concerned with taking punitive measures against humans who may breach, even unwittingly, the performance of algorithms. a surveillance algorithm could activate consequences of this breach in the form of 'a violation algorithm', 'a curfew algorithm' 'a monitoring algorithm', 'an adherence algorithm', 'a credit algorithms'. so we have reached a digital future in which reality mining obliterates the past, mortgages the future and speaks in present tense: a future dominated by the arrogance of the prediction paradigm, and in which humans are penalised if they 'insult the machine', but the machine is protected from violating not just the human body but also violating their privacy, ethics and moral being. as cooley (2013) , in his poem on insulting machines puts it: potential and reality are torn apart as change is confused with progress with slender knowledge of deep subjects -you proceed with present tense technology, obliterating the past and with the future already mortgaged the court of history may find you intoxicated with species arrogance recklessly proceeding without a hippocratic oath. (cooley 2013) whilst the medical and health professionals and data science researchers see covid-19 data as a guide to predict scenarios of infection, fatality, and develop guidelines for safety, the same data are being appropriated by surveillance proponents to promote machine leaning algorithms and apps as instrumental tools for locating, facial recognition, monitoring and tracing people under the cloak of cloak of public safety, national security, fraud detection, and even disease control and diagnosis. as was noted in gill (2020) there are, for example, offers of facial recognition systems for predicting the behaviour of citizens, offers of surveillance drones for 'biometric readings', predictive policing is offered as an effective tool to predict, contact trace and reduce crime rates (e.g. australia's covidsafe), and offers of prediction algorithms (e.g. zegami) to assess the outcome of patient x-rays and diagnose covid-19 virus. as machine learning and data analytics are offered to 'accelerate solutions and minimize the impacts of the virus, help expedite the drug development process, and forecast infection rates, these automation tools also raises ethical issues of data protection, privacy, potential bias in the data, lack of transparency, explainability and accountability. furthermore, this raises questions of potential negative implications for the therapeutic alliance in patient-clinician relationships. gill (ibid) argues that the concern is not just with the automation of behaviour and emotion but also the automation of behavioural interventions and modifications. this automation thereby leads to the exclusion of human engagement to formulate and institute ethical constraints, thereby leads to the exclusion of human interventions from the misappropriation of predictive and affective architectures for high-tech and its market forces for profit. whilst in the 1980s, we faced the challenge of turning 'judgment' to 'calculation', now in 2020s we face the challenge of turning human to data. it is no longer about the exclusion of the social but the exclusion of the human itself. at this stage, we wonder whether we would be able to extricate ourselves from the straight jacket of the fustian exchange of the prediction paradigm in which we feel helpless and abandoned when the computer says 'no'. whilst prediction technologies of behavioural mining, tuning and surveillance are playing havoc with the identities and lives of people, the high-tech companies are building big data commons and behavioural and experience mining laboratories to first create and then satisfy a culture of instant gratification, ranging from credit cards to fast food, that offers immediate pleasure even in the knowledge that it brings long-term pain. in carol ann duffy (ramm 2017) , we learn of the faustian seduction: "i grew to love the lifestyle, / not the life". goethe's story of margareta (gretchen), provides the most poignant episode of the faustian exchange. faust pursues gretchen, seduces her, and then-unwittingly-destroys her and her family. mephistopheles guides his hand but faust's actions are unbearably his own (the demon goads him: "who was it who ruined her? i, or you?"). the gretchen story has become a powerful cultural motif, inspiring elegies such as byron's: her faults were mine -her virtues were her own -i loved her, and destroy'd her!… if i had never lived, that which i love had still been living; had i never loved, that which i love would still be beautiful. (https ://www.bbc.com/cultu re/artic le/20170 907-whatthe-myth-of-faust -can-teach -us) faust tells gretchen (ramm ibid.) : "my sweet, believe me, what's called intellect/is often shallowness and vanity", and almost every iteration of the legend underscores this disenchantment: it is byron's manfred who discerns "the fatal truth, /the tree of knowledge is not that of life". intellectual pursuits have isolated faust, and failed to provide him with wisdom: "the very thing one needs one does not know/and what one knows is needless information". even when the quest for knowledge is successful, it conjures up dark forces, as in frankenstein. like goethe's faust, the proponents and disciples of the prediction paradigm seem to have cast aside their love for scholarship in order to become 'men of action', to tame the civilisation and its social and cultural forces of nature, whose tacit and subsidiary contextual dimensions unsettle their deterministic visions, and fill them with anxiety. their prediction project is beyond human. as cooley (2013) puts it: the diagnosis is serious: a rapidly spreading species' loss of nerve; tacit knowledge is demeaned whilst propositional knowledge is revered. who needs imagination when there are facts ? (cooley 2013) from gill (2020), we learn that the prediction paradigm could neither predict the covid-19 tsunami, nor it could provide any relief or diagnosis to people who suffer from covid-19. just as the tsunami of the virus cannot be controlled without human engagement and intervention (e.g. medical intervention, social distancing), the virus of the prediction paradigm cannot be controlled without social, ethical and moral constraints and interventions. it is worth repeating the argument that within the academic zones of mit and stanford, the prediction paradigm may have been constrained by ethical limits. but once it found its way to silicon valley, it was unconstrained by any ethical limits. further, the covid-19 pandemic has shown us that just as economy is a very narrow way of organising life and deciding who is important and who is not, so is making the digital future as our home a narrow technological way of thinking about what can be, what should be and what ought to be done for the benefit of society. what we have also learnt from covid-19 is that the spread of the virus crosses social, cultural, religious, ethnic and geographical boundaries, and thus can neither be controlled by these boundaries, nor can be abstracted away by quantification or wished or washed away through the technological narrative. so any attempt to externalise the spread of the virus to others or outside sources is not only shirking our social and ethical responsibility to mitigate its impact, but also harms others. for pereira (2019), the problem of the prediction paradigm is not that we have lived with prediction, it is that we are in awe of the power of the machine, and are giving it too much power to automate human behaviour, without social, cultural and legal ethical and moral constraints. this machine learning agency is based on the idea that systems can mine and learn from the huge volume of data, and thereby identify patterns of similarity to make decisions with minimal, if any, human intervention. if this bounded algorithmic agency lacks ethical constraints, then what makes us assured that the prediction paradigm can be tamed by ethical and moral constraints, when it comes to the automation of human behaviour and emotion? the prediction paradigm has highlighted our daily faustian choices. amoore (2020) argues that the injustices of predictive models have been with us for some time. the effects of modelling people's future potential are present in almost all spheres of our lives ranging from predictive policing, visa application, immigration control, children abuse and risk, welfare claims, to student exams, university admission, what we watch, loan applications, and staff recruitment. our life chances-if we get a visa, whether our welfare claims are flagged as fraudulent, or whether we're designated at risk of reoffending-are becoming tightly bound up with algorithmic outputs. for example, she notes that the predictive algorithm developed by the qualifications regulator ofqual for predicting student exam results exemplifies the injustice of the predication paradigm. it not only disregarded the hard work of many young people in a process that ascribed weight to the past performance of schools and colleges, it downgraded the experience of students and teachers. amoore further points out that 'resistance to algorithms has often focused on issues such as data protection and privacy', but the embedding of predicting algorithms in societal domains such as the above, focuses not just on how the data might be used in the future, but how data are being actively used to change our futures. these discriminatory and opaque predictions not only reduce the potential pathways open to people, but limit their life chances. these algorithms illustrate the technical embodiment of a deeply political idea: that a person is only as good as their circumstances dictate. 'in the future, algorithmic injustices could mean people's choices in education, health, criminal justice and immigration are all diminished by a calculation that pays no attention to our individual personhood.' amoore (ibid.) alerts us that it is time to bring to focus the effects of algorithmic injustice for all to see. the danger is that predictive algorithms offer political and policy-makers the allure of definitive solutions and the promise of reducing intractable decisions to simplified outputs. this logic runs counter to democratic politics, which express the contingency of the world and the deliberative nature of collective decision-making. algorithmic solutions translate this contingency into clear parameters that can be tweaked and weights that can be adjusted, such that even major errors and inaccuracies can be fine-tuned or adjusted. this algorithmic worldview is one of defending the "robustness", "validity" and "optimisation" of opaque systems and their outputs, closing off spaces for public challenges that are vital to democracy. the story so far is that the computational model of ai of 1950s, found its rebirth in the prediction paradigm, and consequently we now face challenges of surveillance capitalism. so what went wrong on the way to seeking human-like intelligence? bell (op.cit.) notes that by separating biology from cognition, what was lost was the 'social piece, human piece and biological component'. the earlier ai was just a technical artefact, a logical system based on symbols, engaged in an imitation game that created virtual models of the environment, which could then be projected back onto the world itself. zhouboff (2019) says that on the way, we lost the body. the prediction paradigm first appropriated the outer body by mining human behaviour and human experience, and then hollowed the inner being by mining human emotion in search of an emotion chip. ben medlock (2017) sheds further light on the limitation of the prediction paradigm and inadequacy of its algorithms to seek humanlike ai, without embracing embodiment. he argues that the earlier ai models of symbolic logic such as 'shrdlu', proved 'hopelessly inadequate when faced with real-world problems, where fine-tuned symbols broke down in the face of ambiguous definitions and myriad shades of interpretation.' although the recent shift of ai to machine learning has produced many practical applications that, for example, surpass us at speech recognition, image processing, beat us at chess, jeopardy! and go, and compose pop music, machine learning 'algorithms are a long way from being able to think like us.' we are bodily beings of evolved biology. the human cell, as a biological information processor, is a remarkable piece of networked machinery that has, over centuries, evolved, 'intelligently' adapting and working together to mould us into robust, self-sustaining agents. in asking us to make a 'leap to go from smart, self-organising cells to the brainy sort of intelligence', medlock (ibid.) quotes antonio damasio "we think with our whole body, not just with the brain." he further says that it is the bodily survival in an uncertain world that is the basis of the flexibility and power of human intelligence. this argument suggests that it is questionable whether the prediction paradigm and machine learning approaches will be 'able to capture anything like the richness and diversity of embodied imperative, rooted in the symbiotic relationships of body and the environment. as cooley (2013) puts it: a human enhancing symbiosis ignored whilst a dangerous convergence proceeds apace as human beings confer life on machines and in so doing diminish themselves. your calculus may be greater than his calculus but will it pass the sullenberger hudson river test ? meantime, the virtual is confused with the real -as parents lavish attention on the virtual child whilst their real child dies of neglect and starvation. (cooley 2013) the omission of biology in the creation of the computational paradigm, the separation of the body from the brain, the prediction paradigm's hollowing of the body, and ignoring the symbiosis of body and its environment, has continued to promote and entrench the single story of the universality of the instrumental calculus. weizenbaum (1976) , as early as 1970s, was concerned that instrumental reason is so penetrated in the culture of computation that questions and challenges of human purpose are either ignored or misrepresented, as if every aspect of the real world can be formalised and represented in term of logical calculus. this gave weizenbaum an insight into a fundamental problem; human beings are liable to attribute to the machine, in this case a diagnostic programme in the field of medical care, more intelligence than it possesses. in doing so, we lose our distance, we fail to realize what the limitations are. weizenbaum points out that those who aspire to equating machine intelligence to human intelligence keep convincing themselves that by outplaying human go players, composing music, or creating human-like social robots, machines have either already or are soon going to outsmart human beings. this belief in machine intelligence sees no distinction between the functional machine and the imaginative human being. it seems that in this pursuit of machine intelligence, the validation of human intelligence has been reduced to the display of technological wonders, just as scientific knowledge has been reduced to wonders of data science. in this age of the fascination with big data and prediction, what is at stake is not just the hollowing and loss of the body but also the loss of wisdom in action. whilst the data-information-knowledge-wisdom-action loop allowed for human engagement, the algorithmic jump from data to action has not only eliminated practical wisdom, it has also eliminated human from intelligence as if there were no 'human' in 'intelligence'. as huffington (2018) notes, it is as if humans were simply intelligent machines that could be seamlessly blended with the most intelligent of artificial intelligence with nothing essential lost. what this elimination fails to grasp is that human engagement in action connects self with others, and it is this connectedness that 'gives meaning to life'. further that it is this engagement that 'ultimately determines why technological progress decoupled from wisdom is so dangerous to our humanity.' huffington alerts us to the danger of 'disentangling wisdom from intelligence' and being drowned 'in data and starved for wisdom', and asks us 'to take steps to protect our humanity from the onslaught of technology in every aspect of our lives as we're becoming increasingly addicted to our smartphones and all our ubiquitous screens.' in the pursuit of protecting 'innately human qualities like wisdom and wonder', she quotes harari that up until now, "we humans have built our identity on being homo sapiens, the smartest entities around." but "as we prepare to be humbled by ever smarter machines," harari urges us to "rebrand ourselves as homo sentiens. " we should, however, be mindful that just 'branding' ourselves may be intellectually stimulating, and we need to take serious note of the seductive control exerted by the big tech machine, such as facebook, in turning their users into helpless observers of their own and other lived worlds. the machine first disconnects individuals from their social and cultural contexts, creates a society of individuals dependent upon the machine feedback, in the process seducing individuals dispossessed of their social and cultural skills, and ultimately becoming the only social sanctuary without exit. the machine then plays a similar game with society, expressed in this poem written in the spirit of zhuboff's argument: hail the machine! thou society of individuals-magical gifts of dreams of certainty, hollowed of social and cultural roots-a future of inevitability, dispossession, helplessness. cometh the prediction paradise's seduction of digital behavioural bread crumbs-allure of alignment, checkmate thou! alternative reality-game of surveillance gamification. hail the machine! to the digital future's rendition of humanity. in paying our memorial tribute to mike cooley (march 1934 -sept 2020 , we remember him as the founding chairman of ai&society, the author of the seminal book, architect or bee, the recipient of the alternative nobel prize, and the architect of the human-centred movement. we should be mindful of his warning (cooley 2018) , when he says that the final act of metamorphosis of the artificial is becoming so complete, so technologically elegant, so powerful of calculation, and so intelligent, that, in some respects, we may no longer be able to tell them apart from humans. inspite of this unsettling prediction, cooley (gill 2020) , as ever an optimist, asserts that as architects of our own history, we should have the foresight to rewrite the final script, and circumvent the final act of automation, and avoid the slippery slope of calculation to judgment. cooley urges ai scientists, engineers and practitioners to be architects of the technological future and not let the silicon valleys discover this future for us, when he says that: 'the future is not "out there" in the sense that a coastline is out there before somebody goes to discover it'. it has yet to be built by humans.' why 'ditch the algorithm' is the future of political protest. the guardian decolonising artificial intelligence? ways of knowing delinquent genius: the strange aair of man and his technology prediction paradigm: the human price of instrumentalism the world after coronavirus we're drowning in data but starved for wisdom: we're more than just intelligent machines. thrive global this post originally appeared on aeon should i kill or rather not? what the myth of faust can teach us. bbc culture machine-readable refugees, london review of books-14 computer power and human reason: from judgment to calculation the age of surveillance capitalism publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-264408-vk4lt83x authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: animal models of human viral diseases date: 2017-06-23 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-809468-6.00033-4 sha: doc_id: 264408 cord_uid: vk4lt83x as the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. recent outbreaks of middle east respiratory syndrome corona virus, ebola virus, chikungunya virus, and zika virus illustrate the emerging threats that are encountered. by utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. this is especially true in the advent and implementation of the fda animal rule. although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand disease progression, pathogenesis, and immunologic responses to viral infections in humans. furthermore, to test vaccines and medical countermeasures, animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease course. a good animal model of viral infection should allow assay of many parameters of infection, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most studies in small animal models when possible, and studies in nhps to fill in the remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen free (spf), transgenic and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated to humans. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables collection of larger or more frequent blood samples, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains, thus greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic ferrets are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically, the closest species to humans, thus disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns pertaining to experimentation on nhps along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure the fewest number of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, depending on the pathogen, the route of exposure and the dose should mimic the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on viruses for each family that are of the greatest concern for public health worldwide. norovirus, the genus of which norwalk is the prototypic member, is the most common cause of gastroenteritis in the united states (hall et al., 2013) . there are five distinct genogroups (gi-gv) and numerous strains of norwalk virus, including the particularly significant human pathogens gi.1 norwalk virus, gii.2 snow mountain virus, and gii.1 hawaii virus. in developing countries, norwalk virus, also known as "winter vomiting virus," is responsible for approximately 200,000 deaths annually (patel et al., 2008) . a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants (chen et al., 2009; lutgehetmann et al., 2012; turcios-ruiz et al., 2008) . symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation is normally 1-3 days, with symptoms persisting for 2-3 days (koopmans and duizer, 2004) . viral shedding can range from 6 to 55 days in healthy individuals (atmar et al., 2014) . however, longer illness duration can be indicative of immunocompromised status, with the elderly and young having a prolonged state of shedding (harris et al., 2008; rockx et al., 2002) . interestingly, individuals vary greatly in susceptibility to norovirus infection depending on their fucosyl transferase 2 (fut2) allele functionality and histoblood group antigen status, with type a and o individuals susceptible and types ab and b resistant (hutson et al., 2005) . transmission occurs predominately through the oralfecal route with contaminated food and water being a major vector (atmar and estes, 2001; becker et al., 2000; koopmans and duizer, 2004) . vomiting results in airborne dissemination of the virus with areas of 7.8 m 2 being contaminated and subsequent transmission from oral deposition of airborne particles or contact with contaminated fomites, which can remain contaminated for up to 42 days (makison booth, 2014; tung-thompson et al., 2015) . each vomiting event in a classroom setting elevates the risk of norovirus illness among elementary students with proximity correlating with attack rates (evans et al., 2002; marks et al., 2003) . viral titers in emesis and fecal suspensions are as high as 1.2 × 10 7 and 1.6 × 10 11 ges (genomic equivalent copies per milliliter), respectively and the 50% infectious dose is 1320 ges (atmar et al., 2014) . therefore, outbreaks can be extremely difficult to contain. therapeutic intervention consists of rehydration therapy and antiemetic medication (bucardo et al., 2008; moe et al., 2001) . no approved vaccine or therapeutic is available, and development has been challenging given that immunity is short-lived after infection, new strains rapidly evolve and the correlates of protection are not completely understood (chen et al., 2013) . however, one promising strategy utilized a virus-like particle (vlp)-based vaccine that protected or reduced infection by almost 50% in human volunteers (aliabadi et al., 2015; atmar et al., 2011) . given the relatively benign disease in adults, experimental challenge has been carried out on human volunteers (ball et al., 1999; tacket et al., 2000) . viral titers are determined by shedding in feces and sera with histopathology changes monitored by biopsies particularly of the duodenum. the ph of emesis samples collected containing virus is consistent with viral replication in the small intestine with reflux to the stomach (kirby et al., 2016) . additionally, norwalk virus has been shown to bind to duodenal tissue (chan et al., 2011) . however, this type of research is technically difficult and expensive, and thus other models have been developed. a major hindrance to basic research into this pathogen is the lack of permissive cell culture systems or animal models for norwalk virus. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus are monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity is measured exclusively by assay of viral shedding (rockx et al., 2005) . incidentally, more viruses were needed to create an infection when challenging by the oral route than by the intravenous (iv) route (purcell et al., 2002) . chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route (bok et al., 2011) . although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred and no histopathological changes were detected. the amount and duration of viral shedding was in-line with what is observed upon human infection. as such, chimeric chimpanzee-human antinorovirus neutralizing antibodies have been explored as a possible therapeutic strategy (chen et al., 2013) . a recently identified calicivirus of rhesus origin, named tulane virus, has been used as a surrogate model of infection. unlike norwalk virus, tulane virus can be cultured in cells. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was detected for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease (sestak et al., 2012) . a murine norovirus has been identified and is closely related to human norwalk virus (karst et al., 2003) . however, clinically the virus presents a different disease. the murine norovirus model does not include observable gastrointestinal clinical signs, possibly in part because rodents lack a vomiting reflex. additionally, mice infected with norovirus develop a persistent infection in contrast to human disease (hsu et al., 2006 (hsu et al., , 2007 khan et al., 2009) . porcine enteric caliciviruses can induce diarrheal disease in young pigs, and an asymptomatic infection in adults (wang et al., 2006 . gnotobiotic pigs can successfully be infected with a passaged clinical norovirus isolate by the oral route. diarrheal disease developed in 74% of the animals and virus was detected in the stool of 44% of the animals. no major histopathological changes or viral persistence was noted (cheetham et al., 2006) . calves are naturally infected with bovine noroviruses (scipioni et al., 2008) . experimental challenge of calves by oral inoculation with a bovine isolate resulted in diarrheal disease 14-16 h postinfection. recovery of virus was achieved after 53.5 and 67 h postinfection (otto et al., 2011) . eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), and venezuelan equine encephalitis virus (veev) present with near synonymous symptoms. the majorities of human cases are asymptomatic, but can present as a flu-like illness progressing to central nervous system (cns) involvement to include seizures and paralysis. mortality rates vary among the virus, with the highest reported for eev at 36%-75% followed by weev and lastly veev at less than 1% (ayers et al., 1994; griffin, 2007; steele and twenhafel, 2010) . there are currently no licensed vaccines or therapies but a recent phase 1 clinical trial of a veev dna vaccine resulted in veev-neutralizing antibody responses in 100% of the subjects (hannaman et al., 2016) . mouse models have been developed for numerous routes of infection including cutaneous, intranasal (in), intracranial (ic), and aerosol. eeev susceptibility in mouse models is correlated with age, with younger mice being more susceptible than adults. importantly, eeev pathogenesis is dependent on route of infection with delayed progression upon subcutaneous (subq) exposure (honnold et al., 2015) . newborn mice display neuronal damage with rapid disease progression, resulting in death (murphy and whitfield, 1970) . similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, while inoculation via the subq route causes a pantropic infection eventually resulting in encephalitis (liu et al., 1970; morgan, 1941) . a general drawback to the usage of the mouse model is the lack of vascular involvement during the disease course (liu et al., 1970) . after subq inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h (aguilar, 1970) . the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on day 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14 with brain and mesodermal tissues, such as heart, lungs, liver, and kidney involvement (aguilar, 1970; monath et al., 1978) . intracerebral and in routes of infection resulted in a fatal disease that was highly dependent on dose while intradermal (id) and subq inoculations caused only 50% fatality in mice regardless of the amount of virus (liu et al., 1970) . comparing susceptibility of inbred and outbred strains revealed that cd-1, balb/c, a/j, and c57bl6 mice were all highly susceptible to experimental infection via subq inoculation when challenged prior to 10 weeks old with cns involvement and lethality (blakely et al., 2015) . subq/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans (macdonald and johnston, 2000) . virus begins to replicate in the draining lymph nodes at 4 h postinoculation. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis (charles et al., 1995; steele et al., 1998) . aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death (charles et al., 1995; steele and twenhafel, 2010; steele et al., 1998) . in challenge of c3h/hen mice with high dose veev caused high morbidity and mortality (julander et al., 2008b) . viral titers in brain peaked on day 4 postchallenge and remained elevated until animals succumbed on day 9-10 postchallenge. protein cytokine array performed on brains of infected mice showed elevated il-1a, il-1b, il-6, il-12, mcp-1, ifnγ, mip-1a, and rantes levels. this model was used successfully to test antivirals against veev (julander et al., 2008a) . additionally, a veev vaccine inactivated with 1,5-iodonaphthyl azide v3526 protects against both footpad and aerosol challenge with virulent veev in a mouse model (gupta et al., 2016) . guinea pigs and hamsters have also been developed as animal models for eeev studies (paessler et al., 2004; roy et al., 2009) . guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed in brain lesions in the experimentally challenged animals (roy et al., 2009) . subq inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis, and death. in addition, parenchyma necrosis were observed in the liver and lymphoid organs (paessler et al., 2004) . harlan sprague-dawley hamsters develop viremia and progress to respiratory, gastrointestinal, and nervous system involvement when inoculated via subq route. vasculitis and encephalitis were both evident in this model, which mirrors the human disease clinical spectrum (paessler et al., 2004) . weev is highly infectious to guinea pigs and has been utilized for prophylactic screening (sidwell and smee, 2003) . studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases (zlotnik et al., 1972) . cns involvement was evident with intracerebral, intraperitoneal (ip), and id inoculations (zlotnik et al., 1972) . ip inoculation of weev is fatal in guinea pigs regardless of amount of virus inoculum, with the animals exhibiting signs of illness on day 3-4, followed by death on day 5-9 (nalca, unpublished results) . id, im, or iv inoculations of eeev in nhps cause disease, but does not reliably result in neurological symptoms (dupuy and reed, 2012) . intracerebral infection of eeev produces nervous system disease and fatality in monkeys (nathanson et al., 1969) . the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in nhps when they are inoculated by the peripheral route (wyckoff, 1939) . in and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but are less drastic than intracerebral injections (wyckoff, 1939) . the aerosol route of delivery will result in uniformly lethal disease in cynomolgus macaques (reed et al., 2007) . in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed and nhps became moribund and were euthanized between 5-9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals (steele and twenhafel, 2010) . similar clinical signs and pathology were observed when common marmosets were infected with eeev by the in route (adams et al., 2008) . both aerosol and in nhp models had similar disease progression and pathology as seen in human disease. very limited studies have been performed with nhps. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model could be useful for testing of vaccines and therapeutics against weev (reed et al., 2005) . veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted less than 12 h. secondary fever generally began on day 5 and lasted 3-4 days (gleiser et al., 1961) . veev-infected nhps exhibited mild symptoms, such as anorexia, irritability, diarrhea, and tremors. leukopenia was common in animals exhibiting fever (monath et al., 1974) . supporting the leukopenia, microscopic changes in lymphatic tissues, such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, and focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection (gleiser et al., 1961) . the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. similar to these observations, when cynomolgus macaques were exposed to aerosolized veev, fever, viremia, lymphopenia, and clinical signs of encephalitis were observed but the nhps did not succumb to disease (reed et al., 2004) . a common marmoset model was utilized for comparison studies of south america (sa) and north america (na) strains of eeev (adams et al., 2008) . previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected in with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained asymptomatic and survived until the end of study. chikungunya virus (chikv) is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics centered within africa and southeast asia (griffin, 2007) . the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes. given the widespread endemicity of aedes mosquitoes, chikv has the potential to spread to previously unaffected areas. this is typified by the emergence of disease reported for the first time in 2005 in the islands of south-west indian ocean, including the french la reunion island, and the appearance in central italy in 2007 (charrel et al., 2007; rezza et al., 2007) . the incubation period following a mosquito bite is 2-5 days, leading to a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported (powers and logue, 2007) . individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years old have an increased risk of developing neurological manifestations (arpino et al., 2009) . there is currently no approved vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikv infection by id inoculation. however, it was demonstrated that neonatal mice were susceptible and severity was dependent upon age at infection. six-dayold mice developed paralysis by day 6, and all succumbed by day 12, whereas 50% of 9-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. symptomatic mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications (couderc et al., 2008; ziegler et al., 2008) . an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied with foot swelling and noted inflammation of the musculoskeletal tissue morrison et al., 2011) . adult ifnα/βr knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts (couderc et al., 2008) . icr and cd-1 mice can also be utilized as a disease model. neonatal mice inoculated subq with a passaged clinical isolate of chikv developed lethargy, loss of balance, and difficulty walking. mortality was low, 17 and 8% for newborn cd-1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection (ziegler et al., 2008) . a drawback of both the ifnα/βr and cd-1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised (teo et al., 2012) . a chronic infection model was developed using recombinant activating gene 1 (rag1 −/− ) knockout mice. in this study, mice inoculated via the footpad lost weight in comparison to the control group. both footpad and subq injected mice developed viremia 5-6 days postinfection, which was detectable up to 28 days postinfection. inflammation was evident in the brain, liver, and lung of the subq inoculated animals at 28-56 days postinfection. despite minimal footpad swelling on day 2 postinfection, on day 14 there was severe muscle damage noted at necropsy, which resolved by day 28 (seymour et al., 2015) . golden hamsters serve as another option for small animal modeling. although hamsters do not appear to develop overt clinical symptoms following subq inoculation, viremia developed in the majority of animals within 1 day postinfection with clearance following from day 3 to 4. histologically, inflammation was noted at the skeletal muscle, fascia, and tendon sheaths of numerous limbs. this study was limited in the number of animals utilized, and more work is needed to further develop the hamster model (bosco-lauth et al., 2015) . nhp models of disease include adult, aged, and pregnant rhesus macaques in addition to cynomolgus macaques (broeckel et al., 2015) . differing routes of infection (subq, iv, and im) have been successfully administered, although there is not a clear understanding of the role that route of transmission plays in subsequent pathogenesis and clinical symptoms. typically, viremia is observed 4-5 days postinfection with a correlation between infectious titer and time to viremia observed in cynomolgus but not rhesus (labadie et al., 2010; messaoudi et al., 2013) . fever began at 1-2 days postinfection and persisted for 2-7 days and 3-7 days in cynomolgus and rhesus, respectively and coincided with rash (chen et al., 2010; labadie et al., 2010; messaoudi et al., 2013) . overall blood chemistries changed in conjunction with initiation of viremia, and returned to baseline 10-15 days postexposure (chen et al., 2010) . cns involvement has been difficult to reproduce in nhp models, although it was reported that high inoculum in cynomolgus did result in meningoencephalitis (labadie et al., 2010) . the nhp models have been utilized to conduct efficacy testing on novel vaccines and therapeutics (broeckel et al., 2015) . dengue virus (denv) is transmitted via the mosquito vectors a. aegypti and a. albopictus (moore and mitchell, 1997) . given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to denv. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia (gubler, 2002) . it is estimated that there are 20,000 deaths each year due to dengue hemorrhagic fever (dhf) (guzman and kouri, 2002) . there are four distinct serotypes of denv, numbered 1-4, which are capable of causing a wide clinical spectrum that ranges from asymptomatic to severe with the development of dhf (world health organization, 1997) . incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages following a mosquito bite (balsitis et al., 2009) . typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure (world health organization, 1997) . thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis (gregory et al., 2010) . severe disease has a higher propensity to occur upon secondary infection with a different denv serotype (thein et al., 1997) . this is hypothesized to occur due to antibody dependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in order to further progress toward an effective drug or vaccine, small human cohort studies have taken place. however, to provide statistically relevant results, testing must progress in an animal model. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is considered the optimal range during experimental challenge . denv does not naturally replicate effectively in rodent cells, creating the need for mouse-adapted strains, engineered mouse lines, and a variety of inoculation routes to overcome the initial barrier. several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis (huang et al., 2000; paes et al., 2005; shresta et al., 2004) . a higher dose of an adapted denv strain induced dhf symptoms in both balb/c and c57bl/6 souza et al., 2009) . this model can also yield asymptomatic infections. a mouse-adapted strain of denv 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans (shresta et al., 2006) . passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased and animals died due to vascular leak syndrome (balsitis et al., 2010) . another mouse-adapted strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability (souza et al., 2009) . ic injection of suckling mice with denv leads to death by paralysis and encephalitis, which is rare in human infection (lee et al., 2015; parida et al., 2002; zhao et al., 2014a) . immunocompromised mice have also been used to gain an understanding of the pathogenesis of denv. the most well-defined model is ag129 which is deficient in ifnα/β and γ receptors and can recapitulate dhf/dss if a mouse-adapted strain is utilized yauch et al., 2009) . scid mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (blaney et al., 2002; lin et al., 1998) . df symptoms developed after infection in nod/scid/il2rγko mice engrafted with cd34 + human progenitor cells (mota and rico-hesse, 2011) . rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and labadapted strain of denv 2 (kuruvilla et al., 2007) . a passaged clinical isolate of denv 3 was used to create a model in immunocompetent adult mice. ip injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, ast and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases (costa et al., 2012) . vascular leakage was also observed when c57bl/6 were inoculated with denv 2 (st john et al., 2013) . a murine model was developed that utilized infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of denv 2. infected mosquitoes then fed upon the mouse footpad to allow for transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the number of mosquitoes it was exposed to, with 4-5 being optimal. detectable viral rna was in line with historical human infection data. severe thrombocytopenia developed on day 14. this model is notable in that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus (cox et al., 2012) . nhp models have used a subq inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection (marchette et al., 1973) . the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes (marchette et al., 1980) . throughout these preliminary studies, no clinical disease was detected. in order to circumvent this, a higher dose of denv was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed (onlamoon et al., 2010) . a robust antibody response was observed in multiple studies (marchette et al., 1973; onlamoon et al., 2010) . marmosets also mirror human dengue infection, developing fever, leukopenia, and thrombocytopenia following subq inoculation (omatsu et al., 2011 (omatsu et al., , 2012 . nhps are able to produce antibodies similar to those observed during the course of human infection, making them advantageous in studying ade. sequential infection led to a cross-reactive antibody response which has been demonstrated in both humans and mice (midgley et al., 2011) . this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than previously reported, however, no clinical signs were apparent (goncalvez et al., 2007) . the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937 uganda in (smithburn et al., 1940 . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s (melnick et al., 1951; taylor et al., 1953) and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia (hayes, 1989) . the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area (centers for disease control and prevention, 1999; lanciotti et al., 1999) . since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days (monath and tsai, 2002) . the mortality rate following neuroinvasive disease ranges from 4% to 11% (asnis et al., 2000; hayes, 1989; hubalek and halouzka, 1999; komar, 2000) . the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e., asymptomatic, fever, encephalitis) (pogodina et al., 1983) . thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subq inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia (lieberman et al., 2009; pletnev et al., 2003) . this is mirrored in new zealand white rabbits in that they only develop fever and low levels of viremia following inoculation via footpad (suen et al., 2015) . id inoculation of both marmosets and rhesus macaques did not yield any clinical signs of disease including fever. viremia was detected in both nhp species, but marmosets developed a higher titer for a greater duration than rhesus (verstrepen et al., 2014) . wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and ip routes, resulting in encephalitis and 100% mortality. id route pathogenesis studies indicated that langerhans dendritic cells are the initial viral replication sites in the skin (brown et al., 2007; johnston et al., 1996) . the infected langerhans cells then migrate to lymph nodes and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases (byrne et al., 2001; cunha et al., 2000; diamond et al., 2003; fratkin et al., 2004) . the swiss mouse strain was inoculated ip in order to screen a variety of viral lineages to assess differences in pathogenesis (bingham et al., 2014) . tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared asymptomatic during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms between days 7 and 10 . many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the previously mentioned monkey experiments by pogodina et al. (1983) , persistent wnv infection was found in the brains of hamsters. zika virus recently came to the forefront of public health concerns with the outbreak in brazil at the end of 2015. the clinical disease spectrum is highly variable with reports of a flu-like illness accompanied by rash, guillan-barre syndrome, and microcephaly in newborns (ramos da silva and gao, 2016) . to date, a correlation between gestational age at which exposure to the virus occurs and severity of microcephaly is not fully understood (brasil et al., 2016) . however, a recent study of pregnant women in columbia found that infection with zika virus during the third trimester was not associated with any obvious structural abnormalities of the fetus (pacheco et al., 2016) . transmission of the virus occurs via the bite from an infected a. aegypti or a. albopictus (ramos da silva and gao, 2016) . other reported routes of exposure include sexual transmission and blood transfusion (cunha et al., 2016; d'ortenzio et al., 2016; hills et al., 2016; mccarthy, 2016) . the emergence of this virus with no approved vaccine or therapy, and few diagnostic options demonstrates the utility of well-characterized animal model development. it was first demonstrated in 1956 that experimentally infected mosquitoes could be used to transmit the virus to mice and nhps (boorman and porterfield, 1956) . a129 mice were susceptible to nonadapted zika virus infection following subq inoculation of the limbs. mice began to lose weight 3 days postinfection and met euthanasia criteria by day 6. microscopic lesions within the brain were noted upon necropsy. in conjunction, viral rna was detected in the blood, brain, ovary, spleen, and liver of the infected mice. wild-type 129sv/ev mice were also challenged with no observable clinical disease. however, viral rna was detected at day 3 postinfection in the blood, ovary and spleen, and then remained at detectable levels in the ovaries and spleen on day 7 (dowall et al., 2016) . footpad inoculation of the virus leads to a fatal disease in ag129 mice by day 7 postinoculation with significant histopathological changes in the brain noted at necropsy (aliota et al., 2016) . ag129 mice were also observed to develop neurologic disease by day 6 postexposure (rossi et al., 2016) . immunocompetent mice are resistant to infection via the subq route (rossi et al., 2016) . recently, a mouse model was identified to verify vertical transmission of the virus. pregnant c57 mice were injected either ip or in utero into the lateral ventricle of the fetal brain. ip inoculation induced transient viremia in the pregnant mice on day 1. viral rna was detected in five out of nine placentas on day 3 postinfection. the virus was able to infect the radial glia cells in the fetal brain and leads to a reduction in the cortical neural progenitors . viral exposure via cerebroventricular space/ lateral ventricle of the fetal brain exhibited small brain size at day 5 postexposure in addition to cortical thinning (cugola et al., 2016; li et al., 2016a) . ifnar1 −/− pregnant mice exposed to the virus had nonviable fetuses. in the same study, wild-type mice were given an anti-ifnar antibody prior to and during infection resulting in detectable virus in the fetal head with mild intrauterine growth restriction (miner et al., 2016) . all of these murine studies will further study of the pathogenesis of vertical transmission and the resulting neurological disorders in conjunction with screening novel countermeasures. nhp studies are currently ongoing for animal model development. numerous viruses from the coronavirus (cov) family exist that infect a wide range of animals. six species have been identified that can infect humans. two of these are alpha coronavirues: hcov-229e and hcov-nl63. four are beta coronavirueses: hcov-oc43, hcov-hku1, hcov-sars, and mers-cov. hcov-229e and hcov-oc43 were first detected in the 1960s from the nasal passages of humans with the "common cold" (gaunt et al., 2010) . hcov-nl63, which was first isolated in 2004, causes upper and lower respiratory infections of varying intensity and has been continuously circulating among humans (van der hoek et al., 2006) . hcov-hku1, first isolated in 2002, has been identified more sporadically but also causes respiratory infections (lau et al., 2006) . a significant portion of common cold infections in humans are caused by coronaviruses. in 2002 and 2012, two human coronaviruses, sars-cov and mers-cov, emerged that caused a great deal of alarm since these infections have resulted in nearly 10 and 40% fatality, respectively (assiri et al., 2013; peiris et al., 2004) . the etiologic agent of severe acute respiratory syndrome (sars), sars-cov, emerged in 2002 as it spread throughout 32 countries in a period of 6 months, with 8437 confirmed infections and 813 deaths (roberts and subbarao, 2006; world health organization, 2003) . no additional cases of community acquired sars-cov infection have been reported since 2004. the natural reservoir of sars-cov is the horseshoe bat and the palm civet is an intermediate host (lau et al., 2005) . the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible (pearson et al., 2003; rabenau et al., 2005; rota et al., 2003) . sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 (peiris et al., 2003; wang et al., 2004) . after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough (drosten et al., 2003) . in some sars cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies (tan et al., 2004) . in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses (chen and subbarao, 2007; perlman and dandekar, 2005) . infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection (perlman and dandekar, 2005) . virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues (cheng et al., 2004) . sars-cov can replicate in many species, including: dogs, cats, pigs, mice, rats, ferrets, foxes, and monkeys (roper and rehm, 2009) . no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (∼10%), viral replication, and pathology (roberts et al., 2008) . in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models utilize mice, hamsters, ferrets, and nhps. mouse models of sars-cov typically are inoculated by the in route under light anesthesia (roberts et al., 2005) . young, 6-to 8-week-old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinate, with a peak on day 2 and clearance by day 5 postexposure (mcauliffe et al., 2004) . there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis) . in sars-cov infection of c57bl/6 (b6), also yields reduced weight gain and viral k. viral disease replication in the lungs, with a peak on day 3 and clearance by day 9 (glass et al., 2004) . in contrast, balb/c mice 13-14 months of age show weight loss, hunched posture, dehydration, and ruffled fur on day 3-6 postexposure (bisht et al., 2004) . interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-cov infection similar to those observed for balb/c mice but have lower titers and less prolonged disease. while the aged mouse model is more frequently used then young mice, it is more difficult to obtain large numbers of mice older than 1 year (table 33 .1). a number of immunocompromised knockout mouse models of in sars-cov infection have also been developed. 129svev mice infected with sars-cov by the in route develop bronchiolitis, with peribronchiolar inflammatory infiltrates and interstitial inflammation in adjacent alveolar septae . viral replication and disease in these mice resolves by day 14 postexposure. beige, cd1 −/− , and rag1 −/− mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs (glass et al., 2004) . stat1 ko mice infected in with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths . the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. angiotensin converting enzyme 2 (ace2) and cd209l were identified as cellular receptors for sars-cov, with affinity for the spike (s) protein of the virus (jeffers et al., 2004) . the variations in the ace2 sequence across animal species could partially explain the differences in infection severity (li et al., 2016b; sutton and subbarao, 2015) . since mice in particular have a greater number of sequence differences in ace2, transgenic mice were created that express human ace2 (mccray et al., 2007; netland et al., 2008; yang et al., 2007) . unlike other murine models of sars-cov, mice expressing hace2 had up to 100% mortality, with severity correlating to the level of hace2 expression (tseng et al., 2007) . with high levels of hace2 expression, mice developed a severe lung and brain infection. however, cns k. viral disease infection is only rarely observed in humans infected with sars-cov. syrian golden hamsters (strain lvg) are also susceptible to in exposure of sars-cov. after the administration of 10 3 tcid 50, along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis (roberts et al., 2005) . there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. limited mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred (roberts et al., 2008) . damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal (it) inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge (skowronski et al., 2005) . clinical disease has been observed in several studies excluding one, perhaps due to characteristics of the inoculating virus (kobinger et al., 2007) . sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5%-10% of the lungs, occasionally accompanied by severe pathology within the lungs (martina et al., 2003; ter meulen et al., 2004) . with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or it routes generally results in a very mild infection that resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness but others have rash, lethargy, and respiratory signs and pathology martina et al., 2003; mcauliffe et al., 2004; rowe et al., 2004) . rhesus have little to no disease and only have mild findings upon histopathological analysis (rowe et al., 2004) . agms infected with sars-cov have no overt clinical signs but dad and pneumonitis has been documented (mcauliffe et al., 2004) . viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves, and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were involved in the sars-cov outbreak. it and in inoculation of civets with sars-cov results in lethargy, decreased aggressiveness, fever, diarrhea, and conjunctivitis . leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. squirrel monkeys, mustached tamarinds, and common marmosets have not been susceptible to sars-cov infection (greenough et al., 2005; roberts et al., 2008) . vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine, and have included live-attenuated, killed, dna and viral-vectored vaccine strategies (cavanagh, 2003) . an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease (perlman and dandekar, 2005; weiss and scott, 1981) . as such, immune mice had th2type immunopathology upon sars-cov challenge (tseng et al., 2012) . severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported (weingartl et al., 2004) . additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance (clay et al., 2012) . mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. mouse-adapted sars with uniform lethality was developed from 15 serial passages in the lungs of young balb/c mice (mccray et al., 2007; roberts et al., 2007; rockx et al., 2007) . middle east respiratory syndrome (mers-cov) emerged in saudi arabia and is associated with fever, severe lower respiratory tract infection, and oftentimes renal failure (al-tawfiq et al., 2016; omrani et al., 2015) . mers patients can also occasionally manifest with neurological symptoms. mers-cov infection has a high fatality rate. infections in humans can also be asymptomatic. as of october 2015, there were 1589 confirmed cases and 567 deaths (li et al., 2016b) . bats serve as the likely natural reservoir since virus with 100% nucleotide identity to the index case was isolated from egyptian tomb bats (memish et al., 2013) . spread to humans likely comes from infected dromedary camels (adney et al., 2014; azhar et al., 2014) . the host range for mers-cov is dependent on the binding of the viral s protein to the host receptor, which is human dipeptidyl peptidase four (hdpp4), also known as cd26 (raj et al., 2013) . the expression and distribution of dpp4 in the human respiratory tract has recently been well characterized (meyerholz et al., 2016) . interestingly, dpp4 expression is preferentially localized to alveolar regions, perhaps explaining why mers predominantly manifests as an infection of the lower respiratory tract. humans with preexisting pulmonary disease have increased dpp4 expression in alveolar epithelia. small animals typically used for viral disease research, such as mice, hamsters, guinea pigs, and ferrets are naturally nonpermissive to mers-cov infection due to a low binding efficiency of the viral s protein to the host dpp4 (sutton and subbarao, 2015). in contrast the rhesus macaque and common marmoset have complete homology to human dpp4, allowing productive mers-cov infection to occur falzarano et al., 2014; munster et al., 2013; yao et al., 2014) . new zealand white rabbits can be infected with mers-cov, and virus was isolated from the upper respiratory tract, but there were no clinical symptoms or significant histopathological changes (haagmans et al., 2015) . due to the lack of strong binding affinity of the mers-cov s protein to the murine dpp4 receptor, wildtype mice are not susceptible to mers-cov infection. as such, several approaches have been used to create susceptible murine animal models of mers-cov infection by inducing the expression of hdpp4. one approach utilized an adenovirus vector expressing hdpp4 to transduce mice (zhao et al., 2014b) . these mice developed pneumonia but survived mers-cov infection. in mers-cov infection of mice with global expression of hdpp4 resulted in id 50 and ld 50 values of <1 and 10 tcid 50 , respectively (tao et al., 2016) . thus, mers-cov infection of these transgenic mice can be either sublethal or uniformly lethal depending on the dose. inflammatory infiltrates were found in the lungs and brain stems of mice with some focal infiltrates in the liver as well. another strategy uses transgenic mice expressing hdpp4 under either a surfactant protein c or cytokeratin 10 promoter (li et al., 2016b) . in mers-cov infection in these mice resulted in a uniformly lethal disease characterized by alveolar edema and microvascular thrombosis and mononuclear clear cell infiltration in the lungs. the brain stem was also impacted by the infection. dpp4 expression with an ubiquitously expressing promoter from cytomegalovirus also had a uniformly lethal infection with predominant lung and brain involvement, but numerous other tissues were also impacted and contained virus (agrawal et al., 2015) . common marmosets infected with 5.2 × 10 6 tcid 50 (emc-2012) mers-cov by the combined in, oral, ocular, and it routes capitulate the severe disease in human infections (falzarano et al., 2014) . the animals manifested moderate to severe clinical disease, with interstitial infiltration of both lower lung lobes. two of nine animals became moribund between days 4 and 6. viral rna was detected in nasal and throat swabs, various organs, and in the blood of some animals, indicating a systemic infection. histologically, animals showed evidence of acute bronchial interstitial pneumonia as well as other pathological defects. infection of rhesus macaques with mers-cov results in a mild clinical disease characterized by a transient lung infection with pneumonia. rhesus macaques were inoculated with at least 10 7 tcid 50 (emc-2012) mers-cov either by the it route or a combined in, it, oral, and ocular inoculation . the result was a mild respiratory illness including nasal swelling and a short fever with all animals surviving. viral rna was recovered from nasal swab samples and replicating virus was found in lung tissue . mild pathological lesions were found only in the lungs. radiographic imaging of the lungs revealed interstitial infiltrates, which are signs of pneumonia (yao et al., 2014) . interestingly, mer-cov infection is more severe in marmosets compared to rhesus macaques (falzarano et al., 2014) . this is despite the finding that both species have complete homology with humans within the dpp4 domain that interacts with the viral s protein. other host factors influencing disease severity have not yet been identified. transgenic mouse models expressing hdpp4 are ideal for initial development and screening of mers-cov countermeasures, and marmosets can be used for final selection and characterization. filoviridae consists of three genera, ebolavirus and marburgvirus, and a newly discovered group, cuevavirus (kuhn, 2008) . it is thought that various species of bats are the natural host reservoir for these viruses that have lethality rates from 40% to 82% in humans. there is evidence that the egyptian rousette bat (rousettus aegyptiacus) is the natural reservoir for marburgviruses but may not be for ebolaviruses (jones et al., 2015) . marburg virus (marv) first emerged in 1967 in germany when laboratory workers contracted the virus from agms (chlorocebus aerthiops) that were shipped from uganda. ebolaviruses sudan and zaire (sudv and ebov) caused nearly simultaneous outbreaks in 1976 in what is now the democratic republic of congo (drc). the most recent outbreak of ebov in west africa was by far the largest with over 28,000 suspected, probable and confirmed cases and over 11,000 deaths. bundibugyo virus (bdbv) first emerged in 2007 in bundibugyo, uganda with 56 confirmed cases . two other ebolaviruses are known: taï forest (tafv) (previously named cote d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. filovirus disease in humans is a characterized by aberrant innate immunity and a number of clinical symptoms: fever, nausea, vomiting, k. viral disease arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, and anorexia as well as numerous others (mehedi et al., 2011; wauquier et al., 2010) . approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.) (table 33 .2). natural transmission in an epidemic is through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, im, ip, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures . since filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. one issue to take note of in animal model development of filovirus infection is the impact of particle to plaque-forming unit (pfu) ratios on lethality, wherein it is possible that increasing the dose could actually decrease infectivity due to an immunogenic effect produced by inactive virions in the stock. additionally, the plaque assay used to measure live virions in a stock may greatly underestimate the true quantity of infectious virions in a preparation (alfson et al., 2015; smither et al., 2013a) . immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 interferon response (bray, 2001) . however, wild-type inbred mice are susceptible to filovirus that has been mouse adapted (ma) by serial passage in mice (bray et al., 1999) . marv angola was particularly resistant to adaptation, but after 24 serial passages in scid mice, infection caused severe disease in balb/c and c57bl/6 mice when administered in or ip (qiu et al., 2014) . these mice had pathology with some similarities to infection in humans including lymphopenia, thrombocytopenia, liver damage, and viremia. balb/c mice, which are the strain of choice for ip inoculation of ma-ebov, are not susceptible by the aerosol route (bray et al., 1999; zumbrun et al., 2012a) . for aerosol infection of immunocompetent mice, a panel of bxd (balb/c x dba) recombinant inbred strains were screened and one strain, bxd34, was particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (approximately 100 or 1000 pfu) ( zumbrun et al., 2012a) . these mice developed weight loss of greater than 15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model utilizes a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 µm and a geometric standard deviation (gsd) of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains, such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by ip or aerosol routes (bray, 2001; lever et al., 2012; zumbrun et al., 2012a) . mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection (bray et al., 1999) . liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps (zumbrun et al., 2012a) . while most mouse studies have used ma-ebov or ebov, an ip mouse-adapted marv model is also available (warfield et al., 2007 (warfield et al., , 2009 ). ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds (panchal et al., 2012) . recently, a model was created using immunodeficient nsg [nonobese diabetic (nod)/scid/il-2 receptor chain knockout] mice with transplanted human hematopoietic stem cells from umbilical cord blood. these mice were susceptible to lethal wt (nonadapted) ebov by ip and in exposure (ludtke et al., 2015) . the transplanted mice had all of the cellular components of a fully functional adaptive human immune system and upon ebov (brannan et al., 2015; lever et al., 2012) . interestingly, inoculation of infa/br −/− mice with tafv and restv does not result in clinical signs. yet another strategy uses knockout mice lacking possible receptors for filovirus entry, such as niemann-pick c1 and c2 (npc1 and npc2). npc2 (−/−) mice were fully susceptible to infection with ebov but npc1 (−/−) mice were completely resistant (herbert et al., 2015) . hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models (gowen and holbrook, 2008; herbert et al., 2015) . an ip ma-ebov infection model has been developed in syrian hamsters gowen and holbrook, 2008; herbert et al., 2015; tsuda et al., 2011) . this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, utilizes male 5-to 6-week-old syrian hamsters which are infected with 100 ld 50 of ma-ebov. virus is present in tissues and blood collected on day 4 and all animals succumbed to the disease by day 6. infected hamsters had severe coagulopathy and uncontrolled host immune responses, similar to what is observed in primates. (ebihara et al., 2010) guinea pig models of filovirus infection have been developed for ip and aerosol routes using guinea pigadapted ebov (gp-ebov) and marv (gp-marv) (choi et al., 2012; connolly et al., 1999; twenhafel et al., 2015; zumbrun et al., 2012c) . guinea pig models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. a comparison of ip infection of outbred guinea pigs with guinea pig-adapted marv angola and marv ravn revealed similar pathogenesis (cross et al., 2015) . infection with either strain resulted in features of the disease that are similar to what is seen in human and nhp infection, such as viremia, fever, coagulopathy, lymphopenia, elevated liver enzymes (alt and ast), thrombocytopenia, and splenic, gastrointestinal and hepatic lesions. gp-marv-ravn had a delayed disease progression relative to gp-marv-ang. hartley guinea pigs exposed to aerosolized gp-ebov develop lethal interstitial pneumonia. this is in contrast to subq infection of guinea pigs, aerosol ebov challenge of nhps, and natural human infection (twenhafel et al., 2015) . both subq and aerosol exposure of guinea pigs to gp-ebov resulted in only mild lesions in the liver and spleen. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (less than 1000 pfu) presented doses of airborne gp-marv and more protracted disease is seen in some animals (our unpublished observations) (zumbrun et al., 2012c) . weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e., alt, ast, alkp, and bun) indicating problems with liver and kidney function are also altered late in the disease course. transmission of ebov has been documented from swine to nhps via the respiratory tract (kobinger et al., 2011) . as such, guinea pigs have been used to establish transmission models (wong et al., 2015a,b) . nonexposed guinea pigs were placed in the cages with infected guinea pigs 1 day postexposure to gp-ebov. guinea pigs challenged intanasally were more likely to transmit virus to naive cagemates than those that were exposed by the ip route. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans (dye et al., 2012) . old world primates have been primarily used for development of ip, im, and aerosol models of filovirus infection ( twenhafel et al., 2013) . uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, agms (alves et al., 2010; carrion et al., 2011; davis et al., 1997; hensley et al., 2011a; reed et al., 2007; zumbrun et al., 2012b) . low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. ebov-makona, the strain responsible for the recent large outbreak in west africa, was compared to the "prototype" 1976 ebov strain (marzi et al., 2015) . the disease in cynos was similar for both viruses, but disease progression was delayed for ebov-makona. this delay as well as the lower fatality rate in the 2014 epidemic compared to the 1976 outbreak suggest that ebov-makona is less virulent. the large number of cases in the 2014-15 ebov outbreak brought to light previously underappreciated eye pathology and ocular viral persistence in survivors. while survivors of nhp filovirus infection are infrequent, necrotizing scleritis, conjunctivitis, and other ocular pathology has been observed in ebov-infected animals (alves et al., 2016) . prominent features of the filovirus infections in nhps are onset of fever by day 5 postexposure, viremia, lymphopenia, tachycardia, azotemia, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. petechial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species (zumbrun et al., 2012b) . immunological parameters have been evaluated and t, b, and natural killer cells are greatly diminished as the infection progresses (fernando et al., 2015) . a cytokine storm occurs with rises in ifnγ, tnf, il-6, and ccl2 (fernando et al., 2015) . however, there is also evidence from transcriptional profiling of circulating immune cells that the early immune response is skewed toward a th2 response (connor et al., 2015) . strikingly, animals surviving challenge may have a delay in the production of inflammatory cytokines and chemokines (martins et al., 2015) . clinical disease parameters may have a slightly delayed onset in aerosol models. dyspnea late in infection is a prominent feature of disease after aerosol exposure (zumbrun et al., 2012b) . aerosol filovirus infection of nhps results in early infection of respiratory lymphoid tissues, dendritic cells, alveolar macrophages, blood monocytes, and fibroblastic reticular cells followed by spread to regional lymph nodes then multiple organs (ewers et al., 2016; twenhafel et al., 2013) . a number of pronounced pathology findings include multifocal hepatic necrosis and fibrin accumulation, particularly within the liver and the spleen. for aerosolized marv infection of rhesus, the most significant pathology included destruction of the tracheobronchial and mediastinal lymph nodes (ewers et al., 2016) . lymphocytolysis and lymphoid depletion are also observed (alves et al., 2010) . multilead, surgically implanted telemetry devices are useful in continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection (zumbrun et al., 2012b) . the most recently developed telemetry devices can also aid in plethysmography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. standardized filovirus-infected nhp euthanasia criteria have also been developed to enhance reproducibility for studies that evaluate therapeutic and vaccine countermeasures (warren et al., 2014) . filovirus infection of common marmosets (callithrix jacchus) is also a viable model to study the disease course. respiratory infection of marmosets with marv results in a lethal infection with fever, hemorrhaging, transient rash, disseminated viral infection, increases in liver function enzymes, coagulopathy, hepatitis, and histological lesions particularly in the kidney and liver (smither et al., 2013b) . marmosets are similarly susceptible to infection with ebovkikwit (smither et al., 2015) . thus, ebov or marv infection of marmosets produces features of the disease that are very similar to that of other nhps and humans. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate (rockx et al., 2012) . outbreaks due to nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra virus outbreaks have yet to be reported outside of australia (luby et al., 2009a,b) . hendra was the first member of the genus identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from bat to human. nipah has the distinction of transmission among, although the exact route is unknown (homaira et al., 2010) . the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized as needed for successful transmission (fogarty et al., 2008) . both viruses have a tropism for the neurological and respiratory tracts. the incubation period for hendra virus is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting (hanna et al., 2006) . disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure (playford et al., 2010) . nipah virus has an incubation period of 4 days to 2 weeks (goh et al., 2000) . much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h (lo and rota, 2008). survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions (tan and chua, 2008) . at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because documentation of human cases is at terminal stages. the best small animal model is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died within 4-17 days postinfection. the progression of disease and timeline is highly dependent on dose and route of infection. in inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas ip resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, with the brain having the highest titer. this model is used for vaccination and passive protection studies (guillaume et al., 2009; rockx et al., 2011; wong et al., 2003) . the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge (torres-velez et al., 2008; williamson et al., 2001) . inoculation with hendra virus via the subq route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inoculum has been associated with development of encephalitis and cns lesions. id and in injection does not lead to disease, although the animals are able to seroconvert upon challenge. the inoculum source does not affect clinical progression. nipah virus challenge only causes disease upon ip injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and was present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder (hooper et al., 1997) . ferrets infected with hendra or nipah virus display the same clinical disease as seen in the hamster model and human cases (bossart et al., 2009; pallister et al., 2011) . upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferret's brains, but to a lesser degree than seen in humans. cats have also been utilized as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms (mungall et al., 2007; johnston et al., 2015; westbury et al., 1996) . this model has proven useful for vaccine efficacy studies. squirrel and agms are representative of the nhp models. for squirrel monkeys, nipah virus is introduced by either the in or iv route and subsequently leads to clinical signs similar to humans, although in challenge results in milder disease. upon challenge, only 50% of animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge (marianneau et al., 2010) . agms are very consistent model of both viruses. it inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah viruses, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis rockx et al., 2010) . the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue, and nipah virus can be located in urine . pigs develop a respiratory disease upon infection with both nipah and hendra viruses (berhane et al., 2008; li et al., 2010; middleton et al., 2002) . oral inoculation does not produce a clinical disease, but subq injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah virus can also be transmitted between pigs. nipah virus was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms (weingartl et al., 2005) . within the pig model, it appeared that nipah virus had a greater tropism for the respiratory tract, while hendra for the neurological system. horses are also able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra viruses. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days (marsh et al., 2011; williamson et al., 1998) . animals died within 4 days postexposure and have interstitial pneumonia with necrosis of alveoli (murray et al., 1995a,b) . virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but are nonpermissive to infection (westbury et al., 1995; wong et al., 2003) . suckling balb/c mice succumb to infection if the virus is inoculated intracranially (mungall et al., 2006) . in exposure with nipah does not induce a clinical disease; however, there is evidence of a subclinical infection in the lungs following euthanasia of the mice (dups et al., 2014) . in addition, a human lung xenograph model in nsg mice demonstrated that the human lung is highly susceptible to nipah viral replication and damage (valbuena et al., 2014) . embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection (tanimura et al., 2006) . annually, respiratory syncytial virus (rsv) is responsible for the lower respiratory tract infections of 33 million children under the age of 5, which in turn results in 3 million hospitalizations and approximately 200,000 deaths (nair et al., 2010) . within the united states, hospital costs alone amount to over 600 million dollars per year (paramore et al., 2004) . outbreaks are common in the winter (yusuf et al., 2007) . the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and spreads to the lower respiratory tract. incubation for the virus is 2-8 days. rsv is highly virulent leading to very few asymptomatic infections (collins and graham, 2008) . disease manifestations are highly dependent upon the age of the individual. rsv infections in neonates produce nonspecific symptoms including overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting rsv at this age results in an increased chance of developing childhood asthma (wu et al., 2008) . young children develop recurrent wheezing while adults have exacerbation of previously existing respiratory conditions (falsey et al., 2005) . common clinical symptoms are runny nose, sneezing, and coughing accompanied by fever. mortality rates from rsv in hospitalized children are 1%-3% with the greatest burden of disease seen in 3-4 month olds (ruuskanen and ogra, 1993). hematopoietic stem cell transplant patients, solid organ transplant patients, and copd patients are particularly vulnerable to rsv infection and have mortality rates between 7.3% and 13.3% upon infection (anderson et al., 2016) . although there are almost 60 rsv vaccine candidates which are in preclinical and clinical phases, there is no licensed vaccine available and ribavirin usage is not recommended for routine treatment (american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis, 2006; higgins et al., 2016; kim and chang, 2016) . animal models of rsv were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated rsv (fi-rsv) vaccine. this vaccine induced severe respiratory illness in infants whom received the vaccine and were subsequently infected with live virus (kim et al., 1969) . mice can be used to model rsv infection, although a very high in inoculation is needed to achieve clinical symptoms (jafri et al., 2004; stark et al., 2002) . strain choice is crucial to reproducing a physiological relevant response (stokes et al., 2011). age does not affect primary disease manifestations (graham et al., 1988) . however, it does play a role in later sequelae showing increased airway hyperreactivity . primary rsv infection produces increased breathing with airway obstruction (jafri et al., 2004; van schaik et al., 1998) . virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. a humanized mouse model was recently developed by in inoculation. the challenged mice experienced weight loss and demonstrated a humoral and cellular immune response to the infection (sharma et al., 2016) . cotton rats are useful given that rsv is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after in inoculation (boukhvalova et al., 2009; niewiesk and prince, 2002) . viral replication is 50-to 1000-fold greater in the cotton rat model than mouse model (wyde et al., 1993) . the cotton rats develop mild to moderate bronchiolitis or pneumonia (grieves et al., 2015; prince et al., 1999) . although age does not appear to factor into clinical outcome, it has been reported that older cotton rats tend to take longer to achieve viral clearance. viral loads peak by the 5th day, dropping to below the levels of detection by day 8. the histopathology of the lungs appears similar to that of humans after infection (piazza et al., 1993) . this model has limited use in modeling the human immune response to infection as challenge with the virus induces a th2 response in cotton rats, whereas humans tend to have a response skewed toward th1 (culley et al., 2002; dakhama et al., 2005; ripple et al., 2010) . fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally with rsv via in inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is therefore useful in studying mucosal immunity during infection (gitiban et al., 2005) . ferrets infected by it were found to have detectable rsv in throat swabs up to day 7 postinfection, and positive qpcr up to day 10. immunocompromised ferrets were observed to have higher viral loads accompanied with detectable viral replication in the upper respiratory tract (stittelaar et al., 2016) . chimpanzees are permissive to replication and clinical symptoms of rsv including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms or high levels of viral replication (belshe et al., 1977) . bonnet monkeys were developed an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells (simoes et al., 1999) . the chimpanzee model has been proven useful for vaccine studies (hancock et al., 2000; teng et al., 2000) . sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine rsv (meyerholz et al., 2004) . lambs are also susceptible to human respiratory syncytial infection (olivier et al., 2009; sow et al., 2011) . when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. rsv replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features with humans (plopper et al., 1983; scheerlinck et al., 2008) . the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days (taubenberger and morens, 2008) . the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza (dushoff et al., 2006) . thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing (braun et al., 2007b) . influenza virus replicates in the upper and lower airways, peaking at approximately 48-h postexposure. infection can be more severe in infants and children under the age of 22, people over the age of 65, or immunocompromised individuals where viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis (barnard, 2009; glezen, 1982) . pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza virus itself, accounting for ∼27% of all influenza associated fatalities (alonso et al., 2003; ison and lee, 2010; speshock et al., 2007) . death, often due to ards can occur as early as 2 days after onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation (taubenberger and morens, 2008) . the h5n1 avian strain of influenza, has lethality rates of around ∼50% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus the potential for global spread is a major concern (matrosovich et al., 2004; wang et al., 2016) . h7n9 is another avian influenza a strain that infected more than 130 people and was implicated in 37 deaths. approximately 75% of infected people had a known exposure to birds. there is no evidence of sustained spread between humans but these viruses are of great concern for their pandemic potential (zhang et al., 2013) . the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. (2012) . swine are not frequently utilized but are also a potentially useful model for influenza research since they share many similarities to human anatomy, genetics, susceptibility, and pathogenesis (rajao and vincent, 2015). lethality rates can vary with virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, super infection, and sepsis, severe with ards, and neurologic manifestations (barnard, 2009) . also, models can utilize seasonal or avian strains and have been developed to study transmission, important for understanding the potential for more lethal strains, such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines (gilbert and mcleay, 2008; hagenaars et al., 2008; ortigoza et al., 2012) . inoculation is by the in route, utilizing approximately 60 µl of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with a mmad of <5 µl. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. most inbred laboratory mice lack the mxa gene which is an important part of human innate immune response to influenza infection. the mouse homolog to mxa, mx1 is defective in most inbred mouse strains (staeheli and haller, 1987) . mice with the knocked-in mx1 gene have a 1000-fold higher ld-50 for an influenza a strain (pr8) than wildtype background c57bl/6 mice (grimm et al., 2007) . weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs in influenza infected mice. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature (sidwell and smee, 2000). pulse oximeter readings and measurement of blood gases for oxygen saturation are also used to determine the impact of influenza infection on respiratory function (sidwell et al., 1992) . virus can be isolated from bronchial lavage (bal) fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild to moderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight also frequently occur. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation (o'donnell and subbarao, 2011). to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. h5n1 and the 1918 pandemic influenza virus can cause lethal infection in mice without adaptation (gao et al., 1999; taubenberger, 2006) . h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia ( dybing et al., 2000; gubareva et al., 1998; lu et al., 1999) . as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza virus produce severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia (barnard et al., 2007) . reassortment influenza viruses of the 2009 h1n1 virus and a low-pathogenicity avian h7n3 virus can also induce disease in mice without adaptation . in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by in inoculation of a sublethal dose of a bacterial strain, such as s. pneumoniae or s. pyogenes (chaussee et al., 2011) . morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cagemates can be achieved after infection by the aerosol route and cocaging after 24 h (schulman, 1968). rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes (daniels et al., 2003) . additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets (lowen et al., 2006) . cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies (eichelberger et al., 2004) . cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted (eichelberger et al., 2006; ottolini et al., 2005) . nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats (braun et al., 2007a) . coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans (lambkin et al., 2004; maines et al., 2012; mclaren and butchko, 1978) . like humans, ferrets exclusively express neu5ac, which acts as a receptor for influenza a virus, a feature likely contributing to the susceptibility of ferrets to human-adapted influenza a virus strains (ng et al., 2014) . the glycomic characterization of ferret respiratory tract tissues demonstrated some similarities and some differences to humans in terms of the potential glycan binding sites for the influenza virus (jia et al., 2014) . ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans (van riel et al., 2007) . influenza was first isolated from ferrets infected in with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments (huber et al., 2008; lambkin et al., 2004; maines et al., 2012) . when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by in inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id 50 dropwise to each nostril. however, a larger inoculum volume of 1.0 ml has also been explored as being more appropriate, yielding more severe and consistent respiratory tract pathology, likely because the larger inoculum is more widely distributed in the lower respiratory tract (moore et al., 2014) . video tracking to assign values to activity levels in ferrets can aid ferret studies, eliminating the need for collection of subjective and arbitrary clinical scores (oh et al., 2015) . viral replication in the upper respiratory tract is typically measured by nasal washes, but virus can also be measured in bronchoalveolar lavage fluid using a noninvasive technique (lee et al., 2014) . influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mild to moderately virulent strains (maher and destefano, 2004) . ferrets are more susceptible to influenza a than influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation (zitzow et al., 2002) . however, the localized immune responses within the respiratory tract of ferrets infected with influenza a and b have been characterized and are similar (carolan et al., 2015) . virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirrors that seen in humans (stark et al., 2013) . nonadapted h1n1, h2n2, and h3n2 have mild to moderate virulence in ferrets. the sequencing of the ferret genome has allowed for the characterization of the ferret host response using rnaseq analysis . distinct signatures were obtained depending on the particular influenza strain to inoculate the ferrets. also helpful is the sequencing and characterization of the influenza ferret infectome during different stages of the infection in naïve or immune ferrets (leon et al., 2013) . since influenza infection is particularly devastating to the elderly population, an aged ferret model of h1n1 influenza infection was developed (paquette et al., 2014) . features associated with increased clinical disease are weakened hemagglutinin antibody generation and attenuated th1 responses. pregnant and breastfeeding women and infants are also susceptible to more severe illness from influenza virus. to study this dynamic, a breastfeeding mother-infant ferret influenza infection model was created (paquette et al., 2015) . notably, the mammary gland itself harbored virus and transcript analysis showed downregulation of milk production genes. in support of the development of therapies, the ferret influenza model for pharmacokinetic/pharmacodynamics studies of antiviral drugs as also been developed (reddy et al., 2015) . critical to this model is ensuring pronounced clinical signs and robust viral replication upon influenza infection. strains of low virulence have predominant replication in the nasal turbinates of ferrets. clinical signs and other disease indicators in ferrets are similar to that of humans with mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia (svitek et al., 2008) . replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract of ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater interferon response, transient lymphopenia, and replication in respiratory tract, brain, and other organs (peng et al., 2012; zitzow et al., 2002) . immunocompromised humans have influenza illness of greater duration and complications. immunocompromised ferrets infected with influenza similarly had prolonged virus shedding (van der vries et al., 2013) . interestingly, antiviral resistance emerged in both humans and ferrets with immunocompromised status infected with influenza. alveolar macrophage depleted of ferrets infected with 2009 pandemic h1n1 influenza also had a more severe disease with greater viral replication in the lungs and greater induction of inflammatory chemokines (kim et al., 2013) . a superinfection model resembling that of mice has been developed by in instillation of influenza in 6-to 8-week-old ferrets followed by in inoculation of s. pneumonia 5 days later (peltola et al., 2006) . this typically resulted in otitis media, sinusitis, and pneumonia. transmission models in ferrets have recently met with worldwide media attention and controversy with regard to the study of h5n1 (enserink, 2013; fouchier et al., 2012; herfst et al., 2012; oh et al., 2014) . in general, some subtypes, such as the 2009 h1n1, can transmit easily through aerosol and respiratory droplets (munster et al., 2009) . of concern, h7n9 isolated from humans was more pathogenic and readily transmissible between ferrets by larger respiratory droplets and smaller particle aerosols (kreijtz et al., 2013; richard et al., 2013; zhang et al., 2013) . h5n1 became transmissible by adopting just four mutations, spreading between ferrets in separate cages (imai et al., 2012) . transmission occurs more readily at the height of pyrexia, but for the 2009 h1n1 in particular, can occur before fever is detected (roberts et al., 2012) . ferret-to-ferret transmission of a mouseadapted influenza b virus has also been demonstrated (kim et al., 2015) . since ferrets can be expensive and cumbersome, influenza infection has been characterized and a transmission model developed in the guinea pig; however, this is a newer model with infrequent utilization thus far (lowen et al., 2014) . old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition (murphy et al., 1980) . of old world primates, cynomolgus macaque (macaca fascicularis) is most frequently utilized for studies of vaccines and antiviral drug therapies (stittelaar et al., 2008) . h5n1 and h1n1 1918 infection of cynos is very similar to humans (rimmelzwaan et al., 2001) . cynos develop fever and ards upon in inoculation of h5n1 with necrotizing bronchial interstitial pneumonia . nhps are challenged by multiple routes k. viral disease (ocular, nasal, and tracheal) simultaneously 1 × 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had more than 50% lethality (humans only had a 1%-3% lethality) (kobasa et al., 2007) . ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains, such as h3n2 (o'donnell and subbarao, 2011) . influenza-infected rhesus macaques represent a mild disease model for vaccine and therapeutic efficacy studies (baas et al., 2006) . host microarray and qrt-pcr proved useful for analysis of infected lung tissues. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates, such as squirrel and cebus monkeys (baskin et al., 2009) . domestic pig models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1 (isoda et al., 2006; lipatov et al., 2008) . while pigs infected with influenza may have fever, anorexia, and respiratory signs, such as dyspnea and cough, mortality is rare (van der laan et al., 2008) . size and space requirements make this animal difficult to work with, although the development of minipig models may provide an easier to use alternative. cat and dog influenza models have primarily been utilized to study their susceptibility to h5n1 with the thought that these animals could act as sentinels or could serve to transmit the virus to humans (giese et al., 2008; rimmelzwaan et al., 2006) . these models are not generally used to better understand the disease in humans or for testing vaccines or antivirals. rift valley fever virus (rvfv) causes epizootics and human epidemics in africa. rvfv mainly infects livestock, such as sheep, cattle, goats, etc. after 2-4 days incubation period, animals show signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers (balkhy and memish, 2003) . mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans (abu-elyazeed et al., 1996; mundel and gear, 1951) . after 2-to 6-day-incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever (ikegami and makino, 2011; laughlin et al., 1979; peters and linthicum, 1994) . depending on the severity of the disease when the symptoms start, 10%-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset (ikegami and makino, 2011) . hepatic failure, renal failure or dic, and encephalitis are demonstrated within patients during postmortem examination. live domestic animals especially sheep and goats were used to develop animal models of rvfv (weingartl et al., 2014) . this study indicated that goats were more resistant to the disease compared to sheep. the viremia in goats was lower and had a shorter duration with only some animals developing fever. the susceptibility is influenced by route of infection, breed of animals, the rvfv strain, and growth conditions as well as the passage history. therefore, it might be difficult to establish an animal model with domestic ruminants. mice are one of the most susceptible animal species to rvfv infection. several mouse models including balb/c, ifnar −/− , mbt/pas, 129 and c57bl/6 were exposed to rvfv via parental or aerosol routes of infection (ross et al., 2012) . subq or ip routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice (mims, 1956; smith et al., 2010) . mice start to show signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately following these signs, they become lethargic and generally die 3-6 days postexposure. ocular disease or the hemorrhagic form of the disease has not been observed in mouse models so far (ikegami and makino, 2011) . increased viremia and tissue tropism were reported in mice with (smith et al., 2010) increased liver enzymes and lymphopenia observed in sick mice. aerosolized rvfv causes faster and more severe neuropathy in mice compared to the parental route (dodd et al., 2014; reed et al., 2014) . the liver is a target organ following aerosol exposure and liver failure results in fatality. rats and gerbils are also susceptible to rvfv infection. the rat's susceptibility is dependent on the rat strain utilized for the challenge model and route of exposure. there is also noted age dependence in the susceptibility of rats. while wistar-furth and brown norway strains, and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection via subq route of infection (findlay and howard, 1952; peters and slone, 1982) . similar pathologic changes, such as liver damage and encephalopathy were observed in both rats and mice. the recent study by bales et al. (2012) showed that aerosolized rvfv caused similar disease outcome in wistar-furth and aci rats while lewis rats developed fatal encephalitis which was much more severe than the subq route of infection. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils (anderson et al., 1988) . similar to the rat model, the susceptibility of gerbils was also dependent on age. natural history studies with syrian hamsters indicated that the liver was the target organ with highly elevated alt levels and viral titers (scharton et al., 2015) . lethargy, ruffled fur, and hunched posture were observed in hamsters by day 2 post-subq inoculation and the disease was uniformly lethal by day 2-3 postexposure. this model has been successfully used to test antivirals against rvfv (scharton et al., 2015) . studies thus far showed that rvfv does not cause uniform lethality in a nhp model. ip, in, iv, and aerosol routes have been utilized to develop nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort . monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to humans, such as pathological changes in the liver and hemorrhagic disease. there was no ocular involvement in this model. smith et al. compared iv, in and subq routes of infection in common marmosets and rhesus macaques (peng et al., 2012) . marmosets were more susceptible to rvfv infection than rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subq routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of in challenged marmosets. a recent study by hartman et al. (2014) demonstrated that aerosolized rvfv only caused mild fever in cynomolgus macaques and rhesus macaques, while agms and marmosets had encephalitis and succumbed to disease between days 9 and 11 postexposure. in contrast to other lethal models, the brain was the target organ in agms and marmosets. although no change was observed in ast levels, alp levels were increased in marmosets. little or no change was observed in hepatic enzyme levels in agms. lack of information regarding human disease concerning the aerosol route of exposure makes it difficult to evaluate these animal models. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, appears to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans (ergonul and whitehouse, 2007) . incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorragic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients (mardani and keshtkar-jahromi, 2007; swanepoel et al., 1989) . over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. (fagbami et al., 1975; nalca and whitehouse, 2007; shepherd et al., 1989; smirnova, 1979) . until recently, the only animal that manifests disease is the newborn mouse. infant mice ip infected with cchfv resulted in fatality around day 8 postinfection (tignor and hanham, 1993) . pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies utilizing knockout adult mice were successful to develop a lethal small animal model for cchfv infection (bente et al., 2010; bereczky et al., 2010) . bente et al. infected stat1 knockout mice by the ip route. in this model, after the signs of fever, leukopenia, thrombocytopenia, viremia, elevated liver enzymes and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days postexposure. the second model was developed by using interferon alpha/beta (ifnα/β) receptor knockout mice (ifnar −/− ) (bereczky et al., 2010) . similar observations were made in this model as in the stat1 knockout mouse model. animals were moribund and died 2-4 days after exposure with high viremia levels in liver and spleen. characterization studies with ifnar −/− mice challenged with different routes (ip, in, im, and subq) showed that cchfv causes acute disease with high viral loads, pathology in liver and lymphoid tissues, increased proinflammatory response, severe thrombocytopenia, coagulopathy, and death, all of which are characteristics of human disease . proinflammatory cytokines and chemokines, such as g-csf, ifnγ, cxc-cl10, ccl2 increased dramatically day 3 postchallenge and gm-csf, il-1a, il-1b, il-2, il-6, il-12p70, il-13, il-17, cxcl1, ccl3, ccl5, and tnf-α concentrations were extremely elevated at the time of death/euthanasia. this model is also utilized to test therapeutics, such as ribavirin, arbidol, and t-705 (favipiravir) successfully (oestereich et al., 2014) . experimental vaccines developed for cchf were evaluated in this model provided protection compare to unvaccinated mice (buttigieg et al., 2014; canakoglu et al., 2015, p. 725) . thus, the ifnar −/− mouse model would be a good choice to test medical countermeasures against cchfv, although they have an impaired ifn and immune response phenotype. other laboratory animals, including nhps, show little or no sign of infection or disease when infected with cchfv (nalca and whitehouse, 2007) . butenko et al. utilized agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not show signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. fagbami et al. (1975) infected two patas monkeys (erythrocebus patas) and one guinea baboon (papio papio) with cchfv. whereas all three animals had low-level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia (rabinovich et al., 1972) and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes (blagoveshchenskaya et al., 1975 (blagoveshchenskaya et al., ). shepherd et al. (1989 infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. whereas scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (gerbilliscus brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and mastomys coucha), and syrian hamsters were negative for virus. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed onset of viremia earlier than those infected by the subq or ip routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather rodents (schmaljohn and nichol, 2007) . rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by inhalation of infected rodent saliva, feces, and urine (xu et al., 1985) . human infections can normally be traced to a rural setting with activities, such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention (lednicky, 2003) . the viruses have a tropism for endothelial cells within the microvasculature of the lungs (zaki et al., 1995) . there are two distinct clinical diseases that infection can yield: hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses (nichol, 2001) . hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul (lednicky, 2003) . incubation lasts 2-3 weeks and presents as flu-like in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops which can further progress to shock in approximately 15% patients. overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to development of severe disease. hps was first diagnosed in 1993 within southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus (snv) (centers for disease control and prevention, 1993) . this virus is still the leading cause within north america of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay (padula et al., 2000; stephen et al., 1994) . the first report of hps in maine was recently documented (centers for disease control and prevention, 1993). andes virus (andv) was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans (wells et al., 1997) . the fulminant disease is more lethal than that observed of hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation (peters and khan, 2002) . incubation typically occurs 14-17 days following exposure (young et al., 2000) . unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted (hallin et al., 1996) . syrian golden hamsters are the most widely utilized small animal model for hantavirus infection. hamsters inoculated im with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h prior to death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged snv isolate. no hamsters developed any symptoms during the course of observation. however, an antibody response to the virus that was not dose dependent was determined via elisa. hamsters infected with andv have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andv yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver . normally, snv does not cause a disease in hamsters (wahl-jensen et al., 2007) . but a recent study showed that immunosuppression with dexamethasone and cyclophosphamide in combination causes lethal disease with snv in hamsters (brocato et al., 2014) . the disease was very similar to the disease caused by andv in hamsters. lethal disease can be induced in newborn mice, but does not recapitulate the clinical symptoms observed in human disease (kim and mckee, 1985) . the disease outcome is very much dependent on the age of the mice. younger mice are much more susceptible to virus than the adult mice. adult mice exposed to hanta virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease (seto et al., 2012; wichmann et al., 2002) . knockout mice lacking ifnα/β are highly susceptible to hanta virus infection (muller et al., 1994) . in a study of panel of laboratory strains of mice, c57bl/6 mice were most susceptible to a passaged hanta viral strain injected ip. animals progressed to neurological manifestation including paralyses and convulsions, and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different from that observed in human cases (wichmann et al., 2002) . in a recent study, 2-weekold icr mice was exposed to htnv strain aa57 via the subq route (seto et al., 2012) . mice started to show signs of disease by day 11 postinoculation. piloerection, trembling, hunching, loss of body weight, labored breathing, and severe respiratory disease were observed in mice. studies to develop nhp models were not successful until recently. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported (hooper et al., 2006; mcelroy et al., 2002) . cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease (klingstrom et al., 2002; sironen et al., 2008) . challenge with andv to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. four of six aerosol exposed monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. in a recent study, rhesus macaques were inoculated by the intramuscular route with snv passaged only in deer mice (safronetz et al., 2014) . characteristics of hps disease including rapid onset of respiratory distress, severe pulmonary edema, thrombocytopenia, and leukocytosis were observed in this promising model. viremia was observed 4-10 days prior to respiratory signs of the disease that were observed on days 14-16 postinoculation. with all aspects, this animal model would be very useful to test medical countermeasures against hanta virus. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all of these viruses share common clinical manifestations (mccormick and fisher-hoch, 2002) . lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april (curtis, 2006) . this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5000 deaths (khan et al., 2008) . outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases have sprung up in germany, netherlands, united kingdom, and the united states due to transmission to travelers on commercial airlines (amorosa et al., 2010) . transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex (curtis, 2006) . humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease occurs in 20% of individuals. the incubation period is from 5 to 21 days and initial onset is characterized by flu-like illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease that is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital it is between 15% and 25%. there is no approved vaccine and besides supportive measures, ribavirin is effective only if started within 7 days (mccormick et al., 1986a,b) . the primary animal model used to study lassa fever is the rhesus macaque (jahrling et al., 1980) . aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus mirrored more closely the disease course and histopathology observed in human infection (danes et al., 1963) . iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt (lukashevich et al., 2003) . disease was dose dependent with iv, intramuscular, and subq inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be utilized, and mimic a more natural route of infection (peters et al., 1987) . within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7 and death typically occurred within 10-14 days (lukashevich et al., 2004; rodas et al., 2004) . intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns (hensley et al., 2011b) . this pathogenicity is seen in select cases of human lassa fever (cummins et al., 1992; gunther et al., 2001) . a marmoset model has recently been defined utilizing a subq injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases (carrion et al., 2007) . mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is dependent on the mhc background, age of the animal, and inoculation route (salvato et al., 2005) . stat1 knockout mice inoculated ip with both lethal and nonlethal lassa virus strains develop hearing loss accompanied by damage to the inner ear hair cells and auditory nerve (yun et al., 2015) . guinea pig inbred strain 13 was highly susceptible to lassa virus infection. the outbred hartley strain was less susceptible, and thus strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus (jahrling et al., 1982) . infection with pichinde virus passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death (lucia et al., 1990; qian et al., 1994) . the guinea pig is an excellent model given that it not only results in similar disease pattern, viral distribution, histopathology, and immune response to humans (connolly et al., 1993; katz and starr, 1990) . infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig models. the virus was injected ip resulting in lethargy and anorexia within 6-7 days. virus was first detected at 3 days, and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present (sbrana et al., 2006) . these results were recapitulated with a nonadapted pichinde virus (buchmeier and rawls, 1977; gowen et al., 2005; smee et al., 1993) . the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in subsaharan africa. worldwide, there are approximately 1.8 million deaths per year with over 260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1 (de cock et al., 2011) . hiv targets t-helper cells (cd4+), macrophages, dendritic cells (fields et al., 2007) . acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8+ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4+ t-cells decline to less than 200 cells/µl; thus cell-mediated immunity becomes impaired and the person is more susceptible to opportunistic infections as well as certain cancers. hiv has a narrow host range likely because the virus is unable to antagonize and evade effector molecules of the interferon response (thippeshappa et al., 2012) . humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of an intact and functional human innate and adaptive immune responses (berges and rowan, 2011) . the scidhu hiv infection model has proven useful, particularly in screening antivirals and therapeutics (denton et al., 2008; melkus et al., 2006) . a number of different humanized mouse models have been developed for the study of hiv, including rag1 −/− γc −/− , rag2 −/− γc −/− , nod/scidγc −/− (hnog), nod/scidγc −/− (hnsg), nod/scid blt, and nod/scidγc −/− (hnsg) blt (karpel et al., 2015; li et al., 2015; shimizu et al., 2015) . cd34+ human stem cells derived from umbilical cord blood or fetal liver are used for humanization (baenziger et al., 2006; watanabe et al., 2007) . hiv-1 infection by ip injection can be successful with as little as 5% peripheral blood engraftment (berges et al., 2006) . vaginal and rectal transmission models have been developed in blt scid hu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days postinoculation . in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans (brainard et al., 2009) . importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hivinfected humanized mice and at least some mechanisms of pathogenesis that occur in hiv-infected humans, also occur in the hiv-infected humanized mouse models (baenziger et al., 2006; neff et al., 2011) . the advantage of these models is that these mice are susceptible to hiv infection and thus the impact of drugs on the intended viral targets can be tested. one caveat is that while mice have a "common mucosal immune system," humans do not, due to differences in the distribution of addressins (holmgren and czerkinsky, 2005) . thus, murine mucosal immune responses to hiv do not reflect those of humans. another strategy uses a human cd4-and human ccr5-expressing transgenic luciferase reporter mouse to study hiv-1 pseudovirus entry (gruell et al., 2013) . hiv-1 transgenic (tg) rats are also used to study hiv related pathology, immunopathogenesis, and neuropathology (lentz et al., 2014; reid et al., 2001) . the clinical signs include skin lesions, wasting, respiratory difficulty, and neurological signs. brain volume decreases have been documented and the hiv-1 tg rat is thus used as a model of neuropathology in particular. there are a number of important nhp models for human hiv infection (hessell and haigwood, 2015) . an adaptation of hiv-1 was obtained by four passages in pigtailed macaques transiently depleted of cd8(+) cells during acute infection (hatziioannou et al., 2014) . the resulting disease has several similarities to aids in humans, such as depletion of cd4(+) t-cells (kimata, 2014) . simian immunodeficiency virus (siv) infection of macaques has been widely used as a platform for modeling hiv infection of humans (demberg and robert-guroff, 2015; walker et al., 2015) . importantly, nhps have similar, pharmacokinetics, metabolism, mucosal tcell homing receptors, and vascular addressins to those of humans. thus, while the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero or through breast milk (keele et al., 2009; miller et al., 2005; stone et al., 2009) . there are also macaque models to study the emergence and clinical implications of hiv drug resistance (van rompay et al., 2002) . these models most routinely utilize rhesus macaques (macaca mulatta), cynomolgus macaques (m. fasicularis), and pigtailed macaques (macaca nemestrina). all ages are used, depending on the needs of the study. for instance, use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently employed. female pigtailed macaques have been used to investigate the effect of the menstrual cycle on hiv susceptibility (vishwanathan et al., 2015) . studies are performed in bsl-2 animal laboratories and nhps must be simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied (ebenezer et al., 2012) . exposure in model systems is typically through a single high-dose challenge. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/ deltab670 induces aids in most macaques within 5-17 months (mean of 11 months) (fuller et al., 2012) . peak viremia occurs around week 4. aids in such models is often defined as cd4+ t-cells that have dropped to less than 50% of the baseline values. alternatively, repeated low dose challenges are often utilized, depending on the requirements of the model (henning et al., 2014; moldt et al., 2012; reynolds et al., 2012) . since nhps infected with hiv do not develop an infection with a clinical disease course similar to humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (shivs) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease which progresses to aids, and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1 and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors, thus hiv-1 rt is usually put into siv for such studies (uberla et al., 1995) . sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt, or integrase inhibition (witvrouw et al., 2004) . nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4+ t-cell depletion. additionally, sivmac239 utilizes the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry (veazey et al., 2003) . nhps infected with shiv strains, may not develop aids, but these models are useful in testing vaccine efficacy . for example, rt-shivs and env-shivs are useful for testing and evaluation of drugs that may target the envelope or rt, respectively (uberla et al., 1995) . one disadvantage of the highly virulent env-shiv (shiv-89.6 p), is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops then resolves without further disease progression (humbert et al., 2008) . simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for 3 months, followed by clearance (haigwood, 2009) . a number of routes are utilized for siv or shiv infection of nhps, with iv inoculation the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) 1 month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase susceptibility to infection by atraumatic vaginal instillation (burton et al., 2011) . upon vaginal instillation of 500 tcid 50 of shiv-162p3, peak viremia was seen around 12 days postexposure with greater than 10 7 copies/ml and dropping thereafter to a constant level of 10 4 rna copies/ml at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251 (barouch et al., 2012) . an example of an intrarectal model utilized juvenile (2-year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx (belshan et al., 2012) . here, viremia peaked at approximately 10 days with more than 10 8 copies/ml. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistent high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of haart therapy on virus and t-cells in the male genital tract, adult (3-to 4-year-old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251 and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization (moreau et al., 2012) . pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course (abel, 2009) . importantly, mother-to-infant transmission models have also been developed (jayaraman et al., 2004) . pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected, one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission as well as the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating, and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and trim alleles), differences between challenge strains, and challenge routes (letvin et al., 2011) . nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the long-term clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade (van rompay et al., 2008) . unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study which used an adenovirus-based vaccine approach (buchbinder et al., 2008) . vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv exposure is more difficult to protect than mucosal exposure and is used as a "worst case scenario." however, efficacy at one mucosal route is usually comparable to other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect (bosch and de sanjose, 2002) . there are over 100 human papillomaviruses, with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately one third of papillomaviruses are transmitted sexually. of these, virulent subtypes, such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. up to 35% of head and neck cancers are caused by hpv-16, particularly oropharyngeal cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no wild-type animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist which use animal papillomaviruses in their natural host or a very closely related species (borzacchiello et al., 2009; brandsma, 1994; campo, 2002) . these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines (rabenau et al., 2005) . wild-type inbred mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, orthotopically transplanted or transgenic, but they are often used to look at immunogenicity of vaccines (jagu et al., 2011; oosterhuis et al., 2011) . transgenic mice used for hpv animal modeling typically express the viral oncogenes e5, e6, e7, or the entire early region of hpv-16 from the keratin 14 promoter which is only active in the basal cells of the mouse epithelium (chow, 2015) . cancers in these models develop upon extended estrogen exposure (maufort et al., 2010; ocadiz-delgado et al., 2009; stelzer et al., 2010; thomas et al., 2011) . transgenic mice with constitutively active wnt/b-catenin signaling in cervical epithelial cells expressing the hpb oncoprotein e7 develop invasive cervical squamous carcinomas (bulut and uren, 2015) . the tumors occur within 6 months approximately 94% of the time. another model uses c57bl/6 mice expressing the hpv16-e7 transgene which are then treated topically with 7,12-dimethylbenz(a)anthracene (dmba) (de azambuja et al., 2014) . these mice developed benign and malignant cutaneous lesions. cervical cancers can also be induced in human cervical cancer xenografts transplanted onto the flanks of athymic mice and serially transplanted thereafter ( hiroshima et al., 2015; siolas and hannon, 2013) . a wild-type immunocompetent rodent model uses m. coucha, which is naturally infected with mastomys natalensis papillomavirus (mnpv) (vinzon et al., 2014) . mnpv induces papillomas, keratoacanthomas, and squamous cell carcinomas and provides a means to study vaccination in an immunocompetent small animal model. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which is a very closely related species ( breitburd et al., 1997) . in this model, papillomas can range from cutaneous squamous cell carcinomas on one end of spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) causes florid warty lesions in mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings (johnston et al., 2005) . the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model (nicholls et al., 1999) . these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by application of a 10 µl droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles (nicholls et al., 2001) . some investigators have raised concerns that dogs are not a suitable model for high-risk hpv-induced oral cancer (staff, 2015) . bovine papillomavirus (bpv) has a wider host range than most papillomaviruses, infecting the fibroblasts cells of numerous ungulates (campo, 2002) . bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases, such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, rhesus papillomavirus (rhpv), a sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques is very similar to hpv-16 and is associated with the development of cervical cancer ( ostrow et al., 1990; wood et al., 2007) . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including k. viral disease squirrels (charatan, 2003; khodakevich et al., 1986) . mpxv was discovered during the pox-like disease outbreak among laboratory monkeys (mostly cynomolgus and rhesus macaques) in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak (nalca et al., 2005; sejvar et al., 2004) . it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-to-person spread occurs by large respiratory droplets or direct contact (jeézek and fenner, 1988) . most of the clinical features of human monkeypox are very similar to those of ordinary smallpox (breman and arita, 1980) . after a 7-to 21-dayincubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash (di giulio and eckburg, 2004; jeézek and fenner, 1988) . a typical maculopapular rash follows the prodromal period, generally lasting 1-3 days. the average size of the skin lesions are 0.5-1 cm and the progress of lesions follows the order: macules, papules, vesicles, pustules, umblication then scab, and desquamation and lasts typically 2-4 weeks. the fatality rate is 10% among the unvaccinated population and death generally occurs during the 2nd week of the disease (jeézek and fenner, 1988; nalca et al., 2005) . mpxv is highly pathogenic for a variety of laboratory animals and many animal models have been developed by using different species and different routes of exposure (table 33 .3). due to unavailability of variola virus (smallpox) to develop animal models and similar disease manifestations in humans that are similar, mpxv is one of the pox viruses that are utilized very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, icr mouse, prairie dogs, african dormice, ground squirrels, and gambian pouched rats are highly susceptible to mpxv by different exposure routes (americo et al., 2010; falendysz et al., 2015; hutson et al., 2009; osorio et al., 2009; sbrana et al., 2007; schultz et al., 2009; sergeev et al., 2016; stabenow et al., 2010; tesh et al., 2004; xiao et al., 2005) . cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose dependent mortality after in exposure to mpxv. studies with ip route of challenge indicated a 50fold higher susceptibility to mpxv when compared to in route (americo et al., 2010) . scid-balb/c mice were also susceptible to the ip challenge route and the disease resulted in mortality on day 9 postinfection (osorio et al., 2009) . similarly, c57bl/6 stat1 −/− mice were infected in with mpxv and the infection resulted in weight loss and mortality 10 days postexposure. recently sergeev et al. (2016) showed that in challenge of icr mice with mpxv resulted in purulent conjunctivitis, blepharitis, and ruffled fur in these mice although there was no death. the mouse models mentioned here are very promising for screening therapeutics against poxviruses but testing in additional models will be required for advanced development. high doses of the mpxv by ip or in routes caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels (tesh et al., 2004) . the disease progressed very quickly and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv strains in ground squirrels by the subq route resulted in systemic disease and mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nosebleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. another study by sergeev et al. (2017) showed that in challenge with mpxv caused fever, lymphadenitis, and skin rash in ground squirrels 7-9 days postexposure. mortality was observed in 40% of the animals 13-22 days postexposure (sergeev et al., 2017) . since mpxv was transmitted by infected prairie dogs in the us outbreak, this animal model has been more thoroughly studied and utilized to test therapeutics and vaccines compared to other small animal models ( hutson et al., 2009; keckler et al., 2011; smith et al., 2011; xiao et al., 2005) . studies using in, ip, and id routes of exposure showed that mpxv was highly infectious to prairie dogs, ip infection with the west african mpxv strain caused a more severe disease and 100% mortality than challenge by the in route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to ip route, the in route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in ip-infected animals (xiao et al., 2005) . hutson et al. (2009) african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and most importantly generalized pox lesions. therefore, this model has the utility for testing therapeutics and vaccines against pox viruses. furthermore, mpxv challenged prairie dogs were used to perform in vivo bioluminescent imaging (bli) studies (falendysz et al., 2015) . bli studies showed real time spread of virus in prairie dogs as well as potential routes for shedding and transmission. the african dormouse is susceptible to mpxv by a footpad injection or in routes (schultz et al., 2009) . mice had decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and lung hemorrhage were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. in a recent study, comparison of the disease pathogenesis was performed by using live bioluminescence imaging in the cast/eij mouse and african dormouse challenged with low dose of mpxv (earl et al., 2015) . following in challenge, mpxv dissemination occurred through the blood or lymphatic system in dormice compared to dissemination that was through the nasal cavity and lungs in cast/eij mice. the disease course was much faster in cast/eij mice (earl et al., 2015) . considering the limited availability of prairie dogs, ground squirrels and african dormice, lack of reagents specific for these species, and not having commercial sources of these species, these small animal models are as attractive for further characterization and vaccine, and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal model for mpxv (edghill-smith et al., 2005; johnson et al., 2011; nalca et al., 2010; stittelaar et al., 2006; zaucha et al., 2001) . during our studies using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure (nalca et al., 2010) . complete blood count and clinical chemistries showed abnormalities similar to human monkeypox cases with leukocytosis and thrombocytopenia (huhn et al., 2005) . whole blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. since doses of 4 × 10 4 pfu, 1 × 10 5 pfu, or 1 × 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 × 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, animals succumbed to disease before developing pox lesions. with the low challenge dose, pox lesions were observed but they were few in comparison to the iv model. a recent study also evaluated the cytokine levels in aerosol challenged animals. (tree et al., 2015) . tree et al. (2015) showed that ifnγ, il-1rα, and il-6 increased dramatically on day 8 postexposure the day that death was most likely to occur, and viral dna was detected in most of the tissues. these results support the idea of a cytokine storm causing mortality in monkeypox disease. mpxv causes dose dependent disease in nhps when given by the iv route (johnson et al., 2011) . studies showed that a 1 × 10 7 pfu iv challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an it infection model skips the upper respiratory system and deposits virus into the trachea, delivering the virus directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally (stittelaar et al., 2006) . although a similar challenge dose of it mpxv infection resulted in a similar viremia in nhps to the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to the it route. an intrabronchial route of exposure resulted in pneumonia in nhps (johnson et al., 2011) . delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and it infection models, the number of pox lesions was much less than in the iv infection model. a major downside of the iv, it, and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus to the blood stream or to the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting the subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human varv infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b virus (hbv) is one of the most common infections worldwide with over 400 million people chronically infected and 316,000 cases per year of liver cancer due to infection (lee, 1997) . the virus can naturally infect both humans and chimpanzees (guha et al., 2004) . hbv is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture (lai et al., 2003) . the age at which one is infected dictates the risk of developing chronic disease (hyams, 1995) . acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms of hbv infection last for a few weeks before resolving (ganem and prince, 2004) . after this acute phase, lifetime immunity is achieved (wright and lau, 1993) . of those infected, less than 5% will develop the chronic form of the disease. chronicity is the most serious outcome of the disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than a noncarrier (beasley, 1988) . the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepatitis virus suggest that x protein may be responsible (spandau and lee, 1988). many individuals are asymptomatic until complications emerge related to chronic hbv carriage. chimpanzees have a unique strain that circulates within the population (hu et al., 2000; . it was found that 3%-6% of all wild-caught animals from africa are positive for hbv antigen ( lander et al., 1972) . natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease (prince, 1972) . to date, chimpanzees are the only reliable method to ensure that plasma vaccines are free from infectious particles (prince and brotman, 2001 ). this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially ideal for these studies given that their immune response to infection directly mirrors humans (nayersina et al., 1993) . recent regulations by the national institute of health (nih) and restrictions to use great apes as animal models forced researches to find alternate models for hbv infection. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hbv, none develops hepatic lesions or liver damage as noted by monitoring of liver enzymes (pillot, 1990 ). mice are not permissible to infection, and thus numerous transgenic and humanized lines that express hbv proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease (guha et al., 2004) . recently, the entire genome of hbv was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection (huang et al., 2012) . another model that has been developed is hydrodynamic injection of hbv genomes in the liver of mice (liu et al., 1999; yang et al., 2002) . although this model is very stressful to mice and has liver toxicity, it is successfully used to evaluate antivirals against hbv (mccaffrey et al., 2003) . liver chimeric mouse models are an additional set of surrogate models for hbv infection (dandri and lutgehetmann, 2014) . in these models human hepatocytes are integrated into the murine liver parenchyma (allweiss and dandri, 2016) . this model might be used to test antivirals as well as to study the molecular biology of hbv infection. hbv can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses (mason et al., 1982) . the woodchuck hepatitis virus induces hepatocellular carcinoma (summers et al., 1978) . within a population, 65%-75% of all neonatal woodchucks are susceptible to chronic infection (cote et al., 2000) . a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human carcinogenesis takes much longer (gerin et al., 1989) . the acute infection strongly resembles what occurs during the course of human disease. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage (tennant, 2001) . challenge with virus in neonates leads to a chronic infection while adults only develop the acute phase of disease (buendia, 1992) . a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. the marmot himalayan develops an acute hepatitis with a productive infection (lucifora et al., 2010) . hepatitis d virus (hdv) is dependent upon hbv to undergo replication and successful infection in its human host (gerin, 2001) . there are two modes of infection possible between the viruses: coinfection where a person is simultaneously infected or superinfection in which a chronic carrier of hbv is subsequently infected with hdv (purcell et al., 1987) . coinfection leads to a similar disease as seen with hbv alone; however, superinfection can result in chronic hdv infection and severe liver damage (guilhot et al., 1994) . both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d (ponzetto et al., 1991) . a recently published report demonstrated the use of a humanized chimeric upa mouse to study interactions between the two viruses and drug testing (lutgehetmann et al., 2012) new models ranging from nhps to small animals and representing the disease characteristics in humans are necessary to study viral and host factors that drive disease pathogenesis and evaluate medical countermeasures. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus is a low passage clinical isolate thus animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that occurs in natural disease. in order to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the aforementioned characteristics cannot always be satisfied; however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy fda "animal rule." this rule applies to situations in which vaccine and therapeutic efficacy cannot safely or ethically be tested in humans; thus licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures compared across institutions. this chapter has summarized the best models available for each of the viruses described. the rhesus macaque pediatric siv infection model-a valuable tool in understanding infant hiv-1 pathogenesis and for designing pediatric hiv-1 prevention strategies prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques progress toward norovirus vaccines: considerations for further development and implementation in potential target populations characterization of lethal zika virus infection in ag129 mice experimental in vitro and in vivo models for the study of human hepatitis b virus infection a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice middle east respiratory syndrome coronavirus: current situation and travel-associated concerns aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an ebola virus-infected rhesus macaque (macaca mulatta) with apparent recovery and a delayed time of death 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molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2 −/− gamma c −/− mice choice of inbred rat strain impacts lethality and disease course after respiratory infection with rift valley fever virus rift valley fever: an uninvited zoonosis in the arabian peninsula recombinant norwalk virus-like particles given orally to volunteers: phase i study tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining lethal antibody enhancement of dengue disease in mice is prevented by fc modification animal models for the study of influenza pathogenesis and therapy effect of oral gavage treatment with znal42 and other metallo-ion formulations on influenza a h5n1 and h1n1 virus infections in mice macaque studies of vaccine and microbicide combinations for preventing hiv-1 sexual transmission early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus hepatitis b virus. the major etiology of hepatocellular carcinoma transmission of norwalk virus during football game vpx is critical for sivmne infection of pigtail macaques experimental respiratory syncytial virus infection of four species of primates pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat-1 knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptor-knockout mice the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment hiv-1 infection and cd4 t cell depletion in the humanized rag2 −/− gamma c −/− (rag-hu) mouse model bacterial infections in pigs experimentally infected with nipah virus evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells chimpanzees as an animal model for human norovirus infection and vaccine development a simple technique for infection of mosquitoes with viruses; transmission of zika virus human papillomavirus research: do we still need animal models? human papillomavirus in cervical cancer development of a hamster model for chikungunya virus infection and pathogenesis a neutralizing human monoclonal antibody protects against lethal disease in a 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hpv-associated carcinogenesis the confirmation and maintenance of smallpox eradication a lethal disease model for hantavirus pulmonary syndrome in immunosuppressed syrian hamsters infected with sin nombre virus nonhuman primate models of chikungunya virus infection and disease tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates pediatric norovirus diarrhea in nicaragua efficacy assessment of a cell-mediated immunity hiv-1 vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial variation between strains of hamsters in the lethality of pichinde virus infections hepatitis b viruses and hepatocellular carcinoma generation of k14-e7/n87betacat double transgenic mice as a model of cervical cancer limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody a novel vaccine against crimean-congo 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virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs model systems to study the life cycle of human papillomaviruses and hpv-associated cancers primary severe acute respiratory syndrome coronavirus i nfection limits replication but not lung inflammation upon homologous rechallenge viral and host factors in human respiratory syncytial virus pathogenesis pathogenesis of pichinde virus infection in strain 13 guinea pigs: an immunocytochemical, virologic, and clinical chemistry study pathogenesis of experimental ebola virus infection in guinea pigs transcriptional profiling of the immune response to marburg virus infection the use of a neonatal mouse model to study respiratory syncytial virus infections a model of 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viruses in ferrets lethal crimean-congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone 15 variant development of a murine model for aerosolized filovirus infection using a panel of bxd recombinant inbred mice a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. key: cord-297216-1b99hm1e authors: sariola, salla; gilbert, scott f. title: toward a symbiotic perspective on public health: recognizing the ambivalence of microbes in the anthropocene date: 2020-05-16 journal: microorganisms doi: 10.3390/microorganisms8050746 sha: doc_id: 297216 cord_uid: 1b99hm1e microbes evolve in complex environments that are often fashioned, in part, by human desires. in a global perspective, public health has played major roles in structuring how microbes are perceived, cultivated, and destroyed. the germ theory of disease cast microbes as enemies of the body and the body politic. antibiotics have altered microbial development by providing stringent natural selection on bacterial species, and this has led to the formation of antibiotic-resistant bacterial strains. public health perspectives such as “precision public health” and “one health” have recently been proposed to further manage microbial populations. however, neither of these take into account the symbiotic relationships that exist between bacterial species and between bacteria, viruses, and their eukaryotic hosts. we propose a perspective on public health that recognizes microbial evolution through symbiotic associations (the hologenome theory) and through lateral gene transfer. this perspective has the advantage of including both the pathogenic and beneficial interactions of humans with bacteria, as well as combining the outlook of the “one health” model with the genomic methodologies utilized in the “precision public health” model. in the anthropocene, the conditions for microbial evolution have been altered by human interventions, and public health initiatives must recognize both the beneficial (indeed, necessary) interactions of microbes with their hosts as well as their pathogenic interactions. the anthropocene marks the end of a period characterized by the human triumph of nature, and nowhere is this more prominent than in public health. the heroic era of microbiology made pasteur, koch, lister, and fleming household names, and few have done more for humanity [1] [2] [3] . the lifespan of a newborn parisian was 45 years in 1900; and a century later, a newborn parisian could expect to live another 80 years [4] . much of this increase in life expectancy (as well as the expectation to be healthy) has been due to the ability of public health measures-proper sanitation, food surveillance, antisepsis, and anti-viral vaccination-to remove humans from sources of microbial infection. however, our relationship with microbes and their evolution has changed dramatically since the discovery of antibiotics, and the conquest of polio and other endemic viruses in the 1950s and 1960s. the first part of our changing relationship to microbes involves the western world's manufacturing an environment that is increasingly sterile and characterized by the removal of unplanned and unplannable nature [5, 6] . indeed, nature is no longer our "state of nature." rather, we have continually separated ourselves from nature, separating the "who" of human lives from the "what" of other lives. and immune systems of plants and animals and (2) the microbes' ability to transfer dna horizontally from organism to organism. this article attempts to map out a holobiont perspective to public health. it is important to determine how these new views of microbial evolution-lateral gene transfer and mutualistic symbiosis-might be integrated into public health initiatives. it seems that present initiatives ignore or marginalize these phenomenon and that public health might be served better if they were made more central. two pertinent public health paradigms that have received much publicity in recent years are the "precision public health" (pph) paradigm and the "one health" (oh) paradigm. neither of these appear to take seriously our new appreciation of microbial evolution. precision public health (pph) is the application of genomics technology for population health benefits [34, 35] , and it is the attempt to make public health into a genomic science. pph began in 1997, when the office of public health genomics of the cdc was formed to "transform" population health care into a genomic science "by identifying, evaluating, and implementing evidence-based genomics practices to prevent and control the country's leading chronic, infectious, environmental, and occupational diseases" [36] . pph claims that it would be able to analyze one's genome and then prescribe the appropriate drugs and dietary regimens. however, the original promises that genomic science would find common alleles for common diseases were not fulfilled [37, 38] . genome-wide association studies (gwas) for cardiovascular disease showed that that genes played a negligible role in predicting heart attacks and that human genetic variation accounted for roughly 3% of the variation in blood pressure [39] . moreover, the prediction that a patient would have a heart attack was better made by the number of pushups a patient could do than by genomic analyses [40] . the genes thought to be associated with depression were "lost" when large trials were done [41] ; and deficiencies of the gut microbiome may provide a better account of causation [42] . worse, for any genomic model of public health, was when the genome of the founder of the human genome project, james watson, was analyzed. his dna sequences predicted him to be deaf, blind, growth retarded, and mentally deficient [43, 44] . genes work differently in different people. "phenotypic heterogeneity," wherein the same mutant allele causes different phenotypes in different individuals carrying it, is a well-known phenomenon in medical genetics [45, 46] and a gene that is "normal" in one generation can cause disease in another [47] [48] [49] . nevertheless, the pph got a shot in the arm (to use an old public health metaphor) by the "all of us" project begun at the usa's national institutes of health [50] . its website proclaims this to be a big genome, big data approach to public health, whereby "taking into account individual differences in lifestyle, environment, and biology, researchers will uncover paths toward delivering precision medicine..." pph is getting a shot in the other arm from pharmacogenomics, the study of how responses to drugs are influenced by the genetic makeup of the person receiving the drug. according to kapoor et al. [51] pharmacogenomics, is "one of the cornerstones of precision medicine" and furthermore, is a "significant innovation in health care that possesses the potential to change the paradigm in the practice of medicine, not solely in the way drugs are prescribed, but also in the way drugs are discovered and developed." indeed, precision pharmacogenetics is being touted as a paradigm for third-world health care [52] . however, this population-centered model of genomic healthcare delivery has been criticized [37] as being a salvage attempt to rescue something of value from the numerous extremely expensive genome projects that had been the scientific rage of the late 20th and early 21st centuries. reardon and others [37, 53, 54] claim pph is most likely dangerous fantasy, exacerbating global economic differences, taking the "public" out of "public health," and shifting responsibility for health onto the individual citizen [55, 56] . pph has also been criticized for not recognizing the contributions of the symbiotic microbial genomes [38] . symbiotic relationships with microbes, as will be discussed below, provide essential metabolic pathways for phenotype production (including those for drug metabolism) and over ten-fold the number of different genes than the zygote-derived genome. whereas precision public health works from one privileged level-the genome-up to humans and human communities, the one health paradigm is consciously interdisciplinary and multi-species-it attempts to envision people, animals, and environments as partners in each other's health on several levels [57, 58] . as gibbs [59] (p. 49) notes, "one health is the collaborative effort of multiple disciplines-working locally, nationally, and globally-to attain optimal health for people, animals, and our environment." the one health paradigm, according to friese and nuyts, provides a theoretical basis for research involving nonhumans in public health and used to re-organize relationships between human medicine and animal veterinary medicine so that these two fields communicate in both knowledge and practice [60] . with contributions from such disciplines as ecosystem services and soil microbiology, one health approach also recognizes the role of environments and ecologies in how human and animal health is shaped. these contributions see ecosystems (including symbiotic microbiomes) as providing economic infrastructure benefits that can be calculated as part of any managed change to the environment [61] [62] [63] . however, overall, the implementation of one health is still fixed on protecting humans from zoonotic infections [60] . indeed, the cdc, who, ama, and avma websites stress zoonoses and the fact that most infectious diseases are spread by animals. the environment gets short shrift in these sites, and this deficiency has not gone unnoticed. numerous investigators have documented that the environment does not receive attention or funding in most one health networks [64] [65] [66] [67] . thus, the three components of the one health model are not equal, and the framework is still used to prioritize protection of humans from zoonotic diseases. while the importance of this goal is made obvious in this coronavirus-infused decade, the anthropogenic deterioration of the environment by humans-such as mountain-top coal removal, anthropogenic deforestation, soil microbial deterioration, and reef depletion-are crucial in and of themselves as well as can have enormous effects on public health and do not appear on the one health agenda. only recently have there been calls to put microbes and global climate change under the one health umbrella [68, 69] . some of these initiatives have come under the planetary health [70] , which emphasizes how critical such ecological perspectives are for human health. the planetary health perspective, however, concentrates on the important issues of politics and economics of global health care in the anthropocene, but it does not address the issues the changes in how we perceive microbes. in contrast to the precision public health and one health paradigms, a recently proposed theory holds that microbes such as bacteria are primarily beneficial symbionts of the human body, and their presence is both expected and necessary for normal human health. while pathogenic microbes can cause enormous damage, they are a distinct minority, and public health needs to recognize the other arm of symbiosis-mutualism. this approach, which could revitalize the community-based one health perspective to public health by using the techniques of the molecularly based pph model, is based on the hologenome theory [71] . this model has recently received support by private funding, most notably from the bill and melinda gates foundation [72, 73] . the hologenome theory [74, 75] recasts the individual animal or plant (and other multicellular organisms) as a consortium ("holobiont")-the host plus all its symbiotic microbes. during the past two decades, advances in microbiome research have clearly shown that most animals cannot normally develop, function, or reproduce without the vast numbers of microorganisms that inhabit their bodies [28, 76] . microbes are essential for normal animal development and physiological functioning. for instance, bacteria acquired at birth from the female reproductive tract are critical to the construction of the gut capillaries and epithelia in several vertebrates [77] [78] [79] [80] [81] as well as being critical for the normal development of the vertebrate enteric and cerebral nervous systems [82] [83] [84] . pediatric geneticist barton childs [85] postulated that each person's genetic endowment constitutes a "biochemical individuality" conferred upon us by our genes. patterson and turnbaugh [86] have used the same term to designate the properties of the "hologenome"-the genes we inherit from our parents and our microbes, our germ cells and our germs. while we inherit some 22,000 genes from our parents, we inherit about 8 million different genes from our parents' bacteria [87] . indeed, in some instances, the gut microbiome appears to be critical in drug metabolism. digoxin, cyclophosphamide, and numerous other drugs are each metabolized differently by different populations of microorganisms [88] [89] [90] , giving each person an assortment of genes (and drug-metabolizing phenotypes) that can change with each meal [91] . even the human immune system, so critical in public health, is a holobiont property, and not merely the agency of the host [92] [93] [94] [95] [96] . microbes enter into the body at birth, prior to the maturation of the immune system, and they induce the formation of lymphoid tissues [97] [98] [99] . moreover, these lifelong immune activities are well-regulated only in the continuous presence of microbes, which in turn, constantly regulate the microbes that can stay with the animal [100] [101] [102] [103] [104] [105] [106] . the immune system is a continuously co-constructed property of the holobiont. holobiont public health recognizes that microbes may be pathogenic or beneficial, and that deficiencies in bacteria can cause developmental, immunological, cognitive, and physiological ailments. for instance, kwashiokor, long seen as a protein deficiency disease, manifests as a wasting and anorexic pathology only when certain bacteria are absent [107] . asthma and allergies are also seen to be due to the absence of protective bacteria, which normally are present to be induce anti-inflammatory regulators [108, 109] . in these studies, hanski and colleagues [110] explicitly link environmental health, microbial diversity, and human health. indeed, a new field of dysbiosis is now emerging, including not only infection, but also other conditions that may be caused by deficient or aberrant microbiomes. here, the normal symbiotic relationships that maintain physiological or developmental continuity, have been abrogated. in recent years, science has traced these networks from associations to specific causal changes that can be tested. while many of these experiments have been performed in mice, the same pathways are known to be present in humans. these non-contagious diseases include asthma and allergy [111] , kwashiorkor [107, 112] , obesity [113, 114] , diabetes [115] , ulcerative colitis [116] , depression [42, 117] , and parkinson's disease [118, 119] . these "microbial deficiency diseases," constitute a new and possibly important category of illness. this is not to say that dysbiosis is the only cause of these conditions, but that it is a public health concern that should be investigated. an important conceptual barrier was recently crossed when the gut microbiomes of pregnant mice were demonstrated to be critical for the intrauterine development of the fetus. short-chain fatty acids (such as butyrate and propionate) are products of the gut microbiome's digestion of cellulose. as no mammalian genome contains genes for cellulose digestion, the breakdown of plant material is almost totally accomplished by enzymes produced by microbes. kimura and colleagues [120] demonstrated that propionic acid, derived from the breakdown of fiber by maternal gut microbes, was critical for the normal development of the insulin-producing pancreatic beta cells, the sympathetic neurons that project to the heart, and the gut enteroendocrine cells. without the microbe-derived propionic acid, the adult offspring developed a metabolic syndrome characterized by glucose intolerance, obesity, and insulin resistance. since diet can control the prevalence of microbes, the holobiont model can explain the mechanism whereby eating low-fiber, high-calorie diets during pregnancy predisposes offspring to have metabolic syndrome later in life [121] . to understand the importance of a symbiotic approach to public health, one has to first appreciate the new biological notion of the human body. each of us is a functional entity that includes our zygote-derived cells as well as hundreds of species of microbes. the body is both an organism and a biome containing several ecosystems [28, 76, 101] . once the amnion breaks, and the fetus passes through the birth canal, the newborn becomes colonized by their mother's bacteria [87] . furthermore, mothers' milk contains a special set of nutrients to promote the survival and growth of those bacteria that are important for symbiosis [122] [123] [124] . these symbiotic bacteria will produce short-chain fatty acids and sphingolipids necessary for intestinal peristalsis and homeostasis, peptidoglycans necessary for normal neuron function, lipopolysaccharides necessary for the actions of the immune system, the tripeptides necessary for cardiac physiology, and the digestive enzymes necessary to metabolize plants [28] . remarkably, a third of the small metabolites in the blood are produced or induced by bacteria [125] . nearly all of our peripheral serotonin is induced by gut microbes, where it regulates the maturation of the enteric nervous system and regulates peristalsis [83, 126] . even more remarkable is that such critical symbioses are not only present within humans. rather, the development of most organisms appears to be predicated on interactions between hosts and their symbionts. as mentioned in the above section 1, without microbial symbionts, mice do not form their gut capillaries, their gut-associated lymphoid tissues, their t-and b-cell repertoires, or the proper synaptic connections in their guts and brains. moreover, no vertebrate contains genes that make the enzymes necessary for digesting plant material such as cellulose, hemicellulose, and pectins [127] . these genes are provided by symbiotic microbes in our guts. in mammalian evolution, the entire family of ruminants is made evolutionarily possible by the ability of gut bacteria (acquired at birth) to build the rumen of the stomach and then to ferment grass and grains [128] [129] [130] [131] . thus, symbiosis is a paradigm-changing idea in physiology. we are no longer seen as being "individuals." we are holobionts, and our anatomy, development, immunity, and physiology are intimately linked with that of our microbial components. the importance of the holobiont perspective for public health is that absences of particular microbes may cause dysbioses throughout a population. martin blaser and colleagues [6, 132] have warned that we may need particular microbes for particular functions, and that our obsession with exterminating microbes may be inadvertently killing those bacteria that we need to survive. their data indicate that microbes that used to be prevalent (those, for instance, in barnyards and horse stalls) are becoming rarer. if these microbes are necessary for normal organ, immune, or cognitive development, we will be impaired. rhesus macaques that are bottle-fed, rather than breast-fed, acquire a different population of gut microbes, and this population is not as adequate to develop a functioning immune system that can repel opportunistic infections [133] . in zebrafish, a relatively rare species of bacteria is essential for permitting the expansion of insulin-producing pancreatic cells, thereby protecting these fish against diabetes [134] . this absence of specific bacteria (or their genes) may be crucially important for explaining the increases in allergies and asthma since world war i, and especially, after world war ii. throughout human history, we had constant exposure to barnyard microbes. it was only in the 20th century that they were displaced. the barnyard was not just an attribute of farms. the nineteenth century city, according to raulff [135] (p. 36) "consisted of rows and rows of urban stables." mid 19th c. boston had some 367 stables, each having around eight horses. in contemporary america, only 6% of amish children, whose homes are often adjacent to their barns, have allergy and asthma. about 20% of the genetically similar hutterites, whose farms are not located close to their homes, have allergies, roughly the same as the american population in general [136] . similarly, finnish studies have shown that proximity to the barn is a factor in combatting allergies. a recent study shows that children living in urban homes with barnyard bacteria have much less asthma and allergies than those children living in urban homes with urban bacteria [109] . indeed, two of the bacterial types found in the "rural" homes and missing in "urban" homes were brevibacterium and ruminococaceae, bacteria found in horses and cattle. although the severity of microbiome diversity loss might be most discernable in urban populations [137] , the importance of soil microbiomes for the maintenance of healthy human intestinal microbiomes has recently been emphasized in studies [138] showing that even in rural areas, farming techniques have severely reduced soil microbiome biodiversity. bacterial displacement due to urban living and the absence of animals is only one of the ways that anthropogenic microbial displacement can affect public health. caesarian sections disrupt one of the pathways of maternal kinship. babies receive a protective set of symbiotic microbes from mothers when they pass through the birth canal. in caesarean sections, this transmission is abrogated. babies delivered by c-section were found to be deprived of those microbes that otherwise colonize the infant gut. instead, there were the hospital dwelling microbes that included a substantial number of opportunistic pathogens. moreover, a substantial set of these microbes contained genes associated with antimicrobial resistance [139] . not only were the species of microbes different, but so were their functions. the caesarean-delivered infants had less ability to mount immune responses to common antigens [140] . this may have strong public health implications concerning elective c-sections. the microbes of our gut are critical for "basic neurogenerative processes such as the formation of the blood-brain barrier, myelination, neurogenesis, and microglia maturation." [141] . if this is indeed true, then could microbes also be critical for mental health? what if, in addition to protection against allergies and asthma, bacteria were protecting us against mental health conditions such as schizophrenia, bipolar disease, and autism? several studies now indicate that gut microbes appear to be critical for normal brain development and behaviors in mice [82, [142] [143] [144] [145] [146] [147] [148] . first, mice born from germ-free mothers and who are themselves without microbes have a syndrome that includes obsessive self-grooming and asocial behavior [149] . this behavior is possibly due to the failure of oxytocin-releasing signals from the vagus nerve, and it can be reversed by providing the germ-free mice with lactobacillus reuteri or with microbes from normal mice or from normal humans [141, 150] . germ-free mice given microbes from autistic humans do not show improvement of their symptoms. although human cognitive and affective behaviors cannot be extrapolated for those of mice, a pilot uncontrolled fecal transplant study in humans showed that after two years, the acquisition of normal bowel microbes by autism patients significantly improved their symptoms: from 83% severe autism to 17% severe autism [146] . similar studies in mice and humans have shown that the gut microbiome may be critical in protecting humans from depression [42, [151] [152] [153] . there is therefore reason to test the hypothesis that removal or depletion of normal environmental microbes may be responsible for the increasing percentage of the population diagnosed with cognitive dysfunction. while studies of the effects of microbes on mental health lag behind those studies of microbial involvement in physical health, the relationship of symbiotic microbes to cognitive and affective health and disorder is an area that cannot be ignored. public health must acknowledge that we are not monogenomic individuals. we are consortia of dozens of species per person, integrated together in a complex and dynamically changing network that forms who we are at any given moment. this network is altered by the food we eat, by the food our mother ate, the toxins and medications we are exposed to, and by our daily interactions with other holobionts. our health depends upon other species, making the "one health" perspective more than metaphor. the symbiotic networks of the human holobiont are enmeshed in larger symbiotic networks that sustain the planet. public health would be severely affected if any of the many life support systems on which we depend-including pollinators, soils, and bacteria-fail. indeed, symbiosis is the signature of life on earth. the nitrogen in our soil and atmosphere is made available for protein synthesis by symbioses between rhizobacteria and legumes. the interactions of plant roots and mycorrhizal fungi are critical for plant growth, while endophytic fungi are often necessary to protect the plants against dessication [154] [155] [156] . the coral reef ecosystem is dependent on the symbiosis of algae and the ectoderm of corals, while the marine seagrass ecosystems are sustained by symbioses involving clams and their bacteria. reef-building corals survive through the photosynthesis of their algal symbiont, which enters into the ectoderm of its host and transports over 90% of its photosynthetically derived carbon compounds to the host cells [157] . however, these symbioses, the very symbioses that define the planet, are at risk. these are the analogues of the microbial displacement and extinction that affect human health. although public health is mainly concerned about "human" public health, one readily finds that we cannot separate ourselves from our ecosystems socially, politically, economically, or biologically. coral reefs, for instance, are thought to support 500 million people across 50 nations and contribute nearly a trillion dollars to the world's economy [158] . the great barrier reef, alone, brings 7 billion dollars annually to australian commerce. healthy coral reefs absorb over 95% of a wave's energy, thus protecting the shoreline, preventing nearly a hundred million dollars' worth of flood damage each year. however, the coral that form the critical structure of these reefs must be seen as a holobiont that exists only in a fragile symbiosis between the coral animal and single-celled zooxanthellae algae. the coral animal provides a sunlit, safe, and nutrient-containing environment for the algae; and the algae, living within the animal cells, provide the coral with the sugars it produces by photosynthesis. the coral holobiont can survive only when its symbionts are present to provide the food resources [157, 159] . under stress conditions such as high temperatures, the symbionts are expelled from the corals, leaving the corals "bleached" and undernourished. these corals usually die. as a result of global warming, massive bleaching events and coral die-offs have occurred [160, 161] . we are writing this essay not only in the coronavirus pandemic of 2020 but in the great barrier reef bleaching event of 2020 [162] . over half the corals in the great barrier reef have perished, and some entire reefs have collapsed. the mechanisms for the expulsion of the algal symbiont from its coral host are under investigation, and it appears to be a mutual breakdown of the symbiotic relationship [163] . one hypothesis is that heat disrupts the photosynthetic apparatus of the algae, causing them to produce dangerous hydrogen peroxide radicals. the coral cells defend themselves by expelling the algae or destroying them. another hypothesis is that warmer temperatures permit algae to get the organic nitrogen that allows them to metabolize their sugars without needing the coral, thereby forcing the coral to rely on their own meager carbon reserves [164, 165] . in addition to anthropogenic heating, humans are also affecting symbiosis through domestication. mycorrhizal symbiosis is critical to plant nutrition and, therefore, a necessity for sustainable agriculture. however, artificial fertilization of soil diminishes the mycorrhizal fungi and root symbiosis. martin-robles and colleagues [166] have linked the loss of symbiotic colonization with plant domestication. indeed, failure to colonize is common, making domesticated strains addicted to artificial fertilization [167, 168] . moreover, the lack of myccorhizal fungus may make the domesticated plants more susceptible to pathogenic fungi [169] . we are integrated into these webs, where our nutrition, oxygen, and environmental temperature depend on global symbioses, and microbes are at the base of each of them. the anthropocene has put these relationships in peril. as deborah bird rose [170] wrote, "relationships unravel, mutualities falter, dependence becomes a peril rather than a blessing, and whole worlds of knowledge and practice diminish. we are looking at worlds of loss that are much greater than the species extinction numbers suggest." the vectors of disease are following the sun and following airplane and sea lanes. wastewater, tourism, and trade are circulating microorganisms around the world in a scale never before seen [171] . moreover, global warming is predicted to introduce new microbes from melting permafrost as well as bringing many insect-borne diseases (dengue fever, malaria, lyme disease etc.) into new regions [69] . here, the vector spreads a pre-existing symbiont. these will undoubtedly cause major public health concerns. however, another mechanism of disease can be predicted: when organisms reach new lands, they are capable of finding new symbiotic partners. there is a new anthropogenic mingling going on. as an example, consider the red turpentine beetle, dendroctonus valens, a minor pest species that routinely infects pine trees that have been damaged by weather or fire. like other bark beetles, it is covered by fungi. these fungi digest tree bark, allowing the beetle to have a home and mate. the fungus associated with d. valens is usually leptographium procerum. however, this beetle was introduced from the pacific northwest of america to shanxi province of china in the 1980s. in china, it met other fungi, which are much more potent at digesting wood than the american fungi [172, 173] . these newly acquired fungi can degrade a major host defensive chemical [174] . as a result, over ten million pine trees have been killed by this fungus in china. american officials are worried about a "boomerang effect" [175] . the version of the beetle with its chinese fungi may have been re-imported into the usa. however, the public health services of the various states that might be affected claim they do not have the revenue to test whether this is so. organisms are holobionts, and public health must recognize the webs of symbioses uniting different species of organisms into a collective "individual" and uniting these different individual teams into complex ecosystems. symbiosis takes two major forms-mutualism (cooperative) and parasitism (pathogenic). the emergence of antibiotic drug resistance is the anthropocene effect on parasitic symbiosis. just as anthropogenic changes in the environment have changed the populations of microbes involved in mutualistic interactions with humans, so other anthropogenic changes have increased the prevalence and virulence of parasitic microbes. until the early 20 century, the leading cause of death, world over, was infectious disease. crucial to turning this around were sanitation of water, and the discovery of antibiotics. since their discovery in the 1920s, antibiotics have become the key tool against infections caused by microbes, used across different forms of medicine. by this definition, microbes are understood as pathogenic and parasitic, dangerous, dirty, and damaging the host that they reside in. bodies are seen to be 'at war' against harmful outside invaders and entire disciplines have been hinged on this notion-immunology, clinical medicine, and public health just to mention a few. antibiotics have been the miracle weapon that have been used to tackle the looming threats of bacteria and have been said to have developed contemporary medicine to be the success story that it is today [24] . antibiotics have magnificent power to alleviate symptoms and ensure sterile conditions; they play central roles in basic surgeries, cancer, cesarean birth, and in treating basic infections. they are prescribed against infections by doctors, nurses, pharmacists, dentists, and traditional healers, depending on the contexts, all across the world. there are very few communities left that have not incorporated the use of antibiotics into their basic methods of healing, and research on those communities is tapping to their 'untouched microbiomes' microbiome as an 'oasis' [176] [177] [178] [179] . literature about antibiotic prescription describes how requests for antibiotics reside on all sides of the patient-health care practitioner dyad: patients say that health care practitioners hand out antibiotics liberally and health care practitioners argue that patients demand them [180] [181] [182] . in addition, antibiotics are bought over the counter from pharmacies, and informal markets [183] . antibiotic use is a matter of concern as excessive or unregulated use of antibiotics is connected to the development of drug resistance and while there are few new antibiotics in the pipeline, there is a need to ensure the utility of existing ones [184, 185] . antibiotic use patterns offer insights into how central antibiotics are to public health, as well as the specific practices and contexts that rely on the use of antibiotics. understanding these dynamics also illuminates the effects of pathogenic thinking as well as the myriad ways in which reliance on antibiotics would need to change in order to make space for a holobiont practice of public health. global statistics about antibiotic use show differences between countries that often follow the guidelines of health system efficiency and general national income. since the 2000s, antibiotic use in low-and middle-income countries has considerably increased, while in high-income countries, particularly with those that rely on public rather than private health care, antibiotic use has been reduced. india, pakistan and china are among those countries where use has increased most [186] , while data is unavailable in most african countries [187, 188] . that said, despite the reduction in the high-income countries, antibiotic use in many european countries and the us is still considerably higher per capita than across many african nations [186, 188] . the increase of antibiotic use in low-income countries underscores the utility of antibiotics within lagging health care systems and/or in places where people cannot afford health care. especially in countries where health care access is precarious due to lack of access, poverty, or poorly operating health systems, antibiotics have come to play a central role in how short-term health goals are achieved. for example, work by denyer willis and chandler [189] shows how antibiotics function as a 'quick fix' for well-being. this fix operates on multiple domains: to ensure productivity of humans, animals and crops; hygiene in settings of minimized resources marked by lack of infrastructures; and good health in landscapes scarred by political and economic violence. in short, antibiotic use has come to stand for development and well-being. while use of antibiotics has played a crucial role in helping to increase life expectancy, implementing invasive surgical procedures, and stand in for health care systems where they are otherwise unavailable, the use of antibiotics has accelerated embodied and ecological havoc. a narrow characterization of microbes solely as parasitic and pathogenic enemies rather than as needed and helpful partners contributes to excessive use of antibiotics for humans and animals, where microbes 'refuse' to remain contained in bodies but shift their form by evolving resistance to antibiotics. the heroic narrative of antibiotics is beginning to crumble as microbes push back. mass scale attempts to eradicate bacteria with antibiotics in humans and animals has led to increase of antimicrobial resistance (amr), making it a quintessential anthropocene problem. indeed, the mass scale of antibiotic production, beginning in the 1940s, "quickly became infrastructural to the production of many other things at scale: more health, more meat, more fruit, more surgery, less death, more fertility, in everything from in vitro embryos cultured in antibiotics to fish farming. the scale of production is also the scale of resistance" [24] . the higher-than-expected levels of amr put western medicine in its current form-where antibiotics play central roles-at risk. with antimicrobial resistance, global health literature continues to frame microbes as a threat, now an incurable threat. the most comprehensive report about amr and its future impacts, the so-called o'neill report commissioned by the uk government and the wellcome trust, indicated that 7 million people will die due to complications associated with amr [190] . this report has evoked a flurry of research efforts, systemic interventions, stewardship programmes, and funding to tackle amr. health risks for humans have been extensively documented, with resistance spreading owing to both excessive use of antibiotics for human consumption and the use of antibiotics as part of animal feeding and in husbandry. a key route by which amr spreads is via environmental bacteria that serve as vectors for the resistant genes-lateral gene transfer-which is seen to become a problem when otherwise benign environmental bacteria contribute to the spread of resistance in pathogens [24, 191, 192] . robinson et al. state that this otherwise 'natural' quality of environmental bacteria is exacerbated, for example, by the influx of antibiotic residues from human and animal faeces, and run-offs from hospitals and pharmaceutical manufacturing [193, 194] . a global comparison of socio-economic determinants correlated with amr prevalence offers insights into the crucial roles that developmental and social inequalities play in anthropocene ecology. factors predicting high amr rates are not antibiotic consumption, but, rather, differential access to sanitation, education, and public investment in health care services, as well as the level of corruption in society [195] . the focus, therefore, cannot be simply on clinical bodies, but must broaden to encompass environments including animals and infrastructures on the one hand, and social practices and power on the other. we posit that the notion of plantationocene captures this complexity that transcends the human-more-than-human bodily boundary while taking power structures into consideration. as defined above, the plantationocene constitutes the coercive labor structures and extractive and hierarchical management of planetary resources to feed an ever-growing population [17, 18] . the plantationocene acts here both an analytical and a descriptive term. analytically, plantationocene points to transnational circulations of goods, domination and dominion of people over other people and people over nature, hegemonic colonial legacies, systematisation of farming. haraway et al. [196] point to the historical origins of the term and how relocations of the substances of living and dying around the earth as a necessary prerequisite to their extraction. the logic of the plantation system makes it more efficient to destroy the local labor and import labor from elsewhere. the plantation system is built on the relocation and control of any generative unit, whether plant, animal, microbe, or person [196] . indeed, plantations were the result of one of the most catastrophic public health events in world history-the columbia exchange. a major part of this exchange resulted in the elimination of a majority (perhaps 90%) of indigenous american people by the microbes-rubeola, variola, influenza, rubulavirus, rickettsia, salmonella and bordetella-brought across the atlantic ocean by the european settlers. the great migration of people and crops took place to bring workers to areas whose native populations had perished, especially in the caribbean, where the death rate of indigenous people was probably close to 99% [197] [198] [199] . intensive labour was needed to produce crops in north and south america, and the 'workers' at plantations were slaves shipped from west and central africa-now sites that have the least infrastructure to surveil and control amr, but have the most troubling evidence of amr prevalence [187, 200] . these were also sites of resource extraction as well as subjected to structural adjustments in the 80s by the world trade organisation to privatize health care and social welfare, resulting in poor health care and sanitation infrastructures and overall poverty that now are known to be key factors for the development of amr. the industrial agriculture of the plantationocene may also contribute to the spread of drug resistant microbes. the recent increase in resistant fungicides such as candida auris, that has caused tremendous concern among health practitioners and ecologists alike, is a resistant yeast that has contaminated entire hospitals [201, 202] . its spread has environmental vectors-resistance has developed in connection to the use of fungicides in monocropping [203] . environmental and agricultural practices are thus directly connected to public health concerns. amr with plantationocene underscores that public health needs to re-think its relationships with bacteria and antibiotics-it cannot bracket out environmental extraction, socio-economic injustices and the on-going need for health systems strengthening as factors that create the conditions for why excessive antibiotics are used that lead to antimicrobial resistance. amr by this definition is not an exemplary threat by microbes as is framed in global public health but should be seen as a result of the modernist, eradication approach towards microbes that requires rethinking. health is a negotiation between microbes and hosts. holobiont public health would do well to recognize both the parasitic and the mutualistic branches of symbiosis [204] it would also recognize the two major changes in our scientific knowledge of microbial evolution that have occurred in this century: (1) organisms are holobionts composed of several species, wherein microbes help maintain healthy physiology and resilience; and (2) bacteria can pass genes through horizontal genetic transmission, thereby facilitating the rapid spread of antibiotic resistance through numerous bacterial species. symbionts must be seen as partners and respected as agents with their own agendas. three recent examples of holobiont "management" for public health should be mentioned in this regard. the first concerns the public health against mosquito-transmitted diseases such as dengue, zika, and chikungunya by using wolbachia bacterium to infect aedes egypti mosquitos. wolbachia infects numerous insects, but not these species of mosquitos. however, wolbachia can become a symbiont in these insects, preventing the acquisition or replication of viruses inside their cells. scientists have been able to get wolbachia to grow inside aedes cells, and wolbachia-infected mosquitoes have been released into the wild. where this has happened, there has been significant drops (up to 76%) in reported cases of the vector-transmitted diseases [204] [205] [206] . the second holobiont-informed type of public health involves seeking alternatives to antibiotics and partnering with microbes capable of keeping pathogens in check. if symbionts help protect hosts from pathogenic bacterial infections, then symbiotic microbes would be a good place to start looking for new antibiotics. this is especially true of antibiotics for gram-negative bacteria. the antibiotics currently in use were developed in the 1960s, and several bacterial species have successfully been evolving resistance to them. certain nematode worms are susceptible to the same types of gram-negative bacteria as humans, so imai and colleagues [207] sought out the antibiotics made by the symbiotic strains of bacteria found in the nematode guts. by screening chemicals made by these symbionts, they have isolated darobactin, a modified and crosslinked 7-amino acid peptide. this antibiotic acts by disrupting the cell envelope of the gram-positive pathogens and is largely non-effective in destroying human gut commensals. the experiments further show that this new antibiotic is effective at protecting infected mice given potentially lethal infections of gram-negative bacteria. the third approach recognizes the importance of microbes to the life cycles of parasites and seeks to kill the parasite by killing its symbionts. this approach has worked in eliminating schistosoma mansoni, a filariasis worm that has become resistant to the drugs traditionally used to kill these parasites. a newer treatment strategy has been to use antibiotics (such as doxycyline) against its symbiotic bacteria [208, 209] . once the antibiotic destroys the symbiont, the worms' cells undergo apoptosis and the worms die [210] . a similar strategy is being considered to eradicate the plague locusts that are now devastating east africa. here, a locust-specific fungus might be sprayed on the juvenile locusts as they develop their wings. this fungus would grow inside the maturing insect and consume it from within [211] . we need to be in symbiosis with bacteria on a social, as well as on a corporal level. like the body, we need to be able to distinguish mutualistic from pathogenic microbes and treat them differently. humanity has been given notice. a paper by the alliance of world scientists [212] "puts humanity on notice that the impact of climate change will depend heavily on the responses of microorganisms which are essential for achieving an environmentally sustainable future." public health must take note that we humans are never independent of nature and, therefore, must be expanded to preserve environmental health as well as human and animal health. resilience to perturbations is increased by 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declare no conflict of interest. key: cord-310882-t73xwpaw authors: axin liang, a.; huipeng hou, b.; shanshan tang, c.; liquan sun, d.; aiqin luo, e. title: an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection and determination of human igg date: 2020-09-09 journal: bioelectrochemistry doi: 10.1016/j.bioelechem.2020.107671 sha: doc_id: 310882 cord_uid: t73xwpaw an advanced molecularly imprinted electrochemical sensor with high sensitivity and selectivity for the detection of human immunoglobulin g (igg) was successfully constructed. with acrylamide imprinting systems, surface imprinting on the nanoparticles cufe(2)o(4) targeted at igg was employed to prepare molecularly imprinted polymer, which served as recognition element for the electrochemical sensor. furthermore, the sensor harnessed a molybdenum disulfide (mos(2))@nitrogen doped graphene quantum dots (n-gqds) with ionic liquid (il) nanocomposite for signal amplification. under optimized experimental conditions, the sensor shortened the response time to less than 8 min, and the response was linear at the igg concentration of 0.1–50 ng·ml(−1) with a low detection limit of 0.02 ng·ml(−1) (s/n = 3). our findings suggested that, the sensor exhibited high detectability and long-time stability. the satisfactory results of human serum sample analysis showed that the developed igg sensor had promising potential clinical applications in detecting igg content. to target analytes [27] [28] . many novel nanomaterials can be applied in modifying gce, including mos 2 [29] [30] , metal nanoparticles [31] [32] , graphene quantum dot [33] and carbon black [34] [35] . mos 2 is a typical dichalcogenide, which attracts wide interests in numerous fields, such as hydrogen catalysis and storage, capacitors, and electrochemical device [36] [37] . however, because the conductivity of mos 2 is still lower than that of carbon-based materials, little attention has been paid to the application of mos 2 as an electrode material for sensors [38] . therefore, it is suggested to combine mos 2 with carbon-based materials to show a synergistic effect on the application of electrocatalysis [39] . guochuang huang et al. obtained a layered mos 2 -graphene composite modified electrode with good electrochemical performance, and it was able to detect paracetamol with high sensitivity and selectivity by l-cysteine assisted liquid phase method [40] . tong guo et al. designed a novel three-dimensional (3d) layered mos 2 @graphene functionalized with n-gqds composites, which was used as an enhanced electrochemical hydrogen evolution catalyst [41] . as a result, it is reasonable to speculate that, integrating the layer-structured mos 2 with nitrogen-doped graphene quantum dots (n-gqds) also exerts same function in electrochemical sensors and electrocatalysis. moreover, more and more importance has been attached to ionic liquids (il) during sensor construction due to optimized structures and properties, distinguishable anion/cation combination, high conductivity, remarkable biocompatibility, and good thermostability [42] [43] . ionic liquid is an effective modifier to improve the sensing performance of electrode [44] . these results indicate that the use of ionic liquids can improve reaction sensitivity and promote efficient direct electron transfer of various redox biomolecules. therefore, based on the strong electrostatic-chemical interaction between il and mos 2 @n-gqds and the improvement dispersion by il, the addition of il is expected to further enhance the electrochemical signal in the fabrication of sensor. the recognition element is a vital part in electrochemical sensor. numerous recognition elements are adopted for improving electrochemical sensor selectivity and sensitivity, such as aptamers, phages, antibodies, as well as the molecularly imprinted polymers (mips) [45] [46] [47] [48] . molecular imprinting has been developed as a typical recognition site formation process induced by the template within the polymer [49] . those molecularly imprinted synthetic receptors have exhibited integrated properties, including high affinity, robustness, low production cost, and great specificity. as a result, they may serve as the promising candidate natural receptors [50] [51] [52] . great advances have been attained in the fields of nanotechnology and polymer science, which have strengthened the performances of molecularly imprinted polymer (mip) sensors. many high-quality articles have been published recently to describe mip sensors in determining explosives, abused drug and biomolecules, which facilitate applying such technique in the forensic and medical diagnostic fields [53] [54] [55] . specifically, the magnetic cufe 2 o 4 nanoparticles (nps) have been used as the carriers of the surface-imprinted modified electrochemical sensors, which is ascribed to their superparamagnetism, high specific capacitance, favorable biocompatibility and excellent electroconductibility. our research group prepared a novel electrochemical sensor based on the mips modified cufe 2 o 4 glassy carbon electrode (gce) to electrochemically detect lysozyme [56] . this study aimed to develop an electrochemical sensor to selectively detect igg. the methods used were shown below (schematic 1). firstly, the mos 2 @n-gqds-il nanocomposite with high conductivity was used to modify the gce, so as to amplify all reagents and solvents adopted in this experiment were analytically pure, which were utilized as received. each aqueous solution was prepared with ultra-pure water (18.2 mω cm). a ph meter (sartorius pb-10) was used to determine the solution ph. chi6043e electrochemical workstation (ch instruments, shanghai, china) was adopted for all electrochemical test, where the traditional three-electrode system was employed consisting of a modified gce (diameter, 3.0 mm) working electrode, a platinum wire counter electrode, and an agcl (3 mol·l -1 kcl) reference electrode. shapes and sizes of mos 2 @n-gqds-il nanocomposites were determined by the highresolution h7700 transmission electron microscopy (tem, hitachi, japan) at 100 kv of accelerating voltage using the tecnai f20g2 (fei, netherlands). scanning electron microscopy (sem) was conducted using the inspect f50 microscope (fei, netherlands). afterwards, the ultima iv x-ray diffractometer (rigaku, japan) in the presence of cu kα radiation (λ=1.54178å) was applied for x-ray diffraction (xrd). axin liang, et al. an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg: bioelectrochemistry 5 the uv-1800 (shimadzu, japan) was adopted to obtain the ultraviolet-visible (uv-vis) spectra. serum igg was detected by the bn ii automatic protein analyzer (siemens, german). the 2 mg·ml -1 stock solutions of hsa, bsa and lyz were prepared through dissolution into ultra-pure water and the phosphate buffer solution (pbs), and then preserved at +4℃ within the refrigerator. the pbs (ph = 7.4) containing 5 mmol·l -1 k 3 fe(cn) 6 and 0.1 mol·l -1 kcl was adopted for measuring eis and dpv. the present study prepared n-gqds through hydrothermal treatment with dicyandiamide and citric acid (ca) as reported in references [57] [58] [59] . briefly, 1 g dcd and 2 g ca were added into 5 ml ultra-pure water during the typical synthetic process, followed by transfer to teflon lined autoclave (25 ml), 12 h of heating under the temperature of 180 ℃, and natural cooling of the reactor to ambient temperature. then, the ethanol was added into the product, centrifuged at 5000 r/min for 5 min, washed for 3 times, and dried for 24 h within the vacuum drying oven under the temperature of 60 ℃ to obtain the solid with good water solubility. the modified solution-phase approach assisted by l-cystein was adopted to prepare mos 2 @n-gqds composites, and the final optimal composition of n-gqds and mos 2 was obtained mainly by references and later experimental optimization [60] [61] . specifically, ultra-pure water (50 ml) was added into the 200 ml beaker, followed by transfer of 0.1 g n-gqds and addition of na 2 moo 4 2h 2 o (0.5 g) in it. following 30 min of stirring and ultrasonication, the solution ph was adjusted to 6.5 with the 0.1 mol·l -1 hcl. later, l-cysteine (1.0 g), together with the mixture, was dissolved into ultra-pure water (100 ml), and the resultant mixture was then transferred to the teflonlined stainless steel autoclave (100 ml) and tightly sealed, followed by 36 h heating under the temperature of 180 ℃. later, that autoclave was naturally cooled, centrifuged to collect black precipitates, rinsed by ethanol and ultra-pure water, and dried for 24 h at 60 ℃ within the vacuum oven. mos 2 @n-gqds-il composites were prepared according to the published literature, and the final optimal composition of each component was obtained mainly by references and later optimization [62] . to be specific, 0.04 g [bzmlm]bf 4 was put to 20 ml of 0.01 g ml -1 mos 2 ultra-pure water dispersion at first, and then stirred continuously for 12 h under the temperature of 60 ℃. thereafter, the as-obtained suspension was put to the oil bath at 90 ℃ for 1 h. the 0.22 μm filter membrane was used to filter the resultant dispersion solution, followed by 12 h of drying at 60 ℃ within the vacuum oven (schematic 1). axin liang, et al. an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg: bioelectrochemistry schematic 1 synthesis reaction of mos 2 @n-gqds-il. the cufe 2 o 4 nps were synthesized according to the hydrothermal approach [63, 56] . in brief, 0.408 g cucl 2 ·2h 2 o (2.4 mmol) and 1.296 g fecl 3 ·6h 2 o (4.8 mmol) were immersed into 60 ml ethylene glycol for forming the clear solution. thereafter, 1 g polyethylene glycol (m=20000) and 3.6 g naac were slowly added to the asprepared solution for 1 h, and vigorously stirred under ambient temperature for 24 h to achieve fully dissolved. then, the as-prepared mixture was transferred to the teflonlined stainless steel autoclave (80 ml), heated for 10 h at 200 ℃, and then the autoclave was cooled naturally to ambient temperature. later, the permanent magnet was used to separate those nanohybrid magnetic materials, which were subsequently centrifuged at 4000 rpm, washed with ethanol and ultra-pure water for 3-5 times, and dried for 5 h using the vacuum freeze drier. magnetic mips nps was prepared in the following steps: first of all, 1.4 mmol igg was dissolved into 0.02 mol·l -1 phosphate buffer solution (pbs) (20 ml, ph = 7.4); then, 30 μl maa, 100 mg nipaam, as well as 30 mg aam were added in succession. afterwards, the pre-assembled solution was synthesized through 1 h of stirring. 100 mg cufe 2 o 4 magnetite was added into 0.02 mol·l -1 pbs (20 ml, ph = 7.4) to stir for 20 min. later, 25 mg n,n'-methylene-dipropylene amide (mba) was added into the cufe 2 o 4 -pbs mixture, and the resultant mixture was then put into the pre-assembled solution. typically, the mixture was subjected to 1 h of stirring to prepare the prepolymerized solution, followed by preservation within the three-necked flask. then, the 30% temed (100 μl), together with the 10% wt ammonium persulfate (aps, 20 μl), was put into the polymerization radical initiator for 24 h of reaction at nitrogen atmosphere. later, magnet separation, and repeated washing with ultra-pure water and ethanol were conducted. afterwards, the mixture of acetic acid (aa) (10%, v: v) and acetonitrile (acn) (90%, v: v) was used to wash the mips nps, and template protein was extracted until there was no eluent absorbance at the wavelength of 340 nm. nips nps were prepared using the same procedure without adding igg [17, 29, 57 ]. the mos 2 @n-gqds-il solution (2 mg/ml) was prepared by dissolving mos 2 @n-gqds-il in ultra-pure water. then, mos 2 @n-gqds-il solution (80 μl) was dispersed into mips nps suspension (1 ml) under 30 min of ultrasonication. before preparation, the alumina slurries (0.05 μm) were used to polish the gce, followed by sequential ultrasonic cleaning in acetone and ethanol. later, appropriate mips nps suspension was dripped onto the gce surface, and the solvent was then evaporated to obtain the imprinted gce. non-imprinted mos 2 @n-gqds-il/gce was prepared according to the same procedure except using nips nps instead of mips nps and referred to as the non-imprinted sensor (schematic 2). schematic 2 electrochemical determination of human igg using cufe 2 o 4 nanoparticles molecularly imprinted polymers modified with mos 2 @n-gqds-il. electrochemical measurements were performed in pbs (ph = 7.4) containing 5 mmol·l -1 k 3 fe(cn) 6 and 0.1 mol·l -1 kcl. the volume of solution used was 50 ml. in addition, differential pulse voltammetry (dpv) was measured at a scan potential of -0.2 v to +0.6 v, a pulse amplitude of 50 mv, a pulse width of 0.05 s, and a scan rate of 100 mv/s. in cyclic voltammetry (cv) mode, scan range was from -0.2 v to +0.6 v and scan rate was 100 mv/s. thereafter, the electrochemical impedance spectroscopy (eis) analysis was recorded at an amplitude of 0.005 v, a potential of 0.24 v, and a frequency range of 0.01-100 khz. fig. 1a presents the sem images for the resultant mos 2 @n-gqds-il, which shows the 3d ball-like structure. typically, such a 3d structure increased the contact area with analytes. n-gqds coalescing or overlapping contributed to forming the interlinked conduction network, which also facilitated the quick electron transport during the electrode reaction. as observed from the n-gqds tem images in fig. 1b , the as-prepared n-gqds were the small sheets that had narrow distribution of size. n-axin liang, et al. an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg: bioelectrochemistry 8 gqds had the average diameter of 3.75 nm, which was verified by analyzing the images of 100 individual particles. in comparison with those previously reported gqds (0.5-2 nm in thickness and 15 nm in diameter) [64] , the n-gqds prepared in this study exhibited much lower lateral size while similar height. the representative mos 2 -il tem image is shown in fig. 1c . as observed, the il covalent functionalization offered a molecule layer onto those composites. fig.1d shows the high-resolution tem (hrtem) image for mos 2 @n-gqds-il. clearly, there were a few layers in mos 2 with layered structure, and the distance between two layers was 0.62 nm. obviously, those quantum dots synthesized according to our approach displayed great crystallinity, with a 0.22 nm lattice spacing, which was indexed to the (1 1 2 0) graphene lattice fringes [65] . fig. 1e suggested that, there were only 3 weak diffraction peaks of mos 2 @n-gqds-il composites, which were corresponding to (1 1 0), (1 0 0), and (0 0 2) mos 2 planes, and such results indicated poor mos 2 crystallinity. this phenomenon was ascribed to the fact that, incorporating n-gqds inhibited the growth of layered mos 2 crystal in the hydrothermal process. fig. 1f shows the xrd pattern of n-gqds, the 2 theta angles ranged from 5° to 85°, with a characteristic peak of 22.0°, which was consistent with the graphite structure of n-gqds reported in the literature [66] , indicating that n-gqds were prepared successfully. uv-vis spectroscopy was carried out to characterize the products. fig. 1g revealed that, the characteristic peaks for mos 2 @n-gqds-il composite and n-gqds were observed at 330 nm, respectively. in addition, mos 2 -il had no characteristic peak at 330 nm, indicating that n-gqds were attached to the mos 2 -il. eis is developed as a highly efficient approach to probe into those surfacemodified electrode features [67] [68] [69] [70] . specifically, those impedance spectra are comprised of the linear portion and the semicircle portion. besides, the high-frequency semicircle diameter is corresponding to the resistance of electron transfer (r et ), whereas the low frequency linear part is associated with a diffusion event [71] [72] . fig.2 shows various electrode electron transfer capacities and relevant results. clearly, bare gce had a low semicircle eis at a high frequency; in addition, the linear portion was observed at a lower frequency (curve a), which suggested a quite low r et to the [fe(cn) 6 ] 3-/4redox probe. nonetheless, as observed from the eis analysis for mos 2 @n-gqds-il/gce (curve d), mos 2 @n-gqds/gce (curve c), and mos 2 /gce (curve b), nearly straight lines were seen within the nyquist plot, indicating the little charge transfer impedance of these sensors. further, the electrode process was mainly controlled by the diffusion process, rather than the charge transfer. in addition, the ac impedance spectrum indicated that the bare electrode (curve a) had a high charge transfer impedance, which was remarkably low in the modified sensor, demonstrating that both of them had superb electron conductivity. on the other hand, the above ac impedance spectrum strongly proved the successful modification of mos 2 , mos 2 @n-gqds together with mos 2 @n-gqds-il onto the electrode surface. 6 and 0.1 mol·l -1 kcl (scan rate: 100 mv/s). mips nps, together with the cufe 2 o 4 nps, were investigated using hrtem, so as to verify the distribution of polymer coating, surface and structure. for cufe 2 o 4 nps (fig. 4a) , the spherical shape was observable, and the mean size was 160 nm. in the case of mips nps, (fig. 4b) the cufe 2 o 4 nps were surrounded by an imprinted layer. the imprinted layer had a thin edge of 15 nm, which was conductive to the elution and absorption of the template protein. ft-ir spectra of mips nps before template extraction, mips nps after template extraction and nips nps are presented in fig. 5 . based on those spectra for mips nps as well as nips nps, the fe-o peak was at 581 cm -1 and the broadband was centered at 3392 cm -1 due to o-h stretching overlap. with regard to the ft-ir spectra for mips nps (curve a, b), the peak at 1405 cm -1 was carboxyl, which was used to synthesize cufe 2 o 4 magnetic nanoparticles. comparing the ft-ir spectra for nips nps (curve c), it was discovered the characteristic absorption peak of igg (fig. 5) at 1590 cm -1 amide bond also appeared on the infrared spectra of mips nps before template extraction (curve a). this phenomenon represented that igg was successfully encapsulated, which suggested the successful preparation of imprinted complex. compared the ft-ir spectra for mips nps before template extraction (curve a), there was no 1590 cm -1 band on the infrared spectra of mips nps after template extraction (curve b), indicating the completeness of this extraction. the diverse electrodes were electrochemically characterized by cyclic voltammetric measurements within the pbs containing 5 mmol·l -1 [k 3 fe(cn) 6 ] and 0.1 mol·l -1 kcl. fig. 6 presents the cv distribution of bare gce and different modified electrodes. as shown, bare gce had one pair of reversible redox peaks (curve a). for the electrode modified with mips nps materials before removing the template, it was difficult for the ferrocyanide anions to enter the electrode surface, due to the poor electron transport of the dense membrane. as a result, the intensity of the electrochemical signals was significantly shortened (curve b). after removing the template from the mips nps matrix, the current response sharply increased, indicating that probe molecules diffused to the electrode surface through cavity regeneration (curve d), which invoked the so-called 'gate effect' [73] . at last, the mips nps/mos 2 @n-gqds-il/gce after binding igg (curve c) showed that the peak current decreased relative to curve d. this was called cavity occupation (sites of recognition) by igg. this study investigated influences of experimental variables on oxidation peak current (i p ) for the 1 ng·ml -1 igg on the imprinted gce using the dpv mode, due to its great resolution and sensitivity. the volume and content of the as-prepared mips nps dispersion that was dripped onto gce surface greatly affected the sensor selectivity and sensitivity, as presented in fig. 7a and b . clearly, i p remarkably changed as the volume or content of the asprepared mips nps dispersion altered. typically, i p peaked in the 10 μl mips nps dispersion (3 mg/ml). as the dispersion volume and content increased, the number of igg binding sites elevated. as a result, the presence of a large or low amount of modified mips nps on the sensor surface reduced the igg response. axin liang, et al. an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg: bioelectrochemistry 16 fig. 7 . impacts of the mips nps dispersion content (a) and volume (b). the differential pulse voltammetry (dpv) conditions: pulse width was 0.05 s, pulse amplitude was 50 mv, and scan rate was 100 mv/s, electrolyte solution was pbs (ph = 7.4) with 1 ng·ml -1 igg. error bar = rsd (n = 3). incubation time serves as the vital index to evaluate the sensitivity for the mip sensor. as a result, this study investigated how incubation time affected the mips nps responses by dpv within the pbs (ph = 7.4) containing 5 mmol·l -1 k 3 [fe(cn) 6 ]. thereafter, the molecularly imprinted electrochemical sensor was used to remove those template molecules, followed by immersion with pbs buffer supplemented with 1 ng·ml -1 igg standard solution for various time periods. according to fig. 8 , the peak current quickly shortened within the first 8 min, which indicated that those binding sites of the sensor were able to quickly and effectively adsorb igg molecules. after 8 min, the peak current decreased slowly and remained almost unchanged, which suggested the state of adsorption equilibrium reached by the imprinted sensor. in this study, the adsorption equilibrium peaked in 8 min, and such quick response was ascribed to the surface imprinting. axin liang, et al. an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg: bioelectrochemistry under the optimum condition, the sensor was employed to detect igg in pbs (ph = 7.4) after incubating it for 8 min (fig. 9a) . fig.9b presents a linear calibration curve for igg oxidation within the specific content range (0.1-50 ng·ml -1 ). typically, the mips nps detectability was detected within 0.1-50 ng·ml -1 igg, and the determined lod (s/n = 3) was 0.02 ng·ml -1 . further, that regression equation obtained was i(μa) =1.6211 lgc(ng·ml -1 )+9.1457, (r 2 =0.998). in addition, the comparisons of several reported analytical techniques for detecting human igg are listed in table 1 . as seen, the proposed method has comparable or even better limit of detection and linear concentration range. the research results suggested that this advanced molecularly imprinted electrochemical sensor can be used as an immunosensor for the determination of human igg in serum samples. to verify the reproducibility of our adopted approach, the calibration curves were repeated for three times by measuring the dpv response of a 1 ng·ml -1 igg solution using five independently electrodes under the same conditions. in addition, the values axin liang, et al. an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg: bioelectrochemistry of relative standard deviation (rsd%) were compared between 2% and 5%, and the results suggested acceptable reproducibility of those electrodes. adsorption values for the modified imprinted electrodes on 1 ng·ml -1 igg exhibited no substantial change among various time periods, which verified the method stability. those modified imprinted electrodes were put into the ultra-pure water for 2 weeks under ambient temperature. then, the dpv response was measured every three days. the igg peak current response for the imprinted electrodes was shortened to 93.4%, which indicated the favorable long-term stability and reusability of the electrode. to evaluate the selectivity of the sensor in target identification, several representative interferers that were commonly used in the actual sample detection process were selected in the experiment, so as to detect the sensor selectivity. to be specific, hsa, bsa and lyz were selected as the interferers in the experiment. under the optimal conditions, dpv responses of the proposed mip or nip in different concentrations (0.5, 1, 5, 20 and 50 ng·ml -1 ) igg solutions with or without interferences were assayed. we have obtained the calculation method of imprinting factor and selectivity coefficient through references [78] [79] . the imprinting factor should be determined from the ratio of slopes of calibration plots for mip sensor (0.354 μa/ ng·ml -1 ) ( table 2 ) and nip sensor (0.021 μa/ ng·ml -1 ) ( table 2 ). the imprinting factor was 16.9. moreover, the selectivity coefficient to each interfering compound could be determined as the ratio of the slope of the calibration plot for igg and a given compound including hsa, bsa and lyz (fig.10, table 2 ). and the calculated selectivity coefficient of hsa, bsa and lyz were 7.4, 8.4 and 9.3 respectively. advantageously, the sensor did not respond to hsa, bsa and lyz. consequently, the as-prepared igg biosensor in this study exhibited favorable selectivity for the target igg. such an experimental result showed that the biosensor might be used in accurately measuring igg concentration within practical specimens. for healthy people, the igg content within the serum is 8.0-16.0 mg·ml -1 . in this study, 3 whole blood specimens were obtained from the hospital physic examination center. after complete coagulation, the blood was centrifuged at 3500 r/min for 10 min to collect the upper serum. the blood serum specimens were diluted with pbs at the ratio of 1:200000 prior to analysis. the german siemens bn ii automatic protein analyzer (immune nephelometry) was used to detect serum igg, whereas the values were 25.5, 27.0, and 37.5 ng·ml -1 , separately. the above three specimens were measured with this sensor, and the igg contents were 27.1, 28.3, and 36.7 ng·ml -1 , respectively. the results were compared with the reference values obtained by immune nephelometry method as shown in table 3 . the relative deviation (%) between the electrochemical immunosensors and immune nephelometry method ranged from -6.3% to 2.1%. typically, the igg contents measured using the as-proposed igg sensor were in line with those protein analyzer values. besides, human serum specimens with spiked igg contents were adopted for validating our method. the recovery rates ranged from 94.6% to 101.8%. table 4 present the determined results for 3 distinct specimens and the recovery rates. axin liang, et al. an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg: bioelectrochemistry this study successfully prepares the layered mos 2 @n-gqds-il nanocomposite membrane, and first adopts it as the support matrix to construct the electrochemical sensor for igg. as observed, the mos 2 @n-gqds-il/gce system has markedly increased peak current, demonstrating that the mos 2 @n-gqds-il composite membrane plays a role as a highly effective promoter in enhancing the igg electrochemical performance. in addition, mips nps are then modified onto gce surface to develop the new electrochemical sensor, so as directly determine the igg level. notably, the developed electrochemical sensor can selectively recognize igg, meanwhile, it shows great reliability and accuracy, quick response time, and favorable reproducibility. our research results reveal that the developed igg sensor is successfully used for analyzing igg content in the human serum specimens, which may show promising clinical applications to detect the igg content. data independent analysis of igg glycoforms in samples of unfractionated human plasma elevated cytomegalovirus igg antibody levels are associated with hiv-1 disease progression and immune activation measurement of pre-existing igg and igm antibodies against polyethylene glycol in healthy individuals herpes simplex virus type 2 igg antibodies in sera of umbilical cord as a proxy for placental infection in asymptomatic pregnant women hiv exposed infants vaccinated with a mf59/rgp120 vaccine have higher magnitude anti-v1v2 igg responses than adults immunized with the same vaccine human igg subtype cross-species reactivity to mouse and cynomolgus monkey fcγ receptors interactions between dmpc liposomes and the serum blood proteins hsa and igg new blood tests for antibodies could show true scale of coronavirus pandemic detection of immunoglobulin g1 against rk39 improves monitoring of treatment outcomes in visceral leishmaniasis igg1 fc n-glycan galactosylation as a biomarker for immune activation development of an enzyme-linked immunoassay for the quantitation of influenza haemagglutinin: an alternative method to single radial immunodiffusion immunoassay with single-walled carbon nanotubes as near-infrared fluorescent labels comparison on the anti-interference abilities of immune turbidimetry and immune nephelometry for detecting specific proteins quantum dot loaded liposomes as fluorescent labels for immunoassay enzyme immunoassay and enzyme-linked immunosorbent assay orientation-controlled bioconjugation of antibodies to silver nanoparticles detection of toxoplasma-specific immunoglobulin g in human sera: performance comparison of in house dot-elisa with eclia and elisa electrochemical impedance immunosensor based on gold nanoparticles-protein g for the detection of cancer marker epidermal growth factor receptor in human plasma and brain tissue a novel, label-free immunosensor for the detection of α-fetoprotein using functionalised gold nanoparticles electrochemical impedimetric immunosensor for the detection of measles-specific igg antibodies after measles infections the strategy of nitrite and immunoassay human igg biosensors based on zno@zif-8 and ionic liquid composite film mercapto-propyl)-pyridine hexafluorophosphateionic liquid functionalized gold nanoparticles for igg immunosensing enhancement development of capacitance based immunosensors on mixed self-assembled monolayers preparation of cu 2 o-reduced graphene nanocomposite modified electrodes towards ultrasensitive dopamine detection fabrication of amine-modified magnetite-electrochemically reduced graphene oxide nanocomposite modified glassy carbon electrode for sensitive dopamine determination facile electrochemical sensor for nanomolar rutin detection based on magnetite nanoparticles and reduced graphene oxide decorated electrode an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg mno 2 nanowiresdecorated reduced graphene oxide modified glassy carbon electrode for sensitive determination of bisphenol a high sensitive voltammetric sensor for nanomolarity vanillin detection in food samples via manganese dioxide nanowires hybridized electrode in situ synthesis of mos 2 /graphene nanosheet composites with extraordinarily high electrochemical performance for lithium ion batteries mutually exclusive pmilype and nmilype hybrid electrode of mos2 and graphene for artificial soft touch fingers a selective molecularly imprinted electrochemical sensor with go@cof signal amplification for the simultaneous determination of sulfadiazine and acetaminophen a novel molecularly imprinted polymer decorated by cqds@hbnns nanocomposite and uio-66-nh2 for ultraselective electrochemical sensing of oxaliplatin in biological samples layering of a film of carboxymethyl-botryosphaeran onto carbon black as a novel sensitive electrochemical platform on glassy carbon electrodes for the improvement in the simultaneous determination of phenolic compounds simultaneous determination of paracetamol and levofloxacin using a glassy carbon electrode modified with carbon black, silver nanoparticles and pedot:pss film novel electrochemical sensing platform for ultrasensitive detection of cardiac troponin i based on aptamer-mos 2 nanoconjugates electrochemical determination of 2,4-dichlorophenol by using a glassy carbon electrode modified with molybdenum disulfide, ionic liquid and gold/silver nanorods electrochemical sensing based on layered mos 2 -graphene composites two-step pyrolysis process to synthesize highly dispersed pt-ru/carbon nanotube catalysts for methanol electrooxidation graphene-like mos 2 /graphene composites: cationic surfactant-assisted hydrothermal synthesis and electrochemical reversible storage of lithium an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg layered mos 2 @graphene functionalized with nitrogen-doped graphene quantum dots as an enhanced electrochemical hydrogen evolution catalyst biocompatible ionic liquids: fundamental behaviours and applications ionic liquid-like pharmaceutical ingredients and applications of ionic liquids in medicinal chemistry: development, status and prospects carbon nanotube-ionic liquid composite sensors and biosensors a promising sensing platform toward dopamine using mno2 nanowires/electro-reduced graphene oxide composites molecularly imprinted polymers in electrochemical and optical sensors facile and ultrasensitive determination of 4-nitrophenol based on acetylene black paste and graphene hybrid electrode preparation of molecularly imprinted polymers specific to glycoproteins, glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting fluorescent molecularly imprinted membranes as biosensor for the detection of target protein synthesis of novel monomeric graphene quantum dots and corresponding nanocomposite with molecularly imprinted polymer for electrochemical detection of an anticancerous ifosfamide drug a novel capacitive sensor based on molecularly imprinted nanoparticles as recognition elements rapid recognition and determination of tryptophan by carbon nanotubes and molecularly imprinted polymer-modified glassy carbon electrode molecular imprinted polymer based impedimetric sensor for trace level determination of digoxin in biological and pharmaceutical samples blood heparin sensor made from a paste electrode of graphite particles grafted with molecularly imprinted polymer a novel cufe 2 o 4 nanospheres molecularly imprinted polymers modified electrochemical sensor for lysozyme determination an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg nitrogen-doped graphene quantum dots-based fluorescent probe for the sensitive turn-on detection of glutathione and its cellular imaging nitrogen-doped graphene quantum dotslabeled epitope imprinted polymer with double templates via the metal chelation for specific recognition of cytochrome c highly luminescent s, n codoped graphene quantum dots with broad visible absorption bands for visible light photocatalysts l-cysteine-assisted synthesis of layered mos 2 /graphene composites with excellent electrochemical performances for lithium ion batteries electrochemical sensing based on layered mos 2 -graphene composites a facile and green strategy for the synthesis of mos 2 nanospheres with excellent li-ion storage properties synthesis of magnetic core-shell carbon dots@mfe 2 o 4 (m = mn, zn and cu) hybrid materials and their catalytic properties blue luminescent graphene quantum dots and graphene oxide prepared by tuning the carbonization degree of citric acid graphene quantum dots derived from carbon fibers one-pot synthesis of highly greenish-yellow fluorescent nitrogen-doped graphene quantum dots for pyrophosphate sensing via competitive coordination with eu(3+) ions electrochemical impedance spectroscopy on porous electrodes electrochemical impedance spectroscopy (eis) study of nano-alumina modified alkyd based waterborne coatings differentiation between graphene oxide and reduced graphene by electrochemical impedance spectroscopy (eis) an electrochemical impedance immunosensor based on gold nanoparticle-modified electrodes for the detection of hba1c in human blood a dna electrochemical sensor based on nanogold-modified poly-2,6-pyridinedicarboxylic acid film and detection of pat gene fragment fabrication of a new electrochemical imprinted sensor for determination of ketamine based on modified polytyramine/sol-gel/f-mwcnts@aunps nanocomposite/pencil graphite electrode an advanced molecularly imprinted electrochemical sensor for the highly sensitive and selective detection of human igg gate effect" in molecularly imprinted polymers: the current state of understanding organic-inorganic matrix for electrochemical immunoassay: detection of human igg based on zno/chitosan composite direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor label-free electrochemical impedance spectroscopy biosensor for the determination of human immunoglobulin g layer-by-layer assembly of chemical reduced graphene and carbon nanotubes for sensitive electrochemical immunoassay molecularly imprinted polymer chemosensor for selective determination of an nnitroso-l-proline food toxin electrochemically initiated co-polymerization of monomers of different oxidation potentials for molecular imprinting of electroactive analyte key: cord-312807-8v4r9jij authors: recht, judith; schuenemann, verena j.; sánchez-villagra, marcelo r. title: host diversity and origin of zoonoses: the ancient and the new date: 2020-09-17 journal: animals (basel) doi: 10.3390/ani10091672 sha: doc_id: 312807 cord_uid: 8v4r9jij simple summary: there is a wide variety of diseases caused by bacteria, viruses, and parasites that are transmitted to humans by different routes from other animals. these diseases, known as zoonoses, represent 75% of new or reemerging infectious diseases. there is a considerable impact of these diseases on the economy and health at local and global levels, including zoonotic diseases caused by the ingestion of food and products derived from animals. the wide range of animal species that host these disease-causing organisms include all groups of mammals. birds are the second significant animal group to act as hosts for zoonoses. much progress has been made in understanding disease evolution and animal origin, with important contributions from fields such as paleopathology and analysis of dna, applied to ancient human bone remains. the study of ancient diseases such as brucellosis and tuberculosis benefits from these approaches. more research is needed as new diseases emerge causing pandemics and some previously eradicated reemerge in some regions. global efforts are focused, based on evidence generated by research, on the prevention of new pandemics. abstract: bacterial, viral, and parasitic zoonotic diseases are transmitted to humans from a wide variety of animal species that act as reservoir hosts for the causative organisms. zoonoses contribute an estimated 75% of new or reemerging infectious diseases in humans. all groups of mammals have been shown to act as hosts for transmission of different organisms that cause zoonoses, followed in importance by birds; with both wild and domestic species identified as hosts in specific cases. there has been considerable research progress leading to a better understanding of the host range, animal origin, evolution, and transmission of important zoonoses, including those caused by the ingestion of food and products derived from animals. paleopathology studies of ancient human bone lesions, in combination with ancient dna analysis of the causative pathogen, have contributed to our understanding of the origin of zoonotic diseases, including brucellosis and mycobacterial zoonoses. however, there are still knowledge gaps and new confirmed and potential hosts are reported locally with some frequency. both the economic cost and burden of disease of zoonoses are substantial at local and global levels, as reflected by recent coronavirus pandemics that spread rapidly around the world. evidence-based prevention strategies are currently a global priority increasingly recognized, especially in zoonoses-affected regions. zoonoses, or diseases transmitted to humans from vertebrate animals in both rural and urban settings where humans live, are widespread, with an estimated 60% of all human infectious diseases antimicrobial-resistant isolates. as the leading cause of zoonotic disease in both animals and humans, salmonella was placed in an antimicrobial resistance (amr) "serious threats" category of the usa cdc in 2019 [9] . some strains of salmonella causing infections have developed resistance to antibiotics such as ciprofloxacin, azithromycin, and ceftriaxone, often used to treat patients with severe infections [10] . in the us, campylobacter causes an estimated 1.5 million campylobacteriosis infections annually, of which 29% have decreased susceptibility to antibiotics used to treat these infections, including ciprofloxacin and azithromycin, limiting treatment options especially in low-and middle-income countries [11] . in 2017, a systematic review and meta-analysis of studies on interventions to reduce antibiotic use in food-producing animals compared the presence of antibiotic-resistant bacteria in animals and humans [12] . based on this review, the world health organization (who) launched new guidelines on the use of antimicrobials in these animals, recommending to stop using antibiotics routinely to promote growth and prevent disease [13] . here we present a review on zoonoses host diversity and animal origin of selected zoonoses, with an emphasis on brucellosis and mycobacterial zoonoses. we discuss examples of paleopathology and ancient dna studies contributing to our knowledge on disease origin and evolution, followed by viral zoonoses including recent pandemics as examples of recent and emerging zoonoses. the range of animal hosts of pathogens linked to zoonotic diseases is in most cases impressively diverse. some zoonotic disease outbreak studies have revealed new (sometimes unexpected) animal sources of human infection. hosts may include both wild and domesticated animals, some leading to foodborne zoonoses. monitoring animals for the presence of important pathogens is the focus of public health strategies. aside from pathogens shared by humans with invertebrates (vectors or intermediate hosts for disease transmission), the majority (about 80%) of the reservoirs known for zoonotic diseases are mammalian, followed by avian hosts [1] . among other mammalian major taxa ("orders"), humans share the most pathogens with artiodactyls (frequently in proximity), followed by rodents, carnivorans, and primates. taxonomic identification of source groups for the emergence of zoonotic diseases could help to improve targeting of surveillance and interventions leading to prompt containment and even prevent zoonotic pandemics. however, given the taxonomic breadth reported for the many diseases, pinpointing specific groups and concentrating on them may not be justified. the information on hosts collected in tables s1-s3 reflects how imprecisely and often very generally this is reported, and how vague our knowledge is in most cases. for example, often reports are of "rodents"-this group alone represents more than one-third of recognized mammalian species, with over 2277 of them [14] . bats are the natural reservoirs of a significant number of important viral zoonoses, including probably the recent covid-19 that caused a high impact global pandemic this year (2020), still ongoing and discussed below. it has been hypothesized that bats are unique hosts in this regard, in particular as compared with rodents, the other is a particularly speciose major group (order) of placental mammals besides bats. however, the total number of zoonotic viruses identified in bats (61) was lower than in rodents (68) . there is a higher number of rodents than bat species (2-fold) [14, 15] . a recent report on the largest dataset of the zoonotic virus-reservoir relationships built with avian and mammalian reservoir hosts of 415 viruses aimed at determining whether some animal species are special reservoirs of zoonotic pathogens [16] . the results did not support this view but a host-neutral variation instead; the analysis showed that the proportion of viruses infecting humans showed minimal variability across reservoir taxonomic orders and were in agreement with the number of animal species within each group (including for bats and rodents), with rare reservoir host effects restricted to one or two viral families. the camel has been highlighted in a recent review that showed 19 zoonoses reported in this animal in iran (where raising camels is common with close contact between farmers and camels well as meat and milk consumption) including plague, q fever, campylobacteriosis, tuberculosis, salmonellosis, rabies, mers, and toxoplasmosis ( [17] , section 4). a plague outbreak in southern afghanistan in 2007 manifested as 83 cases of acute gastroenteritis with 17 deaths; the outbreak was linked to the consumption or handling of camel meat [18] . birds are the main reservoir of the influenza a virus (table s2) . pigs, susceptible to infection with both avian and mammalian influenza viruses, may be acting in interspecies viral transmission as "mixing vessels", in which avian and mammalian influenza viruses recombine through a process known as "reassortment" to produce novel strains that can then infect humans [19] . a role for wild migratory birds in the transmission of zoonoses has been acknowledged either as a reservoir host or by dispersing infected arthropod vectors. for example, west nile virus was likely introduced in the usa via infected birds, expanding in 1999-2000 along the atlantic seaboard, a common bird migration route, to reach southern florida to establish an enzootic cycle with mosquitoes and then from 2000 to 2002, expanded westward possibly as a result of elliptical avian migration routes (reviewed in [20] ). migrating birds can also carry one or more ectoparasites such as mites, ticks, fleas, and lice, all arthropods that themselves can carry pathogens; a potential role for migrating birds in dispersing ticks and associated pathogens that cause zoonotic disease has been recently highlighted [21] . gut microbes and obligate ectoparasites such as ticks, fleas, and lice, depending on their animal hosts for transport. in this context, it is remarkable that recent geological events may have had a large impact on the dispersal capacity of both ectoparasites and gut pathogens. the megafauna (animals over 44.5 kg (98 lb) body weight) decline in the late pleistocene/early holocene, which led to a decrease in seeds and nutrients dispersal, may have also caused a reduction in the movement of ectoparasites and generalist fecal microbes, including escherichia coli, to~15% of pre-extinction levels based on reductions after extinction estimated using the average home range for modern and extinct species [22] . in this study, the distance that gut pathogens can travel between consumption and defecation, which is size-dependent, showed the largest declines in the americas and eurasia, however, whether pathogens disappeared with the previous hosts or adapted to new hosts is not known. this subject could be explored by metagenomic analyses of pathogens present in extinct mammal dung [22] . chagas disease is an example of a successful parasitic zoonosis with a wide range (hundreds) of mammal species hosts in south america, transmitted by dozens of triatomine bug species (table 1) . known as american trypanosomiasis, it also owes its name to carlos chagas, a brazilian researcher who first described it in brazil in 1909 as a disease due to the parasite trypanosoma cruzi (named after oswaldo cruz, another brazilian scientist), later shown to parasitize species of mammals from seven "orders" and triatomines from 15 genera [23] . a comprehensive study of reservoirs and wild hosts of t. cruzi. in brazil showed that species of several placental "orders" (artiodactyla, chiroptera, primates, carnivora, rodentia, cingulata, pilosa) and one marsupial (didelphimorphia) were involved in transmission, with four of them (primates, didelphimorphia, chiroptera, and the carnivora species nasua nasua) considered as key reservoir taxa exhibiting higher rates of parasitemia [24] . in latin america, about 16 million people are estimated to be infected with t. cruzi through feces or night bites of the "kissing bug" (triatomines), or through blood transfusion, maternally, or orally through contaminated food that can lead to an acute phase usually without symptoms or very mild [25] . after 10-20 years following the acute phase, however, symptomatic chronic disease can occur, leading to irreversible damage to several organs such as the heart, esophagus, and colon [26] . cattle, bison, african buffalo, cervids, brushtail possums, badgers, kudu can be reservoirs ingestion (unpasteurized dairy products, undercooked meat including bushmeat), inhalation, contamination of breaks in the skin * information sources: [23, 25, [27] [28] [29] [30] ; additional tables with information on many bacterial, viral, and parasitic zoonoses are in the supplementary material (tables s1-s3, respectively). trypanosoma cruzi has been identified in hosts such as armadillos and monkeys (in 1912 and 1924, respectively, by chagas in brazil) [24] , as well as cats and dogs. a recent report of a study lasting two decades (1992-2017) of t. cruzi infection in wild mammals in brazil showed that 17% of mammals were seropositive and 8% with high parasitemia, indicating infectivity potential [30] . a recent study reported a potential effect on chagas disease transmission of oil palm plantations in colombia, where the main vector in the region has been captured and reported to have t. cruzi natural infection, based on vertebrate host analyses of blood meals from nymphs that revealed 18 vertebrate species including pigs, house mouse and opossum [31] . hunting dogs in indigenous mayangna and miskitu populations from nicaragua's remote bosawás biosphere reserve were shown to have t. cruzi antibodies and therefore previous exposure (7/78 sera screened, or 9%), suggesting hunting dogs as the potential zoonotic risk for chagas disease in these communities [32] . the origin of chagas disease is unclear. bats may have been the original reservoirs hosts of t. cruzi, as postulated in the bat-seeding hypothesis, followed by a host switch to a non-volant mammal and then several switches to humans resulting in the diversity of lineages circulating in human populations. two genetic lineages of trypanosomes associated with bats were recently detected in rural areas of southern ecuador [33] , one of which, named tcbat, is t. cruzi-related and was detected in a 5-year-old female in a forest area in northwestern colombia as a mixed infection of t. cruzi i and tcbat genotypes [34] . a recent systematic review on zoonotic diseases that manifest with human febrile illness reported in 53 malaria-endemic countries showed a wide distribution of these diseases causing febrile illness; half of them were bacterial diseases [35] . for a list of selected bacterial zoonoses reflecting the impressive diversity of this group of diseases, see table s1 . next, we discuss two well-known bacteria genera-brucella and myocabacteria-as they represent ancient human diseases for which origins are still highly debated. two major diseases caused by these bacteria are brucellosis and tuberculosis. they affect the skeleton, causing bone pathologies in about 10-15% of individuals who have the disease [36] and can result in similar lesions in affected individuals despite being caused by very different bacterial species. paleopathology studies along with ancient dna analysis of bone lesion remains have been critical in determining the origin for each of these two diseases. the overlapping appearance of affected bones in the spine in both infections have led researchers to initially suspect a tuberculosis lesion, with molecular analyses demonstrating in some cases that the cause was brucellosis. a study that evaluated calcified nodules from a 14th-century skeleton found at an abandoned medieval village in northwest sardinia, italy, initially hypothesized the nodules were due to tuberculosis, but shotgun metagenomics revealed medieval brucella melitensis genome sequences instead [37] . even today, with advanced health technology, there is overlap in the understanding of symptoms and laboratory test results for these two diseases [38] , making differential diagnosis difficult in countries such as india, where they are both endemic [39] . caused by bacteria of the genus brucella (mostly brucella melitensis), brucellosis is a common zoonotic infection resulting in febrile illness in humans, mainly through the ingestion of unpasteurized dairy products and direct contact with infected animals (table 1 ). travelers to endemic areas may get infected by consumption of unpasteurized milk or other dairy products and may be the source of imported cases into their own countries (mostly from infected cheeses consumed by their families), as is the case for most of the acute brucellosis cases in north america and northern europe [29] . re-emergence of zoonotic diseases in countries where they have been previously eradicated has been reported, calling for a need for disease-free countries to remain aware and implement regional monitoring systems. brucellosis has reemerged in bulgaria after 50 years, probably due to the illegal import of infected animals from endemic border countries, as has occurred with bovine brucellosis in france, with possible cross-border brucellosis transmission into europe from middle-eastern countries with the highest incidences of brucellosis worldwide such as turkey and syria (reviewed in [40] ). the exact prevalence of both animal and human disease due to brucellosis is not well known. the economic burden of brucellosis is considerably high in low-income countries in tropical asia and africa [41] . in 2016, out of 1.27 million estimated cases of brucellosis in kenya, 12,004 people died, 96% of whom were livestock keepers [42] . brucellosis can have skeletal manifestations, with bones and joints affected being the most frequent complications occurring in up to 40% of cases [29] . analysis of vertebral lesions and pathological changes described in a 2.4-2.8 million-year-old male skeleton of australopithecus africanus from south africa were interpreted as resulting from initial phases of brucellosis [43] . this disease is an example of human and domestic animal paleopathology studies suggesting brucellosis in ancient bone remains, with most cases involving adult male skeletal individuals showing lumbar vertebrae and sacroiliac joints involved [44] , evidence which combined with ancient dna analysis by pcr have confirmed the presence of brucella dna (reviewed in [45] ). as discussed above, dna detection of brucella dna has been critical in confirming brucellosis in ancient human remains when paleopathology initially suggested tuberculosis. however, brucella bacteria do not preserve as well as mycobacteria, which have a thicker, hydrophobic and more resistant cell wall that has allowed more data to be collected for ancient infection for the latter type of disease-causing bacteria (discussed next). mycobacteria vary in epidemiology, reservoirs, and their ability to cause disease, with groups such as the mycobacterium tuberculosis complex including m. tuberculosis and m. bovis (see bovine tuberculosis below) that infect a large range of mammals including humans via inhalation of droplets containing bacteria that reach the lungs; m. leprae (see leprosy section below); "nontuberculous mycobacteria" [46] . human tuberculosis caused by m. tuberculosis is a well-known disease of unclear origin, with a considerable impact on global health. it affects an estimated 10 million people annually. those living with hiv are more likely to be infected with and die of tuberculosis [47] . the origin of tuberculosis is under debate regarding whether it started as a zoonosis from cattle or not. one hypothesis states that it originated with bovid milk consumption known to occur in the early neolithic in europe [48] . another hypothesis based on biomolecular studies proposes a new evolutionary scenario with human tuberculosis having a human origin, present in early african human populations at least 70,000 years ago and expanding with human migration, especially in the neolithic [49] . in both scenarios, human-animal proximity starting in the neolithic was a critical factor for emergence/expansion, due to close interaction between early domesticates and humans and increased human density. human density in some areas peaked during the industrial revolution when crowded dairy cow populations in densely populated urban settings were the source of milk [48] . although rare, tuberculosis can result in osteological lesions which can help identify, along with molecular analysis, tuberculosis infection in ancient bone remains. these approaches have been applied to materials from both humans and animals. however, molecular genetics applications in these cases have limitations, including cost. human remains from a medieval churchyard in england in which both m. tuberculosis and m. bovis were predicted due to milk and beef consumption at the time, showed only m. tuberculosis dna in human skeletons that displayed morphological symptoms of tuberculosis (reviewed in [48] ). a particular question on ancient dna in this context relates to the origins of tuberculosis in the americas. while today european lineages of m. tuberculosis are found, morphological evidence exists that may support a pre-columbian prevalence of the disease. a recent report on sequencing and analysis of three mycobacterial genomes from peruvian human skeletons dated to 1028-1280 revealed the presence of tuberculosis before european contact in pre-columbian south america [50] , a region with considerable morphological evidence of pre-columbian tuberculosis previously reported. in this study, the three sequenced mycobacterial genomes were most closely related to bacteria belonging to the m. tuberculosis complex that have adapted to seals and sea lions (m. pinnipedii) than those adapted to humans today in europe, asia, or africa. these data suggest that sea mammals may have played a role in tuberculosis transmission to humans across the ocean, an intriguing possibility for a zoonotic origin of new world tuberculosis to be further investigated. bovine tuberculosis resulting from m. bovis infection affects cattle and other mammals and, when transmitted to humans, becomes zoonotic tuberculosis (table 1 ). its overall incidence has decreased due to cattle control and routine pasteurization of milk from cows, as infection happens mostly by consuming unpasteurized milk and dairy products or by wound contact occurring during slaughter or hunting [51] ; occupational exposure of livestock workers may occur via inhalation of aerosol or cough from infected cattle. this form of widespread zoonotic tuberculosis can be fatal, present mainly in africa (causing approximately 3% of all pulmonary tuberculosis cases) and southeast asia. in 2016, out of 9689 cases of bovine tuberculosis in kenya, 1168 people died (about 12%); 70% of the fatalities were livestock keepers [42] . often bovine tuberculosis caused by m. bovis in humans is indistinguishable clinically from human tuberculosis resulting from m. tuberculosis infection; bovine tuberculosis is estimated to account for up to 10% of human tuberculosis cases in some countries [52] . however, accurate information is lacking on the incidence of tuberculosis due to human m. bovis infection from countries with high tuberculosis and hiv prevalence and where populations have direct contact with cattle such as sub-saharan africa [53, 54] . in these low resource settings, it is often assumed (and not tested) that tuberculosis is caused by m. tuberculosis, leading to underestimating the real incidence of zoonotic tuberculosis and underscoring a need to accurately diagnose and treat human tuberculosis caused by m. bovis [55] . m. bovis has a broad host range including domestic and wild animals, with feral maintenance hosts such as badgers (meles meles) in the uk and ireland, the brushtail possum (trichosurus vulpecula) in new zealand, and the white-tailed deer (odocoileus virginianus) in michigan, usa posing a threat for livestock infection as well as disease eradication from cattle (reviewed in [30] ). cases of bovine tuberculosis in humans linked to deer hunting and handling in an endemic white-tailed deer area have been reported in michigan [56] . there is also epidemiological evidence in new zealand that feral ferrets (mustela furo) may be tuberculosis vectors for cattle, and can be used as sentinels for this disease as an alternative to possums (reviewed in [57] ). mycobacterium avium, a causative bacterium of tuberculosis in birds, can also be transmitted to mammals, including livestock. the livestock industry suffers economic losses and trade restrictions due to its incidence. widespread paratuberculosis in animals (also known as jones disease) including bovine paratuberculosis, is an endemic disease especially in developing countries affecting livestock production, zoo, and wildlife animals (reviewed in [58] ). m. avium subspecies can cause paratuberculosis in humans, manifested as inflammatory bowel disease and autoimmune diseases including asthma, insulin-dependent diabetes mellitus, sarcoidosis, rheumatoid arthritis, multiple sclerosis, celiac disease and may also be a contributing factor to crohn's disease [58] . mycobacterium leprae, the main causative agent of leprosy in humans, a chronic disease with skin lesions and peripheral nerve damage mostly spread via a human-to-human transmission with zoonotic transmission from natural reservoirs, which are not clearly understood yet. the first animal reservoir to be discovered was the nine-banded armadillo (dasypus novemcinctus) in the southern united states [59] [60] [61] . this armadillo, which can be naturally infected with m. leprae and shows similar disease presentation as humans systemically, have been used as animal models for leprosy studies by laboratory infection; they present typical plantar ulceration with foot ulcers increasing as the infection progresses (reviewed in [62] ). this species has been recently shown to be a potential reservoir in brazil, where in some areas people hunt and eat them as a dietary source of protein [63] . recently, m. leprae was also isolated from red squirrels on brownsea island in the southern uk [64] . for both nine-banded armadillos and red squirrels reservoirs, related m. leprae strains were found via ancient dna studies in human skeletons from england and denmark dating to medieval times, suggesting a potential european origin of the strains present today in the reservoirs [65, 66] . non-human primates in several regions have been identified as additional m. leprae reservoirs [67] . m. leprae genomes were obtained from a chimpanzee (pan troglodytes) from sierra leone, a cynomolgus macaque (macaca fascicularis) from the philippines, and a sooty mangabey (cercocebus atys) from west africa [67] . marmosets (callithrix jacchus) in brazil, also examined as possible hosts for m. leprae, were not positive by dna analysis although mycobacterial dna (rpob1 locus) was detected [68] . in contrast to the previously discussed zoonoses, which are linked to well-known diseases, viral zoonoses are the cause of the majority of recent human pandemics, suggesting that viruses may evolve more rapidly than other pathogens to adapt to the human host, sometimes leading to the person-to-person transmission without the need for another reservoir, and with transmission enhanced in dense populations and by human travel [1] . many of them are distributed worldwide (table s2 ). here we present examples of recent viral zoonoses, some resulting in widespread pandemics, to showcase the clear involvement of wild animals, which are often debated in the previously introduced cases likely due to the old nature of those diseases. in many areas of central africa in particular, wild meat (known as bush meat) is in high demand. human contact with wild animals during hunting and preparation or consumption of undercooked meat has led to important zoonoses developing. an example may be hiv/aids, which was linked to the butchering of hunted chimpanzees in africa, afterward adapting to human-to-human transmission [69] . the human ebola epidemic in west africa in 2014-2016 caused by the ebola virus was linked to the hunting or handling of infected gorillas and other wild animal carcasses ( [70] , table s2 ). the congo's ebola hemorrhagic fever affects gorillas and chimpanzees; about 5000 gorillas were killed in gabon and the republic of the congo in 2002-2003 by the ebola virus [71] . asia is another example of wild animals trafficking for consumption, especially in china, myanmar, vietnam, and thailand, where "ye wei" ("wild taste" for wild and exotic animals) was associated with social status including in the dynastic eras as well as currently being widely sold in wet markets and restaurants, sourced legally or illegally from the wild or wildlife farms (reviewed in [72] ). important zoonoses such as some caused by coronaviruses (table 2) have also been associated with the consumption of wild meat in markets, two of them in china. the severe acute respiratory syndrome coronavirus (sars-cov) in 2003 and the middle east respiratory syndrome coronavirus (mers-cov) in 2012 each caused a large pandemic, but these were small compared to the one caused by sars-cov-2, a second sars virus recently identified as the cause of covid-19. the sars pandemic caused 8098 cases and 774 deaths and mers resulted in 2494 cases and 858 deaths [73] , while covid-19 has caused, as of 4 july 2020 and only six months after who declared it a pandemic on 11 january, over 11 million cases and slightly over half a million deaths [74] . the animal sources for sars and mers were identified as the civet and dromedary camel, respectively (table 2) . however, these are considered intermediate hosts, as these zoonotic diseases are thought to have originated in bats [75, 76] , subsequently spilling over to intermediate hosts, and eventually jumping to humans [75] . a very recent phylogenetic dating study based on bioinformatic approaches strongly suggests that sars-cov-2 emerged directly, without an intermediate host, from the same horseshoe bat subgenus of sars-like coronaviruses, with both sars-cov and sars-cov-2 diverging at the same time (40-70 years ago) from currently known extant bat virus [77] . nonhuman primates were shown to be an effective yellow fever sentinel system in brazil. yellow fever (table s2) , a reemerging viral zoonotic disease endemic in africa and south america transmitted from vector mosquitoes, often causes outbreaks in both humans and nonhuman primates in brazil. this country has an established and successful yellow fever national surveillance program that includes postmortem nonhuman primate studies for early circulating virus detection and prompt implementation of vaccination and vector control [78] . after a 2017 epizootic occurred in brazil's espirito santo state, 22 deceased nonhuman primates (two howler monkeys (alouatta spp.) and 20 not further identified) were examined with 21 of them showing typical yellow fever features; yellow fever was diagnosed the same year for 150 of 1000 animals tested from southern states of brazil (15% occurrence) [79] . emerging zoonotic diseases causing epidemics have prompted scientists to focus on efforts towards identifying factors involved in disease emergence as well as possible prediction tools, including modeling approaches. recent such modeling used for spatial mapping of hotspots showed that the global distribution of the risk of emerging zoonotic diseases is higher in tropical regions with wildlife biodiversity (especially mammals) experiencing land-use changes [80] . this study, along with previous ones [81] , predicted a higher risk in tropical, developing countries. the authors stated that even though emerging infectious disease events have been predominantly reported in developed countries, this is probably an artifact due to stronger surveillance and reporting systems in these areas. it would be hard to test this idea. the one health multisector approach response to zoonoses threat is an appropriate one, which may need input from additional sectors, such as travel and tourism, social and political scientists, anthropologists, and economists to better plan and implement strategies related to surveillance, capacity building, and risk reduction when addressing, for example, the possible closure of wet markets in communities that rely heavily on them for income and food [82] . there is a wide range of species reported as hosts of zoonoses, making predictions on new and potential reservoirs based on phylogenetic considerations challenging. basic information on which species serve as hosts is lacking for many regions and diseases. although considerable progress has been made in our understanding of these diseases' reservoirs, animal origin, and evolution, with contributions made by paleopathology and ancient dna detection for ancient diseases such as brucellosis and zoonotic tuberculosis. further research is needed to gain insights into mechanisms of disease emergence and transmission. in view of the recent pandemics, research on zoonoses should be prioritized towards developing evidence-based prevention strategies. these should include approaches based on sustainable living and food consumption that address inequality, as commentators across the globe currently discuss prompted by the covid-19 pandemic. supplementary materials: the following are available online at http://www.mdpi.com/2076-2615/10/9/1672/s1. table s1 : selected bacterial zoonoses, table s2 : selected viral zoonoses, table s3 : selected parasitic and other non-bacterial non-viral zoonoses. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection or interpretation of data and the writing of the 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detection of mycobacterium in new world animal reservoirs aids as a zoonosis: scientific and public health implications wild animal mortality monitoring and human ebola outbreaks, gabon and republic of congo ebola outbreak killed 5000 gorillas baby pangolins on my plate: possible lessons to learn from the covid-19 pandemic covid-19 has killed more people than sars and mers combined, despite lower case fatality rate origin and evolution of pathogenic coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat origin evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic surveillance for yellow fever virus in non-human primates in southern brazil outbreak of yellow fever among nonhuman primates global hotspots and correlates of emerging zoonotic diseases global trends in emerging infectious diseases this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord-324295-9c1zxjng authors: bonilla-aldana, d. katterine; jimenez-diaz, s. daniela; arango-duque, j. sebastian; aguirre-florez, mateo; balbin-ramon, graciela j.; paniz-mondolfi, alberto; suárez, jose antonio; pachar, monica r.; perez-garcia, luis a.; delgado-noguera, lourdes a.; sierra, manuel antonio; muñoz-lara, fausto; zambrano, lysien i.; rodriguez-morales, alfonso j. title: bats in ecosystems and their wide spectrum of viral infectious threats: sars-cov-2 and other emerging viruses date: 2020-08-20 journal: int j infect dis doi: 10.1016/j.ijid.2020.08.050 sha: doc_id: 324295 cord_uid: 9c1zxjng bats have populated earth for approximately 52 million years, serving as natural reservoirs for a variety of viruses through the course of evolution. transmission of highly pathogenic viruses from bats has been suspected and linked to a spectrum of emerging infectious diseases in humans and animals worldwide. examples of such viruses include marburg, ebola, nipah, hendra, influenza a, dengue, equine encephalitis viruses, lyssaviruses, madariaga and coronaviruses, involving the now pandemic severe acute respiratory syndrome coronavirus 2 (sars-cov-2). herein, we provide a comprehensive review on the diversity, reservoirs, and geographical distribution of the main bat viruses and their potential for cross-species transmission. j o u r n a l p r e -p r o o f bats have populated earth for approximately 52 million years, serving as natural reservoirs for multiple viruses through the course of their existence 7, 8 . the evolution of their physical, physiological and behavioral characteristics has allowed them to expand to all continents except antarctica, with ecological niches located in urban or rural areas, and especially in caves, mines and some types of foliage 9 . evolutional changes have also determined their current eating patterns and their role within the ecosystem 10 . bats are the only mammals capable of flight; they have nocturnal habits during which they either feed or mate and a layer of short fur that protects them from humidity and cold temperatures 11 . bats belong to the order chiroptera (wings on the upper extremities), with approximately 1,100 species subclassified into two suborders: the megachiroptera and microchiroptera. this classification allows a better understanding of some of their behavioral patterns 8, 12, 13 . bats from the megachiroptera suborder, commonly known as megabats, account for approximately 170 species mainly located in asia, africa and oceania or pacific region ( figure 1 ); their size can vary between 40 and 150 cm with their wings spread, and they weigh 1 kg on average. megabats feed exclusively on fruit, seeds and pollen and their main habitats include caves, mines, trees and some buildings. megachiroptera bats cannot echo localize 8 . the microchiroptera or microbats include over 930 species distributed throughout the entire planet with the exception of some islands and the poles. their size ranges from 4 to 16 cm and feed mostly on flowers and fruit. they possess an echolocation system that allows hematophagous bats to search and capture small prey such as lizards, small mammals, and arthropods. their primary habitats include forests and tropical areas although they are also capable of coexisting with humans in some urban settings 8, 12, 13 . j o u r n a l p r e -p r o o f another notable feature is that microbats can travel long distances of up to 2000 km during migratory season to fulfill their nutritional needs, which is relevant to understand local and intercontinental spread of colonies and coexistence with other animals of the same species. microbats also play a role as pollinators as their feces fertilize and distribute seeds among the areas they inhabit, in addition to serving as plague controllers by feeding on insects, frogs, and rats 8, 14 . bats that coexist within the same geographical area often host common microorganisms 11, 12 . contagion rates depend on contact speed and susceptibility to infections of a specific population 12 . evolutionary processes granted bats with hollow bones to facilitate air maneuvering. this hollowness results in the absence of bone marrow and thus the inability to produce b cells necessary for an efficient immune response, making bats asymptomatic carriers for a long list of viruses 8 . additionally, several species are facultative heterotherms capable of entering profound lethargy during periods of physiological stress to compensate for energy and water deficits, favoring viral persistence 12 . continuous physical contact within bats of the same colony facilitates viral circulation, especially during breeding and migration seasons 15 . the proposed mechanisms of viral transmission between microbats is aerosol release produced by larynx vibrations that occur during echolocation in addition to close contact with other types of secretions such as fecal matter and urine 16 . then, bats carry multiple emerging and reemergin pathogens, especially viral threats ( table 1) . studies from the ecohealth alliance suggest that bats are one of the leading carriers of emerging infectious agents that can potentially affect other mammals, including humans [17] [18] [19] . although the precise reservoir of sars-cov-2 has not been established, a sensible hypothesis is that bats of the genus rhinolophus ferrumequinum could be at fault: chinese studies have reported that sars-cov-2 is very similar to coronaviruses naturally found in bats; however, these viruses are constantly evolving and mutating, making it difficult to pinpoint an exact reservoir ( figure 1 ). since there is no effective treatment or vaccine for covid-19 to date, strong regulations---including isolation, quarantine and social distancing---have been established by many countries in an effort to reduce expansion of the disease given the high person-to-person transmissibility of sars-cov-2, either directly by respiratory droplets with infective particles or indirectly by fluid-contaminated objects. j o u r n a l p r e -p r o o f a study in indonesia identified cov genes in bareback fruit bats, where partial rna-dependent rna polymerase (rdrp) sequences and regions between helicase and rdrp genes were detected and amplified in faeces and tissue samples 22 . another study conducted in zhoushan city, zhejiang province, found that out of 334 bats sampled, approximately 26% were naturally infected with coronaviruses 23 . marburg virus is an rna virus belonging to the filoviridae family (genus marburgvirus) ( further molecular testing conducted on liver, spleen and lung samples of egyptian fruit bat rousettus aegyptiacus reported the presence of marv rna ( figure 1 ). possible routes of transmission include fruit contamination and its consumption by humans or direct contact with bat's infected organs. marv can also spread from human to human through contact with bodily fluids or fomites from sick patients (table 3 ). in humans, the incubation period ranges from 3 to 9 days, and clinical presentation usually involves flulike symptoms, fever between 39 and 40 degrees celsius, conjunctivitis, cramps, cervical lymphadenopathy, and hemorrhagic manifestations. death occurs as a consequence of cardiocirculatory collapse and multiple hemorrhages in the digestive tract and lungs. a study detected a high seroprevalence of antibodies against marburg virus in fruit bats in south africa, with a 19.1% seroconversion rate in recaptured bats 24 ; another study detected marv genome in bats captured in zambia 25 ; and a posterior serosurvey identified filovirus-specific immunoglobulin g antibodies in 71 out of 748 serum samples collected from migratory fruit bats 26 . currently, there is no effective treatment for marv infection other than symptomatic support and rehydration, but some hematologic, immunologic, and pharmacologic actions are under development to improve survival rates. ebola virus also belongs to the filoviridae family and displays a negative-stranded single rna genome. first described in 1976 in the democratic republic of the congo, this notorious virus has decimated populations of gorillas, chimpanzees and humans in africa with mortality rates ranging from 25% to 90% (table 2) . evidence of infection has been reported in three different species of frugivorous bats associated with large outbreaks of ebola hemorrhagic fever in 2014-2015, which later escalated to pandemic proportions resulting in the death of 11,301 people 8, 27 . multiple studies point at bats of the genus myotis as the main reservoir for ebola virus given that these bats carry a copy of viral gene vp35 (table 3) studies in africa analyzed 4,022 blood samples from bats, detecting antibodies against ebola virus in one genus of insectivorous bats and six species of fruit bats 29 . another study conducted in sierra leone identified the complete genome of a new ebola virus, the bombali virus, in free-tailed bats resting inside human dwellings, suggesting potential human transmission 30 . in kenya, researchers also identified the bombali virus in the organs and excreta of free-tailed bats (mops condylurus) 31 . in malaysia, an outbreak in pigs and humans took place between september 1998 and april 1999, affecting 283 people and causing 109 human deaths and the slaughter of more than one million pigs (table 2) . initially, the outbreak was attributed to the japanese encephalitis virus; but later, researchers demonstrated that the causal agent was a virus that belonged to the henipavirus genus, family paramyxoviridae, closely related to the hendra virus. fruit bats (genus pteropus) are the main natural reservoir for nipah virus (niv), while pigs serve as intermediate hosts ( table 3 ). the infection is transmitted from bats to pigs and subsequently from pigs to humans ( figure 1 ). on the island of malaysia, researchers found niv in the urine and saliva of flying foxes (pteropus hypomelanus and pteropus vampyrus). initial circulation of niv likely occurred in late 1997 through contaminated food debris from migrating flying foxes on which pigs fed. fruit bat migration to cultivated orchards and pig farms was a consequence of the lack of fruit during droughts related to el niño phenomenon and wild fires in indonesia. a study conducted in 324 bats (predominantly pteropus, p. vampyrus and p. hypomelanus) from malaysia found that 6.48% had neutralizing antibodies against niv and the virus was subsequently isolated from the urine and fruits consumed by p. hypomelanus. previous human studies showed that most cases had a history of direct contact with live pigs. in humans, the disease can be fatal and is characterized by respiratory and particularly severe neurological manifestations, such as encephalitis and coma. clinical signs in animals may vary, including agitation, spasms, seizures, rapid breathing, and harsh cough. evidence of infection (virus isolation, immunohistochemistry, serology) and neurological involvement has been reported in dogs and horses. transmission studies in australia established that niv could rapidly spread through pigs via oral and parenteral inoculation. neutralizing antibodies were detectable 10-14 days after infection [32] [33] [34] [35] [36] . influenza a viruses (iav) are one of the leading causes of disease in humans, with important animal reservoirs including birds, pigs, and horses that can potentially produce new zoonotic variants (table 2) . (table 3) . notably, small yellow-shouldered bats in central america have been proposed as potential mammalian reservoirs of influenza (figure 1 ). research has shown that bats are susceptible to iav infection. a seroprevalence study identified iav h9 in 30% of fruit bats sampled in africa. bats are believed to have the ability to harbor more genetic diversity of the influenza virus than any other mammal and bird species. j o u r n a l p r e -p r o o f hendra virus, formerly known as equine morbillivirus, is a henipavirus of the paramyxoviridae family for which bats of the genus pteropus serve as main vectors (table 2) . this virus is endemic in australian flying foxes which are currently in danger of extinction and it can be detected in blood, urine, faeces, and fetal and uterine tissue. different studies affirm that hendra virus is horizontally transmitted from bat to bat, but in rare instances vertical transmission has been reported ( figure 1 ). although horses are usually accidental hosts that can contract the virus from these megabats, it has been suggested that the virus could be found in the environment, entering equines through the upper airways and oropharynx. transmission to humans occurs with close contact with infected horses, either through bodily fluids or aerosols; studies have ruled out person-to-person transmission. hendra virus was discovered after an outbreak that killed 20 equines and their trainer in queensland, australia, in 1994 37 . the virus remains viable for approximately four days in bat urine and fruit juice, but fails to survive in temperatures above 37 degrees celsius. the disease has an incubation period of 5-12 days, and its main symptoms are similar to influenza but with a mild, progressive encephalitis. since this infection is uncommon in humans, an effective antiviral has not been developed (table 3) . lyssaviruses are a wide range of pathogens that cause rabies ( table 2 ). the first case of human rabies was reported in ukraine in 1977. hematophagous bat desmodus rotundus, mainly located in latin america ( figure 2) , is considered the primary vector of this disease, which historically has resulted in large economic losses by causing the death of cattle and horses that get infected through bat bites. occasionally, those bites are also seen in human beings (figures 3-4) . lyssaviruses have also been identified in other hematophagous bats such as diphylla ecaudata and diaemus youngi and frugivorous species such as artibeus, planirostris trinitalis, diclidurus albus, hemiderma sp. and phyllostoma superciliatum (table 3) . a colombian study performed on 286 brains from bats of six families and 23 species showed that two species, artibeus lituratus and artibeus planirostris, were positive for the rabies virus 38 . another study conducted in chile reported that 9.5% of 15,000 bats captured were naturally infected 39 as a consequence of the destruction of natural habitats, closer interaction between bats and humans has grown significantly, especially in mining areas 44 . rabies transmission primarily occurs via direct bites or scratches from infected bats 38 ; secondary transmissions in humans takes place through contact with infected pets. these viruses cause acute progressive encephalitis that is inevitably fatal from the onset of clinical signs. initial symptoms are similar to influenza and evolve in a few days to severe neurological involvement that ultimately leads to death 45, 46 . currently, only preventive vaccines are available [47] [48] [49] [50] [51] . dengue virus (denv) belongs to the flaviviridae family, which is transmitted by mosquitoes, most commonly aedes aegypti ( table 2) 52 . there are four denv serotypes (denv-1, denv-2, denv-3 and denv-4) that can cause febrile syndromes in humans. dengue fever is recognized as an epidemic reemerging disease that has affected many countries in recent decades 53-57 . there is evidence that bats could naturally become infected with denv 58 . in mexico, a study evaluated 162 bats and identified denv nucleic acid and anti-denv antibody 59 . another study in costa rica evaluated 12 species of bats, reporting a cumulative seroprevalence of 21.2% (51/241) by prnt and a j o u r n a l p r e -p r o o f prevalence of 8.8% (28/318) in organs tested by rt-pcr 60 . in french guiana, denv nucleic acid was detected in the liver and sera of wild-caught bats 61 . recently in colombia, viral rna was obtained from bat tissues, and a nested-rt-pcr detected amplicons of 143 fragment of the ns5 gene that were then sequenced by the sanger method. in non-hematophagous bats such as carollia perspicillata and phyllostomus discolor captured in ayapel and san carlos (córdoba) respectively, an amplicon corresponding to ns5 was detected; these amplicons showed high similarity with denv-2 57 . yet, the clinical relevance of denv isolation from bats is unclear, as well as the implications in transmission to humans. similarly occurs with other arboviruses, such as madariaga and the equine encephalitis viruses. so far, the have been confirmed as infected hosts, probably reservoirs, but not a direct source for human infections (figure 1 ). the equine encephalitis group involves rna viruses belonging to the togaviridae family, of the alphavirus (table 3 ). these findings suggest that fruit bats from the caribbean region in colombia could be involved in the enzootic cycle of eev 62 (figure 1 ). the madariaga virus is a strain of the eastern equine encephalitis virus. rats and bats presumably serve as the main vectors given the reported seropositivy of brown short-tailed bats (carollia castanea), lanza bats (phyllostomus discolor) and seba's short-tailed bats (carollia perspicillata) 65 . several factors increase the interaction between humans and bats. in most cases, this is due to the intrusion of humans into virgin territories inhabited by bats, fueled by the search of economical resources. this j o u r n a l p r e -p r o o f phenomenon forces bats to adapt to new settings occupied by men, such as buildings, tombs, mines and bridges. additional illegal trading of bats for human consumption and traditional medicine in asia, coupled with poor sanitation and hygiene practices, has facilitated the emergence of zoonotic diseases, some with pandemic potential. financial openness and globalization involve close connections between countries and across continents and can serve as a mean of transportation that accelerates the spread of these emerging diseases. emerging diseases are a global matter of concern, especially during the last decades, and now even more with the occurrence of the 2020 pandemic of covid-19. sars-cov-2 and other emerging pathogens are significantly present in bats. recognizing bats as potential reservoirs or transmission sources for several pathogens that can be a threat to human beings is of upmost importance for global health since many of these conditions may cause severe damage and even led to death, in some cases in a significant proportion of individuals. however, we should not neglect the responsibility of the active role of humans in the invasion of natural habitats and illegal trafficking of bats. the one health approach on this [67] [68] [69] , became critical, the balance of human, animal and environmental health is of utmost importance in the assessment of emerging diseases. we consider that conducting new investigations centered around the role of bats and their ecosystems in the transmission of emerging diseases should be a priority to global health. close contact with infected animals, body fluids such as blood, organs, urine, faeces and aerosols generated during defecation, consuming food contaminated with these viruses, direct bite or scratches from these mammals, or even eating it with poor cooking. work in forests, agriculture and fishing covid-19: zoonotic aspects the next big threat to global health? coronavirus infections reported by promed villamil 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antibody in non-hematophagous bats captured in the central pacific coast of mexico brazil burning! what is the potential impact of the amazon wildfires on vector-borne and zoonotic emerging diseases? -a statement from an international experts meeting progress towards rabies control and elimination in vietnam burden of zoonotic diseases in venezuela during rabies vaccine development by expression of recombinant viral glycoprotein nih. rabies vaccine.drugs and lactation database (lactmed) ecoepidemiological and social factors related to rabies incidence in venezuela during 2002-2004 twenty year experience of the oral rabies vaccine sag2 in wildlife: a global review venezuela: far from the path to dengue and chikungunya control estimating and mapping the incidence of dengue and chikungunya in honduras during 2015 using geographic information systems (gis) covid-19 and dengue fever: a dangerous combination for the health system in brazil dengue in honduras and the americas: the epidemics 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from sars, mers and recent advances to combat this pandemic virus none. key: cord-306056-4jx0u7js authors: sulmasy, daniel p. title: “diseases and natural kinds” date: 2005 journal: theor med bioeth doi: 10.1007/s11017-005-2206-x sha: doc_id: 306056 cord_uid: 4jx0u7js david thomasma called for the development of a medical ethics based squarely on the philosophy of medicine. he recognized, however, that widespread anti-essentialism presented a significant barrier to such an approach. the aim of this article is to introduce a theory that challenges these anti-essentialist objections. the notion of natural kinds presents a modest form of essentialism that can serve as the basis for a foundationalist philosophy of medicine. the notion of a natural kind is neither static nor reductionistic. disease can be understood as making necessary reference to living natural kinds without invoking the claim that diseases themselves are natural kinds. the idea that natural kinds have a natural disposition to flourish as the kinds of things that they are provides a telos to which to tether the notion of disease – an objective telos that is broader than mere survival and narrower than subjective choice. it is argued that while nosology is descriptive and may have therapeutic implications, disease classification is fundamentally explanatory. sickness and illness, while referring to the same state of affairs, can be distinguished from disease phenomenologically. scientific and diagnostic fallibility in making judgments about diseases do not diminish the objectivity of this notion of disease. diseases are things, not kinds. injury is a concept parallel to disease that also makes necessary reference to living natural kinds. these ideas provide a new possibility for the development of a philosophy of medicine with implications for medical ethics. twenty-five years ago, in the pages of this journal, david c. thomasma and edmund d. pellegrino challenged the field by arguing that medical ethics must begin with the philosophy of medicine. 1 before his untimely death, dave let us know that such a comprehensive philosophy of medicine -one that could serve as a foundation for medical ethics -still eluded us. perhaps, he seemed to suggest, it had only become more difficult to achieve. in 1997, he wrote, practice of medicine today, the movement back towards foundations will be difficult indeed. 2 in this essay, dedicated to david's memory, i would like to set forth a possible new direction for pursuing this challenge. as he noted, such an endeavor amounts to swimming upstream against the currents of contemporary ''bioethics.'' despite the difficulties, however, it would appear that substantial new work in analytic philosophy might (surprisingly) lead to the sort of foundation for the philosophy of medicine and medical ethics that david sought. the notion of natural kinds has become very important in contemporary anglo-american philosophy, but scholars have not yet taken seriously how the notion that human beings are a natural kind might have implications for the philosophy of medicine and medical ethics. i will not attempt to lay out all of the implications of this idea in a single essay. rather, i will attempt to explain the notion of a natural kind, and to outline some of the ways in which this notion might help to illuminate a few fundamental questions about disease and health care. in the long run, a philosophy of medicine based centrally upon the notion of the human as a natural kind might be exactly the sort of foundation for medical ethics that david thomasma sought. essences have not been very popular in recent philosophy. positivists, for instance, complained that they had never seen one. the philosophy of medicine has also largely discarded essentialism as a sort of lingering, outmoded aristotelian habit of speech, or as jensen has written, ''the child of a static (pre-darwinian) view of nature.'' 3 jensen goes on to say that, ''a unit of classification is a human construct,'' and argues that since diseases are evolving entities, they cannot be assigned essences, which are necessarily static. 4 the idea that all classifications are human constructs is not new. it traces its origins to 13th century nominalism. but mere nominalism is not radical enough for the 21st century. for the post-modern bioethicist, medical terms are all socially constructed and thus, little more than disguised attempts to assert power and domination over others. thus, one group of postmodern feminists has critiqued the research agenda in women's health, writing that the concept to which the word 'woman' refers is '''crowded with the overdeterminations of male supremacy,' and... contaminated with misogyny and sexism.'' 5 they argue further that 'gender' and 'sex' are also tainted with essentialism. the idea of pure sex difference uninfluenced by social constructions presupposes a mythical time prior to gendering. since it is impossible to be both inside culture and outside it at the same time, sexual differences are always already informed by gender. poststructuralist critiques have demonstrated that women's bodies and their experiences are only knowable through the discourses that constitute them, so both sex and gender are socially constructed. 6 nonetheless, to advance their own political agenda of claiming power for women and righting past injustices, they advocate a cynical appropriation of the essentialist terms 'woman', 'sex', and 'gender' as tools for their movement. we argue that a ''strategic essentialism,'' as advanced by spivak, provides feminists the theoretical ground from which to argue their political agendas. in this formulation, essentialist categories such as woman/women, sex, or gender are used strategically to achieve specific purposes or aims. in spivak's terms, strategic essentialism is the ''strategic use of a positivist essentialism in a scrupulously visible political interest.'' in this sense, the strategy suits the situation, and the essentialized term (i.e., woman) becomes a mobilizing slogan aimed at specific political ends. 7 in an academic climate such as this, small wonder that dave thomasma thought that the movement back to foundationalism would be difficult. and yet, powerful arguments are emerging in recent analytic philosophy that are making the concept of essentialism not only respectable once again, but even changing the antiessentialist received view. consider the work of saul kripke on identity and necessity. 8 kripke has argued that ''identity statements are necessary and not contingent.'' he has used the term ''rigid designator'' to describe what might in an earlier era have been called an essential characteristic of a thing. a rigid designator is ''a term that designates the same object in all possible worlds.'' he does not imply that the thing must exist, of necessity, in all possible worlds, but rather that if it were to exist in any possible world, the rigid designator would designate it in that world. this is not trivial or obscure in its consequences for medical ethics. every time, for instance, that one requests a substituted judgment, the question is framed as a contrary to fact conditional that posits a possible world that is not our own. a clinician asks the daughter of a comatose patient, ''what would your mother, mrs. penelope smith, have wanted in terms of life sustaining treatment if she were able to speak to us today?'' kripke would insist that the mrs. penelope smith to whom we refer in the possible world in which comatose people's thoughts can be understood is not some other person who happens to look like mrs. penelope smith, but is identical with the mrs. penelope smith lying in the bed in the icu. it is this woman's values we are after. it is critically important to note that this routine exercise in clinical ethics, the elicitation of a substituted judgment, hailed by purportedly anti-essentialist bioethicists everywhere, is not even conceptually possible unless there is something akin to the essence of mrs. penelope smith. she is rigidly designated by her name. baruch brody's analysis would also support this interpretation, but on more forthrightly essentialist, aristotelian grounds. 9 brody argues that ''identity across possible worlds is prior to rigid designation,'' 10 and that something must already have been picked out as the same in any possible world before it can be designated as such. kripke's work on naming, identity, and natural necessity has also been complemented and even extended by david wiggins' work on natural kinds. 11 wiggins has developed the notion of ''sortal predicates'' by which entities of a certain kind are picked out, identified, and re-identified over time. he has come to the conclusion that the predicate calculus simply cannot account for much of what is (particularly living things) unless it is enriched by the addition of the concept of a sortal predicate. 12 in other words, one cannot say ''smith is the same man i saw yesterday'' without predicating that smith and the man i saw yesterday belong to the same natural kind (in this case, man). and if this is the case, then wiggins says, we must embrace at least a ''modest'' form of essentialism. these essences are not platonic forms. as wiggins puts it, essences are not ''fancied vacuities parading in the shadow of familiar things as the ultimate explanation of everything that happens in the world. they are the natures whose possession by their owners is the precondition of their owners being divided from rest of reality as anything at all.'' 13 with respect to the philosophy and ethics of medicine, one would think it obvious that at least this sort of modified essentialism would be necessary in order to function in the clinical world. but while this might be obvious to clinicians, it has not been obvious to philosophers. how might the concept of natural kinds bridge the gap between the common sense essentialism of clinicians and the philosophy of medicine? building on the work of putnam, wiggins very clearly, if densely, tells what a natural kind entails: the determination of a natural kind stands or falls with the existence of law-like principles that will collect together the actual extension of the kind around an arbitrary good specimen of it; and these law-like principles will also determine the characteristic development and typical history of members of this extension. 14 diseases and natural kinds i will argue that one cannot understand human disease unless one understands that human beings are a natural kind. this is not to argue that diseases are natural kinds, 15 but that the concept of a disease must make necessary reference to a natural kind. if one is sincerely interested in acquiring knowledge about a subject, rather than beginning with obscure cases proposed as counterarguments in support of all manner of skeptical conclusions, one should begin by studying what is common and paradigmatic. the concept of natural kinds provides a framework for doing so. taking biology, pathology, plant science, human medicine, and veterinary medicine very seriously seems critical to any contemporary discussion of the general notion of disease. in this light, the following points are worth noting: (1) a disease is always a disease of (at least one) living natural kind. for example, powdery mildew is a disease that afflicts members of at least one living natural kind -rosemary bushes (rosmarinus officinalis). but it is not a disease of the human natural kind. human diseases are diseases of members of the human natural kind. (2) diseases can afflict more than one natural kind. plants other than rosemary, such as grapes and roses, can become diseased with powdery mildew. dogs and human beings both can be afflicted with myasthenia gravis. (3) diseases of one natural kind may be caused by another natural kind -e.g. -erysiphe spp. and sphaerotheca spp. are among the natural kinds causing powdery mildew. plasmodium falciparum causes one type of malaria in human beings. but clearly not all diseases are caused by one natural kind invading another. there is no known pathogenic natural kind that causes myasthenia gravis. the important point is that to understand any of the conditions named above as diseases, one must understand how these conditions diseases and natural kinds affect the afflicted individual as the kind of thing that it is. there are law-like principles that determine the characteristic history and typical patterns of development that collect together the actual extension of those individual entities one calls members of the human natural kind. in fact, two arbitrary, good specimens of this natural kind have been picked out, radiologically dissected, and their anatomical features placed on line at the national library of medicine in bethesda, maryland. and amazingly enough, the average man or woman (or even child), is easily able to tell which entities belong to this natural kind (and their sex), in the absence of technical anatomical knowledge and without reference to a political agenda. one can also readily recognize those deviations from the characteristic development and typical history that render some members of this natural kind defective. so, children born with syndactyly (webbed fingers) are defective members of the human natural kind. they are not members of some other natural kind, say dolphins. to say something is a defective x implies that one knows what kind of thing an x is. without at least this much essentialism, medicine would not even be conceptually possible. natural kinds have a natural teleology. to say that they have characteristic patterns of development and typical histories implies a teleology. anthony lisska has called the properties that determine the pattern of development of a natural kind its ''dispositional predicates.'' 16 the successful unfolding of these dispositions over developmental time -a program and a pattern -allows the individual to flourish as the kind of thing that it is. natural kinds have dispositions. this much teleology must be granted. uranium undergoes a characteristic pattern of radioactive decay. various types of stars have dispositional predicates. they develop and change over periods of time that may seem long by human standards, but there is a pattern by which a star's history must unfold temporally if it is to behave as the kind of thing that it is. dispositional predicates seem especially characteristic of living natural kinds. in fact, philippa foot has written that the word 'good' is an attributive adjective, not a predicative adjective, and cannot be understood apart from an understanding of what kind of thing something is. 17 so, for instance, the word 'good', as used in the phrase, ''good roots,'' cannot be understood unless one knows that it is being attributed to a rosemary bush and not to a rhinoceros. having deep roots would not be good for a rhinoceros. natural kinds have natural tendencies. much of the scientific enterprise consists of coming better to understand these natural tendencies. this teleology is natural. it does not imply the anthropomorphizing of things or the ascribing of conscious goal-directed activity to them, nor does it imply that there is a deity that directs their development. that natural kinds have law-like principles that determine how they develop and flourish as the kinds of things that they are is simply a fact about the world as we encounter it. natural kinds are not timeless, changeless platonic entities. the concept of a natural kind is perfectly compatible with biological evolution. that living natural kinds evolve over time is simply one of the law-like generalizations that characterize them. some will go extinct. some may change so dramatically that they become a new natural kind. but for human medicine (as well as veterinary medicine and botany), the slow time frame of evolutionary change is just not significant for clinical pathology and practice. human understanding of the law-like generalizations that determine the characteristic development and typical history of natural kinds may also change over time. but this does not mean that the kind has changed, merely our understanding of it. this understanding has a mind-to-world direction of fit -i.e. -our understanding must be better fitted to the kind; the kind is not shaped to our understanding of it. 18 thus, there is scientific progress in medicine. humoral theories of disease have been abandoned. bloodletting has been replaced by plasmaphoresis. but the gene of mendel and the gene of watson and crick are one and the same gene, now understood in greater depth. genetics is the study of one aspect of the lawlike principles that determine the characteristic development and typical history of living natural kinds. we understand human genetic principles better now than we did 200 years ago. but it is still the same natural kind that is studied and treated by human medicinethe human natural kind. the idea of a natural kind is not reductionistic. the fundamental way that complex systems are understood is through a hierarchical analysis of their structure, recognizing that there is both ''top-down'' and ''bottom-up'' causation along the hierarchical levels. 19 natural kinds can be understood at multiple hierarchical levels of organization, and lower levels of organization are neither individually nor jointly sufficient to explain higher levels of organization, or even sufficient to ask the questions that would be relevant to the understanding of the kind at that higher level of organization. these properties, those that can only be understood in relation to the hierarchical level of organization to which they belong, are said to be ''emergent.'' that is to say, they ''supervene'' only at successively higher levels of organization, but cannot be fully explained by lower levels of organization. thus, physics is insufficient to explain chemistry, chemistry is insufficient to explain biology, biology is insufficient to explain psychology, and psychology is insufficient to explain morality. for example, cell walls contain both quarks and l-mesons, but quarks and l-mesons cannot explain what a cell wall is. this does not preclude the work by which scientists can arrive at partial explanations of various states of affairs of a given natural kind, considered at a given level of organization, by invoking an understanding of one or another state of affairs of lower levels of organization of the kind. thus, at a chemical level, understanding that a base pair mutation results in the substitution of valine for glycine at the 6th position of the b chain of the hemoglobin molecule provides a substantial causal explanation for sickle cell disease. this information is useful and important. it has diagnostic and therapeutic implications. but as an explanation of what sickle cell disease is, it is radically incomplete. one cannot understand this disease without also understanding the biology of plasmodium falciparum, the ecological concept of inclusive fit, population genetics, patterns of migration, the physiology of blood flow, the existence of capillaries, the biology of pain, and the concept of infarction, none of which can be fully reduced to chemistry. the chemical explanation deepens the biological explanation, but only biology and levels of scientific hierarchy above the biological (so-called ''top-down causation'') fully explain the biological phenomena. a moral, rational, living natural kind can be understood, nonreductionistically, at multiple, hierarchically arranged levels of organization at which level-specific properties emerge, featuring both top-down and bottom-up causation. for the human natural kind, these might be sketched out as: evolutionary ecology sociology, economics, history psychology physiology anatomy molecular biology and genetics physics the domain of medicine is primarily the domain of the biology of individual members of the human natural kind -particularly the levels of anatomy, physiology, and psychology. those law-like principles that determine the characteristic development and typical history of members of this natural kind at these hierarchical levels of scientific understanding are the subject of medicine as an applied science. these are the biological principles of the kind. this is not to say that physics and sociology are irrelevant to medicine -patients can develop cancer because of radon exposure and economic factors may help to explain why a particular patient came to be exposed to radon. there is both top-down and bottom-up causation. but medical science cannot claim to be the complete human science. it must have boundaries if it is to be a field of inquiry distinguished from others. and this is determined by its praxis, with its emphasis on caring for the individual with disturbed biology at the level of experience and function. so, for example, molecular biology matters intensely to medicine because it can explain alterations in anatomy, physiology, and psychology and offer possibilities for corrective intervention. there can be ''allied'' sciences of medical sociology, medical economics, public health, and medical ethics. there is also a field of medical engineering, applying physics to medical care. but these fields merit the adjective, 'medical' precisely because they can help to explain or can help produce effective interventions in matters of anatomy, physiology, and psychology. this much being said, i am now ready to offer a formal definition of disease. a disease is a class of states of affairs of individual members of a living natural kind x, that: (1) disturbs the internal biological relations (law-like principles) that determine the characteristic development and typical history of members of the kind, x, (2) in a pattern of disturbance shared with at least one other member of the kind, x. (3) the aim of this classification must be to provide at least a provisional basis for explaining the causes and/or natural history of a disturbance in the internal biological relations of the affected members of x (and, if x is a self-reflective natural kind, can serve as an explanation of the illness of those so affected), (4) and at least some individuals of whom (or which) this class of states of affairs can be predicated are, by virtue of that state, inhibited from flourishing as xs. i must further explicate this fairly dense definition. a. a disease is not a natural kind. it is a classification of a certain state of affairs that can occur in members of particular living natural kinds. b. the ''biological internal relations (law-like principles)'' encompass anatomy, physiology, and psychology. not all variations in the law-like principles that govern natural kinds at these levels of organization are diseases, but only those variations that also meet the other criteria. c. there are no diseases that are unique to particular individuals. individuals may be sick or ill, but until the pattern of disturbance is detected in other members of the kind, it is not a disease. disease is a scientific concept and concerns more than the individual. d. the purpose of disease classification (nosology) is, in the first place, explanatory. even if the disease does not provide a causal explanation for the illness, the purpose of bringing a pattern of disturbance under a particular name is to predict an expected natural history and provide the first step towards explanation. even if the explanation is not scientific by contemporary standards (e.g., possession by demons or angry ancestors), the classification of a pattern of disturbance in the law-like principles that govern the biological organization of a living natural kind as a disease serves an explanatory function. e. the hope of medical practice is that better explanatory knowledge of a disease will provide better treatment. the 19th and 20th centuries rewarded medicine handsomely for pursuing explanatory knowledge of diseases. the eventual payoff was better therapy. but the aim of nosology is fundamentally explanatory, not therapeutic. f. there can be asymptomatic disease. but if a pattern of disturbance in the law-like biological principles that determine the characteristic development and typical history of a living natural kind is to be called a disease, at least some individuals with the disease must be inhibited from flourishing as the kinds of things that they are. for example, prostate cancer at age 80 may be ''incidental'' and never interfere with a man's flourishing. but unless prostate cancer interfered with at least some men's flourishing, it would not be called a disease. g. the telos in this definition is that the individual should flourish as the kind of thing that it is. this is an important difference from christopher boorse's specification of the telos in his definition of disease as survival and reproduction. 20 survival and reproduction might be sufficient for an amoeba to flourish as the kind of thing that it is. but survival is scarcely enough for a rosemary bush, never mind a human being. a rosemary bush might survive a bout of powdery mildew, live a normal lifespan, and reproduce well. but while infected, it is not flourishing as a rosemary bush. likewise, a human being with a coronavirus infection has a disease (the common cold). it is almost inconceivable that this could affect her survival or reproduction. but while infected, she fails to flourish as the kind of thing she is. h. setting as the telos the flourishing of the individual as the kind of thing that it is also explains why it can be controversial to classify as diseases certain patterns of variation in the law-like biological principles that determine the characteristic development and typical history of a living natural kind. in particular, patterns of human behavior are most susceptible to being controversially called diseases. but this does not undermine the definition of a disease. it is only to say that the task of deciding whether to designate as disease a pattern of variation in the lawlike principles that govern a thing as a member of a kind will have some very clear cases. 21 by virtue of being the kinds of things that they are, human beings are incredibly varied -biologically, psychologically, and socially. human beings make choices, some of which preclude one good in order to pursue another. there is, therefore, a subject-relativity to the flourishing of human beings that ought not be confused with subjectivity. 22 thus, human flourishing includes a certain diversity, and this diversity is objectively good. while the purpose of this essay is not to engage in a discussion of medical ethics, it must be stated that this diversity is not morally unbounded. human choices ought not detract from the common good (as integrally understood) 23 and ought not undermine the basic conditions by which human beings flourish as the kinds of things that they are. 24 with minor variations, i accept the by-now classical distinction between disease and illness, 25 and i also accept the distinction between sickness and illness, but with a significant variation on the use of these terms. in sum, i hold that these three words designate three ways of referring to the same state of affairs -individually, socially, and scientifically -corresponding to the terms sickness, illness, and disease, respectively. sickness and illness are not the same as disease. 26 distinctions between these terms have phenomenological and sociological meaning for human beings. while the first two terms, in particular, are largely synonymous in ordinary language, they can be used as technical terms to stipulate different phenomenological and sociological aspects of the lived experience of diseased human beings. sickness, as i use the term, is a state of disturbed internal biological relations that appears (from a non-professional perspective) to be inhibiting the individual from flourishing as the kind of thing that it is. individual members of any living natural kind, whether a rosemary bush or a fruit fly, can be said to be sick. generally, this disturbed state has observable manifestations, but not always. to know that an individual member of a kind is sick, one can observe the pattern of disturbance, and, with at least some knowledge of what it takes for such a kind of thing to flourish, one can make the judgment that the individual is sick. if a crow is walking around in clockwise circles and is not flying, not flapping its wings, not eating, and not cawing, most adult north american observers who know enough about what a crow is and how it typically behaves can make the judgment that the bird is sick. one can also ask for a report (from an individual capable of so reporting) about abnormal sensations or other subjective indications of disturbed biology associated with diminished flourishing. before one hears a report from the individual, however, one can make one's own judgment and simply ask for confirmation, saying, ''you look as if you are sick.'' illness is a socially mediated (but also unscientific) judgment about the exact same state of affairs to which sickness refers. illness connotes the recognition of a pattern, but is not a judgment that is made with an explanatory intention. an individual manifests a pattern of disturbed internal biological relations and appears not to be flourishing (is sick). another observer further adduces that this pattern of disturbance is not unique, and that this judgment has been made regarding other members of the kind. unscientifically, one judges, ''this illness is something that has been going around.'' in a rational, self-reflective, and socially interdependent natural kind such as the human, the individual, subjectively appreciated state that i have called sickness must generally be validated by other member(s) of the kind, thus allowing the ill individual to assume ''the sick role.'' 27 disease serves as an explanatory designation for the same state of affairs. this individual's sickness, the illness that has been ''going around,'' is explained by judging that the individual suffers from a disease, e.g., influenza. human disease is therefore also social, but its aim is scientific -an explanation for the observed biological disturbance that does, scientifically, cause at least some individuals, so afflicted, to fail to flourish as the kinds of things that they are. symptoms are the sensory signals by which a sensible natural kind is alerted that internal biological relations are askew. when symptoms are sensed by self-reflective individuals, such individuals can use these sense data to judge that they suffer from sickness -feeling feverish, nauseated, pruritic, being in pain, etc. many living natural kinds have such signals. many of these signals have biological purpose -e.g. -pain may signal that a behavioral response to avoid the offending stimulus is in order. some are biologically inappropriate, or appropriate in the short-term but not in the long-term. symptoms are ultimately subjective, even if the cause of the symptoms is not. among the members of self-reflective natural kinds, symptoms assume meaning. one author has recently suggested the primary symptom is der unheimliche (a sense of not being at home in oneself). 28 an individual sensing symptoms is often initially uncertain of the veracity of a symptom complex or its meaning. this leads the individual to seek validation by others. when this occurs, the patient enters the sick role and concludes, ''i am ill.'' disease, by contrast, is a class of patterns of actual disturbance in the internal biological relations of members of a living natural kind that can explain the sickness of individuals of whom the disease can be predicated. disease can also explain the sickness or illness of afflicted individuals if the natural kind in question is self-reflective and social. diagnosis is the art by which specialists determine that an individual's illness can be explained by a particular disease. this art requires recognizing a pattern of disturbance that typifies the state of affairs that defines the particular disease. diagnosis is an epistemic project. the data are the symptoms (if the individual has the linguistic capacity to report on internal sensations), and signs. signs are observable manifestations of disturbances in the internal relations or law-like principles that determine the characteristic development and typical history of sick members of the kind to which the individual belongs. signs are what others (e.g., doctors and nurses) observe as the effects of illness. some symptoms cannot be observed and can only be reported, for example, the symptom of seeing yellow halos around lights. some signs are only observed directly by others, and cannot be experienced subjectively, or even observed of oneself directly, such as an asymptomatic change in the surface of the retina. often, signs and symptoms will be intrinsically linked. the same phenomenon can have two sides -it can be experienced as a symptom and observed as a sign. for example, a patient may experience the symptom of feeling feverish. if a doctor observes this patient, the doctor will note that the patient has, perhaps among other signs, a fever. a diagnosis is a disease name that has been judged to be predicable of a state of affairs of a given member of a given natural kind, serving at least as a provisional explanation for the observed pattern of disturbance in the internal, kind-typical, biological relations of the individual. a diagnosis is a judgment that the pattern of disturbance in an individual can be classified as belonging to a named disease. diseases are thus classes of disturbed states of affairs in natural kinds. a diagnosis is a judgment that a certain state of affairs in a particular individual belongs to a certain class. lonergan's distinction between classical and statistical heuristic structures provides an important insight into the ways that diseases are currently understood. 29 from a scientific point of view, there are two basic ways of understanding phenomena -classically and statistically. in classical science, the data converge upon some formula or understanding, such as pv = nrt, or e= mc 2 , or the notion that stars form clusters called galaxies. in statistical science, one appreciates that the phenomenon of interest is itself stochastic, and that one can only understand this phenomenon by coming to know a value such as a mean, from which this inherently non-systematic process cannot systematically deviate. so, for instance, one comes to know that the mean temperature in paris in july is 18°c, or that the mean temperature of the background cosmic microwave radiation in space is 2.725°k. this distinction is also helpful in understanding medical science, some of which is understood via a classical heuristic structure, and some of which is understood via a statistical heuristic structure. so, for instance, classical physiological laws obtained in medicine, such as the henderson-hasselbach equation: ph = pk a þ log ½conjugate base ½weak acid . the data about acid-base balance in the human natural kind converge upon this formula. but other processes can only be understood statistically, such as the fact that the ulnar artery is absent in about 3% of members of the human natural kind. diseases likewise are understood both classically and statistically. pneumococcal pneumonia is an example of a classical disease. the data converge on a particular description of the pattern of disturbance in the internal biological relations afflicting individual members of the human natural kind. this does not mean there will be no variations. in fact, the variation one sees in biology is greater than the variation one sees in physics. there is variation even in physics, and a classical law such as pv = nrt does not describe each measurement of pressure, temperature, molar quantity, and volume, but the for-diseases and natural kinds mula upon which multiple such measurements converge. likewise, the data do converge on a classical description of pneumococcal pneumonia even if it is not true that each and every case of pneumococcal pneumonia is exactly the same. there are also statistical diseases. since human diseases are disturbances in the biological law-like properties that govern the typical history and characteristic development of the human natural kind, it is not the mean value or central tendency but variations from the mean that are of concern to medicine. medicine characterizes some such variations as diseases, to the extent that they also fulfill the other criteria for classifying something as a disease -especially that they inhibit human flourishing. thus, some statistical variations (such as the absence of an ulnar artery) are not classified as diseases, while others (such as hypertension) are classified as diseases. 30 the patterns of reasoning by which one makes the judgment that a particular state of affairs in a particular individual belongs to the class defined by a particular disease are multiple and complexdeductive, analogical, statistical, pragmatic, and more. a full discussion of diagnostic reasoning is beyond the scope of this essay. the philosophical literature on diseases has raged on for decades as a pitched battle between realists and anti-realists. the modest essentialism that accompanies the notion of natural kinds provides the basis for a modified form of realism about disease. the view proposed here may provide a solution to the problems posed by previous defenses of disease realism. diseases are not primary existents. 'systemic lupus erythematosis' does not pick out a primary existent, but a class of states of affairs occurring in members of a natural kind(s). diseases are not natural kinds, but states of affairs. diseases have no essences. saying this does not imply, however, that diseases have no objective basis, or are merely human constructions, or that diseases are merely value judgments. diseases make necessary reference to natural kinds, and natural kinds admit of at least a modest essentialism. it is essentialism about living natural kinds and their natural dispositions that provides the foundation for realism about diseases. the patterns of disturbance that one classifies as diseases are not arbitrary. it is the pattern of disturbed internal biological relation-ships in the natural kind that imposes itself upon the observer; the observer does not impose the pattern upon the affected members of the kind. that is to say, there is a mind-to-world direction of fit -the mind of the observer must conform to the world for the observer's beliefs about disease to be true. the world is the standard by which the observer's beliefs are judged. further, many diseases of one natural kind are caused by other natural kinds, and the pattern of disturbed development of a diseased natural kind may reflect the flourishing of the one causing the disease (e.g., in malaria, the illness of a member of the human natural kind is explained by the characteristic development and typical history of plasmodium falciparum). thus, the essentialism of two natural kinds may necessarily be invoked in defining some diseases. arguments are often proposed in (roughly) the following form: it was once thought that x was a disease. now, we no longer think that x is a disease. therefore diseases are social constructions. 31 such arguments seem superficially to be sound, but they are specious. that the medical community can change its beliefs about disease does not imply that diseases are constructed by physicians according to the mores of their era. an alternative interpretation is that the former belief or the present belief or both are wrong. medical judgments are fallible. but fallibility does not imply subjectivity. for example, it may turn out that edwin hubble was wrong and that the universe is not expanding. but whether hubble was right or wrong depends upon the universe, not upon the mental states of human beings. in thinking about fallibility in medicine, it is important to distinguish two types of fallible judgment about disease -scientific judgments and diagnostic judgments. among scientific judgments about diseases, there are two sub-types, both of which are fallible: (1) one may judge incorrectly that what appears to be a pattern of disturbance in the law-like principles governing the internal biological relations of a natural kind constitutes a disease (i.e., one may be wrong in one's scientific judgment that apparent pattern x is a disease) and (2) one may incorrectly formulate a causal explanation for the pattern of disturbance picked out by a disease name (i.e., one may be wrong in one's judgment about the cause of x). the objective basis for disease classification must be that it makes necessary reference to a natural kind and to disturbances in the internal biological relations that inhibit at least some of the affected members of the kind from flourishing as the kinds of things they are. i will argue that, at the end of the day, this is what settles the question of whether a certain observable pattern constitutes disease. those who offer putative counter-examples, such as the 19th century medical theory that a slave's habitual tendency to try to escape was a disease (''drapetomania''), 32 do not in any way thereby provide the slightest bit of proof that disease is a ''value-judgment.'' the objective basis for deciding this matter is to ask whether a desire no longer to be a slave is a disturbance in the internal biological relations that inhibits afflicted members of the human natural kind from flourishing as the kinds of things they are. if someone once thought this was true of slaves who wanted to escape, this opinion is simply wrong on both counts. it might be understandable that someone would render such an erroneous opinion given the socioeconomic and historical conditions under which physicians who made such judgments lived. but it is an absolute non-sequitor to conclude that this implies that all diseases are value judgments and that therefore appendicitis is simply a human construct. that judgments are fallible does not mean that they have no objective basis. behaviors are particularly susceptible to this sort of error. even today, whether some behaviors (such as homosexuality or substance abuse) are diseases can be hotly contested by various groups. but again, this does not mean that there is no basis for settling the case. the basis for settling this question is the same: whether there is at least a plausible biological explanation and whether the behavior inhibits human beings from flourishing as the kind of things that they are. that we are not 100% settled on what constitutes human flourishing does not mean that there is no basis for making such judgments or that there are not thousands of other clear cut cases (such as appendicitis and malaria) where it is obvious that the observed pattern of kind-atypical activity has a biological basis and that this pattern inhibits human flourishing. medical science can also be mistaken in its causal explanations about diseases. malaria (as its name implies) was once attributed to ''bad air.'' thus, a pattern of disturbance in the law-like characteristics governing the typical development and history of human beings was noted, cases fitting this pattern were carefully classified according to patterns of fever, and each thought to have a particular biological explanation. that explanation made epidemiological sense, and it ultimately fit with the causes of malaria as presently under-stood -anopheles mosquitoes, carriers of the malarial parasites, breed where the air is ''bad.'' doubtless our future understanding of malaria will deepen further than our understanding today. but the ''mal'aria'' of medieval italy and the disease we know today are not two different diseases. they are the same disease, better understood. the fact that we can incorporate previous observations into deeper explanations of the same disease is inconsistent with the assertion that diseases are merely human constructions. throughout the history of medical science, all we have ever tried to do has been to shape our minds better to fit the world as we encounter it. the fact that we have made mistakes does not mean that disease concepts are merely subjectively or intersubjectively chosen values. the ultimate standard of the truth about malaria and other disease concepts is in the world, not in our heads. that is how we can learn that we have been mistaken in our views about diseases. human medical science will establish the criteria for deciding that a particular pattern of disturbance in the biological internal relations of an individual member of the human natural kind falls within the extension of each disease category. one can be mistaken in the criteria that one establishes, because the pattern one is attempting to capture has an objective unity. one discovers that unity; one does not create it. in lonerganian terminology, a disease is a real thing, even if it is not a natural kind. 33 one can progress in one's understanding of a thing. for example, the medical community once judged that a diagnosis of the condition known as lyme disease required manifestations of an oligoarthritis. as more research was conducted, it was noted that oligoarthritis was one stage in this disease, and that earlier stages had different manifestations such as an erythema migrans rash. the pattern of disturbance in the internal biological relations of the human natural kind to which the disease name 'lyme disease' refers did not change. our scientific understanding of lyme disease changed and the diagnostic criteria were adjusted. mistakes are also possible in diagnostic judgments. whether someone does or does not have a disease is fallible judgment, as all physicians (and medical students) know well. mistakes are possible at multiple levels. first one must correctly interpret the observable signs and the communicated symptoms (history and physical). second, one must be knowledgeable about the patterns of signs and symptoms manifested by the diseases as they are classified by the medical science of one's era (differential diagnosis). third, one must be knowledgeable about the appropriate confirmatory tests (diagnostic testing), and sufficiently experienced to use only those that are necessary (diagnostic elegance). fourth, in the setting of uncertainty, one must avoid the extremes of rashness (making a diagnosis before there are sufficient data) and indecisiveness (deferring judgment when sufficient data are at hand). the art of medicine is the mediation between medical science (concerned with universals) and the individual who must be diagnosed and treated. diagnosis is a judgment about individuals. as aristotle has said, the doctor does not cure 'a man' universally taken, except accidentally, but callias or socrates or someone else to whom also the essence of man happens to belong. if, then, someone without the experience has the theory and knows the universal but is ignorant of the individual included under this universal, he will often fail to cure; for it is rather the individual that is curable. (metaphysics 981a 18-25) . 34 the fact that one can be mistaken in applying the science of one's era in making a judgment about whether a particular disease name can be correctly applied to a particular pattern of disturbance in the internal biological relations of an individual member of the human natural kind does not imply that diseases are value judgments. the logic of diagnosis is intensional. the ultimate standard for whether or not one has correctly applied the disease name (as defined by the science of one's day) to a particular patient is the match between that definition and the state of affairs that actually exists in the affected individual member of the human natural kind who is being diagnosed. further, the fact that the standards that are given for defining a disease must, to some extent, be arbitrary and admit of borderline cases, does not mean that diseases are primarily human constructions and value judgments. one must be careful not to confuse the epistemological with the ontological. definition is an epistemic exercise. whatever definition one gives will exclude some cases and include others, because this is what a definition does. giving intensional definitions for ontologically real entities is an inherently fallible enterprise. in a definition of multiple myeloma, for instance, one must arrive at cutoff values, currently set at >30% plasma cells in the bone marrow and >3.5 gms/dl of an igg m-protein. 35 as every experienced clinician knows, some individuals who do not meet these criteria actually have multiple myeloma. however, this only means that the reality to which the definition points is prior to the definition. the definition is open to revision, based on its ability to correctly classify cases as belonging to the recurring pattern of disturbance in internal biological relations of the human natural kind that the definition is attempting to capture. there will be mistakes. some cases will only come to be knowable as the pattern of disturbance in the individual progresses along its own natural history. there will also be borderline cases in which the diagnostic judgment will be difficult. but as anscombe once remarked (in a quote she attributes to samuel johnson), ''the fact of twilight does not mean there is no difference between night and day.'' 36 borderline cases do not suffice to refute objectivity. thus, one must conclude that none of these sources of fallibility in reasoning about diseases provides a philosophical justification for radical skepticism about the realism with which clinicians must undertake the tasks of studying and diagnosing diseases. diseases are objective perturbations in the reality of the natural kinds of which they can be predicated. finally, something must be said about the distinction between disease and injury. i am very sympathetic to the spirit of boorse's defense against those who criticize his theory for failing to make this distinction. 37 this distinction is not fundamental to the notion of disease. however, i do think the distinction has some usefulness and should be maintained by the philosophy of medicine. injury and disease are highly related but distinct concepts. the words are used quite distinctly by both medical laypersons and medical professionals. the sub-specialists who treat diseases (e.g., internists, pediatricians, and family physicians) are different from those who treat injuries (e.g., orthopedists, burn surgeons, and trauma physicians). so, it is probably worthwhile to maintain the distinction. nonetheless, disease and injury are sufficiently related that the definition of injury is really parallel to that already given for disease. like disease, injury makes necessary reference to the notion of a living natural kind. an injury is a state of affairs of an individual member of a living natural kind x, that: (1) disrupts the physical structure or physical integrity that is characteristic of the developmental stage and is typical of the history of members of the kind, x, and (2) this state of affairs inhibits the individual from flourishing as an x. this definition of injury is parallel to the definitions of sickness and illness. that is to say, it is a ''lay'' definition. unfortunately, there is no ready-made cluster of synonyms that one can use to distinguish the various phenomenological and sociological aspects of injury to parallel the vocabulary of sickness, illness, and disease discussed above. instead one can therefore call this lay definition, injury sense 1 . in this sense, one can say, as a medical layperson, in parallel to the notion of sickness, ''my finger is bleeding,'' or ''i hurt my wrist.'' parallel to the sociology i associated with the word, 'illness,' the injured sense 1 individual may seek intersubjective validation. while the same definition applies, there is therefore a usage we can call injury sense 2 . thus, my 4-1/2 year old nephew asks, ''do i have a splinter, uncle danny?'' or someone says, ''you broke your wrist,'' or ''you've burned your hand rather badly.' ' however, there are many specific injuries that are more like diseases than they are like sickness or illness in that they have been named and categorized by medical science. in the absence of synonyms to use as technical terms, one can call this injury sense 3 . this sense of injury can be defined in a manner more formally parallel to the definition of disease. a named injury (injury sense 3 ) is a class of states of affairs of individual members of a living natural kind x, that: (1) disrupts the physical structure or physical integrity that is characteristic of the developmental stage and is typical of the history of members of the kind, x, (2) in a pattern of disruption shared with at least one other member of the kind, x. (3) the aim of this classification must be to provide at least a provisional basis for explaining the causes and/or natural history of this disruption in members of x (and, if x is a self-reflective natural kind, can serve as an explanation of the injury sense 1 of those so affected), (4) and at least some individuals of whom this class of states of affairs can be predicated are, by virtue of that state, inhibited from flourishing as xs. thus the physician says, ''you have a non-displaced colle's fracture,'' or ''this is a second-degree burn injury involving 2.5% of the body surface.'' this is scientific, explanatory vocabulary. the notion of injury sense 3 is parallel to the notion of disease in every way. while it is a different class of states of affairs of the biology of living natural kinds, injuries (sense 2 ), like diseases, can be predicated of any living natural kind. and the concept of injury sense 2 , like the concept of disease, is objective, even if scientific and diagnostic judgments regarding injuries can be just as fallible as those regarding diseases. the notion of natural kinds can be a powerful starting point for the philosophy of medicine. it provides a philosophically credible basis for objectivity about diseases. the modest essentialism that accompanies the notion of a natural kind meets most objections raised in the literature. it provides a basis for discussion of value in disease discourse, while maintaining that such values are objective and inherent in the dispositional predicates of natural kinds in accordance with a theory of natural goodness and the teleology of a thing flourishing as the kind of thing that it is. it provides a basis for disease realism while avoiding the pitfalls of characterizing diseases as natural kinds themselves. it may provide just the sort of foundation for the philosophy of medicine upon which to develop a medical ethics, following the plan david thomasma outlined for us but could not carry out before his untimely death. philosophy of medicine as the source for medical ethics antifoundationalism and the possibility of a moral philosophy of medicine a critique of essentialism in medicine,'' in health, disease, and causal explanations in medicine abstracting women: essentialism in women's health research identity and necessity naming and necessity,'' in semantics of natural language sameness and substance see the debate about the notion of diseases as natural kinds: robert d'amico however, the inference both seem to share, that if disease is not a natural kind then it must be a ''value judgment aquinas' theory of natural law: an analytic reconstruction philippa foot, natural goodness intentionality: an essay in the philosophy of mind on the moral nature of the universe concepts of health,'' in health care ethics: an introduction autonomy, subject-relativity, and subjective and objective theories of well-being in bioethics four basic notions of the common good dependent rational animals: why human beings need the virtues 49-68; and his later refinements of this distinction in ''a rebuttal on health while i accept more or less the same distinction, i am using the terms 'illness' and 'sickness' in the opposite manner as has become standard in the philosophy of medicine (see bjørn hofman since it is preferable to assign to technical terms the use that is closest to ordinary language, i propose a reversal of the standard use of these terms. i will use 'sickness' to refer to the individual's own subjective experience of disturbed internal biological relations, or to an observer's assessment of the disturbed state of an individual. i will use 'illness' to refer to the intersubjectively supported, socially mediated understanding of the same state of affairs. so, we often say that a dog is ''sick,'' but we do not usually say that a dog is ''ill das unheimliche: towards a phenomenology of illness insight: a study of human understanding i am tempted to argue that ''statistical'' diseases are only epidemiological risk factors for classical ones, but i will not explore this hypothesis further in this essay a variation on this fallacious line of argument is the illicit inference that since there are heated arguments in the present about whether certain states such as homosexuality or menopause or aging or infertility are diseases, one must conclude that diseases are socially constructed. this is the tack taken by arthur l. caplan in ''the 'unnaturalness' of aging -give me a reason to live report on the diseases and physical peculiarities of the negro race,'' in health, disease, and illness: concepts in medicine plasma cell neoplasms,'' in principles and practice of oncology war and murder toward a pragmatic theory of disease.'' in what is disease war and murder.'' in war and morality concepts of health.'' in health care ethics: an introduction on the distinction between disease and illness a rebuttal on health the unnaturalness of aging -give me a reason to live.'' in health, disease, and illness report on the diseases and physical peculiarities of the negro race.'' in health, disease, and illness: concepts in medicine is disease a natural kind the foundations of bioethics natural goodness on the triad disease, illness and sickness a critique of essentialism in medicine.'' in health, disease, and causal explanations in medicine identity and necessity.'' in identity and individuation naming and necessity.'' in semantics of natural language theory of natural law: an analytic reconstruction insight: a study of human understanding dependent rational animals: why human beings need the virtues abstracting women: essentialism in womens health research plasma cell neoplasms.'' in principles and practice of oncology on the moral nature of the universe minneapolis definitions of health and illness in the light of american values and social structure.'' in patients, physicians, and illness dis-ease about kinds: reply to damico intentionality: an essay in the philosophy of mind four basic notions of the common good das unheimliche: towards a phenomenology of illness philosophy of medicine as the source for medical ethics antifoundationalism and the possibility of a moral philosophy of medicine autonomy, subject-relativity, and subjective and objective theories of well-being in bioethics sameness and substance key: cord-312247-cza4qsv5 authors: würdinger, t; verheije, m h; raaben, m; bosch, b j; de haan, c a m; van beusechem, v w; rottier, p j m; gerritsen, w r title: targeting non-human coronaviruses to human cancer cells using a bispecific single-chain antibody date: 2005-04-21 journal: gene ther doi: 10.1038/sj.gt.3302535 sha: doc_id: 312247 cord_uid: cza4qsv5 to explore the potential of using non-human coronaviruses for cancer therapy, we first established their ability to kill human tumor cells. we found that the feline infectious peritonitis virus (fipv) and a felinized murine hepatitis virus (fmhv), both normally incapable of infecting human cells, could rapidly and effectively kill human cancer cells artificially expressing the feline coronavirus receptor aminopeptidase n. also 3-d multilayer tumor spheroids established from such cells were effectively eradicated. next, we investigated whether fipv and fmhv could be targeted to human cancer cells by constructing a bispecific single-chain antibody directed on the one hand against the feline coronavirus spike protein – responsible for receptor binding and subsequent cell entry through virus–cell membrane fusion – and on the other hand against the human epidermal growth factor receptor (egfr). the targeting antibody mediated specific infection of egfr-expressing human cancer cells by both coronaviruses. furthermore, in the presence of the targeting antibody, infected cancer cells formed syncytia typical of productive coronavirus infection. by their potent cytotoxicity, the selective targeting of non-human coronaviruses to human cancer cells provides a rationale for further investigations into the use of these viruses as anticancer agents. replicative oncolytic viruses represent new agents with potential utility in cancer therapy. they are aimed to effectively eradicate tumor cells without affecting normal tissues. several different dna and rna viruses are currently being evaluated for their efficacy and selectivity toward cancer cells. 1, 2 so far, there are no reports describing the potential use of coronavirus-based oncolytic agents in cancer virotherapy, despite a number of features that make coronaviruses potentially attractive for this purpose. coronaviruses are positive-strand rna viruses consisting of a nucleocapsid, which contains the approximately 30 kb genome and the nucleocapsid (n) protein, and which is surrounded by an envelope carrying three membrane proteins, spike (s), envelope (e), and matrix (m). of these, the spike glycoprotein s is responsible for virus entry and syncytia formation, as it binds to the cellular receptor and induces membrane fusion. [3] [4] [5] the specific interaction between the amino-terminal spike protein domain s1 and its cognate receptor [6] [7] [8] induces conformational changes in the spike protein domain s2 that trigger the membrane fusion process. these con-formational changes require physical interaction between two heptad repeat (hr) regions, hr1 and hr2, that occur in the s2 domain. prevention of this interaction by using peptides that correspond to these hr1 and hr2 regions can block membrane fusion, by binding of the peptides to their respective counterparts. [9] [10] [11] most coronaviruses exhibit strict species specificity, as determined by the spike-receptor interaction. [12] [13] [14] the coronavirus feline infectious peritonitis virus (fipv), for instance, selectively infects and induces syncytium formation of feline cells via its receptor feline aminopeptidase n (fapn). 6 likewise, the recombinant felinized mouse hepatitis virus (fmhv), 15 a derivative of mouse hepatitis virus (mhv) carrying a chimeric spike of which the ectodomain is from the fipv spike protein, also infects and fuses only feline cells through the fapn molecule. as a consequence of their species restricted tropism, fipv and mhv are nonpathogenic to human cells. however, once the tropism barrier is alleviated, coronaviruses can replicate in cells of different species. 15, 16 thus, fipv and mhv may potentially be converted into specific oncolytic agents for the treatment of human cancer if their spike protein would recognize a receptor on human tumor cells. toward developing recombinant coronaviruses with a selective human tumor tropism, we first set out to investigate the effectiveness of fipv and mhv in eradicating, in vitro, human cancer cells expressing the virus receptor. to allow comparison of the two viruses in the same cells, we assessed fipv and, instead of mhv, the chimeric coronavirus fmhv for their ability to kill human cancer cells artificially expressing fapn. subsequently, we investigated whether fipv and fmhv could be targeted to human cancer cells expressing the epidermal growth factor receptor (egfr), a molecule commonly overexpressed on many types of cancer cells 17 and associated with poor prognosis and response to cancer therapy. 18, 19 for this purpose, we prepared a bispecific single-chain variable fragment (scfv) antibody, binding on one side to the fipv and fmhv spike protein and on the other side to the human egfr. the antibody indeed functioned as a specific targeting device. our findings justify the further development of coronaviruses as oncolytic agents. to determine whether non-human coronaviruses can infect and kill human cancer cells when the host species barrier determined by specific receptor recognition is alleviated, fipv and fmhv were redirected to human cancer cells via the virus receptor fapn. first, the susceptibility of the tumor cell lines caco-2, ovcar-3, hct-8, hela, hepg2, and widr to fipv and fmhv was successfully demonstrated by transient transfection of these cells with an fapn expression plasmid followed by virus inoculation (data not shown). control inoculations on cells not transfected with the fapn expression construct did not show any detectable fipv or fmhv infection, confirming that infection required the appropriate receptor expression. studies of fipv and fmhv propagation, cytotoxicity, and syncytia formation require host cells with stable receptor expression. therefore, hela and ovcar-3 cell lines stably expressing fapn were produced. fipv and fmhv were tested for their growth characteristics in these hela-fapn and ovcar-fapn cells ( figure 1a) . evidently, infection of fapn-expressing human cancer cells with the viruses fipv and fmhv resulted in a typical rapid production of progeny virus. to determine the potency of fipv and fmhv to kill human cancer cells, hela-fapn and ovcar-fapn cells were inoculated at various multiplicities of fipv or fmhv and the cell viability was measured at several time points after infection. fipv and fmhv efficiently killed both types of cells in a dose-dependent manner. the onset of cell death was found to occur as early as 12-24 h after inoculation. following infection at a multiplicity of infection (moi) of 10, both viruses completely eliminated the ovcar-fapn and hela-fapn monolayers within 24 and 36 h, respectively. infections at lower moi apparently required multiple rounds of infection resulting in the death of all or nearly all cells within 60 h ( figure 1b) . furthermore, infected cells clearly showed membrane fusion typical of coronavirus replication (figure 1c ). we also determined whether non-human coronaviruses were able to eradicate human cancer cells in an in vitro solid tumor model. multilayer tumor spheroids offer a useful 3-d model for assessing virus-mediated eradica-tion of tumor tissue. this model has already been employed to study the potency of oncolytic adenoviruses and adeno-associated viruses. 20,21 ovcar-fapn spheroids were established and inoculated with fmhv at 5 â 10 4 plaque forming units (pfu)/spheroid. for comparison, the oncolytic effect of adenovirus type 5 (ad5) at 5 â 10 8 pfu/spheroid was also measured. at several days post-inoculation (p.i.), the cell viability was determined (figure 2a) . at day 5 p.i., a clear decrease in viability was observed for spheroids inoculated with either virus. after 2 days, the ovcar-fapn spheroids infected with 5 â 10 4 pfu fmhv were essentially destroyed, whereas spheroids infected with 5 â 10 8 pfu ad5 were only partially eradicated. it should, however, be noted that the cytolytic and entry mechanisms of coronaviruses and adenoviruses differ distinctly, making a direct comparison not entirely valid. the results were confirmed by light microscopic analysis (figure 2b ). having established that fipv and fmhv can infect and destroy cancer cells once the entry barrier has been overcome, we wanted to develop a general method to target these viruses to a suitable antigen expressed on such cells. to this end, we constructed the bispecific scfv 23f-425, which combines the antigen binding domains from antibodies 23f8.1 and 425, recognizing the fipv s protein and egfr, respectively. the protein was produced by expression in eucaryotic cells. its synthesis and secretion were verified by radiolabeling followed by immunoprecipitation from the cell lysate and culture medium using an anti-myc antibody ( figure 3 ). the results clearly show the synthesis and secretion of the approximately 58 kda bispecific single-chain molecules. to investigate whether scfv 23f-425 could serve as an adapter molecule for fipv and fmhv infection via human egfr, cultures of human cancer cell lines of different tissue origin with confirmed expression of egfr ( figure 4 ) were inoculated with similar amounts of fipv or fmhv in the presence or absence of the bispecific antibody. after 1 h at 371c, the inoculum was replaced by regular culture medium and incubation of the cells was continued for 24 h. the cells were immunostained for coronavirus protein expression. as can be seen in figure 4 , all cell lines tested had become infected with fipv and fmhv in the presence of scfv 23f-425. in contrast, none of the cells stained positive after inoculation with fipv or fmhv that had been preincubated with mock control supernatant (data not shown). similarly, no positive staining was observed when human cancer cells had been inoculated with the control virus mhv in the presence of scfv 23f-425 (data not shown). differences in infection efficiency were observed between different cell lines, with the egfr-high a431 cells showing the highest susceptibility to egfr-targeted coronavirus infection and the egfr-low hepg2 cells being the most poorly infected. on most cell lines, hela cells being the exception, egfr-targeted fipv exhibited a similar infection efficiency as egfr-targeted fmhv. interestingly, infected cells formed syncytia typical for productive coronavirus infection. the formation of infectious progeny virus was confirmed by monitoring -through titration on fcwf-4 cells -the increase in viral targeting non-human coronaviruses to human cancer cells t würdinger et al titers in the medium of a431 cells after inoculation with fipv or fmhv. typical growth curves were obtained, but only after the a431 cells had been inoculated in the presence of bispecific scfv 23f-425 ( figure 5 ). to confirm that the infections of fipv and fmhv established by scfv 23f-425 were indeed mediated by the human egfr protein, we tested the mouse fibroblast cell line nih 3t3 and its human egfr-expressing derivative nih 3t3-hegfr 22 for their susceptibility to fipv and fmhv in the presence and absence of scfv 23f-425. as illustrated in figure 6a further evidence for a specific interaction between the bispecific scfv 23f-425 and human egfr was obtained by studying the effect of the egfr antibody 425 on the scfv 23f-425-mediated coronavirus infection. hela cells were incubated with the 425 monoclonal antibody prior to inoculation of fmhv in the presence of scfv 23f-425. figure 6b shows that infection was blocked almost completely, confirming that a direct interaction with the egfr is required. similarly, the necessity of an interaction between the fipv spike protein and scfv 23f-425 was confirmed. we incubated fmhv with and without anti-fipv spike monoclonal antibody 23f8.1 before adding scfv 23f-425 and inoculating hela cells. again, the anti-s antibody inhibited infection, demonstrating that scfv 23f-425 has to bind to the virus spike protein in order to function as a targeting adaptor ( figure 6b ). several human cancer cell lines that were infected with egfr-targeted fipv or fmhv showed cell-cell fusion typical for coronaviruses (see figure 4 ). syncytium formation is an important determinant for cytotoxicity and spread of coronaviruses. therefore, it was important to investigate if syncytium formation between infected and neighboring cells resulted from undefined interactions or from specific bridging by scfv 23f-425. to this end, a431 cells were inoculated with fmhv in scfv 23f-425-containing medium for 2 h and subsequently cultured for another 22 h in the presence or absence of scfv 23f-425. thus, scfv 23f-425 was either present continuously to support infection and syncytium formation or only briefly to mediate targeted infection. removal of scfv 23f-425 after 2 h significantly reduced syncytium formation by approximately four-fold from 40.874.1 to 11.272.2 nuclei per syncytium (the data represent the average amount of nuclei per syncytium measured in three independent experiments; 7 indicates the standard deviation). hence, scfv 23f-425 did not only promote fusion between the fmhv envelope and target cells, but also fusion between fmhv-infected and neighboring cells. coronaviruses use a class i membrane fusion mechanism, 11 common to a number of enveloped virus families, 23 in which hr regions occurring in the spike protein play an instrumental role. the mechanism involves conformational changes in the spike protein subsequent to receptor binding, resulting in an interaction of the hr1 and hr2 domains, which is necessary to drive the membrane fusion process. this process can be inhibited specifically using peptides corresponding to targeting non-human coronaviruses to human cancer cells t würdinger et al the hr domains as we showed recently for mhv. 11 in order to investigate if the bispecific antibody-targeted fipv and fmhv infections and their induction of syncytia also depend on those conformational rearrangements in the viral spike protein, we tested the sensitivity of these processes to hr-derived peptide. to this end, we prepared a peptide corresponding to the hr1 region of the fipv s protein and initially studied this fhr1 peptide for its ability to block fipv and fmhv infection of feline fcwf-4 cells, which it did. as illustrated for fmhv (figure 7a and b) , addition of fhr1 during or after inoculation of feline fcwf-4 cells abrogated infection and syncytium formation, respectively. next, we determined whether scfv 23f-425-mediated infection and syncytium formation are also sensitive to the fhr1 peptide. to this end, fmhv was targeted toward egfr on a431 cells in the presence or absence of fhr1. as can be seen in figure 7c and d, addition of the peptide reduced both scfv 23f-425-mediated infection and syncytium formation. thus, both processes seem to utilize a membrane fusion process similar to that of the native coronavirus. despite tremendous research efforts over the last decades into the nature of the disease and its causes, and despite the significant new insights acquired, cancer remains one of the most common causes of death. actually, treatment still relies for a major part on classical approaches such as surgery, radiotherapy, and chemotherapy. clearly, novel and creative methods are needed to complement the conventional treatment options. recently, the use of viruses as potential tools for anticancer therapy has gained considerable interest. 1, 2 in this first exploratory study, we demonstrate two important features that make coronaviruses attractive for this purpose, that is, the ability to target these viruses to human tumor cells and their subsequent infection and eradication of these cells. the non-human coronaviruses fipv and fmhv appeared to possess a strong capacity to kill human cancer cells once they are able to enter these cells through an artificially expressed receptor, consistent with earlier observations with mhv by gallagher and co-workers. 6, 24 the observed rapid and efficient eradication of cancer cells can probably be attributed to two coronaviral features. first, coronaviruses are positive-strand rna targeting non-human coronaviruses to human cancer cells t würdinger et al viruses that exhibit a fast, cytoplasmic transcription process leading to rapid virus protein synthesis and progeny virus production. second, the ability of coronaviruses to induce syncytia between infected and noninfected neighboring cells amplifies their cytotoxicity. we found that fipv and fmhv retained these properties in human cancer cells expressing the virus receptor, thus enabling a rapid cytotoxic spread of the virus to surrounding noninfected cancer cells. coronaviruses share some of these attractive characteristics with other enveloped viruses such as a fusogenic mutant of herpes simplex virus 25 and the live attenuated edmonston b vaccine strain of measles virus. 26 however, in contrast to these viruses, the coronaviruses fipv and fmhv are normally incapable of infecting human cells, due to their restricted tropism. thus, their native tropism does not need to be abolished in order to specifically limit their access, hence cytotoxicity, to human cancer cells. the ability to deliberately target viruses to preselected cells is a tremendous challenge with far-reaching implications for all kinds of therapeutic applications. although we were able to demonstrate the principle of retargeting of coronaviruses by exchanging spike ectodomains, 15, 16 neither the detailed structural information nor the knowledge and technology required to purposely redesign the spike for binding to any given antigen are presently available. therefore, as an alternative and also potentially versatile tool, we embarked on the development of a bispecific adapter. thus, to redirect the non-human coronaviruses fipv and fmhv to egfrexpressing cells, we constructed the bispecific antibody molecule scfv 23f-425 that binds to both the feline spike and egfr. the latter was chosen for its frequent overexpression on human cancer cells. inoculation of targeting non-human coronaviruses to human cancer cells t würdinger et al fipv and fmhv onto a number of different egfrexpressing human cancer cell lines of various tissue origins in the presence of scfv 23f-425 resulted in infection, replication, and subsequent formation of syncytia. the targeted infection was completely dependent on the presence of the antibody and its efficiency generally correlated with the levels of egfr expression on the cancer cells. these observations are similar to native coronavirus infections where virus-cell and cellcell fusion efficiencies also correlate with host cell receptor density. 24 the results imply that the bispecific antibody-mediated targeting approach can in principle be applied to direct coronaviruses to any cell surface antigen for which an appropriate antibody (ie hybridoma cell line) is available. the successful application of bispecific adapters for viral tumor therapy will depend, among others, on cell surface antigens that are -preferably -unique to the tumor. useful tumor-specific markers have not yet been identified and future work should reveal whether they occur or can be specifically induced in tumor cells. interestingly, for the attenuated measles virus, it was recently described that receptor density could also be a determinant of preferential tumor killing. 27 this indicates that even overexpression of certain receptors on tumor cells may result in tumor selective infection and subsequent cell killing. this may also be of importance for the targeting of coronaviruses, since similar receptor density dependence has been described to be important for coronavirus infection and syncytia formation. 24 the application also depends on the sufficient, local presence of the adapter. this might be achieved by incorporating the adapter gene sequence into the viral genome to have the virus produce its own targeting device. we have demonstrated this principle recently for the targeting of conditionally replicating adenoviruses. thus, adenoviruses expressing a bispecific scfv for targeting to egfr showed enhanced oncolytic replication in egfr-positive, adenovirus receptor-negative cancer cells. 28 foreign gene expression can also be achieved with coronaviruses as we showed recently after inserting different reporter genes at various positions in the mhv genome. 29 coronaviruses exhibit high mutation rates and are prone to recombination. their application in adaptermediated targeting to tumors will thus raise serious safety questions, particularly regarding the possibility of generating viruses that acquired the capacity to infect human cells independent of targeting devices. these questions will have to be addressed. several options to reduce the risks already exist. one is the use of coronaviruses lacking specific virulence genes; as we showed recently for mhv 30 and fipv, 31 such viruses are strongly attenuated in their natural host. another option is to combine deletion of nonessential virulence genes with genomic rearrangement; reorganization of the order of the structural protein genes, found for mhv to be tolerated without loss of viability, 32 will reduce the risk of generating viable viruses by recombination with circulating field viruses. entry of coronaviruses into cells normally requires binding of the spike to the receptor followed by a series of structural rearrangements in the s protein that eventually lead to the merging of viral and cellular membranes. it appeared that the bispecific antibody-mediated entry, as well as the antibody-mediated induction of syncytia, uses this same fusion mechanism as was illustrated most clearly by the inhibitory effect of the hr1-derived peptide. it will be interesting to find out how this fusion process actually takes place and whether, for instance, the necessary conformational changes in the s protein are induced by its interaction with the antibody or triggered by the binding to the egfr. alternatively, fusion may not be mediated by the spikes that effect the binding but, rather, by the conformational changes induced in the 'free' spikes upon interaction with undefined molecules on the target cells or by the particular conditions experienced when the virus is taken up in endosomes. evidence for the latter mechanism might be obtained by studying whether infection can also be achieved when using a bispecific antibody not binding the virus through the s protein but through one of the other envelope proteins, m or e, as these are not involved in the fusion process. an elegant application of the targeting principle has recently been described for another enveloped virus. in measles virus, the receptor binding and membrane fusion functions are divided over two different envelope proteins, the hemagglutinin (h) and the fusion (f) protein, respectively. when an scfv was carboxy-terminally appended to the type ii membrane glycoprotein h, the virus was successfully targeted to cells expressing the distinguishing receptor. 26, 33 obviously, appending the egfr binding moiety of scfv 23f-425 to the amino terminus of the coronavirus s protein might also expand the targeting possibilities of these viruses. recombinant fmhv 15 and fipv strain 79-1146 34 stocks were produced and titrated in parallel on feline fcwf-4 cells, yielding titers of 5 â 10 6 and 1 â 10 7 tcid 50 /ml (tissue culture infectious dose 50), respectively. wildtype ad5 was produced and titrated on 293 human embryonic kidney cells. the recombinant vaccinia virus vtf7-3 containing the bacteriophage t7 rna polymerase gene was used as a t7 rna polymerase source for the t7 promoter-driven production of bispecific scfv in ost7-1 cells. 35 ost7-1 (obtained from b moss), nih 3t3, hela, ovcar-3, hct-8, caco-2, widr, hepg2, a431 (american type culture collection), and fcwf-4 cells (obtained from nc pedersen) were grown in dulbecco's modified eagle's medium (dmem) (cambrex bio science, verviers, belgium) containing 10% fetal bovine serum (fbs), 100 iu of penicillin/ml, and 100 mg of streptomycin/ml (all from life technologies, ltd, paisley, uk). nih-hegfr cells (nih 3t3-her14, obtained from pmp van bergen en henegouwen) 22 were maintained in dmem containing 10% fbs, 100 iu of penicillin/ml, 100 mg of streptomycin/ml, and 0.25 mg/ ml g418 (life technologies, ltd, paisley, uk). hela-fapn and ovcar-fapn were maintained in the same medium supplemented with 0.5 and 0.25 mg/ml g418, respectively. the hybridoma cell line producing the 23f8.1 monoclonal antibody (mab) against the fipv spike protein 36 was cultured in cd hybridoma medium (all from life technologies, ltd, paisley, uk) . c428, ascitic fluid from an fipv-infected cat (kindly provided by bj haijema), was used as a source of polyclonal antibodies to fipv. the rabbit antiserum k134 raised against purified mhv 37 and the mab r-g-4 directed against the fapn receptor have been described previously. 37, 38 the culture supernatant of the hybridoma cell line 23f8.1 36 was used as a source of the mab 23f8.1 directed against the fipv and fmhv spike. for egfr detection, the mab 425 was used (culture supernatant from the hybridoma cell line 425, atcc). to detect myctagged scfv 23f-425, the anti-myc antibody myc was used (culture supernatant from the hybridoma cell line myc 9e10, atcc). the expression plasmid pcr3-fapn, containing the fapn cdna under the control of the cytomegalovirus promoter, 5 was used to transiently express fapn on different cancer cell lines following transfection with lipofectamine plus reagent (life technologies, ltd, paisley, uk). transfected hela and ovcar-3 cells were also cultured in g418-containing cell culture medium to select for stable transfectants. hela and ovcar-3 g418resistant cells were cloned by two and four rounds of limiting dilution, respectively, and tested for their susceptibility to fipv infection and for fapn expression by fluorescent activated cell sorter (facs) analysis. furthermore, fipv infection was blocked by the anti-fapn antibody r-g-4, confirming that the infections occurred via fapn. an amount of 5 â 10 5 cells/10 cm 2 well was seeded and inoculated the next day with virus at an moi of 5 pfu/ cell for 1 h in serum-free culture medium. the cells were washed three times with pbs, and cultured for up to 48 h. at several time points p.i., the medium was harvested, centrifuged for 10 min at 3000 r.p.m., and stored at à801c until analysis. the amount of virus produced at each time point p.i. was determined by end point titration on feline fcwf-4 cells. an amount of 5 â 10 4 hela-fapn or ovcar-fapn cells was seeded per 0.32 cm 2 well and infected in triplicate with various amounts of fipv or fmhv. at several time points after inoculation, the cells were cultured for 1 h in 10% wst-1 (roche diagnostics gmbh, mannheim, germany), after which the od 450 was measured. viability was expressed relative to uninfected control cells, after subtraction of background values of wst-1 incubated in the absence of cells. three-dimensional multilayer spheroids were produced by incubating 5 â 10 4 ovcar-fapn cells in 0.32 cm 2 wells coated with 100 ml 2% multi purpose agarose (roche diagnostics gmbh, mannheim, germany) in pbs for 24 h on a spinner-platform set at 125 r.p.m., at 371c and 5% co 2 . subsequently, the spheroids were cultured for 7 days at 371c and 5% co 2 , reaching a diameter of approximately 600 mm, before they were used for infection experiments. spheroids were infected in a total volume of 100 ml and cultured for 2, 5, or 7 days, after which they were incubated for 5 h in 10% wst-1. the od 450 was measured directly in spheroid-containing wells. viability was expressed relative to uninfected control spheroids, after subtraction of background values of wst-1 incubated in the absence of spheroids. statistical significance between different groups was determined by the t-test. the 3 linker. in a subsequent pcr, the assembled scfv dna was amplified and restriction sites were added by using the rs primers of the scfv isolation system. the resulting dna fragment contained a 5 0 sfii site and a 3 0 noti site, and was subsequently cloned into pgemteasy and sequenced. the sequence was compared to the sequences of the independent 23f8.1-v h and 23f8.1-v l fragments, and the resulting correct clone was named pgemteasy-23fv h v l . the pgemteasy-23fv h v l plasmid was digested by sfii and noti to isolate the 754 bp scfv 23f, which was ligated into the pcantab derivative pstcf 40 that had been digested by sfii and noti, resulting in pstcf23f. the scfv 425 directed against the egfr was isolated from pstcfs11-425 39, 40 by noti digestion and ligated in a v h -v l configuration into the noti site downstream of the scfv 23f in pstcf23f creating a three ala linker between the two scfv fragments. this resulted in the expression vector pstcf23f-425, which contains the 1587 bp bispecific cdna construct encoding the antispike scfv 23f linked to the anti-egfr scfv 425 in fusion with an amino-terminal igk signal sequence and a carboxy-terminal myc tag under the control of cmv and t7 promoters. to determine whether scfv 23f-425 was produced and secreted into the culture medium, subconfluent monolayers of ost7-1 cells in 2 cm 2 tissue culture dishes were targeting non-human coronaviruses to human cancer cells t würdinger et al inoculated with vtf7-3 at 5 pfu/cell (t ¼ 0 h) and subsequently transfected (t ¼ 1 h) without dna, with pstcf23f-425, or with pstcfs11-425 plasmid dna by using lipofectin (life technologies, ltd, paisley, uk). 39, 40 at t ¼ 5 h, the cells were starved for 30 min in cysteineand methionine-free modified eagle's medium containing 10 mm hepes, ph 7.2, and 5% fbs. the medium was then replaced by 200 ml of similar medium containing 100 mci of 35 s in vitro cell-labeling mixture (amersham pharmacia biotech europe gmbh, germany). after 1 h, the cells were either lysed or the labeling medium on the cells was replaced by 240 ml normal culture medium and incubation continued for 0, 4, or 16 h. the cells were lysed in 300 ml tesv lysis buffer (20 mm tris-hcl (ph 7.3), 1 mm edta, 100 mm nacl, 1 mm pmsf, 1% triton x-100). to the cleared medium, 60 ml of 5 â tesv lysis buffer was added. proteins were immunoprecipitated from the medium or the lysed cells by using the anti-myc antibody diluted 1:10. the immune complexes were adsorbed to pansorbin cells (calbiochem, la jolla, usa) as described previously. 29 equal volumes of the immunoprecipitates were analyzed by sds-page containing 10% polyacrylamide. for the production of scfv 23f-425, subconfluent monolayers of ost7-1 cells were inoculated at an moi of 5 with vtf7-3 (t ¼ 0 h) and transfected (t ¼ 1 h) with pstcf23f-425, or mock transfected (no plasmid dna), by using lipofectin (life technologies, ltd, paisley, uk). the medium was refreshed at t ¼ 4.5 h, harvested at t ¼ 20 h, and centrifuged for 10 min at 3000 r.p.m. to clear it from cell debris. the mock supernatant and the supernatant containing the bispecific scfv were loaded onto a 20% sucrose cushion, centrifuged for 30 min at 13 000 r.p.m. to clear the supernatant from vtf7-3 virus, and stored at à201c in aliquots until use. the supernatant from ost7-1 cells infected with vtf7-3, but not transfected with plasmid dna, was used as a control supernatant. a single batch of scfv 23f-425 and control supernatant was used for all experiments described. to determine the optimal amount of scfv 23f-425 to be used in targeted infections, fipv (moi 5) was preincubated for 1 h with various amounts of scfv 23f-425 and inoculated in a total volume of 500 ml on a431 cells in a 2 cm 2 well. after 1 h at 371c, the inoculum was replaced by regular culture medium and incubation of the cells was continued for 15 h. an immunostaining with serum directed against fipv proteins was performed, after which the stained cells were counted and their numbers plotted against the amount of scfv 23f-425 used. the titration results revealed the optimal amount of bispecific antibody needed for maximal targeting efficiency under these standard conditions to be 200 ml scfv 23f-425. more antibody did not increase but, rather, decreased the level of infection, presumably because excess antibodies in the inoculum may bind to egfr on cells thereby blocking their use by the virus. cells inoculated with fipv preincubated with mock control supernatant remained negative (data not shown). cells were incubated with c428 anti-fipv ascites fluid diluted 1:500, or k134 anti-mhv serum diluted 1:300, followed by goat anti-cat peroxidase (dako, glostrup, denmark) diluted 1:400, or swine anti-rabbit peroxidase (dako, glostrup, denmark) diluted 1:300, respectively. the cells were stained by aec (brunschwig, amsterdam, the netherlands) according to the manufacturer's protocol, and analyzed by light microscopy. to determine whether scfv 23f-425 interacts with the egfr, 2 â 10 5 hela cells per 2 cm 2 well were incubated with or without 500 ml hybridoma supernatant containing mab 425 for 30 min at 41c in order to block the interaction of egfr and scfv 23f-425. next, the cells were inoculated for 15 h at 371c with 1 â 10 5 pfu fmhv preincubated with 200 ml scfv 23f-425 for 1 h at 41c in a total volume of 500 ml. thereafter, the cells were fixed, permeabilized, and immunostained for the presence of fmhv. the number of infected cells was counted by using light microscopy. the interaction of scfv 23f-425 and the spike protein was analyzed by incubating 1 â 10 5 pfu fmhv with or without 200 ml hybridoma supernatant containing anti-fmhv-s antibody 23f8.1. after 1 h of incubation at 41c, 200 ml scfv 23f-425 was added for 1 h of incubation at 41c in a total volume of 500 ml. next, the infection mixes were inoculated on 2 â 10 5 hela cells per 2 cm 2 well for 15 h at 371c. the cells were fixed, permeabilized, and immunostained for the presence of fmhv. again, the number of infected cells was determined by using light microscopy. an amount of 5 â 10 4 a431 cells per 0.32 cm 2 well was inoculated at an moi of 5 for 2 h with fmhv preincubated with 50 ml of scfv 23f-425 for 1 h at 41c. the cells were washed three times with pbs and incubated further in the presence or absence of 50 ml scfv 23f-425. an immunostaining was performed 24 h after infection, and the number of nuclei per syncytium was determined by light microscopy. for the production of the fhr1 peptide corresponding to amino acids 1047-1156 of the fipv spike protein (acc. no. vgih79), a pcr fragment was prepared using as a template the sequence from cdna clone b1, which contains the fipv spike gene. 41 the fhr1 peptide was expressed in escherichia coli, purified, and quantified as described elsewhere. 11, 42 analysis of the fusion mechanisms of coronavirus infection sensitivity of the normal virus-cell fusion process to fhr1 was studied by inoculating feline fcwf-4 with fmhv at an moi of 0.5 in the presence or absence of 0.5 mm fhr1 peptide for 8 h. the effect of fhr1 on the targeted fusion process was analyzed by preparing in parallel two inoculation mixtures by preincubating fmhv with scfv 23f-425 for 1 h at 41c after which 0.5 mm fhr1 peptide was added to one mixture. two cultures of human a431 cells were washed with pbs and targeting non-human coronaviruses to human cancer cells t würdinger et al inoculated at an moi of 5 for 16 h at 371c with the infection mixes. the cells were fixed, permeabilized, and stained for the presence of fmhv using anti-mhv rabbit antiserum k134. the number of infected cells was counted by using light microscopy. the sensitivity of the cell-cell fusion process during normal infection of feline fcwf-4 cells was analyzed by inoculating cells with fmhv at an moi of 0.5 for 1 h at 371c, washing them three times with pbs, and incubating for 7 h in culture medium with or without 0.5 mm fhr1 peptide. for a similar analysis of cell-cell fusion after targeted infection, cultures of a431 cells were inoculated at an moi of 5 with fmhv preincubated with scfv 23f-425 for 1 h at 41c. next, the cells were washed three times with pbs, and incubated for 22 h in culture medium with or without 0.5 mm fhr1 peptide. the cells were fixed, permeabilized, and immunostained for the presence of fmhv proteins. the number of nuclei per syncytium was counted under the light microscope. cytolytic viruses as potential anti-cancer agents rna viruses as virotherapy agents the viruses and their replication the coronavirus surface protein intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a59 the s2 subunit of the murine 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from an oncolytic herpes simplex virus of syncytial phenotype oncolytic measles viruses displaying a singlechain antibody against cd38, a myeloma cell marker high cd46 receptor density determines preferential killing of tumor cells by oncolytic measles virus conditionally replicative adenovirus expressing a targeting adapter molecule exhibits enhanced oncolytic potency on car-deficient tumors coronaviruses as vectors: position dependence of foreign gene expression the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host targeting non-human coronaviruses to human cancer cells t würdinger et al vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization an oncolytic measles virus engineered to enter cells through the cd20 antigen characterization of a feline infectious peritonitis virus isolate cytoplasmic expression system based on constitutive synthesis of bacteriophage t7 rna polymerase in mammalian cells monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus viral protein synthesis in mouse hepatitis virus strain a59-infected cells: effect of tunicamycin differences in virus receptor for type i and type ii feline infectious peritonitis virus a dimeric bispecific miniantibody combines two specificities with avidity targeting of adenoviral vectors through a bispecific single-chain antibody stably expressed fipv peplomer protein induces cell fusion and elicits neutralizing antibodies in mice large-scale production, purification and refolding of the full-length cellular prion protein from syrian golden hamster in escherichia coli using the glutathione s-transferase-fusion system targeting non-human coronaviruses to human cancer cells t würdinger et al we are grateful to tsutomu hohdatsu (kitasato university, towada, aomori, japan) for providing anti-fapn antibody r-g-4, to kathryn v holmes (university of colorado health sciences center, denver, usa) for supplying the fapn expression vector pcr3-fapn, to ed dubovi (cornell university, ithaca, usa) for providing the hybridoma cell line 23f8.1, and to paul mp van bergen en henegouwen (utrecht university, utrecht, the netherlands) for the gift of nih 3t3-her14 cells.this work was supported by the dutch cancer society (uu 2001-2430). victor w van beusechem is supported by a research fellowship of the royal netherlands academy of arts and sciences (knaw). key: cord-328403-139ejlgo authors: ringshausen, f.c.; rohde, g.g.u. title: neue und seltene pneumotrope viren date: 2013-08-15 journal: pneumologe (berl) doi: 10.1007/s10405-013-0675-6 sha: doc_id: 328403 cord_uid: 139ejlgo while acute viral respiratory tract infections are one of the major reasons for the loss of productivity among the general population in industrialized nations, they are one of the top killers among infants worldwide, in particular in low-income countries. with the advances in molecular diagnostics and the introduction of high-throughput screening techniques a variety of novel, so far unknown viruses have been discovered from respiratory secretions. however, the clinical significance is often difficult to determine. this review article provides an introduction to those novel viruses which have been described since the beginning of the millennium and discusses the clinical relevance in the light of current scientific evidence. the viruses covered by the present review are human metapneumovirus, human bocavirus, human coronaviruses oc43, 229e, nl63, hku1, sars and mers, human polyomaviruses ki, mc and wu and human parechoviruses. tiefe atemwegsinfektionen stellen ein weltweit höchst relevantes ge sundheitsproblem dar, das mit sig nifikanter mortalität und morbidität [47] und in deren folge mit außeror dentlichen volkswirtschaftlichen be lastungen einhergeht. während in den industrienationen vor allem äl tere patienten an den folgen tie fer atemwegsinfektionen erkran ken und versterben, sind kinder in entwicklungsländer in besonderem maße betroffen (. abb. 1). aufgrund der stark limitierten ressourcen und der spärlichen datenlage ist die be deutung der neueren viren hier noch weitgehend unbekannt. es zeich net sich jedoch ab, dass auch neue und vermeintlich seltene viren, deren interaktionen mit etablierten viralen pathogenen wie influenzaviren oder dem respiratorischen synzytialvirus (rsv) und mit bakterien (v. a. pneu mokokken) eine bedeutende rolle spielen [8, 40, 42] . in den letzten beiden jahrzehnten wurden durch technische fortschritte eine vielzahl zuvor unbekannter viren in respiratorischen sekreten von patienten mit symptomen tiefer atemwegsinfektionen identifiziert (. tab. 1) und so der begriff des humanen viroms geprägt. diesen technischen fortschritt machte v. a. das mit hoher durchsatzleistung durchgeführte molekulare screening gepoolter klinischer proben mittels polymerase-kettenreaktion ("polymerase chain reaction", pcr), die sequenzierung der zufällig gewonnenen amplifikate sowie deren analyse mit hilfe ausgeklügelter bioinformatischer methoden möglich ("high-throughput deep sequen-cing technology" oder "metagenomic sequencing"; [10]). da diese methoden nicht auf der klassischen viruskultur mit zytopathischem effekt, sondern auf dem alleinigen molekularbiologischen nachweis entsprechender viraler nukleinsäuren basieren, gestaltet sich der beweis der virulenz aufgrund der nichtanwendbarkeit der modifizierten henle-koch-postulate schwierig [38] . die meisten dieser neu entdeckten viren sind nicht anzüchtbar, etablierte tiermodelle existieren nicht, die epidemiologische datenlage ist spärlich und kontrollierte kohortenstudien mit klinisch gut charakterisierten kollektiven fehlen häufig. dies erschwert auch die beurteilung eines "pneumotropismus", so dass sich dieser begriff im folgenden auf den molekularbiologischen nachweis eines virus aus respiratorischen sekreten oder lungengewebe bei entsprechend erkrankten patienten bezieht. das humane metapneumovirus (hmpv) gehört zur familie der paramyxoviren und wurde erstmals 2001 in den niederlanden isoliert [43] polyomaviren sind potentiell onkogene doppelstrang-dna-viren. in den letzten jahren wurde eine vielzahl neuer humaner polyomaviren entdeckt, die sich von den bislang bekannten humanpathogenen vertretern, dem bk-und dem jc-polyomavirus, die als opportunistische erreger der progressiven multifokalen leukenzephalopathie bei immunsupprimierten patienten, einer hämorrhagischen zystitis nach knochenmarktransplantation sowie einer nephropathie nach nierentransplantation gefürchtet sind, genetisch deutlich unterscheiden [9]. im jahre 2007 wurden nahezu zeitgleich in schweden, den vereinigten staaten und australien das ki-(karolinska-institut-) und wu-(washington-university-)polyomavirus (kipyv, wupyv) mittels molekularer techniken in respiratorischen sekreten identifiziert [1, 17]. neben respiratorischem untersuchungsmaterial wurden kipyv und wupyv mittlerweile auch in blut, lymphatischem gewebe, milz, stuhl und verschiedenen tumorgeweben nachgewiesen. die nachweisraten von wupyv sind im allgemeinen höher als die von kipyv (0,4-9% vs. 0,5-5%). kürzlich konnte wupyv in einer deutschen studie unter kindern mit aku-ten atemwegsinfektionen bei 5% der kinder in nasopharyngealen aspiraten (npa) nachgewiesen werden, wobei eine hohe viruslast mit einem virämischen verlauf korrelierte [33] . verläufe mit prolongierter virusausscheidung und hoher viruslast bei immundefizienz [14, 32] sowie nachweise bei patienten mit ambulant erworbener pneumonie sind beschrieben [24] . in einer eigenen studie unter 189 copd-patienten konnten wir wupyv weder in npa noch in sputum nachweisen [36] . auch im falle von kipyv und wupyv sind koinfektionen mit anderen respiratorischen viren in über 50% der fälle anzutreffen. aufgrund der kaum unterschiedlichen nachweisfrequenz bei asymptomatischen individuen wird die assoziation mit einem eigenständigen erkrankungsbild noch bezwei-felt [35] . entsprechend einer kürzlich publizierten studie scheint die seroprävalenz von kipyv und wupyv unter blutspendern in deutschland mit 67 und 89% generell hoch zu sein [34] . while acute viral respiratory tract infections are one of the major reasons for the loss of productivity among the general population in industrialized nations, they are one of the top killers among infants worldwide, in particular in low-income countries. with the advances in molecular diagnostics and the introduction of high-throughput screening techniques a variety of novel, so far unknown viruses have been discovered from respiratory secretions. however, the clinical significance is often difficult to determine. this review article provides an introduction to those novel viruses which have been described since the beginning of the millennium and discusses the clinical relevance in the light of current scientific evidence. the viruses covered by the present review are human metapneumovirus, human bocavirus, human coronaviruses oc43, 229e, nl63, hku1, sars and mers, human polyomaviruses ki, mc and wu and human parechoviruses. the polyomaviruses wupyv and kipyv: a retrospective quantitative analysis in patients undergoing hematopoietic stem cell transplantation. virol j 9:209 detection of wu polyomavirus dna by real-time pcr in nasopharyngeal aspirates, serum, and stool samples high prevalence of antibodies against polyomavirus wu, polyomavirus ki, and human bocavirus in german blood donors no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease no evidence for wu polyomavirus infection in chronic obstructive pulmonary disease frequency and clinical relevance of human bocavirus infection in acute exacerbations of chronic obstructive pulmonary disease viruses and koch's postulates relevance of human metapneumovirus in exacerbations of copd viral pneumonia an outbreak of human metapneumovirus infection in hospitalized psychiatric adult patients in taiwan associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia the global burden of disease: 2004 update isolation of a novel coronavirus from a man with pneumonia in saudi arabia kommentieren sie diesen beitrag auf springermedizin.de 7 geben sie hierzu den beitragstitel in die suche ein und nutzen sie anschließend die kommentarfunktion am beitragsende. digitale formate sind auch in der medizinischen fachpresse im trend, und springer medizin setzt mit seinen angeboten maßstäbe. die fachverlagsgruppe hat für "ärzte zeitung digital", die app-ausgabe der tageszeitung, den renommierten preis "fachmedium des jahres" 2013 in der kategorie "bestes mobiles angebot" erhalten. der preis wird von der deutschen fachpresse in mehreren branchenund sachkategorien verliehen. die deutsche fachpresse ist die marketingund dienstleistungsplattform für alle anbieter von fachinformationen im beruflichen umfeld. mit dem preis werden publikationen gewürdigt, "die beispielhaft für die vielen hochwertigen gedruckten und digitalen informationsangebote aus fachmedienhäusern in deutschland stehen". "wir haben mit der "ärzte zeitung digital" konsequent unsere tageszeitung für ärzte in das digitale zeitalter überführt", kommentiert harm van maanen, executive vice president von springer medizin, die auszeichnung für die fachtageszeitung. die strategie der digitalisierung werde konsequent weiter verfolgt, so van maanen weiter. so soll die app-ausgabe, die bisher weitgehend die inhalte der gedruckten tageszeitung abbildet, eigenständig werden. die ausgabe der "ärzte zeitung" fürs ipad ist erstmals im november 2012 erschienen im september 2013 folgt die version für android-tablets. das angebot vervollständigt die digitalen formate der zeitung, die damit online über aerztezeitung.de, auf smartphones (news app fürs iphone sowie für smartphones optimierte website) und eben auch mit einer eigenen ausgabe für tablets erreichbar ist. key: cord-319646-6cex9gid authors: wu, guoyao title: important roles of dietary taurine, creatine, carnosine, anserine and 4-hydroxyproline in human nutrition and health date: 2020-02-18 journal: amino acids doi: 10.1007/s00726-020-02823-6 sha: doc_id: 319646 cord_uid: 6cex9gid taurine (a sulfur-containing β-amino acid), creatine (a metabolite of arginine, glycine and methionine), carnosine (a dipeptide; β-alanyl-l-histidine), and 4-hydroxyproline (an imino acid; also often referred to as an amino acid) were discovered in cattle, and the discovery of anserine (a methylated product of carnosine; β-alanyl-1-methyl-l-histidine) also originated with cattle. these five nutrients are highly abundant in beef, and have important physiological roles in anti-oxidative and anti-inflammatory reactions, as well as neurological, muscular, retinal, immunological and cardiovascular function. of particular note, taurine, carnosine, anserine, and creatine are absent from plants, and hydroxyproline is negligible in many plant-source foods. consumption of 30 g dry beef can fully meet daily physiological needs of the healthy 70-kg adult human for taurine and carnosine, and can also provide large amounts of creatine, anserine and 4-hydroxyproline to improve human nutrition and health, including metabolic, retinal, immunological, muscular, cartilage, neurological, and cardiovascular health. the present review provides the public with the much-needed knowledge of nutritionally and physiologically significant amino acids, dipeptides and creatine in animal-source foods (including beef). dietary taurine, creatine, carnosine, anserine and 4-hydroxyproline are beneficial for preventing and treating obesity, cardiovascular dysfunction, and ageing-related disorders, as well as inhibiting tumorigenesis, improving skin and bone health, ameliorating neurological abnormalities, and promoting well being in infants, children and adults. furthermore, these nutrients may promote the immunological defense of humans against infections by bacteria, fungi, parasites, and viruses (including coronavirus) through enhancing the metabolism and functions of monocytes, macrophages, and other cells of the immune system. red meat (including beef) is a functional food for optimizing human growth, development and health. the scientific conference entitled "frontiers in agricultural sustainability: studying the protein supply chain to improve dietary quality" hosted by new york academy of sciences highlighted growing controversies on meat consumption by humans in the u.s. (wu et al. 2014) . over the past decades, there have been growing concerns that consumption of red meat (e.g., beef) increases risks for obesity, type ii diabetes mellitus, cardiovascular disease, colon cancer and alzheimer's disease in humans (e.g., nelson et al. 2016; willett et al. 2019) . thus, beef consumption per capita in the u.s. has steadily declined from 95 lb in 1976 to only 60 lb in 2017 (usda 2018) . this has arisen, in part, from a lack of understanding of red meat as a nutritionally important source of functional amino acids (e.g., taurine and hydroxyproline), dipeptides (e.g., carnosine and anserine), and creatine (a metabolite of amino acids) with enormous physiological significance. for example, taurine is a nutritionally essential amino acid for children (particularly preterm infants) and a conditionally essential amino acid for adults (schaffer et al. 2009 ). in addition, dietary supplementation with 4-hydroxyproline improves anti-oxidative function and prevents colitis in the intestine (ji et al. 2018) . furthermore, carnosine and anserine are potent antioxidants (hipkiss and gaunitz 2014) , whereas creatine is both an antioxidant and a major component of energy metabolism in excitable tissues (brain and skeletal muscle) (wyss and kaddurah-daouk 2000) . of note, taurine, carnosine, anserine, and creatine are particularly abundant in beef skeletal muscle but are absent from plants (wu 2013) . also, 4-hydroxyproline is abundant in meat but negligible in many plant-source foods . for comparison, intramuscular concentrations of balenine (β-alanyl-l-3-methylhistidine, an antioxidant and a buffering substance) are high in whale (~ 45 mmol/kg wet weight), relatively low in swine (~ 0.7 to 1 mmol/kg wet weight or 5-8% of carnosine content), and very low in cattle, chickens and sheep (~ 0.05 to 0.1 mmol/kg wet weight; boldyrev et al. 2013; carnegie et al. 1982; harris and milne 1986) . this dipeptide is barely detectable in human and rat muscles (boldyrev et al. 2013 ) and, therefore, is not a focus of the present article. growing evidence shows that taurine, carnosine, anserine, creatine and 4-hydroxyproline play crucial roles in protecting mammalian cells from oxidative stress and injury (abplanalp et al. 2019; avgerinos et al. 2018; seidel et al. 2019; wu et al. 2019) . these results indicate that red meat provides not only high-quality protein for the growth of children but also functional amino acids, dipeptides and creatine for optimal human nutrition and health. however, the public is generally not aware of these beneficial nutrients in meat, including beef (e.g., kausar et al. 2019; uzhova and peñalvo 2019) . to provide accurate and complete information on animal-source foods, it is imperative to review pertinent articles regarding the benefits of dietary taurine, carnosine, anserine, creatine and 4-hydroxyproline on metabolic, retinal, muscle, immunological, and cardiovascular health as well as healthy ageing in humans and animal models. dietary sources of taurine, creatine, carnosine, anserine, and 4-hydroxyproline for humans taurine, creatine, carnosine, anserine, are chemically stable and soluble in water within a physiological range of ph values. they are abundantly present in animal-source foods (such as beef). however, their reported values are highly inconsistent in the literature and may differ by 10-50 fold . for example, the amounts of carnosine in 100 g wet beef meat have been reported to be 14 mg (szterk and roszko 2014) or 1 g (clifford 1922) , and the amounts of anserine in 100 g wet beef meat to be 8.5 mg (thornton et al. 2015) or 67 mg (mateescu et al. 2012) . such large variations in nutrient composition may result, in part, from differences in either beef breeds or analytical techniques. to provide a much-needed database, we analyzed taurine, carnosine, anserine, creatine, and 4-hydroxyproline in cuts from three subprimals (chuck, round, and loin) of beef carcasses selected at three commercial packing plants in the united states (table 1) . these nutrients are abundant in all beef cuts . in contrast, taurine, carnosine, anserine, and creatine are absent from all plants [e.g., corn grains, peanuts, pistachio nuts, potatoes, soybeans, sweet potatoes, wheat flour, and white rice ]. thus, vegetarians are at great risks for the deficiencies of taurine, carnosine, anserine, and creatine, particularly if they table 1 the content of crude protein, amino acids, creatine, carnosine, anserine, and 4-hydroxyproline in beef and plant-source foods adapted from wu et al. (2016) , wu et al. (2020) , and hou et al. (2019) cp crude protein (6.25 × n%), oh-pro 4-hydroxyproline are active in physical exercise (rogerson 2017 ). in addition, most of plant-source foods contained little 4-hydroxyproline and little β-alanine (a precursor of carnosine in humans) (table 1) . these data are useful for nutritionists and medical professionals to make quantitative recommendation for consumption of beef and plant-source foods by humans. biochemistry, physiology, and nutrition of taurine, creatine, carnosine, anserine, and 4-hydroxyproline in humans taurine (2-aminoethanesulfonic acid, a slightly acidic substance) is a sulfur-containing β-amino acid. it was originally isolated from the bile salts (taurocholate, also known as cholyltaurine) of the ox by austrian scientists f. tiedermann and l. gmelin in 1827. taurine is widely distributed in the animal kingdom and to be present in relatively high concentrations in all mammalian and avian tissues, such as blood, intestine, liver, skeletal muscle, heart, brain, kidneys, and retina. a 70-kg person has ~ 70 g taurine (huxtable 1992) . concentrations of taurine in mammalian and avian cells range from 5 to 60 mm, depending on species and cell type (wright et al. 1986 ). human skeletal muscle, heart, retina, and placenta contain 15-20, 28-40, 20-35 , and 20-35 mm taurine, respectively. because of its large mass [e.g., accounting for 45% of body weight (bw) in healthy, nonobese adults], skeletal muscle is the major site (about 70%) of taurine storage in the 70-kg adult. taurine is abundant in the milk of mammals (e.g., 0.4 mm in human, 0.8 mm in mouse, and 2.8 mm in cats) as a physiologically essential nutrient for infants and children (sturman 1993) . dietary taurine is taken up by the enterocyte across its apical membrane via na + -and cl − -dependent transporters, taut, gat2 and gat3 (lambert and hansen 2011; zhou et al. 2012) , as well as pat1 (a h + -coupled, ph-dependent but na + -and cl − -independent transporter), with taut being the major transporter under physiological conditions (anderson et al. 2009 ). taurine exits the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via specific transporters (fig. 1 ). absorbed taurine is not degraded by the intestinal mucosa and, therefore, enters the portal circulation. taurine is transported in blood as a free amino acid for uptake by extra-intestinal tissues (wu 2013) . increasing dietary intake of taurine enhances its concentrations in plasma and tissues, such as skeletal muscle, brain and heart (schaffer et al. 2009; sirdah 2015) . taurine is synthesized from cysteine, a product of methionine catabolism, in the liver of many mammals (wu 2013) . in humans, the rate of taurine synthesis from methionine and cysteine is exceedingly low in comparison with rats (sturman 1993) , because the activity of hepatic cysteinesulfinate decarboxylase (a key enzyme in taurine synthesis) is about three orders of magnitude lower in the young and adult men than that in rats (sturman and hayes 1980) . compared with livestock (e.g., cattle, pigs, and sheep) and poultry (e.g., chickens and ducks), humans also have a very low ability to synthesize taurine at any stage of life. depending on the dietary intake of protein, nutritional status, and hepatic enzyme activity, a healthy adult may synthesize 50-125 mg taurine per day (jacobsen and smith 1968) . under stress or diseased conditions (e.g., heat stress, infection, obesity, diabetes, and cancer), taurine synthesis in the body may be impaired due to the suboptimal function of liver and the reduced availability of the amino acid precursors. infants and children do not sufficiently synthesize taurine despite adequate provision of its precursors in diets (geggel et al. 1985) . in addition, individuals who consume only plant-source foods but no animal products are at increased risk for taurine deficiency, because the precursors of taurine (methionine and cysteine) are present at low concentrations in most proteins of plant origin (e.g., corn, potato, rice, wheat, and vegetables) ). in animal cells, taurine undergoes limited catabolism by taurine:pyruvate transaminase or taurine:α-ketoglutarate transaminase, which catalyzes the transamination of taurine with pyruvate or α-ketoglutarate to form 2-sulfoacetaldehyde (also known as 2-oxoethanesulfonate, 2-hydroxyethanesulfonate, or isethionate) and l-alanine or l-glutamate (read and welty 1962) . these reactions also occur in aerobic and anaerobic bacteria (cook and denger 2006) . in addition, taurine dehydrogenase converts taurine into ammonia plus 2-sulfoacetaldehyde (an oxidation reaction) in aerobic bacteria in the presence of cytochrome c as the physiological electron acceptor (brüggemann et al. 2004) . furthermore, taurine is oxygenated by α-ketoglutarate-dependent taurine dioxygenase to generate sulfite and 2-aminoacetaldehyde in microorganisms (including e. coli eichhorn et al. 1997) . the initial fate of the degradation of the taurine's sulfur atom is sulfite in strictly anaerobic, facultatively anaerobic, or strictly aerobic bacteria (brüggemann et al. 2004 ). thus, taurine catabolism is initiated by transamination, oxidation, or oxygenation in a species and cell-specific manner. in humans, taurine is covalently conjugated with bile acids in the liver to form bile salts, which are then exported by the atp-dependent bile salt export pump out of the hepatocyte and stored in the gallbladder (hofmann 1999) . during feeding, bile salts are secreted from the gallbladder via the common bile duct into the duodenum, where they facilitate the digestion and absorption of dietary lipids. the bile salts are not absorbed by the proximal small intestine (duodenum, jejunum and proximal ileum) due to the lack of their apical transporters, and are resistant to deamidation by pancreatic and mucosal enzymes (including peptidases). instead, after the absorption of dietary lipids, bile salts enter the distal ileum, where a small fraction of them are hydrolyzed (and thus deconjugated) by microbial bile salt hydrolases to form bile acids and taurine (foley et al. 2019) . taurine, the remaining large proportion of bile salts, and bile acids are efficiently absorbed into the enterocyte of the distal ileum by specific transporters (figs. 1, 2) . about 95% of the liver-derived bile salts and bile acids are absorbed primarily by the distal ileum into the portal circulation, and then taken up by hepatocytes of the liver (fig. 2 ). this is known as the enterohepatic circulation for the ileal reabsorption of bile salts and bile acids. only ~ 5% of the liver-derived bile salts and bile acids enter the large intestine during each enterohepatic circulation. thus, it generally takes a prolonged period of time (e.g., months) to induce a taurine deficiency in humans and animals. it is now recognized that taurine plays major roles in human physiology and nutrition, including serving as: (1) a nutrient to conjugate bile acids to form bile salts in the liver that facilitate intestinal absorption of dietary lipids (including lipid-soluble vitamins) and eliminate cholesterol in bile via the fecal route; (2) a major antioxidant, antiinflammatory, and anti-apoptotic factor in the body; (3) a physiological stabilizer of cell membranes; (4) a regulator of modulation of ca 2+ signaling, fluid homeostasis in cells, and retinal photoreceptor activity; (5) a contributor fig. 1 absorption of taurine, creatine, carnosine, anserine, and 4-hydroxyproline by the human small intestine and the transport of the nutrients in blood. dietary collagen is hydrolyzed by proteases, peptidases and prolidase to free amino acids as well as 4-hydroxyproline and its peptides. dietary taurine, creatine, carnosine, anserine, and 4-hydroxyproline are taken up by the enterocyte across its apical membrane via specific transports. inside the cell, taurine, creatinine and anserine are not degraded, some of the 4-hydroxyprolinecontaining peptides are hydrolyzed to 4-hydroxyproline and its peptides, some 4-hydroxyproline is oxidized to glycine, and carnosine undergoes limited catabolism. taurine, creatine, carnosine, anserine, and 4-hydroxyproline, as well as the products of carnosine hydrolysis (β-alanine and histidine) exit the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via specific transporters (wu 2013) . the absorbed nutrients are transported in blood in the free forms for uptake by extra-intestinal tissues via specific transporters. β-ala β-alanine, cat cationic amino acid transporter, cn1 carnosinase-1 (serum carnosinase), cn2 carnosinase-2 (tissue carnosinase), creat1 creatine transporter-1, creat2 creatine transporter-2, gat γ-aminobutyrate transporter, hypd 4-hydroxyproline-containing dipeptides, hypt 4-hydroxyproline-containing tripeptides, oh-pro 4-hydroxyproline, pat1 proton-(h + -coupled) and ph-dependent but na + -and cl − -independent transporter for taurine (low-affinity, high-capacity transporter), pept1 peptide transporter-1, pept2 peptide transporter 2, pht1/2 peptide/histidine transporters 1 and 2, taut taurine transporters. note that the distribution of pht1/2 in tissues is species-specific in that human skeletal muscle expresses pht1 but no pht2, whereas mouse skeletal muscle expresses both pht1 and pht2 to osmoregulation; (6) a key component of nerve and muscle conduction networks; (7) a stimulator of neurological development; and (8) an inhibitory neurotransmitter in the central nervous system (cns) (schaffer and kim 2018) . thus, taurine exerts beneficial effects on cardiovascular (including protection against ischemia-reperfusion injury, maintaining cell membrane structure, and reducing blood pressure), digestive, endocrine, immune, muscular, neurological, reproductive, and visual systems (ito et al. 2014; shimada et al. 2015; seidel et al. 2019) . for example, taurine protects cells and tissues (brain) from the toxicity of reactive oxygen species (ros), excess metals [e.g., nickel (xu et al. 2015) and manganese (ahmadi et al. 2018) ], and ammonia (jamshidzadeh et al. 2017a ) by maintaining the integrity of plasma and organelle (especially mitochondrion) membranes of the cell. conversely, a deficiency of dietary taurine results in retinal degeneration in cats (hayes et al. 1975 ) and children (geggel et al. 1985) that can be corrected with taurine supplementation. furthermore, through the production of n-chlorotaurine by activated human granulocytes and monocytes, taurine contributes to the killing of pathogenic bacteria, fungi, parasites, and viruses (gottardi and nagl 2010) . since 1975, taurine has been used for 45 years as a dietary supplement to improve health in humans and animal models of metabolic syndrome (el idrissi 2019; hayes et al. 1975; ra et al. 2019) . for example, taurine supplementation can prevent diabetic rats from developing cardiomyopathy by inhibiting the expression of the angiotensin ii type-2 receptor (li et al. 2005) , modulating mitochondrial oxidative metabolism (militante et al. 2000) and electron transport activity (jong et al. 2012) , and reducing oxidative stress (schaffer et al. 2009 ). in addition, oral administration of fig. 2 the transport of bile salts from the liver to the duodenum and the return of bile salt from the distal ileum to the liver via the enteralhepatic circulation in humans. conjugated bile acids are exported by the atp-dependent bile salt export pump out of the hepatocyte through its canalicular (apical) membrane into the canaliculus. the bile salts subsequently enter bile ducts, the common hepatic duct, and the gallbladder. during digestion, the bile salts are secreted from the gallbladder to the common bile duct and then the duodenum. in the distal ileum, a fraction of bile salts is hydrolyzed by microbial bile salt hydrolases to form bile acids and taurine or glycine. taurine, glycine and bile salts are efficiently taken up by the enterocytes of the distal ileum via specific transporters. the substances are transported in the blood for uptake by the hepatocyte via its sinusoidal basolateral membrane. during each enteral-hepatic cycle, about 95% of the liver-derived bile salts are reabsorbed to the liver. asbt apical sodium-dependent bile salt/acid transporter (in ileal enterocytes), ba bile acids (unconjugated), bsep bile salt export pump, cba conjugated bile acids, gly glycine, glyt glycine transporters, m3 multidrug resistance protein-3, mbsl microbial bile salt hydrolases, ntcp na + -taurocholate cotransporting polypeptide, oatp organic anion transporting polypeptide family, ostα/β organic solute transporter subunit α/β, pd passive diffusion, tau taurine, taut taurine transporters taurine can ameliorate structural abnormalities of the pancreas (santos-silva et al. 2015) , while improving insulin production by pancreatic β-cells (nakatsuru et al. 2018) and whole-body insulin sensitivity in subjects with hyperglycemia (sarkar et al. 2017) . this role of taurine in diabetic patients are highly significant, because they have a 25% lower concentration of taurine in plasma than normal subjects (sak et al. 2019) and that the number of type-2 diabetic patients is increasing globally (willet et al. 2019) . taurine supplementation may also be needed for cancer patients maintained on total parenteral nutrition, because the concentration of taurine in their plasma is 50% lower than those in healthy subjects (gray et al. 1994) . of particular note, ingestion of 0.4-6 g taurine per day for various days improved (1) metabolic profiles in blood (including reductions in total cholesterol and low-density-lipoprotein cholesterol), and (2) cardiovascular functions in healthy subjects, as well as in patients with overweight, diabetes, hypertension, or congestive heart failure (militante and lombardini 2002; xu et al. 2008 ). in addition, oral administration of 2 g taurine/day for 4 weeks resulted in clinically significant reductions in the frequency, duration, and intensity of muscle cramps in patients with chronic liver disease (vidot et al. 2018) . furthermore, long-term oral administration of taurine (9 or 12 g per day) for 52 weeks can effectively reduce the recurrence of stroke-like episodes in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (melas), a rare genetic disorder caused by point mutations in the mitochondrial dna (ohsawa et al. 2019) . these beneficial effects of oral taurine are summarized in table 2 . waldron et al. (2018) conducted a meta-analysis to evaluate the effects of oral ingestion of taurine at various doses on endurance performance in adult humans who consumed 1-6 g taurine/day in single doses for up to 2 weeks. the authors found that the ingestion of taurine improved overall endurance performance in the subjects. this conclusion is consistent with the recent findings that taurine is a potential ergogenic aid for preventing muscle damage, attenuating muscle protein catabolism, decreasing oxidative stress, and improving performance in subjects with endurance exercise (de carvalho et al. 2017; page et al. 2019; paulucio et al. 2017; waldron et al. 2019 ). creatine (n-[aminoiminomethyl]-n-methyl glycine), "kreas" in greek meaning meat, was discovered by the french chemist michel e. chevreul in 1832 as a component of skeletal muscle in cattle. this neutral, water-soluble substance is abundant in skeletal muscle, heart, brain, and pancreas. a 70-kg adult has about 120 g of total creatine (creatine phosphate plus free creatine), with about 95% of it being in skeletal muscle (casey and greenhaff 2000) . total creatine is about 45% more abundant in white (fast-twitch fibers, type ii) muscle than in red (slow-twitch fibers, type i) muscle (murphy et al. 2001) . in skeletal muscle and brain, creatine stores energy (primarily atp) as creatine phosphate through the action of creatine kinase. in a resting state, about two-third and one-third of total creatine exist as creatine phosphate and creatine, respectively (mcgilvery and murray 1974) . the irreversible loss of creatine as creatinine is 1.7% of the whole-body creatine (brosnan and brosnan 2007) . this means that each day, 98.3% of creatine is continuously recycled to store atp energy via creatine kinase. in human skeletal muscle, the concentration of creatine phosphate is about three to four times that of atp (gray et al. 2011; mcgilvery and murray 1974) . the energy of the γ-phosphate bond (51.6 kj/mol or 12.3 kcal/mol) in one mole of atp is transferred to one mole of creatine for storage in vivo (wu 2018) . in the small intestine of mammals including humans, the apical membrane of its enterocytes absorb dietary (luminal) creatine via na + /cl − -coupled (i.e., sodium-and chloridedependent) creatine transporter-1 (creat1) (santacruz and jacobs 2016) . all the dietary-free creatine (100%) is absorbed by the small intestine of humans into the portal circulation (deldicque et al. 2008) . ingested creatine phosphate, which is also abundant in meat, is hydrolyzed extracellularly by alkaline phosphatases (synthesized and released by enterocytes) into creatine and phosphate before intestinal absorption (fernley 1971) . absorbed creatine undergoes limited phosphorylation (only 1% of ingested creatine) in the intestinal mucosa, and nearly 99% of orally ingested creatine enters the portal circulation (jäger et al. 2011) . creatine is transported in blood as a free substance, and is rapidly taken up by extra-intestinal tissues and cells (e.g., liver, skeletal muscle, brain, kidneys, testes, and other tissues and cell types) via creat1, and by testes and brain also via creat2 (bala et al. 2013) . creat2 protein shares 97% homology with creat1. immunohistochemical analysis has shown that creat1 is mainly associated with the sarcolemmal membrane in all types of skeletal muscle (christie 2007; murphy et al. 2001) . creat1 is widespread in animal tissues, whereas creat2 is primarily present within the testes (snow and murphy 2001). creat1 is essential for normal brain and muscular function as mutations in the gene (slc6a8) result in x-linked mental retardation, severe speech and language delay, epilepsy, autistic behavior, and muscular abnormalities (e.g., hypotonia) (bala et al. 2013; salomons et al. 2001) . increasing dietary intake of creatine adam et al. (1996) enhances its concentrations in plasma and tissues, such as skeletal muscle, brain and heart (derave et al. 2004; wyss and kaddurah-daouk 2000) . the absorption of creatine by the small intestine and its transport in the blood are illustrated in fig. 1 . creatine is a metabolite of three amino acids (arginine, glycine and methionine) via the cooperation of multiple organs, primarily including the liver, pancreas and kidneys (wu 2013) . beef is an abundant source of arginine, glycine and methionine. in contrast, all plant-source foods contain low levels of glycine and methionine, and most of plant-source foods (except for soybean, peanuts and other nuts) also have low levels of arginine ). the pathway of creatine synthesis is initiated by arginine:glycine amidinotransferase, which transfers the guanidino group from arginine to glycine to form guanidinoacetate and ornithine. arginine:glycine amidinotransferase is expressed primarily in the renal tubules, pancreas, and to a much lesser extent in the liver and other organs. thus, the kidneys are the major site of guanidinoacetate formation in the body. the guanidinoacetate released by the kidneys is methylated by guanidinoacetate n-methyltransferase, which is located predominantly in the liver, pancreas, and, to a much lesser extent, in the kidneys to produce creatine (wu and morris 1998) . creatine synthesis is regulated primarily through: (1) changes in expression of renal arginine: glycine amidinotransferase in both rats and humans; and (2) the availability of substrates. dietary intake of creatine and circulating levels of growth hormone are major factors affecting de novo synthesis of creatine (wu and morris 1998) . activities and mrna levels for arginine:glycine amidinotransferase in rat kidney are greatly reduced by hypophysectomy or by feeding a diet containing creatine. neither creatine supplementation nor growth hormone influences the hepatic activity of guanidinoacetate n-methyltransferase in animals. thus, dietary supplementation with creatine helps to spare arginine, glycine and methionine for utilization via other vital metabolic pathways, such as the syntheses of protein, nitric oxide, and glutathione (wu 2013 ). this has important nutritional and physiological significance. a 70-kg healthy adult synthesizes 1.7 g creatine per day from 2.3 g arginine, 1.0 g glycine, and 2.0 g methionine (wu and morris 1998) , which represent 46%, 36% and 87%, respectively, of their daily dietary intakes. this amount of creatine is necessary to replace its daily irreversible loss (1.7 g/day) as creatinine from the subject through the excretion of urine. a greater loss of creatine from the body occurs in response to enhanced muscular activity (kreider et al. 2017; rogerson 2017) , and this amount of creatine should be replenished through enhanced endogenous synthesis there were no side effects for the ingestion of taurine, creatine, carnosine, anserine and hydroxyproline at the indicated dosages on all the studies chf congestive heart failure, hpt and dietary supplementation. for example, cycle ergometer exercise (approximately 45% of maximum o 2 consumption for 90 min) increases the loss of creatine (as indicated by urinary creatinine excretion) by 52% above pre-and postexercise values (calles-escandon et al. 1984) . there are new lines of evidence that endogenous synthesis of creatine is not sufficient for humans under many physiological (e.g., exercise, pregnancy and lactation) and pathological (e.g., tissue injury, ischemia, and diabetes) conditions. first, dietary creatine supplementation aids in increasing the power, strength, and mass of the skeletal muscle in athletes and bodybuilders, especially those who consume little meat (smith et al. 2014) . second, creatine supplementation is beneficial for ameliorating sarcopenia in elderly subjects that is characterized by reductions in the mass and function of skeletal muscle (candow et al. 2014) . third, patients with neurological and muscular disorders can respond well to creatine supplementation with improvements in health, as summarized previously. thus, adequate provision of dietary creatine may be necessary for maintaining homeostasis and optimal health in humans (brosnan and brosnan 2007) , particularly for vegan athletes who generally have low intake of creatine and its precursors (arginine, methionine and glycine) (rogerson 2017 ). creatine undergoes limited catabolism in mammalian cells (wu 2013) . however, creatine is reversibly converted by creatine kinase into creatine phosphate and is irreversibly cyclized into creatinine through spontaneous loss of one molecule of water, as indicated previously. like creatinine, creatine phosphate is also spontaneously converted into creatinine. the breakdown of creatine phosphate results in the losses of phosphate and one molecule of water. creatine kinase exists in the cytosolic and mitochondrial isoforms. this enzyme is not only most abundant in skeletal muscle and brain, but is also present in many tissues and cell types (keller and gordon 1991; trask and billadello 1990) . when healthy adults consume 4.4 g creatine monohydrate once, plasma creatine peaks [762 µm; an 18-fold increase over the 0-min baseline value of about 40 µm (jäger et al. 2007 )] in 60 min (kreider et al. 2017 ) and its half-life is 30 min (jäger et al. 2007 ). thus, creatine is rapidly cleared from plasma, and skeletal muscle is the major sink of creatine. creatinine is excreted in urine. the amount of urinary creatinine is proportional to (and, therefore, an indicator of) skeletal muscle mass in healthy subjects. creatine is essential for energy metabolism in the brain and skeletal muscle (wyss and kaddurah-daouk 2000) . in these two highly excitable tissues that can undergo rapid changes in their membrane potentials for the transport of electrical signals, creatine decreases intracellular calcium concentrations (e.g., by stimulating sarcoplasmic reticulum ca 2+ -atpase in skeletal muscle) and extracellular glutamate concentrations (e.g., through uptake by synaptic vesicles in the brain), and prevents the opening of the mitochondrial permeability transition pore (bender et al. 2005; fortalezas et al. 2018) . creatine also plays an important role in antioxidative and anti-apoptotic reactions, scavenging free radicals, and protection against excitotoxicity in tissues (lawler et al. 2002) . for example, creatine can prevent mitochondrial disorders that are commonly associated with decreased atp production and increased ros production (rodriguez et al. 2007; wyss and kaddurah-daouk 2000) . by delaying the depletion of atp during oxygen deprivation and reducing the availability of ros (balestrino et al. 1999) , pretreatment with creatine can reduce the cardiac and neurological damages induced by ischemia or anoxia and that such treatment can also be useful even after the onset of stroke or myocardial infarction (balestrino et al. 2002; lensman et al. 2006; osbakken et al. 1992; perasso et al. 2013; prass et al. 2007; scheer et al. 2016; shen and goldberg 2012; whittingham and lipton 1981; wilken et al. 1998; zapara et al. 2004) . of note, creatine has been safely and beneficially administered to patients affected by age-related neurological diseases, including parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, long-term memory deficits, alzheimer's disease, and stroke (adhihetty and beal 2008; smith et al. 2014) , as well as patients with pathophysiological conditions, such as gyrate atrophy, post-stroke depression, congestive heart failure, chronic musculoskeletal pain disorders, atherosclerotic diseases, and cisplatin-induced renal damage (genc et al. 2014; hummer et al. 2019; persky and brazeau 2001; wyss and schulze 2002) . through increasing the availability of arginine for the generation of nitric oxide (a killer of pathogenic bacteria, fungi, parasites, and viruses), dietary supplementation with creatine plays an important role in protecting humans from infectious diseases (ren et al. 2018) . athletes and muscle builders are major users of supplemental creatine. this is primarily because (1) both energy metabolism and production of oxidants are enhanced during exercise, and (2) creatine participates in atp turnover in skeletal muscle and is a potent antioxidant. due to the very low rate of creatine loss (1.7%) in the whole-body pool (creatine plus creatine phosphate), dietary supplementation with a low dose of creatine (e.g., 3 g of creatine monohydrate per day for 28 days in 76-kg men) results in a steady accumulation of creatine in skeletal muscle, and the saturation of creatine in the muscle (142 mmol/kg dry matter) occurs by day 28 (hickner et al. 2010; hultman et al. 1996) . for comparison, the content of creatine in the skeletal muscle of adult humans without creatine supplementation is 122 mmol/kg dry matter of muscle (hultman et al. 1996) . thus, based on the current knowledge of creatine metabolism in humans, continuous supplementation with creatine is not necessary to achieve the saturation of creatine in skeletal muscle. in the case of discontinuous supplementation of creatine, such as disruption for short intervals (e.g., 1 or 2 days per week), a longer period of supplementation (e.g., by 5-10 days) should still be sufficient to achieve the saturation of creatine in skeletal muscle. thus, consumption of creatine monohydrate (3 g/day) over a prolonged period of time can beneficially increase the concentration of creatine in skeletal muscle to the saturation level even if the supplement is not taken every single day. when an adult consumes daily 30 g of dry beef that provides 303 mg creatine ) as its sole dietary source, it will take about 240 days to saturate creatine in skeletal muscle. because of increased losses of creatine during active muscular work as noted previously, oral administration of creatine is necessary for maintaining its homeostasis in exercising subjects and this nutritional strategy is beneficial for improving their performance. for example, dietary supplementation with a low dose of creatine (as 3 g of creatine monohydrate per day) to adults can enhance the capacity of skeletal muscle to store energy by 16% [(142 -122)/122 = 16%] (hultman et al. 1996) . the rate of utilization of creatine phosphate by the skeletal muscle of adult humans during intensive exercise is 23 mmol/l of muscle water/min (baker et al. 2010) . this is equivalent to 483 mmol/whole-body skeletal muscle/ min (23 × 70 × 40% × 0.75 = 483) for a 70-kg adult with 21 l of muscle water. through creatine recycling via creatine kinase, ingestion of 3 g of creatine monohydrate can help store 50 kcal energy in the skeletal muscle of a 70-kg person for supporting a 60-min intensive exercise (i.e., 483 mmol creatine phosphate/whole-body skeletal muscle/min × 20/1 42 = 68 mmol = 68 mmol × 12.3 kcal/mol × 0.001 × 60 min = 50 kcal). thus, a small amount of supplemental creatine makes a very significant contribution to energy storage and metabolism in the skeletal muscle of humans to enhance the power and duration of muscular work. in support of this view, dietary supplementation with creatine to young adults decreases oxidative dna damage and lipid peroxidation induced by a single bout of resistance exercise (rahimi 2011) . oral administration of creatine phosphate (20 g/day on days 1-3 and 10 g/day for days 4-42) offers a comparable ergogenic effect to that of equal amounts of creatine monohydrate (peeters et al. 1999) . in the international society of sports nutrition position stand (kreider et al. 2017) , multiple studies have been reviewed to indicate the following. first, there is consistent evidence that oral ingestion of 3 g creatine monophosphate (equivalent to 2.64 g creatine) per day increases intramuscular creatine concentrations. this can be a biochemical basis for improvements in high-intensity exercise performance and training adaptations, as noted previously. second, creatine supplementation may enhance post-exercise recovery, injury prevention, thermoregulation, and rehabilitation, as well as concussion, spinal cord neuroprotection. third, creatine supplementation can improve muscular function and enhancing rehabilitation from injuries in subjects with neurodegenerative diseases (e.g., muscular dystrophy, parkinson's, huntington's disease), diabetes, osteoarthritis, fibromyalgia, aging, brain and heart ischemia, adolescent depression, and pregnancy. fourth, creatine supplementation can enhance anti-oxidative capacity and reduce oxidant-induced tissue injury. thus, creatine is one of the most popular and beneficial nutritional ergogenic aids for athletes (kreider et al. 2017 ). this conclusion continues to be supported by research findings over the past 2 years. for example, wang et al. (2018) reported that creatine supplementation combined with exercise training for 4 weeks improved maximal muscular strength and reduced exercise-associated muscle damage in adults. furthermore, oral consumption of creatine electrolyte improved overall and repeated short duration sprint cycling performance (crisafulli et al. 2018) , as well as anaerobic power and strength in athletes (hummer et al. 2019) . in addition to its ergogenic effect, creatine has also been used for humans to improve cognitive function (avgerinos et al. 2018 ) and reduce traumatic brain injury (balestrino et al. 2016; dolan et al. 2019) . furthermore, creatine supplementation ameliorates skeletal muscle dysfunction, enhances muscle functional capacity in patients with chronic obstructive pulmonary disease on long-term o 2 therapy (de benedetto et al. 2018) , and supports the rehabilitation of tendon overuse injury in athletes (juhasz et al. 2018) . recently, there have been suggestions that creatine may delay or ameliorate sarcopenia in elderly subjects (candow et al. 2019a, b) , reduce fat accumulation in the liver (da silva et al. 2017), improve muscle mass or function in cancer patients (fairman et al. 2019) , and maintain the mass and function of ageing bones (candow et al. 2019a, b) . growing evidence over the past 30 years shows that dietary supplementation with creatine may provide a safe and effective means in the therapeutic intervention of cancers. for example, creatine has an anti-tumor effect in vitro cell cultures and in vivo various transplanted human and rodent tumors (kristensen et al. 1999; lillie et al. 1993; miller et al. 1993 ). in addition, pal et al. (2016) reported that oral administration of creatine (150 mg/kg bw per day) for 10 consecutive days resulted in significant regression of tumor size in sarcoma-180 tumor-bearing mice and improved the overall survival of the animals. furthermore, creatine supplementation (1 g/kg bw per day for 21 days) to walker-256 tumor-bearing rats prevented skeletal muscle atrophy by attenuating systemic inflammation and protein degradation signaling, thereby enhancing their muscle mass and survival (cella et al. 2019) . similar beneficial effects of creatine supplementation have been obtained for humans with cancers (fairman et al. 2019 ). in 1900, the russian biochemist w. gulewitsch discovered an abundant substance in the skeletal muscle of cattle and named this substance carnosine after "caro" or "carnis" (meaning meat in latin), which was identified in 1918 to be a dipeptide, β-alanyl-l-histidine. carnosine is characterized by three ionizable groups: the carboxylic group (pka 2.77), the amino group of the β-alanine residue (pka 9.66), and the imidazole ring in histidine (pka 6.83), with the pka of the whole molecule being 8.25 (tanokura et al. 1976) . thus, at physiological ph (e.g., ph 7.0-7.4), carnosine is present in the zwitterionic form and has a net positive charge. concentrations of carnosine in the skeletal muscle of humans without carnosine or β-alanine supplementation range from 5 to 10 mm (boldyrev et al. 2013) , or 16.7-33.3 mmol/kg dry weight of muscle. for example, the soleus and gastrocnemius muscles of adult males contain 8 and 10 mm carnosine, respectively, or 26.7-33.3 mmol/ kg dry weight of muscle (derave et al. 2007 ). these values are approximately 2, 10 and 20 times greater than those in the skeletal muscle of pigs, rats and mice, respectively (boldyrev et al. 2013) . note that bodybuilders can have carnosine concentrations as high as 15.3 mm or 51 mmol/kg dry weight, with average values being 13 mm or 43 mmol/ kg dry weight (tallon et al. 2005) . the concentrations of carnosine in the olfactory bulb of the brain and the cardiac muscle are comparable to those in skeletal muscle, but those in other tissues (e.g., ~ 0.1 mm in kidneys and adipose tissue) are only 0.1% to 10% of those in skeletal muscle. based on the report that the mean concentrations of carnosine in the skeletal muscles of women and men are 17.5 and 21.3 mmol/ kg dry weight, respectively (mannion et al. 1992) , it can be estimated that a 60-kg women and a 70-kg men have 32 and 45 g carnosine, respectively. in mammals (including humans), about 99% of carnosine is present in skeletal muscle (sale et al. 2010 ). carnosine is negligible or not detectable in the plasma of humans, but is present at low concentrations (2-15 µm) in the plasma of non-primate animals. carnosine is more abundant in the white muscle of humans than in their red muscle (hill et al. 2007 ). based on: (1) the percentages of type i and type ii fibers in skeletal muscle of adult humans, which are 88% and 12%, respectively, in soleus muscle, and 47% and 53%, respectively, in gastrocnemius muscle (hill et al. 2007) , and (2) the concentrations of carnosine in these two muscles (derave et al. 2007) , it can be calculated (0.47x + 0.53y = 10 and 0.88x + 0.12y = 8; x and y are carnosine concentration in type i and ii muscles, respectively) that type i and type ii muscle fibers contain 7.4 and 12.3 mm carnosine, respectively. this means that type ii muscle fiber has 66% more carnosine than type i muscle fiber. such information is important for developing an effective nutritional strategy to enhance muscular carnosine levels and improve muscular strength in athletes and elderly subjects. this is because of the following reasons. first, elite athletes can have up to 80% of type i or type ii muscle fiber, depending on the type of their sports, which is a determinant of their success at competition. for example, a sprinter with 80% of white muscle fibers will have a better performance than somebody with only 30% of white muscle fibers (komi and karlsson 1978) . second, age-related loss of muscle mass results from a decrease in the total number of both type i and type ii fibers and, but secondarily, from a preferential atrophy of type ii fibers (lexell et al. 1988; roos et al. 1997) . therefore, adequate availability of carnosine in skeletal muscle will be beneficial for healthy ageing. dietary carnosine is absorbed by the enterocytes of the small intestine across their apical membrane via peptide transporter-1 (pept1) (fig. 1) . this is an h + -driven process. inside the enterocytes, a limited amount of carnosine is hydrolyzed by carnosinase-2 into β-alanine and histidine (sadikali et al. 1975) . the intracellular carnosine is exported by peptide/histidine transporters 1 and 2 (pht1 and pht2) out of the enterocyte across its basolateral membrane into the lamina propria of the small-intestinal mucosa. pept1, pht1 and pht2 are members of the proton-coupled oligopeptide transporter family (pot-family or slc15), and have a broad specificity for di-and tri-peptides (including carnosine and its methylated analogs). the main difference between pept1 and phts is that the phts can transport l-histidine, in addition to di/tripeptides. within enterocytes, a small amount of carnosine is hydrolyzed by carnosinase-2 (also known as tissue carnosinase), and nearly all of the ingested carnosine enters the portal circulation (asatoor et al. 1970 ). in the blood of humans, carnosine is actively hydrolyzed by carnosinase-1 (also known as serum carnosinase; an enzyme that is synthesized and released from the liver) into β-alanine and histidine, which are then taken up by extra-intestinal cells via specific transporters for beta-amino acids and basic amino acids, respectively. carnosine in plasma, if any, is transported into extra-intestinal tissues and cells via pept2 (peptide transporter 2) as well as pht1 and pht2. pept2, which has a broad specificity for di-and tri-peptides as does pept1, is more widespread than pept1 in extra-intestinal tissues and cells. increasing dietary intake of carnosine enhances its concentrations in skeletal muscle, brain and heart (boldyrev et al. 2013) . ingestion of 4 g synthetic carnosine alone by adult humans does not affect its concentrations in plasma due to high carnosinase-1 activity in plasma that rapidly hydrolyzes carnosine into β-alanine and histidine, but does increase the urinary output of carnosine (gardner et al. 1991) . urinary carnosine accounts for 14% of the ingested amount over a 5-h period and peaks at 2 h after the consumption of carnosine. similarly, within 90 min after the ingestion of 4.5 g carnosine by adult humans, the concentration of carnosine in serum does not change but the concentrations of histidine and β-alanine rapidly increase (asatoor et al. 1970) . this is consistent with the high activity of carnosinase-1 in the plasma of humans to hydrolyze carnosine. interestingly, consumption of beef by men and women can result in an increase in the concentration of carnosine in their plasma that peaks (145 µm) at 2.5 h after intake and returns to the baseline value at 5.5 h after intake (park et al. 2005) . it is possible that some components in beef [anserine, amino acids (e.g., histidine and β-alanine), and copper] inhibit serum carnosinase (bellia et al. 2014; boldyrev et al. 2013) , so that the circulating levels of carnosine are substantially elevated. this can directly supply the dipeptide to extraintestinal tissues (e.g., heart and pancreas) and cells (e.g., red blood cells and immunocytes). in this regard, ingestion of beef as a whole food may be superior to oral administration of synthetic carnosine alone for humans. synthesis of carnosine from β-alanine and histidine is catalyzed by atp-dependent carnosine synthetase (a cytosolic enzyme) (wu 2013) , which is primarily present in skeletal muscle, heart, and certain brain regions (e.g., the olfactory bulb) (drozak et al. 2010; harding and o'fallon 1979) . the sources of β-alanine are diets and endogenous syntheses from the catabolism of aspartate (mainly in bacteria), malonic acid semialdehyde (transamination with glutamate), coenzyme a, pyrimidines, polyamines (wu 2013) . the sources of histidine are diets and the degradation of hemoglobin (which is very rich in histidine), actin, myosin, and other proteins in the body. based on the k d (fractional turnover rate) value of 0.0133/ day for the loss of intramuscular carnosine at the physiological steady state (spelnikov and harris 2019) , the intramuscular concentrations of carnosine in women and men (17.5 and 21.3 mmol/kg dry weight, respectively), skeletal muscle mass (45% of bw), and its dry matter content (30%), it can be estimated that a 60-kg woman and a 70-kg man synthesizes 427 and 606 mg carnosine per day, respectively. the k m values of this enzyme for β-alanine and histidine are 1.0-2.3 mm (ng and marshall 1978) and 16.8 µm (horinishi et al. 1978) , respectively. the value of carnosine synthetase for β-alanine is much greater than or comparable to the intracellular concentration of β-alanine in the brain (0.09 mm) or skeletal muscle (1.0 mm), respectively (wu 2013) . in contrast, the value of carnosine synthetase for histidine is much lower than the intracellular concentration of histidine in the brain (143 µm) and skeletal muscle (404 µm), respectively (wu 2013) . as reported for nitric oxide synthase whose k m value for arginine is much greater in cells than that in assay tubes (wu and meininger 2000) , it is possible that the k m of carnosine synthetase for histidine in vivo is much greater than that (i.e., 16.8 µm) reported for the purified enzyme under in vitro assay conditions, due to complex protein-protein and enzyme-substrate interactions in vivo. availability of β-alanine primarily limits carnosine synthesis by carnosine synthetase in human skeletal muscle and the olfactory bulb, but adequate provision of histidine in diets is also critical for maximal production of carnosine because histidine is not synthesized de novo (hill et al. 2007; sale et al. 2010) . for example, supplementation with β-alanine to humans (e.g., 2-6 g/day) dose-dependently increases the concentrations of carnosine in skeletal muscle by 20-80%, but dietary supplementation with 3.5 g histidine/ day for 23 days has no effect on intramuscular carnosine concentrations in non-vegetarian adults (blancquaert et al. 2017; culbertson et al. 2010) . furthermore, dietary supplementation with 3.2 or 6.4 g β-alanine per day (as multiple doses of 400 or 800 mg) or with l-carnosine (isomolar to 6.4 g β-alanine) per day for 4 weeks augmented intramuscular carnosine concentrations by 42%, 64% and 66% (harris et al. 2006) . these results reveal that histidine is not a limiting factor in carnosine synthesis in adults consuming adequate histidine from animal-source foods. thus, in nonvegetarian humans, consumption of an equal amount of β-alanine and carnosine effectively increases intramuscular carnosine concentrations to the same extent. it is unknown whether dietary intake of histidine limits carnosine synthesis in vegetarians with or without β-alanine supplementation. it is noteworthy that dietary supplementation with β-alanine (6 g/day for 23 days) to adults reduces the concentrations of histidine in their plasma and skeletal muscle by 31% and 32%, respectively, possibly due to reduced intestinal absorption of histidine and increased utilization of histidine for carnosine synthesis by skeletal muscle (blancquaert et al. 2017) . whether this reduction in histidine with β-alanine supplementation has a long-term adverse effect on human health is unknown, but it is prudent to ensure that dietary intake of histidine via supplementation or consumption of histidine-rich foods (e.g., meat) is sufficient. in contrast, oral administration of carnosine increases the concentration of not only β-alanine but also histidine in the plasma of humans (asatoor et al. 1970) , indicating an advantage of the consumption of synthetic carnosine or carnosine-rich foods (e.g., beef) over the consumption of β-alanine alone. within a mammalian species, the synthesis of carnosine is affected by multiple factors, including age, sex, muscle fiver type, muscular activity, and diet (harris et al. 2012) . for example, in a study with 9-to 83-year-old humans, baguet et al. (2012) found that the concentration of carnosine in skeletal muscle increased between 9 and 18 years of age but decreased thereafter with advanced ages. likewise, the content of carnosine in soleus muscle declines with age between 17-and 47-year-old subjects (everaert et al. 2011) . in adult humans, white muscle fibers contain 30-100% more carnosine than red muscle fibers, and men have 22-82% greater concentrations of carnosine in skeletal muscle than women (everaert et al. 2011; mannion et al. 1992) . for example, compared to age-matched women, men have 36, 28 and 82% greater concentrations of carnosine in soleus, gastrocnemius and tibialis anterior muscles, respectively (everaert et al. 2011) . it is possible that androgens enhance carnosine synthesis in skeletal muscle, but the circulating levels of testosterone in healthy adult men do not appear to be related to their intramuscular carnosine concentrations (everaert et al. 2011 ). finally, long-term exercise (e.g., 2 days per week for 8 weeks for sprinters) increases intramuscular carnosine levels in male subjects by 113%, which is associated with a 9% increase in the mean power (e.g., during 30-s maximal cycle ergometer sprinting) was significantly increased following training (suzuki et al. 2004) ; similar results (a 100% increase in intramuscular carnosine levels) were obtained for resistance-trained bodybuilders (tallon et al. 2005) . finally, because plant-source foods contain much lower concentrations of β-alanine and histidine ) than animal products ) and because the formation of β-alanine in vegans is limited (harris et al. 2012) , the synthesis of carnosine in vegans is inadequate and its concentrations in soleus and gastrocnemius muscles are 17% and 26%, respectively, lower than those in omnivores who consume some meat that is an excellent source of both β-alanine and histidine (everaert et al. 2011; harris et al. 2012 ). carnosine is hydrolyzed by carnosinase-2 (a zn 2+ -dependent cytosolic enzyme in tissues) and carnosinase-1 (a mn 2+ -dependent extracellular enzyme in human blood), but not by proteases or dipeptidases, in humans and animals. expression of these two isoforms of carnosinase occurs in a tissue-specific manner. specifically, carnosinase-1 is expressed in the liver, brain, and kidneys but is absent from skeletal muscle and the small intestine (boldyrev et al. 2013; everaert et al. 2013 ). expression of carnosinase-2 in tissues other than the liver, brain, and kidneys is limited shortly after birth and gradually increases to adult levels during adolescence. accordingly, the plasma concentrations of carnosine are 5-10 µm in preterm infants, 3-5 µm in term infants, and absent in healthy adults (asatoor et al. 1970; valman et al. 1971) . in humans, carnosinase-1 that is present in serum at a very high activity is synthesized and secreted mainly by the liver. in contrast, the serum of healthy nonprimate mammals (except for the syrian golden hamster) and birds contains no carnosinase (boldyrev et al. 2013) . thus, carnosine is stable in the plasma of rats and farm animals but not humans (yeum et al. 2010 ). the concentrations of carnosine are 2-15 µm in the plasma of non-primate land animals. in contrast, carnosinase-2 is more widely expressed in mammalian tissues (including skeletal muscle and the small-intestine mucosa of some species), but is absent from the serum or cerebrospinal fluid of mammals or birds (bellia et al. 2014; boldyrev et al. 2013; everaert et al. 2013) . the gastric and colonic mucosae of mammals (including humans, pigs, cattle and rats) contain no carnosinase-1 or carnosinase-2 activity. as noted previously, in humans, dietary carnosine is absorbed intact by the small intestine into the portal circulation, but is rapidly hydrolyzed by serum carnosinase in plasma into β-alanine and histidine; both of the amino acids are reused for carnosine synthesis by skeletal muscle, heart, and the olfactory bulb of the brain (boldyrev et al. 2013; harris et al. 2006 ). in the human skeletal muscle (ph 7.1), which lacks carnosinase-1, the degradation of carnosine by carnosinase-2 is very limited, because its optimal ph 9.5 is much greater than the intracellular ph and its v max is 30 times lower than that of carnosinase activity in the human kidneys (lenney et al. 1985) . this explains the relatively high concentration of carnosine in mammalian skeletal muscle. the mean half-life of intramuscular carnosine in adult humans has been estimated to be 52 days (a range of 46-60 days; spelnikov and harris 2019). this indicates that carnosine has a relatively high degree of biological stability in the body. consistent with this view, the turnover rate of carnosine in adult humans at the physiological steady state is 1.33%/day (spelnikov and harris 2019) . excess carnosine, β-alanine and histidine are excreted from the body via the urine (sale et al. 2010 ). the healthy adult without ingesting carnosine excreted 24 µmol carnosine in 5 h (gardner et al. 1991) . in adult humans consuming 150 g beef or chicken broth, urinary concentrations of carnosine within 7 h after intake were 13-and 15-fold greater, respectively, than the values for no consumption of the meat (yeum et al. 2010) . carnosine has a functional imidazole ring, which can readily donate hydrogen to free radicals for their conversion into non-radical substances (kohen et al. 1988) . such an ability of the imidazole ring is enhanced by the β-alanine moiety in carnosine. the major physiological functions of carnosine include: ph-buffering, activation of muscle atpase to provide energy, metal-ion (copper, zinc and iron) chelation and homeostasis, antioxidant capacity (directly through scavenging ros and peroxyl radicals and indirectly through chelating metals), and protection against lipid peroxidation, protein oxidation, and the formation of advanced protein glycation (by inhibiting protein carbonylation and glycoxidation) and lipoxidation end products (by suppressing lipid peroxidation) (barca et al. 2019; boldyrev et al. 2013; nelson et al. 2019) . the pka value of the imidazole ring of carnosine (6.83) is closer to the intracellular ph in cells than the imidazole ring of free l-histidine (6.04) (wu 2013) . as a positively charged molecule, carnosine can neutralize atp (~ 5 mm, a negatively charged molecule). thus, in skeletal muscle, carnosine (5-10 mm) would be a better buffering molecule than free histidine (0.4 mm). davey (1960) reported that carnosine, together with anserine, accounted for approximately 40% of the ph-buffering capability in the skeletal muscles of rabbits and pigeons. likewise, sewell et al. (1992) found that carnosine contributed about 20% of the buffering in type i muscle fibers and up to 46% in type iib fibers, and these results are consistent with the findings that type i muscle fibers have a lower glycolysis activity and accumulate less lactic acid than type iib muscle fibers. a similar buffering function of carnosine also applies to the olfactory bulb of the brain, which has only 0.14 mm histidine (wu 2013) . the β-alanine moiety of carnosine contributes to a reduction in its oxidation potential, and carnosine is oxidized at a lower oxidation potential than histidine to remove oxidants (kohen et al. 1988 ). thus, carnosine readily forms a charge-transfer complex with oxygen free radicals (e.g., superoxide radical, hydroxyl radicals, peroxyl radicals, and nitric oxide), non-radical ros (e.g., peroxynitrite, hypochlorous acid, singlet oxygen, and h 2 o 2 ), and deleterious aldehydes (e.g., malondialdehyde and formaldehyde) to confer anti-oxidative effect and protect cell membrane and intracellular organelles (e.g., mitochondria) from damage (kohen et al. 1988; pavlov et al. 1993) . besides the antiischemic effect of carnosine on the brain and heart, there is evidence that carnosine can maintain the integrity of the dna molecule, as indicated by studies with telomere (shao et al. 2004 ). the latter is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. specifically, carnosine can reduce telomere shortening rate possibly by protecting telomeres from damage, thereby contributing to the lifeextension effect of carnosine (shao et al. 2004 ). thus, carnosine is beneficial for healthy ageing. other physiological functions of carnosine include the regulation of sarcoplasmic reticulum ca 2+ -release channels and sarcoplasmic reticulum ca 2+ homeostasis in skeletal muscle, activation of its phosphorylase activity to promote glycogen breakdown (johnson et al. 1982) , inhibition of the angiotensin converting enzyme, enhancement of nitric oxide availability in endothelial cells, potentiation of cardiac and skeletal muscle contractilities, serving as a neurotransmitter or a neuromodulator, as well as the modulation of excitation-contraction coupling in skeletal muscle and the activity of the sympathetic nerve innervating the muscle (nagai et al. 2019; berezhnoy et al. 2019) . carnosine also plays a role in the inhibition of the growth and migration but induction of apoptosis of tumor cells, including human glioblastoma cells as well as colorectal and ovarian carcinoma cells (hipkiss and gaunitz 2014; hsieh et al. 2019; iovine et al. 2016) , as well as the suppression of the release of interleukin-6 by lipopolysaccharides plus interferon-γ-activated macrophages (caruso et al. 2017) . most recently, carnosine was reported to influence epigenetic regulation of gene expression in mammalian cells via increased histone acetylation (oppermann et al. 2019). compared with its constituent amino acids, the formation of carnosine can reduce intracellular osmolarity in the skeletal muscle and olfactory bulb, thereby protecting their integrity and function. in addition, being a positively charged dipeptide that is resistant to peptidases, carnosine does not readily exit cells and can be accumulated inside the cells at high concentrations within physiological ranges. through its intracellular actions, carnosine confers a vasorelaxing effect to reduce blood pressure (ririe et al. 2000) , stroke, and seizures (horning et al. 2000) , and carnosine is particularly beneficial for ameliorating ageing-related disorders such as cataract and neurological diseases (cararo et al. 2015; schön et al. 2019 ). thus, carnosine plays an important role in maintaining cell structure and function, as well as whole-body homeostasis and healthy aging, while reducing the risk of oxidative stress-related diseases, such as obesity, diabetes, hypertension, atherosclerosis, alzheimer's disease, stroke and seizures. much research has demonstrated the beneficial effects of dietary supplementation with carnosine on animal models of human diseases (derave et al. 2019 ). these effects include decreased plasma glucose and amelioration of diabetic complications (e.g., nephropathy, ocular damage, and retinopathy) in diabetic mice (lee et al. 2005; pfister et al. 2011 ); reduced lipid oxidation and augmented anti-oxidative capacities [e.g., restoring of blood glutathione and basal activities of antioxidant enzymes in aging rats (aydin et al. 2010a,b; hipkiss and brownson 2000) ] and in pigs (ma et al. 2010 ); amelioration of acetaminophen-induced liver injury (yan et al. 2009 ), thioacetamide-or hyperammonemia-induced liver cirrhosis (aydin et al. 2010a,b; jamshidzadeh et al. 2017b) , and ethanol-induced chronic liver injury in rats (liu et al. 2008) . dietary supplementation with carnosine also results in decreases in malondialdehyde, oxidative stress (including ischemic oxidative stress and cerebral ischemia), and ethanol-induced protein carbonyls in brains (berezhnoy et al. 2019; fedorova et al. 2018 ); amelioration of neurological disorders, including autism spectrum disorder in children (chez et al. 2002; kawahara et al. 2018) ; protection against cardiovascular injury (abplanalp et al. 2019; artioli et al. 2019 ) and bleomycin-induced lung fibrosis in rats (cuzzocrea et al. 2007) ; cardiomyopathy in rats induced by chemotherapeutic agents that cause hydroxyl radical formation and lipid peroxidation (dursun et al. 2011) ; ros-induced renal damage in mice (fouad et al. 2008) ; and promotion of wound healing in rodents (ansurudeen et al. 2012) . through augmenting respiratory burst in neutrophils and their production of ros, as well as modulating the release of the virulent influenza virus from activated neutrophils, oral administration of carnosine and anserine reduces virus dissemination in humans (babizhayev and deyev 2012; babizhayev et al. 2014) . extensive animal experiments have laid a strong foundation for human clinical studies, which indicate beneficial effects of supplementation with carnosine (0.5-2 g/day) for 1-6 months on subjects with chronic diseases (table 2) . for example, oral administration of carnosine has been used to ameliorate syndromes in patients with gastric ulcers (sakae and yanagisawa 2014) , parkinson disease (boldyrev et al. 2008) , schizophrenia (chengappa et al. 2012) , autistic spectrum disorder (chez et al. 2002) , and ocular diseases (babizhayev et al. 2001) . carnosine supplementation has also been shown to attenuate elevated levels of glucose, triglycerides, advanced glycation end products, and tumor necrosis factor-α levels in patients with type-2 diabetes (houjeghani et al. 2018) and in overweight or obese pre-diabetic subjects (de courten et al. 2016; liu et al. 2015) , enhance cardiac output and improve the quality of life in patients with heart failure (cicero and colletti 2017) , as well as renal functional integrity and anti-oxidative capacity in pediatric patients with diabetic nephropathy (elbarbary et al. 2017 ); ameliorate insulin resistance (baye et al. 2019) , and increase leantissue mass in obese or overweight subjects (liu et al. 2015) . exercise is associated with increases in ca 2+ influx into the myofibers of skeletal muscle (regulating myosin-actin cross bridging and action potential along the muscle fiber membrane) and in the production of ros and h + by skeletal muscle. excessive ros and h + must be removed rapidly, for example, by carnosine to sustain muscular activity and atp generation. therefore, dietary supplementation with β-alanine or carnosine improves muscular performance in humans (matthews et al. 2019 ). this notion is further substantiated by several lines of evidence. first, increases in carnosine concentrations in soleus (+ 45%) and gastrocnemius (+ 28%) through oral administration of β-alanine (5 g/day) for 7 weeks enhanced rowing performance in athletes (baguet et al. 2010) . similarly, supplementation with 4-6.4 g β-alanine per day (8 dosing of 0.4 or 0.8 g for each dosing) for 10 weeks to non-vegetarian men (consuming 0.25-0.75 g β-alanine from histidine dipeptides in meat) augmented intramuscular carnosine concentrations by 74% (93% and 57% increase in type i and ii muscle fibers, respectively) and improved their high-intensity cycling performance (hill et al. 2007) . second, dietary supplementation with 2.5 g of a mix of anserine and carnosine (2:1) per day for 13 weeks enhanced cognitive functioning and physical capacity (indicated by the senior fitness test) in the elderly (szcześniak et al. 2014) . third, oral administration of carnosine (500 mg once daily) for 6 months improved exercise performance (indicated by the 6-min walking test, peak o 2 consumption, and peak exercise workload), as well as the quality of life in patients with chronic heart failure (lombardi et al. 2015) . after the discovery of carnosine in beef, scientists analyzed this substance in other animal species. in 1929, n. tolkatschevskaya and d. ackermann independently identified a carnosine-like compound in goose skeletal muscle to be a dipeptide (methyl carnosine; β-alanyl-1-methyl-l-histidine), which was named anserine after the taxonomic name for the goose. this peptide is characterized by three ionizable groups: the carboxylic group (pka 2.64), the amino group of the β-alanine residue (pka 9.49), and the imidazole ring in histidine (pka 7.04), with the pka of the whole molecule being 8.27 (bertinaria et al. 2011) . thus, at physiological ph (e.g., ph 7.0 to 7.4), anserine is present in the zwitterionic form and has a net positive charge. anserine is abundant in the skeletal muscles of birds, certain fish [e.g., salmon, tuna and trout (boldyrev et al. 2013) ], and beef , but is absent from human tissues (including skeletal muscle, heart and brain) (mannion et al. 1992 ). among the animal kingdom, anserine is the major histidine-containing dipeptide in the skeletal muscles of dog, cat, lion, rabbit, agouti, mouse, kangaroo, wallaby, opossum, cod, smelt, marlin, whiting, croaker, tuna, japanese char, salmon and trout (boldyrev et al. 2013) . intramuscular concentrations of anserine in these non-primate species range from 2 mm for opossum to 21 mm for marlin. high concentrations of anserine in the mm range are also present in the brains of the non-primate animals. in contrast, anserine is usually absent from the plasma of healthy adult humans without anserine intake (everaert et al. 2019) and is present at 2-10 µm in the plasma of non-primate animals depending on species (boldyrev et al. 2013) . intramuscular and cardiac concentrations of anserine are affected by a number of factors, including muscle fiber type, muscular contractility, and health status. for example, white muscle fibers contain much more anserine than red muscle fibers, as the concentrations of anserine and carnosine were 2.2-and 2.8-fold higher, respectively, in breast versus thigh muscle . in rat skeletal muscles (longissimus dorsi and quadriceps femoris), the concentrations of carnosine and anserine decrease by 35-50% during senescence, and are 35-45% lower in hypertensive animals than in normotensive ones (johnson and hammer 1992) . similarly, in rat cardiac muscle, the concentrations of total histidine dipeptides decline by 22% during senescence and, are 35% lower in hypertensive animals than in normotensive ones (johnson and hammer 1992) . in humans, dietary anserine is absorbed by the small intestine, transported in blood, and taken up by extra-intestinal tissues as described previously for dietary carnosine (fig. 1) , except that the rates of catabolism of anserine to β-alanine and 1-methyl-histidine by serum carnosinase (carnosinase-1) in plasma and by carnosinae-2 in non-blood tissues are lower than those for carnosine (yeum et al. 2010) . a study with adult humans has shown that oral administration of 2 g anserine/60 kg bw without or with 19.4 g food increased the concentration of anserine in plasma, which peaked (52 and 40 µm, respectively) at 45 min after consumption and returned to the baseline value at 3 h after consumption (kubomura et al. 2009 ). in the absence of food intake, the oral administration of anserine increased the concentration of 1-methyl-histidine in the human plasma, which peaked (125 µm) at 60 min and declined thereafter to ~ 100 µm at 4 h after consumption. when anserine was consumed along with 19.4 g food, because the intestinal absorption of anserine was delayed, the oral administration of anserine increased the concentration of 1-methyl-histidine in the human plasma to the peak value of ~ 100 µm) at 75 min, which remained essentially unchanged at 4 h after consumption. similar results were obtained for β-alanine (yeum et al. 2010) . in adult humans, the t 1/2 of orally administered anserine in plasma is 1.28 or 1.35 h, respectively, without or with food consumption (kubomura et al. 2009 ). thus, in humans, the circulating anserine is cleared rapidly as is carnosine. increasing dietary intake of anserine enhances its concentrations in skeletal muscle, brain and heart (boldyrev et al. 2013 ). humans and other primates do not synthesize anserine. however, non-primate animals can synthesize anserine from β-alanine and histidine at various rates, depending on species (boldyrev et al. 2013) . the atp-dependent pathways require anserine synthetase, as well as carnosine synthetase plus carnosine 1-methyltransferase (wu 2018) . the anserine synthetase pathway is a minor one, because 1-methylhistidine is limited in animal tissues. the carnosine n-methyltransferase is quantitatively important and physiologically active for anserine synthesis in skeletal muscle. because carnosine 1-methyltransferase and guanidinoacetate methyltransferase (the enzyme that converts guanidinoacetate into creatine) compete for s-adenosylmethionine (wu 2013) , there may be a close metabolic relationship between anserine and creatine syntheses in non-primate animals. like carnosine, the homeostasis of anserine in skeletal muscle is controlled by the availability of β-alanine or its degradation via transamination (blancquaert et al. 2017 ). besides using carnosine as a substrate, carnosinase also acts on anserine, but its enzymatic activity is lower for anserine than for carnosine (boldyrev et al. 2013; yeum et al. 2010) . thus, anserine is metabolized in humans and other animals as described previously for carnosine, except that serum carnosinase degrades anserine at a lower rate than carnosine and, therefore, oral administration of anserine or anserinecontaining food (e.g., beef) can transiently increase the concentration of anserine in the human plasma (everaert et al. 2019; yeum et al. et al. 2010) . excess anserine, β-alanine and 1-methyl-histidine are excreted from the body via the urine (sale et al. 2010) . oral administration of 4, 10 and 20 mg anserine/kg bw to young adults dose-dependently increased its urinary excretion, with peak concentrations of anserine in urine at 90 min after intake (everaert et al. 2019) . even in subjects consuming 20 mg anserine/kg bw, the peak concentration of anserine in the plasma was only 3 µm, indicating extensive catabolism of this peptide by serum carnosinase. in adult humans consuming 150 g beef or chicken broth, urinary concentrations of anserine within 7 h after intake were 14-and 243-fold greater, respectively, than the values for no consumption of the meat (yeum et al. 2010) . anserine has physiological functions similar to those of carnosine, including h + buffering, antioxidation, modulation of muscle contractility (e.g., excitation and contraction through transmembrane potential maintenance and electromechanical coupling), and regulation of metabolism (boldyrev et al. 2013; everaert et al. 2019; kohen et al. 1988 ). however, as a methylated metabolite of carnosine, anserine has some biochemical properties that are different from those of carnosine. for example, in contrast to carnosine, anserine does not chelate copper and may not regulate nitric oxide availability in cells (boldyrev et al. 2013) . also, although anserine and carnosine exhibit an equal anti-oxidative activity (boldyrev et al. 1988) , anserine (1 mm), but not carnosine (1 mm), increased the protein and mrna levels of heat shock protein-70 in renal tubular cells treated with 25 mm glucose or 20-100 µm hydrogen peroxide (peters et al. 2018) . furthermore, anserine, but not carnosine, inhibits carnosinase activity (derave et al. 2019 ). thus, anserine may potentiate the action of carnosine in the body. some studies have shown the beneficial effects of dietary supplementation with anserine on animal models of human diseases characterized by oxidative stress. for example, intraperitoneal administration of 0.1 or 1 mg anserine to hyperglycemic rats (250-300 g of body weight) reduced hyperglycemia and plasma glucagon concentrations by suppressing sympathetic nerve activity . similarly, intravenous administration of anserine every 2 days for 6 days to 12-week-old diabetic db/db mice reduced blood glucose concentration by 20%, vascular permeability by one-third, and urinary proteinuria by 50% (peters et al. 2018 ). in addition, kaneko et al. (2017) reported that oral administration of anserine (10 mg/mouse) to 18-month-old aβppswe/psen1de9 mice (a model of alzheimer's disease) for 8 weeks completely recovered the memory deficits, improved pericyte coverage on endothelial cells in the brain, and suppressed glial inflammatory reactions. these results indicate that anserine ameliorates neurovascular dysfunction and improves spatial memory in aged animals. interestingly, the effects of anserine on the renal function seem to be dependent on its doses via actions on the histaminergic nerve. for example, intravenous administration of 1 µg anserine to urethane-anesthetized rats suppressed the renal sympathetic nerve activity, blood pressure, and heart rate, whereas intravenous administration of 1 mg anserine had the opposite effects (tanida et al. 2010) . thus, because of its anti-oxidative and vasodilatory actions, dietary supplementation with anserine-rich chicken meat extracts ameliorated carbon tetrachloride-induced hepatic oxidative stress and injury in rats (peng and lin 2004) , while improving glucose homeostasis and preventing the development of hypertension in stroke-prone spontaneously hypertensive rats (matsumura et al. 2002) . besides animal studies, clinical investigations with humans have demonstrated beneficial effects of anserine on their metabolic, neurological, immunological, cardiovascular and renal functions. for example, oral administration of anserine (10 or 100 mg/45 kg bw in 20 ml of tap water) reduced blood glucose levels during an oral glucose tolerance test in humans . similarly, supplementing anserine plus carnosine (1 g/day, 3:1 ratio, 3 months) to healthy elderly subjects has been shown to preserve verbal episodic memory and brain perfusion (including blood flow in the prefrontal brain (ding et al. 2018; hisatsune et al. 2015; rokicki et al. 2015) , attenuate cognitive impairment (hisatsune et al. 2016; masuoka et al. 2019) , inhibit the production of inflammatory cytokines by peripheral blood mononuclear cells (katakura et al. 2017) , and enhance muscular strength and exercise endurance (hirohiko et al. 2006) . because avian muscle contains a large amount of anserine as noted previously, much evidence shows that consumption of a popular asian food known as chicken essence (chicken meat extract) by humans, particularly elderly subjects, has long been a nutritional means to improve human health and prevent chronic diseases. specifically, oral administration of anserine (0.01-0.6 g/day) to adults can effectively relieve stress and fatigue, ameliorate anxiety, promote post-partum lactation, improve physical capacity and exercise performance, reduce hyperglycemia and hypertension, enhance immunity, prevent ageing-associated neurological (e.g., cognitive and memory) dysfunction and inflammation, and accelerate wound healing szcześniak et al. 2014) . furthermore, oral administration of anserine can enhance the ability of humans to defend against infectious disease, as noted previously. collectively, these results indicate many health benefits of anserine supplementation on multiple systems in humans. tissue distribution of 4-hydroxyproline 4-hydroxyproline (4-hydroxypyrrolidine-2-carboxylic acid; an imino acid; also often referred to as an amino acid) was originally produced from the acid hydrolysates of beef gelatin by e. fischer in 1902. subsequently, 4-hydroxyproline was found to be an abundant constituent of collagen and elastin. studies in the 1960s revealed that 4-hydroxyl-proline is derived from the post-translational hydroxylation of l-proline in proteins (primarily collagen). humans and animals also contain 3-hydroxyproline. in collagen and physiological fluid, the ratio of 4-hydroxyproline to 3-hydroxyproline is 100:1, and these two imino acids exist in the trans form (wu et al. 2019) . the healthy 70-kg adult human contains 15.1% protein (or 10.6 kg protein; wang et al. 2003) , including 3.72 kg collagens (meléndez-hevia et al. 2009 ). in humans and animals, collagen is the most abundant protein comprising 30% and 35% of body protein at birth and in adult life, respectively (wu et al. 2011) . collagen consists of 13.0 proline, 9.01 4-hydroxyproline, 0.09 3-hydroxyproline, and 33 glycine residues per 100 amino acid residues (or 13.3 g proline, 10.73 g 4-hydroxyproline, 0.11 g 3-hydroxyproline, and 25.8 g glycine residues per 100 g collagen). thus, proline plus hydroxyproline accounts for approximately 12.5% (g/g) of proteins in the body, with the ratio of proline to 4-hydroxyproline being 2.25:1 (devlin 1992) . in the gly-x-y repeat of collagen, proline is in the x or y position but 4-hydroxyproline occurs only in the y position. 4-hydroxyproline is abundant in mammalian milk and plasma (mm range) in the peptide form [primarily glycyl-prolyl-4-hydroxyproline (gly-pro-hyp)], and free 4-hydroxyproline is also present in these physiological fluids (µm range). for example, the concentrations of gly-pro-hyp in the plasma of 7-to 21-day-old pigs and sow's milk range from 6 to 10 mm and from 2 to 3 mm, respectively (wu et al. 2019) . for comparison, the concentrations of free 4-hydroxyproline in the plasma of mammals are 10 to 125 µm [e.g., 10-15 µm, adult humans (knight et al. 2006) and 109 µm, 7-day-old pigs (wu et al. 2019) ], depending on diet, species, and age, as well as physiological (e.g., stress) and pathological (cancer) conditions. 4-hydroxyproline-containing proteins (primarily collagens) are hydrolyzed by proteases in the luminal fluids of the stomach (pepsin) and small intestine (trypsin, chymotrypsin, elastase, carboxypeptidases a and b, and aminopeptidase) to yield peptides and free amino acids (wu et al. 2011) . the oligopeptides are further hydrolyzed by peptidases to tripeptides, dipeptides, and amino acids. the mucosa of the small intestine secretes proline peptidase that specifically hydrolyzes proline-containing dipeptides (sjostrom et al. 1973) . some prolyl-4-hydroxyproline (pro-hyp, a product of gly-pro-hyp degradation) is hydrolyzed to proline and 4-hydroxyproline by intestinal prolidase. this multifunctional enzyme possesses a unique ability to degrade imidodipeptides (x-pro or x-hyp) in which a proline or 4-hydroxyproline residue is located at the c-terminal end (lupi et al. 2008 ). thus, hydroxyproline-containing dipeptides and tripeptides in the lumen of the small intestine are transported intact into enterocytes (absorptive epithelial cells) by h + gradient-driven peptide transporters (fig. 1) . in contrast, free proline and 4-hydroxyproline in the lumen are taken up into the cells primarily by the na + -dependent system imino transporter and the system nbb transporter (present on the brush border for the transport of neutral amino acids), as well as the na + -independent system l transporter (brandsch 2006) . inside the enterocyte, some of the 4-hydroxyprolinecontaining tri-and di-peptides undergo hydrolysis by peptidases and/or cytosolic prolidase to form free amino acids, including 4-hydroxyproline. the latter is metabolized locally to form glycine (wu et al. 2019) . the remaining di-and tripeptides exist the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via pht1/2 (fig. 1) . in contrast, proline and 4-hydroxyproline exit the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via specific transporters. some (about 40%) of the absorbed free proline and hydroxyproline are degraded by the intestinal mucosa and the remaining (about 60%) enters the portal circulation (li and wu 2018) . studies with rats have shown that free 4-hydroxyproline and 4-hydroxyproline-containing di-and tri-peptides in the portal vein account for approximately 30% and 70% of the total food-derived 4-hydroxyproline measured in the plasma (osawa et al. 2018) . note that gly-pro-hyp [a major small peptide from dietary collagen hydrolysates (yazaki et al. 2017) ] in the plasma was not determined in the published study (osawa et al. 2018) . these results indicate that small peptides from collagen hydrolysates are absorbed by the small intestine into the portal circulation predominantly as tri-and di-peptides. most of the diet-derived 4-hydroxyproline-containing di-and tri-peptides are not hydrolyzed by the small intestine during the first pass and, therefore, enter the portal circulation (shigemura et al. 2011) . the absolute bioavailability of collagen hydrolysates to the small intestine and extra-intestinal tissues in various amino acid and peptide forms is about 90-95% in humans and animals, depending on species and age. in humans, ingestion of collagen hydrolysates increases the concentrations of at least 11 hyp-containing peptides (pro-hyp, hyp-gly, ala-hyp, ile-hyp, leu-hyp, phe-hyp, glu-hyp, pro-hyp-gly, gly-pro-hyp, ala-hyp-gly, and ser-hyp-gly) in plasma (sato et al. 2019) . a majority of studies have shown that pro-hyp and hyp-gly, which are resistant to peptidases in the human plasma, are the most (about 50%) and second most abundant food-derived 4-hydroxyproline-containing peptides in the plasma of healthy adult humans, rats and mice after consuming collagen hydrolysates (osawa et al. 2018; sato et al. 2019; shigemura et al. 2014 ). the small peptides are cleared rapidly from the blood. the maximum concentration of food-derived 4-hydroxyproline-containing peptides in the plasma of individuals consuming collagen hydrolysates (385 mg/kg bw) is 140 µm (the mean value) at 2 h after administration, with the maximum concentration of free 4-hydroxyproline in the plasma being 120 µm (the mean value) at 1-2 h after administration (ohara et al. 2007 ). interestingly, gly-pro-hyp and pro-hyp are the most and second most abundant small 4-hydroxyproline-containing peptides in the plasma of mice, whereas ala-hyp, gly-pro-hyp and pro-hyp are the most, second most, and third most abundant food-derived 4-hydroxyproline-containing peptides in the plasma of adult men and women, at 1-2 h after the animals and human subjects consumed high amounts of tripeptide (gly-x-x)-containing collagen hydrolysates (0.9-1.8 g/kg bw for mice and 0.3 g/kg bw for humans) (yazaki et al. 2017) . thus, the abundance of 4-hydroxyproline-containing di-and tri-peptides in the plasma is affected by collagen hydrolysate preparations. proline, 4-hydroxyproline, and their small peptides are transported in blood in the free form. they are rapidly taken up by extra-intestinal tissues and cells [e.g., kidneys, skeletal muscle, skin, heart, and immunocytes (yazaki et al. 2017) ] via imino or peptide transporters (wu 2013) . inside these tissues and cells, the 4-hydroxyproline-containing di-and tri-peptides are hydrolyzed by peptidases and/or prolidase to yield their constituent imino acids (4-hydroxyproline or proline) and glycine. increasing dietary intake of 4-hydroxyproline in the free or peptide form enhances the concentrations of 4-hydroxyproline in plasma and tissues, such as the skeletal muscle, colon, joints, and skin (ji et al. 2018; yazaki et al. 2017 ). in fibroblasts, 4-hydroxyproline is formed from the posttranslational hydroxylation of proline residues in proteins, primarily collagen (li and wu 2018) , as noted previously. specifically, proline is incorporated into protein through the intracellular pathway of protein synthesis. thereafter, some proline residues are hydroxylated in the endoplasmic reticulum by prolyl 4-hydroxylase in the presence of oxygen, ascorbic acid, α-ketoglutarate, and fe 2+ to form 4-hydroxyprolyl residues (gorres and raines 2010). hydrolysis of the protein containing hydroxylated proline residues releases 4-hydroxyproline as a free imino acid. other prolyl 4-hydroxylases, including hypoxia-inducible transcription factor α, can convert a limited number of proline residues to 4-hydroxyproline residues in non-collagen proteins (myllyharju and koivunen 2013). relatively little is known about 4-hydroxyproline catabolism in humans or animals. however, there is evidence that the kidneys of adult rats can rapidly take up extracellular 4-hydroxyproline and then convert it into glycine via 4-hydroxyproline oxidase (lowry et al. 1985) . other gibson et al. (2002) . adult humans consume 0.75 g protein/kg bw/ day b yu et al. (1985) . this value refers to the healthy adult consuming 1.5 g protein/day c meléndez-hevia et al. (2009) d the value is estimated from the plasma flux of glycine (22.6 g/day in the 70-kg healthy adult; ginson et al. 2002) and the net conversion of plasma glycine into serine (i.e., 6% of plasma glycine flux; butterworth et al. 1958) e starck et al. (2018) f the average value of 10.1 g/day for the healthy adult consuming 0.75 g protein/day (gibson et al. 2002) and 8.09 g/day for the healthy adult consuming 1.5 g protein/day (yu et al. 1985) g the value was calculated on the basis of the following: (1) the rate of degradation of mature collagens in the extracellular matrix is equal to the rate of net synthesis of collagen (i.e., the rate of collagen secreted from fibroblasts to the extracellular matrix; 96.5 g/day) in the healthy adult human (meléndez-hevia et al. 2009 ); (2) the content of 4-hydroxyproline in collagen is 10.73 g/100 g collagen; wu et al. 2011); and (3) 90% of collagen-derived 4-hydroxyproline is catabolized to form glycine in the healthy adult human (knight et al. 2006 extra-intestinal tissues (e.g., liver, skeletal muscle, and skin) also synthesize glycine from 4-hydroxyproline (hu et al. 2017) . the half-lives of free hydroxyproline (45 to 80 min) and its di-and tri-peptides (25-100 min) in blood are relatively short and vary with the species and age of mammals. for example, we found the half-lives of free 4-hydroxyproline in the blood of 8-and 160-day-old pigs are 46.2 ± 2.8 and 60.5 ± 3.3 min (mean ± sem, n = 6), respectively, as determined by intravenous administration of 4-hydroxyproline (50 mg/kg bw) and the single exponential model of its pharmacokinetics (wu et al. 2007 ). the half-lives of gly-pro-hyp and pro-hyp in the blood of adult humans are estimated to be about 50 and 100 min, respectively, but are about 25 min in mice (yazaki et al. 2017) . similarly, based on the work of kusubata et al. (2015) , the half-life of pro-hyp in the blood of mice is estimated to be about 25 min. the metabolic pathway for glycine synthesis from 4-hydroxyproline, with glyoxylate as the most immediate intermediate (wu et al. 2019) . interestingly, the estimate rate of whole-body glycine synthesis in the 70-kg healthy human adult (8.16 g/day) is comparable to the measured rate of 10.1 g/day (gibson et al. 2002) and 8.09 g/day (yu et al. 1985) in the healthy human adult consuming 0.75 and 1.5 g protein/day, respectively. a small fraction of the glyoxylate is converted into oxalate and glycolate in the liver (knight et al. 2006) . in adult humans, about 95% of the collagenderived 4-hydroxyproline may be converted into glycine, and the remaining 5% oxidized to oxalate and glycolate (knight et al. 2006) . the amounts of 4-hydroxyproline, oxalate and glycolate excreted daily in the urine of adult humans are 3-4, 1-3, and 10-20 mg, respectively. we estimated that milk-borne and collagen-derived 4-hydroxyproline contribute to 14.4% and 31.1%, respectively, of glycine needed by the young pig, whereas milk-borne glycine and non-hydroxyproline amino acids contribute to 24.0% and 30.5%, respectively, of glycine needed by the neonate (wu et al. 2019) . in adult humans, 4-hydroxyproline may contribute to about 60% of total glycine requirement (table 3) . 4-hydroxyproline oxidase is the rate-controlling enzyme in the conversion of 4-hydroxyproline into glycine (wu et al. 2011) and is induced by cortisol (phang et al. 2010) . thus, the circulating levels of hydroxylproline are reduced in animals with fatigue (caused by deprivation of rest and sleep; a physical stress condition) or oxidative stress (kenéz et al. 2018; kume et al. 2015) . as a key component of collagen, proline and 4-hydroxyproline permit the sharp twisting of the collagen helix. this allows for establishing and maintaining the rigid structure of the collagen molecule in connective tissues, particularly skin, tendon, cartilage, bone, blood vessels, and the basement membrane (e.g., the intestinal lamina propria; a thin, fibrous, extracellular matrix of tissue that separates an epithelium from its underlying tissue), as well as protecting other tissues in the body (phang et al. 2010 ). in addition, the presence of 4-hydroxyproline in the gly-x-y collagen peptides reduces chemotaxis and blocks apoptosis in neutrophils, while the hydroxylation of two proline residues in hypoxia-inducible factor-α to form 4-hydroxyproline under normoxic oxygen conditions triggers the proteasomal degradation of the protein to regulate its abundance (wu et al. 2019) . multiple tissues in humans and many animals synthesize glycine from 4-hydroxyproline, as noted previously. this metabolic pathway is not dependent on folate (whose provision in diets is quantitatively low relative to glycine requirement) and, therefore allows for the provision of glycine from the inter-organ metabolism of amino acids (e.g., arginine, glutamine, glutamate, ornithine and proline) other than the tetrahydrofolate-dependent hydroxymethyl transferation of serine. the synthesis of glycine from 4-hydroxyproline plays an important role in maintaining the homeostasis of glycine [an amino acid with enormous nutritional and physiological importance, including heme formation and anti-oxidative reactions (wu 2013) ], as typical diets can meet at most 20% and 14% of daily glycine needs in milk-fed neonates (hou et al. 2015) and adult humans (table 3 ). in addition, glycine is a major limiting factor for the syntheses of glutathione (the most abundant low-molecular-weight antioxidant in cells) (mccarty et al. 2018) , as well as bile salts, purines, heme and creatine in the body (wu 2013) . growing evidence shows that 4-hydroxyproline can scavenge oxidants (phang et al. 2010) , suppress nf-kb activation (ji et al. 2018) , regulate the intracellular redox state, and stimulate the expression of anti-oxidative enzymes in cells (wu et al. 2019) . furthermore, 4-hydroxyproline inhibits the production of hydroxyl radical via the fenton reaction possibly through: (a) formation of coordinate bonds with iron, (b) sequestration of fe 2+ , and (c) interactions with intermediates of the reaction via hydrophobic hydration (milić et al. 2015) . in vitro anti-oxidative effects (e.g., scavenging of radicals and inhibition of lipid peroxidation) have also been demonstrated for the enzymatic hydrolysates of porcine collagen (ao and li 2012) . similarly, 4-hydroxyprolinecontaining collagen hydrolysates reduce inflammation and promote collage synthesis in dermal fibroblasts (offengenden et al. 2018) , while enhancing bone density and strength (watanabe-kamiyama et al. 2010) . finally, as an activator of the apoptotic cascade, oxidation of 4-hydroxyproline by 4-hydroxyproline oxidase to ros can inhibit the growth of cancer cells and promote their death (cooper et al. 2008) , thereby inhibiting tumorigenesis. furthermore, through the action of ros to kill pathogenic bacteria, fungi, parasites, and viruses (vatansever et al. 2013) , 4-hydroxyproline can enhance the ability of humans against infectious diseases. thus, in humans and animals, the generation of 4-hydroxyproline is enhanced in response to oxidative stress as an adaptation mechanism for defense and survival (wu et al. 2019) . research on the health benefits of 4-hydroxyproine has focused on its role in improving joint, skin and bone health (moskowitz 2000; zhang et al. 2011a ) and in preventing gut inflammation in animal models. for example, pro-hyp stimulates the growth and migration of fibroblasts in the mouse skin (shigemura et al. 2009 ) and, therefore, would have important implication for maintaining dermal hydration and accelerating wound healing (li and wu 2018) . furthermore, pro-hyp inhibits the differentiation of chondrocytes into mineralized cells to maintain their normal physiological activity, while increasing glycosamino-glycan content in the extracellular matrix (nakatani et al. 2009 ). consistent with this view, dietary supplementation with 0.3% pro-hyp reduced the breakdown of articular cartilage and ameliorates osteoarthritis in c57bl/6j mice (nakatani et al. 2009 ). likewise, dietary supplementation with 4-hydroxyprolinecontaining collagen peptides (0.2 g/kg bw per day) to mice attenuated uv-b-induced decreases in skin hydration and soluble type i collagen, hyperplasia of the epidermis, and skin damage (tanaka et al. 2009 ). furthermore, oral administration of the collagen hydrolysates enhanced bone density (wu et al. 2004 ) and femur fracture healing in rats (tsuruoka et al. 2007) , as well as cutaneous wound healing in diabetic rats (zhang et al. 2011b ) and injured nondiabetic rats (zhang et al. 2011c) . there is growing interest in the role of 4-hydroxyproline in intestinal health. ji et al. (2018) reported that oral administration of 4-hydroxyproine (1% in drinking water) attenuated dextran sulfate sodium (dss)-induced colitis (characterized by the infiltration of mononuclear cells and colonic mucosal damage) in mice through inhibiting the nf-κb signaling and oxidative stress. likewise, intragastric administration of a 4-hydroxyproline-containing dipeptide (pro-hyp) to dss-challenged mice at the dose of 227 mg/kg bw per day (1 mmol or 131 mg 4-hydroyproline/kg bw per day) for 8 days mitigated dss-induced colitis (zhu et al. 2018) . similarly, intraperitoneal administration of a synthetic 4-hydroxyproline-containing polypeptide [(gly-pro-hyp) 10 ] ameliorated clinical symptoms and histopathological colonic changes in mice with acute dss colitis (heimesaat et al. 2012) . because colitis increases the risk for colon cancer (the third most common type of cancer diagnosed in the us) (healy et al. 2019) , dietary supplementation with 4-hydroxyproline may help to prevent and treat this disease. future studies are warranted to test this novel and important hypothesis. clinical studies with humans have shown tremendous benefits of dietary supplementation with 4-hydroxyprolinecontaining peptides on skin, joint and bone health. for example, in a randomized double-blind, placebo-controlled trial involving 35-55 year-old women, oral administration of 2.5 or 5.0 g of 4-hydroxyproline-containing collagen peptide once daily for 8 weeks enhanced skin elasticity (proksch et al. 2014 ). in addition, daily consumption of collagen hydrolysates enhances the moisture content in the epidermis of women during winter (matsumoto et al. 2006 ) and ameliorates joint pain in subjects with knee osteoarthritis (deal and moskowitz 1999) . likewise, dietary supplementation with collagen hydrolysates (10 g/day) to 40-59 year-old women for 8 weeks increased collagen density in the dermis, while decreasing the fragmentation of the dermal collagen network, with the effects occurring at week 4 and persisting through week 12 (asserin et al. 2015) . furthermore, daily oral administration of 5 g of a collagen-hydrolysate food product (containing 0.1 or 2 g pro-hyp and hyp-gly per kg product) by 35-55 year-old women for 8 weeks resulted in dose-dependent improvements in facial skin conditions, such as skin moisture, elasticity, wrinkles, and roughness (inoue et al. 2016) . of particular interest, 12-month daily oral administration of 5 g collagen hydrolysates (containing 4-hydroxyproline) augmented bone mineral density in postmenopausal women (könig et al. 2018) , suggesting their role in preventing osteoporosis. similarly, dietary supplementation with 4-hydroxyproline-containing collagen hydrolysates (10 g/day) in combination with calcitonin for 24 weeks has shown positive effects on mitigating osteoporosis in postmenopausal women (adam et al. 1996) . the beneficial effects of collagen peptides on humans can result from not only 4-hydroxyproline but also proline and glycine. the latter two amino acids are also abundant in beef ). humans and animals have physiological requirements for taurine, creatine and carnosine (wu 2013) . at present, dietary requirements of taurine, creatine, carnosine, anserine, and 4-hydroxyproline have not been established for humans or animals. the institute of medicine (iom 2006) recommended the dietary requirements of infants, children and adults for the so-called nutritionally essential indispensable) amino acids that are not synthesized by their tissues, but did not recommend dietary requirements for the so-called nutritionally nonessential dispensable) amino acids (e.g., arginine and taurine) that are synthesized de novo by their tissues. this does not necessarily mean that humans have no dietary requirements for taurine, creatine or carnosine. historically, growth or nitrogen balance was the sole criterion to define whether or not an amino acid or a nitrogenous substance was nutritionally essential for animals (hou et al. 2015) . similarly, the criterion for assessing nutritional essentiality of amino acids for humans was a short-term (8-day) nitrogen balance in healthy young adults (rose 1957) . whether an amino acid was classified as nutritionally essential or nonessential is only the matter of definition, and should not reflect the physiological needs of animals or humans for the nutrient wu 2017, 2018) . it must be recognized that short-term nitrogen balance studies are not always sensitive to identify the essentiality of all amino acids for the organisms. for example, nitrogen balance was maintained in normal adult humans fed histidine-free diets for 8 days (rose 1957) , but there are no metabolic pathways for histidine synthesis in mammalian cells (wu 2013) . a negative nitrogen balance did not occur in healthy adults during a short period (e.g., 8 days) of consuming a histidine-free diet because the hydrolysis of histidine-rich hemoglobin in blood and of carnosine in skeletal muscle provides histidine for protein synthesis in tissues at the expense of health. furthermore, it has been known over 75 years that feeding an arginine-deficient diet to adult men for 9 days did not result in a negative nitrogen balance, but decreased sperm counts by ~ 90% and increased the percentage of non-motile sperm ~ 10 fold (holt and albanese 1944) . this striking observation underlines a crucial role of arginine in spermatogenesis and argues that functional needs should be a criterion for the classification of arginine and any other amino acid as an essential or nonessential nutrient in diets . of note, the institute of medicine (2006) stated that dietary arginine was not required by healthy adults because arginine is synthesized via the hepatic urea cycle. however, there is no net synthesis of arginine in the mammalian liver via the hepatic urea cycle (wu and morris 1998) . this demonstrates the importance of ensuring that recommendations for dietary amino acid requirements be based on up-to-date knowledge of new developments in the field of amino acid metabolism. taurine, creatine, and carnosine must be provided in diets for individuals who do not synthesize these nutrients due to inborn errors of metabolism (wu 2013) . by the criterion of maintaining the integrity of tissues (e.g., eyes, heart and skeletal muscle), taurine is clearly a nutritionally essential amino acid for humans without inborn errors of metabolism, particularly the young, as noted previously. based on functional needs, creatine can be classified as a conditionally essential nutrient for humans, particularly athlete vegans who may not have adequate intakes of arginine, glycine and methionine. because glycine intake from typical diets meets < 20% of physiological needs and the endogenous synthesis of glycine may be insufficient for the optimal requirements of healthy adults (table 3) , dietary supplementation is expected to beneficial for their optimal health, particularly under stress conditions and during ageing (wu 2016) . likewise, while a lack of carnosine or anserine in diets is not fatal, consumption of these two dipeptides can improve human health, especially neurological, immunological, retinal, cardiac and muscular functions. furthermore, ingestion of 4-hydroxyproline (an antioxidant and a precursor of glycine) may protect the gastrointestine from oxidative stress and inflammation, thereby possibly reducing risk for stomach, colon and breast cancers as well as possibly other types of cancer. physiological requirement of a healthy 70-kg adult for taurine may be 75 mg/day or 1.07 mg/kg bw per day based on its urinary excretion of 72 mg/day (laidlaw et al. 1988 ). because of their growth and development, infants and children likely have greater physiological requirements for taurine per kg bw than do the human adults. likewise, based on the urinary excretion of creatinine, a 70-kg healthy adult needs 1.7 g creatine/day to meet physiological needs. dietary provision of creatine can spare the use of amino table 4 estimated physiological requirements of the 70-kg adult human for taurine, creatine, carnosine, anserine, and 4-hydroxyproline for optimal health "-" denotes no basal physiological requirement a 62% of the daily glycine intake by the healthy adult consuming 0.75 g protein/day (gibson et al. 2002) nutrient physiological requirement provision of nutrient from 30-g dry weight of beef (mg) beef ( acids for protein synthesis and other important pathways such as the production of polyamines and nitric oxide to improve the function of multiple systems. thus, dietary intake of creatine is beneficial for metabolic health. based on the whole-body turnover rate of 1.33%/day for carnosine and the total amount of carnosine (45.5 g) in a 70-kg man (spelnikov and harris 2019) , it can be estimated that the physiological need for this dipeptide by a 70-kg subject is 606 mg/day. anserine is not a physiological constituent in humans without consuming this dipeptide and, therefore, humans do not appear to have a basal physiological need for it. however, oral administration anserine in the amount of 156 mg/day can enhance whole-body insulin sensitivity in subjects with hyperglycemia ). thus, dietary intake of anserine can improve human health. finally, because 4-hydroxyproline is the post-translational product of protein-bound proline, humans have no dietary requirements for 4-hydroxyproline. however, dietary 4-hydroxyproline can augment intestinal anti-oxidative capacity and endogenous glycine synthesis. based on studies with mice receiving intragastric administration of 227 mg prolyl-hydroxyproline/kg bw per day (1 mmol or 131 mg 4-hydroyproline/kg bw per day) (zhu et al. 2018 ) and the mouse to human conversion factor of 0.08 (fda 2005), we suggest that dietary intake of 18.2 mg 4-hydroxyproline/kg bw per day for adult humans may be beneficial for improving intestinal health and preventing colitis. beef is a rich source of high-quality protein as well as highly bioavailable vitamin b 12 , zinc, and iron (wu et al. 2014) . notably, compared with an isocaloric plant foodbased snack composed of corn, beans and greens, daily supplementation with a 60-g beef (12.8 g protein)-based snack (250 kcal) to plant-source diets containing little or no animal-source protein (mean = 3.1 g animal-source protein/day) for 21 months prevents nutritional growth stunting, while enhancing skeletal muscle mass and improving immune function in 6-to 14-year-old children (grillenberger et al. 2003; neumann et al. 2007 ). an important concept emerging from this review is that beef is an abundant source of taurine, creatine, carnosine, anserine, and 4-hydroxyproline as physiologically important nutrients for infants, children, and adults to maintain their health and prevent chronic diseases. for example, 30 g of dried beef can provide 80.4 mg taurine, which can meet 107% of daily taurine requirement of the 70-kg adult (table 4) . similarly, 30 g of dried beef can provide 608 mg carnosine, which can meet 100% of daily carnosine requirement of the 70-kg adult (table 4) . likewise, meat provides a large amount of creatine, and the consumption of beef can substantially contribute to physiological needs of humans for this nutrient (table 4 ). finally, beef contains abundant anserine and 4-hydroxyproline to improve the health and well-being of the young, adult, and ageing populations. thus, beef is a functional food for humans. there are concerns that consumption of red meat increases risks for chronic diseases or metabolic disorders in humans, including obesity, type ii diabetes mellitus, cardiovascular disease (the number one killer in the developed nations), ageing-related dysfunction and atrophy of organs (particularly, the brain and skeletal muscle), and cancers (willet et al. 2019). however, there is no direct evidence to support this view (johnston et al. 2019; leroy and cofnas 2019) . compelling evidence shows that taurine, creatine, carnosine, anserine, and 4-hydroxyproline, which are all abundant in red meat (e.g., beef, lamb and pork), play an important role in inhibiting oxidative stress (a common trigger of chronic diseases) and inflammation, ameliorating tissue (e.g., brain, heart, skeletal muscle, kidney, liver, gut, eye, and connective tissue) injury, improving metabolic profiles and the health of multiple systems, and enhancing immunity in animals and humans (fig. 3) . these nutrients may help to kill pathogenic bacteria, fungi, parasites, and viruses (including coronavirus) through enhancing the metabolism and functions of monocytes, macrophages and other cells of the immune system. of note, taurine, creatine, carnosine, anserine can inhibit tumorigenesis and promote the apoptosis of tumor (e.g., colon cancer) cells, whereas 4-hydroxyproline prevents colitis and thus possibly reduces risk for colon cancers. thus, red meat is a functional food for optimizing human growth, development and health. as part of a healthy diet, regular consumption of red meat may actually prevent oxidative stress, thereby reducing risks for chronic diseases in humans. future studies with animal models and human subjects are warranted to test this hypothesis. fig. 3 major functions of dietary taurine, creatine, carnosine, anserine and 4-hydroxyproline on improving the health of multiple systems in humans. these beneficial effects of the nutrients are summarized on the basis of available evidence in the current literatrure. some of the effects are tissue-and nutrient-specific. however, because all the systems of the body are integrated, the health of one system can affect that of other systems dietary taurine, creatine, carnosine, anserine, and 4-hydroxyproline (which are all abundant in beef) play an important role in inhibiting oxidative stress (a common trigger of chronic diseases) and inflammation, ameliorating tissue (e.g., brain, heart, skeletal muscle, kidney, liver, and gut) injury, and improving metabolic profiles in animals and humans. such a comprehensive update, along with recent advances in amino acid nutrition and physiology, will be highly informative to educate the public and policy makers about animal-source foods (e.g., beef) for human consumption. the health and ergogenic effects of dietary taurine, creatine, carnosine, anserine, and 4-hydroxyproline are expected to reverse the drastic decline in consumption of red meat (e.g., beef) in the u.s. due to an inadequate understanding of animal-source foods to provide functional amino acids, peptides, and creatine. finally, new knowledge presented herein can be used to guide future research priorities involving meat (including beef) in improving human nutrition, health and well-being. carnosine supplementation mitigates the deleterious effects of particulate matter exposure in mice postmenopausal osteoporosis. treatment with calcitonin and a diet rich in collagen proteins creatine and its potential therapeutic value for targeting cellular energy impairment in neurodegenerative diseases taurine prevents mitochondrial membrane permeabilization and swelling upon interaction with manganese taurine uptake across the human intestinal brush-border membrane is via two transporters: h + -coupled pat1 (slc36a1) and na + -and cl − -dependent taut (slc6a6) carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes amino acid composition and antioxidant activities of hydrolysates and peptide fractions from porcine collagen carnosine in health and disease intestinal absorption of carnosine and its constituent amino acids in man the effect of oral collagen peptide supplementation on skin moisture and the dermal collagen network effects of creatine supplementation on cognitive function of healthy individuals effect of carnosine against thioacetamide-induced liver cirrhosis in rat the effect of carnosine treatment on prooxidant-antioxidant balance in liver, heart and brain tissues of male aged rats management of the virulent influenza virus infection by oral formulation of nonhydrolized carnosine and isopeptide of carnosine attenuating proinflammatory cytokine-induced nitric oxide production n-acetylcarnosine, a natural histidine-containing dipeptide, as a potent ophthalmic drug in treatment of human cataracts l-carnosine modulates respiratory burst and reactive oxygen species production in neutrophil biochemistry and function: may oral dosage form of nonhydrolized dipeptide l-carnosine complement anti-infective antiinfluenza flu treatment, prevention and self-care as an alternative to the conventional vaccination? important role of muscle carnosine in rowing performance the influence of sex, age and heritability on human skeletal muscle carnosine content exogenous creatine delays anoxic depolarization and protects from hypoxic damage: doseeffect relationship role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage potential of creatine or phosphocreatine supplementation in cerebrovascular disease and in ischemic heart disease interaction among skeletal muscle metabolic energy systems during intense exercise slc6 transporters: structure, function, regulation, disease association and therapeutics differences in muscle histidine-containing dipeptides in broilers carnosine modulates the sp1-slc31a1/ctr1 copper-sensing system and influences copper homeostasis in murine cns-derived cells carnosine supplementation reduces plasma soluble transferrin receptor in healthy overweight or obese individuals ) carnosinases, their substrates and diseases creatine supplementation lowers brain glutamate levels in huntington's disease carnosine as an effective neuroprotector in brain pathology and potential neuromodulator in normal conditions synthesis, physicochemical characterization and biological activities of new carnosine derivatives stable in human serum as potential neuroprotective agents effects of histidine and β-alanine supplementation on human muscle carnosine storage carnosine increases efficiency of dopa therapy of parkinson's disease: a pilot study physiology and pathophysiology of carnosine creatine: endogenous metabolite, dietary, and therapeutic supplement enzymes and genes of taurine and isethionate dissimilation in paracoccus denitrificans the absorption of glycine and its conversion to serine in patients with sprue influence of exercise on urea, creatinine, and 3-methylhistidine excretion in normal human subjects creatine supplementation and aging musculoskeletal health variables influencing the effectiveness of creatine supplementation as a therapeutic intervention for sarcopenia effectiveness of creatine supplementation on aging muscle and bone: focus on falls prevention and inflammation carnosine and related peptides: therapeutic potential in age-related disorders ophidine (β-alanyl-l-3-methylhistidine, 'balenine') and other histidine dipeptides in pig muscles and tinned hams carnosine modulates nitric oxide in stimulated murine raw 264.7 macrophages does dietary creatine supplementation play a role in skeletal muscle metabolism and performance? creatine supplementation in walker-256 tumor-bearing rats prevents skeletal muscle atrophy by attenuating systemic inflammation and protein degradation signaling a preliminary, randomized, double-blind, placebocontrolled trial of l-carnosine to improve cognition in schizophrenia double-blind, placebo-controlled study of l-carnosine supplementation in children with autistic spectrum disorders functional insights into the creatine transporter nutraceuticals and dietary supplements to improve quality of life and outcomes in heart failure patients the effect of cold storage on the carnosine content of muscle metabolism of taurine in microorganisms: a primer in molecular biodiversity? a novel function for hydroxyproline oxidase in apoptosis through generation of reactive oxygen species creatine-electrolyte supplementation improves repeated sprint cycling performance: a double blind randomized control study effects of betaalanine on muscle carnosine and exercise performance: a review of the current literature protective effect of orally administered carnosine on bleomycin-induced lung injury dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet the significance of carnosine and anserine in striated skeletal muscle supplementation with qter ® and creatine improves functional performance in copd patients on long term oxygen therapy taurine: a potential ergogenic aid for preventing muscle damage and protein catabolism and decreasing oxidative stress produced by endurance exercise effects of carnosine supplementation on glucose metabolism: pilot clinical trial nutraceuticals as therapeutic agents in osteoarthritis: the role of glucosamine, chondroitin sulfate, and collagen hydrolysate kinetics of 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supplementation on renal functional integrity and oxidative stress in pediatric patients with diabetic nephropathy: a randomized placebo-controlled trial vegetarianism, female gender and increasing age, but not cndp1 genotype, are associated with reduced muscle carnosine levels in humans gene expression of carnosine-related enzymes and transporters in skeletal muscle development and validation of a sensitive lc-ms/ms assay for the quantification of anserine in human plasma and urine and its application to pharmacokinetic study guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers the potential therapeutic effects of creatine supplementation on body composition and muscle function in cancer oxidative status in different areas of the cerebral cortex of wistar rats during focal ischemia and its modulation with carnosine mammalian alkaline phosphatases enzymes bile salt hydrolases: gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract creatine protects against cytosolic calcium dysregulation, mitochondrial depolarization and increase of reactive oxygen species production in rotenone-induced cell death of cerebellar granule neurons protective effect of carnosine against cisplatin-induced nephrotoxicity in mice intestinal absorption of the intact peptide carnosine in man, and comparison with intestinal permeability to lactulose nutritional requirement for taurine in patients receiving long-term, parenteral nutrition effect of free creatine therapy on cisplatin-induced renal damage endogenous glycine and tyrosine production is maintained in adults consuming a marginal-protein diet n-chlorotaurine, a natural antiseptic with outstanding tolerability taurine-supplemented total parenteral nutrition and taurine status of malnourished cancer patients skeletal muscle atp turnover and single fibre atp and pcr content during intense exercise at different muscle temperatures in humans 3964s) food supplements have a positive impact on weight gain and the addition of animal source foods increases lean body mass of kenyan school children the subcellular distribution of carnosine, carnosine synthetase, and carnosinase in mouse olfactory tissues the identification of the n tau-methyl histidine-containing dipeptide, balenine, in muscle extracts from various mammals and the chicken the absorption of orally supplied beta-alanine and its effect on muscle carnosine synthesis in human vastus lateralis determinants of muscle carnosine content retinal degeneration associated with taurine deficiency in the cat screening high-risk populations for colon and rectal cancers the synthetic hydroxyproline-containing collagen analogue (gly-pro-hyp) 10 ameliorates acute dss colitis effect of 28 days of creatine ingestion on muscle metabolism and performance of a simulated cycling road race influence of beta-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity a possible new role for the antiageing peptide carnosine inhibition of tumour cell growth by carnosine: some possible mechanisms efects of carnosine and anserine supplementation on relatively high intensity endurance effect of anserine/ carnosine supplementation on verbal episodic memory in elderly people the continuing importance of bile acids in liver and intestinal disease observations on amino acid deficiencies in man purification and characterization of carnosine synthetase from mouse olfactory bulbs endogenous mechanisms of neuroprotection: role of zinc, copper, and carnosine nutritionally nonessential amino acids: a misnomer in nutritional sciences nutritionally essential amino acids dietary essentiality of "nutritionally nonessential amino acids" for animals and humans composition of polyamines and amino acids in plant-source foods for human consumption l-carnosine supplementation attenuated fasting glucose, triglycerides, advanced glycation end products, and tumor necrosis factor-α levels in patients with type 2 diabetes: a double-blind placebo-controlled randomized clinical trial carnosine suppresses human colorectal cell migration and intravasation by regulating emt and mmp expression the hydroxyproline-glycine pathway for glycine synthesis in neonatal pigs muscle creatine loading in men creatine electrolyte supplement improves anaerobic power and strength: a randomized double-blind control study physiological actions of taurine ingestion of bioactive collagen hydrolysates enhance facial skin moisture and elasticity and reduce facial ageing signs in a randomised double-blind placebocontrolled clinical study l-carnosine dipeptide overcomes acquired resistance to 5-fluorouracil in ht29 human colon cancer cells via downregulation of hif1-alpha and induction of apoptosis the effect of taurine on chronic heart failure: actions of taurine against catecholamine and angiotensin ii biochemistry and physiology of taurine and taurine derivatives comparison of new forms of creatine in raising plasma creatine levels analysis of the efficacy, safety, and regulatory status of novel forms of creatine taurine treatment preserves brain and liver mitochondrial function in a rat model of fulminant hepatic failure and hyperammonemia carnosine ameliorates liver fibrosis and hyperammonemia in cirrhotic rats histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles regulation of muscle phosphorylase activity by carnosine and anserine unprocessed red meat and processed meat consumption: dietary guideline recommendations from the nutrirecs consortium hydroxyproline attenuates dextran sulfate sodium-induced colitis in mice: involvement of the nf-κb signaling and oxidative stress creatine supplementation supports the rehabilitation of adolescent fin swimmers in tendon overuse injury cases mechanisms underlying the antioxidant activity of taurine: prevention of mitochondrial oxidant production anserine (beta-alanyl-3-methyl-l-histidine) improves neurovascular-unit dysfunction and spatial memory in aged aβppswe/psen1de9 alzheimer'smodel mice anserine/carnosine supplementation suppresses the expression of the inflammatory chemokine ccl24 in peripheral blood mononuclear cells from elderly people a review on functional ingredients in red meat products zinc, carnosine, and neurodegenerative diseases discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells lower plasma trans-4-hydroxyproline and methionine sulfoxide levels are associated with insulin dysregulation in horses hydroxyproline ingestion and urinary oxalate and glycolate excretion antioxidant activity of carnosine, homocarnosine, and anserine present in muscle and brain skeletal muscle fibre types, enzyme activities and physical performance in young males and females specific collagen peptides improve bone mineral density and bone 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singlet oxygen scavengers histidine and carnosine histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation characterization of human tissue carnosinase intracerebroventricular administration of creatine protects against damage by global cerebral ischemia in rat should dietary guidelines recommend low red meat intake? what is the cause of ageing atrophy? total number, size, and proportion of different fiber types studied in whole vastus lateralis muscle from 15-to 83-year-old men roles of dietary glycine, proline and hydroxyproline in collagen synthesis and animal growth taurine may prevent diabetic rats from developing cardiomyopathy also by downregulating angiotensin ii type2 receptor expression bioactivities of chicken essence cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhibits growth of a broad spectrum of cancer cells derived from solid tumours beneficial effects of histidine and carnosine on ethanol-induced chronic liver injury a dietary supplement containing cinnamon, chromium and carnosine decreases fasting plasma glucose and increases lean mass in overweight or obese pre-diabetic subjects: a randomized, placebo-controlled trial effects of oral administration oforodispersible levo-carnosine on quality of life and exercise performance in patientswith chronic heart failure hydroxyproline metabolism by the rat kidney: distribution of renal enzymes of hydroxyproline catabolism and renal conversion of hydroxyproline to glycine and serine human prolidase and prolidase deficiency: an overview on the characterization of the enzyme involved in proline recycling and on the effects of its mutations dietary supplementation with carnosine improves antioxidant capacity and meat quality of finishing pigs carnosine and anserine concentrations in the quadriceps femoris muscle of healthy humans effects of anserine/ carnosine supplementation on mild cognitive impairment with apoe4 genetic 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the role of taurine in the pathogenesis of the cardiomyopathy of insulin-dependent diabetes mellitus inhibition of rate of tumour growth by creatine and cyclocreatine role of collagen hydrolysate in bone and joint disease creatine transporter protein content, localization, and gene expression in rat skeletal muscle hypoxia-inducible factor prolyl 4-hydroxylases: common and specific roles topical application of l-carnosine to skeletal muscle excites the sympathetic nerve innervating the contralateral skeletal muscle in rats chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo taurine improves glucose tolerance in stz-induced insulindeficient diabetic mice alignment of healthy dietary patterns and environmental sustainability: a systematic review biochemical characterization of the catecholaldehyde reactivity of l-carnosine and its therapeutic potential in human myocardium meat supplementation improves growth, 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evaluation of intact heart acute taurine supplementation enhances thermoregulation and endurance cycling performance in the heat creatine supplementation with methylglyoxal: a potent therapy for cancer in experimental models quantitation of carnosine in human plasma after dietary consumption of beef taurine supplementation improves economy of movement in the cycle test independently of the detrimental effects of ethanol the mechanism of interaction of carnosine with superoxide radicals in water solutions effect of oral creatine monohydrate and creatine phosphate supplementation on maximal strength indices, body composition, and blood pressure effects of chicken extract on antioxidative status and liver protection under oxidative stress therapeutic use of creatine in brain or heart ischemia: available data and future perspectives clinical pharmacology of the dietary supplement creatine monohydrate protective actions of anserine under diabetic conditions oral carnosine supplementation prevents vascular damage in experimental diabetic retinopathy proline metabolism and microenvironmental stress improved reperfusion and neuroprotection by creatine in a mouse model of stroke oral supplementation of specific collagen peptides has beneficial effects on human skin physiology creatine supplementation decreases oxidative dna damage and lipid peroxidation induced by a single bout of resistance exercise effects of taurine supplementation on vascular endothelial function at rest and after resistance exercise synthesis of taurine and isethionic acid by dog heart slices roles of arginine in cellmediated and humoral immunity vasodilatory actions of the dietary peptide carnosine beneficial effects of creatine, coq10, and lipoic acid in mitochondrial disorders vegan diets: practical advice for athletes and exercisers daily carnosine and anserine supplementation alters verbal episodic memory and resting state network connectivity in healthy elderly adults age-related changes in motor unit function the amino acid requirements of adult man carnosinase activity of human gastrointestinal mucosa the relationship between plasma taurine levels and diabetic complications in patients with type 2 diabetes mellitus oral treatment of pressure ulcers with polaprezinc (zinc l-carnosine complex): 8-week open-label trial effect of beta-alanine supplementation on muscle carnosine concentrations and exercise performance x-linked creatine-transporter gene (slc6a8) defect: a new creatine deficiency syndrome structural correlates of the creatine transporter function regulation: the undiscovered country taurine supplementation ameliorates glucose homeostasis, prevents insulin and glucagon hypersecretion, and controls β, α, and δ-cell masses in genetic obese mice prophylactic role of taurine and its derivatives against diabetes mellitus and its related complications generation of bioactive prolylhydroxyproline (pro-hyp) by oral administration of collagen hydrolysate and degradation 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pretreatment protects against hypoxia-ischemia brain damage in the neonatal rat model oral administration of skin gelatin isolated from chum salmon (oncorhynchus keta) enhances wound healing in diabetic rats oral administration of marine collagen peptides from chum salmon skin enhances cutaneous wound healing and angiogenesis in rats deletion of the γ-aminobutyric acid transporter 2 (gat2 and slc6a13) gene in mice leads to changes in liver and brain taurine contents gelatin versus its two major degradation products, prolyl-hydroxyproline and glycine, as supportive therapy in experimental colitis in mice ethical statement this review article does not require either human consent or the approval of animal use.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-324369-zizyxb6y authors: baptista, joão; stein, mari-klara; klein, stefan; watson-manheim, mary beth; lee, jungwoo title: digital work and organisational transformation: emergent digital/human work configurations in modern organisations date: 2020-06-29 journal: nan doi: 10.1016/j.jsis.2020.101618 sha: doc_id: 324369 cord_uid: zizyxb6y abstract workplace technologies are more central to working in organisations than ever before. these technologies began as instrumental aids to support office work of individuals but have since also become the basis for social interactions and community building in organisations and more recently become able to perform managerial roles with the use of advanced ai capabilities. our call for papers to this special issue invited original studies to go further and advance our thinking on the strategic implications of this layered evolution of workplace technologies on work and the structure of organisations. in this introduction, we synthesise the main themes from the special issue, and also ongoing dialogues with the growing community at the regular ais / ifip 9.1 workshop on the changing nature of work. a key observation is that the work involved in configuring emergent digital/human configurations, is vastly under-reported and poorly understood. paradoxically, this configuring work is the most demanding and critical in the shaping of modern organisations. we suggest that this type of largely invisible work requires engagement beyond the level of execution or even the meaning of work, it requires intervening with third order effects that get to the core of what an organisation is. we highlight the challenges for organisations in dealing with third order change, particularly because these effects are beyond existing frames of reference and require more dynamic and supple responses based on the values, purpose andintent dominantin the organisation – we describe this as structural digital work. leaders that are unable or unwilling to engage with effects at this level, and this type of work, will miss identifying core opportunities and risks associated with digital transformation emerging in organisations. we also reflect on the value of current theories and methods used to research this important and emergent phenomenon. in this special issue, we publish original research on strategic perspectives on the future of work and the digital transformation of modern organisations. our call for papers (baptista et al., 2017a) invited submission of academic studies investigating the adoption of workplace technologies in organisations with an emphasis on implications for strategy and the nature of organising. suggested topics in the call for papers included, understanding the effects of workplace technologies on new dynamics and patterns of work, new structures and ways of organising, new capabilities and work practices, emerging leadership styles, and places and spaces of work with an outlook into the future of organising. we also hoped to use this effort to question the usefulness of extant theories and methods to study the deeper effects of digital workplace technologies on organisations, aiming to contributing with new concepts and theories that better reflect and respond to the emergent dynamics of digital work in modern organisations. we are delighted to introduce four papers 1 that form the basis of this special issue. in this introduction, we discuss the significance https://doi.org/10.1016/j.jsis.2020.101618 1 a few words about the special issue process: the call for papers was published in issue no. 26 of jsis in 2017. interested authors could meet with of these papers and provide a broad view and conceptual foundation for studying the future of work in modern organisations. since we accepted the papers, organisations and the world of work have been pushed further towards digital forms of organising in response to covid-19-related restrictions. this has made the ideas in this special issue even more significant and relevant. we hope that this special issue offers both a meaningful framework for future research on digital work, but also more clarity on the effects of the shift towards digital working in organisations during and after the current crisis. each of the four papers provides a distinct but complementary viewpoint on digital work and emergent dynamics in the workplace. in no particular order, the paper by morton et al. captures the introduction of a new digital platform to support strategy work and conceptualises the emergence of new work by senior leaders, and the structures needed to integrate more voices in strategic activity in the organisation. the paper by rahrovani captures the effort required by community leaders to stabilise and create a convergence of logics of meaning in a digital platform after expanding and opening it up to new members. the paper by grønsund and aanestad reveals the work involved in the gradual and deep integration of advanced algorithmic analysis of open data feeds into strategic business processes and organisational structures. the authors reflect on the impact on individual roles, skills and group capabilities as the organization learnt to control, contextualize and complement automatically generated data. the paper by rossi et al. shows employees crafting workspaces by assembling various digital workplace systems needed to do their day to day work. the paper then conceptualises this effort to create both flexible and stable structures to support the work. review of these four papers reveals how deeply embedded workplace technologies have become within organisations, while also showing how this process creates new types of work and influences the emergence of new structures and capabilities in organisations in response to the evolving nature of these technologies. while early research on digital workplace technologies such as email and intranets, captured the immediate and surface-level effects of these technologies cecez-kecmanovic et al., 1999; bansler et al., 2000; markus, 1994; lee, 1994; butler, 2003) , what we observe now is the introduction of more advanced workplace technologies in organisations, while older technologies are becoming increasingly embedded in the fabric of organisations. the evolutionary use of workplace technologies in organisations over the last two decades has hybridised their use with human activities in organisations, forming complex (benbya et al., 2020) and emergent human-in-the-loop (rai et al., 2019) or meta-human configurations as new forms of socio-technical systems not seen before (lyytinen et al., 2020) . this provides researchers with a challenge to capture the deep effects of workplace technologies in organisations (silva and hirschheim, 2007) and emergent human-technology configurations (suchman, 2012) so we can understand their strategic significance to organisations, leadership and business (dery et al., 2017; heavey et al., tavakoli et al., 2017) . in this introduction, we conceptualise both the surface and deeper effects of workplace technologies on the patterns and nature of work and the structure of organisations, and relate them to the emergence of new types of configurations between humans and technology (suchman, 2012) . from this conceptual perspective, we then analyse the four papers in this special issue to demonstrate the relevance and value of these ideas and concepts. our discussion then provides research directions and we reflect on the value of previous theories to explain the emergent dynamic and mutual shaping effects of workplace technologies on modern organisations. ultimately, we hope to indicate a pathway to develop more relevant theoretical approaches to study the deep effects of advanced technologies to support work in modern organisations. workplace technologies have evolved from basic discrete office applications to connected digital platforms (leonardi et al., 2013) with elements of automation and embedded ai-driven self-learning capabilities in today's digital workplaces (lyytinen et al., 2020; faraj et al., 2018) . this evolution in scope (martini et al., 2009) , complexity and depth of integration into the fabric of organisations is not well understood. further, workplace technologies have largely been neglected and underrepresented in academic research in favour of greater attention to business and enterprise systems, which have also evolved towards distributed platforms. however, while the impact of enterprise systems stems from their interplay with business models, the disruptive effects of workplace technologies on the nature of work and dynamics of organising (baptista et al., 2017b , stein et al., 2013 arise from the co-evolution of these technologies interplaying with the social fabric of organisations (baptista, 2009; clarke and preece, 2005) . we look back at the evolution of workplace technologies to offer a conceptual basis for understanding their evolving significance in organisations. broadly speaking, workplace technologies refer to a range of digital services that enable work within organisations. these range from office applications to integrated smac (social, mobile, analytics and cloud) technologies and smart sensing technologies using enterprise of things, smart agents, workplace robots, and self-learning algorithms (attaran et al., 2020) . we see a gradual layering of progressively more complex workplace technologies within organisations (kane, 2017) , from early workplace technologies based on individual office applications (individual tools layer) to email, intranets, collaboration platforms and social media (group and community layer) and, more recently, to advanced workplace technologies that add sensing devices, ai and cognitive knowledge and collaboration systems, robotic process automation and integrated digital platforms of work (intelligent augmentation layer). research in this area has captured the nature and affordances of workplace technologies (vaast and kaganer, 2013; treem and leonardi, 2012; leonardi and vaast, 2016) but most of this research takes an instrumental view of these technologies by linking features of workplace technologies with more immediate behaviours and practices that contribute to performance, connectedness, knowledge sharing and collective action (rice et al., 2017; saebø et al., 2020; majchrzak et al., 2013; kuegler et al., 2015; von krogh, 2012) . academic research is only recently starting to appreciate the deeper effects on organisations associated with these technologies (riemer et al., 2015; majchrzak et al., 2016; spagnoletti et al., 2015; aral et al., 2013; hutter et al., 2017; baptista et al., 2010) , and taking the first steps to study the distinct nature of a new generation of workplace technologies based on self-determining platforms with artificial intelligence (ai) that can dynamically anticipate and respond to the needs and intentions of workers (schuetz and venkatesh, 2020; lyytinen et al., 2020) . the emerging complex human and machine collaborations (benbya et al., 2020; rai et al., 2019) move the role and nature of workplace technologies beyond merely having an instrumental and supporting role to humans in organisations (riemer and peters, forthcoming) . of course, standalone software applications are still being used for discrete and well defined processes and activities at the local level, such as spreadsheets for inventory management, but it is the way these individual applications become homogenised within a largely automated data-driven "information continents" (lee, 2016a, lamb and davidson, 2005 ) that transforms work, blurring the lines between the performativity of users and the performativity of technology (watson-manheim and klein, 2019) . this highlights the importance of describing and qualifying the distinct nature of these evolving layers of workplace technologies so that we become aware of emerging effects associated with more advanced workplace technologies in organisations. while earlier workplace technologies were instrumental in support of discrete office tasks of individuals, the advent of email, intranets and social media, connected workers to form communities demanding effort to manage conversations and interactions at the social and meaning level (gibbs et al., 2013) . more recently, we have seen the introduction of sophisticated algorithmic features and ai capabilities that leverage information and the features of individual and social workplace technologies to establish patterns of use that aim to anticipate worker and organisational needs and connect people with knowledge, and in some cases perform management functions, raising the possibility of removing the human from the loop. table 1 summarises the functions and characteristics of these three layers of workplace technologies and suggests expected effects on the type of work associated with each layer. in some cases, work in organisations may be aided by multi-layered, evolving technologies, combining instrumental, collaborative and algorithmic features, e.g. in order to collectively work on documents or coordinate project contributions. in other cases, technologies may automate certain tasks to the extent that no human work is needed to perform those tasks, however in those cases, human work then shifts towards effort to integrate automated processes within established activities in the organisation. we wanted to interrogate these complex effects to gain a deeper and clearer understanding of how workplace technologies are changing organisational work, whose work is being changed, and the potential for this to disrupt established structures in organisations. to observe and conceptualise these complex effects, we need to go beyond the direct and first-order effects, and consider second-, and third-order effects of change (bartunek and moch, 1987) . as new types of digital workplace technologies have evolved from supporting functional tasks towards becoming embedded in organisational discourse and meaning, they have impacted on core aspects of the organisation and how it does business.. they have also come to play a heightened role in organisational transformation (vial, 2019) and in the rewiring of the organisation at deeper levelsthis is considered next. drawing on bartunek and moch (1987) , we see first-order effects in the use of workplace technologies as effects that reinforce, enhance and evolve existing practices and understandings of work in organisations. first-order effects are therefore expected, intended and typically incremental effects of workplace technology use that do not change the overarching organisational systems already in place. we conceptualise first-order effects as convergent change (tushman and romanelli, 1985) , that is, a change process occurring within a relatively stable structure, such as when an organisation improves its efficiency and effectiveness without rethinking its core processes (besson and rowe, 2012 p. 104). platforms integrating office applications with advanced social media features, and added sensing technologies to connect and leverage people, knowledge and learning capabilities using central automation and ai capabilities. potential to replace or augment human work and anticipate needs for knowledge and interactions in projects and work activities in organisations. distributed and integrated modular applications used within intelligent platforms that identify patterns and learn from use, with the potential to produce results and evolve without user intervention. partially self-determining and able to perform managerial roles. new types of complex digital work emerge centred on refining the "intelligence" of automated services, demanding managerial insight and effort to meaningfully assemble new work within the core of organisations. new types of digital work emerge centred on sharing information, and managing interactions and social connections, demanding greater effort to integrate individual work in the social fabric of the organisation. individual tools layer standalone applications to support individual office work and facilitate office tasks such as word processing, calculations on spreadsheets, presentation and publishing office applications, text-based emails. instrumental technologies that have specific roles and functions to support discrete office tasks. they are segregated by type and not integrated or connected. new types of digital work emerge centred on completing tasks fast and effectively, demanding limited effort to combine this with existing processes and practices. second-order effects are the result of the first-order effects that shift organisational schemata and social dynamics, and modify patterns of work and interactions. these unintended and typically unexpected effects trigger modifications in the organisational schemata, that is, the organising frameworks that guide cognitions, interpretations and actions of organisational actors (bartunek and moch, 1987 p. 484 ). thus, we conceptualise second-order effects as transforming work where the established ways of thinking and acting are profoundly changing, but still within a given structure. this may happen, for example, when organisations adopt social media and other workplace technologies to stimulate a shift from efficiency-oriented work to innovation-oriented work but do so within their existing operating model. third-order effects occur in reaction to second-order effects. these are effects outside the scope of existing operating reference systems that emerge from the evolving organisational capacity to manage second-order effects and the resultant change in organisational schemata (bartunek and moch, 1987) . third-order effects represent the emergence of entirely new schemata, reshaping views about the nature of work and how it is done, and the corresponding organisational structures (silva and hirschheim, 2007) . by organisational structures, we refer to the fundamental values and governance structures of an organisation or its "deep structure". thus, we conceptualise third-order effects as transforming the organisation where the transformation in established ways of thinking and working, over time constitutes a fundamental transformation in the structure of organisations (besson and rowe, 2012) ; this conceptualisation echoes how recent research defines digital transformation as the (re)defining of the value proposition and identity of an organisation (wessel et al., in press) . the progressive nature of these effects means that we can only observe "effects of effects" over time because of the gradual and incremental sedimentation and hybridisation inherent in the process of mutual shaping between technology and human activities in organisations (baptista, 2009) . it is also becoming apparent that workplace technologies in the intelligent augmentation layer can have deeper and more profound second-and third-order effects because they touch and intervene in the core schemata and structures of organisations, reframing perspectives and shifting established human-technology configurations (suchman, 2012 (suchman, , 2007 , rather than reinforcing established frames of reference and configurations, which would be a first-order effect. emergent human-technology configurations are central to understanding the progressive nature of the first-, second-and thirdorder effects of workplace technologies in organisations. suchman conceptualises these configurations (2012, 2007) as "how humans and machines are figured togetheror configuredin contemporary technological discourses and practices, and how they might be reconfigured, or figured together differently" (suchman, 2012 p.49 ). whilst first-order effects involve rudimentary configurations of human activity and digital technology, second-and third-order effects involve digital and human configurations of much greater complexity and scale, because they dynamically respond to the first-order effects. third-order effects involve changes of a greater magnitude, scale and depth. they reflect work and technology change that reconfigures established human-technology configurations, so they involve organisational transformation beyond improvements to the execution of tasks. drawing on the established notion of human-technology configurations (suchman, 2012 (suchman, , 2007 , we propose that the layered evolution of workplace technologies and their progressively more transformational effects demand a slight shift in thinking and terminology. we suggest instead the use of the term "digital/human configurations". this continues to recognize the relational association between the human and the technology and emphasises the effort and "work" involved in managing these configurations. explaining the notion of "configuration" suchman (2012 p.49) places great emphasis on the "figuring it out together" or "figuration" saying "to figure is to assign shape, designate what is to be made noticeable and consequential, to be taken as identifying". human intervention is therefore at the centre of configuration, but as described above, this effort has evolved in nature (from task execution to managing interactions, to refinement of automated work) and can for many workers become more demanding, less satisfying and, seemingly, less effectual (riemer and peters, forthcoming) as the configurations become more complex and layered. we put digital first in digital/human configurations to denote the emergence of new human digital work to manage these configurations where digital has an unprecedented role while human effort and endeavour is even more critical. further, we indicate the blurring of the lines between the performativity of humans and the performativity of technology (watson-manheim and klein, 2019) by replacing table 2 defining first-, second-, and third-order effects of digital transformation. effects: immediate effects on organisational processes and activities changing the execution of tasks, involving more basic digital/human configurations. effort is necessary at the level of task execution to integrate new configurations with existing ones (effort that deals with questions such as "what is the task and how can we do it better?"). appropriation of layered workplace technologies creates unintended and unexpected changes in patterns and nature of work, leading to fundamental changes in organisational schemata observed only in hindsight. effects: more complex digital/human configurations emerge, stimulating new ways of thinking about the nature of work, with impacts on the meaning of work but within an existing frame of reference. effort is necessary at the level of meaning to "figure out" new configurations with existing ones (effort that deals with questions such as "what is work?"). appropriation of layered workplace technologies and the firstand second-order effects create new understandings in the nature of work and shifts in the deep structure of organisations. effects: emergent digital/human configurations challenge the deep structure of organisations, affecting the core of "what" an organisation is. effort is necessary at deeper levels of intentionality and purpose of the organisation to stabilise new configurations with existing ones (effort that deals with questions such as "what is the organisation?"). the hyphen with a forward slash. table 2 summarises the three orders of effects of digital transformation. this perspective highlights that existing research has perhaps been overly focused on capturing affordances and properties of digital tools entering the workplace, and thus focused on first-order effects of these technologies in organisations. meanwhile, we have been underestimating the second-and third-order effects and the work involved in managing emergent digital/human configurations. this may be partially because these deeper effects are only now becoming more visible but also perhaps because our existing theories and methods have limited ability and scope to capture third-order effects (benbya et al., 2020) . we need theories that better recognise the hybridising and mutual shaping between digital and human aspects of work within organisations to study digital transformation; this need is recognised in the recent call for papers for a special issue of jais on "advancing theoretical perspectives on digital transformation" (markus and rowe, 2020) . these new theories need to be bold about agency in the doing of the digital work. with advanced workplace technologies, elements of platforms can be configured and assembled by humans to then have self-learning capabilities creating self-contained modules that perform tasks and operate independently, only requiring limited input from humans to manage exceptions (schuetz and venkatesh, 2020) . at the same time, human agency increasingly moves from task execution to the sharpening and integration of emergent automated routines in the organisation, such as deciding on variables to be monitored, interpreting what variables mean, looking for biases in results, and training the ai. accordingly, theories should allow for more flexible arrangements of agency in these advanced digital workplace platforms. above, we offered our two-fold conceptual perspective for understanding digital work and organisational transformation. first, we highlighted the different layers of evolving workplace technologies that are expected to fulfil increasingly sophisticated functions in organisations: from instrumental to collaborative to intelligent augmentation. second, we suggested that the layers of evolving workplace technologies can have both surface and deeper effects on all types of workers, patterns of work, and the deep structure of organisations. these effects involve the emergence of ever more complex digital/human configurations and new work to manage these configurations. we now demonstrate the usefulness of this conceptual foundation by analysing the studies covered in the four papers in this special issue. we use the concepts previously developed to analyse the papers in this special issue and capture insights on digital work and the effects of digital transformation on the future of organising. we review the papers by identifying the three orders of effects (convergent change, work transformation and organisational transformation) and the emerging digital/human configurations in each empirical study. we then reflect on the work involved in managing the emerging digital/human configurations in the next section. table 3 summarises our review of the studies, with an extended vision of possible third-order effects based on our framework. as summarised in the table above, the four papers in this special issue provide insight into the emergence of digital/human configurations and the three orders of effects of digital transformation resulting from the mutual shaping between organisations and workplace technologies. in the next section, we explore implications of this for organisations and future research. we now discuss three key implications for research and practice of our analysis of the four papers above. we suggest future research to look into this newly emerging digital/human configuration work, from three different but interrelated perspectives: characterising, recognising, and envisioning. first, inherent characteristics of digital/human configuration need to be identified, studied, and refined as a basis for understanding this phenomenon in organisations. second, we stress the need to recognise digital/ human configuration work within the organisation in order to collectively improve configurations that work fairly and effectively. third, we highlight the need to continuously envision digital/human configuration as part of the structural digital work necessary to guide the development of these emergent new assemblages in organisations. • implication and research direction #1: characterising digital/human configuration work as seen, digital/human configurations are assembled arrangements between digital features and human intent and performative actions within organisations. simple assemblages evolve to be increasingly complex configurations as new and more advanced technologies overlay simpler workplace technologies. these configurations emerge initially to enhance and augment work activities but effort to manage them grows more pronounced as they evolve to become more deeply woven into the activities of individuals and the fabric of organisations. this effort demands human ingenuity and innovation to generate, adapt and sustain productive and meaningful digital/human configurations within organisations. our perspective highlights the intensity of the human effort required to create and stabilise emergent digital/human configurations, and the work involved in maintaining these structures. as seen, individuals are critical in this context. producing meaningful assemblages at the local level demands skilful effort to render features of these digital platforms in relation to local tasks and established structures in organisations. crafting these configurations involves effort at the task level (e.g., making sure new tools are integrated into established practices or automated tools perform properly) and effort at the level of meaning and purpose of work and the organisation (e.g., strategic envisioning of new identity and capabilities). digital/human configurations, therefore, involve effort to create incremental improvements in work at the local and individual levels, but also demand structural interventions deeply rooted in core properties of organisations such as identity, values and strategy (stein et al., 2013) . configuration work at this level is often invisible and may not even be recognized by organisational actors as digital/human configurations: new governance processes to moderate and follow feedback and integrate it into processes of strategic planning and development in the organisation. digital/human configurations: social media becomes integrated within the culture and strategy of the association with the intended objective to signal that members own and are able to shape strategy.digital/human configurations: social media becomes integrated within the culture and strategy of the association with the intended objective to signal that members own and are able to shape strategy. convergent change: strategy includes contributions from members and is seen as more open and shared. transforming work: leaders create structures to manage inclusion and transparency in strategy. transforming the organisation: the association gradually becomes seen as more inclusive and transparent. rahrovani: platform drifting: when work digitalization hijacks its spirit a new community platform is created to mobilize and grow community membership in line with the community's values. social media supports community work that used to be done by leaders in closed meetings. this links members to encourage participation and greater inclusion. social media changes what is seen as critical community work, changing the role of leaders as well as the role of members and volunteers. adoption of the platform creates three types of new digital work: strategy work (positioning social media); infrastructure work (redesigning governance and policies); and aligning work (aligning the different logics of community). participation and inclusiveness become integral to the association supported by more advanced features of the platform and the growing community to include outsiders of the original group. the participative nature of the platform gradually drifts the original logic of cohesion towards a logic of inclusion, leading to rethinking the nature and identity of the community. digital/human configurations: social media features become part of the process to manage the growing community. the open and participatory nature of the platforms becomes integral to the culture and functioning of the community. digital/human configurations: the features of the platform influence and become deeply embedded in the emerging inclusive nature of the community.digital/human configurations: the features of the platform influence and become deeply embedded in the emerging inclusive nature of the community. convergent change: the community grows with greater participation and engagement. transforming work: community work is less centralised and becomes more distributed and participatory. transforming the organisation: the association evolves from a position of cohesion to a position of inclusivity, shifting considerably the nature and identity of the community. grønsund & aanestad: augmenting the algorithm: emerging human-in-the-loop work configurations algorithmic data analysis is used to process open real-time data to provide additional market insights and foresight to clients. the company develops their own algorithms to gain these capabilities and retain market position against new high technology competitors. the existing work of the researcher is automated to a significant extent, but new work emerges to interpret and refine the algorithms and clean data. this includes auditing results against manually created 'ground truth' (contextualizing work), adjusting and altering the algorithm, data acquisition and data cleansing. the algorithm becomes an entity with its own properties and status, and has a team working around it to control and enhance it, requiring new processes, roles, capabilities and culture. this is changing the organisational and business model to compete differently in new open data markets. digital/human configurations: automation of domain expert (researcher) tasks to improve speed and accuracy in sorting and synthesizing data into a trade-table, and integration of results in business processes. digital/human configurations: developing and using the algorithm required assembling teams differently and new roles to manage data and interpret results, including a data scientist. digital/human configurations: algorithms become integral to the functioning and strategic direction of the business and organisation. it is now a strategic asset for the business and integral to the market positioning of the organisation. convergent change: algorithmic processing and analysis of data automates the researcher's work of creating trade-tables. transforming work: researcher work is automated and the work to produce trade-tables at the core of the company is now focused on data cleaning and refining the algorithm.transforming work: researcher work is automated and the work to produce trade-tables at the core of the company is now focused on data cleaning and refining the algorithm. transforming the organisation: there is a new vision of what the organisation's core capabilities and valences are, centred around the new algorithmic capabilities. balancing fluid and cemented routines in a digital workplace a new automated process to select staff for projects is 'cemented' to reduce reliance on project managers find that they need to use additional channels to improve the automated staffing erp-based continuous operation of the automated system and workarounds means that the organisation (continued on next page) 'work'; they may, for example, perceive it instead as unsatisfying exception handling, or error detection in the reconciliation of accounts (riemer and peters, forthcoming) . this suggests the need to better understand different kinds of configuration work within organisations, with future research addressing questions such as: how to observe the emergence of digital/human configurations? who is driving the configuration of digital and human aspects of organisations? what effort and skills are needed to manage evolving digital workplaces? how to surface and manage 'hidden' digital work to configure digital/human assemblages? some of these questions challenge basic assumptions underlying existing theories and research on digital work design, including the well-known aegis of the joint optimization of the social and technical subsystems (mumford, 2006) , which requires these subsystems to be separated first, so that they can then both be specified and described. we need more dynamic and evolutionary theoretical views in order to properly understand digital/human configurations. we also need theories that are able to recognise the exponential effects of layered evolution of workplace technologies in organisations. for example, affordance theory, while capturing the relational aspect of configurations, may be too limiting and static in this context of ongoing evolutionary interplay between human agency and self-learning digital capabilities in modern workplaces. despite strides against this shortcoming which we welcome (strong et al., 2014) , affordance theory isn't well equipped to capture long-term effects that are cumulative and uncertain and result from emergent practices. affordances that emerge in third-order effects are not expected or visible in first-order effects. • implication and research direction #2: recognising digital/human configuration work the continuous assembling work involved in digital/human configurations ultimately leads to third-order effects of organisational transformation and therefore to changes in the understanding of work and shifts in the identity and strategic direction of organisations. this type of strategic configuration work is a new type of work that is often informal, so it is not recognised by organisations despite the significant implications for digital transformation. this type of effort is often invisible, overlooked and mismanaged by organisations, contributing to drift and potential increase in organisational risks. we therefore strongly suggest the need to pay more attention to this strategic configuration work by tracing how and who is managing and reorienting the rendering of workplace technologies within the fabric of organisations. for example, research is needed to capture whether, how and why strategic configuration work requires an entrepreneurial mindset that embraces failure (ries, 2017) , a desire to engage in job crafting (wrzesniewski and dutton, 2001) and perhaps even a kind of hacker mentality (coleman, 2014) , where destruction and creation go hand-in-hand. the role of the workplace equivalent of digital intrapreneurs will be important to consider for organisations looking to successfully manage the process of digital transformation. second-order effects: transforming work third-order effects: transforming the organisation employees and make the process more efficient. this new process is meant to support managers that need to quickly staff projects. the system is embedded in the company's larger erp environment. system. this involves considerable effort to interpret the results and find ways to combine other modules to produce a more useful result for them. develops dynamic balancing of enacted routines to create both stability and fluidity in this process. digital/human configurations: project managers use the automated erp staff module to find new people to staff the projects, speeding this process and reducing human effort involved. digital/human configurations: employees appropriate erp features as well as technologies outside the erp, such as social media and other collaborative tools, to create new workspaces that compensate for restriction of the automated erp system. digital/human configurations: automated staffing processes only work with added services and discretion in combining these services by the project manager. convergent change: erp-based workflow automates the staffing process in the organisation. transforming work: new work is necessary to compensate for the limitations of the automated system in finding best local candidates while also realizing corporate strategic goals. transforming the organisation: automation eliminates tasks but adds more effort by project managers. organisation becomes capable of adopting automated services with a degree of flexibility and discretion. emergent themes across the four papers all four papers describe the use of workplace technologies to facilitate or enhance existing forms of work in the organisations. the technologies seem to be fairly effective in moving organisations forward, such as improving participation or task optimization. however, the use of new technologies also requires effort to integrate new technologies in established practices, resulting in intensification of existing work: open strategizing is more demanding than traditional strategizing; growing communities on social media requires more community work; automated algorithms require oversight; and the automation of staffing processes requires corrections. all cases show that the intensification of work then produces unexpected new forms of work. for example, the work to manage communities leads to very difficult conversations about the nature of the community and its culture, as well as very significant governance work to regulate participation and growth of communities. this is also seen in work to integrate algorithms and automated services in organisations, which demand considerable effort to modify and integrate these systems in established work routines. we therefore see digital work design as one of the main characteristics of this order of effect. the effects of changes in work design means that organisations adapt and modify core capabilities and structures and eventually shift the nature of their identity, culture and strategic position in markets. we see these changes across the papers, although this is more evident in some than in others. the kind of work emerging at this level is deep and structural, not anticipated and reactive. this is managerial work, although it is deeply rooted at the local level, and requires effort to understand firstand second-order effects and react to emergent changes. this new work is structural digital work, which is not often perceived or recognised but is visible when, for example, the logic of communities is influenced by digital media, or core capabilities of automation become prime business assets. as seen, strategic configuration work is deeply rooted in the fundamental structures of organisations, so observing this work requires attention to changes in the values, identity, power, core capabilities and strategy of the organisation. we therefore highlight the importance of managerial care and attention to this critical but often invisible configuration work, and the importance of adjusting leadership and management style and approach to be able to steer its effects in organisations. future research could investigate: how to anticipate, sensitise and steer second-and third-order effects of digital/human configurations? how can organisations adjust strategic capabilities to operate in more fluid digital work environments? what governance models allow for more effective appropriation of digital/human configurations? what is the role of leadership in managing evolving digital/human configurations? this calls for more holistic theories that capture and attribute strategic value to the process of blending of digital and human within organisations, so the asset is not the artefact or the human per se, but instead it is how organisation are able to meaningfully integrate the two. this needs better attention and to be seen as having strategic value. it goes beyond views and concepts of alignment and demands attention to second-and third-order effects as the actual strategic impact of this process. having multi-level theories that see strategic leadership as the capacity needed to meaningfully and tactfully steer the process of digital/human configuration work is, therefore, of great importance. • implication and research direction #3: envisioning digital/human configuration work we have also seen that the widening use and adoption of workplace technologies with intelligent and self-contained capabilities is increasing instances where technologies generate work activities for humans in organisations, shifting the remit of these technologies from helping humans to also performing some limited managerial activities. in this context, digital tools produce work activities for humans, creating new and unique digital/human configurations, characterised by dynamics where technology uses humans rather than humans being users of the technology. weaving these configurations within the wider organisation requires higher level management effort to adjust and meaningfully integrate these more advanced digital/human configurations within the organisation, by for example creating work and formal roles to monitor for misfit and repair when misfits lead to mistakes. some of this work may be interesting, innovative and rewarding for humans (such as in analysing business intelligence output for identifying new strategic directions), but there is also less interesting work needed to train algorithms such as cleaning data and moderating content, work that has been described as unglamorous and underpaid 'ghost work' (gray and suri, 2019) . therefore, we should reflect on the design of the digital work of the future with an emphasis on the ethical distribution of work, responsibility and accountability in digital/ human configurations (gal et al., 2020) . future research could, thus, address questions such as: what is the human role in digital/human configurations? what is the quality (as opposed to quantity) of the work being created for humans? how can we avoid creating profoundly unequal tiers of digital work and combat workplace inequity? this type of envisioning, challenges existing is theories because existing theories tend to favour explanation and description, and distance themselves from speculative and normative theorising. for example, sociomateriality and notions of entanglements lack the very long-term perspective needed for studying effects-of-effects, and often focus on the direct result of the social and the material coming together in a symbiotic entanglement, as if they evolved to optimise each other in a mutually benevolent way. the possibility of malevolent and parasitic entanglements is rarely considered. forward-thinking, speculative theorising (peter et al., 2020) may be one way to engage the is field more widely in envisioning the future of work and organisations that humans will like and thrive in. workplace technologies have evolved from basic discrete office applications in the 80s to the connected digital platforms with elements of automation and embedded ai-driven self-learning capabilities in today's digital workplaces (lyytinen et al., 2020) . this evolution in the scope and depth of integration of these workplace technologies into the fabric of organisations has transformed work but also the structure of organisations evolving from the use of these more advanced platforms of work. in this introduction to the special issue, we explore and conceptualise the deep effects of these human-technology configurations (suchman, 2012) in transforming work practices (stein et al., 2013) and the nature of work (lee, 2016b) and ultimately the strategy and structural arrangements of organisations (baptista et al., 2017b) . we were particularly interested in observing effects that had the potential to fundamentally 'rewire' organisations and work environments (watson-manheim et al., 2002; watson-manheim and belanger, 2007) and to explore the potential of workplace technologies to transform organisations such as by stimulating open participation hautz, 2017; denyer et al., 2011) or by being the catalyst for new organisational capabilities (huang et al., 2013 (huang et al., , 2015 baptista and galliers, 2012) . this introduction first conceptualises genres and types of workplace technologies and then analyses the layered evolution of these progressively more advanced and interconnected technologies in the workplace. we describe the nature and distinct characteristics of the individual layer of office tools, and the characteristics of the second layer of community and collaborative workplace technologies. we then discuss the more recent introduction of intelligent platform-based workplace technologies and discuss the distinct and unique dynamics involved in the configuring of these technologies within organisations. we use this conceptual foundation to make an explicit link between these types of work technologies and organisational transformation and offer a richer and more comprehensive view of the role and effects of workplace technologies in organisations. we suggest that previous research has over-emphasised an instrumental view of these technologies by focusing on their capacity to enable virtual work and teamwork, communication and collaboration. we then conceptualise three orders of effects of workplace technologies on organisations based on bartunek and moch (1987) . first-order effects represent convergent change, the expected and direct effects of workplace technologies on the performance of work. second-order effects affects the roles and the nature of work in organisations. third-order effects involve deeper transformation of the organisation and result in structural changes to the core elements of the organisation, such as its identity, capabilities and strategy. we provide a conceptual basis to structure the effects of workplace technologies at these three levels of change. we suggest that this process of transformation involves the emergence of digital/human configurations, reflecting the assembling of digital features with human intent and their performative within organisations. we highlight that simple configurations evolve to be increasingly complex as new and more advanced technologies overlay simpler workplace technologies. correspondingly, as configurations become more complex the effort required to manage them grows more pronounced, however much of this needed effort remains invisible and mismanaged in organisations. more research is needed on the kinds of organisational actors best placed to do this work, the skills they need and how to productively engage with the unpredictable effects of new technologies and the emerging digital/human configurations. we also highlight the importance of normative and speculative envisioning of future digital/human configurations if the is field is to contribute to the creation of a future of work and organisations worth wanting. we sincerely hope that these considerations are useful not only to guide future research but also to help organisations navigate a path towards digitisation beyond the covid-19 crisis. as discussed, any move towards deeper adoption of workplace technologies will inevitably involve work to configure digital/human assemblages; organisations that properly resource this process are more likely to benefit and adjust to the "new normal" which we hope allows society to rethink work and organising for the benefit of all of us. social media and business transformation: a framework for research technology and organizational change: harnessing the power of digital workplace corporate intranet implementation: managing emergent technologies and organizational practices institutionalisation as a process of interplay between technology and its organisational context of use social media as a driver for new rhetorical practices in organisations paradoxical effects of institutionalisation on the strategic awareness of technology in organisations call for papers: strategic perspectives on digital work and organizational transformation social media and the emergence of reflexiveness as a new capability for open strategy. long range plan first-order, second-order, and third-order change and organization development interventions: a cognitive approach complexity and information systems research in the emerging digital world strategizing information systems-enabled organizational transformation: a transdisciplinary review and new directions an institutional perspective on developing and implementing intranet-and internet-based information systems constructing and using a company intranet: 'it's a very cultural thing hacker, hoaxer, whistleblower, spy: the many faces of anonymous managing the crises in intranet implementation: a stage model social", "open" and "participative"? exploring personal experiences and organisational effects of enterprise2. 0 use. long range plan the digital workplace is key to digital innovation working and organizing in the age of the learning algorithm breaking the vicious cycle of algorithmic management: a virtue ethics approach to people analytics overcoming the ''ideology of openness'': probing the affordances of social media for organizational knowledge sharing ghost work: how to stop silicon valley from building a new global underclass opening up the strategy process -a network perspective open strategy: dimensions, dilemmas, dynamics. long range plan how do strategic leaders engage with social media? a theoretical framework for research and practice. strategic manage reconceptualizing rhetorical practices in organizations: the impact of social media on internal communications communicational ambidexterity as a new capability to manage social media communication within organizations falling short with participation -different effects of ideation, commenting, and evaluating behavior on open strategizing. long range plan the evolutionary implications of social media for organizational knowledge management what's in it for employees? understanding the relationship between use and performance in enterprise social software electronic mail as a medium for rich communication: an empirical investigation using hermeneutic bringing government into the 21st century: the korean digital governance experience the impact of ict on work social media and their affordances for organizing: a review and agenda for research enterprise social media: definition, history, and prospects for the study of social technologies in organizations metahuman systems = humans + machines that learn the contradictory influence of social media affordances on online communal knowledge sharing designing for digital transformation: lessons for information systems research from the use of ict and societal challenges electronic mail as the medium of managerial choice call for papers special issue: envisioning digital transformation: advancing theoretical diversity the story of socio-technical design: reflections on its successes, failures and potential artefacts from the future: engaging audiences in possible futures with emerging technologies for better outcomes next-generation digital platforms: toward human-ai hybrids organizational media affordances: operationalization and associations with media use the robo-apocalypse plays out in the quality, not in the quantity of work from top to bottom: investigating the changing role of hierarchy in enterprise social networks the startup way: how modern companies use entrepreneurial management to transform culture and drive long-term growth research perspectives: the rise of human machines: how cognitive computing systems challenge assumptions of user-system interaction fighting against windmills: strategic information systems and organisational deep structures design for social media engagement: insights from elderly care assistance towards an understanding of identity and technology in the workplace a theory of organization-ehr affordance actualization open strategy: literature review, re-analysis of cases and conceptualisation as a practice social media use in organizations: exploring the affordances of visibility, editability, persistence, and association organisational evolution: a metamorphosis model of convergence and reorientation social media affordances and governance in the workplace: an examination of organizational policies understanding digital transformation: a review and a research agenda how does social software change knowledge management? toward a strategic research agenda communication media repertoires: dealing with the multiplicity of media choices discontinuities and continuities: a new way to understand virtual work conceptualizing hidden human work in a technology intensive work environment unpacking the difference between digital transformation and it-enabled organizational transformation crafting a job: revisioning employees as active crafters of their work key: cord-308857-otsrexqu authors: goel, saurav; hawi, sara; goel, gaurav; thakur, vijay kumar; pearce, oliver; hoskins, clare; hussain, tanvir; agrawal, anupam; upadhyaya, hari m.; cross, graham; barber, asa h. title: resilient and agile engineering solutions to address societal challenges such as coronavirus pandemic date: 2020-05-28 journal: mater today chem doi: 10.1016/j.mtchem.2020.100300 sha: doc_id: 308857 cord_uid: otsrexqu the world is witnessing tumultuous times as major economic powers including the us, uk, russia, india, and most of europe continue to be in a state of lockdown. the worst-hit sectors due to this lockdown are sales, production (manufacturing), transport (aerospace and automotive) and tourism. lockdowns became necessary as a preventive measure to avoid the spread of the contagious and infectious “coronavirus disease 2019” (covid-19). this newly identified disease is caused by a new strain of the virus being referred to as severe acute respiratory syndrome coronavirus 2 (sars cov-2; formerly called 2019-ncov). we review the current medical and manufacturing response to covid-19, including advances in instrumentation, sensing, use of lasers, fumigation chambers and development of novel tools such as lab-on-the-chip using combinatorial additive and subtractive manufacturing techniques and use of molecular modelling and molecular docking in drug and vaccine discovery. we also offer perspectives on future considerations on climate change, outsourced versus indigenous manufacturing, automation, and antimicrobial resistance. overall, this paper attempts to identify key areas where manufacturing can be employed to address societal challenges such as covid-19. coronaviruses (covs) belong to the family coronaviridae which includes four genera: α, β, γ and δ as well as several subgenera and species [1] . sars cov-2 is a β-coronavirus with a single-stranded rna genome of ~30 kb [2] . recent topical research has revealed several new covs (three α-coronaviruses, three new βcoronaviruses, and one previously described α-coronavirus) from bats captured from myanmar and future emergence of new diseases caused by these covs due to change of land use has been speculated [3] . furthermore, newer mutations of the virus that originally spread from wuhan were confirmed as deadlier in some countries compared to others, which has led to added confusion and concern [4] . sars cov-2 was first identified from the outbreak of respiratory illness cases in wuhan city in the hubei province of china. initial reports of the virus were made to the world health organisation (who) on december 31, 2019. this was followed by the who declaring covid-19 as a global health emergency on january 30, 2020 due to rapid spreading, and a later pandemic declaration on march 11, 2020. the disease has quickly engulfed most of the world and has caused severe infection to populations across numerous countries as shown in figure 1 . covid-19 is shorthand for "coronavirus disease 2019" which is caused by the sars cov-2 virus, also called coronavirus in typical usage. viruses cause disease by binding to receptors on cells in the human body and then replicating at a rapid rate which triggers a variety of pathogenic processes. in the case of covid-19, the structures on the surface of the virus bind to receptors in the airway or the lungs of human beings. the lungs may become inflamed, making breathing more difficult. for some people, the infection becomes severe and leads to critical care requirements. the anatomy of sars cov-2, including its internal biological structure of spike protein legs, envelope protein, and membrane protein, surrounding the genomic rna is shown in figure 2 (a). the virus shown in figure 2(a) highlights a large diameter of about 0.1 µm with variations in sizes reported by different researchers. the spike-shaped protein legs that make up a portion of the outer capsule of the virus create the crown-like or corona-like appearance. thus, the name coronavirus. the mechanism of infection due to the surface interactions between the spike protein and the lung cells is depicted in figure 2 (b). this understanding was gathered from the genetic analysis which revealed that the mutations located in the spike surface glycoprotein might induce conformal changes and play an essential role in binding to receptors on the host cell (lungs) and determine host tropisms by leading to possible changing antigenicity [7] . viruses are fundamentally different to bacteria so the classically developed work on nano-structuring and biomimicking surfaces, such as the cicada wing, primarily targeting bacterial killing, should not be confused as a readily available knowledge to kill sars cov-2. physically, a bacterium is larger than a virus (the biggest size of a virus is smaller than the smallest known bacteria). bacteria are living cells which are 100±60 nm capable of prolific reproduction independently, whereas viruses are non-living particles, requiring a host cell for replication. also, bacteria possess a cell-wall engulfing a chromosome, whereas viruses consist of genetic material, either dna or rna, covered by a protein coating. bacterial infections can therefore be treated with high success using antibiotic drugs. although some antiviral medicines are now available, the susceptibility of viruses to react to the treatment rate of medicines is significantly reduced as they are non-living. hence, the only long-term option to eradicate viruses is the development of vaccines that stimulate the natural production of antibodies. studies suggest that the source of sars cov-2 could either be ratg13 from horseshoe bats (rhinolophus affinis), pangolins (manis javanica), or a mix of both. their transmission has occurred by zoonotic mechanisms [6, 7] . however, the coronavirus isolated from pangolins is 99% similar in a specific region of the spike protein, which corresponds to the 74 amino acids involved in the angiotensin-converting enzyme 2 (ace 2) receptor binding domain, which allows the virus to enter human cells to infect them as shown in figure 2 (b). the virus ratg13 isolated from rhinolophus affinis bats is highly divergent in this specific region (only 77 % similarity). this observation indicates that the coronavirus isolated from pangolins can enter human cells, whereas coronavirus isolated from rhinolophus affinis bats is unable to enter human cells. the sars cov-2 has a high transmissible efficiency and covid-19 has high morbidity and mortality. a popular but possibly flawed measure for assessing fatality of disease is the use of deaths/case counts. this measure would yield a fatality rate of 6.6% for covid-19 as of 17th may 2020 [8] . the problem with this measure is that case counts reflect the number of tests that were done rather than infections, and the deaths lag the cases because fatality (if observed) may happen several days after the case is identified. a lag in reporting case numbers and incorrect tests may also occur. an alternative measure is the case fatality rate (cfr), which is the ratio of deaths / (deaths + recovered cases). this measure would yield a fatality rate of 14.6% [9] . the consensus is that the covid-19 disease has high fatality and can exceed the fatality ratio of the century-old "spanish flu", which had a 10% cfr [11] . however, the data analysis of callaway et al. [10] shown in figure 3 suggests that covid-19's cfr is lower than that of mers and ebola and that its infection rate (r0 -the expected number of cases directly generated by one case in a population where all individuals are susceptible to infection) suggests that the infection can spread more easily than other diseases, including seasonal influenza. a further comparison of three different episodes of epidemics and pandemics caused by the family of coronavirus, namely, sars cov-1, mers and sars cov-2, is depicted in table 1 . the exact mechanism of these drugs towards the virus is yet to be completely understood. however, the mechanism where a drug affects the replication of viruses in-vivo is a broadly accepted concept. in general, viruses replicate via protein processing using endosomes within golgi bodies. a drug such as hcq increases the ph of the golgi bodies making them more basic, thus disrupting the integrity of the internal nucleocapsid protein (see figure 2a ). this denatures the protein of the coronavirus rendering it dysfunctional. therefore, the rationale for using hcq is built on the premise that the change in ph brought about by the drug inhibits the endosomal transport necessary to spread the infection, hence the patient recovers. another short-term treatment being implemented by some hospitals across the world is to infuse patients with the antibody-rich blood plasma of people who have recovered from covid19 [13] . this approach has been used during disease outbreaks for over a century. however, the approach carries significant risk in terms of the already immunocompromised patients' immune response after administration, and results may vary between patients. as an early effort of investigation of the problem from scratch, researchers at toho university in japan used high-sensitivity cameras and laser beam guidance experiments to deduce that saliva spray during a sneeze (potentially containing thousands of viruses) can be classified into large vs small droplets or droplets vs aerosols. the droplets, due to their heavier weight, fall off, whereas aerosols remain airborne for a few hours due to their relatively small size. prima facie evidence suggests that the coronavirus has escalated to a pandemic due to the high contagion occurring via this 'airborne' spread model. recently, the possibility of asymptomatic or oligosymptomatic infection has also been highlighted [14] . the aerosols may circulate near an infected patient in an airborne condition depending on the local conditions (airflow rate, humidity, dryness) for up to a few hours, even after the infected person has left the location. hence, the chances of contracting coronavirus are relatively high by merely occupying the same vicinity where an infected person has been or passed through. researchers have experimentally evaluated the stability of sars cov-2 recently, and these comparisons are shown in figure 4 [15, 16] . it is noteworthy that sars cov-2 was stable in the aerosol (airborne) form for up to 3 hours. as opposed to this, the virus appears to be stable on surgical masks or stainless-steel surfaces for up to 7 days and for up to 4 days on smooth surfaces such as glass or currency notes. the same research also shows that the application of hand soap does not immediately deactivate the virus but rather takes up to 5 minutes. therefore, a 5-minute waiting period after a handwash is required before bringing hands in contact with face, mouth or nose. covid-19 is not only present in the airway secretions of infected patients when they sneeze, but also when they simply breathe out or speak. studies have shown that covid-19 is also present in other body fluids of infected people, such as faeces, blood [17], oral fluids, anal secretions [18] , tears [19] , and urine [20] . the virus primarily attacks the respiratory system, however, new evidence shows the virus is not filtered by the kidneys, as traces of virus can be seen in sewage systems [8] . therefore, as a mitigation strategy, testing of these fluids, which can be available and abundant in the wider population can be done for identifying the most effective areas. overall, when people are in close contact with one another, then transmission is more likely. most sources for infection worldwide have happened in an enclosed space, including homes, offices, public transport and restaurants. a second spreading pathway for the virus is through touching a surface or object that has the virus on it and then touching one's own mouth, nose, or eyes. if one is in a wellventilated space with fewer people, even for a longer period, the risk of infection is low. sustained contact with an infected person, even for a short period, even without a cough or sneeze, can spread the infection. by the virtue of disturbing the cell programming, covid-19 possesses the capability to cause a surprise attack on almost any part of the body ranging from lungs, heart, blood vessels, brain, eyes, nose, liver, kidneys and intestine. a schematic representation of this adverse impact is shown in figure 5(a) [21] . recent literature suggests that apart from the airway, the human brain may also be targeted by the virus -see figure 5 (b) and figure 6 [22] . patients have reported having mild (anosmia and ageusia) to severe (encephalopathy) neurological manifestations, and if true, it makes sars cov-2 more lethal. [21] (b) tissue distribution of ace2 receptors in humans [23] (c) possible neuro disorders due to sars cov-2 [22] and (d) life cycle of sars cov-2 in host cells [24] . (figures reprinted with permission) our nasal lining tissue contains a rich number of cell receptors called angiotensinconverting enzyme 2 (ace2), which are favourable sites for the sars cov-2 to attach its spiked protein to, thus paving way for the entrance of the virus inside the body. once attached to the cell, sars cov-2 can change cell programming, replicate itself, and attack new cells at a very rapid pace. within a week of entrance into the body, the virus exhibits effects. during this time, the person may show early symptoms such as fever, dry cough, sore throat, loss of smell and taste, or head and body aches. at this point, the failure of the immunity in defeating the virus means giving the virus a way forward to enter into the respiratory system, where the lungs are attacked. the lungs also have a cell lining rich in ace2 [22] . as the immune system continues to fight the sars cov-2, the supply of healthy oxygen is disrupted. as the disease progresses inside the body, the white blood cells release inflammatory molecules or chemokines, which attack and kill the virusinfected immune cells, leaving the dead cells and pus behind. this affects healthy oxygen transfer in the lungs and is the stage where the patient starts to complain of pneumonia, coughing, fever, and rapid, shallow respiration. acute respiratory distress syndrome may develop. at this stage, the lungs start to be filled with opaque black spots signifying closure of the air pores, and such patients require ventilator support. the survivors of this severity of infection can end up having long-term complications. a few hospitals have successfully applied artificial intelligence to monitor the recovery rate by monitoring the coronascorea general term used to quantify the extent of the blocked pores (in cm 3 ) [25] long-term research measures, advanced manufacturing and metrology have pivotal roles to play in containing a pandemic. specifically, the development of engineering innovations is timely for the control of the covid-19 pandemic. for diagnostic testing to be useful for informing doctors and governments about the true incidence at any one time of coronavirus in the population, it needs to be inexpensive, high in sensitivity and specificity (sensitivity refers to the ability of a test to correctly identify those with the disease or true positive rate, whereas specificity refers to the ability of a test to correctly identify those without the disease or true negative rate) and easy to use for non-experts. as doctors are treating infected patients in this emergent time in an unprepared state, emergent manufacturing measures can help design better testing equipment and thus help in both short and long term to tackle this problem. the most significant challenge now lies in probing early-stage symptoms of covid-19. for a relatively late stage detection in severe cases, chest computer tomography (ct) using x-ray probes (providing >90% sensitivity) has proved to be a more reliable test assay in comparison to the reverse-transcription polymerasechain-reaction (rt-pcr) test and other sensor-based detection methods currently being pursued [27] . since the nervous system and respiratory dysregulation are both likely to co-exist, covid-19 testing should be done by combining brain mri and rt-pcr tests as shown in figure 7(a) [28] . however, caution is required in the analysis of results, such that covid-19 should not be confused with drug-resistant tuberculosis (tb) as both would show symptoms of damages in the lungs. in an attempt to boost the testing in a populous country such as india, the indian council of medical research (icmr) has approved the use of diagnostic machines used for testing drug-resistant tuberculosis under the guidance that the throat/nasal swabs are collected in the viral transport medium [29] . interestingly, the pcr test cannot identify asymptomatic infections or those people who were exposed to or infected with covid-19 in the past and did not suffer from the disease or have recovered from covid-19 and may still be spreading the virus (carriers). therefore, different tests may be required to collate vital information required by government bodies to carry out a risk-benefit analysis to relax lockdown measures to protect their economies. immunodiagnostic kits such as a lab-on-a-chip are being developed for asymptomatic detection of covid-19 [30] . a lab-on-chip (figure 7 (b) works on the principle of detecting the presence of patient-generated antibodies against the virus that causes a specific disease, in this case, covid-19. the test uses a lateral flow immunoassay to assess the presence of an analyte from a patient sample or specimen and detects two types of antibody isotypes: igm and igg. igm antibodies are the first antibodies to appear in response to a novel antigen and imply a more recently initiated infection while igg antibodies are generated later in the course of infection and possess a higher affinity for the target antigen, meaning they are more specifically able to bind the substance which caused the immune response. a test is declared positive if either one or both antibodies are detected during the test, similar to widely used pregnancy tests. the test consists of an anti-human igg coating in the g test line region and an igm coating the m test line region (see figure 7 (b). during testing, the sample (blood, urine etc) reacts with sars-cov-2 antigen-coated gold nanoparticles (aunp) in the conjugation pad of the test cassette. any antibody in the patient sample that recognises the sars-cov-2 antigen binds to the antigen-aunp complex. the mixture then migrates laterally across the membrane by capillary action/lateral flow. as these human antibody/antigen/aunp complexes move across the test lines, they are captured at the anti-human igm 'm' line, the anti-human igg 'g' line, or both, depending on the antibody contents of the specimen. the sample first reaches the anti-human igm antibodies which coat the m line. if the specimen contains igm antibodies to sars-cov-2, a coloured line will appear in the m test line region. next, the sample reaches the anti-human igg antibodies which coat the g line. if a specimen contains igg antibodies to sars-cov-2, the conjugate-specimen complex reacts with anti-human igg. a coloured line appears in the g test line region as a result. the rabbit igg-aunp complexes are captured by the control line (which contains anti-rabbit-igg). this visible line indicates that there has been successful lateral flow across the detection strip. the last check ensures that the sample had enough volume to move across the entirety of the test cassette. only human antibody/sars-cov-2 antigen/aunp complexes will produce a visible red or pink line at the m or g line. other antibodies produce no colour. the accuracy of these lab-on-chip tests is still being debated as it depends on two key parameters, sensitivity and specificity. to date, igg related sensitivity and specificity has been found to be higher than that of igm. a schematic diagram in figure 7(c) and figure 7(d) shows that unlike currently available diagnostic methods, field-effect transistor (fet)-based biosensing devices may have several advantages, including the ability to make highly sensitive and instantaneous measurements using small amounts of analytes [31] . the fet sensor shown in figure 7(d) makes use of a graphene sheet since graphene-based fet biosensors can detect surrounding changes on their surface and provide an optimal sensing environment for ultrasensitive and low-noise detection. from this standpoint, graphene-based fet technology is attractive for applications related to the sensitive immunological diagnosis. graphene as a sensing material is selected, and sars-cov-2 spike antibody is conjugated onto the graphene sheet via 1-pyrenebutyric acid n-hydroxysuccinimide ester [31] (figures reprinted with permission) a group of researchers at mit and harvard are developing coronavirus-identifying sensor embedded face masks. this ongoing work is an extension to their previous work where they developed a low-cost method to detect a zika virus [33] . in this technique, the sensor is made by using genetic material such as the rna or the dna which binds to the viruses. the researchers used a lyophilizer to freeze-dry the genetic material onto the fabric of the mask. the material deposited onto the fabric remains stable for many months (at room temperature) and the detection process starts merely by the presence of moisture (such as saliva). the detection signal is small (in terms of voltage) and can be detected by an additional fluorimeter device that can quantify this signal and emit in form of a fluorescent light. thus, one can expect to see light glowing masks to detect coronavirus in the future. fumigation chambers allow quick disinfection of a person while visiting areas such as hospitals, universities or airports. the aim is to use tubes releasing hydrogen peroxide in a chamber designed to be five-feet wide and seven-feet tall. the fumigation chamber usually seals automatically after the person's entry to avoid any leakage outside of the chamber and has designated sensors facilitating the chamber entry. the disinfection lasts for five seconds. it is to be noted here that hydrogen peroxide has also been recommended by the food and drug administration (fda) to be used as a sterilisation material to decontaminate n95 or n95-equivalent respirators for reuse by health care workers in hospital settings [34] . a populist view is that sunlight and high temperature during peak summers kills the coronavirus, which would help containment of its spread. it is reported that sars cov-2 does not survive beyond 5 min after being exposed to 70ºc [16] . however, it has yet to be ascertained as to how long does sunlight take to deactivate sars cov-2 and at what intensity. a more specific question is whether ultra-violet (uv) light can kill coronavirus? sunlight usually contains three types of uv: uva (320-400 nm), uvb (280-320 nm), and uvc (200-280 nm). uva and uvb can both cause sunburn, however, uvc is shorter and a more energetic wavelength of light. while uvc is effective at destroying genetic material, whether in humans or viral particles, it is filtered by the ozone layer and does not reach the earth's surface. preliminary findings from the national biodefense analysis and countermeasures centre of the usa (which houses a bsl-4 level lab) indicate that "sunlight seems to be very detrimental to the virus… within minutes, the majority of the virus is inactivated on surfaces and in the air in direct sunlight." [35] . research on the use of uv as a treatment is still evolving and the behaviour of sars cov-2 under uv light is unknown. results were relatively favourable for sars cov-1 treatment with uvc [36, 37] . sars cov-1 was efficiently inactivated after 40 minutes of uvc exposure (at about a wavelength of 254 nm), whereas addition of psoralen (a light-sensitive drug) to uva enhances inactivation of the sars cov-1. popular press reports suggest that uv light booths are capable of deactivating coronavirus without any human contact and are proposed to be deployed ( figure 8 ). one must be cautious, since uvc can be dangerous to the skin, causing burns within seconds and harmful to the eyes if observed directly. therefore, risk assessment and associated precautions would need to be deployed that may render this strategy difficult in the short-term. 3.5.1. antimicrobial coatings chouirfa et al. [39] summarised nanomaterials based coatings for antibacterial applications and xing et al. [40] have summarised the potential of natural polymer chitosan as an antimicrobial agent ( figure 9 ). their research on antimicrobial properties of a nanocomposite coating formed by polysaccharide 1-deoxylactit-1-yl chitosan (chitlac) and silver nanoparticles (nag) on methacrylate thermosets showed satisfactory results. figure 9 : antimicrobial mechanism of chitosan-based coating with antimicrobial agents. reprinted with permission from [40] . antimicrobial surfaces are based on three main mechanisms (see figure 10 ): the anti-biofouling mechanism which repels microbes and prevents them from adhering to the surface, the release-killing mechanism where microbes are killed in the nearsurface environment with a release of antimicrobial agents and the contact-killing mechanism where microbes adhere and are killed on the surface [41, 42] . researchers have also experimented with developing long-lasting antimicrobial surfaces to stop spreading pathogenic microbes through commonly touched surfaces or at-risk surfaces and have focused on the use of antimicrobial/antiviral materials such as copper. experimental trials for copper oxide impregnated respiratory protective facemasks have yielded 100% efficiency in eradicating the infectivity of human and avian influenza a virus in simulated breathing conditions [43] . warnes et al. [44] have shown that the surfaces of dry copper alloys are lethal to viruses such as mnv-1 at room temperature, with higher copper content being more time-efficient. generally, the antimicrobial activity of copper is attributed to oxidative behaviour of copper and the solubility properties of copper oxides [45] . more recently, surface texturing of copper via cold spray methods has shown enhanced promise as an antiviral agent [46] . combining the aforementioned antimicrobial contact-killing properties of copper, the anti-adhesion properties of polymeric micelles and the release-kill abilities of chlorine dioxide (clo2), li et al. [47] developed a multifunctional coating viricidal for influenza virus h1n1. this multifunctional coating is composed of clo2 encapsulated in polymeric micelles (with slow on-demand release) on which copper nanoparticles were covalently tethered. other studies have investigated hydrophobic polycationic coatings as antimicrobial coatings [48] [49] [50] . hsu et al. [51] investigated the mechanism by which the n,n-dodecyl,methyl-polyethelenimine (pei) coated surfaces killed the influenza a virus. it was concluded that upon contact with the coated surface, the virus adheres irreversibly and through hydrophobic and electrostatic interactions, the virus' disintegration is then initiated resulting in rna leakage into the solution. the incorporation of pei into protective mask textiles has also been investigated with nearly a 1000-fold improvement in the capture of the t4d bacteriophage virus of escherichia coli b [52] . finally, the direct mechanical action of sharp nanostructures such as darts, blades and spikes as a kind "mechano-cide" has been shown to have some success for bacteria, but remains largely unexplored for viruses [53] . from a contact mechanics perspective, the combined effect of sharp ( somewhat surprisingly, while researchers have investigated bactericidal properties of nanoparticles of silver, yet the current understanding of the interaction of these nanoparticles with viruses is limited. however, some positive results are reported and, based on these reports, this direction of manufacturing research holds a good promise to tackle sars cov-2. previous studies showed that the size of nanoparticle is critical to manifest a viricidal effect. for example, a nanoparticle size of less than 25 nm was found to be effective against tacaribe virus [56] , while a particle size of between 7 to 20 nm worked well against herpes simplex virus (hsv) type 1/2 and human parainfluenza virus type-3 [57] . nakamura et al. [58] reported that materials with immobilised silver nanoparticles possess enhanced microbicidal activities against the virus. these researchers developed a material using silver nanoparticle absorbed on a chitin sheet having a nanoscale fibre-like surface structure shown in figure 11 and obtained favourable results against h1n1influenza a virus. figure 11 : the mechanism of viricidal activity of silver (ag) nanoparticles (np) chitin nanofiber sheets showing strong antimicrobial activity via reactive oxygen species (ros) and silver ions on the substrate. reprinted with permission from [58] . moreover, the unique characteristics of metallic nanoparticles such as high surface to volume ratio, surface-enhanced raman scattering and localized surface plasmon resonance can be utilised for virus detection and therapy via destruction through laser-induced localised hypothermia as well. some of the candidate nanoparticles are metallic and high entropy alloy nanoparticles (agauptpdcu high entropy nanoparticles, feo nps, ag nps, tio2 nps, au nps, ag-au core-shell nps) [59] . for this purpose, uv-visible spectroscopy can be deployed to study the surface plasmon resonance, refractive index, and fluorescence change when nanoparticles interact with virus particles. nanoparticle-based investigation can help detect and inactivate viruses (figure 12 ). specific to coronavirus, graphene oxide sheets with silver nanoparticles (go-ag nps) were reported to inhibit the growth of feline coronavirus (fcov) by up to 25%, in comparison to pure go that inhibited it only up to 16% [60] . additionally, chiral biosensor with self-assembled chiral gold nanohybrids (cau nps) on account of multiple plasmonic scattering showed better detection performance on coronavirus [61] . figure 12 : nanoparticle based therapy for sars cov-2. reprinted with permission from [59] as with all fundamental and translation nanomaterial research, progress is not immediate. caution is required with nanomaterial technologies, as the long-term impact on human health and the environment of free nanoparticles has not been ascertained, and this may ultimately pose greater risk, particularly in the liver and respiratory tract of human beings and in ecosystems. lack of governmental regulation strategies for nanomaterials can also hinder the speed of approval and ultimately the availability in the marketplace. nevertheless, a call has gone out to nanomedicine researchers to utilise their existing knowledge base and translate their technologies towards covid-19, should the outbreak last more than 12 months [62] . filtration efficiency is a strong measure of penetration prevention of aerosols through mask filters and is usually required to be above 98% for surgical face masks [63] . ultrasonic welding is usually deployed to produce filters with sufficient filtration efficiency. both polymer and textile-based masks are popular and can benefit from the adoption of sequential micromachining techniques [64] . a surgical mask (procedure mask) is worn by health professionals during surgery to avoid exposure to aerosols. such masks are not designed to protect the wearer from inhaling airborne bacteria or virus particles and are less effective than respirators, such as n95 or niosh masks which provide better protection due to their material, shape and tight seal. the who laboratory biosafety manual necessitates biosafety level 2 (bsl-2) requirements for non-propagative diagnostic laboratories and bsl-3 for laboratories handling high concentrations of live sars-cov-2. according to the manual and the who biosafety guidance for sars-cov-2, the exhaust air from such laboratories should be discharged through high-efficiency particulate air (hepa) filters. it is worth mentioning that particle collection efficiency of such mechanical filtering methods decreases to about 50% at particle sizes of 0.5 µm due to diffusion and diffusioninterception regimes of particles with sizes in the range from 0.05 up to 1 µm [21] . therefore, hepa filters are not ideal in screening the viruses like sars cov-2 which has a typical diameter from 60 nm to 160 nm [22] . mass production of appropriate filters to screen the virus entry is a micromanufacturing challenge. this could potentially involve technologies such as direct laser micromachining or punch-based microstamping methods. for this purpose, a plasma spraying, or laser micromachining method may be used to obtain a well-suited punch which can be used to stamp (pierce) polymer sheets (e.g., pet), to achieve appropriate filter sizes. another candidate process of subtractive manufacturing is metal anisotropic reactive ion etching with oxidation (mario) [65] . an illustration of how the proposed micromachining strategies can be deployed for scalable fabrication of filters to screen virus scale particles is shown in figure 13 . while self-assembly techniques such as block copolymers have shown promise filtering small viral particles such as human rhinovirus [66] , direct printing methods allow greater engineering control over pore size and spacing, critical to tuning membrane efficiency, fluid resistance and mechanical strength. protection of surrounding or nearby people is important and an infected patient can help to control the spread by wearing a mask, leading to the concept of social distancing suggested by various governments. the safe distance guideline is an important concept especially in populous countries where an assembly of people on the streets can cause the spread to grow exponentially. keeping this in mind, the who urged people to maintain a safe social physical distance of about 3 ft (~1 m), while the centre of disease control and prevention recommends this to be 6 ft (~2 m). these limits are now challenged by recent research that suggests this safe distance should be 23 to 27 ft (7 to 8 m) [69] . the same study also suggested that the currently available commercial masks need to be redesigned. it was suggested that the masks available at present are not suitable for containment of sprayed aerosols (during a sneeze) travelling at the velocity of 108 kmph [70] , as this increases the possibility of escape of viruses through contaminated droplets from edges of the mask making nearby people more vulnerable to contracting the coronavirus. also, chin et al. [16] tested the strength of the virus and suggested that the virus is highly stable at 4ºc as well as in the ph range of 3 to 10. the virus was found to be detectable on a mask surface even after 7 days. these findings suggest a product design strategy to develop more appropriate masks for handling exhalations travelling at high speeds by considering human ergonomics, material aspects, antimicrobial nature and structural aspect. while the design and strategies for developing these new types of masks are underway, a possible technique for deploying the extant fabrication methods would be to merge the two approaches, namely, additive and subtractive manufacturing methods described above. this development would mean that even in the current landscape, a new mask-making strategy would provide more protection than the currently available masks. astm f2100-07 is the relevant international standard that details specifications of materials to be used in medical face masks. however, the current standards do not capture the techniques required to make the mask reusable especially as the current materials are yet to be tested against sars cov-2. as of now, curad antiviral isolation masks making use of a cocktail coating (citric acid, zinc and copper) are available as an option to disinfecting the outer surface of the mask. these are useful for medical professionals as they can come in contact with the virus infected aerosols more frequently. another concept is that of a 'germ trap' surface [71] , which is akin to fooling a virus. the team at the university of manchester, uk identified specific glycoproteins that have carbohydrates attached to their surface, similar to those seen on the surface of the cell of the nasal passages. this study suggests that a glycoprotein biocoating on a snood surface [72] denatures a virus by causing the same interfacial chemical reaction between the spike proteins and carbohydrate cell surface as the one when the coronavirus attaches to the ace2 receptors, but due to unavailability of any physical cells to invade on the snood surface, the virus decomposes and becomes deactivated immediately after landing on the textile surface (see figure 14 ). the efficiency of germ trap coating in achieving its goal is indicated to be 96%. based upon the existing literature, an immediate measure to functionalise the personal protective equipment's (ppes) could be to introduce the use of antimicrobial coatings comprising of proven materials (e.g., zinc and sodium [73, 74] based compounds) on the surface of ppes, especially the shields and visors. such coatings can be deposited by additive methods such as spraying (in 3d) or via roll to roll (r2r) manufacturing. in terms of processing techniques, one limitation of thermal spraying is that it requires heat resistant feedstock materials, limiting the use of most of the chemical species traditionally used to functionalise surfaces, such as polyethylene glycol (peg) [75] . in view of this limitation, a possible research avenue is to develop functional coatings with viricidal properties such that the coating ingredients do not degrade at high temperatures, thus providing a controlled release over time. with this goal in mind, a suitable processing and material matrix availability is crucial to obtain a coating to possess desirable advantages in the medical field. on the other hand, the use of thermal sprayed hydroxyapatite (ha) coatings on orthopaedic implants has been widely adopted in the medical field since the late 1980s [76] . as reported in the early 2000s [77] , the use of thermal sprayed ha coatings on metal implants presents several advantages and a positive potential for its use in the medical field [78] . taking into consideration the status of ha as the standard in thermal sprayed coatings and the decades of research on its behaviour and response to living tissue [79] , the choice of ha as a base material for the development of anti-microbial functional coatings has been most popular to date. at a personal level, deploying smart ppe incorporating viral detection capability into masks, gloves or wearing "viral dosimeter" badges might help monitor the strength of direct transmission vectors as well as environmental exposure rates and accumulation. conversely, masks could collect exhaled viral particles for early detection of infection [70] . detection could be implemented as microfluidic channels functionalized with bioelectronic miniaturized detection schemes [80, 81] with specific virus sensitivity [82, 83] . local area smart sensing filter textiles incorporated into mobile or in-place ventilation systems designed to both filter and detect could also help provide early alert alarms and protection in buildings and other areas of restricted air replacement. the importance of wearing a mask by the infected person and by a healthy person coming in the vicinity of the infected person is ranked on the priority order shown by the cartoon model in figure 15 . safe disposal of materials used in the treatment of infected patients such as gloves, masks, test samples, clothes or the human waste, especially in icu patients who may be closer to death is a challenging task. in addition to the possibility of an infection, this waste could lead to other secondary issues including emergence of a new category of virus/bacteria. hence, safe disposal of biomedical waste is very important. additionally, gaining support from members of the public to adequately dispose of their used personal masks etc. is essential. recently, an absorbent gel with embedded disinfecting material has been developed at sree chitra tirunal institute for medical sciences and technology, india for liquid respiratory and other body fluid solidification and disinfection for the safe management of infected respiratory secretions. this material helps to solidify the liquid respiratory secretions from icu patients or those with copious secretions treated in the wards. the material so collected becomes fit for disposal through the usual incineration system for biomedical wastes. newer guidelines are required to refine the existing risk assessments procedures [84] . big data analytics and extant research utilizes accelerated development of techniques for data mining, machine learning and use of artificial intelligence (ai) that shows promise in the treatment of sars cov-2. as a direct benefit to this approach, a digitalised shadow of the data to study plausible scenarios of certainty of an event becomes easier. as for sars cov-2, genome data now exist offering scope for soft-computing application researchers as well as those involved in ai based research to offer new insights into the area. as an example, the global initiative on sharing all influenza data (gisaid) database (https://www.gisaid.org), made available in march 2020, contains a compilation of over 24,000 sars-cov-2 complete and partial genomes from all over the globe since december 2019 [85] . recently, researchers from cambridge used phylogenetic network on 160 complete genomes of sars cov-2 [85] to predict probable mutation and migration paths of the coronavirus. atomistic modelling and molecular docking [86] can hold the key to a scientific breakthrough to address questions surrounding sars cov-2. the development of vaccines, antibodies and diagnostics is dependent in a large measure on our understanding of the interfacial science between the spike (s) glycoprotein of sars cov-2 and ace2 receptor in human lung cell (as shown earlier in figure 2 ). wrapp et al. [87] obtained a cryo-electron microscopy structure of the sars cov-2 s trimer in the metastable prefusion conformation, showing it undergoes a transient (hide and active states) structural rearrangement. their results relying on surface plasmonic resonance elucidated a stronger affinity of sars cov-2 with ace2 compared to sars cov-1, and shed light on two states, namely, a "down state" or receptorinaccessible and an "up state" or receptor-accessible state shown in figure 16 in yet another interesting study [89] , computer-aided drug screening was used to run a test assay over 10,000 compounds as inhibitors to a key cov enzyme, m pro . ebselen was found to exhibit antiviral activity and illustrate a way forward use of this potentially promising modelling strategy to discover targeted drug and vaccine down: transient hidden state of rbd up: active binding state of rbd development. a molecular docking example shown in figure 16 (b) is yet another effective example of accelerating the drug discovery [88] . recently, liu et al. [90] carried out an aerodynamic analysis by measurement of the rna of sars cov-2. they used pre-sterilized gelatin filters (pore size of 3 μm) to collect the aerosols from different areas of two wuhan hospitals. this study classified the sampling location in three areas: ( surprisingly, the researchers detected maximum stains of sars cov-2 in patients' toilet areas, while the levels were low in the isolation wards and ventilated patient rooms. also, the medical staff areas showed peaks of high concentrations with aerosol size ranging from 0.01 micrometres to 2.5 micrometres and they were neutralised after rigorous sanitisation procedures. the study suggests that there is a likelihood of resuspension of virus-laden aerosols from the surface of medical staff's protective equipment including surgical masks during their removal while having lunch, visiting toilets or breaks etc. the source of these virus suspensions was speculated to be from the direct deposition of patient's respiratory droplets or airborne sars-cov-2 onto the protective apparel. the researchers also found that the sars-cov-2 in aerosol forms had relatively longer residence time, implying the infection remains for a relatively longer time causing further transmissions. this study serves as a practice guide on the requirements and specification after the lockdown procedures are eased to avoid recurrent infection. the exit-strategy after the lockdown must consider carefully the ventilation of offices/classrooms, maximum use of open space (preferable sunlight), regular sanitisation of clothes, hairs and hands, and proper use and disinfection of toilet areas. the data obtained by toho university (figure 17) suggests that laser technology can be employed efficiently to monitor the streamlines and microdroplets movements from a sneeze. this monitoring could be a highly effective lab-scale activity to not just guide the futuristic cfd models but also to efficiently design new masks effective for preventing diseases caused by high velocity spray caused by a sneeze. while engineering and manufacturing will undoubtedly play their part over the coming years, the interventions proposed herein can help avoid the recurrent spread and emergence of new infections. the patients who have survived while battling against covid-19 showed that they have developed antibodies to the virus. antibodies are virus-specific proteins, which have a memory of the exposure to the virus and will recognize the virus on a second exposure. antibodies help prevent future infections by detecting the virus and binding to their surfaces signalling the body's immune system to destroy such viruses or virus-infected cells. while reasons are not clear, common wisdom is that the human body does not have an adequate immune response to hiv. for covid-19, an adequate immune response does exist. therefore, a vaccine is more likely to be created successfully for sars cov-2. additionally, the observation that a large majority of people recover, either without any symptoms or with minimal symptoms such as fever and body ache, also bodes well for the development of a vaccine. three major scenarios are currently being pursued or envisaged in tackling the disease. (i) research and trials on vaccines: clinical trials are being conducted across the globe into novel vaccines and fast-tracking approval processes are being deployed. however, scientists fear that the virus itself, like the flu, can mutate and form different strains. therefore, immunity after immunisation or contracting and surviving covid-19 may only last as short as two years. (ii) therapeutic trials using available drugs: attention is being focused to drug repurposing and advanced formulations, including antimalarial, hiv, and other antiviral and immune-modulating drugs and biomolecules. (iii) nanotechnology: study of molecules that show promise but exhibit poor physiochemical properties or toxicity is currently being formulated across the globe into advanced nanomedicine platforms, translating the knowledge gained in drug targeting and efficacy enhancement from conditions such as cancer and cardiovascular disease. such technologies aim to provide better treatment prognosis for those patients who fall ill with coronavirus related diseases. attention to immunemodulating molecules is growing, as patients who contracted severe forms of covid-19 were reported to experience notorious cytokine storms which ultimately 2 m 1 m lead to their death. ability to suppress such immunological responses could be one key to controlling the progression of the virus from mild to severe infection. lessons learned during this pandemic will also impact the outlook for other viruses. these include governmental pandemic funding allocation, stockpiling of medical protective equipment and emergency aid strategies within and between countries across the globe. as the coronavirus problem increased across the globe in 2020, many questions have been raised regarding what more governments could have done, why they did not act more quickly, and what were the fundamental failures within their policy. over time, addressing these issues will pave the way for a more streamlined and effective future response. in addition to this, contact tracing technology is being heavily invested in across the globe, with the intention that citizens would be able to download mobile applications, which could inform them (and the authorities) if they had been exposed to another person who tested positive to covid-19. whilst early trials seem positive, the longer-term question remains over privacy and ownership of personal data collected by such applications. access to death records inside countries and across continents will allow identification of high-risk population categories which will aid risk prevention. the positive news is that this pandemic has cemented the importance of scientific integrity, the inclusion of scientific advisors into government, and the role of science in society. while this may offer little solace at such an upsetting time, yet, with the power of science, this disease will be managed unlike many other viral threats over the years. currently, vaccine trials are ongoing at various phases of progress in different countries (see table 3 ) and good news may be expected soon. [95] . the world has seen a surge in the use of digital tools being growingly used for teaching, research, consultation, banking and day to day activities. digital health innovation has been a prime example. these approaches culminated in the development of telemedicine consultation. the current situation has also driven many changes in work-life routines such as less travel and digital technology-based remote work. the coronavirus pandemic has brought significant disruption and caused severe emotional, sentimental and financial losses. there is even a chance that life on the planet will never be the same again and many stringent safety measures will continue to be followed in the time to come while using public transport, air travel, or while visiting touristic places. the continued extension of lockdowns (e.g., lockdown 1.0 to lockdown 4.0 in india) has resulted in making people restless when staying indoors. countries who were not very resilient in adapting to the situation even saw disrupted supply chains, disturbed migrant workers, lack of food and other supplies and unavailability of essential items -a situation comparable to a natural disaster. however, every disaster brings forth a new cycle of life and the episode of covid-19 did bring some good things. countries facing severe pollution in air, water, and land saw a life not seen in the last many decades. social websites and digital tools were used heavily to share the beauty of nature. rivers got cleaner, the air became more breathable, and the sky got clearer [96] . as a result of this transition, even climate change and transition in weather were observed in many places and it will be befitting to say that we witnessed how "nature self-heals and mends itself". a prime example of climate change as a result of lockdown can be seen in china, europe and in india (see figure 18 ). india has faced two major challenges for many years in the wake of rapid urbanisation: alarming levels of air pollution and a rapid decrease in the availability of clean water, especially from rivers like the ganga. the government of india had earlier set up a mega project called "namami gange" [97] for freeing the rivers from industrial wastes. the project is of such importance that when addressing the indian community at madison square garden in new york in 2014, the prime minister of india mr narendra modi had said, "if we can clean the ganga, it will be a huge help for 40% population of the country." covid-19 has rejuvenated the ganga and many other rivers around the world, making them cleaner to a point that their water was reported to be potable [98] . the learning from the episode of covid-19 has been that resources offered by mother nature are meant to be used and not to be "exploited". if this simple rule is forgotten, then the world may continue to witness a regular self-healing cycle. on this occasion it was covid-19 but the next time, it could be readjustments in the ecosystem caused by a surge in the carbon emissions. researchers are reporting a decrease in solar activity leading to an increased flux of energetic particles in our galaxy [101] . before it is too late to realize, and the entire human race wipes-off the face of the earth due to the climate change, we must take this pandemic as a wake-up call towards being considerate to the environment. what has apparently come clearer though is that problems like biohazards primarily emerge due to our lifestyle (e.g., consumption patterns including eating habits) or mishandling of biowastes (see figure 19 ). there is an absolute necessity to pay utmost attention to biosafety and implementation of effective iso standards worldwide. health of people is also closely connected to the health of animals and our shared environment. this is because people, animals, plants, and our environment are interdependent [102] and this direction of research considers the concept of "one health". in the wake of the covid-19 pandemic, the world witnessed an all-time-high demand of ventilators, ppe's, masks, bed liners, other essential health supplies and medicines. the pandemic struck everywhere, including the largest manufacturing economiesand pretty much all at once. as a result, most nations experienced manufacturing shocks. the two shocks that have impacted manufacturing worldwide are: • supply shock: the containment measures (such as lockdowns and social distancing) kept the workers away and resulted in reduced productivity and output. • demand shock: customer consumption patterns started changing (e.g., movement away from "non-essentials") and this affected the demand for a large variety of manufactured goods and associated services. as the pandemic spread, a few countries were resilient enough to cope with the pace of transformed supply chain requirements while many others who had earlier (in pre-covid-19 era) aligned themselves to outsourced or cloud-based manufacturing, primarily driven by low-cost, became hugely dependent for their essential supplies on other countries (primarily china the manufacturing related lesson learned from the covid-19 episode is that pandemics can affect manufacturing in several geographical locations simultaneously and therefore can affect an individual nation's surge capacity to deliver essential supplies such as diagnostics, drugs and vaccines to its population. going forward, this lesson will critically affect thoughts on manufacturing strategy and low-cost based outsourcing, and we may see a sustained push towards development of resilient indigenous manufacturing capabilities and a decreasing reliance on low-cost based outsourced manufacturing. a second trend that we perceive is an increasing focus on digitised automation and use of automated production systems including the use of robotics in day to day life as well as deployment of embedded robotics in manufacturing operations. an example of a futuristic automation capability was displayed at the indian institute of technology guwahati who developed remote controlled food delivery robot for covid-19 isolation wards (see figure 20) . the robot was designed for 6 hours run when fully charged. antimicrobial resistance (amr) refers to the resistance of microbes to antimicrobial drugs or "microbial immunity". the who as well as several national health organizations have identified antibiotic resistance as one of the greatest threats to global public health, economic growth, agriculture, economic security and national security. around the globe, people are being admitted to hospitals with infections that do not respond to antibiotic treatment. the problem of amr is rooted in the fact that when microbes are repeatedly exposed to antibiotics, they mutate to produce strains that are resistant to the antibiotic and are therefore able to resist the effects of medication that could successfully treat the microbe. in 2014, lord jim o'neill and his team published a review commissioned by the united kingdom government entitled, "antimicrobial resistance: tackling a crisis for the health and wealth of nations" (the amr review) which is in line to the question that de kraker et al. [104] asked "will 10 million people die a year due to amr by 2050?" the problem of amr is connected to that of covid-19 with respect to the emergence of zoonotic diseases. the trend of overuse and/or misuse of antimicrobials, excess use of certain antibiotics in animals, and pharmaceutical industry pollution can lead to continuous emergence of zoonotic diseases. excessive use of antimicrobials stresses the naturally occurring microbiome and allows for resistant bacteria to become dominant. indeed, research has suggested that amr might spread to humans through food products of animal origin, the environment, and by direct contact in the case of agricultural workers. addressing the issue of amr as well as others shown in figure 21 is thus a global priority as its impact on claiming lives is no less than that of covid-19 [105]. the rapid emergence of the covid-19 pandemic provided minimal time for welldirected resource mobilization, and almost every country was tested for its resilience in its medical and manufacturing abilities during the first half of 2020. the number of tragic health cases pushed organizations such as the fda to allow emergency use authorisation of various drugs without a systematic testing protocol, and among the 70 drugs tried, favipiravir and tocilizumab were rated most effective. amidst the chaotic situation, the growing use of digital tools for professional communications and artificial intelligence to monitor the recovery of covid-19 patients showed some positive outcomes. a major question remains open to date, "how can the symptoms of covid-19 be detected in their early stages?" while more research is needed on the instrumentation and manufacturing sides, there are clear opportunities identified from the available scientific knowledgebase. in relatively advanced stages of the symptoms, rapid measurements by combining chest computer tomography (ct) and reverse-transcription polymerase-chain-reaction (rt-pcr) tests seem to provide a high confidence level in the screening. more work is needed on the sensing side for detection of the virus in the primitive stages. definitive promise has been exhibited using laser technology to monitor the aerosol spread and to detect the rna of the virus and gain a deeper understanding of the aerodynamic properties of emergent viruses. further development of fumigation chambers and ultra-violet chambers seem to hold promise as precautionary measures that can be implemented in public places like malls, airports, theatres and train stations etc. in this paper, we have also highlighted the possibilities of developing novel tools such as lab-on-the-chip, in addition to developing advanced personal protective equipment (ppe) using combinatorial additive and subtractive manufacturing techniques such as roll-to-roll manufacturing and thermal spray. these technologies allow the capability to filter the coronavirus, which has a diameter in the range of 100 ± 60 nm. it is envisioned that in future, wearing masks and social distancing would be mandated to avoid the recurrent spread of the virus. in the long term, micro-manufacturers, medical professionals, metrology engineers, material scientists and virologists will need to work in a more interdisciplinary way to develop modelling informed fabrication strategies. accelerated use of molecular modelling approaches -like molecular dynamics and molecular dockingalso seems to hold promise in drug and vaccine discovery to end this pandemic from its root. as society progresses to meet challenges such as covid-19, many overarching issues will become important. strong measures will need to be implemented to ensure careful handling of biowastes, indigenous capability-based manufacturing focus will become stronger and issues such as antimicrobial resistance will demand increased attention. our hope is that the post-pandemic society will better heed the warning signs from nature and work on making manufacturing systems resilient to 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antimicrobial resistance by 2050? plos medicine authors are very much thankful to the research support provided by the ukri via grants no.: (ep/k503241/1, ep/l016567/1, ep/s013652/1, ep/t001100/1, ep/s036180/1 and ep/t024607/1) as well as the gcrf/ epsrc supported sunrise program (ep/p032591/1). additionally, we acknowledge the support received from h2020 (cost actions (ca18125, ca18224, ca17136 and ca16235) and euramet empir a185 (2018)), royal academy of engineering grant no. iapp18-19\295 (indo-uk partnership), royal academy of engineering grant no. tsp1332 (south africa-uk partnership) and newton fellowship award from the royal society (nif\r1\191571). also, numerical calculations performed on the isambard bristol, uk and archer hpc were made available by the epsrc resource allocation panel (rap). all data in the manuscript will be available through cranfield university open repository. key: cord-317501-yblzopc3 authors: kuhn, philipp; fühner, viola; unkauf, tobias; moreira, gustavo marcal schmidt garcia; frenzel, andré; miethe, sebastian; hust, michael title: recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display date: 2016-06-21 journal: proteomics clin appl doi: 10.1002/prca.201600002 sha: doc_id: 317501 cord_uid: yblzopc3 antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. traditionally, these antibodies are generated by hybridoma technology. an alternative to hybridoma technology is the use of antibody phage display to generate recombinant antibodies. this in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of antibodies against all conceivable molecules. phage display technology enables obtaining human antibodies from naïve antibody gene libraries when either patients are not available or immunization is not ethically feasible. on the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity‐matured antibodies. because the phage packaged dna sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. in this review, an overview of phage display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given. antibodies are essential molecules as tools for basic research [1], diagnostics [2], and therapy [3] . in the past-and still today-polyclonal antibodies were produced as serum in animals like horses [4] . a milestone in antibody generation was the development of hybridoma technology that allows the production of monoclonal antibodies [5] . but the hybridoma technology has drawbacks like limited number of candidates, possible instability of the aneuploid cell lines [6], inability to provide antibodies against highly conserved antigens and most of all its limited application to generate human antibodies [7] . the hybridoma technology essentially gives murine antibodies that can be used for diagnostic or research purposes. however, their therapeutic applications are limited because repeated administration of murine antibodies can cause human anti-mouse antibody reaction, reducing antibody half-life and leading to severe side effects such as anaphylactic shock [8] . a strategy to circumvent these problems is antibody humanization or the use of transgenic animals in which the original antibody gene repertoire is replaced with a human gene repertoire [9] [10] [11] [12] . another strategy is the human hybridoma technology resulting in human antibodies [13, 14] , although this technology has the same problems of the murine hybridoma technology regarding the limitation of the immune system. a technology that overcomes the limitation of the immune system is antibody phage display. since this technology involves an in vitro selection process, it is completely independent of any immune system. the display method most commonly used today is based on the work of georg p. smith on filamentous phage, which infect e. coli [15] . the selection process is called "panning," referring to the gold digger's tool [16] . m13 phage display technology was further developed 1990/91 for antibodies in three places in parallel: heidelberg (germany), cambridge (uk), and la jolla (usa) [17] [18] [19] [20] . two different genetic systems have been developed for the expression of the antibody::piii (phage protein iii) fusion proteins for phage display. first, the antibody genes can be directly inserted into the phage genome fused upstream of the wild-type piii gene [20] . second, most successful systems, uncouple antibody expression from phage propagation by providing the genes encoding the antibody::piii fusion proteins on a separate plasmid (called "phagemid") that contains a phage morphogenetic signal for packaging the vector into the phage particles [18] . no matter the used phage display system, antibody fragments are displayed on the surface of m13 phage and the corresponding antibody gene is packaged in the phage particle. other phage display technologies using phage lambda [21] or t7 [22] are less suitable for the display of antibody fragments. in addition, these phages are lytic phage that aggravates the practical work. the most common antibody formats used for antibody phage display are the single chain fragment variable (scfv) [23] [24] [25] or fragment antigen binding (fabs) [26, 27] . other antibody formats used for phage display are single chain fabs (scfab), human vh domains (dabs), the variable domains of camel heavy chains (vhhs) and immunoglobulins of sharks (ignars) [28] [29] [30] [31] [32] [33] . figure 1 shows an antibody phage and different antibody fragments. for veterinary research, chicken libraries are often used [34, 35] . in contrast to what occurs in humans, the diversity of chicken antibody genes is the result of gene conversion, and the n-and c-terminal parts of chicken's vh and vl are always identical, facilitating antibody gene amplification, and library cloning [36, 37] . also rabbit phage display was used to generate antibodies against pathogens [38] . in the selection process (panning), the antigen is immobilized to a solid surface, like column matrixes [18] , magnetic beads [39] , or plastic surfaces with high protein-binding capacity such as polystyrene tubes or respectively microtitre wells, which is the most common method [40] . an other option is the panning in solution using biotinylated antigens followed by a "pull-down" with streptavidin beads [41] . the vast excess of nonbinding antibody phage will be removed by stringent washing. subsequently, the bound antibody phage will be eluted, e.g. by ph shift or trypsin, and reamplified by infection of e. coli. after infection of the phagemid bearing e. coli with a helperphage, new antibody phage will be produced. the selection cycle will be repeated and the number of antigen-specific antibody phage clones should increase with every panning round. usually 2-3 panning rounds are performed. finally, monoclonal antibody phage or monoclonal soluble antibodies can be identified by, e.g. elisa [42] , immunoblot [40] , or flow cytometry [43] . the antibody fragment genes can be subcloned into any other antibody format, e.g. scfv-fc or igg [23, 27, 42, 44, 45] . a schema of the selection process is given in fig. 2 . regarding the source of genes, antibodies can be selected from two types of libraries: immune libraries and universal libraries. immune libraries are constructed from immunized/infected donors and typically used in medical research to obtain an antibody against a particular target antigen, e.g. an infectious pathogen like ebola virus [46] . an advantage of this kind of library is that the v-genes contain hypermutations and are affinity matured, although its development can be restricted to ethical constraints. the alternative are universal or "single pot" libraries, which includes naïve, semisynthetic and synthetic libraries that are designed to isolate antibody fragments binding to every possible antigen, at least in theory [44, 47] . naïve libraries are constructed from rearranged v genes from b cells (igm) of nonimmunized donors. examples for this library type are the naïve human fab library constructed by de haard and colleagues [26] and the hal scfv libraries [23, 48] . semisynthetic libraries are constructed from unrearranged v genes from pre-b cells (germline cells) [49] or from one antibody framework [50] in which one or several cdrs, but always the cdr h3, are randomized. often used semisynthetic libraries are the tomlinson i and j libraries using one defined framework vh3-23 and kappa ikv1-39 with randomized cdr2 and cdr3 [51] . a combination of naïve and synthetic repertoire was used for the fab310 antibody gene library. in this library, light chains from autoimmune patients were combined with a fd fragment (vh+ch1) containing synthetic cdr1 and cdr2 in the human vh3-23 framework and naïve cdr3 regions, originated from autoimmune patients [27] . fully synthetic libraries are made of human frameworks with randomized cdr cassettes [52] [53] [54] . the theoretical size of these universal libraries is usually higher than 10 10 independent clones [24, 48, [54] [55] [56] . to date, 53 antibodies and antibody conjugates were approved by ema and/or fda (status january 2016) (http://www.imgt.org/mab-db/query.action, development status: phase m in search field) and about 350 antibodies were under development in 2013 [57] . most approved therapeutic antibodies are for cancer and autoimmune diseases and the annual sales of therapeutic antibodies exceeded 50 billion us$ in 2013 [58] . the mechanisms of therapeutic antibodies are manifold and include neutralization of substances, e.g. toxins [59] or cytokines like tumor necrosis factor alpha [60] , blocking of receptors like epidermal growth factor receptor [61] , binding to cells and modulating the host immune system [62] , or combinations of these effects [63] . currently, two recombinant antibodies are approved for the treatment of pathogens or toxins. raxibacumab is a human antibody for anthrax treatment derived from a phage display library from cambridge antibody technology (now medimmune, part of astrazeneca) in cooperation with human genome science (now glaxosmithkline) [64] . the antibody palivizumab for the treatment of respiratory syncytial virus bronchiolitis is a classical humanized antibody [65] . a further antibody, but also not derived from phage display, the clostridium difficile antibody bezlotoxumab is in clinical phase 3 [66] . an overview of recombinant antibodies derived from phage display against bacterial and viral pathogens, eukaryotic pathogens (parasites, fungi), and toxins as well as detailed examples for diagnostic and therapy are given in the next sections. the most therapeutic antibodies against bacterial targets are generated against toxins. these antibodies are described in the section "recombinant antibodies against toxins." the majority of antibodies against bacteria are developed in order to facilitate diagnostics in patients [67, 68] and environmental samples [69, 70] . in general, cultural and microbial detection of bacteria is regarded as standard in diagnostics for many pathogens, e.g. mycobacterium tuberculosis [71] or salmonella typhimurium [72] . since these methods are often time-consuming and require experienced lab personal, high throughput analysis is often limited. real-time pcr measurements have been developed for the diagnostics of many bacteria [73] , offering high sensitivity and specificity of detection. but sample treatment is still needed in many cases, including expensive and complex laboratory devices [74] . an other approach is ms for diagnostic, but this technology needs expensive devices [75] . antibody-based diagnostic like elisa would be more simple and easy to use, also in developing countries. in order to generate antibodies with the desired-binding properties, phage-display has been used to select antibodies on proteins or polysaccharides of chlamydophila psittaci [76] , chlamydia trachomatis [77] , haemophilus influenzae [78] , listeria monocytogenes [79] , mycobaterium bovis [35] , mycobacterium tuberculosis [68, 80, 81] , porphyromonas gingivalis [67] , ralstonia solanacearum [69] , salmonella typhimurium [2, 82] , and yersinia pestis [83] . but even selections on cell lysate or whole cells were performed on mycobacterium avium [84] , bacillus anthracis [70, 85] , moraxella catarrhalis [86] , lawsonia intracellularis [87] , lactobacillus acidophilus [88] , helicobacter pylori [89] , brucella melitensis [90] , and bordetella pertussis [91] . in the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different bacterial pathogens. tuberculosis (tb) is a bacterial infection caused by mycobacterium tuberculosis (mtb), a pathogen with particular cell wall and membrane characteristics that is able to infect and multiply within alveolar macrophages leading to cough, weakness, and fever [92] . in 2013, 9 million people were infected and 1.5 million died worldwide [93] . in order to improve treatment and control disease spread, tb must be diagnosed as early as possible. one prominent target for this purpose is the bacterial antigen 85 (ag85) that is the most abundant protein secreted by mtb [94] . it is a complex of three highly homolog proteins (ag85a, ag85b, and ag85c) with a molecular size of 30-32 kda each [95] . in a novel approach by ferrara et al. [68] monoclonal antibodies were selected against the ag85 complex by combining phage-and yeast-display. first, a naïve scfv-library was preselected against ag85 by phage display, reducing the diversity from ß10 11 unique scfv clones to ß10 5 colony forming units. second, the enriched sublibrary was cloned into a yeast-display system. each clone of the selection output was screened individually for antigen binding by facs. those with the highest antigenbinding signals were sorted and further analyzed. in total 192 clones were sequenced revealing 111 genetically unique scfv clones. further screening assays identified seven antibody pairs, which can detect ag85 down to a concentration of 6.1 nm in the absence of serum and 22.7 nm in its presence. interestingly, none of the antibodies were absolutely specific for one of the ag85 subunits of the complex. all three subunits a, b, and c were bound although not equally. in contrast, fuchs et al. selected five human antibodies against the recombinant ag85b protein from the naïve hal7/8 libraries [81] . three of them bound specifically to the 85b protein in elisa (lod: 5 ng/ml). even sandwich detection of recombinant 85b in elisa (lod: 10 ng/ml) and integration into lateral flow immuno assay (lod: <5 ng/ml) was shown. but antigen detection in mtb cell extracts or culture filtrates was restricted to direct elisa and immunoblot assay. the authors of both papers argued that the selected antibodies must be affinity matured in order to reach required detection limits. successful implementation of this concept was demonstrated by sixholo et al. [80] . chicken antibodies were selected from the semisynthetic nkuku library against a 16 kda recombinant antigen of m. tuberculosis, also known as heat shock protein x (hspx). after four rounds of panning, three scfvs were identified in elisa. the clone with the lowest elisa signal was selected for in vitro engineering using two different approaches. first, a mutant library was generated from the parental scfv by error-prone pcr with a diversity of ß3 × 10 7 unique scfv. this library was selected again on the hspx antigen under more stringent conditions, leading to identification of three mutant scfvs. these contained one or three individual amino acid exchanges, occuring both in cdr and framework regions. all mutants showed increased elisa-binding signals, reaching a signal ß11 times higher when compared to the parental scfv. furthermore, the mutants showed improved association and dissociation kinetics in elisa and spr analysis. in the other approach, the length of the scfv linker was reduced from 15 amino acids to a single glycine residue, forcing tetramerization of antibodies. due to cooperative binding, the apparent functional affinity was improved. in elisa, tetrameric scfv generated a ß14 times higher elisa signal, and spr proved increased association and decreased dissociation rates. porphyromonas gingivalis is one of the major pathogens involved in periodontitis [96] . periodontitis is an inflammatory disease, which causes the loosening and loss of teeth. secreted cysteine proteases like rgpb contribute to disease pathology and represent potential biomarkers for disease detection and progression [97] . skottrup et al. [67] generated a naïve antibody library from camels with a diversity of ß5 × 10 7 clones. a special feature is that the antibodies consist of a single monomeric vhh domain, also called nanobodies. advantages of this format are the small size (ß15 kda), the ease production in e. coli, and the convex paratope that is formed which enables the targeting of cryptic epitopes. the library was selected on immobilized rgpb. one clone was isolated that binds specifically to cell surface displayed and soluble rgpb with an affinity of 362 pm. a detection limit of ß8 × 10 6 cells/ml of saliva was reached when tested by subtractive inhibition elisa. but catalytic activity of rgpb was not inhibited by antibody binding. salmonella typhimurium, is one of the most important pathogens of foodborne gastrointestinal infections [98] . meyer et al. [82] first identified novel immunogenic proteins of s. typhimurium from a genomic library using oligopeptide phage display. in a second step human antibodies were selected against these targets from the naïve hal gene libraries. for diva (differentiating infected from vaccinated animals) vaccine development, s. typhimurium strains lacking a marker protein, e.g. ompd (outer membrane protein d) were developed (diva vaccines) [99] . to allow the discrimination between vaccinated animals and infected animals a diagnostic of this specific marker is necessary. here, meyer et al. [2] generated scfvs against ompd for diagnostics purposes. only some of the generated antibodies were suitable for a competitive elisa using swine serum showing also the difficulties when developing diagnostic assays that are often hampered by the complexity of serum samples. an overview of recombinant antibodies generated by phage display against bacterial pathogens is given in table 1 . up to now, a large panel of antibodies against various viruses has been generated from either naïve or immune libraries using phage display technology. panning against peptides, recombinant viral proteins, or complete virus particles has led to the identification of antibodies directed against human pathogenic viruses such as sin nombre virus [100] , dengue virus [101, 102] , influenza virus [103, 104] , veev [105] , norovirus [106] , sars coronavirus [107] , or hepatitis c [108] from naïve antibody gene libraries. other antibodies were selected from immune antibody gene libraries targeting, e.g. western equine encephalitis virus (weev) [109] , hiv [110, 111] , sars [112] , yellow fever virus [113] , or influenza virus [114, 115] . semisynthetic libraries were also used to generate antibodies specific for influenza virus [116] . beside human and animal viruses, antibodies were also generated against plant viruses [40, 117, 118] . libraries originated from different species have been successfully employed to isolate virus-specific antibodies in the past such as those from macaque [119] , mouse [120] , chimpanzee [121] , llama [122] , chicken [123] , and human origin [124] . most of the virus-specific antibodies have been isolated from libraries in scfv format [125, 126] , although fab [127, 128] and vhh libraries [122] were also successfully used. an interesting approach was used by xiao et al. using the antibody ch2 domain as scaffold to generate binders against gp120 of hiv [129] . different antibody characteristics have an influence on virus binding and neutralization. neutralizing antibodies prevent cell binding of the virus. the anti-gp41 antibody hk20 has a higher neutralizaton rate as scfv or fab compared to igg showing, that the epitope is less accessable for the igg format [130] . another interesting example is the anti-gp41 vhh 2h10. here, a tryptophan in the cdr3 is not relevant for epitope binding, but essential for virus neutralization [131] . in the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different virus groups. vaccinia virus is the prototype virus in the genus of orthopoxvirus. it is a relatively large dna virus with a genome of about 200 kbp [132] . the genus orthopoxvirus includes various species such as monkeypox virus, cowpox virus, and especially variola virus that is the causative agent of smallpox in humans. naturally occurring smallpox has been eradicated in 1977 because of a massive who vaccination program that began in 1967. however, no vaccination of the civilian population is conducted nowadays. the potential threat of intentional release has renewed the search for safe and effective smallpox vaccines as case fatality rates of 30% or more among unvaccinated subjects are reported [133] . because orthopoxviruses are highly related, it is assumed, that immunity against one poxvirus goes along with immunity against most members of the entire virus family [132, 134] . using an immune scfv phage display library constructed from vaccinia virus immunized patients, a panel of human vaccinia-specific antibodies was selected. plaque-reduction neutralization tests revealed that seven of these antibodies neutralized vaccinia as well as cowpox virus in vitro. five of those antibodies additionally neutralized monkeypox virus [124] . other antibodies were generated from a fab immune library derived from vaccinia virus immunized chimpanzee. converted into a chimeric chimpanzee/human igg format, two antibodies displayed high affinities to vaccinia protein b5 (k d of 0.2 and 0.7 nm). antibody 8ah8al was neutralizing in vitro for vaccinia and smallpox virus and proofed to be protective in mice challenged with vaccinia virus even when administered 2 days after challenge. in this model 8ah8al proved to provide significantly greater protection than that of the previously isolated rat anti-b5 antibody 19c2 [135] . vaccinia had to be used as model, because the final confirmation of protection against smallpox is not possible. ebola virus and marburg virus, two filoviruses, cause severe hemorrhagic fever and possess high mortality of up to 90% in humans. in addition to public health concerns associated with natural outbreaks, ebola virus might be a potential agent of biological warfare and bio-terrorism [136] . human antibodies directed against ebola virus were selected from a library originated from patients that recovered from infection in the 1995 ebola virus outbreak in kikwit, democratic republic of congo [137] . several antibodies against various viral proteins such as nucleoprotein, envelope glycoprotein, and secreted envelope glycoprotein have been isolated in this study. one antibody (kz52), specific for envelope glycoprotein, was able to neutralize in vitro as fab (50% neutralization at 0.4 g/ml) and as full igg (90% neutralization at 2.6 g/ml) [46] . follow-up studies were showing effective protection in vivo in a guinea pig ebola challenge model when the antibody was administered up to one hour postviral challenge [138] . interestingly, kz52 was not protective in macaques challenged with ebola even if the antibody was given as a two-dose treatment with the first dose one day prior viral challenge and the second dose 4 days postchallenge [139] . a murine scfv and two shark ignar v immune libraries were generated against inactivated zaire ebola virus to yield various antibodies specific for the viral matrix protein vp40 and the viral nucleoprotein [136] . interestingly, this work represents the first example of a successful targeted ignar v isolation from a shark immune response library. dengue virus (denv), a member of the flaviviridae family, is responsible for at least 100 million symptomatic infections each year and became a major health and economic burden in over 50 countries worldwide [140] [141] [142] . it is a positive strand rna virus with a ß11 kb genome, that compromise a single open reading frame. the four circulating serotypes of dengue virus show approximately 70% sequence homology [128, 141] . fab monoclonal antibodies to dengue type 4 virus were isolated from a chimpanzee immune library. two fab, namely 5h2 and 5d9 neutralized denv-4 efficiently with a titer of 0.24-0.58 g/ml by plague reduction neutralization test [143] . another study selected human scfv antibodies specific to dengue virus envelope protein by panning against recombinant full-length envelope protein and its domain iii [102] . because denv envelope protein is an essential molecule for virion assembly and virus entry, scfvs selected in this study were shown to exhibit inhibitory effects on denv infection in vitro [102] . dengue nonstructural protein 5 (ns5) is essential for viral replication and host immune response modulation, making it an excellent target for dengue-inhibiting antibodies. a naïve human fab-phage library was screened for ns5-specific antibody fragments using various ns5 variants from dengue virus serotypes 1-4 as antigens for panning and characterization [128] . using ns5 from alternating dengue serotypes for each round of panning, this strategy resulted in the identification of two clones that are cross-reactive against all four dengue serotypes. another study selected antibodies using phage display by panning with dengue virus particles directly captured from supernatant of infected vero cells. here, highly serotype-specific antibodies were generated. from a total of nine antibodies, seven were shown to be specific to only one serotype. one dengue-3 selected clone cross-reacted with dengue 1, whereas another clone showed cross-reactivity with all serotypes despite being selected solely on dengue 2 particles. interestingly, all of the obtained antibodies recognized several strains of distinct genotypes within the corresponding serotype [101] . panning against dengue envelope protein resulted in the identification of an antibody (c9) from a mouse/human chimeric fab library that cross-reacts with denv1-3 and neutralizes denv2 in cell-based assays after conversion into full-length igg [141] . besides scfv and fab, variable domain heavy-chain antibodies (vhh antibodies) were also selected using phage display technology. after four rounds of panning on recombinant denv 2 ns1 protein, 20 positive clones were selected. affinity measurements with ns1 revealed a k d value of 2.79 × 10 −8 m for the best vhh antibody p2 [122] . venezuelan equine encephalitis virus (veev), an alphavirus of the togoviridae family, causes equine epidemics but can also cause encephalitis in humans [144, 145] . because this virus is classified as category b agent by the centers for disease control and prevention (cdc), much research has been done to generate neutralizing antibodies against it. antibodies were generated from an immune library from human donors targeting both veev envelope glycoproteins e1 and e2 [146] . the isolated fabs l1a7 and f5 were neutralizing in vitro, with f5 being 300 times more effective than l1a7. subsequently, f5 was converted into full igg format and was employed to generate neutralization-escape variants of veev for epitope mapping. within another study, an immune macaque library was used to generate human-like antibodies [119] . one of these antibodies, scfv-fc tor67-3b4, was protective in mice when administered 6 h postviral challenge with veev trinidiad strains, showing 80-100% survival after a challenge with 100-fold ld 50 . however, scfv-fc tor67-3b4 was not able to neutralize trinidad strain in vitro, but other veev strains instead, showing that neutralization is not mandatory for an in vivo protective antibody [119] . another study describes the selection of antibodies from a human naïve scfv gene library using complete, active veev particles as antigen. in this case, specific detection of the veev strains tc83, h12/93, and 230 by the selected antibodies was proven. remarkably, none of the selected scfv phage clones showed cross-reactivity with alphavirus species of the eastern equine encephalitis virus and weev antigenic complex or with chikungunya virus, making them ideal tools for the immunological detection and diagnostic of alphavirus species [105] . two different scfv antibody libraries were constructed from weev immunized macaques. subcloned as scfv-fc, three antibodies from these libraries specifically bound weev in elisa with little or no cross-reactivity with other alphaviruses and were found to be neutralizing in vitro. in this study, the first antibodies against weev, that were shown to be neutralizing in vitro, were developed. about 1 ng/ml of the best antibody (tor69-3a2) neutralized 50% of 5 × 10 4 tcid50/ml weev [109] . flu is a disease caused by influenza viruses. in the last years, the bird flu (h5n1) and the pandemic swine flu (a variant of h1n1) moved in the research focus. due to many genetic events, such as antigenic drift and shift, new influenza variants will occur in the future and will challenge vaccine and diagnostic development [147, 148] . different groups developed antibodies by phage display against influenza viruses. sui et al. [104] selected antibodies from a nonimmune scfv library against the h5 hemagglutinin ectodomain. hemagglutinin is a trimer and the extracelluar part consists of a stalk domain and a globular head domain [149] . they identified ten antibodies binding to the trimeric h5, but not monomeric h5. interestingly, nine of these antibodies shared the same germline framework (vh1-69). these antibodies were converted into igg1 and were protective in mice with 10 or 15 mg/kg in a prophylactic or therapeutic challenge model, respectively. very remarkably, some antibodies crossneutralized h1, h2, h5, h6, h8, and h9 influenza strains. these are phage display derived antibodies that are candidates for broad-spectrum influenza immunotherapy [104] . rabies is caused by the rabies virus that infects the central nervous system. before louis pasteur developed the rabies vaccination, the disease was always fatal. the current postexposure therapy is based on vaccination-as performed by pasteur in 1885-and polyclonal anti-rabbies immunoglobulins [150] [151] [152] . one hundred forty-seven unique recombinant antibodies against the rabies glycoprotein were selected from two immune scfv libraries [153] . the neutralization of 27 street rabies viruses was tested in vitro and the best neutralizing antibodies were tested in vivo in a hamster rabies infection model. here, the antibody cr4098 showed 100% postexposure protection with 40 iu/kg [154] . this antibody was further analyzed in combination with an other human antibody cr57, derived by somatic cell hybridization technique [155] , in in vitro and in vivo models [152] . the safety of this mab cocktail named cl184 was tested in a clinical phase 1 study [156] and subsequently in phase 2 studies. the antibodies were named foravirumab (cr4098) and rafivirumab (cr57). an overview of recombinant antibodies generated by phage display against viruses is given in table 2 . using phage display technology, a large panel of antibodies against a broad range of eukaryotic pathogens has been generated. these antibodies are directed against pathogenic animals, e.g. taenia solium [157] , protozoa, e.g. cryptosporidium parvum [158, 159] , plasmodium falciparum [160, 161] , or toxoplasma gondii [162] and fungi such as aspergillus fumigatus [41] . not only human pathogens have been in the focus of antibody generation, but also veterinary pathogens like myxobolus rotundus [163] (a fish pathogen) or babesia gibsoni (a dog pathogen) [164] and plant pathogens like aspergillus niger [165] , fusarium verticilloides [166] , or sclerotinia sclerotiorum [167] . most antibodies generated in these studies derive from human antibody gene libraries, but libraries from mouse [168] , chicken [166] , camel [169] , or macaque [41] origin have been also successfully applied in phage display to generate monoclonal antibodies against eukaryotic pathogens. in the following paragraphs detailed examples for the use of phage display for the generation of antibodies against different eukaryotic pathogens are given. aspergillus fumigates is the causative pathogen of allergic bronchopulmonary aspergillosis, chronic necrotizing aspergillosis, saprophytic aspergilloma, and highly lethal invasive aspergillosis, the most important aspergillus-related disease [170, 171] . invasive aspergillosis occurs in immunocompromised individuals for example after hematopoietic stem cell transplantation or solid organ transplantation [172] . due to its severity, an early diagnosis is crucial for a successful treatment. in 2009, schütte et al. [41] reported the generation of several antibodies binding specifically to crf2, an a. fumigates antigen of the glycosyl hydrolase family that is located in the cell wall of the growing hyphae. these antibodies could serve as tools for new diagnostic assays such as serum elisa or histopathological immunofluorescence microscopy. the antibodies described in this study were isolated from two different phage-displayed scfv libraries: one was a macaque immune library with a theoretical diversity of 1.5 × 10 7 independent clones; while the other was human naïve antibody library hal4/7 [23] . both libraries were used in two different panning strategies: the first was performed on recombinant antigen immobilized on immunostrips; the second was carried out in solution with biotinylated antigen. finally, 16 individual scfv clones were selected, with six clones deriving from the naïve library and ten from the immune library. interestingly, all antibodies generated on immobilized antigen bind to linear epitopes, whereas all antibodies generated by panning in solution bind to conformational epitopes. seven of these antibodies bound to their native antigen on growing hyphae of aspergillus fumigatus and did not show crossreactions to other aspergillus species or candida albicans. malaria is a life-threatening protozoal infection of the red blood cells and one of the most common mosquito-borne diseases. currently, an estimated 3.2 billion people in tropical and subtropical countries live at risk of malaria [173, 174] . in humans malaria is elicited by at least five different species of plasmodium, p. falciparum, p. vivax, p. ovale, p. malariae, and p. knowlesi [175] . p. falciparum is responsible for most malaria-related deaths globally, while p. vivax is the most widespread parasite [176] . three different approaches to generate antibodies against p. falciparum using phage display are described. in these studies various structures of different developmental stages of the parasite served as antigens. in a first study roeffen et al. [160] used two scfv immune libraries, constructed from b-lymphocytes from p. falciparum patients with transmission-blocking immunity, to generate antibodies against pfs48/45, a surface protein of p. falciparum that is expressed during zygote-and macrogamete stages. pfs48/45 is a potential vaccine candidate since it is a target of transmissionblocking antibodies that are taken up by the mosquito together with the blood meal and block within the mosquito's intestinal tract the oocyte development [177] [178] [179] . the panning was performed on an extract of gametocytes immobilized in immunotubes and the elution was performed by competition with a mixture of four rat monoclonal mabs, which recognize distinct epitopes on pfs48/45 (epitopes i, iib, iii, and v). interestingly, all selected 14 antibody clones bound to epitope iii of pfs48/45. in 2001, sowa et al. [180] reported the isolation of a human monoclonal antibody against the block 2 region of plasmodium falciparum merozoite surface protein-1 (pfmsp-1) by phage display from a malaria patient derived scfv library. lundquist et al. [161] generated a fab-immune library from leukocytes of 13 adults with acquired immunity to malaria. three individual fabs designated ram1, ram2, and ram3 were isolated from this immune library by panning on a recombinant fragment of the merozite surface protein 3 (msp-3 ). msp-3 is involved in heme binding, and antibodies against this protein promote eradication of the parasite by monocytes [181, 182] . a synthetic peptide of the n-terminal fragment of msp-3 is even used in human clinical trials as vaccine [183] . the antibodies were subcloned into igg1 and igg3 format for further analysis. binding of the antibodies to native parasite protein was demonstrated for all three antibodies in immuno blot and immunofluorescence microscopy. ram1 and ram2 also bound to their antigen in fixed and permeabilized cells in flow cytometry. the igg3 format of ram1 showed in a functional assay (antibody-dependent cellular inhibition assay, acdi) an inhibition rate that is comparable to affinity-purified polyclonal anti-msp-3 211-237 antibodies derived from immune donors. the igg1 format also showed inhibition in the acdi, but lower compared to igg3 [161] . an overview of recombinant antibodies generated by phage display against eukaryotic pathogens is given in table 3 . several toxins are classified by the cdc as category a or b agents (for definition see: www.niaid.nih.gov/topics/ biodefenserelated/biodefense/pages/cata.aspx) that are relevant for diagnostics and therapeutics. they can easily be disseminated and result in high or moderate mortality rates [184] . in this context, the antibody phage display offers a powerful tool for antibody selection and allows the isolation of neutralizing antibodies against complete active toxins or special domains by using different human naïve antibody libraries with high diversity [185] [186] [187] . for toxins, the aim is to find antibodies binding to the cell binding domain of the toxin and neutralize the interaction of this domain with the corresponding cellular target, mainly cell surface proteins. but also antibodies directed against the translocation domain or the enzymatic domain can neutralize the toxicitiy. for the isolation of high-affinity antibodies against specific targets, animals are often immunized with toxoids, nontoxic subunits or selected toxin domains. well suited are macaques for the construction of immune libraries, because macaque v-genes are very similar to their human counterparts [59, 119, [188] [189] [190] [191] . in the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different toxins. so far, antibody phage display was successfully used for antibody selection against a panel of toxins classified as category a agents, such as from clostridium botulinum (botulism) [190, [192] [193] [194] and bacillus anthracis (anthrax) [59] and also against different category b agents, such as staphylococcal enterotoxin b [195, 196] and ricin toxin from ricinus communis [191, 197] . an example for a high-risk microorganism that [198, 199] . especially, serotype a is recognized as the most toxic substance known, showing ld 50 of about 1 ng/kg by intravenous route, about 10 ng/kg by the pulmonary route and about 1 g/kg for the oral route [200] . bonts are composed of a disulfide bond-linked 50 kda light chain and a 100 kda heavy chain. the heavy chain contains two functional domains (hc and hn) that are responsible for toxin uptake into nerve cells by receptor-mediated endocytosis and for the translocation of the light chain across the membrane into the neuronal cytosol. whereas the catalytic domain of the light chain is responsible for the bont toxicity. the current approach for treatment of botulism includes the application of human anti-botulism immunoglobulins, such as babybig, or equine anti-toxin serum. but the human serum stock of babybig is limited and the equine anti-toxin may cause hypersensitivity and serum sickness. in these situations, antibody phage display provides a technology to generate toxinneutralizing antibodies against each serotype. for instance, a macaque immune library was used to isolate neutralizing scfv with nm affinities against the light chain of bont/a [190, 201, 202] , but also antibodies against the heavy chain or other relevant serotypes of bont are of therapeutic interest. phage display technology was also used for isolation of single domain antibodies (vhh) after immunization of a llama with a cocktail of 7 bont toxoids (a-f) [193] . another approach was the generation of a human antibody gene library after inducing a bont/a-specific immune response by in vitro immunization [194] . furthermore, antibody phage display was used to generate antibodies against other clostridial toxins such as those from clostridium tetani or clostridium difficile [188, 203] . anthrax, another serious infectious disease is caused by bacillus anthracis, an aerobic, gram-positive, spore-forming bacterium that is found in soils around the world. bacillus anthracis secrets two toxins: the lethal toxin (lt) and the edema toxin (et) [204] . both are composed of two subunits: the lt consists of the lethal factor (lf), and the protective antigen (pa); while the et is formed by the edema factor, and pa. it was demonstrated that only lt has an essential role in the pathogenesis of anthrax [205] . the subunit pa is the basis of current vaccines and induces the generation of neutralizing antibodies. in combination with antibiotics, commercial monoclonal antibodies against pa, such as raxibacumab, are commonly used for treatment [206] . in 2012, the fda approved raxibacumab to treat inhalational anthrax. due to security issues the use of anti-pa antibodies alone is questionable, since pa could be modified and lose the recognized epitopes while retaining biological activity. an alternative to anti-pa antibodies are antibodies targeting the lf, such as 2lf, which was isolated from an immune library via antibody phage display technology [59] . a combination of an anti-pa antibody with an anti-lf antibody could lead to a synergistic effect and improve the efficacy of the therapy. an example for bacterial toxins classified as category b agent is staphylococcal enterotoxin b (seb) from staphylococcus aureus. this bacterium is a potential causative agent for food-borne illness and produces twenty-one types of staphylococcal enterotoxins that cause symptoms of food poisoning such as abdominal cramps, vomiting, and diarrhea [207, 208] . seb is a single polypeptide of 28 kda and is the most potent toxin secreted by s. aureus. as a superantigen, it stimulates t cells and leads to an overproduction of cytokines, causing clinical symptoms such as fever, hypertension, and in some cases death. phage display was used to generate recombinant antibodies from a murine immune library [196] and to identify the epitope of a seb-specific monoclonal antibody using a peptide phage library [209] . furthermore, a human monoclonal antibody against seb was isolated from a synthetic human antibody gene library that inhibited seb binding to mhc-ii [195] . the phage display technology was also used to isolate antibodies against ricin, which is also classified as category b agent by cdc. ricin is a 61-kda glycoprotein from the castor bean plant (ricinus communis), which consists of two distinct subunits (rta and rtb). rtb is a galactose-and n-acetylgalactosamine-specific lectin that binds to specific sugar residues on the cell surface, allowing internalization of the toxic rta by endocytosis [210] . rta is an rna n-glycosidase that irreversibly inactivates eukaryotic ribosomes, leading to the inhibition of protein synthesis [211] . human-like antibodies were selected by phage display from a macaque immunized with rta. one antibody, 43rca, had a picomolar affinity and neutralized the biological activity of ricin in vitro [191] . furthermore, neutralizing vhh with high affinity was selected from a llama immune library. the best antibody c8 was able to neutralize 100% ricin activity in an in vitro assay using 40 g/ml vhh [197] . in addition to the different toxins that are classified by the cdc as category a or b agents, the number of toxins is almost endless. different animals are known to produce high potential toxins containing a complex composition. for example, tityus serrulatus, known as brazilian yellow scorpion and the most dangerous scorpion in brazil, produces a 61-amino acid peptide, called gamma toxin, which is the major toxic component in the venom [212, 213] . regarding this toxin, a neutralizing antibody was isolated from a human library via phage display and was capable of protecting mice [185] . the same procedure was used for bothrops jararacussu, a venomous pit viper species endemic in south america. by using a human antibody gene library, different antibodies were selected to inhibit the phospholipase activity of the venom in vitro and reduce the myotoxicity in vivo [214] . marine organisms can also produce toxins, e.g. the tetrodotoxin (ttx) from pufferfish. elisa, enzyme-linked immunosorbent assays. here, scfv were selected from a human naïve antibody gene library neutralizing 99% of ttx activity in vitro [215] . an overview of recombinant antibodies generated by phage display against toxins is given in table 4 . antibody phage display allows the generation of recombinant antibodies from different species, including human, llama, camel, macaque, shark, or mice. these antibodies are mainly derived from two types of sources: immune, or naïve libraries. immune libraries should be preferred when immunized animals or convalescent patients are available, offering the chance to directly isolate affinity matured antibodies. if immunization is not possible or ethically not feasible, naïve antibody gene libraries are an alternative. in such an approach, the antibody generation process is not limited by the immune system. using antibody phage display a variety of recombinant antibodies was generated for diagnostics and therapy against bacterial, viral pathogens, and eukaryotic pathogens as well as toxins. man-made antibodies development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms high-avidity human igg kappa monoclonal antibodies from a novel strain of minilocus transgenic mice production of fully human antibodies by transgenic mice human antibodies from transgenic mice development trends for human monoclonal antibody therapeutics human monoclonal antibodies accessing the human repertoire for broadly neutralizing hiv antibodies. mabs filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface antibody-selectable filamentous fd phage vectors: affinity purification of target genes assembly of combinatorial antibody libraries on phage surfaces: the gene iii site a surface expression vector for antibody screening making antibody fragments using phage display libraries phage antibodies: filamentous phage displaying antibody variable domains identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda a human scfv antibody generation pipeline for proteome research application of phage display to high throughput antibody generation and characterization human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library a large non-immunized human fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies generation of high-affinity human antibodies by combining donor-derived and synthetic complementaritydetermining-region diversity domain antibodies: proteins for therapy single chain fab (scfab) fragment single domain camel antibodies: current status camelid immunoglobulins and nanobody technology selection and affinity maturation of ignar variable domains targeting plasmodium falciparum ama1 isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scfvs in inhibition elisas chicken scfvs and bivalent scfv-c(h) fusions directed against hsp65 of mycobacterium bovis chicken immunoglobulin gamma-heavy chains: limited vh gene repertoire, combinatorial diversification by d gene segments and evolution of the heavy chain locus singlechain antibody fragments from a display library derived from chickens immunized with a mixture of parasite and viral antigens recombinant rabbit single-chain antibodies bind to the catalytic and cterminal domains of hiv-1 integrase protein and strongly inhibit hiv-1 replication methodology for selection of human antibodies to membrane proteins from a phage-display library the production of a genus-specific recombinant antibody (scfv) using a recombinant potyvirus protease identification of a putative crf splice variant and generation of recombinant antibodies for the specific detection of aspergillus fumigatus construction of human antibody gene libraries and selection of antibodies by phage display highthroughput screening of single-chain antibodies using multiplexed flow cytometry generating recombinant antibodies to the complete human proteome high level transient production of recombinant antibodies and antibody fusion proteins in hek293 cells recombinant human monoclonal antibodies to ebola virus making antibodies by phage display technology which are the antibodies to watch in 2013? mabs the therapeutic monoclonal antibody market highaffinity, human antibody-like antibody fragment (singlechain variable fragment) neutralizing the lethal factor (lf) of bacillus anthracis by inhibiting protective antigen-lf complex formation tumor necrosis factor alpha drugs in rheumatoid arthritis: systematic review and metaanalysis of efficacy and safety anti-epidermal growth factor receptor monotherapy in the treatment of metastatic colorectal cancer: where are we today? the oncologist cd3-specific antibodies: a portal to the treatment of autoimmunity monoclonal antibody therapy of cancer reduction of respiratory syncytial virus (rsv) in tracheal aspirates in intubated infants by use of humanized monoclonal antibody to rsv f protein mechanisms of protection against clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab diagnostic evaluation of a nanobody with picomolar affinity toward the protease rgpb from porphyromonas gingivalis using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen 85, a tuberculosis biomarker development of specific recombinant monoclonal antibodies against the lipopolysaccharide of ralstonia solanacearum race 3 development and implementation of a single-chain fv antibody for specific detection of bacillus anthracis spores challenges and perspectives in the diagnosis of extrapulmonary tuberculosis detecting non-typhoid salmonella in humans by elisas: a literature review real-time pcr as a diagnostic tool for bacterial diseases sample preparation: the forgotten beginning a systematic review of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry compared to routine microbiological methods for the time taken to identify microbial organisms from positive blood cultures analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from chlamydophila psittaci efficient construction of a large nonimmune phage antibody library: the production of high-affinity human singlechain antibodies to protein antigens human fab fragments specific for the haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression high affinity anti-internalin b vhh antibody fragments isolated from naturally and artificially immunized repertoires improving the characteristics of a mycobacterial 16 kda-specific chicken scfv novel human recombinant antibodies against mycobacterium tuberculosis antigen 85b identification of immunogenic proteins and generation of antibodies against salmonella typhimurium using phage display development of phage-based single chain fv antibody reagents for detection of yersinia pestis nimotuzumab and cetuximab block ligand-independent egf receptor signaling efficiently at different concentrations rugged single domain antibody detection elements for bacillus anthracis spores and vegetative cells phage antibodies obtained by competitive selection on complement-resistant moraxella (branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein isolation of lawsonia intracellularis specific single-chain fv antibody fragments from phage display library using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes generation and characterization of human monoclonal scfv antibodies against helicobacter pylori antigens isolation and expression of recombinant antibody fragments to the biological warfare pathogen brucella melitensis construction and characterization of single-chain variable fragment antibodies directed against the bordetella pertussis surface adhesins filamentous hemagglutinin and pertactin is mycobacterium tuberculosis a closer relative to gram-positive or gram-negative bacterial pathogens? tuberc the antigen 85 complex: a major secretion product of mycobacterium tuberculosis. microbiol a family of cross-reacting proteins secreted by mycobacterium tuberculosis microbial complexes in subgingival plaque the role of gingipains in the pathogenesis of periodontal disease the salmonella enterica pan-genome immunization of pigs to prevent disease in humans: construction and protective efficacy of a salmonella enterica serovar typhimurium live negative-marker vaccine selection and characterization of scfv antibodies against the sin nombre hantavirus nucleocapsid protein selection of phage-displayed human antibody fragments on dengue virus particles captured by a monoclonal antibody: application to the four serotypes human monoclonal single-chain antibodies specific to dengue virus envelope protein neutralizing human monoclonal antibody against h5n1 influenza ha selected from a fab-phage display library structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses development of human antibody fragments using antibody phage display for the detection and diagnosis of venezuelan equine encephalitis virus (veev) isolation of cross-reactive human monoclonal antibodies that prevent binding of human noroviruses to histoblood group antigens potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association genetically engineered singlechain fvs of human immunoglobulin against hepatitis c virus nucleocapsid protein derived from universal phage display library. asian pac human-like antibodies neutralizing western equine encephalitis virus production of recombinant scfv against p24 of human immunodeficiency virus type 1 by phage display technology functional characterization of two scfv-fc antibodies from an hiv controller selected on soluble hiv-1 env complexes: a neutralizing v3-and a trimer-specific gp41 antibody human neutralizing fab molecules against severe acute respiratory syndrome coronavirus generated by phage display antibody responses against wild-type yellow fever virus and the 17d vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza a h5n1 viruses heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza a viruses cucumber mosaic cucumovirus antibodies from a synthetic phage display library generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3 isolation and characterisation of a human-like antibody fragment (scfv) that inactivates veev in vitro and in vivo selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library humanized monoclonal antibodies derived from chimpanzee fabs protect against japanese encephalitis virus in vitro and in vivo development of vhh antibodies against dengue virus type 2 ns1 and comparison with monoclonal antibodies for use in immunological diagnosis a large semi-synthetic single-chain fv phage display library based on chicken immunoglobulin genes the neutralizing human recombinant antibodies to pathogenic orthopoxviruses derived from a phage display immune library protective and therapeutic capacity of human single-chain fv-fc fusion proteins against west nile virus novel phage display-derived h5n1-specific scfvs with potential use in rapid avian flu diagnosis alternative recognition of the conserved stem epitope in influenza a virus hemagglutinin by a vh3-30-encoded heterosubtypic antibody identification and molecular characterization of human antibody fragments specific for dengue ns5 protein a large library based on a novel (ch2) scaffold: identification of hiv-1 inhibitors crystal structure and size-dependent neutralization properties of hk20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41 a gp41 mper-specific llama vhh requires a hydrophobic cdr3 for neutralization but not for antigen recognition vaccination of balb/c mice with escherichia coli-expressed vaccinia virus proteins a27l, b5r, and d8l protects mice from lethal vaccinia virus challenge smallpox as a biological weapon: medical and public health management. working group on civilian biodefense immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox chimpanzee/human mabs to vaccinia virus b5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus isolation and characterisation of ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries ebola virus can be effectively neutralized by antibody produced in natural human infection pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody neutralizing antibody fails to impact the course of ebola virus infection in monkeys identification of human neutralizing antibodies that bind to complex epitopes on dengue virions phage display approaches for the isolation of monoclonal antibodies against dengue virus envelope domain iii from human and mouse derived libraries high affinity human antibody fragments to dengue virus non-structural protein 3 identification of chimpanzee fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin g1 antibody that is highly efficient for neutralization of dengue type 4 virus venezuelan equine encephalitis venezuelan equine encephalitis the first human epitope map of the alphaviral e1 and e2 proteins reveals a new e2 epitope with significant virus neutralizing activity continuing challenges in influenza emerging influenza strains in the last two decades influenza virus hemagglutinin stalkbased antibodies and vaccines the role of vaccination in rabies prevention the prevention and management of rabies comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin the human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants biological characterization of human monoclonal antibodies to rabies virus first administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity development of specific scfv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis single-chain variable fragment antibodies selected by phage display against the sporozoite surface antigen p23 of cryptosporidium parvum single chain variable fragment antibodies selected by phage display against the sporozoite surface antigen s16 of cryptosporidium parvum recombinant human antibodies specific for the pfs48/45 protein of the malaria parasite plasmodium falciparum human recombinant antibodies against plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties construction and characterization of recombinant single-chain variable fragment antibodies against toxoplasma gondii mic2 protein. parasitology a combined phage display scfv library against myxobolus rotundus infecting crucian carp, carassius auratus auratus (l.), in china detection of babesia gibsoni by reaction of phage display single chain antibodies with p50 proteins isolation and characterization of the human monoclonal antibodies c10 in single-chain fragment variable (scfv) format to glucose oxidase from aspergillus niger generation of a highly reactive chicken-derived single-chain variable fragment against fusarium verticillioides by phage display isolation, expression and characterization of two single-chain variable fragment antibodies against an endopolygalacturonase secreted by sclerotinia sclerotiorum isolation from phage display libraries of single chain variable fragment antibodies that recognize conformational epitopes in the malaria vaccine candidate, apical membrane antigen-1 parallel selection of multiple anti-infectome nanobodies without access to purified antigens pulmonary aspergillosis: a spectrum of disease aspergillus fumigatus and aspergillosis patients at high risk of invasive fungal infections: when and how to treat mosquito-borne diseases detection of plasmodium falciparum, p. vivax, p. ovale, and p. malariae merozoite surface protein 1-p19 antibodies in human malaria patients and experimentally infected nonhuman primates plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening challenges in antimalarial drug treatment for vivax malaria control target antigens of transmission-blocking immunity on gametes of plasmodium falciparum biosynthesis of the target antigens of antibodies blocking transmission of plasmodium falciparum sequential expression of antigens on sexual stages of plasmodium falciparum accessible to transmissionblocking antibodies in the mosquito isolation of a monoclonal antibody from a malaria patient-derived phage display library recognising the block 2 region of plasmodium falciparum merozoite surface protein-1 merozoite surface protein-3: a malaria protein inducing antibodies that promote plasmodium falciparum killing by cooperation with blood monocytes plasmodium falciparum merozoite surface protein 3: oligomerization, self-assembly, and heme complex formation phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein 3 antigen. infect. immun antibodies for biodefense isolation and characterization of a human antibody fragment specific for ts1 toxin from tityus serrulatus scorpion human monoclonal scfv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin. asian pac single chain variable fragment antibodies against shiga toxins isolated from a human antibody phage display library a highaffinity macaque antibody fab with human-like framework regions obtained from a small phage display immune library development of human-like scfv-fc neutralizing botulinum neurotoxin e development of neutralizing scfv-fc against botulinum neurotoxin a light chain from a macaque immune library isolation of a human-like antibody fragment (scfv) that neutralizes ricin biological activity genetic and immunological comparison of anti-botulinum type a antibodies from immune and non-immune human phage libraries llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents generation of a recombinant full-length human antibody binding to botulinum neurotoxin a inhibition of toxic shock by human monoclonal antibodies against staphylococcal enterotoxin b construction of a single-chain variable-fragment antibody against the superantigen staphylococcal enterotoxin b development of antiricin single domain antibodies toward detection and therapeutic reagents botulinum toxin as a biological weapon: medical and public health management a novel strain of clostridium botulinum that produces type b and type h botulinum toxins botulism: cause, effects, diagnosis, clinical and laboratory identification, and treatment modalities isolation of a nanomolar scfv inhibiting the endopeptidase activity of botulinum toxin a, by single-round panning of an immune phage-displayed library of macaque origin isolation of nanomolar scfvs of non-human primate origin, cross-neutralizing botulinum neurotoxins a1 and a2 by targeting their heavy chain neutralization of clostridium difficile toxin a with single-domain antibodies targeting the cell receptor binding domain anthrax lethal and edema toxins in anthrax pathogenesis anthrax as a biological weapon, 2002: updated recommendations for management raxibacumab: potential role in the treatment of inhalational anthrax identification and characterization of two novel staphylococcal enterotoxins, types s and t staphylococcal enterotoxin-like toxins u2 and v, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster generation of fab fragment-like molecular recognition proteins against staphylococcal enterotoxin b by phage display technology mechanism of cell entry and toxicity of an affinity-purified lectin from ricinus communis and its differential effects on normal and virus-transformed fibroblasts the mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. the site and the characteristics of the modification in 28 s ribosomal rna caused by the toxins modification of na channel gating by an alpha scorpion toxin from tityus serrulatus scorpion toxins from centruroides noxius and tityus serrulatus. primary structures and sequence comparison by metric analysis human antibody fragments specific for bothrops jararacussu venom reduce the toxicity of other bothrops sp. venoms human scfv that block sodium ion channel activity of tetrodotoxin protein targeting compounds phage-display antibody detection of chlamydia trachomatis-associated antigens isolation of recombinant antibodies directed against surface proteins of clostridium difficile isolation of high-affinity single-chain antibodies against mycobacterium avium subsp. paratuberculosis surface proteins from sheep with johne's disease generation and characterization of a novel recombinant antibody against lmp1-tes1 of epstein-barr virus isolated by phage display production and application of recombinant antibodies to foot-and-mouth disease virus non-structural protein 3abc isolation of recombinant antibodies (scfvs) to grapevine virus b novel camelid antibody fragments targeting recombinant nucleoprotein of araucaria hantavirus: a prototype for an early diagnosis of hantavirus pulmonary syndrome neutralising human recombinant antibodies to human cytomegalovirus glycoproteins gb and gh potent neutralization of hendra and nipah viruses by human monoclonal antibodies four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus screening and evaluation of human single-chain fragment variable antibody against hepatitis b virus surface antigen interference of hcv replication by cell penetrable human monoclonal scfv specific to ns5b polymerase human recombinant antibodies specific for hepatitis c virus core and envelope e2 peptides from an immune phage display library identification of a human epitope in hepatitis c virus (hcv) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-hcvpositive patient nonneutralizing human antibody fragments against hepatitis c virus e2 glycoprotein modulate neutralization of binding activity of human recombinant fabs identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis e virus capsid protein recombinant human fab to glycoprotein d neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses 1 and 2 in vitro directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria cross-reactive hiv-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (r2) with broadly hiv-1 neutralizing antibodies. virology llama antibody fragments recognizing various epitopes of the cd4bs neutralize a broad range of hiv-1 subtypes a, b and c a monoclonal fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse human immunodeficiency virus type 1 strains a human monoclonal antibody neutralizes diverse hiv-1 isolates by binding a critical gp41 epitope human neutralizing human immunodeficiency virus type 2-specific fab molecules generated by phage display a recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza a virus subtype h5n1 fab mabs specific to ha of influenza virus with h5n1 neutralizing activity selected from immunized chicken phage library isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus human monoclonal scfv specific to ns1 protein inhibits replication of influenza viruses across types and subtypes construction of human fab (gamma1/kappa) library and identification of human monoclonal fab possessing neutralizing potency against japanese encephalitis virus neutralizing human fab fragments against measles virus recovered by phage display identification of human single-chain antibodies with broad reactivity for noroviruses development of norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis chimpanzee-human monoclonal antibodies for treatment of chronic poliovirus excretors and emergency postexposure prophylaxis human recombinant puumala virus antibodies: crossreaction with other hantaviruses and use in diagnostics a neutralizing recombinant human antibody fab fragment against puumala hantavirus selection of single chain variable fragments (scfv) against the glycoprotein antigen of the rabies virus from a human synthetic scfv phage display library and their fusion with the fc region of human igg1 the analysis of vh and vl genes repertoires of fab library built from peripheral b cells of human rabies virus vaccinated donors generation and characterization of neutralizing human recombinant antibodies against antigenic site ii of rabies virus glycoprotein single domain antibody multimers confer protection against rabies infection single-chain variable fragment (scfv) antibodies against rotavirus nsp4 enterotoxin generated by phage display selection of single-chain antibodies against the vp8* subunit of rotavirus vp4 outer capsid protein and their expression in lactobacillus casei sars patientsderived human recombinant antibodies to s and m proteins efficiently neutralize sars-coronavirus infectivity chicken single-chain variable fragments against the sars-cov spike protein simian immunodeficiency virus (siv) envelopespecific fabs with high-level homologous neutralizing activity: recovery from a long-term-nonprogressor sivinfected macaque isolation of single-chain antibody fragments against venezuelan equine encephalomyelitis virus from two different immune sources human monoclonal fab antibodies against west nile virus and its neutralizing activity analyzed in vitro and in vivo identification of a wssv neutralizing scfv antibody by phage display technology and in vitro screening recombinant human antibody single chain variable fragments reactive with candida albicans surface antigens differentiation of candida albicans and candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae inhibition of candida albicans adhesion by recombinant human antibody single-chain variable fragment specific for als3p a phagedisplayed chicken single-chain antibody fused to alkaline phosphatase detects fusarium pathogens and their presence in cereal grains singlechain antibodies produced by phage display against the c-terminal 19 kda region of merozoite surface protein-1 of plasmodium yoelii reduce parasite growth following challenge structural and functional characterization of a novel scfv anti-hsp60 of strongyloides sp nanobodies, a promising tool for species-specific diagnosis of taenia solium cysticercosis vhh, bivalent domains and chimeric heavy chainonly antibodies with high neutralizing efficacy for scorpion toxin aahi isolation of single chain variable fragment (scfv) specific for cry1c toxin from human single fold scfv libraries production of human antibody fragments binding to melittin and phospholipase a2 in africanised bee venom: minimising venom toxicity monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice a v h h that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type a molecular characterization of murine humoral immune response to botulinum neurotoxin type a binding domain as assessed by using phage antibody libraries development of human-like scfv-fc antibodies neutralizing botulinum toxin serotype b. mabs in vivo neutralization of botulinum neurotoxins serotype e with heavy-chain camelid antibodies (vhh) a novel multivalent, single-domain antibody targeting tcda and tcdb prevents fulminant clostridium difficile infection in mice selection of nanobodies that block the enzymatic and cytotoxic activities of the binary clostridium difficile toxin phagedisplay derived single-chain fragment variable (scfv) antibodies recognizing conformational epitopes of escherichia coli heat-labile enterotoxin b-subunit production of a single-chain variable fragment antibody against fumonisin b1 a strategy for the generation of specific human antibodies by directed evolution and phage display. an example of a single-chain antibody fragment that neutralizes a major component of scorpion venom detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of trimeresurus mucrosquamatus venom development and characterization of recombinant antibody fragments that recognize and neutralize in vitro stx2 toxin from shiga toxin-producing escherichia coli a single vhh-based toxin-neutralizing agent and an effector antibody protect mice against challenge with shiga toxins 1 and 2 construction of a single chain variable fragment antibody (scfv) against tetrodotoxin (ttx) and its interaction with ttx. toxicon off human monoclonal scfv neutralize lethal thai cobra, naja kaouthia, neurotoxin screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the vibrio parahaemolyticus thermolabile hemolysin a human single-chain variable fragment targeting to vibrio vulnificus rtxa toxin key: cord-331343-qzvwwca9 authors: mason, andrew l.; doucette, karen; wong, gane ka‐shu title: metagenomics and the case of the deadly hamster date: 2008-06-09 journal: hepatology doi: 10.1002/hep.22452 sha: doc_id: 331343 cord_uid: qzvwwca9 nan the article by palacios and colleagues 1 documents three transplant recipients who shared the same donor and succumbed to a novel arenavirus infection. similar events occurred with the very recent infection of two kidney recipients by lymphocytic choriomeningitis virus (lcmv) from a single donor this year, 2 and this complements a report from 2006 detailing other transplant recipients dying from lcmv infection. 3 in the latter lcmv clusters, the timing of infection and viral phylogenetic analysis left little doubt that the infections were derived from a point source. 3 even though there was no evidence of lcmv infection in the two donors, both suffered hemorrhagic intracranial lesions consistent with lcmv infection, and one owned an lcmv-infected pet hamster. 3, 4 the report by palacios and colleagues is particularly notable because of the methods used to discover the novel arenavirus. their exhaustive search started with standard culture techniques for viruses and bacteria and progressed to extensive polymerase chain reaction (pcr) studies using primers complementary to over 30 known viral and bacterial families. 1 the authors then tried panmicrobial microarray analysis with 29,455 oligonucleotide probes reactive to known vertebrate viruses, bacteria, fungi, and parasites, 5 and after drawing a blank with all these studies, they resorted to brute sequencing of all rna in the infected tissue to discover the new virus. 1 this report brings up important issues concerning the role of rodent-derived pathogens in transplant patients. there is also a great deal of interest in the high-throughput sequencing methods that were used to detect the new arenavirus as well as the arrival of metagenomics as the new kid on the block for viral discovery. metagenomics is defined as the sequenced-based analysis of the collective microbial genomes contained in an uncultured environmental sample. 6 the approach has been used for multiple purposes, including the cataloging of ocean microbial communities 7 and the identification of a viral pathogen responsible for honey bee colony collapse. 8 in humans, metagenomic analysis has been used to study viral communities in blood and respiratory secretions, 6 to differentiate bacterial species in gut flora, 9 and to catalog the collective dna and rna viral species in stool samples of healthy subjects 10, 11 and patients with diarrhea. 12 american, european, and asian consortia are now aligned to conduct studies in humans focused on bacteria in the gut and other body cavities, 13 and metagenomics has emerged as the methodology of choice for virus discovery in humans over the last year. 12, 14 for example, merkel cell carcinoma is found in immunosuppressed individuals, and this suggests an infectious etiology. therefore, investigators at the university of pittsburgh sequenced ϳ380,000 transcripts extracted from the rare skin cancer. 14 after biocomputational processing, 2 of the ϳ2400 nonhuman sequences were found to share homology with polyomaviruses. subsequently, sequences from the newly identified merkel cell polyomavirus were detected in 80% of tumor samples and 5% of controls. 14 in essence, the number needed to sequence (nns) in order to discover the novel merkel cell polyomavirus was 1/190,000, which appears cost-prohibitive for current capillary sequencing. in contrast, palacios and colleagues 1 sequenced ϳ94,000 transcripts from infected allografts and found 14 sequences that resembled an old world arenavirus, providing an nns of approximately 1/6700 for their viral agent. both groups used highthroughput pyrosequencing, which is far cheaper than capillary sequencing (fig. 1) . will the brute-force approach of high-throughput sequencing catch on for virus discovery? the answer is undoubtedly yes. the process is unbiased, relatively insensitive to a low copy number, lacks the inherent problem with pcr of widespread contamination, and has the potential to provide a complete representation of all viruses within the environment. 15 on reflection, even houghton's team, cloning hepatitis c virus (hcv) with state-of-the-art molecular complementary dna cloning techniques in the 1980s, eventually had to screen more than 20 million individual clones before they finally identified a gene product reactive to anti-hcv. 16 they would have saved a considerable amount of time and effort if they could have sequenced rna from a few "non-a non-b virus" infected livers (assuming that they could have had access to human genome data that were not available at the time). however, there are additional considerations, such as the copy number of the virus in infected liver samples and the predicted nns required to detect the agent. clearly, the other major issue with metagenomics is the cost. with the marked reduction of sequencing costs predicted in the near future, high-throughput sequencing will become more popular. ironically, most of this technology development is being driven by the global efforts to identify genetic determinants of human disease. 17 an international consortium of american, british, and chinese researchers is promising to sequence 1000 human genomes in the next 3 years. 18 to put these developments in perspective, when one of us (g.k.-s.w.) sequenced the rice genome in 2002, 19 most of the major genome centers were producing high-quality "finished" sequences at $0.10 per base pair. with the latest technologies, however, a similar product can now be created for approximately $0.001 per base pair. the new instruments are quite different from the ones used by the original human genome project, insofar as they do not use electrophoresis to separate the dna molecules. instead, they capture single dna molecules on a solid support, amplify them in situ, perform sequence-dependent enzymatic reactions, and then detect the outcomes to determine sequence content (fig. 1) . given the highly competitive nature of this industry, continuing advances in sequencing technology and falling costs are to be expected. accordingly, it is just a matter of time before metagenomics becomes a practical research technique for determining bacterial and viral contents of clinical samples without the need to culture. 15 let us go back to the plot: what do we know of the novel arenavirus found by palacios and colleagues? the answer is very little, apart from the fact that the 57-year-old male donor died from a cerebral hemorrhage 10 days after returning to australia after a 3-month visit to rural regions of former yugoslavia. 1 genetically, the agent shares close homology with other old world arenaviruses, such as lcmv and the lassa virus, that use rodents as the main reservoir of infection. lassa virus is usually passed from rodent excreta in grain and can also be transmitted from infected patients to healthcare workers. although no wide-ranging lassa virus epidemics have occurred, zoonoses from mice cause 5000 deaths from hemorrhagic fever in western africa per year. 20 depending on the location, a variable proportion (ϳ10%) of the population has serologic evidence of prior lassa virus infection with a calculated ratio of infection to fatality of approximately 1% to 2%. 21 in north america, the seroprevalence of lcmv is approximately 5%, and the estimated fatality in lcmv-infected individuals is much less than 1%, but the mortality is far greater in immunosuppressed and otherwise vulnerable populations. 3, 22 lcmv infection is associated with nonspecific symptoms such as fever, myalgia, and headache, and viremia is limited to the second week of infection in immunocompetent individuals. infrequently, a second phase of disease occurs after convalescence with meningitis and infection disseminated to other organs. 3, 22 therefore, to what extent should we be worried by rodent infections in our transplant patients, and should we be screening donors for these emerging pathogens? we know that about 60% of emerging infectious diseases in humans occur as a result of zoonoses. 23 while it is rare for zoonotic viruses to adapt to become epidemic in humans, there are notable exceptions, such as the severe acute respiratory syndrome (sars) virus epidemic that arose as a result of a zoonosis from bats. 24 on occasion, a zoonosis may become permanently resident in the human population as endemic infection, as observed with human immunodeficiency virus i and human immunodeficiency virus ii derived from chim-panzees and sooty mangabeys, respectively. 25 thankfully, the occurrence of fatal rodent-borne infections in our transplant population is rare. however, is it avoidable? currently, there are no food and drug administration-approved diagnostic tests for lcmv or the newly discovered arenavirus. moreover, the testing of donors by serology or pcr may not be cost-effective or helpful in detecting an acute infection with a limited window of viremia. 22 thus, measures to limit the spread of viruses may provide a degree of protection. pet hamsters have been implicated in lcmv outbreaks, 4, 22 and infection in expectant mothers has been linked to intrauterine death and congenital lcmv infection with severe neurological consequences in the newborn. 26 as a result, our options for controlling the spread of rodent viruses to transplant recipients may be limited to ensuring that our pets are free of known human pathogens. 22 indeed, an outbreak of monkeypox in the midwestern united states from pet rodents 27 underscores the need for implementation of tighter regulations to ensure the absence of zoonotic viruses in pet stores. 22 currently, a detailed donor history is the only screening methodology that we have to identify the potential for donor transmission of infectious agents that cannot be readily detected in serum by food and drug administration-approved diagnostic methods (table 1) . to prevent lcmv transmission, for example, some organ procurement agencies have taken an approach similar to that adopted for avoidance of west nile virus infection, in which donors with encephalitis, meningitis, or flaccid paralysis of undetermined etiology are deferred. 28 an inquiry regarding rodent exposure, such as the acquisition of a new pet within the preceding 6 weeks, may identify potential and generally asymptomatic lcmv infection in a prospective donor. unlike west nile virus, which has a specific geographic distribution and a seasonal variance, lcmv is endemic in rodents and therefore has no predictable pattern. this presents a real problem, as mice are prevalent in most households and are detected in up to 95% of residences, especially in kitchens. 29 we may not have appreciated the full impact of rodent infections on human disease beyond their obvious contribution to dramatic disease outbreaks such as plague, typhus, and hanta virus. 25 more recently, several mouse viruses have been implicated in chronic human disorders, including the endogenous mouse retroviruses, which can be expressed to become infectious agents in other species. for example, the mouse mammary tumor virus, a murine betaretrovirus, has been linked to human breast cancer 30 and primary biliary cirrhosis. 31, 32 mouse gammaretroviruses have also been identified in human samples, such as the xenotrophic murine leukemia virus (mulv)-related virus found in human prostate cancer 33 and mulv sequences found in normal gut flora detected by metagenomic analysis. 11 although the diverse roles of these agents in human disease have yet to be determined and remain a source of controversy, further metagenomic studies are likely to tell us the diversity of rodent microbial agents that can be found in humans. until then, additional vigilance and a detailed donor history for rodent exposure will be required to prevent the introduction of zoonotic infections into our transplant recipients. a new arenavirus in a cluster of fatal transplant-associated diseases rodent virus infected-kidney kills transplant recipient transmission of lymphocytic choriomeningitis virus by organ transplantation lymphocyticchoriomeningitis-virus infection traced to a pet hamster panmicrobial oligonucleotide array for diagnosis of infectious diseases metagenomics: genomic analysis of microbial communities metagenomic analysis of coastal rna virus communities a metagenomic survey of microbes in honey bee colony collapse disorder an obesity-associated gut microbiome with increased capacity for energy harvest metagenomic analyses of an uncultured viral community from human feces rna viral community in human feces: prevalence of plant pathogenic viruses metagenomic analysis of human diarrhea: viral detection and discovery the human microbiome project clonal integration of a polyomavirus in human merkel cell carcinoma viral metagenomics identification of the major non-a non-b hepatitis agent (hepatitis c virus) using a recombinant cdna approach breakthrough of the year. human genetic variation a plan to capture human diversity in 1000 genomes a draft sequence of the rice genome (oryza sativa l. ssp. indica) lassa fever in west african subregion: an overview a prospective study of the epidemiology and ecology of lassa fever lymphocytic choriomeningitis virus-an old enemy up to new tricks global trends in emerging infectious diseases the genome sequence of the sars-associated coronavirus animal origins of human infectious disease lcmv transmission by organ transplantation the detection of monkeypox in humans in the western hemisphere transmission of west nile virus from an organ donor to four transplant recipients mouse allergen. i. the prevalence of mouse allergen in inner-city homes. the national cooperative inner-city asthma study possibilities of a viral etiology for human breast cancer cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis does a betaretrovirus infection trigger primary biliary cirrhosis? identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r462q rnasel variant screening of donor and recipient prior to solid organ transplantation there are currently no approved antifibrotic therapies for liver cirrhosis. we used vitamin a-coupled liposomes to deliver small interfering rna (sirna) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin a. five treatments with the sirnabearing vitamin a-coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose-and duration-dependent manner. rescue was not related to off-target effects or associated with recruitment of innate immunity. receptorspecific sirna delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by ccl 4 or bile duct ligation. the efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis. progressive liver fibrosis, or scarring of the liver, as the common consequence of all chronic liver diseases is a major medical and economic challenge, because there are no effective treatment options except liver transplantation. the current paradigm attributes this fibrogenic response to hepatic stellate cells (hscs), which reside in the subendothelial space of disse and are the major storage site for vitamin a in healthy humans. in response to injury, hscs undergo an activation process in which they lose vitamin a, become highly proliferative, and synthesize abnormal extracellular matrix, including excessive collagen, various proteoglycans, and structural glycoproteins. 1 this activation process and the deposition of extracellular matrix in the space of disse are commonly considered to represent the key pathogenetic events in liver fibrosis. 2 previous studies showed that the collagen-specific chaperone heat shock protein 47 (hsp47)-also termed j6, gp46, collagen-binding 48 kda protein (cb48), collagen binding protein 2 (cbp2), or colligin-plays an essential role in regulating collagen synthesis. it is constitutively expressed with collagens and binds to both helical and nonhelical forms of collagens. 3 the link between hsp47 and collagen production was demonstrated in hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of ␣(i) collagen and fibril structures in mesenchymal tissues. 4 simultaneous activation of both type-i and type-iii collagens with hsp47 expression was also reported in hscs during carbon tetrachloride (ccl 4 )-induced liver fibrosis in rats. 5 conversely, the constitutive overexpression of hsp47 promotes the secretion of collagens in vascular smooth muscle cells. 6 as a molecular chaperone, hsp47 binds closely to procollagen in the endoplasmic reticulum, but dissociates from it in the golgi complex to allow triple helix formation. 7 based on these properties hsp47 has been suggested as plausible molecular target to interfere with fibrogenesis ( fig. 1) . initial proof-of-concept was obtained in experimental models, demonstrating that small interfering rna (sirna) targeting hsp47 inhibited the expression of type-i collagen and the formation of scar tissue. 8, 9 the study by sato et al. 10 targets sirna directed against hsp47 to hscs. the authors used a key feature of hscs, that is, uptake and storage of vitamin a, highlighted by hans popper more than 60 years ago. 11 sato et al. prepared retinol-coupled liposomes carrying sirna against hsp47 and demonstrated binding of retinol binding protein (rbp) to these liposomes (via gel filtration), receptor-mediated uptake of the rbp-coupled complexes by hscs in vitro (via flow cytometry), and suppression of cellular collagen secretion (via sirius red binding assays).using three different models of liver fibrosis (dimethylnitrosamine [dmn], ccl 4 , and bile duct ligation), the study assessed the antifibrotic properties of the sirnacarrying liposomes in vivo at comparatively low doses of 0.75 mg/kg body weight. as shown by immunofluorescence and fluorescence-activated cell sorting analysis of isolated liver cells, the liposomes were predominantly key: cord-304073-f3iwclkm authors: mullick, jhinuk basu; simmons, chelsey s.; gaire, janak title: animal models to study emerging technologies against sars-cov-2 date: 2020-07-27 journal: cell mol bioeng doi: 10.1007/s12195-020-00638-9 sha: doc_id: 304073 cord_uid: f3iwclkm new technologies are being developed toward the novel coronavirus sars-cov-2 to understand its pathogenesis and transmission, to develop therapeutics and vaccines, and to formulate preventive strategies. animal models are indispensable to understand these processes and develop and test emerging technologies; however, the mechanism of infection for sars-cov-2 requires certain similarities to humans that do not exist in common laboratory rodents. here, we review important elements of viral infection, transmission, and clinical presentation reflected by various animal models readily available or being developed and studied for sars-cov-2 to help bioengineers evaluate appropriate preclinical models for their emerging technologies. importantly, applications of traditional mice and rat models are limited for studying sars-cov-2 and development of covid-19. non-human primates, syrian hamsters, ferrets, cats, and engineered chimeras mimic the human infection more closely and hold strong potential as animal models of sars-cov-2 infection and progression of resulting human disease. the novel coronavirus sars-cov-2, which emerged in december 2019 and causes the disease ''coronavirus disease , has galvanized biomedical research to understand, prevent, and treat this life-threatening condition. 43 as with many diseases, animal models are being leveraged to study cellular pathogenesis and transmission of infection, as well as to examine the efficacy of viral vaccines and analyzing herd immunity post-vaccination. however, unique features of sars-cov-2 limit the utility of traditional laboratory animals. sars-cov-2 has a solar-corona-like appearance with spike surface proteins, the characteristic namesake of coronaviruses. these spike proteins have two subunits: the surface unit s1 binds with high affinity to human angiotensin-converting enzyme 2 (ace2) receptors and the transmembrane unit s2, which is then cleaved by human transmembrane serine proteases tmprss1 and tmptss2. 24 ace2 and tmprsss co-expression are essential for sars-cov-2 infection, but mice, rabbits, rats, and guinea pigs do not have ace2 receptors susceptible to sars-cov-2 binding. 49 furthermore, due to their anatomical difference and small size, intranasal viral inoculation of typical small rodent models can result in inhalation and ingestion, thus making it difficult to discriminate between intranasal, oral, and intrapulmonary vaccination 31 and limiting their utility as a model of vaccination against sars-cov-2. here, we discuss alternative rodent models, large animals, and chimeras that may be useful to the bioengineering community working on innovative treatments and vaccinations against sars-cov-2. to investigate sars-cov-2 as with most diseases, no single animal model perfectly matches the clinical profile of sars-cov-2, as observed in humans. a necessary condition for animal models to model the cellular pathogenesis of sars-cov-2 infection is ace2 receptor and serine proteases similar to humans, as discussed above. several larger mammals such as palm civets, pigs, ferrets, cats, and nonhuman primates have ace2 receptors which the virus can recognize. 54 however, many animals known to be susceptible to similar coronaviruses, e.g., chickens, pigs, and dogs, 19 have not been reported to be susceptible to sars-cov-2. given the nuances of each animal model, the choice of animal model for studying covid-19 should vary based on the application. to investigate treatments, for example, models should be considered that show characteristic symptoms similar to humans such as shortness of breath, fever, weight loss, loss of appetite, and pneumonia. 20 since older humans have exhibited increased susceptibility to sars-cov-2 33 models utilizing aged adult animals should be favored. for genomic and pathological studies, viral rna or histopathological tissues need to be obtained, for which animal models should be preferred in whom similar organs as those affected during human pathogenesis are involved. for covid-19, these organs include the respiratory tract, lungs, and gastrointestinal tract. mode of transmission must be similar for projects working to develop personal protective equipment such as respiratory masks or eye protection. in such urgent times, large animal and non-traditional rodent models can be readily utilized without the time delay associated with developing transgenic and chimeric small animal models. large mammals also have the advantage of physiological, anatomical and immunological proximity to humans. 16 their mucosal surfaces can be easily accessed for intranasal and oral vaccine administration and, often being outbred species, may yield more clinically relevant results. a brief overview of readily available animal models of cov-id-19 is provided in table 1 . hamsters are adept at amplifying many viruses and often studied for viral persistence and shedding. 4 the golden syrian hamster (mesocricetus auratus) had been used previously to study other respiratory viruses such as severe acute respiratory syndrome coronavirus (sars), adenovirus, and influenza virus. the structure of the ace2 receptor of hamsters is similar to humans, allowing the spike protein of sars-cov-2 to bind with high affinity and generating preference for the syrian hamster as a model in both sars and sars-cov-2 studies. to mimic human transmission, recent studies challenged male and female syrian hamsters intranasally. 10, 45 the viral loads were found to be highest in the lungs at 2 days post-inoculation (dpi) and were reduced below the detection limits by 7 dpi, when the animals started to recover. the animals showed passive immunization whereby hamsters developed neutralizing antibodies (nabs) by 7 dpi 10 or by 14 dpi. 45 high transmission rate, as shown by transmission to all the naı¨ve co-housed hamsters, did mimic the pathogenesis in humans and was likely through respiratory droplets or oral-fecal contamination. despite high viral loads observed throughout many organs, the hamsters showed only mild symptoms such as weight loss, lethargy, ruffled fur, hunched back posture, and rapid breathing. 10 the main disadvantage of the syrian hamster model was the lack of mortality which did not match the human clinical profile. these studies also used younger hamsters between 4 and 10 weeks of age, likely following previous sars-cov experiments. 39 however, in light of known complications in elderly patients, 33 it may have resembled human disease more closely if aged hamsters were used. overall, the studies show that the syrian hamster is a useful animal model for sars-cov-2 infection especially to study viral replication, shedding, and transmission through the respiratory tract. having anatomical and physiological similarity with the human respiratory system, ferrets (mustula putorius furo) have previously been used for studying sars infection. 56 recent reports suggest affinity of sars-cov-2 for binding to ferret ace2, and the mechanism of transmission correlated with human transmission, which required direct physical contact with potential airborne transmission as viral rna was animal models of sars-cov-2 detected in nasal washes, saliva, blood, fecal, and urine samples. 30, 42 both in humans and ferrets, the virus affected similar organs such as the nasal turbinate, trachea, lung, soft palate, tonsils, intestine, and kidney. 6, 30, 42 clinical signs were mild compared to human patients and included fever and loss of appetite. nabs were detected starting 13 dpi. 42 blanco-melo et al. analyzed the transcriptional regulation of immune modulators to sars-cov-2 in ferret model and inferred that severity of covid-19 in aged population was linked with a severe inflammatory response. 6 they suggest that controlling the sars-cov-2-associated cytokine storm 59 should be the primary focus of treatment. the ferret thus can serve as a useful model for studying the pathogenesis of sars-cov-2 infection in the respiratory tract. ferrets have been considered to be outbred species and hence also used widely in vaccination studies. however recent findings suggest that due to inbreeding, ferrets can be clustered based on geographical locations, where north american and australian clusters have been found to be very low in genetic diversity. 21 researchers using ferrets as a model should consider these issues of the genetic background when interpreting their findings. sars and sars-cov-2 viruses cannot bind to mouse ace2 (mace2) due to differences in key amino acid residues. 49 due to this low sensitivity of mice to sars viruses, several transgenic mice lines were created during the sars epidemic to study its pathogenesis. human ace2 (hace2) genes were expressed under a hybrid chicken beta-actin promoter with cytomegalovirus enhancer 51 or cytokeratin 18 (k18) promoter. 35 the jackson laboratory (bar harbor, me) has recently started re-producing the k18-hace2 strain used to study sars infection, 55 although systemic damage and neuroinflammation still did not represent the human clinical profile. 49 yang et al. developed an efficient model where the endogenous mouse ace2 promoter was used and had tissue distribution similar to the natural hace2 distribution; however, this model still could not mimic the high mortality observed in humans. 58 since both the viruses have a similar mechanism of entry through the ace2 receptor, the developed models are still relevant for sars-cov-2 study. bao et al. used the mace2 promoter line to study the pathogenesis of sars-cov-2 infection. 2 viral rna was detected in the lungs, bronchi, and alveoli, while the wild-type mice failed to be infected by sars-cov-2. the study thus found that the ace2 receptor was essential for sars-cov-2 infection, as expected. soldatov et al. have been working on producing a transgenic line where hace2 and htmprss2 are coexpressed with the mice tmprss2 promoter through gene editing using crispr/cas9. 49 the group hypothesizes that the co-expression will make the model mimic human pathogenesis more closely, particularly in the lungs. other transgenic mice models studied during sars infection can also be helpful for sars-cov-2 studies. one such model is the ace2 knockout mouse, which lacks the ace2 receptor and upon sars infection has been seen to suffer from acute lung failure. 27 tmprss2 knockout mice have shown lower levels of inflammatory cytokines and infiltration by immune cells, leading to less severe tissue damage in the lungs after sars and middle east respiratory syndrome coronavirus (mers) infections, 28 which has pointed researchers to tmprss2 as a potential therapeutic target for serine protease inhibitors. additional strains such as stat1 knockout, balb/c, c57bl/6, and 129s6 mice that were studied for sars may prove to be useful as well. 50 the immune system of feral mice or ''dirty mice'', mice exposed to a diverse group of microbes, have broad t cell distribution that could rapidly control pathogens 18 and render them potential candidates to gain insights into vaccine development for sars-cov-2. as close evolutionary relatives to humans, non-human primates have very similar physiology and immunology to humans 31 and hence greater translational potential. issues with non-human primate models, however, include additional ethical justifications, scarcity of biosafety level-3 facilities, and lack of trained personnel to carry out work. 10 nonetheless, many primate models have yielded helpful insights into sars-cov-2. the rhesus macaque (macaca mulatta) is broadly popular for studying re-infection potentials and designing vaccines. when infected with sars-cov-2, specifically, the rhesus macaque model manifests mild to moderate symptoms of decreased appetite, weight loss, fever, and increased or irregular respiration patterns along with hunched posture. 3, 11, 34, 37 viral rna can be detected throughout the respiratory and gastrointestinal tract, 3, 11, 34, 37, 41 mimicking humans, and also from atypical organs such as spinal cord, heart, skeletal muscles, and bladder 3 ; liver and kidney 11, 14 ; and the spleen. 34 the hallmarks of human sars-cov-2 infection of pulmonary edema and diffused interstitial pneumonia were observed on radiographs throughout these studies. in age-related comparative studies, yu et al. studied the effects of sars-cov-2 infection with three ''young'' (3-5 years) and two ''geriatric'' (15 years) animals. 62 they found geriatric macaques suffered from more severe interstitial pneumonia with more viral load in lungs and anus. the older macaques had viral replication in the entire lung while the younger ones had only in the upper lobes of the lung. another age-related study found that older animals had higher levels of virus-specific antibodies which were detectable early, by 4 dpi. 34 rhesus macaques were also used to investigate uncommon transmission routes; deng et al. inoculated sars-cov-2 through conjunctival and gastric routes. 14 though sars-cov-2 failed to replicate via the gastric route, mild interstitial pneumonia and alimentary canal infection were observed in the conjunctival route challenge. rhesus macaques also have been used to observe if primary infection with sars-cov-2 can protect against re-infections. in these studies, viral loads measured by bronchoalveolar lavage, nasopharyngeal swabs, or anal swabs were found to be reduced manyfold upon reinfection. 3, 11 chandrashekar et al., upon rechallenging the animals, found viral loads were reduced > 100,000-fold. 11 similar results were observed by bao et al., where no clinical symptoms were observed upon reinfection. 3 in a vaccine screening study, yu et al. developed dna vaccine candidates against sars-cov-2 spike protein, which were tested on 35 adult rhesus macaques. 61 after vaccination and upon a second exposure, viral loads were reduced > 1,000-fold. in all studies, animals developed nabs. overall, the rhesus macaque model has been similar in many aspects to the human covid-19 pathogenesis. nonetheless, though studies utilized adult animals up to 15 years old, they could not mimic the high fatality rate of humans. 3, 11, 37 cynomolgus macaques the crab-eating macaque (macaca fascicularis), originally found in southeast asia, had been studied previously for sars. 17 similar to rhesus macaques, when infected with sars-cov-2, geriatric animals showed prolonged viral shedding from the upper respiratory tract in comparison to younger animals through 21 dpi. 40 however, animals infected with sars-cov-2 showed no obvious clinical signs or mortality, even in the geriatric group, 40 which is in contrast to severe systemic responses seen in older macaques for sars. 48 when comparing male and female macaques, no difference in levels of antibodies was observed, though nabs could be detected as early as 4 dpi. 34 lu et al. did report fever and weight loss, and both studies reported diffuse interstitial pneumonia, a common complication in humans. 34, 40 early and prolonged virus shedding of sars-cov-2 compounded with the lack of symptoms highlights an area of concern for community transmission among humans as well. while macaques do not mimic the severity of human symptoms, they may still be helpful to understand disease progression in older and potentially asymptomatic subjects and thus to formulate strategies for containment of covid-19. common marmosets (callithrix jacchus), a new world monkey, were widely used to understand the pathogenesis of and immunization against the deadly mers as their pathophysiology mimicked lethal pneumonia seen in humans. 9 its utility as a model to study sars-cov-2 has not been explored much, though. a single comparative study by lu et al. was found in which six marmosets were intranasally infected with the virus for comparison with other nonhuman primates. 34 viral rna was observed till 14 dpi in blood and nasal, throat, and anal swabs; however, viral-specific antibodies could not be detected in serum, which could be due to lack of marmoset specific antibody detection kit. only one-third of the marmosets had elevated body temperature, lung tissue showed no indication of pneumonia, and no other organs showed the presence of viral rna. overall, the common marmosets were found to be relatively resistant to sars-cov-2 as compared to rhesus macaques and cynomolgus macaques. there has been evidence of infection with sars-cov-2 in tigers at the bronx zoo and isolated reports of transmission from humans to domestic cats. 25 it is helpful therefore, to look into the infectivity of sars-cov-2 on cats to prevent chain of transmission between cats and humans. cats previously had been found susceptible to sars infection and to have the ace2 receptor important in the development of covid-like symptoms. 52 more recently, shi et al. inoculated seven 6-9 months-old ''sub-adult'' cats intranasally with a sars-cov-2 viral strain. 42 viral rna was detected in the nasal turbinates, soft palates, tonsils, tracheas, and the small intestine on 3-6 dpi. the group also found transmission through droplets to the exposed cats. in 70-100 days-old ''juvenile'' cats, by 3 dpi, massive viral lesions were found in the nasal cavity, trachea and lungs, indicating that juvenile cats allow for better replication of the sars-cov-2 virus. nabs were detected in the sub-adult cats between 11 and 12 dpi and in the juvenile cats between 10 and 20 dpi. in a similar study by halfmann et al., inoculated cats showed viral shedding in nasal swab by 1-3 dpi. 22 transmission was also observed in cats that were cohoused. all tested cats were asymptomatic with no clinical symptoms of fever or weight loss. all inoculated and transmission-infected cats produced anti-sars-cov-2 igg antibodies at 24 dpi. 22 in another study, it was reported that 14.7% of cats who were exposed to infected humans harbored antibodies against sars-cov-2 spike protein. 63 the above studies indicate that cats are susceptible to the virus and may harbor it without showing any evident clinical symptoms or mortality. since humans are in close contact with domesticated cats, there is therefore a risk of transmission to humans from asymptomatic feline companion animals. canine ace2 is similar to hace2, 19 and though dogs are known to be infected by some coronaviruses, sars-cov-2 does not seem to be a concern in canines. the first suspected case of human-to-animal transmission of sars-cov-2 was in a pomeranian dog belonging to an infected person in hong kong. 47 generally, isolated cases of sars-cov-2 infection in companion canines have found dogs to remain asymptomatic though viral rna has been found in nasal, oral and rectal swabs. 47 shi et al. inoculated five 3-month-old beagles with sars-cov-2 intranasally, and two un-inoculated beagles were co-housed for transmission studies. 42 while viral rna was observed in rectal swabs of three virus-inoculated dogs by 2-6 dpi, viral rna was not observed in any other organ. by the 14 dpi, two virus-inoculated dogs produced antibodies while the other dogs were found to be seronegative. overall, results from these studies and broad testing of pets 26 indicate low susceptibility for sars-cov-2 among dogs, suggesting a minimal risk of asymptomatic transmission from canine companion animals to humans. the first coronavirus infections were identified in chickens in the 1930s, thirty years before the discovery of the disease in humans. 8 however, the current pandemic-causing coronavirus sars-cov-2 has been reported to be non-infective towards chickens, ducks, and pigs. 42 in both inoculated animals and co-housed uninoculated animals, no viral rna could be isolated from swabs. all animals were also found to be seronegative by 14 dpi. in horses, the enteric equine coronavirus has surged over the last few years, but there is no evidence horses are susceptible to infection with sars-cov-2. 13 while farm animals are unlikely to transmit sars-cov-2, extensive outbreaks have been documented at meat processing plants because of the close working conditions among human workers, suggesting that food production will likely remain a concern throughout the pandemic. 15 despite highly conserved genes broadly, therapeutic and immunization strategies involving adaptive immunity are hard to translate from traditional animal models like mice, rats, and non-human primates into humans. to address these limitations, various strains of immunodeficient mice have been leveraged to create chimeras by transplanting cells and tissues of interest from humans. 12, 38, 44 in particular, engineered chimeras incorporating human immune cells tend to better recapitulate the human immune system, enabling human-specific immune responses to deadly viruses. for example, humanized mice created by injecting human peripheral mononuclear cells (pbmcs) or human cd34+ cells into nod scid gamma (nsg) mice, commonly known as hupbmc-nsg or cd34+ humanized mice, respectively, have been a valuable tool for studying viral infections including hiv, 29 dengue, 36 and hepatitis. 5 as an example, kim et al. created hupbmc-nsg mice and infected with hiv-1 to test the effectiveness of antiviral drugs and the usefulness of nabs. 29 following infection, the authors observed an increase in plasma viral load and a decrease in cd4+ t cells. after the use of an antiviral drug and a nab in hiv-infected hupbmc-nsg mice, they observed a decrease in plasma viral loads and no decline in cd4+ t cells, demonstrating the effectiveness of intervention strategy. although we have not identified reports using humanized mice to study covid-19, success from the aforementioned viral studies highlight the relevance and utility of humanized mice for covid-19 research. cd34+ humanized mice and pbmc humanized mice are available from commercial vendors such as the jackson laboratory, and humanized mice can be further tailored according to research applications. recently, wahl et al. created mice with human lung tissue by subcutaneous transplantation of human fetal lung in nsg mice and later combined these mice with bone marrow-thymic-liver mice, a mouse line with a very robust human immune system. 53 thus, they created a humanized mouse containing both the human immune system and human ''lungs'' that could potentially be used for studying pathogenesis of respiratory viruses like sars-cov-2 and screening for antiviral drugs and therapeutic vaccines. humanized mice, though being the closest replication of the human immune system in a small animal model, come with some limitations. while mice with a degree of ''humanized'' immune function are available commercially, it will take additional effort to develop enhanced variants of humanized mice with a more functional human immune system, e.g. with human leukocyte antigens. 7 unlike readily available small animal models with a competent immune system, it takes a longer time to develop humanized mice and requires additional aseptic handling practices and pathogen-free facilities. nonetheless, humanized mice offer a promising test-bed for rapid development and testing of antiviral drugs, vaccines, and their delivery related to covid 19 research. inspired by the success of using humanized mice to study species-specific response in small animal models, researchers have leveraged the utility of immunodeficient mice to develop chimeras using immune cells from complex animals such as bats. bats are thought to harbor a number of viruses dangerous to humans and other animals without effect, but how viruses and the bat immune system coexist asymptomatically is largely unknown. moreover, sars-cov-2 has~90% similarities to beta-coronaviruses isolated from bats, suggesting bat-to-human transmission was at the nexus of the current pandemic. in vivo studies of bats are limited, though, by challenging breeding conditions and long gestation periods, 23 concerns with capturing large numbers of conserved wild bats, 46 and other challenges of outbred species. most studies on bats are limited to specialized bat cell lines, 1,64 and limits to the availability of these bat-specific cell lines and batspecific antibodies make even in vitro research more challenging than anticipated. to mitigate some of these challenges, a chimera model has been developed by yong et al. that stably expresses the immune system of the bat (eonycteris spelaea) on a mouse background. 60 these ''bat-mice'' have been developed by transplanting bat bone marrow cells into immunodeficient nsg mice. this study showed that~80 to 100 bat-mice can be generated from one bat, thus reducing problems associated with animal numbers. the bat-mice model reconstituted all major immune cells including monocytes, t and b lymphocytes, natural killer cells, and dendritic cells. the model has been found to generate bat specific antibodies in response to antigens as well as resistance to graft rejection when transplanted with bat cells even after 40 weeks. the development of bat-mice has opened up new avenues for research that were previously deemed challenging due to the limited availability of bats for immunological research. a better understanding of how the bat's immune system handles various pathogens, including deadly viruses such as sars-cov-2, will provide valuable insights to strategize the development of effective therapeutic and preventative approaches and additional understanding of bat-tohuman transmission of a variety of viruses. an efficient covid-19 animal model which closely resembles the human clinical picture can accelerate the path for vaccine development and therapeutics as well as shine light on viral pathogenesis and formulation of preventive strategies. for most diseases, mice and other small rodents have been modified to cater to various demands of the research community. an abundant range of reagents and assays are available, too, for such analysis. the problem specific to sars-cov-2, however, has been the dominant role of the ace2 receptor in covid-19 pathogenesis. the virus so far has not been found to bind to the ace2 receptor of any of the commonly used small animals unless genetically modified. genetic modification is a timeconsuming process, and the rapid spread of sars-cov-2 infection and its compounding socioeconomic effects require more rapid solutions. thus, researchers are shifting interest toward readily available larger animal models. many large animals are closer anatomically and physiologically to humans, can be outbred, show many clinical similarities to the human infection, and very importantly have ace2 receptors recognized by the sars-cov-2 virus. there are several drawbacks, however. no animal model has manifested clinical symptoms as severe as observed in humans, especially regarding the mortality rate. only a few studies could be found which involved an aged animal, 34, 40 where even then no fatality was observed. we conjecture that environmental factors could be at play as animals are typically kept in well-controlled indoor environments, whereas patient morbidity correlates with regions of increased air pollution. 57 the results of therapeutic studies on animals housed in sterile conditions should therefore be interpreted cautiously. also, the animal models discussed above have been tested with sars-cov-2 strains which were clinically available from local human subjects. recent reports show as many as fourteen mutated variants of the spike proteins from different geographical locations, some of which are more virulent than the others. 32 studies on one or two specific local strains may be hard to extrapolate to a global scale. as with many large animal studies, most done for sars-cov-2 were limited in the number of animals tested compared to gold-standard preclinical rodent studies, except a large study of dna vaccine candidates on 35 adult rhesus macaques. 61 these limited numbers, coupled with a lack of investigation into the duration of protection afforded by neutralizing antibodies, leave much work in the development and testing of anti-viral treatments and vaccination strategies. drawbacks notwithstanding, a wide range of animal models with ace2 receptors and serine proteases susceptible to sars-cov-2 are available to study emerging technologies in the global fight against covid-19. with the rapid pace of research on this topic, we can expect the development of new animal models with their own advantages and shortcomings and generation of new and better knowledge about the novel disease to arrive every day. generation and characterization of eptesicus fuscus (big brown bat) kidney cell lines immortalized using the myotis polyomavirus large t-antigen the pathogenicity of sars-cov-2 in hace2 transgenic mice lack of reinfection in rhesus macaques infected with sars-cov-2. microbiology animal models: no model is perfect, but many are useful hepatitis b virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and m2-like macrophages imbalanced host response to sars-cov-2 drives development of covid-19 generation of improved humanized mouse models for human infectious diseases coronaviruses in poultry 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pathogenesis, diagnosis, treatment and vaccine planning humanized mice for immune system investigation: progress, promise and challenges pathogenesis and transmission of sars-cov-2 in golden hamsters guidelines of the american society of mammalogists for the use of wild mammals in research infection of dogs with sars-cov-2 exacerbated innate host response to sars-cov in aged non-human primates on the way from sars-covsensitive mice to murine covid-19 model is there an ideal animal model for sars? severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor pathology of experimental sars coronavirus infection in cats and ferrets precision mouse models with expanded tropism for human pathogens receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus hace2 transgenic mouse model for coronavirus (covid-19) research. the jackson laboratory immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets exposure to air pollution and covid-19 mortality in the united states: a nationwide cross-sectional study mice transgenic for human angiotensinconverting enzyme 2 provide a model for sars coronavirus infection the pathogenesis and treatment of the 'cytokine storm' in covid-19 bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system dna vaccine protection against sars-cov-2 in rhesus macaques age-related rhesus macaque models of covid-19 sars-cov-2 neutralizing serum antibodies in cats: a serological investigation unlocking bat immunology: establishment of pteropus alecto bone marrow-derived dendritic cells and macrophages publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this work was supported by the national institute of general medical sciences (nih) through grant r35gm128831 to css. nothing to disclose. key: cord-306671-stc3pbj8 authors: cardona, carol; travis, dominic a.; berger, kavita; coat, gwenaële; kennedy, shaun; steer, clifford j.; murtaugh, michael p.; sriramarao, p. title: advancing one health policy and implementation through the concept of one medicine one science date: 2015-09-01 journal: glob adv health med doi: 10.7453/gahmj.2015.053 sha: doc_id: 306671 cord_uid: stc3pbj8 numerous interspecies disease transmission events, ebola virus being a recent and cogent example, highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a comprehensive scientific manner. the diversity of information gained from the natural, social, behavioral, and systems sciences is critical to developing and sustainably promoting integrated health approaches that can be implemented at the local, national, and international levels to meet grand challenges. the concept of one medicine one science (comos) as outlined herein describes the interplay between scientific knowledge that underpins health and medicine and efforts toward stabilizing local systems using 2 linked case studies: the food system and emerging infectious disease. forums such as the international conference of one medicine one science (icomos), where science and policy can be debated together, missing pieces identified, and science-based collaborations formed among industry, governmental, and nongovernmental policy makers and funders, is an essential step in addressing global health. the expertise of multiple disciplines and research foci to support policy development is critical to the implementation of one health and the successful achievement of global health security goals. numerous interspecies disease transmission events, ebola virus being a recent and cogent example, highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a comprehensive scientific manner. the diversity of information gained from the natural, social, behavioral, and systems sciences is critical to developing and sustainably promoting integrated health approaches that can be implemented at the local, national, and international levels to meet grand challenges. the concept of one medicine one science (comos) as outlined herein describes the interplay between scientific knowledge that underpins health and medicine and efforts toward stabilizing local systems using 2 linked case studies: the food system and emerging infectious disease. forums such as the international conference of one medicine one science (ico-mos), where science and policy can be debated together, missing pieces identified, and science-based collaborations formed among industry, governmental, and nongovernmental policy makers and funders, is an essential step in addressing global health. the expertise of multiple disciplines and research foci to support policy development is critical to the implementation of one health and the successful achievement of global health security goals. los numerosos acontecimientos de transmisión de enfermedades entre especies, de los cuales el virus del ébola es un ejemplo claro y reciente, ponen de manifiesto las complejas interacciones que existen entre la salud humana, animal y medioambiental, así como la importancia de abordar la medicina y la salud de una manera científica e integral. la diversidad de la información obtenida de las ciencias naturales, sociales, conductuales y de los sistemas es fundamental para el desarrollo y fomento sostenibles de enfoques integrados de salud que puedan implementarse a nivel local, nacional e internacional para atender los grandes retos. el concepto de una medicina una ciencia (concept of one medicine one science, comos) esbozado aquí describe la interrelación entre el conocimiento científico que sustenta la salud y la medicina, y los esfuerzos hacia la estabilización de los sistemas locales por medio de dos estudios de casos relacionados: el sistema alimentario y las enfermedades infecciosas emergentes. los foros como la conferencia internacional de una medicina una ciencia (international conference of one medicine one science, icomos), donde se hace posible el debate conjunto de la ciencia y la política, la identificación de eslabones perdidos, y la formación de colaboraciones basadas en la ciencia entre los formuladores y fundadores de políticas industriales, gubernamentales y no gubernamentales, representan un paso decisivo para abordar el tema de la salud mundial. la experiencia de múltiples disciplinas y enfoques de investigación para apoyar el desarrollo de políticas es fundamental para la implementación de una salud y el logro de los objetivos relativos a la seguridad de la salud mundial. human, animal, and environmental health are inextricably linked in our modern, highly globalized society. we expect better health, secure food, clean water, and more comfort from limited natural resources. balancing competing demands of human health, animal health, and sustainable environmental health is a grand challenge of our time. as the distance between human, domestic animal, and wildlife populations narrows and global trade and travel amplify our connectivity, pathogens shared by humans and animals have concurrently emerged. 1 since 1999, among many examples, humans experienced outbreaks of west nile virus in the united states and europe, severe acute respiratory syndrome (sars) virus in china, middle east respiratory syndrome coronavirus (mers-cov) in the middle east, and the emergence of the ebola virus in west africa. shared pathogen exchange is perhaps best illustrated in the case of influenza, where humans serve as the source of influenza a virus (iav) infection of pigs, and, after further evolution of the virus, it returns from pigs to humans, causing new outbreaks. the 2009 influenza outbreak was the most recent example of this cycle of human and animal infection. the failure to address interspecies transmission and ensuing pandemics as a one health issue has created the opportunity for continued emergence of novel iav like h3n2v that emerged as a human pathogen during the agricultural fair season. 2 these events highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a scientific manner that considers all contributing factors. advancement of health at this level of complexity requires integration of medical disciplines spanning individual care to public health, with basic and applied sciences from atomic structure to sociology providing the knowledge base as shown in the figure. the concept of one medicine one science (comos) builds on the idea that the basic biological processes underlying health and disease share common features such that medical knowledge and expertise in one species is relevant and applicable to other species. the scientific approach that expands our understanding of biological processes and the rational basis for medical practice is the same for all species. thus, comos provides common ground to assist the organization of interdisciplinary groups to seek solutions to health challenges at local and global scales. another aspect of comos is development of forums that, in addition to medical and scientific participants, includes industry, governmental and nongovernmental policy makers, and funders to assist in communicating the interconnectedness of human, animal and environmental health to a broad audience so that development of public policies that guide health priorities and investments at the national and international level are informed by the best scientific knowledge. the concept of one health emphasizes the interconnectedness of animal, environmental and human health, and has become a part of the health security and international development lexicon. 3 the 2005 revised international health regulations and 2014 global health security agenda (ghsa) are 2 of many instruments that cement this connection/relationship/interdependence. at the international launch of the ghsa, the united states and 30 other countries, the world health organization (who), food and agriculture organization (fao), and world organisation for animal health (oie) agreed to work to prevent, detect, and respond to global infectious disease threats from the perspective of one health. years before the release of the ghsa, the us agency for international development established the emerging pandemic threats program to "prevent, detect, and control" animal and human pathogens. these activities, which promote one health approaches in the united states and globally, are an important beginning but fall short of providing the robust scientific and culturally informed approach that is needed to address real world challenges. much dialogue on one health has focused on emerging disease surveillance, public health preparedness, and policy issues without connecting these issues to the scientific foundations that underlie pathogen emergence, global health threats, food security, environmental health, social organization, communication, and implementation of health, security, and safety measures. this limitation has resulted in a perceived and apparent separation of science and policy, sometimes diluting the utility of the one health movement. this article builds on the discussions and dialogue about the science of one health and its connection to policy that were held at the international conference on one medicine one science (icomos; www.icomos.umn.edu) in 2014 and to be held again in 2016. 4 in particular, it was concluded that the key roles of food and medicine in addressing health needs of a changing world require the support of rigorous science that empowers informed decision-making and development of useful policies. the goal of icomos is to examine the connections between science and one health policy implementation by (1) focusing on case studies and research that demonstrate the successful integration of human, animal, plant, and environmental health and social and behavioral sciences; (2) examining the science-policy connection by convening panels that include scientists, policy makers, and representatives from private industry; and (3) conducting forward-looking workshops in areas of research, collaboration, and policy formulation. icomos-2014 was focused on 2 of the most important grand challenges of our time-food safety/security and emerging infectious diseases. 4,5 good policy must be based on objective scientific studies that integrate epidemiology, ecology, microbioriginal article ology, social science, and economics to balance the expectation of safe and nutritious food with the need for efficient and profitable production, and sustained environmental health. given that an estimated 870 million people are currently suffering from malnutrition and that the combination of human population growth and consumer preference shifts associated with a rising global middle class are expected to exacerbate this problem, global production and delivery of food for protein, energy, and micronutrients must not just be maintained, it must be increased. 6 in addition, existing land and water resources are already strained and their availability for agriculture will likely decrease, especially as the impacts of climate change shift traditional production regions. the obvious solution is to produce more efficiently, yet the very strategies that promote efficient production of food, such as concentrated farming systems, monoculture cropping, and chemical inputs of fertilizer, pesticides, and herbicides, have unintended consequences that threaten human, ani-mal, and environmental health. 7 we are faced with a paradox in which the demand for increased food production to improve human health today sows the seeds of resource depletion that limit health advances tomorrow. thus, new research on the direct and indirect impacts of food production on human, animal, and environmental health as well as social, organizational, and behavioral sustainability should be a priority. ensuring that sufficient food is not only safely and efficiently delivered to consumers but that it is produced in ways according to local preferences presents another paradox. consumer demand for local and organic production systems actually increases the greenhouse gas footprint of most developed and many developing country food systems, depending on the crop or animal product [8] [9] [10] and also can increase food safety risks relative to today's widely used production systems. 11 those producers that remain local are often small in size, thus limiting benefits of scale, and many use organic methods, which can limit their ability to prevent disease through approaches such as constructing barriers and facilities for containment. the result is increased contact with wildlife, which increases opportunities for the transmission of infectious diseases to crops, livestock, and humans. for instance, interaction between domestic poultry and wild waterfowl reservoirs has resulted in the introduction of new iav, 12 and the movement of h5 hpai from domestic poultry to free-flying bird populations demonstrates that the exchange can be bidirectional, 13 which changes paradigms of transboundary disease spread. the interface between wildlife and agriculture will only increase as more land is converted for agricultural use to meet consumer demands, thus forcing wildlife into shrinking habitats or to adapt to increased contact with humans and livestock. housing food animals inside helps to protect animal health, prevent cross-species disease transmission, and improve production efficiency, but it does not always meet consumer preference for local needs and resources. nontherapeutic (nonclinical) antibiotic use in food animals is a tool that promotes production efficiency, but it has been associated with a rise in antibiotic resistance of human pathogens. however, epidemiological and ecological studies fail to support a causal link; instead, these studies indicate the spread of antibiotic resistance from humans to pigs and chickens. 14 in the future, the world food summit global food strategy of "providing access for all people at all times to sufficient, safe, and nutritious food to maintain a healthy and active life" must be expanded to include considerations for environmental sustainability, animal welfare and non-nutritional aspects of public health. 15 progress toward these goals requires strong linkage of scientific knowledge and discovery that helps both to advance medical practice and health improvement as well as inform health policy development. human population expansion and exploration has increased as never before, and thus attention to the risk of disease emergence at the interface between humans, domesticated animals, and wildlife is increasingly important. while many human pathogens have wildlife reservoirs, 1 the converse is also an issue. humans and their companion animals have triggered epidemic plagues of canine distemper in serengeti lions 16 and morbillivirus in pacific seals 17 while spreading influenza a to food animal species. 18 other dramatic changes include the explosive growth in global travel and trade patterns and legal and illegal trade in live animals, animal products, and wildlife as well as effects of climate change on all of the above. retrospective and emerging analyses may provide insights into these systems. however, focusing on the processes and mechanisms that facilitate pathogen evolution and emergence will help us prepare for unpredictable new outbreaks, thus raising awareness of prevention and control policies that could reduce both the likelihood and magnitude of disease emergence and its resulting effects on animals and humans. single approaches to comprehend and anticipate disease emergence, especially using simplified disease models, are unlikely to produce informative insights involving environmental factors, multiple reservoirs, and complex human-animal-environment interactions. 19 concepts of wildlife reservoirs and spill-over/ cross-species transmission into food animal and human populations followed by expansion and outbreak potential have been developed into mathematical models. although not a predictor in itself, models of zoonotic transmission dynamics have made valuable contributions to inform public health recommendations for control of novel h1n1 influenza and sars and are crucial for understanding pathogen transmission patterns and changes in disease epidemiology. 20 human orthopoxvirus infection is a highly relevant example in which modeling based on rigorously collected data provides prevention strategies for a highly plausible emergent disease. the global human population is increasingly susceptible to smallpox due to the cessation of vaccination programs following eradication. loss of crossreactive immunity has opened an ecological niche that can and has been replaced by related orthopoxviruses, including monkeypox in the congo and novel orthopoxvirus in the country of georgia. 21 emergence of new orthopoxvirus outbreaks with pandemic potential is possible as human-infectious poxviruses circulate in natural reservoirs. the recent discovery of viable smallpox in a us food and drug administration refrigerator on the national institutes of health (nih) campus recently reinvigorated the discussion of both susceptibility and/or risks from infections to smallpox. 22 the specter of a new pandemic, fueled by relatively recent experience with the emergence of aids and new influenza viruses, has driven the us centers for disease control and prevention, the nih-national institute of allergy and infectious diseases, the us department of agriculture, the us agency for international development, and others to develop programs focused on predictive modeling of and early detection and response to novel pathogens that have evolved under a complex set of pressures such as environmental pollution, land use changes, new trade patterns, climatic change, and other anthropogenic factors. the role of medicine in addressing individual and public health needs to be supported by rigorous science that empowers informed decision-making and development of useful policies in the face of unexpected disease threats. the rapid growth and persistence of the ongoing outbreak of ebola virus in west africa presents an unfortunate but perfect opportunity for implementing science-based policy at the crossroads of emerging infectious disease ecology and sustainable food security. experts hypothesize that ebola virus entered the west african population through consumption of original article infected fruit bats, a plausible scenario given local food needs and practices. 23, 24 the ongoing outbreak has stressed social organization and an already thin public health infrastructure to the breaking point in the affected countries. it threatens to exacerbate baseline food insecurity as disease control measures and fear shut down food and agriculture systems, further stressing local food security. 25 this situation makes for the "perfect storm" of grand challenges in the one health arena. frighteningly, while more than 27 000 cases and 11 200 deaths have been recorded as of july 14, 2015, a new ecological niche risk model predicts that 22 million people in 22 central and west african countries are at potential risk for ebola. 26, 27 while the world seems to be stepping up with much-needed scientific, medical, and infrastructure support to this tragedy, broader policies and preparedness are needed for prevention, containment, and response if we are to avoid more extreme human suffering from the virus itself as well as malnutrition, community instability, and other social consequences. addressing the grand challenges of our timesuch as novel disease emergence and food security-is difficult from any perspective. but the universal scientific discovery and exploration approaches that have been applied so effectively to resolve focused problems offer promise in addressing the complex health issues of modern life. a linear refocusing of health research away from disease surveillance and investigation resulting primarily in treatment toward environmental surveillance resulting in prediction and prevention is not enough to inform societal debates on these complex health issues. reducing challenges to simple scenarios allows us to find solutions that do not address the complex problems we face but instead bring on a myriad of unintended and undesirable consequences. grand challenges do not have simple solutions. rather, they represent "wicked problems" that are complex and difficult-to-balance dilemmas with fluid dynamics whose competing components change over time. 28 the creation of forums for convergence on grand challenges where science and policy can be debated together, missing pieces identified, and science-based collaborations stimulated in the presence of industry, governmental and nongovernmental policy makers, and funders is an essential step. the underpinnings of comos-ie, promoting team science that builds on the expertise of different disciplines, stakeholders, and research foci to support policy development-is all the more relevant to the implementation of one health and the successful achievement of global health security goals (figure) . universal scientific discovery and exploration approaches that have been applied so effectively to advance human society will need to be applied to complex health issues of modern life so that the resulting knowledge can inform public policy and decision-making at every level. global trends in emerging infectious diseases swine-to-human transmission of influenza a(h3n2) virus at agricultural fairs one health: scientific and technical review one medicine one science: a framework for exploring challenges at the intersection of animals, humans and the environment one medicine one science and policy households across all income quintiles, especially the poorest, increased animal source food expenditures substantially during recent peruvian economic growth solutions for a cultivated planet the effect of feed demand on greenhouse gas emissions and farm profitability for organic and conventional dairy farms the environmental impact of recombinant bovine somatotropin (rbst) use in dairy production the impact of organic farming on food security in a regional and global perspective food safety and organic meats epizootiology of avian influenza-simultaneous monitoring of sentinel ducks and turkeys in minnesota avian flu: h5n1 virus outbreak in migratory waterfowl sources of antimicrobial resistance world health organization viruses of the serengeti: patterns of infection and mortality in african lions mass die-off of caspian seals caused by canine distemper virus global transmission of influenza viruses from humans to swine nine challenges in modelling the emergence of novel pathogens epidemic dynamics at the human-animal interface vacated niches, competitive release and the community ecology of pathogen eradication safety lapses in us government labs spark debate fruit bats as reservoirs of ebola virus ebola virus disease: from epidemiology to prophylaxis impact of the west african ebola virus outbreak on food security who ebola data and statistics. situation summary mapping the zoonotic niche of ebola virus disease in africa. elife dilemmas in a general theory of planning key: cord-336464-eslgz1ka authors: chomel, bruno b.; belotto, albino; meslin, françois-xavier title: wildlife, exotic pets, and emerging zoonoses date: 2007-01-17 journal: emerg infect dis doi: 10.3201/eid1301.060480 sha: doc_id: 336464 cord_uid: eslgz1ka most emerging infectious diseases are zoonotic; wildlife constitutes a large and often unknown reservoir. wildlife can also be a source for reemergence of previously controlled zoonoses. although the discovery of such zoonoses is often related to better diagnostic tools, the leading causes of their emergence are human behavior and modifications to natural habitats (expansion of human populations and their encroachment on wildlife habitat), changes in agricultural practices, and globalization of trade. however, other factors include wildlife trade and translocation, live animal and bushmeat markets, consumption of exotic foods, development of ecotourism, access to petting zoos, and ownership of exotic pets. to reduce risk for emerging zoonoses, the public should be educated about the risks associated with wildlife, bushmeat, and exotic pet trades; and proper surveillance systems should be implemented. deforestation, development of human habitat, and mining activities have been suggested as risk factors associated with the reemergence of vampire bat rabies in humans in the amazon basin. in 2004, 46 persons died of rabies transmitted by vampire bats, mainly in brazil (22 cases) and colombia (14 cases) ; only 20 human cases of rabies were transmitted by dogs in all latin america (9) . a similar trend was again observed for 2005. when fi rst described in 1957, kyasanur forest disease was restricted to a much smaller area (300 square miles) in india than the actual 2,000 square miles of endemic zone (10) . this tickborne disease occurs in evergreen rain forests interspersed with deciduous patches and clearings for rice cultivation and human habitations. forest workers are particularly at risk; their mortality rates may reach 10%. in 1983, a major epidemic occurred during which several monkeys died, 1,555 humans were infected, and 150 humans died. the outbreak occurred in previously undisturbed forest where some 400 ha were clearcut to establish a cashew tree plantation. most of the human patients were immigrant laborers employed to clear the forest (10) . as many as 1,000 human cases occur each year, and this number has increased in the past 5 years. most cases occur during the dry season (january-may), when nymphal activity is maximal. such a zoonosis is a good example of deforestation and agricultural development leading to human habitat expansion into natural foci of a viral infection. because cleared areas were widely used for grazing of cattle, a major host for adult ticks, these areas favored the proliferation of the tick haemaphysalis spinigera. conversely, the reduction of traditional agricultural land and its replacement with forested areas, home to the main reservoirs and hosts of borrelia burgdorferi, in association with the settlement of persons in periurban areas, led to a considerable increase in human cases of lyme disease in the united states (11) . an estimated 32.4 million wild ruminants, major amplifi ers for adult ixodes scapularis ticks, live in north america (12) . from an estimated 23-40 million white-tailed deer inhabited north america before the arrival of europeans, the deer population was greatly reduced by habitat loss and unrestricted hunting. however, by the mid-20th century, the population was restored throughout north america, and an estimated 14-20 million white-tailed deer are believed to inhabit the united states alone. in many areas of the eastern united states, populations have soared to previously unattained levels (www.aphis.usda.gov/ws/nwrc/is/living/deer.pdf). human activities may also be a source of wildlife infection, which could create new reservoirs of human pathogens. the recent outbreak of tuberculosis caused by mycobacterium tuberculosis in suricats and mongooses was one of the fi rst documented spillovers of a human disease within a wildlife population (13) . banded mongooses were observed feeding regularly at garbage pits and were therefore exposed to human excretions and any infectious material from tuberculosis-infected humans. the emergence of argentine hemorrhagic fever in eastcentral argentina during the 1950s, and its expansion to north-central argentina, has been directly linked to development of agricultural activities (mainly corn growing) that sustain the virus's main reservoir, the corn mouse (calomys musculinus). caused by the junin virus, argentine hemorrhagic fever affects primarily adult male agricultural workers, mainly during the harvest season (14) . in the late 1970s and early 1980s, a rabies epidemic occurred in free-ranging greater kudus (tragelaphus strepsiceros) in namibia (15) . the kudu population had increased considerably in response to favorable conditions and human-made environmental changes. suitable conditions for transmission in the kudu population after initial infection by rabid carnivores are provided by the social behavior of kudus, such as browsing on thorny acacia trees and resultant lesions in the kudus' oral cavity, and excretion of relatively high titers of virus in the saliva of infected animals (15) . the outbreak of nipah virus infection in malaysia during 1998-1999, which caused 265 human cases of viral encephalitis and a 38% mortality rate, was also the result of several major ecologic and environmental changes associated with deforestation and expansion of nonindustrial pig farming in association with production of fruit-bearing trees (16) . such combination led to infection of pigs, which developed respiratory and neurologic symptoms after indirect exposure to infected fruit bats that shed the virus. the sick pigs were a subsequent source of human infection (16) . farming of wild animal species led to reemergence of zoonoses such as bovine tuberculosis in captive deer populations. deer at low population densities on natural range are less likely to be affected to any major extent by disease. however, disease becomes a factor in intensive management of deer (17) . reemergence of zoonotic diseases that had been controlled from their domestic animal reservoirs is also of major concern. wildlife may become new reservoirs of infection and may recontaminate domestic animals; examples include bovine tuberculosis in the united kingdom associated with mycobacterium bovis infection in badgers (meles meles) (18) and brucellosis in outdoor-reared swine in europe that resulted from spillover from the wild boar brucellosis (brucella suis biovar 2) reservoir (19) . wildlife trade provides mechanisms for disease transmission at levels that not only cause human disease out-breaks but also threaten livestock, international trade, rural livelihoods, native wildlife populations, and ecosystem health (7) . worldwide, an estimated 40,000 primates, 4 million birds, 640,000 reptiles, and 350 million tropical fi sh are traded live each year (7) . international wildlife trade is estimated to be a us $6-billion industry (20) . translocation of wild animals is associated with the spread of several zoonoses. rabies was introduced in the mid-atlantic states in the 1970s when hunting pens were repopulated with raccoons trapped in rabies-endemic zones of the southern united states (21) . in eastern europe, raccoon dogs (nyctereutes procyonoides) are becoming a new reservoir for rabies, in addition to the established red fox reservoir, as raccoon dogs have spread into new habitats from accidental release of animals raised for fur trade (22) . brush-tailed possums (trichosurus vulpecula) from tasmania were introduced into new zealand to establish a new species of fur-bearing animals. the translocated population proliferated and is now estimated to be >70 million, of which 3%-30% are possibly infected by m. bovis, a permanent threat to the cattle-and deer-farming industries (21) . translocation of hares from central and eastern europe for sporting purposes led to several outbreaks of tularemia, introduction of b. suis biovar 2 to western europe, and subsequent encroachment of this brucellosis strain into the wild boar population of western europe (19) . during 1993-2003, b. suis biovar 2 infections were reported in >40 outdoor-rearing pig farms in france (19) . illegal trade can also be a possible source of human infection. in march 1994, psittacosis developed in several customs offi cers in antwerp, belgium (23) . a customs officer had been hospitalized with pneumonia 10 days after exposure to parakeets illegally imported by an indian sailor. the risk of contracting psittacosis was 2.8× higher for offi cers exposed to parakeets >2 hours than for those exposed only briefl y. similarly, a highly pathogenic avian infl uenza a h5n1 virus from crested hawk eagles smuggled into europe by air travel has been isolated and characterized (24); fortunately, however, screening of human and avian contacts indicated that no dissemination had occurred. another risk factor related to the emergence of zoonotic diseases from wildlife has been the considerable increase in consumption of bushmeat in many parts of the world, especially central africa and the amazon basin, where 1-3.4 million tons and 67-164 million kilograms, respectively, are consumed each year (7) . the simian foamy virus has been identifi ed as a zoonotic retrovirus that infects people who have direct contact with fresh nonhuman primate bushmeat; this fi nding indicates that such zoonoses are more frequent, widespread, and contemporary than previously appreciated. similarly, new retroviruses, human t-lymphotropic virus types 3 and 4 were found in persons who hunt, butcher, or keep monkeys or apes as pets in southern cameroon (25) . the combination of urban demand for bushmeat (a multibillion-dollar business) and greater access to primate habitats provided by logging roads has increased the amount of hunting in africa, which has increased the frequency of human exposure to primate retroviruses and other disease-causing agents. similarly, several outbreaks of ebola virus in western africa have been associated with consumption of bushmeat, mainly chimpanzees that were found dead (26) . traditional and local food markets in many parts of the world can be associated with emergence of new zoonotic diseases. live animal markets, also known as wet markets, have always been the principal mode of commercialization of poultry and many other animal species. such markets, quite uncommon in the united states and, until recently, in california, are emerging as a new mode of commercialization within specifi c ethnic groups for whom this type of trade assures freshness of the product but raises major public health concerns. the avian infl uenza epidemic, which began in southeast asia in 2003 and recently spread to other parts of the world, is directly related to infected birds sold live in traditional markets. live bird markets facilitate the spread of this avian h5n1 virus by wild birds (27) . similarly, the newly discovered severe acute respiratory syndrome-associated coronavirus was linked to trade of live, wild carnivores, especially civets, in the people's republic of china (2). however, recent data suggest that civets may be only amplifi ers of a natural cycle involving trade and consumption of bats (28) . trichinellosis has long been associated with consumption of undercooked meat from wild animals, such as bears, and now consumption of uncooked meat from deer and wild boar has recently been associated with emergence of severe cases of hepatitis e in hunters in japan (29) . industrialized nations' new taste for exotic food has also been linked with various zoonotic pathogens or parasites, such as protozoa (toxoplasma), trematodes (fasciola sp., paragonimus spp.), cestodes (taenia spp., diphyllobothrium sp.), and nematodes (trichinella spp., anisakis sp., parastrongylus spp.). adventure travel is the largest growing segment of the leisure travel industry; growth rate has been 10% per year since 1985 (adventure travel society, pers. comm.). this type of travel increases the risk that tourists participating in activities such as safaris, tours, adventure sports, and extreme travel will contact pathogens uncommon in industrialized countries. the most commonly encountered rickettsial infection in travel medicine is african tick bite fever, caused by rickettsia africae and transmitted in rural sub-saharan africa by ungulate ticks of the amblyomma genus; >350 imported cases have been reported from several continents during the past few years (30) . most patients are infected during wild game safaris and bush walks. moreover, because ecotourism is becoming increasingly popular with international travelers, more cases of imported rickettsioses are likely to occur in europe, north america, and elsewhere in years to come. cercopithecine herpesvirus 1 (herpes b virus) is an alpha herpesvirus endemic to asian macaques, which mostly carry this virus without overt signs of disease. however, zoonotic infection with herpes b virus in humans usually results in fatal encephalomyelitis or severe neurologic impairment (31) . herpes b virus has been implicated as the cause of ≈40 cases of meningoencephalitis in persons who had direct or indirect contact with laboratory macaques. a survey of workers at a balinese hindu temple, a major tourist attraction where macaques roam free, showed that contact suffi cient to transmit b virus occurred commonly between humans and macaques. furthermore, 31 (81.6%) of 38 macaques at that location had antibodies to herpes b virus (32) . petting zoos, where children are allowed to approach and feed captive wildlife and domestic animals, have been linked to several zoonotic outbreaks, including infections caused by escherichia coli o157:h7, salmonellae, and coxiella burnetii (33) . more than 25 outbreaks of human infectious diseases associated with visitors to animal exhibits were identifi ed during 1990-2000 (33) . in an outbreak of salmonellosis at a colorado zoo, 65 cases (most of them in children) were associated with touching a wooden barrier around the komodo dragon exhibit. salmonella organisms were isolated from 39 case-patients, a komodo dragon, and the wooden barrier. children who did not become infected were more likely to have washed their hands after visiting the exhibit (34) . exposure to captive wild animals at circuses or zoos can also be a source of zoonotic infection. twelve circus elephant handlers at an exotic animal farm in illinois were infected with m. tuberculosis, and 1 had signs consistent with active disease after 3 elephants died of tuberculosis. medical history and testing of the handlers indicated that the elephants had been a probable source of exposure for most of the infected persons (35) . after an m. bovis outbreak in rhinoceroses and monkeys at a zoo in louisiana, 7 animal handlers, previously negative for tuberculosis, had positive test results (36) . exotic pets are also a source of several human infections that vary from severe monkeypox related to pet prairie dogs or lyssaviruses in pet bats to less severe but more common ringworm infections acquired from african pygmy hedgehogs or chinchillas. epidemiologic and animal traceback investigations confi rmed that the fi rst community-acquired cases of monkeypox in humans in the united states (71 cases) resulted from contact with infected prairie dogs that had been housed or transported with african rodents imported from ghana (3) . similarly, an outbreak caused by francisella tularensis type b occurred among wild-caught, commercially traded prairie dogs; f. tularensis antibodies in 1 exposed person documented the fi rst evidence of tularemia transmission from prairie dog to human (37) . african pygmy hedgehogs have been implicated in human salmonellosis cases in the united states and canada (38) . in the united states, the number of commercialized reptiles, especially iguanas, imported per year has increased considerably to ≈1 million. the number of human cases of salmonellosis, especially in very young children, increased dramatically in parallel with iguana pet ownership. the centers for disease control and prevention estimates that ≈7% of human infections with salmonellae in the united states are associated with having handled a reptile. most iguanas have a stable mixture of salmonella serotypes in their intestinal tract and intermittently or continuously shed salmonella organisms in their feces (39) . eight cases of rabies caused by a new rabies virus variant were reported in the state of ceará, brazil, from 1991 through 1998. marmosets (callithrix jacchus jacchus) were determined to be the source of exposure. these primates are common pets; most cases occurred in persons who had tried to capture them, and 1 case was transmitted by a pet marmoset (40) . in 1999, encephalitis was diagnosed in an egyptian rousette bat (rousettus egyptiacus) that had been imported from belgium and sold in a pet shop in southwestern france. the pet bat was infected with a lagos bat lyssavirus and resulted in the treatment of 120 exposed persons (y. rotivel, pers. comm.). emerging infectious diseases have a major effect on human health and can create tremendous economic losses. animals, particularly wild animals, are thought to be the source of >70% of all emerging infections (41) . leading factors for emergence of zoonoses are unbalanced and selective forest exploitation and aggressive agricultural development associated with an exponential increase in the bushmeat trade (8) . similarly, the increase of ecotourism, often in primitive settings with limited hygiene, can be associated with the acquisition of zoonotic agents. therefore, development of appropriate programs for surveillance and for monitoring emerging diseases in their wildlife reservoirs is essential. most animal pathogens for which surveillance programs exist relate to farm animals, and few or no programs are specifi cally aimed at wildlife. two different but complementary approaches are 1) to monitor the pres-ence of specifi cally identifi ed pathogens that have emerged as human pathogens and 2) to investigate in a given wildlife species the presence of known or unknown infectious agents. furthermore, conservation of habitat biodiversity is critical for preventing emergence of new reservoirs or amplifi er species. key measures for reducing the dispersion of emerging zoonoses include sustainable agricultural development, proper education of tourists about the risks of outdoor activities, and better control of the live animal trade (exotic pets, wet markets, bushmeat). public health services and clinical practitioners (physicians, veterinarians) need to more actively educate the public about the risks of owning exotic pets and adopting wild animals. as suggested by kuiken et al. (41) , it is time to form "a joint expert working group to design and implement a global animal surveillance system for zoonotic pathogens that gives early warning of pathogen emergence, is closely integrated to public health surveillance and provides opportunities to control such pathogens before they can affect human health, food supply, economics or biodiversity." major tasks that should be taken by the international community include better integration and coordination of national surveillance systems in industrialized and developing countries; improved reporting systems and international sharing of information; active surveillance at the interface of rural populations and wildlife habitats, especially where poverty and low income increase risks for pathogen transmission; training of professionals, such as veterinarians and biologists, in wildlife health management; and establishment of collaborative multidisciplinary teams ready to intervene when outbreaks occur. dr chomel is director of the world health organization/pan american health organization collaborating center on new and emerging zoonoses at the university of california, davis. his research focuses on zoonotic infections, especially those caused by bartonella spp., in domestic animals and wildlife and their effects on human health. risk factors for human disease emergence conservation medicine and a new agenda for emerging diseases update: multistate outbreak of monkeypox-illinois emerging or reemerging bacterial zoonoses: factors of emergence, surveillance and control diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence the value of wildlife wildlife trade and global disease emergence bushmeat hunting, deforestation, and prediction of zoonoses emergence epidemiologic situation of human rabies in latin america in 2004 the encyclopedia of arthropod-transmitted infections emerging bacterial zoonotic and vector-borne diseases. ecological and epidemiological factors wildlife diseases and population medicine mycobacterium tuberculosis: an emerging disease of freeranging wildlife arenaviruses other than lassa virus rabies in the kudu antelope (tragelaphus strepsiceros) anthropogenic environmental change and the emergence of infectious diseases in wildlife advances in health and welfare of farmed deer in new zealand bovine tuberculosis and badger blame from the discovery of the malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis exotic-pet trade disease risks associated with wildlife translocation projects epidemiological surveillance of rabies in lithuania from 1986 to 1996 a psittacosis outbreak in customs offi cers in antwerp (belgium) highly pathogenic h5n1 infl uenza virus in smuggled thai eagles emergence of unique primate t-lymphotropic viruses among central african bushmeat hunters isolation and phylogenetic characterization of ebola viruses causing different outbreaks in gabon transmission of avian infl uenza viruses to and between humans bats are natural reservoirs of sars-like coronaviruses complete or near-complete nucleotide sequences of hepatitis e virus genome recovered from a wild boar, a deer, and four patients who ate the deer rickettsioses and the international traveler b-virus (cercopithecine herpesvirus 1) infection in humans and macaques: potential for zoonotic disease human exposure to herpesvirus b-seropositive macaques reports of zoonotic disease outbreaks associated with animal exhibits and availability of recommendations for preventing zoonotic disease transmission from animals to people in such settings an outbreak of salmonellosis among children attending a reptile exhibit at a zoo mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants epizootic of mycobacterium bovis in a zoologic park first reported prairie dog-to-human tularemia transmission hedgehog zoonoses prevalence of fecal shedding of salmonella organisms among captive green iguanas and potential public health implications alves araujo fa, de mattos ca public health: pathogen surveillance in animals key: cord-339341-c2o42b5j authors: matibag, gino c.; igarashi, manabu; la porte, ron e.; tamashiro, hiko title: advocacy, promotion and e-learning: supercourse for zoonosis date: 2005-09-01 journal: environmental health and preventive medicine doi: 10.1007/bf02897702 sha: doc_id: 339341 cord_uid: c2o42b5j this paper discusses the history of emerging infectious diseases, risk communication and perception, and the supercourse lectures as means to strengthen the concepts and definition of risk management and global governance of zoonosis. the paper begins by outlining some of the key themes and issues in infectious diseases, highlighting the way which historical analysis challenges ideas of the ‘newness’ of some of these developments. it then discusses the role of risk communication to public accountability. the bulk of the paper presents an overview of developments of the internet-based learning system through the supercourse lectures that may prove to be a strong arm for the promotion of the latest medical information particularly to developing countries. we live in a dangerous world. yet it is also a world far safer in many ways than it has ever been. diseases that only recently were mass killers have been all but eradicated. advances in public health, medicine, environmental regulation, food safety, and worker protection have dramatically reduced many of the major risks we faced just a few decades ago. governance refers to how societies structure responses to the challenges they face. analyses of emerging and re-emerging infectious diseases (eids) have made it clear that national and international societies are confronting increased microbial threats (1) (2) (3) . whether the focus is bioterrorism, hiv/aids, severe acute respiratory syndrome (sars), or avian influenza, germs increasingly pose dangers to human societies. germ governance concerns how societies, both within and beyond national borders, structure their responses to pathogenic challenges (4) . the global nature of the microbial threat requires that governance address the borderless challenges presented by infectious diseases. the emergence of sars is a major global public health threat that requires a coordinated global response in terms of continued and improved surveillance and of research into a number of important public health issues. while much has been learnt about sars since it was brought to international attention in march 2003, there remain many unanswered questions about where it came from, how it spreads, and the effectiveness of public health and other measures employed to control the disease. the overall goal of the "supercourse for zoonosis" is to show the most recent development in the knowledge of sars and other zoonotic diseases such as avian influenza and bovine spongiform encephalopathy (bse), inter alia, which have significant global impact not only on health but also on the economy. the specific objectives of "supercourse for zoonosis" are to develop a set of educational materials for the control of zoonotic diseases, to disseminate them effectively via the internet, to facilitate their use in the prevention and control of the diseases, and to promote human health while minimizing their economic impact. in the light of all these advances, it is most appropriate that all countries remain vigilant, not only with sars, but also to all zoonotic diseases that may toll the productivity of human, animal, ecological, and economical sectors of our daily lives. emerging infections (eis) can be defined as infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range (5) . re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range (2) . the term deliberately emerging refers to both naturally occurring microbial agents such as anthrax (3) , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. about 15 million (more than 25%) of 57 million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis b or c viruses (6) . the burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries (7), and particularly on infants and children (about three million children die each year from malaria and diarrheal diseases alone (6)). in developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities (8) . bacteria and viruses existed long before humans evolved, and bacterial diseases probably co-evolved with each species. many bacterial diseases that we see today have been around for as long as we have, others may have developed later. many examples can be cited in addition to the black death and the 1918 influenza pandemic, such as certain biblical pharaonic plagues and the unidentified plague of athens, which heralded the end of greece's golden age (9) . importation of smallpox into mexico caused 10-15 million deaths in 1520-1521, effectively ending aztec civilization (10, 11) . with the beginning of microbiology, pathogens became apparent. the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases (12) (13) (14) led to enormous progress, notably in the development of vaccines and ultimately of antimicrobials (14) . by the 1950s, which had witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in (15) , and in 1967, the us surgeon general stated that the war against infectious diseases has been won (16) . some experts remained skeptical, aware of the current lessons from history. they were less persuaded by successes than alarmed by failures, such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. the emergence of aids led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases (17) (18) (19) (20) (21) (22) (23) . in the past 25 years, some of the factors that resulted in aids have also led to the re-emergence of historically important diseases such as cholera, diphtheria, trench fever and plague. many re-emergences have been catalyzed by wars, loss of cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants (17) (18) (19) (20) (21) (22) (23) . these infections are those that have not previously been recognized in man. many diverse factors contribute to their emergences (table 1) . numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. at the end of 2004, an estimated 39.4 million (range 35.9-44.3 million) people around the world were living with hiv, including the 4.9 million (range 4.3-6.4 million) people who acquired hiv in 2004. the epidemic claimed an estimated 3.1 million (range 2.8-3.5 million) lives in 2004. sub-saharan africa remains the most affected region and is home to about 65% of the total number of people living with hiv worldwide (18) . before jumping to humans an estimated 60-70 years ago (24) , perhaps through consumption of 'bush meat' from nonhuman primates, hiv-1 and hiv-2 had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, pan troglodytes, and the sooty mangabey, cercocebus atys). but hiv/aids may never have emerged had it not been for disruptions in the economic and social infrastructure in postcolonial sub-saharan africa. increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure, all these promoted sexual practices such as promiscuity and prostitution that facilitate hiv transmission (24) (25) (26) (27) . infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important eis (17, 18) . viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioral factors. farming, the keeping of domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts (17) (18) (19) (20) (21) (22) (23) . examples include the arenavirus hemorrhagic fevers (argentine, bolivian, venezuelan and lassa hemorrhagic fevers) and hantavirus pulmonary syndrome (hps). variant creutzfeldt-jakob disease (vcjd) is another example of a zoonotic disease emerging in humans. it is caused by the human-adapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (bse), commonly known as mad cow disease. the new bse prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. this suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans (28) . infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses. legionnaire's disease, first identified in 1976, is caused by legionella pneumophilia, whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems (18) . campylobacter jejuni and escherichia coli infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. other enteric pathogens, such as the vibrios which cause classic cholera and the zoonotic protozoa cryptosporidium parvum and cyclospora cayetanensis (18), seem to have come from environmental or animal organisms that have adapted to human-to-human 'fecal-oral' transmission through water. some eis come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between 1830 and 1900 (29) . scarlet fever, then the leading cause of death in children, is now rare, but has largely been supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever (31) . streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex. many factors associated with streptococcal virulence have been identified in strains bearing the m1 surface protein as well as in other m protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection (31, 32) . infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. examples include the associations of hepatitis b and c with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of epstein-barr virus with burkitt's lymphoma (largely in africa) and nasopharyngeal carcinoma (in china), of human herpesvirus 8 with kaposi sarcoma, and helicobacter pylori with gastric ulcers and gastric cancer (33) (34) (35) . some data even suggest infectious etiologies for cardiovascular disease and diabetes mellitus (36) , major causes of death and disability worldwide. other associations between infectious agents and idiopathic chronic diseases will inevitably be found. re-emergence is caused by some of the factors that allow for newly emerging infectious diseases, factors such as microbial evolutionary vigor, zoonotic encounters and environmental encroachment. re-emergences or at least cyclical resurgences of some diseases may also be climate-related-for example, the el niño/southern oscillation (enso) phenomenon is associated with resurgences of cholera and malaria (37) . travel has an important role in bringing people into contact with infectious agents (38) . an increase in travelassociated importations of diseases was anticipated as early as 1933, when commercial air travel was still in its infancy (39) . this has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute hemorrhagic conjunctivitis in 1981 (40), by epidemics of meningococcal meningitis associated with the hajj, and more recently by the exportation of epidemic sars (a newly emerging disease) from guangdong province, china, to hong kong, and from there to beijing, hanoi, singapore, toronto and elsewhere (41) . plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide. years of effective use of dichlorodiphenyltrichloroethane (ddt) had led to the abandonment of other mosquito-control programs, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine (42) . research efforts focus on the development of vaccines and new drugs, and on re-establishing public health measures such as the use of bed nets (43) . the remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with hiv infection, which greatly increases the risk of latent mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug resistant and multidrug-resistant strains, and a need for more extensive treatment strategies such as directly observed therapy (44) . it has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. drug resistance, another factor causing microbial and viral re-emergence, may result from mutation or from bacterial acquisition of extraneous genes through transformation or infection with plasmids. sequential emergences of staphylococcus aureus that are resistant to sulpha drugs (1940s), penicillin (1950s), methicillin (1980s) and to vancomycin in 2002 (45)-a last line of antibiotic defense for some multiply drug-resistant bacteria-are troubling. nosocomial enterococcus faecalis became fully resistant to vancomycin by 1988, and then apparently transferred vana resistance genes to co-infecting staphylococci (46) . methicillin-resistant staphylococci are now being isolated from livestock that have been fed with growthpromoting antibiotics (45) , possibly contributing to resistance problems in humans. immune deficiency associated with hiv infection, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than 1% of the world's population), setting the stage for the re-emergence of many opportunistic infections. hiv, which has infected more than 60 million people globally (47) , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesvirus (48) . the simultaneous 1999 emergences of encephalitis due to west nile virus (wnv) in the united states and russia (49, 50) reflect abundances of eclectic vector mosquitoes and avian hosts in these locations. both were probably connected to endemic sites by virus carriage in migratory birds and travelers. the remarkable geographical spread of wnv in the five years since its introduction into the western hemisphere reflects an unfortunate confluence of viral promiscuity and ecological diversity (51) . although humans are dead-end hosts for wnv, the risk of infection is greatly increased by marked zoonotic viral amplification and persistence in the environment. although wnv is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally (52) . usually transmitted by aedes aegypti mosquitoes, dengue has recently been transmitted by aedes albopticus-a vector switch of potential significance with respect to dengue reemergence (52) . dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue hemorrhagic fever (dengue shock syndrome being its highly fatal form). these severe complications are thought to result from the evolution of dengue viruses to escape high population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection (53) . cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto-and zooplankton, algae and crustaceans. such environmental strains seem to act as reservoirs for human virulence genes and to undergo gene transfer events that lead to new strains containing further virulence gene combinations (54) . thus, although cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the 1840s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. influenza a viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts'. these can lead to deadly pandemics, as occurred in 1888, 1918, 1957 and 1968 (55, 56) . in intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts,' which allow them partially to escape human immunity raised by previously circulating influenza viruses. influenza drift is an evolutionary success story for the virus. influenza a has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral hemagglutinin site that attaches to human cells. deliberately emerging microbes are those that have been developed by humans, usually for nefarious use. they include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity (57) . two modern attacks have been well documented. in 1984, an oregon religious cult spiked restaurant salad bars with salmonellae in an attempt to sway a local election (58) . a 2001 anthrax attack (59) , in which a terrorist mailed anthrax-sporefilled letters to prominent figures, including two us senators, resulted in illness in at least 18 people and the death of five of these individuals. the united states, the united kingdom, the russian federation and other nations once had sophisticated offensive bioweapons programs that included the production of weaponized anthrax spores (57) . in japan, the doomsday sect, aum supreme truth, carried out a nerve-gas attack on the tokyo subway in 1995, and made a trial run on an anthrax weapon, using harmless vaccine bacteria as a test (60) . bioterror agents have been grouped into three categories (a, b and c) according to risk (61) . the six category a agents (anthrax, smallpox, plague, tularaemia, viral hemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. category b and c agents include food-borne and water-borne organisms that incapacitate but usually do not kill. risk is the probability that exposure to a hazard will lead to a negative consequence (62) . risk communication is an interactive exchange of information and opinion on risk among risk assessors, risk managers, and other interested parties (63) . humans tend to fear similar things, for similar reasons. scientists studying human behavior have discovered psychological patterns in the subconscious ways we "decide" what to be afraid of and how afraid we should be. any given risk has a set of identifiable characteristics that help predict what emotional responses that risk will trigger. people's perceptions of the magnitude of risk are influenced by factors other than numerical data (64) . the factors influencing risk perception are summarized in table 2 . merely disseminating information without regard for communicating the complexities and uncertainties of risk does not necessarily ensure effective risk communication. wellmanaged efforts will help ensure that messages are constructively formulated, transmitted, and received and that they result in meaningful actions. the seven cardinal rules of risk communication are demonstrated in table 3 . the fundamental goal of risk communication is to provide meaningful, relevant and accurate information, in clear and understandable terms, targeted to a specific audience (63) . the goals of risk communication are summarized in table 4 . it may not resolve all differences between interested parties, but may working in earthquake-prone areas hormone replacement therapy 4. risks perceived to be fairly distributed are more accepted than risks to be unfairly distributed. gun shots (in japan) traffic accidents 5. risks perceived to be natural are more accepted than risks perceived to be manmade. radiation from mobile phones radiation from the sun 6. risks perceived to be statistical are more accepted than risks perceived to be catastrophic. eaten by a shark (catastrophe) heart disease (statistics) 7. risks perceived to be generated by a well-known source are more accepted than risks perceived to be generated by a less known source. private industry government 8. risks perceived to be familiar are more accepted than risks perceived to be exotic. 9. risks perceived to affect adults are more accepted than risks perceived to affect children. asbestos exposure for children asbestos exposure in workplace 10. risks perceived with less uncertainty are more accepted than risks with high uncertainty. new technology conventional technology 11 . risks perceived that could directly affect others are more accepted than risks that could affect oneself. table 3 seven cardinal rules of risk communication* 1. accept and involve the public as a partner. the goal is to produce an informed public, not to defuse public concerns or replace actions. different goals, audiences, and media require different actions. 3. listen to the public's specific concerns. people often care more about trust, credibility, competence, fairness, and empathy than about statistics and details. 4. be honest, frank, and open. trust and credibility are difficult to obtain; once lost, they are almost impossible to regain. 5. work with other credible sources. conflicts and disagreements among organizations make communication with the public much more difficult. 6. meet the needs of the media. the media are usually more interested in politics than risk, simplicity than complexity, danger than safety. 7. speak clearly and with compassion. never let efforts prevent acknowledging the tragedy of an illness, injury, or death. people can understand risk information, but they may still not agree; some people will not be satisfied. * from: covello v and allen f. 1988. (reference no. 67) lead to a better understanding of those differences. it may also lead to more widely understood and accepted risk management decisions. effective risk communication should have goals that build and maintain trust and confidence. it should facilitate a higher degree of consensus and support by all interested parties for the risk management options being proposed. many considerations for effective risk communication, especially those involving the public, can be grouped in a sequence following the systematic approach of the risk communication process. this starts with gathering background and needed information, followed by the preparation and assembly of the message and its dissemination and distribution, with a follow-up review and evaluation of its impact (63) . the general considerations for effective risk communication are demonstrated in table 5 . risk communication efforts and programs need to be evaluated both regularly and systematically to determine their effectiveness and to provide for change when needed. communication aims and objectives need to be clearly stated if an evaluation is to be effective. this could include the proportion of at-risk population to be reached, adoption of appropriate risk reduction practices, and the extent of resolution of the crisis. it is important to learn from both positive and negative risk communication experiences, in order to adjust and improve ongoing communication activities. only through systematic evaluations, which are performed throughout the communication process, can that process be strengthened (63) . globalization of disease prevention lectures, through the supercourse prevention project, is funded by the us national institutes of health (nih). the supercourse is an internet library of lectures on prevention, shared for free by 10,000 table 4 goals of risk communication* 1. promote awareness and understanding of these specific issues under consideration during the risk analysis process, by all participants; 2. promote consistency and transparency in arriving at and implementing risk management decisions; 3. provide a sound basis for understanding the risk management decisions proposed or implemented; 4. improve the overall effectiveness and efficiency of the risk analysis process; 5. contribute to the development and delivery of effective information and education programs, when they are selected as risk management options; 6. foster public trust and confidence in the safety of the food supply; 7. strengthen the working relationships and mutual respect among all participants; 8. promote the appropriate involvement of all interested parties in the risk communication process; and, 9. exchange information on the knowledge, attitudes, values, practices and perceptions of interested parties concerning risks associated with food and related topics. table 6 . japan has associated with the global health network through supercourse japan (66) where a series of lectures in health, environment and sustainable development, epidemiology for decision-making, and zoonosis have so far been developed. the supercourse on health, environment and sustainable development was designed to provide an overview on health and environment in the context of sustainable development for public health students around the world, as well as decision makers, community leaders, scientists and professionals in government and non-governmental organizations, who are interested in health and environmental linkages in sustainable development (66) . the supercourse on epidemiology for decision-making provides a learning resource for students of environmental and occupational epidemiology, and its main purpose is to promote the understanding and application of epidemiology in the prevention of environmental and occupational disease and the promotion of health (66) . the supercourse on zoonosis aims to disseminate rapidly the latest information on animal diseases transmittable to humans. severe acute respiratory syndrome (sars) and bovine spongiform encephalopathy (bse) are some of the lectures discussed which will help in information sharing to developing countries (66) . a joint action plan through the 'the pacific islands forum' was created in 2003. its five priority policy targets are: 1) enhancement of security in the pacific region, 2) creation of a safer and more sustainable environment, 3) improvement in education and human resources development, 4) improvement in health, and 5) promotion of more vigorous and continued trade and economic growth. the countries in the pacific region face a contradictory dilemma: some of the poorest countries have the most expensive telecommunications, yet information and telecommunication technology (ict) need to be extensively utilized. in these circumstances telemedicine, such as for remote clinical and pathological diagnosis, is too expensive and technically demanding to be widely used. since information in the future will be technology-and network-based, it is imperative for "supercourse asia" to fully exploit the potential of ict for health education and health service development in the region. the advantages of this approach are to use the most appropriate, inexpensive, opensource, and low-band ict, and to operate it through active national networking. the supercourse asia network (scan) is an offshoot of this initiative and aims that all children and adults will have equal access to health and education of sufficient quality to empower them to break the poverty cycle, to improve their quality of life (qol), and to participate effectively in national development. the mission is to alleviate poverty and improve health and qol of developing countries in the pacific region through advances in a cost-effective ict-based health educational system. the internet is the most inexpensive and speed efficient means to penetrate the remote places such as the pacific islands. japan, a leader of modern technology in the region, is in the best position to provide these technological and educational tools to contribute to the well-being of people in the region. the scan is a group of primarily academics, united by a common belief: the internet is the best way to disseminate knowledge, especially knowledge about health promotion and prevention. they are working towards facilitating the dissemination of health-related information over the internet and improving teaching in the field of education, social/preventive medicine, public health and epidemiology. the members are voluntary professionals in academia, healthcare, telecommunications, the government, ngos, and other public and private sector organizations who will work together towards developing "supercourse asia." they are not only active members of the scan but also the main contributors of "supercourse asia" lectures, reviewers, translators, and its major user group. the challenges for the scan are 1) how to effectively utilize the national networks among the participating countries and within each participating country that may be geographically remote, 2) how to evaluate the effects of the scan on the advancement of health and qol, and the alleviation of poverty though "supercourse asia" in the region, and 3) how to sustain the network activities for many years to come. currently, the public has become concerned with information and safety, however, gaps between the understanding of safety and assurance are widening. health advocacy and promotion through e-learning are becoming more important in the framework of risk communication for the community. the supercourse lectures will serve as an international platform for sharing information, lectures, and ideas. understand the public perception of the risk through such means as risk surveys, interviews and focus groups expect different people to see the risk differently. preparation/assembly 1. avoid comparisons between familiar risks and new risks, as they may seem flippant and insincere unless presented properly. 2. recognize and respond to the emotional aspects of risk perceptions. speak with sympathy and never use logic alone to convince an audience characterized by emotion express risk in several different ways, making sure not to evade the risk question explain the uncertainty factors which are used in risk assessment and standard setting maintain an openness, flexibility, and recognition of public responsibilities in all communication activities accept and involve the public as a legitimate partner by describing risk/benefit information and control measures in an understandable way. 2. share the public's concern rather than deny it as not legitimate or as unimportant. be prepared to give people's concerns as much emphasis as the risk statistics be honest, frank, and open in discussing all issues if explaining statistics derived from risk assessment, explain the risk assessment process before presenting the numbers coordinate and collaborate with other credible sources evaluate the effectiveness of risk messages and communication channels. 2. emphasize action to monitor, manage, and reduce risk plan carefully and evaluate efforts it is based on the open source model, which allows free redistribution, shows the source code, and allows modifications and derived works this is especially useful for developing countries and minority populations with limited access to resources it aims to overcome the digital divide by improving access in places with low bandwidth internet connection it is a teaching support system -differing from a traditional distance education system it provides timely information for action -one of the greatest advantages of having a regional faculty base with varied areas of expertise which are very important in the field of public health (e.g., lectures on sars from the members in china and singapore, where it is most rampant) it is a "hyper-text comic book format hypertext links from text, images, tables, and pictures take the reader to other relevant supercourse lectures, images, or other websites on the internet oaks sc editors. emerging infections; microbial threats in the united states world health organization. removing obstacles to healthy development global defence against the infectious disease threat. world health organization globalisation of prevention education: a golden lecture factors in the emergence of infectious diseases threats to global health and survival; the growing crises of tropical infectious diseasesan unfinished agenda emerging infectious diseases among indigenous peoples epidemiology of the plague of athens the columbian exchange: biological and cultural consequences of 1492. greenwood, westport: connecticut princes and peasants. smallpox in history. chicago: univ untersuchungen über bacterien. v. die aetiologie der milzbrand-krankheit, begründet auf die entwicklungsgeschichte des bacillus anthracis spreading germs: diseases, theories, and medical practice in 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group a streptococcus: allelic variation, population genetics, and hostpathogen interactions genome sequence of a serotype m3 strain of group a streptococcus: phage-encoded toxins, the highvirulence phenotype, and clone emergence identification if herpesvirus-like dna sequences in aids-associated kaposi's sarcoma microbes and malignancy: infection as a cause of human cancers helicobacter pylori-associated diseases current clinical topics in infectious diseases el niño and health island epidemics epidemiology in relation to air travel acute hemorrhagic conjunctivitis: dealing with a newly emerging disease the severe acute respiratory syndrome two worlds of malaria research toward vaccines against malaria the global situation of mdr-tb methicillin (oxacillin)-resistant staphylococcus aureus strains isolated from major food animals and their potential transmission to humans centers for disease control prevention. staphylococcus aureus resistant to vancomycin-united states joint united nations programme on hiv/aids. aids epidemic update surveillance for aids-defining opportunistic illnesses, 1992-1997. mmwr 48. cdc surveillance summary no. ss-2 the outbreak of west nile virus infection in the new york city area in 1999 outbreak of west nile virus infection west nile virus: epidemiology and ecology in north america dengue and dengue hemorrhagic fever antibody-dependent enhancement of infection and the pathogenesis of viral disease molecular ecology of toxigenic vibrio cholerae the next influenza pandemic: lessons from hong kong a molecular whodunit the chilling true story of the largest covert biological weapons program in the world-told from the inside by the man who ran it a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars investigation of bioterrorism-related anthrax, united states, 2001: epidemiologic findings japanese sect was close to bioterrorism journal says: live anthrax found at office threats in bioterrorism: i. cdc category a agents risk: a practical guide for deciding what's really safe and what's really dangerous in the world around you world health organization. the application of risk communication to food standards and safety matters, a joint fao/ who expert consultation supercourse: epidemiology, the internet, and global health seven cardinal rules of risk communication key: cord-311601-w2jqmpww authors: muzemil, abdulazeez; fasanmi, olubunmi gabriel; fasina, folorunso oludayo title: african perspectives: modern complexities of emerging, re-emerging, and endemic zoonoses date: 2018-10-25 journal: journal of global health doi: 10.7189/johg.08.020310 sha: doc_id: 311601 cord_uid: w2jqmpww nan r ecent events have shown that public health, animal health and national economies have been threatened, globally, by the increased occurrence of emerging and re-emerging infectious diseases (ereids) [1] . specifically, land use change cum agricultural practices, surging human demographic, pathogen evolution (antimicrobial resistance), failure of public health systems, global travel and more global interconnectedness in spatial and temporal dimensions have driven these threats [2] . other aggravating factors include: ecological changes, incursion into previously uninhabited areas, changes in human behavior, environmental degradation, international trade, technology and industry, antimicrobial misuse, and deficiencies in public health infrastructure and decision-making [1] [2] [3] . major ereids − including zoonoses − have been reported in the last two decades including: bovine spongiform encephalopathy, hendra, nipah, severe acute respiratory syndrome (sars), highly pathogenic avian influenza (hpai) h5n1, h5n8 and h7n9 subtypes, west nile fever, pandemic h1n1 influenza, ebola virus disease (evd) and middle east respiratory syndrome coronavirus (mers-cov). many of these diseases have been documented in africa. in africa, 47 of the 54 countries (87%) have reported ereids to the who since 1997 ( table 1) . while several initiatives have been implemented globally to accelerate progress toward a safer world [4] , it is yet not clear whether african countries are ready and capable of handling the magnitude and threats associated with ereids [4] . here, we reviewed plausible reasons and drivers for the upsurge of ereids in africa and proffer some mitigating measures. human populations in african countries have rapidly increased in the last few decades (figure 1 ). population growth has occurred together with a substantial modification of human and pathogen behaviours [2, 4] . for example, hoosegood has identified that societal behaviours eg, union formation and cohabitation, re-marriage and partnering, union instability-widowhood, divorce and separation, fertility and fecundity, and fertility-related decisions significantly impact on and are impacted by hiv and aids in sub-saharan regions particularly, in southern africa [5] . second, urban growth has disrupted wildlife and pathogen ecology [1, 2] . as human-pathogen contacts increase, so does the probability for more outbreaks. pathogens affected include cowpox, lyme disease, nipah, hendra and ebola viruses as well as the group named eskape -which includes enterococci, s. aureus, k. pneumoniae, a. baumannii, p. aeruginosa and en-terobacteria-which explains between 45 and 77% of all african human mortalities [1, 3] . as human-pathogen contacts increase, so does the probability of more outbreaks. to prevent epidemics, it is needed to markedly improve public infrastructures, sanitation, and the health systems. for instance, the public and animal health surveillance systems must transform, so interventions occur within acceptable response times [1, 3, 4] . third, in the 20 th century, the average temperature has increased approximately 0.7°c in the african continent. climate change has predisposed africa to highly vulnerable situations, particularly around internationally shared water resources. consequently, new challenges have emerged, including: border-related conflicts, food security risk due to declines agricultural production, vectorand water-borne diseases, (especially in areas with inadequate health infrastructure), flooding and exacerbation of desertification by changes in rainfall and intensified land use [2] . predictions related to water resources include: (a) decreased rainfalls in portions of the sahel, (b) increased rainfalls in east central africa, (c) increased temperature ranging from 0.2°c to >0.5°c per decade, especially in the semi-arid margins of the sahara and central southern africa [6] . some studies have also suggested that major climate change will influence water resource use, natural resources management and biodiversity, human health, food security, resettlement and infrastructure re-allocation, and desertification [2, 6] . variance in climatic conditions will impact significantly on disease ecology and epidemiology with upsurge in human-animal disease conditions due decreased salinity of the soil which can increase the number of toxic bacteria and breeding sites for mosquitoes and rodents. these challenges can have consequences on international trade and commerce. since the liberalization of trade policies between countries over the past two decades, national economies have grown in leaps and bounds. while such policies have fast-tracked growth forecast for african countries, they also have augmented the risks of emergent and trans-boundary animal and human diseases especially associated with long flights, such zoonotic tuberculosis, influenza viruses, hiv/aids and cholera. because trans-border movements of livestock and/or some commercial practices may bring together disease vectors and humans, human and animal health should be addressed together [1] [2] [3] . fourthly, the rapid expansion in human populations (and consequently, the need to meet the food security needs) has warranted the intensification of animal and crop agriculture. these changes have converted previously fallow lands and forest into arable, agricultural and/or grazing lands. associated with these changes are increased (a) rodents populations, (b) dispersal and redistributions of wild ruminants populations and their ectoparasites, (c) wildlife-livestock-human interactions, and (d) occurrence of diseases like rift valley fever. bodies of evidence have suggested that the rate of future zoonotic diseases will be closely linked to the evolution of the agriculture-environment nexus [1, 2] . it is suggested that, as long as africa (or any other continent) does not address complex interactions -such as those that involve agriculture, the environment, economics, sociology, as well as zoonotic pathogens, disease outbreaks may follow human-driven disruptions, as those observed after major changes in land use, eg, those related with the construction of dams, mines, and intensive agriculture. the fact that pathogens have evolved and keep evolving should be emphasized. microbes such as mycobacterium tuberculosis, enterococcus faecium, enterobacter cloacae, klebsiella pneumoniae, s. aureus, acinetobacter baumanii and pseudomonas aeruginosa have developed multiple resistance mechanisms due to excessive and long-term use of antimicrobials, genetic transfers of resistance genes and selective pressures [2, 3] . endemic antimicrobial-resistant africa will need to prioritize rapid detection, prompt response to ereids, optimize the benefit of geospatial epidemiology in policy decisions and utilize interdisciplinary educational programs. ) . intense human-animal interactions, and consumption of non-certified pathogen-free animal products facilitate the spread of zoonoses pathogens come with heavy clinical and economic burdens, especially in the developing countries. recent review had indicated that endemic infections associated with antimicrobial resistance requires a particular attention because such diseases are linked with approximately 44 to 77% of all annual human deaths in africa [3] . it has been estimated that, by 2050, more lives will be lost due to antimicrobial resistance (amr) than cancer [3] . one major component of antimicrobial resistance is the overuse of antimicrobials in the production of livestock, which are then passed to humans [3] . because vaccines reduce the incidence of infectious diseases (and, therefore, antibiotic use), immunisations might reduce amr [1, 3, 7] . prevention and mitigations: given the numerous and serious issues here identified, african governments need to prioritize efforts aimed at rapid detection and prompt response to emerging or re-emerging pathogens. for example, the critical response time (crt or time available to implement effective epidemic control measures) should be considered in decision-making [1, 5] . that is so because for any intervention to be very effective (≈ 100%), it must be deployed under a realist timeframe; if it requires a longer period of time, it will necessarily be (i) less effective (if not ineffective), and (ii) more expensive [5] . crt may or may not include geo-referenced data. when it lacks geographical data, it becomes much shorter, making useless almost any intervention. thus, the real significance of crt is that it should be expanded (giving decision-makers more time to complete interventions) − which can only be achieved when high-resolution geo-referenced epidemiologic data are analyzed in time and space. thus, to achieve improved epidemic control measures, geographically explicit data should be collected from epidemics, analysed and lessons learnt made available for future interventions. only such (local or regional) data can support scientifically valid decision-making. yet, even recent epidemics have been addressed with ad hoc policies, such as the classic '3-km radius control rings' -which assume all epidemics (ie, all pathogens, all host species, and all local geographies) are identical [1] [2] [3] . failure to consider local bio-geo-epidemiological information has led to widespread dissemination of major epidemics, such as ebola in guinea, liberia, and sierra leone [8] . infectious diseases and zoonoses are extremely expensive to nations both clinically and economically, for example a recent valuation had estimated such costs to include: sars in asia and canada (us$ 30-50 billion), hpai h5n1 globally (us$ 30 billion), worldwide influenza h1n1 (us$ 45-55 billion), ebola in west africa (us$ 10 billion) and zika in latin america and the caribbean (us$ 7-18 billion) [9] , promptly delivered pre-emptive actions and control measures, as well as targeted interventions can significantly reduce burdens associated with these diseases. the creation of interdisciplinary educational programs aimed at local and regional decision-makers involved in disease diagnosis, dissemination, and control, is recommended. such programs could develop and integrate: (i) local data on antimicrobial resistance, (ii) high-resolution, local geo-referenced data, and (iii) site-specific control measures that can be implemented within biologically valid critical response times. one health contributions towards more effective and equitable approaches to health in low-and middle-income countries urbanization and disease emergence: dynamics at the wildlife-livestock-human interface clinical and economic impact of antibiotic resistance in developing countries: a systematic review and meta-analysis the world health report 2007 -a safer future: global public health security in the 21st century the demographic impact of hiv and aids across the family and household life-cycle: implications for efforts to strengthen families in sub-saharan africa working group ii). impacts, adaptation and vulnerability. the third assessment report of the intergovernmental panel on climate change working group ii the role of vaccines in preventing bacterial antimicrobial resistance ebola virus disease in west africa -the first 9 months of the epidemic and forward projections investing in one health: a concerted approach to address shared risks to humans, animals, and the environment acknowledgments: population data from the united nations was utilized in this work. authorship contributions: am initiated the study, contributed to the data and initial draft; ogf contributed to data filtering and analysis; fof reviewed the concept, analyzed data, wrote, edited and reviewed the manuscript and took overall direction of the work. the authors completed the unified competing interest form at www.icmje.org/coi_disclosure.pdf (available upon request from the corresponding author), and declare no conflict of interest. key: cord-330970-6kkqoh7f authors: weiss, robin a title: apes, lice and prehistory date: 2009-02-10 journal: j biol doi: 10.1186/jbiol114 sha: doc_id: 330970 cord_uid: 6kkqoh7f although most epidemic human infectious diseases are caused by recently introduced pathogens, cospeciation of parasite and host is commonplace for endemic infections. occasional host infidelity, however, provides the endemic parasite with an opportunity to survive the potential extinction of its host. such infidelity may account for the survival of certain types of human lice, and it is currently exemplified by viruses such as hiv. hans zinsser's rats, lice and history [1] is a classic in microbiology. written in 1934 and subtitled the biography of a bacillus, it tells the tale of that dreaded disease typhus, its reservoir in rats and its transmission among humans by lice. here, i discuss how we may in the course of prehistory have acquired the lice, and how other infections may, like the typhus bacillus, come to be shared by us and the animal species with which we are in close contact. it is a tale of infidelity that i shall begin with the recent research on lice of david reed and colleagues [2, 3] and of mark stoneking's group [4] who, on the basis of phylogenetic analysis, have speculated that we may have acquired a clade of head lice from another hominid species and pubic lice from gorillas; they have also suggested that lice might help determine the date when humans adopted clothing. i shall examine this unfolding story in the context of what we know about microbial infections, and will look at the promiscuity of viruses through the lens of modern molecular technology; and i will add my own speculation on why naked apes have pubic hair. lice are small, wingless insects that cannot live independently from their hosts ( figure 1 ). they are frequent parasites of birds and mammals, each host species having its own type of louse. humans harbor three kinds of these ectoparasites: head lice, body lice and pubic lice. for much of human history and prehistory blood-sucking lice have been so prevalent that they became part of our everyday language. we speak about feeling lousy and admonish our friends for nit-picking. nits are the eggs of lice, expertly cemented onto hair shafts, as many parents know from painstakingly combing them out of their children's hair. grooming among monkeys and apes is not only a means of social bonding but also a useful way of controlling nits and lice ( figure 2 ). f fa am mi il ly y h he ei ir rl lo oo om ms s a an nd d n ne ew w a ac cq qu ui is si it ti io on ns s writing on infections of humankind, tony mcmichael and i [5] have called those that cospeciated with their hosts 'family heirlooms' and those that crossed over from other hosts in recent evolutionary time 'new acquisitions'. the new acquisitions were initially derived from zoonotic infections but have flourished as self-sustaining infections in the human population. they have diverged from their progenitors in the original host: for example, measles is now distinct from rindepest. the majority of zoonoses, however, remain in their animal reservoirs and, so far as their sojourn in humans goes -even with limited human-tohuman transfer (as with ebola or severe acute respiratory syndrome (sars)) -we can regard them as 'temporary exhibits'. human dna might show 98% similarity to that of chimps, but we share less than 50% of our microbes and parasites with them. ashford [6] argues that the great apes became more specialized forest dwellers at the same time that early hominids explored the savannah, and that human gut parasites resemble those of omnivorous baboons more than those of chimps because humans, like baboons but unlike chimps, are omnivorous. further opportunities for horizontal crossover of microbes and parasites from animals to humans arose when humans spread out of africa. when we domesticated ruminants, and animals such as dogs, cats and rats 'domesticated' us for the rich pickings around human habitation, we acquired many infections from our new neighbors [5, 7] . thus a shared habitat, rather than a shared ancestry, is important for the acquisition of many infections. most human pandemic infections were acquired horizontally very recently on the evolutionary timescale, even though diseases such as typhus, measles and smallpox first occurred in prehistoric times. these new acquisitions originate from evolutionarily distant host species. the influenza pandemic of 1918-1919 came from birds, and the 1968 influenza strain could be an avian-porcine recombinant. today's novel viral infections are more likely to originate from exotic species than from animals that were domesticated long ago [5] . the market for 'bushmeat' has led to ebola virus outbreaks in africa from butchering primates, and to the sars outbreak in china from eating small carnivores, such as civet cats. the primary reservoirs of the ebola filovirus, the sars coronavirus and the nipah paramyxovirus, however, seem to be in fruit bats (flying foxes). lice and nits have been found in textiles, hair and combs excavated from archaeological sites [1, 8] . given that the closest relative of the human head and body louse, pediculus humanus, is p. schaeffi, which infests chimpanzees, one might assume that human and chimp lice have cospeciated with their hosts as family heirlooms ever since they diverged from a common ancestor. this requires, however, that the divergence among hosts and parasites approximates to the same timescale. after all, the closest relative to human immunodeficiency virus type 1 (hiv-1) is the simian immunodeficiency virus of chimpanzees (sivcpz), but it would be facile to suggest that hiv-1 co-evolved with humans, because molecular clock estimates place the most recent common ancestor of the pandemic form of hiv-1 at 75-100 years ago [9, 10] , and this is likely to be close to the time of the species crossover event. hiv-1 has invaded humans at least three times (groups m, n and o), and such is the power of modern forensic dna virology that the precise location in cameroon has been mapped for the chimpanzees carrying the sivcpz most closely related to group m [11, 12] . louse molecular clock. what is more remarkable, however, is that reed et al. [2] found that human lice split into two quite distinct clades, a and b, about 1.18 mya. there is a worldwide clade (which includes both head and body lice) and a new world clade (exclusively head lice). so how can humans harbor two clades of louse that diverged from each other over one million years ago, when that separation is tenfold older than the emergence of homo sapiens? the answer, reed et al. [2] suggested, is that the separation took place around the time of divergence of the ancestors of modern humans from homo erectus. these two hominid lineages then co-existed for about one million years until the demise of h. erectus. when modern humans radiated across asia they might have had contact with h. erectus, just as in more recent millennia h. sapiens met h. neanderthalensis in europe, as dramatized by the nobel laureate william golding [14] . there is no evidence that different human species interbred, but they may well have exchanged ectoparasites. thus, the new world clade of head louse may have crossed horizontally from h. erectus to h. sapiens within the last 100,000 years. zinsser [1] noted that the hair of ancient peruvian mummies and the scalps of pre-columbian native americans contained nits or lice. recent dna analysis of lice from similar remains indicates that they belong to the worldwide clade a, so this clade must have been present in pre-columbian american populations [15] . a third clade of head lice has been delineated in ethiopia and nepal and this clade, c, diverged from clades a and b about 2 mya [16] . if reed et al. [2] were correct to postulate that clade b came from h. erectus, one must wonder in which hominid population might clade c lice have maintained their separate identity. head and body lice used to be designated pediculus capitis and p. corporis but they are now known to belong to the same species, p. humanus [16, 17] . fifty years ago levene and dobzhansky [18] showed that head lice could be trained or adapted to become the rather larger body lice by attaching them to the body in small pill boxes. as we celebrate the 150th anniversary of darwin's origin of species we might recall that it was theodosius dobzhansky, an eminent evolutionary biologist and a russian orthodox christian, who in 1973 famously challenged creationists by declaring that "nothing in biology makes sense except in the light of evolution." his research on lice was no exception. kittler et al. [4] initially reckoned that body lice diverged from head lice approximately 70,000 years ago, but they f fi ig gu ur re e 2 2 nit-picking is an ancient habit, as seen in ( (a a) ) apes (photograph by eric c matthews, reproduced with permission) and in humans as shown in ( (b b) ) a painting by jan siberechts, cour de ferme, 1662. musée des beaux-arts, brussels, belgium. later increased this estimate to 107,000 years ago by correcting an error concerning the original outlier sequence. they postulated that this date of about 100,000 years ago coincided with or followed soon after the origin of clothing, because the naked human body is an inhospitable place for lice to breed. head lice feed on the scalp and breed in hair, whereas body lice feed on the skin but breed in clothing. more extensive phylogenetic analyses [16, 17] indicate that body lice evolved from head lice several times within the worldwide clade a, as they are found in many branches of the cladisitic tree. multiple derivations of body lice from head lice had already been considered by zinsser [1] , and it makes good sense if one considers that clothing was not a single invention. wearing animal pelts fur-side next to the skin would have provided a suitable place for lice to breed before fabrics were developed with the inventions of spinning and weaving. in 17th and 18th century europe, most of the aristocracy and gentry shaved their hair and wore wigs. had this custom arisen to protect them from lice as zinsser [1] suggests? not according to samuel pepys' diary, as he complained more than once about his wig being infested: "thence to my barbers, to have my periwig cleared of its nits." i wonder if they were head or body lice -is a wig hair or clothing? l li ic ce e a an nd d n nu ud di it ty y why naked apes are naked and when we 'lost' our hair has long been disputed, as discussed by desmond morris in the naked ape [19] . rantala [20] suggested that nakedness could have had a selective advantage to rid the body of lice and other ectoparasites, a view also championed by pagel and bodmer [21] , who added that being seen to be free of lice would be a fitness indicator and a good mating strategy. i am rather drawn to the theory first postulated by alister hardy [22] that humans evolved through an aquatic stage, although most anthropologists disparage this hypothesis. yet ashford [6] points out that several parasites specific to humans, such as three schistosoma species, depend for their transmission on our entering water, which again distinguishes us from the great apes. pubic lice are commonly called crabs because of their appearance (figure 1c) (figure 3 ). thus, it seems clear that humans acquired pubic lice horizontally, possibly at the time of the pthirus species' split and probably directly from gorillas. because they were already adapted to the coarse body hair of the gorilla, crabs would have found a suitable niche in human pubic hair. indeed, the diameter of the hair is most likely the key rather than the pubic region, because pediatric infestation of p. pubis is well documented: the crab is then found on the eyelashes of the infant. reed et al. [3] suggest that the most recent common ancestor of the genera pediculus and pthirus was about 12.5 mya, which is earlier than the estimated divergence of gorillas and the chimpanzee-human lineage. so was there duplication and separation of lice in the african anthropoid ape lineage, where they could have occupied separate ecological niches, rather as human head lice and pubic lice do today? although this is an intriguing hypothesis, i was having difficulty envisioning a clear separation of habitats between the groin and other parts of our ancient common ancestor. [23] , all the species of apes, old world monkeys and new world monkeys seem to be less hairy in the pubic region than elsewhere; fur is present but it is short and fine. why do adult humans sport a thick bush of wiry pubic hair, uniquely among primates? it must surely be because we are otherwise naked. it probably serves both a visual and an odorous function, because hair aids the distribution of apocrine scent secretion, like our less visually stunning axillary hair. unlike a beard, pubic hair is not sexually dimorphic, yet it is a feature of sexual maturation. no wonder that pubic lice are said to be the most contagious of all sexually acquired infections. which came first, nakedness or pubic hair? i would postulate that the development of pubic hair was a consequence of the visible nakedness elsewhere on the body. perhaps the acquisition of p. pubis 3.3 mya provides the clue to when hominids developed thick pubic hair, rather as the evolution of body lice is thought to be broadly contemporaneous to the development of clothing. it is noteworthy that the prevalence of infestation by pubic lice seems to be decreasing among women and men who remove their pubic hair using 'bikini wax', rather as the renaissance painters discreetly depilated classical female nudes. a study from the general infirmary, leeds, uk, records the increasing predilection among attendants at a clinic for sexually transmitted infections to undertake extensive pubic hair waxing known as the 'brazilian', leaving only a thin strip of hair. armstrong and wilson [24] noted a significant fall in the incidence of pubic lice among patients, although gonorrhea and chlamydia increased over the same period. thus, there may be a health benefit to this emerging sexual lifestyle, and this finding would also lend support to the notion that nakedness protects humans from ectoparasites. h hu um ma an ns s a as s r re ep po os si it to or ri ie es s o of f a an nc ci ie en nt t i in nf fe ec ct ti io on ns s parasites that have tightly cospeciated with their hosts are, of course, in grave danger of extinction when that host becomes an endangered species. but the 'smart' parasite would gain a whole new lease of evolutionary opportunity if it engaged in occasional 'infidelity', analogous to mutation in dna replication. a dna lineage that does not mutate would have an extraordinarily slow rate of evolution, whereas a super-mutator without repair would provide little opportunity for natural selection before the genotype changed further. thus, a low mutation rate within broad fidelity of dna replication allows for both inheritance and evolution. likewise, total cospeciation dooms the parasite to the fate of the host, whereas the ability to move horizontally to closely related hosts would provide flexibility. parasite jumping between related hosts might occur more frequently than realized because it might easily be overlooked. by this reckoning, the new world clade of head lice formerly faithful to the h. erectus lineage [2] adopted modern humans in the nick of time. hiv-1 might well be another successful case of jumping off a sinking ship, because its former host is not likely to survive for many generations longer in the wild. now that hiv-1 has adopted a new host species, it is enjoying a most successful adaptive radiation and has already colonized around 60 million humans (25 million of whom have died from aids). such crossover events, however, are relatively rare, and only one of the three ape-to-human transfers of hiv-1 has taken off to cause the aids pandemic [11] . it pays the host to place barriers known as restriction factors in the path of potential pathogens. if a species barrier is not recognized by the new invader or is successfully circumvented, the infection can be more virulent in the new host. regarding the intestinal parasites and ectoparasites that specialize in human infestation, ashford [6] pointed out that we not only house two kinds of closely related lice (he meant pediculus head and body lice), but also two species of cimex bedbugs, two of demodex mites and two of taenia tapeworms. he therefore asked if we were once two separate populations that rejoined after a long separation, but ashford did not have dna sequences and molecular clock estimates available to him. can we now view this phenomenon in the same way as the lice, as one of the pair of parasites cospeciating with h. sapiens and the other jumping from non-ancestral archaic humans to the modern human lineage? it would be intriguing to conduct similar molecular phylogenies of ashford's other pairs. ashford [6] finished by stating: "over to the microbiologists: what do the bacteria, viruses and fungi tell us?" there are ample examples both of cospeciation and horizontal transmission. the malaria parasite plasmodium falciparum exemplifies a complex cospeciation between the parasite, its human host and its mosquito vector. anopheles gambiae has also coevolved to be a specialist feeder on humans; by contrast, the other human malaria parasite, p. vivax, has an origin in south east asian monkeys and is transmitted by the more promiscuous culex species. might vivax malaria have first adapted to h. erectus as an intermediate between monkeys and humans? as a virologist, i have felt stimulated [23] to take up ashford's challenge to consider pairs of related human viruses. hiv-1 and hiv-2 are very recent 20th century arrivals, from chimpanzees and sooty mangabey monkeys, respectively. with human t-cell lymphotropic viruses types 1 and 2 (htlv-1 and htlv-2), there have been multiple introductions of htlv-1 from african and asian monkeys and apes [25] . the provenance of htlv-2 is puzzling, as its reservoir is in indigenous american populations and in african pygmies, which are as far apart in h. sapiens as one can find. humans have five distinct polyoma viruses and multiple genital papilloma virus types. the different clades of hepatitis c virus (hcv) have deep roots but there are no known animal relatives to provide an anchor or time calibration. it would be fascinating to learn whether archaic humans harbored hcv variants, but as hcv has an rna genome there is not much hope of gaining direct evidence by sequence amplification from ancient specimens. all members of the herpesvirus family are thought to have strictly cospeciated with their hosts, though i have my doubts. the closest relatives to human herpes simplex virus (an α herpesvirus), cytomegalovirus (β) and epstein-barr virus (γ), seem to be those in the chimpanzee. several simian species have a pair of distinct rhadinoviruses, whereas so far humans are only known to harbor one, kaposi's sarcoma herpesvirus. on the other hand, humans have two herpes simplex viruses (hsvs), types 1 and 2. phylogenetic analysis [26] indicates that hsv-1 and hsv-2 are further apart from each other than hsv-1 is from its chimp ortholog [27] . so where did hsv-2 come from? from its estimated age of divergence from the chimp-human hsv-1 lineage, i would place a bet on horizontal transmission from gorillas or possibly from orang-utans [23] . is it a coincidence that three human parasites acquired horizontally from great apes, namely pubic lice, hiv-1 and speculatively hsv-2, are sexually transmitted? now, before one conjures up a king kong scenario, it should be noted that predators can pick up parasites from their prey. the close contact involved in human ancestors butchering gorillas could have enabled pthirus to jump hosts, rather as bushmeat slaughter practices probably led sivcpz and other retroviruses to invade humans from chimpanzees in modern times [11, 25] . h hu um ma an ns s a as s a a s so ou ur rc ce e o of f i in nf fe ec ct ti io on ns s with the advent of globalization, previously isolated human populations lost nine-tenths of their number to the infections introduced by intrepid migrants and invaders [7] . hernan cortes conquered the mighty aztec empire thanks to smallpox and measles, which the invaders inadvertently introduced to the disease-naive new world peoples [5] . the subsequent population crash was severe; so few indigenous people survived that the lucrative african slave trade was established in order to provide labor in the plantations. this pattern of export of old world diseases was repeated in south america, australia and oceania. as charles darwin remarked in his diary on the voyage of the beagle, "wherever the european has trod death seems to pursue the aboriginal." it is plausible, then, that modern humans could have transmitted lethal infectious diseases to archaic human species. h. erectus might have given us a clade of head lice, but h. sapiens may have conveyed more deadly infections to h. erectus and later to h. neanderthalensis. hard evidence is lacking, and prehistoric legends seldom tell the tale from the point of view of the vanquished, although golding did through the eyes of the neanderthals [14] . to paraphrase darwin, wherever modern humans trod during the pleistocene era, death seemed to pursue archaic humans. there is also a danger today that the surviving great apes may be subjected to a coup de grace from human infections transmitted through jungle safaris and ecotourism. cross-species virulence is well known. myxamotosis resident in american cotton-tail rabbits devastated the european rabbit, and the disappearance of red squirrels in britain wherever american gray squirrels occur is probably due to a pox virus rather than direct competition for habitat. similarly, the α-herpesviruses that have cospeciated with indian and african elephants cause nothing more severe than cold sores in their natural host but each seems to be lethal to the other when the two species are unnaturally housed together in zoos [28] . sivcpz has little effect on chimpanzees but hiv-1 causes aids in humans. thus, it might pay the host to carry a fairly harmless parasite if that infection is lethal to the host's competitors. body lice, and occasionally head and pubic lice, transmit bacterial diseases: typhus (rickettsia prowazekii) [29] , trench fever (bartonella quintana) and relapsing fever (borellia recurrentis). the lice themselves succumb to typhus infection but pass the rickettsia in their feces, which the human then scratches into the skin. typhus is known as 'war fever' and 'jail fever' because it appears in conditions where lice thrive. at the end of rats, lice and history zinsser [1] wrote "typhus is not dead. it will live on for centuries and will break into the open whenever human stupidity and brutality give it a chance." sadly, zinsser's words were prophetic. within a few years, typhus became the major slayer in the nazi concentration camps; typhus broke out among rwandan refugees in burundi in 1994, in bosnia in 1995 and most recently in goma. human brutality is another feature shared with chimpanzees that has survived in the human lineage [30] . it is too bad that we are not closer to the pygmy chimp (bonobo), which evolved a means of conflict resolution between troupes through alpha females engaging in lesbian sex. but that is another story. a ac ck kn no ow wl le ed dg ge em me en nt ts s i am grateful to tim harrison and david reed for commenting on a draft of this paper. r re ef fe er re en nc ce es s zinsser h: rats, lice and history ge en ne et ti ic c a an na al ly ys si is s o of f l li ic ce e s su up pp po or rt ts s d di ir re ec ct t c co on nt ta ac ct t b be et tw we ee en n m mo od de er rn n a an nd d a ar rc ch ha ai ic c h hu um ma an ns s p pa ai pa ar ra a--s si it te es s r re eg ga ai in ne ed d s so oc ci ia al l a an nd d e en nv vi ir ro on nm me en nt ta al l r ri is sk k f fa ac ct to or rs s i in n t th he e e em me er rg ge en nc ce e o of f i in nf fe ec ct ti io ou us s d di is se ea as se es s germs and steel: a short history of everybody for the last 13,000 years b bo od dy y l lo ou us se e r re em ma ai in ns s f fo ou un nd d i in n t te ex xt ti il le es s e ex xc ca av va at te ed d a at t m ma as sa ad da a, i is sr ra ae el l t ti im mi in ng g t th he e a an nc ce es st to or r o of f t th he e h hi iv v--1 1 p pa an nd de em mi ic c s st tr ra ai in ns s d di ir re ec ct t e ev vi id de en nc ce e o of f e ex xt te en n--s si iv ve e d di iv ve er rs si it ty y o of f h hi iv v--1 1 i in n k ki in ns sh ha as sa a b by y 1 19 96 60 0 c ch hi im mp pa an nz ze ee e r re es se er rv vo oi ir rs s o of ve er rs si it ty y a an nd d p ph hy yl lo og ge eo og gr ra ap ph hi ic c c cl lu us st te er ri in ng g o of f s si iv vc cp pz zp pt tt t i in n w wi il ld d c ch hi im mp pa an nz ze ee es s i in n c ca am me er ro oo on n ge en no om mi ic c r re el la at ti io on ns sh hi ip ps s a an nd d s sp pe ec ci ia at ti io on n t ti im me es s o of f h hu um ma an n, c ch hi im mp pa an nz ze ee e, a an nd d g go or ri il ll la a i in nf fe er rr the inheritors. london: faber and faber m mo ol le ec cu ul la ar r i id de en nt ti if fi ic ca at ti io on n o of f l li ic ce e f fr ro om m p pr re e--c co ol lu um mb bi ia an n m mu um mm mi ie es s the naked ape: a zoologist's study of the human animal hu um ma an n n na ak ke ed dn ne es ss s: : a ad da ap pt ta at ti io on n a ag ga ai in ns st t e ec ct to op pa ar ra as si it te es s? ? l le es ss so on ns s f fr ro om m n na ak ke ed d a ap pe es s a an nd d t th he ei ir r i in nf fe ec ct ti io on ns s b br ra az zi il li ia an n t to op pi ic cs s i in n h he er rp pe es sv vi ir ru us s g ge en no om mi ic cs s a an nd d e ev vo ol lu ut ti io on n i is so ol la at ti io on n a an nd d c ch ha ar ra ac ct te er ri iz za at ti io on n o of f a a c ch hi im mp pa an nz ze ee e a al lp ph ha ah he er rp pe es sv vi ir ru us s pr ri im ma at te e t t--l ly ym mp ph ho ot tr ro op pi ic c v vi ir ru us se es s a am mo on ng g c ce en nt tr ra al l a af fr ri ic ca an n b bu us sh hm me ea at t n no ov ve el l e en nd do ot th he el li io ot tr ro op pi ic c h he er rp pe es sv vi ir ru us se es s f fa at ta al l f fo or r a as si ia an n a an nd d a af fr ri ic ca an n e el le ep ph ha an nt ts s e em me er rg gi in ng g a an nd d r re e--e em me er rg gi in ng g r ri ic ck ke et tt ts si io os se es s: : e en nd do ot th he el li ia al l c ce el ll l i in nf fe ec ct ti io on n a an nd d e ea ar rl ly y d di is se ea as se e e ev ve en nt ts s demonic males: apes and the origins of human violence key: cord-323311-xl2fv0qx authors: kahn, r. e.; morozov, i.; feldmann, h.; richt, j. a. title: 6th international conference on emerging zoonoses date: 2012-09-07 journal: zoonoses public health doi: 10.1111/j.1863-2378.2012.01539.x sha: doc_id: 323311 cord_uid: xl2fv0qx the 6th international conference on emerging zoonoses, held at cancun, mexico, 24–27 february 2011, offered 84 participants from 18 countries, a snapshot of current research in numerous zoonoses caused by viruses, bacteria or prions. co‐chaired by professors heinz feldmann and jürgen richt, the conference explored 10 topics: (i) the ecology of emerging zoonotic diseases; (ii) the role of wildlife in emerging zoonoses; (iii) cross‐species transmission of zoonotic pathogens; (iv) emerging and neglected influenza viruses; (v) haemorrhagic fever viruses; (vi) emerging bacterial diseases; (vii) outbreak responses to zoonotic diseases; (viii) food‐borne zoonotic diseases; (ix) prion diseases; and (x) modelling and prediction of emergence of zoonoses. human medicine, veterinary medicine and environmental challenges are viewed as a unity, which must be considered under the umbrella of ‘one health’. several presentations attempted to integrate the insights gained from field data with mathematical models in the search for effective control measures of specific zoonoses. the overriding objective of the research presentations was to create, improve and use the tools essential to address the risk of contagions in a globalized society. in seeking to fulfil this objective, a three‐step approach has often been applied: (i) use cultured cells, model and natural animal hosts and human clinical models to study infection; (ii) combine traditional histopathological and biochemical approaches with functional genomics, proteomics and computational biology; and (iii) obtain signatures of virulence and insights into mechanisms of host defense response, immune evasion and pathogenesis. this meeting review summarizes 39 of the conference presentations and mentions briefly the 16 articles in this special supplement, most of which were presented at the conference in earlier versions. the full affiliations of all presenters and many colleagues have been included to facilitate further inquiries from readers. addition to the summaries later of six presentations on this topic, this special supplement includes an article, monitoring of west nile virus infections in germany by dr. u. ziegler et al. which identified west nile virus (wnv) antibodies in migratory birds, but not in resident birds, in domestic poultry or in local horse populations throughout germany. the wnv antibody-positive species were found in birds that migrate to tropical africa or southern europe; however, wnv-specific rna could not be found in any of the samples. the conference opened with a presentation from professor m. a. diuk-wasser and her colleagues j. simpson and c. m. fosom-o'keefe (all yale school of public health, new haven, ct, usa) and g. molei, p. m. armstrong, and t. g. andreadis (center for vector biology and zoonotic species at the connecticut agricultural experiment station, new haven, ct, usa), ecology of west nile virus in the north-eastern united states. professor diuk-wasser began by noting that west nile virus (wnv) was introduced into new york city in 1999 by unknown means and was now considered endemic throughout the usa, with 29,700 human cases and 1,180 deaths in the usa since 1999. it had been hypothesized that increased biodiversity leads to a decreased risk of exposure to zoonotic pathogens (keesing et al., 2006) . at issue is whether this 'dilution effect' or 'zooprophylaxis' for vector-borne pathogens applies only when vectors are generalist feeders, because the link between host diversity and pathogen transmission might break down when vectors exhibit host preferences. in the north-eastern united states, wnv perpetuates in an enzootic transmission cycle involving culex spp. mosquitoes and virus-competent avian hosts. previous studies had detected that a large proportion of c. pipiens and c. restuans bloodmeals were derived from american robins (turdus migratorius), suggesting a key role for this bird species in the wnv transmission cycle (kilpatrick et al., 2006; molaei et al., 2006) . the new haven-based research team tested for preferential feeding by conducting equal choice experiments (robins versus other bird species) (simpson et al., 2009) and by comparing the proportion of culex spp. bloodmeals acquired from robins to the proportion of robins in the local bird community. both methods indicated preferential feeding for robins. they were also able to identify robin communal roosts as amplification foci in greater new haven (diuk-wasser et al., 2010) . then, through field-informed mathematical modelling, they determined that host preferences were indeed key drivers of wnv transmission and that landscape attributes (such as urbanization) in combination with mosquito abundance and a measure of host community competence were the strongest predictors of pathogen prevalence (simpson et al., 2011) . thus, it was clear that pathogen prevalence and human risk of infection were best predicted by assessing the relative pathogen competence and attractiveness to vectors of all species in the host community, rather than using simple measures of biodiversity. in the next presentation, interactions among multiple tick-borne pathogens in a natural reservoir host, professor fish and his colleagues j. brown, m. fitzpatrick, s. usmani-brown, p. cislo and p. krause (yale school of public health, new haven, ct, usa) stressed that species interactions within a parasite community drive infection risk in a wildlife population (telfer et al., 2010) . at least five tick-borne pathogens are known to be transmitted by ixodes scapularis, the principal vector of lyme disease in the united states: (i) borrelia burgdorferi, an agent of lyme disease; (ii) anaplasma phagocytophilum, an agent of human anaplasmosis; (iii) babesia microti, an agent of human babesiosis; (iv) borrelia miyamotoi, an agent of relapsing fever; and (v) the powassan encephalitis virus. two or more of these pathogens can be transmitted either simultaneously by a single tick or sequentially by successive tick-bites, resulting in 240 different permutations of mixed-infection studies. in the context of pathogen prevalence of ixodes scapularis nymphs, borrelia burgdorferi, has been found in 19.8% of samples from the north-east and mid-western united states, while babesia miroti has been found in 14.7% of samples from block island, rhode island. professor fish explained that several types of co-infections have been explored in an experimental system employing laboratory colonies of i. scapularis ticks and peromyscus leucopus white-footed mice, a natural reservoir host for these pathogens. outcomes of mixed infections in mice have been measured by r o , the fitness parameter and basic reproductive rate which indicates the number of secondary tick infections resulting from a primary infection (levin and fish, 2004) . the observed outcomes of dual mixed infections have been variable with both positive and negative effects on r o , while interactions have been mutual, unidirectional or null. these diverse pathogen interactions play an important role in determining the infection prevalence of host-seeking nymphs in nature, and consequently, in the risk of infection for humans. professor h. henttonen (finnish forest research institute, vantaa, finland) and his team h. leirs, e. r. kallio, k. tersago and l. voutilainen in collaboration with university of antwerp, belgium; university of liverpool, united kingdom; and the universities of helsinki and jyväskylä, finland, studied biome specific rodent dynamics and hanta epidemiologies in europe. their research sought to understand the main biomes and forest coverage in europe, the european hanta viruses and their carriers, and the biome specific dynamics of hanta virus carriers and the biome specific transmission dynamics and epidemiologies. within the bunyaviridae family of viruses, hantaviruses infect rodents (and insectivores) and cause haemorrhagic fever with renal syndrome (hfrs) in humans in the old world and hantavirus cardiopulmonary syndrome (hcps) in the new world. in a large european union project, eden (emerging diseases in a changing european environment, 2011), rodent-borne (robo) viral infections have been studied, along with tick-borne pathogens, leishmaniasis, west nile virus, malaria and rift valley fever. the most important aim of professor henttonen and his colleagues was to clarify the differences in boreal (northern) and temperate europe in the human epidemiology of nephropathia epidemica, by far the most common hantaviral disease in europe, caused by puumala hantavirus (puuv). the population dynamics of the host species, the bank vole, differ greatly in various parts of europe, driven by predation in the north and masting events in the temperate zone. consequently, the causes of rodent fluctuations are different. in addition, the role of landscape patterns (homogenous taiga vs. fragmented temperate forests) in rodent/virus dispersal is significant, as well as local environmental conditions (e.g. temperature and moisture), which affect virus survival outside the host. for example, in room temperature, puuv remains infectious for at least 2 weeks outside the host, and possibly for much longer in cold temperatures and in moist conditions. these research findings are essential for human risk evaluation with regard to both long-term and seasonal occurrence of puuv in the environment. in spite of chronic infection of bank voles and the excretion of puuv in their faeces, urine and saliva, the shedding period is limited, which has significant implications for seasonal transmission dynamics in rodents. thus, within the same host/virus system, biomespecific puuv epidemiologies occur (kallio et al., 2009; tersago et al., 2009) , thereby highlighting the need for geographically comparative studies in europe (metla, 2012) . professor v. sambri and his team, p. gaibani, f. cavrini, a. m. pierro, m. p. landini and g. rossini (all regional centre for microbiological emergencies [crrem] , unit of clinical microbiology, st orsola-malpighi university hospital, bologna, italy) investigated usutu: a novel human pathogenic mosquito-borne flavivirus. this virus belongs to the japanese encephalitis serogroup within the mosquito-borne cluster of the genus flavivirus in the family flaviviridae. first isolated from mosquitoes of the genus culex in south africa in 1959, the usutu virus (usuv) has since been isolated from mosquitoes, rodents and birds throughout sub-saharan africa and europe. the virus is thought to be maintained in nature in a mosquito-bird transmission cycle in areas with a minimum of at least ten hot days >30°c, but no mammalian reservoir has yet been identified. professor sambri pointed out that it was not until september 2009 that usuv was found in the liver of a patient who underwent an orthotropic liver transplant (gaibani et al., 2010) . further study of the plasma and genome sequencing analysis confirmed the presence of usuv viremia. then usuv was detected in the livers of an additional four patients from the same area suffering from acute meningo-encephalitis during 2008/2009. both serological assay and molecular assay have been used as new tools for the diagnosis of usuv infection. thus, it is now clear that usuv is a new emerging flavivirus pathogenic for humans. further studies are required to discover both the geographical distribution of this virus and the mechanisms by which humans acquire the virus. since this conference presentation, there has been increased awareness of the seriousness of usuv (vázquez et al., 2011) . according to the world health organisation (who) and unicef, 1.5 million children under the age of five die from diarrhoea annually (unicef/who, 2009 astroviruses cause infections within the small intestine and are associated with at least 10% of all sporadic cases and >25% of all hospitalized cases. these rapidly evolving, nonenveloped, single-stranded rna viruses can be transmitted directly from infected individuals and animals, and indirectly through contaminated food and water. professor schultz-cherry's laboratory was the first to demonstrate that astroviruses induce diarrhoea by a novel mechanism: they possess an enterotoxin that disrupts intestinal epithelial barrier function independent of cellular damage or an inflammatory response (koci et al., 2003) . this occurs within 24 h post-infection because of reorganization of the tight junction protein occludin and the actin cytoskeleton (moser et al., 2007) . in essence, within a complex pathogenic process, astroviruses cause diarrhoea by increasing intestinal barrier permeability. this is the first evidence showing that a viral coat protein is an enterotoxin. of great interest, the toxin can act independently of species barriers. given the increasing isolation of astroviruses from diverse species, there is increasing evidence that toxicogenic astroviruses could be associated with zoonotic disease. professor m. g. katze (department of microbiology and washington national primate research center, university of washington, seattle, wa, usa) set out a unifying approach to molecular biology in his presentation, systems and computational biology: emerging tools for exploring emerging viruses. he emphasized that modern day virologists and immunologists must do better in their search to understand how a virus kills and how effective vaccines can be developed, especially because traditional virology has yielded surprisingly little information about why some virus strains cause severe diseases while others remain innocuous. he pointed out that the case fatality rate for the 1918 influenza pandemic was about 2.5% and that particular h1n1 virus may have infected as much as one-third of the world's population. issues arise not only in understanding a virus, but also in understanding how hosts respond. for example, the 1918 virus infection resulted in very high expression of inflammatory, antiviral and immune cell genes very early in host infection (kash et al., 2006) . significant progress in overcoming existing and emerging viruses depends on biologists, mathematicians and computer specialists working together within a systems biology paradigm. such research begins with either in vitro studies of virus replication on cell lines or primary cell cultures, moving to nonhuman primate models of virus infection. then samples from the experiments are investigated at multiple time points and conditions; and high throughput data are then examined by data processing to prepare systematic evaluations of different host responses. data integration involving data analysis and modelling of key genes and pathways is then possible, followed by iterative processing of host perturbations and the use of viral mutants to discover specific applications to translational research. such a systems biology approach requires not only continuing experiments with virusinfected experimental systems but also significant efforts to maintain the hardware and software of an extensive laboratory computational infrastructure. it is this computing infrastructure, which permits the laboratory to go quickly from samples to pathway visualization, as the data analysis workflow moves from microarray images to gene expression data to pathway models. the mission of this virolab is to develop steadily over the years to come a virtual laboratory to confront the viruses involved in 14 infectious diseases -influenza, ebola, marburg, hepatitis c, sars-cov, vaccinia, herpes simplex, west nile, hiv-1, siv, measles, lassa, chikungunya and dengue fever. the three key characteristics of this integrated approach to so many infectious diseases are as follows: (i) to use cell culture, primary cells, nonhuman primate and human clinical models to study viral infection; (ii) to combine traditional histopathological, virological and biochemical approaches with functional genomics, proteomics and computational biology (haagmans et al., 2009); and (iii) to obtain signatures of virulence and insights into mechanisms of host defense response, viral evasion and pathogenesis (casadevaill et al., 2011) . for example, with the study of all respiratory viral diseases, a unifying hypothesis is that highly pathogenic respiratory viruses use both unique and common strategies to remodel the host cell to enhance virus replication, regulate disease severity and promote virus transmission (chang et al., 2011) . a highly significant new tool for studying these emerging viruses is next generation sequencing (ngs) which has already 'changed the way we think about scientific approaches in basic, applied and clinical research' to such an extent that 'the potential of ngs is akin to the early days of polymerase chain reaction (pcr), with one's imagination being the primary limitation to its use' (peng et al., 2011) . already, a good understanding of the 'timing' and extent of immune (innate)-mediated injury after virus infection has been achieved. furthermore, molecular 'disease' signatures associated with different pathogens in multiple animal species have been described at micro-rna, mrna, protein level, metabolite and lipid levels. such successful modelling of molecular events has made possible verifiable prediction about key nodes and bottlenecks, enabling the identification of novel host cell drug targets (diamond et al., 2010) . the translational impact of this research, in professor katze's view, will be immense, revealing a completely new and expanded host defense repertoire consisting of non-annotated noncoding rnas. despite all of these achievements, four crucial questions remain unanswered: (i) is systems biology too complicated and too expensive to become the pre-eminent approach in virology and immunology? (ii) are mathematicians and computer scientists up to the challenges? (iii) how will new technologies like next generation sequencing impact virus systems biology research, especially in the context of rna sequencing? (iv) how can new principal investigators best be identified and appointed? (virolab, 2012) . it has long been recognized that the emergence of any zoonoses is a complex process involving 'ecological interactions at the individual, species, community and global scale' (childs et al., 2007, p.2) . this topic began with a presentation from professor a. a. aguirre that focused on the ecological framework in which any zoonotic disease should be considered. the role of bats as an important reservoir host for many dangerous zoonotic pathogens was then considered in some detail (cf. daniels et al., 2007; field et al., 2007; gonzalez et al., 2007; wang and eaton, 2007) . professor a. a. aguirre (department of environmental science and policy, george mason university and executive director, smithsonian-mason global conservation studies program, front royal, virginia, usa) presented emerging zoonotic diseases of wildlife: developing global capacity for prediction and prevention. he began by explaining that conservation medicine and more recently ecohealth have emphasized the need to bridge disciplines, thereby linking human health, animal health and ecosystem health under the paradigm that 'health connects all species in the planet' (aguirre et al., 2002) . in his view, the recent convergence of global problems such as climate change, biodiversity loss, habitat fragmentation, globalization, infectious disease emergence and ecological health demands integrative approaches breaching disciplinary boundaries. the international union for conservation of nature (iucn) maintains a red list of threatened species -an important initiative in view of the 869 animal extinctions that have already occurred, of which 3.7% were caused by disease (smith et al., 2006) . professor aguirre noted that the u.s. agency for international development (usaid) has been a major leader in the global response to the emergence and spread of highly pathogenic avian influenza (hpai). since mid-2005, it has programmed approximately $500 million to build capacities in more than 50 countries for monitoring the spread of hpai among wild bird populations, domestic poultry, and humans, and to mount a rapid and effective containment of the virus when it is found. recent analyses indicate that these efforts have contributed to significant downturns in reported poultry outbreaks and human infections and a dramatic reduction in the number of countries affected. furthermore, the usaid bureau for global health, office of health, infectious disease and nutrition (gh/hidn) recently funded two cooperative agreements, predict and respond, under its avian and pandemic influenza and zoonotic disease program to continue and expand this work. the goal of predict is to establish a global early warning system for zoonotic disease emergence that is capable of detecting, tracking and predicting the emergence of new infectious diseases in high-risk wildlife (e.g. bats, rodents and nonhuman primates) that could pose a major threat to human health. the goal of respond is to improve the capacity of countries in high-risk areas to respond to outbreaks of emergent zoonotic diseases that pose a serious threat to human health. the geographical scope of this expanded effort is directed to zoonotic 'hotspots' of wildlife and domestic animal origins (jones et al., 2008) . predict includes a program of smart (strategic, measurable, adaptive, responsive and targeted) surveillance that focuses on preventing the 'spilling over' from wildlife to humans or to halt these diseases rapidly after that spillover by understanding what factors induce emergence and rapidly identifying ways of prevention, control, and mitigation. the overall aim of smart is to promote an integrated, global approach to emerging zoonoses. this integration requires commitment from a broad coalition of partners and stakeholders including government agencies, universities and non-governmental organizations, collaborating for specific purposes and to generate in the future new international structures able to respond to these emerging zoonoses. with 1.5 billion animals being imported into the united states each year, as well as an extensive international trade in illegal animal exports ) and some 75% of emerging zoonoses worldwide having wildlife origins, professor aguirre stressed that ecohealth has become a necessity, not an optional policy goal. dr. g. a. marsh and his colleague dr. l.-f. wang (australian animal health laboratory [aahl], geelong, victoria, australia) began their presentation, bats: a mixed bag of new and emerging viruses, by pointing out that the ''old'' bat viruses were represented by many zoonotic pathogens, including rabies virus, yellow fever virus, st louis and japanese encephalitis viruses, and west nile virus. now bats have been identified as natural reservoirs for a number of new and emerging viruses -ebola virus, marburg virus, hendra virus and sars-like coronaviruses. there are some 1000 different bat species; and they often roost in high-density colonies of over one million flying mammals, which have, in a very real sense, been travelling for millions of years, exposing themselves to many pathogens; therefore, the resulting complexity is not surprising. key research questions include (i) why do bats seem to be able to co-exist with a great diversity of viruses without showing disease signs? (ii) what triggers the spillover of bat viruses into other animals? (iii) do bats control viral infection differently from other mammals? attempts to isolate viruses from bats have generally been unsuccessful. therefore, in an effort to improve the success rate for virus isolation, dr. marsh and his team have recently developed primary cell culture lines from numerous different species of bats (crameri et al., 2009) . the use of these bat cell lines, in combination with improved sampling techniques, has lead to recent isolation of hendra virus from a number of bat urine samples collected in several locations across queensland, australia, including those associated with human and horse virus spillover events (smith et al., 2011) . furthermore, this henipavirus surveillance program has led to the isolation of a number of novel viruses from two different virus families, whose zoonotic potential is not yet known. in an attempt to understand virus/host interactions, as well as to provide insight into the key factors involved in future spillover events, aahl has launched a number of international collaborative projects in south-east asia and ghana, west africa. c. kohl (sonntag et al., 2009) . the phylogenetic analysis of the genome sequence of bat adv-2 demonstrated a close relationship to canine adenovirus 1 and 2 (cadv-1 and cadv-2) (kohl et al., 2012) . the very similar genome organization supported the hypothesis of a shared ancient ancestor. interestingly, both cadvs are presenting untypical pathological features within the family adenoviridae. these adenoviruses were found to have an unusually broad host range and are causing a rather higher pathogenicity in a variety of carnivore hosts. the untypical pathological features might be understood as signs of a missing adaptation host and could provide a model to study ancient inter-species transmission events. this section of the conference addressed cross-species transmission of selected pathogens. in addition to the summaries below of three presentations on this topic, this special supplement includes an article, epidemiological survey of tryanosoma cruzi infection in domestic owned cats from the tropical southeast of mexico by dr. m. jiménz-coello et al. setting out how a significant public health problem in mexico has been caused by the crossspecies transmission of american trypanosomiasis (at) from triatomine bugs to domestic cats, representing a potential risk to humans. speaking on behalf of an extensive team of collaborators from a number of institutions -c. osborne, p. cryan, t. j. o'shea, l. m. oko, c. ndaluka, c. h. calisher, a. berglund, m. l. klavetter, r. a. bowen and k. v. homes -dr. s. r. dominguez (section on pediatric diseases, the children's hospital, university of colorado school of medicine, aurora, co, usa) began by noting that the first pandemic of the twenty-first century, the deadly sars virus, had its natural reservoir in bats. in his presentation, alphacoronaviruses in new world bats: prevalence, persistence, phylogeny and potential for interaction with humans, he suggested that bat coronaviruses (covs) may well be the ancestors of all group 1 and 2 covs. today bats had become a primary species encountered by humans in terms of potential exposure to significant disease agents. their research was tackling three important unanswered questions: (i) what is the prevalence and diversity of bat covs in new world bats? (ii) do bat covs persist in bat populations and/or individual bats? (iii) what are the potential interactions of infected bats with the human population? a 3-year study (osborne et al., 2011) had collected clinical and environmental samples from bats at 16 rural sites and 5 urban sites throughout colorado, as well as bat carcasses obtained from various counties throughout the state from the colorado department of public health and environment. of the 1,002 faecal or anal swab samples, 75, that is, 7%, were positive for cov rna. the highest prevalence of the virus was in juvenile bats; although rates of prevalence varied from year to year, late spring was the time when the virus peaked. although bat covs persisted within bat populations and their roosts, individually tagged cov-infected bats cleared their infections within 6 weeks without apparent illness. new world bats of the same species in geographically distinct locations and over the course of several years harbour similar covs, and some new world bat covs may be able to infect bats of different genera. strikingly, bats, which had known or potential contact with humans, had a high prevalence of 10-20% of cov infection. it is clear that significant opportunities exist for zoonotic transmission of coronaviruses from bats to humans and vice versa, especially as more than 95 viruses have already been isolated from or detected in bat tissues. noting that many mammalian and avian species in addition to bats are susceptible to coronavirus infection, receptor proteins that include ace2, apn and cea-cam1. the recent emergence of sars coronaviruses from civets, bats and/or other reservoir species into humans depended upon a few amino acid substitutions in the receptor-binding domain (rbd) of s from the animal viruses that allowed them to recognize human ace2 instead of, or in addition to, receptors of their natural hosts (li, 2008) . alphacoronaviruses of pigs, cats, dogs and human coronovirus 229e use apn receptors of the host species, and all four viruses recognize feline apn (tusell et al., 2007) . in contrast, for human alphacoronavirus nl63, the receptor-binding motif (rbm) with its three loops in the rbd binds specifically to human ace2. in the rbds of the cat virus, fipv, professor holmes and her research team predicted three loops structurally similar to the nl63 rbms, and they constructed chimeric fipv rbds containing one, two or three rbms from nl63. receptor-binding assays using enzyme-linked immunoassays (elisa), flow cytometry and co-immunoprecipitation identified three loops (rbms) in fipv rbd that are required for binding to feline apn. furthermore, substitution of only a few key amino acid residues within the rbms of fipv altered apn specificity and viral host range. thus, the emergence of alphacoronaviruses into new host species can occur when spontaneous mutations arise in the rbms that permit binding to variants of the apn receptor protein expressed by different host species. considering the interaction between human and swine h1n1 viruses since 1900, professor h. d. klenk (institute of virology, philipps university, marburg, germany) presented the mechanisms of pathogenicity and host adaptation of influenza viruses in the light of the new h1n1 pandemic. he explained that there was now a clear scientific consensus that wild aquatic birds are the natural hosts for a large variety of influenza a viruses. occasionally these viruses are transmitted from this reservoir to other species, such as chickens, pigs and humans, leading to devastating outbreaks in domestic poultry and the possibility of human influenza pandemics. by the end of february 2010, there had been 15,921 deaths, with the world health organization later confirming cases in 171 countries and territories, with deaths in at least 139 countries and territories before the spread of the h1n1 virus diminished. however, professor klenk set out the evidence to support his view that the pathogenic and pandemic potential of this new h1n1 virus is not yet exhausted. the host range and pathogenicity of any virus are polygenic traits depend on the interaction of different viral proteins with specific host factors. it has long been known that proteolytic activation and receptor specificity of the hemagglutinin (ha) are important determinants for pathogenicity and interspecies transmission, respectively. there is now considerable evidence that ha mutations altering receptor specificity and cell tropism of the 2009 pandemic influenza a virus (h1n1v) are linked to the d222g amino acid substitution and are associated with a particularly severe outcome of infection (liu et al., 2010) . it should be remembered that the viral polymerase has to enter the nucleus of the infected cell to promote replication and transcription of the viral genome. adaptive mutations in polymerase subunits of avian viruses improve binding to importin alpha, a component of the nuclear pore complex in mammalian cells. as a result, nuclear transport of these proteins and efficiency of replication are enhanced. thus, the interaction of the viral polymerase with the nuclear import machinery is an important determinant of host range. some of the structural features typical for avian viruses have been preserved in the polymerase of the 2009 pandemic influenza a virus (h1n1v) suggesting that this virus has the potential to further adapt to humans. recent studies have shown that the ns1 protein, another important determinant of pathogenicity and host range, is sumoylated and that this modification enhances virus growth. interestingly, ns1 of h1n1v is not sumoylated (xu et al., 2011) . taken together, these observations support the view that the pathogenic and pandemic potential of the new virus is not yet exhausted. furthermore, because of the firm evidence of ha polymorphism in position 222, mutants and other mutations with altered receptor specificity will have to be closely monitored. in the subsequent discussion, it was noted that when a virus becomes highly pathogenic, this might block its spread if additional hosts are not readily available. furthermore, the role of co-infection with bacterial inflection was highly relevant in the 1918-1919 influenza pandemic and might well be relevant in a future pandemic. there have been at least three influenza pandemics every century since 1700, with some evidence of earlier epidemics and pandemics after 1500. in the cambridge world history of human disease, a. w. crosby (1993; p. 810) has noted that although the black death and world wars i and ii killed higher percentages of the populations at risk, the 1918-1919 influenza pandemic was possibly 'in terms of absolute numbers, the greatest single demographic shock that the human species has ever received'. the summaries below of seven presentations on this topic highlight the diversity of influenza viruses in north america (cf. nelson et al., 2011) , while other relevant research has been published with respect to swine influenza viruses (sivs) in europe (kyriakis et al., 2011) . considerable research has now been carried out into how the highly pathogenic h5n1 avian influenza virus spreads from wild birds and ducks to chickens and other species, including humans (rabinowitz et al., 2010; ma et al., 2008) . the studies of how influenza viruses can be genetically altered to become more transmissible have become a matter of much controversy palese and wang, 2012) . in addition, to the summaries below, this special supplement includes an article, lessons from emergence of a/goose/guangdong/1996-like h5n1 highly pathogenic avian influenza viruses and recent influenza surveillance efforts in southern china, in which dr. x.-f. wan has considered the emergence and ecology of influenza a viruses in southern china, especially the highly pathogenic h5n1 virus. backed by an extensive team of collaborators, professor a. d. m. e. osterhaus (head, department of virology, erasmus medical centre, rotterdam, the netherlands) began his presentation, emerging and neglected influenza viruses, by explaining the complex aetiology of the influenza a, b and c viruses. while humans can serve as host species for all three viruses, influenza a can also be present in other mammals and avian species, influenza b in seals and influenza c in pigs. the severity of the disease is relatively high with influenza a, moderate with influenza b and low with influenza c, with the prevalence in humans high with both influenza a and b viruses, but lower with influenza c. furthermore, a clear distinction needs to be made between seasonal influenza, avian influenza and pandemic influenza. there are two different mechanisms of host adaptation -sequential mutations and genome reassortment. most recently, the new h1n1 swine flu pandemic outbreak of 2009 drew attention to the speed with which an influenza virus could move around the world. however, the fact that this particular virus was not as virulent as first anticipated proved crucial in confronting the virus, even though it spreads rapidly among humans, unlike the much more virulent h5n1 avian flu virus, from which more than 300 people have died from more than 500 verified cases from 2003 to 2011 (world health organization (who), 2012). although clinical evidence of h5n1 avian influenza appears predominantly in diving ducks, a number of dabbling duck species -mallard, teal, wigeon and gadwall -appear to spread h5n1, generally acquired from wild birds, without showing major signs of disease. the likelihood of a major pandemic linked to h5n1 has not decreased in the last 5 years, even though publicity has certainly decreased. furthermore, professor osterhaus pointed out that the recent h1n1 pandemic influenza outbreak indicated that the scientific community was wrong in its earlier belief that 'a pandemic strain could only arise from a subtype that had not previously been widely disseminated in humans [because] the h1n1 virus has shown that human varieties characterized by different hemagglutinin (ha) molecules may follow separate lines of evolution and may generate potentially pandemic strains within an existing human ha subtype. hence, it is essential to develop methods for estimating how many antigenically different subtypes may reside within each ha type' (cf. rappuoli et al., 2009) . in the light of the continuing prevalence of many subtypes of influenza, there is a critical need for improved monitoring, especially in asia and africa, as part of a move from a reactive to a proactive approach, with greater research into the possibility of developing a universal vaccine. although there are increasing opportunities for virus infections to emerge and spread rapidly in our global society, new tools are being provided by research in molecular biology, epidemiology, genomics and bioinformatics. already early warning systems based on state of the art virus detection techniques, as well as targeted intervention strategies based on data about the mutual virus-host interaction have been instrumental in dealing with numerous viral threats, including sars and avian influenza. the extensive research of the department of virology at erasmus medical centre in rotterdam was highlighted by a further presentation, influenza pneumonia: the role of the alveolar macrophage, given by dr. d. van riel. highly pathogenic avian influenza (hpai) h5n1 virus causes severe, often fatal, pneumonia in humans. the pathogenesis of hpai h5n1 virus is not completely understood, although the alveolar macrophage (am) is thought to play an important role. the am resides in the pulmonary alveolus, the primary site of hpai h5n1 virus replication in humans. it had been shown previously that hpai h5n1 virus attaches abundantly to these am (van riel et al., 2006) . the aim of this study was to determine the response of primary human am to hpai h5n1 virus, seasonal h3n2 virus or pandemic h1n1 virus, and to compare these responses with that of macrophages cultured from monocytes. hpaiv h5n1 infection of am compared with that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% versus up to 84%), lower virus production and lower tnf-alpha induction. infection of am with h3n2 or h1n1 virus resulted in even lower percentages of infected cells (up to 7%) than with hpai h5n1 virus, while virus production and tnf-alpha induction were comparable. in conclusion, this study revealed that macrophages cultured from monocytes are not a good model to study the interaction between am and influenza viruses. furthermore, the interaction between hpai h5n1 virus and am could contribute to the pathogenicity of this virus in humans, because of the relatively high percentage of infected cells rather than virus production or an excessive tnf-alpha induction (van riel et al., 2011 2). one virus of each pair was wild type, while the other carried the h274y na mutation conferring resistance to na inhibitor oseltamivir. within each pair, the wild-type and oseltamivir-resistant virus caused disease of equal severity in ferrets and replicated to comparable virus titers in the upper respiratory tract. then, to assess the fitness of drug-resistant h5n1 influenza viruses, the research team considered virus-virus interactions within the host by co-inoculating ferrets with mixtures of the oseltamivirsensitive and oseltamivir-resistant h5n1 viruses in varying ratios (e.g. 100/0; 80/20; 50/50; 20/80; 0/100). using this novel approach, they demonstrated that the proportion of a/vietnam/1203/2004-h274y clones tended to increase, while the proportion of a/turkey/15/2006-h274y clones tended to decrease. their findings suggest that the h274y na mutation can affect the fitness of two h5n1 viruses differently and is dependent on background na sequence. dr. govorkova pointed out that antigenic and genetic diversity, virulence, the degree of na functional loss of h5n1 virus and differences in host immune response can also contribute to such differences. therefore, the risk of emergence of drug-resistant influenza viruses with uncompromised fitness should be monitored closely and considered carefully in pandemic planning. in a collaboration with c. corzo, k. juleen and m. gramer they initiated an active surveillance program in healthy pigs in multiple sites in 2009, during a period coincident with the emergence of the h1n1 pandemic in humans. their study, active surveillance for influenza viruses in north america, presented an analysis from 12 months of data which indicated that similar viruses can be detected in both active and passive surveillance schemes and that there has been an explosion of diversity in swine influenza viruses (siv) in the united states. not only were a number of pandemic h1n1 infections in swine detected, but a number of pandemic/endemic swine virus reassortants were found, albeit from healthy animals (ducatez et al., 2011) . virologically, the pattern of disease surveillance grounded in the activities of state diagnostic laboratories collecting information from diseased animals is representative; however, epidemiologically this data from diseased animals is not representative. reverse zoonoses have had a huge impact on siv in the united states (vincent et al., 2008) , and the pandemic virus is now endemic. however, in considering whether any particular reassortment causes alarm, it must be acknowledged that there is not yet a good model of risk, so h3, like h1, is going to be found in pigs for some time to come, but the consequences of this diversity in siv are not yet clear. the extensive collaboration now taking place in the study of swine influenza was evident in the presentation vessel pendulum began by explaining the three elements of how swine could be considered as a mixing vessel for influenza a viruses as formally proposed by scholtissek et al. (1985) : (i) swine are susceptible to infection with influenza a viruses from avian and human viruses; (ii) the avian viruses can adapt within the pig, producing novel reassortants; and (iii) these reassortants can then be shed and are infectious to man. the goal of this presentation was to test the first part of the mixing vessel hypothesis, concerned with the susceptibility of swine to avian and human influenza viruses, making use of both mixing vessel studies in pigs and genetic markers to investigate adaptation. dr. lager noted that the emergence of the h5n1 highly pathogenic avian influenza virus that can transmit from avian species directly to man, and the presumption that the 1918 h1n1 influenza jumped from birds to man has expanded our understanding of the swine mixing vessel hypothesis as a potential, but not exclusive, source of human pandemic viruses (taubenberger et al., 2005) . moreover, the emergence of the 2009 pandemic h1n1 virus has re-emphasised swine as a potential source of pandemic virus. in this study, all of the challenge viruses (avian h5, h7, h9) induced a similar effect in pigs; challenge viruses did replicate in pigs; the infections were subclinical with mild pneumonias; most infections resulted in seroconversion; and none of them transmitted to contact controls. this series of studies suggests pigs could be easily infected with avian viruses; however, an adaptation step is needed to generate fit viruses that transmit among swine. parallel studies are currently underway testing the susceptibility of pigs to human seasonal influenza viruses. future studies using reverse genetics could investigate potential genetic markers for adaptation of avian viruses to swine which may provide insight into the interspecies transmission of influenza viruses. a in this study, an attempt was made to recreate the 2009 pandemic virus by co-infecting cells (in vitro) or a group of pigs (in vivo) with eurasian (sp04) and north american triple reassortant (ks07) sivs (ma et al., 2010a) . infected pigs were co-housed with two groups of sentinel animals to investigate virus maintenance and transmission. the origin of each gene segment of viruses was determined, which were isolated from supernatants collected from co-infected cells or nasal swabs and bronchioalveolar fluid samples collected from infected and sentinel animals. different reassortant viruses were identified from co-infected cell lines; however, no virus with the genotype of ph1n1 was found. less reassortant viruses were found in the lungs of co-infected pigs in contrast to those in co-infected cells. interestingly, only the intact ks07 was detected from nasal swabs from the second group of sentinel pigs. these results demonstrated that multiple reassortant events can occur within the lower respiratory tract of the pig; however, only a specific gene constellation is able to be shed from the upper respiratory tract. however, in this study, it was not possible to generate the ph1n1 constellation using co-infection with the techniques described above and previously (ma et al., 2010b) . in . she began by reflecting on the ability of swine to act as a reservoir for many influenza viruses, becoming infected with low mortality, regardless of influenza virus strain. the objective of the study was to further understand the porcine response to influenza and to compare this response to other animals infected with the same virus. to accomplish this objective, they used statistical and functional analysis of global gene expression to compare host transcriptional response during acute infection by a contemporary h1n1 pandemic influenza virus (a/california/04/2009) in swine, non-human primates and mice. using their data, they compared and contrasted the biological pathways most significantly associated with gene expression changes during acute infection across these species. their goal was to leverage data collected in their previous studies (ma et al., 2011; safronetz et al., 2011) to better understand influenza virus pathogenesis through a cross-species analysis that considered three crucial questions: (i) which genes change over the course of acute infection? (ii) what are the top functions altered during infection? (iii) how does functional response compare across the three species? despite challenges to data integration and interpretation, including the differences in transcript representation and annotation on the microarrays for the different species, the researchers found notable differences in response to influenza in the lungs of the three species. although similar functional groups of genes changed with infection in all three species, the nature of that response was species specific. swine exhibited an elevated transcriptional response that tapered by resolution of influenza. mice exhibited a decrease in many acute phase and immune response genes quickly followed by a steady increase in expression. host response in macaques was most pronounced and maintained over time. in considering the transcription of immune-related genes in swine, mice and nonhuman primates, they found that although the number of immune-related genes changing in each species was similar, the precise genes changing were very different, with only 14 immune response genes commonly differentially expressed across all three species. this suggested that the nature of immune response within each species may be quite different. in response to the perennial question after any scientific experiment, ''where do we go from here?'' they offered four ideas: (i) time series analysis could reveal unique response kinetics across species, thereby leading to targeted analysis; (ii) data integration across multiple data types, including transciptomics, proteomics, mirna and ngs could generate a more complex, multidimensional view of response; (iii) as annotation of the different species-specific genomes improves, this information could be integrated into future analyses, making a better understanding of the biological responses to infection possible; and (iv) the gathering of this additional information could empower more precise analysis on what makes each species uniquely susceptible or resistant to influenza. in the firm view of these particular six researchers, studies such as this are necessary for a deeper understanding of influenza pathogenesis and demonstrate the utility of systems biology in the study of emerging viruses. three relevant articles on this topic have been published below, highlighting the global dimensions of both infection and treatment, no matter where the virus first emerges. the need for geographical comparative studies of the emerging hantavirus, puumala hantavirus (puuv), has already been indicated by professor henttonen and his team in their presentation summarized earlier in the opening topic of this meeting review. in a further investigation into the same hantavirus, dr. eckerle and her colleagues have presented an article within this special supplement entitled atypical severe puumala hantavirus infection and virus sequence analysis of the patient and regional reservoir host. in this article, they focus on the difficulties in the diagnosis of and treatment for a single patient and performed virus sequence analysis showing regional clustering in reservoir and host. in their more wide-ranging conference presentation, they investigated cytokine expression in a cohort of patients hospitalized with acute severe hantavirus infection during an epidemic in germany in 2010 (cf. faber et al., 2010) . elevated proinflammatory cytokines during the early phase of disease compared to healthy controls and increase in immunosuppressive tgf-b from early to later phase of disease supported the hypothesis of an immune-mediated pathogenesis of puumala hantavirus (sadeghi et al., 2011) . this finding indicates that the immune status of the host for old-world hantaviruses plays an important role, not only the virus itself. in a further article published in this special supplement, how ebola virus counters the interferon system, a. kühl and s. pöhlmann have reviewed which components of the innate immune system could be effective against the zoonotic transmission of ebola virus (ebov) to humans, which results in severe haemorrhagic fever and high case-fatality rates. their focus is on how the interferon (ifn) system, as a key innate defense against viral infections, is targeted by distinct ebov proteins, and on how specific effector molecules of the ifn system could form a potent barrier against the spread of ebov in humans. finally, in lassa fever in west africa: evidence for an expanded region of endemicity, dr. n. sogoba and his colleagues h. feldmann and d. safronetz have stressed the importance of increased surveillance for lassa virus across west africa. the seven presentations summarized below cover a number of haemorrhagic fever viruses. for example, an important example of a highly contagious and life-threatening haemorrhagic fever virus is crimean-congo haemorrhagic fever virus (cchfv), caused by a tick-borne virus of the bunyaviridae family (elliott, 1990) , first recognized in the crimea in 1944, with an identical virus isolated in the congo in 1956; the incidence and geographical spread of this disease with its high human fatality rate have increased significantly in the past 10 years. however, the causes of this increase are not yet clear (maltezou and papa, 2011) . in the light of the need to develop new therapies and effective, safe vaccines, the next seven research presentations could prove to be of considerable significance, not only for cchfv, but also for the hendra, nipah, lujo and ebola viruses. although these viruses have certain common features in their causes and consequences, each haemorrhagic fever virus needs to be carefully studied as a distinct entity. dr (peyrefitte et al., 2010) . moreover, it has already been shown that cchfv causes liver damage in infected patients and in the animal model (bereczky et al., 2010) . the research objectives were to consider: (i) how does cchfv affect hepatocarcinoma cell lines? (ii) is cchfv able to enter and replicate into these cell lines? (iii) does cchfv modulate the in vitro cellular response? to better understand the cchfv pathogenesis in liver cells, they analysed in vitro the host response induced after cchfv infection in huh7 (unable to produce ifn-beta) and hep-g2 (capable of producing ifn-beta) cell lines. they noticed that while in huh7, cchfv infection elicited at day 3 a cytopathogenic effect, no visible effect was seen in cchfv-infected hepg2. this intriguing feature led them to analyse the viral parameters expecting a differential cellular response. both cell lines were shown to be permissive to cchfv and with a high viral yield as monitored by plaque titration assay, genomic and antigenomic strand quantification. these cchfv-infected hepatocarcinoma cell lines induced only il-8 secretion. in addition, a pro-apoptotic effect was observed in huh7 but not in hepg2. interestingly, no type-i ifn was detected for hep-g2 during the kinetic study, suggesting a strong inhibition of ifn secretion. they concluded that cchfv does enter and replicate in hepatocytes and that hepatocytes could be involved in cchf pathogenesis associated with antigen presenting cells for cchfv dissemination. while cchfv did not induce ifn-beta secretion in hepatocyte cell lines, cchfv did induce the secretion of il-1 in hepatocyte cell lines. furthermore, cchfv induced a higher secretion of il-8 in the apoptotic huh 7 cell line than in the nonapoptotic hep-g2 cell line. thus, this research indicated that il-8 production and apoptosis seemed to be markers of cchfv pathogenesis in hepatocyte cell lines. professor t. w. geisbert (university of texas, medical branch, galveston, tx, usa) presented an evaluation of countermeasures against hendra and nipah viruses in nonhuman primate models. he pointed out that the henipaviruses, hendra virus (hev) and nipah virus (niv) are enigmatic emerging pathogens that can cause severe and often fatal neurologic and/or respiratory disease in both animals and humans. guinea pigs, hamsters, ferrets and cats have been evaluated as animal models of human hev infection. a research team led by professor geisbert recently evaluated african green monkeys as a nonhuman primate model for henipavirus infection and discovered that they are the first consistent and highly susceptible nonhuman primate models of hev and niv infection rockx et al., 2010) . the severe respiratory pathology, neurological disease and generalized vasculitis manifested in both hev-and nivinfected african green monkeys provides an accurate reflection of what is observed in henipavirus-infected humans. these nonhuman primate models were then employed to evaluate several post-exposure treatments including ribavirin (which did not work) and a human anti-henipavirus monoclonal antibody (which was successful). dr the research was motivated by the awareness that neutralizing antibodies are probably the major effectors against this viral infection. the rationale of using rv vectors for the development of a niv vaccine was fourfold: (i) rv-vectored vaccines are not pathogenic regardless of the route of administration or the immune status of the host; (ii) rv-based vaccines are very efficacious even after a single immunization by the oral route; (iii) rv-based vaccines have the ability to target macrophages and dendritic cells, to induce th1 t-cell response and are capable of inducing long-lasting immunity; and (iv) postexposure prophylaxis using recombinant rv vaccines is very effective, even when the cns is already infected (faber et al., 2009a,b) . the niv g gene was inserted into the non-pathogenic rv vectors spbaangas or spbaangas-gas, resulting in spbaangas-ng or the double gas variant spbaan-gas-ng-gas, respectively. further research led to four significant conclusions: (i) there are no detectable amounts of niv g present in recombinant nivg-rv particles; (ii) the presence of an niv g gene does not increase, but rather decreases the pathogenicity of the recombinant viruses; (iii) priming with nivg-rv triggers a strong niv g-specific memory response, which correlates inversely with vaccine concentration used for the priming; and (iv) a single immunization with nivg-rv is probably sufficient to protect against a niv challenge infection. arenaviruses are rodent-borne bisegmented ambisense rna viruses, which include lassa fever virus, lymphocytic choriomeningitis (lcm) and tacaribe the index case for this acute febrile illness virus was a travel agent living on a farm during 2008 in lusaka, zambia, who infected a local cleaner, as well as a paramedic and a nurse in johannesburg, south africa, all of whom died, with the paramedic infecting a further nurse who was treated with ribavirin and survived . the name of the virus originated from the first two letters of the two key cities, lusaka and johannesburg. four of the five infected persons died of haemorrhagic fever-like symptoms paweska et al., 2009 ). viral genome sequencing revealed that this virus differed from other arenaviruses by at least 36% and is highly pathogenic, with a case fatality rate (cfr) of 80% paweska et al., 2009) . in view of the uniqueness and high virulence of lujo virus (ljv), the research team developed a reverse genetics system to study the molecular characteristics of this novel arenavirus. this system will facilitate studies of ljv biology, development of antiviral screening assays and pathogenesis studies in animal models. t. cutts (national microbiology laboratory, public health agency of canada, canadian science centre for human and animal health, winnipeg, manitoba, canada) with his colleagues s. theriault (chief, applied biosafety research program, same centre) and g. kobinger (chief, special pathogens program, same centre) presented cytofixô inactivation of veroe6 cells infected with zaire ebola virus (zebov) both in vitro and in vivo. first, it was pointed out that removing infected tissues from high-containment laboratories requires implementation of a number of different decontamination techniques to render the organism inert and is subject to flexibility according to the laws of the country in which the laboratory is located. according to the canadian biosafety guidelines 4th edition, an organism may be removed from containment once it has been rendered inert, but no procedure is in place to validate these biosafety guidelines, and it is up to the individuals to implement the relevant guidelines (public health agency of canada, 2004, p. 28. chap. 3.1.4). methods such as gamma irradiation, formalin fixation, acetone and methanol permeation, plus the use of various other chemical agents, are common practices to preserve cellular tissue or blood components and to inactivate organisms (elliott et al., 1982; mitchell and mccormick, 1984; preuss et al., 1997; villinger et al., 1999; sanchez et al., 2007) . such methods still raise questions as to their effectiveness or their redundancy. furthermore, these inactivation steps can lead to the alteration of the target organism possibly affecting the qualitative and quantitative results. the focus of the applied biosafety research program was to evaluate and develop technologies and procedures relevant to biocontainment in the context of the laboratory, as well as to prevent unintentional and intentional release of dangerous organisms into the environment. using the commercial product, cytofix/cytoperm tm from bd biosciences, this research sought to inactivate vero e6 cells which had been infected with the deadly zaire ebola virus (zebov). the aim of the research was to determine the effectiveness and duration of cytofix/ cytoperm for fixing the cellular material infected with zebov. the veroe6 cells were infected with the wildtype zebov and a mouse adapted zebov(mazebov) and assayed after a 5-min and 20-min exposure to cyto-fixô followed by neutralization. samples of blood from a non-human primate infected with zebov were drawn at 7 dpi and assayed for effectiveness in the same manner as the in vitro studies with cytofixô. in addition, vero e6 cells infected with mazebov were treated in the same manner and injected into balb/c mice to compose the in vivo studies. cytoxicity and neutralization assays were used to determine the effect (if any) the treatment had on both the virus and the health of host cells. results of the tissue culture tcid50 assay showed that a 5-min exposure to cytofixô inactivated a large portion of the cells containing infectious virions, while after a 20-min exposure, no detectable levels of virus were observed. blood samples from the non-human primates showed similar results to the cell culture assay having no detectable virus from infected cells after 20 min of exposure. in vivo studies with mice showed that both a 5min and 20-min exposure time to cytofixô had a 100% survival rate after 28 days post-infection, while the positive controls succumbed after 4 to7 dpi. because laboratories differ in their preferences of technique, the time of inactivation also varies. what this research demonstrated was the effectiveness of a quick procedure of 20 min for inactivating viruses within cells infected with zebov, thereby rendering organisms safe to remove from containment. has not yet been linked with disease in humans, the presence of antibodies against rebov in people working closely with infected macaques and swine indicates that humans can be infected with this virus (miller et al., 1990; miranda et al., 1991; barrette et al., 2009 ). however, research has been hampered by the fact that the only available disease model for rebov to date has been cynomolgus macaques. seeking new rebov disease models, the research team assessed various rodent models -the balb/c mouse, hartley guinea pig, syrian hamster and stat1 )/) mouse that lacked the signal transducer and activator of transcription 1 (durbin et al., 1996) . although virus replication occurred in guinea pigs and hamsters, progression to disease was only observed upon inoculation of stat1 )/) mice. despite certain drawbacks set out in the journal article, the stat1 )/) mouse can be used to investigate the determinants of differences in pathogenicity in various rebov strains, as well as to assess vaccination and antiviral therapies (miller et al., 1990; miranda et al., 1991; durbin et al., 1996; barrette et al., 2009; de wit et al., 2011) . the unity of human, animal and ecosystem health outlined by professor aguirre, as well as the interactions among multiple tick-borne pathogens in a natural reservoir host set out by professor fish and his research team, both summarized in topic 1 above, highlight the necessity of cross-disciplinary collaboration in studying zoonotic bacterial diseases (daszak et al., 2007, pp. 470-471) . such collaboration is especially important in studying tick-borne infectious disease, which emerged so extensively in the united states during the last three decades of the twentieth century (paddock and yabsley, 2007, p. 290) . now, in an article published in this special supplement, beyond lyme: etiology of tick-borne human disease with emphasis on the southeastern united states, drs. stomdahl and hickling have explained that tick distributions are in flux, especially in the south-eastern united states, requiring health providers to think 'beyond lyme' to identify the specific tick species that bite humans and the different pathogens these ticks carry. in an international context, drs. wood and artsob have set out the increasing importance of travel-associated rickettsioses in their article, spotted fever group rickettsiae: a brief review and a canadian perspective. in a third article published in this special supplement, drs. verma and stevenson present an article on epidemiology of leptospirosis with its one million cases worldwide. in leptospiral uveitis -there's more to it than meets the eye! they hypothesize in detail about how the eye inflammation uveitis is triggered and stress the impact that 'understanding how this bacterium is able to induce this inflammatory process will be a key to the better management and prevention of the disease'. this continuum of basic research leading to understanding a disease and then to managing that disease and finally to preventing it offers a pattern of scientific discovery that is relevant to many other emerging zoonotic diseases. opening his presentation, the foodborne pathogen campylobacter jejuni exploits mammalian host cell receptors and signaling pathways, professor konkel noted that the per cent of c. jejuni isolates that are resistant to antibiotics is continuing to increase and that c. jejuni infections are frequently associated with serious sequelae, including guillain-barré syndrome. it is well understood that infection with c. jejuni is often a consequence of eating foods contaminated with undercooked poultry. however, c. jejuni pathogenesis is a highly complex process that is dependent on many factors including motility, adherence, cell invasion, protein secretion, intracellular survival and toxin production. acute illness, characterized by the presence of blood and leucocytes in stool samples, is specifically associated with c. jejuni invasion of intestinal epithelial cells. dissecting bacteria-host cell interactions are critical to understanding the infection caused by c. jejuni. previous work has shown that maximal invasion of host cells by c. jejuni is dependent on synthesis of the c. jejuni cadf and flpa fibronectin (fn) binding proteins and requires the secreted campylobacter invasion antigens [cia(s)] (larson et al., 2008) . to test the hypothesis that maximal cell invasion requires specific signalling events, binding and internalization assays were performed in the presence of numerous inhibitors of cell signaling pathways. the research team found that c. jejuni cell invasion utilizes components of focal complexes (fcs), as invasion is significantly inhibited by wortmannin (an inhibitor of pi-3 kinase) and pp2 (a c-src inhibitor). they further demonstrated that a wild-type strain of c. jejuni results in the activation of the rho gtpase rac1. these observations are consistent with the proposal that c. jejuni binding to host cell-associated fn and secretion of the cia proteins trigger integrin receptor activation, which in turn promotes intracellular signalling and actin cytoskeletal rearrangement. on the basis of these data, they concluded that c. jejuni utilizes a novel mechanism to promote host cell invasion. the research findings professor konkel presented were recently published in cellular microbiology (eucker and konkel, 2012) . simple, fast and specific tests for pathogen identification are essential for epidemiological investigation of numerous diseases. within the field of immunodiagnostics, a quantitative determination of either antibody or antigen by antigen-antibody interaction can be made by lateral flow tests (also known as a dipstick or rapid tests). dr. e. baranova and her colleagues p. solov'ev, n. kolosova and s. biketov (all state research center for applied microbiology, obolensk, russia) began the presentation, development of lateral flow tests for the fast identification of zoonotic disease agents, by pointing out that lateral flow (lf) tests can be used in the field, as a diagnostic tool that produces results that can be read visually by the naked eye within 20 min after sample application. the creation of an algorithm for the development of an appropriate lf test to identify biopathogens requires the development of a target antigen, obtaining specific antibodies (biketov et al., 2010) and then creating a lf-test formulation to be trial tested. the target antigens must have the ability to induce species-specific antibodies, as well as be characterized by surface localization with multiple epitope presentation on the surface. the antibodies need to have a specificity and sensitivity sufficient for application in the lf detection format, as well as the capacity to be preserved after labelling with gold particles and after immobilization on a surface. over a period of 22 months, the research team developed and tested in the field lf tests for the detection of bacillus anthracis, which causes anthrax, yersinia pestis, which causes bubonic plague, and francisella tularensis, which is the causative agent of tularaemia (or rabbit fever). all three of these lf tests have now been made available as commercial products and are being used throughout russia for the rapid identification of these dangerous pathogens. drs. j. d. trujillo and p. l. nara (center for advanced host defences, immunobiotics and translational comparative medicine, iowa state university, ames, iowa, usa) have developed and validated a new approach to the diagnosis of infectious agents. dr. trujillo explained that they are employing novel polymerase chain reaction (pcr)-based methods for the detection and differentiation of current and emergent mycoplasma species relevant to human and animal medicine and biodefense. their presentation, titled novel sybr ò real-time pcr assay for detection and differentiation of mycoplasma species in biological samples from various hosts, began by explaining the relevance of mycoplasma species, which are endemic, strict or opportunistic pathogens in human and animal medicine. moreover, mycoplasma species are important re-emerging pathogens and foreign animal diseases. importantly, mycoplasma species are difficult to culture or are un-culturable, and thus are difficult to impossible to detect by conventional diagnostic methods. moreover, current pcr methods have limited breath of species detection and differentiation, requiring the use of species-specific assays that are costly and time-consuming. their goal was to develop a pilot mycoplasma genus diagnostic assay to validate the novel application of high-resolution melt (hrm) methodology for rapid, sensitive and cost-effective detection and differentiation of various pathogenic mycoplasma species. dr. trujillo presented the validation and utilization of sybr ò green dye in real-time pcr (qpcr) mycoplasma detection and differentiation assay (panmyco qpcr). this pcr assay utilizes primers specific for this genus (modified from s. c. baird et al., 1999) . this pcr assay results in the generation of small dna fragments of various base pair lengths called pcr amplicons. each amplicon has a melt temperature (tm) that is determined following qpcr. sequence of amplicon representative of the mycoplasma species present defines the melt temperature (tm) and allows for the use of amplicons tm in species identification with limited resolution and excellent sensitivity. the panmyco qpcr assay has similar sensitivity to a conventional nested pcr assay for mycoplasma bovis with a linear detection range of one colony forming unit (trujillo et al., 2009 ). additional work presented described increasing species resolution of this assay, by defining unique melt profiles for each mycoplasma species amplicon utilizing precision melt software from biorad, ca, usa to perform hrm analysis. greater than 30 different species of mycoplasma found in bovine, caprine, ovine, avian and porcine hosts have been characterized with the panmyco qpcr and hrm analysis. occasionally, this testing has resulted in the detection of multiple species in a single sample or discovery of novel or emergent mycoplasma species. this data analysis method allows for the sensitive detection and rapid differentiation of numerous mycoplasma species in many different hosts. dr. trujillo concluded that this novel real-time pcr assay can detect and potentially differentiate all known mycoplasma species. moreover, this presentation demonstrated the novel use of genus-specific sybr green pcr and hrm analysis for the detection, differentiation and discovery of medically important pathogens. several additional translational research projects have been launched to demonstrate the importance and utility of the pan myco qpcr assay in the context of infectious disease surveillance. one translational research project focuses on validation of this novel molecular methodology for field detection assays. there is increasing awareness of the need for improved laboratory investigation, risk assessment, contingency planning and simulation exercises to respond effectively to zoonotic diseases (lipkin, 2008; westergaard, 2008a and 2008b; escorcia et al., 2012) . in view of the need to research into and respond to so many emerging zoonoses, it is relevant to note the fourfold classification of emerging zoonoses proposed earlier by silvio pitlik: type 1: from wild animals to humans (hanta); type 1+: from wild animals to humans, with further human-to-human transmission (aids); type 2: from wild animals to domestic animals to humans (avian flu); and type 2+: from wild animals to domestic animals to humans, with further human-to-human transmission (sars) (kahn et al., 2009: p. 410) . confronting outbreaks of these emerging zoonoses is often possible with an imaginative combination of laboratory investigation and extensive fieldwork (borchert et al., 2011; robinson, 2011) . three distinctive articles appear below on outbreak responses to zoonotic diseases, highlighting the importance of linking together basic research, practical action and an integrated one health-oriented approach. in a. grolla and nine co-authors from eight different institutions in five different countries have explained how two mobile laboratories were set up and capable of running within <24 h of arrival, providing safe, accurate, rapid and reliable diagnostic services as the ebola zaire outbreak began in the democratic republic of the congo. finally, in emerging and exotic zoonotic disease preparedness and response in the united states: coordination of the animal health component, dr. r. l. levings has set out the integrated approach of emergency management and diagnostics, veterinary services, animal and plant health inspection service, united states department of agriculture in the prevention of, the preparedness for, the response to and the recovery from a zoonotic disease outbreak. in all three of these areas -basic research, practical action and an integrated one health-oriented approach -much has been achieved in recent years, but much also remains to be achieved as soon as possible. even when those diseases are not transmitted to humans, there are substantive challenges, as highlighted in the next case study by woods on combating brucellosis in cattle in zimbabwe. in a practical, problem-oriented presentation, dr. p. s. a. woods (veterinary public health section, faculty of veterinary science, university of pretoria, onderstepoort, south africa and university of reading) with r. s. beardsley (pharmaceutical health services research, school of pharmacy, university of maryland, baltimore, maryland, usa) and n. m. taylor (veterinary epidemiology and economics unit, school of agriculture, policy and development, university of reading, reading, united kingdom) asked can we increase farmers' perception of their brucellosis susceptibility to improve adoption of preventive behaviors amongst small-scale dairy farmers in zimbabwe? she explained the background to the problem, presented a model that was used to develop a strategy to confront the disease and then set out the results and recommendations of the research team. brucellosis is an extremely infectious bacterium that causes abortion in cows, different syndromes in other animal species and malaria-like undulant fever, arthritis, depression and epididymitis in people. however, it had been controlled in zimbabwe until 2001 when financial constraints forced the government veterinary services to curtail disease surveillance and discontinue free vaccinations. small-scale farmers did not seek vaccination from other sources, partly because they were unaware of the necessity of vaccination, and also at that time brucellosis was absent from small-scale farming areas. however, uncontrolled cattle movements from 2000 to 2009 linked to invasions of large-scale farms resulted in dispersal of possibly brucella-positive cattle and movement of the disease into small-scale herds. the result was that brucellosis became a potential problem in these herds and now presents a serious zoonotic threat. preventing brucellosis requires movement control to stop brucella-positive cattle entering an area, as well as live vaccine for female calves. although there is no human-to-human spread of the disease, it is essential that people do not handle new-born calves or abortions from brucella-positive cows, nor drink unpasteurized milk from brucella-positive cows (arimi et al., 2005) . in essence, reducing the risk of brucellosis requires that farmers adopt appropriate preventive behaviours, with these control efforts and changes in behaviour being communitydirected in order to be sustainable. it was this stress upon community direction that formed the basis for funding by the wellcome trust to investigate the hypothesis that the level of a farmer's knowledge about brucellosis would influence subsequent preventive behaviour. the approach, based partly on the 'health belief model' (rosenstock et al., 1988) was grounded in the expectation that each small-scale farmer would make health behaviour choices according to individual perceptions about the disease and personal beliefs about their abilities and the costs required to change the risks of their cattle and families acquiring the disease. in this project, the independent variable was the level of an individual farmer's knowledge about brucellosis, while the dependent variables were two key preventive behaviours -decreasing cattle disease by calfhood vaccination and preventing zoonotic disease by milk pasteurization. the research was carried out in partnership with a national network of small-scale dairy cooperatives with all activities conducted with existing local personnel. the aim was to tailor the educational program to the initial knowledge or awareness of each community of farmers, recognizing the considerable difference in knowledge levels between-and within communities. local teams, not outsiders, developed appropriate educational materials, targeting those with the lowest levels of knowledge. completed survey questionnaires indicated a significant relationship between the initial level of farmers' knowledge about brucellosis and their calf brucellosis vaccination practices. the range of brucellosis knowledge among some 210 small-scale farmers in southern zimbabwe was considerable, with 38% of farmers being unaware of the disease, 12% having limited knowledge and 50% having good knowledge. however, even amongst those farmers with a relatively high level of knowledge, 78% of farmers had not vaccinated their calves at the time of the survey. furthermore, there was a disappointingly low uptake of milk boiling despite a significant increase in knowledge about raw milk as a mode of infection for humans. although the information sessions did increase farmers' awareness of the dangers of zoonotic brucellosis, an exaggerated perception of the effectiveness of calf vaccination decreased the likelihood of safe milk practices. this outcome indicated the importance of reaching the women who were responsible for milk and food preparation. ongoing research is investigating whether increasing the role of nurses and environmental health technicians to emphasize human infection and to reach different family members, within a research paradigm which combined veterinary and human medicine, would increase the uptake of milk hygiene practices. there is increasing awareness of the need to balance transparency with carefully designed information disclosure strategies in the face of sudden outbreaks of foodborne diseases (national research council, 2011; taylor, 2011) . both consumers and producers must be rapidly informed of any significant dangers with specific food products; however, considerable misinformation can be spread if laboratory results are incomplete or inconclusive (palm et al., 2012) . recent experience with e. coli-infected sprouts in germany and listeria-infected cantaloupes the united states has highlighted the difficulties in identifying the original source of a disease outbreak, as well as the swiftness with which an unexpected food-borne disease can cause sickness and death (armour, 2011; blaser, 2011; buchholz et al., 2011; frank et al., 2011) . it should be noted that that there was no easily identified zoonotic link in either of these two food-borne diseases derived from bacteria, which killed 29 people in the united states and 50 throughout europe during 2011; however, as professor c. kastner points out later, a significant number of these food-borne diseases do have a zoonotic origin (parker et al., 2011) . two articles linked to this topic are published in this special supplement. first, there is emerging antimicrobial resistance in commensal e. coli with public health relevance by dr. a. käsbohrer and her colleagues. their aim was to assess the prevalence of and trends in antimicrobial resistance through active monitoring programs along the food production chains for poultry, pigs and cattle, as well as to collect isolates for resistance testing and then select certain isolates for further phenotypic and genotypic characterization. the research team found alarming rates of resistance to antimicrobials in zoonotic bacteria and commensals, as set out in their article, which could compromise the effective treatment for human infections. this work provides a basis on which to improve both risk assessment and risk mitigation strategies in the face of the increasing antimicrobial resistance to zoonotic bacteria and parasitic organisms within both humans and animals. second, in american trypanosomiasis infection in fattening pigs from the south-east of mexico, m. jiménz-coello and her colleagues have investigated the extent to which the protozoa trypanosoma cruzi (t. cruzi) is presenting in fattening pigs in yucatan, mexico, threatening parasitic infections in animals destined for human consumption. tackling the question of how to refine national and international strategies to combat food-borne zoonotic diseases, professor c. kastner (food science institute, kansas state university, manhattan, kansas, usa) considered the public health and economic impact of foodborne zoonotic diseases. he began by noting that each year in the united states, according to statistics from the centers for disease control, 48 million people become sick from food-borne diseases, 128,000 are hospitalized and 3,000 die. a significant portion of these diseases have a zoonotic origin, with extensive product recalls and domestic as well as international trade disruptions (fung et al., 2001) . therefore, more than 20 years ago, the us department of agriculture established a food safety consortium (2011) which focuses on food-borne zoonotic diseases involving beef in kansas, pork in iowa and poultry in arkansas. the continuing aim of that consortium is to develop long-term control strategies that identify the critical control points and control technologies, as well as short-term strategies to address incidental contamination, whether accidental or intentional. the us livestock industry in general and kansas in particular are vulnerable to food-borne zoonotic diseases. for example, in kansas, sources of contamination include feed, feedlots (which vary in size from 1,000 to 150,000 head per lot) and packing plants (which vary in size from 3,000 to more than 5,000 head per day per plant). beef processing points where mixing of different ingredients occurs are the most critical points for both incidental and intentional contamination. in the light of these challenges, a biosafety level 3 research facility, the biosecurity research institute (bri) (2011) has been built on the kansas state university campus, to evaluate strategies to detect and control food-borne zoonotic diseases from production through processing. furthermore, in minneapolis, minnesota, ncfpd (national center for food production defense, 2011) has been operational since 2004 as a department of homeland security center of excellence. ncfpd has adopted a systems approach whose goals include to: (i) ensure significant improvements in supply chain security, preparedness and resiliency; (ii) develop rapid and accurate methods to detect incidents of contamination and to identify the specific agent(s) involved; (iii) apply strategies to reduce the risk of food-borne illness because of intentional contamination in the food supply chain and to develop the tools to facilitate recovery from contamination incidents; (iv) deliver appropriate and credible risk communication messages to the public; and (v) develop and deliver highquality education and training programs to develop a cadre of professionals equipped to deal with future threats to the food system. these research centers are essential to minimize the threat of food-borne zoonotic diseases. t. cutts (national microbiology laboratory, public health agency of canada, canadian science centre for human and animal health, winnipeg, manitoba, canada) presented comparative inactivation studies of listeria monocytogenes at room and refrigeration temperatures on behalf of a research team that included b. carruthers, c.-l. cross, s. theriault (chief, applied biosafety research program, same center) and himself. listeria monocytogenes, a non-sporulating, gram-positive bacillus, is found chiefly in ruminants, but can affect all species and causes listeriosis, an infrequent but serious illness that affects the central nervous system of humans and domestic animals (bortolussi, 2008; chan and weidmann, 2009 ). listeriosis can be acquired from the consumption of contaminated foods and has an incubation period ranging from 2 to 70 days (bortolussi, 2008; chan and weidmann, 2009 ). because of this variable incubation period and the fact that listeriosis leads to a mortality rate of 20-30%, the applied biosafety research program at the national microbiology laboratory of the public health agency of canada considered the significance of proper decontamination of listeria in food processing environments (chan and weidmann, 2009 ). the importance of this work is indicated by the fact that somewhere from 1 to 10% of ready-to-eat foods are thought to be contaminated with listeria (public health agency of canada, 2004 and . recently, listeria monocytogenes has gained notoriety because of its ability to grow at the low temperatures, high salt and low ph used in food processing plants (bortolussi, 2008) . therefore, a study was undertaken to determine the bactericidal efficacy of various liquid disinfectants and the effect that low temperatures have on the ability of these disinfectants to inactivate l. monocytogenes at conditions found in food processing plants. at both room and refrigeration temperature (4°), ethanol, javex, su393 and peracetic acid (paa) products outperformed all others. surprisingly, there was no significant variation in performance at room temperature compared with refrigeration temperature. however, as some organisms undergo changes during a temperature shift, it is crucial to test each disinfectant at the temperature at which it will be employed. bleach was found to be effective but is toxic, corrosive and residue forming, while the paa and ethanol compounds do not form residues and are not corrosive. as a result of these studies, major canadian food-processing plants have changed their decontamination procedures and are no longer using quaternary ammonium compounds (quats), which were previously used extensively. positive relations have been built up between companies and laboratories, leading to more relevant laboratory studies and industrial applications (public health agency of canada, 2012). a prion (proteinaceous infectious particle) has been defined as a 'malformed version of a normal cellular protein that apparently ''replicates'' by recruiting normal proteins to adopt its form, [thus becoming] capable of infecting other cells of the same, or a different organism' (prusiner, 2003; thain and hickman, 2004, p. 573) . although two nobel prizes in medicine have been awarded for prion research, to carleton gajusek in 1966 and to stanley prusiner in 1997, the precise nature of the infectious agent remains unclear to such an extent that controversy continues about whether a prion is solely protein (brooks, 2011, pp. 75-100) . whatever the cause, prion diseases are fatal chronic neurological diseases that affect the brains and nervous systems of many mammals, including humans (imran and mahmood, 2011) . prions can be detected in tissues by a number of research techniques, including infective bioassay, animal inoculation, western blot and immunochemistry. it is clear that prions can cause spongiform encephalopathies within both humans and animals (e.g. creutzfeldt-jakob disease, kuru, scrapie, transmissible mink encephalopathy, feline spongiform encephalopathy and bovine spongiform encephalopathy) (blood et al., 2007 (blood et al., , p. 1456 . summaries of the three presentations below offer further insights into the nature of prion diseases. in prion diseases, professor j. j. badiola and dr. c. akin (university of zaragoza, zaragoza, spain) focused on the 1986 outbreak of bovine spongiform encephalopathy (bse) ('mad cow disease') in the united kingdom, which led to a better understanding of the epidemiology and molecular characteristics of the disease. epidemic bse affected mainly the united kingdom, with a total of 184,615 positive animals compared to 5,765 in all other member states of the european union (oie, 2012). control and eradication of transmissible spongiform encephalopathies (tses) became a priority, not only in europe, but throughout the world. in 2000, a reinforcement of the passive surveillance program and the establishment of an active one were established by the european commission for all the european union member states (european commission, 2001) . passive surveillance, focused on animals with clinical signs of the disease, and active surveillance was carried out in the following target groups: healthy slaughtered, fallen stock, emergency slaughtered and animals culled under bse eradication. apart from these measures, specific risk materials (e.g. tonsils, intestines, spleen, spinal cord and skull, including the brain and eyes) were defined and prohibited from being included in the human food chain. moreover, a banning of all meat and bone meal for animal feed was established (european commission, 2009) . the result of these powerful eradication measures has been a rapidly decreasing number of new bse cases, with less than 50 cases detected worldwide in 2010, 45 of which were in the european union (oie, 2012) . the impressive containment of bse in the united kingdom from 35,090 reported cases in 1993 to 11 in 2010 is testimony to the determination with which scientists, politicians, civil servants and farmers have worked together to bring the disease under control. professor c. i. lasmézas (dept of infectology, the scripps research institute, scripps, florida, usa) began her presentation, zoonotic potential of new animal prion diseases: assessment in non-human primates, by noting that the first demonstration of the transmissibility of a prion disease to non-human primates (nhps) was made in 1966 by carleton gajdusek when he transmitted kuru to chimpanzees. since then, animal and human prion diseases have been transmitted to a range of nhps. cynomolgus macaques have shown the highest selectivity with regard to the prion strain by which they can be infected and therefore seem to be the species of choice to assess the risk that any given animal prion strain can be pathogenic for humans (lasmézas et al., 1996) . prions were thought to be very difficult to transmit from one species to another; however, the experience of studying scrapie highlights the difficulties inherent in studying prion diseases in the laboratory. scrapie had been transmitted orally to other ruminants (goats) but only intracerebral inoculations had successfully transmitted scrapie to monkey, mouse or mink. however, the oral transmission of bovine spongiform encephalopathy (bse) to domestic cats in 1990 forced a revision of this earlier belief. transmissions of bse have now occurred orally to sheep, goat, monkey, mink, cheetah, puma, cat and mouse. intracerebral transmission of bse has also occurred to pig. furthermore, intraspecies oral transmission of bse has taken place within numerous speciesmonkey, mink, sheep, goat, cow, hamster and mouse. vcjd (variant creutzfeldt-jakob disease) is a new human disease, which was caused by eating ruminant-derived food products contaminated with bse. vcjd poses a public health problem because of the absence of preclinical diagnostic test, the long incubation periods of prion diseases in humans (possibly extending up to 50 years) and the transmissibility of the disease by blood transfusion. the research team at the french commissariat a l' energie atomique (cea) demonstrated that bovine spongiform encephalopathy (bse) was transmissible to macaques within 3 years with a 100% infection rate and caused a disease indistinguishable from the human variant of creutzfeldt-jakob disease (lasmézas et al., 1996) . this provided a model to study carefully the peripheral pathogenesis of vcjd, the oral infectious dose of bse and evaluate the risk of human-to-human transmission of vcjd by blood transfusion (herzog et al., 2004) . further, the research team used the macaque model to assess the zoonotic potential of emerging forms of bse called l-or h-type. the l-type bse presents with higher pathogenicity to macaques than classical bse (comoy et al., 2008) . therefore, continued precautionary measures remain necessary to protect the human food chain. experiments are ongoing at the national institute of allergy and infectious disease, hamilton, montana, to assess the risk linked to chronic wasting disease that is spreading throughout the usa. the closing acknowledgements of professor lasmézas to 35 other researchers indicated both the complexity and importance of continuing work in prion diseases. furthermore, since the cancun meeting further important research has been published (hamir et al., 2011) . infectivity distribution studies of animals infected with bse prions animals are a matter of considerable importance in seeking to elucidate the route of infectious prions from the gut to the central nervous system (cns) open questions about this lethal journey from the gut to the brain, including where in the gut the disease begins, the initial steps of the neuronal bse pathogenesis, the ascension of bse prions to the brain, the haematogenous spread and the centrifugal contamination of the periphery (buschmann and groschup, 2005; hoffmann et al., 2007) . the scale of the research task was indicated by the fact that 1,400 samples were collected per animal autopsy, leading to some 200,000 frozen samples collected and archived at the friedrich-loeffler-institut. tissue samples were collected from the gut, the central and autonomous nervous system (ans) of the challenged bovines and then examined for the presence of pathological prion proteins (prp sc ). there was some variation among different animals. however, a distinct accumulation of prp sc was observed in the distal ileum, confined to follicles and/or the enteric nervous system, in almost all animals . bse prions were found in the sympathetic nervous system starting from 16 months post-inoculation (mpi) on as well as in the parasympathetic nervous system from 20 mpi on (kaatz et al., 2012) . a clear dissociation of prion infectivity and detectable prp sc deposition was obvious in tongue (balkema. the earliest presence of infectivity in the brainstem was detected at 24 mpi, while prp sc -accumulation was detected first after 28 mpi. in summary, these results deciphered for the first time the centripetal spread of bse prions along the ans to the cns starting already half way during the incubation period. bse prions spread in cattle from the gut to the brain along the sympathetic, parasympathetic and spinal cord routes, possibly in that order of importance. spinal cord involvement may even not be necessary at all, but bse infectivity in the form of prp sc spills over into the periphery already in the pre-clinical phase. the modelling and prediction of emerging zoonoses is a fast-growing field of considerable complexity. of the five papers relevant to this topic, two have been published in full below in this special supplement. dr. g. zanella and her colleagues consider modelling transmission of bovine tuberculosis in red deer and wild boar in normandy, france. their mathematical model of the mycobacterium bovis infection within and between species takes into account the transmission of m. bovis through infected offal -the viscera of animals killed by hunters and left behind. when an animal was hunted in the brotonne forest in normandy prior to 2002, it was eviscerated in situ and only the carcass taken away, with the raw viscera left behind. since 2002, offal disposal has been required in brotonne forest; however, the regulation has not always been observed by hunters (unpublished correspondence with g. zanella, 16-17 december, 2011) an important benefit of mathematical modelling is that it permits consideration of all the elements involved in disease transmission within a population, thereby complementing field data, as well as testing the effects of control measures. thus, the direct transmission of the m. bovis infection within the red deer and wild boar populations can be distinguished from indirect transmission through contaminated offal. the model indicates that offal destruction is the key factor in infection control for both red deer and wild boar. the authors conclude that, in principle, the structure of this model is relevant to the situations where dead animals play an important role in disease transmission between two or more species. in a further article published in this special supplement, constructing ecological networks: a tool to infer risk of transmission and dispersal of leishmaniasis, dr. c. gonzález-salazar and professor c. stephens set out the role of ecological networks as a powerful tool for understanding and visualizing inter-species ecological and evolutionary interactions. taking the example of leishmaniasis in mexico, they show that such networks can be used not only to understand potential ecological interactions between species involved in the transmission of the disease, but also to identify the potential role of the environment in disease transmission and dispersal. strikingly, they show how potential interactions can be inferred from geographical data, rather than by direct observation. their findings have led to the prediction of additional reservoirs in mexico of many new species, including bats and squirrels. the resulting model can be used to understand and map potential transmission risk, as well as construct risk scenarios for the dispersal of leishmaniasis from one geographical region to another. such a risk assessment tool for leishmaniasis will be especially useful in the light of the bill and melinda gates foundation decision in january 2012 to join with 13 major pharmaceutical companies and the world health organization in targeting leishmaniasis as one of the neglected tropical diseases to receive improved drugs, diagnostics, vector control strategies and vaccines (bill & melinda gates foundation, 2012; boseley, 2012) . however, the possibility of new reservoirs suggests it is hard to imagine that leishmaniasis can be completely eradicated. nevertheless, it is increasingly clear that leishmaniasis has a disturbing capacity to jump from species to species, so efforts to control the disease must be given a high priority (unpublished correspondence with c. stephens, february 1, 2012; cf. flanagan et al., 2011) . it is difficult to model and predict the distribution and impact of a new emerging virus. for example, the emergence in november 2011 in europe of a midge-borne virus member of the bunyaviridae family, named schmallenberg virus after the location in germany where it was first detected, has caused serious birth defects in lambs, goats and cattle (ecdc, 2011) . scientists, farmers, veterinarians, public health officials and consumers are all confronted with the uncertainty inherent in facing a new animal pathogen (farmers weekly, 2012) . appropriately, at the same time as this new virus has emerged, the animal health and veterinary laboratories agency (ahvla) has set up a new independent advisory group to evaluate veterinary surveillance in england and wales, although their original intent was in part to consider funding reductions (trickett, 2012) . modelling risk factors for zoonotic influenza infections is challenging because the infections are often rare; the laboratory assays are often difficult and imprecise, and the most definitive studies require intensive resources. this was the view of professor g. c. gray (emerging pathogens institute and college of public health and health professions, university of florida, gainesville, florida, usa) in his presentation, modeling risk factors for zoonotic influenza infections in man: challenges and strategies for success. in particular, serologic detections of these infections in humans may be confounded by crossreacting antibody, waning antibody from the infection of interest, inaccurate matching of the enzootic pathogen and the laboratory strain, laboratory errors and weakly powered statistical comparisons. the underlying question which professor gray and his research team is tackling is: which human, animal and environmental factors predict disease? these three factors can be viewed as a venn diagram with its intricate interactions. like understanding cardiovascular disease, how a person acquires a zoonotic influenza infection is a complex process, and predictive laboratory assays are imprecise. for example, with avian influenza viruses (especially h5n1, popularly known as 'bird flu'), poultry veterinarians, turkey workers, hunters and people without indoor plumbing may be at increased risk of aiv infection but infections are rare. subclinical or mild infections do occur; and occasionally aiv causes severe disease in persons exposed to sick birds. although aiv transmission from human-to-human seems rare, further cohort studies and more sensitive serological assays are needed. a basic scientist often tests hypotheses by: (i) carefully setting up an experimental setting; (ii) isolating confounding factors; and (iii) looking for statistically significant associations with an outcome. such a process is not possible for a number of emerging disease problems such as human infections with swine influenza virus (siv). experimental studies are not possible. hence, epidemiologists must perform observational studies of people most likely to be infected with siv and by looking at possible risk factor associations, infer causality. one must first determine settings where the prevalence of siv in expected to be high and then study those workers. for example, sivs are often endemic in large-scale modern production facilities. risk factors for sow-herd siv seropositivity involve pig density, whether there is an external source of breeding pigs, the total animals on the site and the closeness of barns. similarly, risks factors for finisherherd siv positivity must be considered -the number of siv-positive sows, size of herd, pig farm density and farrow-to-finish type of farm (poljak et al., 2008) . however, siv surveillance in pigs is largely passive and voluntary, so recognizing which pig workers to study is a challenge. detection of siv infections in man often requires a sentinel event (e.g. human illness with pig exposure or sick pigs). as pigs do not always have clinical signs of novel virus infection and often there is no compensation system to protect pig farmers, the pork industry is reluctant to permit the study of their workers for siv infection (gray and baker, 2011) . therefore, these observational studies are currently very difficult. professor gray concluded by pointing out that although there are numerous challenges in conducting epidemiological studies for zoonotic influenza, there are six substantive ways to control confounding variables: (i) design every study carefully; (ii) use non-animal-exposed controls; (iii) employ validated laboratory assay using zoonotic influenza strains; (iv) use multivariate modeling to examine cross-reacting serologic responses due to human viruses and vaccines; (v) consider proportional odds modeling; and (vi) consider employing a second unique serologic test (see gpl, 2012) . with the support of 26 co-authors from 21 different institutions, dr. k. j. linthicum (united states department of agriculture, agricultural research service, center for medical, agricultural & veterinary entomology, gainesville, fl, usa) presented two case studies about forecasting emerging vector-borne diseases. dr. linthicum began by pointing out that global climate variability, often linked to el niño conditions, can be used to forecast emerging vector-borne disease spread in local areas (linthicum et al., 1999) . these forecasts are possible because specific pathogens, their vectors and hosts are sensitive to temperature, moisture and other ambient environmental conditions. with consistent and reliable satellite observations, global sea temperatures, climate and vegetation can be observed. first, temperature plays a major role in its impact on aides aegypti mosquitoes transmitting dengue haemorrhagic fever virus in southeast asia (linthicum et al., 2008) and possibly also on how ae. aegypti transmits chikungunya virus in africa and asia , as well as on how anopheles species mosquitoes transmit p. vivax malaria in the republic of korea. vectorial competence is dependent upon the extrinsic incubation (ei) period in the mosquito vector. the ei represents the time from ingestion of the virus while feeding on a viremic host to the virus arriving in the salivary glands. the shorter the ei period, which occurs during higher ambient temperatures, the greater the vectorial competence (garrettjones and shidrawi, 1969) . if data are available for a specific local area on the daily humanbiting rate (ha) of the mosquitoes, the daily rate of blood feeding (a) and the length of the ei cycle (n), it is possible to calculate vectorial capacity (rattanarithikul et al., 1996) . second, accurate measurements and understanding of how exceptionally heavy rainfall and flooding affects aides and culex mosquitoes and the introduction of virusinfected mosquitoes into susceptible vertebrate hosts enables forecasts to be made about when and where rift valley fever (rvf) will develop in sub-saharan africa and middle east (anyamba et al., 2009) . outbreaks of rift valley fever are known to follow periods of widespread and heavy rainfall associated with the development of a strong inter-tropical convergence zone over eastern africa (davies et al., 1985) . during periods of elevated transmission, there is a significantly increased risk of globalization of these and other arboviruses; however, the forecasting methods described provide 2.5-5 months early warning before an outbreak and provide ample time for disease mitigation before the first cases appear (anyamba et al., 2010) . furthermore, the emergence and expansion of a number of disease vectors (e.g. mosquitoes, mice, locust) often follow the trajectory of the green flush of vegetation in semiarid lands. the ability to predict periods of elevated risk enables better prevention, containment or exclusion strategies to be drawn up to limit globalization of emerging pathogens. thus, it has been possible for the food & agricultural organization (fao) to create a system of alerts -the emergency prevention system for transboundary animal and plant pests and diseases (empress, 2012) . subsequent to dr lithicum's presentation, significant further work has been done to provide a genome-scale overview of gene expression in the malaria-transmitting mosquito anopheles gambiae (maccallum et al., 2011) , as well as to expand the vectorbase website with regularly updated genome information on two other mosquito species, aedes aegypti and culex quinquefasciatus, and numerous other organisms, including the tick species ixodes scapularis (lawson et al., 2009; niaid, 2012) . the ultimate aim of this research is to create a database that will facilitate a systems-level view of gene expression for many different organisms. reflecting on the numerous types of statistical analysis that are used to estimate confidence intervals for proportions in scientific studies, dr. s. guillossou and his colleagues professors h. m. scott and j. a. richt (dept. of diagnostic medicine and pathobiology, college of veterinary medicine, kansas state university, manhattan, kansas, usa) utilized the final presentation of the conference, estimates of low prevalences and diagnostic test estimates: what confidence do we really have? to illustrate the differences, limits and sometimes chaotic behaviour of different statistical approaches. dr. guillossou pointed out that there were more than 15 different methods for determining a 95% confidence interval of a proportion. he stressed that it is always important to report the method of statistical analysis being utilized. in his view, the agresti-coull interval approach presents a satisfactory compromise between computational requirements and coverage probability (newcombe, 1998; brown et al., 2001) . ideally, the effects of coverage probability should be estimated and the most appropriate method chosen before reporting the findings or using proportions as inputs in any epidemiological study. what did this 6th international conference on emerging zoonoses achieve? there was the opportunity to meet old friends and make new friends, to share one's academic work and to reflect on what lies ahead with emerging zoonoses. it is now clear that human medicine, veterinary medicine and environmental challenges are a unity which must be considered under the umbrella of 'one health' (one health initiative, 2012) . viruses are continuing to jump from animals to people with unexpected consequences, because the evolution of any virus is impossible to predict. even the recent relatively mild swine flu virus infected 10% of the human population and killed some 100,000 people globally -far less than would have been the case if the virus had mutated to a more deadly form, as might easily have happened. the reality is, as professor nathan wolfe, professor in human biology at stanford university, has commented: 'as a species, we're not that focused on the things that have the most potential to be devastating to us as a global population, such as viruses. unless people take these things seriously, we're going to look back and say we had all the tools necessary to try to address these risks, and we basically ignored them because they weren't dramatic like a car accident or a hurricane' (geddes, 2011; kahn, 2011; wolfe, 2011) . this conference, many others and the 7th international conference on emerging zoonoses to be held in 2014 in berlin, are aimed at creating, improving and using the tools essential to address the risks of viral contagions in a global society. none. the organizing committee of the conference wishes to acknowledge the excellent services of the conference organizers, target conferences of tel aviv, israel, and the welcome financial contributions of medimmune, boehringer ingelheim vetmedica gmbh, prionics ag, center of excellence for emerging and zoonotic animal diseases (ceezad) and national center for foreign animal and zoonotic disease defense (fazd), as well as the poster prize donated by wiley-blackwell. we are also grateful to the wiley-blackwell staff who have contributed so significantly to this special supplement, especially rachel robinson and peter tubman, as well as to dr. klaus osterrieder for his helpful comments and to the presenters who have approved or improved every summary in this meeting review. this material is based upon work supported by the u.s. department of homeland security under grant award number 2010-st-ag0001. the views and conclusions contained in this supplement are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the u.s. department of homeland security. additional funding has been provided by the kansas bioscience authority. conservation medicine: ecological health in practice prediction of a rift valley fever outbreak 2010: prediction, assessment of the rift valley fever activity in east and southern africa 2006-2008 and possible vector control strategies climate teleconnections and recent patterns of human and animal disease outbreaks risk of infection with brucella abortus and escherichia coli o157:h7 associated with marketing of unpasteurized milk in kenya fallout from listeria outbreak hits walmart: retail detection and identification of mycoplasma from bovine mastitis infections using a nested polymerase chain reaction bse infectivity in the absence of detectable prp(sc) accumulation in the tongue and nasal mucosa of terminally diseased cattle discovery of swine as a host for the reston ebolavirus 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transmission to macaques vectorbase: a data resource for invetebrate vector genomics inference between agents of lyme disease and human granulocytic ehrlichiosis in a natural reservoir host structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections climate and satellite indicators to forecast rift valley fever epidemics in kenya vector-borne diseases -understanding the environmental, human health, and ecological considerations, workshop summary pathogen discovery altered receptor specificity and cell tropism of d222g hemagglutinin mutants isolated from fatal cases of pandemic a (h1n1) 2009 influenza virus the pig as a mixing vessel for influenza viruses: human and veterinary implications pandemic h1n1 virus causes disease causes upregulation of genes related to inflammatory and immune response, cell death, and lipid metabolism in pigs identification and characterization of a highly virulent triple 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virus modification of non-structural protein 1 of influenza a virus by sum01 key: cord-349168-ec5p9b2f authors: domingues, célia p. f.; rebelo, joão s.; dionisio, francisco; botelho, ana; nogueira, teresa title: the social distancing imposed to contain covid-19 can affect our microbiome: a double-edged sword in human health date: 2020-09-16 journal: msphere doi: 10.1128/msphere.00716-20 sha: doc_id: 349168 cord_uid: ec5p9b2f hygienic measures imposed to control the spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and contain covid-19 have proven effective in controlling the pandemic. in this article, we argue that these measures could impact the human microbiome in two different and disparate ways, acting as a double-edged sword in human health. new lines of research have shown that the diversity of human intestinal and oropharyngeal microbiomes can shape pulmonary viral infection progression. here, we suggest that the disruption in microbial sharing, as it is associated with dysbiosis (loss of bacterial diversity associated with an imbalance of the microbiota with deleterious consequences for the host), may worsen the prognosis of covid-19 disease. in addition, social detachment can also decrease the rate of transmission of antibiotic-resistant bacteria. therefore, it seems crucial to perform new studies combining the pandemic control of covid-19 with the diversity of the human microbiome. a mong the main recommendations to fight the covid-19 pandemic are to avoid interpersonal contacts, to disinfect hands upon touching physical surfaces in anthropogenic settings, and to follow strict respiratory etiquette rules to prevent the spread of viral particles and aerosols contaminated with severe acute respiratory syndrome coronavirus 2 (sars-cov-2). quarantine and confinement are also indicated for the prevention of disease transmission in the case of individuals with suspected or confirmed infection. social interconnections between people, however, also lead to the sharing of those microorganisms that have been coevolving with humans and which are very important for human health maintenance, contributing to the control of many diseases and syndromes (1) . the human microbiota engages in symbiotic or mutualistic relationships within the human body. the collection of its genomes-the microbiome-is estimated to account up to 99% of the unique genes in the human body (2) and provides genetic information to perform many complementary functions that are lacking in the human genome, such as helping to break down nutrients and molecules or to stimulate the immune system (3) . changes in the balance of the gut microbiome (dysbiosis) are associated with a greater susceptibility to diseases and opportunistic infections, due to the decrease in the protective microbial load of symbiotic bacteria, which can lead to dysregulation of the immune system and to autoimmune diseases (4) . such changes were also found to be correlated with covid-19 prognosis (5, 6). yet there are no data on the effect of human behavior during the covid-19 pandemic on the gut microbiota. the sharing of microorganisms between humans helps to build up the human microbiome ( fig. 1) . microorganisms belonging to the oral, intestinal, and nasopharyngeal microbiomes can be transferred from person to person in a physical social network. people living in the same home, i.e., sharing a household, are more likely to harbor similar profiles of bacterial lineage diversity in their microbiomes, regardless of their genetic relationships (7) . cohabitation has thus been reported as one of the main factors facilitating the asymptomatic transmission typical of sars-cov-2 (8), accounting for 45% of 793 new cases in portugal in the period from 13 to 21 march 2020, according to the governmental health national service (9) . other forms of social contact were also relevant, namely, in companies (19%) and in nursing homes (11%). it seems reasonable to consider that in the context of group or family confinement, each individual may represent, in epidemiological terms, the entire group, as the individuals may share with each other many microorganisms of the community. although some recent studies suggest that life in the womb is not completely germfree (10) (11) (12) , it has been argued that microorganisms do not colonize the baby (13, 14) and that the human microbiome begins to be enriched following birth and during the first 3 years of life. vaginal birth and breastfeeding provide early contact with maternal microorganisms and help in the establishment of both gut and airway microbiomes, despite not being the main source of microbial diversity in adulthood (15) (16) (17) . skin-to-skin or mucous contacts happening while kissing and hugging are thus important sources of inoculation with human microorganisms from early life (18) . also, contact with surfaces contaminated by humans can provide another important indirect source of colonizing microorganisms from one human microbiome to another. babies and toddlers use their tongues to explore the household environment and, as a result, can ingest a wide variety of new microorganisms, some of which will potentially enrich their microbiota (19) . the systematic disinfection of surfaces and hands can disrupt this indirect source of human microbial inoculation. interrupting the transmission of sars-cov-2 between individuals in a social network through confinement and adherence to rules of hygiene and social distancing has been important to contain covid-19 spread, yet it also decreases the likelihood of sharing other microorganisms of the human microbiota. this decreases the repertoire of functional genes in our "other genome," the gut microbiome (20) , and could entail a loss of functions, exposing humans to disease (fig. 1) . many factors have been driving to a loss of bacterial diversity from one generation to the next in industrialized countries. hygienic measures, vaccination, antibiotic use (21) , and cesarean sections, among other factors, are contributing to a loss of our ancestral microbial heritage (15) . here, we postulate that the lack of contact between humans resulting from the social distance measures recommended for covid-19 might also aggravate this situation and may increase the susceptibility of humans to disease. already in 1969, johanson and colleagues observed differences in the oropharyngeal bacterial microbiota in individuals with severe pneumonia, but those changes were not correlated with antibiotic administration or inhalation therapy, or with the duration of hospitalization (22) . if, on one hand, critical illnesses and intensive care induce changes in the human microbiome, on the other, the changes in the lung and intestine microbiome also modulate critical diseases, as demonstrated in animal models and clinical trials (23) . during lung infection, the induced shift in the microbiomes leads to a positive-feedback loop of inflammation and dysbiosis (23) . the healthy microbiome is closely related to the functioning of the immune system, and changes in the health status of the human host can have drastic effects on the microbiome, and vice versa (24, 25) . dysbiosis in the gut has been associated with many diseases, such as immune diseases like crohn's disease, ulcerative colitis, type 1 diabetes, celiac disease, allergy, and multiple sclerosis, metabolic diseases like obesity or type 2 diabetes and colorectal cancer, and autism (26) . this is of particular relevance in elderly people that have a less diverse gut microbiota (5, 27) . it has been suggested that there is possible cross talk between the lung and the gut microbiota that could influence the outcome of covid-19 (5) . for example, dysbiosis of the gut microbiome can also increase the susceptibility to influenza virus infection in the lungs (5, 28) . we are led to question whether the recommended social distancing measures to prevent sars-cov-2 transmission could increase the number of other serious instabilities. the breaking of the contagion pathways reduces the sharing of microorganisms between people, thus favoring dysbiosis, which, in turn, may increase the poor prognosis of the disease. it has been demonstrated that there is a positive correlation between the diversity of antibiotic resistance genes and the diversity of bacterial virulence genes in human metagenomes (29) . recently, using computer simulations, we have shown evidence that in a social network, bacterial transmission from one person to another is the major factor that explains this positive correlation between the diversity of antibiotic resistance genes and the diversity of virulence genes. therefore, simply because people contaminate themselves in these social networks, we end up with the paradoxical and unwanted situation in which humans with a higher diversity of virulence genes in their metagenomes are precisely those expected to have a high diversity of resistance genes (30) . however, in some cases, antibiotic resistance entails a metabolic burden; hence, after antibiotic treatment ends, there is a decline of resistance by gene loss and competition with sensitive strains (31) . this effect was highlighted in a metagenomic study in which it was observed that that the diversity of genes encoding antibiotic resistance in human intestinal microbiomes increases with age until reaching a limited level (32) . in this context, we trust that there could be a potential beneficial effect of social confinement in decreasing the spread of antibiotic-resistant microorganisms during antimicrobial therapy. this hypothesis needs to be tested experimentally and, if confirmed, could support new recommendations for the use of antibiotics. in this opinion article, we hypothesize that the social distancing imposed for covid-19 prevention might have an impact on the diversity of the human gut microbiome and therefore on human health. we also argue that these measures could have two opposite consequences for covid-19: (i) the loss of biodiversity, if not effectively restored, could be perennial and persist from one generation to the next, potentially driving to disease and a poorer prognostic of covid-19, in a perverse and negative effect; (ii) social isolation and imposed hygiene rules can also lead to a decrease in the transmission of microorganisms and their genes from one individual to another, which might result in the dissociation of the correlation between the diversity of bacterial antibiotic resistance genes and virulence genes (29, 30) . this can be a shorter-term positive effect that may not persist over time. social distancing implemented after the sars-cov-2 pandemic outbreak is therefore a double-edged sword. it might have both negative and positive impacts on the genomic dynamics of the human microbiome, and it is worth studying the implications for public health. the development of these lines of research could help to provide national health systems with a comprehensive analysis of the confinement and social detachment effect on individual and community health. it could also support decision-making and measures to combat the pandemic, namely, in the definition of security protocols to control covid-19 without compromising human health. nevertheless, it is important to balance pandemic control with both the perverse effect of decreasing microbial diversity and the beneficial effect of decreasing the spread of antibiotic-resistant pathogenic bacteria (fig. 1) . targeting microbiota: what do we know about it at present? structure, function and diversity of the healthy human microbiome antibiotics, microbiota, and immune defense effects of antibiotics on human microbiota and subsequent disease gut microbiota and covid-19 -possible link and implications gut microbiota may underlie the predisposition of healthy individuals to covid-19 transmission of humanassociated microbiota along family and social networks clinical characteristics of 24 asymptomatic infections with covid-19 45% das novas infeções contraídas dentro de casa-covid-19 isolation of commensal bacteria from umbilical cord blood of healthy neonates born by cesarean section microbial prevalence, diversity and abundance in amniotic fluid during preterm labor: a molecular and culture-based investigation the placenta harbors a unique microbiome comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota a critical assessment of the "sterile womb" and "in utero colonization" hypotheses: implications for research on the pioneer infant microbiome preserving microbial diversity early-life formation of the microbial and immunological environment of the human airways ecological succession in the vaginal microbiota during pregnancy and birth strain-level analysis of mother-to-child bacterial transmission during the first few months of life can we save our body's ecosystem from extinction? a human gut microbial gene catalogue established by metagenomic sequencing antibiotics and the human gut microbiome: dysbioses and accumulation of resistances changing pharyngeal bacterial flora of hospitalized patients: emergence of gram-negative bacilli the microbiome and critical illness reciprocity in microbiome and immune system interactions and its implications in disease and health potential role of gut microbiota in induction and regulation of innate immune memory gut microbiota diversity and human diseases: should we reintroduce key predators in our ecosystem? gut microbiome and aging: physiological and mechanistic insights collateral effects of antibiotics on mammalian gut microbiomes antibiotic resistance gene diversity and virulence gene diversity are correlated in human gut and environmental microbiomes person-to-person contagion and antibiotic usage are the key factors responsible for the positive correlation between antibiotic resistance gene diversity and virulence gene diversity in human metagenomes seasonality and temporal correlation between community antibiotic use and resistance in the united states antibiotics as both friends and foes of the human gut microbiome: the microbial community approach t.n. was supported by contract alg-01-0145-feder-028824. fundação para a ciência e a tecnologia (fct), i.p., supports ce3c through contract uidp/00329/2020. we thank margarida duarte and eva pinho for critical reading of the manuscript. we declare that no competing interests exist. key: cord-332379-340wczmq authors: pennington, matthew r.; saha, amrita; painter, david f.; gavazzi, christina; ismail, ashrafali m.; zhou, xiaohong; chodosh, james; rajaiya, jaya title: disparate entry of adenoviruses dictates differential innate immune responses on the ocular surface date: 2019-09-13 journal: microorganisms doi: 10.3390/microorganisms7090351 sha: doc_id: 332379 cord_uid: 340wczmq human adenovirus infection of the ocular surface is associated with severe keratoconjunctivitis and the formation of subepithelial corneal infiltrates, which may persist and impair vision for months to years following infection. long term pathology persists well beyond the resolution of viral replication, indicating that the prolonged immune response is not virus-mediated. however, it is not clear how these responses are sustained or even initiated following infection. this review discusses recent work from our laboratory and others which demonstrates different entry pathways specific to both adenovirus and cell type. these findings suggest that adenoviruses may stimulate specific pattern recognition receptors in an entry/trafficking-dependent manner, leading to distinct immune responses dependent on the virus/cell type combination. additional work is needed to understand the specific connections between adenoviral entry and the stimulation of innate immune responses by the various cell types present on the ocular surface. conjunctivitis, a common ocular condition with a range of etiologies, is highly prevalent, affecting approximately 6 million people annually in the united states and accounting for 1% of all primary care office visits [1] [2] [3] [4] . viruses are responsible for up to 80% of conjunctivitis, and human adenoviruses (hadvs) are implicated in up to 65% of all viral cases [5, 6] . adenoviruses are small, non-enveloped viruses with a linear, double-stranded dna genome of approximately 36 kilobase pairs. the seven species (a-g) and more than one hundred genotypes currently in genbank exhibit a broad range of tropisms across the various mucosal surfaces of the body, including those within the respiratory, gastrointestinal, and genitourinary tracts, in addition to cells on the ocular surface. while adenovirus infections are generally acute and self-limiting in immunocompetent patients, they can be fatal in children and immunocompromised individuals [7] [8] [9] . the most severe adenovirus infections of the ocular surface are associated with hadvs of species d (hadv-d) [10] , the species with the largest number of described viruses. the term "ocular surface" broadly refers to the cornea and the conjunctiva (figure 1 ). the principal function of the cornea is to refract incoming light to the lens, where the light is then focused onto the retina for visual discrimination. the cornea is composed of five distinct layers (from anterior to posterior): the epithelium, bowman's layer, stroma, descemet's membrane, and endothelium [11] . the stroma, sometimes called the corneal perhaps due to the relatively self-limiting nature of most conjunctivitis presentations, in which symptoms usually resolve within two to three weeks of onset, very few studies have investigated conjunctival immune responses to virus infection [17] . clinically, however, ocular hadv infection generally presents as one of three highly contagious syndromes: follicular conjunctivitis, pharyngoconjunctival fever (pcf), or epidemic keratoconjunctivitis (ekc) [18] . follicular conjunctivitis is characterized by bulbar conjunctival injection and chemosis, follicular hyperplasia, preauricular adenopathy, and sometimes conjunctival petechiae or frank subconjunctival hemorrhages. pcf appears similar, however, in addition to the ocular signs, is associated with a systemic, flu-like illness [19, 20] . ekc, which is most commonly caused by hadv-d8, -37, -53, -54, -56, and -64, is a severe, hyperacute, and particularly contagious infection [21, 22] . ekc is characterized by acute membranous keratoconjunctivitis and delayed-onset subepithelial corneal infiltrates (seis) (figure 2 ). perhaps due to the relatively self-limiting nature of most conjunctivitis presentations, in which symptoms usually resolve within two to three weeks of onset, very few studies have investigated conjunctival immune responses to virus infection [17] . clinically, however, ocular hadv infection generally presents as one of three highly contagious syndromes: follicular conjunctivitis, pharyngoconjunctival fever (pcf), or epidemic keratoconjunctivitis (ekc) [18] . follicular conjunctivitis is characterized by bulbar conjunctival injection and chemosis, follicular hyperplasia, preauricular adenopathy, and sometimes conjunctival petechiae or frank subconjunctival hemorrhages. pcf appears similar, however, in addition to the ocular signs, is associated with a systemic, flu-like illness [19, 20] . ekc, which is most commonly caused by hadv-d8, -37, -53, -54, -56, and -64, is a severe, hyperacute, and particularly contagious infection [21, 22] . ekc is characterized by acute membranous keratoconjunctivitis and delayed-onset subepithelial corneal infiltrates (seis) (figure 2) . seis, the hallmark feature of ekc, occur in approximately one-third of all ekc cases and may persist or recur for months to years following infection [23] [24] [25] [26] . seis impair vision by physically blocking the passage of light and by disrupting the arrangement of collagen fibrils and other extracellular matrix components of the meticulously organized and normally transparent corneal stroma [27] [28] [29] [30] [31] [32] . clinically, this manifests as reduced vision, foreign body sensation, and photophobia [33] [34] [35] . based on both experimental and clinical evidence, seis form as a consequence of infiltrating leukocytes, recruited from the corneal limbus to the superficial corneal stroma [31, 32] . sei appearance is delayed by up to three weeks from the onset of infection, a time when active viral replication has ceased [36] , which suggests that long term morbidity associated with infection is immune-mediated rather than a result of virus-associated tissue damage. unfortunately, despite frequent outbreaks of adenoviral conjunctivitis microorganisms 2019, 7, 351 3 of 24 and the substantial economic impacts-due to lost work time and expenses associated with medical visits and diagnostic testing-there are currently no specific antiviral therapies for adenovirus ocular infections. further, due to their apparent immunological origin, seis are unresponsive to direct-acting antivirals. the elucidation of the immunopathogenesis of infection and its relationship with the virus replication cycle is crucial to the potential development of future immunomodulatory therapies. seis, the hallmark feature of ekc, occur in approximately one-third of all ekc cases and may persist or recur for months to years following infection [23] [24] [25] [26] . seis impair vision by physically blocking the passage of light and by disrupting the arrangement of collagen fibrils and other extracellular matrix components of the meticulously organized and normally transparent corneal stroma [27] [28] [29] [30] [31] [32] . clinically, this manifests as reduced vision, foreign body sensation, and photophobia [33] [34] [35] . based on both experimental and clinical evidence, seis form as a consequence of infiltrating leukocytes, recruited from the corneal limbus to the superficial corneal stroma [31, 32] . sei appearance is delayed by up to three weeks from the onset of infection, a time when active viral replication has ceased [36] , which suggests that long term morbidity associated with infection is immune-mediated rather than a result of virus-associated tissue damage. unfortunately, despite frequent outbreaks of adenoviral conjunctivitis and the substantial economic impacts -due to lost work time and expenses associated with medical visits and diagnostic testing -there are currently no specific antiviral therapies for adenovirus ocular infections. further, due to their apparent immunological origin, seis are unresponsive to direct-acting antivirals. the elucidation of the immunopathogenesis of infection and its relationship with the virus replication cycle is crucial to the potential development of future immunomodulatory therapies. dogma maintains that hadvs enter host cells via dynamin-dependent, clathrin-mediated endocytosis before trafficking along microtubules to the nucleus for replication [37, 38] . however, recent work has demonstrated that adenoviruses utilize several entry mechanisms, including macropinocytosis [39] [40] [41] and caveolin-mediated pathways [42] . the specific mechanism of entry appears to depend most on the specific pairing of cell and virus type. in some cell types, viruses may exploit more than one pathway with no apparent preference. furthermore, a predominant pathway may be supplanted by another pathway if the former is blocked. redundancy in both entry and subsequent immune responses may be the rule rather than the exception. furthermore, analyses of viral entry may be complicated by the finding that some host signaling proteins that were initially identified as specific to a particular pathway are in fact shared by disparate pathways. innate immune responses to adenoviruses rely on the detection of pathogen-associated molecular patterns (pamps): distinct ligands present on the external surfaces, and nucleic acids of pathogens (but absent in the host) that feature molecular signatures able to be recognized by pattern recognition receptors (prr) on or in infected host cells [43] [44] [45] [46] . due to the specific distribution of these prrs on the cell surface, in endosomes, and in the cytosol, it is expected that adenoviruses utilizing disparate entry and trafficking mechanisms may stimulate specific and unique subsets of prrs, ultimately resulting in unique immune response signatures. consistent with this hypothesis, it was shown that rapidly trafficking adenoviruses replicate more efficiently [47] , but may not stimulate host cytokine responses as effectively as a virus that enters and traffics more slowly [22] . dogma maintains that hadvs enter host cells via dynamin-dependent, clathrin-mediated endocytosis before trafficking along microtubules to the nucleus for replication [37, 38] . however, recent work has demonstrated that adenoviruses utilize several entry mechanisms, including macropinocytosis [39] [40] [41] and caveolin-mediated pathways [42] . the specific mechanism of entry appears to depend most on the specific pairing of cell and virus type. in some cell types, viruses may exploit more than one pathway with no apparent preference. furthermore, a predominant pathway may be supplanted by another pathway if the former is blocked. redundancy in both entry and subsequent immune responses may be the rule rather than the exception. furthermore, analyses of viral entry may be complicated by the finding that some host signaling proteins that were initially identified as specific to a particular pathway are in fact shared by disparate pathways. innate immune responses to adenoviruses rely on the detection of pathogen-associated molecular patterns (pamps): distinct ligands present on the external surfaces, and nucleic acids of pathogens (but absent in the host) that feature molecular signatures able to be recognized by pattern recognition receptors (prr) on or in infected host cells [43] [44] [45] [46] . due to the specific distribution of these prrs on the cell surface, in endosomes, and in the cytosol, it is expected that adenoviruses utilizing disparate entry and trafficking mechanisms may stimulate specific and unique subsets of prrs, ultimately resulting in unique immune response signatures. consistent with this hypothesis, it was shown that rapidly trafficking adenoviruses replicate more efficiently [47] , but may not stimulate host cytokine responses as effectively as a virus that enters and traffics more slowly [22] . however, such a relationship has yet to be fully defined in the context of ocular surface cells. this review will focus broadly on the mechanisms of adenoviral entry and trafficking, the immune responses to adenovirus infection of the ocular surface, and the possible connection between the two. the hadv genome is encapsidated by an icosahedral protein shell (capsid) composed of three major capsid proteins (hexon, penton base, and fiber) and four minor/cement proteins (iiia, vi, viii, and ix). the hexon protein is the most abundant adenovirus protein, with 240 hexon trimers (720 individual hexon proteins) forming the bulk of the capsid structure. each of the 12 capsid vertices is formed by a ring of five penton base proteins, from which a trimeric fiber protein protrudes, ending in its distal fiber knob (reviewed in [48] ). the infection cycle begins with the binding of the fiber knob to a cell surface receptor. most hadvs, except hadv-b and some members of hadv-d, utilize the coxsackievirus-adenovirus receptor (car), which is expressed in most human tissues [49] [50] [51] . typically, hadv-bs bind cd46, a complement regulatory protein, as the primary receptor [52] . the ekc-associated viruses also bind cd46 and additionally bind ganglioside sialic acids, notably gd1a [50, [53] [54] [55] [56] [57] [58] . receptor binding draws the capsid toward the cell membrane, enabling an interaction between an arginine-glycine-aspartic acid (rgd) motif in the outer loop of each penton base protein and cell surface integrins, which serve as secondary receptors. specifically, for hadv-d37, it has been shown that αvβ1 and αvβ3 are utilized for the infection of corneal epithelial cells [59, 60] . the binding of the rgd motif induces conformational changes in the integrins, which mediate downstream intracellular signaling to promote viral entry into the host cell. it is generally thought that specific fiber knob amino acids and the availability of the necessary cellular receptors and integrins determine the tropism of hadvs for the ocular surface [59, 61] . indeed, we recently illustrated positive selection pressure on one particular amino acid in the fiber knob, specifically a lysine or alanine at residue 240, which is critical for corneal tropism and differentiates ekc adenoviruses from non-ekc viruses [62] . for non-ocular, car-utilizing viruses, expression levels of car control entry and nuclear trafficking efficiency (reviewed in [63] ). however, it is important to note that entry into a specific cell type does not guarantee successful trafficking or viral replication. few studies to date have specifically sought to characterize the entry mechanisms for ekc-associated hadvs in ocular cells. most have focused on the non-ekc-associated, species c, specifically hadv-c2 and -5, as well as species b, hadv-b3, -7,-9, and -35 [63] . in addition, immortalized human cell lines, including hela cells, kb cells (subcloned from hela cells), and a549 cells, have been the preferred cell types for hadv-c2 and -5 entry studies due to their support for rapid and robust viral replication observed in these cells [63] . these studies are frequently cited as support for clathrin-mediated endocytosis as the sole or primary means by which adenoviruses enter cells [10] . further work with hadv-cs has led to the idea that subsequent trafficking to the nucleus occurs along the microtubule network in a dynamin-dependent manner [64] [65] [66] [67] . however, recent experimental evidence supports viral utilization of several entry mechanisms, including clathrin-mediated endocytosis [64] , macropinocytosis [39] , and caveolin-mediated pathways [42] . clathrin-mediated endocytosis is the best elucidated route for viral entry, including for adenovirus [64, [68] [69] [70] [71] . this entry process involves the internalization of virions into a double membrane coated pit with triskelion-shaped clathrin proteins, which collectively interact to form a polyhedral lattice surrounding the endocytosed vesicle [64, 72, 73] . for cornea-tropic adenoviruses, there is a general paucity of data on the entry mechanism. however, in agreement with previous studies, we found that hadv-d37 infection of immortalized human corneal epithelial cells was predominantly clathrin-mediated (figure 3a (i)), with a lesser contribution from macropinocytosis (figure 3a (ii)). factors beyond just the clathrin-coated pit are required for clathrin-mediated endocytosis [39] . for example, dynamin, a 100-kda gtpase, plays a critical role in the fission of newly formed vesicles, without which clathrin-mediated endocytosis does not occur [74] . of the three dynamin isoforms, dynamin 2 is expressed in most cell types. it also functions as a microtubule binding protein [75] and as a negative regulator of microtubule stability [76] . the binding of dynamin 2 to microtubules activates its gtpase function, resulting in endosome migration along the cytoskeletal network [77] . clathrin-mediated endocytosis of hadv-c2 and -5 has been shown to require dynamin activity [37, 64, 78] . however, dynamin is not required for the entry of all adenoviruses. for example, hadv-b3 utilizes dynamin-independent endocytosis for rapid entry into epithelial and hematopoietic cells [79] . adenovirus infection of human corneal epithelial cells is highly restricted [42, 80] , and in vitro tropism of specific hadv types for immortalized human corneal epithelial cells very closely matches the known associations between specific hadv types and clinical infection in ekc [22, 81, 82] . corneal epithelial cell infection by tropic hadvs also occurs via a dynamin 2-independent pathway (manuscript in preparation). further, in fibroblasts derived from human corneal stromal keratocytes-also known as human corneal fibroblasts (hcfs)-dynamin 2 activity had no impact on the cellular entry of hadv-d37 but did influence the delivery of viral dna to cell nuclei. specifically, the knockdown of dynamin 2 resulted in increased microtubule acetylation, closer proximity of microtubule organizing centers (mtocs) to cell nuclei, and increased perinuclear hadv-d37 localization [38] . consistent with others' findings that adenovirus accumulation around the mtoc is a prerequisite to nuclear entry [83] , dyamin 2 knockdown in corneal fibroblasts resulted in greater delivery of adenoviral dna into cell nuclei. in parallel experiments, overexpression of dynamin 2 resulted in the opposite effects, including reduced nuclear entry of viral dna [38] . these data confirm a dichotomous role for dynamin in adenoviral entry, dependent on both cell and virus type. dynamin 2 is expressed in most cell types. it also functions as a microtubule binding protein [75] and as a negative regulator of microtubule stability [76] . the binding of dynamin 2 to microtubules activates its gtpase function, resulting in endosome migration along the cytoskeletal network [77] . clathrin-mediated endocytosis of hadv-c2 and -5 has been shown to require dynamin activity [37, 64, 78] . however, dynamin is not required for the entry of all adenoviruses. for example, hadv-b3 utilizes dynamin-independent endocytosis for rapid entry into epithelial and hematopoietic cells [79] . adenovirus infection of human corneal epithelial cells is highly restricted [42, 80] , and in vitro tropism of specific hadv types for immortalized human corneal epithelial cells very closely matches the known associations between specific hadv types and clinical infection in ekc [22, 81, 82] . corneal epithelial cell infection by tropic hadvs also occurs via a dynamin 2-independent pathway (manuscript in preparation). further, in fibroblasts derived from human corneal stromal keratocytes -also known as human corneal fibroblasts (hcfs) -dynamin 2 activity had no impact on the cellular entry of hadv-d37 but did influence the delivery of viral dna to cell nuclei. specifically, the knockdown of dynamin 2 resulted in increased microtubule acetylation, closer proximity of microtubule organizing centers (mtocs) to cell nuclei, and increased perinuclear hadv-d37 localization [38] . consistent with others' findings that adenovirus accumulation around the mtoc is a prerequisite to nuclear entry [83] , dyamin 2 knockdown in corneal fibroblasts resulted in greater delivery of adenoviral dna into cell nuclei. in parallel experiments, overexpression of dynamin 2 resulted in the opposite effects, including reduced nuclear entry of viral dna [38] . these data confirm a dichotomous role for dynamin in adenoviral entry, dependent on both cell and virus type. corneal keratocytes or tert-immortalized human corneal epithelial cells were infected with moi = 10 of cesium chloride purified hadv-d37 for 1 hour at room temperature. cells were washed with pbs, fixed in a fixative solution (2% paraformaldehyde containing 2.5% glutaraldehyde, 0.1 m cacodylate, and 2.5 mm cacl2) for 1 hour, and collected in 2% agarose. the cell pellet was further fixed for 1.5 hours in 2% aqueous oso4 and dehydrated. after dehydration, the cell pellet was embedded in epon and sectioned into 70-90 nm thin sections. the sections were stained with saturated aqueous uranyl acetate, sato's lead stain, and micrographs were taken on a philips cm-10 electron microscope operating at 80 kv and fitted to a ccd camera. (a) electron micrographs of infected tert-immortalized human corneal epithelial cells show that hadv-d37 (white arrows) can enter via clathrin-mediated endocytosis (i) or macropinocytosis (ii). (b) electron micrographs of infected keratocytes showing caveolae (black arrows) associated with hadv-d37 (white arrows) at the cell membrane (i) and inside a caveosome (ii). (c) immunogold staining for caveolin in uninfected (i) and hadv-d37 infected (ii) keratocytes (white arrows indicate virus). figure 3 . electron micrographs of hadv-d37 entering ocular surface cells. corneal keratocytes or tert-immortalized human corneal epithelial cells were infected with moi = 10 of cesium chloride purified hadv-d37 for 1 hour at room temperature. cells were washed with pbs, fixed in a fixative solution (2% paraformaldehyde containing 2.5% glutaraldehyde, 0.1 m cacodylate, and 2.5 mm cacl 2 ) for 1 hour, and collected in 2% agarose. the cell pellet was further fixed for 1.5 hours in 2% aqueous oso4 and dehydrated. after dehydration, the cell pellet was embedded in epon and sectioned into 70-90 nm thin sections. the sections were stained with saturated aqueous uranyl acetate, sato's lead stain, and micrographs were taken on a philips cm-10 electron microscope operating at 80 kv and fitted to a ccd camera. (a) electron micrographs of infected tert-immortalized human corneal epithelial cells show that hadv-d37 (white arrows) can enter via clathrin-mediated endocytosis (i) or macropinocytosis (ii). (b) electron micrographs of infected keratocytes showing caveolae (black arrows) associated with hadv-d37 (white arrows) at the cell membrane (i) and inside a caveosome (ii). (c) immunogold staining for caveolin in uninfected (i) and hadv-d37 infected (ii) keratocytes (white arrows indicate virus). caveolae-dependent cell entry of adenovirus is controversial. caveolae are flask-or omega-shaped lipid raft invaginations of the plasma membrane, with an average diameter of 50-100 nm [84] [85] [86] . caveloae are abundantly present in many cell types including fibroblasts, cardiomyocytes, and adipocytes, but are not common to all eukaryotic cell membranes [87] . caveolins are the major integral membrane proteins of caveolae and include three types: caveolin-1 and -2 are found in most cell types, while caveolin-3 is present only in myocytes [88, 89] . caveolae exist stably at the plasma membrane, but following internalization, they fuse with other caveolae to form larger structures called caveosomes, or they fuse with endosomes in a rab5-dependent manner [90] . while many molecules are needed to initiate the internalization of caveolae, dynamin 2, and src, pkc activation and recruitment of actin appear to be important [91] [92] [93] [94] [95] [96] . caveolae have been implicated in potential entry pathways for a diverse range of viruses, including coronavirus [97] , hepatitis b [98] , polyomavirus [99] , papillomavirus [100] , simian virus 40 [101] , filoviruses [102] , and human immunodeficiency virus [103, 104] , among others [105] [106] [107] [108] [109] [110] [111] . our work has indicated that hadv-d37 enters human keratocytes in vitro using caveolae [42] , while the non-cornea tropic hadv-c2 fails to infect keratocytes altogether. confocal analysis of infected cells revealed robust colocalization of hadv-d37 with caveolin-1, but not lamp1, the latter of which is a late endosomal marker. a general increase in caveolin-1 in lipid rafts as well as increased src phosphorylation was noted in the infected cells. caveolin-rich endosomal fractions were found to contain higher levels of viral dna compared to fractions rich in lamp1. il-8, a cytokine rapidly induced following hadv-d37 infection, was found to be reduced following caveolin-1 knockdown. electron microscopy (em) of the infected cells found multiple flask-shaped vesicles resembling caveolae and caveosome-like structures both contain hadv-d37 virions (figure 3b ). using immunoelectron microscopy, these virus-containing invaginations were also found to express caveolin-1 (figure 3c) . utilizing a novel mouse model of infection, caveolin-1 deficient mice were found to accumulate virus on the cell membranes of corneal stromal cells and exhibit delayed viral entry compared to wild type mice. further, src phosphorylation and expression of cxcl1 (a murine analog of il-8) were both reduced in infected, caveolin-1 knockout mice compared to control mice. collectively, these data support a caveolin-dependent entry pathway for hadv-d37 in the cornea stromal cells. in contrast, in a549 cells, hadv-d37 colocalized with lamp1, which is consistent with an endosomal trafficking pathway in these particular epithelial cells. macropinocytosis is an endocytic process that begins with interactions between a ligand and its host cell receptors, activating a cascade of intracellular signaling and actin rearrangement which drives the formation of cell membrane ruffles on the host cell surface. these ruffles enclose the cargo, forming a large (>250 nm) vesicle known as a macropinosome near the plasma membrane, which then releases the cargo into the cytosol [112, 113] . macropinocytosis may either be constitutively active, as in dendritic cells, or induced. many families of viruses, including herpesviruses [114, 115] , vaccinia viruses [116, 117] , picornaviruses [118] , and some adenoviruses [39] [40] [41] , are known to exploit macropinocytosis for their entry. while clathrin-mediated endocytosis is the predominant entry pathway for hadv-c2 and -5, as discussed above, these viruses can simultaneously utilize macropinocytosis as an alternative entry mechanism. additionally, if clathrin-mediated endocytosis is blocked, macropinocytosis becomes the primary means of entry [39] . in contrast, hadv-b3 and -35 utilize macropinocytosis as their primary entry pathway. macropinocytosis is also variably dependent on dynamin activity. hadv-b3 entry via macropinocytosis does not require dynamin, whereas macropinocytosis of hadv-b35 requires dynamin for entry into the hela-kyoto clonal derivation, but not into the parental hela-atcc cells or wi-38 cells, a diploid human fibroblast cell line derived from lung tissue [41, 79] . in a tert-immortalized human corneal epithelial cell line, as shown by em, macropinocytosis was also utilized for adenovirus entry (figure 3a(ii) ). macropinocytosis is regulated by p21-activated kinase (pak), a serine/threonine kinase, and pretreatment with the pak inhibitor ipa-3 reduced viral gene expression in a dose-dependent manner. interestingly, the sodium-proton exchange inhibitor 5-(n-ethyl-n-isopropyl) amiloride did not block the endocytic uptake of hadv-d37 (manuscript in preparation). further, clathrin knockdown completely abrogated viral entry, while macropinocytosis blocking agents did not. this strongly suggests that clathrin-mediated endocytosis is the dominant pathway in corneal epithelial cells, with macropinocytosis playing a secondary role. additional studies are needed in order to define the molecular entry mechanisms of hadv-d37 and other ekc-associated adenoviruses into ocular surface cells. following adenoviral entry into the cell, for viral replication to occur, the viral genome must reach the nucleus. because intact adenovirions are too large to enter the nucleus through the nuclear pore complex, they must first uncoat to release their genome [119] . mechanical and chemical forces drive this dynamic, tightly-regulated, and irreversible process (reviewed in [120] [121] [122] [123] [124] ). for viruses utilizing clathrin-mediated endocytosis, such as hadv-c2 and -5, uncoating begins with the interaction between the penton base rgd motifs and cellular integrins. subsequent fiber-shedding exposes protein vi (pvi) and the virion core is endocytosed. further, pvi, a 22 kda cement protein, functions as the primary lytic factor for penetration of the endosomal membrane [125] . in contrast, for caveolin-dependent endocytosis and macropinocytosis, the specific adenovirus uncoating pathway is not well-defined. once in the cytosol, the adenovirus capsid core associates with microtubule-based motors, including dynein and kinesin, and is transported to the nucleus via the mtoc [126] [127] [128] . beyond their role in virion transport, dynein and kinesin also exert mechanical forces on the capsid, completing the uncoating process [128] . finally, the viral core particle binds to the nucleoporin nup214 at the nuclear membrane, leading to kinesin-1 mediated disassembly and enabling the viral genome to enter the nucleus [129] [130] [131] . the observation that adenoviral entry, uncoating, and trafficking may vary based on virus and cell type has interesting potential implications for host cell immune responses. all cells are capable, at least to some degree, of stimulating local innate immune and antiviral responses. as discussed above, this is largely mediated through the detection of conserved pamps on or in invading pathogens by host cell prrs, inducing both divergent and convergent signaling cascades and leading to the production of inflammatory mediators [43] [44] [45] [46] . prrs are known to have specific distributions within each cell, unique to cell type, to facilitate the detection of pathogen components based on their cellular location. for example, toll-like receptors (tlrs) 1, 2, 4, 5, and 6 are expressed on the cell surface, while tlrs 3, 7, 8, and 9 are expressed on intracellular endosomal membranes [132] . other prrs, such as the nucleic acid sensors rig-i, aim2, cgas, and the nlrp3 inflammasome complex, are expressed in the cytosol [133] . many of these prrs have been shown to function as sentries to detect adenovirus infection [134] . it follows that the specific pathway of adenoviral entry and trafficking could expose adenoviral pamps to different host cell prrs, inducing cell-and virus-type specific responses. studies have sought to elucidate the connections between entry, trafficking, and innate immune responses by infected cells [44, [135] [136] [137] . in a549 cells, hadv-c5 was shown to move relatively quickly (within 1 hour of infection) from early endosomes to the cell nucleus. in contrast, hadv-d26 and -b35 remained within late endosomes at 2-6 h post-infection. the persistence of virions in late endosomes in human peripheral blood mononuclear cells (pbmcs) was associated with higher expression of ifnα2, il-1β, il-6, mip-1β, and tnfα. pre-treatment with inhibitors of endosomal acidification reduced expression of these factors, supporting viral accumulation in late endosomes as a strong stimulator of innate immune responses [135] . the same findings were reproduced in vivo. vaccination of rhesus macaques with hadv-d26 and hadv-b35 was associated with higher serum levels of pro-inflammatory cytokines and chemokines than with hadv-c5 [136] . in other studies, it was shown that divergent prr activation in different types of immune cells can lead to a convergent interferon (ifn) response. plasmacytoid dendritic cells, a minor subset of the monocyte population found in blood, recognize adenoviral dna in the late endosome in a tlr9 and myd88-dependent manner, leading to the production of ifnα [43] . in conventional dendritic cells, hepatic kupffer cells, and peritoneal macrophages, all of which are more prevalent than plasmacytoid dendritic cells, recombinant adenoviral vectors utilized a tlr-independent pathway to detect cytosolic viral dna and drive ifnα production [44] . while the cytosolic dna sensor has yet to be defined, it was shown that this pathway requires endosomal escape, signaling via sapk/jnk and irf3 [137] . further work is needed to establish specific connections between viral pamps and host sensors during adenoviral infection of ocular surface cells. while the connection between entry, trafficking, and immune responses has not been specifically addressed in the cell types present on the ocular surface, general immune responses to adenovirus have been studied. since the 1940s, it has been understood that the cornea is an immune privileged site capable of mounting immune responses that both protect against insult and preserve the anatomical integrity and visual function of the eye (reviewed in [138, 139] ). as ekc is the ocular adenovirus-associated disease most associated with long term morbidity and vision loss [19, 140] , it is not surprising that the majority of research over the past 70 years has largely focused on immune responses within the cornea. these studies, discussed below, suggest that different ocular surface cells respond in immunologically distinct ways. primary viral replication in corneal epithelial cells results in punctate and geographic epithelial keratitis [141] [142] [143] [144] . corneal epithelial cells produce a variety of cytokines, including ccl20, egf, il-1α, il-6, il-8, and tgf-β1 [145] [146] [147] [148] [149] , in response to different stimuli, though typically at lower levels relative to other corneal cell types. few studies have examined the cytokine responses of corneal epithelial cells to ocular adenoviruses. one study showed that il-1α secreted by hadv-d37 infected epithelial cells can enhance icam-1 expression on endothelial cells to promote lymphocyte entry into the infected cornea, thereby promoting sei formation [150] . in immortalized corneal epithelial cells, although hadv-d37 was able to enter and replicate, our attempts to profile cytokine induction using cytokine arrays failed to identify any elevation of commonly studied cytokines (unpublished data). this is consistent with similar studies performed on corneal epithelial cells with human alphaherpesvirus 1, in which chemokine induction by infected cells was meager in comparison to that by infected corneal fibroblasts [146] . perhaps the study of monolayer epithelial cell cultures in isolation from other cell types, those that are normally present in vivo, is misleading. it is also possible that use of primary corneal epithelial cell cultures and/or a more sensitive cytokine detection methodology would lead to a different conclusion. since the 1950s, it has become clear that the adenovirus-infected corneal stroma is more than just a bystander or mere target of corneal inflammation, as famously suggested by barrie jones [151] . instead, immune responses to adenovirus infection involve the active participation of stromal resident cells, including keratocytes, which are capable of inducing and orchestrating the secretion of specific immune factors. keratocytes are the major cell population of the stroma (figure 1 ) and play important roles in preserving the clarity and highly ordered structure of the stroma [28, 152] . in vitro, cultured keratocytes resemble fibroblasts and have been shown to produce a broad and robust spectrum of cytokines, including ccl11 [153, 154] , ccl20 [149] , cxcl9 [155, 156] , g-csf [157] , gro-alpha/cxcl1 [158] , ifn-γ [154] , il-1α/β [148, 159] , il-6 [147, 154, 160, 161] , il-8 [145, 146, 153, 154, [162] [163] [164] [165] [166] [167] , il-12 [154] , ip-10 [154, 155] , mcp-1/ccl2, rantes [154, 161, 163, [167] [168] [169] [170] , tgf-β1 [148] , and tnfα [160] . additionally, the aforementioned studies found that keratocytes are often more potent producers of pro-inflammatory cytokines than corneal epithelial cells, indicating that they have a considerable role in the orchestration of corneal immune responses. in vitro, adenoviral infected keratocytes express the pro-inflammatory chemokines il-6, il-8/cxcl8, and mcp-1/ccl2, which promote subsequent immune cell infiltration to the cornea and sei formation [31, 164, 171, 172] . however, during natural infection of the eye, it has not yet been demonstrated if adenovirus is capable of reaching the corneal stroma. work from our laboratory has defined the signaling pathways by which these cytokines are stimulated in keratocytes (figure 4) . the prrs tlr2 and tlr9 were shown to mediate cytokine responses to ekc-associated adenoviruses [172, 173] . tlr2 is expressed on the cell surface and may recognize adenoviruses [174, 175] . tlr9 is expressed in the endosome and classically detects unmethylated cpg regions of viral dna [176] . tlr2 and tlr9 act synergistically and the knock-down of both was required to reduce keratitis in a mouse model [173] . however, pro-inflammatory cytokine production, immune cell recruitment, and keratitis, although reduced, were still noted, indicating that there are likely other, as of yet undefined, prrs responsible for mediating the innate immune responses to adenoviruses in hcfs. animal and human clinical studies have shown that seis form in the superficial stroma just below bowman's layer and including the corneal epithelial basement membrane (figure 1 ), areas which are comprised of a variety of immune cells. acutely, sei are comprised of polymorphonuclear leukocytes, but t lymphocytes and dendritic cells have been identified at later time points [24, 25, 29, 30, 32, 184, 185] . neutrophils are the first and by far the most abundant cell type recruited to the cornea, typically within the first 24 hours post-infection in the mouse model, and are a critical pathogenic event in this infection [31, 32, 172, 181] . il-8 is a well-described and highly potent neutrophil chemoattractant and its expression in the adenovirus infected cornea closely correlates to the rapid infiltration of neutrophils [164, 186] . in a three-dimensional culture system incorporating figure 4 . overview of human adenovirus induced cell signaling and downstream immune responses in human keratocytes, highlighting the centrality of src kinase. following engagement with the primary receptor (cd46 or gd1α) by the viral fiber protein and secondary engagement of the penton base with the αvβ1 or αvβ3 integrins, group d adenoviruses are internalized via src-dependent, caveolin-mediated endocytosis. following uncoating and fiber shedding, virions traffic along the microtubule network, through the microtubule organizing center (mtoc), and the viral genome enters the nucleus for replication. adenovirus stimulates cell surface tlr2 and endosomal tlr9, which synergistically activate myd88. myd88 further activates src, which then mediates multiple downstream kinases, leading to nfκb activation. this culminates in the inhibition of apoptosis and expression of pro-inflammatory genes, including il-8 and mcp-1. a similar signaling pathway in human corneal epithelial cells has yet to be elucidated. following the stimulation of tlr2 and tlr9, myd88 is recruited and initiates a signaling cascade that results in the activation of src kinase [173] , a protein-tyrosine kinase which plays key roles in cell growth, division, migration, and survival pathways (figure 4 ) (reviewed in [177] ). src is also a central signaling molecule in keratocytes in response to adenovirus infection [165] and appears to be phosphorylated within minutes of infection by at least two mechanisms. first, myd88 was shown to physically interact with and phosphorylate src, suggesting that tlr activation may directly activate this kinase [173] . second, virus-integrin binding can also lead to src activation [165] . myd88 downstream signaling via irak1/4 and traf6 can also lead to the activation of the same downstream signaling pathways and pro-inflammatory responses as in src activation [178] . src promotes a cell survival pathway by activating the p85 subunit of the phosphoinositide 3-kinase (pi3k), which then activates akt to drive translocation of nfκb p65 to the nucleus. this pathway inhibits caspase 3/7-dependent apoptosis and thereby promotes cell survival, corresponding with increased viral titers, presumably due to prolongation of cell viability [179] . src also activates focal adhesion kinase (fak) within 15 minutes of infection, leading to pi3k activation. it is not known if pi3k activation is dependent on fak or is directly activated by src [180] . src activation induces the expression of il-8 and mcp-1 by activating the mitogen-activated kinases (mapks), including p38, jnk, and erk1/2 [165] . this results in translocation of the p65 and p50 subunits of nfκb to the nucleus, with subsequent binding to and transcriptional activation of the il-8 promoter [181] . src kinase phosphorylates the p38 mapk kinase, leading to iκb phosphorylation, with convergence to the same pathway as erk1/2, resulting in p65/p50 translocation and il-8 expression within 1 hour post-infection [182] . src also phosphorylates mmk7 shortly after infection, leading to jnk and c-jun activation, and subsequent mcp-1 expression [183] . inhibitors of jnk decrease crel protein binding to mcp-1 promoters and reduce mcp-1 expression at 4 hours post-infection [181] . il-8 and mcp-1 are therefore mediated by two distinct pathways, explaining why mcp-1 expression occurs later in infection compared to il-8 [181, 182] . animal and human clinical studies have shown that seis form in the superficial stroma just below bowman's layer and including the corneal epithelial basement membrane (figure 1 ), areas which are comprised of a variety of immune cells. acutely, sei are comprised of polymorphonuclear leukocytes, but t lymphocytes and dendritic cells have been identified at later time points [24, 25, 29, 30, 32, 184, 185] . neutrophils are the first and by far the most abundant cell type recruited to the cornea, typically within the first 24 hours post-infection in the mouse model, and are a critical pathogenic event in this infection [31, 32, 172, 181] . il-8 is a well-described and highly potent neutrophil chemoattractant and its expression in the adenovirus infected cornea closely correlates to the rapid infiltration of neutrophils [164, 186] . in a three-dimensional culture system incorporating primary corneal fibroblasts and extracellular matrix, we were able to mimic human corneal infection by adenoviruses. il-8 was produced by virus infected stromal cells within the cornea facsimile and was bound to heparin sulfate in the facsimile basement membrane, thus presenting a reservoir of chemoattractant for subsequent leukocyte infiltration [187] . similarly, mcp-1 is a chemoattractant for inflammatory monocytes [188] and is thought to be the chemokine responsible for the delayed migration of monocytes in the adenovirus infected cornea [31] . keratocytes are also known to upregulate adhesion molecules following infection, including icam-1/cd54, which are hypothesized to further promote the extravasation of leukocytes into the cornea [165, 167, 189] . in addition, as mentioned earlier, corneal epithelial cell-derived interleukin-1 alpha (il-1α) promotes the expression of icam-1 and vcam-1 on endothelial cells following infection. this allows for transendothelial leukocyte migration and recruitment to the infected cornea [150] . the combination of these cytokines promotes a rapid infiltration of immune cells to the cornea. studies evaluating the effects of corticosteroid treatment on corneal seis have shown that immune cell infiltrate/sei formation is reduced, however both the duration and magnitude of virus shedding are increased [190] [191] [192] [193] [194] . these data suggest that the immune response also serves to limit viral replication. paradoxically, keratitis does not appear to require active viral replication. in the mouse model, uv-inactivated virus was found to be sufficient to induce keratitis, while heat denatured capsid was unable to induce keratitis. this study showed that hadv-d37 viral dna played only a minor role in innate immune responses, while implicating capsid penton base rgd in the onset of clinical keratitis. in further support of this finding, injection of empty capsid directly into the stroma provoked similar cytokine responses, leukocyte infiltration, and clinical disease as intact virus [172] . unadulterated adenovirus does not replicate in the mouse cornea, further supporting the notion that intact and replicating virus is not required for inflammation [32, 195] . it is not known why seis can persist and/or recur for months to years following infection. their presence does not appear to be mediated by viral antigens because, years after infection, adenoviral particles are not apparent by em [184] and adenoviral antigen is not detected by immunofluorescence [185] . other prrs, viral ligands, and cytokines are likely critical to corneal inflammation after adenoviral infection. for example, microarray analysis of hadv-64 infected keratocytes showed upregulation of genes related to cell growth and differentiation, apoptosis and oncogenesis, cell signaling, transcription, and immune responses, including fra1, g-β, lif, mip-2α, thymosin beta 4, and il-8, among others [165, 196] . microarray analysis and transcriptome sequencing of non-ocular cell types infected with different adenovirus types have defined robust changes in innate immune responses. these included the tlr signaling, cell cycle progression, apoptotic, rna binding and processing, and nfκb signaling pathways [197] [198] [199] [200] . it is not known if other cell types present on the ocular surface respond to adenovirus infection in a similar fashion. healthy, uninfected corneas contain several distinct resident populations of antigen presenting cells (apcs). conventional dendritic cells (dcs) and possibly also plasmacytoid dcs are present at the level of the corneal epithelium/basement membrane [13] . macrophages are also found in the anterior corneal stroma [12, 13, 201] . the limbus and corneal periphery contain mature and immature langerhans cells (figure 1 ) [202] . these resident cells stimulate the recruitment of systemic innate (neutrophil, macrophage, and natural killer cells) and adaptive (t-cells) immune responses to the eye [201, [203] [204] [205] [206] [207] . few studies on the role of ocular surface apcs during adenovirus infection have been performed. we recently demonstrated the involvement of myeloid-derived cells in infection using the macrophage fas-induced apoptosis (mafia) mouse. this mouse expresses a membrane bound suicide protein under the control of the myeloid-lineage specific c-fms promoter. following treatment with the fk506 dimerizer ap20187, myeloid cells, including those in the cornea, then undergo apoptosis [208] . ap20187 treated mice were found to have clinically normal corneas; however, following hadv-d37 infection and in comparison to control and vehicle treated mice, ap20187 treated mice showed reduced immune cell infiltration and reduced myeloperoxidase expression, the latter if which is a correlate for neutrophil infiltration [209] . these data suggest that dcs and macrophages are critical in the development of the immunopathology of keratitis following adenovirus infection. despite strong evidence for the role of corneal stromal cells in the pathogenesis of adenovirus keratitis in the mouse model, an understanding of their participation in human adenovirus keratitis remains incomplete. the conjunctiva (figure 1 ) plays critical roles in protecting the ocular surface from infection. it provides antimicrobial protection and lubrication to the ocular surface via the production of tears and mucins, which collectively form the 3 µm thick tear film. the tear film consists of three layers, an innermost mucin layer, consisting of epithelial cell and conjunctival goblet cell-secreted mucins, an aqueous layer secreted by the lacrimal and accessory lacrimal glands, and an outermost lipid layer, secreted by meibomian glands at the eyelid margin. the aqueous and mucin layers mix in a gradient, with the greatest mucin concentration at the epithelial surface and the greatest aqueous concentration just posterior to the lipid layer. the tear film provides an effective chemical and physical barrier due to mucins, lysozymes, soluble iga, and antimicrobial peptides that can entrap and/or destroy invading pathogens, though these functions have primarily been studied for bacteria [210] . as discussed earlier, the conjunctiva also houses a variety of immune cells that are capable of responding rapidly to insults on the ocular surface. the conjunctiva appears to be more susceptible to adenovirus infection than the cornea-many adenoviruses cause conjunctivitis, while only a limited number cause keratitis. additionally, in ekc, conjunctivitis typically precedes keratitis. the evidence is clear that ocular-tropic adenoviruses can infect and replicate in the cells of the human conjunctiva. both fully infectious adenovirus and viral dna are frequently isolated from conjunctival scrapings during human outbreaks [211] [212] [213] [214] . one report showed the persistence of the virus in the tear film and conjunctiva of a subset of patients for up to a decade following primary infection [215] , although the site of viral persistence was not established. interestingly, studies using a rabbit model of human adenovirus infection have demonstrated that hadv-c5 is able to infect and replicate in the acinar epithelial cells of the lacrimal gland, possibly contributing to its detection in the tear film [216] . hadv-d37 replicates efficiently in the chang c conjunctival cell line in vitro [61] , although it is now known that this cell line was established via hela contamination, based on isoenzyme analysis, hela marker chromosomes, and dna fingerprinting [217] . a few studies have focused on the conjunctival immune responses to adenovirus infection. it was shown by oligonucleotide microarray analysis that the infection of primary human conjunctival epithelial cells with low multiplicities of infection of hadv-c5 resulted in the upregulation of cxcl2, cxcl5, cxcl10, cxcl11, and several interferon induced signaling molecules, including irf7 and stat1. this suggests that the infection of conjunctival epithelial cells with adenovirus initiates signaling that would be expected to drive the recruitment of neutrophils, monocytes/macrophages, t cells, nk cells, and dendritic cells to the site of infection [218] . however, these experiments were only performed with hadv-c5, a respiratory virus, and, as discussed, these results cannot be extrapolated to infection by ekc-associated adenoviruses. nevertheless, in ekc, immune rich conjunctival membranes form in response to adenoviral induced inflammation. these membranes contain macrophages, neutrophils, cd4+ and cd8+ t cells, b cells, langerhans cells, and activated dendritic cells, in proportion to the degree and intensity of the inflammation [219] . conjunctival membranes seen in ekc also contain intact adenoviruses and are therefore infectious, as with other ocular secretions in ekc. in the microarray study mentioned above [218] , in addition to their participation in recruiting leukocytes during inflammation, an anti-microbial peptide (defensin)-like role for cxcl10 and cxcl11 was demonstrated against hadv-b3 and hadv-c5, but not against hadv-d8 or hadv-d64. likewise, the β-defensins were found to inhibit respiratory, but not ocular genotypes. it has been suggested that ocular-tropic adenovirus types have evolved immune evasion strategies to avoid host defensins, which are abundantly expressed on the ocular surface [220] . in adenoviral conjunctivitis, it was previously shown that the number of conjunctival lymphocytes, natural killer (nk) cells, and monocytes increases significantly during the acute phase of the infection [221] . generally, nk cells respond rapidly to viral infection by killing virus infected cells and producing cytokines which promote crosstalk between dendritic cells and t cells, thereby inducing t cell differentiation (reviewed in [222] ). mature cd56 dim nk cells that are capable of producing ifnγ are present in the epithelium of healthy conjunctiva [221] . however, upon infection with hadv-d types, mature nk cells are replaced by cd56 bright , immature nk cells. this correlates with the detection of ccl2, ccl3, ccl4, ccl5, cxcl9, and cxcl10 in the tear film, all of which are potent chemokines for immature nk cells. the upregulation of the inhibitory ligand human leukocyte antigen-e on infected epithelial cells and the overall impairment of nk cell responses upon infection with hadv-d types by reducing the expression of activating ligands on the surface of infected epithelial cells) represent additional mechanisms by which hadv-d types actively promote immune escape. notably, non-species d adenoviruses, such as hadv-c3, -e4, and -c5, were unable to induce these responses. this dampening of antiviral responses likely enables enhanced virus replication in the conjunctiva and, thus, subsequent spread to other ocular surface cells [221] . conjunctival epithelial cells express the membrane-associated mucins muc1, muc4, and muc16, which form the protective glycocalyx [223] . mucins are high molecular weight, heavily glycosylated proteins that have been shown to prevent the penetrance of invading pathogens to the eye. further, ocular surface mucins have complex o-and n-glycans with sialyated cores [224] . as mentioned, sialic acid residues on the gd1α ganglioside are one of the primary receptors for cornea-tropic hadv-ds [53, 56] . therefore, sialic acid containing mucins in the tear film and glycocalyx might act as decoy receptors to reduce subsequent infection of ocular surface cells. while sialic acid-based decoy receptors have been evaluated as a therapy for adenovirus infection [225, 226] , the potential for sialic acids present in the normal tear film to limit natural infection has not been evaluated to our knowledge. a study from our laboratory found that hadv-d37 was able to induce ectodomain release of muc16, resulting in decreased glycocalyx barrier function in cultured corneal and conjunctival epithelial cells. in contrast, hadv-d19, which is not associated with ekc, was unable to cleave muc16. this suggests that specific hadv-d types have evolved in their capacity to penetrate the mucin layer to infect the eye. conjunctival goblet cells are another potential mediator of ocular surface immune responses. goblet cells are important for proper tissue homeostasis through the secretion of the major gel-forming mucin muc5ac, but they also produce cytokines [227, 228] . in response to staphylococcus aureus infection, conjunctival goblet cells produce both mucins and il-1β, the latter of which is dependent on the nlrp3 inflammasome [229] . intriguingly, these cells appear to distinguish between commensal and non-toxigenic bacteria, the latter of which does not induce conjunctival goblet cells to initiate an inflammatory response [230] . paradoxically, it was recently shown that the respiratory pathogen hadv-c5, but not the enteric pathogen hadv-f41, preferentially infects goblet cells in human enteroid cultures, suggesting type-specific tropisms for intestinal goblet cells [231] . however, such type-specific tropism and associated immune responses have yet to be investigated for conjunctival goblet cells. dogma maintains that adenoviruses enter host cells by clathrin-mediated endocytosis, uncoat within endosomes, and rapidly traffic to the nucleus for subsequent replication. however, this paradigm was established based on studies utilizing a limited number of virus types and using tumor-derived cell lines. recent work has demonstrated that entry may not always be this straightforward, with adenoviruses entering via diverse and overlapping mechanisms, depending on cell and virus type pairing. due to the cell-specific and varied pattern of the expression of host prrs, divergent entry mechanisms have the potential to lead to the stimulation of immune responses that are distinguishable based on the specific cell and virus pair. such a connection has not been studied for ocular surface cells. we have demonstrated that ekc-associated hadvs enter and traffic differently in corneal epithelial cells than in stromal keratocytes. furthermore, hadvs are known to induce a more diverse and robust cytokine response in stromal keratocytes than in epithelial cells. however, substantial work remains to support or refute the hypothesis that differential entry of adenoviruses to ocular surface cells dictates subsequent immune responses. first, the entry mechanisms of ekc-associated hadvs would need to be defined across a multitude of cell types, including corneal epithelial cells, keratocytes, conjunctival epithelial cells, conjunctival goblet cells, conjunctival fibroblasts, dendritic cells, macrophages, and other immune cell types that are present on the normal ocular surface. it will be important to determine whether ocular-tropic hadvs can productively replicate in these cells. then, the specific immune responses to infection with hadv will need to be defined, along with the connection between cytokine production and entry pathways. such experiments would greatly increase our knowledge of ocular surface immunology and may permit the development of therapies that manipulate immune responses to infection to improve the clinical outcomes of infection. author contributions: m.r.p., j.c., and j.r. conceived the original idea for the manuscript. m.r.p., a.s., and d.f.p. reviewed the literature and prepared the original draft. m.r.p., a.s., d.f.p., c.g., a.m.i., x.z., j.c., and j.r. all assisted in editing and revisions. funding: this work was funded by national institutes of health grants ey013124, ey021558, ey014104, and 5t32ey007145-20, and a senior scientific investigator award grant (to j.c.) from research to prevent 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and c-fms conditional ablation (mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/tlr4-induced corneal inflammation resident corneal c-fms + macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis complexity of the tear film: importance in homeostasis and dysfunction during disease monitoring of adenovirus from conjunctival scrapings in japan during 2005-2006 outbreaks of epidemic keratoconjunctivitis caused by human adenovirus type 8 in the tibet autonomous region of china in 2016 evaluation of adenovirus amplified detection of immunochromatographic test using tears including conjunctival exudate in patients with adenoviral keratoconjunctivitis. graefe's arch immunofluorescent detection of adenovirus antigen in epidemic keratoconjunctivitis evidence for persistence of adenovirus in the tear film a decade following conjunctivitis adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland survey of atcc stocks of human cell lines for hela contamination adenovirus-directed ocular innate immunity: the role of conjunctival defensin-like chemokines (ip-10, i-tac) and phagocytic human defensin-α cellular and tissue architecture of conjunctival membranes in epidemic keratoconjunctivitis defensins and other antimicrobial peptides at the ocular surface dynamic change in natural killer cell type in the human ocular mucosa in situ as means of immune evasion by adenovirus infection evolutionary struggles between nk cells and viruses ocular surface membrane-associated mucins glycosylation pathways of human corneal and conjunctival epithelial cell mucins sulfated glycosaminoglycans as viral decoy receptors for human adenovirus type 37 decoy receptor interactions as novel drug targets against ekc-causing human adenovirus secreted mucins on the ocular surface goblet cells of the conjunctiva: a review of recent findings staphylococcus aureus activates the nlrp3 inflammasome in human and rat conjunctival goblet cells neither non-toxigenic staphylococcus aureus nor commensal s. epidermidi activates nlrp3 inflammasomes in human conjunctival goblet cells adenovirus infection of human enteroids reveals interferon sensitivity and preferential infection of goblet cells key: cord-352832-uih7alib authors: khoury, bassam title: the root causes of covid-19 screech for compassion date: 2020-06-03 journal: mindfulness (n y) doi: 10.1007/s12671-020-01412-8 sha: doc_id: 352832 cord_uid: uih7alib nan sars, bird flu, and now covid-19, caused by a novel coronavirus, are on the rise (smith et al. 2014) . pathogens are crossing from animals to humans, and many can spread quickly to new places. the mechanisms of transmission of viruses such as sars-cov-2 from bats to humans are not yet fully understood but can originate in a direct encounter between bats and humans; for example, during hunting as bats are consumed as food in some corners of the world. another possible route of transmission involves inhalation of infectious particles by humans. these infective aerosols could arise from the secretions (e.g., saliva) of the bats (wong et al. 2007) . a third route for transmission is via a secondary intermediate vertebrate host serving as an amplifying host. this last scenario seems to fit other recent outbreaks of coronavirus-caused disease in humans, such as sars, which arose from cat-like civets, and mers, which arose from camels (andersen et al. 2020) . despite the obvious risk that bat viruses pose to human health, it must be acknowledged that most outbreaks of batborne zoonotic diseases are a consequence of human activities (wynne and wang 2013) . anthropogenic activities such as habitat loss, human encroachment, and the destruction of natural feeding and roosting habitats caused by urban sprawl and agricultural expansion are increasing the interactions between bats, humans, and livestock, thereby heightening the zoonotic potential conferred by those characteristics. researchers have found that disrupting bat habitats and hunting them appears to stress the bats, causing them to shed even more virus in their saliva, urine, and feces, which can then infect other animals (university of california, berkeley, 2020). preliminary links have been established between loss of bat habitat and the spillover of bat viruses into other animals and humans (jones et al. 2013) . it is clear that human actions, such as the hunting of wild animals and mindless destruction of biodiversity, create the underlying favorable conditions for new viruses and diseases such as covid-19, which threaten the existence of humanity itself, so how come humans still engage in destructive behaviors that threaten their own existence? obvious reasons relate to accessing more food sources, exploiting additional land, extending agriculture, and furthering economic growth in order to provide resources for a growing human population. however, such motives, although important, do not explain why humans are failing to balance their needs with the well-being of the planet and animals. to answer this question, it is necessary to turn to buddhist philosophy. in buddhism, the human condition is divided into two states: wholesome (kusala) and unwholesome (akusala) (le duc 2017). unwholesome states lead to suffering and their origins are rooted in greed (rāga), hatred (dosa), and delusion (moha) (kornfield 2011) . greed refers to any form of over-attachment to a sensory object. in this case, greed can be manifested through exploiting the planet, destruction of natural habitats, and taking advantage of animals, without ever attaining satisfaction. hate is the opposite of greed, defined as the drive to avoid, resist, or even destroy things someone does not want. a subtle form of hatred is apathy. apathy can be manifested by a lack of concern to the suffering of others and by justifying it at times as necessary for economic growth. apathy plays a central role in the persistence of the ecological crisis (le duc 2017), the current surge of infectious diseases, and the transmission of viruses, such as the novel coronavirus, from animals to humans. delusion is the inability to perceive reality and phenomena as they really are due to being trapped between the poles of greed and hatred/apathy (khoury 2019) . another form of delusion is ignorance, such as denying empirical scientific facts in order to continue indulging in selfish desires while discounting the well-being of others, including animals and nature. delusion can result from ideological notions supporting, for example, the stance that human beings can exercise absolute domination over nature and animals according to some sort of divine ordination, while denying the impacts of such actions on nature, animals, and therefore humans (le duc 2017). rectifying unwholesome states of mind requires engaging in daily practices of mindfulness and compassion. mindfulness can be defined as an embodied non-judgmental attention and lucid awareness of oneself, others, and the environment (khoury 2018; khoury et al. 2017 khoury et al. , 2019 khoury et al. , 2020 . compassion can be defined as an embodied, integrated state with cognitive, affective, and behavioral dimensions that is characterized by an altruistic attitude, an emphatic concern, and a desire to alleviate suffering in oneself and in others (khoury 2019) . the dalai lama (2005a) included wisdom as an integral part of compassion, writing "genuine compassion must have both wisdom and loving kindness. that is to say, one must understand the nature of the suffering from which we wish to free others (this is wisdom), and one must experience deep intimacy and empathy with other sentient beings (this is loving kindness)" (p. 49). thus, compassion implies non-cruelty and harmlessness towards all sentient beings including animals (finnigan 2017) . empirical studies have shown positive associations between mindfulness and nature connectedness (howell et al. 2011) , ecological sustainability (amel et al. 2009; jacob et al. 2009 ), and ecological behaviors (geiger et al. 2018) . similar studies have shown positive associations between compassion to other human beings and pro-environmental tendencies, including values, intentions, and actions (pfattheicher et al. 2016) , as well as compassion towards animals in lab settings and the well-being of research personnel (lafollette et al. 2020) . according to buddhism, mindfulness and compassion should be taught as part of several complementary perspectives or ethics (shonin et al. 2014) . such perspectives include the concepts of "non-self," "non-attachment," "impermanence," and "inter-connectedness." the term "non-self" refers to the realization that the self has no intrinsic existence (dalai lama 2005b) . "non-attachment" can be defined as the liberation from excessive craving or clinging that is favored by a vision of all objects and living beings as void of any lasting self and by the impermanent nature of all phenomena. "impermanence" refers to the notion that all phenomena are transient occurrences and are subject to decay and dissolution (rinpoche 1998 ). finally, the term "interconnectedness" is used in buddhism to refer to the interconnected nature of all phenomena (called also interbeing) (nhat hanh 1992) . practices of mindfulness and compassion such as concentrative meditation (samatha), insight meditation (vipassana), and loving-kindness meditation (metta), along with nonformal mindfulness practices during daily activities, facilitate the recognition of the interconnectedness between humans, animals, and nature. this interconnectedness implies that the well-being and safety of humans depend on the well-being and safety of animals and of nature. in line with that, planetary health is emerging as a new discipline that focuses on the increasingly visible connections between the well-being of humans, other living things, and entire ecosystems (vidal 2020) . mindfulness and compassion practices rooted in buddhist ethics allow for overcoming greed by cultivating non-attachment, generosity, and humbleness; hatred through generating loving-kindness to all sentient beings and to nature; and delusion by recognizing the realities of impermanence, non-self, and interconnectedness. mindfulness and compassion practices do not preach apathy and non-doing, but rather the opposite. they cultivate a radical commitment to act in order to reduce the suffering of all sentient beings and to approach nature with awareness and kindness. a commitment to act should include the cultivation of social, racial, ethnic, and gender equalities based on a just, mindful, and non-competitive sharing of the natural resources and beauties of the planet. while social and political activism should be a core element of mindfulness and compassion practices, it is paramount that activism is conducted in a totally peaceful, non-violent, and wholesome manner. any use of unwholesome or violent means would defeat the underlying purpose of such a movement. in summary, biological, pharmacological, and medical interventions are highly needed to overcome the current covid-19 pandemic. however, it is essential to understand its root causes in order to undertake a real, sustainable, and profound transformation of our state of mind, moment to moment, and therefore, in our actions towards all sentient beings and the surrounding environment. mindfulness and compassion, when used skillfully and ethically, are among the most influential practices to facilitate change and transformation on an individual, inter-individual, social, environmental, and planetary scale. perhaps, if we start listening to the screeches of bats with awareness and warm heartedness, and responding to them with compassion, we can start to reverse the destruction of the earth and save its sentient habitants, humans, and animals alike. not contain any studies with human participants performed by the author. no official funding was provided. the author has no competing interests. mindfulness and sustainable behavior: pondering attention and awareness as means for increasing green behavior the proximal origin of sars-cov-2 buddhism and animal ethics mindfully green and healthy: an indirect path from mindfulness to ecological behavior nature connectedness: associations with well-being and mindfulness personal and planetary well-being: mindfulness meditation, pro-environmental behavior and personal quality of life in a survey from the social justice and ecological sustainability movement global trends in emerging infectious diseases zoonosis emergence linked to agricultural intensification and environmental change mindfulness: embodied and embedded compassion: embodied and embedded embodied mindfulness. mindfulness la pleine conscience incarnée : un concept unificateur entre les traditions orientales et occidentales de la pleine conscience la dimension interpersonnelle de la pleine conscience bringing home the dharma: awakening right where you are laboratory animal welfare meets human welfare: a cross-sectional study of professional quality of life, including compassion fatigue in laboratory animal personnel essence of the heart sutra: the dalai lama's heart of wisdom teachings the many ways to nirvana buddhist communication of the true roots of the ecological crisis a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? the sun my heart feelings for the suffering of others and the environment: compassion fosters proenvironmental tendencies the emerging role of buddhism in clinical psychology: toward effective integration coronavirus outbreak raises question: why are bat viruses so deadly? bats' fierce immune systems drive viruses to higher virulence, making them deadlier in humans tip of the iceberg': is our destruction of nature responsible for covid-19? the guardian bats as a continuing source of emerging infections in humans rolling updates on coronavirus disease (covid-19) bats and viruses: friend or foe? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. key: cord-337464-otwps68u authors: parray, hilal ahmed; shukla, shivangi; samal, sweety; shrivastava, tripti; ahmed, shubbir; sharma, chandresh; kumar, rajesh title: hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives date: 2020-05-27 journal: int immunopharmacol doi: 10.1016/j.intimp.2020.106639 sha: doc_id: 337464 cord_uid: otwps68u the advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. there are more than 700 antibody-based molecules that are in different stages of phase i/ii/ iii clinical trials targeting new unique targets. to date, approximately more than 80 monoclonal antibodies (mabs) have been approved. a total of 7 novel antibody therapeutics had been granted the first approval either in the united states or european union in the year 2019, representing approximately 20% of the total number of approved drugs. most of these licenced mabs or their derivatives are either of hybridoma origin or their improvised engineered versions. even with the recent development of high throughput mab generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. the recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. this has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. in this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. this review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mabs. antibodies are the glycoproteins produced by the b-cells also known as immunoglobulins, which are present in higher eukaryotes. immunoglobulins are present in either as a soluble form (blood or plasma) or as membrane-bound form (b cell receptors). antibodies are the major component of the humoral immune system that provides protection against the invading pathogens i.e. viruses and bacteria [1] . an antibody is made up of two structural unit's i.e. heavy and light chain. generally, each heavy chain has one variable and three constant regions whereas the light chain has one variable and one constant region. the variable region of antibodies is mainly responsible for its interactions with the invading pathogen and antigen recognition. the antigen-antibody recognition mechanism works like a lock and key fashion. each antibody has a particular paratope (i.e. lock) that binds to a particular antigen (i.e. key). one type of b cell produces one type of antibody against a particular antigen. there are five different types of heavy chains based on the structure of crystallizable fragments (fc) that is attached to the antigen-binding fragments. on the basis of different fc region, antibodies are grouped into five different isotypes i.e; igm, igg, iga, igd, and ige. among all the isotypes igg is the smallest and the most common isotype with the highest therapeutic potential. it makes 70-80% of the total antibodies. iggs have a longer half-life and are permeable to extravascular spaces [2, 3] . https://doi.org/10.1016/j.intimp.2020.106639 received 11 april 2020; received in revised form 6 may 2020; accepted 22 may 2020 antibodies are potentially used for various applications as extraordinary tools in biomedical research for many years. high specificity and selective binding have expanded the scope of antibodies to various applications such as flow cytometry, magnetic cell sorting, immunoassays, therapeutic approaches etc. [4] . antibodies have developed about 40 years ago and have expanded the scope of antibodies to various applications due to their specificity and selective binding ability. these antibodies are classified into two primary subtypes, monoclonal and polyclonal on the means they are basis of their origin from the lymphocytes [5, 6] . both polyclonal and mabs have their advantages and limitations which make them equally suitable for different applications. polyclonal antibodies (pabs) are a pool of immunoglobulin molecules that are secreted by different b cell lineages and react against multiple epitopes of a specific antigen. the pabs are generated by injecting an immunogen into an animal using a prime-boost immunization strategy to produce high titres of antibodies against the particular antigen. after immunization, pabs can be used directly or in the purified form (through affinity column chromatography to remove other serum protein components). polyclonal sera display multiple epitope binding properties which make them an attractive reagent for various purposes, like its use as research or therapeutic reagent either directly or in purified form. the polyclonal serum is widely used for several decades for the treatment of toxin-mediated bacterial and viral diseases [7] . emil adolf von behring was awarded nobel prize in physiology and medicine in 1901 for his work on serum therapy, especially its application against diphtheria, through which he opened doors for new ways of treatment in medical sciences [8, 9] . animal serum-derived therapy has been successfully applied for different medical aspects like overdosing of medication, viral disease (like rabies) and as antitoxins in snakebite envenoming [10] . the beneficial effects of pabs come from its polyclonal nature and biophysical diversity. the poly clonality nature allows targeting multiple sites in a single window of the application and biophysical diversity provides greater stability in environmental changes [11, 12] . despite having beneficial effects of serumderived pabs therapy has several limitations, which need to be evaluated before introducing new interventions. as blood-derived products, intravenous polyclonal immunoglobulins (ivig) have limited availability, batch-to-batch variability; carry the risk of blood-borne disease transmission and only a small fraction of antibodies from the pool of antibodies binds to the target of interest to exert the desired effect. this sometimes results in low specific activity and relatively needs high doses to observe a desired beneficial clinical effect [13, 14] . the other limitation of polyclonal serum is that it cannot be used for the treatment of chronic diseases [15] . due to some of these potential drawbacks of pabs, the way for mab isolation, need, and urgency comes into the light. however, mabs are most suitable and frequently used because of their high sensitivity, specificity, affinity and homogenous nature [16] . monoclonal antibodies are monospecific in nature and produced by identical b cells having high affinity and specificity towards a single epitope of an antigen. in 1975 hybridoma based technology was used to generate the mabs showing very minimal and acceptable batch to batch variation, produced in an indefinite amount continuously. the mabs can be produced against any given epitope present on an antigen or immunogen. moreover, they can be used to detect, purify and characterize the substance of interest. since the development of mab, the scope of antibodies has expanded to various further applications due to their target specificity [17] . this has made mabs a powerful tool in the fields of biochemistry, molecular biology, and medicine. antibody-based biologics are one of the best-selling classes of biomolecules in today's market. advancement in mab generation technologies in recent years has made ease in identification of new target antigens to be explored in diagnostic and therapeutic approaches [18, 19] . several mab generation technologies had been developed over the years, to isolate mabs from immune and non-immune sources using hybridoma, display methods, and more advanced novel mab development technologies like single b cell amplification and culturing methods. unlike the hybridoma method, other methods rely mostly on recombinant production of mabs. each of these technology platforms has their respective advantage, limitations, and applicability [18, 20, 21] . hybridoma technology is the primitive, most fundamental and successful methodology in the field of mab isolation [19] . this technology is quite robust and useful in discovering thousands of antibodies for different applications [22] . the basic practical advantage associated with hybridoma technology is, once the hybridoma clones are established, the production of mab becomes simple and efficient. the antibodies isolated through hybridoma methodology preserves the native pairing of variable and constant regions gene combination, which further supports studies on both direct and indirect functions of a mab. hybridoma technology relies on b cells that are matured in secondary lymphatic organs in response to an antigen. these b cells undergo natural antibody maturation process where the variable region of antibodies diversified by accumulating somatic hypermutations which further results in the selection of high-affinity tight binders [18, 20] . resulting antibodies possess the natural pairing of variable heavy and light chain genes with naturally class-switched matured constant region gene through class switch recombination (csr). such freedom of natural csr is not possible in other mab isolation method that makes hybridoma a unique way to produce naturally matured in vivo antibodies in the laboratory [19] . nevertheless, hybridoma technology is the most preferred technology for mab discovery for in vivo applications. presently there are more than 90% of the antibodies approved by the united states food and drug administration (us fda) are generated by traditional hybridoma technology and are used either directly or in chimeric or humanized versions [19, 23, 24] . a list of these fda approved antibodies has been listed in table 1 . the dominance of this technology has been continued with the recent development of transgenic humanized animals that has opened new avenues for more effective generation of high-quality human antibodies in the modern biotherapeutic era. in this review, we have discussed in detail about hybridoma technology, how it has been expanded to different animal species and their associated biological applications which determine their usage for certain intends. we have also discussed the advancement and challenges associated with hybridoma development, and how it has been overcome in years. georges kohler and cesar milstein in 1975 invented the hybridoma technology, for which they received the nobel prize in 1984 in physiology and medicine [25] . during the same period, herve bazin in louvain-la-nerve belgium created a rat myeloma cell line ir983, allowing the generation of first rat mabs (https://www.synabs.be/2019/ 11/25/hybridoma-vs-display-the-fight-of-the-century/). hybridoma cells are generated via fusion between a short-lived antibody-producing b cell and an immortal myeloma cell. each hybridoma cell constitutively expresses a large amount of one purely specific mab, and favoured hybridoma clones can be cryopreserved for continuous mab production for a long period. hybridoma generation process takes advantage of a host animal's natural ability to generate functional, highly specific and high-affinity mabs [19] . to date, several mabs are developed using this technology and are presently used for diagnosis, prevention, and treatment of different diseases tables 1, 2 and 3. initially, hybridoma technology was limited to murine antigens but with advancement in this field, it is a well-established technology to develop mabs against a vast range of antigens and from different species like rabbits [26] , humans [27, 28] , chickens [29] , goats, sheep [30] , cows [31] , mice [32] , guinea pigs, and rats [33] . choice of animal species especially for mab isolation depends on several factors such as the presence of a homologous protein in the immunized species, table 3 a list of fda approved rabbit mabs that are used in diagnostics. device name availability of suitable fusion partner, the amount of protein or antigen available for immunization, the time required to obtain an antibody response and finally, a purpose for which these mabs are needed. most commonly used hosts for mab production are the mice followed by rabbits. the inbred balb/c strain of mice is usually the right choice and preferably suited for mab isolation [34] . chickens are also considered as a preferred host of choice due to its distinct phylogenetic relationship between the antigen donor and the antibody producer [16, 35] . hybridoma technology has not gained much attention because its success mainly depends on the availability of a suitable fusion partner. in the initial years of hybridoma discovery, technology was limited to mice but in progressive years researchers used this platform for human and rabbit hybridoma development. however major limitation associated with the production of mabs from other species is the instability of hybridoma clones produced with fusion partners from heterologousspecies [18] . this instability resulted in the hybridoma clones are due to the fusion of two cells from different species which leads to chromosomal instability. to overcome this, in the past few years many different strategies have been used to increase the fusion efficiency. generally, there are two types of hybridomas one is homo-hybridomas and second is hetero-hybridomas. in homo-hybridomas both, the igg secreting b cells and fusion partners are from the same species. in hetero-hybridomas the antibody-secreting b cells and fusion partners are from two different species. homo hybridomas are genetically more stable and secrete stable igg as compared to hetero-hybridomas as it gradually lost the chromosomal recombinants during the clonal selection step due to their genetic instability. 3. animal species used for hybridoma development over the years: mouse polyclonal and mabs held the largest market in 2019 as they are more specific and easier to produce in nature. the structural similarities between human and mice antibodies are the prime reason for their high acceptability rate. upgradation and simplicity of mice hybridoma process have made it a more prime reason for their high adoption rate in research and therapeutics [36] . the mice hybridoma technology is a multi-step process that takes advantage of a host animal's natural ability to produce highly specific, high-affinity and fully functional mabs. it involves the development and optimization of specific immunogenic antigen (ag). following the optimization, a host animal is immunized with the ag along with adjuvant for several weeks. the sera from immunized animals are tested for their reactivity and specificity to the immunizing antigen while the animals with high titres of binding antibodies are selected further for splenocytes isolation [32] . the spleen cells are fused with the immortalised myeloma cells in the presence of fusogenic agents like viruses, chemicals and electric pulses. the most common myeloma fusion cell lines are x63-ag 8.6539 [37] and sp2/0-ag 1410 [38] , with the origin from balb/c mouse. the fused cells are then selected on hypoxanthine-aminopterin-thymidine (hat) medium. the myeloma cells are sensitive to hat medium as they lack hypoxanthine-guanine phosphoribosyltransferase (hgprt) gene required for nucleotide synthesis by the de novo or salvage pathways while the unfused b cells die as of short life span. in this process, only the hybrid (b cell-myeloma) survives, as they harbour the functional hgprt gene from the b cells. however, hybrid cells retain the dual properties, antibody secreting property of b cells and continuously growing property (immortality) from myeloma cells. fused or hybrid cells are then screened by "limited dilution cloning" method or with semi solid selective medium to select only those hybridoma that produce antibodies of appropriate specificity. a detailed schematic representation of steps involved in hybridoma production is shown in fig. 1 . production of antibodies by mouse hybridoma technology is quite robust and has been useful to discover thousands of antibodies for different applications. academic and industrial research groups with expertise in the field of antibody isolation, hybridoma methodology continues being the methodology of the first choice, particularly if the goal is to obtain antibodies for analytical purposes. the main associated advantage is that, once the hybridoma clone is isolated, mab production in mouse ascites is simple, efficient and reproducible. these hybridomas can be stored in liquid nitrogen for several years making them virtually immortal [39] . the mouse mabs can be potentially used for diagnostic, therapeutic as well as for other research applications. the first therapeutic antibody that was approved by the food and drug administration (fda) in 1985, developed by murine hybridoma technology was potentially used to reduce the graft rejection in transplant patients [18] . okt3, the first mab to be used in organ transplantation and during the past one decade there has been an extensive experience of its use both for prevention and treatment of organ transplantation rejection. okt3 blocks t cell function by modulating cd3 and the t cell receptor from the t cell surface, functions as an immunosuppressant [40] . since its discovery, okt3 was further improvised in progressive years from chimeric to humanized version, to further reduce adverse effects and to increase immunologic efficiency [41] . the use of murine derived mabs has less therapeutic value as they are entirely from mouse origin and can cause immunogenic reactions in the target host. such limitations have been addressed by chimerization and humanization of antibodies, potentially removing the mouse immunogenic content [42] . several murine mabs has been approved by the fda for use in diagnostic and therapeutic purposes over the years as listed in table 2 . since the discovery of mouse mabs, rabbit hybridoma has been potentially used as a dominant tool in the field of research, diagnostics and therapeutics from several decades [4] . however, a new technology for generating mabs with improved affinity, specificity and having the ability to recognize non-immunogenic rodent's epitopes, are in need as an alternative for the scientific community. the rabbit immune system has been documented as a vehicle for developing antibodies with higher affinity and more diverse recognition of many molecules including phospho-peptides, carbohydrates and immunogens that are not otherwise immunogenic in mouse [24] . antibodies produced in rabbits usually have about 10 to 100 fold greater affinity than those produced by mice. rabbits generate more diverse and complex immune response towards target antigen as compared to human and mice because of gene conversion and somatic hypermutations phenomenon leads towards more mutations in rabbit antibody repertoire [43, 44] . the gene conversion is responsible for introducing mutations and affinity maturation of variable antibody fragments which takes place in double-stranded rearranged v(d)j dna segment of antibody gene via homologous recombination [19, 45] . the rabbit iggs are somewhat simpler than the mouse and human antibodies. rabbit igg has only one subclass i.e. cî³ gene and the majority (90-95%) of light chains are derived from isotype cîº1. only 5% to 10% of the total igg light chains are isotype l. fig. 2 . several efforts were made to generate rabbit mabs after the development of mouse hybridoma technology in the 1970s. due to the favourable properties of rabbit antibodies, many scientific groups tried to develop methods for the generation of rabbit hybridomas. this endeavour was significantly complicated by the absence of rabbit myeloma cell lines. viral transformation of rabbit b cells to generate myeloma-like cell lines also proved to be difficult and rather inefficient [46] . for these reasons, substantial efforts are focused on generating rabbit-mouse hetero-hybridomas. unfortunately, all hetero-hybridomas generated in the early days of hybridoma technology revealed poor fusion efficiency, genetic instability and impaired functional rabbit heavy-and light-chain pairings. in 1988, raybould et al. generated the first stable rabbit-mouse hetero-hybridoma by polyethylene glycol-mediated fusion of rabbit spleen b cells with the mouse myeloma cell line sp2/0-ag14. even though they observed stable rabbit igg expression for several months, other groups observed genetic instability and concomitant decrease of mab secretion [47] . these shortcomings could be partially addressed by extensive efforts to regularly subclone the rabbit-mouse hetero-hybridoma in 1996, weimin zhu and robert pytela, at the university of california, developed an improved rabbit hybridoma fusion partner by repeatedly subcloning. after multiple rounds of subcloning, they selected high fusion efficiency clones based on characteristics like robust growth, morphological characteristics and named it as a new cell line 240e-w, with better fusion efficiency and stability. since then this cell line 240e-w has been further developed and optimized to eliminate endogenous igg and has been used for the production of rabbit mabs for research and commercial applications [48] . the cell line 240e-w was further modified to a superior version 240e-w2. abcam patented this technology to develop highly specific mabs, under the name of ranmab, which has been potentially explored for the production of diagnostic and research antibodies. a large number of rabbit mabs are used in basic laboratory research. rabbit mabs are preferred over the mouse and human mabs in diagnostics and pharmaceuticals applications because of their high specificity and affinity. various rabbit mabs have been approved by the fda to use as in vitro tumour diagnostic tool [49] . a list of fda approved rabbit mabs for diagnostics are listed in table 3 . recently, wei et al. developed an ultrasensitive test for ebola virus diagnostics using carbon nanoparticle-labelled pad with rabbit anti-ebola virus (ebov)-vp40 igg for rapid detection lateral flow test strip for ebola virus [50] . limited success has been gained in the development of rabbit mabs as therapeutic agents but the potential use of rabbit pabs for the prophylaxis and treatment of acute rejection against t cells in organ transplant opened venture to explore the use of humanized rabbit mabs as a therapeutic agent [46] . a company name epitomics has developed a humanized rabbit monoclonal drug candidate and demonstrated in vitro and in vivo efficacies [51] . recent fda approval of humanized rabbit monoclonal single-chain antibody fragment, brolucizumab in 2019 for the treatment of wet age-related macular degeneration (amd) has increased hope and venture of other humanized rabbit monoclonal for therapeutics in near future [52] . a detailed description of these antibodies has been reviewed by mage et al. 2018 [53] . human hybridoma technology which allows the direct generation of human antibodies in a native form, is the most direct effective approach for the production of natural therapeutic and diagnostic antibodies with no additional modifications require [54] . it is believed to be the most promising and convenient technological platform for the isolation of therapeutic mabs. however, success of human hybridoma technology for the therapeutic purposes has been limited since years due to several technical challenges like unavailability of human fusion partners, as most of the fusion partners available are from rodent origin or heteromyelomas. a fusion of human b cells with different fusion partners limits the use of these mabs for therapeutic applications. several hetero-myelomas fusion has been successfully employed for the generation of mabs of human origin for different diseases like hiv [28, 55] , chikungunya [56, 57] , dengue [58] etc. however, such hybridomas are unstable, leads to a loss in the ability of antibody secretion hence there is a challenge in achieving desired pharmacokinetic characteristics of natural human antibodies in terms of distribution, metabolism and excretion [54] . several scientific groups have attempted to develop natural human fusion partner cell lines but limited success stories are reported using these fully human fusion partners. one such example of fusion cell is human karyochi cells which were successfully used to develop complete human stable hybridomas with igg secreting properties for several months. the fusion efficiency of these human karyochi cells was in the range of 10 -5 to 10 -3 with no reports on the endogenous generation of immunoglobulin or chains that can interfere with subsequent synthesis, assembly and purification of mabs. the other major limiting step in the development of human hybridomas is low fusion efficiency of human b cells with the myeloma partner (0.001%) and secondly, the low percentage of circulating antigen-specific b cells in the peripheral blood (0.01%) also limits the use of this technology [59] . frequency of antigen-specific b cells is very rare in circulation therefore, selection of a proper blood donor is critical for the success of this method. acutely infected patients have a higher number of circulating b cells as compared to the convalescent patients. donors showing a high titre of serum binding/neutralizing antibodies may have a higher frequency of peripheral b cells, and an indication of the greater chance of successful production of hybridomas. to overcome these challenges different groups have tried ebv(epstein-barr virus) transformation approach to enrich the population of b cells [60] . the most commonly used cell line b95-8 is a continuous cell line releases high titres of transforming ebv in supernatants [61] . the b95-8 cell line was initiated by exposing marmoset blood leukocytes to ebv fig. 2 . schematic drawing of natural rabbit, mouse, chicken and human igg. generally 150-kda igg comprises of two identical îº or î» light chains paired with two identical heavy chains. the light chain consists of an n-terminal variable domain (vl), followed by one constant domain (cl). the heavy chain consists of an nterminal variable domain (vh), followed by three constant domains (ch1, ch2 and ch3) generally, however, the heavy chain of avian igy contains four constant regions (ch1, ch2, ch3 and ch4). schematic drawing of natural rabbit antibodies in igg format. the~150-kda rabbit igg molecule contains two identical îº (white) or î» (light grey) light chains paired with two identical heavy chains (dark grey). the light chain consists of an n-terminal variable domain (vl), shown with its three cdrs, followed by one constant domain (cl). the heavy chain consists of an n-terminal variable domain (vh), also shown with its three cdrs, followed by three constant domains (ch1, ch2 and ch3). ch1 and ch2 are linked through a flexible hinge region that has the amino-acid sequence apstcskptcp (or apstcskpmcp in an allotypic variant) and anchors three disulphide bridges (orange) of the igg molecule, one for each of the two light-and heavy-chain pairs, and one for the heavychain pair. notably, rabbits have two îº light chains, k1 and k2. the more frequent îº light chain, k1, contains an additional disulfide bridge that links vl and cl. rabbits of the commonly used new zealand white strain have~90% igg-îº (k1),~10% igg-îº (k2) and < 1% igg-î» antibodies. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) extracted from a human leukocyte cell line. b95-8 provides a source for ebv to establish continuous lymphocytic cell lines from human donors. ebv mainly binds with the cells in peripheral blood that contains cd21 receptor and b cells in peripheral blood express these antigens on their surface and activates latent membrane protein (lmp) 2a and lmp1 [62, 63] . due to low antibody-secreting and chromosomal instability characteristics transformed b cells cannot be cultured for a long period. the transformation efficiency of ebv to b cells is low, ranges from 0.1 to 1%, however, the transformation efficiency of b cells in presence of ebv can be increased by the addition of cpg, which acts as toll-like receptor (tlr) 9 agonist [27] . cpg oligo-deoxynucleotides (or cpg odn) are defined as short single-stranded synthetic dna molecules where c refers for a cytosine triphosphate deoxynucleotide ("c") followed by a guanine triphosphate deoxynucleotide ("g"). the "p" denotes to the phosphodiester bond between consecutive nucleotides [64] . in unmethylated form, these cpg motifs act as immune stimulants and are recognized by the pattern recognition receptor (prr) toll-like receptor 9 (tlr9), which is constitutively expressed on immune system cells like b cells. these b cells undergo polyclonal response to cpg dna stimulation by proliferation and differentiation to antibody-producing cells [65] . these transformed b cells are grown for a specific time for the emergence of immortalized cells, which further fused with the fusion partner to establish hybridomas. this cpg activation method has been successfully used to produce mabs against severe acute respiratory syndrome coronavirus (sars-cov) [66] and hiv [27] . the main concern of developing hybridoma for therapeutic purpose is that the final hybrid cells should be free of ebv and other human viruses [67] . the two most common cell lines used for fusion are shm-d3327 and hmma 2.5. the shm-d33; produced by fusing the human myeloma cell line fu-266, clone e-1 (hat sensitive, 8-azaguanine resistant and resistant to g-418 -an antibiotic similar to gentamicin) with the murine myeloma p3x63ag8.653 [68] . this cell line has been used as a fusion partner to stabilize the lymphoblastoid cell lines (lcl's) secreting immunoglobulins to produce mabs against envelope proteins of hiv-1 and parvovirus b19 [55, 69] . hmma 2.5 is a human ã� mouse cell line that was generated by fusing mouse myeloma cell line p3x6ag8.653 with bone marrow mononuclear cells of a patient with iga myeloma [70] . several mabs have been produced using this cell line as a myeloma fusion partner [71] . the other limiting steps in human hybridoma development are fusion efficiency. fusion is mainly performed by three methods (i) chemical agents like polyethylene glycol (peg) (ii) viral agent mediated methods and (iii) electric fusion methods. peg mediated fusion is the most common and traditional method of fusion due to its simplicity and most commonly convenient fusing agent of choice for hybridoma production. peg fuses the plasma membranes of two adjacent mammalian cells by dehydrating the lipid head groups, leading to the asymmetry of the membrane bilayer, favouring fusion of two cells leading to a single cell with two or more nuclei. one major drawback of peg mediated fusion is a generation of non-specific fusion between different kinds of cells [72] . over the year's viral agents have also been used as a successful agent to perform fusion among two different cells. sendai and vesicular stomatitis virus are the two most commonly viruses used as fusion partners. the most efficient and innovative method for cell fusion is electrical cytofusion. this mainly works on the principle of fusion of cells in the presence of high-intensity electric field pulse that causes transient membrane permeabilization. this method has higher fusion efficiency over chemical or viral-based fusion methods. however, electrofusion yields in low fusion efficiency when fusion partner cells are different in size [73] . hmma 2.5 myeloma cell line is found to be the most preferred cell line for electrofusion showing maximum fusion efficiency as compared to other myeloma cells [71] . rems et al., developed a numerical calculation method to fuse cells with shorter, nanosecond (ns) pulses. the performance of this method works on the principle of contact areas between two fusion cells, regardless of their cell size [73] . use of cephalin as fugenic reagent along with emetine and actinomycin d, golestani as selection method dramatically increased the fusion efficiency and recovery (19-34%) [74] . the major advantage of human hybridoma technology is that antibodies produced by this technology are derived from human origin have more therapeutic applications than rodent derived counterparts due to attributable differences between the human and rodent immune systems. due to evolutionary differences between mammalian (e.g. human, mice) and avian species (chickens), the avian/chicken immune system recognizes more epitopes on mammalian proteins as foreign and generates a more vigorous and diverse immune antibody repertoire [35] . phytogenic differences among different species have been illustrated in fig. 3 . as the phylogenetic difference between the immunizing antigen and the immunized animal increases, the immune response increases accordingly. it is, therefore, possible to produce antibodies in chicken that are difficult or impossible to produce in mammals such as g-protein-coupled receptors (gpcrs) against which producing antibodies in rodents is a challenge as of high sequence conservation (> 70%) at the protein level. chickens are emerging as valuable immunization hosts specifically for therapeutic antibody discovery for difficult targets having sequence conservation in mammals. the use of chicken egg yolk for antibody production represents a reduction in animal use (ethical issues) as chicken produces a larger amount of antibodies than laboratory rodents [75] . the advantages of chicken (gallus domesticus) antibodies as diagnostic and therapeutic biomolecules are less characterized than their mammalian counterparts [16] . initially, in 1989 a successful attempt was made to generate chicken hybridomas against newcastle disease virus (ndv) by fusing the peripheral blood lymphocytes (pbl) and thymidine-kinase deficient (tk-) chicken myeloma cells [76] . surprisingly, the secreted antibody hybridomas were initially obtained, but they lose the ability to produce antibody in the culture immediately [77] . to overcome this issue nishinaka s et al., in 1991 developed a new improved fusion cell line r27h4, for the production of chicken mabs [29] . the new cell line was efficient in the development of antibody-producing hybridomas with highly reactive igg secretion ability of 6 months. these cell lines were further improved and several chicken hybridomas were successfully developed [29, 78] . however, chicken hybridoma technology has been explored in limits and most of the investigators prefer to use phage display method over hybridoma for the development of chicken mabs [79] . the main avian antibody isotype igy shares structural and functional homology to mammalian igg and ige isotype counterparts but the difference in a constant region of antibodies, igy has 4 constant regions whereas igg has 3 constant regions [80] [81] [82] fig. 2 . igy present in chicken sera gets passed to the embryo through the egg yolk [83] . egg igy antibodies have been used previously against bacterial and viral infections [84, 85] . humanization of these antibodies can have great potential for biopharmaceutical development [86, 87] . the recent development of transgenic chicken with human immunoglobulin loci has expedited the use of transgenic chicken derived mabs directly for human therapeutic use [88, 89] . these engineered transgenic chickens express antibodies from immunoglobulin heavy and light chain loci containing human variable regions and exhibit normal b cell development raising immune responses to conserved human targets that are non-immunogenic in mice [90] . these transgenic chickens can be potentially used for the development of hybridoma secreting antibodies of human origin. however, like others, the unavailability of robust hybridoma fusion partner limits its potential utility [88] . in recent years different research groups have extensively explored the display method to overcome the limitations of chicken hybridoma. antibodies isolated through hybridoma methodologies have the advantage of being used directly and could be cryopreserved for future uses till an indefinite time, as the fusion partners are myelomas possessing remodelled transcriptional machinery to secrete a continuously large amount of antibodies [91] . advancement in recombinant technology has overcome the challenges by cloning of variable heavy (vh) and variable light (vl) from unstable hybridoma; cloning in transient transfection vectors to produce antibodies in mammalian expression system [92] . currently the development of stable cell lines has advanced the antibody production system by developing stable cell lines from unstable hybridomas to produce consistent antibody production. chinese hamster ovary cell line (cho) is the most preferred cells used for large scale production of mabs. however, the development of stable cell lines is a tedious process that sometimes takes a month to year time where the success of this process depends on random genome integration of transgenes [93] . development of crispr-cas9 in recent years has overcome by immunogenomics reprogramming using plug and play technology, where crispr-cas9 was used to engineer immunogenomics by homology-directed repair to replace endogenous immunoglobulin region by exogenous donor counterpart with the help of guide rna. this technology platform has enabled the rapid generation of full-length antibody-secreting cell lines [94] . a major limitation of hybridoma technology is the lack of suitable fusion partners which limits the use of this technology and limits its applicability to other species. to overcome the problem of suitable fusion partners, the transgenic mice model h-2k b -tsa58 has been developed. h-2k b -tsa58 transgenic mice express the simian virus 40 (sv40) antigens (tag) under the control of mouse major histocompatibility complex h-2k b promotor. this promotor allows differential expression of sv40 antigen in different tissues at various levels. expression of this antigen can be increased by simply increasing the levels of interferons (ifns) [95] . the higher expression level of this antigen is responsible for tumorigenesis and aberrant development. in this approach transgenic mice h-2k b -tsa58 were specifically used to isolate monoclonal against the filamentous phage. transgenic mice were immunized with filamentous phage suspension. splenocytes were recovered from the immunized animals and spleen cells were limited diluted from 6, 24-, 96-well plates. cells from the positive wells were selected to develop monoclonal lines. the main advantage of this technology is that it eliminates the use of fusion partners and can parallelly be used for the development of monoclonal and polyclonal based therapies [96] . the other major challenge associated with hybridoma development is the requirement of purified antigen to generate a specific immune response. in some of the cases, it's a challenging task to purify the antigen. the lack of specific immune reagents for characterization and monitoring of these numerous proteins limits the overall time process for the production of hybridoma. expression and purification of recombinant protein are time-consuming and sometimes not cost-effective. additionally, immunization of animals with these purified recombinant proteins in formulation with adjuvants sometimes leads to alteration of native conformation of these proteins which finally leads to an undesired immune response in animals. in recent years, different approaches have been successfully implemented to overcome these challenges; a novel strategy has been developed to isolate mabs against the native proteins [55] . in this strategy, animals were directly immunized with transiently transfected hek cells that express desire protein on the surface of these cells in a proper folded and glycosylated form. this modified technology is mainly useful for the isolation of mabs against native antigens. the advantage of this technology is that expressed protein on transfected cells closely mimics as it expresses in naturally infected cells. a similar type of strategy was developed by hazen m et al; where they had attempted to isolate monoclonal against the native conformation of extracellular loops & multi transmembrane proteins using dna based immunization strategy in combination with immunomodulatory agents [97] . use of immunomodulators in dna immunization has positively enhanced the immune response and proved to be a successful approach to isolate desired specificity binding mabs to native conformations. kato et al., in 2019 developed a cell-based immunization and screening (cbis) method for isolation of mabs for podoplanin (pdpn) [98] . in this strategy, they immunized the mice with transfected cells overexpressing pdpn and screened the fused hybridoma cells using flow cytometry. these cell-based immunization methods are useful in the isolation of mabs against various membrane proteins which is still difficult to achieve otherwise [56, 57] . the other challenge in hybridoma technology is animal immunization. the overall efficiency of hybridoma depends upon the efficiency of immunization. some factors that affect the efficiency of immunization are; route of administration of antigen, dose, choice of adjuvant, number of boosts and immunization protocol. dna based immunizations are generally more preferred where it is difficult to express fulllength proteins and immune response are mainly targeted towards native or conformational epitopes because the structural integrity of protein is critical for induction of functional mabs. the major routes of dna based delivery are intramuscular, intravenous and intrasplenic. intrasplenic routes are considered to be the most efficient route. a single dose of dna delivery is sufficient to induce antibody responses. antibodies against different proteins can be produced at the same time by immunizing with several nucleic acids encoding for different proteins or single plasmid encoding different subunits of the protein. in such cases multiple booster doses are usually avoided, to reduce immune-dominance amongst antigens. another major challenge in the field of human hybridomas is the requirement of lymphocytes from actively infected patients or who have been exposed to antigen. if the active immune response is absent or not sufficient then the probability of circulating b cells in such cases is very poor or negligible. due to ethical issues and considerations, it is generally not possible to immunize humans. to overcome these limitations li et al., in 2006 have developed a novel and rapid combined ex-vivo immunization strategy with morphogenics platform process for the isolation of therapeutic human mabs. in this strategy, the group has purified b cells (cd19+) and cd4 positive t cells from healthy volunteer blood samples using magnetic bead-based sorting. these b/t cells were cultured in the presence of growth factor and antigen to activate b cells. the pool of these b and t cells were then fused with myeloma fusion partner using electric cyto plus. the fused cells were screened for antigen-specific antibody secreting properties by limited dilution method. the isolated mabs had shown high specificity, biological activity and high affinity. this method avoids the collection and screening of a large number of patient samples which are normally the basic requirement for the generation of therapeutic human hybridomas. it also avoids the risk of potential viral transmission associated with conventional methods where pbmcs are sometimes used from viral infected patients. screening of volunteers and blood cells can be performed before ex vivo immunization and after hybridoma development. this platform technology offers a rapid and cost-effective way for therapeutic human mabs having natural pairing of immunoglobulin genes [99] . after cell fusions between b cells and myeloma cells, protocols of hybridoma technology include multistep screening and cloning processes to identify antigen-specific hybridomas, which is labour-intensive and time-consuming. however, recent advances in robotic screening methods have alleviated this to some extent [88] . the screening process on semi-solid selective medium has made it easy and reduces the overall time in hybridoma production by repeated selection and cloning steps. this screening technology has been used by companies to sale ready to use kit based systems for the development of murine hybridoma. it has also facilitated the use of murine hybridoma technology in a less cumbersome and user friendly. methylcellulosebased semi-solid selective medium is preferentially used in hybridoma selection [56] . technically it avoids the loss of rare clones from an overgrowth of faster-growing cells, which can occur during selection in a liquid medium. the selected clones are further dispersed into a liquid growth medium for screening and expansion. similarly, paul et al recently developed microarray-based screening technology for direct identification of high-affinity clones which avoids the loss of slowgrowing clones. additionally, this approach eliminates the enrichment, isolation, and purification of igg for the characterization process. the crude culture supernatants can be directly used and thus avoids expensive and lengthy screening steps [100] . the screening process has advanced through flow cytometry-based methodology where single cells could be sorted from a bulk mixture of fused hybridoma cells. it has advanced by saving time and labour instead of the traditional multimicro well plate seeding and limiting dilution sub-cloning [101] . in recent years tedious hybridoma screening and cloning processes are replaced with flow cytometry-based sorting methods. these methods avoid effort and time of tedious repeated screening processes. this method could also differentiate igm and igg secreting hybridoma. facs based screening methodology can be applied in any laboratory easily setup as it doesn't require any special reagents [102] [103] [104] . the other alternative approach that some groups had used is the screening of hybridoma supernatant directly by bio-layer interferometry based on disassociation rates to select clones containing high-affinity antibodies for further expansion and subsequent characterization. the main drawback of the elisa based screening method is that clones that express high levels of a low-affinity antibody can give an equivalent signal to clones that express low levels of a high-affinity antibody. as a consequence, superior clones can be overshadowed by inferior clones because elisa method score antibodies based on the binding signal strength and do not provide accurate affinities or dissociation rate constants [105, 106] . current drug approval rates underline the revolutionary effect of fully human mab therapeutics on drug development. antibodies isolated for therapeutic applications from different species excluding human needs a multistep process of humanization and developability through rational sequence optimization [107] . mouse is the most common and preferred progenitor used in mab isolation. to avoid the multistep process of humanization, the concept of transgenic mice harbouring the human antibody repertoire has gain attention, where large human immunoglobulin loci are transferred into the mice germline using yeast artificial chromosome approach [108] . the xenomouse and humab mouse are the first engineered transgenic mice that carry the majority of human vh & vl antibody repertoire [109] . xenomouse transgenic technology was developed by cell-genesya a biotech company (now a part of bristol myers squibb, new york, usa). the first therapeutic antibody developed by this transgenic technology was approved by the fda in 2006 for the treatment of advanced colorectal cancer. the strength of transgenic technology can be evaluated by the recent data on approved mabs. more than 18 fully human mabs developed by transgenic animal-based technology are used for human therapy. a list of fda approved mabs developed by transgenic technology is listed in table 4 . initially, this technology was limited to mice but over the years this technology has been established for other animal models like rabbits, rats, and cows. the success of this technology table 4 a list of fda approved mabs derived from transgenic technology. treatment of colorectal cancer mainly depends on the proper representation of the target antigen to the immune system. in addition, designing a proper immunogen is very critical for success. to avoid the ambiguity of immunogen design in soluble form, genetic immunizations are more preferred over the traditional methods. in contrast to human, mouse possesses very less antibody diversity represents the main limiting step in xenomouse transgenic technology. human antibody repertoire shows diversity more than 10 11 , however, the number of b cells in a mouse is~10 8 only. a single mouse can harbour the only fraction of the antibody repertoire from human antibody [110, 111] . moreover, immunization of a large cohort of mice to increase the diverse response against antigens could be used to overcome the limited antibody diversity. the other transgenic mice technologies, where kymouse & trianni mouse models were developed to represent a more diverse human antibody repertoire that allows the selection of diverse human antibodies and overcomes the limitations of xenomouse [112] . fig. 4 represents an illustration of transgenic antibody technology showing the antibody production route. besides, the other limitation of this technology platform is immune tolerance when attempting to raise an immune response against human targets. a large number of human targets possesses a very high degree of sequence and structural homology, because of this homology the transgenic immune system recognizes these antigens as self-antigen. different groups have tried to overcome the immune tolerance mechanism by adding t cell epitopes to the antigen [89, 113] . the other similar approach tried by different groups to abolish the expression of murine orthologues gene [114] . but the major limitation with these defected mice models is that sometimes these mice suffer from health issues and some of these knockdown genes are necessary for the development of a foetus. recent developments of transgenic rat and fig. 4 . illustration of transgenic antibody technology shows the antibody production route: mouse immunoglobulin gene loci were functionally inactivated in embryonic stem (es) cells by targeted gene deletion used to generate mice homozygous for the necessary deletions. crossbreeding between the transgenic mice (containing both human and mouse antibodies) with mice incapable of producing mouse immunoglobin, resulting in the xenomouse strain which expresses human antibodies but not the mouse antibodies. b cells, isolated from immunized xenomouse, are fused with myeloma cells to produce hybridomas producing human mabs. chicken (omnichicken) models have partially overcome these limitations. the transgenic model-based technology harbours some advantages over the phage display derived antibodies. the antibody developed by transgenic technology requires less development or optimization and thus require a shorter time to reach the product development stage. production of stereo-specific mabs is still a very big challenge. there are hardly any practical technologies available for the generation of stereospecific antibodies, because of hurdles like how to immunize a mouse maintaining the antigen structure intact in the presence of adjuvant. in addition, adjuvants allow more effective sensitization, disrupting the native structure of proteins. however, if an adjuvant is not used, immunization efficiency could be very low. another difficulty is strict selection of stereospecific mab producing sensitized b lymphocytes. although, if the immunization is successful with a native intact antigen, the number of desired sensitized b lymphocytes is extremely small, accounting for very less population of total spleen cells after repeated immunization [72] . most of the established therapeutic mabs have specificity for the antigen targets having primary structures. generally mabs can recognize two types of antigen epitopes which includes linear in the primary structures of proteins and the conformational, dependent on secondary and tertiary structures [115] . stereo-specific mabs recognizing conformational structures of target antigens may thus offer a markedly more versatile approach. besides primary, secondary and tertiary structures, proteins may also exhibit quarterly structures. which are formed by hetero or homo-subunits, providing unique interfacial geometric structures on their complexes. native or conformation-specific mabs are quite reasonable and attractive for future therapeutic purpose. there is need to replace the conventional mabs with stereospecific mabs in the near future for therapeutic medicine as they recognize the 3 dimensional conformation, which intrinsically determine their fundamental biological functions [116] . stereospecific mabs that recognize 3d molecular configuration has advantages over linear epitope-specific mabs that consider only 2d configuration. in general, mabs against primary structures, in target therapeutic antigens with the dominant secondary and tertiary folding are having affinity only with full antigens in restricted areas, as their linear epitope may be masked due to conformational folding. alternatively, target antigens in the immune system can be identified by assuring indigenous structures via immunizing dna of the target antigens expressed on the surface of the cell, allowing healthy and intact structural conformation. this can be linked to membranes, which mimics membrane protein, with a soluble protein harbouring sufficient signal peptides and membrane-penetrating areas that are genetically linked to the 5â�² and/or 3â�² terminals of genes of the desired soluble proteins as nucleic acid sequences to express as a fusion protein [117] . in the emerging diseases like human immunodeficiency viruses-1 (hiv-1), coronavirus -19 (sars-cov2), dengue, and chikungunya, the immunogenic proteins of pathogens harbour complex structural glycoproteins, needs an urgent high-quality stereospecific mabs to control their spread. development of therapeutic antibodies against these pathogenic diseases is aimed to the structural antigens. the complex native structures of envelope proteins on viral surface facilitate the attachment of virus to the host cell, and subsequently entry inside the cell [118] . these native structural proteins induce predominantly cross reactive neutralizing antibodies. the neutralizing antibodies have shown promising results in the protection against pathogens however nonneutralizing antibodies help the virus in evading immune system [119] . in hiv-1, researchers are working to develop broadly neutralizing antibodies targeting conformational epitopes [120] . similar approaches have been taken for other viral antigenic targets [121] . in many of these viral infections "conventional abs" are generated in response to virus infection, but the virus adopts numerous evasion strategies like conformational masking of antigenic targets by glycosylation, high mutation rate etc. [122] . generation of stereospecific mabs are required to display impressive breadth and potency against the conformational proteins. these stereospecific mab productions with promising therapeutic potential can be achieved by inclusion of critical steps like i. dna or soluble protein immunization with native like targets or structural proteins ii. selection of antigen specific myeloma cells and iii. selective fusion of myeloma and b cells to generate hybridoma cells secreting stereospecific mabs. different novel approaches and attempts are being taken to produce the soluble trimers which can display native-like conformational stable structure mimicking with virus surface. these conformational protein structures are promising targets for protein or dna immunization and could subsequently require for the production of stereospecific mabs. one major challenge associated with the use of native like antigens for the development of stereospecific antibodies is use of adjuvants in immunization process. most of the adjuvants usually disrupts the original protein native structure and hence impede its ability to present relevant epitopes or occludes the trimer conformational epitope [115] . in recent years' studies using iscom class of adjuvants in animal preceded by in vitro analyses showed that it has no adverse effect on native trimer conformation or antigenicity [123] . the other way to generate stereospecific antibodies is the direct immunization of mammalian cells expressing cell surface antigens in its native conformation [124] . a number of stereospecific antibodies has been generated against several targets like receptors [125] , ligands [126] , antagonist and chemical compounds [127] . a detailed schematic representation of different approaches used for isolation of stereospecific mab is shown in fig. 5 . the production of stereo-specific mabs could be achieved by tweaking the conventional hybridoma fusion through stereo-specific targeting (sst) technique invented for the first time by tsumoto et al [115] . crucially, it involves a strict selection of the required sensitized b lymphocytes by intact antigens, expressed on myeloma cells, through b-cell receptors (bcrs) and their selective electrofusion (only attached cells can be fused among themselves) to generate a specific hybridoma [128] . sst technology offers selective production, against different protein types, of monoclonal stereospecific antibodies not only for membranous but also for soluble non-membranous antigens. morshed et al recently showed efficiency in activation of the g-protein coupled receptor which holds seven-transmembrane domains by a stereo-specific mab [117] . furthermore, the new promising class of therapeutic mabs, catalytic antibodies are capable of identifying and degrading antigens, has fundamentally demonstrated. hifumi et al. have developed a catalytic antibody to degrade the active site for urease o helicobacter pylori and eliminates the bacterial infection in the mouse [129] . moreover, the catalytic antibodies have proven their utility in suppressing infection of the rabies virus [130] and the influenza virus [131] in vitro and in vivo using human antibody light chains. in addition, they have recently been noted in their ability to effectively reduce the accumulated ã�-amyloid in the mice's brain [132, 133] . medi9447 is a mab that inhibits cd73 (ecto-5â�²nucleotidase) activity on a non-competitive basis and is considered a promising immuno-oncology target [134] . this mab is antagonistic to cd73 employing dual inter-cd73 dimer cross-linking and/or steric blockage mechanisms that prevent the adoption of the cd73 catalytic active conformation [135] . bispecific mabs with stereo-detection may be especially effective for cancer cell detection of membranous antigens which have not been easily detected by conventional linear mabs. in a nutshell, as proteins retain their native conformation in nature, development of stereospecific, alternate forms of bispecific and catalytic mabs, for selective therapy is the founding factor in therapeutic future drugs. the mab has come a long way since the days when unmodified murine mab was explored against the cancer-causing agents. from the last two decades, mabs have been a standard element of cancer therapy, however, with still much room for further improvement in future [64] . the mabs are always preferred over the chemical compound based therapies because of their high specific reactivity and affinity towards the target antigen recognition. they show minimal side effects with favourable pharmacotoxicity and pharmacokinetics properties. the high specificity of mabs towards their targets presents an attractive and successful option for the development of medical treatment and molecular drug targets. [136] . early clinical attempts exploring mab-based therapeutics were very primitive and disappointing 20 years ago, some clinical experts considered the antibody-based therapy treatment for cancer as a failed hypothesis [137] . the first mab which was clinically evaluated against cancer was the murine mab. although there were some fascinating hints that mab therapy could be successful, however, the problems related to the administration of murine mab to humans limited their clinical utility and applications [138] . rise in immune response against the therapeutic mab, very rapid clearance of the mab from the system and suboptimal ability of the murine mab to interact with the human immune system in a manner that led to immune destruction were the challenging tasks. however, some investigators tried continuously to explore the use of mab as a possible cancer treatment. they also evaluated other strategies such as using; igg to target cancer directly, alter the host immune response to cancer, provide cytotoxic substances to cancer, and retarget the cellular immune response towards cancer [64] . over the last twenty years, the effectiveness of antibodies in the treatment of patients with cancer and other deadly diseases has been increasingly recognized, as mentioned in tables 1 and 4. many of these antibodies are specific for antigens expressed by the disease-causing agents itself [139] . in the case of viral targets, mab-based therapeutics have shown limited success. however neutralizing antibodies play an essential part in antiviral immunity and human protection against viral diseases is primarily mediated by the humoral immune response [120, 122] . it is well documented that early administration of mabs in the treatment regimen reduces mortality rate significantly up to 95% [140] . to date, only two mab is licensed for viral infection i.e. respiratory syncytial virus (rsv) and the other one is ibalizumab, which has been recently approved in 2018 for the treatment of hiv positive people with multidrug resistance towards anti-retroviral therapy (art) [18, 141] . several other candidates are at the different stages of clinical trials e.g. leronlimab, an anti-ccr5 igg4 for hiv infection and regn-eb3: a mixture of 3 igg1 mabs for ebola virus infection (https://www. antibodysociety.org/antibodies-to-watch-in-2020-at-pegs-europe/). lack of vaccines against various deadly viral diseases necessitates the development of antibody therapeutics to save the loss of lives and control deadly diseases [142] . however, it is always easy to generate monoclonal to protect the population at the time of the outbreak in a much shorter time as compared to vaccine production. though vaccines are one of the most cost-effective ways to manage infections, vaccines also require time to elicit protective immunity and depend on the host's ability to mount an immune response. a number of a prophylactic vaccine against pathogens such as; herpes simplex virus type-1 (hsv-1) and human immunodeficiency type-1 (hiv-1) have shown protection in animal immunization studies, but, so far, no effective human vaccine against these diseases are available [58, 59] . antigenic drift and high diversity among the emerging pathogens; such as influenza virus and hiv have been reported [60, 61] which further add to the complexity and may lead to vaccine mismatch drop in vaccine effectiveness against circulating serotypes and strains. in developing countries, it is not economically feasible to make a vaccine of every disease because of a lack of awareness of disease burden [62] . the mabs are widely used in the fields of diagnostic, therapeutic and biological applications due to their high specificity and affinity. at present, the majority of mabs approved for therapeutics are humanized or the chimeric versions of mouse mabs and were generated using hybridoma technology. in recent years, these engineered humanize and chimeric antibodies are potentially used to generate different forms of antibody fragments such as scfvs [143, 144] , diabodies [145] , tandom abs [146] , and domain antibodies [147] , pegylated fabs [148] to target novel antigenic sites. technical advancement in the applicability of hybridoma technology to other animal species (other than mice) of different phytogenic origin has led to the development of novel mabs to conserve human antigens. it has opened a new path for therapeutic and diagnostic mabs with high specificity and affinity to poorly immunogenic targets. with the recent development of high throughput mab generation technologies, hybridoma technology is the most favoured method due to its indigenous nature to preserve natural cognate pairing information of antibodies that is lost in other methodologies, reduces the specific diversity of antibodies [149] . advancement in recombinant dna technology methods like chimerization and humanization has increased the potential of hybridoma technology to a great extent. an antibody that undergoes the process of humanization preserves the natural specificity and limits risk of cdrs causing an immune response. the unnatural pairing of antibodies in terms of affinity maturation and recombination pairing in display methods sometimes results in high immunogenic response [19] . all these features have make hybridoma platform as first and most preferred mab isolation technology. recent advances in development of hybridoma cells secreting stereo-specific mabs have opened new avenues of future therapeutics. in comparison with other anti-viral drug treatments, a stereospecific antibody-based therapy could offer potent anti-viral actions by more comprehensive target and potent neutralizing effect. the mab market has shown tremendous increase in the last five years as a diagnostic and therapeutic reagent. the commercial development of therapeutic mabs commenced in the early 1980s, and by 1986 the first therapeutic mab was fda approved for the prevention of kidney transplant rejection. over the years mab market has changed rapidly as a major class of therapeutic agents for the treatment of many human diseases, in terms of global sales revenue for all mab products was~$115.2 billion in 2018. a graphical representation of the global antibody-based therapeutic market trend is represented in figs. 6 and 7 . the global mab therapeutics market is expected to grow at a compound annual growth rate (cagr) of 12.80% and is expected to reach market revenue of around usd 218.97 billion by the end of 2023 [150] . humanized mab accounts for the largest revenue-generating share among antibody-based therapeutics, showing to its widespread acceptance for numerous diseases including cancer, autoimmune diseases, inflammatory diseases, infectious diseases, haematological diseases, and others. the other potential area where mab has shown great success is diagnostics. the mab -based diagnostic reagents potentially identify abnormal cell targets, infectious agents, or elements 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antigen-binding proteins protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain fv analogue produced in escherichia coli diabodies: small bispecific antibody fragments bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics binding activities of a repertoire of single immunoglobulin variable domains secreted from escherichia coli efficacy of a novel pegylated humanized anti-tnf fragment (cdp870) in patients with rheumatoid arthritis: a phase ii doubleblinded, randomized, dose-escalating trial veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas storyid=1096715878&title= global-monoclonal-antibody-therapeutics-market-to-be-worth-usd-21897-billion-by-2023: global monoclonal antibody therapeutics market to be worth usd 218.97 billion by 2023 supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2020.106639. key: cord-320005-i30t7cvr authors: pardo, a. title: the human genome and advances in medicine: limits and future prospects date: 2004-03-31 journal: archivos de bronconeumología ((english edition)) doi: 10.1016/s1579-2129(06)70078-7 sha: doc_id: 320005 cord_uid: i30t7cvr nan on april 14, 2003 , the international human genome sequencing consortium announced the successful completion of its task. the correct sequence of the bases cytosine (c), thymine (t), adenine (a), and guanine (g) in the gene-containing regions of dna had been elucidated with an accuracy of 99.99% for 99% of the euchromatin. this is considered to be the most that can be achieved with current technology, and all that now remains is to sequence the remaining regions, which are more difficult because they include almost 400 highly repetitive dna fragments in addition to the centromeres, the structures that divide chromosomes. the consortium of which the human genome project (hgp) formed a part included 20 centers in 6 countries (china, france, germany, great britain, japan, and the united states of america). this international group chose to announce the completion of the task in april 2003 in order to coincide with the 50th anniversary of the publication, in april 1953, of the paper by watson and crick 1 that first described dna's double helix structure. the hgp's initial objectives were fulfilled 2 years ahead of schedule, and, in addition to compiling a highly accurate sequence of the human genome which has been made freely available and accessible to everyone, the consortium has developed a set of new technologies and has constructed genetic maps of the genomes of various organisms. moreover, this program of scientific investigation is linked to a parallel bioethics program. it is also interesting to note that, thanks to advances in technology, this result was achieved for a cost lower than the initial budget, which estimated that 500 mb would be sequenced annually at a cost of 0.25 dollars per finished base. the final figure was 1400 mb per year at a cost of 0.09 dollars per base. the size and scope of the hgp has also provided valuable lessons about the organization and management of large projects involving international collaboration, and those lessons will no doubt prove useful in the administration of other large scale projects. what was the genesis of this project? what general lessons has it taught us so far? how will it influence medicine? what future prospects, hopes, and fears has it given rise to? what ethical problems does it pose? these are just some of the general questions that this article will attempt to analyze. the genome is the total set of genes carried by an organism, and each gene is a segment of dna's double helix structure containing the recipe for making a polypeptide chain in a protein. a protein may contain a single polypeptide chain, as in the case of insulin, and therefore a single gene will code for this protein, or it may contain more than one chain, as in the case of hemoglobin, so that this protein is encoded by more than one gene. there are around 100 billion (us 100 trillion) cells in the human organism, and each one of these contains a complete genome. this genome is found on the 23 pairs of chromosomes in the cell nucleus. around 1.8 meters of dna containing approximately 3000 million (3 billion us) base pairs is packed into the nucleus of each cell. the genetic code uses groups of 3 dna bases to specify the amino acids that make up the polypeptide chains of proteins, the principal actors in life's drama. one of the first genomes to be completely sequenced was that of simian virus 40 (sv40), which contains 5226 nucleotides. 2 by the beginning of the 1980s, viral genomes containing over 100 000 bases had been sequenced, making it possible for scientists to envisage the possibility of sequencing bacterial genomes containing over 1 000 000 bases. when the idea of sequencing the human genome was first proposed during the mid-1980s, the undertaking seemed hardly feasible using the technology available at that time. however, after various preparatory meetings, the national institutes of health and the department of energy of the usa officially announced on october 1, 1990 the launch of a program to sequence the human genome, and james watson (of watson appointed director of the recently created national center for human genome research. around the same time, the public consortium known as the human genome project was formed, and this organization announced a 15-year plan (from 1990 to 2005) with the following objectives: a) to determine the complete nucleotide sequence of human dna and identify all the genes in human dna (estimated to number between 50 000 and 100 000); b) to build physical and genetic maps; c) to analyze the genomes of selected organisms used in research as model systems (eg, the mouse); d) to develop new technologies; and e) to analyze and debate the ethical and legal implications for individuals and for society as a whole. one of the difficulties that had to be overcome in the task of accurately sequencing the bases that make up the human genome was that approximately 50% of dna is highly repetitive. the strategy adopted by the hgp was to sequence the dna whose location on the chromosomes was already known. 3 however, this strategy was challenged in 1998 by j. craig venter and his team, who had just set up a private company called celera genomics. taking advantage of recent advances in technology, this team proposed an alternative strategy based on cutting the genome into small segments and using a computer to reassemble the sequences by matching the overlapping ends of each fragment. with these innovations, this private consortium announced that they would sequence the human genome in 3 years, in other words, that they would complete the task by 2001. this undoubtedly brought immense pressure to bear on the public group, the hgp, headed since 1992 by francis s. collins, and also gave rise to fears that a private company might control a large part of the human genome through patents. after several unsuccessful attempts to get the private and public sector groups to collaborate, an agreement was reached to simultaneously publish a first draft of the human genome in february, 2001. this draft did not, however, have the degree of precision of the current one. consequently, the hgp consortium published its results in nature 4 in february 2001, and celera did likewise in science. 5 the sequences were subsequently corroborated with a greater degree of reliability, and in april 2003, with the sequence practically complete, the hgp consortium declared the task to be completed. 6, 7 discoveries and surprises one of the surprising facts thrown up by the sequencing of the human genome was that it only contains approximately 30000 genes. owing to its size, it had been estimated that the genome would contain between 50 000 and 100 000 genes. in simple organisms, such as yeasts, the number of genes directly correlates with the size of the genome because most of the information in the genome clearly codes for proteins, and the individual genes have a well-defined beginning and a clear stop point and exit for the messenger rna. it had seemed logical, therefore, that the greater the complexity of the organism, the larger would be the number of genes. however, the sequencing of the genomes of other organisms has yielded unexpected results. for example, the common fruit fly, drosophila melanogaster, has approximately 13 500 genes, fewer than other simpler organisms, such as the earth worm, caenorhabditis elegans, with 18 500 genes, and the mustard plant, arabidopsis thaliana, with around 28000. 8-10 therefore, the human genome only has around 2000 more genes than arabidopsis despite its obviously greater biological complexity. so we have learned that the human genome has fewer genes than expected and also that that the distance separating them is considerable. it has been calculated that the gene density in the human genome is around 12 per 1000000 bases, while in drosophila this figure is 117, and in arabidopsis, 221. it is important to understand that the genes in human dna, as in most eukaryotes, are highly fragmented; in other words, not all of the bases from the beginning to the end of the gene are read to make a protein. the dna in the genes has coding regions, called exons, interrupted by long noncoding sequences, called introns (intergenic regions). these noncoding regions are removed by the process of splicing in the formation of messenger rna, so that the resulting messenger rna is much shorter than the original dna from which it was produced. for example, it has been reported that around 54% to 59% of genes in human chromosomes 14 and 22 undergo alternative splicing-the exons combine in different ways and produce various different proteins. 4, 5 this means that the number and variety of proteins in an organism does not depend solely on the number of genes in the genome, but rather on the way these genes are used. another important question thrown up by the results of the hgp was the following: if only 1% to 2% of the bases in the human genome code for proteins, then what do the rest do? an equivalent part of the noncoding portion of the genome probably contains most of the sequences that regulate the expression of genes, such as the promoters, regions that occur before the beginning of the gene. there are many other elements in the genome that affect the behavior of other components, such as the centromeres and telomeres. finally, a large part of the genome is made up of highly repetitive dna sequences, the function of which is little understood. why are there so many repetitive sequences in the human genome not found in the genomes of invertebrates? many dna sequences seem to have originated as a result of the movement of genetic elements called transposons, segments of dna that can move from one site to another within the genome. it has been postulated that many of the changes that have occurred during the evolution of vertebrates may have been triggered by the action of transposons which jumped to regulating regions and modified the expression pattern of the genes. genome sequencing is a tool that allows us to reconstruct the history of hundreds of millions of years of evolution marked by mutation, that is, the process of exchange and rearrangement of the sequences that has contributed to the formation of new species or has given rise to new genes. the task of solving these puzzles and fitting each piece into its place still presents a huge challenge because clues to our history still lie undiscovered in the noncoding sequences found in each chromosome, the sequences previously considered to be "junk dna." for example, the complete sequencing of the sex-determining y chromosome has revealed some very intriguing facts that have aroused great interest among geneticists and biologists who study evolution. these will be described in general terms in the following section. 11 the 2 human sex chromosomes, x and y, both had their origin in the same ancestral autosome several hundred million years ago, but their sequences diverged through evolution. as a result, sequences identical to those of the x chromosome that permit recombination between the two chromosomes in those regions only exist today in the terminal regions of the y chromosome. however, over 95% of the modern y chromosome has specific regions with no equivalents on another chromosome that would enable recombination during sperm production, and this is a rare example of persistence in the absence of sexual recombination. these regions contain genes that specifically code for testicular proteins as well as highly repetitive sequences which-probably because they are not understoodwere previously considered to be nonfunctional "junk" dna. with the complete sequencing of these regions, it has been found that some of these sequences are palindromic (as in the phrase anita lava la tina); that is, they read the same from left to right as from right to left, on both strands of the double helix. this fact has led to the hypothesis that x-y recombination has been replaced by recombination between the arms of the y chromosome in the regions where the palindromic sequences are located. 12 in this context, the y chromosome reveals great powers of self-preservation, using evolutionary strategies to survive in the absence of recombination with another homologous chromosome. probably one of the greatest expectations generated by the sequencing of the human genome has been the hope that this knowledge might benefit humans through its medical applications. the understanding of the role played by genetic factors in human health and disease will make it possible for us to discover better ways to approach the prevention, diagnosis, and treatment of pathological processes. it is thought that the science of genomics will soon explain the mysteries of the hereditary factors associated with heart disease, cancer, diabetes, schizophrenia, and many other chronic degenerative processes. it is also hoped that it will give us a better understanding of the genetic factors that influence our susceptibility and/or response to various infectious diseases. genomics holds the promise of individualized medicine that can be tailored to each patient's genetic profile. one of the challenging aspects of any analysis of the influence of an individual's genes on the development of certain diseases is ascertaining whether a particular disease is caused by a single gene or the interaction between several genes. it is also essential to understand how the environment influences the expression of such interactions. there are relatively few known diseases that are associated with mutations in a single gene. they include sickle cell anemia and cystic fibrosis. in the case of the gene that causes cystic fibrosis, over 900 different mutations have been identified that affect the function of the protein it encodes. in normal cells, the protein produced by this gene acts as a channel that allows cells to release chloride and other ions. in people with cystic fibrosis, however, this gene has a mutated sequence, and the protein produced is defective so that the cells do not release chloride. the result is an improper salt balance. this gives rise to the production of an abnormally thick mucus which, among other things, obstructs the airways and leads to infections. 13 however, the origin of most human diseases and of the variations in individual responses to drugs is more complex and involves the interrelation between multiple genetic factors, such as genes and the proteins they produce, and nongenetic factors, such as the influence of the environment. although all individuals share dna sequences that are 99.9% the same, each person has a unique genome. the remaining 0.1% is responsible for the genetic diversity between individuals. many differences are due to a variation in a single base pair in a gene. single nucleotide polymorphisms (snps) are variations of a gene that occur because of a change in a single letter (nucleotide) in the dna sequence, for example, the substitution of "cta" for "cca." snps contribute to the differences between individuals. while most of these polymorphisms have no effect, others cause slight differences in certain characteristics that do not affect health, such as physical appearance. others, however, may increase or decrease the individual' s risk of developing certain diseases. this happens, for example, in the case of acquired immune deficiency syndrome (aids). we now know that not all individuals exposed to the type 1 human immunodeficiency virus (hiv) become infected, and that the progression period from infection to aids is highly variable among infected individuals. some patients may develop the disease in 3 years, while others remain asymptomatic for more than 15 years. although the reasons for these differences are not entirely understood, it has recently been discovered that genetic factors play a very important role in the transmission of the virus and progression to disease. there must be 2 co-receptors on the surface of a cell in order for the virus to attach itself effectively and later infect the host cell. the first of these is cd4, the key receptor for t lymphocyte facilitators, and the second is one of the members of the chemokine family of receptors. c-c chemokine receptor 5 (ccr5) is one of the main co-receptors used by the virus to penetrate macrophages and t lymphocytes, so that it plays a critical role in the pathogenic process of aids. several studies have demonstrated that the polymorphic allele ccr5-delta32 (which contains a 32 base pair deletion) has a powerful protective effect in the progression of the hiv infection. 14 similar findings will probably emerge in relation to other diseases, so that in the future we will understand such enigmas as why not all smokers develop chronic obstructive pulmonary disease or lung cancer, or why not everyone who is exposed to avian antigens develops hypersensitivity pneumonitis. scientists have started to compile a catalogue of the common variations in the human population, which includes snps, small deletions and insertions in the coding dna, and other structural differences. part of this database is already available to the public. 15 another important point is that sets of nearby snps on the same chromosome are inherited in blocks. these patterns of snps on a block are known as haplotypes, and certain snps can be used as tags to identify the haplotypes in a block. the elucidation of the complete human genome has given rise to a new project the aim of which is to develop a haplotype map of the human genome called the hapmap. 16 the hapmap locates blocks of haplotypes, and the specific snps that identify them are called snp tags. the international hapmap project was started in 2002 and will be of fundamental importance in examining the genome in relation to phenotypes. it will also be a tool that will enable researchers to identify the genes and genetic variations that affect health and illness. in addition to its use in analyzing the relationship between genes and disease, the hapmap will be a powerful resource for studying the genetic factors that contribute to individual variations in our response to environmental factors, susceptibility to infection, adverse reactions, and response to drugs and vaccines. using only the snp tags, researchers will be able to identify regions on the chromosomes with different distributions of haplotypes in two groups of people, for example, those who suffer from a disease and those who do not. this will also facilitate the development of tests that can predict which medicines and vaccines might be more effective in individuals with particular genotypes for the genes that affect the metabolism of these drugs. the complete sequencing of the genome of an organism is only the first step in the quest to understand its biology. it is still necessary to identify all the genes and ascertain the function of the products expressed by these genes, that is, functional rna and proteins. functional genomics is based on the key premise of the central dogma of molecular genetics, which states that dna sequences are used as templates for the synthesis of rna, and this rna is subsequently used as a template for the synthesis of proteins. 17 moreover, scientists still have to analyze and understand the noncoding regulatory regions and other functional elements of the human genome and of the genomes of other organisms. this has led to the creation of a project called the encyclopedia of dna elements-or encode. the goals of this new project are to identify and map the exact location of all the protein-encoding and non-protein-encoding genes, and to identify other functional elements encoded in the dna sequences, such as promoters and other transcriptional regulatory sequences, as well as determinants of chromosome structure and function, such as origins of replication. the aim is to provide a comprehensive encyclopedia of all these elements in order to help researchers better understand human biology and predict potential disease risks, and to stimulate the development of new therapies for the prevention and treatment of disease. it has been said that the basis for understanding the genome of a mammal is the characterization of the part that is transcribed (ie, the transcriptome) and the identification of the proteins it produces (ie, the proteome). many technologies have been developed to study functional genomics, and foremost among these are the cdna microarrays or dna chips, which have been widely used to explore the expression profiles of thousands of genes simultaneously. 18, 19 this technology has been used to gain a greater understanding of the molecular mechanisms of various diseases, such as, for example, pulmonary fibrosis. idiopathic pulmonary fibrosis belongs to the category of idiopathic interstitial pneumonias and is characterized by the relatively rapid destruction of the lung parenchyma. as a result, some 50% of patients die within 3 years. 20 in a recent study, lung biopsy samples from patients with idiopathic pulmonary fibrosis and other patients with normal lungs were analyzed using this technique of oligonucleotide microarrays. 21 the results showed that gene expression patterns clearly distinguished normal from fibrotic lungs, and that many of the genes that were significantly increased in fibrotic lungs encoded proteins associated with the extracellular matrix and enzymes responsible for its replacement. this study, and others that have investigated various pathological processes, 22 illustrates the analytical power of gene expression in the identification of the molecular pathways involved in disease. the identification of the different groups of genes involved in the pathogenic processes of human disease will also facilitate the discovery of new molecular targets that can eventually be used in the treatment of such diseases. for example, we have recently found in hypersensitivity pneumonitis, an inflammatory lung disease characterized by lymphocytic alveolitis, the exaggerated expression of a chemokine derived from dendritic cells known as ccl18. this chemokine is a powerful attractor of t lymphocytes and, at least theoretically, blocking it for therapeutic reasons could reduce the lymphocyte infiltration that characterizes this disease. 23 other new genomic technologies include: a) toxicogenomics, which studies the genetic basis of an individual's response to environmental factors, such as drugs and contaminants; and b) pharmacogenomics, which deals with the development of drugs designed for specific pathogenic processes that will target specific metabolic pathways. in general terms, the genomic sciences have been defined as those which study genes, their products, and their interactions. one of the earliest objectives of the hgp was to set up a program, called elsi, to analyze the ethical, legal and social implications of genomic sciences. in this context, unesco created the international bioethics committee, and in 1997 published a declaration that states, "recognizing that research on the human genome and the resulting applications open up vast prospects for progress in improving the health of individuals and of humankind as a whole, but emphasizing that such research should fully respect human dignity, freedom and human rights, as well as the prohibition of all forms of discrimination based on genetic characteristics, proclaims the principles that follow and adopts the present declaration." the articles of this declaration 24 deal with the following topics: a) human dignity and the human genome; b) rights of individuals; c) research on the human genome; d) conditions for the exercise of scientific activity; e) solidarity and international cooperation, and f) the promotion of the principles set out in the declaration. article 1 of this universal declaration on the human genome and human rights states: "the human genome underlies the fundamental unity of all members of the human family, as well as the recognition of their inherent dignity and diversity. in a symbolic sense, it is the heritage of humanity." the medical application of the information generated by genetics must be consistent with the general principals of medical ethics: a) beneficence, or acting for the good of individuals and their families; b) doing no harm; c) respecting the autonomy of the individual, that is, allowing individuals to make independent decisions after providing them with information; and d) individual and social justice. genetic information is confidential, and it is the responsibility of institutions and authorities not to interfere without prior consent. however, there are certain circumstances that could justify the intervention of the state, such as those related to public health issues, or the well-founded request of an authority in connection with a judicial investigation. how can we define the limits between what is permitted and what is prohibited, or between privacy and responsibility towards third parties? these are the kind of topics that must be discussed and analyzed by the ethics committees in each country, which should then inform their respective legislators on these issues. other aspects that need to be reported and considered include: privacy and justice in the use of genetic interpretation, nondiscrimination, and the need to distinguish between information that we individually prefer not to know and facts that must be revealed for family or social reasons. closely related to these ethical considerations is the problem of the privatization of knowledge and the granting of patents. for example, the last nucleotide in the genetic code of the coronavirus responsible for severe acute respiratory syndrome had hardly been read when the race had already begun to take control of the intellectual rights to the sequence. in private hands, a patent on a viral sequence could delay or increase the cost of developing a treatment or diagnostic tests for a particular disease. this question has caused concern among biomedical researchers, who are afraid that broad patents on genetic sequences will affect research work in universities and public institutions and will have a detrimental effect on future public health strategies. an example of this is the case of the predictive test for breast cancer, which uses the genes brca1 and brca2. the curie institute in paris has been struggling for the right to continue analyzing these genes at a third of the price currently charged by the genome company myriad genetics (utah, usa), which was granted a european patent for these genes in 2001. molecular biology has implicitly promised to transform medicine by elucidating the smallest details of the mechanisms of life. to the extent that the molecular processes of diseases are revealed, we will, in many cases, be able to prevent them or to design effective cures or individualized treatments. genetic tests will be able to predict an individual's susceptibility to a disease, and the diagnosis of many pathological processes will be much more detailed and specific than it is today. new drugs will be designed based on an understanding of the molecular mechanisms of common diseases, such as diabetes and systemic arterial hypertension, and it will be possible to treat these diseases by focusing on specific molecular targets. in the case of diseases such as cancer, for example, drugs can be adapted to the specific response of the patient and, within a few decades, it will be possible to cure many potential diseases at a molecular level before they develop. most probably these changes will not all occur in the immediate future. it will take us a long time to understand the human genome, the book of our species, with its 23 chapters called chromosomes, each containing thousands of stories known as genes, composed of paragraphs called exons, interrupted by as yet indecipherable messages called introns, written in words called codons, made up of letters called bases. no doubt, access to the exact sequence of the genome will gradually modify, with increasingly greater impact, the practice of medicine in the coming decades, and in this context it is essential that this knowledge and these technologies be immediately incorporated into public and professional education; this is a priority and the task must begin today. prometheus stole fire from the gods for the benefit of mankind; it is up to us to ensure that our new promethean knowledge be used to throw light on many of the mysteries of biology. molecular structure of nucleic acids the genome of simian virus 40 the human genome project: past, present, and future the international human genome sequencing consortium. initial sequencing and analysis of the human genome the sequence of the human genome a vision for the future of genomics research. a blueprint for the genomic era human genome sequencing available at the genome sequence of drosophila melanogaster genome sequence of the rematode c. elegans: a platform for investigating biology. the c. elegans sequencing consortium sequence and analysis of chromosome 5 of the plant arabidopsis thaliana the male-specific region of the human y chromosome is a mosaic of discrete sequence classes abundant gene conversion between arms of palindromes in human and ape y chromosomes identification of the cystic fibrosis gene: cloning and characterization of complementary dna international meta-analysis of hiv host genetics. effects of ccr5-delta32, ccr2-64i, and sdf-1 3'a alleles on hiv-1 disease progression: an international meta-analysis of individualpatient data national human genome research institute. crick f. central dogma of molecular biology medical applications of microarray technologies: a regulatory science perspective chip genético (adn array): el futuro ya está aquí clasificación actual de las neumonías intersticiales idiopáticas gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans uses of expression microarrays in studies of pulmonary fibrosis, asthma, acute lung injury, and emphysema ccl18/dc-ck-1/parc up-regulation in hypersensitivity pneumonitis universal declaration of the human genome and human rights key: cord-315164-nidgnvvi authors: medkour, hacène; amona, inestin; akiana, jean; davoust, bernard; bitam, idir; levasseur, anthony; tall, mamadou lamine; diatta, georges; sokhna, cheikh; hernandez-aguilar, raquel adriana; barciela, amanda; gorsane, slim; la scola, bernard; raoult, didier; fenollar, florence; mediannikov, oleg title: adenovirus infections in african humans and wild non-human primates: great diversity and cross-species transmission date: 2020-06-18 journal: viruses doi: 10.3390/v12060657 sha: doc_id: 315164 cord_uid: nidgnvvi non-human primates (nhps) are known hosts for adenoviruses (advs), so there is the possibility of the zoonotic or cross-species transmission of advs. as with humans, adv infections in animals can cause diseases that range from asymptomatic to fatal. the aim of this study was to investigate the occurrence and diversity of advs in: (i) fecal samples of apes and monkeys from different african countries (republic of congo, senegal, djibouti and algeria), (ii) stool of humans living near gorillas in the republic of congo, in order to explore the potential zoonotic risks. samples were screened by real-time and standard pcrs, followed by the sequencing of the partial dna polymerase gene in order to identify the adv species. the prevalence was 3.3 folds higher in nhps than in humans. more than 1/3 (35.8%) of the nhps and 1/10 (10.5%) of the humans excreted advs in their feces. the positive rate was high in great apes (46%), with a maximum of 54.2% in chimpanzees (pan troglodytes) and 35.9% in gorillas (gorilla gorilla), followed by monkeys (25.6%), with 27.5% in barbary macaques (macaca sylvanus) and 23.1% in baboons (seven papio papio and six papio hamadryas). no green monkeys (chlorocebus sabaeus) were found to be positive for advs. the advs detected in nhps were members of human mastadenovirus e (hadv-e), hadv-c or hadv-b, and those in the humans belonged to hadv-c or hadv-d. hadv-c members were detected in both gorillas and humans, with evidence of zoonotic transmission since phylogenetic analysis revealed that gorilla advs belonging to hadv-c were genetically identical to strains detected in humans who had been living around gorillas, and, inversely, a hadv-c member hadv type was detected in gorillas. this confirms the gorilla-to-human transmission of adenovirus. which has been reported previously. in addition, hadv-e members, the most often detected here, are widely distributed among nhp species regardless of their origin, i.e., hadv-e members seem to lack host specificity. virus isolation was successful from a human sample and the strain of the mbo024 genome, of 35 kb, that was identified as belonging to hadv-d, exhibited close identity to hadv-d members for all genes. this study provides information on the advs that infect african nhps and the human populations living nearby, with an evident zoonotic transmission. it is likely that advs crossed the species barrier between different nhp species (especially hadv-e members), between nhps and humans (especially hadv-c), but also between humans, nhps and other animal species. adenoviruses (advs), members of the adenoviridae family, are dna viruses that naturally infect many vertebrates, including humans and non-human primates (nhps). their name derives from their initial isolation from human adenoids in 1953 [1] . since then, human adenoviruses (hadvs) have been increasingly recognized as major contributors to clinical illness, from mild respiratory infections in young children (known as the common cold) to life-threatening multi-organic diseases in people with weakened immune systems. it is estimated that more than 90% of the human population is seropositive for at least one serotype of advs [2, 3] . illnesses include upper and lower respiratory tract diseases, conjunctivitis, acute hemorrhagic cystitis, meningoencephalitis, diarrhea, intussusceptions, celiac disease, hepatitis, myocarditis and obesity, with certain clinical diseases associated with specific adenoviral species and genotypes [3] . despite their role as pathogens, some hadvs and simian adenoviruses (sadvs) have been used or proposed as tools for vaccine delivery and gene therapy [4] . until now, within the genus mastadenovirus (including all human adenoviruses), 103 human adv genotypes have been assigned in high resolution using genomics (http://hadvwg.gmu.edu/). they have been divided into seven distinct species (human mastadenovirus a to human mastadenovirus g, informally hadv-a to hadv-g) that were originally distinguished by their biological, clinical and restriction enzyme digestion properties and were reconfirmed using omics data. advs within the genus mastadenovirus could infect other mammalian hosts, such as nhps, bats, bovines, canines, deer, dolphins, equines, murines, ovines, swine, sea lions, skunks, squirrels and tree shrews [3] . novel human and animal advs continue to be identified and characterized [5, 6] . in addition, there are advs in four other genera within the adenoviridae family that infect animals. these are atadenovirus, type species: ovine atadenovirus d; aviadenovirus, type species: fowl aviadenovirus a; ichtadenovirus, type species: sturgeon ichtadenovirus a; and siadenovirus, type species: frog siadenovirus a (https://talk.ictvonline.org/taxonomy/). as with humans, adv infections in animals can cause diseases that range from asymptomatic to fatal [3] . for millennia, interactions between humans and their closest living relatives have led to an inherent risk of pathogen transfer. nhps are increasingly implicated as potential sources of emerging zoonotic diseases in humans [7, 8] . indigenous groups that depend on wildlife for survival were exposed to the risk of the transmission of nhp pathogens through hunting, consumption of bushmeat [9] and through other ways; for example, by sharing non-flowing water sources, fruits and plant sources that nhps have used. concerning adenoviruses, virologists have long wondered about the possibility that nhp adenoviruses may one day pose a risk to humans [10] . although adenoviruses are generally considered to be rather host species-specific viruses, canine adenovirus 1 has a wider host range and can infect members of the canidae, mustelidae and ursidae families [11] . the host range and zoonotic potential of simian adenoviruses has more recently become an area of interest [10, 12] . simian adenoviruses (sadvs) have been described in macaques (sadv-1 to sadv-15), african green monkeys (sadv-16 to sadv-18 and sadv-20), baboons (sadv-19) and chimpanzees (sadv-21 to sadv-25) [13] . many more simian advs have been described, like new world monkey and prosimian advs [14] . in 2009, roy s et al. isolated and characterized 30 novel great ape advs from the feces of chimpanzees, bonobos and gorillas, as well as three macaque advs. all of them were captive nhps, held in facilities and zoos in north america. virus isolation was performed, and complete genomes were sequenced and tentatively named sadv-25.2 to sadv-50 [13] . most sadvs have proven to be very similar to hadvs. currently, the naming of a species follows the principle that if an adenovirus species with primate hosts contains at least one type of hadv, the species is called a hadv species, otherwise it is called a sadv species. as a result, most sadvs have been grouped correspondingly into the hadv-b, c, e and g species [13, [15] [16] [17] [18] [19] [20] [21] . in the present study, we sought to investigate the presence and molecular diversity of advs in wild african nhps, including great apes (gorillas and chimpanzees), macaques and other monkeys (baboons, green monkeys), living in close proximity to or outside human settlements. in addition, we assessed the adv infections of a local human population from the republic of congo, who were living near gorillas, in order to evaluate potential zoonotic transmissions. fecal samples were collected and tested by pcr/sequencing, and the adenoviruses were typed based on their dna polymerase partial gene. a human strain was isolated by viral cell culture and described. in senegal, in august 2016, 48 fecal samples of western chimpanzees (pan troglodytes verus) were collected from three sites located within the dindefelo community natural reserve in the kédougou region. site 1: three degraded stool samples (12 • 16 .6291 • e) were collected. for nhps, fresh fecal samples were collected at sleeping sites, feeding sites and places where the primates had been observed. in addition, in 2017, human stool samples were collected after obtaining the verbal consent of all the participants because of their low level of literacy. a total of 38 samples were collected, including 35 from the local population of the village of mbomo, located inside the oknp, and 3 from eco-guards in the llr. all great ape samples were stored in absolute alcohol, and fresh samples and human samples were first stored at −20 • c before being sent from the republic of congo to france for analysis. in the republic of djibouti, 6 fecal samples of hamadryas baboons (papio hamadryas) were collected in 2017. these baboons lived outside the village of oueah, 38 km from the city of djibouti (11 • 29 56.1" n 42 • 51 14.8" e). this collection was carried out in partnership with the center for studies and research of djibouti. in algeria, fecal samples were collected from 69 barbary macaques (macaca sylvanus), with the authorization of the management of the chréa national park (cnp), including 30 samples collected from two sites, the stream of monkeys and the gorges of la chiffa in blida province, 50 km north of algiers (36 • 23 42.9" n 2 • 45 53.6" e), and 39 samples from cap carbon (36 • 46 31.6" n 5 • 06 11.2" e) in the suburbs of béjaïa, 250 km east of algiers. these primates were synanthropic and lived in close contact with the people who provided them with food. no experimentation was conducted on any of the nhps in our study as all fecal samples were collected from the ground. the sampling was non-invasive and did not disrupt any wild animal. all humans in our study were apparently healthy. in addition, the fact that the nhps' feces were not diarrheic may perhaps be some indication of them also being healthy. all collected samples were taken to the ihu méditérranée infection laboratory, 13005 marseille, france, for analysis. they were identified and stored at either −20 • c or −80 • c. viral dna extraction was performed using the qiagen virus mini kit ® v2.0 (qiagen, courtaboeuf, france) on biorobot ez1 (qiagen, courtaboeuf, france), according to the manufacturer's instructions. firstly, about 40 g of each stool was mixed with 360 µl of g2 buffer and 40 µl of proteinase k (qiagen, courtaboeuf, france). this was subjected to a mechanical lysis with tungsten beads (qiagen, courtaboeuf, france), using a fastprep-24tm 5g grinder, before being incubated overnight at 56 • c. then, viral dna was extracted from 190 µl of supernatant with 10 µl of internal control (enterobacteria phage t4). dna was eluted in 130 µl volume, then aliquoted in individual pcr tubes to an amount of 50 µl of pure extracted dna. then, other aliquots of 50 µl of dna were diluted to 1:10, and finally, one-third of 50 µl of dna diluted to 1: 100. dna tubes were stored at −20 • c until use. the dna extraction and dilutions were controlled by qpcr, targeting the phage t4 (table 1) . based on the results of the qpcr for extraction control, dna which had been diluted to 1/10th was chosen for adenovirus testing. first, all samples were screened using qpcr targeting the t4 phages [22] , and after that they were screened by qpcr targeting a conserved region of the hexon gene of all 51 hadv prototypes [23] . the qpcr amplifications were performed in a cfx96 real-time system (bio-rad laboratories, foster city, ca, usa). reactions were carried out in a final volume of 20 µl, containing 5 µl of dna template, 10 µl of master mix roche (eurogentec, seraing, belgium), 0.5 µl each primer per reaction at a concentration of 20 µm, 0.5 µl udg and 0.5 µl of each probe at a concentration of 5 µm. the taqman cycling conditions included two hold steps at 50 • c for 2 min, followed by 95 • c for 15 min and 40 cycles of two steps each (95 • c for 30 s and 60 • c for 30 s). the pcr systems used for the study are detailed in table 1 . each pcr plate contained 96 wells. dna from the cultured adenovirus was included as a positive control and master mixtures as negative controls in each test. all samples were tested by a sensitive and specific nested pcr (table 1) , using outer primers in the first reaction and inner primers in the second. the nested pcr was used for the amplification of the dna polymerase partial gene and the specific diagnostic for adenoviral shedding [13] . for gene amplification, pcr reactions were performed in a total volume of 50 µl, consisting of 25 µl of amplitaq gold master mix, 18 µl of ultra-purified water dnase-rnase free, 1 µl of each primer (20 µm of concentration) and 5 µl of the dna template. in the second amplification, we used only 1 µl of pcr1 product rather than 5 µl of dna and 22 µl of ultra-purified water rather than 18 µl. the thermal cycling conditions for both pcr amplifications were as follows: incubation step at 95 • c for 15 min, 40 cycles (only 30 cycles for the second pcr) of one minute at 95 • c, 30 s for the annealing at 54 • c, 1.5 min (30 s for the second pcr) of elongation time at 72 • c. finally, we included an extension step for five minutes at 72 • c. pcr amplification was performed in a peltier ptc-200 model thermal cycler (mj research inc., watertown, ma, usa). the results of amplification were visualized by electrophoresis on 2% agarose gel. samples that had ct < 35 in qpcr and a good band of nested pcr were selected for sequencing. amplicons were purified using nucleofast 96 pcr plates (macherey nagel eurl, hoerdt, france), as per the manufacturer's instructions, and sequenced using the big dye terminator cycle sequencing kit (perkin elmer applied biosystems, foster city, ca, usa) with an abi automated sequencer (applied biosystems). the obtained electropherograms were assembled and edited using chromaspro software (chromaspro 1.7, technelysium pty ltd., tewantin, australia) and compared with those available in the genbank database compiled by ncbi blast (https://blast.ncbi.nlm.nih. gov/blast.cgi). the sequences obtained were aligned and compared with each other and with those of the advs available in the genbank database. a maximum likelihood method was used to infer the phylogenetic analyses and tree reconstruction was performed using mega software version 7 (https://www.megasoftware.net/). bootstrap analyses were conducted using 1000 replicates. fecal samples from positive nhps and humans for the molecular detection of advs were subjected for virus isolation in cell cultures. the large particles were removed by low speed centrifugation, and the supernatant was filtered through 0.2 µm-pore sized syringe filters. a volume of 100 µl of each filtered sample was inoculated into hep2 and mrc5 cells which had been grown in a mem medium with 10% fetal bovine serum (fbs) for 14 days. cultures were observed every 2 days to detect the appearance of a cytopathogenic effect (cpe). qpcr targeting adenovirus was performed at day 0 and day 14 in each culture to confirm that cpe was related to adenovirus growth. virus isolates collected from the cell lines were analyzed using the molecular approaches described above. the isolated viruses were first analyzed using the same molecular approaches described above and then subjected to next generation sequencing (ngs) for the sequencing of the whole adenovirus genome. whole adenovirus genome analysis was performed using dna extracted from the culture isolate using next generation sequencing (ngs). the genomic dna (gdna) of adv mbo024 was quantified by a qubit assay with the high sensitivity kit (life technologies, carlsbad, ca, usa) to 0.2 ng/µl. the genomic dna was then sequenced with the miseq technology (illumina inc., san diego, ca, usa) with the paired end strategy and was barcoded in order to be mixed respectively with 11 other genomic projects prepared with the nextera xt dna sample prep kit (illumina). to prepare the paired end library, a dilution was performed so that 1 ng of each genome was needed as input to prepare the paired end library. the tagmentation step fragmented and tagged the dna. then, limited cycle pcr amplification (12 cycles) completed the tag adapters and introduced dual-index barcodes. after purification on ampure xp beads (beckman coulter inc., fullerton, ca, usa), the libraries were normalized on specific beads according to the nextera xt protocol (illumina). normalized libraries were pooled into a single library for sequencing on the miseq. the pooled single strand library was loaded onto the reagent cartridge and then onto the instrument, along with the flow cell. automated cluster generation and paired end sequencing with dual index reads were performed in a single 39-h run in 2x250-bp. total information of 54,397 gb was obtained from a 559 k/mm 2 cluster density with a cluster passing quality control filters of 97.1%. within this run, the index representation for adv mbo024 was determined to index 4.8722. the 1,083,204 paired end reads were filtered according to the read qualities. the quality of the sequence data obtained was controlled using the clc genomics workbench v7 (qiagen, https://www.qiagenbioinformatics.com/products/clc-genomics-workbench/) and the assembly was performed by spades v3.13.0 [24] . sequences were trimmed with miseq and trimmomatic [25] software, whereas untrimmed data were processed only using miseq software. to reduce assembly deviations, we used gapcloser software [26] . scaffolds that had a nucleotide number <800 (bp) and scaffolds that had a depth value lower than 25% of the mean depths were removed. the best assembly was selected using different criteria (number of scaffolds, n50, number of n) [27] . after assembly, we applied a mapping program between our genome and that of the reference genomes, human adenovirus 25 (jn226752) and human adenovirus 13 (jn226747), to produce consensus sequences in order to ensure the consistency of the methodology using a scaffold builder [27] with a minimum identity for merging contigs (80%), minimum length for ambiguously mapped contigs (95%) and maximum gap length allowed (5000 nt) (supp. material s1). finally, the genes (e1a, e1b, ix, iva2, e2a, e2b, ptp, l1-l5, e3, e4, u-exon) were analyzed using a homology search engine, the local blast [28, 29] . the sequences were aligned using muscle with default parameters. the dna extraction was validated by the qpcr targeting the t4 phage: all samples tested positive (ct ≤ 35 where we collected gorilla and human samples simultaneously, advs were more frequent in gorillas (35.9%) than humans (10.5%) (4/38) (z test, p-value < 0.0001) (figure 1 , table 2 ). (figure 1 , table 2 ). we succeeded in obtaining 25 sequences of ≈250 bps of adenoviral dna polymerase genes from the stool of nhps: six sequences from gorillas, ten from chimpanzees, seven from macaques and one from baboons from djibouti. four sequences were obtained from human stool samples from the republic of congo. the sequences were compared with each other and with the dna polymerase sequences available in the genbank database (table s1 ). seven sequences from chimpanzees, one from gorillas, one from baboons and seven from macaques exhibited at least a 97% identity with each other and a 99% identity with sadv-38 (fj025922/hb426671). they clustered together and with hadv-e we succeeded in obtaining 25 sequences of ≈250 bps of adenoviral dna polymerase genes from the stool of nhps: six sequences from gorillas, ten from chimpanzees, seven from macaques and one from baboons from djibouti. four sequences were obtained from human stool samples from the republic of congo. the sequences were compared with each other and with the dna polymerase sequences available in the genbank database (table s1 ). seven sequences from chimpanzees, one from gorillas, one from baboons and seven from macaques exhibited at least a 97% identity with each other and a 99% identity with sadv-38 (fj025922/hb426671). they clustered together and with hadv-e members from genbank ( figure 2) . one sequence from a chimpanzee showed a 97% identity with sadv-41.2 (fj025927) and grouped with hadv-b types. one human sequence was 98% identical to a hadv-d type (kf268200). three human and two gorilla sequences were perfectly similar to each other and two other sequences from gorillas showed >97% identity with them. all these sequences clustered together with species hadv-c ( figure 2 ). members from genbank ( figure 2) . one sequence from a chimpanzee showed a 97% identity with sadv-41.2 (fj025927) and grouped with hadv-b types. one human sequence was 98% identical to a hadv-d type (kf268200). three human and two gorilla sequences were perfectly similar to each other and two other sequences from gorillas showed >97% identity with them. all these sequences clustered together with species hadv-c ( figure 2 ). sequence alignment of the dna polymerase gene of human and simian adenoviruses. the evolutionary history was inferred using the maximum likelihood method, based on the tamura three-parameter model. sequences were aligned by the clustalw method and compared with each other as well as with available dna polymerase sequences from genbank. sequences in this study are indicated by the following colors: brown for humans; green for gorillas; blue for chimpanzees; violet for macaques and red for baboons. sequences from genbank are in black. adv types from hadv-c, -b and -e were detected in african nhps and from hadv-c and -d in humans. hadv-c jumped from humans to gorillas and, inversely, hadv-c strains g06 and g07a jumped from gorillas to humans from the republic of congo. the tree with the highest log likelihood (−1102.33) is shown. the percentage of trees in which the associated taxa clustered together is shown next to the branches. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the analysis involved 59 nucleotide sequences. the confidence probability (multiplied by 100) for the inside length of the branch is greater than 0, as estimated by the bootstrap test (1000), which is shown next to the branches. in the republic of congo, we found hadv-c and e members in gorillas and hadv-c and d members in humans, with an abundance of c members in both humans and gorillas (table s1 ). in senegal, hadv-b and e members were detected in chimpanzees, and the e members were the most prevalent ones. adv-e was also found in baboons from djibouti. in algeria, the macaques were infected by adv-e types. in this study, the green monkeys were negative for advs, probably due to the limited number of samples (only four samples). viral isolation was successful in hep2 cells inoculated with a human stool sample with 20 ct in the qpcr control at day 14. no virus was isolated from the nhp stool samples. the mbo024 strain of the whole genome of a 35 kb length showed a 97.6% identity with hadv-25 (jn226752) and a 96.4% identity with hadv-13 (jn226747) from hadv-d detected in a human from the united states of figure 2 . molecular phylogenetic analysis by maximum likelihood method based on a short (250 bps) sequence alignment of the dna polymerase gene of human and simian adenoviruses. the evolutionary history was inferred using the maximum likelihood method, based on the tamura three-parameter model. sequences were aligned by the clustalw method and compared with each other as well as with available dna polymerase sequences from genbank. sequences in this study are indicated by the following colors: brown for humans; green for gorillas; blue for chimpanzees; violet for macaques and red for baboons. sequences from genbank are in black. adv types from hadv-c, -b and -e were detected in african nhps and from hadv-c and -d in humans. hadv-c jumped from humans to gorillas and, inversely, hadv-c strains g06 and g07a jumped from gorillas to humans from the republic of congo. the tree with the highest log likelihood (−1102.33) is shown. the percentage of trees in which the associated taxa clustered together is shown next to the branches. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the analysis involved 59 nucleotide sequences. the confidence probability (multiplied by 100) for the inside length of the branch is greater than 0, as estimated by the bootstrap test (1000), which is shown next to the branches. in the republic of congo, we found hadv-c and e members in gorillas and hadv-c and d members in humans, with an abundance of c members in both humans and gorillas (table s1 ). in senegal, hadv-b and e members were detected in chimpanzees, and the e members were the most prevalent ones. adv-e was also found in baboons from djibouti. in algeria, the macaques were infected by adv-e types. in this study, the green monkeys were negative for advs, probably due to the limited number of samples (only four samples). viral isolation was successful in hep2 cells inoculated with a human stool sample with 20 ct in the qpcr control at day 14. no virus was isolated from the nhp stool samples. the mbo024 strain of the whole genome of a 35 kb length showed a 97.6% identity with hadv-25 (jn226752) and a 96.4% identity with hadv-13 (jn226747) from hadv-d detected in a human from the united states of america. the fifteen recognized genes for advs were identified in the mbo024 strain and compared to the reference adv species; they were close to the hadv-d types (table s2 ). the hadv-mbo024 whole genome was deposited in the genbank database under accession number (lr796137.1). since the recurring influenza scares (h5n1 since 1997 and h7n9 in 2013) and the recent coronavirus outbreaks (sars-cov in 2003, mers-cov in 2010 and sars-cov-2 in 2019), the topic of zoonotic viruses, particularly viruses that can cross the interspecies barrier from their natural host and eventually cause disease in humans, has aroused great interest. for any virus, adaptation to a new host must meet several basic requirements and key events. in this study, we detected several species of advs in african great apes (gorillas and chimpanzees) and monkeys (baboons and algerian macaques), as well as in local people residing near gorillas in the republic of congo. interestingly, the positive rate was 3.3 folds higher in nhps than in humans. more than one third of the nhps and one tenth of the humans in this study carried and excreted advs in their feces. the high prevalence of advs in nhps could be explained by the fact that these animals are repeatedly infected, although these infections would have to be prolonged and frequent to maintain virus circulation and increase their prevalence. there was much less virus excretion in human feces, which may have been due to a more efficient t-cell response to viruses [30] . adv infections have been reported at a high prevalence in wild gorilla and chimpanzee populations, as well as in other great apes [5, 13, [31] [32] [33] [34] . in the present study, the adv-infection rate in gorillas from the republic of congo was 35.9%, which is close to the previously reported rates in free ranging gorillas in the same area (44.9%) [31] or those from loango national park, gabon (48%) [31] . captive gorillas in zoos had a much higher prevalence of shedding (36%-100%) [13] . the results in our senegalese chimpanzee study population (54.2%) were higher than in the cameroon/democratic republic of congo study (40%) [13] and in another population of savannah-dwelling chimpanzees from the issa valley in tanzania (37%) [35] , and they were lower than the reported rate of 68% in the republic of congo [31] . these differences seem to be marginal and could be due to differences in sample collection and conservation conditions, study region, seasonality or degree of anthropogenic disturbance of the habitat, but they are less likely to be due to laboratory differences, since all the studies used the same pcr assay [13] . our results confirm the persistence of adv feces shedding and circulation in african great apes. in addition, the occurrence of advs in macaque feces ranged from 13% to 100%: 13% in oregon-rhesus, 67% covance-rhesus, 88% gtp-cynomolgus and 100% gtp-rhesus [13] . for the first time, we detected the dna of advs in 27% of barbary macaques from algeria (27%). it was also reported that african monkeys could be infected with advs [5] . we found a positive rate of 17% in the sub-saharan monkeys of our study, including 23% in baboons (from senegal and djibouti) and 0% in green monkeys. although this difference in results between the macaques in north africa and the other monkeys in sub-saharan africa was not statistically significant and was related to the difference in sample sizes (69 macaques versus only 13 baboons and 4 green monkeys), the difference was not statistically significant. in addition, macaques already live in contact with tourists, which may be the cause of transmission between the two species. although prevalence statistics are presented here, the reality may be underestimated due to the potential deterioration of the sample. great apes seemed to be more infected (46%) than monkeys (25.6%) and humans (10.5%). on the one hand, apes could be repeatedly infected and, consequently, viruses may be maintained and circulated fluently. on the other hand, the human t-cell response to the viruses is more effective, leading to the limitation of virus shedding. our study revealed a great diversity of advs among african humans and nhps. we identified 25 adv sequences from nhps and four from humans. from nhps, based on the 250 bp of adv dna polymerase gene [13] , we detected the members of three adv species: these were principally hadv-e in 72% of cases, followed by hadv-c in 24% of cases and hadv-b in 4% of cases. these advs have been previously reported [5, 31, 32, 34, 36] . in addition, it could be possible, because of the methodology, that very divergent segments were not picked up by the primers, especially those of monkey advs. therefore, the non-identified advs here could be sadvs or advs from other species which were not amplified by the applied primers. furthermore, the dna polymerase fragment was selected in order to be highly conserved. it is therefore no surprise that it was found to be considerably conserved. as demonstrated in figure 2 , hadv-e members seemed to be well adapted in nhps, since they were present in all nhps in this study, except green monkeys (perhaps because of the small number of samples), and they clustered both with one another and with sadv-e members. although advs are generally considered to be rather host species-specific viruses, there are some exceptions [3] . indeed, hadv-e could be an exception, since african great apes, as well as macaques or other monkeys in our study, carried hadv-e members. for hadv-e type occurrence in this study, wild senegalese chimpanzees were the most numerous hosts (50% of cases), followed by macaques (39%) and then gorillas and baboons (5.5% of cases for each). herein, the hypothesis that nhp adv members of the hadv-e originated from chimpanzees [32, 33] , and because chimpanzees, gorillas and baboons all live in sub-saharan africa, we can suppose that the gorilla and baboon hadv-e types of this study were the result of chimpanzee adv transmission. the evolution of hadv-e viruses and their adaptation to new hosts requires further investigation. hadv-c types were detected in the gorilla feces samples from the llr and oknp and eco-guards as well as in humans living around the oknp. these adv-c members consist of three clades: (i) clade 1: one gorilla isolate from oknp, one gorilla isolate from llr and three human isolates that are fully identical and they are all very similar to sadv-42.2. in addition, two other isolates from llr gorillas had close similarity with sadv-42.1; (ii) clade 2: a gorilla adv from oknp with perfect similarity to hadv-c k67-339 (lc504573); (iii) clade 3: a gorilla adv from llr, closely related to gorilla beringei graueri adv-9 [36] . hadv-c types had already been reported in free ranging gorillas from republic of congo [31] and gabon [33] , wild mountain (gorilla beringei beringei) and grauer's (gorilla beringei graueri) gorillas from rwanda [36] and captive gorillas, as well as captive chimpanzees and bonobos [13] . according to hoppe et al. [32] and other studies [13, 33] , it seems that all the lineages in hadv-c are host specific. nevertheless, we report here that both a gorilla adv (g10) and human advs (mbo025, 058 and ibou01) are related to species hadv-c (figure 2) , which is evidence for cross-species and potential zoonotic transmission. as a result, hadv-c types could have a wider range of hosts rather than being strictly host species-specific. wild gorillas and chimpanzees that do not necessarily come into daily contact with humans carry human-associated adenoviruses. the same was true for the local human population, including the eco-guards that carried simian viruses. this suggests that a kind of "jump" has occurred. a hadv-b type detected here in a senegalese chimpanzee was close to sadv-41.1 which was detected in a captive gorilla [13] . advs belonging to this species have been previously reported in chimpanzees and gorillas from the republic of congo [31] , wild gorillas from gabon [33] and both captive and wild gorillas and chimpanzees [13, 32] . wevers et al. [5] reported hadv-b types in 53 gorillas and six chimpanzees and confirmed for wild individuals that members of hadv-b widely infect great apes. however, viruses of the same host species strayed throughout the clade, and several subclades comprising advs of different hosts were visible [5] . they suggested that human hadv-b originated in great apes. in our study, the hadv-b type that was found in chimpanzees formed a separate lineage from the clade of hadv-b. among human advs, hadv-d is the largest and fastest growing species [5] . we detected hadv-d members in the stool of humans living around the oknp, thus confirming that the species hadv-d originated in humans [32] and so far has been exclusively human specific. nkogue et al. reported four different serotypes in a human population around a gorilla park, highlighting the diversity of advs circulating in humans [33] . furthermore, it has been found that advs from wild chimpanzees clustered in hadv-d and showed a 99% pairwise hexon nucleic acid identity with hadv-15 and a 98% identity with hadv-29 and -30 [5] . as a result of the co-habitation of eco-guards with nhps, the transmission of these viruses from humans to nhps could eventually lead to the extension of hadv-d members in nhp communities. in our study, viral isolation was possible only in a human sample as the nhp samples were not fresh. adv strain mbo024 was identified as a hadv-d member and was found to be almost identical to hadv-25, thus confirming the pcr results. unfortunately, it has not been possible to isolate other advs in humans or nhps in order to better understand their genetic diversity and relationships. in addition to the degraded quality of the nhp samples, failures of the simian virus culture could be related to the lack of a suitable culture system, since the culture was performed using human cell lines. finally, our results show that advs are naturally present among african nhps and the human communities living near them. wild chimpanzees and gorillas of the same population carry a variety of adv genotypes, suggesting that great apes are the origin of many hadvs. these viruses are maintained by the exchange between ape individuals and potentially with human populations that live nearby. one of the strengths of this study was that we analyzed the fecal samples of different nhp species with different origins (north africa and sub-saharan africa) as well as samples from humans living in contact with primates in order to evaluate the potential for zoonotic transmission. moreover, the fact that while the target partial gene seemed to be linked even across species, one organism could be capable of hosting simian and human viruses and recombination events, is not highlighted here, but it is a possibility. we complement and enrich recent knowledge (e.g., [5, 13, 32] ) which has suggested that hadv evolution has been mainly governed by strong, long-term associations with their hominid hosts. adv species (in particular hadv-e) have been found to be represented in several primates, suggesting, most likely, cross-species transmission. moreover, we found evidence of the zoonotic transmission of adenoviruses, particularly in hadv-c members, in the republic of congo. our study reported a rich diversity of adenovirus types in hadv-e, hadv-c and hadv-b among african apes and/or monkeys as well as indigenous human populations (hadv-c and hadv-d). adv shedding was more prevalent in nhps than in humans (infection rate was 3.3 folds higher in nhps compared to humans). hadv-e types were the most predominant ones in nhps regardless of their origins, and they seemed to lack strict host specificity. the advs from nhps that were detected here (especially adv-c members) are common in nhps and human populations living nearby, thus suggesting a zoonotic transmission, meaning that these viruses have switched hosts. this has been especially true for crossing the species barrier between different nhp species (especially in the case of the adv-e members), between nhps and humans, but it may also occur between humans, nhps and other animal species. isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture neutralizing antibodies against 33 human adenoviruses in normal children in rome are adenoviruses zoonotic? a systematic review of the evidence genomic and bioinformatics analysis of hadv-7, a human adenovirus of species b1 that causes acute respiratory disease: implications for vector development in human gene therapy novel adenoviruses in wild primates: a high level of genetic diversity and evidence of zoonotic transmissions first evidence of a new simian adenovirus clustering with human mastadenovirus f viruses integrative approaches to the study of primate infectious disease: implications for biodiversity conservation and global health origins of major human infectious diseases infectious disease risk across the growing human-non human 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molecular detection of novel adenoviruses in fecal specimens of captive monkeys with diarrhea in china rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine pcr tests pring-åkerblom, p. rapid and quantitative detection of human adenovirus dna by real-time pcr spades: a new genome assembly algorithm and its applications to single-cell sequencing trimmomatic: a flexible trimmer for illumina sequence data a tiling path-based gap closer that uses long reads to complete genome assembly combining de novo and reference-guided assembly with scaffold _ builder protein database searches for multiple alignments basic local alignment search tool host immune responses to chronic adenovirus infections in human and nonhuman primates adenovirus and herpesvirus diversity in free-ranging great apes in the sangha region of the republic of congo multiple cross-species transmission events of human adenoviruses (hadv) during hominine evolution molecular epidemiological study of adenovirus infecting western lowland gorillas and humans in and around moukalaba-doudou national park (gabon) chimpanzee adenovirus antibodies in humans, sub-saharan africa adenovirus infection in savanna chimpanzees (pan troglodytes schweinfurthii) in the issa valley adenoviruses isolated from wild gorillas are closely related to human species c viruses we are grateful to clio grimaldier, mboussi vincent, bréchard ludivine, aurelia caputo for their valuable technical assistance in this work. we are grateful to ismail lafri, rahal mohamed and fayçal zeroual, and the parc national de chréa (algeria). we are also grateful to agence congolaise de la the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to publish the results. the following are available online at http://www.mdpi.com/1999-4915/12/6/657/s1, table s1 . adenoviruses identified in the present study in african humans and nhps (sequences of ≈250 bp dna polymerase gene) and their identity with genbank reference sequences; table s2 : mbo024 gene annotation and comparison to other related human mastadenovirus d types. supp. material s1: whole genome mbo024. key: cord-018437-yjvwa1ot authors: mitchell, michael title: taxonomy date: 2013-08-26 journal: viruses and the lung doi: 10.1007/978-3-642-40605-8_3 sha: doc_id: 18437 cord_uid: yjvwa1ot this chapter addresses the classification and taxonomy of viruses with special attention to viruses that show pneumotropic properties. information provided in this chapter supplements that provided in other chapters in parts ii–v of this volume that discuss individual viral pathogens. taxonomy may be defi ned as a logical discipline for the identifi cation and classifi cation of biological entities based on objective, measurable characteristics of relevant entities. useful taxonomic systems should be broadly applicable across diverse types of biological groups. they should also be fl exible, so that new data from technological advances may be integrated into the classifi cation scheme. primary goals of systemic taxonomy, regardless of biological discipline, include the following: • establishing groups (taxa) that refl ect varying degrees of evolutionary relatedness among the different biological entities studied • establishing criteria for assignment of known or unknown clinical isolates to a given group • establishing a clear and unequivocal nomenclature the origins of biological taxonomy are fi rmly rooted in botany and zoology. early taxonomic systems relied on gross characteristics, like biological niche, internal and external morphology, reproductive strategies and compatibilities, and fossil records. the seminal works of the swedish botanist carl linnaeus used a hierarchical scheme to represent biological relatedness and established the simplifi ed binomial system of nomenclature that serves as the basis for modern classifi cation systems. the modern scientifi c classifi cation in biology is designed to describe all biological entities within a hierarchy consisting of the following taxa: a basic assumption for the establishment of such a hierarchy assumes that all biological entities have evolved from a single common cellular life-form. different biological entities have evolved as a result of accumulated changes in dna that have provided survival advantages in different ecological niches. species may be classifi ed on the basis of phylogenetic and evolutionary relatedness: members of a given species are the most closely related, different species within a single genus are more closely related to each other than to a species within a different genus, and so on. newer technologies like microscopy, improved biochemical and physiological analysis, and advanced protein and molecular analytical methods have resulted in an enormous expansion of characteristics that may be studied for the classifi cation of biological entities and validation of taxonomic systems (woese et al. 1990 ). there are a number of excellent texts that discuss the clinical and laboratory aspects of virus biology (knipe and howley 2007 ; richman et al. 2009 ; versalovic 2012 ) . though viruses are certainly "biological entities," they are fundamentally different from the cellular life-forms classifi ed by previous taxonomic schemes. viruses have no autonomous metabolic or replicative ability; they are completely dependent on cellular life-forms. however, within their biological milieu, viruses do replicate and evolve, and they are composed of the same types of organic macromolecules as are cellular lifeforms. because of their intimate relationship with cellular life-forms, it seems legitimate to integrate the schemes for classifi cation of viruses with the schemes used for biological classifi cation of cellular life-forms (lefkowitz 2012 ) . initially, various features, like host range, crossimmunity, clinical disease, and pathologic features, were used to classify viruses. technological advances have led to more detailed and integrated classifi cation, taxonomy, and phylogenetic characterization (evolutionary relatedness) of viruses. sophisticated nucleic acid sequence analysis has emerged as a powerful tool for virus classifi cation and phylogenetic determination, in spite of some limitations (holmes 2008 ; mccormack and clewley 2002 ; zanotto et al. 1996 ) . a robust system for classifi cation of viruses developed by david baltimore has gained wide acceptance (baltimore 1971 ) . classifi cation is based on the genomic nucleic acid used by the virus (dna or rna), strandedness (single or double stranded), and method of replication. the system has been used to defi ne seven classes of viruses: class i: double-stranded dna (dsdna) class ii : single-stranded dna (ssdna) the primary classifi cation of viruses is into species. a virus species is defi ned as a polythetic class of viruses that constitute a replicating lineage and occupy a specifi c ecological niche (international committee on taxonomy of viruses 2002 ). in polythetic classifi cations, group members share a number of characteristics, but no single characteristic is necessary or suffi cient to defi ne members of the group. higher-level taxa are monothetic, i.e., there are characteristics that are necessary and suffi cient to defi ne members of the class. it is important to note that not all viruses can be assigned through all taxonomic levels. virus species may be assigned to a genus or remain unassigned. similarly, a genus may be assigned to a family or subfamily, or remain unassigned, and so on up the taxonomic hierarchy. each genus has a type species . the type species is the virus that necessitated the creation of the genus; it is always linked to the genus. in the most recent publication (2012), the ictv recognized 7 orders, 96 families, 22 subfamilies, 420 genera, and 2,618 species. important characteristics used by the ictv to defi ne and classify viruses within these taxa include the following: • susceptible host range : most viruses have a restricted range of hosts which they are able to infect. • virus structure : the viral genome is surrounded by a protective shell of proteins called a capsid. the capsid may also enclose proteins, like reverse transcriptase or proteins required for organization of the nucleocapsid. a nucleocapsid refers to a viral nucleus surrounded by an intact capsid. the nucleocapsids of certain viruses are also surrounded by an envelope of host-derived membranes. the complete virus particle is referred to as a virion. icosahedral capsids are very common; these quasi-spherical shells are composed of 20 identical equilateral triangles with 30 edges and 12 vertices. icosahedral capsids are very effi cient geometrically (internal volume versus protein content) and genetically (many small sides require fewer and smaller genes to code for capsid proteins). the nucleocapsid proteins of some viruses, like the infl uenza viruses, form helical tubes with the nucleic acid incorporated directly into the helical structure. the nucleocapsids of some viruses are surrounded by envelopes composed of lipid bilayers and host-or viral-encoded proteins. envelopes are typically acquired by budding of the nucleocapsid through a virally modifi ed portion of a specifi c host-cell membrane (plasma, endoplasmic reticulum, golgi, nucleus) . the shape of the virus nucleocapsid or intact virion is usually determined by electron microscopy. the shape and dimensions of the nucleocapsid and intact virion, and the presence or absence of an envelope, are useful characteristics for classifying viruses. • genome : the viral genome is either dna or rna; the nucleic acids may be single or double stranded. the genome size may be expressed in terms of kilobases (kb) for singlestranded genomes or kilobase pairs (kbp) for double-stranded genomes. the sequence of genes of positive-sense ssrna may be directly translated by the host into viral proteins. the sequence of negative-sense ssrna is complementary to the coding sequence for translation, so mrna must be synthesized by rna polymerase, typically carried within the virion, before translation into viral proteins. the sequence of positive-sense ssdna is the same as that of the mrna coding for viral proteins; negative-sense ssdna is complementary to mrna and may be transcribed into mrna for viral protein synthesis. ambisense single-stranded nucleic acids use both positive-sense and negative-sense sequences. the viral nucleic acid may be linear or circular; the nucleic acid may be in the form of a single molecule or broken into two or more segments. in addition to the type of nucleic acid, the size of the viral genome, measured in number of bases or base pairs, is an important characteristic used for classifi cation. • nucleic acid sequence analysis : the analysis of specifi c viral nucleic acid sequences is increasingly used as a powerful tool for taxonomic assignment and assessment of evolutionary relatedness. the utility is greatest for related groups of viruses (lauber and gorbalenya 2012a , b ) , but has been challenging for more divergent groups of viruses. sequence analysis alone has not provided a reliable single criterion on which all viruses may be classifi ed. construction of a universal phylogenetic tree for viruses, as has been proposed for cellular life-forms, may not be possible for viruses. it is not clear that all viruses emerged from a single progenitor virus; there is evidence for multiple, independent origins of existing viruses. phylogenetic analysis using nucleic acid sequences is further complicated by recombination, reassortment, incorporation of host nucleic acid sequences, and other factors (domingo 2007 ; holmes 2011 ) . currently, expert consensus, considering laboratory, phenotypic, clinical, and other characteristics, remains the most accurate and robust method for the classifi cation and taxonomic assignment of viruses. note that the formal names assigned at all taxonomic levels are italicized, while the common names, which are often used clinically, are not italicized. the viruses that have been associated with human infections are shown in table 3 .1 . among the families of viruses able to infect humans and other vertebrate hosts, there are many species that target and cause disease in the lung. these viruses commonly use airborne transmission as an effective mode of transmission between an infected host and a new susceptible host. characteristics of viruses that directly or indirectly cause pulmonary disease are discussed in this section. adenoviridae: adenoviruses are pathogenic for humans and other vertebrate species. a structural protein at each of the 12 of the icosahedral nucleocapsid vertices anchors a rodlike projection with a terminal knob, which interacts with specifi c host surface receptor molecules and which confers the hemagglutination pattern and tissue tropism for the different groups of adenoviruses. the genome encodes ~40 genes (davison et al. 2003a ) , including common genes and species-specifi c genes. genes are grouped into early, delayed early, and late transcribed genes. the genome contains inverted repeat sequences at both ends. sequences of both dna strands are transcribed to mrna; mrna splicing is used for expression of many adenovirus genes. the family adenoviridae has not been assigned to an order. within this family, there are fi ve genera. the seven species that cause human infection are human adenovirus a, b, c, d, e, f, and g , all within the mastadenovirus genus; there are 57 accepted serotypes (buckwalter et al. 2012 ) . endemic respiratory infections are most commonly caused by serotypes of human adenovirus c (the type species of the genus); most epidemic respiratory infections are caused by serotypes within species adenovirus b and adenovirus e . arenaviridae : arenaviruses may cause several hemorrhagic fever syndromes. specifi c rodents are the reservoir for each arenavirus; human disease is incidental and is usually transmitted by infectious aerosols. viruses of this family are enveloped; evenly spaced glycoprotein complexes (a tetramer of viral gp2 with viral gp1 ionically bound as a globular head) are attached to the envelope giving complete virions a studded spherical morphology. complete virions are ~100 nm in diameter, but show signifi cant pleomorphism (range, 60-300 nm). the genome is divided into two segments which are complexed with nucleoproteins (peters 2009 ) . complementary sequences at the 3′ and 5′ ends of each segment result in the formation of two circular nucleocapsids. arenaviruses use both negative-sense and ambisense coding strategies. host ribosomes are often incorporated within the envelope of complete virions. this family of viruses is not assigned to an order. there is one genus, arenavirus , with 25 species that fall into two complexes on the basis of serologic and genetic relatedness. the old world, or african, species include lassa virus (lassa fever) and lujo virus. the new world species include guanarito virus (venezuelan hf), junín virus (argentine hf), and machupo virus (bolivian hf). the type species of the genus arenavirus is lymphocytic choriomeningitis virus . bunyaviridae : bunyaviruses may cause several hemorrhagic fever syndromes. viruses coronaviridae : transmembrane proteins produce blunt projections from the surface of coronaviruses, resulting in a "crown-like" appearance on electron microscopic studies (100-160 nm in diameter). translation of the coronavirus genome is unique and includes production of polyproteins, discontinuous synthesis, overlapping reading frames, ribosomal frame shifting, and post-translational proteolytic processing (marra et al. 2003 ; rota et al. 2003 ; theil et al. 2003 ) . the major structural proteins, spike glycoprotein (s), membrane glycoprotein (m), nucleocapsid phosphoprotein (n), hemagglutinin-esterase glycoprotein (he), and envelope protein (e), are present in all coronaviruses. nonstructural proteins are encoded in 5-10 unique or overlapping reading frames (lai et al. 2007 ). the human coronaviruses are assigned to the order nidovirales , family coronaviridae , and subfamily coronavirinae . there are four genera and three serological groups. relevant viruses include human coronavirus 229e and human coronavirus nl63 of the genus alphacoronavirus (antigenic group i), human coronavirus hku1, betacoronavirus 1 and severe acute respiratory syndrome-related coronavirus of the genus betacoronavirus (antigenic group ii). filoviridae : filoviruses may cause several hemorrhagic fever syndromes. the fi loviruses have a unique threadlike morphology. the helical nucleocapsids are surrounded by an envelope studded by spikes formed by a single type of glycoprotein (gp). the genome consists of a single segment of negative-sense ssrna that encodes for seven proteins (kuhn et al. 2010 ) . the presence of gene overlap for several genes is an unusual feature of fi loviruses. in ebolaviruses, the surface glycoprotein is encoded by two adjacent reading frames. a truncated version (sgp), which lacks the hydrophobic anchor, results from translation of the upstream reading frame only. this protein is secreted from cells and may serve as a decoy for the host's immunological response. the full-length gp is formed only when the rna polymerase misreads a poly-u editing site between the reading frames. the fulllength gp is inserted, as homotrimers, into the host membranes that will form the virion envelope. a helical nucleocapsid is formed by association of the ssrna with nucleoproteins. the nucleocapsid is ~50 nm in diameter, with a central axial space ~20 nm in diameter. the nucleocapsid is attached to the envelope by matrix protein. the complete virions are ~80 nm in diameter, but the virion length may vary from 800 to 10,000 nm. the family filoviridae is assigned to the order mononegavirales . there are two genera within the family ebolavirus and marburgvirus . there are fi ve ebolavirus species, including sudan ebolavirus and zaire ebolavirus (the type species). the genus marburgvirus consists of one species, marburg marburgvirus . humans and nonhuman primates are susceptible to ebolavirus and marburgvirus infection; the host reservoirs for these viruses are unknown. humans may be infected sporadically by presumed contact with the host species or by direct contact with virus containing body fl uids taken from acutely infected humans or nonhuman primates. nosocomial and laboratoryacquired infections are well described. flaviviridae : flaviviruses may cause several hemorrhagic fever syndromes. hepatitis c virus is also a fl avivirus species. flaviviruses are surrounded by an envelope studded with dimers of viral e glycoprotein and m protein which give the mature virion a herringbone appearance with icosahedral symmetry. the genome consists of a single segment of positive-sense ssrna (chambers et al. 1990 ; osatomi and sumiyoshi 1990 ) . cyclization of the genome, through hybridization of rna sequences of the 5′ and 3′ ends of the genome, may be required for mrna synthesis (alvarez et al. 2005 ). there is a long open reading frame that codes for three structural proteins at the 5′ end; downstream of this region are genes for seven nonstructural proteins (thurner et al. 2004 ). the positive-sense genome is directly translated into a large polyprotein, which undergoes intra-and post-translational cleavage. strain evolution and clinical diversity have been driven by a high rate of mutation at replication and through molecular recombination. the nucleocapsid is formed by interaction of genomic rna with capsid proteins. the complete virion has a spherical morphology approximately 50 nm in diameter. this family of viruses is not assigned to an order. there are four genera within the family flaviviridae . within the genus flavivirus , there are 53 species, including dengue virus (simmons et al. 2012 hepatitis c virus (hcv) is the type species of the genus hepacivirus in the family flaviviridae . the physical properties of hcv have not been as well defi ned as other fl aviviruses because there is no effi cient method for in vitro replication of hcv. virion morphology is consistent with other fl aviviruses; complete, enveloped virions have a diameter of 55-65 nm. the single segment positive-sense ssrna is ~9.6 kb in length (hijikata et al. 1991 ) . a single open reading frame is fl anked by highly conserved regions at the 5′ and 3′ ends. cap-independent protein synthesis, typical of flavivirus species, is initiated at an internal ribosomal entry site (ires) within the 5′ untranslated region. this results in synthesis of a polyprotein that undergoes cleavage and further processing during and after translation. a unique and highly conserved sequence upstream of the ires interacts with liver-specifi c microrna and is required for effi cient replication. circulating hcv is associated with host ldl/vldl, which may play a role in delivery of virions to hepatocytes. the error-prone rna polymerase and high replication rate of hcv has resulted in a great genetic diversity and heterogeneity of clinical isolates. hcv isolates can be grouped by genotypic analysis into six groups and many subgroups. there are differences with respect to responses to antiviral therapy among the genotypes, but intrinsic virulence is similar. the vast majority of strains in the united states are genotypes 1a, 1b, and 2, whereas central african strains are almost exclusively genotype 4. hemorrhagic fever (hf) syndromes : viral hemorrhagic fever syndromes may be caused by many species of viruses from four different families: arenaviridae, bunyaviridae, flaviviridae and filoviridae ; all are single-stranded rna viruses. see the discussions above for specifi c information related to these virus families. typical symptoms of viral hemorrhagic fever infection include fever, malaise, hypotension, and coagulation defects. with the exception of dengue, the other hf viral agents are maintained in nonhuman vertebrate hosts; humans are coincidental, dead-end hosts. in dengue, human infection is maintained through a mosquito vector. the epidemiologic distribution of disease refl ects the geographic range of the reservoir host. hf viruses primarily infect dendritic cells, macrocytes, and monocytes, which are present in virtually all tissues and organ systems; parenchymal cells may also be susceptible to infection, depending on the virus. infected cells release mediators that result in marked increased vascular permeability, compromising the function of critical organ systems. suppression of cellular type 1 interferon response is a signifi cant contributor to pathogenesis (habjan et al. 2008 ) . hepadnaviridae: in the family hepadnaviridae , there are two genera, avihepadnavirus (two species) and orthohepadnavirus (four species); hepatitis b virus (hbv), the type species of orthohepadnavirus , is only human pathogen in family. the family hepadnaviridae is not assigned to an order. eight distinct hbv genotypes (a-h) and subtypes can be recognized on the basis of antigenic or sequence variation. the genotypes show geographic and ethnic variability; the hbv genotype infl uences the severity and outcome of disease (garfein et al. 2004 ; lin and kao 2008 ) . the complete, enveloped hbv virion (dane particle) is 42-47 nm in diameter. the icosahedral nucleocapsid (~28 nm in diameter) of the virion contains a single molecule of partially double-stranded dna with a dna-dependent polymerase covalently linked to the 5′ end of the complete dna strand, hepatitis b e antigen (hbeag) and hepatitis b core antigen (hbcag). the nucleocapsid is surrounded by an envelope derived from host-cell membrane and viral envelope proteins, including hepatitis b surface antigen. the genome of hbv is a circular, partially double-stranded dna molecule which is replicated by a unique process of reverse transcription of an rna intermediate. the minus dna strand runs the entire length of the hbv genome; the plus strand covers only about two-thirds of the genome. the genome is replicated by synthesis of a fulllength ssrna transcript (pre-genomic rna), followed by dsdna synthesis by reverse transcription of the ssrna by viral-encoded reverse transcriptase/dna polymerase. all viral proteins are also transcribed from the minus dna strand. there are four overlapping open reading frames, all read in the same direction (liang 2009 ) . herpesviridae : the herpesvirus species associated with human infections (hsv-1, hsv-2, cmv, ebv, vzv, hhv-6, hhv-7, and hhv-8) belong to the family herpesviridae within the order herpesvirales . there are four subfamilies of the herpesviridae : alphaherpesvirinae (5 genera), betaherpesvirinae (4 genera), gammaherpesvirinae (4 genera), and a single genus in an unassigned subfamily. specifi c human herpesviruses are discussed in the sections below. the herpesviruses are double-stranded dna viruses. the icosahedral capsid (~100 nm diameter) is surrounded by an envelope studded by a variety of short glycoproteins. the nucleocapsid is a dense toroid complex with an outer diameter ~70 nm and inner diameter ~18 nm. an irregular "tegument" fi lls the space between the envelope and capsid. depending of the thickness of the tegument layer, complete virions range in size from ~125 to >250 nm. the size and organization of the dsdna genome varies among the species causing human disease (mcgeoch et al. 2006 ) . the genomes of human herpesviruses include unique sequences and repeated sequences. though the genomes are linear in virions, they circularize in the nucleus of infected cells, which is mediated through repeat sequences at both ends of the dsdna genome. for hhv6 and hhv7 (class a genome), a large unique sequence region is fl anked by a region that is repeated at both ends of the linear strand of dsdna. the genome of ebv and the kaposi's sarcoma-associated herpesvirus (class c genome) have smaller left and right terminal repeat sequences, while repeat sequences r1 to r4 divide the unique sequence nucleic acid into four discrete regions. for vzv (class d genome), a large terminal sequence is inverted and inserted into the genome, resulting in a large unique sequence region (ul) and a small unique sequence region (us). hsv-1, hsv-2, and cmv (class e genomes) are the most complex. there are repeat sequence regions at both ends of the linear dsdna molecule. the unique sequence dsdna is divided into ul and us regions by a sequence composed of juxtaposed copies of the terminal repeat sequences inserted in an inverted orientation. typical of dsdna viruses, a large number of proteins are produced by various herpesviruses. the organization of the coding regions is complex, with 3′ and 5′ reading frames, gene overlap, spliced genes, and intron regions. forty genes are conserved among the α-, β-, and γ-herpesviruses. these core genes are divided among seven gene blocks (albà et al. 2001 ) ; within each block the order and polarity of genes are conserved, including genes for gene regulation, nucleotide metabolism, dna replication, virion maturation, envelope glycoprotein synthesis, and capsid, fusion and tegument protein synthesis. diseases caused by human herpesviruses range from systemic to localized infection of virtually all organ systems, although the hostcell range and typical disease characteristics vary by species. a characteristic of herpesvirus infections is latency, which is commonly associated with reactivation and symptomatic infections (e.g., shingles). while active infection with herpesviruses results in the destruction of the infected host cell, latently infected cells remain viable. in latently infected cells, the viral genome forms circularized molecules within the host nucleus with limited expression of viral genes. (gompels et al. 1995 ) of these viruses has the simplest organization and lowest %g + c content compared to the other herpesviruses. reading frames are present on each strand of the dsdna. core herpesvirus proteins are clustered near the center of the strands, while species-specifi c genes are located toward the ends of the strands (braun et al. 1997 ). hhv-6 and hhv-7 are assigned to the genus roseolovirus in the subfamily betaherpesvirinae , family herpesviridae , and order herpesvirales . there are two distinct hhv-6 species: human herpesvirus 6a (the roseolovirus type species) and human herpesvirus 6b . hhv-6b is the agent of exanthem subitum. there is a single hhv-7 species, human herpesvirus 7 , which is also a cause of exanthem subitum. t-lymphocytes are the primary target cell of hhv-6 and hhv-7 viruses. • kaposi's sarcoma-associated herpesvirus : the complete virions of kaposi's sarcoma-associated herpesvirus (kshv) have a diameter ~100 nm. in addition to virusspecifi c proteins, the tegument also carries viral mrnas, probably the result of passive incorporation during the cytoplasmic envelopment process (bechtel et al. 2005 ) . the envelopes of complete virions bear kshv-specifi c glycoproteins. the genome (~170 kbp) has class c organization (russo et al. 1996 ) typical of gammaherpesvirinae . the conserved herpesvirus genes are clustered in four blocks; kshv-specifi c genes are typically distributed in the regions outside and between these blocks (renne et al. 1996 ) . the kshv species designation is human herpesvirus 8 , which is assigned to the genus rhadinovirus within the subfamily gammaherpesvirinae . the virus has tropism for b-lymphocytes and is implicated in all forms of kaposi's sarcoma. four clades, a-d, with distinctive geographical distributions, have been identifi ed by genotypic analysis; the a and c clades cluster together and are most typical for isolates from europe and the united states. • varicella-zoster virus (vzv) : the dense core of vzv is enclosed in an icosahedral capsid (80-120 nm diameter), which is surrounded by an amorphous tegument. the envelope may be derived from multiple types of hostcell membranes during transit from the nucleus through the cytoplasm; specifi c viralencoded glycoproteins are embedded in the envelope of the complete virions, which may be spherical or pleomorphic (180-200 nm in diameter). vzv has a class d dsdna genome (~125 kbp) (clarke et al. 1995 ; davison 1984 ) , resulting in production of two isomeric genomic forms by infected cells through inversion of the us region (ecker and hyman 1982 ) . the genome encodes more than 70 proteins. the organization includes grouping of several genes into single transcription units, genes with overlapping reading frames, and spliced segments (davison and scott 1986 ) . the species designation for vzv is human herpesvirus 3 . it is the type species of the genus varicellovirus within the subfamily alphaherpesvirinae . there is only a single serotype of vzv. for epidemiologic purposes, vzv isolates may be genotyped on the basis of minor differences in dna sequence; different genotypes may be classifi ed as european, japanese, or mosaic (loparev et al. 2004 ). the host range of vzv is restricted to cells of humans or other primates; in humans, vzv has tropism for human t-lymphocytes and establishes latent infection in the cells of the dorsal root ganglia. orthomyxoviridae : infl uenza viruses belong to the family orthomyxoviridae . they are polymorphic; viruses may be spherical (~100 nm diameter) or fi lamentous. complete virions are surrounded by an envelope derived from the host cytoplasmic membrane. viral hemagglutinin and neuraminidase proteins are embedded in the envelope resulting in characteristic 10-14 nm spikes projecting from the surface of virions. in addition to the ha and na protein, m2 protein is embedded into the envelope of infl uenza a viruses; nb and bm2 proteins are embedded into the envelopes of infl uenza b viruses. the matrix protein (m1) is located just below the envelope. the nucleocapsid is composed of viral rna and nonstructural proteins, including ribonucleoproteins and polymerases. the genome of infl uenza viruses is composed of negative-sense ssrna. all viral rna synthesis occurs in the nucleus of the host cell. the a and b infl uenza virus genomes are composed of eight segments, while the infl uenza c virus genome consists of seven segments (hayden and palese 2009 ) . the segments range in size from ~900 to 2,300 nucleotides in length. each segment codes for one or more viral proteins (mccauley et al. 1983 ). the 3′ and 5′ ends of each segment contain noncoding, regulatory regions (fujii et al. 2005 ) . the three largest segments code for various components of rna polymerase; the pb1 segment of infl uenza a virus has a second open reading frame that encodes the pro-apoptotic protein pb1-f2. in infl uenza types a and b, the fourth and sixth segments encode for the surface hemagglutinin (ha) and neuraminidase (na) glycoproteins, respectively. the infl uenza a surface protein m2 is encoded by the seventh segment; infl uenza b surface protein nb is encoded by the sixth segment, while the bm2 is encoded by the seventh segment. the fi fth segment of both a and b infl uenza viruses encodes for the rna-binding nucleoprotein (np). the matrix protein m1 is encoded by the seventh segment of both viruses. the eighth and smallest rna segment of infl uenza a and b viruses encodes for ns1, a multifunctional protein with interferon antagonistic properties and nep/ ns2 protein which is involved in transport of vrnps across the nuclear membrane of the host cell. the names of clinical isolates of human infl uenza isolates include the species of origin, isolation location, number of the isolate, and year of isolation; infl uenza a virus isolates also include the hemagglutinin (h1 to h16) and neuraminidase (n1 to n9) subtypes (atmar and lindstrom 2012 ) . for example, a/california/7/2009 (h1n1), a/victoria/361/2011 (h3n2), and b/ wisconsin/1/2010 viruses were recommended for the 2012-2013 seasonal infl uenza vaccine. large outbreaks have only occurred with h1, h2, and h3 and neuraminidases n1 and n2 viral subtypes. antigenic drift and antigenic shift contribute to reinfection with infl uenza viruses (taubenberger and kash 2010 ) . antigenic drift is caused by a gradual accumulation of point mutations in hemagglutinin and neuraminidase genes, which result in minor antigenic changes in these proteins. antigenic shift is caused by a virus created by reassortment of infl uenza virus rna segments during coinfection of a host, usually with a human infl uenza virus and an avian or swine infl uenza virus or through introduction of a nonhuman infl uenza virus strain into human populations after mutation during a host-species infection creates a new isolate permissive for interspecies transmission. papillomaviridae: the papillomaviruses (pvs) represent a large (and growing) family of viruses that currently includes 30 different genera and 69 species; the taxonomy has undergone signifi cant reorganization in recent years (bravo et al. 2010 ) . the oncogenic potential of human papillomaviruses is well established. pvs are non-enveloped; virions are icosahedral with diameters of 50-55 nm. the capsid contains two structural proteins, l1, the most abundant viral protein, and l2. the pv genome consists of a single molecule of circularized dsdna (zheng and baker 2006 ) . the open reading frames for all viral genes are located on only one of the dna strands, and transcription proceeds in a single direction. there are eight early (e) open reading frames that encode for regulatory proteins that control viral metabolism and dna synthesis. the e6 proteins of high-risk hpv types have anti-apoptotic effects and interfere with p53 regulatory function in infected host cells (howley et al. 1990 ) . two late (l) reading frames encode for synthesis of the structural proteins l1 and l2. epithelial cells of a wide variety of vertebrate hosts are susceptible to papillomavirus infection, but the different host species are only susceptible to species-specifi c viruses. papillomaviruses have been classifi ed according to susceptible host species and the type of disease produced, but comparison of sequence differences of the l1 reading frame has provided a more detailed description of papillomavirus phylogeny (de villiers et al. 2004 ) . the family papillomaviridae is not assigned to an order. human pathogens are clustered within fi ve papillomavirus genera. paramyxoviridae : the paramyxoviruses are enveloped (host cytoplasmic membrane) with an unsegmented negative-sense ssrna genome (15-19 kb) . the viral rna serves as template for synthesis of mrna and for synthesis of antigenomic (positive-sense) rna for synthesis of new viral negative-sense rna for new virions. there are six to ten genes; genes for the six major proteins are linked in the following 3′ to 5′ order: nucleocapsid (n) → phosphoprotein (p) → matrix (m) → fusion (f) → hemagglutinin/neuraminidase (hn) → large polymerase (l). there is an untranslated leader sequence at the 3′ end and untranslated trailer sequence at the 5′ end. the genes are separated by untranslated sequences and do not overlap, with the exception of the m and l genes of human metapneumovirus . translation is initiated at the 3′ end and proceeds directly through to the 5′ end. because the rna polymerase is unstable and may detach at the untranslated regions between genes, there is a gradient in the concentration of gene products from 3′ to 5′. in different species, other proteins are produced by additional small genes, mrna editing, or overlapping reading frames within the p gene. the v and c proteins regulate viral rna transcription and also interfere with host interferon signaling and other aspects of the immune response to paramyxovirus infection (andrejeva et al. 2004 ; durbin et al. 1999 ; swedan et al. 2009 ). formation of the nucleocapsid core is constrained by a required association of one n protein to every six genomic nucleotides (kolakofsky et al. 2005 ; skiadopoulos et al. 2003 ) . the resulting helical structure has a diameter of 18 nm with a 4 nm central core. p proteins (a polymerase cofactor) are attached to this rigid rod and serve as attachment of l proteins, which interact to provide enzymatic activity for rna synthesis. this core structure, rather than free genomic rna, serves as the template for mrna and antigenomic rna synthesis. the paramyxovirus m proteins surround and organize the nucleocapsid and interact with the cytoplasmic tails of transmembrane envelope proteins. the envelope formed from modifi ed host-cell plasma membranes is studded by viral protein complexes, including hn proteins, which mediate virion attachment to target cells, and f protein, which mediates ph-independent fusion of the viral envelope and cell cytoplasmic membrane. the paramyxoviridae are one of the four families within the order mononegavirales and include signifi cant and frequent pathogens of humans and animals. there are two subfamilies: the paramyxovirinae and the pneumovirinae . there are seven genera and thirty-one species in the subfamily paramyxovirinae and two genera and fi ve species in the pneumovirinae subfamily. • henipahviruses: in the subfamily paramyxovirinae , there are two species within the genus henipahvirus : hendra virus (hev) and nipah virus (niv); hev is the type species. henipahvirus virions are pleomorphic (spherical to helical forms). electron microscopy of hendra virus shows a "double-fringe" appearance due to short and long surface projections (hyatt et al. 2001 ) . complete virions range in size from 40 to 1,900 nm in longest dimension. the genome (~18 kb) includes genes typical of paramyxoviridae . long untranslated sequences are attached to the 3′ end of fi ve of the six genes, resulting in the larger genome size of henipahviruses compared to other paramyxoviruses (eaton et al. 2007 ; wang et al. 2000 ) . the p gene also codes for v and w proteins by mrna editing and c protein by a shifted reading frame. henipahviruses are assigned to the family paramyxoviridae and subfamily paramyxovirinae . henipahvirus infections are zoonotic; fruit bats are the presumed reservoir for hendra virus infections, while fruit bats (johara et al. 2001 ) includes two open reading frames; the function of m2-1 protein is undefi ned, while m2-2 protein is a regulator of viral transcription. there is no gene for hemagglutinin-neuraminidase; the product of the g gene serves as the major attachment protein. metapneumoviruses are members of the subfamily pneumovirinae . human metapneumovirus is a species in the genus metapneumovirus . there is a single hmpv serotype, with two antigenic subtypes a and b. • human respiratory syncytial viruses: the envelope of human respiratory syncytial virus (hrsv) is studded with three viral glycoproteins: g protein (the major attachment protein), f protein, and sh protein (a small hydrophobic protein). complete virions are pleomorphic (spherical to fi lamentous forms). the nucleocapsid diameter, 12-15 nm, is smaller than typical for other paramyxoviruses (hall 2001 ) . the hrsv genome is ~15 kb and includes ten genes (collins and wertz 1983 ) . the fi rst eight reading frames are nonoverlapping; the last two genes, m2 and l, overlap by 62 nucleotides. in addition to n, p, and l proteins, m2-1 protein, a transcription factor, is associated with the nucleocapsid. the overall organization of the hrsv genome is similar to other paramyxoviruses. in addition to the typical genes, the hrsv genome includes genes ns1 and ns2 (nonstructural proteins that interfere with interferon induction and signaling), sh, and m2-1 and m2-2 (nonstructural proteins involved in regulation of transcription). there is no nh gene; attachment is mediated by the g gene product. human respiratory syncytial virus is the type species of the genus pneumovirus in the subfamily pneumovirinae . there is a single hrsv serotype, with two antigenic subtypes a and b. (henrickson 2003 ) . the genome of human parainfl uenza viruses is ~15 kb in length with an organization and six reading frames (n, p, m, f, hn, l) typical of the paramyxoviridae (karron and collins 2007 ) . there are no overlapping reading frames. accessory proteins, c (hpiv1 and 3), v (hpiv 2 and 4), and d (hpiv3), are produced by mrna editing of the p gene. n proteins are tightly bound to viral and antigenomic rna; p and l proteins are also bound to the nucleocapsid, forming functional complexes for rna polymerization and processing. human parainfl uenza viruses are assigned to two genera in the subfamily paramyxovirinae . (berns 1990 ). virions are stable in the environment and thought to transmit infection by attachment to specifi c receptors of actively dividing cells. the parvovirus genome is composed of unsegmented ssdna (cotmore and tattersall 1984 ; shade et al. 1986 ; zhi et al. 2004 ) . complete virions of different species may contain negativesense or both negative-and positive-sense dna in various proportions. there are two major reading frames: one encoding capsid proteins and the other coding for nonstructural proteins. noncoding sequences at the 3′ and 5′ ends include complementary sequences which result in the formation of hairpin structures that serve to regulate nucleic acid synthesis (deiss et al. 1990 ) . various host-cell molecules mediate attachment and infection by parvoviruses. erythrocyte p antigen is the major receptor for human parvovirus b19 . viruses are taken up by endocytosis, followed by transport into the host-cell nucleus. viral dna replication depends on host-cell polymerases during the s phase of host-cell replication. human infections are caused by parvovirus b19 and bocavirus (schildgen et al. 2008 ; vicente et al. 2007 ) . the family parvoviridae is not assigned to an order; there are two subfamilies, the densovirinae and the parvoviridae . there are fi ve genera in the subfamily parvoviridae : amdovirus (1 species), bocavirus (2 species including the type species bovine parvovirus ), dependovirus (12 species, including adenoassociated viruses), erythrovirus (4 species including the type species human parvovirus b19 ), and parvovirus (12 species). picornaviridae : infections of the respiratory tract and other organ systems by enteroviruses and parechovirus are well described. enteroviruses were initially classifi ed on the basis of clinical disease and epidemiology, suckling mouse inoculation, replication in cell culture, electron microscopic studies, physical properties, and the vast range of specifi c antigenic differences. the major subgroups were poliovirus, coxsackievirus (a and b), and echovirus. a characteristic of these viruses is their relative stability in acidic media and nonionic detergents. translation of the positive-sense ssrna genome is regulated by a 5′ non-translated region (lindberg and polacek 2000 ) that is covalently linked to protein vpg (virion protein, genome linked); the short 3′ noncoding region is polyadenylated. translation results in synthesis of a single polyprotein, which is cleaved into functional proteins by post-translational processing (nicklin et al. 1987 ; pallansch and roos 2007 ) . there are three functional regions delimited by ribosomal entry sites. the p1 region codes for capsid proteins, while regions p2 and p3 code for nonstructural proteins. capsid proteins vp1, vp2, and vp3 are exposed externally and account for the serological diversity of the viruses. with the advent of molecular phylogenetic analysis, the enteroviruses have been reclassifi ed by the ictv. enteroviruses are in the order picornavirales , family picornaviridae, and genus enterovirus . the enteroviruses have been assigned to 12 species, including human enterovirus a (17 serotypes including coxsackieviruses and enteroviruses), human enterovirus b (56 serotypes, including coxsackieviruses, echoviruses, and enteroviruses), human enterovirus c (the type species; 16 serotypes including coxsackieviruses, all human polioviruses, and enteroviruses), and human enterovirus d (3 enterovirus serotypes). in addition to the enteroviruses, the genus enterovirus also includes 3 rhinoviruses species, human rhinovirus a , b, and c , and more than 100 serotypes. also within the family picornaviridae is the genus parechovirus . human parechovirus is the type species for the genus. there are 14 parechovirus serotypes. polyomaviridae : polyomaviruses may infect a variety of primate and non-primate vertebrate host species; the oncogenic potential of polyomaviruses is well established (white and khalili 2004 ) . sialic acid and/or gangliosides on the hostcell membranes serve as receptors for attachment of human polyomavirus. though these molecules are widespread on human cells, there is a restricted tropism. respiratory epithelial cells and cells of lymphoid origin are the likely targets for initial infection, followed by hematogenous spread to target organs. the virions are non-enveloped; the icosahedral capsids (40-45 nm diameter) are composed of three proteins (vp1, vp2, and vp3), which enclose the circular dsdna genome (~5 kbp). the genome is divided into three regions. the early region encodes for proteins involved in viral processes that occur prior to dna replication, including t (tumor) antigens (benjamin 2001 ) . the late region encodes for proteins involved in processes that primarily occur after dna replication. the early and late regions do not overlap and are transcribed from opposite strands of the viral dna and in opposite directions. a number of viral proteins are encoded as a result of alternative splicing and other posttranslational modifi cations of mrna. polyomaviruses are members of the family polyomaviridae , which is not assigned to an order. there is 1 genus, polyomavirus , and 13 species, including the human pathogens bk polyomavirus and jc polyomavirus and simian virus 40 (type species). retroviridae : the retroviruses are a unique group of viruses, including human immunodefi ciency virus types 1 and 2 and human t-cell leukemia virus type 1; they may infect a wide range of vertebral host species. the human immunodefi ciency viruses and human t-cell leukemia virus 1 are able to cause disease in humans. these rna viruses use a unique replication cycle that uses a "reverse fl ow" of genetic information from rna to dna: viral rna is reverse transcribed and converted into a dsdna copy of the viral genome which is integrated into the host-cell genome. integration of the proviral dna allows the viruses to establish persistent, presumably lifelong, infection. another consequence of insertion of the viral dna is functional mutation of the host genome at the site of insertion which may alter the host gene or regulation of a gene's expression; the oncogenic potential of retroviral infection is well described in humans and other vertebrate host species. the electron microscopic morphology of retroviruses shows a dense nucleocapsid core (cylindrical or cone shaped) (chrystie and almeida 1988 ; gelderblom et al. 1989 ) . viruses are functionally diploid: the core includes two copies of the positive-sense ssrna genome, which are closely complexed with viral nucleoproteins. the sequences of the two ssrna molecules may differ because of errors in transcription of new genomic ssrna molecules during replication. the core also includes several functional viral proteins, including reverse transcriptase, integrase, and protease. the core is surrounded by capsid proteins; the nucleocapsid is surrounded by viral matrix protein. complete virions are surrounded by an envelope derived from virus-modifi ed host-cell cytoplasmic membranes; the envelope is studded by viral glycoproteins. the transmembrane protein extends from the matrix layer through the lipid bilayer to the external surface. the receptorbinding complex is anchored to the external portion of the transmembrane protein. mature virions are spherical (~100 nm diameter). the ssrna genomes of retroviruses are similar to the host-cell mrna. a repeat sequence is present at both ends of the ssrna; the 5′ end is capped and the 3′ end polyadenylated. the order of sequences from the 5′ end to the 3′ end is cap → repeat sequence → unique sequence (u5) → the initiation site for initiation of minus-strand dna synthesis → gag gene → pol gene → env gene → the initiation site for plus-strand dna synthesis → a unique sequence (u3) → repeat sequence → poly(a) sequence. after entry into the cytoplasm of a susceptible cell, double-stranded dna is synthesized by reverse transcription of both copies of the retroviral ssrna. the viral-encoded dna is transported into the nucleus, after which it is integrated into the host's genomic dna. the process of forming new virions is initiated by transcription of the proviral dna. the processed viral rna is exported into the cytoplasm and genes for precursor viral proteins are translated. virions are assembled at the cytoplasmic membrane and then released by budding; fi nal virion maturation occurs by extracellular processing of viral proteins. a characteristic of retroviruses is the high mutation rate and marked genomic heterogeneity of isolates. the major factors that contribute to this phenomenon include (1) error-prone reverse transcription, without proofreading correction, of the infecting virus genome; (2) recombination between the two genomic ssrna strands during reverse transcription; and (3) the very high-level production of progeny viruses from infected cells. retroviruses are not assigned to a taxonomic order. the family retroviridae has two subfamilies. the orthoretrovirinae includes six genera, including deltaretrovirus and lentivirus . htlv-1 is assigned the species name primate t-lymphotropic virus 1 in the deltaretrovirus genus. human immunodefi ciency virus 1 and hiv-2 are named human immunodefi ciency virus 1 (type species) and human immunodefi ciency virus 2 , respectively, in the genus lentivirus (clavel et al. 1986b ). • human immunodefi ciency viruses : the human immunodefi ciency viruses have a conical core surrounded by an envelope derived from viralmodifi ed host-cell cytoplasmic membrane. binding and entry of hiv into susceptible cells requires several specifi c receptors: cd4 (present on host helper t cells, cd4+ macrophages, and some dendritic cells) plus chemokine receptors, including ccr5 and cxcr4 (klatzman et al. 1984 ; simmons et al. 1998 ). the biological properties of hiv-1 isolates depend on the chemokine coreceptor(s) used by the virus (berger et al. 1998 ). isolates that exclusively use cxcr4 are t-cell tropic with rapid replication and syncytium formation. isolates that use ccr5 exclusively are tropic to macrophages, replicate more slowly, and do not induce syncytium formation. isolates that can use either cxcr4 or ccr5 have intermediate phenotypes. the gag , pro , pol, and env genes are translated from full-length mrna transcripts of the proviral genome: gag and env in one reading frame and pro and pol from a second reading frame. in addition, several genes are transcribed from overlapping or unique reading frames, including several spliced gene products. human immunodefi ciency type 1 and 2 viruses evolved from simian viruses (gao et al. 1999 ; peeters et al. 1989 ; daniel et al. 1985 ; marx et al. 1991 ) . these viruses may be distinguished by a number of characteristics, including clinical disease, specifi c antigens, and gene sequences (clavel et al. 1986a ) . hiv-1 isolates may be further characterized into genetic groups and subtypes or clades (wainberg 2004 ) . most hiv-1 isolates are in the m (main) group, which has a number of well-defi ned subgroups and recombinant forms with heterogeneous global distribution; clade b viruses are the predominant isolates in north america and europe (hemelaar et al. 2006 ; osmanov et al. 2002 ) . group o (outlier) strains have mainly been isolated or acquired in western africa. group n (non-m, non-o) and recombinant forms are also most commonly isolated from western africa. • human t-cell leukemia virus type 1 : mature htlv virions have a spherical core, symmetrically placed within the envelope. the host-cell receptor is glut-1, a surface glucose transport molecule (manel et al. 2003 ) . the gag , pro , pol, and env genes are translated from fulllength mrna transcripts of the proviral genome: gag in one reading frame, pro and env from a second, and pol from a third reading frame. in addition, several spliced genes are transcribed from overlapping reading frames. recent and continuing progress to develop and use standardized and widely accepted methods for biological and taxonomic classifi cation of viral pathogens has resulted in improvement in the medical response to viral illnesses. at a very basic level, these systems allow clinicians and scientists to communicate effectively and ensure the comparability of data generated by clinical or basic scientifi c studies. further, accurate and standardized data is critical for understanding issues related to transmission, prevention, and treatment of viral illnesses. establishing phylogenetic similarity to known viral pathogens may allow clinicians to anticipate the clinical behavior of new and emerging viral pathogens, as may be seen when virus mutation results in acquisition of new pathogenic mechanisms, like changes to antigens associated with evasion of the immune response of the host species or changes that allow a viral pathogen to jump from one species into new, susceptible species. as analytical tools improve, even more informative data relevant to clinical and pathologic characteristics of viral pathogens is anticipated. genomewide function conservation and phylogeny in the herpesviridae longrange rna-rna interactions circularize the dengue virus genome the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the inf-beta promoter infl uenza viruses, chap 81. in: versalovik j (ed) manual of clinical microbiology dna sequence and expression of the b95-8 epstein-barr virus genome expression of animal virus genomes rnas in the virion of kaposi's sarcoma-associated herpesvirus polyoma virus: old fi ndings and new challenges a new classifi cation for hiv-1 human herpesvirus 6 the clinical importance of understanding the evolution of papillomaviruses real-time qualitative pcr for 57 human adenovirus types from multiple specimen sources flavivirus genome organization, expression, and replication the morphology of human immunodefi ciency virus (hiv) by negative staining confi guration and terminal sequences of the simian varicella virus genome isolation of a new human retrovirus from west african patients with aids molecular cloning and polymorphism of the human immune defi ciency virus type 2 cdna cloning and transcriptional mapping of nine polyadenylylated rnas encoded by the genome of human respiratory syncytial virus characterization and molecular cloning of a human parvovirus genome isolation of t-cell tropic htlv-iii-like retrovirus from macaques structure of the genome termini of varicella-zoster virus the complete dna sequence of varicella-zoster virus genetic content and evolution of adenoviruses the human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome classifi cation of papillomaviruses cloning of the human parvovirus b19 genome and structural analysis of its palindromic termini the genome sequence of herpes simplex virus type 2 et al (1986) transcriptional map of the measles virus genome functional profi ling of a human cytomegalovirus genome mutations in the c, d, and v open reading frames of human parainfl uenza virus type 3 attenuate replication in rodents and primates henipaviruses, chap 45 virus taxonomy: one step forward, two steps back varicella-zoster virus dna exists as two isomers importance of both the coding and the segment-specifi c noncoding regions of the infl uenza type a virus ns segment for its effi cient incorporation into virions origin of hiv-1 in the chimpanzee pan troglodytes troglodytes factors associated with fulminant liver failure during an outbreak among injection drug users with acute hepatitis b morphogenesis and morphology of hiv. structure-function relations the dna sequence of human herpesvirus-6: structure, coding content, and genome evolution processing of genome 5′ termini as a strategy of negative-strand rna viruses to avoid rig-idependent interferon induction respiratory syncytial virus and parainfl uenza virus infl uenza virus, chap 42 global and regional distribution of hiv-1 genetic subtypes and recombinants in parainfl uenza viruses gene mapping of the putative structural region of the hepatitis c virus genome by in vitro processing analysis evolutionary history and phylogeography of human viruses what does virus evolution tell us about virus origins? association of human papillomavirus types 16 and 18 e6 proteins with p53 ultrastructure of hendra virus and nipah virus within cultured cells and host animals the international code of virus classifi cation and nomenclature nipah virus infection in bats (order chiroptera) in peninsular malaysia t-lymphocyte t4 molecule behaves as the receptor for human retrovirus lav paramyxovirus mrna editing, the "rule of six" and error catastrophe: a hypothesis proposal for a revised taxonomy of the family filoviridae: classifi cation, names of taxa and viruses, and virus abbreviations partitioning the genetic diversity of a virus family: approach and evaluation through a case study of picornaviruses toward genetics-based virus taxonomy: comparative analysis of a geneticsbased classifi cation and the taxonomy of picornaviruses taxonomy and classifi cation of viruses, chap 75. in: versalovic j (ed) manual of clinical microbiology hepatitis b: the virus and disease hepatitis b viral factors and clinical outcomes of chronic hepatitis b molecular analysis of the prototype coxsackievirus b5 genome global identifi cation of three major genotypes of varicella-zoster virus: longitudinal clustering and strategies for genotyping the ubiquitous glucose transporter glut-1 is a receptor for htlv the genome sequence of the sars-associated coronavirus isolation of a simian immunodefi ciency virus related to human immunodefi ciency virus type 2 from a west african pet sooty mangabey structure and function of the infl uenza virus genome the application of molecular phylogenetics to the analysis of viral genome diversity and evolution topics in herpesvirus genomics and evolution bunyaviridae: bunyaviruses, phleboviruses, nairoviruses, and hantaviruses, chap 43 site-specifi c inversion sequences of the herpes simplex virus genome: domain and structural features poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3c and 2a proteinases complete nucleotide sequence of dengue type 3 virus genome rna estimated global distribution and regional spread of hiv-1 genetic subtypes in the year 2000 enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses, chapter 25 isolation and partial characterization of an hiv-related virus occurring naturally in chimpanzees in gabon arenaviruses, chap 44 the size and conformation of kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) dna in infected cells and virions characterization of a novel coronavirus associated with severe acute respiratory syndrome nucleotide sequence of the kaposi sarcoma-associated herpesvirus (hhv8) human bocavirus: passenger or pathogen in acute respiratory tract infections? nucleotide sequence and genome organization of human parvovirus b19 isolated from the serum of a child during aplastic crisis cxcr4 as a functional coreceptor for human immunodeficiency virus type 1 infection of primary macrophages the genome length of human parainfl uenza virus type 2 follows the rule of six, and recombinant viruses recovered from non-poly-hexameric-length antigenomic cdnas contain a biased distribution of correcting mutations respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways infl uenza virus evolution, host adaptation, and pandemic formation mechanisms and enzymes involved in sars coronavirus genome expression conserved rna secondary structures in flaviviridae genomes analysis of the genomic sequence of a human metapneumovirus manual of clinical microbiology human bocavirus, a respiratory and enteric virus hiv-1 subtype distribution and the problem of drug resistance the exceptionally large genome of hendra virus: support for the creation of a new genus within the family parmyxoviridae polyomaviruses and human cancer: molecular mechanisms underlying patterns of tumerogenesis towards a natural system of organisms: proposal for the domains archaea, bacteria and eucarya a reevaluation of the higher taxonomy of viruses based on rna polymerases papillomavirus genome structure, expression, and post-transcriptional regulation construction and sequencing of an infectious clone of the human parvovirus b19 key: cord-336157-aqc9zrrm authors: liang, guodong; gao, xiaoyan; gould, ernest a title: factors responsible for the emergence of arboviruses; strategies, challenges and limitations for their control date: 2015-03-25 journal: emerg microbes infect doi: 10.1038/emi.2015.18 sha: doc_id: 336157 cord_uid: aqc9zrrm slave trading of africans to the americas, during the 16th to the 19th century was responsible for the first recorded emergence in the new world of two arthropod-borne viruses (arboviruses), yellow fever virus and dengue virus. many other arboviruses have since emerged from their sylvatic reservoirs and dispersed globally due to evolving factors that include anthropological behaviour, commercial transportation and land-remediation. here, we outline some characteristics of these highly divergent arboviruses, including the variety of life cycles they have developed and the mechanisms by which they have adapted to evolving changes in habitat and host availability. we cite recent examples of virus emergence that exemplify how arboviruses have exploited the consequences of the modern human lifestyle. using our current understanding of these viruses, we also attempt to demonstrate some of the limitations encountered in developing control strategies to reduce the impact of future emerging arbovirus diseases. finally, we present recommendations for development by an international panel of experts reporting directly to world health organization, with the intention of providing internationally acceptable guidelines for improving emerging arbovirus disease control strategies. success in these aims should alleviate the suffering and costs encountered during recent decades when arboviruses have emerged from their sylvatic environment. despite the announcement of the successful eradication of smallpox in 1979, the last case of rinderpest in 2008 and the current campaigns to eradicate poliomyelitis and measles through mass-immunization programmes, we still face the prospect of emerging or reemerging viral pathogens that exploit changing anthropological behavioural patterns. these include intravenous drug abuse, unregulated marketing of domestic and wild animals, expanding human population densities, increasing human mobility, and dispersion of livestock, arthropods and commercial goods via expanding transportation systems. consequently, the world health organization concluded that acquired immune deficiency syndrome, tuberculosis, malaria, and neglected tropical diseases will remain challenges for the foreseeable future. 1 understandably, the high human fatality rates reported during the recent epidemics of ebola, severe acute respiratory syndrome and middle east respiratory syndrome have attracted high levels of publicity. however, many other rna viruses have emerged or reemerged and dispersed globally despite being considered to be neglected diseases. [2] [3] chikungunya virus (chikv), west nile virus (wnv) and dengue virus (denv) are three of a large number of neglected human pathogenic arthropod-borne viruses (arboviruses) whose combined figures for morbidity and mortality far exceed those for ebola, severe acute respiratory syndrome and middle east respiratory syndrome viruses. for instance, for denv, the number of cases of dengue fever/hemorrhagic fever is between 300-400 million annually, of which an estimated 22 000 humans die. 4 moreover, in the new world, within 12 months of its introduction, chikv caused more than a million cases of chikungunya fever according to pan american health organization/world health organization, with sequelae that include persistent arthralgia, rheumatoid arthritis and lifelong chronic pain. 5 likewise, within two months of its introduction, to polynesia, the number of reported cases exceeded 40 000 6 and is currently believed to be approaching 200000 cases. alarmingly, this rapid dispersion and epidemicity of chikv (and denv or zika virus in oceania) is now threatening europe and parts of asia through infected individuals returning from these newly endemic regions. this is an increasingly worrying trend. for example, in france, from 1 may to 30 november, 2014, 1492 suspected cases of dengue or chikungunya fever were reported. 7 accordingly, this review focuses on the emergence or reemergence of arboviruses and their requirements and limitations for controlling these viruses in the future. arboviruses are transmitted between arthropods (mosquitoes, ticks, sandflies, midges, bugs…) and vertebrates during the life cycle of the virus. 8 many arboviruses are zoonotic, i.e., transmissible from animals to humans. [9] [10] as far as we are aware, there are no confirmed examples of anthroponosis, i.e., transmission of arboviruses from humans to animals. 9-10 the term arbovirus is not a taxonomic indicator; it describes their requirement for a vector in their transmission cycle. [11] [12] humans and animals infected by arboviruses, may suffer diseases ranging from sub-clinical or mild through febrile to encephalitic or hemorrhagic with a significant proportion of fatalities. in contrast, arthropods infected by arboviruses do not show detectable signs of sickness, even though the virus may remain in the arthropod for life. as of 1992, 535 species belonging to 14 virus families were registered in the international catalogue of arboviruses. 12 however, this estimate is continuously increasing as advances in virus isolation procedures and sequencing methods impact on virus studies. whilst many current arboviruses do not appear to be human or animal pathogens, this large number of widely different and highly adaptable arboviruses provides an immense resource for the emergence of new pathogens in the future. in addition to evolving strategies for long-term survival, arboviruses have an enormous choice of arthropod species potentially capable of being infected the predominant species of which appear to be mosquitoes and ticks. approximately 300 types of mosquito can transmit arboviruses. aedes and culex mosquitoes are the species most frequently associated with arbovirus transmission (115 and 105 types of arbovirus, respectively). 12 ticks are also prevalent vectors, 116 different species are currently known to transmit arboviruses. in addition, 25 midge species have been shown to transmit arboviruses, mainly culicoides (24 types) and lasiohelea. sandflies, blackflies, stinkbugs, lice, mites, gadfly, and bedbugs can also transmit arboviruses. 13 this diversity of species and the wide distribution of these transmission vectors explain why arboviruses are so successful in dispersing globally via the mechanisms highlighted earlier. [10] [11] [12] arboviral diseases are primarily associated with specific vectors. however, many other arthropod species, in which viruses have been identified, may be involved in perpetuating the virus life cycle without having been associated with overt disease in humans or animals. for example, wnv is typically mosquito-borne but can be vectored by many different mosquito species and also by ticks and other arthropods. 9-10,12 moreover, japanese encephalitis virus appears to be transmissible by culex, anopheles, and other mosquito species, as well as midges, sandflies and ticks. as a general rule, a specific arthropod species, is likely to predominate during an epidemic. however, if the availability of the vertebrate host, for example birds, becomes limited towards the end of the summer as the birds migrate to warmer countries, the vector might switch its preference to a different vertebrate host. this is consistent with the observation that outbreaks of west nile fever/encephalitis in north america often appear to occur in the late summer, following peak incidence in birds, after they commence their migration to warmer regions. 14 alternatively, aedes aegypti has adapted to the urban environment whereas aedes albopictus occurs more commonly in semi-urban or rural areas. however, these are not hard and fast rules. for example, on the french island of la reunion, where aedes aegypti was not recognizably present, chikv was simultaneously epidemic in both rural and urban environments, and was primarily associated with aedes albopictus. 15 a high proportion of arboviruses associated with human and animal disease circulate in tropical, and subtropical regions, where mosquitoes, and other flying insects, tend to be abundant. however, many arboviruses also circulate amongst wildlife species in temperate regions of the world. despite the global distribution of viruses such as wnv, dengue virus, bluetongue virus and now chikv, most other arboviruses are generally endemic to specific regions of the world. for example, mosquito-borne japanese encephalitis virus is prevalent in india, central and southeast asia, largely due to the prevalence of highly competent culex tritaeniorhynchus mosquito species and intensely farmed pigs which act as amplifying hosts for the virus and, importantly, the mosquitoes. [16] [17] in southeast asia, rice cultivation with enormous areas of paddy fields also attracts migratory birds and provides ideal breeding areas for the mosquitoes which transmit the virus to the birds, ensuring virus dispersion over large areas of asia. 18 in contrast, tick-borne encephalitis virus occurs mainly in northern temperate regions which are the primary habitats of ixodes species ticks (forests etc.). 19 nevertheless, even within this relatively localized distribution of arboviruses, dispersion to distant locations occurs via animal or vector migration. as an example, powassan virus, a close relative of tick-borne encephalitis virus, is found in far east asia and also in canada. 19 in contrast, rift valley fever virus (rvfv) was localized in africa but in 1977, it was introduced to the middle east, where it caused thousands of human infections, with an estimated 598 deaths, during a single epidemic. 20 more than 100 species of arbovirus that cause human/animal or zoonotic diseases have been identified. 12 four virus families, togaviridae, flaviviridae, bunyaviridae and reoviridae, contain most of the arboviruses that cause human/animal diseases. 12 arbovirus infections are not always clinically obvious and often resolve spontaneously after 1-2 weeks. however, some arboviral infections result in high fever, hemorrhage, meningitis, encephalitis, other serious clinical symptoms, and even death. therefore, they cause a large social and economic burden. a summary listing the arboviruses associated with human diseases and their geographic distributions was published previously. 21 arboviruses have evolved a wide variety of strategies to ensure their long-term success, dispersal and survival. they associate with specific arthropods and exhibit distribution characteristics that reflect the environmental preferences of these particular species. however, once an epidemic has run its course, the virus survives via its sylvatic life cycle which may involve a wide variety of species currently not identified. alternatively, arboviruses can be maintained for months or maybe even years in mosquito eggs which remain dormant until the rainy season triggers hatching of the new, healthy but infected mosquito larvae. 21 eggs can also provide a long term reservoir for tickborne arboviruses but in many cases the viruses exploit the extended life cycle of some tick species, surviving for years through the transstadial stages, reproducing at low levels. 21 this long-term survival strategy is also enhanced by non-viraemic transmission during which infected and non-infected ticks co-feed on small animals in the forests. non-viraemic transmission provides an efficient mechanism for transmission of the virus directly between ticks, without necessarily infecting the vertebrate host. whilst co-feeding is taking place, virus infectivity in the tick salivary glands may increase by orders of magnitude presumably increasing virus transmission efficiency between the ticks. 22 a similar non-viraemic co-feeding transmission process involving mosquitoes and blackflies, has also been described for wnv and vesicular stomatitis virus. 23 alternatively some insect-specific flaviviruses exist in the form of dna when they infect insect cells. 24 diverse sequences have been discovered that appear to be related to insect-specific flaviviruses, amplified from mosquitoes, belonging to factors, strategies and challenges for arbovirus control g liang et al 2 the culicine genera culex, aedimorphus, ochlerotatus and/or stegomyia. many of these sequences may represent dna integrations into mosquito genomes. [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] moreover, srnas related to wnv in apparently uninfected culex mosquitoes have also been identified. 35 whilst there is no current evidence that such forms of dna, provide a long-term arbovirus survival strategy, the similarity with other viruses that use dna intermediates and episomal dna should not be ignored. these and other diverse survival strategies provide safe havens for their longterm survival from which they can reemerge to cause epidemics amongst human and animal populations. changing anthropological behaviour, climate change and high mutation frequency are important determinants of arbovirus emergence. arboviruses adapt readily to new susceptible hosts by alteration of receptor specificity, transmission efficiency, antigenicity, and ecological and environmental conditions. humans, livestock and/or domestic animals are not an essential part of this arbovirus life cycle. therefore, unlike, smallpox virus, measles virus or poliovirus, arbovirus disease control based on humans, livestock and/or domestic animals cannot eradicate the arbovirus. consequently, the reservoir for arboviruses in wild species places a limitation on our ability to control disease emergence. for instance, chikv was a zoonotic arbovirus that cycled harmlessly between simians and mosquitoes in the african tropical forests causing localized outbreaks of polyarthralgia in humans. it also occasionally ''escaped'' to asia gradually becoming zoonotic. however, prior to 2005, chikv was rarely an epidemic arbovirus until a mutation occurred in the gene encoding the surface protein of the african strain which increased its capacity to infect, reproduce and be transmitted by the striped asian ''tiger'' mosquito aedes albopictus. 36 coincidentally this mosquito species has gradually dispersed westwards and chikv is now a major global human epidemic pathogen throughout asia. 5 moreover, on 6 december 2013, it was reported to have crossed the atlantic ocean, reaching the french caribbean island of saint martin from where it dispersed to the americas. 37 it also dispersed eastwards from southern asia, reaching polynesia by october 2014. 6 worryingly, chikv is now frequently being introduced into non-endemic europe and northern asia by incoming humans infected in the americas and polynesia. in the americas, wnv, was first reported in new york in august 1999, 38 following a hot and humid summer and many publications describe its emergence and dispersal. 21, [39] [40] [41] in contrast with chikv, the major determinant for the dispersal of wnv, throughout north america, over a period of five years was primarily birds and their associated culex mosquito species. 36 the dispersal and increasing epidemicity of dengue fever/dengue hemorrhagic fever which is confined to humans in the tropics, sub-tropics and southern temperate regions, can generally be attributed to human and aedes aegypti population density increase during the past century, resulting from intensive urbanization and the influence of increased transportation of humans, commercial goods, livestock and major military movements across the oceans. 42 on the other hand, rvfv is enzootic and confined to a wide range of animals, mosquitoes and sandflies throughout africa and the arabian peninsula. generally, the virus circulates without causing major disease outbreaks which usually arise following periods of rainfall when herded ruminants are introduced to rvfv-endemic areas. two important factors are believed to have influenced the epidemiology of rvfv, firstly, major irrigation projects and secondly, the el niño effect. 43 in each case, cited above, a combination of two or more of the factors identified earlier have had an important influence on the appearance of these emerging arboviruses in new territories and/or old territories. other recently emerging arboviruses, not included in this brief outline, include, zika virus in oceania, bluetongue virus and schmallenberg virus in northern europe and bagaza virus in spain. [44] [45] [46] [47] strategies for arbovirus control the concept of arthropod-borne disease transmission was born out of the studies of a physician, josiah clark, 48 and 40 years later developed by carlos finlay 49 who proposed mosquitoes as the agents for transmission of yellow fever. subsequently, empirical methods, such as mosquito eradication, which was used very successfully in cuba, 50 and the development of a yellow fever vaccine has protected millions of humans from potentially fatal infection by yellow fever virus. [51] [52] [53] now, in the 21st century genetically engineered live attenuated vaccines can be manufactured within months, to protect humans against the ravages of pandemic influenza and other virus diseases. moreover, a spectrum of antiviral molecules has been developed to treat humans dying from infection with human immunodeficiency virus and several antiviral drugs have also been developed against other viruses. does this mean that if we develop vaccines and antiviral drugs to prevent or treat humans against infection by pathogenic arboviruses we will resolve the challenges associated with emerging arboviruses? regrettably it is not that simple! it is a remarkable fact that in the future, because of their high mutation rates, many new pathogenic arboviruses will emerge even though they do not currently exist as epidemic strains in the sylvatic environment. it is also becoming clear, from early results of genome sequencing, that mosquitoes carry large numbers of known and unknown viruses that infect humans, primates, mammals, birds, insects, and plants. [53] [54] therefore, should we attempt global eradication of arthropods? the answer is a definite no. this would have a catastrophic impact on the survival of many wildlife species, as first became apparent when dichlorodiphenyltrichloroethane was used widely as a general insect control agent rather than being precisely targeted to relevant mosquito species. 55 however, implementation of temporary localized arthropod control measures during epidemics, for example in high density urbanized areas, can still play an important but transient role in reducing the impact on humans and animals of emerging arboviruses. the breadth and depth of arbovirus surveillance differs regionally, and several areas lack surveillance altogether. there is also a lack of interdisciplinary expertise on arbovirus diseases and understanding their vectors, and epidemiology. in addition, only a small number of arboviral diseases can be prevented using vaccines or specific antiviral drugs, and there are few validated diagnostic reagents, with which to monitor disease progress and control. until such disease control reagents become available, the most effective alternative is to focus on practical procedures to reduce risks of exposure to arthropods. it is clear that we are now entering a period in virological discovery that will reveal a ''pandora's box'' of new viruses with unique characteristics, which circulate in the ecosystem and will potentially lead to the evolution of novel pathogenic arboviruses. future disease control strategies must therefore recognize this and be planned accordingly. what are the major objectives that need to be addressed if we are to win the war against these versatile viruses that appear to have an infinitesimal variety factors, strategies and challenges for arbovirus control g liang et al 3 of strategies to confuse and disorganize our ability to bring them under control? (i) develop vaccines to reduce the incidence of disease caused by known viruses; (ii) develop therapeutic drugs to treat clinical diseases caused by known viruses; (iii) develop unified vector-controlled strategies that will not unduly threaten the survival of wildlife species but locally will reduce the risk of disease in humans and animals; (iv) develop universal teaching/training courses to be taught worldwide, to provide cores of expertise to implement these policies; (v) encourage the strengthening of levels of cooperation between academia and drug and vaccine development companies; (vi) encourage the development of research programmes to understand the underlying mechanisms of arboviral pathogenicity, evolution, emergence and dispersal; (vii) develop, at the international level, public health measures to inform and educate citizens in local arboviral disease control measures, including monitoring and reporting; (viii) implement measures to improve monitoring procedures at borders, harbours, airports to reduce the influx of arthropods to new countries; (ix) develop unified public health strategies for arboviral disease control; (x) simplify the procedures for establishing safety and efficacy of antiviral drugs; (xi) establish an international committee of experts charged with the objective of reviewing global arthropod control strategies; (xii) develop and implement internationally acceptable and user-friendly guidelines for avoiding exposure to the different types of arthropod likely to carry human pathogens. this is undoubtedly an incomplete list of recommendations and probably some recommendations cannot easily be implemented. nevertheless, the list provides the building blocks for a unified strategy on which to develop methods that will reduce the high morbidity and mortality rates due to human or animal arbovirus infections. finally, we also have to recognise that arboviruses and arbovirus-related viruses infect more than just humans, invertebrates 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communities and ecosystems in europe bagaza virus in partridges and pheasants the mississippi valley's great yellow fever epidemic of 1878 propagation of yellow fever: observations based on recent researches application of environmental management principles program for eradication of aedes (stegomyia) aegypti (linneus, 1762) in the republic of cuba yellow fever vaccine safety working group. history of thymoma and yellow fever vaccination broad surveys of dna viral diversity obtained through viral meta genomics of mosquitoes novel virus discovery and genome reconstruction from field rna samples reveals highly divergent viruses in dipteran hosts debating the health effects of ddt: thomas jukes, charles wurster, and the fate of an environmental pollutant this work is supported by grants from national natural science foundation of china (81290342), the ministry of science and technology, china (2011cb504702), development grant of state key laboratory for infectious disease prevention and control (2014sklid103), the eu 7th framework programmes, silver and predemics (grant agreement nos 260644 and 278433, respectively). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. this work is licensed under a creative commons attribution 3.0 unported license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/ factors, strategies and challenges for arbovirus control g liang et al 5 key: cord-340101-n9zqc1gm authors: bzdok, danilo; dunbar, robin i.m. title: the neurobiology of social distance date: 2020-06-03 journal: trends cogn sci doi: 10.1016/j.tics.2020.05.016 sha: doc_id: 340101 cord_uid: n9zqc1gm abstract never before have we experienced social isolation on such a massive scale as we have in response to covid-19. yet we know that the social environment has a dramatic impact on our sense of life satisfaction and well-being. in times of distress, crisis, or disaster, human resilience depends on the richness and strength of social connections, as well as active engagement in groups and communities. over recent years, evidence emerging from various disciplines has made it abundantly clear: loneliness may be the most potent threat to survival and longevity. here, we highlight the benefits of social bonds, choreographies of bond creation and maintenance, as well as the neurocognitive basis of social isolation and its deep consequences for mental and physical health. j o u r n a l p r e -p r o o f 3 are conventionally most concerned about all had much less impact on survival rates. key factors included obesity, diet, alcohol consumption, how much exercise was taken, the drug treatments prescribed, and local air pollution. these authors conducted a follow-up analysis of 70 studies of longevity in older people, which followed ~3.5 million people over an average of ~7 years [16] : social isolation, living alone and feeling lonely increased the chances of dying by about 30%, even after accounting for age, sex and health status. many other studies have shown that social isolation (though not self-reported feelings of loneliness) was a significant predictor of the risk of death. for example, a longitudinal analysis of ~6,500 british men and women in their fifties [17] found that being socially isolated increases the risk that you will die in the next decade by about 25%. quantitative analysis of nearly ~400,000 married couples in the american medicare database revealed that, for men, the death of their spouse increased their own chances of dying in the immediate future by 18%. the death of the husband in turn increased the wife's risk of dying by 16% [18] . similar effects on morbidity rates have been found with respect to social support. a series of elegant prospective studies using data from the framingham heart study [19, 20] found that the chances of becoming happy, depressed or obese were all strongly mirrored by similar changes in the closest friend. there was a smaller significant effect due to the behaviour of the friends' friend. even a just detectable effect was present due to the friend of a friend's friend, but nothing beyond. this contagion phenomenon was especially strong if the friendship was reciprocal (i.e., both individuals listed each other as a friend). if the friendship was not mutual, the social contagion effect was negligible. the investigators also documented a strong effect of "geographical contagion". if you have a happy friend who lives within a mile radius, you are 25% more likely to become happy. and you are 34% more likely to be happy if your next-door neighbour is happy. people who belong to more groups are less likely to experience bouts of depression. such findings emerged from the uk longitudinal study of ageing (elsa) that repeatedly profiled around ~5,000 people from the age of 50 onwards. previous research showed [21] that depressed people reduced their risk of depression at a later time point by almost a quarter if they joined a social group such as a sports club, church, political party, hobby group or charity. indeed, joining three groups individuals were more immersed in their local community and trusted their neighbours more [22] [23] [24] . the causal directionality was difficult to pin down in these cases because of the cross-sectional nature of the data. nevertheless, path analysis provided some indication that intensity of social exchange was the candidate driver. the impetus to access social capital in the wider community [7] extends beyond humans. there is now a wealth of evidence from long-term field studies of wild baboons that socially wellconnected females experience less harassment by other monkeys [7, 23] , have lower levels of cortisol stress hormones [25, 26] , faster wound healing [27] , produce more offspring and live longer [28] [29] [30] [31] . such ramifications of social capital appear to hold up across a diversity of species, including chimpanzees [32] , macaques [33] [34] [35] , feral horses [36, 37] and dolphins [38] . a key underlying reason for these effects, at least in humans, is likely that loneliness directly impairs the immune system, making you less resistant to diseases and infections. research found [39] that freshmen students who reported feeling lonely had a reduced immune system response when they were given a flu vaccine compared to students who felt socially well engaged. moreover, those students with only 4-12 close friends had significantly poorer responses than those with 13-20 friends. these two effects seemed to interact with each other: having many friends (a large social group of nineteen or twenty friends) seems to buffer against a weakened immune response. yet, feeling lonely and having few friends results in a particularly poor immune defence. other investigators [40] used data from the framingham heart study to show that people with fewer contacts in their social network had elevated serum fibrinogen concentrations. in contrast, people enjoying many social contacts had low fibrinogen levels. fibrinogen plays an important role in blood clotting when a blood vessel has been ruptured, as well as facilitating wound healing and tissue repair more generally: high concentrations thus signal poor health. endorphins constitute a core component of the psychoendocrine mechanism underpinning friendship (see box 1). other research found [41] that social bonds stimulate the release of the body's natural killer cells, one of the white blood cells of the innate immune system whose core function is to destroy harmful bacteria and viruses. people who are more socially integrated have better adjusted biomarkers for physiological function, as indexed by lower systolic blood pressure, lower body mass index, and lower levels of creactive protein -the latter being another molecular response to inflammation. this insight was evident in each of four age groups (adolescents, young adults, middle age and old age) based on data from four large longitudinal american health databases [42] . the investigators found that, in j o u r n a l p r e -p r o o f adolescence, lack of social engagement had as big an effect on risk of inflammation as lack of physical activity. in old age, lack of friends had a bigger effect on risk of hypertension than the usually cited clinical causes like diabetes. even more worrying, the effects of social relationships on these physiological measures of good health during adolescence and young adulthood can persist into old age. in a longitudinal study of 267 males, for example, research found [43] that the more socially integrated a child was at six years of age, the lower their blood pressure and body mass index (a measure of fatness) two decades later in their early thirties. this result held up when they controlled for race, body mass index in childhood, parental socioeconomic status, childhood health and extraversion. social isolation may well have pervasive effects on brain connectivity. if rats are socially isolated when young (a condition that would give rise to feelings of loneliness in humans), neural function and plasticity are altered [44] [45] [46] [47] . in particular, episodes of social isolation can irretrievably alter the function of the prefrontal cortex (the part of the brain that is central to managing our social relationships [see below]), as well as its axon myelinisation (the laying down of the fatty sheaths around neurons that enable them to transmit signals faster and more efficiently) [44] . while short periods of loneliness in humans rarely have any long-term adverse outcomes, persistent loneliness escalates the risk of alzheimer's disease and depression [48, 49] . loneliness also leads to poor sleeping habits, with adverse psychological and physiological consequences [50] . the fact that friends can have such dramatic effects on our health and well-being may lead us to suppose that the more friends we have, the better. however, the number of friends and family relationships we can manage at any given time is limited by cognitive constraints to ~150 [51, 52] . there is, however, considerable individual variation, with social network sizes ranging between approximately 100-250. a number of fairly conventional factors are responsible for this variation: age (younger people typically have larger social networks than older people [53] ), sex (females usually have larger social networks than males [53, 54] ; though this does vary with age [55] ), personality (extraverts have larger social networks than introverts [56] ; women who score high on the neuroticism personality dimension have fewer acquaintances than those who score lower [57] ). friendships, however, require the investment of considerable time to create and maintain. the emotional quality of a friendship depends directly on the time invested in a given social link [57] [58] [59] . one prospective study estimated that it takes around 200 hours of face-to-face contact over a three-month period to turn a stranger into a good friend [60] . conversely, the emotional quality of a j o u r n a l p r e -p r o o f 6 relationship will decline rapidly ( figure 1 ) if contact rates drop below those appropriate to the relationship quality [61] . time resources, however, are naturally limited: we devote only around 20% of our day to direct social interaction (excluding business-related interactions), equivalent to about 3.5 hours per day [62] . given that our relationships are not all of equal value to us (friends serve a variety of different functions for us [63, 64] ), we allocate our valuable time across our social network in such a way as to maximise the different benefits that friends of different quality provide [65] . this dynamic results in a specific social fingerprint that is unique to each of us [66] . nonetheless, there are some broadly consistent patterns: a 40% share of our time is devoted to our five closest friends and family, and a further 20% to the ten next closest individuals. in other words, 60% of the 3.5 hours a day we spend in social interaction are devoted to just 15 people. social partners in the outermost layers of the social network each receive just 30 secs of our time a day on average. this gives rise to a very distinctive layering to our social networks, with layers that have a characteristic fractal pattern: the innermost layers of closest friends is very small (typically 5 people) but intense, the outermost ( ~150) very large but more casual [67, 68] . it is that inner circle of five closest friends and family that seems to matter most in terms of the buffering of both loneliness and disease. geographical distance also imposes strong constraints on the organization of friendship. the '30-min rule' provides an empirical reminder that people are less willing to visit friends and family who live more than 30 mins away -no matter whether that involves travel on foot, by bicycle or by car [69] . cutting across this effect is the influence of genetic relatedness: the kinship premium (i.e., the strong mutual benefits that kinship typically affords) incentivizes us to travel an extra mile to maintain contact with family than we are with friends [70] . while the role of close contacts, like friends, is pivotal, other regular contacts can also contribute to one's social capital. previous authors [71] famously claimed that weak -as opposed to strong, or close -ties provide important sources of external information. analyses of information flow in social networks suggest that sources outside the 50 closest friendships offer few benefits [72] . other benefits of interaction with more loose social ties can, of course, include heightened subjective well-being and sense of belonging to the local community [73] . however, as is often the case in such studies, it is crucial to precisely define the meaning of weak versus strong ties, since all weak ties belonged to the same community (a student class). regular interaction with different people at the periphery of social networks can give rise to heightened perceived social and emotional fulfillment in ways that act as psychological buffers [24] , although this might depend on personality or social style [74] . social-affective processes in the presence of others take a different form than during the others' physical absence. already in a nursery, if a baby starts crying, other nearby babies hear the distress signal and typically also start crying by mere emotional contagion. in addition to utterances and prosody, humans tend to align their communication towards each other by imitating vocabulary, grammar, mimics and gestures. for instance, humans tend to unconsciously synchronize their facial expressions even with people who are directing gaze at somebody else [75] . such subliminal motor and emotional resonance is typically found to be intrinsically rewarding [76] . on the positive side, contagion processes can uplift an individual's happiness through people within the close neighborhood, but also miles apart [19] . on the negative side, loneliness also spreads rapidly through an individual's social interaction partners, thus affecting even friends of friends of friends [77, 78] . reading others' faces -impossible during a conventional phone call -may be an evolutionarily conserved means for exchanging pivotal information, which coevolved with the corresponding decoding machinery in brain and behavior responses (see next section). faces offer a plethora of social information about an individuals' sex, age, ethnicity, emotional expression and potentially their intentions and mental state (all of which influence the strength of the bond between two individuals [59] ). throughout development, learning and maturing critically hinge on joint attention of two individuals on the same object [79, 80] . such mentalizing and eye gaze processes have been repeatedly linked to the higher associative and the striatal reward circuitry [79, [81] [82] [83] . some authors even argue that the importance of such facets of interpersonal exchange may explain why humans developed wide and white sclera in the eyes -more easily visible than in most animals [84] . what may lead to greater vulnerability to predators for some species (by making the individual and her intentions more visible and exploitable) may have boosted learning and cooperation in human primates [85] . such evolutionary adaptations facilitate how humans automatically represent the (visual) perspective of nearby others. making statements about objects channels [88] . compared to actual interpersonal encounters, a surprising number of psychological constants exist in how humans entertain and juggle with social relationships in digital environments. for example, the upper bound of ~150 contacts (cf. above), as well as the structure of these networks, appears to hold across both the real world and a variety of virtual online contexts [53, 68, 89, 90] , suggesting that group size in today's society is still orchestrated by the same principles as when we were hunter-gatherers. indeed, several neuroimaging studies [e.g., 51, 91] broadly confirm that our online social networks correlate with the volumes of the same core brain regions that resonate with the size of our offline networks [52, 92] . these constancies suggest that lively virtual social interaction may similarly entrain faculties like memory and concept generation. conversely, paucity of social interaction and loneliness may have deleterious effects on the cognitive and memory systems. it is conceivable that enhancement or decline of cognitive and neural reserve may be mediated by analogous pathways potentially involving dendritic arborization in the hippocampal and prefrontal regions [49] . the need for personalized interactions may already be reflected in the way that stock market traders sometimes add coded numbers to money transfers (e.g., 10,000,467 instead of 10,000,000 shares) as a potential replacement for the recognition of somebody's unique facial identity rather than remaining anonymous [93, 94] . this attractor for a full range of face-to-face cues during social interactions may explain why emojis have become so popular: they replace the important emotional signals in the absence of the ostensive facial cues that we use for the interpretation of utterances in the face-toface environment. these considerations raise the important question how the brain implements toggling between real-world social interactions and virtual or imagined social interaction in the absence of physical contact [79] . the right temporoparietal junction was proposed as a key switching relay between two antagonistic classes of neurocognitive processes: those more anchored in one's current external sensory environment and more stimulus-independent ones relying on internally generated information [95] . this idea was later substantiated by a multi-modal neuroimaging study in 10,000 humans [96] : the right and left temporoparietal junction explained most variation in functional coupling changes between all major brain networks. hence, these two association cortex regions may help mediate shifts of focus from the person in front of you to a person you are texting with on the phone, who is out of sight or touch. taken together, evidence of digital communication suggests that this new medium does not in fact change the general pattern of our social interactions or the numbers of people we contact [68, 89, 90, 97] . the sizes of the layers in our social networks are unchanged by using digital media or virtual communication. also, the frequencies with which we contact certain people in each social j o u r n a l p r e -p r o o f layer are strikingly similar in the online and offline worlds. some digital vehicles, however, lack the communicative richness of real face-to-face interactions: when asked to rate their satisfaction with interactions with their five closest friends each day, participants rated face-to-face and skype interactions as equally satisfying and both as significantly more satisfying than interactions with the same individual by phone, text messaging, sms messaging, email or text-based social media such as facebook [87] . human and non-human primates live in groups mainly to minimize external ecological threats, including predators, raiding by neighbors, and environmental risk. advanced forms of cooperation are rare in non-primate species [98, 99] and probably emerged in non-human primates several million years ago. today, the average humans spends up to 80% of waking hours in the presence of others [100, 101] . investing cognitive resources in keeping track of friends, family and colleagues is highly demanding -more costly than contemplating the physical facts [102, 103] . not only time limits (cf. above) but also neurocognitive limits [e.g., 104] effectively constrain how close one can be to how many individuals. but how is regular social stimulation reflected in neurobiology? in monkeys [105, 106] and in humans [51, 52, 107, 108] , various indices of sociality and measures of social network size are robustly associated with specific regions of the neocortex. these same regions are responsible for processing social information such as predicting others' intentions [109, 110] . at least some of these brain-behavior associations may be cross-culturally consistent in humans, as evidenced by a structural neuroimaging study in the usa and china [111] . whole-brain analyses have repeatedly highlighted a relationship between the ventromedial prefrontal cortex and measures of social network complexity and social competence [92, 105, 110, [112] [113] [114] [115] . the ventromedial prefrontal cortex and striatal nucleus accumbens have been found to play a key role in both social reward behaviors and the amount of social stimulation in humans [113] and other mammals [e.g., 44, 47] . functional neuroimaging has shown that these neural correlates are also implicated in tracking others' popularity status in real-world social networks [116] . similarly, positron emission tomography has shown that, in humans, the density of mu-receptors for betaendorphin, especially in the ventromedial prefrontal cortex, correlates with social attachment style, for which endorphins are more important than other neuropeptides [117] . other evidence, such as in a functional neuroimaging study on maintenance and manipulation of social working memory [104] , has also related the dorsomedial prefrontal cortex to social network properties. there are similar correlations for social cognitive skills like mentalizing that are crucial to maintaining functional social relationships [118] [119] [120] . analyses of social richness and brain morphology in humans tend to identify a neural network involving the prefrontal cortex with several parts of the so-called default mode network as being crucial for managing social networks (e.g., noonan et al., 2018) . this major brain network of the higher association cortex has probably recently expanded in primate evolution [121] . its constituent regions are often thought to support several of the most sophisticated neurocognitive processes [122, 123] . in monkeys, there is evidence that experimental manipulation of social group size results in adaptations in the volume of frontal brain regions, the posterior superior temporal sulcus or temporo-parietal junction, as well as the amygdala and other parts of the limbic system [105, 106] . in humans, there is evidence for structural coupling between social network size measured by number of online friends and parts of the default mode network, including the hippocampus [51] . from a clinical perspective, functional connectivity alterations in the default mode network have been demonstrated as a consequence of feelings of loneliness in younger adults [124] . moreover, the default mode network is especially subject to vulnerability in normal cognitive aging [125] , and is among the main brain circuits to be impacted by neuropathology in alzheimer's disease [126, 127] . complementing higher associative parts of the human social brain [128] , amygdala volume is larger in individuals with more extensive social networks in humans [52, 107] . amygdalar functional connectivity was also reported to increase with canonical brain networks implicated in face perception and approach-avoidance behaviour [107] . indeed, previous authors reported [129] that a patient with complete bilateral amygdala lesions lacked a sense of appropriate personal space vis-ã vis other people (figure 3 ). this patient exhibited no discomfort when at close distances from another person, even to the point of touching the other's nose -despite the fact that their conceptual understanding of people's private physical space was intact. in contrast, healthy individuals typically show amygdala activation in response to close personal proximity. in a similar vein, the grey-matter volume of the amygdala correlated negatively with social phobia [130] . the amygdala may hence be required to trigger the strong emotional reactions normally associated with personal space violations, thus regulating interpersonal distance in humans. such reports on the social brain often seemed to be in conflict about whether they highlight the prefrontal cortex or the amygdala of the limbic system. this apparent discrepancy was reconciled in a recent population neuroimaging study [131] : social traits such as daily exchange with family, friends, and work colleagues were associated with brain morphology in ~10,000 uk biobank participants. particularly prominent findings were reported in the limbic system, where volumes varied consistently with various indicators of social isolation. less socially stimulated participants showed volume effects in various parts of the social brain including the ventromedial prefrontal j o u r n a l p r e -p r o o f cortex and the amygdala, in addition to the nucleus accumbens of the reward circuitry. volume effects in these regions were reported for several markers of brittle social integration, such as living in a socially "emptier" household, knowing fewer individuals with whom to regularly share experiences and concerns, feeling unsatisfied with one's friendship circles, as well as having grown up without brothers or sisters and being unhappy with one's family situation [131] . this analysis also demonstrated wide-ranging sex differentiation in how traits of social isolation are linked to brain morphology. these findings underscore evidence from animals for a sex specific co-evolutionary relationship between the primate brain and social complexity [social brain hypothesis: 132, 133]. the perspective of brain network integration in loneliness was investigated in a seminal neuroimaging study of intrinsic functional connectivity in ~1,000 humans [124] . careful analysis showed that feelings of loneliness especially affect the neural communication strength between the limbic system and the default mode network as well as the communication strength inside of the default mode network. as a particularly discriminatory pattern for loneliness, impoverished functional modularity was found for the default mode network and its interacting brain networks. in contrast, a positive sense of one's meaning in life was linked to strengthened functional differentiation of the canonical network ensemble. the collective evidence led the investigators [124] to argue that the default mode network and its coupling partners represents a neural signature reflecting one's own purpose in life versus social disconnection to others. according to unicef estimates, ~140 million children worldwide live deprived of parents who could provide comfort and support. ~8 million of these children grow up in institutions without the socioemotional context of a regular family. in one of the earliest randomized clinical trials of its kind, orphans raised in institutions were systematically compared to orphans who were later welcomed into a foster home [134] . abandoned children were randomly assigned either to remain under the care of the institution or to transition to the care of foster-parents. their cognitive trajectories were monitored over several years. those children who remained in the institution showed significantly lower development indices and lower iqs [of around 70: 134] than the adopted orphans. being deprived of social bonds with caregivers also led to a pernicious reduction in grey-and white-matter tissue and lower fiber tract integrity as evidenced by brain mri [134] . institutional rearing was also shown to exacerbate the decay of the telomeres in cell nuclei [135, 136] . these protection caps normally prevent chromosome deterioration, which acts like a cellular sand clock of aging. their shortening has major consequences for various biological pathways and health outcomes. the younger the children were when adopted by a foster family, the better the cognitive performance later [137] . impoverished cognitive domains include memory and executive function: for orphans who transitioned to a foster home, some cognitive facets remained below-average throughout later life (e.g., short-term visual memory and attention allocation). other cognitive dimensions (e.g., visual-spatial memory and spatial working memory) caught up with a normal trajectory at age 16 [134] . such unique evidence underlines the fact that lack of socioemotional context in early life severely impedes brain development and maturation of the cognitive repertoire, which can be partially mitigated by developing social bonds to non-genetic parents (see box 2) . early psychosocial deprivation also shows inter-generational effects, which are probably mediated through maternal and epigenetic effects [138] . social isolation in childhood leads to molecular annotations of the genetic strand (such as methylation or phosphorylation of the histones that provide the structure for dna strands) that are passed on to influence how children cope with stress and in turn how they raise their own children. for instance, in rats, socioemotional experience as a pup has an impact on how the rat's own pups later deal with stress and high anxiety levels [139] . epigenetic regulation of gene transcription is involved in how maternal care promotes the rat pup's brain development and cognitive maturation. more licking and grooming by the mother increases protein expression of the grm1 gene in the pup's hippocampus. this up-regulated gene transcription leads to greater availability of glutamate receptor proteins in hippocampal cells for inter-neuronal signaling [140] . in humans, a longitudinal neuroimaging study indeed showed that social support from the mother promotes volume growth trajectories in the hippocampus, and predicts socioemotional development and emotion regulation in early adolescence [141] . in young rhesus monkeys, loss of social contact to the mother leads to behavioral aberrations that last right into adulthood. such social isolation was shown to entail down-regulated dendritic growth in the prefrontal cortex and reduction in gene expression in the amygdala [142] . social adversity undergone by children with institutional upbringing led to disturbed functional connectivity between the prefrontal cortex and the amygdala [143] . such perturbed brain maturation through social deprivation may be mediated by glucocorticoids, which are known to be inhibited by maternal care in primates [144] . hence, maternal care is a critical enrichment of the social environment that promotes maturation, expression of growth hormones, and synaptogenesis in various brain circuits. in contrast, social neglect leads to disturbed social attachment, as well as increased aggression and hyperactivity, often potentially lifelong [145, 146] . how vulnerable an individual is to parental deprivation is subject to complex nature-nurture interactions that are strongly conditioned on personality and overall genetic endowment [147, 148] . rats separated early from their mothers were impaired in adult life in emotion regulation and arousal management [149] . early socioemotional isolation of rat pups had impact on whether these rats later showed healthy responses to stress by mounting adequate cortisol levels [150] . hormones of the hypothalamic-pituitary-adrenocortical (hpa) axis are an important endocrine mechanism of stress neurobiology that plays a key role in social isolation. in baboon monkeys, infant survival is jeopardized for mothers who are more socially isolated and not well integrated in the local communities including ties to sisters, adult daughters, and other mothers [151] . monkey mothers with a thinner social network are less likely to have infants which themselves have high fitness [28] . female baboon monkeys with a larger close social circle of grooming partners have healthy cortisol levels and typically deal better with stressful situations [25, 26, 152] . when one of these strong social bonds is disrupted, such as when a close member of the social group is killed by predators, cortisol titres rise in the blood. such monkeys then tend to seek out new connections to "repair" the lost link in their social network [153] . a lower-than-usual cortisol level in the morning is indicative of extended stress periods in adults [154] . the same diurnal cortisol dynamic is frequently observed in disturbed child-caregiver relationships [155] . in rhesus monkeys, a low hormone response has been observed after repeated separations from the mother. the same observation has been reported for children who were moved between several caregivers. an intact child-caregiver relationship probably provides a stress reserve to adrenoreceptor responses so that children get over stressful episodes quicker [156, 157] . after undergoing adversity in early childhood, such as emotional or physical neglect, maltreatment, or maternal separation, enhancement of the child-caregiver relationship can mitigate the effect of previous hits to the hpa system. early disturbance in important social relationships is linked to dysfunctional cortisol hemostasis in adult life [158] . in some neglected children, ensuing problems and behavioral disruptions can even be exacerbated in adult life [159] . abnormal blood cortisol levels can potentially be prevented, mitigated or restored by family-based therapy and other interventions [160] . nonetheless, dysregulated diurnal cortisol levels are further linked to various mental disorders including major depression, substance abuse, and post-dramatic stress disorder [154] , in addition to stress-induced impact on the immune, cardiovascular, and metabolic systems [161, 162] . further insight into the neurobiology of social isolation has also been derived from rigorous experiments with adult primates (see also box 3). in one study, 20 monkeys were separated from others to live alone for 1.5 years [163] . subsequently, monkeys were re-integrated into social groups of four monkeys housed together. repeated positron emission tomography (pet) scanning revealed increased levels of d 2 receptors in the basal ganglia, which includes key nodes of the reward j o u r n a l p r e -p r o o f circuitry (see above), after being socially housed. this neurochemical adaptation in the monkeys' brain circuitry was apparent after as few as 3 months of social rehabilitation [163] . these authors also reported several differences in respect of social integration and social rank: monkeys of higher rank were groomed more by others. in contrast, subordinate monkeys spent more time by themselves. as a consequence at the behavioral level, the lower-rank monkeys were also significantly more willing to self-administer cocaine, which may also relate to heightened drug abuse in lonely humans [164] . such molecular imaging evidence shows that changing from social deprivation to an environment with constant social stimulation causes neural remodeling in the dopaminergic neurotransmitter pathways in non-human primates, which may be clinically relevant for substance abuse disorders in humans. we are social creatures. social interplay and cooperation have fuelled the rapid ascent of human culture and civilization. yet, social species struggle when forced to live in isolation. the expansion of loneliness has accelerated in the past decade. as one consequence, the united kingdom has launched the 'campaign to end loneliness' -a network of over 600 national, regional and local organizations to create the right conditions for reducing loneliness in later life. such efforts speak to the growing public recognition and political will to confront this evolving societal challenge. these prospects should encourage us to search for means to mitigate possible negative backlash. we offer some suggestions in box 4. additional insight into stress-responsive brain systems is imperative to tailor clinical decision making and therapeutic interventions to single individuals. there is also a dire need for additional longitudinal research on the hpa axis and the cortisol response to psychological stressors. we are grateful to guillaume dumas and tobias kalenscher for valuable comments on a previous version of the manuscript. db was supported by the healthy brains healthy lives initiative (canada first research excellence fund), and by the cifar artificial intelligence chairs program (canada institute for advanced research). primates service their relationships through social grooming. grooming triggers the endorphin system in the brain through a very specific neural system: the afferent ct fibres [165] . these axon bundles have receptors at the base of most hair follicles, have the unusual properties of being unmyelinated (and hence very slow, especially compared to the pain receptors in the skin), with no return motor loop (unlike pain and other proprioceptive neurons), respond to a very specific stimulus (light slow stroking at ~2.5cm per sec) and directly trigger the endorphin reward system [166] . although humans no longer have the full fur covering that encourages social grooming, we still have the receptors and instead use physical contact in the form of touching, stroking, caressing, and hugging as a means for strengthening social ties in our more intimate relationships [167, 168] . physical touch is intimate, and hence limited mainly to close family and friends ( figure 2 ). to bond our wider range of relationships as well as our more intimate ones, humans exploit a number of behaviours that turn out to trigger the endorphin system. these joint activities include laughing [169, 170] , singing [171, 172] , dancing [173, 174] , feasting [22] and emotional storytelling [175] . an important feature of all these behaviours is that behavioural synchrony seems to ramp up the level of endorphin release [174, 176] . in baby primates, close social interaction is not only beneficial, but critical for maturation and resilience. experiments in baby monkeys showed that upbringing in social isolation during the first years causes a variety of social deficits. when separated early from their mothers, baby monkeys showed strong symptoms of social withdrawal: self-hurting behaviour like biting, stereotypical and repetitive motor behaviour, excessive avoidance behavior towards others as well as poor social and maternal skills as adults. when separated later from their mothers, baby monkeys tended to indiscriminately approach unknown monkeys without fear [cf. 177] . reports of human children in some crowded russian and rumanian orphanages painted a strikingly similar picture: socially and emotionally abandoned children showed either forward-j o u r n a l p r e -p r o o f backward rocking tics and social escape or overly strong attachment style, analogous to neglected baby monkeys [cf. 178] . these cases invigorated the then-contested claim that mother-child bonds are indispensable for normal development, and that foster-care parents can compensate many of these needs [134, 160, 179, 180] . disruption of social interplay during critical development impacts negatively on cognitive, verbal, social and motor performance, and predisposes to mental health issues. in other words, early neglect remains measurable in brain and behaviour in later life. the socioemotional dialogue between caregiver and baby is mediated in several important ways. mothers speak to their offspring in "baby talk", which potentially evolved only recently in humans [181] . accompanied by direct face-to-face exchanges, these communication bouts with characteristic vocabulary and prosody promote infant development milestones. the interpersonal stimulation grabs the baby's attention, she gains weight faster, modulates her emotional state, and enhances various health outcomes. mother-infant communication is also delivered through direct skin-to-skin contact [166] . postnatal touching bolsters mother-infant bonding, alleviates anxiety, and provides intrinsic pleasure through endorphin release [182] [183] [184] . throughout life and quite independent of geography, primate societies are orchestrated by the creation, curation, and cultivation of social bonds though purposeful social closeness. among the many consequences of loneliness on body and mind, the scarcity of social contact encourages drug compensation behaviour, such as alcoholism, possibly via non-social rewards triggering dopaminergic neurotransmitter pathways [163] . at the genetic level, loneliness was shown to entail under-expression of anti-inflammatory genes involved in glucocorticoid response and over-expression of genes related to pro-inflammatory immune responses [185] . fortunately for future clinical intervention, loneliness may be a modifiable determinant in healthy aging [11] . as people grow older, the social network typically becomes smaller -naturally diminishing the cognitive stimulation through frequent and intense social interaction on a daily basis, thus potentially reducing the neural reserve. over the last century, the average human lifespan in developed nations has increased by nearly three decades. on the other hand, older people were also reported to show a decline in the capacity to take other people's point of view, as demonstrated in three separate mentalizing tasks [163] . these authors showed that social cognition j o u r n a l p r e -p r o o f deficits were related to decreased neural activity responses in the medial prefrontal default mode network [163] . this capacity is likely to be particularly important when introspecting other people's minds who are not physically present -where social cues like facial expression, mimics, and gestures are missing. both limited social stimulation and weakening social reflection capacities relate to the sense of loneliness in complicated and important ways [13] . once lonely, bias for negative information processing of cues from others hinders social rehabilitation in a downward cycle [4, 186] . many recent studies have corroborated the corpus of empirical evidence that the feelings of loneliness escalate the risk of certain neurological diseases and especially alzheimer's disease in later life [49] . social isolation at massive scale risks creating cohorts of individuals who are socially dysfunctional. it may therefore be important to identify ways of mitigating the worst of the effects so as to alleviate the consequences. the following possible countermeasures may be worth exploring: ï�· one promising intervention would involve creating opportunities where mutual social support relationships (friendships) could develop naturally. you cannot, however, force people to become friends: both parties need to be willing to devote resources to each other in a context where available time budget for social engagement is limited [187, 188] and there are competing friendship interests [66] . however, by providing more opportunities for people to meet in congenial environments, new friendships may blossom. ï�· social neuroscientists [189] undertook a longitudinal intervention study on 332 matched adults who underwent regular training sessions. several months of cognitive training improved empathy for others' affective state or perspective-taking of others' mental state, which resulted in structural remodeling in brain regions belonging to the social brain network, including the frontoinsular network and the default mode network. daily affective training resulted in thickening of the right anterior and mid-insula, with correspondingly enhanced compassion ratings. different training regimes correlated with different brain regions. further research is urgently needed to explore therapeutic interventions using training of social capacities in socially deprived humans. j o u r n a l p r e -p r o o f ï�· one important lesson is that joining clubs can have important benefits in reducing both a sense of loneliness and psychological or psychiatric conditions [21] . one obvious solution is to encourage vulnerable individuals to join social groups and communities that suit their interests and abilities. establishing a wide range of such clubs is likely to be much cheaper than paying for carehomes and prisons. ï�· singing is known to have a dramatic, immediate effect on creating a sense of social engagement and elevating psychological well-being [the "ice-breaker effect" : 171] . vulnerable individuals could be encouraged to join choirs and community singing groups. encouragement and funding may need to be invested in establishing a network of choirs. ï�· use of video-embedded digital communication is likely to gain in importance. this is especially true where family and friendship groups can meet in the same virtual space. the visual component of the interpersonal encounter appears to play a key role in creating a more satisfying experience of digital social media [87] . emotional closeness at the start of the study is set at 0 for both groups. redrawn from [61] . regions. in 1,368 people from several countries, this study investigated the permissibility of social touch [167] . the authors showed that human social touch is particularly dependent on the nature of the relationship. the topography of accepted social touching depends on many factors, including a) emotional relationship, b) type of interpersonal bond including kinship, c) sex, and d) power dynamics. close acquaintances and family members are touched for more different reasons. culture influence, measured in five countries, was small. female, rather than opposite-sex, touch was evaluated as more pleasant, and it was consequently allowed on larger bodily areas. reproduced from [167] . ï�· what further refinements of online digital media might improve people's function in creating and maintain friendships, especially for the housebound? it is insufficiently known which types of modern medium best mimic which neurocognitive facets of real social interaction. ï�· which neurobiological mechanisms explain how the default mode network and its connections to subordinate brain systems support higher social capacities, and their decline in social deprivation? this associative brain network needs to be more completely understood; especially regarding the congruencies and idiosyncrasies between healthy aging trajectories, the experience of social isolation, and vulnerability to neurodegenerative pathologies. in terms of progress towards causal understanding, putting a premium on longitudinal studies holds out unprecedented promise. ï�· across the entire lifespan, to what extent does reduced social stimulation or too few social contacts lead to loss in general capacities of the cognitive repertoire? how much do people struggling with cognitive load have issues maintaining many active social relationships? or both? progress in this chicken-and-egg problem will shed light on the aetiopathology of the loneliness, and usher towards new intervention strategies. the phenotype of loneliness evolutionary mechanisms for loneliness perceived social isolation and cognition the growing problem of loneliness loneliness and attention to social threat in young adults: findings from an eye tracker study the cultural context of loneliness: risk factors in active duty soldiers it is all about who you know: social capital and health in lowincome communities counting on kin: 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research network on early experience and brain development early adverse care romania's abandoned children function of infant-directed speech stroking modulates noxious-evoked brain activity in human infants the communicative functions of touch in humans, nonhuman primates, and rats: a review and synthesis of the infants autonomic cardio-respiratory responses to nurturing stroking touch delivered by the mother or the father social regulation of gene expression in human leukocytes examining the visual processing patterns of lonely adults time as a limited resource: communication strategy in mobile phone networks cognitive resource allocation determines the organization of personal networks structural plasticity of the social brain: differential change after socioaffective and cognitive mental training key: cord-348100-jr923fcu authors: giseke, undine title: covid-19—does social distancing include species distancing? date: 2020-06-08 journal: agric human values doi: 10.1007/s10460-020-10066-0 sha: doc_id: 348100 cord_uid: jr923fcu nan we are disoriented. the most current reason for this is the covid-19 pandemic, but our disorientation actually started long before. let's take the expression we as simply meaning "humankind", and covid-19 for the interaction between viruses and mammals, including humans. right now we are being strongly influenced by global phenomena: the disorientation we've already experienced due to global warming, virtual transformation and the expansion of the technosphere has been increased by the addition of another form, i.e. the immediate disorientation caused by a virus. we are stuck right in the middle, across borders-and responsible. when it comes to climate change we have always been (and still are) caught up in a discussion about whether we are already a part of it, but the coronavirus leaves no doubt about this: not in the way it acts or how enters our bodies, nor in the way it spreads globally. the virus ignores and transcends organic, continental and political boundaries. at the same time it closes borders; images of entire fleets of aircraft on the ground and travel bans are examples of this. these are indeed paradoxical phenomena, which exacerbate our disorientation and force us to immediately deal with an unknown. from a systemic perspective, it could be said that the virus unintentionally uses us as a system chain, with the result being that we have broken many other system chains that were considered untouchable up to now. the dissolving of boundaries and the questioning of existing categories and systematics has preoccupied us for some time now. with the beginning of the new century and the concept of the anthropocene, these issues have gained enormous momentum, both in terms of real life and scientific theory. and now, within a matter of weeks, the virus has become a marker, accelerator and spokesperson for the anthropocene discourse. what does the anthropocene imply? it expresses the idea, in short, that the changes in the earth system caused by humans are comparable to geological forces. humans themselves-the global we-are believed to have become a geological factor. the concept of the anthropocene (crutzen 2002) has been taken up by many disciplines since the 2000's and now encompasses different strands of discourse (dürbeck 2018) . it is undisputed that this new geological period is characterised by an extreme increase in humaninduced, material exchange processes everywhere across the planet. it seems, however, that these calculations were made without considering non-human actors such as viruses. with regards to climate change, we can now directly and physically experience the anthropocene changes of a natural system that was long considered stable. the corona virus has forced us to quickly broaden our perspective. we are becoming conscious of the fact that the spread of the human species and its way of living is not only of a geological magnitude. due to the spread of human-influenced habitats, the interaction between humans and animals is also being intensified, and thus, according to virologists, viruses can more easily change from animal hosts to humans. just under half of the viral species found in mammals have so far been detected in humans, and most of them are a result of zoonosis. the causes that have led to these intensified human-animal interactions are manifold, and they go hand in hand with our globalised, neoliberal way of life and economy. population growth and migration, as well as the restriction of habitats for wild animals through an expansion of human activities are cited by scientist as reasons, as are the intensification of agriculture, changes in our food system, a mobility pattern that has lead to more intercontinental travel, a public health system that is desolate in many areas, and global warming. so how do we proceed? one consequence for the disciplines that design space must be on a higher level and fundamentally question the relationship between the co-existence of humans and other (renn 2017) . while in the anthropocene discourse a shift in the centrality of humans to the deep evolutionary processes of the earth's history, i.e. deep time, and the depth of the geological layers of the earth, i.e. geomorphology or deep ground (wieck and giseke 2018) , has already begun, covid-19 has forced us to significantly broaden our focus. technology and animals should be our family today, demands the feminist, biologist and utopian thinker (haraway 2016) in a very radical, inspiring and hopeful way. in order that such future-oriented thinking is not led ad absurdum by covid-19, we should regard the present situation as an indispensable challenge to radically broaden our frame of thought and to orient and apply our thinking and acting to the creation of connections. where do we stand? isn't it absurd that the amount of chicken bones produced worldwide-which is breathtakingly high-was actually discussed as a marker for the scientific classification of the anthropocene (bennett et al. 2018) . what do our global food systems look like? currently, nearly 22 billion chickens coexist with us on this planet at any given time. the number of individual chicken slaughtered annually is almost three times as high, however. how will we co-exist with non-human species in the future? are dairy cows merely a type of technology for us (gan et al. 2016) ? scientists assume that flying foxes, which are possible carriers of coronaviruses, have played a central role in the settlement of so-called evolutionary hotspots, such as the volcanic islands in the indian ocean. ambivalences of a multi-species entanglement. at the current stage of being directly affected by the virus, we face other urgent issues as well: how do we protect ourselves, how do we slow down the spread of the virus, and how do we show solidarity in the fight against this virus? if we want to achieve co-existence, we must take a different long-term view of our vulnerability. is there a we at all? covid-19 also ultimately reminds us of the manifold unsolved challenges of the porosity and closure of borders between different species, humans, animals and viruses, and of the organisation of space associated with them. it makes us aware of the daily mutual pervasiveness and linking of systems and system chains, intended or not. in questions of nutrition, everyday each of us is, in dependence on the global food system, a contributor and decision maker regarding our direct links to non-human species. this virus makes it clear to us that the times of uninhibited consumption are finally over, and that more space, both mentally and physically, must be given to questions of co-existence. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. the broiler chicken as a signal of a human reconfigured biosphere geology of mankind narrative des anthropozäns. systematisierung eines interdisziplinären diskurses feral technologies: making and unmaking multispecies dumps staying with the trouble. making kin in the chthulucene out there. landscape architecture on global terrain rurbane landschaften. perspektiven des ruralen in einer urbanisierten welt publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.undine giseke (prof.) has been a professor at the technical university berlin, chair of landscape architecture + open space planning since 2003. in 1987, she founded in collaboration with three partners the landscape architecture office bgmr landscape architects. from 2005 to 2014, she led the inter-and transdisciplinary research project >urban agriculture as integrated factor of climate-optimised urban development in casablanca, morocco< (uac project). the project was funded by the bmbf research program megacities from tomorrow. in 2015 she received for her research as well as practical experience the gottfried semper architecture award for sustainable planning and building. key: cord-347884-zpzncgiv authors: galimberti, andrea; cena, hellas; campone, luca; ferri, emanuele; dell'agli, mario; sangiovanni, enrico; belingheri, michael; riva, michele augusto; casiraghi, maurizio; labra, massimo title: rethinking urban and food policies to improve citizens safety after covid-19 pandemic date: 2020-10-08 journal: front nutr doi: 10.3389/fnut.2020.569542 sha: doc_id: 347884 cord_uid: zpzncgiv the ongoing pandemic caused by the coronavirus disease 2019 (covid-19) is literally changing the world. from december 2019 to date, more than 22 million cases have been reported worldwide and global health institutions are acting to slow down the virus transmission and are looking for possible prevention strategies in case of a new outbreak. as in other endemic or pandemic phenomena, the issues mostly covered by scientific and media attention are related to the diagnostic and therapeutic approach of covid-19. however, a still neglected issue regards the adoption of a more systemic approach considering the close connection among the infection, the environment, and human behaviors, including the role of diet and urban management. to shed light on this issue, we brought together a faculty group involving experts in environment and biodiversity, food safety, human nutrition, and behavior, bioprospecting, as well as medical doctors having a deep knowledge of the complex historical relationship between humanity and vector-borne infections. two main aspects emerged from the integrative overview of the current covid-19 pandemic: (i) the scientific community should start sharing social actions and policy advocacy based on the assumption that human health strongly depends upon a sustainable exploitation of natural resources in populated areas; (ii) the specific strategic role of the cities in developing sustainable food systems and promoting healthy dietary patterns. definitely, some priority issues should be addressed to achieve these goals, such as global efforts to increase food safety and security, which would benefit from urban and peri-urban agriculture enhancement, smallholder food producers support, and ecosystem services and local biodiversity maintenance. the ongoing pandemic caused by the coronavirus disease 2019 (covid-19) is literally changing the world. from december 2019 to date, more than 22 million cases have been reported worldwide and global health institutions are acting to slow down the virus transmission and are looking for possible prevention strategies in case of a new outbreak. as in other endemic or pandemic phenomena, the issues mostly covered by scientific and media attention are related to the diagnostic and therapeutic approach of covid-19. however, a still neglected issue regards the adoption of a more systemic approach considering the close connection among the infection, the environment, and human behaviors, including the role of diet and urban management. to shed light on this issue, we brought together a faculty group involving experts in environment and biodiversity, food safety, human nutrition, and behavior, bioprospecting, as well as medical doctors having a deep knowledge of the complex historical relationship between humanity and vector-borne infections. two main aspects emerged from the integrative overview of the current covid-19 pandemic: (i) the scientific community should start sharing social actions and policy advocacy based on the assumption that human health strongly depends upon a sustainable exploitation of natural resources in populated areas; (ii) the specific strategic role of the cities in developing sustainable food systems and promoting healthy dietary patterns. definitely, some priority issues should be addressed to achieve these goals, such as global efforts to increase food safety and security, which would benefit from urban and peri-urban agriculture enhancement, smallholder food producers support, and ecosystem services and local biodiversity maintenance. the ongoing pandemic caused by the coronavirus disease 2019 (covid19) is literally changing the world (1, 2) . from the first documented human patient in wuhan (hubei, people's republic of china) in december 2019, on august 2020, more than 22 million cases have been reported worldwide, of which more than six million still active (1% in serious or critical conditions) and almost 800 k deaths. who and other authorities soon realized that it was no longer possible to contain the virus spread, but only to slow down its transmission and try, at least, to reduce "pressure" on national health systems. as in other endemic or pandemic outbreaks, the issues mostly covered by scientific and media attention are related to the diagnostic and therapeutic approach of covid-19 contagion. however, greater consideration should be given to a systemic approach considering the close connection between this disease, the environment and human behaviors, in a framework of building a safer, more sustainable and healthier world (3) . how is it possible that a virus from a chinese market has spread to other continents so quickly, penetrating the heart of cities and killing the weakest citizens? to shed light on this issue, we brought together a faculty group involving experts in environment and biodiversity, food safety, human nutrition, and behavior, biological activity of natural products as well as medical doctors having a deep knowledge of the complex historical relationship between humanity and vector-borne infections. we believe that unlike the pandemics of the past, the factors triggering the current spread of covid-19 outbreak, should be analyzed not only by scientists and politicians but also by societal stakeholders. many european countries most afflicted with covid-19 have started thinking ahead are now facing with the "phase two" of the covid-19 situation by operating the recovery of industrial and social activities keeping, at the same time, the infection spread as low as possible. in such a context, the linear science, which analyzes those biological variables dealing with the pathogen and its infectivity to find possible solutions [e.g., a vaccine or a therapy (4)], clashes with the "post-normal science" (psn) approach, for which, societal values (e.g., the right to freedom, economic needs, and relational aspects) claim their importance (5) . pns is designed to deal with situations of uncertain facts, values in dispute, high stake and urgent decisions [(6); figure 1 ]. pns should be operated together with responsible research and innovation (rri) tools (https://www.rri-tools.eu/aboutrri) that offer a broad set of strategies to address global challenges throughout the analysis of "real-world complexities' considering new scientific knowledge and technologies, but also the needs of different stakeholder categories. the contrast between these approaches to science is exerting some pressure on governments that even proposed solving the problem of covid-19 with autonomous strategies or hypothesized to reach a sort of herd immunity by sacrificing an entire generation of older people (8, 9) . this situation demonstrates the lack of organization by the modern society to address complex global issues. our contribution aims at supporting the adoption of a pns response to covid-19 pandemic, also considering the geopolitical and social aspects which caused the dramatic susceptibility to such infection. for this reason, we aimed at better addressing two main concepts regarding covid-19 "effect" on cities and citizen-led community responses, to prevent future pandemic events. the first point to discuss is the unexpected permeability of cities to this virus and hypothesize how the spillover from wildlife of such a kind of pathogens can reach urban centers. this evaluation is of primary concern to better define suitable prevention strategies for limiting or blocking the current one and other future pandemic spreads. what characterizes covid-19 contagion, is that it fully took advantage of globalization facilities, enabling rapid spread of the virus across the world. generally, accurate controls are performed on goods transported worldwide but in case of novel pathogens (e.g., the covid-19) diagnostic options are very limited. moreover, when human mobility is a major factor in the spread of infectious diseases, we should acknowledge that screening measures adopted at airports or customs should be implemented (10) . temperature screening alone may not be very effective as it may miss travelers incubating the disease or concealing fever during travel, or it may yield false positives (e.g., having fever of a different cause), therefore it should be accompanied by other health messages, questionnaires and data collection (11) . a different scenario occurs for the trading of food commodities for which strict regulations about quality and safety impose the adoption of analytical tools and innovation technologies to prevent the spread of foodborne pathogens or other contaminants (12, 13) . in most cases, food quality evaluation is based on bioindicators, both at chemical and microbiological level (14, 15) . most diagnostic techniques allow to characterize the internal microbiome and virome (16) (17) (18) (19) . considering covid-19, what can be said with certainty is that such controls did not occur in the wuhan market where the virus started its incredible global spread. this situation further remarks the concept that pandemics are strictly linked to insufficient or absent food safety assessment and disease prevention protocols (e.g., as happening every time ebola viruses outbreak in central africa). it is necessary to remark that the food regulations across countries vary and the quality standards of the same food items produced in different countries are not the same (20, 21). the current covid-19 pandemic, perhaps more than others, highlights that it is necessary to align food security protocols on a global scale since country specific inadequacies may cause serious global consequences. therefore, we believe that to prevent future pandemic outbreaks it is more and more important to consider issues arising from the establishment of supranational risk-governance systems. these typically operate in a framework of compromise between the local governance and local producers needs and the global safety for human health. safety is a priority for all the stakeholders associated with the entire food supply-chain. moreover, after the covid-19 emergency, it is desirable that citizens enhance its awareness toward the topic of food control and the concept of food safety will acquire a stronger social meaning based on "shared values, beliefs and norms that affect human mindset and behavior" (22) . consumers' behavior and choices will help modify food supply-chains safety to prevent zoonoses and reduce other risks for humans. (7)] provides a framework to map systems uncertainty against decision stakes. global citizenship is based on rights to be actively involved in debates, responsibility, shared decisions (following careful risk evaluation), and actions to control and implement shared strategies. with regard to covid-19, we know that a bat species and/or the malayan pangolin have been found to be likely reservoir hosts for the virus; however, the definitive identity of any intermediate host that might have facilitated spillover to humans is still unknown (23, 24) . overall, the identification of the vector has a relatively important value. the central point is that the unceasing exploitation of wildlife and habitat has dramatically increased the risk of exposure to zoonotic diseases, as already and sadly demonstrated for example by hiv, ebola and h5n1 (25) . but how calculating the risk of these phenomena? are stakeholders and citizens aware of the risks? these elements are fundamental for a pns discussion that is necessary to drive the path of food safety. it is time to realize that food safety cannot rely only on the production chain but potential risks to human health and the environment should be considered as well. the increasing advances in scientific and technological tools have now been adopted to assess such risks, thus opening a new era of "prediction" rather than "reaction" to reduce pathogen contamination and foodborne outbreaks (26) . for example, the current trends in food safety research rely on the application of (i) genomic analyses for foodborne pathogen identification and traceability, (ii) geographic information systems (gis) to prevent and predict the spatial spread of pathogens outbreak, (iii) tools adapted from landscape ecology (species distribution and niche modeling) and social network analysis for predicting patterns of disease outbreaks, as well as guidance for interventions, and (iv) meta-analysis tools to confer an overall summary of available study findings, providing generalizable estimates and generating strategic highlights to be used by policymakers and decision makers (26) . overall, risk prevention remains the key factor. the history of pandemics teaches us that almost all recent human pandemics and most of the emerging infectious diseases originated from animals (mainly in wildlife). it is known that species more resistant to human pressure are likely to become the new competent hosts of vector-borne diseases and then to become the most probable spillover agents toward human hosts (27, 28) . furthermore, we must remember that biodiversity perturbation and its trivialization is the main trigger of virus spillover events (29) , as probably happened for covid-19 (figure 2) . given these assumptions, the international food policies concerning food safety should consider biodiversity and ecological interventions to prevent zoonotic spillover events. this would be especially urgent in rural areas, where farming and livestocking often overlap with wildlife species ranges and it has been documented that livestock species usually act as figure 2 | list of the principal pandemic and emerging zoonotic viruses showing human-to-human transmission after the spillover is occurred. summarized host and spillover interface data are provided as in (29) (mouse: directly from wildlife; swine: directly from domestic animals; mosquito: transmission by vector involving wildlife or domestic host respectively; u: unknown). detailed references for each listed virus are provided in (29) . frontiers in nutrition | www.frontiersin.org intermediate hosts of spillover events (e.g., influenza a and sars coronavirus, figure 1 ). for this reason, it is also time to rethink urban areas by projecting proximity buffer zones to prevent direct contact between agricultural/zootechnic activities and natural habitats. finally, the conservation of natural biodiversity and its related species interactions are essential conditions to reduce the risk of spillover events (27) . on the whole, a cooperative work (rri-driven) involving human-health agencies, agricultural authorities, farmers, and natural resource managing institutions, could be essential to promote the global ecological management to avoid the spread of a new putative pandemic "covid-20" or other risky vector-borne pathogens that may adversely affect human health, the environment and economy. our second consideration regards the fragility of citizens, especially the weakest ones such as the elderly, and their sensitivity to diseases. it is now clear that these social categories are the most susceptible to severe covid-19 outcomes, particularly if they already suffer from multiple pathologies. diabetes is the most common comorbidity observed in infected deceased patients in italy, after hypertension (30) . furthermore, recent data showed a high prevalence of obesity (26%) and overweight (41%) in 928 italian patients, median age 65 years, from 76 different italian icus, confirming evidence available so far in the literature supporting impaired immune response to viral infections (31) . therefore, beyond the infectious capacity of this virus, it is important to focus on those elements of modern society which could increase citizens' vulnerability, including diet, lifestyle and environmental factors, strictly linked to morbidity and mortality for all non-communicable diseases (ncds) (32, 33) . although this concept is well-established, today, the global average consumption of healthy foods is substantially lower than the reference dietary intake, whereas overconsumption of highly processed, energy dense, and nutrient-poor foods is increasing (34) . the mediterranean diet is considered by unesco as one of the "intangible cultural heritage of humanity" with multiple health benefits, including fortification of immune defenses. however, epidemiological data on covid-19 would seem to contradict this belief since mediterranean countries (e.g., italy and spain) have the highest number of confirmed covid-19 cases in the world. five years after the expo 2015, dedicated to the theme "feeding the planet, energy for life, " the city of milan, which hosted the event, is under siege by covid-19 pandemic. recent dietary changes within the mediterranean basin, with a decreased consumption of plant foods, increased consumption of fast meals and junk food, and negative health consequences such as rise in obesity rates and in ncds incidence (e.g., diabetes, cardiovascular diseases, and cancer) are partially responsible of this burden (35) . the modern-day change in food choices is the results of lifestyle standardization, enhanced technologies in food production and processing and limited time for culinary activities. this caused for example the progressive erosion of mediterranean food cultures (36, 37) . moreover, environmental emergence, such as water scarcity in most mediterranean countries and land wasting also has deleterious consequences on mediterranean food production. global climate changes have also produced the failure of several crops, fisheries, and livestock productions, and the declining of mediterranean biodiversity and agrobiodiversity does not allow the selection of new varieties and resistant breeds. so, once again the erosion of the environment and biodiversity is closely connected to health risks (38) . how to react to these social and environmental changes showing serious health consequences? the sustainability of the food supply chain is certainly essential (34) . kinnunen et al. showed that less than one-third of the world's population can meet their food demand within a 100-km radius (39) . we should also change our view of food and diet which should no longer, or rather not only, considered an energy source but a reservoir of bioactive molecules beneficial to human health. greater consumption of health-promoting foods and limited intake of unhealthier options are intrinsic to the eating habits of certain regional diets such as the mediterranean diet (32) . healthy dietary patterns positively influence health and promotes the prevention of common non-communicable diseases (ncds), strengthening host community defenses (32, 36) . this concept assumes a particular importance since the over 65 years old citizens could be more at risk of being infected by covid-19, not only for intrinsic conditions due to natural aging processes and comorbidities development, but also for inadequate nutritional status and related inadequate intake of macronutrients (e.g., proteins and healthy fatty acids, like omega-3), micronutrients (e.g., vitamins a, b6, b12, c, d, e, and folate), trace elements (e.g., zinc, iron, selenium, magnesium, and copper) and phytochemicals which are pillars in preventing many chronic degenerative diseases and supporting the immune system (40) . this would seem to be true even for younger patients with metabolic and cardiovascular diseases, showing severe acute respiratory distress syndrome (ards) caused by covid-19 (41) . these considerations are also well-known for past pandemics. a recent retrospective data analysis from the 1918 pandemic flu, showed that nutrition played a consistent role in the severity of the disease and was related to mortality also in younger age groups (42) . more recently, chronic malnutrition has been correlated to high morbidity and mortality during the 2009 influenza pandemic (43) . similarly, malnourished children appear to be at increased risk for viral pneumonia (44) . in dietary recommendation, fat quality has to be addressed (45) , since evidence shows the need to achieve a balance between dietary intake of omega-6 and omega-3 for optimal nutrition (46) , especially in those subjects more vulnerable to malnutrition and "silent inflammation" which disposes to a greater propensity to viral infections (47, 48) . certainly, the presence of an unbalance between pro-and anti-inflammatory lipid mediators has been reported in literature (49) and it is important to remark that science has already given solutions (50) which are not adopted in new diagnostic policies for primary and secondary prevention. moreover, food supplements such as vitamins c and d might also be considered to help both innate and adaptive immune cells (51) (52) (53) (54) . studies on human coronaviruses (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov), have highlighted that secondary metabolites of some plant species seem to inhibit virus proteins, cellular infection, and intracellular replication (55) . extracts of spontaneous plants such as root tubers from rheum officinale baill. (rhubarb), root tubers or vines from polygonum multiflorum thunb., (polygonaceae) showed an inhibitory activity against the interaction of sars-cov s protein with ace2 (56). procyanidins and other secondary metabolites extracted from cinnamomi cortex (cinnamomum cassia j. presl) can reduce the virus infection by interfering with endocytosis (57) . the extract of cimicifuga rhizoma, meliae cortex, coptidis rhizoma, phellodendron cortex, and sophora subprostrata radix showed an ability to inhibit of rna-dependent rna polymerase and/or proteases crucial for coronavirus rna replication (58) . finally, the antiviral activity of licorice (glycyrrhiza glabra l.), containing glycyrrhizin, inhibits replication, absorption and penetration of the sars-cov acting on the early steps of the replicative cycle (59) . in this field, traditional chinese medicine is working actively to identify dedicated compounds to specifically contrast covid-19 infection (60) . this practice relying on biodiversity and known as "bioprospecting, " may be a good strategy to find compounds having a positive effect on human health as well as to find new raw materials to produce novel and fortified foods for modern citizens. recently, di marco et al. (27) suggested that the risk of emerging infectious diseases (eids) is a key aspect for developing suitable policy strategies at the global scale. within this framework, the conservation of biodiversity and food production are two additional pillars that should be considered to prevent drastic environmental changes and the risks of zoonoses, and virus spread. in figure 3 we aimed at further remarking this concept including additional factors strictly related to covid-19 pandemic. from a nutritional point of view, addressing subclinical micronutrient deficiencies is one of the first steps that must be considered to improve resistance to infectious diseases like covid-19 (and other pathogens) (61) . it is therefore recommended that micronutrient testing, such as vitamin d measurement, should be applied in the annual check up of selected individuals at high risk of deficiency (62) . a nutritional approach, ensuring a long-term sustainability, is essential to improve micronutrient status by increasing the availability and consumption of micronutrient-rich foods (63) . besides lifestyle changes, including diet has been shown to positively affect metabolic and cardiovascular diseases, which are the most frequent comorbidities associated to severe covid-19 disease. the spread of inappropriate eating habits and inactivity in western societies, particularly among the younger, "comfortably off " generations, has led to the development of chronic degenerative diseases defined as "comfort" diseases (64) early in life, leading to an increase in premature deaths for ncds. food is readily available in developed countries but there is an evident split-up between scientific evidence, food choices, and dietary patterns of consumers. this, over time has favored the spread of the obesity epidemic and other diet-related diseases. it is time to acknowledge that environmental factors exert a major influence on dietary behavior, primarily by facilitating meals consumption away from home and by minimizing time dedicated to meal preparation and consumption and secondly, making food of poor nutritional quality available on the market and appealing for appearance, taste and price. this burden is exerted by market rules that affect behavior and food choices with scarce public awareness of the potential negative impact on health. it is necessary that science, technology, education, legislation, and community policies combine to create the urban structures and environment required to encourage healthy lifestyle including dietary choices, not just for few, but for everyone (65) . more efforts must be addressed to reduce exposure to ambient air pollution, strongly associated with population density (66), promoting chronic inflammatory state and affecting resilience to infectious diseases not last covid-19 (67) . finally, western medicine generally tends to identify pharmacological molecules that act on specific disease mechanisms; however, human body complexity and individual answers are sometimes underestimated. thus, it could happen that infected patients die more due to comorbidities associated to infection with covid-19 than for covid-19 per se. the time has come to apply a systems biology approach where drugs, foods and lifestyle work in synergy to promote patient healing and prevent further infections, gaining a holistic approach to community health (68) to support the further personalization of health and social care (69) . for these reasons, in our scheme, we stressed the impact of environment and food system of therapeutic approaches. this integrative framework demands both an increased attitude of sharing by the scientific and technical community as well as a social participation since we believe that, as previously anticipated, food safety and human health should be regarded more as a social issue. our final suggestion is to start a frank and open scientific discussion on covid-19 issues and future risks for new pandemic outbreaks, continuing the legacy of expo 2015, declared in the milan urban food policy pact, signed by more than 200 cities in the world (65) . in this document, the strategic role of the cities in developing sustainable food systems and promoting healthy diets is stated, yet acknowledging all the differences in their natural and policy endowments, including economic background and cultural innovation, managing vast public resources, infrastructure, investments, and expertise. these issues will be fundamental especially for those countries that are coming out of covid-19 lockdown restrictions and where it is more expected that political and social contrast will emerge if different stakeholders needs and opinions will not be analyzed and considered for planning the recovery after the pandemic event. we would like to undermine and integrate this overview with few take-home messages that arise from the teachings of this dramatic situation we are experiencing firsthand: (i) food safety is a global issue. the unsafety of local food markets, like the wuhan's one, can exert severe and global impact. (ii) smallholder food producers play a key role in feeding cities, by helping to maintain resilient, equitable, culturally appropriate food systems, and promote sustainable diets. in cities with a high percentage of elderly, local food production should be tailored for specific targets to maintain adequate nutritional status, including fortification of immune system. similarly, in developing countries, smallholder farms should improve the production of local crops rich in macro and micronutrients to improve food security and health of local populations (70) . (iii) acknowledgment that urban and peri-urban agriculture may offer opportunities to protect and integrate biodiversity into urban landscapes and food systems, thereby contributing to synergies across food security, ecosystem services, and human wellbeing. this is very important to prevent the spillover of viruses but also to offer better efficacy of new drugs synthesized to fight future diseases. a unified approach to nutritional screening and assessment is recommended guiding toward integrated panels of biomarkers to investigate the nutritional status and predict future health outcomes of the individual and moving from stratified to personalized to precision nutrition (71) . the whole scientific community should start sharing directions, social actions and policy advocacy recognizing that the health of people is closely connected to the health of biodiversity and ecosystems where they live. in this context, the "one health" approach (https://www. cdc.gov/onehealth/basics/index.html) 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declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer ml declared a past co-authorship with one of the author mr to the handling editor.copyright â© 2020 galimberti, cena, campone, ferri, dell'agli, sangiovanni, belingheri, riva, casiraghi and labra. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-340629-1fle5fpz authors: o’shea, helen; blacklaws, barbara a.; collins, patrick j.; mckillen, john; fitzgerald, rose title: viruses associated with foodborne infections date: 2019-05-21 journal: reference module in life sciences doi: 10.1016/b978-0-12-809633-8.90273-5 sha: doc_id: 340629 cord_uid: 1fle5fpz foodborne pathogens cause acute and chronic health outcomes of very different durations, severity and mortality, resulting in high costs and burdens to society. the issues of food safety and food poisoning are being increasingly emphasised, particularly in developed countries. infection/contamination with many agents i.e., bacterial, parasitic and viral entities can result in foodborne illness. this article will focus mainly on viral agents of infection. a range of different viruses can cause food poisoning/foodborne infection, and infection can result in a myriad of symptoms, ranging from mild, acute disease to chronic, debilitating disease and even death. due to the inherent differences between bacteria and viruses, namely the fact that viruses do not replicate in food, while bacteria do, viruses are frequently difficult to detect. this is compounded by the fact that many of the viruses associated with enteric disease do not replicate in cell culture. these factors can lead to a lag between reporting, detection and analysis of foodborne viruses versus bacterial agents. despite these constraints, it is now evident that there are both well-established and emerging viruses implicated in foodborne infections, and the role of molecular detection and characterisation is becoming increasingly important. in recent years, there has been increasing awareness regarding food safety in terms of food poisoning that results in viral gastroenteritis i.e., stomach flu. it is important to clearly define these terms, for the purpose of this article, prior to focusing on the smaller group of diverse viruses associated with food borne illness. food safety encompasses the handling, preparation, and storage of food to prevent food-borne illness. food poisoning (also referred to as foodborne illness) is caused by eating contaminated food. infectious organisms, which include bacteria, viruses and parasites (or their toxins), are the most common causes of food poisoning. infectious organisms or their toxins can contaminate food at any point of processing or production. contamination can also occur in the home and eateries, if food is incorrectly handled, stored, or cooked. viral gastroenteritis (also known as stomach flu) is an intestinal infection with symptoms including watery (usually nonbloody) diarrhea, abdominal cramps, pain, nausea or vomiting, or both, and sometimes headaches, muscle aches and fever. the most common way to develop viral gastroenteritis (stomach flu) is through contact with an infected person or by ingesting contaminated food, water and water products such as ice. there is a huge annual cost of illness, disease burden and quality-adjusted life year (qaly) loss globally, caused by food-borne pathogens, which has been reported on by many investigators (scallan et al., 2011) . in the western world, the viruses frequently reported as having the highest total cost-of-illness, to date, are norovirus and rotavirus. these viruses will be discussed first. this profile could change, however, as new virus threats are constantly emerging. a variety of different foods and different agents are implicated in foodborne illnesses, and we will elaborate on these in the following sections. many agents cause gastroenteritis, the most common outcome being diarrhea. this can range from mild, self-limiting illness to fatal infection. diarrheal disease is the major cause of illness and death in children in developing countries, while in the developed world it is usually mild, except in the very young, the elderly and the immunocompromised. gastroenteritis (acute gastroenteritis, age) is caused by a variety of different pathogens, including parasites, bacteria and viruses ( table 1) . every day we swallow large numbers of microorganisms in our food and beverages, and from contact with our environment e.g., from fingers. our body's defense mechanisms, [innate (non-specific) and adaptive (specific) defense systems] are very efficient, and the microorganisms rarely succeed in surviving passage to the intestine in sufficient numbers to cause infection. the body's innate (non-specific) defense is the first line of protection and does not distinguish between infectious agents. it includes physical (e.g., skin and mucous membranes) and chemical barriers (e.g., sweat, gastric juices), cellular (e.g., natural killer cells and phagocytosis) and modular defenses (e.g., interferon) and bodily responses (e.g., inflammation and fever). with regard to protection against potential enteric pathogens, the intestinal tract has an abundant microbiota that competes with invading pathogens for space and nutrients. the peristaltic action of the digestive tract encourages movement through the system, deterring colonisation by invading pathogens, with vomiting and diarrhea flushing harmful microbes and their chemical products from the digestive tract. the extremely acidic stomach environment discourages pathogen replication. however, these fortifications are not impenetrable e.g., the enteric virus hepatitis a has the ability to survive and penetrate the body through the gastrointestinal tract (campbell et al., 1999) . in addition, the epithelial barrier of the intestine is embedded with mucous-producing goblet cells. these simple columnar cells secrete gel-forming glycoproteins (mucins), to produce a transparent, impervious barrier (johansson and hannsson, 2013) on the luminal surface. the prevailing mucin of the intestinal tract is muc2 (mucin 2), which is a major structural component of intestinal mucus layer. goblet cells emerge during foetal development at 9-10 weeks of gestation (kim and khan, 2013) and are continuously secreted, migrating from the intestinal glands; crypts of lieberkühn to the lumen of the intestinal tract via the apex of the digestive villi. non-bacterial gastroenteritis and diarrhea is usually caused by viruses. acute viral gastroenteritis (age) is seen in all parts of the world, especially in infants and young children. age ranges from mild and self-limiting to severe, debilitating diarrhea, occasionally resulting in mortality. these illnesses have a huge impact, particularly in parts of asia, africa and latin america, with over 3 million fatalities recorded per annum. age also has major effects on nutritional status and growth. viruses appear to be the commonest causes of gastroenteritis in infants and young children but are not distinguishable clinically from other types of gastroenteritis. some of these viruses are specific for humans, while others are implicated in zoonotic disease. in most instances, infection follows the general rules for faecal-oral transmission. enteric viruses are transmissible by food and water and enter the body through the gastrointestinal tract, thus the viruses are commonly acquired after ingestion of sewage contaminated food or water or from poor hygiene when preparing food. the viruses are shed in high concentrations in faeces and vomit and can remain infectious in the environment for several months. many viruses are difficult or impossible to cultivate in vitro, and tests are unreliable or not developed, thus many cases of non-bacterial acute gastroenteritis viruses are under reported. well established viral pathogens will be discussed in this section. later in the article, emerging viral pathogens associated with food poisoning/foodborne illness/foodborne infections will also be discussed. there are several different viruses involved in foodborne outbreaks. some of these are human viruses that infect and cause illness following ingestion. virus particles are shed in the faeces. to date, the most notable examples in this category are noroviruses and hepatitis a virus. these viruses are different, belonging to different families ( table 2) : noroviruses (formerly called small round structured viruses or srsvs) belong to the family caliciviridae and are small, non-segmented, positive sense, single stranded nonenveloped, icosahedral rna viruses. hepatitis a virus is also a small, non-segmented, positive sense, single stranded nonenveloped icosahedral rna virus, but belonging to the family picornaviridae. hepatitis a virus is slightly smaller and contains a linear genome. is one the five pathogens (namely, salmonella, campylobacter, listeria, toxoplasma, and norovirus) responsible for causing roughly 90% of mean loss due to foodborne illness in the united states (hoffman et al., 2012; scallan et al., 2011) . as outlined above, noroviruses are small, non-enveloped, positive sense rna viruses, within the family caliciviridae. noroviruses have been classified into seven genogroups (gi-gvii) and over 30 genotypes. gii.4 has long been established as a predominant variant, but new variants are continually emerging (lennon et al., 2014) . transmission of the virus may occur through sewage contamination early in the food chain or lack of personal hygiene later in the food chain, particularly when 'ready-to-eat' (rte) food-items are involved. transmission can also occur via by person-to-person contact, environmental contamination, as well as via food-and water-borne transmission. typically, noroviruses cause problems in individuals who have consumed contaminated raw oysters. other implicated foods are fruit and vegetables that have, for example, been irrigated with contaminated water. outbreaks frequently occur in hospitals, nursing homes, restaurants, cruise ships, schools, summer camps, and even at family dinners. in these cases, large numbers of people often eat food handled or prepared by others. the symptoms of norovirus infection vary, but may include nausea, vomiting, abdominal pain or cramps, watery or loose diarrhea, malaise, low-grade fever, muscle pain. signs and symptoms usually begin 12-48 h after first exposure to the virus and last one to three days. shedding of virus can continue for up to two weeks post recovery. some people with norovirus infection may show no signs or symptoms but are still contagious and can spread the virus. there are several recommendations concerning food preparation, disinfection and decontamination, outlined on e.g., the centres for disease control and prevention (cdc) website (see "relevant websites section"). these measures involve practicing good hand hygiene, washing fruit and vegetables and cooking seafood, especially shellfish e.g., oysters, thoroughly, individuals who are ill avoiding contact with others, also food preparation, for at least 48 h after symptoms abate, and decontamination using suitable disinfectants and detergents. some people are particularly vulnerable and should take measure to avoid norovirus infection, especially infants, older adults and people with underlying disease where vomiting and diarrhea can be severely dehydrating and require medical attention. preventative measures e.g., avoiding raw shellfish should be taken in these instances. for many years, rotavirus (rv) has been recognized as a major cause of gastroenteritis in the young of many animal species, including humans (bishop et al., 1973; estes and kapikian, 2007; flewett et al., 1975) . in humans, rv associated disease typically occurs in children less than 5 years of age but rotavirus can infect all ages, including adults (anderson et al., 2012; collins et al., 2015; gunn et al., 2012) . in 2008, an estimated 453,000 deaths worldwide were attributed to rotavirus infection, with most deaths occurring in resource-poor countries (o'shea et al., 2016; tate et al., 2012) . rotavirus belong to the family reoviridae and are medium sized, segmented, double stranded non-enveloped, icosahedral rna viruses. the 11 segments encode six virion proteins (vp1-4, 6-7) and six non-structural proteins (nsp1-6). rotaviruses are classified into 8 antigenically distinct groups and one proposed group (a-i) (mihalov-kovács et al., 2015) , using immunological and phylogenetic analysis of the vp6 gene. rotavirus groups a, b, c, h and i have been identified in mammals, including humans, while rotavirus d-g to date have only been identified in non-human mammal and avian species. a tenth novel rotavirus species has been identified in schreiber's bats (miniopterus schreibersii), provisionally known as rotavirus j (bányai et al., 2018) . within the rva group, the viruses are classified antigenically and genetically, based on the main antigenic determinants, the outer capsid proteins, vp7 and vp4, which specify the g and p serotypes/genotypes, respectively. a whole genome classification system has been adopted for classification of all 11 segments of the rotavirus genome, applying nucleotide homology cutoff values to distinguish genotypes. transmission of rotavirus is predominantly faecal-oral. in humans, rotaviruses are ubiquitous, with 95% of children worldwide being infected by three to five years of age. in infants, prior to the introduction of rotavirus vaccines, rvas could be detected in up to 50%-60% of all childhood hospitalisations due to acute gastroenteritis each year, were estimated to cause 138 million cases of gastroenteritis annually, and 527,000 deaths in children o5 years of age living in developing countries. in other animals, rotavirus disease also occurs in the young of the species and in farm animals, leads to significant economic losses. the symptoms are usually characterised by watery dehydrating diarrhea and vomiting, often accompanied by abdominal cramps and low-grade fever, lasting 6-10 days. subsequent reinfections are associated with mild or subclinical presentations (bishop et al., 1973; velázquez et al., 1996) ; however, such reinfections are important in boosting immunity and maintaining long-term protection from rotavirus disease. due, in part, to the segmented nature of the genome, accumulation of point mutations (genetic drift) and re-assortment (genetic shift) are responsible for the huge genetic heterogeneity of rotaviruses. these mechanisms, in combination with the potential for interspecies (zoonotic and anthroponotic) transmission, can also lead to the emergence of "novel" strains in a given species, with a potential for epidemic or epizootic spread (martella et al., 2010; matthijnssens et al., 2008) . the zoonotic potential of rva has been documented on several occasions (martella et al., 2010) . uncommon human rva genotypes include g5, g6, g8, g10 and g11, in combination with p[3], p[9], p[10], p[11] and p[14] p types, and are generally considered to be animal to human reassortment variants. however, certain animal-like genotypes have become established in human circulation, particularly in developing countries. since its discovery, many attempts have been made to produce effective rotavirus vaccines, with an emphasis on those directed to the prevention of human disease. today, two oral live attenuated vaccines are being used in rotavirus immunisation programmes globally; rotateq™, which is a pentavalent human-bovine reassortment containing human rva derived g1 to g4 and p [8] types within the backbone of the wc3 bovine strain, and rotarix™, a monovalent human g1p[8] live attenuated vaccine. both vaccines are efficacious and endorsed by the who, which recommends the implementation of these vaccines in national immunisation programs (who, 2009) . the introduction of universal mass vaccination (umv) has resulted in a significant decrease in childhood rotavirus infection morbidity and mortality (curns et al., 2010; pendleton et al., 2013; tate et al., 2012; usonis et al., 2012; zeller et al., 2010) . hepatitis or inflammation of the liver can have a number of causes, including medications, toxins, alcohol use and viral infection. there are 5 hepatitis viruses, a-e, but only a and e are transmitted by the oro-faecal route. both viruses are major causes of infectious disease with associated socio-economic consequences caused by morbidity and, in vulnerable groups, mortality. hepatitis a virus (hav), also known as hepatovirus a, belongs to the order picornavirales in the family picornaviridae and is the type species of the genus hepatovirus. it is an icosahedral, non-enveloped virus with a monopartite, positive, single-stranded rna genome. humans and vertebrates serve as natural hosts. there is a single serotype of hav but several genotypes: ia, ib, iia, iib, iiia, iiib primarily in humans and iv-vi, primarily found in non-human primates (cristina and costa-mattioli, 2007) . the virus infects hepatocytes and kupffer cells (liver macrophages). transmission: hav is spread in food, in the water system, by touching contaminated surfaces or by direct contact with an infected person. contamination of food can occur at any stage from farm to fork. according to the who, approximately 1.5 million people are infected each year, although the true infection rate is probably much higher due to the asymptomatic nature of many infections (hepatitis a fact sheet. in: world health organisation: media centre). due to the prolonged infection times, the economic consequences of an outbreak through absenteeism from work can be significant. in developing countries where sanitation is poor, most children are infected while young and are asymptomatic. this gives them immunity and, as such, they cannot become reinfected as adults. in such areas, epidemics of the virus are uncommon but unvaccinated adult visitors from areas where hav is not commonplace are vulnerable. in economically developed countries, epidemics are rare, because good hygiene practices will prevent person-to-person spread. however, in transitional economies, the introduction of improved sanitation may mean that the population is not exposed as children. as such, the introduction of the virus to these susceptible populations of adults can result in significant outbreaks (lemon et al., 2018) . the symptoms: the virus is normally mild without permanent repercussions and is typified by fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, dark urine, clay-coloured stools, joint pain and jaundice (yellowing of the skin and eyes). the development of fulminant hepatitis is rare (less than 0.5% of cases) and can lead to more serious illness, including liver failure and death. severe illness is more common in older people, while in children around 70% are asymptomatic and in those with clinical manifestations the symptoms are generally mild. infected persons may shed infectious virus for several weeks before they show symptoms. in those individuals where symptoms occur, they usually start appearing 4 weeks after exposure, but can occur as early as 2 and as late as 7 weeks following exposure. symptoms usually develop over a period of several days and last less than 2 months, although some people (10%-15%) with hepatitis a can have symptoms for as long as 6 months (jeong and lee, 2010) . hea can survive outside a host cell for several weeks in groundwater and it has been shown to retain infectivity after 92 days at 25°c in seawater. the virus is tolerant of desiccation and freezing and will survive on vegetables throughout the production process until consumption (a critical review of the effect of heat, ph and water activity on the survival of hepatitis a and e viruses -a report to the united kingdom food standards agency july 2014). hea is more resistant to heat than other picornaviruses and the centre for disease control states that temperatures of 85°c for a least 1 min are required for inactivation. hep a is vaccine preventable (ott et al., 2012) . monovalent formalin killed vaccines are available worldwide and live attenuated versions are available in a number of countries. a double dose of vaccine can provide long-term protection and single dose immunisation strategies have been shown to be effective at least in the short and medium term. as well as for active immunisation, the vaccine can be used prophylactically in the first few weeks of infection. human immunoglobulins can also be used for both preventative and post-exposure prophylactic treatment (liu et al., 2009) . post-exposure prophylaxis provides high levels of protection (80%-90%) if provided in the first 2 weeks of exposure. otherwise standard methods for the prevention of food-borne infections apply: ensuring good sanitation and a clean water supply, hand washing and food safety. hepatitis e virus (hev) is a small non-enveloped icosahedral virus, 27-34 nm in size, with a single-stranded positive sense rna genome. the virus belongs to the family hepeviridae, genus orthohepevirus. there are four species in this genus, and the viruses that infect humans belong to the species orthohepevirus a. currently, eight genotypes of orthohepevirus a have been identified. hev1 and hev2 infect humans only, hev3 has a range of known host animals including humans and swine, hev4 infects humans and swine, hev5 and hev6 have been detected in wild boar and hev7 has been detected in camels (sridhar et al., 2017) . transmission: hepatitis e virus (hev) is the most common cause of acute viral hepatitis worldwide (sridhar et al., 2015) . the virus is responsible for endemics and epidemic outbreaks and the who estimate there are 20 million infections and 3.3 million symptomatic cases worldwide per year; in 2015 these led to 44,000 deaths (world health organization, 2017). hev is primarily transmitted through the faecal-oral route via shedding in the faces of infected individuals and subsequent contamination of drinking water. other routes of transmission are consuming raw or undercooked foods from infected animals or shellfish, blood transfusions or vertical transmission from pregnant women to the foetus (wulffen et al., 2018) . the virus has a worldwide distribution but is most prevalent in south and east asia and is associated with areas where sanitation is poor and contaminated faeces can enter the water system. in regions where the virus is endemic, outbreaks are common, usually associated with contaminated drinking water, recurring at intervals of years and may involve thousands of people. such infections are predominantly genotype 1 or 2 (melgaço et al., 2018) . in non-endemic regions, infections were previously associated with international travel, but now the majority of cases are zoonotic genotype 3 or 4 infections from swine or deer. handling of swine and manure of porcine origin is now a public health concern and infectious hev has been isolated from meat products (yugo and meng, 2013) . the symptoms: while hev is primarily a disease of the liver, it has also been associated with non-hepatic diseases such as subacute and monophasic neurological disorders of the peripheral nervous system, acute pancreatitis, glomerulonephritis, mixed cryoglobulinemia, severe thrombocytopenia and haemolytic anaemia (pischke et al., 2017) . the hepatic symptoms of hev are indistinguishable from other forms of acute hepatitis and laboratory diagnosis is required for a definitive diagnosis. the incubation period is approximately 2-6 weeks. hev is characterised by fever, reduced appetite, nausea and vomiting, abdominal pain, and a slightly enlarged, tender liver (hepatomegaly) (kamar et al., 2017) . in rare cases, acute hepatitis e can be severe, resulting in liver failure and death. pregnant women and immunosuppressed people are at high risk of these severe symptoms. in pregnant women mortality rates of 30% have been recorded (perez-gracia et al., 2017) . like hav, infection of young children is often mild or asymptomatic. hev is self-limiting and most cases do not require treatment. for those with acute symptoms or for high risk individuals, hospitalisation is required. the antiviral drug ribavirin may be given, although in pregnant women this must be carefully considered, due to the teratogenic nature of this antiviral compound (krzowska-firych et al., 2017) . a recombinant subunit vaccine to prevent hepatitis e virus infection exists in china but is not licenced elsewhere (nan et al., 2018) . the family adenoviridae contains 5 genera; atadenovirus (8 species) infecting birds, lizards and mammals; aviadenovirus (14 species) infecting birds; ichtadenovirus (1 species) isolated from sturgeon; mastadenovirus (45 species) infecting mammals only, including humans and; siadenovirus (6 species), mostly infecting birds and 1 frog species. virions are non-enveloped, 70-90 nm in diameter with a double-stranded dna genome and an icosahedral capsid. there are 7 human species of human adenoviruses, a-g (lennon et al., 2007) . within these species there are at least 79 subtypes distinguished either by serological differences of by genotypic classification (chen and tian, 2018) . transmission: adenovirus-associated gastroenteritis often occurs in clusters in schools, hospitals or military camps. adenoviruses are spread person-person contact, by coughing and sneezing, by touching contaminated surfaces or by the faecal-oral route (eckardt and baumgart, 2011). adenoviruses infections are not associated with contaminated food, but transmission can occur in water, through public water systems or in swimming pools; the latter are predominately associated with conjunctivitis (rodríguez-lázaro et al., 2012) . symptoms: adenoviruses in humans cause respiratory infections, gastrointestinal disease and conjunctivitis with hemorrhagic cystitis, hepatitis, hemorrhagic colitis, pancreatitis, nephritis, or encephalitis (lynch and kajon, 2016) . adenoviruses infections are often asymptomatic or mild and self-limiting. however, they can be associated with severe morbidity or mortality, with immunocompromised and the young being more susceptible. however, novel or emerging strains have been found to cause mortality in people of all ages such as ad14, which was responsible for fatal respiratory illness in the usa (centres for disease control, 2007) . a number of human adenovirus subtypes can cause gastrointestinal symptoms, but subtypes 40 and 41 from species f are the most commonly associated with age and have been reported to be responsible for 5%-20% of acute gastroenteritis in children (ziros et al., 2015) . adenovirus-associated gastroenteritis is most common in children under 2 years old and is uncommon in adults, accounting for 1.5%-5.4% of cases (eckardt and baumgart, 2011) . the incubation period is around 8-10 days after which diarrhea develops and, in some cases, mild vomiting. fever lasting 2-3 days may develop; severe dehydration is rare (wood, 1988) . currently vaccines against human adenovirus 4 and 7 are being used by the us military, but no vaccines are available for general use (chen and tian, 2018) . as the virus is usually self-limiting in most cases, treatment of adenoviral gastroenteritis is not required. in severe cases, hospitalisation and rehydration may be needed. prevention of adenovirus infection is by standard antiviral hygiene methods; avoiding sharing of eating and drinking utensils, hand washing and avoiding contact with sick individuals, especially in at risk environments such as hospitals. adenoviruses are susceptible to chemical disinfectants but can be resistant to uv irradiation (rodríguez-lázaro et al., 2012) ; in swimming pools, chlorine levels must be adequate. the family astroviridae consists of 2 genera; avastrovirus and mamastrovirus infecting birds and mammals, respectively. virions are non-enveloped, approximately 28-40 nm in diameter with a single-stranded positive sense genome and an icosahedral capsid. the international committee for the taxonomy of viruses (ictv) currently identifies 19 species in the genus mamastrovirus infecting felines, canines, cattle, cervids, rodents, swine, sheep, mink, bats, rabbits, sea lions and dolphins. the viruses that infect humans (referred to as classical hastvs) have traditionally been classified into 8 serotypes, but recently a number of viruses have been detected in humans that are more similar to those from other animals, suggesting cross species transmission (bosch et al., 2014) . transmission: hastvs are predominantly transmitted through the faecal-oral route as well as through drinking water and in sewage. recreational activities in sewage contaminated water bodies is a risk of infection. hastvs are considered to be food-borne viruses with molluscs grown in, or fruits or vegetables irrigated with contaminated water, representing the biggest threat (vu et al., 2017) . as would be expected, poor food hygiene practices may play a role in outbreaks, particularly as asymptomatic carriers are more likely to contribute to infection in the food industry than symptomatic workers. contaminated fomites and surfaces can represent a threat in institutions such as schools and hospitals (abad et al., 2001) . as we are now aware that inter-species infections with astroviruses can occur, and, as such, the zoonotic route should be considered as a potential source of astrovirus infection (bosch et al., 2014) . the symptoms: classical human astroviruses are known to cause mild gastrointestinal disease in children, but symptoms and prevalence are serotype dependant. immunocompromised and elderly patients are also susceptible. serotype 1 is the most common, with a reported level of seropositivity of 90%-100% having been reported in children by 5 years of age (koopmans et al., 1998) . the symptoms are usually watery diarrhea, vomiting, fever, abdominal pain, anorexia, and headache but these are usually mild in nature and self-limiting, typically lasting for 2-4 days (vu et al., 2016) . however, hastvs do cause asymptomatic infections (méndez-toss et al., 2004) and the association of these viruses with disease is not fully understood. while the symptoms are mild, hastvs do contribute to a large proportion of outbreaks of diarrhea, 0.5%-15% of all cases, and often occur as coinfections with other viruses such as noroviruses and rotavirus (de benedictis et al., 2011) . rarer astroviruses have been reported as being responsible for fatal cases of central nervous system infection (fremond et al., 2015) . as with most gastrointestinal infections control of hastvs is by prevention of contamination of food and water and prevention of person to person, or fomite to person spread through hygiene and handwashing. while survival of astroviruses in drinking water is known to be high, chlorination has been shown to be effective in reducing astrovirus viability by alteration of the capsid, resulting in non-infectious virions (abad et al., 1997) and 90% alcohol has been shown to be effective for decontamination of surfaces and hands (kurtz et al., 1980) . no vaccines are available for hastvs. as infections are usually mild and self-limiting, no treatment is normally required. however, those with severe gastroenteritis may require oral or intravenous rehydration. recent emerging epidemic and pandemic virus infections that cause severe disease in humans and that are associated with food production, preparation and food contamination include the coronavirus, severe acute respiratory syndrome (sars-cov), nipah virus, ebola virus and some of the highly pathogenic influenza virus strains, such as the h5n1 subtype. transmission may be by a variety of routes, but often the emergent epidemic is started by contamination of a food source by saliva, urine or faeces from a wild reservoir species or use of a wild animal (bush meat) as the food source and infection through the capture and butchering process (e.g., hiv is thought to have entered the human population by this route). these infections have a low probability of occurring, but a very high impact if they establish infection in domestic animals and humans. the coronaviridae belong to the order nidovirales of single stranded, positive sense rna viruses. within the coronaviridae there are 2 sub-families, of which the coronavirinae contain 4 genera. phylogenetically, sars-cov is within the betacoronavirus genus (ictv, 2017) . in older literature, sars-like covs were assigned to group 2b coronaviruses (lau et al., 2005) . the viruses are roughly spherical (120-140 nm diameter), with an envelope that contains many surface glycoproteins that form a corona (crown) under electron microscopy (masters and perlman, 2013) . although enveloped, the virus is relatively stable especially in faeces and urine for 1-2 days (or longer) at room temperature, and up to 4 days if stool is from diarrhea patients (the ph is higher) (see "relevant websites section"). however, they are sensitive to heat, lipid solvents, oxidizing agents and non-ionic detergents (markey et al., 2013a) . many coronaviruses are associated with respiratory and intestinal disease and can cause severe epidemic gastrointestinal disease in agriculturally important species such as porcine epidemic diarrhea virus in pigs (masters and perlman, 2013) . transmission: is faecal-oral, respiratory by aerosolised secretions. sars-cov was first discovered in 2003, after a pandemic of severe respiratory disease that originated in guangdong province, china. it caused disease in 8098 people and of these, 774 died (see "relevant websites section"). it is now believed that chinese horseshoe bats are the original reservoir host of sars-like covs (lau et al., 2005; li et al., 2005) . bats transmit these viruses, probably via faecal contamination of food sources directly to humans or to intermediate hosts, most notably palm civets and raccoon dogs, and these then transmit virus to humans (guan et al., 2003) . these animals are traded in live animal markets as food and it is thought that aerosolisation of faeces and other body fluids caused respiratory infection of humans (wang et al., 2005) . once sars-cov entered humans, it was spread by respiratory droplets or contamination of surfaces by droplets that were then transferred to the mouth, nose or eyes by touching. there was also a cluster of cases in an apartment block in hong kong that probably arose from sewage aerosolisation into the ventilation system that proceeded to cause respiratory infection of many of the inhabitants (tilgner et al., 2003) . the incubation period is 2-7 days but can be up to 10 days. infection results in symptoms of a high fever, chills, and headache. some people have mild respiratory symptoms from the beginning that progress to lower respiratory involvement with a nonproductive cough, which can lead to hypoxemia and pneumonia. a low proportion of patients (10%-20%) had diarrhea. as stated above, approximately 10% of patients died from the severe respiratory complications, but these were usually older patients or those with other health problems. up to 20% of patients needed mechanical ventilation to survive. the severe sequela could take many days and weeks to develop. shedding in faeces occurs for a long time after symptoms are cleared so contact from soiled material from past patients can be a source of infection (masters and perlman, 2013) . there have been no known cases of sars since 2004 (see "relevant websites section"). however, sars-cov is not eradicated, as similar viruses are still present in their wild-life hosts. therefore, continued vigilance must be used to stop the spread of these viruses from their zoonotic hosts to humans. control of live animal markets and a ban on sales of exotic animals is required (see "relevant websites section"), but difficult to introduce due to the cultural background of the communities in which they are found. there is no vaccine or treatment against the virus (coleman and frieman, 2014) , although several drugs have been developed that could be used in the future (masters and perlman, 2013). nipah virus is a member of the viral family, paramyxoviridae in the genus henipavirus (ictv, 2017). it was originally identified in 1999 in malaysia during an outbreak of encephalitis and respiratory disease in pig farmers and those who had close contact with pigs. its virions are roughly spherical of about 150 nm diameter but filamentous forms can be seen (wang et al., 2013) . it is a relatively unstable virion (markey et al., 2013c) , but can survive well in ph-neutral fruit bat urine (44 days at 22°c), on fruit (up to 2 days) and in artificial date palm sap (de wit et al., 2014; fogarty et al., 2008) . it is however susceptible to desiccation (fogarty et al., 2008) . it is an enveloped virus that is inactivated by most common disinfectants, lipid solvents, and non-ionic detergents (markey et al., 2013c) . transmission is respiratory and oral. the wild-life reservoir host of nipah virus are pteropus fruit bats (chua et al., 2002; yadav et al., 2012) and transmission may be by direct infection of humans, after the virus has passed through an intermediate host e.g., pigs, or from fomites. it is thought that bat saliva or urine is the major source of virus and so food such as partially eaten fruit contaminated with saliva or contamination by faeces and urine may be the source of infection for other hosts. in the malaysian outbreak of 1998-99, it is thought that contaminated fruit from trees adjacent to pig farms encroaching into forest were dropped into enclosures where they were eaten by the pigs or that there was direct contamination of enclosures with urine. the pigs amplified the virus (they also showed clinical respiratory and neurological disease), so that farmers and workers in direct/close contact were infected and were the population where the majority of cases was seen (parashar et al., 2000) . however, in the indian sub-continent, especially bangladesh, where nipah virus infection was first recognized in 2001, the major risk factor for contracting nipah virus is drinking raw palm sap (luby et al., 2006) . there is photographic evidence of fruit bats licking sap from the cuts in the trees from which the sap is collected, and contamination by faeces is seen in or on the pots (khan et al., 2010; rahman et al., 2012) . human infection may be asymptomatic, but there can be acute respiratory disease with fatal encephalitis, leading to mortality rates of 40%-75%. the incubation period is believed to be 4-14 days or longer (see "relevant websites section"). the malaysian outbreak saw more cases of encephalitis than respiratory disease and there was little evidence for human to human transmission. in the indian sub-continent, especially bangladesh, the symptoms of infection manifested more with respiratory disease with fewer cases of encephalitis (luby et al., 2009) . the aerosolisation of respiratory secretions (droplets) and close contact with the sufferer allows respiratory transmission to lead to human to human transmission chains (gurley et al., 2007; yadav et al., 2012) . nipah virus will be maintained in its bat reservoir and so it is unlikely to be eradicated. slaughter and burial of pigs in the affected regions controlled the malaysian and singapore outbreak. routine cleaning and disinfection on pig pens with detergents may reduce exposure of pigs but if an outbreak is suspected, quarantine and culling can reduce the spread of disease (wang et al., 2013) . in bangladesh, the use of bamboo skirts to restrict access of bats to the sites on trees where sap is collected reduces the risk of contamination (khan et al., 2010) . heat treatment of sap also inactivates the virus and this, along with stopping the feeding of raw palm sap to humans and domestic animals reduces the risk of infection and transmission (luby et al., 2009) . continual surveillance for re-occurrences must be in place to monitor possible incursions into humans. there is no vaccine and no specific treatment at present (see "relevant websites section"). influenza viruses are members of the orthomyxoviridae family, and the h5n1 genotype is in the alphainfluenzavirus genus and is of the influenza a species (ictv, 2017). influenza viruses are negative stranded rna viruses that have a segmented genome (shaw and palese, 2013) . influenza a nomenclature uses the 2 genomic segments that encode the envelope glycoproteins, haemagglutinin (h) and neuraminidase (n) because these proteins are major contributors to the pathogenicity of the viruses. the wildlife reservoir for these viruses is wild birds, especially waterfowl, in which a subclinical gastroenteric infection is usually seen. thus, these viruses are often called avian influenza. there are several high pathogenicity avian influenza a (hpai) virus genotypes but one of the most pathogenic for humans and other animals are those in the h5n1 genotype (wright et al., 2013) . with widespread sequencing, the ability to group viruses as 'clades', i.e., those viruses that are derived from a common ancestor, has now allowed a numerical naming system to be instituted. the eurasian-african h5n1 viruses that are circulating and causing human and animal disease can be split into several (≥20) clades (see "relevant websites section"). within all the different clades, the original h5 genotype has remained, despite re-assortment of the other gene segments. the virions are spherical (80-120 nm diameter) or filamentous (up to 20µm long) (noda, 2011) . the virus is present in the faeces of wild waterfowl, water contaminated by their faeces, domestic poultry secretions (respiratory and faeces) and aerosolised respiratory secretions from infected animals. the virion is usually very labile (markey et al., 2013b) but it has been shown that avian influenza virus is infectious for months in low temperature water and for over a week in water at 22°c (hinshaw et al., 1979; markwell and shortridge, 1982) with increased survival when water has neutral to basic ph (7.0-8.5) and low ammonia concentrations (keeler et al., 2014) . it is also stable in frozen lakes (shoham et al., 2012) , allowing maintenance in the environment and infection of waterfowl from year to year. the seasonality of epidemics in humans is affected by temperature and humidity with the virus surviving better at 20°c in low humidity conditions but at 30°c requiring higher humidity. thus, the cool, dry conditions of winter favour transmission in temperate zones and humid, rainy conditions favour transmission in tropical and sub-tropical zones. the presence of salts and proteins in respiratory droplets allows virus survival in aerosolised droplets for up to 1-24 hr (sooryanarain and elankumaran, 2015) . contamination of fomites may also occur allowing transfer by hands. transmission is via inhalation of small aerosol droplets, faecal contamination from poultry and water borne infection. in asia, contamination of the environment by the faeces of waterfowl leads to infection of domestic poultry including chickens, ducks and geese which are often kept in the same environment. this usually causes severe disease and high titres of virus to be secreted from the domestic birds promoting infection of those working with or in contact with them, or in the food chain. farming of poultry and pigs together increases the risk of transmission to pigs and they can amplify and increase the risk of human infection. influenza virus a causes seasonal outbreaks with occasional severe epidemics/pandemics in humans and animals. the asian-african h5n1 lineage is thought to have originated from commercial geese in the guangdong province of china in 1996. this has since been spread across the globe by wild birds and infected people. there is evidence that h5n1 has infected humans directly by drinking duck blood or eating duck meat, however, aerosolisation and inhalation is the more common route (shao et al., 2011; tumpey et al., 2002) . this virus has evidence of direct bird to human transmission without the need for adaptation through a secondary species such as pigs (wright et al., 2013) . in humans, the symptoms it causes are an acute respiratory tract infection with fever and sore throat, cough and malaise. if the virus becomes systemic there may also be vomiting and abdominal pain. h5n1 viruses are highly pathogenic and there is a high case mortality rate with these infections (wright et al., 2013) . reducing the exposure of domestic poultry to wild waterfowl i.e., increased biosecurity reduces the risk of infection of these poultry and so exposure of workers to the virus. quarantine and culling of premises as well as closing live bird markets also reduces the risk of spreading the infection (see "relevant websites section"). in humans, there is a killed vaccine against annual strains of influenza a and b, and a live attenuated influenza a and b nasal spray vaccine. however, none is specifically against the h5n1 strains. there are several antiviral drugs (amantadine, rimantadine, zanamivir, oseltamivir) available to treat those infected with influenza a, but there is the risk of resistance occurring, with some of the circulating h5n1 strains showing resistance to amantadine and rimantadine and oseltamivir resistant strains have been isolated from patients treated with the drug (wright et al., 2013) . infections by severe acute respiratory syndrome (sars) virus, nipah virus (niv), h5n1 virus, hepatitis a virus (hav), hepatitis e virus (hev), adenovirus, astrovirus, norovirus (nov) and rotavirus (rva) in humans and animals are detected by nucleic acid amplification tests and serologic tests. for example, a standardised method based on quantitative real-time pcr (rt-qpcr) for the detection of nov and hep a in food has been developed by the european committee for standardisation (cen) working group (tc 275/wg6/tag 4 -detection of viruses in food) (lees and cen wg6 tag4, 2010) and provides a tool to quantify nov concentration in shellfish. it has been used to demonstrate that the risk of gastrointestinal illness associated with consumption of oysters increases with increasing concentrations of nov genome copies present (lowther et al., 2012) . costantini et al. (2010) demonstrated that a commercially available nov enzyme immunoassay showed excellent specificity but low sensitivity both for outbreaks as well as for samples from sporadic cases. the assay detected 18 of the 21 genotypes evaluated and that at least 10 7 virus particles g −1 of faecal sample were required for a positive signal. this assay may be useful for rapid screening of faecal samples collected during an outbreak of acute gastroenteritis. to detect and quantify hev virus present in environmental and food samples, a rt-qpcr method was developed by jothikumar et al. (2006) . this taqman assay was designed to target a conserved region in orf3, allowing the detection of four different genotypes of hev. a number of commercially elisa (enzyme-linked immunosorbent assay) kits for the hev detection of igm and igg antibodies which can be used early diagnosis of patients suspected for infection with hev, for the screening of blood units and the follow-up of hev-infected patients are also available. a taqman ). this method, when applied to environmental samples, was able to detect rotavirus at a level 2.6 â 10 4 and 2.6 â 10 5 particles/ml in tap water and environmental water, respectively. a competitive rt-pcr sybr green assay was designed based on conserved regions of the vp6 gene of group a rotaviruses, producing a 433 base pair fragment (schwarz et al., 2002) . an in vitro synthesised rna with a 43-base deletion with respect to the wild-type sequence of this fragment was used as an internal control. using these transcripts as templates, 10 rna molecules were amplified and reproducibly detected. using this protocol, the assay could be used to investigate the presence of rotavirus in environmental, food and water samples. immunochromatography tests are also available for detecting rotaviruses in stool samples. a study undertaken by de grazia et al. (2017) compared the performance of two commercially available one-step chromatographic immunoassays that detect both rotavirus and adenovirus. both tests were able to detect the wide range of rva genotypes circulating over the study period (including g1p[8] , g2p[4] , g3, g4, g9 and g12p[8] ). the results of the present study showed a satisfactory efficacy of the two diagnostic tests analyzed using real-time pcr as a reference test. the case fatality rate for niv is estimated to be 40%-75%. in addition, there is no treatment or vaccine available for either people or animals. the main tests used are real time rt-pcr from bodily fluids and antibody detection via enzyme-linked immunosorbent assay elisa (see "relevant websites section"). for example, a taqman assay as described by guillaume et al. (2004) for niv detected a wide range of virus concentrations from 1.2 â l0 5 pfu to 1.2 pfu per reaction, corresponding to a threshold of 200 pfu/ml for rapid, accurate and quantitative diagnosis. the specificity of the niv taqman assay was determined by the absence of amplification using measles and hendra paramyxovirus rna. an antigen capture elisa was developed by chiang et al. (2010) for the viral detection of henipavirus and for the differentiation between niv and hendra virus. this assay allows for the rapid detection and differentiation between the henipaviruses and could be used in any future outbreaks of henipaviruses. astroviruses are classified into two genera: mammalian viruses (mamastroviruses, mastvs) and avian viruses (avastroviruses, aastvs). human astroviruses are found in four mastv species (mastv 1, 6, 8, 9) (pérot et al., 2017) . an rt-pcr assay was designed by finkbeiner et al. (2009) , based on the astrovirus rna polymerase (orf 1 b) that allows for the detection of the eight human astroviruses serotypes found in mastv 1, a common cause of viral gastroenteritis in children. this primer set also detects viruses in mastv 6. these two groups also contain astroviruses from cats, pigs, dogs and rabbits (pérot et al., 2017) . to date, there is no universal pan-astrovirus rt-pcr assay. immunochromatography (ic) tests are also available for detecting astroviruses. an ic test for the detection of astrovirus was evaluated in 44 stool samples of pediatric patients with acute gastroenteritis in japan, during january to march 2007, and it is a rapid method for the detection of astroviruses and may be useful for screening astroviruses during outbreaks of food-borne and person-to-person transmission (khamrin et al., 2010) . a broad-spectrum pcr assay was developed by sibley et al. (2011) for the detection of mastadenovirus (maadv) and atadenovirus (atadv), based on the adenovirus hexon gene. maadv, which comprises human and bovine adenoviruses and a large variety of mammalian adenoviruses. atadv includes bovine adenovirus (badv) as well as adenovirus infecting ducks, goats, sheep, deer, and reptiles (sibley et al., 2011) . this assay has been shown to demonstrate natural badv excretion in urine, badv detection in groundwater, and recombination in adv of livestock origin (sibley et al., 2011) and has the potential to be used in screening samples during outbreaks of food-borne disease. for the detection of h5n1 influenza viruses, the world health organisation (who) (see "relevant websites section") provides updated information on the molecular detection/diagnostic protocols for the surveillance of influenza viruses in humans. for example, the who provides primers and probes sequences for real-time rt-pcr procedures for the detection of: (1) influenza type a viruses (matrix gene). a taqman based assay was designed for the detection of sars and middle east respiratory syndrome (mers) coronaviruses (covs) by noh et al. (2017) , based on the conserved spike s2 region of human sars-cov, mers-cov, and their related bat covs. this assay can detect sars-cov and mers-cov in humans but also several bat covs that are closely related to these viruses in bats. a monoclonal antibody-based capture enzyme immunoassay for the detection of nucleocapsid antigen in sera from patients with sars was developed by che et al. (2004) . this assay used a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen. the sensitivity of the assay was 84.6% in 13 serologically confirmed sars patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). the specificity of the assay was 98.5% in 1272 healthy individuals (1253 of 1272). there was no cross-reaction with other human and animal coronaviruses in this assay. • food can become contaminated by viruses at source and through contaminated food handlers and environments. • good food hygiene and personal hygiene, especially hand washing, are essential to help minimise the spread of these viruses within the food chain. • since foodborne viruses tend to be more resistant to physical and chemical treatments than bacteria, their control represents a challenge for the food industry. • ongoing research is required, for example, current laboratory detection methods can be modified to allow differentiation between e.g., infectious and non-infectious viruses. • there are a number of knowledge gaps in terms of how, for example, nov, hav and hev are transmitted through the food chain, the contribution they make to overall foodborne illness and their survival and elimination from food. • emerging trends indicate that viruses play an important role in foodborne illness. this has implications for the whole of the food chain. potential role of fomites in the vesicular transmission of human astroviruses astrovirus survival in drinking water rotavirus in adults requiring hospitalization virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis human astroviruses acute respiratory disease associated with adenovirus serotype 14 -four states alarmins: awaiting a clinical response sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome vaccine development for human mastadenovirus use of monoclonal antibodies against hendra and nipah viruses in an antigen capture elisa isolation of nipah virus from malaysian island flying-foxes immunology: a short course coronaviruses: important emerging human pathogens changing pattern of rotavirus strains circulating in ireland: re-emergence of g2p [4] and identification of novel genotypes in ireland diagnostic accuracy and analytical sensitivity of ideia norovirus assay for routine screening of human norovirus genetic variability and molecular evolution of hepatitis a virus reduction in acute gastroenteritis hospitalizations among us children after introduction of rotavirus vaccine: analysis of hospital discharge data from 18 us states astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions performance analysis of two immunochromatographic assays for the diagnosis of rotavirus infection foodborne transmission of nipah virus in syrian hamsters fields virology. 2 detection of newly described astrovirus mlb1 in stool samples from children letter: virus diarrhea in foals and other animals henipavirus susceptibility to environmental variables next-generation sequencing for diagnosis and tailored therapy: a case report of astrovirus-associated progressive encephalitis isolation and characterization of viruses related to the sars coronavirus from animals in southern china specific detection of nipah virus using real-time rt-pcr (taqman) molecular characterization of group a rotavirus found in elderly patients in ireland person-to-person transmission of nipah virus in a bangladeshi community sensitive detection of multiple rotavirus genotypes with a single reverse transcription-real-time quantitative pcr assay water-bone transmission of influenza a viruses annual cost of illness and quality-adjusted life year losses in the united states due to 14 foodborne pathogens hepatitis a: clinical manifestations and management a broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection of hepatitis e virus hepatitis e virus infection abiotic factors affecting the persistence of avian influenza virus in surface waters of waterfowl habitats evaluation of a rapid immunochromatography strip test for detection of astrovirus in stool specimens use of infrared camera to understand bats' access to date palm sap: implications for preventing nipah virus transmission goblet cells and mucins: role in innate defense in enteric infections age-stratified seroprevalence of neutralizing antibodies to astrovirus types 1 to 7 in humans in the netherlands hepatitis e -a new era in understanding the action of alcohols on rotavirus, astrovirus and enterovirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats international standardisation of a method for detection of human pathogenic viruses in molluscan shellfish type a viral hepatitis: a summary and update on the molecular virology, epidemiology, pathogenesis and prevention a comparison of the efficiency of elisa and selected primer sets to detect norovirus isolates in southern ireland over a fouryear period (2002-2006): variation in detection rates and evidence for continuing predominance of nov gii.4 genotype prevalence and characterization of enteric adenoviruses in the south of ireland bats are natural reservoirs of sars-like coronaviruses immunoglobulins for preventing hepatitis a comparison of norovirus rna levels in outbreak-related oysters with background environmental levels foodborne transmission of nipah virus transmission of human infection with nipah virus adenovirus: epidemiology, global spread of novel serotypes, and advances in treatment and prevention clinical veterinary microbiology clinical veterinary microbiology clinical veterinary microbiology possible waterborne transmission and maintenance of influenza viruses in domestic ducks zoonotic aspects of rotaviruses full genome-based classification of rotaviruses reveals a common origin between human wa-like and porcine rotavirus strains and human ds-1-like and bovine rotavirus strains prevalence and genetic diversity of human astroviruses in mexican children with symptomatic and asymptomatic infections candidate new rotavirus species in sheltered dogs vaccine development against zoonotic hepatitis e virus: open questions and remaining challenges native morphology of influenza virions simultaneous detection of severe acute respiratory syndrome, middle east respiratory syndrome, and related bat coronaviruses by real-time reverse transcription pcr molecular detection of animal viral pathogens long-term protective effects of hepatitis a vaccines. a systematic review case-control study of risk factors for human infection with a new zoonotic paramyxovirus, nipah virus, during a 1998-1999 outbreak of severe encephalitis in malaysia impact of rotavirus vaccination in australian children below 5 years of age hepatitis e and pregnancy: current state hepatitis e virus: infection beyond the liver date palm sap linked to nipah virus outbreak in bangladesh virus hazards from food, water and other contaminated environments foodborne illness acquired in the united states-major pathogens detection and quantitation of group a rotaviruses by competitive and real-time reverse transcriptionpolymerase chain reaction a brief review of foodborne zoonoses in china orthomyxoviridae persistence of avian influenza viruses in various artificially frozen environmental water types detection of known and novel adenoviruses in cattle wastes via broad-spectrum primers environmental role in influenza virus outbreaks hepatitis e: a disease of reemerging importance hepatitis e virus genotypes and evolution: emergence of camel hepatitis e variants estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis world health organization environmental team characterization of a highly pathogenic h5n1 avian influenza a virus isolated from duck meat the unpredictable diversity of co-circulating rotavirus types in europe and the possible impact of universal mass vaccination programmes on rotavirus genotype incidence rotavirus infection in infants as protection against subsequent infections novel human astroviruses: novel human diseases? epidemiology of classic and novel human astrovirus: gastroenteritis and beyond henipaviruses sars-cov infection in a restaurant from palm civet adenovirus gastroenteritis global use of rotavirus vaccines recommended. who orthomyxoviruses detection of nipah virus rna in fruit bat (pteropus giganteus) from india hepatitis e virus: foodborne, waterborne and zoonotic transmission rotavirus incidence and genotype distribution before and after national rotavirus vaccine introduction in belgium development and evaluation of a loop-mediated isothermal amplification assay for the detection of adenovirus 40 and 41 prevention and control: oie -world organisation for animal health who -first data on stability and resistance of sars coronavirus compiled by members of who laboratory network who information for the molecular detection of influenza viruses who -updated unified nomenclature system for the highly pathogenic h5n1 avian influenza viruses key: cord-335302-6wsx0jby authors: mahy, brian w.j. title: the diversity of viruses infecting humans date: 2011-12-12 journal: biodiversity (nepean) doi: 10.1080/14888386.2006.9712792 sha: doc_id: 335302 cord_uid: 6wsx0jby most human viruses have been discovered through the diseases they cause in animals, plants, bacteria or fungi. recent finds include human bocaviruses, which now seem to have a global distribution, and cause respiratory tract disease in infants, and several new pathogenic human coronaviruses. the sars coronavirus, genetically distinct from all previously known coronaviruses, caused a disease which was highly transmissible and very severe, eventually leading to 8000 cases worldwide with over 800 deaths. many viruses which are transmitted to humans by invertebrates, such as insects or ticks, have the ability to infect and replicate in cells of both vertebrate and invertebrate origin. however human virology is a rapidly expanding field and recent technologies such as the polymerase chain reaction (pcr) amplification system have made it possible to look for previously unrecognized viruses which may or may not be involved in pathogenesis. for example viruses in the genus anellovirus are found in 80% of human blood samples yet do not seem to cause any disease. this paper overviews known human vertebrate viruses, more recent discoveries, and recommends a systematic search for viruses which may already infect the human population but have so far remained undetected. the most recent report of the international committee on taxonomy of viruses (ictv) lists more than 6000 viruses that are classified into 1950 virus species and distributed among more than 391 different taxa (fauquet et al. 2005) . in general, most of these viruses were discovered through the diseases they cause in animals, plants, bacteria or fungi, although since the development of the polymerase chain reaction (pcr) amplification system it has been possible to detect the presence of viruses that do not appear to cause any disease. an example of such viruses can be seen in the genus anellovirus, which contains at least three small circular single-stranded dna viruses that can be found in the blood of humans (biagini et al. 2006) . these viruses are extremely widespread in the human population, with abundant anellovirus dna being detectable in more than 80 % of human blood samples tested yet cause no clinical disease (bendinelli & maggi 2005) . for these reasons, any consideration of the diversity of human viruses must take into account that there has been no systematic search for human viruses that may or may not be associated with disease. humans are vertebrates, and the article by craig pringle in this issue ("the taxonomy of vertebrate viruses") describes the many virus families and species known to infect vertebrates. very few viruses are unique to the human species, and perhaps the only well known example, variola virus, has been eradicated by vaccination and now no longer exists in nature. other potential targets for eradication, hepatitis c virus and human immunodeficiency virus (hiv) seem only able infect humans and certain non-human primates, but no vaccines have been developed so far that might prevent or eliminate these infections. it is also clear that many viruses which are transmitted to humans by invertebrates, such as insects or ticks, must have the ability to infect and replicate in cells of both vertebrate and invertebrate origin. some of these, such as the flavivirus yellow fever virus, cause extremely serious disease consequences in humans, where they target liver cells, with replication in kupffer cells, causing massive necrosis of hepatocytes which leads to jaundice and in the worst cases severe hemorrhagic fever involving the gastrointestinal tract. these pathological features made yellow fever one of the worst plagues in history, particularly affecting the african and south american regions, and perhaps because of the disease severity, the epidemiology of the diversity of viruses infecting humans brian w.j. mahy abstract. most human viruses have been discovered through the diseases they cause in animals, plants, bacteria or fungi. recent finds include human bocaviruses, which now seem to have a global distribution, and cause respiratory tract disease in infants, and several new pathogenic human coronaviruses. the sars coronavirus, genetically distinct from all previously known coronaviruses, caused a disease which was highly transmissible and very severe, eventually leading to 8000 cases worldwide with over 800 deaths. many viruses which are transmitted to humans by invertebrates, such as insects or ticks, have the ability to infect and replicate in cells of both vertebrate and invertebrate origin. however human virology is a rapidly expanding field and recent technologies such as the polymerase chain reaction (pcr) amplification system have made it possible to look for previously unrecognized viruses which may or may not be involved in pathogenesis. for example viruses in the genus anellovirus are found in 80% of human blood samples yet do not seem to cause any disease. this paper overviews known human vertebrate viruses, more recent discoveries, and recommends a systematic search for viruses which may already infect the human population but have so far remained undetected. the disease was worked out and shown to involve mosquito borne transmission as early as 1900, when few viruses had been described (reed 1902) . this led to the development of an effective attenuated yellow fever vaccine for the protection of humans. unfortunately a satisfactory vaccine has not so far been developed for use against a related flavivirus, dengue/ dengue hemorrhagic fever virus, which occurs in four major antigenic types. but dengue fever virus, like yellow fever virus, is spread by aedes aegypti mosquitoes, and so mosquito control methods have benefited the control of both these diseases. in addition to insect borne flaviviruses, there are also serious diseases caused by tick borne flaviviruses, such as kyasanur forest disease virus, found in india, omsk hemorrhagic fever virus, found in russia, and alkhurma hemorrhagic fever virus, found in saudi arabia. the exact mechanisms involved in the infection of insect cells versus tick cells or human cells are not well worked out, but it seems clear that viruses which alternate between infections of arthropod and vertebrate hosts show a remarkable genetic stability during experimental (bilsel et al. 1988) or natural (deubel et al. 1985; charrel et al. 2005 ) transmission cycles. although human viruses have been studied for more than a century, it has become clear in the last 15 -20 years that a large number of new human virus infections have been newly recognized (mahy and murphy 2005) . in some cases a virus has been found in association with a long-recognized disease syndrome such as acute respiratory disease syndrome (ards) caused by the hantavirus sin nombre virus (nichol et al. 1993) , and this led to the discovery of more than 30 related hantaviruses throughout the american continent. these virus infections are spread to humans through a vertebrate (usually a rodent) vector, but until the first one was recognized through epidemiological association and careful molecular analysis, they had remained unsuspected for decades. other new viruses have been recognized because of a new disease they caused in humans, such as the severe acute respiratory syndrome (sars) coronavirus . the disease first appeared in humans in china in 2002, and by 2003 had spread to a number of neighbouring countries, the first virus isolate was made from samples taken from a physician who had died from the disease in vietnam (drosten et al. 2003; ksiazek et al. 2003) . the unusual feature of the sars coronavirus was that although previously known human coronavirus infections (human coronaviruses oc43 and 229e) had long been recognized as causing relatively mild symptoms such as the common cold, genetic analysis of the sars virus showed that it was genetically distinct from all previously known coronaviruses, and caused a disease which was highly transmissible and very severe, eventually leading to 8000 cases worldwide with over 800 deaths. the coronaviruses are currently divided into three genetic groups, with human coronavirus 229e falling into group 1 and human coronavirus oc43 into group 2. group 3 contains only avian coronavirus species. the sequence of the sars coronavirus placed it in group 2 (kim et al. 2006) . studies on the origin of the sars coronavirus are still ongoing: there is recent evidence of a zoonotic origin of the human disease, perhaps from palm civets, but the true natural reservoir of the virus seems most likely to be in a bat species, probably chinese horseshoe bats (lau et al. 2005; li et al. 2005) . what is clear, however, is that careful serological studies revealed no evidence of the presence of the virus in the human population before 2002 (knobler et al. 2004) , when this virus first entered the human population and caused sars. in addition to sars, which was the first major international disease outbreak of the 21 st century, a number of diseases of lesser importance have been newly recognized by the discovery of new virus species related to conventional virus infections. for example, it has been known for many years that the human parvovirus, a small single-stranded dna virus known as b19, was the cause both of a rash in children called erythema infectiosum (fifth disease), and also of aplastic crisis in both children and adults with chronic hemolytic anemia. this was the only known human parvovirus until very recently, when a new parvovirus was discovered to be the cause of lower respiratory tract infections in children. the new virus was discovered by large-scale molecular virus screening of pooled human respiratory tract samples based on host dna depletion, random pcr amplification, large-scale sequencing analysis and bioinformatics. this procedure detected one parvovirus and one coronavirus, both of which were at that time uncharacterized (allander et al. 2005) . the human parvovirus displayed genome nucleotide sequence similarities to known parvoviruses infecting bovine and canine animal species, bovine parvovirus and canine minute virus, which had been placed in the genus bocavirus of the subfamily parvovirinae within the family parvoviridae ( fauquet et al. 2005 ). thus the virus was provisionally named human bocavirus. surprisingly, studies carried out in australia (sloots et al. 2006 ) japan (ma et al. 2006 ) canada (bastien et al. 2006 ) and elsewhere (unpublished) have revealed a significant number of children whose lower respiratory tract disease appears to be caused by human bocavirus infection. the new human coronavirus detected by allander et al. (2005) was found by sequence analysis to be related to another new human coronavirus identified in an elderly man who developed pneumonia in hong kong soon after returning from shenzen, china (woo et al. 2005a) . this virus was named human coronavirus-hku1, since it was isolated in hong kong university. further studies there showed that this virus was also associated with a second case of pneumonia, this time in a 35 year old woman, and a more complete investigation involving 4 hospitals showed that hcov-hku1 virus was responsible for 2.4% of all community-acquired pneumonia (woo et al. 2005b) . most recently, hcov-hku1 virus was found in australian children (sloots et al. 2006) , and in 5 children and one adult in caen, france (vabret et al. 2006 ). probably as a result of the sudden world-wide interest in human coronaviruses generated by the discovery of the sars coronavirus, other new human coronaviruses were found using more conventional approaches. in 1994, a group from the university of amsterdam isolated a new coronavirus from a 7month-old child suffering from bronchiolitis and conjunctivitis, and named the virus hcov-nl63. the virus was genetically distinct from previously reported coronaviruses, but appeared to be a new group 1 coronavirus which was widely spread in the dutch population ( (kaiser et al. 2005) and china (chiu et al. 2005; zhu et al. 2006) . at the same time that the amsterdam group were reporting their findings, a group at yale university discovered a closely related if not identical coronavirus to nl63 which they named new haven coronavirus (hcov-nh) (esper et al. 2005a ). the yale group admitted that their coronavirus was similar and might represent the same species as hcov-nl63. however, in an accompanying paper, esper et al. (2005b) went on to claim an association between hcov-nh and kawazaki disease, a childhood systemic vasculitis long suspected of having a viral origin, though none has ever been found. the importance of this claim stimulated existing kawasaki research groups in california and in taiwan to look for the virus in their clinical specimens, but neither group could confirm any association of either hcov-nh or hcov-nl63 with the disease (shimizu et al. 2005; chang et al. 2006 ). it is clear from these recent discoveries that the sars epidemic catalyzed a great deal of excellent work in what had been a neglected area of virology with the result that we now have more than doubled the number of known human coronaviruses from 2 to at least 5. how many more might still be undetected? in their seminal paper describing the discovery of the new human parvovirus, human bocavirus, allander et al. (2005) suggest that it might be possible to undertake a systematic exploration to describe the "human virome". this might be based not on disease, but given our extensive data bank of virus sequences, it would be possible to carry out a systematic search for the presence of viruses in human tissues. the coronaviruses were investigated because of the intense interest in understanding sars and its origins, but i suggest that an alternative approach might be to focus on viruses known to be important causes of disease in animals, but for which a persistent lifelong infection with no obvious pathological consequences apart from a tenfold rise in lactate dehydrogenase enzyme in the blood. but in aging mice of certain strains a form of poliomyelitis may develop. all arteriviruses, including ldv, primarily infect macrophages. the only known primate arterivirus affects monkeys, causing simian hemorrhagic fever. finally, in addition to the coronavirus genus, the family coronaviridae includes the genus torovirus, whose members are known to infect horses, bovines and carnivores. the discovery of at least one human torovirus has recently been claimed and linked to neonatal necrotizing enterocolitis (lodha et al. 2005) . there is no doubt that human virology is a rapidly expanding field, and technological advances have now made it possible to look for previously unrecognized viruses existing in the human population which may or may not be involved in pathogenesis. recent examples of such newly recognized viruses include the human bocaviruses, which now seem to have a global distribution, causing respiratory tract disease in infants, and several new pathogenic human coronaviruses, as we continue to search for the causes of many human diseases of unknown aetiology, it may be prudent to undertake a more systematic search for viruses which may already infect the human population but have so far remained undetected. cloning of a human parvovirus by molecular screening of respiratory tract samples new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia human coronavirus nl63 infection in canada human bocavirus infection human coronavirus nl63 infections in children: a 1-year study of topley&wilson's microbiology and microbial infections identification of a third member of the anellovirus genus ("small anellovirus") in french blood donors rna genome stability of toscana virus during serial ttransovarial transmission in the sandfly phlebotomus perniciosus lack of association between infection with a novel human coronavirus (hcov), hcov-nh, and kawasaki disease in taiwan low diversity of alkhurma hemorrhagic fever virus, saudi arabia human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong homogeneity among senegalese strains of yellow fever virus identification of a novel coronavirus in patients with severe acute respiratory syndrome detection of human coronavirus nl63 in young children with bronchiolitis evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children association between a novel human coronavirus and kawasaki disease virus taxonomy, eighth report of the international committee on taxonomy of viruses human coronavirus nl63 associated with lower respiratory tract symptoms in early life close relationship between sars-coronavirus and group 2 coronavirus learning from sars. preparing for the next disease outbreak a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sarslike coronaviruses human torovirus: a new virus associated with neonatal necrotizing enterocolitis detection of human bocavirus in japanese children with lower respiratory tract infections of topley&wilson's microbiology and microbial infections a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl63 in children hospitalized with respiratory tract infections in belgium genetic identification of a hantavirus associated with an outbreak of acute respiratory illness recent researches concerning etiology, propagation and prevention of yellow fever by the united states army commission lactate dehydrogenaseelevating virus human coronavirus nl63 is not detected in the respiratory tracts of children with acute kawasaki disease evidence of human coronavirus hku1 and human bocavirus in australian children detection of the new human coronavirus hku1: a report of 6 cases identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia clinical and molecular epidemiological features of coronoavirus hku1-associated community-acquired pneumonia human coronavirus nl63 was detected in specimens from children with acute respiratory infection in beijing, china key: cord-352348-2wtyk3r5 authors: sabroe, ian; dockrell, david h.; vogel, stefanie n.; renshaw, stephen a.; whyte, moira k. b.; dower, steven k. title: identifying and hurdling obstacles to translational research date: 2007 journal: nat rev immunol doi: 10.1038/nri1999 sha: doc_id: 352348 cord_uid: 2wtyk3r5 although there is overwhelming pressure from funding agencies and the general public for scientists to bridge basic and translational studies, the fact remains that there are significant hurdles to overcome in order to achieve this goal. the purpose of this opinion article is to examine the nature of these hurdles and to provide food for thought on the main obstacles that impede this process. it has achieved much, including the sequencing of the entire human genome, but it is already under serious attack for its failure to deliver effective therapies in many areas. this opinion article will provide a subjective view of our understanding of translational research, identify obstacles to its successful development, and propose a series of initiatives to improve the effectiveness of translational research strategies. originating from latin, translation means to 'carry across' . in biomedical research, the goal of translational science is to develop a thorough operational understanding of the human organism in health and disease, with the goal of 'carrying across' this knowledge to alleviate disease and suffering and to improve the quality of human existence. to be translational in medicine we must acquire knowledge from the broad arena of molecular and cellular biology and then apply this knowledge to human disease. the quality of our scientific output (perceived as a change in disease incidence and/or the development of a therapy) is largely dependent on the quality of the input data and the methods for their processing and interpretation, although the process of generating effective translational science is not as linear (that is, from molecules to models to humans) as is often thought. failure to ask the appropriate questions of optimized systems leads to the acquisition of knowledge that might be less relevant than anticipated. further corruption of the process comes as a result of limitations in our models, which are often not fully appreciated (or are simply ignored) at the time of the study. additionally, grant agencies must be sufficiently flexible to allow researchers to follow up on novel observations because many of the most exciting developments arise from unexpected findings. determining what research is intrinsically translational, or has been translated effectively, is therefore surprisingly difficult, and in a healthy global biomedical research environment, translation will continue to mean very different things to different groups. few scientists will see a process through from the conception of a hypothesis to the development of a specific medical therapy. as scientists, we are nonetheless required to measure our performance in terms of our 'translational potential' , particularly when it comes to justifying and generating funding and publications. therefore, in the absence of being able to measure contributions to health directly, we often quantify individual success using surrogate markers (such as publications and their impact, prestige, and funding), which depend on the prevailing concepts of what constitutes importance. however, these markers may be flawed for this purpose. a more global assessment of the output of translational science might consider whether the scientific community has improved specific disease outcomes, quality of life or life expectancy. specific examples of global success might not be as common as we would wish, but increased successes in the areas of organ transplantation or the ability to eradicate diseases such as measles virus highlight our ability to be successful. the improved treatment of many cancers through the combination of good science and high-quality clinical trials of new therapies or combinations of therapies has been strikingly impressive and provides many examples of highly effective translational science 1,2 . there remains, however, a lack of available mechanisms by which to relate our individual contributions to the global progress of translational science, and many factors conspire to impede our progress. the translation of basic scientific research faces a myriad of hurdles, both obvious and occult. these revolve around our understanding of the nature of the translational process, the integration of the outputs of different technological approaches to disease, the use of models, access to tissues and appropriate materials, and the need for support in increasingly complex areas such as ethics and bioinformatics. in addition, owing to technological advances, well-meaning safeguards of personal privacy have been imposed in relation to carrying out research in humans, and these have, in fact, greatly impeded progress in translational studies. problems with the models. entirely appropriate restrictions on what research can be done in humans have contributed to the status quo in which the mouse has become an indispensable model for translational biology; however, it is often not possible to predict biological responses in humans accurately based solely on results obtained from animal models [3] [4] [5] [6] [7] [8] [9] [10] . within immunity, exposure of mice to their own species-specific commensals and pathogenic organisms might contribute to a species-specific immunological phenotype 4,9 that affects the translational relevance. for example, studies of the effects of irak4 (interleukin-1 receptor-associated kinase 4) deficiency have revealed increased identifying and hurdling obstacles to translational research susceptibility to a greater range of pathogens in mice than is observed in humans who are deficient for irak4 . where good potential translational concepts are generated from models, determining how to move to the human can be challenging, as highlighted by difficulties in calculating tolerogenic doses of insulin in the prevention of diabetes 10 . in addition, technical limitations, or the dominance of prevailing models, can sometimes limit the scope of in vivo work, as illustrated by studies of airway disease, where studies in mouse models are focused predominantly on the important t helper 2 (t h 2) component of asthma but are poor models for the contribution of other mediators. mouse models are also complicated by our limited understanding of the phenotypes of human diseases 14, 15 . designing perfect models of diseases that we do not fully understand is a tall order, and chronic diseases that involve life-long interactions between the host and the environment present a particularly difficult challenge [5] [6] [7] [8] . developing truly chronic models of disease is, however, heavily militated against owing to ethical concerns about prolonged suffering. moreover, the typically short-term nature of research funding, where outputs must be deliverable at a reasonable cost and within the time frame of a project grant, hinders the development of chronic models of disease. it is equally clear that work on a single cell line is often poorly predictive of the behaviour of the whole organism. although the use of primary cells from normal tissues can overcome some of these problems, the phenotype of these cells might be altered by their removal from the microenvironmental influences in vivo, and they often show markedly different responses to those of primary cells that are obtained from diseased tissue. therefore, rodent models remain essential and cannot be replaced by in vitro approaches at present, but are an imperfect translational conduit for both biological and practical reasons (such as the difficulty in establishing chronic disease models). other models such as flies and zebrafish (danio rerio) have many advantages for forward genetics and other related studies, but have substantially greater differences to humans than mammalian models. primates are the most compatible mammalian models for immunological research, and have provided invaluable insights into diseases such as hiv, but their broader use is not feasible because of major ethical concerns, as well as other practical and cost-related issues. even these models can have limitations. recently, differential expression of siglecs (sialicacid-binding immunoglobulin-like lectins), which are inhibitory receptors thought to downregulate immune-cell activation, has been noted between apes and humans 16 , and such differences in immune responses between species require careful consideration, as highlighted by the development of a 'cytokine storm' when individuals received an agonistic cd28-specific antibody that did not show the same effects in monkeys 17, 18 . research is being increasingly hindered by bureaucracy. evident at many levels in research management, nowhere is this more of a problem than in the areas of access to human samples and tissues, and in the establishment of clinical collaborations. liaison with industry also provides additional challenges 10 . furthermore, some of the most exciting areas of current research, such as work with embryonic stem cells, are subject to special ethical concerns. even the simplest research involving humans, or archived specimens from humans, is often encumbered by a multistaged, intimidating application process that might dissuade individuals from carrying out the proposed study. overcautious interpretation by ethics committees and regulatory authorities of what is ethical results in restrictive practices that can, for example, prevent re-screening of dna banks for relevant markers and mutations, or apply over-elaborate protection of clinical phenotype data. although it is absolutely clear that robust systems are needed to prevent exploitation of patients or clinical data, it is often unclear how the ethical standards that are applied to the review of proposed studies are developed. apparently arbitrary standards that might be perceived to be more ethical simply because they are more restrictive seem to be ever more on the increase. it is also unethical to excessively hinder research, but this imperative sometimes seems to come second to apparently minor scruples over details such as the methods of recruitment. although the introduction of national standards to ensure timely review of proposals has, in the united kingdom, begun to address these issues, increasingly complex review processes are frustrating and sometimes of questionable ethical value. the area of personal privacy is particularly complex. the majority public view is supportive of the use of some personal data in confidential research 19 . however, although many individuals are less concerned that well-regulated data usage is an invasion of privacy than some ethics committees seem to be, due consideration is needed of the views of the minorities who are more concerned with their privacy. the future potential for apparently confidential genetic research resulting in the identification of individuals through the cross-referencing of dna databases is clearly of concern 20,21 . in addition, governments tend to respond to highly public episodes of research offences or clinical misconduct with legislation. this runs the risk of creating additional administrative burdens for the majority of individual scientists who practise ethically and well, without ensuring the prevention of future wilful misdeeds by a rare minority. increasingly, clinical practice is becoming more defensive and less willing to engage in research as a response to a society that is becoming more risk-adverse, litigious and blame-centred when adverse events occur. this generates further barriers to research in humans and requires a frank reappraisal of what experimentation is acceptable in humans or on samples that are derived from humans. additional problems arise in setting research priorities. without effective public input, we run the risk that research priorities become the whim of changing government focuses, which lack the stable, long-term input necessary for meaningful scientific advancement. although charities and private foundations that are centred on specific diseases provide important contributions, their focus is often too narrow to tackle the wider research agenda. this has led to a culture of increasing financial pressures that are governed by short-term policies, which require rapid outputs, and piecemeal funding, stifling longer term, more open-ended research that might produce real translational progress 10 . for example, political pressures to study agents of bioterrorism or emerging infectious diseases such as sars (severe acute respiratory syndrome) can be welcomed and are clearly important; however, more long-term commitment to studying infections of historical global importance is also essential. with respect to developing long-term goals, the national institutes of health translational biomedicine must be seen by scientists and the wider public in the context of a broad-based body of science that has the potential to contribute, in the near or long term, to the advancement of human health. increased financial and political pressures on scientists to maximize directly measurable patient benefit from research carried out over short time frames runs the risk of destroying a healthy science base and should be actively resisted. a continued and robust engagement of the public is mandatory for scientists, because unless the public values the broad sweep of translational biomedical science, funding will inevitably be diverted away from science or applied only to those areas of research that immediately influence patient care. as scientists, we must learn to articulate not only the promise of science, but also the difficulties that are associated with moving an idea along to the product stage, so that unrealistic expectations of perfect medications for every disease in short time frames are not raised. we must also wrestle the debate on the usefulness of in vivo models away from the more extreme ends of the anti-vivisection movement so that a constructive, rather than confrontational, discussion can evolve. equally, we must foster a culture of research in the curricula of medical students, and promote the goals of translational research to basic science undergraduate and graduate students. we must seek to marry the skills of basic scientists with those of clinicians to convey the idea that strong translational research underlies improved health and is inseparable from the provision of good standards of clinical care. improving our use of the models? in many biomedical fields, a series of debates have highlighted deficiencies in our current in vivo models. strain differences 22 and variance in experimental conditions between research groups pose major challenges in comparing the results derived from different studies. a collaborative investment in phenotyping human disease by clinicians and basic scientists is required for developing robust models. there is clearly an opportunity to define the characteristics, behaviour and relevance of model systems, particularly with a view to standardizing optimal strains and protocols. for example, multiple models exist for common diseases such as asthma 23 and rheumatoid arthritis 24 , although harmonizing protocols and strains for particular features of a disease must avoid the loss of sufficient diversity in order to allow the finding of the unexpected. this challenge could be met by interest groups or individuals working in a particular area, by large national funding agencies and/or international funding initiatives, or alternatively by specialist societies. such debates might facilitate the comparison of data between laboratories and between species, and might highlight the components of specific diseases that are ripe for the development of new in vivo models and protocols (for example, there remains a great need to more effectively model the role of the innate immune system in acute and chronic asthma), broadening the number of disease processes or phenotypes that are modelled in pathology. although much work will continue to focus on the mouse, for some diseases comparative or independent studies in other species will continue to be important. beyond the scope of this article, there are issues ahead with respect to subject selection for early clinical trials and the development of individualized treatment regimes that are based on pharmacogenetic and individual disease phenotypes 25, 26 . figure 1 | developing interactive models. a | in vivo experimentation, perhaps in particular the generation and characterization of knockout mouse strains in experimental models of disease, is often viewed as the gatekeeper between in vitro science and the generation of drug targets that are appropriate for human disease. although central to an effective understanding of immune biology and the role of new candidate drug targets, the predictive value of in vivo experimentation is less than desired, particularly in the context of studies of single knockouts in specific disease models and mouse strains. b | an example of a more holistic network in which multiple lines of evidence allow the refinement of objectives and target relevance in order to increase the chance of successful drug discovery. such approaches reflect the approach of many researchers, but (acknowledging that no branch of science is 'easy') the main difficulties associated with undertaking human-based research run the risk of degrading the quality of data that arise from an integrated scientific approach. the tendency to view translational research as a linear process in which mice are the gateway between basic science and humans does rodent models a great disservice. in particular, the classical route of identifying genes in vitro, followed by generating knockout animals in vivo, has, in general, been poorly predictive of the consequences of targeting specific molecules in humans. advances in medical sciences that have emerged in this manner are vastly outnumbered by those that arise by serendipity or through less predictable routes. a more holistic integration of in vivo models with in vitro science and studies in the human is needed when summarizing the translational relevance of a system, in which in vivo models contribute strongly to an iterative strategy, but are not themselves the final arbiters (fig. 1) . the potential to identify medicines for use in human disease by screening less complex biological systems has long been recognized in the pharmaceutical industry, and there is now considerable interest in the use of model organisms such as caenorhabditis elegans and zebrafish in high-throughput screens for new drugs 27, 28 . it is becoming increasingly evident that these systems can be used to identify therapeutic targets in the immune system 29 . in this way, understanding the biology of pathways and gene products is deferred, and the ability of a compound to intervene in complex biological processes is directly tested, as highlighted in recent studies of calcium-channel antagonists in c. elegans 27 . combining the use of mice and humans in effective strategies. it seems self-evident that research which aspires to influence pathogenesis in humans needs to be carried out on the human system. at the same time, we must also anticipate the potential for doing harm that might accompany any new approaches to treating diseases, which militates against a rapid progression to phase i human trials. nonetheless, the extraordinary difficulties that are associated with carrying out human studies in parallel with animal models has facilitated a culture in which such studies rarely occur, and in which prioritizing research is not driven by the inclusion of such processes. indeed, almost quixotically, work in human tissues and cell lines is sometimes not deemed to be of importance until verified in a mouse with a targeted mutation. bridging this divide requires the scientific community to value more highly the studies that seek to bring mouse and human studies together, and to appreciate that in human studies a lack of phenotype or subtle modifications of processes might be as important as models that generate dramatic phenotypes. from simple co-culture models of normal human tissues and ultimately to the generation of whole organs or representations of whole organs 30 in the laboratory, we are now beginning to produce in vitro human systems that can complement essential work that is currently only achievable in vivo. increasing success with new gene-delivery systems, combined with new technologies such as gene knockdown by rna interference, might allow us to overcome the inability to study humans with targeted gene deficiencies. such models are in their infancy, and cell-culture-based approaches are, in many scientists' views, farther from human biology than techniques that investigate biology in mice. it would seem that both are required, and therefore, a major thrust to develop standardized co-culture models that are based on primary human cells from healthy and diseased subjects would provide a complementary scientific base to our strong expertise in the mouse. our commitment to this development is essential, not only to boost the translation of science to the human, but also to ensure that we honour the principles of reduction, refinement and replacement (minimizing the number of animals that are used and finding alternatives wherever feasible) that are central to all animal experimentation. we are faced with many difficulties in generating appropriate human tissue models, including defining differences in similar cell types that are pathologically relevant (for example, comparing the biology of endothelial cells isolated from the umbilical cord with those isolated from different microvascular beds). we also need to determine the optimal representative culture conditions for primary cells. for example, when should epithelial cells be studied at an air-liquid interface, or when should leukocyte-endothelial interactions be studied under flow? importantly, diseased tissues are often modified by inflammation, genetic traits and epigenetic processes that require further consideration when translating from the biology of health to the biology of disease. nevertheless, we have recently shown that simple co-cultures of primary human cells can in some circumstances replicate inflammatory systems that are observed in vivo in mice 31, 32 ; such co-culture models are becoming increasingly common. the future ability to study systems that resemble organs or complete tissues, and verifying the work of simple co-culture experiments in such systems, offers a future we should not only embrace, but also be actively working towards at every opportunity. it is routine in clinical practice to combine drugs with similar or differing modes of action when treating disease. there is also increasing appreciation of the roles of cellular and molecular networks in the aetiology of disease 33 , a fact that is implicitly recognized by the need for in vivo models in which complex interactions between tissues and cells can be studied without a need to identify the full panoply of the systems involved (box 1). in the context of an inflammatory disease, we have recently proposed that these networks are best described by the terminology of 'contiguous immunity' , whereby, in disease, temporally and spatially contiguous networks comprising multiple processes of innate and adaptive immunity are in continual dialogue and evolution 33 . it is curious, then, that so many studies aim to land a single 'killer blow' on pathological processes, rather than considering the impact of therapeutic combinations. our increasingly deeper understanding of the complexity of the inflammatory process often results in the targeting of downstream components for which there might be redundancy or very specific functionality. the targeting of such systems in combination, although complicated to achieve and less satisfying with respect to the identification of single clear targets, is nonetheless likely to have relevance for successful translation. allowing access to tissues and overcoming ethical anxieties. the diversion of research funding into bureaucracy and the delays in productivity that result from meeting statutory requirements for ethical practice contribute to the erosion of research capacity. research cannot do without governance, but it is disappointing that increasing regulation has not been met by increased support for ethical human-based research, and www.nature.com/reviews/immunol the future ability to study systems that resemble organs or complete tissues â�¦ offers a future we should not only embrace, but also be actively working towards at every opportunity. although many processes conspire to make research harder, few exist to make it easier. a simplification of the regulations and national-level funding that provides templates for ethical applications, together with training and support for investigators, would do much to make this easier. because many projects involve international collaborations, simplified standard international regulations would also greatly facilitate progress. many research projects have conceptually similar goals (such as the tackling of inflammation in a tissue), and generic pre-approved protocols requiring modest local adaptation would obviate many problems. in the united kingdom, the proposed establishment of a single health research board to coordinate government-funded health research provides an opportunity to develop programmes that examine the effects of government regulation on research and mechanisms for overcoming the issues discussed earlier. research on human tissues could be enhanced by improved access to normal and diseased specimens. the establishment of central and devolved services whose function would be to source human tissues in an ethical manner, characterize the donors and tissues anonymously, and make these resources freely available to investigators in research-active countries would transform biological research (indeed, some progress is being made here, but there is still much to be done). an emphasis on dialogue with scientists and flexibility with respect to supporting science would be required to underpin access to these resources by researchers. where materials are scarce, such systems could be supported by expertise in cell-line immortalization. the development of the lung tissue research consortium by the national heart, lung and blood institute is an exemplar of a clinical data and tissue bank that offers enormous potential for research in lung disease; in addition, the development of the uk biobank shows it is feasible for human resources to be sourced ethically in ways that allow relatively broad use in medical research according to need. the proposed tissue banks would promote a positive view of human science working in parallel with mouse biology, which also requires commitment and support from the public to make it work. 'omics'. one area in which large-scale biology is excelling is the use of public-access databases (such as those pertaining to gene expression (genomics) or the production of metabolites (metabolomics)) 34 , but we are missing opportunities to further expand these databases. local and national guidelines define the optimal management of many diseases, but it can be argued that the providers of health care are not given appropriate opportunities to engage with the scientists whose work is developing the future treatments for these diseases. scientific initiatives that are associated with national care plans could drive disease phenotyping and tissue collection for tissue, dna, rna, protein and metabolite databanks with good the difficulty in designing in vitro and in vivo experiments to model human disease has inevitably required and generated simplifications of pathology, often leading to relatively linear models of disease (for example, tissue damage, followed by antigen presentation, the generation of immunological memory and autoantibodies, and a resulting autoimmune disease). in reality, pathology is generated by networks that can exhibit substantial plasticity over the course of a disease. these principles are illustrated in the figure. a disease process -pathology -is represented at the centre of a series of simple conceptual networks, components of which are left intentionally blank to avoid attempting to define specific diseases. each component (or node) within the network might have different roles at different times in a disease, or if active at more than one tissue site, might even have different roles simultaneously in a disease process. therefore, the depicted connections are plastic over time and in individual microenvironments. in a cellular network. pathology can be considered in the context of networks of cells that are recruited or resident at inflammatory sites, whose communication through cytokines and other molecules regulates inflammation. in a cytokine network. pathology is driven by the interrelated actions of cytokines, again forming a dynamic plastic network. in a process network. process behaviour (for example, angiogenesis, scarring and leukocyte recruitment) will contribute differently to pathology at specific tissue locations and different times in the disease. as an illustration, wound-healing responses might contribute to the resolution of normal tissue architecture, the development of fibrosis, or the regulation of inflammatory cell recruitment and survival. depending on the nature and duration of the stimulus driving the woundhealing process, and the location in which it is set, multiple resulting pathological phenotypes are feasible. it is the nature of tissues to contain multiple cells and supporting structures that are physically associated, with many more cells that can transit through or become resident. we have proposed that the immunity seen in pathology rarely falls clearly into categories of innate and adaptive immune, or t helper 1 (t h 1) and t h 2 responses. rather, pathology is generated by a networked interaction that changes over time. the networked relationships of immune pathology might be better described as 'contiguous immunity', in which multiple processes or networks can be operational and in dialogue in the same space (physically contiguous); equally, processes might be linked together in evolving patterns (temporally contiguous). understanding these networks, and, where necessary, developing new models to elucidate and target them, is essential to effective translational biology. ifn, interferon; il, interleukin. public access. good clinical practice not only should focus on ensuring that existing best practice is reliably reproduced, but also should put equal weight to research and development of practice through translational research. it is not just the priority of scientists to engage with clinicians: the imperative is equally strong that clinicians should engage more with basic scientists. in both cases it is important that such engagement is facilitated and supported by national policy. another major challenge is to take the input of a large amount of descriptive data generated by an 'omics' approach and both interpret and reapply it to a translational problem in a hypothesis-driven manner. as we learn to integrate complex data sets with in vitro cell biology and in vivo models, we might begin to generate virtual phenotypes, allowing conclusions to be drawn on the basis of the relatedness of a series of data sets to the questions asked. in essence, all researchers do this when they read published data, but the process is inherently subjective and formalizing such integrated biology could be more objective and so better inform future experiments. we are now experiencing an unprecedented blossoming in available technologies, unparalleled through history, which makes biomedical science extraordinarily exciting. with this comes an ever greater financial burden and increasingly complicated ethical issues. an increasing focus on the need for effective translation from the use of these resources highlights the many obstacles that impede such progress. we have highlighted these difficulties, and have suggested strategies to rejuvenate and maximize our translational potential. tyrosine kinases as targets for cancer therapy adjuvant chemotherapy for breast cancer -30 years later pro: mice are a good model of human airway disease modelling the human immune response: can mice be trusted? modeling allergic asthma in mice: pitfalls and opportunities con: mice are not a good model of human airway disease the mouse trap satisfaction (not) guaranteed: re-evaluating the use of animal models of type 1 diabetes modelling infectious disease -time to think outside the box? lost in translation: barriers to implementing clinical immunotherapeutics for autoimmunity distinct mutations in irak-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections pyogenic bacterial infections in humans with irak-4 deficiency severe impairment of interleukin-1 and toll-like receptor signalling in mice lacking irak-4 phenotypes in asthma: useful guides for therapy, distinct biological processes, or both? evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics loss of siglec expression on t lymphocytes during human evolution differences in immune cell 'brakes' may explain chimp-human split on aids cytokine storm in a phase 1 trial of the anti-cd28 monoclonal antibody tgn1412 national survey of british public's views on use of identifiable medical data by the national cancer registry confidentiality in genome research no longer de-identified mouse model of airway remodeling: strain differences murine models of asthma rodent models of rheumatoid arthritis concordance among gene-expressionbased predictors for breast cancer a genomic strategy to refine prognosis in early-stage non-small-cell lung cancer a small-molecule screen in c. elegans yields a new calcium channel antagonist in vivo drug discovery in the zebrafish a transgenic zebrafish model of neutrophilic inflammation capturing complex 3d tissue physiology in vitro cooperative molecular and cellular networks regulate toll-like receptor-dependent inflammatory responses agonists of toll-like receptors 2 and 4 activate airway smooth muscle via mononuclear leukocytes pulmonary perspective: targeting the networks that underpin contiguous immunity in asthma and copd a bioinformatician's view of the metabolome we thank the many scientists whose stimulating conversations have in some measure been represented in these pages. i. the authors declare no competing financial interests. ian sabroe's homepage: http://www.shef.ac.uk/medicine/staff/sabroe.html lung tissue research consortium: http://www.ltrcpublic.com uk biobank: http://www.ukbiobank.ac.uk access to this links box is available online. key: cord-323380-hm9wd817 authors: helmy, yosra a.; el-adawy, hosny; abdelwhab, elsayed m. title: a comprehensive review of common bacterial, parasitic and viral zoonoses at the human-animal interface in egypt date: 2017-07-21 journal: pathogens doi: 10.3390/pathogens6030033 sha: doc_id: 323380 cord_uid: hm9wd817 egypt has a unique geographical location connecting the three old-world continents africa, asia and europe. it is the country with the highest population density in the middle east, northern africa and the mediterranean basin. this review summarizes the prevalence, reservoirs, sources of human infection and control regimes of common bacterial, parasitic and viral zoonoses in animals and humans in egypt. there is a gap of knowledge conerning the epidemiology of zoonotic diseases at the human-animal interface in different localities in egypt. some zoonotic agents are “exotic” for egypt (e.g., mers-cov and crimean-congo hemorrhagic fever virus), others are endemic (e.g., brucellosis, schistosomiasis and avian influenza). transboundary transmission of emerging pathogens from and to egypt occurred via different routes, mainly importation/exportation of apparently healthy animals or migratory birds. control of the infectious agents and multidrug resistant bacteria in the veterinary sector is on the frontline for infection control in humans. the implementation of control programs significantly decreased the prevalence of some zoonoses, such as schistosomiasis and fascioliasis, in some localities within the country. sustainable awareness, education and training targeting groups at high risk (veterinarians, farmers, abattoir workers, nurses, etc.) are important to lessen the burden of zoonotic diseases among egyptians. there is an urgent need for collaborative surveillance and intervention plans for the control of these diseases in egypt. zoonotic diseases (zd) are those infections that can be naturally transmitted from animals to humans with or without vector [1] . in the past few decades, there has been a rise in the outbreaks of zoonotic diseases which have an enormous socioeconomic impact worldwide, for instance, all foodborne zoonoses occured in a single country costs about $1.3 billion annually [2] . additionally, zd constitute 61% of all and cattle and buffaloes by b. abortus has been frequently reported [53, 54] . in 2016, the prevalence of animal and human brucellosis was 7% and 1.25%, respectively, at a district located in the nile delta with high livestock density [55] . in another serosurvey in east of egypt, 4.42% and 8.91% in private farms and individual cases, respectively, were found positive. brucella melitensis biovar 3 was isolated from seroreactive animals [56] . in the period between 2006 and 2008, the prevalence of brucellosis among sheep, goats and cattle flocks in 18 egyptian governorates were 26.7%, 18.9% and 17.2%, respectively [57] . in humans, up to 70 persons per 100,000 population were found positive to brucella in 2002 and 2003; 70% were male with a median age of 26 years. the infection was associated with close contact to animals and consumption of unpasteurized milk products [58] . escherichia coli is gram-negative facultative bacteria that belongs to the family enterobacteriaceae. although it is normally commensal in nature and animals, many strains are food-and waterborne zoonotic pathogens. some strains like o157 and other enterohemorrhagic e. coli (ehec) cause no discernible disease in their animal reservoirs; however, diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome are not uncommon in humans [59] . in egypt, many studies have shown a high prevalence of e. coli o157 strains in dairy and meat products in different locations [44, 60, 61] . avian pathogenic escherichia coli (apec) infection is responsible for great economic losses in poultry industry. avian e. coli strains from broiler flocks in egypt were genetically similar to e. coli associated with human infection [62] with prevalence rate in chicken visceral and human stool samples of 26.9% and 46.2%, respectively [63] . enteropathogenic e. coli (epec), diffuse-adhering e. coli (daec) and enteroaggregative e. coli (eaec) are associated with infantile diarrhea in egyptian children where 220 enteroadherent e. coli were identified from 729 diarrheic children [64] . in another study, e. coli was isolated from 52.1% hospitalized patients and 60.4% from outpatient clinics with high degrees of resistance to ciprofloxacin and co-trimoxazole [65] . also, there is increasing incidence of carbapenemase-and extended-spectrum β-lactamase-producing enterobacteriaceae in dairy farms and hospital-acquired infections. this represents a major health problem because of the few therapeutic alternatives [66] . listeriosis is one of the foodborne pathogens with high fatality rate. it is caused by listeria monocytogenes, a gram-positive bacterium which is able to grow at temperatures as low as 0 • c challenging the control in human foodstuffs. l. monocytogenes can persist for long periods in the environment or as an asymptomatic infection in adult animals and birds. neurological disorders due to encephalitis are the most common clinical signs in ruminants, in addition to late abortion. consumption of raw milk, cheeses, raw-meat products, poultry and fish is the major sources of infection. human listeriosis is associated with serious invasive illness, particularly in elderly and immunocompromised patients, pregnant women, newborns and infants [67] . in egypt, existence of the organism in water [68, 69] , contaminated food [70] , seafood [71] , milk products and milk samples from apparently healthy buffaloes, cows, she-camels, goats and sheep has been reported [72] [73] [74] [75] . during 2013, listeria spp. was detected in 47.5% of 200 poultry farm samples in egypt [76] . the isolation rates of l. monocytogenes from different localities in egypt were 8% in beef burger, 4% in minced meat and 4% in luncheon meat; while sausage samples were all negative [77] . in humans, l. monocytogenes is considered the most common lethal complication in egyptian patients with liver cirrhosis associated with ascites [78] . detection of the organism in stool samples of hospitalized egyptians was reported in different localities [71, 79] . q fever is a zoonotic bacterial disease with public health implications. the disease is caused by coxiella burnetii, a gram-negative bacteriumthat mostly affect ruminants. the bacterium causes abortion in sheep, goat and cattle and is excreted in infected animal feces, urine, milk, and birth products. people can get infected by inhalation of contaminated materials. c. burnetii causes febrile flu-like illness and pneumonia in poor hygiene settings. the epidemiology of q fever in africa is poorly understood [80, 81] . the epidemiology of c. burnetii in egypt is not well-known [82] . antibodies were detected in egyptian donkeys, goats, sheep, pigs, dogs and rats [83] . q fever may be enzootic in cattle in northern egypt [84] . in 2006 and 2011, c. burnetii antibodies were detected in up to 32.7%, 23.3%, 13.3% and 13% of sheep, goats, camels and cattle samples in different localities in egypt, respectively [82, 85, 86] . however, all seropositive animals were negative for c. burnetii dna by pcr [85] . in 2009, the prevalence of c. burnetii in blood samples collected from domestic and imported livestock slaughtered at the abattoir in central egypt was 4% in buffalo, 8% in sheep, and 70% in camels [87] . the prevalence of antibodies to c. burnetii among egyptians in various locations was 5 to 28% particularly among cattle workers, veterinarians and veterinary assistants and those live in agricultural districts [82, 88, 89] . schistosomiasis is the second most common parasitic infection globally after malaria [90] and considered as one of the neglected tropical diseases (ntds) [91] . schistosomiasis was reported in more than 200 million people in 74 countries and nearly 800 million people are at risk of infection worldwide [90, 92] . humans acquire the infection by contact with or drinking of contaminated water. there are three main species infecting humans including s. mansoni, s. haematobium and s. japonicum complex (s. japonicum and s. mekongi) [93] . infection with schistosoma spp. results in skin rash or itching, flu-like illness and severe intestinal and urinary tract disorders [94, 95] . chronic schistosomiasis can persist for years and lead to neurological complications and death [96, 97] . schistosomiasis is one of the most endemic parasitic diseases in egypt. the earliest case of human schistosomiasis (s. haematobium) occurred more than 5000 years ago in an egyptian adolescent [98] . in addition, s. haematobium calcified eggs were identified in two mummies aged 3000 and 4000 years old of the 20th dynasty [99, 100] . between 1935 and 2010, the infection rate was very high in school-age children and the infection can persist among adults [95, 101, 102] . furthermore, working in agriculture is a risk factor for schistosoma spp. infection [103] . the prevalence of s. haematobium and s. mansoni in egypt ranged between 0.08% and 75% [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] . the highest prevalences of s. haematobium were detected in lower egypt [106, 109] , while the highest prevelance of s. mansoni was detected around central egypt [102, 118, 119] . the overall prevalence of schistosomiasis in egypt declined year by year to reach 10% in 1999, 5% in 2000, 3.5% in 2002, 1.2% in 2006 and 3-10% in 2010 [102, [120] [121] [122] . this continuous decrease was due to the control of schistosomiasis by using praziquantel in mass chemotherapy and due to snail control by using anti-bilharzial chemotherapy [102, 123, 124] . fascioliasis is caused by a liver fluke belonging to genus fasciola (fasciola hepatica and f. gigantica). fasciola is one of the neglected foodborne trematodes that infect ruminants and humans worldwide [125] [126] [127] . it was estimated that fascioliasis, associated with other diseases, especially schistosomiasis, affects at least 2.4 million people in more than 70 countries [128] [129] [130] . it causes gastrointestinal problems and chronic fascioliasis results in jaundice and inflammation of the liver, gallbladder and pancreas [129] . fecal excretion of eggs from infected animals (e.g., cattle, sheep, buffaloes, donkeys and pigs) in fresh-water is the main source of infection. after hatching, larva lodge in a particular type of water snail (the intermediate host). carrier animals are infected by eating metacercaria encysted on leaves of water plants or vegetables [127] . egypt is one of the endemic areas with fascioliasis in the world [131] with annual loss in milk and meat being estimated to be 30% [132] . the climatic factors influence the incidence of fascioliasis and all snail transmitted parasites in egypt [128, 133] . f. gigantica is considered the endogenous fasciolid species in egypt with tropical and subtropical distribution [134] while f. hepatica originated from europe and introduced to egypt through the importation of domestic animals [135, 136] . fasciola spp. infected different animal species in egypt including; sheep, goats, cattle, buffaloes, horses, donkeys, camels and rabbits and the infection rates reach 90% in some areas [137, 138] . the overall prevalence in egypt is unknown because reports show wide variations in infection rates [139] . between 1959 and 2016, the prevalence of fasciola spp. among different animal species in different localities in egypt ranged between 0% and 59% [138] [139] [140] [141] [142] [143] [144] [145] [146] . in 1988, fascioliasis has been reported in approximately all egyptian governorate where up to 60% of cattle and buffaloes and 78% of sheep were positive [139] . since 1980, human fascioliasis was reported in most of the egyptian governorates especially in the nile delta [130, 139] . it was estimated that 830,000 humans are infected and 27 million of persons are exposed to the risk of infection in egypt [147] . between 1958 and 2006, fascioliasis has been reported in humans in different localities in egypt and the prevalence rate was between 2% and 19% [130, 139, [148] [149] [150] [151] [152] [153] . from 1998 to 2002, the prevalence of fascioliasis in the treated endemic areas was reduced from 5.6 to 1.2% [154] . this decrease in the prevalence was continued in 2009 to reach 0% [142] . however, in 2013, the prevalence increased again to reach 8% [155, 156] . triclabendazole is the selective treatment of fascioliasis in specific high-risk age groups such as school children and villagers [154] . cryptosporidiosis is a zoonotic protozoal disease caused by the genus cryptosporidium [157] . cryptosporidium spp. was discovered by tyzzer in mice [158] and the first human case was reported in 1976 [159] . afterwards, more attention was given to cryptosporidiosis since it was determined to cause death in one acquired immune deficiency syndrome (aids) patient [159] . cryptosporidia essentially entered veterinary medicine only in the early 1980s with reports of cryptosporidium-associated neonatal calf diarrhea [160] and established as a primary enteric pathogen [161] . the importance of cryptosporidium as a public health problem began in 1993, when more than 400,000 residents in milwaukee, wi, usa were affected by c. hominis due to the consumption of contaminated drinking water. this was reported as the largest world waterborne outbreak [162] [163] [164] . out of 26 cryptosporidium species, c. hominis and c. parvum are responsible for more than 90% of human cases of cryptosporidiosis [165] , exhibiting acute self-limiting diarrhea in immunocompetent persons and life-threatening diarrhea in immunocompromised persons [166] . it was estimated that 1 to 10% of the developing countries' populations were infected with cryptosporidium particularly among 1-to-9-year-old children and toddlers [167] . reports on cryptosporidiosis in egypt are rare. the highest prevalence rates were reported in rural areas where there is close contact with animals. the accuracy of the reported prevalence rate depends on the used detection method [168] . in animals, from 1999 to 2016 the prevalence rate of cryptosporidium infection ranged between 2% and 69% among different species including cattle, buffalo calves, camels, sheep, goats, lambs, dogs, wild rats and quails. the highest prevalence rates were reported in governorates that located close to the river nile [169] [170] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] [181] . in humans, between 1989 and 2016 cryptosporidium infection has been reported in almost all egyptian governorates with prevalence rates ranged between 3% and 50% or up to 91% in immunocompromised patients and diarrheic children [169] [170] [171] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] . giardiasis is caused by giardia duodenalis (syn. g. intestinalis, g. lamblia) and considered one of the most common intestinal protozoal parasites affecting humans worldwide [198] . it also infects other mammals, including pets and livestock [199] . the high-risk group is children, especially those in daycare settings, orphanages and primary schools [200] with 2.5 million annual cases in the developing countries [201] . infection with giardia spp. results in gastrointestinal disorders [200] . giardia cysts can be transmitted to humans through ingestion of contaminated water or food, but the parasite can be transmitted directly from infected individuals [202] . g. duodenalis has been divided into eight different assemblages (a-h) that have varied host specificities [203] . assemblages a and b have been found to infect humans and other mammals [200] . in egypt, few studies have been done to identify the relation between different assemblages and the presence of symptoms, especially among children and animals [204, 205] . between 2004 and 2016, the prevalence of giardia spp. ranged between 2% and 53% among different animal species including ruminants, dairy cattle, stray cats, wild rats and fish [152, 204, [206] [207] [208] . assemblage e was also more prevalent among ruminants and dairy cattle [204, 206] . in humans, between 2001 and 2017, giardiasis has been reported in different localities of egypt and the prevalence rates ranged between 10% and 75% [152, 192, 204, 206, [209] [210] [211] [212] [213] [214] [215] [216] . the most prevalent g. duodenalis genotypes were assemblage a and b and to lesser extent assemblage e among diarrheic patients in egypt [204] [205] [206] 209, 214, 215, [217] [218] [219] [220] . the prevalence of g. duodenalis was high in rural areas more than in urban areas which was attributed to the higher exposure to multiple risk factors such as poor water supply, poor sanitation, and presence of animals [204] . interestingly, asymptomatic giardia infection can persist for 4 months with 4.5 episodes per child/year in rural areas in egypt [213] . toxoplasma gondii (t. gondii) is one of the most important human zoonotic protozoan parasites, infecting one third of the world's population [221] . infection with t. gondii is prevalent in humans and animals, including poultry [222] . cats are considered the key host in the transmission of t. gondii to humans and other animals as they excrete the environmentally resistant oocysts in their feces [223] . human infections occur through ingestion of food or water containing viable cysts [224] , congenitally, by blood transfusions or organs transplantation [225] . the high risk groups are immunocompromised patients and fetuses whose mothers acquire acute infection during pregnancy [226] . infection with t. gondii is asymptomatic in immunocompetent and primary infected pregnant women; however, severe complication and death have been also reported [224, [226] [227] [228] . in egypt, indoor and outdoor cats are allowed to roam, where they hunt their own food or live on scraps of garbage. therefore, the environment is highly contaminated with oocysts excreted by these cats. this might affect livestock that will later be slaughtered for human consumption [229] . in 1980s, seroprevalence of the parasite ranged between 16% and 59% in stray and domestic cats [230] [231] [232] while in 2008, antibodies were reported in 57% of cats [233] . recently, seroprevalence of t. gondii was up to 98%, indicating high environmental contamination with oocysts [207, 229, 234] . furthermore, t. gondii antibodies were detected in 10% to 62% of ruminants, including cattle, sheep and goat and in equines, including horses and donkey [235] [236] [237] [238] [239] [240] . between 1990 and 2016, seroprevalence of t. gondii in chickens, turkeys and ducks ranged between 9% and 85% in different localities in egypt [241] [242] [243] [244] [245] [246] . therefore, consumption of undercooked poultry meat may be considered a risk factor for toxoplamosis in humans or animals [222] . in humans, there are limited data on the prevalence of t. gondii and associated risk factors in women during pregnancy. in different egyptian localities, between 1993 and 2016, the prevalence of toxoplasma antibodies were ranged between 27% and 68% in pregnant women [236, 239, 245, [247] [248] [249] [250] [251] [252] [253] , 26% in cerebrospinal fluid of meningoencephalitis patients [254] , 59.6% in asymptomatic blood donors [255] . influenza a viruses (iav), members of the rna family orthomyxoviridae, have up to 144 subtypes according to the variation/combination of the surface glycoproteins hemagglutinin and neuraminidase. iav are further classified to human influenza, swine influenza (siv), bat influenza, equine influenza and avian influenza viruses (aiv). siv and aiv transmit from swine or birds to humans, respectively, mostly via direct contact with infected animals. the infection in humans ranges from mild self-limiting respiratory-like illness to death [256, 257] . due to the very low pork production in egypt, swine influenza is not a major zd, although serosurveillance indicated infections of humans in 1979-1980 [258] . on the contrary, aiv are very important zoonotic viruses in egypt. poultry industry in egypt was estimated in 2006 to be one billion birds with several millions of labors. in late 2005, the asian highly pathogenic (hp) aiv of subtype h5n1 was firstly detected in wild migratory birds in a northern egyptian wetland. in february 2006, the virus transmitted to domestic commercial and backyard birds and in march the first human case was recorded. to date, the virus is endemic in egyptian birds causing tremendous economic impact despite the mass vaccination intervention strategy [259] . another aiv subtype is h9n2 which, in poultry, did not cause severe illness unless the infection is complicated by secondary bacterial infection or immunosuppression. the endemic h9n2 in egyptian poultry was firstly detected in 2011 and vaccination is widely used to control the infection [259] . in humans, egypt is the country with the highest recorded cases worldwide. the fatality rate in egypt is lower than the global rate. thus, lower virulence and subsequent adaptation of the virus in human has been assumed. many mutations that enhance virus binding to mammalian type receptors have been studied. subclinical infection in human has been reported as revealed by serological surveillance [258, 259] . nevertheless, a recent study has shown that the virus has not yet acquired the aerogenic transmissibility as naïve ferrets cohoused with inoculated ferrets did not acquire infection [260] . the egyptian h5n1 viruses are highly susceptible to antiviral drugs (oseltamivir), but are resistant to amantadine. infection is usually acquired by intensive contact particularly with backyard birds. women and children are mostly affected. to date, there are 3 human infections by h9n2 viruses reported to the who [261] . subclinical infection in poultry workers and co-infection of poultry and human with h5 and h9 in egypt have been reported. moreover, serosurvey revealed the presence of antibodies against h7 viruses in poultry [262] and in humans [263] , but no virus was isolated, so far [262, 263] . the rabies virus (rabv) belongs to the genus lyssavirus of the rna family rhabdoviridae within the order mononegavirales. rabies is a widespread neurological zoonotic disease of all warm-blooded animals including humans. dogs are the most important host for rabv, however wild carnivores (e.g., fox) and bats are considered reservoirs. infection in humans occurs by direct contact with mucosal surfaces (e.g., bites) or possibly through contamination of wounds with infected materials (e.g., scratches). the virus has a relatively long incubation period (according to the site of bite) which may reach a few months to a year giving the opportunity for immediate vaccination/treatment. each year, about 60,000 fatal human infections are recorded worldwide [264, 265] . in egypt, rabv was recognized before 2300 b.c. [266] . although the notifiable disease is now endemic in many regions throughout the country, there are no recent reported outbreaks of rabv. it is worth mentioning that the canine population in egypt was estimated to exceed three million stray dogs and half a million owned dogs. vaccination of dogs in egypt was usually done by low-egg-passage of the flury strain, which has now been replaced by attenuated tissue culture vaccines [266] . the disease transmitted to humans in egypt mainly by dog bites, however the virus has been detected in other animals: cats, ruminants (e.g., buffaloes), horses, donkeys, rodents and mongooses [266] [267] [268] [269] . in 1970s, the virus was isolated from several rodents and wild mammals including gerbils, foxes and cats [270, 271] . in 1988, naval medical research unit three (namru-3 is a biomedical research laboratory of the us navy located in cairo, arab republic of egypt) isolated street rabv from dogs (n = 9), cats (n = 2), farm animals (n = 2), gerbils (n = 3) and jackal (n = 1). in 1990, an outbreak was reported to the oie. in 1998-1999, there was a record for isolation of rabv from dogs in egypt [272] . in 2012-2014, two studies reported the detection of the virus in the brain samples of water buffaloes (bubalus bubalis) exhibiting fever and/or nervous signs. the infection was acquired mostly after being bitten by a fox. the virus was closely related to other street strains of dogs in egypt, israel and jordan [267, 273] . in 2015, rabv was isolated from brain tissue of an adult female dog. the dog developed hypersalivation, paralysis, and hyperesthesia consistent with rabies symptoms. the virus was genetically similar to canine rabv circulating in egypt [274] . reports of human infection in egypt have been described from 1904 to 2000s [266] and 30 to 40 annual deaths due to rabv was reported [269, 275] . in 1979, a french woman died after corneal transplantation from an egyptian donor [276] . in 1988, namru3 isolated two street rabv from humans [268] . genetically, the egyptian viruses from humans and animals are closely related to those isolated in israel and the me [272] . lastly but not least, the huge number of stray dogs roaming freely with livestock and insufficient vaccination coverage of pets are among the most common hallmarks of the endemic status of rabv in egypt. rift valley fever (rvf) is a vector-borne zoonotic viral disease firstly reported among livestock in rift valley of kenya in the early 1900s. the disease is caused by a rvf virus (rvfv), genus phlebovirus, a member of the rna family bunyaviridae and was first isolated in 1931. the virus infects mosquitoes, which act as a reservoir, while animals and humans are considered amplifying hosts. rvfv infects a wide spectrum of mammals, causing abortion and mortality. in humans, symptoms range from mild fever, muscle pains and headaches to hepatic failure and death. direct contact to infected animal blood, aerosol, drinking unpasteurized contaminated milk, or the bite of infected mosquitoes are the major sources for human infection. vaccination of susceptible animals, restriction of movement (e.g., importation) and reduction or control of mosquitoes' population are the regular control measures. the disease is common in africa and the me [277, 278] . in the last 40 years, egypt reported five large outbreaks: in 1977-1978, 1993-1994, 1997 , 2000 and 2003 and the largest epizootic was in 1977-1978 [279] [280] [281] [282] [283] [284] . the primary introduction of rvfv into livestock in these outbreaks in egypt was mostly linked to importation of animals mainly camels from africa [280, 282, [284] [285] [286] [287] . the virus was isolated from various species of domestic animals (e.g., sheep, cows, buffaloes, camels, goats, horses, and rats) as well as humans [288, 289] .the epizootics of rvf in egypt were reported every year round. the effects of rainfall and river discharge in addition to optimal constellation of interconnected hydrologic, entomologic (high mosquitoes' populations), and social conditions were incriminated in the spread of the virus [286, [290] [291] [292] [293] [294] [295] . moreover, anti-rvfv antibodies were detected in pigs in 2008 [296] , domestic and imported cattle and buffaloes in 2009 [87] and non-immunized dairy cattle from different localities in egypt in 2013-2015 [297] . the reoccurrence of rvf outbreaks from time to time in animals in egypt challenges the importation control check points and the efficacy of the applied vaccination program [280, 281] . in humans, in 1977, rvfv in egypt caused huge outbreaks including 200,000 human infections and 600 deaths [282, [298] [299] [300] [301] [302] [303] [304] . the virus transmitted to eight swedish united nations emergency forces soldiers serving in egypt and the sinai peninsula [305] . possible human-to-human transmission was described [302] , however the infection mostly occurred by handling infected meat and inhaling natural virus aerosols [287, 306] . in 1993, in southern egypt, 600-1500 human infections associated with ocular disease, fever and headache were reported [279, 307] . serological surveillance indicated that workers at abattoir and sewage treatment plants in several governorates of egypt possessed rvfv antibodies without showing clinical signs [308, 309] . moreover, in 2008, rvfv antibodies were detected in~14% of veterinarians and their assistants, butchers and abattoir workers in pigs' abattoir [296] . middle east respiratory syndrome (mers) caused by a newly emerging coronavirus (cov), designated lineage c of betacoronavirus, in the rna family coronaviridae. it was firstly reported in saudi arabia in a patient with respiratory illness in 2012 [310] and transmitted to several countries not only in the middle east (me) but also in africa, asia and europe. the virus is usually transmitted from dromedary camels via direct contact or consumption of milk or medicinal use of camel urine [311] . also, bats and alpacas were considered reservoir hosts [311, 312] . however, limited human-to-human infections have been also reported. the infection can be mild or fatal in those patients with immune system disorders or chronic diseases [313] . in egypt, out of 110 swabs and 52 serums collected in june-december 2013 from clinically healthy imported or locally reared camels in abattoirs, 4 and 48 positive samples were detected, respectively. attempts to isolate the virus in cell culture were not successful. none of 179 samples collected from workers in these abattoirs were positive by rt-pcr [314] . in another study, in june 2013, all serum samples collected from humans, cows, water buffaloes, goats and sheep were negative for mers-cov antibodies. conversely, 94% of serum samples collected from dromedary camels were positive for mers-cov [315] . from june 2014 to february 2016, 2541 sera, 2825 nasal swabs, 114 rectal swabs, 187 milk samples, and 26 urine samples were collected from camels in different sectors in egypt (importation quarantines, markets, abattoirs, free-roaming herds and farmed breeding herds). results revealed 71% seropositivity and 15% of other samples were positive by rt-pcr. seroprevalence was 90% in imported camels and 61% in locally raised camels, likewise rna detection rates were 21% and 12%, respectively. both juveniles and adult camels were positive by 82% and 37% seropositivity and similar rt-pcr detection rates of 15% and 16%, respectively [316] . in humans, from 2012 to 2015, none of nasopharyngeal and oropharyngeal swabs collected from 3364 returning egyptian pilgrims were positive for mers-cov [317] . to january 2017, only one human case was reported from egypt in april 2014 [318] . crimean-congo hemorrhagic fever (cchfv) is a tick-borne virus from the bunyaviridae family. the virus causes severe hemorrhagic fever outbreaks, with a case fatality rate of up to 40%. it is endemic in some african, middle eastern and asian countries. farm animals like sheep, goats, cattle, camels and ostriches can be infected without showing any clinical signs. ticks of the genus hyalomma are the principal vector. humans and animals become infected after being bitten by ticks. also, animal to human transmission through contact with infected animal blood or tissues, particularly at the abattoirs are common. therefore, veterinarians, agricultural workers and slaughterhouse workers are most affected [319] . in egypt in 1976, the antibodies to cchfv detected in 8.8% camels sera and in 23.1% sheep sera but no antibodies were detected in sera from equines (donkeys, horses and mules), pigs, cows, and buffaloes [320] . in 1986-1987, 14% of serum samples collected from imported camels from sudan and kenya into egypt was positive for cchfv antibodies. native livestock including sheep and cows were negative [321] . between september 2004 and august 2005, 3.83% of cattle, 0.38% of water buffalo, 6.3% of sheep and 1.14% of goats were positive for cchfv antibodies [322] . in july 2009, ectoparasites removed from freshly slaughtered cattle, buffalo, sheep and camels imported from sudan and somalia were negative for cchfv except five camels were found to harbor ticks carrying rna from a new cchfv variant [323] . in 2009 in a serosurvey in domestic and imported livestock, only one cow out of 161 was positive while buffaloes, sheep, and camels were negative for cchfv antibodies [87] . no antibodies were detected in sera from humans in 1976 [320] . the only known human infection with cchfv in egypt happened in 1981. an egyptian virologist died after mouth-pipetting a culture of a cchfv isolate that he had brought from iraq [324] . west nile is a remerging zd caused by west nile fever virus (wnv), a flavivirus that belongs to the rna family flaviviridae. the virus was firstly identified in 1937, in uganda. during the last decade, the incidence of wnv increased worldwide. the virus is transmitted by arthropod vectors and the mosquitoes of the genus culex are main reservoirs [325] . the latter feed on birds especially passerines and thus birds become infected. migratory birds thought to be responsible for wide spread of wnv and reintroduction from enzootic to new regions [326] . mosquitoes-birds-mosquitos' infection is the classical transmission cycle in nature. accidentally, human infections occur through biting or dealing with contaminated blood or tissues. the infection in birds is mostly subclinical, while humans exhibit mild, if any, illness to fatal west nile encephalitis particularly in at-risk individuals such as the elderly, immunocompromised and people with chronic illness [327] . seasonal incidence of wnv outbreaks is linked to higher populations of culex mosquitoes during summer months in temperate regions and rainy seasons in the tropics. however, infections of humans have been also associated with other factors (e.g., blood transfusion, occupational exposure, etc.) [327] . the egyptian climate is suitable for the spread of wnv [328] where more than 110 mosquito species and subspecies including culex species were identified [291, 329] . the virus was firstly isolated from mosquitoes in early 1990s [295] . in the period from 1999 to 2002, 15 (0.29%) out of 112,155 examined samples from mosquitoes and sand flies were positive for the wnv. interestingly, the virus transmitted from mosquitoes to sentinel chickens [330] . it is worth mentioning that more than 150 species of migratory birds visit egypt annually in addition to 350 resident species of birds [291, 329] . israeli-like wnv was isolated in white storks in egypt in 1997-2000 suggesting that migrating birds do play a crucial role in geographical spread of the virus [331] . the first serological evidence for human infections with wnv in egypt were reported in 1950 in 22% of children and 61% of adults included in a study conducted by melnick et al. [332] . in 1968, 14.6% of hospitalized febrile children were linked to wnv infection [333] and 5 out of 133 patients suffering from aseptic meningitis or encephalitis possessed wnv antibodies [334] . in 1969, approximately half of 1113 male university students and 3% of 162 patients in different localities had wnv antibodies [335, 336] . between 1984 and 1985, one out of 55 patients with non-specific fever and myalgia was positive for wnv [337] . in 1989, 3% of school children aged 8-14 years [299] and 45.5% out of 180 examined persons in the nile delta were positive for wnv antibodies [338] . wnv had the highest prevalence (54.14%) among other viruses in a serosurvey targeting workers in sewage treatment plants from january to october 1999 [309] . in early 2000s, a dutch 44-year-old female was infected during a holiday in egypt [339] . in 1999-2002, wnv was actively circulating in different areas in egypt causing febrile illness and up to 24% of human serum samples were positive [330] . currently, the disease is mostly underestimated and scarce data are available, so far. in this review, some aspects of viral, bacterial and parasitic diseases with zoonotic importance in egypt were summarized. there is a gap of knowledge about the epidemiology of zoonotic diseases in different localities in egypt, which hinders accurate assessment of the human health burden. surveillance activity is high for some viral diseases such as influenza and mers but is still weak or neglected for others particularly at the human-animal interface. control of diseases in animals depends on the vaccination (e.g., against rvfv, aivs), anti-microbials for bacteria and antiprotozoal medication against parasitic infection. while some pathogens/diseases are exotic in egypt (e.g., mers-cov and rvfv acquired by importation of camels or wnv from wild birds), others are endemic (e.g., aiv, rabies, schistosoma, and fasciola). transboundary transmission of zoonotic agents from egypt to europe, asia and america occurred via different pathways. rabv was reported in the us via falsified vaccination certificate of dogs [274] , in europe via corneal transplantation from an egyptian donor [276] and in asia, probably by dog transfer [272] . also, rvfv infected swedish soldiers on duty in egypt in 1977 and 1978 [305] . egyptian aiv was reported in poultry in neighboring countries most likely due to smuggling of poultry or migratory birds [340] . approximately 100,000 egyptians travel to saudi arabia in pilgrimage, every year, which is an important risk factor for possible introduction or spread of infections [341] . there are several suggestions to improve the control of zoonoses in animals and humans in egypt. enhancing biosecurity and management in animal farms particularly in poultry sectors may reduce the risk of salmonellosis, campylobacteriosis, listeriosis and influenza viruses. multidrug-resistance of bacteria in animals, due to the misuse of antibiotics in the veterinary sector, is an increasing problem in egypt. therefore, regulations for antibiotic application in animals must be enforced to mitigate the serious public health hazard. vaccination as an alternative approach for the control of bacterial infections in animals, vaccination of stray dogs against rabv and regular investigation of cats for toxoplasmosis should be considered. longer quarantine periods or restriction of importation of animals, particularly camels, from endemic countries may be effective to reduce introduction of zoonotic viruses. control of vectors (e.g., mosquitoes), intermediate hosts (e.g., snails) and animal reservoirs (e.g., stray dogs, cats) should be key components in the intervention strategy of zoonoses in egypt. improving, providing and upgrading diagnostic techniques in both veterinary and human medicines are fundamental to early detect and contain zoonotic infections. last but not least, sustainable awareness, education and training targeting groups at high risk 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herzig, volker; cristofori-armstrong, ben; israel, mathilde r.; nixon, samantha a.; vetter, irina; king, glenn f. title: animal toxins — nature’s evolutionary-refined toolkit for basic research and drug discovery date: 2020-06-12 journal: biochem pharmacol doi: 10.1016/j.bcp.2020.114096 sha: doc_id: 316792 cord_uid: 89f8g0m8 venomous animals have evolved toxins that interfere with specific components of their victim’s core physiological systems, thereby causing biological dysfunction that aids in prey capture, defense against predators, or other roles such as intraspecific competition. many animal lineages evolved venom systems independently, highlighting the success of this strategy. over the course of evolution, toxins with exceptional specificity and high potency for their intended molecular targets have prevailed, making venoms an invaluable and almost inexhaustible source of bioactive molecules, some of which have found use as pharmacological tools, human therapeutics, and bioinsecticides. current biomedically-focused research on venoms is directed towards their use in delineating the physiological role of toxin molecular targets such as ion channels and receptors, studying or treating human diseases, targeting vectors of human diseases, and treating microbial and parasitic infections. we provide examples of each of these areas of venom research, highlighting the potential that venom molecules hold for basic research and drug development. venomous animals have taken advantage of the inherent biological complexity of their victims by developing toxins that target one or all of the nervous, musculoskeletal, or cardiovascular systems in order to facilitate prey capture or defense against predators. for example, many predatory venoms contain neurotoxins that disrupt neuromuscular transmission in order to rapidly paralyse envenomated prey [1] . due to the inherent specificity and potency of most venom molecules, venoms have become an invaluable source of modulators to study a wide range of molecular targets, especially ligand-activated [2] [3] [4] and voltage-gated ion channels [5] [6] [7] . while research on many potential drug targets is hampered by the lack of selective modulators, our increasing knowledge of venom pharmacology is beginning to shed light on some of these poorly studied targets (e.g. [8] ). here we provide examples of how venom compounds have been used to study diverse molecular targets and aid our understanding of their role in human disease. we use knowledge gained from approved venom-derived drugs [9, 10] to illustrate how basic research can lead all the way from venom to a human therapeutic. finally, we touch on other potential uses of toxins for human health, such as targeting vectors of human diseases, and development of antimicrobial and antiparasitic drugs (fig. 1 ). during the course of evolution, venom has evolved independently at least 100 times across eight different phyla [11] . it has been estimated that there are more than 220,000 venomous species on the planet, representing ~15% of extant animal biodiversity [12] , spanning invertebrates such as annelids, arthropods, cnidarians, molluscs, nematodes, sea urchins, star fish, and vertebrates such as snakes, lizards, fish, shrews, and platypus [1, [12] [13] [14] . chemically, venoms are complex mixtures of salts, small molecules, peptides, and proteins [15, 16] , with an estimated 20 million compounds in spider venoms alone [17] . most of these compounds are toxins, which are defined as substances that cause dose-dependent pathophysiological injury to a living organism, thereby reducing their viability [18] . the high metabolic cost of venom production [19] has ensured that over the course of evolution only the most potent toxins have prevailed. venoms can therefore be considered natural libraries of highly optimised bioactive compounds [20, 21] . given the vastly different phyletic origins of venomous species and the even greater diversity of their target organisms, it is expected that venom toxins modulate an extremely diverse range of molecular targets (fig. 2) . the dominant components of most venoms are small peptides. the high potency, selectivity and biological stability of many venom-derived peptides has made them particularly attractive from a drug discovery perspective. there are already two fda-approved venom-derived peptides in clinical use -ziconotide, an analgesic drug from the venom of a marine cone snail [22] , and exenatide, an anti-diabetic drug from a venomous lizard [23] -plus numerous others in clinical trials [24, 25] or preclinical development [9, 10] . the larger size of peptides compared to small molecules provides a larger binding surface with their molecular target, typically yielding higher potency and greater target specificity, thereby minimising unwanted side-effects. however, the larger size and polar nature of peptides translates into poor oral bioavailability. other disadvantages of peptides can be poor membrane permeability and low metabolic stability [26] , although these disadvantages can often be overcome by chemical modification [27] [28] [29] . in addition, progress in the chemical synthesis [30] [31] [32] and recombinant production [33, 34] of complex peptides has facilitated cost-effective venom-peptide manufacture. venomous animals have evolved peptide toxins with a huge diversity of molecular frameworks, most of them with tertiary structures stabilised by disulfide bonds [35] . the most well-known of these disulfide-rich frameworks is the ubiquitous inhibitor cystine knot (ick) or knottin motif [36, 37] , which is defined as an antiparallel -sheet stabilised by a cystine knot [38] . a major advantage of ick peptides for drug development is their resistance to proteases, high temperature, and harsh chemicals, which has been attributed to their knotted structure [39] [40] [41] . however, despite our extensive knowledge about a limited number of disulfide frameworks present in animal venoms, there are many families of disulfide-rich venom peptides that remain completely unexplored (e.g. [42] [43] [44] [45] ). further research on these novel disulfide-rich frameworks is therefore required to unleash any potential they might hold for drug development. recent advances in proteomics and transcriptomics have now made it possible to rapidly elucidate the venom proteome of even miniature venomous animals [46] . while these developments have greatly accelerated our understanding of the structure, function and evolution of venom proteins, they have led to a general neglect of non-proteinaceous venom molecules. a wide variety of small molecules have been found in venoms including salts, amino acids, hormones, nucleosides, neurotransmitters, and polyamines [47] [48] [49] . in addition to playing important roles in envenomation [47] , some of these molecules are valuable pharmacological tools; for example, the polyamine toxins found in spider and wasp venoms are potent modulators of glutamate and nicotinic acetylcholine receptors [50, 51] . early toxinological research focused on study of venoms that adversely affect humans, with the aim of understanding their mechanism of action. the molecular targets of these venoms were typically delineated using low-throughput approaches such as in vivo and ex vivo tissue studies, manual patch-clamp recording, or biochemical approaches such as radioligand binding studies [52] . these bioassays can be highly informative because they provide information on integrated tissue or cellular responses that can be used to provide target information [52] . however, they require a priori knowledge about well-defined receptor populations and signalling pathways in target tissues, as well as the availability of selective agonists or antagonists. in addition to providing fundamental insights into the molecular targets and mode of action of venoms and toxins, these traditional approaches yielded key insights into mammalian physiology, as exemplified below. however, these approaches neglected toxins that are of no consequence to human health, those that are present in low abundance in venom, and toxins that act on novel targets that are not represented in conventional bioassays. these neglected toxins may nonetheless offer fundamental insights into human disease, and some may even have therapeutic benefit. accordingly, high-throughput screening approaches have been developed over the past decade to exploit the exceptional biodiversity of animal toxins for drug discovery. since many venoms and toxins exert these biological effects through actions on cell membranes, receptors and ion channels, high-throughput techniques assessing changes in cellular signalling have proven particularly insightful. these include electrophysiological techniques [53] , fluorescent imaging of na + , ca 2+ , k + or clflux [54, 55] , enzyme-linked immunosorbent assays (elisas), as well as assays based on detection of bioluminescence, fluorescence polarisation, fluorescence-resonance energy transfer (fret), bioluminescence resonance energy transfer (bret), and scintillation proximity [56] . invariably, these assays are designed to determine pharmacological activity at specific targets of interest, although each technique has limitations with regard to assay flexibility, throughput, amount of toxin required, and specificity (for a review see [52] ). thus, the choice of assay will depend on whether the intention is to identify the biological target of venom components that exert effects on biological processes either at the level of the cell or organism, or whether the screen is aimed at discovery of novel bioactivity at specific molecular targets or processes of interest. it has been known since the early 1960s that the specific and virtually irreversible blocking action of -bungarotoxin, a venom peptide from the banded krait bungarus multicinctus, at the vertebrate neuromuscular junction is due to functional antagonism of the depolarising effects of acetylcholine [57] . however, the identity of the presumed target of -bungarotoxin, the cholinergic receptor protein, was unclear until a single toxin-bound membrane protein was isolated from electric tissues of the marbled electric ray torpedo marmorata [58] and the electric eel electrophorus electricus [59] . it is now known that acetylcholine receptors (achrs) can be classified into muscarinic (machr) and nicotinic (nachr) subtypes, with the latter subtype being the target of bungarotoxin and numerous other venom peptides. machrs are metabotropic receptors whereas nachrs are pentameric ligand-gated ion channels. the profound impact of -neurotoxins on various biological processes highlighted the importance of nachrs in both physiology and pathology. early medical applications included the surgical use of tubocurarine, a plant-derived toxin that potently inhibits muscle nachrs and is famous for its use as a paralysing agent in poison arrows employed by south american indians [60] . the use of as a tubocurarine as a muscle relaxant allowed patients undergoing surgery to be paralysed without using dangerously high doses of general anaesthetics [61] . more recently, nachrs have gained attention as putative therapeutic targets for a range of human disorders. for example, a number of nachr subtypes are thought to play key roles in chronic pain and inflammation [62] , and several α-conotoxins with good selectivity for these subtypes are being developed as drug leads [63, 64] . the high affinity interaction of α-neurotoxins with nachrs also provided key insights into the structure and mode of action of these receptors [65] , and they remain important pharmacological tools due to their exquisite selectivity for specific achr subtypes. for example, fluorescent derivatives of α-bungarotoxin are excellent probes for identifying α7 nachrs [66] , while subtypeselective α-conotoxins from the venom of marine cone snails can distinguish between nachr subtypes containing common subunits [67, 68] . since ablation of nachr subunits using gene editing approaches would lead to complete loss of all nachr isoforms containing the deleted subunit, α-neurotoxins remain invaluable tools for dissecting the physiological roles of specific nachr subtypes and their contribution to human disease. in addition to being a valuable source of molecules that modulate known drug targets, venoms can also be used to discover new therapeutic targets. for example, the centre of excellence for new target discovery in brazil, which is co-funded by glaxosmithkline, is focused on using venoms to identify and validate new drug targets. one therapeutic area in which venom peptides have been successfully used to identify new drug targets is chronic pain [8, 69] . although the primary role of venom for most animals that possess a venom system is predation, most venomous animals also use venom for defense. some animals, such as bees, caterpillars, stonefish, and stingrays, use venom exclusively for this purpose. in contrast to the diverse mechanisms by which predatory venoms debilitate prey, defensive venoms are largely algogenic, that is they are designed to cause intense pain (as most people that have been stung by a bee or wasp can testify). unlike death, pain induces a lasting memory that can be conveyed to conspecific predators. since pronociceptive venom toxins have evolved to target vertebrate sensory neurons they have the potential to uncover new components of mammalian pain signalling pathways and to serve as pharmacological tools for studying these components [69, 70] . from a screen of venoms that activate mouse sensory neurons, osteen et al. recently isolated two homologous peptides (hm1a and hm1b) from venom of the african tarantula heteroscodra maculata that activated only a subset of sensory neurons [8] . the effect of these peptides was blocked by tetrodotoxin (ttx), a non-selective inhibitor of voltage-gated sodium (na v ) channels. this ultimately led to the discovery that these peptides are highly potent and selective agonists of na v 1.1 channels [8] , which were not previously thought to be involved in pain signalling. na v 1.1 was shown to regulate the excitability of a nerve fibres that transmit mechanical pain signals to the spinal cord, thereby highlighting this ion channel for the first time as a potential analgesic target [8] . moreover, na v 1.1 was shown to be present in sensory neurons that innervate the gut, and its expression was upregulated in a mouse model of irritable bowel syndrome (ibs) [8] . this suggests that compounds that specifically inhibit na v 1.1 might be useful for treatment of ibsrelated gut pain, and indeed na v 1.1 inhibitors were recently shown to reduce mechanical hypersensitivity in several models of chronic visceral pain [71] . an unexpected consequence of the discovery of hm1a and hm1b was their application to dravet syndrome (ds) epilepsy [72, 73] , which is caused by heterozygous loss-of-function mutations in the gene encoding na v 1.1. ds is a pharmacoresistant epileptic encephalopathy characterised by childhood-onset polymorphic seizures, cognitive impairment, psychomotor regression, autistic traits, and increased risk of sudden death [72, 73] . in the central nervous system, na v 1.1 is found in the axon initial segments of gabaergic inhibitory interneurons, and consequently epileptic seizures in ds are thought to result from the reduced inhibitory activity of these neurons [72] . remarkably, in a mouse model of ds, intracerebroventricular infusion of hm1a restored the function of inhibitory interneurons and led to almost complete abolition of seizures within 3 days, which rescued the mice from premature death [72] . in a similar approach to that described above, a screen of snake venoms on somatosensory neurons revealed robust activation of trigeminal ganglia by texas coral snake venom [74] . this activity could only be reproduced when two toxin components were combined to form the high affinity heteromeric mittx complex. subsequent patch-clamp studies revealed the target of mittx to be acid-sensing ion channels (asics). asics are unusual homo-or heterotrimeric channels that are activated by protons and expressed throughout the nervous system [75, 76] . although mittx promotes activation of several asic subtypes, it is ~100-fold more potent at the splice-isoforms asic1a and asic1b compared with other subtypes. texas coral snake envenomation causes intense pain in humans, and indeed mittx elicited robust nocifensive behaviour when injected into the hindpaw of mice. this response was absent in pan-asic1 knockout mice, but present in an asic3 knockout, revealing the contribution of asic1 in peripheral pain sensation. a subsequent study further delineated the specific roles of asic1a and asic1b in different pain pathways using mambalgin toxins isolated from black mamba venom [77] . mambalgins were found to block asics expressed in the central (asic1a, asic1a/2a, and asic1a/2b) and peripheral (asic1b and asic1a/1b) nervous system. intrathecal and intracerebroventricular injection of mambalgins in mice produced potent analgesia that was naloxone resistant and dependent on the presence of asic1a, suggesting an opioid independent mode of action proposed to be via central inhibition of asic1a/2a channels. strikingly, peripheral intraplantar injection of mambalgins in mice produced analgesic effects in wild-type and asic1a knockout mice. this demonstrated for the first time that asic1b-containing channels in rodent nociceptors, as opposed to the splice isoform asic1a, are important for peripheral analgesia. more recent work using an asic1b knockout animal confirmed the role of this subtype in peripheral pain sensing [78] . in this model, peripheral injection of mambalgin had no effect on acid-induced hyperalgesia in asic1b knockout mice. thus, the combined use of genetic techniques and venom toxins (mittx and mambalgins) enabled dissection of the roles of asic1 splice isoforms in pain signalling. in addition to venom peptides, non-peptidic marine toxins have proven to be invaluable tools for elucidating diverse pain pathways. ciguatoxins (ctx) are temperature-stable polyethers produced by various species of gambierdiscus, a benthic dinoflagellate [79] . the consumption of fish with high levels of ciguatoxins present in their flesh and viscera, a result of bioaccumulation, causes ciguatera fish poisoning [80] . these effects are due the action of ctx on ion channels including na v channels [80] . the most potent congener p-ctx-1 has been used to pharmacologically probe neuropathic pain (fig. 3 ). ciguatera is characterised by both gastrointestinal and neurological symptoms including paresthesia, pruritus and cold allodynia, and it can even lead to death in severe cases [81] . the pathognomonic cold allodynia observed in ciguatera patients is also reported in a broad range of clinical indications including in chemotherapy patients, complex regional pain syndrome, and fibromyalgia [82] [83] [84] . defined by sensitivity to innocuous cooling temperatures, cold allodynia is a poorly understood phenomenon. subcutaneous injection of p-ctx-1 into the rodent hindpaw causes robust and concentration-dependent cold allodynia [85] , and therefore it is uniquely positioned to elucidate the molecular basis of cold allodynia. the large family of thermosensitive transient receptor potential (trp) channels plays a critical role in temperature sensation [86] . interestingly, trpm8, the canonical cold sensing channel that is sensitive to menthol, is not associated with p-ctx-1-induced cold allodynia, as trpm8 null mice administered p-ctx-1 display robust pain behaviours in response to innocuous cooling [85] . rather, the combined findings from ex vivo skin-saphenous nerve recordings, in vivo studies and in vitro assays highlighted a role for trpa1 in p-ctx-1-induced cold allodynia [85] . however, trpa1 is not directly activated by p-ctx-1. both ttx-sensitive and ttx-resistant na v channels were responsible for the altered neuronal excitability, which drives a trpa1-dependent change in cold sensing [85] . although the precise role of different trp channel isoforms in cold sensing remains contentious, the use of this marine toxin has proved valuable in delineating these complex mechanisms. p-ctx-1 was also utilised to assess a range of analgesic treatments for cold allodynia. using a suite of selective pharmacological agents including venom-derived peptides (such as analgesic conotoxins; recently reviewed in [87] ), p-ctx-1-induced cold allodynia was found to be dependent on na v 1.6 and na v 1.8 [88] . oxaliplatin-induced cold allodynia was completely blocked by -conotoxin giiia, a na v 1.6 inhibitor from cone snail venom [89] , although intraplantar administration of cn2, a na v 1.6-selective agonist from scorpion venom [90] , elicited spontaneous pain and mechanical allodynia but did not enhance cold sensitivity. the mechanisms underlying neuronal cold sensitivity were further delineated using a class of presynaptic neurotoxins produced by mamba snakes (dendroaspis sp.) (fig. 3) . specifically, in trigeminal neurons, application of αdendrotoxin-k, a blocker of kv1.1 channels, elicited a reversible, concentration-dependent shift in the cold threshold [91] . subtype-selective na v channel toxins have confirmed a crucial role for both na v 1.6 and na v 1.7 in pain signalling (fig. 3) . the na v 1.6 subtype is highly expressed in the central and peripheral nervous system, in particular at the nodes of ranvier [92] . due to the expression of na v 1.6 on motor neurons, na v 1.6 null mice are not viable beyond post-natal day 14 (p14) [93] . this greatly impeded the study of na v 1.6 in peripheral sensory neurons, where its role in excitability was less well defined. the subtype-selective scorpion toxin cn2 was therefore used to probe the role of this channel in nociception. cn2 causes na v 1.6 to open at hyperpolarised (more negative) potentials, and it enhances resurgent currents in large diameter sensory neurons in vitro and increases excitability of medium-diameter a fibres ex vivo [90, 94] . therefore, it follows that in vivo nocifensive behaviours elicited by cn2 administration are primarily mediated by medium-diameter rather than smaller sensory neurons. critically, this demonstrates how toxins can be used to pharmacologically isolate a target of interest (in this case na v 1.6) in the presence of highly similar homologs of the target (na v 1.1, na v 1.7, na v 1.8 and na v 1.9). the toxin od1, isolated from venom of the iranian yellow scorpion odonthobuthus doriae, potently enhances the activity of na v 1.7 by inducing a hyperpolarising shift in the voltagedependence of activation, inhibiting channel fast inactivation, and increasing peak current [95] . injection of od1 into the footpad of mice elicits pain behaviours including licking, lifting and flinching of the injected paw that can be reversed by local or systemic treatment with selective na v 1.7 inhibitors. thus, the analgesic efficacy of na v 1.7 inhibitors can be rapidly profiled in vivo using od1-induced pain behaviour, demonstrating that even toxins with no therapeutic potential can be effective tools for understanding the complexities of peripheral pain sensing [70] (fig. 3 ). snakes are the most well-studied venomous animal, and hundreds of snake-venom proteins have been isolated over several decades. these venom components have improved our understanding of venom toxicity, significantly advanced our the knowledge of hemostatic disease mechanisms, and led to the development of diagnostic agents and life-saving drugs [96] [97] [98] . due to the immense amount of research on snake venoms, we focus here on just a few examples where studies of snake venom have advanced our understanding of hemostasis and associated diseases. studies into the lethal effects of envenomation by the burrowing asp atractaspis engaddensis led to the discovery of a novel peptide named sarafotoxin (sftx). sftx is a potent vasoconstrictor of coronary blood vessels that can induce cardiac arrest [99] [100] [101] . several sftx isoforms were soon isolated from other stiletto snakes [102, 103] , and it was subsequently discovered that the sftxs are in fact orthologs of mammalian vasoconstrictor peptides known as endothelins (ets) [104] . however, a major difference between these peptides is that a protease cleavage site involved in et inactivation is not present in the sftxs [105, 106] , making them more stable in vivo. this allowed the sftxs to be used as molecular tools to study vasoconstriction and expanded the available pharmacological toolbox beyond the ets. the sftxs and ets have both been used to characterise et receptors and downstream signalling pathways [107, 108] . von willebrand factor (vwf) is important for platelet adhesion, and its deficiency in humans leads to a common blood coagulation disorder known as von willebrand disease (vwd). botrocetin is a heterodimeric protein found in venom of the south american pit viper bothrops jararaca and it functions as a platelet-aggregating protein. it was initially named venom coagglutinin [109] as it leads to agglutination and aggregation of platelets by forming a complex with vwf and enhancing its affinity for the platelet receptor glycoprotein ib (gpib). this effect makes botrocetin an excellent diagnostic tool to assay for vwf binding to gpib in diseases such as vwd, or thrombopathies such as bernard-souler disease [110, 111] . botrocetin has also been used as a tool to study the vwf-platelet interaction and its role in disease [112, 113] . unlike other vwf binding proteins such as the antibiotic ristocetin, botrocetin-induced platelet agglutination activity is independent of the vwf multimer size. this is advantageous as it facilitates quantitation of vwf and allows assessment of vwd severity. the combination of botrocetin and ristocetin has allowed identification of missense mutations that cause of type b vwd [114] . a fortuitous observation using viper venom to study neurite outgrowth in healthy and tumorous cells led to the nobel prize in 1986. in studies of the regulation of cell growth, a fraction from mouse sarcoma extracts was shown to induce nerve cell growth in chick embryos [115] . this fraction was termed nerve growth factor (ngf) [116] , and it was shown to be composed of both nucleic acid and protein. snake venom from the cottonmouth viper agkistrodon piscivorus was known to contain phosphodiesterase activity, and thus it was proposed that this venom could degrade the nucleic acid component (which was thought to be a contaminant) and reveal the active ingredient. surprisingly, minute amounts of crude snake venom strongly promoted nerve growth, approximately 3000-6000 times more potently than the ngf-containing sarcoma fraction [117] . comparison of the sarcoma extract and venom contents ultimately led to isolation and characterisation of the proteinaceous ngf. ngf has since been found in several snake venoms from the viperidae, crotalidae, and elapidae families and it has been used widely to study cell growth regulation [118, 119] . the use of snake venom in this case not only led to a serendipitous finding with widespread importance to basic science, but ngf has also been important for elucidating the etiology of diseases such as developmental/growth deformations, tumours, and delayed wound healing. cobra venom factor (cvf) from the venom of cobra (naja sp.) snakes has the intriguing property of disrupting activation of the complement component of the innate immune system. the complement system is comprised of a set of interacting proteins, and it can be activated in three different ways for it to play a role in pathogen clearance. the alternative pathway of activation involves spontaneous hydrolysis of complement factor c3 to c3b, which allows the plasma protein complement factors b and d to bind to c3b and transform it to c3-convertase on the external surface of an invading pathogen membrane. subsequent recruitment of additional components of the complement pathway system leads to assembly of the membrane attack complex, which forms pores in the target cell membrane and that kill or damage the pathogen [120, 121] . cvf is a structural and functional analogue of the human complement factor c3, and it acts in a similar manner to human factors c3b and c3c to activate and deplete the complement cascade [122, 123] . these properties of cvf have made it possible to independently study the role of complement in host defense, immune responses, and pathogenesis of disease. cvf was in fact pivotal to the discovery of complement factor b itself, which facilitated further characterisation of this complex pathway [120, [124] [125] [126] [127] . cvf is not only an immensely useful research tool, but it has recently been used as an immunosuppressant in tissue and organ transplants and cancer therapies [128, 129] , and cvf derivatives are being designed to activate the complement system in disease states where it has been depleted [130, 131] . stroke is the second leading cause of death worldwide [132] , yet therapeutic options are limited to dissolution of blood clots with tissue plasminogen activator (tpa). unfortunately, due to the risk of inducing an intracranial haemorrhage, tpa is used in <5% of stroke patients [133] , and consequently there is an unmet need for compounds that can protect the brain from stroke-induced injury. clinical trials of numerous agents, especially glutamate receptor antagonists, have failed to provide effective neuroprotection in stroke patients, which has spurred interest in better understanding the etiology of ischemia-induced brain injury [133] . spider-venom peptides have been crucial for uncovering the key role of asics in stroke-induced brain damage, and validating these channels as a target for neuroprotective drugs [134] [135] [136] [137] . during ischemic conditions the brain switches from oxidative metabolism of glucose, its primary fuel, to anaerobic glycolysis, which results in lactic acidosis. the brain extracellular ph can fall from ~7.3 to as low as 6.0 in the ischemic core [134, 138, 139] , which is sufficiently acidic to robustly activate proton-gated asics. the asic1a isoform is the dominant subtype in rodent and human brain [140, 141] and it primarily conducts sodium ions, although it is the only asic subtype that is also permeable to calcium (p na /p ca ~15) [75] . it has been proposed that asic1a activation contributes to toxic intracellular calcium overload during stroke via both direct calcium permeability and downstream activation of voltage-gated calcium (ca v ) channels and nmda receptor-gated channels via asic-induced membrane depolarisation [75] . however, the primary mechanism by which asic1a activation leads to neuronal cell death appears to be recruitment and activation of ripk1 [142, 143] , a master regulator of necroptosis. although several small molecules and endogenous ligands of asic1a have been discovered to date, their lack of potency and selectivity limits their use as pharmacological tools for dissecting the role of asics in disease. the first potent and moderately selective asic1a ligand discovered is pctx1, a 40-residue peptide from venom of the trinidad chevron tarantula psalmopoeus cambridgei [144] . pctx1 inhibits homomeric asic1a (ic 50 ~1 nm), asic1a/2b heteromers (ic 50 ~3 nm), and asic1a/2a (35-85% inhibition at 50 nm) channels, and potentiates proton-evoked asic1b currents by over 3-fold control (ec 50 ~25 nm) [144] [145] [146] [147] [148] . the key role of asic1a in stroke-induced brain damage was first highlighted in a seminal 2004 study which showed that genetic ablation of the asic1a-encoding gene reduced brain infarct size by ~60% in a mouse model of ischemic stroke [134] . however, since genetic ablation can give rise to compensatory upregulation of related channels with unknown consequences [149, 150] , these researchers also used "pctx1 venom" (i.e., crude p. cambridgei venom) as a pharmacological tool to validate the genetic studies. they showed that "pctx1 venom" administered 30 min before and after stroke onset reduced infarct size by the same magnitude as genetic ablation of asic1a [134] . however, spider venoms are complex molecular cocktails that modulate an array of ion channels, and the venom of p. cambridgei is no exception. pctx1 constitutes only ~0.4% of total p. cambridgei venom [136] , and other components in this venom are known to target trpv1 and voltage-gated potassium (k v ) channels [151, 152] . this left open the question of whether the observed neuroprotective effects were induced by pctx1, other components in the venom, or a combination of both. to address this question a more recent study used pure recombinant pctx1 to confirm the involvement of asic1a in stroke [136] . structure-activity relationship (sar) studies of pctx1 [153, 154] facilitated design of a weakly active pctx1 mutant, which was not neuroprotective in the same rodent stroke model, thereby strengthening the evidence that asic1a inhibition reduces stroke-induced brain damage. hadronyche infensa proved to be highly neuroprotective in a focal model of ischemic stroke, even when administered 8 h after the stroke onset [137] . in comparison to pctx1, hi1a is a slightly more potent inhibitor of asic1a (ic 50 0.5 nm) and it is more selective, with >2000-fold higher potency for asic1a than homomeric asic1b, asic2a, and asic3 channels. hi1a inhibition of asic1a is much less reversible than pctx1 [137] , and these venom peptides have a different mechanism of action [137, 155] . the different properties of pctx1 and hi1a broaden the pharmacological toolkit for studying asic1a and asic1a-related pathologies. together these two venom peptides have provided striking pharmacological evidence that asic1a is involved in ischemic injuries of not just the brain, but also the heart [156] and kidney [157] . the evolution of drug resistance across bacteria, fungi and parasites has created an unmet need for novel anti-infectives. the 2019 united nations report on antimicrobial resistance estimated that >700,000 people die annually due to drug-resistant infections [158] . while most focus has been on human antibiotic resistance, control of parasitic nematodes in both livestock [159, 160] and companion animals [161, 162] is also seriously threatened by resistance. despite the clear need for new anti-infectives, there is a diminishing pipeline of anti-infective drug candidates in both the human and veterinary spaces [163] [164] [165] , which has created interest in venoms as a potential source of novel anti-infectives. antimicrobial peptides (amps) are found in many animal venoms, and hundreds of examples have been reported with varying activity against fungi and both gram-positive and gram-negative bacteria [166] [167] [168] [169] [170] [171] . venom-derived amps (vamps) are typically small, linear peptides that are rich in cationic residues (lys, his and arg), which facilitates their interaction with anionic membranes [166, 172] . they are often unstructured in aqueous solution but form ordered -helices upon interaction with lipid membranes [172, 173] . many vamps exhibit broad-spectrum cytolytic effects. this can be a double-edged sword for antimicrobial development as it often confers desirable broad-spectrum activity against a wide range of bacterial and fungal pathogens but also cytotoxicity against host cells. for example, the ponericin peptides isolated from the amazonian stinging ant neoponera goeldii have broadspectrum activity against bacteria and fungi, but they are potently hemolytic [174] . likewise, cupiennin 1a from venom of the wandering spider cupiennius salei exhibits non-specific cytolytic activity against bacteria, insects, protozoan parasites, and human cells [175] . mammalian cytotoxicity is typically assessed by screens against red blood cells or mammalian cell lines, but an often overlooked property of cytolytic vamps is that they can also cause pain. for example, venom-peptide ∆-myrtoxin-mp1a from the australian ant myrmecia pilosula has submicromolar activity against bacterial pathogens but it causes mechanical allodynia when injected into mice [176] . melittin, the primary component of honeybee venom, is potently antimicrobial but it induces pain through direct and indirect activation of sensory neurons [177] . hence, future research on vamps should place greater emphasis on screening across a broad range of cell lines and phenotypic readouts, particularly for cytolytic peptides. a small number of vamps show some degree of inherent taxonomic selectivity. for example, bicarinalin from the ant tetramorium bicarinatum is highly active against bacterial and fungal pathogens (minimal inhibitory concentration (mic) of 0.7-25 µm, which is comparable to commercial anti-infectives), with at least 10-fold selectivity over human lymphocytes [178] . it also has similar potency to commercial antibiotics against clinical isolates of the helicobacter pylori, a pathogen that causes stomach ulcers [179] . however, most vamps will require further engineering of their selectivity in order for them to be therapeutically useful [166] , and several strategies have been employed successfully for this purpose. for example, shorter analogues of the scorpion-venom peptides hadrurin and vejovine were engineered with 100-fold improvement in selectivity for escherichia coli over red blood cells [180] . modification of the hydrophobicity and net charge of the wasp-venom peptide mastoparan through selective amino acid substitutions [181] and n-terminal acylation [182] increased its affinity for negatively-charged bacterial membranes and decreased its cytotoxicity towards mammalian cells. although most vamps have not progressed beyond basic in vitro studies, some have proven effective in in vivo infection models. zy4 is a 17-residue cyclised derivative of cathelicidin-bf from venom of the snake bungarus fasciatus with improved potency, selectivity and stability over the parent toxin [183] . in vitro, zy4 kills multidrug-resistant isolates of the human pathogens pseudomonas aeruginosa and acinetobacter baumannii at lower concentrations than commercial antibiotics, and it reduces bacterial load and inflammation in the lungs of mice in a p. aeruginosa infection model, with no reported side-effects [183] . lyetxi-b, an n-terminal acetylated derivative of lyetxi from venom of the wolf spider lycosa erythrognatha, has improved potency and selectivity against e. coli and also reduces both bacteraemia and inflammation in a mouse model of septic arthritis when administered intraarticularly [184] . route of administration is a key issue that must be considered for therapeutic deployment of vamps; traditionally, peptides have poor oral bioavailability [185] , and hence alternative methods of vamp administration such as topical application should be considered. the effectiveness of this approach was demonstrated for kn2-7, a derivative of the peptide bmkn2 from venom of the scorpion mesobuthus martensii, which protected the skin of mice in a staphylococcus aureus in vivo infection model [186] . topical administration can also circumvent or alleviate off-target effects that are evident when the vamp is present systemically. finally, it might be possible to achieve additive or synergistic effects by combining vamps with existing antibiotics by utilising the pore-forming activity of the vamp to enhance antibiotic delivery. for example, garcia et al. found an additive effect when pathogen-specific antibiotics were combined with vamps isolated from the venom of arachnids [187] . viruses continue to pose an enormous threat to global health, as exemplified by the current global pandemic caused by sars-cov-2 [188] . despite intensive intervention efforts, an estimated 770,000 people continue to die annually from human immunodeficiency virus acquired immunodeficiency syndrome (hiv-aids) [189] . many serious viral diseases such as severe acute respiratory syndrome (sars), middle eastern respiratory syndrome (mers), dengue, zika, and ebola lack vaccines or efficacious therapeutic treatments, highlighting the need for novel antivirals. most reported venom toxins with antiviral activity are vamps with broad-spectrum activity; examples include melittin, snake venom pla 2 and l-amino oxidases, and mastoparan derivatives [190, 191] . thus, a key challenge will be engineering these compounds to have selectivity for either the viral particles or the process by which they infect host cells. mastoparan-7, derived from wasp-venom mastoparan, has broad-spectrum activity against lipid-enveloped viruses (including influenza and flaviviruses) through direct insertion and disruption of the envelope, but it remains highly cytotoxic to host cells in vitro [192] . peptides with activity on specific viral protein targets rather than lipids may provide better selectivity. lactarcin-1 from the central asian spider lachesana tarabaevi inhibits replication of dengue virus-2 (ic 50 ~7 µm) by virtue of its ability to inhibit the viral protease ns2b-ns3, but it has low selectivity over mammalian cells [193] . the antibacterial activity of mucroporin, a vamp from the scorpion lychas mucronatus, was improved through histidine and arginine substitutions to create the analog mucroporin-m1 with increased net charge. mucroporin-m1 has improved activity against both gram-positive and gram-negative bacteria [194] , and these modifications also conferred micromolar antiviral activity in vitro (ec 50 1-7 µm) against measles virus and pseudovirus models of the highly pathogenic sars-cov and avian influenza h5n1 viruses [195] . a key limitation of current research on venom-derived antiviral toxins is that few studies have progressed to in vivo testing. one example found that pre-treating vesicular stomatitis virus, a serious pathogen of cattle, with mastoparan-7 significantly improved clinical outcomes in a mouse challenge model through presumed inactivation of viral particles [192] . however, this needs to be confirmed in a more clinically relevant challenge model of viral infection followed by toxin administration to probe efficacy. overall, further research is required to ascertain the potential of venom-derived peptides as viable antiviral drugs. parasites remain one of the most serious threats to human and veterinary medicine, with current treatment options limited by drug resistance, side-effects and limited efficacy [196] . this has stimulated interest in venoms as a source of novel antiparasitic compounds (for a review see [197] ). most such studies have focused on protozoan parasites, which are responsible for a range of human diseases. the most problematic parasitic disease is malaria, which is caused by plasmodium parasites; despite intensive intervention efforts, malaria kills an estimated 435,000 people each year and control is threatened by drug resistance [198] . a further 20 million people worldwide are infected by parasitic protozoans of the genera leishmania and trypanosoma, with approximately 3 million new infections per year [199] . most antiparasitic compounds isolated from animal venoms are small cationic vamps, which again raises the issue of taxonomic selectivity. the vamps mastoparan [200] and melittin [199] inhibit development of trypanosoma cruzi (the causative agent of chagas disease), but selectivity over host cells is low. however, bicarinalin potently inhibits growth of leishmania infantum (mic 1.5 µm), the causative agent of infantile visceral leishmaniasis, with good selectivity over human lymphocytes [178] . remarkably, crotamine, a peptide from venom of the south american rattlesnake crotalus durissus terrificus, crosses infected red blood cell membranes to lyse the plasmodium parasitophorous vacuole, thereby killing the parasite [201] . this may present an opportunity for combination therapy with commercial antimalarials, facilitating drug transport to the parasite. further examples of venom components active against protozoan parasites include trimorphin, a pla 2 from venom of the north american rear-fanged snake trimorphodon biscutatus lambda that inhibits growth of leishmania major [202] , and crovirin, a cysteine-rich secretory protein (crisp) from the rattlesnake crotalus viridis viridis that is active against protozoan parasites at concentrations that are not toxic to host cells [199] . the protozoan toxoplasma gondii, which causes the key zoonotic disease toxoplasmosis, is susceptible to a venom peptide from the marine cone snail conus californicus [203] , while a c-type lectin and a pla 2 from b. pauloensis snake venom impair toxoplasma adhesion and replication [204, 205] . the greatest burden of disability-adjusted life years in the neglected tropical diseases comes from helminth infections, including roundworms (nematoda) and flatworms (platyhelminthes) [189] . these worms cause important parasitic diseases in humans, including onchocerciasis, lymphatic filariasis, and schistosomiasis. about 200 million people worldwide suffer from schistosomiasis, and it is considered the second most socioeconomically devastating parasitic disease after malaria [206] . the burden of parasitic disease is also a key concern in animal health and production, with the worldwide cost of controlling livestock parasites amounting to tens of billions of dollars [207] . despite this clear need for novel anthelmintics, it is an underexplored area of venom research with very few reports of anthelmintic venom toxins. crotamine [208] and ε-latroinsectotoxin from venom of the black widow spider latrodectus mactans tedecimgutattatus [209] both impair growth of the non-parasitic nematode caenorhabditis elegans, but anthelmintic activity has not been reported against parasitic species. more recently, the spider-venom peptide hi1a was shown to inhibit larval development of the barber's pole worm haemonchus contortus, a parasite of sheep production [210] . gomesin, a peptide found in the venom [211] and hemocytes [212] of spiders, has broad-spectrum activity against bacteria, fungi, parasites and tumor cells [213, 214] , and a cyclized version was recently shown to have improved stability and activity against plasmodium in vitro [215] . to our knowledge, no venom-derived antiparasitics have yet been demonstrated to have in vivo efficacy. as for antibacterials, antifungals and antivirals, most venom-derived antiparasitics will likely require engineering to enhance their selectivity over host cells. an alternative strategy to developing venom compounds as anti-parasitic drugs is to instead impair plasmodium development and transmission via transgenic mosquitoes engineered to express the anti-parasitic compound in the mosquito midgut, as recently demonstrated with scorpion-venom peptides [216] . as most research on venom compounds with antibacterial, antiparasitic, or antifungal activity has been performed in the last decade, we expect that more toxins with such activities will be discovered in the near future. most work to date has focussed on human pathogens, but there is a clear need for novel veterinary anti-infectives which could be further explored in future. due to the commonly found cytolytic nature of these peptides, a key challenge across all of these diseases will be engineering analogs with selectivity for the targeted pathogen. arthropods, and in particular hematophagous (blood-sucking) species, are vectors for a wide-range of organisms that can cause human disease [217] . the most pernicious vectors are undoubtedly mosquitoes, which transmit protozoans (e.g. plasmodium species that cause malaria), parasitic nematodes (e.g. brugia species that cause filariasis), parasitic platyhelminths (e.g. trematodes that cause schistosomiases), and viruses (e.g. flaviviruses that cause yellow fever, west nile fever, and dengue) [17, 217, 218] . other important hematophagous disease vectors include triatomine bugs which transmit trypanosomes that cause chagas disease, fleas which transmit the bacterium yersinia pestis that causes bubonic plague, and ticks which transmit a wide variety of viral, bacterial and protozoan pathogens [17, 217] . rather than directly targeting disease-causing organisms, venom toxins could be used instead to target the vectors of these diseases. insects are the dominant vectors of human diseases, and chemical insecticides are the primary means of controlling these insect pests [17, 218] . unfortunately, all chemical insecticides target one of only a handful of molecular targets, and consequently their indiscriminate use has led to widespread resistance [17] . thus, new insecticides with novel modes of action are much sought after. spiders are professional insect killers, and they are the most speciose venomous animal, and consequently their venoms have been intensively studied as a potential source of novel bioinsecticides [17, [217] [218] [219] [220] [221] . as a result, many insecticidal toxins have been isolated from spider venoms, including peptides, proteins and polyamines, but they mostly lack the necessary taxonomic selectivity to be useful as insecticides. an ideal insecticide should be inactive on humans and other vertebrates, as well as beneficial insects such as pollinators and natural predators of the targeted insect pest [221] . this narrow target-organism profile makes insecticide development particularly difficult. however, spidervenom peptides have been isolated with desirable taxonomic selectivity. for example, by carefully counter-screening insect toxins from the venom of australian funnel-web spiders in vertebrate assays, a number of insect-selective neurotoxins were isolated [218] . one of these peptide toxins (gs-/-hexatoxin-hv1a), which has complex polymodal pharmacology, has been developed commercially as a bioinsecticide by vestaron corporation; this peptide is active against a broad range of insect pests but is inactive against bees and vertebrates [221] . in order to target anopheline mosquitoes that transmit malaria, a gene encoding gs-/-hexatoxin-hv1a was engineered into the entomopathogenic fungus metarhizium pingshaense [222] , and this weaponised entomopathogen proved highly successful in controlling malaria vectors in a field trial in burkina faso [223] . an advantage of this approach is that the range of susceptible species is determined by the entomopathogen rather than the venom peptide, thereby allowing deployment of insecticidal venom toxins that would otherwise lack the desired taxonomic selectivity [221, 224] . some insecticidal venom peptides have exceptional taxonomic selectivity. for example, -diguetoxin-dc1a (dc1a), a 56-residue knottin from venom of the desert bush spider diguetia canities [225] , and -theraphotoxin-ae1a, a 39-residue knottin from venom of african tarantula augacephalus ezendami [226] , have potent insecticidal effects on german cockroaches (blattella germanica), without significantly affecting american cockroaches (periplaneta americana). both knottins target insect na v channels, and mutational analyses revealed that the susceptibility of insects to these toxins is determined by small sequence differences in their proposed binding site on the extracellular surface of the domain ii voltage sensor (vsd ii ) [226, 227] . a high-resolution structure of dc1a bound to a cockroach nav channel was recently determined using cryoelectron microscopy [228] , and it not only confirmed that vsd ii is the binding site for dc1a but revealed that the toxin's inactivity against vertebrates [225] can be explained by sequence variations in the vsd ii region of each of the human na v channels. the dc1a-na v channel structure allows predictions to be made about which insects will be susceptible to dc1a based on their na v channel vsd ii sequences, and it should allow use of structure-guided protein engineering to develop toxin analogs with improved potency and/or altered taxonomic selectivity. in addition to highlighting how venom compounds can be used to elucidate disease mechanisms and uncover new drug targets, we want to provide examples of venom compounds that have progressed all the way from basic research to marketed drugs. there are currently six venomderived drugs approved by the fda, plus a snake-venom-derived serine protease (batroxobin; marketed as defibrase®) that is approved for clinical use outside of the usa [9, 10] . two further venom-derived drugs -dalazatide for treatment of autoimmune diseases [24] and tozuleristide for intraoperative imaging of brain tumours [25] -are currently undergoing clinical trials. the examples below demonstrate the potential for developing venom compounds into drugs [229] but, like any class of therapeutics, there are many challenges and the success rate is low [61] . the first venom-derived drug was captopril, an angiotensin-converting enzyme (ace) inhibitor that is used to treat hypertension. the observation that patients envenomated by the lethal brazilian viper bothrops jararaca suffered from hypotension and severe intestinal contractions led to the discovery that the venom inhibits ace [230] [231] [232] . inhibition of ace lowers blood pressure by preventing conversion of angiotensin i to the vasoconstrictive hormone angiotensin ii, and by decreasing degradation of the vasodilatory peptide bradykinin. researchers at squibb isolated six ace inhibitory peptides from b. jararaca venom, one of which (teprotide, 9 residues) was both potent and stable in vivo [230] . detailed sar studies of teprotide ultimately led to a smaller, orally active peptidomimetic known as captopril (d-2-methyl-3-mercaptopropanoyl-l-proline) [230, 232] . captopril was approved by the fda for treatment of hypertension in 1981, and by the mid 1990s it had become a blockbuster drug, reaching peak annual sales of more than usd $1.6 billion [233] . another example of a reptile peptide that was developed into a human drug is exendin-4, a 39residue peptide isolated from venom of the gila monster heloderma suspectum. in striking contrast to captopril, the marketed drug (exenatide) is identical to the peptide found in the lizard's venom. exenatide mirrors the effects of human hormone glucagon-like peptide-1 (glp-1), an incretin that stimulates glucose-dependent insulin secretion, suppresses glucagon secretion, slows gastric emptying, and promotes satiety [234] , but it is much more stable in vivo than human glp-1 with a plasma half-life of 2.4 h [9] . byetta®, a formulation of exenatide that requires twice daily subcutaneous injections, was approved by the fda in 2005 for control of type 2 diabetes, and it achieved worldwide sales of usd $800 million in fiscal year 2009 [9] . in order to improve patient acceptance and compliance, a new formulation was developed in which the peptide is embedded in biodegradable polymeric microspheres that extends the duration of exenatide release to such an extent that therapeutic levels can be maintained with weekly injections [235] . this new formulation (bydureon®) was approved by the fda in 2012, and annual sales have remained steady at usd $500-600 million despite the introduction of competing incretin mimetics. another drug that is an exact copy of a venom peptide is ziconotide, a synthetic version of ωconotoxin mviia, a 25-residue knottin peptide found in venom of the marine cone snail conus magus. ziconotide is used to selectively inhibit ca v 2.2 channels in the spinal cord, thereby preventing the propagation of pain signals from the spinal cord to processing centres in the brain [236] . it was approved by the fda in 2004 for treating patients with chronic pain. ziconotide is administered by intrathecal infusion, which limits its use to patients with intractable chronic pain that have failed on frontline analgesic drugs such as morphine. while ziconotide set a landmark as the first venom-derived drug with a neuronal target, its limited market acceptance due to its mode of administration and neuropsychiatric side-effects [237] has caused venom researchers to focus attention on analgesic targets in peripheral nociceptors rather than central neurons, as outlined in section 4.2. the integration of advanced venom proteomics with next-generation sequencing of venom-gland transcriptomes has made it possible to rapidly determine, at only moderate cost, the complete venom proteome of even miniature venomous animals [46] . as a result, novel venom peptides and proteins, often with unique three-dimensional folds, are being discovered at an unprecedented rate. the coupling of these omics workflows with high-throughput screening technologies [56, 238] is allowing the pharmacology of these compounds to be explored with unprecedented efficiency, often leading to the discovery of toxins with novel pharmacology [8, 74, 77, 239, 240] . all of these advances have combined to create tremendous interest in venoms as a potential source of drugs and bioinsecticides [12, 229] . while there have been some notable successes -there are seven venom-derived drugs in clinical use and several in phase 2/3 clinical trials -the triumphs are scarcer than many predicted. no novel venom-derived drug has been approved by the fda since 2005 [9, 10] , and only one venomderived compound has been commercialised as a bioinsecticide [221] . the reasons for this are manifold. drug development is inherently difficult and the overall success rate is low [61] ; venomderived drug candidates are no different than any other class of therapeutic in this respect, although the overall approval rate for venom-derived drugs entering clinical trials is higher than other classes of therapeutics. most venom researchers are not trained in the nuances of drug development, and this has led to over-hyped claims about the therapeutic potential of venom compounds, often in the absence of in vivo data. we encourage collaboration between venom researchers and those with experience in drug development so that, at a minimum, in vivo efficacy and toxicity can be assessed in validated disease models, using clinically viable routes of administration, before making claims about therapeutic potential. renewed interest from pharmaceutical companies in venoms as source of drug leads and pharmacological tools [241] [242] [243] [244] will help to drive these collaborations. notwithstanding these caveats, there is little doubt that the field of venoms-based drug discovery has rapidly matured over the past decade and is now producing drug leads at an unprecedented rate. advances in cryoelectron microscopy have facilitated determination of the three-dimensional structure of venom compounds bound to large membrane receptors [228, 243, [245] [246] [247] [248] , and this should facilitate the rational design of drug candidates with improved potency and selectivity for their cognate molecular target. we predict that these advances this will lead to significant impact in some disease areas. for example, ion channels are now the third most common human drug target after kinases and g-protein-coupled receptors [249] ; venoms are unquestionably the best natural source of potent ion channel modulators and venom-derived ion channel modulators have already attracted significant interest from pharmaceutical companies [24, [241] [242] [243] [244] . the authors declare that they have no conflicts of interest. figure 1 : fields of application in basic research and human health to which venom compounds have been applied. venom compounds have the potential to be used as pharmacological tools, therapeutics, insecticides, or for targeting vectors of human disease or disease-inducing organisms. botrocetin bound von willebrand factor (vwf) and platelet glycoprotein ib (gpib) (1u0n) [250] ; cobra venom factor (cvf) bound complement factor b (3hrz) [251] ; captopril bound angiotensin converting enzyme (ace) (1uzf) [252] ; triflin bound small serum protein 2 (ssp2) (6imf) [253] ; mittx-α and -β bound acid sensing ion channel 1 (asic1) (4ntw) [254] . (b) cone snail toxin complexes: α-conotoxin imi bound acetylcholine binding protein (achbp) (2byp) [255] ; μconotoxin kiiia bound voltage-gated sodium channel (na v )1.2/auxiliary β2 subunit (6j8e) [256] . (c) spider toxin complexes: pctx1 bound asic1 (4fz0) [257] ; dc1a and tetrodotoxin (ttx) bound na v pas (6a95) [228] ; dktx and resiniferatoxin (rtx) bound transient receptor potential cation channel subfamily v member 1 (trpv1) (5irx) [258] ; protx-ii bound na v 1.7 voltage sensor domain ii/na v ab chimera (6n4i) [243] . (d) gila monster toxin exendin-4 bound glucagon-like peptide 1 receptor (glp-1r) (3c5t) [259] . 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pharmaceutical industry exenatide once weekly for the treatment of type 2 diabetes ziconotide: neuronal calcium channel blocker for treating severe chronic pain do the potential benefits outweigh the risks? an update on the use of ziconotide in clinical practice venomics: a new paradigm for natural products-based drug discovery a distinct three-helix centipede toxin ssd609 inhibits i ks channels by interacting with the kcne1 auxiliary subunit novel venom-derived inhibitors of the human eag channel, a putative antiepileptic drug target potency optimization of huwentoxin-iv on hna v 1.7: a neurotoxin ttx-s sodium-channel antagonist from the venom of the chinese bird-eating spider selenocosmia huwena engineering potent and selective analogues of gptx-1, a tarantula venom peptide antagonist of the na v 1.7 sodium channel structural basis of na v 1.7 inhibition by a gating-modifier spider toxin comprehensive engineering of the tarantula venom peptide huwentoxin-iv to inhibit the human voltage-gated sodium channel hna v 1.7 mechanisms of channel block in calcium-permeable ampa receptors structural basis of ⍺-scorpion toxin action on na v channels structures of human na v 1.7 channel in complex with auxiliary subunits and animal toxins molecular basis for pore blockade of human na + channel na v 1.2 by the μ-conotoxin kiiia a comprehensive map of molecular drug targets the snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation insights into complement convertase formation based on the structure of the factor b-cobra venom factor complex structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin i-converting enzyme crystal structure of the complex between venom toxin and serum inhibitor from viperidae snake x-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a na + -selective channel structures of aplysia achbp complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations molecular basis for pore blockade of human na(+) channel nav1.2 by the μ-conotoxin kiiia structural plasticity and dynamic selectivity of acid-sensing ion channelspider toxin complexes trpv1 structures in nanodiscs reveal mechanisms of ligand and lipid action crystal structure of the ligand-bound glucagonlike peptide-1 receptor extracellular domain structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent k + channel king: conceptualization, supervision king: writing, review & editing, funding acquisition key: cord-324953-3sacf4wu authors: childs, james e.; richt, jürgen a.; mackenzie, john s. title: introduction: conceptualizing and partitioning the emergence process of zoonotic viruses from wildlife to humans date: 2007 journal: wildlife and emerging zoonotic diseases: the biology, circumstances and consequences of cross-species transmission doi: 10.1007/978-3-540-70962-6_1 sha: doc_id: 324953 cord_uid: 3sacf4wu this introduction provides a telegraphic overview of the processes of zoonotic viral emergence, the intricacies of host–virus interactions, and the distinct role of biological transitions and modifying factors. the process of emergence is conceptualized as two transition stages which are common and required for all disease emergence, (1) human contact with the infectious agent and (2) cross-species transmission of the agent, and two transition stages which are not required for emergence and appear unavailable to many zoonotic pathogens, (3) sustained human-to-human transmission and (4) genetic adaptation to the human host. the latter two transitions are presumably prerequisites for the pandemic emergence of a pathogen. the themes introduced herein are amplified and explored in detail by the contributors to this volume. each author explores the mechanisms and unique circumstances by which evolution, biology, history, and current context have contrived to drive the emergence of different zoonotic agents by a series of related events; although recognizable similarities exist among the events leading to emergence the details and circumstances are never repetitive. the process of zoonotic disease emergence can be understood by coupling knowledge of how zoonotic viruses have evolved and are maintained among their wildlife hosts, transmitted across a species barrier to cause productive infection in a taxonomically distinct secondary host, initiate a pathologic process causing disease, and, by repetitive infection within the secondary host species, result in incident morbidity or mortality of sufficient magnitude to be detected and characterized as a novel health concern of local, regional, or global significance (see the chapter by childs, this volume). obviously, we possess no such knowledge for any zoonotic virus or zoonotic disease, but casting the emergence process in this context underscores how disciplinary boundaries are blurred; advances require approaches spanning the spectrum of biological inquiry, and solutions to imminent threats require approaches unbounded by the notion of specific scientific discipline. the emergence process involves ecological interactions at the individual, species, community, and global scale. the dynamic circumstances and relative importance of the participants reflect the evolutionary context in which zoonotic agents have become accommodated to, and been accommodated by, their reservoir hosts (h r s) (see the chapters by cleaveland et al. and by holmes and drummond, this volume) , the diversity of reservoir species involved, their geographic ranges and the local dispersion of host and pathogen populations. in turn, historical factors have modified and blurred traditional patterns of species distribution, abundance, and diversity, and are continually transforming the landscape of opportunity on which zoonotic viruses with their h r s mingle with novel, potentially susceptible secondary host species (h s )s (see the chapters by daszak et al., field et al., regnery, and wang and eaton, this volume) . the current historical circumstances are unprecedented in their efficiency for continually shuffling an expanding repertoire of zoonotic viruses and hosts, introducing them in novel ecologic circumstances to a wealth of previously unavailable and unexplored niches. within the last decade, the accelerated pace of rapid translocations of infected h r s or h s s have heralded a sea change in how we view the public health threat posed by zoonotic viruses (childs 2004) , as testified by the emergence of sars coronavirus (sars cov) ; see the chapter by wang and eaton, this volume) , influenza a subtype h5n1 (peiris et al. 2004 ; see chapter by webby et al., this volume), west nile virus (wnv) (lanciotti et al. 1999) , nipah virus (niv) (chua et al. 1999 ; see the chapter by field et al., this volume) , and monkeypox virus ; see the chapter by regnery, this volume). inherent in the term "cross-species transmission" (or spillover) is the ability for a foreign virus, once introduced into an individual of a h s population, to complete the virus infectious cycle: (1) adsorption, penetration, and uncoating, or separation of the viral nucleic acid from the capsid; (2) transcription, translation, and replication, and; (3) assembly and release (nayak 2000) . binding and entry into permissive h s cells is mediated by common or related cellular receptors. additional bouts of infection following virus release from infected cells lead to the dissemination of virus throughout the host's tissue(s), precipitating, as a byproduct, pathologic alterations in the individual h s identifiable as symptomatic disease. the pathogenic course of infection and disease within the secondary host (h s ) may bear little correspondence to the infectious process and outcome within the reservoir host (h r ). oral lesions caused by herpesvirus b (cehv-1) infection among individual macaques of the h r are transmogrified into an often fatal (~70%) meningoencephalitis in the human h s (huff and barry 2003) . hantaviruses cause subclinical infections or subtle behavioral changes with limited pathology in individual rodents of species constituting the virus-specific h r s (hinson et al. 2004; llyubsky et al. 1996 ; see the chapter by klein and calisher, this volume), accompanied by no notable loss of fitness (childs et al. 1989 ). however, these subtleties are lost in the severe and often fatal hemorrhagic fever with renal syndrome (hfrs) and hantavirus pulmonary syndrome (hps), developing in the human h s after virus spillover (zaki et al. 1995; tsai 1987) . no matter how different the disease course among the human h s , the pathologic component of intra-h r transmission is highly relevant when considering strategies to prevent human infection rather than treating post-spillover disease (see the chapter by daniels et al., this volume) . ignoring the intricacies of zoonotic virus transmission among wildlife h r s guarantees that solutions springing from a traditional anthropocentric disease-treatment/vaccine-preventative approach will consider a limited universe of defensive prevention targets and generate a restricted arsenal of intervention tools . the ecologic process of zoonotic disease emergence can be schematized by four transition stages (fig. 1 ) , of which only the first two are prerequisites for emergence: (1) contact between infectious propagules originating from the wildlife h r with individuals of a susceptible h s and (2) cross-species transmission, a transition subsuming the complex interactions of the virus infectious cycle within the h s (nayak 2000; childs 2004 ). these first two transitions may require a mediating host such as an arthropod vector (h v ) or an intermediary vertebrate host (h i ); these additional host populations are readily accommodated by the modular emergence schema (fig. 1 ). the latter two transition stages demarcate a change in the interrelationship of host and virus ( fig. 1): (3) sustained transmission of the once zoonotic introduction virus between members of the new h s , subsequent to, and independent of, new spillover events, and (4) genetic adaptation and phenotypic changes accompanying sustained intra-h s transmission. once sustained transmission occurs within the human host, evolutionary adaptation between virus and host can transform the once zoonotic virus into a distinctive new virus with a new human h r . the new virus associated with humans must be quantitatively and qualitatively different from ancestral strains in genetic and phenotypic characters, in order to designate the emergence of a new biological entity. with hiv and pandemic influenza subtypes, the qualities of the newly adapted viruses to humans are readily apparent in terms of host preference and host pathogenicity (hahn et al. 2000; claas 2000) . with sars cov infecting humans, the specific genetic changes are less clear-cut (song et al. 2005) , most probably because the transmission of sars cov was curtailed early its relationship to the new human host. support for this conclusion is based on the genetic differences accrued by sars covs sustained through multiple generations of human-to-human transmission as compared with those viruses with shorter interhuman passages . some viruses are capable of sustained human-to-human transmission with minimal or no genetic change [i.e., sars cov; see the chapter by wang and eaton, this volume, although limits to genetic adaptation within humans may be imposed by the requirement for an intermediate vector or extensive prior adaptation to a specific reservoir host (gould et al. 2003) ]. the arboviruses, yellow fever virus, and the four dengue serotypes circulate in a human-tohuman transmission cycle mediated by anthropophilic h v s after introduction by bridging h v s feeding on infected primate h r s (de silva et al. 1999; wolfe et al. 2001; monath 1989; downs 1982) ; these viruses appear closely related to the wild type viruses circulating in sylvatic cycles, although regional variation is apparent (bryant and barrett 2003) . viral adaptation to the human h r appears in most cases to be critical to developing a virus with pandemic potential (mims 1991 (mims , 1995 . the introduction of avian-like gene segments into preexisting, aerosol-transmitted, human influenza a viruses, or alternatively, the introduction of key genetic components into preexisting avian viruses (see the chapter by webby et al., this volume) may be prerequisite to pandemic influenza a emergence (claas 2000) . the emergence of sars into the human population was accompanied by strong and rapid positive selection of different subtypes of virus as indicated by comparisons of sequence data from humans and from palm civets and rhinolophid bats, putative h r s, or intermediate hosts (h i s) for sars cov (lau et al. 2005; song et al. 2005 ; see the chapter by wang and eaton, this volume). to capture the rate at which outbreaks spread among hosts, epidemiologists have relied upon the reproduction potential, r 0 , as a measure of the expected number of secondarily infected and infectious hosts produced during the infectious period of a single infected host when introduced into a freely mixing population of susceptible individuals (halloran 1998) . the relative fitness defined by r 0 is a composite of three terms c , the contact rate or number of contacts per unit time, p , the transmission probability per contact, and d , the duration of infectiousness (see the chapter by real and biek, this volume). examples of zoonotic viruses taking alternative paths to emergence, with highly variable r 0 s are discussed below. the underlying feature distinguishing modifying factors (fig. 1 , right panel) from transition stages in zoonotic virus emergence (fig. 1, left panel) is that the former requires the substrate provided by the latter on which to act. modifying disease emergence can occur at the local and regional level or as a pandemic depending on the nature of the pathogen and the influence of modifying factors. modifying factors are largely responsible for driving the magnitude and geographic scope of an emergent event, but by themselves are insufficient to lead to disease emergence. although only a single population source for a zoonotic pathogen is indicated, the reservoir host (h r ) population, the schema is modular and readily accommodates inclusion of vector (h v ) populations and intermediate vertebrate host (h i or h s 1 ) populations antecedent to spillover to humans (see fig. 1 in the chapter by childs, this volume). transition stages, with the exception of contact (transition 1) and cross-species transmission (spillover; transition 2), are not strictly hierarchical in the emergence process. transition stages are shown to the left of the population boxes and the two transitions required for emergence (contact and spillover) are shaded gray. in the center are two population boxes, the top shaded box indicating a h r population, in which a zoonotic virus or some other zoonotic pathogen circulates, and the bottom shaded box indicating the secondary host population (h s ) affected by pathogen spillover (assumed in most instances to be humans). the graded shaded pyramid within the h s population indicates that emergence often proceeds through a gradient of human population sizes and social connectivity. spillover and human transmission chains in remote villages (apex of pyramid) can lead to spread to urban centers (base of pyramid), at which point a pathogen is assumed to have access to the entire h s population demarcated by the h s box. to the left of the population boxes are fig. 1 continued examples of modifying factors. contact and spillover are sufficient to result in disease emergence at the local, regional, or even continental scale through reiterative introductions, as exemplified by zoonotic diseases such as rabies or west nile fever. the two solid black lines with arrowheads leading from the h r then directly through the h s to local or regional emergence (the first emergence box) represent reiterative events as a pathway to emergence. two other transitions not essential for emergence, but critical to pandemic disease emergence, require sustained intra-h s transmission of the zoonotic pathogen (transition 3) and, potentially, adaptation to the human host (e.g., sars coronavirus). sequential human-to-human transmission of a zoonotic pathogen at the local and regional scales is indicated by the series of broken white lines in the h s pyramid. evolutionary forces can transform a zoonotic agent into a genetically distinct agent adapted to a new reservoir host, establishing the former h s as a new h r (e.g., siv to hiv; avian influenza to pandemic human influenza). this transition and the modifying factors associated with the geographical location and context of the initial disease outbreak (white arrows in shaded h s cone) can ultimately precipitate a pandemic emergence (e.g., hiv and pandemic influenza). the process of emergence for any zoonotic pathogen can fail at a minimum of three points, indicated by the labeled dashed lines with arrowheads leading out of the h s population box to the right. zoonotic pathogens may fail to initiate cross-species infection following exposures (top dashed line), fail to generate any additional infections within the h s (second dashed line), or experience epidemic fadeout when sustained human-to-human transmission fails and r 0 decreases below unity (third dashed line). the initial transition to sustained intra-h s transmission is prone to failure when populations are sparse or social connectivity is limited. (modified from childs 2004) factors alter the likelihood of a transition occurring and drive the geographic spread and determine the magnitude of morbidity and mortality resulting from a particular instance of emergence. abiotic factors alter the potential for contact between h r and h s populations, or infectious intermediaries, and modulate the potential for spillover; zoonotic diseases highly dependent on abiotic factors are often labeled environmentally driven epizootics (allen and cormier 1996) . on a global scale, climate change has been increasingly linked to instances of zoonotic disease emergence, with el nino southern oscillation (enso) providing the largest interannual signal of climate variation (wang et al. 1999 ). one hypothesized mechanism by which enso triggers increased incidence of zoonotic disease among humans, is through a chain of sequentially induced events referred to as a trophic cascade (polis et al. 2000) , ultimately leading to increased numbers of individuals among h r or h v populations and increasing the risk of human exposure to a zoonotic pathogen (nicholls 1986; kelly-hope et al. 2004; bi and parton 2003; glass et al. 2002; anyamba et al. 2001 ). enso events have been correlated with increased risk of hps and plague in the southeastern united states (glass et al. 2002; parmenter et al. 1999) , increased infection by ross river virus in australia (lindsay and mackenzie 1997; kelly-hope et al. 2004) , and arthropod-vectored bartonella bacilliformis and visceral leishmaniasis in south america (chinga-alayo et al. 2004; franke et al. 2002) . local weather conditions, potentially driven by global climate variation, have been repeatedly shown to influence the emergence of zoonotic and vector-borne viruses. drought can serve to amplify enzootic transmission of st. louis virus (shaman et al. 2002) and possibly japanese encephalitis (hanna et al. 1999; mackenzie et al. 2002) and ebola viruses (pinzon et al. 2004 ; see the chapter by gonzalez et al., this volume), ultimately placing humans at higher risk for spillover. the converse, excessive rainfall, can increase breeding populations of h v s, driving enzootic transmission levels of western equine encephalomyelitis virus, ross river virus, and rift valley fever virus to heightened levels, and ultimately increasing zoonotic virus spillover to humans (lindsay et al. 1993; wegbreit and reisen 2000; linthicum et al. 1999 ). intrinsic biotic and evolutionary factors enhance the ability of certain zoonotic viruses, notably those with rna genomes (cleaveland et al. 2001; dobson and foufopoulos 2001 ; see the chapters by cleaveland et al. and by holmes and drummond, this volume) , to cross species barriers. viruses with high replication rates, high mutation rates, and increased potential for recombination or reassortment may more readily adapt to new fitness landscapes and become transmitted among humans to emerge as pandemic threats (burke 1998; nichol et al. 2000) ; examples include hiv and subtypes of influenza a (hahn et al. 2000; claas, 2000 ; see the chapter by webby et al., this volume). the intrinsic genetic variability in susceptibility to infectious diseases within the human h s (segal and hill 2003) is further modulated by an individual's cumulative life experience and history of infection by various pathogens, reflected by acquired immunological memory or, possibly, an individual's ancestry and evolutionary imprint of prior exposure to pathogens (gillespie 1975; lipsitch and sousa 2002) . furthermore, immunologic function and the susceptibility of individual humans to infection and disease are dynamic and vary with factors such as nutritional status and age (boelle et al. 2004) . strong evolutionary forces may be in play in circumstances where zoonotically acquired viruses are intermittently maintained among small and sparsely distributed human populations where r 0 may hover close to unity. in theory, virus evolution is affected by socially structured host populations, such as where some human populations are aggregated in remote villages, with limited opportunity for social interchange. models of virus transmission which assume homogeneous or freely mixing populations are of limited use in such circumstances. in such settings, modest increases in the number of generations of human-to-human transmission sustained by a new virus prior to fadeout (fig. 1 , second terminal dotted line) improves the likelihood of virus evolution to a higher average r 0 , and hence emergence (antia et al. 2003) . additionally, sparsely distributed populations where contact rates, c , between infectious and susceptible individuals are low can support bistable evolutionary dynamics. one trend leads to the rapid evolution of increasingly virulent viruses. when viruses of relatively low virulence are transmitted among dispersed metapopulations of hosts, the result can be a cluster of infected individuals surrounding an index case, which is rapidly transformed into a semi-impermeable barrier of immune individuals (boots et al. 2004) , effectively terminating additional transmission. virulent viruses causing lethal infections leave no immune survivors to block transmission and, in the course of removing infected individuals, further enhance the sparseness of the existing social structure. in situations where viruses of relatively low virulence circulate, the introduction of a highly virulent virus strain, either through an infected immigrant or from viral recombination, can alter the evolutionary trajectory of virus-host adaptation favoring selection for increasing virulence and an alternative, evolutionarily stable situation (white et al. 2002; boots et al. 2004 ). extrinsic biotic interactions, such as natural or human-assisted translocations of infected or latently infected individuals of h r or h v species have played an exaggerated role in the rapid emergence of zoonotic diseases within the last few years. monkeypox transported with african rodents destined for the us pet trade (centers for disease control and prevention 2003; see the chapter by regnery, this volume), globe-trotting humans infected with sars-cov ; see the chapter by wang and eaton, this volume), domestic dogs incubating rabies accompanying human colonialists (see the chapter by nel and rupprecht, this volume), and the stowaway mosquito, bird, or human infected with wnv (lanciotti et al. 2002) bear witness to the growing problems of a shrinking interconnected world (see the chapter by daszak et al., this volume) . mosquito-borne viral diseases have resulted from the introduction of exotic viruses into indigenous local populations of mosquitoes previously not involved as vectors (lanciotti et al. 2002) , in addition to the establishment and spread of exotic mosquito species harboring viruses into new geographic locations (lounibos 2002; mackenzie et al. 2004 ). however, not all biological invasions or disease introductions survive to cause epidemics, as was the case with sars and monkeypox in north america. in contrast, wnv was an entirely different matter. the rapid establishment and spread of wnv in north america was nearly assured by the presences of indigenous species of competent wild bird h r s and mosquito h v s. certain bird species sustained wnv viremias of sufficient titer and duration to infect blood-feeding "bridge vectors" (turell et al. 2001; komar et al. 2003) , maintaining transmission to humans and spreading wnv as migrating birds followed traditional flyways (peterson et al. 2003) . the same extrinsic phenomena of a community of seemingly preadapted and widely available potential h v s and h r s in conjunction with the biogeography of avian migration aided the introduction and spread of wnv in europe and the middle east (malkinson and banet 2002) . by an alternate route of introduction, wind-blown infected mosquitoes may have introduced japanese encephalitis virus (jev) into northern mainland australia in 1998 (ritchie and rochester 2001) . as indicated above, many of the most important and widely cited factors modifying the scope and scale of zoonotic disease emergence are anthropogenic in origin; a few examples are described to highlight their importance and their distinctiveness from required transition stages. human population growth and modern agricultural practices have enticed human settlers into clearing patches within ecosystems of maximally high biodiversity, such as tropical rain forests, converting substantial areas into cultivated fields and pastures (patz et al. 2004; logiudice et al. 2003) . commercial farming operations inserted into clearings in forest habitats juxtapose and intermingle humans and livestock with native animal populations (kock et al. 2002; daszak et al. 2001 ; see the chapter by field et al., this volume) , and, coincidentally, with whatever zoonotic pathogens exist within these natural nidi (pavlovsky1957). in many instances, the cleared land has been used for irrigated agriculture, resulting in an increase in vectorborne diseases such as jev as mosquitoes and water bird h r s are brought in close proximity to domestic pigs in nearby villages (morse 1995; keiser et al. 2005) . dams are built to maintain water for human consumption and for use in irrigated agriculture, but they too may lead to increased zoonotic disease emergence as they provide the milieu for intermingling mosquito vectors and reservoir hosts of arboviruses as well as the spread of other diseases such as schistosomiasis. modern agricultural practices have also provided the mechanism by which bovine spongiform encephalopathy emerged in the united kingdom in the early 1980s (pattison 1998) . species now linked by domestication to homo sapiens provide rich fodder for evolutionary forays by zoonotic viruses into potential new hosts. the emergence of zoonotic viruses among humans or domestic livestock where our species has drifted into preexisting sylvatic foci of zoonotic viruses is driven by local circumstance, history, and serendipity. the role of livestock, such as horses and pigs, can be pivotal in a transmission chain leading to human infection, as illustrated by the henipaviruses (see the chapters by daniels et al. and field et al., this volume) . niv and hev first jumped the species barrier to infect pigs and horses, respectively, and only then were transmitted by these h i s to humans (barclay and paton 2000; chua et al. 1999 ). however, these two viruses also demonstrate the importance of transmissibility in the h i s influencing the ultimate emergence of human disease; niv was readily transmitted among pigs while hev was rarely transmitted among horses or from horse to human (see the chapter by daniels et al., this volume) . rabies virus associated with domestic dogs incubating infection and transported with humans was the likely source of endemic cycles of rabies involving most terrestrial mammals in north and south america and in many areas of africa (childs et al. 2002; smith et al. 1992 ; see the chapter by nel and rupprecht, this volume). in addition to causing an estimated 50,000 human deaths annually, rabies virus associated with domestic dogs have driven naïve indigenous populations of african wild dogs ( lycaon pictus ) and ethiopian wolves ( canis simensis ) to the threshold of extinction and caused declines among other large carnivore populations (roelke-parker et al. 1996; sillero-zubiri et al. 1996; gascoyne et al. 1993; chapman 1978 ; see the chapter by nel and rupprecht, this volume). other domesticated species have become efficiently enlisted as h i s or h r s, in a bridging process leading to human disease. swine production management practices have improved the efficacy of this economically important livestock species as an amplifying h i for jev and niv transmission to humans (daniels et al. 2002; singh and jamaluddin 2002; mohd nor et al. 2000 ; see the chapter by field et al., this volume) . swine may also serve as the mammalian mixing vessel for influenza a viruses of domestic and wild birds, offering the opportunity for avian viruses to obtain the complement of genes required for their sustained transmission within mammalian hosts, such as humans (suarez et al. 2002; gibbs et al. 2001 ; see the chapter by webby et al., this volume). significant changes in the demography of global human populations during the past five decades have been driven not only by population growth, but by changes in population distribution and social structuring brought about by migration, the ongoing movement of persons from rural to urban environments and the resettlement of refugees. the concentration of humans in the urban environment has given rise to mega-cities where a large proportion of persons may live in substandard conditions in marginal areas, sometimes referred to as shanty towns, surrounding the urban core. the crowded living conditions within shanty towns are further degraded by poor sanitation and lack of water; these conditions have been associated with the emergence of diseases, notably those involving vector-transmitted pathogens (gratz 1999; gubler 2002; mackenzie et al. 2004) . urban and periurban changes in land use have altered the availability and quality of habitat available to wildlife, and ecological changes in resource availability have in instances increased the potential for human-animal-vector interactions. later chapters illustrate how ecological changes have influenced the abundance and accessibility of wildlife species serving as reservoir hosts for different pathogens, leading to the emergence of zoonotic pathogens associated with pteropid bats (see the chapter by field et al., this volume) and white-tailed deer (see the chapter by paddock and yabsley, this volume). perhaps the most influential and certainly the most infamous anthropogenic modifiers driving the emergence process have been those enhancing social connectivity through road construction (larkin 2000) , railroads, and, the crown jewel of rapid modern transport, jet plane-assisted travel ( fig. 1; childs 2004 ; see the chapter by daszak et al., this volume) . nowhere has the role of rapid transportation been more evident than with sars cov (table 1 ) , where a presumed focus of human infection in the wet markets of guangzhou, guangdong province, china, where live animals or their products are available for purchase, was transformed into a global health problem affecting 27 nations on every populated continent (heymann 2004 ; see the chapter by wang and eaton, this volume). the human sars cov appears to have been inadvertently transported to a wet-market, along with an infected h i or h r , on a journey destined to end with human consumption (bell et al. 2004) . wildlife farming and an immense network of illegal national and international trade in wildlife has been fueled by human demands for wildlife products of unusual culinary or putative medicinal properties (bell et al. 2004 ). these cultural propensities enriched the range of opportunities for novel host/zoonotic virus interchange, but alone, as with rapid transport of persons, would not have resulted in a case of sars without the biological capabilities of the virus to readily establish spillover. modern medical practices requiring the widespread use of needles, increased application of immunosuppressive therapies, organ transplant, and blood transfusions have contributed substantially to the spread and emergence of zoonotic pathogens (institute of medicine 2003; see the chapter by paddock and yabsley, this volume). in certain exceptional instances, medical technology has permitted zoonotic viruses, generally limited in their capacity for humanto-human transmission, to flirt briefly with the transmission route prerequisite to pandemic emergence (fig. 1) . illustrative of this phenomena were instances of wnv and rabies virus transmission from infected donors to susceptible recipients receiving blood transfusion (wnv) and organ and tissue transplants the schema for emerging diseases (fig. 1 ) emphasizes viral interactions within the newly colonized secondary host, of which humans may be but one of several susceptible species (h s. . .n ). the process outlined is similar to the schema developed to characterize biological invasions by nonindigenous species (kolar and lodge 2001) . the transition states proposed for emerging diseases and those for invasive species are largely parallel: (1) contact with infectious propagules aligns with the nonindigenous species in a transport pathway to a foreign shore; (2) cross-species transmission aligns with the nonindigenous species surviving transport and being introduced into a foreign environment; (3) sustained intra-h s transmission of a zoonotic virus aligns with the establishment (self-perpetuation) of the invasive species within the new environment; and (4) sustained intra-h s transmission accompanied by evolutionary adaptation of the once zoonotic virus to a new h r , prior to emergence, aligns with adaptive radiation and spread of the invasive species beyond the local site of introduction (grant et al. 2001 ). the potential terminating points in the process of virus emergence or biological invasion (broken arrows leading outside of the h s population block in fig. 1 ) are consequences of similar circumstances. failure to cross the species barrier (spillover) aligns with "fails in transport"; failure to sustain transmission, with a transmission potential, r 0 <1, aligns with "fails to establish"; and interruption of sustained intra-h s transmission, an average r 0 <1, aligns with "noninvasion" by the nonindigenous species. differences between disease emergence and biological invasion exist, as transitions leading to disease emergence are not strictly hierarchical. reiteration of contact and spillover (fig. 1, transitions 1 and 2) at sufficiently high levels can suffice for a disease to emerge, but if an invading species never moves and perishes at the site of its introduction, even if repeatedly introduced to the site, further establishment and spread, prerequisites of invasion, is precluded. in addition to biological factors which establish the setting in which zoonotic pathogens may emerge (see the chapters by cleaveland et al. and daszak et al., this volume) , the emergence of a zoonotic agent within human or animal populations must be detected by humans. too often the presence of a zoonotic agent is first identified by the presence of disease in humans, and surveillance for disease emergence is largely restricted to identifying incident cases of disease in humans rather than monitoring infection or disease among wildlife h r s or h i s (see the chapters by childs, by merianos, and by stallknecht, this volume). the challenges present to designing programs aiming to disrupt transmission of a zoonotic pathogen within a wildlife reservoir host population prior to spillover and disease emergence are discussed in the chapters by childs and by stallknecht in this volume. altering the environmental unit being invaded produces a radically different schema. the invasion process in fig. 1 has an organismal or medical orientation, which can be transformed to a population or community orientation by regarding humans, rather than a zoonotic pathogen, as the invasive species. human invasion of new habitats and new environments is a frequently cited factor in the emergence process of viral zoonoses (morse 1995; institute of medicine 2003) . where native h r s and h v s and their co-evolved viral pathogens exist in natural foci (pavlovsky 1957) , enhanced opportunities for novel ecologic interactions await. initial instances of emergence have proven unpredictable as exemplified by hps resulting from transmission of hantaviruses maintained by sigmodontine rodent h r s in north america (monroe et al. 1999) , hiv resulting from transmission of sivs circulating among nonhuman primate h r s in west africa (apetrei et al. 2004) , and niv and hev viruses from pteropid bat h r s in asia and australia (field et al. 2001 ). herein, we stress the organismal-medical orientation, humans colonized or invaded by zoonotic viruses. human intrusion into novel environments is regarded as an anthropogenic factor which modifies the likelihood of contact and spillover transitions occurring. however, without the preexisting sylvatic zoonotic cycle, human invasion alone would not engender the first case of illness along the path to emergence. insights as to why and how certain zoonotic viruses appear predisposed to spillover and the various paths they take in the emergence process, are to be gleaned by examining the evolutionary history and current context of where and how zoonotic viruses exist and just how they become identified as etiologic agents of human disease (see the chapter by childs, this volume). predisposing biological characteristics include evidence of multiple h r s (dobson and foufopoulos 2001; cleaveland et al. 2001 ; see the chapters by cleaveland and by holmes and drummond, this volume), high replication rates, high mutation rates, and the potential for homologous or heterologous recombination, which reach maxima in zoonotic viruses with rna genomes (holland et al. 1982; arias et al. 2001) . two zoonotic viruses with histories of reemergence are rabies virus and wnv, both of which depend solely, with the exception of rare instances mentioned above, on repetitive contact and spillover between infected h r s or infected h v s (wnv) for their transmission to the human h s . although the rna genomes of these two zoonotic viruses are markedly different in terms of organization, polarity, and replication strategy, both viruses show evidence of reduced positive selection , even where established within novel h r s or h v s . the term "evolutionary generalists" has been applied to both viruses as they share, to some extent, the requirement of being able to infect and multiply within cells belonging to different species and, in the case of vector-borne wnv, the need to infect and multiply within avian, mammalian, and insect h r s, h v s, and h s s. the rare instances of human-to-human transmission of these viruses are epidemiologically insignificant (dietzschold and koprowski 2004; iwamoto et al. 2003) . although humans are, with few exceptions, incidental hosts for zoonotic viruses emerging from sylvatic transmission cycles, a few zoonotic arboviruses can be maintained by human-to-human transmission mediated by anthropophilic vectors in urban settings where large populations of humans and competent h v s coexist, particularly in environments with poor sanitation and overcrowding. yellow fever virus (wolfe et al. 2001; de silva et al. 1999) and dengue virus serotypes (kuiken et al. 2003; ksiazek et al. 2003 ) are arboviruses where major epidemics are associated with urban transmission cycles rather than sporadic spillover from sylvatic h r s and h v s, and dengue serotypes have become endemic among some suitably large human populations in asia (gubler 2002) . rabies virus crosses mammalian orders and species and can establish sustained transmission within new h r s (badrane and tordo 2001) , as has been observed on several occasions where bat-associated variants of rabies virus have achieved temporary sustained transmission among terrestrial carnivores, such as red foxes ( vulpes vulpes ) and striped skunks ( mephitis mephitis ) (daoust et al. 1996; engeman et al. 2003) . the maintenance of rabies virus, considered a single species of lyssavirus , serotype 1/genotype 1, is achieved as a myriad of distinct viral variants maintained within different specific mammalian h r s, rather than a homogeneous virus infecting multiple h r s; control or elimination of rabies in a specific h r may be achieved but the diversity of host-virus dyads is a formidable buffer against any overall elimination scheme. epidemics of rabies virus are sustained when there are sufficient individuals of the primary h r (s) to sustain intra-h r transmission, with coincidental spillover to h s s by reiterative introductions by inoculation of infectious virus in saliva. as rabies is fatal among most mammalian species, population declines among the principal h r generally coincide with declines in incidental rabies epizootics among h s s (gordon et al. 2004; wandeler et al. 1974) . epizootics can reemerge at periodic intervals as h r populations recover above the critical threshold density ( k t ) required to sustain virus transmission at r 0 >1 (anderson et al. 1981; childs et al. 2000; coyne et al. 1989) . in an analogous manner, epidemics caused by wnv involve reiterative introductions of infectious virus by any of a number of competent mosquito h v (s). wnv readily infects at least three classes of vertebrates (avia, mammalia, reptilia) and mosquito species, and some species of ticks (gould et al. 2003; komar et al. 2003; lvov et al. 2004; sardelis et al. 2002; turell et al. 2001) . although wnv appears to be reasonably homogeneous in regions of north america over time, geographic clustering of genetically similar strains is detectable and certain epizootiologically dominant genetic clades have emerged, some with shorter extrinsic incubation periods within north american vectors ebel et al. 2004 ). subtleties associated with host-vector-virus relationships are being uncovered, such as the greater frequency of flavivirus recombination among mosquito h v s compared to tick h v s (twiddy and holmes 2003) . the temperature and humidity requirements for survival and breeding of mosquito vectors, and the demand for temperature-sensitive extrinsic viral incubation within h v s, drive the strong seasonal transmission dynamics of wnv and other arboviral diseases. with the onset of cold temperatures in temperate zones, wnv transmission ceases and epidemics of human disease desist (woodring et al. 1996) . that sars-cov is new to science is not in question. however, the origin of sars-cov as a human pathogen arising from direct cross-species transmission of a preexisting, but previously unknown virus (gibbs et al. 2004; holmes and rambaut 2004 ; see the chapters by holmes and drummond and by wang and eaton, this volume) or as a virus formed from the recombination of existing mammalian and avian coronaviruses (rest and mindell 2003; zhang et al. 2004 ) has been the subject of debate. sars-cov is sufficiently distinct in genetic sequence from previously known coronaviruses ) that a long history of preexistence with its natural h r population is surmised (parashar and anderson 2004) . recently, coronaviruses related to sars-cov have been amplified by pcr from three communal cave-dwelling species of the genus rhinolophus in the family rhinolophidae. genome sequence analysis indicated that sars-like coronaviruses among these bats have an almost identical genome organization to those of sars-covs isolated from humans or civets ; see the chapter by wang and eaton, this volume). these data suggest that bats serve as the h r of sars-cov and that palm civets served as a h s1 in a chain of events leading to infection of humans as secondarily infected h s2 . sars-cov's global emergence may be an extraordinary example of a relatively unmodified zoonotic virus, successfully sustained by intrahuman transmission. however, increasing data suggest sars-cov was a virus rapidly adapting to its new human host and the rapid and effective public health response terminating its transmission halted an evolutionary dramas in the making (see the chapter by wang and eaton, this volume). genetic sequence data indicate that strong positive selection accompanied sars-cov's emergence and that distinctive human-associated changes in the genome distinguish virulent sars-cov from isolates of virus from palm civets (song et al. 2005) . additional data indicate genetic changes were accompanying longer chains of human-to-human transmission. heterogeneous viral sequences recovered from a single patient's samples indicate the degree of viral variation available for selection within the individual human. these findings are compelling evidence of evolutionary events underway, presaging the emergence of a virus with a unique genetic signature associated with its human host. whatever the exact origin of sars-cov, the genetic endowments of this virus facilitated cross-species infection. an evolutionary history that includes viral preadaptation permitting infection to occur among a broad range of h r s and h s s is suggested by sars cov's ability to infect a range of mammalian orders (ng 2003; song et al. 2005; bell et al. 2004) . such preadaptation can be assumed to have endowed sars-cov with a suite of traits readily adaptable for establishing sustained intrahuman transmission (riley et al. 2003; isakbaeva et al. 2004) . pathogenesis within the novel human host's respiratory tissues offered an efficient means for sustained transmission by expressed droplets, or possibly aerosol . the biological properties of sars-cov, the human behaviors and societal practices which increased the likelihood of contact and spillover and the rapid transport of already infected individuals drove the trajectory of the emergence of this global health problem. the distinctions and critical interactions between required biological transitions and modifying factors are clearly illuminated by this emergence process (fig. 1, table 1 ). fortunately, sars-cov was effectively controlled by tried and true public health measures serving to increase social distance and diminish infectious contacts, c , perhaps curtailing a rapid evolutionary path toward a r 0 sufficiently high to bypass these methods (song et al. 2005; antia et al. 2003; fraser et al. 2004) . the zoonotic viruses leading to potentially uncontrolled, pandemic health problems have adopted unique qualities associated with their sustained transmission within the human host. adaptation to the human host may be mediated by viral preadaptation to a genetically similar intermediate host, as is hypothesized to occur among swine for avian-adapted influenza a subtypes (see the chapter by webby et al., this volume). permissive cells for subtype a influenza virus replication in the pig's respiratory track have cell-surface glycoprotein receptors recognized by some avian-adapted viruses, in addition to some human-adapted influenza viruses (basler et al. 2001 ). in the case of hiv, the h r for sivs giving rise to hiv-1 was our closest living genetic relative, the chimpanzee ( pan troglodytes troglodytes ) (gao et al. 1999) , and the h r for hiv-2 was a sooty mangabey ( cerocebus atys ) (hirsch et al. 1989 ), a respectfully close relative of the order primates. spillover was enhanced by the enormous population of candidate viruses within the genetically heterogeneous "quasispecies" of viruses present in the infected h r at spillover. sustained intrahuman transmission was accompanied by viral adaptations to the human host, readily detectable and quantifiable by sequence changes in the rna genome and marked by qualitative phenotypic changes identifiable by host species preference and pathogenic interactions (hirsch et al. 1989; gao et al. 1999 ). evaluations of the genetic relatedness of hiv-1 and hiv-2 to sivs circulating among nonhuman primates has led to wide acceptance that these distinctive human lentiviruses, now globally distributed in humans, originated from cross-species transmission in the not so distant past, perhaps within the first half of the twentieth century (may et al. 2001) . the human lentiviruses, hiv-1 and hiv-2, have evolved and escaped from remote african settings on at least eight independent occasions to emerge as distinctive genetic subtypes responsible for regional or pandemic human disease (apetrei et al. 2004; b. hahn, personal communication) . these recognized cases of emergence are certainly not the first instances where sivs have successfully crossed species and evolved as distinctive hivs of humans. early emergences were likely restricted to local occurrences in remote locations where human contact rates, c , and population size were insufficient to support a r 0 > 1, even if infections were of a long duration, d ; such occurrences would be highly prone to transmission fadeout (fig. 1) . road construction, automotive transport, and, perhaps, reuse of nonsterile needles (gisselquist 2003) were presumably key anthropogenic factors increasing the level of social connectivity by providing hiv-infected individuals access to larger aggregates of susceptible hosts in cities (apetrei et al. 2004 ). the number of sivs described among nonhuman primates in africa as of 2004 was approximately 40 (zhuang et al. 2002) . the biological capacity of lentiviruses includes rapid replication, high mutability, and the highest recorded rates of recombination known in virology. knowledge of the frequency of potential opportunities for siv spillover, based on transmission of a zoo of diverse simian foamy viruses during encounters between monkeys, apes, and human hunters in west africa ) indicate transmission of blood-borne retroviruses is not rare. these facts highlight two important features of emergence; first, emergence is a process, not an event; second, the probability of new genetic lineages of human hivs arising approximates unity. the same lessons apply to numerous other conditions which make up the body of this volume. environmentally driven epizootics a case of severe monkeypox virus disease in an american child: emerging infections and changing professional values population dynamics of fox rabies in europe the role of evolution in the emergence of infectious diseases climate-disease connections: rift valley fever in kenya the history of sivs, aids: epidemiology, phylogeny and biology of isolates from naturally siv infected non-human primates (nhp) in africa molecular intermediates of fitness gain of an rna virus: characterization of a mutant spectrum by biological and molecular cloning host switching in lyssavirus history from the chiroptera to the carnivora orders hendra (equine morbillivirus) sequence of the 1918 pandemic influenza virus nonstructural gene (ns) segment and characterization of recombinant viruses bearing the 1918 ns genes animal origins of sars coronavirus: possible links with the international trade in small carnivores el nino and incidence of hemorrhagic fever with renal syndrome in china epidemiological evidence of higher susceptibility to vcjd in the young large shifts in pathogen virulence relate to host population structure comparative phylogenies of yellow fever isolates from peru and brazil evolvability of emerging viruses multistate outbreak of monkeypox-illinois investigation of rabies infections in organ donor and transplant recipients rabies: decimation of a wolf pack in artic alaska zoonotic viruses of wildlife: hither from yon effects of hantaviral infection on survival, growth and fertility in wild rat ( rattus norvegicus ) populations of baltimore maryland predicting the local dynamics of epizootic rabies among raccoons in the united states public health surveillance and the molecular epidemiology of rabies the influence of climate on the epidemiology of bartonellosis in ancash peru fatal encephalitis due to nipah virus among pig-farmers in malaysia pandemic influenza is a zoonosis, as it requires introduction of avianlike gene segments in the human population diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence mathematic model for the population biology of rabies in raccoons in the mid-atlantic states japanese encephalitis virus cluster of rabies cases of probable bat origin among red foxes in prince edward island canada anthropogenic environmental change and the emergence of infectious diseases in wildlife genetic variation among temporally and geographically distinct west nile virus isolates united states serologic evidence for an epizootic dengue virus infecting toque macaques ( macaca sinica ) at polonnaruwa sri lanka rabies transmission from organ transplants in the usa emerging infectious pathogens of wildlife history of epidemiological aspects of yellow fever identification of a novel coronavirus in patients with severe acute respiratory syndrome genetic and phenotypic variation of west nile virus population monitoring in support of a rabies vaccination program for skunks in arizona the natural history of hendra and nipah viruses impact of the el nino/southern oscillation on visceral leishmaniasis brazil factors that make an infectious disease outbreak controllable origin of hiv-1 in the chimpanzee pan troglodytes troglodytes rabies in african wild dogs ( lycaon pictus ) in the serengeti region tanzania the phylogeny of sars coronavirus the haemagglutinin gene, but not the neuraminidase gene, of 'spanish flu' was a recombinant natural selection for resistance to epidemics emergence of the hiv type 1 epidemic in the twentieth century: comparing hypotheses to evidence satellite imagery characterizes local animal reservoir populations of sin nombre virus in the southwestern united states two rabies deaths after corneal grafts from one donor west nile virus update: a new route of transmission is found temporal dynamics of rabies in a wildlife host and the risk of cross-species transmission origins, evolution, and vector/host coadaptations within the genus flavivirus a population founded by a single pair of individuals: establishment, expansion, and evolution emerging and resurging vector-borne diseases the global emergence/resurgence of arboviral diseases as public health problems aids as a zoonosis: scientific and public health implications concepts of infectious disease epidemiology japanese encephalitis in north queensland the international response to the outbreak of sars in wounding: the primary mode of seoul virus transmission among male norway rats an african primate lentivirus (sivsm) closely related to hiv-2 rapid evolution of rna genomes viral evolution and the emergence of sars coronavirus genetic constraints and the adaptive evolution of rabies virus in nature b-virus ( cercopithecine herpesvirus 1) infection in humans and macaques: potential for zoonotic disease microbial threats to health; emergence, detection, and response transmission of west nile virus from an organ donor to four transplant recipients effect of irrigated rice agriculture on japanese encephalitis, including challenges and opportunities for integrated vector management el nino southern oscillation and ross river virus outbreaks in australia wildlife and pastoral society--shifting paradigms in disease control progress in invasion biology: predicting invaders experimental infection of north american birds with the new york 1999 strain of west nile virus a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states complete genome sequences and phylogenetic analysis of west nile virus strains isolated from the united states hunting and logging linked to emerging infectious diseases severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats climate and satellite indicators to forecast rift valley fever epidemics in kenya bats are natural reservoirs of sars-like coronaviruses vector-borne viral diseases and climate change in the australian region: major concerns and the public health response ross river virus isolations from mosquitoes in arid regions of western australia: implication of vertical transmission as a means of persistence of the virus historical intensity of natural selection for resistance to tuberculosis sars transmission pattern in singapore reassessed by viral sequence variation analysis histopathology of peromyscus leucopus naturally infected with pathogenic ny-1 hantaviruses: pathologic markers of hps infection in mice the ecology of infectious disease: effects of host diversity and community composition on lyme disease risk invasions by insect vectors of human disease west nile virus and other zoonotic viruses in russia: examples of ermerging-reemerging situations japanese encephalitis as an emerging virus: the emergence and spread of japanese encephalitis virus in australasia emerging flaviviruses: the spread and resurgence of dengue japanese encephalitis and west nile viruses the role of birds in the ecology of west nile virus in europe and africa infectious disease dynamics: what characterizes a successful invader the origin of major human infections and the crucial role of personto-person spread virology research and virulent human pandemics nipah virus infection of pigs in peninsular malaysia yellow fever genetic diversity and distribution of peromyscus -borne hantaviruses in north america factors in the emergence of infectious diseases virus morphology, replication, and assembly possible role of an animal vector in the sars outbreak at amoy gardens emerging viral diseases a method for predicting murray valley encephalitis in southeast australia using the southern oscillation transmission of the severe acute respiratory syndrome on aircraft severe acute respiratory syndrome: review and lessons of the 2003 outbreak incidence of plague associated with increased winter-spring precipitation in new mexico the emergence of bovine spongiform encephalopathy and related diseases unhealthy landscapes: policy recommendations on land use change and infectious disease emergence human diseases with natural foci (translated from russian by d rottenberg) migratory birds modeled as critical transport agents for west nile virus in north america trigger events: enviroclimatic coupling of ebola hemorrhagic fever outbreaks when is a trophic cascade a trophic cascade sars associated coronavirus has a recombinant polymerase and coronaviruses have a history of host-shifting transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions wind-blown mosquitoes and introduction of japanese encephalitis into australia a canine distemper virus epidemic in serengeti lions ( panthera leo ) characterization of a novel coronavirus associated with severe acute respiratory syndrome vector competence of three north american strains of aedes albopictus for west nile virus genetic susceptibility to infectious disease drought-induced amplification of saint louis encephalitis virus florida rabies and mortality in ethiopian wolves ( canis simensis ) nipah virus infection in swine epidemiologic and historical relationships among 87 rabies virus isolates as determined by limited sequence analysis cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human isolation from turkey breeder hens of a reassortant h1n2 influenza virus with swine, human, and avian lineage genes hemorrhagic fever with renal syndrome: clinical aspects vector competence of north american mosquitoes (diptera: culicidae) for west nile virus the extent of homologous recombination in members of the genus flavivirus rabies in wild carnivores in central europe. i. epidemiological studies el nino and the related phenomenon southern oscillation (enso): the largest signal in interannual climate variation relationships among weather, mosquito abundance, and encephalitis virus activity in california: kern county 1990-1998 epidemiological consequences of a pathogen having both virulent and avirulent modes of transmission: the case of rabbit haemorrhagic disease virus reduced positive selection in vector-borne rna viruses sylvatic transmission of arboviruses among bornean orangutans naturally acquired simian retrovirus infections in central african hunters natural cycles of vector-borne pathogens evidence of airborne transmission of the severe acute respiratory syndrome virus hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease testing the hypothesis of a recombinant origin of the sars-associated coronavirus human immunodeficiency virus type 1 recombination: rate, fidelity, and putative hot spots key: cord-318061-xe8lljz0 authors: overgaauw, paul a.m.; vinke, claudia m.; van hagen, marjan a.e.; lipman, len j.a. title: a one health perspective on the human–companion animal relationship with emphasis on zoonotic aspects date: 2020-05-27 journal: int j environ res public health doi: 10.3390/ijerph17113789 sha: doc_id: 318061 cord_uid: xe8lljz0 over time the human–animal bond has been changed. for instance, the role of pets has changed from work animals (protecting houses, catching mice) to animals with a social function, giving companionship. pets can be important for the physical and mental health of their owners but may also transmit zoonotic infections. the one health initiative is a worldwide strategy for expanding collaborations in all aspects of health care for humans, animals, and the environment. however, in one health communications the role of particularly dogs and cats is often underestimated. objective: evaluation of positive and negative one health issues of the human–companion animal relationship with a focus on zoonotic aspects of cats and dogs in industrialized countries. method: literature review. results: pets undoubtedly have a positive effect on human health, while owners are increasing aware of pet’s health and welfare. the changing attitude of humans with regard to pets and their environment can also lead to negative effects such as changes in feeding practices, extreme breeding, and behavioral problems, and anthropozoonoses. for the human, there may be a higher risk of the transmission of zoonotic infections due to trends such as sleeping with pets, allowing pets to lick the face or wounds, bite accidents, keeping exotic animals, the importation of rescue dogs, and soil contact. conclusions: one health issues need frequently re-evaluated as the close human–animal relationship with pet animals can totally differ compared to decennia ago. because of the changed human–companion animal bond, recommendations regarding responsible pet-ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conforming the biology of the animal are required. education can be performed by vets and physicians as part of the one health concept. the one health initiative or concept is a worldwide strategy that recognizes that public health is connected with animal health and the environment. it concerns multidisciplinary collaboration between physicians, veterinarians, environmental scientists, public health professionals, wildlife experts, and many others [1, 2] . with a multisectoral and transdisciplinary approach, public health threats can be better monitored and controlled. the resulting synergism enhances the knowledge of how diseases, known as zoonotic diseases, can be shared between animals and people with the goal of this article is based on an existing post-graduate course for veterinarians, vet technicians, family doctors, midwifes, and specialists such as pediatricians which explores healthy human-animal relationships. the existing evidence-based knowledge contained in this course has been actualized by performing a literature search to add new relevant publications. a literature search was conducted through 2 march 2020, using the national library of medicine's pubmed for the terms "one health" and "companion animals"; "pet ownership"; "households" and "pets"; "dogs" or "cats" or "pets" and "mental" or "physical health" or "children"; "animal assisted therapy"; "dogs" or "cats" and "nutritional problems" or "overweight" or "obesity" or "homemade" or "raw meat diets"; "dogs" or "cats" and "behavior problems" or "aggression" or "fear" or "anxiety" or "abnormal repetitive behavior"; "dogs" or "cats" and "breeding" or "genetic problems"; "dogs" or "cats" and "zooanthroponoses"; "pets" and "anthropomorphism"; "dogs" or "cats" or "exotic animals" or "rescue dogs" or "soil" and zoonoses. for some topics the internet was accessed and used as reference if additional information was not available as scientific publication. the authors selected articles that described pivotal and novel insights in the different topics. all searches were carried out without filters. the titles of all found articles were screened for relevance to the topic, and appropriate titles were assessed and selected based on their abstracts. if a selected article was a review, it was read and relevant citations were used to find primary literature on the subject. additional studies were found using the bibliographies of selected articles. occasionally, reviews were directly used as sources, mostly to convey background information that is not in the core focus of this article. original articles in english and different national languages (dutch, german, french, spanish, if available) were included. specific searches were made for citations dated after the year 2000 to ensure more recent literature on the topic had not been missed. a pet or companion animal is an animal that lives in or around the house and is fed and cared for by humans. until the 1960s, pets were mainly kept as utility animals, for example as draft dogs or watch dogs or for pest control when it comes to cats. due to major changes that have taken place in society since the second world war, such as increased leisure time and prosperity, but also individualization of humans, animals are nowadays kept as pets and are regarded by many owners as valued family members, e.g., over 90% in the united kingdom [13] . pet ownership is still increasing in many industrialized countries and these animals are more often considered a member of the family [5] . even in china, a country where pets were banned in urban areas until 1992, pet ownership has grown quite rapidly in the major cities. the rate of pet ownership of all usa households increased from 57.6% to 59% in 2018. dogs continue to dominate in popularity among american households. approximately 38% of households nationwide owned a dog, bringing the population of pet dogs to nearly 77 million, while 25% of households owned cats, with a total population of 58 million [14] . in 2018, an estimated 80 million european households owned at least one pet animal; 24% of households owned dogs and 25% owned cats [15] . there are 85 million pet dogs and 104 million pet cats in europe which is a 15% increase of dogs within 8 years (74 million dogs in 2010) and a 22% increase of cats (85 million in 2010) [16] . pet cats in europe thereby are more popular than dogs. an explanation of the higher popularity of cats may be the number of single-person households in the eu that rose on average by 2.0% per annum between 2006 and 2016 to 32.5%, and the growth of two-family households grew by 1% to 31% in this period [17] . also, dual-earner families are widespread as a result of quite a steep growth in female employment over the past two decades [18] . when animals live with humans, they too benefit from human interaction. over the past decades, animal welfare has evolved to recognize that animals are sentient beings capable of experiencing positive and negative emotions. the social and ethical dimensions of animal welfare, which are concerned with how human society morally regards and treats non-human animals, are also increasingly being recognized [19] . in dutch law, the intrinsic value of kept animals is expressly incorporated and used as a guiding ethical principle that forms the basis of any further legislation. the intrinsic value is thereby defined that animals are sentient beings that can feel pain and discomfort. therefore, they should be kept free of stress, pain, disease, hunger, thirst, and should be able to show natural behavior known as the five freedoms of r. brambell 1965 [20] . in industrialized countries animal keepers, their owners, are legally required to provide such circumstances and can be prosecuted if they infringe the law. of course, in the field there is animal abuse, negative animal welfare conditions, and animal diseases. however, in general, caretaking for companion animals is nowadays performed at a high level. new insights into animal behavior has had its influence on the general public. for example, owners are aware or are told by vets and pet shops that rabbits should not be kept alone but at least in pairs due to their need for social contact [21] . there are various reasons to keep pets, such as love, warmth, and companionship. companion animals have an important emotional value, and promote the socialization of the lonely elderly because they facilitate additional contact with people. pets form a goal in life, reduce stress, and ensure that the owner keeps physically active. around 95% of dog owners and 93% of cat owners expressed that owning their pet makes them happy and 44% of owners selected this as one of the reasons they got their pet in the first place [22] . however, the function of companion animals consists of more than just providing a socializing being. studies show other benefits of having a pet, such as the positive effect on individuals' mental and physiological health status. most research addressing the health benefits of pet ownership show reductions in distress and anxiety, decreases in loneliness and depression, and increases in physical condition [23] . the positive benefits to human health from interacting with animals, focusing on the companion animal, have also be described with the term "zooeyia" [24] . in fact, 63% of owners agreed that having a pet makes them physically healthier, with dog owners more likely to agree, most probably because dog owners exercise more (85% agreeing, compared to 41% of cat owners). besides, 84% of owners agreed that having a pet makes them mentally healthier. expressed reasons are the non-judgmental nature of their pets, their playfulness, or physical contact [22] . another demonstrated positive influence is the blood pressure and heart rate lowering effects that occurs when stroking a friendly-looking dog or even being in the presence of a friendly animal, while it is not necessary to own a pet to obtain these stress-moderating benefits [23] . many studies have demonstrated the association between pet ownership and cardiovascular health and dog owners appear to have a significantly greater chance of survival after a heart attack compared to people without pets [25] [26] [27] . pets can therefore play an important role in reducing absenteeism and visits to family doctors or the hospital [28] . it has been estimated that pet ownership saved australia $1 billion in 1994 [29] , while it may reduce the use of the national health service (nhs) in the uk to the value of £2.45 billion per year [30] . dogs also play an increasing role as co-therapist or as supporter for people with psychological or physical disabilities. the benefits of these animal-assisted activities are improved mood, decreased physiological distress, depression, dementia, and loneliness [31, 32] . examples include resident or visiting dogs in prisons, nursing homes [33] , mental institutions, and hospitals where they can reduce patient anxiety in a hospital emergency department [34] , reduce pain perceptions in children after surgery [35] , or calm young patients at a pediatric dental clinic [36] . since dogs have extremely sensitive noses, they are used for several purposes such as tracking, bomb detection, and search and rescue. in recent years, canine olfaction has also been more recognized as a diagnostic tool for identifying pre-clinical disease status, such as diabetes (ketones), different forms of cancer, and infections from biological media samples [37] . animal-assisted therapies can act as co-therapies to facilitate psychotherapy or to provide specific types of therapeutic interventions such as improving motor skills or behavior [23] . such interventions were effective in improving the state of children or adults with or at risk of developing mental disorders such as attention deficit hyperactivity disorder (adhd), post-traumatic stress disorder (ptsd), or autism spectrum disorder (asd) [38] [39] [40] , and for the treatment of ptsd in military veterans [41, 42] . assistance or service animals are trained to perform tasks for the benefit of individuals who have disabilities such as hearing loss, physical disabilities, emotional disabilities, seizure disorders, or diabetes [23] . finally, a wide range of emotional health benefits from childhood pet ownership has been identified, particularly for those suffering from low self-esteem and loneliness. there is evidence of an association between pet ownership and educational and cognitive benefits, increased social competence, social networks, social interaction, and social play behavior [43, 44] . significantly less absenteeism from school through sickness among children who live with pets has also been reported [13] . having a dog or cat in the house during the first year of life may protect against childhood asthma and allergy [45, 46] . it can therefore be concluded that companion animals contribute significantly toward the public health, but also increasingly, the health of individually challenged persons through animal-assisted interventions [47] . providing companion animals with feed by humans, has been considered an advantage for companion animals in their relationship with humans. however, the feeding practices can also have a negative impact on companion animals [48, 49] . obesity in cats and dogs is a disease which is rapidly increasing with significant and lifelong implications for animal welfare. although no universally accepted definition of canine and feline obesity exists, the american veterinary medical association defined obesity being more than 30% above the ideal weight of an animal. overweight is defined as 10%-20% above the ideal weight. using body condition scores, it has been estimated that in the united states, 54% of dogs and 59% of cats are obese or overweight. a study in the united kingdom reported 65% of adult dogs and 37% of juvenile dogs as being obese or overweight [50, 51] . overweight dogs are more likely to be diagnosed with, e.g., urinary tract diseases. obese and overweight dogs are at risk developing orthopedic disorders and hypothyroidism [52, 53] . obese cats are at higher risk for developing urinary tract disease, diabetes mellitus, and neoplasia [54, 55] . other diet-related problems in companion animals can be caused by the changed feeding behavior of humans, e.g., by providing companion animals with bone and raw feed (barf) or vegan diets. risks for companion animals associated with barf or vegan diets are the presence of microbial hazards, insufficient nutrition, and in raw meat diets the presence of risk materials like thyroid tissue. through contact with their animals, it is possible that risks could even develop for owners. there could be an increased risk of human salmonellosis because of the presence of salmonella spp. in the diet which can spread to humans through diet leftovers or by contact with animal feces. recently a review was published on the risks of barf feeding [56] . the authors concluded that the data for the nutritional, medical, and public health risks of raw feeding are fragmentary, but they are increasingly forming a compelling body of formal scientific evidence. publications were found reporting the presence of escherichia coli o157, salmonella typhimurium, campylobacter spp., and antibiotic resistant bacteria in the feed. nutritional problems, such as calcium/phosphorous imbalances and specific vitamin deficiencies [57] are also reported. moreover, homemade diets are inherently susceptible to nutritional imbalances and deficiencies [58] . awareness about climate change, public health and animal welfare has incited a major change in dietary choices among many individuals. the number of vegans in the world keeps growing, even quadrupling from 150,000 to 600,000 individuals between 2014 and 2019 in affluent countries such as the uk [59] . the popularity of veganism goes beyond the scope of the human diet, as more people are interested in the possibility of feeding their companion animal a vegan diet than ever before. to create animal-free complete cat food requires replacing nutrients in animal-based materials with plant-based materials. different sources are used such as corn, rice, peas, soy, potato, and different oils and seeds. any further nutrients that are missing from plant-based materials, such as taurine and carnitine, are replaced with synthetically produced versions [60, 61] . feeding trials using vegan animal food are either not performed due to testing costs or kept private due to the highly competitive vegan pet food market [62] . additionally, they reported testing 24 vegetarian diets for cats and dogs and found that one was lacking protein and six did not meet all amino acid concentration requirements. vegan animal food may not contain meat, but it does contain grains, soy, and corn. plant-based products, such as grains, can be a source of health problems because of the presence of mycotoxins, for example [63] . warm, humid storage conditions can lead to the formation of mycotoxins such as aflatoxins, produced by the fungi aspergillus flavus and aspergillus parasiticus. many regular animal feeds also contain plant-based products, therefore the negative impact of feeding vegan diets to companion animals, especially obligate carnivores such as the cat or the ferret, seems therefore more related to diet insufficiency than to microbial health risks. additionally, addressing behavioral problems, the "free" provision of food might fulfil the consumptive part of feeding behavior of our companion animals but does not fulfil the appetitive phase. especially this phase of feeding patterns can have consequences for the companion animals' mental health and may result in behavioral problems if appetitive physical and mental challenges remain chronically absent in the human-animal relationship. in the human-companion animal bond, pets may develop abnormal behavior, including excessive aggression, fear and anxiety, or even abnormal repetitive behavior. abnormal repetitive behavior (arbs) were first noticed in zoo-, shelter-, and laboratory animals: all animals housed under stimulus-poor conditions and with limited space. however, companion animals can develop arbs as well, if the individual's adaptive capacity is exceeded due to, e.g., a lack of social contact, physical exercise, mental challenges, and in uncontrollable and unpredictable environments (e.g., separation, mistreatment, or inadequate application of cages). arbs can either be classified in stereotypies or compulsive disorders [64] with stereotypies generally defined as unvarying repetitive behavior patterns with no obvious goal or function [65] . the terminology of compulsive disorders is preferably chosen for repetitive behavior patterns that are goal-directed and show variability in the repetitive (motor) patterns [66, 67] . under chronic conditions without possibilities to adapt (cope), companion animals may develop stereotypies or compulsive disorders like, e.g., tail chasing, polyphagia, compulsively self-directed licking and/or biting the coat [68] , or feather pecking in parrots [69] . self-directed patterns can result in serious degrees of alopecia, lick granuloma, or even self-inflicted injuries (auto-mutilation) with a risk of infection. two main reasons underlie the development of arbs in our companion animals. first of all, a lot of companion animals are social species eager for social contact. in the human-companion animal bond, the need for social contact with either conspecifics and/or humans [70, 71] can remain unfulfilled if owners work from nine to five, five days a week with the pet staying alone at home on a daily basis. on the other hand, a cat which is originally a solitary hunter with a complex dynamic social structure may start overgrooming or house soiling in the presence of another cat in the territory. such situations might occur in multi-cat households, in the presence of neighboring cats, and in in-stable grouped housing conditions in shelters [72, 73] . secondly, most companion animals are species that are eager for mental and physical challenges on a daily basis. the lack of foraging opportunities, the appetitive phase of feeding behavior [74] might be another reason for the possible development of arbs in companion animals. foraging is often regarded as a high priority behavior [75, 76] , i.e., an internally motivated behavioral pattern that should be performed, or otherwise may induce a state of chronic stress, which may result in behavioral pathology like arbs as described in many other animal species [77] [78] [79] . foraging patterns may include walking, running, jumping, nose pushing, digging, and overseeing the area, all active patterns that imply the daily need for physical exercise and mental challenges in most of our companion animals. nonetheless, our pets mostly, if not always, get their food for free with minimal foraging challenges, except for going out 3-5 times a day. for some individuals (and especially some dog breeds, e.g., malinois, border collies, and pit bull terriers) [80, 81] , situations and contexts with limited challenges can make them more vulnerable to the development of arbs. as well as arbs, other problematic behavior may develop in our companion animals, and the prevalence of some of this behavior is even higher than that of arbs, for example excessive interspecific and/or intraspecific aggression, fear, and anxiety. at what moment, and which type of problem behavior may develop, depends on the intermingled factors of, e.g., genetics, early life experiences (maternal-child bonding, weaning, socialization [82] ), daily environment, and multiple factors in and around the human-companion animal bond. the history of breeding animals goes back to a time when humans and animals shared each other's habitat. dogs originally have been selectively bred to support human needs, such as hunting, herding, obedience, guarding, rescuing, and for companionship. this artificial selection has generated a large number of dog breeds, displaying a large variation of behavior, size, head shape, coat color, and coat texture [83, 84] . unfortunately, in the last 100 years, intensive selection for extreme looks and a narrow gene pool of many breeds has interfered in the genetic make-up of dogs, leading to unfavorable anatomy (extreme large, or extreme "teacup" small), and genetic predisposition to numerous health, welfare, and behavioral problems [85] . over 700 inherited disorders and traits have already been described in the domestic dog [86] . one type of dog with a distinct dysmorphology is the brachycephalic dog. brachycephalic dogs are characterized by a large head and round face due to a shortened muzzle, a high and protruding forehead, and widely spaced large eyes. these facial features fit the concept of baby schema ("kindchenschema") proposed by konrad lorenz [87] . infantile (cute) faces are biologically relevant stimuli for rapidly and unconsciously capturing attention and eliciting positive or affectionate behavior, including the willingness to care [88] . the appeal of brachycephalic animals has led to specimens that are the so-called "over-typed" dogs and cats with a too short nose, excessively protruding eyes, too straight angulations, etc. breeding animals with this type of severe skull and muzzle abnormalities leads to physical and physiological hardship and limits their natural behavior [89, 90] . this violates their integrity and is a big risk for their welfare. selectively breeding animals in order to express specific traits does not only alter existing animals, but also creates new ones, turning animals into an instrument for human use [91] . the bambino sphynx cat is an example of so called "mutant breeding", where breeders deliberately stack in two steps the recessive inheriting mutations, which leads to hairlessness in sphynx cats, on the dominant inheriting (lethal) mutation responsible for the shortened legs of the munchkin cat. the lack of hair in combination with short legs interferes with the normal physiology of the cat with regard to the manner of movement, thermoregulation, and skin health. one may argue that artificial selection in exchange for money, status, or aesthetic reasons violates the animal's dignity and integrity [92] . conclusively, artificial selection for excessive traits can have direct consequences for individual health and welfare, may obstruct and prevent a pet from fulfilling its behavioral needs, and conflicts with the current moral way of thinking on animal dignity and integrity. pet animals are not only perceived in 80%-90% as family members or partners but are almost treated like humans. in one study, up to 62% of owners agreed with the statement "my dog is more important to me than any human being" [93] . this kind of behavior is the result of the attribution of human cognitive processes and emotional states to animals, such as feelings of happiness, love, or guilt. people believe that animals have awareness, thoughts, and feelings. this behavior is called anthropomorphism, personification, or humanization and can also be applied to plants, gods, or objects. anthropomorphism appears to be caused by the perceived similarity between humans and animals and the extent to which people have developed an affectionate bond with their dogs and cats [94] . human empathy provides the basis for the attribution of empathy to other animals, as well as attributions of the communicative ability of other animals [95] . anthropomorphistic behavior can be harmless, such as talking to pets, which many owners do and one of the reasons for this may be the unique ability of humans to recognize facial expressions. talking to pets is also found to be linked to social intelligence [94] . however, it can lead to animal welfare problems when the feelings of owners no longer match the needs and the intrinsic values of their animal. examples of this are designer dog clothes, animal perfumes, and jewelry, thought the use of protective coats in colder climates for small, short-haired breeds, e.g., chihuahuas, is considered useful. the large number of obese pets can also be partly attributed to anthropomorphism. studies from north america, europe, and australia to determine what proportion of animals, mainly dogs, are overweight or obese reported prevalences of between 22%-44% [96] . in 2018, an estimated 60% of cats and 56% of dogs in the usa were overweight or obese [50] . when the owner takes a treat with coffee, it is believed that the dog should also get it. even chocolate treats are given, when these are potentially fatal for dogs and cats. this also applies to a good meal that is shared with the pet. dog owners who did not consider obesity to be a disease, maybe because the facial features of their pets fit the concept of baby schema [87] , were more likely to have obese dogs [97] . an often-unrecognized risk for pets is reverse zoonotic disease transmission, the so-called zooanthroponosis. a review on this subject reported 56 articles dealing with human to animal disease transmission [98] . most of the articles dealt with bacterial pathogens but also viral, parasitical, and fungal pathogens were studied in these publications. animals reported to have been infected or inoculated with human diseases included wildlife, livestock, companion animals, and other animals or animals not explicitly mentioned. the majority of the studies focused on human to wildlife transmission with an emphasis on mycobacterium spp. for companion animals, mrsa-infection was especially reported but m. tuberculosis, influenza a, and candida albicans were also discussed. for all groups of animals, microsporum spp. and trichophyton spp. were identified as infectious agents originating from humans [99] . recent publications report a different kind of zooanthroponosis: the transmission of high-risk, multidrug-resistant pathogens from humans to animals [100] . a major issue mentioned is the transmission of high-risk clones of extended-spectrum beta-lactamase (esbl) producing bacteria including escherichia coli, enterobacter cloacae, and klebsiella pneumonia [100, 101] . the transmission of carbapenem-resistant ndm-5 producing e. coli from previously hospitalized humans to dogs has also been suggested [102] . transmission of hospital acquired antibiotic resistant bacteria from human patients to their pets has been confirmed, such as the vim-2 producing pseudomonas aeruginosa st233 strain in brazil [103] . this increased transmission of high-risk multidrug-resistant pathogens from humans to animals was related to the closer relationships between humans and companion animals. some authors doubt the generalized pet-effect on human mental and physical health because of conflicting results that are prevalent in this area of science and the lack of publication of negative results [13, [104] [105] [106] . the majority of research evidence was also considered inconclusive due to methodological limitations such as reliance on self-reports, small sample sizes that may not be representative of the general population, homogeneous populations, varying research designs, narrow range of outcome variables that were examined, and the use of cross-sectional designs that do not consider long-term health outcomes [107] [108] [109] . other studies found for example that pet ownership was associated with a higher incidence of heart attacks and readmissions in heart attack patients instead of a lower incidence [110] or that pet owners had higher diastolic blood pressure than those without pets [111] . müllersdorf (2010) showed that pet owners had better general health but suffered more from mental problems such as anxiety, insomnia, and depression, than those who did not own pets [112] . other studies failed to support earlier findings that pet ownership is associated with a reduced use of general practitioner services [113] or psychological or physical benefits on health for community dwelling older people [114] . negative effects of pet ownership include dog and cat bites or scratches, the spreading of disease (zoonoses), and fall injuries, caused by falling or tripping over dogs and cats [115] . allergic reactions may be a consequence of animal contact and affect 15%-30% of individuals (often genetically) predisposed [116] . allergies relating to more uncommon pets such as fish, birds, and amphibians seem to be increasing in prevalence [117] . other studies prove that pet ownership in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis symptoms in children aged 6-10 years. therefore, advice to avoid or to specifically acquire pets for primary prevention of asthma or allergic rhinitis in children should not be given [118] . there are also less-positive effects that pets can have on health. more excessive forms of anthropomorphism became clear in our study for the presence of zoonotic parasites in healthy dogs and cats. fifty percent of owners allow pets to lick their faces. sixty percent of the pets visit the bedroom; 45-60% (dogs-cats) are allowed on the bed, and 18-30% (dogs-cats) sleep with their owner in bed. six percent of pets always sleep in the bedroom. of the cats, 45% are allowed to jump onto the kitchen sink [119] . this means that in addition to the detected zoonotic parasites (the hazard), there was a significant potential exposure to these pathogens. in addition to parasites, other pathogens such as bacteria, viruses, and fungi can also be transmitted by animals by direct contact through biting, licking, scratching, sneezing or coughing, handling pets or their body fluids or secretions and by indirect contact through contaminated bedding, food, water, or bites from an arthropod vector [120] . not every individual will develop symptoms after being infected with a zoonosis. this is the result of various factors such as the causative pet species, housing, the degree of contact and contamination, the ability of a micro-organism to cause disease in humans and animals, but especially due to the degree of immunity of the recipient. in order to assess the risk of disease transmission from pets it is important that the nature and frequency of contacts between pets and their owners or other people are evaluated [120] . we traditionally know that young children (age < 5 years), the elderly (age ≥ 65 years), patients with an impaired immunity, and pregnant women that carry a fragile fetus are at more than average risk of becoming ill after an infection. moreover, they may have more severe disease, have symptoms for a longer duration, or develop more severe complications compared to other patients. young children (notably those aged 3-5 years) and some people with developmental disabilities often have suboptimal hygiene practices or higher risk contact with animals which further increases risk [121] . in children, hand-to-mouth behavior is part of their natural development and they mouth their fingers and other objects. in a meta-analysis, the average indoor hand-to-mouth frequency ranged from 6.7 to 28.0 contacts/hour and the average outdoor frequency ranged from 2.9 to 14.5 contacts/hour. the lowest value was attributed to the 6-to 11-year-olds and the highest to the 3-to < 6-month-olds [122] . fifteen percent of dog owners and 8% of cat owners always wash their hands after contact with the animals [119] . in addition, due to improved healthcare in recent decades, the group of immunocompromised patients has increased sharply. this includes, for example, patients with diabetes, post-splenectomy, after placement of implants and patients being treated with chemotherapy or immunosuppressants. the risk groups are also referred to as yopis (young, old, pregnant, and immune suppressed). patient surveys and epidemiological studies suggest that the occurrence of pet-associated zoonotic disease is low overall [121] . many of these pathogens are not reportable and presumably underdiagnosed or not recognized by family doctors due to the general, mostly flu-like, symptoms. therefore, any reported frequency of such infections is likely underestimated. to get a better picture of zoonotic risks, a risk analysis is required where a risk score can be calculated using exposure, contagiousness of the infection, and its consequences in the human. in addition, the disease burden of an infection for the population can be calculated and expressed in disability adjusted life years (dalys). this quantifies the health loss based on two components: the life years lost due to premature death and secondly the proportional loss of quality of life as a result of the disease. there is a trend towards closer physical contact between owners and their pets or their environment which poses an increased risk of transmission of zoonotic pathogens. these trends (a general direction in which something is developing or changing) will be explained. our publication [119] showed that a high percentage of pets were allowed in the bedroom and in bed, with 6% always sleeping in bed with their owner. is that a problem? already from a hygienic point of view, it is not advisable to sleep with animals or take them into bed. they do not pay attention to where they are walking outside and do not wipe their feet after arriving home. dogs like to roll in carcasses and both dogs and cats regularly lick the anus and thereafter the fur. in a pilot study with 28 healthy dogs and 22 healthy cats that slept with their owners, we tested 68% of the dogs (19) and 32% of the cats (7) positive for enterobacteriaceae on the fur or footpads. fleas and flea larvae were found on 14% of pets [123] . as a result of our publication, chomel investigated in 2011 whether transmission of infections by sleeping with pets and licking the face could be found in literature. he reported that also in the usa, france and the uk, a relatively large number of pets slept in bed with their owner (14%-33% of dogs and 45%-60% of cats) [124] . similar results of sleeping with pets were also reported from canada (26% of pets slept with children), the czech republic (45%), and qatar (63.3%) [125] [126] [127] . chomel found bacterial infections such as yersinia pestis (plague), bartonella henselae (cat scratch disease), methicillin-resistant staphylococcus aureus, and sometimes fatal bite wound infections such as capnocytophaga canimorsus and pasteurella multocida. furthermore, parasite infections such as cheyletiella spp. were reported. feline cowpox is a rare viral infection, but it can be transferred to the human after direct contact. both animals and humans reveal local exanthema on arms and legs or on the face. in most cases the disease is self-limiting, but immunosuppressed patients can develop a lethal systemic disease resembling smallpox [128] . it can be concluded that, although uncommon with healthy pets, the risk of transmission of zoonotic agents by close contact between pets and their owners through bed sharing is real and has even been documented for life threatening infections such as plague [129, 130] . although pets do not transmit arthropod-borne diseases to people (e.g., lyme borreliosis, ehrlichiosis, anaplasmosis), they do bring zoonotic disease vectors such as ticks and fleas, in close proximity to people, e.g., when they are sleeping with their animals [121] . while fleas are considered a vector of bartonella henselae (the causative agent of cat scratch disease) tickborne diseases are reported as increasing as ticks expand their ranges [131, 132] . with an estimated 65,000 cases a year, lyme borreliosis is responsible for the largest disease burden of any vector-borne disease in the european union [133] . another increased risk associated with close contact with fur is when it is contaminated with zoonotic parasite eggs. especially with echinococcus multilocularis (fox tapeworm) or e. granulosus (hydatid worm, or dog tapeworm). these eggs are immediately infective and may cause serious health problems in the human, many years post infection [134, 135] . despite a low prevalence of infectious (embryonated) eggs of toxocara spp. on dog's fur, the potential zoonotic risk should not be disregarded [136] . the same risk is applicable for the persistence of sporulated toxoplasma gondii oocysts in dogs' fur [137] . in relation to this, it is noteworthy that many publications report striking increases of ringworm, a common zoonotic fungal skin infection in mainly children caused by microsporum spp., trichophyton spp. or arthroderma spp., where the presence of pets is always mentioned [94] . however, nowhere has it been suggested that close contact with infected pets in bed increases the infection risk [138, 139] . rodents or rabbits are mainly infected with t. mentagrophytes, while m. canis is primarily found in dogs and cats. infection occurs by direct or indirect contact with infected hair, scales, or materials. infected animals may be asymptomatic carriers without clinical signs. examples are 16% m. canis carriage in a study of european cats [140] , 27% in suspected brazilian cats [141] , and the isolation of t. mentagrophytes dermatophytes from 4% of clinically healthy rabbits and 17% of guinea pigs in dutch pet shops [142] . licking the face of humans by mainly dogs is an expression of their naturally submissive, positive social behavior. the owner is recognized by the dog as the dominant superior in the ranking. in a pack of dogs, submissive dogs lick their dominant counterparts at the corners of the mouth from a typical submissive attitude [143, 144] . owners apparently allow this as a token of affection from their pet. such behavior is more common in young animals and has been considered as attention-seeking or care-soliciting gestures. it indicates to the owner the strength of the social bond between dogs and people [120] . licking or nudging of veterans by service dogs may help take their mind off any negative thoughts, emotions, or memories that they might be experiencing [145] . on the internet, many images can be found of mainly dogs, but also cats and even rats licking their owner's face. various studies show that around 40%-50% of owners allow this [119, 120] . the question is whether this is harmful due to the potential transmission of infections. the review by chomel (2011) shows in the literature that infections, especially pasteurella spp. and capnocytophaga canimorsus, were reported to have transmitted to humans by dogs, cats, kittens, and rabbits [124] . pasteurella multocida meningitis has been reported in 36 infants where 87% had been exposed directly or indirectly to the oropharyngeal secretions of household dogs or cats through licking or sniffing [146] . zoonotic transmission via this route is also assumed for various other pathogens such as gastric helicobacter spp. [147] , periodontal pathogens [148] , and bartonella henselae the etiological agent in cat scratch disease. the b. henselae bacteria may cause ocular complications, including parinaud oculoglandular syndrome, a severe eye infection. the route of infection is unknown, although direct conjunctival inoculation, most likely with infected flea feces, seems to be most plausible [149] . knowing, however, that b. henselae is present in up to 40% of cat saliva [150] , it is more plausible that salivary fluid could be rubbed directly into the eye from the skin after been licked by a cat. there are several anecdotal reports of infections in mainly young children that were transmitted by being licked. one recent example is an 8-month-old baby that presented with fever and preseptal cellulitis with purulent discharge. the causative agent was surprisingly corynebacterium bovis, a bacterium that is normally found in bovine mastitis. it became clear that the dog was frequently allowed to lick the baby's face and was fed on raw meat [151] . there is an ineradicable belief among a large part of the public that the licking of human wounds by dogs can disinfect them and that the saliva thereby has healing properties [152] . in addition, it is regularly reported that a dog's tongue is believed to be sterile. this is of course not the case and the oral flora of a dog comprises hundreds of species (including pathogenic) bacteria, fungi, and viruses [153, 154] . various wound healing saliva components have indeed been demonstrated in human and animal studies [155, 156] . the bactericidal effects of male and female dog saliva facilitate the hygienic function of maternal licking of the mammary and anogenital areas by protecting newborns from fatal coliform enteritis caused by e. coli and neonatal septicemia caused by streptococcus canis. however, the saliva is only slightly, and non-significantly, bactericidal against wound bacteria such as coagulase positive staphylococci and pseudomonas aeruginosa [157] . capnocytophaga canimorsus and pasteurella multocida are common commensals in the oral cavity of dogs, cats, and other species [158, 159] . transmission has been reported after the licking of mucous membranes or open wounds [160] [161] [162] . in patients at high risk, severe wound infections, sepsis, disseminated intravascular coagulation, or death can occur. patients with immunodeficiency, splenectomy, or alcohol dependence are at a particularly increased risk of infection with c. canimorsus [159] . even immunocompetent persons who have been licked by a dog can develop fatal sepsis [163] . a further negative effect of companion animal ownership is, of course, accidents inflicted on humans by these animals. these accidents can involve tripping over a cat or being dragged by an enthusiastic dog, but in literature, most evidence points towards biting and scratching incidents. dog biting and cat scratching incidents can cause physical health problems both at the time of infliction but also afterwards by triggering trauma-related secondary infections. dog biting incident reports are numerous, and numbers vary from country to country. in the uk, 18.7 dog bites per 1000 population per year were reported [164] , while a commission in the netherlands reported in 2008, 150,000 bite accidents per year in a population of 16 million (9 events per 1000 inhabitants) [165] . children are especially vulnerable to dog bites. the majority of dog bites occurred in children 5 years of age or younger (68.0%) and almost all (89.8%) of the dogs were known to the children [166] . recently, a systematic review has been published that analyzed more than 26,000 bites from the literature of the past 30 years about the risk of bites relative to specific breeds of dogs, combining bite incidence with bite severity [167] . the analysis by breed revealed that pit bulls were responsible for the highest percentage of reported bites across all studies (22.5%), followed by mixed breed (21.2%), and german shepherds (17.8%). dog bite incidents can result in medical treatment, hospitalization and even death. in the netherlands it was calculated that from the 150,000 dog bite victims per year, around 50,000 seek medical attention and 230 are hospitalized [165] . between 3% and 30% of dog bites become infected and complications become more severe when infection occurs. more than 100 species of bacteria have been isolated from bacterial infections of dog bites, suggesting that most oral flora of dogs have the potential to be pathogenic [168] . the top 3 pathogens found are pasteurella, staphylococcus, and streptococcus. in literature, specific attention is given to wound infections caused by capnocytophaga canimorsus, because this bacterium is seen as the relatively deadliest pathogen. it was suggested that only 2% of dog bite wounds contained capnocytophaga spp. [154] while others reported infection percentages of 4.2% [169] . wound infection with this bacterium can lead to severe complications like septicaemia, meningitis, osteomyelitis, peritonitis, endocarditis, pneumonia, purulent arthritis, and disseminated intravascular coagulation. c. canimorsus septicaemia has been associated with 30% mortality. the true number of c. canimorsus infections is probably largely underestimated due to the fastidious growth of the organism. however, infected dog bites in predisposed persons should be taken seriously especially after splenectomy [170] . cat bite incidents occur less frequently. in only 5%-10% of reported bite incidents in australia, cats are to blame. the long incisor teeth inflict less severe superficial wounds but because of the penetrating effect, joint and tendon infections more easily occur. in a review it is reported that 28%-80% of cat bites become infected mostly by pasteurella multocida [169] . the bacterium bartonella henselae can also be transmitted by cats through biting incidents but the transmission of this bacterium is much more related to a cat scratch accident. even less frequently reported animal biting incidents are those inflicted by rodents (2% of cases in australia). these bites have an infection rate of approximately 10%. this could result in rat bite fever in humans, an infection with streptobacillus monoliformis or spirillum minus, characterized by the triad of fever, rash, and arthritis [169] . cat scratch disease (csd) was first described in a french boy in 1931 and is a common, often self-limited, disease that usually presents as tender lymphadenopathy caused by bartonella henselae [171] . the cat is considered the primary reservoir for this bacterium, with infected fleas and ticks serving as vectors and humans and dogs as accidental hosts. vector transmission of this bacterium occurs via two primary routes: inoculation of bartonella contaminated arthropod feces via animal scratches, most often cat scratches, or by self-inflicted contamination of wounds induced by the host scratching arthropod bites [172] . immunocompromised human hosts (kidney transplant patients or patients with hiv) are especially susceptible to infection. in these individuals, the disease may be present as a more disseminated form with hepatosplenomegaly, meningoencephalitis, or angiomatosis [173] . an increasing number of pocket pets and exotic pets are kept by humans. numbers of ornamental birds in europe are estimated as 50 million, fish tanks 15.5 million (300 million ornamental fish), small mammals 27 million, and reptiles 8 million [15] . these animal species can also be a source of many zoonotic diseases, especially in young children and immunocompromised individuals. most cases of these conditions are not serious, and deaths are very rare but some of these diseases can be life threatening, such as rabies, rat bite fever infections, and plague [174] . however, there is also a trend to keep more unusual exotic animals, legally or illegally. these are "wild" animals that are kept in the home such as bats, foxes, skunks, raccoons, meerkats, prairie dogs, kinkajous, sloths, monkeys, apes, prosimians (mammals), parrots, mynah birds, finches (birds), crocodiles, turtles, tortoises, lizards, snakes (reptiles), frogs, toads, newts, salamanders (amphibians), fish, eels, rays (fish), crabs, crayfish, snails, insects, spiders, and millipedes (invertebrates) [175] . even fruit bats are kept as pets [176] and it is known that bats harbor a higher proportion of zoonoses than all other mammalian orders, including rabies-like viruses that are highly pathogenic for people. [177] . another example of a zoonosis is monkeypox following the importation of prairie dogs in the usa [178] . most reptiles and amphibians such as turtles, lizards, and frogs carry salmonella bacteria in their gut, in most cases without visible signs of infection. the infection may cause symptoms of sickness, diarrhea and fever in humans [179] . zoonotic transmission of salmonella infections causes an estimated 11% of salmonellosis annually in the united states. in cases involving pet turtles, almost half (45%) of infections occurred in children younger than 5 years [180] . salmonella infections are often transferred by feeder rodents [181] and outbreaks highlight the importance of improving public awareness and education in countries who receive imported reptiles [182] . it is advised to exclude reptiles, amphibians, rodents, exotic species, baby poultry, and raw animal-based pet food items from the households of patients at high risk. in lower risk households, an understanding of the risk of salmonellosis and other pet-associated zoonoses and preventive hygiene measures is needed. the pet trade in general, with its high turnover and diversity of available species, creates a reservoir for pathogens originating from all over the globe. reptiles account for approximately 10% of live animal shipments imported to the united states [182] . importation of live reptiles and amphibians for commercial purposes is for a great part unregulated at eu level except cites and customs regulations [176] . in a risk assessment study, the five pathogens with the highest public health risk caused by the import of exotic animals were salmonella spp., crimean-congo hemorrhagic fever virus, west nile virus, yersinia pestis, and arenaviruses. the risk via legally imported animals was considered low, but substantial for illegally imported animals due to the unknown health status of the animals [183] . avoiding exposure to exotic pet pathogens in the home is difficult and best achieved by not keeping them in the first place. otherwise, it is advised to always wash the hands immediately and thoroughly after contact with exotic pets and after handling raw (including frozen or defrosted) mice, rats and chicks. children should be supervised so that they do not put their mouths close to or kiss exotic animals. reptiles and other animals should be kept out of rooms in which food is prepared and eaten. the number of abandoned and homeless dogs and cats in europe is estimated to be over 100 million. countries with more than 1 million abandoned animals are italy, romania, russia, and ukraine [184] . there is an increasing trend to rescue and import dogs from countries with stray animal problems, in europe often from southern or eastern europe and in the us from puerto rico, the dominican republic, mexico, the middle east, turkey, china, and korea [185] . many charities and independent groups are involved to rescue dogs and seek adoption in more animal-friendly countries [186] . new owners primarily choose to adopt from abroad based on a desire for a particular dog they had seen advertised and on concern for its situation, while some were motivated by previously having been refused dogs from local rescues [187] . in the usa 85% of all household dogs were neutered and today there are no longer enough dogs being born in the usa annually to replace the approximately 8 million dogs that die each year. developing countries have hundreds of millions of street dogs available for export, for example egypt has an estimated 15 million, india, 30 million, and afghanistan, 100 million [188] . in 2006, more than a decade ago, the centers for disease control (cdc) estimated annual dog imports at around 287,000. today the number of imported dogs is estimated to be more than a million a year [189] . imported dogs are reintroducing diseases and parasites that were previously eliminated in the usa [190] . in the usa new and lethal strains of distemper and canine influenza as a result of imported rescue dogs were reported as well as canine brucellosis, rabies, and vector-borne diseases like ehrlichiosis, heartworm, babesiosis, and leishmaniasis [188, 191] . in many southern european countries, there is also a risk of exposure to diseases not encountered in the northern, importing, countries. animals could be infected with anaplasma phagocytophilum, babesia canis, brucella canis, borrelia burgdorferi, dirofilaria immitis (heartworm), dirofilaria repens (subcutaneous worm), echinococcus multilocularis (fox tapeworm), echinococcus granulosus (hydatid or dog tapeworm), ehrlichia canis, hepatozoon canis, leishmania infantum, linguatula serrata (tongue worm), onchocerca lupi, rabies, rickettsia conorii, strongyloides stercoralis, and thelazia callipeda. except b. canis, e. canis, and h. canis, the infections are zoonotic and regularly reported [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] [202] . most of these are transmitted by ticks, sand flies, or mosquitoes that are non-endemic in the receiving countries, but a reservoir of infections has been created and the risk is that vectors will become present as result of climate change. [203] . animals that are infected with rabies or echinococcus spp. may infect people directly. the importation of dogs from endemic, predominantly mediterranean, regions to northern europe, as well as travelling with dogs to these regions carries a significant risk of acquiring an infection. pet owners are therefore advised not to travel with dogs and to seek the advice of their veterinarian prior to importing a dog from an endemic area or travelling to such areas [193] . for this reason, esccap developed maps on their website featuring european countries and regions with advice on endemic parasites, diseases and recommended treatments when travelling with dogs [204] . a three-year european union funded project entitled callisto (companion animal multisectorial interprofessional and interdisciplinary strategic think tank on zoonoses), has investigated zoonotic infectious diseases transmitted between companion animals and humans and food producing animals [176] . the committee advised that special attention should be given to stray cats and dogs. stray dogs, in particular, may pose serious health and welfare problems for humans and animals [81] , including the transmission of zoonotic diseases such as rabies. consideration should be given to controlling companion animal movement between areas of the eu endemic for particular zoonoses and areas that are not currently endemic for that disease. reliable figures are not available, probably because of the fact that many voluntary organizations are responsible for saving and transporting these rescue animals. such data are essential in order to be able to quantify the actual risks of zoonotic diseases attributable to companion animals and to develop sustainable interventions to prevent transmission to humans and livestock [176] . as long as there are no official guidelines, to prevent the spread of (zoonotic) diseases to new countries, the time, expense, disease risk, and the implications of adopting a dog from abroad should all be carefully considered before importation. as an alternative, the conditions for native dogs could be improved by supporting local charities to organize neutering campaigns and rehoming programs, to build local animal shelters and to improve attitudes towards dogs and their living conditions [205] . cats and dogs harbor the enteric nematodes toxocara canis and toxocara cati, and cats are the final host for the protozoal parasite toxoplasma gondii. these parasites can be transmitted to humans because they have an oral-fecal transmission cycle. humans can be infected by ingestion of infective toxocara spp. eggs or toxoplasma oocysts from contaminated soil (gardens, sandpits, and playgrounds) [206, 207] . a recent meta-analysis of data from published records indicates that public places are often heavily contaminated with a pooled global prevalence of toxocara eggs of 21% [208] . both parasites are considered by cdc as part of five neglected parasitic infections. these infections are considered neglected because relatively little attention has been devoted to their surveillance, prevention, and/or treatment. the diseases that they cause have been targeted as priorities for public health action based on the number of people infected, the severity of the illnesses and the ability to prevent and treat them [209] . tens of millions of people worldwide are estimated to be exposed to, or infected with, toxocara spp. and recent findings suggest that the effect of toxocarosis on human health is increasing in some countries. almost one fifth (19%; 1.4 billion individuals) of the world's human population is seropositive to toxocara. the highest seroprevalence rates were found in africa (mean: 37.7%) and the lowest in the eastern mediterranean region (mean: 8.2%) [210] . toxocara larvae migrate into the body of the human to several organ systems with a preference for the central nervous system (brain, eyes). human toxocarosis can manifest itself as syndromes known as visceral larva migrans, ocular larva migrans, neurotoxocarosis, and covert or common toxocarosis [211] . asthma is one of the most common chronic respiratory diseases worldwide, with a negative impact on the quality of life and socio-economic status of patients. two decades ago, the first evidence was published that suggested that toxocara infection is a neglected risk factor for childhood asthma [212] . the finding that children infected with toxocara spp. are more likely to have asthma compared to non-infected children was recently confirmed in a systematic review and meta-analysis [213] . cognitive or developmental delays in children or young adults who become infected is of particular concern. toxocarosis appears to be associated with decreased cognitive function [214, 215] . the annual toxoplasma oocyst burden measured in community surveys has been reported as up to 434 oocysts per square foot (4670 per square meter) and is greater in areas with loose soil, that cats like to use to cover their feces in gardens, children's play areas, and especially sandboxes, also called sandpits and sand piles [207] . because a single oocyst can possibly cause infection, this oocyst burden represents a major potential public health problem. an estimated one third of the world's population harbor anti-toxoplasma antibodies. due to keeping pigs indoors, more education and awareness, the prevalence of the disease in the usa and europe declined by 50% over the last decades [216] . during an acute invasion of toxoplasma parasites there is mild to major tissue damage without clinical symptoms (latent toxoplasmosis). the most important form is congenital toxoplasmosis when a woman receives her first exposure to toxoplasma during pregnancy. in early pregnancy, this can lead to abortion or to malformations that are not compatible with life shortly after birth. congenital infections may also be characterized by mental retardation and ocular defects. acquired infection after birth may result in clinical symptoms such as lymphadenitis, fever, and malaise and probably leads to a clinically symptomatic disease state more frequently than the congenital condition, with an estimated incidence of 30% of all ocular toxoplasmosis cases [216] . playing in a sandbox is also found to be a predominant risk factor for s. typhimurium salmonellosis in children aged 4-12 years [217] . this can be the result of fecal contamination of the sand by dogs and cats that have been fed raw meat (see section 5.1.1.). young children are especially at risk as they put their hands or other objects in their mouths every 2-3 min [208] . it has also been reported that children ingest a median of 40 mg of soil per day and that one child consumed 5-8 g of soil per day on average [218] . it is therefore advisable not to let children play in public places or on playgrounds with loose sand, but only in sandboxes that can be covered. furthermore, washing hands after playing outside is important and fingernails must be trimmed to prevent sand being left behind. in this context, a strange trend can be observed as young children play in mud baths on 29 june "as a way to connect and celebrate the natural joys of playing in the mud". this international mud day originated in 2008 and was initiated by an australian pedagogue who had observed this phenomenon during a visit to nepal [219] . if the origin of the soil needed for producing the mud is unknown, there is of course an infection risk for the above discussed parasites. to prevent the transmission of zoonotic diseases from pets, risk analysis is of great value. this starts with an assessment of the potential zoonoses in an area, depending on the endemicity (the hazard, h). hazard characterization also includes prevalence in animals (the reservoir), virulence for man, transmission routes, and survival of the agent in the environment. the second step is exposure assessment (e). who is exposed to the potential hazard and for how long or how often? how much of the potential pathogen is needed to become a health risk? this inevitably is directly related to human behavior in relation to their pets. the third step is to assess the impact of getting infected (i). how serious is the disease, what is the chance of complications, and what economic consequences may be expected (e.g., labor hours lost)? each of the parameters can be ranked in classes from negligible to the most serious possibility. ranking is based on literature data, own observations (measuring), or experts' opinions. the final risk assessment can be achieved by multiplying the outcome of hazard characterization, exposure assessment and impact (h × e × i = a number). the outcome can be compared with other zoonotic agents and a ranking of significance can be made [220] . there is one important parameter to reduce the risk of contracting a zoonotic infection that can directly be influenced, which is exposure. recommendations are particularly targeted to households with very young children, the elderly, pregnant women, or immunocompromised patients. they are based on reducing exposure to hazards and involve four categories of advice (table 1) . table 1 . recommendations to prevent the transmission of zoonotic pathogens from pets [121, 221] . • washing the hands thoroughly -after animal contact, at least before eating and drinking and before preparing food or drinks -after handling raw pet food -after handling pet habitats or equipment -after cleaning up feces -after removing soiled clothes or shoes reflecting on the changed human-companion animal bond, it can be concluded that pets undoubtedly have a positive effect on human health. conversely, the human-pet bond seen nowadays is facing many challenges, putting pet welfare under more pressure due to issues such as anthropomorphism, which mainly results in obesity, breeding on extreme appearance rather than health, behavioral problems connected to unfulfilled species specific mental and physical needs, and the provision of inadequate food because owners mistakenly think they feed more naturally. with regard to the negative effects of pets this article attempts to give an impression of increasing trends in the human-companion relationship that can be observed in society, which appears to increase the risk of transmission of infection between pets and humans. it is mainly a consequence of the increasing contacts between humans and pets and with pathogens secreted by animals in the shared environment. more than 6 out of every 10 known infectious diseases in people can be spread from animals, and 3 out of every 4 new or emerging infectious diseases in people come from animals [222] . the recent pandemic of the covid19 coronavirus (sars-cov-2), that may be originated from bats, is a good example of a recent emerging zoonotic infectious disease. a few cats and dogs have tested positive but are not considered as a source of infection for people. the proportion of zoonotic human disease that is attributable to pets is largely unknown. reports about the frequency of such infections are likely underestimated [120] ; however, the risk of infection is relatively small for many zoonoses and the severity of the disease is often limited. a person's age and health status may affect their immune system, and thereby increase his or her chances of getting a disease from animals. pregnant women should avoid contact with pet rodents, reptiles, cat feces, and raw meat to prevent infection of the unborn child, abortion, or birth defects. if symptoms occur in immunocompetent, non-pregnant persons between 5 and 64 years of age, these are mainly of a general nature such as diarrhea or flu-like symptoms. physicians do not regularly ask about the presence of pets or pet contact, nor do they discuss the risks of zoonotic diseases with patients, regardless of the patient's immune status, which means that many cases of zoonotic diseases go undiagnosed. the general public and people at high risk of pet-associated disease are not aware of the risks associated with high risk pet practices or recommendations to reduce them. since unfamiliarity with hazards reinforces fear, communication plays an important role in this. veterinarians play a key role in education regarding risk reduction by giving advice about responsible pet ownership and the required preventive hygiene. healthcare providers such as family doctors, school doctors, and pediatricians can also provide information about safe pet ownership. physicians should ask as part of the medical history about eventual contact with pets, particularly with patients at high risk [120] . the "one health" initiative aims to reduce this professional gap between vets and physicians [2] . when giving recommendations to prevent zoonotic transmission, one of the often-made remarks is that people nowadays are already too hygienic. it is assumed that there is a protective influence of postnatal infection and that it might be lost in the presence of modern hygiene. this belief is based on the hygiene hypothesis that was formulated in 1989 [223] . it was observed that hay fever in young adults was inversely related to the number of siblings in their family. this hypothesis focused exclusively on allergic conditions as a result of the modern way of life and the assumption that modern hygiene was reducing contact with bacteria. another view is that some chronic inflammatory disorders have increased over the last decades as a result of decreased frequency of infections due to pathogenic organisms [224] . another related theory is the "old friends" mechanism that is based on the positive influence of gut parasites, non-pathogenic environmental bacteria (saprophytes, pseudocommensals), and gut commensals or microbiota. however, decreased exposure to these microorganisms is not the only reason for the increasing frequency of allergies and chronic inflammatory disorders in industrialized countries. nowadays there is more information about the immunological roles of skin, oral mucosa, and gut microbiota as well as helminths and the influence these have on the immune system [225] . gut flora may be modified due to diet, obesity, hygiene, antibiotics, but also to psychological stress, vitamin d deficiency, and pollution [225] [226] [227] . pollution also has a significant effect on the development of several respiratory problems and diseases. not only due to outdoor pollution such as fine dust, harmful solids, liquids, or gases [228] , but also due to indoor molds as result of insufficient ventilation in energy efficient homes [229] . finally, there is a clear increase in the allergen production of house dust mites and pollen leading to more exposure and sensitization in susceptible individuals [230] . all together it must be clear that there is much more known about other causes of increasing allergies worldwide than simply excessive cleanliness as suggested in the hygiene hypothesis. regarding field infections with helminths such as trichuris trichiura in early life, these are associated with a reduced prevalence of allergies later in life and infants of helminth-infected mothers have a reduced prevalence of eczema. hookworm infections in developing countries are associated with a reduced prevalence of asthma [231] . the rate of eczema in such countries was found to be about five times higher in infants whose mothers had never had helminths compared with persistent helminth-infected mothers [232, 233] . helminths are nowadays even used under controlled conditions to stimulate immunity. examples are trichuris suis therapy for crohn's disease and necator americanus larvae to treat crohn's disease and other autoimmune disorders [234] . there are no clinically apparent childhood infections found to be associated with protection from allergic disorders [235] . it can even result in an opposite effect in the case of toxocara infection by children after soil contact. a recent meta-analysis showed that children infected with toxocara spp. are more likely to have asthma compared to non-infected children [213] . this parasite and asthma both have elevated immunoglobulin e (ige) levels and eosinophilia in common. that means that precautions should be taken in children to prevent soil contact not only to prevent toxocara infection, but also to prevent acquired ocular toxoplasmosis. there is no need, therefore, to stimulate the contraction of pathogenic bacteria or helminths to achieve a healthy gut microbiota and to reduce allergic conditions. recommendations based on the hygiene hypothesis should preferably be based on results from controlled studies to prevent unintentional negative effects. it is both humans and companion animals who experience negative effects of a changed human-companion animal bond. the education given by vets to their clients should therefore also focus on preventing these negative health and behavioral effects. for instance, by giving science-based advice on feeding practices. in general, regulating authorities should encourage the development of enforcement criteria for breeding dogs and cats to reduce health and welfare risks. pets undoubtedly have a positive effect on human health and well-being, while owners are increasingly aware of pet health, welfare, and well-being. anthropomorphism, also resulting in behavioral problems and breeding on appearance rather than health, and trends such as keeping exotic animals and importing rescue dogs may result in an increased risk of contracting zoonotic infections. recommendations regarding responsible pet ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conform the biology of the animal, are key in preventing such negative aspects of the human-animal bond. there is no need to stimulate unhygienic practices following the hygiene hypothesis. education can be performed by vets and physicians as 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imported eyeworm infection in dogs: a new threat for the united kingdom international dog travelling and risk for zoonotic onchocerca lupi strongyloides stercoralis infection in imported and local dogs in switzerland: from clinics to molecular genetics recent advances on dirofilaria repens in dogs and humans in europe. parasites vectors potential risk posed by the importation of ticks into the uk on animals: records from the tick surveillance scheme available online: www.esccap.org/travelling-petsadvice points to consider when importing a rescue dog from abroad veterinary and public health aspects of toxocara spp toxoplasma oocysts as a public health problem toxocara eggs in public places worldwide-a systematic review and meta-analysis neglected parasitic infections human toxocariasis-a look at a neglected disease through an epidemiological 'prism' human toxocariasis relationship between allergic manifestations and toxocara seropositivity: a cross-sectional study among elementary school children toxocara spp. infection and risk of childhood asthma: a systematic review and meta-analysis reduced cognitive function in children with toxocariasis in a nationally representative sample of the united states association between toxocariasis and cognitive function in young to middle-aged adults ocular toxoplasmosis: an update risk factors for salmonella enteritidis and typhimurium (dt104 and non-dt104) infections in the netherlands: predominant roles for raw eggs in enteritidis and sandboxes in typhimurium infections how much soil do young children ingest: an epidemiologic study celebrating international mud day dogs and transmission of infection to man, respected member of the family? healthy pets. available online: www zoonotic diseases. available online: www hay fever, hygiene, and household size microbes, immunoregulation, and the gut 99th dahlem conference on infection, inflammation and chronic inflammatory disorders: darwinian medicine and the 'hygiene' or 'old friends' hypothesis metabolic and metagenomic outcomes from early-life pulsed antibiotic treatment current understanding of the human microbiome environmental and health impacts of air pollution: a review. front. public health 2020, 8, 14 abridged version of the awmf guideline for the medical clinical diagnostics of indoor mould exposure house dust mite allergy under changing environments interactions between helminth parasites and allergy the impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood immunologic profiles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection an update on the use of helminths to treat crohn's and other autoimmune diseases infections presenting for clinical care in early life and later risk of hay fever in two uk birth cohorts this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-319933-yp9ofhi8 authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: chapter 38 animal models of human viral diseases date: 2013-12-31 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-415894-8.00038-5 sha: doc_id: 319933 cord_uid: yp9ofhi8 abstract as the threat of exposure to emerging and reemerging viruses within a naive population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. by using animal models in this endeavor, the response to viruses can be studied in a more natural context to identify novel drug targets, and assess the efficacy and safety of new products. this is especially true in the advent of the food and drug administration's animal rule. although no one animal model is able to recapitulate all the aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to be considered before an animal study are the route of challenge, species of animals, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand the disease progression, pathogenesis, and immunologic responses in humans. furthermore, to test vaccines and medical countermeasures, well-developed animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease. a good animal model of viral infection should allow many parameters of infection to be assayed, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations, and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for initially testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for the pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most experiments performed in small animal models when possible, and nhps only used to fill in remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen-free (spf), transgenic, and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables larger or more frequent blood samples to be collected, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains; thus, greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic animals are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically the closest species to humans; thus, disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns of experimentation on nhps, along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure that the least amount of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, route of exposure and the dose should be as close as possible to the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are also often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on the viruses for each family that are the greatest concern for public health worldwide. poliovirus (pv) is an enterovirus in the picornavirus family and causes poliomyelitis. 1 humans are the only natural host for the virus, but a number of nhp species are also susceptible. all three serotypes of pv cause paralytic disease, but it is relatively rare with only 1-2% of infected individuals ultimately developing paralysis. humans typically acquire and transmit the virus by the oral-fecal route, although transmission by aerosol droplets may also be possible. 2 the virus replicates in the oropharyngeal and intestinal mucosa, made possible by the resistance of pv to stomach acids. 3 cd155 expression in peyer's patches and m cells suggest that these cell types may be important during initiation of infection. 4 replication at extraneural sites 38 . in vivo models of viral diseases affecting humans precedes invasion into the central nervous systems (cnss), when it occurs. two effective vaccines, the salk killed polio vaccine delivered by the intramuscular route and the sabin live attenuated polio vaccine delivered orally, have been used very successfully to eliminate the disease from most parts of the world. 5 the world health organization has led a long and hard-fought global polio eradication campaign, with much success, but full eradication has not yet been achieved. since 2003, between 1000 and 2000 cases of pv infection are reported worldwide each year. 6 thus, animal models are also needed to test new vaccine approaches that could be used toward eradication of polio in the areas where it still persists. additionally, the recent focus of work with pv animal models has been fraught with urgency, as to gain understanding of pv pathogenesis before the eradication effort is complete and work with this virus ceases. animal models for the study of pv consist of nhp models and mouse models. mice are susceptible to certain adapted pv strains: p2/lansing, p1/lsb, and a variant of p3/leon. mice infected intracerebrally with p2/lansing develop disease with some clinical and histopathological features resembling that of humans. 7 wild-type mice are not susceptible to wildtype pv; however, the discovery of the pv receptor (cd155) in 1989 led to the use of hcd155 transgenic mice as a model of pv infection. 8 these mice are not susceptible to pv by the oral route and must be exposed intranasally or by intramuscular infection to induce paralytic disease. 9 interestingly, hcd155 mice that have a disruption in the interferon (ifn)-a/b receptor gene are susceptible to oral infection. 10 this finding has given rise to speculation that an intact ifn-a/b response may be responsible for limiting infection in the majority of individuals exposed to pv. thus, mouse models have proven to be very useful in gaining a better understanding of pv disease and pathogenesis. rhesus macaques are not susceptible to pv by the oral route, but they have been used extensively to study vaccine formulations for safety and immunogenicity, for monitoring neurovirulence of the live attenuated sabin vaccine, and in the past for typing pv strains. 11 bonnet monkeys are also susceptible to oral inoculation of pv, which results in the gastrointestinal shedding of virus for several weeks, with paralysis occurring in only a small proportion of animals. consistent paralytic disease can be induced in bonnet monkeys (macaca radiata) through exposure to pv by infection into the right ulnar nerve (at the elbow), resulting in limb paralysis that resembles human paralytic poliomyelitis both clinically and pathologically. 12 as such, bonnet monkeys can be used to study pv distribution and pathology and the induction of paralytic poliomyelitis or provocation paralysis. 13 hepatitis a virus causes jaundice, which is a public health problem worldwide. the incubation period lasts from 15 to 45 days with an average of 28 days. transmission between humans occurs by the oral-fecal route, person-to-person contact, or ingestion of contaminated food and water. 14 hepatitis a virus causes an acute and self-limited infection of the liver with a spectrum of signs and symptoms ranging from subclinical disease, to jaundice, fulminant hepatitis, and in some cases death. 15, 16 the disease can be divided into four clinical phases: (1) incubation period, during which the patient is asymptomatic but virus replicates and possibly transmits to others. (2) prodromal period, which might last from a few days to a week with patients generally experiencing anorexia, fever (<103 f), fatigue, malaise, myalgia, nausea, and vomiting. (3) icteric phase, in which increased bilirubin causes characteristic dark brownish colored urine. this sign is followed by pale stool and yellowish discoloration of the mucous membranes, conjunctiva, sclera, and skin. most patients develop an enlarged liver, and approximately 5-15% of the patients have splenomegaly. (4) convalescent period, with resolution of the disease and recovery of the patient. rarely, during the icteric phase, extensive necrosis of the liver occurs. these patients show a sudden increase in body temperature, marked abdominal pain, vomiting, jaundice, and the development of hepatic encephalopathy associated with coma and seizures, all signs of fulminant hepatitis. death occurs in 70-90% of patients with fulminant hepatitis. 16 experiments showed that hepatitis a causes disease only in humans, chimpanzees, several species of south american marmosets, stump-tailed monkeys, and owl monkeys via the oral or intravenous (iv) routes. [17] [18] [19] [20] it is known that cynomolgus macaques are infected with hepatitis a virus in the wild. 21 amado et al. used cynomolgus macaques (macaca fascicularis) for experimental hepatitis a infections. 17 the animals did not exhibit clinical signs of disease, but viral shedding was observed in saliva and stool as early as 6 h postinoculation (pi) and 7 days pi, respectively. although mildto-moderate hepatic pathology was observed in all macaques, seroconversion and mildly increased alanine aminotransferase (alt), an enzyme associated with liver function, were observed in some of them. because this study had a very small group of animals (four macaques), the data should not be considered as conclusive, and more studies are needed to better define the cynomolgus macaque model. although hepatitis a virus is transmitted by the oralfecal route, studies in chimpanzees and tamarins showed that the iv route was much more infectious than oral route was. there was no correlation between dose and development of clinical disease for either species or experimental routes, and similar to cynomolgus macaques, none of these species showed clinical signs of disease. 20 inoculation of common marmosets (callithrix jacchus) with hepatitis a virus did not produce clinical signs of disease as seen in other nhp models. 22, 23 liver enzyme levels increased on day 14 pi, and monkeys had measurable antihepatitis a antibodies by day 32 pi. an experimental study with cell culture-adapted hepatitis avirus in guinea pigs challenged by oral or intraperitoneal routes did not result in clinical disease, increase in liver enzymes, or seroconversion. 24 viral load was detected in stool and serum between days 14 and 52 and 21 and 49 days, respectively. liver pathology showed mild hepatitis. furthermore, histopathology indicated that virus replicated in extrahepatic tissues such as spleen, regional lymph nodes, and intestinal tract. in summary, none of the animal models for hepatitis a infection is suitable for studying pathogenesis of the virus because all clinical and most of the laboratory parameters remain within normal range or only slightly increased after the infection. one possibility is to test the safety of vaccines against hepatitis a virus in those models with demonstrable viral shedding. noroviruses, of which norwalk is the prototypic member, are responsible for up to 85% of reported food-borne gastroenteritis cases. in developing countries, this virus is responsible for approximately 200,000 deaths annually. 25 a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants. 26, 27 symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation normally is for 1-3 days, with symptoms enduring for 2-3 days. 28 viral shedding is indicative of immunocompromised status within an individual with the elderly and young having a prolonged state of shedding. 29 transmission occurs predominately through the oral-fecal route with contaminated food and water being the major vector. 30 a major hindrance to basic research into this pathogen is the lack of a cell culture system. therefore, animal models are used not only to determine the efficacy of novel drugs and vaccines but also for understanding the pathogenesis of the virus. therapeutic intervention consists of rehydration therapy and antiemetic medication. 31 no vaccine is available, and development of one is expected to be challenging given that immunity is short lived after infection. 32 nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus can be monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity are measured exclusively by assay of viral shedding. 33 it was determined that more virus was needed to create an infection when challenging by the oral route than when challenging by the iv route. chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route. although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred, and no histopathological changes were detected. the amount and duration of viral shedding were in line with what is observed upon human infection. 34 a recently identified calicivirus of rhesus origin, named tulane virus, was used as a surrogate model of infection. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was achieved for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease. 35 a murine norovirus has been identified and is closely related to human norwalk virus. however, clinically, the viruses present a different disease. the murine norovirus does not induce diarrhea nor vomiting and can develop a persistent infection in contrast to human disease. [36] [37] [38] porcine enteric caliciviruses can induce diarrheal disease in young pigs and an asymptomatic infection in adults. 39 gnotobiotic pigs can successfully be infected with a passaged clinical noroviruses isolate orally. diarrheal disease developed in 74% of the animals, and 44% were able to shed virus in their stool. no major histopathological changes or viral persistence was noted. 40 calves are naturally infected with bovine noroviruses. experimentally challenging calves with an oral inoculation of a bovine isolate resulted in diarrheal disease [14] [15] [16] the link between equine cases and the human disease was confirmed in 1938 by observing 30 cases of fatal encephalitis in children living in the same area as the equine cases. during this outbreak, eeev was isolated from the cnss of these children as well as from pigeons and pheasants. 44 eeev primarily affects areas near salty marshes and can cause localized outbreaks of disease in the summer. the enzootic cycles are maintained in moist environments such as coastal areas, shaded marshy salt swamps in north america (na), and moist forests in central america and south america (sa). 45 birds are the primary reservoir, and the virus is transmitted via mosquitoes. furthermore, forest-dwelling rodents, bats, and marsupials frequently become infected and may provide an additional reservoir in central america and sa. despite known natural hosts, the transmission cycles in these animals are not well characterized. 44 reptiles and amphibians have also been reported to become infected by eeev. eeev pathogenesis and disease have been studied in several laboratory animals. as a natural host, birds do not generally develop encephalitis except pheasants or emus, in which eeev causes encephalitis with 50-70% mortality. 46 young chickens show signs of extensive myocarditis in early experimental infection and heart failure rather than encephalitis is the cause of death. 47 besides the heart, other organs such as pancreas and kidney show multifocal necrosis. additionally, lymphocytopenia has been observed in the thymus and spleen in birds. 45 eeev causes neuronal damage in newborn mice, and the disease progresses rapidly, resulting in death. 48 similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, whereas inoculation via the subcutaneous route causes a pantropic infection eventually resulting in encephalitis. 49, 50 guinea pigs and hamsters have also been used as animal models for eeev studies. 51, 52 guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed and resulted in brain lesions in these animals. 52 subcutaneous inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis and death. in addition, parenchyma necroses were observed in the liver and lymphoid organs. 51 intradermal, intramuscular, or iv inoculations of eeev in nhps cause disease but does not always result in symptoms of the nervous system. intracerebral infection of eeev results in nervous system disease and fatality in monkeys. 53 the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in monkeys when they are infected by the peripheral route. 54 intranasal and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but less drastic than those caused by intracerebral injections. 54 the aerosol route of infection also progresses to uniformly lethal disease in cynomolgus macaques. 55 in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed, and nhps became moribund and were euthanized between days 5 and 9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals. 56 similar clinical signs and pathology were observed when common marmosets were infected with eeev by the intranasal route. 57 both aerosol and intranasal nhp models had similar disease progression and pathology as those seen in human disease. a common marmoset model was used for comparison studies of sa and na strains of eeev. 57 previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected intranasally with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained healthy and survived the challenge. epizootics of viral encephalitis in horses were previously described in argentina. more than 25,000 horses died from western equine encephalitis virus (weev) in the central plains of the united states in 1912. 58 weev was first isolated from the brains of horses during the outbreak in the san joaquin valley of california in 1930. although it was suspected, the first diagnosis of weev as a cause of human encephalitis occurred in 1938, when the virus was recovered from the brain of a child with fatal encephalitis. 44 in horses, the signs of disease are fever, loss of coordination, drowsiness, and anorexia, leading to prostration, coma, and death in about 40% of affected animals. 59 weev also infects other species of birds and often causes fatal disease in sparrows. weev infection occurs throughout western na and sporadically in sa as it circulates between its mosquito vector and wild birds. 44 chickens and other domestic birds, pheasants, rodents, rabbits, ungulates, tortoises, and snakes are natural reservoirs of weev. 60,61 weev has caused epidemics of encephalitis in humans, horses, and emus, but the fatality rate is lower than that for eeev. 62 predominately young children and those older than 50 years demonstrate the clinical symptoms of the disease. 63 severe disease, seizures, fatal encephalitis, and significant sequelae are more likely to occur in infants and young children. 64, 65 typically, the disease progresses asymptomatically with seroprevalence in humans being fairly common in endemic areas. species used to develop animal models for weev are mice, hamsters, guinea pigs, and ponies. studies with ponies resulted in viremia in 100% of the animals 1-5 days pi. fever was observed in 7 of 11 animals, and six exhibited signs of encephalitis. 44 after subcutaneous inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h. 66 in suckling mice, the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on days 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14, and both brain and mesodermal tissues such as heart, lungs, liver, and kidney were involved. 66, 67 intracerebral and intranasal routes of infection resulted in a fatal disease that was highly dependent on dose, while intradermal and subcutaneous inoculations caused only 50% fatality in mice regardless of the amount of virus. 49 studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases. 68 cns involvement was evident with intracerebral, intraperitoneal, and intradermal inoculations. 68 weev is highly infectious to guinea pigs. 69 intraperitoneal inoculation of weev is fatal in guinea pigs regardless of virus inoculum, with the animals exhibiting signs of illness on days 3-4, followed by death on days 5-9 (nalca, unpublished results). very limited studies have been performed with nhps. the intranasal route of infection causes severe, lethal encephalitis in rhesus macaques. 54 reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model would be useful for testing of vaccines and therapeutics against weev. 70 venezuelan equine encephalitis virus (veev) is maintained in nature in a cycle between small rodents and mosquitoes. 45 the spread of epizootic strains of the virus to equines leads to high viremia followed by a lethal encephalitis, and tangential spread to humans. veev can easily be spread by the aerosol route making it a considerable danger for laboratory exposure. in humans, veev infection causes a sudden onset of malaise, fever, chills, headache, and sore throat. 45, 71, 72 symptoms persist for 4-6 days, followed by a 2-to 3-week period of generalized weakness. encephalitis occurs in a small percentage of adults ( 0.5%); however, the rate in children may be as high as 4%. neurologic symptoms range from nuchal rigidity, ataxia, and convulsions to the more severe cases exhibiting coma and paralysis. the overall mortality rate in humans is <1%. 45 laboratory animals such as mice, guinea pigs, and nhps exhibit different pathologic responses when infected with veev. the lymphatic system is a general target in all animals infected with cns involvement variable between different animal species. the disease caused by veev progresses very rapidly without showing signs of cns disease in guinea pigs and hamsters. mortality is typically observed within 2-4 days after infection and fatality is not dose dependent. 44 veev infection lasts longer in mice, which develop signs of nervous system disease in 5-6 days and death 1-2 days later. lethal dose in mice changes depending on the age of mice and the route of exposure. 56 in contrast to guinea pigs and hamsters, the time of the death in mice is dose dependent. mortality is observed generally within 2-4 days after infection and fatality is not dose dependent. subcutaneous/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans. 73 virus begins to replicate in the draining lymph nodes at 4 h pi. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis. 74, 75 aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure do. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death. 56, 74, 75 intranasal challenge of c3h/hen mice with high dose veev caused high morbidity and mortality. 76 viral titers in brain peaked on day 4 postchallenge and stayed high until animals died on day 9-10 postchallenge. protein cytokine array done on brains of infected mice showed elevated interleukin (il)-1a, il-1b, il-6, il-12, monocyte chemoattractant protein-1 (mcp-1), ifng, mip-1a, and regulated and normal t-cell expressed and secreted levels. this model was used successfully to test antivirals against veev. 77 xi. viral disease veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted <12 h. secondary fever generally began on day 5 and lasted 3-4 days. 78 veev-infected nhps exhibited mild symptoms such as anorexia, irritability, diarrhea, and tremors. leucopenia was common in animals exhibiting fever. 79 supporting the leucopenia, observed microscopic changes in lymphatic tissues such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection. 78 the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. cynomolgus macaques develop similar clinical signs including fever, viremia, lymphopenia, and encephalitis upon aerosol exposure to veev. 80 chikungunya virus is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics mainly within africa and southeast asia. 45 the virus is transmitted by aedes mosquitoes. given the widespread endemicity of aedes mosquitoes, chikungunya virus has the potential to spread to previously unaffected areas. this is typified by the emergence of disease for the first time in 2005 in the islands of the southwest indian ocean, including the french la reunion island, and the appearance in central italy in 2007. 81, 82 the incubation period after a mosquito bite is 2-5 days followed by a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported. 83 individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions, including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years have an increased risk of developing neurological manifestations. 84 there is no effective vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikungunya virus infection by intradermal inoculation. however, it was demonstrated that neonatal mice were susceptible, and severity was dependent upon age at infection. six-day-old mice developed paralysis by day 6, and all died by day 12, whereas 50% of nine-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. infected mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications. 85, 86 an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied by foot swelling and noted inflammation of the musculoskeletal tissue. 87, 88 adult ifna/br knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts. 85 imprinting control region (icr) cd1 mice can also be used as a disease model. neonatal mice subcutaneously inoculated with a passaged clinical isolate of chikungunya virus developed lethargy, loss of balance, and difficulty in walking. mortality was low, 17% and 8% for newborn cd1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection. 86 a drawback of both the ifna/ br and cd1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised. 89 long-tailed macaques challenged with a clinical isolate of the virus developed a similar clinical disease to humans. initially, the monkeys developed high viremia with fever and rash. after this period, viremia resolved and virus could be detected in lymphoid, liver, meninges, joint, and muscle tissue. the last stage mimicked the chronic phase in which virus could be detected up to two months after infection, although no arthralgia was noted. 90 dengue virus is transmitted via the mosquito vectors aedes aegypti and aedes albopictus. 91 given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to dengue virus. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia. 92 it is estimated that there are 20,000 deaths each year caused by dengue hemorrhagic fever (dhf). 93 there are four serotypes of dengue virus, numbered 1-4, which are capable of causing a wide spectrum of disease that ranges from asymptomatic to severe with the development of dhf. 94 incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages after a mosquito bite. 95 typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure. 94 thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis. 96 severe disease has a higher propensity to occur upon secondary infection with a different dengue virus serotype. 97 this is hypothesized to occur due to antibodydependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is therefore the optimal range to be used during challenge. a comprehensive review of the literature regarding animal models of dengue infection was recently published by zompi et al. 98 several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis. [99] [100] [101] a higher dose of an adapted dengue virus strain induced dhf symptoms in both balb/c and c57bl/6. 102, 103 this model can also yield asymptomatic infections. a mouse-adapted (ma) strain of dengue virus 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans. 104 passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased, and animals died due to vascular leak syndrome. 105 another ma strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability. 103 intracranial injection of suckling mice with dengue virus leads to death and has been used to test the efficacy of therapeutics. 106 scid mice engrafted with human tumor cells develop paralysis upon infection, and are thus not useful for pathogenesis studies. 107,108 df symptoms developed after infection in nod/scid/il2rgko mice engrafted with cd34 ã¾ human progenitor cells. 109 rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and laboratory-adapted strain of dengue virus 2. 110 a passaged clinical isolate of dengue virus type 3 was recently used to create a model in immunocompetent adult mice. interperitoneal injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose-dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, aspartate aminotransferase (ast) and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases. 111 a novel model was developed that used infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of dengue virus type 2. infected mosquitoes then fed upon the mouse footpad to allow for the transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the amount of mosquitoes it was exposed to, with four to five being optimal. detectable viral rna was in line with what is observed during human infection. severe thrombocytopenia developed on day 14. this model is intriguing given that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus. 112 nhp models have used a subcutaneous inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection. 113 the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes. 114 throughout these preliminary studies, no clinical disease was detected. to circumvent this, a higher dose of dengue virus was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed. 115 further development would allow this model to be used for testing of novel therapeutics and vaccines. although primates do not develop disease upon infection with dengue, their immune system does produce antibodies similar to those observed during the course of human infection. this has been advantageous in studying ade. sequential infection led to a crossreactive antibody response, which has been demonstrated in both humans and mice. 116 this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than was previously reported; however, no clinical signs were apparent. 117 the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. japanese encephalitis virus ( jev) is a leading cause of childhood viral encephalitis in southern and eastern asia and is a problem among military personnel and travelers to these regions. it was first isolated from the brain of a patient who died from encephalitis in japan in 1935. 118 culex mosquitoes, which breed in rice fields, transmit the virus from birds or mammals (mostly domestic pigs) to humans. the disease symptoms range from a mild febrile illness to acute meningomyeloencephalitis. after an asymptomatic incubation period of 1-2 weeks, patients show signs of fever, headache, stupor, and generalized motor seizures, especially in children. the virus causes encephalitis by invading and destroying the cortical neurons. the fatality rate ranges from 10% to 50%, and most survivors have neurological and psychiatric sequelae. 119, 120 jev virus causes fatality in infant mice by all routes of inoculation. differences in pathogenesis and outcome are seen when the virus is given by intraperitoneal inoculation. 121 these differences depend on the amount of virus and the specific viral strains used. the biphasic viral multiplication after peripheral inoculation is observed in mice tissues. primary virus replication occurs in the peripheral tissues and the secondary replication phase in the brain. 122 hamsters are another small animal species that are used as an animal model for jev. fatality was observed in hamsters inoculated intracerebrally or intranasally, while peripheral inoculation caused asymptomatic viremia. studies with rabbits and guinea pigs showed that all routes of inoculation of jev produce asymptomatic infection. 123 serial sampling studies with 12-day-old wistar rats inoculated intracerebrally with jev indicated that jev causes the overproduction of free radicals by neurons and apoptosis of neuronal cells. 124 following a study in 2010 by the same group, showed that although cytokines tumor necrosis factor (tnf)-a, ifn-g, il-4, il-6, il-10, and chemokine mcp-1 increased gradually and peaked on days 10 pi with jevin rats, the levels eventually declined, and there was no correlation with the levels of cytokines and chemokines and neuronal damage. 125 intracerebral inoculation of jev causes severe histopathological changes in brain hemispheres of rhesus monkeys. symptoms such as weakness, tremors, and convulsions began to appear on days 6-10, with indicative signs of encephalomyelitis occurring on days 8-12 postinfection for most of the animals followed by death occurring on days 8-12 postinfection for most of the animals followed by death. 126 although intranasal inoculation of jev results in fatality in both rhesus and cynomolgus monkeys, peripheral inoculation causes asymptomatic viremia in these species. 123, 127 west nile virus west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937. 128 after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s 129, 130 and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia. 131 the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area. 132, 133 since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days. 134 the mortality rate after neuroinvasive disease ranges from 4% to 11%. 131, [135] [136] [137] the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. although many early laboratory studies of wn encephalitis were performed in nhps, mice, rat, hamster, horse, pig, dog, and cat models were used to study the disease. [138] [139] [140] [141] [142] [143] [144] inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e. asymptomatic, fever, encephalitis). 141 thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subcutaneous inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia. 145, 146 wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and intraperitoneal routes, resulting in encephalitis and 100% mortality. intradermal route pathogenesis studies indicated that langerhans dentritic cells are the initial viral replication sites in the skin. 147, 148 the infected langerhans cells then migrate to lymph nodes, and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases. [149] [150] [151] [152] tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared normal during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms by days 7-10. 143 many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the above-mentioned monkey experiments by pogodina et al., persistent wnv infection was found in the brains of hamsters. the etiologic agent of severe acute respiratory syndrome (sars), sars-coronavirus (cov), emerged in 2002 as it spread throughout 32 countries in a period of 6 months, infecting >8000 people and causing nearly 800 deaths. 153, 154 the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible. 155 although other members of the family usually cause mild illness, sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 years. 156, 157 after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough. 158 in some sars, cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies. 159 in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses. 160 infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection. virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues. 161 sars-cov can replicate in many species, including dogs, cats, pigs, mice, rats, ferrets, foxes, and nhps. 162 chinese palm civets, raccoon dogs, and bats are possible natural hosts. no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (w10%), viral replication, and pathology. 163 in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models use mice, hamsters, ferrets, and nhps (table 38 .1). mouse models of sars-cov typically are inoculated by the intranasal route under light anesthesia. young, 6-to 8-week old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinates, with a peak on day 2 and clearance by day 5 postexposure. there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis). intranasal sars-cov infection of c57bl/6 (b6) also yield reduced weight gain and viral replication in the lungs, with a peak on day 3 and clearance by day 9. 171 in contrast, balb/c mice 13-14 interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-covinfection similar to those observed for balb/c mice but have lower titers and less prolonged disease. one problem is that it is more difficult to obtain large numbers of mice older than 1 year. a number of immunocompromised knockout mouse models of intranasal sars-cov infection have also been developed. 129svev mice infected with sars-cov by the intranasal route develop bronchiolitis, with peribronchiolar inflammatory infiltrates, and interstitial inflammation in adjacent alveolar septae. 172 viral replication and disease in these mice resolve by day 14 postexposure beige, cd1ã�/ã�, and rag1ã�/ã� mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs. signal transducer and activator of transcription-1 (stat1) ko mice infected intranasally with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths. the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. syrian golden hamsters (strain lvg) are also susceptible to intranasal exposure of sars-cov. after the administration of 10 3 tcid 50 (tissue culture infective dose), along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis. there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. some mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred. 163 damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge. 167 clinical disease has been observed in several studies. sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5-10% of the lungs, occasionally accompanied by severe pathology within the lungs. 173 with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or intratracheal routes generally results in a very mild infection, which resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness, but others have rash, lethargy, and respiratory signs and pathology. 170 rhesus have little to no disease and only have mild findings upon histopathological analysis. agms infected with sars-cov have no overt clinical signs, but dad and pneumonitis have been documented. viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were thought to be involved in the sars-covoutbreak. intratracheal and intranasal inoculation of civets with sars-covresults in lethargy, decreased aggressiveness fever, diarrhea, and conjunctivitis. 174 leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. common marmosets have also been shown to be susceptible to sars-cov infection. 175 vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine have used live-attenuated, killed, dna and viral-vectored vaccine strategies. 176 an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease. 177 as such, immune mice had th2-type immunopathology upon sars-cov challenge. 178 severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported. 179 additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance. 180 mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. ma sars and human ace2 transgenic mice are available. 181 all mammals experimentally or naturally exposed to rabies virus have been found to be susceptible. this highly neurotropic virus is a member of the lyssavirus genus and is transmitted from the bite of an infected animal to humans. 182 the virus is able to replicate within the muscle cells at the site of the bite, and then travel to the cns. once reaching the cns by retrograde axonal transport, the virus replicates within neurons creating inflammation and necrosis. the virus subsequently spreads throughout the body via peripheral nerves. 183 a typical incubation period is 30-90 days, and is highly dependent upon the location of the bite. proximity to the brain is a major factor for the onset of symptoms. the prodromal stage lasts from 2 to 10 days and is when the virus initially invades the cns. flu-like symptoms are the norm in conjunction with pain and inflammation at the site of the bite. subsequently, there are two forms of disease that can develop. in 80% of cases, an individual develops the encephalitic or furious form. this form is marked by hyperexcitability, autonomic dysfunction, and hydrophobia. the paralytic, or dumb form, is characterized by ascending paralysis. ultimately, both forms result in death days after the onset of symptoms. once the symptoms develop, there is no proven effective therapy. in the developing world, death is caused by the lack of access to medical care including postexposure prophylaxis. in na, fatal cases result because of late diagnosis. 184 syrian hamsters have been challenged with rabies virus intracerebrally, intraperitoneally, intradermally, and intranasally. all animals died as a result of the exposure, although intracerebral and intranasal inoculation led to only the furious form depicted by extreme irritability, spasms, excessive salivation, and cries. the virus used had been isolated from an infected dog brain and passaged in swiss albino mice. animals inoculated by intracerebral injection develop disease within 4-6 days, whereas all other routes of entry develop disease within 6-12 days. 185 this model has been used to study and test novel vaccine candidates. 186 mice have been extensively studied as an animal model for rabies. it was shown that swiss albino mice intracerebrally injected with a virus isolated from a dog developed only the paralytic form of disease 6 days after the initial challenge. balb/c mice are universally susceptible to intracerebral injection of rabies virus within 9 days. disease symptoms include paralysis, cachexia, and bristling appearing 1-3 days before death. 187 a more natural route of infection via peripheral injection into masseter muscles was tested on icr mice. these mice developed neurological signs including limb paralysis, and all died within 6-12 days. 188 icr mice have been instrumental in analyzing novel vaccines and correlates of protection. 189 this line of mice was also used to assess the value of ketamine treatment to induce coma during rabies infection. 190 another mouse line used is the p75 neurotrophin receptor-deficient mouse. this mouse developed a fatal encephalitis when inoculated intracerebrally with the challenge virus standard. 191 bax-deficient mice have also been used to determine the role of apoptotic cell death in the brain during the course of infection. 192 a viral isolate from silver-haired bats can also be used in the mouse model. this strain is advantageous given that it is responsible for the majority of deaths in north na. 193 early death phenomenon is typified by a decrease to time of death in a subset of individuals and animals that have been vaccinated and subsequently exposed to rabies. 194 this trend has been demonstrated experimentally in swiss outbred mice and primates. 195, 196 cynomolgus and rhesus were both infected with passaged rabies virus to create an nhp model. a high titer of virus was needed to induce disease, but exposure was found to not be universally fatal. the animals that survived beyond 4 weeks within the experiment did not develop clinical disease nor succumb to infection. primates that did develop disease refused food and had progressively less activity until death. this lasted from 24 h up to 4 days, with all animals with symptoms dying within 2 weeks. 196 bats have been experimentally challenged with rabies. vampire bats, desmodus rotundus, intramuscularly injected with a bat viral isolate displayed clinical signs including paralysis in half of the population of the study animals. of those who did develop disease, the duration was 2 days, and incubation period ranged from 7 to 30 days. regardless of disease manifestation, 89% of challenged animals died. 197 skunks can be challenged intramuscularly or intranasally with either challenge virus strain or a skunk viral isolate. interestingly, the challenge virus strain more readily produced the paralytic form, whereas the street form of rabies developed into the furious form. however, the challenge strain virus resulted in a shorter incubation period of 7-8 days in comparison to 12-14 days seen with the street virus. 198 filoviridae consists of two well-established genera, ebola virus and marburg virus (marv) and a newly discovered group, cuevavirus 199 (table 38 200 two other ebola viruses are known; ta㯠forest (tafv; previously named cote (circumflex over the 'o') d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. the disease in humans is characterized by aberrant innate immunity and a number of clinical symptoms such as fever, nausea, vomiting, arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, anorexia, and numerous others. 201 approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.). 199 natural transmission in an epidemic is thought to be through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, intramuscular, intraperitoneal, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures. 202 because filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 ifn response. 203 however, wild-type inbred mice are susceptible to filovirus that has been ma by serial passage. 204 balb/c mice, which are the strain of choice for intraperitoneal inoculation of ma-ebov, are not susceptible by the aerosol route. 205 for aerosol infection of immunocompetent mice, a panel of bxd (balb/ c ã� dba) recombinant inbred strains were screened, and one strain, bxd34, was shown to be particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (w100 or 1000 pfu). these mice developed weight loss of >15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model uses a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 mm and a geometric standard deviation of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by intraperitoneal or aerosol routes. 206 mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection. liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps. although most mouse studies have used ma-ebov or ebov, an intraperitoneal ma marv model is also available. 207 ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds. 208 hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models. 209 an intraperitoneal ma-ebov infection model has been developed in syrian hamsters. 210 this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, uses male 5-to 6-week-old syrian hamsters that are infected with 100 ld50 of ma-ebov. virus is present in tissues and blood collected on day 4, and all animals succumbed to the disease by day 6. detailed accounts of this model have been presented at international scientific meetings by ebihara and feldmann et al. but have not been reported in a scientific journal at the time of writing this chapter. 211 guinea pig models of filovirus infection have been developed for intraperitoneal and aerosol routes using guinea pig-adapted ebov (gp-ebov) and marv (gp-marv). 212, 213 guinea pigs models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent (hourly) intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. hartley guinea pigs exposed to aerosolized gp-marv or gp-ebov become moribund at times comparable to that of nhps, generally succumbing to the infection between 7 and 12 days postexposure. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (<1000 pfu) presented doses of airborne gp-marv, and more protracted disease is seen in some animals. 213 weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e. alt, ast, alkaline phosphatase (alkp), and blood urea nitrogen) indicating problems with liver and kidney function are also altered late in the disease course. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans. 214 old world primates have been primarily used for the development of intraperitoneal, intramuscular, and aerosol models of filovirus infection. uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, in agms and marmosets. [215] [216] [217] [218] [219] low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. prominent features of the infections are onset of fever by day 5 postexposure, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. clinical disease parameters may have a slightly delayed onset in aerosol models. petichial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species. dyspnea late in infection is a prominent feature of disease after aerosol exposure. a number of pronounced pathology findings include multifocal necrosis and fibrin lesions, particularly within the liver and the spleen. lymphocytolysis and lymphoid depletion are also observed. multilead, surgically implanted telemetry devices are useful in the continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection. the most recently developed telemetry devices can aid in plethysomography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate. 220 outbreaks caused by nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra outbreaks have yet to be reported outside of australia. 221, 222 hendra was the first member of the genus to be identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from human to human or bat to human. nipah has the distinction of being able to be transmitted by humans, although the exact route is unknown. 223 the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized to be needed for successful transmission. 224 both viruses have a tropism for the neurological and respiratory tract. hendra virus incubation period is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting. 225 disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure. 226 nipah virus has an incubation period of 4 days to 2 weeks. 227 much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h. 228 survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions. 229 at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because observed human cases are all at terminal stages. the best small animal representative is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died xi. viral disease 38 . in vivo models of viral diseases affecting humans within 4-17 days postinfection. the progression of disease and timeline are highly dependent on dose and route of infection. intranasal inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas intraperitoneal resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, while the brain was the most affected organ. this model has been used for vaccination and passive protection studies. [230] [231] [232] the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge. 233, 234 inoculation with hendra virus via the subcutaneous route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inocula have been associated with the development of encephalitis lesions. intradermal and intranasal injections do not lead to disease, although the animals are able to seroconvert upon challenge. inoculum source does not affect clinical progression. nipah virus challenge only develops disease upon intraperitoneal injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and found to be present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder. 235 ferrets display the same clinical disease as seen in the hamster model and human cases. 236, 237 upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferrets' brains, but to a lesser degree than seen in humans. cats have also been used as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms. 238, 239 this model has proven to be useful in a vaccine challenge model. squirrel and agms are representative of the nhp models. within the squirrel monkeys, nipah virus is introduced by either the intranasal or iv route and subsequently leads to clinical signs similar to that in humans, although intranasal challenge results in milder disease. upon challenge, only 50% animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in the lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge. 240 agms have been found to be a very consistent model of both viruses. intratracheal inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis. 241, 242 the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue and nipah virus can be located in urine. 243 pigs have been investigated as a model as they develop a respiratory disease upon infection with both nipah and hendra. [244] [245] [246] oral inoculation does not produce a clinical disease, but subcutaneous injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah can also be transmitted between pigs. nipah was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms. 247 within the pig model, it seemed that nipah had a greater tropism for the respiratory tract, while hendra for the neurological system. horses also are able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days. 248, 249 animals died within 4 days postexposure and were found to have interstitial pneumonia with necrosis of alveoli. 250, 251 virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but found to be nonpermissive to infection. 232, 252 suckling balb/c mice succumb to infection if the virus is inoculated intracranially. 253 embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection. 254 respiratory syncytial virus is responsible for lower respiratory tract infections of 33 million children under the age of 5 years, which in turn results in three million hospitalizations and approximately 200,000 deaths. 255 within the united states, hospital costs alone amount to >600 million dollars. 256 outbreaks are common in the winter. 257 the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and further spreads to the lower respiratory tract. incubation for the virus is 2-8 days. respiratory syncytial virus is highly virulent leading to very few asymptomatic infections. 258 disease manifestations are highly dependent upon the age of the individual. primary infections in neonates produce nonspecific symptoms including the overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting the virus at this age results in an increased chance of developing childhood asthma. 259 young children develop recurrent wheezing, whereas adults exacerbate previous respiratory conditions. 260 common clinical symptoms are runny nose, sneezing, and coughing accompanied with fever. mortality rates in hospitalized children are 1-3% with the greatest burden of disease seen in 3-4-month-olds. 261 there is no vaccine available, and ribavirin usage is not recommended for routine treatment. 262 animal models were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated respiratory syncytial virus vaccine (fi-rsv). this vaccineinduced severe respiratory illness in infants who received the vaccine and were subsequently infected with live virus. 263 mice can be used to model disease, although a very high intranasal inoculation is needed to achieve clinical symptoms. 264, 265 strain choice is crucial to reproducing a physiological relevant response. 266 age does not affect primary disease manifestations. 267 however, it does play a role in later sequelae showing increased airway hyperreactivity. 268 primary infection produces increased breathing with airway obstruction. 264, 269 virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. cotton rats are useful given that it is a small animal disease model. the virus is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after intranasal inoculation. 270, 271 it has been reported that viral replication is 50-to 1000-fold greater in the rat model than in the mouse model. 272 the rats develop mild-to-moderate bronchiolitis or pneumonia. 273 although age does not seem to factor in clinical outcome, it has been reported that older rats tend to take longer to achieve viral clearance. viral loads peak by the fifth day, dropping to below the levels of detection by 8. the histopathology of the lungs seems to be similar to that in humans after infection. 274 this model has limited usage in modeling the human immune response to infection as challenge with the virus creates a th2 response, whereas humans tend to skew toward th1. [275] [276] [277] fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally via intranasal inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is thought to be useful in studying mucosal immunity during infection. 278 chimpanzees are permissive to replication and clinical symptoms of respiratory syncytial virus including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms nor high levels of viral replication. 279 bonnet monkeys were also tested and found to develop an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells. 280 the chimpanzee model has proven to be useful for vaccine studies. 281, 282 sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine respiratory syncytial virus. 283 lambs were also found to be susceptible to human respiratory syncytial infection. 284, 285 when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. during the course of disease, viral replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features between them and humans. 286, 287 the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days. 288 the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza. 289 thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing. 290 influenza virus replicates in the upper and lower airways, peaking at approximately 48 h postexposure. infection can be more severe in infants and in children under the age of 22 years, people over the age of 65 years, or immunocompromised individuals in whom viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis. 291 pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyrogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza itself, accounting for approximately 27% of all influenza-associated fatalities. 292 death, often due to ards can occur as early as 2 days after the onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation. 288 the h5n1 avian strain of influenza has lethality rates of approximately 60% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus, the potential for global spread is a major concern. 293 the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. 294 lethality rate can vary with the virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, superinfection and sepsis, severe with ards, and neurologic manifestations. 290 also, models can use seasonal or avian strains and models have been developed to study transmission, important for understanding the potential for more lethal strains such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines. 295 inoculation is by the intranasal route, using approximately 60 ml of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with an mmad of <5 mm. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease, but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. mice lack the mxa gene, which is an important part of the human innate immune response to influenza infection. the mouse homolog to mxa, mx1, is defective in most inbred mouse strains. 296 weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature. 297 pulse oximeter readings and measurement of blood gases of oxygen saturation are also used to determine the impact of influenza infection on respiratory function. 298 virus can be isolated from bronchial lavage fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild-tomoderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight are also frequently present. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation. 299 to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. however, h5n1 and the 1918 pandemic influenza virus can cause lethal infection without adaptation. 300 h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia. 301 as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza produces severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia. 302 in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by intranasal inoculation of a sublethal dose of a bacterial strain such as s. pneumoniae or s. pyrogenes. 303 morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cage mates can be achieved after infection by the aerosol route and cocaging after 24 h. 304 domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans. [305] [306] [307] ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans. 308 influenza was first isolated from ferrets infected intranasally with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments. 309 when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by intranasal. inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id50 dropwise to each nostril. influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mildly to moderately virulent strains. 310 ferrets are more susceptible to influenza a than to influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation. 311 virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirror that seen in humans. nonadapted h1n1, h2n2, and h3n2 have mild-to-moderate virulence in ferrets. strains of low virulence have predominant replication in the nasal turbinates. clinical signs and other disease indicators are similar to that of humans with a mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia. 312 replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater ifn response, transient lymphopenia, and replication in respiratory tract, brain, and other organs. 313 old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models, which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition. 314 of old world primates, cynomolgus macaque (m. fascicularis) are most frequently used for studies of vaccines and antiviral drug therapies. 315, 316 h5n1 and h1n1 1918 infections of cynos are very similar to those in humans. 317 cynos develop fever and ards upon intranasal inoculation of h5n1 with necrotizing bronchial interstitial pneumonia 318 nhps are challenged by multiple routes (ocular, nasal, and tracheal) simultaneously 1 ã� 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had >50% lethality (humans only had a 1-3% lethality). ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains such as h3n2. 299 influenzainfected rhesus macaques represent a mild disease model pathogenesis for vaccine and therapeutic efficacy studies. 319 other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates such as squirrel and cebus monkeys. 320 rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes. 321 additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets. 322 cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies. 323, 324 cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted. 325, 326 nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats. 327 coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic pig influenza models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1. 328, 329 although pigs infected with influenza may have fever, anorexia, and respiratory signs such as dyspnea and cough, mortality is rare. 330 size and space requirements make this animal difficult to work with, although the development of minipig (ellegaard gottingen) models may provide an easier-to-use alternative. incubation period, animals exhibit signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers. 331 mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans. 332,333 after 2-6 days of incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever. [334] [335] [336] depending on the severity of the disease when the symptoms start, 10-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset. 334 hepatic failure, renal failure or disseminated intravascular coagulation (dic), and encephalitis are demonstrated within patients during postmortem examination. mice are one of the most susceptible animal species to rvfv infection. subcutaneous or intraperitoneal routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice. 337, 338 mice start to exhibit signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately after these signs are observed, they become lethargic and generally die 3-6 days postexposure. ocular diseases or hemorrhagic form of the disease has not been observed in mice models so far. 334 increased viremia and tissue tropism were reported in mice with 338 increased liver enzymes and lymphopenia observed in sick mice. rats and gerbils are also susceptible to rvfv infection. rats' susceptibility is dependent on the rat strain used for the challenge model. there was also noted an age dependence in susceptibility of rats. although wistar-furth and brown norway strains and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection. 339, 340 similar pathologic changes such as liver damage and encephalopathy were observed in both rats and mice. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils. 341 similar to the rat model, the susceptibility of gerbils was also dependent on age. so far, studies showed that rvfv does not cause uniform lethality in an nhp model. intraperitoneal, intranasal, iv, and aerosol routes have been used to develop the nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort. 342 monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to those seen in humans such as pathological changes in liver and hemorrhagic disease. there was no ocular involvement in this model. recently, smith et al. compared iv, intranasal, and subcutaneous routes of infection in common marmosets and rhesus macaques. 343 marmosets were more susceptible to rvfv infection than were rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subcutaneous routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of intranasally challenged marmosets. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, seems to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans. 344 incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorrhagic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients. 345, 346 over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. [347] [348] [349] [350] until recently, the only animal that manifests disease is the newborn mouse. infant mice infected with cchfv intraperitoneally caused fatality around day 8 postinfection. 351 pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies using knockout adult mice were successful to develop a lethal small animal model for cchfv infection. 352, 353 bente et al. infected stat1 knockout mice by the intraperitoneal route. in this model, after the signs of fever, leucopenia, thrombocytopenia, viremia, elevated liver enzymes, and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days of postexposure. the second model was developed by using ifn-alpha/beta (ifna/b) receptor knockout mice. 353 similar observations were made in this model as in the stat1 knockout mouse model. the animals were moribund and died 2-4 days after exposure with high viremia levels in the liver and spleen. other laboratory animals, including nhps, show little or no signs of infection or disease when infected with cchfv. 348 butenko et al. used agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not exhibit signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. in 1975, fagbami et al. infected two patas monkeys (cercopithecus erythrocebuserythrocebus) and one guinea baboon (papio papio) with cchfv. 347 although all three animals had low level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia, 354 and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes. 355 shepherd et al. infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. 349 although scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (tatera brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and m. coucha), and syrian hamsters were negative. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed the onset of viremia earlier than those infected by the subcutaneous or intraperitoneal routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather by rodents. 356 rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by the inhalation of infected rodent saliva, feces, and urine. 357 human infections can normally be traced to a rural setting with activities such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention. 358 the viruses have a tropism for endothelial cells within the microvasculature of the lungs. 359 there are two distinct clinical diseases that infection can yield; hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses. 360 hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul. 358 incubation lasts two to three weeks and presents as flulike in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops, which can further progress to shock in approximately 15% patients. the overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to the development of severe disease. hps was first diagnosed in 1993 within the southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus. 361 this virus is still the leading cause within na of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay. 362, 363 the first report of hps in maine was recently documented. 361 andes virus was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans. 364 the fulminant disease is more lethal than that observed for hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation. 365 incubation typically occurs 14-17 days after exposure. 366 unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted. 367 vaccine development has been hampered by the vast diversity of hantaviruses and the limited number of outbreaks. 368 syrian golden hamsters are the most widely used small animal models for hantavirus infection. hamsters inoculated intramuscularly with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h before death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged sin nombre isolate. no hamsters developed any symptoms during the course of observation. although an antibody response to the virus that was not dose dependent was determined via an enzymelinked immunosorbent assay. hamsters infected with andes virus were found to have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andes virus yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver. 369 lethal disease can be induced in newborn mice but does not recapitulate the clinical symptoms observed in human disease. 370 adult mice exposed to hantaan virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease. 371, 372 knockout mice lacing ifn-a/b were found to be highly susceptible to hantaan virus infection. 373 in a study looking at a panel of laboratory strains of mice, c57bl/6 mice were found to be most susceptible to a passaged hantaan viral strain injected intraperitoneally. animals progressed to neurological manifestation including paralyses and convulsions and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different than that observed in human cases. 372 nhps have been challenged with new world hantaviruses; however, no clinical signs were reported. 374, 375 cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease. 376, 377 challenge with andes virus to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. aerosol exposure led to 4 of 6 monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all these viruses share common clinical manifestations. 378 lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april. 379 this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5,000 deaths. 380 outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases sprung up in germany, the netherlands, the united kingdom, and the united states due to transmission to travelers on commercial airlines. 381 transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex. 379 humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease can occur in 20% of individuals. the incubation period is from 5 to 21 days, and the initial onset is characterized by flulike illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease, which is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital, it is between 15% and 25%. there is no approved vaccine, and besides supportive measures, ribavirin is effective only if started within 7 days. 382, 383 the primary animal model used to study lassa fever is the rhesus macaque. 384 aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus more closely mirrored the disease course and histopathology observed in human infection. 385 iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt. 386 disease was dose-dependent with iv, intramuscular, and subcutaneous inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be used, and mimic a more natural route of infection. 387 within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7, and death typically occurred within 10-14 days. 388, 389 intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns. 390 this pathogenicity is seen in select cases of human lassa fever. 391, 392 a marmoset model has recently been defined using a subcutaneous injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated, and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases. 393 mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is completely dependent upon the major histocompatibility complex (mhc) background and age of animal along with the route of inoculation. 394 guinea pig inbred strain 13 was found to be highly susceptible to lassa virus infection. the outbreed hartley strain was less susceptible, and thus, strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus. 395 infection with pichinde virus that has been passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death. 396, 397 the guinea pig is an excellent model given that it not only results in similar disease pattern as humans but also the viral distribution is similar along with the histopathology and immune response. 398, 399 infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig model. the virus was injected intraperitoneally resulting in the animals becoming lethargic and anorexic within 6-7 days. virus was first detected at 3 days and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all the animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present. 400 these results were recapitulated with a nonadapted pichinde virus. [401] [402] [403] globally, diarrheal disease is the leading cause of death with rotavirus being one of the main etiological agents responsible. according to the world health organization, rotavirus alone is responsible for a third of all hospitalization related to diarrhea and 500,000-600,000 deaths per year. 404 the virus is very stable due to its three-layer capsid, which allows it to be transmitted via the oral-fecal route, depositing itself in the small intestine. rotavirus is highly contagious, and only 10 viruses are needed to cause symptomatic disease. 405 the host determinant with the greatest influence on clinical outcome is age. neonates typically are asymptomatic, which is suggested to be due to the existence of maternal antibodies. hence, the most susceptible age group is 3 months to 2 years, coinciding with a drop in these protective antibodies. 406 within this age range, children will develop noninflammatory diarrhea. virus replicates in the intestinal villus enterocytes resulting in their destruction and malabsorption of needed electrolytes and nutrients. symptoms of disease include watery, nonbloody diarrhea with vomiting, fever, and potentially dehydration that lasts up to a week. 407 there is a short episode of viremia during the course of infection. 408 mice can be used as both an infection and disease model depending upon age at challenge. mice <14 days old develop disease, whereas older mice are able to clear the infection before the onset of symptoms. 409 this halts the study of active vaccination against disease in the infection model. in the adult mouse model, the course of the infection is monitored via viral shedding within the stool. 410 infant mice, specifically balb/c, receiving an oral inoculation of a clinical strain of virus developed diarrhea within 24 h postinfection, and 95% of those exposed developed symptoms within 72 h postinfection. symptoms lasted from 2 to 4 days with no mortality. viral shedding was at its peak at 24 h and lasted up to 5 days. there were noted histopathological changes within the small intestine localized to the villi that was reversible. 407 within the adult mouse model, oral inoculation of a mouse rotavirus strain showed viral shedding by 3 days lasting up until 6 days postinfection. 410 these mouse models have been used to study correlates of protection and therapeutic efficacy including gastro-gard ã� . 409, 411, 412 rats can also be used as disease models depending upon the strain of rat. 413, 414 suckling fischer 344 rats were exposed to a simian strain of rotavirus orally. the rats were susceptible to diarrheal disease till they were 8 days old with age determining the length of viral shedding. 415 rats have mainly been used to study the correct formulation for oral rehydration. these rodents are large enough to perform in situ intestinal perfusions. within these studies, 8-day-old rats were infected with a rat strain of rotavirus by orogastric intubation. within 24 h postinfection, the rats developed diarrhea, at which point the small intestine was perfused to compare differing solutions of oral rehydration. 416 gnotobiotic pigs are also used given that they can be infected with both porcine and human strains. 417 they are susceptible to developing clinical disease from human strains up to 6 weeks of age. they allow for the analysis of the primary immune response to the virus given that they do not receive transplacental maternal antibodies and are immune competent at birth. 418 another advantage of this model is that the gastrointestinal physiology and mucosal immune system closely resemble that of humans. 419 this model has been useful in studying correlates of protection. gnotobiotic and colostrum-deprived calves have also been used as an experimental model of rotavirus infection. they are able to develop diarrhea and shed live virus. 420 gnotobiotic lambs can also develop clinical disease upon oral inoculation with clinical strains. 421 infant baboons, agms, and rhesus macaques have all proven to be infection models with severity measure by viral shedding. 422, 423 retroviridae human immunodeficiency virus type 1 the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in sub-saharan africa. worldwide, there are approximately 1.8 million deaths per year with >260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1. 424 hiv targets t-helper cells (cd4ã¾), macrophages, and dendritic cells. 425 acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8ã¾ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4ã¾ t-cells decline to <200 cells per microliter; thus, cell-mediated immunity becomes impaired, and the person is more susceptible to opportunistic infections and certain cancers. humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of intact and functional human innate and adaptive immune responses. 426 the scidhu hiv infection model has proven to be useful, particularly in screening antivirals and therapeutics. 427 a number of different humanized mouse models have been developed for the study of hiv, including rag1ã�/ã�gcã�/ã�, rag2ã�/ ã�gcã�/ã�, nod/scidgcã�/ã� (hnog), nod/ scidgcã�/ã� (hnsg), nod/scid blt, and nod/ scidgcã�/ã� (hnsg) blt. cd34ã¾ human stem cells derived from the umbilical cord blood or fetal liver are used for humanization. 428 hiv-1 infection by intraperitoneal injection can be successful with as little as 5% peripheral blood engraftment. 429 vaginal and rectal transmission models have been developed in blt scidhu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days pi. 430 in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans. 431 importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hiv-infected humanized mice, and at least some mechanisms of pathogenesis that occur in hiv-infected humans also occur in the hiv-infected humanized mouse models. 432 the advantage of these models is that these mice are susceptible to hiv infection, and thus, the impact of drugs on the intended viral targets can be tested. one caveat is that although mice have a "common mucosal immune system," humans do not, due to the differences in the distribution of addressins. 433 thus, murine mucosal immune responses to hiv do not reflect those of humans. there are a number of important nhp models for human hiv infection. simian immunodeficiency virus (siv) infection of macaques is widely considered to be the best platform for modeling hiv infection of humans. importantly, nhps have similar, pharmacokinetics, metabolism, mucosal t-cell homing receptors, and vascular addressins to those of humans. thus, although the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through the use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero, or through breast milk. [434] [435] [436] there are also macaque models to study the emergence and clinical implications of hiv drug resistance. 437 these models most routinely use rhesus macaques (macaca mulatta), cynomolgus macaques (macaca fasicularis), and pigtailed macaque (macaca nemestrina). animals of all ages are used, depending on the needs of the study. for instance, the use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently used. studies are performed in bsl-2 animal laboratories, and nhps must be of simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied. 438 challenges may be through a single high dose. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/deltab670 induces aids in most macaques within 5-17 months (mean of 11 months). 439 peak viremia occurs around week 4. aids in such models is often defined as cd4ã¾ t-cells that have dropped to <50% of the baseline values. alternatively, repeated low-dose challenges are often used, depending on the requirements of the model. 440, 441 because nhps infected with hiv do not develop an infection with a clinical disease course similar to that in humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (simian-human immunodeficiency virus or shiv) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease, which progresses to aids and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1, and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors; thus, hiv-1 rt is usually put into siv for such studies. 442 sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt or integrase inhibition. 443 nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4ã¾ t-cell depletion. additionally, sivmac239 uses the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry. 444 nhps infected with shiv strains may not develop aids, but these models are useful in testing vaccine efficacy. for example, rt-shivs and env-shivs are useful for the testing and evaluation of drugs that may target the envelope or rt, respectively. 442 one disadvantage of the highly virulent env-shiv (shiv-89.6 p) is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops and then resolves without further disease progression. 445 simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for three months, followed by clearance. 446 a number of routes are used for siv or shiv infection of nhps, with iv inoculation being the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than does the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) one month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase the susceptibility to infection by atraumatic vaginal instillation. 447 upon vaginal instillation of 500 tcid50 of shiv-162p3, peak viremia was seen around 12 days postexposure with >10 7 copies per milliliter and dropping thereafter to a constant level of 10 4 rna copies per milliliter at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251. 448 an example of an intrarectal model used juvenile (2year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx. 449 here, viremia peaked at approximately 10 days with >10 8 copies per milliliter. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistently high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of highly active antiretroviral therapy on virus and t-cells in the male genital tract, adult (3-to 4-year old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251, and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization. 450 pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course. 451 importantly, mother-to-infant transmission models have also been developed. 452 pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected; one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission and the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and terminal-repeat retrotransposon in miniature (trim) alleles), differences between challenge strains, and challenge routes. 453 nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the longterm clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade. 454 unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study that used an adenovirus-based vaccine approach. 455 vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv is more difficult to protect than mucosal and is used as a "worst-case scenario." however, efficacy at one mucosal route is usually comparable to that at other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect. 456 there are >100 human papillomaviruses (hpvs), with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately a third of these are transmitted sexually. of these, virulent subtypes such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist that use animal papillomaviruses in their natural host, or a very closely related species. 457, 458 these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines. 459 wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which are a very closely related species. 460 in this model, papillomas can range from cutaneous squamous cell carcinomas on the one end of the spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) cause florid warty lesions in the mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings. 461 the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model. 462 these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by the application of a 10 ml droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles. 463 bovine papillomavirus (bpv) has a wider host range than do most papillomaviruses, infecting the fibroblasts cells of numerous ungulates. 458 bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques, rhesus papillomavirus, is very similar to hpv-16 and is associated with the development of cervical cancer. 464 mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, but they are often used to look at immunogenicity of vaccines. 465, 466 herpesviridae please see chapter 25. monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including squirrels. 467, 468 mpxv was discovered during the pox-like disease outbreak among laboratory java macaques in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with the continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and the western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak. 469, 470 it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-toperson spread occurs by large respiratory droplets or direct contact. 471 most of the clinical features of human monkeypox are very similar to those of ordinary smallpox. 472 after a 7-to 21-day incubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash. 471, 473 typical maculopapular rash follows the prodromal period generally lasting 1-3 days. the average size of the skin lesions is 0.5-1 cm, and the progress of lesions follows the order macules through papules, vesicles, pustules, umblication then scab and desquamation and lasts typically 2-4 weeks. fatality rate is 10% among the unvaccinated population and death generally occurs during the second week of the disease. 469, 471 mpxv is highly pathogenic for a variety of laboratory animals, and so far, many animal models have been developed by using different species and different routes of exposure (table 38. 3). because of the unavailability of variola virus to develop animal models and resulting disease manifestations in humans that are similar, mpxv is one of the pox viruses that are used very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, prairie dogs, african dormice, ground squirrels are highly susceptible to mpxv by different exposure routes. [474] [475] [476] [477] [478] [479] [480] [481] cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose-dependent mortality after intranasal exposure to mpxv. studies with the intraperitoneal route of challenge indicated a higher susceptibility to mpxv with an almost 50-fold less ld50 when compared to the intranasal route. 474 scid-balb/c mice were also susceptible to the intraperitoneal challenge route, and the disease resulted in mortality day 9 postinfection. 476 similarly c57bl/6 stat1 ã�/ã� mice were infected intranasally with mpxv, and the infection resulted in weight loss and mortality 10 days postexposure. mice models mentioned here are very promising for screening of therapeutics against pox viruses, but testing in additional models will be required for advanced development. high doses of mpxv by intraperitoneal or intranasal route caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels. 480 the disease progressed very quickly, and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv in ground squirrels by subcutaneous route resulted in systemic disease and the mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nose bleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. because in the us outbreak the virus was transmitted by infected prairie dogs, this animal model has recently been studied much further and used to test therapeutics and vaccines compared to other small animal models. 475, 481, 491, 492 studies using intranasal, intraperitoneal, and intradermal routes of exposure showed that mpxv was highly infectious to prairie dogs. by using the west african mpxv strain, the intraperitoneal route caused a more severe disease and 100% mortality than challenge by the intranasal route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to the intraperitoneal route, the intranasal route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in intraperitoneally infected animals. 481 recent studies by hutson et al. used intranasal and intradermal infections with west african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and generalized pox lesions. 475 therefore, this model can be used for testing therapeutics and vaccines against pox viruses. the african dormouse is susceptible to mpxv by the foodpad injection route or intranasal route. 478 mice exhibited decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and hemorrhage in lungs were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. considering the limited availability of ground squirrels and african dormice, lack of reagents to these species, and resemblance to hemorrhagic smallpox disease, these models are not very attractive for further characterization and vaccine and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal models for mpxv. 482, 483, 485, 489, 490 during our studies by using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure. 482 complete blood count and clinical chemistries showed abnormalities similar to those of human monkeypox cases with leukocytosis and thrombocytopenia. 493 whole-blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. because doses of 4 ã� 10 4 , 1 ã� 10 5 , or 1 ã� 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 ã� 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, before animals can develop pox lesions, they succumbed to disease. with the low challenge dose, pox lesions were observed, but they were few in comparison to the iv model. mpxv causes dose-dependent disease in nhps when given by the iv route. 490 studies showed that with 1ã� 10 7 pfu iv, challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an intratracheal infection model deposits virus into the trachea, delivering directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation by skipping the upper respiratory system. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally. 489 although a similar challenge dose of intratracheal mpxv infection resulted in a similar viremia in nhps than with the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to that after intratracheal delivery. an intrabronchial route of exposure resulted in pneumonia in nhps. 490 delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and the intratracheal route of infection models, the number of pox lesions was much less than in the iv route of the infection model. a major downside of the iv, intratracheal and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus into the blood stream or into the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human variola virus (varv) infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is decided to be used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b is one of the most common infections worldwide with >400 million people chronically infected and 316,000 cases per year of liver cancer due to infection. 494 the virus can naturally infect both humans and chimpanzees. 495 hepatitis b is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture. 496 the age at which one is infected dictates the risk of developing chronic disease. 497 acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms last for a few weeks before resolving. 498 after this acute phase, life time immunity is achieved. 499 of those infected, <5% will develop the chronic form of disease. chronicity is the most serious outcome of disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than in a noncarrier. 500 the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepadna virus suggest that x protein may be responsible. 501 many individuals are asymptomatic until complications emerge related to chronic carriage. chimpanzees have a unique strain that circulates within the population. 502, 503 it was found that 3-6% of all wild-caught animals from africa are positive for hepatitis b antigen. 504 natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease. 505 to date, the use of chimpanzees provides the only reliable method to ensure that plasma vaccines are free from infectious particles. 506 this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially attuned to these studies given that their immune response to infection directly mirrors humans. 507 other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hepatitis b, none develop hepatic lesions or liver damage as noted by monitoring of liver enzymes. 508 mice are not permissible to infection, and thus, numerous transgenic and humanized lines that express hepatitis b proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease. 495 recently, the entire genome of hepatitis b was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection. 509 hepatitis b can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses. 510 the woodchuck hepatitis virus was found to induce hepatocellular carcinoma. 511 within a population, 65-75% of all neonatal woodchucks are susceptible to chronic infection. 512 a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human infection takes much longer. 513 the acute infection strongly resembles what occurs during the course of disease in humans. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage. 514 challenge with virus in neonates leads to a chronic infection, while adults only develop the acute phase of disease. 515 a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. it was found to develop an acute hepatitis with a productive infection. 516 hepatitis d is dependent upon hepatitis b to undergo replication and successful infection in its human host. 517 there are two modes of infection possible between the viruses: coinfection in which a person is simultaneously infected or superinfection in which a chronic carrier of hepatitis b is subsequently infected with hepatitis d. 518 coinfection leads to a similar disease as seen with hepatitis b alone; however, superinfection can result in chronic hepatitis d infection and severe liver damage. 519 both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d. 520 a recently published report demonstrated the use of a humanized chimeric mouse to study the interactions between the two viruses and drug testing. 521 the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus be a low passage clinical isolate; thus, animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that which occurs in natural disease. to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the above characteristics cannot always be satisfied, however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy the food and fda's "animal rule." this rule applies to situations in which vaccines and therapeutics cannot safely or ethically be tested in humans; thus, licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures to be compared across institutions. this chapter has summarized the best models available for each of the viruses described. fields' virology pathogenesis of poliomyelitis: reappraisal in the light of new data one hundred years of poliovirus pathogenesis expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut present status of attenuated live-virus poliomyelitis vaccine world health organization. polio global eradication initiative annual report pathogenesis of human poliovirus infection in mice. ii. age-dependency of paralysis the poliovirus receptor protein is produced both as membranebound and secreted forms poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: agedependent paralysis and a mucosal route of infection establishment of a poliovirus oral infection system in human poliovirus receptor-expressing transgenic mice that are deficient in alpha/beta interferon receptor macaque models of human infectious disease ulnar nerve inoculation of poliovirus in bonnet monkey: a new primate model to investigate neurovirulence experimental poliomyelitis in bonnet monkey. clinical features, virology and pathology hepatitis a in day-care centers. a community-wide assessment persistence of hepatitis a virus in fulminant hepatitis and after liver transplantation acute liver failure: redefining the syndromes experimental hepatitis a virus (hav) infection in cynomolgus monkeys (macaca fascicularis): evidence of active extrahepatic site of hav replication experimental hepatitis a virus (hav) infection in callithrix jacchus: early detection of hav antigen and viral fate animal models of hepatitis a and e relative infectivity of hepatitis a virus by the oral and intravenous routes in 2 species of nonhuman primates wild malaysian cynomolgus monkeys are exposed to hepatitis a virus histopathological and immunohistochemical studies of hepatitis a virus infection in marmoset callithrix jacchus intragastric infection induced in marmosets (callithrix jacchus) by a brazilian hepatitis a virus (haf-203) experimental hepatitis a virus infection in guinea pigs systematic literature review of role of noroviruses in sporadic gastroenteritis norovirus infection as a cause of diarrhea-associated benign infantile seizures outbreak of necrotizing enterocolitis caused by norovirus in a neonatal intensive care unit progress in understanding norovirus epidemiology natural history of human calicivirus infection: a prospective cohort study foodborne viruses: an emerging problem pediatric norovirus diarrhea in nicaragua clinical immunity in acute gastroenteritis caused by norwalk agent experimental norovirus infections in nonhuman primates chimpanzees as an animal model for human norovirus infection and vaccine development experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses molecular characterization of three novel murine noroviruses persistent infection with and serologic cross-reactivity of three novel murine noroviruses [comparative study research support, n.i.h., extramural research support gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease porcine enteric caliciviruses: genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs infection of calves with bovine norovirus giii.1 strain jena virus: an experimental model to study the pathogenesis of norovirus infection an epizootic of equine encephalomyelitis that occurred in massachusetts in 1831 eastern equine encephalomyelitis virus: epidemiology and evolution of mosquito transmission vaccines and animal models for arboviral encephalitides fields virology studies on avian encephalomyelitis. ii. flock survey for embryo susceptibility to the virus the occurrence in nature of "equine encephalomyelitis" in the ring-necked pheasant eastern equine encephalitis virus infection: electron microscopic studies of mouse central nervous system a comparative study of the pathogenesis of western equine and eastern equine encephalomyelitis viral infections in mice by intracerebral and subcutaneous inoculations influence of age on susceptibility and on immune response of mice to eastern equine encephalomyelitis virus the hamster as an animal model for eastern equine encephalitiisdand its use in studies of virus entrance into the brain pathogenesis of aerosolized eastern equine encephalitis virus infection in guinea pigs eastern equine encephalitis. distribution of central nervous system lesions in man and rhesus monkey encephalomyelitis in monkeys severe encephalitis in cynomolgus macaques exposed to aerosolized eastern equine encephalitis virus pathology of animal models of alphavirus encephalitis common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains arbovirus investigations in argentina historical aspects and description of study sites western encephalitis in illinois horses and ponies medically important arboviruses of the united states and canada the ecology of western equine encephalomyelitis virus in the central valley of california an epizootic attributable to western equine encephalitis virus infection in emus in texas epidemiologic observations on acute infectious encephalitis in california, with special reference to the 1952 outbreak neurologic, intellectual, and psychologic sequelae following western encephalitis. a follow-up study of 35 cases louis encephalitis; preliminary report of a clinical follow-up study in california pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus necrotizing myocarditis in mice infected with western equine encephalitis virus: clinical, electrocardiographic, and histopathologic correlations the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone 15 variant viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control aerosol exposure to western equine encephalitis virus causes fever and encephalitis in cynomolgus macaques venezuelan equine encephalitis recovery of venezuelan equine encephalomyelitis virus in panama. a fatal case in man role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis mechanism of neuroinvasion of venezuelan equine encephalitis virus in the mouse comparative neurovirulence and tissue tropism of wild-type and attenuated strains of venezuelan equine encephalitis virus administered by aerosol in c3h/hen and balb/c mice c3h/hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection treatment of venezuelan equine encephalitis virus infection with (-)-carbodine studies on the virus of venezuelan equine encephalomyelitis. i modification by cortisone of the response of the central nervous system of macaca mulatta experimental studies of rhesus monkeys infected with epizootic and enzootic subtypes of venezuelan equine encephalitis virus aerosol infection of cynomolgus macaques with enzootic strains of venezuelan equine encephalitis viruses chikungunya outbreaksdthe globalization of vectorborne diseases infection with chikungunya virus in italy: an outbreak in a temperate region changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus chikungunya and the nervous system: what we do and do not know [review] a mouse model for chikungunya: young age and inefficient type-i interferon signaling are risk factors for severe disease an animal model for studying the pathogenesis of chikungunya virus infection chikungunya virus arthritis in adult wild-type mice a mouse model of chikungunya virus-induced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence mouse models for chikungunya virus: deciphering immune mechanisms responsible for disease and pathology chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages aedes albopictus in the united states: tenyear presence and public health implications epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century dengue: an update dengue haemorrhagic fever: diagnosis, treatment, prevention, and control tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic areadpuerto rico risk factors in dengue shock syndrome animal models of dengue virus infection manifestation of thrombocytopenia in dengue-2-virus-infected mice liver injury and viremia in mice infected with dengue-2 virus early activation of natural killer and b cells in response to primary dengue virus infection in a/j mice induction of tetravalent protective immunity against four dengue serotypes by the tandem domain iii of the envelope protein essential role of platelet-activating factor receptor in the pathogenesis of dengue virus infection murine model for dengue virus-induced lethal disease with increased vascular permeability lethal antibody enhancement of dengue disease in mice is prevented by fc modification inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type-2 replication genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells study of dengue virus infection in scid mice engrafted with human k562 cells dengue virus tropism in humanized mice recapitulates human dengue fever dengue virus infection and immune response in humanized rag2(ã�/ã�) gamma(c)(ã�/ã�) (rag-hu) mice a model of denv-3 infection that recapitulates severe disease and highlights the importance of ifn-gamma in host resistance to infection mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice studies on the pathogenesis of dengue infection in monkeys. 3. sequential distribution of virus in primary and heterologous infections studies on dengue 2 virus infection in cyclophosphamide-treated rhesus monkeys dengue virus-induced hemorrhage in a nonhuman primate model an in-depth analysis of original antigenic sin in dengue virus infection monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention the arboviruses: epidemiology and ecology screening of protective antigens of japanese encephalitis virus by dna immunization: a comparative study with conventional viral vaccines immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-japanese encephalitis virus (chimerivax-je) as a live, attenuated vaccine candidate against japanese encephalitis studies on host factors in inapparent infection with japanese b encephalitis: influence of age, nutrition and luminal induced sleep on the course of infection in mice relation of the peripheral multiplication of japanese b encephalitis virus to the pathogenesis of the infection in mice field's virology free radical generation by neurons in a rat model of japanese encephalitis sequential changes in serum cytokines and chemokines in a rat model of japanese encephalitis experimental infections of monkeys with langat virus. i. comparison of viremia following peripheral inoculation of langat and japanese encephalitis viruses intranasal infection of monkeys with japanese encephalitis virus: clinical response and treatment with a nuclease-resistant derivative of poly (i).poly (c) a neurotropic virus isolated from the blood of a native uganda isolation from human sera in egypt of a virus apparently identical to west nile virus isolation of west nile virus from culex mosquitoes the arboviruses: epidemiology and ecology centers for disease control & prevention. outbreak of west nile-like viral encephalitisdnew york origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states clinical virology the west nile virus outbreak of 1999 in new york: the flushing hospital experience nile feverda reemerging mosquito-borne viral disease in europe west nile viral encephalitis experimental infection of cats and dogs with west nile virus experimental infection of horses with west nile virus pathogenesis of west nile virus encephalitis in mice and rats. 1. influence of age and species on mortality and infection study on west nile virus persistence in monkeys experimental infection of pigs with west nile virus persistent west nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections experimental encephalitis following peripheral inoculation of west nile virus in mice of different ages immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys molecularly engineered live-attenuated chimeric west nile/dengue virus vaccines protect rhesus monkeys from west nile virus tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates phenotypic changes in langerhans' cells after infection with arboviruses: a role in the immune response to epidermally acquired viral infection? interleukin-1beta but not tumor necrosis factor is involved in west nile virusinduced langerhans cell migration from the skin in c57bl/6 mice profound and prolonged lymphocytopenia with west nile encephalitis innate and adaptive immune responses determine protection against disseminated infection by west nile encephalitis virus spinal cord neuropathology in human west nile virus infection animal models for sars world health organization. the world health report 2003d shaping the future stability and inactivation of sars coronavirus the severe acute respiratory syndrome clinical manifestations, laboratory findings, and treatment outcomes of sars patients identification of a novel coronavirus in patients with severe acute respiratory syndrome profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers the immunobiology of sars* viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome sars vaccines: where are we? animal models and vaccines for sars-cov infection replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice severe acute respiratory syndrome (sars): a year in review koch's postulates fulfilled for sars virus pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques macaque model for severe acute respiratory syndrome mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice resolution of primary severe acute respiratory syndromeassociated coronavirus infection requires stat1 virology: sars virus infection of cats and ferrets civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates pneumonitis and multi-organ system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus immunopathogenesis of coronavirus infections: implications for sars immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets primary severe acute respiratory syndrome coronavirus infection limits replication but not lung inflammation upon homologous rechallenge a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice rabies re-examined rabies and other lyssavirus diseases overview, prevention, and treatment of rabies studies of rabies street virus in the syrian hamster live attenuated rabies virus co-infected with street rabies virus protects animals against rabies spread and pathogenic characteristics of a g-deficient rabies virus recombinant: an in vitro and in vivo study biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique intracerebral vaccination suppresses the spread of rabies virus in the mouse brain human rabies therapy: lessons learned from experimental studies in mouse models experimental rabies virus infection of p.75 neurotrophin receptor-deficient mice apoptosis in experimental rabies in bax-deficient mice emerging pattern of rabies deaths and increased viral infectivity effective protection of monkeys against death from street virus by post-exposure administration of tissue-culture rabies vaccine a model in mice for the pathogenesis and treatment of rabies a model in mice for the study of the early death phenomenon after vaccination and challenge with rabies virus experimental rabies infection and oral vaccination in vampire bats (desmodus rotundus the distribution of challenge virus standard rabies 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fever viruses protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the syrian hamster model of lethal ebola virus infection pathogenesis of filoviruses in small animal models program abstr 5th int symp filoviruses pathogenesis of experimental ebola virus infection in guinea pigs characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation postexposure antibody prophylaxis protects nonhuman primates from filovirus disease aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques a small nonhuman primate model for filovirus-induced disease pathology of experimental ebola virus infection in african green monkeys. involvement of fibroblastic reticular cells pathogenesis of marburg hemorrhagic fever in cynomolgus macaques a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques recent progress in henipavirus research: molecular biology, genetic diversity, animal models transmission of human infection with nipah virus recurrent zoonotic transmission of nipah virus into humans nipah virus outbreak with person-to-person transmission in a district of bangladesh henipavirus susceptibility to environmental variables hendra virus infection in a veterinarian human hendra virus encephalitis associated with equine outbreak clinical features of nipah virus encephalitis among pig farmers in malaysia the emergence of nipah virus, a highly pathogenic paramyxovirus nipah virus encephalitis acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection a golden hamster model for human acute nipah virus infection histopathologic and immunohistochemical characterization of nipah virus infection in the guinea pig a guinea-pig model of hendra virus encephalitis the lesions of experimental equine morbillivirus disease in cats and guinea pigs a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a recombinant hendra virus g glycoproteinbased subunit vaccine protects ferrets from lethal hendra virus challenge vertical transmission and fetal replication of nipah virus in an experimentally infected cat susceptibility of cats to equine morbillivirus experimental infection of squirrel monkeys with nipah virus development of an acute and highly pathogenic nonhuman primate model of nipah virus infection a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment experimental nipah virus infection in pteropid bats (pteropus poliocephalus) bacterial infections in pigs experimentally infected with nipah virus experimental inoculation study indicates swine as a potential host for 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ferret model by an intranasal virosome-formulated influenza subunit vaccine local innate immune responses and influenza virus transmission and virulence in ferrets regional t-and b-cell responses in influenza-infected ferrets human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus [comparative study the ferret: an animal model to study influenza virus pathogenesis of avian influenza a (h5n1) viruses in ferrets severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction neuropathology of h5n1 virus infection in ferrets evaluation of three strains of influenza a virus in humans and in owl, cebus, and squirrel monkeys a computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from h5n1 infection evaluation of 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study influenza pathogenesis and immunity the cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/7/04 (h5n1) in different species of birds and mammals domestic pigs have low susceptibility to h5n1 highly pathogenic avian influenza viruses [comparative study research support animal models in influenza vaccine testing rift valley fever: an uninvited zoonosis in the arabian peninsula prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt rift valley fever the occurrence of human cases in johannesburg the pathogenesis of rift valley fever epidemic rift valley fever in egypt: observations of the spectrum of human illness crc handbook series in zoonoses rift valley fever virus in mice. i. general features of the infection the pathogenesis of rift valley fever virus in the mouse model the susceptibility of rats to rift valley fever in relation to age inbred rat strains mimic the disparate human response to rift valley fever virus infection the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis experimental rift valley fever in rhesus macaques development of a novel nonhuman primate model for rift valley fever crimean-congo hemorrhagic fever: a global perspective crimean-congo hemorrhagic fever the clinical pathology of crimean-congo hemorrhagic fever experimental congo virus (ib-an7620) infection in primates crimean congo hemorrhagic fever: a global perspective viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection a comparative study of the crimean hemorrhagic faverdcongo group of viruses [comparative study ribavirin efficacy in an in vivo model of crimean-congo 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phylogenetic relationships with human and other primate genotypes antibody to hepatitis-associated antigen. frequency and pattern of response as detected by radioimmunoprecipitation hepatitis and blood transfusion perspectives on hepatitis b studies with chimpanzees hla a2 restricted cytotoxic t lymphocyte responses to multiple hepatitis b surface antigen epitopes during hepatitis b virus infection primates in the study of hepatitis viruses transfer of hbv genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice asymmetric replication of duck hepatitis b virus dna in liver cells: free minus-strand dna a virus similar to human hepatitis b virus associated with hepatitis and hepatoma in woodchucks effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection hepadnavirusinduced liver cancer in woodchucks animal models of hepadnavirus-associated hepatocellular carcinoma hepatitis b viruses and hepatocellular carcinoma hepatitis b virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model animal models of hepatitis delta virus infection and disease experimental hepatitis delta virus infection in the chimpanzee expression of the hepatitis delta virus large and small antigens in transgenic mice experimental hepatitis delta virus infection in the animal model humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation key: cord-342054-1u2fkwx3 authors: funaro, ada; gribaudo, giorgio; luganini, anna; ortolan, erika; lo buono, nicola; vicenzi, elisa; cassetta, luca; landolfo, santo; buick, richard; falciola, luca; murphy, marianne; garotta, gianni; malavasi, fabio title: generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune b cells date: 2008-11-12 journal: bmc biotechnol doi: 10.1186/1472-6750-8-85 sha: doc_id: 342054 cord_uid: 1u2fkwx3 background: human monoclonal antibodies (mabs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective. human cytomegalovirus (hcmv) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. the available therapeutic armamentarium (e.g. hcmv hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mab may be a decisive alternative in the prevention of primary and re-activated hcmv infections in these patients. results: the purpose of this study was to generate neutralizing mab against hcmv from the immunological repertoire of immune donors. to this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human b-lymphocytes are stimulated with tlr9-agonists and il-2; second, after both additives are removed, the cells are infected with ebv. using this strategy we obtained 29 clones secreting igg neutralizing the hcmv infectivity; four among these were further characterized. all of the mabs neutralize the infection in different combinations of hcmv strains and target cells, with a potency ~20 fold higher than that of the hcmv hyperimmune globulins, currently used in transplant recipients. recombinant human monoclonal igg1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. conclusion: the technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mabs for immunotherapeutic uses. antibodies constitute the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines [1] . most of the growing number of antibodies entering the clinical trials are human [2] and are derived from phage-display technology [3] or from transgenic mice that express human immmunoglobulin genes [4] . however, the best mabs for clinical applications derive from natural human antibodies generated as a result of the in vivo immune response because they i) are products of the human and not animal repertoire, and ii) are of human origin, thus minimizing the risks of reactivity against self-antigens. lastly, iii) passive immunotherapy with human igg can confer immediate protection without the side effects linked to the use of chimeric or humanized mabs containing animal-derived amino acid sequences. furthermore, considerable evidence indicates that antibodies represent a new, although historically validated, approach to the development of therapies against bacterial and viral pathogens that causes disease in individuals with impaired immune response and/or for which there are few or no available drugs [5] [6] [7] [8] . ebv has long been used to immortalize human b-lymphocytes in vitro and to isolate human mab [9] [10] [11] , but the method was not routinely embraced due to its poor efficiency and because the resulting transformed cells are frequently unstable and fail to grow or secrete igg. agonists of toll receptor 9 (tlr9), e.g., cpg odn 2006 (synthetic oligodeoxynucleotides that contain immunostimulatory deoxycytidyl-deoxyguanosine motifs), have been reported to improve ebv infectivity and cloning efficiency [12] , however, there is no clear consensus as to the best conditions for their use and which subset of b-lymphocytes should be used. we applied the ebv immortalization procedure in the presence of different amounts of cpg with or without il-2 to 3 healthy donors selected on the basis of the high titer of cmv-specific igg in the blood and we obtained an immortalization efficiency not comparable to that recently reported [12] . therefore we re-evaluated all the steps crucial to cell immortalization, and established an optimized procedure of isolating human mabs from the repertoire of immune donors. the resulting method is highly reproducible, effective and rapid. further, it was validated by the successful isolation of clones of human b-lymphocytes that secrete high levels of igg that bind and neutralize the human cytomegalovirus (hcmv). hcmv is a ubiquitous opportunistic virus that infects 50-90% of adult human populations and persists for the life of the human host after primary infection. hcmv infection of an immunocompetent individual generally results in subclinical disease; by contrast, hcmv infection of immunocompromised individuals may cause lethal infections [13] . indeed, hcmv remains the single most impor-tant pathogen in hematopoietic stem cell transplantation [14] as well as, in organ transplantation [15] so that effective neutralizing mabs would make a major contribution to eliminate hcmv in these patients. the isolation of fully human mab from those generated during the natural human immune response is likely to identify the most effective therapeutic antibodies [6, 16] . many approaches have been used to generate human mab [17] , however, an efficient and reproducible process to isolate human mab with desired specificity from the natural repertoire is not available. agonists of tlr9 have been reported to improve ebv infectivity and cloning efficiency [12] . however, it is known that tlr9 agonists, such as cpg odn 2006, protect against a wide range of viral pathogens [18] [19] [20] . with this in mind, we reasoned that the presence of cpg during the ebv infection might exert negative effects and we decided to separate activation and ebv infection in two sequential phases. we developed a technology for raising human mabs for clinical applications based on two discrete steps (sequential, figure 1 ): first, purified human b-lymphocytes are treated with cpg2006 and il-2. next, after both additives are removed, the activated cells are infected with ebv in a distinct step. the new process was compared to a method using cpg2006 and il-2 in the presence of ebv [12] (combined) and a method using outline of the procedure for comparing ebv transformation methods figure 1 outline of the procedure for comparing ebv transformation methods. overview of the procedure for comparing the populations of ebv-transformed cells according to the sequential, combined, and basic methods. ebv alone (basic) (figure 1 ). different experimental settings were compared in pools of cells derived from 5 healthy donors, to minimize donor variability. the results clearly demonstrated that activation of cd22 + b-lymphocytes before ebv exposure leads to improved immortalization, as highlighted by the viability of cells following ebv transformation which is significantly higher than that obtained with both combined and basic conditions (figure 2a) . the short-term viability of b lymphocytes immortalized following the basic procedure is higher than those of b lymphocytes treated with the combined procedure. this is attributable to the polyclonal activation elicited by ebv itself which is not paralleled by an efficient immortalization. indeed, it is well documented that exposure of human b-lymphocytes to ebv results in cell death of a proportion of cells and that b-lymphocytes are activated but not immediately immortalized following exposure to ebv [21] [22] [23] . removal of the activating agents before immortalization significantly improves growth and viability of cd22 + blymphocytes allowing for a higher proportion of cells to be immortalized. indeed, when stimulation and ebv infection are performed separately rather than simultaneously, or with no stimulation at all, the cells exhibit increased proliferation potential and improved viability. in contrast, the combined presence of activating agents and ebv has adverse effects on both cell viability and ebv infectivity (figure 2a , b). the negative effects of the com-bined method on viability are surprising, given that cpg2006 induces robust polyclonal activation and proliferation of b-lymphocytes [24, 25] and that these effects are significantly enhanced by il-2 [26] . it has been reported that tlr-9 triggering resulted in higher transformation rates of b cells infected with ebv [12] but a comparison between ebv transformation in the presence of cpg2006 and ebv and after sequential exposure of b cells to cpg2006 and separately to ebv, was not investigated. the results obtained using different experimental setting on the same cell population clearly demonstrated that cpg2006 and il-2 used in combination with ebv exert negative effects on cell transformation. it is conceivable that the detrimental effects are related to the anti-proliferative activity of anti-viral cytokines induced by cpg2006 [27, 28] . indeed, the significant levels of tlr9 expressed by resting b-lymphocytes are further up-regulated by cpg2006 itself. increased tlr9 expression correlates with increased responsiveness to the ligand, indicating a positive feedback loop in which cpg2006 enhances its own effects [29] . kinetic studies demonstrated that optimal concentrations of cpg2006 combined with il-2 provide maximal cell stimulation after 2-5 days (data not shown). these observations are in line with cell-cycle analyses demonstrating that, in the same time frame, more than quantitative and qualitative comparison of different ebv transformation methods figure 2 quantitative and qualitative comparison of different ebv transformation methods. a) the b-lymphocyte subsets were prepared using the sequential, combined or basic methods, as outlined in fig. 1 . ten days after exposure to ebv, samples of each population were compared for the total cell number (black bars), and for the number of viable cells (white bars) measured microscopically by trypan blue dye exclusion. data are expressed as % of initial b cells exposed to ebv and represent the means ± s.d. of 5 cell counts for each condition. results are representative of two independent experiments. p < 0.001: sequential vs. combined; basic vs. combined (white bars); p < 0.05: sequential vs. basic (white bars) and sequential vs. combined; and basic vs. combined (black bars). b) ten days after infection with ebv, the cells prepared by the sequential, combined, or basic method were analyzed to identify viable lymphoblasts by propodium iodide (pi) exclusion (top panels) and cd23 expression (bottom panels) by flow cytometry. for each population of cells, the viable lymphoblasts (gated in regions r2, top panels) were defined as those with relatively high forward scatter (horizontal axes) and negative for pi fluorescence (vertical axes). r2 gated cells were then analyzed for cd23 expression. fluorescence was analyzed with a facscalibur flow cytometer and cellquest software (becton dickinson). background fluorescence was determined with a fitc-labeled, isotype-matched negative control mab. 10,000 events were measured for each condition. 95% of b cells enter the cell cycle independently from their memory or naïve phenotype [24, 30] . the sequential method does not modify the expression of cd21, which is the receptor for ebv [31] . the next step was to correlate the efficiency of ebv infection to the expression of cd23, which is directly upregulated by ebna2, one of the earliest markers expressed by ebv-infected b-lymphocytes [32] , it is expresses at high levels on ebv-transformed b cells [33] and its expression correlates with igg secretion [34] . the results demonstrated that cd23 expression (the number of positive cells and mean fluorescence intensity) is higher on cells exposed to the polyclonal activators and ebv separately than on those exposed simultaneously ( figure 2b ). the b-lymphocytes representing the whole repertoire of a selected donor immortalized with the sequential method show a significantly higher viability than those obtained with the combined method, and in consequence are easily clonable by limiting dilution, either in the presence or in the absence of polyclonal activators, such as cpg2006 and il-2. tlr9 expression is increased after ebv infection. thus bona fide, the addition of cpg2006 during the cloning phase enhances cell growth. however, we observed that cloning efficiency may be independent of cpg2006 and il-2 in some donors, suggesting that it is also dependent on the genetic background or physiological state of the donor. this issue warrants further investigation. the presence of cpg2006 and il-2 during the cloning phase may also have practical relevance for screening procedures. for instance, trace amounts of cpg2006 completely block hcmv infection in vitro, hampering screening by functional assays [20] . moreover, cpg2006 is a potent inducer of cytokines, such as il-12 and ifn-γ, potentially interfering with a number of bioassays [35] . the validity of the sequential method was evaluated by using it in a project to produce neutralizing human mabs against hcmv, a leading cause of morbidity and mortality in immunocompromised individuals and the most serious pathogenetic agent in transplant patients [15, 36, 37] . in the absence of other therapies such as vaccines [38] , these patients are treated with antivirals, which are often associated with considerable side effects and the emergence of drug-resistant strains. hcmv is also the leading viral cause of congenital infection associated to significant birth defects and neurological damage for which no effective therapies are available before birth [13, 39] . hyperimmune globulins specific for hcmv are beneficial in both conditions for the prevention and treatment of these infections [40, 41] . further, transfer of memory b cells from immune animals to severely immunodeficient recipients confers long-term protection from lethal murine cmv infection. this indicates that the humoral immune response is effective against cmv infection in the absence of a t cell-mediated response [42] . natural human mabs with stronger neutralizing activity than that of currently available immunoglobulin preparations may be a decisive alternative in the prevention of primary and re-activated hcmv infections in immunosuppressed patients, or during high-risk pregnancies [43] . using the sequential method, we obtained natural antibodies neutralizing hcmv infectivity by immortalizing b-lymphocytes from a healthy donor (cmv5) and from a patient recovering from acute hcmv infection (cmv7). high titers of anti-hcmv-specific igg coupled with the virus-neutralizing activity of the sera were the criteria for selecting the two donors from a larger panel ( table 1 ). the immortalized b-lymphocytes were cloned by limiting dilution. after 3 weeks, clusters of cells growing in wells were appreciable and the igg concentration in the clone supernatants was 10-40 μg/ml/10 6 cells, as determined by means of an immunoenzymatic method, measuring igg concentrations in the media normalized for cell densities. clones producing hcmv-specific igg were identified by different primary screening assays. in a first experiment a total of 1,664 clones were tested either by their binding to specific hcmv envelope glycoprotein domains (such as those from gb or gh, which have been shown to induce neutralizing antibodies) [44] , or by their ability to neutralize hcmv infectivity in vitro [45, 46] . the effect on hcmv infectivity was determined by an hcmv microneutralization assay based on the evaluation of the ability of clone supernatants to interfere on the infectivity of hcmv ad169 strain (a laboratory strain from atcc, cod. vr-538) in human embryo lung fibroblasts (helf). this strategy yielded 44 clones producing mabs specific for the gb, and 15 for gh. nine of these displayed a neutralizing activity of > 40%. those supernatants that were negative in the gb and gh elisas, or in the neutralization assay were also screened in an elisa that detects human igg that bind to hcmv virion proteins extract (beia-cmv), and 8 samples were further selected by this screening assay. in a second experiment, the immortalized blymphocytes were cloned by limiting dilution and after 3 weeks, 324 clones were screened directly for their neutralizing activity. of these, 20 produced igg with a neutralizing activity of > 40%, among these, 2 wells were found to bind gb, while the remaining 18 did not bind in either the gb or gh domains. four clones has been chosen as promising candidates for future clinical applications on the basis of their intrinsic characteristics: 10b7 and 8c10 showed a very strong binding to gb glycoprotein and 1f7 to gh glycoprotein; 26a1 has the strongest neutralizing activity. all clones were expanded in the absence of cpg2006, il-2 and a feeder layer, and further characterized. mabs10b7 and 8c10 proved to be specific for the hcmv envelope glycoprotein gb, mab 1f7 for the hcmv envelope glycoprotein gh and mab 26a1 recognized a viral antigen not yet characterized, as inferred from the reactivity on the hcmv virion proteins extract ( table 2 ). all of the antibodies neutralized the infectivity of the hcmv ad169 laboratory strain in human helf as well as that of the vr1814 clinical isolate in human umbilical vein endothelial cells (huvec), indicating that they are neither strain nor cell-specific. lastly, none of the antibodies bound the helf or huvec (data not shown). the specificity of the hcmv neutralizing activity was confirmed by the lack of significant neutralization of herpes simplex virus (hsv)-1 in vero cells (data not shown). clone stability was tested by maintaining the cells for 3 months in serum-free medium without cpg2006 or other additives, and the mabs recovered in the supernatant at a concentration of 20-50 μg/ml. all purified igg maintained their neutralizing activity; mab 26a1 demonstrated a potent neutralizing capacity, with a calculated inhibitory concentration 50 (ic 50 ) of 0.92 μg/ml against ad169 in helf (fig. 3a) , and of 1 μg/ml against vr1814 in huvec (fig. 3b) . the potency of 26a1 against both hcmv strains was ~20 fold higher than the reference anti-hcmv igg (ic 50 of 16 μg/ ml), currently used in transplant recipients. the inhibitory activity of mab 26a1 against the whole viral replicative cycle was confirmed by plaque reduction neutralization assay ( figure 3c ). the mechanism of virus neutralization is subject of ongoing studies. the broad and robust neutralizing activity of mab 26a1 encouraged us to generate recombinant igg suitable as prophylactic or therapeutic tools in clinical applications. to this aim, cells were submitted for 5' race pcr amplification, the pcr products of two amplification reactions were cloned using a eco ri restriction site in a sequencing vector and used for transforming top10 e. coli. a single vh and vl sequence was obtained from each cell culture, confirming that each original culture was clonal ( figure 4 ). the sequences encoding the vh and vl regions of mab 26a1 were cloned in expression vectors for the expression of mab 26a1 variable regions as recombinant igg1. the recombinant mab 26a1 confirmed the neutralizing activity of the natural mab 26a1 (figure 3 ). the sequential method is also effective when used with frozen cells. indeed, it was possible to select antibodies neutralizing hsv-1 and hsv-2 infectivity from frozen pbmcs from an hsv and hiv co-infected individual (not shown). neutralizing activity of natural (n) and recombinant (m) purified 26a1 igg in summary, this study provides proof of concept that good-quality, fully human mabs with desired specificity and biological activity can be generated with the sequential method. the strengths of this approach are: i) it allows the selection of human monoclonal igg to a variety of antigens, from a small sample of fresh or frozen peripheral blood, ii) it is rapid, iii) screening can be performed using a variety of assays, including functional assays, iv) the mabs of interest can be easily produced from the original clone as recombinant proteins suitable for clinical applications, and v) the generation of iggsecreting polyclonal populations can be considered as a library of antibody-secreting cells that can be used to select mabs with specificities not considered when cells were immortalized. the technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mabs for immunotherapeutic uses. it can be applied to the isolation of natural mabs from individuals affected by a variety of diseases, ranging from infectious diseases to cancer and autoimmune diseases. it allows the isolation of a large repertoire of human mabs with desired specificities to be selected from a small (~8 ml) volume of peripheral blood, and can be used with either fresh or frozen b-lymphocytes. moreover, direct cloning by limiting dilution and robust igg production allow the generation of panels of mabs with different specificities, thus allowing the development of mab cocktails for immunotherapy [47] . after informed consent was obtained, peripheral blood (8-10 ml) was collected from healthy blood donors or infectious disease patients. serum was screened for reactivity with hcmv glycoproteins and for hcmv neutralizing activity. peripheral blood mononuclear cells (pbmcs) were purified from heparinized peripheral blood by density gradient centrifugation on ficoll/hypaque (amersham pharmacia, milan, italy). cd22 + lymphocytes were purified by magnetic selection (miltenyi biotec, bologna, italy) from pbmcs pooled from 5 healthy donors, then cd22 + cells were divided into three aliquots that were exposed to different ebv-based methods for immortalization. for the basic process, cd22 + /igg + lymphocytes were isolated by depletion of igm + cells with a moflo ® high-performance cell sorter (dakocytomation, glostrup, denmark) and cultured (10 6 /ml) in 24 well plates in iscove's modified dulbecco's medium (imdm) supplemented with l-glutamine, 100 μg/ml streptomycin and 100 u/ml penicillin, non-essential amino acids and 10% fbs (complete medium) for 15 h, in the presence of ebv-containing supernatant (from b95-8 marmoset lymphoma cells, atcc, washington, dc, cat. no. crl-1612) 1:1 (v/v) at 37°c and 5% co 2 . the cells were then washed c. sera were tested in the hcmv microneutralization assay using the ad169 strain and helf [48] . numbers indicate dilution of serum required for 50% inhibition of hcmv infectivity. the number of viable lymphoblasts was measured microscopically by trypan blue dye exclusion. where indicated, the dna intercalating, fluorescent dye propidium iodide (pi, sigma aldritch) was used with a facscalibur flow cytometer and cellquest software (becton dickinson, franklin lakes, nj) to assess cell viability. viable cells were defined as those with a high forward and orthogonal scatter, characteristic of lymphoblasts, and excluding pi. min at 4°c with anti-human cd23-fitc (becton dickinson, milano, italy). after washing, fluorescence was analyzed using a facscalibur flow cytometer and cellquest software. cd23 staining was evaluated only on the viable lymphoblast population, gated using forward/orthagonal scatter and pi staining, as described above. background staining was determined with a fitc-conjugated isotypematched negative control mab. the number of cells analyzed was 10,000. the microneutralization assay was adapted from a previously reported technique [48] . the effect of b cell supernatants (or purified igg) on hcmv infectivity was measured by staining the hcmv immediate early antigens (iea, ie1+ie2) by indirect immunoperoxidase. iea-positive nuclei were counted under the microscope. b cell supernatants containing irrelevant igg antibodies were used as a negative control and a commercial preparation of human igg, purified from the serum of hcmv seropositive patients (cytotect; biotest, dreieich, germany), as a positive control, respectively. positivity was defined as ≥ 40% inhibition of iea + cells, compared to negative control wells. hcmv microneutralization assays were also performed with the endotheliotropic hcmv vr1814, a derivative of a clinical isolate recovered from a cervical swab of a pregnant woman [49] , and huvec, as described above. experiments were performed with cells at passage 2-6. plaque reduction assay was performed as reported [50] . selected experiments were done with igg purified on protein a columns (biorad, milano, italia) from serum-free supernatants (natural igg). antibodies were tested using the elisa assay for detection of igg binding to total proteins extract from hcmv virions (beia cmv igg quant kit; bouty, milano, italy) according to the manufacturer's instructions. the anti-hcmv recombinant gb igg elisa was purchased from biotest, ag (dreieich, germany) and used according to the manufacturer's instructions. the elisa makes use of the recombinant autologous interstrain fusion antigen cg3, corresponding to a combination of the gb antigenic domain 2 (ad2) from hcmv strains ad169 (swissprot acc. no. p06473) and towne (swissprot acc. no. p13201). the ad2 region contains a site (amino acids 70-81) conserved in different viral strains, that has been shown to be recognized by neutralizing antibodies [51, 52] . the gb-gst antigen was purchased from biodesign (saco, me, usa) and contains a gb immunodominant region fused to gst that reacts with hcmv-positive sera. it was used in elisa assays according to the manufacturer's instructions. the anti-hcmv recombinant gh-specific elisa was performed using the recombinant gh-gst antigen coated onto plastic. gh-gst corresponds to an in-frame fusion between the gh amino terminal region (amino acids 16-144) from the hcmv strain vr1814 and gst. the amino terminus of gh contains a linear antibody binding site between residues 34-43 that is recognized by neutralizing antibodies [44, 53] . the gh gene segment was pcr amplified from vr1814 dna with the following primers: vr1814 gh f: 5'-agtcatggatccctccttagtcac-ctca-3'; vr1814 gh r: 5'-actgatctcgaggcct-tcagctgctgc-3'. the 400 bp pcr product was then digested by xhoi and bamhi, the restricted fragment purified by agarose gel elecrophoresis and cloned into the pgex 4t3 expression vector (amersham) that had been restricted by xhoi and bamhi and gel purified. the recombinant gh-gst fusion protein was then expressed in e. coli bl21 and purified from the bacterial cell lysate on the basis of gst affinity. the variable regions of heavy chain (vh) and light chain (vl) of mab 26a1 were sequenced according the technology established by fusion antibodies ltd. briefly, frozen cells pellets (~5 × 10 4 cells) were used for extracting total rna with stat-60 rna extraction reagent. cdna was produced by reverse transcription with an oligo(d)t primer. pcr reactions were set up to amplify vh and vl regions with a mix of igg-and igk/λ specific primers, respectively. the pcr products of two amplification reactions were cloned using a eco ri restriction site in a sequencing vector (pcr2.1; invitrogen) and used for transforming top10 e. coli cells, following the manufacturer's instructions. a consensus sequence was determined from at least ten clones. the resulting dna sequences were aligned and translated into protein sequences enabling the generation of consensus dna and protein sequence for vh 26a1 and vl 26a1. the vh 26a1 and vl 26a1 protein sequences were compared and aligned with sequences present in the genomequest, geneseq, and ebi databases. the cdrs characterizing vh 26a1 and vl 26a1 protein sequences were predicted by the imgt database [54] . to generate a recombinant igg1, a consensus 26a1 vh sequence was amplified by pcr with the following primers: 5'-ttttttagatctcaccatgaacatactgtggag-catgctc-3'; 5'-ttttttagatcttgaggagacggtga ccagggttcc-3'. the primers contained a bglii restriction site (underlined) for ligation into the higg expression vector already containing human igg1 heavy chain constant domains. the in-house designed bicistronic mammalian retroviral expression vector pretmex (fusion antibodies ltd. belfast, ireland) was a dual promoter vector allowing expression of both antibody chains. the vh domain was cloned into the bamh1 site of multiple cloning site (mcs) 1 of the vector. a complete 26a1 recombinant vector was confirmed by dna sequencing. cho dg44 cells were adapted to serum-free suspension culture and seeded at 1 × 10 6 cells/ ml in 125 ml spinner flasks. 300 μg of plasmid dna was mixed with 900 μg of linear 25 kda pei and used to transfect the cell culture. the medium was harvested after 6 days. recombinant mab was purified with a protein g column on an akta prime chromatography unit following the manufacturer's standard programme (recombinant igg). values are expressed as means ± sd. data were analyzed for significance using a one-way analysis of variance (anova) with bonferroni post-test correction for multiple comparisons. data were considered significant at p < 0.05. therapeutic antibodies for human diseases at the draw of the twenty-first century monoclonal antibody successes in the clinic selecting and screening recombinant antibody libraries human antibodies from transgenic animals antibodies for the prevention and treatment of viral diseases passive antibody therapy for infectious diseases prophylactic and therapeutic efficacy of human monoclonal antibodies against h5n1 influenza human monoclonal antibodies by immortalization of memory b cells eb virus-induced b lymphocyte cell lines producing specific antibodies human monoclonals from antigen-specific selection of b lymphocytes and transformation by ebv synergy between anti-cd40 mab and epstein-barr virus in activation and transformation of human b lymphocytes an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus the human cytomegalovirus high risk of death due to bacterial and fungal infection among cytomegalovirus (cmv)-seronegative recipients of stem cell transplants from seropositive donors: evidence for indirect effects of primary cmv infection acceleration of allograft failure by cytomegalovirus human antibodies for immunotherapy development generated via a human b cell hybridoma technology potent antibody therapeutics by design local delivery of cpg oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication, but not entry, of herpes simplex virus type 2 immunotherapeutic uses of cpg oligodeoxynucleotides phosphorothioatemodified oligodeoxynucleotides inhibit human cytomegalovirus replication by blocking virus entry innate apoptosis of human b lymphoblasts transformed by epstein-barr virus: modulation by cellular immortalization and senescence epstein-barr virus provides a new paradigm: a requirement for the immediate inhibition of apoptosis steps involved in immortalization and tumorigenesis in human b-lymphoblastoid cell lines transformed by epstein-barr virus cpg motifs in bacterial dna trigger direct b-cell activation delineation of a cpg phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo activation of human b cells by phosphorothioate oligodeoxynucleotides interferon-alpha/beta receptor-mediated selective induction of a gene cluster by cpg oligodeoxynucleotide immune activation suppresses initiation of lytic epstein-barr virus infection the tolllike receptor repertoire of human b lymphocytes: inducible and selective expression of tlr9 and tlr10 in normal and transformed cells mechanism and function of a newly identified cpg dna motif in human primary b cells determination of the role for cd21 during epstein-barr virus infection of b-lymphoblastoid cells epstein-barr virus-induced b-cell transformation: quantitating events from virus binding to cell outgrowth b cell activation and the establishment of epstein-barr virus latency cell surface phenotyping and cytokine production of epstein-barr virus (ebv)-transformed lymphoblastoid cell lines (lcls) a minimal human immunostimulatory cpg motif that potently induces ifn-gamma and ifn-alpha production severe graft rejection, increased immunosuppression, and active cmv infection in renal transplant prophylaxis and treatment of cytomegalovirus disease in recipients of solid organ transplants: current approach and future challenges location, location, timing: analysis of cytomegalovirus epitopes for neutralizing antibodies preconceptional primary human cytomegalovirus infection and risk of congenital infections passive immunization during pregnancy for congenital cytomegalovirus infection intravenous immunoglobulin: appropriate indications and uses in hematopoietic stem cell transplantation protection from cmv infection in immunodeficient hosts by adoptive transfer of memory b cells the growth and potential of human antiviral monoclonal antibody therapeutics antibody-mediated neutralization of infectivity a model for neutralization of viruses based on antibody coating of the virion surface the antiviral activity of antibodies in vitro and in vivo recombinant human polyclonal antibodies: a new class of therapeutic antibodies against viral infections a rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus human cytomegalovirus immediate-early messenger rna in blood of pregnant women with primary infection and of congenitally infected newborns human cytomegalovirus stimulates cellular ikk2 activity and requires the enzyme for productive replication assembly of conformationdependent neutralizing domains on glycoprotein b of human cytomegalovirus an elisa using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus the dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific imgt/v-quest, an integrated software program for immunoglobulin and t cell receptor v-j and v-d-j rearrangement analysis the authors would like to thank am gianni, m di nicola and a lazzarin for providing blood samples from cmv + patients, al horenstein for assistance with antibody purification, and g gerna for providing the hcmv vr1814 strain. gerrit hagens and guido poli are acknowledged for helpful discussion and intellectual contributions. this work was funded, in part, by grants from ribovax biotechnologies sa (geneva, switzerland), from miur (italian ministry for university and scientific research) (prin and 60% projects), from ricerca sanitaria finalizzata e ricerca scientifica applicata (regione piemonte), from compagnia sanpaolo (torino, italy) and from firms (international foundation for research in experimental medicine). a. funaro, m. murphy and g. garotta are inventors on a pct patent application (published as wo 2007/068758) describing the technology. a funaro, g. gribaudo and s. landolfo are inventors on unpublished patent applications describing specific hcmv-neutralizing antibodies that were isolated using the described technology. all patent applications are filed in the name of ribovax biotechnologies sa (petit-lancy, switzerland).data were analyzed for significance using a one-way analysis of variance (anova) with bonferroni post-test correction for multiple comparisons. data were considered significant at p < 0.05. af, ggr, mm and gga designed the study. al carried out the neutralization assays, nlb carried out the immunoassays, eo participated to the analysis and interpretation of data. ev, lc. selected antibodies neutralizing hsv-1 and hsv-2, sl and lf contributed to design the study. rb prepared the recombinant 26a1 igg, and af and fm wrote the paper. all authors read and approved the final manuscript. key: cord-306904-8iteddug authors: uversky, vladimir n title: unreported intrinsic disorder in proteins: building connections to the literature on idps date: 2014-12-12 journal: intrinsically disord proteins doi: 10.4161/21690693.2014.970499 sha: doc_id: 306904 cord_uid: 8iteddug this review opens a new series entitled “unreported intrinsic disorder in proteins.” the goal of this series is to bring attention of researchers to an interesting phenomenon of missed (or overlooked, or ignored, or unreported) disorder. this series serves as a companion to “digested disorder” which provides a quarterly review of papers on intrinsically disordered proteins (idps) found by standard literature searches. the need for this alternative series results from the observation that there are numerous publications that describe idps (or hybrid proteins with ordered and disordered regions) yet fail to recognize many of the key discoveries and publications in the idp field. by ignoring the body of work on idps, such publications often fail to relate their findings to prior discoveries or fail to explore the obvious implications of their work. thus, the goal of this series is not only to review these very interesting and important papers, but also to point out how each paper relates to the idp field and show how common tools in the idp field can readily take the findings in new directions or provide a broader context for the reported findings. disorder. this series serves as a companion to "digested disorder" which provides a quarterly review of papers on intrinsically disordered proteins (idps) found by standard literature searches. the need for this alternative series results from the observation that there are numerous publications that describe idps (or hybrid proteins with ordered and disordered regions) yet fail to recognize many of the key discoveries and publications in the idp field. by ignoring the body of work on idps, such publications often fail to relate their findings to prior discoveries or fail to explore the obvious implications of their work. thus, the goal of this series is not only to review these very interesting and important papers, but also to point out how each paper relates to the idp field and show how common tools in the idp field can readily take the findings in new directions or provide a broader context for the reported findings. recent years evidence an exponential increase in the number of papers on intrinsically disordered proteins (idps), indicating that the phenomenon of protein intrinsic disorder is becoming a popular research topic. this point is also reflected in the articles of the "digested disorder" series published in this journal. [1] [2] [3] to make this point stronger some interesting numbers are provided below. pubmed search (as of august 03, 2014) for ((intrinsically disordered) or (natively unfolded) or (intrinsically unstructured) or (intrinsically unfolded) or (intrinsically flexible protein)) returned 2,490 hits. restricting this search to the past one and a half years only (i.e., searching for ((intrinsically disordered) or (natively unfolded) or (intrinsically unstructured) or (intrinsically unfolded) or (intrinsically flexible protein)) and ("2013/01/ 01"[date -publication]: "3000"[date -publication])) gives 659 hits. curiously, although papers on idps (2,490) constitute just 0.045% of all the protein-related papers (5, 569, 481) published during the past 115 years, the fraction of idprelated papers increases to 0.18% if only publications of the past year and a half are taken into account. it is of interest to compare these trends with the related tendencies of the research on proteins in general. protein-related papers (5, 569, 481) constitute »23% of all the papers published during the past 115 y and annotated in pubmed (24, 068, 783) . however, this number decreases to »21% if only papers published during the past year and a half are taken into account (in 2013-2014, the overall number of published papers is 1,719,568; the number of papers dealing with proteins is 362,612). this suggests that an idp is a new rising star in the field of protein science. despite this steady and sure increase in the appreciation of protein intrinsic disorder, there are still numerous instances when this concept is overlooked, or missed or simply ignored. unfortunately, such "missed disorder" phenomenon continues to be rather common in the modern literature. the original goal of the first article in the new "unreported disorder in proteins" series was to find several papers published in different journals that talk about idps (or hybrid proteins containing both ordered and disordered regions) not recognizing that they are talking about such proteins, to show how consideration of intrinsic disorder can be used to strengthen conclusions and draw relationships to prior discoveries in the field. however, analysis of recent publications in just 2 journals revealed that the "missed disorder" happened to be essentially more abundant than it was originally expected. in fact, as of august 3, 2014, of 677 papers published during 2013-2014 in the journal of molecular biology, 609 were dealing with proteins, with 87 papers being dedicated to the idps or hybrid proteins. papers were considered to be dealing with intrinsic disorder if their texts contained at least a single mention of disorder, flexibility, conformational flexibility, or unfoldedness in relation to the protein of interest or any of its regions. obviously, this is a very relaxed and inclusive approach, since simple mentioning of disorder or conformational flexibility is not sufficient for the detailed elucidation of the functional role of disorder/flexibility. the direct pubmed search for the idprelated papers published in the journal of molecular biology using the search criteria ((intrinsic disorder) or (intrinsically disordered) or (natively unfolded) or (intrinsically unstructured) or (intrinsically unfolded) or (intrinsically flexible protein)) and ("2013/01/01" [date -publication]: "3000"[date -publication]) and ("journal of molecular biology"[journal]) generated just 17 hits. analysis of the table of content of this journal combined with a brief computational analysis of related proteins revealed that there are at least 22 more papers, research subject of which are idps. analogous analysis of the publications in biochemistry during the 2013-2014 provided comparable data: of 1455 published papers, 25 were "officially" dedicated to intrinsic disorder or to intrinsically disordered/natively unfolded/intrinsically unstructured/ intrinsically unfolded/intrinsically flexible proteins. mining of papers published in biochemistry during 2013-2014 revealed that some 189 papers contained at least a single mention of "conformational flexibility" or "intrinsic disorder" or "disordered protein" or "disordered peptide" or "disordered region" or "natively unfolded" in relation to the protein of interest or any of its regions. furthermore, based on the combination of literature mining and a brief computational analysis 20 "hidden gems" were found; i.e., papers that missed protein disorder. the mentioned 42 papers dealing with the unreported intrinsic disorder and their related idps or hybrid proteins containing ordered and intrinsically disordered regions are briefly outlined below. table 1 contains all the proteins considered in this review. proteins are arranged according to the increase in the extent of their disorder evaluated as percentage of the residues predicted to be disordered (i.e., possessing disorder scores above 0.5) by pondr ò vsl2, which is among the more accurate disorder predictors. 4 table 1 shows that the extent of unreported intrinsic disorder in 143 proteins ranges from 10% to 100%, indicating that all these proteins belong to the category of moderately or highly disordered proteins; i.e., proteins with the disorder content ranging from 10% to 30% and from 30% to 100%, respectively. table 1 also shows that a very significant fraction of such proteins with overlooked disorder (>75%) belongs to the "highly disordered" category. the order of sections in this review crudely follows the order of proteins in table 1 . when several proteins are discussed in a paper, the corresponding section within the text is placed at a position ascribed by table 1 to a protein with the lowest disorder content. the corresponding sections are organized in the following way: first, a brief description of the paper is provided; then, the biological importance of the related proteins is discussed; next, the results of the computational and bioinformatics analyses of the disorder status of a given protein (or set of proteins) are represented to show how consideration of intrinsic disorder can enhance conclusions of a paper. eukaryotic translation initiation factors 4a, 4b and 4g (eif4a, eif4b, and eif4g) andreou & klostermeier investigated the peculiarities of the combined action of 3 eukaryotic translation initiation factors, eif4a, eif4b, and eif4g, in rna unwinding needed to resolve secondary structure elements from the 5 0 -untranslated region of mrnas to enable ribosome scanning. 5 a set of eukaryotic translation initiation factors (eifs) is involved in the complex, multi-stage process of initiation of mrna translation in eukaryotes. in the first step of this process, the eif4f complex binds to the m7 gppp cap on the 5 0 -end of the mrna. this eif4f complex consists of the cap-binding protein eif4e, the scaffolding protein eif4g, and the deadbox helicase eif4a. 6, 7 at the next steps of the translation initiation, the eif4f complex recruits the 43s ribosomal preinitiation complex which scans the 5 0untranslated region in 5 0 -to-3 0 direction in search for the translation start codon. 8, 9 one of the important points in this process is the resolution of secondary and tertiary structures in the mrna, which is typically done by the dead-box helicase eif4a, which possesses rna-dependent atpase and atp-dependent rna helicase activities. 10 these 2 activities of eif4a are enhanced by some auxiliary proteins, such as eif4b and eif4g that act synergistically stimulating the eif4a helicase activity in the mrna scanning process. 5 figure 1 illustrates the disorder propensities of the 3 proteins related to the mrna unwinding. disorder was evaluated by a family of pondr predictors. here, scores above 0.5 correspond to disordered residues/regions. pondr ò vsl2b is one of the most accurate standalone disorder predictors, 11 pondr ò vl3 possesses high accuracy in finding long idprs, 12 pondr ò vlxt is not the most accurate predictor but has high sensitivity to local sequence peculiarities which are often associated with disorderbased interaction sites, 13 whereas pondr-fit represents a metapredictor which, being moderately more accurate than each of the component predictors, is one of the most accurate disorder predictors. 14 the various predictors often give different predictions of disorder for the same protein, perhaps leading so some confusion when trying to understand the implications of this and the following figures. these differences arise because of different computational methods and especially because different training sets were used. despite the differences, the overall disorder prediction trends are typically similar for each protein. in agreement with the notion that catalytic functions require specific and ordered structure, eif4a, with its atpase and atp-dependent rna helicase activities, is predicted to be mostly ordered protein (see fig. 1a ; uniprot id: p10081). the mostly ordered nature of this enzyme is further supported by the fact that almost entire sequence is seen in the crystal structure (see red structure at fig. 1b , pdb id: 2vso), except to the residues 1-11, 126-135, 351-356, and 394-395. on the contrary, the auxiliary proteins eif4b and eif4g are predicted to possess long disordered regions (see figs. 1c and 1d; uniprot ids: p34167 and p39935, respectively). eif4g is also predicted to have an ordered region that coincides with its middle or eif4a interacting domain (residues 571-854). this region was cocrystallized with the eif4a factor (see blue structure at fig. 1b) . curiously, several eif4g regions were missing in structure of the eif4a-eif4g complex (e.g., residues 571-576, 583-596, 686-688, 717-729, 803-811, and 853-854). long intrinsically disordered regions are important for functions of eif4b and eif4g. in fact, according to anchor, 15 human plasminogen activator inhibitor 1 (pai-1) florova et al. investigated the mechanisms of the stabilization of human plasminogen activator inhibitor 1 (pai-1, also known as serpin e1) during the formation of the transient ternary "molecular sandwich" complex (msc) containing pai-1, vitronectin (vn), and the target enzyme. 17 major players involved in the formation of this complex are pai-1, which is a major endogenous inhibitor of plasminogen activators, a cell adhesive glycoprotein vn, and a proteinase inhibited by pai-1. an important feature of pai-1 is its ability to exist in 2 forms, a metastable active conformation with solventaccessible reactive center loop (rcl) and thermodynamically stable, inactive, latent conformation, where rcl is spontaneously inserted to the middle of the b-sheet b of pai-1. 17 this "magic" chameleonlike rcl is located in the 331-350 region. figure 2a shows that although human pai-1 (uniprot id: p05121) is predicted to be mostly ordered, its rcl is located within grayish area with a mean disorder score close to 0.5, suggesting that this region is characterized by noticeable intrinsic mobility. perumal et al. reported that a specific interaction between the bacteriophage t4 uvsw helicase and the t4 singlestranded dna (ssdna) binding protein gp32 is required for enhancement of the uvsw dna unwinding function. 18 curiously, uvsw interact with gp32, both in the presence and absence of dna, through the c-terminal acidic tail of the gp32 protein. in the absence of this interaction, the ssdna annealing and atp-dependent translocation activities of uvsw are severely inhibited when gp32 coats the ssdna lattice. however, when uvsw and gp32 do interact, uvsw is able to efficiently displace the gp32 protein from the ssdna. the tail-less gp32 inhibits activities of uvsw on dna in the presence of gp32. 18 the uvsw helicase, a member of the sf2 superfamily of helicases 19 (i.e., enzymes that use atp to carry out mechanical work related to the unwinding of duplex dna structures and reorganization of rna secondary structures 20 ) , is one of the 3 helicases encoded by the bacteriophage t4, 21 where it plays a number of roles in a variety of dna repair and recombination pathways. [22] [23] [24] [25] [26] [27] the involvement of this helicase in dna replication is achieved via the uvsw-mediated unwinding of r-loop structures generated at the replication origin by the host rna polymerase. 22 uvsw (uniprot id: p20703) activity involves the generation and consumption of single stranded dna (ssdna). in agreement with the overall low disorder predisposition (see fig. 3a ), almost entire sequence of uvsw was successfully crystallized (fig. 3b , pdb id: 2oca). naked ssdna rarely occurs within t4infected cells as they are immediately coated with gp32 ssdna binding protein, which serves to protect it against degradation by endonucleases. the gp32 is also thought to coordinate the many activities necessary to carry out dna replication, recombination and repair by recruiting and modulating the activity of the enzymes involved in these processes. 28 the bacteriophage t4 gp32 (uniprot id: p03695) is a 301 residue-long protein. the crystal structure of the gp32 central region (residues 22-239) is known (see fig. 3c ). 29 gp32 binds preferentially to ssdna and destabilizes double-stranded dna. it is involved in dna replication, repair and recombination, dinds ssdna as the replication fork advances and stimulates the replisome processivity and accuracy. the n-terminal region of gp32 is important for the cooperative binding of gp32 monomers on ssdna, whereas the c-terminal acidic tail is involved interaction with the replicative dna polymerase, the primase and helicase loader protein. [30] [31] [32] also, this c-terminal region (residues 254-301) was shown to play a crucial role in controlling the d-loop unwinding capability of uvsw, since the presence of a mutant gp32 protein lacking the acidic tail (delta254-301, gp32-a) led to a significant reduction in the unwinding of the d-loop substrate. 18 fig. 3d shows that a significant portion of the c-terminal half of this protein is predicted to be mostly disordered (residues 200-301). curiously, this disordered region includes the functionally important c-terminal tail that defines the ability of gp32 to modulate the uvsw activity. pilf and pilq of the pseudomonas aeruginosa type iv pilus system some bacteria and archaea contain specific cell envelope-spanning biomolecular machines, type iv pili (t4p), which are used by bacteria and archaea to interact with the environment. pilus biogenesis is controlled by the assembly of the outer membrane pilq secretin channel through which the pili are extruded. koo et al. investigated the roles of the pseudomonas aeruginosa type iv pili (t4p) pilotin protein pilf in the outer membrane pilq secretin channel targeting, oligomerization, and function. 33 pilf belongs to the class 1 pilotins, which are a-helical proteins containing several tetratricopeptide (tpr) motifs, that are comprised of approximately 34amino acid and are able to form a superhelical fold that mediates proteinprotein interactions in both prokaryotes and eukaryotes. 33 secretins (including pilq sectretin) possess a conserved c-terminal region, containing the secretin domain that is putatively embedded into the outer membrane, and a variable, system-specific n-terminal region. 34, 35 figure 4a shows that the p.aeruginosa pilf (uniprot id: q51385) is predicted to be a hybrid protein possessing both ordered and disordered regions. curiously, about 2/3 of this protein possesses disorder scores close to 0.5. this grayish area is likely to be characterized by high conformational plasticity and is predicted to contain 4 aibss, residues 5-16, 41-46, 76-81 and 111-115. similarly, pilq (uniprot id: p34750) is predicted to be a hybrid protein (see fig. 4b ) with several disorder-based binding sites (residues 101-107, 120-122, 245-251, 502-505, and 533-547). bonet et al. analyzed the molecular mechanisms of interaction of the 14-3-3z protein with several integrin proteins. 36 the authors provided a detailed biophysical characterization of the cytoplasmic tails of a4, b1, b2 and b3 integrins binding to 14-3-3z and showed that binding affinities and interaction modes of different integrins with this 14-3-3 are rather different. furthermore, although many structural features of these interactions are similar to other known 14-3-3 complexes, the binding exhibits specific features involving secondary sites. particularly, in addition to a canonical binding mode for the a4 phospho-peptide, some residues outside the consensus 14-3-3z binding motif of this integrin were shown to be essential for an efficient interaction. although a short b2 phospho-peptide is sufficient for high-affinity binding to 14-3-3z, the authors also found novel 14-3-3z/integrin tail interactions that were independent of phosphorylation. 36 the members of the 14-3-3 family are highly conserved acidic proteins of »30 kda that are abundantly expressed in all eukaryotic cells. in human, there are 7 14-3-3 isoforms (b, g, e, h, s, t and z) forming homodimers or heterodimers. these proteins regulate and control many signaling pathways, such as cytoskeletal dynamics programmed cell death, and cell cycle progression, and are involved in the pathogenesis of several human diseases, such as cancer and neurological disorders. 37,38 although 14-3-3 proteins are considered as phosphor-serine/threonine binding modules possessing 2 consensus recognition motifs, rsxpsxp and rxf/ yxpsxp, 39 some other binding modes are known including interaction with some unphosphorylated motifs. 40 ,41 x-ray structures of various 14-3-3 isoforms and their numerous complexes are known providing considerable knowledge on the various binding modes. 42, 43 curiously, a detailed analysis of more than 200 binding partners of 14-3-3 proteins showed neither structural nor functional relatedness in this group of proteins. 44 however, bioinformatics analysis established that >90 % of the 14-3-3 interactors contain disordered regions and that almost all 14-3-3-binding sites are located inside disordered regions. 44 among various partners of 14-3-3 proteins are membrane-spanning receptors, integrins, which are involved in numerous biological functions, such as cell adhesion, migration and differentiation and are associated with a wide-range of diseases. 45, 46 integrins are heterodimers formed by aand b-subunits composed of large extracellular (ecto) domains, trans-membrane domains and short (13-70 residues) c-terminal cytoplasmic domains, 47 which are flexible tails acting as hubs for numerous integrin-based protein-protein interactions. [48] [49] [50] these cytoplasmic tails of the 2 integrin subunits also mediates the bidirectional inside-tooutside and outside-to-inside signaling, where signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, and where integrin affinity for the extracellular ligands can also be controlled by intracellular factors. 47 figure 5a represents a structure of the complex between the 14-3-3z protein (blue cloud) and a phosphorylated peptide from the b2 integrin tail (red chain). 51 it is seen that similar to many other 14-3-3 complexes, the b2 integrin tail is bound in a highly extended form. to illustrate interactivity of the b2 integrin, figure 5b shows the results of the analysis of this protein by string. here, settings were chosen to find binding partners with the highest confidence (0.9). finally, figure 3c shows some disorder-based alignments of the c-terminal tails used in the work of bonet et al., 36 namely residues 1001-1032 of human integrin a4 (black line, uniprot id p13612), residues 752-801 of human integrin b1 (black line, uniprot id p05556), residues 724-769 of human integrin b2 (black line, uniprot id p05107), residues 742-788 of human integrin b3 (black line, uniprot id p05106), and residues 747-798 of human integrin b7 (black line, uniprot id p26010). figure 5c clearly shows that all these integrin tails involved in the specific interaction with the 14-3-3z protein are mostly disordered. in the review by rajsbaum et al., an important family of proteins, tripartite motif (trim) proteins is introduced together with the multitude of their functional roles in the innate antiviral immunity. 52 since there are more than 70 distinct members in the family of human trim proteins, it is physically impossible to consider all of them even very superficially. the feature that links all these proteins together is the fact that they share 3 conserved n-terminal domains: a really interesting new gene (ring) domain, one or 2 b-boxes (b1/b2) and a coiledcoil domain. [53] [54] [55] [56] in addition to immunerelated functions many trim proteins are involved in a wide range of biological activities, such as transcriptional regulation, apoptosis, cell differentiation, development, oncogenesis, ubiquitin e3 ligases, and e3 ligases for other ubiquitinlike molecules such as sumo and the ifn-inducible protein isg15. 57 computational analysis (see table 1 ) revealed that all members of the human trim family are extensively disordered. helbig and beard overviewed the ability of viperin to modulate conditions within the cell and to interfere with proviral host proteins in order to create an unfavorable environment for viral replication. 58 viperin is one of the few products of numerous interferon-stimulated genes (isgs) possessing direct antiviral activity, being able to limit a broad range of viruses and to play an emerging role in modulating innate immune signaling. this is a highly species conserved protein consisting of 3 distinct domains: a variable n-terminal domain that contains an amphipathic helix and a leucine zipper region, a highly conserved central domain containing a "radical sam domain," and a conserved c-terminal critical for the antiviral properties of viperin against a number of viruses. 58 the authors showed that viperin plays a role in innate immune signaling, limits different viruses through both direct inhibition of replication and interference with viral budding/release, and disrupts the actin cytoskeleton to increase infectivity of human cytomegalovirus (hcmv) in a known example of evolutionary escape of hcmv from the antiviral properties of viperin. 58 viperin is able to interact with the 5 host proteins fpps, tfp, irak1, vap-a and traf6 and the 3 viral proteins denv ns3, hcv ns5a and hcmv vmia in order to accomplish these diverse biological functions. 58 figure 6a represents the results of the string 59 -based evaluation of viperin interactivity. although it has been emphasized that "it is unusual for viperin to be able to interact with such a divergent range of other proteins and to potentially mediate quite distinct cellular functions", 58 the mystery of such "unusual" polyfunctionality is naturally solved by the presence of extensive disorder in this protein (see fig. 6b ; uniprot id: q8wxg1). sze et al. overviewed available information on the sterile a motif and histidineaspartic domain (hd) containing protein 1 (samhd1), which is a member of the unique group of host restriction factors that limit retroviral replication at distinct stages of the viral life cycle. 60 samhd1 is a deoxynucleoside triphosphate triphosphohydrolase responsible for the degradation of the deoxynucleoside triphosphates (dntp) into their deoxynucleosides (dn) and inorganic triphosphates, thus depleting the cellular dntp pool required for cellular dna polymerase. 61 activity of this protein is modulated by post-translational modifications, cell-cycle-dependent functions and cytokine-mediated changes, as well as via interaction with the vpx accessory protein. 60 samhd1is a predominantly nuclear protein composed of 2 functional domains, the sterile a motif (sam) domain involved in protein-protein and samhd1-nucleic acid interactions, 62 and the hd domain containing the enzymatic sites crucial for its triphosphohydrolase activity, rna binding and nuclease activity. 63 another functionally important region is located at the c-terminus of samhd1, where a v-domain capable of interaction with the hiv-2/sivsm vpx accessory protein is located (residues 595-626). 64 based on the intrinsic disorder propensity analysis (fig. 2b) , samhd1 (uniprot id: q9y3z3) is expected to be a predominantly ordered protein possessing disordered n-and c-terminal tails (residues 1-100 and 580-626). obviously, intrinsic disorder in the c-terminal tail is important for the samhd1 interaction with the hiv-2/sivsm vpx accessory protein. interaction between the major bacterial heat shock chaperone groesl and an rna chaperone cspc in their recent paper, lenz & ron described a novel interaction between the major heat shock chaperone groesl of e. coli (hsp60) with an rna chaperone (cspc) leading to the cspc proteolysis needed for the transient nature of the heat shock response. 65 protein chaperones and proteases have multiple functions in controlling the wellbeing of a cell, acting as protein quality control that enables the cells to cope with the unfolding and aggregation of proteins. 66, 67 cspc is a member of the cold shock protein (csp) family that consists of 7 small homologues with high affinity to single-strand nucleic acid and that serve as rna chaperones. [68] [69] [70] [71] it has been shown that cspc is degraded during heat shock and that interaction of cspc with the major protein chaperone groesl plays a role in the enhanced, temperature-dependent proteolysis of cspc. 65 the fact that cspc is able to interact with groesl (which is known to bind partially unfolded and misfolded protein species) is a clear indication that cspc does not possess rigid structure, at least under the conditions favoring such interaction. the likely explanation for this binding is in the potential intrinsically disordered nature of cspc. in fact, many protein and rna chaperones were shown to be intrinsically disordered or hybrid proteins possessing both ordered and intrinsically disordered regions/ domains. 72 in agreement with this observation, figure 2c represents the pondr plots for the cspc from e. coli (uniprot id: p0a9y6) and shows that a significant portion of this small proteins (up to 35%) is predicted to be disordered. kalli et al. used multiscale molecular dynamics simulations to define the interaction mechanisms of phosphatase and tensin homolog (pten; uniprot id: p60484) and of the pten domain of ciona intestinalis voltage sensitive phosphatase (ci-vsp; uniprot id: q4w8a1) with phosphatidylinositol phosphate (pip)-containing lipid bilayers. 73 the authors revealed that the association of the pten with such bilayers involves the formation of an initial electrostatics-driven encounter complex between the protein and bilayer followed by reorientation of the protein to optimize its interactions with pip molecules in the membrane. 73 pten is a cytosolic enzyme that can interact with the inner leaflet of the plasma membrane and, being bound to membrane, catalyzes dephosphorylation of pi(3,4,5)p 3 to ptdins(4,5)p 2 . 74 pten has 4 domains, an n-terminal pip 2 -binding module, a phosphatase domain (pd), a c2 domain, and a c-terminal tail. several recent studies indicated that both nand c-tails of this protein are intrinsically disordered. [75] [76] [77] furthermore, it has been emphasized that post-translational modifications, conserved eukaryotic linear motifs, and molecular recognition features are present in the disordered c-tail of pten, enhancing protein-protein interactions of this protein needed for the various cellular functions of pten. 75 domain swapped pukovnik xis singh et al. determined the structure of pukovnik xis by x-ray crystallography. 78 xis is the recombination directionality factor that serves as a dna bending machine that difines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-dna architectures. 79 mycobacterium phage pukovnik (which is a relative of phage l5) contains a small xis protein (56 residues) that binds cooperatively to attr dna at specific x1-x4 binding sequences. similar to l5, pukovnik xis stimulates integrase-mediated excision and inhibits integration. 80, 81 the presence of both xis and intergrase is needed for the formation of an attr intasome. 78 the cooperative binding of several xis proteins to dna is driven by a wingedhelix motif and relies on the use of contacts between a central loop and the wing motif to mediate interactions between xis proteins when bound to dna leading to the formation of a micronucleoprotein filament with a modest bend. 78, 82, 83 singh et al. showed that in the dnabound form, 5 individual pukovnik xis subunits stack onto each other through an extensive array of protein-protein interactions forming a filament with left-handed superhelical twist. 78 stacking of xis subunits within the filament is rather regular, and each monomer exhibits a twist of 40 and an »60 bending angle. within the asymmetric unit, the filament region contains 4 xis protomers whereas the fifth protomer is positioned adjacent to the filament. this "outside" protomer forms the domain-swapped complex with the xis protomer 3. this domain-swapped xis 59 string produces the network of predicted associations for a particular group of proteins. the network nodes are proteins. the edges represent the predicted functional associations. an edge may be drawn with up to 7 differently colored lines -these lines represent the existence of the 7 types of evidence used in predicting the associations. a red line indicates the presence of fusion evidence; a green line -neighborhood evidence; a blue lineco-occurrence evidence; a purple line -experimental evidence; a yellow linetext mining evidence; a light blue line -database evidence; a black lineco-expression evidence. 59 (b) intrinsic disorder propensity of the human viperin. disorder propensity was evaluated by a set of predictors from the pondr family, pondr ò fit, vsl2b, vl3, and vlxt. scores above 0.5 correspond to disordered residues/regions. pair is created due to a dramatic rearrangement within the loop connecting b1 and b2 (residues 36-39) allowing the c-terminal residues 40-56 of one subunit to interact with the n-terminal 35 residues of a neighboring xis monomer. 78 it has been emphasized that the most important protein-protein interactions mediating the filament formation are formed by residues 51-54 of pukovnik xis that make extensive contacts with the wing of the adjacent monomer and deletion of which greatly diminishes dna binding affinity and abolishes excision. 78 therefore, although the xis filament is composed of identical subunits, the individual xis protomers are involved in very different interactions and form very different interfaces. to further illustrate this point, figure 7a represents a crystal structure of the pukovnik xis pentamer (pdb id: 4j2n) and 4 pairs of different dimers found within this pentameric filament. figure 7b shows that pukovnik xis (uniprot id: b3vgi6) is predicted to contain substantial amount of intrinsic disorder (note scale of the yaxis), including disordered n-and c-terminal tails. therefore, it is likely that the intrinsically disordered nature determines the ability of this small protein to be involved in a complex net of proteinprotein and protein-dna interactions, including its ability to form a wide array of regular and domain-swapped dimers. li et al. provided a detailed characterization of the functional mechanisms of interesting hsp90 co-chaperones, human aryl hydrocarbon receptor (ahr) interacting protein (aip) and aip like 1 (aipl1). 84 aip and aipl1 share 49% sequence identity, contain an n-terminal fkbp-like prolyl peptidyl isomerase (ppiase) domain (which is inactive in both proteins) followed by a tetratricopeptide repeat (tpr) domain. in addition, aipl1 harbors a unique c-terminal proline-rich domain (prd). 84 the authors showed that aip is inactive as a chaperone, whereas aipl1 exhibits chaperone activity and prevents the aggregation of non-native proteins, suggesting that prd is crucial for the chaperone function of this protein providing a means for efficient binding of aipl1 to non-native proteins. 84 since aipl1 possesses decreased affinity to hsp90, the c-terminal prd plays a role of a negative regulator of the aipl1-hsp90 interaction. 84 figure 8a shows that the major portion of the human aipl1 (n-terminal 75%) containing fkbp-like prolyl peptidyl isomerase and tpr domains is predicted to be mostly ordered (uniprot id: q9nzn9), whereas the c-terminal domain is predicted to be highly disordered. on the other hand, figure 8b indicates that the human aip (uniprot id: o00170) is expected to be mostly ordered. in agreement with these disorder predictions, 3d structures are known for the ppiase fkbp-type domain of human aip (residues 2-166, pdb id: 2lkn) and for its tpr domain (residues 173-330, pdb id: 4aif). the intrinsically disordered nature of the c-terminal prd of the human aipl1 possessing chaperone activity is in accord with earlier observations that chaperones are often either entirely disordered or contain long disordered regions. 72, 85 basic residues in the activated protein c (apc) exosite takeyama et al. provided a detailed description of the interaction between the activated protein c (apc) and factor (f) viiia. 86 the authors showed that the basic residues located within the 39-, 60-, and 70-80-loops of apc constitutes an exosite that contributes to the binding of fviii and therefore are important for the subsequent proteolytic inactivation of fviii. 86 apc, which is also known as vitamin k-dependent protein c, anticoagulant protein c, autoprothrombin iia, and blood coagulation factor xiv, is a single chain vitamin k-dependent zymogen for a plasma serine protease that upon activation by the thrombin-thrombomodulin complex down regulates the coagulation cascade by limited proteolysis of fva and fviiia. [87] [88] [89] analysis of the crystal structure of human apc (pdb id: 1aut) revealed that there are 3 surface loops (39, 60, and 70-80) , rich in basic residues, located in the protease domain of apc near the active site pocket. 86, 90 fviiia contains acidic c-terminal sequences that may potentially provide interactive sites for the basic exosite of apc. 86 figure 8c represents the results of the computational disorder analysis in human apc (uniprot id: p04070) and shows that mentioned loops enriched in basic residues are predicted to be disordered or very flexible, thereby providing an interesting mechanistic plane for the molecular basis of apc recognition and binding of fviii. the escherichia coli primosomal dnat protein molecular mechanisms of the oligomerization of e.coli primosomal dnat protein are uncovered in the study by szymanski et al. 91 the authors showed that the removal of the short c-terminal region dramatically affects the oligomerization process and instead of the trimer, the isolated n-terminal domain of dnat forms a dimer. 91 priming of the dna strand during the replication process is catalyzed by a multiprotein-dna complex known as the primosome. 92, 93 the translocation of this complex along the dna is fueled by ntp hydrolysis. besides being responsible for the synthesis of short oligoribonucleotide primers used to initiate synthesis of the cdna strand, primosome plays a role in the restarting of the stalled replication fork at the damaged dna sites. 93, 94 the assembly of the primosome is driven by an essential replication protein in escherichia coli, the dnat protein, where, the primosome assembly is initiated by recognition of a specific primosome assembly site (pas) of the replicating dna or the damaged dna site by the pria protein, or the prib protein-pria complex, followed by the association of the dnat and the pric protein. [92] [93] [94] this primary dna-protein complex is a scaffold, specifically recognized by the dnab helicase-dnac protein complex, which results in formation of the preprimosome. next, the preprimosome is recognized by the primase, and a functional primosome is formed. the dnat protein is crucial for the specific entry of the dnab helicase into the primosome complex. [92] [93] [94] the dnat monomer consists of the large, n-terminal core domain that includes the first 161 residues of the protein and a small c-terminal region containing the 18 remaining amino acids. 91 disorder analysis of this protein (uniprot id: p0a8j2) revealed that although the majority of the dnat is predicted to be ordered, the crucial for trimerization cterminal tail is expected to be completely disordered (see fig. 8d ). this observation is in agreement with recent notion that disordered protein tails are commonly involved in a wide array of important functions. 95 platelet endothelial cell adhesion molecule 1 (pecam-1) tourdot et al. dedicated their research to the analysis of the effect of the sequential phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (itims) on function of the platelet endothelial cell adhesion molecule-1 (pecam-1). 96 pecam-1 is a dual itim-containing receptor of the ig superfamily that is capable of inhibiting immunoreceptor tyrosine-based activation motif (itam)induced activation of b cells, t cells, and mast cells, as well as gpvi/fcrg chainmediated platelet activation. 97 the inhibitory properties of pecam-1 require phosphorylation of both of its itims located in the vicinity of tyrosine residues at positions 663 (vqy 663 tev) and 686 (tvy 686 sev). 96 curiously, the itims of pecam-1 are not phosphorylated in resting cells but are phosphorylated upon cellular activation. furthermore, these pecam-1 itims are phosphorylated sequentially, with phosphorylation of y 663 depending on prior phosphorylation of y 686 . 98 this sequential process of the pecam-1 phosphorylation starts with the phosphorylation of the c-terminal itim by src family kinases, which enables phosphorylation of the n-terminal itim of pecam-1 by other src homology 2 domain-containing nonreceptor tyrosine kinases (nrtks). 96 figure 8e shows that although human pecam-1 (uniprot id: p16284) is predicted to be mostly ordered, the residues, phosphorylation of which is crucial for its function (y 663 and y 686 ), are located within the highly disordered c-terminal tail. mouse serine/arginine-rich splicing factor 1 (srsf1) aubol et al. studied the molecular mechanisms of the phosphorylation of sr splicing factors, which are the proteins possessing regions enriched in arg-ser dipeptide repeats known as rs domains, by a specific serine kinase srpk1. 99 phosphorylation of rs domains is crucial for the regulation of the activities of the sr splicing factors. rs domains of sr proteins can range from 50 to over 300 residues in length, and the arg-ser dipeptide repeats can vary in both length and position. srpk1 phosphorylates the prototype sr protein, serine/arginine-rich splicing factor 1 (srsf1), via a directional mechanism where 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. 99 figure 8f shows that srsf1 (uniprot id: q6pdm2) is predicted to be a highly disordered protein. the disordered nature of this splicing factor is in agreement with recent bioinformatics studies, which showed that human 100 and yeast spliceosomes 101 are enriched in intrinsic disorder, and abundant disordered regions are crucial for functions of numerous auxiliary protein factors involved in the formation of this ribonucleoprotein complex. 100, 101 the potential functionality of the disordered regions found in mouse srsf1 was further supported by anchor-based analysis, 15 102 replication of viruses requires breaching the plasma membrane of the host cell. the entry of the enveloped viruses relies on the specialized fusion proteins. there are 3 major classes of viral fusion proteins, all containing a fusion peptide that inserts into the cytolemma, thereby anchoring the 2 membranes side by side. 106 human ifitms block the entry of viruses from each of the 3 classes of viral fusion proteins, 102 by preventing viral-host membrane fusion subsequent to viral binding and endocytosis. 104, 107 in humans, there are 5 ifitms (ifitm1, 2, 3, 5, and 10; corresponding uniprot ids: p13164, q01629, q01628, a6nnb3, and a6nmd0). these proteins each contain 2 hydrophobic membrane-associated domains separated by a conserved intracellular loop. all of them also contain the cytosolically-located n-terminal domain (ntd) of various length. 102 figure 9 shows that the ntds of all 5 human ifitms are predicted to be intrinsically disordered. the likely functional role of these disordered tails is reflected in the fact that the longest ntd of ifitm10 contains 3 aibss, residues 8-29, 51-64, and 71-82. shorter tails of other human ifitms might also be involved in protein-protein interactions since all of them contain characteristic "dips," which are commonly associated with the existence of specific molecular recognition features, morfs, which are short foldingprone fragments of disordered regions that are able to fold (at least partially) at interaction with specific binding partners. [108] [109] [110] [111] [112] chameleon region undergoing a-helix to b-strand transition in the mutant form of the type 2 ryanodine receptor domain a (ryr2a) linked with a heritable cardiomyopathy using the solution nmr spectroscopy and x-ray crystallography, amador et al. studied the effects of mutations associated with arrhythmogenic right ventricular dysplasia type 2 (arvd2) on the structure and dynamics of the n-terminal domain of the mouse ryanodine receptor ryr2a (uniprot id: e9q401). 113 ryanodine receptors (ryrs) are the largest tetrameric ca 2c release channels (»2.2 mda) of the sarcoplasmic reticulum in the electrically excitable cells. here, ryrs resides in close proximity to plasma membrane voltagegated dihydropyridine receptors (dhprs) and respond to the dhpr activity through a direct interaction and/or indirect ca 2c sensitivity, propagating sarcoplasmic reticulum luminal ca 2c release, thereby playing an integral role in excitation-contraction coupling in skeletal muscle cells and cardiomyocytes. 114, 115 mutations in the ryr genes are associated with several heritable human diseases, with most disease-associated mutations making ryrs hypersensitive to activating stimuli such as ca 2c on either the luminal or the cytosolic side of the receptor. in one of such mutations, the removal of exon 3 in ryr2 (ryr2ad3) results in a 35-residue deletion (n57-g91) that does not destabilize the protein. 116, 117 earlier structural analysis revealed that the b-trefoil fold of this domain is rescued by the transformation of the part of the preceding region of unknown structure (residues 88-109) into the missing b-strand leading to the noticeable increase in the thermal stability of the ryr2ad3 domain. 117 using solution nmr analysis, amador et al. showed that this "part of the preceding region of unknown structure" corresponds to a previously unresolved a 2helix (residues 95-104) in the wild-type ryr2a. 113 curiously, this a-helix undergoes structural transformation to a b-strand, thereby rescuing the b-strand deleted in the ryr2ad3 due to the removal of 3 in ryr2 gene. therefore, the authors revealed that this newly discovered a 2 -helix corresponds to the region of missing electron density in the earlier crystal structures of the protein, is rather dynamic, exhibits a greater backbone rmsd compared to the remaining secondary structure elements, and is able to undergo an a-to-b transition after the deletion of residues n57-g91 in ryr2ad3. 113 in other words, applying nmr to look at the crystallographically invisible region, an important chameleon region was discovered. although this chameleon region is relatively small, it is really mighty, with at least some mightiness being attributed to its intrinsically disordered nature. rodriguez et al. used complementary biophysical techniques for examining the atp-induced conformational switching of the isolated e subunit of the f 0 f 1 atp synthase from the thermophile bacillus ps3 (te). 118 the authors showed that atp binding induces large-scale conformational transition consistent with formation of stable helical structure in the isolated te. 118 based on the complimentary hydrogen/deuterium exchange (hdx) mass spectrometry analysis it has been concluded that this transition is accompanied by a pronounced stabilization in the vicinity of the atp-binding pocket. 118 structurally, the e subunit consists of a b-sandwich and 2 c-terminal helices, a1 and a2. 119 curiously, this protein is able to undergo dramatic structural rearrangements from a compact structure in the non-bound form (see fig. 10a ; pdb id: 1aqt) 119 to a highly extended open form in a complex with other subunits of the bacterial f 1 complex (see fig. 10b ; pdb id: 3oaa). the ability of the e subunit to undergo large conformational change associated with binding to the f 1 complex is determined by the presence of long idpr at the c-terminal region (see fig. 10c ; uniprot id: p0a6e6). simon et al. investigated functional peculiarities of the candida albicans cyclin capcl5. 120 the authors showed that the activation of the cyclin-dependent kinase pho85 that induces phosphorylation of the transcription factor cagcn4 is controlled via the specific binding of capcl5 to pho85. furthermore, it is shown that capcl5 induces its own phosphorylation at 2 adjacent sites in the n-terminal region of the protein and that this phosphorylation causes degradation of the cyclin in vivo, whereas in vitro studies indicated that this phosphorylation was accompanied by a loss of specific substrate recognition thereby representing a novel mechanism for limiting cyclin activity. 120 activity of cyclin-dependent kinases (cdks) is regulated via binding of ancillary subunits, the cyclins, 121 which also participate in targeting the kinase to specific substrates. 122 similar to other members of the cdk family, a yeast (saccharomyces cerevisiae) cdk pho85 can bind up to 10 different pho85 cyclins (pcls), 123 which defines the ability of this cdk to phosphorylate different substrates leading to a wide range of pho85 functions in cellular regulation. among various regulatory functions of pho85 complexed with different cyclins, is the pho85/pcl5-driven phosphorylation of the bzip transcription factor gcn4 leading to its degradation. 124 curiously, it was shown that cyclin pcl5 coevolved with its substrate gcn4, and in the pathogenic yeast candida albicans, degradation of cagcn4 depends on capcl5. 125 mutagenic analysis of the capcl5 (uni-prot id: q5ak08) revealed 2 potential cdk phosphorylation sites located in the n-terminal segment, at positions 38 and 43. 120 figure 11 shows that both of these phosphorylated sites are located within the disordered region, providing further illustration for an important notion stating that sites of posttranslational modifications in proteins are commonly located within the disordered regions. 126 nuclear localization of the alternatively spliced sirtuin-2 isoform rack et al. described a discovery of a new alternatively spliced isoform of the human sirtuin-2. 127 the new isoform results from skipping of exons 2-4 in the sirt2 gene. 127 sirtuin-2 is a member of a large family of protein-modifying enzymes, nad cdependent deacetylases, which are highly conserved throughout bacteria, archaea, and eukaryotes, and are heavily involved in modulation of various cellular processes ranging from metabolic homeostasis, to cell cycle control, to development, and to chromatin organization. [128] [129] [130] [131] [132] [133] in their turn, the numerous activities of sirtuins are regulated by alternative splicing, modulation of expression levels, posttranslational modifications or subcellular compartmentalization. [134] [135] [136] [137] in humans, there are 7 sirtuin homologues with distinct cellular locations. for example, sirt1, sirt6 and sirt7 are primarily localized to the nucleus, whereas sirt3, sirt4 and sirt5 are mitochondrial proteins, and sirt2 is believed to have a predominant cytosolic localization. 138 earlier, 4 variants of human sirt2 gene were reported, with alternative splicing affecting the n-terminal part of the sirtuin-2 protein in 2 isoforms (residues 1-37 are missing in the isoform-2 and the residues 1-38 (maepdpshpletqagkv-qeaqdsdsdseggaaggeadm) are substituted by residues mplaecpsc-rclssfrsv in the isoform-3), and with the isoform-4 possessing changes at the c-terminus, where residues 266-271 (vqpfas) are substituted by grglag, and residues 272-389 are missing. these isoforms contain a leucine-rich nuclear export signal (nes) within their n-terminal region which mediate their cytosolic localization 139 where they have numerous functions. a recent finding a new isoform-5 with the predominant nuclear localization helped to resolve an apparent contradiction between the predominant cytosolic localization of this protein and the existence of multiple nuclear functions. 127 the newly discovered alternative splicing event results in the substitution of the codons for amino acids 6-76 of the full-length orf of isoform-1 by an arginine codon in a new isoform. this isoform-5 is predominantly localized in the nucleus, does not exhibit detectable deacetylase activity, but is properly folded and retains the ability to bind p300. 127 it has been established that the protein segments affected by alternative splicing are most often intrinsically disordered, suggesting that alternative splicing enables functional and regulatory diversity while avoiding structural complications associated with the deletion of portions of the well-folded proteins. 140 in line with these observations, later studies revealed that the tissue-specific protein segments produced by alternative splicing often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. 141 also, it was proposed that alternative splicing of intrinsically disordered regions containing linear interaction motifs and/or post-translational modification sites results in complete rewiring of protein interactions. 142 figure 12 shows that in agreement with these earlier observations the alternatively spliced isoform-5 of human sirtuin-2 differs from the canonical isoform (uniprot id: q8ixj6) by lacking the intrinsically disordered nterminal tail (cf. figs. 12a and 12b) . curiously, the anchor analysis 15,16 of the human sirtuin-2 revealed that this alternative splicing event removed 2 aibss (located at the residues 1-15 and 36-49 of the isoform-1). therefore, functionality of siruin-2 is modulated by alternative splicing that removes potential binding sites from the disordered region of this protein. parker et al. investigated the role of the amino acid sequence at the c-terminal region of human semaphorin-3 in interaction of this protein with neuropilin-1. 143 the neuropilin-1 is a member of the family of type i transmembrane receptors that are essential in development, homeostasis, and pathogenesis being involved in the coordination of several important volume 2 issue 1 intrinsically disordered proteins signaling events in the cardiovascular and nervous system. the members of the class iii semaphorin (sema3) family of axon guidance molecules are among the ligands neuropilins (nrps). ligand binding of nrps is a subject of the intensive research due to the involvement of these transmembrane receptors in various pathogenic conditions. it has been established that all known nrp ligands require a c-terminal arginine (c r ) for binding to a conserved pocket in the nrp b1 domain. [144] [145] [146] [147] to better understand the mechanism of interaction between the nrps and their ligands and to understand the role of residues upstream of the c r , parker et al. synthesized a library of semaphorin-3 derived peptides. this study revealed that the c-1 residue (i.e., residue preceding the c r ) serves the critical role of positioning the c r and c-2 residues to promote concurrent nrp binding. 143 bioinformatics analysis revealed that the analyzed sequence (wdqkkprnrr) is a part of long intrinsically disordered tail that spans over the last 100 residues of human semaphorin-3 (uni-prot id: q13275, see fig. 9f ). malashkevich et al. reported the x-ray crystal structure of the unmodified 3 glu-ocn form of bovine osteocalcin. 148 this is an important contribution since it adds a crucial structural information on osteocalcin, which is a small, abundant noncollagenous protein synthesized by osteoblasts, and that typically contains 3 g-carboxyglutamic acid (3 gla-ocn) residues generated posttranslationally in a vitamin k-dependent process 149,150 by the vitamin k-dependent (vkd) carboxylase. 151 earlier, based on the circular dichroism study it has been concluded that in the presence of ca 2c , the 3 glu-ocn molecule contained significantly less a-helical structure than the 3 gla-ocn form. 152 furthermore, it was shown that ca 2c no longer binds to decarboxylated, unmodified osteocalcin 153 malashkevich et al. revealed that the crystal structure of the thermally decarboxylated bovine osteocalcin contained residues 17-47, was rather similar to 3 gla ca 2c -ocn, consisted of 3 a-helices surrounding a hydrophobic core, and contained c23-c29 disulfide bond between 2 of the helices but did not contain bound ca 2c . 148 this is an interesting finding since earlier work clearly showed that in their apo-forms, modified and non-modified osteocalcin from different sources are mostly disordered. 148 the explanation for this apparent contradiction can be derived from the analysis of conditions used for the 3 glu-ocn crystallization, where protein in 20 mm nacl and 10 mm cacl 2 (ph 7.0) was mixed with the reservoir solution containing 2.5 m ammonium sulfate and 0.1 m bis-tris propane (ph 7.0) in 1:1 ratio. 148 therefore, high ionic strength solution was used to partially neutralize the high anionic charge in the helical regions of osteocalcin. although this approach permitted better realization of the a-helix-forming potential of the nonmodified osteocalcin, the crystallization conditions are clearly non-physiological. sasi et al. investigated the peculiarities of the regulation of expression of the inducible heat shock protein hspa1a at the transcription level by several transcription factors. 154 hspa1a, together with hspa1b are the members of the human inducible heat shock protein family (hsp70), altered expression of which has been attributed to the pathogenesis of various diseases, such as cancer, cardiovascular diseases, and neurodegenerative disorders. [155] [156] [157] [158] it is known that the heat shock response-related proteins (including hsp70) can be expressed during normal conditions (e.g., during the cell growth and development) or can be induced by various pathological conditions, such as infection, inflammation, and protein conformation diseases. sasi et al. show that transcription factors hsf-1, creb, nf-y, and nf-kb synergistically regulate expression of the heat-shock-induced gene hspa1a. 154 the initiation of the heat shock response is manifested by the activation of the heat shock transcription factors (hsfs), a family of related transcription factors which, in mammals, is composed of hsf-1, -2, -3, -4, -5, -y and -x. 159 camp response element binding protein (creb) is the camp-regulated transcription factor that has been shown to stimulate target gene expression often via the associating with the coactivator paralogs p300 and creb binding protein (cbp). [160] [161] [162] complex formation between creb and cbp/p300 requires protein kinase a (pka)-mediated phosphorylation of creb at ser-133. 160 the nuclear transcription factor y (nf-y) is a sequence-specific dna-binding protein that recognizes the y box, which is a promoter element common to all major histocompatibility complex class ii genes. 163 also, nf-y is known to interact with a ccaat box, which is one of the most common elements in eukaryotic promoters, found in the forward or reverse orientation. 164 furthermore, nf-y has been reported to play a key role in the basal expression of many hsps through this ccaat box. 165, 166 nf-y is heterotrimeric transcription factor composed of 3 components, nf-ya (subunit a, uni-prot id: p23511), nf-yb (subunit b, uniprot id: p25208), and nf-yc (subunit g, uniprot id: q13952), where the dimerization of nf-yb and nf-yc is a prerequisite for the nf-ya association and dna binding. 167 finally, the transcription factor nuclear factor-kappa b (nf-kb; uniprot id: 19838) is a key player in an intracellular signaling cascade regulating many inflammatory mediators. 168 nf-kb is a common transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. nf-kb is a homo-or heterodimeric complex formed by the rel-like domain-containing proteins rela/p65, relb, nf-kb1/p105, nf-kb1/p50, rel and nf-kb2/p52, and the heterodimeric p65-p50 complex. figure 13 represents the results of the multiparametric evaluation of intrinsic disorder propensities of hsf-1 (a, uniprot id: q00613), creb (b, uniprot id: p16220), nf-ya (c, uniprot id: p23511), nf-yb (d, uniprot id: p25208), nf-yc (e, uni-prot id: q13952), and nf-kb (f, uniprot id: p19838) and shows that these proteins are predicted to be highly disordered. the high levels of functional intrinsic disorder is a "family signature" of the transcription factors in general. 169 furthermore, the recent analysis of the abundance and importance of idprs in functions of various hsfs clearly indicated that the heat shock response requires hsf flexibility to be more efficient. 170 also, the importance of intrinsic disorder in functions of various proteins involved in the innate immune response (including nf-kb) has been recently analyzed. 171 phosphorylation sites in melanopsin blasic et al. analyzed the peculiarities of phosphorylation of the c-terminal domain of the photopigment melanopsin possessing 37 serine and threonine sites that are potential sites for phosphorylation by a g-protein dependent kinase (grk). 172 melanopsin is a member of the g protein coupled receptor (gpcr) family that undergoes light-dependent phosphorylation that is involved in deactivation of the photoresponse. blasic et al. revealed that of the 37 phosphorylation sites, a small cluster of 6 or 7 sites in the proximal region of the c-tail is critical for mediating deactivation. 172 figure 14a shows that mouse melanopsin (uniprot id: q9qxz9) is predicted to have long disordered c-tail (residues 380-521). this is a rather expected output since phosphorylation sites are known to be preferentially located within the idprs. [173] [174] [175] human defensins a recent review of wisons et al. is dedicated to the antiviral activities of human defensins. 176 these peptides with a wide range of antimicrobial activities are important components of the innate immune system. among various antiviral mechanisms of human defensins are direct targeting of viral envelopes, glycoproteins, and capsids, inhibition of viral fusion and post-entry neutralization, inhibition of viral replication via disruption of host intracellular signaling and binding and modulation of host cell surface receptors, and augmentation and altering of adaptive immune responses. 176 of the 2 types of defensins, aand b-defensins, found in human, significant knowledge is accumulated about the molecular mechanisms of the a-defensin action, whereas molecular details on the b-defensin activities are more disperse. 176 defensins are small (»29-129 amino acids) cationic, amphipathic polypeptides with a predominantly b-sheet structure stabilized by 3 disulfide bonds. 176 human a-defensins are typically shorter than b-defensins, with a-defensins staying in a range of 29-39 residues and some b-defensins can being as long as 129 residues. search of uniprot for human defensins produces about 40 hits, of which 6 entries were a-defensins. defensins are produced in a form of proprotein containing a signal peptide and/or a propeptide. figure 15 represents disorder profile for several representative members of the human defensin family and shows that all proproteins possess noticeable amount of intrinsic disorder, the content of which varies significantly between different defensins. it is likely that the presence of noticeable disordered tails plays a role in the maturation of defensins since sites of the proteolytic attack are commonly located within regions of intrinsic disorder. 177 it is also possible that these disordered tails serve as entropic bristle domains 178 burke et al. investigated the roles of the phosphorylation of the c-terminal domain of the retinoblastoma protein (rbc) in interaction of rb with the e2f transcription factor and related regulation of the rb activities in growth suppression. 179 deregulation of the broad-functioning tumor suppressor retinoblastoma protein (rb) is associated with several human cancers. 180, 181 among numerous functions ascribed to this important protein is a negative regulation of cell division at the g 1 -s transition of the cell cycle, 182 where rb forms a growth-repressive complex with e2f transcription factors 183 in a phosphorylation-dependent manner. burke et al. showed that there are at least 2 different mechanisms by which rbc phosphorylation inhibits e2f binding. here, phosphorylation of s788 and s795 weakens the direct association between the rbc and the marked-box domains of e2f, whereas phosphorylation of s788, s795, s807 and s811 induces an intramolecular association between rbc and the pocket domain, which overlaps with the site of e2f transactivation domain binding. 179 figure 14b shows that human retinoblastoma-associated protein (uniprot id: p06400) is predicted to have numerous disordered regions, including long disordered tails. the longest disordered region is the c-terminal tail (residues 760-928) that contains all the phosphorylation sites discussed above. according to the anchor analysis, 15 www.landesbioscience.com e970499-23 intrinsically disordered proteins are typically found in regions of intrinsic disorder. 126, 174, [185] [186] [187] the tetratricopeptide repeat (tpr) motifcontaining protein lgn and its binding partner frmpd1 (ferm and pdz domain containing 1) pan et al. described structural peculiarities of the human lgn-frmpd1 complex. 188 to this end, a crystal structure of the complex between the 15-350 fragment of human lgn containing 8 tetratricopeptide repeat motifs (uniprot id: p81274) and the 901-951 fragment of human frmdp1 (uniprot id: q5syb0) was solved at 2.4 a resolution. 188 in this lgn-tpr/frmpd1 complex, almost the entire length of lgn-tpr was well resolved, and most residues of the frmpd1 fragment were clearly resolved adopting an extended conformation that occupied most of the concave channel formed by the 8 tpr motifs and buried a total of 2541 a 2 surface area. 188 the noticeable exceptions were the 11 residues in the loop connecting the aa and ab of tpr3 (amino acids 153-163) and the 6 residues at the c terminus (amino acids 345-350) in the lgn-tpr, and the nterminal 8 residues (amino acids 912-919) and the last 2 residues at the c terminus (amino acids 937-938) of the frmpd1 fragment, all of which were missing in the structure of the lgn-tpr/frmpd1 complex. 188 leu-gly-asn repeatenriched protein lgn/ ags3 in mammals (pins, or partner of inscuteable in drosophila neuroblasts) plays a number of crucial roles in regulation of cell polarity and spindle orientation during cellular differentiation and self-renewal in multicellular eukaryotes development. [189] [190] [191] structurally, lgn consist of 8 tetratricopeptide repeat (tpr) motifs in its n-terminal half and 3 or 4 goloco motifs (also referred to as g-protein regulatory or gpr motifs) in the c-terminal half. [192] [193] [194] both of these motifs are crucial for protein-protein interactions, 195, 196 with each goloco motif of pins/lgn being capable of binding to gdp-bound gai 197 leading to stable cortical localization of pins/lgn, 198 and with the tpr motifs possessing multiple binding partners. the canonical tpr motif is a 34-amino-acid protein-protein interaction module multiple copies of are found in a wide range of proteins with diverse functions such as cell cycle regulations, gene transcription and splicing processes, protein trafficking, and protein folding. 195, 199 figure 15a shows that human lgn protein is involved in a wide range of protein-protein interactions, clearly serving as an important hub protein. this interactivity analysis is done by string. 59 frmpd1 protein (ferm and pdz domain containing 1) is known to serve as a regulatory binding partner of ags3, with a short fragment of frmpd1 (amino acids 901-938) being shown to bind to the 8 tpr motifs of ags3. 200 also, a 50residue fragment (amino acids 901-951) of frmpd1 was shown to bind to lgn with a k d »1 mm. 188, 194 curiously, besides the mentioned above regions of missing electron density in the lgn-tpr/frmpd1 complex figures 15b and 15c shows that fulllength lgn and frmpd1 proteins both belong to the category of hybrid proteins possessing some ordered domains and significant number of intrinsically disordered protein regions (idprs). in fact, the goloco motif containing c-terminal half of human lgn (residues 350-684) and the major portion of frmpd1 (residues 511-1390) are predicted to be mostly disordered by all the computational tools used in this study. overall, more than 40% of the lgn residues and more than 50% of the frmpd1 residues are predicted figure 16 . (a) evaluation of the interactivity of the human lgn by string, which is the online database resource search tool for the retrieval of interacting genes that provides both experimental and predicted interaction information. 59 string produces the network of predicted associations for a particular group of proteins. the network nodes are proteins. the edges represent the predicted functional associations. an edge may be drawn with up to 7 differently colored lines -these lines represent the existence of the 7 types of evidence used in predicting the associations. a red line indicates the presence of fusion evidence; a green line -neighborhood evidence; a blue lineco-occurrence evidence; a purple line -experimental evidence; a yellow linetext mining evidence; a light blue line -database evidence; a black lineco-expression evidence. 59 to be disordered, which clearly places these proteins to the category of highly disordered proteins. furthermore, both proteins are expected to have numerous disorder-based interaction sites. these can be identified by anchor, 15,16 a computational tool that relies on the pair-wise energy estimation approach developed for the general disorder prediction method iupred, 201, 202 and is based on the hypothesis that long regions of disorder contain localized potential binding sites that cannot form enough favorable intra-chain interactions to fold on their own, but are likely to gain stabilizing energy by interacting with a globular protein partner. 15, 16 here the term anchor-indicated binding site (aibs) is used to identify a region of a protein suggested by the anchor algorithm to have significant potential to be a binding site for an appropriate but typically unidentified partner protein. this analysis revealed that there are 10 aibss in lgn (residues 370-379, 436-452, 491-499, 508-513, 531-537, 545-553, 566-573, 596-607, 629-638 and 661-671), whereas frmpd1 contains 23 aibss (residues 534-554, 609-617, 622-636, 652-662, 704-715, 747-779, 792-804, 828-878, 896-909, 932-949, 958-967, 983-1019, 1023-1077, 1087-1100, 1106-1114, 1119-1129, 1138-1171, 1192-1220, 1245-1250, 1290-1297, 1312-1319, 1336-1342, and 1424-1431) . of special interest is the fact that the 901-951 fragment of human frmdp1 used in the mentioned structural analysis 188 is predicted to overlap with 2 aibss (residues 896-909 and 932-949). this is a clear indication that intrinsic disorder plays a role in the formation of the lgn-tpr/frmpd1 complex. davenport et al. reported a crystal structure of human sirtuin-1catalytic domain (residues 234-510) in complex with its c-terminal regulatory segment (ctr, residues 641-665). 203 in this structure (see fig. 17a , pdb id: 4ig9), the catalytic nad c -binding domain adopts the canonical sirtuin fold, whereas ctr forms a b-hairpin structure complementing the b-sheet of the catalytic domain and covering an essentially invariant hydrophobic surface. 203 biological significance of the sirtuin family was outlined in the previous section. human sirtuin-1 (sirt1) is implicated in a wide range of human diseases due to the fact that this enzyme plays multiple roles in cellular processes ranging from energy metabolism to cell survival via it ability to deacetylate a wide range of substrates, such as p53, nf-kb, foxo transcription factors, and pgc-1a. [204] [205] [206] deacetylation activity of sirt1 was shown to be affected by various regions located within the long n-and c-termini that flank the sirt1 catalytic domain. 207, 208 figure 17b illustrates that in agreement with a recent bioinformatics study revealing that n-and c-terminal segments of sirtuins in all known organisms are expected to be intrinsically disordered, 209 both termini of human sirt1 (uniprot id: q96eb6, residues 1-250 and 500-747) are predicted to be mostly disordered, whereas the central catalytic domain is mostly ordered. the anchor analysis 15, 16 revealed that human sirt1 contains 14 aibss located at the residues 1-33, 43-49, 53-125, 133-169, 184-193, 220-225, 498 -503, 521-528, 549-574, 588-593, 618-624, 637-672, 691-706, and 710-744. it is important to emphasize here that the ctr (residues 641-665) used in the mentioned above structural analysis of human sirt1 203 is completely embedded within one of the aibss (residues 637-672). the endosome-associated deubiquitinase amsh davies et al. performed a systematic mutational analysis to elucidate the molecular mechanisms of activation of amsh [associated molecule with a src homology 3 domain of signal transducing adaptor molecule (stam)], a deubiquitinating enzyme (dub) with exquisite specificity for lys63-linked polyubiquitin chains and recruitment of this protein to the endosomal sorting complexes required for transport (escrt) machinery. 210 the fact that the mutations amsh were implemented in microcephaly capillary malformation (mic-cap) syndrome in children 211 further reiterates the importance of this study. amsh is a member of the jamm (jab1/mpn/mov34) family of dubs involved in the regulation of ubiquitin signaling by catalyzing the hydrolysis of isopeptide (or peptide) bonds between ubiquitin and target proteins or within polymeric chains of ubiquitin. 212 the catalytic activity of amsh is stimulated upon binding to stam, which is a member of the escrt-0 complex. here, the amsh-stam interaction is conducted through the binding of the sh3 binding motif of amsh to the sh3 domain of the stam. 213 davies et al. used in their analysis the catalytic domain of amsh (residues 219-424). 210 figure 14c represents the results of the disorder analysis of human amsh (uniprot id: o95630) and shows that this protein contains a long idpr (residues 90-250) thereby illustrating that the n-terminal part of the analyzed catalytic domain is predicted to be disordered. tonb is the e.coli inner membrane protein, which possesses a polyproline motif (residues 70-81) that may span the length of the periplasmic space 216 and a globular c-terminal domain (residues that interacts with the transporters. 217, 218 figure 18a shows that the n-terminal half of the periplasmic domain of tonb protein (uniprot id: p02929) is predicted to be highly disordered, whereas its c-terminal domain possesses significant amount of order. figure 18b illustrates an important point that the dimer formed by the truncated periplasmic domain (residues 150-239) is a highly intertwined and elongated structure with noticeable domain swapping. a crystal structure of the tonb-fhua complex is shown in figure 18c . curiously, although the entire periplasmic domain of tonb was used in the crystallization experiment, residues 39-157 and 236-239 are not seen in the resulting structure, clearly indicating that these regions are likely to preserve mostly disordered structure even in the bound form of tonb. the disordered n-terminal and c-terminal tails clearly have functional importance since they contains aibss, 3 at the n-terminus (residues 39-65, 92-95, and 124-164) and one at the c-terminus (residues 232-239). one more aibs is predicted at residues 171-188. stark et al. performed functional analysis of the important human chaperone, dnaj homolog subfamily a member 1 (dnaja1), and solved solution structure of its j-domain (residues 1-67). 219 human dnaja1 has multiple important functions and act as protein chaperone, regulator of androgen receptor signaling, and activator of the dnak protein. furthermore, levels of the human dnaja1 serve as a biomarker for pancreatic cancer, since the expression of this protein in pancreatic cancer cells is downregulated fold5-. 220 nmr analysis revealed that the structure of the human dnaja1 j-domain consists of 4 a-helices, residues 17-21 (a1), 29-42 (a2), 52-65 (a3), and 68-75 (a4) (see fig. 19a ; pdb id: 2m6y). 219 on the contrary, the evaluation of intrinsic disorder propensity of the full-length protein suggested that dnaja1 (uniprot id: p31689) possesses significant amount of disorder throughout its n-and c-terminal tails (see fig. 19b ). curiously, by anchor analysis, there are 3 disorderbased binding sites in dnaja1, residues 26-31, 85-93, and 392-397, with binding site #2 being overlapped with a2. structure and functions of the purinerich element binding protein b (purb) in a recent comprehensive study, romora et al. showed that the purine-rich element binding protein b (purb) serves as a suppressor of myofibroblast differentiation and acta2 repression. 221 pura and purb are members of a small family of nucleic acid-binding proteins that interact with purine-rich ssdna or rna sequences homologous to the so-called pur element originally described in eukaryotic gene flanking regions and origins of dna replication. [222] [223] [224] figure 14d shows that human purb protein (uniprot id: o35295) is predicted to possess significant amount of functionally important intrinsic disorder. importance of the long disordered regions in this protein is supported by finding 10 aibss (residues 1-6, 18-27, 47-75, 87-95, 99-106, 135-137, 142-144, 186-201, 243-256, and 277-287) . in agreement with these predictions, the authors showed that several regions of purb were (residues 41-112, 125-210, and 125-303) were intrinsically unstable. 221 the host restriction factor tetherin hotter et al. provided a comprehensive review of the antiviral role of one of the host restriction factors (i.e., specific cellular antiviral factors that inhibit retroviral replication at different steps of the viral life cycle), human tetherin. 225 among various antiviral mechanisms ascribed to tetherin are the inhibition of the release of diverse enveloped viruses by tethering them to the cell surface, induction of an inflammatory response, activation of nf-kb, action as an innate immune sensor of viral infections, activation of immune response through interactions with the immunoglobulin-like transcript 7 (ilt7, lilra4). 225 as commonly seen in the virus-host arm race, effective antiviral strategies of the host are counterbalanced by the development of novel invasive strategies, and various viruses have evolved antagonists against this host restriction factor, such as accessory hiv-1 (human immunodeficiency virus type 1) vpu protein that counteracts the effects of tetherin. 225 tetherin is a type ii transmembrane protein that contains both an n-terminal transmembrane region and a c-terminal glycosyl-phosphatidylinositol (gpi) anchor. 226 structurally, human tetherin exists as a disulfide-linked parallel homodimer, which is formed via the extracellular part that is involved in the formation of a canonical coiled-coil structure, where a long a-helix contains 3 cysteines that form disulfide bonds with the corresponding cysteines of a second tetherin molecule. 227 it is believed that tetherin-caused viral retention relies on this unusual architecture, where one transmembrane anchor may remain in the cellular plasma membrane, whereas the other is able to stick to the viral membrane. 228, 229 figure 20a represents the results of the multi-tool disorder predictions for human tetherin (also known as bone marrow stromal antigen, uniprot id: q10589) and shows that this protein is expected to be mostly disordered. this is not a very surprising observation since coiled-coil proteins are expected to be intrinsically disordered in their monomeric states and fold to a helical structure at the formation of coiled-coil structure caused by the interaction with corresponding partners. 230 cyclin d1/cdk2 complex to clarify some uncertainties in previous studies regarding formation and activities of the cyclin d1/cyclin-dependent kinase 2 (cdk2) complexes jahn et al. utilized a novel p21-pcna fusion protein and p21 mutant proteins to understand the mechanisms of the cyclin d1/cdk2 complex formation. 231 the authors show that p21 serves as an important scaffolding protein, which is required for the formation of the functional cyclin d1/cdk2 complex, since cyclin d1 and cdk2 failing to complex in its absence. 231 figure 20b shows that the scaffolding protein p21 (which is a cyclin-dependent kinase inhibitor 1, uniprot id: p38936) is predicted to be mostly disordered and packed with disorder-based binding sites. in fact, there are 5 aibss in this relatively small protein, residues 36-41, 68-79, 100-105, 109-123, and 145-164. using this approach, the authors were able to identify a novel cdk2 substrate, polypyrimidine tract binding protein-associated splicing factor (psf). figure 20c shows that psf (uniprot id: p23246) is predicted to have highly disordered tails. anchor analysis 15, 16 revealed that intrinsic disorder is crucial for the psf human and mouse apolipoproteins a-i nguen et al. analyzed mechanistic peculiarities of the interaction between the high density lipoprotein (hdl) particles and human or mouse apolipoprotein a-i (apoa-i). 232 the authors established that the c-terminal domains (ctds) of human and mouse apoa-i proteins are the major players facilitating interactions of these proteins with hdl. curiously, human ctd, being a bit more hydrophobic than mouse ctd, binds to the hdl particle with higher affinity. on the other hand, the isolated n-terminal helix bundle domains (residues 1-190) of human and mouse apoa-i proteins binds hdl poorly. 232 apoa-i stabilizes discoidal hdl particles by forming a double-belt structure 233, 234 and plays a major role in the reverse cholesterol transport pathway. 235, 236 apoa-i contains a globular n-terminal domain (residues 1-43) and a lipid-binding c-terminal domain (residues 44-243). 234 in the belt-like structure of the smallest discoidal hdl, 2 apoa-i molecules wrap around a small patch of bilayer containing 160 lipid molecules. the c-terminal domain of each monomer is ring-like, curved, planar amphipathic a-helix with the hydrophobic surface curved toward the lipids. 234 figure 21 shows that both human and mouse apoa-i proteins (uniprot ids: p02647 and q00623) are predicted to be mostly disordered, with mouse protein possessing a bit more disordered structure. according to the anchor analysis, there are 3 aibss in both proteins (residues 15-21, 158-164, and 221-230 in human protein, and residues 13-18, 173-204, and 207-230 in mouse apoa-i). figure 21c represents a structure of the human apoa-i dimer in a complex with lipids (pdb id: 3k2s). this model was built by uniting several synergistic experimental techniques, such as small angle neutron scattering (sans) with contrast variation, isotopic deuteration of selected macromolecule components, and hydrogen/ deuterium exchange tandem mass spectrometry (hd-ms/ms). 237 in agreement with the results of disorder predictions, the apoa-i dimer represents a highly intertwined double superhelix with a minimal number of intrachain contacts, which is stabilized mostly by the interchain interactions. in other words, this complex is characterized by a very large interface area, and, therefore, according to gunasekaran et al. 238 it belong to the category of 2-state dimers, where the monomers are unfolded in the unbound state and fold simultaneously with the complex formation. function and regulation of mavs jacobs and coyne provided an important overview of the mitochondrial antiviral signaling protein (mavs, also known as ips-1/visa/cardif). 239 mavs is the mitochondrially-located innate immune signaling adaptor regulating and coordinating signals received from 2 independent cytosolic pathogen recognition receptors to induce antiviral genes. mavs can be regulated by host cell factors that inhibit mavs signaling by direct protein-protein interactions, by altering mitochondrial properties or dynamics, or by post-translational modifications. 239 the tip of the mavs c-terminus contains a mitochondrial intermembrane region (residues 535-540) needed for the interaction with the mitochondrial membrane. according to uniprot, human mavs (uniprot id: q7z434) is involved in a wide array of protein-protein interactions. among established mavs binding proteins are: ddx58/rig-i, ifih1/mda5, traf2, traf6, c1qbp, irf3, fadd, ripk1, chuk, ikbkb, hcv and hepatitis gb virus b ns3/4a proteases, hhav protein 3abc, nlrx1, psma7, trafd1, pcbp2, ips1, itch, cyld, src, dhx58/lgp2, ddx58/rig-i, ikbke, tmem173/mita, ifit3, tbk1 , and human respiratory syncytial virus (hrsv) ns1 protein (http://www. uniprot.org/uniprot/q7z434). mavs contains multiple alternatively spliced isoforms, sites of posttranslational modifications, and a proline-rich region (residues . in other words, mavs satisfies all the major criteria to be considered as an intrinsically disordered protein. figure 22a represents the vast interactome of mavs evaluated by string. figure 22b shows that human mavs is predicted to be a highly disordered protein. anchor analysis 15 khasnis et al. analyzed the mechanisms and functional outputs of the phosphorylation/dephosphorylation events at thr696 and thr853 sites of the mypt1 protein, which is a regulatory subunit of the myosin light chain phosphatase (mlcp). 240 mlcp is a cytoskeleton-associated protein phosphatase-1 (pp1) that serves as a rhoa/rock effector, regulating dynamic reorganization of the cytoskeleton crucial for cell motility. the authors also showed that the c-terminal domain of human mypt1 (residues 495-1030) was responsible for the binding to the n-terminal portion of myosin light meromyosin. 240 promiscuous interactability of mypt1 is illustrated by figure 23a representing the results of string analysis. figure 23b shows that the very significant part of mypt1 (uniprot id: o14974) is expected to be intrinsically disordered, with longest disordered region covering »72% of protein length (residues 290-1030) and containing 18 disorder-based binding sites (residues 318-338, 374-403, 406-419, 434-454, 472-477, 492-506, 548-564, 579-588, 626-649, 666-673, 697-712, 754-774, 780-815, 853-860, 887-892, 897-917-932-943, and 1021-1030) . clearly, intrinsic disorder is crucial for function of this protein. li et al. investigated structural peculiarities of the core polypeptide of the inner membrane gerd lipoprotein from geobacillus stearothermophilus. 241 some bacteria are able to form endospores in response to adverse growth conditions. 242, 243 spores formed as a result of such sporulation process are extremely resistant to various environmental insults thereby providing the bacteria with an important means to exist in the metabolically dormant state indefinitely and remain viable for hundreds of years without water or nutrients. 244, 245 restoration of the favorable conditions triggers spore germination leading to the fast (within minutes) "awakening" of the normal metabolism in bacteria followed by outgrowth to generate growing cells. 243, 244, 246 this awakening is controlled by the specific nutrient sensors, cognate germinant receptors (grs) located in the inner membrane of the spore. gerd is one of such bacterial cognate germinant receptors that trigger spore germination in the presence of specific nutrients called germinants in the environments of the spores. 241 li et al. have determined the crystal structure of the 121-residue core domain of the geobacillus stearothermophilus gerd protein (gerd ) that lacks the n-terminal signal and lipobox sequences (residues 1-28), the h01 and h02 helices (residues , and the c-terminal acidic tail (residues [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [193] [194] [195] . 241 by a set of biophysical techniques, this gerd 60-180 was shown to form a stable, well-ordered 3helix bundle in solution. in crystal structure, gerd 60-180 trimer consists of 3 parallel polypeptide chains twisted into a superhelical, right-handed rope (pdb id: 4o8w). 241 in this elongated trimer, each of the individual gerd 60-180 chains forms 8 helices (h1 to h8) linked by short turns. structures of the individual chains can be superimposed on each other except to the loosely packed h8 helices. helices h1-h7 in each gerd 60-180 chain twist around the central axis to form 2 complete turns of a right-handed supercoil with a pitch of »44a . figure 24a represents crystal structure of the gerd 60-180 trimer as a molecular surface and ribbon diagram and also shows the highly extended structure of one of the gerd 60-180 monomers. 241 analysis of the overall shapes of the highly intertwined and extended monomers within the gerd 60-180 trimer suggests that the formation of this trimer represents an example of the folding-uponbinding process. in agreement with this hypothesis, figure 24b shows that the core domain of the geobacillus stearothermophilus gerd protein (uniprot id: q5l3q1) is mostly disordered. prokaryotic ubiquitin-like protein (pup), the pup ligase pafa and bacterial proteasome forer et al. investigated the interaction between the prokaryotic ubiquitin-like protein (pup), the proteasome regulatory subunit mpa (mycobacterium proteasome atpase), and proteasome accessory factor a (pafa), an enzyme that forms an isopeptide bond between the g-carboxylate of a glutamate at the c-terminus of pup and the e-amine of a substrate lysine. 247 the authors show that pup is involved in simultaneous interaction with both mpa and pafa, and that mpa forms a complex with pafa, suggesting that pafa and the proteasome acts as a modular machine for the tagging and degradation of cytoplasmic proteins. 247 figure 25a represents a crystal structure of 3 pup molecules bound to the mpa hexamer (pdb id: 3m9d), and the results of disorder prediction for the mycobacterium tuberculosis pup (uniprot id: p9whn5) are shown in figure 25b . pup is obviously a highly disordered protein that partially folds at binding. in fact, of 68 residues used in the crystallization experiment, only 31 are visible in structure, whereas remaining 37 residues (1-20 and 52-68) are located within the regions of missing electron density. according to the anchor analysis, pup possesses 2 disorder-based binding regions, residues 1-15 and 34-64. curiously, forer et al. suggested that pup has 2 binding sites for rafa located at residues 38-47 and 51-58, and that the first rafa binding site is overlapped with the region responsible for the mpa binding (residues 21-51). 247 the nucleolar pict-1/gltscr2 protein borodianskiy-shteinberg et al. studied self-association of the human protein interacting with carboxyl terminus-1 (pict-1), also known as the glioma tumor suppressor candidate region 2 gene product (gltscr2), a nucleolar protein with unknown function. 248 pict-1/ gltscr2 is conserved among eukaryotes and is assumed to be essential for preimplantation embryogenesis and embryonic stem cell survival and proliferation. 249 it was suggested that pict-1 might act as a potential tumor suppressor, being involved in interaction with the cterminal region of the tumor suppressor phosphatase and tensin homolog (pten), promoting its phosphorylation and stabilization. 250 it was also suggested that pict-1 can function as an oncogene through the inhibition of p53. 249 figure 26a represents the results of the evaluation of the interactivity of the human pict-1/gltscr2 (uniprot id: q9nzm5) by string and shows a dense interaction network of this intriguing protein. figure 26b provides an explanation for the binding promiscuity of pict-1 by showing that this protein is predicted to be highly disordered. recognition of modified trna by the hiv-1 nucleocapsid protein p7 and related peptides spears et al. studied the molecular mechanisms underlying specific recognition of highly post-transcriptionally modified human trna lys3 uuu (which is the primer for hiv replication) by the hiv-1 nucleocapsid protein p7. 251 it is known that p7 recruits htrna lys3 uuu from the host cell by binding to and remodeling the trna structure. an intrigue here is in the fact that htrna lys3 uuu is one of the most uniquely processed trnas that has a broad spectrum of chemically different post-transcriptional modifications playing crucial roles in regulation of the conformation and function of this trna during protein synthesis. 252 using phage display approach, spears et al. showed that p7 contains a specific htrna-lys3 uuu -binding region whose interaction with fully modified trna can be mimicked by the 15-and 16-amino acid peptides with the signature sequence of r-w-q/n-h-x 2 -f-pho-x-g/a-w-r-x 2 -g (where x can be most amino acids, and pho is any hydrophobic residue). 251 hiv-1 nucleocapsid protein p7 is a 55residues-long protein containing 2 zinc finger domains flanked by basic amino acids required for interaction with nucleic acids. 253, 254 in addition to the discussed above capability of p7 to specifically recognize htrna lys3 uuu , the major function of p7 is to bind specifically to the packaging signal of the full-length viral rnas and to deliver them into the assembling virion. 255 as it is a highly charged basic protein, p7 binds single-stranded nucleic acids nonspecifically. consequently, it coats the genomic rna protecting it from nucleases and compacting viral rna within the core. protein p7 also serves as an rna chaperone that enhances several nucleic acid-dependent steps of viral life, such as melting rna secondary structures, promoting dna strand exchange reactions during reverse transcription, [256] [257] [258] and stimulating integration. 259 in the nmr solution structure of p7, regions corresponding to the 2 zinc fingers (residues 15-28 and 36-49) possess wellresolved structures, whereas residues 1-13, 32-34, and 52-55 were highly dynamic and did not converge to the unique conformations. 260, 261 recent intrinsic disorder propensity analysis revealed that p7 is a highly disordered protein, with regions corresponding to the zinc fingers predicted to be more ordered than the remainder of the protein and identified as potential a-morfs. 262 this article represents a set of papers dealing with interesting and important proteins with diverse functions from various organisms. the only uniting theme for all these studies is the notion that they represent noticeable disorder overlooks. in fact, all proteins studied there contain intrinsic disorder. the amount of disorder ranges, and in some proteins only relative short regions are disordered or highly flexible, whereas some other proteins are almost entirely disordered. however, very often this disorder has functional implementations, and consideration of studied proteins in light of intrinsic disorder often provides interesting mechanical clues on the molecular mechanisms of their action. we hope that the readers will find this new series useful, since it might increase the awareness of the scientific community about importance of intrinsic disorder for protein function. we also hope to see submissions from readers who found some important intrinsic disorder overlooks in published articles. obviously, papers in this seris will range in their size, and we envision several types of submissions, such as comprehensive reviews of unreported disorder in papers published in particular journals (i.e., similar to this review), reviews of unreported disorder in specific areas of protein-related research, as well as brief notes about overlooked disorder in individual papers. the biggest hope though is that this series will become obsolete, and protein intrinsic disorder will find its way to be robustly present in the scientific literature. no potential conflicts of interest were disclosed. digested disorder: quarterly intrinsic disorder digest digested disorder, issue #2: quarterly intrinsic disorder digest digested disorder, issue #3: quarterly intrinsic disorder digest comprehensive comparative assessment of in-silico predictors of disordered regions eif4b and eif4g jointly stimulate eif4a atpase and unwinding activities by modulation of the eif4a conformational cycle new initiation factor activity required for globin mrna translation the mechanism of eukaryotic translation initiation and principles of its regulation molecular mechanism of scanning and start codon selection in eukaryotes mrna helicases: the tacticians of translational control the dead-box helicase eif4a: paradigm or the odd one out exploiting heterogeneous sequence properties improves prediction of protein disorder predicting intrinsic disorder from amino acid sequence sequence complexity of disordered protein pondr-fit: a meta-predictor of intrinsically disordered amino acids prediction of protein binding regions in disordered proteins anchor: web server for predicting protein binding regions in disordered proteins remarkable stabilization of plasminogen activator inhibitor 1 in a "molecular sandwich" complex interaction of t4 uvsw helicase and single-stranded dna binding protein gp32 through its carboxy-terminal acidic tail crystallographic and nmr analyses of uvsw and uvsw.1 from bacteriophage t4 dna helicases: new breeds of translocating motors and molecular pumps analysis of the dna translocation and unwinding activities of t4 phage helicases uvsw protein regulates bacteriophage t4 origin-dependent replication by unwinding r-loops dna helicase requirements for dna replication during bacteriophage t4 infection the t4 phage uvsw protein contains both dna unwinding and strand annealing activities processive and unidirectional translocation of monomeric uvsw helicase on single-stranded dna bacteriophage t4 uvsw protein is a helicase involved in recombination, repair and the regulation of dna replication origins the phage t4 protein uvsw drives holliday junction branch migration repetitive lagging strand dna synthesis by the bacteriophage t4 replisome crystal structure of a replication fork single-stranded dna binding protein (t4 gp32) complexed to dna the gene 59 protein of bacteriophage t4. characterization of protein-protein interactions with gene 32 protein, the t4 single-stranded dna binding protein characterization of bacteriophage t4-coordinated leading-and lagging-strand synthesis on a minicircle substrate identification and mapping of protein-protein interactions between gp32 and gp59 by cross-linking functional mapping of pilf and pilq in the pseudomonas aeruginosa type iv pilus system structure and function of pilq, a secretin of the dna transporter from the thermophilic bacterium thermus thermophilus hb27 secretins: dynamic channels for protein transport across membranes characterization of 14-3-3-zeta interactions with integrin tails 14-3-3 proteins-a focus on cancer and human disease 14-3-3 proteins as potential therapeutic targets cantley lc. the structural basis for 14-3-3:phosphopeptide binding specificity binding of 14-3-3 protein to the plasma membrane h(c)-atpase aha2 involves the three c-terminal residues tyr(946)-thr-val and requires phosphorylation of thr(947) atm-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the x-ray crystal structures of all human 14-3-3 isoforms structural basis of 14-3-3 protein functions intrinsic disorder is a key characteristic in partners that bind 14-3-3 proteins integrins: bidirectional, allosteric signaling machines integrin signalling at a glance the tail of integrin activation integrin cytoplasmic domain-binding proteins mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails the final steps of integrin activation: the end game beta2 integrin phosphorylation on thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding trimmunity: the roles of the trim e3-ubiquitin ligase family in innate antiviral immunity a unique bipartite cysteinehistidine motif defines a subfamily of potential zincfinger proteins a novel zinc finger coiled-coil domain in a family of nuclear proteins a novel protein sequence motif related to the zinc finger trim/rbcc, a novel class of 'single protein ring finger' e3 ubiquitin ligases the role of viperin in the innate antiviral response the string database in 2011: functional interaction networks of proteins, globally integrated and scored a molecular dynamics simulation-based interpretation of nuclear magnetic resonance multidimensional heteronuclear spectra of alpha-synuclein dopamine adducts hiv-1 restriction factor samhd1 is a deoxynucleoside triphosphate triphosphohydrolase nuclease activity of the human samhd1 protein implicated in the aicardi-goutieres syndrome and hiv-1 restriction contribution of sam and hd domains to retroviral restriction mediated by human samhd1 hiv/simian immunodeficiency virus (siv) accessory virulence factor vpx loads the host cell restriction factor samhd1 onto the e3 ubiquitin ligase complex crl4dcaf1 novel interaction between the major bacterial heat shock chaperone (groesl) and an rna chaperone (cspc) interplay between the heat shock response and translation in escherichia coli proteolysis in the escherichia coli heat shock response: a player at many levels the cold-shock response-a hot topic recent developments in bacterial coldshock response cold-shock response and cold-shock proteins rna remodeling and gene regulation by cold shock proteins the role of structural disorder in the function of rna and protein chaperones interactions of phosphatase and tensin homologue (pten) proteins with phosphatidylinositol phosphates: insights from molecular dynamics simulations of pten and voltage sensitive phosphatase the tumor suppressor, pten/mmac1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate intrinsic disorder in pten and its interactome confers structural plasticity and functional versatility the pten long n-tail is intrinsically disordered: increased viability for pten therapy pathological unfoldomics of uncontrolled chaos: intrinsically disordered proteins and human diseases the structure of xis reveals the basis for filament formation and insight into dna bending within a mycobacteriophage intasome determinants of directionality in lambda site-specific recombination control of directionality in l5 integrase-mediated site-specific recombination identification and characterization of mycobacteriophage l5 excisionase structure of the cooperative xis-dna complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly regulation of directionality in bacteriophage lambda site-specific recombination: structure of the xis protein unique proline-rich domain regulates the chaperone function of aipl1 flexible nets of malleable guardians: intrinsically disordered chaperones in neurodegenerative diseases variable contributions of basic residues forming an apc exosite in the binding and inactivation of factor viiia molecular events that control the protein c anticoagulant pathway regulation of blood coagulation by the protein c system the mechanism of inactivation of human factor v and human factor va by activated protein c the 2.8 a crystal structure of gla-domainless activated protein c the escherichia coli primosomal dnat protein exists in solution as a monomer-trimer equilibrium system assembly of the primosome of dna replication in escherichia coli requirements for replication restart proteins during constitutive stable dna replication in escherichia coli k-12 replication fork reactivation downstream of a blocked nascent leading strand the most important thing is the tail: multitudinous functionalities of intrinsically disordered protein termini immunoreceptor tyrosine-based inhibitory motif (itim)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: a case study involving pecam-1 signal transduction pathways mediated by pecam-1: new roles for an old molecule in platelet and vascular cell biology residues within a lipid-associated segment of the pecam-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation splicing kinase srpk1 conforms to the landscape of its sr protein substrate intrinsic disorder in the human spliceosomal proteome malleable ribonucleoprotein machine: protein intrinsic disorder in the saccharomyces cerevisiae spliceosome ifitms restrict the replication of multiple pathogenic viruses the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm1 is a tight junction protein that inhibits hepatitis c virus entry mechanism of membrane fusion by viral envelope proteins ifitm3 inhibits influenza a virus infection by preventing cytosolic entry predicting disordered regions from amino acid sequence: common themes despite differing structural characterization predicting binding regions within disordered proteins coupled folding and binding with alpha-helix-forming molecular recognition elements analysis of molecular recognition features (morfs) mining alpha-helix-forming molecular recognition features with cross species sequence alignments type 2 ryanodine receptor domain a contains a unique and dynamic alpha-helix that transitions to a beta-strand in a mutant linked with a heritable cardiomyopathy involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle interactions between dihydropyridine receptors and ryanodine receptors in striated muscle crystal structures of the nterminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations van petegem f. the deletion of exon 3 in the cardiac ryanodine receptor is rescued by beta strand switching atpinduced dimerization of the ff epsilon subunit from bacillus ps3: a hydrogen exchange-mass spectrometry study crystal structure of the epsilon subunit of the proton-translocating atp synthase from escherichia coli phosphorylation of the cyclin capcl5 modulates both cyclin stability and specific recognition of the substrate principles of cdk regulation cyclin specificity: how many wheels do you need on a unicycle? a family of cyclin-like proteins that interact with the pho85 cyclin-dependent kinase regulation of the transcription factor gcn4 by pho85 cyclin pcl5 coevolution of cyclin pcl5 and its substrate gcn4 understanding protein non-folding constitutive nuclear localization of an alternatively spliced sirtuin-2 isoform crosstalk between components of circadian and metabolic cycles in mammals sirtuins as regulators of metabolism and healthspan sirt2 maintains genome integrity and suppresses tumorigenesis through regulating apc/c activity the mammalian sir2alpha protein has a role in embryogenesis and gametogenesis the conserved role of sirtuins in chromatin regulation impaired dna damage response, genome instability, and tumorigenesis in sirt1 mutant mice regulation of sirtuin function by posttranslational modifications characterization of the murine sirt3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short sirt3 isoforms distinct regulation of mitochondrial localization and stability of two human sirt5 isoforms transcription levels of sirtuin family in neural stem cells and brain tissues of adult mice evolutionarily conserved and nonconserved cellular localizations and functions of human sirt proteins interphase nucleo-cytoplasmic shuttling and localization of sirt2 during mitosis alternative splicing in concert with protein intrinsic disorder enables increased functional diversity in multicellular organisms tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks alternative splicing of intrinsically disordered regions and rewiring of protein interactions effect of c-terminal sequence on competitive semaphorin binding to neuropilin-1 furin processing of semaphorin 3f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin structural basis for ligand and heparin binding to neuropilin b domains structural basis for selective vascular endothelial growth factor-a (vegf-a) binding to neuropilin-1 new prospects in the roles of the c-terminal domains of vegf-a and their cooperation for ligand binding, cellular signaling and vessels formation x-ray crystal structure of bovine 3 glu-osteocalcin direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue characterization of a gamma-carboxyglutamic acid-containing protein from bone the vitamin k-dependent carboxylase calcium-dependent alphahelical structure in osteocalcin a method for decarboxylation of gamma-carboxyglutamic acid in proteins. properties of the decarboxylated gamma-carboxyglutamic acid protein from calf bone coordinated transcriptional regulation of hspa1a gene by multiple transcription factors: crucial roles for hsf-1 plasma levels of hsp70 and anti-hsp70 antibody predict risk of acute coronary syndrome heat shock protein 70 kda: molecular biology, biochemistry, and physiology heat shock proteins in breast cancer progression-a suitable case for treatment? impaired heat shock response in cells expressing full-length polyglutamineexpanded huntingtin the heat shock factor family and adaptation to proteotoxic stress phosphorylated creb binds specifically to the nuclear protein cbp activation of camp and mitogen responsive genes relies on a common nuclear factor nuclear protein cbp is a coactivator for the transcription factor creb a multiplicity of ccaat box-binding proteins a survey of 178 nf-y binding ccaat boxes a ccaat box confers cell-type-specific regulation on the xenopus hsp70 gene in oocytes hsp-cbf is an nf-y-dependent coactivator of the heat shock promoters ccaat boxes sequence-specific transcription factor nf-y displays histone-like dna binding and h2b-like ubiquitination role of the nf-kappab signaling pathway and kappab cis-regulatory elements on the irf-1 and inos promoter regions in mycobacterial lipoarabinomannan induction of nitric oxide intrinsic disorder in transcription factors hsf transcription factor family, heat shock response, and protein intrinsic disorder intrinsic disorder in proteins involved in the innate antiviral immunity: another flexible side of a molecular arms race identification of critical phosphorylation sites on the carboxy tail of melanopsin the importance of intrinsic disorder for protein phosphorylation intrinsic disorder-based protein interactions and their modulators the structural and functional signatures of proteins that undergo multiple events of post-translational modification antiviral mechanisms of human defensins identification of intrinsic order and disorder in the dna repair protein xpa sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression multiple mechanisms for e2f binding inhibition by phosphorylation of the retinoblastoma protein c-terminal domain cellular mechanisms of tumour suppression by the retinoblastoma gene molecular mechanisms underlying rb protein function cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product the retinoblastoma protein copurifies with e2f-i, an e1a-regulated inhibitor of the transcription factor e2f identification, analysis, and prediction of protein ubiquitination sites functional anthology of intrinsic disorder. 1. biological processes and functions of proteins with long disordered regions functional anthology of intrinsic disorder. 2. cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions functional anthology of intrinsic disorder. 3. ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins structural and biochemical characterization of the interaction between lgn and frmpd1 analysis of partner of inscuteable, a novel player of drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization spindle orientation during asymmetric cell division mechanisms of asymmetric stem cell division lgn/minsc and lgn/numa complex structures suggest distinct functions in asymmetric cell division for the par3/minsc/ lgn and galphai/lgn/numa pathways inscuteable and numa proteins bind competitively to leu-gly-asn repeat-enriched protein (lgn) during asymmetric cell divisions structural basis for interaction between the conserved cell polarity proteins inscuteable and leu-gly-asn repeat-enriched protein (lgn) the tetratricopeptide repeat: a structural motif mediating protein-protein interactions return of the gdi: the goloco motif in cell division mammalian pins is a conformational switch that links numa to heterotrimeric g proteins distinct roles of galphai and gbeta13f subunits of the heterotrimeric g protein complex in the mediation of drosophila neuroblast asymmetric divisions tpr proteins: the versatile helix the pdz and band 4.1 containing protein frmpd1 regulates the subcellular location of activator of g-protein signaling 3 and its interaction with g-proteins the pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins iupred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content structural and functional analysis of human sirt1 aging and disease: connections to sirtuins sirtuins in aging and disease sirtuins in aging and agerelated disease peptide switch is essential for sirt1 deacetylase activity sirt1 contains n-and c-terminal regions that potentiate deacetylase activity genealogy of an ancient protein family: the sirtuins, a family of disordered members mechanism of recruitment and activation of the endosome-associated deubiquitinase amsh mutations in stambp, encoding a deubiquitinating enzyme, cause microcephaly-capillary malformation syndrome mpnc, a putative catalytic motif found in a subset of mpn domain proteins from eukaryotes and prokaryotes, is critical for rpn11 function activation of the endosome-associated ubiquitin isopeptidase amsh by stam, a component of the multivesicular body-sorting machinery the eukaryotic linear motif resource elm: 10 years and counting monomeric tonb and the ton box are required for the formation of a high-affinity transporter-tonb complex the proline-rich domain of tonb possesses an extended polyproline ii-like conformation of sufficient length to span the periplasm of gram-negative bacteria outer membrane active transport: structure of the btub:tonb complex structure of tonb in complex with fhua, e. coli outer membrane receptor structure and function of human dnaj homologue subfamily a member 1 (dnaja1) and its relationship to pancreatic cancer proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma structural basis of multisite single-stranded dna recognition and acta2 repression by purine-rich element binding protein b (purbeta) the hela pur factor binds single-stranded dna at a specific element conserved in gene flanking regions and origins of dna replication sequence of cdna comprising the human pur gene and sequencespecific single-stranded-dna-binding properties of the encoded protein the pur protein family: genetic and structural features in development and disease emerging role of the host restriction factor tetherin in viral immune sensing bst-2/hm1.24 is a raft-associated apical membrane protein with an unusual topology structural and biophysical analysis of bst-2/tetherin ectodomains reveals an evolutionary conserved design to inhibit virus release running in reverse: the structural basis for translocation polarity in hexameric helicases immunoelectron microscopic evidence for tetherin/bst2 as the physical bridge between hiv-1 virions and the plasma membrane intrinsic disorder of the extracellular matrix assembly, activation, and substrate specificity of cyclin d1/cdk2 complexes interactions of apolipoprotein a-i with high-density lipoprotein particles the structure of human lipoprotein a-i. evidence for the "belt" model a detailed molecular belt model for apolipoprotein a-i in discoidal high density lipoprotein high density lipoprotein structure-function and role in reverse cholesterol transport high-density lipoprotein heterogeneity and function in reverse cholesterol transport double superhelix model of high density lipoprotein analysis of ordered and disordered protein complexes reveals structural features discriminating between stable and unstable monomers mechanisms of mavs regulation at the mitochondrial membrane reconstituted human myosin light chain phosphatase reveals distinct roles of two inhibitory phosphorylation sites of the regulatory subunit, mypt1 structural and functional analysis of the gerd spore germination protein of bacillus species spores of bacillus subtilis: their resistance to and killing by radiation, heat and chemicals germination of spores of bacillales and clostridiales species: mechanisms and proteins involved spore germination how do spores germinate? the ger receptor family from sporulating bacteria bacterial proteasome and pafa, the pup ligase, interact to form a modular protein tagging and degradation machine the nucleolar pict-1/gltscr2 protein forms homo-oligomers regulation of the mdm2-p53 pathway and tumor growth by pict1 via nucleolar rpl11 regulation of pten phosphorylation and stability by a tumor suppressor candidate protein amino acid signature enables proteins to recognize modified trna human trna(lys3)(uuu) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism mutations of basic amino acids of ncp7 of human immunodeficiency virus type 1 affect rna binding in vitro charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in rna packaging and infectivity hiv-1: fifteen proteins and an rna effect of mutations in the nucleocapsid protein (ncp7) upon pr160(gagpol) and trna(lys) incorporation into human immunodeficiency virus type 1 human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral dna synthesis by inhibiting tar-dependent self-priming from minus-strand strong-stop dna mutations in hiv reverse transcriptase which alter rnase h activity and decrease strand transfer efficiency are suppressed by hiv nucleocapsid protein human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates mg2c-dependent dna integration in vitro nucleocapsid zinc fingers detected in retroviruses: exafs studies of intact viruses and the solution-state structure of the nucleocapsid protein from hiv-1 determination of the structure of the nucleocapsid protein ncp7 from the human immunodeficiency virus type 1 by 1h nmr protein intrinsic disorder as a flexible armor and a weapon of hiv-1 key: cord-313173-0u4s5y20 authors: ten have, h.a.m.j. title: sheltering at our common home date: 2020-08-25 journal: j bioeth inq doi: 10.1007/s11673-020-10014-x sha: doc_id: 313173 cord_uid: 0u4s5y20 the current covid-19 pandemic has reactivated ancient metaphors (especially military ones) but also initiated a new vocabulary: social distancing, lockdown, self-isolation, and sheltering in place. terminology is not ethically neutral but reflects prevailing value systems. i will argue that there are two metaphorical vocabularies at work: an authoritarian one and a liberal one. missing is an ecological vocabulary. it has been known for a long time that emerging infectious diseases are associated with the destruction of functioning ecosystems and biodiversity. ebola and avian influenza viruses have been significant warnings. obviously, this pandemic will not be the last one. as the planet is our common home, the major metaphor to explore is sheltering at this home. catastrophic risk for the future (global priorities project 2016). in 2003, it was estimated that since 1980 more than thirty-five new infectious diseases have emerged in humans; one every eight months (smolinski et al. 2003) . since then the list has only grown (for example, the severe acute respiratory syndrome (sars) coronavirus, swine influenza (h1n1 and other sub-types), the middle east respiratory syndrome (mers) coronavirus, ebola virus disease (evd), and the zika flavivirus). most of these outbreaks have been localized but the human immunodeficiency virus (hiv), sars, and avian influenza (h5n1 and other sub-types) should have been warnings that globalization can easily occur. already in 2005, the who launched its global influenza preparedness plan, urging countries to make national bio-preparedness plans, and many countries did so (who 2005) . furthermore, it has been known for some time that infectious diseases are promoted by environmental degradation as a result of biodiversity loss and climate change. destruction of ecosystems is shrinking the wildlife habitat and increasing contacts between wildlife and human beings. it is estimated that zoonotic pathogens cause 60 per cent of emerging infectious diseases in humans (jones et al. 2008; daszak et al. 2004) . the global threat of pandemics therefore does not emerge spontaneously as a natural event but is the product of human behaviour. it is a consequence of the human way of life and exploitation of the planet. destruction of biodiversity creates the conditions for the emergence of new viral diseases (quammen 2012) . although much is unclear, studies indicate that bats are the reservoir hosts for the novel coronavirus sars-cov-2 causing covid-19, while pangolins might be possible hosts. bats are very common mammals. they harbour around thirty different coronaviruses but in fact many more viruses, mostly unknown. pangolins are the most frequently trafficked mammals, especially in china, used as a food source and for traditional medicine. they have been traded in the wet market in wuhan where the infection emerged (lam et al. 2020) . live animal markets, industrial livestock farming, trade in wild animals, and consumption of wildlife meat bring virus reservoirs in close contact with human beings. the only way to prevent future zoonotic diseases is to study the origin of pandemics. instead of waiting for the next pandemic, epidemiological research should be expanded and stringent measures taken to prevent the "jumping" of viruses to humans. what is missing in the pandemic management responses so far is the ecological perspective that pandemics are related to the current economic global order which assumes a separation of humans and nature and regards nature as a resource to be exploited and commodified. the absence of an ecological perspective is highlighted in the images and words associated with covid-19. sheltering in place is a notion from the cold war. when there is an imminent nuclear threat, people should take refuge in a small, interior room, lock all windows and remain indoors, or move to special shelters. this usually takes a few hours, not days or weeks. (american red cross, 2003) . the concept is also used for biological and chemical threats, as well as for extreme weather events. the basic idea is to wait until the worst is over; then go outside and resume normal activities. it assumes a specific view of disasters: they are sudden events with immediate but often localized impact such as tsunamis and tornadoes. pandemics, however, are gradual disasters; they have slow impact, and move across the globe, potentially affecting everyone. the difference is important for the degree of preparedness. since pandemics come in waves, not all regions and countries are simultaneously affected. this leaves time for preparation. it also means that sheltering will be extended for weeks perhaps months, not knowing how long it will provide security. during a pandemic, sheltering at home seems a more acceptable term. it avoids connotations of mandatory quarantine or isolation. it is restricting freedom of movement but appeals to the responsibility of individuals. contrary to shelter-in-place, sheltering at home is more lenient since it allows to go out for essential business and walking, keeping physical distance to other people. the notion of "home" generally has positive connotations. in distinction to "place" it is not a neutral location. it is where people live together in a space of intimacy and privacy, often regarded as a haven or refuge, a secure place to retreat and feel comfortable, a setting for caring relationships and conviviality (mallet 2004) . at home means more than residing in a specific place. of course, not all homes are the same (e.g. nursing homes), not all homes are safe (e.g. domestic abuse), and not everybody has a home. now that more than half of the world population is confined to their homes, philosopher gaston bachelard reminds us that beingat-home is more fundamental than activities such as working (bachelard 2014) . nevertheless, the emphasis on sheltering and isolation reflects an individualistic perspective, assuming that people can easily withdraw from social interactions. in many cases, connections with environing conditions cannot be severed. for numerous people, especially in low-and middle-income settings, this is not an option. also, poor people in affluent countries cannot shelter but have to expose themselves in order to subsist. the image of sheltering at home has become incorporated in popular discourse. previously used in epidemiology, new words (e.g. self-isolation, social distancing, and lockdown) are now disseminated to advance images of control and containment. they visualize the spread of the virus, what we can do to avoid infection and to become aware of the effects of our behaviour. these images are associated with the war metaphor dominating current policies. the disease is regarded as a threat, the virus as enemy, leaders as commanders, and vaccines as new weapons. today's metaphor is associated with the older discourse of bio-invasion. since the 1990s, many countries have developed policies to protect native biodiversity against invasive species. non-native species are considered as aliens, enemies; we need to act to protect nature. the assumption is that nature and humans can be separated. nature, and wildlife in particular, is regarded as a source of danger. however, bio-invasion is impossible to prevent. when mobility and interconnectedness are the hallmarks of globalization, bio-invasion is a global phenomenon par excellence since borders are irrelevant. the same applies to new, "emerging" viruses. viral diseases have been with us since the beginning of humanity. they are now more easily disseminated through global traffic. the point is that these "new" viruses do not simply "emerge" as natural events. their impact is the result of human activities. the military metaphor is currently applied in most countries. the effect is reinforcement of boundaries. first, between inside and outside, enemies and friends. it blames people for "bringing in the virus." second, individuals and social context are disconnected. terminology is not ethically neutral but reflects prevailing value systems. when the virus moved across the world, some words (e.g. mandatory quarantines, containment zones, home confinement) were less often used in liberal societies. while the military metaphor is predominant, it is articulated in distinct policies: authoritarian and liberal ones. both appeal to different normative frameworks which are often mixed in practical approaches. for example, "lockdown" and "quarantine" emphasize state measures beyond individual rights and freedoms. on the other hand, "self-isolation" appeals to individual responsibility. the term "social distancing" is typically neoliberal; it does not recognize the communal nature of many societies. what is at stake is physical (not social) distancing, while in many countries social connections and solidarity have grown. emphasizing self-interest risks that other persons are seen as threats. many countries, especially in the west, struggle with the balance between authoritarian and liberal policies. they are concerned with the protection of human rights and civil liberties. the discussion about the use of surveillance technologies and their impact on privacy is an example (gostin, friedman, and wetter 2020) . since both authoritarian and liberal policies are animated by the military metaphor, they share the same urge to control. they reflect a system of governance that administers, fosters, and secures life by controlling the population and disciplining the individual. this application of "biopower" is closely linked to the ideology of neoliberalism in emphasizing internal regulation by autonomous subjects rather than external force and pressure (foucault 2008; kakuk 2017) . even in authoritarian countries, stringent policies will not succeed if they do not appeal to the responsibility of citizens. emphasizing individual responsibility and empowerment, however, neglects the social, political, and economic dimensions of human life. it narrows bioethical discourse since it is difficult to conceptualize the significance of the context in which problems arise and to develop practices based on relationality and connectedness which are articulated in global bioethics with an ecological vocabulary. interconnectedness and interdependency articulate that humans cannot be separated from the surrounding world, not only society and culture but also the natural world of animals and plants. the starting point for bioethical discourse is therefore not individual autonomy but the broader context in which individuals are embedded. self-isolation in this perspective is not merely protecting oneself but first of all protecting fellow citizens. the experience of togetherness is not restricted to humans but involves all forms of life, even viruses. regarding humans as part of an interconnected web of life means moving from an anthropo-centric to biocentric approach with a relational concept of the self, dependent on biodiversity. this shift has been advocated by many environmental ethicists as well as in indigenous worldviews (rolston 1988; johnson 2020) .the ecological perspective implies that the military language of the pandemic is distorting the human embeddedness in the natural world. emphasizing the antagonism between humans and the virus as enemy, blames nature for diseases ignoring that diseases emerge due to environmental degradation as a result of human exploitation of biodiversity. it also disregards that microbes are inhabitants of our world; they have been and will be always there. rather than eradication, cohabitation will be required. the military metaphor encourages the search for "magic bullets" that can attack and eliminate the virus. it also creates an atmosphere of secrecy, suspicion, and mistrust where facts, findings, and potential weapons are disputed and concealed. another consequence of the ecological perspective is a broader notion of health. human health, animal health, and healthy ecosystems are linked. human beings cannot be healthy when the planet is not healthy. the implication is that governance should be global. it should be focused on health as one, as a planetary concern. applying the vision of one health means monitoring and surveilling human connections with animals, specifically in the bioindustry. another implication is that attention should not merely focus on morbidity and mortality but on prevention. future pandemics will emerge if environmental destruction and loss of biodiversity are not addressed. global interconnectedness implies that citizens in one country will be exposed to diseases when they emerge in another country. the ecological perspective therefore stresses the need for solidarity. this is not just an ethical requirement, but a medical necessity. closing borders, restricting travel, and concentrating on national interests had only a limited effect on the dissemination of covid-19. public health as a common good is essential for the well-being and survival of humanity. it will require cooperation and collective action, especially in the early stages of a pandemic disease when the caseload is still manageable and secondary prevention is possible. in an ecological perspective, vulnerability to infectious diseases is not confined to specific individuals, populations, or nations. while covid-19 and its impact will be temporary, the effect of climate change is already there and will continue, requiring sustained action for a very long time (guterres 2020) . both global threats are interpreted from a similar perspective: humans are not embedded in nature. the earth is a resource that can be commodified and exploited; it is not regarded as a living organism that creates and nourishes life (humans, animals, and microorganisms). the fact that many of us are now confined to our homes is hopefully an incentive to realize that we all share a common home. it demonstrates not merely interconnectedness of human beings manifested in society and culture but furthermore their embeddedness in the natural world. what affects the health of the planet will unquestionably deteriorate human health. concepts such as "one health" and "planetary health" articulate the connection between healthcare and earthcare. they make clear that the focus of bioethics should go beyond individual health and that effective policies should be based on collective action (ten have 2019). given the interconnection between health and biodiversity, the notion of sheltering at our common home is most appropriate in the current circumstances. the earth is our home, as stated in the preamble of the rio declaration (united nations 1992) . like all homes, this common home has dark sides, clearly noticeable today. but humans have no other dwelling place. our actions can destroy this place but they can also turn it into a home where everyone feels safe and secure. facing the covid-19 pandemic is an opportunity to make ourselves at home in the world and to preserve our common home. fact sheet on shelter-in-place the poetics of space conservation medicine and a new agenda for emerging diseases the birth of biopolitics: lectures at the collège de france global catastrophic risks. stockholm: global challenges foundation responding to covid-19: how to navigate a public health emergency legally and ethically press conference by secretary-general antonio guterres at united nations headquarters indigenous jingle dress dancing goes "viral" on social media to help heal the world. cbc news global trends in emerging infectious diseases 2017. bioethics and biopolitics. theories, applications and connections identifying sars-cov-2 related coronaviruses in malayan pangolins understanding home. a critical review of the literature spillover. animal infections and the next human pandemic environmental ethics: duties to and values in the natural world microbial threats to health: emergence, detection, and response united nations. 1992. rio declaration on environment and development. united nations general assembly a / c o n f . 1 5 1 / 2 6 who global influenza preparedness plan. the role of who and recommendations for national measures before and during pandemics publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-332569-af8oq2d6 authors: friedman, henry; ator, nancy; haigwood, nancy; newsome, william; allan, james s.; golos, thaddeus g.; kordower, jeff h.; shade, robert e.; goldberg, michael e.; bailey, matthew r.; bianchi, paul title: the critical role of nonhuman primates in medical research date: 2017-08-23 journal: pathog immun doi: 10.20411/pai.v2i3.186 sha: doc_id: 332569 cord_uid: af8oq2d6 the sponsors of this report endorse carefully regulated research with nonhuman primates. this research is essential to learning about the biology, treatment and prevention of diseases and conditions that cause human suffering. research with nonhuman primates (nhps) -monkeys for the most part -has led to critical health advances that have saved or improved millions of human lives. while nhps account for just one-half of one percent of animals in current medical research, it is no exaggeration to say they are essential to our ability to find cures for cancer, aids, alzheimer's, parkinson's, obesity/diabetes, and dozens of other diseases that cause human suffering and death. research with monkeys is critical to increasing our knowledge of how the human brain works and its role in cognitive, motor, and mental illnesses such as alzheimer's, parkinson's, and depression. this research is also fundamental to understanding how to prevent and treat emerging infectious diseases like zika and ebola. nhp research is uncovering critical information about the most common and costly metabolic disorder in the u.s. -type 2 diabetes -as well as the obesity that leads to most cases. without nhp research, we lose our ability to learn better ways to prevent negative pregnancy outcomes, including miscarriage, stillbirth, and premature birth. this research is also helping scientists to uncover information that makes human organ transplants easier and more accessible, literally giving new life to those whose kidneys, hearts, and lungs are failing. news headlines tout medical breakthroughs. breakthrough sounds dramatic, and to someone hearing about how the virus that causes polio is being used to put an aggressive form of brain cancer into remission, it is indeed. but as the scientists involved in that cancer research-and research into every other area of medicine-will tell you, breakthroughs might be dramatic, but they are never sudden. a well thought-out and structured process is behind virtually every medical breakthrough and the discovery process probably took decades or more. every step in the process was essential to the next, from basic research to human clinical trials. monkeys are often involved at the later stage of the processwhat is called translational or applied research. here all of the knowledge accumulated earlier is applied to specific medical questions such as: will this vaccine protect a pregnant woman (and her baby) from zika infection? and is the vaccine likely to be safe? research scientists are like detectives solving mysteries. we want to know why. more importantly, we want to save lives. but monkeys also play a vital role in basic science research that can come decades earlier. basic nhp research in the 1970s helped scientists understand the inner workings of the basal ganglia, the part of the brain that coordinates movement. those early findings led to the "breakthrough" 30 years later in which deep brain stimulation is used to reduce involuntary movements of parkinson's disease. see more breakthroughs linked to nhp research in appendix a. regardless of where it occurs in the scientific discovery process, research with monkeys is highly regulated (see appendix b). scientists use monkeys only when no other research model can provide the required information. while rodents are used extensively and are extremely helpful in answering many basic research questions, their usefulness is limited by differences from primates in their lack of sophisticated brain structures, less developed immune systems and motor skills, and differences in how their metabolism functions, among other traits. to cite an example, rodent brains are very different from human brains. the rodent lacks the prefrontal cortex specialization that is found in monkeys and humans. this difference limits the applicability of rodent studies in relation to studies of injury in the human brain. nhps are also the main animals that allow quick response and research into emerging viruses, like zika. what scientists learn about zika itself, as well as what they learn about the best use of monkeys in zika studies, they will apply to studies of future emergent diseases. and with recent history as a guide (zika, ebola, middle east respiratory syndrome [mers], sars, pandemic flu, etc.), we should expect more infectious disease outbreaks in the near future. modified poliovirus is being tested as a way to help the body's immune system see and destroy glioblastomas, the deadliest type of brain cancer. glioblastomas can double in size every two weeks and can be deadly within months of diagnosis. the new treatment has led to complete remission in two glioblastoma patients and investigations are ongoing. this type of research, called immunotherapy, uses the body's natural defense -the immune system -to destroy cancer cells. the harmless form of poliovirus is injected into glioblastoma tumors where it attaches to the cancer cells. the immune system recognizes the poliovirus as a dangerous invader and attacks -killing it and the cancer along with it. current studies in monkeys are helping to find ways to help wounded soldiers and stroke victims regain their independence after losing limbs or the ability to control them. this is the most promising therapy i've seen in my career. it was 18 years from the earliest research until the first study in humans. monkey research was essential in mapping out how to get the poliovirus through the brain and inside the cancerous tumors. the national institutes of health and the food and drug administration also mandated testing the treatment first in monkeys to be sure the modified poliovirus would be harmless to humans. doctors now are designing research to use this approach in treating many forms of cancer -including breast and prostate cancers -since the same receptor on the glioblastoma tumors that allows the poliovirus to attach itself also is found on virtually every cancerous tumor in humans. scientists are looking for vaccines that can prevent hiv infection and treatments leading to a cure. just 20 years ago medical advances changed the disease from a death sentence into a chronic, manageable disease. drugs that keep the virus in check now give millions of hiv-infected people hope for a long and productive life. drug therapies effectively prevent hiv disease, but scientific advances in hiv are still needed. people with well-treated hiv still face more health problems than those without hiv [1, 2] . they age faster, too. doctors estimate people with hiv are at least 5 to 14 years older than their chronological age [3, 4] . monkeys are crucial to ongoing hiv research because of the combination of their unique biology among animals and their longevity, which is key in hiv studies that take from months to years to complete. their similar biology helps scientists understand hiv disease, infection routes, the potential for vaccine-induced protection, and even an hiv cure. an experiment reported in early 2016 looked at preventing mother-tochild hiv transmission [5] . after being exposed to simian-human immunodeficiency virus (shiv), which is similar to hiv, infant monkeys with early stage infection were treated with human antibodies to block the infection. all of the monkeys in this experiment had no detectable virus in their blood or any of their tissues at the end of six months of observation. in another experiment reported this year, rhesus macaques infected with another hiv-like virus were treated with standard anti-hiv medications plus an experimental drug that stimulates the immune system [6] . at the end of the study, 90 days after both medications were stopped, two monkeys showed no detectable virus in their bloodstream. this immune system stimulator was tested earlier in nhps infected with chronic hepatitis b, leading to current research in humans with this potentially deadly infection. [7] the value of studying monkeys is scientifically proven. hiv is no longer an impending death sentence. several years ago, this young woman was diagnosed with a deadly brain tumor the size of a tennis ball. when conventional treatment didn't work her doctors suggested a new treatment developed with primate research. she agreed. today, she's cancer free. these studies hold promise for protecting babies from hiv infection and for finding a cure for those already infected, but much more research with monkeys will be needed to get there. in human clinical studies, a fundamental question is, "do the potential benefits of this treatment outweigh the potential risks?" this question takes on added meaning when the study is in pregnant women. researchers must not only consider the risks and benefits to the pregnant woman, but also to her developing fetus and ultimately to the child [8] . but how do researchers even begin to define these risks and benefits before human clinical trials? the answer is research with monkeys, since their fetal and placental development is uniquely similar to humans. researchers are working with macaque monkeys to understand the impact of zika, the latest virus to emerge as a global threat. zika infection in pregnant women can cause microcephaly, a condition where the child is born with a small head due to abnormal brain development. it also appears to cause stillbirth, miscarriages, and fetal growth restriction. these problems all appear to be rooted in how the zika virus affects the developing fetus and the placenta, which nourishes the baby in its mother's womb. the zika virus infects monkeys just as it does humans, and both experience the disease in the same way. researchers can study pregnant monkeys much as an obstetrician follows a woman's pregnancy -they can take blood, monitor fetal development through ultrasounds, and collect amniotic fluid. they can then test vaccines and drugs with the hope of protecting the fetus. no other animal model allows for this entire spectrum of study and application of the findings to pregnant women. more than 120,000 people in the u.s. are waiting for organ transplants and 22 of them die every day [9] . it is all the more tragic, then, when an organ transplant fails. this failure, or rejection, is caused when the recipient's immune system sees the new organ as "foreign" and attacks it. to reduce the chance of organ rejection, transplant patients receive drugs to suppress their immune system. but the drugs come with a risk of toxicity and increase the risk of other problems, including development of cancers and infections resulting from a weakened immune system. research with monkeys is focused on achieving transplant tolerancewhere the body's immune system does not see the new organ as foreign, thus eliminating the need for immunosuppressive drugs. while scientists have already made great strides in kidney transplant tolerance, they understand that tolerance is organ specific, so knowledge about the kidney primate research will help us learn new ways to prevent miscarriage, stillbirth, and premature babies. understanding all brain diseases relies on us understanding how the healthy brain works during normal functioning. may not transfer to the heart, lungs, liver, pancreas/pancreatic islets, or other types of transplants. transplant tolerance also differs by species. in other words, what works in a mouse may not work in a pig, and what works in a pig may not work in a monkey. scientists learned about kidney transplant tolerance by starting with mice and then working up through swine and eventually into monkeys and humans. the same process is underway now for many other types of transplants. how does the brain work? no question could be more important for understanding human behavior and mental health, and for acquiring new information about the triggers in the brain that cause psychiatric, movement and other neurological diseases. the u.s. national institutes of health-supported brain initiative has developed a plan for improving our knowledge in these areas and research with monkeys and other species is critical to its success [10] . scientists are mapping the activity of the billions of neurons deep inside the brain -the special cells that transmit the signals that drive thinking, mood, movement, and much more. by tracking neuron activity in monkeys while they are performing new tasks, scientists can actually see what parts of the brain are involved in sending the signals that take in, process, and store the newly acquired information. what is unique to -or at least greatly enhanced by -the use of monkeys in this research is the range of cognitive behaviors that can be studied, the amount and precision of the data that can be collected, and the relevance of that data to human behavior and mental activity. seeing what is happening in a healthy monkey brain helps scientists understand what has gone wrong when a human brain is no longer working as it should. this type of research has relevance to parkinson's disease and other movement disorders, all forms of dementia, including alzheimer's, and behavioral and psychiatric problems from alcoholism and attention-deficit disorder to bipolar disorder and autism. you see someone walking haltingly, dragging one leg behind him, or sitting with one arm draped listlessly on a table and immediately know he has had a stroke. scientists learned long ago that it's not the muscles that are at fault; it's the nerve impulses inside the brain that have been affected. alzheimer's and other dementias cost the u.s. $236 billion each year [11] . monkeys have certain traits and characteristics that make them essential and irreplaceable in medical research. they're the "bridge to the clinic." a combination of scientific breakthroughs in neuroscience, computer processing and robotics has led to development of "brain-machine interfaces" -devices that allow humans to interact with their environment with prosthetic arms when they have lost the use of their own. brain-machine interfaces translate signals in the brain into directions to move prosthetic arms. this area of research shows enormous promise for humans who are paralyzed, such as injured veterans, or those with brain damage and paralysis due to stroke. as nhps and humans have similarly developed brains and movements, experiments in monkeys have been vital to moving this field forward both conceptually and technically. nhps are essential to vaccine research. among research animals, they alone can reproduce the entire biological process of the infections being studied. they allow researchers to monitor for information that is vital in understanding human infectious diseases -such as how a virus or bacterium reproduces inside the body, what symptoms it causes, and how the body's immune system responds to attack the invader. among the viruses currently in vaccine research trials is respiratory syncytial virus, or rsv -the most common cause of lower respiratory tract infections in u.s. infants and small children [12] . there is no treatment for rsv, [13] which hospitalizes nearly 60,000 u.s. children under age 5 every year and sends 2.1 million more to the doctor [14] . vaccine research with monkeys is evaluating the safety of potential rsv vaccines in infants. other viruses under study include ebola and marburg, which can cause extreme bleeding that leads to death; the mosquito-borne dengue and zika viruses, capable of causing massive epidemics; and mers plus the dangerous h5 and h7 bird flu strains, all of which have very high death rates. lowering blood pressure is vitally important to individuals and our society. high blood pressure is a major factor in heart disease -the number one killer in the u.s. and the world [15] . and it's not just heart disease; high blood pressure leads to stroke, kidney damage, memory problems, and many other illnesses [16] . decades ago, researchers made a breakthrough discovery that longterm blood pressure regulation is nearly identical in humans, baboons, brain-machine interfaces can help paralyzed veterans interact with their environment. the top 3 benefits of our human/monkey research partnerships? safety. efficacy. and greater predictability. scientists recently discovered that baboons share another unique trait with humans -a characteristic in their red blood cells that can lead to salt-sensitivity and an inherited form of hypertension that is particularly difficult to treat. current research is looking for new targets to control this type of high blood pressure. research with monkeys provides another key benefit -lifespan. high blood pressure becomes more common as we age and researchers are able to work with older baboons to gain essential information about the mechanisms driving this age-based increase -vital to the health of our aging population. type 2 diabetes develops in monkeys just as it does in humans, even following the same age patterns, that is to say, more disease as we get older (one-fourth of u.s. seniors have diabetes) [17] . nhps with diabetes even develop the same complications that are common in humans: eye disease, kidney disease, nerve damage and pain, and blood vessel disease, among others [18] . nhps and humans have very similar systems that regulate blood sugar. for example, the structure and function of the group of cells in the monkey pancreas (called islets) that produce insulin are very similar to human islets. the islets in mice, rats, pigs, and other animals share some similarities with humans, but there are important differences, making monkeys a critical model for developing treatment and prevention methods, and for testing new therapies for people with diabetes. type 2 diabetes and the u.s. obesity epidemic are linked -obesity is a contributing factor to the condition. more than a third of u.s. adults are obese and another third are overweight [19] . as with diabetes research, monkeys provide a critically important study model for human obesity. monkeys that are fed a diet similar to the typical american diet respond like humans, gaining weight and later progressing to type 2 diabetes. researchers are examining the role of gastrointestinal proteins called glucagon-like peptides in the development of obesity in bonnet macaques. bonnet macaques are unique among nhps because they have a strong genetic predisposition to obesity. this research is looking for obesity treatments that will be as effective as invasive bariatric surgery, but with far less risk. in conclusion, because nhps are the most readily available models with the greatest psychological and genetic similarities to humans, they play an indispensable role in the process of medical research and development. nonhuman primates are the ideal model for testing new therapies for people with diabetes, including the artificial pancreas, drugs and devices. all of our ability to address diseases in applied research comes from efforts in basic science research. william newsome, phd, neurobiologist studying how â�¢ components of blood and plasma discovered. â�¢ ability to diagnose and treat typhoid fever. â�¢ modern anesthesia. â�¢ mumps virus discovered. â�¢ treatment of rheumatoid arthritis. â�¢ discovery of the rh factor, blood-typing knowledge critical for safe blood transfusions. â�¢ development of polio vaccine. â�¢ development of antipsychotic medication chlorpromazine and its tranquilizing derivatives. â�¢ cancer chemotherapy. â�¢ development of yellow fever vaccine. â�¢ mapping of the heart's connections to arteries. â�¢ development of german measles vaccine. â�¢ therapeutic use of cortisone for reducing inflammation and allergy symptoms. â�¢ corneal transplants. â�¢ development of treatment and prevention of radiation sickness. â�¢ development of measles, mumps, and rubella (mmr) vaccine. â�¢ discovery of the biochemical cause of depression. â�¢ transmissibility of human prion diseases, such as creutzfeldt-jacob disease, discovered. â�¢ treatment of leprosy. â�¢ procedures to restore blood supply in the brain. â�¢ interaction between tumor viruses and genetic material. â�¢ understanding of slow viruses, which linger in the nervous system. â�¢ understanding of the inner workings of the basal ganglia, the part of the brain that coordinates movement. â�¢ discovery of mechanisms of opiate withdrawal and the anti-withdrawal effects of clonidine. â�¢ development of cyclosporine and other anti-rejection drugs helpful for organ transplants. â�¢ processing of visual information by the brain. â�¢ identification of physiological and psychological co-factors in depression, anxiety and phobias. â�¢ treatment of malnutrition caused by food aversion following chemotherapy. â�¢ treatment of congenital cataracts and "lazy eye" in children. â�¢ first animal model for research on parkinson's disease, enabling doctors to more accurately research human parkinson's disease. â�¢ heart and lung transplant to treat cardiopulmonary hypertension. â�¢ first hepatitis b vaccine. â�¢ development of rhesus monkey model for hiv/aids. â�¢ addition of taurine to infant formulas. taurine is necessary for normal eye development. â�¢ first treatment of naturally diabetic nhps with a hormone-like insulin stimulus that is now in wide use both for diabetes and obesity treatment (glp-1 agonist). without continued nonhuman primate research, many important translational discoveries in the field of transplantation will never happen. these preclinical investigations are critical to the many patients who are currently waiting for transplantation and the many others who are facing the possibility of organ rejection after transplantation. â�¢ estrogen discovered to control an enzyme key to making serotonin, the brain chemical that regulates mood. represents first step to providing effective medications for depression at the end of the menstrual cycle, and postpartum and postmenopausal depression. â�¢ demonstration of the effectiveness of early administrationof azt to prevent or treat hiv infection. thanks to this, hiv-infected mothers can give birth to hiv-free babies. â�¢ demonstration in monkeys of the high efficacy of the hiv drug tenofovir to prevent or treat infection. â�¢ lead toxicity studies help u.s. fight childhood lead exposure. â�¢ ongoing development of a one-dose transplant drug to prevent organ rejection. â�¢ first controlled study to reveal that even moderate levels of alcohol are dangerous in pregnancy. â�¢ breakthroughs in understanding the mechanisms of puberty and disorders of puberty. â�¢ primate embryonic stem cells studied extensively for the first time, advancing efforts to better understand reproduction and genetic disorders. â�¢ control of intimal hyperplasia, a complication of coronary bypass surgery. â�¢ parent to child lung transplants for cystic fibrosis. â�¢ nhps shown to naturally develop diabetes, which is the same disease as in humans, thus opening the path to research for new treatments. â�¢ naturally regenerative mechanism discovered in the mature nhp brain, spurring new research toward curing alzheimer's and other degenerative brain disorders. â�¢ development of anthrax vaccine. â�¢ development of life-saving medications for lupus. â�¢ gene that boosts dopamine production and strengthens brain cells used to successfully treat monkeys showing symptoms of parkinson's disease. â�¢ monkey model developed to study the effects of malaria in pregnant women and their offspring. â�¢ nhps are prime model for development of hiv treatments and potential vaccines. â�¢ insulin-treated diabetic patients live longer, fuller lives. â�¢ the most common and debilitating complications of diabetes can now be studied in nhps. â�¢ high blood pressure is treated to prevent heart attack, stroke, and kidney failure. â�¢ patients can receive hip replacements and are no longer reliant on wheelchairs. â�¢ people with degenerative eye diseases are able to see more clearly. â�¢ better medications improve lives of people with severe depression, bipolar disorder, and other psychiatric illnesses. â�¢ better pre-and postnatal care protects children. â�¢ earlier diagnoses and better treatments help those with polycystic ovary syndrome, endometriosis, and breast cancer. â�¢ improved treatments help more men survive prostate cancer. â�¢ secondhand smoke shown to affect prenatal, neonatal and child lung development, cognitive function and brain development. â�¢ exposure to wildfire smoke adversely affects development of the immune system. â�¢ better understanding of the effects of bpa, a chemical found in plastic, on prenatal development improves health of children and adults. for research funded by any public health service entity, such as the national institutes of health (nih) or the national science foundation, the nih: g establishes public health service policy on humane care and use of laboratory animals. [20] g mandates use of the guide for the care and use of laboratory animals, which is issued by the national academy of science institute for laboratory animal research. this guide addresses day-to-day aspects of caring for laboratory animals. [21] g mandates that every institution appoints an institutional animal care and use committee (see below). the usda's animal plant and health inspection service enforces the animal welfare act with unannounced compliance inspections of all regulated entities using animals in research, testing or teaching at least yearly [22] . the association for the assessment and accreditation of laboratory animal care international is an independent non-government accrediting organization. while voluntary, this accreditation includes broader requirements than the regulations. this demonstrates research facilities want to go "above and beyond" in the care of animals [23] . every institution involved in nonhuman primate research is required by the animal welfare act as well as public health service policy to appoint and empower an institutional animal care and use committee [24] that reviews and approves the research. scientists must justify their use of primates and also explain why alternative forms of research (for example, studying cells or using computer simulations) are not able to achieve their scientific goals. they must also confirm that their research does not unnecessarily duplicate previous research [25] . the number of nonhuman primates used in research is less than 1%. but its impact on human health is enormous. all others geriatric syndromes in older hiv-infected adults the end of aids: hiv infection as a chronic disease methylome-wide analysis of chronic hiv infection reveals five-year increase in biological age and epigenetic targeting of hla acceleration of age-associated methylation patterns in hiv-1-infected adults early short-term treatment with neutralizing human monoclonal antibodies halts shiv infection in infant macaques gilead announces data from new preclinical study evaluating an investigational tlr7 agonist in siv-infected monkeys gs-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees ethical issues related to the inclusion of pregnant women in clinical trials (i) american transplant association. facts and myths national institutes of health. what is the brain initiative? available from alzheimer's disease facts and figures respiratory syncytial virus infection (rsv) american lung association. diagnosing and treating rsv the burden of respiratory syncytial virus infection in young children heart disease and stroke statistics-at-a-glance high blood pressure (hypertension) statistics about diabetes the rhesus monkey (macaca mulatta) manifests all features of human type 2 diabetes national institute of diabetes and digestive and kidney diseases public health service policy on humane care and use of laboratory animals. revised committee for the update of the guide for the care and use of laboratory animals; institute for laboratory animal research. guide for the care and use of laboratory animals animal welfare act inspections. at 1.usa.gov/1xcaq7m american association for the accreditation of laboratory animal care. the aaalac international accreditation program office of research integrity. how to work with your institutional animal care and use committee (iacuc) good laboratory practice for nonclinical laboratory studies the authors wish to acknowledge the following experts who participated in conference calls that served as the basis for the paper's content and/or who provided comments during drafting: key: cord-333405-ji58jbct authors: morens, david m.; folkers, gregory k.; fauci, anthony s. title: the challenge of emerging and re-emerging infectious diseases date: 2004-07-08 journal: nature doi: 10.1038/nature02759 sha: doc_id: 333405 cord_uid: ji58jbct infectious diseases have for centuries ranked with wars and famine as major challenges to human progress and survival. they remain among the leading causes of death and disability worldwide. against a constant background of established infections, epidemics of new and old infectious diseases periodically emerge, greatly magnifying the global burden of infections. studies of these emerging infections reveal the evolutionary properties of pathogenic microorganisms and the dynamic relationships between microorganisms, their hosts and the environment. merging infections (eis) can be defined as "infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range" 1 . eis have shaped the course of human history and have caused incalculable misery and death. in 1981, a new disease -acquired immune deficiency syndrome (aids) -was first recognized. as a global killer, aids now threatens to surpass the black death of the fourteenth century and the 1918-1920 influenza pandemic, each of which killed at least 50 million people 2, 3 . of the 'newly emerging' and 're-emerging/resurging' diseases that have followed the appearance of aids (fig. 1) , some have been minor curiosities, such as the 2003 cases of monkeypox imported into the united states 4 , whereas others, such as severe acute respiratory syndrome (sars), which emerged in the same year 5 , have had a worldwide impact. the 2001 anthrax bioterrorist attack in the united states 6 falls into a third category: 'deliberately emerging' diseases. eis can be expected to remain a considerable challenge for the foreseeable future. here we examine the nature and scope of emerging and reemerging microbial threats, and consider methods for their control. we emphasize that emergence results from dynamic interactions between rapidly evolving infectious agents and changes in the environment and in host behaviour that provide such agents with favourable new ecological niches. about 15 million (>25%) of 57 million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis b or c viruses 7 (fig. 2) . the burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries 8 , and particularly on infants and children (about three million children die each year from malaria and diarrhoeal diseases alone 7 ). in developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities 9 . eis have been familiar threats since ancient times. they were once identified by terms such as ȕșȓȗș ȝ (loimos) 10 , and later as 'pestilences' , 'pestes' , 'pests' and 'plagues' . many examples can be cited in addition to the black death and the 1918 influenza pandemic, such as certain biblical pharaonic plagues and the unidentified plague of athens, which heralded the end of greece's golden age 11 . the age of discovery, starting in the fifteenth century, was a particularly disastrous period with regard to the spread of infectious diseases. importation of smallpox into mexico caused 10-15 million deaths in 1520-1521, effectively ending aztec civilization 12, 13 . other amerindian and pacific civilizations were destroyed by imported smallpox and measles [13] [14] [15] [16] [17] . historians have referred to these events as apocalypses 16 and even as genocide 15 . for centuries, mankind seemed helpless against these sudden epidemics. but the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases [18] [19] [20] led to enormous progress, notably the development of vaccines and ultimately of antimicrobials 20 . in fact, the era of the identification of microbes had barely begun 18 when optimists at the end of the nineteenth century predicted the eradication of infectious diseases 21 . by the 1950s, which witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in 22 , and in 1967, the us surgeon general stated that "the war against infectious diseases has been won" 23 . some experts remained sceptical, aware of recurrent lessons from history. they were less persuaded by successes than alarmed by failures such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. richard krause, then the director of the us national institute of allergy and infectious diseases, warned in 1981 (ref. 24) that microbial diversity and evolutionary vigour were still dynamic forces threatening mankind. as krause was completing his book the restless tide 24 , aids -one of history's most devastating pandemics -was already insidiously emerging. the emergence of aids led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases [25] [26] [27] [28] [29] [30] [31] . in the past 25 years, some of the factors that resulted in aids have also led to re-emergences of historically important diseases such as cholera, diphtheria, trench fever and plague. many re-emergences have been catalysed by wars, loss of social cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants [25] [26] [27] [28] [29] [30] [31] . the challenge of emerging and re-emerging infectious diseases the classification of eis as 'newly emerging' , 're-emerging/resurging' or 'deliberately emerging' is useful because the underlying causes of emergence and the optimal prevention or control responses frequently differ between the groups. newly emerging infections are those that have not previously been recognized in man. many diverse factors contribute to their emergences (see box 1); these include microbial genetic mutation and viral genetic recombination or reassortment, changes in populations of reservoir hosts or intermediate insect vectors, microbial switching from animal to human hosts, human behavioural changes (notably human movement and urbanization), and environmental factors. these numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. any discussion of recent eis must begin with the human immunodeficiency virus (hiv) that causes aids. hiv has so far infected more than 60 million people worldwide 33 . before jumping to humans an estimated 60-70 years ago 34 , perhaps as a consequence of the consumption of 'bush meat' from non-human primates, hiv-1 and hiv-2 had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, pan troglodytes, and the sooty mangabey, cercocebus atys). but hiv/aids might never have emerged had it not been for disruptions in the economic and social infrastructure in post-colonial sub-saharan africa. increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure all promoted sexual practices, such as promiscuity and prostitution, that facilitate hiv transmission [34] [35] [36] [37] . such complex interactions between infectious agents, hosts and the environment are not unique to the epidemiology of hiv/aids. the examples cited below further illustrate how changes in population density, human movements and the environment interact to create ecological niches that facilitate microbial or viral adaptation. some infectious agents that have adapted to non-human hosts can jump to humans but, unlike hiv, are not generally transmitted from person to person, achieving only 'dead end' transmission. infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important eis 25, 26 . examples include the arenavirus haemorrhagic fevers (argentine, bolivian, venezuelan and lassa haemorrhagic fevers) and hantavirus pulmonary syndrome (hps). viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioural factors. farming, keeping domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts [25] [26] [27] [28] [29] [30] [31] . the first epidemic of hps, detected in the southwestern region of the united states in 1993 (ref. 38) , resulted from population booms of the deer mouse peromyscus maniculatis, in turn caused by climate-related and recurrent proliferation of rodent food sources. increased rodent populations and eventual shortages of food drove expanded deer mouse populations into homes, exposing people to virus-containing droppings. the 1998-1999 malaysian nipah virus epidemic 39 further illustrates the influence of human behaviours and environmental perturbations on newly emerging human infections. pigs crammed together in pens located in or near orchards attracted fruit bats whose normal habitats had been destroyed by deforestation and whose droppings contained the then-unknown paramyxovirus. virus aerosolization caused infection of pigs, with overcrowding leading to explosive transmission rates and ultimately to infections in pig handlers. variant creutzfeldt-jakob disease (vcjd) is another example of a zoonotic disease emerging in humans. vcjd is caused by the humanadapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (bse) 40 figure 1 global examples of emerging and re-emerging infectious diseases, some of which are discussed in the main text. red represents newly emerging diseases; blue, re-emerging/resurging diseases; black, a 'deliberately emerging' disease. adapted, with permission, from ref. 23. epizootic/vcjd epidemic, primarily affecting great britain, probably resulted from the now-abandoned practice of supplementing cattle feed with the pulverized meat and bones of previously slaughtered cattle. bse itself is suspected to have emerged because of even earlier use of cattle feed containing the agent of sheep scrapie, a prion disease recognized by farmers more than 250 years ago 41 . alarmingly, the new bse prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. this suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans 40 . the recent reports of variant strains of the bse prion 42 suggest that the bse agent could be a more serious threat than other animal prions. infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses, as well as certain non-zoonotic diseases, which are discussed below. for example, legionnaires' disease, first identified in 1976, is caused by legionella pneumophila, whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems 26 . campylobacter jejuni and shiga-toxin-producing escherichia coli (e. coli o157:h7 and other agents of haemolytic-uraemic syndrome) infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. other enteric pathogens, such as the vibrios causing classical cholera (re-emerging; see below) and serogroup o139 cholera, and the zoonotic protozoa cryptosporidium parvum and cyclospora cayetanensis 26 , seem to have come from environmental or animal organisms that have adapted to human-to-human 'faecal-oral' transmission through water. some eis come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between 1830 and 1900 (ref. 44) . scarlet fever, then the leading cause of death in children, is now rare, but has been largely supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever 45 . when new microbes are discovered, their emergences as disease-causing pathogens may be delayed. for example, in 1883, robert koch was unable to show that the newly discovered koch-weeks bacillus caused serious disease. more than a century later, a fatal ei dubbed brazilian purpuric fever was linked to virulent clonal variants of haemophilus influenzae biogroup aegyptius (the koch-weeks bacillus) 46 . although the bases of emergences of new and more severe diseases caused by s. pyogenes and h. influenzae biogroup aegyptius are not fully known, in both cases complex microbial genetic events are suspected. the distinctive clonal variants associated with severe h. influenzae biogroup aegyptius disease have been shown by pcr (polymerase chain reaction)-based subtractive genome hybridization to contain not only a unique plasmid, but also unique chromosomal regions, some of which are encoded by bacteriophages 47 . this research has narrowed the search for virulence determinants to unique proteins, some of which may have been acquired from other organisms by horizontal gene transfer. streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex than for h. influenzae biogroup aegyptius. many factors associated with streptococcal virulence have been identified in strains bearing the m1 surface protein as well as in other m protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection 48, 49 . infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. examples include the associations of hepatitis b and c with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of epstein-barr virus with burkitt's lymphoma (largely in africa) and nasopharyngeal carcinoma (in china), of human herpesvirus 8 with kaposi sarcoma, and of helicobacter pylori with gastric ulcers and gastric cancer [50] [51] [52] . some data even suggest infectious aetiologies for cardiovascular disease and diabetes mellitus 53 , major causes of death and disability worldwide. other associations between infectious agents and idiopathic chronic diseases will inevitably be found. re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range. re-emergence is caused by some of the factors that cause newly emerging infectious diseases, such as microbial evolutionary vigour, zoonotic encounters and environmental encroachment. re-emergences or at least cyclical resurgences of some diseases may also be climate-related -for example, the el niño/southern oscillation (enso) phenomenon is associated with resurgences of cholera and malaria 54 . the impact of both new and re-emerging infectious diseases on human populations is affected by the rate and degree to which they spread across geographical areas, depending on the movement of human hosts or of the vectors or reservoirs of infections. travel has an important role in bringing people into contact with infectious agents 55 . an increase in travel-associated importations of diseases was anticipated as early as 1933, when commercial air travel was still in its infancy 56 . this has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute haemorrhagic conjunctivitis in 1981 (ref. 57) , by epidemics of meningococcal meningitis associated with the hajj, and more recently by the exportation of epidemic sars (a newly emerging disease) from guangdong province, china, to hong kong, and from there to beijing, hanoi, singapore, toronto and elsewhere 5 (fig. 3) . the persistent spread of hiv along air, trucking, drug-trafficking and troop-deployment routes is a deadly variation on this theme [35] [36] [37] . plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide (fig. 2) . years of effective use of dichlorodiphenyltrichloroethane (ddt) led to the abandonment of other mosquito-control programmes, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine 58 . research efforts focus on the development of vaccines 59 and new drugs, and on re-establishing public health measures such as the use of bed nets. tuberculosis is one of the most deadly re-emerging diseases (fig. 2) . the discovery of isoniazid and other drugs initially led to effective tuberculosis cures, empty sanitoria and the dismantling of public health control systems in developed nations. consequently, by the 1980s, when tuberculosis had re-emerged in the era of hiv/aids, local and state health departments in the united states lacked field, laboratory and clinical staff and so had to reinvent tuberculosiscontrol programmes 25 . the remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with aids, which greatly increases the risk of latent mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug-resistant and multidrug-resistant strains 60 , and a need for more expensive treatment strategies such as directly observed therapy. it has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. 63 . immune deficiency associated with aids, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than 1% of the world's population), setting the stage for the re-emergence of many opportunistic infections. hiv, which has infected more than 60 million people globally 33 , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesviruses 64 . breakthroughs in cancer therapy and in immunosuppressive therapies used to treat immune-mediated diseases and for transplantation 65, 66 can also leave patients susceptible to opportunistic infections. human organ transplantation adds a further risk of infection with undetected pathogens in donor tissues, and transplantation of animal organs introduces the risk of transmission to humans of animal microbes 67 . the emergence of zoonotic and vector-borne diseases can also be associated with human behaviours and environmental perturbation. although west nile virus is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally 71 . a 2001-2002 epidemic in hawaii -fortunately without fatalities -is a reminder that dengue has also re-emerged in locations once considered to be dengue-free. usually transmitted by aedes aegypti mosquitoes, dengue has recently been transmitted by aedes albopictus -a vector switch of potential significance with respect to dengue re-emergence 71 . in the americas, including many us southern states, a. albopictus has been spreading into areas where a. aegypti mosquitoes are not found, and persisting for longer seasonal periods, putting tens of millions more people at risk of dengue infection. dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue haemorrhagic fever (dengue shock syndrome being its highly fatal form). these severe complications are thought to result from the evolution of dengue viruses to escape high insight review articles 246 nature | vol 430 | 8 july 2004 | www.nature.com/nature population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection 72 . cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto-and zooplankton, algae and crustaceans. such environmental strains seem to act as reservoirs for human virulence genes (for example, genes for the phage-encoded cholera toxin and the toxin-coregulated pilus (tcp) factor associated with attachment), and to undergo gene transfer events that lead to new strains containing further virulence gene combinations. these result in periodic cholera emergences that cause epidemics and pandemics 73 . thus, although the disease we know as cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the 1840s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. influenza a viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts' . these can lead to deadly pandemics, as occurred in 1888, 1918, 1957 and 1968 (refs 74, 75) . in intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts' , which allow them partially to escape human immunity raised by previously circulating influenza viruses. influenza drift is an evolutionary success story for the virus. influenza a has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral haemagglutinin site that attaches to human cells. it remains something of a mystery how zoonotic influenza viruses mix with each other and with human strains to acquire the additional properties of human virulence and human-to-human transmissibility. before 1997, mild cases of human disease associated with avian influenza viruses were occasionally reported 76 . these events have become more frequent, sometimes resulting in severe cases of disease and death. avian influenza has recently made dead-end jumps to humans -for example, the 1997 hong kong outbreak of the newly emergent h5n1 influenza, the 2003 h7n7 epidemic in the netherlands, the 2003-2004 h5n1 and h7n3 epizootics in asia and elsewhere, and occasional cases of h9n2 disease (fig. 4) . meanwhile, back-switches of human h3n2 viruses have emerged in pigs, from which both doubly mixed (pig-human) and triply mixed (pig-human-avian) viruses 74, 75 have been isolated. such enzootic/zoonotic mixing is suspected to have occurred in the influenza pandemic of 1918-1920, which was caused by an h1n1 virus with an avian-like receptor-binding site 77 . the predicted virulence genes of this virus are now being sought from 85-year-old pathology specimens and from frozen corpses 78 . the implications of interspecies genetic mixing for future influenza pandemics are troubling. although much remains speculative about how influenza viruses emerge and spread, it seems clear that the process is driven by prolific and complex viral evolution (genetic reassortment and mutational 'drift'), interspecies mixing and adaptation, and ecological factors that bring humans into contact with animals and each other. by whatever means new influenza virus pandemic strains emerge, they eventually reach a critical threshold of human transmission beyond which epidemic and pandemic spread follows mathematically predictable patterns. the dynamics and determinants of such epidemic development have been studied since the nineteenth century for several infectious diseases. for influenza, both historical and prospective epidemics have been described or predicted using deterministic and stochastic mathematical models, often with surprising accuracy when compared with actual epidemic data. more complicated mathematical models that describe how diseases spread by means other than personto-person aerosol transmission have generally been less successful in describing and predicting epidemics, but have nonetheless been helpful in planning public health responses to epidemics caused by hiv 79 , vcjd 80 and other diseases. mathematical modelling is also used to determine the impact of emerging epidemics. for example, it has been difficult to estimate overall influenza mortality because fatal infections are often neither diagnosed nor accurately recorded in hospital records and death certificates, especially in the elderly. recent epidemiological attempts to obtain improved influenza mortality estimates from seasonal excess mortality data 81 have indicated that influenza mortality may be insight review articles nature | vol 430 | 8 july 2004 | www.nature.com/nature 247 greater than was previously suspected, because influenza deaths are frequently coded under seemingly unrelated categories such as cardiovascular diseases. the same approaches also show that other influenza-like deaths may actually be due to other agents, such as respiratory syncytial virus (rsv), a common childhood virus that in the past decade has emerged as a major cause of adult mortality 81 . deliberately emerging microbes are those that have been developed by man, usually for nefarious use. the term 'deliberately emerging' refers to both naturally occurring microbial agents such as anthrax 6 , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. deliberately emerging microbes include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity 82 . as concepts, bioterrorism and biowarfare are probably not new. the alleged catapulting of plague-ridden corpses over enemy walls in the 1346 siege of caffa (the modern crimean port of feodosia, ukraine) and the dispatch of smallpox-impregnated blankets to indians by british officers in the seven years war (1754-1763) have frequently been cited as examples of bioterrorism or biowarfare 83, 84 . two modern attacks have been well documented. in 1984, an oregon religious cult spiked restaurant salad bars with salmonellae in an attempt to sway a local election 85 . a 2001 anthrax attack 6 , in which a terrorist mailed anthrax-spore-filled letters to prominent figures, including two us senators, resulted in illness in at least 18 people and the death of five of these individuals. public alarm was elevated by the knowledge that bacillus anthracis is a common and easily obtainable enzootic and soil organism found in laboratories worldwide, and that scientific technology had increased its lethality: the spores had been weaponized by being concentrated, finely milled and packed with a dispersal agent to increase their capacity to disseminate 82 . the united states, the united kingdom, the soviet union and other nations once had sophisticated offensive bioweapons programmes that included the production of weaponized anthrax spores 82 . soviet scientists continued to produce large quantities of organisms adapted for biowarfare and bioterror -among them the agents of smallpox, plague, tularaemia and marburg virus -for several years after their signing of the bioweapons and toxins treaty convention in 1972, which forbade such activities 82 . by 1987, the soviet programme was annually producing 5,000 tonnes of weaponized anthrax spores, packing them into warheads and other delivery devices 82 . before the 2001 anthrax attacks 6 , the us scientific community had for several years been bolstering its biodefence research capacity. the anthrax attacks greatly accelerated this expansion as part of a national defence plan, which includes efforts to provide a knowledge base for the development of effective countermeasures against agents of bioterror, such as diagnostics, therapeutics and vaccines, and to translate this knowledge into the production and delivery of such measures 86 . bioterror agents have been grouped into three categories of risk 87 . the six category a agents (anthrax, smallpox, plague, tularaemia, viral haemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. category b and c agents include food-borne and water-borne organisms that incapacitate but usually do not kill. infectious diseases will continue to emerge and re-emerge, leading to unpredictable epidemics and difficult challenges to public health and to microbiology and allied sciences. surveillance and response, the key elements in controlling eis, be they naturally occurring or deliberately engineered, depend on rapid clinical diagnosis and detection and containment in populations and 89 , which along with state and local health departments and other agencies have been making significant strides in national surveillance-response capacity. the enormous influx of us government-funded research resources (largely through the national institutes of health) and public health resources (mainly through the cdc, and state and local public health agencies) in response to the increased threat of a bioterrorist attack 86 will fortify the response capabilities related to all eis. however, it is clear that surveillance and other activities that traditionally fall within the domain of public health are not in themselves sufficient to adequately address the problem of eis. of critical importance are basic, translational and applied research efforts to develop advanced countermeasures such as surveillance tools, diagnostic tests, vaccines and therapeutics 86 . genomics, proteomics and advances in nanotechnology 90 are increasingly being exploited in diagnostic, therapeutic and microbial research applications, and in rational drug and vaccine design. direct and computational structural determination 91 , prediction of protein-protein interactions between microorganisms and drugs, and sophisticated bioinformatics techniques support research in all of the above areas. these technologies have led to numerous advances in real-world utility against eis, most notably in the development of more than 20 antiretroviral drugs that can effectively suppress hiv replication. where they are available and properly used in hiv-infected individuals, these medications have dramatically reduced hiv morbidity and mortality 92 . gene-and protein-based microarrays can be used to detect pathogen signals, to monitor resistance to anti-infective agents, to characterize host gene responses to recent infections, and to facilitate the development of new drugs and vaccines 93 . basic and applied research together have provided promising new vaccine platforms, such as recombinant proteins, immunogenic peptides, naked dna vaccines, viral vectors of extraneous genes encoding immunogenic proteins (including chimaeras), replicons and pseudovirions 94 . many novel vaccine candidates are now being developed against eis such as hiv, ebola virus, west nile virus, dengue, the sars coronavirus, tuberculosis and malaria. of particular note are novel tuberculosis vaccines that recently entered clinical trials -the first time in more than 60 years that new approaches to vaccination for tuberculosis have been assessed in humans 95 . chimaeric flavivirus vaccines for west nile virus, dengue and japanese encephalitis virus are effective in animal models and are in various stages of clinical testing 95 . our growing understanding of the human immune system is also helping to accelerate vaccine development. this is especially true in the case of innate immune responses, which are evolutionarily older, less specific and faster-acting than the adaptive responses that have been the traditional targets of vaccines 96 . as we learn more about innate immunity and its relationship with the adaptive immune system, opportunities to create more effective vaccine adjuvants will emerge. for example, synthetic dna sequences that contain repeated cpg motifs mimic the stimulatory activity that bacterial dna fragments exert on the innate immune system. these sequences show promise as vaccine adjuvants that accelerate and augment immune responses 97 . we can anticipate more progress of this kind as we continue to delineate the complex interactions between innate and adaptive immune responses. the sequencing of the human genome, the genomes of six other animals, including the mouse, and those of microbial vectors and microbes themselves (for example, p. falciparum and its mosquito vector, anopheles gambiae), have elevated microbiology to a wholegenome level. the ability to sequence microbial genomes in a few days 98 or less, and to examine host-vector-microbe interactions at both the genome level and at the tertiary protein structural level, will help us to understand the molecular mechanisms that underlie the pathogenesis of infectious disease and host defences, including resistance and immune evasion. these advances will facilitate the development of new countermeasures. other fertile areas of research include the use of geographical information systems 99 and satellite imaging to support field study and epidemic prevention (for example, predicting hps and rift valley fever epidemics in indigenous areas by satellite imagery of water and vegetation related to animal reservoir and vector prevalence). underlying disease emergence are evolutionary conflicts between rapidly evolving and adapting infectious agents and their slowly evolving hosts. these are fought out in the context of accelerating environmental and human behavioural alterations that provide new ecological niches into which evolving microbes can readily fit. it is essential that broadly based prevention strategies, as well as new and improved countermeasures (that is, surveillance tools, diagnostics, therapeutics and vaccines), be continually tested, refined and upgraded, requiring a strengthened relationship between public health and basic and clinical sciences. the challenge presented by the ongoing conflict between pathogenic microorganisms and man has been well summarized by a noted champion of the war on eis, joshua lederberg: "the future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled our wits versus their genes" 29 . the global scientific and public health communities must confront this reality not only with wit, but also with vision and sustained commitment to meet a perpetual challenge. in figure 2 of this review, the pie chart was drawn incorrectly, with the wedge sizes not in proportion to the total size. the correct figure is shown below. asthma and chronic obstructive pulmonary disease 3.0 million factors in the emergence of infectious diseases the wordsworth encyclopedia of plague and pestilence updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic centers for disease control and prevention. multistate outbreak of monkeypox -illinois the severe acute respiratory syndrome investigation of bioterrorism-related anthrax, united states, 2001: epidemiologic findings threats to global health and survival: the growing crises of tropical infectious diseases -an "unfinished" agenda emerging infectious diseases among indigenous peoples ǽșȓȗșȕșȍȓȋ -sive, pestis nuperae apud populum londinensem grassantis narratio historica epidemiology of the plague of athens the columbian exchange: biological and cultural consequences of 1492 princes and peasants. smallpox in history ch island populations of the pacific the gifts of civilization. germs and genocide in hawai'i agents of apocalypse: epidemic disease in the colonial philippines measles in fiji, 1875: thoughts on the history of emerging infectious diseases die aetiologie der milzbrand-krankheit, begründet auf die entwicklungsgeschichte des bacillus anthracis. beiträge zur biologie der pflanzen spreading germs: diseases, theories, and medical practice in britain the greatest benefit to mankind: a medical history of humanity from antiquity to the present the extermination of infectious diseases the evolution and eradication of infectious diseases infectious diseases: considerations for the 21st century the restless tide: the persistent challenge of the microbial world (national foundation for infectious diseases committee on emerging microbial threats to health. emerging infections. microbial threats to health in the united states committee on emerging microbial threats to health in the 21st century. microbial threats to health in the united states: emergence, detection and response emerging and re-emerging infectious diseases: a multidisciplinary perspective emerging and re-emerging infectious diseases infectious history emerging infectious diseases in the 21st century emerging and re-emerging infectious diseases human frontiers, environments and disease the origins of acquired immune deficiency viruses: where and when? phil population migration and the spread of types 1 and 2 human immunodeficiency viruses the aids knowledge base slowing heterosexual hiv transmission a novel hantavirus associated with an outbreak of fatal respiratory disease in the southwestern united states: evolutionary relationships to known hantaviruses nipah virus: a recently emergent deadly paramyxovirus variant creutzfeldt-jakob disease and the acquired and transmissible spongiform encephalopathies nützliche und auf die erfahrung gegründete einleitung zu der landwirthschaft identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic creutzfeldt-jakob disease scrapie and chronic wasting disease severe streptococcal infections in historical perspective emerging infections brazilian purpuric fever: evolutionary genetic relationships of the case clone of haemophilus influenzae biogroup aegyptius to encapsulated strains of haemophilus influenzae identification and characterization of genomic loci unique to the brazilian purpuric fever clonal group of h. influenzae biogroup aegyptius: functionality explored using meningococcal homology group a streptococcus: allelic variation, population genetics, and host-pathogen interactions genome sequence of a serotype m3 strain of group a streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma microbes and malignancy: infection as a cause of human cancers helicobacter pylori-associated diseases current clinical topics in infectious diseases vol el niño and health epidemiology in relation to air travel acute haemorrhagic conjunctivitis: dealing with a newly emerging disease two worlds of malaria research toward vaccines against malaria the global situation of mdr-tb staphylococcus aureus resistant to vancomycin -united states methicillin (oxacillin)-resistant staphylococcus aureus strains isolated from major food animals and their potential transmission to humans the crisis in antibiotic resistance surveillance for aids-defining opportunistic illnesses, 1992-1997. mmwr 48 (cdc surveillance summary no. ss-2) infections in patients with cancer undergoing chemotherapy: aetiology, prevention, and treatment impact of current transplantation practices on the changing epidemiology of infections in transplant recipients xenotransplantation: public health risks -patient vs. society in an emerging field the outbreak of west nile virus infection in the new york city area in 1999 outbreak of west nile infection west nile virus: epidemiology and ecology in north america dengue and dengue haemorrhagic fever antibody-dependent enhancement of infection and the pathogenesis of viral disease molecular ecology of toxigenic vibrio cholerae the next influenza pandemic: lessons from hong kong a molecular whodunit avian influenza viruses infecting humans structure of the uncleaved human h1 haemagglutinin from the extinct 1918 influenza virus the origin of the 1918 pandemic influenza virus: a continuing enigma potential impact of low-efficacy hiv-1 vaccines in populations with high rates of infection short-term projections for variant creutzfeldt-jakob disease onsets mortality associated with influenza and respiratory syncytial virus in the united states the chilling true story of the largest covert biological weapons program in the world -told from the inside by the man who ran it (random house document zur geschichte des schwarzen todes. mitgetheilt und eingeleitet smallpox and the indians in the american colonies a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars biodefence on the research agenda: the world needs new and creative ways to counter bioterrorism threats in bioterrorism: i. cdc category a agents world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) preventing emerging infectious diseases: a strategy for the 21st century (department of health and human services protein structure prediction and structural genomics guidelines for using antiretroviral agents among hiv-infected adults and adolescents new technology platforms in the development of vaccines for the future the jordan report: 20th anniversary adjuvants of immunity: harnessing innate immunity to promote adaptive immunity recent advances in the discovery and delivery of vaccine adjuvants the value of complete microbial genome sequencing (you get what you pay for) the authors thank r. m. krause for helpful discussions, and j. weddle for graphic design. the authors declare that they have no competing financial interests.insight review articles key: cord-334353-nc2jhemz authors: murphy, thérèse; whitty, noel title: is human rights prepared? risk, rights and public health emergencies date: 2009-05-08 journal: med law rev doi: 10.1093/medlaw/fwp007 sha: doc_id: 334353 cord_uid: nc2jhemz nan public health security is . . . the activities required, both proactive and reactive, to minimize vulnerability to acute public health events that endanger the collective health of national populations. global public health security widens this definition to include acute public health events that endanger the collective health of populations living across geographical regions and international boundaries . . . .[g]lobal health security, or lack of it, may also have an impact on economic or political stability, trade, tourism, access to goods and services and, if they occur repeatedly, on demographic stability. 3 the other term that requires some explanation is 'public health emergency legal preparedness'. stated shortly, this is all about having the right laws in place and then using them in the right way in a time of public health emergency. 4 in other words, it is about both legal preparedness for, and response to, public health emergencies -it is both proactive and reactive. more generally, it can be said to be an essential part of both public and global public health security, and a subset of public health emergency preparedness. having identified these basics, we move to the purpose of this article. the aim is to present a human rights lawyer's perspective on both public health emergency preparedness and, more generally, the turn towards securing public health through securitisation. we develop this perspective by asking the following question: is human rights prepared for public health emergency preparedness? we are not confident that it is. in what follows, we explain why we take this view and we also sketch out a preliminary agenda designed to encourage greater human rights preparedness. we focus in particular on the importance of thinking through how risk and its relationship to rights is being framed, and also how this relationship ought to be framed. one response to public health emergency preparedness might go as follows: even if there is an abundance of new terms, there is nothing new about the link between health and security. the detailed version 3 ibid., at 1. 4 220 medical law review [2009] of this response would run as follows: focusing first on national security, the effect of disease on military strength and preparedness is, without doubt, an age-old concern. infectious diseases can also be a source of indirect harm to national security given their potential to cause 'political and economic damage in countries in which a state has vital security, foreign policy, and trade interests'. 5 turning next to economic security, it is clear that here too there is a longstanding link to health. the first international health regulations (1969) and their predecessors, the international sanitary regulations (1951) , were explicit about this link: they aimed to 'ensure the maximum security against the international spread of diseases with a minimum interference with world traffic'. 6 finally, a third, more general, link between health and security arises from the fact that infectious diseases can 'erode governance capacities' and undermine 'a population's confidence and trust in the political leadership and system'. 7 clearly, then, there are longstanding connections between health and security. but that does not mean that the contemporary linkage between health and security is nothing new. to explain why we think there is something new in play, and to provide background for our argument in the latter parts of the article, this section sketches a history of the relationship between security and health. in particular, drawing on recent developments in international law and policy, it aims to show that health has come to be 'looked at through a new lens'. 8 we begin in 1994, the year in which the united nations development programme (undp) called for attention to be directed towards the achievement of 'human security', 'an idea . . . likely to revolutionize society in the 21st century'. 9 for undp, traditional concepts of security were, on the one hand, too focused on protecting states from 'external aggression, or as protection of national interests in foreign policy or as global security from the threat of nuclear holocaust' and, on the other, not focused enough on 'the legitimate concerns of ordinary people who sought security in their daily lives'. 10 undp saw 'human security' as a way to address this imbalance. it argued that human security could help to protect people from both chronic threats (such as hunger) and sudden, harmful disruptions of their daily lives. 11 six years later, in 2000, another less-than-conventional definition of security emerged. this time it came from a more surprising source: the un security council, which held a session in january 2000 on peace, security and hiv/aids, focusing in particular on the impact of aids in africa. 12 the security council's conjunction of peace, security and hiv/aids was novel. so too was its depiction of security and its explicit reference to the pioneering nature of its own position: it emphasised that by discussing hiv/aids as a security threat, it was 'exploring a brand-new definition of world security' and establishing 'a precedent for security council concern and action on a broader security agenda'. 13 11 september 2001 (or 9/11) is the next crucial date in this short history. as lucia zedner has explained, '[t]he events of that day alter the landscape of security irrevocably'. 14 very shortly thereafter, anthrax letters, sent using the us postal service, affected 22 people, of whom five died. as a core component of its response to the events of 2001, the united states committed to a range of legislative and regulatory activity designed to improve the preparedness of its public health law. for example, within weeks of september 11, the centers for disease control and prevention (cdc) had commissioned a draft model state emergency health powers act and, as of 2009, the united states is scheduled to have its first national health security strategy, designed to augment the extant strategies covering, respectively, the national security of the united states and homeland security. 15 strongly worded rhetoric on health and security has run alongside these reforms. typically, this rhetoric invokes the importance of experienced an outbreak of plague, causing 56 reported deaths; and second, a cholera epidemic killed up to 50,000 of the approximately 700,000 refugees who had fled to the democratic republic of the congo to escape the crisis in rwanda: see, respectively, dt dennis, 'plague in india ' (1994) the united states is only one among numerous jurisdictions pursuing public health emergency preparedness. put bluntly, 'vulnerability is universal '. 17 in 2003, another global health security concern emerged: severe acute respiratory syndrome (sars), a new and serious infectious disease. sars began to spread internationally in february 2003, approximately two months after the global outbreak alert and response network (goarn)-a multi-partner network of agencies and technical institutions established by who 18 -had detected a confirmed influenza outbreak in the guangdong province of china. on 12 march 2003, who issued its first global alert about the new infectious disease. three days later, it issued a second alert, naming the disease, offering guidance to health professionals and public health authorities, and alerting international travellers to the spread of the disease. within four months, however, transmission of sars had been interrupted in all affected countries and, on 5 july 2003, who announced that the outbreak had been contained. in total, 8,098 cases of sars were recorded in 26 countries, with 774 documented deaths (hospital staff were most affected). there were also serious financial implications due to disruption of travel, tourism, trade and production; who's estimate is that the outbreak cost us $12.3 billion in the asian countries affected. 19 the sars outbreak and its containment produced a range of responses. 20 china, for example, was criticised for its delay in reporting 16 cases and an initial lack of cooperation with who. who was criticised by canada for its unilateral issuance of travel advice to persons proposing to travel to toronto, the city outside asia worst affected by the outbreak. 21 meanwhile, in canada, delays and wrangling between the ontario government and the federal government over funding to provide compensation for individuals may have undermined the quarantine scheme by leaving people with financial incentives to break quarantine. 22 as part of quarantine and isolation measures, some jurisdictions, including canada, adopted policies involving heavy limitations on individual rights. 23 who has said that these control schemes were responsible for the interruption of transmission of the disease within four months of the announcement of the outbreak, but the use of measures that severely restricted individual freedoms was deeply controversial. the year 2003 is, however, notable for more than the sars outbreak and its containment. it was also the year in which the commission on human security, co-chaired by sadako ogata and amartya sen, issued its final report. 24 the commission labelled illness, disability and avoidable death as 'critical pervasive threats' to human security; more generally, it harnessed the language of security to highlight the ongoing neglect of social and economic rights. one year on, in 2004, 'comprehensive collective security'-described as a 'new and broader understanding' of international security-provided the overall vision behind the report of the un secretary general's high-level panel on threats, challenges and change. 25 the panel prescribed an improvement in public health systems, arguing that: ur best defence against this danger lies in strengthening public health' and he seemed to indicate that he supported an expanded role for the security council in the event of an 'overwhelming outbreak of infectious disease that threatens international peace and security'. 29 a few months later, in may 2005, the world health assembly, the highest decision-making body of who, adopted a revised set of international health regulations (ihr (2005)). 30 these regulations were welcomed by the then director general of who as a 'major step forward for international health'. 31 they took effect in june 2007, 32 binding who member states on an opt-out basis. 33 they contain 66 articles, organised across ten parts, and a total of nine annexes, and, crucially, they are significantly different from their predecessors, the international health regulations (1969). the new regulations take what has been described as an '"all risks" approach', 34 encompassing any emergency with repercussions for international health security, including 26 ibid., at para 47. 27 ibid., at para 19. 28 ibid., at para xx. 29 outbreaks of emerging and epidemic-prone diseases, outbreaks of food borne disease, natural disasters, and the accidental or deliberate release of pathogens, or chemical or radio-nuclear materials. 35 their purpose is to: prevent, protect against, control and provide a public health response to the international spread of disease in ways that are commensurate with and restricted to public health risks, and which avoid unnecessary interference with international traffic and trade. 36 to give effect to this, they place obligations on member states with respect to surveillance and response capacities; states are also obliged to notify who of events within their territories that may constitute a 'public health emergency of international concern'. 37 but what of human rights? do they feature in the new ihr? put shortly, the answer is yes. article 3(1) contains the first reference to human rights: it states that the implementation of the ihr is to be 'with full respect for the dignity, human rights and fundamental freedoms of persons'. thereafter, human rights crop up in a number of articles. article 42, for example, provides that all health measures must be applied in a transparent and non-discriminatory way. informed consent and information privacy feature in several articles, including article 23 which provides that states parties must not apply health measures such as vaccination, medical examination or isolation to international travellers without 'prior express informed consent', save in circumstances where there is 'evidence of an imminent public health risk'. 38 article 45(1) provides that states parties must treat personal health information in a confidential manner 'as required by national law'. more generally, the new ihr feature requirements concerning any limitations on rights that are familiar from international human rights law. so, for example, who recommendations to states parties, and health measures implemented by states parties in response to identifiable risks, must be 'no more invasive or intrusive to persons than reasonably available alternatives that would achieve the appropriate level of health 35 see article 1 for definitions of the terms 'public health risk' and 'public health emergency of international concern'. 36 article 2. article 3(4), however, emphasises that states parties have 'the sovereign right to legislate and to implement legislation in pursuance of their health policies', noting that in so doing they should uphold the ihr. 37 see, respectively, articles 5, 13 and annex 1, and article 6 and annex 2. 38 if there is such evidence, then international travellers may be advised or compelled to submit to control measures such as vaccination, quarantine and isolation. protection'. 39 the ihr also give who a new power to use information about disease outbreaks provided by unofficial sources (for example, non-governmental organisations or individual scientists). 40 significantly, who has described this new power as a 'revolutionary departure from previous international conventions and regulations'. 41 so, how should a human rights lawyer respond to the panoply of new linkages between health and security and, more specifically, the increasing focus on public health emergency preparedness? in this section of the article, we outline two possible responses. we use broad strokes, not close detail: the aim is to provide a sense of the most likely human rights responses and to flag up why, in our view, they point to a need for greater human rights preparedness. the first possible human rights response to public health emergency preparedness is to embrace it, even acclaim it. this response will see public health being brought 'in from the cold'-its profile raised and increased resources directed towards it-as a result of the new linkage between health and security. preparedness, it will suggest, is a 'win-win'. specifically, investment in countering biothreats offers not just the possibility of protection against bioterrorism, but also enhanced public health given that 'the more research in weaponized diseases that takes place, the more innovations for disease prevention will be found'. 42 investment in hospitals, it will argue, should also receive a boost as a result of the new focus on preparedness: after all, 'if a bioweapon is released and people get sick, they will go to hospital first'. 43 faced with historical evidence of abuse committed in the name of public health, and of a traditionally hands-off response by the judiciary when faced with challenges to public health measures, 44 this first response is likely to counter-claim that human rights have been explicitly built-in as part of the ihr. it may also argue that states parties 39 article 43(1) re states parties; see also articles 23 and 31. as regards who recommendations, see article 17. 40 see articles 9, 10 and 11. who is required to seek official verification from the state concerned before taking action. it can share the information with other states if the affected state does not cooperate with verification and control efforts, 'when justified by the magnitude of the public health risk'. 41 above n 2, at xv. 42 jackson, above n 16, at 8. 43 ibid., at 17. 44 this first response will probably also argue that the overall response to hiv/aids shows that the linkage between health and human rights is now both accepted and embedded as best practice. the ascent of human rights in the popular imagination and in national, and international, legal and political orders might also be invoked. and, linked to this, it is likely that emphasis will be placed not just on the emergence of a 'right to human security' but also the fact that, today, neither states nor private actors (such as pharmaceutical companies) can afford to ignore human rights. putting that another way, this first response will point out that, today, rights are a potential risk for states and private actors. we think that this first response will also make claims focused on 'what works'. two particular claims spring to mind. the first addresses international co-operation: it will counter the argument that state sovereignty is an insurmountable obstacle to co-operation with evidence from the sars outbreak in 2003-in particular, who's unilateral issuance of a travel advisory and, relatedly, the fact that canada was 'a model of transparency in its reporting and public information, of determination in its contact tracing'. 46 in short, and in the words of the then executive director of communicable diseases at who, the argument will be that: the outbreak of sars in 2003 and its successful global containment are testimony to a new way of working internationally for the public good. 47 there is another possible version of the 'what works' argument, which focuses on drawing-out the rights potential of the language of security. it will run as follows: if, as seems to be the case in the counter-terrorism context, security and human rights are increasingly represented as in 45 conflict, with rights struggling to maintain popular confidence, why not use security-specifically, a right to security-as a way to bolster human rights? this argument will then go on to emphasise that states' obligations vis-à -vis positive rights (that is, economic, social and cultural rights) could also be bolstered by emphasising the importance of human security. there is though a second, very different, human rights response to public health emergency preparedness and the wider securitisation of health. as we see it, its defining stance will range from anxiety to outright opposition, and it absolutely will not share the optimism of the first response. for instance, faced with the latter's claims on 'win-win', its position is likely to be that preparedness distorts public health priorities, with negative consequences everywhere, but especially in developing states. securitisation, it will argue, compromises the public's health as it clambers after public health as security. moreover, even if one accepts the skewing of attention towards preparedness, isn't it the case-it will argue-that the preparedness project is out of balance? why does investment in neglected diseases continue to lose out so heavily in terms of funding? and, if hospital 'surge capacity' is a core issue, why hasn't there been more investment in public hospitals? claims of a new, co-operative world order will also be met with incredulity. co-operation on preparedness is essential and is embedded in the ihr, but it is also incredibly fragile, as was made clear in 2006 -07 when indonesia temporarily stopped sharing samples of the human avian influenza virus with who. 48 the cessation was prompted by the indonesian government's anger at what it saw as the commercial exploitation of developing countries. the catalyst for this was the announcement by an australian pharmaceutical company that it had developed a vaccine and could manufacture enough to protect the australian public within six months. the company was one of a number that had received indonesian samples from who. indonesia pulled out of sample-sharing with who, insisting that who change the rules to stop commercial abuse of poorer countries. it also allegedly entered into an arrangement with a us-based pharmaceutical company to provide samples in return for affordable access to any vaccine that might be developed. it is very likely that this second grouping of human rights advocateslet us call them linkage-sceptics-will also take a very different stance on the contemporary standing of human rights. it is true, the sceptics will agree, that human rights are built into the ihr. ultimately, however, article 3 of the ihr reserves the sovereign right of states parties to legislate for the public good, upholding the purpose of the ihr and sound science. moreover, the human rights provisions in the ihr have 'little to say about protection of livelihoods and food security, or potential health impacts that might result from the distortion of public health priorities towards global surveillance'. 49 the linkage-sceptic is also likely to point to a problem arising from a less obvious source: the health and human rights movement itself. this movement encompasses two different approaches to public health and human rights, one that forthrightly acknowledges the potential tension between public health necessity and human rights, and another that rejects the assumption of such a tension and instead sees the pursuit of public health and human rights as inextricably linked. one consequence of this has been vigorous intra-movement debate about the extent and inevitability of conflicts and trade-offs between public health and liberty. 50 this debate is both inevitable and deeply useful, but there is a problem in that, in an 'age of preparedness', robust debate amongst rights advocates about the pros and cons of trade-offs could be read in a way that puts rights at risk. threats to human rights-and indeed, human rights as a threat-are nothing new. the hype, or 'myth', of rights is a claim that is familiar from empirical socio-legal work on rights. problems with rights have also been emphasised by rights-sceptics. 51 the point though is that, lately, a new link between human rights and risk has emerged, largely as a result of post 9/11 rhetoric about life in a 'time of crisis'. its position, in crude terms, is that exceptional times mandate exceptional measures to deal with risk. as liora lazarus and benjamin goold have pointed out, 'the idea that certain human rights can be "turned off" when necessary' has acquired remarkable power; it is now widely regarded as a 'thoroughly reasonable reaction to the dangers allegedly faced by democratic societies'. 52 and, as they go on to emphasise: the exceptionalism argument has become pivotal, so much so that liberals and human rights organisations must either rebut claims 49 in this environment, human rights advocates debating the relationship between public health and human rights need to be extremely careful. 54 the ascent of post 9/11 exceptionalism is also reason to be deeply wary of upbeat, optimistic assessments concerning the relationship between security and human rights. for the linkage-sceptic, the hope that has been invested in the idea of a 'right to security' is misplaced, not least because this right could take shape as an argument against human rights. think, for example, of the claim that human rights are no more than a tool used and abused by 'minorities', especially 'dangerous, violent minorities'. this tends to be accompanied by another claim: namely, it is the right to security of 'law-abiding, decent people' to live safe and secure from crime and violence that has been neglected and needs shoring up. 55 moreover, these claims do not crop up only in the context of terrorism. as jonathan montgomery has pointed out, in the uk, discussions on the criminalisation of disease transmission have been fed by metaphors of disease carriers 'as markedly different from "normal" members of society', encouraging 'people to think of the transmission of disease as the province of evil criminals, not people like them'. specifically: those being prosecuted are [now] demonized less for their disease status than as sexual predators, and coverage shows a strong racial overtone. . . . aids is no longer presented as a gay plague but an african one. even more clearly the protection of victims from aggressors has emerged as the rationale for criminalization, obscuring public health issues. the innocence of the victims and the evil of the aggressor are stressed. 56 in our view, the sceptic's argument makes a great deal of sense. put bluntly, amidst the new 'rights revolution', which is characterised by a 53 ibid. med.l.rev. defensiveness in human rights advocacy and, its correlate, an anti-rights sentiment that queries the legitimacy of rights in a time of alleged crisis, there are no guarantees that the securitisation of public health, the right to security or human security will be good for human rights. 57 moreover, we know from history that, in the past, terrible abuses have been committed in the name of public health. 58 we have a potential problem, however, because in addition to agreeing with the sceptic's argument, we also agree with at least some of the optimist's argument. in particular, we cannot see how being 'against security' is a viable stance. in what follows, we try to address our split reaction by thinking through how human rights could engage more effectively with security. in this section, we focus on moving towards human rights preparedness in the 'age of preparedness'. in particular, we focus on risk, an increasingly common thread in contemporary debates about security. we ask: how does the identification and management of public health risks, tied explicitly in the ihr to the use of scientific principles and evidence, fit with human rights? we argue that, in order to answer this, human rights advocates need to direct greater critical attention towards prevailing interpretations of the relationship between risk and rights. 59 we propose two frames of enquiry: first, risk within rights and, secondly, rights as risk. thinking through these frames is, we suggest, an essential foil to the now-conventional mode of thinking about risk and rights which fixates on risk versus rights. as its name suggests, the first frame-risk within rights-emphasises risk as a component of human rights law. it emphasises, in particular, that the prevention of (future) harm can be interpreted within the existing framework of human rights. 60 risk-emerges from a now-dominant feature of contemporary governance: the assessment and management of risk. governments and organisations (whether national or international, and public, private or public-private in character) are expected to identify and prioritise the range of risks (financial, legal, political, reputational, regulatory, etc.) to which they are exposed. the particular point we want to draw out from this is that managing risk means managing the risk of rights. moreover, this risk is not limited to legal risk, that is, ( potential) claims and litigation for violation of human rights obligations. rather, because human rights are more than human rights law, rights as risk also encompasses the potential for human rights consciousness (as manifested, for example, in a public campaign) to disrupt the interests and overall standing of governments and organisations. a. risk within rights in many respects, 'risk' is not new ground for human rights lawyers. the terminology of risk was not widely used in the past, but analysing how threats to public health could be dealt with in a manner compatible with the normative frameworks of human rights instruments is an exercise that should be familiar to human rights lawyers. international instruments, including the 1950 european convention on human rights (echr) and the 1966 international covenant on civil and political rights (iccpr), enable states parties to derogate from certain human rights in the event of public emergencies that threaten 'the life of the nation'. 61 at other times, limitations on non-absolute rights are permitted, provided certain conditions are met. for example, interference with article 8 echr on the right to privacy is justified if it is legally prescribed, serves a legitimate purpose and can be proved to be 'necessary in a democratic society'. for the european court of human rights, the principle of proportionality is a key component of the latter 'democratic necessity' test. 62 more broadly, although the intensity of review in individual contexts (for example, interference with the right to liberty or privacy), and the exact legal terminology and method used, will differ, common judicial approaches towards questions of justification must recognize that liberty is part of our communal interests, along with public health . . . .' 61 as regards the iccpr, see also the 1984 siracusa principles on the limitation and derogation provisions in the iccpr. see, also, the 'intervention ladder' proposed by the nuffield council on bioethics in its recent report on public health, above n 23. 62 or necessity can be found across jurisdictions. 63 it is simply not correct therefore to claim that human rights law fails to deal with risk, or that a choice always has to be made between risk and rights. it is possible to identify-through the structure and interpretive principles of human rights instruments-the means to mediate the relationship between risk and rights. putting that another way, the framework of human rights law can, and already does, address issues of risk. until very recently, however, the identification of a 'risk within rights' approach did not register either among human rights lawyers or in the public imagination more generally. one explanation for this is that, for understandable reasons, lawyers did not pay much attention to the meaning, and role, of risk. 64 it was not a focus of critical concern (even though it was present in 'everyday' legal contexts-for example, identifying risk factors and making predictive judgements in relation to detention, imprisonment, mental health, child protection, asylum or deportation). instead, it was for the most part accepted as an uncontroversial component of the expert, scientific evidence put forward in support of one's legal arguments. 65 but a change has taken place due to the new societal emphasis on (in)security and public protection. significantly, in some jurisdictions, the new post 9/11 national security context has generated strong demand for greater prioritisation of risk 66 -even if this comes at the expense of established human rights norms. 67 the risk and rights relationship, in other words, is being framed as risk or rights-and rights seem to be at a substantial disadvantage. there is though a new critical counter-terrorism scholarship which is fighting this dangerous turn. in our view, its arguments need to be read very closely by those working on public health emergency preparedness, not least because of the effect that a 'risk or rights' approach 63 could have on the interpretation, and use, of the ihr and the design, interpretation and use of national preparedness measures. three insights from critical counter-terrorism scholarship merit particular attention. first, it should not be assumed that the human rights argument has been won: the reasons why human rights need to be protected have to be explained again and again, without fail. arguments, especially official ones, which insist that human rights norms have no place in times of emergency mean that counter-argument needs to be seen as constant requirement. 68 secondly, defending human rights in an era of heightened security-consciousness entails not just closer engagement with the contemporary politics of security, but also recognition of the value of security. 69 and, thirdly, a human rights spotlight on the language and practices of risk assessment and management is essential in order to evaluate the legitimacy of responses to public health threats. in thinking about risk within rights in the context of public health emergency preparedness, these points suggest a range of strategies. for example, the use of coercive measures-such as quarantine-requires particularly strong justification. these measures infringe liberty and privacy rights and, if mistakes are made, healthy individuals are put at serious risk of infection. the threat of quarantine can lead individuals to delay seeking diagnosis and treatment. it can also provoke or compound discrimination and stigmatisation of particular individuals and groups. and, as lucia zedner has pointed out, the use of risk categories to target certain groups can create a further problem: at one level, it makes perfect sense to target security measures at those deemed most to threaten. yet an inherent danger of selectivity is that precisely because it imposes restrictions only on targeted sections of the population, it is less likely to invoke the natural political resistance generated by burdens that affect us all. med.l.rev. critical counter-terrorism scholarship should also be seen as relevant in developing an account of risk-based evidence and its relationship to human rights. some public health commentators, discussing coercive measures, have begun to advocate a direct linkage between risk expertise and human rights standards: an approach, which might enable the formulation of a body of public health ethics acceptable to human rights jurisprudence is to introduce into the language of ethics and rights the notion of evidence-based assessment of risk. 71 this approach, they argue, could help to bring consistency and coherence to the 'balancing [of ] the common public health good with individual rights'. 72 for this to be true, critical counter-terrorism scholarship suggests that two particular problems will need to be addressed: the first concerns the terminology of balance; the second concerns the nature and role of risk-based evidence in legal processes. the metaphor of balance-which is now widely used in debates about appropriate legal responses to the risk of terrorism-has the potential to undermine human rights. as andrew ashworth has argued, the imagery of balancing assumes a 'hydraulic relationship between human rights safeguards and the promotion of security': specifically, 'as one goes up the other must go down, and vice versa'. 73 balancing, unlike proportionality, 'involves a broad brush, and sometimes opaque, analysis aimed at a resolution of the interests at stake and the rights involved'. it uses a 'utilitarian analysis of the rights and public interest goals in question, giving no significantly greater weight to rights than to security measures'. 74 proportionality, in contrast, is more protective of human rights: specifically, under the proportionality approach, there is a presumption that rights restrict public interest goals. rights outweigh goals that are not legitimate and, where goals are legitimate, any measures giving effect to them must be suitable, the least restrictive possible, and proportionate between the effects of the measures and the objectives to be achieved. 75 71 coker and others, above n 59, at 612. 72 ibid., at 613 (emphasis added). the second issue is risk-based evidence. a striking feature of contemporary security debates, in the terrorism and public health fields, is the increased reliance on risk-based evidence and expertise. human rights scholars have argued that, if risk evidence is to be used to justify limitations on rights, the evidence of threats to security must be disclosed and scrutinised according to identifiable legal norms. 76 clearly, where there is litigation, it is the judges' approaches to risk that will be crucial. historically, many types of expert evidence pertaining to public or national security were viewed as non-justiciable. and, where judicial intervention did occur, the actual level of scrutiny was often minimal and deferential to government assertions and experts. 77 today, however, what we have labelled as 'risk within rights' is beginning to be openly articulated by the judiciary in security contexts. specifically, in some jurisdictions, in response to concerns about the nature, and direction, of recent antiterrorism legislation and practice-including preventative detention, surveillance and deportation of suspected individuals-judges have started to engage openly, and more critically, with the relationship between risk and human rights. 78 this is a welcome development. but a wider and deeper engagement will be needed in order to dislodge the zero-sum representation of risk and human rights, and among other things this will require an exploration of the tendency to think of risk as an expert, rational or scientific knowledge, and human rights as a value-based legal knowledge. 79 we shift now to the complex terrain of 'rights as risk'. as noted earlier, the prompt for this second frame is that risk management has become a pre-eminent concern for governments and organisations (whether public, private or public -private in character)-to quote michael power, we are in the midst of 'the risk management of everything'. 80 in this section, we focus on organisational risk, emphasising that this includes management of the risk of rights, and we consider what this might mean for the project of human rights preparedness. 81 as we explain below, we take the view that a human rights engagement with 'rights as risk' is both necessary and undeniably complex. organisational risk is an umbrella term covering the possible risks affecting an organisation (including, for example, government departments, regulatory bodies and pharmaceutical companies). the organisational risks that have to be managed vary widely and are generally classed as reputational, financial, legal, political and operational in nature. the regulatory space in which the organisation operates will be a key factor. influences, demands and obligations can be exerted from numerous directions-for example, from international institutions, governments, state regulatory bodies, courts, professional associations, corporations, shareholders, trades union, litigants, ngos, the public and the media. 82 human rights are a particularly complex organisational risk. they encompass, but are not limited to, legal risk: that is, ( potential) claims and litigation for human rights law violations. rather, because human rights are more than human rights law, rights as an organisational risk are also about the capacity of human rights consciousness (as manifested, for example, in community group protests) to adversely affect the interests of the organisation. furthermore, for an organisation, risks exist in both engaging with, and rejecting, human rights. michael likosky, discussing the imperative of governments and private companies to manage human rights risks in joint projects, emphasises that organisations' risk-mitigation strategies will vary. for example: do they address the underlying human rights problem itself, making a project more respectful of human rights? do they discredit the ngo or community group campaign? do they negotiate with one ngo but not with another? do they assuage the concerns 80 in the public health emergency field, a wide range of influences, demands and obligations have the capacity to lead to the construction of rights as a risk. indeed, the cross-sector and cross-jurisdiction nature of public health emergencies (especially those of 'international concern') creates an especially complex governance landscape: distinct histories, cultures and agendas will be colliding in times of crisis when immediate responses are expected. in what follows, we outline a range of reasons why, from an organisational risk perspective, rights are likely to seen as a risk in the field of public health emergency preparedness. one obvious source of risk is the mounting number of human rights instruments and, as a result of increased litigation and the migration of legal arguments, the worldwide expansion of human rights-referencing case law. 84 the campaign to expand corporate liability for human rights violations is another source: although corporate opposition to new legally binding duties persists, this campaign has encouraged companies to commit to improved self-regulation. 85 another source is the human rights demands found within the conditional loan agreements between international organisations (such as the world bank) and recipient governments, or within the contractual and public procurement rules relating to the providers of public goods and services. 86 global health governance is also characterised by an increasingly influential role for hybrid and non-state actors, including ngos (such as médecins sans frontières), public-private partnerships (such as the global alliance for vaccines and immunisation (gavi)), and private foundations (such as the gates foundation). indeed, in evidence to a uk parliamentary committee, david fidler stated that: increasingly, the gates foundation is the first place people will pick up the phone to call; not the who. in fact, someone told me -and i do not know if this is true -that bill gates is now going to fly to indonesia to help intervene in that controversy over virus sharing. something has changed here. 87 in federal states, the legal relationship between federal and local government may constitute rights as a risk in the public health emergency field. on the one hand, a strong states' rights tradition can hamper, or be used as an excuse by, federal government in developing particular national preparedness measures. on the other hand, however, where federal government seeks aggressively to assert its power and interfere with protected human rights, a tradition of strong states' rights could be a bulwark against such abuse. 88 the levels of public trust in a particular national culture will also influence rights as risk perspectives. 89 lesley jacobs, in a study of the divergent uses of quarantine in hong kong, shanghai and toronto during the sars crisis, attributes the more extensive use of quarantine in toronto to the particular legal consciousness of senior public health officials: 'health security was weighed much more heavily than rights concerns . . . whereas in hong kong and shanghai there was much more of an even balance'. 90 but, even though there was dissent in toronto, courts and human rights bodies were not used to raise concerns about rights violations. one federal public health official has speculated that: the belief that the decisions about sars by senior public officials, provided that they had a legal basis, would be made fairly was so deeply ingrained among the public that there was little need to question or scrutinize those decisions. 91 jacobs's explanation for the non-use of courts is that legal avenues of redress might not be seen as appropriate 'where the health security of the community is at stake'. 92 from an organisational risk perspective, this phenomenon could be used as a reason to dismiss rights as a risk and, more specifically, to downgrade the need for rights protection in 87 house of lords select committee, above n 45, at 381. 88 medical law review [2009] preparedness planning and implementation. this, however, would be counter-productive. as james childress and ruth gaare bernheim have argued, 'public justification, deliberation, and other relationshipbuilding activities may be more important for biopreparedness than state power because they maintain and nurture civic ideals, cooperation, and trust'. 93 moreover, childress and gaare bernheim's argument can be extended to encompass the role of the private sector in sustaining public trust during an emergency. to give just one example: an employer's policy on matters such as job security has a very real capacity either to aid or to hinder the extent of voluntary compliance with quarantine requirements. but it is not just national cultures that merit attention in thinking about rights as risk: organisational cultures also 'shape how human rights are framed, interpreted and institutionalised'. 94 article 3(1) of the ihr states that implementation of the regulations is to be 'with full respect for the dignity, human rights and fundamental freedoms of persons'. the extent of internalisation of human rights within who is, therefore, crucial. little, however, is known about this as the organization has not been the focus of human rights or, indeed, regulation scholarship. 95 but it is obvious that the range of internal staff responses will play a significant role in the design, implementation and monitoring of any human rights-based policies. we can also speculate that any internal divisions on human rights will both increase who's own exposure to organisational risk and affect the handling of the specific human rights matter in question. consider, for example, galit sarfaty's study of interpretations of human rights at the world bank. comparing world bank investment projects relating to hiv/aids prevention in russia and saint lucia, sarfaty speculates on the reasons why different framings of the human rights dimension were adopted in each case: one reason may be that the project team was dominated by staff who prefer a cost-benefit analysis to a legal approach, and who concluded that funds would be better spent on other preventative projects. she also highlights two different core approaches to human rights among world bank staff: she labels the first 'instrumental' and the second 'intrinsic'. staff who adopt an intrinsic framework draw on either moral or legal imperatives and, overall, they see human rights in normative terms as an end in themselves. the instrumentalists, in contrast, see rights as a means to an end, using a functionalist, economics-driven rationale to determine whether, and how, human rights have value in any given case. we believe that thinking about 'rights as risk' should be a key aspect of human rights preparedness. human rights advocates need to recognise the growing significance of organisational risk and, in particular, the ways in which organisational culture intertwines with risk and rights. in addition, human rights needs to reflect on the emergence, from within its ranks, of the 'human rights risk strategist'. likosky, discussing public -private partnership infrastructure projects in different countries, highlights how governments and other organisations may aim to 'advance human rights interests for their own ethical or strategic reasons', and may therefore enter into negotiations and alliances with certain ngos. 97 human rights 'risk consultants', drawn from legal and other backgrounds, may also be employed by governments and organisations, to offer expertise on particular projects and human rights campaigns. looking specifically at the public health emergency context, it can be argued that who's new power under the ihr to use information about disease outbreaks provided by unofficial sources gives governments an added incentive to work with ngos and this, in turn, gives ngos increased influence. the end result is likely to be a mixture of human rights agendas, including-and this is the key point-human rights advocates adopting instrumentalist strategies and language (for example, urging the need to avoid 'business risk') in order to promote human rights protection. there is a lot for human rights advocates to address here. one question that needs to be asked is: are human rights risk strategists at work in public health preparedness projects and, if so, in what ways? are they, for example, picking up on the argument made by francis that it is not merely questions of justice within pandemic planning that demand attention but also the justice of pandemic planning: the triage choices in pandemic planning for the distribution of vaccines and antivirals are open, coordinated, and institutionally adopted. perhaps this is one reason why they have drawn so much attention. . . . no doubt there are other explanations, too, for the apparent assumption that devoting resources to pandemic planning is just. . . . nonetheless, there are serious questions of justice to be asked about the allocation of extensive resources to pandemic threats. 98 and, as they go on to point out, questions concerning the 'justice of' and 'justice within' pandemic planning are not unrelated. put shortly, if basic health care infrastructure is in a state of neglect or unavailable, if health professionals are in short supply or are not trusted by the population, and if there is inequality in access to primary care, then global surveillance, surge capacity and, more generally, preventing and coping with a pandemic will be all the more difficult. 'pandemic myopia', in other words, is a seriously flawed approach to pandemic planning 99 -which is surely something that human rights advocates (whether they see themselves as 'risk strategists' or not) need to be arguing as strongly and clearly as possible. there are other, broader questions too. these stem from the fact that rights as risk is not home-ground for human rights advocacy. put bluntly, it smacks of bad faith, of working on 'their' terms, not on 'ours'. being 'at the table', if that course is chosen, is not likely to be at all easy in practice. the counter-argument to this is that human rights advocacy has always been complex. 100 think, for example, of the diversity of human rights advocates (such as the us christian right at the united nations using human rights to argue against rights to sexual and reproductive health). 101 and it cannot be denied that human rights 'victories' have sometimes had unexpected, negative consequences: on occasion, they have been used by governments as a basis from which to claim that action has been taken and no further governmental action is required. it can also be argued that if deeper engagement with human rights by governments and other organisations (even if primarily through the lens of risk management) is a significant contemporary dynamic, then human rights advocates have little choice but to respond in some fashion. and it should not be forgotten that risk-based indicators and compliance strategies have also been growing areas of interest within human rights work. 102 lastly, there is, of course, the possibility that an organisation that starts out with an instrumentalist mindset towards human rights might ultimately internalise them to beneficial effect. overall, then, human rights advocates should not-and probably cannot-avoid engaging with rights as risk. one of the conclusions in the report of a recent uk parliamentary committee on infectious diseases and global health governance is that who needs additional funding 'if it is to be able to respond effectively to threats on behalf of the international community'. 103 this statement is a reminder of the importance, and the fragility, of public health emergency preparedness. it is likely that it will also be read, at least by some, as a rebuke to individuals, like us, who set out to critique preparedness when it is currently so precarious in practice. critiquing public health emergency preparedness is undeniably hard. who, in the end, would choose to be 'against preparedness'-especially when one considers the extreme human costs of recent public health emergencies? we are not against it, however. our argument throughout has been that critique is essential to preparedness and that, to date, critique from a human rights perspective has been in short supply. we have suggested a preliminary agenda to address the human rights gap, focusing in particular on the frames 'risk within rights' and 'rights as risk', and we assigned the name 'human rights preparedness' to the project as a whole. we concluded by turning inwards, emphasising that this project carries risks for human rights itself. specifically, critiquing preparedness could well involve self-critique as human rights advocacy works towards a more differentiated analysis of how security should be engaged and what it means to do human rights today. putting that another way, human rights preparedness-and, in particular, engagement with 'rights as risk'-is likely to be human rights without a safety net. human rights, terrorism and risk: the role of politicians and judges civil liberties law: the human rights act era see the discussion of uk and canadian national security cases in t poole regulation and administrative constitutionalism designs on nature: science and democracy in europe and the united states risk and human rights: ending slopping out in a scottish prison legal knowledges of risk' in law commission of canada risk, rights and public health emergencies infrastructure and human rights (cup a common law of human rights? transnational judicial conversations on constitutional rights the un human rights norms for corporations: the private implications of public international law between light and shadow: the world bank, the international monetary fund and international human rights law the new corporate accountability: corporate social responsibility and the law (cup risk, rights and public health emergencies pandemic planning and distributive justice in health care on the need to take account of social justice in pandemic planning, especially the needs and interests of the most disadvantaged, see the bellagio statement of principles globalizing family values: the christian right in international politics risk, rights and public health emergencies key: cord-337218-risqto89 authors: chu, ellen w.; karr, james r. title: environmental impact, concept and measurement of date: 2013-02-05 journal: encyclopedia of biodiversity doi: 10.1016/b978-0-12-384719-5.00253-7 sha: doc_id: 337218 cord_uid: risqto89 environments on earth are always changing, and living systems evolve within them. for most of their history, human beings did the same. but in the last two centuries, humans have become the planet's dominant species, changing and impoverishing the environment for all life on earth and even decimating humans' own cultural diversity. contemporary cultural worldviews that have severed humans' ancient connections with the natural world, along with consumption and population growth, have deepened this impoverishment. understanding, measuring, and managing human environmental impacts – the most important of which is the impoverishment of living systems – is the 21st century's greatest challenge. all organisms change their environment as they live, grow, and reproduce. over millennia, organisms evolve to contend with changes in their environment. those that do not adapt go extinct. those that survive are molded by evolution and biogeography as the environment changes. even unusual or seemingly catastrophic events, like tidal waves from earthquakes, are an integral part of the ecological contexts to which organisms adapt over long time spans. some organisms, like beavers and elephants, change their surroundings so dramatically that they have been called ecosystem engineers. beaver dams alter the flow of rivers, increase dissolved oxygen in downstream waters, create wetlands, and modify streamside zones. african elephants convert wooded savanna to open grassland by toppling trees as they browse. change brought about by living things, including ecosystem engineers, has been slow and incremental in evolutionary terms. ecosystem engineers evolve along with other inhabitants of their environments, developing ways to coexist; like other environmental components, ecosystem engineers, and their effects are part of an evolving ecological context. in contrast, human effects since the industrial revolutionincluding many that may be invisible to a casual observeroutpace the capacity of living systems to respond. in evolutionary terms, these effects are recent and outside the experience of most organisms. over the past two centuriesbarely more than two human lifetimes -humans have profoundly altered living and nonliving systems everywhere. for the first time in the earth's history, the environmental impact of one species, homo sapiens, is the principal agent of global change. understanding the environmental impacts of human actions is one of modern science's greatest challenges. understanding the consequences of those impacts, and managing them to protect the well-being of human society and other life on earth, is humanity's greatest challenge. the human evolutionary line began in africa about 7 million years ago (ma). it took some 5 or 6 million years (my) for protohumans to spread from africa to asia and then to europe. these early humans, like other primates, made their living by seeking food and shelter from their environment, gathering plant foods, and hunting easy-to-kill prey. sometimes, they also experienced threats from their environment, including accidents, droughts, vector-borne diseases, and attacks from predators. at this stage, with relatively low population densities and limited technologies, humans were not ecosystem engineers. by some 50,000 years ago, however, humans had learned to use fire; developed complex tools, weapons, and language; and created art. on local scales, these modern humans were very much ecosystem engineers. sometimes their enhanced abilities to make a living outstripped their local environment's capacity to provide that living, and they caused local ecological disruptions. on several continents, for example, humans hunted large mammals to the point where many, such as the marsupial lion of australia, went extinct. as humans became more efficient at exploiting their local environments, they spread farther. by 13,000 years ago, modern humans had spread to all continents and many islands across the globe. then, about 10,000 years ago, people began to domesticate plants and animals. instead of searching for food, they began to produce food. food production changed the course of human and environmental history. domestication of plants and animals enabled people to adopt a sedentary lifestyle. as detailed by geographer and ecologist diamond (1997 diamond ( , 2002 , populations grew as agriculture developed, because larger sedentary populations both demanded and enabled more food production. local ecological disruptions became more numerous and widespread and more intense. with animal domestication, contagious diseases of pets and livestock adapted to new, human hosts. diseases spread more quickly in crowded conditions; inadequate sanitation compounded the effects. from agriculture, civilization followed and with it, cities, writing, advanced technology, and political empires. in just 10,000 years, these developments led to nearly 7 billion people on earth, industrial societies, and a global economy founded on complicated technologies and fossil fuels. humans have emerged as ecosystem engineers on a global scale. the ecological disruptions we cause are no longer just local or regional but global, and we have become the principal threat to the environment. yet despite today's advanced technologies, humans are as dependent on their environments as other organisms are. history, not just ecology, has been very clear on this point. from the old kingdom of egypt more than 4000 years ago to the culture that created the huge stone monoliths on easter island between 1000 and ad 1550 to the 1930s dust bowl of north america, civilizations or ways of life have prospered and failed by using and (mostly unwittingly) abusing natural resources. in old kingdom egypt, the resource was the valley of the nile, richly fertilized with sediment at each flooding of the river, laced with canals and side streams, blessed with a luxuriant delta. agriculture flourished and populations swelled, until unusually severe droughts brought on the civilization's collapse. on easter island, the resource was trees, which gave polynesians colonizing the island the means to build shelter, canoes for fishing the open waters around the island, and log rollers for moving the ceremonial stone monuments for which the island is famous. deforestation not only eliminated the humans' source of wood, but also further deprived the already poor soil of nutrients and made it impossible to sustain the agriculture that had sustained the island's civilization. on the dry great plains of north america, settlers were convinced that rain would follow the plow, and so they plowed homestead after homestead, only to watch their homesteads' soils literally blow away in the wind. in these cases and many others, human civilizations damaged their environments, and their actions also worsened the effects on their civilizations of climatic or other natural cycles. yet in each case, a human culture was operating precisely the way it had evolved to operate: the culture of old kingdom egypt enabled its people to prosper on the nile's natural bounty, but prolonged, unprecedented drought brought starvation and political disorder. easter islanders thrived and populated the island until its resources were exhausted. dust bowl farmers lived out their culture's view of dominating and exploiting the land for all it was worth. the inevitable outcome for all three cases was a catastrophe for the immediate environment and the people it supported -not only because the people were unprepared to cope with dramatic natural changes in their environments but because their own actions magnified the disastrous effects of those changes. quoting an apt bit of cynical graffiti, historical philosopher wright (2004, p. 107 ) sums up what he calls the hazards of human progress this way: ''each time history repeats itself, the price goes up.'' indeed, as the second decade of the 21st century begins, humans are ecosystem engineers on a planetary scale, and our global civilization threatens the life-sustaining capacity of all of earth's environmental ''spheres'': • geosphere (lithosphere): earth's crust and upper mantle, containing nonrenewable fossil fuels, minerals, and nutrients that plants require. the activities of plants, animals, and microorganisms weather mineral soils and rocks, create organic soils, and alter erosion and sedimentation rates. humans mine minerals, metals, and gems; extract fossil fuels including coal, oil, and natural gas; and increase erosion and sedimentation by removing or altering natural plant cover through agriculture, logging, and urbanization. • atmosphere: the thin envelope of gases encircling the planet. living systems modify the atmosphere, its temperature, and the amount of water it contains by continually generating oxygen and consuming carbon dioxide through photosynthesis and affecting the amount and forms of other gases. humans release toxic chemicals into the air and alter the climate by raising the atmospheric concentration of greenhouse gases, such as carbon dioxide and methane, through the burning of fossil fuels in motor vehicles, ships, airplanes, and power plants. • hydrosphere: earth's liquid surface and underground water; its polar ice caps, oceanic icebergs, and terrestrial permafrost; and its atmospheric water vapor. living systems alter the water cycle by modifying the earth's temperature and the amount of water plants send into the atmosphere through a process called evapotranspiration. humans build dams, irrigation canals, drinking-water delivery systems, and wastewater treatment plants. they use water to generate electricity; they mine groundwater from dwindling underground aquifers for farming as well as drinking; they alter the flows of surface waters for everything from transportation to the mining of gold; they drain wetlands to gain land area and abate waterborne diseases. modern human interference in global climate is likely to disrupt the entire planetary water cycle. • biosphere: the totality of earth's living systems, that part of the earth inhabited by living organisms. life on earth emerged 3.9 billion years ago and has sustained itself through changes in form, diversity, and detail since then. no planet yet discovered supports complex life as we know it on earth. as predators, humans have decimated or eliminated wild animal populations worldwide. as domesticators of animals and plants, humans have massively reshaped landscapes by cutting forests, burning and plowing grasslands, building cities, desertifying vast areas, and overharvesting fish and shellfish. human actions have precipitated a spasm of extinctions that today rivals five previous mass extinctions caused by astronomical or geological forces, each of which eliminated more than 70% of species then existing. humans themselves may be thought of as a sphere within the greater biosphere: the ethnosphere, or the sum total of all thoughts and intuitions, myths and beliefs, ideas and inspirations brought into being by the human imagination since the dawn of consciousness. in the words of anthropologist davis (2009, p. 2) , who coined and defined the term in 2002, ''the ethnosphere is humanity's greatest legacy. it is the product of our dreams, the embodiment of our hopes, the symbol of all we are and all that we, as a wildly inquisitive and astonishingly adaptive species, have created.'' but, davis notes, just as the biosphere is being severely eroded, so too is the ethnosphere, but at a much faster pace. today, the scientific consensus is that h. sapiens -a single species -rivals astronomical and geological forces in its impact on life on earth. the first step in dealing with the present impact of human activity is to correctly identify the nature of humanity's environmental impact, concept and measurement of relationship with the environment and how human actions affect that relationship. many people still see the environment as something people must overcome, or they regard environmental needs as something that ought to be balanced against human needs (for example, jobs vs. the environment). most people still regard the environment as a provider of commodities or a receptacle for waste. when asked to name humanity's primary environmental challenges, people typically think of running out of nonrenewable raw materials and energy or about water and air pollution. environmental research and development institutions focus on ways technology can help solve each problem, such as fuel cells to provide clean, potentially renewable energy or scrubbers to curb smokestack pollution. even when people worry about biodiversity loss, they are concerned primarily with stopping the extinction of species, rather than with understanding the underlying losses leading up to species extinctions or the broader biological crisis that extinctions signal. these perspectives miss a crucial point: the reason pollution, energy use, extinction, and dozens of other human impacts are important is their larger impact on the biosphere. ecosystems, particularly their living components, have always provided the capital to fuel human economies. when populations were small, humans making a living from nature's wealth caused no more disruption than other species. but with nearly 7 billion people occupying or using resources from every place on earth, humans are overwhelming the ability of other life-forms to make a living and depleting the planet's natural wealth. at this point in the planet's history, one species is compromising earth's ability to support the living systems that evolved here over millions of years. the systematic reduction in earth's capacity to support life -which woodwell (1990) termed 'biotic impoverishment' -is thus the most important human-caused environmental impact. at best, the ethics of this impact are questionable; at worst, it is jeopardizing our own survival. the connection between biotic impoverishment and extinction is intuitively obvious. by overharvesting fish, overcutting forests, overgrazing grasslands, or paving over land for cities, we are clearly killing other organisms outright or eliminating their habitats, thereby driving species to extinction and impoverishing the diversity of life. but biotic impoverishment takes many forms besides extinction. it encompasses three categories of human impacts on the biosphere: (1) indirect depletion of living systems through alterations in physical and chemical systems, (2) direct depletion of nonhuman life, and (3) direct degradation of human life (table 1) . identifying and understanding the biological significance of our actions -their effects on living systems, including our own social and economic systems -is the key to developing effective ways to manage our impacts. humans affect virtually all the physical and chemical systems life depends on: water, soils, air, and the biogeochemical cycles linking them. some human-driven physical and chemical changes have no repercussions on the biota; others do, becoming agents of biotic impoverishment. humans probably spend more energy, money, and time trying to control the movement and availability of water than to manage any other natural resource. in the process, we contaminate water, move water across and out of natural basins, deplete surface and groundwater; modify the timing and amount of flow in rivers, straighten or build dikes to constrain rivers, and alter natural flood patterns. we change the amount, timing, and chemistry of fresh water reaching coastal regions, and we dry up wetlands, lakes, and inland seas. our demands are outrunning supplies of this nonrenewable resource, and the scale of our transformations risks altering the planetary water cycle. physical alterations of the earth's waters, combined with massive industrial, agricultural, and residential pollution, have taken a heavy toll on nonhuman aquatic life. by 2005 as much as one-fifth of the world's coral reefs had been destroyed, and only 30% remained healthy. globally, the number of oceanic dead zones, where little or no dissolved oxygen exists, tripled during the last 30 years of the 20th century. the biota of freshwater systems has fared no better. a 4-year survey of the freshwater fishes inhabiting malaysian rivers in the late 1980s found only 46% of 266 known malaysian species. some 40% of north america's freshwater fishes are at risk of extinction; two-thirds of freshwater mussels and crayfishes and one-third of amphibians that depend on aquatic habitats in the united states are rare or imperiled. humans use at least 54% of the earth's accessible water runoff, a figure that is likely to grow to 70% by 2025. by then, more than a third of the world's population could suffer shortages of fresh water for drinking and irrigation. groundwater aquifers in many of the world's most important cropproducing regions are being drained faster than they can be replenished: a study published in 2010 found that the rate of groundwater depletion worldwide had more than doubled from 1960 to 2000. natural flood regimes, as in the nile river basin, no longer spread nutrient-rich silt across floodplains to nourish agriculture; indeed, the high dam at aswan traps so much silt behind it that the nile delta, essential to egypt's present-day economy, is slumping into the sea. whole inland seas, such as the aral sea in uzbekistan, are drying up because the streams feeding them contain so little water. in addition to eliminating habitat for resident organisms, the sea's drying is bringing diseases to surrounding human populations. indeed, diseases caused by waterborne pathogens are making a comeback even in industrialized nations. in the past five or six decades, the number of large dams on the world's rivers grew more than seven times, to more than 40,000 today. the mammoth three gorges dam across china's yangtze river, completed in 2006, created a 660-km-long serpentine lake behind it. the dam displaced more than 1 million people and may force the relocation of another 4 million from the reservoir region, which, at 58,000 km 2 , is larger than switzerland. the dam has greatly altered ecosystems on the yangtze's middle reaches, compounding perils already faced by prized and endemic fish and aquatic mammals. the sheer weight of the water and silt behind the concrete dam raises the risk of landslides and strains the region's geological structure, while water released from the dam eats away at downstream banks and scours the bottom. and by slowing the flow of the yangtze and nearby tributaries, the dam drains the river's ability to flush out and detoxify pollutants from upstream industries. not just dirt, soil is a living system that makes it possible for raw elements from air, water, and bedrock to be physically and chemically assembled, disassembled, and reassembled with the aid of living macro-and microorganisms into the green cloak of life above ground. accumulated over thousands of years, soil cannot be renewed in any time frame useful to humans alive today, or even to their great-grandchildren. humans degrade soils when they compact it, erode it, disrupt its organic and inorganic structure, turn it too salty for life, and cause desertification. urbanization, logging, mining, overgrazing, alterations in soil moisture, air pollution, fires, chemical pollution, and leaching out of minerals all damage or destroy soils. thanks to removal of vegetative cover, mining, agriculture, and other activities, the world's topsoils are eroded by wind and water ten to hundreds of times faster than they are renewed (at roughly 10 tons ha à1 year à1 ). soils constitute the foundation of human agriculture, yet agriculture, including livestock raising, is the worst culprit in degrading soils. agricultural practices have eroded or degraded more than 40% of present cropland. over the last half century, some 24,000 villages in northern and western china have been overrun by the drifting sands of desertification. besides topsoil erosion, the damage includes salting and saturation of poorly managed irrigated lands; compaction by heavy machinery and the hooves of livestock; and pollution from excessive fertilizers, animal wastes, and pesticides. living, dead, and decomposing organic matter is the key to soil structure and fertility. soil depleted of organic matter is less permeable to water and air and thus less able to support either aboveground plants or soil organisms. the linkages between soil's inorganic components and the soil biotanaturalist wilson's (1987) ''little things that run the world''are what give soil its life-sustaining capacity. a clear-cut forest patch whose soil biota has been damaged beyond recovery can no longer sustain trees, no matter how many are planted; another clear-cut patch whose soil community is intact -especially the close associations among fungi and tree rootswill support new tree growth. destroying soil biota unleashes a whole series of impoverishing biotic effects both below and above ground. in 1962, rachel carson's landmark book, silent spring, alerted the world to the pervasiveness of synthetic chemicals produced since world war ii. as many as 100,000 synthetic chemicals are in use today. true to one company's slogan, many of these have brought ''better living through chemistry,'' providing new fabrics and lighter manufacturing materials, antibiotics, and life-saving drugs. but industrial nations have carelessly pumped chemicals into every medium. chemicals -as varied as prescription drugs flowing out of sewage plants, pesticides, heavy metals, and cancer-causing by-products of countless manufacturing processes -now lace the world's water, soil, and air and the bodies of all living things, including humans. chemicals directly poison organisms; they accumulate in physical surroundings and are passed through and, in many cases, concentrated within portions of the food web. chemicals cause cancer, interfere with hormonal systems, provoke asthma, and impair the functioning of immune systems. they have intergenerational effects, such as intellectual impairment in children whose mothers have eaten contaminated fish. what's more, over half a century of pesticide and antibiotic overuse has bred resistance to these chemicals among insects, plants, and microbes, giving rise to new and reemerging scourges. many chemicals travel oceanic and atmospheric currents to sites far from their source. sulfur emissions from the us midwest, for example, fall to earth again as acid rain in europe, killing forests and so acidifying streams and lakes that they, too, effectively die. china's burning of soft coal sends air pollution all the way to northwestern north america; the heavy haze hanging over china's chief farming regions may be cutting agricultural production by a third. chlorofluorocarbons (cfcs), once widely used as refrigerants, have damaged the atmospheric ozone layer, which moderates how much ultraviolet radiation reaches the earth, and opened ozone holes over the arctic and antarctic. but even more alarming is an unprecedented acidification of the oceans that has only recently attracted the attention of major scientific research consortiums. acid added to the world ocean by human activity has lowered the ocean's ph; it is lower now than it has been in 20 my, which translates into a 30% increase in sea-surface acidity since industrialization began. the future of marine life in an ocean acidifying at this speed and intensity looks bleak. as the concentration of hydrogen ions rises, the calcium carbonate in the shells or skeletons of organisms such as tropical corals, microscopic foraminifera, and mollusks begins to dissolve; further, more hydrogen ions combine with the calcium carbonate building blocks the organisms need, making it harder for them to extract this compound from the water and build their shells in the first place. although many of the most obviously deadly chemicals were banned in the 1970s, they continue to impoverish the biosphere. polychlorinated biphenyls -stable, nonflammable compounds once used in electrical transformers and many other industrial and household applications -remain in the environment for long periods, cycling among air, water, and soils and persisting in the food web. they are found in polar bears and arctic villagers; they are implicated in reproductive disorders, particularly in such animals as marine mammals, whose long lives, thick fat layers where chemicals concentrate, and position as top predators make them especially vulnerable. the agricultural pesticide ddt, sprayed with abandon in the 1940s and 1950s, even directly on children, had severely thinned wild birds' eggshells by the time it was banned in the united states. populations of birds such as the brown pelican and bald eagle had dropped precipitously by the 1970s, although they have recovered enough for the species to be taken off the us endangered species list, the bald eagle in 2007 and the brown pelican in 2009. reproduction of central california populations of the california condor, in contrast, continues to be threatened by ddt breakdown products, which, 40 years after the pesticide was banned, are still found in the sea lion carcasses the birds sometimes feed on. carson's book revealed the real danger of chemical pollutants: they have not simply perturbed the chemistry of water, soil, and air but harmed the biosphere as well. the list of chemicals' effects on living things is so long that chemical pollution equals humans' environmental impact in most people's minds, yet it is just one form of biotic impoverishment. all the substances found in living things -such as water, carbon, nitrogen, phosphorus, and sulfur -cycle through ecosystems in biogeochemical cycles. human activities modify or have the potential to modify all these cycles. sometimes the results stem from changing the amount or the precise chemistry of the cycled substance; in other cases, humans change biogeochemical cycles by changing the biota itself. freshwater use, dams, and other engineering ventures affect the amount and rate of river flow to the oceans and increase evaporation rates, directly affecting the water cycle and indirectly impoverishing aquatic life. direct human modifications of living systems also alter the water cycle. in south africa, european settlers supplemented the treeless native scrub, or fynbos, with such trees as pines and australian acacias from similar mediterranean climates. but because these trees are larger and thirstier than the native scrub, regional water tables have fallen sharply. human activity has disrupted the global nitrogen cycle by greatly increasing the amount of nitrogen fixed from the atmosphere (combined into compounds usable by living things). the increase comes mostly from deliberate addition of nitrogen to soils as fertilizer but also as a by-product of the burning of fossil fuels. agriculture, livestock raising, and residential yard maintenance chronically add tons of excess nutrients, including nitrogen and phosphorus, to soils and water. the additions are often invisible; their biological impacts are often dramatic. increased nutrients in coastal waters, for example, trigger blooms of toxic dinoflagellates -the algae that cause red tides, fish kills, and tumors and other diseases in varied sea creatures. when huge blooms of algae die, they fall to the seafloor, where their decomposition so robs the water of oxygen that fish and other marine organisms can no longer live there. with nitrogen concentrations in the mississippi river two to three times as high as they were 50 years ago, a gigantic dead zone forms in the gulf of mexico every summer; in summer 2010 this dead zone covered 20,000 km 2 . the burning of fossil fuels is transforming the carbon cycle, primarily by raising the atmospheric concentration of carbon dioxide. with other greenhouse gases, such as methane and oxides of nitrogen, carbon dioxide helps keep earth's surface at a livable temperature and drives plant photosynthesis, but since the industrial revolution, atmospheric carbon dioxide concentrations have risen nearly 40% and are now widely thought to be disrupting the planet's climate. in addition, the effects of catastrophic oil spills like the one that followed the april 2010 explosion of the deepwater horizon drilling rig in the gulf of mexico -and the effects of the chemicals used to disperse the resulting plumes of oil -may reverberate for decades. in its 2007 report, written and reviewed by more than 3500 scientists, the intergovernmental panel on climate change (ipcc) concluded that climate warming was ''unequivocal'' (p. 2), that most of the average global warming over the previous 50 years was ''very likely due to anthropogenic ghg [greenhouse gas] increases'' (p. 5), and that this warming has likely had ''a discernible influence at the global scale on observed changes in many physical and biological systems'' (p. 6). the concentrations of heat-trapping gases in the atmosphere are at their highest level in more than 200,000 years. the 20th century in the northern hemisphere was the warmest of the past millennium; the first decade of the 21st first century, and the first 7 months of 2010, were the warmest on record. higher global temperatures set in motion a whole series of effects, making the study of climate change, and of humans' role in it, complex and controversial. spring arrives at least 1 week earlier in the northern hemisphere. polar glaciers and ice sheets are receding. the arctic is warming twice as fast as the rest of the planet, and arctic sea ice melted at a near-record pace in 2010. with the sun heating the newly open water, winter refreezing will take longer, and the resulting thinner ice will melt more easily the following summer. the large-scale circulation of global air masses is changing and, with them, the large-scale cycles in ocean currents, including the periodic warming and cooling in the tropical pacific ocean known as el niñ o and la niñ a. as a result, the distribution, timing, and amount of rain and snow are also changing, making the weather seem more unpredictable than ever. unusually warm or cold winters, massive hurricanes like those that devastated the us gulf coast in 2005, and weatherrelated damage to human life and property are all predicted to increase with global warming. in the united states in 2005, weather-related damage totaled nearly $97 billion. where other nutrients are not limiting, rising carbon dioxide concentrations may enhance plant photosynthesis and growth. rising temperatures may shift the ranges of many plants and animals -both wild and domestic -rearranging the composition and distribution of the world's biomes, as well as those of agricultural systems. the resulting displacements will have far-reaching implications not only for the displaced plants and animals but also for the goods and services humans depend on from these living systems. from their beginnings as hunter-gatherers, humans have become highly efficient, machine-aided ecosystem engineers and predators. we transform the land so it produces what we need or want; we harvest the oceans in addition to reaping our own fields; we cover the land, even agricultural land, with sprawling cities. all these activities directly affect the ability of other life-forms to survive and reproduce. we deplete nonhuman life by eliminating some forms and favoring others; the result is a loss of genetic, population, and species diversity. we are irreversibly homogenizing life on earth, in effect exercising an unnatural selection that is erasing the diversity generated by millions of years of evolution by natural selection. one species is now determining which other species will survive, reproduce, and thereby contribute the raw material for future evolution. in the 1930s, so many sardines were scooped from the waters off monterey's cannery row in california that the population collapsed, taking other sea creatures and human livelihoods with it; after rebounding somewhat in the first decade of the 2000s, the species has still not recovered fully. according to the us national marine fisheries service, nearly 80% of commercially valuable fish of known status were overfished or fished to their full potential by 1993. atlantic commercial fish species at their lowest levels in history include tuna, marlin, cod, and swordfish. overfishing not only depletes target species but restructures entire marine food webs. marine mammals, including whales, seals, sea lions, manatees, and sea otters, were so badly depleted by human hunters that one species, steller's sea cow (hydrodamalis gigas), went extinct; many other species almost disappeared. in the 19th century, russian fur traders wiped out sea otters (enhydra lutris) along the central california coast. with the otters gone, their principal prey, purple sea urchins (stronglyocentrotus purpuratus), overran the offshore forests of giant kelp (macrocystis pyrifera), decimating the kelp fronds and the habitat they provided for countless other marine creatures, including commercially harvested fishes. thanks to five decades of protection, marine mammal populations began slowly recovering -only to face food shortages as regional marine food webs continue to unravel because of fishing, changing oceanic conditions, and contamination. timber harvest has stripped vegetation from the amazonian rainforest to mountainsides on all continents, diminishing and fragmenting habitat for innumerable forest and stream organisms, eroding soils, worsening floods, and contributing significantly to global carbon dioxide emissions. in the northern hemisphere, 10% or less remains of old-growth temperate rainforests. the uniform stands of trees usually replanted after logging do not replace the diversity lost with the native forest, any more than monocultures of corn replace the diversity within native tallgrass prairies. a great deal of human ecosystem engineering not only alters or damages the habitats of other living things but often destroys those habitats. satellite-mounted remote-sensing instruments have revealed transformations of terrestrial landscapes on a scale unimaginable in centuries past. together, cropland and pastures occupy 40% of earth's land surface. estimates of the share of land wholly transformed or degraded by humans hover around 50%. our roads, farms, cities, feedlots, and ranches either fragment or destroy the habitats of most large carnivorous mammals. mining and oil drilling damage the soil, remove vegetation, and pollute marine areas. grazing compacts soil and sends silt and manure into streams, where they harm stream life. landscapes that have not been entirely converted to human use have been cut into fragments. in song of the dodo, writer quammen (1996) likens our actions to starting with a fine persian carpet and then slicing it neatly into 36 equal pieces; even if we had the same square footage, we would not have 36 nice persian rugs -only ragged, nonfunctional fragments. and in fact, we do not even have the original square footage because we have destroyed an enormous fraction of it. such habitat destruction is not limited to terrestrial environments. human channelization of rivers may remove whole segments of riverbed. in the kissimmee river of the us state of florida, for example, channelization in the 1960s transformed 165 km of free-flowing river into 90 km of canal, effectively removing 35 km of river channel and drastically altering the orphaned river meanders left behind. wetlands worldwide continue to disappear, drained to create shoreline communities for people and filled to increase cropland. the lower 48 united states lost 53% of their wetlands between the 1700s and mid-1980s. such losses destroy major fish and shellfish nurseries, natural flood and pollution control, and habitat for countless plants and animals. the mosaic of habitats in, on, or near the seafloor -home to 98% of all marine species -is also being decimated. like clear-cutting of an old-growth forest, the use of large, heavy trawls dragged along the sea bottom to catch groundfish and other species flattens and simplifies complex, structured habitats such as gravels, coral reefs, crevices, and boulders and drastically reduces biodiversity. studies reported on by the national research council of the us national academy of sciences have shown that a single tow can injure or destroy upward of two-thirds of certain bottom-dwelling species, which may still not have recovered after a year or more of no trawling. habitat fragmentation and destruction, whether on land or in freshwater and marine environments, may lead directly to extinction or isolate organisms in ways that make them extremely vulnerable to natural disturbances, climate change, or further human disturbance. ''the one process ongoing y that will take millions of years to correct,' ' wilson (1994, p. 355) admonishes us, ''is the loss of genetic and species diversity by the destruction of natural habitats. this is the folly our descendants are least likely to forgive us.'' both deliberately and unwittingly, humans are rearranging earth's living components, reducing diversity and homogenizing biotas around the world. the present, continuing loss of genetic diversity, of populations, and of species vastly exceeds background rates. at the same time, our global economy is transporting species worldwide at unprecedented scales. the globe is now experiencing its sixth mass extinction, the largest since the dinosaurs vanished 65 ma; present extinction rates are thought to be on the order of 100-1000 times those before people dominated earth. according to the millennium ecosystem assessment, a 5-year project begun in 2001 to assess the world's ecosystems, an estimated 10-15% of the world's species will be committed to extinction over the next 30 years. approximately 20% of all vertebrates, including 33% of sharks and rays, are at risk of extinction. at least one of every eight plant species is also threatened with extinction. although mammals and birds typically receive the most attention, massive extinctions of plants, which form the basis of the biosphere's food webs, undermine lifesupport foundations. the mutualistic relationships between animals and plants, particularly evident in tropical forests, mean that extinctions in one group have cascading effects in other groups. plants reliant on animals for pollination or seed dispersal, for example, are themselves threatened by the extinction of the animal species they depend on. not surprisingly, some scientists view extinction as the worst biological tragedy, but extinction is just another symptom of global biotic impoverishment. ever since they began to spread over the globe, people have transported other organisms with them, sometimes for food, sometimes for aesthetic reasons, and most often inadvertently. with the mobility of modern societies and today's especially speedy globalization of trade, the introduction of alien species has reached epidemic proportions, causing some scientists to label it biological pollution. aliens -zebra mussels (dreissena polymorpha) and tamarisks, or saltcedar (tamarix spp.), in north america; the red sea sea jelly rhopilema nomadica and the common aquarium alga caulerpa taxifolia now choking the mediterranean sea; and leidy's comb jelly (mnemiopsis leidyi) of northeastern america in the black sea, to name just a few -are present everywhere, and they usually thrive and spread at the expense of native species. on many islands, for example, more than half the plant species are not native, and in many continental areas the figure reaches 20% or more. the costs of such invasions, in both economic and ecological terms, are high. in the united states, for example, annual economic losses due to damage by invasive species or the costs of controlling them exceed $137 billion per year -$40 billion more than the nation's losses from weather-related damage in 2005. furthermore, alien invasions cause extinctions and, when added to other extinctions and the deliberate monocultures of agricultural crops, homogenize biotas still more. introduced species are fast catching up with habitat fragmentation and destruction as the major engines of ecological deterioration. humans have been manipulating their crop plants and domesticated animals for 10,000 years or so -selecting seeds or individuals and breeding and cross-breeding them. the goal was something better, bigger, tastier, hardier, or all of the above; success was sometimes elusive, but the result was crop homogenization. of the myriad strains of potatoes domesticated by south american cultures, for example, only one was accepted and cultivated when potatoes first made it to europe. the new crop made it possible to feed more people from an equivalent area of land and initially staved off malnutrition. but the strain succumbed to a fungal potato blight in the 1800s. had more than one strain been cultivated, the tragic irish potato famines might have been averted. in the last few decades of the 20th century, people began to manipulate genes directly using the tools of molecular biotechnology, even cloning sheep and cows from adult body cells. us farmers routinely plant their fields with corn whose genetic material incorporates a bacterial gene resistant to certain pathogens. more than 40 genetically altered crops have been approved for sale to us farmers since 1992, with genes borrowed from bacteria, viruses, and insects. the united states accounts for nearly two-thirds of biotechnology crops planted globally. worldwide in 2003, 67.6 million hectares in 18 countries on six continents were planted with genetically modified crops, as compared with 1.7 million hectares in six countries in 1996 -a 40-fold expansion in 8 years. biotechnologists focus on the potential of this newmillennium green revolution to feed the growing world population, which has added almost 1 billion people in the past decade alone. but other scientists worry about unknown human and ecological health risks; these concerns have stirred deep scientific and public debate, especially in europe, akin to the debate over pesticides in rachel carson's time. one worrisome practice is plant genetic engineers' technique of attaching the genes they want to introduce into plants to an antibiotic-resistant gene. they can then easily select plants that have acquired the desired genes by treating them with the antibiotic, which kills any nonresistant plants. critics worry that the antibiotic-resistant genes could spread to human pathogens and worsen an already growing antibioticresistance problem. another concern arises from allergies humans might have or develop in response to genetically modified foods. although supporters of genetic engineering believe that genetically altered crops pose few ecological risks, ecologists have raised a variety of concerns. studies in the late 1990s indicated that pollen from genetically engineered bt corn can kill monarch butterfly caterpillars. bt is a strain of bacterium that has been used since the 1980s as a pesticidal spray; its genes have also been inserted directly into corn and other crops. ecologists have long worried that genetically engineered plants could escape from fields and cross-breed with wild relatives. studies in radishes, sorghum, canola, and sunflowers found that genes from an engineered plant could jump to wild relatives through interbreeding. the fear is that a gene conferring insect or herbicide resistance might spread through wild plants, creating invasive superweeds that could potentially lower crop yields and further disturb natural ecosystems. in fact, herbicide-resistant turf grass tested in oregon in 2006 did escape and spread, and transgenic canola has been appearing throughout the us state of north dakota, which has tens of thousands of hectares in conventional and genetically modified canola. according to the scientists who discovered the transgenic escapees growing in north dakota -far from any canola field -the plants are likely to be cross-pollinating in the wild and swapping introduced genes; the plants' novel gene combinations indicate that the transgenic traits are stable and evolving outside of cultivation. genetically engineered crops do confer some economic and environmental benefits: for farmers, higher yields, lower costs, savings in management time and gains in flexibility; for the environment, indirect benefits from using fewer pesticides and herbicides. but it is still an open question whether such benefits outweigh the potential ecological risks or whether the public will embrace having genetically modified foods as staples in their diet. human biotic impacts are not confined to other species; human cultures themselves have suffered from the widening circles of indirect and direct effects humans have imposed on the rest of nature. over the past hundred years, human technology has cut both ways with regard to public health. wonder drugs controlled common pathogens at the same time that natural selection strengthened those pathogens' ability to resist the drugs. reservoirs in the tropics made water supplies more reliable for humans but also created ideal environments for human parasites. industrialization exposed human society to a remarkable array of toxic substances. although man's inhumanity to man has been both fact and the subject of discourse for thousands of years, the discussions have mostly been removed from any environmental context. few people today regard social ills as environmental impacts or humans as part of a biota. but diminished societal well-being -whether manifest in high death rates or poor quality of life -shares many of its roots with diminished nonhuman life as a form of biotic impoverishment. the intersection of the environment and human health is the core of the discipline known as environmental health. among the environmental challenges to public health are the direct effects of toxic chemicals; occupational health threats, including exposures to hazardous materials on the job; and sanitation and disposal of hazardous wastes. exploitation of nonrenewable natural resources -including coal mining, rock quarrying or other mining operations, and petroleum extraction and refining -often chronically impairs workers' health and shortens their lives. farmworkers around the world suffer long-term ills from high exposures to pesticides and herbicides. partly because of increased air pollution, asthma rates are rising, particularly in big cities. synthetic volatile solvents are used in products from shoes to semiconductors, producing lung diseases and toxic wastes. nuclear weapons production starting in world war ii, and associated contamination, have been linked to a variety of illnesses, including syndromes neither recognized nor understood at the time and whose causes were not diagnosed until decades afterward. the grayish metal beryllium, for example, was used in weapons production and was found decades later to scar the lungs of workers and people living near toxic waste sites. infectious diseases have challenged human populations throughout history, playing a significant role in their evolution and cultural development over the past 10,000 years, with 75% of human diseases linked to wildlife or domestic animals. the 20th century brought major successes in eradicating such infectious diseases as smallpox, polio, and many waterborne illnesses. but toward the century's end, emerging and reemerging diseases were again reaching pandemic proportions. infectious diseases thought to be on the wane -including tuberculosis, malaria, cholera, diptheria, leptospirosis, encephalitis, and dengue fever -have begun a resurgence. in addition, seemingly new scourges -ebola virus, hantavirus, hiv/aids, lyme disease, and west nile fever -are also spreading, often, it appears, from wild animal hosts to humans as people encroach further upon previously undisturbed regions. a number of studies over the decade before 2010 show complex connections between biodiversity and disease: biodiversity loss often increases disease transmission, as in lyme disease and west nile fever, but diverse ecosystems also serve as sources of pathogens. overall, however, the studies indicate that preserving intact ecosystems and their endemic biodiversity often reduces the prevalence of infectious diseases. human migrations -including their modern incarnation through air travel -also accelerate pathogen traffic and launch global pandemics, such as the 2003 outbreak of severe acute respiratory syndrome and the 2009 swine flu outbreak caused by the h1n1 virus. even something as simple and apparently benign as lighting can become an indirect agent of disease. artificial lighting, especially in the tropics, for example, can alter human and insect behavior in ways that speed transmission of insect-borne diseases, such as chagas's disease, malaria, and leishmaniasis. in addition, especially in highly developed countries such as the united states, diseases of affluence and overconsumption are taking a toll. heart disease is the number one cause of death in the united states; overnutrition, obesity, and diabetes due to sedentary, technology-driven lifestyles, particularly among children, are chronic and rising. one estimate put the share of us children considered overweight or obese at one in three. this rise in obesity rates has been stunningly rapid. as recently as 1980, just 15% of adults were obese; by 2008 the rate had hit 34%, and two-thirds of americans are now considered either overweight or obese. although not conventionally regarded as elements of biodiversity, human languages, customs, agricultural systems, technologies, and political systems have evolved out of specific regional environments. like other organisms' adaptive traits and behaviors, these elements of human culture constitute unique natural histories adapted, like any natural history, to the biogeographical context in which they arose. yet modern technology, transportation, and trade have pushed the world into a globalized culture, thereby reducing human biological and cultural diversity. linguists, for example, are predicting that at least half of the 7000 languages spoken today will become extinct in the 21st century. with the spread of euro-american culture, unique indigenous human cultures, with their knowledge of local medicines and geographically specialized economies, are disappearing even more rapidly than the natural systems that nurtured them. this loss of human biodiversity is in every way as troubling as the loss of nonhuman biodiversity. the effects of environmental degradation on human quality of life are another symptom of biotic impoverishment. food availability, which depends on environmental conditions, is a basic determinant of quality of life. yet according to the world health organization, nearly half the world's population suffers from one of two forms of poor nutrition: undernutrition or overnutrition. a big belly is now a symptom shared by malnourished children, who lack calories and protein, and overweight residents of the developed world, who suffer clogged arteries and heart disease from eating too much. independent of race or economic class, declining quality of life in today's world is manifest in symptoms such as increased asthma in the united states caused by environmental contaminants and the high disease rates in the former soviet bloc after decades of unregulated pollution. even with explicit legal requirements that industries release information on their toxic emissions, many people throughout the world still lack both information and the decision-making power that would give them any control over the quality of their lives. aggrieved about the degraded environment and resulting quality of life in his homeland, ogoni activist ken saro-wiwa issued a statement shortly before he was executed by the nigerian government in 1995 saying, ''the environment is man's first right. without a safe environment, man cannot exist to claim other rights, be they political, social, or economic.'' kenyan maathai (2009, p. 249) , 2004 winner of the nobel peace prize, has also written, ''[i]f we destroy it, we will undermine our own ways of life and ultimately kill ourselves. this is why the environment needs to be at the center of domestic and international policy and practice. if it is not, we don't stand a chance of alleviating poverty in any significant way.'' having ignored this kind of advice for decades, nations are seeing a new kind of refugee attempting to escape environmental degradation and desperate living conditions; the number of international environmental refugees exceeded the number of political refugees around the world for the first time in 1999. environmental refugees flee homelands devastated by flooding from dam building, extraction of mineral resources, desertification, and unjust policies of national and international institutions. such degradation preempts many fundamental human rights, including the rights to health, livelihood, culture, privacy, and property. people have long recognized that human activities that degrade environmental conditions threaten not only the biosphere but also humans' own quality of life. as early as 4500 years ago in mesopotamia and south asia, writings revealed an awareness of biodiversity, of natural order among living things, and of consequences of disrupting the biosphere. throughout history, even as civilization grew increasingly divorced from its natural underpinnings, writers, thinkers, activists, and people from all walks of life have continued to see and extol the benefits of nature to humans' quality of life. contemporary society still has the chance to relearn how important the environment is to quality of life. it is encouraging that the united steelworkers of america in 1990 released a report recognizing that protecting steelworker jobs could not be done by ignoring environmental problems and that the destruction of the environment may pose the greatest threat to their children's future. it is also encouraging that the 2007 nobel peace prize was awarded to a political figure and a group of scientists for their work on climate change. making a living from nature's wealth has consistently opened gaps between haves and have-nots, between those who bear the brunt of environmental damage to their home places and those who do not, and between the rights of people alive now and those of future generations; these disparities too are part of biotic impoverishment. inequitable access to ''man's first right'' -a healthy local environment -has come to be known as environmental injustice. environmental injustices, such as institutional racism, occur in industrial and nonindustrial nations. injustice can be overt, as when land-use planning sites landfills, incinerators, and hazardous waste facilities in minority communities, or when environmental agencies levy fines for hazardous waste violations that are lower in minority communities than in white communities. less overt, but no less unjust, is the harm done to one community when unsound environmental practices benefit another, as when clear-cut logging in the highlands of northwestern north america benefits logging communities while damaging the livelihoods of lowland fishing communities subjected to debris flows, sedimentation, and downstream flooding. the plight of the working poor and the disparities between rich and poor are also examples of biotic impoverishment within the human community. according to the united nations research institute for social development, the collective wealth of the world's 358 billionaires equaled the combined income of the poorest 2.4 billion people in 1994. forbes magazine put the number of billionaires in early 2010 at 1011, with a total worth of $3.6 trillion. in the united states during the last decade of the 20th century, the incomes of poor and middle-class families stagnated or fell, despite a booming stock market. the center on budget and policy priorities and the economic policy institute reported that between 1988 and 1998, earnings of the poorest fifth of american families rose less than 1%, while earnings of the richest fifth jumped 15%. by the middle of the first decade of the 21st century, americans' income inequality had become the widest among industrialized nations, with the wealthiest 20% of the population holding 85% of the wealth. the wealthiest americans continued to prosper even during the global recession late in that decade, while the less well-off kept losing ground. but perhaps the grossest example of human and environmental domination leading to continued injustice is the creation of a so-called third world to supply raw materials and labor to the dominant european civilization after 1500 and the resulting schism between today's developed and developing nations. developing regions throughout the world held tremendous stores of natural wealth, some of it -like petroleum -having obvious monetary value in the dominant economies and some having a value invisible to those economies -like vast intact ecosystems. a 2010 united nations study (teeb) estimated that even today, earth's ecosystems account for roughly half to 90% of the source of livelihoods for rural and forest-dwelling peoples; the study calls this value the gross domestic product (gdp) of the poor. dominant european civilizations unabashedly exploited this natural wealth and colonized or enslaved the people in whose homelands the wealth was found. but the dominant civilizations also exported their ways of thinking and their economic models to the developing world, not only colonizing places but also effecting what wangari maathai has called a colonization of the mind. although dominant 21st century society tends to dismiss ancient wisdom as irrelevant in the modern world, perhaps the cruelest impoverishment of all is the cultural and spiritual deracination experienced by exploited peoples worldwide. exploitation of poor nations and their citizens by richer, consumer countries -and in many cases by the same governments that fought for independence from the colonists while adopting the colonists' attitudes and economic models -persists today in agriculture, wild materials harvesting, and textile and other manufacturing sweatshops. in the mid-1990s, industrial countries consumed 86% of the globe's aluminum, 81% of its paper, 80% of its iron and steel, 75% of its energy, and 61% of its meat; they are thus responsible for most of the environmental degradation associated with producing these goods. most of the actual degradation, however, still takes place in developing nations. as a result, continuing environmental and social injusticeenvironmental and social impoverishment perpetrated by outsiders and insiders alike -pervades developing nations. such impoverishment can take the form of wrenching physical dislocation like the massive displacements enforced by china's three gorges dam. it can appear as environmental devastation of homelands and murder of the people who fought to keep their lands, as in the nigerian government-backed exploitation of ogoniland's oil reserves by the shell petroleum development corporation. after saro-wiwa's execution, the ogoni were left, without a voice, to deal with a scarred and oilpolluted landscape. despite great advances in the welfare of women and children over the past century, poverty still plagues both groups. children from impoverished communities, even in affluent nations, suffer from the lethargy and impaired physical and intellectual development known as failure to thrive. poverty forces many children to work the land or in industrial sweatshops; lack of education prevents them from attaining their intellectual potential. this impoverishment in the lives of women and children is as much a symptom of biotic impoverishment as are deforestation, invasive alien organisms, or species extinctions. little by little, community-based conservation and development initiatives are being mounted by local citizens to combat this impoverishment: witness maathai's green belt movement, which began with tree planting to restore community landscapes and provide livelihoods for residents, and the rise of ecotourism and microlending (small loans made to individuals, especially women, to start independent businesses) as ways to bring monetary benefits directly to local people without further damaging their environments. ultimately, one could see all efforts to protect the ethnosphere and the biosphere as a fight for the rights of future generations to an environment that can support them. only during the last two decades of the 20th century did environmental issues find a place on international diplomatic agendas, as scholars began calling attention to -and governments began to see -irreversible connections between environmental degradation and national security. british scholar myers (1993) , noting that environmental problems were likely to become predominant causes of conflict in the decades ahead, was one of the first to define a new concept of environmental security. national security threatened by unprecedented environmental changes irrespective of political boundaries will require unprecedented responses altogether different from military actions, he warned. nations cannot deploy their armies to hold back advancing deserts, rising seas, or the greenhouse effect. canadian scholar homer-dixon (1999) showed that environmental scarcities -whether created by ecological constraints or sociopolitical factors including growing populations, depletion of renewable resources such as fish or timber, and environmental injustice perpetrated by one segment of a population on another -were fast becoming a permanent, independent cause of civil strife and ethnic violence. he found that such scarcity was helping to drive societies into a self-reinforcing spiral of dysfunction and violence, including terrorism. environmental and economic injustices worldwide leave no country immune to this type of threat. typically, diplomacy has stalled in conflicts over natural resources: arguments over water rights have more than once held up israeli-palestinian peace agreements; fights over fish erupted between canada and the united states, spain, and portugal. in contrast, in adopting the montreal protocol on substances that deplete the ozone layer in 1987, governments, nongovernmental organizations, and industry successfully worked together to safeguard part of the environmental commons. the treaty requires signatory nations to curb their use of cfcs and other ozone-destroying chemicals and has been, according to former united nations secretary general kofi annan, perhaps the most successful international agreement to date. if scientists have learned anything about the factors leading to biotic impoverishment, they have learned that the factors' cumulative effects can take on surprising dimensions. as scholars like fagan (1999) and diamond (2005) have chronicled, the multiple stresses of global climatic cycles such as el niñ o, natural events like droughts or floods, resource depletion, and social upheaval have shaped the fates of civilizations. societies as far-flung as ancient egypt, peru, the american southwest, and easter island prospered and collapsed because of unwise management of their environments. the city of ubar, built on desert sands in what is now southern oman, literally disappeared into the sinkhole created by drawing too much water out of its great well. in modern sahelian africa, a combination of well digging and improved medical care and sanitation led to a threefold population increase; sedentary ways, heavy taxes imposed by a colonial government, and an impoverished people took the place of a nomadic culture evolved within the desert's realities. during the first decade of the 21st century, numerous natural disasters befell nations around the world: wildfires in australia, bolivia, canada, and russia; flooding in china, india, romania, and west africa; devastating hurricanes and typhoons in the caribbean, philippines, taiwan, and southeastern united states; catastrophic landslides and floods in china, guatemala, pakistan, and portugal; and destructive earthquakes in chile, china, haiti, indonesia, and pakistan. neither the rains nor the earthquakes were caused by human activity, but the cumulative effects of human land uses and management practices -from dikes separating the mississippi from its floodplain to deforestation in haiti -made the losses of human life and property much worse than they might have been otherwise. the ultimate cause of humans' massive environmental impact is our individual and collective consumptive and reproductive behavior, which has given us spectacular success as a species. but the very things that enabled humans to thrive in nearly every environment have magnified our impacts on those environments, and the technological and political steps we take to mitigate our impacts often aggravate them. too many of us simply take too much from the natural world and ask it to absorb too much waste. for most of human history, people remained tied to their natural surroundings. even as agriculture, writing, and technology advanced, barriers of geography, language, and culture kept humans a diverse lot, each group depending on mostly local and regional knowledge about where and when to find resources necessary for survival. their worldviews, and resulting economies, reflected this dependency. for example, in northwestern north america starting about 3000 years ago, a native economy centered on the abundance of salmon. at its core was the concept of the gift and a belief system that treated all parts of the earth -animate and inanimate -as equal members of a community. in this and other ancient gift economies, a gift was not a possession that could be owned; rather, it had to be passed on, creating a cycle of obligatory returns. individuals or tribes gained prestige through the size of their gifts, not the amount of wealth they accumulated. this system coevolved with the migratory habits of the salmon, which moved en masse upriver to spawn each year. because the indians viewed salmon as equals to themselves, killing salmon represented a gift of food from salmon to people. fishers were obligated to treat salmon with respect or risk losing this vital gift. the exchange of gifts between salmon and humans -food for respectful treatment -minimized waste and overharvest and ensured a continuous supply of food. further, the perennial trading of gifts among the people effectively redistributed the natural wealth brought each year by fluctuating populations of migrating fish, leveling out the boom-and-bust cycles that usually accompany reliance on an uncertain resource. in modern times, the gift economy along with the egalitarian worldview that accompanied it, has been eclipsed by a redistributive economy tied not to an exchange of gifts with nature but to the exploitation of nature and to the technologies that enhance that exploitation. nature became a resource for humans, rather than an equal to humans. in economic terms, natural resources fell under the heading of 'land' in an economic trinity comprising three factors of production: land, labor, and capital. land and resources, including crops, became commodities -expendable or easily substitutable forms of capital -whose value was determined solely by their value in the human marketplace. in 1776 adam smith published his famous inquiry into the nature and causes of the wealth of nations, in which he argued that society is merely the sum of its individuals, that the social good is the sum of individual wants, and that the market (the so-called invisible hand) automatically guides individual behavior to the common good. crucial to his theories were division of labor and the idea that all the factors of production were freely mobile. his mechanistic views created an economic rationale for no longer regarding individuals as members of a community linked by ethical, social, and ecological bonds. about the same time, fueling and fueled by the beginnings of the industrial revolution, the study of the natural world was morphing into modern physics, chemistry, geology, and biology. before the mid-19 century, those who studied the natural world -early 19 century german biogeographer baron alexander von humboldt and his disciple charles darwin among them -took an integrated view of science and nature, including humans. both scientists regarded the understanding of the complex interdependencies among living things as the noblest and most important result of scientific inquiry. but this integrated natural philosophy was soon supplanted by more atomistic views, which fit better with industrialization. mass production of new machines relied on division of labor and interchangeable parts. like automobiles on an assembly line, natural phenomena too were broken down into their supposed component parts in a reductionism that has dominated science ever since. rushing to gain indepth, specialized knowledge, science and society lost sight of the need to tie the knowledge together. disciplinary specialization replaced integrative scholarship. neoclassical economics, which arose around 1870, ushered in the economic worldview that rules today. a good's value was no longer tied to the labor required to make it but derived instead from its scarcity. a good's price was determined only by the interaction of supply and demand. as part of 'land,' natural resources therefore became part of the human economy, rather than the material foundation making the human economy possible. because of its doctrine of infinite substitutability, neoclassical economics rejects any limits on growth; forgotten are the classical economic thinkers and contemporaries of von humboldt, including thomas malthus and john stuart mill, who saw limits to the growth of human population and material well-being. consequently, the 19th and 20th centuries saw the rise to dominance of economic indicators that fostered the economic invisibility of nature, misleading society about the relevance of earth's living systems to human well-being. among the worst offenders in this regard are gross national product (gnp) and its cousin, gdp. gnp measures the value of goods and services produced by a nation's citizens or companies, regardless of their location around the globe. gdp, in contrast, measures the value of goods and services produced within a country's borders, regardless of who generates those goods and services. in effect, both gnp and gdp measure money changing hands, no matter what the money pays for; they make no distinction between what is desirable and undesirable, between costs and benefits. both indicators ignore important aspects of the economy like unpaid work or nonmonetary contributions to human fulfillment -parenting, volunteering, checking books out of the library. worse, the indicators also omit social and environmental costs, such as pollution, illness, or resource depletion; they only add and do not subtract. gdp math adds in the value of paid daycare or a stay in the hospital and ignores the value of unpaid parenting or being cared for at home by friends. it adds in the value of timber sold, but fails to subtract the losses in biodiversity, watershed protection, or climate regulation that come when a forest is cut. efforts have been made in the past few decades to create less blinkered economic indicators. social scientists herman daly and john cobb in 1989 developed an index of sustainable economic welfare, which adjusts the united states' gnp by adding in environmental good things and subtracting environmental bad things. public expenditures on education, for example, are weighted as ''goods'' while costs of pollution cleanup, depletion of natural resources, and treating environment-related illnesses are counted as 'bads.' unlike the soaring gdp of recent decades, this index of sustainable economic welfare remained nearly unchanged over the same period. still other work aims to reveal nature's worth in monetary terms by assigning dollar values to ecological goods and services. a 1997 study by ecologist david pimentel and colleagues calculated separate values for specific biological services, such as soil formation, crop breeding, or pollination; by summing these figures, these researchers estimated the total economic benefits of biodiversity for the united states at $319 billion -5% of us gdp at the time -and for the world at $2928 billion. a 2000 analysis by pimentel and colleagues reported that the approximately 50,000 nonnative species in the united states cause major environmental damage and reparation costs amounting to $137 billion a year. as part of the united nations international year of biodiversity in 2010, several studies have translated the value of the world's ecosystems into dollar values. one report estimated the worth of crucial ecosystem services delivered to humans by living systems at $21-72 trillion per year -comparable to a world gross national income of $58 trillion in 2008. another study reported, among other things, that as many as 500 million people worldwide depend on coral reefs -valued between $30 billion and $172 billion a year -for fisheries, tourism, and protection from ocean storms and high waves, services threatened by warmer and more acidic seas. although a monetary approach does not create a comprehensive indicator of environmental condition, it certainly points out that ecological values ignored by the global economy are enormous. the us census bureau predicts that by 2012, the global human population will top 7 billion -having grown by nearly 1 billion people during the decade between the first and second editions of this encyclopedia. from the appearance of h. sapiens about 200,000 years ago, it took humans until 1804 to reach their first billion, 123 years to double to 2 billion, and 33 years to achieve 3 billion. human population doubled again from 3 to 6 billion in about 40 years -before most post-world war ii baby boomers reached retirement age. even with fertility rates declining in developed countries, china, and some developing countries where women are gaining education and economic power, and with pandemics like aids claiming more lives, the census bureau predicts that world population will reach 9 billion by 2044. humans appropriate about 40% of global plant production, 54% of earth's freshwater runoff, and enough of the ocean's bounty to have depleted 63% of assessed marine fish stocks. in energy terms, one person's food consumption amounts to 2500-3000 calories a day, about the same as that of a common dolphin. but with all the other energy and materials humans use, global per capita energy and material consumption have soared even faster than population growth over the past 50 years. now, instead of coevolving with a natural economy, global society is consuming the foundations of that economy, impoverishing earth's living systems, and undermining the foundations of its own existence (figure 1 ). for most of the 20th century, environmental measurements, or indicators, tracked primarily two classes of information: counts of activities directed at environmental protection and the supply of products to people. regulatory agencies are typically preoccupied with legislation, permitting, or enforcement, such as the numbers of environmental laws passed, permits issued, enforcement actions taken, or treatment plants constructed. resource protection agencies concentrate on resource harvest and allocation. water managers, for example, measure water quantity; they allocate water to domestic, industrial, and agricultural uses but seldom make it a priority to reserve supplies for sustaining aquatic life, to protect scenic and recreational values, or simply to maintain the water cycle. foresters, farmers, and fishers count board-feet of timber, bushels of grain, and tons of fish harvested. governmental and nongovernmental organizations charged with protecting biological resources also keep counts of threatened and endangered species. as in the parable of the three blind men and the elephanteach of whom thinks the elephant looks like the one body part he can touch -these or similar indicators measure only one aspect of environmental quality. counting bureaucratic achievements focuses on actions rather than on information about real ecological status and trends. measurements of resource supply keep track of commodity production, not necessarily a system's capacity to continue supplying that commodity. and measuring only what we remove from natural systems, as if we were taking out the interest on a savings account, overlooks the fact that we are usually depleting principal as well. even biologists' counts of threatened and endangered species -which would seem to measure biotic impoverishment directly -still focus narrowly on biological parts, not ecological wholes. enumerating threatened and endangered species is just like counting any other commodity. it brings our attention to a system already in trouble, perhaps too late. and it subtly reinforces our view that we know which parts of the biota are most important. society needs to rethink its use of available environmental indicators, and it needs to develop new indicators that represent current conditions and trends in the systems humans depend on ( table 2) . it particularly needs objective measures more directly tied to the condition, or health, of the environment so that people can judge whether their actions are compromising that condition. such measures should be quantitative, yet easy to understand and communicate; they should be cost-effective and applicable in many circumstances. unlike narrow criteria tracking only administrative, commodity, or endangered species numbers, they should provide reliable signals about status and trends in ecological systems. ideally, effective indicators should describe the present condition of a place, aid in diagnosing the underlying causes of that condition, and make predictions about future trends. they should reveal not only risks from present activities but also potential benefits from alternative management decisions. most important, these indicators should, either singly or in combination, give information explicitly about living systems. measurements of physical or chemical factors can sometimes act as surrogates for direct biological measurements, but only when the connection between those measures and living systems is clearly understood. too often we make assumptionswhen water managers assume that chemically clean water equals a healthy aquatic biota, for example -that turn out to be wrong and fail to protect living systems. as environmental concerns have become more urgent -and governmental and nongovernmental organizations have struggled to define and implement the concept of sustainable development -the effort has grown to create indicator systems that explicitly direct the public's and policymakers' attention to the value of living things. moving well past solely economic indexes like gdp, several indexes now integrate ecological, social, and economic well-being. the index of environmental trends for nine industrialized countries, developed by the nonprofit national center for economic and security alternatives, incorporates ratings of air, land, and water quality; chemical and waste generation; and social system economic system natural system figure 1 relationships among the natural, social, and economic systems on earth. human economies may be thought of as icing atop a two-layer cake. the economic icing is eroding the human social and natural layers beneath it, threatening the foundation and sustainability of all three systems. modified from karr jr and chu ew (1995) ecological integrity: reclaiming lost connections. in: westra l and lemons j (eds.) perspectives in ecological integrity, pp 34-48. dordrecht, netherlands: kluwer academic. energy use since 1970. by its 1995 rankings, environmental quality in the united states had gone down by 22% since 1970, while denmark had declined by 11%. in 2000, world leaders, supported by the united nations development programme, defined a set of eight millennium development goals to be attained by 2015, which combine poverty, education, employment, and environmental sustainability. they include human rights and health goals -such as universal primary education, gender equality, and combating aids and other diseases -as well as ensuring environmental sustainability. in the decade since the program began, progress has been made in reducing poverty; expanding educational opportunities for children, especially in africa; slowing deforestation; and providing better water for many people, among others. but progress controlling climate change, halting biodiversity loss, and achieving gender equality has lagged. a later environmental performance index spearheaded by yale and columbia universities ranks 163 countries on 25 performance indicators in 10 well-established policy categories to determine whether and how well countries are meeting established environmental goals. this index incorporates measurements addressing environmental stresses to human health plus ecosystem health and natural resource management, collectively referred to as ecosystem vitality. the categories representing environmental health include the environmental burden of disease and the effects of air and water quality on human health. those representing ecosystem vitality include air and water resources for ecosystems; biodiversity and habitat; and climate change, among others. although hampered by data gaps and the inability to track changes in countries' performance through time, the index provides a sense of countries' relative performance with regard to the environmental challenges faced by all. top performers in the 2010 rankings included iceland, switzerland, costa rica, and sweden. the united states ranked 61st well below japan and nations in the european union. a resource-accounting approach pioneered in the 1990s by geographers rees and wackernagel (1996) translates humans' impact on nature, particularly resource consumption, into a metaphorical ecological footprint. this accounting estimates the area required by a city, town, nation, or other human community to produce consumed resources and absorb generated wastes; it then compares the physical area occupied by a city or country with the area required to supply that city or country's needs. the 29 largest cities of baltic europe, for example, appropriate areas of forest, agricultural, marine, and wetland ecosystems that are at least 565-1130 times larger than the areas of the cities themselves. according to the global footprint network, national ecological footprints in 2010 ranged from a high of 10.7 hectares per person for the united arab emirates to 0.4 hectares per person for timor-leste and 0.6 for afghanistan and bangladesh. the united states' ecological footprint -8.0 hectares per person -tied for fourth among 152 nations with populations of at least 1 million. one-hundred and four of these nations operate under ecological deficits; that is, their 6. cumulative effects (frequency of catastrophic natural disasters; costs of weather-related property damage; human death tolls; government subsidies of environmentally destructive activities such as fishery overcapitalization, below-cost timber sales, water projects, and agricultural supports; replacement costs for ecological services; pricing that reflects environmental costs; ''green'' taxes; rise in polycultural agricultural practices; number of organic farms) a these indicators have been or could be used to monitor status and trends in environmental quality, including dimensions of biotic impoverishment. without a full spectrum of indicators, however, and without coupling them to direct measures of biological condition, only a partial view of the degree of biotic impoverishment will emerge. consumption exceeds the biological capacity of their lands and waters to provide needed resources and absorb their wastes. at their present rates of consumption, these nations are therefore overexploiting either their own resources or those of other nations. by ecological footprint accounting, raising the nearly 7 billion people on earth in the early 21st century to living standards -and thus ecological footprints -equal to those in the united states would require four planets more than the only one we have. clearly, humans are consuming more resources, and discarding more waste, than earth's living systems can produce or absorb in a given time period. this gap is the global sustainability gap the world now faces. most environmental indexes and accounting systems are still human centered; they still do not measure the condition of the biosphere itself. we may know that biodiversity's services are worth huge sums of money and that our hometown's ecological footprint is much bigger than the town's physical footprint, but how do we know whether specific actions damage living systems or that other actions benefit them? how do we know if aggregate human activity is diminishing life on earth? to answer this question, we need measures that directly assess the condition of living systems. biological assessment directly measures the attributes of living systems to determine the condition of a specific landscape. the very presence of thriving living systems -sea otters and kelp forests off the central california coast; salmon, orcas, and herring in pacific northwest waters; monk seals in the mediterranean sea -says that the conditions those organisms need to survive are also present. a biota is thus the most direct and integrative indicator of local, regional, or global biological condition. biological assessments give us a way to evaluate whether monetary valuations, sustainability indexes, and ecological footprints are telling the truth about human impact on the biosphere. biological assessments permit a new level of integration because living systems, including human cultures, register the accumulated effects of all forms of degradation caused by human actions. direct, comprehensive biological monitoring and assessment has been done for many aquatic systems; measures are less developed for terrestrial systems. the index of biological integrity (ibi), for example, was developed in 1981 to assess the health of streams in the us midwest and has since helped scientists, resource managers, and citizen volunteers understand, protect, and restore rivers in at least 67 countries worldwide (karr 2006) . borrowing from the same page as more recent sustainability indexes, ibi takes the concept behind economic indexes like gdp or the consumer price index -that of multiple indicators integrated into a multimetric index -and applies it to animals and plants in bodies of water or other environments. the specific measurements ( table 3 ) are sensitive to a broad range of human effects in waterways, such as sedimentation, nutrient enrichment, toxic chemicals, physical habitat destruction, and altered flows. the resulting index combines the responses of biological parts such as species, as well as processes such as food web dynamics, to human actions. indexes of biological integrity have been developed for a number of aquatic and terrestrial environments; the most widely used indexes for assessing rivers examine fishes and benthic (bottom-dwelling) invertebrates. these groups are abundant and easily sampled, and the species living in virtually any water body represent a diversity of anatomical, ecological, and behavioral adaptations. as humans alter watersheds and water bodies, changes occur in taxonomic richness (biodiversity), species composition (which species are present), individual health, and feeding and reproductive relationships. sampling the inhabitants of a stream can tell us much about that stream and its landscape. biological diversity is higher upstream of wastewater treatment plants than downstream, for example, whereas, at the same location, year-toyear variation is low (figure 2) . biological sampling can also reveal differences between urban and rural streams. for instance, samples of invertebrates from one of the best streams in rural king county, in the us state of washington, contain 27 kinds, or taxa, of invertebrates; similar samples from an urban stream in the city of seattle contain only 7. the rural stream has 18 taxa of mayflies, stoneflies, and caddisflies; the urban stream, only 2 or 3. when these and other metrics are combined in an index based on invertebrates, the resulting benthic ibi (b-ibi) ranks the condition, or health, of a stream numerically ( table 4) . a benthic ibi can also be used to compare sites in different regions. areas in wyoming's grand teton national park where human visitors are rare have near-maximum b-ibis. streams with moderate recreation taking place in their watersheds have b-ibis that are not significantly lower than those with no human presence, but places where recreation is heavy are clearly damaged. urban streams in the nearby town of jackson are even more degraded, yet not as bad as urban streams in seattle. nation-specific biological assessments also can be and are being done. the us environmental protection agency (2006), for example, performed a nationwide survey of stream condition using an ibi-like multimetric index. the survey found that 28% of us stream miles were in good condition in table 3 biological attributes in two indexes of biological integrity comparison with least-disturbed reference sites in their regions, 25% were in fair condition, and 42% were in poor condition (5% were not assessed). the agency has been expanding this effort to include other water resource types, including coastal waters, coral reefs, lakes, large rivers, and wetlands. since 2000, the heinz center (2008) has published two editions of its report on the state of us ecosystems, which seek to capture a view of the large-scale patterns, conditions, and trends across the united states. the center defined and compiled a select set of indicators -specific variables tracking ecosystem extent and pattern, chemical and physical characteristics, biological components, and goods and services derived from the natural world -for six key ecosystems: coasts and oceans, farmlands, forests, fresh waters, grasslands and shrublands, and urban and suburban landscapes. among the many conclusions of the 2008 report were that the acreage burned every year by wildfires was on the increase; nonnative fish had invaded nearly every watershed in the lower 48 states; and chemical contaminants were found in virtually all streams and most groundwater wells, often at levels above those set to protect human health or wildlife. on the plus side, ecosystems were increasing their storage of carbon, soil quality was improving, and crop yields had grown significantly. the massive international un millennium ecosystem assessment (2005) remains the gold standard for synthesizing ecological conditions at a variety of scales. from 2001 through 2005, the project examined the full range of global ecosystems -from those relatively undisturbed, such as natural forests, to landscapes with mixed patterns of human use to ecosystems intensively managed and modified by humans, such as agricultural land and urban areas -and communicated its findings in terms of the consequences of ecosystem change for human well-being. the resulting set of reports drew attention to the many kinds of services people derive from ecosystems, specifically, supporting services, such as photosynthesis, soil formation, and waste absorption; regulating services, such as climate and flood control and maintenance of water quality; provisioning services, such as food, wood, and nature's pharmacopoeia; creek near portland, oregon (united states), taxa richness differed little between years but differed dramatically between sites upstream of a wastewater outfall and sites downstream. (b) taxa richness also differed between two creeks with wastewater outfalls (tickle and north fork deep) and one creek without an outfall (foster). all three streams flowed through watersheds with similar land uses. fig. 2 for graphs of selected b-ibi metrics at these sites. and cultural services from scientific to spiritual. in addition, the reports explicitly tied the status of diverse ecosystems and their capacity to provide these services to human needs as varied as food and health, personal safety and security, and social cohesion. even while recognizing that the human species is buffered against ecological changes by culture and technology, the reports highlighted our fundamental dependence on the flow of ecosystem services and our direct responsibility for the many faces of biotic impoverishment. among other findings, the assessment found that 60% of the services provided by ecosystems are being degraded at levels that derail efforts to stem poverty, hunger, and disease among the poor everywhere. government subsidies that provide incentives to overharvest natural resources are a leading cause of the decline in renewable natural resources. such declines are not limited to coral reefs and tropical forests, which have been on the public's radar for some time; they are pervasive in grasslands, deserts, mountains, and other landscapes as well. moreover, the degradation of ecosystem services could grow worse during the first half of the 21st century, effectively blocking achievement of the united nations' eight millennium development goals. the core message embodied in ecological, especially biological, assessments is that preventing harmful environmental impacts goes beyond narrow protection of clean water or clear skies, even beyond protecting single desired species. certain species may be valuable for commerce or sport, but these species do not exist in isolation. we cannot predict which organisms are vital for the survival of commercial species or species we want for other reasons. failing to protect all organisms -from microbes and fungi to plants, invertebrates, and vertebrates -ignores the key contributions of these groups to healthy biotic communities. no matter how important a particular species is to humans, it cannot persist outside the biological context that sustains it. direct biological assessment objectively measures this context and is fundamental to advancing the kinds of syntheses like those of the millennium ecosystem assessment. every animal is alert to dangers in its environment. a microscopic protozoan gliding through water responds to light, temperature, and chemicals in its path, turning away at the first sign of something noxious. a bird looking for food must decide when to pursue prey and when not, because pursuit might expose the bird to predators. the bird might risk pursuit when it is hungry but not when it has young to protect. animals that assess risks properly and adjust their behavior are more likely to survive; in nature, flawed risk assessment often means death or the end of a genetic line. humans, too, are natural risk assessors. each person chooses whether to smoke or drink, fly or take the train, drive a car or ride a motorcycle and at what speeds. each decision is the result of a partially objective, partially subjective internal calculus that weighs benefits and risks against one another. risk is a combination of two factors: the numerical probability that an adverse event will occur and the consequences of the adverse event. people may not always have the right signals about these two factors, however, and may base their risk calculus on the wrong clues. city dwellers in the united states generally feel that it is safer to drive home on a saturday night than to fly in an airplane, for example. even though the numerical odds of an accident are much higher on the highway than in the air, people fear more the consequences of an airplane falling out of the sky. human society also strives to reduce its collective exposure to risks. governments seldom hesitate to use military power to defend their sovereignty or, albeit more reluctantly, their regulatory power to reduce workplace risks and risks associated with consumer products like automobiles. but people and their governments have been much less successful in defining and reducing a broad range of ecological risks, largely because they have denied that the threats are real. policies and plans generated by economists, technologists, engineers, and even ecologists typically assume that the lost and damaged components of living systems are unimportant or can be repaired or replaced. widespread ecological degradation has resulted directly from the failure of modern society to properly assess the ecological risks it faces. like the fate of old kingdom egypt or easter island, our civilization's future depends on our ability to recognize this deficiency and correct it. risk assessment as formally practiced by various government agencies began as a way to evaluate the effects of toxic substances on human health, usually the effects of single substances, such as pollutants or drugs, from single sources, such as a chemical plant. during the 1990s, the focus widened to encompass mixtures of substances and also ecological risks. ecological risk assessment by the us environmental protection agency (1998) asks five questions: is there a problem? what is the nature of the problem? what are the exposure and ecological effects? (a hazard to which no one or nothing is exposed is not considered to pose any risk.) how can we summarize and explain the problem to affected parties, both at-risk populations and those whose activities would be curtailed? how can we manage the risks? even though these are good questions, ecological risk management has not made any visible headway in stemming biotic impoverishment. its central failing comes from an inability to correctly answer the second question, what is the nature of the problem? our present political, social, and economic systems simply do not give us the right clues about what is at risk. none of society's most familiar indicatorswhether gdp or number of threatened and endangered species -measure the consequences, or risks, of losing living systems. if biotic impoverishment is the problem, then it only makes sense to direct environmental policy toward protecting the integrity of biotic systems. integrity implies a wholeness or unimpaired condition. in present biological usage, integrity refers to the condition at sites with little or no influence from human activity; the organisms there are the products of natural evolutionary and biogeographic processes in the absence of humans. tying the concept of integrity to an evolutionary framework provides a benchmark against which to evaluate sites that humans have altered. directing policy toward protecting biological integrity -as called for in the united states' clean water act, canada's national park act, and the european union's water framework directive, among others -does not, however, mean that humans must cease all activity that interferes with a ''pristine'' earthly biota. the demands of feeding, clothing, and housing billions of people mean that few places on earth will maintain a biota with evolutionary and biogeographic integrity. rather, because humans depend on living systems, it is in our interest to manage our activities so they do not compromise a place's capacity to support those activities in the future; that capacity can be called ecological health. ecological health describes the preferred state of sites heavily used for human purposes: cities, croplands, tree farms, water bodies stocked for fish, and the like. at these places, it is impractical to set a goal of integrity in an evolutionary sense, but we should avoid practices that damage these places to the point where we can no longer derive the intended benefits indefinitely. for example, agricultural practices that leave behind saline soils, depress regional water tables, and erode fertile topsoil faster than it can be renewed destroy the land's biological capacity for agriculture; such practices are unhealthy in both ecological and economic terms. biological integrity as a policy goal redirects our focus away from maximizing goods and services for the human economy toward ways to manage human affairs within the bounds set by the natural economy. it begins to turn our attention away from questions such as, how much stress can landscapes and ecosystems absorb? to ones such as, how can responsible human actions protect and restore ecosystems? in contrast to risk assessment, striving to protect biological integrity is more likely to lead away from mandated corrective actions in the form of technological fixes for environmental problems and toward practices that prevent ecological degradation and encourage ecological restoration. leopold (1949, pp. 224-225) , in a sand county almanac, was the first to invoke the concept of integrity in an ecological sense: ''a thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community. it is wrong when it tends otherwise.'' managing for biological integrity requires the kind of ethical commitment inherent in leopold's words. we are called to curb consumerism and limit population size, to embrace less-selfish attitudes toward land stewardship, and to understand that the biosphere matters. instead of calling on human technical and spiritual wellsprings to manage resources, we have to call on them for managing human affairs. we have to find and use appropriate measurements for all the factors contributing to biotic impoverishment, be they climate change, overharvesting, agriculture, or environmental injustice. measurement of environmental impact founded on the evolutionary idea of integrity allow us to directly assess biotic condition and to compare that condition with what might be expected in a place with little or no human influence. at least then we can make an informed choice: continue with activities that degrade biotic condition or think of an alternative. the ecological world is a complex, variable, and often unpredictable system. when managing for ecological risks, people and their governments need to expect the unexpected and develop formal, yet flexible means of coping with environmental surprises. rather than plunge ahead with projects entailing ecological risks because they can be done, decision makers should follow the precautionary principle, which holds that regulators should act to prevent potential environmental harm even in the absence of certainty. it acknowledges the existence of uncertainty rather than deny it, and it includes mechanisms to safeguard against potentially harmful effects. although inappropriate ecological risk assessment and management are more often the norm today, modern institutions are capable of recognizing ecological threats correctly and responding to them in time, as they did with the montreal protocol. a decade after the agreement's adoption, satellite measurements in the stratosphere indicated that ozone-depleting pollutants were in fact on the decline. given the treaty's success, some policy experts have suggested using it to help curb global warming. specifically, negotiators at the 2010 annual meeting of signatory parties were considering a proposed expansion of the ozone treaty to phase out the production and use of industrial chemicals called hydrofluorocarbons, which have thousands of times the global warming potential of carbon dioxide. even though the montreal protocol was not designed to fight climate change, policymakers in favor of the new proposal say that it could and should be used to achieve broader environmental objectives. early in the 20th century, two sciences of ''home maintenance'' began to flourish: the young science of ecology (from the greek oikos, meaning home) and a maturing neoclassical economics (also from oikos). ecology arose to document and understand the interactions between organisms and their living and nonliving surroundings -in essence, how organisms make a living in the natural economy. in fact, ernst haeckel, who coined the term in the 1860s, defined ecology in an 1870 article as the body of knowledge concerning the economy of nature. neoclassical economics, in contrast, reinforced humans' self-appointed dominion over nature's wealth. it brought unparalleled gains in societal welfare in some places, but it also divorced the human economy from the natural one on which it stands (see figure 1) . by now it is clear to economists and ecologists alike that human actions and their effects have reached scales unprecedented in the history of life. we have altered earth's physical and chemical environment, changed the planet's water and nutrient cycles, and perturbed its climate. we have unleashed the greatest mass extinction in 65 my and distorted the structure and function of nonhuman and human communities worldwide. in trying to make our own living, we have jeopardized the earth's capacity to sustain other species and our own species as well. we are losing the bio in biosphere. now, faced with these unprecedented losses, we need to understand -not deny -the ecological consequences of what we do. we urgently need a new science and art of home maintenance, one that sees the human species' role as ecosystem engineer for what it has become and reconnects human and natural economies. this new science can help us understand and correctly interpret the consequences of human-driven change. by using indicators that measure what matters for sustaining living systems, this new science can make nature visible again and shed new light on the value of the ancient heritage we share with the larger biosphere. it can help us reunite the fragments of our worldview and re-create ethical, social, and ecological bonds that were put aside two centuries ago in the name of progress. and such a science can enable us to reengineer our own social, political, and economic institutions instead of ecosystems. this we must donow -before we impoverish the biosphere and ourselves for all time. the wayfinders: why ancient wisdom matters in the modern world guns, germs, and steel: the fates of human societies evolution, consequences and future of plant and animal domestication collapse: how societies choose to fail or succeed floods, famines, and emperors: el niño and the fate of civilizations the state of the nation's ecosystems: measuring the lands, waters, and living resources of the united states environment, scarcity, and violence intergovernmental panel on climate change (ipcc) (2007) climate change seven foundations of biological monitoring and assessment a sand county almanac: and sketches here and there the challenge for africa. new york: pantheon books. millennium ecosystem assessment (2005) ecosystems and human well-being: synthesis ultimate security: the environmental basis of political stability song of the dodo: island biogeography in an age of extinction the economics of ecosystems and biodiversity: mainstreaming the economics of nature: a synthesis of the approach, conclusions and recommendations of teeb. united nations environment programme guidelines for ecological risk assessment wadeable streams assessment: a collaborative survey of the nation's streams, epa 841-b-06-002 our ecological footprint: reducing human impact on the earth the little things that run the world the earth in transition: patterns and processes of biotic impoverishment a short history of progress key: cord-346308-9h2fk9qt authors: kaur, rajwinder; yadav, bhoomika; tyagi, r.d. title: microbiology of hospital wastewater date: 2020-05-01 journal: current developments in biotechnology and bioengineering doi: 10.1016/b978-0-12-819722-6.00004-3 sha: doc_id: 346308 cord_uid: 9h2fk9qt the study of hospital wastewater (hww) microbiology is important to understand the pollution load, growth of particular pathogenic microbes, shift and drift in microbial community, development and spread of antibiotic resistance in microbes, and subsequent change in treatment efficiencies. this chapter investigates the potential microbes such as bacteria, viruses, fungi, and parasites present in hww along with the diseases associated and methods of treatment used. due to the indiscriminate release of antibiotics from hospitals, hww serves as a hotspot for emergence of antibiotic-resistance genes (args) and antibiotic-resistance bacteria. this chapter discusses the args occurrence in hww, their prevalence in the environment, the molecular tools used for identification, and different mechanisms of horizontal gene transfer. thus better understanding of the microbiology of hww could further help in development of advanced treatment technologies for effective removal of microbes and their bioproducts (toxins and infectious nucleic acid) from hww and contaminated water. wastewater includes any type (e.g., from agriculture, domestic means, industries, human excretion, commercial sectors, pharmaceuticals, healthcare units) of water which quality has been deteriorated under anthropogenic influence [1] . hospital wastewater (hww) is quite different from the wastewater discharged from other sources and is hazardous and infectious. it consists of wide range of several micro-and macropollutants, discharged from operation (surgery) rooms, wards, laboratories, laundry, polyclinics, research units, radiology, and medicine and nutrient solutions used in microbiology laboratories [2] . the micro-and macropollutants include radioactive isotopes, pharmaceuticals, stock cultures, heavy metals, media, pathogens, drugs, cotton particles, disinfectants, and chemical compounds [3] . the contraceptive-rich pharmaceuticals present in hww were reported to be associated with effects of endocrine disruption, for instance, exposure to pharmaceutical waste containing estrogen or androgen caused sex reversals in fishes and thus, reproductive impairment [4] . the hospital effluent discharged directly into the ponds has caused eutrophication, which is evident by oxygen depletion, dense algal blooms, and death of aquatic animals [1] . the discharge of wastewater depends upon the capacity of hospital and generally water varying from 400 to 1200 l/day/bed is consumed by the hospitals [5] . the hww with many microbes and emerging infectious particles such as prions, viroids, and toxins is hazardous for the environment, and ultimately human health. however, in many countries, hww is directly discharged into sewage water without pretreatment, it then undergoes treatment along with the municipal wastewater but the treatment could not be sufficient to remove micropollutants from hww [6] . moreover, the pharmaceutical compounds present in hww undergo biological transformation and form conjugate compounds, those toxicity could be even higher than the present metabolite [7] . thus wastewater pretreatment methods designed for municipal wastewaters are not able to remove the pharmaceutical-related micropollutants or conjugate compounds, which are generally present in low concentration than other macropollutants (chemical oxygen demand, biological oxygen demand, phosphorus, and nitrogen) and thus, could not be removed using conventional wastewater treatment plant (wwtp) operations [8] . moreover, the pathogens present in hww could cause microbial population imbalance in existing and operating municipal wwtp and metals or heavy metals present in the hww can affect the biological processes like nutrient removal. the heavy metals are in fact nonbiodegradable as compared with other organic pollutants and thus move to other pollutant sources [9] . the hww can act as an ideal growth medium for various pathogenic microbes including bacteria, viruses, fungi, and parasites. the wastewaters from hospitals also consist of several resistant bacteria and antibiotic residues as well, which could inhibit the growth of susceptible bacteria, thereby increasing the population of resistant bacteria in the receiving water. resistant bacteria discharged into environment either act as vectors to carry a transmissible gene or as the reservoirs for antibiotic-resistance genes (args) that could pose a threat to public health [10] . in addition, fungi which have capability to grow at faster rate and can spread their spores to external environment also pose a serious threat to environment and human health [11] . the absence of specific pretreatment technologies for hww also increased the frequency of gastroenteric viruses in aquatic bodies [12] . the direct discharge of hww into municipal wastewater containing disease-causing parasites has also increased the risk of skin infections and other harmful diseases in humans [13] . the specific and advanced technologies are required to be developed for hww treatment to prevent the release of harmful contaminants into the environment. therefore the microbial community analysis of hww is utmost importance to assess the pollution load and to develop the specific treatment methods for protection of the environmental health. thus authors explain about the various pathogens present in hww, args, and tools used to study the args. moreover, metagenomics, a recent approach used to study different microbial communities and gene-specific identification, are also explained in this chapter. in addition, horizontal gene transfer (hgt) approach, which can efficiently contribute to spreading of args, is discussed briefly. the hww poses a serious threat to humans with respect to contagiousness and drastic spread of infective diseases in healthcare units, society, hospital employees, and environment [5] . different solvents, pharmaceuticals, and radionuclides are used in hospitals for purpose of diagnostics, disinfection, and research. after the drug consumption, various drugs remain nonmetabolized or partially assimilate in the human body and thus excrete as such and end up into wastewater. the residual quantities of disinfectants used for treatment of skin microbial infection and to disinfect instruments and surfaces of hospitals, also end up in the hww, resulting in an increase in the population of pathogenic microbes. the pathogenic microflora present in hwws also come from medical devices, atmosphere, and water used in the hospital practice, and the pathogens are released mainly in the form of excreta of patients [14] . therefore hww consists of a mixture of pathogenic microbes including bacteria, fungi, yeasts, algae, viruses, protozoa, parasites, and bacteriophages. the effluent from hospitals is usually discharged and treated with domestic wastewaters without any prior treatment [15] . the pathogens in the receiving water, if untreated, survive for a long time in soil or water and enter into the food chain causing infectious diseases and health risks to human beings [16] . due to the economic reasons and lack of resources for analysis of actual pathogens, certain bacteria have been used as indicators for contamination of water since decades [17] (box 4.1). however, it has to be considered that removal of coliforms, which is indicator of fecal contamination, could not be directly correlated with removal of viruses, pathogens, fungi, protozoa, or other bacteria from water (samples) [17] . in addition, the indicator bacteria such as escherichia coli and clostridium perfringens when compared with pathogenic bacteria, protozoa and viruses are more sensitive to inactivation through processes such as natural competition, wastewater treatment, and high temperature [18] . traditional detection of pathogenic bacteria involves selective culture media and biochemical characterization methods. these techniques are inexpensive and simple; however, sampling error, time consumption (5à11 days), tedious, and monospecific detection (detection of only one type of pathogen) are the major limitations [17] . the enzyme-linked immuno sorbent assay (elisa) is used for laboratory diagnostics of different pathogens. the polymerase chain reaction (pcr) has been adapted in many ways: nested pcr, multiplex pcr, real-time pcr, fluorescence, and digital pcr [19] (box 4.2) for analysis of waterborne pathogens. criteria for selection: • should be present only in contaminated water, not in uncontaminated ones • should not be able to grow and proliferate in water • should be present in intestinal tract of warm blooded animals • should have similar survival pattern as pathogens present outside the host • should be easily detectable • should be useful for all types of water • should be relatively cheap to use total coliforms are higher in number than any other pathogens. the subgroup fecal coliforms indicate fecal contamination of water and thus, indicate the presence of other pathogens as well, which are lower in number, hard to detect. however, lower number of pathogens are enough to cause morbidity and mortality to humans. they are detectable by inexpensive cultural methods and do not pose any health risk to laboratory workers. however, there are few limitations of fecal indicator bacteria, which have been discussed in the text. biochemical tests, pcr, and sequencing [32] (continued) bed-linens, or through infected patients [39] . the hospital-acquired infections resulted in increase of methicillin-resistant s. aureus (mrsa) isolates. the mrsa is associated with infections of skin and soft tissues and has been a global threat for human health. in the united states, 72,444 cases of mrsa infections were reported in 2014 [40] . e. coli and pseudomonas aeruginosa are also commonly found in hww along with other nosocomial pathogens including candida albicans, klebsiella proteus, and enterobacter species [37] . most of the times, e. coli is harmless and is important part of human intestinal tract, but some of the strains are pathogenic to humans. the e. coli 0157:h7 is related to many outbreaks of water and food-borne illness and also responsible for 63,000 hemorrhagic colitis (bloody diarrhea) cases in the united states annually [41] . a study reported on hww of brazil also confirmed the presence of other bacterial species including citrobacter freundii, klebsiella ornithinolytica, proteus mirabilis, pantoea agglomerans, and serratia rubidacea [28] . many c. freundii infections have been reported in bloodstream such as septic shock, pneumonia, hypothermia, oliguria, and thrombocytopenia [42] . another bacterium p. mirabilis, which is found in hww, is responsible for urinary tract infections and is further accompanied by development of kidney stones [43] . p. agglomerans mostly affects plants but also causes opportunistic infections in immunocompromised individuals causing skin infections [44] . an important prospect about fluctuations of pollution was discussed [44] , according to which, the microbial load present in wastewater was directly correlated to several hospital activities. the higher microbial load was present in hww during the day period as compared with evening and early morning. another study [44] was also conducted on hww collected during early hours (eh) and late hours (lh) of the day in abia state, nigeria. the microbiological analysis revealed the presence of higher total bacterial count (7.9 3 10 4 cfu/ ml) at lh as compared with that (6.7 3 10 4 cfu/ml) at eh of the day. the similar results were also observed for total coliform count, which was also higher (2.2 3 10 3 cfu/ml) at lh as compared with that (1.8 3 10 3 cfu/ml) at eh due to the fact that hospital activities at night time were more restricted than day time. according to the recent study, the hww samples were collected from three hospitals in different cities of romania [35] . three hospitals (h1, h2, and h3) were having different numbers of beds and inhabitants. the microbial community analysis results revealed that wastewater from h1 was dominant by 97.27% of the proteobacteria phylum, while from h2 was dominant by 44.7% of proteobacteria phyla followed by firmicutes (33.68%), bacteroidetes (16.4%), and actinobacter (4.9%) and from h3 was dominant by 71.4% of proteobacteria, bacteroidetes (13.7%), and firmicutes (13.1%) [35] . the similar taxonomic composition was also reported in various studies using wastewater collected from different hospital facilities, nonhospital medical facilities, and municipal treatment plant [26, 45, 46] . the dominant nature of proteobacteria was correlated to their presence in human feces, long-term survival ability in wastewater, and exposure to several antibiotics present in hww. moreover, the dominant nature of firmicutes could be correlated to their capacity to survive in extreme environmental conditions and high contaminant levels [47] . in 2018 buelow et al. [48] detected abundant bacterial taxa to be different from urban and suburban wwtp influents and comprised of camphylobacteraceae, aeromonadaceae, and carnobacteriaceae. hww also consisted of several different members of human gut microbiota such as of genus streptococcus and family ruminococcaceae due to the sampling location in close proximity of the hospital sanitation areas as compared with wwtp influent. these microbes are not well suited to survive in such complex environment, which results in progressively decreased levels of these bacteria in urban wwtp influent [48] . another study compared the relative abundances of the most abundant bacterial orders in wwtp for 7 years and showed that the wwtp environment was dominated by phyla actinobacteria, bacteriodetes, and proteobacteria, and the wastewater community was highly stable and unique to its environment [49] . hospital sewage also contained high levels of anaerobic bacteria including genera such as bifidobacteriales, clostridales, bacteroidales that were likely to originate from the human gut [1] . the release of antibiotics from hospitals results in creating a selection pressure on bacteria and as a result, effluents from hospitals contain high numbers of resistant bacteria as well as antibiotic residues. prevalence and spread of antibiotic-resistance bacteria (arb) in the environment are a major problem worldwide due to improper antibiotic usage and lack of effective hww management systems. therefore arb and args are particularly studied among the hospital contaminants. for example, one study reported e. coli from hospital effluent to be multiresistant toward ampicillin, streptomycin, sulfamethoxazole, cephalosporin, ciprofloxacin, and tetracycline [29] . the hospital water environment includes potable water, faucets, wastewater drainage systems, and effluents can be the reservoir of nosocomial pathogens (arb such as mdr enterobacteriaceae, acinetobacter baumannii, and pseudomonas species), thus, increasingly dominating the hww microbiome [50] . commonly found isolates such as p. aeruginosa, a. baumannii, and enterobacteriaceace in hospital effluents have shown carbepenem resistance and have disseminated around the globe [50] . baricz et al. [51] reported antimicrobial effect of a crude bacterial extract of janthinobacterium lividum against mdr bacteria of both clinical and environmental origin, for example, mrsa, methicillin susceptible s. aureus, enterococci, and enterobacteriaceae [51] . such studies not only help to provide promising candidates for development of new antimicrobials but also to propose new or improved treatment technologies to reduce the burden of antimicrobial resistance (amr) in environment. the microbiological analysis of several hwws showed the prevalence of fungal species along with bacterial and coliform species. the fungi have simpler nutritional requirements and have higher capability to grow at lower water activity as compared with bacteria [52] . moreover, the fungal populations can easily spread their spores to external environment, hence affecting the human beings directly [11, 52] . this is the reason for its prevalence in hospital and clinical environment because if healthcare facilities are even considered free from fungus, but nature and environment factors such as temperature, moisture, and nutrients could provide easy and favorable conditions for the extensive growth of fungal species in storage containers holding clinical waste. the fungal infections could range from moderate to fatal depending upon the infection site as well as immune system of the affecting individual [53] . the prevalence of invasive fungal species has been reported to increase since three decades, due to increase in number of immunocompromised patients. the moderate fungal infections include athlete's foot and ring worm infections (cutaneous infections) in immunocompetent patients and life-threating infections include mucosal and systemic infections [53] . the fungal isolates, associated diseases, and treatment methods reported in various studies are presented in table 4à2 . the study reported by neely and orloff [54] stated the prevalence of aspergillus species in the hospital waste collected from the united states and even worse than that, this species was capable enough to survive for more than a month on the hospital waste. the longer survival of aspergillus can result in reoccurrence of fungi and has higher chance to cause disease. moreover, many spores have capacity to remain dormant under adverse conditions and then again develop into fungi, when conditions become favorable. the other fungal species were also detected but the survival capacity of mucar ( . 20 days), paecilomyces (11 days), and fusarium (10 days) species was lower than that of aspergillus species. the survival efficiency of different fungal species is related to the presence of specific structure of spores. the spores of aspergillus species are spiny and rough and thus capable enough to adhere to any type of waste and can survive longer time. however, spores of fusarium species are smooth and thus have less capability to adhere and survive. the wastewater collected from nigeria revealed the presence of total fungal count of 3.5 3 10 3 cfu/ml during the day time activities in the hospital [72] . the most prevalence fungal species in the hww were aspergillus (33.3%), followed by candida (28.7%), cryptococcus (14.9%), and penicillium (14.2%) species. the fungal diversity (29 species) was reported to be higher for waste samples collected from hematology section of malaysia hospital [72] . moreover, gloves waste consisted of the highest number of fungal species (19 species) among all the different types of waste collected from hospital. the high fungal count of 7 3 10 5 cfu/ml was also reported in soil collected from hospital dumpsite, which was correlated with the presence of high organic material present in the hospital waste. the fungi are heterotrophic microorganism, which have capacity to consume organic compounds from the surrounding environment [72] . the common fungal species identified in hospital waste were penicillium rubrum, penicillium viricadum, trichothecium roseum, rhizopus nigricans, aspergillus flavus, aspergillus parasiticus, microsporum canis, and aspergillus niger. among them, a. niger was found to be dominant fungal species with 34.5% due to the fact that it released variety of enzyme such as amylases, pectinases, and proteases and had capacity to utilize number of organic compounds as a substrate [73] . a. flavus and a. niger can cause disease named as aspergillosis and are considered as pathogens for both humans and animals. they can cause either bronchopulmonary (coughing and wheezing) or invasive aspergillosis (affects the lungs and could spread to throughout the body) [74] . more than 200,000 cases and 30%à95% mortality rate have been reported for aspergillosis infections worldwide. the nonpathogenic black bread mold called r. nigricans was also found in abundance with 27.5%. m. canis can cause infection in upper and dead layer of skin of domestic animals, including dogs and cats and later on the humans can also get affected by the infection [75] . other fungal species including t. roseum, p. viricadum, and p. rubrum are nonpathogenic fungus, which can only affect the humans with weak immune system. the curvularia species have also been reported to be present in hospital waste and can cause rare infections in the respiratory tract and cornea of human beings. curvularia lunata is also responsible for eumycetoma disease caused in farmers of chandigarh and rajasthan. most of the agricultural regions use azole fungicides (imidazole, metalaxyl, tebuconazole, and propiconazole). therefore the farmers responded poorly to antifungal treatment and were referred for long-term therapies [76] . the few fungal species of fusarium (1.5%) and trichoderma (2.2%) have also been reported in hospital waste, but infection caused by these fungal species is difficult to cure [77] . over 1.2 billion people worldwide are reported to be suffered from several fungal infections and 1.5à2 million people die due to fungal infections each year, which are far superior than malaria or tuberculosis death rate [76] . antifungal drugs are mostly used for treatment of fungal infections as they target plasma membrane, sterol biosynthesis, dna biosynthesis, and β-glucan biosynthesis of different fungi. however, since past decades, the increased use of antifungal drugs resulted in development of resistance, leading to increase in morbidity and mortality. the drug resistance could be due to several reasons such as alterations of the drug target site, increased efflux of drugs, and biofilm formation. although several studies have reported, antifungal drugs resistance is not at the same level as resistance in bacteria against antibiotics. other reasons of high mortality rate with fungal infections include availability of limited treatment options against invasive fungal diseases and less susceptibility of immunocompromised patients to antifungal drugs [78] . for instance, in the united states, until 2013, there were reports of 19,262 organ transplants, which resulted in increased susceptibility to fungal infections in immunocompromised patients [78] . in addition, adverse effects of antifungal drugs and lack of effective antifungal therapies are the other reasons, which necessities the development of novel treatment strategies and next-generations of antifungal drugs. the development of ultrahigh-throughput screening techniques could help in advent of novel antifungal drugs by providing rapid, effective, and economical drug screening. presently, the viral infections are in forefront as compared with pathogenic diseases caused by bacteria, fungi, and parasites (table 4à3) . moreover, the detection, analysis, characterization, and epidemiology of virus is entirely different from bacteria because the bacterial indicators used to represent water contamination cannot actually represent viral contamination. it has been reported that each of the material present in the hospital waste can carry viruses, and thus, viruses are able to survive for 5à8 days [78] . the viral hepatitis is very common and leading disease. moreover, human immunodeficiency virus infections, hepatitis b, and hepatitis c are among the deadly infectious diseases, transmitted by direct contact of blood from infected to another. however, these are yet easy to prevent, once detected at early stages and obligatory precautions are followed by patients [5] . the samples collected from different wwtps have been analyzed for detection and characterization, and even research has been focused on removal of viruses from wastewater [79] . however, in spite of all the health risks associated with viruses, very few research [5] has been conducted specifically for hww, and thus, requires special attention due to the fact that, it is one of the main sources to spread the pathogenic and deadly viral diseases. the modern techniques of molecular biology including pcr assays offer several advantages such as specificity, sensitivity, and wider data analysis for easy and faster detection of viruses. the occurrence of rotavirus a, norovirus genogroup i and ii, human adenovirus (hadv), and hepatitis were detected in samples collected from two different hospital wwtps. the load of rotavirus and hadv present in hospital wwtp was 2 log cycle higher as compared with that present in urban sewage wwtp [80] . the hadv consists of double-stranded linear dna (fig. 4à1) and comes under category of nonenveloped viruses, which makes them resistant to heat, dry, and acidic conditions [78] . the adenovirus consists of outer capsid and inner core with several histone proteins. the elongated fiber proteins on the surface of the virus interact with receptor of host cell such as coxsackie and adenovirus receptor and mcp and start infection in the host cells. after interaction between virus fiber proteins and host cell receptors, uncoating of virus particles takes place resulting in dissolution of viral protein in the endosome. ultimately, the translocation of adenovirus takes place with microtubules through cytoplasm toward the nucleus [81] . hadv is mostly profound in patients with weak immune system and acute lower respiratory disease [82] . seventy-nine types of hadv have been reported [83] , and out of them, hadv 40/41 is mostly prevalent in aquatic systems such as sea water, sewage, rivers, surface water, and drinking water [12] . the hadv is placed among the contaminant candidate list for drinking water by u.s. environment protection agency and is a real factor of concern for the human health [84] . it can cause several diseases in humans including ocular infections, conjunctivitis, genitourinary infections, pharyngoconjunctival fever, hemorrhagic cystitis, exanthema, encephalitis, and pneumonia [85] . the hadv is more frequently found in wastewater than any other enteric virus. the hadv is reported to be transmitted through fecal-oral transmission, aerosol droplets, and contaminated materials. it can survive for the extended period of 3à8 weeks in the environment without host. due to its stable and persistence nature in aquatic systems, it could also be considered as an indicator of fecal contamination as suggested by various studies [86, 87] . the prevalence of hadv species varies according to the hospital environment. according to prado et al. [80] , the hadv 40/41 (species f) was among the most prevalence genotype found during molecular characterization of viruses, detected during analysis of samples collected from two different hospital wwtps. the species f come from the hospitalized children reported with acute gastroenteritis disease. the other reported hadv strains include species c and d, which are associated with conjunctivitis and respiratory tract infections [80] . the frequency of hadv has also been tested for the samples (102 in number) collected throughout year from hospital wwtp located in tunisia city, tunisia and 64% samples were detected positive for hadv and most prevalent species were species f (hadv 40/41) [12] . in the similar way, the prevalence of hadv was detected in samples collected from different regions of the world such as 92.1% in poland, 76.9%à92.3% in greece, 92% in norway, and 100% in brazil, while samples collected from morocco (45.5%), italy (21.6%), and taiwan (27.3%) consisted of low prevalence of hadv [78] . the high and low prevalence of hadv reported in various countries could be collaborated by the fact that the circulation of virus varied according to the geographical regions and epidemiological community profile. however, whether it was high or low prevalence, still presence of hadv questions the ability of wwtps to remove the gastroenteric viruses and highlights the urgency for development of effective environmental control programs and innovative hww treatment plants before discharging the hww into the sewage system. the detection of hadv includes cell culture method, antigen detection, and pcr method. serology is also used sometimes to detect adenovirus-specific antibodies. there is no effective treatment against hadv, essential precautions should be taken to control infections, which include washing of hands, disinfection of instruments as well as application of infection control protocol in hospitals against adenovirus to prevent outbreaks [88] . the rotavirus consists of 11 segmented double-stranded rna and its surface is surrounded by three layers: the outer capsid, inner capsid, and central core. there are seven rotavirus groups that have been reported from a to g and studies have revealed that groups a, b, and c affect both humans and animals, while d to g are reported to cause infection mainly in animals. the dual (binary) classification system has been used for detailed classification of rotavirus on the basis of two outer capsid proteins; the glycoprotein vp7 and protein vp4 (fig. 4à2) , where the glycoprotein vp7 defines for the stereotype g and protein vp4 defines for stereotype p [89] . the viral attachment to the host cell surface is by glycoprotein vp7 or surface protein vp8. after interaction with host cell, calcium-dependent endocytosis takes place and thus, uncoating of the particle occurs, resulting in release of double layer of the viral particle into cytoplasm. further, transcription and translation takes place in the cytoplasm and viral proteins are synthesized by cellular ribosomes. after assembly, virus particle is released from the infected cell through cell lysis, and infection is further spread into the host [90] . rotavirus is associated with around 111 million cases of acute gastroenteritis in the newborn babies and children across the world, and 352,000à592,000 deaths have been reported for children with age of ,5 years globally. after infection and replication, rotavirus is discharged into surface water, groundwater, drinking water, and wastewater, able to transmit effectively through water due to its stable and resistant nature under adverse environmental conditions [91] . the rotavirus can survive at ambient temperature of 30 cà35 c and can exist on inanimate objects for 60 days without host. the rotavirus is mostly prevalent in humans and 11%à42% of positive samples are associated with group a rotavirus [92, 93] . the detection methods of rotavirus include electron microscopy, elisa, passive particle agglutination tests, polyacrylamide gel electrophoresis, and rt-pcr. the vaccination with safety and efficacy profile for rotavirus a has been developed and licensed in 2006 after the withdrawn of first ever vaccination, which was associated to cause intestinal intussusceptions in children [94] . the prevalence of rotavirus in different seasons (summer, autumn, and winter) was detected by collecting 60 different samples in the period of 9 months from hww located in shiraz, iran. enzyme immunoassays (eias) were performed and positive specimens were further investigated with nested-pcr by various primers. moreover, the virus concentration method such as the pellet and two-phase is reported to enhance the concentration of virus by 50-to 100-fold before applying eia. about 15 samples (25%) were found to be positive for rotavirus a and the highest prevalence was detected in autumn with frequency of 46.67% followed by 33.33% and 20% of prevalence in winter and spring, respectively [95] . other studies also confirmed the prevalence of rotavirus a in cold weather as compared with other seasons [96] . the molecular characterization of rotavirus further revealed the predominance of g1 genotype during various clinical investigations followed by mixed genotype of g1g4, which is associated with frequent water contamination and further generation of novel strains by genetic reassortment. moreover, various studies have reported the prevalence of rotavirus in treated wastewater as well, which is a real concern for public health [95] . the studies on rotavirus highlight the significance of environmental surveillance tools for detection of novel genotypes and to further analyze the distribution and treatment of the hazardous effects caused by rotavirus in human beings. the norovirus belongs to the family caliciviridae, and is reported to be nonenveloped and contains a positive sense, single-stranded rna, which is organized into three open reading frames (orfs). the three orfs encode three different proteins; orf1 is of size 5 kb and encodes nonstructural (ns) polyproteins, orf2 is having 1.8 kb size and encodes structural capsid protein vp1, while orf3 with size of 0.6 kb encodes a vp2 protein to maintain the stability of capsid protein [97] . the norovirus life cycle has been presented in fig. 4à3 . the interaction of norovirus with the host cell surface takes place through histoblood group antigens. after release of the vpg-linked rna genome of the virus into cytoplasm of host cell, the viral rna translation takes place. once the translation of the orf1 polyprotein is done, further co-and posttranslation processing occurs by ns6 protease of virus, which results in the release of ns proteins for the formation of replication complex. thereafter the viral replication takes place by rna-dependent rna polymerase using de novo and vpg-dependent mechanism. furthermore, the replicated genomes are translated and further packed into the capsid for assembly of virion and exit [98] . the norovirus is divided into seven genotypes, among them three genotypes gi, gii, and giv are responsible for infection in humans, and gii with genotype 4 (gii.4) is a predominant infectious virus associated with 18% of the diarrheal diseases and gastroenteritis infections around the world. in addition to gii.4, various other non-gii.4 containing viral strains are also responsible for gastroenteritis infections, for instance, the gastroenteritis emerged in southeast asia in 2014à15 was associated with g11.17 viral strain. norovirus is associated with 677 million gastroenteritis cases and 210,000 deaths annually around the world [99] . the emission of waterborne norovirus is caused by contamination of water systems including drinking water, surface water, mineral water, and groundwater [100] . as the infectious individuals shed the virus into the water system, the samples collected from hww are considered useful for study of norovirus epidemiology at population level [99] . the transmission of norovirus is through fecalàoral route from person to person or animal to person by contamination food or water intake. depending on the surface temperature and conditions, the norovirus can survive for longer period outside the host. it has capacity to survive for weeks on the hard surface, until 12 days on contaminated fabrics and for years in the contaminated still water [101] . the norovirus infection in humans can be detected by real-time pcr, multiplex gastrointestinal platforms, eias, and genotyping as well. the development of real-time pcr for detection of norovirus is considered as significant advancement with various environmental applications. the prevalence of norovirus was studied by ibrahim et al. [100] , where 102 samples were collected form 5 different basins of hospital wwtp located in tunisia. the 65% of the samples were positive for norovirus gii followed by norovirus g1, which was detected in 1% of the samples collected from wastewater, while frequency of norovirus was lowered moving from one basin to another. however, there is requirement of tertiary wastewater treatment before recycling of water for bathing and other purposes. different studies have revealed the prevalence of norovirus in different seasons, for instance, studies on hww collected from italy and sweden have showed occurrence of norovirus gii in spring and summer seasons [102, 103] . however, the study from china has revealed the prevalence of norovirus in winter conditions, which could be explained by variability in temperature during different seasons, immunity level of the host, humidity as well as socioeconomic conditions of different countries [104] . the emerging recombinant norovirus has been analyzed by illumine miseq and sanger sequencing technique by collecting different clinical and wastewater samples within australia and new zealand. the prevalence of pandemic variant (gii.pe/gii.4) was found in sydney during 2014 and 2015, while decline in pandemic virus was observed in 2016 with emergence of five new recombinant strains. these new viruses were held responsible for emergence of gastroenteritis outbreaks during november 2016 [99] . the use of new generation sequencing tools has advanced and reformed the genome sequencing of different pathogens present in wastewater. the norovirus has been responsible for 210,000 deaths per year. in 2016 world health organization (who) identified norovirus as a priority disease for vaccine development. however, several vaccines are still in trails phase and preclinical stages and no licensed vaccine has been available for norovirus infection treatment. the cost effectiveness, target population, and public acceptance are the major considerations for vaccine development [105] . the public awareness about the norovirus and prevention measurement should be spread for protection of people from norovirus infection due to lack of treatment available. hepatitis a virus (hav) is a nonenveloped virus, belongs to the picornaviridae family, and has positive sense single-stranded rna (fig. 4à4) . the rna consists of only one orf, which encodes a large polyprotein [106] . there are six genotypes of the virus reported and genotype i, ii, and iii are infectious to human population and are further subgrouped into a and b, while iv, v, and vi affects other primates than human beings [107] . there is a genetic nucleotide variation of 7.5% in each of the subgenotypes, which has been evaluated with pcr by vp1/vp2a junction present in 168 nucleotide fragment [108] . the interaction of the hav with host cell involves endocytic pathway. the host cell receptor involved in interaction with virus is tim-1 (havcr1) and it is reported to cycles between plasma membrane and lysosome of host cell with calthrin-mediated endocytosis. the replication, translation, and assembly of virus occurs inside the cytoplasm of host cell [108] . the genetic analysis of hav could be correlated with the outbreaks of viral infections and transmission mode as well. the main transmission mode for the virus is fecalàoral route, which could be directly transmitted through one human to another or indigestion of contaminated food as well [109] . the hav has capacity to survive for months outside the host body, it can survive through freezing temperature but can be killed, when exposed to high temperature of .80 c. the geographical distribution of the virus has been mainly present in india, africa, middle east, central america, and south america. the hav is considered as one of the most important food-borne pathogens and is a major cause of hepatitis in humans with reported cases of around 1.5 million globally. it has been estimated that half of the hepatitis cases are related to hav. the wastewater coming from the hospital could be emerging source of hav due to the fact that human excretion contains enormous amount of virus; moreover, virus is highly resistant to harsh environmental conditions and is able to survive for longer periods [110] . the hav is also detected in rivers, raw or treated water, and dam water as well [111] . these viruses are a real cause of concern for human health; moreover, there are no strict regulations in existence related to monitoring of these viruses in environment and water resources [112] . the consumption of contaminated food has also been directly linked with outbreak of hav at several places. for instance, there were 397 reported cases of hav in michigan between august 2016 to october 2017 with 15 fatalities and 320 hospitalization. the outbreak was linked to consumption of raw scallops served in sushi chain. in another case at hawaii, 292 cases suffering with hepatitis a infections were reported between june and october 2016, which were linked to contaminated frozen strawberries [113] . the prevalence of hav was detected in different clinical samples collected over a period of 2 years from patras and alexandroupolis located in greece. the nested real-time pcr revealed the presence of 100% genotype-1 and particularly subgenotype a [114] . the predominance of genotype-1 and subgroup a has also been reported in wastewater samples collected from brazil [80] . the prevalence of virus was also reported to be higher during the lower number of cases reported for hepatitis a, which could be directly correlated to the shedding of virus into feces. the detection of igm antibodies and anti-hav in blood is related to hepatitis a infection. the presence of anti-hav in blood indicates infection or past vaccination. the hav vaccination within 2 weeks of infection is the prevention method for hav infection and includes two-dose series for individuals above age of 12 months. the vaccination includes inactivated virus and is safe for immunocompromised persons [113] . the hww is heavily loaded with pathogens and discharged directly into aquatic bodies could be directly evident by skin infections and intestinal parasites. around the globe, millions of people are affected by deadly parasites infections, for instance, 2.8 million suffer from giardiasis, 50 million from amebic dysentery, 740 million affected by hookworm infections, 795 million detected with trichuriasis, and 1.2 billion are infected with ascariasis [13] . these parasites are mostly transmitted through fecalàoral route under poor hygienic conditions, contaminated water and food sources, and poor wastewater disposal practices. the parasites in their ineffective stages such as cysts, eggs, and oocysts survive under environmental adverse conditions and through many wastewater treatments processes due to the presence of protective outer layer. therefore parasites have capability to survive in wastewater for extended time period in comparison to viruses and bacteria [115] . the samples collected from hospital sewage treatment plant located in zaria, nigeria contained several eggs, cysts, and oocytes of various parasites. about 1648 eggs, cysts, and oocysts were present per liter of wastewater. ascaris lumbricoides was the common parasite found in hww with 307 eggs per liter (18.67%) of wastewater followed by 287 eggs (17.42%) of taenia spp., 253 eggs (15.35%) of schistosoma spp., 176 eggs of toxocara spp., 135 eggs (8.19%) of ancylostoma spp., 130 eggs (7.89%) of cryptosporidium parvum, and 58 eggs (3.52%) of giardia lambila. moreover, 92 eggs (5.58%) of trichuris and hymenolepis spp. were also found in hww. the cysts of entamoeba histolytica and a. lumbricoides were found in many studies and remained viable for longer period of time in pond effluents that was further used to irrigate raw vegetables, thus, entering the food chain and then directly to humans [13] . c. parvum causes the disease cryptosporiadiasis and g. lambila causes giardiasis, both could be life threatening if found in persons with weak immune system. the children diarrhea in many cases is caused by the cryptosporidium species [116] . taenia species can cause cysticerocosis in humans that can infect muscles, brain and can ultimately cause onset seizures in adults, while it can also cause bovine cysticerocosis in cattle after consumption of contaminated water. the main symptoms of parasitic infection include nausea, vomiting, malabsorption, diarrhea, and stomach cramps [117] . therefore the wastewater should be treated before discharge into water bodies otherwise they could be a great risk to public health. antibiotics are one of the most successful and important drugs used in therapeutic applications and the indiscriminate use of these compounds has made their way into the environment. the overuse and misuse of antibiotics not only in human therapeutics but also in veterinary, agriculture, and aquaculture applications [118] has led to the emergence of args and arb compromising or decreasing the effect of antibiotic compounds as they are becoming resistant to multiple drugs thus, causing a major concern. some studies have shown that resistant bacteria can also be present where antibiotic concentrations are low, saying that subinhibitory concentrations can also promote resistance among bacteria as similar to concentrations found in some aquatic and soil environments [118] . just as natural antibiotics have existed for billions of years, args are also ancient [119] . there have been occurrences of args in places where anthropogenic activities are minimal such as genes encoding for β-lactamase in a remote alaskan soil suggesting the environment to be a reservoir of args [120] . antibiotic-resistant bacteria have been reported from lechuguilla cave, new mexico which has been isolated for more than 4 million years including some strains which were resistant to over 14 different antibiotics [121] and in ancient permafrost samples where communities harbored resistance mechanisms minimum 5000 years ago [122] . such studies improve our understanding of the prevalence of resistance genes in environments much prior to human use of antibiotics. within past years, pieces of evidence have shown mobilization of these resistance genes from the environment into pathogenic bacteria causing health risks to humans and animals and also, demonstrating a link between environmental and clinical resistance [123] . moreover, due to the introduction of antibiotics from various human activities, the environment has turned into a reactor of arb and args contributing to the evolution and spreading of resistant genes. the phenomenon of emergence and spread of args has increased so intensely that 70% of all hospital-acquired infections show resistance toward at least one family of antibiotics. hww represents an important source of args and arb and such effluents are highly hazardous due to its infectious and toxic features [78] . traditionally, resistance was viewed as a healthcare problem but now nonclinical environment has also been reported as an important factor in the dissemination of resistance genes [124] . a wide range of antibiotics and args are being released from the hospitals and urban wastewaters which are received by wwtps [125] . the wwtp also serves as a hotspot in the emergence and spreading of args and arb in the ecosystem [126] . even after the treatment, some of the antibiotics and args are still not completely removed and being released into the receiving water bodies making aquatic ecosystems an ideal place for acquisition and spread of such genes. commensal bacteria of humans and animals also constitute a reservoir of such resistance genes which can enter the environment through sewer systems or use of animal manure [127, 128] . other than wwtp effluents, args can reach the soils, sediments, surface water and groundwater bodies including drinking water systems [129, 130] by surface runoff or infiltration of water that has been used for agricultural purpose [118] . it has been estimated by who that arbs are responsible for 25,000, 23,000, and 38,000 deaths per year in european union, the united states, and thailand, respectively [131] . there has been increasing resistance in many human pathogens such as e. coli, a. baumannii, enterococcus sp., klebsiella pneumonia, s. aureus, p. aeruginosa, serratia marcesens, citrobacter sp., and other enterobacter species, and there have been a great number of growing reports concerning the prevalence and dissemination of these pathogens into various environmental settings [132à134]. knapp et al. [135] reported args from different major classes of antibiotics tested from 1940 to 2008 had significantly increased since 1940, along with tetracycline args around 15 times more abundant than in the 1970s [135] . the resistant gene can be specific to one antibiotic, for example, ciprofloxacin or a group of antibiotics such as β-lactums and as a result, hundreds of args are being detected [136] . genes resistant to antibiotics are commonly observed for aminoglycoside (aac, aad, aph), chloramphenicol (cat, cml), sulfonamide (suli, sulii, suliii, sula), trimethoprim (dfra, dfrb), quinolone (qnra, b, s), tetracycline (teta-e, g, h, j, y, z, etc.), vancomycin (vana, vanb), macrolide (erma-c, e, f, t, v, x), β-lactum, and penicillin (bla, meca, pena) antibiotics in different environments [124, 136, 137] . infections caused by resistant strains are difficult to deal and have led to increasing rates of infections, higher hospital costs, and high rates of morbidity and mortality, for example, strains producing extended-spectrum β-lactamase (esbl) are responsible for higher rates of mortality [138] . there is a widespread occurrence of args originating from different settings that has contributed to a web of resistance among humans, animals, and the environment and, even after their treatment, they can be still present and can contribute to their spread in the environment. furthermore, there still exists an unexplored pool of genes that may have the potential to be used as args and may be passed to pathogenic bacteria. various hypothesis have been given for the mechanisms of resistance genes to traverse wwtp and their influence by the treatment process [139] (fig. 4à5) . thus antibiotic resistance development includes a broader impact on the environment and human health rather than just a local health issue and has to be addressed. the current risk assessment is inadequate and requires advanced biological risk assessment evaluations to detect the proliferation of args and antibiotics. this includes minimizing the resistance emergence and spread in the environment and their transmission to humans. to achieve this, it is necessary to achieve goals such as defining resistance in environmental samples and standardizing testing in those samples which will further require establishment of more comprehensible databases to combine both environmental and clinical metadata. it would help to understand relationships between resistomes of different settings and would improve the risk assessment of args and arb to further develop control strategies [140] . the need for screening of args present in bacteria is important for the optimal antimicrobial therapy to treat infections in patients and this need is increasing with increasing resistance among bacteria. detection will also predict the spread of resistant organisms and genes throughout the environment. a diversity of detection methods is available for both phenotypic as well as genotypic determination of args in isolates. antibiotic resistance is a selectable phenotype and can be detected using growth inhibition assays using disc diffusion method, broth dilution, gradient strips, or other methods to determine the minimum inhibitory concentration (mic) of antibiotics [141] . the mic is calculated for each isolate and based on the results, isolate can either be susceptible or resistant to the antibiotic [142] . however, there remain certain problems associated with this method, such as gradation of resistance, time-consuming (can take several weeks for slow-growing bacteria such as mycobacterium tuberculosis), and culture-dependent method, relate to only concentration of antibiotics (difficult to detect low-level resistance), and give no information about dissemination of resistance via mobile genetic elements (mges) [141, 143] . to overcome these disadvantages, genotypic or molecular characterization methods such as pcr, hybridization techniques including microarray, and whole genome sequence (wgs) are used extensively to determine specific resistance genes and providing results within hours. genotypic characterization has led to the identification of args in fastidious bacteria which expresses few phenotypic features, for example, tropheryma whipplei, that causes whipple's disease showed mutations in gyra and parc gene owning to fluoroquinolones resistance [144] . the wwtp compartments like influent, activated sludge, effluent consist of contrasting conditions including different concentrations of metals, antibiotics, etc. changing stress concentrations may act as drivers for microbial community and resistomes thus, changing biomass per volume and persistence/enrichment of arb this can result in a strong shift in the whole microbial community composition and antibiotic-resistance subset these changes are expected to correlate with the changes of resistome. contigs analysis allows further identification of marker genes for mobile genetic elements finally, horizontal gene transfer may help in the transfer of resistance genes and may act on evolutionary timescales the use of a plethora of pcrs such as standard, real-time, multiplex pcr to detect the presence of genes encoding resistance to aminoglycoside, chloramphenicol, macrolide, β-lactum, penicillin, trimethoprim, and tetracycline in bacteria is quite common [136, 145, 146] . isothermal amplifications using loop-mediated isothermal amplification [147] pcrs are very rapid, performed at one constant temperature and have been developed to detect esbls genes and carbapenemases genes in bacteria but they cannot be used in multiplex to detect several genes simultaneously. very recently, a paper-based chip which is integrated with lamp and a switch molecule for fluorescent detection of args has been developed to allow more convenient and efficient detection especially in resource-limited conditions [148] . many other detecting and genotyping techniques based on the pcr have been developed to identify args such as restriction fragment length polymorphism-pcr, mismatch amplification mutation assay-pcr, or pcr-single strand conformation polymorphism that have been used to detect gyra mutations in quinolone-resistant isolates [149] . schwartz et al. [150] used pcr technique to detect vana (vancomycin-resistance gene), meca (methicillin-resistant gene), and ampc (ampicillin-resistant gene) in wastewater, surface water, and drinking water biofilms [150] . recently both pcr and rt-pcr have been used across europe for detection of plasmid-mediated mcr-1 genes harboring resistance against colistin [151à154] (table 4à4) . multiplex rt-pcr has been used to identify staphylococci from blood samples by targeting meca and other species-specific genes [158] . another example of using multiplex pcr is in the detection of prevalent esbls genes in e. coli that encoded mainly bla shv , bla tem , bla ctx-m , and bla oxa [156] . it is also possible to devise and use multiplex pcr to detect 35 genetically diverse resistance genes for [136] tetracycline thus, making it one of the most widely used techniques [159] . however, these methods have low-throughput, sometimes gives false results, mostly ignore potential reservoirs of resistance that are nonculturable bacteria, and depend on primers that leave less room for discovery of novel genes. molecular hybridization is one of the oldest molecular techniques that have been used to detect the presence of specific args and meanwhile several improvements have been done on probe designs and synthesis. southern hybridization demonstrated tet and class-i integrons can be cotransferred from isolates present in soil to e. coli and/or pseudomonas putida [160] . pcr-southern blot assays were used frequently and reported tetracycline (tet39) and sulfonamide (sulii) resistance genes in acinetobacter species from fish farms in thailand [157] . probes can be labeled with a variety of reporters including radioactive and nonradiolabeled systems. one nonradiolabeled method is fluorescence in situ hybridization that has been used for rapid detection of macrolide, clarithromycin, linezolidresistances [136] . high-throughput dna microarray is another technique that has been successfully used to detect args in the test organism in comparison to a reference strain and works with high speed and high delicacy. in this technique, probes specific to the particular gene are spotted on a solid substrate (e.g., glass slide). dna is labeled and hybridized, and the specific targetprobe duplexes are detected. comparison of genomic diversity in a large number of test isolates can be done for which wgs is not available [145] . it enables detection of a large number of single genes, mutations, mges and also can characterize strain at the molecular level [161] . it has been used for antimicrobial genes detection in a diverse range of bacteria [162] . glass-based microarray has been developed for the detection of 17 tetracyclineresistance genes and one β-lactamase gene in multiple bacteria that used microarray probes c. 550-base pair pcr products [134] . perreten et al. [163] developed a rapid and efficient screening of gram-positive bacteria using microarray for the presence of 90 args which was further improved by additional oligonucleotides for detecting 117 args [163, 164] . similarly, alere microarrays have been developed capable of detecting 75 clinically relevant antibiotics and can be used in routine diagnostics laboratories [165] . some of its disadvantages include, low detection limit, high cost (due to use of platforms with probes of short oligonucleotides or pcr products as well as fluorescent dyes) and problems in dealing with complex samples [166, 167] . just like pcr and microarray, wgs is another potential method to detect genes and mutations conferring antibiotic resistance. the main advantage is its ability to use and detect different targets simultaneously and to subtype-specific gene variants. one can possibly add new targets to the database for analysis and can perform rapid in silico reanalysis of sequenced isolates. the presence of args within the wgs data has to be determined for which, comprehensive databases containing relevant target dna is required along with the use of appropriate bioinformatics methods for obtaining information about the wgs data. some of the databases that have been used for detection of args in curated wgs data are resfinder, which is a web server and uses blast for identification of args [168] , comprehensive antibiotic-resistance database that uses blast and resistance gene identifier as two analysis options [169] , antibiotic-resistance gene-annotation that gives user an opportunity to use local blast in bio-edit software and analysis can be done without web interface [170] , and antibiotic-resistance gene database that is a manually curated database unifying publicly available information on args such as resistance profile, ontology, mechanism of action, and much more [171] . other knowledge resources of amr include antibioticresistance genes online, collection of antimicrobial peptides, database of antimicrobial activity and structure of peptides, antimicrobial peptide database, and bacmet which differ from each other on the basis of the knowledge on molecular level of args they reflect and the scope of resistance mechanism they cover [172] . however, one major drawback of using molecular techniques for arg detection is that new emerging resistances against some antibiotics may be overlooked as observed for ndm-1 gene encoding resistance to carbapenem antibiotics which was first isolated using phenotypic methods and then characterized on the basis of genotype [141] . thus, within the number of both phenotypic and genotypic methods, there are limitations involved with each one and one can perform a combination of these screening methods to monitor resistance. thus the use of all these techniques helps in predicting the resistance genes, setting up control measures in hospital infections, adapt to a specific therapy and using them routine detection tools [173] . the molecular analysis tools such as pcr, quantitative pcr, and 16s rrna analysis have provided the in-depth knowledge about microbial communities present in wastewater since decades [174] . however, requirement of gene-specific primers, and species-specific approach used by these tools limit their activity for detection of certain targeted microbes providing incomplete information about microbial communities present in wastewater [175] . recently, metagenomics analysis has been introduced, which overcomes the limitations related with conventional molecular analysis tools and is able to generate hundreds to thousands of sequences, providing complete profile of microbial communities present in unknown samples, thus high abundance of potential microbes are detected [176] . the metagenomics analysis consists of four steps; genetic material isolation followed by genetic material cloning, construction of library, and the analysis of genetic material from the metagenomics library. metagenomics along with search engine for amr [177] can analyze the unknown samples collected from environment and can provide full-length arg data. the hww has been reported to have two overexpressed β-lactam-resistance genes (bla ges and bla oxa ) as compared with the water collected from other aquatic bodies, which could be correlated with antibiotic usage over the time in hospitals and discharge of the residues of antibiotics in the wastewater [176] . the hww that is derived from clinical speciality ward is determined to be major spot for the amr. the wastewater derived from different wards of the hospital and the final effluent of hospital was tested and compared for resistance genes. the wastewater has a high abundance of class a β-lactamase resistant genes (i.e., bla kpc , bla ctx-m , bla shv , bla tem ) and the wastewater from clinical ward also consists of high level of bla kpc-2 genes (142, 200 x/gb), encoding for carbapenem resistance. moreover, there was presence of assembled scaffolds of incq and incf plasmids having quinolone-resistance genes (qnrs1, qnrs2) and the class a β-lactamase gene (bla tem-1 ) in wastewater, which further helps in proliferation of amr [178] . metagenomic functional selection has been integrated with deep metagenomic sequencing to trace validated args in different environments. studies have identified wwtp resistome consisting of novel args thus, throwing the limelight on wwtp reservoir of uncharacterized resistance genes using these selections. using such metagenomic methods relates the number of clinically relevant resistant genes to overall functional resistant genes and thus, helps in improving the risk classification in environments [49] . metagenomics strategy also revealed the association of isolated p. aeruginosa from hww with amr frequency. the isolated strain was selected as a bio-indicator, due to its ability to survive and colonize in harsh environments and it was used to assess its viability, antibiotic susceptibility, and diversity in hww. the samples collected during different treatment steps of hww revealed that each treatment step was able to decrease the bacterial population by four logarithm cycle; however, the antibiotic resistance profile did not decrease at each treatment level, while, on the contrary, there was increase in amr microbes and genes as well. the microorganism was able to remain survived in the spore form during different treatment steps and again transform into vegetative form when released into surviving environment [179] . the healthcare clinics and hospitals, where antibiotic use is more common among the major contributor to disseminate the pathogenic microorganism into the environment. in addition, shortgun metagenomics analysis of hww collected from turkey revealed the presence of more than 100 antibiotic resistance determinants and most common genes belongs to aminoglycosides and β-lactameses. the prediction of high resistome diversity in hww raises an alarm for health concern of human population. an important step in coping with the serious problem of emergence and dissemination of args is to understand the pathways for resistance gene spread. the ability of bacteria to respond to selective pressures and new environments can be explained by the acquisition of new genes by cells using horizontal transfer methods. studies have shown around 75% of the genes in each genome have been transferred by hgt during evolution [180] . many resistance genes are located on mges such as plasmids, transposons also known as jumping genes, and integrons that are capable of capturing and expressing gene cassettes [181] , which are responsible for antibiotic resistance that functions as vectors for their dissemination [182] . horizontal acquisitions might be neutral or have deleterious effects in the chromosome or confer a selective advantage to the host. other than mutations, hgt has been one of the methods which are most responsible for the dissemination of args among different bacterial species. it has caused the spread of antibiotic resistance from commensal and environmental bacteria to pathogenic species. the args transfer by hgt exists much back before the use of antibiotics by humans, for example, oxa β-lactamase genes that confer resistance against β-lactam antibiotics mobilized from chromosomes to plasmid millions of years ago [183] . but the rate of the resistance gene transfer has increased tremendously due to selective pressure caused by antibiotic use by humans in the last few decades. bacteria employ three mechanisms for hgt, that is, conjugation, transformation, and transduction (fig. 4à6) . conjugation is said to have the greatest influence over the spread of args. conjugation requires a physical contact between two cells via pili or adhesions to transfer the genetic material. this mechanism has been found in bacterial cells with an exception of agrobacterium species that uses hgt in plants. the conjugative machinery encoded by the genes on plasmids or by integrative conjugative elements on chromosomes facilitates this process. args are mostly associated with conjugative elements like plasmids and transposons, and the transfer of these elements is most likely to occur by conjugation because it provides a more efficient way to enter the recipient cell and gives better protection in the environment [119] . conjugation can be of various types such as (1) transfer of a selftransmissible conjugative plasmid, for example, rp4 plasmid of e. coli. (2) mobilization of nonself-transmissible plasmids by the action of conjugative plasmid, for example, incq plasmid rsf1010. (3) cointegration of two different circular plasmids to fuse to become one. (4) conjugated transposons which may facilitate mobilization of plasmids or cointegration. the mges conferring agrs transfer have been found in bacteria ranging from soil, water ecosystems to food, and human pathogens [185] . the transfer of the conjugative elements between bacteria over distant phylogeny indicates the emergence of multiresistance between different reservoirs [186] . bla ctx-m esbl genes have been disseminated to various plasmids within enterobacteriaceae and other pathogens and can be found in various geographical locations [187] . the conjugation of plasmids has caused dissemination of args worldwide that encode resistance to β-lactamases, carbapenemases, quinolones, aminoglycosides, colistin, sulfonamide, tetracyclines, and other classes of drugs. conjugation has been also reported between bacteria and eukaryotic cells and is observed in various environments such as soil, aquatic, marine sediment, wastewater, and activated sludge [124] . the second mechanism is transformation, in which the cells take up the naked dna from the environment. for transformation to take place, there are certain prerequisites like release and persistence of extracellular dna in the environment, the recipient cells must be competent, and the dna translocated must be stabilized by integration into the genome or by recircularizing into self-replicating plasmids [188] . the process takes place in two steps: first, the dna substrate is transferred from surface to the cytoplasmic membrane which is mediated by type ii secretion system, type iv secretion system, and type iv pili; and second, is the transport of dna across the cytoplasmic membrane using cytoplasmic membrane channels [189] . with an exception of neisseria gonorrhoeae that is a naturally transformable bacteria which responds to environmental conditions [188] , most species develop competence under autoinducers, peptides, or stressful conditions, for example, aminoglycoside and fluoroquinolone antibiotics sublethal concentrations induce competence in streptococcus pneumoniae and legionella pneumophila [190] . mao et al. [191] developed a method to extract extracellular and intracellular dna from water and sediments. they obtained a higher concentration of extracellular dna than intracellular dna in sediments from a river basin that serves as a major arg reservoir that could be transferred to indigenous bacteria through natural transformation [191] . also, it has been shown in pneumococcal genome that the conjugated transposon disseminates via transformation in addition to conjugation [192] . transduction is the process by which dna is transferred with the help of bacteriophages. bacteriophages can transfer dna sequences like chromosomal dna, mges such as plasmids, transposons and genomic islands that are advantageous to their bacterial hosts as well as serves in improving the survival of bacteriophages [193] . transduction can be generalized where random host dna is incorporated during cell lysis and specialized, where the prophage excises itself from the host genome and also incorporates flanking host dnas [194] . args transfer by transduction has also been reported in various bacteria, for example, β-lactamase gene transfers by p1-like bacteriophages in e. coli [195] , erythromycin resistance transfer by phage between clostridium difficile strains [196] , tetracycline and gentamycin resistance between enterococci [197] , or transfer of resistance plasmids in msra [198] . bacteriophages can have broad host range, that is, between different species or among different taxonomic groups [197] thus, indicating dissemination of args via transduction in the environment, a common mechanism. other mechanisms such as gene transfer agents (gtas) and cell fusion have also been described. gtas are delivery systems that carry random pieces of host genome in capsids and get integrated into the host chromosome. the amount of dna that gta contain is insufficient to encode their proteins counterparts, unable to self-propagate thus, they do not necessarily carry gta-encoding genes that is an important distinction from transduction mechanism [199] . the first gta discovered was in rhodopseudomonas capsulate called as rcgta and they were able to transfer antibiotic resistance to bacteria by a mechanism similar to transduction which does not require cells contact and was resistant to dnases [200] . apart from the evidence of gta shown in various members of rhodobacterales, for example, ruegeria pomeroyi which contain a complete rcgta-like cluster of these genes, roseovarious nubinhibens and ruegeria mobilis also showed rates of transfer of antibiotic resistance around 10 6 -fold higher than rates of transformation and transduction when added to microbial communities [199] . mcdaniel et al. [201] reported gta frequencies to be much higher, around a million times higher the frequency of transformation and transduction measured previously in the marine environment [201] . there are several advantages of gtas over other mechanisms such as protection of dna from environmental factors, transfer not constrained to cellàcell contact, and they contain random dna from host rather than most of the bacteriophage dna as observed in transduction. another mechanism, cell fusion has been observed in haloferax and sulfolobus species [202, 203] . symmetric and bidirectional cell fusion has been observed in haloferax volcanii. studies have shown interspecies gene exchange in halophilic archaea where cells fuse forming a diploid state containing two different chromosomes of parental cells thus, facilitating genetic exchange and recombination followed by separation of hybrid parental cells [202] . although new mechanisms are continuously being developed by bacteria and identification, mapping of these resistance mechanisms provides us with knowledge of sources of resistance and helps in designing new antimicrobial drugs. the prions are infectious protein particles present in brain, which are responsible for degenerative neurological disorders. in humans, the prnp gene present on chromosome 20 produces the prp protein and prions results in transformation of prp protein into abnormal disease causing isoform [204] . these pathogenic prions cause the functional disruption of neural cells resulting in cell death and leading to memory problems, trouble with movement and personality changes as well. the prions can result in many infectious diseases such as creutzfeldtjakob disease (cjd), variably protease-sensitive prionopathy, gerstmann-sträussler-scheinker disease, scrapie, variant cjd, fatal insomnia, and kuru. these transmissible disease impacts number of mammalian species, including sheep and goats, cattle, deer, moose, elk, and humans. the scrapie and cjd are of particular concern because they can be transmitted horizontally and they remain infectious in the environment for very long time [205] . for instance, the scrapie infection was persistent even after burial in soil for 3 years. the cjd disease is most common and deadly human prion disease and can cause infection in three ways: sporadic, genetic, and acquired. most of the times (85%à90%), cjd occurs sporadically and approximately 1à1.5 million people are affected annually, because there is no cure available for cjd and other prion diseases [206] . the recently developed methods for detection of prion infectivity include protein misfolding cycling amplification, quake-induced conversion, and quantitative tandem mass spectrometric techniques [207] . however, the study of prions is highly challenging due to the fact that the high-resolution structure of prion isotherm is still to be determined and change in structure directly affect infectivity and pathology of disease. prions are likely to enter through live, infected hosts, and can be shred through saliva, urine, feces, mucus, and blood; and they enter in wastewater through hospital effluents, research facilities, homes, slaughterhouses, and mortuaries [208] . the wwtps are not able to treat prions, for instance, after entering into municipal wwtp, the prions bind to sewage sludge, survive through anaerobic digestion and are further present in treated biosolids. these biosolids are further used for land application, which results in their introduction into environment. thereafter they can be swept by wind and contaminated water with prions could further migrate into lakes, rivers, and oceans and thus, directly affecting the humans, aquatic life, and animals as well [209] . viroids are small (b250à400 nucleotides), single stranded, circular rna particles, which are distinguished from viruses by smaller size, lack of capsid and also they do not encode any protein. they have capacity to reproduce and are mostly known to cause infection in plants; however, hepatitis δ, which cause infections in humans, has many similar properties with viroids and could be related to viroids [210] . a total of 29 viroids species has been identified, which are grouped into avsunviroidae and pospiviroidae. the viroids are mainly infectious for plants such as tomatoes, cucumber, avocados, potatoes, apples, coconut palms, and chrysanthemums and are responsible for million dollars' loss in agriculture revenue each year. the most commonly reported viroid plant diseases caused by viroid include apple fruit crinkle, tomato chloric dwarf, and chrysanthemum chlorotic mottle [211] . the yellowish curled leaves of plants are due to pairing of viroid rna with messenger rna of plants resulting in interference of proper translation. some of the viroids cause devastating effects, for instance, coconut cadongàcadong viroid has caused lethal effects in coconut palms in philippines, resulting in loss of 40 million coconut palm [212] . other reported effects of viroids include epinasty, rugosity, necrosis, chlorosis, stem shortening, color alteration of fruits, flowering, and ripening delays. the microarrays are identified as a significant tool for detection of many viroids simultaneously and even has ability to detect emerging or established viroid in new host. potent protein toxins such as tetanus and botulinum toxins, anthrax toxin, epsilon-toxin, and enterotoxin can cause some most significant diseases in humans and animals. these protein toxins are most common virulence factors encoded by plasmids in clostridium and bacillus species, for example, tetanus toxin plasmid and conjugative toxin plasmid of c. perfringens. extracellular vesicles (evs) are also infectious particles said to be produced by all types of microorganisms. these evs can contain nucleic acids, toxins, lipoproteins, adhesins, nutrient scavenging factors, and enzymes that can play major role in pathogenicity in hww. such vesicles from gram-positive bacteria, mycobacteria, and fungi contain virulence factors that can elicit host immune responses. for example, crytococcus neoformans evs have glucuronoxylomannan (capsular polysaccharide) which is a virulence factor [212] . evs from gramnegative bacteria originate from outer membrane and are called outer-membrane vesicles which have been associated with cytotoxicity, virulence, invasion of host cells, and antibiotic resistance proteins. in addition to these particles, certain factors and conditions may further increase the virulence of evs, for example, a study indicated the role of iron-limiting conditions in the virulence of mycobacterium evs. these emerging infectious particles are not easily removed by conventional treatment technologies. thus there is a requirement of more novel methods and techniques to deal with such infectious particles. therefore these particles have to be readily analyzed for their presence and have to be studied for their removal from hww due to their potential negative effects on human and animal life. the recent years have witnessed the emphasis toward management of hww and various studies focused on the microbial communities present in wastewater have increased immensely. the pathogenic microbes present in hww have affected the human health since decades and antibiotic resistance microbes are also increasing significantly with time. the development of resistance toward antibiotics has been observed worldwide and has challenged both public and animal health. the use and release of various antibiotic agents in different settings have not only led to the prevalence of args in the environment but also spread and emergence of resistant bacteria. this has caused increased resistance in human pathogens and thus, making infections caused by them difficult to deal with, leading to higher mortality rates. however, the surveillance of its spread and prevalence in environment is limited and has to be expanded more due to the broad 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virus taxonomy: ninth report of the international committee on taxonomy of viruses detection of coconut cadang-cadang viroid (cccvd) in oil palm by reverse transcription loop-mediated isothermal amplification (rt-lamp) key: cord-341155-3d64mso0 authors: slots, jørgen; slots, henrik title: bacterial and viral pathogens in saliva: disease relationship and infectious risk date: 2010-12-07 journal: periodontol 2000 doi: 10.1111/j.1600-0757.2010.00361.x sha: doc_id: 341155 cord_uid: 3d64mso0 nan research on the infectious aspects of dental diseases has focused on the internal development and the pathogenicity of dental biofilms, and comparably little attention has been given to the source of the biofilm microorganisms. odontopathic bacteria exist in saliva before colonizing dental surfaces, and a better understanding of the acquisition of salivary pathogens may lead to new approaches for managing dental diseases. human viruses are also frequent inhabitants of the human mouth, and their presence in saliva may be caused by the direct transfer of saliva from infected individuals, a bloodborne infection of the salivary glands, infection of the oral mucosa, or serumal exudates from diseased periodontal sites. it has long been recognized that saliva can contain potential pathogens in quantities sufficient to infect other individuals (152) . the classic example of a serious infection contracted through saliva is epstein-barr virus-induced mononucleosis, which colloquially is termed the ôkissing diseaseõ. an example from the past is the ôno spittingõ signs to prevent inhalation of the tubercle bacillus from spit specimens on street pavements and public floors. dental clinics implement stringent infection-control measures to protect personnel and patients from pathogens in spatter, mists, aerosols, particulate matter or contaminated instruments (36) and, in the future, may adopt fully automated test systems to identify salivaborne pathogens of oral and medical diseases (72, 144) . the potential of salivary biomolecules to aid in the diagnosis of various conditions ⁄ diseases is a topic of current interest (209) . highly sensitive and specific molecular detection methods have greatly facilitated the search for salivary molecules of diagnostic value. polymerase chain reaction (pcr)-based assays can detect a large array of pathogens in saliva with no interference from pcr inhibitors (139) , and even more efficient identification techniques are rapidly emerging (97, 132) . a major advantage of salivary testing is the ease by which diagnostic samples can be collected by health professionals, by the individuals themselves, or by parents for young children. salivary sampling is painless and involves fewer health and safety issues than venepuncture, especially in patients with hemorrhagic diseases or virulent bloodborne pathogens. used as diagnostic aids, salivary biomolecules can identify a variety of cancers, illicit and prescription drug use, hereditary disorders and hormonal irregularities (87) . salivary testing can also screen for infection with the human immunodeficiency virus (hiv) (206) , herpesviruses (144, 172) , hepatitis viruses (144) , measles virus (70) and other pathogenic viruses and bacteria (discussed later). some biomarkers in saliva exhibit significant intrasubject fluctuations and may have limited diagnostic utility (189) . salivary microbial assays to assess the presence or the risk of dental diseases are premised on the idea that (i) whole saliva is the immediate source of oral biofilm bacteria, and saliva and dental biofilms tend to harbor similar relative levels of odontopathogens, and (ii) high salivary counts of odontopathic bacteria infer a high risk for dental disease and for pathogen transmission between individuals, and a decrease in the salivary count of pathogens can serve as an indicator of therapeutic effectiveness. periodontal disease severity may be ascertained by the salivary level of periodontal pathogens or host-response markers (67, 130, 146, 193, 214) , and the periodontopathic bacteria may be acquired from the infectious saliva of close family members (17) . caries risk is assessed by the levels of mutans streptococci and lactobacilli in stimulated saliva (94, 96) , and salivary transmission of cariogenic bacteria frequently occurs from the mother to her child (92, 100) . yeasts can be part of oral biofilms and cause candidosis and other oral diseases (157, 210) , especially in hiv-infected individuals, and candida albicans transmission between spouses can take place through saliva (26) . comparatively few parasites colonize the mouth, but systemic parasitic infections can affect the oral cavity (e.g. leishmaniasis), and oral protozoa may be more common than once thought (21) . this review article presents evidence that pathogenic bacteria and viruses can be present in saliva at levels that pose a disease risk for individuals with whom saliva is exchanged. emphasis is placed on the salivary route of transmission of periodontopathic bacteria and herpesviruses, and on the relationship between these infectious agents and periodontitis. the salivary presence of viral pathogens of rare, but serious, medical diseases is also reviewed. periodontitis and dental caries are infectious diseases, but the exact causes and their relative importance is still a matter of research. the search for etiological factors is closely connected to the question of how to avoid dental diseases. the consensus viewpoint of the scientific community is that specific bacteria cause both periodontitis and dental caries. this understanding has prompted the pursuit of microbiological methods to diagnose, prevent and treat dental infections. the periodontopathic microbiota has been studied for the purpose of developing more effective diagnostic tests and treatments (12, 165) . as periodontopathic bacteria also colonize the tongue dorsum and other nondental sites (43, 131) , and can be transferred via saliva to close family members (17) , periodontitis therapeutic measures ought to target periodontal pathogens in the whole mouth, not only in dental biofilms, and may even include entire family units in order to prevent cross-infection. umeda et al. (193) compared the presence of six species of periodontopathic bacteria in whole saliva and subgingival plaque from 202 subjects. each study subject contributed a whole saliva sample and a paper point sample pooled from the deepest perio-dontal pocket in each quadrant of the dentition, and the test bacteria were identified using a 16s ribosomal rna-based pcr assay (15) . a statistical relationship was found between the presence of porphyromonas gingivalis, prevotella intermedia, prevotella nigrescens and treponema denticola in whole saliva and in periodontal pocket samples, and in the event of disagreement, the organisms were more frequently present in whole saliva than in periodontal pockets (p < 0.01). the oral presence of aggregatibacter actinomycetemcomitans and tannerella forsythia was not reliably detected by sampling either whole saliva or periodontal pockets. other studies also found that a salivary sample alone did not identify all individuals infected with a. actinomycetemcomitans (176, 200) . taken together, a sample of whole saliva seems to be superior to a pooled periodontal pocket sample for detecting oral p. gingivalis, p. intermedia, p. nigrescens and t. denticola, but samples of both whole saliva and periodontal pockets may be needed in order to detect oral a. actinomycetemcomitans and t. forsythia with reasonably good accuracy. the reason for this is that a. actinomycetemcomitans and t. forsythia can persist in nondental sites, as best demonstrated in fully edentulous individuals (55, 194) . umeda et al. (192) also investigated risk factors for harboring a. actinomycetemcomitans, p. gingivalis, t. forsythia, p. intermedia, p. nigrescens and t. denticola in periodontal pockets, in whole saliva, or in both sites (i.e. orally). the study subjects included 49 african-americans, 48 asian-americans, 50 hispanics and 52 caucasians living in los angeles. periodontal probing depth was positively associated with all six study bacteria. african-americans were at increased risk (compared with caucasians) for harboring p. gingivalis in saliva [odds ratio (or) 2.95] and orally (or 2.66), and at reduced risk for harboring t. denticola orally (or 0.34). asian-americans showed an increased risk for harboring a. actinomycetemcomitans in periodontal pockets (or 6.63) and for harboring p. gingivalis in periodontal pockets (or 5.39), in saliva (or 5.74) and orally (or 5.81). hispanics demonstrated an increased risk for harboring a. actinomycetemcomitans in periodontal pockets (or 12.27) , and for harboring p. gingivalis in periodontal pockets (or 6.07), in saliva (or 8.72) and orally (or 7.98). age was positively associated with the prevalence of a. actinomycetemcomitans orally (or 1.18), and with p. gingivalis in saliva (or 1.20) and orally (or 1.20). the male gender was a risk factor for harboring p. intermedia in periodontal pockets (or 2.40), in saliva (or 3.31) and orally (or 4.25) , and for harboring p. nigrescens in saliva (or 2.85). the longer the subjects had resided in the usa, the greater the decrease in detection of a. actinomycetemcomitans orally (or 0.82). former smokers demonstrated a decreased risk for harboring a. actinomycetemcomitans in saliva (or 0.23), and current smokers displayed an increased risk for harboring t. denticola in periodontal pockets (or 4.61) . current and passive smokers revealed less salivary p. nigrescens than nonsmokers (127) . in sum, the study found a relationship between the presence of periodontopathic bacteria in whole saliva and in periodontal pockets, and pointed to the importance of genetic or environmental factors in the colonization of these pathogens. salivary tests for periodontitis may show increased accuracy if supplementing infectious disease variables with ethnic and social factors and with smoking habits (177) . studies have evaluated the salivary route of transmission of periodontopathic bacteria. transmission of periodontal pathogens from person to person depends on the salivary load of pathogens in the donor subject and various ecological factors in the recipient (16) . an early epidemiologic study found that members of the same family were infected with a. actinomycetemcomitans strains of the same biotype and serotype (213) . however, even in families with individuals heavily infected with a. actinomycetemcomitans, some family members did not harbor the organism, attesting to a relatively poor transmissibility of a. actinomycetemcomitans (213) . a study based on bacterial typing by means of the arbitrarily primed pcr method revealed an interspousal transmission of a. actinomycetemcomitans in 4 ⁄ 11 (36%) of married couples and of p. gingivalis in 2 ⁄ 10 (20%) of married couples (17) . parent-to-child transmission of a. actinomycetemcomitans took place in 6 ⁄ 19 (32%) families, whereas p. gingivalis was not transmitted from parent to child in any of the families studied (17) . similarities in the profile of periodontal bacteria have also been shown for 6-to 36-month-old children and their caregivers (186) . a review article described horizontal transmission between spouses to be 14-60% for a. actinomycetemcomitans and 30-75% for p. gingivalis, and vertical transmission to be 30-60% for a. actinomycetemcomitans and to occur only rarely for p. gingivalis (197) . the intrafamilial transmission of a. actinomycetemcomitans and p. gingivalis may in part explain the familial pattern of some types of periodontitis (13) . also, periodontal treatment and marked suppression of periodontopathic bacteria in members of a periodontitis-prone family may diminish the risk of transferring the pathogens and the disease to uninfected family members. the major cariogenic bacteria are mutans streptococci in incipient dental caries and lactobacilli in advanced caries lesions (95) , perhaps in combination with other bacteria of the dental biofilm (1, 142) . after adjusting for age and ethnicity, 6-to 36-month-old children with high levels of streptococcus mutans were found to be five times more likely to have dental caries than children with low levels of the bacterium (117) . recent large-scale microbiological studies have linked s. mutans to crown caries in children and adolescents (1, 42) and to root caries in elderly patients (142) . herpesviruses have been statistically associated with severe dental caries, but their role, if any, in the caries process remains obscure (38, 212) . an intrafamilial transfer of s. mutans was first suggested in the 1980s (23, 47) . transmission of cariogenic bacteria from the mother to the young child is particularly common, although the organisms also may be acquired from a spouse or from outside the family (98) . more recent studies have found a similar profile of cariogenic bacteria in young children and their caregivers (186) , and molecular typing studies have provided additional evidence of a transmission of mutans streptococci from mother to child (92, 100) . caries-free twins have a more similar oral microflora than twins that are caries-active, and hereditary factors seem to influence the colonization of oral bacterial species that protect against dental caries (41) . the finding of a relatively unique cariogenic microflora has a practical implication. routine testing for elevated caries risk, based on the salivary level of mutans streptococci (>1,000,000 per ml saliva) and lactobacilli (>100,000 per ml saliva), has been performed in sweden for more than 30 years (46, 94) . repeat swabbing of teeth of young children with 10% povidone-iodine can reduce the number of mutans streptococci (22) and the incidence of caries (106) . suppression of high levels of s. mutans in the mother may delay or prevent the establishment of the organism in her child (91) . a variety of bacterial pathogens of medical diseases can be present in the oral cavity and may be transmitted to individuals in close contact with the host (45) . medical pathogens are mostly detected in the mouth during the acute phase of the nonoral infection, but the organisms can also occur in the saliva of clinically healthy subjects. streptococcus pyogenes (beta-hemolytic group a streptococcus) is the cause of a variety of human diseases ranging from mild illnesses of the skin or throat (pharyngitis or ôstrep. throatõ) to severe invasive infections, including necrotizing fasciitis (flesheating disease), septicemia, toxic shock syndrome, erysipelas, cellulitis, acute postinfectious glomerulonephritis, rheumatic fever and scarlet fever (178) . s. pyogenes normally resides in the throat and is one of the most common medical pathogens in the saliva. an asymptomatic carriage stage of s. pyogenes was detected in approximately 10% of adults and 25% of children, and in as many as 60% of subjects during large outbreaks of streptococcal pharyngotonsillitis (178) . beta-hemolytic group a streptococci were found in 20% of pharyngeal samples and in 5% of saliva samples of young schoolchildren in new zealand, with a suggestion of a child-to-child transmission of the organism (185) . members in the same household of a patient with pharyngotonsillitis frequently harbor the same strain of beta-hemolytic group a streptococcus, indicating an intrafamilial transmission of the bacterium (58) . haemophilus influenzae can cause acute bronchitis and exacerbations of chronic obstructive pulmonary disease, as well as meningitis in children and other serious diseases (124) . despite the availability of highly effective vaccines since the early 1990s, 100,000s of unvaccinated children die every year from h. influenzae-related disease (208) . the organism resides in the pharynx and is rarely recovered from the saliva of healthy individuals (88) . it can reach quantities of 10 3 -10 8 ⁄ ml in the sputum of patients with lower respiratory tract infections and purulent sputum (61) . staphylococcus spp., pseudomonas spp. and acinetobacter spp. are also potential pathogens in respiratory (and other) diseases. these bacteria were detected in the oral cavity of 85% of hospitalized patients in brazil (216) and in subgingival sites of periodontitis patients in the usa (145, 174) . periodontal staphylococci occurred with highest proportions in younger individuals, and periodontal gram-negative bacilli were found mostly in older subjects (174) . staphylococci can also be prominent in the microbiota of failing dental implants (78) . gram-negative bacilli are frequent inhabitants of the oral cavity of individuals in developing countries, where the bacteria are probably acquired through contaminated potable water (8, 79, 175) . meningococcal invasive disease (septicemia and ⁄ or meningitis in association with hemorrhagic rash) is a life-threatening condition that primarily affects young children. meningococcal disease can also occur in teenagers, and is more common in collage ⁄ university students than in the general population (or 3.4) (190) . although neisseria meningitidis resides in the nasopharynx and in the tonsils, and is much less common in saliva (129), intimate kissing, especially with multiple partners, constitutes a risk factor for meningococcal disease (or 3.7) (190) . fortunately, the prevalence of meningitis caused by n. meningitidis, h. influenzae type b and streptococcus pneumoniae has decreased markedly after the introduction of vaccines against these bacteria (89) . neisseria gonorrhoeae (which causes gonorrhea) and treponema pallidum (which causes syphilis) can produce acute and chronic oral infections. gonorrhea is a widespread disease worldwide, with an estimated 600,000 new cases each year in the usa (103) . although oral gonorrhea is relatively rare, the literature describes more than 500 cases of oropharyngeal gonorrhea (20) . syphilis is re-emerging in many countries, especially in hiv-infected individuals and among men who have sex with men, and oral sex is often reported to be the route of t. pallidum transmission (37, 168, 198) . infants have contracted syphilis by the mouth-to-mouth transfer of prechewed food from actively infected relatives (215) . dentists can play an important role in the control of sexually transmitted diseases by identifying signs and symptoms of gonorrhea and syphilis and making appropriate referrals for treatment. tuberculosis remains a serious disease worldwide (68) . in 2005, there were an estimated 8.8 million new cases of tuberculosis, with 7.4 million occurring in asia and sub-saharan africa, and 1.6 million people died of tuberculosis, including 195,000 with hiv infection (114) . mycobacterium tuberculosis can be identified in the whole saliva of almost all tuberculosis patients (54) and of some nontuberculous individuals (101) , and has been recovered from alginate dental impressions (140) . the us centers for disease control has identified the personnel of a dental-care facility to be at increased risk for infection with m. tuberculosis (35) and has updated the tuberculosis infection control guidelines for dental clinics (40) . helicobacter pylori can cause gastritis, peptic ulcers and gastric adenocarcinoma (143) . the organism resides primarily in the human stomach and may colonize about 50% of the worldõs population. large quantities of h. pylori can be recovered from vomitus, and the bacterium can also be detected in saliva, especially in subjects suffering from gastric ulcer. however, published data on the occurrence of h. pylori in the mouth vary greatly (143) , perhaps because the oral carriage of h. pylori is population dependent or is only transient (50) . the transmission route is mainly from the mother, or an older sibling, to younger children. both gastro-to-oral and oral-tooral transmission are considered important. legionella pneumophila is the cause of legionellosis (legionnaireõs disease), a severe type of pneumonia with multisystem failure, and of pontiac fever, a self-limiting influenza-like illness (114) . the natural reservoir for l. pneumophila and other legionella species is aquatic habitats. legionellae have been isolated from sputum and other body fluids and sites (123) . l. pneumophila has also been recovered from dental unit water in england, germany and austria (184) , and from 8% of dental units in the usa (18) . however, no evidence exists to incriminate dental units as a significant source of legionellosis. herpesvirus species comprise the most prevalent viral family in human saliva and are important periodontopathic agents (173) . eight herpesvirus species, with distinct biological and clinical characteristics, can infect humans: herpes simplex virus-1 and -2, varicella-zoster virus, epstein-barr virus, human cytomegalovirus, human herpesvirus-6, human herpesvirus-7 and human herpesvirus-8 (kaposiõs sarcoma virus) (171) . herpesviruses establish a lifelong persistent infection, and some herpesvirus species infect as many as 90% of the adult population. the clinical outcome of a herpesvirus infection ranges from subclinical or mild disease to encephalitis, pneumonia and various types of cancer. herpesviral infections in the oral cavity may give rise to asymptomatic and unrecognized shedding of virions into saliva, or to diseases of the oral mucosa or the periodontium (171, 179) . a recent article reviewed acute herpesviral infections in the oral cavity of children (156) . herpesviruses exhibit a biphasic infection cycle involving a lytic, replicative (ôproductiveõ) phase and a latent, nonproductive phase (171) . the replicative phase involves expression of viral regulatory and structural proteins, and the formation of infectious virion particles (172) . the ability to switch between replicative and latent states ensures viral transmissibility between individuals as well as a permanent infection of the host. following the initial infection, herpesviruses preferentially exist in a state of latency in sensory ganglion cells (herpes simplex viruses and varicella-zoster virus), b-lymphocytes (epstein-barr virus, herpesvirus-8), or monocytes and t-lymphocytes (cytomegalovirus and herpesviruses-6 and -7). herpesvirus conversion from a latent form to lytic replication can occur spontaneously or be caused by environmental stimuli, chemical agents and physical and psychosocial stress events, as found in adults with an abusive early-childhood history, astronauts in space flight, students before important academic exams, elite athletes in intensive training and subjects with work-related fatigue ( table 1 ). reactivation of an oral herpesviral infection can be estimated by a rise in herpesvirus salivary counts or a significant increase in herpesvirus-specific salivary antibodies. immunocompetent individuals usually experience herpesvirus re-activation lasting for only a few hours or days (112) , which is probably too short a time period to initiate or exacerbate clinical disease. however, the egress of herpesvirus virions into saliva poses a risk for infecting individuals in intimate contact. by contrast, immunosuppressive conditions ⁄ diseases and long-term medications may result in the re-activation of oral herpesviruses that continues for an extended period of time and may pose a pathogenetic risk for the infected individual. the immune system of older persons may fail to control a latent varicella-zoster infection, resulting in herpes zoster outbreaks (29), or may not protect effectively against epstein-barr virus and cytomegalovirus re-activation (180) . the herpesvirus infection in such persons may be characterized as chronically re-activated instead of latent. the great majority of systemically healthy adults continually shed herpesvirus dna into saliva. herpes simplex virus-1 dna was detected in saliva in quantities up to 2.0-2.8 · 10 6 ⁄ ml (102, 118) . epstein-barr virus dna copies in saliva can reach levels of 10 8 ⁄ ml (76), 1.6 · 10 9 ⁄ ml (155), 7.1 · 10 5 ⁄ ml (181) and 2.2 · 10 6 per 0.5 lg of dna (202) . as the epstein-barr virus salivary count only decreased moderately after large-volume mouth gargles and rinses, or after normal swallowing every 2 min, a large quantity of the virus must constantly enter the saliva (76) . however, the salivary epstein-barr virus load can vary by as much as 4-5 logs over the course of several months, which complicates the categorizing of (76) . cytomegalovirus dna was detected in the saliva of 61% of immunocompetent and immunocompromised subjects (65) , and could reach salivary dna copy counts of 4.2 · 10 4 ⁄ ml (155) . herpesvirus-6 and herpesvirus-7 may occur in saliva, with prevalences exceeding 95% and in quantities of several million dna copies ⁄ ml (118) . salivary herpesvirus-8 dna, in quantities of 2.0-7.3 log 10 copies ⁄ ml, was detected in 61% of asymptomatic, immunocompetent men who have sex with men (32) , and in 37% of zimbabwean women with kaposiõs sarcoma, but not in women without the disease (99) . varicella-zoster virus dna is present at a low prevalence and in quantities of <1,100 copies ⁄ ml in the saliva of both healthy and hiv-infected individuals (205) . table 2 shows the association between salivary herpesviruses and periodontitis. a periodontal dual infection of herpesviruses and pathogenic bacteria gives rise to enhanced cytokine release and immune signaling dysregulation (27, 104, 187) , and tends to be associated with more severe periodontitis than a periodontal infection involving solely bacteria (172) . herpes simplex virus-1 may contribute to periodontitis in a subset of individuals (173) , and the virus was identified in whole saliva of 24% of patients with chronic periodontitis (71) . in the same group of patients, herpes simplex virus-1 dna was present in 16% of subgingival samples and in 8% of peripheral blood samples (71) . herpes simplex virus dna was found in the saliva of 84% of patients with overt herpetic lesions (144) . epstein-barr virus dna has been detected in whole saliva of 79% of periodontitis patients and 33% of gingivitis patients (155) , and in 49% of periodontitis patients and 15% of healthy individuals (82) . a correlation was found between salivary and subgingival levels of epstein-barr virus in one study (48) but not in another study (84) . as high quantities of salivary epstein-barr virus dna can be recovered from fully edentulous patients (155) , the occurrence of the virus in saliva may not be a reliable indicator of its subgingival level or of the periodontitis disease status. cytomegalovirus periodontal active infection is closely linked to aggressive periodontitis (173) . cytomegalovirus dna was detected in the saliva of 50% of periodontitis patients, but was not found in the saliva of gingivitis patients or complete denture wearers, suggesting that salivary cytomegalovirus originates mainly from periodontitis lesions (155) . also, cytomegalovirus dna from infected breast milk appeared in the saliva of infants at 4 months of age, peaked 4-10 months after birth, and thereafter decreased or became undetectable (122) . to sum up, a great proportion of salivary herpeviruses are shed from periodontal disease sites. as periodontal treatment can markedly reduce subgingival (73, 162) and salivary (82, 162) herpesvirus dna counts, the establishment of a healthy periodontium may diminish the risk of intersubject herpesvirus transmission and of herpesvirus-related diseases. the close relationship between some herpesvirus species and periodontitis also argues for examining the potential of using herpesvirus salivary counts to indicate periodontal disease risk. infectious mononucleosis is caused by a primary infection with epstein-barr virus, and predominantly by epstein-barr virus type 1 (44) . approximately 10% of mononucleosis-like disease is attributable to cytomegalovirus. the epstein-barr virus infects b-lymphocytes, which gives rise to the strong t-lymphocyte response that is characteristic of mononucleosis. clinical signs of infectious mononucleosis are long-lasting fever, tonsillopharyngitis, lymphadenopathy, fatigue, and occasionally splenomegaly, liver involvement and pericarditis (199) . oral signs are sore throat, palatal petechiae and enlarged lymph nodes in the throat and neck. the epstein-barr virus is transmitted through direct contact with virus-infected saliva, such as with kissing, and rarely via the air or blood. young adults with a primary epstein-barr virus infection can rapidly clear the virus from the blood but not from the oropharynx (19) . however, individuals who are already infected with the epstein-barr virus (and cytomegalovirus) are not at risk for infectious mononucleosis, even when exposed to individuals with the disease. other diseases have been linked to salivary herpesviruses (table 2) . relationships have been found between bellõs palsy (idiopathic peripheral facial paralysis) and an active herpes simplex virus-1 infection (3), between oropharyngeal lesions of the ramsay hunt syndrome and varicella-zoster virus (62, 144) , and between hiv infection and epstein-barr virus (74) and herpesvirus-8 (33) . young children with exanthem subitum acquired the disease from their mothers who excreted the causative herpesvirus-6 into saliva (121) . human immunodeficiency virus infection is a potent herpesvirus re-activator, as demonstrated by a strong correlation between decreasing cd4 cell counts in hiv-infected patients and increasing rates of herpesvirus re-activation (34) . an hiv infection is frequently associated with the salivary presence of several re-activated herpesvirus species (table 3 ). in the mode of synergism, herpesviruses (196) , p. gingivalis (83) and other periodontal bacteria (81) may resides in the buccal epithelial cells of hiv-infected subjects (134) , and can be transmitted horizontally from an hiv-infected mother to her young children (66, 111) but, despite the possibility of in utero infection (28) , vertical transmission of the virus is uncommon in infants born to an hiv-positive mother (111) . herpesvirus-8 can also be transmitted by oral sex. deep kissing was an independent risk factor (odds ratio of 5.4) for transmitting herpesvirus-8 from hiv-seropositive men to hiv-seronegative men, (134) . taken together, the saliva of hiv-infected persons is a risk factor for the transmission of several virulent herpesvirus species, and patients receiving haart cannot be assumed to be less infectious for herpesviruses than individuals not receiving haart. oral mucositis is an important complication of immunosuppressive radiotherapy, chemotherapy and radiochemotherapy (163) . the mucositis may involve herpesviruses, bacteria and yeasts, individually or in combination (163) . bone marrow and stem cell transplantation has been associated with oral cytomegalovirus re-activation (148) , and renal allograft transplantation has been associated with oral cytomegalovirus re-activation (128) and oral herpesvirus-8 re-activation (9) . also, although not studied in the oral cavity, corticosteroid immunosuppressive treatment may trigger the re-activation of herpesvirus species (14, 49, 164, 211) . viruses of serious medical diseases can be present in saliva at levels sufficient to be transmitted from person to person through close (within 2 meter) or intimate contact (table 4) . moreover, viral pathogens can be transferred to humans by animals or insects (table 4) , or from humans to animals and then later transferred back into humans (56) . viruses in saliva may infect the periodontium and exacerbate periodontal disease. human papillomaviruses are frequent inhabitants of the oral mucosa of normal adults (188) and have been found to occur in the saliva of 25% of healthy individuals (154) . papillomavirus dna was detected in 26% of gingival biopsies from periodontitis lesions (80) , and in as many as 92% of biopsies of cyclosporin-induced gingival hyperplasia from renal transplant recipients (30) . papillomavirus type 16 is associated with a subset of oropharyngeal squamous cell carcinomas (171) , and quantitative measurement of salivary papillomavirus-16 dna has shown promise for early detection of recurrence of head and neck squamous cell carcinoma (39) , and for surveillance of premalignant oral disorders (183) . papillomavirus dna was identified in the saliva of 10% (5) and 41% (154) of oral squamous cell carcinoma patients, and in the saliva of 35% of hiv-positive individuals (5) . a spouse had a 10-fold higher risk of acquiring a persistent oral papillomavirus infection if the other spouse had a persistent oral papillomavirus infection, a finding that is consistent with the oral route of papillomavirus transmission (149) . the likelihood of contracting an oral papillomavirus infection increases with increasing numbers of open-mouthed kissing partners and oral sex partners (52) , and papillomavirus-positive oral tumors are strongly linked to multiple oral sex partners (53) . the current prophylactic papillomavirus-6 ⁄ 11 ⁄ 16 ⁄ 18 vaccine, designed to prevent cervical cancer, generates an oral antibody response and will probably also reduce the incidence of papillomavirus-related diseases of the mouth (153) . human immunodeficiency virus is transmitted through sexual contact or by contaminated needles and blood, but only exceptionally rarely through saliva. a recent study provided compelling evidence that three infants acquired hiv ⁄ acquired immunedeficiency syndrome (aids) after receiving prechewed food (64) . the hiv-infected caregivers had bleeding gingiva while masticating food for the infants, and thus blood, not saliva, was probably the vehicle for hiv transmission in the three cases reported. in fact, submandibular ⁄ sublingual gland secretions contain mucin molecules that normally will prevent infection and transmission of hiv by the oral route (75) . thus, as is the case for hiv and for other viruses, saliva is not merely serving as a passive transport medium, but can significantly affect the efficiency of pathogen transmission and the course of disease. fortunately, anti-retroviral drugs have turned hiv infection into a manageable condition with a greatly reduced morbidity. the proviral dna of human t-cell lymphotropic virus type i, an oncogenic retrovirus, was detected in whole saliva of 77% of mashhadi-born iranian jews with viral myelopathy (4) . this finding may suggest the potential for a salivary transmission of human t-cell lymphotropic virus type i and may possibly help to explain the relatively high rate of myelopathy in the elderly mashhadi-jewish population. the human t-cell lymphotropic virus type i can also be present in the saliva of asymptomatic carriers of the virus (4). hepatitis viruses (designated a through g) cause the majority of cases of acute and chronic hepatitis and liver damage worldwide. hepatitis ranges pathologically from asymptomatic or mild disease to fulminant liver failure. hepatitis a and hepatitis e viruses are transmitted by water contaminated with feces (fecal-oral route), produce acute infections and do not induce a chronic carrier state. a high incidence of hepatitis a and hepatitis e viral infections occurs in countries with poor sanitary standards. hepatitis a virus rna was detected in the saliva of 50% of patients during a hepatitis a outbreak (11) . a study in cynomolgus monkeys found that the tonsils and salivary glands acted as extrahepatic sites for early hepatitis a virus replication and constituted potential sources for saliva-transmitted infection (10) . hepatitis b virus is parenterally transmitted and is frequently associated with chronic viremia. hepatitis b virus dna was found at concentrations of >10 5 copies ⁄ ml of saliva in 15% of patients with chronic hepatitis b (195) . that concentration may be sufficient to permit horizontal transmission of the virus, and perhaps some of the 20% of hepatitis b patients, who contract the disease without a known origin of the infection, may have acquired the hepatitis b virus by salivary transfer (195) . chronic hepatitis c affects more than 170 million people worldwide, and the hepatitis c virus persists in 80% of the infected individuals, where it can give rise to liver inflammation, liver cirrhosis and hepatocellular carcinoma (135) , and perhaps to periodontitis, sjögrenõs syndrome, oral lichen planus and sialadenitis (171) . hepatitis c virus rna was present in the saliva of 39-72% of subjects with chronic hepatitis (113, 133, 144, 204) , and was detected in 59% of gingival crevice fluid specimens from viremic patients (113) . the gingival crevice fluid was identified as the major source for salivary hepatitis c virus (113) . twenty-seven percent of spouses of individuals with chronic hepatitis c revealed antibodies against three hiv-positive infants (9-39 months old) were fed with premasticated food: two children by an hiv-infected mother with oral bleeding; and one child by an hiv-positive aunt (the mother was hiv-negative) the infants were not breastfed and perinatal transmission of hiv was previously ruled out. viruses of respiratory diseases are usually transmitted through coughs or sneezes that release large quantities of high-velocity droplets into the air, and the risk of cross-infection through salivary exchange is comparatively small. children with respiratory disease revealed respiratory viruses (respiratory syncytial virus, influenza virus, parainfluenza virus, adenovirus) in 74% of oral specimens and in 77% of nasopharyngeal specimens (150) , and respiratory syncytial virus rna in 76% of salivary samples (201) . although present in saliva (150) , influenza virions may not be infectious because of the anti-influenza virus activity of salivary glycoproteins (207) . the severe acute respiratory syndrome (sars) corona virus, the etiological agent of a highly lethal type of pneumonia, was detected in the saliva of each sars patient studied, and was present in quantities up to 6.38 · 10 8 copies ⁄ ml (203) . a dental clinic located in a sars-affected region must institute strict infectioncontrol measures in order to prevent cross-infection with the sars virus (158) . measles and rubella are rare diseases in vaccinated populations, but still occur in unvaccinated persons, commonly in developing countries. the causative viruses are spread through respiration, and can be present in saliva in high numbers during disease outbreaks (2, 126) . ebola is a viral hemorrhagic febrile disease that can cause death in 2-5 days. ebola virus was detected in the saliva of all 24 patients with a positive ebola diagnosis (60) , and transmission of the ebola virus through oral exposure has been demonstrated in nonhuman primates (85) . merkel cell carcinoma is a highly lethal neuroepithelial tumor of the skin, and at least some merkel cell carcinomas appear to be caused by a newly discovered polyomavirus. the merkel cell polyomavirus is found in relatively high numbers in respiratory secretions and in the saliva of patients with merkel cell carcinoma (107) , possibly exposing close individuals to a risk of infection. humans can contract serious viral diseases through zoonotic transfer ( table 4 ). the rabies virus resides in dogs, foxes, cats, vampire bats and other animals, and is transmitted to humans through the bite of a rabid animal. rabies virus rna was identified in 88% of salivary samples from humans with an ante-mortem diagnosis of rabies (125) . hantaviruses cause hemorrhagic fever with renal syndrome (in eurasia) or cardiopulmonary syndrome (in the americas), and rodent-to-human transmission usually occurs by the inhalation of aerozolized viruscontaminated rodent excreta. however, the andes hantavirus infects the secretory cells of human salivary glands and can be detected in the saliva after onset of disease symptoms, suggesting that the virus also may be transmitted by human-to-human contact (77, 137) . nipah virus, a paramyxovirus with a reservoir in fruit bats, can cause respiratory disease and severe encephalitis in humans. a study in bangladesh concluded that 50% of nipah patients acquired the virus through salivary transmission from person to person (108) . some viral diseases in tropical and subtropical parts of the world are acquired through insect bites. dengue fever, caused by a mosquito-borne flavivirus, afflicts more than 100 million subjects annually. patients with dengue fever revealed the dengue virus genome in saliva during the acute phase of the infection (120, 141) . crimean-congo hemorrhagic fever virus is transmitted by tick bites or by contact with the blood or tissues of infected patients and livestock. the genome of the crimean-congo hemorrhagic fever virus was detected in the saliva of five of six patients with confirmed disease (24) , increasing the likelihood of a human-to-human transmission. our knowledge of infectious agents in the human oral cavity has expanded greatly in recent years, mainly as a result of molecular techniques that can identify and quantify oral bacteria and viruses with great accuracy. several oral and medical pathogens occur in saliva at levels that are sufficient to infect close individuals, and contact with saliva may be a more important mode of pathogen transmission than previously realized. the rising awareness of the infectious potential of saliva raises challenging questions about the safety of intimate (ôdeepõ or ôopen mouthedõ) kissing contact. the risk of cross-infection by salivary transfer may not be trivial and needs to be studied further. the type of pathogenic agents that can retain infectiousness in saliva and that are efficiently spread by saliva needs to be identified and controlled. current knowledge of the oral ecology may form the basis for more efficient treatments of bacterial and viral infections around teeth and of the oral mucosa. the finding of major periodontopathic bacteria in nondental sites, especially on the tongue, argues for antimicrobial treatment of the entire oral cavity, not only of dental biofilms (151) . virtually all periodontal patients can benefit from treatment with antiseptics active against bacteria and herpesviruses, such as sodium hypochlorite and povidone-iodine (169) , and selective patients may benefit from treatment with systemic antibacterial (170) and antiviral (182) medications. effective periodontal therapy includes professional administration of a battery of well-tolerated antimicrobial agents, each exhibiting high activity against periodontal pathogens and delivered in ways that simultaneously affect pathogens residing in different oral ecological niches [i.e. chlorhexidine or dilute sodium hypochlorite (bleach) for general oral disinfection, povidone-iodine for subgingival irrigation, and systemic antibiotics to reach microorganisms within periodontal tissue and in difficult-to-reach subgingival and extra-dental sites]. the follow-up maintenance program should have a strong anti-infective emphasis, and may include patient-administered subgingival irrigation with dilute sodium hypochlorite and oral rinsing with sodium hypochlorite or chlorhexidine two to three times per week. full-mouth disinfection may also reduce the risk for cross-infection of oral pathogens between individuals in close contact. however, in the final analysis, most chronic infectious diseases such as periodontitis and dental caries will be defeated on a mass-scale only by employing effective, safe and inexpensive vaccines. vaccines may be prophylactic, therapeutic, or a combination of both. perhaps a vaccine that reduces the infectious load without actually eliminating the infectious agent is sufficient to arrest or prevent dental and other oral diseases. vaccination studies on herpesviruses and some oral bacteria have yielded occasional successes in animal models, but a number of human trials have failed to show adequate efficacy. vaccine development has been difficult because of the heterogeneity, variability and poor immunogenicity of the outer surface components of many infectious agents. nonetheless, despite the setbacks, vaccines against herpes zoster virus and oncogenic papillomaviruses were recently approved for clinical use by the us food and drug administration. effective and safe vaccines against oral infectious diseases constitute one of the most important needs in dentistry. bacteria of dental caries in primary and permanent teeth in children and young adults confirmation of rubella within 4 days of 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hiv-1 seropositive persons the role of crude human saliva and purified salivary muc5b and muc7 mucins in the inhibition of human immunodeficiency virus type 1 in an inhibition assay the dynamics of ebv shedding implicate a central role for epithelial cells in amplifying viral output sensitivity of andes hantavirus to antiviral effect of human saliva comparative biology of chronic and aggressive periodontitis vs. peri-implantitis subgingival microbial profiles in chronic periodontitis patients from chile, colombia and spain marginal periodontium as a potential reservoir of human papillomavirus in oral mucosa oral bacteria induce a differential activation of hiv-1 promoter in t cells, macrophages, and dendritic cells detection of epstein-barr virus in saliva by real-time pcr reactivation of latent hiv-1 infection by the periodontopathic bacterium porphyromonas gingivalis involves histone modification detection of epstein-barr virus and human cytomegalovirus in blood and oral samples: 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study of transmission of mutans streptococci detection of kaposiõs sarcoma-associated herpesvirus in oral and genital secretions of zimbabwean women demonstration of mother-to-child transmission of streptococcus mutans using multilocus sequence typing detection of hepatitis b virus and mycobacterium tuberculosis in korean dental patients asymptomatically shed recombinant herpes simplex virus type 1 strains detected in saliva gonorrhea: update. oral surg oral med oral pathol oral radiol endod cytokine responses against periodontal infection: protective and destructive roles hepatitis c-contamination of toothbrushes: myth or reality? topical antimicrobial therapy in the prevention of early childhood caries quantitative detection of merkel cell virus in human tissues and possible mode of transmission transmission of human infection with nipah virus epstein-barr virus (ebv) dna in saliva and ebv serology of hiv-1-infected persons with and without hairy leukoplakia shedding of cytomegalovirus and herpesviruses 6, 7 and 8 in saliva of human immunodeficiency virus type 1-infected patients and healthy controls evidence for horizontal and not vertical transmission of human herpesvirus 8 in children born to human immunodeficiency virus-infected mothers rapidly cleared episodes of herpes simplex virus reactivation in immunocompetent adults detection of hepatitis c virus rna from gingival crevicular fluid and its relation to virus presence in saliva respiratory tract infections and pneumonia stress-induced subclinical reactivation of varicella zoster virus in astronauts epstein-barr virus reactivation associated with diminished cell-mediated immunity in antarctic expeditioners dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6-to 36-month-old children effect of prophylactic valacyclovir on the presence of human herpesvirus dna in saliva of healthy individuals after dental treatment high prevalence of multiple human 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meningococcal transmission detection of multiple pathogenic species in saliva is associated with periodontal infection in adults the microbiota on different oral surfaces in healthy children molecular microbial diagnosis detection of hepatitis c virus-rna in saliva from chronically hcv-infected patients mucosal shedding of human herpesvirus 8 in men pathophysiology of hepatitis c virus infection and related liver disease incidence of epstein-barr virus in astronaut saliva during spaceflight hantavirus rna in saliva from patients with hemorrhagic fever with renal syndrome epstein-barr virus shedding by astronauts during space flight clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary epstein-barr virus infection in children incidence of viable mycobacteria tuberculosis on alginate impressions in patients with positive sputum detection of dengue virus in saliva and urine by real time rt-pcr bacterial profiles of root caries in elderly patients infections of the esophagus and the stomach reliable detection and quantitation of viral nucleic acids in oral fluid: liquid phase-based sample collection in conjunction with automated and standardized molecular assays staphylococci in human periodontal diseases giannobile wv. identification of pathogen and host-response markers correlated with periodontal disease quantitation of hepatitis c virus rna in saliva and serum of patients coinfected with hcv and human immunodeficiency virus quantitative determination of cmv-dna in saliva of patients with bone marrow and stem cell transplantation using taqman-pcr natural history of oral human papillomasvirus infections in female and male partners: a prospective finnish hpv family study use of throat swab or saliva specimens for detection of respiratory viruses in children the use of pvp-iodine as an adjunct to nonsurgical treatment of chronic periodontitis quantitative studies on the salivary flora antibody responses in oral fluid after administration of prophylactic human papillomavirus vaccines human papillomavirus in saliva of patients with oral squamous cell carcinoma periodontitis lesions are the main source of salivary cytomegalovirus oral viral infections of children oral mucosal fungal infections severe acute respiratory syndrome and dentistry: a retrospective view epstein-barr virus specific salivary antibodies as related to stress caused by examinations human cytomegalovirus salivary antibodies as related to stress academic stress, immunological reaction, and academic performance among students of nursing and physiotherapy periodontitis lesions are a source of salivary cytomegalovirus and epstein-barr virus oral mucositis: a challenging complication of radiotherapy, chemotherapy, and radiochemotherapy: part 1, pathogenesis and prophylaxis of mucositis reactivation of human herpesvirus (hhv) family members other than hhv-6 in drug-induced hypersensitivity syndrome microbial testing in periodontics: value, limitations and future directions investigating the concurrent presence of hcv in serum, oral fluid and urine samples from chronic hcv patients in faisalabad early childhood stress is associated with elevated antibody levels to herpes simplex virus type 1 the re-emergence of syphilis in the united kingdom: the new epidemic phases selection of antimicrobial agents in periodontal therapy systemic antibiotics in periodontics oral viral infections of adults herpesviral-bacterial interactions in periodontal diseases human viruses in periodontitis age and sex relationships of superinfecting microorganisms in periodontitis patients subgingival microflora of advanced periodontitis in the dominican republic actinobacillus actinomycetemcomitans in human periodontal disease: a crosssectional microbiological investigation genetic and environmental risk factors for chronic periodontitis and aggressive periodontitis oral herpetic infections (hsv 1-8) chronic herpesvirus reactivation occurs in aging relationship between porphyromonas gingivalis, epstein-barr virus infection and reactivation in periodontitis patient with severe periodontitis and subgingival epstein-barr virus treated with antiviral therapy progressive increase of human papillomavirus carriage rates in potentially malignant and malignant oral disorders with increasing malignant potential risk of exposure to legionella in dental practice a longitudinal study of lancefield group a streptococcus acquisitions by a group of young dunedin schoolchildren similarity of the oral microbiota of pre-school children with that of their caregivers in a population-based study cytokine regulation of immune responses to porphyromonas gingivalis high prevalence of human papillomaviruses in the normal oral cavity of adults within-subject variability in repeated measures of salivary analytes in healthy adults risk and protective factors for meningococcal disease in adolescents: matched cohort study cytokine secretion and latent herpes virus reactivation with 28 days of horizontal hypokinesia risk indicators for harboring periodontal pathogens the utility of whole saliva to detect the oral presence of periodontopathic bacteria do periodontopathogens disappear after full-mouth tooth extraction? paired, quantitative measurements of hepatitis b virus dna in saliva, urine and serum of chronic hepatitis b patients herpes simplex virus and hiv-1: deciphering viral synergy transmission of periodontal bacteria and models of infection syphilis epidemiology in sweden: re-emergence since 2000 primarily due to spread among men who have sex with men infectious mononucleosis and epstein-barr virus occurrence of aggregatibacter actinomycetemcomitans in brazilian indians from umutina reservation, mato grosso, brazil excretion patterns of human metapneumovirus and respiratory syncytial virus among young children multiple epstein-barr virus infections in healthy individuals detection of sars-associated coronavirus in throat wash and saliva in early diagnosis high serum hepatitis c virus (hcv) rna load predicts the presence of hcv rna in saliva from individuals with chronic and acute hcv infection low prevalence of varicella zoster virus and herpes simplex virus type 2 in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral therapy. oral surg oral med oral pathol oral radiol endod post-marketing surveillance team. post-marketing surveillance of oraquick whole blood and oral fluid rapid hiv testing multiple components contribute to ability of saliva to inhibit influenza viruses haemophilus influenzae type b (hib) salivary diagnostics powered by nanotechnologies, proteomics and genomics candida biofilms and oral candidosis: treatment and prevention disseminated varicella infection in a child receiving short-term steroids for asthma taqman real-time quantification of epstein-barr virus in severe early childhood caries actinobacillus actinomycetemcomitans in human periodontal disease. prevalence in patient groups and distribution of biotypes and serotypes within families the clinical value of salivary biomarkers for periodontal disease nonvenereal transmission of syphilis in infancy by mouth-to-mouth transfer of prechewed food prevalence of potential bacterial respiratory pathogens in the oral cavity of hospitalised individuals key: cord-336117-hit4kza8 authors: heymann, d.l.; reinhardt, k. title: emerging infections, the international health regulations, and macro-economy date: 2014-02-27 journal: encyclopedia of health economics doi: 10.1016/b978-0-12-375678-7.00624-6 sha: doc_id: 336117 cord_uid: hit4kza8 when breaches in the species barrier between animals and humans result in crossover of animal infections to humans, the result is an emerging infectious disease. emerging infections have the potential to cause severe human illness and death, and to spread among human populations and across geographic borders. direct costs associated with emerging infections are often high, caused by prolonged patient management, hospitalization, and convalescence. indirect costs are caused by absenteeism from work, death, and permanent removal from the workforce. there may be additional costs if precautionary prevention measures are required, based on the known or potential risk. these costs result from culling and prohibited trade in animals and animal-based products if infected animals are a continuing threat to humans; or from postponement or cancellation of travel and tourism if transmission in the general population is uncontrolled. emerging infections sometimes cause panic and/or fear because of misunderstanding of the risk, and can lead to actions that result in economic loss similar to that caused by legitimate public health precautionary measures. they include reactions such as avoiding animal-based foods mistakenly thought to be the source of infection; unnecessary cancelling travel plans to avoid geographic areas where there is a perception of risk of infection; and/or unwarranted culling of animals or destruction of animal-based foods. if the misunderstanding is transmitted widely by the press or through social media networks, the negative economic impact can be amplified. economic loss caused by these perceived risks can generally not be disentangled from the losses causes by public health measures that may be in place, but it is thought that at times they add significantly to the economic burden. the international health regulations (ihr) were designed to respond to the international spread of infectious disease outbreaks in a manner that prevents unnecessary negative economic impacts. agreed by all member states of the world health organization in 2005, they are a legally binding global agreement with the specific purpose of ensuring maximum security against the international spread of diseases, with a minimum interference in world traffic and trade. they reflect current understanding of infectious disease risks, especially those that emerge at the animal/human interface; the realization that infectious diseases cannot be stopped from entering countries at borders; the vast increase in access to information through the internet; and globalization that increases the speed with which infectious diseases spread. under the ihr, countries are able to work transparently with who and its scientific experts and collaborating laboratories to conduct joint risk assessments of public health events such as outbreaks of infectious diseases; to make evidence-based recommendations to help prevent or control their international spread; and, by providing valid and transparent information to national focal points, to help prevent unnecessary panic and misunderstanding about risk. by the end of 2009, the year in which mexico first reported human infections with the h1n1 influenza a virus that then spread globally to cause a pandemic, 70 715 mexicans had been reported with confirmed h1n1 infection of whom 1316 (b5%) had died. during this same period, though there were no official travel or trade bans from th,1e mexican government or international bodies such as the world health organization (who), mexican tourism and trade in pork decreased both nationally and internationally. temporal decreases in output from the pork industry contributed to a pork trade deficit of an estimated us$27 million, whereas an estimated loss of one million overseas visitors translated into an estimated economic loss of approximately us$2.8 billion. the economic losses related to h1n1 outbreak in mexico were clearly influenced by the unfounded perception by tourists and travel agencies that the risk of becoming infected with h1n1 was somehow greater in mexico than elsewhere, even though the virus had spread throughout the world; and by a misunderstanding among pork trade partners that the pandemic was being amplified by infected pigs, despite the fact that it was human to human transmission, in which swine played no role, that was responsible for the global spread of the pandemic. in other countries, there were official recommendations, apparently based on this same misunderstanding that also caused negative economic impact. in egypt, for example, slaughter of pigs was ordered by the egyptian government early in the pandemic, even though the h1n1 virus had already been demonstrated to be highly transmissible from human to human, and despite the recommendation of the world health organization for animals that culling of pigs was not scientifically justifiable. countries around the world were affected as the h1n1 pandemic spread, and economies suffered. in spain, for example, the direct economic impact of illness from h1n1 influenza on health services utilization, and indirect costs from work absenteeism, for example, has been estimated at h6236.00 per hospitalized patient. in canada, it is estimated that the cost of the increased patient load to hospitals caused by h1n1 between april and december 2009 was canadian$ 200 million. the world bank predicts that a pandemic caused by a different influenza virus, the highly infectious and virulent avian (h5n1) influenza virus, could cost the world economy as much as us$800 billion a year from direct patient costs, and indirect costs from lost lives, travel, and trade. the h5n1 virus is currently continuing to cause disease among poultry, but is only able to infect humans sporadically when they come in contact with infected chickens. as influenza viruses are highly unstable, however, the h5n1 influenza virus could mutate or combine with other influenza viruses circulating in nature to a form that spreads easily from human to human, resulting in an influenza pandemic with much higher mortality than the h1n1 pandemic. to prevent such a scenario, attempts are being made to eliminate the h5n1 virus by culling entire flocks of infected poultry, mainly chickens. this precautionary measure, recommended by the world health organization (who) and the food and agriculture organization, is causing lost revenue and poultry-replacement costs that have been estimated to be in billions of us dollars. emerging infections such as h1n1 and h5n1 influenza are the newly identified infectious diseases in humans caused by viruses that have breached the species barrier between an infected animal and a human. they are by definition new, and sometimes they are called novel infections. as they are new they are poorly understood, and their full potential to cause disease and death in humans is not known. unlike influenza, there are other emerging infections that cause human disease but are unable to spread from human to human. economic cost associated with these infections is due to patient management and decreased work productivity while sick; and if there is death, from the lost years of work. an example is rabies: humans are infected by the bite of a rabiesinfected animal, become sick and die, but do not spread the infection to other humans unless an organ is obtained from them postmortem, and grafted into another human. the direct cost of treating persons exposed to rabies has been estimated (conservatively) to be us$40 in sub-saharan africa and us$49 in asia, a cost that equals 5.8% and 3.9%, respectively, of the average annual per capita gross national income. additional indirect costs attributed to persons with rabies occur because of death and permanent removal from the workforce. it is estimated that the economic impact from rabies each year in the united states is approximately $300 million, where an average of two human infections occur each year. bovine spongiform encephalopathy (bse) is another example of an emerging infection that does not spread from human to human. bse, or mad cow disease, was identified in the united kingdom (uk) during the 1980s. to rid cattle populations of infection, precautionary culling of herds with infected cattle was required. when it was understood that humans could be infected with bse from cattle and cattle products in 1996, culling activity increased, and the economic loss in the uk during the following year was estimated to be us$1.5 billion. countries that had imported cattle from the uk also culled infected herds at a considerable economic loss. another emerging infection -monkeypox virus in the united states -was caused by human contact with infected prairie dogs bought as pets. the prairie dogs had been infected with the monkeypox virus in pet shops by other animals imported from west africa as exotic pets. the outbreak was stopped and there were no deaths. though the overall direct costs for diagnosing and managing illness were not calculated, nor were the costs from the bans on pet sales by pet shops and their furnishers, they were significant to health insurance companies and to the trade in animals as pets. occasionally, an infection emerges at the human/animal interface, is able to spread easily from human to human, and then becomes endemic in human populations with long-term associated economic cost. hiv is one such emerging infection. thought to have crossed the species barrier from nonhuman primates to humans sometime during the early twentieth century, it is spread from human to human mainly by intimate sexual contact. owing to the long, symptom-free incubation period, hiv had already spread throughout the world's population by the time it was first identified in 1981. since then the cumulative economic impact of aids on gdp has been estimated by various economists with a wide range of costs -one of these, the estimated direct costs in 2009 to achieve universal access to treatment and care alone was us$7 billion. an outbreak of an emerging infection, severe acute respiratory syndrome virus, occurred in the guangdong province of china in late 2002. in china, sars spread from infected persons to other family members and to health workers, and from them to others in the community, causing an outbreak associated with severe illness and death. in february 2003, when sars was still unrecognized as a new and emerging infection in china, it crossed the border from the guangdong province to hong kong in a doctor, who had been treating patients with sars. he himself had become sick, and during a one-night stay in a hong kong hotel spread sars to other hotel guests. before they had any major symptoms, infected hotel guests travelled by plane to other asian countries, north america, and europe where they became sick and spread infection to others. sars had never before been seen in humans. there were thus no vaccines, medicines, or predetermined measures that could be used for its control. as the virus continued to spread from human to human, there was concern that like hiv, it would become yet another endemic infection, sustaining itself indefinitely in humans. precautionary measures to prevent international spread of the infection were immediately recommended by the who -it was first recommended that persons who were ill with similar symptoms and contact with geographic areas where outbreaks were occurring defer their travel until they were well. these precautionary measures caused a decrease in international air travel from geographic areas where outbreaks were occurring. concern and panic ensued, however, among populations from other geographic areas as well -clearly demonstrated in a decrease in passenger movements through international airports. the precautionary prevention measures recommending that persons who were ill with sars-like symptoms postpone travel resulted in a decrease of passengers who were ill, but many well passengers perceived the risk of travel as being great. this resulted in a steady decrease in passenger movement, clearly shown in figure 1 passenger movements at the hong kong international airport decreased soon after the outbreak was announced. when sars spread throughout a major housing complex in hong kong, among persons who had not been in contact with each other it was hypothesized that sars might be spreading through an environmental factor such as an insect or water in addition to face to face contact. this led to stronger precautionary recommendations -to postpone or cancel travel to areas where outbreaks of sars were occurring and a human contact could not be identified as a source of infection for each person with sars. when who made this stronger precautionary recommendation on 2 april, a sustained decrease in passenger movement occurred in hong kong throughout the month of april and until 23 may, when the who removed the precautionary travel advisory. overall, hong kong international airport had had an approximate decrease of 70% in passenger movements in april 2003 compared with april 2002, and aircraft movements decreased by an estimated 30%. in april 2003, the number of flights cancelled each day was approximately 164, representing more than 30% of all flights cancelled, and resulting in an estimated loss in landing fees of a minimum of $3.5 million per day. during this same period, income from restaurants, hotels, and retail sales decreased because of panic and misperception of the risk among the hong kong population that resulted in decreased consumer activity. figures 2-4 provide clear examples of the decreases in economic activity that occurred. the sars outbreak caused ended in july 2003, with 8096 reported cases from 37 countries of which 1706 (21%) were fatal. the asian development bank estimated the economic impact of sars at approximately us$18 billion in east asiaaround 0.6% of gross domestic product. however, fortunately recovery was rapid once international spread had been stopped. attempts to limit the international spread of infectious diseases were first recorded in venice in the fourteenth century when quarantine was used to keep ships and individuals at land border crossings in isolation for 40 days in an attempt to stop the spread of plague. quarantine was widely used during the following centuries to attempt to limit the spread of plague and other diseases such as cholera, yellow fever, and smallpox; and during the nineteenth century a series of sanitary conferences within and between europe and the americas focused on these same four diseases demonstrated the concern. in the early twentieth century these sanitary conferences were broadened, under the league of nations, to include all its member states. in 1969, the who member states had agreed to a set of regulations aimed at ensuring the maximum security against the international spread of diseases with a minimum interference with world traffic. the ihr 1969 were revised in 2005, incorporating many of the lessons learned during the sars outbreak, and now ensure broader disease coverage, and in addition require countries to develop core capacities in public health laboratory and epidemiology in order to detect and respond to diseases where and when it occurs, and before it spreads internationally (box 1). several disease threats have occurred since the revision of the ihr, including the h1n1 pandemic in 2009. the risk assessment when h1n1 first emerged was conducted by who and the ihr emergency committee. though who recommendations based on this risk assessment clearly stated that travel and trade should continue as before, irrational trade and travel measures were imposed by several countries as described earlier in this article, and they resulted in the consequent economic losses described. the outbreak of escherichia coli (e. coli) that caused hemolytic-uremic syndrome in germany occurred after the revision of the ihr as well. though the outbreak resulted in an unexpected direct economic burden on the german health system, it also resulted in a severely negative economic impact on the european agricultural sector. initial laboratory testing wrongly suggested that the outbreak was associated with consumption of salad greens and tomatoes imported from various countries neighboring germany, and with consumption of cucumbers imported from spain. once this link was published in the mass media, the market for cucumbers fell and spanish farmers began to experience losses of up to an estimated us$ 200 million per week. polish, dutch, and italian farmers had similar losses, and german vegetable farmers had a drop in real income of 2.8%. at the same time, russia banned vegetable imports from the entire european union (eu), an annual 600 million euro market for eu farmers. as the outbreak investigation continued, however, it became clear that the outbreak was linked to ingestion of bean sprouts from an organic farm in lower at the world assembly in may 2003 based on lessons being learned from the ongoing sars outbreak, a resolution was agreed by member states of the who that helped to speed up the revision of the international health regulations (ihr). the ihr first agreed by the who member states in 1969, and had as a goal maximum prevention of the international spread of infectious diseases with minimal interruption of world traffic and trade. by setting out certain border requirements, and targeting four infectious diseases -cholera, plague, yellow fever, and smallpox (removed after eradication in 1980) -it was hoped that these four diseases could be stopped at international borders. however, countries often did not report these diseases when they occurred because of fear of irrational trade and travel measures and the severe negative economic impact that could occur. in addition, as knowledge about emerging infections grew, it became clear that there were other infectious diseases of equal or greater potential for international spread than those that were covered by the ihr, and a revision of the ihr was begun in the late 1990s. by the time of the sars outbreak, a new way of detecting and responding to infectious disease outbreaks had been developed by who as a precursor to the revision of the ihr, and it was these ways of working that led to the coordinated global response to sars. one of the major lessons learned was that strong national disease detection and response systems were of great importance in order that countries could detect and response to infectious disease outbreaks where and when they occur, thus preventing human suffering and death, and minimizing the risk of international spread. this concept was incorporate into the revised ihr that now required all countries to develop a minimum core public laboratory and epidemiology capacity in order to detect and respond to outbreaks when and where they occur. the revised ihr also continue a requirement for reporting of disease outbreaks, and the requirement has been broadened to reporting all public health events of international concern (pheic) after risk assessment using a decision tree provided in the ihr. however, gives cause for hope that the revised ihr do indeed offer a means of ensuring maximum security against the international spread of infectious diseases, while minimizing interference with travel and trade. reports of persons infected with this newly identified sars-like virus were made to the who from countries treating patients with origins in the kingdom of saudi arabia, qatar, jordan, and the united arab emirates. the initial reports originated at the time of religious pilgrimage for hajj. an irrational response could have caused great confusion and a heavy economic and spiritual loss to pilgrims and to saudi arabia, which has increased its investment each year to provide for the health security of pilgrims. an immediate and transparent risk assessment was made under the framework of the revised ihr, and the risk was then communicated widely. the hajj was unaffected by the reports of the risk assessment, and surveillance of hajj pilgrims for severe respiratory symptoms was conducted during the pilgrimage and after pilgrims had returned to their home country. only time will tell whether the new ways of working and communicating risk under the ihr will continue to help prevent unnecessary panic and confusion when an outbreak occurs and spreads internationally; and prevent the irrational reaction that increases their negative economic impact. e. coli cucumber scare: spain angry at german claims e. coli: russia bans import of eu vegetables erd policy brief no. 42 -potential economic impact of an avian influenza pandemic in asia. asian development bank sars: a global response to an international threat assessing the economic impact of public health emergencies in international concern: the case of sars. globalization, trade and heath working papers series. geneva: world health organization the world bank key: cord-028721-x6f26ahr authors: nistal, manuel; paniagua, ricardo title: non-neoplastic diseases of the testis date: 2020-06-22 journal: urologic surgical pathology doi: 10.1016/b978-0-323-01970-5.50014-2 sha: doc_id: 28721 cord_uid: x6f26ahr nan testicular biopsy 665 infertility and chromosomal anomalies 686 other syndromes associated with hypergonadotropic hypogonadism 692 secondary idiopathic hypogonadism 694 hypogonadism secondary to endocrine gland dysfunction 697 infertility secondary to physical and chemical agents 705 infertility in patients with spinal cord injury 708 orchitis 708 histiocytosis with testicular involvement 712 non-neoplastic diseases of the testis manuel nistal, ricardo paniagua chapter 12 embryology and anatomy of the testis embryology sexual differentiation is the result of complex genetic and endocrine mechanisms that are closely associated with the development of both the genitourinary system and the adrenal glands. formation of the bipotential gonad and, subsequently, of the ovaries and testes, depends on gene expression in both sex and autosomal chromosomes. testes secrete steroid and peptidic hormones that are necessary for the development of inner and outer male genitalia. these hormonal actions are mediated by specifi c receptors that are transcriptional regulators. alteration of these genetic events leads to sexual dimorphism involving the inner and outer genitalia, and can also hinder the development of other organs. 1 chromosomal gender is established at fecundation with formation of an egg with either a 46xy (male) or a 46xx (female) karyotype. each chromosomal constitution initiates a cascade of genetic events leading to the development of female (ovaries) or male (testes) gonads (gonadal gender). hormonal secretions from the ovaries or testes are essential for the development of external genitalia (phenotypic gender). the relationship between the individual and the environment determines the social gender. there are multiple genes involved in the formation of the undifferentiated gonad. the two most important for the proper formation of the bipotential gonad are wt1 (wilms' tumor gene) and nr5a1 ( fig. 12-1 ). wt1 contains 10 exons located on chromosome 11p13, with two alternative splicing loci in introns 5 and 9. intron 9 splicing can lead to the inclusion or exclusion of three amino acids (kts: lysine, threonine and serine), giving rise to kts+ or kts− isoforms. an adequate kts+/kts− balance is crucial for normal expression of the gene. translation of this gene may generate up to 24 isoforms with several zinc-fi nger domains. this gene is expressed mainly in the kidneys and gonads, and mediates the transition from stroma to epithelium and morphogenetic differentiation (inhibits those genes that encode proliferative factors and activates those that enhance epithelial differentiation). wt1 gene anomalies lead to a wide variety of phenotypes; deletions are associated with minimal genitourinary alterations and predisposition to develop wilms' tumor. [2] [3] [4] missense heterozygous mutations give rise to denys-drash syndrome (complete or partial 46xy gonadal dysgenesis, renal disease of early onset with diffuse mesangial sclerosis, and wilms' tumor (omim 19408) ). 5 loss of the kts+ isoform accounts for frasier's syndrome (46xy gonadal dysgenesis, renal disease of late onset and absence of wilms' tumor (omim 136680)). 6 nr5a1 gene product is termed sf-1 (steroidogenic factor 1). the gene has seven exons in chromosome 9q33.3, and is expressed in the urogenital ridge that forms the gonads and adrenal glands. sf-1 promotes the expression of the anti-müllerian hormone (amh) and joins elements that regulate upstream the amh gene. sf-1 is fi rst detected in the developing sertoli cells of sex cords, but later is mainly localized in leydig cells. 7 a heterozygous deletion causes a female phenotype in patients with 46xy, adrenal failure during the fi rst weeks of extrauterine life, persistence of normal müllerian structures, and gonads consisting of poorly differentiated tubules embedded in abundant connective tissue. these patients do not respond to hcg stimulation. 8 in 46xx patients, ovarian development is not modifi ed by sf-1 mutations, and they present with adrenal failure only. 9 lim-1 is another gene involved in the formation of the bipotential gonad and kidneys. it was recently identifi ed in mice that bore homozygous deletions and presented alterations in both organs. 10 fgf-9 (fi broblastic growth factor 9) has also been related to gonadal development. both gonosomal and autosomal genes mediate the progression of the bipotential gonad toward testicular differentiation. the signal is triggered by the sry gene on the distal portion of the short arm of the y chromosome (sexdetermining region of the y chromosome; yp11.3), also called tdf (testis determining factor gene). 11 this gene stimulates the differentiation of sertoli cell precursors and germ cells, is responsible for the production of the anti-müllerian hormone, 12 and regulates other genes of the downstream cascade. these are either activated or inhibited by other genes in such a way that dozens of genes are involved in testicular differentiation. 13 the sry gene contains a single exon that encodes a 204 amino acid protein whose central part (79 amino acids) encodes a dna-binding domain termed hmg (high mobility group). immunohistochemical studies have demonstrated expression of the sry gene in the nuclei of both sertoli cells and germ cells, 14 suggesting that this gene acts in somatic cells of genital ridge and germ cells. sry works with the amh promoter gene and also regulates steroidogenic hormone expression. 15 sry mutations produce pure gonadal dysgenesis (swyer's syndrome) or true hermaphroditism; the karyotype of patients with the male phenotype lacking y chromosome is either 46xx sry+ (80%) or 46xx sry− (20%), and all have male external genitalia, testes, azoospermia and no müllerian structures. some 46xx srypatients have sox-9 duplication. 16 following discovery of the sry gene, the knowledge about genes involved in gonadal formation advanced experimentally with knockout mice and the study of human syndromes. now, there are numerous reported genes (including sox-8, sox-9, dax-1, lhx-9, lim-1 and dmrt-1) that encode associated transcription factors. sox-8 and sox-9 (sryy box 8 and 9 or sry hmg-box gene 9 ) are related to autosomal genes. sox-9 is on chromosome 17q24,3q25,1 and is expressed after sry expression in the same cell type (the pre-sertoli cell). 17 this gene is also essential for the development of the cartilaginous extracellular matrix. in the mouse gonad, sox-9 inhibits testicular development or sertoli cell marker expression, and the gonad acquires an ovarian pattern. 18 sox-9 haploinsufficiency (loss of a functional allele) causes camptomelic dysplasia (a syndrome characterized by abnormal formation of cartilage) and a 46xy constitution with female phenotype, 17, 19 whereas sox-9 duplication results in 46xx patients with male phenotype. 20 sox-8 is other cofactor in amh regulation and acts by protein-protein interaction with sf-1. experimental models show that sox-9 dysfunction results in replacement by sox-8 expression via a feedback mechanism. 21 dax-1 (dosage-sensitive sex-reversal, adrenal hyperplasia, x-linked) gene is involved in the development of testes, ovaries, and adrenal glands. dax-1, on x chromosome, is expressed during ovarian formation and inhibited by sry during testicular formation. duplication of the dax-1 region in xp21 results in 46xy gonadal dysgenesis. 22, 23 conversely, dax-1 mutations decrease gene expression, resulting in absence of adrenal cortex and hypogonadotropic hypogonadism; 10 testicular determination is normal. deletions in chromosomes 9p 24 and 10q 25 are associated with the female phenotype in 46xy individuals. chromosome 9p deletions are also associated with facial malformations, premature closure of the frontal suture, hydronephrosis, and delayed development. deletions of two genes (dmrt1 and dmrt2) on chromosome 9p24.3 may be found in 46xy females. terminal deletions in chromosome 10q are associated with genital malformations, multiple phenotypic anomalies, and mental retardation. in the fourth week of gestation, the urogenital ridges appear as two parallel prominences along the posterior abdominal wall. these give rise to two important pairs of structures: the genital ridges arising from the medial prominences, and the mesonephric ridges from the lateral prominences. the genital ridges are the fi rst primordium of the gonad and stand out as a pair of prominences about the midline. in 30-32-day embryos, each genital ridge is lateral to the aorta and medial to the mesonephric duct ( fig. 12-2) . the celomic epithelium forming the genital ridges grows as cordlike structures to create the primary sex cords. immediately beneath the celomic epithelium there are several mesonephric ductuli and glomeruli ( fig. 12-3) . the origin of the gonadal blastema results from the junction of two cell types: epithelial cells from the celomic epithelium and mesenchymal cells from the mesonephric region, 26,27 although experimental data are confl icting. one of the earliest effects of sry expression is induction of mesonephric cell migration toward the genital ridge. 28, 29 histochemical studies revealed that an early event is also disruption of the celomic epithelium basal lamina, permitting the migration of these epithelial cells inside the gonad. if chromosomal constitution is xy, these cells give rise to sertoli cells. 30 cells derived from the celomic epithelium are recognized by their pale cytoplasm, large size, embryology and anatomy of the testis the rete testis originates from mesonephric remnants of sex cords that are in continuity with the seminiferous cords. the connection between the testis and the mesonephros becomes progressively thinner ( fig. 12-4) . the testis has a round transversal section, and remains located between two suspensory ligaments: the cranial and the caudal, the latter of which gives rise to the gubernaculum. the development of the urogenital tract begins at the stage of the undifferentiated gonad, with the appearance of two different pairs of ducts: the wolffi an and the müllerian. the wolffi an ducts are formed in the mesonephros in the third week of gestation, when the cranial region of the segmented intermediate mesoderm gives rise to 10 pairs of tubules (the nephric tubules) that are metamerically arranged. these tubules form the pronephros. on each side of the body, the tubules converge to form a longitudinal duct that opens in the celomic cavity. in the fourth week, the pronephros disappears and is replaced by another tubular system (derived from the intermediate mesoderm, which is not segmented) that forms the mesonephros. the medial ends of the mesonephric tubules do not open to the celomic cavity but are connected to glomeruli at one end and the wolffi an duct at the other. at the end of the second month of gestation, the mesonephros is replaced by the metanephros or defi nitive kidney. however, in the male, the most caudal mesonephric tubules and the wolffi an duct persist. the former give rise to the ductuli efferentes, and the latter forms the ductus epididymidis, the ductus deferens, the seminal vesicle, and the ejaculatory duct. both müllerian ducts originate from a longitudinal invagination of the celomic epithelium in the anterolateral aspect of the genital ridge. the cranial end of each duct is a funnel that opens in the celomic cavity. each duct runs parallel and lateral to the respective wolffi an duct and, as they pass caudally, the müllerian duct crosses over the wolffi an duct and lies medial to it. finally, the two müllerian ducts fuse into the uterovaginal duct. this elongates caudally up to the posterior and ovoid euchromatic nucleus. the cells of mesonephric origin are darker and have a mesenchymal pattern. initially, the genital ridges are devoid of germ cells. in the third week, primordial germ cells appear in the extraembryonal mesoderm lining the posterior wall of the yolk sac near the allantoic evagination. they are ovoid, measuring 12-14 µm in diameter, and are easily detected histochemically by a high content of alkaline phosphatase. the nuclei are spherical and possess one or two prominent central nucleoli. the cytoplasm contains mitochondria with tubular cristae, lysosomes, microfi laments, lipid inclusions, numerous ribosomes, and abundant glycogen granules. attracted by chemotactic factors, the primordial germ cells migrate along the mesenchyma of the mesentery and reach the genital ridge by 32-35 days. the seminiferous cords arise from the gonadal blastema. 31,32 many germ cells reach the seminiferous cords, but some degenerate during migration. the seminiferous cords are delimited from the stroma by a basement membrane 33 and lose their connection to the celomic epithelium, which reduces its depth to one or two cell layers only. the intercordal mesenchyma, composed chiefl y of cells that migrated from the mesonephric stroma, differentiate later into myoid cells, leydig cells, fi broblasts, and blood vessels. 34 up to the sixth week, the gonads appear similar, although the incipient testes have more numerous blood vessels, more abundant stroma, 35 and a higher total dna content, suggesting more rapid growth. sertoli cells arise from somatic sex cord cells. these cells differentiate at the end of the seventh week from the somatic cells in the cords, develop adherent junctions between them and a basal lamina on the other cord surface, and begin to express amh. 36 in the eighth week, leydig cells differentiate from the intercordal gonadal blastema, 37 and immunohistochemical detection of 3β-hsd is apparently the fi rst step in this process. leydig cell development peaks during the 18th week, and numbers subsequently decrease progressively. 38 aspect of the urogenital sinus, forming the müllerian tubercle. the wolffi an ducts terminate at either side of this tubercle. the remaining structures of the male genital system are derived from the urogenital sinus. epithelium with endodermal origin forms the prostate, the urethra, and the bulbourethral and periurethral glands. the primitive urogenital sinus derives from the cloaca, a structure that appears at the end of the fi rst month and which consists of a dilation of the terminal portion of the primitive posterior intestine. the cloaca is closed by the cloacal membrane. in the third week, mesenchyma proliferates in the outer aspect of the cloacal membrane to form the cloacal folds and the cloacal eminence. in the sixth week, the cloacal folds enlarge to form the genital (or urethral) tubercle. external to the genital folds, another mesenchymal thickening develops into the genital prominences or genital swellings. in the fi fth week, a septum forms, dividing the cloaca into two compartments. the anterior compartment is the primitive urogenital sinus that is covered by the urogenital membrane. the posterior compartment is the anorectal canal, covered by the anal membrane. the primitive urogenital sinus then divides into two new compartments: superior and inferior. the superior compartment is the vesicourethral canal that later forms the urinary bladder and the urethra. the inferior compartment is the defi nitive urogenital sinus that will develop later according to the gender. the development of the male genital system is directly infl uenced by the action of multiple hormones, including anti-müllerian hormone (amh), dihydrotestosterone (derived from testosterone), and the pituitary hormones folliclestimulating hormone (fsh) and luteinizing hormone (lh) . amh (müllerian inhibitory substance; mis), 39 secreted by the sertoli cells, is a glycoprotein polymer consisting of two identical 72 kda subunits linked by disulfi de bonds. [40] [41] [42] it belongs to the tgf-β family and is synthesized as a 560 amino-acid precursor protein with proteolytic cleavage at 109 amino acids from the c terminal. cleavage is necessary to activate the hormone. amh is encoded by a 2.75 kb gene that comprises fi ve exons and is located on the p13.2 region of chromosome 19. amh is secreted by somatic cells only in both sexes: sertoli cells in males and granulosa cells in females. it is detected by 6-7 weeks of gonadal development (8-9 weeks of gestation), probably as soon as germ cells make contact with pre-sertoli cells, a week before the müllerian ducts lose their responsiveness. 43, 44 amh is at high concentration in the second trimester, but drops precipitously in the third trimester. 45 levels rise again during the fi rst year after birth, are detectable during infancy and childhood, and fi nally drop defi nitively to undetectable levels at the onset of puberty. the secreted amount of amh is inversely correlated to the degree of sertoli cell maturation. amh regulation is incompletely understood. its expression is controlled by steroidogenic factor 1 (sf-1), also called ad4bp, 46 which is an orphan nuclear receptor that acts as a transcriptional regulator of all steroidogenic genes. amh regulates sry expression, which in sertoli cells is detected immediately before amh expression. 47 during puberty, amh is negatively regulated by androgens. 48 amh acts on the testis, genital tract, and extragenital structures. it causes involution of the ipsilateral müllerian duct. action begins at the caudal testicular pole and progresses rapidly. in adults, remnants of the müllerian ducts include the appendix testis at the cranial end and the prostatic utricle (verumontanum) at the caudal end. amh also stimulates development of the tunica albuginea, formed by insertion of mesenchyma between the celomic epithelium and primordial sex cords. this mesenchyma is also the origin of collagenized connective tissue, with deposition of collagen fi bers in several layers that parallel the testicular surface. 49 amh also hinders the entry of spermatogonia in meiosis. 50 the best-known function of amh in the extragonadal system is the maturation of fetal lungs. 51 testosterone is synthesized by the leydig cells. these fi rst appear among the sex cords in the eighth week of gestation, and their number increases to 48 million per pair of testes by the 16th week, 52 occupying about 50% of the testicular volume ( fig. 12-6 ). the relative number of leydig cells decreases from the 16th to the 24th week, owing to rapid enlargement of the testis during this period. however, the absolute number of leydig cells remains constant. from the 24th week to birth, the number of leydig cells decreases to 18 million per pair of testes. testosterone synthesis begins after the 56th day of gestation. testosterone secretion is regulated by hcg and lh concentrations. hcg peaks between weeks 11 and 17 and drops markedly thereafter; hcg-dependent testosterone is the most important determinant of genital differentiation. wolffian duct differentiation occurs only as a response to the testosterone secreted by the ipsilateral testis. this secretion stimulates differentiation of the ductus epididymidis, ductus deferens, and seminal vesicle. anomalies in androgen synthesis lead to incomplete masculinization and cryptorchidism. dihydrotestosterone (dht) derives from testosterone by the action of the enzyme 5α-reductase and is responsible for differentiation of the prostate and the development of the external genitalia, male urethra, penis and scrotum. it induces fusion of the labioscrotal folds in the middle plane to form the scrotum and the middle scrotal raphe. the urethral folds become fused to form the penile urethra. the genital tubercle enlarges to form the glans penis. an ectodermal invagination of the glans tip forms the terminal portion of the urethra. the urogenital sinus gives rise to the urinary bladder, prostatic urethra, and prostate. 53 the initial effects of dht (labioscrotal fusion) occur on approximately day 70; the urethral groove is closed on about day 74; and the external genitalia are completely developed by week 20. the actions of these hormones occur at precise moments in development. failure in the amount or timing of secretion or in the responsiveness of target tissues causes most of the malformations found in intersex conditions. 52 fsh and lh both play an important role in the last months of gestation. lh appears in the fetal circulation during the 10th week and peaks by the 18th, decreasing progressively and slowly thereafter until birth. lh chiefl y regulates androgen production during the second half of fetal life. fsh is an essential mitogen for sertoli cells that reach the highest mitotic ratio at the end of fetal life ( fig. 12-7) . 54, 55 testicular descent testicular descent is the result of hormonal and mechanical actions that are not fully understood. three steps are recognized: nephric displacement, transabdominal descent, and inguinal descent. in nephric displacement, the gonad detaches from the metanephros in the seventh week of gestation. transabdominal descent occurs in the 12th week and consists of the displacement of the testis towards the deep inguinal ring. inguinal descent occurs between the seventh month and birth. 56 clinically, the term testicular descent often refers only to this last step, in which the testis passes from the abdominal cavity to the scrotum. testicular descent is directed by the gubernaculum testis, a structure that appears in the sixth week as an elongate condensation of mesenchymal cells extending from the genital ridge to the presumptive inguinal region. 57, 58 at this level in the abdominal wall, the gubernaculum cells persist as a simple mesenchyma while the remaining abdominal wall cells differentiate into muscle. these mesenchymal cells give rise to the inguinal canal. thus, the testis lies on a continuous column of mesenchyma limited by the cranial testicular ligament in the upper pole and by the plica gubernaculi that joins the testis to the future scrotal region in the inferior pole. the periphery of this mesenchymal tissue is invaded by the processus vaginalis, which develops from a peritoneal pouch that grows into this mesenchyma. once the inguinal canal and the plica gubernaculi are formed, development slows. in the seventh month the processus vaginalis undergoes active growth, the cremasteric muscle develops from the mesenchyma outside the processus vaginalis, and the distal end of the gubernaculum enlarges markedly. gubernacular enlargement occurs from the 16th to the 24th weeks of gestation period and is caused by hyperplasia, hypertrophy, and the absorption of a great volume of water by the glycosaminoglycans of the matrix. 59 the tissue is reminiscent of wharton's jelly of the umbilical cord. by this time, the testis-epididymis complex is pear-shaped and its largest component is the gubernaculum. the testis and epididymis slide through the inguinal canal behind the gubernaculum. simultaneously, development of the processus vaginalis is completed and the gubernaculum begins to shorten, the epididymis develops further, and the testicular blood vessels and vas deferens lengthen. 60 testicular descent is a complex process integrating several essential factors, including normal function of the hypothalamopituitary-testicular axis, normal development of abdominal musculature, gubernaculum and the processus vaginalis, 61, 62 and a testis with normal endocrine function. the critical role of normal hormonal function is supported by clinical and experimental observations: destruction of the hypophysis in laboratory animals impedes testicular descent; anencephalic fetuses usually have undescended testes; many cryptorchid patients have transitory neonatal hypogonadotropic hypogonadism; and some undescended testes descend after treatment with human chorionic gonadotropin or gonadotropin-releasing hormone. adequate intra-abdominal pressure is another requisite. 63, 64 in the prune-belly syndrome, bilateral cryptorchidism is associated with urologic malformations and absence of the abdominal wall musculature. in a variant of this syndrome, termed pseudo-prune-belly syndrome, there is a positive correlation between the development of the abdominal wall musculature and testicular descent. development of the processus vaginalis also plays a critical role in testicular descent. this structure grows within the gubernaculum; if it is partially replaced by fi brous tissue, the testis will follow other directions in its descent and end in an ectopic location. if fi brous tissue completely replaces the gubernaculum, the processus vaginalis and cremasteric muscle fail to develop fully, and descent of the testis is mechanically blocked. 62 the hormonal requirements for testicular descent are not clear. 65 the most important factor in transabdominal descent is the androgen-independent peptide insulin-like factor 3 (insf-3), a member of the relaxin-insulin family that is produced by fetal leydig cells. this peptide stimulates gubernaculum cells to initiate gubernaculum swelling, a necessary step for the initiation of testicular descent. 66 mutations in insl-3 gene or its receptors lgrb-8 (leucine-rich repeatcontaining g protein-coupled receptor 8) or great (g protein-coupled receptor affecting testicular descent) interfere with transabdominal descent and cause cryptorchidism. 67, 68 amh and androgens are also involved in the gubernaculum swelling reaction; androgens also facilitate regression of the cranial suspensory ligament. uncertainty exists regarding the mechanism of inguinoscrotal descent and its hormonal control. androgens and the genitofemoral nerve are two factors strongly implicated in these processes. the role of androgens on the gubernaculum is very limited, because this structure has neither muscular cells 69 nor androgen receptors at the time of testicular descent. androgenic effects are explained by the hypothesis of the genitofemoral nerve. 70 androgens appear to act on the nucleus of the genitofemoral nerve in the spinal cord rather than directly on the gubernacula, producing masculinization of the neurons that form this nucleus 71 (these neurons are much more numerous in males than in females) and secreting great amounts of calcitonin gene-related peptide (cgrp). in rats, cgrp causes rapid rhythmic contractions of the gubernaculum and it has been suggested that the gubernaculum might have embryonic cardiac muscle cells. however, it is also possible that cgrp acts on the cremasteric muscle that develops within the gubernaculum and is innervated by the genitofemoral nerve. this hypothesis is supported by the observation of neurogenic atrophy of this muscle in cryptorchid patients. 72 other factors involved in testicular descent are estrogens and epidermal growth factor (egf). during the fi rst trimester of gestation, mothers of cryptorchid infants have free estradiol serum concentrations that are signifi cantly higher than those of controls. 73 experimental studies have shown that estradiol diminishes gubernacular swelling and stabilizes müllerian ducts. it has been proposed that estradiol inhibits the cell proliferation that causes gubernaculum swelling. 74, 75 egf may facilitate testicular descent throughout the placental-gonadal axis. maternal egf levels increase just before fetal masculinization occurs. 76 the placenta has an elevated concentration of egf receptors, and placental stimulation by egf might stimulate hcg production, which may also stimulate fetal leydig cells to produce androgens; hypothetically, these and/or other factors may determine testicular descent. after birth, the gubernaculum and processus vaginalis regress. the gubernaculum is replaced by fi brous tissue that forms the scrotal ligament. the cephalic segment of the processus vaginalis atrophies after testicular descent. an exaggerated resorption of the processus vaginalis with pulling up of the testis may induce a testis that had descended normally to ascend, resulting in cryptorchidism. 77 from birth to puberty the testis is a dynamic structure, an important consideration in interpreting biopsies from children. all testicular components undergo waves of proliferation and differentiation prior to puberty. 78 three waves of germ cell proliferation occur: during the neonatal period, infancy, and puberty. 79 the last gives rise to complete spermatogenesis. there also are three waves of leydig cell proliferation (fetal, neonatal, and pubertal); the last corresponds to the pubertal wave of germ cell proliferation. the testis at birth the newborn testis has a volume of about 0.57 ml 80 and is covered by a thin tunica albuginea from which the intratesticular septa arise. these divide the testis into lobules containing the seminiferous tubules and testicular interstitium ( fig. 12-8) . the seminiferous tubules measure 60-65 µm in diameter, with no apparent lumina, and are fi lled with sertoli cells and germ cells. sertoli cells are the most abundant, with 26-28 cells per tubular cross-section ( fig. 12-9) . 81 they form a pseudostratifi ed cellular layer and have elongated to oval nuclei with darker chromatin than that of mature sertoli cells, as well as one or two small peripheral nucleoli. the cytoplasm contains abundant rough endoplasmic reticulum, several golgi complexes and numerous vimentin fi laments, and expresses inhibin b (fig. 12-10) . no specialized intercellular junctions appear between sertoli cells, but desmosome-like junctions are present between sertoli cells and germ cells. 82 two types of germ cell are present at birth: gonocytes and spermatogonia. gonocytes are usually located near the center of the tubules, with voluminous nuclei and large central nucleoli. 82 gonocyte migration is probably facilitated by cell adhesion molecules such as p cadherin, which is expressed by sertoli cells of immature testes. 83 spermatogonia are mainly located on the basal lamina, and possess smaller nuclei and less cytoplasm than gonocytes; the nucleoli are peripheral and very small. at birth, most spermatogonia correspond to the adult type a (see discussion on the adult testis below) ( fig. 12-11) . the testicular interstitium contains fetal leydig cells that resemble adult leydig cells but lack reinke's crystalloids ( fig. 12-12) . 84, 85 additionally, mast cells, macrophages, and hematopoietic cell are present. 86 the fi rst wave of testicular development occurs during the neonatal period and involves germ cells and leydig cells. these changes are caused by a signifi cant increase in secretion of both fsh and lh during the third postnatal month. 87-89 testicular weight and volume increase. lh stimulates the leydig cells to produce testosterone, 90,91 which stimulates the transformation of gonocytes to spermatogonia of the ad type ( fig. 12-13) . afterwards, some of these in some normal testes at this age, meiotic primary spermatocytes and round spermatids are observed ( fig. 12-15 ). this spermatogenic attempt fails and many degenerate germ cells may be present. 94, 95 the testis continues to produce amh (by sertoli cells) 96 and inhibin b. 97 amh modulates the number and function of leydig cells by regulating differentiation of their mesenchymal precursors and the expression of steroidogenic enzymes. 98 inhibin b plays a role in fsh inactivation during infancy. the cause of this second wave of germ cell proliferation is unknown; there is no elevation of fsh or lh serum concentrations between 6 months and 10 years of life. after the sixth year, there is a slight increase in adrenal androgens, but testicular testosterone levels increase only after the 10th year. 99, 100 by the third year, most leydig cells have degenerated: from a peak of about 18 million at birth, only 60 000 remain by the age of 6 years. at this age, testosterone levels embryology and anatomy of the testis divide to form ap spermatogonia (see discussion on the adult testis below). six months after birth, gonocytes are absent, coinciding with the loss of fetal germ cell markers (placental alkaline phosphatase and c-kit). paraganglia are often observed in epididymides and spermatic cords from newborns. this is not surprising, as paraganglia are the main source of catecholamine before birth ( fig. 12-14) . 92 the testis in infancy from the sixth month to approximately the second half of the third year of life, the testis is in a resting period; this quiescence is broken by the second wave of germ cell proliferation. 93 the number of ap spermatogonia increases, and b spermatogonia (derived from ap spermatogonia) appear. are similar to those of girls, 99 and most androgens are of adrenal origin. at about 9 years of age, the third and defi nitive wave of spermatogenesis begins, 101 coinciding with a signifi cant elevation of lh. this is followed by additional increases in the level of this hormone between 13 and 15 years of age. lh induces fi broblast-like leydig cell precursors to differentiate into mature leydig cells. 102 by the end of puberty, the population of leydig cells per testis has risen to about 786 million. 103 leydig cells secrete androgens, which, together with the rise in fsh between 11 and 14 years of age, cause sertoli cell maturation, germ cell development, and the appearance of tubular lumina ( fig. 12-16) , 103 increasing the size of the testes between the ages of 11.5 and 12.5 years of life. 104 at 13.5 years, before the testis reaches adult size, spermatozoa are present, secondary sex characteristics are completely developed, and the epiphyses close. 105 testicular biopsy in children is useful for diagnosing those with ambiguous genitalia, a history of leukemia or lymphoma whose testes underwent a rapid enlargement, or precocious testicular maturation of unknown cause. in other situations, the value of testicular biopsy is less established. for example, the value of biopsy of cryptorchid testes during orchidopexy is controversial. evaluation of biopsies of the prepubertal testis should involve the assessment of several features, including tunica albuginea thickness, mean tubular diameter, and the number of germ cells, sertoli cells, and leydig cells. the most frequent anomalies of the tunica albuginea include thin, poorly collagenized tunica albuginea with abnormal tubules typical of testicular dysgenesis (see the section on male pseudohermaphrodites with müllerian remnants, below); well-collagenized tunica albuginea containing ectopic seminiferous tubules, a frequent fi nding in cryptorchidism; and poorly collagenized tunica albuginea containing ovocytes characteristic of true hermaphroditic ovotestes. the mean tubular diameter is an excellent indicator of the development of the seminiferous epithelium. in the prepubertal testis, tubular diameter depends principally on the sertoli cells and thus indicates whether they are adequately stimulated by fsh. tubular diameter varies throughout, being smallest in the end of the third year of life, slowly enlarging up to 9 years of age, and rapidly enlarging thereafter up to 15 years ( fig. 12-17) . the most frequent abnormality in the prepubertal testis is a low mean tubular diameter. this is seen in undescended testes as well as in hypogonadotropic or hypergonadotropic hypogonadism. in the latter, the lesion results from anomalous sertoli cell responsiveness to fsh. 106 there are three levels of severity of low tubular diameter: slight tubular hypoplasia (up to 10% reduction in relation to the diameter normal for the age); marked tubular hypoplasia (from 10% to 30% reduction); and severe tubular hypoplasia (more than 30% reduction). many testicular biopsies show malformed seminiferous tubules that vary from straight or branched tubules up to ring-shaped. these are megatubules formed by either tight spiral or bell-shaped tubules. the presence of these malformations suggests the child will be infertile in adulthood. diffuse increase in mean tubular diameter may be unilateral or bilateral. unilateral increase is found in monorchidism (compensatory testicular hypertrophy) and some testes that are contralateral to cryptorchid testes. most frequently, diffuse enlargement occurs with benign idiopathic macroorchidism or macroorchidism associated with fragile x chromosome, familial testotoxicosis, hypothyroidism, or different forms of precocious puberty. focal increases in mean tubular diameter are usually associated with precocious maturation of the seminiferous epithelium layers, and occur at the periphery of some sertoli cell and leydig cell tumors. germ cells can be counted in two ways: calculation of the number of cells per tubular cross-section, or determination of the tubular fertility index. the former counts the number of germ cells in a light microscopic fi eld and divides this by the number of cross-sectioned tubules in the same fi eld. in the fi rst 6 months of postnatal life the normal testis has two germ cells per cross-sectioned tubule. this number drops to 1.5 at the end of the fi rst year and to 0.5 at the end of the third year. the number of germ cells increases to 1.8 cells at the age of 3-4 years, which coincides with the appearance of spermatocytes in some tubules. the tubular fertility index refl ects the percentage of tubular sections containing germ cells. in newborns, 68% of tubular sections contain at least one germ cell. from birth to 3 years this decreases to 50%, followed by a progressive increase to 100% at puberty. 93 if the numbers of gonocytes and spermatogonia are calculated separately, it is possible to determine when the transformation of gonocytes to spermatogonia occurs. the most accurate measure is calculation of total germ cell numbers per testis. this is more diffi cult because it requires morphometric assessment of intratubular volume and careful clinical measurement of the three axes of the testis. congenital decrease of germ cells occurs in numerous conditions, including trisomies 13, 18, and 21, some forms of primary hypogonadism such as klinefelter's syndrome, anencephaly, many cryptorchid testes, and in patients with posterior urethral valves and severe obstruction of the urinary ducts. 107 an increased number of germ cells may be seen at the periphery of germ cell tumor, gonadal-stromal tumor, and paratesticular sarcoma. at the periphery of leydig cell tumor, seminiferous tubular cellular maturation may be complete. three levels of severity of germinal hypoplasia are recognized: slight (tubular fertility index >50), marked (tubular fertility index between 50 and 30), and severe (tubular fertility index <30) ( fig. 12-17 ). marked and severe germinal hypoplasia is usually associated with marked or severe tubular hypoplasia, in most cases resulting from tubular dysgenesis. it also is useful to determine whether the seminiferous tubules devoid of germ cells are randomly distributed. if they are grouped, they probably belong to the same lobule or group of lobules that never will develop normally. other germ cells observed are multinucleate or hypertrophied spermatogonia and gonocyte-like cells; these latter may require immunohistochemical studies to exclude intratubular germ cell neoplasia. the number of sertoli cells per tubular cross-section varies during childhood as a result of slow proliferation from 4 years to 12 years 108 and the redistribution of sertoli cells as the seminiferous tubules become longer and broader. the pseudostratifi ed cellular pattern characteristic of sertoli cells at birth changes slowly to a columnar pattern at puberty . testicular biopsies may reveal hypoplasia or hyperplasia of sertoli cells; hyperplasia is usually pronounced and a sign of tubular dysgenesis, often detected during the fi rst year of life or the beginning of puberty. 109 some biopsies reveal one or several tubular sections containing sertoli cells with eosinophilic and granular cytoplasm that is positive to cd68 and α 1 -antitrypsin. these oncocytic changes are the result of lysosomal accumulation. 110 calculation of leydig cell numbers during childhood is diffi cult because at this age the population is scant. 102 semi-thin sections or immunohistochemistry to detect testosteronecontaining cells may be helpful. 111 selection of the appropriate denominator to express the leydig cell population is another problem. the most frequent measures are leydig cell number per tubular section, per unit area, or total number per testis. 104 low numbers of leydig cell are observed in undescended testes, hypogonadotropic hypogonadism, some variants of male pseudohermaphroditism caused by a defect in the lh receptor, and in anencephalic fetuses. high numbers of leydig cells occur in congenital leydig cell hyperplasia, 112 triploid fetuses, 113 variants of precocious puberty, several syndromes such as leprechaunism and beckwith-wiederman syndrome, and in most male pseudohermaphroditisms. an apparent increase in loose connective tissue is found in patients with marked tubular hypoplasia; in addition, disordered thick fusiform cell bundles are seen in patients with androgen insensitivity. other alterations include the presence of excessively developed lymphatic vessels (lymphangiectasis), focal hematopoiesis, leukemic infi ltration, and the presence of cells similar to those of the adrenal cortex (tumors of the adrenogenital syndrome). embryology and anatomy of the testis the adult testis is an egg-shaped organ that hangs in the scrotum from the spermatic cord, the retroepididymal surface, and the scrotal ligament. mean weight in caucasian men is 21.6 ± 0.4 g for the right testis and 20 ± 0.4 g for the left. mean testicular diameter is 4.6 cm (range, 3.6-5.5 cm) for the longest axis and 2.6 cm (range, 2.1-3.2 cm) for the shortest. [114] [115] [116] [117] testicular volume varies from 15 to 25 ml. the tunica albuginea and interlobular septa make up the connective tissue framework of the testis. the tunica albuginea consists of three connective tissue layers and an outer surface covered by mesothelium. from the outer to the inner layers, the amount of collagen fi bers decreases while the number of cells increases. the fi bers and cells in the two outermost layers form planes parallel to the testicular surface; cell types include fi broblasts, myofi broblasts, and mast cells. myofi broblasts are more numerous in the posterior portion of the testis. the thickness of the tunica albuginea increases with age from 400-450 µm in young men to more than 900 µm in elderly men. 118 it acts as a semipermeable membrane that produces the fl uid of the vaginal cavity. the presence of many contractile cells showing high concentrations of gmp suggests that the tunica albuginea undergoes impulses of contraction and relaxation. these cells might regulate testicular size 119 and favor the transport of spermatozoa into the epididymis. 120 the innermost layer, the tunica vasculosa, consists of loose connective tissue containing blood and lymphatic vessels. the interlobular septa consist of fi brous connective tissue with blood vessels supplying the testicular parenchyma. the interlobular septa divide the testis into approximately 250 pyramidal lobules with their bases at the tunica albuginea and vertices at the mediastinum testis. each lobule contains two to four seminiferous tubules and numerous leydig cells. 121 adult seminiferous tubules are 180-200 µm in diameter and 30-80 cm long. the total combined length of the seminiferous tubules is about 540 m (range, 299-981 m). 122 they are highly convoluted and tightly packed within the lobules. the seminiferous tubules comprise about 80% of testicular volume. the tubular lining of germ cells and sertoli cells is surrounded by a lamina propria (tunica propria) ( fig. 12-18 ). sertoli cells are columnar cells that extend from the basal lamina to the tubular lumen, with 10-12 cells per crosssectioned tubule. they are easily identifi ed by their nuclear characteristics. the nucleus is located near the basal lamina and has a triangular shape with indented outline, pale chromatin, and a large central nucleolus ( fig. 12-19 ). charcot-böttcher's crystals and lipid droplets often are visible in the cytoplasm. [123] [124] [125] [126] ultrastructurally, sertoli cells have characteristic nucleoli, plasma membranes, and cytoplasmic components. the nucleolus has a tripartite structure with a round fi brillar center, a compact granular portion, and a three-dimensional net composed of intermingled fi brillar and granular portions. [127] [128] [129] the plasma membrane has two types of intercellular junction which develop at puberty: junctions between adjacent sertoli cells, and junctions between sertoli cells and germ cells. 130 the inter-sertoli cell junctions are tightjunction complexes. the adjacent cytoplasm has numerous actin fi laments and parallel-arranged smooth endoplasmic reticula cisternae. in adjacent plasma membranes there are adhesion molecules, including connexin-43. between the plasma membrane and the adjacent endoplasmic reticulum cisterna there are many molecules, including those required for actin fi lament anchorage, vinculin, zonula occludens-1, plakoglobin, and radixin. the inter-sertoli cell junctions are the morphologic basis for the blood-testis barrier and divide the seminiferous epithelium into two compartments: the basal compartment (which contains spermatogonia and newly formed primary spermatocytes) and the adluminal compartment (which contains meiotic primary spermatocytes, secondary spermatocytes and spermatids). these junctions permit each compartment to have its own microenvironment for spermatogenic development. [131] [132] [133] the sertoli cell-germ cell junctions persist from the primary spermatocyte stage through spermatozoon release. these junctions are desmosomes and gap-type junctions. the adhesion among sertoli cells and germ cells is mediated by n-cadherin. these junctions have also occasionally been observed between spermatogonia. 134 sertoli cell cytoplasm contains abundant smooth endoplasmic reticulum, elongated mitochondria, annulate lamellae, lysosomes, residual bodies, glycogen granules, microtubules, vimentin fi laments around the nucleus , 135 actin fi laments in both inter-sertoli cell junctions and ectoplasmic specializations that surround germ cells, 136 lipid droplets in amounts that vary with the seminiferous tubular cycle, 137 charcot-böttcher crystals (structures several micrometers long, formed of multiple parallel laminae of protein), and scant rough endoplasmic reticulum and ribosomes. 138 the number of sertoli cells decreases with age, from about 250 million per testis in young men to 125 million in men over 50 years. 139, 140 there is a positive correlation between the number of sertoli cells and daily sperm production. 141 sertoli cells are the target of fsh 142, 143 and androgen action ( fig. 12 -21) . 144 in adulthood, they produce testicular fl uid through an active transport mechanism, and synthesize multiple products to ensure the nutrition, proliferation and maturation of germ cells, to stimulate other cells such as leydig cells and peritubular cells, 145 and to contribute to hormonal regulation (inhibin secretion) (table 12 -1). the transport of small molecules (<600-700 da) such as pyruvate, lactate, and probably choline from the sertoli cell, to germ cells occurs through gap junctions. large or small soluble molecules are transported by proteins that are synthesized by the sertoli cell, and include androgen-binding protein, transferrin, ceruloplas-embryology and anatomy of the testis min, sulfated glycoproteins, α 2 -macroglobulin, and γ-glutamyl transpeptidase. 146 activin and inhibin are sertoli cell-secreted proteins that induce the proliferation and differentiation of germ cells. whereas activin stimulates fsh production and, subsequently, spermatogonial proliferation, inhibin b inhibits fsh secretion, and is an important marker of spermatogenesis. 147 other sertoli cell secretions are interleukins, mainly il-1, 148 and growth factors such as transforming growth factor-β (tgf-β), insulin growth factors 1 and 2 (igf-1 and igf-2), and seminiferous growth factor (sgf) or stem cell factor (scf). some of these growth factors, such as tgf-α, tgf-β, and igf-1, are involved in the regulation of leydig cell function. other secreted substances include clusterin, the steroid 3-α-4-pregnen-20-one (3hp), and prostaglandin d synthase (table 12-2) . sertoli cells are also involved in migration of differentiating germ cells towards the tubular lumen. this movement leads to a continuous remodeling of the plasma membrane and requires synthesis of several proteases, including urokinase, tissue-type plasminogen activator, cyclic protein 2, collagenase iv, other metalloproteins, and several antiproteases, such as cystatin c, tissue inhibitor of metalloproteinase type 2, and α 2 -macroglobulin. 149 the sertoli cell also regulates germ cell apoptosis by the production of fas-ligand, which binds to the fas-ligand receptor (apo-1, cd95) in germ cell plasma membranes. in addition, sertoli cells possess receptors for several factors such as the nerve growth factor (ngf) produced by spermatocytes and young spermatids, emphasizing the complexity of the sertoli cell-germ cell relationship. sertoli cells also produce some steroid hormones (estradiol and testosterone) and several components of the seminiferous tubule wall, including laminin, type iv collagen, and heparin sulfate-rich proteoglycans. the germ cells of the adult testis include spermatogonia, primary and secondary spermatocytes, and spermatids . spermatogonia there are two types of spermatogonia: a and b. type a are about 12 µm in diameter, rest on the basal lamina, and are surrounded by the cytoplasm of the adjacent sertoli cells. the nuclei of type a spermatogonia are spherical, contain several peripheral nucleoli, and have four different patterns: ad (dark), ap (pale), al (long), and ac (cloudy). 150, 151 the cytoplasm of these spermatogonia contains a moderate number of ribosomes, small ovoid mitochondria joined by electron-dense bars, and lubarsch's crystals. these are several micrometers long and are composed of numerous 8-15 nm parallel fi laments intermingled with ribosome-like granules. ad spermatogonia are thought to be stem cells in spermatogenesis. some of them replicate dna and, during replication, acquire the al pattern. afterwards, they divide to make another ad (maintaining the stem cell reservoir) and an ap spermatogonium. during replication, ap spermatogonia become ac and then divide to form two type b spermatogonia. [152] [153] [154] type b spermatogonia are the most numerous, and their contact with the basal lamina is less extensive than that of ultrastructural studies show coarse chromatin masses in which synaptonemal complexes and sex pairs may be present. the nucleolus acquires a peculiar appearance, with segregation of the fi brillar and granular portions. associated with the nucleolus is the round body that contains proteins but no nucleic acids. 128 in the pachytene spermatocyte, homologous chromosomes are completely paired, and on electron microscopy the chromatin masses appear larger and less numerous than in the zygotene spermatocyte. in the diplotene spermatocyte, paired homologous chromosomes begin to separate and remain joined by the points of interchange (chiasmata); neither synaptonemal complexes nor sex pairs are observed. the diakinesis spermatocyte shows maximal chromosome shortening and the chiasmata begin to resolve by displacement towards the chromosomal ends. the nuclear envelope and the nucleolus disintegrate. the spermatocyte completes the other phases of the fi rst meiotic division (metaphase, anaphase and telophase), forming two secondary spermatocytes; the fi rst meiotic division lasts 24 days. 156 secondary spermatocytes are haploid cells, smaller than primary spermatocytes, and show coarse chromatin granules and abundant rough endoplasmic reticulum cisternae. 157 these cells rapidly undergo the second meiotic division and within 8 hours give rise to two spermatids. the newly formed spermatids differ from secondary spermatocytes, having smaller nuclei with homogeneously distributed chromatin. spermiogenesis the transformation of spermatids into spermatozoa is called spermiogenesis. during this process pronounced changes occur in the nucleus and cytoplasm. 158 the nucleus becomes progressively darker and elongated. 159 the cytoplasm develops the acrosome and fl agellum, 160 the mitochondria cluster around the fi rst portion of the spermatozoon tail, and the remaining cytoplasm is phagocytosed by sertoli cells. 161, 162 by electron microscopy, there are four tran-sient stages of spermatid development: golgi, cap, acrosome, and maturation. these correspond to those defi ned by light microscopy of nuclear morphology: sa, sb, sb 1 , sb 2 , sc, sd 1 and sd 2 . 163, 164 these phases may be grouped as early (or round) spermatids that comprise the stages with round nuclei (sa and sb), and as late (or elongated) spermatids that comprise the stages with elongated nuclei (sc and sd). mature spermatids (sd 2 ) are the spermatozoa that are released into the tubular lumen (spermiation). all the germ cells derived from the same stem cell remain interconnected by cytoplasmic bridges that ensure synchronous maturation during the spermatogenic process. 165 cycle of the seminiferous epithelium at fi rst glance, the arrangement of the germ cells in the seminiferous tubules appears disorderly. however, closer study reveals that these cells are grouped into six successive associations, designated i-vi. in contrast to other mammals, in humans the volume occupied by each association is small, so that several associations may be observed in the same tubular cross-section. stereological studies have shown that the successive associations are organized helically along the length of the seminiferous tubule. 126, [165] [166] [167] each association persists for a specifi c number of days (i, 4.8 days; ii, 3.1 days; iii, 1 day; iv, 1.2 days; v, 5 days; and vi, 0.8 days), and each successively transforms into the following one. finally, at the end of association vi, the cycle is repeated; the spermatogenic process requires 4.6 cycles. 168 because each cycle lasts 15.9 days, the transformation of spermatogonium into spermatozoon takes 74 days (fig. 12-22) . the succession of different associations probably depends on cyclic sertoli cell activity. cyclic changes in the mitochondria, rough endoplasmic reticulum, golgi complex, lysosomes, and lipid droplets have been reported. [169] [170] [171] this cyclic activity is probably regulated by germ cell signals. 172 the yield of human spermatogenesis is lower than that of embryology and anatomy of the testis the six different germ cell associations of the seminiferous tubules and the sequence of spermatogenesis. completion of spermatogenesis requires more than four cycles and lasts for approximately 74 days. each association is indicated by roman numerals with its corresponding duration. ad: dark type of a spermatogonia; ap: pale type of a spermatogonia; b: b spermatogonia; i: interphase primary spermatocyte; l: leptotene primary spermatocyte; z: zygotene primary spermatocyte; p: pachytene primary spermatocyte; ii: secondary spermatocyte (only in stage vi). s a , s b1 , s b2 , s c , s d1 , and s d2 represent the progressive stages of spermatid differentiation into spermatozoa. most mammalian species, including primates, with maximal cell degeneration occurring at the end of meiosis. 173 the seminiferous tubule is surrounded by a 6 µm thick lamina propria (tunica propria) consisting of a basement membrane, myofi broblasts, fi broblasts, collagen and elastic fi bers, and extracellular matrix. 174, 175 the basement membrane measures 100-200 nm in thickness, and displays three layers: lamina lucida (beneath the sertoli cells), lamina densa (basal lamina), and lamina reticularis (a discontinuous layer containing fi bers). the basal lamina contains laminin, type iv collagen, entactin (nidogen), and heparan sulfate. 176 external to the basal lamina there are fi ve to seven layers of fl attened, elongated peritubular cells that have important secretory functions (table 12-3) . 177 the cells forming the three to fi ve innermost layers are myofi broblasts containing numerous actin, myosin, and desmin fi laments. these cells play an important role in the rhythmic tubular contractions that propel spermatozoa toward the rete testis. 178, 179 the two outermost cell layers consist of fi broblasts without desmin fi laments, and with less actin and myosin than the myofi broblasts. collagen fi bers are present among the peritubular cells and are abundant between the basal lamina and the peritubular cells. elastic fi bers are located mainly at the periphery of peritubular cells. because elastic fi bers appear at puberty, their absence in adults is a sign of tubular immaturity or dysgenesis. 180 the extracellular matrix contains proteoglycans and fi bronectin. in addition, the tubular wall contains capillaries and leydig cells. these are very similar to the interstitial leydig cells and are named peritubular leydig cells. the most important functions of myofi broblasts are contraction of seminiferous tubules and control of sertoli cells. 181 myofi broblasts have α and β adrenergic and muscarinic receptors. 182 contractility depends on several factors produced in the testis (endothelin-1, vasopressin, oxytocin, and tgf-β) and prostaglandins. relaxation can be facilitated by the no/cgmp system because myofi broblasts are also able to synthesize nitric oxide. sertoli cell control by myofibroblasts is facilitated by the production of p-mod-s, which activates aromatase activity, inhibin production, and the secretion of androgen-binding protein and transferrin. the interstitium between the seminiferous tubules contain leydig cells, macrophages, neuron-like cells, mast cells, blood vessels, lymphatic vessels, and nerves, accounting for 12-20% of testicular volume. 183 the most numerous connective tissue cells are fi broblasts and myofi broblasts. the former are also known as interstitial dendritic cells or cd34-positive stromal cells. they display a network around the seminiferous tubules and leydig cells, and also form the outermost layers of the tubular wall. 184 this distribution begins in fetal life. some of these cells are in contact with typical macrophages, so it has been suggested that they might be involved in immune surveillance. myofibroblasts, in addition to their presence in the inner layer of the tubular wall, are numerous in the tunica albuginea. leydig cells are distributed single or in clusters, and form about 3.8% of testicular volume. most are in the testicular interstitium, although they may also be found in the tubular tunica propria, mediastinum testis, tunica albuginea, epididymis, and spermatic cord. extratesticular leydig cells are usually seen within or near nerve trunks. [185] [186] [187] leydig cells have spherical eccentric nuclei with one or two eccentric nucleoli and prominent nuclear lamina. the cytoplasm is abundant, eosinophilic, and contains lipid droplets and lipofuscin granules (residual bodies) ( fig. 12-23 ). reinke's crystalloids are found only in the leydig cells of adults and, although it was believed that these crystals were present exclusively in humans, they have also been observed in the wild bush rat. reinke's crystalloids are up to 20 µm long and 2-3 µm wide, consisting of a complicated meshwork of 5 nm fi laments with a trigonal lattice arrangement. depending on the plane of section, three basic aspects of this lattice can be discerned. frequently, the crystalloids display pale lines, considered to be potential planes of cleavage. the fi laments are grouped into 19 nm-wide hexagons visible on cross-section. in some areas there are aggregates of electron-dense, rodshaped structures. some leydig cells contain other types of paracrystalline inclusion, the most common of which consists of multiple parallel-folded laminae. 188 leydig cells contain abundant well-developed smooth endoplasmic reticulum, pleomorphic mitochondria with tubular cristae, lysosomes, and peroxisomes. leydig cells react with antibodies to s100 protein and neuron-specifi c enolase. 189 leydig cells immunoreact to lh receptors, 3-β-hydroxysteroid dehydrogenase (3-β-ηsd), relaxin-like factor, 190 inhibin, and ghrelin. 191 relaxin-like factor, also known as insulin-like factor 3 (insf-3), is a peptide that is involved in testicular descent and can be found in serum. its concentration is a maker of the leydig cell functional status. as occurs with testosterone, insf-3 production is associated with that of lh. 192 leydig cells immunoreact with calretinin, a 29 kda calcium-binding protein that has a buffering effect to avoid abnormal increases in intracellular calcium. 193 calretinin is a more sensitive marker than inhibin, albeit less specifi c ( fig. 12-24) . 194 leydig cells also contain vegf and its two receptors (flt-1 and kdr), and endothelin and its two receptors (α and β). vegf and endothelin are involved in paracrine and autocrine control of leydig cells. leydig cells near seminiferous tubules show immunoreactivity for glial fi brillar acid protein (gfap) 195 (fig. 12-24 ). the demonstration of several substances that are characteristic of nerve cells, such as substance p, neurofi lament triplet proteins (nf-l, nf-m and nf-h), and the ultrastructural observation of microtubules, intermediate fi laments, and clear and dense core vesicles, qualifi es leydig cells for inclusion within the family of the diffuse endocrine system or paraneurons. 196, 197 leydig cells of the adult testis originate from fi broblastic precursor cells at puberty under lh stimulation. 198 experimental studies in rats have shown that adult leydig cells differentiate from peritubular cells (myofi broblasts and blood capillary pericytes). precursor leydig cells are reminiscent of neural stem cells because they express nestin and eventually acquire properties of neurons and glial cells. 199 the human testis contains about 200 million leydig cells. this number decreases with age: the testes of 60-year-old men contain about half as many as those of 20-year-old men. [202] [203] mitotic fi gures are seen occasionally in normal leydig cells. 204 leydig cells are the target cell of lh, in response to which they produce testosterone and other androgens necessary for the maintenance of spermatogenesis and many structures of the male genital tract, as well as other tissues such as bone, muscle, and skin. [205] [206] [207] [208] testosterone acts on the sertoli cells, either directly 209 or via the p-mod-s factor secreted by the myofi broblasts in the tunica propria. [210] [211] [212] leydig cells also secrete numerous non-steroidal factors, including oxytocin, which acts on myofi broblasts and stimulates seminiferous tubule contraction; β endorphin, which inhibits sertoli cell proliferation and function; egf, which regulates spermatogenesis; and other factors with less known actions, such as angiotensin, pro-opiomelanocortin, and α-melanotropic stimulating hormone (table 12-4) . together with sertoli cells, peritubular cells, and endothelial cells, leydig cells produce nitric oxide, which has a relaxing effect on smooth muscle. 213 leydig cells are associated with cholinergic and adrenergic nerve fi bers. 186 varicosities containing synaptic vesicles in the proximity of leydig cells and nerve endings in direct contact with leydig cells have been reported, although the functional signifi cance of this innervation is unknown. 214, 215 macrophages, neuron-like cells, and mast cells macrophages are a normal component of the testis 216-218 and can be classifi ed into two groups: resident and activated. resident macrophages are an essential cell type of the testicular interstitium (about 25% of interstitial cells in mouse testis). 219 in young adult men, there is one macrophage per 10-15 leydig cells, and this number increases with age. macrophages are closely related to leydig cells and play a role in proliferation and differentiation of leydig cell fi broblastic precursors. 220 interaction between macrophages and leydig cells is an example of paracrine function. in the rat, testicular macrophages produce 25-hydroxycholesterol (25-hc) and express 25-hydroxylase, which transforms cholesterol into 25-hc. 221,222 activated macrophages produce interleukins 1 and 6 (il-1 and il-6), tumor necrotizing factor-α (tnf-α), and transforming growth factor-α (tgf-α). immunohistochemical techniques have demonstrated neuron-like cells in the testicular interstitium. 223 these cells are an important source of intratesticular cate-cholamines, which appear to be increased in some disorders such as the sertoli cell-only syndrome, and hypospermatogenesis. mast cells are a normal component of the testicular interstitium, where they are often found near blood vessels. their number increases in several diseases. 224 the testis is supplied by the testicular artery, which arises from the abdominal aorta. in the spermatic cord, the testicular artery gives rise to two or three branches that obliquely penetrate the tunica albuginea testis and to multiple branches that run along the intralobular septa of the testis. 225 these centripetal arteries lead to the mediastinum testis. along their course, the centripetal arteries give off branches that abruptly reverse direction; these are called centrifugal arteries. at puberty, both the centripetal and the centrifugal arteries develop a pronounced spiral architecture. 226,227 the centrifugal arteries develop additional branches in the testicular interstitium, giving rise to arterioles and capillaries that form intertubular plexuses, some of which are apposed to the tunica propria. 228, 229 capillaries are of the continuous type, except for the seminiferous tubule capillaries, which are partially fenestrated, 230 and their endothelial cells are similar to those of brain capillaries, with scant pinocytosis, intercellular junctions of the fascia adherens type, and low permeability. the mediastinum testis is poorly vascularized. the inner two-thirds of the testicular parenchyma is drained by veins that follow the interlobular septa to the mediastinum testis (centripetal veins). the outer third is drained by veins that lead to the tunica albuginea (centrifugal veins). both centripetal and centrifugal veins join to form the pampiniform plexus, which drains the testis via the spermatic cord. lymphatic vessels are poorly developed in the testis and limited to the tunica vasculosa and interlobular septa, 231 where they accompany arterioles and venules. prelymphatic vessels have been reported in the interstitium and probably drain interstitial fl uid into the true interlobular lymphatic vessels. efferent innervation of the testis is mainly supplied by neurons of the pelvic ganglia, where contralateral and bilateral neural connections occur. postganglionic nerve fi bers enter the testis via the pelvic nerves, extend throughout the tunica vasculosa, and follow the interlobular septa to reach the interstitium. these nerve fi bers end in the wall of arterioles, the wall of seminiferous tubules, and the leydig cells. 232 adrenergic nerve fi bers innervate the tunica albuginea and the blood vessels of the tunica vasculosa. 233 peptidergic nerve endings are uncommon. afferent nerve endings form corpuscles similar to those of meissner and pacini in the tunica albuginea. the rete testis is a network of channels and cavities that connects the seminiferous tubules with the ductuli efferentes. differences in the confi guration and size of channels and cavities distinguish three portions of the rete testis: septal (intralobular), composed of the tubuli recti; mediastinal, composed of a network of interconnected channels; and extratesticular, composed of dilated cavities (up to 3 mm in diameter) termed the bullae retis. the tubuli recti are short tubules (0.5-1 mm long) that connect the seminiferous tubules to the mediastinal rete, although some seminiferous tubules may connect directly to the mediastinal rete, principally those in the central region of the testis. the tubuli recti are lined by cuboidal epithelium. there are approximately 1500 tubuli recti (or their analogous seminiferous tubule segments). the tubuli recti in the cranial, central, and anterior testis are perpendicular to the mediastinal rete testis channel into which they drain, and those in the caudal testicular region are parallel to their respective channels. the transitional segments between the seminiferous tubules and the tubuli recti are formed by modifi ed sertoli cells. 234 the epithelium of the mediastinal rete testis consists of fl attened cells interspersed with small areas of columnar cells. both cell types have single centrally located cilia and numerous microvilli on their free surfaces, and contain keratin and vimentin fi laments. 235 there are interdigitations between adjacent cells. the epithelium rests on a basal lamina, surrounded by a layer of myofi broblasts and a more peripheral layer of fi broblasts and collagen and elastic fi bers. the rete channels and cavities are traversed by the chordae rete, columns from 15 µm to 100 µm long and from 5 µm to 40 µm wide, arranged obliquely to the long axis of the cavity. the chordae consist of fi brous connective tissue with fi broblasts and are covered by fl attened epithelium; the widest contain capillaries. the rete testis probably has the following functions: damping differences in pressure between the seminiferous tubules and ductuli efferentes; reabsorption of protein and potassium from tubular fl uid; and, occasionally, phagocytosis of spermatozoa. anorchidism refers to the absence of one (monorchidism) or both testes (testicular regression syndrome). monorchidism is estimated to occur in about 4.5% of cryptorchid testes, 236 40% of the testes that are impalpable in physical examination, 237 or 1 in 5000 males. bilateral anorchidism occurs in approximately 1 in 20 000 males. 238 monorchidism the hormonal pattern in prepubertal patients with monorchidism does not differ from that of normal children, whereas children lacking both testes have elevated levels of gonadotropins and fail to respond to stimulation with hcg. [238] [239] [240] although the hcg stimulation test is often positive in children with bilateral cryptorchidism, it is negative in some children with bilateral intra-abdominal cryptorchidism and this further complicates the differential diagnosis between anorchidism and cryptorchidism. 241 for unknown reasons, the left testis is more frequently absent (68.7%) than the right. in such cases the contralateral scrotal testis undergoes compensatory hypertrophy and its volume increases to more than 2 ml. 242 compensatory hypertrophy has also been reported in association with abdominal cryptorchid testis. 243 the absence of testicular parenchyma should be confi rmed before diagnosing monorchidism. at exploration, the fi nding of a vas deferens ending near or in a hypoplastic epididymis is not suffi cient for the diagnosis of monorchidism. the only acceptable fi nding is blind-ending spermatic vessels. if inguinoscrotal exploration fails to identify these vessels, intra-abdominal exploration is required to insure against an undescended testis and avoid the development of a testicular tumor. 224 all remnants found at exploration should be removed. 245 testicular regression syndrome testicular regression syndrome refers to a variety of conditions, including agonadism, anorchidism, testicular agenesis, rudimentary testes, hypoplastic testes, and embryonal testicular dysgenesis. 246 each of these syndromes shares a complete absence or involution of both testes 247 but differ in the time of testicular disappearance during development. the most frequently observed are swyer's syndrome (see discussion on gonadal dysgenesis below), true agonadism, rudimentary testes, bilateral anorchidism, vanishing testes syndrome, and leydig cell-only syndrome (table 12-5) . true agonadism (46xy gonadal agenesis syndrome) patients with true agonadism have ambiguous external genitalia, fusion of the labia, and a short vagina, refl ecting very early testicular regression (between the eighth and 12th weeks of embryonal development). the internal genitalia consist of a uterus and two uterine tubes, although both müllerian and wolffi an derivatives may be absent. no gonads (not even in an ectopic location) are found. patients are phenotypically girls, and the male gender may be discovered only at the time of referral for other symptoms. 248 both sporadic and familial cases with associated extragenital anomalies have been reported. in some cases the cause is a heterozygous mutation of wt1. 249 in most familial cases inheritance is either recessive autonomic or x-linked, and the cause seems to be either unknown anomalies in the wt1 gene or known anomalies in other genes involved in development. 250 a sry molecular defect has never been observed. 251 agonadism may be associ-ated with several syndromes, including those of pagod (hypoplasia of lungs and pulmonary artery, agonadism, omphalocele/diaphragmatic defect, dextrocardia), 252 kennerknecht, 253 seckel, 254 and charge. 255 rudimentary testes syndrome patients with rudimentary testes have a normal male phenotype. müllerian remnants are absent and wolffi an derivatives usually are found. the testes are cryptorchid and very small, less than 0.5 cm long. seminiferous tubules are few ( fig. 12-25 ). the testicular regression occurs between the 14th and 20th weeks of gestation. this syndrome has been reported in several members of the same family, 256 suggesting genetic transmission, but this is not a constant feature. 257, 258 congenital bilateral anorchidism congenital bilateral anorchidism occurs in 1 in 20 000 newborns. the patients have male external genitalia, but the internal genitalia consist only of normal wolffi an derivatives without müllerian derivatives, suggesting that the testes were present and functionally active up to approximately the 20th week of gestation. patients have male external genitalia with hypoplasia of both the scrotum and penis. the karyotype is the normal male. the disorder may be associated with other malformations, such as anal atresia, rectourethral and rectovaginal fi stula, and urinary exstrophy. patients diagnosed at adulthood have male phenotype, androgen insuffi ciency symptoms, and elevated levels of both fsh and lh. 259, 260 familial incidence in some cases suggests sry gene mutation, but this has not been confi rmed. 261, 262 vanishing testes syndrome this term refers to the disappearance of one or both testes between the last months of intrauterine life and the beginning of puberty. [263] [264] [265] as testicular regression occurs after the seventh month, exploration fi nds the vas deferens in the inguinal canal or high in the scrotum; it may be accompanied by the epididymis and, less frequently, by testicular remnants consisting of small groups of seminiferous tubules ( fig. 12-26 ). patients lacking both testes develop hypergonadotropic hypogonadism after puberty, with gynecomastia, infantile phallus, hypoplastic scrotum, and impalpable prostate. the condition is usually secondary to a perinatal scrotal torsion, 266 although rarely there is a genetic cause. 267, 268 leydig cell-only syndrome patients with leydig cell-only syndrome have agonadism without eunuchoidism and a normal male phenotype, although meticulous surgical exploration fails to fi nd testicular remnants. study of serial sections from the spermatic cord reveals clusters of leydig cells. 269 detection of testosterone in spermatic vein blood indicates that these ectopic leydig cells are functionally active and synthesize testosterone in amounts suffi cient to induce a rudimentary male phenotype but insuffi cient to support the complete development of secondary sex characteristics. the morphology of spermatic cord remnants is similar in monorchidism and testicular regression syndrome occurring after the 20th week of gestation. [270] [271] [272] grossly, a small, fi rm mass is found at the end of the cord ( fig. 12-27 ). histologic examination reveals vas deferens, epididymis, or small groups of seminiferous tubules in 69-83% of cases. 273 vas deferens is the most constant fi nding (79%), followed by epididymis (36%) and seminiferous tubules (5-13%). the spermatic vessels are abnormally small in 83% of cases. 245, 274 areas of dystrophic calcifi cation, hemosiderin deposition, and giant cell reaction may be found within the mass in place of the testis. other fi ndings include arterial and venous vessels (88%), fat (44%), and nerves that may resemble traumatic neuroma (56%). the minimal requirement to diagnose vanishing testis is to fi nd either a vascularized fi brous nodule with calcifi cation or hemosiderin, or a fi brous nodule with cord elements. 275 it has been proposed that removal of the testicular nubbin in this syndrome may not be required because the percentage of seminiferous tubules is very low and the presence of germ cells low, and thus the probability of a tumor is minimal. 276, 277 the general recommendation is scrotal exploration as a fi rst step, reserving laparoscopy for cases in which either the atrophic remnant cannot be identifi ed during scrotal exploration or has a patent vaginal process. 266 the histologic fi ndings suggest that most cases of unilateral and bilateral anorchidism are produced during the fetal period after the testis has inhibited the müllerian ducts and induced differentiation of wolffi an duct derivatives. two hypotheses account for the disappearance of the testes: primary anomaly of the gonad; and atrophy secondary to a vascular lesion such as thrombosis or intrauterine torsion. the presence of macrophages with hemosiderin and dystrophic calcifi cation supports the latter. absence of one testis may be associated with malformations of the urogenital system, such as absence of the kidney, cystic seminal vesicles, and ipsilateral renal dysgenesis. 278, 279 this clinical term refers to diverse conditions (klinefelter's syndrome, hypogonadotropic hypogonadism, rudimentary testes syndrome, bilateral cryptorchidism, etc.) that share small testicular size. 280, 281 a peculiar case is presented by some patients with kenny-caffey syndrome: short stature, cortical thickening and medullary stenosis of long bones, delayed closure of anterior fontanelles, hypoparathyroidism, and several ocular alterations. fsh serum levels are elevated, but only in some cases, whereas lh and testosterone are normal. adult testes are small, with seminiferous tubules showing complete but diminished spermatogenesis. leydig are hyperplastic. unlike patients with the rudimentary testes syndrome, microorchidism patients have a normal-sized penis and no epididymal or prostatic atrophy. 282 polyorchidism is a rare condition, with approximately 100 reported cases. 283, 284 it was fi rst described in a postmortem study in 1880, 285 and the fi rst case treated surgically and confi rmed histologically was reported in 1895. 286 although three testes are the most common, 287 four testes have been reported in six patients, [288] [289] [290] [291] [292] and fi ve in one case but without histologic confi rmation. 293 age of diagnosis varies from newborn to 74 years, with a mean of 17 years. testicular duplication is usually an incidental fi nding during surgery for inguinal hernia, cryptorchidism, or testicular torsion, but has also been detected in patients with infertility or unexplained fertility after bilateral vasectomy. 294 the extra testis is often intrascrotal (75%) and less frequently inguinal (20%), abdominal, 295 or retroperitoneal (5%). 296, 297 duplication is three times more frequent on the left than on the right. 298 high-resolution ultrasound is the appropriate diagnostic technique. 284, 299 testicular maldescent (40%), inguinal hernia (30%), hydrocele, varicocele, and contralateral cryptorchidism are the most frequently associated anomalies. [300] [301] [302] testicular torsion (13%) 303 and testicular cancer (5.4%) are occasional complications. although the extra testis may be histologically normal, 304-306 usually it is not, 300, 307 and displays lesions such as sertoli cell-only tubules, hypospermatogenesis, or maturation arrest. the lack of spermatogenesis has been attributed to the anomalous location of the testis and the absence of communication between the testis and excretory ducts. 308 the embryologic origin of polyorchidism remains uncertain, and the following have been proposed to account for the variety of fi ndings in different cases ( fig. 12-28 the clinical differential diagnosis of polyorchidism includes most of pathologic conditions that enlarge the scrotum and spermatic cords: spermatocele, hydrocele, cysts and tumors of the spermatic cord, crossed testicular ectopia, adrenal cortical ectopia, and splenogonadal fusion. orchidectomy used to be the treatment of choice for all atrophic and non-scrotal testes. today, most surgeons undertake fi xation of the testis to the scrotal pouch and the re-creation of a 'simple testis' if it is permitted by the anatomical condition and malignancy has been precluded. this treatment may allow spermatogenesis as well as additional psychologic and cosmetic benefi ts. 313 intrascrotal rhabdomyosarcoma, testicular teratoma, and seminoma have been reported in patients with polyorchidism. 314, 315 macro-orchidism may be uni-or bilateral and be associated with chromosomal anomalies or endocrine alterations. an increase in the testicular parenchyma occurs in several conditions, 316 including congenital leydig cell hyperplasia, compensatory hypertrophy, benign idiopathic macroorchidism, bilateral megalotestes with low gonadotropins, fragile x chromosome, and the testicular hypertrophy observed in juvenile hypothyroidism. congenital leydig cell hyperplasia is uncommon and may be diffuse or nodular. the diagnosis of diffuse leydig cell hyperplasia requires quantifi cation of leydig cells by morphometry, using normal newborn testes as controls ( fig. 12-29 ). nodular leydig cell hyperplasia is characterized by the presence of non-encapsulated leydig cell nodules in the mediastinum testis, adjacent testicular parenchyma and connective tissue among the ductuli efferentes ( fig. 12-30) . the differential diagnosis of nodular leydig cell hyperplasia includes intratesticular adrenal rests and bilateral leydig cell tumor. except for patients with adrenogenital syndrome, intratesticular adrenal rests are rare. these rests are encapsulated, with the exception of the adrenogenital tumors, and consist of radially arranged cells with vesicular nuclei and small nucleoli displacing the rete testis or seminiferous tubules. leydig cell tumors may be bilateral, poorly circumscribed, and surrounded by testicular parenchyma, features making it diffi cult to distinguish from leydig cell hyper-plasia. however, leydig cell tumors are rarely congenital, whereas those occurring at infancy often induce precocious maturation of the adjacent seminiferous tubules and early macrogenitosomia. leydig cell hyperplasia is caused by large quantities of hcg entering the fetal circulation. diabetic mothers, particularly those with hypertension, may develop hyperplacentosis; the resulting edema in the placental villi alters the vascular permeability and allows the passage of hcg to the fetus. congenital leydig cell hyperplasia decreases rapidly during the fi rst months of postnatal life, after maternal human chorionic gonadotropin is gone. combined diffuse and nodular leydig cell hyperplasia occurs in several malformative syndromes, such as beckwith-wiederman, leprechaunism, triploid fetuses, fetuses with rh isoimmunization, 317 and in several complications of pregnancy. compensatory hypertrophy has been observed in monorchidism, 318 cryptorchidism 319 ( fig. 12-31) , varicocele, 320 and after testicular injury. hypertrophy persists and may increase during childhood and puberty, but ceases thereafter; the hypertrophied testis then becomes normal or remains slightly enlarged. 321, 322 the degree of hypertrophy is determined by three factors: the volume of the remaining testicular parenchyma, the age at which the injury occurred, and the functional ability of the descended testis. 323 compensatory hypertrophy results from an alteration in the hypophyseal hormonal feedback mechanism, followed by an increase in secretion of fsh, evidence that the contralateral testis is normal. in monorchidism, the testis is initially normal. 237 when a 50% reduction of testicular mass occurs (probably before birth), the endocrine feedback changes and the resulting secretion of fsh (before or immediately after birth) causes accelerated growth of the contralateral testis. in cryptorchidism, the reduction in testicular mass is less severe than in monorchidism, and the scrotal testis may also be abnormal, inducing a lesser compensatory hypertrophy. compensatory hypertrophy develops between birth and 3 years of age, and the testis may reach a volume twice normal when the other testis is absent. 243 some prepubertal and pubertal patients have pronounced unilateral 324 or bilateral [325] [326] [327] testicular hypertrophy in the absence of other pathologic fi ndings. this probably results from hormonal receptivity in the testicular parenchyma. morphometric studies have shown that the testicular enlargement is chiefl y due to an increase in the length of the seminiferous tubules, although increases in tubular diameter and sertoli cell numbers have also been observed. elevated fsh serum levels, reported in some cases, or hyperactive fsh receptors might be the cause of the excessive sertoli cell proliferation and the lengthening and thickening of seminiferous tubules. [328] [329] [330] in addition, leydig cell hyperplasia and defi cient spermatogenesis are frequent fi ndings in adult life. as the development of the two testes may be asynchronous during puberty, some unilateral macroorchidisms may represent cases in which these differences are unusually exaggerated. about 2% of adults with fertility problems have enlarged testes, with volumes over 25 ml, and low levels of fsh, lh, testosterone, prolactin, and estradiol. 331 despite the important hormonal changes, sperm concentrations and total numbers of spermatozoa are higher than normal. low fsh levels may be attributable to increased inhibin secretion because the number of sertoli cells is elevated in these testes, but no explanation for the reduction in the other hormone levels has been found. fragile x chromosome is the best-known form of inherited mental retardation, with an incidence of 1 in 1500 males and 1 in 2500 females. 332 in addition to facial dysmorphia (large ears, prognathism, high forehead, and arched palate), macroorchidism (martin-bell syndrome) is often an associated fi nding. [333] [334] [335] [336] [337] the impaired gene (fmr1 gene) is mapped to xq27,3 which is genetically fragile. the gene alteration is due to a lengthening of a trinucleotide cgg repeat that results in fmr1 gene silencing. if the cgg sequence is repeated fewer than 200 times, the disorder is considered a premutation and males show no symptoms; if the number of repetitions exceeds 200, mutation is complete and all show the disorder. [338] [339] [340] in men with this syndrome, the average testicular volume is more than 70 ml (four times greater than normal). the penis also is larger than normal, and both anomalies are apparent in infancy. the scrotum is also enlarged and prematurely pigmented. this precocious genital development is diffi cult to explain because the hypothalamopituitary axis is normal, but it may be caused by increased sensitivity to stimulation by fsh. 341 testicular biopsies from adults may be normal or show interstitial edema and hypospermatogenesis ( fig. 12-32) . usually, there is normal testicular parenchyma with focal reduced spermatogenesis and sertoli cell hyperplasia ( fig. 12 -33) or tubules containing only immature sertoli cells. morphometry indicates that testicular enlargement is chiefl y the result of lengthening of seminiferous tubules. 328 the low number of spermatids is attributed to atrophy caused by compression of the seminiferous epithelium by marked increase in intratubular fl uid. 342 meiotic anomalies have been excluded. 343 the fragile x syndrome is second in frequency only to down's syndrome as a cause of mental retardation. [344] [345] [346] however, this chromosomal anomaly is not always associated with mental retardation or macroorchidism, and there are men with fragile x syndrome who are otherwise normal. 347 the terms 'fragile x-negative martin-bell syndrome' or 'mental retardation-macro-orchidism' refer to x-linked (mrmo) or xlmr+mo patients who have the martin-bell syndrome phenotype but do not present the fragile x site. the gene responsible for this disorder is mapped to xq12-q21. 348 starts. 353, 358, 359 the etiopathogenesis has been explained by three hypotheses: an increase in gonadotropin secretion caused by trh stimulation of gonadotropic cells; 360,361 a direct tsh effect on the testis due to the structural similarity between tsh receptors and fsh receptors present in the testis; 362 and a lack of steroid hormones that are required for testicular maturation (in their absence, sertoli cell proliferation is excessive, giving rise to testicular enlargement). [363] [364] [365] [366] precocious puberty precocious puberty is defi ned by onset of secondary sex characteristics at a chronologic age that is below the mean middle age for the population. for practical purposes, this is considered to be before 8 years of age in girls and 9 years in boys. the incidence is estimated at between 1 in 5000 and 1 in 10 000, with a female:male ratio higher than 20 : 1. in boys, the fi rst symptom is rapid testicular enlargement followed by growth of pubic and axillary hair, enlargement of the penis, and acceleration of skeletal growth. 367 according to hypothalamopituitary-gonadal axis function, precocious puberty can be classifi ed into three groups: central or gonadotropin-dependent, which results from the activation of this axis; peripheral or gonadotropinindependent, mediated by sex steroid hormones secreted by the testis or adrenal glands; and a mixed group that fi rst appears as peripheral precocious puberty and thereafter, because of the secondary response of the hypothalamus, becomes gonadotropin dependent. other possible causes of precocious puberty are hypoprolactinemia, pituitary tumor, and alteration of testicular steroid metabolism. central precocious puberty (cpp) central precocious puberty, also known as true precocious puberty, is isosexual. it is the most common form of precocious puberty in girls and accounts for more than 50% of cases in boys. the age of presentation is between 4 and 10 years. 368 the cause is only known in 60% of cases; most are related to lesions in the central nervous system, whereas the others are usually idiopathic. lesions in the central nervous system that causes cpp share alterations of specifi c areas, including the posterior hypothalamus (eminencia media and tuber cinereum), mammillary bodies, the bottom of the third ventricle, or the pineal gland. 369 testicular hypertrophy appears associated with fsh-secreting pituitary adenoma, 349 hyperprolactinemia, hypoprolactinemia, and hypothyroidism. 350, 351 the most frequent association of testicular hypertrophy is with hypothyroidism. children with hypothyroidism often show testicular enlargement without virilization. 350 about 80% have macroorchidism, 352 most have elevated fsh levels, and half have increased lh levels. 353, 354 testosterone levels are normal during infancy. the response of fsh and lh to gnrh is altered and no pulsatile lh release occurs ( fig. 12-34 ). 355 testicular biopsies before puberty show an accelerated development of the testis with pubertal maturation of seminiferous tubules but not leydig cells. testicular biopsies in untreated adults show tubular and interstitial hyalinization with few leydig cells. 356, 357 testicular size in this type of macroorchidism diminishes as soon as the substitutive therapy • hereditary diseases as neurofi bromatosis and tuberous sclerosis. children with type i neurofi bromatosis often have also optic pathway tumors. • cerebral irradiation, as occurs in hypothalamopituitary selective irradiation, 378 prophylactic irradiation in children with acute lymphoblastic leukemia, 379 and irradiation of cerebral tumor that is far from the hypothalamopituitary region. the diagnosis of central precocious puberty is easy if the hormonal fi ndings show elevated gonadotropin levels (both basal values and in response to gnrh), associated with high testosterone levels and an increase in either lh/fsh ratio or in lh and fsh values after stimulation with gnrh agonists. however, in some cases it is necessary to measure nocturnal lh secretion to fi nd secretion pulses before a dynamic test can reveal the pubertal pattern. knowledge of the etiology in males has improved with the use of ct and mri. 380, 381 one of the most important contributions of these techniques is the fi nding of a high number of hamartomas in children with precocious puberty. [382] [383] [384] these lesions, also known as gangliocytomas, consist of abnormally located neurons and glial cells. lesions are usually multiple, small, and located on the hypothalamus between the anterior part of the mammillary body and the posterior part of the tuber cinereum. these neurons contain lhrh-positive neurosecretory granules, suggesting that this hormone can be released into the blood draining the hypophyseal portal system and reach the gonadotropic cells. 385 precocious puberty owing to cerebral tumors usually occurs with advanced stage of the tumor, preceded by cerebral symptoms such as hydrocephaly, papillary edema, or psychic alterations. the same occurs when precocious puberty results from cerebral infl ammation or cerebral malformation. although pineal gland tumor is rare in children, 30% produce precocious puberty, principally in boys. this tumor is usually a teratoma or non-parenchymatous tumor that destroys the pineal gland, hindering its antigonadotropic action and initiating puberty. 386 in contrast, pinealocytederived tumor secretes great amounts of melatonin that delay the onset of puberty. peripheral precocious puberty (ppp) peripheral precocious puberty is also known as precocious pseudopuberty. it may be caused by a primary testicular disorder, a lesion in other endocrine glands, or hormonal treatment. primary testicular disorders causing precocious pseudopuberty include familial testotoxicosis, functioning testicular tumor, excessive aromatase activity, or leydig cell hyperplasia with focal spermatogenesis. the principal secondary anomalies include adrenal cortical anomaly (congenital adrenal hyperplasia, virilizing tumor of the adrenal, and nelson's syndrome), and lesion secondary to hcg-secreting tumor (hepatoblastoma accounts for half of precocious pseudopuberty cases, and testicular germ cell tumor and the tumors of the retroperitoneum, mediastinum, and pineal gland are responsible for the other half of cases). 387 familial testotoxicosis: gonadotropin-independent precocious puberty (gipp) or familial male-limited precocious puberty (fmpp) familial testotoxicosis is a form of male sexual precocity characterized by early differentiation of leydig cells and the initiation of spermatogenesis in the absence of stimulation by pituitary gonadotropin. this is a primary testicular abnormality with autosomal dominant inheritance. 388, 389 ultrastructural studies confi rm an adult leydig cell pattern and complete spermatogenesis, although many spermatids are abnormal. 390 the cause of familial testotoxicosis is a constitutive activating mutation of the lh receptor gene. 391 this gene comprises 11 exons and has been mapped to 2p21. hormonal measurements show elevated serum levels of testosterone, and low levels of dihydroepiandrosterone sulfate, androstenedione, 17-hydroxyprogesterone, gonadotropin-releasing hormone (grh), and lh, as well as absence of a pulsatile pattern. in addition, serum levels of inhibin b appear elevated before the normal age of onset of puberty. 392 in some patients, a mutation in lh receptor induces leydig cell adenoma. 393 precocious puberty secondary to functioning testicular tumor a syndrome of precocious puberty can be the result of different tumors, including leydig cell tumor, sex cord tumor, adrenal cortex virilizing carcinoma, and extratesticular hcg-secreting germ cell tumor. leydig cell tumor may cause precocious puberty. the testis is enlarged owing to tumor growth and maturation of the seminiferous tubules adjacent to the tumor; such maturation results from androgen secretion by tumor cells (fig. 12-35) . in most cases, the contralateral testis is not enlarged. 394, 395 sex cord tumor with annular tubules and large cell calcifying sertoli cell tumor may give rise to precocious pseudopuberty that is isosexual (development of musculature and axillary and pubic hair) and heterosexual (gynecomastia). this precocious testicular maturation and the development of the tumor itself cause testicular enlargement. it has been suggested that tumor cells stimulate leydig cells to produce androgens that are aromatized to estrogens by the tumor cells themselves, thus accounting for the clinical symptoms. these tumors are frequently observed in peutz-jeghers syndrome 396,397 and carney's complex. 398 most infants with adrenal cortex virilizing tumors have small testes, but some cases of testicular hypertrophy have also been observed. 399 testicular development in these cases is attributed to adrenal androgenic action on seminiferous tubules. 400 in untreated (or maltreated) congenital adrenal hyperplasia, both testes can be enlarged because they contain growing masses of adrenal cortex-like cells. 401 a similar condition is observed in nelson's syndrome. testicular enlargement is modest in paraneoplastic precocious pseudopuberty secondary to hepatoblastoma 402 or extratesticular hcg-secreting germ cell tumor, although nodular or diffuse precocious maturation has been occasionally reported. 403 precocious pseudopuberty secondary to excessive aromatase activity biosynthesis of c18 estrogens from c19 androgens occurs by three consecutive oxidative reactions that are catalyzed by an enzymatic complex known as estrogen synthetase or aromatase. 404 this complex has two components: p450 arom (a product from the cyp19 gene located on 15p21.1), 405 which joins c19 substrate and catalyzes the insertion of oxygen in c19 to form c18 estrogens; and nadph-cytochrome p450 reductase, a ubiquitous fl avoprotein that conveys reducing equivalents to any form of cytochrome p450 it meets. aromatase is in the endoplasmic reticulum of estrogensynthesizing cells and expressed in placenta, ovarian granulosa, sertoli cells, leydig cells, adipose tissue, and several central nervous system regions, including the hypothalamus, amygdala, and hippocampus. excessive aromatase causes excessive conversion of androgens to estrogen, 406 and is a heterogeneous genetic disorder with an autosomal dominant inheritance. the disorder leads to heterosexual precocious pseudopuberty with gynecomastia in males, and to isosexual precocity and macromastia in females. ultimately, patient stature is short because of the potent ability of androgens to accelerate epiphyseal closure. most males are fertile and have normal libido. 407 generally, the inhibitory estrogenic effect on testicular function is less than that observed with estrogen-producing tumors or in patients treated with exogen estrogens. excessive aromatase caused by p450 mutation induces alterations in both males and females. in females lacking estrogens owing to desmolase defi ciency, excessive aromatase leads to pseudohermaphroditism and progressive virilization at puberty; conversely, pubertal development is normal in males. in children, fsh and lh levels and gonadotropin response to gnrh are normal, suggesting that the role of estrogens in pituitary regulation is weak during infancy. 408 in both genders, epiphyseal closure is delayed and a eunuchoid habitus results. adult males have small testes, severe oligozoospermia, and complete asthenozoospermia; fsh and lh levels are high, testosterone levels are normal, and serum estrogen levels are very low. all patients with excessive aromatase have short stature, with continuing linear growth into adulthood, unfused epiphyses, osteoporosis, bilateral genu valgum, and eunuchoid proportions. the testes show macroorchidism with normal testicular consistency in some cases, 409 and are small with severe oligozoospermia and 100% immotile spermatozoa in other cases. 410 a syndrome similar to that of excessive aromatase production is found in patients with estrogen resistance caused by disruptive mutations of the er gene. these patients show macroorchidism, elevated testosterone levels, and increased levels of fsh, lh, estradiol, and estrona. 411 precocious pseudopuberty secondary to leydig cell hyperplasia with focal spermatogenesis this entity can present with clinical symptoms similar to those of a functioning leydig cell tumor; this is a precocious pseudopuberty with ipsilateral testicular enlargement. 412 the testes contains hypertrophic leydig cell nests in association with normal spermatogenesis. no tumoral mass is seen. leydig cells do not contain reinke's crystalloids and do not compress the seminiferous tubules. there is a clear delimitation between tubules with spermatogenesis and infantile immature tubules. the differential diagnosis between this entity and leydig cell tumor with precocious pseudopuberty is based on the histological pattern. open excisional testicular biopsy is recommended; if there is leydig cell tumor, or the diagnosis by frozen section is not conclusive, removal is advisable. 413 there are no data to suggest that this hyperplasia might develop into leydig cell tumor. mixed precocious puberty the best known form is the mccune-albright syndrome (mas), characterized by the association of 'coffee and milk' pigmentary lesions in the skin, bone lesions (polyostotic fi brous dysplasia), enlarged testes, prepubertal size of the penis, and absence of pubic and axillary hair. although testicular enlargement is usually bilateral, unilateral macroorchidism may be the fi rst symptom. 414 an interesting fi nding is that the onset of testicular maturation is induced by the testis itself, which produces steroid secretion due to autonomous hyperfunction of sertoli cells without evidence of leydig cell involvement. 415 this secretion causes early maturation of the hypothalamopituitary-testicular axis and, subsequently, true precocious puberty. 416 serum levels of testosterone are low, but those of inhibin b and amh are abnormally increased. this syndrome is caused by mutations that activate the gnas-1 gene, which encodes the α subunit of the trimeric g-protein. because mutations are lethal in the uterus, those subjects producing amh bear a mosaicism chromosomal constitution for this defi ciency. a testis is ectopic when it is in a location outside the normal path of descent. unlike cryptorchid testes, ectopic testes are nearly normal in size and are accompanied by a spermatic cord that is normal or even longer than normal, and by a normal scrotum. 417 testicular ectopia is classifi ed according to location; [418] [419] [420] [421] [422] in decreasing order of frequency, the major types are: • interstitial or inguinal superfi cial ectopia. this is the most frequent form and may be confused with inguinal cryptorchidism. after passing through the outer genital opening, the testis ascends to the anterosuperior iliac spine and remains on the aponeurosis of the major oblique muscle. these testes often are more nearly normal histologically than are cryptorchid testes. • femoral or crural ectopia. after passing through the inguinal canal, the testis lodges in the high crural cone in scarpa's triangle. • perineal. the testis is located between the raphe and the genitocrural fold. • transverse or crossed ectopia. both testes descend through the same inguinal canal and lodge in the same scrotal pouch. each possesses its own vascular supply, epididymis, and vas deferens. in addition, there is ipsilateral hernia. [423] [424] [425] [426] [427] [428] [429] [430] between 20% and 40% of patients with this ectopia have persistent müllerian duct syndrome [431] [432] and show a high incidence of testicular germ cell tumor. 433 • pubopenile ectopia. the ectopic testis is on the back of the penis near the symphysis pubis. 434 • pelvic ectopia. the testis is in the pelvis, usually in the depth of douglas' cul-de-sac. • other unusual testicular ectopias include retroumbilical, craniolateral to the inner inguinal opening between the outer and inner oblique muscles, and subumbilical. 435 rarely, the testis and its spermatic cord may protrude through a defect in the scrotal skin, a condition called testicular exstrophy. 436 the term testicular dislocation refers to testes that secondarily disappear from the scrotum and lodge around the superfi cial inguinal ring, within the inguinal ring, or inside the abdominal cavity as a result of testicular trauma. the formation of canalicular and intra-abdominal dislocation requires the presence of previous inguinal hernia. 437 testicular fusion is a rare anomaly characterized by fusion of the testes to form a single structure, usually in the midline. each has its own epididymis and vas deferens. this anomaly is often associated with other malformations, such as fusion of the adrenal glands or horseshoe kidney. cystic dysplasia of the testis is a congenital lesion characterized by cystic transformation of an excessively developed rete testis that may extend to the tunica albuginea of the opposite pole. 438 to date, fewer than 40 cases have been reported. 439, 440 the seminiferous tubules may be dilated and atrophic; this is more evident after puberty. ultrasound images are characteristic. 441, 442 cysts arise in the septal and mediastinal rete testis ( fig. 12 -36); they are interconnected and contain acellular, eosinophilic, periodic acid-schiffpositive material. they are lined by cuboidal cells that resemble those of the normal rete testis. [443] [444] [445] the connective tissue between the cysts is scant and histologically similar to the interstitial connective tissue. there may be small groups of cysts limited to the region of the mediastinum testis, or cysts extending throughout the entire testis. in extensive cases, residual seminiferous tubules occupy only a small crescent beneath the tunica albuginea and the testis is grossly spongy. cystic dysplasia occurs in normally descended and cryptorchid testes in children and adults, and may affect one or both testes. 446 in adults, the residual parenchyma often shows complete tubular sclerosis or hypospermatogenesis with intratubular accumulation of spermatozoa and leydig cell pseudohyperplasia. in most cases the epididymis is altered. 447 the head of the epididymis is small and contains few ductuli efferentes with irregular, usually dilated lumina. the ductus epididymidis is dilated, has an atrophic epithelium, and thick connective tissue replaces the muscular layer ( fig. 12-37) . testicular cystic dysplasia is frequently associated with severe anomalies of the urinary system. renal agenesis, [446] [447] [448] [449] renal dysplasia, 446 hydroureter, and urethral stenosis 450 have been reported ipsilateral to cystic dysplasia. the clinical differential diagnosis should consider all cystic testicular lesions impairing prepubertal testes, including epidermoid cyst, cystic teratoma, juvenile granulosa cell tumor, testicular lymphangiectasis, and simple cyst of the testis. 451 the presence of ipsilateral renal anomalies during ultrasound exploration provides an important diagnostic clue. 452 previously, orchidectomy was the treatment of choice, but testis-sparing surgery 453 is now recommended. 454, 455 the etiology and pathogenesis of cystic dysplasia are uncertain. given that the rete testis is a mesonephric derivative and most of the associated renal malformations are apparently caused by failure in the induction of renal blastema by the mesonephros, cystic dysplasia is considered to be the result of an abnormal mesonephros. during childhood, the normal rete testis has no lumina, and these form during puberty. the adult rete testis is a conduit for the passage of tubular fl uid and spermatozoa and also actively reabsorbs part of this fl uid while adding ions, proteins and steroids to it. malfunction of the rete testis cells may cause the formation of excessive fl uid of abnormal composition, resulting in a condition morphologically similar to cystic dysplasia of the rete testis induced in fowl by sodium intoxication or the administration of the saltretaining hormone deoxycorticosterone acetate. gonadoblastoid testicular dysplasia refers to an abnormally differentiated testicular parenchyma beneath the tunica albuginea. 456 the anomaly consists of large tubular or nodular structures within a dense stroma, reminiscent of ovarian stroma ( fig. 12-38 ). each structure is composed of three cell types: cells with vesicular nuclei and vacuolated cytoplasm; cells with hyperchromatic nuclei; and germ cell-like cells. the former two types are arranged at the periphery, forming a pseudostratifi ed epithelium. the third type resembles fetal spermatogonia and are fewer in number. these structures contain eosinophilic, periodic acid-schiff-positive material, similar to call-exner bodies ( fig. 12-39 ). there may be continuity between these structures and normal seminiferous tubules. the differential diagnosis includes conditions showing anomalous seminiferous tubules at the gonadal periphery, including testicular dysgenesis and gonadoblastoma. testicular dysgenesis also presents tubular or cord-like structures, but these are differentiated (some form true seminiferous tubules) and may also be present within a poorly collagenized tunica albuginea; patients with testicular dysgenesis are male pseudohermaphrodites with müllerian remnants. gonadoblastoma usually appears in a streak gonad or dysgenetic gonad and contains granulosa-sertoli cells and germ cells that are similar to those of dysgerminoma or seminoma; these cells are absent in gonadoblastoid testicular dysplasia. several cases with this disorder have been reported in patients with walker-warburg syndrome. 457, 458 this disorder refers to the presence, in an adult testis, of one or several foci of infantile (immature) seminiferous tubules. each group of tubules appears well delimited but unencapsulated. nodule size varies from microscopic to 5 mm. on section, each nodule is distinguished by its whitish color. sertoli cell nodule is found in most adult cryptorchid testes, regardless of when the testes descended. it is also present in 22% of normal scrotal testes in some series, 459 and is an occasional fi nding in males with idiopathic infertility. the seminiferous tubules have a prepubertal diameter and may be anastomotic. the epithelium is columnar or pseudostratifi ed, devoid of lumina, and usually consists only of sertoli cells ( fig. 12-40 ). the cells have elongated hyperchromatic nuclei with one or several peripherally placed small nucleoli. 459 the interstitium varies from scant to well collagenized. leydig cells are usually absent in these areas and, if present, their numbers are low. study of serial sections reveals continuity between some of these tubules and normal tubules. sertoli cell nodule changes with advancing age. the sertoli cells produce large amounts of basal lamina that protrudes inside the hypoplastic tubules. in transverse and oblique sections, these protrusions might be misinterpreted as intratubular accumulations of basal lamina material ( fig. 12-41 ). this material can undergo calcifi cation to form microliths. immunohistochemical study reveals two basic components of the basal lamina (collagen iv and laminin), confi rming its extracellular origin; the protrusions consist mainly of laminin, whereas collagen iv delimits the outer profi le of the seminiferous tubules. so, while the amount of collagen iv is uniform around the tubules, the depth of laminin varies within the same tubule. tubular hypoplasia is assumed to be a primary testicular lesion, and refers to the presence of seminiferous tubules that are unable to undergo pubertal development despite the same hormonal stimuli of adjacent normal tubules. this dysgenesis includes immature sertoli cell pattern, low inhibin secretion, absence of androgen receptors, 460 and lack of maturation of peritubular myoid cells that fail to synthesize elastic fi bers. the presence of hypoplastic zones in a testicular biopsy is an adverse prognostic sign for fertility. the differential diagnosis includes tubular hamartoma in androgen insensitivity syndrome, sex cord tumor with annular tubules, and mixed atrophy of the testis. tubular hamartoma in androgen insensitivity syndrome is multiple, similar to the hypoplastic zones of tubular hypoplasia; however, the sertoli-like cells of hamartoma have spherical nuclei (rather of elongated nuclei), form a cuboidal epithelium, and contain numerous leydig cells among the tubules (see androgen insensitivity syndrome, below). sex cord tumor with annular tubules may present with multiple foci of intratubular neoplasia, similar in distribution to that of hypoplastic zones; however, sex cord tumor appears in undescended testes, and in patients with peutz-jeghers syndrome, and consists of cuboidal or spherical cells that express cytokeratins that are not expressed in hypoplastic tubules. it is possible that hypoplastic tubules contain some germ cells that may be spermatogonia or gonocytes. there are scant spermatogonia that fail to display signs of maturation or proliferation. also, some of the tubules contain intratubular undifferentiated germ cell neoplasia that usually also appears in the adjacent, non-hypoplastic seminiferous tubules. the histologic picture is similar to that of gonadoblastoma, but such a tumor can be easily excluded because it arises in malformed gonads (gonadal dysgenesis and testicular dysgenesis) characteristic of intersex stages, unlike patients with tubular hypoplasia. congenital testicular lymphangiectasis is characterized by abnormal and excessive development of lymphatic vessels in the tunica albuginea, mediastinum testis, interlobular septa, and testicular interstitium. [461] [462] [463] ultrastructurally these dilated vessels are similar to normal lymphatic capillaries, although some are markedly dilated and the testicular interstitium is slightly edematous ( fig. 12-42 ). testicular lymphangiectasis occurs in both cryptorchid and scrotal testes; in one of the latter cases, the patient had noonan's syndrome. the disease does not seem to affect the seminiferous tubules, and low numbers of spermatogonia and reduced tubular diameters are observed only in cryptorchid testes. the epididymis and spermatic cord are not affected, and congenital testicular lymphangiectasis is not associated with pulmonary, intestinal, or systemic lymphangiectasis. during fetal life, lymphatic vessels are visible only immediately beneath the tunica albuginea and in the interlobular septa. 464 during childhood, the number and size of the septal lymphatic vessels decreases; 465 by adulthood they are inconspicuous. 466 in lymphangiectasis, the septal lymphatic vessels are large and often massively dilated. testicular lymphangiectasis occurs only in the childhood testis, suggesting that these dilated vessels undergo involution at puberty or that pubertal development of the seminiferous tubules masks the lymphangiectasis. one exceptional case of epididymal lymphangiectasis, with dilated epididymal blood vessels, was reported in a 59-year-old man. 467 the vessels distort the architecture of the ductuli efferentes, which in turn become irregularly dilated by mechanical compression. other hamartomas of the testis include hamartoma of the rete testis and smooth muscle hamartoma. hamartoma of the rete testis is a disordered proliferation of tubular structures in a loose connective tissue. 468 cystic transformation of the rete testis associated with proliferation of smooth muscle cells and abundant myxoid stroma was reported in a 26-year-old man. 469 smooth muscle hamartoma is located in the inferior testicular pole, the cauda of the epididymis, and the proximal segment of the vas deferens ( fig. 12-43) , 470 and is similar to that reported in the digestive and respiratory tracts. 471, 472 smooth muscle hyperplasia also occurs in the androgen insensitivity syndrome, forming nodules up to 1 cm in diameter. the muscular proliferation is located in the lower testicular pole, and involves the tunica albuginea and adjacent soft tissues. this infrequent fi nding has been observed in newborns and consists of gonadal blastema in otherwise normal testes. the blastema is located in the vicinity of the upper testicular pole, near the implantation of the caput of the epididymis, displays a crescent shape, and extends throughout the depth of the tunica albuginea and the adjacent testicular parenchyma. the blastema consists of epithelial cords of cells or solid masses in continuity with the mesothelium (fig. 12-44 ). these cells are intermingled with others that are larger, with pale cytoplasm, vesicular nuclei, and prominent nucleoli. the blastematous epithelial cells display immunoreactivity for vimentin, laminin, type iv collagen, and cytokeratin; the expression of the latter in the most superfi cial cells is similar to that of mesothelial cells and decreases in intensity in the deeper cells. this suggests that these may be pre-sertoli cells. the cord-like structures are delimited by laminin and type iv collagen. the second larger cell type is immunoreactive for placenta-like alkaline phosphatase (plap) on the surface, suggesting that it is related to the gonocyte. leydig cells have not been observed among the cords of gonadal blastema. the differential diagnosis of gonadal blastema ectopia is with ovotestes. the small size of the gonocytes distinguishes them from ovocytes, which are several times larger. in addition, no intersex condition is observed. the presence of seminiferous tubules within the tunica albuginea is rare and usually an incidental histologic fi nding. 473 ectopic tubules are present in approximately 0.8% of pediatric autopsies and 0.3% of adult autopsies. the lower incidence in adults may be explained by proportionally less sampling. the lesion ranges from microscopic size to a few millimeters in diameter, and may be visible as minute bulges in which multiple small vesicles protrude through a thin tunica albuginea. 474 histologically there are groups of seminiferous tubules in the tunica albuginea, sometimes accompanied by leydig cells. in children, the ectopic tubules appear normal ( fig. 12-45 ), whereas in adults they are usually slightly dilated, although some may be hyalinized. serial sections reveal continuity with the intraparenchymatous seminiferous tubules. ectopia of the seminiferous tubule is probably congenital, although it has been found in elderly men. 475 it does not appear to be the result of trauma. the malformation probably arises in the sixth week of gestation, when the primordial sex cords have formed and are branching toward the gonadal surface, and the developing testes is covered by only one to three layers of celomic epithelium. later, the tunica albuginea forms around the sex cords and under the celomic epithelium. failure of insertion of the tunica albuginea between the sex cords and celomic epithelium may entrap seminiferous tubules. ectopia differs from testicular dysgenesis, a distinctive form of male pseudohermaphroditism with müllerian remnants. numerous features, characteristic of ectopic seminiferous tubules, distinguish it from other conditions, including normal thickness and collagenization of the tunica albuginea, absence of interstitial tissue resembling ovarian stroma (characteristic of testicular dysgenesis), and clear delimitation of the tunica albuginea and testicular parenchyma (see discussion on male pseudohermaphroditism with müllerian remnants, below). in a unique case, there were multiple clusters of seminiferous tubules in the wall of a hernia sac that accompanied an undescended testis removed from an adult man. the ectopic tubules were not surrounded by tunica albuginea and were similar to those in cryptorchid testicular parenchyma with only dysgenetic sertoli cells. leydig cells occur normally in the testicular interstitium (interstitial leydig cells) and in the wall of the seminiferous tubules (peritubular leydig cells). however, clusters of leydig cells are often observed in other locations in the testis, or in the epididymis or spermatic cord. 476 ectopic leydig cells may be found in the interlobular septa, 477-479 rete testis, tunica albuginea, [480] [481] [482] or within hyalinized seminiferous tubules. 478, [483] [484] [485] intratubular leydig cells are found only in tubules with advanced atrophy and marked thickening of the tunica propria, including the tubules in adult cryptorchid testes, those of men with klinefelter's syndrome, and in some other primary hypogonadisms ( fig. 12-46 ). immunohistochemical studies suggest that the endocrine function of these leydig cells is low. 486 several theories have been offered to account for these ectopic cells, including in situ differentiation, migration from the testicular interstitium, and trapping of peritubular leydig cells in the tunica propria during its thickening. 487 leydig cells are commonly found in the epididymis 487 and spermatic cord; 488,489 26 of 64 autopsies had such foci. 490 extratesticular leydig cells usually form small groups within or adjacent to nerves ( fig. 12-47) . 477, 490 the occurrence of ectopic leydig cells in the albuginea, epididymis, or spermatic cord may account for the rare cases of leydig cell tumor in these paratesticular structures. ectopic leydig cells should not be misinterpreted as tumor cells (infi ltration or metastasis) when malignancy of a testicular leydig cell tumor is suspected. other rare forms of ectopia are found within and outside the testis. intratesticular ectopia includes adrenal cortical ectopia, osseous and adipose tissue heterotopia, and ectopia of the ductus epididymidis. extratesticular ectopia includes splenic ectopia (splenogonadal fusion), hepatic ectopia (hepatotesadrenal cortical ectopia may be important in two conditions that develop tumoral masses: adrenogenital syndrome and nelson's syndrome. tumors in adrenogenital syndrome appear in 8.2% of patients with congenital adrenal hyperplasia, appearing as bilateral testicular masses of synchronous growth. these tumors consist of well delimited but non-encapsulated yellow nodules, several centimeters long, composed of large microvacuolated cells. the cause seems to be prolonged stimulation by elevated acth secretion. the differential diagnosis includes leydig cell tumor. the diagnosis of tumors in adrenogenital syndrome is supported by a family or personal history of salt-lost syndrome or hypertension, demonstration of 11 β-hydroxysteroids (a specifi c marker for adrenal cortex) in spermatic vein blood, or a rapid positive response of tumor to corticoid treatment. nelson's syndrome occurs in patients who, after adrenalectomy for treatment of cushing's syndrome, develop an acth-secreting pituitary adenoma. these patients may develop testicular tumor growth similar to that in adrenogenital syndrome. most nelson's syndrome tumors do not respond to dexametasone treatment. cartilaginous heterotopia may be found in the caput of the epididymis and has been attributed to metaplasia of metanephric rests. osseous heterotopia (testicular osteoma) is a metaplasia occurring in areas of the testicular parenchyma with fi brosis or ischemia. 491 adipose metaplasia is frequent in undescended testis, elderly men, and those with cowden's syndrome ( fig. 12-48 ). 492 groups of tubular formations that resembles the epididymis have been reported inside the testicular parenchyma in testes with marked tubular atrophy, and probably represent a rare form of metaplasia. 493 testicular descent is not always complete at birth, and about 3.2% of full-term newborns have incompletely descended testes. most of these descend within 3 months, and only 0.8% of infants have incompletely descended testes 12 months after birth. spontaneous testicular descent is exceptional after the fi rst year. in recent decades, a signifi cant increase in the incidence of cryptorchidism has been detected. 494 only 5% of patients with impalpable testes are actually devoid of testes. other causes include true cryptorchidism, testicular ectopia, and retractile testes. true cryptorchidism includes abdominal, inguinal, and high scrotal testes that cannot be moved to the scrotum. ectopic testes are those located out of the normal path of testicular descent; the most frequent site is the superfi cial inguinal pouch. other rare locations of ectopia include the abdominal wall, the upper thigh, the perineum, and the base of the penis. retractile testes may be moved to the scrotum at exploration and account for about one third of undescended testes. patients with true cryptorchidism account for about 25% of cases of empty scrotum. these testes most frequently are found in the inguinal canal or upper scrotum; arrest within the abdomen is less frequent. cryptorchidism is slightly more frequent on the right than the left, and in approximately 18% of cases is bilateral. there is a family history of cryptorchidism in 14% of cases. 495 the cryptorchid testis is usually smaller than the contralateral one, and this difference is often discernible at 6 months of age. 496 one-third of cryptorchid testes are soft. several conditions are predictive of high risk of cryptorchidism, including increased maternal age, maternal obesity, pregnancy toxemia, bleeding during late pregnancy, and smoking, tallness, subfertility antecedents, cesarean birth, low birthweight, preterm newborn, twin birth, hypospadias 497 and other congenital malformations, and children born from september to november, and in may and june. 498, 499 of these associations, low birth weight seems to be the most important. 500 there are two types of cryptorchidism: congenital and acquired. this cryptorchidism is caused by anomalies in anatomic development or hormonal mechanisms involved in testicular descent (described above). impalpable undescended testes are infrequent because the transabdominal phase follows the simple mechanism of relative movement of the testis, whereas displacement of the ovary is more complex. 501 conversely, palpable undescended testes are more frequent because the second phase of testicular descent is more complex. unilateral cryptorchidism may be caused by androgen failure, which leads to either an ipsilateral lesion in the development of genitofemoral nerve neurons or a defect in cgrp release that hinders normal migration of the gubernaculum. a normally descended testis may become cryptorchid and locate even in the abdominal cavity. two categories of acquired undescended testis have been described. the postoperative trapped testis 502 is a normally descended testis that leaves the scrotal pouch after surgery owing to an inguinal hernia or hydrocele. [503] [504] [505] this iatrogenic cryptorchidism occurs in 1.2% of children after herniotomy. adherence of the testis or the cremasteric muscle to the surgical incision causes testicular ascent when the incision heals and undergoes retraction. spontaneous ascent from unknown causes. various mechanisms have been proposed, including inability of the spermatic blood vessels to grow adequately, 506 anomalous insertion of the gubernaculum, 507 failure in reabsorption of the vaginal process 508, 509 and failure in postnatal elongation of the spermatic cord. 510, 511 the spermatic cord measures 4-5 cm at birth and reaches 8-10 cm at 10 years of age. this growth does not occur if the peritoneal-vaginal duct has become a fi brous remnant. the cause might be a defect in postnatal cgrp release by the genitofemoral nerve. 501, 512, 513 pathogenesis the most frequent fi ndings in congenital and acquired cryptorchidism at infancy are decreased germ cell numbers and diminished tubular diameter. 514, 515 there are multiple causes of testicular maldescent, including anatomical anomalies of the gubernaculum testis, hormonal dysfunction (hypogonadotropic hypogonadism), mechanical impairment (insuffi cient intra-abdominal pressure, short spermatic cord, underdeveloped processus vaginalis), dysgenetic (primary anomaly of the testis), and heredity. most cryptorchidism appears to be caused by either a defi cit of fetal androgens or an excess of maternal estrogens. androgen insuffi ciency seems to be slight and transient because anomalies other than hypoplasia of the epididymis are not seen. elevated maternal estrogens level could cause diminution of fsh secretion by the fetal pituitary, inducing low müllerian-inhibiting hormone production that would hinder testicular descent. 516 three mechanisms seem to be involved in the process: • primary testicular anomaly. cryptorchid testes may bear an anomalous germ cell population, as suggested many years ago. 517 more than 40% of cryptorchid patients have a marked decrease in the tubular fertility index, 518 in the normal testis there is transient formation of spermatocytes at 4-5 years of age. this meiotic attempt is probably an androgenic event that does not occur in cryptorchid testes and agrees with the characteristic low numbers of spermatogonia in the prepubertal age. 523 prepubertal testes undescended testes are usually smaller than the contralateral ones. this difference is already significant at 6 months of age. 524, 525 although there have been a number of biopsy studies in the fi rst years of life, there is no agreement about the severity of damage or the time of its onset. 523, 526, 527 based on the tubular fertility index (tfi) and mean tubular diameter (mtd), most testicular biopsies from cryptorchid testes of children can be classifi ed into one of three groups: • type i (testes with slight alterations). the tubular fertility index is higher than 50, and the mean tubular diameter is normal or slightly (<10%) decreased. approximately 31% of cryptorchid testes are in this group ( fig. 12-49 ). • type ii (testes with marked germinal hypoplasia). tubular fertility index is between 30 and 50, and mean tubular diameter is 10-30% lower than normal. the spermatogonia are distributed irregularly and most are in tubular sections that are grouped in the same testicular lobule. these testes comprise approximately 29% of cryptorchid testes ( fig. 12-50 ). 528 • type iii (testes with severe germinal hypoplasia). tubular fertility index is less than 30, and mean tubular diameter less than 30% of normal. many of the spermatogonia are giant with dark nuclei (fig. 12 -51). these testes often contain ring-shaped tubules, induced by increased temperature. 527 testes with type ii or iii lesions bear variable degrees of dysgenesis that, in addition to germ cells, involve sertoli cells, peritubular myofibroblasts, and leydig cells. the dysgenesis of these other cell types is evident only after puberty. in about 25% of cases the contralateral scrotal testis also has histologic lesions of variable severity. this fi nding supports the hypothesis of a bilateral defect in many cases of unilateral cryptorchidism. microdeletions in the long arm of the y chromosome are present in 27% of patients with corrected unilateral cryptorchidism who present with azoospermia or severe oligospermia. 530 these fi ndings are similar to those observed in patients with azoospermia or severe idiopathic oligospermia. unilateral cryptorchidism with a normal contralateral testis could be due to an end-organ failure. 531 in cryptorchidism secondary to spontaneous ascent, lesions are similar to megatubules (with or without eosinophilic bodies or microliths) ( fig. 12-52) , and focal granular changes in the sertoli cells ( fig. 12-53 ). the testicular interstitium is wide and edematous. these comprise about 40% of cryptorchid testes. about 8% of tests with type i lesions show many multinucleated spermatogonia (with three or more nuclei) ( fig. 12-54 ). 529 the seminiferous tubules of testes with types ii or iii lesions have a thickened lamina propria during childhood and, at puberty, sertoli cell hyperplasia. 526 patients with bilateral cryptorchidism have a higher incidence of type ii and iii lesions than those with unilateral cryptorchidism. type i lesions are comparable to those seen in experimental cryptorchidism; normal testes in which lesions were those of congenital cryptorchidism, whereas in cryptorchidism secondary to herniotomy, germ cell depletion is slight 532 and becomes important only after 5 years of age. 533 adult testes most pubertal and adult cryptorchid testes have anomalies in all testicular structures. the seminiferous tubules have decreased diameters and defi cient spermatogenesis. in decreasing order of frequency, the most common germ cell lesions are tubules with sertoli cell and spermatogonia-only pattern; tubules with sertoli cells (dysgenetic) only; tubular hyalinization; and mixed atrophy. the lamina propria has scant elastic fi bers and increased collagen fi bers. 534 sertoli cells are present in increased numbers and do not mature normally except in tubules with germ cells (fig. 12-55 ). 528, 535 often, groups of tubules containing only sertoli cells with a prepubertal pattern (very small diameter and total absence of maturation) are present and are considered hypoplastic, dysgenetic, or hamartomatous. areas of apparent leydig cell hyperplasia are frequent, and many of these cells contain vacuolated lipid-laden cytoplasm ( fig. 12-56 ). the rete testis is hypoplastic in most cases and is lined by columnar epithelium with rare areas of fl attened cells. cystic dilation is common, and adenomatous hyperplasia has been found in some cases. near the rete testis, the testicular parenchyma frequently contains metaplastic fat. in some cryptorchid testes, several tubular segments are destroyed by infl ammation that probably has an autoimmune cause (focal orchitis). 536 epididymal tubules are poorly developed and peritubular tissue is immature. blood fl ow is associated with testicular histology. for example, testicular volume, histologic pattern, and testicular artery resistive index are lower in undescended testes than in controls, and testicular artery resistive index is inversely proportional to testicular histology score in undescended testes. 537 there is also an apparent correlation between testicular size, spermiogram, and hormone levels. assuming that a signifi cant reduction in testicular size (>12 ml) is only observed in 9.3% of cases, and that serum levels of fsh, lh, and testosterone are normal, an inverse correlation is seen between fsh and testicular volume, sperm concentration, sperm motility, and normally shaped sperm. in addition, there is a direct relation between testicular volume and sperm concentration, sperm motility, and normally shaped sperm. these fi ndings indicate the cause of tubular impairment in young men operated on in childhood for cryptorchidism. 538 obstructed testes are located in the superfi cial inguinal pouch (denis-browne pouch) and are considered ectopic by some authors and cryptorchid by others. 539, 540 histologic studies reveal that most obstructed testes bear the same lesions as true cryptorchid testes. type i lesions are observed in half, type ii in more than one-third, and the remainder show type iii lesions. the higher proportion of type i lesions suggests a better prognosis than in true cryptorchid testes. some authors assume that retractile testes are normal and exclude them from studies of cryptorchidism. 541, 542 however, these testes may present important lesions and many consider them to be a form of cryptorchidism. [543] [544] [545] retractile testes may not always be movable to the lower scrotum (70-75 mm from the pubic tubercle) and in 50% of cases are smaller than scrotal testes. approximately 50% of retractile testes remain high after age 6 years, when cremasteric activity declines. 546 retractile testes have a 32% risk of becoming ascending or acquired undescended testes. the risk is higher in boys younger than 7 years, or when the spermatic cord is tight or inelastic. 547 during childhood, tubular diameter and tubular fertility index decrease. 544 adults with retractile testes that descended spontaneously but late may be fertile 548 or infertile. 549 usually there is germ cell atrophy that varies in severity from lobule to lobule. 544 regular examination of retractile testes is advisable during childhood and, if complete testicular descent does not occur, orchidopexy is indicated. most cryptorchid patients have a patent processus vaginalis, and 65-75% have a hernia sac, although most hernias are not clinically visible. urologic anomalies are present in 10.5% of patients, the most frequent being hypospadias, complete duplication of the urinary tract, non-obstructive ureteral dilatation, kidney malrotation, and posterior urethral valves. cryptorchidism is more frequent in patients with microcephaly, myelomeningocele, bifi d spine, omphalocele, gastroschisis, micropenis, and imperforate anus. cryptorchidism may appear isolated or associated with congenital anomalies, endocrine dysfunction, chromosomal disorders, or intersex conditions. thus, cryptorchidism is found in the kallmann, prader-willi, klinefelter, noonan, smith-lemli-opitz, aarskog-scott, rubinstein-taybi, prune belly, and caudal regression syndromes, anomalies of the androgen receptor, absence of anti-müllerian hormone, charge association, and trisomies 13, 18, and 21. sperm excretory duct anomalies occur in 9-36% of cryptorchid patients, 550, 551 and are classifi ed into three types: 552 • ductal fusion anomalies (25% of cases). these consist of anomalous fusion of the caput of the epididymis to the testis or segmental atresia of the epididymis and vas deferens. this is chiefl y associated with intraabdominal or high scrotal cryptorchid testes. • ductal suspension anomalies (59% of cases). the caput of the epididymis is attached to the testis, whereas the corpus and the cauda of the epididymis are separated from the testis by a mesentery. a variant consists of an excessively long cauda of the epididymis that descends along the inguinal duct to the scrotum. • anomalies associated with absent or vanishing testes (16% of cases). cryptorchidism is part of the testicular dysgenesis syndrome. this consists of abnormal testicular development that predisposes to cryptorchidism, hypospadias, spermatogenetic alterations, and testicular cancer. the association of these disorders with cryptorchidism has been corroborated by numerous clinical, epidemiological and genetic studies. the least severe form of this syndrome is a defect in spermatogenesis; the most severe is testicular cancer. a constellation of histologic lesions is common in the testes of men with testicular dysgenesis; these lesions include sertoli cellonly pattern, mixed atrophy, hypoplastic tubules (sertoli cell nodules), microlithiasis, malformed tubules, granular changes in sertoli cells, nodular leydig cell hyperplasia, and intratubular germ cell neoplasia. it is assumed that there is a prenatal development of the lesions as a result of several genetic, environmental, or endocrine disruptor factors that would interfere with the estrogen/androgen ratio. [553] [554] [555] [556] the main complications of cryptorchidism are testicular cancer, infertility, testicular torsion, and psychological problems. approximately 0.8% of 1-year-old males have cryptorchidism, and about 10% of testicular cancer patients had cryptorchidism. the risk of testicular cancer in cryptorchid males is four to10 times higher than that of the general population. testes with elevated number of multinucleated spermatogonia seem to have a higher risk of cancer and adulthood. 557 about 5% of biopsies in children contain cells similar to those seen in undifferentiated intratubular germ cell neoplasia, and these cells may evolve toward germ cell tumor ( fig. 12-57 ). 558 the most frequent tumor in undescended testes is seminoma. 559, 560 regardless of timing, orchidopexy does not reduce the risk of cancer, although it facilitates early detection as the intrascrotal testis is palpable. one in fi ve testicular tumors arises in properly descended testes contralateral to cryptorchid testes, suggesting that there is a primary bilateral testicular anomaly in cryptorchidism. intra-abdominal testes also have a higher incidence of tumors. 560 infertility infertility is the most frequent problem caused by cryptorchidism. in a series of patients with infertility, nearly 9% had cryptorchidism. 561 infertility is infl uenced by several factors, including bilaterality, number of germ cells, location and size of the testis, and age at time of orchidopexy. the most important risk factors are bilaterality and germ cell number. only 16% 562 to 25% 563 of men with bilateral cryptorchidism have normal sperm counts (20 million/ml or more). the highest sperm counts occur with testes in the superfi cial inguinal pouch. patients with bilaterally impalpable testes are usually azoospermic. 563 fertility rates in unilateral cryptorchidism vary from 25% to 81%. 564 the number of germ cells per cross-sectioned tubule is the most important prognostic factor. patients with no increase in inhibin b during the postoperative period usually have a low number of spermatogonia per cross-sectioned tubule and a low tubular fertility index. in unilateral cryptorchidism, fertility depends on the number of spermatogonia in the contralateral testis. however, if the number of germ cells per cross-sectioned tubule in the cryptorchid testis is lower than 1% of normal, the risk of infertility is 33%. in bilateral cryptorchidism the risk of infertility rises from 75% to 100% when one or both testes have less than 1% of germ cells per cross-sectioned tubule. neither the preoperative location of the testis in patients with unilateral cryptorchidism nor the small size of the testis at the time of orchidopexy is relevant for fertility. [565] [566] [567] an important fertility factor is the permeability of sperm excretory ducts. the age at orchidopexy may also infl uence fertility, although this has not been proven. in patients over 4 years of age orchidopexy does not enhance fertility. 568, 569 benefi t of testicular biopsy in patients with cryptorchidism testicular biopsies of infantile testes at orchidopexy are useful for determining baseline germ cell status and whether surgery should be completed with hormonal treatment. 570 however, even if biopsy supplies important data, it is not considered a routine procedure. even in the best cases when the number of spermatogonia is nearly normal, spermatogenesis may never occur owing to defi cient spermatogonium development during childhood, failure of spermatogenesis at puberty, and, if complete spermatogenesis occurs, this might be associated with obstruction of sperm excretory ducts. in childhood, the chance of a biopsy fi nding an occult cancer or precancer is low because intratubular germ cell neoplasia is not diffusely distributed throughout the testis. testicular biopsy is recommended in patients with intra-abdominal testes, abnormal external genitalia, or abnormal karyotype. 571 the situation is different in adults because intratubular germ cell neoplasia is present in 2-3% of cases and is diffuse. 572, 573 when intratubular germ cell neoplasia is detected in a child, further examination of the testis and rebiopsy after puberty are recommended. 574 in adults, if intratubular germ cell neoplasia is unilateral orchidectomy should be performed, but if it is bilateral, radiation may be used to eradicate the neoplasia while maintaining leydig cell function. 575 testicular microlithiasis (tm) is characterized by the presence of numerous calcifi cations diffusely distributed throughout the testicular parenchyma. the number and size of the calcifi cations often is great enough to be detected radiographically or by ultrasound. 576 isolated microliths have been reported in undescended testes, prepubertal klinefelter's syndrome, male pseudohermaphroditism, and otherwise normal children and patients studied for other diseases. 577 in adults, microliths are frequently observed in cryptorchid and ex-cryptorchid testes, 578 seminiferous tubules located at the periphery of germ cell tumor, 579 infertile patients, [580] [581] [582] and in some patients complaining of orchialgia 583, 584 or testicular asymmetry. 585 testicular microlithiasis occurs in 0.3% of cryptorchid testes and is slightly more common in prepubertal than adult testes. in adults, it usually is diagnosed when men seek help for infertility, pain, or testicular asymmetry. 581 microlithiasis has been observed in 1.4-2% of testicular echographies of different disorders. 586, 587 in infertile patients the incidence is slightly higher. microlithiasis is present in 35% of testis having a malignant tumor. 588 ultrasound studies reveal two types of microlithiasis: classic tm, in which the number of microliths is fi ve or more; and limited tm, when there are fewer than fi ve microliths ( fig. 12-58 ). the incidences of tm in these studies are lower than 1% in infants, 5.6% in the general population aged between 18 and 35 years (bilateral in 66% of patients showing microliths, 589 0.68-4.1% in patients with other disorders, 586,587,590-592 from 4.6% 593 to 20% 594 in subfertile patients, 9.52% in ex-cryptorchid testes, 595 and more than 30% in adult testes with germ cell tumors). 588,596-599 several cases of testicular microlithiasis have also been observed in infant testes with germ cell tumor or gonadal stroma tumor. 600, 601 the incidence is higher in whites than in blacks. pain is the most common clinical symptom in patients without a palpable testicular mass, and has been attributed to dilation of seminiferous tubules secondary to obstruction by microliths. microliths are made by hydroxyapatite, according to x-ray diffraction studies 602 and raman spectroscopy. 603 in the prepubertal testis, microliths are surrounded by a double layer of sertoli cells and measure up to 300 µm in diameter. when they are very large, the seminiferous epithelium may be destroyed and the microlith is surrounded by peritubular cells (fig. 12-59 ). testes with microliths have subnormal mean tubular diameters and tubular fertility index. 604 in adult testes with microliths there is incomplete spermatogenesis. some seminiferous tubules with microliths are cystically dilated (fig. 12-60 ). microliths arise as extratubular eosinophilic bodies that mineralize and pass into the tubular lumina. 605 microlithiasis may be a disorder of the tunica propria. also, testicular microlithiasis is occasionally associated with pulmonary microlithiasis and with calcifi cations in the parasympathetic nervous system. 606, 607 the association of microlithiasis and testicular cancer is controversial. 608, 609 although the development of testicular cancer has been observed in several patients whose testicular microlithiasis had been previously diagnosed by ultrasound studies, [610] [611] [612] [613] [614] it is also thought that patients with testicular microlithiasis not associated with other disorder do not require any follow-up. 615 when microlithiasis is associated with infertility the incidence of cancer varies according to the unilaterality or bilaterality of microlithiasis: 594 subfertile patients with unilateral microlithiasis show no intratubular germ cell neoplasia, whereas this is present in 20% of those with bilateral microlithiasis. the risk of malignancy is higher in classic than in limited tm. 616 the nexus between microlithiasis and cancer does not seem to be the predisposition of one disorder towards the other but rather the predisposition of both to develop in abnormal testes. this may also explain the association between microlithiasis and infertility. yearly ultrasound examination, perhaps with testicular biopsy, is recommended in those with testicular microlithiasis associated with cryptorchidism, infertility, atrophic testes, or contralateral testis bearing germ cell tumor. 617 microlithiasis also occurs in the rete testis or sperm excretory ducts. epididymal rupture and extravasation of microliths into the interductal tissue may cause a histiocytic reaction resembling malakoplakia ( fig. 12-61 ). the disorder is asymptomatic and not associated with testicular cancer. 618 gonadal dysgenesis refers to disorders characterized by amenorrhea and streak gonads in phenotypically female patients. in adults, streak gonads are elongated masses of fi brous tissue resembling ovarian stroma ( fig. 12-62 ). they may contain hilar cells and rete or epithelial cords with variable degrees of maturation, and may result from failure in gonad formation, failure of gonadal differentiation to ovary, or failure of gonadal differentiation to testis. some streak gonads contain a few ovocytes or primordial follicles, but all germ cells disappear at puberty. patients with streak gonads have a hypoplastic uterus and fallopian tubes. four types of gonadal dysgenesis have been described: 46xy pure, 46xx pure, 45x0, and mixed. 46xy gonadal dysgenesis (swyer's syndrome) is characterized by female phenotype, absence of turnerian stigmata, and female external genitalia, sometimes with fused labia majora, a hypertrophic clitoris, and hypospadias. the breasts develop at puberty. sexual infantilism persists in adulthood, and eunuchoidism and amenorrhea appear. these patients have elevated serum gonadotropin levels and low serum estradiol. there are two types of gonadal dysgenesis: complete and incomplete. patients with the complete type have female external genitalia and classic streak gonads, although cases with ovarian tissue have been reported. the cause is unknown in about 80% of cases, 619 and is due to alterations in the sry gene in the remainder (a mutation in 10-15% of cases, and a sry deletion as a result of an aberrant x/y interchange in 10-15%). 620 the consequence of failure is very early gonadal alteration (sixth to eighth week of gestation). with the subsequent absence of müllerian inhibiting factor, testosterone, and dihydrotestosterone, a female phenotype develops. patients with incomplete 46xy gonadal dysgenesis have ambiguous external genitalia and variable degree of development of the müllerian and wolffi an structures. although they have streak gonads, testicular development is usually observed. this gonadal dysgenesis does not seem to be caused by sry alterations. 621 these fi ndings suggest that in the fi rst type ovarian differentiation was canceled, and that in the second type testicular differentiation failed. the fi rst is similar to the gonad of 45x0 turner's syndrome, whereas the second resembles the gonad of mixed gonadal dysgenesis. 622 the clitoromegaly may be caused by androgens secreted by hyperplastic leydig cells in the streak gonad. some patients with 46xy gonadal dysgenesis present with extragonadal anomalies and multiple syndromes, including camptomelic dysplasia and renal disorder, 623 myotonic dystrophy and terminal renal disease, 624 without 631 facial anomalies or short stature, 632 renal insufficiency and wilms' tumor (denys-drash syndrome), the combination of cleft palate, micrognathia, kyphosis, scoliosis, and clubfoot (gardner-silengo-wachtel syndrome or genitopalatocardiac syndrome), 633 pterygium multiple syndrome, 634 graves' disease, 635, 636 and congenital universalis alopecia, microcephaly, cutis marmorata, and short stature. 637, 638 most cases are sporadic, 639 although the syndrome has been reported in several members of the same family, [640] [641] [642] [643] and several forms of inheritance (x-linked, autosomal recessive, and male-limited autosomal dominant) have been proposed. 644 in addition to infertility, patients with 46xy gonadal dysgenesis have a high risk of germ cell tumor. this risk is about 5% in the fi rst decade of life, and 25-30% overall, [645] [646] [647] [648] and, thus, prophylactic gonadectomy is recommended. patients with 46xx gonadal dysgenesis have normal stature, female phenotype, well-developed external genitalia, and hypoplastic ovaries rather than streak gonads ( fig. 12-65 ). the anomaly is usually detected when patients present with primary amenorrhea or infertility. this syndrome is sporadic and familial, and it may be linked to recessive autosomal inheritance. 649, 650 patients have no predisposition to gonadal neoplasia. associated somatic anomalies such as neurosensory hearing loss (perrault's syndrome) are rare. some familial cases have shown a balanced translocation of the x chromosome (from the long arm to the short arm) 651, 652 or between chromosomes 1 and 11. 653 because the development of ovarian follicles requires fsh, mutations have been sought in the fshr gene. mutations have been detected in familial cases and also in unrelated patients, 654, 655 whereas other patients have shown no mutations in this gene. 656 the incidence of tumors in these patients is very low, and the most common is dysgerminoma. [657] [658] [659] this is one of the most common chromosomal anomalies (from 1/2500 to 1/5000 in female newborns), 660 although 99% of zygotes with this karyotype are aborted in the fi rst stages of embryonal development. 661 patients with 45xo gonadal dysgenesis have characteristic stigmata of turner's syndrome, including short stature, pterygium coli, lymphedema, and cardiac malformations. the external genitalia are female and infantile; the gonads are typical streak gonads. today, turner's syndrome is defi ned by the combination of physical features and the complete or partial absence of one of both x chromosomes, frequently associated with mosaicism. turnerian stigmata may be classifi ed into four groups: 662 skeletal anomalies such as cubitus valgus, shortening of the fourth metacarpal and madelund's deformity characteristic of leri-weill dyschondrosteosis; soft tissue anomalies such as webbed neck, low posterior hair line, and puffy hands and feet; visceral anomalies such as aortic coarctation, horseshoe kidney, polycystic kidney, urethral stenosis and vesicourethral refl ux; and miscellaneous anomalies such as nevus pigmentosus. 663 during embryonic life, these gonads show normal germ cell numbers up to the third month, when germ cell proliferation ceases. 665, 666 ovogenesis stops in meiosis i, usually before the pachytene stage. the cause seems to be generalized meiotic pairing errors with the start of an apoptotic mechanism to avoid the formation of abnormal gametes. 667 massive apoptosis of ovocytes occurs between the 15th and the 20th weeks. 668 surviving germ cells disappear throughout fetal life, and their numbers at birth are usually low ( fig. 12-66 ). 669 patients with mosaicism have fewer anomalies than pure 45x0 individuals; 12% have menstruation (compared to 3% of pure 45x0 patients), and 18% have breast development (compared to 5% of pure 45x0 patients). in 10-20% of 45x0 patients the sry gene is demonstrable by in-situ hybridization. it has been proposed that patients with sry expression should undergo gonadectomy, because this gene is also a marker of gonadoblastoma. 670 these patients may develop gonadoblastoma, dysgerminoma, and mixed germ cell tumor. 670, 671 mixed gonadal dysgenesis is characterized by the presence of a streak gonad and a contralateral testis (often cryptorchid) or streak testis (see discussion on male pseudohermaphroditism with müllerian remnants, below). true hermaphroditism is a disorder of gonadal differentiation characterized by the presence in the same individual of both testicular and ovarian tissue. this condition is rare and usually diffi cult to diagnose, so only 25% of male hermaphrodites are diagnosed before age 20. 672 failure to recognize this disorder may lead to surgical intervention for hernia repair or orchidopexy. most hermaphrodites raised as males display symptoms for the fi rst time at puberty because of breast development 673 (95% of hermaphrodites have some degree of gynecomastia), periodic hematuria 674 (if they have a uterus ending in the urinary tract), or cryptorchidism. 675 hermaphrodites raised as females initially present with irregular menstruation or clitoromegaly. true hermaphroditism should be suspected in all children with ambiguous sex characteristics ( fig. 12-67 ). the gonads of these patients are ovotestes, ovaries, or testes, with all possible combinations. 676 true hermaphroditism can be (1) unilateral, if there are both testicular and ovarian tissues (forming one ovotestes or two separated gonads) on one side, and a testis or an ovary in the other side; if there is no gonadal tissue in this latter side, unilateral hermaphroditism is incomplete; (2) bilateral, if testicular and ovarian tissues are present on both sides of the body; and (3) alternate, if there is a testis on one side, and an ovary on the other side. ovotestis is the most frequent gonadal type in true hermaphroditism. it is more frequent on the right side and is located in the abdomen (50% of cases), labioscrotal folds, inguinal canal, or the external inguinal ring. the ovotestis has a bilobated or ovoid shape ( fig. 12-68 ). in the bilobated ovotestis the testis and ovary are connected by a pedicle, whereas in the ovoid ovotestis the ovarian tissue forms a crescent capping the testicular parenchyma. the proportion of ovary to testis varies widely ( fig. 12-69 ). at adulthood, the ovarian follicles mature and corpora lutea or corpora albicantia may be seen. the seminiferous tubules rarely develop complete spermatogenesis. the interstitium usually contains leydig cells. ovotestis is associated with a fallopian tube in 65% of cases, and with a vas deferens in the remainder. if the patient has ovotestis/ovary, a completely developed uterus is present. if the patient has bilateral ovotestis (13%), uterine agenesis is frequent (fig. 12-70 ). 677 the testis of hermaphrodites is most often on the right side (60%) and is located anywhere from the abdomen to the scrotum. these testes have low tubular fertility indices during childhood. after puberty, the seminiferous tubules remain small, often containing only dysgenetic sertoli cells, similar to the tubules of cryptorchid testes. incomplete spermatogenesis has been reported, but complete spermatogenesis is exceptional. the ovary of hermaphrodites is most frequently on the left side (63%) and usually is hypoplastic with few primordial follicles. however, in occasional patients the ovary is histologically and functionally normal. the most frequent karyotype is 46xx (60%), followed by several mosaicisms (33%) which, in decreasing order of frequency, are 46xx/46xy, 46xy/47xxy, 45x0/46xy, 46xx/47xxy. the 46xy karyotype is the least common (7%). there is variation in the incidence of some karyotypes around the world. mosaicism is found in 40.5% of european cases, but in only 21% of north america cases. conversely, most african true hermaphrodites (97%) have 46xx karyotype. the karyotype 46xy is rare and its frequency is similar in europe, asia, and north america. 678, 679 most cases are sporadic, and families with several affected members also have 46xx males. this fi nding suggests that both genetic anomalies are alternative forms of a single genetic defect. 680 the following mechanisms 681, 682 have been proposed to explain the occurrence of testicular parenchyma: true hermaphroditism 46xx, a hidden mosaicism with a cell line having a y chromosome; transfer from a y chromosome fragment (including sry gene) to the x chromosome; autosomal mutation of variable penetrance; and x-linked mutation coupled with rare x inactivation or x mutation that permits testicular differentiation in the absence of sry. some 46xx hermaphrodites with sry-negative leukocytes are positive for this gene in dna from the testicular parenchyma in the ovotestis. 683 over 22 pregnancies in true hermaphrodites have been reported, 684 in contrast to the exceptional cases of paternity. ovules may arise from the ovotestes or the ovary. management of true hermaphroditism depends on the patient's age at the time of diagnosis, the nature and location of the gonads, and the developmental stage of the external genitalia. although bilateral castration may be justifi ed in order to avoid the risk of neoplasia, gonadal preservation may be desirable until adulthood. in this case, if the patient is raised as a girl, puberty will occur spontaneously and there is a small chance of fertility. 685 however, the high risk of malignancy (estimated at 4.6%) should be taken into account. the most frequent tumors are gonadoblastoma, dysgerminoma, and yolk sac tumor. 676 the risk of cancer may be reduced if some precautions are taken, including removal of the testis if it has not descended and surveillance of the residual gonad with periodic ultrasound studies, especially in cases of chromosomal mosaicisms. normal male development requires adequate differentiation of the testes in the fetal period, synthesis and secretion of testicular hormones, and proper response of target organs to these hormones. anti-müllerian hormone produced by sertoli cells inhibits the development of müllerian derivatives that would otherwise form the uterus and fallopian tubes. testosterone produced by leydig cells stimulates differentiation of the wolffi an ducts into male genital ducts. the conversion of testosterone into dihydrotestosterone by the enzyme 5α-reductase ensures the development of male external genitalia. alterations in these processes may cause male pseudohermaphroditism. androgen synthesis defi ciencies these autosomal recessive syndromes are characterized by an error in testosterone synthesis that results in incomplete or absent virilization. cholesterol is the source for the synthesis of androgens, estrogens, and other steroid hormones through multiple steps. first, the steroidogenic acute regulatory protein (star) generates cholesterol into mitochondria; star gene mutations cause congenital lipoid adrenal hyperplasia. second, within mitochondria, the cholesterol sidechain cleavage enzyme p450scc transforms cholesterol into pregnenolone; a disorder in this enzyme is rare because it is highly lethal in embryonic life. third, pregnenolone undergoes 17α-hydroxylation by microsomal p450c17; defi ciency in 17α-hydroxylase causes female sexual infantilism and hypertension. fourth, 17-oh-pregnenolone is converted into dhea by 17,20-lyase activity of p450c17. the ratio of 17,20-lyase to 17α-hydroxylase activity of p450c17 determines the ratio of c21 to c19 steroids produced. the ratio is regulated by at least three factors, including the electrondonating protein p450 oxidoreductase (por), cytochrome b5, and serine phosphorylation of p450c17. mutations in por are present in the antley-bixler skeletal dysplasia syndrome as well as a variant of polycystic ovarian syndrome. figure 12 -71 shows the enzymes involved in the abovementioned steps. the enzyme 3β-hydroxysteroid dehydrogenase transforms dhea to androstenedione, and the enzymatic complex called aromatase transforms androstenedione into estrone and testosterone into estradiol. in some patients cholesterol synthesis is also impaired, and congenital adrenal hyperplasia is superimposed on androgen defi ciency. defi cient testosterone synthesis may result from abnormalities in the enzymes involved in pregnenolone formation (congenital lipoid adrenal hyperplasia), including 3β hydroxysteroid dehydrogenase, 17αhydroxylase, 17,20-desmolase, and 17β-hydroxysteroid dehydrogenase ( fig. 12-71 ). congenital lipoid adrenal hyperplasia is the most severe form of congenital adrenal hyperplasia. 686 the disorder is characterized by a defi cit in steroid hormone synthesis in the adrenal cortex and gonads, producing a female phenotype with severe salt-loss syndrome. conversion of cholesterol to pregnenolone requires the enzymes 20α-hydroxylase, 20,22desmolase, and 22α-hydroxylase. failure of any of these leads to defi cits in cortisol, aldosterone, and testosterone. 687 the enzymatic defect is usually is caused by a defi cit in the steroidogenic acute regulatory (star) protein; in other cases, the defi cit is in p450ssc. the mitochondrial protein star promotes cholesterol transfer from the outer to inner mitochondrial membrane, where cholesterol serves as a substrate for p450scc and initiates steroidogenesis. more than 35 different mutations in the star gene have been identifi ed. 690 as a result, cholesterol is not converted to pregnenolone, which is required for the synthesis of mineralocorticoids, glucocorticoids, and sex hormones. the disorder is rare in most countries, but is common in japan, korea, and the arabian countries. 691, 692 patients usually present with salt-losing crisis in the fi rst 2 months of life. 693, 694 in most cases, males have female or ambiguous external genitalia and a blind-sac vagina, hypoplastic wolffi an derivatives, absence of müllerian structures, and cryptorchidism. 695 the adrenals usually appear enlarged and contain lipid accumulations, 696,697 but these diminish with age and the adrenals shrink. in the testes, lipid accumulations may be present or absent in leydig cells 686, 696, [698] [699] [700] [701] [702] or sertoli cells. 703 an 8-year-old child had partially hyalinized seminiferous tubules with sertoli cell-only pattern. 704 the testes of pubertal patients are usually normal for age. 701, 702 intratubular germ cell neoplasia has been reported in one case. 705 most patients die from adrenal insuffi ciency. survivors have female phenotype 704 and require the administration of glucocorticoids, mineralocorticoids, and gonadal steroids. 703 3β-hydroxysteroid dehydrogenase defi ciency patients with this defect have two main problems: salt-loss syndrome produced by reduced aldosterone secretion, and incomplete virilization. 706 at puberty, virilization increases and gynecomastia develops. 707, 708 the enzyme 3bhsd catalyzes the conversion of 5-3βhydroxysteroids such as pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone into respectively 4-3β-ketosteroid, progesterone, 17-hydroxyprogesterone, and androstenedione. 709 there are two 3bhsd genes located on the p11-p13 region of chromosome 1. the type i gene is expressed in the placenta, kidney, and skin, whereas the type ii gene 3bhsd is expressed only in the gonads and adrenal glands. complete absence of the 3bhsd gene is lethal; therefore, most reported cases have only partial 3bhsd deficits. [710] [711] [712] it is assumed that these defi cits account for 10% of cases of congenital adrenal hyperplasia. the classic form of salt-losing 3bhsd defi cit is diagnosed in the fi rst months of life because of insuffi cient aldosterone synthesis and subsequent loss of salt. the other 3bhsd defi cit, without salt loss, is due to mutations in the type ii 3bhsd gene 708 and its diagnosis may be delayed until puberty. severe forms of 3bhsd defi ciency are associated with defi cits in aldosterone, cortisol, and estradiol. symptoms may vary widely, as enzymatic activity in the adrenal gland is not the same as in the testis. most patients show salt loss and adrenal crisis; they have incomplete masculinization and may develop spontaneous puberty and gynecomastia. 706, 707, 713 patients with mild forms have normal genitalia and normal mineralocorticoid levels. some patients have only hypospadias 714 or micropenis. 715 the testes are smaller and softer than normal. 17α-hydroxylase defi ciency the cause of defi cits in the enzymes 17α-hydroxylase and 17,20-lyase are mutations of the cyp17 gene that encodes cytochrome p450c17. 716 the cyp17 gene is located on chromosome 10q24-q25, 717 and 50 different mutations have been described. 718 p450c17 catalyzes the 17α-hydroxylation of pregnenolone to 17ohpregnenolone and of progesterone to 17α-oh-progesterone. this enzyme also catalyzes 17,20-lyase activity, transforming 17oh-pregnenolone to dhea. the classic form of 17αhydroxylase defi cit is caused by severe defi ciencies in cyp17; less severe defects give rise to the isolated 17,20-lyase defi cit. 17α-hydroxylase defi cit impairs the synthesis of both cortisol and testosterone. 719 low cortisol levels stimulate acth secretion, causing hypersecretion of aldosterone precursors and the development of hypokalemic hypertension and male pseudohermaphroditism in males. 720 patients usually have hypospadias and develop gynecomastia at puberty. 721 the enzyme 17,20-desmolase cleaves the side chain of 17-hydroxypregnenolone and 17hydroxyprogesterone to form dehydroepiandrosterone and androstenedione, respectively. varying degrees of 17,20desmolase defi ciency are seen, resulting in varied development of external genitalia, ranging from female phenotype to virilization with microphallus, bifi d scrotum, and perineal hypospadias. in childhood, the testes contain reduced numbers of spermatogonia (figs 12-72, 12-73) . 722, 723 the cause may be mutations in one of the genetic loci encoding p450c17, fl avoprotein or or b5. 724 17β-hydroxysteroid dehydrogenase defi ciency this enzyme transforms androstenedione into testosterone and also converts estrone into estradiol. the enzymatic defects are sexlinked. most patients have female phenotype at birth and are raised as girls, but at puberty undergo virilization. 725 one or both testes may be cryptorchid or are located in the labia majora. normal spermatogenesis has never been observed. the most common testicular patterns are hypoplasia or absence of germ cells and leydig cell hyperplasia. 726 the germ cell injury was initially attributed to cryptorchidism, but it is now thought to be a primary testicular lesion because even very young patients lack germ cells. 727 this defi cit is due to mutations in the hsd17b3 gene located on 9q22. 728, 729 leydig cell hypoplasia this variant of male pseudohermaphroditism is defi ned by insuffi cient testosterone secretion 422 and the following characteristics: predominance of female external genitalia; absence of male secondary sex characteristics at puberty; absence of uterus and fallopian tubes and the presence of epididymis and vas deferens; 46xy karyotype; lack of response to human chorionic gonadotropin stimulation; absence of an enzymatic defect in testosterone synthesis; and small undescended testes that are gray and mucous on section. [730] [731] [732] [733] age at diagnosis varies from 4 months to 35 years. the syndrome is sporadic and familial. 734, 735 the best-known cause of leydig cell hypoplasia is inactivating mutation of the lh receptor in these cells. [736] [737] [738] during fetal life, there is an inadequate response to placental hcg initially and to pituitary lh subsequently. phenotypes vary widely according to the presence of complete or partial loss of receptor function. these changes range from male pseudohermaphroditism with female external genitalia (type i of leydig cell hypoplasia) to male phenotype with micropenis, hypospadias, pubertal delay, and primary hypogonadism (type ii of leydig cell hypoplasia). in type i hypoplasia, the testes contain small seminiferous tubules with sertoli cells, spermatogonia, and thickened basement membranes. leydig cells are rare or absent, in contrast to leydig cell hyperplasia seen in other types of male pseudohermaphroditism, such as those arising from defects in androgen synthesis or androgen action on peripheral tissues. 739, 740 leydig cell hypoplasia accounts for low serum testosterone levels, lack of virilization, and lack of spermatogenesis. the absence of müllerian derivatives suggests a normal function of sertoli cells, which synthesize müllerian inhibiting factor. in type ii hypoplasia, adult testes show maturation arrest of spermatogonia and a few incompletely differentiated leydig cells. 741, 742 resistance to androgen stimulation is the cause of several syndromes with phenotypes varying from complete testicular feminization 743 to normal male. 744, 745 these syndromes are caused by partial or complete lack of response of the target organs to androgens 746 due to the absence, diminution, or impairment of androgen receptors or postreceptor anomaly. 740 the gene for the androgen receptor is located on the x chromosome (xq11-q12), and x-linked transmission occurs in two-thirds of cases. the karyotype is usually 46xy, but 47xxy and several mosaicisms have been observed. 747 these syndromes affect 1 : 20 000-1 : 40 000 newborns. the diverse phenotypes associated with androgen insensitivity may be classifi ed as: complete androgen insensitivity syndrome (cais) or testicular feminization syndrome; partial androgen insensitivity syndrome (pais) or partial testicular feminization syndrome, which includes the syndromes of lubs, gilbert-dreyfus, reifenstein, and rossewater; and mild androgen insensitivity syndrome (mais), infertile men with light androgen insensitivity, and kennedy's disease. complete androgen insensitivity syndrome (complete testicular feminization syndrome) this form of male pseudohermaphroditism is characterized by female phenotype with testes. complete testicular feminization syndrome is rarely diagnosed during childhood except in patients who present with hernia, inguinal tumor, or with a family history of pseudohermaphroditism. primary amenorrhea is the principal presentation in adults. the testes may be in the abdomen, inguinal canal, or labia majora, and during the fi rst year of life may be normal histologically except for reduced tubular diameter and low tubular fertility index. after the fi rst year, decreased germ cell numbers become evident and the few remaining spermatogonia are concentrated in clusters of seminiferous tubules. the testicular interstitium contains numerous spindle cells arranged in bundles, and during the fi rst year of life has leydig cells with abundant eosinophilic or vacuolated cytoplasm. at puberty, patients have female external genitalia, a short blind-ended vagina, feminine breast development; and scarce pubic and axillary hair. serum testosterone is at the normal male level and lh is markedly increased. in adults, the testes vary in size from small to large, are tan-brown, and contain small seminiferous tubules without lumina which usually contain only sertoli cells. 748, 749 in one-third of patients both sertoli cells and spermatogonia are present. 750 ultrastructurally, sertoli cells lack charcot-böttcher crystals and annulated lamellae; inter-sertoli cell specialized junctions are not well developed, and in cryofracture studies the arrangement of membrane particles has an immature pattern. 751 leydig cells are abundant, but few contain reinke's crystalloids. often, there are areas resembling ovarian stroma in the testicular interstitium. in about two-thirds of cases the testes contain grossly visible white nodules that stand out from the surrounding testicular parenchyma (figs 12-74, 21-75). histologically, the nodules consist of clusters of small seminiferous tubules with immature sertoli cells, hyalinized lamina propria, numerous leydig cells, and an absence of elastic fi bers ( fig. 12-76) . these have been referred to as sertoli-leydig cell hamartoma. about 25% of testes have sertoli cell adenoma, sometimes very large, consisting of tubules resembling infantile testis but lacking in germ cells and peritubular myofi broblasts. no leydig cells are present between the tubules (figs 12-77, 12-78). 752 other benign tumors include sertoli cell tumor (large cell calcifying sertoli cell tumor and sex cord tumor with annular tubules), leydig cell tumor, leiomyoma, and fi broma. 746 approximately 60% of cases have small cystic structures closely apposed to the testes, and about 80% of patients have thick bundles of smooth muscle fi bers resembling myometrium near the testis. true myometrium has been demonstrated in only one case. hypoplastic fallopian tubes are present in about one-third of cases. in about 70% of patients the epididymis and vas deferens are rudimentary; the only explanation for this is residual activity of the mutated androgen receptor. 753 approximately 10% of testes from patients with testicular feminization syndrome develop cancer. the frequency increases with age, but tumors rarely appear before puberty. these tumors include intratubular germ cell neoplasia ( fig. 12-79) , 749 several types of germ cell tumor, 750, 754 and sex cord tumor. 441 thus, the gonads should be removed immediately after puberty. 755 partial androgen insensitivity syndrome (partial testicular feminization syndrome) the phenotype of patients with partial testicular feminization varies from normal female to normal male. the disorder includes four classic syndromes: lubs' syndrome, 756 characterized by partial fusion of labioscrotal folds, a defi nitive introitus, clitoromegaly, pubic and axillary hair, and poor breast development; 757 gilbert-dreyfus syndrome, characterized by progressively greater male phenotypic features that include small phallus, hypospadias, incomplete development of wolffi an derivatives, and gynecomastia; 758 reifenstein's syndrome, characterized by hypospadias, weak or absent virilization, testicular atrophy, gynecomastia, azoospermia, and infertility; 759 and rosewater-gwinup-hamwi syndrome, characterized by infertile men whose only abnormal feature is gynecomastia. 760 mild androgen insensitivity syndrome spermatogenesis requires high levels of intratesticular testosterone. a minor form of androgen insensitivity may be observed in some patients with male phenotype who present with infertility. 761 the frequency of androgen resistance among azoospermic and oligozoospermic men is estimated at about 19% 762 or lower. 763, 764 some patients have lost exon 4 765 or mutated exons 6 764 or 7. 766 kennedy's disease kennedy's disease (spinal and bulbar muscular atrophy, sbma) is an x-linked recessive disorder of the adult male 767,768 characterized by loss of motor neurons in the spinal cord and brain stem and associated with less important loss of sensory neurons and atrophy caused by skeletal muscle denervation. 767, 769 disease onset around 20 years of age includes muscular weakness, cramps, and fasciculations. 770 in most cases the male reproductive system is impaired. [770] [771] [772] the testes may be normal in the initial stages of the disease, and many patients are fertile; however, with progression, there is onset of secondary testicular atrophy and gynecomastia. testosterone levels are decreased in some cases. the disease results from mutations in the fi rst exon of the androgen receptor (ar) gene. 773 the smba gene, located on xq11-12, has expansion of a repetitive cag sequence in exon a. the number of cag repeats is 21 (range, 17-26) in control men and more than 40 in men with kennedy's disease. 768, [774] [775] [776] [777] this disorder is a variant of male pseudohermaphroditism caused by a lack of the enzyme 5α-reductase with failure of conversion of testosterone to dihydrotestosterone. 778 in patients with the 46xy karyotype there are two isoenzymes: isoenzyme 1 is encoded by the gene srd5a, located on 5p15, and isoenzyme 2 is encoded by the gene srd5a2 on 2p23. most reported cases result from defects in srd5a2. 779 many mutations in different exons have been reported. [780] [781] [782] [783] [784] during childhood, patients have a clitoriform penis, bifi d scrotum, urogenital sinus, and testes in the inguinal canal or labioscrotal folds (fig. 12-80) . müllerian derivatives are absent. at puberty they acquire the male phenotype, with development of the penis and scrotum. adults have erections, ejaculations, and normal libido, scant body hair and a thin beard, a very small prostate, and lack of temporal hairline recession (male pattern baldness). serum levels of fsh, lh, and testosterone are increased, but dihydrotestosterone is decreased. 785, 786 the disorder is autosomal recessive and has been observed in many consanguineous families from the dominican republic. 787 this group of male pseudohermaphrodites is characterized by the presence of müllerian derivatives and unilateral or bilateral testicular dysgenesis. these two features depend on anti-müllerian hormone gene mutations and end-organ insensitivity. [788] [789] [790] [791] in normal development, anti-müllerian hormone is responsible for inhibition of the ipsilateral müllerian ducts and collagenization of the tunica albuginea. patients with defi cient secretion of this hormone may also have androgen defi ciency. three variants of defective müllerian duct regression have been reported: mixed gonadal dysgenesis, dysgenetic male pseudohermaphroditism, and persistent müllerian duct syndrome. mixed gonadal dysgenesis (asymmetric gonadal differentiation) is characterized by the presence of a testis on one side of the body and a streak gonad on the other. 792 if the gonads are intra-abdominal, the labioscrotal folds may appear as either normal labia or empty scrotal sacs (fig. 12-81 ). in the former, the syndrome cannot be recognized in the newborn unless a peniform clitoris is present. if the gonad is descended, it is usually a testis. müllerian derivatives such as fallopian tubes are usually associated with streak gonad (95% of cases), but may also be associated with testicular tissue (74%). ipsilateral to the testis there is one epididymis and one vas deferens. on the contralateral side, no gonad or a streak gonad and a fallopian tube are present. a hypoplastic uterus and a poorly developed vagina are frequent fi ndings. this syndrome accounts for about 15% of intersex conditions. some patients are raised as males, although their external genitalia are usually ambiguous as a result of fetal virilization. the penis is clitoriform, and the urethra opens in the perineum. most have cryptorchid testes and are raised as girls, becoming virilized at puberty. infertility is a common symptom. 793 the etiology is heterogeneous: 794 one-third of patients have turnerian features, in accordance with the presence of the 45x0/46xy karyotype in more than 50% of patients. other observed karyotypes are 46xy and 45x0/47xyy. approximately 81% of patients have one y chromosome. mutation in the sry gene has not been found. 795 the testes can show two different patterns: testicular dysgenesis and streak testis. testicular dysgenesis is characterized by a tunica albuginea that varies in width and is reminiscent of ovarian stroma by the storiform distribution of cells and fi bers; there are also malformed seminiferous tubules (fig. 12-82 ) that are small, usually lack lumina, and contain only immature sertoli cells. in adults, spermatogenesis has been observed occasionally. the testicular interstitium contains increased numbers of leydig cells. streak testes are complex gonads in which testicular dysgenesis is associated with a fi brous streak. most of the gonad consists of a testis showing the characteristic lesions of testicular dysgenesis. in a pole of the gonad, or in continuity with it, there is a fi brous streak whose structure may correspond to any of the varieties mentioned above (fig. 12-83 ). this peculiar gonad can also be observed in some dysgenetic male pseudohermaphrodites as well as in the persistent müllerian duct syndrome. in these cases, the streak contains no ovocytes. light microscopy indicates a wide spectrum of testicular lesions, ranging from those of patients with 46xy pure gonadal dysgenesis to true hermaphroditism. differentiation of the ovocyte-containing streak testis and ovotestis remains controversial. 796, 797 the testes in mixed gonadal dysgenesis are incapable of müllerian duct inhibition and allow complete differentiation of wolffi an derivatives, virilization of external genitalia, and, in most cases, testicular descent. the risk of germ cell neoplasia reaches 50% in the third decade of life, usually beginning with gonadoblastoma. the testes should be removed after puberty. dysgenetic male pseudohermaphroditism is a disorder of sexual differentiation characterized by bilateral dysgenetic testes or streak testis, persistent müllerian structures, and cryptorchidism. this syndrome is considered a variant of mixed gonadal dysgenesis ( fig. 12-84) . 791, 798 the karyotype may be 46xy or 45x0/46xy, and turnerian stigmata may be present. the uterus and fallopian tubes are present and both are usually hypoplastic (fig. 12-85 ). 799 the testes show lesions characteristic of testicular dysgenesis, with few germ cells during childhood (fig. 12-85) . 799 in adults, spermatogenesis is poorly developed and the testicular interstitium shows leydig cell hyperplasia. about 25% of patients develop gonadoblastoma. 800 persistent müllerian duct syndrome has many names, including male with uterus, tubular hermaphroditism, persistent oviduct syndrome, and hernia uteri inguinalis. 801 it is a rare form of pseudohermaphroditism, with müllerian derivatives in an otherwise phenotypically normal male, and is the most characteristic form of isolated anti-müllerian hormone defi ciency. the molecular basis of this syndrome is heterogeneous. three hypotheses have been proposed, including a defect in anti-müllerian hormone synthesis, caused by mutation in the anti-müllerian hormone gene (45% of cases); resistance of target organs to this hormone, caused by mutation in the receptor ii for this hormone (39% of cases); and failure in the action of this hormone immediately before the eighth week of gestation (16% of cases). 802 although the external genitalia are male, one (35% of cases) or both testes (75% of cases) are cryptorchid. the syndrome usually also includes inguinal hernia contralateral to the undescended testis, with a uterus and fallopian tubes within the hernia sac (figs 12-86, 12-87) . 803 several cases with transverse testicular ectopia and persistent müllerian duct structures have been reported. 804, 805 patients usually have inguinal hernia, but others have cryptorchidism, infertility, 806 and testicular tumor. 807 in childhood, the testes have a low tubular fertility index and decreased tubular diameter. in adults, the tunica albuginea is variably thickened, contains connective tissue resembling ovarian stroma, and may contain tubular structures -alterations typical of testicular dysgenesis. the seminiferous tubules are usually atrophic and hyalinized. tubules with reduced spermatogenesis or patterns suggesting mixed atrophy (seminiferous tubules with spermatogenesis intermingled with sertoli cell-only tubules) have also been reported. the leydig cells appear hyperplastic. azoospermia or oligozoospermia are common, and paternity is exceptional. 808 the syndrome is sporadic or familial, with autosomal recessive or x-linked inheritance. 809, 810 these patients have a higher risk of testicular tumor than that attributed to cryptorchidism, 811 and all types of germ cell tumor have been observed. 812, 813 other forms of male pseudohermaphroditism of the dysmorphic syndromes associated with incomplete virilization of external genitalia, the best-known are those of rsh, denys-drash, wagr, opiz, camptomelic dysplasia, atr-x, gardner-silengo-wachtel, meckel, branchioskeletal-genital, down's, and other trisomies. rsh (smith-lemli-opitz) syndrome is a malformative recessive autosomal syndrome caused by mutations in the gene encoding for 7-dehydrocholesterol reductase (dhcr7), responsible for the synthesis of cholesterol from its immediate precursor 7-dehydrocholesterol. [814] [815] [816] the disorder is common in europe and rare in other countries. 817 the most severe form is lethal before birth. fetuses show postaxial oligodactyly (instead of polydactyly) and sometimes severe hydrops. 818 non-lethal forms are characterized after birth by severe growth failure; a semi-obtunded state; absence of psychomotor development; microcephaly; congenital cataracts; peculiar facies; broad anteriorly rugose alveolar ridges with cleft palate, edema of the nape of the neck, and unilobulate lungs; male pseudohermaphroditism or female external genitalia in 46,xy patients; postaxial polydactyly of the hands and feet; congenital heart defects; and renal anomalies. 819 hepatic and renal insuffi ciencies are frequent. 820 the less severe forms in the male have genital anomalies (70%) varying from normal genitalia to severe hypospadias with or without cryptorchidism, and numerous small anomalies whose collection characterizes the syndrome. most patients also show mental retardation and severe behavioral problems. 821 the dhcr7 gene maps to chromosome 11q12-13. its product is a microsomal, membrane-bound protein. many different missense, nonsense, and splice-site mutations as well as duplications and deletions have been reported. [822] [823] [824] [825] [826] [827] prenatal diagnosis is possible by relating ultrasound and cytogenetic studies and carrying out a biochemical analysis in the second trimester in those pregnant women who have low levels or no conjugated estriol. 828 in denys-drash syndrome, male pseudohermaphroditism is associated with nephroblastoma and renal insufficiency. 829 the pseudohermaphroditism is usually either mixed gonadal dysgenesis, dysgenetic male pseudohermaphroditism, 46xy pure gonadal dysgenesis, or true hermaphroditism. 830 the most common nephropathy is diffuse mesangial sclerosis. 831 most patients have mutations in the wt-1 gene, 832 which is expressed in the genital ridge in the sixth week of gestation and gives rise to either streak gonads or testicular dysgenesis, but, if a delay in testicular determination occurs, normal testes are formed. 833 the term wagr syndrome refers to wilms' tumor, aniridia, genital anomalies, and mental retardation. prevalence is estimated at between 0.75% and 2% of wilms' tumor patients. the syndrome is related to the syndrome of denys-drash and that of frasier (a variety of 46,xy gonadal dysgenesis). 834, 835 all have in common mutations in the wt-1 gene located on chromosome 11 (11p13). wt-1 product is a transcription factor expressed in different tissues that participates in embryogenesis and cell differentiation. mutations lead to the production of an anomalous protein that causes alterations in renal function, gonadal anomalies, and the loss of tumor suppressor function. six variants of alleles have been described: isolated wilms' tumor, mesothelioma, isolated diffuse mesangial sclerosis, denis-drash syndrome, frasier syndrome, and wagr syndrome. frasier syndrome is caused by mutations in the donor zone of the intron 9 link, with the subsequent loss of the +kts isoform (the patient has an imbalance in kts isoforms), whereas large deletions or loss of genetic material that comprises the wt-1 gene and other contiguous genes (pax6 or an) lead to the wagr syndrome. 836, 837 patients with opitz's syndrome are mainly boys with hypertelorism and, in the severe forms, unilateral or bilateral lip cleft, laryngeal cleft, severe dysphagia with more or less life-threatening aspiration, hypospadias and, occasionally, imperforate anus. the most important internal anomalies are those in the tracheobronchial tree, cardiovascular system (defects in cardiac septation), and gallbladder, with a subjacent defect of the developing embryonal ventral midline. the syndrome is genetically heterogeneous and consists of two entities that were described as different in the past: ados, or autosomal dominant opitz syndrome or g syndrome 838 with a mutated gene that maps to 22q11.2; and xlos, or x-linked opitz syndrome or bbb syndrome 839 with a mutated gene that maps to xp22.3. 840, 841 camptomelic dysplasia is an autosomal dominant syndrome with multiple osseous malformations. patients have 46xy karyotype and external genitalia that are ambiguous or female. gonadal histology varies from testes to dysgenetic ovaries with primary follicles. the cause is a haploinsufficiency of sox9, located on 17q. 842 the incidence of gonadoblastoma is low. atr-x syndrome is characterized by mild α-thalassemia, mental retardation, facial dysmorphism, and hypospadias. 843, 844 the disorder is x-linked, and is caused by mutation in the art-x gene (synonymous xnp, hx2). 845 testicular biopsy to diagnose infertility began in the 1940s, 846, 847 and most of the diagnostic terms used today were created then. 848 these terms are usually descriptive and, except for a few (normal testes, sertoli cell-only tubules, tubular hyalinization, for example), do not specify the degree of tubular abnormality that is evaluated by each pathologist subjectively. the terms maturation arrest and hypospermatogenesis have been applied to biopsies in more than 50% of cases of infertility, 849-851 but the criteria for these vary widely among pathologists. two forms of maturation arrest have been described: spermatogenic arrest, and spermatocytic arrest, or its equivalent, meiotic arrest. true spermatogenic arrest is rare because germ cell maturation usually does not arrest at the level of a defi ned germ cell type. 852 to avoid confusion the term irregular hypospermatogenesis has been proposed 853 for testicular biopsies with decreased numbers of germ cells, subclassifi ed as slight, moderate, or severe. however, this diagnosis is of little help to clinicians. the reported frequency of spermatocytic (meiotic) arrest in infertile men varies from 12% 854 to 32.1% 855 and is present in one or both testes of about 18% of oligozoospermic or azoospermic patients. 856 if observed in only one testis, the contralateral testis may show histologic changes ranging from normal spermatogenesis to hyalinized tubules. disorganization of the seminiferous tubular cell layers is another frequent diagnosis in testicular biopsies, 848,857,858 but this term is rejected by many pathologists. actual disorganization of the seminiferous tubular cells is unlikely and has not been demonstrated in ultrastructural studies. in most cases, the apparent disorganization is an artifact induced by handling or fi xation. 859, 860 the term tubular blockage was introduced by meinhard and co-workers 858 for testes with at least 50% of seminiferous tubules devoid of a central lumen and showing spatial disorganization of germ cells. this morphology was found in 28% of testicular biopsies from infertile men, mainly those with obstructive azoospermia. 861 although this appearance can result from improper fi xation, 862 the accumulation of sertoli cells and immature germ cells in the centers of tubules suggests a specifi c lesion, a variant of germ cell sloughing. diagnostic confusion decreased the interest and trust of urologists and andrologists in the study of testicular biopsies. subsequent studies attempted to correlate semen spermatozoa concentration with testicular size and biochemical fi ndings such as serum levels of fsh, and testicular biopsies were undertaken in only a limited number of oligozoospermic and azoospermic patients. 859, 862, 863 however, these studies were also discouraging because fsh was found to correlate poorly with numbers of spermatozoa in the semen but better with numbers of spermatogonia in the seminiferous tubules, 864 and normal numbers of spermatozoa can be produced by relatively small testes whereas some large testes have no spermatogenesis. in recent years, serum levels of inhibin b have been shown to have a positive correlation with spermatozoon numbers and serum fsh level. 865, 866 the development of morphometry caused a resurgence of interest in biopsies. many semiquantitative 853,867-869 and quantitative [870] [871] [872] [873] [874] [875] studies were carried out. the greatest achievements of these studies were enhancement of the reproducibility of results and better evaluation of the reversibility of lesions. morphometry emerged as the best method to objectively evaluate the seminiferous tubular cells. 876 the scoring method of johnsen, 868 estimation of the germ cell/ sertoli cell ratio for each germ cell type, 871 and calculation of germ cell number per unit length of seminiferous tubules 870 are reliable and useful. several methods are available to evaluate the leydig cell population, including the mean number of cells per seminiferous tubule and per cell cluster; the mean number of leydig cell clusters per seminiferous tubule; the ratio of leydig cell area to seminiferous tubule area; 877 and the ratio of leydig cells to sertoli cells. 878 these methods have shown that the appearance of leydig cell hyperplasia described in many conditions is false, and that true leydig cell hyperplasia is extremely rare. optimal interpretation of testicular biopsies depends on the surgical technique by which the tissue sample is taken, the care and delicacy with which the specimen is manipulated, and proper fi xation and processing of the tissue. the size of the biopsy should not be greater than a grain of rice: that is, no diameter should be greater than 3 mm. this amounts to about 0.12% of testicular volume (normal volume is approximately 20 ml). the biopsy should be bilateral because in more than 28% of patients the fi ndings differ between the testes. at the time of biopsy, the testicular axes should be measured as the basis of quantitative studies. the tissue should be taken opposite to the rete testis through a 4-5 mm incision in the tunica albuginea. this parenchyma herniates through the incision and can be carefully snipped off. if only light microscopy is to be performed, the specimen should be fi xed in bouin's fl uid for 24 hours. if electron microscopy is indicated, a small biopsy fragment should be fi xed in glutaraldehyde-osmium tetroxide or a similar fi xative. to perform meiotic studies, testicular biopsy should be processed by air-drying or surface-spreading methods. the examination of testicular biopsies includes qualitative and quantitative evaluation of the testis and correlation between the biopsy and spermiogram. light microscopy immediately reveals whether the lesion is focal or diffuse. if focal, the percentage of tubules showing each lesion (sertoli cell-only, hyalinization, tubular hypoplasia, etc.) should be calculated. it is useful to evaluate elastic fi bers with a special stain because this highlights groups of small tubules that may be missed with hematoxylin and eosin. a minimum of 30 cross-sectioned tubules should be studied (this is usually possible when fi ve or six histological sections are available). the diameter of each tubule should be measured, and the number of spermatogonia, primary spermatocytes, young spermatids (also called round spermatids or s a + s b spermatids), mature spermatids (also called elongated or s c + s d spermatids), sertoli cells, and, in some cases, peritubular cells counted. the presence of tubular diverticula, 879 the most frequently observed lesions are sertoli cell-only tubules, tubular hyalinization, alterations in spermatogenesis in either the adluminal or the basal compartments of seminiferous tubules, and mixed tubular atrophy. sertoli cell-only syndrome includes all azoospermias in which the seminiferous epithelium consists only of sertoli cells. to better understand this syndrome, it is necessary to consider the morphological and functional changes induced in the sertoli cell by hypophyseal gonadotropin secretion during puberty. during childhood, sertoli cells are pseudostratifi ed and their nuclei are dark, small, and round or elongated, with regular outlines and one or two small peripherally placed nucleoli. the cytoplasm lacks specialized organelles. 881 adult sertoli cells have characteristically pale, triangular nuclei with irregular, indented outlines. the nucleoli are large and have tripartite structures. the cytoplasm contains abundant smooth endoplasmic reticulum and specialized structures, including annulate lamellae, charcot-böttcher crystals, and specialized junctional complexes with other sertoli cells. the pubertal increase in length and width of the seminiferous tubules replaces the infantile pseudostratifi ed pattern with a simple columnar distribution. five variants of the sertoli cell-only syndrome are identifi ed by sertoli cell morphology, the degree of development of the seminiferous tubules, and the presence or absence of interstitial lesions. 882 these variants are designated by the appearance of the predominant sertoli cell population: immature sertoli cells, dysgenetic sertoli cells, adult sertoli cells, involuting sertoli cells, and dedifferentiated sertoli cells (fig. 12-88) . each type is associated with other tubular and interstitial alterations (table 12-7) . the most frequent types of sertoli cell-only syndrome in infertility patients are dysgenetic sertoli cells, adult sertoli cells, and involuting sertoli cells. the clinical manifestations are similar, including normal external genitalia, welldeveloped secondary male characteristics, azoospermia, elevated serum fsh level, normal or elevated serum lh level, and normal or slightly low testosterone. these clinical and histologic features were long thought to constitute a single syndrome, del castillo's syndrome, but recent ultrastructural, histochemical, immunohistochemical, and cytogenetic studies have shown that this results from a variety of syndromes that may have primary or secondary causes (table 12-7) . [883] [884] [885] [886] [887] some patients with the adult or dysgenetic sertoli cellonly syndrome variants have a few spermatozoa in their spermiograms. this discrepancy between oligozoospermia and the biopsy histology is caused by the presence of some seminiferous tubules with complete spermatogenesis elsewhere in the testicular parenchyma. sertoli cell-only syndrome with immature sertoli cells sertoli cells in adult testes with this variant of sertoli cell-only syndrome have an immature prepubertal appearance with pseudostratifi cation. the number of cells per cross-sectioned tubule is greater than normal. other tubular and interstitial features suggest immaturity, including small tubular diame-ters (<80 µm), tubules lacking central lumina, thin lamina propria lacking elastic fi bers, and interstitium lacking mature leydig cells. [888] [889] [890] this syndrome is caused by a defi ciency of both fsh and lh which begins in childhood and is responsible for the lack of maturation of the sertoli cells, tubular walls, and interstitium. subsequently, there is no renewal or differentiation of germ cells, and these eventually disappear. when these patients are treated with hormones, the biopsy may show some degree of spermatogenesis or thickening and hyalinization of the tubular basement membrane. sertoli cell-only syndrome with dysgenetic sertoli cells dysgenetic sertoli cells begin pubertal differentiation but variably deviate from normal maturation, so that the morphology of dysgenetic sertoli cells differs among tubules and even among sertoli cells within the same tubule. nuclei usually have both mature features (pale chromatin and a centrally located, tripartite nucleolus) and features of immaturity (ovoid or round shape; regular outline; and small, dense chromatin granules) (fig. 12-89) . 891 in addition to vimentin, sertoli cells immunoexpress anti-müllerian hormone (amh) 892 and cytokeratin 18. 893 immunoreaction to these two substances is assumed to be a sign of immaturity, as under normal conditions it is not detected after puberty. other signs of immaturity are poor development of the hematotesticular barrier 894 and the absence of tubular lumina. tubular lumina are very small or absent in most dysgenetic sertoli cell-containing tubules, because the ability to produce testicular fl uid is greatly reduced. sertoli cell numbers per cross-sectioned tubule are very high, and mean tubular diameter is lower than 120 µm. the tubular walls have few elastic fi bers, 534 and most tubules show a variable degree of tunica propria hyalinization. completely hyalinized tubules are frequent. the testicular interstitium contains a variable number of leydig cells (normal, decreased, or apparently increased), many of which are pleomorphic with abundant paracrystalline inclusions. 895, 896 most patients have normal or slightly subnormal testosterone level and elevated levels of fsh and lh. this syndrome can be observed in men with cryptorchid testes, at the periphery of germ cell tumors, in men with idiopathic infertility, 897 and in men with y chromosome anomalies. 898 sertoli cell-only syndrome with mature sertoli cells in this variant, most sertoli cells appear mature but are present in increased numbers (14 ± 0.8 per cross-sectioned tubule). the seminiferous tubules have small diameters, but are still larger than in the two variants described above, and central lumina are visible. the cytoplasm contains abundant vacuoles that communicate with the tubular lumina ( fig. 12-90 ). the lateral cell surfaces have many unfolding and extensive specialized junctions with other sertoli cells (from the basement membrane to the apical cytoplasmic portion). lipid droplets, usually derived from phagocytosis of spermatid tubulobulbar complexes and dead germ cells, are scant. 884 vimentin fi laments are abundant in the basal and perinuclear cytoplasm. 899 the lamina propria is normal or slightly thickened. leydig cells are normal. serum testosterone level is normal or nearly normal, and fsh and lh levels are elevated. [900] [901] [902] this syndrome is probably caused by failure of migration of primordial germ cells from the primitive yolk sac to the gonadal ridge. 903 this failure may be due to a deletion in the azfa region in yq11 904 or a mutation in the genes that encodes c-kit or its ligand (stem cell factor), responsible for migration, proliferation, and survival of germ cells. testes with this variant of sertoli cell-only syndrome have numerous changes. sertoli cell nuclei may have lobulated shapes with irregular outlines, coarse chromatin granules, and inconspicuous nucleoli. seminiferous tubules have central lumina, decreased diameters, and variable thickening of the basement membrane (fig. 12-91) . elastic fi bers are present in normal or diminished amounts. leydig cells are variably involuted. this syndrome may be a primary disorder or secondary to irradiation or cytotoxic therapy, such as cancer chemotherapy or treatment for nephrotic syndrome. 905 it is not usually possible to determine the etiology from the biopsy fi ndings alone. changes in the tubular walls are more pronounced in patients with a history of cyclophosphamide treatment, combination chemotherapy, or radiotherapy. the testicular interstitium may be fi brotic in patients treated with cis-platinum or cyclophosphamide. 906 some syndromes with involuting sertoli cells, mainly those associated with decreased number of elastic fi bers, probably express a primary testicular anomaly with involuting and dysgenetic sertoli cells within the same tubule. the presence of immature-appearing sertoli cells in the tubular wall is thickened and contains elastic fi bers, increased amounts of collagen fi bers, and elevated numbers of peritubular cells as a result of tubular shortening. mean tubular diameter is markedly decreased to less than 90 µm. the testicular interstitium contains few leydig cells, and these appear dedifferentiated or contain an increased amount of lipofuscin. this variant has been observed in surgical specimens from patients receiving androgen deprivation therapy for prostatic cancer, estrogen treatment for transsexuality, and cancer chemotherapy with cis-platinum. there is a correlation between the degree of sertoli cell dedifferentiation and the dose and timing of treatment with estrogens or anti-androgens. brief treatment induces germ cell loss and inconspicuous sertoli cell changes; long-term treatment causes pronounced sertoli cell changes, including initial nuclear rounding followed by nuclear elongation and the development of dark chromatin masses. 907 eventually, the nuclei come to resemble those of infantile sertoli cells, including pseudostratifi cation. at the same time, the tubules become hyalinized and peritubular cells increase whereas leydig cells disappear. 908, 909 estrogens act on the pituitary by inhibiting lh secretion, and on leydig cells. 910 the action of gonadotropin-releasing hormone agonist analogs is only on the pituitary, whereas cis-platinum acts only on the testis. the most common causes of tubular hyalinization include dysgenetic hyalinization, hormonal defi cit, ischemia, obstruction, infl ammation, and physical or chemical agents. the differential diagnosis is given in table 12 -8. dysgenetic hyalinization dysgenetic hyalinization is a diffuse lesion in which most tubules are uniformly hyalinized (fig. 12-92) . tubules lack seminiferous tubular cells and have a reduced number of peritubular cells. the few preserved tubules usually contain only sertoli cells, although rarely a few with spermatogenesis are present. dysgenetic hyalinization is seen in klinefelter's syndrome, testes that remain cryptorchid through puberty, and some hypergonadotropic hypogonadisms associated with myopathy. focal lesions are seen in mixed atrophy of the testis. tubular hyalinization is pronounced in klinefelter's syndrome, and from infancy the seminiferous tubules are small, containing reduced numbers of sertoli cells and few or no spermatogonia. at puberty, the dysgenetic sertoli cells fail to infertility mature and soon disappear. the tubules collapse, giving the appearance of phantom tubules. 911 peritubular cells fail to differentiate and their number is low. 912 they form a discontinuous ring around the hyalinized tubules and are incapable of synthesizing elastic fi bers and other components of the lamina propria. dysgenesis also involves the interstitium: leydig cells exhibit a characteristic adenomatous pattern, although their total number is decreased. the morphology of the leydig cell is not uniform, and there are shrunken, normal, and large forms. most contain reduced amounts of lipofuscin granules and lipid droplets. reinke's crystalloids are uncommon, and paracrystalline inclusions are abundant. 896 in spite of the hyperplastic adenomatous appearance of the leydig cells, testosterone secretion is markedly decreased, and the resulting hypogonadism is the most important clinical feature of klinefelter's syndrome. tubular hyalinization in the cryptorchid testis is also dysgenetic. however, in contrast to the atrophic collapse seen in klinefelter's syndrome, cross-sections of the hyalinized tubules in cryptorchidism are targetoid. this results from the arrangement of the peritubular cells into two layers, suggesting an atrophic process that has evolved over a longer period than in klinefelter's syndrome, or a lower degree of dysgenesis. 913 elastic fi bers are diminished. 534 in the interstitium leydig cells appear hyperplastic, forming large aggregates, although their absolute numbers are decreased. leydig cell pleomorphism is less intense than in klinefelter's syndrome. many leydig cells have abundant vacuolated cytoplasm. whereas tubular hyalinization in klinefelter's syndrome is secondary to the effect of pubertal gonadotropin secretion on dysgenetic tubules, tubular hyalinization in cryptorchidism probably results from the effect of increased temperature on the dysgenetic tubules. however, other mechanisms are also involved in cryptorchid tubular hyalinization, including obstruction of sperm excretory ducts (anomalies in these ducts are frequent in cryptorchidism) and ischemia (principally in testes that could only be incompletely descended by surgery). hyalinization caused by hormonal defi cit hormonal defi cit causes diffuse tubular hyalinization, although the tubules may be recognized for a time as cellular cords surrounded by hyaline material. sertoli cell, a few spermatogonia, and rare primary spermatocytes may be identifi ed in these cords. when hyalinization is complete, only the elastic fi bers in the lamina propria indicate the structure of the previously normal adult testis. peritubular myofi broblasts decrease in number and form a ring at the periphery of the lamina propria. leydig cells disappear as hyalinization progresses, and the few that remain have pyknotic nuclei and shrunken cytoplasm with abundant lipofuscin granules. this process manifests clinically as postpubertal hypogonadotropic hypogonadism and is usually caused by a lesion in or near the pituitary, such as pituitary adenoma, craniopharyngioma, and trauma to the cranial base or sella turcica (see discussion on hypogonadotropic hypogonadism in this chapter). ischemic hyalinization ischemic atrophy is usually caused by torsion of the spermatic cord, vascular injury during inguinal surgery, 914 polyarteritis nodosa, and severe arteriosclerosis. 915 except for cases caused by torsion of the cord, these patients usually are not referred to infertility clinics. torsion of the spermatic cord often is not listed as a cause in large series of infertile patients. however, follow-up of men with torsion reveals marked alteration in their spermiograms. several hypotheses have been offered to explain the low number of sperm produced by the contralateral normal testis; the most promising include response to the release of antigens by the ischemic testis, and primary lesions of the contralateral testis 916 (see discussion on testicular torsion in this chapter). testicular anoxia caused by torsion rapidly produces severe lesions that are irreversible without adequate treatment. eight hours after torsion, there is intense hemorrhagic infarction of the seminiferous tubular cells. chronic anoxia leads to tubular hyalinization and loss of leydig cells (fig. 12-93) . testicular atrophy secondary to inguinal hernia surgery occurs in 0.03-0.5% of patents in the fi rst repair, and in 0.8-5% in surgery for recurrent hernia. atrophy is most frepostobstructive hyalinization obstruction of sperm excretory ducts may cause atrophy of seminiferous tubules. in order to produce tubular hyalinization, the obstruction must be close to the testis because the ductuli efferentes in the caput epididymis absorb about 90% of tubular fl uid and protect the testis from excessive intratubular pressure. obstructive tubular hyalinization is usually focal and secondary to varicocele and other disorders involving dilation of the channels of the rete testis. these may be congenital, as in epididymis-testis dissociation, or acquired, as in rete testis dilation secondary to epididymal atrophy caused by arteritis, arteriosclerosis, or androgen insuffi ciency. obstructive tubular hyalinization also occurs in the seminiferous tubules at the periphery of the testis in patients who have had orchitis. 917 obstructive hyalinization has a mosaic distribution: lobules of completely hyalinized tubules are intermingled with lobules of normal tubules (fig. 12-94 ). the diameter of the hyalinized tubules is not as small as in other causes of hyalinization, and the tubules occasionally contain sertoli cells. in the centers of many of the tubules there is a small lumen or vacuole, the latter in the cytoplasm of a residual sertoli cell. 918 the lamina propria is thick and contains hypertrophic peritubular cells and abundant extracellular material. finally, the peritubular cells dedifferentiate and only fi broblasts remain. 919 the interstitium contains a normal number of leydig cells forming small clusters, some of which are among hyalinized tubules. this is not seen in other patterns such as ischemic hyalinization. in addition, dilated veins with eccentrically hyalinized walls can be seen in testes associated with varicocele. this lobular pattern of tubular atrophy causes a peculiar ultrasound image which has been described as a striated pattern. 920, 921 postinfl ammatory hyalinization many infections of the testis cause irreversible lesions in the seminiferous tubules. in bacterial infection the epididymis is usually involved, resulting in obstructive azoospermia. in viral infection the testis is often affected, even without symptoms. two types of viral orchitis often cause infertility, including mumps orchitis and coxsackie b orchitis. tubular atrophy caused by viral infection has a mosaic topography in which hyalinized and normal tubules are intermingled. in fully hyalinized tubules, the only recognizable cells are peritubular cells that form an incomplete, peripheral ring around the hyalinized material. the presence of elastic fi bers in these tubules distinguishes this from dysgenetic hyalinization. leydig cells form clusters of variable size, but their total number is normal. in bacterial infection the pattern of tubular hyalinization is variable. tubular atrophy of unknown etiology may be caused by an autoimmune response. this appears to occur in hypogonadism associated with disorders in other endocrine glands, such as addison's disease associated with gonadal insufficiency; adrenal-thyroid-gonadal insuffi ciency; and the association of diabetes, hypogonadism, adrenal insuffi ciency, and hypothyroidism. the testicular lesions are morphologically similar to those seen in the seminiferous tubules at the periphery of germ cell tumor and in testes with burn-out germinal cancer. in the initial stages of hyalinization associated with germ cell neoplasm, the tubules are small, contain intratubular germ cell neoplasia and dedifferentiating sertoli cells, and the lamina propria is infi ltrated by macrophages, lymphocytes, and plasma cells. in the fi nal stages, the intratubular cells have degenerated, the infl ammation has disappeared, and the seminiferous tubules are replaced by areas of hypocellular or acellular fi brosis (fig. 12-95) . it should be noted that autoimmune hyalinization is not the most common type of hyalinization associated with testicular tumors: the obstructive, ischemic, and dysgenetic variants are more common. radiation and a wide variety of chemicals cause tubular hyalinization. lengthy cancer chemotherapy combined with infertility radiotherapy invariably causes hyalinization. children's testes are more sensitive to radiation than those of adults. radiation for testicular leukemia frequently causes tubular hyalinization. in addition, radiation induces dense interstitial fi brosis and loss of peritubular cells, obscuring the borders between the interstitium and the tubules. this makes the tubules hard to see in hematoxylin-eosin-stained sections. leydig cells are atrophic and decreased in number. ischemia secondary to radiation-induced vascular injury also contributes to hyalinization. in tubular hyalinization associated with cancer chemotherapy, in addition to the direct toxicity of drugs on seminiferous tubular cells (see discussion on sertoli cell-only syndrome with involuting sertoli cells in this chapter), nutritional defi ciencies cause hypogonadotropic hypogonadism. 922, 923 histophysiological studies have distinguished two compartments in the seminiferous tubules: basal and adluminal. the blood-testis barrier separates these, and each contains different cell types with diverse hormonal and nutritional requirements. on this basis, lesions may be classifi ed as involving only the adluminal compartment or both the basal and the adluminal compartments. the following discussion of spermatogenic lesions uses this new concept of tubular pathophysiology, conserving as much as possible of the classic terminology. this category includes all infertile testes with normal numbers of spermatogonia per cross-sectioned tubule, normal or decreased numbers of spermatocytes and young spermatids, and variable numbers of adult spermatids. a descriptive term for this disorder is immature germ cell sloughing. a few immature germ cells are normally found in the lumina of the seminiferous tubules, 923 a fi nding that correlates with their presence in the ejaculates of fertile men. 924 when these cells make up more than 4% of the cells in the ejaculate, it is abnormal and the result of premature sloughing of spermatids and, in some cases, of spermatocytes. 925, 926 some authors have attempted to establish a correlation between the number of sloughed immature germ cells and the severity of lesions of the seminiferous tubules using light 927 and electron 928 microscopy. lesions in the adluminal compartment are classifi ed according to the most abundant type of germ cell whose maturation is arrested and which then sloughs: young spermatids, late primary spermatocytes, or early primary spermatocytes ( fig. 12-96) . young spermatid sloughing young spermatid sloughing is present when the ratio of elongated (s c + s d ) spermatids to round (s a + s b ) spermatids is lower than normal. the implication of this pattern is that many round spermatids are incapable of further differentiation and are sloughed ( fig. 12-97) . late primary spermatocyte sloughing in this condition, spermatogenesis develops normally up to the level of interphase primary spermatocytes, and these are present in normal numbers. afterwards, these spermatocytes degenerate without achieving meiosis and slough into the tubular lumen. all types of spermatid are greatly reduced in number. when biopsies of these testes are not properly fi xed, the seminiferous tubules may have a target-like appearance, with numerous cells in the lumen. this appearance sometimes has been referred to as tubular blockage. another descriptive term, spermatogenic arrest, also has been applied to this morphology. the latter term is inadequate in most cases, because some spermatids are present, and the number of primary spermatocytes is usually not increased as would occur if the transformation of spermatocyte into spermatid were blocked (fig. 12-98 ). late spermatocyte sloughing is a more accurate term for this condition and is preferred. primary spermatocyte sloughing occurs at the pachytene or diplotene stage of meiosis. early primary spermatocyte sloughing this lesion is characterized by the presence of a normal number of spermatogonia and decreased numbers of primary spermatocytes (fig. 12-99 ). the seminiferous tubules may contain a few spermatids. the term early primary spermatocyte sloughing does not necessarily imply an early meiotic lesion, which is quite rare. 856, 926 rather, it refers to the sloughing of newly formed spermatocytes. the sertoli cells may show vacuolation of the apical cytoplasm as an expression of germ cell loss. this lesion is more severe than that in testes with late primary spermatocyte sloughing, and is considered to result from failure of the sertoli cells to maintain the adluminal compartment. etiology the mechanisms causing adluminal compartment lesions can be classifi ed into obstructive and nonobstructive. obstruction is present in more than 70% of cases, and is characterized by variability of involvement among lobules and the presence of at least two of the following abnormalities: enlargement of tubular diameter and a lumen with remarkable differences among lobules; sertoli cells with adherens germ cells protruding into the lumen, giving an indented outline; intense apical vacuolation of sertoli cell cytoplasm; accumulation of spermatozoa in the lumen of some tubules; or number of spermatids s c + s d is higher that that of s a + s b (see testicular lesions resulting from obstruction of sperm excretory ducts). 929 the three levels of severity of adluminal compartment lesions emphasized by the terms young spermatid sloughing, later primary spermatocytes sloughing, and early primary spermatocyte sloughing, depend on the degree (total or partial) of obstruction and the level of sperm excretory duct obstruction: as the obstruction gets nearer to the testis, the greater the severity. obstruction may be extratesticular (epididymis, vas deferens, and ejaculatory ducts) or intratesticular (rete testis or any level of the seminiferous tubule length). the most frequent causes of extratesticular excretory duct obstruction are vasectomy, infl ammation (epididymitis, prostatitis), mucoviscidosis (congenital bilateral absence of vas deferens), and testis-epididymis dissociation. rete testis obstruction. varicocele is the most frequent cause of obstruction of the rete testis. more than 50% of testes with varicocele patients also often have spermatozoa with characteristically elongated heads with thin bases. 930 initially, abnormalities are confi ned to the testis ipsilateral to the varicocele, but eventually both testes are affected, although abnormalities are more severe in the ipsilateral testis. elevated pressure in the pampiniform plexus is transmitted to the veins within the testes, principally to the centripetal veins that cross the testicular mediastinum and drain most of the testicular parenchyma ( fig. 12-100 ). 931 the dilated centripetal veins compress the intratesticular sperm excretory ducts, explaining the mosaic distribution of the tubular lesions. 932 seminiferous tubule obstruction. obstruction at the level of the seminiferous tubules can be dysgenetic or post-orchitic. a dysgenetic cause may be suspected in specimens with a mosaic distribution of lesions and seminiferous tubules with small diameters, thickened lamina propria, and an unusual seminiferous tubular cell layer consisting of cuboidal sertoli cells and spermatozoa that clog the lumina (fig. 12-101) . the diagnosis is confi rmed if study of serial sections demonstrates continuity between these tubules and those with conserved spermatogenesis. the structure of seminiferous tubules has been observed with scanning microscopy at such points of continuity. 858, 933 tubular stenosis appears to be due to a primary anomaly of sertoli cells and peritubular cells. post-orchitic obstruction should be suspected in cases of tubular atrophy with a mosaic pattern without dysgenetic tubules or varicocele. some patients have a history of orchitis associated with parotiditis; 934 in others the only fi ndings are oligozoospermia and small testes. testicular biopsy, sampling only the testicular periphery, reveals only the consequences of obstruction, lesions similar to those observed with varicocele. however, some postinfl ammatory changes should also be present, including hyalinized tubules, dilated tubules lined by cuboidal sertoli cells, or complete spermatogenesis. occasionally, there is modest perivascular or peritubular infl ammation and angiectasis. 935, 936 about 30% of testes with lesions in the adluminal compartment have no obstruction, and most have primary anomalies of germ cells. this claim is supported by the following: pronounced decrease of germ cell type when the preceding type is greatly increased in number; normal correlation between the number of mature spermatids in biopsy and number of spermatozoa in the spermiogram; and the presence of numerous malformed germ cells in the adluminal compartment. decrease in the number of a germ cell type may be so important that spermatogenesis is arrested, with subsequent azoospermia. in some cases, maturation arrest is only partial and results in severe oligozoospermia. this maturation arrest is observed mainly in primary spermatocytes and young spermatids. primary spermatocyte sloughing may also be owing to meiotic anomalies ( fig. 12-102 ). the observation of increased numbers of spermatocytes arrested in preleptotene-leptotene 926 or, more frequently, pachytene 856 suggests the diagnosis. the lesion is always bilateral. spermatocytes arrested in pachytene are usually increased in size and later degenerate. in addition, some spermatids have large, diploid, spherical, hyperchromatic nuclei. the anomaly does not always affect all spermatocytes, and then a higher number of spermatids are produced. 856 young spermatid sloughing not associated with obstruction may be due to either meiotic anomalies or defective spermiogenesis. the former gives rises to the appearance of many multinucleate, polyploid, hyperchromatic young spermatids. in the second cause, young spermatids are incapable of transforming into mature spermatids, and only round spermatids appear in the ejaculate. lesions in the basal and adluminal compartments of seminiferous tubules are the most frequent histological fi ndings in testicular biopsies from infertile men. these testes may be classifi ed into two major subgroups: hypospermatogenesis and spermatogonial maturation arrest ( fig. 12-103) . hypospermatogenesis: types and etiology hypospermatogenesis is defi ned as a reduced number of spermatogonia and primary spermatocytes, with primary spermatocytes outnumbering the spermatogonia. most seminiferous tubules contain few spermatids. about 8% of patients with hypospermatogenesis have focal tubular hyalinization. 937 two variants of hypospermatogenesis have been quantitatively distinguished: pure hypospermatogenesis, and hypospermatogenesis associated with sloughing of primary spermatocytes. pure hypospermatogenesis is defi ned as a proportionate decrease in the number of all types of germ cell. the number of spermatogonia per cross-sectioned tubule is less than 17 and usually more than 10. the number of primary spermatocytes is equal to or higher than that of spermatogonia. the number of round spermatids is higher than that of primary spermatocytes, and the number of elongated spermatids is similar to that of spermatogonia ( fig. 12-104) . hypospermatogenesis associated with primary spermatocyte sloughing is characterized by two features: low numbers of spermatogonia and primary spermatocytes (with spermatocytes more numerous than spermatogonia), and degeneration and sloughing of many primary spermatocytes. the remaining spermatocytes give rise to the few spermatids observed in the tubules (fig. 12-105) . etiology of hypospermatogenesis. hypospermatogenesis may result from hormonal dysfunction, congenital germ cell defi ciency, sertoli cell dysfunction, leydig cell dysfunction, infertility androgen insensitivity, exposure to chemical or physical agents, and vascular malfunction. hormonal dysregulation. although complete spermatogenesis may be observed in men with low levels of fsh and lh, the production of a normal number of spermatozoa requires normal gonadotropin levels. hypospermatogenesis has been reported in patients with abnormal pulsatile secretion of fsh and lh, 938 low gonadotropin secretion, 939 biologically inactive gonadotropins, mutation in the gonadotropin β subunit, 940 inactivating mutation of fsh receptor gene, 941 hyperprolactinemia, and adrenal and thyroid dysfunction (see discussion on hypogonadisms secondary to endocrine gland dysfunction in this chapter). congenital germ cell defi ciency. biopsy of cryptorchid patients after orchidopexy reveals that spermatogonia proliferation is decreased and germ cell development is insufficient in adulthood even if the number of spermatogonia was normal in infancy. is it likely that this poorly understood primary anomaly of germ cells is present in some cases of hypospermatogenesis. sertoli cell dysfunction. for many years, primary germ cell defi ciency was considered the most common cause of hypospermatogenesis; today, it is known that sertoli cell failure is the cause of many cases of germ cell defi ciency. this conclusion is based on several fi ndings. sertoli cells in many infertile patients are markedly abnormal, with an increase in the number of glycogen granules 942 and acid phosphatase activity; 884 a decrease in the number of lipid droplets; and alterations in the cytoskeleton, 943 the nucleus, 944 and cytoplasmic organelles. 945 in some cases sertoli cells have abnormal maturation, with elongated nuclei containing coarse clumped chromatin instead of triangular-shaped nuclei with pale chromatin. anomalies in sertoli cell fsh receptors may be present in idiopathic oligozoospermia associated with elevated levels of fsh. 946 serum inhibin b concentration may be used as a marker to estimate sertoli cell function. 947 leydig cell dysfunction. testosterone synthesis by leydig cells is necessary for normal spermatogenesis, 948 and abnormal leydig cell function is a frequent fi nding in idiopathic oligozoospermia. [949] [950] [951] leydig cell dysfunction should be suspected when the cells appear diffusely hyperplastic. patients have elevated serum lh level with depletion of rapid-release testosterone, revealing a lack of early response of leydig cells to gonadotropin-releasing hormone stimulation. the ratio of testosterone to lh in the plasma indicates the degree of leydig cell dysfunction. decreased ratio with normal testosterone level suggests compensated dysfunction. patients with a ratio of less than 1 : 5 and normal other parameters may have complete spermatogenesis. 951 androgen insensitivity. some patients with severe oligozoospermia or azoospermia have a defect in androgen receptor responsiveness, similar to that in reifenstein's syndrome. [952] [953] [954] the abnormality may arise from a genetic defect in the eight exons that code for this receptor, mapped to xq11-12, 955 or from post-translational errors. 956, 957 this defect is also referred to as infertile male syndrome and mild androgen insensitivity, and the patients have male phenotype with somatic features of slight androgen defi cit. 958 histologically, the testis is similar to that observed with leydig cell dysfunction or mixed atrophy, although the mechanism causing the leydig cell hyperplasia is quite different (fig. 12-106) . peripheral resistance to testosterone action alters regulation of the hypothalamohypophyseal-testicular axis, and lh and testosterone levels are elevated. androgen insensitivity causes between 10% 959 and 40% 960 of all cases of severe oligozoospermia or azoospermia. in such cases spermatogenesis improves with the administration of tamoxifen citrate, 960 clomiphene citrate, or androgen therapy. 961, 962 calculation of the index of androgen insensitivity can be helpful: plasma lh (miu/ml) × plasma testosterone levels (ng/ml). in patients with androgen insensitivity, the index is higher than 200 (normal is about 102). physical and chemical agents. the number of chemicals implicated in infertility increases daily. a detailed history is invaluable in evaluating these patients. the same is true of physical agents such as prolonged exposure to heat, ionizing radiation, or microwave radiation. 963 etiology of hypospermatogenesis associated with primary spermatocyte sloughing. most testes with primary spermatocyte sloughing have varicocele, and this is commonly associated with infertility. [964] [965] [966] [967] varicocele is found in 15% of the general population, and is present in 30-40% of infertile men. the mechanism by which varicocele affects fertility is unknown. clinical varicocele may occur without a testicular lesion (or only phlebectasis), and subclinical varicocele may be associated with severe spermatogenic lesions. increased testicular temperature 968, 969 and compression of intratesticular sperm excretory ducts by dilated veins 932 are the most plausible mechanisms. in other cases, primary spermatocyte sloughing results from anomalies of primary spermatocytes and spermatids, suggesting a meiotic anomaly. finally, in some patients the cause may be the presence of involuting sertoli cells. spermatogonial maturation arrest spermatogonial maturation arrest is a disorder defi ned by the presence of fewer than 17 spermatogonia per cross-sectioned tubule and even fewer primary spermatocytes. spermatids are usually absent. there have been attempts to correlate the etiology of spermatogonial maturation arrest with the sertoli cell type present. 970 immature sertoli cells are characteristic of hypogonadotropic hypogonadism and some syndromes with androgen insensitivity (fig. 12-107) . mature sertoli cells, if their presence is unilateral, are observed in varicocele, epididymitis, and ipsilateral testicular traumatism, but if they appear in both testes the etiology is unknown. involuting sertoli cells are usually present bilaterally; some cases are idiopathic, whereas others are associated with a history of alcoholism or chemotherapy. dedifferentiated sertoli cells are found in spermatogonial maturation arrest caused by gonadotropin inhibition in treatment with estrogen, -releasing hormone agonist, or anti-androgen. 971 mixed atrophy is a descriptive term for the coexistence, in the same testis, of tubules containing only sertoli cells and tubules with complete or incomplete spermatogenesis. 972 this disorder includes patchy failure of spermatogenesis and partial del castillo's syndrome. the extent of sertoli cell-only tubules varies widely. tubules with spermatogenesis may be normal or partially atrophic. tubular hyalinization is occasionally seen (fig. 12-108) . mixed atrophy is more common than suggested by the literature, and many cases are included under other diagnoses, such as 'hypospermatogenesis with a severe germ cell depletion in such a way that some sertoli cell-only tubules are seen,' 859 and 'sertoli cell-only syndromes with focal spermatogenesis.' 973 serial sections from testes with mixed atrophy reveal that the two different types of tubule are grouped according to their histologic pattern, suggesting that the distribution is by testicular lobules. in cases of mixed atrophy, the percentage of tubules with spermatogenesis, the degree of spermatogenic development in the tubules, and the type of sertoli cell present should be reported. correlation of the fi rst two with the spermiogram gives an indication of prognosis, and the sertoli cell types identifi es the nature (primary or secondary) of the lesion. 974 mixed atrophy (probably primary) is observed in idiopathic infertility, cryptorchidism (even if orchidopexy was done at infancy, in both the cryptorchid and the contralateral descended testis), retractile testes, macroorchidism, intravaginal torsion of the spermatic cord (in both twisted and contralateral testis), and chromosomal anomalies such as down's syndrome, 47/xyy karyotype, 46/xx karyotype, infertility giant y chromosome, klinefelter's syndrome with chromosomal mosaicism, partial androgen insensitivity, and some male pseudohermaphrodites. secondary mixed atrophy may be seen in patients undergoing chemotherapy, corticoid therapy, 975 or in those with a history of viral orchitis. in addition to anomalies in the seminiferous tubules, examination of the biopsy should include a description of the morphology of the germ cells. giant spermatogonia are a normal component of the seminiferous epithelium. these cells may be altered spermatogonia in the s or g 2 phases of the cell cycle. they rest on the basal lamina and have pale cytoplasm and an ovoid nucleus measuring at least 13 µm in diameter. the frequency of these cells in normal and infertile men is about 0.65 cells per 50 cross-sectioned tubules, although their number is usually higher in mixed atrophy. these cells should not be mistaken for intratubular germ cell neoplasia; they are also present in normal numbers in tubules at the periphery of germ cell tumor ( fig. 12-109 ). 976 multinucleate spermatogonia are a common fi nding in cryptorchid testes that were surgically corrected, infertile patients, and old men. nuclei of both ad and ap spermatogonial types may be seen within the same cell. normally, spermatogonia are present only in the transition zone between the seminiferous tubule basal layer and the tubuli recti. dislocated spermatogonia have been found throughout the testis in old age, 977 in infertile patients with a variety of lesions, after long-term estrogen therapy, 978 and in seminiferous tubules with intratubular germ cell neoplasia. 979 megalospermatocytes are large primary spermatocytes arrested in the leptotene stage ( fig. 12-110) 980 that exhibit asynapsis of chromosomes. 981 joined by cytoplasmic bridges, they form small groups. these cells may be clones of synchronously degenerating spermatocytes. 982 they are frequently found in elderly men and are a non-specifi c fi nding in infertile patients. the presence of spermatids with multiple nuclei (from 2 to 86) is frequent is old age. 983 similar cells with fewer nuclei have also been reported in infertility due to cryptorchidism, 984 hyperprolactinemia, and idiopathic infertility ( fig. 12-111 ). there are at least four teratozoospermic syndromes that may be easily identifi ed by testicular biopsy, although in most the diagnosis previously relied on morphologic study of the spermiogram: (1) round-headed spermatids (characteristic of spermatozoa lacking acrosomes) (fig. 12-112) , (2) s c + s d spermatids with a very elongated head (characteristic of varicocele) (fig. 12-113) , (3) macrocephalic s c + s d spermatids whose dna content suggests an anomaly in the fi rst meiotic division, and (4) s c + s d spermatids with voluminous eosinophilic cytoplasmic droplets (syndrome of spermatozoa with short thick fl agella 985 or fi brous sheath dysplasia). in some patients, s a + s b spermatids rest in these initial phases of spermiogenesis and eventually become sloughed in the tubular lumina. 986 in other testes there are macrocephalic s c and s d spermatids with anomalous dna content, suggesting an anomaly in the fi rst meiotic division. ultrastructural study of spermatozoa is sometimes necessary to determine the cause of male infertility. a number of mor-phologically abnormal spermatozoa are present in all semen samples, including those from fertile men, but abnormal spermatozoa are very numerous in infertile patients. ultrastructural study is advised in all cases of asthenozoospermia, in teratozoospermia when the number of spermatozoa showing the same morphological anomaly is high, and in cases with apparently normal spermatozoa that fail to fertilize in vitro. 987 the classifi cation of ultrastructural anomalies in spermatozoa is based on light microscopy fi ndings 988 of lesions in the head and tail. anomalies of the spermatozoal head these are defi ned by changes in the shape of the head, and usually involve both the nucleus and the acrosome. some anomalies, such as pear-shaped, candle-shaped, or egg-shaped heads, 989,990 are regarded as minor variants of normal. more signifi cant abnormalities are the elongated, microcephalic, macrocephalic, and crater-defect forms. the most frequent abnormal head shape is elongated with a narrow base (tapered head spermatozoa). this anomaly is frequently associated with varicocele. 991 microcephalic spermatozoa have spherical (globozoospermia) or irregularly shaped heads. the former have spherical nuclei with poorly condensed chromatin and lack acrosomes, postacrosomal sheaths, and a nuclear ring (fig. 12-114 ). most cases are sporadic, but this lesion was also reported in two pairs of infertile brothers. 992, 993 microcephalic spermatozoa with irregularly shaped heads have small and irregularly shaped acrosomes that usually are not in contact with the nucleus. this anomaly may be congenital, as in aarskog-scott syndrome, 994 or secondary to heat exposure or hashish smoking. in both types of microcephaly loss of connection between the acrosomal vesicle and the spermatozoal head is attributed to a defi ciency in basic proteins of the sperm perinuclear theca that promotes nuclear envelope organization and adhesion of the acrosomal vesicle. 995 acrosin is reduced or absent in spermatozoa lacking acrosomes and those with small acrosomes. 996 motility may be infertility normal. the occurrence of aneuploidy 997 and disomy of sex chromosomes 998, 999 in some cases should be evaluated before performing intracytoplasmic sperm injection (icsi). the cause of round-headed spermatozoa might be the lack of golgi-associated protein known in male mice as golgi-associated pdz-and coiled-coil motif-containing protein (gopc). this protein is principally localized in the trans-golgi region in round spermatids, and its loss produces globozoospermia. the primary defect consists of an inability of acrosomal vesicles to fuse to each other to create the acrosome. 1000 macrocephalic spermatozoa (macronuclear spermatozoa) have enlarged, irregularly shaped heads and defi cient chromatin condensation. there are two types (multiple tails 1001, 1002 and afl agellate), both of which have abnormal dna content (many are tetraploid), suggesting a meiotic anomaly. 1003, 1004 irregularly shaped spermatozoa are characterized by irregularity in the shape of the nucleus or acrosome. 1005 in the crater defect syndrome, there is invagination of the nuclear envelope in which the acrosome penetrates. the tail is morphologically normal, and motility is only slightly reduced. in spermatozoa with spoon-shaped nuclei, the defect is probably genetic. other anomalies include double-headed spermatozoa with two nuclei sharing a single acrosome. 1006 anomalies in the spermatozoal tail spermatozoal tail anomalies are classifi ed as generalized anomalies of the tail or anomalies in defi ned tail components, such as the connecting piece, the axoneme, or the periaxonemal structure. 1007 generalized anomalies in the tail cytoplasmic remnants. the presence of cytoplasmic droplets is normal during spermiogenesis. an elevated number of spermatozoa with cytoplasmic droplets in semen is associated with premature sloughing of spermatozoa, as occurs in varicocele, and should not be misinterpreted as spermatozoa with excess residual cytoplasm. 1008 these spermatozoa are very often abnormal and the residual cytoplasm may be located around the intermediate piece or surrounding the head. these spermatozoa also have other fl agellar anomalies. bent tail. a bend in the tail may occur at the level of the connecting piece or the intermediate piece. in bends of the connecting piece, the tail is laterally implanted and forms an angle with a nucleus that displays a thin base. bends of the intermediate piece are associated with cytoplasmic droplets, malposition of mitochondria, and loss of the parallel arrangement of the dense outer fi bers. coiled tail. spermatozoa with a coiled tail are a frequent fi nding in centrifuged semen, but they may also be a true abnormality. these spermatozoa have a perinuclear cytoplasmic remnant containing a fl agellum that is coiled around the nucleus and along the middle or principal pieces (fig. 12-115 ). this is frequently associated with abnormalities of the periaxonemal structures. tail stump (short-tail spermatozoa). the presence of many spermatozoa with short, thick tails in semen represents a well-defi ned teratozoospermic syndrome. 1009 ultrastructural examination reveals hypertrophy and hyperplasia of the fi brous sheath, 1010 hence this syndrome has also been termed 'fi brous sheath dysplasia.' 1011 additional axonemal malformations, including absence of the central pair of microtu-bules ( fig. 12-116) 1012 and, less frequently, lack of dynein arms, are observed in 50% of cases. about 24% of patients have respiratory disease, such as rhinosinusitis, bronchitis, and bronchiectasis from an early age. similar fi ndings have been reported in the cilia of the upper respiratory tract, and thus a relationship between fi brous sheath dysplasia and immotile cilia syndrome has been assumed. clinical presentation may be sporadic or familial. the cause of fi brous sheath dysplasia and the subsequent lack of motility in these spermatozoa is probably related to the occurrence of deletions in akap3 and akap4 genes, as well as the absence of akp4 protein in the fi brous sheath. 1013 multiple tails. the presence of more than two tails is associated with macrocephalic spermatozoa. 1014 sperm tail agenesis. teratozoospermia with 100% sperm tail agenesis has been reported in patients with a high degree of consanguinity. these spermatozoa also have defects in chromatin condensation and residual cytoplasmic droplets. 1015 anomalies of the connecting piece anomalies of the connecting piece are classifi ed as acephalic spermatozoa, defi cient organization of the connecting piece, and separation between the head and the tail. acephalic spermatozoa are known as 'pin-headed,' although they lack a true head; the small cephalic knob-like thickening is actually a cytoplasmic droplet with a variable degree of mitochondrial organization giving rise to a variable degree of motility. 1016 this anomaly is due to an early failure in spermiogenesis. it may be familial in some cases. 1017, 1018 spermatozoa with defi cient organization of the connecting piece have narrowing at this level, with loss of alignment of the head and fl agellum axes. spermatozoa with a separated head and fl agellum, known as decapitated and decaudated spermatozoa, are also the result of an anomaly in spermiogenesis, but the separation between heads and tails can occur during spermiation or at any level of the sperm excretory ducts. 1019, 1020 anomalies in axoneme abnormalities of the axoneme are classifi ed as numerical anomalies, microtubular ectopia, and immotile cilia syndrome. the most common numerical anomalies are the absence of one or both microtubules of the central pair and complete lack of the axoneme. spermatozoa lacking the central microtubule pair also lack the central sheath and are immotile, although they are normal by light microscopy. familial cases have been reported. 1021 this anomaly may be associated with ciliary dyskinesia. 1022 immotile cilia syndrome (primary ciliary dyskinesia) 1023 refers to patients having low mucociliary clearance associated with otitis, sinusitis, bronchitis, bronchiectasis, and immotile spermatozoa. most patients have the same defect in the axoneme and cilia of the respiratory mucosa. the frequency of this syndrome is estimated at between 1 in 20 000 and 1 in 60 000 men. clinical symptoms consist of reduced clearance of ciliary mucus in the airway, with onset at infancy. in order to prevent the later development of bronchiectasis, ultrastructural study of the respiratory mucosa is advisable if other disorders have been excluded, including cystic fi brosis, allergy and other immune disorders, α 1antitrypsin defi ciency, and cardiovascular and metabolic diseases. 1024 the most frequent anomalies of this syndrome are the absence of microtubule doublets and peripheral junctions, the central microtubule pair, the outer dynein arms, the central junctions, the two dynein arms, and the inner dynein arm plus the peripheral junctions ( fig. 12-117) . spermatozoa lacking the two dynein arms or the peripheral junctions are immotile. reduced motility is seen in spermatozoa with only one dynein arm. kartagener's syndrome is a variant of the immotile cilia syndrome characterized by the classic triad of situs inversus, bronchiectasis, and chronic sinusitis. the syndrome has autosomal recessive inheritance 1025 and is found in 20-25% of patients with situs inversus. 1026 anomalies in periaxonemal structures periaxonemal abnormalities include mitochondrial sheath defects, 1027 malposition of the annulus, alteration in number, shape, or length of the outer dense fi bers, and absence, thickening, or disruption of the fi brous sheath. 1011, 1028 many cases of asthenozoospermia, present in 30% of infertile men, may be attributable to defi cient mitochondrial function, possibly caused by mutations in their dna. 1029 abnormalities of the dense fi bers are associated with deficient motility. abnormalities of the fi brous sheath include, in addition to the abovementioned dysplasia of the fi brous sheath, absence of the fi brous sheath, and redundant fi brous sheath material associated with a defi cit or lack of mitochondria. 1030 the three defects are probably inherited. the incidence of intratubular germ cell neoplasia (igcn) in infertile patient is 0.4% in england, 1031 0.7% in spain, 1932 0.73% in germany, 1033 and 1.1% in denmark. 1034 a higher risk occurs in patients with severe oligozoospermia (fewer than 10 million spermatozoa per milliliter), azoospermia associated with unilaterally or bilaterally diminished testicular volume, 1035 a history of testicular maldescent, 1036, 1037 or unilateral testicular cancer. 1038 the cells of igcn are located in seminiferous tubules with decreased tubular diameter and lacking spermatogenesis. these cells are large and have pale cytoplasm and large and irregularly outlined nuclei, with one or several prominent nucleoli. they stain intensely with periodic acid-schiff and express placenta-like alkaline phosphatase, c-kit, and the cell adhesion molecule cd44. 1039 a reduction in the number or absence of leydig cells is infrequent in infertility, and only occurs in hypogonadotropic hypogonadism secondary to lh defi cit and in patients infertility with biologically inactive lh. leydig cell hyperplasia is very common, 1040 and has been observed in klinefelter's syndrome, cryptorchidism, male pseudohermaphroditism, minor androgen insensitivity, infertility secondary to leydig cell dysfunction, varicocele, after treatment with 5αreductase inhibitors or non-steroidal anti-androgens, and in some elderly men. such hyperplasia may give rise to hypoechoic or hyperechoic images that may be misdiagnosed as tumor. 1041 there is a close relationship between testicular dysfunction and elevated mast cell numbers in the testis. an increase in interstitial and peritubular mast cells occasionally occurs in infertile patients. 1042, 1043 this increase is higher than that observed in infl ammatory or neoplastic process. 1044 daily administration of ketotifen, an antihistamine-like drug with a mast cell-stabilizing effect, signifi cantly improves the spermiogram parameters in some patients. 1045 for effective therapy, it is important to know whether or not the azoospermia or oligozoospermia is the result of obstruction. 863, 1046 obstructive azoospermia and oligozoospermia azoospermia caused by obstruction is usually easily diagnosed, but this determination is more diffi cult with oligozoospermia. obstruction of the ductal system should be suspected when there are more than 20 mature spermatids (s c + s d ) per cross-sectioned tubule and fewer than 10 million spermatozoa in the spermiogram (fig. 12-118) . 1047, 1048 obstructive azoospermia is implicated in 7.4-14.3% of cases of male infertility. obstruction is classifi ed as proximal, distal, and mixed, according to the distance from the testis to the point of obstruction in the ductal system. proximal obstruction obstruction is considered proximal when the lesion lies between the seminiferous tubules and the distal end of the ampulla of the vas deferens. epididymal obstruction, principally of the caput-corpus transition zone, accounts for 66% of cases. rarely, there is a defective connection between the rete testis and epididymal ductuli efferentes. because the seminal vesicles are normal, men with proximal obstruction have a normal volume of semen (the testicular contribution to semen is about 5% of the total volume). when obstruction is in the cauda of the epididymis, epididymal markers, including carnitine, glycerophosphorylcholine and α-glycosidase are low. 1049 the nearer the obstruction is to the caput of the epididymis, the higher the level of these markers. distal obstruction distal obstruction is located between the ampulla of the vas deferens and the junction of the ejaculatory ducts and urethra. these patients present with sacral, perineal, or scrotal pain on ejaculation. rectal examination often reveals enlarged seminal vesicles. the volume of semen is low and consists of watery fl uid that fails to coagulate. seminal vesicle secretions are lacking. the concentration of prostatic secretions, such as acid phosphatase and citric acid, is increased owing to the lack of semen dilution. vasography may help in diagnosis, as higher segments fail to fi ll. 1050 transrectal ultrasonography is the most accurate imaging modality for the diagnosis of ejaculatory duct obstruction. needle aspiration of seminal vesicle fl uid may show spermatozoa that have entered the seminal vesicles by refl ux. mixed obstruction mixed obstruction refers to lack of patency of the vas deferens or the epididymis and alterations in the ejaculatory ducts or seminal vesicles (low ejaculate volume, and absence of fructose). the most frequent cause is mucoviscidosis. one-third of patients with congenital bilateral absence of the vas deferens have agenesis or hypoplasia of the seminal vesicles. the cause of epididymal obstruction in patients with anomalies of the prostate-vesiculo-deferential junctions is diffi cult to determine. etiology of obstructive azoospermia obstructive azoospermia may be caused by congenital or acquired lesions. congenital azoospermia the most frequent anomalies associated with congenital azoospermia are testis-epididymis dissociation, epididymal malformation in cryptorchidism, bilateral absence of the vas deferens, congenital unilateral absence of the vas deferens associated with pathology of the contralateral testis or its sperm excretory ducts, seminal vesicle agenesis, and ejaculatory duct obstruction (table 12-9) . agenesis of all mesonephric duct derivatives. agenesis of all mesonephric duct derivatives is a rare disorder that gives rise to varied anatomical anomalies, depending on the stage of embryonic development at which the mesonephric duct derivatives disappear. if failure occurs before the fourth week the ipsilateral kidney and ureter are absent, although the testis may be present, or there may be other renal anomalies. if failure occurs in the fourth week, and the ureteral bud is already formed, the ureter and kidney may develop normally. if failure occurs between the fourth and the 13th weeks, there is a variable constellation of anomalies that most frequently include normal development of the testis and globus major and hypoplasia of the other excretory duct segments, or agenesis of an excretory duct segment (epididymis, vas deferens, or seminal vesicle). epididymal anomalies. the most frequent epididymal anomalies are absence of the epididymis, testis-epididymis dissociation, defective connection of the vas deferens and epididymis, epididymal cyst, and anatomical abnormalities of the epididymis. complete absence of the epididymis is frequent in monorchidism and anorchidism. the epididymis is replaced by a small mass of cellular connective tissue with abundant blood vessels at the blind end of the vas deferens. partial absence of the epididymis is more frequent than complete absence. absence of the corpus of the epididymis gives rise to a characteristic malformation called bilobated epididymis. this varies from simple strangulation to complete separation of the caput and cauda. these anomalies are often associated with absence of the vas deferens. testis-epididymis dissociation is found in 1% of cases of obstructive azoospermia and is usually associated with cryptorchidism. defects in connection between the ductuli efferentes and the ductus epididymidis are rarely complete. in the incomplete form, some of the fi ve to 30 ductuli efferentes in the epididymis are short and end blindly. epididymal cysts usually arise from blind-ended ductuli efferentes and contain spermatozoa. these spermatoceles retain their epithelial lining, although it becomes atrophic ( fig. 12-119 ). spermatozoa may be obtained from these cysts. some epididymal cysts arise from embryonic remnants, do not contain spermatozoa, and are lined by columnar or pseudostratifi ed epithelium. wolffi an cyst, unlike müllerian cyst, is immunoreactive in the apical border of epithelial cells with cd10. 1051 cyst lined by clear cells with or without papillae raises concern for von hippel-lindau disease. 1052 large epididymal cyst requires removal and must be excised with great care to avoid damaging the ductuli efferentes and resulting in obstruction. epididymal cyst is present in about 5% of males, and the incidence is high (21%) in those exposed to diethylstilbestrol during gestation. the incidence of epididymal cyst in those with hepatorenal polycystosis is similar to that in the general population. 1053 anomalies in epididymal confi guration, altering its shape and location, are frequent in men with cryptorchidism and uncommon with descended testes. the most common malformations are elongated epididymis, angulated epididymis, and free epididymis. elongated epididymis is found in approximately 68% of undescended testes. the length of the epididymis may be several times that of the testis, and, in abdominal or inguinal cryptorchidism, the epididymis extends several centimeters below the testis. angulated epididymis is characterized by a long epididymis that has a sharp bend in the corpus with or without stenosis. with free epididymis, all or part of the epididymis is unattached to the testis. the most common variant is epididymis with free cauda. vas deferens anomalies. the most frequent anomalies are of the vas deferens are congenital absence, segmental aplasia, ectopia, duplication, diverticula, and crossed dystopia. 1054 congenital absence is defi ned as unilateral or bilateral absence of either the whole vas deferens or only a segment. obviously, azoospermia occurs with bilateral absence. the frequency of this malformation varies among populations. at autopsy, the prevalence is 0.5%, but the clinical incidence infertility is 1-1.3% in infertile men 1055 and 10-25% in patients with obstructive azoospermia. unilateral complete absence is three times more frequent than bilateral absence, and absence of only a segment is even more frequent. the affected segment may be absent or reduced to a fi brous cord. absence of the vas deferens may be associated with other malformations of the sperm excretory ducts or urinary system. the most frequent malformations of the excretory ducts are absence of the ejaculatory ducts (33% of cases) and, less frequently, absence of the seminal vesicles. about 71% of patients with bilateral absence of the vas deferens have partial aplasia of the epididymis. the most frequent malformations of the urinary system are absence of the ipsilateral kidney and other renal anomalies. complete or partial absence of the vas deferens occurs frequently in patients with cystic fi brosis. persistent mesonephric duct consists of the ureter joined to the vas deferens, forming a single duct that opens in an ectopic orifi ce between the trigone and the verumontanum. this malformation may be associated with cystic transformation or absence of the seminal vesicle. the kidney may be normal or dysplastic. anomalies of seminal vesicle and ejaculatory duct. the most frequent anomalies are agenesis of the seminal vesicles or ejaculatory ducts, cyst of the seminal vesicle, and ectopic opening of the ureter into the seminal vesicle. the last is the most common and often is associated with ipsilateral renal dysplasia. acquired azoospermia infl ammation and trauma are the main causes of acquired azoospermia. epididymitis is a frequent cause; chlamydia trachomatis 1056, 1057 and escherichia coli are the most common infectious causes in developed countries. 1958 infections with neisseria gonorrheae and mycobacteria are also implicated, and non-specifi c epididymitis is important. 1059 apart from elective vasectomy, the most frequent traumatic causes of azoospermia are surgical accidents during herniorrhaphy in chidren, 1060 orchidopexy, varicocelectomy, hydrocelectomy, deferentography, 1061 and removal of epididymal cyst. obstructive azoospermia may also result from blockage of the ejaculatory ducts following transurethral resection, or as a result of chronic urethral catheterization. lesions of the testis and epididymis may result from obstructed sperm excretory ducts, depending on the location, origin (congenital or acquired), and duration of the obstruction. location of obstruction obstruction at the level of the ampulla of the vas deferens, seminal vesicles, or ejaculatory ducts does not usually cause signifi cant lesions in the testis or epididymis. more proximal obstruction at the level of the vas deferens, epididymis, or testis-epididymis junction usually causes severe lesions in both the sperm excretory ducts and the testicular parenchyma. obstruction of the vas deferens causes increased pressure within the ductus epididymis. as a result, epididymal lumina dilate, the epithelium atrophies, and fl uid containing few spermatozoa and some spermiophages accumulates in the lumen (fig. 12-120) . the most dilated epididymal segment is the caput. the ductuli efferentes often become cystically dilated and fi lled with spermatozoa and macrophages. from reabsorption and lysosomal degradation of this protein-rich fl uid, the epithelium accumulates lipofuscin granules or aquires apical eosinophilic granules (paneth cell-like change). 1062 rupture of the vas deferens gives rise to microgranulomas and ceroid granuloma (fig. 12-121 ). macrophages and lymphocytes are often present in the intertubular connective tissue. 1063 the most frequent testicular lesions in proximal obstruction involve the adluminal compartment, and are the result of the negative effect of hydrostatic pressure on the seminiferous tubular cell layers and, in particular, on the sertoli cell (figs 12-122-12-124) . etiology of obstruction obstruction secondary to congenital absence of the vas deferens usually causes little testicular injury, mainly dilation of the seminiferous tubules and an increase in the number of mature (s c + s d ) spermatids. 1064 lesions resulting from vasectomy are more important. increased intraluminal pressure in the epididymis 1065 may give rise to pain (late post-vasectomy syndrome). 1066 testicular lesions depend on the surgical technique used: they are slight if the proximal end of the vas deferens is not ligated or sperm granuloma forms at the site of vasectomy. the spermatogenic rhythm in the testis is slower than before vasectomy, and lesions characteristic of testicular obstruction develop, including thickening of the lamina propria and fi brosis of the interstitium. 1067, 1068 in testicular obstruction secondary to herniorrhaphy in infancy, testicular lesions are mild. testicular lesions may be important if the epididymis is damaged by hydrocelectomy, and consist mainly of primary spermatocyte sloughing. in addition to these lesions, hyalinized tubules may be observed when obstruction is caused by infl ammation. in acquired obstruction the testicular lesions worsen with time. obstruction in the caput of the epididymis leads to disappearance of all germ cells in the adluminal compartment of seminiferous tubules. the tubules become dilated and sertoli cells appear vacuolated. testicular alterations after vasectomy may not be related to the duration of the obstruction but rather to the initial injury, and may disappear with time as the intraluminal pressure decreases. 1069 however, if a signifi cant amount of time has elapsed after vasectomy, the possibility of attaining a normal spermiogram with vasovasostomy is very low. vasal patency is restored in most cases of reanastomosis, but paternity rates are markedly lower (25-51%) 1069 than normal (85%). 1070 some azoospermic patients have testicular biopsy with minimal histologic abnormality or minor tubular dilation without detectable excretory duct obstruction. these fi ndings are characteristic of two main conditions: young's syndrome, and alterations in spermatozoal transport. young's syndrome young's syndrome is defi ned by the following constellation of fi ndings: azoospermia, sinusitis, bronchitis or bronchiectasis, and normal spermatozoal fl agella. 1071 the incidence is probably higher than that recorded in the literature, and young's syndrome should be suspected in all patients with obstructive azoospermia without a history of epididymitis or scrotal trauma. these patients have a lesion at the junction of the caput and corpus of the epididymis that gives the epididymis a characteristic gross appearance. the caput of the epididymis is distended, the ductuli efferentes contain yellowish fl uid and numerous spermatozoa, and the remaining epididymal segments are normal. the ductus epididymidis is blocked by thick fl uid. 1072 young's syndrome should be distinguished from other causes of infertility also associated with chronic sinusitis and pulmonary infections, including ciliary dyskinesia and cystic fi brosis. ciliary dyskinesia consists of morphological, biochemical, and functional alterations in cilia and fl agella, and includes infertility several diseases such as the immotile cilia syndrome, kartagener's syndrome, and miscellaneous syndromes characterized by imperfectly defi ned abnormalities of cilia and fl agella. 1073 in young's syndrome, sinusitis and pulmonary infections develop in childhood and stabilize or improve in adolescence; in other conditions, the pulmonary damage increases with age and the cilia and fl agella are ultrastructurally abnormal. 1074 alterations in spermatozoon transport normally, spermatozoa detach from the sertoli cells and are transported through the intratesticular and extratesticular excretory ducts, where they are stored, mainly in the cauda of the epididymis, and fi nally released from the corpus by ejaculation or eliminated by phagocytosis. only about 50% of spermatozoa are ejaculated. whereas the release of spermatozoa from the corpus is intermittent, their transport through the sperm excretory ducts is continuous. transport is accomplished by the myofi broblasts in the wall of the seminiferous tubules and ductuli efferentes and the smooth muscle cells in the wall of the ductus epididymidis and vas deferens. these cells cause peristaltic contraction, propelling spermatozoa along the length of the epididymis in a mean of 12 days (range, 1-21 days). the walls of the seminiferous tubules and extratesticular excretory ducts are under hormonal and neural control. the myofi broblasts in the seminiferous tubules have oxytocinic, α 1 -β-adrenergic, and muscarinic receptors. unmyelinated nerve fi bers penetrate the tubular lamina propria, pass among the myofi broblasts, and end near the sertoli cells. 1075 along their length these nerve fi bers have varicosities containing sympathetic vesicles. the ductus epididymis is innervated by sympathetic adrenergic nerve fi bers that end among the smooth muscle cells. several hormones, including oxytocin, endothelin-1, vasopressin, and prostaglandins, act on the musculature of the ductus epididymis. the peristaltic contractions begin in the caput and propagate toward the cauda. the frequency and amplitude of contractions vary from region to region, being higher in frequency near the caput and of maximal amplitude in the initial portion of the cauda. the progressive increase in amplitude parallels the progressive increase in thickness of the muscular wall and the requirement for greater force to propel the fl uid as it becomes progressively more viscous with a higher concentration of spermatozoa. the distal portion of the cauda is unusually at rest because it is the main reservoir of spermatozoa between ejaculations. several times daily, vigorous contractions of the distal cauda impel the spermatozoa from the cauda toward the vas deferens. 1076 several drugs that favor contraction of the muscular wall (α 1 blocking and f 2α prostaglandins) have been successfully used in the treatment of alterations in the spermatozoon transport. 1077 knowledge of the incidence of chromosomal abnormalities in male infertility has progressed in parallel with advances in technology: karyotypic studies in peripheral blood, meiotic and chromosomal studies of testicular biopsies, analysis of chromosomes in spermatozoa, and analysis of dna in blood and spermatozoa for the detection of chromosome y deletions. 1078 the incidence of chromosomal anomalies in infertile men is 2.2-6.6%, whereas in the general population it is lower than 0.5%. the frequency of chromosomal abnormalities increases with the decrease in number of spermatozoa in the ejaculate. 1079 klinefelter's syndrome genetic and clinical aspects klinefelter's syndrome is characterized by an abnormal number of x chromosomes and primary gonadal insuffi ciency. the original description was of a man with eunuchoidism, gynecomastia, small testes, mental retardation, and elevated level of serum gonadotropins. 1080 the frequency of this syndrome varies according to the population studied: 1 in 1000 to 1 in 1400 surviving newborns; 1 in 100 patients in mental institution; 3.4 in 100 infertile men; and 11% of patients who are azoospermic. 1081 in 80% of cases, the karyotype is 47xxy. the remaining 20% have chromosomal mosaicism with at least two x chromosomes. the most common are xy/xxy, xy/xxxy, xx/ xxy, xxy/xx/xy, xy/xo/xxy, xx/xxy/xxxy, and xxxy/ xxxxy. the 47xxy lesion is due to non-disjunction in sex chromosome migration during the fi rst or second meiotic division of the spermatocyte or ovule, or during the fi rst meiotic division of the zygote. 1082 study of the xg antigen in blood revealed that the extra x chromosome is from the mother in 73% of cases. advanced maternal age increases the incidence of children with the 47xxy karyotype. in 47xxy patients, the most common clinical fi ndings are: 1083 • eunuchoid phenotype with increased stature. the increased height is due to a disproportionate lengthening of the lower extremities. the ratio of span to height is less than 1. • incomplete virilization. this is variable and ranges from normal development to absence of secondary sex characteristics. • gynecomastia, usually bilateral, present in 50% of patients. • mental retardation. other commonly associated conditions include chronic bronchitis; varicose veins; cervical rib; kyphosis; scoliosis or pectus excavatum; and a high incidence of hypothalamic, hypophyseal, thyroid, and pancreatic dysfunction. 1084 the external genitalia usually are normally developed. the testes are usually less than 2.5 cm long, although in some cases of chromosomal mosaicism they are of normal size. 1085 the incidence of cryptorchidism is low in 47xxy patients but increased in mosaicism. 1086 supernumerary x-chromosome material is associated with a reduction of gray matter in the left temporal lobule, a fi nding correlated with verbal and language defi cits. 1087 histologically, the testes show the classic picture of tubular dysgenesis with small hyalinized seminiferous tubules lacking elastic fi bers and pseudoadenomatous clustering of leydig cells (figs 12-125, 12-126 ). 1080 most biopsies show some tubules with a few sertoli cells. 1088 these cells may be dysgenetic (pseudostratifi ed distribution of nuclei that are dark and elongate and contain small peripherally placed nucleoli in tubules without apparent lumina). sex chromatin may only be observed in dysgenetic sertoli cells. 1089 this suggests that either there is testicular mosaicism of the x chromosome, or that both x chromosomes are heterochromatinized. in mosaicism, sertoli cell-only tubules may be more numerous than hyalinized ones. the reduced testicular volume gives an appearance of leydig cell hyperplasia, 1090 although quantitative studies have shown that the total number of leydig cells is lower than normal. 1091 many of the leydig cells are pleomorphic and some are multivacuolated. immature fi broblast-like leydig cells may be present. the abnormally differentiated leydig cells have nuclei with coarse masses of dense chromatin, deep unfolding of the nuclear envelope, multiple paracrystalline inclusions instead of reinke's crystalloids, multilayered concentric cisternae of smooth endoplasmic reticulum, large masses of microfi laments, and scant lipid droplets. 1092 sex chromatin is apparent in 40-70% of leydig cells. leydig cell function is insuffi cient and androgen levels are less than 50% of normal. basal fsh and lh are markedly increased. 1084, 1093, 1094 in a few patients the testicular damage is less severe, with some tubules showing spermatogenesis and less prominence of leydig cells. 1095 exceptionally, complete spermatogenesis and even paternity have been reported. 1096 the xy/xxy karyotype is the most frequent variant of klinefelter's syndrome with chromosomal mosaicism. in this condition, the clinical abnormalities may be attenuated. gynecomastia is present in 33% of cases, compared to a frequency of 55% in men with the 47xxy karyotype. azoospermia is found in 50% of cases (93% in xxy men). the testes are larger and spermatogenesis is more developed in men with xxy ( fig. 12-127) . patients with the 47xxy karyotype who have spermatozoa in seminiferous tubules are bearers of 46xy spermatogonia and also of 47xxy spermatogonia, whereas those who have no spermatozoa have 47xxy spermatogonia only; these 47xxy spermatogonia may include some spermatozoa with 23x or 23y chromosomal complement, elevated numbers of both 24xy and 24xx spermatozoa, and also a high frequency of spermatozoa with 21 disomy; this could be an important risk for gonosomy 1097 and also for trisomy 21. 1098 genetic counseling is advisable in patients seeking intracytoplasmic sperm injection therapy. genetic diagnosis before implantation of the zygote or prenatal diagnosis have been recommended, except for parents who assume the risk of gonosomy. the incidence of the 48xxyy karyotype is estimated to be 0.04 per 1000 live births. [1099] [1100] [1101] [1102] this karyotype may be associated with aggressive character, antisocial behavior, more severe mental retardation, and a higher frequency of congenital malformations than the 47xxy karyotype. men with the 48xxyy karyotype also have characteristic dermatoglyphics with an increase in arches, a decrease in total fi nger ridge count, and ulnar triradiuses associated with changes in the hypothenar region. 1103 concentric lamellae of smooth endoplasmic reticulum in leydig cells are a characteristic fi nding (fig. 12-128 ). 1104 men with the 48xxxy or 49xxxyy karyotype often have skeletal malformations, principally radioulnar synostosis, and cryptorchidism. 1105 in addition to the characteristic symptoms of 47xxy klinefelter's syndrome, 1106 men with the 49xxxxy karyotype have other abnormalities, including severe mental retardation, hypoplasia of external genitalia, cardiac malformations, radioulnar synostosis, microcephaly, and a high arched palate. 1107 association of klinefelter's syndrome with malignancy patients with klinefelter's syndrome have a higher incidence of malignancy than the general population. the association was fi rst discovered with breast carcinoma, 1108 which had an incidence 20 times greater than in the general male population, 1109 and is related to hormonal stimulation. 1110 although testicular germ cell tumor is rare in these patients, 1111 extragonadal germ cell tumor is 30-40 times more frequent than in the general population. most occur in the mediastinum (about 71%) and are less frequent in the pineal gland, central nervous system, and retroperitoneum. the most frequent types are teratoma and choriocarcinoma; embryonal carcinoma and seminoma are rare. [1112] [1113] [1114] the extragonadal origin of germ cell tumors has been attributed to abnormal germ cell migration from the yolk sac. the high incidence has been attributed to elevated hormone levels and chromosomal anomaly. 1115 in a patient with the xy/xxy chromosomal mosaic and bronchogenic carcinoma, cultured xxy fi broblasts transformed three times more frequently when exposed to sv40 virus than did fi broblasts from normal men. 1116 other tumors reported in patients with klinefelter's syndrome (lymphoma, leukemia, bronchogenic carcinoma, urothelial carcinoma of the bladder, adrenal carcinoma, prostatic adenocarcinoma, testicular leydig cell tumor, and epidermoid cyst) do not appear to have a higher incidence than in the general population. [1117] [1118] [1119] [1120] occurrence of klinefelter's syndrome in childhood early identifi cation of this syndrome is possible with systematic cytogenetic study of newborns with positive sex chromatin or mental retardation. 1121 several clinical symptoms suggest klinefelter's syndrome. initial symptoms include decreased muscle tone, delayed speech, and poor language skills with an increased incidence of reading diffi culties and dyslexia. 1122 later, there may be recognition of mental retardation, 1123 psychiatric problems, excessive stature for age, disproportionately long legs, micropenis, and small testes. [1124] [1125] [1126] [1127] androgen defi ciency is an early fi nding. 1128 testicular biopsy reveals scant or absent germ cells. quantitative studies indicate that the number of germ cells in 47xxy fetuses is signifi cantly lower than in normal 46xy fetuses. the seminiferous tubules have reduced diameter, particularly those devoid of germ cells. the number of sertoli cells per cross-sectioned tubule is reduced. megatubules, ring-shaped tubules, and intratubular eosinophilic bodies are common (fig. 12-129) . in some cases of klinefelter's syndrome associated with down's syndrome, tubular hyalinization is observed in childhood. 1129 the interstitium is wide and contains few leydig cell precursors. if one testis is undescended, its histology does not differ from that of the contralateral testis. the testicular pattern remains constant through childhood. 1130 at puberty, before maturation of the tunica propria occurs, the seminiferous tubules rapidly hyalinize and leydig cell precursors differentiate into leydig cells. 1131 association of klinefelter's syndrome with precocious puberty although precocious puberty is not a characteristic fi nding in klinefelter's syndrome, karyotyping in older boys with mental retardation, gynecomastia, small testes, and precocious puberty is advisable. in most cases, the cause of precocious puberty is a hcg-secreting germ cell tumor in the mediastinum. 1132 infrequently, precocious puberty is idiopathic, and only in isolated cases is there a hamartoma in the third ventricle. 1133 klinefelter's syndrome is often associated with pituitary disorders such as panhypopituitarism 1134 or incomplete hypopituitarism. 1135 defi cits in fsh, 1136 lh, 1137 or both 1138, 1139 have been reported. the cause of this association is unknown, and diverse etiologies such as trauma, immunologic disorders, and genetic defi ciencies have been postulated. alternatively, it may be due to exhaustion of pituitary gonadotropin-secreting cells after years of gonadotropin-releasing hormone stimulation. 1135 in patients defi cient in both gonadotropins, testicular biopsy shows diffuse tubular hyalinization and a marked reduction in or absence of leydig cells. the histological picture is similar to that of hypogonadotropic hypogonadism occurring after puberty, except for the presence of isolated tubules containing only dysgenetic sertoli cells and absence of elastic fi bers in the hyalinized tubular wall (fig. 12-130) . 1139 biopsy of patients with a defi cit only in fsh is similar to that of the dysgenetic sertoli cell variant of the sertoli cell-only syndrome, although some hyalinized tubules are present. the testicular biopsy of patients defi cient only in lh resembles that of men with classic 47xxy klinefelter's syndrome. 46xx males the 46xx karyotype may be present in three phenotypes: male phenotype, including normal external genitalia; male pseudohermaphrodites, with a variable degree of ambiguity in external genitalia, ranging from hypospadias to micropenis; and true male hermaphrodites. 46xx males with male phenotype and normal external genitalia men with the 46xx karyotype having male phenotype and normal external genitalia have clinical features similar to those of klinefelter's syndrome, including small testes, small or normal penis, azoospermia, gynecomastia, and minimal development of secondary sex characteristics. however, these men have harmonious body proportions, normal or slightly low stature, and normal intelligence. 1140 the incidence of 46xx males varies from 1 : 10 000 to 1 : 25 000 live births, accounting for about 0.2% of infertile men. 1141, 1142 males with 46xx karyotype have hypergonadotropic hypogonadism with elevated serum levels of fsh and, to a lesser degree, elevated lh, with normal or slightly decreased testosterone. familial cases have been reported. 1143 during childhood, biopsy of 46xx males reveals decreased numbers of germ cells. 1144, 1145 biopsies from adults show one of three patterns: histology similar to that of 47xxy men, including diffuse tubular hyalinization with prominent leydig cells; 1146 sertoli cell-only tubules; 1147, 1148 and both patterns intermingled with less prominent leydig cells. the last is the most frequent ( fig. 12-131) . ultrastructural studies reveal an increase in intermediate fi laments, absence of annulate lamellae in sertoli cells, 1149 absence of reinke's crystalloids, and abundance of intracytoplasmic and intranuclear paracrystalline inclusions in leydig cells. 1147 46xx males with ambiguous external genitalia some patients with the 46xx karyotype have ambiguous external genitalia or hypospadias and are assumed to have a variation of male pseudohermaphroditism. 1150 these males, together with true hermaphrodites, may be found in the same family, suggesting that both disorders are different manifestations of the same genetic defect. the origin of 46xx males may be diffi cult to determine. however, as testicular differentiation requires genes located on the y chromosome, 46xx males have been classifi ed by cytogenetics as those having the sry gene, those lacking the sry gene, and xx/xy mosaicism. males with the sry gene comprise 80% of 46xx males. 1151 it is likely that this occurs when the genetic material from infertility the short arm of the y chromosome is translocated to the x chromosome. 1152 during paternal meiosis, the homolog pseudoautosomal regions of chromosomes x and y interchange the terminal portions of their short arms, giving rise to an x chromosome with the sry gene but lacking the azoospermia factor. [1153] [1154] [1155] [1156] [1157] [1158] alternatively, the sry region may be inserted in an autosome. 1159 most 46xx patients who are sry positive have a normal male phenotype. about 10% of 46xx males are sry negative and most have ambiguous genitalia. some patients have a normal male phenotype 1161 and only infertility. 1162 although sry is assumed to be the most important regulator factor of testicular determination, these patients may have mutation of one of the downstream non-y testis-determining genes. [1163] [1164] [1165] [1166] about 10% of 46xx males have xx/xy mosaicism or other karyotype with the chromosomal complement y. in these cases, detection of the specifi c dna sequences of y chromosome may be diffi cult because this chromosome may be only in some tissues and in a small number of cells. 1160 47xyy syndrome the 47xyy syndrome was fi rst described in 1961 in the father of a girl with down's syndrome. 1167 the only clinical fi ndings were excessive height and pustular acne. study of other cases suggests that these men are predisposed to a psychopathic personality and antisocial behavior, although most have a normal personality and are socially adapted. the incidence of 47xyy patients is estimated to be 0.01% of the general population, 0.7-0.9% of men in prison, and 1.8% of sexual homicide criminals. 1168 the extra y chromosome originates from non-disjunction during the paternal second meiotic division. in the past decade, many cases have been diagnosed prenatally. from birth, the patients have weight, stature and cephalic circumference above mean values and a higher risk for delayed language and/or motor development. about 50% of children have psychological and psychiatric problems such as autism; although their intelligence is normal, many patients are referred to special education programs. 1169 as adults, they have normal external genitalia and secondary sex characteristics. fertility is reduced, 1170 although many have been fathers. usually, testicular biopsy reveals mixed atrophy characterized by tubules with spermatogenesis associated with sertoli cell-only tubules (fig. 12-132) . 1171, 1172 those tubules with spermatogenesis may show normal spermatogenesis or have lesions in the adluminal or basal compartments. in these tubules, many xxy spermatocytes degenerate during meiosis. about 64 % of pachytene cells have three sex chromosomes. 1173 the number of normal spermatozoa in the ejaculate is low. there is a high incidence of both yy and xy spermatozoa and disomy 18. the variability in germ cell development is apparently due to elimination of germ cells that could not pair their sex chromosomes during the fi rst or second meiotic divisions 1174 or, later, during the round spermatid stage. 1175 spermatocytes that succeed in forming trivalent chromosomes are initially viable. 1176 the ultimate trivalent chromosome segregation yields aneuploid and euploid cells in equal numbers. sertoli cell-only tubules are attributed to either spermatogonial damage by substances released from degenerated spermatocytes 1177 or absence of testicular colonization by primordial germ cells. these men have normal serum levels of testosterone and lh. the latter may be slightly increased in 47xyy men with severe spermatogenic alterations. 1178 47xyy men with mosaicism (47xyy/46xy) have a higher risk of fathering children with hyperdiploid chromosomal constitution, and spermatozoa should be studied genetically to evaluate the risk of intracytoplasmic sperm injection. 1179 men with three and four y chromosomes have been reported. men with the 48xyyy karyotype are tall and have normal male phenotype, slight mental retardation, azoospermia and, during childhood, frequent infections of the upper respiratory tract. 1180 testicular biopsy shows sertoli cell-only tubules, severe hyalinization of tubular basement membrane, and diffuse leydig cell hyperplasia. the chromosomal complement of parents can be normal. 1181 men with 49xyyyy also have no signifi cant phenotypic abnormalities (except for cases of chromosomal mosaicism). slight mental retardation, infertility, and antisocial behavior are the most signifi cant clinical fi ndings. 1182 rarely patients have facial dysmorphism and various skeletal abnormalities. 1183 the y chromosome is essential for gender determination and spermatogenesis, and abnormalities often lead to infertility. the relationship between y chromosome abnormalities and infertility is best understood in azoospermic men with alterations in yq11, the distal region of the euchromatic part of the long y arm, the location of a male fertility gene complex called azoospermia factor. infertility may result from deletion of any of four subregions in which the azoospermia factor has been divided (azfa, azfb, azfc and azfd). 1184, 1185 the best-known y chromosome genes involved in spermatogenesis are rbm. daz, dffry, cdy, smcy, and zfy. six different partial deletions of this region have been found in azoospermic patients (table 12monocentric deleted yq chromosome partial deletion of the distal portion of the yq11 euchromatic region is associated with azoospermia owing to loss of the azoospermia factor. these men have normal external genitalia except for small testes, 1187 normal testosterone and lh serum levels, and increased fsh serum level. the most frequent histological fi nding is sertoli cell-only pattern, although many other patterns have been reported. 1188 the number of leydig cells is normal or increased. these fi ndings suggest that the azoospermia factor is required for early spermatogenesis. 1189 if the breakpoint of yq11 is proximal to the centromere, patients are short because the gene that controls stature is close to that for the azoospermia factor. 1190 dicentric yq isochromosomes sterility is frequent in men with dicentric yq isochromosomes. 1191 this anomaly is usually associated with a 45x cell line. the proportion of this line varies between patients and between cell types (fi broblasts or lymphocytes). when the point of breakage and fusion of the two y chromosomes is in the distal region yq11, and the second centromere is inactivated, the y isochromosome is normal in size but does not stain with quinacrine, and thus is called non-fl uorescent y chromosome (ynf). as the breakpoint is in the yq11 region, the azoospermia factor function is altered. development of external genitalia varies from ambiguous to normal, and is probably related to the extent of xo present. 1192 testicular biopsies are similar to those of men with monocentric deleted yq chromosomes (fig. 12-133) . 1193, 1194 ring y chromosome men with ring y chromosomes have normal male phenotype, azoospermia, and, in some cases, short stature. most have mosaic karyotype with a 45x line. in some cases, testicular biopsy resembles that of men with monocentric deleted yq chromosome, but in others there is premeiotic arrest of spermatocyte maturation. 1195 this is attributed to diffi culty in pairing the x and y chromosomes during meiosis. many patients have deletion of some azf regions. 1196, 1197 y/y translocation chromosome patients with this anomaly have small soft testes and primary spermatocyte maturation arrest owing to defective pairing of the x and y chromosomes. the karyotype may be mosaic with a 45x line. 1198 translocation of y chromosome to x chromosome most frequently this translocation is cytogenetically undetectable, and patients present with infertility and are found to have 46xx karyotype. 1199 the phenotype is similar to that of men with klinefelter's syndrome except for shorter stature, absence of mental retardation, and smaller teeth. testicular biopsy shows sertoli cell-only pattern. men with cytogenetically detectable translocations have short stature, small testes, tubular hyalinization, and prominent clustered leydig cells similar to klinefelter's syndrome. autosomal translocation of y chromosome translocation of the distal heterochromatic portion of the y chromosome to the short arm of an acrocentric chromosome occurs occasionally. the most frequent are translocations to chromosomes 5, 18, 13, 15, and 22. the fertility of these men depends on the point of breakage. 1200, 1201 if this occurs in the yq12 heterochromatic region, the patient has a male phenotype and is fertile. if the point of breakage is in the yq11 region, the patient is infertile and has small testes. seminiferous tubules may show only sertoli cells, spermatogenetic arrest in early stages of meiosis, or an infantile pattern. 1202, 1203 interstitial microdeletion in yq11 yq11 microdeletion is the most frequent congenital cause of infertility. the frequency of y chromosome microdeletion in infertile patients varies widely (1-35%). 1204 in azoospermic men, the frequency is between 18% 1205 and 37%. 1206 in oligozoospermic males the incidence drops. most microdeletions are in the azfc subregion. 1207, 1208 testicular biopsy shows only sertoli cells, maturation arrest, or mixed atrophy. there is no correlation between site of azf subregion alteration and histological pattern. 1209 there is no exact correlation between genotype and phenotype, 1209 but most microdeletions in azfa are associated with azoospermia, most microdeletions in azfb are associated with maturation arrest, and most microdeletions of azfc are associated with spermatid maturation arrest or mixed testicular atrophy. partial deletion of azfc has a mild effect on fertility. 1210 external genitalia in 46xy patients with duplication of distal xp vary from male, ambiguous, to female, and gonadal dysgenesis is frequent. if the patient has male genitalia, these are usually hypoplastic with hypogonadotropic hypogonadism and, frequently, multiple congenital anomalies and mental retardation. 1211 males with translocation of the x chromosome to an autosome may have disturbed spermatogenesis with subfertility or infertility. 1212,1213 47xxx males show mental retardation, gynecomastia, normal stature, hypoplastic scrotum, a well-confi gured but small penis, small testes, and poorly developed pubic hair. serum testosterone levels are very low. seminiferous tubules appear severely hyalinized. 47xxx males result from an abnormal x-y interchange during paternal meiosis and x-x non-disjunction during maternal meiosis. 1214 there have been many reports on the relationship between autosomal anomalies and infertility, although the causes are not fully understood because the same anomaly is associated with infertility in some patients but not in others. robertsonian translocations are found in 0.7% of infertile men (8.5% higher than in the normal population) and are more frequent in oligozoospermic than in azoospermic men. the most frequent translocations are 13;14 and 14;21. the incidence of reciprocal translocations in infertile patients is 0.5% (0.1% in the general population) and increases to 0.8% in patients with azoospermia or severe oligozoospermia. 1215 the most frequent in infertile men are 11;22 and 17;21. paracentric and pericentric inversions (except for the pericentric inversion of the heterochromatic region in chromosome 9) are eight times greater in infertile patients (0.16%) than in the general population. the highest risk for infertility occurs in the pericentric inversion of chromosome 1. 1216, 1217 the most common testicular lesions in men with autosomal anomalies are spermatogonial maturation arrest, primary spermatocyte sloughing sometimes associated with hypospermatogenesis, and sertoli cell-only pattern. 1218 the only autosomal anomaly with prolonged survival is down's syndrome. in addition to trisomy of chromosome 21 and the characteristic appearance, patients with down's syndrome usually have cryptorchidism, small testes, hypoplasia of the penis and scrotum, and hypospadias. 1219 adults have oligozoospermia or azoospermia secondary to primary testicular defi ciency. levels of fsh and lh are elevated, but testosterone is normal or slightly diminished. 1220 isolated cases of paternity have been reported. 1221, 1222 in utero, there is marked delay in germ cell development. 1223 histologic studies of prepubertal testes at autopsy reveal decreased tubular diameter and tubular fertility index. eosinophilic bodies or microliths may be present in some tubules (fig. 12-134 ). adult testes have defi cient spermatogenesis and mixed atrophy, with some tubules showing complete spermatogenesis and others containing sertoli cellonly pattern. 1224 hypergonadotropic hypogonadism is found in several myopathies (myotonic dystrophy and progressive muscular fig. 12-134 prepubertal testis in down's syndrome. there are megatubules, ring-shaped tubules, and small tubules. germ cell number is very low in all these tubules. eosinophilic bodies or microliths are present in some tubules. dystrophy) and dermopathies (bloom's, rothmund-thomson, werner's, cockayne's, and tay's syndromes), with testicular histology that resembles that of klinefelter's syndrome. hypogonadism is also observed in noonan's syndrome, cerebellar ataxia (with milder testicular lesions), and a miscellaneous group of syndromes with variable histological fi ndings. 1225 myotonic dystrophy accounts for approximately 30% of men with muscular disorders, and about 80% have testicular atrophy. the estimated incidence is 1 in 8000 live births. the abnormality involves the distal muscles of the extremities. in addition, patients may have premature baldness, posterior subcapsular cataracts, cardiac conduction defects, impotence, gynecomastia (rarely), and dementia (at later stages). myotonic dystrophy is an autosomal dominant inherited disease with variable penetrance. two loci are associated with the disease phenotype: dm1 in 19q13.3, and dm2 in chromosome 3. mutation in dm1 results in a serine/threonine protein kinase defi ciency that causes expansion of a ctg repeat (from 50 to several hundred repeats) located on the 3′-untranslated region of the dystrophy myotonic-protein kinase gene. the number of repeats is positively correlated to severity of the disease and negatively correlated to age of clinical onset. [1226] [1227] [1228] dm2 is caused by a mutation in 3q21.3 of the znf9 gene and accounts for cctg-repeat expansion (from 75 to 11 000 repeats) in intron 1 of this gene. the common clinical symptoms are due to gain of function of rna mechanism in cug and ccug repeats altering cellular function, including alternative splicing of various genes. 1229 the severity of the disease increases in the successive generations. 1230 the number of ctg repeats is not associated with male subfertility. 1231 hypogonadism is hypergonadotropic in most cases and is not related to the number of ctg repeats. 1232 testicular lesions probably begin late because 65% of patients are fathers. testicular biopsy shows different degrees of severity, ranging from nearly normal to fully hyalinized seminiferous tubules, with the number of leydig cells varying from increased to decreased. in some patients the hypogonadism is hypogonadotropic, and the testes show an infantile pattern. infertility may be the fi rst symptom of myotonic dystrophy. 1233 progressive muscular dystrophy is a multisystemic x-linked disease. it is usually associated with gonadal atrophy caused by a defective locus in chromosome 19. patients rarely live more than 20 years. the incidence is approximately 1 in 4000 live births. in both duchenne and becker forms the cause is a defect in the dystrophin gene. 1234, 1235 bloom's, rothmund-thomson, and werner's syndromes are caused by a homozygous defect in human receq helicases in chromosome 15. of the fi ve members of this gene family (recq1, blm, wrn, recq4, and recq5), three produce autosomal recessive inherited diseases. mutations of blm have been identifi ed in patients with bloom's syndrome, wrn has been shown to be mutated in werner's syndrome, and mutations of recq4 have been associated with rothmund-thomson syndrome. 1236, 1237 despite the close genetic origin of the three syndromes, symptoms are very different. bloom's syndrome is characterized by short stature, narrow face with prominent nose, facial 'patchy' skin color changes that become more marked with sun light exposure, and increased susceptibility to respiratory diseases, cancer and leukemia. severe oligozoospermia and azoospermia are common. leydig cell function is conserved. 1238 rothmund-thomson syndrome presents with poikiloderma, juvenile cataracts, sparse hair, short stature, skeletal defects, dystrophic teeth and nails, and hypogonadism. these patients are predisposed to cancer and osteogenic sarcoma. 1239 werner's syndrome (progeria) is characterized by short stature, prematurely graying hair, baldness, cataracts, atrophy and calcifi cation of muscle and fat, wrinkling of the skin, keratosis, osteoporosis, telangiectasis, atheroma, diabetes mellitus, gynecomastia, and hypergonadotropic hypogonadism. the lifespan of fi broblasts and other cells is shortened in this syndrome. the mutation is in the recq3 helicase gene. cockayne's syndrome is a rare autosomal recessive neurodegenerative disorder. signs and symptoms include infantile failure to thrive, short stature, poorly developed trunk, premature aging, neurological alterations, retinitis pigmentosa, optic atrophy, cataract, deafness, microcephaly, micrognathia, photosensitivity, delayed eruption of primary teeth, congenital absence of some permanent teeth, partial macrodontia, atrophy of the alveolar process and caries, limited articular movements in elbows, knees, and fi ngers, 1240 abnormally small eccrine glands, 1241 and hypergonadotropic hypogonadism. it may be caused by two gene mutations: cnk1 (ercc8) and ercc6, located respectively on chromosomes 5 and 10, and causing two variations of cockayne's syndrome, including cs-a, secondary to a ercc8 mutation, and cs-b with ercc6 mutation. cs-b patients have hypersensitivity to ultraviolet light secondary to a dna repair defect. 1242 tay's syndrome (trichothiodystrophy) has two presentations: ibsd (ichthyosis, brittle hair, impaired intelligence, short stature) and ibisd (photosensitivity, ichthyosis, brittle hair, impaired intelligence, short stature). in both forms, patients have decreased fertility. one case of hypergonadotropic hypogonadism has been reported. 1243 noonan's syndrome is characterized by multiple malformations reminiscent of turner's syndrome, including short statute, pterygium coli, and cubitus valgus, although there is normal male karyotype. the disease has an incidence of 1 in 1000 to 1 in 2500 live births and autosomal dominant inheritance, with sporadic occurrence in about 50% of cases. a locus for dominant forms has been mapped to 12q24.1. 1244 mutation in ptpn11 (protein-tyrosine phosphatase, nonreceptor-type 11) accounts for half of cases, although similar germline mutations also cause leopard's syndrome and certain pediatric hematopoietic malignancies. 1245 cryptorchidism is present in about 70% of cases and is usually bilateral. during childhood, testicular biopsy shows a low tubular fertility index. puberty is often delayed, and, at adulthood, hypogonadotropic or hypergonadotropic hypogonadism occurs. ultrastructural studies reveal morphologic anomalies in germ cells. 1246 although spermatogenesis is generally impaired, some patients have been fertile (fig. 12-135) . cerebellar atrophy may be associated with hypogonadism. patients are infertile and have moderate ataxia without endocrine disorder. infertility is due to morphological abnormalities of spermatozoa caused by decreased expression of map2 (the most important microtubule-associated protein), and a defect in erythroid ankyrin. 1247, 1248 many other syndromes also present with primary hypogonadism. the best known are alström's, weinstein's, borjenson-forssman-lehmann, marinesco-sjögren, richards-rundle, robinow's, and silver-russell syndromes. hypogonadotropic hypogonadism or hypogonadism of hypothalamo-hypophyseal origin is classifi ed according to whether the hypothalamo-hypophyseal failure occurs before or after puberty. eunuchoidism, present only in the former group, is the basis of the distinction. the most frequent types of hypogonadism caused by hypothalamo-hypophyseal failure are those caused by a defi cit of gonadotropinreleasing hormone, bioinactive fsh and lh, defi cit in growth hormone, those associated with prader-willi syndrome, and laurence-moon-rozabal-bardet-biedl syndrome. the onset and maintenance of the hypothalamo-hypophyseal-gonadal axis is due to pulsatile gonadotropin-releasing hormone (gnrh) secretion by neurons of the nucleus arcuatus hypothalamus, with release into the pituitary portal system and subsequent stimulation of gonadotropinreleasing hormone receptors on the surface of gonadotropinsecreting cells. the gnrh gene is located on 4q13. 1249 patients with gnrh defi cit have partial or complete absence of gnrh-induced pulsatile lh secretion, and normalization of pituitary and gonadal secretions after exogenous gnrh administration. imaging studies of the hypothalamo-hypophyseal region are normal. clinical symptoms vary with age at presentation (congenital or acquired) and severity (complete or partial defi cit). clinical presentations include delayed puberty, idiopathic hypogonadotropic hypogonadism (isolated gonadotropin defi cit), kallmann's syndrome, isolated fsh defi cit, and isolated lh defi cit (fertile eunuch syndrome). constitutional delayed puberty is assumed to be a minor form of gnrh defi cit, 1250 and is characterized by delayed sexual maturation in otherwise healthy males. patients are short and usually have a family history of delayed puberty. puberty usually begins at 13-14 years of age and progresses over 2 years. if a 14-year-old boy has not begun pubertal changes (testicular enlargement, growth in height, and development of secondary sex characteristics), delayed puberty should be suspected. 1251 simple pubertal delay that is overcome naturally in a short time without treatment must be distinguished from hypogonadotropic hypogonadism. the latter should be suspected when any of the following symptoms are present in the patient or his family: a midline defect, anosmia, or pubic hair without testicular development. hormone assays may also assist in diagnosis. if a patient between 16 and 18 years old has prepubertal gonadotropin levels, he probably has hypogonadotropic hypogonadism. a variant of hypogonadotropic hypogonadism, isolated gonadotropin defi cit is characterized by defects in the synthesis or release of fsh and lh; other hypophyseal functions are normal. patients have eunuchoid phenotype, with small testes and penis, scanty body hair and beard, a high-pitched voice, and poorly developed muscles. presentation may be sporadic, autosomal dominant, autosomal recessive, or xlinked. the cause might be a mutation in the gnrh receptor gene. 1252 patients have very low levels of fsh, lh, testosterone, and estrogen. clomiphene citrate treatment fails to stimulate hormonal secretion. 1253 pulsatile administration of gnrh is useful to promote both androgen production and spermatogenesis. the lh-leydig cell-testosterone axis is normal in most cases, but normalization of the fsh-sertoli cell-inhibin axis is not achieved in all cases. basal inhibin levels higher than 60 pg/ml and absence of cryptorchidism are favorable predictor factors for the acquisition of normal testicular size and acceptable spermatogenesis. 1254 testicular biopsy reveals an immature pattern. the seminiferous tubules have neither lumina nor elastic fi bers (fig. 12-136 ). sertoli cells are immature, and no differentiated leydig cells are seen. spermatogonia are rare. in some patients the pattern is similar to that of sertoli cell-only testes with immature sertoli cells. 1255 hypogonadism associated with anosmia is also known as maestre de san juan, 1256 kallman, 1257 abnormalities include olfactory bulb agenesis, cryptorchidism, mental retardation, color blindness, facial asymmetry, nerve deafness, epilepsy, shortening of the fourth metacarpal, tarsal navicular fi brous dysplasia, familial cerebellar ataxia, diabetes mellitus, hyperlipidemia, gynecomastia, cleft lip, maxillary or palate, unilateral renal aplasia, and cardiovascular abnormalities. the syndrome may be xlinked or autosomal. the gene for the x-linked form is mapped to xp22.3 and may have different mutations (termed kal-x, kalig-1, and admlx), complete deletion, and point mutations. this gene encodes the protein anosmina-1, which is similar to other nerve cell adhesion molecules and is involved in axonal growth and development. kal protein, secreted by mitral cells, permits the passage of olfactory neurons into the olfactory bulbs and is lacking in kallmann's syndrome. this failure also inhibits migration of neuroblasts from the olfactory epithelium to the hypothalamus to form gnrh-secreting neurons. 1259 the autosomal dominant presentation (occurring in 10% of cases) is due to loss of function of fi broblastic growth factor receptor 1 (fgfr1). 1260 interaction between kal1 and fgfr1 is required for neuronal migration. 1261 patients are classifi ed into two groups according to the partial or complete absence of gnrh. partial absence of gnrh is diagnosed by the presence of spontaneous pulses of lh, fsh, and testosterone during a 24-hour period. complete absence is diagnosed by the absence of spontaneous pulses of lh, fsh, and testosterone during a 24-hour period. these patients show an increase in fsh only after gnrh administration. 1262 testes are histologically infantile; the tubules have a small diameter, lack lumina, and contain immature sertoli cells and isolated spermatogonia. 1263 the interstitium is wide and consists of acellular connective tissue with no recognizable leydig cell precursors (fig. 12-137 ). 1264 autopsy studies in patients with anosmia and hypogonadism reveal agenesis of the olfactory bulbs that may be partial or complete and unilateral or bilateral, together with an apparently normal hypophysis and normal or hypoplastic hypothalamus. this syndrome is the least severe form of holoprosencephaly-hypopituitarism complex, a spectrum of developmental anomalies associated with impaired midline cleavage of the embryonic forebrain, aplasia of the olfactory bulbs and tracts, and midline dysplasia of the face. testicular seminoma has been reported in a patient with anosmia with hypogonadotropic hypogonadism. 1265 this rare syndrome is characterized by azoospermia or oligozoospermia in normally virilized patients with normal sexual potency. serum levels of lh and testosterone are normal, but fsh levels are very low or undetectable. the clomiphene stimulation test gives variable results. the gnrh test induces a normal response only of lh. mutations in the fsh-β gene are exceptional. 1266, 1267 testicular biopsy shows maturation arrest at the spermatocyte level, hypospermatogenesis, or partial sertoli cell-only pattern. 1268 gonadotropin treatment increases spermatozoal numbers in most cases, and fertility may be induced. isolated lh defi ciency, also known as pasqualini's or fertile eunuch syndrome, 1269, 1270 is characterized by hypogonadism secondary to lh defi cit with preservation of spermatogenesis. patients have eunuchoid habitus, small testes, decreased libido, female distribution of pubic hair, and a high-pitched voice. other frequent fi ndings include gynecomastia, anosmia, ocular lesions, and pituitary tumor. 1271 fsh level is normal, but lh and testosterone levels are very low. mutations in the lh-β subunit gene 1272 and the gnrh receptor have been reported. 1273 the clomiphene test is usually negative, and gnrh stimulation increases lh and, to a lesser degree, fsh. testicular biopsy shows seminiferous tubules with normal or slightly decreased diameters and complete spermatogenesis; however, the number of all germ cell types is below normal. leydig cells are rare or absent (fig. 12-138) . maintenance of spermatogenesis in the absence of leydig cells and serum testosterone can only be explained by assuming the occurrence of testosterone secretion suffi cient for spermatogenesis but not to be detectable in the blood. in addition to adequate hypothalamic function, spermatogenesis requires that fsh and lh are biologically active. lh is a heterodimer, composed of two subunits: α (common to fsh and lh) and β (specifi c for lh). the genes for the β subunit are on 19q13.32. if both alleles are mutated for this subunit, the lh produced in biologically inactive although it may be detectable in standard hormone assay. homozygous patients have elevated serum level of lh and low testosterone levels, lack of puberty, and infantile testes. heterozygous patients are only infertile. 1274 patients with mutation in the β subunit of the fsh gene are oligozoospermic or azoospermic. 1275 activating and inactivating mutations of gonadotropin receptor genes have been reported. activating mutation of the lh/human chorionic gonadotropin receptor gene causes familial precocious puberty (see discussion on familial testotoxicosis, below). inactivating mutation of this gene causes male pseudohermaphroditism (see discussion on leydig cell hypoplasia in this chapter). inactivating mutation of the fsh receptor gene produces only mild spermatogenetic lesions, emphasizing the relative value assumed for fsh in spermatogenesis. activating muta-tion of this gene gives rise to spermatogenesis even in the absence of pituitary function. patients with isolated growth hormone defi cit and those with resistance to growth hormone action may have delayed puberty and hypogonadotropic hypogonadism. 1276 some patients with spermatogenetic maturation arrest or idiopathic oligozoospermia have a relative defi cit of growth hormone. this hormone probably acts on the testis by stimulating local secretion of insulin-like growth factor-1, which cooperates with testosterone. prader-willi syndrome is characterized by hypogonadism, obesity, muscular hypotonia, mental and physical retardation, and acromicria. 1277 other frequent fi ndings include strabismus and non-insulin-dependent diabetes mellitus. the incidence is estimated at between 1 in 12 000 and 1 in 15 000 newborns in 25 000 live births, and is higher in males. patients have low serum levels of lh, testosterone, estradiol, and inhibin b, and high levels of fsh. these hormonal fi ndings suggest the occurrence of a mixed form of central (low lh) and peripheral (low inhibin b and high fsh) hypogonadism. 1278 the penis and testes are hypoplastic, and cryptorchidism is present in about 70% of cases (bilateral in 45% of cases) (fig. 12-139 ). 1279 during infancy and childhood, the testes have reduced tubular diameters; adults have an infantile pattern. 1280 this syndrome is caused by an anomaly of chromosome 15, usually in the 15p11-12 band. other chromosomal anomalies include robertsonian translocations, reciprocal translocations, small supernumerary metacentric chromosomes, and partial deletion of the long arm of chromosome 15. this syndrome is a pleiotropic disorder characterized by obesity, infantilism, short stature, diabetes insipidus, mental retardation, retinitis pigmentosa, polydactyly, and syndactyly. it is more frequent in males than in females. men with this syndrome are infertile, and about 74% show hypogonadism. the testes are prepubertal, the scrotum is hypoplastic or bifi d, and the penis is small. cryptorchidism is found in 42% of males, and is bilateral in 28%. at least 11 genes responsible for this syndrome have been cloned, and it is probable that additional genes are involved. the function of the products of these gene is to mediate and regulate microtubule-based transport processes. 1281, 1282 hypogonadotropic hypogonadism associated with dermatologic diseases several dermatopathies are associated with hypogonadotropic hypogonadism, including ichthyosis and johnson's neuroectodermic syndrome. most cases of ichthyosis associated with hypogonadism are x-linked. about 15% of these patients have cryptorchidism, small testes, micropenis, and high risk of testicular cancer. the cause is a defective microsomal enzyme, steroid sulfatase, causing the accumulation of cholesterol sulfate that hinders sloughing of the cornifi ed layer of the epidermis. the gene responsible for this enzyme is mapped to xp22,3. some of these patients also have anosmia or hyposmia owing to involvement of the neighboring genes, causing a contiguous gene defect. 1283 johnson-mcmillin neuroectodermic syndrome is a rare autosomal dominant disorder characterized by alopecia, hypogonadotropic hypogonadism, anosmia or hyposmia, deafness, prominent ears, microtia and/or atresia of the external auditory meatus, and a pronounced tendency to dental caries. 1284 hypogonadism associated with ataxia is rare. most patients are the offspring of a consanguineous marriage. inheritance is autosomal recessive. the most frequent syndromes are louis-bar's syndrome (ataxia-telangiectasia) and friedreich's ataxia. ataxia-telangiectasia is the most common inherited ataxia and is characterized by cerebellar ataxia that starts in infancy and develops progressively; mucocutaneous telangiectasis; anomalies of the immune system that cause pulmonary infection; hypersensitivity to ionizing radiation owing to impairment of dna repair; and a high risk of lymphoid neoplasia. the gene responsible is on 11q22-q23.1. 1285 this ataxia results from inactivation of the a-t mutated (atm) kinase, a critical protein kinase that regulates the response to dna double-strand breaks by selective phosphorylation of a variety of substrates. 1286 friedreich's ataxia is a neurodegenerative disorder characterized by degeneration of dorsal root ganglia and spinocerebellar tracts. hypertrophic myocardiopathy is also observed in many of these patients. the incidence is estimated at 1 in 40 000 children. it is caused by defects in the gene encoding frataxin, a protein required for vesicular traffi c in cell and synaptic transmission. 1287 about 95% of patients are homozygous for an unstable trinucleoid (gaa) expansion in intron 18 of stm7 on 9q13. the normal gene has up to 35 or 40 triplet repeats, whereas patients with this ataxia carry 70 to more than 1000 gaa triplets. 1288 the normal gene has seven to 22 gaa repeats, whereas the mutated gene has over 120 repeats. the extent of the expanded allele is directly proportional to the severity of disease, early onset of disease, and development of cardiac abnormalities. other ataxias associated with hypogonadism are kearns-sayre, boucher-neuhauser, and gordon-holmes syndromes. hypogonadotropic hypogonadism may also be present in carpenter's, biemond's, fraser's, and moebius' syndromes, and in patients with mental retardation. maintenance of spermatogenesis requires the harmonious cooperation of several endocrine glands and proper functioning of other tissues. symptomatic endocrinopathy is present in only 1.7% of infertile men, but over 9% of infertile patients have abnormalities in their endocrine studies. 1289 hypogonadism may be present in disorders involving the hypothalamus-hypophysis, thyroid, adrenals, pancreas, liver, kidney, and gastrointestinal tract, and may be associated with aids, chronic anemia, obesity, lysosomal and peroxisomal diseases, and neoplasia. hypogonadotropic hypogonadism can also be found in some (usually women) who perform rigorous sports (long-distance runners, swimmers, dancers, and rhythmic gymnasts). 1290 hypopituitarism hypogonadism may result from destruction of the hypothalamus or hypophysis by primary or secondary hypothalamic tumor; granulomatous disease ( fig. 12-140 ); fracture infertility of the cranial base; radiotherapy for malignancy of the nasopharynx, central nervous system, or the eye orbit; pituitary adenoma and cyst; aneurysm of the inner carotid artery; and chronic and nutritional disease. many of these processes cause panhypopituitarism with varied symptoms. 1291 clinical manifestations of hypogonadism in patients with pituitary lesions vary according to time of onset (childhood, or after puberty). in prepubertal hypopituitarism the testes retain an infantile appearance into adulthood, and there is rarely proliferation of spermatogonia and the development of primary spermatocytes. biopsy shows variable hyalinization of tubules. in postpubertal hypopituitarism the appearance ranges from complete spermatogenesis to tubular hyalinization ( fig. 12-141) . the presence of elastic fi bers in tubular walls indicates that pubertal maturation occurred before the development of hypopituitarism. leydig cells have pyknotic nuclei and retracted cytoplasm with abundant lipofuscin. in some patients, recovery of spermatogenesis occurs after administration of human chorionic gonadotropin. 1292 there are cases in which pituitary adenoma secretes both fsh and lh, inducing testosterone hypersecretion and an elevated sperm count. 1293 fsh-secreting pituitary adenoma associated with large testes and increased serum inhibin concentration has been reported. 1294 hyperprolactinemia prolactin inhibits gnrh secretion and hence fsh and lh secretion. in addition, prolactin has a direct inhibitory effect on androgens in target tissues. in men, hyperprolactinemia causes impairment of spermatogenesis, impotence, loss of libido, and depressed serum testosterone. 1295 some patients seek treatment because of oligozoospermia and infertility. hyperprolactinemia is also associated with dysfunction of prolactin receptors. 1296 spermiograms usually show oligozoospermia and an elevated level of fructose, 1287 although not all males with hyperprolactinemia have subnormal testicular function. 1298 testicular biopsy reveals variable testicular atrophy. the most frequent lesion is in the tubular adluminal compartment, with degenerative changes in the apical cytoplasm of sertoli cells, sloughing of young spermatids, 1297 and increased lipid droplets in leydig cells. 1299 in boys, two different conditions associated with abnormal prolactin secretion have been reported: hyperprolactinemia, testicular enlargement, and primary hypothyroidism; and prolactin defi ciency, obesity, and enlarged testes. infertility caused by thyroid gland malfunction is rare but reversible. it accounts for about 0.5% of male infertility testicular function is impaired more by hypo-than by hyperthyroidism. patients with hyperthyroidism may have gynecomastia, impotence, and infertility. levels of fsh and lh serum are normal or increased, with elevated sex hormonebinding globulin, increased testosterone concentration, reduced non-sex hormone-binding globulin-bound testosterone, and little or no change in free testosterone. 1300, 1301 in graves' disease there is a pronounced inhibition of gonadal steroidogenesis. 1302 in patients with hyperthyroidism, spermatozoa may be normal or reduced in number, and in both cases progressive motility is low. prepubertal hypothyroidism may impair testicular function by causing precocious or delayed puberty. in delayed puberty, hypothyroidism leads to hypogonadotropic hypogonadism, with testes showing incomplete maturation arrest and, in severe myxedematous hypothyroidism, hydrocele. 1303 in experimental hypothyroidism, testicular enlargement is frequently associated with increased spermatid production. 1304 primary hypothyroidism in adults causes hypergonadotropic, hypogonadotropic, or normogonadotropic hypogonadism, 1305 but testicular function is rarely impaired and patients are usually infertile. 1306 the cause of testicular damage is decreased gonadotropins or hyperprolactinemia. 1307 children with hypothyroidism usually have precocious pseudopuberty. 1308 about 11% of infertile patients reportedly have subclinical adrenal dysfunction, but the true incidence is probably lower. adrenal disorders most frequently associated with infertility are adrenal hypoplasia, adrenal hyperplasia, and adrenal carcinoma. congenital adrenal hypoplasia with hypogonadotropic hypogonadism is an x-linked recessive disorder that gives rise to adrenal insuffi ciency in the fi rst months of life. in later presentations, patients have cryptorchidism and delayed puberty. 1309 the responsible gene, dax1 on xp21, is expressed in the adrenals, testes, pituitary, and hypothalamus. the resulting hypogonadism may be either pure or mixed (hypophyseal and testicular). in the last case, hypogonadism is partial. 1310 testicular biopsy from one adult with adrenal hypoplasia showed an apparent primary lesion, including tubules with dysgenetic sertoli cells and others with spermatogonial maturation arrest in associated with hypertrophy and hyperplasia of leydig cells. 1311 infertility is frequent in patients with minor forms of congenital adrenal hyperplasia. those with defi ciency of 21hydroxylase 1312 or 11β-hydroxylase usually have complete spermatogenesis but with reduced numbers of all germ cells. the characteristic histologic fi nding is decreased numbers of leydig cells. [1313] [1314] [1315] [1316] in untreated patients, the testes become enlarged by 'tumors' of the adrenogenital syndrome that consist of cells similar to adrenal cortical cells ( fig. 12-142 ). [1316] [1317] [1318] [1319] adrenal cortical carcinoma adrenal carcinoma is often associated with excessive secretion of several hormones, causing hyperaldosteronism, cushing's syndrome, virilization, or feminization. virilizing tumors in infancy have their own characteristics, which differ from those of the same adult tumors as the infantile form may be associated with other disorders, such as hemihypertrophy and beckwith-wiederman syndrome, may be included in the spectrum of 'families with cancer predisposition' (mutations in p53 gene), and produce precocious pseudopuberty syndrome. in adults, adrenal carcinoma may cause marked spermatogenic depletion owing to the conversion of large amounts of dehydroepiandrosterone produced by the tumor into estrogen. feminizing tumor in infancy causes gynecomastia and pubic hair development. 1320 feminizing tumor presents more striking clinical characteristics, including progressive loss of secondary sex characteristics and feminization due to elevated estrogen. testicular atrophy results from the inhibitory effect of estrogen on pituitary gonadotropins. similar symptoms may be observed in patients with prostatic carcinoma treated with estrogens ( fig. 12-143 ) and in other conditions with excessive estrogen production, such as sertoli cell or leydig cell tumor. patients with cushing's syndrome or diseases that require long-term corticoid therapy, such as ulcerous colitis, rheumatoid arthritis, or asthma, have reversible reduction of fertility. the explanation for this is that most testicular receptors for corticoids are in leydig cells, and thus glucocorticoids are powerful inhibitors of testosterone synthesis. alterations in the carbohydrate, lipid and protein metabolism characteristic of diabetes mellitus involve the genital system, although most diabetic patients are fertile. gonadal impairment depends on the type of diabetes and the time of disease onset (infancy and childhood, puberty, or adulthood). 1321, 1322 testicular lesions in newborns with diabetic mothers are discussed in the section on congenital anomalies of the testis. 317 puberty may be delayed in diabetic patients, although the cause is unknown. other gonadal alterations appear at puberty, and diabetic men who have not been adequately treated may be infertile and have sexual dysfunction. serum levels of fsh, lh, and testosterone are decreased. 1323 spermiograms reveal low numbers and poor motility of spermatozoa. 1324 prolactin levels are increased and testosterone levels low or near normal. the seminiferous tubules have reduced diameters, thickening of the lamina propria, and alterations in the adluminal compartment. these consist of degenerative changes in the sertoli cell apical cytoplasm and sloughing of immature germ cells. the major lesion is in the interstitial connective tissue and leydig cells. small interstitial blood vessels show diabetic microangiopathy characterized by enlargement and duplication of the basal lamina, pericyte degeneration, and endothelial cell alterations. the number of fi broblasts infertility and the amount of collagen and ground substance in the interstitial connective tissue are increased. 1325 leydig cells are decreased in number and show increased amounts of lipid droplets and lysosomes, accounting for the reduced function of these cells. the tubular lesions are attributed to low serum testosterone, probably owing to defi cient leydig cell stimulation by insulin (or a decrease in insulin-dependent fsh) and abnormal carbohydrate metabolism of sertoli cells. sexual dysfunction is present in more than half of patients and consists of impotence, decreased libido, disorders of intercourse, and retrograde ejaculation. the causes of impotence are multiple, including microangiopathy and macroangiopathy, hormonal defi ciencies, psychological factors, and autonomic neuropathy affecting the parasympathetic system. neuropathy is probably chiefl y responsible for erectile failure in diabetic men. 1326 alterations in sperm excretory ducts may be associated with diabetes. the most frequent are enlarged seminal vesicles and calcifi cation of both seminal vesicles and vasa deferentia. calcifi cations are found in the muscular layers and display a concentric arrangement (fig. 12-144 ). 1327 although cystic fi brosis (mucoviscidosis) was recognized as a disease prior to 1940, its effects on the male genital system were not recognized until the 1970s. this may be explained by improvements in medical care during childhood, allowing the survival of many patients to adulthood, and the recognition of cystic fi brosis in patients who had been diagnosed with chronic bronchitis and hepatic or digestive dysfunction. in the us, cystic fi brosis is the most lethal congenital disease, with a prevalence of 1 in 2500 children, and a carrier status of 1 in 25 white men. 1328 lesions in sperm excretory ducts involve (in decreasing order of frequency) the vas deferens (congenital bilateral absence, unilateral absence), ejaculatory ducts (bilateral obstruction), epididymis (diffuse or segmental hypoplasia), and seminal vesicles (incomplete development). thus, it appears that most patients with cystic fi brosis have infertility due to obstruction. 1329, 1330 histologic studies in children, even at an early age, reveal that the vas deferens and ductus epididymis are absent or reduced to small ductuli with reduced or absent lumina and thin, poorly muscular walls (fig. 12-145 ). the testes are normal during childhood, but show hypospermatogenesis and spermatid malformations by adulthood. the spermiogram is characteristic of obstructive azoospermia, with acid ph, decreased semen volume and fructose concentration, and increased citric acid and acid phosphatase. 1331 the disease is a genetic disorder with autosomal recessive inheritance. the impaired gene (cystic fi brosis gene) is on chromosome 7 (7q31), 1332 and encodes a protein termed cystic fi brosis transmembrane regulator (cftr). alterations in this protein cause cystic fi brosis. although more than 800 mutations of this gene have been identifi ed, 1333 the most frequent mutation in caucasians is d-f508, responsible for 70% of cases. congenital bilateral obstructive azoospermia secondary to bilateral absence of the vas deferens, even in the absence of other symptoms, is often a forme fruste of cystic fi brosis. 1334 before initiating treatment for infertility, the possibility that the patient is a carrier of the cystic fi brosis gene should be evaluated. 1335 malformation of the genital system plays the most important role in infertility in cystic fi brosis. 1336 the lesions begin in the 10th week of gestation, when the wolffi an duct forms the sperm excretory ducts. 1337 variable penetrance of the cystic fi brosis gene accounts for the diversity of malformations affecting different regions of the male genital system. the liver has a primary role in metabolism, detoxifi cation, and excretion of sex steroid hormones. chronic hepatic failure damages the hypothalamo-hypophyseal-testicular axis, and subsequently all related endocrine glands. hypogonadism is frequent in the fi nal stages of severe chronic liver diseases, including alcoholism, non-alcoholic liver disease, and hemochromatosis. the association of testicular atrophy with gynecomastia and hepatic cirrhosis is well known and is referred to as silvestrini-corda syndrome. 1338, 1339 alcohol has a direct toxic effect on leydig cells. acute alcoholic intoxication suppresses serum testosterone in voluntary non-alcoholic men and laboratory animals. chronic alcohol ingestion, even in the absence of cirrhosis, causes hypogonadism, with symptoms of leydig cell failure, including testicular atrophy, infertility, decreased libido, impotence, and reduced size of the prostate and seminal vesicles. 1340 chronic alcoholic patients with cirrhosis also have symptoms of hyperestrogenism, including gynecomastia, female escutcheon, and female fat distribution pattern. most chronic alcoholic men, with or without cirrhosis, have signifi cant testicular lesions. the seminiferous tubules have reduced diameters, thickened lamina propria, and decreased or absent germ cells. leydig cells are reduced in number and contain abundant lipofuscin granules ( fig. 12-146 ). the epididymis becomes atrophic, mainly in the ductuli efferentes, owing to androgen deprivation. the epithelium of the rete testis becomes cuboidal or columnar due to estrogens. the spermiogram correlates with the variability of histologic fi ndings, usually showing a marked reduction in the number and motility of spermatozoa and an increase in the percentage of morphologically abnormal spermatozoa. 1341, 1342 about 20% of patients initially have an increase in serum testosterone; with advanced disease, testosterone level decreases. the initial increase is due to an elevation in sex hormone-binding globulin concentration and reduced testosterone metabolism by the liver. 1343 serum estrogen level also increases owing to increased conversion of testos-terone into estrogen in peripheral adipose and muscular tissue. 1344 non-alcoholic liver disease impairs gonadal function according to the severity of the disease. 1345 patients have decreased levels of total and biologically active free testosterone. hormonal alterations are not as severe as in alcoholic patients, emphasizing the direct action of alcohol on leydig cells. in α 1 -antitrypsin defi ciency testicular function and fertility are conserved; only in advanced stages of the disease do minor biochemical alterations occur. 1346 in alagille's syndrome (intrahepatic biliary duct hypoplasia), hypogonadism is associated with cholestasis, frequent vertebral, cardiac, and facial malformations, and mental retardation. hypogonadism is manifest by small testes, delayed puberty, and, in adults, lack of germ cell development. hereditary hemochromatosis is the most frequent genetic disease in the northern hemisphere and results from excessive iron absorption and accumulation in multiple tissue and organs, leading to cirrhosis, diabetes, hypogonadism, and arthralgia. four types of hereditary hemochromatosis have been reported. 1347 type 1, the most frequent, is caused by mutation in the hfe gene (c282y), leading to increased intestinal absorption of iron, supersaturation of iron deposits, and damage in multiples organs. the type i hereditary hemochromatosis gene (hfe) is located on the short arm of chromosome 6, 1348,1349 is present in 85-100% of hemochromatosis patients with northern european ancestry, and its protein product is mainly expressed in the epithelium of lieberkühn crypts. this protein interacts with the transferrin receptor, reducing its affi nity for iron-bound transferrin; therefore, hfe becomes a negative regulator of transferrinbound iron uptake. type 2 gene is a juvenile form that expresses before the age of 30 years in both sexes, and is associated with severe cardiomyopathy and hypogonadism. 1350 the type 2 hemochromatosis locus is on chromosome 1q21, but this gene has not yet been isolated. 1351, 1352 type 3 is on chromosome 7q22, impairs the transferrin 2 receptor, and its consequences are similar to those of type 1 receptor defect. type 4 is autosomal dominant, on 2q32, and affects the basolateral iron carrier ferroportin 1, resulting in iron deposition in macrophages. types 1, 2, and 3 have recessive autosomal inheritance and show a similar distribution pattern of iron deposits. in these three types, alteration of gonadal function has also been reported. iron homeostasis depends on many genes that act in a coordinated manner, and their exact function is not well known. it is assumed that normal individuals absorb 1-2 mg/day of iron, whereas homozygous patients with hereditary hemochromatosis absorb up to 3-4 mg/day. once iron deposits become saturated (cells of liver, pancreas, hypophysis, heart, adrenals, and gastric mucosa), the toxic effects of iron cause dysfunction of the liver (cirrhosis and cancer in 5-10% of patients), the pancreas (diabetes in 80% of patients), the heart (myocardiopathy), musculoskeletal system (arthritis), and hypophysis (hypogonadism) (fig. 12-147) . hypogonadism may be the fi rst sign of disease when it starts in adult life. 1353 with age, hypogonadism becomes hypogonadotropic, with low serum levels of testosterone, lh, and fsh in more than 40% of patients, 1354 except if early treatment is initiated. 1355 the most frequent fi ndings are testicular atrophy with diminished tubular diameter, tubular wall thickening, a progressive decrease in spermatogenesis, and increased lipofuscin granules in leydig cells. the cause of these testicular disorders might be preferential deposition of iron in gonadotropic cells. 1356 iron deposits are not observed in the testis. hypogonadism decreases after aggressive therapy. 1357 polycystic renal disease in adults is a dominant autosomal disorder that appears with 1 in 1000 frequency in the general population. patients with this disease comprise 10% of end-stage renal failure cases. 1358 infertility is common, even before the beginning of renal insuffi ciency. oligoteratozoospermia and necrospermia are frequent fi ndings. 1359, 1360 serum levels of fsh, lh, prolactin, testosterone, and estradiol remain normal for a long time before the onset of renal insuffi ciency. the causes of spermiogram alterations have been related to partial obstruction of ejaculatory ducts (based on fi nding cystic dilations in seminal vesicles in 60% of patients) or seminal vesicle cyst. 1361 the incidence of these two disorders in patients with polycystic renal disease is very high compared to andrological patients without this disease (5.2%). 1362 chronic renal insuffi ciency is associated with disturbed endocrine function in the pituitary, thyroid, parathyroids, and testes. the associated sexual dysfunction consists of erectile impotence, diminution of libido and semen volume, oligozoospermia or azoospermia, and infertility. in children, skeletal development and puberty are delayed. 1363 hormonal studies reveal elevated levels of fsh, lh, and prolactin, but testosterone levels are low. 1364 testicular biopsy shows seminiferous tubules with reduced diameters and reduced or absent germ cells (fig. 12-148) . 1365, 1366 the interstitium contains a normal number of leydig cells and increased numbers of macrophages. additionally, patients with chronic renal insuffi ciency due to glomerulonephritis have thickening of the tubular lamina propria and decreased number of leydig cells. patients with end-stage renal disease who undergo dialysis show calcifi cations in several organs and tissues, including the male genital system (epididymidis, tunica albuginea, and cavernous tissue) in 87% of cases, and, in isolated cases, calcifi cation of the testicular parenchyma and microlithiasis. 1367 elevated serum levels of phosphorus, increased calciumphosphorus product, severe hyperparathyroidism secondary to other disorders, older age, and prolonged time on dialysis contribute to this disorder. uremic calcifi cation is a cell-mediated process in which elevated levels of tgf, vitamin k-dependent proteins such as osteocalcin and atherocalcin, and defects in calcium-regulatory proteins such as fetuin are implicated. 1368 when these patients are dialyzed, accumulations of urate and oxalate crystals are deposited in the rete testes and ductuli efferentes. these crystals are deposited beneath the epithelium and often sloughed into the lumen. reactive changes in the rete testis, including cystic transformation, are frequent (see disorders of the rete testis). 1369 the cause of gonadal dysfunction is unclear and probably involves several factors, including impaired testicular steroidogenesis, 1370 reduced clearance of pituitary hormones, 1371 and secretory defects of the pituitary and hypothalamus. 1372 dialysis does not improve testicular function. the response to renal transplantation is not immediate and is related to the glomerular fi ltration rate. patients with rates lower than 50 ml/min develop atrophy of the seminiferous tubular cells. 1370 hypogonadism is a frequent fi nding in men with celiac disease, and results in clinical symptoms in 5-10% of untreated patients. celiac disease causes infertility in some cases. spermiograms show reduced motility and numerous morphologic anomalies in spermatozoa. hormonal studies show elevated serum fsh levels in more than 25% of men with celiac disease. lh also is increased in more than 50% of these men. the response of fsh and lh to gnrh stimulation is excessive. the cause of this pituitary derangement is unknown. sperm anomalies are not always corrected by a gluten-free diet. studies in patients with ulcerative colitis and regional enteritis reveal a low sperm count, impaired motility, and ultrastructural alterations, including nuclear pleomorphism and chromatin malcondensation and decondensation. zinc defi cit may be responsible for these alterations in crohn's disease. 1373 the alterations apparently are related to the extent of the intestinal lesions and the severity of symptoms. 1374 patients with ulcerative colitis treated with salazopyrine, 1375 mesalazine 1376 or fasalazine 1377 present with signifi cant impairment of spermatogenesis and subfertility. spermiogram parameters improve when treatment ceases. more than 17% of hiv-infected men have hypogonadism, 1378 which can be observed even in those whose viral replication is under control and show normal numbers of cd4 lymphocytes. patients frequently develop 'early andropause,' marked by dysregulation of the hypothalamopituitary-testicular axis. 1379 hypogonadism is more frequent in hiv-infected men with wasting syndrome, and therefore these patients should undergo screening for hypogonadism and, if necessary, physiologic androgen replacement therapy. [1380] [1381] [1382] [1383] the incidence of hypogonadism in males with aids is estimated to be 50%. 1384, 1385 according to autopsy studies this increases to 100% in the 3-24 months prior to death. 1386 histological studies reveal that 28% have complete but quantitatively abnormal spermatogenesis, and the remainder have spermatocytic arrest or sertoli cell-only pattern. patients with chronic anemia requiring multiple transfusions develop iron deposits in the pituitary and polyglandular insuffi ciency, with atrophy of the thyroid, adrenals, and testes ( fig. 12-149) . the most frequent conditions are βthalassemia and sickle cell anemia (see fig. 12-119) . β-thalassemia is an autosomal dominant disease with three types: thalassemia trait (heterozygous β-thalassemia), intermediate thalassemia, and major β-thalassemia. the cause is mutation in the β-globin gene resulting in ineffective erythropoiesis, hemolysis, and anemia. nearly 20% of patients with major thalassemia have delayed puberty, [1387] [1388] [1389] and 69% have hypogonadotropic hypogonadism. 1390 gonadal dysfunction persists in most patients after healing of the thalassemia. 1391 sickle cell anemia is an autosomal recessive disorder with a constellation of fi ndings resulting from abnormal synthesis of hemoglobin, with over 90% of hemoglobin being type a. most patients have hypogonadotropic hypogonadism. 1392 the majority of people in developed countries are currently overweight, and the incidence of obesity seems to be increasing. infertility is frequently associated with obesity. very obese males have increased levels of serum estradiol and decreased levels of free testosterone and inhibin b. 1393 testosterone reduction is not followed by a compensatory increase in gonadotropins, resulting in hypogonadotropic hypogonadism. 1394, 1395 testicular abnormalities begin with the adluminal compartment and later involve the basal compartment; also, there are leydig cell atrophy, cuboidal metaplasia of the rete testis, and epididymal atrophy. there are three types of autoimmune polyglandular insufficiency syndrome. type i is defi ned by the presence of at least two of three characteristic features: addison's disease, hypoparathyroidism, and chronic mucocutaneous candidiasis. the aire gene (autoimmune regulator), responsible for type l disease, is on 21q22.3, 1396,1397 and the disorder is recessive autosomal. hypergonadotropic hypogonadism is frequent. 1398 patients with type i syndrome have antibodies against many autoantigens, intracellular enzymes including the p450 side-chain cleavage enzyme, 17α-hydroxylase 1399, 1400 and 21-hydroxylase, glutamic acid decarboxylase 65, aromatic l-amino acid decarboxylase, tyrosine phosphataselike protein ia-2, tryptophan hydroxylase (tph), tyrosine hydroxylase, and cytochrome p450 1a2. 1401 type ii autoimmune polyglandular syndrome is characterized by the presence of diabetes mellitus, hyperthyroidinfertility ism, hashimoto's thyroiditis, addison's disease, vitiligo, alopecia, pernicious anemia, and hypogonadism (listed in decreasing order of frequency). type iii syndrome includes thyroiditis, diabetes mellitus, pernicious anemia, and vitiligo or alopecia. about 14% of patients have hypogonadism owing to autoimmune destruction of the testis or pituitary gonadotropin-secreting cells (fig. 12-150) . 1402, 1403 there are at least four diseases caused by metabolic deposits in lysosomes or peroxisomes associated with testicular alterations, including fabry's disease, adrenal leukodystrophy, wolman's disease, and cystinosis. fabry's disease is an x-linked metabolic disorder characterized by intralysosomal deposits of globotriaosylceramide (gb3) owing to α-galactosidase defi ciency. clinical symptoms begin with painful neuropathy and progressive renal, cardiovascular, and cerebrovascular dysfunction. all endocrine glands may accumulate gb3 as a result of welldeveloped vasculature and low rate of cell proliferation. 1404 testes and sperm excretory ducts are always damaged. some alterations, including those of endothelial cells, smooth muscle cells, and fi broblasts, are non-specifi c; others, such as those of myofi broblasts, leydig cells, and epididymal epithelium, are specifi c (figs 12-151, 12-152 ). spermatogenesis is defi cient. 1405 enzyme replacement therapy with recombinant human α-galactosidase eliminates existing glycosphingolipid deposits and blocks new ones, and is thus recommended for implementation as soon as possible after diagnosis. [1406] [1407] [1408] adrenoleukodystrophy (adrenal testicular myeloneuropathy) this disorder is caused by mutation in the adrenoleukodystrophy gene on xq28. 1409 mutation at this site produces three peroxisomal diseases: adrenoleukodystrophy, adrenomyeloneuropathy, and addison's disease. adrenoleukodystrophy is characterized by progressive demyelinization of the central nervous system, usually in children and young adults, often with adrenal insuffi ciency and testicular failure. peroxisomal β-oxidation is defi cient and, as a result, very long-chain fatty acids accumulate inside peroxisomes in many tissues, causing the signs and symptoms of the disease. 1410, 1411 adrenomyeloneuropathy begins at a later age (about 30 years) with progressive paraparesis, peripheral neuropathy, and adrenal cortical failure. males usually have gonadal dysfunction with oligozoospermia or azoospermia and hypergonadotropic hypogonadism. 1412 testicular atrophy develops slowly, the seminiferous epithelium disappear, and leydig cells contain characteristic cytoplasmic lamellar inclusions, with similar inclusions in adrenal cortical cells and cerebral cells. 1413 wolman's disease is a rare inherited lysosomal disease characterized by a defi cit in acid lipase/cholesteryl ester hydrolase. the genetic mutation has been mapped to 10q23.2-q23.3. 1414 complete enzymatic defi ciency (wolman's disease) causes death in infancy as a result of the accumulation of cholesterol esters and triglycerides in numerous organs such as the liver, adrenal cortex, and intestines. 1415 partial defi ciency is known as cholesteryl ester storage disease, and the testis accumulates triglycerides and cholesterol in leydig cells and, to a lesser degree, in interstitial macrophages. delayed disruption of spermatogenesis by this storage disease probably accounts for the frequent lack of fertility problems in men with this disease. 1416 early treatment of children with wolman's disease by transplantation of umbilical cord blood-derived stem cells may successfully restore acid lipase level in some. 1417 cystinosis is an autosomal recessive metabolopathy characterized by alterations in cystine transport from the lysosomes to the cytosol that results in intralysosomal accumulation of cystine. there are several genes responsible, all on chromosome 17p13. cystine storage occurs in all body tissues. deposits in the renal parenchyma cause the main complication of cystinosis, namely renal insuffi ciency (nephropathic cystinosis). patients also develop hypergonadotropic hypogonadism. testicular involvement may be massive, with interstitial macrophages fi lled with cystine crystals that are visible by polarized light. 1418 niemann-pick disease consists of a heterogeneous group of inherited recessive autosomal diseases characterized by deposition of lipids in macrophages and other tissues. there are four reported types (a, b, c, d). the most common, type a, results from excessive storage of sphingomyelin owing to a mutation in the acid sphingomyelin gene that encodes a lysosomal hydrolase, located on 11p15.1-4 region. 1419 interstitial macrophages in the testes have wide eosinophilic, granular cytoplasm. ultrastructural studies reveal a large number of lysosomes fi lled with laminate bodies. physical and chemical agents may impair testicular function by direct action on the pituitary, the testis, or the sperm excretory ducts. in the pituitary, damage to gonadotropic cells may be caused by estrogen. in the testes, gonadotoxic agents may selectively impair a select cell type, but later, global dysfunction occurs. for example, there is direct toxicity to sertoli cells by phthalates used as plasticizers, nitroaromatic compounds intermediate in the production of dyes and explosives, and γ-diketones used as solvents. direct toxicity on spermatogenesis is seen wtih ionizing radiation. many drugs that impair epididymal fl uid or spermatozoon transport damage sperm excretory ducts, with subsequent loss of fertility. 1420 the relationship between infertility or subfertility and certain professions or exposures to environmental agents is well known. 1421 adverse effects of the following agents on spermatogenesis has been demonstrated: organic solvents such as chlorinated solvents, aromatic solvents and varnishes, degreasers, thinners, and adhesives; this is also the case with carbon disulfi de exposure; pesticides such as ddt, linuron, and polychlorinated biphenyls; 1422 heavy metals such as lead, cadmium, mercury, and copper; industrial wastes such as dioxins and ethylene dibromide; phthalates and polyvinyl chloride; oral contraceptives; exposure to radiation or high temperature; and recreational drugs and doping. there is also a long list of potentially harmful agents that disrupt testicular function. 1423 carbon disulfi de is used as a solvent in the production of rayon. continuous exposure is toxic to the nervous system, and causes a decrease in spermatogenesis and libido and an increase in fsh and lh serum levels. 1424, 1425 dibromochloropropane dibromochloropropane is used as a soil fumigant to control nematodes. lengthy exposure causes oligozoospermia, azoospermia, increased fsh and lh levels, and y-chromosome non-dysjunction. 1426 of the two natural forms of lead, organic and inorganic, the inorganic form is more dangerous. exposure to inorganic lead by workers in smelting, battery, and stained-glass plants causes direct spermatogenic damage. 1427 patients have asthenospermia, teratozoospermia, and oligozoospermia. 1428, 1429 oral contraceptive manufacture workers in pharmaceutical plants using synthetic estrogens and progestins develop hyperestrogenism with gynecomastia, decreased libido, and impotence. 1430 neonatal exposure of males to diethylstilbestrol may induce cryptorchidism, testicular hypoplasia, epididymal cyst, and severe anomalies in semen production. 1431 there is increasing evidence to suggest that estrogen-like effects are produced by a variety of naturally occurring estrogens (so-called phytoestrogens) and numerous synthetic compounds such as phthalates, 1432 pesticides, 1433 and polychlorinated biphenyls. 1434 the principal methods of contact with potential endocrine-disrupting compounds is dietary ingestion of milk, fi sh, meat, fruits and vegetables, or environmental exposure. 1435 the increasing incidence of cryptorchidism, hypospadias, testicular cancer, and poor semen quality may be related to the negative infl uence of environmental factors on the testis during fetal life. the term 'testicular dysgenesis syndrome' has been proposed to designate this constellation of putative syndromes. 1436 estrogen exposure in utero may disrupt development of the testes and the entire male reproductive tract. estrogen may hinder fsh secretion by the fetal pituitary, and also interfere with subsequent sertoli cell proliferation, and hence the secretion of amh required for the regression of müllerian ducts. persistence of müllerian derivatives is associated with lack of testicular descent. changes in amh secretion may also account for altered germ cell proliferation during fetal life. exposure to high concentrations of estrogen might compromise testosterone production as well as masculinization of external genitalia (hypospadias) and inguinal descent of the testis (cryptorchidism). abnormal development of sertoli cells and low germ cell numbers could cause diminished spermatozoon production and infertility. 1437 marijuana decreases sperm density and motility and increases the number of morphologically abnormal spermatozoa. 1438 cocaine induces apoptosis in the rat testis ( fig. 12-153 ). 1439 about 20% of injection drug users have low serum testosterone levels. consumption of more than 80 g alcohol per day adversely affects spermatogenesis in two-thirds of patients. 1440 women smoking more than 20 cigarettes per day have fertility problems, neonatal and perinatal mortality, miscarriage, and congenital malformations. 1441 abuse of anabolic steroids by athletes causes hypogonadotropic hypogonadism and transient azoospermia. 1442 ionizing radiation causes alterations in spermatogenesis and hormonal regulation of the testes. some patients recover fertility a few years after exposure. 1443 the effects of non-ionizing radiation are less severe; however, reduced libido and reduced numbers of spermatozoa have been reported in men exposed to microwaves. 1444 heat normal intratesticular temperature is 31-33°c, about 4-6°c lower than core body temperature. conditions causing higher testicular temperature, such as varicocele and cryptorchidism, also cause testicular damage, with decreased numbers of spermatozoa and an elevated percentage of sper-matozoa with abnormal forms and low motility. 1445, 1446 primary spermatocytes at the end of the pachytene stage are most sensitive to heat. the mechanism by which heat produces testicular lesions is unknown; hyperthermia affects the activity of enzymes such as ornithine decarboxylase 1447 and carnitine acetyl transferase, 1448 both necessary for metabolism and proliferation of the seminiferous tubular cells. 1449 the synthesis of dna and rna by germ cells also depends on temperature. dna synthesis by spermatogonia and preleptotene primary spermatocytes is higher at 31°c than at 37°c. rna and protein synthesis are normal at temperatures between 28°c and 37°c, but decrease markedly at 40°c. 1450 testicular trauma is especially frequent among athletes. trauma results in a wide variety of lesions, including contusion with or without hematocele, rupture, dislocation, and eventually spermatogenetic alteration that may lead to infertility. dislocation involves the displacement of one or both testes to a non-scrotal location 1451, 1452 such as the inguinal canal, abdominal cavity, acetabular area, or distant locations such as the perineum, subcutaneous tissues, or superfi cial to the outer oblique fascia. 1453, 1454 spermatogenetic recovery by orchidopexy has been successfully performed up to 13 years after bilateral traumatic dislocation. 1455 sexual dysfunction is found in 25-50% of patients who are treated for cancer. 1456 testicular cancer, hodgkin's disease, and leukemia are the most frequent malignancies during the reproductive years. therefore, preservation of fertility requires careful selection of less gonadotoxic therapeutic regimens; if paternity is planned, cryopreservation of semen before treatment may be considered. the most destructive treatments for gonadal function are radiation therapy and alkylating agents. 1457 the testicular parenchyma is one of the most radiosensitive tissues of the body, and the germ cells are the most radiosensitive cells of the testis. experimental irradiation of volunteers with a single dose revealed that late spermatogonia (ap and b) are more radiosensitive than early (ad) spermatogonia. ap and b spermatogonia may be destroyed with doses as low as 0.3 gy (1 gy = 100 rad), whereas ad spermatogonia tolerate doses higher than 4 gy. type a spermatogonia, spermatids, and spermatozoa are respectively 100, 200, and 10 000 times less radiosensitive than b spermatogonia. doses higher than 6 gy produce a sertoli cellonly pattern. leydig cells tolerate up to 8 gy and sertoli cells up to 60 gy, although sertoli cells show ultrastructural alterations and increased phagocytosis of germ cell remnants after low doses of radiation. even with optimal protection, the contralateral testis absorbs from 0.2 to 1.4 gy in adjuvant therapy for rectal cancer 1458 or when the opposite testis is irradiated, 1459 a dose suffi cient to cause temporary azoospermia. likewise, irradiation of iliac or inguinal lymph nodes for hodgkin's disease or other forms of lymphoma exposes the testes to about 5 gy. 1460 restoration of testicular function is time-dependent, 1461 requiring at least 2 years. 1462 fertility in thyroid cancer patients who received radioiodine-131 ( 131 i) therapy decreases briefl y, but infertility is not permanent. 1463 electromagnetic radiation from cell phones impairs spermatozoon motility according to one study. 1464 prepubertal testes also are sensitive to radiation therapy. patients treated for wilms' tumor may have delayed puberty and, at adulthood, oligoospermia or azoospermia with elevated levels of fsh; this fi nding suggests that leydig cells are also damaged. a special case is that of children with acute lymphoblastic leukemia involving the testis. radiotherapy with doses of 20-25 gy, either alone or with chemotherapy, causes irreversible damage to the seminiferous tubules and leydig cells. these patients develop azoospermia and hypogonadotropic hypogonadism with low serum testosterone ( fig. 12-154 ). widespread use of cytotoxic chemotherapy has created a number of adverse side effects, including gonadotoxicity. combination chemotherapy makes it diffi cult to ascertain which specifi c agent is responsible for azoospermia and leydig cell dysfunction. comparative studies of chemotherapy for acute lymphoblastic leukemia, 1465 extragonadal solid tumors, 1466 hodgkin's disease, 1467 ewing's sarcoma, and other soft tissue sarcomas 1468 in children and pubertal boys have shown that alkylating agents cause the most severe testicular damage. alkylating agents destroy the seminiferous tubular cells and induce tubular atrophy, shrinking the testis and increasing fsh serum concentration. 1469 these agents also impair leydig cell function, causing low testosterone, normal or elevated serum levels of lh, and an exaggerated response of lh to gnrh administration. 1470 testicular damage may be increased by combination with other agents (fig. 12-155 ). cyclophosphamide appears to be responsible for the greatest number of permanent or temporary cases of azoospermia after chemotherapy. this agent acts directly on the spermatogenic stem cells, 1468 and recovery depends on the number of surviving cells. in children, cyclophosphamide reduces seminiferous tubule diameter and germ cell numbers; in the residual spermatogonia nuclei are enlarged. puberty may progress, even during treatment, and the adult testis may show a sertoli cell-only pattern. 1465 in adults, cyclophosphamide treatment may cause irreversible testicular damage. administered alone, a dose of 20 000 mg/m 2 produces permanent azoospermia in 50% of men. if cyclophosphamide is administered with doxorubicin, vincristine, dacarbazine, or dactinomycin (drugs that alone do not cause azoospermia), doses of 7500 mg/m 2 cause azoospermia in 50% of patients. fludarabine, used for the treatment of chronic lymphocytic leukemia, produces testicular damage with diminution of ejaculate volume, oligozoospermia, increase in serum levels of fsh and lh, and decreased testosterone level. dna in spermatozoa is markedly abnormal, an effect that persists for several months. 1471 procarbazine, used to treat hodgkin's disease, causes permanent azoospermia in 30% of patients, even when not combined with alkylating agents. 1472 patients treated with a combination of cyclophosphamide and procarbazine in the copp protocol (cyclophosphamide, vincristine, procarbazine, and prednisone) do not recover spermatogenesis even if the cyclophosphamide dose does not exceed 4800 mg/m 2 . chemotherapy without both alkylating agents and procarbazine, such as the abvd (dexorubicin, bleomycin, vinblastine and dacarbazine) or vbm (vinblastine, bleomycin and methotrexate) regimens, produces reversible azoospermia in 36% of patients. the alternating use of mopp (mechlorethamine, vincristine, procarbazine and prednisone) and fig. 12-154 testis from a 26-year-old patient who, at the age of 9 years, underwent surgery followed by radiotherapy for paratesticular rhabdomyosarcoma. the testicular biopsy shows post-irradiation lesions, including germ cell aplasia and peritubular and interstitial fi brosis. abvd treatments causes testicular dysfunction in 87% of patients, but spermatogenesis recovers in 40%. 1473 patients with germ cell cancer who received chemotherapy with bep regimens (cisplatinum, etoposide, and bleomycin) become azoospermic 7-8 weeks after starting treatment. when the total doses reaches 600 mg/m 2 , infertility is irreversible; at lower dosages, fertility might be recovered over a period of about 2 (50% of patients) to 5 (80%) years, 1474 although a high percentage of spermatozoa with dna abnormalities persists. 1475 an important consideration in patients with testicular cancer or hodgkin's disease is the existence of testicular dysfunction before treatment. in some series 1476 dysfunction is present at diagnosis in more than 50% of patients; its cause is unknown. proposed mechanisms include primary germ cell defi ciency, release of toxic substances by tumor cells, and alteration in the hypothalamo-hypophysealtesticular axis. sexual function is often lost in patients who undergo bilateral retroperitoneal lymph node dissection for nonseminomatous testicular cancer. up to 90% lose antegrade ejaculation, although libido, erection, and orgasm are normal. loss of antegrade ejaculation results from the removal of or injury to sympathetic ganglia and the hypogastric nervous plexus during surgery. unilateral surgery, especially if the left side is not operated on, reduces this complication. 1477, 1478 hypospermatogenesis sometimes occurs after surgery for rectal cancer, perhaps due to vascular compromise. spinal cord injury is a frequent fi nding, with more than 10 000 cases annually in the us, mostly in young adults. 1479 fertility is impaired in 90% of males with spinal cord injury. the major sexual dysfunctions in these patients are the lack of erection and ejaculation and poor semen quality. [1480] [1481] [1482] [1483] [1484] [1485] failure of ejaculation occurs in 95% of patients. semen may be obtained by means of vibratory stimulation of the penis or electroejaculation in more than 90%, but its quality is low, with increased numbers of dead spermatozoa, markedly low motility, and reduced fertilization rate. [1486] [1487] [1488] possible explanations include genitourinary tract infection, endocrine anomaly, and impaired spermatogenesis. recurrent infection occurs in 60-70% of patients. compared to controls, a signifi cant increase in the numbers of neutrophils and macrophages occurs, with a marked increase in the production of reactive oxygen species. 1489, 1490 this fi nding and the presence of elevated cytokine levels 1491 are assumed to be involved in pathogenesis. endocrine anomalies are transient, and hormonal levels return to normal after a few months. more than 50% of patients have abnormalities of the adluminal compartment of the seminiferous tubules, with variable degrees of immature germ cell sloughing; 1482 in 50% of patients the number of mature spermatids per cross-sectioned tubule is less than 10 (normal >21). possible etiologies include an increase in testicular temperature due to vascular dilation, or an alteration in scrotal thermoregulation secondary to impaired sympathetic innervation from prolonged wheelchair restraint; alteration in sperm transport secondary to nerve injury, resulting in sperm stagnation in seminal vesicles, a hostile environment that normally is devoid of spermatozoa; 1493 and abnormal composition of seminal fl uid, causing deterioration of spermatozoa that in the epididymis and ductus deferens had good motility. 1494 more than 25% of patients with spinal cord injury have brown-tinged semen in some ejaculations. 1495 although the cause is unknown, it might be related to seminal vesicle dysfunction. when spermatozoa cannot be obtained by electroejaculation or vibratory stimulation, vasal aspiration or testicular biopsy are recommended. most patients have at least a few mature spermatids in some seminiferous tubules; therefore, testicular sperm extraction followed by intracytoplasmic sperm injection is a reasonable consideration in azoospermic patients. 1492 infectious agents may reach the testis and epididymis through blood vessels, lymphatics, sperm excretory ducts, or directly from a superfi cial wound. infection transmitted through the blood mainly affects the testis and causes orchitis, whereas infection ascending through the sperm excretory ducts usually causes epididymitis. acute infl ammation is accompanied by enlargement of the testis or epididymis. the tunica albuginea is covered by a fi brinous exudate, and the testicular parenchyma is yellow or brown. bacterial infection may cause abscess. in some cases the infection begins to heal, with the deposition of granulation tissue and fi brosis; in others, the infection may persist as an active process for a long time, resulting in chronic orchidoepididymitis. the most frequent causes of viral orchidoepididymitis are mumps virus and coxsackie b virus. other viral infections that occasionally cause acute orchitis include infl uenza, infectious mononucleosis, echovirus, lymphocytic choriomeningitis, adenovirus, coronavirus, bat salivary gland virus, smallpox, varicella, vaccinia, rubella, dengue, and phlebotomous fever. subclinical orchitis probably occurs during other viral infections (fig. 12-156) . before vaccination was commonly used, mumps orchidoepididymitis complicated 14-35% of adult mumps cases and was bilateral in 20-25% of cases. nevertheless, miniepidemics still occasionally occur. 1496, 1497 as expected, the incidence remains high in countries where vaccination is not obligatory. 1498 in about 85% of cases of mumps orchitis the epididymis is also involved, but epididymal involvement alone is rare. 1499 clinical symptoms of orchitis usually appear 4-6 days after symptoms of parotiditis, but orchitis may also appear without parotid involvement. 1500 testicular involvement is multifocal, and consists of acute infl ammation of the interstitium and seminiferous tubules. the tubular lining is destroyed, and eventually only hyalinized tubules and clusters of leydig cells remain. 1501 with time, the testes shrink and become soft. if the infection is bilateral the patient is usually infertile, with severe oligozoospermia or azoospermia, although biopsy may reveal the presence of mature spermatids in some tubules, allowing sperm extraction for paternity. 1502 if only one testis was affected, the sperm concentration may be normal or slightly decreased and fertility is maintained. occasionally the testicular damage is so severe that testicular endocrine function is impaired, causing hypergonadotropic hypogonadism, with low testosterone levels and regression of secondary sex characteristics. mumps orchidoepididymitis is infrequent in childhood. most bacterial orchitis is associated with bacterial epididymitis. orchitis secondary to suppurative epididymitis caused by escherichia coli is most common. 1503 on light microscopy, the tubules are effaced by intense acute infl ammation. chronic orchitis with microabscesses is caused by e. coli, streptococci, staphylococci, pneumococci, salmonella enteritidis, 1504 and actinomyces israeli. 1505, 1506 in some cases of chronic bacterial orchitis, the testis contains an infl ammatory infi ltrate consisting of numerous histiocytes with foamy cytoplasm (xanthogranulomatous orchitis) (fig. 12-157) , 1507 similar to that of idiopathic granulomatous orchitis but lacking intratubular giant cells. rarely, as in whipple's disease, large numbers of bacilli are present in histiocytes in the interstitium, vascular walls, and seminiferous tubules. the most frequent complications of pyogenic bacterial orchidoepididymitis are scrotal pyocele and chronic draining scrotal sinus. small fragments of testicular parenchyma may be eliminated through the scrotal skin, known clinically as fungus testis. another complication is testicular infarct, resulting from compression or thrombosis of the veins of the spermatic cord, in the scrotal neck, or the superfi cial inguinal ring. most cases of chronic orchidoepididymitis are associated with granulomas in the testis. specifi c causes may require special stains, cultures, or serologic tests, and include tuberculosis, syphilis, leprosy, brucellosis, mycoses, and parasitic diseases. in sarcoidosis and idiopathic granulomatous orchitis, the agent is unknown. the incidence of tuberculous orchidoepididymitis declined after the development of effective antibiotics, but it has recently undergone a resurgence among people who have emigrated from countries with a high incidence of the disease and the increasing population of immunologically compromised patients. most cases of tuberculous orchidoepididymitis are associated with involvement elsewhere in the genitourinary system. 1508 tuberculous epididymitis is usually the result of ascent from tuberculous prostatitis, which in turn is often secondary to renal or pulmonary tuberculosis. the pattern of spread is different in children: more than half have advanced pulmonary tuberculosis, and the testis is infected through the blood. 1509 more than 50% of patients with renal tuberculosis develop tuberculous epididymitis, and orchitis occurs in approximately 3% of patients with genital tuberculosis, usually secondary to epididymal tuberculosis. it has been suggested that some cases of tuberculous orchidoepididymitis are sexually transmitted. 1510 tuberculous orchidoepididymitis occurs mainly in adults: 72% of patients are older than 35 years, and 18% are over 65 years. the signs and symptoms may be mild, consisting only of testicular enlargement and scrotal pain. in such cases, fever is infrequent and constitutional symptoms may be absent. 1511 histologically, there are typical caseating and noncaseating granulomas that destroy the seminiferous tubules and interstitium (figs 12-158, 12-159 ). in immunosuppressed patients, the granulomas consist of epithelioid histiocytes and a few lymphocytes with rare giant cells. acid-fast bacilli tend to be more numerous in immunosuppressed patients. similar lesions may be observed in orchidoepididymitis caused by bacillus calmette-guérin, which is usually used for intravesical instillation in patients with vesicular urothelial carcinoma. 1512 syphilis syphilitic orchitis may be congenital or acquired. in congenital orchitis, both testes are enlarged at birth. the histological fi ndings are similar to those of the interstitial orchitis of acquired syphilis. if diagnosis is delayed until puberty, the testis often shows retraction and fi brosis. in adults, acquired orchitis is a complication of the tertiary stage of syphilis and has two characteristic histologic patterns: interstitial infl ammation and gumma. early in the disease, patients with interstitial orchitis have painless enlargement. grossly, the parenchyma is gray with translucent areas. histologically, plasma cells are abundant. the infl ammation begins in the mediastinum testis and testicular septa, later extending through the parenchyma as the seminiferous tubules lose their cellular lining and undergo sclerosis. initially, the arteries show an obliterans type of endarteritis. small gummas may be observed. eventually, the infl ammation subsides and is replaced by fi brosis. the epididymis is usually not affected. gummatous orchitis is characterized by the presence of one or several well-delineated grossly gray-yellow zones of necrosis. 1513 histologically, ghostly silhouettes of seminiferous tubules are visible within the gumma, surrounded by infl ammation consisting of lymphocytes, plasma cells, and scattered giant cells. in most cases spirochetes may be demonstrated histochemically with warthin-starry silver stain, but the most specifi c diagnostic technique is genetic testing. the testis may be infected in patients with lepromatous or borderline leprosy. frequent involvement of the testis in lepromatous leprosy results from the low intrascrotal temperature that promotes growth of the bacilli. orchitis is usually bilateral, although the degree of involvement may differ between the testes. occasionally, testicular involvement may be the sole indication of the infection, and the diagnosis may be made by testicular biopsy. 1514 the histologic fi ndings in the testis vary with the duration of the infection. initially, there is perivascular lymphocytic infl ammation and interstitial macrophages that contain numerous acid-fast bacilli. later, the seminiferous tubules undergo atrophy, the leydig cells cluster, and blood vessels show endarteritis obliterans. finally, the testis is replaced by fi brous tissue with a few lymphocytes and macrophages containing acid-fast bacilli. most patients with lepromatous leprosy are infertile, even if the orchitis was clinically mild. 1515, 1516 brucellosis brucellosis is common in some parts of the world, including the middle east. 1517, 1518 orchitis occurs in some patients and may be the fi rst sign of disease. brucellosis should be suspected when testicular enlargement occurs in patients with undulating fever, malaise, sweats, weight loss, and headache. 1519 occasionally this may mimic testicular tumor. histologically, there is a dense lymphohistiocytic infl ammation with occasional non-caseating granulomas in the interstitium. the seminiferous tubules are infi ltrated by infl ammatory cells and undergo atrophy. diagnosis is made by clinical and laboratory fi ndings, including blood culture, the bengal rose test, and high brucella agglutination titers, 1520, 1521 or by real-time polymerase chain reaction assay of urine. 1522 sarcoidosis is a systemic granulomatous disease of unknown etiology that preferentially affects young black adults. the genitourinary tract is involved in only 0.5% of clinical cases and 5% of autopsy cases. fewer than 30 cases of primary epididymal involvement have been reported, and about 12 of these also involved the testis. 1523, 1524 isolated testicular involvement is exceptional. 1523, 1525, 1526 testicular sarcoidosis is usually unilateral and nodular. 947 it is often asymptomatic and found at autopsy. 1527 the testis contains non-caseating granulomas similar to sarcoid granulomas at other locations. before diagnosing testicular sarcoidosis, other granulomatous lesions should be excluded, including tuberculosis, sperm granuloma, granulomatous orchitis, and seminoma. seminoma often has an intense sarcoid-like reaction, and examination of multiple histologic sections may be necessary to fi nd diagnostic foci of seminoma. an association of mediastinal sarcoidosis and testicular cancer has been reported. 1528 genital involvement of sarcoidosis may be the cause of intermittent azoospermia that benefi ts from corticoid therapy. 1529 malakoplakia is a chronic infl ammatory disease that was initially described in the bladder 1530 and subsequently in many other organs. the testes (alone or together with the epididymis) are involved in 12% of cases involving the urogenital system. 1531, 1532 grossly, the testes are enlarged and have a brown-yellow parenchymous discoloration, 1533 often with abscesses. malakoplakia causes tubular destruction that is associated with a dense infi ltrate of macrophages with granular eosinophilic cytoplasm that often contains michaelis-gutmann bodies (fig. 12-160) . 1534, 1535 the differential diagnosis includes idiopathic granulomatous orchitis and leydig cell tumor. infl ammation in idiopathic granulomatous orchitis includes intratubular multinucleate giant cells; in malakoplakia it is diffi cult to identify the tubular outlines, and giant cells are usually absent. leydig cell tumor is not usually associated with infl ammation, but may contain mononucleated or binucleated cells with abundant eosinophilic cytoplasm. reinke's crystalloids are identifi ed in up to 40% of cases of leydig cell tumor but absent in malakoplakia, and michaelis-gutmann bodies are absent. fungal orchitis is rare; most cases are associated with blastomycosis, coccidiomycosis, histoplasmosis, and cryptococcocis. 1536 the genital tract may be involved in widespread blastomycosis. in decreasing order, the organs most frequently affected are the prostate, epididymis, testis, and seminal vesicles. grossly, there often are small abscesses that may have caseous centers. fungi measuring 8-15 µm in diameter with double refringent contours are present in the giant cells in granulomas and stain positively with periodic acid-schiff and methenamine silver stains. coccidioidomycosis is endemic in california, the southwestern united states, and mexico, and may present as epididymal disease after remission of systemic symptoms. 1537 the granulomas are similar to those of tuberculosis and contain 30-60 µm sporangia with endospores that stain with periodic acid-schiff. dissemination of histoplasmosis and cryptococcosis frequently occurs after steroid therapy and may give rise to granulomatous orchitis with extensive necrosis. 1538 histoplasma capsulatum measures 1-5 µm in diameter and may be demonstrated with silver stain. cryptococcus is identifi ed by its thick wall that stains with mucicarmine. most parasites that reach the genital tract, such as phyllaria and schistosoma, are in the spermatic cord, and testicular lesions are secondary to vascular injury. 1539 testicular infection has also been reported in patients with visceral leishmaniasis, congenital and acquired toxoplasmosis ( fig. 12-161) , 1540 echinococcus infection, 1541 and orchitis due to trichomonas vaginalis. idiopathic granulomatous orchitis is a chronic infl ammatory condition of older adults (mean, 59.2 years). the most prominent clinical symptom is testicular enlargement, suggesting malignancy. 1542 most patients have a history of scrotal trauma, 66% have symptoms of urinary tract infection with negative cultures, and 40% have sperm granuloma in the epididymis. an autoimmune etiology has been suggested. the testis is enlarged, with a nodular cut surface and areas of necrosis or infarction. there are two histologic forms, according to whether the lesion is predominantly in the tubules (tubular orchitis) or the interstitium (interstitial orchitis). in tubular orchitis, germ cells degenerate and the sertoli cells have vacuolated cytoplasm and vesicular nuclei. plasma cells and lymphocytes infi ltrate the walls of the seminiferous tubules, forming concentric rings. multinucleated giant cells are present in the tubular lumina and sometimes in the interstitium (fig. 12-162 ). vascular thrombosis and arteritis are common. in interstitial orchitis, the infl ammation is predominantly interstitial. ultimately, tubular atrophy and interstitial fi brosis prevail in both forms, which may arise from different immune mechanisms. 1543 tubular orchitis histologically resembles experimental orchitis caused by injection of serum from animals with orchitis, whereas interstitial orchitis resembles orchitis produced by the transfer of cells from immunized animals. the differential diagnosis of idiopathic granulomatous orchitis is infectious orchitis caused by bacteria, spirochetes, fungi, or parasites. a useful clue in the tubular form is the presence of giant cells within seminiferous tubules. the occurrence of focal lymphoid cell infi ltrates in the testicular interstitium is common in infertile patients, 1544, 1545 patients who have undergone surgery for bilateral inguinal hernia, 1546 vasectomized patients who developed post-infection obstruction, 1547 after testicular piercing, 1548 and cryptorchidism. 1549 infl ammatory infi ltrates usually involve the seminiferous tubules, and this suggests the disorder is due to an immunologic response (fig. 12-163 ). pseudolymphoma is a benign reactive process with a lymphoid cell proliferation so intense that it may be mistaken for lymphoma. testicular pseudolymphoma consists of infl ammatory infi ltrates with numerous lymphocytes and plasma cells that partially or totally destroy testicular parenchyma. 1550, 1551 the differential diagnosis includes lymphoma, various forms of orchitis, and seminoma. the diagnosis of lymphoma may be excluded by the lack of atypia and polyclonal nature of the infl ammation. syphilitic orchitis also contains a plasma cell-rich infl ammatory infi ltrate, but pseudolymphoma does not have other characteristic features of syphilitic orchitis, such as endarteritis obliterans; spirochetes cannot be demonstrated by special stains. the lack of granulomas or signifi cant numbers of macrophages, together with the negative results of specifi c histochemical stains, also helps to exclude idiopathic granulomatous orchitis, tuberculosis, leprosy, sarcoidosis, and fungal infection. finally, although the presence of a prominent infl ammatory infi ltrate and, in many cases, numerous lymphoid follicles, may suggest the diagnosis of seminoma, the presence of seminoma cells should be easily demonstrated with best's carmine stain, periodic acid-schiff, or placenta-like alkaline phosphatase. the term plasma cell granuloma 1152 refers to a reactive process characterized by the presence of polyclonal adult plasma cells that are absent in testicular plasmacytoma. 1553 sinus histiocytosis with massive lymphadenopathy (rosai-dorfman disease) is a benign proliferation of macrophages that uniquely contain numerous lymphocytes in their cytoplasm. the disease was reported in a kidney and testis of a patient in remission from malignant lymphoma in association with monoclonal iga gammopathy, 1554 and in a second patient with diabetes mellitus who had been previously treated for pulmonary tuberculosis. 1555 increased numbers of interstitial macrophages may also be observed in more than two-thirds of autopsies from adult patients, but the cause is unknown. one condition associated with this disorder is treatment with hydroxyethylstarch plasma expander. in this lesion, the interstitial macrophages stand out by virtue of their large size and multivacuolated cytoplasm, suggesting thesaurosis. there is no evidence of mucin glycoproteins, proteoglycans, starch, lipids, glycogen, or foreign body material. most patients have no clinical symptoms other than pruritus and persistent erythrema. 1556 epididymitis nodosa is a proliferation of small irregular ducts whose epithelium lacks the characteristic features of the epididymal epithelium. the disorder is associated with infl ammation and fi brosis, similar to vasitis nodosa. 1557 in several tissues, including the testis, amiodarone is concentrated up to 300 times its plasma level, 1558 causing testicular atrophy and increased serum levels of fsh and lh in some patients. 1559 the incidence of epididymitis during amiodarone therapy varies from 3% to 11%, 1560, 1561 and more than 35 cases (in several cases involvement was bilateral) have been reported, although there are probably many others. 1562, 1563 the disorder may occur at any age. 1564 when amiodarone dosage is reduced to 300 mg/day the epididymitis heals within a few weeks. 1565 autopsy studies show focal areas of fi brosis and lymphoid cell infi ltrates not related to infection. recognition is important to avoid unnecessary antibiotics or aggressive surgery. this term describes a lesion located in the epididymal head characterized by non-infectious necrosis with polypoid masses of infl amed granulation tissue in peripheral ductal structures. granulomas containing multinucleated giant cells present within efferent ductuli or form sperm microgranulomas with ductal neoformation similar to that of epididymitis nodosa. the cause is unknown, but may result from ischemia. 1566 the terms 'testicular calculus' and 'stone in the testis' have been used to describe a lesion characterized by the presence of nodular testicular calcifi cation that is not related to ischemia, orchitis, vasculitis, hematoma, or tumor. 1567, 1568 the testicular arteries may be affected by systemic disorders such as schönlein-henoch purpura, 1569 wegener's disease, 1570, 1571 kogan's disease, 1572 behçet's disease, 1573 relapsing polychondritis, rheumatoid arthritis, and dermatomyositis, but the most frequent involvement is with polyarteritis nodosa. 1574 approximately 80% of patients with polyarteritis nodosa have testicular or epididymal involvement, 1575 but only 2-18% are diagnosed during life. rarely, testicular or epididymal polyarteritis nodosa is the fi rst manifestation of the disease. in these cases the symptoms may suggest orchitis, epididymitis, testicular torsion, or tumor. [1576] [1577] [1578] the testis usually shows arterial lesions in different stages of evolution, including fi brinoid necrosis, infl ammatory reaction, thrombosis, or aneurysm. the parenchyma initially has zones of infarction ( fig. 12-164 ). histologic and immunohistochemical fi ndings similar to those of polyarteritis nodosa may occasionally be observed in the testis or the epididymis without lesions elsewhere; this condition is referred to as isolated arteritis of the testis and epididymis, 1579 and differs from classic polyarteritis by a lack of vascular thrombosis, aneurysm, or infarct. the etiology of isolated arteritis is unknown, but the prognosis is excellent. 1580 the histologic fi ndings of necrotizing arteritis in the testis or epididymis should be followed by clinical, hematologic, and biochemical studies to exclude systemic arteritis. 1581, 1582 torsion of the spermatic cord is the most frequent cause of testicular infarct, followed by trauma, incarcerated inguinal hernia, epididymitis, and vasculitis. spermatic cord torsion is a surgical emergency. if repair is delayed more than 8 hours, testicular viability is usually compromised. this disorder may appear at any age, but the other testicular and epididymal lesions peaks of maximal incidence are the perinatal period and puberty. 1583 factors that predispose to testicular torsion are anatomical anomalies in testicular suspension and abnormal position of the testis. many men with testicular torsion have an abnormally high refl ection of the tunica vaginalis, giving rise to the deformity known as 'bell-clapper.' other anomalies include elongated mesorchium, separation between the epididymis and testis, and absent or very elongated gubernaculum. the frequency of testicular torsion is higher in cryptorchid and retractile testes than in normal testes. there are two classic anatomic forms of testicular torsion: high (supravaginal or extravaginal) and low (intravaginal). each appears at a different age. extravaginal torsion typically occurs in infancy and childhood, whereas intravaginal torsion is more frequent at puberty and adulthood. neonatal torsion is bilateral in 12-21% of cases. 1584 most torsion observed on the fi rst of life is intrauterine. 1585 pubertal and adult torsion causes testicular pain that may radiate to the abdomen or other sites. about 36% of patients have a previous history of pain or swelling in one or both testes. the differential diagnosis includes all causes of acute scrotum. 1586, 1587 torsion causes hemorrhagic infarction of the testis ( fig. 12-165 ). in old neonatal torsion, the histological fi ndings are so advanced that only collagenized tissue containing calcium and hemosiderin deposits is seen. in adults, three degrees of histological lesion may be distinguished. 1588 degree i (26.5% of adult twisted testes) is characterized by edema, vascular congestion, and focal hemorrhage. seminiferous tubules are dilated, with sloughed immature germ cells, apical vacuolation of sertoli cells, and dilated lymphatic vessels. 1589 degree ii (26.5% of testes) has pronounced interstitial hemorrhage and sloughing of all germ cell types in the seminiferous tubules. the lesion is more severe in the center of the testis, and thus biopsy might provide erroneous information (fig. 12-166 ). degree iii lesions (45% of testes) are characterized by necrosis of the seminiferous tubular cell layers. there is often a correlation between the time interval of torsion and the degree of the histologic lesion. 1590 degree i appears in torsion of less than 4 hours' duration, degree ii in torsion of between 4 and 8 hours, and degree iii in torsion of more than 12 hours. nevertheless, there are some exceptions that could probably be related, among other factors, to the number of twists in the torsed spermatic cord (degrees of testicular rotation). the testicular salvage rate, defi ned as testicular growth and development that refl ects the age of the patient and the contralateral testis, is around 50% in all cases of testicular torsion. 1591 testes that do not bleed into the albugineal incision within 10 minutes are assumed to be non-viable and should be removed. 1592 little attention has been paid to intermittent testicular torsion. early orchiopexy may save these testes, but after surgery, the testis becomes small and excessively mobile, and most have the bell-clapper deformity. 1593 seminiferous tubules are devoid of germ cells and have hyalinized walls. some adults with untreated testicular torsion develop lipomembranous fat necrosis of the spermatic cord. 1594 patients seek help for pain in the high scrotum. at this level, there is a small nodule that corresponds to remnants of the twisted testis. the epididymis and proximal spermatic cord characteristically contain fat necrosis (fig. 12-167) . adults with prior spermatic cord torsion often consult for infertility. the mechanism causing spermiogram alteration is controversial, and three hypotheses have been proposed: • autoimmune process. it has been suggested that the ischemic injury breaks the blood-testis barrier, and antigens released from the necrotic germ cells activate macrophages and lymphocytes in the interstitium, stimulating the formation of antibodies against these antigens. these antibodies that enter in the blood circulation may presumably damage the contralateral testis. 1595 • alterations in microcirculation. after testicular torsion, blood fl ow decreases in the contralateral testis, causing an increase in the characteristic products of hypoxia, such as lactic acid and hypoxanthine. 1596 intense apoptosis involving mainly spermatocytes i and ii has been observed. 1597 long-term effects are yet unknown. • primary testicular lesions. many twisted testes have lesions that cannot be formed in a few hours, such as hypoplastic tubules, microlithiasis, and focal spermatogenesis. in addition, more than half of biopsies of the contralateral testis show marked spermatogenetic lesions. 1598 these fi ndings suggest that torsion occurs in testes with congenital lesions. trauma 1599 and lesions of the vessels of the spermatic cord may also cause testicular infarct. ischemic atrophy is a risk of inguinal surgery, including herniorrhaphy, varicocelectomy, hydrocelectomy, and descent of cryptorchid testis ( fig. 12-168 ). the incidence of atrophy after inguinal herniorrhaphy varies from 0.06% in primary herniorrhaphy 1600 to 7.9% after surgery for recurrent herna, 1601 depending on the diffi culty and extent of the hernia. atrophy occurs in some cases of thrombosis of the vena cava or spermatic artery. 1602 focal infarction of the testis is associated with polycythemia, sickle cell disease, trauma, 1603, 1604 and laparoscopic inguinal hernia repair. focal infarction may also be spontaneous. clinical symptoms of testicular infarct mimic testicular tumor. color doppler ultrasound reveals the diagnosis in most cases. 1605 cystic malformation of the tunica albuginea and testicular parenchyma was fi rst described in the 19th century, 1606 and was long considered rare and mainly present in the tunica albuginea. 1607, 1608 with the systematic use of ultrasonography, the incidence of cysts has been found to be much higher: 1609 non-neoplastic cysts are found in 2.1% 1610 to 9.8% 1611 of testes. 1612, 1613 cyst of the tunica albuginea is usually an incidental fi nding in patients in the fi fth or sixth decade of life. it is located in the anterolateral aspect of the testis and may be unilocular or multilocular, 1614 ranging from 2 to 4 mm and containing clear fl uid without spermatozoa. the cyst may be embedded within the connective tissue of the tunica albuginea, protrude from the inner surface of the tunica albuginea into the testicular parenchyma, or protrude from the outer surface forming a blue lump in the tunica albuginea. the epithelium lining the cyst may be simple columnar or stratifi ed cuboidal, and is supported by a thin layer of collagenized connective tissue. the columnar epithelium usually includes some ciliated cells, 1615 and the cuboidal epithelium is composed of two layers of non-ciliated cells (fig. 12-169) . cyst of the rete testis is identifi ed by a distinctive epithelial lining of areas of fl attened cells intermingled with areas of tall columnar cells. spermatozoa are frequently found within the cyst, 1616 and hence the cyst is also called intratesticular spermatocele. 1617 it may be associated with cystic transformation of the rete testis and multiple epididymal cysts. rete testis cyst is not always attached to the rete and may be found at a distance. simple cyst of the testis constitutes the remaining intraparenchymal cyst. it is usually lined by cuboidal epithelium and contains no spermatozoa. 1618, 1619 simple cyst ranges other testicular diseases from 2 nm to 18 mm in diameter. 1620, 1621 the disorders occurs at any age, from 5 months to 80 years, with a bimodal distribution with peaks at 8 month and 60 years. 1622 it may occur bilaterally, 1623 and may present as two cysts in the same testis. 1624 origin of the three types of testicular cyst is uncertain. previously, traumatic 1625 and infl ammatory 1626 origins were attributed to tunica albuginea cyst, but most now believe that they are derived from embryonal remnants of the mesonephric ducts 1615, 1627 or mesothelial cells embedded in the tunica albuginea during embryogenesis. 1614, 1628, 1629 simple cyst of the testis may also have a mesothelial origin, but it is possible that some arise from ectopic rete testis epithelium. these cysts are unrelated to epidermoid cyst, differing in the ultrasonographic 1630,1631 and histologic features (see discussion on cystic dysplasia and testicular tumors in the section on hamartomatous testicular lesions). ultrasound studies indicate that testicular cyst has little potential for growth. 1630,1632 currently, excision is recommended only in children when the cyst may impair testicular development. 1633 dysgenesis of the rete testis is characterized by inadequate maturation and persistence of infantile or pubertal characteristics in adults. 1634 this disorder is frequent in undescended adult testes. the lesion involves the rete testis segments referred to as septal, mediastinal, and extratesticular. there is poor development of the cavities and their epithelial lining, which becomes cuboidal or columnar instead of fl attened with areas of columnar cells. the lumina of the rete testis cavities may be completely absent (simple hypoplasia) or, conversely, undergo microcystic dilation (cystic hypoplasia). in a few cases, the rete testis develops papillary, cribriform, or tubular formations (adenomatous hyperplasia). the epithelium of the rete testis is usually fl attened, with scattered areas of columnar cells. in estrogen-treated patients, those with chronic hepatic insuffi ciency, functioning tumor that secretes estrogens or human chorionic gonadotropin, and other disorders that are described as hyperplasia of the rete testis it may undergo diffuse transformation into tall columnar epithelium. except for the latter group, metaplasia of the rete testis seems to be an estrogen-dependent process, and estrogen receptors are present in the rete testis epithelium. 1635 acquired cystic transformation of the rete testis is common, and its incidence increases with age and associated disorders. 1636 ultrasound 1637 ultrasound ,1638 and magnetic resonance 1639 studies reveal characteristic images that may suggest malignancy. the lesion has three forms: simple, associated with epithelial metaplasia, and with crystalline deposits. simple cystic transformation consists of dilated cavities with normal epithelium. it results from obstruction of the epididymis or the initial portion of the vas deferens due to ischemia (aging men); compression by epididymal and spermatic cord tumor, or by congestive veins in varicocele; infl ammation in patients with previous epididymitis; malformation (testis-epididymis dissociation, malformed epididymis and absence of the vas deferens); 1640 or iatrogenic causes (surgery for epididymoectomy or removal of epididymal cyst) (fig. 12-170) . 1641 cystic transformation with epithelial metaplasia is a frequent fi nding at autopsy. 1369 its development is probably due to the concurrence of sperm excretory duct obstruction and conditions involved in increased serum estrogen levels, such as chronic liver insuffi ciency. another possible cause is infl ammation involving the rete testis. cystic transformation with crystalline deposits has also been called cystic transformation of the rete testis secondary to renal insuffi ciency. 1642 it is a bilateral lesion of adult testes characterized by the concurrence of three fi ndings: cystic transformation of the rete testis, cuboidal or columnar metaplasia of its epithelium, and the presence of urate and oxalate crystalline deposits that may be recognized by polarized light. the lesion is pathognomonic of dialyzed patients with chronic renal insuffi ciency. crystalline deposits are initially formed beneath the epithelia of the rete testis and ductuli efferentes; later they protrude into the lumina, where they are fi nally released. infl ammation is absent or slight, although a few giant cells and small fi brotic areas are often seen (figs 12-171, 12-172 ). this lesion is characterized by diffuse or nodular proliferation of tubular or papillary structures that are derived from the rete testis 1643 and are observed in cryptorchid or normally descended testes. cases have been reported in newborns, children, and adults. 1644 adenomatous hyperplasia in newborn and infantile testes consists of enlargement of the mediastinum testis by cordlike or tubular structures derived from the rete testis. the lesion may extend up to one-third of testicular volume. despite excessive development of the rete testis, the normal connections with seminiferous tubules and efferent ductuli remain. presentation may be unilateral or bilateral. unilateral presentation is associated with cryptorchidism or vanishing testis. bilateral cases may also present with bilateral renal dysplasia. efferent ductuli may show luminal dilation and irregular outlines. the etiopathogenesis might be similar to that of cystic dysplasia of the testis. 1644 adenomatous hyperplasia in adults is usually an incidental fi nding at autopsy, 1645 in cryptorchid testes, 1646 or in testes with germ cell tumor. the rete testis epithelium forms nonencapsulated nodular outgrowths or a diffuse pattern. nodule size may be large enough to suggest tumor. the epithelium consists of cuboidal cells with ovoid nuclei, deep nuclear folds, and peripheral nucleoli. atypias and mitotic fi gures are lacking (fig. 12-173) . the ultrastructure and immunophenotype of the epithelium are similar to those of the normal rete testis. spermatozoa may be seen inside the cavities in some cases, suggesting that such a proliferation is connected with the seminiferous tubules. most of the testes show a certain degree of seminiferous tubular atrophy. in incidental autopsy cases the etiology is unknown, although it may be related to hormonal or chemical agent effects. [1647] [1648] [1649] in cryptorchid testes and with many testicular tumors, the most probable cause is a primary anomaly that is part of the testicular dysgenesis syndrome. 1650 adenomatous hyperplasia should be distinguished from three entities: rete testis pseudohyperplasia, which appears in atrophic testes; primary rete testis tumor; and metastasis of adenocarcinoma. in pseudohyperplasia, lesions are focal, microscopic, and usually located in the septal rete, although the mediastinal rete shows few or no alterations. benign rete testis tumor such as adenoma (solid and papillary variants) fig. 12-171 changes in the rete testis associated with dialysis. dilation of the rete testis and initial portion of the ductuli efferentes can be observed. crystalline structures, mainly rhomboidal in shape, accumulate inside and outside the tubules. other testicular diseases and cystoadenoma are isolated and focal, 1651 whereas rete testis hyperplasia is diffuse. adenocarcinoma of the rete testis is a tumor that displays numerous mitotic fi gures and infi ltrates adjacent structures. 1652 metastasis of prostatic adenocarcinoma may be excluded because these metastases alter the rete testis architecture and are immunoreactive for prostatic acid phosphatase and psa. this reactive lesion is characterized by the presence of intracytoplasmic accumulation of hyaline eosinophilic globules in the epithelial cells of the rete testis. the epithelium may be hyperplastic, but does not contain mitotic fi gures or nuclear atypia. the globules are up to 15 µm in diameter ( fig. 12-174 ). this lesion is associated with tumor and infl ammatory processes occurring near the mediastinum testis, and can be observed in association with 75% of mixed testicular germ cell tumors, 47% of seminomas, and 20% of non-germ cell testicular tumors, such as epididymal tumor that infi ltrates the testis (adenomatoid tumor). 1653 yolk sac tumor infi ltrating the rete testis may closely resemble this type of rete testis hyperplasia. positive immunoreactions for α-fetoprotein and placenta-like alkaline phosphatase, as well as nuclear atypia, are helpful to distinguish germ cell neoplasia from this rete testis hyperplasia. 1654 this lesion, described as nodular proliferation of calcifying connective tissue in the rete testis, is characterized by the presence of multiple nodules that originate from the rete testis lining and subjacent connective tissue, protruding into the channels of the rete testis. these consist of cellular connective tissue covered by several layers of a fi brin-like material, which in turn is covered by rete testis epithelium. the nodules may be totally or partially calcifi ed (fig. 12-175 ). 1655 the lesion is an incidental fi nding at autopsy in patients with impaired peripheral perfusion. selective location of the lesion in the walls of the cavities and chordae rete testis is probably related to poor vascularization of these structures. the etiopathogenetic mechanism may 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spermatogenesis in the human testis computeraided threedimensional reconstructions of the arrangement of primary spermatocytes in human seminiferous tubules the cycle of the seminiferous epithelium in man morphometrical analysis of sertoli cell ultrastructure during the seminiferous epithelial cycle in rats stage-specifi c signals in germ line differentiation control of sertoli cell phagocytic activity by spermatogenic cells changes in the lipid inclusions/ sertoli cell cytoplasm area ratio during the cycle of the sertoli cell of the human seminiferous epithelium interaction between germ cells and sertoli cells in the testis apoptosis of male germ cells, a generalized or a cell typespecifi c phenomenon? ultrastructure and function of the lamina propia of mammalian seminiferous tubules the peritubular tissue in the normal and pathological human testis: an ultrastructural study basement membrane regulation of sertoli cells the lamina propia of vertebrate seminiferous tubules: a comparative light and electron microscopic investigation contractile cells in human seminiferous tubules peritubular myoid cells of human and rat testis are smooth muscle cells that contain desmin-type intermediate fi laments elastic tissue in the limiting membrane of the human seminiferous tubules the biology of myofi broblasts evidence for contractility of the human seminiferous tubule confi rmed by its response to noradrenaline and acetylcholine agerelated variations in seminiferous tubules in men. a stereologic evaluation distribution and role of cd34-positive stromal cells and myofi broblasts in human normal testicular stroma giant interstitial cells and extraparenchimal interstitial cells of the human testis morphological relationship between testicular nerves and leydig cells in man sertoli cells and leydig cells in man ultrastructure of leydig cells in human ageins testes neuron-specifi c enolase-like inmunoreactivity in human leydig cells relaxin-like factor: a highly specifi c and constitutive new marker for leydig cells in the human testis ghrelin and reproduction: a novel signal linking energy status and fertility a novel circulating hormone of testis origin in humans calretinin is expressed in the leydig cells of rat testis calretinin, a more sensitive but less specifi c marker than α-inhibin for ovarian sex cord-stromal neoplasms: an immunohistochemical study of 215 cases barrier properties of testis microvessels the leydig cell of the human testis -a new member of the diffuse neuroendocrine system sertoli and leydig cells of the human testis express neurofi lament triplet proteins isolation of human leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture progenitor cells of the testosterone-producing leydig cells revealed attrition of the human leydig cell population with advancing age effect of ageing on the volume, structure and total leydig cell content of human testis stereological analysis of leydig cell ultrastructure in aged humans multinucleate leydig cells in normal human testes mitosis in adult human leydig cells leydig cell numbers, daily sperm production and serum gonadotropin levels in ageing men agerelated change in numbers of other interstitial cells in testes of adult men: evidence bearing on the fate of leydig cells lost with increasing age testosterone and spermatogenesis. identifi cation of stage-specifi c, androgen-regulated proteins secreted by adult rat seminiferous tubules regulation of the semininiferous epithelium changes in surface area and number of leydig cells in relation to the 6 stages of the cycle of the human seminiferous epithelium actions of the testicular paracrine factor (p-mod-s) on sertoli cell transferrin secretion througout pubertal development diaphragmatic hernia in denys-drash syndrome sex reversal and diaphragmatic hernia in phenotypically female sibs with normal xy chromosomes clinical expression and sry gene analysis in xy subjets lacking gonadal tissue pagod syndrome: eighth case and comparison to animal models of congenital vitamin a defi ciency familial occurrence of agonadism and multiple internal malformations in phenotypically normal girls with 46,xy and 46,xx karyotypes, respectively: a new autosomal recessive syndrome low birth-weight, microcephalic malformation syndrome in a 46,xx girl and her 46,xy sister with agonadism: third report of the kennerknecht syndrome or autosomal recessive seckel-like syndrome with previously undescribed genital anomalies agonadism in a 46,xy patient with charge association the syndrome of rudimentary testes: occurrence in live siblings identical twins discordant for the 'rudimentary testes' syndrome rudimentary testes syndrome revisited congenital anorchism: diagnostic and therapeutic aspects congenital bilateral anorchia: clinical, hormonal and imaging study in 12 cases pcr analysis and sequencing of the sry sex determining gene in four patients with bilateral congenital anorchia is bilateral congenital anorchia genetically determined? the vanishing testis syndrome. indications for conservative therapy the vanishing testis the vanishing testis is the vanished testis always a scrotal event? bilateral anorchia: discordance in monozygotic twins an analysis of the genetic factors involved in testicular descent in a cohort of 14 male patients with anorchia report of 11 cases with discussion of etiology and pathogenesis hyperplasia of spermatic cord nerves: a sign of testicular absence signifi cance of testicular biopsies in cryptorchidism in children human monorchism: a clinicopathological study of unilateral absent testes in 65 boys testicular regression syndrome a pathological study of 77 cases testicular regression syndrome: a clinical and pathologic study of 11 cases laparoscopic and histologic evaluation of the inguinal vanishing testis histological evaluation of the testicular nubbin in the vanishing testis syndrome ureteral ectopia into cystic seminal vesicle with ipsilateral renal dysgenesis and monorchia renal and testicular agenesis in a patient with darier's disease findings: small testicles klinefelter's syndrome diagnosed three years after surgery for mediastinal teratoma kenny-caffey syndrome and microorchidism polyorchidism: case report and review of literature polyorchidism: sonographic and magnetic resonance image fi ndings a case of supernumerary testis polyorchidism presenting as retractile testes polyorchidism: evaluation by mr polyorchidism: an unusual case polyorchidism: a strange anomaly with unsuspected properties bilateral double by testis: evaluation magnetic resonance imaging one man with fi ve testes: report of case triorchidism with normal spermatogenesis: an unusual cause for failure of vasectomy abdominal polyorchidism: a case report and review of the literature ultrasound of polyorchidism: case report and literature review sonographic features of polyorchidism polyorchidism: case report and literature review polyorchidism discovered as testicular torsion associated with undescended atrophic contralateral testis. a surgical solution revisión y aportación de un nuevo caso polyorchidism discovered as testicular torsion polyorchidism with normal spermatogenesis polyorchidism with normal spermatogenesis polyorchidism with normal spermatogenesis and equal sized testes. a theory of embryonal development testiculo supernumerario. comunicación de un caso y revisión de la literatura polyorchidism: case report and review of the literature polyorchidism: a case report polyorchidism: report of a case polyorchidism in a newborn: case report and review of the literature polyorchidism: a case report and review of the literature polyorchidism and seminoma in a child a case of polyorchidism with testicular teratoma signifi cance of testicular size measurement in andrology. ii correlation of testicular size with testicular function congenital leydig cell hyperplasia evidence in favor of the mechanical (intrauterine torsion) theory over the endocrinopathy (cryptorchidism) theory in the pathogenesis of testicular agenesis compensatory hypertrophy of testicle in unilateral cnyptorchidism impact of varicocele on testicular volume in young men: signifi cance of compensatory hypertrophy of contralateral testis plasma lh and fsh response to lrh in boys with compensatory testicular hypertrophy followup of boys with unilateral compensatory testicular hypertrophy testicular volume during adolencence unilateral testicular hypertrophy: an apparently benign occurrence without cryptorchidism benign bilateral testicular enlargement benign macroorchidism in a pubescent boy idiopathic macroorchidism macro-orchidism: light and electron microscopic study of four cases idiopathic benign bilateral testicular enlargement in a pubertal boy: a case report and review of literature macroorchidism: a case report endocrine and spermatological characteristics of 135 patients with bilateral megalotestis the fragile x premutation: new insights and clinical consequences a marker x chromosome a pedigree of mental defect showing sex-linkage x-linked mental retardation associated with macroorchidism inherited congenital normofunctional testicular hyperplasia and mental defi ciency. a corroborative study x-linked mental defi ciency megalotestes syndrome xlinked mental retardation with macroorchidism and the fragile site at xq 27 or 28 familial-x-linked mental retardation with a marker x chromosome and its relationship to macroorchidism a recognizable syndrome of sex-linked mental retardation, large testes and marker x chromosome gonadal function in men with the martin-bell (fragile x) syndrome spermatogenesis in two patients with the fragile x syndrome. i. histology: light and electron microscopy spermatogenesis in two patients with the fragile x syndrome x-linked mental retardation and an x-chromosome marker population incidence and segregation ratios in martin-bell syndrome study of a family with a fragile site of the x chromosome at xq 27-28 without mental retardation testicular size in fetal fragile x syndrome a family with mental retardation, variable macrocephaly and macroorchidism, and linkage to xq12-q21 testicular enlargement and elevated serum inhibin concentrations occur in patients with pituitary macroadenomas secreting follicle stimulating hormone juvenile hypothyroidism with testicular enlargement hipotiroidismo y maduración testicular precoz thyroid hormone and male gonadal function sexual maturation in juvenile hypothyroidism hypothalamic-pituitary gonadal axis in boys with primary hypothyroidism and macroorchidism thyroid hormones: their role in testicular steroidogenesis male hypogonadism in hypothyroidism: a study of six cases macroorchidism and testicular fi brosis associated with autoimmune thyroiditis juvenile hypothyroidism and precocious testicular maturation acquired hypothyroidism with muscular hypertrophy and precocious testicular enlargement regulation of gonadotropin-releasing hormone (gnrh) gene expression in hypothalamic neuronal cells hypothyroidism-induced macroorchidism: use of a gonadotropin-hormone agonist to understand its mechanism and augment adult stature a potential novel mechanism for precocious puberty in juvenile hypothyroidism high neonatal triiodothironine levels reduce the period of sertoli cell proliferation and accelerate tubular lumen formation in the rat testis, and increase serum inhibin levels increased numbers of sertoli and germ cells in adult rat testes induced by synergistic action of transient neonatal hypothyroidism and neonatal hemicastration tri-iodothyronine directly affects rat sertoli cell proliferation and differentiation neonatal hypothyroidism causes delayed sertoli cell maturation in rats treated with propylthiouracil: evidence that the sertoli cell controls testis growth central precocious puberty. an overview of diagnosis, treatment, and outcome precocious puberty precocious puberty sexual precocity precocious puberty caused by a suprasellar arachnoid cyst. analysis of 6 cases the endocrine spectrum of arachnoid cysts in childhood growth, puberty and hypothalamicpituitary function in children with suprasellar arachnoid cyst aspects étiologiques cliniques et biologiques des pubertés précoces d'origine centrale precocious puberty following severe head trauma precocious puberty after traumatic brain injury endocrine disorder as the only sign of chronic 'nonhypertensive' hydrocephalus precocious puberty after hypothalamic and pituitary irradiation in young children precocious and premature puberty associated with treatment of acute lymphoblastic leukaemia endocrine function and morphological fi ndings in patients with disorders of the hypothalamopituitary area: a study with magnetic resonance the radiological approach to precocious puberty mr imaging features in hypothalamic hamartoma: a report of three cases and review of literature boys with precocious puberty due to hypothalamic hamartoma: reproductive axis after discontinuation of gonadotropinreleasing hormone analog therapy etiology of central precocious puberty in males: the results of the italian study group for physiopathology of puberty hypothalamic hamartoma: a source references of luteinizing-hormone-releasing factor in precocious puberty mixed germ cell tumour of the pineal region: a case report hcg-secreting pineal teratoma causing precocious puberty: report of two patients and review of the literature puberty without gonadotropins: a unique mechanism of sexual development gonadotropinindependent familial sexual precocity with premature leydig and germinal cell maturation (familial testotoxicosis): effects of a patent luteinizing hormone-releasing factor agonist and metroxyprogesterone acetate therapy in four cases testicular changes in gonadotropin-independent familial male sexual precocity. familial testotoxicosis gonadotropin-independent precocious puberty due to luteinizing hormone receptor mutations in brazillian boys: a novel constitutively activating mutation in the fi rst transmembrane helix activating mutations in the lh receptor gene: a human model of non fsh-dependent inhibin production and germ cell maturation mutational analysis of the luteinizing hormone receptor gene in two individuals with leydig cell tumors tumor de células de leydig con pseudopubertad precoz leydig cell tumor in a child with spermatocyte maturation and no pseudoprecocious puberty an aromatase-producing sex-cord tumor resulting in prepubertal gynecomastia sertoli cell tumour in a boy with peutz-jeghers syndrome carney complex: the complex of mixomas, spotty pigmentation, endocrine overactivity, and schwannomas virilising adrenal cortical tumours in children virilizing adrenal cortical carcinoma with hypertrophy of spermatic tubules in childhood bilateral testicular tumours in congenital adrenal hyperplasia: a continuing diagnostic and therapeutic dilemma hepatoblastoma presenting as isosexual precocity. the clinical importance of histologic and serologic parameters gonadotropin-secreting pineal teratoma causing precocious puberty the p450 gene superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci human aromatase: cdna cloning, southern blot analysis, and assignment of the gene to chromosome 15 familial hyperestrogenism in both sexes: clinical, hormonal, and molecular studies of two siblings the aromatase excess syndrome is associated with feminization of both sexes and autosomal dominant transmission of aberrant p450 aromatase gene transcription aromatase defi ciency caused by a novel p450arom gene mutation: impact of absent estrogen production on serum gonadotropin concentration in a boy aromatase defi ciency in male and female siblings caused by a novel mutation and the physiological role of estrogens effect of testosterone and estradiol in a man with aromatase defi ciency estrogen: consequences and implications of human mutations in synthesis and action primary testicular abnormalities causing precocious puberty leydig cell tumor, leydig cell hyperplasia, and adrenal rest tumor focal lobular spermatogenesis and pubertal acceleration associated with ipsilateral leydig cell hyperplasia mccune-albright syndrome in a boy may present with a monolateral macroorchidism as an early and isolated clinical manifestation macroorchidism due to autonomous hyperfunction of sertoli cells and g(s)alpha gene mutation: an unusual expression of mccune-albright syndrome in a prepubertal boy mccune-albright syndrome in a male child: a clinical and endocrinologic enigma anomalies of the testicle preperitoneal ectopic testis: a case report ectopia epidídimo-perineal testículo ectópico perineal perineal testicle crossed ectopic testis: a case report and review of the literature crossed ectopic testis. case report and review transverse testicular ectopia crossed ectopic testis with common vas deferens crossed testicular ectopia detected by laparoscopy transverse testicular ectopia: a case report crossed testicular ectopia in association with double incomplete testicular descent two rare genital abnormalities: crossed testicular and scrototesticular ectopia transverse testicular ectopia associated with persistent müllerian duct syndrome. a case report the persistent müllerian duct syndrome: a rare cause of cryptorchidism mixed germ cell tumor after bilateral orchidopexy in persistent müllerian duct syndrome with transverse testicular ectopia two rare cases of ectopic testes subumbilical ectopic testis exstrophy of the testis testicular dislocation after scrotal trauma cystic dysplasia of the testis: a unique anomaly studied by microdissection cystic dysplasia of the rete testis. case report cystic dysplasia of the rete testis associated to cryptorchidism: a case report cystic dysplasia of the testis: sonographic and pathologic fi ndings cystic testicular lesions in the pediatric population the human rete testis the rete testis in man: ultrastructural aspects the mammalian rete testis. a morphological examination cystic displasia of the testis: light and electron microscopic study of three cases cystic dysplasia of the testis associated with multicystic dysplasia of the kidney ectasia of the rete testis with ipsilateral renal agenesis cystic dysplasia of the testis cystic dysplasia of testis coffi n cm. cystic testicular lesions in the pediatric population cystic dysplasia of the testis with ipsilateral renal agenesis. a case report and review of the literature conservative managemet of cystic dysplasia of the testis testicular cystic dysplasia: evaluation of 3 new cases treated without surgery cystic dysplasia of rete testis associated with ipsilateral renal agenesis. case report fetal gonadoblastoid testicular dysplasia gonadoblastoid testicular dysplasia in walker-warburg syndrome fetal gonadoblastoid testicular dysplasia: a focal failure of testicular development frequency of so-called hypoplastic or dysgenetic zones in scrotal and otherwise normal testes androgen receptor expression in sertoli cells as a function of seminiferous tubule maturation in the human cryptorchid testis congenital testicular lymphangiectasis congenital testicular lymphangiectasis in noonan's syndrome congenital testicular lymphangiectasis in children with otherwise normal testes macromicroscopischeskoe is sledavanie vnutriorgannoi limfaticheskoi sistemy muzhskoi polovoi zhelezy [macro/ microscopic study of intraorgan lymphatic system of male gonad in man distribution and fi ne structure of the lymphatic system in the human testis lymph vascular system of the interstitial tissue of the testis as revealed by electron microscopy epididymal lymphangiectasis tumors and cysts of the paratesticular region complex multilocular cystic lesion of rete testis, accompanied by smooth muscle hyperplasia, mimicking intratesticular leydig cell neoplasm smooth muscle hyperplasia of the testicular adnexa clinically mimicking neoplasia. clinicopathologic study of sixteen cases myoepithelial hamartoma of the small bowel: report of a case focal muscular hyperplasia of the trachea ectopic seminiferous tubules in the tunica albuginea of normal and dysgenetic testes pseudocysts of the tunica albuginea: benign invasion by testicular tubules development of the testis from birth to puberty testosterone immunoexpression in human leydig cells of the tunica albugiena testis and spermatic cord. a quantitative study in normal fetuses, young adults, elderly men and patients with cryptorchidism giant interstitial cells and extraparenchymal interstitial cells of the human testis the infi ltrative activity of leydig cells relation of leydig cells in the human testicle to the tubules and testicular function a histological study of extraparenchymal leydig-like cells über die zwischenzellen des hodens zur pathologischen anatomie der leydig zelle leydig cells within the lamina propria of seminiferous tubules in four patients with azoospermia ectopic leydig cells in seminiferous tubules of an infertile human male with a chromosomal aberration leydig cells within the spermatogenic seminiferous tubules immunohistochemical and quantitative study of interstitial and intratubular leydig cells in normal men, cryptorchidism, and klinefelter's syndrome über das verhalten von hoden und nebenhoden bei angeborenem fehlen des ductus deferens, zugleich zur frage des vorkommens von zwischenzellen in menschliche nebenhoden sur l'existence de glands sympathicotropes dans l'ovaire et le testicule humains; leur rapport avec la glande interstitielle du testicule zur ultrastrusktur der leydigzellen im funiculus spermaticus des menschen histogenesis of human extraparenchymal leydig cells presentación de un caso y revisión de la literatura testicular lipomatosis in cowden's syndrome age-related epididymis-like intratesticular structures: benign lesions of wolffi an origin that can be misdiagnosed as testicular tumors male reproductive disorders in humans and prenatal indicators of estrogen exposure. a review of published epidemiological studies testicular cancers occurring in brothers with cryptorchism anatomical, morphological and volumetric analysis: a review of 759 cases of testicular maldescent risk factor patterns for cryptorchidism and hypospadias cryptorchidism: a registry based study in sweden on some factors of some possible etiological importance maternal and neonatal risk factors for cryptorchidism risk factors for cryptorchidism and hypospadias abnormalities of testicular descent surgical outcome of orchidopexy ii. trapped and ascending testes iatrogenic cryptorchidism resulting from hernia repair iatrogenic ascent of the testis: an under-recognized complication of inguinal hernia operation in children iatrogenic ascent of the testis: an underrecognized complication of inguinal hernia operation in children testicular descent and ascent in the fi rst year of life late presentation of cryptorchidism: the etiology of testicular re-ascent ascent of the testis: fact or fi ction incomplete disappearance of the processus vaginalis as a cause of ascending testis undescended testis: congenital or acquired? spontaneous ascent of the testis acquired undescended testis does proximal genitofemoral nerve division induce testicular maldescent or ascent in the rat? association between testicular microlithiasis, testicular cancer, cryptorchidism and history of ascending testis the ascending testis and the testis undescended since birth share the same histopathology elevated placental estradiol: a possible etiological factor of human cryptorchidism histologic observations in cryptorchidism: the congenital germinal-cell defi ciency of the undescended testis histologic classifi cation of undescended testes cytophotometric dna quantifi cation in human 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unusual subset of cryptorchidism: possible end organ failure ascent of the testis in children impaired germ cells in secondary cryptorchid testis after herniotomy elastic fi bers in tunica propria of undescended and contralateral scrotal testes from cryptorchid patients hyperplasia and the immature appearance of sertoli cells in primary testicular disorders focal orchitis in undescended testes: discussion of pathogenetic mechanisms of tubular atrophy undescended testes in adults: clinical signifi cance of resistive index values of the testicular artery measured by doppler ultrasound as a predictor of testicular histology testicular function in men treated in childhood for undescended testes histologic lesions in undescended ectopic obstructed testes is a testis located at the superfi cial inguinal pouch (denis browne pouch) comparable to a true cryptorchid testis? histologic maldevelopment of unilaterally cryptorchid testes and their descended partners anatomical, morphological and volumetric analysis: a review of 759 cases of testicular maldescent les testicules oscillants. forme degradée de cryptorchidie? infertility in adult males with retractile testes changes in the volume and histology of retractile testes in prepubertal boys the retractile testis retractile testis -is it really a normal variant? bilateral retractile testes -subsequent effects on fertility effect of cryptorchidism and retractile testes on male factor infertility: a multicenter, retrospective, chart review epididymal anomalies associated with hydrocele/hernia and cryptorchidism: implications regarding testicular descent congenital deformities of the testis and epididymis signifi cance of epididymal and ductal anomalies associated with undescended testis insulin-like factor 3 gene mutations in testicular dysgenesis syndrome: clinical and functional characterization testicular dysgenesis syndrome: possible role of endocrine disrupters ethnic differences in occurrence of tds -genetics and/or environment? granular changes in sertoli cells in children and pubertal patients erythropoietin may reduce the risk of germ cell loss in boys with cryptorchidism early orchiopexy: prepubertal intratubular germ cell neoplasia and fertility outcome testicular cancer and cryptorchidism cryptorchidism and testicular neoplasia testicular maldescent and infertility histology of testicular biopsies taken at operation for bilateral maldescended testes in relation to fertility in adulthood semen analysis in patients operated on for impalpable testes fertility in cryptorchidism: an overview in 1987 paternity and hormone levels after unilateral cryptorchidism: association with pretreatment testicular location no relationship of testicular size at orchiopexy with fertility in men who previously had unilateral cryptorchidism fertility potential: a comparison of intra-abdominal and intracanalicular testes by age groups in children surgical mangeament of undescended testis: retrospective study of potential fertility in 274 cases orchiopexy and infertility: a critical long-term retrospective analysis the importance of mini-puberty for fertility in cryptorchidism cryptorchidism: aspects of fertility references and neoplasms. a study including data of 1,335 consecutive boys who underwent testicular biopsy simultaneously with surgery for cryptorchidism experience of screening for carcinoma-in-situ of the testis among young men with surgically corrected maldescended testes distribution of carcinoma-in-situ in testes from infertile men carcinoma-in-situ of the testis: aneuploid cells in semen cryptorchidism and testicular neoplasia testicular microlithiasis: sonographic features with pathologic correlation testicular microlithiasis in a child with torsion of the appendix testis testicular microlithiasis occurring in a postorchidopexy testis microcalcifi cations in testicular malignancy: diagnostic tool in occult tumor? testicular microlithiasis with sterility testicular microlithiasis in male infertility microlitiasis testicular asociada a infertilidad painful testicular lithiasis testicular microlithiasis: diagnosis associated with orchialgia idiopathic testicular microlithiasis. ultrastructural study testicular microlithiasis in a uk population: its incidence, associations and follow-up testicular microlithiasis is associated with testicular pathology testicular microlithiasis -a possibly premalignant condition the prevalence of testicular microlithiasis in an asymptomatic population of men 18 to 35 years old testicular microlithiasis: prevalence and tumor risk in a population referred for scrotal sonography testicular calcifi cation and microlithiasis: association with primary intra-testicular malignancy in 3,477 patients testicular microlithiasis, a premalignant condition: prevalence, histopathologic fi ndings, and relation to testicular tumor seminal profi le of subjects with testicular microlithiasis and testicular calcifi cations bilateral testicular microlithiasis predicts the presence of the precursor of testicular germ cell tumors in subfertile men testicular nicrolithiasis and cryptorchidism: ultrasound analysis after orchidopexy testicular microlithiasis. a benign condition with a malignant association testicular microlithiasis: a review and its association with testicular cancer testicular microlithiasis heralding mixed germ cell tumor of the testis in a boy signifi cance of testicular microlithiasis yolk sac tumor and testicular microlithiasis testicular microlithiasis in children: sonographic features and clinical implications identifi cation of seminiferous tubule aberrations and a low incidence of testicular microliths associated with the development of azoospermia raman spectroscopic analysis identifi es testicular microlithiasis as intratubular hydroxyapatite testicular calcifi cation in a 4-year-old boy the origin of testicular microliths testicular microlithiasis in 2 children with bilateral cryptorchidism pulmonary alveolar microlithiasis with involvement of the sympathetic nervous system and gonads testicular microlithiasis and concomitant testicular intraepithelial neoplasia increased risk of carcinoma in situ in patients with testicular germ cell cancer with ultrasonic microlithiasis in the contralateral testicle testicular carcinoma in a patient with previously demostrated testicular microlithiasis testicular microlithiasis and subsequent development of metastatic germ cell tumor detection of testicular microlithiasis by sonography the interval of development of testicular carcinoma in a patient with previously demonstrated testicular microlithiasis surveillance of testicular microlithiasis? results of an uk based national questionnaire survey testicular microlithiasis: us follow-up testicular microlithiasis as a predictor of intratubular germ cell neoplasia microlithiasis of the epididymis and the rete testis three novel sry mutations in xy gonadal dysgenesis and the enigma of xy dysgenesis cases without sry mutations a novel postzygotic nonsense mutation in sry in familial xy gonadal dysgenesis mutations in sry and wt1 genes required for gonadal development are not responsible for xy partial gonadal dysgenesis pathology of 46,xy pure gonadal dysgenesis: absence of testis differentiation associated with mutations in the testis-determining factor xy gonadal dysgenesis: genetic heterogeneity based upon clinical observations, h-y antigen status and segregations analysis chronic renal disease, myotonic dystrophy, and gonadoblastoma in xy gonadal dysgenesis renal failure with xy gonadal dysgenesis: report of the second case a syndrome of chronic renal failure and xy gonadal dysgenesis in young phenotypic females without genital ambiguity atypical presentation of denys-drash syndrome in a female with a novel wt1 gene mutation molecular analysis of frasier syndrome: mutation in the wt1 gene in a girl with gonadal dysgenesis and nephronophthisis frasier syndrome: a rare syndrome with wt1 gene mutation in pediatric urology xy siblings with inadequate virilization and cns defi ciency testicular dysgenesis and mental retardation in two incompletely masculinized xysiblings analysis of the testisdetermining gene sry in patients with xy gonadal dysgenesis the gardner-silengo-wachtel or genitor-palato-cardiac syndrome: male pseudohermaphroditism with micrognathia, cleft palate, and conotruncal cardiac defects xy gonadal dysgenesis associated with a multiple pterygium syndrome phenotype xy pure gonadal dysgenesis: a case with graves' disease a case of gonadal dysgenesis, breast development, graves' disease, and low bone mass alopecia universalis congenita, xy gonadal dysgenesis and laryngomalacia: a novel malformation syndrome alopecia congenita universalis, microcephaly, cutis marmorata, short stature and xy gonadal dysgenesis: variable expression of el-shanti syndrome syndromal (and nonsyndromal) forms of male pseudohermaphroditism familial ovarian dysgerminomas (swyer syndrome) in females associated with a new familial syndrome of 46 xy gonadal dysgenesis with anomalies of ectodermal and mesodermal structural familial xy gonadal dysgenesis familial xy gonadal dysgenesis xy gonadal dysgenesis: genetic heterogeneity based upon clinical observations, h-y antigen status and segregations analysis the x linked recessive form of xy gonadal dysgenesis with high incidende of gonadal cell tumors: clinical and genetic studies the relationship of neoplasia to disorders of abnormal sexual diferentiation xy gonadal dysgenesis: evidence for autosomal dominant transmission in a large kindred gonadoblastomas in 5 patients with 46xy gonadal dysgenesis familial 46,xx gonadal dysgenesis pure gonadal dysgenesis xx and xy: observations in fi fteen patients gonadal dysgenesis in a patient with an x; 3 translocation: case report and review gonadal dysgenesis, intra-x chromosomal insertion, and possible position effect in an otherwise normal female 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localization of the human cyp17 gene (cytochrome p450 (17 alpha)) to 10q24.3 by fl uorescence 'in situ' hybridization and simultaneous chromosome banding a novel point mutation in p450c17 (cyp17) causing combined 17alpha-hydroxylase/17,20-lyase defi ciency towards a unifying mechanism for cyp17 mutations that cause isolated 17,20-lyase defi ciency 17 alpha-hydroxylation defi ciency in man endocrine studies in male pseudohermaphroditism in childhood and adolescence desmolase defi ciency male pseudohermaphroditism consistent with 17-20 desmolase defi ciency pitfalls in characterizing p450c17 mutations associated with isolated 17,20-lyase defi ciency severe 46,xy virilization defi cit due to 17β-hydroxysteroid dehydrogenase defi ciency 17 ketosteroid reductase defi ciency in an adult patient without gynecomastia but with female psychosexual orientation absent spermatogenesis despite early bilateral orchidopexy in 17-ketoreductase defi ciency substitution mutation c268y causes 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appearance the diversity of reproductive tract abnormalities in males with cystic fi brosis postsurgical changes in the testis: a diagnostic dilemma acquired cystic transformation of the rete testis secondary to renal failure adenomatous hyperplasia of the rete testis adenomatous hyperplasia of the rete testis. a review and report of new cases displasia quística del testículo: anomalia en la diferenciación del parénquima testicular por probable fallo en la conexión entre los conductos de origen mesonéfrico y los cordones testiculares adenomatous hyperplasia of the references rete testis: report of two cases glandular changes in the rete testis: metastatic tumour or adenomatous hyperplasia? [letter tumors and cysts of the paratesticular region adenomatous hyperplasia of the rete testis. a clinicopathologic study of nine cases primary testicular lesions are associated with testicular germ cell tumors of adult men adénome du rete testis adenocarcinoma of the rete testis: case report, ultrastructural observations, and clinicopathologic correlates infarcted adenomatoid tumor: a report of fi ve cases of a facet of a benign neoplasm that may cause diagnostic diffi culty rete testis hyperplasia with hyaline globule formation. a lesion simulating yolk sac tumor nodular proliferation of calcifying connective tissue in the rete testis: a study of three cases key: cord-335311-l73hsik0 authors: chan, conrad e. z.; lim, angeline p. c.; macary, paul a.; hanson, brendon j. title: the role of phage display in therapeutic antibody discovery date: 2014-08-18 journal: int immunol doi: 10.1093/intimm/dxu082 sha: doc_id: 335311 cord_uid: l73hsik0 phage display involves the expression of selected proteins on the surface of filamentous phage through fusion with phage coat protein, with the genetic sequence packaged within, linking phenotype to genotype selection. when combined with antibody libraries, phage display allows for rapid in vitro selection of antigen-specific antibodies and recovery of their corresponding coding sequence. large non-immune and synthetic human libraries have been constructed as well as smaller immune libraries based on capturing a single individual’s immune repertoire. this completely in vitro process allows for isolation of antibodies against poorly immunogenic targets as well as those that cannot be obtained by animal immunization, thus further expanding the utility of the approach. phage antibody display represents the first developed methodology for high throughput screening for human therapeutic antibody candidates. recently, other methods have been developed for generation of fully human therapeutic antibodies, such as single b-cell screening, next-generation genome sequencing and transgenic mice with human germline b-cell genes. while each of these have their particular advantages, phage display has remained a key methodology for human antibody discovery due its in vitro process. here, we review the continuing role of this technique alongside other developing technologies for therapeutic antibody discovery. antibodies are one of primary effector molecules of the human immune system and bind their target antigen through their complementarity-determining regions (cdrs) displayed on the fab (antigen-binding fragment) portion of the antibody (fig. 1) . the cdrs encode unique structural diversity and provide antibodies with the ability to recognize a wide variety of targets. the protective efficacy of an antibody can be achieved through binding alone if it results in neutralization of a function of the target or may require additional immune functions provided by the fc (crystallizable fragment) portion of the antibody molecule with receptors found on immune cells or complement proteins in serum. antibody fc region functionality can trigger or augment a number of different host defence mechanisms including phagocytosis, release of cytotoxic or immunomodulatory biomolecules and the complement cascade and formation of the membrane attack complex. these properties potentially make antibodies extremely potent molecules for therapy and they have been developed into an important class of drugs for the treatment of numerous cancers, autoimmune and infectious diseases (1) . display of properly folded proteins on phage through fusion with phage coat proteins was initially conceived as a means to identify the sequence of the protein target which binds to an antibody (2) . ironically, one of the most common uses of phage display today, almost 30 years after its initially development, is to discover antibodies against selected protein targets. originally developed by greg winter et al. at the mrc laboratory of molecular biology and richard lerner, carlos barbas et al. at scripps research institute, the basic concept that a vast antibody repertoire could be displayed on phage with their genetic sequences packaged within has allowed for the robust screening of a variety of target antigens and rapid recovery of specific antibody sequences for recombinant expression (3) (4) (5) . today, a wide variety of antibody phage display libraries exist, with the antibodies displayed either as fabs, scfvs (single-chain variable fragments) or sdabs (single-domain antibodies) in fusion with typically the gene iii coat protein (6,7) (fig. 1 ). the small size and solubility of the phage particle (up to 10 13 particles/ml) has allowed repertoire sizes of up to 10 11-12 to be efficiently displayed and manipulated, which improves the likelihood of finding suitable antibodies (8) . these antibody sequences can be obtained from natural sources (animal or human) or synthetically constructed [usually a mix of pre-defined framework regions (frs) with randomized sequences in the cdrs] and isolation of specific sequences is carried out by repeated enrichment against the target of interest. the defining attribute of all phage display libraries is the physical linking of antibody phenotype (specificity and affinity) with genotype (sequence) via the phage particle-this allows for in vitro selection on immobilized antigen or whole cells (fig. 2) . as this method by-passes the immune system, the usual factors that influence antibody development and limit diversity based on those that can be isolated in animals are not applicable. as such, antibodies can be isolated against targets that have low immunogenicity in animals (nonproteinaceous antigens), to regions of a protein not usually targeted due to the presence of immunodominant epitopes elsewhere, or to hydrophobic targets that are challenging for in vivo manipulation (9) (10) (11) . as such, phage display offers high potential for discovery of novel therapeutic antibodies that cannot be generated naturally through in vivo immunization or infection. furthermore, the ability to display synthetic repertoires devoid of any negative selection has made this technique a key method for the generation of potent fully human antibody therapeutics against self-antigens (12). nixon et al. provide an in-depth review of the clinical development of such drugs (13). each antibody comprises two heavy and two light chains each of which have four and two immunoglobulin domains, respectively. the first domain is variable and determines specificity (vl and vh) while the second domain of the light chain (cl) and the second to fourth domains of the heavy (ch1-3) are constant across all antibodies of the same isotype. the light chain and first two domains of the heavy chain form the fab, which is the portion expressed on the phage. the last two domains of the heavy chain form the fc and are responsible for immune function through engagement of receptors on immune cells. heavy and light chains are linked through a single disulfide bond (orange) between the cl and ch1 domains and the two heavy chains have multiple disulfide bonds at the hinge region between the ch1 and ch2. an scfv consists of just variable light and variable heavy domains joined by a flexible polypeptide linker while an sdab as the name implies is only a single immunoglobulin (usually vh) domain which is sufficient for binding. (b) variable domain genetic structure and construction of a natural phage display library. each variable domain consists of three hypervariable cdrs interspersed between the more conserved frs. the immunoglobulin domain folds such that the cdrs are brought together to form the antigen-binding surface at the tip of the fab. degenerate primers (arrows) are used to amplify the entire variable heavy and light chains (or alternatively variable and first constant domain) from a source of b cells and cloned in-frame with the phage coat protein (usually gene iii) into e. coli to produce an fab, scfv or sdab library. the rest of the phage genome is supplied through replication defective helper phage to produce antibody-displaying phage. general strategy for phage panning. polyclonal phage expressing recombinant antibodies on their surface is applied to target antigen presented as either immobilized on a magnetic bead, polystyrene surface or on the surface of a whole cell. phage carrying antigen-binding fab bind and non-specific fab are removed through stringent washing. antigen-bound phage is eluted off either typically through ph change or protease digestion and re-infected into e. coli, from which a new library enriched for antigen-binding clones can be made. after several cycles, the library would be sufficiently enriched so that the individual clones can be isolated from e. coli stock, expressed as monoclonal phage, tested, sequenced and the specific antibodies expressed recombinantly. another key attribute of phage display is that selection can be far more rapid as compared to the natural immune response, and in our laboratory, we have been able to identify monoclonal antibodies (mabs) in as short a period as 1 week (14) . this is particularly useful for generation of antibodies against novel or genetically modified pathogens in an emergency outbreak scenario. in recent years, however, alternative antibody discovery systems have been developed, such as high-throughput testing of individual b cells and next-generation sequencing of b-cell repertoires. we review here the use of phage display against various types of targets, such as self-antigens, non-protein targets and infectious disease agents, in relation to on-going developments in both phage display and other antibody discovery technologies. in the human immune system, naive b cells targeting selfantigens are deleted by negative selection and immunization in a different host is therefore required to generate specific antibodies against human targets. however, the resultant antibodies, even after humanization by replacement of constant regions or of frs with human equivalents, are usually more immunogenic than a human-derived antibody (15) . while this can result in increased toxicity, the primary concern is the potential for reduced clinical efficacy of the therapy which can occur with development of a humoral immune response against the antibody. for self-antigens, such as tumour cell markers, phage display is an ideal technique for the generation of fully human antibodies due to the absence of tolerance mechanisms. semi-synthetic or synthetic libraries, whose sequences have not been negatively selected for any antigen, are usually the first choice. an additional advantage of these libraries is that design features can be incorporated to allow facile affinity maturation, such as cdrs flanked by restriction sites for easy replacement or appropriate consensus primers for targeting of specific cdrs for directed mutagenesis (16) . several fully human synthetic library-derived mabs have been approved by the us food and drugs administration or completed clinical trials. the first of these was adalimumab, an anti-tnf-α antibody developed by cambridge antibody technologies for the treatment of rheumatic arthritis and crohn's disease (17, 18) . subsequently, other antibodies were developed for the treatment of cancer and autoimmune disease such as necitumumab (non-small cell lung cancer), ramucirumab (gastrointestinal cancers) and belimumab (systemic lupus erythematosus) (12, 19, 20) . many others are currently at advanced stages of clinical development and may reach market approval within the next few years (21, 22) . as mabs gain popularity as a form of alternative drug therapy, the use of phage displayed antibody libraries has also gained a reputation as the premier technology for antibody discovery. semi-synthetic libraries, which were the first to be made, typically consist of frs sourced from naturally occurring antibodies and cdr regions synthesized from degenerate oligonucleotides (23, 24) . in many instances, to simplify construction, only the cdr3 region was fully synthetic. subsequently, libraries with fully synthetic variable sequences were introduced, with the fr based on consensus sequences from natural antibodies but optimized for high expression on phage in escherichia coli to reduce the effect of variable expression of the human framework sequences on selection. the first fully synthetic human combinatorial antibody libraries (hucal) generated to produce therapeutic antibodies was reported by morphosys in 2000 with their first version of the hucal (16) . several versions of the hucal has since been constructed that incorporate additional features such as improved phage elution by disulphide bond cleavage, selection for in-frame clones, additional cdr3 loop length and diversity and optimization for expression in mammalian cell culture through removal of unwanted potential post-translational modification sites (25, 26) . these modifications have resulted in an increase in the number of antibodies isolated per target antigen. there are several antibodies generated from the morphosys libraries currently in various stages of clinical development; a recent example being the potent anti-cancer activity of omp-18r5, with activity in multiple preclinical human tumour models (27) . this antibody targeting the wnt pathway was isolated from the hucal gold library and is currently in phase 1 clinical testing. while synthetic libraries have allowed the development of human antibodies with reduced immunogenicity relative to humanized animal antibodies against self-antigens, the cdrs of these libraries can also be immunogenic as often the sequences are not naturally present in the human antibody repertoire (28) . with this in mind, another approach to build synthetic libraries has been to generate novel variable region sequences by overlapping pcr of individual amplified germline cdrs with respective fr (29) . while allowing generation of novel sequences that target self-antigens, it also prevents the generation of synthetic sequences that could contain high levels of t-cell epitopes and be strongly immunogenic. indeed, antibodies obtained from this library had a proportion of t-cell epitopes no higher than that of naturally occurring antibodies. an antibody against icam-1 was isolated from this library and shown to have potent anti-myeloma activity in vivo (30) . over the years, semi-synthetic and fully synthetic libraries have shown utility beyond targeting of self-antigens, as these libraries are also capable of generating antibodies of high affinity against a wide range of target antigens (16, 24, 25) . one such high-affinity antibody which was recently approved for clinical use is raxibacumab for the treatment of inhalational anthrax (12) . extremely high diversity synthetic libraries have also been generated with the goal of providing higher affinity antibodies than currently approved therapeutic antibodies, thus circumventing the need for further improvement by affinity maturation. hoet et al. at dyax corp reported the isolation of very high-affinity antibodies through synthetic diversity introduced into the key antigen-contact sites of cdr1 and cdr2 within the heavy chain in combination with naturally occurring diversity in the heavy chain cdr3 and light chains from donors with autoimmune diseases (31) . their human antibody phage display libraries have produced multiple drug candidates currently in different phases of clinical development. non-protein targets: carbohydrates, lipids and posttranslationally modified proteins while antibodies are typically raised against proteins, the first therapeutic antibodies in clinical use in the early 20th century, prior to the discovery of antibiotics, were actually polyclonal preparations that recognized the carbohydrate coat of pathogenic bacteria such as neisseria meningitidis and streptococcus pneumoniae. these were raised in animals against inactivated whole bacteria for treatment of severe bacterial infections and each preparation was effective only on bacterial strains with homologous carbohydrate structures (32, 33) . this lead to a realization that antibody recognition of the coat structure was an important correlate for efficacy, and the functional classification of bacteria into serotypes for proper matching of antibody and bacterial strain. the requirement for multiple polyclonal antibodies for treatment of even one bacterial species due to large numbers of serotypes was one of the reasons for the switch to more broadly acting antibiotics once they became available (33) . nonetheless, their effective use is an indication of the therapeutic potential of non-protein targets. to generate high-affinity antibodies against such targets, a strong immune response is required which is usually stimulated through conjugation with a protein carrier, delivery with an adjuvant such as lipid a or as an antigen complex with proteins such as whole inactivated bacteria (34) (35) (36) (37) . even so, the antibodies obtained can often be low affinity igm due to the inability to engage the mechanisms required for affinity maturation and class switching, hence limiting the utility of animal immunization for antibody generation (36, 38) . on the other hand, phage display has been used to produce anti-carbohydrate antibodies of reasonable affinity in the low nanomolar range (39) . in particular, a phage library constructed with the heavy chain cdr3 enriched for basic residues to improve binding to negatively charged carbohydrates produced anti-carbohydrate antibodies that had relatively high affinity (k d ≈ 50 nm) and excellent specificity (40) . although no anti-carbohydrate antibodies have been approved for routine clinical use, such antibodies have been shown to be able to target both carbohydrate-rich tumour antigen and bacterial lipopolysaccharides, the latter having been used on occasion to treat severe cases of sepsis (41, 42) . anti-lipid/lipoprotein antibodies are fairly uncommon in nature and in humans, they are usually associated with metabolic, autoimmune and neurodegenerative diseases; perhaps unsurprisingly given the key role lipids play in these processes. cerebrospinal fluid and serum antibodies against myelin basic protein, sulfatide and oxidized lipids have been found in patients with multiple sclerosis, and serum antibodies against neuronal gangliosides are directly implicated in the pathology of the peripheral neuropathy guillain-barré syndrome (43) (44) (45) . anti-oxidized low-density lipoprotein antibodies on the other hand may play a role in the development of atherosclerosis (46) . isolation of such antibodies via phage display and the use of recombinant versions in animal models of disease may help determine their pathological role and potentially reverse its effects (47) . for example, in anti-phospholipid syndrome, antibodies against phospholipid-binding proteins such as β2-glycoprotein i are considered to be a major driver of pathology through inappropriate complement fixation. the use of phage display to obtain human antibodies of the same specificity has enabled the development of blocking antibodies that are defective in fixing complement which were shown to inhibit the effect of endogenous antibodies in animal models (48) . in another example, with certain animal models of atherosclerosis, a high-titre antibody response to the malondialdehyde low-density lipoprotein immunogen also appears to correlate with reduced disease severity, suggesting that such antibodies could be used therapeutically (49) . phage display can also be used to develop anti-idiotypic antibodies; these have been shown to have therapeutic potential by binding and blocking the effects of naturally occurring autoantibodies (50) . using phage display, we and others have also been able to generate anti-lipid antibodies even against pure lipids completely insoluble in aqueous solution, through enrichment against crystalline microdroplets formed following evaporation of organic solvents (9, 10, 51) . while having potential for use as diagnostic reagents to identify targeted lipids in crude mixtures, it remains unclear whether such antibodies can recognize lipids in their natural biological environment and the therapeutic potential of anti-lipid antibodies generated through such modalities remains an open question. another type of target uniquely suited to phage display is proteins with post-translational modifications such as glycosylation or phosphorylation. in such cases, the approach is to engineer a specific recognition domain into a particular cdr region which significantly increases the likelihood that the recovered antibody will recognize the modification in addition to the protein epitope (52) . for recognition of phosphorylated peptides, a phosphate-binding motif from a naturally occurring antibody was used to generate a mutant antibody library from which antibodies binding specifically to either phosphoserine, phosphotyrosine or phosphothreonine were identified. the cdr amino acids responsible for phosphorylated amino acid recognition were identified and then used to generate a second set of libraries which preserved these structures but had the rest of the cdr randomized; these libraries were then used to screen phosphorylated peptide targets. a similar strategy was used to identify glycan-binding antibodies based on a motif isolated from the anti-hiv antibody 2g12, which binds the hiv-1 glycoprotein gp120 primarily through its glycosylation (53) . a key advantage of phage display is the in vitro nature of the antibody identification process, as this allows for selection methodologies not possible with in vivo antibody generation. by carefully controlling the selection and screening conditions on immobilized antigen, for example, by the presentation of specific antigen conformations or the inclusion of competitors for direct selection towards specific targets or epitopes, the generation of antibodies can be biased towards desired regions of the target (54) (55) (56) . moreover, binding affinity and on and off rates of recovered antibodies can be optimized independently according to the selection strategy employed, enabling tailoring to the desired biomedical application (57, 58) . this can be further enhanced during recombinant expression as full antibodies where various immunoglobulin isotypes and constructs with alternative functionality can be easily generated. another screening modality available to phage display is the targeting of antigen presented on the surface of whole cells or tissue. this is especially useful for membrane-bound antigen such as cell surface receptors and transmembrane proteins whose structure is not easily recapitulated by free antigen and is particularly useful for tumour-specific surface antigens. however, this approach is complicated by the diversity of antigens on the cell surface which may result in selection of phage against common surface epitopes (59). one means to overcome this is to leverage on unique biological properties of the target antigen. for example, signalling receptors such as her2, epidermal growth factor receptor and p75 neurotrophin receptor, cycle from the cell surface to the interior and recovery of phage from the cell interior has allowed specific phage enrichment (60) (61) (62) . however, most cell types will carry multiple membrane proteins capable of internalizing bound phage. an alternative and complementary means for selection is to pan against alternating cell types expressing the same target antigen to prevent selection of common surface antigens; this can be achieved through the use of mammalian or yeast expression vectors for expression of recombinant antigen on a suitable host cell (62, 63) . in addition to these in vitro techniques, phage library approaches with in vivo binding modalities have also been explored. first described for organ targeting using peptide phage libraries where the library was injected intravenously into mice and the phage were subsequently rescued from the brain and kidneys (64) , the technique has also been successfully carried out in tumour-bearing mice (65) . in recent years, similar methodologies have also been employed with antibody phage libraries not only to identify potentially therapeutic antibodies but also for determining the accessibility of a given target (66) , further expanding the boundaries of phage display technology. today, almost all widely accessible commercial libraries are based on non-immune repertoires of high complexity to enable use in the selection of antibodies against a virtually unlimited number of target antigens. the latest state-of-art libraries reach complexities of up to 10 11 -10 12 clones and thus may even exceed the size of individual human cell repertoires (67, 68) . while high-affinity antibodies can be isolated from these libraries, occasionally the affinity of the obtained antibodies is low as the genes are obtained from naive b cells that have not yet undergone affinity maturation. in such cases, where the affinity required for therapeutic efficacy is higher than that of the antibody obtained, in vitro affinity maturation may be performed. this is often accomplished by introducing random or specific mutations in the cdrs or the exchange of whole heavy or light chain domains by chain shuffling (69) (70) (71) (72) (73) . recently, li et al. described an alternative phage display approach for affinity maturation of a natural human antibody msl-109 targeting human cytomegalovirus (cmv), where each of the cdrs was allowed to vary in only one amino acid residue at a time (69) . higher affinity antibodies were isolated which had improved cmv neutralization potency when expressed as fab fragments in bacterial cells. in another novel application of in vitro phage library screening, single antibodies capable of dual epitope recognition have also been developed. carried out by bostrom et al., a subset of solvent exposed residues of the light chain cdrs of the anti-her2 antibody herceptin were randomized to generate a highly diverse combinatorial phage library (10 10 particles) (74) . by subjecting the library to selection for vegf binding while maintaining her2 binding, dual antigen-binding clones were identified which inhibited both vegf and erb-b2-mediated cell proliferation in vitro and tumour progression in mouse models (74) . such 'two-in-one' antibodies may represent a paradigm shift and provide exciting new opportunities for therapeutics. in contrast to lipids, carbohydrates and self-antigens, highaffinity antibodies against pathogen-derived protein antigens are easily generated through immunization or during the course of a natural infection (75, 76) . present at high levels during the course of the immune response due to secretion from short-lived plasma cells and plasma blasts, lower levels are maintained in the serum by secretion from long-lived plasma cells and usually have therapeutic or prophylactic efficacy. this was put to good use in the preantibiotic era through serum therapy, where patients were given pathogen-specific polyclonal serum raised in animals (33) . more recently, human-derived antibodies have been shown to be efficacious against a number of highly pathogenic diseases such as severe acute respiratory syndrome (sars) coronavirus, ebola and h5n1 avian influenza (77) (78) (79) (80) . recovery of such naturally occurring antibodies from convalescent patients is clearly of prime importance for developing drugs against pathogens to which there are no other effective treatments. in addition, they provide a wealth of information on the humoral immune response, its duration and maintenance over time, and how it can be effective, or in some cases, ineffective, against infection (81, 82) . to capture these antibodies, immune libraries can be constructed relatively quickly, allowing identification of the high-affinity antibodies that can be used to generate diagnostic reagents and also tested for their therapeutic potential. in addition, novel in vitro screening strategies can be employed to isolate rare broadly neutralizing antibodies, as has been highlighted in the hunt for antibodies against pandemic influenza. kashyap et al. reported the generation of combinatorial antibody libraries from the bone marrow of five survivors of a h5n1 avian influenza outbreak in turkey yielding >300 unique antibodies against h5n1 viral antigens, several of which were shown to have broadly neutralizing properties across various h5 subtypes (83) . similarly, throsby et al., using combinatorial display libraries constructed from human memory b cells elicited by seasonal influenza vaccination, isolated 13 antibodies exhibiting broad heterosubtypic neutralizing activity against antigenically diverse h1, h2, h5, h6, h8 and h9 influenza subtypes (84) . the most potent antibody was also protective in mice when given before and after lethal h5n1 or h1n1 challenge and the epitope was localized to the highly conserved therapeutic antibodies from phage display 653 membrane-proximal stem of the ha1 and ha2 subunits of hemagglutinin (84, 85) . using a combination of panning strategies against the hemagglutinin from several antigenically distinct h5n1 isolates to bias-selection of antibodies towards more conserved domains of the protein, we were able to isolate similar antibodies to that described by throsby et al. from a non-immune fab phage display library (11) . the selection of such antibodies from even non-immune phage displayed libraries highlights the potential of these readily available libraries for the development of therapeutically relevant antibodies against emerging pathogens of high concern and may enable more rapid development of therapies against novel emergent pathogens in the future. the main competition for phage libraries in the area of cancer and autoimmune disease therapeutics where self-antigens are the primary target are transgenic mice whose immunoglobulin germline genes have been replaced with human equivalents. this technology, developed in the 1990s, has been driven mainly by two biotech companies-mederax and abgenix-which licence the humab and xenomouse strains, respectively (86) (87) (88) . during the course of v(d)j recombination, these reassemble into fully human antibodies although they are carried on murine b cells and the mice can generate functional immune responses (including somatic hypermutation) during immunization, from which antibody-secreting hybridomas can be made. some of these antibodies are already approved for clinical use and more are in the clinical pipeline (12, 89) . one disadvantage of these first-generation strains appears to be the use of human constant regions, which has been suggested to affect downstream signalling in b cells and result in defective immune responses against particular antigens; newer strains of transgenic mice have been developed that have only the variable regions replaced and utilize murine constant regions (90) . currently, more antibodies in clinical use or trials are derived from transgenic mice as compared to phage display, but this may not be an accurate comparison of their utility as the technology for transgenic mice was developed several years ahead of the first truly diverse synthetic antibody libraries (12) . additionally, as antibody generation is governed by the rules of the immune response, their utility for generating high-affinity antibodies against epitopes common to humans and mice, non-protein targets, or antibodies with novel binding characteristics such as those achieved by in vitro selection under non-physiological conditions, is limited. for isolation of antibodies from human subjects, while phage display was the first technique suitable for the screening of large numbers of b cells, in recent years, additional techniques of similar robustness and efficacy have been developed. these fall into two main categories: analysis of single b cells through in vitro immortalization, activation into antibody-secreting cells, or recovery of their individual antibody transcripts; or alternatively, high-throughput analysis of polyclonal antibodies. the latter approach can be carried out at either the proteome or transcriptome level through mass spectrometry of digested peptides or next generation sequencing of harvested b-cell mrna, respectively. (80, (91) (92) (93) (94) . both these techniques have one main advantage over phage display; they allow elucidation of the heavy and light chain pairings and frequencies of the b cells of interest. as such, they may be more useful for characterizing the dynamics of humoral immune responses (95) . on the other hand, they require significant investment in specialist equipment, such as a high-speed fluorescenceactivated cell sorter (facs) or next generation sequencer, as well as robotic handling equipment if large numbers of cells are to be screened (77) . phage display does not require any such equipment and can be performed with basic microbiological and molecular biology apparatus with the exception of an electroporator if high efficiency cloning is required (67) . another advantage is that a phage library once created is usually sufficient for numerous screenings and can be stored almost indefinitely, while the various omics procedures involve destruction of the sample and single b-cell cultures, unlike murine hybridomas, do not allow for indefinite antibody production (96) . even antibodies produced by b cells immortalized with epstein-barr virus can gradually lose affinity due to re-activation of somatic hypermutation (p. a. macary, unpublished data). hence, while these techniques require a finely honed screening strategy only available with well-characterized pathogens, phage display may be more suited towards investigation of poorly understood or novel pathogens where multiple screening targets and conditions need to be investigated. in addition, sideby-side comparison of phage display and next generation sequencing produced completely different sets of antibodies that were attributed to the poor expression of particular antibodies on phage. nevertheless, the antibodies produced by either technique had comparable specificity and affinity (97) . while for some applications, such as isolation of antibodies from patients, phage display may be complemented or substituted with other techniques, an area where phage display remains critical is for directed evolution of antibodies for increased stability under non-physiological conditions of ph and temperature-such selection is simply not possible in vivo. neither are other in vitro alternatives such as cell surface display (mammalian, yeast or bacterial) or ribosome display feasible due to their lower resistance to high temperatures or extreme ph. phage can tolerate temperatures of up to 60°c and a ph range of 2-11 (98) . table 1 provides a summary comparison of the advantages and disadvantages of these various in vitro techniques. phage display is also likely to remain useful for discovery of antibodies against non-protein targets, evolution of dual-binding antibodies and for affinity maturation, due to the limitations of the natural immune system. however, antibodies against non-protein targets such as lipids and carbohydrates remain a fairly unexplored area, partially due to the lack of validated therapeutic targets aside from bacterial membrane surface carbohydrates. one area that could be of particular interest is lipids involved in autoimmune and neurodegenerative disorders, for which there is some evidence that antibodies could have both protective and pathological roles and which a therapeutic antibody designed to suppress immune function, e.g. of the igg4 isotype, could have benefits. for regular protein antigens, there may still be one particular niche for phage display based on its strengths: the rapid development of therapeutic antibodies against emerging diseases or a genetically modified pathogen in an emergency situation. antibodies are a natural and well-studied component of the immune system and use of a naturally derived antibody should have minimal side effects and thus be ideal for emergency use. in such a situation, rapid antibody isolation is critical. phage display allows for the screening of an extremely large collection of antibodies of up to 10 11 sequences, far more than that possible by next generation sequencing or single b-cell analysis, thus improving the chance of isolating rare antibodies. even if an immune library is required, rapid construction and screening is possible using cloning protocols such as megawhop which do not require prior digestion of pcr immunoglobulin sequences and new panning strategies that enable effective enrichment over only one cycle of selection (102, 103) . in addition, vectors have been developed that can express antibodies both on phage as well as in mammalian cell culture as full length igg through the use of intron-mediated differential splicing without any need for sub-cloning, which enables rapid highthroughput expression analysis of the full length antibody (104) . as such, phage display is likely to remain a key technique for antibody discovery for the foreseeable future. isolation of naturally paired vh-vl immunoglobulin genes that have gone through affinity maturation. lacks post-translational modification of antibody fragments. requires optimization due to relative instability of rnaribosomal complex. smaller library size compared to phage/ribosome display. labour intensive and technically challenging. smallest library size. affinity maturation can be performed in vitro. can be used to select for highaffinity binders. can be used to select for high-affinity binders during facs sorting. not required. therapeutic antibodies from phage display 655 the use of antibodies in the treatment of infectious diseases filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface phage antibodies: filamentous phage displaying antibody variable domains generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda assembly of combinatorial antibody libraries on phage surfaces: the gene iii site selecting and screening recombinant antibody libraries selection of non-aggregating vh binders from synthetic vh phage-display libraries high affinity, developability and functional size: the holy grail of combinatorial antibody library generation novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis generation and characterization of a novel recombinant antibody against 15-ketocholestane isolated by phage-display neutralizing human monoclonal antibody against h5n1 influenza ha selected from a fab-phage display library development trends for human monoclonal antibody therapeutics drugs derived from phage display: from candidate identification to clinical practice comparison of the efficiency of antibody selection from semi-synthetic scfv and non-immune fab phage display libraries against protein targets for rapid development of diagnostic immunoassays immunogenicity of engineered antibodies fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen technology evaluation: adalimumab, abbott laboratories antibody-based therapeutics to watch in 2011 antibody phage display libraries: contributions to oncology beyond natural antibodies: the power of in vitro display technologies in vitro display technologies reveal novel biopharmaceutics selection and application of human single chain fv antibody fragments from a semisynthetic phage antibody display library with designed cdr3 regions isolation of high affinity human antibodies directly from large synthetic repertoires hucal platinum, a synthetic fab library optimized for sequence diversity and superior performance in mammalian expression systems the human combinatorial antibody library hucal gold combines diversification of all six cdrs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies wnt pathway inhibition via the targeting of frizzled receptors results in decreased growth and tumorigenicity of human tumors the immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the cdr regions recombining germline-derived cdr sequences for creating diverse singleframework antibody libraries a human icam-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity serum therapy revisited: animal models of infection and development of passive antibody therapy synthesis and characterization of hapten-protein conjugates for antibody production against small molecules conjugate vaccines-a breakthrough in vaccine development characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of helicobacter pylori lipopolysaccharide the binding sites of monoclonal antibodies to the non-reducing end of francisella tularensis o-antigen accommodate mainly the terminal saccharide isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. expression, purification, and characterization of recombinant single chain antibodies engineering antibody heavy chain cdr3 to create a phage display fab library rich in antibodies that bind charged carbohydrates isolation and characterization of antibodies against three consecutive tn-antigen clusters from a phage library displaying human single-chain variable fragments treatment of gram-negative bacteremia and septic shock with ha-1a human monoclonal antibody against endotoxin. a randomized, doubleblind, placebo-controlled trial. the ha-1a sepsis study group lipid microarrays identify key mediators of autoimmune brain inflammation peripheral neuropathies and anti-glycolipid antibodies combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and ebv antigen human-derived anti-oxidized ldl autoantibody blocks uptake of oxidized ldl by macrophages and localizes to atherosclerotic lesions in vivo anti-beta(2)gp-i and antiprothrombin antibodies generated by phage display a non-complement-fixing antibody to β2 glycoprotein i as a novel therapy for antiphospholipid syndrome hyperimmunization of apo-e-deficient mice with homologous malondialdehyde low-density lipoprotein suppresses early atherogenesis efficacy of intravenous immunoglobulin (ivig) affinity-purified anti-desmoglein anti-idiotypic antibodies in the treatment of an experimental model of pemphigus vulgaris characterization of 11-dehydro-thromboxane b2 recombinant antibody obtained by phage display technology nature-inspired design of motif-specific antibody scaffolds a strategy for phage display selection of functional domain-exchanged immunoglobulin scaffolds with high affinity for glycan targets directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries directed selection of mip-1 alpha neutralizing ccr5 antibodies from a phage display human antibody library directing phage selections towards specific epitopes tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity improved affinity selection using phage display technology and off-rate based selection selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library a novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75 toward selection of internalizing antibodies from phage libraries selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library internalizing cancer antibodies from phage libraries selected on tumor cells and yeast-displayed tumor antigens organ targeting in vivo using phage display peptide libraries vascular ligand-receptor mapping by direct combinatorial selection in cancer patients proteasome activator complex pa28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies construction of large naïve fab libraries modelling the human immune response: performance of a 1011 human antibody repertoire against a broad panel of therapeutically relevant antigens in vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity crystal structure of an in vitro affinity-and specificity-matured anti-testosterone fab in complex with testosterone. improved affinity results from small structural changes within the variable domains in vitro antibody affinity maturation targeting germline hotspots antibody affinity maturation by chain shuffling variants of the antibody herceptin that interact with her2 and vegf at the antigen binding site appearance of peripheral blood plasma cells and memory b cells in a primary and secondary immune response in humans duration of humoral immunity to common viral and vaccine antigens a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants ebola virus can be effectively neutralized by antibody produced in natural human infection an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus the human immune response to dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity neutralizing antibodies derived from the b cells of 1918 influenza pandemic survivors combinatorial antibody libraries from survivors of the turkish h5n1 avian influenza outbreak reveal virus neutralization strategies heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells antibody recognition of a highly conserved influenza virus epitope functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice antigenspecific human antibodies from mice comprising four distinct genetic modifications functional expression and germline transmission of a human chromosome fragment in chimaeric mice human antibodies from transgenic animals mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice monoclonal antibodies isolated without screening by analyzing the variablegene repertoire of plasma cells rapid cloning of high-affinity human monoclonal antibodies against influenza virus ex vivo characterization and isolation of rare memory b cells with antigen tetramers rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array profiling human antibody responses by integrated single-cell analysis an in vitro model of differentiation of memory b cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization antibody isolation from immunized animals: comparison of phage display and antibody discovery via v gene repertoire mining stability engineering of antibody single-chain fv fragments harnessing phage and ribosome display for antibody optimisation engineering antibodies by yeast display single b cell antibody technologies a general strategy for antibody library screening via conversion of transient target binding into permanent reporter deposition megawhop cloning: a method of creating random mutagenesis libraries via megaprimer pcr of whole plasmids a dual host vector for fab phage display and expression of native igg in mammalian cells the authors declared no conflicts of interest. key: cord-351665-6gwb900b authors: sarkar, priyanka; debnath, nirmal; reang, demsai title: coupled human-environment system amid covid-19 crisis: a conceptual model to understand the nexus date: 2020-08-18 journal: sci total environ doi: 10.1016/j.scitotenv.2020.141757 sha: doc_id: 351665 cord_uid: 6gwb900b abstract the world today is dealing with a havoc crisis due to the pervasive outbreak of covid-19. as a preventive measure against the pandemic, government authorities worldwide have implemented and adopted strict policy interventions such as lockdown, social distancing, and quarantine, to curtail the disease transmission. consequently, humans have been experiencing several ill impacts, while the natural environment has been reaping the benefits of the interventions. therefore, it is imperative to understand the interlinked relationship between human society and the natural environment amid the current crisis. herein, we performed a meta-analysis of existing literature reporting the various impacts of covid-19 on human society and the natural environment. a conceptual model was developed to portray and address how the interaction of the existing elements of both sub-components of the coupled human-environment system (ches) – human society and natural environment – are impacted by the government interventions. results revealed a suite of positive and negative impacts of covid-19 on both the sub-components. our model provides an explicit impression of the complex nexus of ches amid the current crisis. the proposed conceptual model could help in understanding the complex nexus by identifying the route of short-term impacts of covid-19 measures and thus may aid in identifying priority areas for discussion and planning in similar other crises as well. coupled human-environment system (ches) represents a complex, dynamic, interconnected, and integrated system in which humans and the natural environment interact with each other (turner et al. 2003; liu et al. 2007; galvani et al. 2016) . in recent centuries, humans have been remarkably intervening with the environment to fulfill the demands of the growing population and rapid economic development (myers and patz 2009) . such a plethora of anthropogenic interventions poses long-term repercussions i.e., extreme climatic events and other natural calamities, food, and water scarcity, increased exposure to infectious diseases, population displacement, etc. on the human society (myers and patz 2009; galvani et al. 2016 ). therefore, understanding the complex ches is important for recognizing and addressing the vulnerability of the situation on human society, and natural ecosystems amid any local or global crisis (turner et al. 2003) . the world today is dealing with a havoc crisis due to the pervasive outbreak of coronavirus disease 2019 . the vicious covid-19 is an infectious disease caused by a new 2020), and exhibits a higher human to human transmissibility (chan et al. 2020) . the pandemic has posed a great threat to the global-public health and economic recession and is still ongoing (bogoch et al. 2020; wu et al. 2020) . so far, no proven pharmaceutical treatment has been developed to combat the pandemic (singhal 2020). given its severe impact, the world health organization (who) has declared covid-19 as a public health emergency of international concern on january 30, 2020. as a preventive measure against the pandemic, the government authorities worldwide have implemented and adopted strict policy interventions to maintain social (physical) distancing and curtail the transmission of covid-19. almost one-quarter of the global population is now confined within their homes, experiencing several negative/ill impacts in terms of socioeconomic and psychological well-being. on the contrary, there are several hidden benefits of the interventions on the environment or natural world. therefore, it is imperative to understand and appreciate the mutually-affective relationship between human society and the natural environment amid the current crisis (kumar 2020a ) and identify the priority areas for designing necessary action plans for a balanced state. although, few studies have highlighted the impacts of covid-19 and relevant policy actions on the environment, and social aspects like economic and health consequences (hevia and neumeyer 2020; lin et al. 2020; muhammad et al. 2020; sharma et al. 2020; zambrano-monserrate et al. 2020) , the complexity of ches amid the current crisis has not been well understood. unpredictability and vulnerability of ches require a case or situation-specific assessment to consider appropriate relationships of variables with a set of standardized methods (polsky et al. 2003; turner et al. 2003) . in this regard, our study attempted to understand the processes, responses, and feedbacks within the complex ches amid the crisis through a conceptual model. such models are valuable communication tools that represent the current knowledge of a system and illustrate its complex interactions in a simplified way (gross 2003; imgraben j o u r n a l p r e -p r o o f journal pre-proof et al. 2019) . conceptual models may thus assist in identifying the priority areas that require further research or monitoring and build a basis for discussion and planning (roman and barrett 1999) . the specific objectives of the study were to (i) perform a meta-analysis of existing literature reporting various impacts of covid-19 on human society and the natural environment, and (ii) develop a conceptual model to illustrate and understand the complex nexus of ches amid the pandemic. our conceptual model could be helpful in clearly portraying the complex and coupled nature of the system amid the current crisis. thus, aid in identifying the priority areas and intrigue the discussion for further planning towards mitigating the effects of the current-as well as similar crisis. we performed a systematic review of peer-reviewed scientific articles on the impact of covid-19 on human society and the natural environment worldwide written in english using the isi web of science (wos) -core collection database published until april 30, 2020 (with an open initial date). a literature search was conducted using a combined search string with two topic fields. search strings in the first topic field included different terms denoting covid-19 outbreak, restrictions and their impacts ("covid-19", 'coronavirus', the conceptual model was developed based on the information collected from the literature review, and the previous experience and expertise of the first author on model development -'expert-based models' approach (ferrier et al. 2016 ). this was followed by brainstorming amongst all the authors to improvise the model. during the entire process of model development, authors' practical experiences and in-depth understanding of the context of ecological and environmental studies, and associated socio-economic factors were advantageous. the literature review revealed a suite of impacts (positive and/or negative) of covid-19 and lockdown/restrictions on human society and the natural environment (table 1) on both human society and the environment were also recorded (table 1) . the proposed conceptual model comprises of two modules, (i) drivers of change: covid-19 (direct driver) and lockdown/restrictions (indirect driver), and (ii) our focal point: coupled human-environment system (ches) (fig. 1) . drivers of change refer to the factors that directly or indirectly cause changes in nature and its components, anthropogenic assets, and good quality of life (ipbes, https://ipbes.net/glossary/driver). the conceptual model illustrates how the different elements of two sub-systems: human society and natural environment, interact with one another -directly and/or indirectly, through forming a network of associations. the relationships between the elements of the two sub-systems are depicted by arrows, which also shows the direction of impacts, positive and/or negative ( fig. 1 ). for example, for the sub-system: human society, limited transportation had a positive impact in containing the disease, thus lowering infection & death. similarly, limited transportation due to lockdown resulted in reduced fuel consumption, generating positive impacts on the sub-system: natural environment, characterized by reduced-air pollution (lower conc. of co 2 , co, nox, pm 2.5 , pm 10 ) and environmental noise, leading to flourished biodiversity/wildlife. the conceptual model also displayed two feedbacks -(i) between the primary and secondary drivers -covid-19 and lockdown/restrictions, and, (ii) between covid-19 and the health workers. to illustrate, the spreading of the virus has mandated the implementation of lockdown inducing grief to human society worldwide, which on the other hand, had a positive impact on suppressing the pandemic. covid-19 crisis is considered one of the worst pandemics in history which has further complicated the entire coupled human-environmental system. lower adaptability of human society to various socio-environmental crises arises mainly due to the poor understanding of the complex and interconnected nature of the human-environment system (dearing et al. 2006 ). hence, understanding the complexity of any system is pressing to avoid any environmental ensue on a regional or global scale (roberts et al. 2002) . conceptual models assist in envisioning the complex interactions in a simplified manner and recognize priority areas for the implementation of necessary management strategies. our conceptual model explicitly illustrates the impacts of covid-19 and lockdown/restriction -directly or indirectlyagainst the pandemic on the various elements of the intricate and coupled humanenvironment system and/or the feedbacks amongst them as described below: as depicted in the model, covid-19 has a direct negative impact on human health owing to its high risk of infection and death. the fatal impact of covid-19 on global human health has surpassed the number of infections and deaths caused by its ancestors and is still accelerating (bogoch et al. 2020; chan et al. 2020; lin et al. 2020; wu et al. 2020) . grech (2020) suggested that the pandemic may lead to half a billion deaths i.e., ~ 6% of the global population or more; likely due to the absence of a concrete approved treatment to combat the pandemic (singhal 2020). the pandemic has led to the increased generation of tons of medical/healthcare wastes several folds when compared to before the disease outbreak (adb j o u r n a l p r e -p r o o f journal pre-proof calma 2020; isdm 2020; saddat et al. 2020) . as highlighted in the model, increased generation of medical wastes, in turn, led to unmanageable medical waste triggering other human health risks (alverson 2020; jiangtao and zheng 2020; zambrano-monserrate 2020). for example, health-& sanitation workers, rag pickers, trash cleaners, etc. are at high risk of infection due to close contact with the patients and/or unmarked medical wastes such as discarded masks, gloves, etc. (mallapur 2020; saddat et al. 2020 ). in addition, the model also depicts the existence of feedback between covid-19 and health workers. for instance, while the health workers are at the front line of the pandemic outbreak, their exposure to the virus has put them at the risk of infection and death, coupled with other health risks such as fatigue, occupational burnout, psychological distress, etc. making them vulnerable to the current crisis. as of april 12, 2020, the who reported that the pandemic has already hit over 22,000 health workers across 52 countries (the economies times 2020a). on the contrary, health workers stand as an important potential barrier to minimize the risk of covid-19 infections and death as feedback and contribute largely to the wellbeing of the global public. the pandemic has plunged the entire world into a looming global economic recession (corlett et al. 2020; giles et al. 2020; ozili and arun 2020) . as depicted in the model, the the model depicts a direct negative impact of lockdown on human society in terms of inducing poverty and food insecurity/crisis. however, the impact of lockdown on the poor and wealthy sections of society has been disparate. for instance, while the period of total lockdown has been easier for the rich and middle-class society to pull through with assured incomes, health insurance, adequate spaces at home to maintain physical distancing & running water supplies, daily sustenance of the weaker section of the society has been very miserable. insecure sources of income for billions of the poor people worldwide due to loss of jobs amid lockdown as discussed in section 4.1.2 have severely affected their livelihoods pushing them towards extreme poverty. in addition to the instability of food availability due our model highlights both positive and negative impacts of lockdown due to the limitedtransportation and movement. the model depicts the positive impact of lockdown through limiting movement/transportation, thus ceasing all events -social, political, sports, academic, and other gatherings (breeding grounds of the virus) to maintain social distancing and curtail the disease transmission as mandated by government authorities worldwide (financial express 2020). more than 3.9 billion people or half of the world's population are currently under containment (sandford 2020) . consequently, the lockdown has posed a negative impact on the psychological resilience of people (homes et al. 2020; li et al. 2020; qiu et al. 2020; wang et al. 2020) . however, it is worth mentioning that homestay due to limited movement may likely bring a positive impact in terms of enhancing the family bonding. as shown in the model, paralyzed transportation due to lockdown has negatively impacted the global economic recession. for instance, lockdown against covid-19 has caused the shutdown of national borders in nearly 100 countries which have dropped air travel by 96% j o u r n a l p r e -p r o o f journal pre-proof (wallace 2020) , causing a loss of us$ 113 billion to the air travel industry (appleton 2020). the tourism industry has also come to halt, causing 1% global economic contraction as reported by the un (the statesman 2020). similarly, the lockdown has also hit the railway industry in terms of financial loss as reported by western railways, govt. of india (the economic times 2020). limited transportation has also caused unemployment and job insecurity for people involved in the private transport sector, which would ultimately affect their psychological resilience. . esa also reported the changing density of harmful gases emitted due to fossil fuel burning (child 2020) . in addition, the carbon emissions in china, the epicenter of the covid-19 pandemic, has dropped by ~ 25% over four weeks at the beginning of 2020 (child 2020) . in northern india, the residents now could view the himalayan mountain range due to increased visibility, which otherwise has been concealed by pollution for ~ 30 years (child 2020) . the air quality index (aqi) in delhi and ncr's, india, has reduced to 60, which otherwise scores an aqi of 600 during the smoggy winter months (the economic times 2020). while in venice, italy, restrained tourism industries have improved the water quality of winding canals (child 2020) . moreover, paralyzed transportation has also plummeted environmental noise due to the slowdown of traffic (gibney 2020, schuster 2020). all these factors are likely to contribute to the flourishing and liberation of biodiversity and wildlife. our model also shows an indirect positive impact of improved air quality in containing the disease transmission and death. air pollution is known to have a strong association with a high incidence of various respiratory infections (cipolla et al. 2018; silva et al. 2014; zhang et al. 2020) , and higher mortality rates (lelieveld et al. 2015) . evidence shows high cases of covid-19 in highly polluted areas of china, italy, and the united states, the countries with higher cases (pansini and fornacca 2020) . in addition to the impact of covid-19 in terms of infection and death as discussed in section 4.1, extended lockdown and stay-at-home regulations against the pandemic has associated human health risks such as weight gains due to sedentary lifestyle, psychological/behavioral changes, etc. (lippi et al. 2020) parivaar, govt. of india, https://www.gangaaction.org/actions/issues/solid-waste/). all these activities are very likely to contribute to flourishing biodiversity/wildlife. the shutdown of offices, business centers, industries, and other workplaces due to the pandemic (muhammad et al. 2020, richards and rickard 2020) has both negative and positive impacts on the ches as identified in the model. nickle (2020) suggested that 86% of industry members such as the growers, shippers, retailers have been reported to be affected due to the lockdown. this instigated a high negative impact through a looming global economic recession, leading to unemployment/job insecurity and lower psychological resilience. on the contrary, the model also identifies the positive impact of the shutdown of workplaces on the natural environment. for example, reduced generation of industrial and commercial wastes contributed to the improvement of air quality, creating a similar situation like halted transportation as discussed in section 4.3. in addition, the shutdown of the industrial sector that releases a huge quantity of pollutants has contributed to improving the air and water quality as identified in the model. for some years now, the river ganga and yamuna of india were considered amongst the most polluted water bodies. however, due to the lockdown, the rivers have been reported to appear cleaner and brighter owing to the temporary shutdown of the chemical industries; the major source of river pollution in india (times of india 2020; the economic times 2020c). all these factors are likely to contribute to enhancing the overall environmental quality and flourishing biodiversity/wildlife. benefits for the wildlife in terms of higher reproductive success, less migration, and lower mortality rates (ro 2020) . seismologists suggest that such noise reductions have resulted in less seismic noise, or vibrations in the earth's crust by ~ one-third compared to pre-lockdown levels (ro 2020) . moreover, decreased noise in oceans due to halted cruises is likely to decrease the production of stress hormones in sea fauna (koren 2020) . as shown in the model, humans are also likely to be beneficial given the harmful physical effect of chronic noise such as high stress, disrupted sleep, high blood pressure, cognitive impairment in children, hearing loss, heart disease, etc. (ro 2020). owing to the indirect positive impact of lockdown on the natural environment as discussed above, nature is reviving, thereby contributing to flourished wildlife and biodiversity as depicted in the model. due to the improved water quality in rivers, many of the rare animals have been spotted in the places not seen earlier. for example, the indian gangetic dolphins (one of the four freshwater dolphin species in the world; an iucn endangered species) could be spotted more in vikramshila gangetic dolphin sanctuary, bihar, india, as a result of limited human activity along the river ganga due to lockdown (khan 2020) . the presence of dolphins is a bio-indicator of a healthy river ecosystem (khan 2020) and hence, the lockdown in human society has turned to be a 'blessing in disguise' for the dolphins. elsewise, degradation of water quality in indian rivers due to dumping of municipal and industrial wastes is known to have a negative impact on human health and other aquatic fauna (ganga action plan, govt. of india, https://www.gangaaction.org/actions/issues/solid-waste/). das (2020) reported mass nesting of the endangered olive ridley sea turtles at rushikulya rookery in odisha, india due to the shutdown of touristic activities. in thailand, restriction of fishing and touristic activities has favored the increased spawning of rare leatherback sea turtles (a vulnerable iucn species) (the guardian 2020). basu (2020) reported that restricted j o u r n a l p r e -p r o o f journal pre-proof transportation due to nationwide lockdown has led to flourishing bird diversity in kolkata, india. our model identified the negative impacts of lockdown on education. for instance, government authorities worldwide have mandated shutdown of academic sectors including schools, colleges, and universities in 166 countries to maintain social distancing to curtail the transmission (corlett et al. 2020) . as a result, more than 1.52 billion students, and nearly 60.2 million teachers are no longer in the classroom (un 2020), postponing and/or canceling of examinations and subsequent delay in graduating. nevertheless, the lockdown of academic institutions has provided an opportunity and platform for digital education and online/elearning. yet, the negative impacts of lockdown on education is more overpowering on its positive aspect. on the other hand, we identified both positive and negative impacts of lockdown on research. for instance, the lockdown has frozen many laboratories, and field-based research, thus halting the ongoing research and instigation of new research as well (corlett et al. 2020 ). moreover, travel restrictions have canceled and/or postponed many national and international conferences, meetings, and research stays. yet, the lockdown has also provided ample time for research and innovations (paital et al. 2020) , and to collaborate among the experts globally. given the complexity of ches, the emergence of unprecedented uncertainties is inevitable for a pandemic like covid-19 which was not evidenced in the last century (who 2020). uncertainties mainly arise due to limited current knowledge about the virus and how people j o u r n a l p r e -p r o o f journal pre-proof across the globe modify their behaviors in response to the pandemic (chater 2020) . hence, uncertainty can accelerate fear, stress, panic, anxiousness, loss of trust amongst human society, making us powerless over the direction of life (robinson and smith 2020; who 2020) . in absence of the covid-19 vaccine, it is uncertain if the virus will flare up as restriction gets relaxed; or, whether the lockdown & personal safety measures in addition to contact-tracing and testing, might be able to stamp it out. over time, the ongoing restrictions due to the current crisis are likely to surge the uncertainty over the various elements of human society such as the economy, health, psychological well-being, etc. as discussed in section 4.1.1, governmental intervention such as strict lockdown has potentially impeded the spreading of the virus saving billions of lives worldwide. however, this has come at the cost of doomed economy and income loss due to the shutdown of industry and transportation sectors, etc. as discussed in section 4.1.2, creating a 'health-wealth trade-off'. in the opposite scenario, the relaxation of lockdown would involve a reverse trade-off where the economy recovers at the expense of an increased threat of mass contamination and death. it is therefore uncertain or unclear to decide the best policy measure yet as covid-19 would take a toll either way. this scenario is likely to be worse especially in the developing countries due to the differences in population structure, fiscal capacity, healthcare capacity, higher prevalence of "hand-to-mouth'' households, and the size of the informal sector (alon et al. 2020) . herein, the quantification of trade-offs for a better understanding of the current crisis is urgent for the decision-makers to develop effective policy measures that account for different resource allocation strategies (daher and mohtar 2015) . such quantitative analyses may also assist in understanding how the different policy response priorities to the current pandemic should differ in the developed and developing nations (alon et al. 2020) . another important instance worth mentioning amid the current crisis is the differential scenario of water security in the developed and developing world. until the discovery of j o u r n a l p r e -p r o o f journal pre-proof medicine or vaccine to control the disease, the who has recommended basic protection measures such as the frequent washing of hands and the use of face masks and hand gloves to curtail/spread the virus infection, in addition to maintaining social (physical) distancing. while frequent hand washing is well-practiced in the rich/developed societies/countries having better availability of safe water, millions of people especially in the poor countries/societies are highly susceptible to the virus due to the lack of safe water supply (ndaw 2020). however, in a world with the frequent outbreak of similar pandemics, it is uncertain if only the countries/communities with low/no access to safe water would be affected more. there is a high chance of water scarcity due to water overuse for domestic and hygiene-related practices to prevent or suppress the potential pandemics throughout the world irrespective of its economic stability (kumar 2020b; rohila 2020) . the abatement of air pollution due to lockdown is another instance with high uncertainty. although the emission of co 2 has reduced due to the lockdown/restrictions as discussed in section 4.1.4, such short-term drops in gas emissions are likely to have very little impact on the overall co 2 concentration in the atmosphere given its relentless pile-up and longer residence in the atmosphere. as mentioned in section 4.1.9, flourished biodiversity due to governmental interventions has been one of the bright sides of the current crisis for the natural environment. however, it is uncertain if such liberation would be favorable for the wildlife in the long run. as wild animals venture into settlement areas and human-modified landscapes in search of food and resources, unexpected threats such as human-animal conflicts might arise. this may favor the poachers and opportunistic delinquents as the lockdown prevails, ultimately leading to conservation threats. in addition, the toll on the global economies might also reflect in reduced funding for wildlife conservation and management. illegal encroachment of forested lands is also likely to arise when the forest officials split their time between regular duties and j o u r n a l p r e -p r o o f journal pre-proof helping out with the covid-19 situation. another instance could be the risk for biodiversity and natural habitat by forcing poor people to adopt hunting and illicit lopping of trees for daily livelihood sustenance as a result of the loss of employment as mentioned in section 4.1.7. in-depth and country-tailored assessment & quantification of such uncertainties through developing multiple scenarios for both the developed and developing nations is therefore important to better understand the long-term impacts of such crises on ches. future studies may consider quantifying the trade-offs and uncertainties of the current crisis on ches to assist the decision-makers for better policy-making process. overall, our study demonstrates how the different policy actions i.e., lockdown, socialdistancing, quarantine, etc. against covid-19 has changed the way ches interacts and influences each other. although the crisis has instigated severe ill impacts on human society, it has proved to be a 'blessing in disguise' for the natural environment as discussed in sections 4.1.6, 4.1.8, 4.1.9. restricted/regulated anthropogenic interventions against covid-19 have given rise to a better natural environment for which governments and scientists worldwide have been investing time and money for decades. on the contrary, the current situation urges for the adoption of preventive measures to mitigate ill effects faced by human society as discussed in sections 4.1. 1-4.1.5, 4.1.7, & 4.1.10 . however, the recent situation does not reflect sustainable earth, given the overpowering negative impact of the current crisis on human society. thus, even if the natural environment is benefited due to the ongoing crisis, several uncertainties persist as discussed in section 4.2. the situation is likely to reverse once the lockdown is withdrawn, and both the sub-systems would be affected -the worst-case scenario. over the centuries, humans have imposed dazzled pressure on nature and experienced its repercussions from time to time. thus, in both cases, the benefits are shortterm, which is likely to lead to a pessimistic future where both the sub-system of ches is negatively affected. thus, with a prolonged crisis, it is likely that the aspects of ches would j o u r n a l p r e -p r o o f give rise to several trade-offs and many more uncertainties as discussed in section 4.2. our study, therefore recommends that new policies should aim to convert the short-term gains into long-term benefits, creating an optimistic post-coronavirus world, beneficial for both the sub-systems. given the fact that the human sub-component of ches is experiencing the worst impacts of the crisis, policy actions should primarily focus on mitigating the ill impacts faced by human society. in this regard, our model could help identify the priority areas to understand the vulnerability of the current situation. however, incorporating the perspective of the local stakeholders and policymakers is important while developing a conceptual model. this allows covering the gaps and variations in external political and economic forces on the regional and local environment, which is a limitation of the present study given the sensitivity of the current crisis. the covid-19 crisis might be an 'eye-opener' for humanity. the current predicament reflects that nature has the potential to revive itself given that the anthropogenic interventions are checked. therefore, it is high time to understand and appreciate the complexity of ches and adopt appropriate measures towards tackling the current crisis while maintaining harmony with nature. the main purpose of the study was to propose a conceptual model to portray and address how the interaction of the existing elements of both sub-components of ches -human society and natural environment -are impacted by the various governmental interventions i.e., lockdown, social distancing, quarantine, etc. towards combating the crisis. the merit of our model is that it comprises all possible elements of ches and provides an explicit impression of complex ches amid the crisis. the proposed conceptual model provides an insight into the intricate linkage of ches and helps in understanding the j o u r n a l p r e -p r o o f journal pre-proof complex nexus by identifying the route of short-term impacts of covid-19 measures. thus, our model may be considered as a baseline for further studies and may serve as a precursor towards building quantitative modeling. the model thus may aid in policymaking by identifying the priority areas for discussion and planning in similar other crises as well. future studies may focus on forecasting the long-term impacts of the current crisis through developing scenarios considering the different components of the ches identified in the present study. the first author would like to acknowledge the ipbes and bpbes experts from the 'são dutheil, f., baker, j.s., navel, v., 2020. covid-19 as a factor influencing air pollution?. j o u r n a l p r e -p r o o f how should policy responses to the covid-19 pandemic differ in the developing world? 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indirect effects of covid-19 on the environment short-term effects of ambient pm1 and pm2.5 air pollution on hospital admission for respiratory diseases: case-crossover evidence from shenzhen, china j o u r n a l p r e -p r o o f key: cord-340971-e42g37la authors: lehrer, robert i.; bevins, charles l.; ganz, tomas title: defensins and other antimicrobial peptides and proteins date: 2007-05-09 journal: mucosal immunology doi: 10.1016/b978-012491543-5/50010-3 sha: doc_id: 340971 cord_uid: e42g37la nan endogenous antimicrobial peptides are widely distributed among vertebrates. most are amphiphilic, polycationic molecules with an α-helical, cystine-stabilized β-sheet, or proline-rich structure. they represent elements of a robust, ancestral animal immune system that predates the advent of lymphocytes and immunoglobulins. secreted and cell-associated antimicrobial peptides enable their hosts to resist incursions by potential pathogens. from a pathogen's perspective, these peptides present a series of barriers to evade or overcome. in humans (and other mammals), defensins and cathelicidins are the principal antimicrobial peptides of neutrophils and epithelial cells. many mucosal surfaces are bathed by antimicrobial proteins, including lysozyme, lactoferrin, secretory leukoprotease inhibitor (slpi), and secretory phospholipase a2. all defensins have a largely β-sheet structure and contain three intramolecular cystine disulfide bonds. the smallest, θ-defensins, are circular peptides that contain 18 residues, α-defensins have between 29 and 35 residues, and β-defensins have up to 45 (fig. 6.1) . to date, more than 100 α-defensin, β-defensin, and θ-defensin molecules have been reported, and humans express six different α-defensins and at least four βdefensins. additional human defensin/defensinlike genes exist in five different chromosomal loci, but their expression and properties remain to be characterized. α-defensins occur in the neutrophils of humans, rats, rabbits, guinea pigs, and hamsters, but not in those of mice, horses, pigs, and sheep. although the small intestinal paneth cells of mice express at least 6 selsted et al. 1992 ) and possibly as many as 20 α-defensins (ouellette and selsted 1996) , only two very dissimilar defensin species are found in human paneth cells (mallow et al. 1996) . two atypical α-defensins exist in rabbit kidney, but their functions remain uncertain (bateman et al. 1996; wu et al. 1998) . thus far, with the exception of rabbit alveolar macrophages, mononuclear phagocytes are not known to produce appreciable amounts of α-defensins. although many human epithelial cells constitutively express human β-defensins (hbd-1), this peptide is particularly abundant in the distal renal tubules and in other parts of the genitourinary tract. hbd-2, originally recovered from psoriatic skin (harder et al. 1997) , is induced in various epithelia by inflammatory signals. in the epidermis, it is present in very low amounts unless induced by interleukin-1 (il-1) .the hbd-3 gene was found by genomic searching (jia et al. 2001) and the corresponding peptide was isolated from psoriatic skin (harder et al. 2001) . hbd-4 is highly expressed in the testis (epididymis) and less abundantly in the gastric antrum (garcia et al. 2001b) . since about 50 different β-defensin genes have been identified in the mouse (scheetz et al. 2002) , sorting out their functions and relationships to human biology is unlikely to be a simple task. through its interaction with this chemokine receptor (yang et al. 1999) . the θ-defensins, initially discovered in rhesus macaque neutrophils, have a remarkable structure, so far unique among mammalian peptides. these 18-amino acid (aa) peptides with three intramolecular disulfide bonds have a cyclic amide backbone (tang et al. 1999; leonova et al. 2001) , formed from two nonapeptides by a novel posttranslational ligation reaction. the nonapeptides are encoded by two θ-defensin genes very similar to conventional α-defensins except that they are shortened by a "premature" stop codon, and contain only three cysteines. either homodimers or heterodimers can be joined to form a mature θ-defensin peptide. many different defensin genes have been sequenced. the myeloid α-defensin (defa) genes have similar layouts, with three exons that correspond approximately to the 5′-untranslated region, signal sequence/propiece, and mature defensin region (linzmeier et al. 1993) .the genes for hd-5 and 6, αdefensins expressed predominantly within the human intestine, have only two exons as do the defb genes that encode β-defensins, suggesting that the evolution of α-defensins involved both deletional and insertional events. although dissimilar in their genomic sequence, the genes for hd-5 and hd-6 show considerable sequence similarity in their proximal 5′-flanking regions, which presumably contain elements involved in their tissue-specific expression (mallow et al. 1996; salzman et al. 2003) . the defa genes encoding human myeloid and enteric α-defensins are clustered on human chromosome 8 (linzmeier et al. 1999) . the human defb genes that encode hbd-1, hdb-2, and hdb-3 contain two exons and are all within a few hundred kilobases from the myeloid α-defensin locus on chromosome 8p23 (liu et al. 1997; peng et al. 2001) . this region is also home to a deft pseudogene that is highly homologous to the genes encoding the α-defensins of rhesus monkeys. θdefensin (deft) genes evidently arose in an old world monkey when the mutation of a preexisting α-defensin gene placed a premature stop codon within exon 3 of a defa gene. additional defensin and defensinlike genes have been identified in four other chromosomal loci, but their tissue expression and biology remain to be characterized . a review of the antimicrobial activity of defensins against gram-positive and gram-negative bacteria is available (lehrer et al. 1993) . briefly, defensins are effective broadspectrum microbicides, especially when present in high local concentrations or acting in low-ionic strength media. high local concentrations occur within phagolysosomes, in the capillary-like lumen of small intestinal crypts, and at interfaces between effector and target cells. rabbit defensins np-1 and np-2 bind with high affinity to pseudomonas aeruginosa pao1, forming small surface blebs and permeabilizing its outer membrane. this process, sometimes called "self-promoted uptake" (sawyer et al. 1988) , allows the polycationic peptide molecules to displace divalent cations that link adjacent lipopolysaccharide (lps) molecules. the consequent permeability changes allow otherwise excluded molecules, such as lysozyme, to enter the periplasmic space and attack the bacterium further. viljanen and coworkers (viljanen et al. 1988 ) noted that human defensins increased the outer membrane's permeability to hydrophobic probes (e.g., rifampin) in p. aeruginosa, escherichia coli, and salmonella typhimurium. bactericidal concentrations of hnp-1 permeabilized the outer and inner membranes of e. coli, causing immediate and simultaneous cessation of macromolecular synthesis and respiration (lehrer et al. 1989a) . permeabilization of microbial cell membranes is a hallmark of defensin-mediated antimicrobial activity. especially with human defensins, such permeabilization is prevented by treatments that inhibit the target cell's metabolism, growth, or transmembrane protonmotive force (lehrer et al. 1989a) . defensins permeabilize artificial phospholipid membranes only when a transmembrane electronmotive force of sufficient magnitude and correct polarity is applied (kagan et al. 1990 ). membrane conductance increases as the second to fourth power of the defensin concentration, suggesting that two to four defensin molecules interact to form a channel in this system. wimley and associates (wimley et al. 1994) examined interactions between human defensins and lipid bilayers. they found that hnp-2 (net charge, +3) bound to and permeabilized unilamellar vesicles composed of anionic phospholipids, but not those formed of electroneutral phospholipids. data from experiments with entrapped markers of chapter 6 defensins and other antimicrobial peptides and proteins 97 fig. 6 .2. a dimer of human α-defensin hnp-3. the figure is based on coordinates obtained from x-ray crystallography (hill et al. 1991) . different sizes suggested that multimeric hnp-2 formed aqueous pores with a maximum diameter of ~25 å. if such pores formed in bacterial membranes, they should allow passage of defensin monomers and even dimers (~26 × 15 × 15 å) to more interior sites. certain bacteria are intrinsically resistant to defensins, including neisseria gonorrhoeae (qu et al. 1996a) , burkholderia cepacia, burkholderia pseudomallei brucella spp. (martinez de tejada et al. 1995) . what can make bacteria resistant has been partially explained by identifying mutations that increase bacterial susceptibility to antimicrobial peptides. studies on the intracellular enteric pathogen s. typhimurium revealed the importance of the two-component regulatory system, phop/phoq. phoq is a magnesium-binding sensor kinase that responds to ionic and other environmental changes by phosphorylating phop, a dna-binding protein that can activate numerous genes, including some needed for resistance to antimicrobial peptides and intracellular survival (miller et al. 1990; chamnongpol et al. 2003) . these include important aspects of lps structure (ernst et al. 2001) , and expression of an outer membrane protease that cleaves, and presumably inactivates, certain antimicrobial peptides (guina et al. 2000) . studies with staphylococcus aureus associated its relative resistance to defensins and other cationic antimicrobial peptides to the modification of its membrane phospholipids with l-lysine (kristian et al. 2003) and of its lipoteichoic acids with d-alanine (collins et al. 2002) . both modifications reduce electrostatic binding of the peptides to the staphylococcal surface. n. gonorrhoeae may owe its defensin-resistance to an energy-dependent "mtr" efflux pump (shafer et al. 1998) . these findings suggest that interactions with cationic antimicrobial peptides may have profoundly influenced bacterial evolution. in summary, the effect of defensins on microbes can be envisioned to occur in stages. electrostatic adsorption to anionic sites on or near the target cell's membrane or (for certain defensins) to sugars in microbial glycoproteins or lps result in locally high concentrations that promote defensin aggregation and multimer formation. insertion of defensins into microbial membranes is assisted by their positive charge and by the target cell's transmembrane potential. the ensuing membrane disruption and channel formation permit intracytoplasmic entry of defensins and allow essential microbial components to leak out. unless repaired, these events lead to irreversible target cell injury. mammalian defensins can kill various fungal pathogens, including candida spp., cryptococcus neoformans, and hyphae and germinating spores of rhizopus oryzae and aspergillus fumigatus (reviewed in lehrer et al. 1993) . in recent years, the antifungal properties of defensins have been studied more by botanists and agriculturists than by immunologists. this reflects the importance of fungi as plant pathogens (thomma et al. 2002) and the likely utility of transgenic plants. plant defensins, like those of mammalian origin, probably owe their antifungal properties to an ability to induce membrane permeabilization (thevissen et al. 1999 ). both human and rabbit defensins show activity against mycobacteria, including m. tuberculosis (miyakawa et al. 1996; sharma et al. 2000) and m. avium-intracellulare (ogata et al. 1992) , even in the presence of divalent cations or physiologic salt concentrations. although the plasma membrane of m. tuberculosis is the principal target for hnp-1 action, its dna may be a secondary target (sharma and khuller 2001) . human neutrophils can ingest and kill both m. avium (hartmann et al. 2001) and m. tuberculosis, and the latter is apparently subdued by nonoxidative mechanisms (kisich et al. 2002) . in vivo, mycobacteria that evaded or survived the attentions of neutrophils would likely find a defensin-free sanctuary once resident within a macrophage, because the only mammalian macrophages known to contain significant quantities of defensins are alveolar macrophages of the rabbit. in a rabbit model of syphilis, large local amounts of defensins were seen during the first 24 hours. this subsided, but reappeared on days 10 to 16, when healing began (borenstein et al. 1991) .the nichols strain of treponema pallidum was neutralized by rabbit defensins in vitro and in vivo. borrelia burgdorferi, the causative agent of lyme disease, is also susceptible to defensins (lusitani et al. 2002) . the effect of defensins on protozoans has received little study, at least in vertebrates. human α-defensin hnp-1, mouse paneth cell defensin-2 and defensin-3 (cryptdins), and rabbit α-defensin np-2 killed trophozoites of giardia lamblia, especially when the concentrations of sodium chloride and divalent cations were low (aley et al. 1994 ). both α-defensins (zhang et al. 2002) and θ-defensins (cole et al. 2002) can protect cells from infection by hiv-1 in vitro. persons infected with human immunodeficiency virus-1 (hiv-1) whose α,β cd8 t cells maintain the ability to produce and release α-defensins may exhibit relative resistance to developing acquired immune deficiency syndromedefining (aids-defining) symptoms, compared with hivinfected subjects whose t cells are defective in this regard (zhang et al. 2002) . θ-defensins act by blocking an early step in the uptake process, and their ability to do so correlates with their ability to bind gp120 with high affinity (cole et al. 2002) . α-defensins also bind gp120 with high affinity, but their mechanisms of action against hiv-1 has not yet been defined. α-defensins also protect cells from infection by herpes simplex virus (hsv) types 1 and 2 in tissue culture media (daher et al. 1986 ), by interfering with an early step in the infection process (sinha et al. 2003) . human and rabbit α-defensins also neutralized vesicular stomatitis and influenza a/wsn virus, but lacked significant activity against cytomegalovirus, reovirus, and echovirus (daher et al. 1986 ). among the nonenveloped viruses, only adenoviruses have so far been reported to be susceptible to defensins (gropp et al. 1999; bastian and schafer 2001) . purified human defensins kill various normal and tumor cell targets in a concentration and time-dependent fashion (reviewed in lehrer et al. 1993 ) and show synergistic activity when combined with sublytic concentrations of hydrogen peroxide. defensin molecules bound to mammalian target cells with biphasic kinetics, similar to those with candida albicans. k562 cells exposed to 20 μg/ml of radiolabeled hnp-1 bound approximately 4.3 × 10 9 defensin molecules per cell. this initial binding did not cause cytolysis and was comparable in defensin-resistant and defensin-sensitive target cells. after 5 to 10 minutes, sensitive targets became permeable to trypan blue (molecular weight = 960 da) and manifested enhanced transmembrane ion flux. membranebound defensin molecules became progressively more difficult to dislodge by adding serum, and after 3 to 4 hours, the cells began releasing 51 cr-labeled cytoplasmic components. strand breaks and adenosine triphosphate-ribosylation (adp-ribosylation) were first detected in k562 and raji targets 6-8 hours after incubation with hnp, and these increased to maximal levels by 18 hours. dna was not degraded into nucleosome-sized fragments. other experiments suggested that the initial defensin-induced pores were voltage dependent and that defensin-mediated injury to mammalian cells depended on an energized target cell membrane. since defensins are bound by α2-macroglobulin and other normal serum components, it is not surprising that defensins were minimally, if at all, cytotoxic in the presence of serum. whether and where defensins are cytotoxic in vivo is not known. although most studies of defensins have examined their antimicrobial properties, considerable evidence suggests that some defensins also play other roles, including immunomodulation, hormonal regulation, opsonization, and stimulating wound repair. human neutrophil defensins are chemotactic for human monocytes (territo et al. 1989) , t-cells (chertov et al. 1996) , and immature dendritic cells (yang et al. 2000) in vitro, so that their release from neutrophils could provide a signal to mobilize immunocompetent mononuclear cells. defensins also induce the synthesis of interleukin-8 (il-8), a c-x-c cytokine, by human airway epithelial cells, possibly providing a mechanism to recruit additional neutrophils to sites of inflammation (van wetering et al. 1997) .the α-defensins of human neutrophils were shown to act as adjuvants to enhance systemic igg antibody response (lillard et al. 1999) . murine β-defensin mbd-2 acted as an adjuvant for tumor immunization, by providing a costimulatory signal via the toll-like receptor 4 (biragyn et al. 2002) . it is not yet clear whether these activities are shared by other defensins. rabbit np-3a ("corticostatin") and certain other α-defensins bound reversibly to the adrenocorticotropic hormone (acth) receptor of rat adrenal cells in vitro and inhibited acth-stimulated steroidogenesis (zhu and solomon 1992) . rabbit defensins np-1 and np-2 greatly enhanced the ability of rabbit alveolar macrophages to ingest bacteria and fungi under serum-free conditions (fleischmann et al. 1985) . defensins were mitogenic for fibroblasts (murphy et al. 1993) , and small daily injections of rabbit defensins accelerated wound healing in rats (kudriashov et al. 1990 ). guinea pig defensins were reported to release histamine from rat mast cells in vitro (yamashita and saito 1989) . most normal mucosal surfaces contain considerably more epithelial cells than neutrophils or macrophages. notable exceptions include the gingival crevices that surround teeth, which are bathed in high concentrations of neutrophils and their components. neutrophil defensin concentrations as high as 1 mg/ml have been detected in such fluids (t. ganz and k. miyasaki, unpublished) and could play an important role in controlling local microbial proliferation. in other locations, neutrophils and macrophages are rapidly recruited to epithelial surfaces when the primary mucosal defenses are overwhelmed by microbial invaders. the important function of these secondary defenses is illustrated by the particularly severe impact of neutropenia on the integrity of oral and gastrointestinal mucosae and the characteristic involvement of periodontal tissues in congenital or acquired neutrophil disorders. each human neutrophil contains several thousand cytoplasmic granules that are divided into three principal subtypes, called primary (or azurophil), secondary (or specific), and tertiary. these subtypes can be distinguished by buoyant density, when they are made during the neutrophil's maturation, their response to secretagogues, and their composition. about 30% to 50% of the protein content of the azurophil granules consists of defensins. relatively small amounts of defensins are released extracellularly after phagocytosis or secretagogue exposure, as the peptides are preferentially delivered phagosomes that contain opsonized bacteria (joiner et al. 1989 ). this targeted delivery of defensins not only places them where they are most needed, but it also helps minimize any potential cytotoxicity to host tissues. the high concentrations of defensins in human neutrophils are not unique: α-defensins are similarly plentiful in the granules of rabbit neutrophils (~10-15 μg/million cells) and rabbit alveolar macrophages (~2 μg/million cells). although myeloid defensins (hnps) 1-4 are most abundant in neutrophils, hnp-1 is also highly expressed by natural killer (nk) cells (obata-onai et al. 2002; agerberth et al. 2000) and can be produced by other lymphocytes (agerberth et al. 2000) . although human blood neutrophils contain about 5 μg of defensins per million cells, defensin mrna is not detectable in northern blots of mature neutrophils, indicating that defensin synthesis is restricted to the neutrophil's bone marrow precursors. myeloid α-defensins are synthesized as 90-95-aa prepropeptides with a generally well-conserved signal sequence, followed by an anionic propiece and the cterminally placed defensin domain. nevertheless, nearly all of the defensin in neutrophil azurophil granules is found to be completely processed to 29-30-aa mature peptides . posttranslational defensin processing has been studied in metabolically labeled hl-60 and cml cells (valore and ganz 1992) . their earliest defensin intermediate contained 75 aa and arose by cotranslational removal of the 19-residue signal sequence. considerable quantities of this 75-aa form were secreted into the medium, and the remainder was proteolytically processed over 20 hours via a 56-aa intermediate into the mature 29-aa and 30-aa defensins. mature human neutrophils also contained minor amounts (0.25% of total defensins) of incompletely processed 39-aa, 34-aa, and 32-aa defensins (harwig and ganz 1992) . this pattern of synthesis and posttranslational processing was substantially reproduced when the hnp-1 defensin cdna was transduced into defensinless murine 32d and 32d cl3 cells (liu and ganz 1995) . these transgenic lines accumulated mature defensin in acidified vacuoles corresponding to lysosomes or immature granules. in contrast, two transgenic, defensin-producing nonmyeloid cell lines (an embryonic nih 3t3 cell line and the pituitary adenoma cell line att-20) failed to process the 75-aa prodefensin, indicating that the requisite processing pathway may be tissue specific. to study the role of the propiece in preprohnp-1, a series of in-frame deletions between the signal peptidase site and the aminoterminus of the mature defensin region (aa 21-64) was constructed, packaged, and transduced into the 32d cl3 granulocytic cell line. deletions in the aminoterminal two-fifths of the propiece had only minor effects on defensin biosynthesis and did not interfere with accumulation of mature defensin in the granules of 32d cl3 cells. deletions in the carboxyterminal three-fifths of the propiece diminished net defensin synthesis, blocked constitutive secretion of prodefensin, and interfered with defensin accumulation in cytoplasmic granules. these effects were reproduced by the smaller deletion δ 40-51 , which contains a highly conserved secondary structure. therefore, propiece residues 40-51 appear to be essential for subcellular trafficking and sorting of human neutrophil α-defensins (liu and ganz 1995) . instead of containing α-defensins like those found in human, rabbit, rat, hamster, and guinea pig neutrophils, bovine neutrophils contain an impressive array of β-defensin peptides . whereas myeloid α-defensins have free amino termini, about half of the β-defensins isolated from bovine neutrophils had a pyroglutamate residue at their amino terminus. this moiety characteristically results from enzymatic modification of an amino-terminal glutamine residue and could confer resistance to proteolytic cleavage by local proteases or alter other biologic properties. the β-defensin peptides from neutrophils manifest antibacterial activity against gram-positive and gram-negative bacteria, but because different in vitro assay conditions were used, direct comparison of the relative activity of bovine epithelial and neutrophil β-defensins is not yet possible. β-defensins ("gallinacins") were also isolated from the leukocytes of chickens (harwig et al. 1994 ) and turkeys (evans et al. 1994) . two of the three chicken gallinacins differed at only three amino-acid residues and had comparable antibacterial and antifungal activities. the third gallinacin differed at over half of the amino acids and exerted antibacterial activity, but lacked the antifungal activity seen with the other two (harwig et al. 1994) . the β-defensins of turkeys showed good activity against c. albicans, salmonella enteriditis, and campylobacter jejuni, but were relatively ineffective against pasteurella multocida at 16 μg/ml, the highest peptide concentration tested. they did not neutralize infectious bronchitis virus, an enveloped coronavirus (evans et al. 1995) . in normal chickens, the expression of gallinacin-3 was especially prominent in the tongue, bursa of fabricius, and trachea. it also occurred in other organs, including skin, esophagus, air sacs, large intestine, and kidney. the tracheal expression of gallinacin-3 increased significantly in chickens experimentally infected with haemophilus paragallinarum ). despite continual entry of microorganisms from swallowed food and oral secretions, the normal small intestine contains only a sparse resident flora. why? although multiple factors contribute, the ability of intestinal enterocytes and paneth cells to secrete antimicrobial molecules is likely to play a significant role. through their differential antimicrobial activities, these molecules could contribute to selecting the normal intestinal flora, which may afford protection from pathogenic bacteria that enter the intestine. since the intestinal epithelium stem cells reside in the intestinal crypts, secretion of paneth cell defensins into the crypt lumen (figure 6. 3) could provide a protective barrier for this vital proliferative compartment. paneth cells, first described over 100 years ago, are located at the base of the crypts of lieberkühn. they are more abundant in the region of the ileum and jejunum and less so in the duodenum. the high density of paneth cells in the distal small intestine might signify a role in preventing adverse consequences from reflux of colonic flora into the ileum. paneth cells contain an extensive endoplasmic reticulum and golgi network and are filled with numerous, apically located eosinophilic secretory granules whose secretion is stimulated by cholinergic agonists or the entry of bacteria or lipopolysaccharide into the intestinal lumen (satoh 1988; qu et al. 1996b ). ouellette and colleagues (ouellette et al. 1989 ) identified an α-defensin among several prominently expressed rna messages in the postnatal mouse small intestine. because this mrna was expressed in paneth cells at the base of small intestinal crypts, the corresponding peptides were named "cryptdins." cryptdin mrna levels were normal in germfree and nude mice, suggesting that expression was independent of t-cell signals or acquisition of the intestinal flora. independently, two α-defensin genes (hd-5 and hd-6) expressed in human paneth cells were detected bevins 1992, 1993) . in contrast to the six characterized murine enteric defensin genes, which had very high (85%) nucleotide similarity, hd-5 and hd-6 were not as closely related, presumably having duplicated and diverged before their murine counterparts . the striking difference in murine and human enteric defensin gene numbers remains enigmatic. paneth cells also secrete larger antimicrobial polypeptides, including lysozyme and secretory phospholipase a2 (see sections on "identification of enteric α-defensins in paneth cells" and "developmental regulation of enteric α-defensin expression" in this chapter). antimicrobial activity of enteric α-defensins native mature defensins (cryptdins) have antibacterial and activity comparable with myeloid defensins (selsted et al. 1992; eisenhauer et al. 1992 ) and can kill the protozoan parasite giardia lamblia (aley et al. 1994) . recombinant human small intestinal defensin hd-5 was effective against listeria monocytogenes, s. typhimurium, and c. albicans (porter et al. 1997b; ghosh et al. 2002) . it showed minimal cytotoxic activity against human intestinal cell lines (porter et al. 1997a) . morphologically identifiable paneth cells appear in the mouse intestine immediately before birth. thereafter, murine α-defensin mrna levels and defensin immunoreactivity increase gradually and reach adult levels by the fourth postnatal week, paralleling expansion of the paneth cell population. mice reared in a germfree environment express comparable levels of enteric α-defensins (ouellette et al. 1989; putsep et al. 2000) . in humans, a sensitive rt-pcr assay can detect mrna encoding human enteric defensins hd-5 and hd-6 at 13.5 weeks' gestation, close to the time that morphologically distinguishable paneth cells can be identified by electron microscopy. at approximately 24 weeks' gestation, hd-5 and hd-6 mrna was detected in paneth cells of the small intestinal crypt by in situ hybridization. by northern blot analysis, the levels were approximately 100-fold lower than in adults. the ability to detect human enteric defensin mrna prenatally indicates that its expression is at least partially constitutive and governed by a developmental program that operates without direct stimulation by microbes or their products. like their hematopoietic counterparts, intestinal αdefensins are initially synthesized as prepropeptides. however, unlike the myeloid defensins, paneth cell defensins are stored as propeptides, exclusively so in humans (porter et al. 1998; ouellette et al. 2000; ghosh et al. 2002) . the posttranslational processing of these epithelial α-defensins appears to be an important regulatory step in the generation of bioactive peptides in the small intestine. however, the pathways of processing appear to be quite different in mice and humans. in mice, the matrix metalloprotease matrilysin (matrix metalloproteinase 7) has been identified as an essential enzyme in the processing of small intestinal α-defensins . in humans, the orthologous small intestine α-defensins are processed to mature peptides during or after secretion into the small intestinal lumen by the serine protease trypsin (ghosh et al. 2002) . in both species, the proteases mediating this α-defensin processing are expressed by paneth cells. the hypothesis that intestinal defensins contribute to the regulation of microbial flora in the small intestine received support from studies in matrilysin-deficient mice. matrilysin (matrix metalloproteinase 7) is expressed specifically in paneth cells. in mice with a targeted disruption of the intestinal prodefensin-processing protease, matrilysin, paneth cell defensin precursors were not processed to active mature peptides. the mice had increased susceptibility to intestinal infections with gram-negative organisms . detailed analysis of secretions from the isolated crypts of normal and matrilysin-deficient mice indicated that the antimicrobial activity of crypt secretions was largely the result of defensins and that it was greatly impaired in matrilysin-deficient mice (ayabe et al. 2000) . in another model, the transgenic expression of human defensin-5 in the paneth cells of mice increased their resistance to orally administered s. typhimurium but, as might be expected, did not protect against infection by the peritoneal route (saltzman et al., 2003) . in the aggregate, these models point to a central role for paneth cell defensins as regulators of small intestinal flora and as enteroprotective molecules. bovine tracheal extracts contain an abundant peptide called "tap," or tracheal antimicrobial peptide, that exhibited antimicrobial activity in vitro against klebsiella pneumoniae, s. aureus, and c. albicans (diamond et al. 1991) . sequence analysis revealed similarity of this molecule to defensins expressed in leukocytes and to its designation as the first epithelial β-defensin. tap expression was found in the pseudostratified columnar epithelial cells of conducting airway and nasal mucosa (diamond et al. 1993) . a hallmark of tap expression, like several more recently described epithelial β-defensins of humans and mice, is inducible expression in response to bacterial lps and some inflammatory cytokines russell et al. 1996; diamond et al. 2000) . a canonical nf-kb recognition site in the 5′-flanking region of the tap gene was important in the transcriptional regulation of this gene by bacterial products (diamond et al. 2000) . in vivo inoculation of pathogenic bacteria into the bovine lung caused a rapid and dramatic increase in β-defensin expression in the conducting airway epithelium, which was not seen in the adjacent control lobe . in bovine tracheal epithelial cells, β-defensin induction by lps appears to be dependent on epithelium-derived cd14, a well-characterized lps-binding protein . this suggests that local expression of cd-14 might provide epithelial cells with the capacity to recognize bacterial products at mucosal surfaces and initiate local defense responses such as antimicrobial peptide production. several additional sites of β-defensin expression were identified in cattle, including squamous epithelial cells of the tongue (schonwetter et al. 1995) and epithelial cells of the small intestine and colon (tarver et al. 1998 ). mrna encoding a bovine lingual β-defensin ("lap") was markedly increased in epithelia surrounding naturally occurring tongue lesions, suggesting an integral role for antimicrobial peptides in local inflammatory responses. the antimicrobial activity of lap and tap was very similar, and lap expression was enhanced in bovine trachea after lps or tnf-α application . in mice, epithelial expression of β-defensins is also observed in the mucosal epithelium of the respiratory and digestive tracts (morrison et al. 1999; bals et al. 1999; jia et al. 2000) . similar to humans, mice express a noninducible βdefensin in kidney tubule cells and mucosal epithelia of several organ systems (huttner et al. 1997) . recent studies of mice rendered deficient in this epithelial β-defensin through targeted homologous recombination have shown a minimal phenotype (moser et al. 2002; morrison et al. 2002) . further studies of these mice may be necessary to elucidate the functional role of this β-defensin. a systematic characterization of the peptides present in human blood ultrafiltrates provided the first evidence for the existence of hbd-1, the first characterized human β-defensin (bensch et al. 1995) . hbd-1 shared the nine invariantly conserved amino acids present in β-defensins from bovine and avian cells. although free hbd-1 was present in nanomolar concentrations in human plasma, an abundant hbd-1 message occurred in the kidney and vagina (bensch et al. 1995) . several hbd-1 peptides, generated by differential posttranslational proteolysis at the n-terminus, were the predominant cationic peptides in urine. when tested, using urine as a growth medium, they displayed antibacterial activity against e. coli. in situ hybridization and immunostaining localized their site of production to the distal tubules of the kidney as well as the various epithelia of the female genital tract (valore et al. 1998) . hbd-1 is also present in human conducting-airway epithelia (mccray and bentley 1997) as well as in epithelia of many other tissues and in certain glands (zhao et al. 1996) . expression of hbd-1 by lung epithelia is developmentally regulated, in a manner reminiscent of the α-defensins found in rabbit lung macrophages (mccray and bentley 1997) . circumstantial evidence implicates the impaired function of hbd-1, consequent to high bronchial fluid salinity (smith et al. 1996) , as a contributing factor in respiratory tract colonization by p. aeruginosa in cystic fibrosis patients (goldman et al. 1997) . hbd-2 was first detected as an abundant cationic peptide and an antimicrobial component in the flaking epidermis of patients with psoriasis (harder et al. 1997) . it is produced by differentiated keratinocytes in the epidermis under the influence of inflammatory signals, principally il-1 (liu et al. 1998 (liu et al. , 2002 and secreted in lamellar bodies into the space between keratinocytes, along with the lipids that make the epidermis impermeable to water . there, hbd-2 reaches concentrations sufficient for antimicrobial activity (liu et al. 2002) . hbd-2 is active against a broad range of microbes, with the significant exception of the skin pathogen s. aureus (harder et al. 1997; liu et al. 2002) . hbd-3 was detected more or less simultaneously by genomic methodologies (jia et al. 2001; garcia et al. 2001a) and by direct extraction of the peptide from psoriatic skin (harder et al. 2001) . hbd-3 is one of the most cationic defensins known, with a charge of +11 at neutral ph. unlike hbd-2, hbd-3 is active against s. aureus and even b. cepacia, at least under low ionic strength conditions.the skin and the tonsils appear to have the highest levels of expression, but hbd-3 is detectable in other epithelial and nonepithelial tissues as well. the mechanisms of its regulation have not yet been reported. genomic analysis indicates that many additional human β-defensin genes and related genes exist, but they remain to be characterized at the peptide level . the α-defensin and β-defensin families almost certainly arose by divergence from an ancestral premammalian defensin and not by convergent evolution. this conclusion is supported by the proximity of human α-defensin and β-defensin genes within 150 kb on chromosome 8p23 (liu et al. 1997) . defensinlike molecules not assignable to either defensin family have been identified in invertebrates. hemocytes of the horseshoe crab, tachypleus tridentatus, contain an antibacterial peptide ("big defensin") whose cterminal 37 residues have a cysteine structure resembling that of mammalian β-defensins and a primary sequence similar to rat myeloid α-defensins (saito et al. 1995) . when the unusually long, 35-residue amino-terminal extension of big-defensin was cleaved by tryptic digestion, both the defensinlike domain and the amino-terminal extension had antimicrobial properties. an extremely defensinlike antiviral peptide was also identified in the sea anemone, anemonia sulcata (driscoll et al. 1989) . the primary structural resemblance between avian βdefensins (gallinacins) and certain cytostatic (marquardt et al. 1988 ) and myotoxic peptides in snake venoms (fig. 6.4) also suggests that the progenitors of β-defensins had potential to become weapons of offense, as well as of host defense. the same can be said about the antimicrobial insect defensins, which show striking structural similarity to certain toxic peptides found in scorpion venom (cociancich et al. 1993) . given such findings, it is reasonable to postulate the antiquity of defensins as host-defense molecules. an evolutionary relationship between vertebrate defensins and the structurally more distant defensins of insects and plants is possible, but lacks convincing supporting data. calprotectin, a member of the widespread calcium-binding s-100 protein family, is present in remarkably high concentration in the cytoplasm of human neutrophils (brandtzaeg et al. 1995) . the calprotectin molecule is composed of light (mrp8) and heavy (mrp14) subunits. although not secreted from intact neutrophils, calprotectin release from dead and dying neutrophils creates high concentrations of the protein in inflammatory or abscess fluids and in the intestinal tract lumen of patients with inflammatory bowel disease. calprotectin is also found in reactive tissue macrophages, in nonkeratinizing squamous epithelia, and in reactive epidermal cells. the c-terminal portion of calprotectin's heavy chain, mrp14, is identical to a peptide called neutrophil immobilizing factor, "nif." calprotectin concentrations of 50-250 μg/ml inhibit growth by s. aureus, s. epidermidis, and e. coli. even lower concentrations (4-32 μg/ml) inhibit growth by c. albicans, possibly by depriving the candida cells of zinc. calprotectin purified from rat inflammatory peritoneal cells was markedly cytotoxic for mitogenstimulated lymphocytes and for various tumor cell lines, in fig. 6.4 . from serpent's tooth to chicken soup. myotoxin a is a myotoxic peptide from the venom of crotalus viridis viridis, the prairie rattlesnake (fox et al. 1979) . svgap stands for snake venom growth arresting peptide (marquardt 1988) , and is described in u.s. patent 4774318. gal-1 is the chicken β-defensin gallinacin-1, originally isolated from chicken heterophils (harwig et al. 1994 ) and later found in chicken soup (lehrer unpublished) . the disulfide pairing patterns (1:5, 2:4, 3:6) of myotoxin a (fox et al. 1979 ) and avian β-defensins (harwig, unpublished) are identical. which it induced apoptosis. epithelial cell calprotectin might protect host cells from microbes that invade the cytoplasm directly or by lysing phagosomal membranes. the murine mrp8 protein (also called cp10 or s100a8) is chemotactic for myeloid cells but this property is not shared by human calprotectin. homozygous disruption of mrp8 (s100a8) gene in mice causes embryonic death by day 9.5 but mrp14 (s100a9)-deficient mice are viable and healthy (manitz et al. 2003 ) even though their neutrophils lack both mrp8/14 partners. initial studies of these mice detected only very mild defects in neutrophil cytoskeletal organization and migration, but the activity of these neutrophils in microbicidal assays has not been reported. lysozyme was discovered over 75 years ago by fleming, who called it a "remarkable bacteriolytic ferment" and did not patent it-or penicillin, which he discovered a few years later. this very cationic 14.3-kda protein enzymatically attacks the β1-4 glycosidic bonds between n-acetylglucosamine and n-acetylmuramic acid, which stabilize bacterial peptidoglycans. gram-positive bacteria susceptible to the lytic action of lysozyme include bacillus subtilis, bacillus megaterium, and micrococcus luteus (nee lysodeicticus). lysozyme also exerts bactericidal activity by nonenzymatic, nonbacteriolytic mechanisms. it is a remarkably abundant component of phagocytic leukocytes, and also of tears, saliva, and many other secretions. the detection of abundant lysozyme in paneth cell granules yielded the first clue that these intestinal epithelial cells were important in host defense (peeters and vantrappen 1975) . most gram-negative bacteria are resistant to lysozyme, except under very low ionic strength conditions. this is caused largely by the protective effects of their outer membrane, which covers and masks their peptidoglycan layer. lysozyme may function most effectively in conjunction with other antimicrobial factors, especially those capable of permeabilizing the outer membrane. recent experiments in mice deficient in lysozyme-m showed delayed killing of the lysozymesensitive m. luteus, but the major abnormality in these mice was the highly exaggerated inflammatory response to these bacteria and to peptidoglycan. peptidoglycan is a potent inflammatory stimulus, in part through its binding to tlr-2 and by peptidoglycan recognition proteins. whatever lysozyme may contribute to antibacterial activity, it appears to be critically important for eliminating bacterial peptidoglycan, thus extinguishing the strong signal to innate immunity and inflammation generated by this characteristic bacterial macromolecule. lactoferrin is a 78-kda single-chain glycoprotein that can bind one or two molecules of ferric iron. it is found in various secretions, including tears, milk, and semen, and also in the secretory granules of neutrophils. intestinal cells and mononuclear phagocytes have surface receptors for lactoferrin, but do not synthesize it. certain bacteria (e.g., n. gonorrhoeae, neisseria meningitidis, and moraxella catarrhalis) also possess lactoferrin receptors and use them to acquire iron. lactoferrin or lactoferricin-treated gram-negative bacteria release lps, develop electron-dense "membrane blisters," and become sensitized to lysozyme-all consistent with outer membrane damage. lactoferrin binds bacterial lipopolysaccharide with high affinity and also binds various other proteins. exposure of lactoferrin to pepsin (its normal fate when ingested in milk) releases an antimicrobial polypeptide, "lactoferricin" comprising the amino-terminal lactoferrin residues (tomita et al. 1994) . lactoferricin kills many bacteria and c. albicans, but is ineffective against proteus and serratia species and p. cepacia. lactoferrin also inhibits microbial adherence to and invasion of epithelial cells. lactoferrin has a higher affinity for iron than its plasma homolog transferrin, and its iron binding is less affected by acid ph. these characteristics may allow it to function as an iron-sequestering substance in mucosal secretions and inflammatory fluids. because iron is an essential metal for all living organisms, sequestration of iron is an effective means of inhibiting microbial growth. iron starvation also interferes with bacterial biofilm formation. even under conditions that otherwise promote biofilm formation, lactoferrin-exposed bacteria remain in the planktonic form, presumably allowing the more mobile bacterial population to disperse and reach iron sources (singh et al. 2002) . phospholipase a 2 enzymes remove fatty acids from the middle (sn-2) carbon atom of phosphoglycerides. several phospholipase a 2 enzymes occur in mammalian cells, but only the 14-kda secretory pla 2 (spla 2 ) has potent microbicidal properties, principally against gram-positive bacteria (harwig et al. 1995; weinrauch et al. 1996) . spla 2 differs from pancreatic pla 2 structurally and in preferring phospholipids prominent in bacterial membranes [e.g., phosphatidylglycerol (pg) and phosphatidylethanolamine (pe)] over phosphatidylcholine as substrates. pla 2 is present in granules of human neutrophils and small intestinal paneth cells. spla 2 is released from neutrophils exposed to secretagogues, such as phorbol esters or calcium ionophores, and by paneth cells exposed to cholinergic agonists, bacteria, or lps. serum spla 2 levels are greatly elevated in patients with sepsis and rise sharply within 3 hours after the experimental administration of lipopolysaccharide to uninfected patients. high concentrations of spla are also present in human tears. the antimicrobial specialization of this phospholipase is probably a result of its unusually high net positive charge and a cluster of positively charged residues near its n-terminus that facilitate its interaction with bacteria and access to its phospholipid target (weiss et al. 1991) . slpi is a 107-aa (12.7-kda) nonglycosylated peptide with a bipartite structure. it is very abundant in many human secretions, including seminal plasma, cervical mucus, nasal secretions, and tears. slpi has received many alternative names, including human seminal plasma inhibitor-i (husi-i), cervix uteri secretion inhibitor (cusi), bronchial secretory inhibitor (bsi), and bronchial mucus inhibitor (mpi). mice deficient in slpi show impaired wound healing (ashcroft et al. 2000; zhu et al. 2002) , but their response to infection has not been reported yet. in vitro, slpi shows low-level activity against bacteria and fungi (hiemstra 2002) and may also have anti-hiv activity. if the high concentrations of slpi in many epithelial secretions compensate for its low intrinsic potency, its antimicrobial activity could be biologically important. slpi is synthesized as a 132-aa prepropeptide with a 25aa signal peptide. each of its two structurally similar domains contains four intradomain disulfide bonds in the "four-disulfide-core" pattern also present in wheat germ agglutinin and neurophysin.the c-terminal domain of slpi (residues 55-107) shows homology to the second domain of chelonianin, a basic protease inhibitor from red sea turtle, and is responsible for the molecule's prominent antiprotease activity (masuda et al. 1995) . slpi holoprotein and its nterminal domain (residues 1-54) have microbicidal properties (hiemstra et al. 1996) . other protease inhibitors with antimicrobial properties include aprotinin, a 58-aa protease inhibitor in bovine mast cells (pellegrini et al. 1996) , and enap-2, a four-disulfide core protein found in equine granulocytes (couto et al. 1992) . multifunctionality may be a common attribute of other proteins involved in host defense. eosinophils, as well as other myeloid and epithelial cells, contain cationic antimicrobial proteins belonging to the ribonuclease family (rosenberg and domachowske 2001) . in humans, eosinophil cationic protein (ecp) and eosinophil-derived neurotoxin (edn) are among the principal components of eosinophil granules. ecp is broadly antimicrobial (lehrer, 1989b) and toxic to helminths (hamann et al. 1987 (hamann et al. , 1990 ), but edn is nearly inactive in these assays. both ecp and edn show antiviral activity (rosenberg and domachowske 2001) against respiratory rna viruses: respiratory syncytial virus (rsv) and a related murine virus. new members of the antimicrobial ribonuclease family likely to be important in defending body surfaces include rnase 7, an abundant component of human epidermis (harder and schroder 2002) and angiogenin 4, an antimicrobial protein of murine paneth cell granules (hooper et al. 2003) . the specific role of these proteins in host defense remains to be defined. bactericidal permeability-increasing protein (bpi) (elsbach and weiss 1998) is found in the azurophil (primary) granules of human and rabbit neutrophils, but like defensins, may be absent from mouse neutrophils. it is a 55-kda cationic protein and a member of a family of lipid-binding proteins, two of which, bpi and lipopolysaccharide-binding protein (lbp) avidly bind to bacterial lipopolysaccharide. in vitro, bpi is specifically active against selected gram-negative bacteria at concentrations as low as nanomolar, and this activity is wholly contained in a 25-kda amino terminal fragment. the mechanism of activity of bpi against bacteria depends on the initial high-affinity interaction with lps in the outer membrane. the resulting rapid permeabilization of the outer membrane is followed by a slower process that culminates in the disruption of the inner membrane of gram-negative bacteria with attendant loss of viability (wiese et al. 1997 ). more recently, bpi was also identified as a component of human eosinophil granules and as a lipoxin-inducible protein in mucosal epithelia (canny et al. 2002) . in vitro studies suggest that bpi contributes to the killing of gram-negative bacteria in isolated epithelia and neutrophils. the neutrophils of humans, pigs, cattle, sheep, mice, and other mammals contain a variety of structurally diverse antimicrobial peptides collectively called "cathelicidins." these peptides were grouped together because they are synthesized at the carboxy-terminal portion of a precursor containing a highly conserved domain, approximately 100 amino acids long, called "cathelin" (zanetti et al. 1995; ganz and weiss 1997) . some of these peptides have amphipathic α-helical structures, others form compact, defensin-like β-sheets stabilized by intramolecular cystine disulfide bonds, and still others are remarkably rich in proline, arginine, or tryptophan residues.the genes for cathelicidins contain four exons, the first three of which encode most of the conserved cathelin domain. exon 4 specifies the last few cathelin residues, including the cleavage site and the mature peptide.the 5′ flanking sequences of this gene family contain motifs for binding nf-κb, il-6, gm-csf, and nf-1, suggesting that synthesis responds to mediators generated early during infection. the sole known human cathelicidin is named hcap18 or fall39/ll37 (agerberth et al. 1995; cowland et al. 1995; larrick et al. 1995) . unlike defensins, which are fully processed before they are stored in the azurophil granules of the neutrophil, the human cathelicidin peptide is stored in the specific (secretory) granules as hcap18, a cathelin-containing, 140-residue, 17-kd propeptide. during or after secretion, hcap18 undergoes proteolytic processing by proteinase 3 (sorensen et al. 2001) to liberate ll-37, the 37-residue, α-helical antimicrobial peptide at its c-terminus (gudmundsson et al. 1996) . the analogous proteolytic processing of bovine cathelicidins (probac5 and probac7) and porcine cathelicidins (proprotegrins 1-3) is mediated by trace amounts of neutrophil elastase (panyutich et al. 1997; scocchi et al. 1992) . mrna for hcap18 is found in testis (malm et al. 2000) , inflamed keratinocytes (frohm et al. 1997; agerberth et al. 1995) , and airway epithelia (bals et al. 1998) . in vitro, ll-37 displayed both lps binding and microbicidal activities (agerberth et al. 1995) against e. coli. the abundance of ll-37 in neutrophil specific granules is about a third that of lactoferrin or lysozyme, the principal proteins of specific granules . in addition to microbicidal activity, certain cathelicidins also have chemoattractant activity. for example, the human cathelicidin ll-37 is chemotactic for neutrophils, monocytes, and t cells, but not for dendritic cells. this activity is likely mediated through binding to the formyl-peptide receptorlike 1 receptor (de et al. 2000) . the proline-rich porcine cathelicidin pr-39 can induce upregulation of syndecans, heparan sulfate proteoglycans involved in the repair process at the site of skin wounds (gallo et al. 1994) . although the pig has a large number of cathelicidin genes, the most active antimicrobial peptides in porcine skin wounds are protegrins, secreted from neutrophils as proprotegrins and activated by proteolytic cleavage by neutrophil elastase (panyutich et al. 1997) . in vitro, inhibition of neutrophil elastase by specific inhibitors blocked the conversion of proprotegrins to protegrins and largely ablated the stable antimicrobial activity secreted by porcine neutrophils (shi and ganz 1998) . the application of neutrophil elastase inhibitor to porcine wounds decreased the concentration of mature protegrin in wound fluid and impaired the clearance of bacteria from wounds. the deficit could be restored by supplementing the wound fluid with synthetic protegrin in vitro or in vivo (cole et al. 2001 ). thus protegrins act as natural antibiotics that contribute to the clearance of microbes from wounds. strong evidence for the antibacterial function of cathelicidins in the skin comes from studies of dermal infections in mice with an ablated gene for cramp (cathelin-related antimicrobial peptide). cramp, normally the main murine cathelicidin (nizet et al. 2001) , is the murine homolog of human hcap18. in cramp-knockout mice, the skin lesions caused by experimental group a streptococcus inoculation were larger lesions and had higher numbers of viable bacteria than those seen in control mice. as in pigs, infiltrating neutrophils appeared to be the chief source of cathelicidins in inflamed skin. epithelial cells also produced cathelicidins, but it is not clear if their production was quantitatively significant. how or if epithelial cathelicidins undergo proteolytic processing remains to be defined. many of our previous and ongoing studies have been supported by grants from the national institutes of health, including: ai 32234, 32738, and 50843 to c. b.; ai 37945 and 22837 to r. i. l.; and hl46809, ai46514, and ai48167 to t. g. the continuing support of the will rogers institute is gratefully acknowledged. we thank ken miyasaki for his artistic rendering of the human defensin dimer, all our past and present collaborators for putting up with us, and antimicrobial peptides for being there. fall-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis the human antimicrobial and chemotactic peptides ll-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations killing of giardia lamblia by cryptdins and cationic neutrophil peptides secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing secretion of microbicidal α-defensins by intestinal paneth cells in response to bacteria the peptide antibiotic ll-37/hcap-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface mouse beta-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs human alpha-defensin 1 (hnp-1) inhibits adenoviral infection in vitro the isolation and characterization of a novel corticostatin/defensin-like peptide from the kidney hbd-1: a novel beta-defensin from human plasma human enteric defensin genes: chromosomal map position and a model for possible evolutionary relationships toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2 contribution of rabbit leukocyte defensins to the host response in experimental syphilis biosynthesis of granule proteins in normal human bone marrow cells: gelatinase is a marker of terminal neutrophil differentiation the leucocyte protein l1 (calprotectin): a putative nonspecific defence factor at epithelial surfaces lipid mediator-induced expression of bactericidal/ permeability-increasing protein (bpi) in human mucosal epithelia mg2+ sensing by the mg2+ sensor phoq of salmonella enterica identification of defensin-1, defensin-2, and cap37/azurocidin as t-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils purification and characterization of a scorpion defensin, a 4kda antibacterial peptide presenting structural similarities with insect defensins and scorpion toxins inhibition of neutrophil elastase prevents cathelicidin activation and impairs clearance of bacteria from wounds retrocyclin: a primate peptide that protects cells from infection by t-and m-tropic strains of hiv-1 staphylococcus aureus strains lacking d-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice enap-2, a novel cysteine-rich bactericidal peptide from equine leukocytes hcap-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules direct inactivation of viruses by human granulocyte defensins ll-37, the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (fprl1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells tracheal antimicrobial peptide, a cysteinerich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cdna airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells determination of the three-dimensional solution structure of the antihypertensive and antiviral protein bds-i from the sea anemone anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing cryptdins: antimicrobial defensins of the murine small intestine role of the bactericidal/permeabilityincreasing protein in host defence salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity isolation of antimicrobial peptides from avian heterophils antimicrobial activity of chicken and turkey heterophil peptides chp1, chp2,thp1, and thp3 opsonic activity of mcp-1 and mcp-2, cationic peptides from rabbit alveolar macrophages amino acid sequence and disulfide bond assignment of myotoxin a isolated from the venom of prairie rattlesnake (crotalus viridis viridis) the expression of the gene coding for the antibacterial 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of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa loss of s100a9 (mrp14) results in reduced interleukin-8-induced cd11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro snake venom growth arresting peptide the outer membranes of brucella spp. are resistant to bactericidal cationic peptides pharmacological activity of the c-terminal and n-terminal domains of secretory leukoprotease inhibitor in vitro human airway epithelia express a beta-defensin characterization of defensin resistance phenotypes associated with mutations in the phop virulence regulon of salmonella typhimurium in vitro activity of the antimicrobial peptides human and rabbit defensins and porcine leukocyte protegrin against mycobacterium tuberculosis a novel mouse beta defensin, defb2, which is upregulated in the airways by lipopolysaccharide characterization of the mouse beta defensin 1, defb1, mutant 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a source of intestinal lysozyme identification and isolation of the bactericidal domains in the proteinase inhibitor aprotinin discovery of new human betadefensins using a genomics-based approach nmr solution structure of murine ccl20/mip-3 alpha, a chemokine that specifically chemoattracts immature dendritic cells and lymphocytes through its highly specific interaction with the betachemokine receptor ccr6 localization of human intestinal defensin 5 in paneth cell granules broadspectrum antimicrobial activity of human intestinal defensin 5 isolation of human intestinal defensins from ileal neobladder urine germ-free and colonized mice generate the same products from enteric prodefensins susceptibility of neisseria gonorrhoeae to protegrins secretion of type ii phospholipase a2 and cryptdin by rat small intestinal paneth cells eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha a novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin effect of live and heat-killed bacteria on the secretory activity of paneth cells in germ-free mice interaction of macrophage cationic proteins with the outer membrane of pseudomonas aeruginosa genomics-based approaches to gene discovery in innate immunity the solution structures of the human beta-defensins lead to a better understanding of the potent bactericidal activity of hbd3 against staphylococcus aureus epithelial antibiotics induced at sites of inflammation discovery of five conserved beta-defensin gene clusters using a computational search strategy proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins 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membrane the potent anti-staphylococcus aureus activity of a sterile rabbit inflammatory fluid is due to a 14-kd phospholipase a2 conversion of pig pancreas phospholipase a2 by protein engineering into enzyme active against escherichia coli treated with the bactericidal/permeability-increasing protein mechanisms of action of the bactericidal/permeability-increasing protein bpi on endotoxin and phospholipid monolayers and aggregates regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores rk-2: a novel rabbit kidney defensin and its implications for renal host defense purification, primary structure, and biological activity of guinea pig neutrophil cationic peptides beta-defensins: linking innate and adaptive immunity through dendritic and t cell ccr6 human neutrophil defensins selectively chemoattract naive t and immature dendritic cells cathelicidins: a novel protein family with a common proregion and a variable c-terminal antimicrobial domain contribution of human alpha-defensin 1, 2, and 3 to the anti-hiv-1 activity of cd8 antiviral factor widespread expression of beta-defensin hbd-1 in human secretory glands and epithelial cells gallinacin-3, an inducible epithelial betadefensin in the chicken isolation and mode of action of rabbit corticostatic (antiadrenocorticotropin) peptides conversion of proepithelin to rpithelins. roles of slpi and elastase in host defense and wound repair key: cord-351905-tjcyvkcv authors: mummah, riley o.; hoff, nicole a.; rimoin, anne w.; lloyd-smith, james o. title: controlling emerging zoonoses at the animal-human interface date: 2020-09-18 journal: one health outlook doi: 10.1186/s42522-020-00024-5 sha: doc_id: 351905 cord_uid: tjcyvkcv background: for many emerging or re-emerging pathogens, cases in humans arise from a mixture of introductions (via zoonotic spillover from animal reservoirs or geographic spillover from endemic regions) and secondary human-to-human transmission. interventions aiming to reduce incidence of these infections can be focused on preventing spillover or reducing human-to-human transmission, or sometimes both at once, and typically are governed by resource constraints that require policymakers to make choices. despite increasing emphasis on using mathematical models to inform disease control policies, little attention has been paid to guiding rational disease control at the animal-human interface. methods: we introduce a modeling framework to analyze the impacts of different disease control policies, focusing on pathogens exhibiting subcritical transmission among humans (i.e. pathogens that cannot establish sustained human-to-human transmission). we quantify the relative effectiveness of measures to reduce spillover (e.g. reducing contact with animal hosts), human-to-human transmission (e.g. case isolation), or both at once (e.g. vaccination), across a range of epidemiological contexts. results: we provide guidelines for choosing which mode of control to prioritize in different epidemiological scenarios and considering different levels of resource and relative costs. we contextualize our analysis with current zoonotic pathogens and other subcritical pathogens, such as post-elimination measles, and control policies that have been applied. conclusions: our work provides a model-based, theoretical foundation to understand and guide policy for subcritical zoonoses, integrating across disciplinary and species boundaries in a manner consistent with one health principles. zoonotic pathogens are a major threat to global health, both through their on-going contributions to disease burden and their potential contributions to the emergence of novel pandemic pathogens [1] . zoonotic spillover is defined as transmission of a pathogen from an animal host to a susceptible human and is the source of diseases from monkeypox to plague to leishmaniasis. risk of zoonotic spillover is driven by many ecological, epidemiological, and behavioral factors across scales [2] [3] [4] . the combination of animal ecology, human behavior, and environmental conditions can lead to cross-species transmission and, thus, requires a onehealth perspective to evaluate and respond to outbreaks of disease. beyond the complexity of the zoonotic spillover process, zoonotic pathogens differ greatly with respect to their efficiency of human-to-human transmission [5, 6] . transmissibility between humans is described by the reproductive number, r 0 , which is defined as the average number of secondary cases caused by a single infected individual in an entirely susceptible population [7] . some zoonotic pathogens face preexisting immunity in the population, and are governed by the effective reproductive number, r eff , which is the average number of secondary cases in a population with both susceptible and immune individuals [8] [9] [10] . for simplicity, we will use r throughout this study to refer to r 0 or r eff , in either case representing the efficiency of human-to-human transmission before further control measures are considered. it is useful to classify zoonoses by their transmissibility among humans, as captured by their r value [6] . for pathogens with r = 0, like west nile virus or rabies virus, transmission only occurs through spillover, and the pathogen is unable to transmit between humans. when r is between 0 and 1, the pathogen is subcritical and causes self-limiting outbreaks, as for monkeypox virus, nipah virus, or some avian influenza viruses. supercritical pathogens with r > 1, such as pandemic influenza or ebola virus, can cause epidemics or pandemics in the human population. subcritical zoonoses have been understudied by infectious disease modelers, likely because they do not align with dominant modeling frameworks, they require integration of animal and human dynamics, and they presently lack pandemic potential [6] . modeling effort on these systems has been particularly sparse for questions of disease control and its economic components, and the interplay between spillover and human-to-human transmission in driving the epidemiology of subcritical pathogens. some improvements have been made in the last decade, especially in methods for estimating r for subcritical pathogens [11] [12] [13] [14] [15] , but there is still a lack of theory to guide control efforts [16] . this paper aims to address this gap. we note that our findings extend directly to non-zoonotic pathogens such as postelimination measles, where r < 1 due to herd immunity, and geographic importation plays the role of spillover. our findings also relate to the on-going covid-19 pandemic caused by sars-cov-2, where r < 1 in some settings due to social distancing, contact tracing, quarantine and isolation, and other measures, and again geographic importation acts like spillover to introduce new cases. our work also applies equally to pathogens that transmit directly or via arthropod vectors. control measures for subcritical zoonoses can be classified into three functional groups according to the modes of transmission they aim to reduce: prevention of spillover, reduction of human-to-human transmission, and control of both spillover and human-to-human transmission jointly (table 1) . because subcritical pathogens cannot cause epidemics and every outbreak is triggered by a spillover event, public health policy may naturally focus on spillover prevention. however as r rises toward 1, an increasing proportion of cases are caused by human-to-human transmission (fig. 1) . this leads to open questions about how to target control measures. should control be targeted at animal-tohuman transmission, human-to-human transmission, or both? furthermore, for pathogens that spill over infrequently, implementing controls that focus on human-tohuman transmission when there are no active outbreaks does not seem cost effective. would a reactive strategy which switches from preventing zoonotic spillover to reducing human-to-human transmission be more effective? this decision space gets more complicated when economic costs of control are considered, especially because measures to reduce different types of transmission may differ in cost. similarly, there are often known host or environmental factors that influence spillover risk. epidemiological risk factor studies can define these factors so high-risk groups can be identified. for example, individuals in contact with dromedary camels and hunting or handling bushmeat are at higher risk for transmission of middle east respiratory syndrome (mers) coronavirus and simian retroviruses, respectively [63, 64] . can targeted reduction of spillover in these high-risk groups be an effective control measure for subcritical pathogens? this study presents a general theory to build intuition and give evidence-based guidelines for effective control of subcritical zoonoses (or other subcritical pathogens). our framework reveals general principles to aid policymakers faced with difficult decisions and resource constraints and can be adapted to specific pathogens and settings to guide concrete decisions and support allocation of finite resources. for a subcritical zoonosis, the total incidence of infection in the human population arises from a mixture of primary and secondary cases. we assume that zoonotic spillover events (primary cases) occur at some characteristic total rate λ z in a population of interest, e.g. λ z might represent 100 spillover events per year in a given administrative region. each human case is then capable of transmitting the infection to cause secondary cases, with the reproductive number r denoting the expected number of secondary cases per infected human. it is possible to model the stochastic dynamics of transmission in the human population using a branching process [65] [66] [67] or similar formulation. for the present analysis, it is sufficient to focus on the expected dynamics of these models. the first generation of transmission (i.e. those infected by the index case) will have r cases, on average. each of these cases will infect another r cases, so the second generation will have r 2 cases, and so on. thus the mean number of cases in each minor outbreak, including the primary case that triggers the outbreak, is given by a geometric series, and for r < 1 [68] : the expected total incidence rate i is then given by the product of the spillover rate and the mean number of cases associated with each spillover event: proportion of cases infected by human sources as shown above (eq. 1), when r < 1, the expected number of cases that result from each introduction is 1/(1-r). because there is one primary case per introduction, the proportion of cases that are primary is given by the reciprocal of this quantity and is equal to (1-r). thus, for zoonotic pathogens with r < 1, the expected proportion of cases infected by humans is r. we consider the effect on total incidence of control measures that reduce spillover rates, human-to-human transmission, or both. let c z be the factor by which control measures reduce spillover rates, and c r be the factor by which control measures reduce the reproductive number in the human population. when these reduction factors equal 1, there is no impact on transmission; when they equal 0, that mode of transmission is halted completely. the total incidence under control, i c , is then: for control measures that affect both modes of transmission equally, such as protective vaccination of humans, c z = c r = c and this expression simplifies accordingly. in settings where it is necessary to choose between measures to reduce zoonotic spillover (via c z ) and measures to reduce human-to-human transmission (via c r ), we can compare the total incidence when each measure a human-to-human transmission has been reported in nosocomial settings but is rare fig. 1 the expected source of infection for cases is determined by the reproductive number for human-to-human transmission is in place. the point where the two strategies are equivalent is given by: since the zoonotic spillover rate enters both sides linearly, the preferred strategy in a given context is governed by the reproductive number. rearranging these expressions, we can find the value of the reproductive number where the two strategies are equivalent: when r > r switch , the strategy to reduce human-tohuman transmission will yield a greater reduction in incidence. to incorporate the influence of differing cost, we consider how the effectiveness of control measures varies with effort using a simple model for the principle of diminishing returns on investment (fig. 2 ). this corresponds to many public health settings where heterogeneity in the structure, accessibility and compliance of populations means that the incremental cost of expanding coverage rises as coverage rises. we model this phenomenon by setting the effectiveness of control measures to be a declining exponential function of resources invested, r. to reflect the different costs of different control strategies, we introduce a factor α to scale the return on investment for reducing spillover relative to reducing human-to-human transmission: substituting these expressions into eq. 5 and solving for the value of the reproductive number r switch at which the strategies are equivalent, we find: r switch is the value of r above which control measures should be targeted at human-to-human transmission, for a given level of resource investment. the curves in fig. 3 are generated by plotting 1c z (or equivalently, 1 − e −αr ) versus r switch parametrically as a function of r, for different values of the relative cost α (which are shown as different line types). for a given reduction factor (c) describing the effectiveness of control, the greatest reduction in incidence is obtained from control measures such as vaccination that simultaneously reduce both zoonotic spillover and human-to-human transmission. when this is not possible and when resources are constraining, it may be desirable to implement control measures in a reactive manner. here we analyze the impact of control policies that focus on reducing spillover as a default but switch their focus to reducing human-to-human transmission when an outbreak has been detected. we assume that the switch occurs after k generations of transmission among humans, reflecting inevitable delays in case identification and policy implementation. if control is imposed after the first generation of human-to-human transmission has occurred (k = 1), then the first generation will have expected size r and subsequent generations will have expected size c r r. thus, the expected number of cases in the minor outbreak will be: in the supplement we show that the general form, for a delay of k generations, will be: thus the total incidence under reactive control strategy, with reduction factors c r and c z , is: this expression was used to plot the green curves in fig. 3 with c r = c z . for most zoonotic pathogens, zoonotic spillover risk is not fixed across the population due to variation in ecological, epidemiological, and behavioral factors. to explore how heterogeneous spillover risk might influence the choice of disease control strategy, we analyze a model with two defined groups with different risk of zoonotic infection. we define p as the proportion of the population in the group with higher risk of zoonotic spillover. let λ h be the spillover rate for the high-risk group and λ l be the spillover rate for the low risk group. then the total spillover rate λ z can be written as pλ h + (1 − p)λ l . thus, in the absence of control measures, the incidence is given by: analysis of control measures for subcritical zoonoses with risk groups we now consider the effect of different control measures when there are defined high-risk and low-risk spillover groups. like before, we can apply a general spillover reduction term, c λ , that applies to both risk groups to reduce the two spillover rates equally. the total incidence is then: in settings with defined risk groups, interventions that target the high-risk group are an attractive strategy for reducing overall incidence. to model control measures targeted at the high-risk spillover group, we define c h to fig. 3 impacts of different control measures on incidence of a zoonotic infection with initial value of r (i.e. before control) between 0 and 1. panel a illustrates the effects of control on the total incidence expected in a focal population, whereas panel b shows the proportional reduction in expected incidence when compared to the incidence level without control (black line). in a, the black line shows how the expected total incidence increases nonlinearly with r, for a fixed rate of zoonotic spillover. colored lines show the total incidence that would result from interventions that cause 50% reductions in spillover transmission (red), human-to-human transmission (blue), or both types of transmission (purple). green lines show the incidence resulting from a reactive intervention strategy, where effort is focused on reducing spillover transmission but is shifted to reducing human-to-human transmission once an outbreak is detected. the three green lines show the total incidence resulting when control is shifted after one, two, or three generations of transmission among humans, respectively from top to bottom be the factor by which control measures reduce spillover rates in the high-risk group. the total incidence under such a targeted control policy is then: we assume that targeted control makes more efficient use of resources, in proportion to the size of the highrisk group. thus to calculate the effect of targeted control in our cost-benefit analyses, we multiply the resources invested by a factor 1/p. the reduction factor c h is thus a function of resources invested, r, the factor α that reflects the relative cost of reducing spillover versus reducing human-to-human transmission, and the proportion of high-risk individuals in the population, p: different combinations of targeted spillover reduction, universal spillover reduction, and efforts to reduce r give rise to incidence expressions similar to eqs. (10) and (13) . table s1 contains the full list of equations that were used to plot the curves in figs. 6 and 7. our analysis of control measures for subcritical zoonoses is guided by strikingly simple predictions, derived from basic theory for outbreak dynamics, about the expected proportion of all human cases infected by other humans versus by animals ( fig. 1; eq. 1 ). the relative importance of these transmission routes in any system is governed by the efficiency of human-to-human transmission, as quantified by r. for subcritical zoonoses with r < 1, the expected number of cases that result from each introduction (including the 1 primary case) is 1/(1-r), and thus the proportion of cases infected by humans is r. when r > 1, endemic circulation of the pathogen in the human population is possible. averaging over many introductions, secondary cases from successful outbreaks greatly outweigh the primary cases, including instances where introductions go extinct, and effectively all cases are from human sources. for a given rate of zoonotic spillover, the expected total incidence level depends strongly on the prevailing value of r, with expected outbreak sizes rising sharply as r approaches 1 (fig. 3a) . accordingly, disease control interventions exhibit marked differences in effectiveness as a function of r, in both absolute and proportional terms (fig. 3a,b) . because the total incidence scales linearly with the spillover rate (eq. 2), measures that reduce spillover transmission have a fixed proportional impact, regardless of r (fig. 3b) . measures to reduce human-tohuman transmission have limited impact when r is low, compared to measures reducing zoonotic spillover, but this situation is reversed dramatically as r approaches 1 (fig. 3) . when comparing measures that reduce either type of transmission by 50% (i.e. comparing c z = 0.5 to c r = 0.5), spillover-reducing measures are preferred for r values up to 0.67, then measures reducing human-tohuman transmission are preferred above this point (eq. 5, fig. 3a) . the most effective control measures are those, like vaccination, that reduce both routes of transmission by a given amount. when vaccines are not available, as is initially the case for many emerging pathogens, a reactive strategy that targets spillover then switches to human-to-human transmission once an outbreak is detected can be almost as effective, even if the switch is delayed for several generations of transmission (fig. 3a,b) . we then considered how various control strategies perform under different scenarios of resource investment, where resources govern the effectiveness of control measures via our assumption of diminishing returns on investment (fig. 2) . two findings stood out. first, control measures that focus strictly on reducing human-tohuman transmission will never reduce incidence to zero (fig. 4) . even with very high resource investment, when human-to-human transmission is halted entirely, primary cases are undiminished. however, at lower resource levels, measures to reduce human-to-human transmission can be cost-effective, depending on the epidemiological context (fig. 4) . in low transmissibility settings, investing resources into reducing human-tohuman transmission is only barely better than doing nothing. as r increases, though, we see a growing range of resource levels where reducing r is more effective than reducing spillover. when r = 0.9, this difference is large and persists throughout almost the full range of incidence reductionyet only spillover reduction can drive incidence to zero (fig. 4) . unsurprisingly, if it is possible to reduce both modes of transmission at the same cost as reducing one, then this is always the most cost-effective strategy. notably, though, the reactive strategy is nearly as cost-effective, given the costs of reducing cross-species transmission and human-to-human transmission are equal. for most emerging zoonoses a vaccine is unavailable, and in many settings reactive measures may not be practical due to logistical challenges, unavoidable delays, or shortcomings in surveillance. in these settings a choice must be made between reducing zoonotic spillover or reducing human-to-human transmission, and we can determine which strategy would be most effective for a given r value. the preferred strategy depends on the level of resources available, which we quantify here by the proportional reduction in spillover that is achievable if all resources are devoted to spillover control (fig. 5) . the curved solid line marks the boundary between optimal strategies, assuming it is equally expensive to implement spillover reduction or human-to-human transmission reduction (i.e. α = 1). this line corresponds to eq. 7 for r switch , plotted parametrically as a function of resource investment (i.e. with zero investment and c z = 1 at the bottom, and infinite investment and c z = 0 at fig. 4 impacts of control measures with varying resource investments on incidence of a zoonotic infection with r between 0 and 1. each panel shows a different r (before control) value. the black lines show the incidence under no control. colored lines show the change in total incidence that would result from increasing investment for interventions that cause reductions in spillover transmission (red), human-to-human transmission (blue), or both types of transmission (purple). the green line shows the incidence for increasing investment resulting from a reactive intervention strategy, where effort is focused on reducing spillover transmission but is shifted to reducing human-to-human transmission once an outbreak is detected (detection after two generations of transmission is shown). controls measures targeting spillover transmission are assumed to be equally costly as measures targeting human-to-human transmission, i.e. α = 1 fig. 5 policy guidance whether incidence will be reduced more by focusing on reducing spillover transmission or human-to-human transmission, for different values of r (before control) and the reduction in spillover that is achievable given resource constraints. the solid line shows the boundary between preferred strategies when costs of the two types of control are equal, as defined by eq. (5). the dashed and dotted lines show how the boundary shifts due to differences in relative cost (each line is labeled by the relative cost of reducing spillover by a given proportion compared to the cost of reducing r by the same proportion) the top). when r is low or resource levels are high (fig. 5 -shaded in orange), it is preferable to cut off zoonoses at the source by focusing control efforts on reducing cross-species spillover. as r approaches 1, it becomes more effective to reduce human-to-human transmission, as a diminishing fraction of cases are attributed to spillover. yet in order to drive total incidence to zero, in the limit of high resource investment, it is necessary to focus on spillover reduction. if the costs of the strategies are not equal, the tradeoff line shifts (fig. 5, dotted and dashed lines) . the greater the cost of reducing spillover transmission relative to reducing human-to-human transmission, the more the tradeoff curve moves to the left, indicating that targeting human-to-human transmission would be a better use of resources for lower r valuesthough spillover reduction always becomes preferable as we aim to push incidence levels toward zero (figs. 4 and 5) . conversely, if reducing spillover transmission is substantially cheaper than reducing human-to-human spread, then spillover reduction remains the preferred strategy for values of r approaching 1 (fig. 5) . in many settings, there are identifiable groups at elevated risk for zoonotic spillover risk, and these high-risk groups present an attractive focus for targeted control measures. to incorporate a risk structure for spillover in our model, we assumed that the high-risk group composed a fixed proportion (here p=0.10) of the population, and varied the rate ratio of zoonotic infection (λ h / λ l ) between the high-and low-risk groups. under no control, total incidence grows nonlinearly with increasing r, as in fig. 2a , and also rises as the relative risk of spillover in the high-risk group increases (fig. 6a) . the latter effect is a simple reflection of increased total spillover in the population, as we treat λ l as constant. considering different control strategies, we find that the broad hierarchy of strategies as described above is remarkably robust to heterogeneities in spillover risk, while the epidemiological parameters shape the potential benefit of targeted control. we first observe that untargeted control policies, such as general awareness campaigns aimed at reducing spillover in the whole population or human-to-human transmission, are unaffected by the defined risk groups (fig. 6b & c) . in contrast, a targeted strategy to reduce spillover, such as improved biosafety protocols among people who have high-risk contacts with animals, will have varying impact depending on the risk ratio (fig. 6d ), but as with universal spillover reduction (fig. 6b) , this impact does not exhibit any dependence on r. we also note that the overall incidence reduction appears lower for targeted control than for universal spillover control, but this is because this plot assumes equal reduction factors (c = 0.5) for all control measures; under this assumption, control measures are inevitably more impactful when applied to the whole population versus a high-risk group of just 10% of the population. considering mixed strategies that combine targeted control of the high-risk spillover group with general control of human-to-human transmission (fig. 6e-h) , we see that the benefits of targeted control for total incidence reduction are greater for higher risk ratios, but this difference diminishes as r approaches 1 and human-to-human transmission dominates the epidemiology. as in fig. 3b , the benefits of including human-to-human transmission controls depend on the delay before initiation. for longer delays the curves resemble the targeted spillover control in fig. 6d for low values of r, since when r is low many transmission chains do not last long enough to be affected by reduced human-to-human transmission. in contrast, under joint programs with no delay (fig. 6e) , the benefits of control are seen immediately as r increases. finally, we consider targeted control measures under different resource scenarios, to explore the possible benefits of efficiently reducing spillover in the high-risk group. again, we see a tradeoff between strategies preferred at modest resource levels and those preferred when resources are not limiting (fig. 7) . among strategies that only reduce spillover transmission (red and orange lines in fig. 7) , targeted control shows considerable benefits at low resource levels, particularly for high risk ratios and higher values of r. yet targeted strategies are less effective at high resource levels, since they do not reduce spillover in the low-risk group, and hence can never reduce incidence to zero. a similar pattern is found for mixed strategies, where targeted joint or reactive approaches (i.e. high-risk spillover reduction followed by a switch to reducing human-to-human transmission once an outbreak is underway) are the most effective control policies at low resource levels, particularly when r > 0.5 and the risk ratio is high, but are incapable of reducing incidence to zero even at high levels of investment. in many other epidemiological settings, such as when r is 0.5 or lower and when risk ratios are near 1, any benefits to targeted control are imperceptible and tend to be outweighed by the disadvantages of allowing spillover to the low-risk group to continue unchecked. implementing efficient, cost-effective control measures is crucial for the control of emerging infectious disease, both to reduce the disease burden of human cases and to minimize the opportunities for pathogen adaptation, international spread, or other adverse events. however, for subcritical pathogens that exhibit low transmissibility among humans, it is not obvious whether control efforts should focus on reducing primary cases arising from spillover from external reservoirs or reducing secondary cases arising from human-to-human transmission. using a simple mathematical model, we developed a theoretical framework to guide decisions about how to target resources under scenarios with different pathogen transmissibility and risk group structure. we focused on the relative impacts achievable in resource-constrained settings, as well as the maximum benefits that could be obtained when resource investment was high. our work is framed in the context of zoonotic infections, where introductions arise via cross-species spillover from animal reservoirs, but our findings translate fully to other scenarios where outbreaks of subcritical pathogens are seeded by introductions from outside. this includes vaccine-preventable diseases such as measles in postelimination settings where herd immunity has reduced r below 1, or pathogens such as methicillin-resistant staphylococcus aureus (mrsa) in hospitals that exhibit inefficient nosocomial transmission [39, 40, 69] . geographic importation from endemic regions serves as 'spillover' for measles or covid-19, introducing the pathogen into areas where it was previously eliminated or brought under control. similarly, community introduction of mrsa into hospitals serves as the spillover mechanism prior to transmission within the hospital. we found that the optimal focus of control measures for subcritical pathogens depends primarily on the human-to-human transmissibility of the pathogen, as fig. 6 impacts of different control measures on the total incidence of a zoonotic infection with r (before control) between 0 and 1 with a varying ratio of high and low spillover rates. panel a shows how total incidence increases with r for varying ratios of high-to-low zoonotic spillover. panels b-e illustrate proportional reduction in incidence for controls that would cause 50% reductions in all spillover transmission (b), human-to-human transmission (c), spillover transmission into the high-risk group (d), or jointly high-risk spillover and human-to-human transmission (e). panels f-h show the proportional reduction in incidence given a reactive strategy that first targets high-risk spillover and then switches to reducing human-to-human transmission after 1, 2, or 3 generations of transmission, respectively. note that longer delays cause the results to resemble panel d over increasing ranges of r values, since at low r many transmission chains don't last multiple generations. the proportion of high-risk individuals in the population was set to 0.10 summarized by the reproductive number r (fig. 3) . for pathogens with the lowest transmissibility among humans (r near zero; e.g. h7n9 avian influenza), measures to reduce zoonotic spillover are most effective. thus for these zoonoses, strategies such as awareness campaigns to reduce contact with reservoir host animals or animals found dead, infection control in live animal markets, culling infected reservoir populations, and removing rodents from homes (table 1) will be most effective in reducing human cases. for pathogens with greater transmissibility among humans (e.g. postelimination measles, sars-cov-2, or ebola), reducing human-to-human transmission becomes more effective. in such scenarios, preferred control methods will include providing personal protective equipment in high-risk settings such as hospitals, awareness campaigns to reduce unprotected contact with sick individuals, and strengthened surveillance for improved case tracking and faster case isolation. of course, the strongest control strategies would act to reduce zoonotic spillover and human-to-human transmission at the same time, as with a protective vaccine. where this option is not available (or is cost-prohibitive to deploy widely in advance of an outbreak), we found that a reactive strategy could achieve nearly the same effect without substantially greater investment. such a strategy would have a baseline emphasis on reducing zoonotic spillover, but when a spillover or subsequent outbreak is discovered, the fig. 7 impacts of different control measures with varying resource investment on the total incidence of a zoonotic infection with r (before control) between 0 and 1 with different ratios of high-to-low spillover rates. columns (left to right) show increasing values of r. rows (top to bottom) represent an increasing ratio of spillover rates in high-risk versus low-risk groups (λ h /λ l ). the black lines indicate total incidence under no control. colored lines represent the reduction in incidence for increasing resource investment. these scenarios were explored earlier in fig. 4 but now include the added comparison of targeted versus universal spillover control for all strategies emphasis would shift locally to reducing human-tohuman transmission. strategies could include an awareness campaign that focuses on reducing interactions with known animal hosts, such as not touching dead animals in the forest, shifting to increased contact precautions and active surveillance to detect human cases quickly to reduce human-to-human transmission once an outbreak is detected [27] . unsurprisingly, many existing control policies designed by public health professionals align broadly with the recommendations of our model. for pathogens with low r such as h7n9 avian influenza, our work advises an emphasis on preventing spillover. this is consistent with current public health control measures for h7n9 avian influenza, such as market disinfection or cessation of live poultry trade, which focus on reducing contact with birds and lowering risk of cross-species transmission [17] [18] [19] . in contrast, lassa virus has a higher r value estimated near 0.7 and, thus, has a higher expected proportion of cases which arise from human-to-human transmission. public health policy for lassa fever has recently focused on preventing nosocomial transmission between humans [34] [35] [36] . in some settings, r changes through time due to shifts in population immunity or other factors, and priorities for disease control should change accordingly. for example, r for monkeypox has increased over the decades since the cessation of widespread smallpox vaccination around 1980 [28, 70] , and the historic emphasis on spillover transmission should be re-examined in light of changing circumstances. similarly, r for measles has risen as childhood vaccination rates have dropped [39, 71] . public health systems frequently deal with resource constraints, in terms of finances and human or institutional capacity [72] . exploring the effects of resource investment on the impacts of control on overall human incidence, our work illustrates potential trade-offs between higher cost effectiveness at low investment versus the ability to reduce incidence to zero at high investment (fig. 4) . at low investment levels, the best simple strategies are those which follow the priorities laid out above, i.e. focusing on spillover if r is low, or on human-to-human transmission if r is higher. however, as resource levels increase, the limitations of these simple priorities become clear, because strategies that omit the reduction of spillover cannot ever reduce human incidence to zero as they do not decrease the number of primary cases. therefore, it is necessary to incorporate spillover reduction in any policy hoping to drive incidence to zero. when all types of interventions are assumed to be available with the same cost, then joint approaches that reduce both animal-to-human transmission and human-to-human transmission are most effective for a given resource level. when resources do not permit reduction of both transmission modes simultaneously, practitioners must decide which transmission mode is more important to control. our analysis provides guidance as to which transmission method is best to control as a function of resource investment and r, accounting for possible differences in cost (fig. 5) . while further work is needed to characterize the cost-efficacy curves for control measures in particular systems, in order to implement this approach, this analysis provides a foundation for rational cost-benefit analysis to support disease control policy. zoonotic spillover risk is heterogeneous in the human population, since some groups have more frequent or riskier exposures to zoonotic reservoirs due to cultural or occupational factors [5] . our analysis demonstrated that targeted spillover control in these high-risk groups offers the potential for markedly greater reductions in incidence, relative to control efforts spread across the entire population, when resources are limited. however, these targeted strategies are limited in impact as investment levels rise, since they don't reduce the spillover rate in the low-risk group so they can never drive incidence to zero. if there is negligible risk in the low-risk group or if the low-risk group receives low-intensity control as a side benefit of the targeted control (for instance, via an awareness campaign focused on the highrisk group but available to all), then the targeted control strategy would remain the most efficient option. ultimately, the desirability of targeting the high-risk spillover group depends on the epidemiological context (r value), resource level, and risk ratio between the risk groups. it is important to recognize that there are often substantial challenges in identifying and quantifying risk factors for spillover, to support a rational decision about targeting. for zoonoses that spill over very infrequently and unpredictably, such as ebolaviruses, coronaviruses including sars-cov-2 and mers, or some hantaviruses, the necessary data are hard to acquire and uncontrolled variation among spillover events can obscure patterns. in settings where the majority of individuals engage in potentially high-risk activities, yet spillover events are sporadic due to variation at other levels (e.g. in infection prevalence in the animal reservoir), it can be difficult to ascertain how the magnitude of risk is split across specific risk factors [2] . for example, in populations which nutritionally or economically rely on bushmeat, the majority of individuals can be exposed to multiple animal species through multiple modes of contact (e.g. hunting, food preparation, and cooking), which all present a potential risk of transmission [73] . the resulting difficulties in determining risk factors, and identifying distinct high-risk groups, can further complicate the implementation of targeted control measures. several caveats should be borne in mind in interpreting our analysis, which point to opportunities for further research. for the sake of clarity, our model is based on expected incidence levels under different scenarios, but stochastic variation can be large so individual outbreaks could differ substantially from our predictions. we also assumed stationarity (i.e. no changes in the model parameters through time), ignoring behavior change of affected populations or on-the-ground factors that can impede control measures [74] . we did not account for superspreading, or for variation in transmissibility across spatial or social contexts, both of which can have dramatic effects in the early phase of outbreaks [65, 75] . our analysis incorporated heterogeneities in spillover risk but did not address different risk groups for ongoing human-to-human transmission. such a scenario could arise due to age-structured mixing or susceptibility, or from other risk factors such as occupational exposure in health-care settings, and could have important effects on outbreak dynamics [76, 77] . factors giving rise to immune compromise, such as human immunodeficiency virus (hiv) infection, are another potential source of heterogeneity in both modes of transmission [78, 79] . the existence of distinct risk groups for human-to-human transmission would offer another important opportunity for targeted control measures and warrants further investigation. all of the above complexities could be addressed via more complex analytic methods, such as multitype branching processes, or via stochastic simulation analyses. our cost-benefit model is theoretical in nature and aims for simplicity rather than realism. we used a phenomenological model to represent diminishing returns on investment, but we do not properly account for complexities in scaling up control measures in space or time. for the reactive strategy, we assumed a clean and immediate switch between reducing spillover and reducing human-to-human transmission with no overlap and no lapse in control. in real-world settings, ramping up (or terminating) control measures is inevitably more complicated and a reactive strategy would likely entail delays and changing effectiveness through time. we also assumed there is no additional cost associated with switching strategies, but this is unlikely to be true due to the resources involved in initiating any program. while our theoretical analysis enabled a first exploration of the general cost-effectiveness of subcritical disease control, more intensive case studies are needed for specific pathogens. subcritical zoonotic pathogens exist at the animalhuman interface, and little policy guidance exists on the most effective ways to implement controls. in this study we present a framework to think systematically about controlling subcritical zoonoses, considering the relative importance of reducing animal-to-human spillover versus human-to-human transmission. our work shows how the relative effectiveness of these strategies depends on epidemiological context and highlights a trade-off between cost-effectiveness at low resource levels and the potential to reduce incidence to zero as investment increases. our findings illustrate core principles for evidence-based control of subcritical zoonoses and 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methods for estimating r0 from the size distribution of subcritical transmission chains using routine surveillance data to estimate the epidemic potential of emerging zoonoses: application to the emergence of us swine origin influenza a h3n2v virus quantifying transmission of emerging zoonoses: using mathematical models to maximize the value of surveillance data. biorxiv opportunities and challenges in modeling emerging infectious diseases transmission potential of influenza a(h7n9) virus, china at the frontier of the global battle against emerging infections: surveillance and management of avian influenza a (h7n9) in guangdong province effect of closure of live poultry markets on poultry-to-person transmission of avian influenza a h7n9 virus: an ecological study a systematic review of the comparative epidemiology of avian and human influenza a h5n1 and h7n9 -lessons and unanswered questions public reactions to the 2013 chinese h7n9 in fl uenza outbreak: perceptions of risk , 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democratic republic of the congo the differential impact of violence on ebola virus disease transmission: a mathematical modeling study of the 2018-2019 outbreak in the democratic republic of the congo invasion dynamics in spatially heterogeneous environments characterizing the transmission potential of zoonotic infections from minor outbreaks detecting differential transmissibilities that affect the size of self-limited outbreaks hiv-1/parasite co-infection and the emergence of new parasite strains antiretroviral drugs for tuberculosis control in the era of hiv publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to acknowledge seth blumberg for early input on the theory of the paper. received: 8 january 2020 accepted: 9 july 2020 key: cord-354325-r73datur authors: berger, mitchell; shankar, vidya; vafai, abbas title: therapeutic applications of monoclonal antibodies date: 2002-07-31 journal: the american journal of the medical sciences doi: 10.1097/00000441-200207000-00004 sha: doc_id: 354325 cord_uid: r73datur abstract researchers have sought therapeutic applications for monoclonal antibodies since their development in 1975. however, murine-derived monoclonal antibodies may cause an immunogenic response in human patients, reducing their therapeutic efficacy. chimeric and humanized antibodies have been developed that are less likely to provoke an immune reaction in human patients than are murine-derived antibodies. antibody fragments, bispecific antibodies, and antibodies produced through the use of phage display systems and genetically modified plants and animals may aid researchers in developing new uses for monoclonal antibodies in the treatment of disease. monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of asthma, autoimmune diseases, cancer, poisoning, septicemia, substance abuse, viral infections, and other diseases. i n 1975, kohler and milstein revolutionized the field of immunology by developing monoclonal antibodies (mabs). since that time, many mabs have been developed for use in diagnostic procedures and in immunotherapy. ever since it was observed that the therapeutic use of heterologous mabs elicited immunogenic responses in humans, significant research efforts have been devoted toward creating chimeric and humanized antibodies for use in human patients. major achievements have been in the production of mabs in transgenic plants and animals. the use of phage display libraries has created customized antibodies with defined affinity and specificity. this review describes how rodent, chimeric, and humanized antibodies have each been used, with varying degrees of success to treat cancer, septicemia, autoimmune disorders, and infectious diseases. we also describe here recent applications of antibody engineering, such as the use of bispecific antibodies and antibody fragments in immunotherapy. von behring and kitasato discovered in 1890 that the serum of vaccinated persons contained certain substances, which they termed "antibodies." in 1895, they treated diphtheria with an antiserum raised against the toxin. on the basis of research on tetanus toxin and trypanosome parasites, ehrlich proposed in 1900 the "side-chain theory" of antibody formation, which hypothesized that physiologically active substances, including toxins, attach to cell surface receptors that are produced in response to toxin-cell interactions and then ejected from the cells into the bloodstream, leading to circulating antibodies. 1, 2 not until the 1950s, however, did scientists' understanding of antibodies become sufficient to lay the foundation for the development of mabs. jerne 3 postulated in 1955 a theory of natural selection for antibody formation. animals vaccinated with an antigen were expected to produce several distinct antibodies against several epitopes of the antigen. frank macfarlane burnet subsequently refined and expanded jerne's theory. 4 burnet's "clonal selection theory," as it is generally known, postulates that cells specific for synthesizing 1 type of antibody are spontaneously generated due to random somatic mutations during the maturation of the immune system and that these cells proliferate when exposed to an antigen. at about the same time, porter isolated fragment antigen binding (fab) and fragment crystalline (fc) from proteolytically cleaved rabbit ␥-globulin. 5 until the 1960s, antibody-producing cells were difficult to maintain in culture, because they died after a few days. in addition, only polyclonal antibodies could be obtained. in 1964, littlefield 6 developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymi-dine (hat) selection media. in 1975, khler and milstein 7 provided the most outstanding proof of the clonal selection theory from results of heterokaryons-cell hybrids formed by the fusion of normal and malignant cells. twenty-five years after kohler and milstein produced the first monoclonal antibodies, dramatic progress has been made in using antibodies for diagnostic purposes, but the uses of mabs to treat disease have-until recently-remained somewhat limited. 8, 9 however, as this review indicates, the potential of mabs to aid in the treatment of a wide range of diseases is now beginning to be realized. antibodies are y-shaped proteins composed of peptides called heavy and light chains, the ends of which vary from antibody to antibody. each combination of heavy and light chains binds to a particular antigenic site. these glycoprotein chains are folded into domains of about 110 amino acids that become twisted into the immunoglobulin (ig) fold and are stabilized by disulfide bonds. structurally, each ig molecule consists of 2 50-kd heavy chains and 2 25-kd light chains, linked by disulfide bonds. human immunoglobulins are divided into 5 classes or isotypes based on the amino acid composition of their heavy chains: ␣, ␦, ⑀, ␥, and , denoted iga, igd, ige, igg, and igm, respectively. there are 2 kinds of light chains, (k) and (l), which are common to all 5 classes. four subclasses of igg (igg1, igg2, igg3, and igg4) and 2 subclasses of iga (iga1 and iga2) exist, each with a distinct function. secretory iga exists in dimeric form held together by a j chain and is associated with a secretory component that helps it pass through the cell membrane. 10, 11 each chain has a constant domain to bind host effector molecule and a variable domain to bind to the target antigen. light chains have 1 variable (v l ) and 1 constant domain (c l ), whereas heavy chains have 1 variable (v h ) and either 3 (␣, ␦, and ␥ chains) or 4 constant domains (⑀ and heavy chains) depending upon the isotype class. each variable domain contains 3 regions known as "hypervariable loops," also known as complementarity-determining regions (cdrs), that identify the antigen. the other amino acids in the variable (fv) domain are known as framework residues and act as a scaffold to support the loops. the v l and the v h , the c h 1 and the c l , and the 2 c h 3 domains are paired; the 2 c h 2 domains have carbohydrate side chains attached to them and are not paired. the folded constant domains may be homologous among different species, allowing hybrid domains (eg, mouse-human) to be produced. the variable domain confers specificity and affinity. the cdr amino acid sequences are extremely variable and play a large role in interac-tion with the targeted antigen. the chain type, region, and distance from the amino terminus characterize the domains. thus, c h 2 domain refers to the second constant domain of the heavy chain. upon digestion with papain, the antibody molecule is cleaved on the amino-terminal side of the disulfide bridges into 2 identical fabs and an fc fragment, whereas pepsin cleaves the antibody on the carboxy-terminal side of the disulfide bridges into 1 f(ab') 2 fragment containing both the arms of the antibody, and many small pieces of the fc fragment. currently, the following are available: whole antibodies, enzymatically produced 50-kd fab fragments, engineered 25-kd single-chain fv (scfv) antibodies consisting of the v h and v l connected by a flexible peptide linker, diabodies (noncovalent dimers of scfv), minibodies (scfv-c h 3 dimers), and heavy chain iggs found in species of the camelidae family, which are devoid of light chains and are referred to as v hh . the first mab described by kohler and milstein was created by the fusion of murine myeloma cells with murine-antibody-secreting lymphocytes. 7, 8 myeloma cells are immortalized b lymphocytes capable of secreting homogeneous antibodies. the immortal myeloma cell lacks the enzyme hypoxanthine guanosine phosphoribosyl transferase (hgprt) and is sensitive to the hat media. however, a hybrid cell known as a hybridoma, generated by the fusion of myeloma cell and an antibody-producing b cell, can survive in the hat media. the spleen b lymphocytes contribute the hgprt gene to the hybrid cell and, hence, unfused myeloma cells and spleen cells die in the hat media. the conventional method of generating mabs is the hybridoma technology in which spleen cells from immunized mice are fused with murine myeloma cells. whereas the myeloma cell imparts immortality to the hybridoma allowing cells to be cultivated indefinitely, the immune spleen b-cell confers antigen specificity. because each hybridoma is derived from a single cell, the cells within a hybridoma cell line are identical and make the same antibody molecule with same antigen-binding site and isotype, hence it is called a mab. among several excellent reviews that detail mab production with hybridomas is a recent review by dean and shepherd. 12 initial attempts to bypass the mouse to make human mabs involved fusion of human immune spleen lymphocytes with nonsecreting human myeloma partners to obtain hybrid cells that continually secrete a specific antibody. however, poor fusion of human myelomas, unsatisfactory performance of the hybrid cells, and the difficulty in accessing immune lymphocytes have prevented success. although heteromyelomas-which are fusions of hu-man and mouse myelomas-work better, these hybrids are usually unstable. attempts to use mouse myeloma cells to create hybrids and derive human mabs led to the loss of human chromosomes and the inability to make human igs. 13 unfortunately, in vitro immunization is limited by its inability to produce a secondary response and by the absence of the affinity maturation process that occurs in vivo. 13 affinity maturation process is a complex phenomenon, a consequence of intense bcell proliferation, somatic hypermutation of ig variable domain genes, and selection for b cells with high-affinity antigen binding, all occurring in the specialized microenvironment of the germinal center within the lymphoid tissue. thus, the search for an ideal fusion partner for generating human mabs has been difficult, which is why the epstein-barr virus (ebv) technique for immortalization of b lymphocytes is preferred. but immortalized b cells do not always replicate exactly the in vivo antibody response, because the tissues from which these cells were selected and the manipulations to which they are subjected to in the laboratory may alter the antibody specificity. 14, 15 protocols for the preparation of ebv virus, b cells, and cell fusion have been described elsewhere. 14, 16, 17 methods used for large-scale production of mabs may include the generation of ascites tumors in mice or in vitro mammalian cell culture fermentation by using bioreactors and continuous perfusion culture systems. 18, 19 the key issues in scale-up productions are the growth media, fermenter size, fermentation time, and purification procedures. 19 purification or downstream processing is accomplished by chromatography, fragmentation, conjugation with chelating agents, ultrafiltration, and controlled precipitation. 18 a more recent technique for producing antibodylike molecules uses what is known as the phage display library. it involves the construction of v h and v l gene libraries and their expression on the surface of a filamentous bacteriophage. developed in the 1990s, the phage display method requires repeated "panning" or screening of different antibodies based on their affinity for a specific antigen. antibody genes are linked to bacteriophage coat protein genes and the bacteriophages with the fusion genes are used to infect bacteria to create the phage display library. the resulting bacteriophages express the fusion proteins and display them on their surface, and the phage display library comprises recombinant phages, each displaying a different antigenbinding site on its surface. the phage expressing an antigen-binding domain specific for a particular antigen can be detected and isolated by binding to the surface coated with that antigen. libraries of v h and v l genes may be generated from nonimmunized donors, immunized donors who have an immune response against a particular antigen or from a synthetic library consisting of antibody fragments. 20, 21 one promising way to increase antibody yield or develop new antibodies may be by using genetically altered animals and plants. abgenix, a company in fremont, ca, has developed the transgenic "xeno-mouse," in which the mouse antibody-producing genes have been inactivated and functionally replaced by approximately 90% of the human ig gene loci in germline configuration, coding for the heavy and light chains. 22, 23 upon immunization with any specific human or nonhuman antigens, the "xeno-mouse" generates mabs, which are fully human igs, with high affinities and antigen-binding specificities. "xenomouse" strains producing specifically igg1, igg2, or igg4 isotypes have also been created to generate panels of diverse and highly specific mabs. kirin brewery company, japan, has developed another transgenic mouse known as the "trans-chromo" mouse. the endogenous igh and igg loci of the "trans-chromo" mouse were inactivated, but it harbors 2 individual human chromosome fragments, derived from human chromosomes 2 and 14, that contain whole human ig light-and heavy-chain loci, respectively. 24 these mice are capable of producing every subtype of fully human ig, including iga and igm. in these transgenic mouse models, human antibodies with high affinity to an immunized antigen are naturally selected by the murine immune system via an affinity maturation process, and thereby show increased diversity of the mabs. transgenic mice may be a suitable alternative to chimeric or humanized antibody production or the use of phage display systems to create less immunogenic or novel antibodies. 25 for instance, transgenic mice that express mabs to coronavirus in their milk have been developed. 26, 27 because ruminant animals, such as cows, goats, and sheep, produce relatively large amounts of milk, genetically-modified members of these species could also be used to produce large quantities of therapeutic proteins, including mabs. 28 plants may be a potential source of recombinant proteins, including mabs. 29 -31 plant virus vectors, such as the tobacco mosaic virus, may be used to make mabs. transgenic tobacco plants may also be used for large-scale production of recombinant iga, which is used in passive mucosal immunotherapy. 30, 31 this mab could be added to toothpaste to effectively protect against bacteria that cause tooth decay. 31, 32 recombinant antigens obtained from plants may also have therapeutic applications. for instance, attempts to immunize mice with escherichia coli heat labile enterotoxin b produced in transgenic tobacco and potato plants have proved promising. 33 hepatitis b viral surface protein produced in transgenic tobacco plants has been shown to be immunogenic in mice. 32 the genes coding for murine malignant b-cell specific markers are inserted into tobacco mosaic virus to cultivate an immunogenic protein in tobacco plants that may eventually be used in developing a vaccine against non-hodgkin lymphoma. 34, 35 other immunotherapeutic proteins under development include norwalk virus capsid proteins produced in tobacco and potatoes, cholera toxin and ct-b produced in potatoes, hepatitis b antigen produced in tobacco, anti-human igg used to detect nonagglutinating antibodies produced in alfalfa, and humanized anti-herpes simplex virus (hsv)-2 grown in soybeans. 36 -39 the food and drug administration (fda) considers antibodies to be "biopharmaceuticals"; as such, mab applications are regulated by the agency's center for biologics evaluation and research (cber) and the center for drug evaluation and research. 40, 41 the fda has created a "points to consider" document advising manufacturers of factors to consider in the production and testing of mabs intended for human use and identified information that should appear in investigational new drug or biologics license applications. 40 the "points to consider" document serves to "indicate the agency's current thinking on mab products for human use." 40 the agency's recommendations are aimed at protecting human health because viruses and cellular dna from antibody-producing cells with malignant phenotypes may be integrated into host cells after transformation. accordingly, among the points to consider are several steps-such as taking care to ensure purity of immunoconjugates and demonstrating the ability of any purification scheme to remove adventitious agents-designed to prevent contamination of the final product by human pathogens. 40 manufacturers must also adhere to animal care standards and detail steps to prevent contamination of cell culture. the points to consider document includes a list of normal human tissues used in cross-reactivity testing, tests for murine viruses, and organs to be considered in dosimetry estimates. the fda recognizes that because of species differences, animal models expressing the antigen of interest or cross-reactive epitopes are not always available. the agency has also published a guidance entitled s6 preclinical safety evaluation of biotechnology derived pharmaceuticals based on international conference of harmonization technical requirements. 41 before beginning phase 1 clinical studiesconducted to assess the safety of the drug and its mechanism of action-the fda recommends that researchers conduct in vivo and in vitro testing of the mab to assess cross-reactivity with human tissues or non-target-tissue binding. preclinical safety testing aims to evaluate the immunogenicity, crossreactivity, stability and effector functions of mabs. the metabolism, carcinogenicity, and genotoxicity of the product should also be evaluated. 41 guidance has also been published for mabs used as reagents in drug manufacturing. 42 the guidance emphasizes biological safety, performance characteristics of the reagent, and potential presence of residual amounts of the reagent in the final product. the fda general principles for testing and manufacturing are broadly applicable to many classes of antibodies, including those produced by phage display systems or transgenic plants and animals. depending on the expression system being used, other fda guidance documents, such as cber's points to produce biologicals should also be referenced. [43] [44] [45] humanizing monoclonal antibodies rodent mabs with excellent affinities and specificities have been generated using conventional hybridoma technology, but their use in clinical medicine is limited due to the immune responses they elicit in humans. for instance, the human antimouse antibody (hama) response can compromise the clinical effectiveness of murine mabs. although hama responses are directed against the murine constant regions, which represent the major antigenic features of the mouse ig, significant responses are also directed toward the murine variable regions. as a result, patients may mount an immune response against the injected murine antibodies, leading to allergic or immune complex hypersensitivities, rapid clearance of the antibody, and reduced clinical efficacy. 13,46 -51 initially, morrison and colleagues 48,52 introduced chimeric mabs in 1984, which showed several advantages over unmodified rodent antibodies. generally, chimeras combine the human constant regions with the intact rodent variable regions, replicating the rodent antibody variable regions by pcr and then cloning them into eukaryotic expression vectors containing human constant regions. 49 ideally, this allows better interaction with human effector cells and the complement system. because the fc region has little influence on the structure of the fv region, the chimeric constructs' affinity and specificity are virtually unchanged, and both rodent and chimeric antibodies cause apoptosis at a similar rate and intensity against target cells in vitro. 50, 51, 55 although chimeric antibodies have helped solve some of the problems associated with the use of rodent mabs, they still show significant immunogenicity in humans; because of their approximately 30% mouse sequence, they cause an human-antichimeric antibody response. humanized antibodies containing only the cdrs of the rodent variable region grafted onto the human variable region framework have been introduced to overcome these deficiencies. 53 the early work on recombinant, chimeric, and rodent/human antibodies happened during the mid 1980s and, by the late 1980s, greg winters and his colleagues demonstrated that a functional human-like antibody could be created by grafting the antigen-binding cdrs from variable domains of rodent antibodies onto human variable domains. numerous humanized antibodies have now been designed and constructed, and many are currently being evaluated in clinical trials. 13, 14, 39, 47, 54 efficient procedures for constructing humanized antibodies have been developed. 13, 14, 47, 55 the first step is to clone and sequence the complementary dnas (cdnas), coding for the variable domains of the mouse antibody to be humanized. the mouse hybridoma cell line is grown in an appropriate culture medium, and cells are harvested for rna isolation. polymerase chain reaction (pcr) primers that hybridize to the 5' ends of the mouse leader sequences and to the 5' ends of the mouse constant regions are designed for cloning light chain variable regions and heavy chain variable regions. cdna is synthesized from total rna, followed by pcr amplification with light and heavy chain specific primers. positive bacterial colonies containing mouse variable regions are then screened. construction of a chimeric antibody involves modifying the cloned mouse leader-variable regions at the 5'-and 3'-ends, using pcr primers to create restriction enzyme sites for convenient insertion into expression vectors, to incorporate sequences for efficient eukaryotic translation, and to incorporate splice-donor sites for rna splicing of the variable and constant regions. the adapted mouse light and heavy chain leader-variable regions are inserted into vectors containing, for example, human cytomegalovirus enhancer and promoter for transcription, a human light or heavy chain constant region, a neomycin gene for selection of transformed cells, and the simian virus 40 origin of replication in cos cells. these vectors are designed to express chimeric or reshaped human light and heavy chains in mammalian cells. the design and construction of an engineered human antibody require an analysis of the primary amino acid sequences of the mouse variable regions to identify the residues most critical in forming the antigen-binding site. a structural model of the mouse variable region is built on the basis of homology to known antibody variable regions. the framework regions (frs) of the new variable regions are modeled on frs from structurally similar immunoglobulin variable regions. the design process involves selecting human light and heavy chain variable regions that will serve as templates for the construction of a reshaped human antibody. the mouse cdrs are then joined to the frs from selected human variable regions. the primary amino acid sequences are then carefully analyzed to ascertain whether they would recreate an antigen-binding site that mimics the original mouse antibody. within the frs, the amino acid differences between the mouse and the human sequences are examined, and the relative importance of each amino acid in the formation of antigen-binding site is evaluated. minimum changes in the frs are desirable and should closely match the sequences from natural human antibodies. any potential glycosylation site in the frs of either mouse or human sequence needs to be identified and its influence on antigen binding considered. the dna sequences coding for the reshaped human variable regions, either made synthetically or based on an existing sequence that is very similar to the newly designed reshaped human variable region, are modified by pcr with specially designed oligonucleotide primers. the human variable regions together with their leader sequences are then cloned into a mammalian expression vector that already contains human constant regions. each human variable region is linked to the desired constant region via an intron. preliminary expression and analysis of the reshaped human antibodies are done by cotransfection of mammalian cell-expression vectors, 1 coding for human light chain and 1 coding for human heavy chain. the vectors will replicate in the cos cells and transiently express and secrete reshaped human antibodies. the concentration of the antibody produced can be analyzed by using an enzyme-linked immunosorbent assay. specific changes in the amino acids of the framework region may also be required to preserve the orientation and structure of the rodent cdr required for binding. computer modeling using databases containing human variable genes will identify sequences homologous to the rodent v regions. 13, 47 a computer model of the rodent fv can identify the non-cdr residues that interact with the cdr sequences, and choices can be made regarding which residues need to be included in the variable region. antibodies humanized in this way may have binding affinities up to one-third greater than the corresponding murine antibodies. 49 allergenicity is also significantly reduced. about 20 to 40% of patients exhibit a hama reaction to murine antibodies, whereas only about 7% have a similar reaction to humanized antibodies. 47,53,56 -58 humanization of mabs still has several practical difficulties. first, a detailed knowledge of the antibody structure and function is required. second, methods for efficient construction of humanized mabs are limited. third, unpredictable immunogenicity may result when a new amino acid sequence is introduced to balance affinity retention. fourth, the antibody repertoire is limited to the animal in which the progenitor mab originated. fifth, the rodent mab producing hybridoma must be isolated and thoroughly characterized. however, obstacles to humanization are gradually being surmounted. karpas et al 15 reported creating a hat-sensitive and ouabain-resistant human myeloma cell line that can fuse with human lymphoblast cells. a hat-sensitive subline of the myeloma cells that secreted only light chains was fused with ebv-transformed white blood cells that produced igg mabs to hiv-1 gp41. eventually, a clone was isolated that was polyethylene glycol-resistant and would not revert when placed in hat medium. standard polyethylene glycol fusion protocol was followed to fuse the myeloma cells with both ebvtransformed white cells and fresh white cells obtained from the peripheral blood of adults and tonsil cells from 2 children. the authors reported promising rates of hybridoma formation, stable ig production, and high yield of secreted antibodies compared with antibody-producing cell lines from mouse myeloma cells. the authors have now developed 40 hybridomas that have been secreting cells for more than 5 months. a human myeloma cell line may eventually prove useful in creating mabs against certain autoimmune diseases and cancers. this technique may also make it easier for other researchers to generate human mabs for use in therapy. antibodies may act directly when binding to a target molecule by inducing apoptosis, inhibiting cell growth, mimicking or blocking a ligand, or interfering with a key function. 53, 59 in addition, antibodies may modulate or potentiate drugs or other therapies. the antibody may itself act as an effector-as in antibody-dependent cellular cytotoxicity (adcc) or antibody-dependent complement-mediated cascade-or it may involve effector elements such as cytotoxins, enzymes, radioactive isotopes, signals for other parts of the immune system, and/or cytotoxic drugs. 49,50,60 -62 adcc occurs if fc regions on the antibodies are recognized by receptors present on cytotoxic cells, such as natural killer cells, macrophages, granulocytes, and monocytes. complement-mediated cytotoxicity ensues if the antibody binding prompts a complement cascade to occur. active immunotherapy can be accomplished if the antigen can provoke a long-lasting t-cell response, which may be achieved by administering whole cancer cell extracts or by using small antigenic peptides isolated from tumors in experimental patients or animals. 58 for instance, mabs mimicking breast cancer-specific antigens elicit anti-idiotype antibodies; this is another way of creating active immunity, which can lead to a humoral auto antibody-like immune response. 58 the use of bispecific molecules that recognize antigens on both the target cell and effector cell can increase adcc. the most effective mechanisms are blockade of a crucial ligand or growth factor or adcc in which tumor cells are killed by fc receptorbearing cytotoxic effectors. cell killing by adcc is proportional to the amount of antibody bound to a cell, whereas the blockade of an essential growth factor may not show effects until most of its receptor is saturated. a higher antigen expression by the target cell will increase antibody binding and subsequent adcc. but high receptor expression will also make it difficult to prevent the binding of a cytokine or ligand at minimum threshold. 53, 59, 63 with a bispecific antibody, the specificity of mab is combined with the cytotoxicity of immune effector cells, for instance, to neutralize a tumor. 9, 45, 63, 64 bispecific antibodies link the tumor cell directly to the killer cell via cytotoxic trigger molecules, such as t-cell receptors or fc receptors, leading to lysis and/or phagocytosis by the effector cells. although bispecific antibodies can enrich effectors at the tumor site and activate tumor bound effector cells by enabling cross-linking between effector and target cells, they can cause system-wide immune activation because of t-cell receptor cross-linking. bispecific antibodies can also mediate cellular cytotoxicity via various effector cells, including phagocytes, natural killer cells, and t-lymphocytes. another way to use the binding properties of antibodies is by conjugating antibodies to cytotoxic drugs, radioisotopes, or toxins. 49, 58, 63, 65, 66 techniques for conjugation have been described in several recent reviews and articles. 49, 58, 67, 70 mabs can be conjugated to chemotherapeutic drugs such as doxorubicin, mitomycin, and methotrexate. chemotherapeutic agents constitute cytotoxic or cytostatic drugs that can be conjugated to antibodies; thus far, however, these drugs have shown poor specificity for target cells and frequently lead to toxicity. 55, 62, 65 often, antibodies may lose reactivity upon conjugation with such agents. immunotoxins used in cancer therapy are conjugated antibodies that combine plant (ricin, gelonin, saponin, abrin, pokeweed antiviral protein) or bacterial (diphtheria toxin and pseudomonas toxin a) toxins with antibody specificity. 52, 59, 62, 63, 65, 66, 68, 69 toxins may inhibit protein synthesis even at picomolar concentrations. natural toxins, such as diphtheria toxin, can act at low concentrations, enter cells, and disrupt key cellular processes. 69 enzymes may also be conjugated to antibodies in antibody-directed enzyme-prodrug therapy. 55, 61, 62, 66, 71 an enzyme that can convert a prodrug to an active form may be conjugated to an antibody and targeted to a desired location using the antibody-binding region. they may more effectively localize tumors than chemical conjugates, and a prodrug is converted to an active drug by the enzyme at the target cells. another therapeutic technique relies on using antibody fragments, which may be produced by the traditional method (proteolytic digestion with papain) or with newer methods that may reduce damage to the binding sites. 66, 71 some newer methods attempt to construct mimetics that combine multiple cdrs from several antibodies into single molecules with molecular weights substantially lower than single chain fragments. because of their smaller size, antibody fragments may be able to penetrate tissues and tumors more readily and be less immunogenic than whole mabs. however, although antibody fragments may have better penetration than whole antibodies, their shorter half-lives may compromise clinical usefulness. 68 heteropolymerized antibodies have recently been developed by chemically linking a mouse igg mab, specific to a complement receptor site (cr1) on the human or nonhuman primate erythrocyte, to a second mouse igg mab that is specific to the targeted antigen. 72 the heteropolymer technique is based on immune adherence, in which antibody-antigen immune complexes bind to a complement receptor on the red blood cell, facilitating the phagocytosis of these complexes. 72 once introduced into the patient, the heteropolymers will bind to both the red blood cells and the targeted antigen. the antigen-antibody heteropolymer complexes are then transported to the liver and spleen, where the complexes are destroyed by macrophages, whereas the red blood cells return to the circulation unharmed. [72] [73] [74] [75] a wide variety of conditions, of both autoimmune and foreign origin, may potentially be treated with this method, including hiv infection, systemic lupus erythematosus, marburg virus infection, and myasthenia gravis. 76 -78 although mouse antibodies are currently being used with this technique, chimeric mousehuman antibodies could also be constructed. 72 it may also be possible to use the heteropolymers as "sentinels" by injecting them before antigen exposure. in 1 recent study, multiple infusions of heteropolymers provided nonimmunized monkeys with protection against an antigen (⌽174) for as long as 2 weeks. 79 when milstein and kohler announced their isolation of a mab in 1975, many thought mabs would provide an effective cancer treatment. however, early clinical trials were disappointing. 80 -81 producing mabs to tumor antigens is a complex process. first, proteins peculiar to human cancer cells are identified; then, mice are injected with human tumor cells (antigen) to stimulate an immune response. after about 30 days, the mouse lymphocytes are removed and fused with myeloma cells to isolate and reproduce hybrid cells specific to a predetermined antigen. rodent antibodies alone have been used in several trials, but the clinical effects are often minimal because of poor activation of human effector or complement cells by the fc region of the murine antibody and the accompanying hama responses. 55, 57 substituting the rodent fc region with a human fc fragment may help overcome these effects. 81 with the discovery of proto-oncogenes, secondgeneration trials in the 1990s turned to more specific tumor antigens that could be potential targets. 58 in 1994, mab 17-1a, an antibody to epithelial cell surface antigen expressed on human colorectal carcinomas, was approved for the identification of adenocarcinomas; mab17-1a may reduce the mortality and occurrence rate of colorectal cancer. 82, 83 in 1997, rituximab, a mouse-human chimeric anti-cd20 antibody, was approved for the treatment of non-hodgkin b cell lymphoma. 53, 83 rituximab binds to cd20 antigen on b cells and b cell tumors and then elicits a natural immune response that can kill malignant cells. recent animal trials have shown the potential of mab therapy in cancer. 52 increases in epidermal growth factor (egf) receptor expression have been found on many human cancers, and a fully human anti-egf receptor igg2 mab has been shown to inhibit human cancer growth in vitro and in vivo. 22 other efforts are focusing on antibody to her2 antigen in breast cancer. patients with cancer often mount an immunologic response to tumor-associated antigens with increases in cytotoxic lymphocytes and antibodies. 14 several tumor-reactive antibodies have been cloned against melanomas, colon carcinomas, ovarian, breast, and lung tumors. often, these antibodies cross-react with other malignant tissues or cell lines. 14 recently, trastuzumab (herceptin), a humanized mab that binds directly to the c-erb2 protein (her2), has been approved by the fda for the treatment of breast cancer. 52,84 -89 herceptin can be used to treat metastatic breast cancer in patients whose tumors overexpress the her2 protein (about 30% of breast cancer patients) and may be used in conjunction with other therapies such as paclitaxel (taxol). 89 antibody was developed by humanizing murine mab 4d5 by inserting antigen-binding regions of mab 4d5 into the framework of a consensus human igg1, resulting in rhumab-her2 or trastuzumab. 85 cancer cells have antigens that are specific to the tumor (tumor-specific antigens) or are present in greater concentration than normal (tumor-associated antigens). antibodies may eliminate target cells by complement action or through adcc. the variable region of the antibody recognizes and attaches to a specific antigen and the constant region, then joins with an effector cell capable of killing the targeted cancer cell. when the antigen and antibody bind, precipitation and agglutination may isolate the complex. anti-idiotype mabs that mimic-tumor associated antigens also may be used in cancer therapy. 82 the idiotype network hypothesis, formulated by lindemann and jerne, suggests that because each mature b cell secretes an antibody with unique antigenbinding specificity in the variable domain (referred to as the idiotype), anti-idiotype mabs can be generated and used as surrogate antigens or vaccines for immunization against the tumor. 82, 90 a murine anti-idiotype mab, aca125, which mimics the tumor-associated antigen ca125, was recently found to induce a specific anti-anti-idiotypic immune response in 28 of 42 patients with platinum-pretreated recurrent ovarian cancer enrolled in a phase i/ii clinical trial. 90,91 a positive immune response was associated with statistically significant (p ͻ 0.0001) prolonged survival times (19.9 ϯ 3.1 months in patients shown to have an anti-antiidiotypic response compared with 5.3 ϯ 4.3 months in patients who were anti-anti-idiotypic negative). 90, 91 in addition, peripheral blood lymphocyte mediated lysis of ca-125 expressing tumor cells increased in 9 of 18 patients after vaccination with the mab. 91 despite the use of a murine antibody, both this study and a previous phase i study involving patients with ovarian cancer found minimal side effects. 90, 91 promising early results have also been demonstrated in patients with advanced colorectal carcinoma who received a murine anti-idiotype mab that mimics an epitope of carcinoembryonic antigen (ceavac) 82, 90 and in patients with malignant melanoma who received an anti-idiotype mab (trigem) that mimics disialoganglioside gd2 92 despite promising developments, however, there are still several obstacles to effective cancer therapy with mabs. problems with the tumor, the mab, or conjugate characteristics all continue to challenge researchers. 52, 63, 84 thus far, chemotherapeutic mab therapies have faced obstacles due to the poor ability of agents to affect tumor cells preferentially over healthy cells, the intrinsic insensitivity of many tumors to these drugs, and the rapid development of resistance in tumor cells. 65, 66, 93, 94 mabs often decrease the size of tumors, but rarely lead to complete remission of solid tumors. 59 at the cellular level, mabs at low doses ideally should bind with excellent affinities. because no antigens have been identified that are expressed exclusively on tumor cells, cross-reactivity of mabs must be examined with histochemical tests on tissue sections or in animal models. careful testing must also be done to ensure that the effector molecules such as drugs, toxins, or isotopes in the conjugates do not inadvertently target healthy cells. although humanized antibodies have greater affinity than murine antibodies, this potential has yet to be translated into improved clinical outcomes against cancer. 52 however, recent success using the xenomouse technology to develop a fully human igg2 mab specific to epidermal growth factor receptor indicates the potential for future advances in this area. 22 recombinant immunotoxins, fusion proteins containing the fv of a mab and a bacterial toxin, are under development for cancer therapy. because immunotoxins exploit only the variable region-binding function of the mab, in theory just a fragment of the binding region, such as the bivalent f(ab')2 fragment, monovalent fab, scfv, or disulfide-stabilized fv fragments may be used as opposed to the larger and probably more immunogenic whole antibody. disulfide-stabilized fragments may be more stable or have higher affinity for the antigen than scfv, in which the linker may interfere with binding or fail to stabilize the fragment. 66 recombinant immunotoxins derived from pseudomonas enterotoxin have been shown to be active against lymphomas, solid tumors, and leukemias. 66 because binding specificity and affinity are the key elements, it is not known which form of the immunoconjugate is usually more effective in treating human tumors; some animal tests suggest that the whole antibody, with its longer half-life and bivalent binding, is more effective than the antibody fragments with decreased binding affinity. bispecific antibodies and single chain fvs are being developed that may be more effective because they clear faster from nontumor tissues and more deeply penetrate tumors than whole antibodies. 49, 64, 70 bispecific antibodies may be used to target tumor vascular endothelial cells, thereby limiting the tumor's blood supply and possibly inhibiting its spread. 70 advances in radiolabeling have allowed immunoconjugates to be delivered to cells and showed promise in clinical trials. 14, 63, 80, 95, 96 radioimmunotherapy uses a radiolabeled mab to deliver radioactive isotopes to targeted cells. radioisotopes such as iodine-131 and yttrium-90, which are ␤ emitters, can cause damage not only to the bound cell but also to cells adjacent to tumor cells that antibodies may not be able to reach within the tumors. lack of knowledge about the appropriate dose, biodistribution, and shedding of antigen hinders use of radioisotopes. radiolabeled mabs may also affect normal cells, depending on the extent to which reticuloendothelial cells expressing fc receptors bind to the constant regions of intact antibody molecules. using antibody fragments or constructs may modify this nonspecific uptake. 63, 68, 82, 93 radiotherapy exerts most of its effect by emitting a low dose, which exponentially decreases continuous radiation, but the antibody per se may have a cytotoxic effect. the duration of radiotherapy is determined by the half-lives of the antibody and the isotope used. the success of radioimmunotherapy is affected by the specificity, affinity, dose, and immunoreactivity of the antibody, heterogeneity of antigen expression, diffusion rate, tumor volume, blood supply and tumor location, dose rate effects, and variability in dose deposition. the choice of radionuclide, selection of the chelate used to link the mab to the radionuclide, and the mab selected are critical in development of radiolabeled mabs. 80 suitable radio nuclides include yttrium-90, iodine-131, and copper-67. iodine-131 was the first radioisotope to be used in treatment of hodgkin disease and other lymphomas. 68 yttrium-90 is potentially useful for lymphoma therapy because it decays with ␤, but not ␥, emissions that may kill other tumor cells in a cross-fire effect. 68 the energy released by yttrium is 5 times higher than that of iodine-131, which degrades rapidly after uptake into a tumor cell, causing toxicity. 80 in addition to their uses in radiotherapy, radiolabeled mabs can also be used to diagnose cancer. 18, 56, 57, 82, 94 radioactive isotopes linked with mabs may help localize tumors in a form of diagnostic imaging called immunoscintigraphy. for instance, oncoscint, a mab coupled to indium-111, may be used to detect an antigen (tag-72) found on colorectal adenocarcinomas. 56 mabs may potentially be used to suppress the immune system after transplant or to induce tolerance to transplanted organs or tissues. thus far, however, only antibodies to cd3 and cd25 are licensed for clinical use. 97 okt3, a murine igg2a antibody to human cd3, and antibodies to cd25 (il-2 receptor) have been used to reduce allograft rejection. 97 graft-versus-host disease (gvhd) is a complication of allogeneic stem cell transplant that occurs despite histocompatibility testing and use of cyclosporine and its analogs. 98 gvhd is a frequent cause of illness or death in allogeneic transplant patients and occurs when alloselective donor t cells recognize and interact with major and minor histocompatibility antiigens in the host, leading to cytokine release. mab therapy against gvhd is most effective when it is administered after a bone marrow transplant but before gvhd development by targeting t-cells before their activation. 99 for instance, 1 target is the interleukin-2 receptor ␣-chain (il-2r␣ tac protein or cd25), whose expression is a crucial step in the activation of alloreactive t cells. 97, 100 recently, it was reported that daclizumab, a humanized anti-il-2a antibody, was an effective complement to dual immunosuppression therapy in renal transplant patients. 101 because only activated cells express il-2, antibodies to this cytokine might inhibit t cells during allograft rejection and prevent generation of cytotoxic t cells. the development of hama response in patients and the decreased effectiveness of murine mab relative to human mab have thus far compromised the effectiveness of this approach. however, humanized anti-tac mabs, with better human effector functions, may survive longer in vivo and may prove less immunogenic than its murine counterpart. short-term cd4 antibody therapy may also contribute to long-term acceptance of skin and islet allografts, even after gaining immunocompetence. 102 more generally, the ability of mabs to induce tolerance to transplanted tissues and self-antigens may hold great therapeutic potential. 97, 102 mabs to cd4 and cd8 have been studied, although thus far these have not resulted in clinical success. 97 depletion or blockade of t cells by antibodies may facilitate tolerance by preventing t cells from attacking the graft, and recent t cell depletion studies in primates have proven promising. 97 mab treatments for other complications after transplants are also under study, including potential treatment of posttransplant lymphoproliferative disorder with the anti-cd20 mab rituximab and the use of anti-lfa-1 mab (odulimomab) to protect against ischemia-reperfusion injury after kidney transplants. [103] [104] [105] daclizumab, a humanized antibody that targets the anti il-2 receptor, may reduce the risk of acute rejection of a renal transplant and also lower cytomegalovirus infection rates among transplant recipients. 106 the inflammatory bowel disorder known as crohn disease has also been treated with mab therapy. 107 a chimeric igg1k antibody, infliximab (remicade), acts by binding to soluble and transmembrane tumor necrosis factor ␣ (tnf␣), preventing it from binding to its receptors on activated macrophages. the anti-tnf␣ antibody may provide relief to patients with moderately to severely active crohn's disease. in addition, the fda recently approved infliximab for the treatment of rheumatoid arthritis in combination with methotrexate. 108 -111 high levels of ige may cause bronchial hyperresponsiveness, a risk factor for asthma. [112] [113] [114] im-therapeutic applications of monoclonal antibodies mune responses mediated by ige are important in the pathogenesis of allergic asthma. in 1 recent study with subjects reporting moderate to severe allergic asthma, twice-weekly injections of recombinant humanized anti-ige antibody (rhumab-e25), which forms complexes with free ige and blocks its interaction with mast cells and basophils led to a fall in serum ige levels and slightly decreased asthma symptom scores relative to the placebo group. 112 patients receiving anti-ige were able to reduce reliance on corticosteroids. although the study must be interpreted cautiously, it is nonetheless a promising step in finding more effective asthma therapies. 112, 114, 116 approximately 400,000 cases of sepsis in the us and about 25,000 cases in the uk are reported each year. 116, 117 the mortality rate could be as high as 40 to 70%, depending on the population studied. 117, 118 mabs have been targeted to tnf␣ and tnf␣ receptors, which are key elements of the inflammatory response. unfortunately, this may inhibit many cytokines, which can impair the patients' ability to fight infection and increase the risk of secondary sepsis; also inhibiting a few cytokines may not be sufficient. although mabs targeted toward components of the host immune system, such as individual receptors or mediator cells, help in the treatment of septic shock, targeting the bacterial endotoxin or lipid a of gram-negative bacteria with mabs may be more efficacious. lipopolysaccharide endotoxin, a component of the outer cell membrane of gramnegative bacteria, consisting of a highly variable o-linked polysaccharide chain, an r core region, and lipid a, which in turn is composed of a glucosamine disaccharide backbone substituted with amide-and ester-linked long-chain fatty acids, is believed to be important in many cases of septic shock and septicemia, and can trigger an inflammatory cascade that can cause serious injury and even death. 119, 120 lipid a is linked by the core region to the o-linked side chain, and variations in the o-linked side chains result in an enormous diversity among gram-negative bacteria, making adoptive immunotherapy against the o-linked antigenic determinants difficult. 114 directing antibodies to the core region or lipid a, which tend to be more conserved, would allow for therapy against a diverse array of gram-negative bacteria. favorable results with polyclonal j5 antibody have spurred attempts to develop a mab. 116, 117 a murine igm mab, e5 (xoma, berkeley, ca) binds to an epitope on lipid a of e coli j5. a human igm, ha-1a (centoxin) is derived from a heterohybridoma fused from spleen cells of a patient vaccinated with e coli j5 before splenectomy. 116, 117, 119, 121 both the antibodies have thus far shown mixed success in patients with sepsis and neither is currently cleared by the fda for therapeutic use. because patients with bacteremia often have endotoxemia, ha-1a, an igm antibody to endotoxin may be effective against endotoxin in the bloodstream as well. unfortunately, the high cost of antibody ($3500/dose in europe) combined with the uncertainty that the sepsis is caused by gram-negative bacteria has hindered the widespread use of ha-1a. 112, 115, 117 although it is less efficacious, the only other option may be to target tnf␣, but 1 recent study reported a high (54.1%) rate of adverse reactions in patients receiving an anti-tnf␣ antibody. 118 the antibody was no more effective than the placebo given to control subjects. however, research on a mab for the treatment of septicemia continues. cytomegalovirus (cmv) causes serious illness affecting the immunocompromised, such as patients with aids and those undergoing organ transplants. infection rates may be up to 75% in those negative for cmv who receive kidneys from seropositive patients. 122 cmv infection can result in retinitis and gastroenteritis in hiv-infected patients and may also cause chronic intrauterine infection. 122, 123 up to 40,000 cases of congenital cmv infection are reported each year; mental retardation and hearing loss may occur in about 25% of these cases. 122 currently, there is no vaccine against cmv. ganciclovir, foscarnet, and (s)-1-[3-hydroxy-(2 phosphonylmethoxy)propyl]cytosine are some of the potential treatments for cmv infection. 122 another mode of treatment is via administration of anti-cmv hyperimmunoglobulin, derived from pooled sera of cmvseropositive persons. passive immunization has been shown to reduce the severity of cmv and prevent mother-to-infant transmissions. 122 moreover, antibodies may be able to clear the virus from infected tissues, a function previously thought to be exclusive to cytotoxic t lymphocytes. many physicians use a combination of antiviral agents and immunoglobulins in patients at risk for cmv infection. mabs may also decrease the amount of antiviral agents required for treatment. mabs against murine cmv polypeptides have been shown to be protective in animal models. 124 although mabs may act synergistically with foscarnet or ganciclovir in vitro, it is unclear whether this advantage could be extrapolated to in vivo conditions. experiments in which murine cmv is used as a model system suggest that high concentrations of antibody, along with ganciclovir or (s)-1-[3-hydroxy-(2 phosphonylmethoxy)propyl]cytosine, synergistically inhibit the growth of murine cmv in cell culture, whereas lower antibody concentrations produce only an additive effect. however, antibody ad-ministered along with ganciclovir in severe combined immunodeficient mice also produced only an additive effect. 123 the extent to which a mab can protect the host from cmv infection cannot reliably be predicted from its immunoreactivity, neutralizing titers in vitro, or from its antigen-binding data, because the mechanism of action of a mab in vitro and in vivo may be entirely different. 124 if an antibody is used along with drugs, not only is the treatment efficacy improved but also the side effects of a high drug concentration may be avoided. for instance, 25 mg of ganciclovir/kg of body weight, along with an antibody, was almost as effective as 50 mg/kg ganciclovir administered alone in cmv-infected severe combined immunodeficient mice. 124 the msl109 antibody, a human igg1 directed against human cmv glycoprotein, is marginally synergistic in inhibiting cmv in vitro when conjugated to ganciclovir or foscarnet. in a recent trial involving 209 aids patients with active cmv retinitis, the rate of retinitis progression was similar in the group receiving the antibody (60 mg, intravenous) for 2 weeks and the placebo control group. 125 surprisingly, mortality increased in the msl-109 group, and the poor performance of msl-109 in this trial may have been due to the inability of the antibody to neutralize the established cmv infection in aids patients or its failure to cross the blood-ocular barrier in sufficient quantities. liver transplant patients are also at risk of cmv infection and may require treatment with the murine mab okt3, an anti-t cell antibody that is directed to cd3 antigen, along with ganciclovir to mitigate transplant rejection. 126 human mabs neutralizing cmv were tested for their safety and pharmacokinetics over a period of 3 to 73 days in bone marrow transplant recipients and were found to be safe and efficacious, as evidenced by a steady increase in the neutralizing activity. 127 also, a human anti-cmv mab designated c23 that was purified from hybridomas generated by the fusion of human lymphocytes with mouse myeloma cells has virus neutralization titers about 1000-fold higher than those produced after conventional human ␥ globulin used in humans. 128 a humanized mab has been developed that binds to the 86-kd glycoprotein, gpul75 (gh), of cmv, recognizes a variety of virus strains, and neutralizes clinical isolates of cmv; it could be used as a potential agent for the prevention or treatment of cmv infections in humans. 129 respiratory syncytial virus (rsv) causes serious lower respiratory tract disease requiring considerable supportive care, administration of humidified oxygen, and respiratory assistance. the fda approved the use of a humanized mab against rsv, which afflicts mostly infants and children younger than 24 months of age. 130, 131 known as palivizumab, the antibody (produced by synagis and medimmune, inc.) treatment showed a 55% reduction in rsv infection in hospitalized patients. 130, 131 the american academy of pediatrics' committee on infectious diseases has published recommendations on the use of this antibody in children at risk for rsv infection. 130, 131 mabs have also been used in the therapy of hsv infections. the incidence of neonatal herpes is 30 to 50% in babies born vaginally to mothers with primary infection but only 1 to 3% in babies born to mothers with recurrent infections. 132 this difference is attributed to the passive transfer of protective antibodies to the fetus. antibodies to hsv-1 may also confer partial protection against type 2 strains. based on this observation, the potential of mab therapy against herpes has been studied for more than a decade. experiments in mice, for instance, indicate that microgram quantities of antibodies may promote healing of corneal opacity and blepharitis caused by herpes simplex. 133 however, attempts to confer immunity in humans and other higher animals by passive immunization alone have been disappointing, because antibodies to hsv are restricted to only a few specific epitopes. 133 among the 11 glycoproteins expressed by the herpes virus on its surface, 5 glycoproteins (gb, gd, gh, gk, and gl) are thought to be crucial to infection. the initial step in hsv infection seems to be the binding of gc to heparan sulfate proteoglycans followed by the binding of gb, and the binding of gb and gc is stabilized by gd. gh is involved in initiation of viral fusion and other glycoproteins may help in expanding the fusion process. evidence that neutralizing antibodies to gh, gd, and gb prevent membrane fusion but not viral attachment is consistent with this model. 133 most human antibodies are directed against gd and gb epitopes. mabs can target at least 7 different antigenic sites on hsv glycoprotein d in the murine model. 133, 134 glycoprotein d is necessary for virus replication in tissue culture. yamamoto et al 135 reported the antibody-dependent cellular cytotoxicity effect of hs1, a neutralizing mab to glycoprotein b of hsv, in athymic nude mice inoculated with hsv intracutaneously. when hs1 is administered, the skin lesions healed in 50% of the mice given the antibody. also, latent infection in the ganglia was prevented in mice that survived, as evidenced by the failure to detect hsv upon co-cultivation with vero cells. administration of hs1 after development of zosteriform lesions (5 to 9 days after infection) reduced the virus in the ganglia and prolonged survival time; however, the disease was not totally avoided and eventually the mice succumbed to the disease. the antibody therapy could prevent or decrease the severity of herpetic keratitis, iritis, and blepharitis after corneal infection by hsv. 136, 137 thus, mabs could potentially be used to prevent congenital herpes and sexual transmission of the herpes virus. 47, 133, 138, 139 currently, no treatments exist for the hemorrhagic fever caused by the filovirus ebola. recently, a team of researchers at the us army's medical research institute identified protective antibodies directed against epitopes on ebola virus membraneanchored glycoprotein. some of the antibodies protected mice as long as 2 days after exposure; doses were equivalent to acceptable (3 to 5 mg/kg) human levels. antibody specificity is important, because some mabs studied bound to ebola zaire but not to the ivory coast or sudan serotypes. much research remains to be done before the potential of antibodies in the treatment of ebola will become realized. 140 toxins are poisonous proteinaceous substances. antibodies were first clinically used in the 1970s for protection against the toxin digitalis. 48, 102 today, antidigitalis immunotoxicotherapy is a standard therapy. digoxin is produced from sheep immunized with digoxin-serum albumin conjugate. igg antibody specific for digoxin is purified and cleaved into fabs, because the smaller molecular mass of the fragments allows faster renal clearance and more rapid action than the whole antibody. the fab fragments bind to circulating digitalis molecules and generate a complex that is unable to bind to receptors. adverse effects are rare, and the immunotherapy is generally effective against digoxin and digitoxin within 1 hour. 120, 141 however, because polyclonal antibodies may be difficult to produce and may cause hypersensitive reactions, researchers have attempted to develop humanized mabs against digoxin and other toxins. 141 for example, 1 group has used a transgenic mouse to produce hybridomasecreted human mabs against digoxin. 141 more recently, the effect of goat anti-colchicine igg antibodies has been studied in mice exposed to a lethal intraperitoneal dose of anti-inflammatory colchicines, and preliminary studies in a 25-yearold woman with colchicine poisoning showed promise. 102, 141 attempts have also been made to use drug-specific goat antibody fragments to treat anti-tricyclic antidepressant (tca) overdose. 120, 141 antidepressant overdose is the most common cause of intentional drug overdose in the us. however, lethal doses of tricyclics are 10-to 100-fold higher than those of colchicine, digoxin, and snake venom, which require higher antibody doses (up to several g/kg) to reverse the toxicity. 120, 142 high-affinity tricyclic antidepressant-specific mabs have been shown to reverse the cardiovascular toxicity of antidepressant desipramine in rats and prolong their survival, and tricyclic antidepressant-specific fab fragments, along with sodium bicarbonate, a standard treatment for tricyclic overdose, also minimizes the required dose of antibody. 142 the use of smaller antibody fragments, such as single chain fv fragments, half the size of a fab, may also be a promising approach because the single chain fragment retains affinity and has shorter half-life in the body. monoclonal antibodies may also help to alleviate poisoning due to environmental contamination. hexachlorobiphenyl, paraquat, atrazine, 3,5,6-trichloro-2-pyridinol, the chief degradation byproduct of the insecticides chloropyrifos, triclopyr, and chloroprifosmethyl, and other chemicals may soon be detected and remediated with the help of antibodies. 139, [143] [144] [145] antibodies may also be used in the treatment of poisoning due to paraquat, hexachlorobiphenyl, domoic acid [amnesic shellfish poisoning], and other contaminants that are otherwise difficult to remove from the body or surrounding environment. [145] [146] [147] [148] the primary target of many abused drugs is the central nervous system (cns), and immunotherapy against such chemicals must be able to penetrate the cns. 149 pcp or phencyclidine (angel dust) is a type of arylcyclohexamine that affects multiple sites in the brain. 150, 151 it is linked to violent psychotic episodes that are similar to schizophrenia. 149 treatment is difficult because there is no known antagonist identified, pcp has a high volume of distribution, and about 95% of it is cleared after being metabolized. 151 recently, mabs have been described that could bind to cocaine or pcp and act like sponges in the bloodstream to prevent them from reaching the brain. 152 it has been shown that a single dose of antibody reduced pcp effects for up to 2 weeks in animals, which might be equivalent to a several months in humans. high-affinity antibody 6b5 fragments against pcp function better in that the fragments with bound pcp are more quickly cleared from the body than the whole antibodybound pcp. 153 the crystal structure of the antibody fragment complexed with pcp has been studied in detail. 154 antibody fragments are also preferable to whole antibodies in the treatment of pcp addiction because of their lower antigenicity and improved pharmacokinetics. 149, 151 anti-idiotype antibodies could potentially be produced against the drug-mab binding site and mimic drug structural features, although these antibodies do not cross the bloodbrain barrier. 151 by increasing protein binding in the vascular compartment and lowering the drug's volume of distribution, the antibody acts like a pharmacokinetic antagonist. 150 evidence that the antibody can also reverse effects of other potent arylcyclohexylamine drugs suggests that antibody medications can be used to treat different classes of drugs. 150, 151 an anti-pcp igg has been produced from a hybridoma cell line using both ascites and bioreactor methods, and the pharmacological and immune specificity of the antibody has been confirmed by administering the anti-pcp fragment to rats in three different studies without producing observable effects in behavior. 155 active immunotherapy against methamphetamine addiction is also being contemplated. 156 antibody therapy for cocaine addiction is also a possibility, especially with the development of catalytic antibodies having enzyme-like characteristics that can be used to inactivate drugs. 151, [157] [158] [159] antibodies with esterase activity have been successfully mimicked, and because cocaine is believed to be metabolized by in vivo esterases, such antibodies would be useful in the treatment of cocaine addiction. 151 catalytic antibodies probably would not be as useful as high-affinity anti-cocaine antibodies, but could be used in medical emergencies or as part of a withdrawal therapy. 151 potential antibody applications against cocaine addiction have been tested in rats with a vaccine approach by (1) attaching cocaine molecules to a carrier protein for immunizing rats to produce antibodies against cocaine and (2) humanizing anticocaine antibodies derived from rats and producing them in bacteria. 152, 160 a catalytic mab (15a10) has recently been reported to attenuate cocaine's cardiovascular effects in mice. 155, 159 conclusion about 200 years have elapsed since edward jenner vaccinated a young child against smallpox. since that time, the field of immunology has evolved at a rapid pace and has yielded many critical developments. although vaccination has thus far proven to be the most cost-effective method of preventing diseases worldwide, the development of mabs that use the specificity of immunological responses is one of the most successful applications of immunology to date. chimeric and humanized antibodies have reduced the risk of allergenicity from exposure to nonself antibodies and heightened the clinical effectiveness of mab treatments. developments in radiology and pharmacology have allowed radiolabeled and immunoconjugated antibodies to be produced. antibody fragments, heteropolymers, and bispecific antibodies are now available in addition to whole mabs. these promising developments may soon allow mabs to be used to treat afflictions as varied as substance abuse, cancer, asthma, viral infection, septicemia, and poisoning. as heddy zola observed in 1995, "the therapeutic application of mabs, whilst still limited in scope, promises to break its substantial shackles and realize the potential forecast by its proponents." 46 on immunity: with special reference to cell life ehrlich's passion: the origins of his receptor immunology the natural selection theory of antibody formation a modification of jerne's theory of antibody production using the concept of clonal selection the hydrolysis of rabbit gamma globulin and antibodies with crystalline papain selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants continuous cultures of fused cells secreting antibody of predefined specificity with the benefit of hindsight antibodies in diagnostics-from immunoassays to protein chips igg subclasses-a review biological activities of immunoglobulins of different classes and subclasses preparation of rodent monoclonal antibodies by in vitro somatic hybridization monoclonal antibodies in clinical applications monoclonal antibodies: the second generation. herndon (va): bios scientific a human myeloma cell line suitable for the generation of human monoclonal antibodies the optimal technological approach to the development of human hybridomas continuous production of monoclonal rheumatoid factor by ebv-transformed lymphocytes clinical use of monoclonal antibodies production of clinical grade monoclonal antibodies. presentation at international business communications fifth annual antibody production & downstream processing conference introduction to antibody engineering and phage display natural and designer binding sites made by phage display technology development of abx-egf, a fully human anti-egf receptor monoclonal antibody, for cancer therapy antibody engineering via genetic engineering of the mouse: xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing ig heavy and loci and expression of fully human antibodies the trimera mouse: generating human monoclonal antibodies and an animal model for human diseases lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus transgenic mice secreting coronavirus neutralizing antibodies into the milk the future of transgenics. regulatory affairs focus expression and assembly of a full-length monoclonal antibody in plants using a plant virus vector characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans generation and assembly of secretory antibodies in plants exploring transgenic plants as a new vaccine source oral immunization with a recombinant bacterial antigen produced in transgenic plants tobacco can be good for you. the new york times rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain fv epitopes in tobacco plants production of antibodies in transgenic plants a humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes production of a diagnostic monoclonal antibody in perennial alfalfa plants using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases points to consider in the manufacture and testing of monoclonal antibody products for human use guidance for industry. s6 preclinical safety evaluation of biotechnology-derived pharmaceuticals center for biologics evaluation and research and center for drug evaluation and research, us food & drug administration points to consider in the production and testing of therapeutic products for human use derived from transgenic animals points to consider in the production and testing of new drugs and biologicals produced by recombinant dna technology points to consider in the characterization of cell lines used to produce biologicals. center for biologics evaluation and research, us food & drug administration monoclonal antibodies: the second generation human antibodies by design genetically engineered antibody molecules engineering antibodies for therapy recombinant antibodies: alternative strategies for developing and manipulating murine-derived monoclonal antibodies monoclonal antibody-based therapy for solid tumors: an overview monoclonal antibodies: innovations in diagnosis and therapy unconjugated monoclonal antibody therapy of lymphoma monoclonal antibodies: the second generation. herndon (va): bios scientific preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by cdr grafting the promise and pitfalls of monoclonal antibody therapeutics radiolabeled monoclonal antibodies monoclonal antibody-based therapy of breast cancer monoclonal antibodies in cancer therapy antibody-based therapies for hodgkin's disease principles of antibody therapy reconstruction of monoclonal antibodies by genetic engineering genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents immunotherapeutic perspective for bispecific antibodies monoclonal antibodybased therapy of cancer recombinant fv immunotoxins and fv fragments as novel agents for cancer therapy and diagnosis photoreduction of monoclonal antibodies for conjugation and fragmentation monoclonal antibody-based therapies of leukemia and lymphoma immunotoxin therapy of lymphoma: studies with anti-b4-blocked ricin the use of bispecific antibodies in tumor cell and tumor vasculature directed immunotherapy production of fab fragments of monoclonal antibodies using polyelectrolyte complexes use of heteropolymeric monoclonal antibodies to attach antigens to the c3b receptor of human erythrocytes: a potential therapeutic treatment bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model the primate erythrocyte complement receptor (cr1) as a privileged site: binding of immunoglobulin g to erythrocyte cr1 does not target erythrocytes for phagocytosis antigenbased heteropolymers facilitate, via primate erythrocyte complement receptor type 1, rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys quantitative studies of heteropolymer-mediated binding of inactivated marburg virus to the complement receptor on primate erythrocytes antigen-based heteropolymers, a potential therapy for binding and clearing autoantibodies via erythrocyte cr1 infusion of bispecific monoclonal antibody complexes into monkeys provides immunologic protection against later challenge with a model pathogen radioimmunotherapy of interleukin-2r alpha-expressing adult t-cell leukemia with yttrium-90-labeled anti-tac monoclonal antibody therapy of cancer colorectal cancer as a model for immunotherapy advances in cancer immunotherapy antibody-targeted immunotherapy for treatment of malignancy first mab approved for treatment of metastatic breast cancer breast cancer biology blossoms in the clinic response of metastatic breast cancer to trastuzumab anti-her2 antibody and heregulin suppress growth of her2-overexpressing human breast cancer cells through different mechanisms tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents are solid tumor anti-idiotype vaccines ready for prime time? immunological 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rheumatoid arthritis monoclonal antibody therapy in rheumatoid arthritis treatment of allergic asthma with monoclonal anti-ige antibody. rhu mab-e25 study group treatment of allergic asthma with monoclonal anti-ige antibody anti-ige therapy for asthma treatment of allergic asthma with monoclonal anti-ige antibody monoclonal antibodies in sepsis and septic shock treatment of gram-negative bacteremia and septic shock with ha-1a human monoclonal antibody against endotoxin. a randomized, double-blind, placebo-controlled trial double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock. norasept ii study group adoptive immunotherapy of gram-negative sepsis: use of monoclonal antibodies to lipopolysaccharide antibodies proposed as therapeutic agents anti-endotoxin monoclonal antibodies human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries effects of monoclonal antibody combined 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cocaine. i. monoclonal antibody 15a10 and the reinforcing effects of cocaine in rats evaluation of anti-cocaine antibodies and a cocaine vaccine in a rat selfadministration model we thank carolyn m. black and the national center for infectious diseases editorial office for the critical review of this manuscript. key: cord-331288-elnwn7l5 authors: grützmacher, kim; keil, verena; leinert, vera; leguillon, floraine; henlin, arthur; couacy‐hymann, emmanuel; köndgen, sophie; lang, alexander; deschner, tobias; wittig, roman m.; leendertz, fabian h. title: human quarantine: toward reducing infectious pressure on chimpanzees at the taï chimpanzee project, côte d'ivoire date: 2017-01-17 journal: am j primatol doi: 10.1002/ajp.22619 sha: doc_id: 331288 cord_uid: elnwn7l5 due to their genetic relatedness, great apes are highly susceptible to common human respiratory pathogens. although most respiratory pathogens, such as human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv), rarely cause severe disease in healthy human adults, they are associated with considerable morbidity and mortality in wild great apes habituated to humans for research or tourism. to prevent pathogen transmission, most great ape projects have established a set of hygiene measures ranging from keeping a specific distance, to the use of surgical masks and establishment of quarantines. this study investigates the incidence of respiratory symptoms and human respiratory viruses in humans at a human‐great ape interface, the taï chimpanzee project (tcp) in côte d'ivoire, and consequently, the effectiveness of a 5‐day quarantine designed to reduce the risk of potential exposure to human respiratory pathogens. to assess the impact of quarantine as a preventative measure, we monitored the quarantine process and tested 262 throat swabs for respiratory viruses, collected during quarantine over a period of 1 year. although only 1 subject tested positive for a respiratory virus (hrsv), 17 subjects developed symptoms of infection while in quarantine and were subsequently kept from approaching the chimpanzees, preventing potential exposure in 18 cases. our results suggest that quarantine—in combination with monitoring for symptoms—is effective in reducing the risk of potential pathogen exposure. this research contributes to our understanding of how endangered great apes can be protected from human‐borne infectious disease. due to their genetic relatedness, great apes are highly susceptible to common human respiratory pathogens. although most respiratory pathogens, such as human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv), rarely cause severe disease in healthy human adults, they are associated with considerable morbidity and mortality in wild great apes habituated to humans for research or tourism. to prevent pathogen transmission, most great ape projects have established a set of hygiene measures ranging from keeping a specific distance, to the use of surgical masks and establishment of quarantines. this study investigates the incidence of respiratory symptoms and human respiratory viruses in humans at a human-great ape interface, the taï chimpanzee project (tcp) in côte d'ivoire, and consequently, the effectiveness of a 5-day quarantine designed to reduce the risk of potential exposure to human respiratory pathogens. to assess the impact of quarantine as a preventative measure, we monitored the quarantine process and tested 262 throat swabs for respiratory viruses, collected during quarantine over a period of 1 year. although only 1 subject tested positive for a respiratory virus (hrsv), 17 subjects developed symptoms of infection while in quarantine and were subsequently kept from approaching the chimpanzees, preventing potential exposure in 18 cases. our results suggest that quarantine-in combination with monitoring for symptoms-is effective in reducing the risk of potential pathogen exposure. this research contributes to our understanding of how endangered great apes can be protected from human-borne infectious disease. taï national park. they set out to habituate chimpanzees-to accustom them to human presence-in order to study their behavior (boesch & boesch-achermann, 2000) . their work has significantly contributed to our understanding of chimpanzee culture and cognition as well as human evolution. moreover, boesch and boesch-achermann made important observations on illness, on the disappearance of individual chimpanzees, and obtained biological samples that paved the way for future investigations of disease (boesch, 2008; leendertz et al., 2006) . these later investigations have led to the identification of numerous pathogens. not only do they account for pathogens occurring naturally in the chimpanzees (e.g., stlv and sfv) and in the habitat (e.g., ebola and bacillus cereus bv anthracis), but also those introduced by humans, such as the human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv) (boesch, 2008; calvignac-spencer et al., 2012; formenty et al., 1999; gogarten et al., 2014; köndgen et al., 2008; köndgen, schenk, pauli, boesch, & leendertz, 2010; le guenno et al., 1995; leendertz, boesch, junglen, since 1999 , tcp experienced at least six major respiratory disease outbreaks of human origin and with losses of up to 19% of the chimpanzee communities (köndgen et al., 2008) . other projects have also published data on human respiratory pathogens infecting and, ultimately, killing several wild, habituated great apes (grützmacher et al., 2016; kaur et al., 2008; palacios et al., 2011) . such findings emphasize the inherent risk of proximity. in all of the reported outbreaks, the viruses detected were either hrsv or hmpv: two common human paramyxoviruses that generally cause only mild symptoms in healthy human adults (falsey and walsh, 2000; webe, mulholland, & greenwood, 1998) . people can easily be unaware of their infectiousness because they may still feel physically fit enough to visit the great apes. in short, the risk of transmission is often overlooked. although the issue of whether humans should interfere with naturally occurring disease outbreaks in protected species is controversial, it is commonly understood that disease transmission from humans should be mitigated (gilardi et al., 2015) . it is the ethical responsibility of humans entering great ape habitats to help minimize the costs of habituation, including potential changes in behavior, alterations of the habitat, and the sickness and loss of individuals. indeed, since 1992, the tcp has gradually implemented preventative steps to reduce the risk of exposing chimpanzees to human pathogens. the first steps included rules not to enter the forest when ill and to remove all garbage. two years later, additional rules to bury human feces and to bring back all food remains from the forest were established and complemented by veterinary collaboration. in 1999, a minimum viewing distance of 5 m was implemented (later extended to 7 m) alongside requirements that all humans be vaccinated against yellow fever, tuberculosis, measles, and poliomyelitis. vaccination (boesch, 2008) . following the last major outbreak in 2009, the quarantine rules were adjusted to a 5-day period of quarantine in a separate quarantine camp for all humans intending to visit the chimpanzees (gilardi et al., 2015) . additionally, international travelers have to spend a minimum of 2 days in côte d'ivoire before entering quarantine. researchers and field assistants alike now enter the quarantine on day 1 (qd1). they must remain in a designated area in the quarantine camp without direct contact to people who have already cleared quarantine, anybody from the outside, or anybody in a different quarantine stage (the quarantine camp is divided into two separate sections to allow for two independent parties). after four nights, should no symptoms develop, the respective person clears quarantine on the 5th day (qd5). however, if any symptoms are detected, the person exits quarantine and starts over with qd1 after all symptoms have subsided. if anyone falls sick during quarantine, then everyone in quarantine at the same time also has to start over with qd1 (see figure 1 ). however, 5 days do not cover the longest possible incubation periods-the time between infection and symptom onsetof most human respiratory pathogens (see table 1 ). but, with the exception of measles (against which humans have to be vaccinated in order to visit habituated chimpanzees at tcp), at least 50% of this period is covered for all other relevant viruses. against this backdrop, the goal of this study is to determine the number of humans falling ill during quarantine. furthermore, the risk of potential exposure to human pathogens is assessed by testing sick humans to detect common human respiratory viruses they brought to the habituation site, and by randomly testing apparently healthy humans in the beginning and at the end of quarantine to assess the possibility of excreting hrsv and hmpv, the two most relevant viruses for wild great apes. the current study was performed at the tcp in côte d'ivoire. at the tcp, the quarantine camp is separate from the research camps with a designated kitchen, shower, and toilet. a driver supplies subjects in quarantine with food, wearing a facemask on delivery and leaving the food outside the building with no personal contact (for a more detailed description of quarantine protocols at tcp see gilardi et al., 2015) . when a subject falls ill, he or she is taken home to their village, or in case of international visitors (including researchers and film-makers), the subject is physically separated within the quarantine facilities until symptoms cease and quarantine can begin again at qd1. if symptoms are more severe, subjects are taken to the local hospital for treatment. overview of the quarantine system in the taї chimpanzee project and time from infection to observation of symptoms for hrsv and hmpv. y-axis: parametric estimates of the incubation period; p (symptomatic) = cumulative percentage of cases developing symptoms, and thus, shedding hrsv or hmpv by a given day under the estimates for the log-normal distribution (lessler et al., 2009) . the red line represents hrsv and the orange line represents hmpv to exemplify the earliest and latest possible scenario. whereas the blue dotted area illustrates the window covered through the quarantine, the red area covers the remaining risk. the staff health program of tcp includes yearly health checks at the local hospital, were a tuberculosis skin test and vaccinations against yellow fever, measles, poliomyelitis, and meningococcal disease are and qd5. in total, 262 were selected for testing. this sample selection was systematized to achieve an even seasonal distribution, wherein a minimum of 20 samples were tested for each month of the 1-year research period, and to best assess the risk of potential pathogen exposure. thus, the selection included 14 samples, collected from subjects with symptoms of illness (n = 18) plus randomly selected subjects with corresponding samples from qd1 (n = 110) and qd5 (n = 101) (when possible), and samples from subjects tested on a different day of quarantine (due to various constraints) or who had been in contact with sick individuals (n = 33). signs of illness were generally mild respiratory symptoms, including sore throat, runny nose, or cough. subjects in quarantine are expected to self-report symptoms; however, the respective camp manager and "veterinarian in charge" also oversee the quarantine process. swabs were stored and transported in liquid nitrogen and later kept in −80°c freezers. three hundred microliters of nuclease-free water were added to each sample and vortexed thoroughly before extraction. anthropogenic respiratory disease poses a serious risk to habituated wild great apes with high morbidity and considerable mortality (boesch, 2008; kaur et al., 2008; köndgen et al., 2010; leendertz et al., 2006; palacios et al., 2011) . its mitigation is therefore, an ethical responsibility for habituation projects (gruen, fultz, & pruetz, 2013) . however, preventing people from exposing wild habituated great apes to human pathogens is a challenge. in terms of potential pathogen exposure, both people from overseas who come to visit habituation sites as well as local staff on such projects routinely pose a risk to the health of wild great apes (muehlenbein & ancrenaz, 2009) . people who come to visit wild great apes from overseas are more likely to have been exposed to a variety of pathogens throughout their travel (e.g., in airports or airplanes) (mangili & gendreau, 2005) . additionally, the stress of travel and changes in climate make them more susceptible to infection, leading to disease within few days after arrival at the destination where the great apes are encountered (muehlenbein & ancrenaz, 2009 ). despite these different risk factors, eco-tourists frequently lack information about the relevance of their own health for the wildlife they intend to visit and, thus, may not take precautions or report illness (muehlenbein & ancrenaz, 2009 ). finally, local project staff can also pose an increased risk when they come into frequent contact with children in their villages-a subpopulation with high respiratory disease prevalence (walker et al., 2013) . the results show that international visitors represented 7 (39%) and local staff represented 11 (61%) of symptomatic subjects. however, only two of the seven international visitors became ill shortly after their arrival in côte d'ivoire and the remaining five rather mirror the health of permanent project staff in contact with the local diversity of pathogens. the detection of hrsv in only one out of 262 tested samples (including the 14 samples from people showing symptoms of respiratory disease) points toward a rather low prevalence of humans excreting hrsv and hmpv, when entering the tcp research area. although the possibility of false-negative results exists, it seems likely that if a person excretes these viruses in any relevant quantity-a quantity that would be sufficient for transmission to chimpanzees-it is likely to be detectable with the methods used here. of course, the negative results for asymptomatic people are not surprising. for some respiratory viruses, however, pathogen excretion might start before the first symptoms occur. for example, 1-8% of influenza infectiousness occurs prior to illness onset (lau et al., 2010) . cases of asymptomatic shedding have been reported for hrsv and hmpv-the most common causative agents of respiratory disease outbreaks in wild habituated great apesbut this is believed to be a rather rare occurrence (falsey, erdman, anderson, & walsh, 2003) . that said, hmpv has recently been reported at two different great ape field sites in 1 out of 24 and 1 out of 42 apparently healthy staff members (grützmacher et al., 2016) . although asymptomatic infections must be taken into account as a risk factor, the absence of symptoms also limits the physical spread (e.g., the absence of cough or nasal discharge limit the excretion of the pathogen). thus, strict hygiene rules should be sufficient to counteract the potential risk posed by apparently healthy people, especially those who have passed through quarantine. in this study, 13 subjects with respiratory symptoms (including cough, nasal discharge, or a sore throat) were negative for all viruses tested. that pathogens were not detected despite the observed symptoms may indicate that the symptoms were caused by viruses not included in our pcr panel or be due to the fact that symptoms do not always correlate well with viral particle excretion. these negative results can also be due to the detection limit of diagnostic methods (here pcr). furthermore, symptoms indicating respiratory disease can be caused by primary or secondary bacterial infections, such as with streptococcus pneumoniae. importantly, despite their involvement in mortalities as secondary infections (chi et al., 2007; köndgen et al., 2011; palacios et al., 2011), we did not test for potentially pathogenic bacteria. we did not do so mainly because such bacteria are often carried within the commensal flora of healthy humans, which makes it impossible to link a positive finding to the symptoms observed. this is a common problem for diagnostics in human medicine as well (bartlett, 2011; bosch, biesbroek, trzcinski, sanders, & bogaert, 2013) . beyond viruses and bacteria, the most frequent causative agents for acute respiratory illness, certain parasites, or funguses are also capable of triggering symptoms. more generally, infectious agents are not the only cause of respiratory symptoms (boutayeb, 2006) . non-infectious etiologies include allergies, asthma, chronic obstructive pulmonary disease (copd), cancer, air pollution, wildfire smoke, and smoking. while most of these causes are unlikely sources for the symptoms observed in this study-there was no particular air pollution or forest fire during the study period and neither allergies, asthma, nor any other chronic respiratory disease were reported by any of the subjects-it is impossible to entirely rule them out. from a practical point of view, however, preventing the spread of viruses is a bigger challenge because the particle size is smaller and aerosolization is more likely. therefore, hygiene measures preventing viral spread will also likely prevent the transmission of bacterial pathogens. even though a causative pathogen could only be determined in a single case, the delays through quarantine led to development of symptoms within isolation in 17 cases. without the quarantine procedure, these subjects would have already entered the forest, possibly approaching the chimpanzees and interacting with other people in the camps. notably, the one incidence in which a person developed symptoms after clearing quarantine was the very same incidence where hrsv was found. therefore, the infectious subject had not yet approached the chimpanzees and all contact persons were returned to quarantine. hrsv has a median incubation period of 4.4 days (95% ci: 3.9-4.9) and 95% of infected individuals will develop symptoms by 6.3 days (95% ci: 5.2-7.3) after infection (lessler et al., 2009) . while a 5-day quarantine does not cover the complete incubation period, it does cover 71% of the hrsv incubation period (83% for hmpv), and thus, lowers the risk considerably. quarantine has rather high costs and is a time intensive measure, so length needs to be decided carefully. extra space must also be made available to have physically separated facilities. additionally, people are very limited in the work they can do while in quarantine and lose time that could otherwise be spent doing valuable fieldwork-which is expensive for the project. yet, from a purely preventive health point of view, a 6-day quarantine would be even better as it would cover 95% of people developing symptoms of hrsv, and 100% for hmpv-the only two viruses ever found in respiratory disease outbreaks among wild great apes (kaur et al., 2008; köndgen et al., 2010; palacios et al., 2011) . in addition, the challenging process of quarantine has the positive side effect of limiting exchanges with surrounding villages, which in itself lowers the risk of project staff becoming infected. if logistics allow, then adding this one additional day to the quarantine process should be strongly considered. observations of outbreak frequency at the tcp are not high enough to statistically show that the frequency has declined since the implementation of the described quarantine procedure. that said, the patterns strongly suggest beneficial impacts from this procedure. before the implementation of quarantine, tcp experienced six major outbreaks between 1999 and 2006. since its implementation, the tcp has only experienced one further outbreak (in 2009), one that started when the quarantine was ignored (leendertz, unpublished data) . this underscores the argument that following quarantine rules is essential. it also demonstrates that all measures taken only result in risk reduction and emergency protocols must be kept in place for any outbreak intervention. our data and observations clearly suggest that quarantine does not replace monitoring for symptoms-they need to be used synergistically. in conclusion, it is important to note that single measures are insufficient for obtaining ethically acceptable risk reduction. several hygiene measures need to be combined: the implementation of quarantine and symptom monitoring with keeping a minimum distance of 7 m and wearing surgical masks (especially as chimpanzees do not follow distance rules) (gilardi et al., 2015; köndgen et al., 2008) . moreover, through monitoring and diagnostic investigation we can further characterize the real risk while assuring that the preventive measures are being followed. we thank the ivorian authorities for their long-term support, especially diagnostic tests for agents of community-acquired pneumonia the chimpanzees of the taï forest: behavioural ecology and evolution why do chimpanzees die in the forest? the challenges of understanding and controlling for wild ape health viral and bacterial interactions in the upper respiratory tract the double burden of communicable and noncommunicable diseases in developing countries origin of human t-lymphotropic virus type 1 in rural cote d'ivoire new streptococcus pneumoniae clones in deceased wild chimpanzees development of a pcr-based assay for detection, quantification, and genotyping of human adenoviruses respiratory syncytial virus infection in adults human metapneumovirus infections in young and elderly adults ebola outbreak in wild chimpanzees living in a rainforest of côte d'ivoire best practice guidelines for 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ten-year molecular and epidemiological analysis in germany genotyping of measles virus isolates from central europe and russia molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons diagnostic approach for the differentiation of the pandemic influenza a (h1n1) v virus from recent human influenza viruses by real-time pcr sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses global burden of childhood pneumonia and diarrhoea. the lancet respiratory syncytial virus infection in tropical and developing countries how to cite this article: grützmacher k, keil v, leinert v, et al.human quarantine: toward reducing infectious pressure on chimpanzees at the taï chimpanzee project, côte d'ivoire. am j key: cord-279255-v861kk0i authors: dhama, kuldeep; khan, sharun; tiwari, ruchi; sircar, shubhankar; bhat, sudipta; malik, yashpal singh; singh, karam pal; chaicumpa, wanpen; bonilla-aldana, d. katterine; rodriguez-morales, alfonso j. title: coronavirus disease 2019–covid-19 date: 2020-06-24 journal: clin microbiol rev doi: 10.1128/cmr.00028-20 sha: doc_id: 279255 cord_uid: v861kk0i in recent decades, several new diseases have emerged in different geographical areas, with pathogens including ebola virus, zika virus, nipah virus, and coronaviruses (covs). recently, a new type of viral infection emerged in wuhan city, china, and initial genomic sequencing data of this virus do not match with previously sequenced covs, suggesting a novel cov strain (2019-ncov), which has now been termed severe acute respiratory syndrome cov-2 (sars-cov-2). although coronavirus disease 2019 (covid-19) is suspected to originate from an animal host (zoonotic origin) followed by human-to-human transmission, the possibility of other routes should not be ruled out. compared to diseases caused by previously known human covs, covid-19 shows less severe pathogenesis but higher transmission competence, as is evident from the continuously increasing number of confirmed cases globally. compared to other emerging viruses, such as ebola virus, avian h7n9, sars-cov, and middle east respiratory syndrome coronavirus (mers-cov), sars-cov-2 has shown relatively low pathogenicity and moderate transmissibility. codon usage studies suggest that this novel virus has been transferred from an animal source, such as bats. early diagnosis by real-time pcr and next-generation sequencing has facilitated the identification of the pathogen at an early stage. since no antiviral drug or vaccine exists to treat or prevent sars-cov-2, potential therapeutic strategies that are currently being evaluated predominantly stem from previous experience with treating sars-cov, mers-cov, and other emerging viral diseases. in this review, we address epidemiological, diagnostic, clinical, and therapeutic aspects, including perspectives of vaccines and preventive measures that have already been globally recommended to counter this pandemic virus. o ver the past 2 decades, coronaviruses (covs) have been associated with significant disease outbreaks in east asia and the middle east. the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) began to emerge in 2002 and 2012, respectively. recently, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causing coronavirus disease 2019 (covid19) , emerged in late 2019, and it has posed a global health threat, causing an ongoing pandemic in many countries and territories (1) . health workers worldwide are currently making efforts to control further disease outbreaks caused by the novel cov (originally named 2019-ncov), which was first identified in wuhan city, hubei province, china, on 12 december 2019. on 11 february 2020, the world health organization (who) announced the official designation for the current cov-associated disease to be covid-19, caused by sars-cov-2. the primary cluster of patients was found to be connected with the huanan south china seafood market in wuhan (2) . covs belong to the family coronaviridae (subfamily coronavirinae), the members of which infect a broad range of hosts, producing symptoms and diseases ranging from the common cold to severe and ultimately fatal illnesses, such as sars, mers, and, presently, covid-19. sars-cov-2 is considered one of the seven members of the cov family that infect humans (3) , and it belongs to the same lineage of covs that causes sars; however, this novel virus is genetically distinct. until 2020, six covs were known to infect humans, including human cov 229e (hcov-229e), hcov-nl63, hcov-oc43, hcov-hku1, sars-cov, and mers-cov. although sars-cov and mers-cov have resulted in outbreaks with high mortality, others remain associated with mild upper-respiratory-tract illnesses (4) . newly evolved covs pose a high threat to global public health. the current emergence of covid-19 is the third cov outbreak in humans over the past 2 decades (5) . it is no coincidence that fan et al. predicted potential sars-or mers-like cov outbreaks in china following pathogen transmission from bats (6) . covid-19 emerged in china and spread rapidly throughout the country and, subsequently, to other countries. due to the severity of this outbreak and the potential of spreading on an international scale, the who declared a global health emergency on 31 january 2020; subsequently, on 11 march 2020, they declared it a pandemic situation. at present, we are not in a position to effectively treat covid-19, since neither approved vaccines nor specific antiviral drugs for treating human cov infections are available (7) (8) (9) . most nations are currently making efforts to prevent the further spreading of this potentially deadly virus by implementing preventive and control strategies. in domestic animals, infections with covs are associated with a broad spectrum of furthermore, it acts as a critical factor for tissue tropism and the determination of host range (45) . notably, s protein is one of the vital immunodominant proteins of covs capable of inducing host immune responses (45) . the ectodomains in all covs s proteins have similar domain organizations, divided into two subunits, s1 and s2 (43) . the first one, s1, helps in host receptor binding, while the second one, s2, accounts for fusion. the former (s1) is further divided into two subdomains, namely, the n-terminal domain (ntd) and c-terminal domain (ctd). both of these subdomains act as receptorbinding domains, interacting efficiently with various host receptors (45) . the s1 ctd contains the receptor-binding motif (rbm). in each coronavirus spike protein, the trimeric s1 locates itself on top of the trimeric s2 stalk (45) . recently, structural analyses of the s proteins of covid-19 have revealed 27 amino acid substitutions within a 1,273-amino-acid stretch (16) . six substitutions are located in the rbd (amino acids 357 to 528), while four substitutions are in the rbm at the ctd of the s1 domain (16) . of note, no amino acid change is seen in the rbm, which binds directly to the angiotensinconverting enzyme-2 (ace2) receptor in sars-cov (16, 46) . at present, the main emphasis is knowing how many differences would be required to change the host tropism. sequence comparison revealed 17 nonsynonymous changes between the early sequence of sars-cov-2 and the later isolates of sars-cov. the changes were found scattered over the genome of the virus, with nine substitutions in orf1ab, orf8 (4 substitutions), the spike gene (3 substitutions) , and orf7a (single substitution) (4) . notably, the same nonsynonymous changes were found in a familial cluster, indicating that the viral evolution happened during person-to-person transmission (4, 47) . such adaptive evolution events are frequent and constitute a constantly ongoing process once the virus spreads among new hosts (47) . even though no functional changes occur in the virus associated with this adaptive evolution, close monitoring of the viral mutations that occur during subsequent human-to-human transmission is warranted. the m protein is the most abundant viral protein present in the virion particle, giving a definite shape to the viral envelope (48) . it binds to the nucleocapsid and acts as a central organizer of coronavirus assembly (49) . coronavirus m proteins are highly diverse in amino acid contents but maintain overall structural similarity within different genera (50) . the m protein has three transmembrane domains, flanked by a short amino terminus outside the virion and a long carboxy terminus inside the virion (50) . overall, the viral scaffold is maintained by m-m interaction. of note, the m protein of sars-cov-2 does not have an amino acid substitution compared to that of sars-cov (16) . the coronavirus e protein is the most enigmatic and smallest of the major structural proteins (51) . it plays a multifunctional role in the pathogenesis, assembly, and release of the virus (52) . it is a small integral membrane polypeptide that acts as a viroporin (ion channel) (53) . the inactivation or absence of this protein is related to the altered virulence of coronaviruses due to changes in morphology and tropism (54) . the e protein consists of three domains, namely, a short hydrophilic amino terminal, a large hydrophobic transmembrane domain, and an efficient c-terminal domain (51) . the sars-cov-2 e protein reveals a similar amino acid constitution without any substitution (16) . the n protein of coronavirus is multipurpose. among several functions, it plays a role in complex formation with the viral genome, facilitates m protein interaction needed during virion assembly, and enhances the transcription efficiency of the virus (55, 56) . it contains three highly conserved and distinct domains, namely, an ntd, an rna-binding domain or a linker region (lkr), and a ctd (57) . the ntd binds with the 3= end of the viral genome, perhaps via electrostatic interactions, and is highly diverged both in length and sequence (58) . the charged lkr is serine and arginine rich and is also known as the sr (serine and arginine) domain (59) . the lkr is capable of direct interaction with in vitro rna interaction and is responsible for cell signaling (60, 61) . it also modulates the antiviral response of the host by working as an antagonist for interferon (ifn) and rna interference (62) . compared to that of sars-cov, the n protein of sars-cov-2 possess five amino acid mutations, where two are in the intrinsically dispersed region (idr; positions 25 and 26) , one each in the ntd (position 103), lkr (position 217), and ctd (position 334) (16) . besides the important structural proteins, the sars-cov-2 genome contains 15 nsps, nsp1 to nsp10 and nsp12 to nsp16, and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14) (16) . all these proteins play a specific role in viral replication (27) . unlike the accessory proteins of sars-cov, sars-cov-2 does not contain 8a protein and has a longer 8b and shorter 3b protein (16) . the nsp7, nsp13, envelope, matrix, and p6 and 8b accessory proteins have not been detected with any amino acid substitutions compared to the sequences of other coronaviruses (16) . the virus structure of sars-cov-2 is depicted in fig. 2 . sequence percent similarity analysis. we assessed the nucleotide percent similarity using the megalign software program, where the similarity between the novel sars-cov-2 isolates was in the range of 99.4% to 100%. among the other serbecovirus cov sequences, the novel sars-cov-2 sequences revealed the highest similarity to bat-sl-cov, with nucleotide percent identity ranges between 88.12 and 89.65%. meanwhile, earlier reported sars-covs showed 70.6 to 74.9% similarity to sars-cov-2 at the nucleotide level. further, the nucleotide percent similarity was 55.4%, 45.5% to 47.9%, 46 .2% to 46.6%, and 45.0% to 46.3% to the other four subgenera, namely, hibecovirus, nobecovirus, merbecovirus, and embecovirus, respectively. the percent similarity index of current outbreak isolates indicates a close relationship between sars-cov-2 isolates and bat-sl-cov, indicating a common origin. however, particular pieces of evidence based on further complete genomic analysis of current isolates are necessary to draw any conclusions, although it was ascertained that the current novel sars-cov-2 isolates belong to the subgenus sarbecovirus in the diverse range of betacoronaviruses. their possible ancestor was hypothesized to be from bat cov strains, wherein bats might have played a crucial role in harboring this class of viruses. splitstree phylogeny analysis. in the unrooted phylogenetic tree of different betacoronaviruses based on the s protein, virus sequences from different subgenera grouped into separate clusters. sars-cov-2 sequences from wuhan and other countries exhibited a close relationship and appeared in a single cluster (fig. 1 ). the covs from the subgenus sarbecovirus appeared jointly in splitstree and divided into three subclusters, namely, sars-cov-2, bat-sars-like-cov (bat-sl-cov), and sars-cov (fig. 1) . in the case of other subgenera, like merbecovirus, all of the sequences grouped clinical microbiology reviews than italy. a john hopkins university web platform has provided daily updates on the basic epidemiology of the covid-19 outbreak (https://gisanddata.maps.arcgis.com/ apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6) (238) . covid-19 has also been confirmed on a cruise ship, named diamond princess, quarantined in japanese waters (port of yokohama), as well as on other cruise ships around the world (239) (fig. 3) . the significant events of the sars-cov-2/covid-19 virus outbreak occurring since 8 december 2019 are presented as a timeline in fig. 5 . at the beginning, china experienced the majority of the burden associated with covid-19 in the form of disease morbidity and mortality (65), but over time the covid-19 menace moved to europe, particularly italy and spain, and now the united states has the highest number of confirmed cases and deaths. the covid-19 outbreak has also been associated with severe economic impacts globally due to the sudden interruption of global trade and supply chains that forced multinational companies to make decisions that led to significant economic losses (66) . the recent increase in the number of confirmed critically ill patients with covid-19 has already surpassed the intensive care supplies, limiting intensive care services to only a small portion of critically ill patients (67) . this might also have contributed to the increased case fatality rate observed in the covid-19 outbreak. the novel coronavirus was identified within 1 month (28 days) of the outbreak. this is impressively fast compared to the time taken to identify sars-cov reported in foshan, guangdong province, china (125 days) (68) . immediately after the confirmation of viral etiology, the chinese virologists rapidly released the genomic sequence of sars-cov-2, which played a crucial role in controlling the spread of this newly emerged novel coronavirus to other parts of the world (69) . the possible origin of sars-cov-2 and the first mode of disease transmission are not yet identified (70) . analysis of the initial cluster of infections suggests that the infected individuals had a common exposure point, a seafood market in wuhan, hubei province, china (fig. 6 ). the restaurants of this market are well-known for providing different types of wild animals for human consumption (71) . the huanan south china seafood market also sells live animals, such as poultry, bats, snakes, and marmots (72) . this might be the point where zoonotic (animal-to-human) transmission occurred (71) . although sars-cov-2 is alleged to have originated from an animal host (zoonotic origin) with further humanto-human transmission (fig. 6 ), the likelihood of foodborne transmission should be ruled out with further investigations, since it is a latent possibility (1). additionally, other clinical microbiology reviews potential and expected routes would be associated with transmission, as in other respiratory viruses, by direct contact, such as shaking contaminated hands, or by direct contact with contaminated surfaces (fig. 6) . still, whether blood transfusion and organ transplantation (276) , as well as transplacental and perinatal routes, are possible routes for sars-cov-2 transmission needs to be determined (fig. 6 ). from experience with several outbreaks associated with known emerging viruses, higher pathogenicity of a virus is often associated with lower transmissibility. compared to emerging viruses like ebola virus, avian h7n9, sars-cov, and mers-cov, sars-cov-2 has relatively lower pathogenicity and moderate transmissibility (15) . the risk of death among individuals infected with covid-19 was calculated using the infection fatality risk (ifr). the ifr was found to be in the range of 0.3% to 0.6%, which is comparable to that of a previous asian influenza pandemic (1957 to 1958) (73, 277) . notably, the reanalysis of the covid-19 pandemic curve from the initial cluster of cases pointed to considerable human-to-human transmission. it is opined that the exposure history of sars-cov-2 at the wuhan seafood market originated from humanto-human transmission rather than animal-to-human transmission (74) ; however, in light of the zoonotic spillover in covid-19, is too early to fully endorse this idea (1). following the initial infection, human-to-human transmission has been observed with a preliminary reproduction number (r 0 ) estimate of 1.4 to 2.5 (70, 75) , and recently it is estimated to be 2.24 to 3.58 (76) . in another study, the average reproductive number of covid-19 was found to be 3.28, which is significantly higher than the initial who estimate of 1.4 to 2.5 (77) . it is too early to obtain the exact r 0 value, since there is a possibility of bias due to insufficient data. the higher r 0 value is indicative of the more significant potential of sars-cov-2 transmission in a susceptible population. this is not the first time where the culinary practices of china have been blamed for the origin of novel coronavirus infection in humans. previously, the animals present in the liveanimal market were identified to be the intermediate hosts of the sars outbreak in china (78) . several wildlife species were found to harbor potentially evolving coronavirus strains that can overcome the species barrier (79) . one of the main principles of chinese food culture is that live-slaughtered animals are considered more nutritious (5) . after 4 months of struggle that lasted from december 2019 to march 2020, the covid-19 situation now seems under control in china. the wet animal markets have reopened, and people have started buying bats, dogs, cats, birds, scorpions, badgers, rabbits, pangolins (scaly anteaters), minks, soup from palm civet, ostriches, hamsters, snapping turtles, ducks, fish, siamese crocodiles, and other animal meats without any fear of covid-19. the chinese government is encouraging people to feel they can return to normalcy. however, this could be a risk, as it has been mentioned in advisories that people should avoid contact with live-dead animals as much as possible, as sars-cov-2 has shown zoonotic spillover. additionally, we cannot rule out the possibility of new mutations in the same virus being closely related to contact with both animals and humans at the market (284) . in january 2020, china imposed a temporary ban on the sale of live-dead animals in wet markets. however, now hundreds of such wet markets have been reopened without optimizing standard food safety and sanitation practices (286) . with china being the most populated country in the world and due to its domestic and international food exportation policies, the whole world is now facing the menace of covid-19, including china itself. wet markets of live-dead animals do not maintain strict food hygienic practices. fresh blood splashes are present everywhere, on the floor and tabletops, and such food customs could encourage many pathogens to adapt, mutate, and jump the species barrier. as a result, the whole world is suffering from novel sars-cov-2, with more than 4,170,424 cases and 287,399 deaths across the globe. there is an urgent need for a rational international campaign against the unhealthy food practices of china to encourage the sellers to increase hygienic food practices or close the crude live-dead animal wet markets. there is a need to modify food policies at national and international levels to avoid further life threats and clinical microbiology reviews economic consequences from any emerging or reemerging pandemic due to close animal-human interaction (285) . even though individuals of all ages and sexes are susceptible to covid-19, older people with an underlying chronic disease are more likely to become severely infected (80) . recently, individuals with asymptomatic infection were also found to act as a source of infection to susceptible individuals (81) . both the asymptomatic and symptomatic patients secrete similar viral loads, which indicates that the transmission capacity of asymptomatic or minimally symptomatic patients is very high. thus, sars-cov-2 transmission can happen early in the course of infection (82) . atypical clinical manifestations have also been reported in covid-19 in which the only reporting symptom was fatigue. such patients may lack respiratory signs, such as fever, cough, and sputum (83) . hence, the clinicians must be on the look-out for the possible occurrence of atypical clinical manifestations to avoid the possibility of missed diagnosis. the early transmission ability of sars-cov-2 was found to be similar to or slightly higher than that of sars-cov, reflecting that it could be controlled despite moderate to high transmissibility (84) . increasing reports of sars-cov-2 in sewage and wastewater warrants the need for further investigation due to the possibility of fecal-oral transmission. sars-cov-2 present in environmental compartments such as soil and water will finally end up in the wastewater and sewage sludge of treatment plants (328) . therefore, we have to reevaluate the current wastewater and sewage sludge treatment procedures and introduce advanced techniques that are specific and effective against sars-cov-2. since there is active shedding of sars-cov-2 in the stool, the prevalence of infections in a large population can be studied using wastewater-based epidemiology. recently, reverse transcription-quantitative pcr (rt-qpcr) was used to enumerate the copies of sars-cov-2 rna concentrated from wastewater collected from a wastewater treatment plant (327) . the calculated viral rna copy numbers determine the number of infected individuals. the increasing reports of virus shedding via the fecal route warrants the introduction of negative fecal viral nucleic acid test results as one of the additional discharge criteria in laboratory-confirmed cases of covid-19 (326) . the covid-19 pandemic does not have any novel factors, other than the genetically unique pathogen and a further possible reservoir. the cause and the likely future outcome are just repetitions of our previous interactions with fatal coronaviruses. the only difference is the time of occurrence and the genetic distinctness of the pathogen involved. mutations on the rbd of covs facilitated their capability of infecting newer hosts, thereby expanding their reach to all corners of the world (85) . this is a potential threat to the health of both animals and humans. advanced studies using bayesian phylogeographic reconstruction identified the most probable origin of sars-cov-2 as the bat sars-like coronavirus, circulating in the rhinolophus bat family (86) . phylogenetic analysis of 10 whole-genome sequences of sars-cov-2 showed that they are related to two covs of bat origin, namely, bat-sl-covzc45 and bat-sl-covzxc21, which were reported during 2018 in china (17) . it was reported that sars-cov-2 had been confirmed to use ace2 as an entry receptor while exhibiting an rbd similar to that of sars-cov (17, 87, 254, 255) . several countries have provided recommendations to their people traveling to china (88, 89) . compared to the previous coronavirus outbreaks caused by sars-cov and mers-cov, the efficiency of sars-cov-2 human-to-human transmission was thought to be less. this assumption was based on the finding that health workers were affected less than they were in previous outbreaks of fatal coronaviruses (2) . superspreading events are considered the main culprit for the extensive transmission of sars and mers (90, 91) . almost half of the mers-cov cases reported in saudi arabia are of secondary origin that occurred through contact with infected asymptomatic or symptomatic individuals through human-tohuman transmission (92) . the occurrence of superspreading events in the covid-19 outbreak cannot be ruled out until its possibility is evaluated. like sars and mers, covid-19 can also infect the lower respiratory tract, with milder symptoms (27) . the basic reproduction number of covid-19 has been found to be in the range of 2.8 to 3.3 based on real-time reports and 3.2 to 3.9 based on predicted infected cases (84) . coronavirus infection in humans is commonly associated with mild to severe respiratory diseases, with high fever, severe inflammation, cough, and internal organ dysfunction that can even lead to death (92) . most of the identified coronaviruses cause the common cold in humans. however, this changed when sars-cov was identified, paving the way for severe forms of the disease in humans (22) . our previous experience with the outbreaks of other coronaviruses, like sars and mers, suggests that the mode of transmission in covid-19 as mainly human-to-human transmission via direct contact, droplets, and fomites (25) . recent studies have demonstrated that the virus could remain viable for hours in aerosols and up to days on surfaces; thus, aerosol and fomite contamination could play potent roles in the transmission of sars-cov-2 (257) . the immune response against coronavirus is vital to control and get rid of the infection. however, maladjusted immune responses may contribute to the immunopathology of the disease, resulting in impairment of pulmonary gas exchange. understanding the interaction between covs and host innate immune systems could enlighten our understanding of the lung inflammation associated with this infection (24) . sars is a viral respiratory disease caused by a formerly unrecognized animal cov that originated from the wet markets in southern china after adapting to the human host, thereby enabling transmission between humans (90) . the sars outbreak reported in 2002 to 2003 had 8,098 confirmed cases with 774 total deaths (9.6%) (93) . the outbreak severely affected the asia pacific region, especially mainland china (94) . even though the case fatality rate (cfr) of sars-cov-2 (covid-19) is lower than that of sars-cov, there exists a severe concern linked to this outbreak due to its epidemiological similarity to influenza viruses (95, 279) . this can fail the public health system, resulting in a pandemic (96) . mers is another respiratory disease that was first reported in saudi arabia during the year 2012. the disease was found to have a cfr of around 35% (97) . the analysis of available data sets suggests that the incubation period of sars-cov-2, sars-cov, and mers-cov is in almost the same range. the longest predicted incubation time of sars-cov-2 is 14 days. hence, suspected individuals are isolated for 14 days to avoid the risk of further spread (98) . even though a high similarity has been reported between the genome sequence of the new coronavirus (sars-cov-2) and sars-like covs, the comparative analysis recognized a furin-like cleavage site in the sars-cov-2 s protein that is missing from other sars-like covs (99) . the furin-like cleavage site is expected to play a role in the life cycle of the virus and disease pathogenicity and might even act as a therapeutic target for furin inhibitors. the highly contagious nature of sars-cov-2 compared to that of its predecessors might be the result of a stabilizing mutation that occurred in the endosome-associated-protein-like domain of nsp2 protein. similarly, the destabilizing mutation near the phosphatase domain of nsp3 proteins in sars-cov-2 could indicate a potential mechanism that differentiates it from other covs (100) . even though the cfr reported for covid-19 is meager compared to those of the previous sars and mers outbreaks, it has caused more deaths than sars and mers combined (101) . possibly related to the viral pathogenesis is the recent finding of an 832-nucleotide (nt) deletion in orf8, which appears to reduce the replicative fitness of the virus and leads to attenuated phenotypes of sars-cov-2 (256) . coronavirus is the most prominent example of a virus that has crossed the species barrier twice from wild animals to humans during sars and mers outbreaks (79, 102) . the possibility of crossing the species barrier for the third time has also been suspected in the case of sars-cov-2 (covid19) . bats are recognized as a possible natural reservoir host of both sars-cov and mers-cov infection. in contrast, the possible intermediary host is the palm civet for sars-cov and the dromedary camel for mers-cov infection (102) . bats are considered the ancestral hosts for both sars and mers (103) . bats are also considered the reservoir host of human coronaviruses like clinical microbiology reviews hcov-229e and hcov-nl63 (104) . in the case of covid-19, there are two possibilities for primary transmission: it can be transmitted either through intermediate hosts, similar to that of sars and mers, or directly from bats (103) . the emergence paradigm put forward in the sars outbreak suggests that sars-cov originated from bats (reservoir host) and later jumped to civets (intermediate host) and incorporated changes within the receptor-binding domain (rbd) to improve binding to civet ace2. this civetadapted virus, during their subsequent exposure to humans at live markets, promoted further adaptations that resulted in the epidemic strain (104) . transmission can also occur directly from the reservoir host to humans without rbd adaptations. the bat coronavirus that is currently in circulation maintains specific "poised" spike proteins that facilitate human infection without the requirement of any mutations or adaptations (105) . altogether, different species of bats carry a massive number of coronaviruses around the world (106) . the high plasticity in receptor usage, along with the feasibility of adaptive mutation and recombination, may result in frequent interspecies transmission of coronavirus from bats to animals and humans (106) . the pathogenesis of most bat coronaviruses is unknown, as most of these viruses are not isolated and studied (4) . hedgehog coronavirus hku31, a betacoronavirus, has been identified from amur hedgehogs in china. studies show that hedgehogs are the reservoir of betacoronavirus, and there is evidence of recombination (107) . the current scientific evidence available on mers infection suggests that the significant reservoir host, as well as the animal source of mers infection in humans, is the dromedary camels (97) . the infected dromedary camels may not show any visible signs of infection, making it challenging to identify animals actively excreting mers-cov that has the potential to infect humans. however, they may shed mers-cov through milk, urine, feces, and nasal and eye discharge and can also be found in the raw organs (108) . in a study conducted to evaluate the susceptibility of animal species to mers-cov infection, llamas and pigs were found to be susceptible, indicating the possibility of mers-cov circulation in animal species other than dromedary camels (109) . following the outbreak of sars in china, sars-cov-like viruses were isolated from himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) found in a live-animal market in guangdong, china. the animal isolates obtained from the live-animal market retained a 29-nucleotide sequence that was not present in most of the human isolates (78) . these findings were critical in identifying the possibility of interspecies transmission in sars-cov. the higher diversity and prevalence of bat coronaviruses in this region compared to those in previous reports indicate a host/ pathogen coevolution. sars-like coronaviruses also have been found circulating in the chinese horseshoe bat (rhinolophus sinicus) populations. the in vitro and in vivo studies carried out on the isolated virus confirmed that there is a potential risk for the reemergence of sars-cov infection from the viruses that are currently circulating in the bat population (105) . the disease caused by sars-cov-2 is also named severe specific contagious pneumonia (sscp), wuhan pneumonia, and, recently, covid-19 (110) . compared to sars-cov, sars-cov-2 has less severe pathogenesis but has superior transmission capability, as evidenced by the rapidly increasing number of covid-19 cases (111) . the incubation period of sars-cov-2 in familial clusters was found to be 3 to 6 days (112) . the mean incubation period of covid-19 was found to be 6.4 days, ranging from 2.1 to 11.1 days (113) . among an early affected group of 425 patients, 59 years was the median age, of which more males were affected (114) . similar to sars and mers, the severity of this ncov is high in age groups above 50 years (2, 115) . symptoms of covid-19 include fever, cough, myalgia or fatigue, and, less commonly, headache, hemoptysis, and diarrhea (116, 282) . compared to the sars-cov-2-infected patients in wuhan during the initial stages of the outbreak, only mild symptoms were noticed in those patients that are infected by human-to-human transmission (14) . the initial trends suggested that the mortality associated with covid-19 was less than that of previous outbreaks of sars (101) . the updates obtained from countries like china, japan, thailand, and south korea indicated that the covid-19 patients had relatively mild manifestations compared to those with sars and mers (4). regardless of the coronavirus type, immune cells, like mast cells, that are present in the submucosa of the respiratory tract and nasal cavity are considered the primary barrier against this virus (92) . advanced in-depth analysis of the genome has identified 380 amino acid substitutions between the amino acid sequences of sars-cov-2 and the sars/sarslike coronaviruses. these differences in the amino acid sequences might have contributed to the difference in the pathogenic divergence of sars-cov-2 (16) . further research is required to evaluate the possible differences in tropism, pathogenesis, and transmission of this novel agent associated with this change in the amino acid sequence. with the current outbreak of covid-19, there is an expectancy of a significant increase in the number of published studies about this emerging coronavirus, as occurred with sars and mers (117) . sars-cov-2 invades the lung parenchyma, resulting in severe interstitial inflammation of the lungs. this is evident on computed tomography (ct) images as ground-glass opacity in the lungs. this lesion initially involves a single lobe but later expands to multiple lung lobes (118) . the histological assessment of lung biopsy samples obtained from covid-19-infected patients revealed diffuse alveolar damage, cellular fibromyxoid exudates, hyaline membrane formation, and desquamation of pneumocytes, indicative of acute respiratory distress syndrome (119) . it was also found that the sars-cov-2infected patients often have lymphocytopenia with or without leukocyte abnormalities. the degree of lymphocytopenia gives an idea about disease prognosis, as it is found to be positively correlated with disease severity (118) . pregnant women are considered to have a higher risk of getting infected by covid-19. the coronaviruses can cause adverse outcomes for the fetus, such as intrauterine growth restriction, spontaneous abortion, preterm delivery, and perinatal death. nevertheless, the possibility of intrauterine maternal-fetal transmission (vertical transmission) of covs is low and was not seen during either the sars-or mers-cov outbreak (120) . however, there has been concern regarding the impact of sars-cov-2/covid-19 on pregnancy. researchers have mentioned the probability of in utero transmission of novel sars-cov-2 from covid-19-infected mothers to their neonates in china based upon the rise in igm and igg antibody levels and cytokine values in the blood obtained from newborn infants immediately postbirth; however, rt-pcr failed to confirm the presence of sars-cov-2 genetic material in the infants (283) . recent studies show that at least in some cases, preterm delivery and its consequences are associated with the virus. nonetheless, some cases have raised doubts for the likelihood of vertical transmission (240) (241) (242) (243) . covid-19 infection was associated with pneumonia, and some developed acute respiratory distress syndrome (ards). the blood biochemistry indexes, such as albumin, lactate dehydrogenase, c-reactive protein, lymphocytes (percent), and neutrophils (percent) give an idea about the disease severity in covid-19 infection (121) . during covid-19, patients may present leukocytosis, leukopenia with lymphopenia (244), hypoalbuminemia, and an increase of lactate dehydrogenase, aspartate transaminase, alanine aminotransferase, bilirubin, and, especially, d-dimer (244) . middle-aged and elderly patients with primary chronic diseases, especially high blood pressure and diabetes, were found to be more susceptible to respiratory failure and, therefore, had poorer prognoses. providing respiratory support at early stages improved the disease prognosis and facilitated recovery (18) . the ards in covid-19 is due to the occurrence of cytokine storms that results in exaggerated immune response, immune regulatory network imbalance, and, finally, multiple-organ failure (122) . in addition to the exaggerated inflammatory response seen in patients with covid-19 pneumonia, the bile duct epithelial cell-derived hepatocytes upregulate ace2 expression in liver tissue by compensatory proliferation that might result in hepatic tissue injury (123) . coronavirus can cause disease in several species of domestic and wild animals, as well as humans (23) . the different animal species that are infected with cov include horses, camels, cattle, swine, dogs, cats, rodents, birds, ferrets, minks, bats, rabbits, snakes, and various other wild animals (20, 30, 79, 93, 124, 125, 287) . coronavirus infection is linked to different kinds of clinical manifestations, varying from enteritis in cows and pigs, upper respiratory disease in chickens, and fatal respiratory infections in humans (30) . among the cov genera, alphacoronavirus and betacoronavirus infect mammals, while gammacoronavirus and deltacoronavirus mainly infect birds, fishes, and, sometimes, mammals (27, 29, 106) . several novel coronaviruses that come under the genus deltacoronavirus have been discovered in the past from birds, like wigeon coronavirus hku20, bulbul coronavirus hku11, munia coronavirus hku13, white-eye coronavirus hku16, night-heron coronavirus hku19, and common moorhen coronavirus hku21, as well as from pigs (porcine coronavirus hku15) (6, 29) . transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and porcine hemagglutinating encephalomyelitis virus (phev) are some of the coronaviruses of swine. among them, tgev and pedv are responsible for causing severe gastroenteritis in young piglets with noteworthy morbidity and mortality. infection with phev also causes enteric infection but can cause encephalitis due to its ability to infect the nervous system (30) . bovine coronaviruses (bocovs) are known to infect several domestic and wild ruminants (126) . bocov inflicts neonatal calf diarrhea in adult cattle, leading to bloody diarrhea (winter dysentery) and respiratory disease complex (shipping fever) in cattle of all age groups (126) . bocov-like viruses have been noted in humans, suggesting its zoonotic potential as well (127) . feline enteric and feline infectious peritonitis (fip) viruses are the two major feline covs (128) , where feline covs can affect the gastrointestinal tract, abdominal cavity (peritonitis), respiratory tract, and central nervous system (128) . canines are also affected by covs that fall under different genera, namely, canine enteric coronavirus in alphacoronavirus and canine respiratory coronavirus in betacoronavirus, affecting the enteric and respiratory tract, respectively (129, 130) . ibv, under gammacoronavirus, causes diseases of respiratory, urinary, and reproductive systems, with substantial economic losses in chickens (131, 132) . in small laboratory animals, mouse hepatitis virus, rat sialodacryoadenitis coronavirus, and guinea pig and rabbit coronaviruses are the major covs associated with disease manifestations like enteritis, hepatitis, and respiratory infections (10, 133) . swine acute diarrhea syndrome coronavirus (sads-cov) was first identified in suckling piglets having severe enteritis and belongs to the genus alphacoronavirus (106) . the outbreak was associated with considerable scale mortality of piglets (24,693 deaths) across four farms in china (134) . the virus isolated from the piglets was almost identical to and had 95% genomic similarity with horseshoe bat (rhinolophus species) coronavirus hku2, suggesting a bat origin of the pig virus (106, 134, 135) . it is also imperative to note that the sads-cov outbreak started in guangdong province, near the location of the sars pandemic origin (134) . before this outbreak, pigs were not known to be infected with bat-origin coronaviruses. this indicates that the bat-origin coronavirus jumped to pig by breaking the species barrier. the next step of this jump might not end well, since pigs are considered the mixing vessel for influenza a viruses due to their ability to be infected by both human and avian influenza a viruses (136) . similarly, they may act as the mixing vessel for coronaviruses, since they are in frequent contact with both humans and multiple wildlife species. additionally, pigs are also found to be susceptible to infection with human sars-cov and mers-cov, making this scenario a nightmare (109, 137) . it is only a matter of time before another zoonotic coronavirus results in an epidemic by jumping the so-called species barrier (287) . the host spectrum of coronavirus increased when a novel coronavirus, namely, sw1, was recognized in the liver tissue of a captive beluga whale (delphinapterus leucas) (138) . in recent decades, several novel coronaviruses were identified from different animal species. bats can harbor these viruses without manifesting any clinical disease but are persistently infected (30) . they are the only mammals with the capacity for self-powered flight, which enables them to migrate long distances, unlike land mammals. bats are distributed worldwide and also account for about a fifth of all mammalian species (6) . this makes them the ideal reservoir host for many viral agents and also the source of novel coronaviruses that have yet to be identified. it has become a necessity to study the diversity of coronavirus in the bat population to prevent future outbreaks that could jeopardize livestock and public health. the repeated outbreaks caused by bat-origin coronaviruses calls for the development of efficient molecular surveillance strategies for studying betacoronavirus among animals (12) , especially in the rhinolophus bat family (86) . chinese bats have high commercial value, since they are used in traditional chinese medicine (tcm). therefore, the handling of bats for trading purposes poses a considerable risk of transmitting zoonotic cov epidemics (139) . due to the possible role played by farm and wild animals in sars-cov-2 infection, the who, in their novel coronavirus (covid-19) situation report, recommended the avoidance of unprotected contact with both farm and wild animals (25) . the live-animal markets, like the one in guangdong, china, provides a setting for animal coronaviruses to amplify and to be transmitted to new hosts, like humans (78) . such markets can be considered a critical place for the origin of novel zoonotic diseases and have enormous public health significance in the event of an outbreak. bats are the reservoirs for several viruses; hence, the role of bats in the present outbreak cannot be ruled out (140) . in a qualitative study conducted for evaluating the zoonotic risk factors among rural communities of southern china, the frequent human-animal interactions along with the low levels of environmental biosecurity were identified as significant risks for the emergence of zoonotic disease in local communities (141, 142) . the comprehensive sequence analysis of the sars-cov-2 rna genome identified that the cov from wuhan is a recombinant virus of the bat coronavirus and another coronavirus of unknown origin. the recombination was found to have happened within the viral spike glycoprotein, which recognizes the cell surface receptor. further analysis of the genome based on codon usage identified the snake as the most probable animal reservoir of sars-cov-2 (143) . contrary to these findings, another genome analysis proposed that the genome of sars-cov-2 is 96% identical to bat coronavirus, reflecting its origin from bats (63) . the involvement of bat-derived materials in causing the current outbreak cannot be ruled out. high risk is involved in the production of bat-derived materials for tcm practices involving the handling of wild bats. the use of bats for tcm practices will remain a severe risk for the occurrence of zoonotic coronavirus epidemics in the future (139) . furthermore, the pangolins are an endangered species of animals that harbor a wide variety of viruses, including coronaviruses (144) . the coronavirus isolated from malayan pangolins (manis javanica) showed a very high amino acid identity with covid-19 at e (100%), m (98.2%), n (96.7%), and s genes (90.4%). the rbd of s protein in cov isolated from pangolin was almost identical (one amino acid difference) to that of sars-cov-2. a comparison of the genomes suggests recombination between pangolin-cov-like viruses with the bat-cov-ratg13-like virus. all this suggests the potential of pangolins to act as the intermediate host of sars-cov-2 (145) . human-wildlife interactions, which are increasing in the context of climate change (142) , are further considered high risk and responsible for the emergence of sars-cov. covid-19 is also suspected of having a similar mode of origin. hence, to prevent the occurrence of another zoonotic spillover (1), exhaustive coordinated efforts are needed to identify the high-risk pathogens harbored by wild animal populations, conducting surveillance among the people who are susceptible to zoonotic spillover events (12) , and to improve the biosecurity measures associated with the wildlife trade (146) . the serological surveillance studies conducted in people living in proximity to bat caves had earlier identified the serological confirmation of sars-related covs in humans. people clinical microbiology reviews living at the wildlife-human interface, mainly in rural china, are regularly exposed to sars-related covs (147) . these findings will not have any significance until a significant outbreak occurs due to a virus-like sars-cov-2. there is a steady increase in the reports of covid-19 in companion and wild animals around the world. further studies are required to evaluate the potential of animals (especially companion animals) to serve as an efficient reservoir host that can further alter the dynamics of human-to-human transmission (330) . to date, two pet dogs (hong kong) and four pet cats (one each from belgium and hong kong, two from the united states) have tested positive for sars-cov-2 (335) . the world organization for animal health (oie) has confirmed the diagnosis of covid-19 in both dogs and cats due to human-to-animal transmission (331) . the similarity observed in the gene sequence of sars-cov-2 from an infected pet owner and his dog further confirms the occurrence of human-to-animal transmission (333) . even though asymptomatic, feline species should be considered a potential transmission route from animals to humans (326) . however, currently, there are no reports of sars-cov-2 transmission from felines to human beings. based on the current evidence, we can conclude that cats are susceptible to sars-cov-2 and can get infected by human beings. however, evidence of cat-to-human transmission is lacking and requires further studies (332) . rather than waiting for firmer evidence on animal-to-human transmission, necessary preventive measures are advised, as well as following social distancing practices among companion animals of different households (331) . one of the leading veterinary diagnostic companies, idexx, has conducted large-scale testing for covid-19 in specimens collected from dogs and cats. however, none of the tests turned out to be positive (334) . in a study conducted to investigate the potential of different animal species to act as the intermediate host of sars-cov-2, it was found that both ferrets and cats can be infected via experimental inoculation of the virus. in addition, infected cats efficiently transmitted the disease to naive cats (329) . sars-cov-2 infection and subsequent transmission in ferrets were found to recapitulate the clinical aspects of covid-19 in humans. the infected ferrets also shed virus via multiple routes, such as saliva, nasal washes, feces, and urine, postinfection, making them an ideal animal model for studying disease transmission (337) . experimental inoculation was also done in other animal species and found that the dogs have low susceptibility, while the chickens, ducks, and pigs are not at all susceptible to sars-cov-2 (329) . similarly, the national veterinary services laboratories of the usda have reported covid-19 in tigers and lions that exhibited respiratory signs like dry cough and wheezing. the zoo animals are suspected to have been infected by an asymptomatic zookeeper (335) . the total number of covid-19-positive cases in human beings is increasing at a high rate, thereby creating ideal conditions for viral spillover to other species, such as pigs. the evidence obtained from sars-cov suggests that pigs can get infected with sars-cov-2 (336). however, experimental inoculation with sars-cov-2 failed to infect pigs (329) . further studies are required to identify the possible animal reservoirs of sars-cov-2 and the seasonal variation in the circulation of these viruses in the animal population. research collaboration between human and animal health sectors is becoming a necessity to evaluate and identify the possible risk factors of transmission between animals and humans. such cooperation will help to devise efficient strategies for the management of emerging zoonotic diseases (12) . rna tests can confirm the diagnosis of sars-cov-2 (covid-19) cases with real-time rt-pcr or next-generation sequencing (148, 149, 245, 246) . at present, nucleic acid detection techniques, like rt-pcr, are considered an effective method for confirming the diagnosis in clinical cases of covid-19 (148) . several companies across the world are currently focusing on developing and marketing sars-cov-2-specific nucleic acid detection kits. multiple laboratories are also developing their own in-house rt-pcr. one of them is the sars-cov-2 nucleic acid detection kit produced by shuoshi biotechnology (double fluorescence pcr method) (150) . up to 30 march 2020, the u.s. food and drug administration (fda) had granted 22 in vitro diagnostics emergency use authorizations (euas), including for the rt-pcr diagnostic panel for the universal detection of sars-like betacoronaviruses and specific detection of sars-cov-2, developed by the u.s. cdc (table 1) (258, 259) . recently, 95 full-length genomic sequences of saras-cov-2 strains available in the national center for biotechnology information and gisaid databases were subjected to multiple-sequence alignment and phylogenetic analyses for studying variations in the viral genome (260) . all the viral strains revealed high homology of 99.99% (99.91% to 100%) at the nucleotide level and 99.99% (99.79% to 100%) at the amino acid level. overall variation was found to be low in orf regions, with 13 variation sites recognized in 1a, 1b, s, 3a, m, 8, and n regions. mutation rates of 30.53% (29/95) and 29.47% (28/95) were observed at nt 28144 (orf8) and nt 8782 (orf1a) positions, respectively. owing to such selective mutations, a few specific regions of sars-cov-2 should not be considered for designing primers and probes. the sars-cov-2 reference sequence could pave the way to study molecular biology and pathobiology, along with developing diagnostics and appropriate prevention and control strategies for countering sars-cov-2 (260) . nucleic acids of sars-cov-2 can be detected from samples (64) such as bronchoalveolar lavage fluid, sputum, nasal swabs, fiber bronchoscope brush biopsy specimen, pharyngeal swabs, feces, blood, and urine, with different levels of diagnostic performance (table 2) (80, 245, 246) . the viral loads of sars-cov-2 were measured using n-gene-specific quantitative rt-pcr in throat swab and sputum samples collected from covid-19-infected individuals. the results indicated that the viral load peaked at around 5 to 6 days following the onset of symptoms, and it ranged from 10 4 to 10 7 copies/ml during this time (151) . in another study, the viral load was found to be higher in the nasal swabs than the throat swabs obtained from covid-19 symptomatic patients (82) . although initially it was thought that viral load would be associated with poor outcomes, some case reports have shown asymptomatic individuals with high viral loads (247) . recently, the viral load in nasal and throat swabs of 17 symptomatic patients was determined, and higher viral loads were recorded soon after the onset of symptoms, particularly in the nose compared to the throat. the pattern of viral nucleic the results of the studies related to sars-cov-2 viral loads reflect active replication of this virus in the upper respiratory tract and prolonged viral shedding after symptoms disappear, including via stool. thus, the current case definition needs to be updated along with a reassessment of the strategies to be adopted for restraining the sars-cov-2 outbreak spread (248) . in some cases, the viral load studies of sars-cov-2 have also been useful to recommend precautionary measures when handling specific samples, e.g., feces. in a recent survey from 17 confirmed cases of sars-cov-2 infection with available data (representing days 0 to 13 after onset), stool samples from nine cases (53%; days 0 to 11 after onset) were positive on rt-pcr analysis. although the viral loads were lower than those of respiratory samples (range, 550 copies per ml to 1.21 ϫ 10 5 copies per ml), this has essential biosafety implications (151) . the samples from 18 sars-cov-2-positive patients in singapore who had traveled from wuhan to singapore showed the presence of viral rna in stool and whole blood but not in urine by real-time rt-pcr (288) . further, novel sars-cov-2 infections have been detected in a variety of clinical specimens, like bronchoalveolar lavage fluid, sputum, nasal swabs, fibrobronchoscope brush biopsy specimens, pharyngeal swabs, feces, and blood (246) . the presence of sars-cov-2 in fecal samples has posed grave public health concerns. in addition to the direct transmission mainly occurring via droplets of sneezing and coughing, other routes, such as fecal excretion and environmental and fomite contamination, are contributing to sars-cov-2 transmission and spread (249) (250) (251) (252) . fecal excretion has also been documented for sars-cov and mers-cov, along with the potential to stay viable in situations aiding fecal-oral transmission. thus, sars-cov-2 has every possibility to be transmitted through this mode. fecal-oral transmission of sars-cov-2, particularly in regions having low standards of hygiene and poor sanitation, may have grave consequences with regard to the high spread of this virus. ethanol and disinfectants containing chlorine or bleach are effective against coronaviruses (249) (250) (251) (252) . appropriate precautions need to be followed strictly while handling the stools of patients infected with sars-cov-2. biowaste materials and sewage from hospitals must be adequately disinfected, treated, and disposed of properly. the significance of frequent and good hand hygiene and sanitation practices needs to be given due emphasis (249) (250) (251) (252) . future explorative research needs to be conducted with regard to the fecal-oral transmission of sars-cov-2, along with focusing on environmental investigations to find out if this virus could stay viable in situations and atmospheres facilitating such potent routes of transmission. the correlation of fecal concentrations of viral rna with disease severity needs to be determined, along with assessing the gastrointestinal symptoms and the possibility of fecal sars-cov-2 rna detection during the covid-19 incubation period or convalescence phases of the disease (249) (250) (251) (252) . the lower respiratory tract sampling techniques, like bronchoalveolar lavage fluid aspirate, are considered the ideal clinical materials, rather than the throat swab, due to their higher positive rate on the nucleic acid test (148) . the diagnosis of covid-19 can be made by using upper-respiratory-tract specimens collected using nasopharyngeal and oropharyngeal swabs. however, these techniques are associated with unnecessary risks to health care workers due to close contact with patients (152) . similarly, a single patient with a high viral load was reported to contaminate an entire endoscopy room by shedding the virus, which may remain viable for at least 3 days and is considered a great risk for uninfected patients and health care workers (289) . recently, it was found that the anal swabs gave more positive results than oral swabs in the later stages of infection (153) . hence, clinicians have to be cautious while discharging any covid-19infected patient based on negative oral swab test results due to the possibility of fecal-oral transmission. even though the viral loads in stool samples were found to be less than those of respiratory samples, strict precautionary measures have to be followed while handling stool samples of covid-19 suspected or infected patients (151) . children infected with sars-cov-2 experience only a mild form of illness and recover immediately after treatment. it was recently found that stool samples of sars-cov-2-infected children that gave negative throat swab results were positive within ten days of negative results. this could result in the fecal-oral transmission of sars-cov-2 infections, especially in children (290) . hence, to prevent the fecal-oral transmission of sars-cov-2, infected covid-19 patients should only be considered negative when they test negative for sars-cov-2 in the stool sample. a suspected case of covid-19 infection is said to be confirmed if the respiratory tract aspirate or blood samples test positive for sars-cov-2 nucleic acid using rt-pcr or by the identification of sars-cov-2 genetic sequence in respiratory tract aspirate or blood samples (80) . the patient will be confirmed as cured when two subsequent oral swab results are negative (153) . recently, the live virus was detected in the selfcollected saliva of patients infected with covid-19. these findings were confirmative of using saliva as a noninvasive specimen for the diagnosis of covid-19 infection in suspected individuals (152) . it has also been observed that the initial screening of covid-19 patients infected with rt-pcr may give negative results even if they have chest ct findings that are suggestive of infection. hence, for the accurate diagnosis of covid-19, a combination of repeated swab tests using rt-pcr and ct scanning is required to prevent the possibility of false-negative results during disease screening (154) . rt-pcr is the most widely used test for diagnosing covid-19. however, it has some significant limitations from the clinical perspective, since it will not give any clarity regarding disease progression. droplet digital pcr (ddpcr) can be used for the quantification of viral load in the samples obtained from lower respiratory tracts. hence, based on the viral load, we can quickly evaluate the progression of infection (291) . in addition to all of the above findings, sequencing and phylogenetics are critical in the correct identification and confirmation of the causative viral agent and useful to establish relationships with previous isolates and sequences, as well as to know, especially during an epidemic, the nucleotide and amino acid mutations and the molecular divergence. the rapid development and implementation of diagnostic tests against emerging novel diseases like covid-19 pose significant challenges due to the lack of resources and logistical limitations associated with an outbreak (155) . sars-cov-2 infection can also be confirmed by isolation and culturing. the human airway epithelial cell culture was found to be useful in isolating sars-cov-2 (3). the efficient control of an outbreak depends on the rapid diagnosis of the disease. recently, in response to the covid-19 outbreak, 1-step quantitative realtime reverse transcription-pcr assays were developed that detect the orf1b and n regions of the sars-cov-2 genome (156) . that assay was found to achieve the rapid detection of sars-cov-2. nucleic acid-based assays offer high accuracy in the diagnosis of sars-cov-2, but the current rate of spread limits its use due to the lack of diagnostic assay kits. this will further result in the extensive transmission of covid-19, since only a portion of suspected cases can be diagnosed. in such situations, conventional serological assays, like enzyme-linked immunosorbent assay (elisa), that are specific to covid-19 igm and igg antibodies can be used as a high-throughput alternative (149) . at present, there is no diagnostic kit available for detecting the sars-cov-2 antibody (150) . the specific antibody profiles of covid-19 patients were analyzed, and it was found that the igm level lasted more than 1 month, indicating a prolonged stage of virus replication in sars-cov-2-infected patients. the igg levels were found to increase only in the later stages of the disease. these findings indicate that the specific antibody profiles of sars-cov-2 and sars-cov were similar (325) . these findings can be utilized for the development of specific diagnostic tests against covid-19 and can be used for rapid screening. even though diagnostic test kits are already available that can detect the genetic sequences of sars-cov-2 (95), their availability is a concern, as the number of covid-19 cases is skyrocketing (155, 157) . a major problem associated with this diagnostic kit is that it works only when the test subject has an active infection, limiting its use to the earlier stages of infection. several laboratories around the world are currently developing antibody-based diagnostic tests against sars-cov-2 (157). chest ct is an ideal diagnostic tool for identifying viral pneumonia. the sensitivity of chest ct is far superior to that of x-ray screening. the chest ct findings associated with covid-19-infected patients include characteristic patchy infiltration that later progresses to ground-glass opacities (158) . early manifestations of covid-19 pneumonia might not be evident in x-ray chest radiography. in such situations, a chest ct examination can be performed, as it is considered highly specific for covid-19 pneumonia (118) . those patients having covid-19 pneumonia will exhibit the typical ground-glass opacity in their chest ct images (154) . the patients infected with covid-19 had elevated plasma angiotensin 2 levels. the level of angiotensin 2 was found to be linearly associated with viral load and lung injury, indicating its potential as a diagnostic biomarker (121) . the chest ct imaging abnormalities associated with covid-19 pneumonia have also been observed even in asymptomatic patients. these abnormalities progress from the initial focal unilateral to diffuse bilateral ground-glass opacities and will further progress to or coexist with lung consolidation changes within 1 to 3 weeks (159). the role played by radiologists in the current scenario is very important. radiologists can help in the early diagnosis of lung abnormalities associated with covid-19 pneumonia. they can also help in the evaluation of disease severity, identifying its progression to acute respiratory distress syndrome and the presence of secondary bacterial infections (160) . even though chest ct is considered an essential diagnostic tool for covid-19, the extensive use of ct for screening purposes in the suspected individuals might be associated with a disproportionate risk-benefit ratio due to increased radiation exposure as well as increased risk of cross-infection. hence, the use of ct for early diagnosis of sars-cov-2 infection in high-risk groups should be done with great caution (292) . more recently, other advanced diagnostics have been designed and developed for the detection of sars-cov-2 (345, 347, (350) (351) (352) . a reverse transcriptional loopmediated isothermal amplification (rt-lamp), namely, ilaco, has been developed for rapid and colorimetric detection of this virus (354) . rt-lamp serves as a simple, rapid, and sensitive diagnostic method that does not require sophisticated equipment or skilled personnel (349) . an interactive web-based dashboard for tracking sars-cov-2 in a real-time mode has been designed (238) . a smartphone-integrated home-based point-of-care testing (poct) tool, a paper-based poct combined with lamp, is a useful point-of-care diagnostic (353) . an abbott id now covid-19 molecular poct-based test, using isothermal nucleic acid amplification technology, has been designed as a pointof-care test for very rapid detection of sars-cov-2 in just 5 min (344) . a crispr-based sherlock (specific high-sensitivity enzymatic reporter unlocking) diagnostic for rapid detection of sars-cov-2 without the requirement of specialized instrumentation has been reported to be very useful in the clinical diagnosis of covid-19 (360) . a crispr-cas12-based lateral flow assay also has been developed for rapid detection of sars-cov-2 (346) . artificial intelligence, by means of a three-dimensional deep-learning model, has been developed for sensitive and specific diagnosis of covid-19 via ct images (332) . tracking and mapping of the rising incidence rates, disease outbreaks, community spread, clustered transmission events, hot spots, and superspreader potential of sars-cov-2/covid warrant full exploitation of real-time disease mapping by employing geographical information systems (gis), such as the gis software kosmo 3.1, web-based real-time tools and dashboards, apps, and advances in information technology (356-359). researchers have also developed a few prediction tools/models, such as the prediction model risk of bias assessment tool (probast) and critical appraisal and data extraction for systematic reviews of prediction modeling studies (charms), which could aid in assessing the possibility of getting infection and estimating the prognosis in patients; however, such models may suffer from bias issues and, hence, cannot be considered completely trustworthy, which necessitates the development of new and reliable predictors (360) . recently emerged viruses, such as zika, ebola, and nipah viruses, and their grave threats to humans have begun a race in exploring the designing and developing of advanced vaccines, prophylactics, therapeutics, and drug regimens to counter emerging viruses (161) (162) (163) 280) . several attempts are being made to design and develop vaccines for cov infection, mostly by targeting the spike glycoprotein. nevertheless, owing to extensive diversity in antigenic variants, cross-protection rendered by the vaccines is significantly limited, even within the strains of a phylogenetic subcluster (104) . due to the lack of effective antiviral therapy and vaccines in the present scenario, we need to depend solely on implementing effective infection control measures to lessen the risk of possible nosocomial transmission (68) . recently, the receptor for sars-cov-2 was established as the human angiotensin-converting enzyme 2 (hace2), and the virus was found to enter the host cell mainly through endocytosis. it was also found that the major components that have a critical role in viral entry include pikfyve, tpc2, and cathepsin l. these findings are critical, since the components described above might act as candidates for vaccines or therapeutic drugs against sars-cov-2 (293) . the majority of the treatment options and strategies that are being evaluated for sars-cov-2 (covid-19) have been taken from our previous experiences in treating sars-cov, mers-cov, and other emerging viral diseases. several therapeutic and preventive strategies, including vaccines, immunotherapeutics, and antiviral drugs, have been exploited against the previous cov outbreaks (sars-cov and mers-cov) (8, 104, (164) (165) (166) (167) . these valuable options have already been evaluated for their potency, efficacy, and safety, along with several other types of current research that will fuel our search for ideal therapeutic agents against covid-19 (7, 9, 19, 21, 36) . the primary cause of the unavailability of approved and commercial vaccines, drugs, and therapeutics to counter the earlier sars-cov and mers-cov seems to owe to the lesser attention of the biomedicine and pharmaceutical companies, as these two covs did not cause much havoc, global threat, and panic like those posed by the sars-cov-2 pandemic (19) . moreover, for such outbreak situations, the requirement for vaccines and therapeutics/drugs exists only for a limited period, until the outbreak is controlled. the proportion of the human population infected with sars-cov and mers-cov was also much lower across the globe, failing to attract drug and vaccine manufacturers and clinical microbiology reviews producers. therefore, by the time an effective drug or vaccine is designed against such disease outbreaks, the virus would have been controlled by adopting appropriate and strict prevention and control measures, and patients for clinical trials will not be available. the newly developed drugs cannot be marketed due to the lack of end users. the s protein plays a significant role in the induction of protective immunity against sars-cov by mediating t-cell responses and neutralizing antibody production (168) . in the past few decades, we have seen several attempts to develop a vaccine against human coronaviruses by using s protein as the target (168, 169) . however, the developed vaccines have minimal application, even among closely related strains of the virus, due to a lack of cross-protection. that is mainly because of the extensive diversity existing among the different antigenic variants of the virus (104) . the contributions of the structural proteins, like spike (s), matrix (m), small envelope (e), and nucleocapsid (n) proteins, of sars-cov to induce protective immunity has been evaluated by expressing them in a recombinant parainfluenza virus type 3 vector (bhpiv3). of note, the result was conclusive that the expression of m, e, or n proteins without the presence of s protein would not confer any noticeable protection, with the absence of detectable serum sars-cov-neutralizing antibodies (170) . antigenic determinant sites present over s and n structural proteins of sars-cov-2 can be explored as suitable vaccine candidates (294) . in the asian population, s, e, m, and n proteins of sars-cov-2 are being targeted for developing subunit vaccines against covid-19 (295) . the identification of the immunodominant region among the subunits and domains of s protein is critical for developing an effective vaccine against the coronavirus. the c-terminal domain of the s1 subunit is considered the immunodominant region of the porcine deltacoronavirus s protein (171) . similarly, further investigations are needed to determine the immunodominant regions of sars-cov-2 for facilitating vaccine development. however, our previous attempts to develop a universal vaccine that is effective for both sars-cov and mers-cov based on t-cell epitope similarity pointed out the possibility of cross-reactivity among coronaviruses (172) . that can be made possible by selected potential vaccine targets that are common to both viruses. sars-cov-2 has been reported to be closely related to sars-cov (173, 174) . hence, knowledge and understanding of s protein-based vaccine development in sars-cov will help to identify potential s protein vaccine candidates in sars-cov-2. therefore, vaccine strategies based on the whole s protein, s protein subunits, or specific potential epitopes of s protein appear to be the most promising vaccine candidates against coronaviruses. the rbd of the s1 subunit of s protein has a superior capacity to induce neutralizing antibodies. this property of the rbd can be utilized for designing potential sars-cov vaccines either by using rbd-containing recombinant proteins or recombinant vectors that encode rbd (175) . hence, the superior genetic similarity existing between sars-cov-2 and sars-cov can be utilized to repurpose vaccines that have proven in vitro efficacy against sars-cov to be utilized for sars-cov-2. the possibility of cross-protection in covid-19 was evaluated by comparing the s protein sequences of sars-cov-2 with that of sars-cov. the comparative analysis confirmed that the variable residues were found concentrated on the s1 subunit of s protein, an important vaccine target of the virus (150) . hence, the possibility of sars-cov-specific neutralizing antibodies providing cross-protection to covid-19 might be lower. further genetic analysis is required between sars-cov-2 and different strains of sars-cov and sarslike (sl) covs to evaluate the possibility of repurposed vaccines against covid-19. this strategy will be helpful in the scenario of an outbreak, since much time can be saved, because preliminary evaluation, including in vitro studies, already would be completed for such vaccine candidates. multiepitope subunit vaccines can be considered a promising preventive strategy against the ongoing covid-19 pandemic. in silico and advanced immunoinformatic tools can be used to develop multiepitope subunit vaccines. the vaccines that are engineered by this technique can be further evaluated using docking studies and, if found effective, then can be further evaluated in animal models (365) . identifying epitopes that have the potential to become a vaccine candidate is critical to developing an effective vaccine against covid-19. the immunoinformatics approach has been used for recognizing essential epitopes of cytotoxic t lymphocytes and b cells from the surface glycoprotein of sars-cov-2. recently, a few epitopes have been recognized from the sars-cov-2 surface glycoprotein. the selected epitopes explored targeting molecular dynamic simulations, evaluating their interaction with corresponding major histocompatibility complex class i molecules. they potentially induce immune responses (176) . the recombinant vaccine can be designed by using rabies virus (rv) as a viral vector. rv can be made to express mers-cov s1 protein on its surface so that an immune response is induced against mers-cov. the rv vector-based vaccines against mers-cov can induce faster antibody response as well as higher degrees of cellular immunity than the gram-positive enhancer matrix (gem) particle vector-based vaccine. however, the latter can induce a very high antibody response at lower doses (167) . hence, the degree of humoral and cellular immune responses produced by such vaccines depends upon the vector used. dual vaccines have been getting more popular recently. among them, the rabies virus-based vectored vaccine platform is used to develop vaccines against emerging infectious diseases. the dual vaccine developed from inactivated rabies virus particles that express the mers-cov s1 domain of s protein was found to induce immune responses for both mers-cov and rabies virus. the vaccinated mice were found to be completely protected from challenge with mers-cov (169) . the intranasal administration of the recombinant adenovirus-based vaccine in balb/c mice was found to induce long-lasting neutralizing immunity against mers spike pseudotyped virus, characterized by the induction of systemic igg, secretory iga, and lung-resident memory t-cell responses (177) . immunoinformatics methods have been employed for the genomewide screening of potential vaccine targets among the different immunogens of mers-cov (178) . the n protein and the potential b-cell epitopes of mers-cov e protein have been suggested as immunoprotective targets inducing both t-cell and neutralizing antibody responses (178, 179) . the collaborative effort of the researchers of rocky mountain laboratories and oxford university is designing a chimpanzee adenovirus-vectored vaccine to counter covid-19 (180) . the coalition for epidemic preparedness innovations (cepi) has initiated three programs to design sars-cov-2 vaccines (181) . cepi has a collaborative project with inovio for designing a mers-cov dna vaccine that could potentiate effective immunity. cepi and the university of queensland are designing a molecular clamp vaccine platform for mers-cov and other pathogens, which could assist in the easier identification of antigens by the immune system (181) . cepi has also funded moderna to develop a vaccine for covid-19 in partnership with the vaccine research center (vrc) of the national institute of allergy and infectious diseases (niaid), part of the national institutes of health (nih) (182) . by employing mrna vaccine platform technology, a vaccine candidate expressing sars-cov-2 spike protein is likely to go through clinical testing in the coming months (180 (298) . the process of vaccine development usually takes approximately ten years, in the case of inactivated or live attenuated vaccines, since it involves the generation of long-term efficacy data. however, this was brought down to 5 years during the ebola emergency for viral vector vaccines. in the urgency associated with the covid-19 outbreaks, we expect a vaccine by the end of this year (343) . the development of an effective vaccine against covid-19 with high speed and precision is the combined result of advancements in computational biology, gene synthesis, protein engineering, and the invention of advanced manufacturing platforms (342) . the recurring nature of the coronavirus outbreaks calls for the development of a pan-coronavirus vaccine that can produce cross-reactive antibodies. however, the success of such a vaccine relies greatly on its ability to provide protection not only against present versions of the virus but also the ones that are likely to emerge in the future. this can be achieved by identifying antibodies that can recognize relatively conserved epitopes that are maintained as such even after the occurrence of considerable variations (362) . even though several vaccine clinical trials are being conducted around the world, pregnant women have been completely excluded from these studies. pregnant women are highly vulnerable to emerging diseases such as covid-19 due to alterations in the immune system and other physiological systems that are associated with pregnancy. therefore, in the event of successful vaccine development, pregnant women will not get access to the vaccines (361) . hence, it is recommended that pregnant women be included in the ongoing vaccine trials, since successful vaccination in pregnancy will protect the mother, fetus, and newborn. the heterologous immune effects induced by bacillus calmette guérin (bcg) vaccination is a promising strategy for controlling the covid-19 pandemic and requires further investigations. bcg is a widely used vaccine against tuberculosis in high-risk regions. it is derived from a live attenuated strain of mycobacterium bovis. at present, three new clinical trials have been registered to evaluate the protective role of bcg vaccination against sars-cov-2 (363) . recently, a cohort study was conducted to evaluate the impact of childhood bcg vaccination in covid-19 pcr positivity rates. however, childhood bcg vaccination was found to be associated with a rate of covid-19-positive test results similar to that of the nonvaccinated group (364) . further studies are required to analyze whether bcg vaccination in childhood can induce protective effects against covid-19 in adulthood. population genetic studies conducted on 103 genomes identified that the sars-cov-2 virus has evolved into two major types, l and s. among the two types, l type is expected to be the most prevalent (ϳ70%), followed by the s type (ϳ30%) (366) . this finding has a significant impact on our race to develop an ideal vaccine, since the vaccine candidate has to target both strains to be considered effective. at present, the genetic differences between the l and s types are very small and may not affect the immune response. however, we can expect further genetic variations in the coming days that could lead to the emergence of new strains (367) . there is no currently licensed specific antiviral treatment for mers-and sars-cov infections, and the main focus in clinical settings remains on lessening clinical signs and providing supportive care (183) (184) (185) (186) . effective drugs to manage covid-19 patients include remdesivir, lopinavir/ritonavir alone or in a blend with interferon beta, convalescent plasma, and monoclonal antibodies (mabs); however, efficacy and safety issues of these drugs require additional clinical trials (187, 281) . a controlled trial of ritonavirboosted lopinavir and interferon alpha 2b treatment was performed on covid-19 hospitalized patients (chictr2000029308) (188) . in addition, the use of hydroxychloroquine and tocilizumab for their potential role in modulating inflammatory responses in the lungs and antiviral effect has been proposed and discussed in many research articles. still, no fool-proof clinical trials have been published (194, 196, 197, (261) (262) (263) (264) (265) (266) (267) (268) (269) (270) (271) (272) . recently, a clinical trial conducted on adult patients suffering from severe covid-19 revealed no benefit of lopinavir-ritonavir treatment over standard care (273) . the efforts to control sars-cov-2 infection utilize defined strategies as followed against mers and sars, along with adopting and strengthening a few precautionary measures owing to the unknown nature of this novel virus (36, 189) . presently, the main course of treatment for severely affected sars-cov-2 patients admitted to hospitals includes mechanical ventilation, intensive care unit (icu) admittance, and symptomatic and supportive therapies. additionally, rna synthesis inhibitors (lamivudine and tenofovir disoproxil fumarate), remdesivir, neuraminidase inhibitors, peptide (ek1), antiinflammatory drugs, abidol, and chinese traditional medicine (lianhuaqingwen and shufengjiedu capsules) could aid in covid-19 treatment. however, further clinical trials are being carried out concerning their safety and efficacy (7) . it might require months to a year(s) to design and develop effective drugs, therapeutics, and vaccines against covid-19, with adequate evaluation and approval from regulatory bodies and moving to the bulk production of many millions of doses at commercial levels to meet the timely demand of mass populations across the globe (9) . continuous efforts are also warranted to identify and assess viable drugs and immunotherapeutic regimens that revealed proven potency in combating other viral agents similar to sars-cov-2. covid-19 patients showing severe signs are treated symptomatically along with oxygen therapy. in such cases where the patients progress toward respiratory failure and become refractory to oxygen therapy, mechanical ventilation is necessitated. the covid-19-induced septic shock can be managed by providing adequate hemodynamic support (299) . several classes of drugs are currently being evaluated for their potential therapeutic action against sars-cov-2. therapeutic agents that have anti-sars-cov-2 activity can be broadly classified into three categories: drugs that block virus entry into the host cell, drugs that block viral replication as well as its survival within the host cell, and drugs that attenuate the exaggerated host immune response (300) . an inflammatory cytokine storm is commonly seen in critically ill covid-19 patients. hence, they may benefit from the use of timely anti-inflammation treatment. anti-inflammatory therapy using drugs like glucocorticoids, cytokine inhibitors, jak inhibitors, and chloroquine/hydroxychloroquine should be done only after analyzing the risk/benefit ratio in covid-19 patients (301). there have not been any studies concerning the application of nonsteroidal anti-inflammatory drugs (nsaid) to covid-19-infected patients. however, reasonable pieces of evidence are available that link nsaid uses with the occurrence of respiratory and cardiovascular adverse effects. hence, as a cautionary approach, it is better to recommend the use of nsaids as the first-line option for managing covid-19 symptoms (302) . the use of corticosteroids in covid-19 patients is still a matter of controversy and requires further systematic clinical studies. the guidelines that were put forward to manage critically ill adults suggest the use of systemic corticosteroids in mechanically ventilated adults with ards (303) . the generalized use of corticosteroids is not indicated in covid-19, since there are some concerns associated with the use of corticosteroids in viral pneumonia. stem cell therapy using mesenchymal stem cells (mscs) is another hopeful strategy that can be used in clinical cases of covid-19 owing to its potential immunomodulatory capacity. it may have a beneficial role in attenuating the cytokine storm that is observed in severe cases of sars-cov-2 infection, thereby reducing mortality. among the different types of mscs, expanded umbilical cord mscs can be considered a potential therapeutic agent that requires further validation for managing critically ill covid-19 patients (304) . repurposed broad-spectrum antiviral drugs having proven uses against other viral pathogens can be employed for sars-cov-2-infected patients. these possess benefits of easy accessibility and recognized pharmacokinetic and pharmacodynamic activities, stability, doses, and side effects (9) . repurposed drugs have been studied for treating cov infections, like lopinavir/ritonavir, and interferon-1␤ revealed in vitro anti-mers-cov action. the in vivo experiment carried out in the nonhuman primate model of clinical microbiology reviews common marmosets treated with lopinavir/ritonavir and interferon beta showed superior protective results in treated animals than in the untreated ones (190) . a combination of these drugs is being evaluated to treat mers in humans (miracle trial) (191) . these two protease inhibitors (lopinavir and ritonavir), in combination with ribavirin, gave encouraging clinical outcomes in sars patients, suggesting their therapeutic values (165) . however, in the current scenario, due to the lack of specific therapeutic agents against sars-cov-2, hospitalized patients confirmed for the disease are given supportive care, like oxygen and fluid therapy, along with antibiotic therapy for managing secondary bacterial infections (192) . patients with novel coronavirus or covid-19 pneumonia who are mechanically ventilated often require sedatives, analgesics, and even muscle relaxation drugs to prevent ventilator-related lung injury associated with human-machine incoordination (122) . the result obtained from a clinical study of four patients infected with covid-19 claimed that combination therapy using lopinavir/ritonavir, arbidol, and shufeng jiedu capsules (traditional chinese medicine) was found to be effective in managing covid-19 pneumonia (193) . it is difficult to evaluate the therapeutic potential of a drug or a combination of drugs for managing a disease based on such a limited sample size. before choosing the ideal therapeutic agent for the management of covid-19, randomized clinical control studies should be performed with a sufficient study population. several classes of routinely used antiviral drugs, like oseltamivir (neuraminidase inhibitor), acyclovir, ganciclovir, and ribavirin, do not have any effect on covid-19 and, hence, are not recommended (187) . oseltamivir, a neuraminidase inhibitor, has been explored in chinese hospitals for treating suspected covid-19 cases, although proven efficacy against sars-cov-2 is still lacking for this drug (7) . the in vitro antiviral potential of fad-approved drugs, viz., ribavirin, penciclovir, nitazoxanide, nafamostat, and chloroquine, tested in comparison to remdesivir and favipiravir (broad-spectrum antiviral drugs) revealed remdesivir and chloroquine to be highly effective against sars-cov-2 infection in vitro (194) . ribavirin, penciclovir, and favipiravir might not possess noteworthy in vivo antiviral actions for sars-cov-2, since higher concentrations of these nucleoside analogs are needed in vitro to lessen the viral infection. both remdesivir and chloroquine are being used in humans to treat other diseases, and such safer drugs can be explored for assessing their effectiveness in covid-19 patients. several therapeutic agents, such as lopinavir/ritonavir, chloroquine, and hydroxychloroquine, have been proposed for the clinical management of covid-19 (299) . a molecular docking study, conducted in the rna-dependent rna polymerase (rdrp) of sars-cov-2 using different commercially available antipolymerase drugs, identified that drugs such as ribavirin, remdesivir, galidesivir, tenofovir, and sofosbuvir bind rdrp tightly, indicating their vast potential to be used against covid-19 (305). a broadspectrum antiviral drug that was developed in the united states, tilorone dihydrochloride (tilorone), was previously found to possess potent antiviral activity against mers, marburg, ebola, and chikungunya viruses (306) . even though it had broad-spectrum activity, it was neglected for an extended period. tilorone is another antiviral drug that might have activity against sars-cov-2. remdesivir, a novel nucleotide analog prodrug, was developed for treating ebola virus disease (evd), and it was also found to inhibit the replication of sars-cov and mers-cov in primary human airway epithelial cell culture systems (195) . recently, in vitro study has proven that remdesivir has better antiviral activity than lopinavir and ritonavir. further, in vivo studies conducted in mice also identified that treatment with remdesivir improved pulmonary function and reduced viral loads and lung pathology both in prophylactic and therapeutic regimens compared to lopinavir/ritonavir-ifn-␥ treatment in mers-cov infection (8) . remdesivir also inhibits a diverse range of coronaviruses, including circulating human cov, zoonotic bat cov, and prepandemic zoonotic cov (195) . remdesivir is also considered the only therapeutic drug that significantly reduces pulmonary pathology (8) . all these findings indicate that remde-sivir has to be further evaluated for its efficacy in the treatment of covid-19 infection in humans. the broad-spectrum activity exhibited by remdesivir will help control the spread of disease in the event of a new coronavirus outbreak. chloroquine is an antimalarial drug known to possess antiviral activity due to its ability to block virus-cell fusion by raising the endosomal ph necessary for fusion. it also interferes with virus-receptor binding by interfering with the terminal glycosylation of sars-cov cellular receptors, such as ace2 (196) . in a recent multicenter clinical trial that was conducted in china, chloroquine phosphate was found to exhibit both efficacy and safety in the therapeutic management of sars-cov-2-associated pneumonia (197) . this drug is already included in the treatment guidelines issued by the national health commission of the people's republic of china. the preliminary clinical trials using hydroxychloroquine, another aminoquinoline drug, gave promising results. the covid-19 patients received 600 mg of hydroxychloroquine daily along with azithromycin as a single-arm protocol. this protocol was found to be associated with a noteworthy reduction in viral load. finally, it resulted in a complete cure (271) ; however, the study comprised a small population and, hence, the possibility of misinterpretation could arise. however, in another case study, the authors raised concerns over the efficacy of hydroxychloroquine-azithromycin in the treatment of covid-19 patients, since no observable effect was seen when they were used. in some cases, the treatment was discontinued due to the prolongation of the qt interval (307) . hence, further randomized clinical trials are required before concluding this matter. recently, another fda-approved drug, ivermectin, was reported to inhibit the in vitro replication of sars-cov-2. the findings from this study indicate that a single treatment of this drug was able to induce an ϳ5,000-fold reduction in the viral rna at 48 h in cell culture. (308) . one of the main disadvantages that limit the clinical utility of ivermectin is its potential to cause cytotoxicity. however, altering the vehicles used in the formulations, the pharmacokinetic properties can be modified, thereby having significant control over the systemic concentration of ivermectin (338) . based on the pharmacokinetic simulation, it was also found that ivermectin may have limited therapeutic utility in managing covid-19, since the inhibitory concentration that has to be achieved for effective anti-sars-cov-2 activity is far higher than the maximum plasma concentration achieved by administering the approved dose (340) . however, ivermectin, being a host-directed agent, exhibits antiviral activity by targeting a critical cellular process of the mammalian cell. therefore, the administration of ivermectin, even at lower doses, will reduce the viral load at a minor level. this slight decrease will provide a great advantage to the immune system for mounting a large-scale antiviral response against sars-cov-2 (341). further, a combination of ivermectin and hydroxychloroquine might have a synergistic effect, since ivermectin reduces viral replication, while hydroxychloroquine inhibits the entry of the virus in the host cell (339) . further, in vivo studies and randomized clinical control trials are required to understand the mechanism as well as the clinical utility of this promising drug. nafamostat is a potent inhibitor of mers-cov that acts by preventing membrane fusion. nevertheless, it does not have any sort of inhibitory action against sars-cov-2 infection (194) . recently, several newly synthesized halogenated triazole compounds were evaluated, using fluorescence resonance energy transfer (fret)-based helicase assays, for their ability to inhibit helicase activity. among the evaluated compounds, 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-iodophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol and 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-chlorophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol were found to be the most potent. these compounds were used for in silico studies, and molecular docking was accomplished into the active binding site of mers-cov helicase nsp13 (21) . further studies are required for evaluating the therapeutic potential of these newly identified compounds in the management of covid-19 infection. monoclonal antibodies (mabs) may be helpful in the intervention of disease in clinical microbiology reviews cov-exposed individuals. patients recovering from sars showed robust neutralizing antibodies against this cov infection (164) . a set of mabs aimed at the mers-cov s protein-specific domains, comprising six specific epitope groups interacting with receptor-binding, membrane fusion, and sialic acid-binding sites, make up crucial entry tasks of s protein (198, 199) . passive immunization employing weaker and strongly neutralizing antibodies provided considerable protection in mice against a mers-cov lethal challenge. such antibodies may play a crucial role in enhancing protective humoral responses against the emerging covs by aiming appropriate epitopes and functions of the s protein. the cross-neutralization ability of sars-cov rbd-specific neutralizing mabs considerably relies on the resemblance between their rbds; therefore, sars-cov rbd-specific antibodies could cross-neutralized sl covs, i.e., bat-sl-cov strain wiv1 (rbd with eight amino acid differences from sars-cov) but not bat-sl-cov strain shc014 (24 amino acid differences) (200) . appropriate rbd-specific mabs can be recognized by a relative analysis of rbd of sars-cov-2 to that of sars-cov, and cross-neutralizing sars-cov rbd-specific mabs could be explored for their effectiveness against covid-19 and further need to be assessed clinically. the u.s. biotechnology company regeneron is attempting to recognize potent and specific mabs to combat covid-19. an ideal therapeutic option suggested for sars-cov-2 (covid-19) is the combination therapy comprised of mabs and the drug remdesivir (covid-19) (201) . the sars-cov-specific human mab cr3022 is found to bind with sars-cov-2 rbd, indicating its potential as a therapeutic agent in the management of covid-19. it can be used alone or in combination with other effective neutralizing antibodies for the treatment and prevention of covid-19 (202) . furthermore, sars-cov-specific neutralizing antibodies, like m396 and cr3014, failed to bind the s protein of sars-cov-2, indicating that a particular level of similarity is mandatory between the rbds of sars-cov and sars-cov-2 for the cross-reactivity to occur. further assessment is necessary before confirming the effectiveness of such combination therapy. in addition, to prevent further community and nosocomial spread of covid-19, the postprocedure risk management program should not be neglected (309) . development of broad-spectrum inhibitors against the human coronaviral pathogens will help to facilitate clinical trials on the effectiveness of such inhibitors against endemic and emerging coronaviruses (203) . a promising animal study revealed the protective effect of passive immunotherapy with immune serum from mers-immune camels on mice infected with mers-cov (204) . passive immunotherapy using convalescent plasma is another strategy that can be used for treating covid-19-infected, critically ill patients (205) . the exploration of fully human antibodies (human single-chain antibodies; huscfvs) or humanized nanobodies (single-domain antibodies; sdab, vh/vhh) could aid in blocking virus replication, as these agents can traverse the virus-infected cell membranes (transbodies) and can interfere with the biological characteristics of the replicating virus proteins. such examples include transbodies to the influenza virus, hepatitis c virus, ebola virus, and dengue virus (206) . producing similar transbodies against intracellular proteins of coronaviruses, such as papain-like proteases (plpro), cysteinelike protease (3clpro), or other nsps, which are essential for replication and transcription of the virus, might formulate a practical move forward for a safer and potent passive immunization approach for virus-exposed persons and rendering therapy to infected patients. in a case study on five grimly sick patients having symptoms of severe pneumonia due to covid-19, convalescent plasma administration was found to be helpful in patients recovering successfully. the convalescent plasma containing a sars-cov-2specific elisa (serum) antibody titer higher than 1:1,000 and neutralizing antibody titer more significant than 40 was collected from the recovered patients and used for plasma transfusion twice in a volume of 200 to 250 ml on the day of collection (310) . at present, treatment for sepsis and ards mainly involves antimicrobial therapy, source control, and supportive care. hence, the use of therapeutic plasma exchange can be considered an option in managing such severe conditions. further randomized trials can be designed to investigate its efficacy (311) . potent therapeutics to combat sars-cov-2 infection include virus binding molecules, molecules or inhibitors targeting particular enzymes implicated in replication and transcription process of the virus, helicase inhibitors, vital viral proteases and proteins, protease inhibitors of host cells, endocytosis inhibitors, short interfering rna (sirna), neutralizing antibodies, mabs against the host receptor, mabs interfering with the s1 rbd, antiviral peptide aimed at s2, and natural drugs/medicines (7, 166, 186) . the s protein acts as the critical target for developing cov antivirals, like inhibitors of s protein and s cleavage, neutralizing antibodies, rbd-ace2 blockers, sirnas, blockers of the fusion core, and proteases (168) . all of these therapeutic approaches have revealed both in vitro and in vivo anti-cov potential. although in vitro research carried out with these therapeutics showed efficacy, most need appropriate support from randomized animal or human trials. therefore, they might be of limited applicability and require trials against sars-cov-2 to gain practical usefulness. the binding of sars-cov-2 with ace2 leads to the exacerbation of pneumonia as a consequence of the imbalance in the reninangiotensin system (ras). the virus-induced pulmonary inflammatory responses may be reduced by the administration of ace inhibitors (acei) and angiotensin type-1 receptor (at1r) (207) . several investigations have suggested the use of small-molecule inhibitors for the potential control of sars-cov infections. drugs of the fda-approved compound library were screened to identify four small-molecule inhibitors of mers-cov (chlorpromazine, chloroquine, loperamide, and lopinavir) that inhibited viral replication. these compounds also hinder sars-cov and human covs (208) . therapeutic strategies involving the use of specific antibodies or compounds that neutralize cytokines and their receptors will help to restrain the host inflammatory responses. such drugs acting specifically in the respiratory tract will help to reduce virus-triggered immune pathologies in covid-19 (209) . the later stages of coronavirus-induced inflammatory cascades are characterized by the release of proinflammatory interleukin-1 (il-1) family members, such as il-1 and il-33. hence, there exists a possibility that the inflammation associated with coronavirus can be inhibited by utilizing anti-inflammatory cytokines that belong to the il-1 family (92) . it has also been suggested that the actin protein is the host factor that is involved in cell entry and pathogenesis of sars-cov-2. hence, those drugs that modulate the biological activity of this protein, like ibuprofen, might have some therapeutic application in managing the disease (174). the plasma angiotensin 2 level was found to be markedly elevated in covid-19 infection and was correlated with viral load and lung injury. hence, drugs that block angiotensin receptors may have potential for treating covid-19 infection (121) . a scientist from germany, named rolf hilgenfeld, has been working on the identification of drugs for the treatment of coronaviral infection since the time of the first sars outbreak (19) . the sars-cov s2 subunit has a significant function in mediating virus fusion that provides entry into the host cell. heptad repeat 1 (hr1) and heptad repeat 2 (hr2) can interact and form a six-helix bundle that brings the viral and cellular membranes in close proximity, facilitating its fusion. the sequence alignment study conducted between covid-19 and sars-cov identified that the s2 subunits are highly conserved in these covs. the hr1 and hr2 domains showed 92.6% and 100% overall identity, respectively (210) . from these findings, we can confirm the significance of covid-19 hr1 and hr2 and their vital role in host cell entry. hence, fusion inhibitors target the hr1 domain of s protein, thereby preventing viral fusion and entry into the host cell. this is another potential therapeutic strategy that can be used in the management of covid-19. other than the specific therapy directed against covid-19, general treatments play a vital role in the enhancement of host immune responses against the viral agent. inadequate nutrition is linked to the weakening of the host immune response, clinical microbiology reviews making the individual more susceptible. the role played by nutrition in disease susceptibility should be measured by evaluating the nutritional status of patients with covid-19 (205) . for evaluating the potential of vaccines and therapeutics against covs, including sars-cov, mers-covs, and the presently emerging sars-cov-2, suitable animal models that can mimic the clinical disease are needed (211, 212) . various animal models were assessed for sars-and mers-covs, such as mice, guinea pigs, golden syrian hamsters, ferrets, rabbits, nonhuman primates like rhesus macaques and marmosets, and cats (185, (213) (214) (215) (216) (217) (218) . the specificity of the virus to hace2 (receptor of sars-cov) was found to be a significant barrier in developing animal models. consequently, a sars-cov transgenic mouse model has been developed by inserting the hace2 gene into the mouse genome (219) . the inability of mers-cov to replicate in the respiratory tracts of animals (mice, hamsters, and ferrets) is another limiting factor. however, with genetic engineering, a 288-330 ϩ/ϩ mers-cov genetically modified mouse model was developed and now is in use for the assessment of novel drugs and vaccines against mers-cov (220). in the past, small animals (mice or hamsters) have been targeted for being closer to a humanized structure, such as mouse dpp4 altered with human dpp4 (hdpp4), hdpp4-transduced mice, and hdpp4-tg mice (transgenic for expressing hdpp4) for mers-cov infection (221) . the crispr-cas9 gene-editing tool has been used for inserting genomic alterations in mice, making them susceptible to mers-cov infection (222) . efforts are under way to recognize suitable animal models for sars-cov2/covid-19, identify the receptor affinity of this virus, study pathology in experimental animal models, and explore virus-specific immune responses and protection studies, which together would increase the pace of efforts being made for developing potent vaccines and drugs to counter this emerging virus. cell lines, such as monkey epithelial cell lines (llc-mk2 and vero-b4), goat lung cells, alpaca kidney cells, dromedary umbilical cord cells, and advanced ex vivo three-dimensional tracheobronchial tissue, have been explored to study human covs (mers-cov) (223, 224) . vero and huh-7 cells (human liver cancer cells) have been used for isolating sars-cov-2 (194) . recently, an experimental study with rhesus monkeys as animal models revealed the absence of any viral loads in nasopharyngeal and anal swabs, and no viral replication was recorded in the primary tissues at a time interval of 5 days post-reinfection in reexposed monkeys (274) . the subsequent virological, radiological, and pathological observations indicated that the monkeys with reexposure had no recurrence of covid-19, like the sars-cov-2-infected monkeys without rechallenge. these findings suggest that primary infection with sars-cov-2 could protect from later exposures to the virus, which could help in defining disease prognosis and crucial inferences for designing and developing potent vaccines against covid-19 (274). in contrast to their response to the 2002 sars outbreak, china has shown immense political openness in reporting the covid-19 outbreak promptly. they have also performed rapid sequencing of covid-19 at multiple levels and shared the findings globally within days of identifying the novel virus (225) . the move made by china opened a new chapter in global health security and diplomacy. even though complete lockdown was declared following the covid-19 outbreak in wuhan, the large-scale movement of people has resulted in a radiating spread of infections in the surrounding provinces as well as to several other countries. large-scale screening programs might help us to control the spread of this virus. however, this is both challenging as well as time-consuming due to the present extent of infection (226) . the current scenario demands effective implementation of vigorous prevention and control strategies owing to the prospect of covid-19 for nosocomial infections (68) . follow-ups of infected patients by telephone on day 7 and day 14 are advised to avoid any further unintentional spread or nosocomial transmission (312) . the availability of public data sets provided by independent analytical teams will act as robust evidence that would guide us in designing interventions against the covid-19 outbreak. newspaper reports and social media can be used to analyze and reconstruct the progression of an outbreak. they can help us to obtain detailed patient-level data in the early stages of an outbreak (227) . immediate travel restrictions imposed by several countries might have contributed significantly to preventing the spread of sars-cov-2 globally (89, 228) . following the outbreak, a temporary ban was imposed on the wildlife trade, keeping in mind the possible role played by wild animal species in the origin of sars-cov-2/covid-19 (147) . making a permanent and bold decision on the trade of wild animal species is necessary to prevent the possibility of virus spread and initiation of an outbreak due to zoonotic spillover (1) . personal protective equipment (ppe), like face masks, will help to prevent the spread of respiratory infections like covid-19. face masks not only protect from infectious aerosols but also prevent the transmission of disease to other susceptible individuals while traveling through public transport systems (313) . another critical practice that can reduce the transmission of respiratory diseases is the maintenance of hand hygiene. however, the efficacy of this practice in reducing the transmission of respiratory viruses like sars-cov-2 is much dependent upon the size of droplets produced. hand hygiene will reduce disease transmission only if the virus is transmitted through the formation of large droplets (314) . hence, it is better not to overemphasize that hand hygiene will prevent the transmission of sars-cov-2, since it may produce a false sense of safety among the general public that further contributes to the spread of covid-19. even though airborne spread has not been reported in sars-cov-2 infection, transmission can occur through droplets and fomites, especially when there is close, unprotected contact between infected and susceptible individuals. hence, hand hygiene is equally as important as the use of appropriate ppe, like face masks, to break the transmission cycle of the virus; both hand hygiene and face masks help to lessen the risk of covid-19 transmission (315) . medical staff are in the group of individuals most at risk of getting covid-19 infection. this is because they are exposed directly to infected patients. hence, proper training must be given to all hospital staff on methods of prevention and protection so that they become competent enough to protect themselves and others from this deadly disease (316) . as a preventive measure, health care workers caring for infected patients should take extreme precautions against both contact and airborne transmission. they should use ppe such as face masks (n95 or ffp3), eye protection (goggles), gowns, and gloves to nullify the risk of infection (299) . the human-to-human transmission reported in sars-cov-2 infection occurs mainly through droplet or direct contact. due to this finding, frontline health care workers should follow stringent infection control and preventive measures, such as the use of ppe, to prevent infection (110) . the mental health of the medical/health workers who are involved in the covid-19 outbreak is of great importance, because the strain on their mental well-being will affect their attention, concentration, and decision-making capacity. hence, for control of the covid-19 outbreak, rapid steps should be taken to protect the mental health of medical workers (229) . since the living mammals sold in the wet market are suspected to be the intermediate host of sars-cov-2, there is a need for strengthening the regulatory mechanism for wild animal trade (13) . the total number of covid-19 confirmed cases is on a continuous rise and the cure rate is relatively low, making disease control very difficult to achieve. the chinese government is making continuous efforts to contain the disease by taking emergency control and prevention measures. they have already built a hospital for patients affected by this virus and are currently building several more for accommodating the continuously increasing infected population (230) . the effective control of sars-cov-2/covid-19 requires high-level interventions like intensive contact tracing, as well as the quarantine of people with suspected infection and the isolation of infected individuals. the implementation of rigorous control and preventive measures together might control the r 0 number and reduce the transmission risk (228) . clinical microbiology reviews considering the zoonotic links associated with sars-cov-2, the one health approach may play a vital role in the prevention and control measures being followed to restrain this pandemic virus (317) (318) (319) . the substantial importation of covid-19 presymptomatic cases from wuhan has resulted in independent, self-sustaining outbreaks across major cities both within the country and across the globe. the majority of chinese cities are now facing localized outbreaks of covid-19 (231) . hence, deploying efficient public health interventions might help to cut the spread of this virus globally. the occurrence of covid-19 infection on several cruise ships gave us a preliminary idea regarding the transmission pattern of the disease. cruise ships act as a closed environment and provide an ideal setting for the occurrence of respiratory disease outbreaks. such a situation poses a significant threat to travelers, since people from different countries are on board, which favors the introduction of the pathogen (320). although nearly 30 cruise ships from different countries have been found harboring covid-19 infection, the major cruise ships that were involved in the covid-19 outbreaks are the diamond princess, grand princess, celebrity apex, and ruby princess. the number of confirmed covid-19 cases around the world is on the rise. the success of preventive measures put forward by every country is mainly dependent upon their ability to anticipate the approaching waves of patients. this will help to properly prepare the health care workers and increase the intensive care unit (icu) capacity (321) . instead of entirely relying on lockdown protocols, countries should focus mainly on alternative intervention strategies, such as large-scale testing, contract tracing, and localized quarantine of suspected cases for limiting the spread of this pandemic virus. such intervention strategies will be useful either at the beginning of the pandemic or after lockdown relaxation (322) . lockdown should be imposed only to slow down disease progression among the population so that the health care system is not overloaded. the reproduction number (r 0 ) of covid-19 infection was earlier estimated to be in the range of 1.4 to 2.5 (70); recently, it was estimated to be 2.24 to 3.58 (76) . compared to its coronavirus predecessors, covid-19 has an r 0 value that is greater than that of mers (r 0 ͻ 1) (108) but less than that of sars (r 0 value of 2 to 5) (93) . still, to prevent further spread of disease at mass gatherings, functions remain canceled in the affected cities, and persons are asked to work from home (232) . hence, it is a relief that the current outbreak of covid-19 infection can be brought under control with the adoption of strategic preventive and control measures along with the early isolation of subsequent cases in the coming days. studies also report that since air traffic between china and african countries increased many times over in the decade after the sars outbreak, african countries need to be vigilant to prevent the spread of novel coronavirus in africa (225) . due to fear of virus spread, wuhan city was completely shut down (233) . the immediate control of the ongoing covid-19 outbreaks appears a mammoth task, especially for developing countries, due to their inability to allocate quarantine stations that could screen infected individuals' movements (234) . such underdeveloped countries should divert their resources and energy to enforcing the primary level of preventive measures, like controlling the entry of individuals from china or countries where the disease has flared up, isolating the infected individuals, and quarantining individuals with suspected infection. most of the sub-saharan african countries have a fragile health system that can be crippled in the event of an outbreak. effective management of covid-19 would be difficult for low-income countries due to their inability to respond rapidly due to the lack of an efficient health care system (65) . controlling the imported cases is critical in preventing the spread of covid-19 to other countries that have not reported the disease until now. the possibility of an imported case of covid-19 leading to sustained human-to-human transmission was estimated to be 0.41. this can be reduced to a value of 0.012 by decreasing the mean time from the onset of symptoms to hospitalization and can only be made possible by using intense disease surveillance systems (235) . the silent importations of infected individuals (before the manifestation of clinical signs) also contributed significantly to the spread of disease across the major cities of the world. even though the travel ban was implemented in wuhan (89) , infected persons who traveled out of the city just before the imposition of the ban might have remained undetected and resulted in local outbreaks (236) . emerging novel diseases like covid-19 are difficult to contain within the country of origin, since globalization has led to a world without borders. hence, international collaboration plays a vital role in preventing the further spread of this virus across the globe (237) . we also predict the possibility of another outbreak, as predicted by fan et al. (6) . indeed, the present outbreak caused by sars-cov-2 (covid-19) was expected. similar to previous outbreaks, the current outbreak also will be contained shortly. however, the real issue is how we are planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or even sooner (fig. 7) . several years after the global sars epidemic, the current sars-cov-2/covid-19 pandemic has served as a reminder of how novel pathogens can rapidly emerge and spread through the human population and eventually cause severe public health crises. further research should be conducted to establish animal models for sars-cov-2 to investigate replication, transmission dynamics, and pathogenesis in humans. this may help develop and evaluate potential therapeutic strategies against zoonotic cov epidemics. present trends suggest the occurrence of future outbreaks of covs due to changes in the climate, and ecological conditions may be associated with humananimal contact. live-animal markets, such as the huanan south china seafood market, represent ideal conditions for interspecies contact of wildlife with domestic birds, pigs, and mammals, which substantially increases the probability of interspecies transmission of cov infections and could result in high risks to humans due to adaptive genetic recombination in these viruses (323) (324) (325) . the covid-19-associated symptoms are fever, cough, expectoration, headache, and myalgia or fatigue. individuals with asymptomatic and atypical clinical manifestations were also identified recently, further adding to the complexity of disease transmission dynamics. atypical clinical manifestations may only express symptoms such as fatigue instead of respiratory signs such as fever, cough, and sputum. in such cases, the clinician must be vigilant for the possible occurrence of asymptomatic and atypical clinical manifestations to avoid the possibility of missed diagnoses. the present outbreak caused by sars-cov-2 was, indeed, expected. similar to clinical microbiology reviews previous outbreaks, the current pandemic also will be contained shortly. however, the real question is, how are we planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or perhaps sooner? our knowledge of most of the bat covs is scarce, as these viruses have not been isolated and studied, and extensive studies on such viruses are typically only conducted when they are associated with specific disease outbreaks. the next step following the control of the covid-19 outbreak in china should be focused on screening, identification, isolation, and characterization of covs present in wildlife species of china, particularly in bats. both in vitro and in vivo studies (using suitable animal models) should be conducted to evaluate the risk of future epidemics. presently, licensed antiviral drugs or vaccines against sars-cov, mers-cov, and sars-cov-2 are lacking. however, 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origin and continuing evolution of sars-cov-2 covid-19: the race for a vaccine all authors substantially contributed to the conception, design, analysis, and interpretation of data and checking and approving the final version of the manuscript, and we agree to be accountable for its contents.this compilation is a review article written, analyzed, and designed by its authors and required no substantial funding to be developed.all authors declare that there are no existing commercial or financial relationships that could, in any way, lead to a potential conflict of interest. with 25 years of research and teaching experience in the areas of microbiology, immunology, virology, public health, medicine, and biomedicine as an eminent researcher, he has developed several diagnostics, vaccines, immunomodulatory modules, and hypotheses to counter infectious diseases of animals, poultry, and public health concerns. he has to his credit 600 publications, 6 books, and 65 book chapters. dr. dhama has been recognized as an extremely productive researcher in the journal nature. he has been honored with 50 best paper awards and other recognitions. he is an naas (national academy of agricultural science, india) associate and has worked as nodal officer, wto, and member, wildlife health specialist group (iucn). he is actively serving as editor-in-chief, co-eic, editor, and member, editorial board, of nearly 20 scientific journals. his google scholar h-index is 47 and scopus h-index is 31. sharun khan, m.v.sc., is currently working as a researcher in the stem cell laboratory, division of surgery, icar-indian veterinary research institute, izatnagar, india. his area of interest is regenerative medicine with a focus on understanding cell biology and molecular pathways involved in the maintenance and differentiation of stem cells originating from different tissues. he has particular interest and knowledge in the fields of veterinary medicine, pharmacology, infectious diseases of animals, wildlife diseases, diagnosis and therapy of animal diseases, nutrition, and biomedicine. with excellent academic records, he has received awards and recognitions (fellowships and scholarships) and participated in national and international workshops, training programs, and courses. he has a keen interest in learning excellent scientific writing skills and has published 30 papers, including in international journals of repute. he is highly enthusiastic about gaining knowledge of advancements in educational and scientific research areas.ruchi tiwari is currently working as assistant professor in the department of veterinary microbiology, college of veterinary sciences, duvasu, mathura, india. she is currently pursuing her ph.d. (hons) degree from duvasu. with an excellent academic record and 10 years of research and teaching experience, she has expertise in the field of diagnosis, prevention, and control of important livestock/poultry diseases/pathogens having public health significance, along with particular reference to veterinary microbiology, immunology, ethnoveterinary medicine, alternative and complementary therapies, and bacteriophage therapy. dr. tiwari has published 150 research/review articles and 5 book chapters. she has been honored with the young scientist award, best paper awards (10) key: cord-349163-q52upndx authors: luo, guangxiang (george); gao, shou‐jiang title: global health concerns stirred by emerging viral infections date: 2020-02-14 journal: j med virol doi: 10.1002/jmv.25683 sha: doc_id: 349163 cord_uid: q52upndx emerging viral infections continue to pose a major threat to global public health. in 1997, a highly pathogenic avian influenza a (h5n1) virus was found to directly spread from poultry to humans unlike previously reported transmission routs of human-to-human and livestock-to-human, stirring a grave concern for a possible influenza pandemic. this article is protected by copyright. all rights reserved. emerging viral infections continue to pose a major threat to global public health. in 1997, a highly pathogenic avian influenza a (h5n1) virus was found to directly spread from poultry to humans unlike previously reported transmission routs of human-to-human and livestock-to-human, stirring a grave concern for a possible influenza pandemic. 1 several other avian influenza a virus subtypes (h7n9, h9n2, and h7n3) were also associated with human disease, raising an alarm that all subtypes of influenza a virus circulating in domestic and wild birds and livestock can potentially spill over to humans, resulting in pandemics. [2] [3] [4] in 1999, a newly emerged paramyxovirus termed nipah virus was identified as the cause of a severe encephalitis outbreak occurred in malaysia and singapore. 5 human influenza a h5n1 virus related to a highly pathogenic avian influenza virus human infection with a novel avian-origin influenza a (h7n9) virus human infection with influenza h9n2. the lancet human illness from avian influenza h7n3, british columbia nipah virus: a recently emergent deadly paramyxovirus koch's postulates fulfilled for sars virus reassortment of pandemic h1n1/2009 influenza a virus in swine middle east respiratory syndrome fever with thrombocytopenia associated with a novel bunyavirus in china the pathogenesis of ebola virus disease anticipating the international spread of zika virus from brazil outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan updates on wuhan 2019 novel coronavirus epidemic potential of large 'first generation' human-to-human transmission of 2019-ncov recent advances in the detection of respiratory virus infection in humans emerging coronaviruses: genome structure, replication, and pathogenesis coronavirus infection and immune responses homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission to humans the 2019-new coronavirus epidemic: evidence for virus evolution a pneumonia outbreak associated with a new coronavirus of probable bat origin mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigenpresenting cells key: cord-350286-n7ylgqfu authors: giri, rajanish; bhardwaj, taniya; shegane, meenakshi; gehi, bhuvaneshwari r.; kumar, prateek; gadhave, kundlik; oldfield, christopher j.; uversky, vladimir n. title: when darkness becomes a ray of light in the dark times: understanding the covid-19 via the comparative analysis of the dark proteomes of sars-cov-2, human sars and bat sars-like coronaviruses date: 2020-04-03 journal: biorxiv doi: 10.1101/2020.03.13.990598 sha: doc_id: 350286 cord_uid: n7ylgqfu recently emerged coronavirus designated as sars-cov-2 (also known as 2019 novel coronavirus (2019-ncov) or wuhan coronavirus) is a causative agent of coronavirus disease 2019 (covid-19), which is rapidly spreading throughout the world now. more than 9,00,000 cases of sars-cov-2 infection and more than 47,000 covid-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. world health organization (who) has declared the sars-cov-2 spread as a global public health emergency and admitted that the covid-19 is a pandemic now. the multiple sequence alignment data correlated with the already published reports on the sars-cov-2 evolution and indicated that this virus is closely related to the bat severe acute respiratory syndrome-like coronavirus (bat sars-like cov) and the well-studied human sars coronavirus (sars cov). the disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of sars-cov-2, bat sars-like, and human sars covs by analysing the prevalence of intrinsic disorder in their proteins. according to our findings, sars-cov-2 proteome contains very significant levels of structural order. in fact, except for nucleocapsid, nsp8, and orf6, the vast majority of sars-cov-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (idprs). however, idprs found in sars-cov-2 proteins are functionally important. for example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all sars-cov-2 proteins were shown to contain molecular recognition features (morfs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. the results of our extensive investigation of the dark side of the sars-cov-2 proteome will have important implications for the structural and non-structural biology of sars or sars-like coronaviruses. significance the infection caused by a novel coronavirus (sars-cov-2) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current covid-19 pandemic. no in-depth information on structures and functions of sars-cov-2 proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. our study provides the first comparative analysis of the orderand disorder-based features of the sars-cov-2 proteome relative to human sars and bat cov that may be useful for structure-based drug discovery. intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs)), in order to better understand an interplay between the ordered and disordered components of the proteome. in classical structure-function-paradigm, it is believed that a unique, stable, and well-defined 3-dimensional structure is a prerequisite for a protein to accomplish its unique biological function. although this notion dominated scientific minds for over the hundred years, eventually an idea of the presence of functional intrinsic disorder in proteins came to the attention of the structural biologists. according to this "heretic" viewpoint, a noticeable amount of biologically active proteins (of protein regions) fail to fold into the well-defined structures and instead remain disordered, existing as highly dynamic ensembles of rapidly interconverting conformations under the physiological conditions. these proteins and protein regions are known now as intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs), respectively. the propensity of being functional intrinsically disordered proteins (similar to the propensity of forming unique biologically active structures of ordered proteins) is determined by the amino acid sequences [11] [12] [13] . idps exhibit their biological functions in numerous biological processes commonly associated with cellular signalling, gene regulation, and control by interacting with their physiological partners [14] [15] [16] [17] [18] . these functions of idps and idps are regulated by their protein-protein, protein-rna, protein-dna interactions [19, 20] . molecular recognition features (morfs) are the regions in idps implicated in regulation of idps function by protein-protein interactions and serve as the primary stage in molecular recognition. zhang and colleagues have reported the genomic sequence of sars-cov-2 with genbank accession number nc_045512 having 29,903 nucleotides. the virus was isolated from the bronchoalveolar lavage fluid of a patient, went through a circle or renaming, from novel wuhan seafood market pneumonia virus to deadly wuhan coronavirus, to the 2019 novel coronavirus (2019-ncov) or the wuhan-2019 novel coronavirus (wuhan-2019-ncov, and was eventually named sars-cov-2 by the who [21] . it is known that the idps/idprs are present in all three kingdoms of life, and viral proteins often contain unstructured regions that have been strongly correlated with their virulence [22] [23] [24] [25] . in this report, we investigated the disordered side of the sars-cov-2 proteome using a complementary set of computational approaches to check the prevalence of idprs in various sars-cov-2 proteins and to shed some light on their disorder-related functions. we also have comprehensively analyzed idprs among the closely related viruses, such as human sars cov and bat sars-like cov. furthermore, we have also identified protein functions related to protein-protein interactions, rna binding, and dna binding from all three viruses. since these three viruses are closely related, our study provides important means for a better understanding of the sequence and structural peculiarities of their evolution. we believe that this study will help the structural and non-structural biologists to design and perform experiments for a more in-depth understanding of this virus and its pathogenicity. this also will have long-term implications for developing new drugs or vaccines against this currently unpreventable infection. sequence retrieval and multiple sequence alignment. the protein sequences of bat cov (sars-like) and human sars cov were retrieved from uniprot (uniprot ids for individual proteins are listed in table 1 ). the translated sequences of sars-cov-2 proteins [genbank database [26] (accession id: nc_045512.2)] were obtained from genbank. we used these sequences for performing multiple sequence alignment (msa) and predicting the idprs. we have used clustal omega [27] for protein sequence alignment and esprit 3.0 [28] for constructing the aligned images. for the prediction of the intrinsic disorder predisposition of cov proteomes, we have used multiple predictors, such as members of the pondr ® (predictor of natural disordered regions) family including pondr ® vls2 [29] , pondr ® vl3 [30] , pondr ® fit [31] , and pondr ® vlxt [32] , as well as the iupred platform for predicting long (≥30 residues) and short idprs (<30 residues) [33] . these computational tools predict residues/regions, which do not have the tendency to form an ordered structure. residues with disorder scores exceeding the threshold value of 0.5 are considered as intrinsically disordered residues, whereas residues with the predicted disorder scores between 0.2 and 0.5 are considered flexible. complete predicted percent of intrinsic disorder (ppid) in a query protein was calculated for every protein of all the three viruses from outputs of six predictors. the detailed methodology has been given in our previous reports [34, 35] . and disopred3 [42] . the protein residues with anchor, morfpred, and disopred3 score above the threshold value of 0.5 and morfchibi_web score above the threshold value of 0.725 are considered morf regions. idprs facilitate interactions with rnas and dnas and regulates many cellular functions [43] . thus, for predicting the dna binding residues in cov proteins, we have used two online servers: drnapred [19] and disordpbind [43] . for rna binding residues, we used pprint (prediction of protein rna-interaction) [44] and disordpbind [43] . the mean values of the predicted percentage of intrinsic disorder scores (mean ppids), that were obtained by averaging the predicted disorder scores from six disorder predictors (supplementary table 1 -3) for each protein of sars-cov-2 as well as human sars, and bat cov are represented in table 1 . * these sequences are based on genome annotations conducted by wu et al. [45] . proteins and their ppids are coloured to reflect their disorder status (ordered -blue, moderately disorderedpink, highly disordered -red) figures 2a, 2b , and 2c are 2d-disorder plots generated for sars-cov-2, human sars and bat cov proteins, respectively, and represent the ppid pondr-fit vs. ppid mean plots. based on their predicted levels of intrinsic disorder, proteins can be classified as highly ordered (ppid < 10%), moderately disordered (10% ≤ ppid < 30%) and highly disordered (ppid ≥ 30%) [46] . from the data in table 1 , figures 2a, 2b , and 2c, as well as the ppid based classification, we conclude that the nucleocapsid protein from all three strains of coronavirus possesses the highest percentage of the disorder and is classified as highly disordered protein. orf3b protein in bat cov, orf6 protein in sars-cov-2, human sars, and bat cov, and orf9b protein in human sars and sars cov belong to the class of moderately disordered proteins. while the structured proteins, namely, spike glycoprotein (s), an envelope protein (e) and membrane protein (m) as well as accessory proteins orf3a, orf7a, orf8 (orf8a and orf8b in case of human sars) of all three strains of coronavirus are ordered proteins. orf14 and orf10 proteins also belong to the class of ordered proteins. in ch-cdf plot of the proteins of (d) sars-cov-2 (e) human sars and (f) bat cov, the y coordinate of each protein spot signifies distance of corresponding protein from the boundary in ch plot and the x coordinate value corresponds to the average distance of the cdf curve for the respective protein from the cdf boundary. in order to further investigate the nature of the disorder in proteins of sars-cov-2, human sars, and bat cov, we utilized the combined ch-cdf tool that uses the outputs of two binary classifiers of disorder, charge hydropathy (ch) plot and cumulative distribution function (cdf) plot. this helped in retrieving more detailed characterization of the global disorder predisposition of the query proteins and their classification according to the disorder "favours". the ch plot is a linear classifier that differentiates between proteins that are predisposed to possess extended disordered conformations that include random coils and premolten globules from proteins that have compact conformations (ordered proteins and molten globule-like proteins). the other binary predictor, cdf is a nonlinear classifier that uses the pondr ® vlxt scores to discriminate ordered globular proteins from all disordered conformations, which include native molten globules, pre-molten globules, and random coils. the ch-cdf plot can be divided into four quadrants: q1 (bottom right quadrant) is an area of ch-cdf phase space that is expected to include ordered proteins; q2 (bottom left quadrant) includes proteins predicted to be disordered by cdf and compact by ch (i.e., native molten globules and hybrid proteins containing high levels of both ordered and disordered regions); q3 (top left quadrant) contains proteins that are predicted to be disordered by both ch and cdf analysis (i.e., highly disordered proteins with the extended disorder); and q4 (top right quadrant) possesses proteins disordered according to ch but ordered according to cdf analysis [34] . figures 2d, 2e and 2f represent the ch-cdf analysis of proteins of sars-cov-2, human sars, and bat cov and show that all the proteins are located within the two quadrants q1 and q2. the ch-cdf analysis leads to the conclusion that all proteins of sars-cov-2, human sars, and bat cov are ordered except nucleocapsid protein, which is predicted to be disordered by cdf but ordered by ch and hence lies in q2. molecular recognition features (morfs) are short interaction-prone disordered regions found within idps/idprs that commence a disorder-to-order transition upon binding to their partners [47, 48] . these regions are important for protein-protein interactions and may initiate an early step in molecular recognition [48] . in this study, we have analyzed and compared morfs (protein-binding regions) in sars-cov-2 with human sars and bat cov. the results of this analysis are summarized in table 2 , which clearly shows that most of the sars-cov-2 proteins contain at least one morf, indicating that disorder does play an important role in the functionality of these viral proteins. in addition to protein-protein interactions/protein-binding functions, idps and idrs also mediates functions by facilitating their interactions with nucleotides such as dna and rna [20, 49] . therefore, we have used a combination of two different online servers for locating protein residues that are showing the propensity to bind with dna as well as rna. nucleotide-binding residues in proteins of three studied coronaviruses are listed in supplementary coronaviruses encode four structural proteins, namely, spike (s), envelope (e) glycoprotein, membrane (m), and nucleocapsid (n) proteins, which are translated from the last ~10kb nucleotides and form the outer cover of the covs, encapsulating their single-stranded genomic rna. s protein is a large multifunctional protein forming the exterior of the cov particles [50, 51] . it forms surface homotrimers and contains two distinct ectodomain regions known as s1 and s2. in some covs, the s protein is actually cleaved into these subunits, which are joined non-covalently, whereas an additional proteolytic cleavage within the n-terminal part of the s2 subunit that takes place upon virus endocytosis generates spike proteins s2'. subunit s1 initiates viral infection by binding to the host cell receptors, s2 acts as a class i viral fusion protein that mediates fusion of the virion and cellular membranes and thereby promotes the viral entry into the host cells, whereas s2' serves as a viral fusion peptide [52, 53] . spike binds to the virion m protein through its c-terminal transmembrane region [54] . belonging to a class i viral fusion protein, s protein binds to specific surface receptor angiotensin-converting enzyme 2 (ace2) on host cell plasma membrane through its n-terminal receptor-binding domain (rbd) and mediates viral entry into host cells [55] . the s protein consists of an n-terminal signal peptide, a long extracellular domain, a singlepass transmembrane domain, and a short intracellular domain [56] . a 3.60 å resolution structure (pdb id: 6acc) of s protein from human sars complexed with its host binding partner ace2 has been obtained by cryo-electron microscopy (cryo-em the biophysical analysis reported in previous study has also revealed that the s protein from sars-cov-2 has a higher binding affinity to ace2 than s protein from human sars [58] . which has been calculated by averaging the disorder scores from all six predictors is represented by a short-dot line (sky-blue line) in the graph. the light sky-blue shadow region signifies the mean error distribution. the residues missing in the pdb structure or the residues for which pdb structure is unavailable are represented by the grey-coloured area in the corresponding graphs. (e) aligned disorder profiles generated for spike glycoprotein from sars-cov-2 (black line), human sars (red line), and bat cov (green line) based on the outputs of the pondr ® vsl2. msa analysis among all three coronaviruses demonstrates that s protein of sars-cov-2 has a 77.71% sequence identity with bat cov and 77.14% identity with human sars (supplementary figure s1a) . all three s proteins are found to have a conserved c-terminal region. however, the n-terminal regions of s proteins display noticeable differences. given that there is significant sequence variation rbd located at the n-terminal region of s protein, this might be the reason behind variation in its virulence and its receptor-mediated binding and entry into the host cell. according to our intrinsic disorder propensity analysis, s protein from all three covs analysed in this study are highly structured, as their predicted disorder propensity lies below 10% ( table 1 ). in fact, the mean ppid scores of sars-cov-2, human sars cov, and bat cov are calculated to be 1.41%, 1.12%, and 1.85%, respectively. figures 3b, 3c , and 3d represent the intrinsic disorder profiles of s proteins from sars-cov-2, human sars and bat cov obtained from six disorder predictors. finally, figure 3e shows aligned disorder profiles of s proteins from these covs and illustrates remarkable similarity in their disorder propensity, especially in the c-terminal region. it is of interest to map known functional regions of s proteins to their corresponding disorder profiles. the maturation of s protein requires specific posttranslational modification (ptm), proteolytic cleavage that happens at two stages. first, host cell furin or another cellular protease nicks the s precursor to generate s1 and s2 proteins, whereas the second cleavage that takes place after the viral attachment to host cell receptors leads to the release of a fusion peptide generating the s2' subunit. in human sars cov, the first and second cleavage site is located at residues r 667 and r 797 , respectively, whereas in bat cov, the corresponding cleavage sites are residues r 654 and r 784 . as it follows from figure 3 , these cleavage sites are located within the idprs. in human sars cov s protein, fusion peptide (residues 770-788) is located within a flexible region, is characterized by the mean disorder score of 0.232±0.053. similarly, in bat cov s protein, fusion peptide (residues 757-775) has a mean disorder score of 0.320±0.046. s protein contains two heptad repeat regions that form coiledcoil structure during viral and target cell membrane fusion, assuming a trimer-of-hairpins structure needed for the functional positioning of the fusion peptide. in human sars cov s protein, heptad repeat regions are formed by residues 902-952 and 1145-1184, which have mean disorder scores of 0.458±0.067 and 0.353±0.062, respectively. the analogous situation is observed for the s protein from bat cov, where these heptad repeat regions are positioned at residues 889-939 (0.44±0.11) and 1132-1171 (0.353±0.062). another functional region found in s proteins is the receptor-binding domain (residues 306-527 and 310-514 in human sars cov and bat cov, respectively) containing a receptor-binding motif responsible for interaction with human ace2. in human s protein of human sars cov this motif (residues 424-494) is not only characterized by structural flexibility, possessing a mean disorder score of 0.30±0.16, but also contains a disordered region (residues 461-466). since s protein is known as spike glycoprotein, it contains numerous glycosylation sites. due to rather close similarity of disorder profiles of s proteins analysed here, we can assume that all the aforementioned indications of the functional importance of disorder and flexible regions in s proteins from sars cov and bat cov are also applicable to sars-cov-2 s protein. finally, table 2 shows that s protein from sars-cov-2 contain one morf region at its cterminal (residues 1265-1272) by morfchibi_web, two morf regions ((residues 2-6) & (residues 819-823)) by morfpred, and one morf region at n-terminal (residues 1-10) by disopred3. these results indicating that intrinsic disorder is important for its interaction with binding partners. interestingly, the n-terminal region of s protein (residues 1-10) from all three viruses are observed to be a disorder-based protein binding region by two predictors (morfpred and disopred3). n-terminal morf displays its role in viral interaction with host receptor and c-terminal morf displays its role in m protein interaction and viral assembly. moreover, morf region mainly lies in the n-and c-terminal regions suggesting a possible role during cleavage as well. in addition to protein-binding regions, s protein also shows many nucleotide-binding residues. tables 9, 10, and 11 shows that numerous rna binding residues predicted by pprint in all three viruses and a single rna binding residue were predicted by disordpbind in human sars. further, drnapred and disordpbind predicted the presence of many dna binding residues in s protein of all three viruses. these results signify the role of s protein functions related to molecular recognition (protein-protein interaction, rna binding, and dna binding) such as interaction with host cell membrane and further viral infection. therefore, identified idps/idprs and residues/regions from s protein crucial for molecular recognition can be targeted for disorder-based drug discovery. envelope (e) protein is a small, multifunctional inner membrane protein that plays an important role in the assembly and morphogenesis of virions in the cell [59] [60] [61] . e protein consists of two ectodomains associated with n-and c-terminal regions, and a transmembrane domain. it homo-oligomerize to form pentameric membrane destabilizing transmembrane (tm) hairpins to form a pore necessary for its ion channel activity [62] . figure 4a shows the nmr-structure (pdb id: 2mm4) of human sars envelope glycoprotein of 8-65 residues [63] . msa results illustrate ( figure 4b ) that this protein is highly conserved, with only three amino acid substitutions in e protein of sars-cov-2 conferring its 96% sequence similarity with human sars and bat cov. bat cov shares 100% sequence identity with human sars. mean ppid calculated for sars-cov-2, human sars, and bat cov e proteins are 5.33%, 6.58%, and 6.58% respectively ( table 1) . the e protein is found to have a reasonably well-predicted structure. our predictions suggest that the residues of n-and c-terminals are displaying a higher tendency for the disorder. the last 18 hydrophilic residues (residues 59-76) have been reported to adopt a random-coil conformation with and without the addition of lipid membranes [64] . literature suggests that the last four amino acids of the c-terminal region of e protein containing a pzd-binding motif are involved in protein-protein interactions with a tight junction protein pals1. our results support literature as we identified long n-terminal region of approximately 30 residues long as disorder-based protein binding region in all three viruses (see table 2, supplementary table 7 and 8). pals1 is involved in maintaining the polarity of epithelial cells in mammals [65] . respective graphs in figures 4c, 4d , and 4e show the predicted intrinsic disorder profiles for e proteins of sars-cov-2, human sars, and bat cov. we speculate that the disordered region content may be facilitating the interactions with other proteins as well. in agreement with this hypothesis, table 2 shows that in e protein from sars-cov-2, the c-terminal domain serves as protein-binding region. we found that the residues from 45-75 is a long morf in e proteins of all three viruses as predicted by morfchibi_web ( table 2, supplementary table 7 and 8). as aforementioned, these randomly-coiled binding-residues at c-terminus may gain structure while assisting the protein-protein interaction mediated by e protein. one more morf region (residues [26] [27] [28] [29] [30] in the transmembrane domain was observed by disopred3 in the e protein of all three viruses. since these residues are the part of ion channel, we speculate that these residues do specific interactions and may be guiding the specifi functions of ion channel activity. few rna binding residues by pprint and disordpbind and several dna binding residues by drnapred are predicted for e protein in all three viruses. assembly by interacting with the nucleocapsid (n) and e proteins [66] [67] [68] . protein m interacts specifically with a short viral packaging signal containing coronavirus rna in the absence of n protein, thereby highlighting an important nucleocapsid-independent viral rna packaging mechanism inside the host cells [69] . it gains high-mannose n-glycans in er, which are subsequently modified into complex n-glycans in the golgi complex. glycosylation of m protein is observed to be not essential for virion fusion in cell culture [70, 71] . cryo-em and tomography data indicate that m forms two distinct conformations, a compact m protein having high flexibility and low spike density, and an elongated m protein having a rigid structure and narrow range of membrane curvature [72] . some regions of m glycoproteins might serve as important dominant immunogens. although no structural information is available for the full-length m protein as of yet, a short peptide of the membrane glycoprotein (residues 88-96) from human sars cov was co-crystallized with a complex between a-2 alpha chain of the hla class i histocompatibility antigen and β2microglobulin (pdb id: 3i6g) [73] . figure 5a shows that within this complex, the cocrystallized m protein region exists in an extended conformation. m protein of sars-cov-2 has a sequence similarity of 90.1% with bat cov and 89.6% with human sars m proteins ( figure 5b ). our analysis revealed that the intrinsic disorder levels in m proteins of sars-cov-2, human sars cov, and bat cov are relatively low since these proteins show the ppid values of 2.70%, 1.36%, and 1.36% respectively. this is in line with the previous publication by goh et al. on human sars hku4 where they found the mean ppid of 4% using additional predictors such as topidp and foldindex along with the predictors used in our study [74] . figures 5c, 5d , and 5e represent per-residue disorder profiles generated for m proteins of sars-cov-2, human sars cov, and bat cov and show that with the exception to their n-and c-terminal regions, these proteins are mostly ordered. the last 20 residues of mers-cov m protein are important for intracellular trafficking and contains a determinant that localizes it into the golgi network [75] . our results in table 2 illustrates that the disordered c-tail of the m protein is predicted to have disorder based protein-binding region and therefore can serve as a binding site for its specific partner required for its localization inside the host cell. a long morf region (residues 186-220) at the c-terminal of m protein in all three viruses were observed by morfchibi_web. two morf regions (one at n-terminus (residues 1-16) and one at c-terminus (residues 205-221)) was observed by disopred3 in human sars and bat cov. however, single morf (residues 117-132) observed in sars-cov-2 by disopred3. morfpred also predicts a short morf at c-terminus of sars-cov-2 (residues 214-222), human sars (residues 214-221), and bat cov (residues 211-221) ( table 2, supplementary tables 7 and 8) . furthermore, the m protein from all three viruses displays strong tendency to bind with rna (as predicted by pprint and disordpbind) and dna (as predicted by drnapred and disordpbind) (see supplementary tables 9, 10 , and 11). our understanding on m protein of covs (idps and morf at c-terminus and molecular recognition) elucidates its crucial role in interaction with the n and e proteins for viral assembly. nucleocapsid (n) protein: nucleocapsid (n) protein is one of the major viral proteins playing several significant roles in transcription, and virion assembly of coronaviruses [76] . it binds to viral genomic rna forming a ribonucleoprotein core required for the rna encapsidation during viral particle assembly [77] . sars-cov virus-like particles (vlps) formation has been reported to depend upon either m and e proteins or m and n proteins. for the effective production and release of vlps, co-expression of e or n proteins with m protein is necessary [78] . n protein of human sars consists of two structural domains, the n-terminal rna-binding domain (ntd: 45-181 residues) and the c-terminal dimerization domain (ctd: 248-365 residues) with a disordered patch in between these domains. n protein has been demonstrated to bind viral rna using both ntd and ctd [79] . figure 6a1 displays the nmr solution structure of the ntd of human sars cov nucleocapsid protein (45-181 residues) (pdb id: 1ssk) [80] . figure 6a2 shows an x-ray crystal structure of the ctd of human sars cov nucleocapsid protein (270-366 residues) (pdb id: 2gib) [81] . a model of the domain organization of the n-protein from sars-cov-2 is shown in figure 6b . the 419 amino acid-long n protein of sars-cov-2 shows a percentage identity of 88.76% with n protein of bat cov n protein and 89.74% with human sars n protein (supplementary figure s1b) . our analysis revealed that the n proteins of coronaviruses contain the highest levels of intrinsic disorder (see figure 6 and table 1 ). in fact, n proteins from sars-cov-2 human sars cov, and bat cov are characterized by the mean ppid of 64.91%, 71.09%, and 65.80%, respectively. in accordance with the previously evaluated intrinsic disorder predisposition [74] , n protein is highly disordered in all three sars viruses analysed in this study ( table 1) . graphs in figures 6c, 6d , and 6e depict the disorder profiles of sars-cov-2, human sars cov, and bat cov nucleocapsid proteins and show that their n-and c-terminal regions are completely disordered, and all three proteins also contain the central unstructured segment. as expected, the intrinsic disorder predisposition of the n protein of sars-cov-2 is remarkably similar to that for the n protein of human sars cov as reported in a previous study [74] . this is further supported by figure 6f , where pondr ® vsl2-generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. it is clear that in n-proteins, the n-and c-termini and a log central segment are completely disordered. figure 6c shows that in the n protein from sars-cov-2, residues 1-57, 64-102, 145-162, 166-289, and 362-422 are found to be disordered. many of these residues are lying within the ntd and ctd regions, and which, due to their structural plasticity, were not crystallized in human sars cov n protein. sars-cov-2 has a disordered segment from 168-289 residues while human sars has predicted to have an unstructured segment from 145-289 residues. overall, all three n proteins are found to be highly disordered. the n protein from human sars cov has one phosphorylation site (residue s 177 ) and several regions with compositional biases, such as ser-rich (residues 181-213), poly-leu, poly-gln, and ploy-lys (residues 220-225, 240-245, and 370-376), all predicted to be disordered. similarly, in n protein from bat cov, s 176 is phosphorylated, and this protein has ser-rich, poly-leu, and ploy-lys regions (residues 176-206, 219-224, and 369-375, respectively), all of which are disordered. it has been reported to interact using the central disordered region with m protein, hnrnp a1, and self n-n interaction [82] [83] [84] . the middle flexible region is also responsible for its rna-binding activity [85] . deletion of 184-196 residues, 169-308 residues, 161-210 residues of n abolishes its multimerization, rnabinding capacity, and hnrnp a1 interactions respectively. supplementary table 7 and 8, and table 2 shows that n protein is heavily decorated with numerous morfs, suggesting that this protein is a promiscuous binder. long disorder-based protein bonding regions at nand c-terminus of n protein of all three viruses were observed by all four predictors (morfchibi_web, anchor, morfpred, and disopred3). indeed, this is the single protein where we found many morfs as compared with the other structural, non-structural and accessory proteins of covs. the morfs present in these regions may mediate the abovementioned interactions of n proteins. figure 6a2 represents another important disorderrelated functional feature of the n protein. in fact, the ctd homodimer shown there is characterized by highly intertwined morphology, which is typically a result of bindinginduced folding [86] [87] [88] , indicating that a very significant part of ctd gains structure during dimerization. we identified numerous rna binding residues in all three viruses using pprint server. this finding supports the function of n protein as it interacts with genomic rna for a ribonucleoprotein core formation which is crucial step for rna encapsidation during viral particle assembly. in addition, drnapred and disordpbind predicts multiple dna binding residues for n protein in sars-cov-2, human sars, and bat cov. the long flexible (idprs) regions at n and c-terminus of sars-cov-2 have long protein-binding as well as nucleotide-binding regions that may have important role in its interaction with viral rna. these flexible regions can be targeted to inhibit interaction of n protein with viral genomic rna. literature suggests that some viral proteins are translated from the genes interspersed in between the genes of structural proteins. these proteins are known as accessory proteins, and many of them are proposed to be involved in viral pathogenesis [89] . proteins orf3a and orf3b. orf3a is a multifunctional protein with the molecular weight of ~31 kda that has been found to localize in different organelles inside the host cells. also referred to as u274, x1, and orf3, the gene for this protein is present between the s and e genes of the sars-cov genome [90] [91] [92] . the homo-tetrameric complex of orf3a has been demonstrated to form a potassium-ion channel on the host cell plasma membrane [93] . it performs a major function during virion assembly by co-localizing with e, m, and s viral proteins [94, 95] . orf3b protein can be found in the cytoplasm, nucleolus, and outer membrane of mitochondria of the host cells [96, 97] . in huh 7 cells, its over-expression has been linked with the activation of ap-1 via erk and jnk pathways [98] . transfection of orf3b-egfp leads to cell growth arrest at the g0/g1 phase of vero, 293, and cos-7 cells [99] . orf3a induces apoptosis via caspase 8/9 directed mitochondrial-mediated pathways, while orf3b is reported to affect only the caspase 3-related pathways [100, 101] . on performing msa, results of which are shown in figure 7d , we found that orf3a protein from sars-cov-2 is slightly evolutionary closer to the orf3a of bat cov (73.36%) than to the orf3a of human sars cov (72.99%). graphs in figures 7a, 7b , and 7c depict the propensity for disorder in orf3a proteins of novel sars-cov-2, human sars cov, and bat cov (sars-like), respectively. mean ppids in these orf3a proteins are 9.1% (sars-cov-2), 8.8% (human sars), and 6.2% (bat cov (sars-like)). orf3a of sars cov-2 shows protein-binding regions at its n-terminus (by morfchibi_web (residues 1-6), morfpred (residues 7-12), and disopred3 (residues [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ) and at c-terminus (by morfchibi_web (residues 261-268) and morfpred (residues 259-263)) ( table 2) . similarly, orf3a of human sars and bat cov also shows morfs at n-and c-terminus with the help of morfchibi_web and morfpred (supplementary tables 7 and 8 ). these protein-binding regions in orf3a may have role in its co-localization with e, m, and s viral proteins. apart from morfs, it also displays several nucleotide-binding residues in all three viruses (see supplementary tables 9, 10 , and 11). in fact, this represents maximum number of rna and dna binding residues as compared with all other accessory proteins. these results indicate that the idps/idprs of this protein could be utilized in molecular recognition (protein-protein, protein-rna, and protein-dna interaction). according to the intrinsic disorder predisposition analysis of orf3b proteins, their mean ppid values in sars-cov-2, human sars cov, and bat cov are 0%, 7.1%, and 23.1% respectively, as represented in figures 8a, 8b , and 8c. msa results ( figure 8d ) demonstrate that orf3b of sars-cov-2 is not closer to orf3b protein of human sars and orf3b protein of bat-cov, having a sequence similarity of only 54.6% and 59.1%, respectively. as we can see in table 2 , there is not a single morf found in orf3b of sars-cov-2. however, for human sars we identified three morfs (residues 32-37, 41-70, and 125-153) and for bat cov one morf at n-terminus (residues 1-38) by morfchibi_web server. protein orf6. orf6 is a short coronavirus protein with just 63 residues. also known as p6, this membrane-associated protein serves as an interferon (ifn) antagonist [102] . it downregulates the ifn pathway by blocking a nuclear import protein, karyopherin α2. using its c-terminal residues, orf6 disrupts karyopherin import complex in the cytosol and, therefore, hampers the movement of transcription factors like stat1 into the nucleus [102, 103] . it contains a ysel motif near its c-terminal region, which functions in protein internalization from the plasma membrane into the endosomal vesicles [104] . another study has also demonstrated the presence of orf6 in endosomal/lysosomal compartments [104, 105] . msa results demonstrate that (figure 9d) , sars-cov-2 orf6 is closer to orf6 protein of human sars cov, having a sequence similarity of 68.85% than to the orf6 of bat cov (sars-like) (67.21%). novel sars-cov-2 orf6 is predicted to be the second most disordered structural protein, with ppid of 22.95%, and with especially disordered cterminal region. our analysis of the intrinsic disorder predisposition using six predictors revealed the mean ppid in orf6 proteins of sars-cov-2, human sars, and bat cov to be 22.95%, 20.63%, and 20.63%, respectively ( table 1) . graphs in figures 9a, 9b and 9c illustrate that orf6 proteins from all three studied coronaviruses are expected to be moderately disordered proteins with the high disorder content in their c-terminal regions. these disordered regions are important for the biological activities of orf6. as aforementioned, this hydrophilic region contains lysosomal targeting motif (ysel) and diacidic motif (ddee) responsible for binding and recognition during translocation [104] . however, the n-terminal region does not contain a noticeable disorder. the 1-38 residues of the n-terminal region of human sars cov orf6 was shown to be α-helical and embedded in the membrane, although orf6 is not a transmembrane protein [106] . a long morf region ((residues 26-61 in sars-cov-2), (residues 31-63 in human sars), and (residues 30-60 in bat cov)) is also present at cterminus of orf6 proteins which are tabulated in table 2 , and supplementary tables 7 and 8. no predictor other than morfchibi_web has located morfs in this protein. supplementary table 9 , 10, and 11 shows nucleotide-binding residues in orf6 of all three viruses. it represents very few rna binding residues by pprint and few dna binding residues by drnapred. orf7a and orf7b proteins. alternatively called u122, orf7a is a type i transmembrane protein [107, 108] . it has been proven to localize in er, golgi, and peri-nuclear space. the presence of a krkte motif near the c-terminal region is needed for importing this protein from the er to the golgi apparatus [107, 108] . orf7a contributes to viral pathogenesis by activating the release of pro-inflammatory cytokines and chemokines, such as il-8 and rantes [109, 110] . in another study, overexpression of bcl-xl in 293t cells blocked the orf7a mediated apoptosis [111] . on the other hand, orf7b is an integral membrane protein that has been shown to localize in the golgi complex [112, 113] . the same reports also confirm the role of orf7b as an accessory as well as a structural protein in sars-cov virion [112, 113] . figure 10d represents the 1.8 å x-ray crystal structure of the 14-96 fragment of the orf7a from human sars cov (pdb id: 1xak) and demonstrates the compact seven-stranded topology of this protein, which is similar to that of the ig-superfamily members [114] . importantly, in this crystal structure, residues 82-96 constituted the region with missing electron density, indicating high structural flexibility of this segment. in line with this hypothesis, the nmr solution structure of the 16-99 fragment of the orf7a from human sars cov (pdb id: 1yo4) showed that residues 81-99 are highly disordered [115] . at the domain level, the structure of the orf7a protein includes a signal peptide, a luminal domain, a transmembrane domain, and a short cytoplasmic tail at the c-terminus [95, 114] . we found that 121-residue-long orf7a protein of sars-cov-2 shares 89.26% and 85.95% sequence identity with orf7a proteins of bat cov and human sars cov, respectively ( figure 10e) . on the other hand, the orf7b of sars-cov-2 is found to be closer to orf7b of human sars than to orf7b of bat cov, showing sequence identities of 81.40% and 79.07%, respectively (see figure 11d ). as can be observed from table 1 , our disorder predisposition analyses resulted in the overall ppid for orf7a proteins of 1.65% for sars-cov-2, 0.82% for bat cov and 0.82% for human sars cov. mean ppids estimated for orf7b proteins are 9.30% for sars-cov-2, 4.55% for bat cov and 4.55% human sars cov. figures 10a, 10b , and 10c represent the residues predisposed for disorder in orf7a proteins of sars-cov-2, human sars cov, and bat cov, respectively. table 2 shows that orf7a protein is expected to have several morfs indicating the potential involvement of this protein in disorder-dependent proteinprotein interactions. at the n-terminus, we observed one morf region (residues 1-10) with the help of disopred3 in all three viruses. in addition to protein binding regions, orf7a also contains several rna and dna binding residues. analysis also represents, orf7b proteins from all three viruses have low disorder content, likewise, they are not predicted to contain any morf by any of the predictors used in this study ( table 2, supplementary table 7 and 8). although the orf7b does not contain protein-binding regions, it was found to contain nucleotide (rns and dna) binding regions in the protein figures 11a, 11b , and 11c depict the residues predisposed for disorder in orf7b proteins of sars-cov-2, human sars cov, and bat cov, respectively. according to our analysis, both proteins in all three studied coronaviruses have a mostly ordered structure. proteins orf8a and orf8b. in animals and isolates from early human infections, the orf8 gene codes for a single orf8 protein. however, in late infections, more specifically, at middle and late stages, a 29 nucleotide deletion in the orf8 gene led to the formation of two distinct proteins, orf8a and orf8b containing 39 and 84 residues respectively [116, 117] . both proteins have conformations different from that of the longer orf8 protein. it has been reported that overexpression of orf8b resulted in the downregulation of e protein while the proteins orf8a and orf8/orf8ab have no effect on the expression of protein e. also, orf8/orf8ab was found to interact very strongly with proteins s, orf3a, and orf7a. orf8a interacts with s and e proteins, whereas orf8b protein interacts with e, m, orf3a and orf7a proteins [118] . the disorder-based protein binding regions of this protein identified in this study may have important role in interaction with other proteins. orf8 protein found in early sars-cov-2 isolates having 121 residues and according to our analysis, it shares a 90.05% sequence identity with orf8 protein of bat cov ( figure 12c) . furthermore, figures 12a and 12b show that there is no intrinsic disorder in both orf8 proteins from sars-cov-2 and bat cov. therefore, these two proteins predicted to be completely structured having a mean ppid of 0.00%. in orf8a and orf8b proteins of the human sars, the predicted disorder is estimated to be 2.56% and 2.38%, respectively (table 1) . graphs in figures 13a and 13b illustrate the presence of some disorder near the n-and c-terminals of orf8a and orf8b proteins. table 2 shows the identified morf regions in orf8 of sars-cov-2. it shows three morf regions (residues 1-5, 26-52, and 69-91) by morfchibi_web and one morf region (residues 1-10) by disopred3. in human sars, the n-terminus of both orf8a (residues 1-39) and orf8b (residues 1-83) was found to be morf by morfchibi_web server (supplementary table 7) . further, four proteinbinding regions (residues 26-53, 70-91, 98-104, and 113-130) were identified by morfchibi_web server in bat cov (supplementary table 8 ). apart from protein-binding, orf8 of sars-cov-2, orf8a and orf8b of human sars, and orf8 of bat cov also comprise several nucleotide-binding residues (see supplementary table 9 , 10, and 11). this protein is expressed from an alternative orf within the n gene through a leaky ribosome binding process [119] . inside the host cells, orf9b enters the nucleus, which is a cell cycle-independent process and represents a passive entry. this protein was shown to interact with a nuclear export protein receptor exportin 1 (crm1), using which it translocate out of the nucleus [120] . our morfs analysis shows the presence of disorderbased protein binding regions in orf9b protein which may have role in its interaction with crm1 and further translocation outside the nucleus. a 2.8 å resolution crystal structure of orf9b protein from human sars cov (pdb id: 2cme) shows the presence of a dimeric tent-like -structure along with the central hydrophobic amino acids ( figure 14d) . the published structure has the highly polarized distribution of charges, with positively charged residues on the one side of the tent and negatively charged on the other [121] . based on the sequence availability of accession id nc_045512.2, the translated protein sequence of orf9b is not reported for the sars-cov-2 as of yet. however, based on the report by wu and colleagues [45] , the sequences of the sars-cov-2 are already annotated. therefore, we took the corresponding amino acid sequences from that study and conducted the intrinsic disorder analysis. according to the msa, results shown in figure 14e , orf9b protein from sars-cov-2 shares 73.2% identity with human sars and 74.23% identity with bat cov. our idp analysis ( table 1) shows that orf9b from human sars is a moderately unstructured protein with a mean ppid estimated to 26.53%. as depicted in figure 14a , 14b, and 14c, disorder mainly lies near the n-terminal end 1-10 residues and 28-40 residues near the central region with a well-ordered inner core of human sars orf9b protein. the x-ray crystal structure of orf9b has a missing electron density of the first 8 residues and 26-37 residues near the central region. this indicates that the corresponding regions are disordered, which are difficult to crystallize due to their highly dynamic structural organization. sars-cov-2 orf9b protein with a mean ppid of 10.31% has an n-terminal (1-10 residues) predicted disordered segment. orf9b of bat cov is shown to have an intrinsic disorder content of 9.28%, comparatively lower than that of the human sars orf9b protein. morfs lies in the n-terminal region of orf9b proteins ( table 2, supplementary table 7 and 8). in the absence of other viral proteins, its first 41 residues have been demonstrated to induce membranous structures similar to dmvs [106] . the available crystal structure also has the missing electron density in the n-terminal region suggests that these flexible amino acids are likely to interact with host lipids. the first 3-29 residues of sars-cov2 are identified as disorder-based protein binding region that may have role in its interaction with host lipids and formation of dmvs. supplementary tables 9, 10, and 11 represents nucleotide-binding residues for orf9b of sars-cov-2, human sars, and bat cov. the newly emerged sars-cov-2 has an orf10 protein of 38 amino acids. orf10 of sars-cov-2 has a 100% sequence similarity with orf10 of bat cov strain bat-sl-covzc45 [21] . however, we did not conduct the disorder analysis for orf10 from the bat-sl-covzc45 strain, since all our studies reported here are related to a different strain of bat cov (reviewed strain hku3-1). therefore, we report here only the results of disorder analysis for the orf10 protein from sars-cov-2, according to which this protein has a mean ppid of 0.00% (see also figure 15 for disorder profile of orf10). this protein contains a morf from 3-7 residues at its n-terminus as predicted by morfchibi_web. further, we found its tendency to nucleotides and found the presence of few rna binding sites, however, it does not contain dna binding residues. protein orf14. this is a 70-amino-acid-long uncharacterized protein of unknown function, which is present in human sars and bat cov. in sars-cov-2, orf14 is a 73-amino-acidlong protein. according to the msa, orf14 of sars-cov-2 has 77.1% identity with human-sars and 72.9% identity with bat cov as represented in figure 16d . we have performed the intrinsic disorder analysis to see the peculiarities of the distribution of disorder predisposition in this protein. figures 16a, 16b, and 16c show the resulting disorder profiles of orf14 of sars-cov-2, human sars cov, and bat cov. although these proteins have calculated mean ppid values of 0.00%, 2.86%, and 0.00% respectively, figure 16 shows that they have flexible n-and c-terminal regions. this protein can use intrinsic disorder or structural flexibility for protein-protein interactions since it possesses morfs. it mainly contains morfs at n-and c-terminal regions as tabulated in (table 2, supplementary table 7 and 8). it was also found to contain several rna and dna binding residues (supplementary table 9, 10, and 11) . these results indicating its vital role in protein function related to molecular recognition such as protein-protein, protein-rna, and protein-dna interaction. in coronaviruses, due to ribosomal leakage during translation, two-third of the rna genome is processed into two polyproteins: (i) replicase polyprotein 1a and (ii) replicase polyprotein 1ab. both contain non-structural proteins (nsp1-10) in addition to different proteins required for viral replication and pathogenesis. replicase polyprotein 1a contains an additional nsp11 protein of 13 amino acids, the function of which is not investigated yet. the longer replicase polyprotein 1ab of 7073 amino acids accommodates five other non-structural proteins (nsp12-16) [122] . these proteins assist in er membrane-induced vesicle formation, which acts as sites for replication and transcription. in addition to this, non-structural proteins work as proteases, helicases, and mrna capping and methylation enzymes, crucial for virus survival and replication inside host cells [122, 123] . global analysis of intrinsic disorder in the replicase polyprotein 1ab table 3 represents the ppid mean scores of 15 non-structural proteins (nsps) derived from the replicase polyprotein 1ab in sars-cov-2, human sars cov, and bat cov. these values were obtained by combining the results from six disorder predictors (see supplementary table s4-s6) . figures 17a, 17b , and 17c represent the 2d-disorder plots of the nsps coded by orf1ab in sars-cov-2, human sars cov, and bat cov, respectively. based on the mean ppid scores in table 3 , figures 17a, 17b, 17c , and taking into ppid based classification [46] , we conclude that none of the nsps in sars-cov-2, human sars cov, and bat cov are highly disordered. the highest disorder was observed for nsp8 proteins in all three coronaviruses. both nsp1 and nsp8 are moderately disordered proteins (10% ≤ ppid ≤ 30%). we also observed that nsp2, nsp3, nsp5, nsp6, nsp7, nsp9, nsp10, nsp15, and nsp16 have less than 10% disordered residues and hence, belong to the category of mostly ordered proteins. other non-structural proteins, namely, nsp4, nsp12, nsp13, and nsp14 have negligible levels of disorder (ppid < 1%), which tells us that these are highly structured proteins. the ch-cdf analysis of the nsps from sars-cov-2, human sars and bat cov have been represented in figures 17d, 17e , and 17f respectively. it was observed that all the nsps of the three coronaviruses are located within the quadrant q1 of the ch-cdf phase space, indicating that all the nsps are predicted to be mostly ordered. replicase polyprotein 1ab. the longer replicase polyprotein 1ab is a 7,073 amino acid-long polypeptide, which contains 15 non-structural proteins listed in table 3 . nsp1, nsp2, and nsp3 are cleaved using a viral papain-like proteinase (nsp3/pl-pro), while the rest of nsps are cleaved by another viral 3c-like proteinase, nsp5/3cl-pro. we mapped the cleavage sites of the replicase 1ab polyprotein from human sars cov to the disorder profile of this polyprotein. figure 18 represents the results of this analysis by showing zoomed-in regions surrounding all the cleavage sites with few residues spanning at both terminals. interestingly, we observed that all the cleavage sites are largely disordered, suggesting that intrinsic disorder may have a crucial role in the maturation of individual non-structural proteins. as the nsps of human sars cov are evolutionary close to the nsps of sars-cov-2, we hypothesize that the cleavage sites in the sars-cov-2 replicase 1ab polyprotein are also intrinsically disordered or flexible. to shed more light on other implications of idprs, the structural and functional properties of nsps and their predicted idprs are thoroughly described below. this protein acts as a host translation inhibitor as it binds to the 40s subunit of the ribosome and blocks the translation of cap-dependent mrnas as well as mrnas that uses the internal ribosome entry site (ires) [124] . figure 19d shows the nmr solution structure (pdb id: 2gdt) of human sars nsp1 protein (13-128 residues), whereas residues 117-180 were not included in this structural analysis [125] . sars-cov-2 nsp1 shares 84.44% and 83.80% sequence identity with nsp1s of human sars cov and bat cov, respectively. its n-terminal region is found to be more conserved than the rest of the protein sequence ( figure 19e ). mean ppids of nsp1s from sars-cov-2, human sars cov, and bat cov are 12.78%, 14.44%, and 12.85%, respectively. figure 19a , 19b, and 19c represent the graphs of predicted per-residue intrinsic disorder propensity of these nsp1s. according to the analysis, the following regions are predicted to be disordered: sars-cov-2 (residues 1-7 and 165-180), human sars cov (residues 1-5 and 165-180), and bat cov (residues 1-5 and 165-179). nmr solution structure of nsp1 from human sars revealed the presence of two unstructured segments near the n-terminal (1-12 residues) and c-terminal (129-179 residues) regions [125] . the disordered region residues) at c-terminus is important for nsp1 expression [126] . based on sequence homology with human sars cov nsp1, the predicted disordered c-terminal region of sars-cov-2 nsp1 may play a critical role in its expression. alanine mutants at k164 and h165 in the cterminal region of nsp1 protein is reported to abolish its binding with the 40s subunit of the host ribosome [127] . in conjunction with this data, several morfs are present in the unstructured segments of nsp1 proteins. these regions are tabulated in table 2, and supplementary tables 7 and 8 . this protein functions by disrupting the host survival pathway via interaction with the host proteins prohibitin-1 and prohibitin-2 [128] . reverse genetic deletion in the coding sequence of nsp2 of the sars virus attenuated little viral growth and replication and allowed the recovery of mutant virulent viruses. this indicates the dispensable nature of the nsp2 protein for sars viruses [129] . the sequence identity of the nsp2 protein from sars-cov-2 with nsp2s of human sars cov and bat cov amounts to 68.34% and 68.97%, respectively (supplementary figure s2a) . we have estimated the mean ppids of nsp2s of sars-cov-2, human sars cov, and bat cov to be 5.17%, 2.04%, and 2.03% respectively (see table 3 ). the per-residues predisposition for the intrinsic disorder of nsp2s from sars-cov-2, human sars cov, and bat cov are depicted in figures 20a, 20b , and 20c. according to this analysis, the following regions in nsp2 proteins are predicted to be disordered, residues 570-595 (sars-cov-2), residues 110-115 (human sars), and residues 112-116 (bat cov). as listed in table 2 , and supplementary tables 7 and 8, human sars cov does not contain morf while sars-cov-2 and bat cov have an n-terminally located morf region predicted by morfchibi_web. nsp3 is an almost 2,000-residue-long viral papain-like protease (plp) that affects the phosphorylation and activation of irf3 and therefore antagonizes the ifn pathway [130] . it was also demonstrated that nsp3 works by stabilizing nf-inhibitor further blocking the nf-pathway [130] . figure 21d represents the 1.85 å resolution x-ray crystal structure of the catalytic core of nsp3 protein from human sars cov (pdb id: 2fe8), which was obtained by andrew and colleagues [131] . this structure consists of the residues 723-1036 of nsp3. the structure revealed folds similar to a deubiquitinating enzyme in-vitro deubiquitinating activity of which was found to be efficiently high [131] . nsp3 protein of sars-cov-2 contains several substituted residues throughout the protein. it is equally close with both nsp3 proteins of human sars and bat cov sharing respective 76.69% and 76.31% identity (supplementary figure s2b) . according to our results, the mean ppids of nsp3 proteins of sars-cov-2, human sars, and bat cov are 7.40%, 7.91%, and 7.78% respectively ( table 3) . graphs in figures 21a, 21b , and 21c portray the tendency of nsp3 proteins of sars-cov-2, human sars, and bat cov for the intrinsic disorder. nsp3 proteins of all three studied sars viruses were found to be highly structured and characterized by rather similar disorder profiles. this is further supported by figure 21e , where pondr ® vsl2-generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. according to the mean disorder analysis (see figures 21a, 21b, and 21c) , nsp3 proteins are predicted to have the following idprs, sars-cov-2 (1-5, 105-199, 1221-1238), human sars (102-189, 355-384, 1195-1223) and bat cov (107-182, 352-376, 1191-1217) . the first 112 residues in nsp3 represent a ubiquitin-like globular fold while 113-183 residues form the flexible acidic domain rich in glutamic acid. it is thought to bind and ubiquitinate viral e protein using the n-terminal acidic domain [132, 133] . this unstructured segment has many morfs predicted by anchor and morfpred servers which may facilitate the protein-protein interaction ( table 2) . interestingly, nsp3 of all three viruses was found with highest number of rnabinding residues (supplementary tables 9, 10, and 11) . nsp4 has been reported to induce the formation of the double-membrane vesicles (dmvs) with the co-expression of full-length nsp3 and nsp6 proteins for optimal replication inside host cells [134] [135] [136] . it localizes itself in ermembrane, when expressed alone but is demonstrated to be present in replication units in infected cells. it was observed that nsp4 protein contains a tetraspanning transmembrane region having its n-and c-terminals in the cytosol [137] . no crystal or nmr solution structure is reported for this protein as of yet. nsp4 protein of sars-cov-2 has multiple substitutions near the n-terminal region and has a quite conserved c-terminus (supplementary figure s2c) . it is found to be closer to nsp4 of bat cov (81.40% identity) than to human sars nsp4 (80%). mean ppids of nsp4s from sars-cov-2, human sars, and bat cov are estimated to be 0.80%, 0.60%, and 0.60% respectively. the low level of intrinsic disorder is further illustrated by figures 22a, 22b , and 22c. with ppids around zero, nsp4 were classified as highly structured proteins, which, however, contain some flexible regions. likewise, table 2 shows the presence of only nand c-terminal morfs which possibly assist in cleavage of nsp4 protein from long polyproteins 1a and 1ab. also referred to as 3cl-pro, nsp5 works as a protease that cleaves the replicase polyproteins (1a and 1ab) at 11 major sites [138, 139] . x-ray crystal structure with 1.5 å resolution (pdb id: 5c5o) obtained for human sars cov nsp5 is shown in figure 23d . here, 3cl-protease is bound to a phenyl-beta-alanyl (s, r)-n-declin type inhibitor. another crystal structure resolved to 1.96 å revealed a chymotrypsin-like fold and a conserved substrate-binding site connected to a novel α-helical fold [140] . recently, the x-ray crystal structure (resolution 2.16 å) was solved for the sars-cov-2 nsp5 in complex with an inhibitor n3 (pdb id: 6lu7) ( figure 23e ). nsp5 protein is found to be highly conserved in all three studied cov viruses. sars-cov-2 nsp5 shares a 96.08% sequence identity with human sars nsp5 and 95.42% with nsp5 of bat cov (supplementary figure s2d) . therefore, it not surprising that our analysis demonstrated the identical mean ppid values of 1.96% for nsp5s from sars-cov-2, human sars, and bat cov ( table 3) . the predicted per-residue intrinsic disorder propensity of sars-cov-2, human sars, and bat cov nsp5s are presented in figures 23a, 23b , and 23c, respectively. as the graphs depict, nsp5s have several flexible regions and n-terminally idpr of six residues. due to the low flexibility of this protein, a single morf predicted by morfchibi_web is present in the n-terminal region (residues 3-8) in nsp5s of all three viruses (table 2, supplementary tables 7 and 8) . further, the identified nucleotide-binding residues in nsp5 of all three viruses are tabulated in supplementary tables 9, 10, and 11 . non-structural protein 6 (nsp6). nsp6 protein is involved in blocking er-induced autophagosome/autolysosome vesicle formation that functions in restricting viral production inside host cells. it induces autophagy by activating the omegasome pathway, which is normally utilized by cells in response to starvation. sars nsp6 leads to the generation of small autophagosome vesicles thereby limiting their expansion [141] . nsp6 of sars-cov-2 is equally close to nsp6s from both human sars and bat cov, having a sequence identity of 87.24% ( figure 24d ). according to our analysis, mean ppids for nsp6s are calculated to be 1.03%, 1.03%, and 1.03% for sars-cov-2, human sars cov, and bat cov, respectively. figures 24a, 24b, and 24c show the corresponding graphs of intrinsic disorder tendency of nsp6s from sars-cov-2, human sars cov, and bat cov and demonstrate that these proteins are highly ordered and show low flexibility. as it is a membrane protein, nsp6 proteins are predicted to have only a single morf near the nterminal region (residues 1-19 in sars-cov-2, residues 1-22 in human sars, and residues 1-21 in bat cov) by the disopred3 server (table 2, supplementary tables 7 and 8) . the role of these protein-binding regions for the induction of autophagy is need to be elucidated. nsp7 and 8) . the ~10 kda nsp7 helps in primaseindependent de novo initiation of viral rna replication by forming a hexadecameric ring-like structure with nsp8 protein [142, 143] . both non-structural proteins 7 and 8 contribute 8 molecules to the ring-structured multimeric viral rna polymerase. site-directed mutagenesis in nsp8 revealed a d/exd/e motif essential for the in vitro catalysis [142] . figure 25d depicts the 3.1 å resolution electron microscopy-based structure (pdb id: 6nur) of the rdrp-nsp8-nsp7 complex bound to the nsp12. the structure identified conserved neutral nsp7 and nsp8 binding sites overlapping with finger and thumb domains on nsp12 of the virus [144] . we found that nsp7 of sars-cov-2 share 100% sequence identity with nsp7 of bat cov and 98.80% with nsp7 from human sars (figure 25e) , while sars-cov-2 nsp8 is closer to nsp8 of human sars (97.47%) than to nsp8 of bat cov (96.46%) ( figure 26d ). due to the high levels of sequence identity, mean ppids of all nsp7s were found to be identical and equal to 9.64%. both sars-cov-2 and human sars nsp8 proteins were calculated to have a mean ppid of 23.74% and, for nsp8 of bat cov mean disorder is predicted to be 22.22%. figures 25a, 25b , and 25c display the intrinsic disorder profiles for nsp7s, whereas figures 26a, 26b , and 26c represent the predicted intrinsic disorder propensity of nsp8s. as our analysis suggests, nsp7s might have a well-predicted structure, while nsp8s are moderately disordered. nsp8s are predicted to have a long idpr (residues 44-84) in both sars-cov-2 and human sars, and a bit shorter idpr in bat cov (residues . furthermore, sars-cov nsp7 using its n-terminus residues (v11, c13, v17, and v21) forms a hydrophobic core with nsp8 residues (m92, m95, l96, m99, and l103). additionally, h-bonding takes place between nsp7 q24 and nsp8 t89 residues [143] . these amino acids are the part of morfs predicted in nsp7 and nsp8 proteins. the results are tabulated in both supplementary tables 9, 10 , and 11). nsp9 protein is a single-stranded rna-binding protein [145] . it might protect rna from nucleases by binding and stabilizing viral nucleic acids during replication or transcription [145] . our results on nucleotide-binding tendency of nsp9 shows the presence of several rna binding and few dna binding residues in nsp9 of sars-cov-2, human sars, and bat cov (supplementary tables 9, 10, and 11) . presumed to evolve from a protease, nsp9 forms a dimer using its gxxxg motif [146, 147] . figure 27d shows a 2.7 å crystal structure of the homodimer of human sars nsp9 (pdb id: 1qz8) that identified a unique and previously unreported for other proteins, oligosaccharide/oligonucleotide fold-like fold [145] . here, each monomer contains a coneshaped β-barrel and a c-terminal α-helix arranged into a compact domain [145] . nsp9 of sars-cov-2 is equally similar to nsp9s from both human sars and bat cov, having a percentage identity of 97.35%. the difference in three amino acids at 34, 35 and 48 positions accounts for these similarity scores ( figure 27e ). as calculated, the mean ppids of nsp9s of sars-cov-2, human sars cov, and bat cov are 7.08%, 7.96%, and 7.08% respectively. figures 27a, 27b , and 27c depict the predicted intrinsic disorder propensity in the nsp9 protein from sars-cov-2, human sars, and bat cov. according to our analysis, all three nsp9s are rather structured but contain flexible regions. nsp9 contains conserved residues (r10, k52, y53, r55, r74, f75, k86, y87, f90, k92, r99, and r111) of positively charged side chains suitable for binding with the negatively charged phosphate backbone of rna and aromatic side-chain amino acids providing stacking interactions [145] . these residues are a part of multiple disorder-based binding sites predicted by morfchibi_web server ( table 2, supplementary table 7 and 8) . nsp10 performs several functions for sars-cov. it forms a complex with nsp14 for dsrna hydrolysis in 3′ to 5′ direction and activates its exonuclease activity [148] . it also stimulates the methyltransferase (mtase) activity of nsp14 required during rna-cap formation after replication [149] . figure 28d represents the x-ray crystal structure of the nsp10/nsp14 complex (pdb id: 5c8t) [150] . in agreement with the results of previous biochemical experimental studies, the structure identified important interactions with the exon (exonuclease domain) of nsp14 without affecting its n7-mtase activity [148, 149] . sars-cov-2 nsp10 protein is quite conserved having a 97.12% sequence identity with nsp10 of human sars and 97.84% with nsp10 of bat cov (figure 28e) . mean ppids of all three studied nsp10 proteins are found to be 5.04%. figures 28a, 28b , and 28c represent disorder profiles of nsp10s and signify the lack of long idprs but presence flexible regions in these proteins. furthermore, [11] [12] [13] [14] [15] [16] [17] [18] was predicted by morfpred server. interestingly, the sars-cov nsp10 residues f16, f19, and v21 form van der waals interactions with many of the nsp14 amino acids [150] and one residue (f16) is located in morf region which we have identified. furthermore, many nucleotide-binding residues which are found in all three viruses (supplementary table 9 , 10, and 11) and above-mentioned residues are not found to interact with dna/rna. in coronaviruses, nsp12 is an rna-dependent rna polymerase (rdrp). it carries out both primer-independent and primer-dependent synthesis of viral rna with mn 2+ as its metallic co-factor and viral nsp7 and 8 as protein co-factors [151] . as aforementioned, a 3.1å resolution structure of human sars nsp12 in association with nsp7 and nsp8 proteins (pdb id: 6nur) has been reported using electron microscopy ( figure 25d ). nsp12 has a polymerase domain similar to "right hand", finger domain (398-581, 628-687 residues), palm domain (582-627, 688-815 residues) and a thumb domain (816-919) [144] . sars-cov-2 nsp12 protein has a highly conserved c-terminal region (supplementary figure s2e ). it is found to share a 96.35% sequence identity with human sars nsp12 and 95.60% with bat cov nsp12. mean ppid values for all three nsp12s are estimated to be 0.43% (table 3) . figures 29a, 29b , and 29c show that although these proteins are mostly ordered, they have multiple flexible regions. as rdrp protein is observed to be mostly structured, significant morfs in disordered regions are not found ( table 2, supplementary table 7 and 8). nsp13 functions as a viral helicase and unwinds dsdna/dsrna in 5' to 3' direction [152] . recombinant viral helicase expressed in e.coli rosetta 2 strain was reported to unwind ~280 bp per second [152] . figure 30d represents a 2.8 å x-ray crystal structure of human sars nsp13 (pdb id: 6jyt) [153] . this helicase contains a 19-20 loop on 1a domain, which is primarily responsible for its unwinding activity. furthermore, the study revealed an important interaction of nsp12 with nsp13 that further enhances its helicase activity [153] . the 601-amino-acid-long nsp13 of sars-cov-2 is almost completely conserved, as it shares 99.83% with nsp13 of humans sars and 98.84% with nsp13 of bat cov (supplementary figure s2f) . in accordance with our results, the mean ppids of all three nsp13 proteins are estimated to be 0.67%. figures 30a, 30b , and 30c show that nsp13s contain multiple flexible regions but do not possess significant disorder. as expected, being a low disorder protein nsp13 does not contain any morf region and not a single bindingregion is located by any server used in all three viruses ( table 2, supplementary table 7 and 8). it has many nucleotide-binding residues (rna and dna) which are tabulated in supplementary tables 9, 10, and 11. nsp14 is a multifunctional viral protein that acts as an exoribonuclease (exon) and methyltransferase (n7-mtase) in sars coronaviruses. it's 3' to 5' exonuclease activity lies in the conserved dedd residues related to exonuclease superfamily [154] . its guanine-n7 methyltransferase activity depends upon the s-adenosyl-lmethionine (adomet) as a cofactor [149] . as aforementioned, nsp14 requires nsp10 for activating its exon and n7-mtase activity inside the host cells. figure 28d depicts the 3.2 å crystal structure of human sars nsp10/nsp14 complex (pdb id: 5c8t), where amino acids 1-287 form the exon domain and 288-527 residues form the n7-mtase domain of nsp14. a loop (residues 288-301) is essential for its n7-mtase activity [150] . figure s2g) . mean ppid values for nsp14s from sars-cov-2 and human sars is calculated to be 0.38%, while the nsp14 from bat cov has a mean ppid 0.57%. predicted per-residue intrinsic disorder propensity of nsp14s from sars-cov-2, human sars, and bat cov is represented in figures 31a, 31b , and 31c, respectively. as can be observed from these plots and corresponding ppid values, all nsp14s are found to be highly structured. likewise, table 2 shows nsp14 contains two protein binding regions (residues 8-13 and 441-445) predicted by the morfpred server in all three viruses. as shown in supplementary tables 9, 10, and 11, the orf14 represents multiple nucleotide-binding residues. nsp15 is a uridylate-specific rna endonuclease (nendou) that creates a 2′-3′ cyclic phosphates after cleavage. its endonuclease activity depends upon mn 2+ ions as co-factors. conserved in nidovirus, it acts as an important genetic marker due to its absence in other rna viruses [155] . figure 32d represents a 2.6 å crystal structure of uridylate-specific nsp15 (pdb id: 2h85) that was deduced by bruno and colleagues using x-ray diffraction [156] . the monomeric nsp15 has three domains: nterminal domain (1-62 residues) formed by a three anti-parallel -strands and two α-helices packed together; a middle domain residues) that contains an α-helix connected via a 39-amino-acid-long coil to an ordered region containing two α-helices and five -strands; and a c-terminal domain (192-345 residues) consisting of two anti-parallel three -strand sheets on each side of a central α-helical core [156] . the nsp15 is found to be quite conserved across human sars and bat covs. sars-cov-2 nsp15 shares an 88.73% sequence identity with nsp15 of human sars and 88.15% with nsp15 of bat cov (supplementary figure s2h) . calculated mean ppids of nsp15s from sars-cov-2, human sars, and bat cov are 1.73%, 2.60%, and 2.60%, respectively. similar to many other non-structural proteins of coronaviruses, nsp15s from sars-cov-2, human sars, and bat cov are predicted to possess multiple flexible regions but contain virtually no idprs (see figures 32a, 32b, and 32c) . similarly, no significant disorderbinding regions are predicted in nsp15 proteins ( table 2) . sars-cov-2 contain one morf (residues 9-13) predicted by morfpred server. human sars do not have a single morf while bat cov possesses two very short binding regions (supplementary table 7 and 8). supplementary table 9 , 10, and 11 depicts the presence of many rna binding residues and few dna binding residues in nsp15 of all three viruses. nsp16 protein is another mtase domain-containing protein. as methylation of coronavirus mrnas occurs in steps, three proteins nsp10, nsp14, and nsp16 acts one after another. the first event requires the initiation trigger from nsp10 protein, after which nsp14 methylates capped mrnas forming cap-0 (7me) gpppa-rnas. nsp16 protein, along with its co-activator protein nsp10, acts on cap-0 (7me) gpppa-rnas to give rise to final cap-1 (7me)gpppa(2'ome)-rnas [149, 157] . a 2 å x-ray crystal structure of the human sars nsp10-nsp16 complex is depicted in figure 33d (pdb id: 3r24) [158] . the structure consists of a characteristic fold present in class i mtase family comprising of α-helices and loops surrounding a seven-stranded β-sheet [158] . nsp16 protein of sars-cov-2 is found to be equally similar to nsp16s from human sars and bat cov (93.29%) (supplementary figure s2i) . mean ppids for nsp16s from sars-cov-2, human sars, and bat cov are 5.37%, 3.02%, and 3.02%, respectively. in line with these ppids values, figures 33a, 33b, and 33c show that nsp16s are mostly ordered proteins containing several flexible regions. correspondingly, no significant morfs are present in this protein ( table 2, supplementary table 7 and 8). a single morf (residues [151] [152] [153] [154] [155] [156] were found with the help of morfpred in all three viruses. further, several rnabinding and few dna-binding residues are also identified (supplementary table 9 , 10, and 11). replicase polyprotein 1a. since replicase polyprotein 1a contains non-structural proteins 1-10 identical to those found in replicase polyprotein 1ab, we did not perform their disorder analysis separately. however, replicase polyprotein 1a has one additional non-structural protein designated as nsp11. nsp11 is a small uncharacterized protein cleaved from the replicase polyprotein 1a. this small protein with unknown function requires experimental insights to further characterize this protein. the intrinsic disorder predicting software used in this study requires amino acid sequences, which are at least 30-residue long. therefore, because of their short sequences (just 13 residues) nsp11s from all three studied coronaviruses were not checked for the intrinsic disorder, disorder-based protein binding regions, and nucleotide-binding residues. based on the msa outputs, nsp11 from sars-cov-2 was found to have a sequence identity of 84.62% with nsp11s from human sars and bat cov (figure 34 ). the emergence of new viruses and associated deaths around the globe represent one of the major concerns of modern times. despite its pandemic nature, there is very little information available in the public domain regarding the structures and functions of sars-cov-2 proteins. based on its similarity with human sars cov and bat cov, the published reports have suggested the functions of sars-cov-2 proteins. in this study, we utilized information available on sars-cov-2 genome and translated proteome from genbank, and carried out a comprehensive computational analysis of the prevalence of the intrinsic disorder in sars-cov-2 proteins. additionally, a comparison was also made with proteins from close relatives of sars-cov-2 from the same group of beta coronaviruses, human sars cov and bat cov. our analysis revealed that in these three covs, the n proteins are highly disordered, possessing the ppid values of more than 60%. these viruses also have several moderately disordered proteins, such as nsp8, orf6, and orf9b. although other proteins have shown lower disorder content, almost all of them contain at least some idprs, and all cov proteins analysed in this study definitely have multiple flexible regions. importantly, our study provides novel information on presence of intrinsic disorder at the cleavage sites of the replicase 1ab polyprotein of covs. this observation confirms the crucial role of idprs in maturation of individual proteins. we also established that many of these proteins contain disorder-based binding motifs. since idps/idprs might undergo structural transition upon association with their physiological partners, our study generates important grounds for better understanding of the functionality of these proteins, their interactions with other viral proteins, as well as interaction with host proteins in different physiological conditions. this will also guide structural biologists to carry out a structure-based analysis of sars-cov-2 proteome to explore the path for the development of new drugs and vaccines. the periodical outbreaks of pathogens worldwide always remind the lack of suitable drugs or vaccines for proper cure or treatment. in 2003, nearly 750 deaths were reported due to the sars outbreak in more than 24 countries. but this time, the outbreak of wuhan's novel coronavirus (sars-cov-2) has quickly surpassed this number, indicating more causalities soon. the lack of accurate information and ignorance of primary symptoms are major reasons, which cause many infection cases. although efficient transmission from human to human is confirmed, the actual reasons for fast sars-cov-2 spread are still unknown, but some assumptions were made by researchers and chinese authorities. the fast spread of sars-cov-2, covid-19 pandemic, and associated introduction of quarantine also have made major impacts on economy and education worldwide due to several restrictions, such as limited transportation, restrained or frozen traveling, halted attendance of mass events, the introduction of distant teaching and learning, etc. due to advancements in sequencing techniques, the full genome sequence of sars-cov-2 was made available in a few days of the first infection report from wuhan, china. however, massive subsequent research needs to be done to identify the actual cause of sars-cov-2 infectivity and to design suitable treatment in the coming future. certain possibilities can be explored with the available information. the mutational pressure study on this virus will be very interesting to see if this virus transforms from bat sars to human sars to sars-cov-2. more in-depth experimental studies using molecular and cell biology techniques to establish structurefunction relationships are required for a better understanding of the functioning of sars-cov-2 proteins. additionally, based on the sequence homology and information on proteinprotein interactions, the associated viral and host proteins should be explored, for finding means suitable for limiting replication, maturation, and ultimately pathogenesis of this virus. although structural biology techniques (so-called rational drug design) can be used in drug development utilizing high throughput screening of compounds virtually or experimentally, the applicability of these techniques is limited by the presence of intrinsic disorder in target proteins. therefore, the thorough disorder analysis of three coronaviruses conducted in this study will help structural biologists to rationally design experiments keeping this information in mind. authors contribution: rg: conception and design, interpretation of data, writing, and review of the manuscript, and study supervision. vnu: conception and design, acquisition and interpretation of data, writing, and review of the manuscript. ms, tb, pk, brg, kg: acquisition and interpretation of data, writing of the manuscript. table 6 . evaluation of intrinsic disorder in non-structural proteins of bat cov. table 7 : predicted morf residues in human sars proteins. supplementary table 8 : predicted morf residues in bat cov proteins. supplementary table 9 : predicted nucleotide-binding residues in sars-cov-2 proteins. supplementary table 10 : predicted nucleotide-binding residues in human sars proteins. supplementary table 11 : predicted nucleotide-binding residues in bat cov proteins. supplementary figures s1. multiple sequence alignment of structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit 3.0. figure s1a . msa of sars-cov-2, human sars, and bat cov spike glycoproteins. figure s1b . msa of sars-cov-2, human sars, and bat cov nucleoproteins. supplementary figure s2 . multiple sequence alignment of non-structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit 3.0. figure s2a . msa of sars-cov-2, human sars, and bat cov nsp2 proteins. figure s2b . msa of sars-cov-2, human sars, and bat cov nsp3 proteins. figure s2c . msa of sars-cov-2, human sars, and bat cov nsp4 proteins. figure s2d . msa of sars-cov-2, human sars, and bat cov nsp5 proteins. figure s2e . msa of sars-cov-2, human sars, and bat cov nsp12 proteins. figure s2f . msa of sars-cov-2, human sars, and bat cov nsp13 proteins. figure s2g . msa of sars-cov-2, human sars, and bat cov nsp14 proteins. figure s2h . msa of sars-cov-2, human sars, and bat cov nsp15 proteins. figure s2i . msa of sars-cov-2, human sars, and bat 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sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane enhancement of murine coronavirus replication by severe acute respiratory syndrome coronavirus protein 6 requires the n-terminal hydrophobic region but not c-terminal sorting motifs a putative diacidic motif in the sars-cov orf6 protein influences its subcellular localization and suppression of expression of cotransfected expression constructs the n-terminal region of severe acute respiratory syndrome coronavirus protein 6 induces membrane rearrangement and enhances virus replication characterization of a unique group-specific protein (u122) of the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/x1 and 7a/x4 proteins through nf-kappab activation chemokine upregulation in sars-coronavirus-infected, monocyte-derived human dendritic cells induction of apoptosis by the severe acute respiratory syndrome coronavirus 7a protein is dependent on its interaction with the bcl-xl protein the orf7b protein of severe acute respiratory syndrome coronavirus (sars-cov) is expressed in virus-infected cells and incorporated into sars-cov particles 7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase structure and intracellular targeting of the sars-coronavirus orf7a accessory protein solution structure of the x4 protein coded by the sars related coronavirus reveals an immunoglobulin like fold and suggests a binding activity to integrin i domains the 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8 molecular evolution of the sars coronavirus during the course of the sars epidemic in china the human severe acute respiratory syndrome coronavirus (sars-cov) 8b protein is distinct from its counterpart in animal sars-cov and down-regulates the expression of the envelope protein in infected cells severe acute respiratory syndrome coronavirus accessory protein 9b is a virion-associated protein sars-cov 9b protein diffuses into nucleus, undergoes active crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus the crystal structure of orf-9b, a lipid binding protein from the sars coronavirus mechanisms and enzymes involved in sars coronavirus genome expression biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation novel -barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus identification of residues of sars-cov nsp1 that differentially affect inhibition of gene expression and antiviral signaling coronavirus nonstructural protein 1: common and distinct functions in the regulation of host and viral gene expression severe acute respiratory syndrome coronavirus nonstructural protein 2 interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling the nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-b signaling severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme nuclear magnetic resonance structure of the n-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus the envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles mobility and interactions of coronavirus nonstructural protein 4 two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals a novel mutation in murine hepatitis virus nsp5, the viral 3c-like proteinase, causes temperature-sensitive defects in viral growth and protein processing structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus nsp6 restricts autophagosome expansion the sars-coronavirus nsp7+nsp8 complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension insights into sars-cov transcription and replication from the structure of the nsp7-nsp8 hexadecamer structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a singlestranded rna-binding subunit unique in the rna virus world variable oligomerization modes in coronavirus non-structural protein 9 severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth rna 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex in vitro reconstitution of sars-coronavirus mrna cap methylation structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex biochemical characterization of a recombinant sars coronavirus nsp12 rna-dependent rna polymerase capable of copying viral rna templates mechanism of nucleic acid unwinding by sars-cov helicase delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp13 upon atp hydrolysis discovery of an rna virus 3'->5' exoribonuclease that is critically involved in coronavirus rna synthesis major genetic marker of nidoviruses encodes a replicative endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein 15 define an endoribonuclease family coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'o)-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-o-methylation by nsp16/nsp10 protein complex key: cord-353609-no3mbg5d authors: vandegrift, kurt j.; wale, nina; epstein, jonathan h. title: an ecological and conservation perspective on advances in the applied virology of zoonoses date: 2011-04-15 journal: viruses doi: 10.3390/v3040379 sha: doc_id: 353609 cord_uid: no3mbg5d the aim of this manuscript is to describe how modern advances in our knowledge of viruses and viral evolution can be applied to the fields of disease ecology and conservation. we review recent progress in virology and provide examples of how it is informing both empirical research in field ecology and applied conservation. we include a discussion of needed breakthroughs and ways to bridge communication gaps between the field and the lab. in an effort to foster this interdisciplinary effort, we have also included a table that lists the definitions of key terms. the importance of understanding the dynamics of zoonotic pathogens in their reservoir hosts is emphasized as a tool to both assess risk factors for spillover and to test hypotheses related to treatment and/or intervention strategies. in conclusion, we highlight the need for smart surveillance, viral discovery efforts and predictive modeling. a shift towards a predictive approach is necessary in today’s globalized society because, as the 2009 h1n1 pandemic demonstrated, identification post-emergence is often too late to prevent global spread. integrating molecular virology and ecological techniques will allow for earlier recognition of potentially dangerous pathogens, ideally before they jump from wildlife reservoirs into human or livestock populations and cause serious public health or conservation issues. generated concern for the environment and thrust ecologists into a new political field where preserving the integrity of our global ecosystems was the priority [18] . even so, the society for conservation biology was not established until 1985 [19] . as a part of this transition, ecology shifted from a descriptive science to one of prediction, reflecting the hope that ecologists might mitigate changes which can have negative impacts upon the ecosystem. ecologists have branched out into the study of parasites and disease as it has become increasingly apparent that parasites are inextricably linked to the ecology of their hosts and environments, to the point where they have been a driving force in the evolution of sexual reproduction and in the shaping of biodiversity [20, 21] . over the past 30 years, disease ecologists have developed the study of parasites and pathogens in the wild. this knowledge has been synthesized into mathematical models which describe the dynamic properties of ecosystems and predict how parasites and pathogens flow through them. [22, 23] . these models are becoming more commonly integrated into epidemiological studies that seek to predict outbreaks or periods of time when cross-species spillover risk is highest. parallel to this progress, the field of virology, particularly the subfields of molecular virology and viral evolution, have also been burgeoning, largely due to advances in technology that have made molecular assays and genetic sequencing more accessible to a greater number of scientists. the development of high-throughput sequencing has greatly increased our ability to efficiently detect known viruses as well as to discover new types of viruses, thereby improving our understanding of viral diversity, pathology and evolution. this increased capacity has spawned the development of new fields of study. for example, phylodynamics allows researchers to determine the origin of circulating viruses in space and time. mutations among viral strains can be used to investigate interactions among host species as well as long-range host movement via corridors and flyways. phylodynamic analyses can also inform livestock management practices, as was the case with foot and mouth disease in the united kingdom [24] . conducting viral surveillance in animal reservoirs and invertebrate vectors can help explain circulation within host species; observed patterns of zoonotic transmission; and even allow for the prediction of periods of increased risk of zoonotic transmission (e.g., rift valley fever and rainfall [25] ; west nile virus (wnv) and american robin (turdus turdus) migration [26] ; as well as hantavirus in mice [27, 28] ). understanding viral ecology in wildlife reservoirs and identifying high-risk human-wildlife interfaces is especially critical in the context of ever increasing globalization, whereby transportation networks facilitate rapid spread of pathogens well beyond bounds where traditional epidemiological methods can be effective [29] [30] [31] . the 2009 influenza a (h1n1) pandemic spread from the presumptive point of emergence in la gloria mexico to new zealand in just under a month [32] while sars radiated from guangdong, china to 26 different countries within several months [29] . the negative impacts of emerging infectious diseases are not limited to humans. indeed, wildlife conservationists have documented several mass mortality events in other animal species. western lowland gorillas (gorilla gorilla gorilla) have been decimated by ebola virus [33] and an especially virulent calicivirus, rabbit hemorrhagic disease virus, spread through both domestic and wild rabbit populations, resulting in tens of millions of deaths [34] . in some instances the viruses have attenuated, while in others the animal populations have been brought to the brink of extinction. importantly, the risk from disease to humans and animals should not be separated. the global transportation network facilitated the introduction of infected vectors (e.g., mosquitoes) into new york and wnv caused both avian and human mortality, and this virus has subsequently spread across the united states [26] . table 1 . definitions of key terms used in the text. the formation of a hybrid population through the mixing of two ancestral, or long-separated, populations. a method for estimating historical population dynamics from a sample of sequences without assuming a predefined demographic model. coalescent theory a mathematical framework which describes the distribution of gene trees in populations. it provides mathematical methods for connecting demographic or ecological models with a phylogenetic tree. demography is the statistical study of populations. in the field of ecology, demography encompasses the study of the size, structure and distribution of populations, and spatial and/or temporal changes in them in response to birth, migration, aging and death. however, here we use a more rigid definition of demography-as the pattern and rate of population growth. the number of breeding individuals in an idealized population that would show the same amount of dispersion of allele frequencies under random genetic drift, or the same amount of inbreeding, as the natural population under consideration. the analogue of ne for viruses, the ‗effective number of infections' is related to the number of infected host individuals and to the number of new transmission events [35] . a discipline which uses next-generation sequencing technologies to characterize the entirety of genomic material found in environmental samples. the molecular clock is derived from the hypothesis that sequence evolution, while random, occurs at stable rate such that the time since the divergence of two or more sequences can be estimated. recent ‗relaxed molecular clock' analysis can account for variation in the rate of sequence evolution through time or between lineages. the relations among a set of sequences showing which shares a most recent common ancestor with other sequences. the study of the principles and processes governing the geographical distribution of genealogical lineages. in the field of population genetics, population structure is defined as the absence of random mating within a population. this is the definition used here. in ecology, population structure is defined by several key parameters including number of individuals in a population, age distribution of individuals, probabilities of survival (or mortality), and rates of fecundity. a process that occurs in segmented viruses by which one or more segments ‗swap' to create a new viral genome. this drives the process of antigenic shift in influenza a viruses. the process by which new genotypes are created by the combination of distinct lineages. in sexual organisms it occurs during meiotic division, by the exchange of dna between different chromosomes or ‗crossing-over'. viral recombination occurs during viral replication and is an important factor in viral evolution (for more details see worobey and holmes, 1999 [36] ). mutation of a virus such that it changes ‗back' to its wild-type state. the timing of an organism's schedule of reproduction and death. species with long life histories, also known as ‗k' strategists, tend to have low reproductive rates, stable populations, long generation times and long lifespans. where the parasite population is not randomly distributed among hosts, such that the variance is greater than the mean. the macroparasites in a host population are often best described by the negative binomial distribution such that a minority of hosts possess the majority of the parasites. r 0 (basic reproduction number) in the case of viruses and other microparasites, r 0 is the average number of secondary infections which an infection produces. as such it is a measure of parasite fitness. a host species that can independently maintain a disease and act as a source of infection to other host species. infection in reservoirs is usually more persistent and less harmful than that of other hosts. zoonotic disease a disease transmissible from animals to humans or vice versa. globalization, host ecology, host-virus dynamics, climate change, and anthropogenic landscape changes all contribute to the complexity of zoonotic viral emergence and disease, and create significant conservation and public health challenges. comprehensive and collaborative scientific approaches that transcend disciplinary boundaries are necessary to address these challenges. it is the goal of this paper to review new methods for understanding viral dynamics and illustrate how and when these techniques can be used by not only public health officials, but also disease ecologists and conservation biologists. the phylodynamic paradigm, established in 2004 [37] , exemplifies the power of a multidisciplinary approach. it unites the ecological and evolutionary study of viruses and builds upon advances in sequencing technologies and coalescent theory, by which gene genealogies are reconstructed backward in time [38] . the analysis of phylogenetic trees enables researchers to address many of the primary questions posed by disease ecologists (figure 1 ). in some cases this approach can provide an estimate of a virus's basic reproductive number (r 0 ), which is a measure of parasite fitness [39] . phylodynamics has far-reaching applications for the control of viruses in both human and animal populations, in addition to being vital to our understanding of the interconnectedness between them. phylodynamic studies can be used to identify reservoir species as well as defining the spatial and temporal origin of emerging infectious diseases [37] . they can also help to elucidate how these viruses spread following their emergence [39] . firstly, chains of viral transmission can be extrapolated from the branching topology of phylogenetic trees. one example of the utility of this approach is with rabies virus. rabies causes thousands of human deaths a year in africa and has been implicated in the decline and local extinction of several populations of african wild dog (lycaon pictus) [40, 41] . lembo et al. analyzed sequences of rabies virus from the serengeti, revealing that domestic dogs were the reservoir of the virus and that they had transmitted it to other resident carnivore populations on repeated occasions [42] . this work has applicability in that it can be used to design efficient and effective vaccination strategies, both to alleviate current distress and prevent future outbreaks [40] . secondly, by mapping the geographical origin of each sequence onto the nodes of phylogenetic trees, the geographical origin of a virus might be identified. wallace et al. (2007) [43] used this phylogeographic approach to identify guangdong province, china as the most parsimonious origin of highly pathogenic h5n1 strain of avian influenza and to delineate the most likely pathways of viral spread [43] . however, a recent bayesian analysis of this data did not support the conclusion that h5n1 had dispersed from guangdong to indonesia [44] . instead, the bayesian analysis suggested it had spread to indonesia from guangxi or hunan in china. this example demonstrates that different statistical techniques may yield different conclusions. as yet, neither the bayesian nor the frequentist method is universally considered to be superior and there is much room for improvement as statistical phylogeography develops as a field. phylogeographic tools have also been applied by walsh et al. (2005) [45] to locate the putative origin of the zaire strain of ebola virus. in an attempt to resolve the controversy over the time of emergence and spreading trajectory of ebola in the congo basin, they then used spatial data and two different tests for the impact of selection on the virus genome [45] . where a virus is expanding in range, as the zaire strain of ebola virus appears to be, using a ‗landscape genetics' approach may help identify geographical barriers to viral spread and help identify vulnerable human or wildlife populations lying in the path of infection [46, 47] . these phylogenies may reflect transmission chains, however sampling must be sufficient for them to do so, while recombination may obscure ‗true' relationships between viral sequences. (b) simple molecular clock theory, predicated on the neutral theory of molecular evolution [48] assumes that mutation occurs at a constant rate over time, thus the time that has elapsed since a pair of virus strains diverged from a common ancestor may be quantified. methods that account for differences in the evolutionary rates of different strains, and for variation in these rates through time, have been recently developed [49] . here variants represented by thick lines evolve much faster than those represented by thin lines. (c) using a phylogeographic approach, the location at which a sequence was sampled may be mapped onto the viral phylogeny and the likely spreading trajectory of the virus inferred. while parsimony approaches have been popular, powerful bayesian methods that account for uncertainty of dispersal process and historical phylogeny have been developed to reconstruct viral dispersal events [44] . crosses on the phylogeny represent such viral dispersal events, in this example. (d) coalescent theory provides the basis for many phylodynamic approaches. here, circles on the same row represent temporally simultaneous infections. working back from sampled infections (red circles), lineages can be traced back to the most recent common ancestor (black circle) via hypothetical, unsampled ancestors (grey circles). the time it takes for sampled lineages to coalesce is dependent on a variety of variables (i.e., viral effective population size, population structure, selection, stochastic infection die-out and recombination). a variety of methods are available to test for selection and recombination. phylodynamic analyses are not without limitations. dense and representative sampling at a scale equivalent to epidemiological surveys is required to fulfill the potential of phylodynamics for understanding epidemics of rapidly-evolving viruses [51] . the construction of phylogenetic trees from these viral sequences may be complicated by recombination and, in the case of segmented viruses, reassortment of viral genomes [36] . as a result of these processes, the genes on a single viral sequence may have very different origins (see the discussion of the different origins of the hemagglutinin and neuraminidase segments of h5n1 avian influenza in lemey et al. 2009 [44] ). therefore, concatenated analysis of multiple genes may be confounded. the ability to construct phylogenies is further limited by the total viral genetic information available. genbank is a vast public database that contains records of genetic sequences; however, its usefulness is dependent upon the willingness and/or ability of individuals and organizations to submit viral sequences. governments and industrial institutions may be reluctant to report sequences of economically important viruses (i.e., avian influenza) due to the potential negative economic impacts that may ensue. although phylodynamics is currently encumbered by the aforementioned factors, there is hope for progress. advancements in coalescent theory will help us to deal with the phylogeny construction problems caused by recombination and reassortment. they will also facilitate better utilization of genomic and spatial data, provided these advancements are also accompanied by a simultaneous increase in computing power, which is also currently limiting. the utility of phylodynamics is not limited to questions of interest to virologists and disease ecologists. this approach may also inform investigations of host population biology and, in so doing, aid in the development of conservation policy. host molecular markers (e.g., microsatellites, mitochondrial dna) are used by conservation biologists and ecologists to infer population structure, historical demography and other critical features of wildlife populations [52] and have proved particularly powerful when analyzed in combination (i.e., [53, 54] ). recently, it has been demonstrated that the pathogens of host populations might also be useful to this end. research using helminths and bacteria has revealed patterns of ancient human migration and dispersal [55, 56] identified ancient refuges of rodent and bird taxa [57, 58] and shown that there was past contact between contemporary non-sympatric bat species [59] . however, there have been few attempts to utilize viruses to this end (save [60, 61] ). it is surprising that viruses have not been used more for the inference of host population biology since some of their characteristics make them ideal for doing so. most viruses have large population sizes and short generation times, and many replicate using a highly error-prone rna-dependent rna polymerase, causing them to accumulate many more mutations (nucleotide changes) per unit time than the host genomes [39, 62] . consequently, viruses may provide information about host demographics on a shorter timescale than molecular markers of the host. one of the signature tools of phylodynamics, the bayesian skyline plot, might also be utilized to infer changes in historical population size of the host. these plots incorporate the use of a molecular clock and coalescent theory to infer historical changes in virus population sizes without assuming a predefined demographic model [63] . it is important, however, that the timescale over which the evolutionary dynamics of the virus population can be reliably reconstructed is appropriate for the parameters of interest in the host population. unfortunately, the very characteristics that make viruses useful for estimating host population structure and demography may also impede the analyses. multiple substitutions can occur quickly in the viral genome and this will obscure the host population's actual evolutionary history. meanwhile, variations in the transmission mechanisms of viruses (vertical vs. horizontal) can alter the ability to accurately infer a virus' relationship to a host population. cross-species transmission is also problematic in that it can cause pathogen phylogenies to inaccurately reflect the history of their hosts [62] . therefore, before the genetic information contained within a virus population can be used to infer the population structure and demography of the host, it is critical to test for congruence in the evolutionary history of the host and virus populations. this is accomplished by statistically comparing the respective phylogenies within the relevant timescale. viruses with high host specificity have a greater likelihood of exhibiting such congruence. feline immunodeficiency virus (fiv) is known for high host specificity and is, thus far, the only virus to have been used to elucidate changes in host population structure and size. from a phylogenetic analysis of fivpco, the fiv type specific to the cougar (puma concolor), biek, drummond, and poss (2006) [61] inferred that the north american population of cougars became subdivided during the last century but subsequently expanded in both size and range. subsequently, antunes et al. (2008) [60] used the distribution of fiv ple subtypes in the serengeti to infer that recent admixture has occurred between the region's lion (panthera leo) populations. these recent changes in felid population size and structure could not have been inferred from host genetic data. there is great potential for the further use of this technique by conservation biologists and ecologists, and for it to complement existing methods which utilize host genetic data. host genetic markers are used to define management units for conservation purposes [64] . many ‗flagship' endangered species have long life histories [65] , a feature that correlates with both extinction risk in certain regions [66] and difficulty in reconstructing recent demographic history from molecular markers. because of the latter, the use of viral genetics to define management units may be an important avenue of exploration. in addition to aiding the definition of management units, viral data could be used to analyze the consequences of management activities and other environmental changes on target species. where viral genetic diversity exists in a spatially heterogeneous distribution, viral movement patterns could be used to study the migratory behavior of animals (as macroparasites have been [67, 68] ). as such, researchers could monitor the use of wildlife corridors and the efficacy of control measures aimed at limiting the range of a host species [69, 70] . where viruses can be readily amplified from non-invasively collected samples (see [71] ), the above objectives could be achieved in a cost effective manner with minimal disturbance of the study species. viruses with specific transmission routes may also serve as proxies for behaviors related to transmission (i.e., sexually transmitted diseases). similarly, where cross-species transmission occurs, viruses might be indicative of types of sustained, direct contact between different, sympatric taxa which facilitate such transmission, for example predator-prey interactions [72] . at the broader ecosystem level, inferences about long-term evolutionary processes might also be made by examining the phylogeographic structure of numerous host and virus populations of a region (see [73] ). a major hurdle for both virologists and ecologists is defining the biodiversity of life. at present, scientists do not know the actual number of mammal species, much less the diversity of viruses they harbor [2] . indeed, the diversity of viruses known to infect the house mouse (mus musculus), a staple in biomedical research, is not yet completely known. recent advances in genetic sequencing, including high-throughput sequencing and other -next-generation sequencing‖ techniques, as well as masstag pcr and microarray multiplex assays [74, 75] have made the study of microbial diversity feasible [76] . these technologies have facilitated a movement from classical virology, where the focus was on disease etiology, toward a broader discipline that considers the rest of the viral diversity or -the virosphere‖. metagenomic studies have used next-generation sequencing to study biodiversity in substrates such as ocean water and soil [77, 78] . metagenomics has also been used to screen human and animal clinical samples in order to determine etiologic agents of disease or to describe the microbial flora normally present in a vertebrate host-in many cases the result has been the discovery both of novel pathogens and novel associations between clinical disease and agent [79] [80] [81] [82] . as technology becomes more affordable, and thus accessible, there will be increasing opportunities to ask large-scale questions such as: how does the virosphere vary across space and time? how does it vary across species? can we use this to define risk of cross-species transmission or to inform conservation efforts? and how might co-infections with these undiscovered viruses influence the dynamics of the more well known viruses? the paucity of information about viral diversity within a host poses problems for research progress. it is difficult to understand viral pathogenesis and transmission without completely understanding the dynamics of co-infections. indeed, it is currently difficult to ascribe a host's symptoms to an individual virus with any certainty because a virus' actions, and even its ability to infect the host, could be a function of another (possibly undetected) co-habitant of the host. evidence of interactions between co-infecting species has been clearly demonstrated [83, 84] and it will be critically important to elucidate the interactions that occur between multiple pathogens as well as the combined effects they may have on a host's immune system. broadening our understanding of the diversity of pathogens that exist in human and animal hosts through wildlife and domestic animal surveillance will significantly improve our ability to recognize novel zoonotic agents in the context of a disease outbreak. phylogenetic information obtained from comparative sequence analyses can improve our understanding of the impact of sequence mutation on virulence, as well as inform decisions about vaccine development. a final noteworthy benefit of viral discovery efforts is that these techniques should be important for identifying candidates for future vaccines as a virus's most worthy competitor is often another virus. from a health perspective, vaccination is arguably the most important technology that has arisen from the study of viruses. vaccination offers a direct means of intervening in a host-pathogen system and it has become routine in many parts of the world. efforts to this end have resulted in the eradication and/or control of smallpox, polio, mumps, measles, rubella and most recently, rinderpest. vaccines take several forms including live-attenuated viruses; inactivated whole viruses; inactivated toxins and viral protein subunits, and these are often delivered in combination. while live-attenuated vaccines have been predominant, a new generation of techniques including gene delivery and nano-technologies are being used to develop highly-efficacious and safer vaccines, that have less risk of reversion [85] . new types of administration methods are also being developed with oral, aerosolized and nasal vaccines currently on the market. these less invasive administration techniques decrease labor costs associated with administration and offer increased capacity for mass-dispersal of vaccines to both humans and free-ranging wildlife [86] . vaccination campaigns aimed at both protecting threatened species and decreasing public health risks via animal vaccination have taken place. swiss health officials were pioneers in this field, using oral vaccines to control rabies in wild red foxes (vulpes vulpes) [87, 88] . these vaccines were inserted into chicken heads which were distributed in the wild beginning in 1978 [88] . as can be observed from the supplemental movie (video s1), their initial barrier approach evolved into a large-scale treatment of infected areas and resulted in rabies being successfully pushed back to and then eliminated from the swiss alps [87, 88] . following the success of these trials, campaigns were conducted in western europe [89] and canada [90] with similar results, though the situation in the united states has proven more challenging. other successful vaccination examples include canine distemper virus in black-footed ferrets (mustela nigripes; [91] ) and ethiopian wolves (canis simensis; [92] ), as well as rabies in florida panthers (felis concolor coryi; [93] ) and african wild dogs [94, 95] . a caveat to this success is that there is growing evidence that vaccination against a specific strain of pathogen can result in inadvertent selection for related co-infecting strains. thus vaccination can influence the dynamics of a pathogen [96] . the possibility of inadvertent viral strain selection highlights the importance of understanding the long-term evolutionary and ecological consequences of vaccination. indeed, where threatened or endangered animals are concerned, mishaps may prove disastrous. attenuated canine distemper vaccines did not provide immunity to critically endangered black-footed ferrets, while the use of a live canine distemper virus vaccine resulted in clinical distemper arising in one of the few remaining populations [97] . ideally, long-term clinical trials with suitable animal models might avert these problems. these trials should also be used to provide an a priori understanding of how vaccination might shape future evolutionary processes. in contrast to the ferret experience, efforts with the endangered ethiopian wolf serve as an example of a successful vaccination program. wolf populations were suffering severe mortality due to rabies and distemper acquired from the wild dogs that shared their home range [98] . on the basis of a spatially explicit individual-based model, which indicated rabies could be controlled in dogs given just over 60% coverage [99] , knobel et al. executed an intensive vaccination plan [92] . both the extent and duration of outbreaks in the treated areas were limited and, although monitoring and continued vaccination are required, the situation appeared to be under control in 2008 [92] . wildlife vaccination campaigns are also being investigated as tools to limit public health risks. tsao et al. vaccinated mice in an effort to break the cycle of lyme disease and reduce the risk of emergence in human populations, in which it causes tens of thousands of deaths per year in the us [59, 100] . in the same vein, griffing et al. [101] have tested the efficacy of vaccinating american robins to interrupt the wnv transmission cycle. this species can absorb up to 75% of the potentially infective mosquito bites in early spring and is thus a key host in the wnv system [102] . targeted vaccination of this single species could potentially result in herd immunity and reduce the risk of human infection as well as decreasing wildlife mortality. these works exemplify how ecological knowledge can be used to identify and exploit some of the heterogeneities which so often dominate the dynamics of pathogens. while the lasting efficacy of wildlife vaccination efforts has yet to be demonstrated with either endangered species or in breaking the transmission cycle of human pathogens, an increasing number of researchers are drawing attention to systems where it seems feasible [99, 103] ; demonstrating that intricate knowledge of host and virus ecology can greatly reduce the amount of vaccine coverage that is necessary to control these viruses. the problems entailed by the sheer number of viruses, viral resistance, the explosive potential for spread, and the economic burden, make it clear that currently available vaccination methods do not provide a sustainable solution for either human or animal disease. the unambiguous indication is that researchers need to work towards the goal of developing a predictive framework where risk can be defined for different scenarios and not only to rank pathogens, and species, but also, places and times of year that can be identified as more or less precarious for global health. pending questions include: which geographic areas will experience more disease and conservation problems? which areas pose the highest risk for pandemic spread of pathogens? what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission events? and what are the relative roles of genetic relatedness and contact rate for transmission? some modeling work and reviews of historic data have been informative [104, 105] , but novel uses of phylogenies of both viruses and hosts (as discussed above) provide promise for progress to this end, especially when coupled with high quality surveillance data. once we have this information, scientists will be able to design -smart surveillance‖ strategies whereby valuable vaccine resources can be efficiently targeted and efficiently distributed. ecological studies can effectively inform conservation as well as public health policy. gaining knowledge of reservoir host ecology can be critical for the development of eradication strategies. most viral disease systems are dominated by heterogeneities and identifying and understanding these can be crucially important when trying to interrupt the chain of events that leads to persistence. ecological studies of wnv have shown how forest fragmentation and decreased biodiversity can alter transmission among avian hosts as well as to humans [106] . likewise, researchers have used satellite imagery to identify habitat characteristics that accurately predict the prevalence of sin nombre virus [107, 108] , a hantavirus that uses the deer mouse (peromyscus maniculatus) as a reservoir host and it occasionally infects and kills humans [107] . these studies epitomize the type of effort scientists will need to successfully fight viral pathogens in the future. however, piecing together emerging disease and conservation problems ex posto facto is only of limited value. increased pathogen surveillance and ecosystem process monitoring may provide the insight necessary to mitigate problems before they become serious human health or conservation concerns. this is especially the case for zoonotic viral pathogens where the reservoir hosts are known and a targeted approach is feasible. rodents rank as the number one reservoir of emerging and re-emerging zoonotic viruses [9] . conveniently, these small mammals also present a manageable system for studying disease dynamics [109] [110] [111] . individuals can be marked and sampled individually through time. their locations as well as their contacts with other individuals can be measured. as such, wild populations of rodents can be valuable as model disease systems to address relevant questions like: are there key hosts for transmission? how does prevalence vary seasonally or over time? what is the contact rate between the reservoir and humans? how do these pathogens flow through populations? the answers are of critical importance because they provide an indication of when and where there is increased risk of a zoonotic event whereby a human becomes infected, or when a species becomes at genuine risk of extinction. by monitoring and manipulating wild populations, one might also be able to identify factors that may increase a pathogens chance of emerging. for instance, what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission? and what are the relative roles of genetic relatedness and contact rate for transmission? long-term monitoring and surveillance in reservoirs will also enlighten us to the kind of aggregations and other heterogeneities that exist through time and that and can be exploited with efficient vaccination campaigns. we are experiencing a global increase in the rate of emerging viral zoonoses, which are primarily driven by anthropogenic activities such as land-use change, agricultural intensification, and driven by global travel and trade [112] . in order to adequately understand, predict and ultimately interrupt the processes by which zoonoses cross the species barrier from their natural reservoirs to humans, and then become established as human pathogens, comprehensive scientific studies that use the tools of ecology, virology, microbiology, and epidemiology are needed [113] . the study of disease ecology has become an established discipline with advances in both the formulation of new theory as well as the integration of molecular virological techniques that provide important information about epidemiology, ecology and viral evolution, all of which has been applied to both health and conservation [114] [115] [116] . because ecological systems are rife with heterogeneities and often have non-intuitive processes underlying their dynamics, it is critically important for scientists to use a comprehensive approach to understanding the population processes of an ecosystem before successful intervention strategies can be developed or implemented. admittedly this is a daunting task and it is often the case that scientists need to operate with less than complete information. where this is the case, a modeling approach is necessary to identify key processes that allow successful interventions. technological advances in molecular virology and genetics, as well as the expanded use of mathematical models in epidemiology and disease ecology have dramatically changed our ability to manage both conservation and health. finally, it is only with this type of interdisciplinary approach that considers free-ranging wildlife, domestic animals and humans as inextricable components of a single disease system, that progress can be made that will satisfy both conservation and public health needs. this is the essence of conservation medicine [117] and indeed we believe a -one health‖ approach to infectious disease is necessarily the way forward. supplemental movie (video s1). are we in the midst of the sixth mass extinction? a view from the world of amphibians how many species? philos the future of biodiversity the impact of infection and disease on animal populations: implications for conservation biology disease and conservation emerging viruses emerging infectious diseases of wildlife-threats to biodiversity and human health risk factors for human disease emergence host range and emerging and reemerging pathogens rates of evolutionary change in viruses: patterns and determinants 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low-coverage vaccination strategies for the conservation of endangered species an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle mosquito landing rates on nesting american robins (turdus migratorius). vector borne zoonotic dis vaccination of wildlife to control zoonotic disease: west nile virus as a case study transmission dynamics and prospects for the elimination of canine rabies global trends in emerging infectious diseases ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses land use and west nile virus seroprevalence in wild mammals satellite imagery characterizes local animal reservoir populations of sin nombre virus in the southwestern united states ecology of hantaviruses and their hosts in north america. vector borne zoonotic dis chain reactions linking acorns to gypsy moth outbreaks and lyme disease risk could parasites destabilize mouse populations? the potential role of pterygodermatites peromysci in the population dynamics of free-living mice, peromyscus leucopus predicting the emergence of human hantavirus disease using a combination of viral dynamics and rodent demographic patterns anthropogenic environmental change and the emergence of infectious diseases in wildlife collaborative research approaches to the role of wildlife in zoonotic disease emergence ecology of infectious diseases in natural populations the ecology of wildlife diseases conservation medicine and a new agenda for emerging diseases this work was supported in part by nih/nsf -ecology of infectious diseases‖ awards from the john e. fogarty international center (grant 3r01-tw005869-05s1) and an nih award (k08ai067549) from the national institute of allergy and infectious diseases. we thank c. j. wale, b. wale and h. ewing for their help in proofing the manuscript. key: cord-335567-ssnvr6nj authors: berry, michael; gamieldien, junaid; fielding, burtram c. title: identification of new respiratory viruses in the new millennium date: 2015-03-06 journal: viruses doi: 10.3390/v7030996 sha: doc_id: 335567 cord_uid: ssnvr6nj the rapid advancement of molecular tools in the past 15 years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. the inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. in 2001, this led to the discovery of human metapneumovirus (hmpv) and soon following that the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (hcov) nl63 and hcov-hku1. human bocavirus, with its four separate lineages, discovered in 2005, has been linked to acute respiratory tract infections and gastrointestinal complications. middle east respiratory syndrome coronavirus (mers-cov) represents the most recent outbreak of a completely novel respiratory virus, which occurred in saudi arabia in 2012 and presents a significant threat to human health. this review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since 2001. viral infections of the upper and lower respiratory tract are among the most common illness in humans. children and infants bear the major burden of infection, typically presenting with 5 to 6 episodes annually [1] . these infections are often associated with significant patient morbidity and related mortality. for this reason, urtis and lrtis represents the leading cause of death in children younger than five years of age worldwide [2, 3] ; this accounts for approximately 4 million deaths annually [4] . acute respiratory tract disease is the leading cause of hospitalization in children and febrile episodes in infants younger than three months of age [5, 6] . bacteria only represent approximately 10% of all upper respiratory tract infections with the subsequent 90% of infections caused by respiratory viruses [7] . despite the viral aetiological origin of most respiratory infections, antibiotics are often prescribed in the treatment of such diseases [8] , exacerbating antibiotic abuse. the morbidity and fiscal implications associated with respiratory infections are significant, with approximately 500 million cases reported in the united states alone each year with subsequent direct and indirect costs to the us economy estimated at $40 billion annually [2] . the burden of respiratory tract infections is increased in patients with chronic comorbidities or clinical risk factors including asthma [9] , chronic obstructive pulmonary disease (copd) [10] , young, elderly [11] and immunocompromised [12, 13] . the viruses primarily associated with upper respiratory tract infections commonly include rhinoviruses, enteroviruses, adenoviruses, parainfluenza viruses (piv), influenza viruses, respiratory syncytial viruses (rsv) and coronaviruses [3, 8, 14, 15] . in recent years six new human respiratory viruses have been reported including human metapneumovirus (hmpv) [16] , bocavirus and four new human coronaviruses including severe acute respiratory syndrome coronavirus (sars-cov), human coronavirus nl63 (hcov-nl63), hcov-hku1 and middle east respiratory syndrome coronavirus (mers-cov). this review will detail these newly discovered and emerging respiratory viruses. coronaviruses affect a diverse group of animal hosts, and cause a plethora of diseases in animals including progressive peritonitis, acute and chronic hepatitis, gastroenteritis, nephritis, and encephalitis [17] . in humans coronavirus infection results in respiratory tract complications with varying degree of severity and have been associated with gastroenteritis. four human coronaviruses (hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1) are endemic in the human population and are mainly associated with mild, self-limiting respiratory illnesses. another two human coronaviruses, namely sars-cov and mers-cov cause severe respiratory syndromes and present a significant threat with their high fatality rates. the first human coronaviruses were identified in the 1960s by tyrell and bynoe who passaged a virus, named b814, in human embryonic tracheal organ cultures. when this virus was inoculated intranasally into human volunteers, common cold-like symptoms were produced [18, 19] . hamre and procknow (1966) later isolated a virus, which they were able to grow in tissue culture, from subjects presenting with symptoms of the common cold. the virus was later named human coronavirus 229e (hcov-229e) [20] . seroepidemiological studies have shown that 40% of wild animal traders and 20% of people responsible for the slaughtering of animals in the region where human sars was thought to originate, were seropositive for sars-cov, although all cases were asymptomatic. this indicates that these people were previously exposed to a sars-like-cov, which resulted in asymptomatic infection [38] . sars presents as an atypical pneumonia [42, 43] , with pneumocytes being the primary target of infection. infection results in haemorrhagic inflammation in most pulmonary alveoli with alveolar thickening, diffuse alveolar damage, desquamation of pneumocytes, formation of hyaline membranes and multinucleated pneumocytes with capillary engorgement and microthrombosis [44] . approximately 60% of patients deteriorated in the second week of infection, presenting with persistent fever, dyspnoea and oxygen desaturation [29] . approximately 20%-30% of patients were subsequently admitted to intensive care, where mechanical ventilation was necessary [45] . a surprising finding with the sars outbreak was that it was not as great a threat to infants and children [33, 46] . clinical presentation was less severe in infants and no children aged between 1 and 12 required intensive care or mechanical ventilation [47, 48] . this is in sharp contrast to the age related burden of other respiratory infections and the underlying biological mechanism remains unclear [49] . a family of viruses that were previously understood to cause mild, self-limiting upper respiratory tract infections was showcased by the sars-cov outbreak to be a significant threat to global public health. the economic losses brought on by the sars pandemic was estimated to be in the region 40 billion dollars [50] with hong kong bearing a significant proportion of the losses. tourism, entertainment and restaurant industries in the area recorded up to 80% loss in business. the pressure on healthcare facilities in affected areas was substantially exacerbated by the spread to healthcare workers. several hospitals were forced to close to new admissions as large numbers of staff became infected with sars-cov. to view this as an isolated incidence would be naï ve and the potential for the emergence and re-emergence of new and existing infectious agents poses a probable risk. understanding the sars-cov outbreak has provided immense knowledge and an excellent model to replicate in the event of further outbreaks of communicable diseases. in 2003 a 7 month old child presenting with bronchiolitis and conjunctivitis was screened for several respiratory viruses to identify the causative agent, with all diagnostics yielding negative results. the group led by lia van der hoek then used a modified cdna amplified restriction fragment-length polymorphism (cdna-aflp) technique (virus-discovery-cdna-aflp or vidisca), to identify the causative agent. briefly, the technique utilizes reverse transcription-pcr of viral rna with subsequent restriction digest of the cdna using frequently cutting restriction enzymes. since the restriction sites are selected and therefore known, the resultant "sticky ends" can be ligated into anchors for amplification and sequencing with specific primers. the results showed highest sequence similarities with known coronaviruses, but with significant sequence divergence indicating the discovery of a new coronavirus species, later named human coronavirus nl63 [51] . at about the same time, two other independent groups identified essentially the same virus [52, 53] . shortly after the van der hoek paper [51] , a novel coronavirus that replicated efficiently in tertiary monkey kidney and vero cells, was retrospectively isolated from a nose swab sample collected in 1988 from an 8 month-old boy presenting with pneumonia. this virus was reported to be similar to hcov-nl63 and named hcov-nl [52] . in 2005, also reported the identification of a novel coronavirus isolated in new haven, connecticut, which was named hcov-nh. this novel virus was identified by pcr which was adapted to amplify a conserved region within the replicase 1a or pol gene [53] . subsequent sequence analysis showed that these three viruses were essentially the same virus or variants thereof [53, 54] . it is obvious that hcov-nl63 has been circulating in the human population since before 1988 [52] . in fact, molecular clock analyses have shown that hcov-nl63 and hcov-229e diverged from a most recent common ancestor, in a zoonotic event, approximately 563 to 822 years ago [55] . hcov-nl63 later diverged into two lineages with subsequent recombination of the two lineages during co-infection. this frequent recombination has given hcov-nl63 a mosaic structured genome with multiple recombination sites [56] . a 71 year old male patient from china was admitted to hospital with pneumonia in january 2004. viral cultures, rt-pcr and direct antigen detection from nasopharyngeal aspirate were all negative for respiratory viruses. a pan-coronavirus rt-pcr targeting a conserved region of the pol gene confirmed the presence of a coronavirus however attempts to culture the causative agent were all unsuccessful. sequencing the gene segment amplified by the pan-coronavirus assay indicated a high homology to other viruses of the βcov genus including hcov-oc43 but of novel origin. the human coronavirus, termed hcov-hku1, was later isolated from another female patient. efforts to culture the virus had posed complicated and the complete genome was isolated, amplified and sequenced directly from rna extracted from a nasopharyngeal aspirate [57] . successful propagation of the virus was achieved recently in human ciliated airway epithelial cells [58] but culturing of hcov-hku1 still remains a daunting task. the presence of the he gene further characterizes hcov-hku1 as belonging to the βcov genus. since the discovery of hcov-hku1, its prevalence has shown a global distribution and retrospective analysis on stored nasopharyngeal aspirates have confirmed its existence since 1995 [59] ; however, phylogenetic analysis suggests a much earlier divergence. june 2012 saw the most recent emergence of a completely novel strain of human coronavirus. a sputum sample was collected from a 60 year old male patient presenting with severe respiratory disease in the dr. soliman fakeeh hospital in jeddah, saudi arabia. viral assays frequently used could not identify any aetiological agent responsible for the disease. the sputum sample was sent to dr. ron fouchier at the erasmus medical college in rotterdam, netherlands, where the virus was identified as a novel coronavirus, provisionally termed hcov-emc (human coronavirus erasmus medical college). the patient later succumbed to the disease with acute pneumonia and subsequent renal failure [60] . a retrospective study further traced the virus back to april 2012 where an outbreak of pneumonia, resulting in two fatalities, occurred in health care workers in an intensive care unit in zarqa, jordan [61] . since the initial discovery, several new isolates were identified and described in scientific literature, databases and media under various names. this provoked the convening of the coronavirus study group to introduce a naming convention and avoid confusion within the research field, health care authorities, governments and general public. the name middle east respiratory syndrome coronavirus (mers-cov) was coined and has been widely accepted by the discoverers of the virus and pioneers of the field, the who and saudi ministry of health [61] . primary infections, to which all cases were linked directly or indirectly, occurred in middle eastern countries including saudi arabia, qatar, jordan, oman and united arab emirates and subsequently spread to united kingdom, tunisia, france, italy and germany with egypt and the united states recently reporting their first laboratory confirmed cases [62, 63] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [64] and only reported in close, sustained contact [65] such as within families [66] , healthcare workers [67] and as nosocomial transmission [68] , especially if the patient represents with comorbidities. there has been no evidence to suggest sustained community transmission [62] . the ever increasing case numbers and countries affected by the disease however suggests mers-cov does represent potential for widespread, global outbreak [69] . from the first reported case in june 2012, until 7 february 2014, a total of 182 cases had been reported with 79 fatalities. according to the latest figures available from the who, as of 11 june 2014, the virus had resulted in a total of 699 laboratory confirmed cases with a total of 209 fatalities [70] . these statistics indicate that within a 4 month period case numbers had more than trebled suggesting the virus is far from controlled. the fatality rate of 30% is also substantially increased in patients with comorbidities, with a large number of reported cases being identified in immunocompromised patients or those with underlying disease [68, 71] and a severe threat of nosocomial transmission has been demonstrated [72] . clinical presentation is similar to sars and includes a spectrum of respiratory diseases with the most common symptoms including cough, fever and gastrointestinal symptoms [69] before progressing to pneumonia [68] . acute respiratory distress syndrome (ards), renal failure, pericarditis and disseminated intravascular coagulation have also been reported [69] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [64] and only reported in close, sustained contact [65] such as within families [66] , healthcare workers [67] and as nosocomial transmission [68] , especially if the patient presents with comorbidities. there has been no evidence to suggest sustained community transmission [62] . the ever increasing case numbers and countries affected by the disease however suggests that mers-cov does represent potential for widespread, global outbreak [69] . the inefficient spread between infected patients strongly suggests zoonotic transmission, however over two years on from the initial discovery of mers-cov it is still unclear where it originated from and how it infects humans. during the outbreak of sars-cov the identification of the wet markets of china as the probable source greatly contributed to the control of the disease [38] . this highlights the importance in identifying the origins of disease outbreak. phylogenetic analysis places it in the same genus as sars-cov but within a new lineage, 2c. the closest detectable relatives suggests origins from bat coronavirus species with relative high sequence homology between bat-cov hku4 and hku5 in china [73] , vm314 in the netherlands [74] and a recently discovered isolate from south africa [75] . assuming that bats also form the immediate source is improbable and as with sars-cov an intermediate animal host species for transmission to humans is of greater likelihood [61] . high levels of neutralizing antibodies, viral rna and infectious viruses have been discovered in dromedary camels suggesting a potential for camels to form the source for human transmission [76] [77] [78] [79] . a recent study identified identical single nucleotide polymorphisms in a sequence of mers-cov isolated from an infected patient and camel, which was cared for by the patient. phylogenetic analysis of these two sequences supported the conclusion that transmission occurred between patient and camel, however the direction of transmission could not be confirmed [80] . it has since been proven that a patient who succumbed to mers-cov infection obtained the virus directly from his camel herd. the direction of transmission was confirmed as camel-to-human by serological analysis [81] . these facts strongly contribute to an increasingly popular hypothesis that mers-cov infections in humans are directly acquired from camels, however very few patients have had reported contact with camels. many residents of the arabian peninsula frequently consume unpasteurized camel milk and this has been suggested as a potential source as the virus has been detected and can remain viable for prolonged periods in milk [82, 83] . the who, saudi arabia and qatar have recently issued warnings and recommended against consuming unpasteurized camel milk and to be cautious when interacting with dromedary camels [84] . the outbreak of sars-cov in 2003 and mers-cov less than 10 years later highlights the significant threat of coronaviruses to humans and confirms that the sars outbreak was not an isolated incident. with the ever increasing diversity of animal coronavirus species, especially within bats, the likelihood of recombination leading to future outbreaks is high and the threat of potential pandemics is real as highly pathogenic coronaviruses continue to spill over from zoonotic sources into the human population. misdiagnosis of these outbreaks pose a further substantial threat to healthcare workers with nosocomial spread to other patients putting further pressure on an already strained healthcare system. understanding the dynamics and molecular characteristics of human coronaviruses currently in circulation and how they emerge, infect and cause disease, we can be better prepared for future pandemics allowing for improved response, management and treatment of related conditions. the clinical presentation of human coronaviruses clearly follows two distinct lines of progression. sars-cov and mers-cov present with severe respiratory complications and often multisystem involvement with renal failure and enteric symptoms. the high fatality rates associated with these infections is not reflected in the remaining four human coronaviruses, hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1. the clinical importance of sars-cov and mers-cov are evident and discussed in detail in previous sections allowing for the presentation of the involvement of the remaining non-severe human coronaviruses and there implications in human health. the clinical presentation of these four non-severe human coronaviruses is largely identical and indistinguishable symptomatically, commonly presenting with rhinorrhoea, sore throat, cough and fever [85, 86] . majority of infections are associated with self-limiting upper respiratory tract disease or "the common cold" but can also present with high morbidity outcomes of the lower respiratory tract including bronchiolitis, pneumonia, [87] [88] [89] , asthmatic exacerbations [9] acute exacerbations of chronic obstructive pulmonary disease (copd) [10] and croup in hcov-nl63 infected patients [90] . a report by esper et al. found a correlation between hcov-nl63 infections and kawasaki disease [91] , although other studies could not replicate the association [92] [93] [94] . febrile seizures have been reported for most human coronaviruses but the significance of hcov-hku1 is alarming with one study indicating that 50% of patients infected with hcov-hku1 experience febrile seizures [9] . human coronaviruses affect all age groups [85, 86] but elicit more serious disease in young, elderly and immunecompromized [87, 95, 96] , frequently resulting in hospitalization. reports on the prevalence of human coronaviruses and their association with upper and lower respiratory tract disease vary but range between 3.3% and 16% [85, 86, 95, 97] . over 70% of the general public has seroconverted towards all four non-severe human coronaviruses with primary infection shown to occur in childhood [98] and reinfection occurring throughout life [95] . given the high prevalence of respiratory infections, human coronaviruses represent a substantial disease burden which is exacerbated by the high implications of healthcare workers in coronavirus outbreaks [97] . high rates of co-infection with other respiratory viruses are commonly reported [85, 86, 95, 99] . viruses frequently associated with co-infection include enterovirus, rhinovirus and piv [100] however reports of co-infection with two human coronaviruses are limited. dijkman et al. recently demonstrated that hcov-oc43 and hcov-nl63 may elicit immunity that protects against hcov-hku1 and hcov-229e, respectively [101] . clinical progression and outcomes of disease in patients presenting with co-infection are however similar to patients presenting with mono-infection [85, 95] . there is also no substantial difference in coronaviral load between co-infected and mono-infected patients. no substantial difference in disease progression in co-infected versus mono-infected patients has been reported and therefore understood to have little impact; however, the role in facilitation of infection of one respiratory virus by another is still speculative [95] . all four human coronaviruses are endemic worldwide but the prevalence, regional distribution and pathogenicity of individual human coronaviruses is unclear and highly subjective on study conducted with parameters including population sampled, respiratory sample collected, sensitivity of diagnostic assay used, region where study was conducted and duration of the study playing a substantial role in epidemiological findings. a common finding in majority of studies conducted is the increased prevalence of human coronaviruses during winter months in temperate climates [95, 97] . however even this has shown to be geographically dependent with a spring/summer predominance in subtropical and tropical climates [9, 102] . biennial outbreaks are frequently reported [85, 95, 99, 100] for all strains of non-severe human coronaviruses. in 2001 a previously undiscovered virus was identified in 28 epidemiologically distinct patients in the netherlands. patient symptoms were similar to those infected with rsv and, several patients required hospitalization and mechanical ventilation. viral isolates were cultured in tertiary monkey kidney (tmk) cells and cytopathic effects caused by the virus were largely identical to those caused by rsv. electron microscopy of infected cell supernatants revealed paramyxovirus-like particles; however, rt-pcr assays to detect known paramyxoviruses were all negative. the low stringency of the assays used indicated a currently unknown, genetically distinct virus. a rap-pcr assay was then utilized to obtain sequence information of the unknown virus and fragments amplified by the rap-pcr allowed for further sequencing of the 3'-end of the genome. based on the sequence homology and gene organization, the unidentified virus displayed closest homology with avian pneumovirus, but to be a tentative new member of the metapneumovirus genus and the first virus in the genus to infect humans, provisionally termed human metapneumovirus (hmpv) [16] . symptomatic differentiation between hmpv and other respiratory viruses cannot be made as there is a significant overlap in clinical presentation [103, 104] . the most common presentation of hmpv in children includes complications of the upper respiratory tract with rhinorrhoea, cough and fever [105] . acute otitis media is also frequently reported [106, 107] and conjunctivitis, rash, diarrhea and vomiting are reported but infrequently [103] . bronchiolitis, pneumonia, croup and asthmatic exacerbations are the most frequently associated lower respiratory tract complications [108] and viral load is directly associated with disease severity [109] . hmpv infection in the young and elderly frequently requires hospitalization and fatalities have been reported in the elderly [110, 111] . an increased morbidity in elderly patients with a delayed clearance of symptoms has been reported and is likely related to the age related impairment of the innate and adaptive immunity [103] or an over stimulated immune response leading to inflammation [112] . elderly patients requiring hospitalization most frequently present with acute bronchitis, copd exacerbations, pneumonia and congestive heart failure [113] . in healthy adults asymptomatic infections or cold-and flu-like symptoms are the most prevalent presentation [114] . the pathogenesis of hmpv infection is strongly affected by bacterial coinfections with pneumococcus. one study has shown that administration of a conjugate pneumococcal vaccine is sufficient to reduce the incidence of hmpv infection of the lower respiratory tract and the incidence of clinical pneumonia in both hiv positive and negative patients [115] . these finding suggest that the incidence of hospitalizations in hmpv infections may be decreased by vaccination with a conjugate pneumococcal vaccine. another case report of severe respiratory failure was found to be caused by coinfection with hmpv and streptococcus pneumonia in a 64 year old patient [116] . both in vitro and in vivo studies have shown that infection with hmpv facilitates adhesion of pneumococcal bacteria, which may provide an explanation for the coinfection with pneumococcal strains and hmpv [117] . viral coinfections between hmpv and rsv have been reported, but remain a contentious issue. the typical seasonal overlap of the two viruses has been suggested to promote viral coinfection. one study reported a 10-fold increase in risk of admission to an intensive care unit in pediatric patients coinfected with rsv and hmpv and associated the dual infection as capable of augmenting severe bronchiolitis [118] . other studies do not support this finding and further report a decreased correlation between hmpv-rsv coinfections and hospitalization and additionally lists dual infection, along with breastfeeding, as having protective effects [119] . although hmpv was only discovered in 2001, it has been shown by phylogenetic analysis to have been in existence for approximately 50 years [120, 121] . soon after the discovery of hmpv, it was evident that two lineages, a and b, existed. these two lineages were further subdivided into two sublineages per lineage, a1-a2 and b1-b2 [16] . a recent report analyzing sequence divergence of the attachment and fusion surface glycoproteins indicates the presence of five sublineages, namely a1, a2a, a2b, b1 and b2 [122] . from long term retrospective studies it was evident that these lineages are not restricted to certain locations or times and that multiple lineages can exist in the same location and period [108, 123] . it has also become evident that old sublineages may be replaced by new variants [103] . disease progression or varying clinical outcomes related to different lineages of hmpv has become a contentious issue. several studies have reported that lineage a presents with more severe clinical outcomes [124] [125] [126] where the same is reported for lineage b by other groups [127, 128] . it has been further reported that there is no difference in disease outcomes related to the two lineages [108, 129, 130] . between 7% and 19% of all cases of respiratory infections in children are caused by hmpv, in both hospitalized and outpatients [108, 131, 132] and has been reported to be the second most frequently identified virus in respiratory tract infections [133] . extrapolation of consensus data suggests a total of 20 000 hospital and one million clinic visits annually in the us among children younger than 5 [131] . children hospitalized with hmpv infections are also more likely to present with pneumonia or asthma and required longer stay in intensive care units with supplemental oxygen, when compared to other respiratory viruses [131] . seroprevalence studies indicates that 100% of young adults are seropositive for hmpv with stable neutralizing titres, which further suggests that reinfection occurs throughout life [16] , with a potential for genetic variation between clades promoting reinfection [108] . hmpv has a worldwide distribution and affects all age groups but predominantly affects young, elderly and immunocompromised patients [111] , with children younger than five years of age being most susceptible to infection [134] . children and adults with underlying or chronic conditions such as asthma, chronic lung disease, congenital heart disease, cancer or copd are more likely to be hospitalized with hmpv infection [131] . infection with hmpv occurs throughout the year but seasonal prevalence in late winter and spring has been observed and coincides with the peak of rsv infection [131, [135] [136] [137] . the first human bocavirus (hbov) was discovered in 2005 from nasopharyngeal aspirates of 282 patients with unresolved lower respiratory tract infections in sweden. researchers utilized a novel technique which included steps of dnase treatment to exclude contaminating, or non-viral, nucleic acids followed by pcr amplification by nonspecific primers. the pcr-products were subsequently cloned with large-scale sequencing of the clones. bioinformatic analysis of generated sequence data yielded the discovery of a new parvovirus with a high homology to bovine and canine minute parvoviruses. the genus name bocavirus was in fact derived from the species infected by the known virus strains, namely bovine and canine. the new virus was named hbov1 and was the first virus to be discovered by molecular virus screening [138] . three additional species of hbov were later discovered in 2010 and added to the genus; these were named hbov2, hbov3 and hbov4 [139] [140] [141] . hbov1 is a respiratory pathogen affecting all regions of the globe and is associated with approximately 2%-19% of all upper and lower respiratory tract conditions [142] [143] [144] . hbov1 productively infects human airway epithelium cell cultures and leads to damage of airway epithelial cells [145] [146] [147] , which supports clinical observations that infection does result in respiratory disease. in contrast, hbov2-4, are found in the gastrointestinal tract and hbov2, and possibly hbov3, are associated with gastroenteritis [139, [148] [149] [150] . interestingly, hbov2 is the only enteric bocavirus to be isolated from nasopharygeal aspirates and may, therefore, also be associated with respiratory disease [151, 152] . hbov1 is detected in all age groups, but predominantly in young children between the ages of 6-24 months [143, 153, 154] and is rarely detected in adults [155] [156] [157] [158] [159] . transmission and infection occurs throughout the year, but predominantly during winter and spring months [158, [160] [161] [162] . seroprevalence studies suggest that maternal antibodies, which provide protection, are present in infants younger than 2 months of age [163, 164] , after which seropositivity decreases with low levels of detection until 6-12 months. virtually 100% of children aged 6 are seroconverted for hbov1 and as reinfection occurs throughout life this remains into adulthood [143, [163] [164] [165] [166] . the presence of the three enteric bocaviruses does however complicate the findings of seroconversion as cross-reactivity does exist [166] . as with many respiratory viruses, clinical differentiation with hbov1 infection is not possible by symptomatic presentation [8] . common features of infection of the upper respiratory tract include common cold-like symptoms with cough, rhinorrhoea and acute otitis media [167] . infection of the lower respiratory tract in children is associated with pneumonia, acute wheezing, asthmatic exacerbations and bronchiolitis [160, 162, [168] [169] [170] , but life-threatening complications are rare with hbov1 infection [143] . although hbov1 has been isolated from stool samples, there is no statistical evidence to associate hbov1 with gastrointestinal disease [139] . hbov1 has not only been found in the upper and lower respiratory tract and gastrointestinal specimens, but also in urine samples, serum, saliva, and tonsils [143] . rather than having a role in disease pathogenesis, this viraemia and systemic spread may be a feature common to all parvoviruses as they require proliferating host cells for replication [138] . interestingly, hbov appears to be more than just a respiratory or gastrointestinal virus. in a recent study, hbov was identified in 18.3% of lung (n = 11/60) and 20.5% of colorectal (n = 9/44) tumors screened. unfortunately, the study did not investigate whether the hbov genomes were in fact incorporated into the host genome as reported for other known parvoviruses. therefore, based on their observations as well as previous studies on other parvoviruses, the authors speculate that hbov could contribute to the development of some lung and colorectal tumors. however, they do also acknowledge that these tumors could simply be providing an optimal environment for hbov replication and more conclusive studies are required to resolve this issue [171] . hbov1 has been associated with a prolonged period of persistence in the mucosa of the respiratory tract. this prolonged presence has possibly led to a high frequency of coinfection found with hbov1 infections of the both the upper and lower respiratory tract [142, 157, 158] . the high rate of detection of multiple respiratory viruses within up to 83% of respiratory specimens, and the presence of asymptomatic hbov1 infections, does complicate the determination of the actual pathogenic role of hbov [143] . high viral load is statistically associated with symptoms [172] and this may therefore be a better indication of coinfections which are related to disease severity or symptomatic presentation. it has been further suggested that patients presenting with viraemia are better candidates to assess the symptoms of disease when compared to investigations of respiratory secretions [172] . the effects and mechanisms of latency, persistence, reactivation and reinfection are however poorly understood and therefore its effects on coinfection and its contribution to active disease cannot be accurately stated [173] . the etiological agents of 12%-39% of lower respiratory tract infections still remain to be identified [138, 158] . these results may vary significantly depending on the sensitivity of the diagnostic assay used, respiratory site sampled and even geographical location of the study but it does suggest that many uncharacterized respiratory pathogens could still remain elusive awaiting discovery. the vast improvements in molecular techniques within the past decade have however led to the discovery of four previously circulating respiratory viruses and also the rapid characterization of two completely novel viruses, namely sars-cov and mers-cov. all these viruses have varying but significant impact on human health and the potential for outbreak of completely novel, emergent respiratory viruses, seen with sars and mers, poses their own unique threats. lessons learned from these viruses, and others currently in circulation, provide health care authorities and scientists with suitable expertise and knowledge to rapidly identify and combat novel respiratory viruses and as our techniques improve we will be in a position to characterize those viruses that are currently 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pickles, raymond; baric, ralph; garcia, j victor title: acute sars-cov-2 infection is highly cytopathic, elicits a robust innate immune response and is efficiently prevented by eidd-2801 date: 2020-09-24 journal: res sq doi: 10.21203/rs.3.rs-80404/v1 sha: doc_id: 348301 cord_uid: bk80pps9 all known recently emerged human coronaviruses likely originated in bats. here, we used a single experimental platform based on human lung-only mice (lom) to demonstrate efficient in vivo replication of all recently emerged human coronaviruses (sars-cov, mers-cov, sars-cov-2) and two highly relevant endogenous pre-pandemic sars-like bat coronaviruses. virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. our results indicate that bats harbor endogenous coronaviruses capable of direct transmission into humans. further detailed analysis of pandemic sars-cov-2 in vivo infection of lom human lung tissue showed predominant infection of human lung epithelial cells, including type ii pneumocytes present in alveoli and ciliated airway cells. acute sars-cov-2 infection was highly cytopathic and induced a robust and sustained type i interferon and inflammatory cytokine/chemokine response. finally, we evaluated a pre-exposure prophylaxis strategy for coronavirus infection. our results show that prophylactic administration of eidd-2801, an oral broad spectrum antiviral currently in phase ii clinical trials for the treatment of covid-19, dramatically prevented sars-cov-2 infection in vivo and thus has significant potential for the prevention and treatment of covid-19. the recently emerged human pandemic coronavirus severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the causative agent of coronavirus disease (covid)-19, has spread to six continents resulting in substantial morbidity and mortality worldwide 1 . bats serve as a natural reservoir for coronaviruses and are the presumed source of sars-cov-2 and the highly pathogenic human coronaviruses sars-cov and middle east respiratory syndrome (mers)-cov 2 . transmission of coronaviruses from bats to other species is well-documented and adaptation in an intermediary host can facilitate their transmission to humans 2 . while it is possible that sars-cov-2 was transmitted to humans via an intermediate host such as pangolins, phylogenic analysis indicates that the sars-cov-2 lineage has circulated in bats for decades and evolved in bats into a virus capable of replicating in human cells 3 . thus, bats are a potential reservoir for coronaviruses with human pandemic potential that can be directly transmitted to humans. given the repeated and accelerating emergence of highly pathogenic coronaviruses, it has become increasingly important to monitor and characterize coronaviruses circulating in bats and to identify the viral determinants of human infection, disease, and global spread as well as to develop effective therapeutic interventions. animal models are useful in studying highly pathogenic human coronaviruses, the emergence potential of zoonotic coronaviruses, and to evaluate novel inhibitors for their ability to control coronavirus infection [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . however, human coronaviruses do not replicate in mice without either extensive adaptation of the virus or genetic modi cation of the host by genetic editing of the receptor or by introducing the individual human receptor genes for each virus [4] [5] [6] [7] [8] [9] [10] [11] 14, 15 . although existing rodent models have made several important contributions to our understanding of coronavirus infection and pathogenesis, none of these models possess the diverse set of primary human cells present in the human lung that can serve as targets for viral infection 16 . here, we show that human lung-only mice (lom), immune de cient mice implanted with authentic human lung tissue 17 , allow for the in vivo study of all recently emerged human coronaviruses (sars-cov, mers-cov, sars-cov-2) in a single platform that permits direct comparison of experimental outcomes. using lom, we also established that bats harbor novel coronaviruses capable of e cient replication in human lungs without prior adaptation. in addition, we performed an in-depth in vivo analysis of acute sars-cov-2 infection in the human lung. our results revealed robust sars-cov-2 replication, pathogenesis and sustained activation of the innate host immune response. finally, we used this platform for the in vivo evaluation of eidd-2801, an orally administered broad spectrum antiviral currently in phase ii clinical trials for covid-19 treatment, to prevent sars-cov-2 infection. our results show that eidd-2801 e ciently prevented sars-cov-2 infection in vivo strongly supporting its progression in clinical development for covid-19. lom are constructed by subcutaneous implantation of a small piece of human lung tissue into the back of immune de cient mice (fig. 1a) . previously, we demonstrated that the human lung tissue expands to form a highly vascularized palpable implant 17 (fig. 1a) . these lung implants contain human broblast, epithelial, endothelial, and mesenchymal cells that form highly relevant lung structures including cartilaginous and non-cartilaginous bronchial airways lined with ciliated and non-ciliated epithelium, alveolar sac structures, and extensive vasculature 17 (extended data fig. 1a,b) . we also showed that the human lung tissue of loms supports replication of a diverse set of emerging and clinically relevant human pathogens including zika virus, human cytomegalovirus, respiratory syncytial virus and mers-cov 17 . recently emerged human coronaviruses have used at least two different receptors to gain entry into host cells, human angiotensin converting enzyme-2 (ace2) and dipeptidyl peptidase 4 (dpp4). sars-cov and sars-cov-2 use human ace2 as a receptor while mers-cov uses dpp4 18-22 . these differences in receptor usage in uence viral tropism, pathogenesis and disease progression 23 . infection of lom with mers-cov resulted in robust virus replication and infection of human epithelial, endothelial and mesenchymal cells in vivo 17 . these ndings were consistent with the broad cellular distribution of its receptor, dpp4 24 . here, we rst evaluated the potential of lom to serve as a single platform to study all known recently emerged human coronaviruses and the potential of endogenous bat coronaviruses for human emergence. we rst con rmed that ace2, the receptor for sars-cov and sars-cov-2 in human cells, was present on the surface of human epithelial cells (cytokeratin 19+) in the human lung tissues of lom (extended data fig. 1c,d) . next, lom were inoculated with recently emerged coronaviruses sars-cov, mers-cov, or sars-cov-2 (extended data table 1 ). lom supported replication of all these viruses in vivo. speci cally, sars-cov and sars-cov-2 infection resulted in mean virus titers of 1.76x10 8 and 2.42x10 7 pfu/g respectively at 2 days post-infection (fig. 1b) . viral nucleoprotein antigen was abundantly observed in the human lung tissues of sars-cov and sars-cov-2 infected lom (extended data fig. 2a ). consistent with our previous results 17 , we also observed robust replication of mers-cov in the human lung tissues of lom with mean titers of 4.79x10 8 pfu/g in lom human lung tissues at 2 days postinfection (fig. 1b) and abundant viral antigen (extended data fig. 2a) . pre-pandemic bat coronaviruses wiv1-cov and shc014-cov have high sequence homology to sars-cov, use ace2 to infect human cells, and grow modestly in primary human airway cultures on liquid interface 4, 5 . lom were inoculated with wiv1-cov or shc014-cov and virus titers in human lung tissues measured 2 days post-infection (extended data table 1 ). wiv1-cov and shc014-cov e ciently replicated in the human lung tissue of lom with mean titers of 1.58x10 7 and 1.48x10 7 pfu/g, respectively (fig. 1b) and viral antigen was readily detected in human lung tissues (extended data fig. 2b ). collectively, these results demonstrate that lom serve as single platform where all newly emerged coronaviruses sars-cov, mers-cov, and sars-cov-2 replicated e ciently in human lung tissue. importantly, the fact that sars-like bat coronaviruses wiv1-cov and shc014-cov also replicated e ciently in the lom platform indicates that bats harbor coronaviruses that are potentially capable of direct transmission to humans, thus bypassing the need for further adaptation in an intermediary host. given the state of the current covid-19 pandemic and the urgent need to develop therapeutic and preventative approaches to control and prevent infection, we evaluated replication of sars-cov-2 in lom in detail. human lung tissues of lom were inoculated with sars-cov-2 and titers of replication competent virus determined 2, 6, and 14 days post-exposure (fig. 1c , extended data table 2 ). high titers of replication competent virus were noted at all time points although they were highest 2 days postinfection (fig. 1d) . the distribution of virus-infected cells was determined with rnascope (viral rna) and immuno uorescence microscopy (viral nucleoprotein). virus infection was widely distributed throughout the tissue with large numbers of cells positive for viral rna (fig. 1e ) and nucleoprotein (fig. 1f ). costaining with a human cytokeratin 19 antibody demonstrated that sars-cov-2 predominantly infects human epithelial cells in the lung (fig. 1g ). viral antigen was not detected in human cd34 expressing (endothelial) cells, and only a few human vimentin expressing (mesenchymal) cells were positive for viral nucleoprotein (fig. 1g) . to identify the epithelial cell types in the lung tissue that are susceptible to sars-cov-2 infection, we further identi ed infected cells by assessing co-localization of viral nucleoprotein with antibodies against acetylated alpha-tubulin iv (ciliated cells), cc10 (club cells), ht1-56 (alveolar type i [at1] pneumocytes), and pro-sp-c (alveolar type ii [at2] pneumocytes) (fig. 1h) . we were able to clearly identify virus antigen in cells which expressed pro-sp-c or acetylated alpha-tubulin iv; we did not detect virus antigen in ht1-56 or cc10 positive cells (fig. 1h) . these results demonstrate that sars-cov-2 has limited tropism in the lung with at2 pneumocytes and ciliated airway epithelial cells being the predominant lung cells infected by virus. to evaluate the cytopathogenic effects of sars-cov-2 during acute infection of human lung tissue in lom, we used a combination of histological analysis and electron microscopy. histopathologic analysis revealed several features of early diffuse alveolar damage that have been described in lung tissues of covid-19 patients including the accumulation of proteinaceous exudate and brin in alveolar spaces, desquamation of pneumocytes, multi-nucleated cell formation, and the appearance of brin thrombi in small vessels (fig. 2 ) 25-27 . proteinaceous exudate, including large globules of protein material, was observed in alveolar spaces, which overlapped with areas of virus accumulation (fig. 2a,b) . as early as 2 days post-infection, desquamation of pneumocytes was also noted; there were a large number of virally infected cells fully detached or detaching from the alveolar basement membrane into the alveolar space ( fig. 2c,d) . infected multi-nucleated cells were also observed (fig. 2c) . while the formation of hyaline membranes was not noted, brin was detected in alveolar spaces (fig. 2e) . importantly, we observed multiple occluded vessels containing brin thrombi as reported in the lungs of covid-19 patients ( fig. 2f ,g) [25] [26] [27] . electron microscopy demonstrated the normal architecture and integrity of uninfected at2 pneumocytes that were present in human lung tissue obtained from lom two days post-infection ( fig. 2h ). in contrast, at2 cells containing virus particles in the same sample had swollen mitochondria with loss of matrix and cristae as well as rough endoplasmic reticula with distended cisternae, protein accumulation, and virus particles (fig. 2i) . degenerative sars-cov-2 infected at2 cells detached from the alveolar basal membrane could also be observed in the alveolar luminal space (fig. 2j ). higher magni cation revealed subcellular accumulation of virus containing vesicles indicative of virus replication and egress. virions with electron dense nucleocapsids and distinctive crown-like spikes were observed (fig. 2i,j) . consistent with previous reports in human airway epithelial cell cultures and portmortem lung samples 26,28 , virions produced by human lung cells were pleomorphic in size (69 to 112 nm) and shape. despite the extensive damage in icted in the lung tissue by the virus, the endothelium in the majority of blood vessels was intact with tight junctions, numerous pinocytotic vesicles, and normal mitochondria and endoplasmic reticulum (fig. 2k ,l). virions were not detected within endothelial cells in agreement with a lack of infection as per our immuno uorescence analysis ( fig. 1g and fig. 2k ,l). however, pleomorphic virions were present in capillary lumen surrounded by brillar protein deposits and cell debris (fig. 2k,l) . together, these results demonstrate that acute sars-cov-2 infection of lom closely resembles infection of human lung in humans and is highly cytopathic resulting in signi cant injury to the fragile alveolar lung structures. to determine the effect of sars-cov-2 infection on human gene transcription, we performed rnasequencing analysis of human lung tissues collected from animals 2, 6 and 14 days post-infection. abundant viral transcripts were detected in infected lung tissue, ranging from 0.55% to 3.6% of the total reads at 2 days post-infection (extended data table 3 ). viral transcripts were still abundant but lower at 6 days and 14 days post-infection (extended data 3). sequencing data was consistent with previously identi ed canonical sars-cov-2 transcripts 29 and con rmed maintenance of the furin cleavage site throughout the course of infection in vivo. analysis of human gene transcripts revealed 1,504 differentially expressed cellular genes between naïve and infected human lung tissue at 2 days postexposure, the peak of infection (fig. 3a , supplementary tables 1 and 2) (adjusted p value <0.05 after correcting for multiple testing). of these, 1,043 genes were up-regulated and 461 genes were down regulated in the infected human lung tissue relative to non-infected lung tissue (fig. 3a , supplementary tables 1 and 2) . notably, numerous interferon-stimulated genes (isgs) and in ammatory cytokine genes, including pro-in ammatory cytokines genes il6, cxcl8 (il-8), cxcl10 (ip-10), tnf, and ccl5 (rantes) were potently induced in infected lung tissue (supplementary tables 1 and 2) . we also observed dramatic upregulation of ifnb1 expression (>1,000 fold) at 2 days post-exposure, suggesting that this cytokine plays a key role in the antiviral response to sars-cov-2 (supplementary tables 1 and 2 ). gene set enrichment analysis (gsea) showed over 840 gene pathways signi cantly upregulated (p<0.05) including response to type 1 interferon (p=0.0011), response to virus (p=0.0010), innate immune response (p=0.0010), cytokine mediated signaling (p=0.0010), cytokine production (p=0.0010), response to stress (p=0.0010), in ammatory response, (p=0.0010), nik nf-kb signaling (p=0.0011), acute in ammatory response (p=0.0035), regulation of cell death (p=0.0030), and coagulation pathways (p=0.0453) (fig. 3b ). complement activation, which contributes to sars-cov pathogenesis in mouse models 14 , was also increased (p=0.0470) (fig. 3b) . importantly, analysis of host gene expression at later time points demonstrated a sustained upregulation of antiviral and in ammatory genes that in some instances (e.g. isg15, ifitm1, tnf, cxcl9) persisted for up to 14 days post-infection (last time analyzed) (fig. 3c ,d, extended data table 4, supplementary tables 1 and 2 ). these results demonstrate that acute sars-cov-2 infection causes a potent and sustained upregulation of innate immune responses in virus-infected human lung tissue. currently, there is no vaccine to prevent sars-cov-2 infection or effective therapy to treat patients with covid-19. the ribonucleoside analog β-d-n 4 -hydroxycytidine (nhc) has been shown to broadly inhibit coronavirus infection in vitro in human airway epithelial cell cultures, with potent activity against sars-cov-2 as well as sars-cov, mers-cov, and bat sars-like and mers-like coronaviruses 7 . we therefore tested the ability of prophylactic eidd-2801 (also known as mk-4482), the oral pro-drug of nhc, to inhibit sars-cov-2 replication in vivo. for this purpose, lom were administered eidd-2801 starting 12 h prior to sars-cov-2 exposure and every 12 h thereafter (fig. 4a , extended data table 5 ). our results show that eidd-2801 had a dramatic effect on virus infection, signi cantly reducing the number of infectious particles in the human lung tissue of eidd-2801 treated animals in two independent experiments (fig. 4b ,c) by over 100,000 fold (fig. 4c,d) . furthermore, in contrast to eidd-2801 treated mice, abundant cell debris and nucleoprotein positive cells could be readily observed in the alveolar lumen of vehicle control treated mice consistent with the extensive pathogenic effects in icted on the lung by sars-cov-2 (fig.4e,f) . these results demonstrate that prophylactic administration eidd-2801 is highly effective at preventing sars-cov-2 infection and pathogenesis in vivo. the zoonotic transmission of the pathogenic sars-cov-2 resulted in a pandemic that has in icted signi cant morbidity and mortality as well as dire world-wide economic and social consequences. herein, we describe a unique model for the in vivo study of human coronavirus infection particularly well suited to model distal human lung virus infection. our results demonstrate replication of all known recently emerged human coronaviruses in lom. importantly, our results demonstrate that wiv1-cov and shc014-cov, two pre-pandemic endogenous bat viruses can replicate relatively e ciently in loms suggesting that coronaviruses circulating in bats have future pandemic potential without the need for further adaptation to the human host. we also show that interestingly, an analysis of post-mortem covid-19 patient lungs 12 did not reveal increased ifnb1 expression and it has been shown in vitro that its expression is blocked during sars-cov infection 33, 34 . these results suggest that in human lung tissue ifnb1 gene expression is induced during the acute phase of sars-cov-2 infection. we also observed increased expression of several human cytokine genes in lom infected with sars-cov-2. a substantial number of these cytokines were also increased in the serum of covid-19 patients and post-mortem lung tissue samples further establishing the similarities between lom and human infection with sars-cov-2 12, 35 . currently, there is no vaccine to prevent or therapeutics to treat covid-19. pre-exposure prophylaxis approaches to infectious diseases have proven to be highly e cacious and can contribute to reduce the risk of infection. the continued global spread of the virus and its associated morbidity and mortality are strong incentives to the development of prevention strategies for covid-19. in this regard, nhc was shown to have broad activity against human and bat coronavirus infection in vitro 7 . in addition, prophylactic and therapeutic administration of its oral pro-drug eidd-2801 reduced sars-cov and mers-cov replication and pathogenesis in mice 7 . here we show that prophylactic administration of eidd-2801 e ciently prevents sars-cov-2 infection in vivo highlighting its potential utility as an effective prophylactic agent against sars-cov-2 and other past and future zoonotic coronaviruses. there are some limitations of our study including the fact that lom do not possess the human nasal airway structures that are thought to be early sites of sars-cov-2 replication in humans 36 . since lom do not have an autologous human adaptive immune system they re ect the direct effect of viruses on their targets and bystander cells as well as their innate immune response to infection. collectively, our results indicate that lom re ect the pathogenic effects in icted by sars-cov-2 on the human lung and demonstrate their utility as a single in vivo platform to evaluate and compare the replication and pathogenesis of past, present, and future pre-emergent, epidemic, and pandemic coronaviruses accelerating the development and testing of pre-exposure prophylaxis agents such as eidd-2801. immunohistochemistry was performed as previously described 17 . brie y, xed (10% formalin) human lung tissues collected from coronavirus-infected lom were para n embedded and sectioned (5 um human lung tissues collected from mice were xed in 10% formalin and para n embedded. immuno uoresence staining of 5 um tissue sections was performed as previously described 17 . brie y, following depara nization and antigen retrieval (diva decloaker), tissue sections were incubated with a 10% normal donkey serum solution with 0.1% triton x-100 in 1x pbs to block non-speci c binding. tissue sections were then incubated overnight with primary antibodies at 4°c followed by incubation with uorescent conjugated secondary antibodies (supplementary table 7 ). primary antibodies were directed against sars nucleoprotein and human cytokeratin 19, cd34, vimentin, acetylated alpha-tubulin iv, cc10, ht1-56, and pro-sp-c (supplementary table 7 ). background auto uorescence was then quenched using a 0.1% sudan black b solution in 80% ethanol prior to staining with dapi. slides were mounted and then imaged using an olympus bx61 upright wide-eld microscope using volocity software (version 6.3) with a hamamatsu orca rc camera. appropriate negative controls without primary antibodies were also imaged using the same exposure time as matching stained sections. whole image contrast, brightness, and pseudocoloring were adjusted using imagej/fiji (version 2.0.0-rc-69/1.51w) and adobe photoshop tissue pieces were washed in deionized water, dehydrated in ethanol, and placed through two exchanges of propylene oxide before in ltration and embedment in polybed 812 epoxy resin (polysciences an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2 a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic origin and evolution of pathogenic coronaviruses an interactive web-based dashboard to track covid-19 in real time precision mouse models with expanded tropism for human pathogens resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function a mouse-adapted model of sars-cov-2 to test covid-19 countermeasures complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate imbalanced host response to sars-cov-2 drives development of covid-19 lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus the pathogenicity of sars-cov-2 in hace2 transgenic mice a mouse model for mers coronavirus-induced acute respiratory distress syndrome a novel coronavirus from patients with pneumonia in china pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer dysregulation of immune response in patients with covid-19 in wuhan sars-cov-2 reverse genetics reveals a variable infection gradient in the respiratory tract viruses were directly injected into the human lung tissue of loms and lung tissue collected either 2, 6, or 14 days post-exposure for virus titer determination and/or analysis by histology, electron microscopy, or rna-seq. lung tissue (advanced bioscience resources) subcutaneously into the upper and lower back of male and female 12-21 week old nod.cg-prkdc scid ll2rg tm1wjl /szj mice [nsg mice; the jackson laboratory] as previously described 17 . engraftment of lung tissue was assessed by palpation and by 8 weeks post-surgery animals were ready for experimentation. mice were housed and maintained by the division of comparative medicine at the university of north carolina-chapel hill shc014-cov, and wiv1-cov were derived from infectious virus clones and were prepared and titered on vero e6 (sars-cov, shc014-cov, and wiv1-cov) or vero ccl81 cells (mers-cov) (american type culture collection) as previously described 4,5,9,17 . a clinical isolate of sars-cov-2 (2019-ncov/usa-wa1/2020) was obtained from the reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus galloylglucoses of low molecular weight as mordant in electron microscopy. i. procedure, and evidence for mordanting effect star: ultrafast universal rna-seq aligner salmon provides fast and bias-aware quanti cation of transcript expression positive cells, brown) of human lung tissue of lom administered eidd-2801 (n=8 analyzed) or control vehicle (ctrl, n=8 analyzed) at 2 days post-exposure (scale bars acknowledgements: we thank current and past members of the garcia laboratory for technical assistance. the authors also thank technicians at the unc animal histopathology and laboratory animal medicine core and division of comparative medicine. we also thank the unc school of medicine bioinformatics and analytics research collaborative (barc) for providing technical support and k. author contributions: aw, cej, wy, mk, and cd constructed lom. aw contributed to experimental design, data interpretation, data presentation, and manuscript writing, coordinated the study, and the preparation of the manuscript. leg performed the virus inoculations, animal necropsies, virus tittering, processing of lung tissues for rna extraction, and contributed to the experimental design, planning, data analysis, data interpretation, and manuscript writing. cej performed immuno uorescence and h&e analyses, wy performed immunohistochemistry and h&e analyses, and mk performed the in-situ hybridization analysis of lom human lung tissues. khd, as, srl, and kg assisted with the in vivo experiments with coronavirus-infected loms. vjm in conjunction with kkw performed the electron microscopy analysis.fba assisted with the pathohistological analysis. hl, hmk, tz, pog, performed the rna-sequencing analysis. epb and cdj contributed to design of rna-sequencing experiments. rjp assisted with the immuno uorescence analysis and contributed to experimental design, data interpretation, data presentation, and manuscript writing. rsb conceived and designed experiments and contributed to data interpretation and manuscript writing. jvg conceived, designed and coordinated the study, conceived and designed experiments, and contributed to data interpretation, data presentation, and manuscript preparation.competing interests: the authors have no competing interests. supplementary information is available for this paper at correspondence: correspondence to j. victor garcia. further information on research design is available in the nature research reporting summary linked to this paper.data availability: gene-expression data are available at the gene expression omnibus (geo) repository (accession: gse155286). all other data is available in the manuscript or the supplementary materials. key: cord-339382-ii4xurmr authors: bachofen, claudia title: selected viruses detected on and in our food date: 2018-03-21 journal: curr clin microbiol rep doi: 10.1007/s40588-018-0087-9 sha: doc_id: 339382 cord_uid: ii4xurmr purpose of review: the purpose of this review is to provide an update on recent literature and findings concerning selected foodborne viruses. two groups of viruses were selected: (a) the most important viruses contaminating food, based on numbers of publications in the last 5 years and (b) viruses infecting sources of food that might have an impact on human health. recent findings: important foodborne viruses such as norovirus, hepatitis a and rotavirus are usually “only” contaminating food and are detected on the surface of foodstuffs. however, they are threats to human public health and make up for the majority of cases. in contrast, the meaning of viruses born from within the food such as natural animal and plant viruses is still in many cases unknown. an exception is hepatitis e virus that is endemic in pigs, transmitted via pork meat and is recognised as an emerging zoonosis in industrialised countries. summary: even though the clinical meaning of “new” foodborne viruses, often detected by next generation sequencing, still needs clarification, the method has great potential to enhance surveillance and detection particularly in view of an increasingly globalised food trade. every form of live harbours its own range of viruses [1] . no wonder, they are also present on and in our food, being it of animal or vegetable origin, and are consumed on a regular basis [2] . considering this fact, only relatively few outbreaks or cases of disease due to foodborne viruses are reported-in contrast to bacterial infections [3• ]. this may be largely due to inability of these viruses to infect humans and/or inactivation during food processing. however, there are also several reasons that hamper recognition and reporting. foodborne viruses are highly diverse but the clinical signs they cause are usually not specific for the single virus and often rather general such as diarrhoea and malaise making diagnosis challenging and tedious. in addition, in case of human-to-human transmission after uptake of the virus, the initial foodborne origin may not be recognisable anymore if it is not extremely obvious. furthermore, lacking awareness of clinicians [4] and a limitation of concerted surveillance programmes for foodborne viruses may contribute to underreporting [5] . interestingly, the most important foodborne viruses regarding case numbers and economic impact are not really borne from within sources of food but are contaminating the surface of foodstuffs. examples are large outbreaks of hepatitis a from frozen berries or norovirus present on salad and vegetables [6•, 7] . often, the people handling and processing food as well as contaminated water play a key role in transmitting viruses onto food [8] . on the other hand, our foodstuffs originate from living organisms that carry their own set of viruses. hence, there is also a spectrum of viruses present within our food. the role of these viruses on our health is less studied and many viruses have only recently been detected by next generation sequencing (ngs). new diagnostic methods may improve detection of foodborne viruses dramatically, allowing for more efficient surveillance programmes and enhancing our knowledge on the importance of viruses in food [9•] . in contrast to traditional diagnostic methods, metagenomic approaches by ngs are untargeted and require no specific knowledge of the viral genome. for bacteria, conserved genetic markers such as 16s rrna allow for amplicon sequencing and subsequent taxonomic analysis resulting in an efficient representation of the bacterial abundance in a sample [10] . since there are no conserved genetic markers present in different virus families, metagenomic analysis of viral populations is based on untargeted shotgun sequencing. basically, all nucleic acid present in a sample is sequenced concurrently and comparison of the resulting nucleotide or deduced protein sequence to existing databases allows identification of all viruses that are somewhat similar to existing database entries. thanks to ngs, the range of foodborne viruses detected on and in food is constantly increasing. however, a fully comprehensive overview is beyond the scope of this article. therefore, a selection had to be made. due to the lack of systematic surveillance on most foodborne viruses, it was not possible to use case numbers as selective parameter. therefore, the number of pubmed entries in the last 5 years was taken as selective criterium (status on the 7th december 2017). the classics-viruses contaminating food (table 1) norovirus of the 558 articles matching to the search terms "foodborne" and "virus", 272 comply with "foodborne" and "norovirus". undoubtedly, norovirus (nov) ranks top among the most important causes of acute gastroenteritis in humans worldwide. impressive annual numbers of up to 21 million illnesses, 71,000 hospitalisation and 800,000 deaths that are accounted to nov in the usa alone show the importance of this virus for human public health [11•] . norovirus is a genus within the family of caliciviridae. virus particles are 27-37 nm in diameter and unenveloped which accounts for the high tenacity and resistance to disinfection. the genome is a single-stranded positive-sensed rna of 7.5-7.7 kb length and contains three open reading frames (orf) [12] . propagation of nov in cell culture is notoriously difficult. successful cultivation requires for example the addition of enteric bacteria expressing strainsuitable histo-blood group antigen (hbga) [13] or stem cellderived, nontransformed human intestinal enteroid monolayer cultures with addition of bile [14] . due to these difficulties, the recently published iso standard method for nov in food is based on quantitative rt-pcr [15] . beside of humans, several mammal species were shown to harbour noroviruses, such as pigs, cattle, rodents, dogs, cats, bats and even sea mammals such as harbour porpoises and sea lions [16] . while nov are mostly species-specific, human noroviruses were detected in pigs and cattle in canada [17] and some porcine nov isolates are genetically closely related to human nov [18] . zoonotic transmission may hence be possible. however, due to the high contagiosity, the most important mode of transmission is directly from human-to-human or indirectly via the contaminated environment. particularly vulnerable are people on cruise ships, schools and other situations of dense clustering, even if hygiene standards are high. based on genotype profiles, only 14% of all nov outbreaks seem to be attributed to contaminated food [19] . the most relevant source of food contamination with nov seems to be infected humans handling food rather than environmental contamination [20, 21] . however, outbreaks due to contaminated fruit (e.g. raspberries and strawberries [22, 23] ), leafy vegetables [24] and particularly oysters [25] show that the combination of faecally contaminated water and uncooked foods is particularly dangerous. thanks to molecular tracing of outbreaks and the acquired knowledge on sources and ways of transmission measures such as efficient disinfection and water treatment are increasingly taken to reduce the risk of foodborne nov infections [26] . the second most frequently reported foodborne virus in pubmed is hepatitis a virus (95 reports in the last 5 years). however, in contrast to nov infection, hepatitis a (hav) is a vaccinable disease. hav is a member of the family picornaviridae and belongs to the genus hepatovirus. the virus particles come in two versions: naked, unenveloped icosahedral virions of 27 nm diameter that are shed in faeces and pseudo-enveloped virions that are found in the blood of infected people and in cell cultures [27] . the who estimates that hepatitis a virus (hav) accounted for 14 million foodborne illnesses and 27.731 death in 2010 [28••] . however, the prevalence and incidence vary significantly around the world. based on the level of anti-hav antibodies, endemicity is grouped into low (< 15%), intermediate (15-50%) and high (> 50%). particularly high endemicity is observed in sub-saharan africa and india, while western europe, usa and australia have low levels [29] . however, the disease burden in high endemicity regions is comparably low [30] . this "paradox of hepatitis a risk" is due to the fact that in endemic areas children are infected at early age, e.g. in sub-saharan africa, 90% of the 10-year-olds are antibody positive [31] but the infection in young children is often asymptomatic or atypical and triggers a robust immunity [32] . in contrast, in regions with low endemicity, an increase in more severe clinical outbreaks can be observed due to a high degree of susceptible, non-vaccinated and older people. population movements and globalised markets play an important additional role [32] . on the third place of the most frequently reported foodborne viruses is hepatitis e virus (hev) with 65 publications. hev has gained a lot of attention in recent years. hepatitis e may clinically be indistinguishable from hepatitis a but hepatitis e virus (hev) is taxonomically unrelated to hav. hev is the most important member of the family hepeviridae and the genus orthohepevirus. interestingly, similar to hav, hepatitis e virus particles are unenveloped when excreted in faeces but pseudo-enveloped in blood and cell culture [33] . hev viruses infecting humans belong to the species orthohepevirus a, whereas b, c and d infect birds, rodents and bats respectively [34] . hepatitis e viruses of the orthohepevirus a species are further divided into the genotypes 1-8. only the genotypes 1 and 2 are considered primary human pathogens that are transmitted by faecal-oral route. the genotypes 3 and 4 that are recognised as important zoonotic viruses are discussed in the second part of the review. the unenveloped viruses show high tenacity and can be detected in sewage and waste water [35] . even though the main route of transmission is considered direct contact or faecal contamination of drinking water, hev-1 and 2 strains may also be foodborne pathogens in developing countries with limited access to sanitation, hygiene and health systems. while the infections are usually self-limiting and often subclinical in immunocompetent people, these figures may rise to 10-30% in pregnant women and immunocompromised individuals [36] . insufficient immune response to the infection may lead to chronic disease and virus shedding [37] . however, in western europe, infection with hev-1 or 2 is usually considered an imported or travel disease [36] . the fourth foodborne virus to be mentioned (36 entries) is rotavirus. named after the wheel-like appearance they show on the electron microscope, rotaviruses (rv) are particularly dangerous for young animals and humans. the most important rv infecting mammals is the serogroup a. the who estimates that globally 215,000 children under 5 years died in 2013 due to rotavirus infection; nearly half of them in india, nigeria, pakistan and the democratic republic of the congo [38] . however, this figure was up to 528.000 in the year 2000. a vaccine against rotavirus is on the market since 2006 and has significantly reduced the death rates due to rotavirus in children, e.g. by 65% in mexico [39] . rotaviruses belongs to the family reoviridae and form an own genus. they are genetically highly diverse, forming up to eight serogroups [40] . rva can be detected in many animal species and is an economically important pathogen in suckling pigs and calves but is usually species-specific. reports of zoonotic transmissions exist but the most important sources of human infection are humans [41, 42] . foodborne transmission is mainly a problem in developing countries where also water contamination is frequent. in a recent surveillance study in columbia, 20% of analysed municipal drinking water samples were rvcontaminated [43] . however, rv have also been detected in 13% of mussels in southern italy 2014-2015 [44] . human adenoviruses (hadv), on the fifth place with 19 reports, are often involved in gastroenteritis (ge) in children, where they are considered the second most frequent cause of ge after rotaviruses [45] . however, they may also cause respiratory signs and eye infections. hadvs belong to the genus mastadenovirus within the family adenoviridae. to date, there are seven species of human adenoviruses known (human mastadenovirus a-g) (ictv 9th report (2011)). the genus is non-segmented, linear double-stranded dna of 35-36 kb length and the approximately 90 nm large virions are unenveloped, making them very resistant to environmental impacts [46] . spread may be directly from person to person or via the faecal-oral route. contaminated recreational and irrigation water seems to be a frequent source of infection [45] . contaminated water may also lead to contamination of food. for example, human adenoviruses were recently detected on fresh parsley using a metagenomic approach [47] and are often contaminating shellfish [48] . no zoonotic transmission has been reported as adenoviruses are usually speciesspecific [49] . the last virus showing over ten pubmed entries related to "foodborne" in the last 5 years is the human sapovirus (12 publications) . belonging to the caliciviridae family (like nov) and forming an own genus, it is a small (27-40 nm) unenveloped rna virus. similar to nov, sapoviruses (sav) which were only detected in 1977 are highly resistant to unfavourable environmental conditions. currently, human and animal savs are classified into 15 genogroups based on the vp1 protein [50] . as hadvs, sapovirus outbreaks are often reported in kindergartens, nurseries and schools. the outbreaks are of sporadic nature and are reported world-wide but particularly many reports are from japan, where the virus was first studied and named after the city of origin of the first type strain: sapporo [51] . transmission is mainly direct faecal-oral or via contaminated environmental surfaces. however, several reports show food as source of infection such as catered box lunches in a junior high school or food served at a wedding hall in japan [52] . even though porcine sav are regularly detected in porcine faeces, transmission to humans and zoonotic outbreaks have not been reported to date [53] . however, inter-genogroup recombination events of sav in pigs have been reported and highlight the high genetic diversity and evolution rate of sav [54] . other potentially foodborne viruses that may cause disease in humans but have lower numbers of publications are human astroviruses (astroviridae), aichivirus/human kobuvirus (picornaviridae) and enteroviruses (picornaviridae) [55•] . astroviruses were even the most frequently detected viruses in mussels in southern italy [44] . however, the clinical importance of these viruses is not completely clear, and literature is controversial. they show worldwide spread and are detected in faeces of healthy individuals and may hence be part of the normal flora of the gastrointestinal tract [56, 57] . however, there are also reports of severe outbreaks of acute gastrointestinal disease, often in children under 5 years [58, 59] . enteroviruses in particular are frequently detected in environmental samples and waste water used for irrigation [60] . it seems that most mammal species harbour their own astro-, entero-and kobuviruses [56, 61] . while interspecies transmissions seem to occur [56, 62] , their frequency and consequences for human health require further investigations. the "newbies"-viruses intrinsically present in food (table 2) the vast majority of humans rely on plants as major source of food. metagenomic analysis of romaine and iceberg lettuce has shown the presence of wide range of plant and animal/ human viruses already on the field [63•]. it has also been shown that nov can be internalised by some plants such as lettuce and strawberries through the root system and can therefore be present inside the plant and fruits rather than just contaminating the surface [64, 65] ; similar to the internalisation and retention of acid resistant viruses in water filtering molluscs [66] . even though being internalised, nov, such as most animal and human viruses, is not able to infect or replicate in plant cells [67] . however, there are numerous viruses specifically infecting plants. plant viruses are frequently detected in human and animal stool samples [68] , but even though some virus families, like bunyaviridae, rhabdoviridae and reoviridae, contain plant-, animal-and human viruses, no plant virus is known to be a human pathogen [69] . however, a report on the detection of antibodies against pepper mild mottle virus (pmmov) in patients with abdominal pains and virus positive stool samples points to interaction of the human immune system with this plant virus [70•] . furthermore, some plant viruses are known to infect and persist in insect-vectors and one of them, tomato spotted wilt virus, a member of the genus tospovirus of the bunyaviridae family, was even shown to replicate in human cell lines [71] . due to the high genetic elasticity of their rna genome and the broad (plant-) host range, tospoviruses are indeed discussed to have the potential to cross the kingdom-barrier [69] . in contrast to crossing the barrier between the plant and animal kingdom, it is far easier for viruses to cross species barriers. exposure to animal viruses, mainly of other mammals, represents therefore a greater risk for human health. since viruses are heat sensitive, raw or undercooked foodstuffs such as meat, milk and dairy products are the most likely sources for uptake of infectious animal viruses. only recently, the range of viruses intrinsically present in these products is being studied, mainly using ngs, and has revealed several "new" viruses. the most emerging zoonotic foodborne viruses in third world countries may well belong to the hepatitis e genotypes 3 and 4. hev-3 and 4 strains infect humans, but the reservoir is thought to be in several animal species, whereof the pig plays the most important role for foodborne transmission. hev-3 is distributed worldwide and represents the predominating genotype in europe while hev-4 is mainly reported from china, japan, india and indonesia [74•] but sporadic cases and virus detection have also occurred in italy [75] , france [76] and belgium [77] . with seroprevalence rates of 46-100% in many countries, hev seems to be endemic in piggeries and is also present in wild boar [74•] . in pigs, the virus causes only a subclinical disease with mild mostly microscopic lesions in the liver. virus is shed for 3-7 weeks in faeces and transmission between animals is via the faecal-oral route [78] . the first foodborne transmission of hev was observed in japan where identical virus sequences were found in a diseased patient and in sika deer meat [79] . additional reports, linking human cases to grilled wild boar meat in japan and pig meat in spain supported the initial finding. salines et al. (2017) [80••] have summarised the reports of hev contaminations in meat products and found highest virus prevalence in pork liver pàtés (47% in canada and 36% in brazil) and drycured sausages containing pig liver such as the figatelli from corsica in france (30%). other sources of infection for humans are game meat (deer and probably rabbit) and food potentially contaminated by pig/wild boar faeces such as wild herbs [81] . human-to-human transmission may occur but seems to be rare; however, blood transfusion has been reported as a transmission pathway [82] . in humans, most infections seem to be subclinical; the most severe clinical sings are observed in older males and patients with pre-existing liver diseases or increased alcohol consumption [83, 84] . a meta-analysis of 432 studies on the hev seroprevalence in humans in europe from 2003 to 2015 showed a wide range from 0.6 to 52.5%. contact to swine/wild animals had a significant influence as well as the type of assay used and the geographical area with highest mean values in france (32%) and lowest in italy (7.5%) [85•] . the surveillance report of the european centre for disease control (ecdc) that includes data of 22 eu/eea member states (accounting for > 90% of the total eu/eea population) provides data on confirmed cases of hev for 2005-2015 and shows an annual three-fold increase in the number of annual cases between 2011 and 2015 [86•] . however, hev-3 was already present in german wild boars as early as 1995/1996 [87] and in india in 1988 [88] as the analysis of archived samples has shown. therefore, it may well be that the disease and its zoonotic aspect has been largely overlooked for decades and acute cases regarded as hepatitis a. only recently, routine diagnostic tools are available and awareness in clinicians has grown. similar to havnet for hepatitis a, hevnet which was initiated 2017 aims at concerting and harmonising hev surveillance in europe [86•] and governs a hev sequencing repository for molecular tracing of outbreaks as well as a public hev online typing tool (http://www.rivm.nl/mpf/typingtool/ hev/). however, while research and surveillance of the disease in humans is increasing, many aspects of the virus life cycle and epidemiology in pigs are still unknown. due to the lack of clinical signs, hev has not been of veterinary importance so far. however, in order to fully understand and counteract the disease in humans, this gap needs to be closed. another animal product that may contain viruses is milk. while it is well known that several important bacterial diseases can be attained by consuming raw milk or dairy products such as brucellosis, tuberculosis and listeriosis, not so much is known about transmission of zoonotic viruses by milk. an exception is the confirmed transmission of tick-borne encephalitis virus (tbev) via goat milk leading to human infection [89, 90] . a study in a high tbe risk region in poland showed 22.2% of sheep milk positive for tbev by rt-pcr followed by goat milk (20.7%) and cow milk (11.1%) [91] . importantly, even though tbev and other related tick-borne flaviviruses, being enveloped rna viruses, are not generally considered particularly resistant to environmental influences, they may survive low ph conditions such as present in the stomach. however, while langat virus, a close relative to tbev, survived conditions present during cheese production, it was completely inactivated by pasteurisation [92] . raw milk cheese, particularly of sheep and goats may thus present a source of infection for the potentially lethal tbev and probably other tick-borne flaviviruses. while foodborne hev and tbev clearly represent a threat for human public health, the role of several other viruses of animal origin detected in food still needs to be assessed. zhang et al. (2014) [93••] have used ngs to search for viruses present in beef, pork and chicken bought in supermarkets in san francisco. the muscle tissue they analysed was explicitly taken from the centre, not the surface of the meat pieces. interestingly, all viruses found were small, unenveloped dna viruses, mostly of the parvoviridae family. in pork, they found sequences of four different parvovirus species of three parvovirus genera. parvoviruses are among the smallest dna viruses (latin "parvus" = small) with a diameter of only 18-28 nm and are particularly resistant to disinfection and environmental conditions. they are frequently detected in faeces of diverse domesticated and wild animal species and humans, and may range from asymptomatic to highly virulent causing mainly enteric diseases [94] . even though they are usually species-specific, interspecies transmission is occurring as in the case of canine parvovirus-2 that is thought to originate from cats [95] . interestingly, porcine parvovirus was also detected in beef which may point to an interspecies transmission [93••] . other dna viruses detected in beef and pork are torque teno viruses (ttv) of the anelloviridae family and members of the circoviridae family (e.g. porcine circovirus-2 in pork) [93••] . for ttv alone, no clear association with disease, but a role in rendering other infections (e.g. by circoviruses) more virulent is discussed in pigs [96] . however, newer studies have not observed a correlation of post-weaning multisystemic wasting syndrome (pmws) caused by porcine circovirus-2 and the presence of ttv [97] . interestingly, human and animal ttvs are often detected in sera by ngs, even in healthy individuals [98, 99] ; this may also be the reason why they are present in meat. porcine circovirus-2 (pcv-2) is endemic in pigs and associated with several clinical syndromes. it is also present in skeletal muscle tissue of infected pigs which was shown to be infectious for other pigs after oral uptake [100] . no antibodies against pcv-2 were detected in humans so far but a certain risk for immunocompromised humans after receiving porcine xenotransplants is discussed [101] . pcv-2 as well as parvovirus may also originate from live attenuated vaccines that are routinely used in piggeries [102] . however, even though infectious pcv-1 was detected as a contaminant of a human rotavirus vaccine, no seroconversion was detected in the vaccinated children [103] . in the analysed chicken, sequences matching to six different types of gyroviruses were detected [93••] . they belong to the family of circoviridae but are structurally similar to anelloviridae and are small unenveloped viruses with a circular dna genome of about 2.3 kb. the most prevalent and world widely distributed representative of this genus is the chicken anaemia virus (cav) that infects and induces apoptosis in erythropoietic and lymphatic progenitor cells leading to anaemia and immunosuppression in young chicken. vaccines against this virus are routinely used in poultry farms [104] . cav sequences have been detected in faeces of children and cats but due to the low copy numbers it is assumed that they are of dietary origin without infecting human cells [105] . even though several gyroviruses with high similarity to cav were detected on the skin, in the blood and in faeces of immunocompromised humans, no antibody response against cav was observed [106] . in contrast to gyrovirus, antibodies against an animal polyomavirus (bpyv) have been reported in humans. polyomaviridae are relatively small (50 nm) unenveloped viruses with a circular, double-stranded dna genome of 5 kb length and consist of the genera alpha-, beta-, gamma-, deltapolyomavirus. polyomaviruses have shown to persist in the organs of humans and many animal species, usually without causing overt clinical sings. however, in immunocompromised patients, an association with oncogenesis through transformation of infected cells has been proposed [107] . since this transformation is due to integration of viral dna into the host genome-particularly in non-permissive infections-the relatively high seroprevalence against bovine polyomavirus (bpyv-1) in people handling cattle such as farmers, vets and abattoir workers was of some concern [108] . based on epidemiological data, it was also speculated that bpyv in red meat might have an etiological role in the development of colorectal cancer in humans regularly consuming beef [72] . furthermore, the presence of a novel bpyv-2 in beef was recently shown by metagenomic analysis [93••] . however, while bpyv seems to be highly prevalent in cattle and may be transmitted to humans, no disease has yet been clearly attributable to the infection neither in cattle nor in man [108] . another virus that is contradictorily discussed regarding its clinical meaning in humans after foodborne transmission is bovine leukaemia virus (blv). blv is endemic in cattle in many countries where it causes leukosis in 2-5% of infected animals [109] . its presence in meat and dairy products has been shown [110] and buehring et al. (2003) [111] found antibodies against blv capsid antigen in 74% of human serum samples using immunoblotting. there are also several reports claiming an association of breast or colon cancer with blv [73, 112] . however, data from a recent study in china did not detect any blv genome or antibodies against blv in women with or without breast cancer even though the virus-and antibody prevalences of cows in the analysed regions were high [113] . in addition, being a member of the retrovirus family, blv is highly susceptible to inactivation and it is unlikely to survive gastric conditions. however, more data are required to conclude on the foodborne zoonotic potential of blv. even viruses prone to inactivation by low gastric ph levels and proteases may cause foodborne infection if uptake occurs via a non-oral route. occupational groups like abattoir worker, vets and butchers may become infected thorough animal blood, body fluids and excretions via skin lesions, mucous membranes or by inhalation of aerosols [55•] . it is assumed that several important human viruses such as hiv, sars coronavirus and ebola were and still are crossing the species barrier not primarily through the consumption of meat but mainly during the butchering process of animals carrying the viruses such as primates and bats [114, 115] . similarly, live poultry markets and culling of infected birds was shown to be a significant risk factor for human infection with h5n1 influenza virus [116, 117] . the classic foodborne pathogens such as nov, hav and rotavirus make up the majority of clinical cases in humans. these viruses are adapted to humans and are efficiently transmitted between humans, making it often difficult to untangle foodborne and direct human-to-human transmission. furthermore, in case of relatively "novel" foodborne viruses such as astroviridae or sapoviruses, it is often difficult to differentiate between "commensal" viruses, opportunistic pathogens and real threats for human public health. ngs has made it possible to non-specifically screen food for viruses and has shown that many intrinsic plant and animal viruses are also present in food products. the finding of several types of circo-and parvoviruses in beef, pork and poultry shows that also pure muscle tissue of apparently healthy animals harbours a surprising range of viruses [93••] . the high tenacity of these small unenveloped dna viruses means that they can also be present in processed meat. this is supported by own observations, where ngs sequencing reads obtained from a dry cured pork sausage covered not only the whole genome of hev [118] but also of porcine circo-and parvoviruses as well a plant virus (pepino mosaic virus) in high depth (unpublished data). it is not sure if these viruses remained infectious in the sausage. however, the hev sequence was identical to the virus isolated from a patient with acute hepatitis who consumed this type of sausage [118, 119] . hence, new diagnostic tools have 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the role of food and the food system in the transmission and spread of ebolavirus origins of hiv and the aids pandemic circulation of avian influenza h5n1 in live bird markets in egypt avian influenza virus (h5n1): a threat to human health complete genome sequences of two swiss hepatitis e virus isolates from human stool and raw pork sausage new subcluster of hev genotype 3 strains linked to the first confrmed swiss case of foodborne hepatitis e infection key: cord-337058-rhu5hp9t authors: snyder, brian f. title: the genetic and cultural evolution of unsustainability date: 2020-04-06 journal: sustain sci doi: 10.1007/s11625-020-00803-z sha: doc_id: 337058 cord_uid: rhu5hp9t anthropogenic changes are accelerating and threaten the future of life on earth. while the proximate mechanisms of these anthropogenic changes are well studied (e.g., climate change, biodiversity loss, population growth), the evolutionary causality of these anthropogenic changes have been largely ignored. anthroecological theory (aet) proposes that the ultimate cause of anthropogenic environmental change is multi-level selection for niche construction and ecosystem engineering. here, we integrate this theory with lotka’s maximum power principle and propose a model linking energy extraction from the environment with genetic, technological and cultural evolution to increase human ecosystem carrying capacity. carrying capacity is partially determined by energetic factors such as the net energy a population can acquire from its environment and the efficiency of conversion from energy input to offspring output. these factors are under darwinian genetic selection in all species, but in humans, they are also determined by technology and culture. if there is genetic or non-genetic heritable variation in the ability of an individual or social group to increase its carrying capacity, then we hypothesize that selection or cultural evolution will act to increase carrying capacity. furthermore, if this evolution of carrying capacity occurs faster than the biotic components of the ecological system can respond via their own evolution, then we hypothesize that unsustainable ecological changes will result. since the neolithic demographic transition, human societies have often lived along a continuum of unsustainability (ponting 2007; gowdy and krall 2013) , as depicted in fig. 1 . 1 at the most extreme end of this continuum is socio-ecological "collapse", defined here as a rapid decline in the population and/or development of a society due to a combination of ecological and social factors (cumming and peterson 2017) . civilizations that may have experienced socio-ecological collapse in whole or part due to their over-extraction of resources include the mayans, romans, greeks, the civilizations of easter and pitcairn islands, the norse colony in greenland, the cahokia in the american midwest, and the anasazi (lopinot and woods 1993; ponting 2007; hughes 2011; kennett and beach 2013) . in these cases, human systems may have expanded beyond their ability to absorb energy and materials from the environment, and these resource constraints interacted with socio-political conflict to generate socio-ecological collapse. the precise mechanisms and the relative weight of social versus ecological factors is debated in these and other cases (butzer 2012; butzer and endfield 2012; boersema 2015; cumming and peterson 2017) , and we do not imply that every case of social collapse is primarily resource driven. instead, we simply note that human populations have repeatedly experienced collapse and in many of these cases, environmental and resource constraints are implicated. while these cases of permanent collapse are particularly notable, humans have also spent a great deal of their history in a state of socio-ecological crisis. we define socio-ecological crisis as a situation in which human populations have expanded close to or beyond their ability to gather energy and materials from their environment which imperils the population and its standard of living. 2 socio-ecological crisis may lead to the collapse, but societies might also be resilient. examples of socio-ecological crisis might include the 1828 years (out of 2000) between 108 bce and 1910 ce in which there was a famine in at least one province in china; the european famine of 1315-17 in which the population resorted to cannibalism; or the finish famine of 1696-97 in which a quarter of the population perished (ponting 2007) . in each case, crisis did not lead to collapse, but for our purposes, it is only important that so much of recorded human history has been spent in a state of crisis. in addition to states of socio-ecological crisis and socioecological collapse, human populations have spent much of the remainder of our post-paleolithic history in a state of unsustainability. we define unsustainability as a growth phase in which humans extract increasing quantities of energy and materials from the environment and use them for both maintenance and growth. unsustainability is associated both with economic (daly 1991) and population growth (ehrlich and ehrlich 2009 ) and may lead to socio-ecological crisis. we might consider the growth phase of the ancient greek (hughes 2011) or contemporary western civilizations to exist in a state of unsustainability. given our history and our contemporary society, we might wonder why human civilization, since the neolithic revolution seems to be dominated by periods of unsustainability punctuated with periods of socio-ecological crisis and collapse (ponting 2007; gowdy and krall 2013) ? the purpose of this paper is to provide a conceptual model for understanding why humans may be predisposed to environmental unsustainability, socio-ecological crisis, and collapse. anthroecological theory (aet) hypothesizes that human social and cultural evolution is the ultimate cause of the ecological crises currently damaging earth systems (ellis 2015; ellis et al. 2018) . according to aet, human damage to earth systems is a consequence of socio-cultural niche construction which has evolved via a multi-level group selection process. in this understanding, humans have acted as ecosystem engineers and niche builders since at least the agricultural revolution, and perhaps since their burning of ancient savannahs for corralling game. this ecosystem engineering led to progressively greater "social scales" and population size, requiring ever-increasing levels of ecosystem engineering. here, we attempt to merge aet with lotka's maximum power principle (lotka 1922) and concepts from evolutionary ecology. lotka argued that natural selection acted so that organisms sought to maximize the rate at which it extracted energy from the environment; h.t. odum later named this hypothesis the maximum power principle (sciubba 2011) . if lotka was correct, then the ecosystem engineering and niche construction described by aet can be understood as a consequence of the maximum power principle acting via natural selection. while this energetic view is similar to ellis's aet model, we use energy extraction as a target of selection and as a metric of environmental load. this formalization might allow novel tests and models of aet. in the remainder of the paper, we first review the available literature on the evolution of unsustainability including prior approaches from agriculture, evolutionary psychology, and aet. we then describe our conceptual model of energetic aet and its relationship to the evolution of carrying capacity. finally, we discuss the implications of energetic aet for our understanding of two contemporary issues in socio-ecology and the paper ends with conclusions. as in the present work, gowdy and krall (2013 , 2014 attempted to understand the cause of human unsustainability. they argued that human ultra-sociality (called eusociality in the sociobiological literature) evolved as a consequence of the neolithic demographic transition and that this ultrasociality was the ultimate cause of the anthropocene's environmental crisis. to gowdy and krall, the ultra-social nature of human groups allowed for a shift in the primary level of selection from the individual level to the group level. thus, "with the transition to agriculture the group as an adaptive unit comes to constitute a wholly different gestalt driven by the imperative to produce surplus (gowdy and krall 2014, 139) ." this imperative to produce surplus leads to environmental crisis in gowdy and krall's model, but the same phenotype could also be understood as an expression of lotka's maximum power principle in our model. of course, gowdy and krall were not the first to link agriculture and socio-ecological crisis. thomas malthus' classic work on agriculture and population, and the responses to it, are to our knowledge, the first studies of the energetic sustainability of human societies. in is 1798, an essay on the principle of population, malthus observed that population grew exponentially, while improvements in agricultural yields grew linearly at most (malthus and pullen 1989) . the result was what has been called a malthusian catastrophe in which the human demand for food energy exceeds our ability to extract it from the environment with consequent decreases in population size, standard of living, or both. phrased in the language of the present paper, malthus argued that the rate of increase of energy extraction from the environment was principally resource (rather than technology) limited and that this resulted in socio-ecological crisis when the rate of population growth exceeded the rate of energy extraction growth. malthus' ideas have been challenged many times since the eighteenth century, perhaps most notably by esther boserup (2014). boserup argued that agricultural productivity was principally limited by the technology and labor humans dedicated to agriculture. in her view, as population density rose, farming practices shifted through "agricultural intensification" which would serve to increase yields at the cost of increased labor. phrased in the language of the present paper, such a change would amount to technological and cultural evolution towards increasing energy extraction from the environment. this increasing energy extraction could then, contra malthus, support an exponentially growing population. since boserup's work, the green revolution increased staple crop productivity via new crop varieties and natural gas-made fertilizer. this revolution was able to ameliorate the potential socio-ecological crisis associated with the rapid population growth in the developing world in the latter half of the twentieth century (ehrlich and ehrlich 2009 ). thus, consistent with the outlines of boserup's thesis, humans were able to use technology to increase their energy extraction from the environment, this technologically-mediated energy extraction supported increased population size and occurred commensurate with a cultural shift (evolution) that allowed for increasing technological use in agriculture (possas et al. 1996) . this process is consistent with the energetic aet hypothesis developed here. while boserup and malthus provide important and contrasting lenses through which to understand the role of agriculture in human population, and while gowdy and krall identify agriculture as a critical turning point in human evolution, agriculture is no longer the predominant energy source for humans (smil 2017) . thus, we seek to build a conceptual evolutionary model of the human socio-ecological system that is consistent with these insights from agricultural systems but is more evolutionary and more general and incorporates extra-somatic energy, defined as energy that is used by humans but not used in direct human metabolism (price 1995) . furthermore, we propose a positive feedback that is missing from prior models of the energetics of socioecological systems, and we also integrate a ratchet-effect that causes the evolution of humans towards socio-ecological crisis to be unidirectional. where gowdy and krall (2013, 2014) saw agriculture as the key to understanding human unsustainability, van vugt et al. (2014) saw the roots of unsustainability in the paleolithic, before the invention of agriculture. using an evolutionary psychological approach, van vugt et al. argued that ancestral selection in paleolithic humans created psychological conditions that make humans ill prepared to living sustainably. for example, they argue that relative values-valuing status rather than absolute quantities of goods-would have been adaptive in prehistory, but has become maladaptive for sustainability in the contemporary world. in the present paper, we understand the traits described by van vugt et al. (2014) as pre-adaptations that are exploited by cultural evolution. aet proposes that humans are predisposed for unsustainability through their biological and cultural evolution. specifically, aet proposes that human unsustainability has evolved via a multi-level selection process (see waring et al. (2015) for a relevant overview of multi-level selection) acting on the genome and occurring in concert with selective and non-selective mechanisms acting on culture and technology. in aet, this process results in a species that is prone to niche construction and ecosystem engineering, and the scale of these processes continues to increase as the population rises. this increasing scale coupled with human propensity for niche construction leads to human unsustainability (ellis 2015; ellis et al. 2018) . here, we hypothesize that humans have evolved to increase their carrying capacity (scale) by increasing their rate of energy extraction from the environment. this hypothesis is identical to that of aet, with the exception that it emphasizes the cause of the process (maximization of energy extraction) rather than the mechanism (niche construction and ecosystem engineering). that is, niche construction and ecosystem engineering are two possible mechanisms by which humans maximize their extraction of energy from the environment. because the substrate of this evolution is cultural and technological as well as genetic, it outpaces the ability of the ecological system to evolutionarily respond. the human and non-human ecological systems are thus locked in a form of red queen coevolution (van valen 1973; stenseth and smith 1984) , but human technology evolves faster than the biotic ecological system's ability to keep up. the red queen hypothesis is often invoked to describe coevolutionary systems in which one species coevolves to keep up with selective pressure from another species or species group and in which there is a difference in the evolutionary rate of the coevolutionary partners (e.g., pathogens and their hosts). here, we adapt the red queen to include coevolution between a species (humans) and the biotic components of the ecosystem (the biosphere). thus, we hypothesize that as human population size increases, human populations demand more energy from the environment. this places a selective pressure on the biotic environment, because energetic resources are limited and because of the 2nd law of thermodynamics, energetic resources are zero-sum; that is, if humans consume energy from the environment, there is necessarily less energy available for other organisms. however, the human population evolves via techno-cultural evolution, which is far faster than the biotic environment can evolve via genetic evolution. thus, the analogy between a fast-evolving parasite (humans) and a slow-evolving host (the biotic environment) is apt. note that we do not intend to imply that the environment itself coevolves with human techno-cultural evolution, but that the biotic populations that compose the ecosystem evolve, with consequent impacts on the abiotic environment (but see lovelock and margulis 1974 , levin 1998 , swenson et al. 2000 . while the conceptual differences between aet and the present hypothesis are modest, there are important differences in their implications. ellis's aet proposes that humans are somewhat unique, because we are expert ecosystem engineers and niche builders. the present hypothesis suggests the alternative. humans, just like all other species, maximize their energy extraction from the environment. just like all other species, humans are opposed in this process by a coevolutionary response in the biotic components of the ecosystem. humans are unique only, because the rate of cultural evolution in energy extraction exceeds our biotic environment's coevolutionary response. in this view, human unsustainability is a result of our uniquely advanced culture coupled with our non-unique drive towards for energy maximization; any species that developed a similar rate of cultural evolution would behave similarly. niche building and ecosystem engineering (ellis 2015) or agriculture (gowdy and krall 2014) are simply the means by which we maximize energy extraction from the environment. natural selection works to maximize the number of offspring an individual contributes to a following generation (i.e., fitness). however, energy and fitness are closely linked, because fitness is the result of a physiological process by which energy is captured from the environment and converted into offspring. that is, growth and reproduction at either the group or individual level require energy input from the environment coupled with high entropy waste output to the environment. this physiological understanding of fitness has been well studied in non-humans and is the foundation for much of life history theory [e.g., (weiner 1992 , garland jr and carter 1994 , ricklefs and wikelski 2002 ]. in this context, pianka (1970) argued that, "…natural selection will usually act to maximize the amounts of matter and energy gathered per unit time." brown et al. (1993) likewise offered an energetic definition in which fitness is "reproductive power, or the rate of conversion of energy into offspring." this reproductive power was taken to be a function of both the rate of assimilation of energy from the environment and the rate of conversion of energy to offspring (but see (kozlowski 1996) ). based on brown et al.'s definition, fitness (f) of individual i is a function of energy extracted (x) from the environment and the efficiency of energy conversion to offspring (e): where f is offspring number per lifetime, x is in calories extracted by the individual over its lifetime, and e is offspring produced per calorie. in this model, fitness is on the individual level, but identical logic applies at the group level, assuming heritable group variation in x or e. this physiological understanding of fitness is tautological when applied to non-humans, because the only source of energy non-human organisms use is somatic. for example, applied to a lion, eq. 1 simply argues that the lion's offspring number is the total number of calories it eats multiplied by its offspring number divided by the number of calories it eats. however, both x and e can be selected independently, given heritable variation. that is, lions could be selected for better foraging ability (increased total energy gain from the environment), or for more efficient foraging, digestion, metabolism, etc. (increased efficiency). unlike non-humans, humans can use extra-somatic energy sources (e.g., fossil fuels), and can convert those energy sources into genetic fitness. for example, humans can burn savannas to harvest game (scherjon et al. 2015) , burn forests to create terra preta (glaser et al. 2001) , and reform fossil fuels to create fertilizer; all of these uses of energy use non-trophic energy to indirectly increase fitness. thus, for humans x and e are not limited by photosynthesis and respiration as they are in non-human populations. we hypothesize that humans have been selected for increased x and e and that this may have occurred through a combination of individual selection acting on genetic/ behavioral traits that favor high individual x, as well as group selection favoring groups which, due to their genes, culture, or technology, more rapidly absorb energy from the environment and convert that energy to fitness. we hypothesize that this selection has acted to increase both somatic x (e.g., food) and extra-somatic x that can contribute to fitness (e.g., fossil fuels). we hypothesize that humans have been preferentially selected to maximize x rather than e, that this is the cause of our predilection for socio-ecological crisis, and that this preferential selection on x occurs because of thermodynamic limits on the change in e. furthermore, even selection for increased resource efficiency can lead to increased environmental impacts. as resources are more efficiently converted into fitness, population size increases which in turns leads to increased absolute resource use. this is analogous to jevons' paradox and the ideas of steady state economics which propose that even increased energy efficiency can result in increased environmental impacts through increased economic growth (daly and cobb jr 1994) . at the individual level, we can see the impacts of selection to maximize energy extraction in human social behavior (penn 2003) . as we accumulate more energy from the environment, demonstrated socially in the form of material possessions, we improve our status in the group and presumably improve our mating success (kruger 2008; sundie et al. 2011 ). this might apply both to indirect material indicators of energy gain (e.g., objects) as well as direct physical indicators of energy gain [e.g., body mass as an indicator of male attractiveness (swami and tovée 2005) ]. of course, the aggregate accumulation of material possessions and status comes at an environmental cost. we use energy and materials to create and obtain status bearing objects (cars, houses, jewelry, clothes, etc.), and this resource use results in high entropy wastes output to the environment. thus, one way to understand our socio-ecological crisis is as a result of ancestral selection acting at the individual level and favoring social dominance expressed via the accumulation of embodied energy (odum 1996) . this adapted drive to accumulate material and embodied energy is a preadaptation for environmental extraction which is exploited by later cultural evolution (van vugt et al. 2014) . genetic selection might also operate at a group level (wilson and sober 1994) . in this case, groups of individuals that can more effectively accumulate resources outcompete groups that cannot or do not. if there is heritable (genetic) variation in the efficiency of resource extraction or resource use, these traits would be selected for (we consider nongenetic mechanisms in the next section). as in eq. 1, the fitness of the group is a function of the group's ability to extract energy from the environment and the group efficiency of conversion. for example, groups of hunter-gatherers that hunt more effectively due to genetic mutations in social intelligence might be selected over groups without such traits, even if the trait exhibits a cost at the individual level. see henrich (2004) for a discussion for both the potential and limits of genetic group selection. we might view human social organization in general in this lens: social organization exists to maximize the extraction of energy from the environment to the group and individual (x), and the efficiency of the conversion of extracted energy into offspring (e). this is identical to the claim that social organization exists to maximize the fitness of the group (wilson and sober 1994) and/or the individuals which compose the group (nowak et al. 2010) , given an energetic definition of fitness. what is unique is the implication. if social organization acts to maximize group fitness (or the fitness of individual group members), and if fitness is dependent on energy extraction from the environment, then social organization results in increasing energy extraction from the environment. again, either increased x or e leads to environmental unsustainability, and thus social organization is an evolutionary cause of the socioecological crisis. multi-level selection theory argues that the phenotypes, particularly social phenotypes, observed in nature can be understood as the result of a selective process that acts on both individual and higher levels of social organization simultaneously. in multi-level selection theory, it is the balance of variance and selective pressures at the group and individual levels that determine the genotypes and phenotypes that result. multi-level selection is increasingly used in biology as a means of explaining insect social behavior nowak et al. (2010) safarzyńska et al. 2012 , waring et al. 2017 , brooks et al. 2018 . one of the traditional weaknesses of multi-level selection theory is that ancestral human groups seemed poorly suited to maintaining the genetic variance between groups required for group-level selection (henrich 2004). however, selection is not limited to genetic transmission. cultural group selection posits that culture, rather than genes, is the source of heritable variation on which selection acts at the human group level. because there are differences in the transmissibility and variance generation between cultural and genetic inheritance, the maintenance of cultural variation across groups is more plausible (henrich 2004; bell et al. 2009; waring et al. 2015) , thus the attraction of group-level cultural selection as a means of explaining human traits. table 1 describes the levels of selection acting to create human unsustainability and speed with which they act. the strength of selection is a function of the heritable variation in a trait and the trait's association with fitness (henrich 2004; freeman and herron 2007) . the speed of selection is a function of its strength and the generation time (van valen 1973) . cultural group selection can be more rapid than genetic forms of selection, because cultural change does not need to wait for biological reproduction to change phenotypic ratios, and new cultural innovations can be added to the population through non-random guided mutation (perreault 2012; brooks et al. 2018) . we hypothesize that genetic, individual level selection for behavioral and psychological traits that increase x serve results in pre-adaptations for a form of gene-culture coevolution (feldman and laland 1996; ambrose 2010) that results in technology that further increases x. technologicalcultural evolution spreads via cultural group selection (or other non-genetic means of transmission) (cavalli-sforza and feldman 1981; mesoudi et al. 2004; claidière et al. 2014; jordan 2014) . as a result, technology might change far more quickly than natural systems can respond via natural selection (wilson 2012) . in species without technology or a technological culture, changes in x and e will occur on evolutionary time and within a biological system that will evolve in response to any evolution. that is, in the absence of culture and technology, all of the biotic components of the system are limited to gradual changes in gene frequencies over time such that the rate of change in one species can be adapted to by another species. in humans, cultural evolution may have been too fast for other species to respond (perreault 2012) . thus, another way to understand our socioecological crisis is as a result of our ability to increase k by rapid, non-genetic processes. this rapid increase in technology creates an unsustainable situation in which the human system changes faster than the biotic components of the nonhuman system can co-evolutionarily respond. by analogy to the genetic definition of evolution (change in gene frequencies over time), consider technocultural evolution to be the change in the frequency of use of a specific technology over time. this frequency can change extremely rapidly. for example, the frequency of automobile use in the early twentieth century increased from 10,000 vehicles registered in the u.s. in 1900, to 10 million vehicles 20 years later (nakicenovic 1986 ). we hypothesize that societies that heavily employed automobiles might have experienced greater economic and population growth in this period than those that did not, extracted more resources from the environment, and increased their carrying capacity. thus, in the early twentieth century, the u.s. increased x through a change in technology and this change occurred far more rapidly than the non-human system could respond via coevolution. this technologically mediated increase in x in the early twentieth century u.s. resulted in an increased survival and reproduction in the u.s., resulting in increased population growth. however, as population and affluence grew, new sources of energy were required to support the larger population and its energetic (e.g., economic) expectations. despite the challenge of maintaining the product of x and e during the twentieth century, the u.s. did so. several factors contributed to this capability, but as the twentieth century progressed the increasing population increased the number of minds that could be dedicated to increasing x and e. this increased the rate of innovation which increased the rate of energy extraction, which increased the ability of the society to support the population. this positive feedback loop is at the core of the energetic aet hypothesis (fig. 2 ). the result of genetic and cultural evolution for increased x is lotka's maximum power principle (lotka 1921 (lotka , 1922 . the maximum power principle states that open systems tend to maximize the rate at which they can absorb useful energy from the environment. systems do not necessarily maximize energy gain, nor do they minimize energy loss, but instead systems maximize the useable power (energy divided by time) absorbed from the environment (hall 2004) . this is similar to the argument expressed in the preceding sections, because x is a rate (energy per lifetime). therefore, to say evolution acts to maximize x is to say selection acts to maximize power from the environment, consistent with lotka's principle. note that odum and pinkerton (odum and pinkerton 1955; odum 2007 ) expanded lotka's work and their model might draw different conclusions about efficiency than those presented here (hall et al. 1986 ). here, we use the maximum power principle sensu lotka rather than odum. lotka (1922) writes: if sources are presented, capable of supplying available energy in excess of that actually being tapped by the entire system of living organisms, then an opportunity is furnished for suitably constituted organisms to enlarge the total energy flux through the system … in every instance considered, natural selection will so operate as to increase the total mass of the organic system, to increase the rate of circulation of matter through the system, and to increase the total energy flux through the system, so long as there is presented a unutilized residue of matter and available energy" (147-148) thus, lotka imagines a system which absorbs exergy x from its environment and sustains n subsystems. if x increases, lotka argues that n will change to take advantage of the increased x. humans have been able to repeatedly increase x via technology and as a result, we increase n. according to lotka, we are expected to continue to do so, "so long as there is presented a unutilized residue of matter and available energy." unsustainability arises, because humans have evolved to employ increasingly sophisticated means of finding new unutilized sources of matter and energy. empirical tests of the maximum power principle are limited, but extant (hall 2004; cai et al. 2006; delong 2008; li et al. 2013) as are criticisms (månsson and mcglade 1993) . perhaps the most relevant example of the maximum power principle comes from the neolithic revolution. bowles (2011) compared the energy efficiency of hunter gatherers to early farmers and found that the efficiency in terms of energy extracted per unit of labor was higher for huntergatherers than for farmers (see also ponting 2007; smil 2017) . this led bowles to conclude that non-energetic factors explain the transition to agriculture. however, according to lotka, natural selection favors maximum power (energy per unit time) rather than maximum efficiency (energy per unit input). early farmers were able to extract more energy from the environment per unit time than hunter gatherers and this explains the success of the neolithic revolution. that is, in a given year (time), a farming society could extract more energy than a hunter-gatherer society (ponting 2007; smil 2017 ) and could convert this energy to fitness. thus, the transition to agriculture is consistent with the maximum power principle even while being inconsistent with explanations based on labor or energy efficiency. lotka stipulated that just because natural selection will act to maximize power flow through a system, evolution will not necessarily follow due to a lack of variance in the phenotypes leading to increased power flow; he called this variance "generating influences". we propose that human technology and culture is the primary generative influence that allows humans to obey the maximum power principle. in nonhumans, metabolic systems are highly conserved across species such that the same molecular pathways are present in diverse taxa. respiration, for example, is similar in plants, fungi and animals. this conservation is due to the lack of generative influence (variance) in respiration efficiency and results in a thermodynamic limit to the efficiency by which one generation can create the next generation. as a result, the evolution of e is limited. likewise, in non-humans, the evolution of x is limited by coevolution such that every adaptation that increases x in one species acts as a selective pressure on another species. using non-somatic energy, humans have freed x and e from these limitations, however, we hypothesize that there is more variation in the ability to capture energy flow into the system than in the efficiency of energy conversion. energy efficiency is limited by thermodynamic limits, while energy extraction is, because of rapid evolution through culture and technology, freed from the effects of coevolutionary responses. in other words, it has been easier for the system to evolve towards energy extraction than to evolve to energy efficiency. for example, we might expect that the rate of increase of global wood extraction will exceed the rate of increase in wood-fired energy efficiency. similar hypotheses could be generated for other fuels. human carrying capacity (k) is not a fixed quantity, but an emergent property of the earth system of which humans are a part (sayre 2008; chapman and byron 2018) . that is, k is not exogenous to humans but determined by the technology and behavior of humans interacting with climate, biodiversity, and biogeochemical cycles. over the past several centuries, humans have repeatedly increased k using technology to increase our extraction of energy and materials from the environment and to more efficiently convert those energy and materials into fecundity and survival. thus, selection has acted on individuals and groups to preferentially favor those groups that more rapidly increase the group-experienced k. in other words, carrying capacity is a group-level phenotype that is an important target of selections and, in humans, emerges from the interaction of genes, culture, technology and environment. we hypothesize that if there was heritable cultural or genetic variation in social ability to increase k, humans will have been selected towards increased k. that is, in the absence of top-down regulation, human populations have been selected to overcome bottomup regulation via genetic and cultural evolution. more formally, assume a discrete population growth system in which generation t + 1 is composed only of the offspring of generation t (that is, generation t yields generation t + 1 and dies). in this case where n t is the population size at time t and f is fitness as defined in eq. 1. thus, population size is simply the sum of all individual fitness. substituting from eq. 1: that is, the population in the next generation is the sum of all of the energy extracted by all members of the population, multiplied by each individual's conversion efficiency. assuming the population is at k, n t equals n t-1 , and k equals n t . therefore at the individual level, we assume that there is heritable (genetic) variation in either x or e. as this heritable variation is selected for, k increases, because they are positively related. at the group level, we suppose that each group experiences a local k, and a group-level x and e, and that there is heritable, between-group variation in x and e. in this case, groups with higher x or e would increase k, thereby increasing their population size. the increased population size relative to other groups implies might allow for social dominance which could increase the rate of spread of the genes and technologies used to increase x. the ability of a species to shift its carrying capacity is critical. if k is exogenously determined, selection cannot act to increase the extraction of materials and energy from the environment, because this has already been maximized. however, if k is endogenously determined, that is, if a species can determine its k through culture and technology, then we hypothesize that there may be cultural or genetic selection towards increasing k. the increase in k would, in turn, allow the population to expand to this new k, again placing a selective pressure on the population. however, the population is now larger with greater collective cognitive power and even better adapted at increasing k. thus, we might expect the rate of increase of k to increase over time ( fig. 3 ; see meyer and ausubel (1999) ). this is analogous to an increase in the total frequency of mutation in a population as population size increases. as the size of a population increases, the probability of any given beneficial mutation increases in each generation. likewise, as the size of the human population increases, the probability of innovation also increases as the number of brains that may be dedicated to innovation increases. to illustrate the argument, we built a simple simulation population model that tracks population growth, energy extraction and the evolution of carrying capacity. the model is based on a standard discrete population growth model in which energy extraction from the environment replaces carrying capacity. thus where r is the intrinsic growth rate and is equal to f from eq. 1, x max is the energetic carrying capacity of the system and x t is the total population energy extraction. in other words, x max and x t substitute for k and n t in a standard discrete population growth model (as in eqs. 3 and 4) . fig. 3 rate of change of k decreases as population size increases due to increasingly rapid technological innovation is determined by both by the energy available in the environment and the technology used to capture that energy. therefore, x max can change via technological innovation. we propose that the change in x max is a linear function of population size; however, we also propose that x max is more likely to change as x t approaches x max . that is, as the total energy extracted by the population nears the maximum energy available for extraction, it creates a selective pressure to increase x max . therefore, we define the probability of innovation, p, at time t as the coefficient 0.01 indicates that we assume a 1% chance of innovation per person per generation. the model generates a random integer between 0 and 100 and compares the random number to p t . if p t exceeds the random number, innovation occurs and x max increases by 10%; otherwise x max stays constant. changes to x max are assumed to be positive and permanent. substituting eqs. 1 and 3 into eq. 5 and solving for x t yields where f and e are as defined in eq. 1, above. for this illustration, we assume that e is fixed at 4 × 10 -7 offspring per kcal and x i is fixed at 5 × 10 6 kcal per generation. these assumptions yield an r (or f) of 2. results of 10 simulations are depicted in fig. 4 . unsurprisingly, the energetic carrying capacity, x max , increases over time, indicating increasing environmental load on the however, the rate of increase of x max also increases over time. figure 5 shows the mean x max from the simulations depicted in fig. 3 , with a best-fit exponential equation. the fact that the relationship between generation number and x max is exponential, suggests that the rate of increase in energy extraction will itself increase over time. unless the biotic ecosystem can co-evolve to limit this increasing energy extraction, an unsustainable situation will result. note that this model does not include top-down factors such as parasites and pathogens that may act to limit human populations. parasites and pathogens may represent one way in which the biotic system can co-evolutionarily "keep up" with the techno-cultural evolution of the human system. the model we presented is not intended to explain socioecological collapse per se, but rather the human propensity towards unsustainability and socio-ecological crisis, which may lead to socio-ecological collapse. nonetheless, some of the critiques of the concept of socio-ecological collapse may be help clarify our argument. tainter (2006) argued that the role of ecological change in socio-ecological collapse is historically unsupported. instead, tainter attributes the collapse of ancient societies principally to social failures. for example, tainter ascribes the collapse of the sumerian population around ur in the late third millennia b.c.e. to poor irrigation practices combined with a failure of leadership to recognize the worsening agricultural situation. thus, tainter might disagree that population growth and environmental energy extraction were the primary causes of the collapse of sumer. however, phrased in the context of carrying capacity evolution, tainter is suggesting that the carrying capacity declined due to salinization of the soils, which was in turn a consequence of poor soil management. from our perspective, the decline in k was only possible, because k had been inflated by unsustainable irrigation practices. that is, societies use technology to increase x max (and thus k) and their populations grow accordingly. in some cases, societies might use this new technology to extract too many resources and the population might overshoot k with negative consequences; deforestation or prey extinction might be examples. in other cases, populations may not overshoot k, but k might decrease below the population size, because the system used to increase x was unsustainable (e.g., dryland agriculture in sumer). in either case, the "ecological" in socio-ecological collapse is critical. we propose that human populations face two problems related to carrying capacity evolution. first, continued increases in k may become increasingly difficult due to thermodynamic constraints, eventually leading to a situation in which the human population exceeds carrying capacity and the population collapses. second, increases in k may not be permanent and may depend on unsustainable extraction of energy and embodied energy from the environment. in this case, the population may not overshoot k but may nonetheless exceed k when k declines and experience collapse. butzer (2012) proposes socio-ecological collapse begins with economic and fiscal decline over decadal to centennial timescales, coupled with and precipitating economic crises at decadal timescales. butzer proposes that many of these economic and fiscal conditions are ecologic, for example, declining agricultural productivity and anthropogenic degradation, while others are socio-economic (economic depression; foreign attacks). however, from the perspective of the energetic version of aet described above, nearly all of the preconditions and triggers described by butzer are energetic and ecologic. this is due to the thermodynamic linkage between economic activity and energy (georgescu-roegen 1975; daly 1991) . that is, all economic activity implies the dissipation of energy so that when butzer identifies "economic depression" as a trigger for socio-ecological collapse, this implies that human societies are no longer able to extract energy from the environment and convert that energy into economic activity as they had in the recent past. similarly, when butzer identifies "foreign attacks" as a stimulus for collapse, the energetic-aet model proposed here sees invasion and conflict as a mechanism for extracting increasing quantities of energy from the environment and thereby increasing k. energetic-aet has implications for the ecomodernism versus technological pessimism debate and the interconnected environmentalist's paradox. the environmentalist's paradox is the observation that human wellbeing seems to be improving in the face of declining natural system wellbeing. raudsepp-hearne et al. (2010) both defined the environmentalist's paradox and evaluated four potential explanations for it. the four non-mutually exclusive potential explanations were that: (1) human well-being has been measured improperly; (2) human well-being is dependent only on food-related ecosystem services which have increased; (3) technology has decoupled human well-being from ecosystem services; and (4) time lags have insulated contemporary populations from future declines. while a complete critique of their analysis is beyond the scope of the present article, raudsepp-hearne et al. argued that human well-being has not been evaluated improperly but could not reject the other three hypotheses. alternatively, energetic-aet proposes that humans have been able to avoid the negative social impacts of environmental perturbation by increasing their energy demand on the environment. contra explanation (3), technology has not decoupled human dependence on ecosystem services, but shifted which ecosystem services we depend on and increased our dependence on them. specifically, humans have used energy subsidies in the form of fossil fuels, renewables and nuclear energy to compensate for declines in energy flows from natural systems. for example, humans use energy in natural gas to reduce nitrogen for fertilizer production. human wellbeing increases, because energy extraction has increased faster than population growth such that per capita primary energy consumption increased from 1336 kg of oil equivalent per person in 1971-1920 kg of oil equivalent per person in 2014. consequently, human wellbeing has increased. the suggestion that humans typically live on a continuum of unsustainability as depicted in fig. 1 may seem inconsistent with the observed sustainability of some indigenous societies (trosper 2002; campbell and butler 2010) . nonetheless, it is a consequence of the present hypothesis. to phrase the energetic aet hypothesis in the language of game theory, we hypothesize that the evolutionary stable strategy of human societies is maximized and increasing rates of energy extraction. cases of sustainability in which societies fail to increase their energy extraction from the environment-as in some indigenous societies-are hence viewed as temporary, evolutionarily unstable strategies that will be outcompeted and replaced by more extractive strategies. this suggests a testable hypothesis: that societies with higher, less sustainable rates of energy extraction have outcompeted those with lower, more sustainable rates of energy extraction. note that evolution in general, and cultural evolution specifically, is value-neutral (sommers and rosenberg 2003) , so while it may be troubling to view sustainable cultures as evolutionarily unstable, this does not falsify the hypothesis. the model discussed here assumes human carrying capacity is controlled by bottom-up factors, specifically energy extraction from the environment (hopfenberg 2003) . as the global sars coronavirus-2 pandemic of 2020 demonstrates, humans are also susceptible to top-down population regulation by pathogens, as human pathogens may be one of the few parts of the ecosystem that can evolve as rapidly as human techno-culture. thus, human carrying capacity may be limited both by energy gain from the environment and by disease. future researchers may attempt to integrate disease evolution into the feedback loop, as depicted in fig. 2 . all else equal, as population density increases, pathogen virulence may be expected to increase (van baalen and sabelis 1995; lively 2006; mennerat et al. 2010; borovkov et al. 2013) , suggesting that pathogens may place a constraint on the feedback loop in fig. 2 . however, the evolution of pathogen virulence is complex and depends on ecological and genetic factors in both the pathogen and the host and the history of interactions between the two (knolle 1989; antia et al. 1994; levin 1996) . furthermore, the germ theory of disease, vaccination, and the development of antibiotic represent techno-cultural changes in how many human societies responded to pathogens, and this change may be positively associated with population density and energy extraction from the environment. thus, it may be interesting for future scholars to explore if pathogens act to limit population growth and energy extraction, or if technological innovation and population density have acted to remove the ability of pathogens to function as top-down population regulators. understanding how societies transition from unsustainable to sustainable states may be one of the most important questions in environmental social science in the twenty-first century. while such research is ongoing, less effort has focused on understanding why human societies are generally unsustainable in the first place. we propose that human societies are prone to unsustainability, because they have evolved to maximize their rate of energy extraction from the environment through a 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conspicuous consumption as a sexual signaling system male physical attractiveness in britain and malaysia: a cross-cultural study artificial ecosystem selection archaeology of overshoot and collapse northwest coast indigenous institutions that supported resilience and sustainability the dynamics of multiple infection and the evolution of virulence a group selection perspective on economic behavior, institutions and organizations a new evolutionary law evolutionary naturally green: harnessing stone age psychological biases to foster environmental behavior the coevolution of economic institutions and sustainable consumption via cultural group selection a multilevel evolutionary framework for sustainability analysis physiological limits to sustainable energy budgets in birds and mammals: ecological implications reintroducing group selection to the human behavioral sciences an evolutionary theory of largescale human warfare: group-structured cultural selection the author is grateful to leslie ruyle, jason lang, and two anonymous reviewers for their invaluable feedback on this manuscript. ambrose sh (2010) coevolution of composite-tool technology, constructive memory, and language: implications for the evolution of modern human behavior. curr anthropol 51:s135-s147 antia r, levin br, may rm (1994) key: cord-349177-8h25qj9y authors: khan, naazneen; de manuel, marc; peyregne, stephane; do, raymond; prufer, kay; marques-bonet, tomas; varki, nissi; gagneux, pascal; varki, ajit title: multiple genomic events altering hominin siglec biology and innate immunity predated the common ancestor of humans and archaic hominins date: 2020-06-18 journal: genome biol evol doi: 10.1093/gbe/evaa125 sha: doc_id: 349177 cord_uid: 8h25qj9y human-specific pseudogenization of the cmah gene eliminated the mammalian sialic acid (sia) neu5gc (generating an excess of its precursor neu5ac), thus changing ubiquitous cell surface “self-associated molecular patterns” that modulate innate immunity via engagement of cd33-related-siglec receptors. the alu-fusion-mediated loss-of-function of cmah fixed ∼2–3 ma, possibly contributing to the origins of the genus homo. the mutation likely altered human self-associated molecular patterns, triggering multiple events, including emergence of human-adapted pathogens with strong preference for neu5ac recognition and/or presenting neu5ac-containing molecular mimics of human glycans, which can suppress immune responses via cd33-related-siglec engagement. human-specific alterations reported in some gene-encoding sia-sensing proteins suggested a “hotspot” in hominin evolution. the availability of more hominid genomes including those of two extinct hominins now allows full reanalysis and evolutionary timing. functional changes occur in 8/13 members of the human genomic cluster encoding cd33-related siglecs, all predating the human common ancestor. comparisons with great ape genomes indicate that these changes are unique to hominins. we found no evidence for strong selection after the human–neanderthal/denisovan common ancestor, and these extinct hominin genomes include almost all major changes found in humans, indicating that these changes in hominin sialobiology predate the neanderthal–human divergence ∼0.6 ma. multiple changes in this genomic cluster may also explain human-specific expression of cd33rsiglecs in unexpected locations such as amnion, placental trophoblast, pancreatic islets, ovarian fibroblasts, microglia, natural killer(nk) cells, and epithelia. taken together, our data suggest that innate immune interactions with pathogens markedly altered hominin siglec biology between 0.6 and 2 ma, potentially affecting human evolution. prior to the mid-1990s, the extreme similarity of human and chimpanzee protein sequences suggested that phenotypic differences were primarily due to differences in gene regulation (king and wilson 1975) . the first definitive exception was a fixed loss-of-function genomic mutation unique to the human lineage, in the gene-encoding cmp-neu5ac hydroxylase (cmah) (chou et al. 1998) , an event mediated by an alu-alu fusion (hayakawa et al. 2001) . this mutation eliminated biosynthesis of the common mammalian sialic acid (sia) n-glycolylneuraminic acid (neu5gc) and caused accumulation of its precursor n-acetylneuraminic acid (neu5ac), radically changing cell surface extracellular glycosylation throughout the body in the hominin lineage. the mutation was dated to >2 ma by multiple methods (chou et al. 2002; hayakawa et al. 2006) . additional examples emerged a few years later, including the forkhead box protein p2 (foxp2) (enard et al. 2002 (enard et al. , 2009 ), a myosin heavy chain family member (mhy16) (currie 2004; stedman et al. 2004) , and examples of gene duplication followed by adaptive evolution (johnson et al. 2001) . along with biomedical considerations (varki 2000) , such discoveries lead to sequencing of the chimpanzee genome (chimpanzee sequencing and analysis consortium 2005) . there are now many more defined genetic differences between humans and our closest evolutionary cousins, involving not only gene expression (enard et al. 2002; fujiyama et al. 2002; watanabe et al. 2004; calarco et al. 2007; kehrer-sawatzki and cooper 2007; cruz-gordillo et al. 2010; otto et al. 2014; atkinson et al. 2018 ) but also other distinct genomic changes ranging from massive (megabase) structural variation to differences in gene copy numbers, de novo genes, pseudogenization (o'bleness et al. 2012; ruiz-orera et al. 2015) , human-accelerated regions in noncoding regions, and microrna genes (eddy 2001; mello and conte 2004; kim 2005; ruiz-orera et al. 2015) . this study focuses on human genes involved in the biology of sialic acids. all living cells are covered with complex arrays of glycoconjugates, with sialic acids occupying the majority of terminal positions on such glycan chains in animals of the deuterostome lineage (including echinoderms and vertebrates) (gagneux et al. 2015) . these acidic, nine-carbon backbone amino-sugars play important roles in cell-cell and cell-matrix interactions, as well as in host-pathogen interactions . the hominin-specific loss-of-function mutation in cmah mentioned above eliminated biosynthesis of neu5gc, which was a target for selective cell recognition by various nonhuman pathogens and toxins (kyogashima et al. 1989; martin et al. 2005; campanero-rhodes et al. 2007; deng et al. 2014; alisson-silva et al. 2018) . it is reasonable to speculate that exogenous pathogen recognition could have initially driven the cmah loss-of-function, generating a polymorphism as the recessive loss-of-function mutant allele rose in frequency. however, this deletion was then fixed in an ancestral hominin population $2-3 ma (chou et al. 2002) , possibly by directional selection against neu5gc via female immunity during reproduction, a mechanism demonstrated in vivo using transgenic mice that carry the same mutation in their cmah gene as humans (ghaderi et al. 2011) . given the likely timing of these events, we speculated that anti-neu5gc immunity of hominin females immunized against neu5gc by increased contact with neu5gc-rich vertebrate animal prey might have contributed to the origins of the genus homo $2 ma (wood and boyle 2016; bergfeld et al. 2017) . early discovery of some additional human-specific changes affecting sialic acid biology (brinkmanvan der linden et al. 2000; gagneux et al. 2003; sonnenburg et al. 2004; hayakawa et al. 2005; nguyen et al. 2006 ) and system-wide genomic and biochemical comparisons of sialic acid biology among primates and rodents (altheide et al. 2006 ) suggested a possible "hotspot" in human sialic acid evolution (varki 2009 ). with the availability of more human genomes (1000 genomes project consortium et al. 2015 , a recent study showed the patterns of genetic variation of 55 sialic acid biology-related genes in modern human populations did not significantly deviate from neutral expectations and were in fact not significantly different among genes belonging to different functional categories (moon et al. 2018) . sialic acid binding ig-like lectin (siglecs) are type i transmembrane proteins with an n-terminal immunoglobulin (ig)-like-v-set domain that mediates sialic acid recognition, and a variable number of ig-like-c-2 type domains (angata, hingorani, et al. 2001) . siglecs often have a cytoplasmic tail with one or more immunoreceptor tyrosine-based inhibitory motifs that can suppress immune cell activation. alternatively, they can recruit adaptor proteins with immunoreceptor tyrosine-based activating motifs. although siglecs likely have multiple functions, one prominent role appears to be recognition of endogenous sialylated glycans as self-associated molecular patterns, suppressing reactions of innate immune cells against self (varki 2011) . activating siglecs have a positively charged arginine or lysine in their transmembrane domains that can recruit dap12 and activate cellular immune responses against pathogens mimicking endogenous sialic acids (schwarz et al. 2017 ). cd33-related siglecs are rapidly evolving and among this family, nine inhibitory (hsiglec-3 and hsiglec-5 to hsiglec-12) and two activating members (hsiglec-14 and hsiglec-16) have been characterized in humans. recently, an article published from our group showed variable presence or absence of functional changes in the cd33rsiglec cluster in 26 mammalian species including great apes (khan et al. 2020) . with the availability of genomes from both living great apes (prado-martinez et al. 2013; xue et al. 2015; kronenberg et al. 2018 ) and extinct archaic hominins (reich et al. 2010; meyer et al. 2012; prufer et al. 2014) , we now systematically reassess the previous discoveries from multiple groups and also report on several new findings. overall, we find that multiple complex changes in genes involving sialic acid biology beyond the initial cmah mutation did indeed occur but are mostly confined to the cd33rsiglec gene cluster on chromosome 19 (angata, hingorani, et al. 2001) , encoding cd33rsiglecs, prominent on innate immune cells (varki and angata 2006) . overall, we found that multiple changes in the cd33rsiglec gene cluster are common in all human populations, postdate the common ancestor with the chimpanzee/ bonobo lineage, but predate the common ancestor with neanderthals and denisovans. such multiple complex changes in this gene cluster appear to be associated with altered expression of these genes, not just confined to innate immune cells, but also in other unexpected human cell types, some associated with diseases that appear to be uniquely human. the great ape genome data (total number 147) for cmah and cd33rsiglecs were derived from three publications (prado-martinez et al. 2013; xue et al. 2015; de manuel et al. 2016; kronenberg et al. 2018 ). these great ape genomes were mapped to human reference genome (grch37/hg19) retrieved from ucsc genome browser, using burrows-wheeler aligner and further processed with picard tool to remove duplicates and variant were called using genome-analysis toolkit. moreover, the variants in variant call format of chimpanzee, bonobos, gorilla, and orangutan were visualized in integrative genome viewer with their respective reference genome (pantro4, gorgor3, and ponabe2) and annotation file. all archaic hominins raw and processed files were obtained from the max planck institute for evolutionary anthropology (reich et al. 2010; castellano et al. 2014; prufer et al. 2014; slon et al. 2018 ) (http://cdna. eva.mpg.de/neandertal). bed coordinates of siglecs and cmah genes were provided for each human and ape lineage as supplemental (chimpanzee, gorilla, and orangutan) (supplementary file 1, supplementary material online). additionally, bed coordinates of additional polymorphism present in cd33rsiglecs with allele frequency in great ape population were also provided as supplemental (supplementary file 2, supplementary material online). the genes involved in sialic acid biology (67 genes) were overlapped with regions displaying signatures of ancient selective sweeps (peyregne et al. 2017) . the method used to identify these signatures of archaic selection relies on a hidden markov model to detect extended regions in the genome where the neanderthal and denisovan lineages fall outside the human variation (meyer et al. 2012; prufer et al. 2014 ). this method can only detect events that occurred between the split of modern and archaic humans around 0.5 ma (prufer et al. 2017 ) and the split of modern human populations from each other around 0.2 ma (schiffels and durbin 2014) . for events with a selective advantage of 0.5% or larger, with an origin of the beneficial mutation as old as 600,000 years ago, the false positive rate of the method is lower than 0.1% and its true positive rate is larger than 65% (peyregne et al. 2017) . we also note that signals of positive selection are not detectable if the selection coefficient is smaller than 0.1%. the ensembl database (release 82) (aken et al. 2016 ) was used to annotate each gene with hg19 coordinates from transcription start to transcription end. furthermore, l1cam, pecam, cmah, siglec13, siglec16, and siglec17 were excluded because they fall either in filtered regions not considered for the ancient sweep screen or could not be mapped to hg19 coordinates. as regulatory regions may also have been the target of positive selection, we extended the start and end coordinates 1 mb upstream and downstream, respectively. if a neighboring gene was within 1 mb, we only extended the coordinates until 5 or 1 kb from the transcription start or end of this neighboring gene. in order to test whether the lack of signatures of ancient selection is statistically significant, the candidate regions of ancient selection were randomly placed in the genome and the random placements of all regions were iterated 1,000 times, counting how often no overlap with genes involved in sialic acid biology was detected. the depletion in selection is not significant (356 sets never overlap; p value ¼ 0.356). paraffin sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol and trisbuffered saline-tween (tbst). following this, endogenous binding sites and peroxidases were blocked with 1% bovine serum albumin/tbst and 0.3% h 2 o 2 /tbst. this was followed by blocking of endogenous biotin and then heat-induced antigen retrieval was performed in citrate buffer ph 6. primary antibodies (mouse anti-siglec-7 and rabbit anti-siglec-13) or mouse igg were then overlaid at optimal dilutions, and slides were incubated overnight at 4 c in a humid chamber. specific binding was detected using a biotinylated antimouse or anti-rabbit (for siglec-13), followed by horseradish peroxidase (hrp) streptavidin and then biotinyl tyramide enhancement and then hrp streptavidin. substrate color was developed, and nuclei were counterstained with hematoxylin and the slides were aqueous mounted for viewing and digital photomicrography using an olympus bh2 microscope with an olympus magnafire camera. multiple derived, fixed, or polymorphic genomic siglec variants are present in all modern human populations table 1 summarizes events and frequencies in human populations based on the 1000 genomes project data and compares these with archaic hominin and great ape genomes. all human genomic changes in cd33-related siglecs (affecting 8 out of 13 members of this class of genes) were present across modern human populations. genome-wide association studies identified a derived cd33-linked allele (rs3865444(a), rs12459419(t)) that is protective against late onset alzheimer's disease (schwarz et al. 2016) . the linked single-nucleotide polymorphism was found to be variable across human populations with highest frequency in native american populations (48%) and lowest in african populations (5%). the previously reported alu-mediated human siglec13 deletion (wang, mitra, secundino, et al. 2012) and siglec17 (siglec-p3) pseudogenization due to open reading frame disruption and mutation of a critical arginine residue in its v-set domain (wang, mitra, secundino, et al. 2012) appear to be fixed in all humans. siglec12, siglec14, and siglec16 show polymorphic pseudogenization in variable frequencies across human populations, as detailed in table 1. siglec12 also harbors a human-universal mutation of the critical arginine residues in the v-set domain (arg -> cys, first v-set domain) abrogating its ability to recognize sialic acid. an additional siglec12 inactivation mutation (rs16982743) was found at an overall 18.6% frequency, highest in africans (37%) and lowest in east asians (5%). another siglec12 polymorphism, that is, frameshift (rs66949844) averages 59% (highest in native americans and lowest in east asians) in human population. additionally, based on the two linked alleles present in siglec16 (rs12611411(t) and rs12984584(c)), the pseudogene variant (siglec16p) is present in higher frequency than the functional allele (siglec16) in human populations (wang, mitra, cruz, et al. 2012) . because these siglec clusters are undergoing multiple gene conversions, some regions are highly prone to low mapping quality and hence some variants are not well defined, for example, siglec14 fusion-deletion, which is highly prevalent in east asian populations (yamanaka et al. 2009; ali et al. 2014 ). due to the lack of high coverage sequence data, these regions have not been well genotyped in nonhuman genomes. the cmah exon deletion is confirmed to be universal to humans. our previous human-ape comparisons involved the incomplete draft of a single chimpanzee genome, a few additional incomplete great ape sequences and a small number of human genomes (chimpanzee sequencing and analysis consortium 2005). thus, it remained possible that the genomic changes were actually a common feature of such genes across all these taxa. the availability of newly annotated versions of great ape genomes (prado-martinez et al. 2013; xue et al. 2015; de manuel et al. 2016; kronenberg et al. 2018) now allows for much better control against ascertainment bias, by comparing human-specific polymorphisms with multiple different genomes for each ape species, capturing potential polymorphisms in the latter as well (sullivan et al. 2017) . we observed that great ape genomes share none of table 1 detailed the mutations observed in human populations. instead, we only observed independent changes in critical arginine residues in v-set domain of siglec5/14, rendering them unable to recognize sialic acids (table 1) . although the essential arginine change (arg -> his) in siglec5 was found to be polymorphic in chimpanzee and gorilla, sequences in orangutan were not recognized due to low mappability. the siglec14 essential arginine change (arg -> his) was found to be polymorphic in chimpanzee, bonobo, and gorilla. however, sequences of siglec14 in orangutan were not determined in most of the species due to low coverage, except for one that harbors an essential arginine change to tyrosine (arg -> tyr). the cmah gene is functional in all great apes as expected, given that both types of sialic acids (neu5ac and neu5gc) are present in all these species (muchmore et al. 1998 ). overall, human genomes harbored a far greater number of structural/functional changes in cd33rsiglec genes (see fig. 1 and supplementary file 2, supplementary material online). to further understand the timing of these human genomic changes of these human genomic variants, we looked for their presence in archaic human genomes, that is, neanderthal and denisovan. the exon deletion leading to loss-of-function of the cmah gene is shared by these two archaic genomes, as expected based on the existing estimates of fixation of the mutation over 2 ma (hayakawa et al. 2001) . notably, almost all other human cd33rsiglecs variants (except for cd33-linked variants protective against late onset alzheimer's disease) were also present in these archaic hominin genomes in variable frequency (listed in table 1 and fig. 1 ) placing their origin before the human-neanderthal common ancestor ($0.6 ma) (prufer et al. 2017; hajdinjak et al. 2018 ). due to unavailability of well annotated sequences, the siglec14 fusion/deletion could not be determined in archaic hominins. the presence of all genomic siglec changes in human populations indicates that they predate the common ancestor of modern humans $0.2-0.3 ma (hublin et al. 2017) . consistent with this observation, the recent article by moon et al. (2018) found that the patterns of genetic variation of most cd33rsiglec genes did not significantly deviate from neutral expectations, and the few that did significantly deviate from neutrality experienced either soft sweeps or populationspecific hard sweeps (moon et al. 2018) . using a method that allows detection of selection in deeper time ($0.5 ma) (peyregne et al. 2017) , we also found no evidence of strong selection after the ancestors of modern and archaic humans (neanderthal-denisovan) split from each other around 500,000 years ago. as selection may also target neighboring regulatory regions, we defined regulatory domains around each gene and looked for overlaps with candidate sweep regions. using these extended gene coordinates, we again found no overlap with candidate regions for selective sweeps, suggesting that none of the genes involved in sialic acid biology exhibits strong signatures of selection more recently than 0.5 myr in the common ancestral population of all modern humans. the description of extended lineage sorting (els) is provided in figure 2. in addition to these multiple, polymorphic complex genomic changes in the human cd33rsiglec gene cluster, there appear to be unusual (derived) human-specific expression patterns of cd33rsiglecs (in nonhemopoietic cells) in locations such as placental trophoblast (siglec6) (kang et al. 2011) , ovarian fibroblasts (siglec11/16) (wang et al. 2011) , amniotic epithelium (siglec5/14) (ali et al. 2014) , and microglia (siglec11/16) (hayakawa et al. 2005) . in each of the above instances, we have previously reported human-specific expression and lack of expression in chimpanzee tissue samples (in some instances also other available great ape tissues). with regard to the recent report of siglec7 expression in human pancreatic islets (yamaguchi et al. 2017) , we now show that such expression is missing in chimpanzee pancreatic islets (meyer et al. 2012) ; top rectangle to modern humans (bottom rectangles) at any given position along their genomes. there are two types of genealogies: the archaic falls either outside the modern human variation (external genealogy) or inside (internal genealogy). the internal genealogy is the most frequent in the genome. however, if a mutation spread among the ancestors of modern humans (blue dot) more recently than the population split with the ancestors of archaic humans, the archaic will be expected to fall outside the modern human variation in the genomic region around the mutation that has not been unlinked by recombination (blue rectangles). this region is expected to be large if the mutation was positively selected and spread quickly in the population, not leaving enough time for recombination to break its linkage with other mutations (black dots). (b) time scales for the signatures of selection in humans. the tree represents the simplified population history of neanderthals, denisovans, and modern humans (prufer et al. 2017) . the colors indicate the time scales investigated by the els scan (red) and other methods (blue) used to detect events of positive selection on the modern human lineage. ( fig. 3) . we also found that siglec-13, which is deleted in hominins, is highly expressed in chimpanzee intestinal epithelium and skin ( fig. 3 ). current and previously published data are summarized in table 2. although we obviously cannot detect tissue-specific expression in neanderthals or denisovans, it is reasonable to consider the possibility that these unusual human-specific expression patterns arose during the course of the large-scale changes affecting the cd33rsiglecs gene cluster during the proposed "hotspot" and would thus have already existed in both of those extinct relatives. recent availability of sequences of many modern human genomes (1000 genomes project consortium et al. 2015 , archaic hominins (green et al. 2010; reich et al. 2010 reich et al. , 2011 meyer et al. 2012 meyer et al. , 2016 castellano et al. 2014; prufer et al. 2014 prufer et al. , 2017 sawyer et al. 2015; kuhlwilm et al. 2016; slon et al. 2018) , and great apes (prado-martinez et al. 2013; xue et al. 2015; de manuel et al. 2016 ) provides a tool for understanding the role of evolution in shaping genetic architecture and allowing us to identify the genetic basis of phenotypic traits of various species (haffter et al. 1996; bryk and tautz 2014; valenzano et al. 2015; castellano and munch 2020) . our earlier studies noted multiple genomic changes affecting sialic acid biology, and we suggested the possibility of a hotspot in humans affecting these pathways compared with our closest evolutionary relatives (altheide et al. 2006) . however, the limited availability of annotated and high coverage genomes at the time left the question unresolved, and our suggestion has been recently tested by others (moon et al. 2018 ), who could not find evidence for strong selective sweeps in current human populations. we herewith show that apart from the fixed cmah null mutation and increased expression of st6gal1 (gagneux et al. 2003) , most of the human-specific changes affecting sialic acid biology are found in the siglec gene cluster on chromosome 19, and that although great ape genomes do not show many changes in this cluster, almost all the human changes are also found in archaic genomes of neanderthals and denisovans (reich et al. 2010; castellano et al. 2014; prufer et al. 2014; slon et al. 2018; bokelmann et al. 2019 ). in keeping with this overall conclusion, there was no evidence for strong selective pressure in this cluster after $0.6 ma, when the human lineage diverged from neanderthal-denisovan common ancestor. with regard to timing and order of changes, the most likely possibility is that the cmah mutation first dramatically altered sialic acids throughout the body, possibly initiating a series of events that resulted in siglec cluster changes. the complex and dynamic relationships between host and pathogens is often characterized by the red queen hypothesis, whereby slowly evolving hosts have to keep changing to keep pace with the pressure exerted by more rapidly evolving pathogens. sialic acids form one primary interface, "the molecular frontier" in this evolutionary arms race. the loss-of-function of cmah and the changes in st6gal1 expression undoubtedly led to multiple alterations of human-pathogen interactions. the direct consequence was the change in human cell surfaces, leading to overexpression of neu5ac, escaping neu5gcspecific pathogens, and favoring human pathogens that interact with neu5ac. there are many other known and possible biological consequences of human neu5gc loss (varki 2009; okerblom and varki 2017) , including protection from various neu5gc-recognizing pathogens such as malaria caused by plasmodium reichenowi in african great apes (martin et al. 2005) , escherichia coli k99 gastroenteritis (kyogashima et al. 1989) , transmissible gastroenteritis coronavirus (schwegmann-wessels and herrler 2006), and simian virus 40 (campanero-rhodes et al. 2007 ). conversely, cmah loss likely made humans susceptible to neu5ac-preferring pathogens such as vibrio cholerae (causative agent of the human-specific disease cholera) (alisson-silva et al. 2018) and typhoid fever caused by a secreted bacterial toxin that (deng et al. 2014) . another human-specific pathogen streptococcus pneumoniae recognizes free neu5ac released from human cells by its secreted sialidase (hentrich et al. 2016 ). yet another secondary consequence appears to have been the evolution of pathogens that express neu5ac on their surfaces as a way to engage cd33related siglecs and downregulate innate immune responses in the human host. again, the existence of many human-specific pathogens that display neu5ac supports this scenario (angata 2018) . for example, group b streptococcus type iii, interacts with siglec-9 through sialylated cps and inhibits inflammatory response by neutrophils (carlin et al. 2009 ). likewise, nontypeable haemophilus influenzae expressing sialic acid on its lipooligosaccharide binds to siglec-5 and reduces cytokine production by myeloid cells (angata et al. 2013) , and the e. coli k1 strain that causes meningitis in neonates and infection in urinary tract, engages siglec-11, and escapes killing (hayakawa et al. 2017) . also, as expected, nonhuman primate siglec-9 prefers binding to neu5gc, whereas human siglec-9 prefers binding to neu5ac (sonnenburg et al. 2004) . likewise, human siglec-5 and cd33 prefer binding to neu5ac, compared with the baboon cd33, which strongly prefers neu5gc (padler-karavani et al. 2014 ). these findings imply that siglecs underwent adaptation in new environments to "catch up" with the changes in human sialome caused by the hominin cmah mutation. some human siglecs still prefer binding to neu5gc (angata 2018) , perhaps due to incomplete adaptation to the derived, human neu5ac-dominant sialome. although these genomic changes are ancient, evidence also suggests that existing human polymorphisms are associated with several diseases such as chronic obstructive pulmonary disease, asthma, alzheimer's disease, and meningitis (yamanaka et al. 2009; gao et al. 2010; ali et al. 2014; schwarz et al. 2016 ). in addition, human siglec-xii and chimpanzee siglec12 are expressed on macrophages and luminal epithelia (mitra et al. 2011) . however, human siglec-xii harbors a universal mutation (r122c) that makes the protein unable to recognize sialic acid. interestingly, chimpanzee siglec12 and human siglec-xii with its arginine experimentally restored strongly prefer neu5gc (mitra et al. 2011) . these results suggest a scenario where siglec-12 lost an endogenous ligand and is thus being eliminated from the population. prior studies have reported human-specific siglec expression changes in placenta (siglec-6) (brinkmanvan der linden et al. 2007 ), brain microglia (cd33/siglec11/16) (hayakawa et al. 2005; schwarz et al. 2016) , amniotic epithelium (siglec-5/14) (ali et al. 2014) , ovarian fibroblasts (siglec11/16) (wang et al. 2011), and nk cells (siglec-17) (wang, mitra, secundino, et al. 2012) . we here add to these expression differences by showing that siglec-7 is upregulated in human but not chimpanzee pancreatic islets, and that the siglec13 deletion resulted in a loss of siglec-13 expression from human epithelia. many of these expression changes could represent secondary consequences of multiple genomic changes that occurred in this gene cluster earlier in the hominin lineage. overall, it is likely that the selective pressures driving all these changes were most prominent sometime after the split from the common ancestor of human and chimpanzee, but before the split from the human-neanderthal-denisovan common ancestor. taken together, our data suggest that innate immune encounters with pathogens markedly altered hominin siglec biology between 0.6 and 2 ma, potentially affecting human evolution. notably, this is the time period when a variety of changes were occurring in genus homo, including exploration of new habitats (white et al. 1993; semaw et al. 1997; demenocal 2011) , striding bipedalism and running (bramble and lieberman 2004; lieberman 2015) , and scavenging and hunting, involving butchery of animal carcasses with stone tools (semaw et al. 1997; o'connell et al. 2002; mcpherron et al. 2010; sayers and lovejoy 2014; harmand et al. 2015; baird et al. 2016) , activities that may have increased risk of injury, novel infections, and use of fire (smith et al. 2015) . in this regard, it is also interesting that human-like elimination of cmah in mice enhances running ability (okerblom et al. 2018) as well as macrophage activation ). supplementary data are available at genome biology and evolution online. a global reference for human genetic variation the ensembl gene annotation system siglec-5 and siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group b streptococcus human evolutionary loss of epithelial neu5gc expression and species-specific 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in the placenta copy number variants and selective sweeps in natural populations of the house mouse (mus musculus domesticus). front genet global analysis of alternative splicing differences between humans and chimpanzees n-glycolyl gm1 ganglioside as a receptor for simian virus 40 group b streptococcus suppression of phagocyte functions by protein-mediated engagement of human siglec-5 population genomics in the great apes patterns of coding variation in the complete exomes of three neandertals initial sequence of the chimpanzee genome and comparison with the human genome a mutation in human cmp-sialic acid hydroxylase occurred after the homo-pan divergence inactivation of cmp-n-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution extensive changes in the expression of the opioid genes between humans and chimpanzees human genetics: muscling in on hominid evolution chimpanzee genomic diversity reveals ancient admixture with bonobos anthropology. 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cmp-nacetylneuraminic acid hydroxylase gene a human-specific gene in microglia fixation of the human-specific cmp-n-acetylneuraminic acid hydroxylase pseudogene and implications of haplotype diversity for human evolution coevolution of siglec-11 and siglec-16 via gene conversion in primates streptococcus pneumoniae senses a human-like sialic acid profile via the response regulator ciar new fossils from jebel irhoud, morocco and the pan-african origin of homo sapiens positive selection of a gene family during the emergence of humans and african apes preeclampsia leads to dysregulation of various signaling pathways in placenta understanding the recent evolution of the human genome: insights from human-chimpanzee genome comparisons maximum reproductive lifespan correlates with cd33rsiglec gene number: implications for nadph oxidasederived reactive oxygen species in aging microrna biogenesis: coordinated cropping and dicing evolution at two levels in humans and chimpanzees high-resolution comparative analysis of great ape genomes ancient gene flow from early modern humans into eastern neanderthals escherichia coli k99 binds to n-glycolylsialoparagloboside and n-glycolyl-gm3 found in piglet small intestine human locomotion and heat loss: an evolutionary perspective evolution of human-chimpanzee differences in malaria susceptibility: relationship to human genetic loss of n-glycolylneuraminic acid evidence for stone-tool-assisted consumption of animal tissues before 3.39 million years ago at dikika, ethiopia revealing the world of rna interference a high-coverage genome sequence from an archaic denisovan individual nuclear dna sequences from the middle pleistocene sima de los huesos hominins siglec12, a human-specific segregating (pseudo)-gene, encodes a signaling molecule expressed in prostate carcinomas examination of signatures of recent positive selection on genes involved in human sialic acid biology a structural difference between the cell surfaces of humans and the great apes loss of siglec expression on t lymphocytes during human evolution evolution of genetic and genomic features unique to the human lineage male strategies and plio-pleistocene archaeology biochemical, cellular, physiological, and pathological consequences of human loss of n-glycolylneuraminic acid human-like cmah inactivation in mice increases running endurance and decreases muscle fatigability: implications for human evolution loss of cmah during human evolution primed the monocyte-macrophage lineage toward a more inflammatory and phagocytic state genome sequencing of chimpanzee malaria parasites reveals possible pathways of adaptation to human hosts rapid evolution of binding specificities and expression patterns of inhibitory cd33-related siglecs in primates detecting ancient positive selection in humans using extended lineage sorting great ape genetic diversity and population history the complete genome sequence of a neanderthal from the altai mountains a high-coverage 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of domain-specific functional adaptation in a sialic acidbinding receptor myosin gene mutation correlates with anatomical changes in the human lineage an evolutionary medicine perspective on neandertal extinction the african turquoise killifish genome provides insights into evolution and genetic architecture of lifespan a chimpanzee genome project is a biomedical imperative multiple changes in sialic acid biology during human evolution since there are pamps and damps, there must be samps? glycan "self-associated molecular patterns" dampen innate immunity, but pathogens can mimic them siglecs-the major subfamily of i-type lectins symbol nomenclature for graphical representations of glycans evolution of siglec-11 and siglec-16 genes in hominins specific inactivation of two immunomodulatory siglec genes during human evolution expression of siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology dna sequence and comparative analysis of chimpanzee chromosome 22 new discoveries of australopithecus at maka in ethiopia hominin taxic diversity: fact or fantasy mountain gorilla genomes reveal the impact of longterm population decline and inbreeding chemical synthesis and evaluation of a disialic acid-containing dextran polymer as an inhibitor for the interaction between siglec 7 and its ligand deletion polymorphism of siglec14 and its functional implications associate editor: naruya saitou we thank members of the varki key: cord-327063-ea7a1xfl authors: dhama, kuldeep; patel, shailesh kumar; sharun, khan; pathak, mamta; tiwari, ruchi; yatoo, mohd iqbal; malik, yashpal singh; sah, ranjit; rabaan, ali a.; panwar, parmod kumar; singh, karam pal; michalak, izabela; chaicumpa, wanpen; martinez-pulgarin, dayron f.; bonilla-aldana, d. katterine; rodriguez-morales, alfonso j. title: sars-cov-2 jumping the species barrier: zoonotic lessons from sars, mers and recent advances to combat this pandemic virus date: 2020-08-02 journal: travel med infect dis doi: 10.1016/j.tmaid.2020.101830 sha: doc_id: 327063 cord_uid: ea7a1xfl coronavirus disease 2019 (covid-19), caused by sars-cov-2 (severe acute respiratory syndrome coronavirus-2) of the family coronaviridae, appeared in china in december 2019. this disease was declared as posing public health international emergency by world health organization on january 30, 2020, attained the status of a very high-risk category on february 29, and now having a pandemic status (march 11). covid-19 has presently spread to more than 215 countries/territories while killing nearly 0.62 million humans out of cumulative confirmed infected asymptomatic or symptomatic cases accounting to almost 15 million as of july 22, 2020, within a short period of just a few months. researchers worldwide are pacing with high efforts to counter the spread of this virus and to design effective vaccines and therapeutics/drugs. few of the studies have shown the potential of the animal-human interface and zoonotic links in the origin of sars-cov-2. exploring the possible zoonosis and revealing the factors responsible for its initial transmission from animals to humans will pave ways to design and implement effective preventive and control strategies to counter the covid-19. the present review presents a comprehensive overview of covid-19 and sars-cov-2, with emphasis on the role of animals and their jumping the cross-species barriers, experiences learned from sarsand mers-covs, zoonotic links, and spillover events, transmission to humans and rapid spread, and highlights the new advances in diagnosis, vaccine and therapies, preventive and control measures, one health concept along with recent research developments to counter this pandemic disease. in the 21 st century, we have faced a few deadly disease outbreaks caused by pathogenic viruses such as bird flu caused by avian influenza virus h5n1, swine flu caused by reassorted influenza virus h1n1 pandemic 2009 (h1n1pdm2009), severe acute respiratory syndrome (sars) caused by sars-cov (coronavirus), the middle east respiratory syndrome (mers) caused by mers-cov [1] [2] [3] , ebola [4] , zika [5, 6] , nipah virus infections, and the most recent threat [7] , coronavirus disease 2019 (covid19 ) that has been posed by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) of the family coronaviridae, genus betacoronavirus [8] [9] [10] [11] [12] [13] [14] . the sars-cov-2 virus emerged from the city of wuhan, hubei province, china, during december 2019, was declared as public health international emergency by the world health organization (who) on january 31, 2020. consequently, it was categorized in a high-risk category on february 29, 2020, and gained the pandemic status on march 11, 2020 . the disease emerged from wuhan, china, as its epicentre, which moved later to italy, then the usa, and brazil. subsequently, within a short time interval of six months, it has affected nearly 215 countries/territories and claimed near to 0.62 million human deaths out of cumulative confirmed infected asymptomatic or symptomatic cases accounting to almost 15 million. sars-cov-2 has very adversely affected the usa, brazil, india, russia, south africa, peru, mexico, chile, spain, the united kingdom (uk), iran, pakistan, saudi arabia, italy and other countries. the disease incidences are lower in children than adults but exhibit all symptoms of a disease like adults [15] . the lessons learned from earlier threats of sars, mers and the present covid-19 pandemic situations warrants designing and implementing some modified plans and strategies to combat emerging and zoonotic pathogens that could pose pandemic threats/risks while taking away many human lives [11, [16] [17] [18] [19] [20] [21] [22] . researchers and health agencies across the world are putting high efforts to contain/restrain the spread of this deadly disease. they are pacing to develop potential vaccines and therapeutics/drugs [23, 24] . evidence from the initial outbreak indicates earlier cases had links to huanan wholesale seafood market in china [25] and further isolation of sars-cov-2 from different samples of the area (people, animals, birds, discharges, soil, structures) suggests the involvement of intermediate hosts [26] . recently, a literature of review has pointed out the possible potential role of the animal-human interface, zoonotic links and spillover events towards the origin of sars-cov-2/ covid-19 [11, 20, [27] [28] [29] [30] [31] . in the past couple of decade's animal origin viral diseases, especially bats-linked, have increased many folds in humans with noted cross-species transmissions. although many of the illnesses are linked with bats still information on their ecological behaviour, molecular aspects are limited, which could lead to more viral outbreaks shortly [32] . the ongoing covid-19 pandemic has emphasized the importance of understanding the evolution of natural hosts in response to viral pathogens. in a recent study on ace2 receptors, the gene was found under intense selection pressure in bats and positive selection in other selected mammalian hosts [33] . the sars-cov-2 is also thought to have originated from bats, just like sars-cov and mers-cov. civets and dromedary camels are considered as the intermediate host of sars-and mers-cov, respectively, from where they were transmitted to humans [34] . the understanding of genomic signatures of sars-cov-2 with other covs is must for strategic planning through identifying natural or intermediate hosts. using genomic and protein data in a natural vector method (alignment-free approach), phylogenetic analysis revealed the possible transmission path originates from bats to pangolins to humans [35] . however, the likely source of virus origin and the intermediate host of sars-cov-2 are yet to be identified. initially, when the novel virus emerged in china, a hypothesis was put forward, claiming the recent recombination event as the cause of the sars-cov-2 emergence . nevertheless, the phylogenetic and recombination analysis performed within the subgenus of sarbecovirus demonstrated that the novel virus shows discordant clustering with bat-sars-like coronavirus (ratg13) sequences thus rejecting the possibility of a recent recombination event [36] . previously, it was found that the continuous passaging of mers-cov in non-susceptible cells that express viral receptors led to the accumulation of mutations in the spike protein gene. this paid attention to the potential of coronaviruses like mers-cov to undergo mutations that enhance viral entry into novel animal species, thus resulting in cross-species transmission [37] . the covid-19 outbreak is still associated with several unanswered questions like the possibility of shedding of the virus before the onset of clinical signs, whether the transmission is limited to only through respiratory droplets, the possibility of an intermediate host that is responsible for zoonotic spillover, and the possible transmission characteristics [20, 38] . hitherto studies report that the spillover risk remains high from zoonotic viruses and on the same lines a study from north america proposed a hypothesized conceptual model demonstrating sars-cov-2 spillover from humans to naive wildlife host species through the gastrointestinal route where stool from covid-19 infected patient contaminates water bodies and reaches to wildlife hosts [39] . besides, the pandemic imposed a massive blow on the chinese economy, which is not going to heal soon [40] . instead of the current situation, singapore's prime minister lee hsien loong rightly said that the virus might have started in china. however, it does not respect nationality or race. it does not check your passport before it goes into your body, and anybody can be infected. hence, all suspected people need to be tested and quarantined [41] . further research exploring the sars-cov-2 associated zoonosis and mechanisms accounting for its initial transmission from animals to humans, will lead to sort out the spread of this virus as well as design and develop appropriate prevention and control strategies to counter covid-19. the present comprehensive manuscript presents an overview on covid-19, an emerging sars-cov-2 infectious disease while focusing mainly on the events and circumstantial evidences with regards to this virus jumping the species barriers, sharing a few lessons learned from sars-and mers-covs, zoonotic spillover events (zoonosis), acquiring transmission ability to infect humans, and adopting appropriate preventive and control measures [42] . it also highlights the recent advances in sars-cov-2 diagnosis (see supplement material s1), vaccine, drugs and therapies (see supplement material s2), which could aid to counter and restrain this emerging virus at the face of pandemic situations, as well as prevention and control (see supplement material s3). sars-cov-2 is an enveloped virus measuring approximately 50-200 nm in diameter with a single strand positive-sense rna genome ranging from 26 to 32 kilobases in length [43, 44] . it has club-shaped glycoprotein spikes in the envelope, giving it a crown-like or coronal appearance [25] . the genome sars-cov-2 is comprised of 5′ untranslated region (5′ utr) that includes 5′ leader sequence, open reading frame (orf) 1a/b (replicase genes), spike (s) protein, envelop (e) protein, membrane/matrix (m) protein, and accessory proteins (orf 3,6,7a, 7b, 8 and 9b), nucleoprotein (n), and 3′ untranslated region (3′ utr) in their sequence [45] . it has a 50% genetic identity to mers-cov and 80% to sars-cov [43, 46] . the receptor-binding domain (rbd) of virus spikes helps in binding to cellular receptor angiotensin-converting enzyme 2 (ace-2) [47, 48] . orf and rbd of sars-cov-2 may have a role in elucidating cellular interactions and cross-species transmission mechanisms [49] . receptor binding motifs (rbm) have a role in interaction with human receptors, human to human transmission, and cross-species transmission as gln493 provides favourable interaction, and asn501 shows compatibility with human ace-2 [47] . besides, sars-cov-2 has superior transmission competence in comparison to the sars-cov, leading to a continuously increasing number of confirmed cases [50] . sars-cov-2 has the potential to survive in the environment for several days [51] . though believed to be sensitive to environmental factors and alcohol-based sanitizers, bleach, and chloroform, the sars-cov-2 can survive in wet surroundings for days and in closed air conditions up to 12 hours [26, 52] . survival of sars-cov-2 varies with the nature of the surface (glass, fabric, metal, plastic, or paper), environment, and virus load. it can survive on surfaces for hours to several days. it can survive in aerosols for up to 3 hours and on plastic for up to 72 hours [26, 53] . the initial clinical picture of the covid-19 was pneumonia of unknown origin as the first clinical cases were presented with signs of pneumonia [38] . later it was diagnosed as sars-cov-2 infection that was associated with severe pneumonia. hence, initially named as novel coronavirus pneumonia (ncp) [54] . as the outbreak proceeded, a series of cases were produced, developing a wide range of clinical signs with few remaining asymptomatic being in the early incubation stage of the disease. thus covid-19 is characterized by three major patterns of the clinical course of infection, including mild illness producing upper respiratory signs, non-life-threatening pneumonia, and severe pneumonia with acute respiratory distress syndrome (ards) [55, 56] . initially, mild signs appear for 7-8 days, followed by rapid deterioration and ards. it can be mild to moderate in 80% of affected cases, including pneumonia and non-pneumonia cases. in comparison, 13.8% are severe cases, including dyspnea, respiratory distress, hemoptysis, gastrointestinal infection, liver, central nervous system, and lung damage cases [57, 58] . critical cases account for 6.1% and include respiratory failure, septic shock, and multiple organ failure/dysfunction cases. few cases remain asymptomatic and include cases that can become any of the above during infection [26] . thus, the symptoms can be nonspecific and can range from no symptoms (asymptomatic) to severe pneumonia [26] . in this context, a study concluded that the covid-19 is probably overestimated, as around 2.6 million people succumb to respiratory diseases every year in comparison to approximately 400,000 deaths due to the sars-cov-2 infection [59] . the typical clinical signs of covid-19 are fever, chills, cough, fatigue, and chest distress [60, 61] . fever and cough are considered as the most common symptoms in covid-19 patients [62] , followed by headache, dyspnea, sore throat, hemoptysis, myalgia, diarrhoea, nausea, and vomiting are also observed [61, 62] . some patients have shown rhinorrhea, chest pain [25] , nasal congestion [26] , anorexia, pharyngalgia, and abdominal pain [63] . furthermore, neurological symptoms like anosmia and ageusia are also reported as significant clinical symptoms of covid-19 [64] . the characteristic of covid-19 is attacking the lower respiratory tract and producing signs of upper respiratory distress, including rhinorrhea, sneezing, and sore throat [49] . the clinical presentation of individuals infected with sars-cov-2 revealed upper respiratory tract infection, viremia, viral shedding from the nasopharynx, and stool along with the development of nausea, vomiting or diarrhoea after antiviral treatment [65] . on diagnostic imaging using computed tomography (ct scan) and radiography (x-ray) bilateral pneumonia, ground-glass opacity, multiple mottling, pneumothorax, infiltration, consolidation or bronchoinflation sign has been noted in many cases of covid-19 [25, 49, 66] . previously, sars-cov was found to infect the brainstem heavily [67] . even though fever is considered as the most common symptom associated with covid-19 infection, a large proportion of the patients do not express fever during the initial hospital admission [68] . the different transmission routes of sars-cov-2 infections have not yet been entirely ascertained, and are still under investigation. both direct and indirect pathways of transmission are being explored [69] . similar to sars and mers, sars-cov-2 is predominantly spread via the respiratory route [70] . person to person transmission is the main reason for community and global spread. the initial estimated reproduction number (rovalue) of covid-19 was assessed to be from 0.8 to 2.4 in december 2019, which later has been increased to a mean value of 2.6 (range 2.1-5.1) [71] . human-to-human transmission is by face-to-face contact with a sneeze or cough, or from contact with secretions of infected people [56, 70] . nevertheless, the infectivity of other secretions and excretions are not fully understood and may require further study [45] . aerosol and plastic surfaces can sustain virus for hours to days [53] . travelling of infected people is considered as the main reason for the global spread of covid-19 [20, 56] . although the asymptomatic and mild cases are the major hurdle in the evaluation of the real number of infected people, the genuine data on travellers returning from affected countries or areas may prove crucial in estimating the disease incidence [72] . the possible occurrence of super-spreading events is very high at large gatherings, and suspension of gathering during a pandemic may prove crucial in reducing the overall transmission [73] . close contact with any person within 6 feet of the covid-19 patient or anyone having direct contact with secretions of covid-19 patients [74] may set up the infection. unlike sars-cov, most transmissions in covid-19 are during the prodromal period when the infected individuals produce large quantities of virus in the upper respiratory tract, move/travel, and usually work thus spreading virus before illness develops [56] . the rapid spread of covid-19 among the susceptible population can be due to the wide variation in illness degrees that results in a missed diagnosis. heavy viral load in asymptomatic cases and nosocomial transmission is spreading covid-19 unknowingly [75] . recently, high viral load was detected in the sputum of convalescent patients, pointing out the possibility of prolonged shedding of sars-cov-2 even after recovery. this finding, along with the fact that asymptomatic persons can also act as the potential source of infection, may warrant a reassessment in the transmission dynamics of the covid-19 outbreak [76] . the presence of viral nucleic acids in faeces is an important finding, thereby increasing the possibility of faecal-oral transmission. however, symptoms may or may not be manifested [77] . this was found to be the unique feature of covid-19 and was lacking in the previous sars and mers outbreaks. in a study conducted by the chinese cdc, it was found that the majority of patients (80.9%) infected with covid-19 infection were either asymptomatic or had mild pneumonia [56, 66] . furthermore, all forms of sexual contacts have been reported to pose a significantly high risk of disease transmission through respiratory aerosols and fomites. however, to date, no evidence of sexual transmission is available, and further investigation is required in this direction [78] . disease severity is higher in older individuals, especially males with immunocompromised conditions and comorbidities like diabetes, asthma, or cardiovascular diseases [26, 66] . these are considered to be vulnerable to the sars-cov-2 infection. predisposition increases under risk environments where transmission of the virus from affected persons or contaminated fomites to unaffected ones becomes feasible. it was earlier noted that covs are not common to affect immunocompromised patients like other some viral infections (influenza, rhinovirus, adenoviruses, to name a few). the current pandemic has shown sars-cov-2 to affect more lethally than young patients, mainly destroying the lung tissues [79] . till now, evidence regarding the higher susceptibility of pregnant women in comparison to non-pregnant women lacks in covid-19. also, there is no evidence of vertical transmission (mother to fetus/baby transmission) of covid-19 infection [80] . a case study reporting the birth of a healthy infant by a sars-cov-2 infected woman suggests that mother-to-child transmission is unlikely in the case of covid-19. the study also pointed out that on the delivery day, all the samples tested negative except for sputum, which proved positive [81] . however, as per one most recent report, neonates have been found positive for sars-cov-2, indicating the possibility of vertical transmission from infected mothers to their progeny, thus rendering newborns into a high-risk group owing to their immature immune system [82] . individuals harbouring sars-cov-2 may remain asymptomatic for the incubation period [83] . different from sars-cov and mers-cov infection, the median incubation period of covid-19 was found to be four days [68] . the median period from the development of signs to death was 14 days [84] . the case fatality rate (cfr) of covid-19 was found to be lower than mers and sars [62] . however, current disease dynamics with the involvement of many more countries or areas may change the future mortality rate. the recent analysis suggests that the total fatality rate of covid-19 is calculated at 3.46% [75] . however, italy experienced the worst cfr of more than 9% with older people and males suffering from multiple comorbidities as primary victims [85] . sars-cov-2 has shown characteristics of efficient replication in the upper respiratory tract, causing the less abrupt onset of clinical signs just like the common cold and unlike sars-cov [70] . it can also replicate in the lower respiratory tract as has been noted in cases without pneumonia but having lesions in the lungs on radiological examination [70] . the pathogenesis mechanisms of covid-19 are yet to be fully elucidated. however, both cellular and humoral immune responses against sars-cov-2 or its antigenic structures like spike protein (s) are believed to be of importance [86, 87] with disturbed levels of inflammatory mediators playing a mediating role [66] . following receptor binding with angiotensin-converting enzyme 2 (ace2) through receptor binding motif (rbm) of the receptor-binding domain (rbd) of s1 subunit of the sars-cov-2 spike glycoprotein (s), virus gains entry in host cells [47, 88, 89] . s2 subunit helps in the fusion of viral and hosts cell membranes [47, 90] . sars-cov-2 produces cytopathic effects in respiratory and gastrointestinal surface epithelial cells [91] . these include multinucleated syncytial cells, abnormally enlarged pulmonary cells, infiltration with mononuclear cells, lymphocytes infiltration in pulmonary organs, fibrinous exudation, and hyaline deposition [66] . cytokine storm is believed to be involved in this inflammatory pathophysiology of the covid-19 patients producing lung lesions and systemic symptoms [66] . elevated levels of tnf-α, il1b, ifnγ, ip10, gcsf, mip1a, and mcp1, may have stimulated t-helper-1 (th1) cells leading to this inflammatory cascade [66] . however, levels of anti-inflammatory mediators (il4, il10) were also increased, indicating t-helper-2 (th2) stimulation, which suppresses inflammation, unlike what happens in sars [66] . a study documented that the nucleic acid of sars-cov-2 detected in the faecal samples was as accurate as of that of pharyngeal samples obtained from infected patients. moreover, the patients tested positive for sars-cov-2 in stool showed no gastrointestinal symptoms and had no relation to the severity of lung infections [92] . one of the significant clinical signs of covid-19 patients during the initial presentations was gastrointestinal symptoms. hence, the involvement of git in pathogenesis needs to be explored. the significant laboratory findings include lymphopenia, increased values of erythrocyte sedimentation rate, c-reactive protein, lactate dehydrogenase, and decreased oxygenation index [61] . an increase in proinflammatory cytokine and a decrease in antiinflammatory cytokines have also been noted [66] . viral isolation has been achieved from bronchoalveolar lavage of affected persons; however, in the case of pregnant women, serum, faeces, urine, breast milk, umbilical cord blood, placenta, and amniotic fluid were found to be negative for sars-cov-2 [81] . at the same time, the sputum was tested positive [82] . the presence of abnormal coagulation parameters in patients with severe novel coronavirus pneumonia was associated with poor prognosis. the non-survivor patients had higher levels of d-dimer, and fibrin degradation product (fdp) along with longer activated partial thromboplastin time and prothrombin time compared to survivors at the time of admission [93] . though clinical manifestations, pathological changes, and diagnostic laboratory findings can unravel the disease nature helping in devising therapeutic modalities, however, for epidemiological aspects and future prevention and control, simultaneous tracing of the origin and explaining the spillover events can prove beneficial. the sars-cov-2 has first been reported from the pneumonia patients of the wuhan city in hubei province of china. these patients were involved in trading at a wet animal market in the huanan area. it is believed that sars-cov-2 is introduced from the animal kingdom to human populations during november or december 2019, as revealed from the phylogeny of the genomic sequences from the initially reported cases [94] . the spillover of sars-cov-2 from animals to humans took place at the beginning of december 2019 [56] , and the clinical cases appeared around ending december [46, 95] . genetic analysis showed that this novel virus is closely related to bat covs and is similar but distinct from the sars virus [43] . several evidences based on genome sequences, the homology of the ace2 receptor, and the presence of single intact orf on gene 8 indicate bats as a natural reservoir of these viruses. however, an unknown animal is yet to be unravelled as an intermediate host [43, 46, 47, 49, 56] . initial investigations on animal source origin of sars-cov-2 have inconclusively revealed snakes [27] , pangolins, and turtles [96] . the rapid spread of covid-19 followed the initial animal to human spillover through human-to-human transmission. genetic epidemiology had revealed that the spread from the beginning of december when the first cases were retrospectively traced in wuhan was mainly by a human-to-human transmission and not due to continued spillover [56] . these species cross jumping, spillover, and rapid transmission events are linked to viral characteristics, host diversity, and environmental feasibility. coronaviruses being rna viruses have high mutation rates that, besides creating new strains, enable them to adapt to a wide range of hosts. hence, based on genome sequences, all known human covs have emerged from animal sources [97] . this seventh member of the human cov has also been isolated initially from the pneumonia patients who were having direct or indirect links to the huanan seafood market in wuhan china, wherein other animals were also being sold [98] . these include a 49-year-old lady retailer in this wet animal market, a 61-year-old frequent visitor to this market, and a 32-year-old man [74, 98] . further, isolation of the sars-cov-2 from the environmental samples around this market, including people, animals, soil, discharges, or structures, strengthens the claims of involvement of hosts either as a reservoir or intermediate [26, 52, 99] . recently, a pomeranian dog as a probable intermediate host was identified; however, such reports are yet to be validated, and research is underway to explore the emergence of this infectious disease at the animal-human interface [29, 99] . multiple substitutions were observed in ace2 receptors of a dog [96] . in this context, the pomeranian dog of the infected owner found positive for covid-19 suggest the permissiveness of the species for sars-cov-2 as a result of species jumping [99, 100] . among the fifteen dogs tested from different households with confirmed human covid-19 cases in hong kong sar, two dogs were found to be infected with sars-cov-2. the diagnosis was made using quantitative rt-pcr, serology, and viral genome sequencing. virus isolation was also done from the samples obtained from one dog. the genetic sequences of viruses obtained from the two dogs were identical to the ones that were detected from their human cases indicating human-to-dog transmission [101] . moreover, a study reported that the sars-cov-2 might infect the cats and further transmitted by the infected cat to other cats [102] . one cat was tested positive for sars-cov-2 in france that showed mild respiratory and digestive signs. the cat was tested positive by rt-qpcr on the rectal swab, and serological analysis identified the presence of antibodies against sars-cov-2. genome analysis further confirmed that the sars-cov-2 isolated from the cat belongs to the phylogenetic clade a2a seen in french human indicating humanto-animal transmission [103] . this is not the first time that a domestic cat has been found susceptible to zoonotic coronavirus. during the 2003 sars-cov outbreak, domestic cats were tested positive for sars-cov that were living near sars infected humans [21, 104] . even though experimental evidence indicates the possibility of sars-cov-2 transmission from infected to a susceptible cat close, sars-cov-2 transmission between cats or cat-tohumans are not reported under natural conditions [21] . furthermore, along with dogs and cats, the zoo animals like tigers and lions were also reported to get the sars-cov-2 infection and exhibit clinical signs such as vomiting, diarrhoea, dry cough, breathing difficulty and wheezing [105, 106] . spillover of sars-cov-2 was also reported in mink farms of netherlands, further increasing the concern of transmission to humans. outbreaks of sars-cov-2 were reported in two mink farms holding 12,000 and 7500 animals. the virus is suspected to be introduced by a farmworker having covid-19 [107, 108] . host-pathogen interactions and pathogenesis determine the severity and expression of disease [30, [109] [110] [111] . adaptation over time reduces the severity of infection as happened with hcovs; however, the emergence of novel viruses or strains due to genetic alterations or recombinations can enhance hardness producing novel diseases like covid-19 [111, 112] . evolutionarily, the balance of viral-human interaction and immune response against virus enables adaptation, thereby persistence in a host without severe or symptomatic disease when the aggravated pathogenesis results in mortality. hence, loss of sustainable hosts and transmission to novel hosts becomes inevitable for future sustainability [111, 112] . a pathogen cannot kill all its hosts, and for future sustainability, it adapts to some suitable host or spills over to a new host. sars-cov-2 has been implicated to be originated from animals, and associated with animal linkages, spillover events, cross-species barrier jumping and zoonosis [9, 20, 27, [29] [30] [31] 113] . since the beginning of 2002 till the end of 2019, three coronaviruses viz. sars-cov, mers-cov, and sars-cov-2 have caused havoc in the human population globally and will continue to do so. earlier identified betacoronaviruses (sars-cov and mers-cov) were reported in guangdong province of china in november 2002 and saudi arabia in 2012, respectively [114] . sars-cov-2 is the third zoonotic betacoronaviruses recognized in this century. however, the cfr of the sars-cov-2 is lower to date when compared with sars and mers. it should not be overlooked as many asymptomatic cases may remain undiagnosed due to the unavailability of diagnostic kits in china. with nearly 0.62 million deaths till the preparation of the manuscript, sars-cov-2 is proven to be deadliest as far as the number of deaths is concerned in comparison with sars-cov and mers-cov with 774 and 858 associated deaths, respectively [115, 116] . earlier, covid-19 was linked with the exposure to the huanan seafood market. however, individuals with no history of exposure above were also diagnosed with the illness, further supporting the human to human spread through droplets produced by cough and sneeze [66] . the spread of covid-19 that occurred with a high pace and lack of transparency in reporting the disease by the chinese health ministry and failure in the timely implementation of preventive measures has been considered as the primary contributor as stated earlier in sars [19, 117] . both sars-cov and sars-cov-2 showed prominent similarities in their pathogenesis and epidemics. in both cases, bats were considered as the natural host, and the cold temperature and low humidity in cold, dry winter provided conducive environmental conditions that promoted the survival of the virus in the environment [62] . further, moriyama et al. [118] assessed the significance of the environmental factor on host immune system targeting innate and adaptive both responses in the respiratory tract. zoonotic spillover is the transmission of pathogens to humans from vertebrate animals [119] . at present, these spillovers are of significant concern as in the past, many spillovers in the form of nipah, hendra, ebola, sars, mers, and ongoing covid-19 involving many animal species like pigs, horses, monkeys, camels, civets, among others, were documented. bovine covs have been reported to infect children and thus possess zoonotic potential [9, 13, 120, 121] . spillover is governed by the interaction of viral-specific proteins like s protein and host ace2 receptor [30, 31, 111, 112] . these s proteins have rbd in covs, which contain receptor binding motifs (rbm) that help in specific binding to host ace2 receptors [47, 122] . mutations in amino acid sequences of rbds results in a change in specificity of a receptor, interaction and binding, hence alteration in transmissibility, pathogenicity and cross-species jumping with a predisposition to novel and more severe diseases [110, 123] . in the case of sars-cov-2, rbd of s protein has 10-15 times affinity ace2r [88, 110] . it has furin recognition sequence "rrar" at the s1-s2 cleaving site that represents a functional site for the cellular serine protease tmprss2 thus increasing the efficiency of transmission and contagiousness [88, 90, 110] . in addition to enhanced binding affinity, electrostatic complementarity and hydrophobic interactions are critical to enhancing receptor binding and escaping antibody recognition by the rbd of sars-cov-2, thereby further increasing transmission capability and contagiousness [110] . a detailed investigation regarding the emergence of new coronavirus, host range, and transmissibility is crucial to understand such pandemics shortly. the literature revealed that before the appearance of sars-cov and mers-cov, human coronavirus (hcov) strains like hcov-nl63, hcov-229e, hcov-oc43, and hcov-hku1 were the covs strains producing mild infections in humans. however, their natural ancestral hosts were of animal origin, like bats for hcov-nl63, and hcov-229e and rodents were natural hosts for hcov-oc43 and hku1. these four hcovs were initially of low pathogenicity. to enhance the pathogenicity, they used intermediate hosts such as cattle for hcov-oc43 (natural host was rodent), and alpacas for hcov-229e (bats were natural host) and this way acquired the ability to infect human beings with serious health hazards [124] . added to the involvement of bats and pangolins, the recent reports revealing sars-cov-2 infection in cats, dogs, tigers, lions and minks have raised concerns over this virus affecting multiple animal species, and also points out towards the incidences of reverse zoonosis [31, 102, 106, 108, 125, 126] . the ferrets, cats, and primates are suggested to be good candidates for susceptibility to sars-cov-2 [123, 127] . covid-19 research and surveillance in companion and pet animals, livestock animals, zoo animal species, wildlife animal species as well as their handlers, veterinarians, and owners need to be enhanced during the pandemic, which would help to follow better integrated one health strategies [128] and appropriate preventive and mitigation to counter sars-cov-2 effectively [29, 126, [129] [130] [131] [132] [133] . significance of covid-19 monitoring and implementation of suitable public health measures among workers involved in meat and poultry processing facilities/industries has been emphasized, which would protect them as well as aid in preserving the critical meat and poultry production infrastructure and the meat products [134] . the involvement of intermediate hosts in maintaining and transmitting the virus to susceptible host predisposes humans to novel covs leading to the emergence of new diseases in humans. the currently ongoing sars-cov-2 / covid-19 pandemic has put on hold the entire world [111, 135] . the covs have frequently been associated with animal and human diseases and have a zoonotic interface [97, 111] . usually, one or more types of animal hosts are involved in the transmission cycle of covs to humans [30, 97] . that can be natural host, reservoir host, intermediate host or definitive host [31] . bats have been the natural hosts for human covs of alphacoronavirus (hcov-nl63, hcov-229e) and betacoronavirus (sars-cov, mers-cov, sars-cov-2) genera whereas for betacoronavirus members hcov-oc43 and hcov-hku1, rodents are the natural hosts. genome sequence analysis has revealed bats as a natural host for sars-cov-2 [30, 97] . in natural or reservoir hosts, covs adapts well, however, being unstable rna viruses, they keep multiplying continuously without producing disease thereby enabling persistence or survivability and accumulation of mutations over the time resulting in the emergence of newer and novel strains of viruses [97, 111, 136] . these unique strains or viruses occasionally spill over to other species including animals or humans, adapting to their body systems and hence broaden the biological host range for evolutionary sustainability; however, results in epidemiological widening of disease sphere as well [25, 31] . this transmission and adaptation scenario initiates a host-pathogen response resulting in the novel usually severe diseases that can at times be fatal in initial stages or over extended periods until virus pathogen adapts to host or the host develops sufficient immune defence [137, 138] . it has been reported that almost all hcovs have originated from animals like bats (sars-cov, mers-cov, hcov-nl63, and hcov-229e) and rodents (hcov-oc43 and hku1) [139, 140] . additionally, covs have been reported to infect several species of domestic and wild animals either clinically or subclinically [27, 28, 30, 141] . cattle, horses, camels, swine, dogs, cats, birds, rabbits, rodents, ferrets, mink, bats, snakes, frogs, marmots, hedgehogs, malayan pangolin along with other wild animals may serve as a reservoir host of coronavirus [9, 27, 44, 124, [142] [143] [144] [145] [146] [147] . in the context of sars-cov-2, snakes, pangolins and bats have been suspected as intermediate hosts since the first cases of covid-19 had links to huanan sea food market where different animals, birds, and wild animals were being sold along with seafood items [27, 28, 43, 109, 147, 148] . coronaviruses have been reported to cause salivary, enteric and respiratory infections in laboratory animals (mice, rat, guinea pig, and rabbit) and urinary tract infection, respiratory illness and reproductive disorder in poultry [9, 142] . in bovine, canine, feline and swine covs infections have resulted in diarrhoea, enteritis, respiratory illness, gastro-intestinal affections and nervous symptoms [120, 121, [149] [150] [151] . coronavirus, namely-sw1, has been reported in captive beluga whale using a panviral microarray method [152] . among all the assumptions on animal hosts as the intermediate host, genomic and evolutionary information from pangolins reveals the highest closeness to the sars-cov-2 than any other host covs isolates [153] . the spike protein, the main target of many studies searching for a cure of covid-19, has been found highly similar to sars-cov-2 and, thus, could serve as a surrogate system for further evaluations [153] . bats are the natural reservoir host of many covs. as reported earlier, 7 out of 11 alphacoronaviruses and 4 out of 9 betacoronaviruses as per the international committee on taxonomy of viruses (ictv) classification were solely originated from bats [97, 154] . according to the literature, bats have been regarded as a potential wildlife reservoir whereas civets and dromedary camels as intermediate hosts of sars-cov and mers-cov, respectively [155, 156] . the bat coronavirus, batcov ratg13, has shown higher relatedness to sars-cov-2 at the whole genome level and spike gene in particular [153] . coexistence and frequent recombination between highly diversified and prevalent bat sars-related coronaviruses (sarsr-cov) and coronaviruses may suggest the probable emergence of novel viruses shortly [157, 158] . benvenuto et al. [159] analyzed the whole genome sequences of different covs using fast unconstrained bayesian approximation (fubar) to understand the evolutionary and molecular epidemiology of sars-cov-2. the authors concluded that sars-cov-2 clustered with sequences of bat sars-like covs with a few mutations in nucleocapsid and spike glycoprotein, suggesting its probable transmission from the bats [159] . bats, especially horseshoe bats (rhinolophus spp.), are considered to be the known reservoirs of sars-related covs. since the bat origin, covs have always caused outbreaks in humans, studying the diversity and distribution of coronavirus populations in the bats will help to mitigate future outbreaks in humans and animals [160] . interestingly, bats play a crucial role in all the spillovers mentioned above, indicating their importance in the emergence of new viruses. the reason behind the emergence and broad host range of covs in the past and present might be due to unstable rna-dependent rna polymerase (rdrp), lack of proof-reading ability, high frequency of mutations in the receptor-binding domain of spike gene and genetic recombination [140, 161, 162] . bat covs have high diversity and great potential of spillover in different animal species, as reported earlier in civet cat and dromedary camel, leading to well-known pandemics sars and mers, respectively along with the recent spillover in pigs resulted in swine acute diarrhoea syndrome (sads). however, spillover resulted in the emergence of sads-cov, which showed a 95% genomic identity with bat coronavirus, which led to severe mortality with 24,693 deaths in neonatal piglets [160] . fortunately, it did not excel in the form of the third pandemic, and no human cases were reported till date. the spillover responsible for ongoing covid-19 is still under investigation and a matter of great concern for the researchers all around the globe. based on resampling similarity codon usage (rscu), snakes (bungarus multicinctus and naja atra) were suggested as wildlife reservoirs of sars-cov-2 and reported to be associated with the cross-species transmission [27] and later it was disapproved by other researchers [163, 164] . unfortunately, to date, the intermediate host of the sars-cov-2 is abstruse what results in its escalation in the human population around the globe. in this context, analyzing the interaction between the asn501 site in rbd of spike glycoprotein of sars-cov-2 and the residue at 41 sites of ace2 receptor of different hosts (pangolins, turtle, mouse, dog, cat, hamster and bat) revealed that tyrosine has higher receptor binding affinity than histidine suggesting pangolins and turtle be closer than bats to humans and maybe the probable intermediate hosts of sars-cov-2 [96] . however, this hypothesis was also contradicted by li et al. [28] based on an insertion of the unique peptide (prra) in the sars-cov-2 virus, which was lacking in covs from pangolins. moreover, sars-cov-2 showed higher similarity to the betacov/bat/yunnan/ratg13/2013 compared to the ones that were isolated from the pangolins, thereby denied the direct link of the virus from pangolins. however, further studies are required to confirm the role of pangolins in sars-cov-2 spread to humans. the receptor-binding domain of the spike protein of sars-cov interacts with the host receptor ace2 facilitating its potential of cross-species, as well as human-to-human transmission [47] . similarly, the spike protein of sars-cov-2 was reported to recognize ace2 receptors expressed in fish, amphibians, reptiles, birds, and mammals and has a more robust binding capacity (affinity) in comparison to sars-cov [89] . this suggests their involvement as probable natural and intermediate hosts [161] , which may further help in the selection of animal models for epidemic investigation and preventing its spread [47] . bat origin covs have been found to cross the species barrier that favoured their transmission via recombination/mutations in the rbd. the evidence of a virus outbreak that occurred in chinese pig farms suggests its possible cross-species [160, 165] . also, murine cells were found permissive for sars-cov after substitution of his353 with lys353 in the ace2 receptor of a mouse, which suggests the role of residue changes in the cross-species and human-to-human transmission [166] . mutation in residues at position 479 and 487 of receptor binding motif (rbm) of sars-cov was reported to play a role in civet-to-human and human-to-human transmission, respectively [167, 168] . the covs are more prone to recombination and mutations leading to variable host range, and resemblance of receptors in various hosts results in cross-species jumping [31, 112, 169] . genetic divergence due to these genetic alterations results in the evolution of newer viral strains having altered virulence, tissue tropism, and host range [170, 171] . moreover, the presence of threonine at position 487 was reported to enhance the binding affinity of rbm for the ace2 receptor of civet and humans [172] . however, many sarsrelated coronaviruses (sarsr-cov) have been reported in bats and used ace2 receptors for entry into a host cell, which showed its potential to infect humans directly without any intermediate host [173] . in addition to this, no direct transmission of sarsr-cov is reported from bats to humans to date. however, seropositivity on a serological investigation of individuals without prior exposure to sars-cov residing near bat caves in china revealed likely infection of humans by bat sarsr-cov and related viruses [174] . besides, the interspecies transmission potential of sarsr-covs is due to the orf8 gene [175] . as per reports, the sars-cov emerged via recombination of bat sarsr-covs, was transmitted to farmed civets along with other mammals, and these infected civets spread the virus to market civets. the virus was reported to undergo mutations in infected market civets before its spillover to humans. similarly, the mers-cov circulated for 30 years in camels before the pandemic [97, 176] supporting the hypothesis that after species jumping the exogenous viruses opted for adaptation to the environment and host before spillover to humans [177] . moreover, the possible spillover of other circulating bat sarsr-covs to humans from mammalian hosts soon is highly anticipated. the cross-species jumping and adaptation are determined by the presence of specific receptors on host tissues (like ace 2 receptor for hcov-nl63, sars-cov and sars-cov-2, dipeptidyl peptidase-4 for mers-cov, human aminopeptidase n for hcov-229e, 9-oacetylsialic acids for hcov-oc43, hcov-hku1) which help in binding and entry of the virus into host cells [30, 31] . these receptors are present in various body systems in animals and humans, including respiratory and gastrointestinal systems [30] . reservoir host animals including bats and rodents possess these receptors which are similar to those present in camels, masked palm civets (paguma larvata), or bovines, that act as an intermediate host for different covs [30, 110, 111] . presence of some of these receptors in humans like ace2 or dpp4 makes them vulnerable to cov infection like sars-cov and mers-cov causing sars and mers infections, respectively [31, 168] . the mers-cov spike was found to possess the capacity for adapting to species variation in the host receptor dpp4 [37] . the mechanism expressed by mers-cov in adapting to infect cells of new species might be present in the other coronaviruses. ace2 has also been found as a binding receptor for sars-cov-2 [47] . the species-specific variations in the host receptors limit the interaction with cov spike protein, and this is responsible for the development of the species barrier that prevents spillover infection. snakes, civets, and pangolins are considered as the potential intermediate hosts of covid-19. however, further confirmation is required by tracking the origin of the virus. this is critical for preventing additional exposure to this fatal virus [178] . the probability of the sars-cov-2 spread during incubation and convalescent period has been suggested [76] . as per reports, presence of covs has been observed in respiratory droplets, body fluids and inanimate objects with the ability to remain infectious for nine days on contaminated surfaces resulting in its risk of self-inoculation via mucous membranes of the eyes, mouth or nose [179] [180] [181] . nosocomial, as well as human-to-human transmission, have been reported to occur via virus-laden aerosols, contaminated hands or surfaces, and close community contact with an infected person [66, 182, 183] . the ocular route has been reported in the human-to-human transmission of sars-cov-2, as observed in sars-cov, suggesting the involvement of different ways other than the respiratory tract [184, 185] . later on, the probability of the faecal-oral route for potential transmission of the virus was also suggested [186] . the metatranscriptome sequencing of sars-cov-2 in the bronchoalveolar lavage fluid (balf) of infected individuals resulted in polymorphism in few intra-hosts variants, suggesting the in vivo evolution of the virus thereby affecting its virulence, transmissibility, and infectivity [187] . an overview of coronaviruses jumping the cross-species barriers, zoonotic covs transmitted from bats to animals before spillover to humans, and possible prospects for further transmission to mammalian hosts is depicted in figure 1 . the first two decades (2000-2020) of 21 st century have proven a nightmare for the countries around the globe considering the coronavirus zoonosis, including the ongoing crisis of covid-19 which has involved entire fields of global [188, 189] . the countries affected severely by previous covs were even not evolved entirely from the effects of sars and mers when the covid-19 struck almost the entire world. novel coronavirus sars-cov-2 has shaken all the sectors of the countries irrespective of being developed or underdeveloped including healthcare system, economics, trade, infrastructure, service and production sectors [190, 191] . being a zoonotic disease with still unknown intermediate host, undisclosed features of a novel viral pathogen, unclear modes of transmission and ecological aspects, less explored pathogenesis and substantial morbidity and considerable mortality, the safety of all is a matter of great concern, and thus the involvement of various authorities was sought since the inception of disease [26, 31, 192, 193] . the first time the need for one health concept has risen to a level that authorities in various countries implemented coordinated approaches between medical, veterinary, public health, wildlife, food safety, environmental departments and so on [193] [194] [195] . that involved acquiring suggestions, diagnosis and prevention and treatment measures and their implementation in collaboration. non-medical staff in association with the medical staff was employed for initial screening, quarantine, contact tracing when the expertise of molecular biologists or technicians from various disciplines was used in the laboratory diagnosis. medical staff provided the cure and management of the patients when the public health departments, including public health engineering, municipality, food and supplies ensured sanitation, hygiene, food supply and safety. imposing of lockdown was provided by security personnel's and the transport department facilitated the movement of stranded people. thus, this crises management strategy involved various agencies directly or indirectly. however, as the animal, human and environmental health is linked to one another, the prime and future efforts should primarily focus on all these aspects. in addition to regular hand hygiene, respiratory etiquette, social/physical distancing, use of personnel protective equipment (ppe) and food safety recommendations, one health approach encompasses the role of veterinary, medical and environmental specialists for the prevention and control of current covid-19 crises and investigating the animal origin of covid-19, regulating and limiting the sale and farming of wildlife species for food and taking a one health approach to food systems feeding the world for the prevention of future pandemics [193, 196, 197] . considering the contagiousness of the virus, discouraging the working of affected individuals, public health hygiene strategies, and social distancing has been recommended as preventive measures [26, 198] . food hygiene and safety, as recommended by oie [193] and usda [199] , should be followed. as the viral survivability has been demonstrated on various surfaces [53] hence disinfection by using recommended disinfectants is necessary [200] . environmental hygiene and cleanliness are also essential [200] . interaction with animals and improper utilization of animal products during an outbreak should be avoided [193] . though the one health involves mainly public health, animal health and environmental experts, however, for the successful management of current crises and future prevention and control requires the participation of all concerned sectors having a role in public health measures, identifying clinical cases, diagnosis, contact tracing, proper infection control in various settings, isolation, quarantine, cure and management, public awareness, facilitation of infrastructure and other facilities through local administrations [26, 195] . as the human covid-19 cases are on the rise due to efficient human-to-human transmission, there is a subsequent rise in the natural infections of covid-19 among the companion and wild animal species owing to the spillover. this is mainly because of the specific biological and virological characteristics of coronaviruses that gives them the ability to easily cross-species barriers [130] . even though animal-to-human transmission is not reported in covid-19, 'one health' approach is necessary to control this pandemic virus a schematic illustration of covid-19 clinical signs, modes of transmission, important diagnostic methods, and advances in vaccine development along with salient prevention and control strategies are presented in figure 2 . with the rising number and worldwide spread of covid-19, the need for global efforts rely heavily on the investigations carried out at infection sites to trace different aspects of this novel coronavirus outbreak. one of the critical facets and the earliest research must involve determining the root cause, origin, and source of this emerging infectious disease. shreds of evidence have revealed various cross-species jumping or spillover from animals to humans of these zoonotic coronaviruses. detailed serological investigation of all domestic and wild animals residing in the proximity to humans is of utmost necessity to know and prevent likely spillover of many other bat-related covs in the future. rapid detection of spillovers above will only be possible by the implementation of an effective and robust surveillance system for circulating viruses with high zoonotic potential in animals. besides, detection of a pathogen while crossing the species barrier to start circulation among humans and prevention of human-to-human transmission in early-stage may prove crucial in termination of a probable epidemic or pandemic. application of 'one health' concept involving medical, veterinary, wildlife, public health, and other related professionals may help in infection tracing, exploring risk factors and predisposition, minimizing risk to susceptible ones, and finally devising better prevention and control strategies. in the initial stages of the covid-19 outbreak, the steps taken for implementing stringent control and 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and emerging viral diseases time to intensify zoonoses surveillance in brazil outbreak of novel coronavirus (sars-cov-2): first evidences from international scientific literature and pending questions severe acute respiratory syndrome coronavirus on hospital surfaces avian influenza and sialic acid receptors: more than meets the eye? the lancet infectious diseases transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents emerging threats from zoonotic coronaviruses-from sars and mers to 2019-ncov ocular tropism of respiratory viruses. microbiology and molecular biology reviews 2019-ncov transmission through the ocular surface must not be ignored first case of 2019 novel coronavirus in the united states genomic diversity of sars-cov-2 in coronavirus disease 2019 patients emerging zoonoses: a one health challenge covid-19: first severe pandemic of the 21st century coronavirus outbreak will set back india's growth recovery covid-19: a look into the modern age pandemic biosafety and biosecurity approaches to restrain/contain and counter sars-cov-2/covid-19 pandemic: a rapid-review oie. questions and answers on the covid-19 calling for a covid-19 one health research coalition from sars to covid-19: a previously unknown sars-related coronavirus (sars-cov-2) of pandemic potential infecting humans -call for a one health approach ohc. covid-19 and one health 3 steps to help prevent another animal-to-human virus pandemic prevention cfdca. covid-19 and animals usda ensures food safety during covid-19 outbreak disinfectants for use against sars-cov none. key: cord-211735-qqm4fbor authors: gulec, fatih; atakan, baris title: mobile human ad hoc networks: a communication engineering viewpoint on interhuman airborne pathogen transmission date: 2020-11-02 journal: nan doi: nan sha: doc_id: 211735 cord_uid: qqm4fbor pathogens such as viruses and bacteria play a vital role in human life, since they cause infectious diseases which can lead to epidemics. recent coronavirus disease 2019 epidemic has shown that taking effective prevention measures such as wearing masks are important to reduce the human deaths and side effects of the epidemic. it is therefore requisite to accurately model the spread of infectious diseases whose one of the most crucial routes of transmission is airborne transmission. the transmission models in the literature are proposed independently from each other, at different scales and by the researchers from various disciplines. thus, there is a need to merge all these research attempts. to this end, we propose a communication engineering approach that melts different disciplines such as epidemiology, biology, medicine, and fluid dynamics in the same pot to model airborne pathogen transmission among humans. in this approach, we introduce the concept of mobile human ad hoc networks (mohanets). this concept exploits the similarity of airborne transmission-driven human groups with mobile ad hoc networks and uses molecular communication as the enabling paradigm. the aim of this article is to present a unified framework using communication engineering, and to highlight future research directions for modeling the spread of infectious diseases among humans through airborne pathogen transmission. in this article, we first review the airborne pathogen transmission mechanisms. then, the mohanet is given with a layered structure. in these layers, the infectious human emitting pathogen-laden droplets through air and the exposed human to these droplets are considered as the transmitter and receiver, respectively. moreover, the experimental methods for the proposed approach are reviewed and discussed. throughout the history, epidemics caused by infectious diseases have been a major threat to human life. epidemic diseases such as black plague, smallpox, spanish flu and recent coronavirus disease 2019 (covid-19) gave rise to millions of human deaths. in addition, epidemics can induce mental disorders in humans and recessions in the world economy due to prevention and control measures such as lockdown. owing to these facts, it is essential to understand and accurately model the spread of infectious diseases among humans. the interhuman spread of infectious diseases occur via direct contact and airborne transmission 1 where pathogens are transferred from an infectious human to a susceptible one. the authors are with the department of electrical and electronics engineering, izmir institute of technology, 35430, urla, izmir, turkey. 1 here, transmission is employed synonymously with contagion rather than its usage in communication engineering. in airborne transmission, these pathogens (viruses, bacteria, fungi, and so on) are carried by large droplets and aerosols (droplet nuclei) which are emitted via breathing, speaking, coughing and sneezing [1] . throughout this article, we use the term droplet to refer to both large droplets and aerosols together. as for the airborne pathogen transmission, it is not fully unraveled how its mechanisms operate between two humans, for example, it is still a matter of debate whether large droplets or aerosols are more infectious. in addition, the mobility and interplay of people during their daily life makes the problem of modeling infectious disease spread in an epidemic more chaotic. as people displace, there exist dynamic human groups exchanging pathogens among each other. due to their mobility, humans form different groups in an ad hoc fashion as their smart phones do in a mobile telecommunication network. actually, a human emitting expiratory droplets is an information source [2] . when these emitted information carrying droplets are received by another human through sensory organs, we can consider there exists a communication path between them. hence, an analogy between human groups and mobile telecommunication networks can be established, since they both possess an intermittent connectivity which is detailed later. by utilizing this analogy, we propose an approach to modeling interhuman airborne pathogen transmission with communication engineering perspective where mobile humans forming a group are considered as a mobile human ad hoc network (mohanet). in a mohanet, the infectious human is the transmitter (tx), the susceptible human is the receiver (rx) and pathogen-laden droplets are information carriers propagating in the communication channel, that is, air. here, molecular communication (mc) employing chemical signals instead of electrical signals emerges as an enabler paradigm for the communication among humans due to its biocompatibility with the human body and multiscale applicability. on the other hand, researchers from many disciplines work separately in different scales to reveal the mechanisms of airborne pathogen transmission and model the behavior of epidemics. in fluid dynamics literature, researchers focus on the propagation of pathogen-laden droplets and their interactions with air [1] . biologists deal with the survival of airborne pathogens in macroscale [3] and their interactions with the human cells in microscale [4] . furthermore, the medical literature conducts researches in cellular level to discover new drugs which cure the infectious diseases. in a larger scale, epidemiology literature focuses on the epidemic data to develop mathematical models for the spread of epidemics in time and space [5] . however, there is a need to merge all of these research efforts in a unified framework. communication engineering approach can provide this framework by combining micro-and macroscale modeling issues. in this way, researchers will be able to utilize theoretical tools of communication theory in order to model the complicated nature of airborne pathogen transmission. in the remainder of this article, we first review the airborne pathogen transmission mechanisms and the motivation to use mc as the enabler communication paradigm. then, the communication engineering approach which merges different disciplines. in this approach, the layered architecture of mohanet is presented in detail and open research issues are discussed. finally, we give the existing and possible experimental techniques and conclude the article. this section provides a brief overview for the main issues of the airborne pathogen transmission mechanisms. then, the roles of molecular signals in the transfer of pathogens among humans are discussed. a. overview of main issues on airborne pathogen transmission 1) respiratory activity and droplet size: pathogen-laden droplets are emitted to the air from an infected human via respiratory activities such as coughing, sneezing, speaking and breathing. these activities have different initial droplet velocities allowing different propagation distances. for instance, the initial velocities for coughing and breathing are about 10 m/s [1] and 2.4 m/s [6] , respectively. therefore, a cough can infect people at a greater distance than breathing in still air. furthermore, the expiratory droplets are defined according to their diameters where aerosols and large droplets are assumed to have smaller and larger diameters than the size range 5-10 µm, respectively [1] . while speaking, sneezing, and coughing release more large droplets into the air, breathing mostly contains aerosols. 2) air distribution: in addition to the initial velocity, emitted droplets are influenced by the airflows, similar to a mc channel with drift. in outdoor environments, winds carry the droplets and dilute the concentration of pathogens via dispersion. therefore, it is less probable to get infected in outdoor environments. however, in indoor environments such as hospitals, offices or residential buildings, airflows generated by ventilation systems are critical for the spread of pathogens due to the circulation of air in bounded conditions. furthermore, personalized ventilation and exhaust systems are proposed as advanced ventilation systems to diminish the infection risk [1] . these air distributions are required to be considered for realistic indoor airborne transmission models. 3) posture, relative orientation, distance and movement of the human: for short distances, the posture, that is, standing, sitting or lying position, and the relative orientation of the infected and susceptible persons are important for the infection risk as shown in fig. 1 . for instance, a doctor can reduce the exposure from an infected lying patient in a hospital ward via a standing posture and sideways orientation instead of face-to-face orientation [1] . furthermore, a walking person can increase the infection risk in a closed and ventilated room by increasing the dispersion of the droplets [7] . another important factor that influences the infection risk is the relative distance of the humans which is also referred as the social distance. surely, the infection risk decreases, as the relative distance between two people increases. the temperature difference between the human body surface and the surrounding air generates a thermal plume which is a buoyancy-driven upward flow of the surrounding air. as illustrated in fig. 1 , this thermal plume leads to a convective boundary layer (cbl) around the human body, which should be taken into account for the movement of the droplets in the breathing zone [8] . this upward flow can change the channel impulse response via generating an upward drift for the pathogens during the reception into the human body. 5) survival of pathogens: subsequent to a respiratory activity, all of the emitted pathogens may not survive. in [3] , it is shown that more than 80 percent of the influenza viruses cannot survive within one minute. however, these survival rates are severely influenced by environmental factors such as temperature and relative humidity (rh). while increasing temperature decreases survival rates of the pathogens due to its effect at molecular levels, increasing rh results in decreasing evaporation of droplets [9] . the decreasing number of pathogens results in a time-varying channel due to the dependence on the previous number of pathogens. via the aforementioned respiratory activities, a human can transfer pathogen-laden droplets to another human. this type of transfer (or communication) among humans is investigated in the medical literature where pheromone-based molecular signals are studied for the interaction of humans. in [10] , it is proposed that pheromones secreted from the axillary apocrine glands of women living in close proximity provides a synchronization in their menstrual cycle. hence, molecular signals may give rise to some biological responses in human organism. as given in the next section, the transfer of the pathogen-laden droplets which cause infection can be considered in the context of mc. in this section, we present a framework with communication engineering perspective to model the spread of infectious diseases through airborne pathogen transmission. furthermore, open research issues are given. as shown in fig. 2 , the proposed framework merges all of the multiscale research efforts in various disciplines such as fluid dynamics, biology, medicine, and epidemiology under the umbrella of communication engineering. mc emerges as the key paradigm that connects the studies among different disciplines in macro-and microscales. first, the mohanet is introduced through a layered architecture as depicted in fig. 2 . layers are associated with different disciplines from µm to km scale in this architecture where each layer sends its output to a upper layer. the first layer is defined as the physical layer where the infectious human (tx) emits pathogen-laden droplets through the communication channel (air) as illustrated in fig. 1 . the next layer is the reception layer which takes place at the susceptible human (rx) and includes two sublayers, that is, outer and inner reception sub-layers. the outer reception sub-layer comprises the interactions of the facial sensory organs with the droplets and inner reception sublayer provides the details about the interactions of pathogens with the biological cells in the human body. the networking layer where infectious diseases spread among different people is given at the top of the mohanet architecture. here, methods from mobile telecommunication networks literature are exploited and the outputs of the lower layers are employed rather. the details of this layered architecture are introduced as follows. a. physical layer 1) transmitter: in a mohanet, an infected person is considered as a tx and her/his respiratory activities determine the tx parameters such as initial droplet velocities and droplet size distribution [2] . the respiratory activities which are mentioned earlier can be classified as impulsive (sneezing and coughing) and continuous (breathing and speaking) emission signals. for continuous emissions, the frequency of the human exhalation is an influential factor for the transmission models. however, it is crucial to characterize speaking, since it is not always periodic and has more complex patterns than breathing. in addition, the respiratory organs such as nose or mouth affect the direction of the emitted signals. for example, the infection risk increases, when the tx uses the mouth instead of nose [1] . furthermore, the convective boundary layer (cbl) of the human body, posture and relative orientation should be taken into account for accurate tx models. as mentioned earlier, the upward flow stemming from the cbl can affect the direction of the emitted pathogens in a tx model. 2) channel: from the viewpoint of communication engineering, the channel is the physical medium between the tx and rx including the boundary conditions. as shown in fig. 2 , channel modeling in the physical layer requires knowledge from fluid dynamics and biology due to the airdroplet interaction and survival of pathogens, respectively. the propagation dynamics of droplets can be examined under two subheadings depending on whether there is an external airflow or not. a) still air: in indoor environments such as residential buildings, it is generally assumed that there is no airflow, if there is not any ventilation system. after the emission of pathogen-laden droplets with an initial velocity, they are subject to newtonian mechanics during their interaction with the air. emitted droplets can be modeled as a cloud consisting of droplets and air particles. the movement of this cloud can be defined as a two-phase flow where these phases represent the gaseous state of air and liquid state of droplets [11] . due to gravity, large droplets may fall earlier to the ground with respect to aerosols and evaporation can shrink the size of the droplets. as mentioned earlier, the temperature of the air and evaporation influence the survival rates of the pathogens. for continuous emissions, this fact can affect the channel memory, which is crucial for channel modeling. furthermore, initial velocities of droplets determined by respiratory activities can figure 3 . two-layered receiver. give rise to short-term laminar and turbulent flows. these flows fade out as the distance between the tx and rx increases. b) windy air: for windy outdoor environments and indoor environments with airflows such as ventilation or wind arising from the open doors and windows, airflows dominate the propagation of droplets rather than other factors given for still air environments. the airflow which carries the pathogenladen droplets can be examined by advection and diffusion mechanisms. briefly, advection results from the airflow velocity and diffusion depends on the turbulent eddies during the mass transfer [12] . it should be noted that molecular diffusion related with the thermal energy of molecules is negligible in macroscale. in order to calculate the concentration of droplets in time and space, deterministic and stochastic approaches which are based on differential navier-stokes and continuity equations are employed. for certain initial and boundary conditions, the solutions of these equations for deterministic concentration are known as gaussian plume for steady-state and gaussian puff model for transient analysis [12] . actually, the concentration and velocity of droplets are random processes whose mean values are represented by these deterministic solutions. thus, stochastic differential equations are obtained which are non-trivial to solve as a closed form expression. therefore, these equations are mostly solved by numerical methods using eulerian and langrangian approaches [12] . in addition, indoor ventilation types such as under floor air distribution, mixing, displacement, and downward ventilation should be incorporated into these airflow models. for example, downward ventilation can reduce the infection risk by diluting the dispersion of droplets [1] . a human gets infected, when the transmitted pathogens are received into the body. as shown in fig. 2 , the reception layer covers the issues related to biology and medicine in microscale where mc is utilized for the interactions of pathogens with the human body. the reception of these pathogens by the exposed human (rx) have not been well investigated, although there are myriads of theoretical, experimental and clinical studies for the propagation of pathogens. to this end, we propose a two-layered rx as shown in fig. 3 and detailed below. the reception of pathogenladen droplets occur in the eyes [13] , mouth and nose for many pathogens such as influenza virus [1] . hence, we define the first step of reception as the outer layer sensing for the reception via facial sensory organs as illustrated in fig. 3 . the whole surface of the human face is also important for the reception, since an infection may occur by touching the face contaminated with pathogens and these organs consecutively. pathogen-laden droplets emitted via a respiratory activity propagate as a mixture of droplets and air particles, which can be represented as a cloud [11] . this cloud is affected by the momentum due to the initial velocity of droplets, gravity and buoyancy stemming from the temperature difference of the mouth and ambient air. according to this model, the trajectory of the cloud is given in fig. 4 for a scenario that the tx emits pathogens by coughing. here, there is no airflow in the channel and, the tx and rx are standing toward each other at the same height as illustrated in fig. 1. fig. 5 gives the change of the number of droplets in the cloud by taking settling and reception of droplets into account. the cross-section of the rx is assumed to cover a circular area including eyes, mouth and nose at the outer layer as illustrated in fig. 3 . at this point, an analogy with the communication systems can be established by considering the infected state of the rx as symbol 1 and no infection as symbol 0. this reception is accomplished by a detection according to a threshold value (γ = 80) indicating the number of droplets required to become infected, as given in fig. 5 . γ is a critical parameter in the airborne tranmission model, since it depends on the strength of human's immune system. to this end, biomedical data of humans such as body mass index, glucose level and whether or not having chronic diseases can be employed to estimate γ. in addition to these issues in the outer layer, the posture, relative orientation and cbl of the rx should be taken into account for an accurate receiver model as considered for the tx. furthermore, the reception of pathogen-laden droplets at the outer layer with different types of masks is an open issue to be investigated. 2) inner reception layer: as shown in fig. 3 , pathogens actually enter human body at the cellular level and increase their population. for example, viruses replicate themselves by inserting their genetic material (dna or rna) into human cells in two ways: they can bind their fusion (or spike) protein on specific receptor sites on the human cell or they can enter by using endosomes like a trojan horse [4] . their binding sites can have different concentrations in different parts of the body. for instance, severe acute respiratory syndrome coronavirus-2, which causes covid-19, binds to angiotensin converting enzyme-2 receptors which are mostly found at upper respiratory tract [14] . while large droplets are effective in upper respiratory tract, aerosols can reach down to alveoli in lower respiratory tract. hence, the droplet size can be effective to determine the infection risk according to the type of the disease. moreover, the viruses diffuse among human cells, bind to receptors and copy their genetic material in a random way. all of these issues at the inter-and intracellular level need to be modeled for an accurate transmission model for the spread of infectious diseases in mohanets. these modeling efforts can also contribute to drug and vaccine developments. what we examine up to here in lower layers of the mo-hanet architecture is about the transmission of infectious diseases between two humans. however, these transmissions occur many times in an epidemic, which requires a perspective to handle the population as a connected group, that is, a network. in the networking layer, the details of the mohanet architecture are presented in order to model the spread of infectious diseases in a large scale (km) within the communication engineering framework as shown in fig. 2. 1) mobile human ad hoc networks: in epidemiology literature, each human, that is, a node, can be represented as susceptible (s), exposed (e), infectious (i) or recovered (r) according to the seir-based models in the infectious disease modeling approaches [5] . according to the disease type, different combinations of these node types can be employed for the models such as sir or sirs. for example, covid-19 is suitable to use all the node types due to a non-infectious incubation period. in the literature, the number of these node types are modeled by ordinary differential equations where the number of the nodes can be deterministic or a stochastic process. the transition among different types of nodes (s,e,i,r) are defined with certain rates which are obtained by fitting statistical epidemic data. in experimental studies, these data are obtained by oral surveys or exploiting wireless sensor network technology [5] . it is noteworthy that very few studies model the spatial change of the epidemic rather than its temporal change. by utilizing the widespread sir model, a mohanet is given in fig. 6 which gives both the spatial and temporal changes. as the time elapses, the number of nodes may alter and the nodes can make transitions between states such as s, i or r. for example, a susceptible node can become infected, if it is in the transmission range of an infectious node or an infectious node can recover after a certain period. 2) transmission types in mohanets: as illustrated in fig. 6 , three transmission types are defined for the propagation of pathogen-laden droplets from the infectious nodes to the susceptible nodes as follows: • point-to-point transmission includes the communication between two nodes where the infectious and susceptible nodes are the tx and rx, respectively. • multicast transmission is the scheme that one infectious node spreads the disease to more than one node within its communication range. • multiple-access transmission comprises the scenario where a susceptible node is exposed to pathogen-laden droplets from multiple infectious nodes. humans are susceptible to infectious diseases in indoor places such as public transportation vehicles, shopping malls or offices. however, this is not the case that is encountered continuously. instead, the risk to get infected is intermittent due to the mobility of humans. as people displace, their smart phones can communicate opportunistically with each other as they are in the communication range. the same type of networking is also used in many applications such as wireless sensor, vehicular, and flying ad hoc networks. these dynamically changing structures defined as mobile ad hoc networks (manets) enable communication using the infrastructure at their location without a dedicated router. therefore, a mohanet can be resembled as a specific type of manet, that is, a delay tolerant network (dtn) in which an end-to-end link among the nodes may not always exist. the nodes in a dtn store their data and wait until they find a suitable connection. by considering this waiting delay, the routing algorithms in dtns provide the path to the desired user. similarly, an infected human can store its pathogens until finding a susceptible human to infect via airborne transmission. hence, we propose that opportunistic routing protocols such as epidemic or spray and wait can be adopted to model the spread of the infectious diseases. interestingly, epidemic routing protocol which is a reference method for routing in manets was already inspired by the mechanism of infectious disease spread during an epidemic [15] . in order to observe and model the airborne transmission mechanisms among humans, experimental setups and computer simulations can be employed. in this section, we present and discuss how the performance of the proposed methods in different layers of the mohanet architecture can be evaluated. in physical and reception layers, the emulation of breathing, coughing and sneezing in experimental setups are realized by respiratory machines or thermal manikins which can be heated to change their temperature. these devices emit tracer gases including droplets. the concentration of droplets is measured by air samplers or via imaging techniques such as particle image velocimetry which gives the velocity and directions of droplets [1] . moreover, sprayer-based mc systems can also be used instead of respiratory machines, manikins and air samplers. albeit reliable results can be obtained by the physical experiments regarding the consideration of droplet-air interaction and airflows, the collected data have a low-resolution in space and time and experimental devices are expensive. therefore, computational fluid dynamics (cfd) simulations are employed to evaluate the airborne transmission mechanisms with a high spatiotemporal resolution and less cost [1] . however, the simulation software programs are based on navier-stokes equations which lack the capability to model all of the effects during the transmission realistically. these experimental techniques and cfd simulations can be employed to model the airborne pathogen transmission with communication engineering perspective for various scenarios between two humans. in a larger scale, for example, in a crowded city, it is essential to model the spread of infectious diseases with an approach that takes into account the interaction of people and their mobility in both time and space. the movement patterns of humans can be simulated by synthetic models such as random waypoint model or tracebased models which rely on real mobility data of mobile nodes as applied in manets. the adapted routing protocols for mohanets can also be evaluated in time and space by employing these mobility models according to the scenario via network simulation software. this article presents a framework to model airborne pathogen transmission with a communication engineering perspective. first, airborne pathogen transmission mechanisms are reviewed and mc is utilized to model the propagation and reception of this transmission. the concept of mohanet is proposed to handle the infectious disease spread modeling problem by using a layered structure in macro-and microscales. furthermore, simulation techniques and experimental methods to model airborne pathogen transmission are reviewed and discussed. throughout the article, open research issues possessing the potential for development opportunities are given. the efforts to model the infectious disease spread via airborne pathogen transmission with a novel approach given in this article has the potential for a holistic viewpoint. this communication engineering viewpoint can bring different disciplines such as fluid dynamics, medicine, biology and epidemiology together for accurate predictions about the spread of infectious diseases. hence, the most proper intervention method (lockdown, social distancing, wearing masks, and so on) can be chosen and how it will be applied can be determined to stop the epidemics in an effective way. airborne spread of expiratory droplet nuclei between the occupants of indoor environments: a review communication through breath: aerosol transmission survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids how viruses invade cells dynamics of infectious diseases close contact behavior in indoor environment and transmission of respiratory infection performance of "ductless" personalized ventilation in conjunction with displacement ventilation: impact of disturbances due to walking person (s) human convective boundary layer and its interaction with room ventilation flow mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence regulation of ovulation by human pheromones a molecular communication perspective on airborne pathogen transmission and reception via droplets generated by coughing and sneezing air dispersion modeling: foundations and applications the severe acute respiratory syndrome a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemic routing for partially connected ad hoc networks turkey as a research/teaching assistant under the supervision of assoc. prof. dr. barış atakan. his research interests include micro and macroscale molecular communications and molecular networks he is currently an associate professor with the department of key: cord-338468-c0jv3i1t authors: kanduc, darja title: from anti-sars-cov-2 immune responses to covid-19 via molecular mimicry date: 2020-07-16 journal: antibodies (basel) doi: 10.3390/antib9030033 sha: doc_id: 338468 cord_uid: c0jv3i1t aim: to define the autoimmune potential of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. methods: experimentally validated epitopes cataloged at the immune epitope database (iedb) and present in sars-cov-2 were analyzed for peptide sharing with the human proteome. results: immunoreactive epitopes present in sars-cov-2 were mostly composed of peptide sequences present in human proteins that—when altered, mutated, deficient or, however, improperly functioning—may associate with a wide range of disorders, from respiratory distress to multiple organ failure. conclusions: this study represents a starting point or hint for future scientific–clinical investigations and suggests a range of possible protein targets of autoimmunity in sars-cov-2 infection. from an experimental perspective, the results warrant the testing of patients’ sera for autoantibodies against these protein targets. clinically, the results warrant a stringent surveillance on the future pathologic sequelae of the current sars-cov-2 pandemic. it is well known that coronavirus (re)infections have a pathologic immune potential. indeed: • progressive immune-associated injury is a hallmark of sars infection [1] and, since 2003 [2] , the association with hyper-immune inflammation and systemic immunopathology in the sars-cov-infected host has been well illustrated [2] [3] [4] [5] ; • in 2008, it was reported [6] that prior immunization with sars-cov nucleocapsid protein n causes severe pneumonia in mice infected with sars-cov; • in 2012, immunization with sars coronavirus vaccines was found to lead to lung immunopathology on challenge with the sars virus [7] ; • in 2016, agrawal et al. [8] demonstrated that immunization with inactivated middle east respiratory syndrome (mers) coronavirus vaccine leads to lung immunopathology on challenge with live virus. moreover, the authors documented that immunopathology with sars-cov vaccines occurred for whole-virus vaccines, subunit vaccines, different inactivation methods, different preparation substrates, and with recombinant surface spike protein. finally, the authors also underscored that, even if studies with vector vaccines point to the nucleoprotein n as responsible for the immunopathological effects and indicate that the s protein might be free of risk, nonetheless, also recombinant spike protein induced the immunopathology [6] [7] [8] [9] . however, the molecular and cellular basis for how sars-covs might impact on the host immune system resulting in dysfunctional immune responses, severe morbidity, and mortality remain obscure [1, 10] . following the current sars-cov-2 pandemic, cross-reactivity was proposed as a mechanism that might explain the immunopathology associated with the coronavirus infection [11] . the rationale is that the sharing of peptide motifs between sars-cov-2 and human proteins might evoke immune responses capable of hitting not only the virus but also the human proteins with consequent autoimmune pathologic sequelae in the human host. as a matter of fact, the study [11] called attention to a vast and specific peptide sharing between sars-cov-2 spike glycoprotein and alveolar surfactant-related proteins, in this way addressing the issue of why sars-cov-2 so heavily attacks the respiratory system. in the wake of such results, in order to validate (or, as well, invalidate) the cross-reactivity hypothesis, investigation was expanded here by analyzing the peptide sharing between the human host and immunoreactive epitopes that are also present in sars-cov-2. actually, the mere sharing of peptide sequences between pathogens and human proteins might be of little significance whether it remained sterile of cross-reactive autoimmune reactions. on the contrary, a peptide sharing between immunoreactive pathogen-derived epitopes and human proteins would assume the value of a proof of concept of cross-reactivity as a mechanism contributing to the pathogen-induced diseases. in this scientific framework, using the hexapeptide as an antigenic and immunogenic unit [12] [13] [14] [15] , immunoreactive epitopes that are present in sars-cov-2 were analyzed for matches with the human proteome. the results confirm a vast peptide commonality that involves human proteins implied in pulmonary insufficiency, neurological disorders, cardiac and vascular alterations, pregnancy dysfunctions, multiple cancers and anosmia, among others. analyses were conducted on an immunome composed of 233 linear epitopes that were experimentally validated, present in the proteins of sars-cov, and cataloged in the iedb [16] . the experimentally validated 233 epitopes have been described in detail by ahmed et al. [17] , map identically in sars-cov-2 proteins, and are listed in supplementary materials table s1 . the hexapeptide was used as a measurement unit to define minimal epitopic sequences. indeed, 5-6 amino acids (aa) can form a sufficient minimal determinant for epitope-paratope interaction [12, 13] . likewise, t-cell epitopes contain a core of 5-6 aa that are involved in antigenicity and immunogenicity [13] [14] [15] and are flanked by nh 2 -and cooh-terminus aa that act as anchor motifs. the sars-cov-2 epitope sequences were dissected into hexapeptides which overlapped each other by five aa residues. the resulting 733 hexapeptides were analyzed for occurrence(s) within human proteins using the pir peptide match program [18] . human proteins involved in the hexapeptide sharing were analyzed for function/diseases using uniprotkb, pubmed, and omim resources. the expected value for hexapeptide sharing between two proteins can be calculated by considering the number of all possible hexapeptides, n. since in a hexapeptide each residue can be any of the 20 aa, the number of all possible hexapeptides n is given by n = 20 6 = 64 × 10 6 . then, the number of the expected occurrences is directly proportional to the number of hexapeptides in the two proteins and inversely proportional to n. assuming that the number of hexapeptides in the two proteins is n and neglecting the relative abundance of aa, we obtain a formula derived by approximation, where the expected number of hexapeptides is 1/n or 20 −6 . table 1 reports that 230 out of the 733 hexapeptides composing the analyzed 233 immuno-reactive epitopes occurred among 460 human proteins. many of these shared sequences recurred more times. for instance, the zinc finger protein znf265-a splice factor that is important in renin mrna processing and stability [19] -shares the hexapeptide srsssr with the viral epitope sqassrsssr (iedb id: 60380). the hexapeptide srsssr recurs three times in the zinc finger protein, exactly at aa positions: 211-216, 241-246, and 256-261. then, including multiple occurrences, on the whole the hexapeptides shared with the human proteins amount to 505. for reasons of synthesis, table 2 reports the distribution of the shared hexapeptides relatively to a sample (n = 58) of sars-cov-2 epitopes. in the viral epitope sequences, the hexapeptides shared with human proteins are marked in capital letter format. the distribution of the shared hexapeptides throughout the entire set of 233 sars-cov-2 epitopes is described in supplementary materials table s2 . table 2 documents that numerous immunoreactive sars-cov-2 epitopes are composed mostly or, in many instances, uniquely of peptide sequences shared with human proteins. as an example, among the many, the epitope iedb id 4936, which is present in the viral nucleocapsid protein and corresponds to the aa sequence ategalntpk, results from the consecutive succession/overlapping of 6 mers present in human proteins too. taken together, table 1 , table 2 , and table s2 highlight the unexpectedness of the peptide overlap between the human proteome and the immunoreactive viral epitopes derived from iedb, described by ahmed et al. [17] and analyzed here. from a mathematical point of view, if one considers that the probability of a hexapeptide to occur in 2 proteins is~20 −6 (or 1 out of 64,000,000 or 0.000000015625), then the number of hexapeptides (namely, 505, including multiple occurrences) shared between the sars-cov-2 immunome and human proteins is surprisingly high and can be explained only on the basis of evolutionary processes [20] . in the context of autoimmunity, table 1 , table 2 , and table s2 concretize the effective possibility of cross-reactions between sars-cov-2 and the human proteome, given the fact that the immunological information unit in terms of both immunogenicity and antigenicity is contained in a space formed by 5-6 aa residues [12] [13] [14] [15] . as quantified in table 1 , hexapeptides from immunoreactive viral epitopes occur across 460 human proteins. the 460 human proteins are listed and synthetically described in supplementary materials table s3 . the 460 human proteins are involved in metabolic, developmental, and regulatory cellular functions and-when mutated, modified, deficient or, however, improperly functioning-may lead to altered functions and more or less severe pathologies in the human organism. obvious reasons of space prevent a protein-by-protein analysis of all of them and, here, only a few proteins (given by the uniprot name in italic and shared peptides in parentheses) and the related pathologies that could arise in case of cross reactivity are dealt with as follows. molecular mimicry between sars-cov-2 spike glycoprotein and alveolar surfactants-related proteins have already been described [11] . additional proteins that-if hit-can alter lung functions are: mothers against decapentaplegic homolog 9 protein (qassrs), alterations of which may lead to pulmonary hypertension with proliferating endothelial cells in pulmonary arterioles, right ventricular failure, and death [21] ; • phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (ikdlpk) that is deficient in pulmonary vascular endothelial cells of patients with acute respiratory distress syndrome [22, 23] ; • adenylate cyclase type 9 (kqlssn) that is expressed in multiple cells of the lung with expression highest in airway smooth muscle [24] ; • acetylcholinesterase (avlqsg) where imbalances in the neurotransmitter acetylcholine relate to neurological conditions, such as alzheimer's disease, parkinson's disease, and myastenia gravis; irreversible inhibition of acetylcholinesterase may lead to muscular paralysis, convulsions, bronchial constriction, and death by asphyxiation [25] . • endoribonuclease dicer (assrss) which is linked to pleuropulmonary blastoma [26] ; • protein naked cuticle homolog 1 (llpsla), downregulation of which is linked to poor prognosis in non-small-cell lung cancer and breast invasive ductal carcinoma [27, 28] ; • downregulated in multiple cancers 1 (aaeira) is not expressed in multiple human cancers [29] ; • ubiquitin carboxyl-terminal hydrolase bap1 (llsvll) relates to tumor predisposition syndrome; linked to mesothelioma [30] [31] [32] ; • in addition, the tumor suppressor proteins pinin (ssrsss) [33] , tripartite motif-containing protein 35 (sfkeel) [34] , unconventional myosin-ixb (llpsla) [35] , and cyclin-d-binding myb-like transcription factor 1 (aalqip) [36] . • low-density lipoprotein receptor-related protein 8 (lallll), alterations of which can lead to myocardial infarction [37] ; • dol-p-glc:glc(2)man(9)glcnac(2)-pp-dol alpha-1,2-glucosyltransferase (tltlav) is implicated in susceptibility to the long qt syndrome [38] ; • presenilin-2 (tlacfv) relates to dilated cardiomyopathy and heart failure [39] ; • nesprin-1 (llsagi) is involved in dilated or hypertrophic cardiomyopathy [40, 41] : • nuclear receptor coactivator 6 (pslatv) can cause dilated cardiomyopathy [42] ; • latent-transforming growth factor beta-binding protein 3 (lallll) can associate with skin thickening, cardiac valvular thickening, tracheal stenosis, and respiratory insufficiency [43] . • ephrin-b3 (lallll) is involved in blood pressure control and vascular smooth muscle cell contractility [44] ; • endoglin (lvlsvn) is required for normal structure and integrity of adult vasculature [45] ; • elastin (fgagaa) is a major structural protein of tissues, such as aorta and nuchal ligament, and relates to cutis laxa disease, may also, although rarely, lead to pulmonary artery stenosis, aortic aneurysm, bronchiectasis, and emphysema [46, 47] ; • filamin-a (pyrvvv), alterations of filamin-a can cause disorders related to the vascular system and to a large phenotypic spectrum of disorders such as deafness, urogenital defects, malformations, intestinal obstruction, constipation, recurrent vomiting, and diarrhea [48] ; • glomulin (rimasl) is crucial in vascular morphogenesis-especially in cutaneous veins [49] . • tumor necrosis factor receptor superfamily member 1a (gttvll) may associate with fever, abdominal pain, localized tender skin lesions, myalgia, and reactive amyloidosis as the main complication [50, 51] . • thrombomodulin (agaalq), a well-known natural anti-coagulant that acts as a linker of coagulation and fibrinolysis, is involved in disseminated intravascular coagulation, is a predictor of risk of arterial thrombosis and is essential for the maintenance of pregnancy [52] [53] [54] . • in addition, cross-reactive peptide sharing involving additional proteins can further affect the level of thrombomodulin. indeed, cross-reactions with retinoic acid receptor rxr-alpha (lgfstg) and prostaglandin g/h synthase 2 (tvllke) might completely eliminate thrombomodulin from blood circulation because (1) retinoic acid receptor rxr-alpha promotes the thrombomodulin gene transcription [55] and (2) prostaglandin g/h synthase 2 stimulates the expression of functionally active thrombomodulin in human smooth muscle cells [56] . adding up to such pro-thrombotic scenario, it is also worth of mention the potential cross-reactivity with tyrosine-protein kinase jak2 (llddfv) that is involved in myelofibrosis, myeloid leukocytosis, and thrombocytosis with excessive platelet production resulting in increased numbers of circulating platelets, hemorrhages, and thrombotic episodes [57] [58] [59] . • neuronal pas domain-containing protein 2 (assrss), which is highly polymorphic in autism spectrum disorder patients [60, 61] ; • circadian locomotor output cycles protein kaput (assrss) relates to bipolar disorder [62] ; • adenosine receptor a1 (vlppll), where sleep is significantly attenuated by the loss of adenosine a1 receptor expression [63] ; • bdnf/nt-3 growth factors receptor (sanlaa), alterations may cause temporal lobe epilepsy, memory impairment, anorexia nervosa, bulimia, alzheimer's disease [64] [65] [66] ; • calcium/calmodulin-dependent protein kinase kinase 2 (pslatv) is linked to disturbances of higher cognitive functions, such as working memory and executive function. as well as schizophrenia [67] ; • endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (lafllf) can cause mental retardation [68] ; • glutaminase kidney isoform, mitochondrial (lqelgk) can associate with epileptic encephalopathy, infantile cataract, skin abnormalities leukocytoclasia at the surface of the dermis, focal vacuolar alterations, hyperkeratosis, parakeratosis, glutamate excess, and impaired intellectual development, global developmental delay, progressive ataxia, and elevated glutamine [69] [70] [71] ; • mitochondrial glutamate carrier 1 (rlqslq) relates to neonatal myoclonic epilepsy [72] . moreover, and remarkably, the voltage-gated calcium channel gamma subunits 2, 3, 4, 6, and 8 contain epitopic hexapeptides (table 3) . voltage-gated calcium channels control cellular calcium entry in response to membrane potential changes [73, 74] , and gamma subunits 2, 3, 4, and 8 regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (or ampa receptor or ampar) and are collectively known as transmembrane ampar regulatory proteins (tarps) [75] . ampars are involved in the fast synaptic transmission in the central nervous system and are altered in many psychological and neurological disorders such as schizophrenia, depression and parkinson's disease [76] . table 3 . hexapeptide sharing between sars-cov-2 epitopes and tarps. lsagif, srsssr gamma-2 subunit lsagif gamma-3 subunit srsssr gamma-4 subunit lgagcf gamma-6 subunit srsssr gamma-8 subunit as a final note, this results section closes with one disorder that is one of the first symptoms to appear in covid-19, namely, anosmia. the role of anosmia as a first symptom is clearly justified by the fact that seven olfactory receptors (i.e., proteins related to the smell) [77] share hexapeptides with the viral epitopes (table 4 ). this study shows that hexapeptides from immunoreactive epitopes present in sars-cov-2 are widespread among a high number of human proteins. such a peptide sharing implies the possibility of cross-reactions and, consequently, as discussed in the results (section 3), of a vast phenotypic constellation of diseases, from pneumonia and neurological disorders to cardio-vascular alterations and coagulopathies. hence, this study appears to offer scientific hints to explain the clinical fact that sars-cov-2 infection is capable of triggering so many and so different pathologies in so many and so different organs of the human host [78] . on the whole, the data indicate that-besides possible virus-induced multi-organ direct cytopathic effects and other possible pathogenic mechanisms-self-reactive antibodies may be at the root of the pathologic scenario that accompanies sars-cov-2 infection. in fact, previous research focusing on sars-cov, which similarly to sars-cov-2 causes a respiratory failure syndrome, showed that anti-spike protein iggs can cause lung damage by directly affecting inflammatory mechanisms, promoting the release of pro-inflammatory cytokines, such as il-8, as well as macrophagic tissue infiltration [79] . a similar effect of sars-cov-2 on inflammatory response has already been proposed [80] . then it appears reasonable to hypothesize that autoantibody-mediated macrophagic and complement activation and autoantibody-dependent cell-mediated cytotoxicity mechanisms might be some of the mechanisms behind the well-documented multi-organ damage in covid-19, thus representing one of many possible links between adaptive and innate immunity in the pathogenesis of the disease. moreover, as reviewed by vabret et al. [81] , it has been shown that high antibody response can associate with more severe clinical cases. this had also been seen in the previous sars-cov-1 epidemic, where neutralizing antibody titers were found to be significantly higher in deceased patients compared to patients who had recovered [82] and might lead to suspect antibody-dependent enhancement phenomena. of note, the present data also indicate that most possibly the damage caused by sars-cov-2 might not end with the end of the pandemic. indeed, the peptide sharing between sars-cov-2 epitopes and specific tumor suppressor proteins, and, in general, many tumor-related proteins [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] , theoretically predicts, in the absence of current clinical data, that a morbidity/mortality increase in various cancers might follow the current sars-cov-2 pandemic. as a conclusive note, it is mandatory also to observe that this study largely undervalues the potential risk of cross-reactivity between sars-cov-2 immunome and human proteins. indeed, analyses were conducted on linear epitopes and used the hexapeptide as an immune unit probe. then, if one considered conformational epitopes and used the pentapeptide as a minimal immune determinant [13, 83] , the number of viral versus human commonalities would increase exponentially. given this premise, the present results call researchers and clinicians for a common research effort to study the autoimmune pathogenicity connected to the anti-sars-cov-2 immune response and suggests, once more, that using entire antigens in anti-sars-cov-2 vaccine formulations might lead to autoimmune manifestations and adverse events [84] . actually, the present data further support the fundamental concept that only "non-self" peptides can lead to safe and efficacious immunotherapies [85] [86] [87] . supplementary materials: the following are available online at http://www.mdpi.com/2073-4468/9/3/33/s1, table s1 : sars-cov-2 immunome consisting of 233 immunopositive linear epitopes, assembled from iedb [16] , described by ahmed et al. [17] , and listed according to iedb id number. table s2 : hexapeptide sharing between 233 epitopes present in sars-cov-2 and human proteins. table s3 : list and short description of 460 human proteins that share hexapeptides with the 233 sars-cov-2 epitopes. funding: this research received no funding. the author declares no conflict of interest. human immunopathogenesis of severe acute respiratory syndrome (sars) lung pathology of fatal severe acute respiratory syndrome an interferon-gamma-related cytokine storm in sars patients pathogenesis of severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with inactivated middle east 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comparative biochemistry of viruses and humans: an evolutionary path towards autoimmunity molecular genetic characterization of smad signaling molecules in pulmonary arterial hypertension endothelial p110γpi3k mediates endothelial regeneration and vascular repair after inflammatory vascular injury role of the pi3-kinase signaling pathway in trafficking of the surfactant protein a receptor p63 (ckap4) on type ii pneumocytes adenylyl cyclase type 9 gene polymorphisms are associated with asthma and allergy in brazilian children actions of drugs on the autonomic nervous system dicer1 mutations in familial pleuropulmonary blastoma down-regulation of nkd1 increases the invasive potential of non-small-cell lung cancer and correlates with a poor prognosis nkd1 down-regulation is associated with poor prognosis in breast invasive ductal carcinoma identification of dmc1, a novel gene in the toc region on 17q25.1 that shows loss of expression in multiple human cancers the nuclear deubiquitinase bap1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma germline bap1 mutations predispose to malignant mesothelioma germline mutations in bap1 predispose to melanocytic tumors characterization of the gene encoding pinin/drs/mema and evidence for its potential tumor suppressor function hls5, a novel rbcc (ring finger, b box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor myo9b is a key player in slit/robo-mediated lung tumor suppression the hdmp1 tumor suppressor is a new wt1 target in myeloid leukemias an lrp8 variant is associated with familial and premature coronary artery disease and myocardial infarction a kcr1 variant implicated in susceptibility to the long qt syndrome mutations of presenilin genes in dilated cardiomyopathy and heart failure analysis of selected genes associated with cardiomyopathy by next-generation sequencing novel nesprin-1 mutations associated with dilated cardiomyopathy cause nuclear envelope disruption and defects in myogenesis perturbation of ncoa6 leads to dilated cardiomyopathy mutations in ltbp3 cause acromicric dysplasia and geleophysic dysplasia the role of grip1 and ephrin b3 in blood pressure control and vascular smooth muscle cell contractility endoglin, a tgf-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1 cutis laxa arising from frameshift mutations in exon 30 of the elastin gene (eln) isolated supravalvular aortic stenosis: functional haploinsufficiency of the elastin gene as a result of nonsense-mediated decay vascular and connective tissue anomalies associated with x-linked periventricular heterotopia due to mutations in filamin a mutations in a novel factor, glomulin, are responsible for glomuvenous malformations germline mutations in the extracellular domains of the 55 kda tnf receptor, tnfr1, define a family of dominantly inherited autoinflammatory syndromes 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peptides in the design of vaccines immunogenicity, immunopathogenicity, and immunotolerance in one graph from hbv to hpv: designing vaccines for extensive and intensive vaccination campaigns worldwide key: cord-346331-d0s028wl authors: lackey, kimberly a.; pace, ryan m.; williams, janet e.; bode, lars; donovan, sharon m.; järvinen, kirsi m.; seppo, antti e.; raiten, daniel j.; meehan, courtney l.; mcguire, mark a.; mcguire, michelle k. title: sars‐cov‐2 and human milk: what is the evidence? date: 2020-05-30 journal: matern child nutr doi: 10.1111/mcn.13032 sha: doc_id: 346331 cord_uid: d0s028wl the novel coronavirus sars‐cov‐2 has emerged as one of the most compelling and concerning public health challenges of our time. to address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical and public health communities has rapidly engaged to collectively find answers and solutions. one area of active inquiry is understanding the mode(s) of sars‐cov‐2 transmission. although respiratory droplets are a known mechanism of transmission, other mechanisms are likely. of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. however, there is limited published literature related to vertical transmission of any human coronaviruses (including sars‐cov‐2) via human milk and/or breastfeeding. results of the literature search reported here (finalized on 17 april 2020) revealed a single study providing some evidence of vertical transmission of human coronavirus 229e; a single study evaluating presence of sars‐cov in human milk (it was negative); and no published data on mers‐cov and human milk. we identified 13 studies reporting human milk tested for sars‐cov‐2; one study (a non‐peer‐reviewed preprint) detected the virus in one milk sample, and another study detected sars‐cov‐2 specific igg in milk. importantly, none of the studies on coronaviruses and human milk report validation of their collection and analytical methods for use in human milk. these reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses (in particular, sars‐cov‐2) during breastfeeding are discussed. the global pandemic caused by the sars-cov-2 virus is one of the most compelling and concerning global health crises of our time. fortunately, this pandemic has rapidly mobilized the full range of expertise represented by researchers, clinicians and public health officials. although our understanding of the biology, clinical implications and strategies for mitigation continues to evolve, one issue that has received limited attention is the implication of this pandemic for infant feeding practices. this lack of attention has resulted in mixed messages regarding guidance about optimal infant feeding practices (e.g., american academy of pediatrics, 2020; centers for disease control and prevention, 2020a; world health organization, 2020a; united nations children's fund [unicef] , 2020) and a consequent lack of confidence about best approaches to infant feeding in the face of this growing pandemic. even when a mother is positive for covid-19, the world health organization (who) recommends breastfeeding be initiated within 1 h of birth, exclusive breastfeeding be continued for 6 months and breastfeeding be continued for up to 2 years. they suggest use of appropriate respiratory hygiene, hand hygiene and environmental cleaning precautions. the unicef recommends that covid-19-positive mothers continue breastfeeding while applying precautions, such as wearing a mask and handwashing before and after feeding (unicef, 2020) . the u.s. centers for disease control and prevention (cdc) neither recommends nor discourages breastfeeding but advises that decisions be made by the mother and family in consultation with their health care providers (centers for disease control and prevention, 2020a) . they recommend that during temporary separation (should that occur), mothers who intend to breastfeed should express their milk using proper hand hygiene and that the expressed milk should be fed to the newborn by a healthy caregiver. further, if a mother and newborn do room-in and the mother wishes to feed at the breast, the cdc recommends that she should wear a facemask and practice hand hygiene before each feeding. it is well established that viral transmission through human milk can occur (jones, 2001; lawrence & lawrence, 2004) . notable examples include human immunodeficiency virus (hiv; black, 1996; ziegler, johnson, cooper, & gold, 1985) , cytomegalovirus (cmv; stagno & cloud, 1994) and human t-cell lymphotropic virus type 1 (htlv-1; boostani, sadeghi, sabouri, & ghabeli-juibary, 2018) . perhaps the most prominent example of mother-to-child viral transmission via breastfeeding is hiv infection, during which higher milk and serum viral loads are associated with an increased risk of transmission (davis et al., 2016; semba et al., 1999; willumsen et al., 2003) . the risk of postnatal infection for breastfed infants of hiv+ mothers is ≈10-20% over the first 2 years of life without the use of antiretroviral therapies (art; dunn, newell, ades, & peckham, 1992; nduati et al., 2001) . however, compared with mixed feeding, exclusive breastfeeding is associated with lower risk of transmission of hiv infection to infants (coutsoudis et al., 2001; iliff et al., 2005) . in many high-income nations, breastfeeding is contraindicated in the case of maternal hiv infection with or without maternal art (e.g., american academy of pediatrics, 2012; centers for disease control and prevention, 2020b) . conversely, in low-and-middle-income nations, infant mortality from malnutrition and infectious disease may outweigh the risk of acquiring hiv via vertical transmission during breastfeeding. as such, breastfeeding is recommended (world health organization, 2016) . with respect to cmv, it is estimated that 60-70% of breastfed infants of cmv-seropositive mothers become infected with cmv (dworsky, yow, stagno, pass, & alford, 1983; minamishima et al., 1994) . the risk of cmv infection in neonates is highest in preterm and very low birthweight (less than 1,500 g) infants (hamprecht & goelz, 2017; lanzieri, dollard, josephson, schmid, & bialek, 2013) . a small percentage of infected infants develop a severe complication known as cmv sepsis-like syndrome, which can be fatal (fischer et al., 2010) . nonetheless, breastfeeding is not contraindicated in cmv-seropositive women with healthy, term infants for htlv-1, breastfeeding is considered the major route of infection for infants (moriuchi, masuzaki, doi, & katamine, 2013) . htlv-1 infection is lifelong, and although most infected individuals remain asymptomatic, approximately 10% develop severe disease, including adult t-cell leukaemia, a highly aggressive and usually fatal malignancy (rosadas & taylor, 2019) . some organizations and agencies list maternal htlv-1 as a contraindication for breastfeeding (american academy of pediatrics, 2012; centers for disease control and prevention, 2019a), whereas others do not (world health organization, 2009 ). human coronaviruses are enveloped, positive-sense, singlestranded rna viruses first described in 1965 (tyrrell & bynoe, 1965) . there are seven identified strains known to infect humans. four of the strains (alphacoronaviruses 229e, nl63 and oc43 and betacoronavirus hku1) are ubiquitous in humans and cause the common cold. there is limited evidence that one of these (229e) may be vertically transmitted from mothers to infants, although the mechanism remains unclear (gagneur et al., 2008) . the presence of 229e in neonatal gastric samples suggests that one possible mechanism for infection is through human milk, although this study did not evaluate human milk specifically (gagneur et al., 2008) . in light of the emergence of the novel coronavirus sars-cov-2, several issues related to human milk and coronavirus infection demand immediate attention, the first and foremost being whether or • very little is known about coronaviruses in human milk and whether breastfeeding is a possible mode of vertical transmission. • limited, weak evidence suggests that some coronaviruses (including sars-cov-2) may be present in human milk, but these studies do not report methods of sample collection and validation of reverse transcription polymerase chain reaction (rt-pcr) assays for human milk. • nothing is known about the timing of the antibody response in human milk to sars-cov-2 infection. • future research should utilize validated methods and focus on both potential risks and protective effects of breastfeeding. not the virus is present in human milk produced by infected or exposed women. of particular interest in this context are (1) the potential role that breastfeeding could play in vertical transmission of sars-cov-2 from women to infants via human milk and (2) the potential protective effects of targeted antibodies and other immunoprotective components in human milk against covid-19. the goal of this review was to evaluate the published evidence regarding the presence of this and other human coronaviruses in human milk. we used a variety of databases to identify relevant literature published as of 17 april 2020, and the list of databases and search terms used can be found in table 1 . it is noteworthy that in addition to using standard scientific databases (e.g., pubmed), we also used a general google search and a search of preprint servers to identify reports that had not yet been published in refereed journals (i.e., grey literature). any research in which human milk was collected and tested for a human coronavirus was included in this review. see addendum in the supporting information online. this review does not constitute human subject research, and as such, ethical approval was not required. the deadliest of the human coronaviruses to date is mers-cov, which emerged in saudi arabia in 2012. the disease caused by mers-cov, middle eastern respiratory syndrome (mers), is characterized by severe respiratory illness with symptoms of fever, cough and shortness of breath. mers-cov is a betacoronavirus, and the case fatality rate of mers is 34% (mahase, 2020) . there are no reports of the presence or absence of mers-cov in human milk. however, there are reports of the presence of mers-cov in the milk of dromedary camels (camelus dromedaries; conzade et al., 2018; hemida et al., 2015; reusken et al., 2014) , and there is one report of a human likely infected through the consumption of raw (unpasteurized) camel milk (memish et al., 2014) . in camel milk samples spiked with currently, there is one report in which human milk was tested for sars-cov (robertson et al., 2004) and two reports of human milk being tested for sars-cov antibodies (robertson et al., 2004; stockman, lowther, coy, saw, & parashar, 2004 ). robertson and colleagues described a woman infected during the second trimester of pregnancy (19 weeks). a single milk sample was collected 131 days after the onset of symptoms, but no additional detail on the collection methodologies was provided. milk was submitted to the cdc, where it was analysed using reverse transcription polymerase chain reaction (rtsearch terms, databases and preprint servers used to identify existing literature reporting the possibility of vertical transmission of coronaviruses from mother to infant during breastfeeding as of 17 april 2020 the infant in this study was never tested for sars-cov infection. the first trimester of pregnancy (7 weeks the novel coronavirus sars-cov-2 was named after sars-cov due to its shared sequence homology (77.9%; kim et al., 2020) and similar clinical characteristics. the first reported cases of sars-cov-2 infection emerged in late 2019 in china. although the current estimated case fatality rate for covid-19 (the disease caused by the sars-cov-2 virus) is much lower than those of sars and mers at roughly 2% (mahase, 2020) , the spread of this pathogen has been much more rapid and extensive. at the time of writing, there were 13 studies (seven case studies and six case series; three of which were preprints or preliminary reports that had not been formally peer-reviewed; table 2 ) reporting direct testing of milk produced by women infected with sars-cov-2 dong et al., 2020; fan et al., 2020; liu, wang, li, et al., 2020; liu, wang, zhang, et al., 2020; salvatori et al., 2020; wang et al., 2020; yu et al., 2020; wu et al., 2020) or by women whose infants were infected kam et al., 2020; yuehua et al., 2020) . in total, 48 milk samples produced by 32 women had been tested; all but one sample (wu et al., 2020; non-peer-reviewed preprint) were negative for the presence of the virus. two milk samples produced by a single woman were tested for sars-cov-2 specific antibodies; igg but not igm was identified in both samples non-peer-reviewed preprint) . a description of the relevant characteristics for the women and infants in these studies can be found in table 2 . investigators conducting nine of the 13 studies analysed milk samples collected at birth or shortly thereafter, reporting only findings in colostrum or transitional milk. those same nine studies reported on the milk produced by women who were infected during the third trimester of pregnancy, whereas the other four reported findings from milk produced by mothers of infants infected at 1.5, 3, 6 and 13 months of age kam et al., 2020; yu et al., 2020; yuehua et al., 2020) . for the infants born to women infected during pregnancy, most were immediately separated from their mothers post-delivery and were not breastfed for the duration of the period observed in their respective reports. fourteen of the 32 infants described in these reports were born via caesarean section; only two were specified as vaginal births. repeated milk samples, collected up to 27 days apart, were analysed for eight of the women. all the studies were conducted in china cui et al., 2020; dong et al., 2020; fan et al., 2020; liu, wang, li, et al., 2020; liu, wang, zhang, et al., 2020; wang et al., 2020; yuehua et al., 2020 wu et al., 2020) , singapore (kam et al., 2020) or italy (salvatori et al., 2020) . pneumonia were collected after their first lactation' and that milk was collected following who guidelines, but they did not provide a citation for this collection method. all milk tested negative for the virus, but no information was provided on the methods used for analysis. in a report by liu, wang, zhang, et al. (2020) , milk produced by two women was tested. one woman was 34 years old and at in a case series by liu, wang, li, et al. (2020) , milk produced by 10 women infected during late pregnancy was tested via rt-pcr for sars-cov-2; all samples tested negative. it is noteworthy that this report included data from 19 women, but milk was collected from only 10 of them. the authors did not specify for which of the women milk was collected. none of the 19 infants reported in this study tested positive for sars-cov-2 via rt-pcr. the only detail available on collection or testing methods is that rt-pcr was used to test the samples and that 'milk was collected after the first lactation' . despite their results, the authors concluded that delivery should occur in an isolation room and that infants be separated from infected mothers. li et al. (2020) described a 30-year-old woman at 35-week gestation who was positive for sars-cov-2 and who delivered a male infant via emergency caesarean section. the infant was tested immediately upon delivery via oropharyngeal swab, which was negative. after delivery, the infant was kept in isolation away from his mother. milk was collected immediately after delivery and on days 2 and 3 post-partum; all samples were negative. again, no information on the collection or testing methods for the milk sample is available in this report. the infant's breastfeeding status was not specified in the report, but it is presumed that he was at least partially breastfed as the mother was producing milk at 6-month post-partum. d study presented data from three women but only presented data on the milk produced by two women. e study presented data from 19 women but only presented data on the milk produced by 10 women. f study presented data from 13 women but only presented data on the milk produced by three women. while the previous reports focused on infected women, there are also three case studies focused on infected infants. in these studies, milk produced by the infants' mothers was tested for sars-cov-2. the youngest of these infants was reported by cui et al. (2020) . after being exposed to infected family members, the 55-day-old female was admitted to the hospital with symptoms of covid-19 and diagnosed based on clinical data and exposure history. the infant was 'mixed fed'. her mother's milk was collected on the first three consecutive days of her hospitalization; all were negative for sars-cov-2. no information on the collection or testing methods for the milk sample is included in this report. yuehua et al. (2020) reported on a 3-month-old, breastfed female who was hospitalized and tested via throat swab for sars-cov-2; the swab was positive. a single milk sample was collected from the infant's mother; it tested negative. the authors provided no information on the collection or testing methods for the milk. importantly, this infant developed symptoms of covid-19 7 days before her parents became ill. as such, one possibility is that she was infected first and passed the infection to them. another case report on a mature milk sample comes from singapore (kam et al., 2020) . this report is particularly interesting as the infant had no symptoms but was hospitalized and tested because his caregivers were all hospitalized with covid-19, and there was no one to care for him. the infant was 6 months old and presumably at least partially human milk fed as a sample of milk was successfully collected from his mother. despite being asymptomatic, a nasopharyngeal swab taken from the infant was positive for sars-cov-2. the authors reported that milk produced by the mother on a single day tested negative for the virus but did not specify how many samples were taken. this report provided no data on the methods used for the collection and analysis of these sample(s). cov and sars-cov-2, there remains much to be learned about their modes of transmission. respiratory droplets are a documented source of the virus (world health organization, 2020b), but other sources such as breastfeeding and/or human milk may exist. the primary purpose of this review was to examine the evidence (or lack, thereof) for the vertical transmission of sars-cov-2 from mother to infant via breastfeeding considering what is known about other human coronaviruses. we also examined the evidence presented in the same reports related to maternal/infant antibody production to the virus. in total, we identified 14 studies that had tested human milk for human coronaviruses directly. thirteen of these studies were newly published reports on sars-cov-2 and human milk, which collectively encompassed 48 milk samples. all but one of these samples tested negative for sars-cov-2, and that result was reported in a non-peerreviewed, online preprint. we identified no comparable data for mers; a single case report for sars, which yielded a negative result for the presence of the virus but positive results for antibodies specific to sars-cov; and no reports of human milk tested for other human coronaviruses. there was one report of antibody tests in milk specific to sars-cov-2, which identified igg but not igm . the single milk sample that was supposedly positive for sars-cov-2 (wu et al., 2020) is clearly anomalous to the larger body of evidence. importantly, since no methodologies were provided and the study has not been published in a refereed journal, any interpretation of this result must be made with extreme caution. this underscores the fact that it is critical for the research community to focus its efforts on analysing appropriately collected human milk using laboratory methods that have been validated and optimized for the human milk matrix. in addition, results should be submitted to high-quality, refereed journals for peer review. only then can methods and results be rigorously assessed by scientists with the knowledge base needed to judge them. the dearth of high-quality evidence substantially compromises the ability to effectively respond to this pandemic and provide guidance to some of the most vulnerable individuals: pregnant and lactating women and infants. limited and weak data suggest mers may be present in camel milk, but the relevance to sars-cov-2 in human milk is unclear. notably, reusken et al. (2014) reported that milk analysed in the camelid studies was not collected aseptically; rather, samples were obtained according to local milking customs. as such, it is possible that the presence of mers-cov in camel milk could be due to contamination from the milker, the calf or the environment, rather than milk representing an endogenous source of the virus. this is likely an issue with all the studies on sars-cov-2, where only one reported cleaning of the breast prior to sample collection (wu et al., 2020) . however, the limited data available on all three of these viruses (and human coronaviruses, in general) leave many questions unanswered with respect to the role, if any, of human milk in vertical transmission of coronaviruses. one possible reason that most of the rt-pcr results for the milk samples tested were negative is that the methods used were neither designed nor validated for human milk. milk is a complex matrix containing substantial fat, dnases (babina, kanyshkova, buneva, & nevinsky, 2004) , rnases (das, padhy, koshy, sirsat, & rich, 1976; mccormick, larson, & rich, 1974; ramaswamy, swamy, & das, 1993) and other pcr inhibitors (abu al-soud, jonsson, & radstrom, 2000; al-soud & radstrom, 2001; schrader, schielke, ellerbroek, & johne, 2012) . of note is the fact that commonly used silica columnbased rna isolation methods are designed for a limited sample volume, and as such are not suitable for more voluminous liquid samples. thus, validation of methods using human milk is needed (see box 1). in addition, other than general statements about the timing of collection (e.g., 'milk was collected after the first lactation') and brief descriptions of the rt-pcr assays used for nasal and throat swabs, none of the studies to date has described the methods of collection or how the milk was handled and stored in any detail. in addition, nothing is known about stability of sars-cov-2, if present, in human milk and how quickly (or at what temperature) it must be frozen to preserve fidelity. information on sample collection, handling and storage is critical to evaluating whether the negative results described in these studies could be due to inadequate methods used. some factors to consider when validating methods for human milk testing of coronaviruses. • method of milk collection: use of manual milk expression versus electric pump, cleaning procedures of breast and pump, partial versus full breast expression and foremilk versus hindmilk. • sample handling and storage: container material, temperature and duration of refrigeration/freezing. • assay validation: nucleic acid extraction protocols, amplification protocols, reagent selection, proper positive and negative controls and fresh versus frozen milk. • viral quantification and viability: infectious dose and biologically relevant concentrations. another possibility is that there is low abundance of the virus in human milk, and it is often not captured in the limited samples tested so far. for example, in the report on other human coronaviruses by gagneur et al. (2008) , 159 maternal-infant dyads were tested (including 161 infants, two sets of twins). in this report, 229e was present in both maternal and infant samples in only two dyads. additionally, in the milk of dromedary camels, mers-cov appears to be present at very low abundance (reusken et al., 2014) . this suggests the possibility that a very low viral load in milk might also lead to an inflation of false negatives. limited evidence from two patients suggests that sars-cov-2 shedding in respiratory samples peaks at 6 days after onset of symptoms (pan, zhang, yang, poon, & wang, 2020) , indicating that timing of sample collection also plays an important role in virus detection. only one study has investigated antibodies in milk specific to sars-cov-2 non-peer-reviewed preprint) , and these researchers identified igg in one milk sample produced by a woman at 13-month post-partum. although limited to a single study, this finding combined with a large body of literature documenting targeted antibodies in human milk indicates that there may be a protective effect of breastfeeding when the mother is covid-19 positive. the infant in this study was older than all the other infants described here, was likely not exclusively breastfed (based on reported age) and likely had a more mature immune system than the youngest infants described in other reports. still, further investigation into this finding is a critical next step in understanding how breastfeeding and/or the infant's con(robertson et al., 2004) . together, these observations suggest that infant infection may occur in utero but that the virus may simply be absent from the upper respiratory tract immediately after birth and therefore undetectable on pharyngeal swabs. very recent work has demonstrated that, like sars-cov and human coronavirus nl63 (hofmann et al., 2005) , angiotensinconverting enzyme 2 (ace2) is one of the receptors used by sars-cov-2 to enter host cells (w. h. li et al., 2003; yan et al., 2020) . ace2 is expressed across many body sites and tissue types, including the oral cavity (e.g., tongue and oral mucosa) and in mammary tissue . if mammary epithelial cells express this receptor, then it follows that viable virus could exist in milk. if it does, then the introduction of virus-containing human milk could represent a mechanism of entry for sars-cov-2 and covid-19 infection for infants. another observation worth considering is that, in at least one of the reports (yuehua et al., 2020) , the infant was infected and symptomatic 7 days prior to the infant's parents. this suggests the possibility that a 'reverse' vertical transmission from infant to mother could occur, a phenomenon that has been observed for other pathogens, such as hiv (belitsky, 1989; little et al., 2012) and ebola virus (sissoko et al., 2016) . one possible mechanism for maternal infection in this case is through retrograde flow, where milk and saliva move back into the mammary gland from the infant's mouth during suckling (ramsay, kent, owens, & hartmann, 2004) . although this mechanism is speculative, it represents a possible route whereby an infant could theoretically transfer a pathogen it has encountered in the environment to the mother. it is also possible that maternal infection could occur through other mechanisms, such as infant respiratory droplets (world health organization, 2020a) or via faecal matter . to date, all reports on sars-cov-2 and human milk have originated in asia (china and singapore) or europe (italy). although this limited geography makes sense given the fact that the initial epicentre of this pandemic was in asia and this was followed by a large outbreak throughout italy, studies from other globally representative populations are needed to make definitive conclusions regarding the possible presence and/or role of sars-cov-2 in human milk. additionally, the importance of a coordinated, international effort by scientists, clinicians and public health officials to elucidate answers to the many remaining questions related to sars-cov-2 and breastfeeding cannot be overemphasized. human milk is the gold standard for infant feeding. however, confidence as to its safety and best practices around breastfeeding during maternal covid-19 infection has been compromised by the lack of rigorous evidence as to whether sars-cov-2 can be vertically transmitted in milk and/or during breastfeeding. as such, there exists an immediate need to rapidly generate rigorous evidence for the role substantial interdisciplinary research on this topic is required and should be performed rigorously and rapidly to best inform policies regarding early feeding choices and clinical management of breastfeeding mothers infected with sars-cov-2 and their infants. to understand the role of human milk and sars-cov-2 infection, the following points must be rapidly addressed. • optimization of human milk collection and storage protocols for sars-cov-2 research. • validation of assays for identification of sars-cov-2 rna and sars-cov-2-specific immune components in human milk. • multinational population studies documenting presence or absence of sars-cov-2 virus and immune factors (including antibodies) in milk produced by infected women, women with infected infants and women who have been exposed to sars-cov-2; if the virus is identified in milk, its viability must be verified. • multinational population studies documenting (or not documenting) risk of covid-19 infections in breastfed versus nonbreastfed infants whose mothers are covid-19 positive. • research delineating implications of skin-to-skin breastfeeding versus consumption of pumped human milk. identification and characterization of immunoglobulin g in blood as a major inhibitor of diagnostic pcr purification and characterization of pcr-inhibitory components in blood cells policy statement: breastfeeding and the use of human milk initial guidance: management of infants born to mothers with covid-19 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(2020b). modes of transmission of virus causing covid-19: implications for ipc precaution recommendations viral shedding of covid-19 in pregnant women high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding structural basis for the recognition of sars-cov-2 by full-length human ace2 breast milk-fed infant of covid-19 pneumonia mother: a case report a case of three-month-old infant with new coronavirus infection antibodies in infants born to mothers with covid-19 pneumonia postnatal transmission of aids-associated retrovirus from mother to infant sars-cov-2 and human milk: what is the evidence? matern child nutr the authors acknowledge funding for other covid-19 and human milk related projects, including the generous gift by the family larsson-rosenquist foundation (flrf) in support of uc san diego's momi core; the bill & melinda gates foundation; the national instikey: cord-356062-7q5n4t97 authors: nan title: cumulative pharmacological activity index volumes 1-30 date: 2005-12-31 journal: studies in natural products chemistry doi: 10.1016/s1572-5995(05)80101-2 sha: doc_id: 356062 cord_uid: 7q5n4t97 publisher summary this chapter lists the important subjects on pharmacological activity that are discussed in the publication studies in natural products chemistry, volumes 1–30, such as abdominal constriction test, acanthoic acid, acetaminophen, parkinson's disease, photodynamic activity, prostaglandins, and oleanolic acid. the terms are mentioned along with the page numbers in which they are discussed in the publication. use of psycotria colorata in 30:205 19(4--+ 3)-abeo-8 c~, 13(s)-epoxylabda-4(18), 14-diene 29:102 activity in ebv bioassay system 29:102 19-nor-abieta-4( 18),8,11,13-tetraen-7one 29:100 activity in ebv assay system 29:100 abieta-8,11,13-trien-7-one 29:99 activity in ebv assay system 29:99 18-nor11, 7a, 15triol 29:99 activity in ebv assay system 29:99 11, 15, activity in ebv assay system 29:99 abieta-8,11,13-triene-7o~,l 5,18-triol 7acetate 29:99 activity in ebv assay system 29:99 abietic acid 29:99 activity in tpa assay system 29:99 activity in crg (+)-5-epi-acetomycin 10:447 antitumor activity of 10:447 2(x-acetoxy-14-hydroxy-15-iso-valeroyloxy-9-oxo-costunolide 29:89 activity in nfkb assay system 29:89 activity in tnf(x assay system 29:89 9cx-acetoxy-4 ~, 15-epoxymiller-1 ( 10)zenolide 29:89 activity in nfkb assay system 29:89 3~-acetoxy-813-isobutyryloxy-reynosin 29:87 activity in mam-2 assay system 29:87 activity in dif assay system 29:87 15-acetoxy-9 ~-hydroxy-8 [3-methacryloyloxy-14-oxo-acanthospermolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-9cx-methacryloyloxy-8 ~hydroxy-14-oxo-acantho-spermolide 29:89 activity in nfkb assay system 29:89 3-acetoxycostunolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-eremantholide b 29:89 activity in nfkb assay system 29:89 activity in tnf~ assay system 29:89 9~-acetoxy-miller-1 ( 10) 30:191, 192, 203 development of 30:192, 203 use of 30:191 analgesic effects 26:401, 403, 404, 406, 416, 418; 30:205, 208 5:512,747;10:78, 117;11:134;12:398;14:145;20:245, 712 ;21:21,108,109,190,207,213,214, 230,237,262,266,267,275,284,601, 615 ;23:41,126,164,235,258,355,468, 375,472,473,531,534,800;24:573, 856,857;26:226,1002;28:140,688; 30:328 11:130;12:192; 17:283 ;21:188,202,223,239,364,437; 23:649;24:953-954 21:186,192,197, 203,206,213,214,218,221,226, 227,233-235,223,238,240,389, 391,399,404,405,435,599-662; 22:626;23:3,4,271,341,344,348, 468,473,798,801354,368 ;26:227, 228,553,1098;28:4;66,473,475,686; 30:327,738,629 against candida albicans 30:327,629 from hypericum roeperanum 30:629 of (e)-8(17), 12-1abdadiene-15,16-dial 23:798 of (e)-8b, 17-epoxylabd-12-ene-15,16-deol 23:798 of (e)-8 [3,17-epoxylabol-12-ene-15,1619:511,748;21:132, 133,149,252,269,664,665 ;23:316, 533,534,687,704 ;24:742,743 ;30:226, 396,397 13:647;17:137,376;18:775;21:130, 134,153,152,312,591,594,690,616, 656,657;22:93,119,307,345,361; 23:158,297,512,602,832;25:43; 26:340,341,343,391,398,400,401, 403,404,406,415,418,482,556,558, 741,759,1035 ;27:660,858 ;28:200, 257,293 ;671,844;30:206,207,224, 225,292,320,689 21:674,744; 26:229,790,813,887,789,790,791, 800,805,807,810,811,821,828,830 -microbial activity 5:752;10:443; 15:327-339;18:776-778;21:153, 235,257,401,571,573,591,596,597, 599,606;22:626;23:94,153,180,233, 237,249,262,268,267,346,353,522; 24:64,69,206,330,331-334,402,403, 404,406,417,418,443,470,485,859, 1091,1092,1189;26:64,330,401,402, 1189;27:807;28:62,224,257,293; 28:698 22:308,321,345 ;23:395,534,535,742, 772,773,845 ;25:59;30:173,224,225, 289,320,321,524,561,743,745; 26:250,255,509,556,672,763,768, 887,1005,1006;28:4,16,17,63,179, 257,293 1:275,316-320-408; 17:17 ;21:137,405,420,435,436,638, 704,733;22:676;23:74,96,126,132, 139,395,445,522,696,721; 24:272-275 ;24:288,845,847,849; 25:43,430,431,438,450-453;26:56, 482,706;27:803;28:211,517,519,559, 560,566;30:3,28,29,55,56,58,59,64, 65,224,314,395,589,690,777,840 :461,563;10:608, 609,619,620; 17:146 ;21:132,148, 150,393,435,600,615,662,690,718; 22:307,361 ;23:233,268,233,346,395, 473,512,531,703;25:114;26:169,222, 224,226,344,452,741,759;27:482; 28:257,293 ;30:224,324-326,393,394, 395,398,401-405,407,410-412 132,148,162, 223,236,238,263,265,269,284,297, 366,388,390,410,419,421,431,571, 578,591,598,601,603,620,694 ;23:63, 93,95,108,111,129,126,107-152,153, 158,179,267,271,285,395-453, 455-485,491,497,642-643 ;739;24:25, 799,845 ;27:178,377,431,421,443, 693,778,801,924 ;24:353-376; 27:185-225,332,659;28:109,212,228, 229,340;29:743 ;30:4,26,172,175, 200,224,303,310,395,404,483,484, 485,495,496,497,499,562,588,603, 637,831,840,843, acaricidal enzymatic activity 24:995-998 tx-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in pkc assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 ~-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 of (-)-deoxypodophyllotoxin 30:589 of (-)-hemone 30:590 of (-)-nymphone 30:590 of (-)-yatein 30:590 of (+)-corytuberine 30:589 of (+)-epiaschantin 30:590 of (+)-epimagnolin 30:590 of (+)-epiyangambin 30:590 of (+)-hernovine 30:589 of (+)-laurotetanine 30:589 of (+)-magnoflorine 30:589 of (+)-malekulatine 30:590 of (+)-n-formyldehydroovigerine 30:589 of (+)-n-formylnornantenine 30:589 of (+)-n-formylovigerine 30:589 of (+)-n-hydroxyhemangerine 30:589 of (+)-n-methylhemangerine 30:590 of (+)-ovigerine 30:590 of (+)-ovihernangerine 30:590 of (+)-vateamine-2' [3-n134,136,150, 174,257,259,260,262,263,265,267, 269,270,271,273,274,284,311,364, 410,634,643,656,661,690,704,744; 23:96,97,187,218,238,249,250,253, 255,272,274,277,290,292,299,308, 317,701,274 ;24:294,352,740,867; 26:212-213,1185 as anti-fungal agents 24:406 2 [3,5-epoxy-5,10-dihydroxy-6c~ange lo y-lo xy-9 [3-is obutylo xygermacran-8ot, 12-olide 29:89 activity in inos assay system 29:89 activity in nfkb assay system 29:89 7 a, 8 c~-ep oxy-6 or-hydro xyabieta-9(11), 13-dien-12-one 29:99 activity in ebv assay system 29:99 9,10or-epoxy-9,10-seco-abieta4-o-phenyl-2-propionyl-neu5ac-cx2me 27:123 4-t-butyl-dimethyl-silyl ether 27:127 5-azido-5-deamino-neu5ac-c~2me 27:119 5-n-benzyloxycarbonyl-neu-cx2me 27:119 5-n-propionyl-neu-c~2me 27:119 7-deoxy-neu5 ac-c~2me 27:123,127 8-amino-8-deoxy-neu5 ac 27:121,122 8-azido-8-deoxy-neu5ac 27:121,122 8-tosyl-neu5acme ester 27:121,122 9-amino-9-deoxy-neu5 ac 27:121,122 9-amino-9-deoxy-neu5ac-c~2me 27:119 9-azido-9-deoxy-neu5 ac 27:121,22 9-azido-9-deoxy-neu5ac-c~2me 27:119 9-o-acetyl-neu5 ac-( ,590 activity of (-)-deoxypodophyllotoxin in 30:589 activity of (-)-hemone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-activity of hydroxyhernangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2'13-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy12-hydroxy -6,7-seco-abieta-8,11,13trien-6,7-dial 29:99 activity in ebv assay system 29:99 7-hydroxy-6-methoxy-1-methyliso22:510,512,513,516,518,519, 521,522,525-529,531,534 achyranthes aspera l 9,459,503,753,754,311; 17:313,441 ;20:108-113,273,613; 24:215,228-232,503-509,739-798; 26:229,790,813,887;30:560,562,567, 570,590 chemo-differentiation therapy 30:498 of acute monocytic leukemia chemopreventive activity 30:591,597, 766 in mouse mammary organ culture model 30:591 in vitro 30:597 in vitro bioassays for 30:591 of chlorella vulgaris 30:766 ofisoquinoline alkaloids 30:597 of lignans 30:597 chemopreventive agent 26:219 genkwanin as chemopreventive efficacy 27:386 of limonene carcinogenesis 27:386 chemoprophylaxis 26:674 chemosensitizers 30:595 antimalarial drugs as 30:595 chemotaxonomic markers 26:1128 chemotherapeutic agent 30:5,330,742 atovaquone 30:330 for colon cancer 30:5 for multi-drug resistant cancers 30:5 for ovarian cancer 5 for taxol-resistant breast cancer chemotherapeutic intervention 30:397 targets for chemotherapy drug 27:436 antitumor activity of 27:436 chest pain 30:203 use of ginseng root in 30:203 childhood disintegrative disorder (cdd) gram-negative bacteria 30:693 maytenus species against gram-positive bacteria 23:68;30:629, 693 actamycin against 23:68 antimicrobial activity of 30:629 inhibitor maytenus species against grandiflorolic acid 29:100 activity in ebv bioassay system grandiflorolic acid angelate 29:100 activity in ebv bioassay system grandisin 26:230 trypanosomicidal activity of grandmal seizures granulocyte phagocytosis 26:47,55 ofjenisseensosides c 26:47,55 ofjenisseensosides d green tea 27:417,420 biological activity of 757 effect of general analogues on pgf2 na-induced contraction griseofulvin 24:934 as anti-fungal agents grob-type fragmentation 24:728,731 growth 30:61 of tumor cells 30:61 growth inhibition 17:142 growth inhibitor activity 7:407 guaiazulene 5:363,369 as an anti-inflammatory agent guidimacrin 27:831 antitumor mechanism 584 neurodegeneration 30:224 reperfusion injuries 30:224 respiratory disorders 30:224 role of free radicals in 30:224 human growth hormone human hek 293 cell line 30:781 biological response of human hl-60 leukemia cells 30:496 differentiation of 30:496 inhibition of 30:496 proliferation of human leukemia cells 18:269 human leukocyte elastase 16:727 inhibitor human leukocyte elastase (hle) 30:830,831,843 homologous sequences of 30:831 plasminogen activator (u-pa) 30:830 human lung carcinoma 23:290;30:5 discodermolide against human lung carcinoma) 30:691 phenolic triterpenes against 30:691 human medulloblastoma 23:290 human melanoma 23:255 human mesangial cell proliferation 30:308 in vitro human monoblastic leukemia cells 30:498 effect of hydroxyurea (hu) 30:498 growth inhibition of 30:498 human monoblastic leukemia u937 cells 30:498 growth inhibition of human neuroblastoma nbla-n-5 cells tamoxifen activity against 12:390 human neuroblastoma sh-sy-5y cells 12:390 human neutrophils 12:390 human neutropil protein kinase c 12:389 human onchocerciasis 12:9 ivermectin for human papilloma virus 30:394 pathogenesis of 30:394 cause of hepatocellular carcinoma human papilloma virus (hpv) 23:97 human papilloma virus type 11 30:406 microbicidal compound against human papilloma virus type 40 30:406 microbicidal compound against 30:406 human platelets 12:390 study of protein kinase c in 12:390 by staurosporine 12:390 human rotavirus (hrv) 30:406,412 cause of dehydrating gastroenteritis 30:406 member of 30:406 reoviridae type of human serotonin transporter gene (slc6a4) 30:376 regulation of neuronal activity human spermatozoa motility 21:675 human synovial pla2 25:697 inhibitor anti-glycemic effects 24:902-904 anti-hyperglycemic effects of -o-methyl-20-hydroxyecdysone activity in 29:28 pinnatasterone activity in 29:28 polypodine b 2-o-cinnamate activity in muscarinic achr antagonists 21:56 muscarinic antagonist 22:19 muscarinic receptor 21:95 muscarinic receptor antagonists 22:734 muscarinic-l,2-receptor 21:95 muscle pain 24:898 aconitine for relieving activation effects of 24:906 medicinal uses of 24:906 protein kinase c activation effects 24:906 muscular p-388 (murine lymphocytic leukemia) 30:588 activity of (-)-deoxypodophyllotoxin against 30:588 activity of (-)-yatein against p-388 lymphocytic leukemia cell 25:764 p388 mouse leukemia 12:390 staurosperine activity against p-388 murine leukemia cells 21:411 pachyman 5:288,317 antitumor activity of 5:317 pachyrrhizus erosus 24:779,822-826 anti-feeding activity of 24:825 pachyrrhizus palmatilobis 24:822 biological activity of 24:823 paclitaxel 240 (+)-acetoxypinoresinol dimethyl ether as 26:241 arctigenin as 26:241 arctigenin methyl ether as 26:241 chamigrenal as 26:244 cinnamophilin as 26:240 dehydroschisandrol a as 26:245 denudatin as 458 fptase inhibition by 24:458 paw oederma 30:207 inhibition of 30:207 2-pectenotoxin 19:580 cytoxic activity of 19:580 pectinasic pectolinaringenin 30:725,748 antispasmodic activity of 30:748 pedunculariside 29:84 activity in cox assay system 29:84 activity in epp assay system pelargonidin (anthocyanidin) 29:585 effects on lela 29:585 effects on penates sp. 28:713 as ~-glucosidase inhibitor 28:713 penicillium turbatum 11:194 antibiotic a 26771b by 11:194 penstemide 7:441 activity against p-388 lymphocytic leukemia penstemon rosseus 24:838 anti-fungal activity pentacyclic triterpenes 30:207 protein kinases inhibition by 564 in lipid autoxidation 3,4,6-p enta-o-gallo yl-~ -d-g luc o s e (galloyl ester) 29:589 effects on 229 biological activity of 28:229 8-prenyleriodictyol 28:229 biological activity of 28:229 prenylflavones 28:225 anti-human immunodeficiency virus (hiv) activity of 28:226 prenylfiavonoid 30:207 antinocicieptive effect of antitumor promoting activities of 29:720 as cytomegalovirus protease inhibitors 29:720 gastro-intestinal problems 29:720 use in hodgkins disease 29:720 use in infections 29:720 use in lupus 29:720 use in osteomyelitis 29:720 use in parkinson's disease 29:720 use in psoriasis 29:720 use in respiratory problems 29:720 use in skin ulcerations 29:720 use in syphilis 29:720 6-prenylnaringenin prevention by chitosan 27:436 of myelotoxicity 27:436 primary biliary cirrhosis 25:463 primary granulosa cells 25:269 apoptosis in 25:269 primary solid-tumor growth 30:64 effect of triterpenoids on 30:64 pristimarin 30:692 cytotoxic activity of prl1 & prl2 proproteins 29:592 effects on cysteine protease 851 probes 27:332 activity of 27:346 procurcumenol 29:90 activity in tnfct assay system procyanidin b 1 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b2 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b-5 3,3'-di-o-gallate 29:580 effects on ace procyanidin c2 (trimeric flavan-3-ol) 29:580 effects on ace 29:580 procyanidin polymer (flavan-3-ol polymer) 29:580 effects on ace prodrug monotherapy 21:157 natural anthracyclines for 21:157 prodrugs 21:157 of natural anthracyclines pro-inflammatory cytokines 25:461 pro-inflammatory substances 30:206 in chronic painful inflammatory diseases 30:206 proliferation 30:368,378 effect of serotonergic neurotransmission on 30:368 serotonin action on 30:378 prolysine 25:388 prophylactic effects pro-nociceptive prostaglandins 30:192 in inflammation pro-nociceptive transmitter 30:194 release inhibition of 30:194 prooxidant activities 30:745 in thiobarbituric acid assays 30:745 of chemiluminescence 30:745 of tert-butyl hydroperoxide 30:745 prooxidant property 30:524 of carotenoids 30:524 prophylactic drugs prostaglandin e2-dependent flavonoids cytoprotection effect pgh2) 25:595 prostaglandin inhibitors 30:192 clinical use of 30:192 use in rheumatism 30:192 use in osteoarthritis 30:192 use in headache 30:192 use in dental surgery anticancer clavulones 16:366 as antiviral compound 24:541-544 as pain mediator 30:192 in mast cells 30:192 nociceptive effect of prostate cancer prevention 30:525 role of lycopene protease inhibitor proteins from plants 29:596 effects on metallo protease inhibitors 24:487,488 567 cereal bifunctionals as 29:567 chymotrypsin as 29:568 destructive potential of 29:568 in alzheimer's disease 29:567 in angiogenesis 29:567 in cancer inflammatory disease 29:567 in protozoal infection 29:567 in viral infection 29:567 kunitz as 29:567 mustard family of 29:567 of metallocarboxypeptidase 29:567 ofserine protease 29:567 pepsin as 29:568 phytocystatins as 29:567 potato type 29:567 proteins as 29:567 serpin as 29:567 trypsin as 29:568 proteases 24:1005;29:567 enzymatic activity 24:1005 in apoptosis 29:567 in blood clotting 29:567 in cell division 29:567 in digestion 29:567 in extracellular matrix digestion 29:567 in inflammatory responses 29:567 protein kinase a (pka) 192 in regulation of ionotropic receptors protein kinase activity 28:677 ofxestoquinolide protein kinase c (pkc) activator 30:70 protein kinase c inhibition 24:573; 25:46,488 by xestocylamine 24:573 protein kinase inhibition 30:207 by pentacyclic triterpenes protein phosphatase inhibitors 27:874 protein phosphatases 25:707 inhibitor of 25:707 protein synthesis 30:407 inhibitor of 30:407 protein transduction domain (ptd) proteinaceous receptor 25:371 proteinase activity 21:150 protein-coupled receptor 27:821 g-proteine kinase c activity 21:638 proteins protein-tyrosine kinase activity 27:842 offlavonoid aglycones 27:842 of glycosides 27:842 ofkoelreuteria henryi protein-tyrosine kinase inhibitory activities proteolytic activity 30:841 proteolytic systemin inactivation protium kleinii 30:206 anti-inflammatory effect of 30:206 antinociceptive effect of 30:207 protocatechuic acid (3,4-dihydroxybenzoic acid) 29:589 effects on proton pump inhibitors (ppis) 25:612 proton-translocating nadh:q oxidoreductase 28:435 high-affinity inhibitors protozoocidal activity 30:329,741,742 against leishmania 30:329 against plasmodium 30:329 against tryponosoma 30:329 genus baccharis 30:741 in vitro 30:329 of neo-clerodane diterpenoids 30:742 ofquinone derivative 30:329 protozoocidal compounds 30:740 use of proxyelocytic leukaemia (hl-60) prunella vulgaris 30:401 antiviral activity of psammaplin a (bisprasin) 28:693 effects on bacillus subtilis psammaplysilla purpurea 28:693 antimicrobial activity of 28:693 as tyrosine kinase inhibitor pseudobersana mossambicensis 20:478 bioactive steroids from pseudoguaiane 11 a,13-dihydrohelenalin acetate 29:90 activity in cro assay system 627 against cytomegalo virus 30:627 against human immunodeficiency virus-1 30:627 against influenza virus 30:627 as virucidal agents 30:627 retroviral activity of 7:421 pseudolaric acid b 13:653 pseudomonas aeruginosa 30:739 antibacterial activity against 30:739 antifungal activity against pseudorabies virus 30:404 in vitro replication of 30:404 role ofheparin 30:404 psii inhibitors 26:368,371 psk in mitosis 25:377 psk-receptor 25:378 psk-a activity 25:378 psoriasis 30:484 use of lc~,25(oh)2d3 30:484 psychiatric disorders 30:369 anorexia 30:369 bipolar disorder 30:369 bulimia 30:369 effect of circadian activity on 30:369 obsessive compulsive disorder 30:369 panic disorder 30:369 schizophrenia 30:369 seasonal effective disorder 30:369 unipolar depression 30:369 psychodelic drugs 26:820 cocaine as 26:820 morphine as 26:820 semisynthetic lsd as 26:820 psychovegetative disorders 22:643,684 psycotria colorata 30:205 to relieve abdominal pain puberulin a 26:243,244 as paf-induced inhibitor pulegone 29:83 activity in am assay system 29:83 pulmericin 16:299 antifungal activity of 16:299 antileukemic activity of 16:299 cytotoxic activity 16:299 pulmonary vascular injury 30:70 role of serine protease in 30:70 role of thrombin pumiliotoxin alkaloids 27:238,239 alio-ptx 323 b alkaloid 27:249,250 biological activity of punta toro virus 30:411 treatment of 30:411 purifying blood 26:50 herniaria hirsuta in 26:50 purine receptors putative receptor proteins 25:398 putative satiety factor 24:910 effect of cholecystokinin 24:910 pyranocoumarin 27:429 biological activity pyranoid carbasugars 29:466 as cellular messengers 29:467 as insulin release mediators 29:467 biological activities of 29:466 effects on glycosidase enzyme 29:467 effects on glycosyltransferase enzyme 29:467 galactosyltrans ferase inhibition by pyridoacridine alkaloids 28:639 cytotoxic effects on kb cells 524 glycosidase inhibitors of 10:524 pyrrolomycin antibiotics 25:794 pytressin-induced coronary spasm antileukaemic activity of 4'-quercetagetin (6-hydroxy quercetin hexahydroxyflavone) (flavonol) 29:573 effects on 4'-pentahydroxyflavone) 29:578;30:208,749 analgesic effect of quercetin 3-o-c~-l-arabinopyranoside 28:64 anti-oxidant activity quercetin 3-o-c~-l-rhamnopyranoside 28:64,65 anti-oxidant activity of quercetin 3-o-[3-d-galactopyranoside 28:64 anti-oxidant activity o-galloyl)-glucoside 29:580 effects on ace quercetin-3-o-ot-l-rhamnopyranoside 28:64 anti-oxidant activity quercitrin (quercetin-3-rhamnoside) 29:581 effects on ace quillaia 26:55 of adjuvant activity 26:55 quinidine 25:347 quinine 25:344 290 antitumor activity of 10:117 inhibitor of dna quinoline drugs 25:348 quinoline drug family quinoline-based antimalarials 25:344 quinoline-ring antimalarial drugs 25:327 quinolines 25:327 quinolizidine derivatives 27:284,285 testing against mycobacterium tuberculosis 541-544 as antiviral quinone methides 30:694 antiplasmodial activity of 30:694 quinones rabidaea platyphylla 22:513 for epilepsy 22:513 rabies virus 30:409 inhibitor of 30:409 rabies virus infection 30:409 in chicken-embryo-related cells 30:409 rabies virus replication 30:409 in nerve fibers rachitic bone healing potencies 484 of vitamin d2 30:484 of vitamin d3 30:484 of vitamin d4 30:484 of vitamin d5 30:484 of vitamin d6 30:484 of vitamin d7 radical scavenging activity 23:362 of coumarin 23:335 radioligands 30:814 from brain membrane synaptosomes ranitidine 25:612,617 ranp-triggered signal transduction raphe nuclei 30:368 role in cognitive functions ras oncogenes 22:621 rat brain synaptosomes 30:807 p-affinity rat mammary carcinogenesis model 30:592 cancer chemopreventive activity rat serum vitamin d-binding protein (dbp) 30:494 binding assay for rebaudioside a 29:101 activity in tpa bioassay system tpa bioassay system 8-receptor 22:296 ~-receptor 22:296 8-receptor affinity 30:814 binding properties of 30:814 ~t-receptor affinity 30:814 binding properties of 30:814 of glycopeptide 30:814 8-receptor agonists k-receptor agonists 30:797,806 pharmacological activity of -receptor agonists 30:797 pharmacological activity of 30:797 8-receptor antagonists 30:797 pharmacological activity of k-receptor antagonists 30:797 pharmacological activity of 30:797 ~t-receptor antagonists 30:797 pharmacological activity of 30:797 receptor binding 27:377 receptor binding assay 30:810 in vitro 30:810 mouse vas deferens (mvd) activity in 30:810 of guinea-pig ileum (gpi) 30:810 receptor binding properties 30:815 of tyr-d-ala-phe-[[3-d-glc(oac)4]tyr-pro-ser-nh2 30:815 of tyr-d-ala-phe-asp-val-val-gly-nh2 (deltorphin c) 30:815 of oac)4]-gly-nh2: 30:815 of tyr-d-ala-phe-gly-tyr-pro-ser-nh2 (deltorphin c) 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr([3-d-glc)-gly-nh2 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr c )-val-vai-gly-nh2 receptor tyrosine kinase (rtk) 25:512 receptors 27:363;30:421 and cell-cell adhesion 30:421 and cell-cell communication 30:421 antigenic determinants of 30:421 for bacteria 30:421 ofchiral proteins 27:363 with enzymatic activity 27:824 without enzymatic recombinant prourokinase (pro-uk) 30:839 as fibrinolytic enzymes recombinant tissue-type plasminogen activator 30:839 as fibrinolytic enzymes 30:839 red wine 27:430 biological activity of 27:430 redox enzymes redox inhibitors 30:225 of 5-1ipoxygenase repandusic acid (hydrolysable tannin) 29:573 effects on hiv-1 protease 29:573 repellent properties 28:395 of amblyomma variegatum 28:395 reperfusion injuries 30:224 role of free radicals rescorcinolic lipids 30:165-167 effects on enzymatic activity 30:165-167 interaction with proteins reserpine 25:538 as immunosuppressive drug resistance-modifiers 30:595 antimalarial drugs as resistant plasmodium species 30:595 in chloroquine sensitivity resorcinolic lipids 30:119,158,160,162-164 as antifungal fluids 30:160 as growth reulators 30:162-163 as hair restoration-lotion 30:160 used in gingival infections respiratory ailments 24:847 respiratory depression 30:799 due to analgesic alkaloid 30:799 respiratory disorder 30:224 role of free radicals in 30:224 respiratory infection 30:408 respiratory syncytial virus 30:395 respiratory toxins 24:1002,1063 respiratory tract infections 26:50 herniaria hirsuta in 26:50 response decay mechanism 25:491 response modifiers resveratrol 27:430 biological activity of 27:430 resveratrol 28:582 effects in llc-bearing mice 28:582 effects on tumor 597 treatment of 30:597 use of hernandia moerenhoutiana in 30:597 use of hernandia nymphaeifolia 596 antimalarial activity of 30:595 antiplasmodial activity of 30:596 as platelet aggregation inhibitor 30:594 effect on antiplasmodial activity 30:596 retinal 29:111;30:520,521 activity in am bioassay system 29:111 in visual signal transduction 30:520 retinoic acid 29:111 activity in am, ebv, skin-1, odc bioassay systems 29:111 retinoic acid 30:493,498,521 in cell differentiation 30:521 in development 30:521 in growth regulation 30:521 retinoid retinol 29:111 activity in am retinol acetate 29:111 activity in am bioassay system retinol palmitate 29:111 activity in am bioassay system 29:111 retroviral enzymes 30:397 inhibitor of 408 causative agent of immunodeficiency syndrome 30:394 inhibitor of rett's disorder (rtt) 30:372,373,384 by mutations in mecp2 gene 30:373 genetic basis of 30:373 reutericyclin 28:127 as antibiotic reverse transcriptase 30:226,395,397 role in hiv replication cycle 30:226 role in viral replication rhazinilams 29:362 as microtubule poisons rheedia gardneriana 30:207 antinocicieptive effect of 30:207 rheumatism 26:50 herniaria hirsuta in 26:50 rheumatism 30:192,691 use of non-steroidal antiinflammatory drugs (nsaids) 30:192 use of prostaglandin inhibitors 30:192 rheumatoid arteriosclerosis 30:70 rheumatoid arthritis 21:152;22:310; 26:170,486;30:70,204,225 in treatment of cph82 26:170 in rats 30:204 reagent-induced 30:204 treatment of 30:225 use of kalopanax pictus rhoifolin (5, 4'-dihydroxy -7-rhamnosyl-glucosyl-flavone) 29:573 effects on hiv-1 protease 29:573 rhombenone 24:456 fptase inhibition by ricinoleic acid 30:193 analgesic action of 30:193 rickettsial pathogens 28:394 cowdria ruminantium as rifamycin-type macrolides 24:545,546 antiviral microbial-derived compound 24 rift valley fever infection 30:411 treatment of rimantidine 27:108 for treatment of influenza in infections 814 activity of deltorphin b in 30:807 of glycopeptide 30:814 rna virus 30:394 arenavirus type of 30:394 bunyavirus type of 30:394 coronavirus 30:394 influenza virus 30:394 measles virus 30:394 parainfluenza virus 30:394 picornavirus type of 30:394 rabies virus 30:394 reovirus type of 30:394 rotavirus type of rna-dependent rna polymerase activity 30:407 virion-associated rna-directed dna-polymerases 29:127 inhibition by sf-9 cell line 29:33 activity in mam-3assay system 29:100 antileukemic activity 8:18 sarcoptes mites 28:410 cause of human scabies 28:410 symptoms sarcoptes scabiei 28:409 cause of mange mites 28:409 cause of scabies sargasm horneri 30:400,405 anti-cmv activity of 30:405 polysaccharide (ps) from 30:400 sarkomycin 8:150 as antitumor agent 8:150 satratoxins 30:743 antiviral activity of 204 scavenging effects 23:443 of tannic acid 23:443 sch 207278 24:451 fptase inhibition by 24:451 schisandra 26:183 display platelet activating factor antagonist activity 26:183 effect on cardiovascular system 26:183 effect on heart rate 26:183 for treatment of hepatitis schisandrol a 26:245 as paf-induced inhibitor schistosomiasis (bilharzia) 7:405 schistosomicidal activity 1:545 schizonticidal drug 26:837 schizophrenia 101 activity in crg bioassay system 29:101 from scoparia dulcis scropolioside a 29:84 activity in tpa assay system 29:84 scrovalentinoside 29:84 activity in tpa assay system scutellaria baicalensis 30:55,56,69, 254,289 in allergric inflammatory disease 431 sdb 21:695 inhibitory effect of 21:695 sdc 21:695 inhibitory effect of 21:695 sdz-249-665 30:201 antinociceptive activity of 30:201 anti-hyperalgesic activity of 30:201 seasonal affective disorder 30:369 effect of circadian activity on 30:369 seaweeds 30:404 anti-cmv activity of activity in ebv assay system 29:100 1,2-secoemetine derivatives 6:485 amoebicidal activity of 6:485 secoiridoids 26:330 antimicrobial activity of 26:330 second messenger 25:516 secondary metabolites 28:3,432,617 acaricidal activity of 28:432 biological activity of 28:617 secondary metastatic tumor growth 30:64 effect of triterpenoids on 599 arnica as 22:559 artocarpus heterophyllus as basilicum polystachyon for 22:514 sedative activity 21:673 sedative in convulsions 22:518 crocus sativus l. for 22:518 sedative in epilepsy 22:535 withania somnifera l. as 22:535 g-selective agonist 30:807 dermorphin 30:807 8-selective antagonist tyr-tic-phe-nh2 selective antitumor activity 15:355 selective cytotoxicity 13:648 8-selective opioid agonist 30:817 deltorphin b 30:817 g-selective opioid peptide 30:802 8-selective opioid peptide of ici 19944 30:811 ofk opioids 30:811 8,g-selectivity 30:819 in vivo 30:819 of glyco analogues semilicoisoflavone b 28:229 biological activity of 28:229 semiochemicals semisynthetic lsd 26:820 as psychodelic drug 10-dihydro xy-7-methoxy-naphtho sendai virus infection sensory neurons 30:194 hyperpolarisation of 30:194 8er264 inhibitors serine 27:850 activitiy of serine 30:836 c~2-macroglobulin inhibition by serine protease inhibitor proteins 29:617 arrowhead pis api-a & api-b as cp-thionin as 29:617 effects on barley malt cysteine endoproteinases 29:617 effects on chymotrypsin 29:617 effects on subtilisin 29:618 effects on subtilisin eleusine double-headed try-~x-amylase inhibitor i-2 as 29:617 from brassica nigra 29:618 from brassica napus 29:618 from cassia fistula 29:617 from eleusine coracana 29:617 from hordeum vulgare 29:617 from phaseolus angularis from sagittaria sagittifolia 29:617 from sinapis arvensis 29:618 from vigna unguiculata 29:617 hordeum lipid transfer proteins as 840 blood clotting factors as 29:570 cathepsin g as 29:570 chymase as 29:570 chymotrypsin as 29:570 granzymes as 29:570 in angiogenesis 29:570 in blood clotting 29:570 in cytosolic proteolysis 29:570 in digestion 29:570 in inflammation 29:570 in proprotein processing 29:570 in tissue remodelling 29:570 kallikrein as 29:570 plasmin as 29:570 prolyl endopeptidases as 29:570 role in blood coagulation 30:70 role in platelet activation 30:70 role in pulmonary vascular injury serine proteases (subtilases) 25:388 cysteine proteinase inhibitor 25:370 poly phenol oxidase 25:370 serine proteinase inhibitor serine proteinase inhibitor i 25:370 as defense proteins serine-threonine-specific receptor protein kinases serotonergic innervations 30:377 autoradiographic imaging 30:377 in cerebral cortex 30:377 in neonatal rats by immunohistochemical techniques 30:377 physiological role of 377 serotonergic neurons 30:368,376 in raphe nuclei 30:376 role in 30:regulation of neurogenesis 627 as hyperforin inhibitor 30:627 as neurotransmitter 30:367 effect on synaptogenesis 30:378 in apoptosis 30:367 in cell proliferation 30:367 in presynaptic neuron 30:381 morphogenic properties of 30:367 neurochemical connection of 30:368 receptor activity of -ht) receptors 30:376,378, 382,383 in brain 30:383 in limbic brain regions 30:383 pharmacological aspects of serotonin (5-ht) re-uptake inhibitors 30:368 mechanism of serotonin (5-ht) synthesis 30:370,376, 377 in autistic children serotonin (5-ht) 1ar receptor antagonists 30:378 in posmatal treatment -ht)sa receptor 30:375 in brain development 30:375 in phenotypic behavioral irregularities 30:375 in purkinje cells 30:375 mrna expression of 30:375 serotonin action 30:378 on apoptosis 30:378 on maturation 30:378 on neuronal functioning 30:378 on proliferation serotonin agonists 25:531 anticomplementary activity of 25:46 role of cyclic amp-dependent protein kinase 25:46 delayed-type allergy suppressant activity of 25 serotonin synthesis inhibitor 30:378 parachlorophenylalanine as serum tc 27:420 biological activity of 27:420 serum transaminase 27:415,417,420 biological activity of 27:415 serum transaminase activity 25:469 of curcumia longa l serum triglyceride 27:404 biological activity sesquiterpene lactone 7:426 sesquiterpene quinones 5:429 antimicrobial activity of 5:429 cytotoxic activity of sesquiterpenoids drimane-type synthesis 29:127 avian myeloblastosis virus (amv) inhibition by 127 by jauch 29:127 sex pheromone sexual potency 28:4 ofbroussoflavonol g 28:4 sgot 30:292 activities of 30:292 sgpt 30:292 activities of 21:654 shikimic acid derivatives 29:478 antitumoral activity of 29:479 shinpterocarpin 28:229 biological activity of 28:229 sialic acids 27:103 biological function of 27:103 model for sialyltransferase activity 16:81 side effect 30:194,799 of analgesic alkaloid 30:799 of opioids sigmoidin a 28:229 biological activity sigmoidin b 28:229 biological activity of 28:229 signal reception 30:377 in autistic children 30:377 signal transduction mechanisms 25:518 signal transduction pathways signal transductors/activators of transcription (stats) 25:519 silandrin 26:255 as antihepatotoxic agent 26:255 silchristin 26:255 as antihepatotoxic agent 26:255 sildenafil 30:155 used for erectile dysfunction silymonin 26:255 as antihepatotoxic agent 26:255 simocyclinon 29:319 antibiotic activity of 29:319 cytostatic effects of 29:319 sindbis virus 17:135 sinomenine 25:472,476 hepatoprotective effect glycoside) 29:590 effects on pep 29:590 skin cancer 5:747 skin diseases 30:732 skin inflammation 30:323 anthrones used for 30:323 skk moth 1:704-706 juvenile hormone from slaframine 27:252 biological activity slaframine alkaloids 27:250,255 as muscarinic agonist 27:255 sleeping sickness smenoquinone 5:434,425 antimicrobial activity of smooth muscle relaxation 24:875-925 as biological action 24:875 of kampo medicines snake bite 30:691 treatment of 30:691 use of crossopetalum gaumeri 30:691 sobrerol 29:83 activity in mam-2 assay system 29:83 activity in gst assay system 29:83 activity in ras assay system 29:83 social phobia sodium salt of caffeic acid tetramer 24:742,743 anti-hiv activity of 24:742 soilborne fungi 21:181 bioactive metabolites from soilborne phytopathogens 21:182 biological control of 21:182 solandelactones 24:455 fptase inhibition by solanidane-induced teratogenicity 23:573 solid tumors neovasularization 25:593 somatostain 25:265 as a~ adenosine agonists somatostatin receptor 21:72;22:26; 25:530 type of g-protein-linked receptor 25:530 sonodione 30:566 from hernandia sonora 30:566 sores 30:616 tricyclic acylphloroglucinols sorivudine 24:474,486 as anti-viral agent 24 sorocein f 28:230 biological activity of 28:230 sos chromotest 22:623 soyabean saponins 25:222,223 hypocholesterdemic effects soyasaponins 27:398 anti-obesity action of 27:398 biological activity of 27:399 soybean trypsin inhibitor (sbti) spasmolytic activity 23:358 ;28:257,293; 30:264,561,748 of hernandia moerenhoutiana 30:561 ofhernandia voyronii 30:561 of imperatorin 23:350,358 of plant polyphenols 28:257,293 of thymus satureioides 30:264 on guinea pig ileum 30:748 on smooth muscles specionin 10:425 antifeedant activity of 10:425 spermicidal activity 26:53 of gypsophila paniculata sphingolipid receptor ca 2+ channels 25:535 sphingolipid receptors 25:534 sphinxolide 10:153;17:17 antitumor activity of 17:17 spider mite 1:702 hatching inhibitor spiroalkyl analogs 28:357 biological activity spirulina platensis 30:399,404,408 anti-influenza activity of 30:408 biological activity of 30:404 calcium spirulan from 30:399 polysaccharide (ps) from spleen/adipose tissue weight 30:56,57, 66 1203 effect of fucoidan on 30:56,57 effect of oleic acid on 30:65 spontaneous apoptosis 26:926 spontaneous metastasis squarroside a 26:31,50 immuno-modulatory effect of 26:31 from vaccaria segetalis squash family serine protease inhibitors 29:614 bd-ti-ii as 29:614 cmti-i as 29:614 cmti-iii as 29:614 cmti-iv as 29:615 cpgti-i as 29:615 cpti-ii as factor xiia, kallikrein, trypsin 29:615 effects on lysyl endopeptidase 29:614 effects on trypsin 29:614 effects on trypsin 29:615 effects on trypsin 29:616 effects on xa, xiia, kallikrein, plasmin, trypsin 29:614 elti-i as 29:615 from bryonia dioica 29:614 from citrullus vulgaris 29:614 from cucumis melo 29:614 from cucumis melo 29:614 from cucumis sativus 29:614 from cucurbita maxima 29:614 from cucurbita maxima 29:614 from cucurbita pepo 29:615 from cucurbita pepo 29:615 from ecballium elaterium 29:615 from echinocystis lobata 29:615 from lagenaria leucantha 29:615 from luffa acutangula 29:615 from luffa cylindrica cucurbitaceae) 29:616 from tricosanthes 29:616 hmti-i as 29:614 lati as 29:615 lati-ii as 29:615 lldti-i as 29:615 lldti-ii as 29:615 mcei-i as stachenone 29:101 activity in ebv bioassay system 29:101 standishinal 29:100 activity in ebv assay system 29:100 standishinal diacetate 29:100 activity in ebv assay system staphyllococcus aureus 30:693 minimal inhibitory concentration (mic) of staphyllococcus epidermidis 30:693 minimal inhibitory concentration (mic) of staphyllococcus saprophyticus 30:693 minimal inhibitory concentration (mic) of staphyllococcus warnieri 30:693 minimal inhibitory concentration (mic) of staphylococcus aureus 30:628,739 antibacterial activity against 30:739 antibacterial properties of 30:628 antifungal activity against starfish fertilisation inhibitor 28:712 callyspongin b as 28:712 stavudin 24:474,486,488 as anti-viral agent 24:474 steganes 29:360,399 cytotoxicity of 29:360,399 tubulin assembly inhibition by 29:360 antitubulin activity of 29:399,400 microtubules assembly inhibition by sterculia urens roxb. 30:405 antiviral activity of 30:405 steroid hormones 27:823 steroid saponins 7:426 teratogenic metabolites of 7:21-24 toxic cardiac active steroids sterol biosynthesis 25:303 inhibitor of 25:303 6a-sterol sulfate 28:702 cytotoxicity against sterol sulfates haplosamate a 28:703 as hiv-1 integrase inhibitor 28:703 sterols 25:43 antitumor activity of 25:43 antiinflammatory activity of 25:43 stevia stevia extract 27:307,310,314 for diabetics stevia leaves 27:315 immunological activity stevia plant 27:315 allergenic activity steviol 27:304,305,307,312 acute toxicity tests of 27:307 effect on fertility 27:312 mutagenicity tests with tpa bioassaysystem 29:101 activity in skin-1 bioassay system 29:101 acute toxicity tests of 27:307 allergenicity problems of 27:315 chronic toxicity studies of 27:306,307,308 effect on fertility 27:309,310 for phenylketonuria 27:315 mutagenicity tests for stigmasterol 30:208,505,570,719 analgesic effect of stilbene derivatives 27:405,409;28:579 biological activity of 27:405,409 in llc-bearing mice 28:579 tumor growth inhibition by 28:579 stimulatory 24:845 biological activity 24:845 stimulant agent 23:642 ofteucrium sp stimulus-response mechanisms 25:515 streptococci 29:535 pathogenicity of 29:535 serological groups streptokinase/streptodomase 25:271 streptolygidin 28:130 antibiotic activity of 28:130 as bacterial rna polymerase 28:inhibitor 130 as terminal dna transferase striga asiatica 30:162 infection with ofbioactive metabolites 21:251 of scopadulan-type diterpenoids structure activity relationship studies (sar) 30:195,494,800 of anandamide 30:196 of aspirin 30:195 ofopioid analogues 30:800 of vitamin d analogues 30:493 structure dereplication 29:658 bioactive natural product database in 29:670 of melanin inhibition strychnine 25:530 as immunosuppressive drug 25:530 strychnopentamine 26:1062 anticancer activity strychnos bisindole 26:1062 anticancer activity strychnos monoindole 26:1061 anticancer/protozoal subacute toxicity 27:307 from animals 27:307 of stevioside 588 effects on furin 29:588 sudhl-4-1ymphoma 21:138 sugar cane cystatins 29:593 effects on cysteine proteases 29:593 suicide inhibition 27:348 of 8'-hydroxylase 27:348 suicide inhibitors 9 sulfatobastadin 13 28:695 as endothelin a receptor inhibitor sulfide-containing pyrroloiminoquinones 28:685 as antileukaemic agents sulfur-containing cyclic peptides 28:678 biological activities of 28:678 cytotoxicity of sulphated polysaccharide (ps) 30:398, 400,405 anti-cmv activity of 30:405 anti-hiv activity of 30:398 antiviral activity against hiv 30:400 effect on viral replication 30:400 from marine sources 30:400 inhibitory effect of 30:398 mechanisms of action sulphated polysaccharide (ps) extracts 30:399 inhibitory activity of sulphonylurea drugs 25:534 sunflower cystatin phytocystatin 29:593 effects on cathepsin h ficin 29:593 effects on papain 29:593 sunflower multicystatin 29:593 effects on papain supercritical fluid extraction 21:576 superoxide dismutase superoxide dismutase activity 21:669 suppressing activity 30:308 on cells proliferation zizyphus saponin i as 27:41 zizyphus saponin ii sweroside 25:471,476 serum alt activity of 25 symbiotic marine microorganisms 23:185 bioactive metabolites 23:185 sympathetic ganglia 30:782 blockade of 30:782 sympathetic nerve activity 30:787 sympathomimetic amines 12:411 synapic potential mediation 28:318 by non-nmda receptors 28:318 synaptic actions 30:381 of serotonin synaptic dopamine transporters 25:539 synaptic glycine transporters 25:539 synaptic serotonin transporters 25:539 synaptogenesis 30:368,377 effect of serotonergic neurotransmission on 30:368 role in rodents 30:377 role of serotonin in 30:377 syndrome type-la 26:1130 adipose tissue distribution as 26:1130 cerebellar dysfunction as 26:1130 liver insufficiency as 26:1130 peripheral neuropathy as 26:1130 psychomotor retardation as synergistic activity 21:673 synergistic effect 21:608,611; 30:498,596 ofhervelines b 30:596 ofhervelines c 30:596 of retinoids 30:498 of vitamin d derivatives 30:498 synergistic interaction 21:698 of acyclovir(acv) 21:698 ofganciclovir (gcv) 21:698 with ganciclovir synergistic purgative actions 30:306 of rheinanthrone 30:306 of aloe-emodin-anthrone 30:306 synthetic pharmacological agents 30:224 antioxidant activity of 30:224 synthetic podolactones 28:484 allelopathic activity synthetic psychotropic agents 24:1093 syringaresinol 24:741 anti-platelet aggregation activity of systemic wound response protein 25:402 systemin activity 25:373 assay for ]-val-val-gly-nh2 30:815 receptor binding properties of receptor binding properties tyr-pro-ser-nh2 (deltorphin c) 30:815 receptor binding properties of -d-glc)-gly-nh2 30:815 receptor binding properties of oac)4]-gly-nh2 30:815 receptor binding properties of -d-glc)-tyr-pro-ser-nh2 30:815 receptor binding properties of -d-g lc )-v a 1-v al-gly-nh2 30:815 receptor binding properties of tyrosinase inhibitory activity 21:587 tyrosine kinase tyrosine-specific kinase 19:178 inhibitor of tyr-tic-nh2 30:809 activity in functional bioassay tyr-tic-phe-nh2 30:806 as 5 selective antagonist 30:806 u2 66 myeloma cell 25:273 ucn-01-ucn-02 12:386 antitumor activity of 12:395 protein kinase inhibitor ulapualides a 19:609 antifungal activity unsaturated carbapyranoses 29:475 as enzyme inhibitors 29:475 as herbicidal 29:475 biological properties of 29:475 unsaturated ketonucleosides 4:253 tumor inhibition by u-plasminogen activator (u-pa) 30:844 urdamycin a 11:134 antifungal activity of 11:134 antitumor activity ureterolithiasis-drug-therapy 27:382 urinary tract infections 26:50 herniaria hirsuta in 26:50 urine retention 30:194 as opioid's side effect 30:194 uroterpenol 29:83 activity in mam-2 assay system 29:83 activity in ras assay system ursane triterpene 29:585 effect on chy 29:585 ursolic acid 28:18,40;29:588 effects on hiv-1 protease 29:575 effects on lela 29:588 inhibition of hiv-1 protease dimerization by ursolic acid methyl ester (ursene triterpene) 29:575 effects on hiv-1 protease 29:575 usambarensine 26:1062 anticancer activity of 26:1060 uterine tumor 30:27 effect of pironetin derivatives in 30:27 uusambarine 26:1062 anticancer activity uvaol (triterpene) 29:588 effects on urs-12-ene-3,28-diol) (ursene triterpene) 29:575 effects on vaccaria segetalis 26:50 imunomodulatory effect of 50 vaccination 30:408 for respiratory infection as neuraminidase inhibitors 29:487 as c~-amylase inhibitor 29:486 c~-glucosidase inhibitors of 7:46;10:518 yeast a-glucosidase inhibitor of 10:518 valinuoctins 24:459 fptase inhibition by valiolamine 13:189,195 yeast c~-glucosidase inhibitor valiolamines 29:484 as glucosidase inhibitor 29:484 biological potential of 29:484 in clinical trials 29:484 in treatment of diabetes 29:484 valproic acid 22:507 vancomycin 25:791 vancoresmycin 28:150 activity against gram-positive bacteria vanilloid receptor agonist 30:202 olvanil as 30:202 capsaicin as 30:202 structure of vanilloid receptor antagonist 30:201, 202 capsazepine as 30 varicella-zoster virus 30:394 cause of varroajacobsoni 28:387 toxicity of varroa mites 28:383 vascular physiological action 30:55,69 of natural products vasodilating activity 21:379 vasodilator 23:358 scoparone as vasodilator effect 25:595 of acetylcholine vasodilatory activity 30:275 ofsatujera obovata vasoprotective agent 22:443 vasoprotective effects 28:302 of green tea vateamine-2' [3-n-oxide 30:567, 590,595 activity in human cancer cell line 30:589 activity in murine cancer cell line 30:589 as platelet aggregation inhibitor 30:594 cytotoxic effect of 30:589 veinotonic activities veraguensin 26:230 trypanosomicidal activity verapamil 25:534,673 as caz+-channel blocker 25:487 vermisporin 28:125 antimicrobial activity vernonia amygdalina 28:400 tick toxicity of 28:400 verrucarins 30:743 arenavirus junin 30:743 herpes simplex virus type ii (hsv-2) verrucosin 26:228 ofantiviral activity 26:228 vertebrate toxicity 21:98 vesicant activity vesicular glutamate transporters 25:538 vesicular monoamine transporter type 2 (vmat2) inhibitor vesicular stomatitis virus (vsv) 30:743,752 replication of 30:743 vesititol 28:224 anti-microbial activity of 28:224 vestitol 28:243 anti-helicobacter pylori activity of 28:243 veterinary medicine 28:383 for ectoparasites control 28:383 vetiver grass 28:399 for controlling ticks viagra (sildenafil) 25:541 vicolides 29:89 activity in ctn bioassay system 458 anticancer activity of 14:805 viral diseases 30:732 viral dna 30:226 integration of 12:332;21:98,193, 263,270,312,398,430,585,589,591, 594,643,659,670,673,675,690;23:59, 216,808;26:217;28:481 ;30 :629 against bone resorption 21:720, 721 against h+,k+-atpase 21:719 against staphyllococcus aureus 30:629 mechanism action of 30:225 of (-)-(8,8a-di-epi-swainsonine 12:325 of (-)-swainsonine 12:327 o f (+) cyasterone activity in 29:32 2-deoxy-20-hyroxyecdysone activity in 29:32 14-deoxymuristerone a activity in 29:32 2 [3,3 [3-dihydro xy-513-chole st-7en-6-one activity in 29:32 3 [3,14a-dihydroxy-5 [3-cholest-7en-6-one activity in 29:32 ecdysone activity in 29:32 313-hydro xy-5 [3-cho le st-6-one activity in 29:32 20-hydroxyecdysone activity in 29:32 inokosterone activity in 29:32 kc cell bioassay as 29:30 makisterone a activity in 29:32 muristerone a activity in 29:32 podecdysone a activity in 29:32 ponasterones activity in 29:32 213,313,513,14 a-tetrahydro x ycholest-7-en-6-one activity in 29:32 2[3,3 ~ ,25-tridihydroxy-513cholest-6-one activity in 29:32 viticosterone e activity in 29:32 insect control 21:611 insect juvenile hormone analogues 14:391-397 from thujone 14:391-397 insect pheromones 11: [415] [416] [417] via diisopropylethanediol esters 11:415-417 insect repellant 5:757 insecticidal activity 18:229,704;21:252, 254,262,266,267,269,270,271,278, 285,379,595,611;23:666;667,670, 677,678;24:25,799,845,866;26:231, 232,426,471,479,485;28:477; 30:153-154 activity of (-)-deoxypodo phyllotoxin in 30:589 activity of (-)-hernone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-hydroxyhemangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2' [3-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy1:467,468;2:278,279,286, 289;6:108;7:103,110,121-123; 17:235,244 ;29:87 ;30:193,206 activity in ebv assay system 29:87 activity in skin 1 assay system 29:87 activity in crg assay system 29:87 activity in cro assay system 29:87 activity in aa assay system 29:87 activity in brk assay system 29:87 activity in cps assay system 29:87 activity in htm assay system 29:87 activity in paf assay system 26:3,4,10,13,15, 18,27,30,42,54,57;30:407 anti-inflammatory activity of 26:27 antiviral activity of 30:407 saponins 1:305; 402; 7:155, 156, 190, [426] [427] [428] [429] [430] [431] [432] 434, 435; 18:649 ajugasterone c activity in 29:28 amarasterone a activity in 29:28 amarasterone b activity in 29:28 cyasterone activity in 29:28 ponasterone a 2p-glucoside activity in 29:28 pterosterone activity in 29:28 rubrosterone activity in 29:28 sengosterone activity in 29:28 stachysterone c activity in 29:28 capitasterone activity in 29:28 sarcophytol a 29:100 activity in ebv assay system 29:100 activity in colon-2 assay system 29:100 activity in liver-2 assay system 29:100 activity in lung-1 assay system 29:100 activity in mam-3 assay system 29:100 activity in pancr assay system 29:100 activity in skin-2 assay system 29:100 sarcophytol b 8:18;29:100 activity in skin-2 assay system 29:100 use of platycodon grandiflorum key: cord-022889-lv6fy6e6 authors: dávalos, alberto; henriques, rossana; latasa, maría jesús; laparra, moisés; coca, maría title: literature review of baseline information on non‐coding rna (ncrna) to support the risk assessment of ncrna‐based genetically modified plants for food and feed date: 2019-08-07 journal: nan doi: 10.2903/sp.efsa.2019.en-1688 sha: doc_id: 22889 cord_uid: lv6fy6e6 this report is the outcome of an efsa procurement (np/efsa/gmo/2016/01) reviewing relevant scientific information on ncrna and on rna interference(rnai) that could support the food and feed risk assessment of ncrna‐based genetically modified (gm) plants. information was retrieved through key words and key questions covering the stability and degradation of ncrnas after oral ingestion, the passage of ncrnas from food and feed to human and animal organs and tissues via the gastrointestinal tract and other barriers, as well as the potential effects on the gastrointestinal tract, the immune system or the entire organism.full description of the strategy used for the literature search and for studies selectionis provided and the number of retrieved publications is reported. this report is divided into four partsdiscussing the kinetics of exogenous ncrnas in humans and animals, with focus on ingested ncrnas (part 1); the possible effects of ncrnas on the gastrointestinal tract (part 2), systemically(part 3)and on the immune system (part 4). this report suggests that some plant ncrnas (e.g mirnas and sirnas) show higher stability as compared to other ncrnas due to peculiar chemical characteristics (2’‐o‐methylation at 3’ end).however, ingested or administered ncrna must overcome many extracellular and cellular barriers to reach the intended target tissue or functional location in sufficient amount to exert any biological effect. literature data indicate that chemically unmodified and unformulated ncrnas exhibit very low stability in the gastrointestinal tract and in biological fluids and, in general, do not elicit major biological effects.this report also provides an overview of the rna content in plant‐derived foods and diets and discusses the controversies on the presence of dietary exogenous rnas in the biological fluids of humans and animals and their effects. finally, gaps in the scientific literature are highlighted and recommendations provided 1. introduction this scientific report is the result of a contract awarded by the european food safety authority (efsa) to the madrid institute of advanced studies (imdea)-food, under the contract title: "literature review of baseline information on non-coding rna (ncrna) that could support the food/feed risk assessment of ncrna-based gm plants" (contract number np/efsa/gmo/2016/01). for the literature review of the available scientific information on foreign (exogenous) ncrnas that could support the risk assessment of ncrna-based gm plants, the following tasks were defined by efsa. based on available scientific literature (narrative review) to review the kinetics profile of foreign (exogenous) ncrna in humans and animals (experimental animals, livestock and pets). this to be based on information from research and development of ncrnas intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area) and focused primarily on the oral route of administration of ncrna, with detailed information on the absorption, distribution, metabolism and excretion of ncrna molecules. other routes of administration, however, not to be excluded. based on available scientific literature (narrative review), to review the effects (physiological, paraphysiological, and pathological) of foreign (exogenous) ncrnas on the gi tract and annex glands (e.g. liver, pancreas, salivary glands) in humans and animals (experimental animals, livestock and pets). this to be based on information from research and development of ncrna molecules intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area). based on available scientific literature (narrative review), to provide information on the possibility of systemic effects of foreign (exogenous) ncrnas in humans and animals (experimental animals, livestock and pets) with focus on gastrointestinal barrier to absorption, and other barriers as relevant (e.g. placenta). based on available scientific literature (narrative review), to assess the plausibility of effects on the immune system of humans and animals (experimental animals, livestock and pets) of foreign (exogenous) ncrnas. gm plants based on silencing approaches by rnai (through ncrna expression) are developed for food and feed purposes and assessed by efsa within the eu gmo regulatory framework. 1 the team considered that pursuing the four tasks defined by efsa to support the risk assessment of gm plants in this regulatory context required gathering preparatory baseline information on ncrna and refinement of the scope of the tasks. therefore, further elaboration on the terms of reference provided by efsa has been conducted and it is described below. it should be emphasized that the available information on ncrnas which could be possibly relevant to food and feed safety, overlaps with and is barely distinguishable from the broader information on rnas. information on rna in general has therefore been included in the search as warranted; when possible, the relevance of distinguishing peculiar ncrnas features is described. the term "foreign ncrna" or "exogenous ncrna" used in this report refers to ncrna molecules to which humans/animals can be exposed through the diet or via therapeutic treatment. similarly, "foreign rna" or "exogenous rna" refers to rna molecules to which humans/animals can be exposed through the diet or via therapeutic treatment. part 1: kinetics of exogenous ncrna in humans and animals (efsa task 1) efsa task 1: based on available scientific literature (narrative review) to review the kinetics profile of foreign (exogenous) ncrna in humans and animals (experimental animals, livestock and pets) . this to be based on information from research and development of ncrnas intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area) and to be focused primarily on the oral route of administration of ncrna, with detailed information on the absorption, distribution, metabolism and excretion of ncrna molecules. other routes of administration, however, not to be excluded. to address efsa task 1, preparatory baseline information is provided i) on general features of plant ncrnas including their function, movement in the plant, biogenesis and degradation (section 3.1.1), ii) on their stability (i.e. for how long ncrna molecules retain their original structure, and how they resist degradation over time and in various conditions, both inside and outside the plant), and turnover, also in comparison with turnover of endogenous ncrna in animals (section 3.1.2). if necessary, comparisons between the ncrnas class and other rna classes are made. general information on rnas used/intended for use as therapeutics (including ncrnas) is provided, expanding on the chemical modifications necessary to avoid degradation and to achieve a target effect after administration (section 3.1.3). lessons from rna-based therapeutics as regards the pharmacokinetics of exogenous rna are discussed, with a focus on non chemically modified (naked)ncrnas. in addition, aspects of the pharmacodynamics of rnas as learnt by therapeutics are addressed (section 3.1.4). a description of cellular uptake of rna (in general and with focus on ncrnas), and of the many barriers represented by human/animal cells after oral ingestion is addressed insection 3.1.5. further relevant information on dietary exposure to plant rnas (i.e. exposure to plant rnas following dietary consumption) by different diet types, and presentation of the gaps in the literature on dietary plant rna exposure is provided in section 3.1.6. efsa task 2: based on available scientific literature (narrative review), to review the effects (physiological, paraphysiological, pathological) of foreign (exogenous) ncrna on the gastrointestinal tract and annex glands (e.g. liver, pancreas, salivary gland) to address efsa task 2 preparatory baseline information is provided on i) the gi tract barriers to ingested exogenous rnas, including ncrnas (section 3.2.1); ii) experience in rna-based therapeutics for oral (gi) administration, with focus on delivery of naked (non chemically modified) ncrnas (section 3.2.2). biological effects of dietary exogenous ncrnas (plant and nonplant origin) on the gi tract and its annex glands is presented in section 3.2.3. www.efsa.europa.eu/publications 8 efsa supporting publication 2019: en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to address efsa task 3, preparatory baseline information is provided on the molecular pathways relevant for the uptake of exogenous ncrnas by human and animal cells and the intracellular trafficking that follows, with description of the effects by exogenous ncrnas once they reach the specific subcellular location (section 3.3.1). to exert systemic biological effects, in most cases the ingested exogenous rna must enter the systemic circulation to reach the target tissues. information is therefore provided on the landscape of exogenous rnas in biological fluids and tissues from humans and animals, with focus on ncrna when possible (section 3.3.2). a description of possible systemic biological effects of dietary exogenous ncrnas is provided in section 3.3.3, with details on tissue barriers (i.e. placenta, brain) encountered. a detailed review is made of the contradictory information on the systemic effects exerted by plant-derived exogenous ncrnas. finally, studies on the safety and in general on risk assessment on ncrnas from gmos are reviewed (section 3.3.4). efsa task 4: based on available scientific literature (narrative review), to assess the plausibility of effects on the immune system of humans and animals (experimental animals, livestock and pets) of foreign (exogenous) ncrna molecules. to address efsa task 4, preparatory baseline information is provided on ncrna mediated regulation of the immune system in humans and animals. the many pathways of sensing exogenous rnas, both at the endosomal compartment and in the cellular cytosol, and the mechanism of triggering the immune system are reviewed (section 3.4.1). an overview is done of the plausibility of biological effects of exogenous plant ncrnas on the regulation and function of the immune system upon uptake by mammalian cells (section 3.4.2). in addition, since the gut microbiota influences the immune system, a review on the possible effects of exogenous ncrnas as gut microbiota modulators was considered relevant and it is provided (section 3.4.3). in the final section of this report, concluding remarks inform on the gaps existing in the scientific literature, and highlight areas needing further investigations to better understand and support the food and feed risk assessment of ncrna-based gm plants. an extensive search was done to identify as many studies as possible relevant to the literature review questions. to include all possibly relevant information multidisciplinary databases and information resources were explored. unpublished research reports ("grey literature") including dissertations, thesis or other scientific reports were also included, which were retrieved using general search engines (i.e. google). the following information sources, databases or search engines ( table 2) were used for the literature review: pubmed (biomedical), world wide science (multidisciplinary), web of science (multidisciplinary), springerlink (multidisciplinary), scopus (multidisciplinary), scielo (multidisciplinary) and biorxiv (preprint biology). finally, the publications reporting the outcome of two efsa procurements aiming respectively at investigating and summarising the state of knowledge on the mode-of-action of dsrna and mirna pathways, the potential for non-target gene regulation by dsrna-derived sirnas or mirnas, the determination of sirna pools in plant tissues and the importance of individual sirnas for silencing 6 ; and reviewing relevant scientific information on rna interference that could serve as baseline information for the environmental risk assessment of rnai-based gm plants ) 7 were also used. www.efsa.europa.eu/publications 11 efsa supporting publication 2019: en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the methodology used for this literature review process and report preparation followed three basic principles: i) methodological rigour and coherence in study retrieval and selection; ii) transparency; and iii) reproducibility. to ensure that these principles were implemented in this literature review process, the search methodology design, study selection and data and results presentation follow, whenever possible, most of the methods and techniques described in the efsa guidelines on application of systematic review methodology for food and feed safety assessments to support decision-making (efsa, 2010) . 7 the following section describes the general key steps followed in the literature search methodology; detailed descriptions of the literature search methodology for each topic are also provided in this section. to find a comprehensive set of scientific literature related to exogenous ncrnas and their biological effects on a target organism, either local or systemically, key questions were identified and key words were used as search queries for each task section according to a pre-defined review process. to provide a fit-for-purpose literature search, the team defined key questions based on the efsa tender specifications and their interpretation as above described. to support this phase, the team identified key elements of the questions considering the pico approach of efsa (2010). 8 the key elements identified include the population, in this case humans or animals (experimental animals, livestock and pets) as defined by the terms of reference of efsa. the intervention was identified as exogenous rnas (focusing on ncrnas). the comparator was identified as the control or reference group (not exposed to exogenous rnas) in most studies, whenever possible. the comparator could also be identified as the modified (either chemically or biologically) version of the exogenous rnas when referring to the stability or pharmacokinetic of the exogenous rna. the outcome is the biological effect (if any) as a response to the intervention. other specific key questions are determined for every part of the specific tasks defined by efsa. evidence is generally scarce for most of the topics covered by this scientific review, and in some cases the information was available from not directly related studies (i.e. pharmaceutical industry studies). the literature review was helpful in identifying both knowledge gaps and unexpectedly relevant studies or evidence not previously known to exist. these knowledge gaps are used to make informed proposals for future research designs (section 4.2). the key methodological steps in the literature search, selection and review process are shown in figure 1 . three phases were defined in this process. the preparatory phase started with team discussions of topics to be included in the literature review. during this phase key elements addressing the efsa tender specifications and their interpretation as above described were identified and key questions were defined. the key questions aimed to to assist the literature search in order to produce a comprehensive overview of the topic that best fits tender specifications. the bibliography search and selection phase included several steps. the initial search in bibliographic databases and search engines was done using task-specific key words. the key words used for the search were derived from the efsa tender specifications and their interpretation (tasks 1-4) and were complemented with other key words identified in the preparatory phase. identified key words are detailed for each task in section 2. the final phase of review and final selection involved two additional steps, i.e. a detailed review of all relevant previously selected documents and a systematic evaluation of the final selected studies. a detailed review of the selected documents was done for studies with full text availability (either freely available or available for purchase) and, based on this, a final selection was made of full-text studies most relevant to answer the key questions for tasks (1-4) and apt for review in this report. a systematic evaluation of these final documents was done to develop a comprehensive overview of the topic. conflicting or contradictory results were reported in a neutral way and supported by references and the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. citations of all available evidence, both from the published and grey literature. retracted papers were carefully identified and noted. finally, each section of the narrative review was assessed by one reviewer. if more specific data was needed because of missing or novel information, additional bibliographic research was performed. additional key words that could directly address the specific data needed were identified and used to run a new specific search. the search among full-text studies was focused on obtaining only specific missing or required data (e.g. quantitative data on rnas levels in plants, earlier studies on exogenous rna exposure to mammalian cells). this new specific search generally represented less than 1% of the literature included in this report and it was performed only once for certain topics. finally, to reduce the risk of introducing bias into the literature review process and to assure reproducibility in the review methodology the following general approach was applied: when the search of the identified "key words" in different databases resulted in a myriad of duplicate documents, manual removal of these duplicates was performed in each section by each team expert; when relevant studies that might answer the identified key questions were identified after the first search run, these were used to further refine the literature search, for example refining the keywords; each selected full-text study to be included in the result section was manually examined by the team. to identify literature relevant to the topic, the methodology described in section 2.2.1.2 ( figure 1 ) was applied, with adaptations. a multiple-step approach was followed based on the expertise of the team members who carried out research in the area. following a first search based on identified keywords, a careful selection of retrieved review studies was done, followed by a search of specific publications referred to in these reviews. additional specific key words were identified, and an additional search was run. this search retrieved a relatively small number of studies; therefore, team members reviewed all these to determine which were relevant to the topic. duplicates, irrelevant or erroneously assigned publications were removed. team members also cross-checked each others' findings, sharing discussing the results of their searches. the following areas were investigated. the purpose of this search was to provide general information on rnas function, movement in the plant and aspects of biogenesis and degradation, with a focus on ncrna (either small or long). by comparing animal and plant ncrna pathways, the aim was to identify similarities between animal and plant ncrna processing machineries that would indicate the possibility of ingested plant-derived ncrnas being processed in animals. in addition, this section was organized to avoid details that were outside the scope of this literature review in the topic of plant ncrna biology. this serves as an introductory chapter allowing easier integration of the knowledge presented in later chapters. the following combination of key words or phrases was used in some of the databases indicated above: plant small rnas; plant and sirna; plant and mirnas; plant and lncrnas; plant and circrnas; plant rna and movement. www.efsa.europa.eu/publications 14 efsa supporting publication 2019: en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the initial search using the above-mentioned key words or phrases yielded a large number (≈50 000) of documents ( table 3) , most of which were not relevant to the topic. particularly, the databse web of science retrieved documents not directly related to the topic. to assist and refine the search, studies were further filtered by their relevance to the key questions identified: -what type of ncrnas are described in plants? -what are the main differences between mammalian and plant ncrnas? -what is the function of plant ncrnas? -what is known about the movement of plant ncrnas? title and abstracts were evaluated, and studies further refined using the key questions. detailed study of the most relevant full-text documents answering the key questions, retrieved 173 documents which were finally selected as relevant to give an overview of plant ncrnas. search was then refined by using other key words including "half-life", "turnover", "fate" or "degradation". the identified studies were then manually evaluated by title and abstract and their number further limited due to their low relevance to the below identified key questions for this specific subchapter: -are plant ncrnas more stable than animal (mammalian) ncrnas? -if so, what makes a plant ncrna more stable than its animal (mammalian) counterpart? -why is it relevant to consider ncrna stability outside the organism of origin (plant)? -are certain types of plant ncrnas more or less stable than others? -what is known about the half-life of ncrnas? documents on the stability of rnas (not only ncrnas) were further screened for relevance to the question. most of the studies in the literature describe the stability of a specific class of intracellular rnas (i.e. mrnas). the stability of rnas other than ncrnas (e.g. mrna, trna), unless are used as model of general ncrna stability, is outside the scope of this literature review, since it is very unlikely they would be used as baseline information for food/feed risk assessment of ncrna-based gm plants. these keywords were specifically selected since they describe the kinetic profile of exogenous ncrnas in human and animals. the word "pharmacokinetics" was included in the search because it is a general term that retrieves data on absorption, distribution, metabolism or excretion of any molecule, particularly those intended for use as therapeutics (pharmaceutical/medicine areas). the keywords "increased intestinal permeability" or "increased plasma clearance" are intended to retrieve any study indicating changes in these parameters. although a large number of publications unrelated to ncrnas were expected, these keywords were chosen to initially explore the general aspects of physiological or pathological conditions associated with modification in these parameters. the search was further refined after this initial exploration. the search of the above-mentioned key words on different databases yielded ≈27.000 entries ( table 6) . a quick exploration of titles and abstracts suggested that (as expected) a number were unrelated to or were not useful to appropriately address the topic. it is worth noting that the search in the previous section (section 2.2.2.1.) indicated that the chemical modifications generally introduced for rna-based therapeutics are normally absent in nature. scientific data that could serve as a baseline to support risk assessment of ncrna-based gm plants should contain primarily information on ncrnas resembling those present in the nature or in gm plants. therefore, studies on naked exogenous rnas were analysed to prepare this narrative review. to promote comparisons between natural and synthetic rnas, a very few examples of slightly chemically modified rnas were included. heavily chemically modified rnas were not considered due to their unlikely existence in nature. only in vivo studies were considered, both in animals (mammals) or humans. studies were further evaluated for their relevance to answer the key questions identified for this section: -what are the pharmacokinetic properties of rna-based therapeutics? -what routes of administration have been used for rna-based therapeutics? -what is it known about the pharmacodynamic properties of rna-based therapeutics? -do disease conditions modify the pharmacokinetic properties of rnas when administered to mammals? the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after detailed evaluation of the full-text reports, 119 documents meeting the described criteria were finally selected for review. understanding whether and how exogenous rnas are taken up by cells and what barriers are encountered is relevant to study the possible absorption and systemic exposure to ncrnas introduced by food and feed. this topic is reviewed in this dedicated section and excluded from the section on the pharmacokinetics of exogenous rnas (section 2.2.2.1. above). to search and select the relevant studies concerning the uptake of exogenous rnas in mammals, the following combination of key words was used in some of the databases listed above: rna and cellular uptake; ncrna and cellular uptake; mirna and cellular uptake; lncrna and cellular uptake. these key words were used to screen for studies where "cellular uptake" was mentioned in the title or abstract for any type of rnas. it was also decided to include specific types of ncrnas including "mirna" or "lncrna" to expand the search to literatures where the more general term "rna" was not necessarily used. the initial search of scientific documents using the above keywords yielded ≈7000 documents ( table 7) . these were explored by their relevance (title and abstract), and only studies addressing naked ncrnas were considered for full text analysis, since highly chemically modified rnas are not common in nature. to address the reference terms for task-1 of this procurement, and after expert discussion for this section (preparatory phase) key questions were identified and the studies then further filtered for their relevance to the these: -are exogenous rnas taken up by mammalian cells? -how does exogenous rnas uptake in mammalian cells occur? -what are the biological barriers to consumed rnas? -what are the biological barriers for the exogenous rnas after absorption and entering into the mammalian circulatory system? only studies where exogenous rnas were evaluated were included in the literature review. after detailed evaluation of full-text reports and expert judgement on their relevance to efsa task 1, 39 documents were finally selected for review. to select relevant studies for plant rna exposure, the following combination of keywords were used: plant and rna content; plant and mirna content; plant and sirna content; plant and lncrna content; plant and dsrna content; rna intake and vegetarians; vegetables/fruit intake and vegetarians. these specific keywords were used because the wording "rna (ncrna, mirna, sirna, dsrna, lncrna) content" would yield most of the documents related to quantities of rnas in a specific substrate. these were combined with "plant" to retrieve only those studies where plants were used to evaluate rna content. it was also decided to use "rna intake", "vegetable intake" or "fruit intake" to specifically refer to consumption, and to combine this with "vegetarian" to search only for studies where comparisons are made between vegetarian and other types of diets. alternative wordings including "rna amount" or "vegan" were used to search for alternative information. using the above keywords, ≈3300 studies were retrieved (table 8) during the initial search. to more appropriately cover this section and to refine the search, the following key questions were identified during the preparatory phase: -what is the general amount of rnas naturally occurring in edible plants? -what percentage of total plant rnas are ncrnas, mirnas, sirnas, lncrnas or dsrnas? -does exposure to rnas change in different diet types? -do changes in rnase activity, due to pathologies or human polymorphisms, influence dietary exposure? titles and abstracts were evaluated, and studies further filtered using the key questions above. the search was also refined to focus on those studies where quantitative rnas data were mentioned. only in vivo exposure studies were used (either epidemiological or experimental). common types of diets were only considered (i.e. vegetarian, vegan or omnivorous). for rna exposure by different diet types, studies where comparative analysis of at least two different diets (one of which was the general population diet type) were mainly considered. in all cases, full-text studies were evaluated to screen for relevance to answer the identified key questions. a detailed study of the full-text documents resulted in the identification of 39 studies most relevant to address this specific topic of task-1, and which were reviewed for this section of the report. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to identify literature relevant to the topic, the methodology described in section 2.2.1.2 (figure 1 ) was applied. during the preparatory phase key questions were identified to assist in the literature search. the document search was first done using specific key words and then refined according to the key questions. detailed analysis of the full-text documents was done by team members with expertise in the area. search and analysis results were discussed collectively among team members. the following areas were investigated: in this subsection, there is a general description of the biological barriers within the gi tract that exogenous rnas would need to overcome to exert a biological effect, either locally or systemically. to select relevant studies, the following combination of key words or phrases were used: "gastrointestinal tract anatomy"; "gastrointestinal tract physiology"; intestinal transport pathway; physicochemical properties of rnas. an exploration of titles and abstract of the yielded entries (table 9 ) suggested that a large number of documents were unrelated to the topic. only one key question was identified in the preparatory phase: -what are the biological barriers within the gastrointestinal tract to ingested exogenous rnas? the search for relevant studies addressing this key question was based mainly on team member judgement. detailed study of the most relevant full-text documents answering the key question retrieved 21 documents which were finally selected as relevant to this section of the report. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this search was intended to collect data from rna-based therapeutics employing oral administration (and other gi routes) which could support the search on the possible biological effects of plant dietary exogenous rna and ncrnas on the gi tract and its annex glands. the initial search was done using the following key words on the different databases: ncrna oral delivery; rna oral delivery; sirna oral delivery; aptamer and oral; sirna gastrointestinal delivery; mirna gastrointestinal delivery; oral exosome rna; extracellular vesicle and gastrointestinal. these key words were selected to search for any documents related to rnas and oral administration. any information on oral administration was considered relevant. to expand the search, different terms referring to different types of ncrnas (mirna, aptamer, sirna or others), and a synonym for "administration", i.e. "delivery", were included. the initial search using the above key words or phrases yielded a small number (≈800) of documents ( table 10 ). the different databases generated similar number amounts of publications, most of which were duplicates. using the key word "lncrnas" in combination with the other key words did not yield any information relevant to the topic. indeed, just the search using "lncrna exosome" identified fewer than 100 documents, but none was relevant to oral delivery or gi administration. the wording "dsrna and oral" produced ≈300 documents, but none of them was relevant to mammalian organisms. the documents were selected by relevance, evaluating title and abstract. in this section, only studies in animals (mammals) or humans were considered. when replacing the word "delivery" with "administration", ≈8000 additional documents were identified, but almost 100% were unrelated to the review topic. since most of the documents in the literature describe minimally modified rnas for oral use, all chemically modified rnas were considered in this section. these can provide baseline information to support food/feed risk assessment of ncrna-based gm plants. to assist and refine the search, studies were further filtered for their relevance to the key question identified: is the oral route of administration relevant for rna-based therapeutic development? -what types of formulation (delivery vehicles) are tested for rna-based therapeutics for the oral route of administration or other gastrointestinal tract routes of administration? the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. -do exogenous ncrnas (naked rnas) resist the conditions of the gastrointestinal tract environment? -could ncrnas-based therapeutics -delivered by oral administration or other gi tract routes of administration-exert a biological effect in the gi tract or its annex glands? -are there any natural vehicles for oral administration of exogenous ncrnas? from the initial ≈8000 entries identified using the above key words and after filtering for relevance to the key question with evaluation of titles and abstracts and detailed study of the full-text reports, 110 documents were selected for review in this section. twenty (20) of these documents reports selected examples of local effects of nucleic acids-based drugs (mainly exogenous rnas) within the gi tract, using oral administration. since efsa required a literature review also including livestock species, an additional specific search was run for birds and fish. this search was intended to compile information on administration of exogenous ncrnas in either fish or birds, regardless of administration method, exerting any biological effects in vivo. the initial search was done using the following key words on the different databases: exogenous rna and fish; exogenous rna and bird; rna oral delivery and fish; rna oral delivery and bird; ncrna administration and fish; ncrna administration and bird. to expand the search, different terms referring to different types of ncrnas were used (sirna, mirna, circrna, or dsrna). the initial search using the key words or phrases produced a small number (≈500) of documents (table 11 ) in most of the databases. to assist and refine the search, only one key question was identified during the preparatory phase for this subsection: -are exogenous ncrnas also used to exert rnai effects in fish or birds? from the initial ≈500 entries identified using the above key words, and after filtering for relevance to the key question, and evaluating titles and abstracts and detailed study of the full-text reports, 18 documents were selected for the review in this subsection. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this chapter provides baseline information supporting food/feed risk assessment of ncrnas-based gm plants. in the effort to cover the widest amount of literature containing relevant scientific data on exogenous ncrnas and their possible biological effects, the following key words or phrases were used in the search: dietary plant non coding rnas; food plant non coding rnas; exogenous plant non coding rnas; diet plant micrornas; food plant mirnas; breast milk exosomes; breast milk mirna; breast milk ncrnas; breast milk lncrnas; breast milk rna; dietary exogenous rna. these key words were selected to cover most, if not all, available studies on the topic ( table 12) . the search of other common examples of exogenous ncrnas (non-plant origin) consumed by oral intake was focused on breast milk ncrnas, due to their relevance to human nutrition (table 13) . almost all documents yielded by the three databases (pubmed, scopus or web of science) were duplicates. in this section, studies reporting local effects (in the gi tract or its annex glands when consuming exogenous ncrnas) were preferentially (but not exclusively) evaluated. other databases including the preprint server for biology (biorxiv), scielo or search engines (i.e. google) were also used. the initial search in some databases using the above key words yielded a number of entries (table 12 and 13) not exceeding ≈2600 documents. because this specific topic is especially salient to future understanding of the possible food/feed risk assessment of ncrnas-based gm plants, all the identified studies were further evaluated by title and abstract and their relevance to answering the identified key questions: does the literature describe the effect of exogenous plant-origin ncrnas when consumed orally? -does the literature describe negative results or contradictory results regarding the possible biological effect of ncrnas when consumed orally? -are ncrna stability properties under gi tract conditions normally evaluated? -are exogenous ncrnas evaluated quantitatively? -are exogenous ncrnas consumed in an amount sufficient to exert a local biological effect?are there other common examples of exogenous ncrnas (other than plant-origin) that could exert a biological effect on the gi tract and its annex glands when ingested orally? local effects (in the gi tract or its annex glands) are mainly described in this section. the possible mechanisms of plant exogenous ncrnas cellular uptake, their intracellular trafficking and systemic biological effects (including plasma levels) are reviewed in another section (section 3.3.). of the initial ≈2500 documents retrieved using the above key words, and after filtering for relevance to the key questions by evaluating titles and abstracts, and detailed study of the full-text reports, 64 documents were selected for the review in this subsection. from these, 29 documents were selected for plant-origin ncrnas and 35 for non-plant origin (breast milk) ncrnas. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in line with the methodology described in section 2.2.1.2. (figure 1 ), a multiple-step approach based on the expertise of the team members responsible for this section was applied to identify literature relevant to the possible systemic effects of exogenous ncrnas. the document search was first done using specific key words in the databases and refined based on the appropriate key questions. detailed analysis of the full-text documents was done by team members and the results discussed collectively. the below areas were investigated. the search objective was to obtain information on basic molecular cellular uptake pathways of exogenous ncrna, including proteins involved in this process (if any). a review was also done on several other aspects of intracellular trafficking, once an exogenous ncrna is internalized, and how it exerts a biological effect. tissue barriers to exogenous ncrna function were also investigated. the content of this section deepens on molecular pathways previously described in section 3.1.5. to assist the search the following key words were used in the different databases: exogenous ncrna and mechanism and absorption; ncrna and mechanism and absorption; sidt1 (sid 1 transmembrane family member 1) and ncrna; sidt2 (sid 1 transmembrane family member 2) and ncrna; ncrna and uptake; intracellular trafficking and ncrna. these key words and terms were selected to identify any document related to the mechanisms of uptake and intracellular trafficking of ncrnas exerting a biological effect. the search was expanded by adding and combining different terms for exogenous ncrnas (see table 14 ), including the general term "rna", www.efsa.europa.eu/publications 25 efsa supporting publication 2019: en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. or specific ncrnas such as "mirna". the initial search using the above key words or combinations thereof produced a moderate number of documents (table 12 ). the different databases generated similar amounts of documents, most of which were duplicates. the largest proportion of the references was obtained using the key wording "ncrna and uptake" (≈2500). however, most of these had no relation to this specific section. the search was refined by filtering for relevance to answering the identified key questions: is there any clear transport of exogenous ncrnas into mammalian cells? -are orthologous of environmental rnas transporters in lower organisms (i.e. sid family of proteins) involved in the mammalian exogenous ncrna mechanism of uptake? -what molecular mechanisms are involved in exogenous ncrnas trafficking inside the cell? -what is the cellular fate of exogenous ncrnas once inside the cell? -what are the specific tissue barriers to exogenous ncrna function? very few documents evaluate the molecular mechanism of exogenous ncrnas uptake, intracellular trafficking and function. from the initial ≈3500 documents retrieved using the above key words, and after evaluation of their titles and abstracts and filtering for relevance to the key question, less than 50 documents initially seemed relevant to the topic and 33 were finally selected for review after detailed study of the full-text reports. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this subsection is intended to retrieve available information on the presence of exogenous rnas in human and animal biological fluids (i.e. blood, plasma, serum or any other biological fluids) and tissues. the search focused on the presence of dietary plant-derived exogenous ncrnas, due to their relevance to the overall literature review. to cover the largest possible portion of the relevant literature, the following key words were used in the search: exogenous rna and human plasma; exogenous rna and serum; plant ncrna and human tissue; plant lncrna and human plasma; plant mirna and blood; plant mirna and tissue; plant rna and tissue. to expand the search, the above key words were used in different combinations for the same search (i.e. blood, serum or plasma for biological fluids) ( table 15 ). the general wording "rna" was also incorporated into some searches. specific types of ncrnas were also added, including "mirna" or "lncrna", to avoid missing documents possibly related to these specific molecule types. the search using these key words or phrases in different databases yielded more than ≈20.000 publications (table 15) . a quick exploration of the titles and abstracts suggested that only few studies were related to the review topic. only one key question was identified as relevant which was related to the presence or absence of evidence of exogenous ncrnas in biological fluids and tissues. since these findings are important for the risk assessment of ncrna-based gm plants, the key question was presented in its positive and negative forms as follow: are there studies describing the presence of exogenous rnas in the biological fluids and tissues of humans and/or animals? -are there studies contradicting the presence of exogenous rnas in the biological fluids and tissues of humans and/or animals? after a detailed assessment of the full-text, 20 documents were finally selected for review. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this search primarily aimed to gather information relevant to understand the possibility of systemic effects of dietary exogenous ncrnas after intake (oral administration) by humans and/or animals (mammals). information on the possible passage through specific biological barriers (i.e. placenta or brain) was also searched. the search was guided by the following key words to identify studies providing any data on the possibility of systemic effects of dietary exogenous ncrnas: plant ncrna and diet; plant exogenous rna and diet; plant rna and diet, plant environmental rnai and diet; plant rna and placenta; plant rna and brain; dietary rna and cross kingdom. these key words were used to guarantee that every single document potentially providinginformation on the possibility of systemic effects of exogenous ncrnas was found. to widen the search, other wording such as "environmental rnai", "cross kingdom" or "mobile rnas" were used. although the wording "systemic effects" was not used in the key words during the search, the above key words were used to retrieve any documents in which a biological effect (local or systemic) might be reported. different wording for the term ncrnas was also used (including lncrna, circular rna, mirna, or dsrna). the initial search in some databases using the above key words produced a small number of entries (table 16) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. approximately ≈1900 documents were initially identified using the above key words (table 16) . after filtering for their relevance to the key question by detailed assessment of the full-text reports, 48 documents were finally selected for review. within this specific subsection, the selected documents are mainly grouped as studies that "support the evidence" or studies that "contradict the evidence" of systemic biological effects of dietary exogenous ncrnas in humans and animals (mammals). although not specifically included in the scope of this literature review, search for toxicological effects of dietary exposure to exogenous ncrnas in humans or animals is relevant to risk assessment. this search was aimed at identifying information on possible toxicological effects of plant-derived exogenous ncrnas following food/feed consumption. the initial search was conducted using the following key words in the different databases: the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. few documents were initially retrieved and after a quick evaluation of titles and abstracts, it became evident that very little information is available in this area. therefore, different terms were used to refer to exogenous ncrnas, including circular rna, lncrna and mirnas. the search using the above key words yielded a small number of documents (table 17) . only in vivo studies were considered, both in animals (mammals) and humans (if available), with focus on dietary plant-origin exogenous ncrnas. the key questions identified to refine the literature search for this specific topic were: -is there evidence of any toxicological effects of plant-derived dietary exogenous ncrnas? -are the toxicological effects related to rnai-mediated interaction with the human or animal genome? -are there possible interactions related to unintended rnai-mediated gene regulation? approximately 700 documents were identified using the above key words. after filtering for relevance to the key questions, and assessing the full-text reports, only 12 documents were finally selected for review. a systematic and comprehensive review and collection of the literature relevant to the topic was performed with the aid of the team members' expertise. an extensive search using key words was initially done on multidisciplinary databases to avoid publication bias, followed by a grey literature search using general search engines. the resulting documents were screened by title and abstract to determine relevance to the topic and refined using the identified key questions. after full-text analysis of the documents, irrelevant documents were eliminated. all stages of the screening process were independently assessed and, in case of uncertainty, discussed with other experts to avoid personal biases. the methodology followed the key steps described in section 2.2.1.2. (figure 1 ) and a total of 91 papers were identified, investigating the areas below described. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the final goal of this search was to identify relevant information on ncrnas-mediated processes associated with innate and adaptive immune responses. this section serves as an introduction to the studies presented later, and it is based on the results of the seach described in the next paragraph. sixty-four (64) documents were reviewed for this section. the search was aimed primarily at gathering state-of-the-art knowledge on the possible biological effects of exogenous (plant) ncrnas on the regulation and function of the immune system. to cover this specific section, a combination of the following key words was used in the different databases: exogenous rna and immune and human/mice/rat; exogenous plant rna/mirna/sirna/lncrna/dsrna/transgenic rna and immune function/inmmunity and human/mice/rat; genetically modified plants and immune function/immunity and human/mice/rat; exogenous plant rna/mirna/sirna/lncrna/dsrna and primary cells and lymphocyte/monocyte/macrophage; exogenous rna/genetically modified plants and immune function/immunity and zebrafish. the key words were designed to identify all publications that use in vivo, including non-mammalian animal models, and in vitro ncrnas testing to evaluate the influence on tolerance processes as well as immune cell function. the search also included publications on in silico methods. the different key words were entered one by one into the search field 'topic' of the databases ( table 2 ). the search was designed to identify documents including methods that could use non-mammalian animal models (i.e. zebrafish embryos). all search terms within one key word were combined by the operator 'or' and 'and' copied in a second or third search field. 'review' and 'meeting' documents were excluded. a search for grey literature was done in general search engines (i.e. google) using the different key words in the 'search' function of the respective websites. all the information on in vivo studies, using mammalian and/or non-mammalian organisms, and in silico studies was collected. these key words were chosen because this section aimed to describe the possible effects that exogenous ncrnas, specifically of plant dietary origin, might exert on the immune system of humans and animals. the search yielded ≈2255 entries (table 18) . publications were excluded if one of the following criteria was met: the biological effect could not be unambiguosly attributed to ncrnas; the publication addressed immunodeficiency or autoimmunity; the publication reported no immunity (innate or adaptive)-associated endpoint or it is not specific for immune function and/or homeostasis evaluation; evaluation of exposure of and/or effect on cells other than immune cells (e.g. epithelial cells); the study was unable to measure processes related to immune function and/or homeostasis; the study reported data on potentially immunocompetent cells not fully differentiated as mature immune cell; no ncrnabased plant was tested in the publication; and the publication did not address plant-derived exogenous ncrna. the unexpectedly low number of publications retrieved using the key words proved a challenge in the selection process. for example, selection for in vivo studies produced only 53 articles after title/abstract screening. to expand the number of documents, additional key word searches were included and less conservative criteria were set for the title/abstract screening. retrieval of grey literature was also based on information containing "preliminary results" or on-going projects that could possibly answer the key questions. www.efsa.europa.eu/publications 31 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. studies were further evaluated for their relevance to answering the key questions. the key questions identified using the above key words were: -which test methods or approaches are available to investigate the possible role of plant-derived exogenous ncrnas on the immune system of humans or animals? -what human or animal immune response(s), innate or adaptive, are possibly associated with administration of plant-derived exogenous ncrnas? -which mediators in humans and animals have been identified and/or ascribed to immune response(s) possibly mediated by administration of plant-derived ncrnas? -which innate immunity mediators (i.e. chemokines, cytokines) have been identified in humans and animals as associated to effects of plant-derived ncrnas? -which human or animal cells (i.e. lymphocytes, macrophages, myeloid) are possibly involved in immune responses mediated by plant-derived ncrnas? after the selection of the full-text documents, publications were excluded if not providing sufficient information (i.e. endpoint, exposure time), if investigating a mechanism other than immune-related processes, or if describing an operational procedure or guideline rather than a research study. from the initial ≈2255 documents identified using the above key words, and after filtering for the relevance to the key questions and detailed study of the full-text reports, 91 documents were finally selected for review in the three sections of part 4 (efsa task 4). of these, 22 documents were selected for review in this section. gut microbiota is important for the function of the immune system, therefore the purpose of this additional search was to provide general information on the effect of dietary exogenous ncrnas on the gut microbiota and the possible consequent secondary modulation of human or animal immune systems. this would serve as an introduction to identifying information gaps and experimental needs for future studies relevant to food/feed risk assessment of ncrna-based plants. selected full-text studies were analysed for their relevance to answering the following key question: -is there evidence for influence of exogenous plant ncrnas on the gut microbiota related to the immune system? five documents were finally selected for review in this section. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. results are presented narratively. when appropriate, tables and figures are included to allow better understanding of the topic covered by the literature review. some sections of this review provide only limited amount of quantitative analysis due to the nature of the available evidence and the key questions identified for each subchapter. at the end of each subchapter, a short summary presents the general points identified in the reviewed literature. gaps in the literature and open questions identified in the field are presented in a subchapter of certain sections. these gaps in the literature may serve as suggestions for further research in each topic. part 1: kinetics of exogenous ncrnas in humans and animals (efsa task 1) ncrnas are transcripts that are not translated into any functional peptides or proteins. these transcripts include structural (transfer, ribosomal, small nuclear, and small nucleolar rnas) and regulatory ncrnas. the latter comprise both small and long ncrnas that can regulate gene expression by acting on chromatin, transcription, rna processing, rna stability and translation. this part of the report introduces general features of regulatory ncrnas including their function and aspects of their biogenesis and degradation. it also establishes possible comparisons between animal and plant ncrna pathways. this can be considered background information to clarify the possibility of plant ncrna biological effect in humans and animals that ingest food/feed from ncrna-expressing gm plants. in addition, ncrna movement inside the plant is also described. specific details on the structural ncrnas and the broad topic of plant ncrna biology are outside the scope of this literature review. following an extensive literature search as described above and based on the team expert decisions on the topic, this section is a review of 173 scientific documents. small ncrnas (srnas) srnas are common to both plants and animals. two major classes of srnas are found in eukaryotes: micrornas (mirnas) and small interfering rnas (sirnas). they function as regulators of endogenous genes or as defenders from invasive nucleic acids. they are characterized by the double-stranded nature of their precursors, and differ from the less abundant piwi-interacting rnas (pirnas) which derive from fragmentation of single-stranded rnas (juliano et al., 2011) , and are primarily found in animals; they have not been described in plants. pirnas exert their functions in germ and stem cells through interaction with piwi-proteins (juliano et al., 2011) . by contrast, both mirnas and sirnas are bound to argonaute (ago) proteins, where they identify 'target' rnas by base-pairing interactions, and act in both somatic and germ cells. all srnas in plants are modified at the 3' -terminus by 2'-o-methylation, including mirnas and sirnas, which lack this modification in animals. 2'-o-methylation is essential to conferring stability and protection from 3' uridylation and degradation (borges and martienssen, , 2015) . mirnas and sirnas are distinguished by their origin and biogenesis (figure 2) . mirnas are derived from processing of single-stranded precursors with a hairpin structure, whereas sirnas are generated from long, fully complementary double-stranded rna (dsrna) precursors (carthew and sontheimer, 2009 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endonucleases (dcls) that excise rna precursors into short double-stranded fragments, 20-24 nucleotide (nt) long, with 2-nt 3' overhangs; and argonaute proteins (agos) that engage these duplexes and support their silencing activies based on target complementarity (carthew and sontheimer, 2009 ). further details on sirnas and mirnas are provided below. sirnas several different classes of sirnas with specialized biological roles have been identified in plants. the most abundant class consists of heterochromatic sirnas (hetsirnas), which are usually 23-24 nt long and originate from the repetitive and intergenic regions in the chromosome and transposable elements. hetsirna are crucial in dna methylation and chromatin modification through a process known as rnadirected dna methylation (daxinger et al., 2009) . hetsirnas are processed by dcl3 nuclease and preferentially loaded into ago4. mature ago4-sirna complex can interact with complementary noncoding nascent rna polymerase triggering the recruitment of dna methyltransferase (domains rearranged methyltransferse 2) to silence target loci at the transcriptional level via dna methylation and repressive chromatin modifications (vaucheret, 2006; lee and carroll, 2018) . these hetsirnas may account for approximately half of a plant's total mass of srnas. another class of sirnas are naturalantisense transcript sirnas (natsirnas) which can be generated from dsrna precursors through hybridization of independently-transcribed complementary rna strands (vaucheret, 2006) . in addition, secondary sirnas generated as a "secondary effect" of mirna-mediated target cleavage are found in plants (axtell, 2013; vaucheret, 2006) . the mirna-mediated cleaved target is occasionally used by rna-dependent rna polymerase (rdr) to produce secondary sirnas, which can either give rise to a phased set of sirnas (phasirnas) or trans-acting sirnas (tasirnas) with the ability to target genes different from their loci of origin. this secondary pool of sirnas can greatly amplify and sustain a systemic silencing throughout the organism. recognizable rdr-encoding genes are present in the genome of many rnai-competent eukaryotes, with the notable exceptions of insect and vertebrate species in which these secondary sirnas are absent (carthew and sontheimer, 2009 ). the lack of these secondary sirnas might have a positive effect on specificity (as sirna amplification can lead to the silencing of multiple transcripts, specifically if they share a highly conserved sequence or a common exon) and a negative effect on the amplification of rna silencing in these species. plants have more diversified and specialized sirna-based pathways than other organisms, which are thought to contribute to plant plasticity . it is generally accepted that these pathways evolved as a cellular defence mechanism against rna viruses and transposable elements, which were later adapted to regulate the expression of endogenous genes. this is consistent with the fact that most small rna classes have a recognized role in defence responses, as well as in epigenetic regulation, and that plants have larger and repetitive genomes (borges and martienssen, 2015) . the diversification of srna-directed silencing pathways in plants occurred through the expansion of the rdr-polymerases, dcl and ago proteins. rdr genes are found in rna viruses, plants, fungi, protists and some animals, but are absent in flies, mice and humans; this is consistent with the fact that the vast majority of srnas in humans are mirnas (kurzynska-kokorniak et al., 2015) . absence of rdr activity may also justify the lack of intercellular spreading of rna silencing in vertebrates, whereas systemic silencing is a phenomenon widely reported in plants and nematodes. dicer or dicer-like proteins in plants constitute a four-member gene family, whereas vertebrates have only one member (mukherjee et al., 2013) . the diversification of agos has resulted in development of distinct gene-silencing processes based on differential ago affinities to small-rna duplexes (borges and martienssen, 2015) . www.efsa.europa.eu/publications 34 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mirnas mirnas are well-studied srnas deeply conserved over long evolutionary distances, even between the plant and animal kingdoms. however, there are substantial differences between these two kingdoms with regard to mirna biogenesis, and mechanism and scope of mirna-mediated gene regulation (jones-rhoades et al., 2006) . detailed pathway comparisons and species information are described elsewhere . transcription of mirnas is typically performed by rna polymerase ii, and transcripts are capped and polyadenylated. in animals, most mirnas are derived from longer hairpin transcripts (pri-mirna) by the consecutive processing by the rnase iii-like enzymes drosha and dicer, whereas in plants only dicer (particularly dcl1) is responsible for mirna processing. thus, the biogenesis of most mirnas in plants occurs in the nucleus, whereas in animals it requires the sequential cleavage of pri-mirna in the nucleus and then in the cytoplasm by distinct rnaseiii enzymes (rogers and chen, 2013) . once processed, one strand of the hairpin duplex is loaded into an ago protein to form the core of the mirna-induced silencing complexes (miriscs). miriscs silence the expression of target genes predominantly at the post-transcriptional level (ptgs). the targets to be silenced are recognised through base-pairing interactions between the loaded mirna and mrna target, which contains a partially or fully complementary sequence (huntzinger and izaurralde, 2011) . plant mirnas recognize fully or nearly complementary binding sites, which are generally located within the open reading frames (orfs) of the mrna target. of importance is that mirna nt 9-12 are usually engaged in base pairing, which allows target cleavage by ago proteins (between nt 10 and 11). by contrast, animal mirnas recognize partially complementary binding sites, which are generally located in 3' utrs. in both plants and animals, complementarity to the 5' end of the mirna (the 'seed' sequence, containing nt 2-7) is a major determinant in target recognition and is sufficient to trigger silencing. for most mirna-binding sites complementarity is limited to the seed sequence (seed-matched sites) or to the seed sequence plus mirna nucleotide 8. however, in some rare cases complementarity to the 3' region of the mirna might contribute to target recognition, particularly when the mrna has a weak seed match. even for these sites, however, mirna nucleotides 9-12 generally bulge out, preventing endonucleolytic cleavage by agos. in both animals and plants the mirna 5' terminal nucleotide is buried in the mid domain of agos and is not available for pairing with the target (huntzinger and izaurralde, 2011) . therefore, there is an important difference in complementarity between plant and animal mirnas and their targets; this is less extensive in animals than in plants. in both animals and plants mirnas can move from cell-tocell. while in mammalian cells mirnas and other types of rnas can be transferred through secretory vesicles (ruvkun, 2008; valadi et al., 2007) , in plants they mostly use a different mechanism (kobayashi and zambryski, 2007) (see section 3.1.1.4. for details). however, recent evidences also suggest that plants can send small rnas in extracellular vesicles to pathogens to silence virulence genes (cai et al., 2018) . regarding the silencing mechanism, mirnas were initially thought to inhibit translation in animals and to predominantly promote target endonucleolytic cleavage in plants. however, recent evidence has changed this view by showing that mirnas can trigger translational repression and mrna destabilization in both kingdoms. in both plants and animals, the current evidence suggests that target mrna degradation provides a major contribution to silencing by mirnas (huntzinger and izaurralde, 2011 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. next generation sequencing approaches have revealed a new transcriptional landscape in which many novel lncrnas have been identified. these are transcripts longer than 200 bp without any clear protein coding potential. lncrnas are transcribed either from intergenic regions (lincrnas), introns (incrnas) or the opposite strand to protein-coding genes or other lncrnas; they are thus natural antisense transcripts (nats) (ariel et al., 2015; chekanova, 2015; shafiq et al., 2016; yamada, 2017) . as a confirmation of their widespread nature and possible biological relevance, lncrnas have been identified in plants, fungi and animals. in fact, lncrnas are involved in diverse biological processes across the eukaryotes, ranging from regulation of mating type in yeast to the pluripotency of embryonic stem cells in mammals (zofall et al., 2012; flynn and chang, 2014) . in plants, lncrnas play key roles in flowering time regulation, gene silencing, root organogenesis, seedling photomorphogenesis, and reproduction (ariel et al., 2015; chekanova, 2015; shafiq et al., 2016; yamada, 2017) . although plant lncrna biological characterization is still limited, detailed analysis of over 200 arabidopsis thaliana transcriptome data sets identified 40,000 putative lncrnas, including approximately 30,000 nats and over 6000 lincrnas. both in plants an mammalians (jin et al., 2013; liu, j et al., 2012) these lncrnas do not show association with small rnas and are also expressed at lower levels (30-fold to 60fold less) than mrnas. plant nat-lncrnas can overlap completely (60%) or have complementary sequences in the 5' or 3' regions of mrnas. they accumulate in a tissue-specific manner and many are modulated in respond to biotic or abiotic stresses, suggesting fine-tuning regulatory roles wang et al., 2015; ben amor et al., 2009; xin et al., 2011) . nat-lncrnas also accumulate under specific environmental conditions such as light exposure, and their expression overlaps with accumulation of histone acetylation marks, suggesting some transcription regulation effect . in plants there are other types of lncrnas. such is the case of the lncrnas involved in: 1) the rna-dependent dna methylation silencing pathway (rddm); and 2) the lncrnas generated from phasloci which serve as precursors of 21-nt and 24-nt secondary phased phasirnas in many plant genomes (zhai et al., 2015; fei et al., 2013; zheng et al., 2015) . however, the function of most plant lncrnas is still unknown (yamada, 2017; shafiq et al., 2016; ariel et al., 2015) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. many lncrnas in mammals and yeast originate from specific genomic locations such as the regions around transcription start sites (tsss), enhancer regions, intron splicing sites, and transcription termination sites. most of the studied lncrnas are expressed around the tss, including exosomesensitive yeast cryptic unstable transcripts (cuts) and stable un-annotated transcripts (suts) (xu et al., 2009) , and upstream antisense rnas (uarnas) (flynn et al., 2011) , among others. many mammalian non-polyadenylated lncrnas also correspond to divergently transcribed, exosome-sensitive ernas mapped to enhancer regions (andersson et al., 2014) ; so far plant ernas have not been reported. although most plant lncrnas are transcribed by rna pol ii, there are two plant-specific rna polymerases, pol iv and pol v, that also produce lncrnas (wierzbicki et al., 2008; li et al., 2014; yamada, 2017) . like many yeast and mammalian lncrnas, most plant lncrnas are polyadenylated, although non-polyadenylated lncrnas do exist (heo and sung, 2011; shin and chekanova, 2014; kim and sung, 2017; andersson et al., 2014) . in fact, hundreds of non-polyadenylated lncrnas induced by specific abiotic stresses were identified in arabidopsis . the expression of lncrna is regulated at transcriptional level and in combination with the pathways involved in their biogenesis, 3' end processing and degradation. the main 3'-5' exoribonuclease complex may regulate lncrna levels since various groups of polyadenylated ncrnas were originally identified in arabidopsis exosome mutants (chekanova et al., 2007) . rna exosome is an evolutionary conserved cellular rna processing/degradation complex (lange and gagliardi, 2011). some of these lncrnas originate from the tsss of protein-coding genes, resembling cuts, or they overlap with the 5' ends of protein-coding transcripts and extend into the first intron (chekanova et al., 2007) . gene expression can be regulated by lncrnas (either in cis or in trans) by sequence complementarity or homology with other rnas or dna, and/or by their structure; lncrnas can thus form specific scaffolds or platforms for assembly of specific complexes (chekanova, 2015) . most lncrnas regulate transcription. in animals this can be achieved by: 1) modulation of transcription factor dna-binding activity; 2) control of rna pol ii pausing; or 3) recruitment of chromatin modellers, which will ultimately affect chromatin topology and nuclear organization (bonasio and shiekhattar, 2014) . in plants, there are two lncrnas (hid1, apolo) that associate with chromatin, promote loop formation, and modulate transcription ), (ariel et al., 2015 (figure 3 ). some lncrnas act at the post-transcriptional and translational level (chekanova, 2015; jabnoune et al., 2013) . specifically, lncrnas can act as "decoys" or mimics by blocking certain rnas and/or dnas to access their protein regulators (e.g. ips1) (franco-zorrilla et al., 2007; huang et al., 2011) . bioinformatics analyses have also identified other putative mirna target mimics in animals that act similarly to plant mirna sponges (chekanova, 2015) . another plant lncrna (asco) also acts as a decoy of nuclear speckle rna binding proteins leading to different alternative splicing events and consequent altered root development (bardou et al., 2014) . other lncrnas with biological function are the enhancerrnas (ernas), described in yeast and mammals, but still unknown in plants. these ernas can act in cis as scaffolds to recruit co-activators and thus promote chromosome looping between enhancer and promoter regions; they can interact with other lncrnas to form specific chromosome structures to control gene expression. ernas are regulated by the exosome which can affect either rna synthesis or degradation in these regions (pefanis et al. 2015) . the exosome can also protect ernas from genomic instability by resolving r loops, which are stable rna-dna triplexes naturally formed during transcription, which can persist in regions that have divergent transcription, eventually becoming deleterious (skourti-stathaki and proudfoot, 2014 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. action of ernas and exosome can control gene expression and nuclear organization in these enhancer regions. interactions between lncrna and chromatin modifiers can be either dependent or independent of srnas. in animals they are srna-independent and occur via the trithorax complex (h3k4 trimethylation) ; or prc2 (polycomb repressive complex 2, that regulates h3k27 methylation) (tsai et al., 2010) . in plants they are -srna-dependent, with the rna-dependent dna methylation pathway (rddm) (matzke and mosher, 2014 ) (see below).further details on specific aspects are provided below. the rddm pathway seems to be a plant-specific pathway that relies on lncrnas transcribed from pol iv that will produce 24 nt sirnas. in parallel, lncrnas produced by pol v act as a scaffold that can then recruit the sirna-ago complex by sequence complementarity (matzke and mosher, 2014) . however, pol v-dependent lncrnas are difficult to identify probably due to their low accumulation rates. nevertheless, the few identified lncrna are non-polyadenylated and can be either tri-phosphorylated or capped at their 5' end (wierzbicki et al., 2008) . in addition, pol v also seems to collaborate with pol ii to promote rddm (zheng et al., 2009) . another group of regulatory ncrnas that are pol iv -rdr2 dependent were identified at intergenic regions overlapping mostly with transposons or sequence repeats. these ncrnas are also non-polyadenylated and at their 5' end have a monophosphate instead of 5' tri-phosphate or cap (zheng et al., 2009) . the arabidopsis exosome is involved in metabolism or processing of these lncrnas generated by pol iv, v and also some from pol ii (chekanova, 2015) . also worth mentioning is that the exosome is constituted by different subunits that are functionally diverse and can affect metabolism of smrnas and dna methylation (chekanova, 2015) . lncrnas and regulation of flowering some of the best characterized plant lncrnas are those involved in regulation of flowering upon "vernalisation" (the need of plants to experience a period of cold -winter-to enable flowering in spring). therefore, transgenic plants expressing any of these lncrnas may have biotechnological value. to assess the future relevance of these lncrnas it is important to understand the molecular mechanisms behind their function, since they are quite different from those of srnas (sirnas and mirnas).the best studied lncrnas in this process are: 1) coldair (heo and sung, 2011) ; 2) coldwrap (kim and sung, 2017); 3) coolair (swiezewski et al., 2009) and asl (shin and chekanova, 2014) . all these lncrnas modulate the flclocus, which encodes a transcription factor able to repress flowering (berry and dean, 2015) . interestingly, these four lncrnas originate from different regions within the flclocus (promoter, first intron, and 3' end antisense) suggesting a function in cis. they all contribute to prevent flc transcription by recruiting the prc2 silencing complex and the accumulation of repressive chromatin marks (crevillén and dean, 2011; csorba et al., 2014; rosa et al., 2016; sun et al., 2013sza; swiezewski et al., 2009; liu, f et al., 2007) . of interest is that certain aspects of this regulation are similar to that of the mammalian lncrnas hotair and xist. it has been proposed that some of these lncrnas could directly bind to prc2 components, but findings showing that mammalian prc2 can bind very strongly to unrelated rnas (davidovich et al., 2015) question the need for specific lncrnas in this recruitment step. the study of components of the arabidopsis exosome (rrp6l1 and rrp6l2) revealed their role in coolair and asl expression or processing, although this seemed to occur independently of the exosome core complex. both rrp6ls could process the 3' end of coolair and promote asl accumulation, similarly to xist in humans (shin and chekanova, 2014; ciaudo et al., 2006 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. regulate the levels of different lncrnas and probably act in different mechanisms to silence flc (chekanova, 2015) . lncrnas and r-loop formation coolair transcript levels are decreased due to r-loop formation at its promoter (sun et al., 2013b) , which could promote exosome recruitment through the 3' end processing pathway (chekanova, 2015) . this function is also seen in mammals where the exosome also resolves/degrades deleterious r loops (pefanis et al.) . surprisingly, in mutants affected in r loop formation, coolair may accumulate together with flc, suggesting that this regulation is still not fully understood (chekanova, 2015) . lncrnas and nuclear architecture animal lncrnas are involved in tethering rna, dna and proteins, and thus affect nuclear 3d structure (engreitz et al., 2013; quinodoz and guttman, 2014; hacisuleyman et al., 2014) . in plants, extensive evidence supports a similar role, either within the rddm pathway (moissiard et al., 2012) , in the regulation of flowering time (hepworth and dean, 2015) or in auxin signalling and root development (ariel et al., 2014; ariel et al., 2015) . plant and animal lncrnas can be intergenic, intronic or natural antisense transcripts, depending on their location in the genome. although only a few lncrnas have been characterized in detail, they are known to have different modes of action, ranging from chromatin modifications including prc2 recruitment, to promotion of translation, mirna target mimicry, hijacking splicing factors and formation of chromatin loops. adapted from (ariel et al., 2015) . not all processes occur simultaneously. different types of lncrnas are shown schematically. circrnas earlier studies of rnas structure proved that viroids (existing as uncoated rna molecules and are known to infect plants) have circular rna (circrna) molecules. the nature of covalently closed circular rna molecules was determined due to: i) the inability to phosphorylate at the 5'-terminus; ii) resistance to metaperiodate oxidation or borohydride reduction of the 3'-terminal ribose; or iii) resistance to venom phosphodiesterase degradation (sanger et al., 1976) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. al., 1990), plants (daros and flores, 1996) , and mammals (nigro et al., 1991) including humans memczak et al., 2013) , and across the eukaryotic spectrum. circular rnas' lack of coding potential was identified early on by the inability of circular mrna (kozak, 1979) or rna (konarska et al., 1981) to assemble/adhere to eukaryotic ribosomes. circular rnas are also unable to be translated in plants extracts . as with other ncrnas, circrnas in plants exhibit tissue-specific expression, have a considerable number of isoforms, alternative backsplicing (canonical and noncanonical) and alternative circularization patterns (lu et al., 2015; sun et al., 2016; wang et al., 2016; darbani et al., 2016) . the biogenesis of circrnas is a conserved feature in animal and plant cells. transcribed by rna polymerase ii and backsplicing reactions of pre-messenger rnas, they occur in the spliceosome and require a repeated sequence and rna-binding proteins. spliceosome formation is initiated by sequential assembly of small nuclear ribonucleoproteins onto a specific region of the pre-mrna downstream of the 5' donor splice site (dinucleotide gt) and upstream of the 3' acceptor site (dnucleotide ag). thus, exonic or intronic circrnas are generated depending on the initial small nuclear ribonucleoprotein binding site (lee et al., 2017; sun et al., 2016) . although their mechanisms of action in plants are still unclear, some studies in rice suggest that they do not imply any significant enrichment (as occurring in humans) for mirna target sites, and that circular rna and its linear form may act as a negative regulator of its parental gene (lu et al., 2015) . other studies have shown that fluctuations in circrnas do not correlate with the levels of their parentalloci encoded linear transcripts (darbani et al., 2016) . some circrnas have also been shown to contain putative mirna binding sites lu et al., 2015) and have been identified as mirna sponges (zuo et al., 2016) . by binding to mirnas, and consequently repress their function, circrnas act as mirna sponge to regulate the response to stress . recent data suggest that circrnas in plants may affect plant response to abiotic and biotic stress. for example, circrnas may be involved in chilling injury in tomato (zuo et al., 2016) , or may respond to imbalances in iron and zinc (darbani et al., 2016) . in another study, tan and colleagues (tan et al., 2017) showed that overexpression of a tomato circrna derived from phytoene synthase 1 (psy1), reduced psy1 mrna abundance, and lycopene and β-carotene accumulation. this was likely due to the continuous highly expressed circrna and/or the low abundance of linear rna from the overexpression vector. similar results were reported for another circrna derived from the phytoene desaturase gene (tan et al., 2017) . their role in developmental processes has also been established (cheng et al., 2017) , including their possible role in different aspects of development and senescence in arabidopsis cheng et al., 2017) . other biological processes such as photosynthesis (dou et al., 2017) or mitochondrion function (darbani et al., 2016) have also been proposed as involving circrnas. circrnas are a popular topic in animal research because of their potential as post-transcriptional regulators (memczak et al., 2013; piwecka et al., 2017; guarnerio et al., 2016; hansen et al., 2013; ashwal-fluss et al., 2014) , their recognized function as mirna sponges, as sponges for rna-binding proteins or their competition with linear splicing; and their role as diagnostic markers (zhao et al., 2017a; zhao et al., 2017b) . indeed, circrnas have also been found in biological fluids including saliva (bahn et al., 2015) , seminal fluid (dong et al., 2016) and plasma . research in plants is just emerging and functional studies are still lacking. it is unknown if this type of novel ncrnas could resist gastrointestinal tract conditions, due to their expected high stability, and have a biological effect on an organism. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ncrnas can regulate many biological processes through their impact on gene expression by acting on chromatin structure and affecting transcription, rna processing, rna stability and translation. among these, srnas are produced by cleavage of dsrnas intermediates, either from hairpin precursors (mirnas) or from the synthesis of dsrnas by rdrs (sirnas); most lncrnas regulate gene expression without being processed. despite size and biogenesis differences, most ncrnas share sequence-specific inhibitory functions. important differences are present between plants and other organisms in srnadirected silencing pathways, which are highly diversified and specialized in plants through the expansion of rdr, dcl and ago proteins that mediate srnas biogenesis and function. the vast majority of small regulatory rnas in humans and animals are mirnas. in contrast to plants, no evidence for systemic spreading of rna silencing in humans and vertebrates have been found. although very few plant lncrnas have been studied in detail, they seem to share with their animals' counterparts the ability to recruit chromatin modifiers and thus regulate gene expression. in the same context, very few studies are available on plant circrnas. their functional characterization will bring novel insights into their possible role as a novel class of noncoding regulators. when considering the potential presence of plant ncrnas in food or feed it is important to determine the mobility potential of these molecules inside the source plant (figure 4 ). there are several comprehensive reviews (chitwood and timmermans, 2010; dunoyer et al., 2013) addressing sirna/mirna movement in detail. it has been hypothesiezed that srnas may move throughout the plant. although the initial model would suggest non-cell autonomous (one whose action extends beyong the cell producing the signal) rna silencing by srnas, other intermediates could also account for this signal: dsrnas produced by rdr activity (plant specific), or fold back rna acting as silencing trigger (dunoyer et al., 2007; dunoyer et al., 2005; himber et al., 2003 and smith et al., 2007) . these intermediates could also diffuse from one cell to another and be processed again by dcl4, which has been associated with 21nt sirna movement (dunoyer et al., 2005) . in detail, secondary sirnas processed from dsrna by dcl4 could move cell-tocell to propagate silencing by signal amplification. it has been shown that dcl4 expression from phloem companion cells is critical to the short-term spread of the silencing effect of a sirna duplex. this was reported in a study in which the viral suppressor p19 was found to be expressed in phloem companion cells and able to bind to 21nt small dsrna (vargason et al., 2003) . dcl4-dependent sirna movement has also been shown to occur within certain organs, such as leaves, to create gradients (chitwood et al., 2009; levine et al., 2007) . other dcls-dependent sirnas are involved in sirna movement and these can also be sorted into different risc complexes (montgomery et al., 2008; mi et al., 2008) . other examples of mobile sirnas could be the ta-sirnas and dcl4-dependent 21nt sirnas, also acting through ago1. dcl3 processing of long dsrna substrates generated by pol iv (a plant-specific rna polymerase) and rdr2 (another plant-specific protein) leads to production of 24nt sirnas that are incorporated into ago4 and will transcriptionally silence transposons (chapman and carrington, 2007; mi et al., 2008) . in fact, sirnas derived from transposons or methylated regions in the genome have been shown to be associated with chromatin silencing. these 24-nt mobile sirnas could then promote epigenetic changes transmissible to following generations. when transgenes are expressed in plants, the generated dsrnas can be processed by dcl3 and dcl4. another dcl, dcl2, processes dsrnas into 22nt sirnas which can induce gene silencing of viral origin or from transgene expression (deleris et al., 2006) . sirnas may also function as mobile signals able to promote epigenetic modifications. grafting experiments with roots from dcl2dcl3dcl4 mutants and shoots expressing gfp sirnas could the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. indicate that sirnas 21 to 24 nt long move throughout the plant, but still unclear if in free or proteinbound form (i.e. bound to ago or other proteins) (molnar et al., 2010) . within the plant, sirnas are known to move in a source-to-sink direction and into growing meristems (molnar et al., 2010; palauqui et al., 1997; schwab et al., 2006) . their accumulation in reproductive cells could promote epigenetic changes in subsequent generations. in fact, slotkin et al. (slotkin et al., 2009) showed that dcl3dependent sirnas generated by transposon activation in the pollen vegetative nucleus can silence transposons in pollen sperm cells. these cells are then able to transmit genetic material to the following generation. a similar effect may occur if maternally-derived sirnas in the endosperm moved to the embryo and influence genome integrity or even lead to the creation of new epialleles (molnar et al., 2010) . similar findings of sirnas silencing transposable elements in the animal germ line have also been reported in an animal cell line and tetrahymena (malone and hannon, 2009 ). considering that plant development relies more on positional effects than cell lineage, the role of mirnas in the control of several developmental processes has to be strictly regulated at the mobility level (chitwood et al., 2009; levine et al., 2007) . for example, as small rnas diffuse into regions of low mrna (targets) expression, it eliminates target molecules therein, but cannot affect regions of high mrna levels (levine et al., 2007) . as mentioned previously, dcl1 processes imperfect hairpins into 21nt mirnas, with a 5' u nucleotide incorporated into ago1, and cleavage the mrna targets. ago10 can also incorporate these mirnas (brodersen et al., 2008) . while sirna movement is better understood, this is not the case for mirna movement. mirnas can be isolated from phloem sap (mir395, mir398, mir399) but it is unclear if they can leave the phloem (buhtz et al., 2008; pant et al., 2008; yoo et al., 2004) . some reports support both endogenous mirna and artificial mirna movement (chitwood et al., 2009; schwab et al., 2006) . it has been shown that mirnas can move from below the shoot apical meristem into the meristematic layers (chitwood et al., 2009) , and, similarly to sirna movement, from the phloem towards the root meristems (molnar et al., 2010) . although initial evidence suggested sirna mobility, with only dcl4 responsible for this, it is now accepted that almost every known rnai pathway in plants has non-cell autonomous activity. movement has been shown for srnas 1) of viral origin; 2) induced by transgene expression; 3) ta-sirnas; 4) mirnas and 5) repeat-associated sirnas (dunoyer et al., 2005; carlsbecker et al., 2010; chitwood et al., 2009; molnar et al., 2010; slotkin et al., 2009) . however, it is still unclear if srnas can move in a free state or associated with specific proteins, and in a single or double strand form. whereas some reports suggest that the srna molecule movement happens in a duplex form, the strand bias found in the srnas present in dcl2dcl3dcl4 grafted roots suggests that single strand sirnas can also diffuse (molnar et al., 2010) . unlike in animals, in which sirnas can move from cell-to-cell through secretory vesicles (ruvkun, 2008; valadi et al., 2007) , in plants the size of small rna would allow cellto-cell movement through plasmodesmata channels connecting different plant cells (kobayashi and zambryski, 2007) . this movement seems to occur in a source-to-sink direction, and could be regulated in some tissues through the formation of sirnas-ago complexes (molnar et al., 2010; schwach et al., 2005) . www.efsa.europa.eu/publications 42 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. plant small rnas (srnas) can move either cell-to-cell or long distance. cell-to-cell movement occurs through plasmodesmata (green arrows) that allow spreading of srnas from the cell where they are generated to 10-15 neighbouring cells (a). this cellto-cell movement can be extended over the surrounding cells by signal amplification (b). in this case, srna targets will be converted into new dsrnas by the combined action of dcls and rdrs. several grafting experiments have demonstrated that 21-24nt sirnas can move over long distances in the plant (c). whether they move as ssrna, dsrna or associated with proteins is still unclear. adapted from (dunoyer, p. et al., 2013) , (melnyk et al., 2011) and (molnar et al., 2010) . in terms of mirna mobility, it is still unclear if all mirnas, or only a subset, can diffuse. different mobility could be associated with different tissues/structures, and/or the size and stability of the passenger strand mirna (a.k.a. mirna*, star strand). in addition, mirna movement regulation could involve subcellular compartmentalization (by nuclear export proteins such as hasty (park et al., 2005) ) and sequestration via ago proteins (e.g. mir165/6 can be sequestered by ago10 and affect shoot apical meristem differentiation (liu et al., 2009; tucker et al., 2008) ). most of the information is available for srnas since they represent the most studied class of ncrna to date. in addition to the role of srna movement when considering ingestion of transgenic plants expressing altered levels of srnas, it is also important to address the issue of their biogenesis and turnover to assess their temporal availability when ingested. this is especially important since transgenic plants expressing rna interference constructs will accumulate the different srna intermediary forms (pri-mirnas, pre-mirnas, mature mirnas, drnas, sirnas), which may also be processed outside the plant when ingested. several reviews published in high-impact journals (rogers and rogers and chen, 2013; or scientific reports the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. view of the processes of mirna maturation and degradation in plants. it is important to consider the homology between the mirna biogenesis pathways in plants and animals as well as mirna stability. this knowledge allows understanding 1) whether any aspect of animal metabolism or other biological processes could be modulated by plant mirnas; and 2) if mirnas can survive for long periods after ingestion. as typical pol ii transcripts, pri-mirnas are stabilized by addition of a 5' 7-methylguanosine cap (xie et al., 2005) and a 3' polyadenylated tail (jones-rhoades and bartel, 2004; zhang et al., 2005) . mirna genes are also subjected to transcription factor regulation and can be alternatively spliced. the size of these transcripts varies and their processing normally occurs in a base-to-loop direction (rogers and . in plants, there are multiple dcl endonucleases that possess dexd/h-box rna helicase, duf283, paz, tandem rnaseiii, and dsrna-binding domains. dcls cleave the dsrna precursor generating a 2-nucleotide 3' overhang (margis et al., 2006) . processing of the pri-mirna requires at least two catalytic cycles to free the mirna:mirna* duplex. in animals, this requires sequential cleavage of the pri-mirna in the nucleus first and then in the cytoplasm by different rnase iii enzymes. the biogenesis of most mirnas in arabidopsis requires dcl1 (park et al., 2002; reinhart et al., 2002) , although other dcls may be involved, but are not essential (xie et al., 2004; gasciolli et al., 2005) . in contrast to animals, all plant mirna processing steps occur in the nucleus (papp et al., 2003) . dcl1 activity will result in mature mirnas of 20-21 nucleotides (nt), although variations in size can occur (rogers and chen, 2012) . confirming its fundamental biological relevance, dcl1 mutants are lethal (golden et al., 2002) . other dcl proteins that function in different srna biogenesis pathways might also have minor roles in mirna biogenesis. for instance, several mirna genes are partially processed into 23-25-nucleotide mature mirnas species whose accumulation depends on dcl3 (vazquez et al., 2008) . in the absence of dcl3, mature 22-nucleotide mirnas can accumulate due to dcl2 activity. in contrast, processing of mir822, mir839, and mir869 depends primarily on dcl4, probably as a consequence of highly complementary fold-backs in these pri-mirnas (ben amor et al., 2009; rajagopalan et al., 2006) . dicer cleavage of animal pri-mirnas is facilitated by the action of dsrnabinding domain (dsrbd) proteins (drbs) (jiang et al., 2005; parker et al., 2006) . in arabidopsis, there are five drbs that have been shown to bind dsrna in vitro (hiraguri et al., 2005) . the best studied member of this family is hyl1 (hyponastic leaves 1) which is involved in pri-mirna processing, but also in pri-mirna intron splicing (laubinger et al., 2008; szarzynska et al., 2009) . hyl1 seems to increase the accuracy of dcl1 processing of most, but not all, pri-mirnas, suggesting that other mechanisms may also be involved (liu, c et al., 2012) . from the same family, drb2 and drb4 are also involved in pri-mirna processing, by associating with dcl4 (rogers and . another protein involved in mirna biogenesis is serrate (se), which can interact with hyl1 and dcl1 (lobbes et al., 2006; machida et al., 2011) . like hyl1, se also seems to increase dcl1 pri-mirna cleavage efficiency (dong et al., 2008) . of interest is that these two proteins (hyl1 and se) can be regulated by phosphorylation. although hyl1 phosphorylation can decrease its activity (manavella et al., 2012) , the biological relevance of this modification in se is not known (rogers and . another regulator, dawdle (ddl), a phosphothreonine-binding forkhead associated (fha) domain protein, seems to stabilize pri-mirnas, possibly by direct binding (yu et al., 2008) . like se and hyl1, ddl can also interact with dcl1, but does not seem to be involved in pri-mirna processing. ddl most likely facilitates dcl1 access or recognition of pri-mirnas. in addition, ddl can recruit phosphorylated se or hyl1 to the pri-mirna. the human homologue of ddl, snip1, seems, among other functions, to be involved in mirna biogenesis and interacts with drosha (yu et al., 2008) . ddl could therefore be an evolutionarily www.efsa.europa.eu/publications 44 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. conserved factor in mirna biogenesis. other examples of conserved srna biogenesis machinery include ago, dicer, hen1 and exportin 5 (yu et al., 2008) . two components form the cap structure of rna pol ii transcripts in plants: cap-binding proteins (cbps) 20 and 80. there is substantial overlap among cbp20, abh1/cbp80, and se mutants in terms of: 1) the specific pri-mirnas affected; 2) the specific mrna splicing defects observed, and 3) a bias for accumulation of first introns (laubinger et al., 2008) . this connection between pri-mirna processing and pre-mrna splicing seems to converge at se and cbps. in fact, splicing defects are also observed in cbp20 and abh1/cbp80 in plants. in animals, the cap and its associated cap-binding complex are essential for the correct splicing of the first intron (lewis et al., 1996) . the connection between pri-mirna processing and splicing also converges in tough (tgh), a g-patch domain rna-binding protein. the tgh mutant accumulates pri-mirnas in vivo and has been shown to affect processing of pri-mirnas into 21-nucleotide mature mirnas (ren et al., 2012) . tgh may have either a structural or a regulatory role in pri-mirna processing. tgh binds both pri-and pre-mirnas in vivo, but this probably occurs via association with the loop structure since tgh binds ssrna but not dsrna in vitro (ren et al., 2012) . tgh paralogs have been described in metazoans (calderon-villalobos et al., 2005) , but their roles in mirna biogenesis are still unknown. a human tgh paralog is reported in spliceosomal preparations (jurica et al., 2002) . a conserved connection may exist between tgh and splicing and possibly pri-mirna processing (rogers and chen, 2012). mirna processing seems to occur in specific subnuclear regions. hyl1, se and dcl1 can make pairwise interactions that occur in certain subnuclear particles (fang and spector, 2007) . tgh can also interact with dcl1, hyl1, and se in subnuclear foci (ren et al., 2012) . these foci seem to be zones of functional pri-mirna processing (fang and spector, 2007; fujioka et al., 2007; manavella et al., 2012) . some of these components (dcl1, hyl1, se, tgh) also co-localize with components of the splicing machinery, further strengthening the connection between the two processes (calderon-villalobos et al., 2005; fang and spector, 2007; fujioka et al., 2007) . processed mirna:mirna* duplex will leave the nucleus to carry out its function in the cytoplasm. several components have been implicated in this transport and function and are described below. nucleo-cytoplasmic transport hasty (hst) is a member of the importin-ß family of arabidopsis that accumulates in the nuclear periphery (bollman et al., 2003) . hst is a paralog of human exportin-5 involved in pre-mirna transport. hst can participate in dcl1-dependent mirna:mirna* duplex export (zeng and cullen, 2004) . but this is probably not the sole mechanism for mirna nucleocytoplasmic transport, since passive diffusion via the nuclear pore may also occur (jacob et al., 2007; park et al., 2005) . ema1/sad1 is another importin-ß family protein involved in mirna function. ema1 is an orthologue of human importin-8 which facilitates nuclear import of ago2 and reduces its association with target mrnas (weinmann et al., 2009) . it is still unclear if ema1 1) sequesters mirnas; 2) is involved in transport of other risc loading factors or; 3) affects loading of certain mirnas into risc (rogers and . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after leaving the nucleus the mirna:mirna* duplex is loaded into ago, a risc component. the arabidopsis ago family consists of 10 members that associate with small rnas possessing specific 5' nucleotides (mi et al., 2008; takeda et al., 2008) . in plants, the slicer activity of agos has been shown for ago1, ago2, ago4, ago7, and ago10 (rogers and . ago1 seems to have a predominant role since it preferably associates with 5'u small rnas (most common 5' nucleotide in mirnas) (mi et al., 2008; takeda et al., 2008; vaucheret et al., 2004) . however, other agos can also associate with specific mirnas depending on mirna length or even their accumulation in different tissues (ebhardt et al., 2010; rogers, kestrel and chen, xuemei, 2013) , suggesting a certain flexibility in this association. in plants, mirna guide strands generally accumulate in higher levels than mirna* strands, although the exact mechanism for strand selection is still not fully understood (rogers and chen, 2012). hyl1 (drb) seems to be involved in this process (eamens et al., 2009; manavella et al., 2012) . in animals, loading of sirnas into risc requires the cytosolic drb protein loq, but loading of mirnas into risc requires dicer rather than loqs (liu et al., 2003; liu, x et al., 2007) , suggesting different mechanisms in plants and animals. hyl1 is predominantly nuclear, therefore its association with the mirna:mirna* duplex suggests that 1) hyl1 may be transported to the cytoplasm together with the processed mirna duplex; or 2) loading may also occur in the nucleus. in addition, other risc loading mechanisms can exist (eamens et al., 2009 ). in animals, sirna passenger strand unwinding requires ago2 slicer activity; mirnas duplexes are unwound by a slicing-independent mechanism (matranga et al., 2005) . in plants, ago1 slicer activity removes sirna passenger strand but this function is not required for mirna passenger strand removal (carbonell et al., 2012; iki et al., 2010) . as occurs in animals, plant mirnas use an alternative slicerindependent unwinding mechanism. ago1 loading seems to require heat shock protein 90 (hsp90) too (iki et al., 2010) . in addition, via hsp90, ago1 is able to interact with squint (sqn) and the phosphatase pp5 (iki et al., 2012) . these interactions seem to regulate ago1 loading and consequently risc activity. ago1 phosphorylation has been detected in arabidopsis (de la fuente van bentem et al., 2008) in manner similar to human ago2. apparently, phosphorylated ago2 exhibits reduced srna binding (rüdel et al., 2011) . phosphoregulation by pp5 or other proteins may modulate arabidopsis ago1 loading capacity. unlike in animals, in which mirna biogenesis occurs in the nucleus and cytoplasm, the mirnas in plants are generated in the nucleus in a mostly dcl-1-dependent manner. studies confirm that several of the major components are evolutionary conserved, such as the dicer, ago, hen1 and exportin 5 paralogs involved in nucleocytoplasmic transport of the mirna:mirna* duplex. however, shared pathway components among the different species do not reflect totally identical processes, such as, loading of mirnas into risc which occurs differently in plants and animals. one interesting common aspect remains the pri-mirna processing and pre-mrna splicing which seems to occur both in plants and animals. www.efsa.europa.eu/publications 51 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section 2.2.2.1 and following the methodology described in section 2.2.1.2., a total of 46 publications were selected as relevant to the stability and turn-over of non-coding rnas. of these, only four analyse ncrnas stability at a genome-wide scale, and one (enuka et al., 2016) simultaneously addresses the stability of several circrnas. using these studies, the stability of small ncrnas, lncrnas and circrnas can be compared. while earlier studies report the degradation of plant nucleic acids when exposed to hard conditions (i.e. cooking), six recent studies suggest that some ncrnas (i.e. mirnas) from plants can resist these conditions. turnover of ncrna molecules in plants and mammals depends both on their stability and degradation rate and it is described that chemical modifications can increase stability of different types of srnas (e.g. sirnas, mirnas). in mammalian cells, artificially introduced 2-o-methyl groups can stabilize sirnas without affecting their rna interference activity (czauderna et al., 2003) . considerable work has been done to address the issue of mirna modifications and how these affect their stability. among these, 2'-o-methylation has been identified as relevant. plant mirnas have a naturally occurring methyl group on the 3' nucleotide ribose ( figure 5 ). in this case, methylation does not require guide rnas, since hen1 (hua enhancer 1) can methylate mirna:mirna* duplexes. this hen1-dependent 2'-omethylation on the 3' terminal ribose is a mg2+ dependent methylation mechanism that will ultimately stabilize mirnas (abe et al., 2010; molnar et al., 2007; yu et al., 2005; yang et al., 2006) .the hen1 mg2+-dependent methylation mechanism relies on its two dsrbds binding the substrate duplex, and the la motif-containing domain interacting with the 5' end of the substrate strand (huang et al., 2009 ). this methylation step occurs after dcl processing creates the mirna:mirna* duplex but before duplex www.efsa.europa.eu/publications 54 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. unwinding and selection of the mirna guide strand. however, it is still not clear whether hen1 can methylate free mirnas or can bind hyl1, dcl1 and ago1, although it has been shown to interact with hyl1 and dcl1. in animals, the hen1 homolog has a different structure since it does not possess the dsrbd and la motif-containing domains. therefore, the animal hen1 homolog methylates singlestranded srnas associated with ago or piwi proteins (saito et al., 2007; horwich et al., 2007) . the degradation of ncrna molecules in plants and mammals is mediated by 3' nucleotidyl transferases and exoribonucleases. the arabidopsis hen1 suppressor1 (heso1) belongs to the dna polymerase β gene family (zhao et al., 2012; ren et al., 2012) . in other organisms, heso1 putative homologs are ribonucleotidyl transferases that are able to add specific nucleotides to the 3' end of different rnas (martin and keller, 2007) . arabidopsis heso1 can also add nontemplated nucleotides to the 3' end of unmethylated mirnas in vitro and exhibits a preference for uridine (ren et al., 2012; zhao et al., 2012) . analysis of hen1 and heso1 mutants indicates that this 3' oligo uridylation triggers degradation of mirnas (ren et al., 2012; zhao et al., 2012) . the arabidopsis family of small rna degrading nucleases (sdns) of 3'-5' exonucleases has similarity to the yeast rex exonucleases. sdn1 has 3'-5' exonuclease activity versus short ssrnas but is not active against longer or double-stranded substrates (ramachandran and chen, 2008) . in plants, 3' truncated mirnas can be modified by the addition of 3' oligonucleotide tails (see above) (ibrahim, fadia et al., 2010; li, j et al., 2005; lu et al., 2009 ). in addition, these 3' truncated mirnas also seem to associate with ago1. this interaction delays their degradation and allow addition of the 3' oligonucleotide tail leading to their degradation at a later stage (zhao et al., 2012) . whereas 2'-o-methylation may protect against 3' oligouridylation by heso1 (see above) and consequent targeting to degradation, this modification would not protect against sdn1. it is possible that sdn1 can promote mirna 3' truncation and then facilitate subsequent 3' polyuridylation by heso1. however, after 3' polyuridylation sdn1 would be unable to degrade these tailed mirnas, suggesting the existence of other nucleases (ramachandran and chen, 2008) .for instance, the addition of 3' polyuridylated tails in mirnas seems to attract exosome-mediated degradation in algae (ibrahim, f. et al., 2010) , but in arabidopsis this regulation has not been addressed in detail (rogers and rogers, kestrel and chen, xuemei, 2013) . another possible mechanism would include loss of ago1 association and protection of mirnas with long 3' tails due to heso1 activity. in addition, 3' polyuridylation may act as a nucleases recruitment platform, thus promoting mirna degradation. nevertheless, 3' polyuridylation and nuclease processing seem to be interconnected events that will ultimately result in degradation of the tagged mirnas. the overall turn-over of rnas is mainly described using half-life studies in both plants and animals. most of the available reports assessing ncrna half-life focus on the role of 3' truncation, 3' uridylation and consequent degradation (see above) as the main processes regulating mirna turnover in plants. however, a recent report identifies the enzymes (sdn1 and sdn2 exonucleases) responsible for 3' truncation in vivo and its relationship with 3' tailing (uridylation). also of importance, this report shows the opposite role of ago1 and ago10 in mir165/6 degradation by sdns, specifically sdn1. ago10 promotes mir165 degradation by sdn1, an unexpected role for a plant ago which has been associated with a protecting role in mirna stability (yu et al., 2017) . this function of ago10 (enhancing mir165 degradation) keeps mir165 out of the stem cell niche in the shoot apical meristem (where ago10 is not expressed). accumulation of ectopic mir165 in the shoot apical meristem fails to maintain the stem cell population. this kind of mechanism is thus needed to clear mir165 since this particular mirna can move between cell layers (see mirna movement section, above). another report evaluates the activity of nucleotidyl transferases in 3' uridylation of mirnas and in vitro assays indicated a fast enzymatic process (tu et al., 2015) . also, xrn -2 nucleases that affect mir homeostasis in c. elegans do not www.efsa.europa.eu/publications 55 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. seem to process mature plant mirs but rather the by-products of pri-mirna processing (ji, lijuan and chen, xuemei, 2012) . in summary, there are no specific studies on mirna half-life in plants. the reports mentioned here evaluate mirnas levels at steady state in different mutants, for example, when either the exonucleases and/or nucleotidyl transferases involved in mirna turnover are deleted. the half-life of mirnas in mammals has been addressed in studies of dicer1 ablation in mouse embryonic fibroblasts showing that mirnas are highly stable inside the cell. indeed, turnover assays revealed that the average mirna half-life is 119h (i.e. ≈5 days) (figure 6 ). although some mirnas have a shorter half-life, these data generally indicate that mirnas are much more stable (≈10x more) than messenger rnas. (gantier et al., 2011) . several features have been described that might explain this high half-life for mirnas. for instance, partial pairing of mirna and the mrna target site generally produces translational repression, while extensive pairing of mirna and mrna produce a mrna cleavage (pasquinelli, 2010) . in the former case the mirna remains stable, but in the latter it degrades. further studies in mammalian cells have also shown that argonaute proteins stabilize mature mirnas in a slicing-independent manner, increasing mature mirna stability (winter and diederichs, 2011). for specific mirnas, it has been found that certain rna-binding proteins (i.e. roquin) can enhance mature mirna-146 stability by reducing its 3' end uridylation (srivastava et al., 2015) . while intracellular levels of mirnas are mostly affected by cell division (ghosh et al., 2015; gantier et al., 2011) , mirna activity and turnover can also be controlled by subcellular distribution of microribonucleoproteins; that is, polysome sequestration contributes to an increase of cellular mirna levels, but without an increase in mirna activity (ghosh et al., 2015) . plant small rna duplexes can be 2'-o-methylated at their 3' terminal ribose by hen1, loaded into ago and protected from uridylation and degradation by 3'-5' exonucleases. ago1-bound unprotected small rnas, however, are uridylated by heso1 and degraded by sdn1. adapted from (borges and martienssen, 2015) and (ji and chen, 2012) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. widespread variation of mirna decay in slow-dividing primary macrophages. bone marrow derived macrophages from when analysing lncrnas stability in mammals at a genome-wide scale, lncrnas half-life generally varies over a wide time range, comparable to, although on average less than, that of mrnas (clark et al., 2012) . in a study of all transcripts, including mammals, it was found that the range of half-lifes for ncrnas (all types) was similar to that of the mrna half-life (tani et al., 2012) . indeed, a large number of ncrnas transcripts have a half-life of less than 4h (tani et al., 2012) , which agrees with the < 2h half-life for hundreds of unstable lncrnas (clark et al., 2012) . however, although there are lncrnas with extreme stability (half-life > 16h) they are still much less stable than mirnas (gantier et al., 2011) (see above), but more stable than protein-coding rnas (wang, l et al., 2014) . circrnas have a covalently closed loop structure with neither 5'-3' end nor a polyadenylated tail, which confers high resistance to rna exonuclease or rnase r treatment when compared to that of their linear sequence counterparts (memczak et al., 2013) . indeed, earlier studies of the structure of rnas from viroids, which were found to be circular, suggested that the nature of covalently closed circular rna molecules confers certain properties including resistance to metaperiodate oxidation or borohydride reduction of the 3'-terminal ribose; the inability to phosphorylate at the 5'-terminus; or resistance to venom phosphodiesterase degradation (sanger et al., 1976) . in mammals, some studies have evaluated the stability of circrnas compared to that of their linear isoform derived from the same host gene. for example, circrnas were found to be less abundant and dynamic than their counterparts (enuka et al., 2016) . calculating the half-life of 60 circrnas and their linear counterparts, enuka et al., 2016 showed that the media half-life of circrnas of mammary cells (18.8-23.7h) was at least 2.5 times longer than the median half-life of their linear counterparts (4.0-7.4h). this suggests that cirrnas are generally more stable and static compared to linear species (enuka et al., 2016) (figure 7 frame a). in a stability study on some circrnas it was found that while the associated linear transcripts exhibited a half-life of <20h, the circular rna isoforms were highly stable, with transcript half-life exceeding 48h (jeck et al., 2013) . in plants, covalently circularized exogenous rna incubated with wheat embryo cell extract in vitro have shown that the circular rnas exhibited considerable stability compared to the linear version (makino et the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. al. , 2006) . while linear rna degraded within the first few minutes, its circular counterpart was stable for more than 80 min in vitro (figure 7 frame b). by contrast, circularization dramatically reduced the translation capability of the rna, which was restored to some extent after linearization. plant srnas are naturally methylated at their 3' nucleotide ribose. this modification depends on hen1 and protects srna from degradation. unmethylated or truncated srnas can be uridylated by heso1 and thus be targeted for degradation by 3'-5' exonucleases of the sdn family. although ago binding to mirnas was believed to protect them from degradation, recent reports suggest that ago10 in particular may promote mirna degradation by sdns. differently from the plant situation, animal srnas are not methylated. a) mcf10a cells were metabolically labelled using 4su, for 1 or 2 h. the rna was then extracted, biotinylated and purified on streptavidin magnetic beads. flow-through rna was also collected. next, rna was reverse transcribed and quantified using highthroughput real time pcr (fluidigm). the half-lives of 60 circrnas and their corresponding linear counterparts are shown. halflife values were calculated from two samples, labelled with 4su for 1 or 2 h and then averaged. all data were corrected for any bias introduced due to low uridine (short length) of rna species (see 'materials and methods' section). the circrnas (red dots) and their linear counterparts (blue dots) were sorted from high to low according to the difference between their half-lifes. error bars represent standard errors. source: from (enuka et al., 2016) . b) stability of circular rnas in the wheat germ cell-free translation system. a denaturing polyacrylamide gel separating the synthesized circular and linear rnas with a (gaaa) 16 sequence after incubation with the wheat embryo extract for the indicated time periods. untreated rna is in lane n. positions of circular, linear, and ribosomal rnas are indicated as c, l, and rrna, respectively, on the right. source: from . compelling evidence supports the significant contribution of srnas to communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. (baulcombe, 2015; (ghag et al., 2014; koch et al., 2013; vega-arreguín et al., 2014; helber et al., 2011) . srnas or dsrnas, efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. for example, can be transferred from plant to pests such as insects that eat leaves or nematodes that infect roots. in fact, transgenic plants can be created that express dsrna homologous to essential genes of insect pests or nematodes and thus control them (baum et al., 2007; fairbairn et al., 2007; mao et al., 2007) . thus, this silencing transfer mechanism is very relevant for the food and feed risk assessment of ncrna gmo plants, highlighting the need to evaluate the stability of ncrnas outside the plant. earlier studies suggested that food nucleic acid content was hydrolysed when cooked (colling and wolfram, 1987) . most of ncrnas, including srnas and lncrnas have been discovered in the last 20 years, and thus new data has been added to the body of knowledge. in a study of srna stability under cooking conditions, plant mirnas were detected after cooking although at significantly lower levels when compared to fresh plant tissues (see table 19 ) (zhang, lin et al., 2012) . differences were observed for mirnas from different food/feed sources (rice, cabbage, wheat or potato), ranging from nearly undetectable values to almost 80% persistance of mirnas after cooking. levels of mirnas were monitored with a stem-loop quantitative reverse transcription polymerase chain reaction (qrt-pcr) assay using the u6 snrna for normalization, a commonly used housekeeping gene in mirnas analyses. the same study found that most of the plant mirnas and mammalian mirnas could survive under acidic conditions that mimic the gastric acidic environment. of note is that the degradation rate of mammalian mirnas under acidic conditions was similar to that of their synthetic form (without 2'-omethylated 3' ends), whereas plant mirnas had a much slower degradation rate compared with their synthetic form (without 2'-o-methylated 3' ends), suggesting that methylation had a protective effect on the stability of plant mirnas (table 20) . another study reported that endogenous mirnas can be detected by qpcr analysis in rice and soybean plant materials after storage, processing and cooking (philip et al., 2015) . for processing and cooking, soybeans were soaked in rnase-free water with 0.25% w/v nahco 3 overnight at 4 ºc, then separated from the soaking liquid, rinsed in fresh rnase-free water, and then boiled in rnase-free water for 80 www.efsa.europa.eu/publications 59 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. min until they became soft in texture. by constrast, a synthetic mirna showed a significantly higher susceptibility to simulated food processing conditions as compared to plant mirnas, as well as high molecular weight rnas (total rna). the synthetic mirna used for comparisons was the cel-lin-4, which is a caenorhabditis elegans mirna. the synthetic mirna was synthesized without 2'-o-methylated 3' ends (philip et al., 2015) . these results suggest that the methylation and the small size of plant mirnas make them more resistant to degradation than synthetic (not chemically modified) mirnas. in addition, the study showed significant plant mirna stability in a simulated digestion system for 75 min prior to absorption or transport into the blood stream. however, this study provided most of the quantitative data from q-pcr analysis without normalization, and data should be considered as relative compared to the treated control in the experiments. studying the stability of plant srna in vitro, liang and colleagues exposed total rna (5 µg), isolated from brassica oleracea, to freshly drawn serum from mice. in serum, a large amount of srnas survived after 24h of incubation at 37ºc, but after 36h only about 5% of the rna could be detected. the authors also exposed the plant rna molecules to fecal suspensions at 37 ºc. after 4h incubation significant amounts of srnas were still detectable, but after 18h rna molecules were undetectable . suggesting that srnas were more resistant to degradation in the presence of serum suspension than in fecal suspensions. these results were produced by gel electrophoresis of srnas, which is known to be less sensitive than qrt-pcr or rna sequencing methods. this study also reports that, when orally ingested, plant mir172 (chosen because it is the most highly enriched plant mirna from b. oleracea) can pass intact through the gastrointestinal tract in mice (a maximum of 4.5% recovered from the stomach of some individuals), and can be detected in the blood, liver, spleen and kidney. levels of mir172 were monitored by qrt-pcr using a taqman probe, which can distinguish differences of one nucleotide. since only one mirna was evaluated, the above findings cannot be generalized to other mirnas. other studies have reported mirnas to be stable in watermelon and mixed fruit juices produced by simple extraction without additives . plant mirnas were detected by stem-loop qrt-pcr using taqman probes and some were validated by northern-blot analysis. to reduce the nonspecificity signal of qrt-pcr, no-template controls were used. yang and colleagues studied artificial in vitro digestion systems that simulate mammalian gastric and intestinal conditions, and found that mir2911 was between 10-100 fold more stable than other mirnas . this effect was observed for cabbage extracts when compared to either the 2'o-methylated or the non-modified form. detection was also done by qrt-pcr. the same research group reported recently that several plant mirnas, including mir172, were degraded during storage at 4 ºc in cabbage extracts obtained by mechanical maceration . degradation of these mirnas correlated with that of 26s rrna, whereas mir2911 accumulation was increased over 600-fold after 24 hours of storage, and then gradually decreased over a span of 7 days. mirna (mir2911) increasing in macerated tissues (rather than degrading) is uncommon for most mirnas. indeed, the authors indicated that, in terms of genesis and amplification, mir2911 is an atypical mirna, which could explain the disparity in serum detection with respect to canonical plant mirnas. thus, mir2911 was shown to be derived from 26s rrna and processed in a dcl1-independent manner, meaning that when 26s rrna is degraded mir2911 is produced. this study presents the possibility that certain ncrnas can have non canonical features for synthesis or stability, and should be evaluated individually. in another recent study, the in vitro stability of curcuma longa mirna 14 (clo-mir14) was assessed. when exposed to foetal bovine serum (fbs) for up to 48h, the mir14 exhibited high resistance . in summary, food nucleic acid content are generally hydrolysed during processing, with some studies suggesting that mirnas from plants may be more resistant to degradation than synthetic or animal www.efsa.europa.eu/publications 60 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mirnas due to endogenous modifications (2'-o-methylated 3' ends). however, these findings are not consistent with mirna stability studies in the animal gastrointestinal tract, and further research is required. although there are many studies on plant ncrnas stability (see 3.1.2.1.), these mainly focus on ncrna stability within the plant itself. mammalian ncrna stability differs from that of plants because lack the natural 2'-o-methylation at the 3' ends occurring in plant ncrnas, mostly in mirnas (yu et al., 2005) . very few reports were identified in which plant ncrna stability is investigated in a mammalian organism ) (see sections 3.2. and 3.3.) . understanding the molecular basis that confers plant ncrna stability is of vital importance and needs to be further evaluated to better inform on the relevance of plant ncrna for food and feed risk assessment. for instance, modifications plant mirnas not occurring in mammals, such as the methyl group located on the 3' nucleotide ribose, could hypothetically have an impact on plant mirnas stability in mammalian systems due to the lack of the appropriate enzymes for their recognition and degradation, or due to increased stability to mammalian rnases . should this be the case, plant mirnas could manifest a much longer than expected half-life in mammalian organisms when compared to mammalian mirna, which could increase the probability of encountering target molecules. the literature contains descriptions of possible in vitro interaction of plant mirnas with mammalian mirna silencing complexes which may trigger target repression chin et al., 2016) . however, this hypothesis needs to be experimentally validated since there are gaps in the literature. moreover, it needs to be determined if sufficient levels of ingested plant mirnas reach a target cell to determine an effect in mammalian cells. for instance, in mammalian studies it has been suggested that the threshold for target gene regulation is between 1000 and 10000 copies of mammalian mirna per cell . accumulation of plant exogenous ncrnas has not yet been reported, but recent next generation sequencing studies have reported a plethora of plant mirnas and other rnas in circulating in the human organism (see section 3.3). the biological significance of their presence is still unknown. it remains unknown if under certain pathological conditions (i.e. compromised intestinal permeability or renal function) exposure to these plant ncrnas could change. all these gaps in the literature would need to be experimentally validated. to determine the half-life of plant ncrnas in mammalian cells would require experiments utilizing labelled plant ncrnas, first transfected into mammalian cells (in vitro studies) and then orally administered to experimental animals (in vivo studies). the markers used for these experiments could be radioactive probes or labelling molecules of very low molecular weight, such as biotin or fluorescein, to prevent distorting the actual size of the plant ncrna under study. due to their small size, this is important when studying mirnas stability. there is evidence that certain plant ncrnas can induce silencing in some eukaryotic pathogens, pests, parasites or symbiotic microorganisms as a defence strategy. in return, various pathogens develop mechanisms to evade this defence system, proving the existence of a two-way sirnas traffic between pathogens and their plant hosts. plant ncrnas stability outside the plant and in animal systems may play a role in cross-kingdom effects but has received limited attention. the resistance of plant ncrna to the gastrointestinal environment or processing has been partly investigated but more research would be needed. efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following the methodology described in the section 2.2.1.2 and based on a literature search as described in section 2.2.2.1.3, a total of 178 documents were selected as relevant to describing the state of the art in the field of ncrnas used/intended for use as therapeutics in humans (pharmaceuticals/medicine area). this constitutes valuable preparatory material on the pharmacokinetics (and pharmacodynamics) of ncrna, as required by task 1. of these publications at least 130 indicate that to ensure the biological activity of exogenous rnas for therapeutic requires numerous chemical modifications and delivery methods. although composed mainly of only four basic building blocks, rnas form simple to very complex structures. its intrinsic base-pair property, enhanced by the ability of the extra hydroxyl groups in the ribose sugar to form hydrogen bonds, are the foundations of its diverse structure and functions (lu et al., 2016) . rna can fold into various secondary structures including stem, loop, bugle, pseudoknots, gquadruplex and kissing hairpin. it can perform a wide range of biological functions, ranging from regulation of gene expression at various levels to catalysing chemical reactions which closely depend on the ribonucleotide chain's spatial structure. other rnas, such as ribozymes (doudna and cech, 2002) , can form tertiary structures with catalytic activity. certain rnas can also undergo transformation between alternative structures, depending for instance on binding to specific ligands or environmental changes, as is the case of riboswitches (mironov et al., 2002) . riboswitches are noncoding rna structures located within mrnas that bind endogenous ligands to regulate gene expression or rna splicing events (cheah et al., 2007) . the sirna mechanism of action involves dicing of endogenous dsrnas from longer rnas transcripts by dicer and loading into the protein complex of the rna-induced silencing complex (risc). argonaute (ago) proteins are the catalytic core of the risc complex in plants and animals (tolia and joshua-tor, 2007) . one strand (the passenger strand) is discarded and the guide strand is paired to a complementary mrna sequence via the risc complex. gene silencing can be achieved mainly through post-transcriptional gene silencing (ptgs) or transcriptional gene silencing (tgs). in ptgs mechanisms, the mrna undergoing translation can be sequence-specific cleaved by the risc complex and degraded when the target mrna is perfectly complementary to the sirna. when the interaction is partial or only limited complementarity exists, translational repression and rna degradation occurs, which is a mechanism exerted by mirnas. the mechanism of action for mirna differs somewhat. the mirnas are endogenous encoded singlestranded rnas of about 22 nt long that are important post-transcriptional regulators of gene expression by pairing through a sequence-specific complementary binding to the 3' utr of the target mrna. this is usually mediated by a sequence of 2-8 nucleotides, known as the seed region, at the 5' end of the mature small rna (bartel, 2009). however, mirnas can also bind to other mrna sites including 5' utr or protein coding exons (forman and coller, 2010) . depending on the degree of sequence complementarity, the interaction will result in mrna degradation and/or translational repression (ameres et al., 2010; bartel, 2009) , with destabilization of target mrna being the predominant efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mechanism to reduce protein output exerted by mammalian micrornas (guo et al., 2010) . most animal mirnas are transcribed by rna polymerase ii into a long primary transcript (pri-mirna) containing a stem-loop structure. the pri-mirna is then processed by a multiprotein complex containing the nuclear rnase iii drosha into a ≈70 nt long hairpin-shape precursor (pre-mirna) and exported to the cytoplasm via an exportin-5 and ran-gtp-dependent mechanism. the cytoplasmic rnase iii dicer further processes the pre-mirna by removing the terminal loop to produce a small rna duplex, containing the functional microrna and the passenger (*) strand (mirna/mirna*). the duplex mirna is then separated and the functional mirna incorporated into ago proteins within the rna-induced silencing complex (risc), guiding it to the target mrna to induce its translational repression or mrna destabilization (krol et al., 2010; bartel, 2009 ). in 1978, zamecnik first reported the use of an exogenous synthetic oligodeoxynucleotide complementary to the rous sarcoma virus 35s rna as a potent inhibitor of protein translation (stephenson and zamecnik, 1978) and virus replication (zamecnik and stephenson, 1978) . since then, the ability to use exogenous (synthetic) agents to control gene expression has revolutionized many aspects of biological research and catalysed the development of a new promising class of exogenous molecules to treat human diseases. however, despite the obvious promise of this approach, progress has been slow because of the need to overcome myriad technical hurdles, particularly those related to the pharmacokinetics, pharmacodynamics and delivery of antisense molecules. indeed, as most antisense-based potential therapies have not yet produced significant clinical results, very few rnabased or dna-based antisense drugs have been approved for clinical use. in 1998, the u.s. food and drug administration (fda) approved the first aso-based therapeutics for the treatment of a human disease. in 1999, fomivirsen (marketed as vitravene) was approved for clinical use by the european medicines agency (ema) for the european market for treatment of cytomegalovirus (cmv) retinitis in immunocompromised patients. fomivirsen is a synthetic 21-mer aso (dna) that binds complementary to the sequence of the mrna encoding the ie2 protein from cmv. the first rna-based therapeutic approved for clinical use was pegaptanib (marketed as macugen), which was approved by both the fda (in 2004) and ema (in 2006), for treatment of neovascular (wet) age-related macular degeneration. pegaptanib is also the only approved single strand rna aptamer drug, and it targets the vascular endothelial growth factor as an antagonist. in both cases, fomivirsen and pegaptanib, the route of administration is intravitreal injection, and the unique properties of this route provide benefits in terms of pharmacokinetic and immunological response. in 2013, the fda (but not the ema) approved the second rna-based therapeutic, mipomersen (marketed as kynamro) for treatment of homozygous familial hypercholesterolemia. mipomersen is a dna-rna aso that binds to the mrna of apolipoprotein b (apob), thus reducing apob protein and concomitantly ldl cholesterol levels. antisense oligos like fomivirsen (dna) and mipomersen (dna-rna) act by binding to a complementary sequence of a mrna, thus preventing production of its encoded protein. nusinersen (marketed as spinraza) approved by the fda (in 2016) and ema (in 2017) as an orphan-drug for treatment of spinal muscular atrophy is a rnabased aso that increases the production of the full-length survival motor neuron 2 (smn2) protein. eteplirsen ( the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (bobbin and rossi, 2016) and many others, which suggests that exogenous rnas can be delivered to humans and used to modify gene function, protein accumulation and treat human diseases. the ability of nucleic acids, both dna and rna, to form duplexes by base complementarity, has been used to develop oligonucleotide-based drugs for gene silencing. in principle, an oligonucleotide binds a target rna through watson-crick base pairing and exerts gene silencing through different mechanisms (kole et al., 2012) . a single strand antisense oligonucleotide (aso) binds to the mrna, which is also single-stranded, and is translated into proteins. different molecular mechanisms to either block gene expression or degrade the rna duplex formed by watson-crick base pairing have been described. cleavage of the rna strand of dna•rna hybrids is predominantly mediated by the enzyme rnase h (walder and walder, 1988) , which is an abundant enzyme in both the nucleus and cytoplasm of eukaryotic cells (wu et al., 2004; ten asbroek et al., 2002) . asos can also bind rna and block ("steric blockers") gene expression rather than facilitating rna cleavage (dias et al., 1999) . also considered to be within the aso family are peptide nucleic acids (pnas) or phosphorodiamidate morpholino oligomers (pmos or "morpholinos"). their modification usually acts through this mechanism (michel et al., 2003) , which in some cases exerts high efficacy in vivo (iversen et al., 2003; kole et al., 2012) . exogenous rna molecules or asos can alternatively target and bind a splicing site on the target pre-mrna and thus modify its exon content (mayeda et al., 1990; skordis et al., 2003) , which results in the production of alternative splicing products with potential applications in exon skipping therapy (goemans et al., 2011) . because many of these asos contain dna with multiple modifications, they are not discussed here in detail. the present literature review focuses mainly on ncrna and particularly on exogenous ncrnas. the discovery of rna interference (rnai) (fire et al., 1998) increased the interest in using exogenous chemically-synthesised rnas for silencing genes in mammals, and promoted their potential usage in human therapeutics (song et al., 2003) . to achieve therapeutic results, rnai can be administered via delivery to the cell of small exogenous rna duplexes -including short interfering rnas (sirnas) (zuckerman and davis, 2015), mirnas mimics (bouchie, 2013), short hairpin rnas and dicer substrate rnas (dsirnas) (foster et al., 2012; kim et al., 2005) -to the cell for further processing into an rna interfering silencing complex. although double-stranded rnas have been widely used for sirna therapy, single-stranded sirna (ss-sirna) is capable of activating the rnai pathway and exerting rnai effects (holen et al., 2003; lima et al., 2012; prakash et al., 2015) . moreover, singlestranded mirna mimics have also been shown to activate the mirna pathway (chorn et al., 2012) . in principle, then, both dsrnas and ss-sirnas can trigger gene silencing of complementary messenger rna sequences (holen et al., 2003) . other small rnas with imperfect matches have also been described to repress mrna translation (saxena et al., 2003) . for mirna therapeutics, different pharmacological tools have been developed which involved either inhibiting or enhancing mirna function (van rooij and olson, 2012; janssen et al., 2013) . approaches to inhibiting mirna function include small-molecule inhibitors; mirna masking; mirna sponges; and aso, such as anti-mirs, locked-nucleic acids (lna), or cholesterol-modified antagomirs (krutzfeldt et al., 2005) . the latter's complementarity allows them to bind to mirnas, inducing duplex formation or mirna degradation. the lna therapeutic approach successfully led to the first mirna-based clinical trials for the treatment of hepatitis c (miravirsen; santaris pharma, denmark) with promising results (janssen et al., 2013) . there are also approaches that can be utilized to enhance mirnas function called "mimic". these include small-molecule activators of mirna expression and mirna mimics, which are www.efsa.europa.eu/publications 66 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. exogenous mirnas delivered by several methods aiming to repress the function of their endogenous targets. they are also referred to in the literature as "mirna replacement therapy". as proof of concept, a synthetic version of mir-34a (mrx34, mirna therapeutics), delivered using a liposomal delivery formulation, was the first mirna to enter a clinical trial for cancer (beg et al., 2016) . the literature also describes other types of rnas that can exert physiological functions similar to that of mirnas and sirnas. for example, a 30-nt long guide hairpin rna (ghrna), that might not require ago2 or dicer, has been shown to exert mirna or sirna activity in vivo (ohno et al., 2016) . this novel rna interference technology may act as a novel platform for rna-based therapy. indeed, systemic or local injection of the ghr-form mirna-34a (ghr-34a) suppressed tumour growth in a mouse model of ras-induced lung cancer. understanding the manifold mechanisms of rna function requires detailed knowledge of the rna tertiary structure (magnus et al., 2014) , which can lead to therapeutic development (digiusto et al., 2010; strobel et al., 2015) . indeed, different experimental (lu et al., 2016; weinreb et al., 2016) and computational methods (magnus et al., 2014) have been developed to predict rnas structure and interactions. the capacity of rna to fold in various ways can generate unique three dimensional secondary structures capable of specific molecular recognition of their target cognate (zhou and rossi, 2016; tuerk and gold, 1990) . aptamers are short single-strand rna molecules (20-100 nt) with a defined structure that can specifically bind to a molecular target via tertiary structures. aptamers can be rationally designed using selex technology (tuerk et al., 1992) , and can therefore be incorporated into chemically modified rnas with high nuclease resistance properties suitable for animal and clinical studies (sullenger and nair, 2016). unlike other ncrnas therapeutics, aptamers can target soluble extracellular proteins or extracellular domains of cell-surface receptors. the latter characteristic is unique and can be used to deliver sirnas, mirnas or other compounds to target tissues, binding them to a cell-specific aptamer (zhou and rossi, 2014) . aptamer therapeutics have rapidly progressed to clinical studies, and some are already in phase 3 clinical trials (sullenger and nair, 2016; zhou and rossi, 2016) . the unique properties of aptamers led to development of the first therapeutic aptamer and the first rna-based drug approved for clinical use, pegaptanib. however, aptamer and other ncrnas therapeutics are not free of adverse effects (lincoff et al., 2016) . when compared to sirna, dsirna was found to be comparable in potency and duration of effect. however, dsirna was found to be more immunostimulatory when compared to the shorter sirnas (foster et al., 2012) . preclinical studies suggest that when administered in lipid nanoparticles (lnp) dsirnas can exert biological effects in different mouse models with tumours of diverse origin (ganesh et al., 2016) . although preliminary data in nonhuman primates suggest acceptable tolerance for this specific lnp formulation (ganesh et al., 2016) , the dsirna therapeutic arena still needs to demonstrate its efficacy and safety in humans. catalytic rnas, the so-called ribozymes, are another family of rnas for which therapeutics have been actively developed (kobayashi et al., 2005) , reaching advanced clinical trials (burnett and rossi, 2012; mitsuyasu et al., 2009 ), but this review will not focus on this type of rna molecules. unmodified nucleic acids have been shown to possess limited stability in biological media and are subject to rapid enzymatic-mediated degradation (braasch et al., 2004; . there are three major classes of intracellular rna-degrading enzymes (houseley and tollervey, 2009) (a.k.a. ribonucleases or rnases): (i) endonucleases acting within the rna chain, ii) 5' exonucleases and iii) 3' exonucleases degrading rna from the 5' or 3' end respectively. very large amounts of nuclear rnas efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. are rapidly degraded (brandhorst and mcconkey, 1974) , this being considered either as translation noise (struhl, 2007) or part of the biological function of ncrnas. instead, most cellular rnas are modified to resist to exonuclease degradation (ren et al., 2014) . most genomes encode a plethora of rnases, often with overlapping activities, making redundancy a general feature of rna degradation systems, presumably with the goal of enhancing the overall efficiency and robustness of these degradation pathways (houseley and tollervey, 2009). normal physiological degradation of endogenous rna molecules or degradation of endogenous rna molecules with defects in processing, folding or assembly are not addressed in this literature review. abundant rnase activity has been described in different tissues, including human body fluids (blank et al., 1981) (weickmann and glitz, 1982) and with differential catalytic properties (leimoni et al., 2003) . different tissues contribute to body fluid rnases (neuwelt et al., 1978) , supporting the cellular defence system against, for instance, viruses (barrangou et al, 2007 )(gupta et al., 2013 . the mammalian ribonuclease a family seems to contribute to this host defence by exerting antimicrobial activity (harder and schroder, 2002; dyer and rosenberg, 2006) . rnase 7, for instance, is a rnase a superfamily member with potent ribonuclease activity that is secreted by epithelial tissues including skin, respiratory tract, genitourinary tract, and the gut (harder and schroder, 2002) . the abundant rnase a produced in humans very probably functions to reduce rna contamination, whether endogenous or exogenous, preventing entry into unwanted rna-processing pathways (houseley and tollervey, 2009). as above described, unmodified rnas (naked) are generally unstable in biological systems, due to the large amount of ubiquitously expressed nucleases. thus, several chemical modifications have been tested for exogenous rna developed for in vivo therapies to increase resistance to nucleases, enhance binding affinity, facilitate cellular uptake, improve the pharmacokinetic and pharmacodynamics profiles, and reduce immunological response or toxicity (wan and seth, 2016; bennett et al., 2017) . there are several sites of an exogenous ncrna molecule susceptible to chemical modification ( figure 8 ) without interfering with its ability for base-paring or enhancing its drug-like properties. the several sites on rnas that can be modified include the base, the sugars, and the backbone. this allows them to conjugate with a wide variety of molecules. different chemical modifications have been tested in the context of developing rna-based therapeutics aimed at increasing the pharmacological properties of exogenous rnas and allowing their successful use in different pathologies/targeted therapies. the following section briefly reviews the types of rna modifications included in different studies in the available literature. nucleobase modifications can affect base-paring properties, binding affinity or specificity for the target mrna, as well as exert adverse effects due to competition with natural nucleotide pools or interfere with polymerases (wan and seth, 2016). replacement of adenosine in the sirna guide strand with adenosine analogues has been found to modify immunomodulatory activity. watson-crick face-localized n-ethylpiperidine triazole modification has been found to be less reactive than the hoogsteen edge modification, and the 5' end was more effective at blocking cytokine production than those placed at the 3' end (valenzuela et al., 2015) . c-5 substitution in pyrimidines in the rna guide strand increase thermal stability of sirna duplexes. indeed, smaller 5-methyl group substitutions resulted in better sirna activity (terrazas and kool, 2009). major groove modifications have also been found to increase the serum stability of sirnas (terrazas and kool, 2009). 7-position purine modifications, where the major groove edge (i.e. the hoogsteen face) is modified, have also been tested. thiazole modifications of guide strand sirna at position 15 is well tolerated in sirnas, but 7-ethynyl at this location reduces efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. potency (ibarra-soza et al., 2012) . several other nucleobase modifications have been evaluated to modify thermal stability of the sirna duplex, hydrogen bonding and sterics, or off-target effects (peacock et al., 2011) . although not exactly a base modification, movement of the nucleobase from c-1 to another position on the ribose (i.e. isonucleosides) in sirnas has been evaluated (zhang, j et al., 2012) . while maintaining binding capacity to rna and stability toward nucleases, passenger strand modifications with isonucleoside at the 3' or 5' terminals can retain the silencing activity and minimize the passenger strand specific off-target effect (zhang, j et al., 2012) . chiral inversion of the natural d-forms (spiegelmers) of rna has also been tested in aptamers, leading to their clinical evaluation (ludwig et al., 2017) . increase nuclease resistance modify plasma protein binding prevent rapid renal excretion improve pharmacokinetics enhance rna-binding affinity enhance thermal stability increase nuclease resistance enhance rna-binding affinity enhance cellular uptake reduce renal filtration enhace delivery to certain tissues modulate protein binding enhance specific cellular uptake increase possibility of formulations for specific tissues/mucosal mebranes ability to traverse biological barriers using extracellular vesicles 5´to 3d irection sites susceptible for modification on an rna molecule. exogenous rnas for therapeutic use can be subjected to different chemical modifications without interference on their base-pairing ability and enhancing their drug-like properties. conjugates at the 5´-end or the 3´-end may have the same objectives. earlier studies showed that the 2'-oh of the rna ribose sugar is not required for sirna activity (chiu and rana, 2003) and 2'-modifications influence the sugar to adopt a 3'-endo sugar pucker due to the gauche effects between the o 4'-and 2'-substitutes, improving affinity properties (egli et al., 2005) . therefore, the furanose ring structure at the 2' position of rnas has been extensively modified to efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. achieve pharmacological properties including increasing nuclease resistance, stability and efficacy (egli et al., 2005) . the 2'-o-methyl (2'-ome) or 2'-o-methoxyethyl (2'-moe) modifications increase resistance to nucleases and duplex melting temperature (lamond and sproat, 1993; prakash and bhat, 2007) , and have been widely used to develop mirnas (meister et al., 2004) , sirnas (judge et al., 2006; choung et al., 2006) or aptamer-based (burmeister et al., 2005) therapies. for example, 2'-ome in sirna has been shown to selectively protect the particularly vulnerable 5'-end of the guide strand against exonucleolytic degradation in human blood serum (hoerter and walter, 2007) . moreover, 2'-ome is a naturally occurring modification of rna found in trna and other small rnas. 2'-ome has also been used to modulate rna splicing ( toxicity related to lna modifications has also been described (swayze et al., 2007) . although sugar-modified oligonucleotides or exogenous rnas, including sirnas or antimirs, are more effective rna inhibitors than their unmodified counterparts, they are still susceptible to serum in addition, the phosphorothioate linkage promotes plasma protein binding (srinivasan et al., 1995; wan and seth, 2016; sands et al., 1994) . this increases pharmacokinetic benefits by reducing rapid renal clearance (sands et al., 1994) and facilitating tissue delivery, binding to cell surface proteins and cellular uptake (miller et al., 2016; overhoff and sczakiel, 2005 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. binding affinity (tm) for its target complementary nucleic acids (≈0.4ºc/linkage) (freier and altmann, 1997) including mrnas and mirnas (krutzfeldt et al., 2007) . thus, the use of a mixture of phosphodiester and phosphorothioate bonds may be preferred over all phosphorothioate bonds for in vivo applications (ghosh et al., 1993; krutzfeldt et al., 2007) . also, the phosphorothioathe linkage confers chirality at phosphorus and might be relevant for sirna activity (jahns et al., 2015) . although initial studies have shown exogenous homologous rna uptake by cell suspension, there was very rapid degradation after uptake (shanmugam and bhargava, 1966) . duplex rnas may be more stable than single-stranded rnas, even in the absence of phosphorothioate modifications (braasch et al., 2003) . indeed, 2'-ome hairpin-designed antimir oligonucleotides do not seem to require phosphorothioate modification to exhibit in vitro activity (lennox and behlke, 2010). minimally-modified phosphodiester aso has been reported to have less (brigui et al., 2003) , similar or superior activity than its complete phosphorothioate counterparts (uhlmann et al., 2000) , but sugar modifications in phosphodiester backbones can compensate the loss of activity (monia et al., 1996) . some adverse effects related to phosphorothioate modifications, compared to their phosphodiester backbone counterparts, have been described in aso and sirnas, including nonspecific protein binding (brown et al., 1994) , the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (dsrna). alpha-tocopherol conjugated sirnas can be delivered to liver tissue (nishina et al., 2008) or the brain when administered locally (uno et al., 2011) . other lipid conjugates of rnas have been evaluated including bile acids, long-chain fatty acids, and medium-chain fatty acids (murakami et al., 2015; lorenz et al., 2004; wolfrum et al., 2007) . conjugation with specific peptides has also targeted sirnas to specific tissues (hsu and mitragotri, 2011; yamayoshi et al., 2009) . another approach includes conjugation with high-molecular-weight polyethylene glycol (peg) to overcome renal filtration. this strategy was used in rna aptamers either approved (macugen) or in advanced clinical trials for ocular disorders (zhou and rossi, 2016; drolet et al., 2016) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. rna delivery to a target tissue for appropriate and specific cellular uptake is currently a main hurdle in rna therapeutics. several synthetic non-viral methods for rnas delivery have been developed for local and systemic rna therapeutics ( multiple studies have suggested that exosomes, naturally-released extracellular vesicles, can deliver rnas (mrnas, mirnas, lncrnas) to recipient cells (zhang, l et al., 2015; . due to their ability to traverse biological barriers, the use of exosomes or exosome-mimetic nanovesicles for rna delivery is an open field for future rna therapeutics (lunavat et al., 2016; zhou et al., 2016) . indeed, preclinical studies suggest that exosome-mediated delivery of rnas is feasible in vivo (didiot et al., 2016; el-andaloussi et al., 2012) . different rnas have been found in milk exosomes from different species, including humans zhou, q et al., 2012) , but whether these vesicle-protected exogenous rnas are bioavailable is still in debate (zempleni et al., 2017) . it is important to note that most of the above described chemical modifications are not found in nature and have been developed generally to increase the drug-like properties of rnas and other nucleotides. to the best of our knowledge there is no evidence of clinical trials using unconjugated naked or unmodified exogenous rna for human use with any administration route. efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. early studies suggested that rnas were poor drug candidates due to their relatively high instability, relatively short half-life in vivo, immunomodulatory effects and the many hurdles to their cellular absorption caused by their negative charge. however, several improvements in stabilization chemistry have been identified in recent decades. indeed, a number of chemical modifications and conjugation strategies have improved their rna-binding affinity, in vivo nuclease stability and pharmacokinetic and pharmacodynamic properties. an exogenous rna can be amenable for chemical modification without interfering with its base-pairing ability. several parts of the exogenous rna molecule can be chemically modified including the nucleobase, the backbone or the ribose (sugar). rnas can also be conjugated with a variety of molecules which can exert different biological properties. in addition to these, chemical and biological approaches can be applied to deliver exogenous rna to cells or specific tissues. although many aspects are still to be fully understood in the field of chemical modification of exogenous rnas for therapeutic use, several exogenous rnas (sirnas, aptamers, mirnas, etc.) have entered advanced clinical trials for human use. adams the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. www.efsa.europa.eu/publications 77 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section 2.2.1.2 and 2.2.2.1., a total of 119 papers were reviewed on the pharmacokinetics of foreign exogenous ncrnas used/intended to be used as therapeutics (hereafter referred as exogenous ncrnas). this section focuses on the pharmacokinetics of exogenous ncrnas (naked, i.e. nonchemically modified or minimally chemically modified) other than those consumed orally from plants; these will be covered in chapter 3.3. moreover, only in vivo studies, in mammals and humans, are reviewed here. of the 119 documents included here, 15 were used for review of ncrna pharmacokinetics in disease and other conditions, which may be relevant to risk assessment considerations. although not specifically requested by the mandate, information on pharmacodynamics aspects of ncrnas was considered. pharmacodynamics is the study of a drug's effect on an organism, meaning its biological effects and other aspects of its xenobiotic action, (efficacy, potency, and toxicity) and this is considered relevant for ncrnas (see 1.3). pharmacokinetics is dedicated to determining the fate of substances administered to a living organism. it describes the trajectory of a xenobiotic (in this case exogenous ncrna) after delivery into an organism, and encompasses from the movement of administration, to its movement through the organism, to its complete elimination; in other words, absorption, distribution, metabolism and excretion. a drug's pharmacokinetics depends on an individual's physiology (patient-related factors, i.e. renal function, sex, age) or pathology (i.e. renal failure, hepatic failure), and drug chemical properties. pharmacokinetic relevant parameters include: dose, the amount of drug administered at a single point in time; dosing interval, the time between drug dose administration; c max , the peak plasma concentration of a drug post administration; t max , the time to reach c max ; elimination half-life, the time required for the drug concentration to reach half its original value; area under the curve (auc), the integral of the concentration-time curve (after a single dose or in steady state); and clearance, the volume of plasma from which a substance is completely removed per unit time. pharmacokinetics modelling is done using noncompartmental or compartmental methods; the former estimate exposure to a drug by estimating the auc of a concentration-time graph, and the latter estimate the concentration-time graph using different kinetic models. considering an organism as a homogeneous compartment (monocompartmental model) implies that a drug is distributed equally throughout the entire body (tissues and fluids). therefore, blood plasma drug concentrations are a true reflection of drug concentration in other fluids or tissues, and drug elimination is directly proportional to its concentration in the organism. not all tissues in an organism receive the same blood supply and different drugs have different characteristics (i.e. variation in drug passage through natural barriers, such as the brain blood barrier, due to a drug's biochemical properties), meaning other models are sometimes needed. the two-compartmental model assumes that there is a compartment where distribution is more rapid (central compartment) and another compartment www.efsa.europa.eu/publications 82 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (peripheral compartment) consisting of organs with a lower relative blood flow, which consequently exhibits a slower drug distribution rate. saturation of the enzymes responsible for drug metabolisation, the presence of active transmembrane transport mechanisms independent of drug plasma concentration, and many other factors not taken into account by the two-compartmental model, require the use of multi-compartmental models. very few studies have focused on the pharmacokinetic profile of exogenous rnas (naked or unmodified), with most of the knowledge on the absorption, distribution, metabolism and excretion profile of ncrnas coming from the development of antisense oligonucleotides therapeutics (dirin and winkler, 2013) . scaling dosing from preclinical animal studies to humans (i.e. based on body weight or surface area) is challenging and apparently depends on the mechanism of clearance. for example, the pharmacokinetics of a naked sirna formulated into polymer-based nanoparticles in mice, rats, monkeys and humans exhibited blood c max shortly after intravenous administration and rapid elimination across all species in correlation with the body weight (zuckerman et al., 2014) . the pharmacokinetic profile investigated during the development of exogenous rna therapeutics has shown to be influenced by the route of administration, as described below. a comprehensive summary of available information of in vivo studies and related pharmacokinetics of exogenous rnas is provided in table 21 . most studies on exogenous ncrnas have focused on iv administration. studies in mice have shown that naked unconjugated sirna (400 µg/kg) injected intravenously disappears from peripheral blood 1 min after administration, restricting the likelihood of accumulation to peripheral organs (gao et al., 2009 ). similar results were found following iv administration of naked sirna ( 123 i labelled), which distributed to the kidney and liver within the first minute, peaking in the kidney, liver and bladder within the first 5 minutes (braasch et al., 2004) . although distribution of sirna ( 125 i labelled) decreased markedly after 24h, it persisted in the kidney and liver up to 72h, and lower concentrations were observed in the lung, spleen and heart (braasch et al., 2004) . in rats, sirnas were distributed to the kidney and excreted into the urine one hour after injection (van de water et al., 2006) . exogenous mirna iv administration has also been tested in animal models and entered human clinical trials. in this case (mirna mimics), they are either chemically modified dsrnas formulated in different nanoparticles (xue et al., 2014) to preferentially target a tissue (trang et al., 2011), or incorporated into naturally circulating extracellular vesicles (bala et al., 2015) . for example, mirnas mir-34 and let-7b formulated in a neutral lipid emulsion have been iv administered to mice and have shown to exert a biological effect (kasinski et al., 2015) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. administration site for less than one day, while in a chitosan-formulated version it persisted for up to 7 days (ma et al., 2014) . peptide-modified and chemically modified sirnas applied topically to the skin have been found to exert a local biological effect, as compared to the sirna alone (hsu and mitragotri, 2011) . mirnas formulated with transfection agents or nanocomplexes have also been injected intradermally (srivastava et al., 2017) , or subcutaneously (urgard et al., 2016) , and demonstrated to exert local biological effects. intranasal administration of sirnas in rats for brain targeting has resulted in very low levels of radioactive sirnas in the brain or plasma when naked sirnas were administered compared to a formulated version (perez et al., 2012) . mirnas administered by intranasal injection using transfection reagents reached the dorsal root ganglia and the olfactory bulb (cheng et al., 2015) and, using another nanovector delivery system through intranasal administration (as drops), it reached the brain, exerting a local biological effect (zhuang et al., 2016) . due to ncrnas susceptibility to degradation, very few studies have focused on exogenous ncrna administration in the gi tract. non chemically modified naked sirna administered by oral gavage to female mice (78 µg in total), was found intact and at high levels (analyzed by northern blot and by quantitative pcr analysis) 1 and 5 hours after dosing in the stomach, small intestine and colon when formulated with chitosan nanoparticles (ballarin-gonzalez et al., 2013) . by contrast only low sirnas levels were observed in the stomach, intestine and colon of mice 1 hour after oral administration of the non formulated naked sirna. at this timepoint intact sirna was almost exclusively present in the stomach while partial degradation products were observed in the proximal and distal regions of the small intestines. further reduction of sirna levels was observed 5 hours after administration, with traces only found in the colon (ballarin-gonzalez et al., 2013) . orally administered sirnas, formulated using specific vehicles including thioketal nanoparticles (20 µg kg -1 d the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. counterparts were present in all epithelial cells (martirosyan et al., 2016) . oral administration of exogenous exosomes containing mirnas of bovine origin has been shown to attenuate arthritis in mice models. bovine milk derived extracellular vesicles were administered daily by oral gavage starting at week 5 till week 15 after birth and arthritis was induced by collagen gavage. animals receiving exosomes ameliorated the clinical condition, although no clear mechanisms for this effect have been described . enteral (large intestine) sirna administration for therapeutic gene silencing targeting the liver (via the lymphatic route) has also been described, but using chemically modified and formulated sirnas (murakami et al., 2015) . enzyme-and ph-responsive microencapsulated nanogels are also being developed for oral sirna delivery, as reported in recent in vitro tests (knipe et al., 2016) . naked sirna formulated in a water-in-oil microemulsion has been administered via the rectal mucosa to mice along 12 days (300-600 µg sirna/kg, 8 administrations) and has reached the brain where exerted a biological effect through downregulation of the prion protein expression (lehmann et al., 2014) . prion infected mice presented an improvement of some neuropathological conditions after receiving the formulated sirna following the rectal mucosa route of administration. being the eye the target for the first rna-based therapy, sirna intraocular administration has been investigated. intravitreally injected naked sirna (slightly modified, dtdt) distributed throughout the eye (vitreous, iris, retina, retinal pigment epithelium and sclera) of rabbits when administered at 2 mg/eye, and the pattern of ocular distribution was similar in male and female rabbits ( . conjugated or formulated sirnas have also been tested in animal models (zhang et al., 2014; janout et al., 2014) . intravitreal injection of mirnas has been described in the literature using either naked mirnas in mice , rats (qin et al., 2016) or using transfection reagent in rats (mcarthur et al., 2011) or exosomes in rats (mead and tomarev, 2017) . some studies have evaluated ip administration of naked sirnas (slightly modified, dtdt) in animal models (wilcox et al., 2006; shimizu et al., 2010) . the half-life of ip administered naked sirna (dtdt) was found to be ≈11 times lower than the formulated version (perepelyuk et al., 2016) . sirna (dtdt) was also quickly excreted into the urine compared to a formulated version (shimizu et al., 2010) . in a comparison of a formulated (complexed) and naked sirna after ip administration in mice it was found that after 30 min the formulated version was detectable in muscle, kidney, liver and tumour tissue, while the naked sirna was completely absent (urban-klein et al., 2005) . while not a common administration option, chemically modified sirnas formulated in ph-responsive nanoparticle complexes have been administered in the submandibular gland of mice (by retroductal injection) and found to exert rnai effects (arany et al., 2013) . the monitoring of the distribution of the chemically modified sirna 3 h post-administration indicated that the formulated sirna was internalized www.efsa.europa.eu/publications 85 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in the cytoplasm of duct cells via the endosomal pathway. at 6 h post-administration, the signal was concentrated in the acinar compartment as well. after 24 h, ≈40% of the submandibular gland cells were positive for the sirna. the delivery of the sirna formulated in nanoparticles was more efficient compared to that of naked sirna (arany et al., 2013) . in another study, iv administration of chemically modified sirna (2.5 mg/kg) was shown to accumulate remarkably in the submandibular gland just 30 min after injection, and had a half-life in this tissue of ≈22.7 h, but did not have a rnai biological effect . in contrast, direct local injection of liposome-encapsulated sirna (0.3 mg/kg) into the submandibular gland was shown to have strong gene-silencing effects, while naked sirna injection (2 mg/kg) did not . few studies have focused on this administration route. senn and colleagues compared central nervous system administration of sirna vs. aso (senn et al., 2005) . compared to phosphothioate and 2'methoxyethyl protected aso (up to 50 µg), naked (slightly-modified, dtdt) sirna (50 µg) administered to rats via icv was not found in any brain region, including the area around the injection site. in contrast, 3 h after aso injection it was distributed in the thalamus and hypothalamus. the naked sirna clearly exhibited very poor distribution and uptake in the brain compared to the aso. although aso exhibited a capacity to reach different brain structures, its administration at a total dose of 150 µg over two days produced no rnai effect. in the same context, icv administration of sirna (up to 100 µg/rat/day) on two consecutive days did not exert rnai effects. however, in another study, a sirna chemically modified to allow for delivery in the absence of transfection reagents (accell sirna) was administered via the icv route at 5 µg/rat. the sirna was incorporated into different types of neurons, although not glia, where it exerted a biological effect in regions like the cortex, the caudate subregion of the striatum and in the pyramidal cells of the hippocampal ca1 subregion (nakajima et al., 2012) . in a different study, a mirnas mimic (chemically modified double-stranded rnas) administered by icv (3 pmol/g body weight, mixed with cationic lipid dotap) was found to increase forty fold the level of this mirna in the brain (stary et al., 2015) . several chemically modified mirnas (agomirs) were also found to be active when administered via this route in rats (huang et al., 2015; ge et al., 2015) . a double-stranded modified pre-mirnas was also reported as active in rats (davis et al., 2011), but a naked mirna (double-stranded 22-bp rna) injected by icv (100 pmol) in this study was unable to increase its levels or exert a biological effect, suggesting that the naked mirna had a higher susceptibility to rnase degradation (davis et al., 2011) . lipid-complexed naked sirna is reported to be absorbed by mouse epithelial and lamina propria cells of the vagina and ectocervix (palliser et al., 2006) . however, uncomplexed and unmodified sirnas were immediately degraded (<15 min) when exposed to genital wash fluid (wu et al., 2009) , suggesting high rnase activity in the cervicovaginal secretion. also, slightly modified but still naked sirna (dtdt) was unable to exert a clear biological effect (zhang et al., 2006) . in summary, different administration routes have been tested during ncrna therapeutics development. the administration route used for exogenous ncrna delivery clearly influences pharmacokinetic profile of chemically synthesized ncrna. no matter the administration route, it seems that naked and unformulated rnas rapidly degrade and exert very limited or no biological activity. very few studies have evaluated the distribution profile of administered naked oligonucleotides in organs and tissues or compared it to the distribution profile of their chemically modified counterparts. using the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. imaging techniques in baboons (positron emission tomography -pet -and [ 18 f]-labelling) it was shown that naked oligonucleotides are poor drug candidates due to limited distribution into organs and tissues and very rapid elimination (tavitian et al., 1998) . compared to 2'-o-methyl or phosphorothioatemodified oligonucleotides, naked oligonucleotides were found in the heart, liver and kidney and, to a lower extent, in other tissues 5 min after iv injection. at that timepoint, naked oligonucleotides were already found in the bladder suggesting that naked oligonucleotides are very rapidly eliminated compared to 2'-o-methyl or phosphorothioate ones. moreover, one hour after injection, almost all naked oligonucleotides were found in the bladder or residually in the kidney, while the phosphorothioate oligonucleotides were found largely in the liver and kidney, and the 2'-o-methyl ones were in an intermediate distribution (tavitian et al., 1998) . kinetics studies with radiolabelled oligonucleotides also showed a dramatic reduction of naked oligonucleotides in the plasma within the first 10 min, while 2'-o-methyl and phosphorothioate-modified ones showed a much slower reduction during the first 60 min. naked sirna have been reported to distribute to the kidneys and bladder of mice 5 min after iv administration (braasch et al., 2004; naeye et al., 2013) . intravenous administration of 50 mg kg -1 partially-modified unconjugated sirna (partial phosphorothioate backbone and 2'-o-methyl sugar modification) or cholesterol-modified sirna in mice resulted in a broader tissue distribution (liver, heart, kidney, adipose and lung) of the cholesterol-conjugated sirna 24 h after injection (soutschek et al., 2004) . in another study, naked sirna (free sirna) iv administered (retro-orbitally) was found in the kidney 30 min after injection (naeye et al., 2013) . ip administration of synthetic sirna formulated with low molecular weight polyethylenimine resulted in its presence in muscle, liver, kidney and tumour tissues 30 min after injection, while the naked counterpart was completely absent (urban-klein et al., 2005) . in rats, chemically modified naked sirnas were distributed in a similar manner to those of 2'-o-methyl or 2'-f modified sirnas (viel et al., 2008) , even though the circulation time of the 2'-f was increased. in other studies with rats, iv injection of sirna (chelated to indium-111) resulted mainly in renal distribution 1 hour after exposure (van de water et al., 2006) . some liver distribution of sirnas have been also reported in mice, when using either the two phosphodiester rna strands duplexes or one phosphodiester strand and one phosphorotioate strand sirna duplex (braasch et al., 2004) . orallyadministered naked sirnas (78 µg sirna) were found to be present in the stomach, proximal and distal small intestine and colon tissues 1 hour after administration but at very low levels, further lowering 5 hours after administration (ballarin-gonzalez et al., 2013) . the same sirna formulated with chitosan nanoparticles was found to distribute systemically and reach the liver, spleen and kidney (ballarin-gonzalez et al., 2013) . in an experimental model of arthritis in mice, iv administered naked sirna (150 µg) was found to distribute to arthritic joints immediately after injection, but disappeared within 12 hours, compared to that of specific formulations (komano et al., 2012) . overall, these finding suggest that naked sirna, although distributing at very low levels and with short residence times compared to other formulated counterpart versions, does distribute to the gi tract when administered orally or other tissues when administered systemically. www.efsa.europa.eu/publications 87 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. formulation type (i.e. cationic lipids, neutral lipids, or others) can drive preferential distribution to specific tissues. as described for certain mirnas, a neutral lipid emulsions (nle) formulation has been shown to distribute to the lung after iv injection (20 µg) (trang et al., 2011) . the lack of cationic lipids in the nle seems to bypass some of the shortcomings that can be attributed to the charge, including formation of aggregates in biofluids, filtration by the liver, adherence to the endothelium or uptake by scavenging macrophages. the result is excellent distribution to lung tissues (trang et al., 2011) . extracellular vesicles (exosomes)-formulated synthetic mir-155 was found to distribute to liver, adipose, lung, muscle and kidney tissues 10 min after iv injection (bala et al., 2015) . administration of exosomeloaded mir-155 resulted in its detection by isolated hepatocytes and liver mononuclear cells, suggesting cellular uptake (bala et al., 2015) . in summary, naked oligonucleotides, including ncrnas, rapidly distribute to the liver, lung and kidney after iv injection. within the first minutes they are found in the bladder, suggesting rapid renal elimination. chemical modifications or formulations increase their distribution to other tissues or delay clearance. exosome-loaded rnas have recently been shown to be an alternative tool for distribution to different tissues. ncrnas by oral administration seem to distribute to the gi tract or, if formulated, systemically. there are few studies which have evaluated the metabolism of exogenous rnas administered to animals or humans, most of which only investigate their degradation. naked sirnas seem to be rapidly degraded when administered iv (viel et al., 2008) , decreasing by ≈50% within the first 3 min, although certain chemical modifications (i.e. 2'f, 2'ome) can slightly increase this time. stability of naked sirna was reduced (<5 min) compared to that of other chemically modified or formulated sirna when administered by iv injection in mice (gao et al., 2009) . indeed, incubation of naked sirna with plasma or foetal bovine serum causes a very rapidly degradation of the genetic material (jiang et al., 2012; braasch et al., 2004; urban-klein et al., 2005) . unmodified aptamers in general are also very rapidly degraded (≈10 min) (zhou and rossi, 2016) . when naked sirna is administered as formulated (i.e. nanoparticles) it was shown that the renal filtration barrier can separate the sirna from its carrier (naeye et al., 2013; zuckerman et al., 2012) . in the eye, sirnas are degraded by endonucleases without preferences for one side of the duplex (as observed also for chemically modified sirnas) (beverly et al., 2006) . early in vitro studies also showed that exogenous rnas taken up by cells are rapidly degraded (shanmugam and bhargava, 1966) , and exogenous rna injected into the blood is rapidly degraded to nucleosides, ribose phosphate and free bases (sved, 1965). the renal filtration barrier owns an effective size cut-off of ≈40 å (batsford et al., 1987; bohrer et al., 1978) , but is also influenced by net molecular charge discrimination (brenner et al., 1978) . this is supposed to facilitate rapid renal clearance of small molecules and free sirna (zuckerman et al., 2012; naeye et al., 2013) since aso, sirnas and aptamers are commonly smaller than this cut-off (huang et al., 2010) . by comparing naked sirnas and ribose-modified sirnas (2'-o-methyl or 2'f) using pet scans in rats and mice, the kidney was identified as the main organ for sirna elimination (viel et al., 2008) . however, it is worth noting that also the liver participates in sirna elimination. in other studies, sirna appeared to accumulate in the bladder shortly after administration (hatanaka et al., 2010) . this was also the case for modified sirnas (lna) compared to their versions conjugated with peg administered in mice, in which their levels peaked in the bladder 5 min post injection and they were found in the urine as soon as 1 min after injection (iversen et al., 2013) . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. some studies have described rapid elimination of naked sirna and renal excretion after iv injection at doses including 50 µg (jiang et al., 2012) and 400 µg/kg (gao et al., 2009) in mice or rats (van de water et al., 2006) . other studies using dynamic imaging techniques or radiolabelled sirnas have confirmed that most of the naked sirna (free sirna) administered to mice was found in the bladder after just 1 min up to 10 min, faster than the formulated counterparts (naeye et al., 2013; zuckerman et al., 2012) . the literature mostly addresses chemically modified rnas (dyke et al., 2006) in clinical trials (zhou and rossi, 2016) . the small size of rna aptamers renders them susceptible to rapid clearance by renal filtration which has driven efforts to increase their size by conjugation for therapeutic use (healy et al., 2004; haruta et al., 2017) . overall this indicates that naked exogenous small rnas are rapidly cleared from systemic circulation and excreted in the urine. iv injection of a cocktail of four different plant-based small rnas (mir2911, mir168a, mir156a, and mir161; 5 pmol each) showed them to be rapidly cleared from circulation (<5 min) . the exception was mir2911, which peaked 5 min after injection compared to the other mirnas administered at equal dosages, and then dramatically decreased at 30 min. when compared to other small rnas, anomalous or atypical stability and genesis were proposed for this specific mirna. efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. although scarcely described, there may be several conditions in which, theoretically, the pharmacokinetic properties of exogenous rnas could be modified. increased intestinal permeability has been described in different conditions including consumption of nonsteroidal anti-inflammatory drugs, celiac disease, crohn's disease or other intestinal inflammatory disease (oman et al., 1996; sundqvist et al., 1980; michielan and d'inca, 2015) . for instance, absorption may be altered in cases where vasculature permeability of the gut is increased, as it has been observed in murine models of ageassociated inflammation (jeong et al., 2017) . increased intestinal permeability ("leaky gut") has been described in alcoholism, contributing to the extra-intestinal tissue damage caused by alcohol (bjarnason et al., 1984) , including liver damage (keshavarzian et al., 1999) . similarly, leaky gut has been linked to liver steatosis in obese patients ( theoretically, leaky gut may increase the possibilities of passively absorbing larger amounts of exogenous rnas with consequent release into the circulatory system. however, very few studies are available in which sirnas, aptamers, mirnas or other exogenous ncrnas have been tested under these conditions. absorption of naked sirna formulated into nanoparticles following intrarectal administration was tested in mice models of dextran sulphate sodium (dss) induced colonic inflammation. uptake of sirna (observed mainly in epithelial cells, lamina propria lymphocytes, and cells from the mesenteric lymph nodes including dendritic cells and t cells) was increased in mice suffering from acute colitis compared to uptake (in the same cell types) under healthy conditions (frede et al., 2016) , suggesting that inflammatory conditions may alter ncrna absorption. although there are no studies using naked unmodified exogenous ncrnas, this topic deserves further attention. acute kidney injury or chronic kidney disease are important public health issues. the kidneys play a key role in elimination of xenobiotics and metabolism products and thus renal clearance should be considered in drug discovery and drug interaction research (feng et al., 2010) . very few studies have focused on the clearance of exogenous ncrnas in pathological renal conditions. for example, plasma clearance of chemically modified sirna administered by iv injection (10 mg/kg) was slightly reduced in renally impaired rats than in normal animals (thompson et al., 2012) . although sirna concentration in residual kidney tissue of partially nephrectomised animals was slightly greater than the mean in normal animals, the differences did not result in appreciably greater sirna distribution into other organs (thompson et al., 2012) . in general, similar results, but using asos, were observed in rats after cisplatin-induced renal injury (masarjian et al., 2004) . also in a study of an aso against mirnas, it was recently reported that in end stage renal disease patients, only relatively modest increases in aso plasma levels were observed when compared to control subjects (http://regulusrx.com/wpcontent/uploads/2016/11/2016-aasld-rg-101-pk-and-safety-in-esrd-vs-normal.pdf). also using asos, distribution within the kidney was found to be altered in a mice model with chronic kidney disease (ckd) compared to controls (donner et al., 2018) . moreover, concentration of 2`-moe asos in the liver of mice with ckd was elevated as compared to mice without ckd, suggesting that a reduction of renal function and aso excretion can potentially influence the systemic delivery of asos (donner et al., 2018). www.efsa.europa.eu/publications 94 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. liver disease may also lead to alterations in drug pharmacokinetics, and thus the potential effects of hepatic impairment are considered in drug development (allen et al., 2012) . although exogenous ncrnas have not been analysed quantitatively, one study showed that chemically modified and formulated sirnas in lipid nanoparticles were found to be distributed and similarly accumulated in pathological or normal liver tissue from mice (yu et al., 2012) . other physiological, pathological or lifestyle factors, such as aging (cohen, 1986) and exercise activity (van baak, 1990) , may influence the pharmacokinetics of exogenous molecules. however, no information is yet available regarding exogenous rnas and their behaviour under these conditions. pharmacodynamics refers to the relationship between the concentration of a drug at the site of action and the resulting effect, including the time course and intensity of therapeutic and adverse effects. there is scarce data on the pharmacodynamics of naked unmodified exogenous ncrnas. some information is available on chemically modified rnas (or nuclease-stabilized rnas, the latter being outside the scope of this literature review). for example, in a mouse model of viral hepatitis the administration (hydrodynamic injection) of naked or chemically modified (methyl-modified) sirnas was associated with clear dose-related effects of various rnai constructs (peng et al., 2005) . in this study the naked sirnas had the highest inhibitory effects, but these rapidly declined (shorter-lasting inhibitory effects); in contrast, the methyl-modified sirna duplexes were more stable inside cells and exerted their effect over a longer time period (peng et al., 2005) . non-compartmental analysis of rna therapeutics is used in most preclinical studies when analysing pharmacokinetic and pharmacodynamic parameters. for example, a non-compartmental model was used to estimate the properties of intravitreal injection of slightly chemically modified sirna (dtdt) (dejneka et al., 2008) . also, in a mirna mimic therapy by iv administration of a liposomal formulation, a non-compartmental model was used to evaluate different parameters (beg et al., 2016) . systemic delivery of naked sirna has generally failed to produce significant biological effects (gene silencing). no biological effects have been reported when naked sirna (1 µg/eye) was administered by intravitreous injection in rats . few studies report gene silencing effects following local administration, although some of the tested rnas were slightly chemically modified (i.e. dtdt overhangs). intrathracheal administration of naked (but slightly modified, dtdt) sirna was effective through gene silencing effects in a mouse model of haemorrhagic shock and sepsis (perl et al., 2005) . in non-human primates, naked (but slightly modified, dtdt) and unformulated sirna administered intranasally was effective in severe acute respiratory syndrome sars coronavirus infection reducing viral load and host symptoms (li et al., 2005) . it looks that naked ncrnas require proper formulation to attain efficacy. naked sirna administered intranasally to mice was effective in reducing viral replication when complexed with lipofectamine (fulton et al., 2009) . naked sirna formulated in ph-triggered nanoparticles exerted gene silencing effects when administered by iv injection in mice (3 mg/kg) (kolli et al., 2013) . a formulation of naked sirnas using cyclodextrin-containing polycations and transferrin, as a targeting ligand for delivery to transferrin receptor-expressing tumor cells, also showed gene silencing effects when administered by iv injection (2.5 mg/kg) in a mouse model (hu-lieskovan et al., 2005) . when administered orally efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (20 µg/kg) to a mice model of thioglycollate-elicited inflammation, a formulated sirna reached macrophages in the peritoneum, spleen, liver and lung, exerting a sirna effect (aouadi et al., 2009) . a liposome-formulated naked mirna iv administered twice a week was efficacious in clinical trials (beg et al., 2016) . a cationic liposome formulated dicer-substrate sirna (dsirna) (a solid dsirna core in an envelope of cationic lipids and cholesterol) was found to be efficacious in mice when administered by iv injection (3 mg/kg) (ganesh et al., 2016) . preliminary results within this formulation demonstrated a tolerability profile in non-human primates upon repeated administration (ganesh et al., 2016) . when comparing the naked sirna and to a highly formulated sirna in cyclodextrin-containing polycations with surface peg and transferrin as the targeting ligand, only the formulated version (2.5 mg/kg iv) was effective in reducing the expression of the ews-fli1 gene and tumour engraftment, while the naked version exerted no biological effect (hu-lieskovan et al., 2005) . similarly, in a mouse model of concanavalin a-induced hepatitis a reduction of hepatic injury by liver-specific induction of rna interference was observed upon iv administration a of galactose-conjugated liposome nanoparticles (gal-liponp) bearing naked sirna, while no effects were noted following administration of the unformulated naked sirna (jiang et al., 2012) . in a collagen-induced arthritis mouse model and using systemic administration, naked sirna (dtdt) was used against tnf-α, and was completely unable to repress tnf-α mrna expression, whereas a formulated version repressed it (komano et al., 2012) . in a mouse tumour model, intraperitoneal injection of naked sirna exerted no rnai effects, while its complex-formulated version did (urban-klein et al., 2005) . while developing a model to study sirna pharmacodynamics, intramuscular administration (10 µg) of naked sirna (chemically stable) in mice showed no effects without use of electroporation (stevenson et al., 2013) . there are several ongoing clinical trials using sirnas, rna aptamers, mirnas and other noncoding rnas intended for use in humans to treat diseases including cancer, cardiovascular disease, inflammation, ocular disease and others (zuckerman and davis, 2015; li and rana, 2014; bobbin and rossi, 2016; zhou and rossi, 2016; sullenger and nair, 2016) . most of these ncrnas are chemically modified or formulated. adverse effects were identified in some of these studies. the delivery component of the formulation (i.e. liposomes), rather than the naked sirna component, was primarily responsible for the adverse effects observed in different preclinical toxicity studies (zuckerman et al., 2014) . for the mir-34a mimic, adverse effects in humans have been reported including acute renal injury, back pain and fatigue, all likely attributed to the liposomal carrier rather than the mirna (beg et al., 2016) . liposomerelated toxicities are considered due to activation of the complement and have been well characterized with the most frequent symptoms observed being flushing, rash, dyspnoea, chest pain, back pain and subjective distress (szebeni et al., 2011) . in non-human primates, repeated iv administration of escalating doses of a non chemically modified sirna led to increased il-6 and inf-gamma levels, as well as kidney and liver toxicity at the highest dose tested (27 mg/kg) (heidel et al., 2007) . in several clinical trials on rna therapeutics, dexamethasone premedication was required to reduce infusion-related adverse effects or reactions (i.e. hypersensitivity, flushing, oedema, etc.) (beg et al., 2016) . for some sirnas used in the clinic, a predosing hydration protocol has been used to protect the kidneys (zuckerman et al., 2014), thus reducing in humans the toxicities observed in animals. there are also animal studies in which no toxic effects are reported. for instance, when highly formulated naked sirnas were administered by iv route at 2.5 mg/kg, no apparent liver or kidney toxicity, immunogenicity-related toxicity or organ damage were detected in a murine model of metastatic ewing's sarcoma (hu-lieskovan et al., 2005) . no histological toxic effects were seen in liver, kidney, lung, heart and spleen in the same model following repeated treatment with the same formulation the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (twice weekly for 4 weeks) (hu-lieskovan et al., 2005) . the same results were obtained for the naked sirna administered at the same dose and for the same duration (hu-lieskovan et al., 2005) . in addition, when administered daily via oral gavage (200 µg sirna/kg) for 20 days in mice, sirna nanoparticles in galactose modified trimethyl-cysteine conjugates were reported to have no histological toxicity in major organs (brain, heart, kidney, lung, liver or spleen) (han et al., 2014) . since there are so few studies in the current literature on the pharmacokinetics of naked/unmodified rna molecules, this section included studies of other, minimally-modified, exogenous rnas to compare their pharmacokinetic and pharmacodynamic properties to that of naked rnas, when possible. the findings suggest that naked or unmodified rnas are rapidly cleared from circulation and have been reported in the kidney and urine immediately after administration (generally intravenously). in general, no major gene silencing effects have been observed for naked rnas when compared to chemically modified or formulated ones. this lack of efficacy has probably discouraged studies investigating the toxicity and safety of naked unmodified rnas. while limited studies have reported minor biological effects of certain naked rnas in animal models (very much dependent on the route of administration), the lack of studies in humans prevents scientists from understanding the kinetic profile of exogenous rnas and developing effective therapeutics. finally, scientific literature on toxicological or safety studies provides no evidence for concern about the systemic toxicity of naked exogenous small rnas under normal physiological conditions. allen the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section 2.2.2.1. and following the methodology described in the section 2.2.1.2, a total of 39 papers were selected as relevant to reviewing the topic of rnas uptake. information on naked (non chemically modified) exogenous rnas is mainly presented in this section. of these publications, 15 support the uptake of exogenous ncrnas. one study reports no intestinal absorption of rna (baintner and toth, 1986) , and three others suggest that rna is degraded before absorption (loretz et al., 2006; o'neill et al., 2011; sonoda and tatibana, 1978) . uptake of plant exogenous rnas, after oral intake is not included here, and will be reviewed in chapter 3.3; and uptake of exogenous rnas from the pharmaceutical/medicinal area is described in section 3.1.4. herein, uptake refers to passage of the molecule (in this case a ncrna) through the cellular membrane as a part of the absorption process. an important feature for the possible biological effect that exogenous rnas, including ncrnas, may have within an organism is their cellular uptake. because of their size and negative charges, exogenous rnas cannot easily passively cross cell membranes (see below). however, earlier studies suggested that exogenous rnas, either homologous (shanmugam and bhargava, 1966; galand and ledoux, 1966; sirdeshmukh and bhargava, 1983) or heterologous (i.e. from e. coli, yeast or others) (natural academy of sciences, 1960; herrera et al., 1970; galand et al., 1966) , could be taken up by mammalian cells (bensch and king, 1961) . intact, rna may be present inside the cell but is reported to degrade rapidly in some studies (herrera et al., 1970; shanmugam and bhargava, 1966) . extracellular ribonuclease activity seems to inhibit uptake (shanmugam and bhargava, 1966) or function (niu et al., 1961) of exogenous rnas. uptake of exogenous rna is apparently not due to altered membrane permeability, impaired cell viability, or ribonuclease degradation of the macromolecule during incubation experiments (herrera et al., 1970) . radioautographic evidences (radioactive assays) have shown the uptake of macromolecular rnas by normal and neoplastic mouse cells, but degradation was also shown to occur prior to or after rna uptake (schwarz and rieke, 1962) . although intracellular degradation may occur, early studies also described biological actions in response to uptake of exogenous rnas (niu et al., 1961; niu et al., 1962; 1960; esposito, 1964) , suggesting that once taken up by a cell, rna could eventually exert a biological effect. studies on other types of cellular uptake of exogenous rnas, such as transfer rnas, either homologous or heterologous (depending on donor rna and recipient cell type) have been conducted (gallagher et al., 1972; sakai and cohen, 1977; crooke et al., 1971) . however, as noted in rna therapeutics development (see section 3.1.3.1), the large number of barriers encountered by rnas from intake to cell uptake needs to be considered when evaluating possible exogenous rna bioavailability (figure 10 ). the gi tract offers a large surface area potentially relevant for ncrnas cellular uptake and following systemic release, since it contains a large variety of cell types at different maturational levels (e.g. stem cells in the crypts of lieberkühn) located only a few microns from an extensive capillary network (lozier et al., 1997) . however several barriers exist that could prevent ncrnas uptake by mammalian cells (o'neill et al., 2011) . for example, in a study conducted in neonatal pigs, no intestinal absorption of rna was observed (baintner and toth, 1986), suggesting possible rna degradation or an inability to cross the intestinal barrier. gastrointestinal barriers can be distinguished in extracellular and cellular barriers and are presented below. efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. orally ingested exogenous ncrna would have to pass different barriers in the human gi tract. for clarity, only the general barriers are shown. gi barriers to the uptake of orally ingested rnas. the first environment encountered by any orally ingested rna is the oral cavity, where lytic enzymes such as amylase or lipase are secreted in the saliva, and the ph ranges from 5.6 to 7.9. the stomach is a more acidic environment (ph 1.5-1.9) (dressman et al., 1990) , and nucleic acids are known to be denatured and depurinated (hydrolysis of nucleic acids to release purine bases) over time in acidic gastric fluid (loretz et al., 2006) . the gastroenteric fluid flow and peristaltic activity in the gi tract could reduce the contact time between ncrnas and the epithelial layer, therefore diminishing the opportunities for cellular uptake, while nuclease enzymes present in the gi lumen could degrade nucleic acids before any cellular uptake (o'neill et al., 2011) . indeed, purines and pyrimidines in nucleic acids are believed to be absorbed mainly in the form of nucleosides (sonoda and tatibana, 1978) . the gut flora, predominant in the distal ileum and in the large intestine, produces a range of enzymes that could degrade ncrnas as well. the gi tract is lined by a viscous sticky layer of mucus entrapping foreign particles before reaching the underlying epithelium (figure 11) . this "trapping" effect could be mediated by mucus composition, which confers a net negative charge to the mucous layer. since nucleic acids present a net negative charge as well, this mucus could represent an electrostatic barrier difficult to overcome by rnas, and particularly ncrna, if not properly delivered or chemically modified. furthermore, mucus turnover efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (secretion, shedding and discard) happens in a relatively short time period (50-270 min) (lehr, 1991), preventing rnas from traversing the mucous layer and to reach the underlying epithelium. in conditions such as ulcerative colitis this mucus layer can be reduced or missing in areas of acute inflammation (pullan et al., 1994) , which would allow nucleic acids to reach the epithelial cell layer. another barrier is the glycocalyx, a glycoprotein and polysaccharide layer (400-500 nm thick) associated with the apical cellular membrane of enterocytes (frey et al., 1996) . this layer prevents the access of certain viruses, bacteria and particles (such as rnas) to the underlying plasma membrane by acting as a size-selective diffusional barrier. the intestinal mucosa is structured as a three-layer barrier: a single layer of epithelial cells, the lamina propria ( furthermore, the epithelial cells (enterocytes), which represent approximately 90% of the epithelium, have a short lifetime of 5-7 days, and are continuously shed and replaced (quastler and sherman, 1959; jung et al., 2000) . the enterocyte apical membranes are characterized by a high number of microvilli, structures approximately 1 mm long and 50 nm wide (johnson, 1987) . since endocytosis occurs primarily at the base of microvilli (jung et al., 2000) , particles with a diameter greater than 50 nm could not be efficiently endocytosed (uduehi et al., 1999) . m-cells (cells dedicated to trans-cellular transport and associated to intestinal lymphoid follicles) are fewer in number than enterocytes, but, due to their role in the transport of foreign material from the lumen to the immune cells of the lamina propria, are characterized by a high endocytic activity. moreover, m-cells have microfolds in their apical membrane, and are covered by a reduced glycocalyx and mucous layer. this determines a low breakdown capacity (e.g. low alkaline phosphatase activity, and a lower number of lysosomes) so intracellular degradation of ncrnas could be spared. conversely the capillary network underlying the m-cells is less dense and less permeable (o'neill et al., 2011) . although the literature has described these cellular barriers when studying several molecules, there is little specific data (o'neill et al., 2011) on the relevance of these to exogenous rna molecules. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. assuming that ncrnas could overcome the gi barriers and reach the circulatory system, they would still encounter other biological barriers before reaching the appropriate machinery to exert any effects. significant degradation occurs of sirna duplexes (non chemically modified) in 15 min when they are incubated in foetal calf or human sera (haupenthal et al., 2006; urban-klein et al., 2005) . sirna in human plasma is rapidly degraded, with nearly 75% degraded within 2 min (layzer et al., 2004) . nuclease activity in plasma and tissues is relevant to degrading rnas, including ncrnas. the major activity in plasma is a 3' exonuclease, but cleavage of internucleotide bonds can also take place (juliano et al., 2009) . encapsulation methods, which protect rnas from degradation, include exosomes, microvesicles and apoptotic bodies, all of which are extracellular vesicles distinguished by size, biogenesis and cargos (ela et al., 2013; van niel et al., 2018) . exosomes, for instance, contain various species of rna, conferring them protection against degradation and providing them a vehicle for cellular uptake of rnas by endocytosis (cui et al., 2017). the reticuloendothelial system (res) is comprised of phagocytic cells, including circulating monocytes and tissue macrophages, whose physiological function is to clear the body of foreign pathogens, remove cellular debris generated by tissue remodelling, and clear cells that have undergone apoptosis. additionally, the res plays an important role in uptake and clearance of individual "free" oligonucleotides. phagocytic cells of the res, particularly the abundant kupffer cells in the liver and splenic macrophages, express a number of cell surface receptors, including integrins and scavenger receptors, that are potentially involved in uptake, although the role of scavenger receptors in uptake of free oligonucleotides is somewhat controversial (juliano, 2016). following internalization of an oligonucleotide, the phagosome fuses with lysosomal compartments, where the contents are subjected to enzymatic degradation by proteases and hydrolases that operate efficiently in the low-ph lysosomal environment. tissues macrophages are most abundant in the liver and spleen, and both these tissues receive high blood flow (juliano et al., 2009) . the capillary lumen is surrounded by a layer of endothelial cells interlinked by adherence junctions (vecadherin containing) and by tight junctions (occluding and claudin-containing) and tightly adherent to the underlying extracellular matrix. this structure forms a barrier between blood and the parenchymal space. molecules in the blood can be transported across the endothelial barrier by two routes. paracellular transport occurs through the junctions between cells and is limited to molecules of approximately 6 nm diameter or less; only micrornas would be able to transit this barrier by this method. caveolar-mediated transcytosis carries large proteins across the endothelium within vesicles of about 70 nm. in some tissues, such as liver and spleen, there are gaps or fenestrations of 100-200 nm diameter between the endothelial cells, which allow egress of larger molecules (juliano, 2016). endothelial permeability is also increased in sites of inflammation and in some tumours, in which increased permeability is associated with fenestrations between the endothelial cells that result from rapid and disorganized angiogenesis in the tumour. this is known as the epr effect (enhanced permeability and retention), although some argue that this is mostly due to poor lymph flow in the area the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. and many minutes to several hours for the elimination phase; (ii) oligonucleotides are accumulated in most tissues, particularly in kidney and liver, but not in the central nervous system (probably due to the blood brain barrier); (iii) the major route of elimination is via the kidneys; and (iv) although the most detailed studies have been performed in rodents, the pharmacokinetic behaviour of the modified oligonucleotides in humans is similar to that observed in lower animals (juliano et al., 2009) . the pharmacokinetics and biodistribution of sirna duplexes is similar to that of single-stranded antisense molecules, with the highest uptake in the kidney followed by the liver (juliano et al., 2009 ). sirna and uncharged oligonucleotides do not bind extensively to plasma proteins, and are thus cleared by the kidney much more readily than modified oligonucleotides and tend to accumulate at lower levels in tissues (juliano, 2016) . typically, molecules with sizes of 3-6 nm or less can be ultrafiltered by the kidney; micrornas could therefore be rapidly excreted by the renal route, unlike long ncrnas. in fact, sirna molecules intravenously injected can be visualized in renal filtrate within seconds of injection (molitoris et al., 2009) . plasma sirna concentrations are reported to decline by more than 90% within 30 min (relative to levels at 5 min post dose) and decline by more than 98% within 2h (thompson et al., 2012) . only about 1-2% of the iv administered dose is absorbed by tissues and the majority of this uptake occurs in the kidney (thompson et al., 2012) , most of it being cleared from tissues within 30 h of administration . to reach their target, ncrnas must enter mammalian cells and arrive at the appropriate subcellular location (the nucleus or the cytoplasm, depending where the target molecules are located). like other large, polar and charged biological macromolecules, ncrnas are internalized by endocytosis and then trafficked through multiple membrane-bound intracellular compartments. the ability of ncrnas to reach their targets depends on both cellular internalisation (uptake) and intracellular trafficking. (figure 12 ). there are five major classes of endocytosis: (i) the clathrin-coated pit pathway; (ii) the caveolar pathway; (iii) the noncaveolar, clathrin-independent pathways (clic pathways); (iv) phagocytosis (mainly taking place in "professional phagocytes" such as macrophages and granulocytes); and (v) macropinocytosis (in which internalized macromolecules are simply dissolved in the ambient medium) (juliano et al., 2009). non-coding rnas (ncrnas) can enter cells via different endocytic pathways, including micropinocytosis. internalized ncrnas can traffic from early endosomes (ee) to late endosomes (le) and to lysosomes. ncrnas must escape endosomal organelles to reach the cytosol and nucleus and act on the target molecules. some proteins may direct ncrna to non-productive pathways. the micropinocytosis pathway may represent a non-productive pathway for naked ncrnas. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the first four classes often involve a cell surface receptor and are collectively termed receptor-mediated endocytosis. these pathways are mainly utilized in cellular delivery of oligonucleotides. in general, receptor-mediated endocytosis includes three major steps. (i) receptor binding and internalization represents the primary barrier for oligonucleotide transport; the ligand-receptor binding determines which target cells and tissues oligonucleotides are delivered to. (ii) sequential intracellular trafficking leads oligonucleotides into a variety of low ph endomembrane compartments, including early/sorting endosomes, late endosomes/multivesicular bodies, and lysosomes. in some cases, receptors/ligands can traffic to the golgi complex. in many instances, receptor and ligand are dissociated in the low ph endosome environment. vesicular trafficking can prevent ncrnas from reaching their targets, for example, by sorting to secretory or lysosomal vesicles which may lead to export of ncrnas out of cells or degradation in the lysosomes. (iii) ncrnas must exit from the endosome to reach the site of action in the cytoplasm or nucleus. endosomal trapping represents an important barrier for ncrnas final functional objective. on the other hand, oligonucleotides are able to continuously shuttle between the nucleus and the cytoplasm mediated by nuclear pore structures, and do not require classical nuclear localization signals (juliano et al., 2009 ). the available data clearly describe several barriers to be considered when evaluating exogenous rna absorption, circulation, cellular uptake or intracellular trafficking. exogenous rna must overcome numerous biological barriers to reach its intended target within mammalian cells. considering oral administration as the most relevant for exogenous plant ncrnas, the first major limiting step to exogenous rna bioavailability is the gi tract itself. the gi tract encompasses both extracellular and cellular barriers. the extracellular barriers include the presence of enzymes in the lumen, such as amylase or nucleases, a harsh environment with ph ranging from 1.5 to 7.9, and a net negative charged mucous layer with a very rapid turnover (50-270 min). the cellular barriers include a single layer of epithelial cells, the lamina propria and the muscularis mucosa, constituting the threelayer intestinal mucosa barrier. ncrnas could cross the epithelium between cells but this is limited by the presence of tight junctions. pore size in the human intestine would prevent passage of all ncrnas other than mirnas, the smallest ncrnas. ncrnas could also cross this epithelium through transcytosis by traversing the cells; this mechanism implies exposure to intracellular nucleases, recycling of ncrnas back to the lumen and nuclear uptake. many biological barriers and environmental conditions between the gi epithelium and the target tissue are then encountered by ingested rnas. ncrnas would be exposed to nucleases both in the plasma and in the target tissues. once inside the circulatory system, these rnas would be subjected to distribution and elimination, accumulating mostly in the liver and kidney. due to their small size, mirnas are especially rapidly cleared by the kidney, unlike long ncrnas. therefore, a very small percentage of rnas could actually be absorbed by tissues. furthermore, ncrnas would need to escape the reticuloendothelial system, the function of which is to clear the organism of foreign molecules. then rnas must cross the vascular endothelial barrier to reach the target tissue. for ncrnas to exert their functions they must enter the target cells. rna cellular uptake is achieved by endocytosis after which they would enter the intracellular trafficking system through multiple membrane-bound compartments. ncrnas would need to exit these compartments to reach their functional sublocation within the cell, be it the cytosol or the nucleus, while simultaneously escape degradation in the lysosomes. efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. seeds, corn kernels and rice grains, all of which are common food and feed, and numerous endogenous plant srnas are reported to have perfect complementarity to genes in humans, and other animals . however studies in mammals and humans with sirna indicates that the exposure levels required to produce regulatory effects exceed the levels achieved following ingestion . all animal and plant-related foods and feeds contain naturally occurring coding (i.e. mrnas) and ncrnas. early (srivastava, 1965; lassek and montag, 1990 ) and more recent studies ) have estimated that the total rna content in plants is about 1 mg/g plant tissue. plants contain an array of ncrnas, including highly abundant transfer rnas (trnas) and ribosomal rnas (rrnas), single-stranded antisense rnas, and the mirnas and sirnas that trigger rnai and their precursor dsrnas . long dsrnas from non-plant, exogenous sources are particularly common in plants, including plants used for food and feed, due to infection from rna-containing viruses. animalderived foods are generally richer in rna than plant-derived foods, and are likely to contribute significantly to overall rna consumption (jonas et al., 2001) . of the 1 mg/g tissue of total rna content in plants, relative percentages (by weight) of the major rna forms are approximately 80% rrna, 3-5% mrna, and 10-15% trna, with srnas accounting for less than 5% of total rna in plants . srnas are present at levels of up to 1.61 µg/g of conventional soybean grain (average = 0.66 µg/g grain) and comparable amounts are present in the grains of conventional corn and rice . in tobacco plants engineered to overexpress a dsrna under the control of a constitutive promoter, sirnas levels in leaves reached about 1.5% of total rna (chau, b. l. and lee, k. a., 2007) . total exposure to construct-derived srnas from a putative biotech soybean product was estimated to be 45 µg/kg/day in the general population and 82 µg/kg/day for children aged six and under . using these same assumptions for rna expression levels in sweet corn, consumption of corn at the highest possible rate would result in an estimated intake of 106 µg/kg/day in the general population and 170 µg/kg/day for children aged in the absence of encapsulation or chemical stabilization to prevent degradation or without the addition of penetration enhancers, the absorption of rna -including sirnas -across the gi tract is described as negligible (akhtar, 2009; jain, 2008) . one study suggests that activity could be possible for certain highly expressed plant mirnas following food intake (zhang et al., 2012a) . this study reports that in mice fed a diet consisting entirely of cooked rice (i.e. human equivalent of about 33 kg/day of cooked rice) , several rice mirnas were detectable in mouse serum and liver. some of the results presented in this study were contested in later studies (chen et al., 2013) . petrick et al. clearly stated the unlikelihood of achieving such high concentrations of mature plant mirnas in the serum, plasma and organs of humans or animals via food intake since the high levels of rice administered the experimental animals did not reflect anticipated dietary exposure levels. in another study, it was concluded that plant mirnas identified in animal srna sequencing data can originate from artefacts of the sequencing process (zhang et al., 2012b) . even in the case of 20-mer oligonucleotides modified to increase their stability, oral bioavailability in rats was only 0.1% (nicklin et al., 1998) and intestinal absorption was less than 1% in a model that bypasses the gastric acidic environment. most of the labelled oligonucleotide was associated with the luminal epithelial cell membrane and very little efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. was localized intracellularly (khatsenko et al., 2000) . regarding the possibility of dietary sirnas and other dsrnas effects on gastrointestinal tissues, studies with radiolabelled or immunostained dna oligonucleotides demonstrate that these are primarily located extracellularly within the intestinal tract (lumen and luminal wall) (khatsenko et al., 2000; nicklin et al., 1998) , and are thus presumed to be minimally absorbed (negligible bioavailability, <2%). it has also been observed that absorption decreased as the oligonucleotide length increased (from 6 to 22 nt) (khatsenko et al., 2000) . the only published quantitative data on srnas content in plant-derived foods ) (detailed above) indicate that 0.6 µg of srna is obtained per gram of soybean, rice seeds or corn kernels. as total rna content in plant-derived foods appears to be around 1 mg/g tissue (lassek and montag, 1990), srnas could therefore represent 0.1-0.2% of plant seed materials. the amount of sirnas in plants genetically modified to carry hairpin transgenes is estimated at about 50 ng sirna/340 µg total rna/g tobacco leaf tissue (chau, bess l. and lee, kevin aw, 2007) . the resulting estimated human dietary exposure to plant small rnas from rna-based biotech crops was derived from this (table 22 ). . (b) assuming 100% of the specific food is consumed from a biotech crop, with total rna levels of ≈1 mg/g grain and 1.5% of these small rnas derived from the transgene. for details see . these doses are significantly lower than those required to elicit adverse effects in rodents or monkeys (800 or 1000 mg/kg of chemically-stabilized sirna, respectively) by iv administration (thompson et al., 2012) . moreover, high mirna concentration is required to target suppression, and levels below 100 copies/cell mirnas have little regulatory capacity (mullokandov et al., 2012) . according to snow et al., dietary plant mirnas are present at less than one copy per cell in target organs assuming reliable quantification . in another study the calculated dietary exposure in humans of a specific dsrna (dvsnf7) was estimated to be around 3.8 x 10 -5 µg/kg/day in the us population (petrick et al., 2016b) . the same study reported that 100 mg dvsnf7/kg/day in a 28-day repeated dose oral (gavage) toxicity study in mice did not produce any toxic effects (petrick et al., 2016a) . levels for this specific dsrna in this biotech crop were reported to be ≈ 0.104 x 10 -3 µg/g grain tissue on a dry weight basis for corn food, and ≈ 55.1 x 10 -3 µg/g in the whole plant on a dry weight basis . of note is that there is little information on the impact of intended rnai in gm plants on the coding and non-conding plant rna. knowledge of which other rnas are modified, or how other rnas are compensated or desregulated in the gm plant needs to be experimentally evaluated for each specific case. different studies have evaluated the amount of plant-derived foods consumed by the general population. in a study of cohort of women in the uk, the difference between the mean intake of overall fruit and vegetables consumption was three times greater in high consumers vs. low consumers (pollard et al., 2001) ; vegans and vegetarians were the highest consumers of fruit and vegetables. in an efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. evaluation of the flemish population it was observed that vegans, while having the lowest total energy intake compared to omnivores, have the highest total fruit and total vegetables consumption. the intake of fruit, nuts and/or legume about twice in vegans, using either the healthy eating index (clarys et al., 2014) or total mean intake (clarys et al., 2013) . in the epic cohort study, which includes ten european countries, mean daily consumption was between 269 and 103 g/day vegetables and between 453 and 121 g/day fruit (agudo et al., 2002) . although not homogeneous among countries and centres, the ratio between the highest and lowest consumers was around 2.5 for vegetables and 3 for fruit (agudo et al., 2002) . countries in southern europe had the highest vegetables and fruit consumption. for example, consumption of vegetables and fruit in healthy adults in spain (epic data), considered higher than most european countries and the usa, was 273 g (3.4 servings) of vegetables and 348 g (4.4 servings) of fruit (agudo et al., 1999) , equal to ≈600 g/d vegetables and fruit. in the united states (us) the total vegetable and fruit intake has increased slightly (up to ≈4.5 servings/day) in all consumer categories from the late 1990s (krebs-smith and kantor, 2001) and remained stable over the last decade (rehm et al., 2016) . actual quantities, however, are around 2.4 servings/day (rehm et al., 2016) , that is 1.12 for fruit and 1.19 for vegetables (eaton et al., 2013) . in cup-equivalents, this is 1.7 cup equivalents for vegetables and 1.0 cup equivalent for fruit (moore and thompson, 2015) . mean total vegetable intake in the us for vegetarians is reported to be around 250 g/day and that for non-vegetarians as 197 g/day. total fruit intake was around 261 g/day for vegetarians and 159 g/day for non-vegetarians (haddad and tanzman, 2003) . in other words, there is less than two-fold difference in the amount of fruit and vegetables consumed between vegetarians and non-vegetarians in the general us population. in a specific population (i.e. the adventist health study) in the us, daily mean consumption of fruits was higher (≈424 g/day) for vegans than for other types of vegetarians or non-vegetarians (≈319 g/day). the same trend was observed for daily mean consumption of fruit which was higher in vegans (≈483 g/day) than other vegetarians or non-vegetarians (≈298 g/day) (orlich et al., 2014). based on these data, dietary exposure to rnas from plants can in general be assumed to be higher in vegans and vegetarians than omnivores. although total rna content in plant-derived foods varies (lassek and montag, 1990), a person consuming an estimated daily dose of total fruit and vegetables ≈600 g/day would theoretically ingest 600 mg of total rnas assuming about 1 mg/g of plant tissue. this value agrees with the dietary rna intake, which typically ranges from 0.1-1 g/person/day (jain, 2008) . considering that srnas represent up to 0.2% of plant seed materials ), estimated daily intake in the general population would be around 1.2 mg srnas. since vegans and vegetarians normally consume higher amounts of vegetables and fruits (see above), they could be exposed to an average of three times more plant exogenous rna (3.6 mg/day). no pharmacokinetics studies were identified in these groups. changes in rnase activity can occur in physiological or pathological conditions, this possibly impacting exposure after dietary intake. for instance, increased serum rnase activity has been observed in pancreatic carcinoma, pancreatitis, and renal failure (peterson, 1979 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. et al., 2011) . human argonaute 2 (ago-2) is a catalytic core component of rnai machinery. ago-2 mediated gene silencing has been proposed to be linked to the mitogen activated proteine kinase signalling pathway (zeng et al., 2008) , which is activated in response to cellular stress. since various kinds of stress on human cells induce formation of stress granules, ago-2 can be recruited to these granules, modifying its intracellular localization and the efficacy of rnai activity (detzer et al., 2011) . ago-2 is thus regulated at both the transcriptional and post-translational levels, suggesting that ago-2 levels can influence cellular mirna activity (adams et al., 2009 ). it has also been proposed that inactivation of mitochondria could lead to a strong decrease in mirna-mediated rnai efficiency, and, to a lesser extent, to sirna-mediated rnai (huang et al., 2011) . due to the interaction of p-bodies with mitochondria, reduced ago-2 activity could be due to changes in location of endogenous ago-2 from pbodies (huang et al., 2011) . association of ago-2 with cytoplasmic rna granules is known to regulate the translational repression activity of the protein, but phosphorylation of certain residues may prevent this inhibition, allowing the protein to remain active in the cytoplasm (lopez-orozco et al., 2015) . overall, it seems clear that ago-2 activity can be modified by cellular stress conditions. however, whether this can influence the function or the exposure to dietary ncrnas remains unknown and would benefit from further research. most studies have been focused on mirnas since these ncrnas were the first to be described. these studies have consequently extended to transgenic dsrnas as these have increasingly used to induce gene silencing. as mentioned previously, little is known about dietary exposure to other ncrnas. given the role of lncrnas in plant physiology, it is important to better understand human and animal exposure to these ncrnas. further research is needed on how transgenic ncrnas affect expression levels of other ncrnas, and other rnas such as mrnas. alteration of the expression levels of certain ncrnas within the plant may modify the levels of other ncrnas due to putative compensatory circuits and codifying rnas. this in turn could lead to changes in protein and enzymatic content, with putative consequent alteration of the modified plant's nutritional value. controlling for these changes would require complete transcriptome sequencing, and comparison of rna levels in unmodified to those of the altered plant . another point to consider is whether special diets (e.g. vegetarian or vegan) modulate ncrnas uptake throughout the gi tract, possibly leading to increased exposure. humans and animals have been continuously exposed to naturally-occurring plant rnas. estimated amounts of ingested plant ncrnas contrast with the much higher doses administered to experimental animals and in clinical trials to humans. although some reports on the presence of plant mirnas in serum, plasma and organs of humans or animals do not agree, the extremely low reported concentrations prevent them from being functional, even in the case of rnas modified artificially to augment their bioavailability. humans following special diets, such as vegetarians and vegans, have high intake of plant rnas, but their increased estimated exposure to such rnas is barely on average 3fold greater than in omnivorous diets. there are no specific studies determining the effects of increased dietary exposure to plant ncrnas. adams bd, claffey kp, white ba. 2009. argonaute-2 expression is regulated by epidermal growth factor receptor and mitogen-activated protein kinase signaling and correlates with a transformed phenotype in breast cancer cells. endocrinology 150, 14-23. efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to exert a biological effect, exogenous ncrnas need to overcome the physical and biological barriers present in both the gi tract and the circulatory system, reach the target tissue, and enter the appropriate intracellular pathway at sufficient dose. this section describes the obstacles and challenges encountered by exogenous ncrnas following oral intake. following an extensive literature search, and based on expert judgement in the topic, 21 publications were reviewed for this section. rna molecules are large, hydrophilic and negatively charged. even the smallest rna molecules (mirnas) have a high molecular weight (∼13 kd) and their net charge is negative (akhtar and benter, 2007a; akhtar and benter, 2007b) . due to these physicochemical properties, rna molecules are highly water soluble with low membrane permeability, thus falling into class iii of the biopharmaceutical classification system (fda, 2000; amidon et al., 1995) . like many other class iii compounds, they are not absorbed at all or only to a very small degree. being highly charged species, rna molecules resist partitioning across lipid bilayers, and higher molecular weights effectively restrict their movement by function of the tight junctions present in the gi tract epithelium (tillman et al., 2008) . in vitro and in vivo preclinical studies have assessed the use of medium chain fatty acids (6-12 carbon atoms), as well as bile salts, as absorption enhancers to cross the intestinal mucosa (gonzalez ferreiro et al., 2002; tillman et al., 2008; raoof et al., 2002) . orally introduced rna molecules encounter relevant obstacles in the gi tract, these precluding their absorption and activity. in general oligonucleotides are rapidly degraded in the harsh biological milieu of the acidic stomach and enzyme-rich gi tract, as described previously, and show poor transcytosis across the gut (akhtar, 2009) . in humans the ph in the gi lumen can vary from 1 in the stomach to 8 in the intestine (gamboa and leong, 2013) . exposure to these ph values can cause ph-induced oxidation or de-amination of rna, resulting in loss of activity (pridgen et al., 2015) . in addition, nucleases present in the gi lumen for digestion of biological molecules enzymatically degrade rna molecules too (gamboa and leong, 2013; kriegel et al., 2013) . the presence of a rich bacterial population, mostly in the intestines (the human microbiome project consortium, 2012) may also contribute to degradation of these molecules, as bacterial degradation of ribonucleic acids through the secretion of extracellular nucleases has been early described (nishimura, 1960; eaves and jeffries, 1962) . finally, if the above-mentioned obstacles are overcome, rna molecules still face the intestinal extrinsic and intrinsic barriers (see also 3.1.5). the extrinsic barrier consists of the mucus layer covering the epithelial cells. this is a complex hydrogel material composed of proteins, carbohydrates, lipids, salts, antibodies, bacteria, and cellular debris (o'neill et al., 2011; pridgen et al., 2015) . it consists of loosely and firmly adherent layers that vary in thickness along the gi tract and can fluctuate based on diet. the mucus is secreted by cells presenting a rapid cell turnover, approximately 2-5 days in the small intestine in humans (gamboa and leong, 2013; o'neill et al., 2011; atuma et al., 2001) . among other functions, the mucus layer protects www.efsa.europa.eu/publications 118 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. epithelial surfaces by trapping pathogens and foreign particulates, and rapidly clearing them. penetration of this mucus barrier is necessary to reach the absorptive epithelial cells. several mucuspenetrating and mucoadhesive materials can be used to enable penetration of molecule to the mucus layers, increasing residence time and contact of the delivered molecules to the epithelium (pridgen et al., 2015) . the intrinsic barrier consists of the epithelial cell monolayer, constituting the greatest barrier to material from the intestinal lumen to the lamina propria and bloodstream. cells maintain this barrier by forming tight junctions, whose permeability can be modulated through specific combinations of different proteins (o'neill et al., 2011) . there are several possible pathways across the intrinsic (epithelial) barrier (figure 13 ).  the transcellular pathway passes through the apical and basolateral cell membranes, as well as the cytoplasm. this pathway is very restrictive to the passive flow of hydrophilic solutes such as rna molecules, because of the lipid bilayer membrane and its impermeability to large molecules. transport mechanisms for this pathway can be passive for hydrophobic molecules, or active with membrane pumps for specific molecules such as ions.  the paracellular pathway is the major passive permeation pathway and allows diffusion of small molecules in the space between epithelial cells. the tight junctions regulate permeability of this pathway based on molecules' size and charge.  finally, transcytosis is an active transport pathway that relies on molecule-specific receptors guiding the molecule through the cell without entering degradation pathways. because of their large hydrodynamic size, macromolecules such as rnas are restricted to this pathway (pridgen et al., 2015) . the m cell transcytosis pathway is the most extensively studied for oral delivery of rnas. this pathway, which is used to transport antigens across the epithelium for immune surveillance, is attractive because m cells lack mucus secretion and have a sparse glycocalyx, among other features. however m cells are closely associated with immune cells in the lamina propria and peyer's patches, such as dendritic cells and macrophages, this from one side limiting the ability of the delivered molecule to reach the bloodstream and on the other side increasing the risk of triggering an immune response (florence, 2004; kriegel et al., 2013; pridgen et al., 2015) . moreover, m cells only make up a small percentage (5-10%) of the non-absorptive epithelium in humans. the surface properties of m cells, as well as the number of peyer's patches vary among species, which could make difficult extrapolating animal model data to humans (pridgen et al., 2015) . an additional obstacle is represented by the immune system, which is intimately associated with the epithelium. numerous types of immune cells patrol the lamina propria, including t cells, macrophages and dendritic cells. if the ingested rna molecules reach the bloodstream, they must also evade the mononuclear phagocytic system (reischl and zimmer, 2009; pridgen et al., 2015) . the presence of endogenous and luminal nucleases, together with gi ph conditions, in general determine rapid degradation and low rna molecules' biological half-life (bhavsar and amiji, 2007) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the paracellular pathway allows diffusion of molecules in the space between epithelial cells and is regulated by intercellular tight junctions. the transcellular pathway passes through the apical and basolateral cell membranes and cytoplasm. it is restricted to hydrophobic molecules or molecules transported by membrane pumps. the transcytosis pathway is an active transport system that relies on molecule-specific receptors guiding the molecule through the cell escaping degradation pathways. transcytosis pathways are found in both epithelial and m cells. from pridgen et al, 2015. transepithelial transport: paracellular, transcellular and transcytosis pathways. first-pass elimination occurs when a compound is metabolised between its site of administration and the site of sampling for drug concentration measurement. this greatly affects the bioavailability of orally administered compounds since the concentration of the active substance reaching systemic circulation is decreased. the liver is usually assumed to be the major site of first-pass metabolism of an orally administered drug, but other sites are the gi tract, blood, vascular endothelium and lungs (pond and tozer, 1984) . in fact, the obstacles previously described for rna molecule absorption in the gi tract can be considered a first-pass effect. rna molecules reaching the circulatory system must avoid filtration by the kidneys, accumulation in non-target reticuloendothelial system (res) of the liver, kidney, lungs and spleen, degradation by endogenous nucleases, aggregation with proteins in the serum, and uptake by non-targeted cells such as phagocytes (kriegel et al., 2013; reischl and zimmer, 2009 ). once cellular uptake has taken place in the potential target tissue (surmounting the obstacle of crossing the vascular walls and the lipid bilayer cellular membranes), nucleic acids have to undergo efficient intracellular trafficking to ultimately produce the intended pharmacological effects. this includes endosomal transportation (escaping endolysosomal degradation), cytoplasm release, and efficient incorporation into the relevant intracellular pathways such as risc loading (for sirna, mrna, and mirna) or nuclear uptake (for lncrna) kriegel et al., 2013; pouton and seymour, 2001) . www.efsa.europa.eu/publications 120 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. rna molecules are large, hydrophilic and negatively-charged molecules. to achieve effect via the oral route, these molecules need to overcome the physical and biological barriers present in both the gi tract, where they encounter variable and harsh conditions along with degrading nucleases, and the circulatory system. rna molecules must reach the target tissue, escaping endo-lysosomal degradation, and enter the appropriate intracellular pathway in sufficient doses to exert their effects. www.efsa.europa.eu/publications 121 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a literature search as described in section 2.2.2.2. and based on the methodology described in section 2.2.1.2, a total of 110 documents were selected as relevant to a general review of rna-based therapeutics administered by gi route. of these documents, at least 24 report the use of specific vehicles for gi administration of exogenous ncrnas (i.e. nanoparticles). six of these documents simultaneously evaluated formulated exogenous rnas and their naked version, and reported either rapid degradation under gi conditions, or reduced or absence of biological effect of the naked version compared to the formulated one (table 23) . twenty (20) of these documents provide specific examples of local effects of rna-based drugs administered orally. in addition, 18 papers were selected as relevant to inform on administration of exogenous ncrnas in either fish or birds and possibly exerting biological effects. orally delivered rna has been used as a dietary supplement or empirical therapeutic agent since 1960s (jain, 2008). for example, yeast rna supplementation as a source of dietary ribonucleic acids has been used in different animals (choudhury et al., 2005; jha et al., 2007) including mammals (sukumar et al., 1999; heaf and davies, 1976) and in human studies (gianotti et al., 2002; tepaske et al., 2001) . in human studies, up to 4 g per day (in divided doses) of exogenous rnas have been administered (for 8-12 days), producing an increase in plasmatic uric acid levels (zollner and grobner, 1971; zollner, 1982) . the absence of attempts to study the gi absorption of pure rna in these studies suggests that its effects, if any, could be related to rna molecules as a source of nucleotides. indeed, nucleotide supplementation in rats has been shown to affect the healing of ulcerative conditions, ameliorating indomethacin-induced ileitis and aggravating the severity of dextran sulfate sodium (dss)-induced colitis (sukumar et al., 1997 , sukumar et al., 1999 . also, a study of body fluid composition of rats orally administered yeast rna, mixtures of its constituent nucleosides or its constituent bases and ribose was performed (heaf and davies, 1976) . the authors showed that ingestion of rna increased intestinal levels of ribose, inorganic phosphate, uridine, pseudouridine, uracil, inosine or uric acid. the effect of orally administered mixed nucleosides on blood and urine composition was similar to that of rna, while some differences were noted for some equivalent mixture of free bases (heaf and davies, 1976) . this study showed that the dietary rna-phosphate passed to the urine from the gut, suggesting that most of the rna-ribose was probably metabolized (heaf and davies, 1976) . it has been shown in a murine model of staphylococcus aureus infection that the oral administration of rna was ineffective while the intraperitoneal nucleoside-nucleotide mixture was more effective in maintaining host resistance against bacterial infection (adjei et al., 1993) . supplementation with nucleotides has also been shown to exert biological effects, including expression regulation of certain genes (sanchez-pozo and gil, 2002; gil, 2002) , although the mechanisms of action are still unknown. it is generally assumed that nucleotides are not essential nutrients, but under certain conditions (i.e. low dietary intake, tissue needs increased or stress) dietary nucleotides may play a role as conditionally essential nutrients (jung and batal, 2012; carver and walker, 1995) . in this context, dietary supplementation with yeast rna promoted ulcer healing in a rat model of indomethacin-induced ulcerative ileitis (sukumar et al., 1997) . similar effects were observed when nucleosides and nucleotides were administered intravenously (veerabagu et al., 1996) . in both cases, the possible mechanism of action was partly due to increased cell proliferation in the damaged tissue (sukumar et al., 1997; veerabagu et al., 1996) . similar effects were observed when rna was administered orally or its constituents (nucleotides) were injected iv, suggesting that the effects are due to the absorbed nucleotides. dietary nucleotide supplementation in formula-fed infants efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. has been shown to improve gut microbiota composition (singhal et al., 2008) . in studies on certain fish model, supplementation with dietary rna at a range of concentrations (0.4-0.8% of nucleic acids in the diet compared to the non-suplemented control group) enhances immunological response (choudhury et al., 2005; jha et al., 2007) . in human studies, postoperative enteral diet supplementation with rna, arginine and omega-3 fatty acids has been shown to modulate the postoperative immune response after surgery for upper gastrointestinal cancer (senkal et al., 1995) , overcoming more rapidly the immunologic depression after surgical trauma (kemen et al., 1995) . also in humans, oral supplementation with rna (from yeast) during 5 days before surgery improved host defence in patients undergoing cardiac surgery (tepaske et al., 2001) or gi cancer surgery (gianotti et al., 2002) . in both cases, rna supplementation was also accompanied with arginine and omega-3 fatty acids administration. in summary, the specific contribution of orally administered rna to these effects cannot be ascertained. the instability of orally administered rna molecules and the presence of a large array of rnaases within the gi tract (see sections 3.1.3 and 3.2.1) suggest low absorption, if any. exposure to a highly active enzymatic environment in the gi tract, extreme ph conditions, and the existence of a mucosal epithelial barrier are the main challenges for oral delivery of rna therapeutics (martirosyan et al., 2014) . although a considerable amount of food rna is ingested, which varies widely between individuals but is typically in the range 0.1-1 g/person/day, it is assumed it is degraded and absorbed in minimal amount (jain, 2008) . however, further data are required to document the extent to which this conclusion pertains to rna molecules with complex structures. to overcome the multiple barriers encountered by exogenous rnas for oral delivery, several strategies in the laboratory or clinical trials have been developed (see 3.2.2.1 below). the oral administration route is advantageous because it increases patient compliance and comfort over injection, provides for simple, repeatable administration, and offers a large surface area for absorption (forbes and peppas, 2012) . rna delivery may also benefit from advances in the oral delivery of aso for animal (raoof et al., 2004; raoof et al., 2002; . indeed, polymeric nanoparticles containing alginate, dextran, chitosan, polyethylenimine, polyethylene glycol, polylactide, yeast derived β-glucans and/or several other natural or chemically synthesized molecules have been tested by the pharmaceutical/medicine field. the literature describes several strategies for the oral delivery of rna oligonucleotides. the target is to stabilize and protect rnas during gi transit, enable cellular uptake in the intestine, and in some cases, promote endosomal escape and increase biological action. thioketal nanoparticles (tkns) were formulated from a polymer, poly-(1,4-phenyleneacetone dimethylene thioketal) that degrades selectively in response to reactive oxygen species ( the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (plga) nanoparticles were also tested in the same study, but with no efficacy in diminishing tnfα expression. to obtain efficient mucus transportation, targeted cellular uptake and endosomal/lysosomal escape, different nanoparticles have been tested ( nanoparticles of galactosylated trimethyl chitosan-cysteine containing sirna against map4k4 were tested and found to target activated macrophages in colonic tissue (zhang et al., 2013b) . compared to nanoparticle-protected sirnas, naked sirnas were completely degraded by the intestinal fluids. when administered orally (250 µg sirna/kg) for 6 days, nanoparticles improved the dss-induced model of colitis in mice (zhang et al., 2013b) . other ternary polymeric nanoparticles formed by thiolated trimethyl chitosan with tripolyphosphate have been tested in mice and found to efficiently deliver sirnas to the intestine and other tissues when administered orally (zhang et al., 2013a the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (4%, 13% and 21%) were also evaluated in trimethyl chitosan-cysteine conjugate nanoparticles . in a mouse model of ulcerative colitis, the mannose density of 4% was the most effective for sirna knockdown of tnfα following oral administration (30 µg/kg) of 2'-o-methyl-modified tnfαspecific sirna duplex . the use of supramolecular self-assembly nanoparticles (ssnps) -which contain oleyl trimethyl chitosan, poly(γ-(4-(((piperidin-1-yl)ethyl)amino)methyl)benzyl-lglutamate), oleyl-peg-mannose, oleyl-peg-cysteamine, and sodium tripolyphosphate -with specific functions of mucoadhesion, transepithelial permeation, membrane penetration and active targeting have also been tested ( however, not all nanoparticle types can efficiently deliver rnas. in a study evaluating the ability of unassisted epithelial entry of nucleic acids as a consequence of nanocarrier contact with mucus, antisense oligonucleotides but not sirna (naked) were shown to be delivered to mouse intestine when formulated with nanocarriers composed by chitosan (martirosyan et al., 2016). multi-compartmental formulations -consisting of one or more internal compartments surrounded by protective external compartments -have been developed to surmount the many barriers to oral rna delivery (kriegel et al., 2013; . one example is the "nanoparticles in the microspheres oral system" (nimos), a solid-in-solid multi-compartmental system (kriegel et al., 2013) . gelatin nanoparticles entrapped in poly(epsilon-caprolactone) microspheres were prepared carrying tnfα sirna (slightly chemically modified dtdt) to treat dss-induced colitis. oral administration of sirna (1.2 mg/kg) to female balb/c mice decreased colonic tnfα expression and supressed the expression of proinflammatory cytokines (kriegel, c and amiji, m, 2011) . when combined with sirna against cyclin d1 (naked sirna, also at 1.2 mg/kg/day), the silencing effects were more potent than with tnfα sirna alone (kriegel c and amiji mm, 2011) . dectin-1 recognizes beta-1,3 and beta-1,6 linked glucans rich particles and intact yeast and is particularly expressed on the monocyte/macrophage and neutrophil lineage (herre et al., 2004) . β-1,3-d-glucan has been used to deliver exogenous rna targeting macrophages (aouadi et al., 2009 ). delivery of β-1,3-d-glucan sirna particles containing 20 µg/kg unmodified sirna by oral gavage to mice for 8 days reduced map4k4 expression in different macrophages from different tissues and protected them from lipopolysaccharide-induced lethality (aouadi et al., 2009 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the cd40 gene was the target of the antigen-presenting dendritic cells and oral administration of the recombinant yeast. short hairpin rna delivered in lactobacillus casei have also been tested to target the intestine (kuwahara et al., 2007) . other organisms, including attenuated salmonella typhi, were used as vectors to deliver rnas, even ribozymes (bai et al., 2011) or sirna (jiang et al., 2007) , using oral administration in mice to target either local or systemic effects (bai et al., 2011; jiang et al., 2007) . the the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. extracellular vesicles are a heterogeneous family of vesicles delimited by membranes. these can be classified by their size as i) exosomes (30-100 nm in diameter), which originate from the endosomal system, ii) microvesicles (100-2000 nm) formed by budding out of the plasma membrane, and iii) apoptotic bodies (>1000 nm) formed by blebbing of the plasma membrane during apoptosis (reviewed in yanez-mo et al., 2015; colombo et al., 2014) . increasing evidence suggests that extracellular vesiclemediated communication can take place in vivo, but the nature of the extracellular vesicles involved in these effects remains to be clarified (reviewed in tkach and thery, 2016; pitt et al., 2016) . exosomes, which are formed in multivesicular compartments, are secreted when these compartments fuse with the plasma membrane (kowal et al., 2014; abels and breakefield, 2016) . exosomes have been shown to contain or transport a myriad of molecules including proteins, carbohydrates, lipids, and a variety of genetic material including dna, mrna and ncrnas (reviewed in choi et al., 2015; abels and breakefield, 2016) . exosomes are also described as participating in intercellular communication (costa-silva et al., 2015; pironti et al., 2015; nojima et al., 2016) , delivering their parental cell-derived molecular cargo to a recipient cell. some of these processes are thought to be mediated by ncrnas, including mirnas thomou et al., 2017) . detailed information on how all these biological processes occur is outside the scope of this review. given the above characteristics of exosomes, these vesicles hold promise as delivery vehicles for therapeutics (munagala et al., 2016; van der meel et al., 2014) . indeed, exosomes have been used to deliver sirna to different tissues when administered systemically, including the brain (alvarez-erviti et al., 2011; cooper et al., 2014; didiot et al., 2016) . mirnas have also been delivered via exosomes to different tissues including xenograft breast cancer tissue (ohno et al., 2013), brain and other tissues when administered systemically or locally (zhang, d et al., 2017) . interestingly, oral administration of exosomes or extracellular vesicles has also been described in animal models agrawal et al., 2017; oliveira et al., 2017; oliveira et al., 2016) . however, their biological effects were not related to transport of ncrnas. some extracellular vesicles have been shown to resist digestion under in vitro simulated gi conditions (benmoussa et al., 2016; , particularly those from bovine milk (vashisht et al., 2017) . moreover, milk exosomes seem to exhibit cross-species tolerance and are not described to induce adverse immune and inflammatory responses (munagala et al., 2016) . however, whether exosomes or other extracellular vesicles resist the harsh in vivo conditions of the gi tract and the digestive process remains poorly described and needs to be studied (tomé-carneiro et al., 2018) . exosome-like nanoparticles from plants have been isolated from different species including ginger, grape, grapefruit and carrot (mu et al., 2014; ju et al., 2013; zhang et al., 2016) . some authors indicated that the approximate amount of exosome-like nanoparticles in these edible plants were reported to be ≈350 mg/100 g edible plant (mu et al., 2014) , while other authors reported only ≈50 mg/kg ginger . most of these studies reported the presence of rnas, either small or large, including ncrnas (mu et al., 2014; zhang et al., 2016; ju et al., 2013) . the amount of rna present in these exosome-like nanoparticles was reported to be ≈ 4 µg rna/100 mg exosomelike nanoparticles for grape and grapefruit, and ≈ 12 µg rna/100 mg exosome-like nanoparticles for ginger and carrot (mu et al., 2014) . the in vivo biological effects of these nanovesicles have been assessed. oral delivery of ginger nanovesicles during 7 days at a dose of 0.3 mg/day was found to target the colon, to be taken up by epithelial cells and macrophages, and to reduce dss-induced colitis in mice . treatment for 18 weeks with the same dose also exhibited antiinflammatory activity in a mouse model of chronic colitis . ginger-derived lipid vehicles have also been generated from ginger lipids and loaded with sirna-cd98 (zhang m et al., www.efsa.europa.eu/publications 130 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 2017). oral administration of these ginger-derived lipid vehicles targets the colon tissue and they are effective in treatment of induced ulcerative colitis in mice. grapefruit exosomes at a dose of 10 mg/kg per day for 7 days also ameliorated the dss-induced colitis targeting intestinal macrophages . when administered orally at a dose of 2 mg of nanovesicles/day, grape exosomes-like nanoparticles were also found to protect mice from a dssinduced model of colitis by induction of intestinal stem cells (ju et al., 2013) . local intestinal macrophages and stem cells from the small and large intestine were also found to be the target of these exosome-like nanoparticles when administered by oral gavage (2 mg/day) (mu et al., 2014) . even though mirnas were reported to be present in these exosome-like nanovesicles (ju et al., 2013; mu et al., 2014; zhang et al., 2016) , the biological effects of these ncrnas were not evaluated. whether these mirnas resist the gi tract conditions remains unknown. a main hurdle in rna-based therapeutics is the successful delivery of naked rna (non-chemically modified) molecules to specific (target) tissues in the gi tract. the literature contains few studies in which exogenous rnas are administered in vivo using routes of administration other than oral, and most are in the field of inflammatory bowel disease. an amphiphilic cationic cyclodextrin (cd) vector was developed for a complex sirna against tnfα, where the complex was concentrated to 50 µg/100 µl in 5% glucose (mccarthy et al., 2013) . cd.tnfα.sirna was found to be efficient against dss-induced colitis in c57bl/6 mice treated twice (day 2 and 4 post-dss) by intrarectal administration with the test solution (100 µl). cd.sirna administration reduced tnfα and il-6 expression as compared to the non-silencing sirna control or the naked tnfα sirna (not delivered in formulated cyclodextrin). enteral delivery of sirnas for systemic effect was also tested. a nuclease resistance and chemically modified sirna against apolipoprotein b was conjugated with α-tocopherol and was administered (10 mg/kg) as lipid nanoparticles in the large intestine of mice. the lipid nanoparticle was composed of mixed micelles comprised of linoleic acid and peg-60 hydrogenated castor oil. the sirna efficiently reached the liver and other tissues using the chylomicron-mediated pathway via the lymphatic route (murakami et al., 2015) . delivery of sirna into hepatocytes was markedly reduced when the sirna was not bound to α-tocopherol, indicating that conjugation with α-tocopherol was essential to this delivery system using this route. a calcium phosphate (cap) core coated with sirnas and encapsulated in poly(d,l-lactide-co-glycolide acid) (plga) nanoparticles containing an outer layer of polyethyleneimine (pei) was developed for intrarectal delivery (frede et al., 2016) . in a dss-induced model of colonic inflammation, intrarectal administration (12 µg/day) of nanoparticles containing 114 ng sirna against either tnfα, ip-10 or kc from day 2 to 5 after 4% dss treatment, ameliorated the colitis. indeed, while intestinal epithelial cells, dendritic cells, macrophages and t cells could uptake the tnfα sirna nanoparticles, reduction of tnfα was only detected in epithelial cells and t cells (frede et al., 2016) . the stability of sirna within the cap/plga/pei nanoparticles against enzymatic nucleases under colonic conditions was evaluated. while the sirna-loaded cap/plga/pei nanoparticles were detectable after 1 h incubation at 37ºc with colonic fluid or homogenate, the same sirnas not formulated in nanoparticles was not detected and was assumed to be degraded. even after exposure to colonic homogenate for 1 h at 37ºc, the nanoparticles retained biological activity. since the supernatant of the homogenate subsequently incubated with a cell line, still reduced the expression of tnfα by 50% (frede et al., 2016) , suggesting that sirna-loaded nanoparticles retain its biological activity compared to that of nacked sirnas (not formulated). www.efsa.europa.eu/publications 131 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. other alternatives for rna delivery have been tested. ultrasound-mediated rna delivery has been efficiently achieved in mice (schoellhammer et al., 2017) . chemically modified sirna against tnfα (100 ng sirna in 200 µl water) was delivered in the rectum using ultrasound exposure to 40 khz for 0.5 s and after a 6-day treatment tnfα was efficiently repressed in colonic tissue of a mouse model of induced colitis. in the same study, a mrna (≈ 950 kda) complexed in lipid nanoparticles (lnps) was administered in the colon (300 µl lnps solution) with ultrasound and found to be successfully translated locally in the colonic tissue (schoellhammer et al., 2017) . oral administration of exogenous rnas to exert a biological effect, either locally or systemically, is successful if combined with specific delivery technologies, such as vehicles developed for pharmaceutical/medical purposes. nanoparticles have been designed to release their cargo in specific regions of the gi tract (i.e. where inflammation occurs) or to resist the gi conditions. in contrast to other routes of administration, where normally highly chemically modified rnas are used, oral delivery normally involves use of naked rna or minimally modified rnas formulated with complex delivery vehicles. achieving the local desired effect (i.e. targeting inflammation in the colon) has contributed to the development of delivery technologies and the usage of other routes of administration different than the oral as described above. however, the available literature also suggests that naked or unmodified exogenous rnas are rapidly degraded when exposed to the gi conditions without incorporation into delivery vehicles. extracellular vesicles are natural lipid particles released by many cell types with the potential to mediate cell-to-cell communication. these extracellular vesicles have a tremendous potential as delivery vehicles for therapeutics. however, there are still very few studies evaluating their resistance to the harsh conditions of the gi tract following oral administration. knowledge of their biological effects as transporters of exogenous ncrnas is still very limited. nucleic acid-based therapeutics have the potential to treat numerous diseases by correcting abnormal expression of specific genes. as indicated previously, the oral route of drug administration poses serious delivery challenges due to the gi degrading environment, as well as the need to overcome other barriers preventing nucleic acid delivery. because of these obstacles, efforts in developing oligonucleotide-based therapeutics using the oral route have focused on local gastrointestinal delivery, i.e. directly delivering the drug to the target tissue for localized effects, and, therefore, increasing local bioavailability and maintaining doses low while diminishing possible effects to non-target tissues kriegel et al., 2013) . crohn's disease and ulcerative colitis are the two principal forms of ibd, characterised by being a chronic relapsing inflammatory condition of the gi tract. the pathogenesis of ibd is dependent on the interaction between local immune and environmental factors in genetically-susceptible individuals (o'neill et al., 2011) . inflammation in crohn's disease is characterised by high production of the cytokines interferon (ifn)-gamma, il-17 and tnf-alpha, representing a t helper1 (th1)-th17 response. in ulcerative colitis, the immune response is characterised by th17 and an atypical th2, with high production of il-5, il-10 and il-13 (o'neill et al., 2011) . conventional treatment consists of antiinflammatory and immune-suppressive drugs but, while some medications are effective to combat inflammation in the acute phase, they are ineffective in maintaining remission due to toxicity, dependency and higher relapse rates (kriegel, c and amiji, m, 2011) . the goal for ibd treatment is local delivery of therapeutics to intestinal immune cells. initial approaches involved il-10 supplementation www.efsa.europa.eu/publications 132 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. due to its huge potential in blocking proinflammatory cytokines and inflammatory tissue damage. but human clinical trials have not proven that systemic il-10 supplementation can help prevent or improve the ibd symptoms, probably due to il-10 low mucosal levels . separate studies have used gene therapy to increase il-10 mucosal availability by engineering il-10 plasmid dna, and achieving more success in rodent models (barbara et al., 2000; steidler et al., 2000; lindsay et al., 2003; bhavsar and amiji, 2008) . deregulation of tumour necrosis factor (tnf)-alpha production, a proinflammatory cytokine primarily produced by activated macrophages along with other cell types, is associated with the onset and progression of a number of inflammatory diseases (such as ibd, rheumatoid arthritis), alzheimer's disease and psoriasis . the harmful effects of tnf-alpha are thought to be mainly mediated by activation of nf-kappab, therefore regulating expression of over 200 genes. systemic blockage of tnf-alpha is accompanied by several side effects that can be avoided if local inhibition occurs. rnai-mediated local suppression of tnf-alpha can be beneficial for overcoming many of these side effects. different groups have developed various delivery systems for sirna molecules targeting tnf-alpha expression in the gi tract, assaying them both on cells and several rodent models for ibd (wilson et al., 2010; kriegel, c and amiji, m, 2011; kriegel, c and amiji, mm, 2011; laroui et al., 2011; yin et al., 2013; he et al., 2013b, a; he et al., 2015) . even though therapeutic rnai development is in its infancy, current approaches and tools for delivery of these biomolecules via the oral route are improving. in fact, recent reports describe the effects of an oral smad7 antisense oligonucleotide in a randomized, placebo-controlled, double-blind, phase 2 clinical trial in patients with crohn's disease (monteleone et al., 2016; monteleone et al., 2015) . although in this instance the biomolecule used is an antisense oligonucleotide and not a sirna, the design of the oral formulation could be very similar. iron is an essential metal required for numerous physiological functions, but in excess it is a well-defined risk factor in the pathogenesis of several diseases, including cardiovascular and neurodegenerative diseases. since there is no recognised active pathway of iron excretion, disposal of excess iron from the body is the primary therapeutic goal of treating patients with iron overload. use of iron chelators is limited due to nonspecific distribution in non-target tissues, which results in a number of serious side effects and toxicity . another way to treat iron excess is by limiting absorption of exogenous iron. the divalent metal transporter 1 (dmt1) protein plays a well-established role in iron absorption. the primary site of dmt1 activity is the intestinal epithelium. dmt1 expression is regulated in response to body iron levels, with expression enhanced when iron stores are low and, conversely, reduced when iron stores are high. oral delivery of sirna-encapsulated nimos to selectively suppress intestinal dmt1 could decrease intestinal uptake of dietary iron, thereby mitigating iron overload and preventing iron-mediated toxicity . celiac disease is caused by a t-cell mediated immune response in the small intestine against deamidated cereal gluten peptides modified by the enzyme transglutaminase 2 (tg2). the only way to prevent celiac symptoms is strict adherence to a gluten-free diet. the pathophysiology of celiac disease involves a combination of environmental, genetic and immunological factors. among several immune mediators, enzyme tissue tg2 and the proinflammatory cytokine interleukin-15 (il-15) have emerged as key players in promoting inflammatory responses against dietary gluten. given their important role in the pathophysiology of celiac disease, sirna mediated silencing of intestinal tg2 and il-15 (i.e. by oral administration using nimos) may result in neutralizing proinflammatory effects and could therefore alleviate disease symptoms . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. colorectal cancer is one of the most common forms of cancer, and rnai therapy has great potential in the treatment of local intestinal cancers. however, rnai therapy faces major challenges since the genotypic footprints of the various cancers differ widely, even from individual to individual. depending on the tumour, several different sets of genes may be misexpressed, making it extremely difficult to determine a common/unique "culprit" gene. moreover, mice and rats do not generally spontaneously develop colon cancer, so induction of tumour growth is required. but these developed animal models do not reflect the genetic changes observed in human tumours (o'neill et al., 2011) . genetic mouse models that spontaneously develop intestinal cancer have also been developed. mutations in the apc gene are associated with development of colon cancer as well as a condition called familial adenomatous polyposis (fap). fap is an inherited condition characterised by development of numerous polyps in the colon. some of these polyps can subsequently become malignant adenocarcinomas (o'neill et al., 2011) . regarding fap and human gene therapy, one study explored oral delivery of escherichia coli expressing shrna against beta-catenin, and found significant silencing activity in healthy mice (xiang et al., 2006) . the gi tract provides the possibility of acting at systemic level by targeting local resident cells, such as m cells. m cells can take up encapsulated biomolecules to be phagocytosed by macrophages. in fact, aouadi et al. reported that glucan encapsulated sirna particles (gerps) loaded with sirna were phagocytosed by the underlying gut-associated lymphatic tissue (galt) macrophages and then translocated to distal organs of the reticuloendothelial system such as the liver, spleen and lung (aouadi et al., 2009) . another report claims functional cd40 shrna expression delivered into dendritic cells in mice by oral administration of recombinant yeast (xu et al., 2016) . in this manner, the targeted cells located in the intestine can undergo sirna-mediated gene silencing and migrate into tissues throughout the body. when successfully administered, rnai is a very useful therapeutic strategy for efficiently modulating gene expression. orally administered local rnai therapeutics have great potential due to the increasing bioavailability of the therapeutic biomolecule at the targeted tissue or cell type, coupled with simultaneous evasion of systemic side effects and immunogenic reactions. most advanced studies are focused on inflammatory bowel disease, although other potential diseases are being explored. the current main challenge is development of suitable encapsulation methods to protect the rna molecules from the harsh and varying conditions of the gi tract. the literature on rna delivery in fish and birds is scarce, with some examples of dietary delivery of exogenous rnas. in birds, most of the studies have been done using central nervous system administration of sirnas. for example, in the zebra finch bird, sirnas (three 19-25 nt long sequences against tyrosine kinase b, trkb) administration via direct injection into the brain was effective in reducing the volume of the high vocal centre by diminishing the robust nucleus of the arcopallium and the relative number of cells within it (beach et al., 2016) . in another study, direct administration of sirnas in the brain of white-crowned sparrows reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations (ubuka et al., 2013; ubuka et al., 2012) . in the japanese quail, central administration of the same sirnas against gonadotropin-inhibitory hormone the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. sirnas for rna interference in chicken embryos has also been used to target marek's disease virus in vivo (chen et al., 2009 ). in fish models, systemic exosome-mediated sirna delivery to zebrafish resulted in effective crossing of the brain blood barrier and localization in the brain in vivo (yang t et al., 2017) . exosome-delivered sirnas decreased fluorescence-labelled tumour cells in the brain more than fourfold by inhibiting vascular endothelial growth factor (vegf) in a xenograft zebra fish brain tumour model (yang t et al., 2017) . these results suggested that brain endothelial exosomes could be used to deliver exogenous sirnas to the target site in the brain for treatment of brain cancer. the proposed higher delivery efficiency, lower immunogenicity, and better compatibility than existing foreign rna carriers have promoted the possible use of exosomes for exogenous rna delivery (mei et al., 2018) . several other studies have evaluated systemic delivery of sirnas to fish animal models, including delivery to the heart using peg-pla nanoparticles (diao et al., 2015) or using a neutralized non-charged polyethyleniminebased system . the literature describes other examples of sirna delivery into zebrafish embryos to inhibit specific gene function (gruber et al., 2005) . in the context of other types of ncrnas, direct injection of mir-26a mimic rnas at the one-cell stage of zebrafish embryos resulted in inhibition of formation of the caudal vein plexus by means of targeting the endothelial cell bone morphogenic protein smad1 signalling (icli et al., 2013) . also in the zebrafish model, injection of a mir-722 mimic into embryos at the one-cell stage to deliver mir-722 ubiquitously resulted in protection against sterile inflammation (hsu et al., 2017) . dsrna microinjection administration to zebrafish embryos (1-2 cell stage) at a concentration of 15-60 pg rna/embryo resulted in rna interference, an effect that was diminished when ssrna was used (wargelius et al., 1999) . in cultured european sea bass, intramuscular injection (7 weeks, 40 µg of dsrna per dose) followed by in vivo electrically mediated dsrna delivery resulted in a reduction of myostatin gene expression (terova et al., 2013) . delivery of exogenous rnas has also been tested in lower aquatic organisms. in the crustacean red claw crayfish cherax quadricarinatus, systemic injection of sequence specific dsrnas against viral protein b2 of macrobrachium rosenbergii nodavirus (mrnv) was conducive to rnai effects that were able to functionally prevent and reduce mortality in infected individuals (hayakijkosol and owens, 2012) . dsrnas (intramuscular injection) have been used to induce antiviral immunity in invertebrates as they can also recognize dsrna as a virus-associated molecular pattern, resulting in activation of an innate antiviral response (robalino et al., 2004) . oral administration of encapsulated dsrnas (inactivated bacteria expressing dsrna) in the prawn macrobrachium rosenbergii fed twice daily at a rate of 5% total biomass for 7 days resulted in their protection against white tail diseases caused by mrnv (naveen kumar and karunasagar, 2013) . dsrnas designed against the capsid and b2 genes of mrnv reduced the mortality of prawns by ≈70% due to sequence specific-mediated silencing of the viral genes (naveen kumar and karunasagar, 2013) . injection of viral protein 28-targeted dsrna (≥12 µg dsrna/g shrimp body weight) into shrimp resulted in a high level of 22 nt sirnas mapping across the entire white spot syndrome virus genome after virus challenge, which resulted in protection against the virus (nilsen et al., 2017) . in planarians, ingestion of bacterially-expressed dsrnas or direct microinjection into the animals can inhibit gene expression through rna interference (newmark et al., 2003) . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mongersen, an oral smad7 antisense oligonucleotide, and crohn's disease. n engl j med 372, 1104-1113. mu j, zhuang x, wang q, jiang h, deng zb, wang b, et al. 2014 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 1927 -1943 . zollner n. 1982 . purine and pyrimidine metabolism. proc nutr soc 41, 329-342. zollner n, grobner w. 1971 . influence of oral ribonucleic acid on orotaciduria due to allopurinol administration. z gesamte exp med 156, 317-319. following a literature search as described in section 2.2.2.2 and based on the methodology described in section 2.2.1.2, a total of 64 documents were selected as relevant to reviewing the effect of dietary exogenous ncrnas on the gi tract and annex glands. very few studies have focused on the gi tract and their annex glands. four documents specifically evaluated the resistance of plant-derived srnas to simulated in vitro digestion system, ex vivo digestion system or in vivo digestion samples (philip et al., 2015; yang, et al., 2017a; and reported that the percentage of unaltered mirnas after digestion might be below 0.01%. due to their relevance in early human nutrition, literature on breast milk ncrnas was chosen to describe the biological effects of dietary exogenous ncrnas of non-plant origin, for which 35 full-text reports were studied and reviewed. zhang et al. were the first to report on the transfer of mirnas from food plants to the mammalian circulatory system causing effects on recipient cells . this study reported the detection by high-throughput sequencing of plant srnas in human serum, as well as in tissues of the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. humans, mice and calves. the plant mirnas were distinguished from human mirnas by their 2'-omethyl modification, which made them resistant to periodate whereas the human mirnas having free 2' hydroxyl were periodate sensitive. it was then assumed that the identified plant mirnas were coming from the food uptake. two rice mirnas named osa-mir156a and osa-mir168a were particularly abundant in human serum samples, since rice is common in human diets. additional in vitro experiments showed that these plant mirnas can endure conditions that mimic the acidic environment of the gut, suggesting that plant mirnas can survive being eaten and digested, pass through the mammalian gi tract and reach target organs. the authors also showed that diet-derived plant mir168a can target the low density lipoprotein receptor adapter protein 1 (ldlrap1) mrna based on sequence complementarity. this mirna was able to reduce the ldlrap1 protein level in the blood and liver of mice fed rice, and eventually increase their plasma low-density lipoproteins (ldl) content. this publication raised questions about whether food-derived srnas could play an active role in human/animal health. however, it was controversial and its results were questioned due to nutritional imbalances in the test-diet studies or lack of rnai-mediated modulation of ldlrap1 protein levels in mouse liver (witwer and hirschi, 2014; chen et al., 2013; . scientists from the monsanto and miragen companies were unable to reproduce these findings . measuring ldlrap1 protein levels in the liver of mice fed with mir168a-containing rice diets by elisa instead of western-blot analysis, dickinson and colleagues did not detect any protein change modulated by mirna168a-diets. although changes in the ldl plasma levels were indeed detected, the authors concluded that ldl increases resulted from nutritional imbalances rather than from a mir168 consumption mediated effect. additionally, plant mirnas including the highly abundant and stable osa-mir168a were not detected in liver and plasma samples of mice fed under different rice-based regimes by srna sequencing with the hiseq illumina system . other independent research groups failed to detect dietary plant mirnas in plasma and tissues when conducting controlled feeding studies in humans and in animal models using different detection methods, such as real time quantitative pcr (rt-qpcr), droplet digital pcr (ddpcr) and next generation sequencing (ngs) analysis . these studies provide contradicting evidence of mirnas dietary absorption. in all the attempts, plant mirnas were detected at substantial levels in the diets but were undetectable in animal fluids and tissues. snow and colleagues studied three plant mirna species (mir156a, mir159a, and mir169a) highly abundant in fruits routinely ingested by a group of ten healthy athletes. examination of the athletes' plasma by rt-qpcr analysis using taqman probes revealed high levels of endogenous mirnas but not the three dietderived plant mirnas . the same results were obtained with mice fed vegetarian diets rich in these 3 mirnas. furthermore, using a mir21 null mutant mouse they showed that none of the dietary mirna including mirna21 was detected in blood and body tissues, suggesting that dietderived mirnas were not absorbed or maintained in mouse body fluids or organs. another group reported negative results on dietary mirna absorption in non-human primates . using qrt-pcr analysis and the same timeframe of as in zhang's report a, plant-specific mirnas were undetectable in tissues from macaques given a vegetarian diet. another feeding study in humans showed no significant differences in the levels of the plant mir167a and mir824 in the plasma after consumption of broccoli sprouts highly rich in these two mirnas (baier et al., 2014) . more recently, a different research group using by ngs analysis failed to detect plant mirnas in the plasma of healthy volunteers that normally consume olive oil . the possibility that the plant rnas detected in human tissues and body fluids in some studies were contaminants remains a serious concern . by searching the composition of srna-seq data generated by the zhang's team from amphioxus animals, www.efsa.europa.eu/publications 142 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. it was found that the amphioxus data contained rice mirnas as previously reported for human serum. given that amphibia have an exclusive algae-based diet, this strongly suggests that the rice mirnas in both studies are the result of samples contamination . spurious detection of such sequences could arise due to contamination during sample handling, library preparation or sequencing, or result from errors in data analysis. since next-generation sequencing is very sensitive, just a few molecules could cause a false positive detection. more recently a comprehensive meta-study surveying for the presence of cross-species mirnas in 824 sequencing data sets from various human tissues and body fluids indicated that xenomirs (cross-species mirnas) are technical artefacts rather than the results of dietary intake . a bioinformatics study identified plant mirnas in human and porcine milk exosomes . by searching publicly available ngs datasets, the authors identified several plant mirna species, most of them belonging to evolutionarily conserved and highly abundant mirna families. the target prediction suggests that these mirnas may interact with mrnas coding several transcription factors, protein receptors, transporters and immune-related proteins, thus potentially influencing biological functions in humans. of note is that the plant mirna profiles found in mammalian breast milk were similar to the composition in human blood presented by zhang and colleagues . liang and colleagues reported positive results on the absorption of food-derived mirnas from feeding studies in mice using cabbage (brassica oleracea) . they showed that mir172, the most abundant mirna in cabbage, was detected in blood and various organs after cabbage consumption. however, there was no absolute quantification of mir172 or a description of an endogenous control for normalization of qrt-pcr data. a more recent study by zhang's group described how plant mirna2911 from honeysuckle (lonicera japonica) inhibited influenza a virus replication in mice when entering through the gi tract, and they hypothesized that this mirna might be the active component in the traditional chinese medicine based on honeysuckle herb to treat influenza infection . the authors demonstrated that mirna2911 is highly stable in honeysuckle decoction by solexa sequencing and northern blot analysis. they also showed that mir2911 levels increased in mouse peripheral blood and lung following continuous honeysuckle decoction intake (drinking) or gavage feeding, as measured by qrt-pcr using taqman mirna probes. additionally, luciferase reporter assays and in vivo experiments showed that mir2911 was able to target the influenza a virus, and consequently inhibited its replication and reduced mice mortality. the same authors reported that plant mirnas can be found in human umbilical cord blood and amniotic fluid, transferred from mother to the foetus, and that these influence foetal development and health . they showed that mir2911 content increased in the maternal plasma and foetal liver of mice fed honeysuckle. a fluorescently labelled sirna was used to trace the transplacental transmission through feeding. furthermore, they showed that after feeding mice with different amounts of sirnas targeting the alphafetoprotein mrnas the levels of this protein were down regulated in foetal tissues. these results suggest that dietary sirnas delivered from the mother to the foetus could regulate foetal gene expression. more recently, the anti-proliferative effect of a plant mirnas on breast tumours was demonstrated in feeding experiments in mice (chin et al., 2016) . the plant mir159, particularly abundant in broccoli, was found in sera from women, and its levels were inversely correlated with breast cancer morbidity and progression. in human sera, mir159 was detected in extracellular vesicles by qrt-pcr using a taqman probe. transfection of breast cancer cells with a synthetic mimic mir159 was shown to reduce their proliferative growth by targeting the transcription factor 7 mrnas. more importantly, oral administration of synthetic mir159 significantly inhibited the growth of xerograph breast tumours in mice. these results agree with a previous report in which oral administration of a cocktail of three www.efsa.europa.eu/publications 143 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. tumour suppressor mirnas that were 2'-o-methylated to mimic plant mirnas reduced colon tumour burden . additional research is needed to address discrepancies among different studies and verify whether plant srnas derived from diet are absorbed at functional levels in animal species. most of the available data suggests either that gastrointestinal absorption of dietary plant mirnas does not occur in healthy consumers or mirnas levels in blood and tissues of consumers are too low to have a biological impact. however, the biological activity of food mirnas may be more dependent on their accumulation in exosomes than on their abundance per se, because of their reported low concentration. development of sensitive sensors to help to detect the functionality of low-level absorbed mirnas would facilitate assessment of the effects of dietary mirnas in consumers. future studies are needed to establish how exogenous ncrnas absorption and tissue distribution occurs, as well as to address their bioavailability and their biological function. reported that increased mir2911 was detected in sera and urine after consumption of particular foods (i.e. honeysuckle), and disappeared 48 h after the honeysuckle was removed from the diet; this supports the idea that these small rnas were of dietary origin. the role of kidney damage in mirna retention in the animals was also evaluated. using two models of acute renal failure (baliga et al., 1999) , i.e. the cisplatin (a chemotherapeutic agent) and the glycerol-induced models, it was observed that only mice receiving cisplatin exhibited measurable levels of dietary mirnas in sera and urine. this suggests that kidney damage alone did not lead to enhanced mirna retention in the mouse circulatory system . of interest is that cisplatin treatment, but not glycerol treatment or honeysuckle feeding, disrupted the organization of small intestine epithelial cells as shown by histological analysis. in this study, yang et al. used droplet pcr to demonstrate that the amplified products were likely to be specific and not due, for example, to nonspecific amplification of endogenous rnas . whether particular foods and/or alterations in intestinal permeability could improve the capacity to absorb small rnas from the diet or influence the biological effect within the gi tract, annex glands and systemically is poorly described, but still relevant for risk assessment of exogenous ncrnas in a subset of population. no studies specifically report the biological effects of dietary exogenous plant ncrna on the gi tract. only a few studies (described above) report the possible biological effects in their annex glands, including the liver . for gastrointestinal tissues or the liver, liang et al. reported that icr mice (a strain of albino mice) fed plant total rna (10-50 µg) extracted from brassica oleracea showed detectable levels of mir172 in the stomach (up to ≈30000 copies), intestinal (up to ≈16000 copies) and faecal content (up to ≈12000 copies), with the maximum amount present at 2 to 4 h after feeding . estimation of the proportion of orally administered rna that survived digestion suggested that the stomach contained 0.4-4.5%, the intestines 0.2-2.4% and the faeces 0.3-1.8 %, while the blood contained about 0.2-1.3% and the spleen about 0.04-0.38% . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. which was similar to that of their synthetic form (without 2'-o-methylated 3' ends) . whereas plant mirnas had a much slower degradation rate compared to their synthetic form without 2'-o-methylated 3' ends. it is important to note that extreme ph conditions are not the only physicochemical or biological condition within the gi tract (see section 3.2.1.1. for details). using a simulated human digestion system in vitro, philip et al. evaluated the resistance of soybean and rice mirnas to simulated gastric fluid early stage digestion (philip et al., 2015) . the study found continual survivability of plant mirnas in an in vitro simulated digestion system for over 75 min without any significant decrease in their levels (philip et al., 2015) . mirna levels have been analysed qualitatively by real-time pcr using taqman mirna assays. stability of the plant-derived small rna mirna2911 was also assessed using an artificial in vitro digestion system that simulates mammalian gastric and intestinal conditions . yang et al. compared three samples which contained a) 1 ml cabbage extract with 10 pmol plant-derived mir2911 (measured by qrt-pcr), and 10 pmol spiked-in synthetic 2'-o-methylated mir168a and artificial mirna termed c7; b) 1 ml phosphate buffered saline (pbs) with 10 pmol each of synthetic 2'-o-methylated mir2911, mir168a and c7; and c) 1 ml pbs with 10 pmol each of synthetic mir2911, mir168a and c7 without the 2'-o-methylation. time course analysis during both gastric and intestinal phase digestion showed that most mirnas displayed modest resistance in the acidic gastric environment (up to 60 min), while in the intestinal phase the levels of all mirnas were drastically reduced after five minutes (figure 14) . regardless of synthesis origin and 2'-o-methylation, the digestive stability of mir2911 was up to 100-fold higher than that of the other mirnas. the most stable form of mir2911 was the plant-derived form in the cabbage extract, since 0.044% survived, compared to 0.0059% for the 2'-o-methylated form, and 0.0037% for the non-modified form. similar results were observed when 10 pmol of synthetic 2'-o-methylated mir2911, mir168a and c7 were digested in vitro using the ex vivo intestinal fluids ( figure 14) . indeed, mir2911 appeared to be more stable after 2-hour digestion than the other tested mirnas. finally, by directly measuring mir2911 levels in vivo in the small intestines of mice fed the plant-based diet (daily dietary mir2911 intake 10 pmol) it was observed that this particular mirna reached level more than 100-fold higher than those observed in the animals consuming a single 400 pmol dose of the synthetic mir2911. mir168a, a mirna also present in the plant-based diets, was also detected in the small intestines at a level 100 times lower than that of mir2911 . using transgenic arabidopsis lines expressing the artificial mirna amir-rice, the murine mirna mmu-mir-146a or their controls, yang et al. studied gastrointestinal digestion and bioavailability of transgenic mirnas (yang et al., 2017b) . levels of transgenic mirnas in plants (qrt-pcr quantification) were similar (22.9 and 26.3 fmol/g of fresh weight for amir-rice and mmu-mir-146a, respectively) to that of srna mir2911 (18-19 fmol/g in fresh shoot tissue). however, in diets stored at room temperature, the abundance of amir-rice and mmu-mir-146a decreased gradually by ≈10-fold (≈1 fmol/g diet) while that of the srna mir2911 increased ≈85fold after 24h. in vitro gastric and intestinal simulated digestion of the synthetic forms of the plantbased transgenic mirnas showed that amir-rice and mir2911 had similar digestive stability, while mmu-mir-146a was significantly less stable. the surviving percentage in the intestinal phase after 75 min was 0.39% for mmu-mir-146a compared to ≈1.11% for mir2911 or amir-rice (figure 15 , frame a). the surviving percentage in the in vivo assays (gavage-fed 400 pmol of synthetic srnas) further decreased the stability of the mirnas, reducing their level to 0.0014% for amir-rice and 0.00045% for mir2911 (figure 15, frame b) . however, the levels of mirnas in the small intestines were higher for mir2911 (238 fmol/mouse) than amir-rice (2.7 fmol/mouse), while mmu-mir-146a levels were undistinguishable from the host mouse mirna (figure 15 , frame c). amir-rice was not found in serum (neither are mirnas reported in previous studies and present in transgenic plants) while mir2911 was found only in the serum (28.5 fm, or 1.3 x 10 7 copies per mouse) of mice fed the plant transgenic diets (yang, j. et al., 2017b) www.efsa.europa.eu/publications 145 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. altogether, these results suggest that the plant-derived mir2911 might be more stable than other plantbased small rnas. whether this exceptional stability could be generalized to other ncrnas is unknown. however, recent evidence, including some studies evaluating mir2910 and mir2911, suggests that there may be some misidentified alternative mirnas . indeed, several other rarer but consistently mapped plant mirnas also have 100% or near 100% matches to human transcripts or genomic sequences (i.e. rrna), and some do not map to plant genomes at all, suggesting artefactual results of plant mirnas in mammalian sequencing datasets. this emphasizes the need for rigorous filtering strategies when assessing possible dietary exogenous mirnas . indeed, the small rna mir2911 is derived from 26s rrna and does not undergo canonical mirna processing, as it does not depend on dcl1 (yang, j. et al., 2017a) . mir2911 is also inefficiently assembled into the risc complex in human cells and modestly regulates gene expression. this suggests that this exogenous srna is different from the canonical plant-based mirnas (yang, et al., 2017b) . these results highlight the need to evaluate other ncrnas as they could originate from precise processing at the 5' or 3' end of mature or precursor rnas generating other abundant small rnas (lee et al., 2009) . also, the rna degradome is a crucial component of the total cellular rna pool of plants (nowacka et al., 2013) . they can be stable degradation intermediates present in a high copy number, or they can derive from various rna species including trna, rrna, mrna and snrna (addo-quaye et al., 2008; nowacka et al., 2013) . among the few examples in the literature of possible biological effects of exogenous non-plant origin ncrnas, milk has been studied because it constitutes a rich source of secreted mirnas. human breast milk is an essential source of nutrition for new-borns. in addition to its nutritional function, breast milk contains myriad biologically-active components that influence the development of the the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. more than 87% of the 234 unique mirnas identified in porcine milk exosomes (gu et al., 2012) or 75% in other studies (chen et al., 2014) . in human exosomes, the ten most abundant mirnas account for 62% of the 602 unique mirnas identified . lncrnas previously reported as being important for developmental processes were found in human breast milk extracellular vesicles (karlsson et al., 2016) . maternal factors such as the mother's diet seem to influence mirna expression in the breast milk fat globule (munch et al., 2013) or exosomes (sun j, 2017) . maternal weight (xi et al., 2016 ) also seems to influence mirna expression. certain mirnas found in breast milk have also been found in placenta tissue (munch et al., 2013) , although their physiological role is unknown. other studies have shown that colostrum mirnas (early lactation period) are more abundant than in the later lactation period (gu et al., 2012; xi et al., 2016; sun et al., 2013) . bovine milk mirnas isolated from the colostrum whey fraction were more abundant than in the mature milk whey fraction (izumi et al., 2012) . immunerelated mirnas expression is reported to be higher in colostrum than mature milk (izumi et al., 2012; na et al., 2015) . in vitro studies of human milk mirnas using either rnase digestion, freeze-thaw cycles, low ph or other gi conditions have shown that these mirnas are relatively highly stable (kosaka et al., 2010; liao et al., 2017) . similarly high stability of mirnas was found when using one or all of the above mentioned hard conditions for milk from cow (hata et al., 2010; izumi et al., 2012 ), pig (gu et al., 2012 or panda . indeed, it has been proposed that rna in milk is present in microvesicles and is protected from rnases and the other gi conditions by surrounding membranes (hata et al., 2010; gu et al., 2012; . however, technological conditions such as pasteurization (golan-gerstl et al., 2017) have been shown to reduce mirnas abundance in cow and goat milk by up to ≈40%, or up to ≈60% for certain mirnas (howard et al., 2015) , and in human exosome (personal communication, faseb conference 2017). microwave heating also reduces the amount of certain mirnas (howard et al., 2015) , while ultrasonication influences exosome membrane integrity and thus increases cargo instability (sun et al., 2013) . among dairy products, mirnas concentrations varied considerably, but were generally lower than the concentration in pasteurized whole milk (howard et al., 2015) . the exception was fresh cheese ("queso fresco") dip, which contained higher concentrations of mirnas than observed in pasteurized milk (table 24 ). in vitro studies using mirnas isolated from breast milk exosomes, either human or bovine, were found (liao et al., 2017) , and crl1831, k562 or lim1215 (golan-gerstl et al., 2017) . when subjected to proteinase k treatment or when the vesicle structure was destroyed, uptake or transport of these exosomal mirnas was compromised (kusuma et al., 2016; wolf et al., 2015; sun et al., 2013) . different studies have attempted to quantify the amount of rnas present in breast milk. rna content in microvesicles isolated from cow milk has been estimated to be ≈1700 ng for colostrum and ≈980 for mature milk when isolated from 6 ml sample (hata et al., 2010) . in human milk, total rna was estimated to be ≈2600 ng/10 6 cells of the milk cell fraction and ≈30 ng/µl fat from milk fat (alsaweed et al., 2016c) . exogenous plant mirnas have also been found in panda milk exosomes , the whole milk or exosomes of human breast milk the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. et al., 2012). however, levels of mirnas or total rnas were very low (golan-gerstl et al., 2017; alsaweed et al., 2016c) compared to that of breast milk. indeed, alsaweed et al. reported that the total amount of rna found in bovine milk-based infant formula was ≈0.1 ng/µl formula (45 human mirnas detected) as compared to 69.9 ng/µl for a soy-based formula (22 human mirnas detected) (alsaweed et al., 2016c). in terms of possible breast milk mirna absorption, plasma levels of mirnas were found to be higher in colostrum-fed piglets compared to mature milk-fed piglets (gu et al., 2012) . in humans, plasma mirnas (mir-29b and mir-200c) increased when different doses of cow milk were consumed (baier et al., 2014) . in contrast to the studies above, the literature also describes several studies that show either different or contradictory results regarding uptake of exogenous rnas from breast milk. auerbach et al. re-analysed a study in which mir-29b-3p and mir-200c-3p were found in plasma after bovine milk ingestion by healthy humans (baier et al., 2014) and found no significant altered mirna levels after milk ingestion (auerbach et al., 2016) . these results were confirmed by qpcr and rna-sequencing. several technical issues including low mir-29b expression, use of control mirnas (normalization), and variation level may have contributed to these discrepancies (auerbach et al., 2016) . piglets fed cow milk for 4 weeks showed very low levels of cow-specific mirnas in the bloodstream, which were compared to the levels measured from animals fed maize, which also showed similar counts of cow-specific sequences , suggesting a lack of transfer of exogenous small rnas. using a transgenic mouse model that overexpresses mir-30b precursor in the mammary epithelial cells, and thus increases levels of mir-30b in milk (≈134-fold), no differences were found in blood, liver, small intestine, kidney or lung tissues of pups from these dams when compared to pups from wild type dams (laubier et al., 2015) . interestingly, the level of mir-30b was ≈30-fold higher in the stomach of mice consuming the transgenic milk. oral ingestion of 0.125 µg and 0.25 µg of total rna from porcine milk exosomes administered daily during three weeks to mice, significalty increased villus height and crypt depth of the duodenum and jejunum relative to the control group, suggesting an effect improving the development of the gi tract in mice . using genetic knock-out (ko) mouse models for mirnas mir-375 and mir-200c/141, title et al., evaluated the uptake of maternal-milk derived mirnas using the foster mother offspring exchange model to prevent the confounding effects of mirnas derived from tissues of suckling offspring . wild type (wt) offspring consuming mir-375 ko milk at day 3 exhibited basal levels of mir-375 in milk from the stomach, which likely comes from stomach epithelial cells. at day 14 of suckling, it was confirmed that most mir-375/mir-200c came from the milk itself rather than the offspring www.efsa.europa.eu/publications 149 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. genotype, because there was no significant difference between wt pups and 375ko pups receiving wt milk. evaluating different parts of the gi tract (jejunum, ileum and colon), no evidence of uptake was observed because there was no measurable increase in mir-375 levels in enterocytes of 375ko pups receiving wt milk compared to 375ko milk. moreover, no changes in plasma, liver or spleen levels of mir-375 were observed in 375ko pups receiving wt or 375ko milk . similar effects were observed at day 3 of treatment and upon evaluating mir-200c on day 14. when the fate of milkderived mirnas downstream of the stomach was analysed, the levels of mir-375 in the intestinal content of 375ko pups receiving wt milk was seen to dramatically decrease compared to that of the stomach contents. these data suggest that milk mirnas are being degraded by the digestive system (since no evidence of uptake was observed). indeed, when the spike in cell-mir-39 was tested, it was degraded more rapidly than milk mir-375, suggesting that milk mirnas exhibited a certain degree of resistance (perhaps because of their exosome content). but less than 10% of mir-375 copies remained after 2 h incubation of intestinal content, suggesting a possible digestive enzymatic degradation of the milk-derived (exosomes) mirnas . observational studies also suggest that human breast milk mirnas are not related to preventing atopic dermatitis in infancy 3 months postpartum . in general, rna contamination has been proposed as the main source of controversy in mirnas studies reported in the field of breast milk (bagci and allmer, 2016) . the literature contains very few studies on the biological effects of dietary exogenous ncrnas in the gi tract and its annex glands. indeed, these few studies only report a biological effect in the liver. the available literature suggests that dietary exogenous plant small ncrnas seem to resist the harsh conditions of the gi tract. however, the stability of plant mirnas under gi conditions in vitro, ex vivo or in vivo is low. moreover, these studies suggest that the percentage of surviving mirnas in the gi tract in the best-case scenario is around ≈1%, although this depends on the specific small ncrnas evaluated. several studies report the presence of dietary exogenous mirnas in breast milk of different mammals, including humans. some protected mirnas (i.e. transported in extracelluar vesicles) have been found to be stable under in vitro degradative conditions. while some evidence suggests a possible absortion through oral feeding, other studies indicates a very limited biovailability or lack of accumulation within the gi tract or its annex glands. recent evidence supports the significant contribution of srnas to communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. this silencing transfer phenomenon is very relevant to food and feed risk assessment of ncrna gm plants. although this section (efsa task 3) will focus on studies related to the possible trafficking of ncrnas between plants and humans and animals, studies supporting the movement of rna-silencing signals between plants and pathogens are also reviewed. virus-induced gene silencing represents the model of gene silencing in the host by exogenous sirnas. in this case, the sirna specificity determinant is derived from viral rnas. typically, with a discrete length of 22 nt they form perfect duplexes which are produced in plants by dcl2 and dcl4. virusinduced gene silencing is a very effective defence system, and, consistently with the normal dynamics of host-pathogen interactions, all viruses encode silencing suppressors as a counter defence (baulcombe, 2015) . www.efsa.europa.eu/publications 153 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. cross-kingdom rnai implies that a translocation of gene silencing signals occurs between hosts and these organisms. this implies a two-way traffic of sirnas between pathogens and their plant hosts. several studies report that rnas produced in a host plant can be transferred to a fungus or oomycete, symbiont or pathogen, to induce rna silencing in the interacting microorganism (ghag et al., 2014; koch et al., 2013; vega-arreguín et al., 2014; helber et al., 2011) . transgenic plants carrying a rnai construct, usually with a sense-intron-antisense palindromic structure that produces dsrnas and sirnas, can induce silencing of the targeted transcripts in the interacting organism. furthermore, treatment of fungal conidia with dsrnas of essential genes led to fungal growth inhibition, demonstrating rna interference from environmental silencing signals (koch et al., 2013) . conversely, the rna produced in a fungus can affect a host plant's defence system (weiberg et al., 2013) . remarkably, scientists have developed an effective disease control strategy, called host-induced gene silencing (higs), by generating transgenic plants that express exogenous rnai triggers to successfully silence essential genes in pathogens and pests (weiberg et al., 2015) . in this context, transgenic plants expressing dsrnas or hairpin rnas targeting vital fungal genes of fusarium sp. developed resistance to these important phytopathogens (ghag et al., 2014; koch et al., 2013) . small rnas or dsrnas can also be transferred from plant to pests, such as insects eating leaves or nematodes infecting roots. indeed, transgenic plants that express dsrna homologous to essential genes of insect pests or nematodes became resistant to these specific parasites through the silencing activity produced within the target organism when srnas expressed from the plant transgenes is consumed (baum et al., 2007; fairbairn et al., 2007; mao et al., 2007) . many aspects of these cross-kingdom rna interference phenomena are still poorly understood. one of them is how these rna molecules 'travel,' sometimes over long distances through diverse cellular boundaries between plants and interacting organisms. these silencing signals may utilize conserved cell-to-cell as well as systemic rnai pathways present in plants and animals, and may also use organismspecific pathways. rna-protective factors such as agos, other rna-binding proteins, or encapsulation into extracellular vesicles likely play important roles in protecting mobile rnas against degradation during transport (weiberg et al., 2015; lefebvre and lecuyer, 2017) . another important question is how these srnas use the target cell rna interference machinery to convey the silencing effect. based on current knowledge, rna-mediated gene silencing seems to be an ubiquitous phenomenon that exists in almost all eukaryotes, and which always follows the principle of complementary nucleotide base-pairing between regulatory srna and mrna sequences (weiberg et al., 2015) . despite the tremendous differences present in the structural features of regulatory rnas, and the completely unrelated or highly divergent rna gene-silencing mechanisms and pathways that have evolved in diverse organisms, complementary sequence matches seem to be sufficient enough to trigger cross-kingdom gene silencing, as exemplified by plants-parasites, bacteria-to-worms and other lower organism interactions (weiberg et al., 2015) . following a literature search as described in section 2.2.1.2 and based on the methodology described in the section 2.2.2.3, a total of 33 documents were selected for review of the topic molecular mechanisms of exogenous ncrna uptake and function. the uptake of exogenous ncrnas is described from the molecular mechanisms point of view (i.e. the presence of a specific transporter, if any). efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. although plant-derived exogenous ncrnas are the main topic of this subsection, some other examples of general molecular mechanisms of exogenous ncrnas uptake, intracellular trafficking and function are included as relevant to understanding the possible fate of plant-derived exogenous ncrnas. other general aspects of rnas uptake are reviewed in chapter 3.1.5, and the uptake of exogenous rnas from the pharmaceutical/medicinal area is covered in section 3.1.4. as previously mentioned in section 3.1.5, cellular uptake of ncrnas presents great challenges due to the physicochemical properties of these molecules. their size and negative charges prevent their easy entry into mammalian cells. cell membranes pose a major barrier to ncrnas entrance since entry into cells is tightly controlled and regulated (mcerlean et al., 2016) . the anionic lipophilic bilayer prevents entry of macromolecular anionic nucleic acids in their naked form, both restricting their binding to and passive diffusion across these lipophilic cell membranes . furthermore, even though their internalization may depend on the endocytic pathway, endosomal entrapment and lysosomal degradation can be major issues because of the reduced accessibility of ncrnas to their sites of action (nucleus, cytoplasm or mitochondria) (won et al., 2011; . many different carrier molecules have been developed to achieve the entrance of ncrnas (mostly mirnas and dsrnas) into target cells (bolhassani, 2011; lindgren and langel, 2011; mao et al., 2010; ragelle et al., 2014; rudzinski and aminabhavi, 2010; shum and rossi, 2016) . exogenous ncrnas must be carried by polymers, synthetic cationic lipids or cell-penetrating peptides neutralizing the negative charges of the nucleic acids. most of these cationic lipids tend to form liposomes when dispersed in an aqueous phase, such as blood. endocytosis has been suggested as the main pathway for this nucleic acids-cationic lipid complex internalization by the cell (el ouahabi et al., 1997) . most reports suggest that so-called cell-penetrating peptides (cpps) bind initially to negatively-charged proteoglycans at the cell surface and are internalized into endosomes (juliano et al., 2012) . there are multiple pathways of endocytosis (see also section 3.1.5). i. the clathrin-coated pit pathway is the archetype of endocytosis pathways. cell surface receptors and their associated ligands interact with adapter proteins and accessory factors, clustering the receptors into specialized membrane areas subtended by a network of clathrin triskelions. the clathrin network is invaginated by means of membrane curvature promoting specialized proteins, giving rise to a clathrin-coated endosome. this endosome quickly uncoats, generating an uncoated vesicle that will begin its intracellular journey (juliano et al., 2012) . ii. the caveolar pathway implies the presence of small cell membrane invaginations rich in cholesterol and sphingolipids containing caveolin1. cavins, which coat proteins helping to stabilize caveolar structures, are present in these invaginations. there is controversy as to whether caveolae generate independent intracellular vesicles or whether they remain as tubular structures linked to the plasma membrane; however, some data suggest that caveolae generate vesicles able to participate in intracellular traffic, generally presenting a smaller size than other forms of endocytotic vesicles (juliano et al., 2012) . iii. a number of clathrin-and caveolin-independent pathways have been described. these pathways are often defined in terms of the morphologies of the vesicles they generate or in terms of the cargo that is preferentially internalized. for instance, the flotillin pathway involves the presence of flotillin-rich membrane microdomains, where flotillins are membrane-inserted proteins that may be involved in ordering lipid domains and subsequent endocytosis, similar efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to caveolin (juliano et al., 2012) . another such clathrin-and caveolin-independent pathway is the clic/geec pathway, which seems to be particularly important for fluid phase endocytosis. clic/geec stands for clathrin and dynamin independent carriers/gpi-ap enriched early endosomal compartments. this pathway gives rise to high-volume tubular endosomes rich in gpi-proteins and that typically contain fluid phase markers such as dextrans (juliano et al., 2012) . additional clathrin-and caveolin-independent pathways exist, some involving dynaminmediated disjunction of vesicles from the plasma membrane (juliano et al., 2012) . iv. macropinocytosis is a process by which cell protrusions pinch off large volumes of extracellular fluid and is therefore an important pathway in fluid phase endocytosis. it is also involved in internalization of clustered, activated receptor tyrosine kinases. v. phagocytosis and entosis are large-volume internalization mechanisms as well. they come into play in specialized cells or unusual circumstances but do not play much of a role in oligonucleotides processing in most cell types (juliano et al., 2012) . vi. the actin cytoskeleton plays an important role in most of the endocytotic processes described above, although certain arenaviruses enter cells by a pathway independent of clathrin, caveolin, dynamin and actin. it has been reported that phosphorothioate antisense oligonucleotides seem to enter the cells by this pathway as well (juliano et al., 2012) . there have been many attempts to identify endogenous receptors for antisense or sirna molecules. however, no direct evidence for their involvement in oligonucleotide trafficking has been provided (juliano et al., 2012) . integrins of the beta-2 subclass as well as scavenger receptors have been suggested as candidates, but this is controversial. toll-like receptor (tlr) family members seem to be the most convincing examples of cellular receptors for oligonucleotides; tlr9 binds dna having cpg motifs, tlrs7/8 binds single-stranded rna, while tlr3 binds double-stranded rna. although these tlrs are usually found within endosomes rather than at the cell surface, in some cases they seem to be able to assist in accumulation of oligonucleotides by cells (juliano et al., 2012). one interesting candidate as a receptor for oligonucleotides is the mammalian homolog of the doublestranded rna (dsrna) transport protein sid-1 found in caenorhabditis elegans (feinberg and hunter, 2003) . the human homolog of this protein, sidt1, has been described as facilitating rapid contactdependent intercellular small rna transfer (elhassan et al., 2012) and as selectively binding long doublestranded rna (li, w et al., 2015) . another member of the sid1 transmembrane family, sidt2, has been shown to take up extracellular double-stranded rna in drosophila s2 cells (mcewan et al., 2012) via endocytosis, although in mammalian cells this protein has been located in lysosomal membranes (jialin et al., 2010) . in fact, some authors have proposed that sidt2 could mediate cellular rna degradation inside lysosomes through a novel type of autophagy called rnautophagy (aizawa et al., 2016) . another research group recently reported that sidt2 is required to transport internalized dsrna from endocytic compartments into the cytoplasm for immune activation (nguyen et al., 2017), while others have described that this protein mediates "gymnosis", facilitating uptake of naked single-stranded oligonucleotides into living cells (takahashi et al., 2017) . a more recent report indicates that both sidt1 and sidt2 not only do not transport rna, but are involved in cholesterol transport (mendez-acevedo et al., 2017) . once an oligonucleotide has entered a cell in an endosome, it encounters a complex maze of intracellular pathways leading to multiple destinations and regulated by an intricate protein machinery. key subcellular membrane bound compartments include early and recycling endosomes, late www.efsa.europa.eu/publications 156 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endosomes/multi-vesicular bodies, lysosomes, the golgi apparatus and the endoplasmic reticulum. intracellular trafficking is not a random process, but rather a carefully orchestrated choreography that allows the cells to transport endogenous and exogenous materials to the most appropriate places. for nucleotides to exert their function, they need to leave the membrane bound compartments and access the cytosol and/or nucleus (juliano et al., 2012) . knowledge as to how endocytotic cargos are delivered to subcellular compartments is still partial. many of the internalization pathways previously described converge at the stage of early endosomes, raising the question of how the differentially internalized components traffic to different destinations. certain evidences suggest that membrane domains originating from different internalization pathways maintain their identity within early endosomes, thus providing the means for specific sorting and trafficking to distinct downstream destinations (juliano et al., 2012) . all membrane traffic proceeds through the same basic steps: i) a coated vesicle is pinched off from a larger donor membrane compartment; ii) the vesicle uncoats, allowing display of the tethering and fusion proteins; iii) the vesicle is carried to its destination along "tracks" provided by actin-or tubulin-based cytoskeletal structures; iv) the vesicle recognizes its target membrane compartment using tethering proteins and then utilizes snare proteins to complete the fusion process and deliver membrane and contents to the target compartment (juliano et al., 2012) (figure 16 ). ncrnas must escape from the endomembrane compartments and reach the cytosol to exert their function. as mentioned above, intracellular trafficking involves a highly dynamic flux of membrane vesicles engaged in a multitude of fusion and disjunction events. there are a few key points in these processes to be considered when designing non-viral oligonucleotide delivery strategies: 1) fusion involves localized stress on the fusion partners, including formation of non-bilayer lipid domains; 2) non-bilayer regions of membranes can be much leakier than bilayer regions; 3) many enveloped viruses fuse with cells via specialized membrane interacting proteins that, while differing in sequence, act in a manner similar to cellular snare proteins; in many cases these proteins can also induce increments in membrane permeability. therefore, there is an intrinsic relationship between the fusion events inherent to intracellular trafficking and transient leakage of vesicular contents. thus, the innate activity of oligonucleotides taken up by cells is likely due to a modest amount of continuous leakage from endomembrane compartments spontaneously occurring during intracellular trafficking (juliano et al., 2012) . some ncrnas function is exerted in the nucleus, although nuclear entry may not be the rate-limiting step for oligonucleotide action. studies have shown that oligonucleotides, particularly those with phosphorothioate backbones, are able to continuously shuttle between the nucleus and cytoplasm. this is an active process mediated by nuclear pore structures but does not require classic nuclear localization signals. for phosphodiester oligonucleotides, both passive diffusion and active transport have been described as nuclear entry mechanisms (juliano et al., 2012) . in terms of the uptake and trafficking of "free" or "naked" oligonucleotides, the co-existence of both productive and non-productive uptake routes has been suggested. the non-productive pathway seems to involve trafficking to lysosomes, while the pathway that results in rnase h-dependent antisense effects eventually leads to interaction with cellular pre-mrna (koller et al., 2011) . other studies have involved so-called "gymnotic" uptake of antisense oligonucleotides modified with lna (locked nucleic acid) moieties (stein et al., 2010; zhang et al., 2011) . subcellular distribution studies surprisingly suggested that the deoxy lna compounds became associated with p-bodies that are usually thought to be sirna action sites (stein et al., 2010) . unlike the study performed with phosphorothioate www.efsa.europa.eu/publications 157 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. oligonucleotides in which the concentrations used were in the nanomolar range, the antisense effects of "naked" lna required micromolar concentrations (juliano et al., 2012). as described in section 3.1.5, exogenous ncrnas must overcome a series of barriers to be functional at systemic level. in addition to the extracellular barriers specifically related to oral absorption, defensive humoral and cellular barriers efficiently prevent the intrusion of exogenous entities into the organism (jeong et al., 2007) . systemically-administered nucleic acids are rapidly degraded by nucleases in a few minutes and, even in nucleic acid drug therapy using positively-charged polyplexes, they readily interact with serum components to form larger aggregates, resulting in rapid clearance by res or phagocytes (jeong et al., 2007) . if the nucleic acids survive in the blood stream, they must still reach the appropriate tissues and enter the target cells. in systemic nucleic acid therapy, continuous endothelial walls in the microvasculatures are one of the main barriers limiting the access of oligonucleotides to target cells. in certain pathological cases, such as cancer, the presence of leaky vasculatures in the vicinity of highly penetrating solid tumours allows nanoparticulates to penetrate and accumulate in tumours due to the enhanced permeation and retention effect (jeong et al., 2007) . caveolae-mediated transcytosis has been considered one of the important mechanisms of macromolecules transport across the endothelium (schnitzer, 2001) . another possible limiting barrier is the extracellular matrix consisting of various proteoglycans, which are proteins covalently cross-linked with carboxylic or sulphated glycosaminoglycans (gags) (ruponen et al., 2003) . since gags, such as heparin sulphate, chondroitin sulphate and hyaluronic acid, are polyanions, they may repel the negatively-charged oligonucleotides, affecting mobility of the nucleic acids in the tissue extracellular matrix and therefore limiting their access to target cells. in the case that oligonucleotides reach the target cell membrane, they must enter the cell. the first barrier is the anionic plasma membrane itself, and the second is cellular uptake via endocytosis (see section 3.1.5). without a specific ligand-receptor interaction mechanism, the entrance of ncrnas results in a lack of cell specificity (jeong et al., 2007) . once taken up by cells, the nucleic acids are localized within the endosomal compartments, where the ph rapidly drops to about 5 by the action of membrane-bound atp-driven proton pumps. the endosomes mature to lysosomes where the oligonucleotides can be degraded by various enzymes (jeong et al., 2007) . as previously indicated, any surviving nucleic acids must then escape from the endosome to exert their functional effects. in nucleic acid drug therapy, certain endosomal disruptive agents, such as lysomotropic chloroquine, fusogenic peptides and ph-sensitive neutral lipid, are used to facilitate or promote endosomal escape through membrane disruption (jeong et al., 2007) . after endosomal escape, ncrnas should move through the cytoplasm to encounter their molecular targets, or to the peri-nuclear space where nuclear translocation takes place if the nucleus is the target compartment. passive diffusion of high-molecular weight macromolecules is limited in the cytoplasm. a complex network of microfilaments and microtubules, highly concentrated proteins and various subcellular organelles provide the cytoplasm with a high fluid phase viscosity and mesh-like structure with 300 to 400 å pores from, making the diffusion process size-dependent (jeong et al., 2007) . in fact, microinjection of plasmid dna into the cytoplasm showed that normal dna was practically immobile in the cytoplasmic space (dowty et al., 1995) . however, dna molecules with less than 250 base pairs seem to undergo free diffusion in the cytoplasm (lukacs et al., 2000) , suggesting that small oligonucleotides (i.e. mirnas) do not face limitations regarding their intracellular trafficking, contrary to large ncrnas (i.e. lncrnas) which might encounter more restrictions reaching the peri-nuclear space. finally, some ncrnas need to enter the nucleus to be functional. the nuclear envelope consists of a double-layer membrane with nuclear pores. the nuclear pore complex allows free diffusion of ions and small molecules, but restricts passage of macromolecules with molecular efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. weights greater than 60 kda, unless accompanied by a nuclear localization signal peptide (nls) (jeong et al., 2007) , which is not the case with lncrnas oligonucleotides are initially accumulated in an endomembrane compartment (the donor compartment, e.g. early endosomes) and are then trafficked by shuttle vesicles to various other endomembrane compartments (the recipient compartment, e.g. the trans-golgi). the first step (1) involves disjunction ('pinching off') of a shuttle vesicle under the influence of a coat protein as well as other accessory proteins. at this stage there are non-bilayer regions at the junction between the membranes of the donor compartment and the shuttle vesicle. this provides an opportunity for some oligonucleotides to escape into the cytosol. step 2 involves uncoating of the vesicle; and rab proteins can contribute to this step. step 3 comprises movement of the shuttle vesicle toward its destination along cytoskeletal tracks. motor proteins such as various myosins (for the actin system) or dyneins or kinesins (for the microtubular system) propel the vesicle. rab proteins are involved in forming the appropriate linkages to the cytoskeleton. step 4 entails recognition of the recipient ('target') compartment by the shuttle vesicle. tether proteins work with rab proteins to provide interaction specificity while v-snare proteins in the vesicle membrane interact with t-snare proteins in the recipient compartment membrane to provide firm bridging, as well as contributing to specificity. in step 5 the snare proteins undergo major conformational changes, and, with the assistance of accessory proteins, trigger fusion of the shuttle vesicle membrane with the membrane of the recipient compartment. at this stage non-bilayer regions exist at the junction between shuttle and recipient membranes potentially allowing oligonucleotide escape. reprint with permission from (r l juliano, x ming, o nakagawa. cellular uptake and intracellular trafficking of antisense and sirna oligonucleotides. bioconjugate chemistry 23:147-157). copyrigth (2012) american chemical society. figure 16 : proposed mechanism of vesicular trafficking of oligonucleotides some research has been done on cellular uptake, trafficking and tissue distribution of oligonucleotides without specific targeting or carrier mechanisms. in terms of tissue distribution, certain cell types exhibit a preferential in vivo uptake, particularly kidney proximal tubule cells and liver kupffer cells for phosphorothioate (ps) antisense compounds and sirna (juliano et al., 2012) . however, only one report has studied systemic level uptake and function of orally administered mirnas . these authors studied intestinal uptake of maternal milk-derived mirnas by new-born mice employing genetically-modified models to distinguish endogenous mirnas from milk-derived exogenous mirnas. their analysis of the intestinal epithelium, blood, liver and spleen revealed no evidence for mirna www.efsa.europa.eu/publications 159 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. uptake, even though milk in lactating mothers has been shown to be a particularly rich source of secreted mirnas. sequencing and microarray analysis of mirnas in the milk of various mammalian species has led to the discovery of hundreds of mirnas in cow, pig, rat and human milk, which are derived from exosomes and cellular components. furthermore, their study revealed rapid degradation of milk mirna in intestinal fluid, indicating that mirnas in the milk play a nutritional rather than a generegulatory role in new-born mice . there are certain tissues containing special physical structures that can function as additional barriers to ncrnas entry. such is the case of the central nervous system (cns), where delivery of nucleic acids is particularly challenging because of its anatomical and physiological complexities. the cns is protected by the blood-brain barrier (bbb), which consists of tightly joined capillary endothelial cells, restricting access of large molecules into the brain. the cns extends to include the spinal cord, which is protected by the blood-cerebrospinal fluid barrier (bcsfb), made up of choroid plexus epithelial cells limiting the free diffusion of molecules into the cerebrospinal fluid (csf). the bbb and the bcsfb express numerous transporters and receptors which contribute to the transfer of essential nutrients such as glucose and amino acids into the cns. within the cns, different cell types exist including neurons, astrocytes and other glial cells. entry of nucleic acids into neurons is notoriously difficult for unclear reasons, although it is likely related to their post-mitotic nature, as well as the complex structures and intricacies of neuronal networking (o'mahony et al., 2013) . for instance, variances in the uptake of sirna lipoplexes (liposome and nucleic acid complexes) were reported at different parts of the neuron structure, with greater uptake efficiency at the cell soma compared to the neuritis, which may occur because of different membrane compositions in these domains (o'mahony et al., 2013) . circumventing the bbb is only possible by direct administration of ncrnas to a target region of the brain or by administration to the spinal cord where trans-synaptic retrograde transport to the brain is possible. some delivery systems exploit the receptor-mediated uptake of molecules such as transferrin, lactoferrin and insulin receptors by attaching a ligand for the receptor to the non-viral delivery system, or promoting a transient mechanical disruption of the bbb (o' mahony et al., 2013) . in certain neurological diseases, and in particular in brain cancers, anatomical and physiological changes occur that can impact bbb integrity and the cns accessibility. delivery to the csf to bypass the bbb presents its own obstacles, including rapid clearance from the csf, the bcsfb and limited diffusion from the csf into the brain parenchyma (o'mahony et al., 2013) (figure 17) . another specialized barrier is the placenta. the placenta serves as the interface between the maternal and foetal circulatory systems, and regulates the transfer of oxygen, nutrients and waste products. when exogenous substances are present in the maternal bloodstream, the extent to which these affect the foetus is determined by transport and biotransformation processes in the placental barrier. this barrier is formed by fusion of the outer blastocyst trophoblast cells facing the uterine epithelium into multinucleated syncytiotrophoblast. proliferation of syncytiotrophoblast is mediated by the fusion of precursor cytotrophoblast cells. maternal blood in the intervillous space and foetal blood in the villous capillaries are separated by a continuous layer of syncytiotrophoblast, a discontinuous layer of cytotrophoblast cells, basal lamina, connective tissue, and foetal endothelial cells (al-enazy et al., 2017) . the placenta undergoes several changes as pregnancy progresses. overall, the placental barrier becomes thinner over time, the distance separating the maternal circulation from the foetal circulation decreasing from 50-100 µm in the second month to only 4-5 µm at term. passive diffusion across the placenta is favoured for lipophilic substances, allowing them to cross the phospholipid bilayer. smaller compounds tend to cross the placenta more readily, with compounds having a molecular weight less than 500 da crossing it most easily (al-enazy et al., 2017) . since nucleic acids are polarized, negatively charged molecules, it is unlikely they would be able to cross the placenta by passive diffusion. nevertheless, the placental syncytiotrophoblast is capable of efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endocytosis (al-enazy et al., 2017) . no reports refer to exogenous ncrnas entering the placental barrier. however, the presence of placental mirnas has been described in the blood circulation of pregnant women. these mirnas have been reported to be actively secreted into microvesicles and exosomes. these specific exosomal mirnas may be important for embryo implantation, playing a significant role in the intercellular communication pathways that potentially contribute to placentation and development of maternal-foetal vascular exchange (mitchell et al., 2015) . since exosomes can cross the blood brain barrier, meaning they can cross several layers of cells, it has been speculated that exosomes could directly move from maternal to foetal tissues, and vice-versa, during pregnancy (record, 2014). the the molecular mechanisms of exogenous ncrna cellular uptake have been inferred from studies performed when developing strategies for nucleic acid delivery in therapeutics. few studies have been done with "naked" oligonucleotides, and even in these cases, the oligonucleotides were chemically modified. these molecular mechanisms of cellular uptake include the different types of endocytosis mechanisms known to date. the existence of receptor-mediated uptake of oligonucleotides through the sidt1 and sidt2 proteins has also been described, although their function has been contradicted since they have been described as cholesterol transporters. among the numerous challenges ncrnas must overcome once taken-up by the cell, escaping the endosomal compartments is the most relevant and significant, although reaching their site of action is also crucial to exerting their function. specialized barriers such as the blood-brain barrier or the placental barrier present further obstacles to overcome. www.efsa.europa.eu/publications 163 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. zhang y, qu z, kim s, shi v, liao b, kraft p, et al. 2011 . down-modulation of cancer targets using locked nucleic acid (lna)-based antisense oligonucleotides without transfection. gene ther 18, 326-333. zhou g, zhou y, chen x. 2017 following a literature search as described in section 2.2.1.2. and based on the methodology in section 2.2.2.3., a total 20 documents were selected for reviewing the landscape of exogenous rnas in biological fluids of human and animals. using public databases of rna sequencing datasets, several documents report the application of in silico bioinformatics analysis (table 25 ) clearly describing the widespread presence of exogenous ncrnas in biological fluids(from multiple sources, including diet) wang et al., 2013; beatty et al., 2014; yeri et al., 2017; . however, four documents suggest that exogenous ncrnas present in the public datasets may represent contamination from the laboratory environment since their abundance is extremely low, or they are of non-dietary origin, or could originate from technical artefacts or contamination bagci and allmer, 2016) . five documents report the development of bioinformatics resources to either predict transportable mirnas, predict functional analysis, or list and compare the exogenous mirnas found in samples chiang et al., 2015; zhang et al., 2016; zheng et al., 2017) . the generalized use of highly parallelized next generation (nexgen) sequencing technologies has promoted the use of sequencing to study complex biological systems, including human and animal biological fluids and tissues. initial studies evaluating the possible presence of exogenous ncrnas in biological fluids were done in silico using public databases of rna-seq results. several studies report the presence of exogenous rnas (i.e. dietary plant ncrnas) in the biological fluids of humans and animals. initial studies of the srnas spectra in human plasma showed that a small fraction of sequence reads from plasma mapped to human mirnas (≈1.5%) or human transcripts and human genome sequences (≈11%) . this fraction increased by up to ≈60% when a higher sequence mismatch tolerance was allowed (under two mismatch allowance). surprisingly, a significant number of the unmapped reads aligned with various bacterial and fungal sequences (i.e. ribosomal rnas and trnas). also, many processed reads mapped to common food items, the most abundant being rna sequences from corn (zea mays) and rice (oryza sativa). compared to data from a serum sample from a chinese individual, the sequence abundance between corn and rice was reversed , suggesting the influence of dietary habits. other sequences from other foods in human plasma samples included soybean (glycine max), tomato (solanum lycopersicum), grape (vitis vinifera) and others. including exogenous mirnas, various common household insects were identified . treating plasma samples with dnase, protease, triton x-100, and additional rnase reduced the total number of reads compared to the addition of rnase alone, suggesting that some of the exogenous rna molecules are probably associated to circulating protein and/or lipid complexes . in the same study, a lower percentage of exogenous sequences was observed in mouse plasma samples compared to human samples . another study evaluating a larger number of human plasma samples (n=183 young male athletes) reported exogenous srnas from different bacteria, but very few srnas were consistently detected in all samples (yeri et al., 2017) . in a study on public srna datasets from various tissues from mammals, chicken and insects, plant mirnas were found in 63 out of 83 datasets analysed, with mir-168 being extremely over-represented efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. . in 19 datasets (2 humans, 14 mice, 1 pig and others) the plant mirnas reads ranged from ≈0.053-0.456% of the total animal mirna reads. insects fed with plant foods containing mir-168, did not show accumulation of mir-168 when evaluated by northern-blot. however, in a very few samples sequencing data detected plant mirnas not present in the food source, including mir-1507, in addition to mir-168 at a moderate read, suggesting that plant mirnas observed in some public srna databases may be artefactual and due to sequencing methodology . plant mir-168a from monocots was also found to be the most abundant exogenous plant mirna, in most cases, in other srna sequencing datasets studies with levels typically <1% of human mirnas . but when analysis was performed in the absence of any known sources of plant contamination, plant mirnas were not detected. this suggests that precaution should be taken to prevent cross-contamination of the samples due to the high sensitivity of next-generation sequencing methods . other human public srna sequencing datasets have also been questioned for the presence of exogenous plant mirnas . of 410 analysed human plasma samples, 1301 plant mirnas were found when compared to the genome of 5 different plant model organisms (arabidopsis thaliana, triticum aestivum, oryza sativa, zea mays, and brachypodium distachyon). at least one read was present in selected samples. peu-mir-2910, found in all 410 samples, is a singular mirna found in selected samples with more than 1000 counts. mir-2910 is found in zea mays and conserved in fruits and vegetables including melon, sorghum, tomato, tea and oil palm. other highly-expressed plant mirnas found in up to hundreds of copies in selected samples include peu-mir-2916 (found in 379 samples), peu-mir-2914 (found in 359 samples), and tae-mir-2018 (found in 353 samples) . plant mir-159 (found in 32 samples) and mir-168a (found in 7 samples) were detected in relatively small amounts (less than 4 copies). using bioinformatics tools, the same study showed that seed sequences of mir-2910 and mir-2916 are similar to hsa-mir-4259 and mir-4715-5p for the former and has-mir-4652-5p for the latter, and thus potentially target several gene targets of their human mirna counterparts . other studies have also reported in mouse plasma samples the presence of exogenous rnas matching exogenous species including bacteria, fungi, viridiplantae and food items . these included mir-166d and mir-168a (corn or rice as the most likely source), and mirnas from worms and insects . abundant non-human rnas were found in other healthy individuals human plasma samples (n=3) assigned to metazoan, bacteria or fungi (beatty et al., 2014) . several exogenous srnas were found that could potentially derive from food items including fragaria vesca, medicago truncatula, lotus japonicus, glycine max, arabidopsis thaliana, and solanum lycopersicum (beatty et al., 2014) . twelve public srna sequencing databases were analysed for the presence of plant exogenous mirnas. breast milk exosomes from humans (4 samples, 86.37 million reads) and pigs (8 samples, 179.9 million reads) were analysed. although 17 plant mirna species belonging to 11 mirna families (6 samples) were found, they showed low abundance (<25 read counts for the highest expressed mirna). zma-mir-168a, zma-mir-156a, ath-mir-166a, ath-mir-319b and ptc-mir-319d showed the highest abundance levels, while in humans only 2 samples contained 35 plant mirnas from 25 mirna families (<194 read counts, for the highest expressed mirna, average of 2 samples). ath-mir-166a, pab-mir-951, pc-mir-472a, bdi-mir-168, aly-mir-167d, osa-mir-444b.2 and zma-mir-156a showed the highest abundance level in human samples . however, bagci and allmer reanalysed the data from lukasik and zielenkiewicz study and found that exogenous plant-derived mirnas in milk are likely to be contamination (bagci and allmer, 2016) . in a later observational study, lukasik et al. evaluated plant mirnas mir-166a, mir-156a, mir-157a, mir-172a and mir-168a and reported the presence of plant mirnas in human (n=6) breast milk, both in whole milk and exosomes . out of 5 mirnas, only 2 were found in exosomes (mir-168a and mir-156a). while the human has-mir-148a was found in all samples in a concentration range of 6.5-50 pm for whole milk efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. and 1.2-12.5 pm in exosomes fractions, plant-derived mirna mir168a was found in all samples at a concentration of 300-700 fm (≈100-fold higher than other plant mirnas) . compared to endogenous mirnas, the levels of exogenous plant mirnas were rather low and varied significantly between the samples . kang et al. analysed public sequencing srnas datasets from various human tissues and body fluids (n=824), including 215 from serum and plasma samples, 37 from exosomes, 180 from cerebrospinal fluids, 93 from liver, 197 from blood cells and 102 from brain . of note is that exogenous mirnas (xenomirs) were only found in 17% of the tissue samples (from 392 total samples), with several studies being completely void of any xenomirs. xenomirs were absent from most human tissues samples, or present at very low abundances (with a median of 3.5 read counts). no enrichment of xenomirs was found in tissues relevant to dietary intake (i.e. hepatocytes and blood cells). by contrast, out of 432 human body fluids datasets, xenomirs were found in 69% of samples, although at very low levels (median of 5 read counts). even though the blood brain barrier separates the cerebrospinal fluid from the bloodstream, xenomirs were found in comparable quantities in both , suggesting no depletion (as expected by the presence of the barrier) of xenomirs in body fluids separated from the main bloodstream. moreover, the lack of co-occurrence of xenomirs in cerebrospinal fluid and serum of the same individuals, do not support that xenomirs detected in cerebrospinal fluid samples have entered through the bloodstream, and thus do not support a dietary origin of xenomirs. the most common xenomirs in body fluid samples belonged to the clades for rodents (34%), dicots (29%), insects (13%) and euphyllophytes (11%), suggesting that xenomirs composition does not reflect human food sources. from 10 billion sequence reads (all samples), xenomirs were in low abundance, comprising only 0.001% of the total reads (40,055). the same study reported a controlled feeding trial in rats and piglets (see section 3.3.3 below for details). shu et al. (2015) used bioinformatics tools to characterize the structural characteristics of exogenous mirnas that can contribute to cross-species transportation and thus be transferred into human circulation . thirty-four thousand (34.000) mirnas sequences were compiled from 194 species (from five kingdoms including animal, plantae, fungi, protista and viruses), including dietary mirnas (5217 mirnas) from 15 types of common food species such as cow milk, breast milk, tomato, grape, and apple. using a support vector machine (smv)-based feature elimination strategy, 1102 features based on sequence, structure, and physicochemical properties were analysed to distinguish human circulating mirnas from the remaining sequences. predictions were made for 221 features (categorized into 8 groups) to better predict transportable mirnas in human circulation. known human plasma mirnas were ranked among the top of the list (dominated by animalia origin). only 14 dietary mirnas were ranked among the top 500 mirnas, five of which have a sequence identical to that in humans (bta-mir-487b, bta-mir-421, bta-mir-216, gga-mir-29a-3p, gga-mir-20b-5p) . while only one mirna from plantae ranked among the top 500 mirnas, 30 plantae mirnas ranked in the top 1000 as possibly transportable , suggesting that animal-borne mirnas may be subject to more significant absorption in humans than in plant mirnas . it is important to note that this study of evaluated 26,705 animalia mirnas and 7645 plantae mirnas, whether this discrepancy in the original number of evaluated mirnas may have biased the smv-based classifiers to mammalian transportable mirnas was not addressed. in the same context, a database called "dietary mirna database" that describes different dietary mirna parameters and functional analysis has been described and is freely available. additional databases for exogenous mirna discovery using rna-seq data are also available in the scientific community . using bioinformatics tools (i.e. rnahybrid database), other studies have also predicted the potential function of dietary mirnas in the human body . in a bioinformatics study of 25 plant mirnas, some similar functions between human and plant target genes were identified, such as ion transport and stress response . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analysing 563 public srna sequencing datasets from humans (382), mice (128), pig (6), chicken (4), fly (8), c. elegans (16) and many other nematodes, protozoa or prokaryota, zheng et al. developed a database (exo-mirexplorer) for exploring and comparatively analysing exogenous mirnas (zheng et al., 2017) . two hundred and thirty-seven (237) plant-derived mirnas were detected in 382 sequencing samples. common mirna families included mir-168, mir-156, mir-166, mir-167 and mir-172. mir-168a was the most frequently found mirna, and was detected in 95 human samples from 11 different studies (zheng et al., 2017) . the average abundance of mir-168a was 22,114.9 reads per million. in total, mir-168a was found in 155 samples from multiple species. mir-156a is another frequently observed exogenous mirna from plants, and was detected in 65 samples with an average abundance of 86.69 read per million (zheng et al., 2017) . regarding other biological fluids, exogenous rnas of bacterial origin were found in 204 (urine) and 46 (saliva) samples from young male athletes analysed for srna-seq (yeri et al., 2017) . plant small rna mir-2911 was detected in urine of mice fed honeysuckle decoction (yang et al., 2015b) . mir-168a has been also detected in serum when honeysuckle was supplemented with 400 pmol synthetic mir-168a. mir-2911 and mir-168a were detected in the urine of mice that had received cisplatin treatment (cisplatin administration produces acute renal failure) and consumed a honeysuckle decoction alone or supplemented with synthetic mir-168a. the increased detection of plant small rnas was attributed to disruption of the organization of small intestine epithelial cells produced by cisplatin treatment or longterm consumption (pre-fed) of honeysuckle (yang et al., 2015b) , by mechanisms as yet unknown. mir-2911 levels in urine reached ≈260 fm. in tissues, the overall percentage of exogenous sequences in human lung tissue (normal lung rna samples), was found to be less than 1% . in spermatozoa samples, using public srna sequences datasets, tosar et al. found high amounts of exogenous sequences in three samples . however, in their own analysis of three spermatozoa samples they found no detectable traces of any plant mirnas, suggesting that exogenous mirnas present in the public datasets may have been caused by laboratory environment contamination . in terms of the possible passage of plant mirnas through the brain-blood barrier, kang et al. analysed 180 samples from cerebrospinal fluid (csf) (from one study), which is separated from the bloodstream by the brain-blood barrier, and 102 brain samples (from 7 studies) . in general, xenomirs were absent from most of the human tissue samples, and, when present, they were found at very low abundance (17% of analysed tissues). although the brain-blood barrier would relatively protect the brain from dietary molecules, xenomirs were present in similar fractions in the exposed liver and in the relatively protected brain samples , suggesting, according to the authors, a nondietary origin . xenomirs were present in more body fluids samples (≈69% of samples) than tissue samples (≈17% of samples) and were identified in comparable proportions in the serum and cfs. this study does not provide support for the dietary origin of xenomirs (including plant exogenous rnas) meaning it may originate from technical artefacts . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in addition to mirnas reported by the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. very few studies have reported the influence of dietary or physiological factors on levels of exogenous ncrnas in biological fluids. in a small study (n=3 per group), wang and colleagues compared some abundant exogenous mirnas species identified in plasma samples from healthy subjects, colorectal cancer and ulcerative colitis patients . although ulcerative colitis patients apparently had increased levels of circulating mirnas of certain species (i.e. from a bantam family), a large variability of mirnas levels were observed among subjects of the three groups , suggesting that health/disease status may influence the presence of circulating mirnas in human plasma. tissue injury produced by excessive consumption of common pharmaceutical drugs can also influence exogenous rnas levels in plasma . wang et al. showed that acetaminophen overdose in a mouse model produced liver injury, and an increase of rna concentration in plasma compared to the control group was observed (from ≈200 to ≈600 ng/ml of total rna 8h after treatment). plasma ncrnas levels were affected by acetaminophen overdose, both endogenous and exogenous. plantderived exogenous mirnas levels were reduced, and this drop in plant rna was related to the observation that mice lose appetite after acetaminophen injection , suggesting the influence of diet on plasma plant-derived mirna levels. levels of exogenous rnas can be affected in case of drug-induced kidney damage. sera and urine from mice treated with cisplatin, a chemotherapeutic agent also producing small intestines epithelial cells disruption, showed increased plant exogenous ncrnas (i.e. plant mirnas), not observed in untreated mice (yang et al., 2015b) . the effect was not observed in a glycerol-induced model of acute renal failure (which does not affect small intestine organization). detection levels of exogenous plant mirnas, including mir-2911 and mir-168a, were below background levels in mice pre-fed with chow diet devoid of honeysuckle. however, when mice were pre-fed with honeysuckle and honeysuckle was administered (alone or mir-168a-supplemented) dose-dependent levels of mir-168a was detected in serum and elevated serum levels of mir-2911 were observed (yang et al., 2015b) . the study shows that long-term honeysuckle feeding potentiates absorption of dietary mirnas through a mechanism that likely differs from cisplatin treatment, suggesting that particular diets can modify the capacity to absorb small rnas, and that altered or damaged guts resulting from illness and/or therapeutic treatment may enhance dietary rna uptake (yang et al., 2015b) . while comparing the presence of exogenous srnas in three human plasma samples, beatty et al. reported that the largest proportion of reads matching plant sequences were found from one individual who reported following a vegetarian diet (beatty et al., 2014) , suggesting that the diet can influence levels of exogenous srnas of food origin. in another study comparing human breast milk for mirnas derived from plants, no significant differences were observed for exosomal breast milk plant-derived mirnas (mir-168a and mir-156a) between vegetarian or non-vegetarian volunteers . in the whole milk an increase in plant mirna mir-168a was observed in non-vegetarian versus vegetarian diet volunteers . although some studies (see above) have reported the presence of exogenous rnas in human and animal circulation and tissues, very few have focused on quantitative data relevant to risk assessment. wang et al. estimated the concentration of rna in plasma to be <0.3 pm based on an average of 100 ng/ml of rna in plasma with an average of 100 nt in length and a selected rna (i.e. mirna) sequence representing less than 0.01% of the total rna population. after 7 days feeding of herbals, yang et al. reported an increase of mir-2911 in the sera of mice from 25 fm (5-fold higher than control chow-fed animals) following ginseng intake to 207 fm (39-fold higher than control chow-fed animals) following honeysuckle intake . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the lack of enrichment of plant-derived small ncrnas in samples to be analysed by rna-sequencing (i.e. through periodate oxidation) might also influence the low enrichment of these exogenous sequences in biological fluids. however, although 2'-o-methyl modification of synthetic rna oligonucleotides 3'-ends can affect ligation activity of the t4 ligase in srna library preparation and may potentially result in srna sequencing bias and under-representation of srna with 2'-o -methyl 3'-ends in quantification experiments (munafo and robb, 2010) , others suggest a broad capability to detect plant mirnas during the sequencing procedure of small rna library construction . the available literature clearly suggests the widespread presence of exogenous rnas from multiple species, including plants, microbiota and many others, in human and animal biological fluids. evidence for plant-origin ncrnas, possibly relevant to risk assessment of ncrna-based gm, suggests that plantderived mirnas can be found in the biological fluids of humans and animals. whether these sequences are directly derived from dietary sources has not been clearly demonstrated. their overall low abundance, lack of enrichment in tissues most exposed to dietary changes, and their high variation between samples suggest that some may have originated as technical artefacts or contamination, as indicated by some authors. although few reports were available in this area, recent evidence also suggests that the levels of exogenous plant ncrnas in biological fluids may be influenced by diet, subject physiological or pathological conditions, or certain therapeutic treatments. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a literature search as described in section 2.2.1.2. and based on the methodology described in the section 2.2.2.3., a total of 48 papers were selected as being relevant to review of the systemic effects of dietary exogenous ncrnas. very few of these studies (14) provide supporting evidence (in favour) that dietary exogenous ncrnas can be detected in biological samples and/or exert a biological effect after dietary ingestion. by contrast, 10 studies provide contradicting evidence (against) that dietary exogenous ncrnas are either bioavailable or exert a systemic biological effect. one study provides evidence of the possible transfer of small ncrnas through the mammalian placenta . in addition, following a literature search as described in section 2.2.1.2. and based on the methodology described in the section 2.2.2.3, 12 papers were selected as relevant to review toxicological effects of dietary exposure to exogenous ncrnas in humans or animals. the first report of systemic effects of dietary exogenous mirna in animals was published in 2012 by . plant mirnas were detected in human chinese healthy donors, among which mir-156a and mir-168a were highly expressed. these mirnas were also detected in calves and mice. plant mirna levels were relatively lower, but at a similar concentration range as endogenous mammalian mirnas present in serum. periodate oxidation, which oxidize mirnas but not plant mirnas (which are 2'-o-methyl modified), confirmed that these mirnas were genuine plant mirnas as most mammalian mirnas in human sera were oxidized and failed to be sequenced by solexa . the plant mirnas mir-168a and mir-156a were also detected in different mice tissues, while plant mir-166a was not, although it was present in the sera. both mir-168a and mir-156a were primarily detected in microvesicles in c57bl/6j mouse plasma. foods were found to contain the plant mirnas mir-168a, mir-156a and mir-166a, including a chow mice diet at a concentration of 0.43, 0.54 and 0.66 fmol/g, respectively. fresh rice contained between 3 to 10-fold higher amounts than the chow diet. plant mirnas were detected in other foods including chinese cabbage, wheat and potato. plant mirnas were found to be stable in cooked foods. plant mir-168a and mir-156a were higher in the sera and liver of mice after 6h feeding with fresh rice. mir-168a also increased in the stomach and small intestines, but not in the kidney, of mice fed a chow diet or fresh rice. mice fed with total rna extracted from rice or synthetic mir-168a (methylated) showed an increased level of mir-168a in serum and liver 3h after feeding. in vitro stability assays suggested that plant mirnas had a much slower degradation rate than their synthetic form (without 2'-o-methylated 3'ends), suggesting that methylation has a protective effect on the stability of plant mirna . of note efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. is that mir-168a targets the liver-enriched gene low-density lipoprotein receptor adapter protein 1 (ldlrap1), which plays a role in facilitating the removal of ldl from the circulatory system (garcia et al., 2001) . direct binding of mir-168a to exon 4 of ldlrap1, which is in the orf (open reading frame), was demonstrated by luciferase reported assays. transfection of both pre-mir-168a and mature mir-168a resulted in decreased ldlrap1 protein expression (but not that of mrna) in hepg2 cells. moreover, transfection of the intestinal epithelial caco-2 cell line with mir-168a produced microvesicles with plant mir-168a which can be taken up by hepg2 and reduce ldlrap1 protein expression. mir-168a seemed to be associated to ago2 protein and other cells like 293t cells which were also able to package mir-168a in microvesicles and then deliver mir-168a into hepg2 . injection (iv) of antisense oligonucleotide against mir-168a into mice during fresh-rice feeding reduced mir-168a levels in the liver and reversed ldlrap1 repression. mice were finally fed with fresh rice for 7 days to evaluate the physiological function of food-enriched in plant mir-168a. serum and liver mir-168a levels were induced already at 1 day of feeding and the liver ldlrap1 protein was repressed after 1, 3 and 7 days. consequently, ldl levels in mouse plasma were significantly elevated on days 3 and 7 after fresh rice feeding. administration of an antisense oligonucleotide against mir-168a reduced plasma levels of mir-168a, repressed reduction of ldlrap1 in the liver and blocked the rice-induced elevation of plasma ldl levels, suggesting that elevation of fresh-rice derived mir-168a in the mouse liver specifically decreased liver ldlrap1 expression and that this caused elevated ldl levels in mouse plasma (figure 18 ). this study provides the first overall evidence of cross-kingdom regulation of gene expression through dietary plant mirnas. the authors further tested a mature mammalian mir-150 added to the chow diet for mice, and reported an increase in serum and liver levels of mir-150 after three days , leading to downregulation of liver c-myc expression . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. by analysing western human sera samples from 42 female stage ii-iii breast cancer patients, chin et al. detected multiple plant mirnas from arabidopsis thaliana and soybean (glycine max), but with sparse counts (chin et al., 2016) . among the detected plant mirnas, ath-mir-159a, gma-mir-159a-3p and gma-mir-159e-3p had the highest counts (≤37 read counts) and were over 6 times more abundant than other plant mirnas. circulating mir-159 was more abundant in female healthy donors (n=6) than breast cancer patients (n=30) as analysed by qrt-pcr. moreover, circulating mir-159 was less abundant in patients who relapsed with metastatic disease, suggesting that human serum mir-159 levels inversely correlate with breast cancer incidence and progression (chin et al., 2016) . mir-159 in human serum was detected in an extracellular vesicle-enriched serum fraction obtained by ultracentrifugation and was resistant to sodium periodate oxidation, suggesting its plant origin due to 2'-o-methylation of the 3' end (which makes plant mirnas more resistant to periodate oxidation). in situ hybridization confirmed the presence of mir-159 in tumour samples, suggesting that mir-159 in human serum is capable of entering breast tissue (chin et al., 2016) . the authors analysed several plant food items for the presence of mir-159 and found that broccoli is particularly rich in this mirna and most of this mirna was still present after cooking (boiling for 25 min, >50% remained stable, compared to fresh). in vitro studies showed that synthetic mir-159 (both double-stranded and 2'-o-methylated) reduced cell proliferation in breast cancer cell lines but not the non-cancerous breast cell line. extracellular vesicles from healthy human donors also have a similar effect. luciferase assays determined that mir-159 targets transcription factor 7 (tcf7) and binds to two different sites within the 3' utr. tcf7 is a transcription factor of the wnt signalling pathway and is upregulated in breast cancer. in vitro assays also showed that mir-159 inhibits breast cancer cell growth by targeting tcf7. oral gavage of synthetic 2'-o-methylated mir-159 (25 mg/kg mir-159 or scramble control oligos, daily) reduced tumour growth from day 16 of treatment until the end of xenograph experiments (5 weeks) (figure 19) , an effect that was lost when tumour cells stably overexpress tcf7 without the 3' utr (chin et al., 2016) . mir-159 was detected in xenografts of tumours by in situ hybridization analysis. overall, these experiments suggest that plant mirnas can inhibit cancer growth in mammals. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. liang et al. evaluated and quantified 16 plant mirnas in human plasma after consuming a single dose of watermelon juice or a mixture of fruits (liang et al., 2015b) . using taqman probe-based qrt-pcr assays and appropriate non-template controls, sixteen plant mirnas including ath-mir-156a, mir-157a, mir-158a, mir-159a, mir-160a, mir-162a, mir-163a, mir-166a, mir-167a, mir-168a, mir-169a, mir-172a, mir-390a, mir-528, mir-824 and mir-894 were evaluated and a standard curve generated for quantification. mirnas concentration ranged from 6.66 pm for mir-163a to 39,846.54 pm for mir-894 in watermelon juice, and from 0.01 pmol/kg for mir-166a to 55,192.46 pmol/kg for mir-894 in the fruit mixture. watermelon juice was orally administered to nine healthy male adult volunteers and plasma samples were taken 0.5, 1, 3, 6 and 9 h after drinking 2.5 l of juice. different mirnas followed different peak time or appearance kinetics in plasma after administration (liang et al., 2015b) . using six plant mirnas ( table 26 ) that showed typical kinetic absorption curves, the total amount of plant mirnas absorbed by the body was calculated. using the plant mirnas concentration in watermelon juice and the areas under the plasma concentration-vs-time curves (auc) of plant mirnas, the total uptake amount, total absorption amount and absorption rate for each plant mirna was calculated (table 25) . absorption rate ranged from 0.04% to 1.31%. mir-156a, mir-162a and mir-168a were found almost exclusively encapsulated in microvesicles. mirnas were validated in watermelon juice or plasma by northern blotting. using the same protocol, a fruit eating study was performed. each subject consumed an equal portion of watermelon, apple, banana, orange, grape, mango and cantaloupe (2.5 kg in total). while mirnas concentration varied, the kinetics of plant mirnas after fruit administration appeared similar to the kinetics after juice administration (liang et al., 2015b) . the authors also suggest that the real concentrations of plant mirnas may be underestimated and, for instance, the real concentration of mir-156a in the watermelon juice might be six times higher and its plasma levels four times the previously calculated (table 25) concentration (liang et al., 2015b) . adapted from (liang et al., 2015b) in a comment (correspondence) to the editor, the results of above article were criticized as being either a result of technical artefacts or a contamination problem, due to the presence of mir-528 in monocots and not in the dicot watermelon (witwer, 2015) . in a reply letter, the original authors indicated that mir-258 also exists in dicots and that the mirnas display a dynamic physiological kinetic absorption curve in human plasma, supporting the concept of uptake of food-derived mirnas by humans and animals (liang et al., 2015a) . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analysing mir-2911 by qrt-pcr in dried flowers and herbs used in chinese medicines or as tea, yang et al. reported different levels in different products including: sophora (≈6736 fmol/g), honeysuckle (≈5000 fmol/g), chamomile (≈5037 fmol/g), blue mallow (≈1127 fmol/g), ginseng flowers (≈278 fmol/g) and as low as 0.2 fmol/g in hibiscus . herbal feeding of male mice for 7 days increased plasma circulating levels of mir-2911 compared to that of chow-fed animals. honeysuckle increased 39-fold (207 fm) in sera levels, while chamomile increased 27-fold (147 fm), sophora 22-fold (120 fm), lavender 13-fold (71 fm), blue mallow increased 12-fold (64 fm), ginseng increased 5-fold (25 fm) and no significant changes were detected in hibiscus (3 fm). post-feeding urine levels of mir-2911 ranged from 160-fold higher (264 fm) for honeysuckle, and 82-fold higher (135 fm) for chamomile and to 17-fold higher (28 fm) for lavender . however, when chemically synthesized mir-2911 (2'-o-methylated, plant-specific) was administered after single dose feeding of 400 pmols, mice serum levels were elevated roughly 1-fold within 30 min after gavage but decreased to background levels ≈ 1h after gavage . mir-2911 was not associated with ago2 in sera and remained in the unbound fraction. clearance of mir-2911 and other plant-derived synthetic mirnas (mir168a, mir156a and mir161) were analysed in mice by tail vein injection of 50 fmols. sera collected from mice (5min, 30min, 1h, 3h and 24h post injection) showed that most mirnas were rapidly cleared from circulation. compared to the other mirnas administered at equal doses, mir-2911 levels were substantially higher at 5 min after injection but after 3 h the apparent clearance of all srnas tested was complete . in the same study, gut microbiota did not seem to influence mir-2911 absorption despite its 100-fold increase in their microbiome titre after honeysuckle consumption. honeysuckle feeding of mice was also found not to influence gut permeability, discarding its possible absorption due to changes in gut permeability . rapeseed (brassica campestris) bee pollen was evaluated for the possible absorption of plant mirnas in male mice after pollen oral feeding (10 g/kg). mirnas were analysed by srna sequencing after 3 or 6 h in mice serum (200 µl). from ≈ 10 m clean reads, only 132 reads were plant mirnas, which belonged to 34 mirnas. of the plant mirnas, bna-mir-166a (35 reads) and bna-mir-159 (22 reads) showed the highest levels in mouse blood and were mapped to the rapeseed genome. the rest of the plant mirnas had ≤ 8 reads, of which 23 mirnas had ≤ 2 reads. both mirnas, mir-166a and mir-159, were also detected in the rapeseed bee pollen as evaluated by qrt-pcr, with higher abundance for mir-166a. serum levels of mir-166a after 6h of rapeseed bee pollen ingestion versus a control were ≈4 pm for treated animals while in chow diet fed animals it was ≈1.5 pm, suggesting that plant mirnas from rapeseed bee pollen can be absorbed by mice by oral ingestion and detected in systemic circulation . evaluated herbal medicine honeysuckle mirnas before and after decoction (boiling for 30 min, ≈0.2 g/ml water). from ≈10 m reads, ≈0.5 m reads of 148 mirnas were obtained, from which osa-mir-166g, peu-mir-2911 and peu-mir-2914 contained more than 10.000 copy numbers. most mirnas were degraded by decoction (i.e. mir-166 g degraded from 222.051 to 315 reads), while mir-2911 remained high (11.322 reads) reaching 70% of total mirnas in the final honeysuckle decoction. concentration analysis of mir-2911 showed a reduction from 0.34 pmol/g in honeysuckle to 0.06 pmol/ml in decoction when analysed by qrt-pcr, or from ≈0.40 pmol/g to 0.08 pmol/ml when assessed by northern-blot . compared to other plant mirnas, the apparent high resistance to boiling of mir-2911 was also supported by its high resistance to rnase treatment, which was dependent on its unique sequence and high gc content. oral administration (n=6 per group) of a single dose (500 µl) of honeysuckle decoction (mir-2911 concentration ≈0.12 pmol/ml) resulted in a maximum peak level in mice plasma and lung tissues 6h after ingestion (i.e. plasma levels increased from 242.0 to 1189.2 fm). most plasma mir-2911 was found in cell-derived microvesicles and associated efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to the ago2 complex. continuous drinking of decoction during three days (0.06 pmol/ml, ≈4 ml/day) increased plasma levels from 250.5 to 831 fm, while no changes were observed in the control mice (from 215.8 to 253.3 fm). levels of mir-2911 in lung and liver tissues increased from 8 to 10-fold, while the intestines and kidneys remained unchanged. synthetic mir-2911 administration (1 nmol/ml, 100 µl given once per mouse, n=5 per group) resulted in an increase at peak (3h) from 239.3 to 1655.7 fm in plasma and also an increase at peak (6h) in lung tissue, suggesting overall that atypical dietary mirna mir-2911 is absorbed and delivered to tissues . studying its possible biological function, zhou et al. found influenza virus h1n1 encoded mrnas targets of mir-2911, i.e. virus-encoded proteins pb2 and ns1, which are relevant for viral replication (zanin et al., 2017) . luciferase activity assays confirmed that mir-2911 binds to pb2 and ns1, which is lost by point-mutation of mir-2911 binding sites of the individual viral gene sequences. in vitro h1n1 replication was reduced by synthetic mir-2911 or total rna extracted from honeysuckle decoction. the inhibitory effect was abolished by co-transfection with anti-mir-2911 antagomir. moreover, no effects in viral replication were observed when pb2 and ns1 mutant virus were used with synthetic mir-2911 or honeysuckle decoction rnas . in vivo administration of honeysuckle decoction or synthetic mir-2911 (0.1 nmol per day) by gavage one day before and during 7 days after inoculation reduced weight loss and viral titre in lung tissues. these results were abolished when the anti-mir-2911 antagomir was used concomitantly. moreover, weight loss and high viral titre were not affected when pb2 and ns1 mutant virus were used, and synthetic mir-2911 or honeysuckle decoction administered . the authors also showed similar effects for synthetic mir-2911 or honeysuckle decoction with other influenza viruses, including h5n1 and h7n9, tested in vitro and in vivo (figure 20) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. regarding this particular mirna, yang et al. observed a different bioavailability of plant-derived mir-2911 vs. its synthetic 2'-o-methylated counterpart . oral administration of cabbage extract containing 12.5 pmol of mir-2911 per 500 µl gavage volume was compared with a 32-fold higher dose of synthetic 2'-o-methylated mir-2911 (400 pmoles per mouse). after gavage, mice plasma levels of cabbage mir-2911 reached an approximately 2-fold increase (≈23 fm) versus that of synthetic mir-2911 (≈10 fm), suggesting that mir-2911 derived from plants is more bioavailable than synthetic rnas . whether this observation can be generalized to other dietary plant mirnas is unknown. the authors also showed that mir-2911 was not present in exosome vesicles that were proteinase-k resistant in circulation . du et al. evaluated the presence of srnas in hong jing tian (hjt, rhodiola crenulata), which is a chinese herbal medicine. male c57bl/6j mice were fed with hjt rnas by gavage for three days (250 µg/kg per day) and lung tissue collected at 12, 24, or 48 h (n=3 mice per time). the srna sequences of lung tissues (top eight ranked) that were also present in the plant hjt had ≈200k read counts . one of the small rna (hjt-srna-m7) was effective in reducing in vitro transfected gene expression vectors for α-sma, fibronectin and collagen type i α1 (col1a1) in a tgf-β1 induced fibrosis model. although the type of srna is not clearly described, hjt-srna-m7 targets the 3' utr of these three genes. using the hjt-srna-m7 agomir (cholesterol-conjugated srna), by intratracheal administration (8 mg/kg in 100 µl of saline) (n=10 mice per group), the authors showed that fibrosis was reduced in a bleomycin-induced fibrosis mouse model . due to the relevance of liposomes in delivering rna molecules, the authors evaluated the possible formation of liposomes during decoction (boiling for 35 min) with plant lipids. they found that phosphocholine lipid molecules were also present in the decoction and suggested that liposomes may be formed that might facilitate the entry of rnas from plants to human cells and tissues; this was not tested . in another study, mlotshwa et al., described oral administration of a cocktail of tumour suppressor mirnas (normally downregulated in colon cancer) and a reduction of tumour burden in the apc min/+ mouse (c57bl/6j) model of colon cancer . mir-34a, mir-143 and mir-145 were chemically synthesized with the same mice sequence, but with a methyl group on the 2' position of the ribose at the 3' end, which is a chemical characteristic of plant mirnas (see section 3.1.1). for 28 days (starting at 5 weeks of age), mice (n=7 per group) received either total plant rna spiked with the mix of the three mirnas, total plant rna alone (from a. thaliana) or water. oral dose was 61 µg of total plant rna alone or plus 4.5 to 7.4 µg (corresponding to 0.7 to 1.0 nmole) of each species of tumour suppressor mirnas. tumour burden was dramatically reduced in mice receiving the mirna mixture. moreover, mice receiving the plant rna alone also showed a reduced tumour burden, although it was not statistically significant. after periodate oxidation, intestinal levels of mir-34a were 10-fold higher than that of the group receiving water alone . mir-143 and mir-145 intestinal levels failed to be detected due to high background of endogenous mice mirnas after periodate oxidation. although the study does not directly evaluate the mirnas produced by the plant, mirnas designed to mimic small rnas produced in plants seemed to be taken up by the digestive tract of apc min/+ mice upon ingestion and exert a biological effect . oral ingestion of total rna isolated from brassica oleracea (50 µg) resulted in the detection of plant mir-172 (the most highly enriched exogenous plant mirna in b. oleracea) in blood of icr mice between 2 h and 72 h after feeding . mir-172 was also detected in the spleen (0.38-0.04% recovered of orally administered mir-172), and merely detected in the liver or kidney. no evidence of sex differences was reported . at lower doses of total rna (30 µg or 10 µg), no mir-172 was detected in the blood of mice . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. using pigs as experimental animals, luo et al. evaluated the detection of plant-derived mirnas in the serum and tissues after feeding fresh maize to pigs (sus scrofa). jinhua female pigs consumed a fresh maize diet and water ad libitum for 7 days (n=3), and blood and different tissue samples were obtained for plant-derived mirna analysis. eighteen maize mirnas were evaluated (16 were detected) in serum and tissues, and exhibited relatively low abundance in the pancreas and longissimus dorsi muscle tissue . periodate oxidation treatment of the samples confirmed the detection of zma-mir-164a-5p, zma-mir-167e-5p, zma-mir-168a-5p, zma-mir-319a-3p, and zma-mir-408a-3p (while the rest were completely degraded), suggesting that these mirnas are bona fide plant mirnas (figure 21 ). in a separate experiment, female pigs were fed one meal with fresh maize (1 kg per pig) after overnight fasting. serum was collected at different times (i.e. 0, 1, 3, 6, 12, 18 and 24h). after 24h, pigs were provided with fresh maize diet ad libitum, and serum collected at 1, 3, and 7 days and tissue collected after sacrifice at 7 days. the serum levels of some of these mirnas (mir-164a-5p, mir-166a-3p, mir-167e-5p, mir-168a-5p and mir-319a-3p) gradually increased after one feeding of fresh meal, reaching peak values at 6 or 12 hours within the first 24 hours and maintaining stable detectable levels (by qrt-pcr) in the serum following 7 days of access to fresh maize. the changes in mirna expression at all measured time-points in most cases were modest (≈2 to 3-fold increase). the five mirnas were primarily present in serum exosomes (≈58.2% of concentration in the serum) ( figure 21 ). using bioinformatics, 50 potential targets were determined within the porcine mrnas for zma-mir-164a-5p. using in vitro luciferase assays with porcine kidney cells, the authors showed that zma-mir-164a-5p binds to three of these potential target genes including cspg4, otx1 and plagl2, an interaction lost when their mutant constructions were used, suggesting overall that dietetically-absorbed mirnas can target endogenous porcine mrnas . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. several of the above-mentioned studies have reported the presence of dietary plant mirnas in circulation associated to extracellular vesicles or exosomes (chin et al., 2016; liang et al., 2015b; . this could be relevant for their possible biological effects, as extracellular vesicles can deliver a cargo to several tissues, including crossing the blood-brain barrier, and other biological barriers such as the placenta (matsumoto et al., 2017; yang, t et al., 2015; jayabalan et al., 2017) . their presence in microvesicles could modify their function, for example, it has been described that in some cases delivered naked mirnas cannot exert the same effects as those exerted by delivered exosomal mirnas (thomou et al., 2017) . also, exosomes as therapeutic drug carriers and delivery vehicles are gaining relevance in therapeutics (aryani and denecke, 2016; ha et al., 2016) . recently, it has been described that edible plant-derived exosome-like nanoparticles can be isolated from these plants (ju et al., 2013) , can transport ncrnas (i.e. mirnas) (mu et al., 2014) and can exert a biological effect (raimondo et al., 2015) , highlighting their role as therapeutic nanoparticles . it is still unknown if these exosome-like nanoparticles contribute to the resistance of ncrnas in the harsh conditions of the gi tract and to the absorption of plant ncrnas or the formation of exosomes circulating in biological fluids. regarding other biological barriers, only one study has reported on the transfer of small ncrnas through the placenta. li et al. found that exogenous plant mirnas were consistently detected in the umbilical cord blood and amniotic fluid of healthy chinese pregnant woman . this belongs to foetal circulation system, suggesting that mirnas may transfer through the placenta to the foetal side. three hours after gavage feeding of synthesized mature mirnas of the influenza virus to pregnant mice (at last 14-day pregnant and with mature placenta) increased mirnas levels were detected in the maternal plasma. an increase of exogenous mirnas levels in the foetal liver was also observed . to determine if mature plant mirnas can pass through the placenta and efficiently enter foetal organs, the traditional chinese herbal honeysuckle was tested. mice were gavage fed with 0.5 ml honeysuckle decoction (0.25 pmol/mice of mir-2911) and maternal plasma and foetal liver collected 3 h after gavage feeding. plant mir-2911 levels were elevated ≈3.5-fold in the maternal plasma (reaching 180 fm), ≈2-fold in the placenta and ≈2.5-fold in the foetuses, suggesting that plant mirna mir-2911 in honeysuckle can efficiently transfer through the placenta to enter the foetal liver (figure 22) . the amount of circulating mir-2911 increased only in microvesicles, suggesting that circulating mir-2911 was primarily present in microvesicles and that transplacental transmission may involve a microvesiclesmediated pathway . the same study also tested gavage fed synthetic sirnas (5 nmol), which were found in the plasma and foetal liver. protein targeting of the sirna was dramatically reduced, suggesting that exogenous sirnas delivered from the mother to the foetus by trasnplacental transmission may regulate foetal gene expression. direct injection of microvesicles loaded with sirnas also reduced their mrna target in the foetus liver, suggesting overall that exogenous small ncrnas can be transplacentally transmitted from the mother to the foetus. although outside the scope of this review, other non-plant derived exogenous ncrnas have also been reported to exert systemic effects in animals. bovine milk-derived extracellular vesicles (bmevs) were orally administered to il-1ra -/mice on the balb/c background, which spontaneously develop polyarticular arthritis, from week 5 to week 15. drinking water was also supplemented and administered to a collagen-induced arthritis (cia) dba/1j mice model starting at week 1 before immunization to day 40. mice receiving the higher dose of 28 x 10 6 bmevs (≈1200 micrograms/ml) showed a considerable delay in disease onset in the il-1 ra -/mice model. moreover, in the cia model the higher dose administered in drinking water 14.3 x 10 6 /ml (≈115 microgram/ml) reduced arthritis incidence and severity, accompanied by reduced expression of inflammatory cytokine il-6 and mcp-1 . although bmevs contained mirnas mir-30a, mir-223 and mir-92a, it is unclear if they are efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. responsible for the observed biological effects. in a similar study, oliveira et al. administered bmevs (particle concentration of 4.7 x 10 6 /ml or 14.3 x 10 6 /ml) in the drinking water to female dba/1j mice for 7 weeks and observed an increase in osteocyte numbers. moreover, the highest concentration of bmevs increased the percentage of woven bone compared to the pbs group, and a reduction of the adipocyte area in the bone marrow (oliveira et al., 2016) . although the bmevs also contained mir-29a (oliveira et al., 2016) , its implication in the biological effects is unknown. exogenous plant-derived ncrnas can pass the placenta and reach the foetus. by analysing small rnas components in royal jelly, honey, beebread and pollen collected during the cabbage (brassica campestris) flowering stage, zhu et al. found that honeybee srnas were present at a far higher level in royal jelly than in beebread and pollen, while the abundance of cabbage small rnas gradually increased from royal jelly to honey, beebread and pollen . several mirnas were detected in the samples, the plant mirnas occurring at far higher concentrations. moreover, beebread and polled mirnas were at a much higher concentration and their composition of individual mirnas was highly similar. the 16 plant representative mirnas with the highest concentration were mir-156a, mir-157a, mir-158a, mir-160a, mir-162a, mir-166a, mir-166g, mir-167a, mir-168a, mir-172a, mir-172c, mir-390a, mir-397a, mir-403, mir-824 and mir-845a. the same analysis in samples collected during the camellia (camellia japonica) flowering stage showed similar results and 13 out of the 16 enriched mirnas were similar, suggesting that plant mirnas in beebread and pollen may not be very diverse between different sources . absolute concentration of the selected plant mirnas ranged from <0.1 fmol/µg of total rna (almost undetectable) in royal jelly and honey, to a maximum of ≈10 fmol/µg total rna in pollen and beebread. feeding experiments to larvae with either rna purified from cabbage pollen (levels of mirnas similar to natural beebread mirnas levels) or a efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. synthetic mix of the 16 plant mirnas, increased whole body accumulation of the 16 representative plant mirnas and developed worker bee-like characteristics (reduced weight and size, extended pre-adult development time and decreased ovary size). ninety-six honeybee genes were predicted to be targeted by the 16 plant mirnas, some of which are known to influence the developmental fate of honeybees (i.e. apis millifera tor, amtor). luciferase assays showed that mir-162a directly targets aptor mrna. dietary supplementation of larvae food with synthetic mir-162a decreased amtor mrna levels in honeybees and showed reduced body weight, size and ovary size . similar experiments were performed in drosophila melanogaster (dm) with total pollen rna, a synthetic mirna pool or mir-162a alone, and in all cases similar phenotypes were obtained (delayed drosophila development, reduced final size, ovary size and fecundity). dmtor was also a target of mir-162a. overall, this study implies that plant rnas can be transmitted between species of different kingdoms. the literature also describes some in vitro studies of plant mirnas and their possible effects on mammalian cells. for example, exposure (24h) of plant mirnas extracted from a glycyrrhiza uralensis decoction to human pbmcs produced apparent cell aggregation and increased cell number and hla-dr+ cell proportion (shao et al., 2015) . in silico studies have also attempted to predict possible plant mirnas with potential targets in humans . as described before (see section 3.1), in contrast to mammalian mirnas, plant mirnas possess a 2'-omethylation at the 3'-end that stabilizes and provides resistance to rnases and oxidation. this characteristic has been used to improve the quantification method for mature exogenous mirnas from plants in biological mammalian matrices . thus, periodate oxidation and βelimination were introduced to evaluate the origin of exogenous mirnas. male c57bl/6 mice were fed with corn/soy-based rodent chow diet for 2 weeks. plant mir156a, mir164a and mir167a were evaluated. these mirnas were detected in abundant amounts in a corn/soy-based chow diet, but after periodate oxidation and β-elimination 30.6%, 38.4% and 23.2% of mir156a, mir164a and mir167a were determined to be in methylated forms (2.0, 0.19 and 21.4 fmol/g of diet, respectively). the control diet showed no differences between the diet and non-template control ct values after periodate oxidation/β-elimination. this study also suggests the use of exogenous rnas (spike-in), both methylated and un-methylated forms of mirnas, to ensure appropriate periodate oxidation/β-elimination . no detectable levels of plant mirnas were found in plasma, liver or faeces samples from the mice fed the corn/soy-based chow diet, since no differences occurred between the diet and the non-template control after periodate oxidation/β-elimination . this suggests that false positive results can be resolved using treatment with periodate oxidation/β-elimination. plant mirnas from extra virgin olive oil (evoo) and beer have also been evaluated by small rna sequencing. however, very few sequences (≤16 reads) were identified, and most aligned to soybean and orange . a feeding study of a single dose ingestion of 40 ml of evoo in healthy adult volunteers (n=5) was performed. mirnas analysis at baseline and after 2h ingestion was performed. no plant mirnas were detected in plasma after single ingestion of evoo . only one read count was found for other species mirna vvi-mir3623-3p from grape, while two read counts were found from medicago trunculata, grape or brachypodium distachyon when 2 mismatches were accepted . this suggests that beer or evoo do not contain mirnas that contribute to dietary intake, even though mirnas were reported in the pulp of olive (olea europaea) (donaire et al., 2011) . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. dickinson et al. evaluated a controlled mouse feeding study with rice-containing chow diets (modified ain93-g with 40.8% of rice or rice-based with 75% of rice) or with a purified synthetic chow devoid of plant grain or forage for 1, 3 and 7 days (figure 23) . the "synthetic chow" had negligible levels of osa-mir-168a, the "balance rice chow" (40.8% of rice) contained 227 reads, while the rice grain contained 279 reads. the concentration of osa-mir-168a in each diet was 0.0025, 54 and 114 fmol/g of diet for synthetic chow, balance rice chow and rice chow, respectively. plasma and liver analysis of mice fed the diets did not reveal measurable uptake of any rice grain mirnas, including osa-mir-168a. indeed, fewer than ten reads were detected in five out of eight samples from mice fed on rice-containing chow and four out of five samples from mice fed on synthetic chow . since the synthetic chow contained no grain or forage from plants, this low number for rice mirnas-mappable reads can only be explained by sequence errors or cross-contamination. in agreement with , the authors indicated that animals fed a chow containing high levels of uncooked rice (%) had significantly increased plasma low-density lipoproteins (ldl) levels at 3 and 7 days after treatment initiation. however, this increase in ldl was not observed in the balanced rice/chow group (40.8% rice), which contained 54 fmol/g osa-mir-168a. the increase of ldl was attributed to nutritional imbalances between the test and control groups . ldlrap1 protein was also evaluated by elisa in mouse liver samples, and no changes in levels were observed in any group at any time, suggesting that dietary intake of osa-mir-168a (from 54 to 114 fmol/g) does not produce rnai-mediated modulation of ldlrap1 protein levels in mouse liver . efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. a commercially available plant-based, plant mirna-rich substance containing soy and fruit materials, but not animal products, was administered by gastric gavage (≈5% of estimated blood volume for each animal) to male macaques (macaca nemestrina). relative abundance of plant mirnas mir-160, mir-156, mir-166, mir-167, mir-168 and mir-172 were analysed by qrt-pcr in the plant-based dietary substance. for instance, for mir-160 a consistent and efficient amplification through at least 35 pcr cycles was detected. plant mirnas were analysed in plasma samples of macaques (n=2) before and after 1, 4 and 12 h oral gavage of plant rich mirnas. qrt-pcr results indicated late amplifications of some plant mirnas, but results were highly variable. mir-172 did not amplify before 42 cycles, and mirs-166, -167, and -168 had a median cq greater than 35. mir-168 non-template controls also showed regular amplification within the same cq range. mir-160 exhibited a tendency to increase after ingestion, but was highly variable, and cq cycles were greater than 35. this was not the case for endogenous animal mirnas or the spike-in control, supporting the case that the late apparent amplification of plant mirnas in plasma was non-specific . moreover, droplet digital pcr analysis showed that counts from plasma were generally very low and of variable intensity, as occurs in non-specific amplifications. this even occurred in mir-160 (plant mirna with relatively high apparent count by qrt-pcr), which showed large number of counts but with variable intensity plots ("rain" appearance) (figure 24) , and is consistent with non-specific amplification . overall, the study (though only 2 subjects were included) suggests that the detection of plant mirnas in plasma does not support a general and consistent uptake of dietary plant mirnas . : substantial levels of mirna in oral diets consumed by macaque but negligible steady-state presence of diet-derived mirnas in plasma or organ tissues. snow et al. (2013) analysed the presence of exogenous plant mirnas after regular intake of a western diet containing fruits in healthy human subjects, and mir-21 in plasma or organ tissues of mir-21 -/mice . the conserved plant mirnas mir-156a, mir-159a and mir-169a were highly the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. expressed in many fruits common in the human diet such as ripened avocado, apple, banana, orange and cantaloupe. mir-156a and mir-159 reached up to 1000,000 copy numbers/mg food. the conserved animal mirna mir-21 was expressed at substantial levels (≈100,000 copies/mg food) in meat but not in fruits. mouse vegetarian and soy-enriched diets also contained high quantities (up to 1000,000 copy numbers/mg food) of mir-156a and mir-159a, but not mir-21. in contrast, mouse diets enriched with animal products contained elevated levels of mir-21, suggesting that conserved plant and animal mirnas are commonly present in oral diets for various animal organisms. although the dietary record of 7 of the 10 healthy adults reported the intake of fruits replete in mir-156a, mir-159a and mir-169a the day prior to plasma harvest, these plant-derived mirnas were undetectable in plasma samples. following 4-week feeding with a custom animal lard diet containing mir-21 no detectable levels of mir-21 were found in plasma samples of mir-21 -/mice, while high quantities of this mirna in plasma were seen in control wild type mice fed the same diet . in tissues (liver, lung, kidney and stomach) mir-21 was either not detected or detected at exceedingly low levels corresponding to less than one copy of mir-21 per cell (assuming that the mammalian cell expresses roughly 10 pg of total rna). wild type-mice showed only a non-significant trend toward increased plasma levels of mir-156a after consuming either a vegetarian or soy-enriched diet when compared to mice fed a lard diet for the same period (4-week) ( figure 25) ; however, levels were very low. indeed, mir-156a was detected in organ tissues (liver, lung, kidney and stomach) but at levels of less than one copy of mirna per cell and regardless of diets. mir-159a and mir-169a were not detected at all. fresh avocado containing three specific plant mirnas was also fed to mice (4.5 g per mouse over 24 h). even though the three mirnas were found in the stomach content of mice at high levels, very low levels were found in plasma and organ tissues, reaching levels lower than one copy mirna per cell in tissues. this provides no evidence of substantial steady-state presence of these plant mirnas in organ tissues after dietary intake . assuming that, on average, at least 100 copies per cell (>100 copies/pg srna per cell) are necessary to achieve canonical target gene repression (brown et al., 2007) , at least 10 15 copies of a given plant mirna must be ingested and delivered for widespread biological activity in humans (the average human body contains ≈10 13 cells). based on the concentration in ripe cantaloupe (≈6 x 10 6 copies mir-156a/mg fruit), an individual would need to consume 1,670 kg of cantaloupe just to release this number of copies in the gi tract . in terms of srnas, the literature describes the threshold for target gene regulation to be between 1000 and 10000 copies of mammalian mirna per cell . in an analysis of activity for hundreds of mirnas, only the most abundant mirnas in cells were found to mediate targeted repression and a large amount of mirnas had no discernible activity (mullokandov et al., 2012) . this suggests that only a minimum amount of mirnas need be reached to exert a biological activity, although this will also depend on the amount of target transcripts. a study mentioned above the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analyses of other common plant mirnas reported by others to be bioavailable were not successful. this implies that although the transgenic mirnas had resistance to degradation comparable to that of mir-2911 and high expression levels in plants, they were not readily bioavailable . huang (huang et al., 2018) analysed the possible bioavailability of orally-ingested corn mirnas in mice when incorporated into a rodent diet and consumed for 14 days. in an initial experiment, male c57bl/6 mice (n=10 per group) received either water (control), random nucleotides (25 µg, equivalent to the same amount of small rnas) or purified small rnas isolated from corn kernel (25 µg, equivalent to the same amout of random nucleotides). in the second experiment, mice received either a control diet (ain-93m), ain-93 + 3% autoclaved corn kernel powder or ain-93 + 3% fresh corn kernel powder. mirna levels in the isolated rna contained 56.03 pg mir-156a, 13.2 pg mir-164a, and 1215.63 pg mir-167a per 25 µg corn srna isolates. the diet supplement with fresh corn powder contained 173 pg mir-156a, 65 pg mir-164a, and 515 pg mir-167a per gram of diet. autoclaving the corn powder at 121ºc for 30 min reduced the amount of initial mirnas by more than 96%. in the first study, liver and whole blood sample mirnas (analysed by qrt-pcr), showed no differences in ct values between the three groups. after periodate oxidation followed by β-elimination, no differences were detected between the no-template control group and the experimental groups. similar observations were made in the liver and whole blood in the second study using fresh corn powder. also, after periodate oxidation followed by β-elimination, no differences were detected between the no-template control group and the experimental groups in the liver and blood samples. analysis of corn mirnas in cecal and faecal samples www.efsa.europa.eu/publications 186 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. showed similar results after periodate oxidation, although certain minor differences remained between the no-template control group and the experimental groups. in all cases, less than 0.1% of total the mirna tested in both studies was recovered from the faecal samples. moreover, analysis of the mirnas in the content collected from different parts of the mouse gi tract from the animal studies administered via gavage or dietary intake showed that calculated recovery accounted for less than 0.3% of the content originally ingested in the stomach, less than 0.01% of that originally ingested in the intestine and faeces, and less than 0.01% of that originally ingested in the colon and cecum (huang et al., 2018) . further experiments using in vitro digestion system suggested that after the gastric phase, over 97% of corn mirna mir-167a from the diet or from the extract were degraded, suggesting overall that significant degradation of corn mrnas occurred during digestion, which resulted in minimal uptake of corn mirnas after oral intake (huang et al., 2018) . witwer re-analysed the plasma data evaluated by liu et al., and found that only one putative plant mirna mapped above a median cut-off (200 reads), which was the plant mirna peu-mir-2910 . this implies that all rnas -including previously reported exogenous mirnas (xenomirs) such as mir-159a, mir-168a and the plant ribosomal degradation fragment mir-2911 -are below the level of background noise. it is not clear if mir-2910 is a real mirna as it is found in the highly conserved and expressed subunit rrna of plants . moreover, the mir-2910 sequence is not only a fragment of plant rrna, but also has a 100% coverage and 100% identity match with human 18s rrna, while others do not map to plant genomes at all . perhaps more analyses are needed when assessing possible xenomirs. after in silico xenomirs analysis using public sequencing datasets (see section 3.3.2 for details), kang et al. performed controlled feeding studies to detect the transfer of exogenous plant mirnas to rat blood or from bovine milk sequences into piglet blood . adult rats (n=3 per group) followed a 28-day controlled feeding study with supplementation in three different diets: monocot plant material (rice); dicot material (potatoes); and husbandry chow containing a defined mixture of grains, cereals, vitamins, minerals and fats. small rnas were isolated from serum and sequenced. most samples showed either a lack of plant xenomirs in rat serum, or three or less read counts in certain samples . these extremely low numbers suggested a probable false positive result. in the same study, piglets were fed either cow milk or soy milk for 4 weeks followed by 7 weeks of feeding with maize. few cow-specific sequences were found (15 reads) in the piglets fed cow milk, while piglets fed maize as well did show cow-specific sequences (21 reads). this suggests likely false positive results. both studies provide no evidence of dietary transfer of xenomirs . the abovementioned studies are summarized in table 27 . although outside the scope of this literature review, pollen plant mirnas ingested as part of a typical diet of adult honey bees were not found to be robustly transferred across the epithelial barrier under normal conditions (masood et al., 2016) . for example, despite the high levels of plant mir-156a in pollen or its detected levels in the bee digestive tract (0.5 copies per 10 pg total rna in the midgut), less than 0.1 copy per 10 pg total rna was found in abdominal tissue (masood et al., 2016) . www.efsa.europa.eu/publications 187 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. large amount of plant mirnas mir-156a, mir-159a and mir-169a detected in vegetarian, soy-enriched, or avocado diets (i.e. ≈1 x 1010 copies for vegetarian soy/diets or ≈1.1 x 109 copies for avocado diet of mir-156a after 24h), but very low levels found in plasma and less than 1 copy per/cell in tissues. after 4 weeks mir-21 rich diet in mir-21 ko mice, no plasma levels and less than one copy of mir-21 per cell detected in tissues. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after periodate oxidation, no major differences observed in mir-156a, mir-164a, or mir-167a among treatment, control groups or no-template control in liver and blood. in cecal and fecal samples no major differences observed among control and treated groups. mirna analysis in the gi content collected from different parts of mouse showed that the recovery (calculated) account for less than 0.3% of originally ingested in the stomach, less than 0.01% of originally ingested in the intestine and feces, and less than 0.01% of originally ingested in colon and cecum. in vitro digestion experiments suggested that after the gastric phase, over 97% of corn mirna mir-167a from the diet or from the extract is degraded. (huang et al., 2018) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. exogenous plant mirnas found in umbilical cord blood and amniotic fluid of healthy chinese pregnant women. plant mir-2911 increased in plasma of the mothers, placenta and foetus liver 3h after oral gavage (2.5 nmol/mice). exogenous sirna gavage (5 nmol) increased plasma levels of mother and foetus liver. protein target in foetus repressed. direct injection of microvesicles loaded with sirnas also increased its levels in foetal liver and reduced mrna expression of its target. exogenous small ncrnas can be transplacentally transmitted from mother to foetus. n.d., not determined. ko, knock out; wt, wild type; mirna, microrna; srna, small rna; dsrna, double-stranded rna; col3a1, collagen type i α1; α-sma, alpha smooth muscle actin; hjt, hong jing tian; www.efsa.europa.eu/publications 195 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). the available evidence presents several examples of systemic effects of plant-derived exogenous ncrnas when administered orally. however, there are also several reports that do not support these findings and contradict the hypothesis of cross-kingdom regulation. in either case, the essential question concerning the existence of this possibility is still heavily debated. important aspects such as the precise mechanism of transport of plant ncrnas from food to the circulatory system, the amount of exogenous ncrnas reaching tissues or the molecular mechanism of cellular uptake need to be understood. it remains unknown if such a transfer could be modulated in particular context (i.e. specific diet, disease status, medication). the available evidence also suggests that plant ncrnas can directly target mammalian genes through rnai effects when they are exposed in in vitro systems. without considering other physiological parameters, if a synthetic plant mirna is exposed to its mammalian target, it can effectively bind by base complementarity and exert a biological effect (i.e. gene repression). however, whether the exogenous plant ncrna can bypass all the biological barriers to reach and interact with the possible target remains to be clarified. apparently, exogenous plant-derived ncrnas are found in exosomes or macrovesicles. it is not yet known how they reach these types of structure in biological fluids, or if novel plant exosome-like nanoparticles participate in any of these processes. www.efsa.europa.eu/publications 201 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). immune cells were mainly from studies in human and mouse isolated lymphocytes. however, these studies were descriptive/not comprehensive, reporting specific effects of ncrnas in proinflammatory cytokine production using human cells, and would not allow identifying specific endpoints that can be used to evaluate changes in the regulation of immune function and homeostasis by ncrnas. retrieval of grey literature using the key words 'thesis plant rna immune' provided numerous results (i.e. google = 510.000), mostly regarding the function of mirnas and rna silencing in plant immunity. for instance, a document could be chosen based on title selection because it included the terms 'plant, double-stranded rna and systemic immunity' (https://uknowledge.uky.edu/plantpath_etds/21). however, this study was not further considered since it refers to plant immunity. after analysing the documents and refining by key questions, a large number of screened publications were discarded because they refer to the role of endogenous ncrna (human and animals) as well as viral infections in relation to the immune function, which is outside the scope of this literature review. as an example, within 1358 original in vivo publications related to the key words, only 30 studies were considered relevant for the proposed review questions. in summary, from the literature search, 91 papers were finally selected as relevant to review of the effects of ncrnas on immunity. very few studies analyse the direct relationship of exogenous plant ncrna with immune function and homeostasis of humans or animals. albeit controversial, exogenous ncrnas derived from plants and microorganisms have also been described in human blood (detailed in section 3.3.2.). overall, the results indicate that there is scarce information on the specific impact of plant ncrnas on activation of specific immunocompetent cells. of these 91 references, 64 documents were used for reviewing the general features of ncrnas in immune function and immunity in humans and animals (section 3.4.1.), 22 were used for the effect of exogenous plant ncrnas on immune system (section 3.4.2.) and 5 for the additional section on the effects of exogenous ncrnas on gut microbial composition (section 3.4.3.) ncrnas have been shown to play important roles in immune cell development and function in normal and disease conditions (ansel, 2013) . human and mouse studies demonstrate that mirnas play a critical role in the regulation of immune cell development and their function. there is also evidence of the relevance of neonatal mirna-mediated immune activation (kosaka et al., 2010) . here, mirna molecules of maternal origin appear to be stable in the infant's gut conditions, allowing dietary intake and transfer of mirna (kosaka et al., 2010) . ncrna have also been identified as regulators of metabolic shifts within microbial communities (paul et al., 2015) . thereby, ncrnas may significantly influence the critical roles of gut microbiota on health and disease. this part of the literature review (efsa task 4) presents the current knowledge on the role of exogenous ncrna molecules on the immune system of humans and animals and aims to provide information on the possible effets of dietary ncrnas on the regulation and function of the immune system. in addition, since the gut microbiota influences the immune system, a review on the possible effects of exogenous ncrnas as gut microbiota modulators is provided this chapter provides background information on the modulatory effects of ncrna on the immune system of humans and animals and serves as a general introduction to explore the possibility of dietary plant ncrnas effect on the immune system after dietary intake. sixty-four (64) documents were finally used to review this section. when activated, the immune system tightly regulates innate and adaptive response(s), aiming at restoring homeostasis, and preventing autoimmunity. the 'amplitude' (i.e. intensity and duration) of the www.efsa.europa.eu/publications 202 efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). interaction with the t-(tcr) and b-(bcr) cell receptors determines cell fate decisions. in the periphery, regulatory t cells (tregs) play a key role in restraining the activity of mature b and t (th1, th2 and th17) cells, and preserving tolerance mechanism(s). these responses depend on transcriptional and epigenetic regulation including regulation by mirnas. emerging systems for measuring mirna activity during immune activation revealed the complex network of genes that may be simultaneously targeted by mirnas to tune distinct cell fate decisions (wells et al., 2017) . rnas exhibit an intrinsic property of base pairs that is crucial in defining their structure and, thereby, their physiological role (lu et al., 2016) . plant mirnas are 2'-o-methyl modified on their terminal nucleotide, making them more difficult to be ligated to the cloning adapter. the inhibitory effect of 2'-o-methyl modification at the 3' end of the rna substrate for the mammalian nuclease family member nef-sp has been described (silva et al., 2017) . as described elsewhere, most mammalian mirnas require 'pre-processing steps' to become physiologically active. nucleotide sequence complementarity appears as a critical feature driving the predominant result exerted by mirnas. notably, it has been reported that a modification pattern involving alternating 2'-o-methyl rna bases improves dsirna in vitro stability and evades activation of the innate immune system (collingwood et al., 2008) . these data were obtained with isolated peripheral blood mononuclear cells (pbmcs) as a mixed 'lymphocyte' population of immune receptors that are encountered in vivo. however, the authors do not provide any molecular mechanism for the observed effects. sirnas have been shown to be potent stimuli of interferon- production by plasmocytoid dendritic cells (hornung et al., 2005) . this sirna technology induced systemic immune responses in the same range as the toll-like receptor (tlr)-9 ligand 'cpg', including activation of t cells and dendritic cells in spleen. notably, immunostimulation by sirna was absent in tlr7-deficient mice. there is a lack of data related to the effects of plant ncrnas on pge2 production, implicated in immunosuppression by tlr3 via cox2. several distinct classes of lncrnas are transcribed from different dna elements or are derived from long primary transcripts with noncanonical rna processing pathways, generating new rna species with unexpected formats. these lncrnas can be processed by several mechanisms, including ribonuclease p (rnase p) cleavage to generate mature 3' ends, capped by small nucleolar rna (snorna)-protein (snornp) complexes at their ends, or the formation of circular structures. recently, it was reported that several lncrnas mediate their regulatory effects through binding to specific rna-binding proteins . in addition, expression of lncrnas has been defined as highly cell-type specific (guttman et al., 2010; washietl et al., 2014) . this high specificity has been reported to be critical for the development and activation of immune lineages. however, lncrnas-mediated regulation of innate immune activation (i.e. il-6 production) has not been tested for function in vivo, but only evaluated in vitro (roux et al., 2017) . the regulation of inflammatory mediators or cytokines by lncrnas is still poorly understood. synthetic nucleic acids, such as dsrnas, have been reported to be recognized by toll-like receptor (tlr)-3 and can stimulate the innate immune system and trigger a type i interferon response (marques et al., 2006; mian et al., 2013) . in plants, it has been described that dsrna with 5' overhangs contributes to endogenous and antiviral rna silencing pathways (fukunaga and doudna, 2009) . earlier reports defined a structural basis (i.e. 3' overhangs) to cause nonspecific effects on the ds-rna-activated signalling pathways through the interferon responsive factor (irf)-3 (marques et al., 2006) . similarly, immune activation by the subcutaneous route with a viral mimic dsrna (poly i:c) of c57bl/6 mice during neonatal early or late brain development differentially affects depression-related behaviours developed in adolescent and adult mice (majidi-zolbanin et al., 2014) . the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). specific molecular interactions of the exogenous rna triggering immune responses have been reported. several studies have demonstrated the role of components of innate immunity, including toll-like receptors (tlrs), the retinoic acid-inducible gene i/melanoma-differentiation factor 5 (rig-i/mda5) and the interferon response effectors protein kinase r (pkr) and rnase l, as well as mirnas in the recognition of viral or synthetic dsrna (malathi et al., 2007; urcuqui-inchima et al., 2017) . tlrs are a family of proteins recognizing different pathogen and damage-associated molecular patterns, pamps and dmaps, respectively. rig-i/mda5, pkr and rnase l constitute cytosolic rna sensing proteins. the pathways responsible for these defined signalling processes can be largely separated (figure 26) , despite the existence of some degree of crosstalk between them where the engagement of interferon regulatory factors is a common feature. tlrs play an essential role in innate immune responses in mammals. in humans, ten different tlrs recognize distinct molecular patterns, where tlr-3, -7, -8 and -9 stimulate innate immune responses upon interaction with nucleic acids weber et al., 2012) . tlr3 is located on the plasmatic cell membrane, while tlr-7, -8 and -9 display endosomal localization. it has been reported that tlr3 recognizes dsrna, viral or the synthetic poly i:c, and sirna has also been identified (in the presence or absence of delivery systems) as a relevant ligand . notwithstanding, rna interference is considered an unlikely mechanism to be engaged for viral sensing (cullen, 2006) . gurich rna from viruses or synthetic single-stranded (ss) oligoribonucleotides displays an apparent preference for stimulating human tlr7/8-mediated immune effects (heil et al., 2004; forsbach et al., 2008) . a minimum of four nucleotides, uugu, found in the gu-rich region are reported as necessary to stimulate cytokine responses. endogenous or exogenous rnas are internalized into the endosomal compartment. the rna sensor system is composed of i) several innate immune 'toll-like' receptors (tlrs) located at the endosomal compartment that trigger different signalling pathways engaging adaptor distinct molecules (myd88 and trif), and ii) a cytosolic system (rig-i/mda-5) that coordinates its signalling with tlrs. antigen processing will lead to rearrangement of mhc molecules to activate the adaptive immune response(s). type i interferons (ifn-α/β) are expressed rapidly after infection and serve as a link between the innate and adaptive immune responses. : schematic representation of the eukaryotic rna sensor (single-stranded rna and short/long double-stranded rna) and its relation to innate immunity. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). two classes of ssrna motifs have been described that either preferentially activate tlr8-mediated or tlr7/8-mediated immune responses. au-rich oligoribonucleotides stimulate tlr8-but not tlr7mediated immune activation (forsbach et al., 2008) . rna motifs activating tlr8-associated signalling fail to induce ifn- from tlr7-expressing plasmacytoid dendritic cells but induce the secretion of th1like and proinflammatory cytokines from monocytes or myeloid dendritic cells. in addition, rna motifs activating the tlr7/8-associated signalling pathway stimulate cytokine secretion from both tlr7-and tlr8-positive immunocytes (forsbach et al., 2008) . differences in the protein sequence of tlr-8 between different mammals (human, monkey, chimpanzee, cattle, porcine, mouse, and rat) can be found, and species-specific recognition of ssrna via tlr7/8 had been described (heil et al., 2004) . the tlr8-specific rna sequences are unable to trigger cytokine responses from mouse, rat, and porcine immune cells (forsbach et al., 2008) . a dual function has also been suggested for the murine coreceptor cd14 to enhance tlr-3, -7 and -9 signalling (baumann et al., 2010; lee et al., 2006) . however, further studies showed evidenced that human cd14 did not appear to function as a co-receptor for tlr-3 or tlr9 (weber et al., 2012) .the innate immune system detects rna lacking nucleoside methylation (m5c, m6a and m5u) or otherwise (i.e. pseudouridine or 2'-o-methyl-u) modified as a mechanism to selectively trigger immune responses to nucleic acids from necrotic tissues or of exogenous origin. a recent systematic study on transcriptional and, especially, post-transcriptional regulation of stressresponsive lncrnas in oryza sativa showed that hundreds of lncrnas with down-regulated polyadenylation are highly conserved in stresses . in the ascidian ciona intestinalis a novel alternative polyadenylation signal activated by the prototypical tlr4 agonist (i.e. lps) has been described (vizzini et al., 2016) . moreover, alternative polyadenylation has been identified as a regulatory mechanism during the innate antiviral immune response in macrophages (jia et al., 2017) . these authors showed an enrichment of polyadenylated genes and mrna abundance change in tlrs as well as rig-i-like receptor, jak-stat and apoptosis-related signalling pathways. studies conducted on the crystal structure of the sensing tlr-3 ectodomain allowed definition of dsrna recognition of at least 40 bp (liu et al., 2008; leonard et al., 2008) . it was concluded that tlr3 assembles on dsrna as stable dimers and that the minimal signalling unit is one tlr3 dimer. recognition by secondary structure 'alone' has been reported, independently of the sequence motif, by tlr3. this dsrna length is larger than the typical helix occurring in normal mirnas and sirnas. tlr-7 and -8 have been found to recognize single-stranded rna (ssrna). the existence of a similar double horseshoe structure for tlr-7, -8 and -9 upon activation has been proposed, although their binding modes to their ligands remain unclear . often, in a partial view, tlr7 is considered as the ssrna-sensing tlr and as complementary to tlr3. however, higher structural features have been identified as important aspects in tlr7 rna sensing (jöckel et al., 2012) . in addition, small molecules that may be viewed as nucleoside analogues are able to activate tlr7 and/or tlr8 (hemmi et al., 2002; lee et al., 2003) . it has not been defined if the recognition mode for small molecules, including srnas, resembles that of tlr7 for rna recognition. besides tlrs, rig-i is a cytosolic multi-domain protein sensing viral rna associated to n-terminal caspase activation and recruitment domains (cards) to transmit signalling but preventing it in the absence of rna. the identification of rig-i/mda5 as cytoplasmic sensors for dsrna, associated to facilitators such as pkr and 2'-5'-olygoadenylate synthetase, suggests that additional 'aspects' (i.e. dependence on atp) other than dsrna structure or short size influence innate immune responses to srnas (i.e. sirnas and mirnas). these aspects remain unclear. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). current information on mirnas points to clear differences in cellular recognition of endogenous and exogenous dsrna. here, 5'-triphosphate and dsrna represent molecular patterns enabling rig-i to discriminate exogenous from endogenous rna (myong et al., 2009) . dsrna products as short as 21-23 bp are sensed by rig-i independently from the 3'-termini (marques et al., 2006) . similarly, the ifninducible 2'-5'-olygoadenylate synthetase (oas) requires dsrna that must be at least 15 nucleotides long for activity, and no modification of the 2′-hydroxyl group is tolerated (sarkar et al., 1999) . the oas family requires synthesizing di-, tri-, and tetrameric 2'-5'-oligoadenylates (2-5a), which in turn bind and activate rnase activation of rnase l together with pkr, irf3 and c-jun-n-terminal kinases, constituting pro-apoptotic mediators in response to dsrna. moreover, human oas may also enhance rig-i mediated signalling by mimicking polyubiquitin . further structural and mechanistic studies identify a key role of domain duplication in the oas family, thus revealing different functions of oas-1 and oas-3 in sensing dsrna (donovan et al., 2013; . rna interference (rnai) as well as antisense oligonucleotides and aptamers have been postulated to exert immune stimulatory effects in mammals (agrawal and kandimalla, 2004; cullen, 2006; mustonen et al., 2017) . however, at present, there is still an important open debate about agents exerting physiologically relevant functions at systems level biology (cullen, 2006; kleinman et al., 2008) . moreover, clinical trials of sirna demonstrated that the only population carrying the 412ff coding variant for tlr3 was protected from sirna-induced cytotoxicity (kleinman et al., 2008) . this study concluded that, because multiple cells express surface tlr3, the therapeutic approach with sirna might induce unanticipated vascular or immune effects. here, anti-angiogenic innate immunity triggered by sirna was found to occur without the induction of inf-α/β via trif activation being biased toward nf-ĸb rather than irf-3. side effects, for example, the immunosupressive effects of t regulatory cell function are inhibited by ifn-α-2β (yu et al., 2016) , are still only vaguely understood in association with exogenous ncrnas administration. naturally occurring mammalian rnas do not display a stimulatory potential on innate immune responses through tlr-3, -7, -8 and -9 in dendritic cells and tlr-expressing cells (karikó et al., 2005) . by contrast, it has been shown that major innate immune responses to chemically synthesized sirnas are mediated by tlr7 and/or tlr8. notably, replacement of uridines with their 2'-modified counterparts in the sirnas, reduced immune activation (sioud, 2010; barik and lu, 2015) . studies conducted on synthetic sirnas have shown results directly dependent on the nucleotide sequence (judge et al., 2005 (judge et al., , 2006 hornung et al., 2005) , and not to silencing effects of a target gene. putative immunostimulatory motifs allowing definition of ifn-α induction by β-gal control sirna or its constituent sense (gu-rich) and antisense ssrna oligonucleotides (judge et al., 2005) have been identified. these studies were performed in pbmcs, and therefore lack a clear demonstration of these effects at the organism level. intravenous administration of encapsulated sirna induced substantial dose-dependent (1-50 g sirna) ifn-α responses in outbred institute for cancer research (icr) inbred mice. in this line, reports also exist of increased hepatotoxicity of asos containing locked nucleic acid modifications (swayze et al., 2007) . in addition, asos carrying 2'-o-methoxyethylribose modifications showed no cytotoxic effects but were able to reduce targeted mrna. by analysis of sequence variants of either ss or ds sirna, uridine-rich immunostimulatory motifs within sirna were identified in human peripheral blood cells via tlr7 (sioud, 2010; jurk et al., 2011; judge et al., 2006) . there is a report of sequence-and targetindependent angiogenesis suppression induced by sirna via tlr3 (kleinman et al., 2008) . a minimum length for dsrna of about 21-nucleotide or 23-nucleotide luc sirna, but not truncated versions the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). suppressed by choroidal neovascularization have been observed in wild-type mice (kleinman et al., 2008) . recently, it has been established that nuclease-resistant aptamers can play a 'dual' role in immune signalling pathways (gefen et al., 2017; rajagopalan et al., 2017) . these aptamers seem to possess a unique feature acting as both agonist and antagonist depending on their degree of oligomerization (nozari and berezovski, 2017) . aptamers are ss oligonucleotides (70 -100 nucleotides) capable of recognizing, in a specific manner and with high affinity, several types of target molecules by means of a three-dimensional folding of their chain. preclinical studies in murine models have highlighted distinct regulatory points that may be influenced by oligonucleotide aptamers, including clusters of differentiation (cd) (pastor et al., 2013) , cell-to-cell communication such as immunoglobulins (gefen et al., 2017) and interleukin (il) signalling (rajagopalan et al., 2017) . the cd molecules can act in several ways, often acting as receptors or ligands important to the cell initiating signal cascades or serving as adhesion molecules. here, cd28 is one of the main co-stimulatory receptors responsible for proper activation of t lymphocytes that could be manipulated to modulate the immune response (pastor et al., 2013) . further studies reported enhancement of t cell functions by specific t cell immunoglobulin-3 aptamers through negative regulation of ifn- secretion by cd4 + and cd8 + cells (gefen et al., 2017) . positive effects attributed to oligonucleotide aptamer have also been associated to attenuation of il-2 signalling in cd8 + cells (rajagopalan et al., 2017) . while sirna and dsrna in cultured cells have been shown to trigger an interferon response (alexopoulou et al., 2001; sledz et al., 2003) , the administration of naked (dtdt) synthetic sirnas (2.5 mg/kg) to mice (either intraperitoneally or iv via low pressure or high pressure injection) induced no interferon response (heidel et al., 2004) . this suggests that sirna administration in vivo may elicit little or no immune response. also, modification of nucleotides within rna molecules was shown to reduce immune response (durbin et al., 2016) , which is relevant when applying nucleotide modifications to rna therapeutics. for example, n-6-methyladenosine (m6a) and pseudouridine seems to reduce the retinoic acid inducible gene i triggering of the innate immune signalling (durbin et al., 2016) . thus, rna modification seems to influence recognition of exogenous rnas, for example, ribose 2'-o-methylation of mrna at the 5'-end provides a molecular signature for the discrimination of self and non-self rna (züst et al., 2011) . prior to immune cells performing their immunological action on target cells, they need to receive a suitable 'maturation' signal to enhance clonal expansion and acquire the effector function. activation signals occur in peripheral lymphoid organs through a concerted interaction with mhc molecules. the major histocompatibility complex (mhc) regulates the cell-mediated immune response(s). there are recent reports on the regulatory role of mirnas on mhc class i and ii molecules (wongfieng et al., 2017; xie et al., 2017) . for instance, mhc class i chain related protein b present in natural killer (nk) cells was associated to cell-mediated antitumor immune response through engagement with the nkg2d receptor (xie et al., 2017) . the expression level of this receptor has been shown to be regulated by exogenous mir-30c mimics in nk cells . based on the temporal and spatial regulation which determines the expression patterns of mirnas, that suggestion was made for the participation of complex regulatory networks composed of several transcription factors influencing the expression level of 'each' mirna. the mhc class ii complex has been identified as a target in mammalian mirnas regulation (wongfieng et al., 2017) . in addition, some lncrnas have been identified as candidates to be translated into peptide fragments (guttman et al., 2013) suggesting, as several other 'indirect' lines of evidence, the the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). participation of ncrnas-derived peptides on mhc complexes (yewdell, 2007; mellman et al., 2011) . peptides bound to mhc molecules serve as recognizing fragments of antigen for the tcr. here, it has been shown that mirnas can influence the amplitude of tcr signalling modulating t cell fate decisions towards survival and maturation wells et al., 2017) . here it is worth noting the recently defined dual role of mirna in maintenance of the naive phenotype of cd8 t cells or clonal expansion, where modulation of let-7 mirnas levels is required (wells et al., 2017) . in a similar way to mhc class ii for t cells, bcr determines the fate of developing b cells allowing their adequate maturation or the development of self-reactive b cells leading to immunodeficiency of b cell malignancy. the mirnas set threshold for lymphocyte development targets several genes in the pi3k pathway (simpson and ansel, 2015) . it is also important to highlight that pi3k displays an inhibitory interaction on tlrs-induced production of il-12, therefore limiting excessive th1 polarization and suggesting a potential immunoregulatory role for mirnas. a few relatively recent studies address the potential regulatory role of 'specific' mirnas in treg functions (jeker et al., 2012; warth et al., 2015) . these studies have shown that inhibition of mirna-10a in c57bl/6j mice led to reduced foxp3 expression dependent on the levels of mediators such as tgf and/or retinoic acid (ra) (jeker et al., 2012) . another study reported induction of treg cell differentiation regulated by a mirna 'network' influencing the promoting factor mtor (warth et al., 2015) . these studies revealed that the underlying molecular mediators responsible for the mirna regulation of t cell signalling pathways remain unknown. unlike animals, plant mirnas have not been associated to tgf nor ra. intersecting the rapidly emerging field of treg function, it has been discovered that ra controls both the homing and differentiation of treg. in addition, computational and systems biology approaches have identified mtor as a potential pathway to explain the cross-kingdom mirnas-mediated regulatory potential of camptotheca acuminata ). although many reports can be found regarding the immunomodulatory role of endogenous and viral ncrnas, very few evaluated the impact of plant ncrnas on immune function and homeostasis when ingested/absorbed by animals and/or humans. thus, this section was reviewed using 19 scientific documents. however, no studies were found that directly evaluate the effect of plant exogenous ncrna and their effects on the immune system following dietary intervention trials. recent reports highlight the shortcomings of existing mirna measuring systems (i.e. qpcr) in comparison to sequencing procedures to explore mirnas in biological fluids chen et al., 2016a) . these studies point out the considerable number of unmapped ncrnas as a major factor limiting the use of qpcr techniques. notably, the abundance level of identified exogenous rna sequences in biological fluids (i.e. human plasma and breast milk, and serum from mice) was rather low. in addition, the role of food matrix on ncrna delivery to their cellular or tissue targets has not been defined. although the identification of exogenous ncrnas in biological samples led to considerations on their role as cross-kingdom regulators of immune-related processes, the impact (i.e. influence and regulatory role) of exogenous plant ncrnas on immune function and homeostasis is inferential. exogenous rnas sequences have been found in plasma , serum (chen et al., 2016b) and breast milk ) from humans and/or animals. these studies reported the presence of ncrnas in biological fluids, although their impact on immune function remains to be fully clarified. the recent discovery of blood circulating rnas originating from foods seems to indicate that certain dietary plant mirnas can be absorbed at significant levels . however, the potential contribution of distinct 'plant food matrices' on ncrnas the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). absorption has not been addressed as it has been described for viral rnas (seljelid et al., 1973; zou et al., 2010) . the existing studies show that adequate antigen-processing processes at the intestinal level can be avoided by ncrnas bound to coadjuvant molecules (i.e. polycations, cationic lipids, phospholipids) (seljelid et al., 1973; zou et al., 2010; vertzoni et al., 2004) . notably, phospholipid homeostasis is central in the response to toll like receptors (tlrs) activation through a type i interferon 'autocrine-paracrine' loop (song et al., 2015) . in biological samples, rna sequences have been reported from most common gm food matrices, including corn (zea mays), rice (oryza sativa japonica group), soybeans (glycine max), tomato (solanum lycopersicum) and grape (vitis vinifera) . the identification of rice mir-168a at fm concentration level in various mouse tissues may indicate the possibility of a cellular active uptake system for circulating rna . these data are in agreement with the role of defined food matrices as effective delivery systems protecting ncrnas from interaction with macrophages, dendritic cells, b-lymphocytes, and t-lymphocytes found in peyer's patches and other sites of gut-associated lymphoid tissue. however, several other studies contradict these results (see section 3.3.3. for details). enrichment of mirnas with targets related to immune response has been observed in colostral milk . colostrum represents a primordial food driving the first immunization of offspring and provides the nutritional needs of their immature organs in the earliest stages of life. the facts that immune-related mirnas from host, and exogenous plant mirnas (from bamboo diet to milk exosomes of giant panda) targeting synapse organization, neuron migration or axon guidance are enriched in giant breast milk of panda (restricted to a diet primarily comprising bamboo) indicate that breast milk may facilitate dietary intake of plant mirnas by infants for possible regulation of postnatal development ). very few studies address feeding plant ncrnas to animals (i.e. weaning, adult and old age) to evaluate induction of specific adaptive immune response(s) (i.e. th1, th2 or th17). similarly, there is a lack of data on the impact of exogenous plant ncrnas on expression of mhc-ii molecules to assess the potential allergenicity of these rnas. additionally, the different immune response(s) induced by the administration of distinct exogenous plant ncrnas and well-known weak or strong immune inducers by different routes (intraperitoneal or intragastric) have not been tested. understanding of the allergenic functions of exogenous plant ncrnas is comparatively meagre in relation to proteins. the homology of distinct ncrnas could elicit different immune response(s) as proteins showing homology values up to 86% on their amino acid sequences induce different igg1 and igg2 responses (adel-patient et al., 2011) . for example, the literature search identified one study on safety assessment of the possible effects of gm-triticale on the immune system using a murine c57bl/6 model (krzyżowska et al., 2010) . immunoblotting analysis in serum showed significantly increased il-2 levels, but not those of ifn-γ. it was concluded that multigenerational use of feeds for rodents containing the gm-triticale leads to expansion of the b cell compartment in the secondary lymphoid organs not associated to allergic response(s) (krzyżowska et al., 2010) . however, this study did not report the ncrna content in gmtriticale. efforts over the last decade have focused on elucidating the role of ncrnas in immune function. although no immunogenic potential has been found, ncrnas implications in pathways regulating the production of inflammatory mediators (witwer et al., 2010) , the development of hematopoietic cells (satpathy and chang, 2015) , regulation of tlrs signalling (carpenter et al., 2013) and changes in t cells needed to support clonal expansion and growth have been highlighted. papers on ncrnas-mediated regulation of the amplitude and quality of immune response(s) could not be found. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). at the moment, knowledge on the role of ncrnas in immune responses at systems-level remains incomplete. earlier reports defined significant differences in the effects of dsrna (poly i:c) in innate immune responses as a function of 'length' (0.5-1.5 kb and 2.0-8.0 kb), depending on cell type (i.e. splenocytes, pbmcs, raw264.7 and thp-1) (mian et al., 2013) . notably, it was reported that the 'shorter' dsrna caused higher immunomodulatory effects. poly i:c, a synthetic analogue of viral dsrna, has long been known as a strong inducer of innate immune responses by interacting with tlr3 in the endosome. following ligand recognition, specific ligand cascades involving interferon regulatory proteins and nuclear factor kappa b (nf-b) are activated to produce inflammatory mediators (démoulins et al., 2009) . similarly, signal-specific lncrnas have been reported in dendritic cells (dcs) stimulated with tlr4 agonists, 80% of which clustered with nf-b signalling components (guttman et al., 2009) . further approaches also identified upregulated responses in a long intergenic noncoding rna (lincrna) in response to tlr-1, -2, -7 and -8, but not tlr-3 in macrophages (carpenter et al., 2013) . specifically, lincrna-cox2 was found to mediate both activation and repression of immune response genes dependent on interactions of lincrna-cox2 with heterogeneous nuclear ribonucleoprotein a/b and a2/b1. a further study determined that lincrna-cox2 plays a key role as a coactivator of nf-b for the transcription of late-primary inflammatory response genes in macrophages through modulation of epigenetic chromatin remodelling (hu et al., 2016) . due to the relevance of gut microbiota in immune system development and homeostasis, the possibility of modulating the gut microbiota through dietary exogenous ncrnas was reviewed. as described in section 1.3, during the preparatory phase this novel topic was identified as relevant to this literature review. this additional section serves as a general introduction to emphasize the relevance of the possible modulation of the immune system by dietary exogenous ncrnas through the gut microbiota, and to identify gaps pointing to needs for future studies relevant to food/feed risk assessment of ncrnabased gm plants. specific details of the possible mechanisms of action, if any, are outside the scope of this review. due to the novelty of this topic and the lack of information on the scientific literature, only 5 documents were reviewed for this section. the intestinal tract harbours a complex community (microbiota), which has an enormous impact not only on the nutritional, but also immune status of the host. a balanced gut microbiota composition confers benefits to the host, while microbial imbalances are associated with metainflammatory disorders. the dynamic processes initiated upon birth define the microbiota composition that co-evolves with the host and its environment. thus, colonization of the intestine in early life seems particularly important as it represents one of the major environmental stimuli for immune system maturation. particularly, breast feeding is crucial to influencing early intestinal colonization. as previously indicated, development of emerging 'next generation sequencing technologies' has allowed identification of a significant fraction of the circulating rna in human plasma that originated from exogenous bacterial and fungi species . this study, based on microarray profiling results, reported that plasma can contain ncrnas from common foods influencing the expression profile of a number of genes in the cells. however, several other studies suggest that the quantitative amount of ncrnas are irrelevant for influencing gene expression (see section 3.3.3. for details). though the interaction between humans and their environments, particularly microbes, raises the possibility of some sort of feedback signalling process, it is still not fully understood. the literature search showed how few publications address the role of ncrnas in shaping gut microbiota. emerging the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). evidences point out interactions between environmental factors and inter-species gene regulation facilitating host control of the gut microbiota choi et al., 2017) . however, the impact of exogenous plant ncrnas on gut microbiota modulation remains elusive. increasing evidence demonstrates that a significant number of ncrnas are found in the extracellular media, and it is apparent that mammalian cells release srna that might carry functions which transcend the confines of single cells (valadi et al., 2007) . few studies have identified changes in mirna expression in human stool. overall, they provide an inconsistent association between their level and dietary habits and lifestyle (tarallo et al., 2014; . in this context, the survival of exogenous (orally administered) plant mirnas in faeces from mice has been observed . although the amount of particular mirnas such as mir-172 that survived the gi tract reached a maximum of 4.5%, this study does not support consistent survival rates for distinct ncrnas. a recent study identified mirnas secreted by intestinal epithelial cells in intestinal contents and supports their role in modulating gut microbiota composition . this study reports the potential of host mirnas to enter bacteria and co-localize with microbial nucleic acids. similarly, exogenous plant mirnas that were present in animal faeces were indicated as being primarily acquired orally . recently, bacterial rnas comparable in size to mirnas have been identified that could serve as signalling molecules mediating bacteria-to-human interaction (choi et al., 2017) . these data were obtained from outer membrane vesicles produced by periodontopathogens and were able to exert 'slight' immunosuppressive effects on t cells cytokine production (il-5, il-13 and il-15). mirna expression levels and amount in stool appear to be modulated by diet (micro-and macronutrients, phytochemicals) without excluding the possibility that the results were affected by other lifestyle factors; however, the influence of gm plant exogenous ncrnas has not yet been exhaustively studied in animals or humans. distinct innate immune mediators participating in the recognition and discrimination between the distinct nucleic acids have been identified; thus, additional definition of the dynamic interplay between the different 'sensors' (i.e. tlrs, rig-i/mda5, the interferon response effectors protein kinase r and rnase l) in response to ncrnas have to be clarified. the chemical modifications observed in plant ncrnas highlight the need to address the detection and measurement of exogenous plant ncrnas in body fluids, as well as the sensitivity and specificity of these rnas to cellular and tissue targets. although many reports focusing on synthetic ss-and dsrna mimics can be found in the literature, scientifically assessed direct hazardous impacts of food and feed on fauna and flora are scarce and conflicting. notably, there is a need to evaluate the effects of exogenous plant ncrnas at the system biology level and in human trials. current knowledge on the immune functions of exogenous ncrnas is comparatively lacking. most studies use cell models based on isolated cells that could not mimick the whole set of players that determine immune response(s), which are necessary for mechanistic studies, but need approach cell-to-cell communication in target tissues. additionally, these studies should focus on the relationship of immune responses to production of ifns since tlrs signalling converge on the production of this immune regulatory factor. to better understand the impact of exogenous ncrnas in host microbiota composition and diversity, further studies are needed to establish the microbial targets and their effect on physiological conditions. understanding the basic biological aspects of plant-derived exogenous ncrna (including their half-life and stability), gathering information on human and animal exposure by diet to these molecules, their subsequent bioavailability and possibility to exert local or systemic effects, and reviewing the experience from the development of rna-based therapeutics are relevant aspects for the risk assessment of ncrnabased gm food and feed. the literature includes very few studies evaluating the half-life of plant-derived ncrnas. while no specific studies on plant mirnas and lncrnas half-life were found in this review, the studies on plant circrnas half-life suggest that they exhibit considerable stability as compared to linear rnas (section 3.1.2.1). in mammals, the few reports related to ncrnas half-life suggest that mirnas and circrnas are generally much more stable than mrna, while lncrnas have a half-life like that of mrna. however, when assessing the stability of plant ncrnas outside the plant (section 3.1.2.2.), compelling evidence exists that plant mirnas are highly stable under different conditions including food storage, processing, cooking, or simulated digestion. moreover, they seem to survive after long incubation in serum, or are detected in the gastric content of mice, suggesting that plant mirnas are more resistant to degradation than synthetic or animal mirnas. this high resistance has been attributed to the unique characteristics of plant mirnas (and sirnas), in contrast to mirnas from mammalian and other organisms; the 2'-omethylation at their 3' end which confers plant mirnas more resistance to degradation (section 3.1.1.1). efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). the experience from development of ncrna therapeutics (section 3.1.3) shows that unmodified nucleic acids (i.e. rnas) exhibit very low stability in biological media and are subjected to rapid nuclease mediated degradation. indeed, a plethora of rnases are encoded within most genomes, often with overlapping activities, making redundancy a general feature of rna degradation systems. consequently, different chemical modifications or conjugations have been introduced to potential rna-based therapeutics (section 3.1.3.2) to improve their rna-binding affinity, their in vivo nuclease stability and their pharmacokinetic and pharmacodynamic properties. the available literature also suggests that naked or unmodified rnas parenterally administered (e.g. iv) are rapidly cleared from circulation and their presence in the kidney and urine has been reported immediately after administration (section 3.1.4). similarly, following oral administration naked or unmodified exogenous rnas are rapidly degraded when exposed to the gi harsh conditions, and different delivery vehicles for exogenous rnas have been developed. in general, and in contrast to chemically modified or formulated rnas, no major biological effects have been observed for naked rnas, which has probably limited further evaluation in humans (section 3.1.4.7). although certain disease conditions may influence the pharmacokinetics of exogenous rnas, there are no studies that specifically evaluate this for naked unmodified exogenous ncrnas. to exert a biological effect, an exogenous ncrna first needs to reach the intended target tissue in sufficient quantity levels, this implying the necessity to overcome many biological barriers (section 3.1.5). considering the oral intake as the main possible entry point of exogenous plant ncrnas into both animals and humans, the first major barrier is the mammalian gi tract, which encompasses a group of extracellular and cellular barriers. extracellular barriers include the presence of different enzymes (i.e. nucleases), the harsh environment and a net negative charged mucous layer. the cellular barriers include a three-layer (epithelial cells or enterocytes, the lamina propria and the muscularis mucosa) barrier known as the intestinal mucosa. possible ncrnas passage between cells is limited by the presence of tight junctions and the pore size in the human intestine would prevent the passage of all but mirnas, which are the smallest ncrnas. although crossing the cells by transcytosis may be possible, this mechanism implies facing new intracellular barriers, such as nucleases, recycling of ncrnas back to the lumen and nuclear uptake. however, m-cells, present in the gut epithelium interspersed between enterocytes, present certain characteristics that would make them more amenable for rna uptake. a second barrier is represented by a set of obstacles during the passage from the intestine to the target tissue, which encompasses plasma and tissue nucleases. once in the circulatory system, exogenous ncrnas are subjected to distribution and elimination. for example, small rnas would be rapidly cleared by the kidney due to their small size. furthermore, ncrnas need to escape the reticuloendothelial system, the function of which is to clear foreign pathogens. finally, they would have to cross the vascular endothelial barrier to reach the target tissue, facing hurdles like those in the enterocytes. packaging or incorporation of ncrnas into extracellular transport systems (i.e. exosomes/microvesicles or inclusion in ribonucleoprotein complexes) would confer certain resistance to nucleases and other barriers. ncrnas must enter the target cells to exert their functions. the molecular mechanisms of exogenous ncrna cellular uptake have been inferred from studies performed when developing strategies for nucleic acids delivery as therapeutics and is also largely derived from invertebrates, with little data reported in mammals. descriptions exist of receptor-mediated uptake of oligonucleotides through the sidt1 and sidt2 proteins, although this is now in doubt since they have been described as cholesterol transporters. due to the charged nature of rnas, cellular uptake could be achieved by endocytosis after which they would enter the system of intracellular trafficking through multiple membrane-bound compartments. ncrnas must exit these compartments to reach their functional sublocation within the efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). cell, either the cytosol or the nucleus, while simultaneously escaping degradation in the lysosomes. specialized barriers such as the blood-brain barrier or the placental barrier represent additional obstacles to overcome. while rna content in plant-derived foods varies (≈1 mg/g of tissue), it could be estimated that when consuming a daily dose of total fruit and vegetables of ≈600 g/day, humans theoretically ingest 600 mg of total rnas (dietary intake of plant rna may typically range from 0.1-1 g/person/day) (section 3.1.6.1). humans following special diets, such as vegetarians and vegans, have higher intake of plant origin rnas, but estimated exposure to such rnas implies only a 3-fold increase on average (section 3.1.6.2). it has been estimated that small rnas make up far less than 5% of total rna in plants, suggesting a very low exposure to plant ncrnas under normal conditions. whether sufficient levels of exogenous plant ncrnas can be consumed in the diet to exert a biological effect is also another aspect that needs to be evaluated. some examples in the literature suggest that for certain plant mirnas (i.e. mir-156a) a person would need to consume 1670 kg of cantaloupe to reach the 100 copies of mirna per cell possibly determining an effect. these aspects should be evaluated for each plant ncrna (on a case basis), considering that some plant-derived mirnas have shown unexpectedly high resistance to degradation and greater bioavailability. the available literature indicates a widespread presence of exogenous rnas (including plant derived ncrnas, i.e. small rnas) in the biological fluids of humans and animals (section 3.3.2). whether these rnas are derived from dietary intake is unclear. their generally low abundance and lack of enrichment in tissues mostly exposed to dietary changes (i.e. liver) suggest that some of may have be originated as technical artefacts or through contamination. very few studies address biological effects of dietary exogenous ncrnas in the gi tract and its annex glands (e.g. liver) (section 3. can directly target mammalian genes through rnai effects, but still clarification is needed if exogenous plant ncrnas can overcome all the biological barriers to reach and interact with their possible targets. exogenous plant-derived ncrnas have been found in exosomes or macrovesicles. how they reach these types of structures in biological fluids is unknown. in summary, supporting and contradicting evidence concerning the existence of systemic effects of dietary plant-derived exogenous ncrnas is heavily debated. important aspects such as the precise mechanism/s of transport of plant ncrnas from food into the systemic circulation, the amount of exogenous ncrnas reaching tissues or the molecular mechanisms of cellular uptake need to be determined. while several in vitro studies cover the participation of exogenous ncrnas in the dynamic network to modulate innate immunity responses, few tackle the immunomodulatory effects of plant ncrnas in adaptive immune response (section 3.4). none of these studies have considered the potential impact of exogenous dietary plant-derived ncrnas at the systemic level. since the first publication suggesting a possible cross-kingdom effect by dietary ncrna (through rice mir-168a) the knowledge on exogenous ncrnas, and particularly plant-derived exogenous ncrnas, on efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). their fate when ingested has increased. however, the knowledge useful to support the assessment of dietary ncrna such as ncrna-based gm food and feed still faces several gaps. an important aspect needing further investigation is the stability of plant ncrnas. the meagre available literature contains a very few studies of the half-life of plant ncrnas (i.e. mirnas, lncrnas and circrnas) both inside the plant cell and outside the plant. specifically, the stability of circrnas should be assessed due to their apparently high stability. moreover, very few reports focus on assessment of plant ncrnas stability in mammalian systems, when ingested. understanding the molecular basis that confers plant ncrnas stability is of great importance and needs to be further evaluated. for instance, plant mirnas and sirnas carry modifications that do not occur in mammals. these modifications, such as the presence of a methyl group located on the 3' nucleotide ribose, could hypothetically have an impact on the plant mirnas stability in mammalian systems/cells due to either the lack of the appropriate enzymes for recognition and degradation of plant mirnas by mammalian cells, or the increased stability to degradation by mammalian rnases. the circularized structure of plant circrnas would hypothetically confer them increased stability to mammalian rnases. if this is the case, plant mirnas and circrnas could exhibit a much longer half-life in mammalian systems than expected, especially when compared to mammalian mirna or circrnas. this would increase their chances of reaching the appropriate target tissue and encountering suitable target molecules within the cells. the half-life of plant ncrnas within mammalian cells is also an important aspect to consider. experiments are needed with labelled plant ncrnas, first in transfected mammalian cells (in vitro studies) and then orally administered to experimental animals (in vivo studies). radioactive probes or molecules of very low molecular weight, such as biotin or fluorescein, should be used, in order to prevent distorting the actual size of the studied plant ncrna; this is important when studying mirnas stability due to their small size. another aspect possibly relevant for risk assessment is to understand whether and how ingested gm plant-derived ncrnas can affect the expression levels of other ncrnas and other rnas, due to putative compensatory circuits and codifying rnas. whole transcriptome sequencing, comparing the rna levels of unmodified (wild type) to those of modified (transgenic) plants coul be useful to this aim. although available information suggests that many biological barriers exist in mammalian system to ingested plant exogenous ncrnas preventing these to exert a possible local or systemic effect, several gaps are still present. putative pathways of dietary exogenous ncrnas after ingestion and the many transporters potentially involved (i.e, in cellular uptake, or barrier passage transport pathways) in the transit from the gut to the target tissues should be characterized using standard molecular biology gainof-function and loss-of-function approaches. also, the many routes of mammalian intracellular trafficking to the specific intracellular target should be studied. to this aim, ncrnas isolated from human diets should be used preferentially, since most of the incomplete current knowledge comes from the rna-therapeutics field or is derived from the invertebrate world. which molecular mechanisms support the possible passage through specialized barriers (i.e. blood-brain barrier or the placental barrier), is another critical aspect requiring further studies. while there are in vitro studies describing the interaction between plant mirnas and mammalian mirna silencing complexes possibly leading to target repression, these findings need to be experimentally validated in vivo. therefore, more studies are needed to understand the possibility of processing plant ncrnas in mammalian cells. it is also unknown if ingested plant mirnas reaches or needs to reach a necessary level to exert a biological effect. for instance, studies in mammals suggest that the threshold for target gene regulation is >100 copies per cell for a certain study; however, another study suggests around 1000 to 10.000 copies of mammalian mirna per cell. clearly, a minimum amount of small rnas efsa supporting publication 2019:en-1688 the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). would be required to achieve biologically relevant effects on gene expression. on a case by case basis it would be useful to evaluate the amount of consumption of a given ncrna in a normal diet. it is also relevant to study if exposure to these plant ncrnas could change under certain pathological conditions (i.e. compromised intestinal permeability or renal function) or dietary patterns (i.e. vegetarian or vegan diets). although tissue accumulation of dietary plant exogenous ncrnas has not been reported to date, it seems clear that plant mirnas and other exogenous rnas are present in biological fluids from humans and animals. the biological significance of their presence is still unknown and needs to be addressed. methods that do not require amplification for detection should be used. more quantitative approaches to determine the levels of plant-derived exogenous ncrnas should be determined both in biological fluids and tissues to understand the magnitude of presence. for example, this could be partially obtained from studies reporting the presence of exogenous rnas in biological fluids using public small rna-seq databases. in the case of plant-derived mirnas, the use of sodium periodate oxidation of samples could be a good start to verifying the presence of genuine plant-derived mirnas from diet in human and animal biological fluids. the immunomodulatory effects of exogenous rnas have been widely described in the literature. however, there is no in vivo information regarding the immunomodulatory effects of dietary exogenous ncrnas. these types of studies should be performed both in vitro and in vivo to determine if the different types of ncrnas consumed in the diet can affect the immune system. in the case of target gene repression by plant ncrnas investigations in specific animal models could be considered. expression levels of other ncrnas and rnas, which may be modified due to putative compensatory circuits could be evaluated in these models, as well as information on the stability and half-life of the specific ncrna in specific cells and systemically following ingestion. genome-wide high-resolution mapping of exosome substrates reveals hidden features in the arabidopsis transcriptome genome-wide identification of circular rnas in arabidopsis thaliana a lariat-derived circular rna is required for plant development in arabidopsis pattern formation via small rna mobility small rnas are on the move literature review of baseline information on rnai to support the environmental risk assessment of rnai-based gm plants identification of circular rnas from the parental genes involved in multiple aspects of cellular metabolism in barley a group i plant intron accumulates as circular rna forms with extensive 5' deletions in vivo toward a consensus on the binding specificity and promiscuity of prc2 for rna a stepwise pathway for biogenesis of 24-nt secundary sirnas and spreading of dna methylation site-specific phosphorylation profiling of arabidopsis proteins by mass spectrometry and peptide chip analysis hierarchical action and inhibition of plant dicer-like proteins in antiviral defense characterization of stress-responsive lncrnas in arabidopsis thaliana by integrating expression, epigenetic and structural features identification and characterization of human testis derived circular rnas and their existence in seminal plasma the rna-binding proteins hyl1 and se promote accurate in vitro processing of pri-mirna by dcl1 genome-wide discovery of circular rnas in the leaf and seedling tissues of arabidopsis thaliana intra-and intercellular rna interference in arabidopsis thaliana requires components of the microrna and heterochromatic silencing pathways dicer-like 4 is required for rna interference and produces the 21-nucleotide small interfering rna component of the plant cell-to-cell silencing signal plant mobile small rnas the arabidopsis thaliana doublestranded rna binding protein drb1 directs guide strand selection from microrna duplexes naturally occurring variations in sequence length creates microrna isoforms that differ in argonaute effector complex specificity the xist lncrna exploits three-dimensional genome architecture to spread across the x chromosome identification of nuclear dicing bodies containing proteins for microrna biogenesis in living arabidopsis plants phased, secondary, small interfering rnas in posttranscriptional regulatory networks antisense rna polymerase ii divergent transcripts are p-tefb dependent and substrates for the rna exosome long noncoding rnas in cell-fate programming and reprogramming target mimicry provides a new mechanism for regulation of microrna activity location of a possible mirna processing site in smd3/smb nuclear bodies in arabidopsis partially redundant functions of arabidopsis dicer-like enzymes and a role for dcl4 in producing trans-acting sirnas short integuments1/suspensor1/carpel factory, a dicer homolog, is a maternal effect gene required for embryo development in arabidopsis oncogenic role of fusion-circrnas derived from cancer-associated chromosomal translocations topological organization of multichromosomal regions by the long intergenic noncoding rna firre natural rna circles function as efficient microrna sponges vernalization-mediated epigenetic silencing by a long intronic noncoding rna flowering locus c's lessons: conserved chromatin switches underpinning developmental timing and adaptation transitivity-dependent andindependent cell-to-cell movement of rna silencing specific interactions between dicer-like proteins and hyl1/drb-family dsrna-binding proteins in arabidopsis thaliana phosphate utilization efficiency correlates with expression of low-affinity phosphate transporters and noncoding rna, ips1, in barley gene silencing by micrornas: contributions of translational repression and mrna decay cyclophilin 40 facilitates hsp90-mediated risc assembly in plants in vitro assembly of plant rna-induced silencing complexes facilitated by molecular chaperone hsp90 a rice cis-natural antisense rna acts as a translational enhancer for its cognate mrna and contributes to phosphate homeostasis and plant fitness the nuclear pore protein attpr is required for rna homeostasis, flowering time, and auxin signaling dicer-1 and r3d1-l catalyze microrna maturation in drosophila molecular insights into mirna processing by arabidopsis thaliana serrate covalent circularization of exogenous rna during incubation with a wheat embryo cell extract small rnas as guardians of the genome fast-forward genetics identifies plant cpl phosphatases as regulators of mirna processing factor hyl1 the evolution and diversification of dicers in plants passenger-strand cleavage facilitates assembly of sirna into ago2-containing rnai enzyme complexes circular single-stranded rna replicon in saccharomyces cerevisiae rna-directed dna methylation: an epigenetic pathway of increasing complexity intercellular and systemic movement of rna silencing signals circular rnas are a large class of animal rnas with regulatory potency sorting of small rnas into arabidopsis argonaute complexes is directed by the 5' terminal nucleotide morc family atpases required for heterochromatin condensation and gene silencing small silencing rnas in plants are mobile and direct epigenetic modification in recipient cells specificity of argonaute7-mir390 interaction and dual functionality in tas3 trans-acting sirna formation evolution of animal and plant dicers: early parallel duplications and recurrent adaptation of antiviral rna binding in plants literature review of baseline information to support the risk assessment of rnai-based gm plants systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions microrna399 is a long-distance signal for the regulation of plant phosphate homeostasis evidence for nuclear processing of plant micro rna and short interfering rna precursors nuclear processing and export of micrornas in arabidopsis carpel factory, a dicer homolog, and hen1, a novel protein, act in microrna metabolism in arabidopsis thaliana rde-4 preferentially binds long dsrna and its dimerization is necessary for cleavage of dsrna to sirna rna exosome-regulated long non-coding rna transcription controls super-enhancer activity prediction of trans-antisense transcripts in arabidopsis thaliana genome-wide identification of long noncoding natural antisense transcripts and their responses to light in arabidopsis identification and characterization of circrnas in pyrus betulifolia bunge under drought stress analysis of noncoding transcriptome in rice and maize uncovers roles of conserved lncrnas associated with agriculture traits a long noncoding rna maintains active chromatin to coordinate homeotic gene expression arabidopsis noncoding rna mediates control of photomorphogenesis by red light identification of circular rnas and their targets in leaves of triticum aestivum l. under dehydration stress importin 8 is a gene silencing factor that targets argonaute proteins to distinct mrnas noncoding transcription by rna polymerase pol ivb/pol v mediates transcriptional silencing of overlapping and adjacent genes expression of arabidopsis mirna genes genetic and functional diversification of small rna pathways in plants identification and characterization of wheat long non-protein coding rnas responsive to powdery mildew infection and heat stress by using microarray analysis and sbs sequencing bidirectional promoters generate pervasive transcription in yeast functions of long intergenic non-coding (linc) rnas in plants widespread noncoding circular rnas in plants a systemic small rna signaling system in plants wavy leaf1, an ortholog of arabidopsis hen1, regulates shoot development by maintaining microrna and trans-acting small interfering rna accumulation in rice vigs, higs and figs: small rna silencing in the interactions of viruses or filamentous organisms with their plant hosts control of coleopteran insect pests through rna interference the expanding world of small rnas in plants genome-wide analysis of long noncoding rna stability structural variations and stabilising modifications of synthetic sirnas in mammalian cells circular rnas are long-lived and display only minimal early alterations in response to a growth factor host-delivered rnai: an effective strategy to silence genes in plant parasitic nematodes analysis of microrna turnover in mammalian cells following dicer1 ablation polysome arrest restricts mirna turnover by preventing exosomal export of mirna in growth-retarded mammalian cells a versatile monosaccharide transporter that operates in the arbuscular mycorrhizal fungus glomus sp is crucial for the symbiotic relationship with plants the drosophila rna methyltransferase, dmhen1, modifies germline pirnas and single-stranded sirnas in risc uridylation of mature mirnas and sirnas by the mut68 nucleotidyltransferase promotes their degradation in chlamydomonas circular rnas are abundant, conserved, and associated with alu repeats regulation of small rna stability: methylation and beyond methylation protects mirnas and sirnas from a 3' end uridylation activity in arabidopsis assessing the survival of exogenous plant microrna in mice effective detection and quantification of dietetically absorbed plant micrornas in human plasma adenylation of plant mirnas covalent circularization of exogenous rna during incubation with a wheat embryo cell extract silencing a cotton bollworm p450 monooxygenase gene by plant-mediated rnai impairs larval tolerance of gossypol rna-specific ribonucleotidyl transferases oral delivery of sirna and antisense oligonucleotides toxicogenomics of non-viral drug delivery systems for rnai: potential impact on sirna-mediated gene silencing activity and specificity nonviral delivery of synthetic sirnas in vivo a theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability oral nucleic acid therapy using multicompartmental delivery systems the adherent gastrointestinal mucus gel layer: thickness and physical state in vivo polymeric nano-and microparticle technologies for oral gene delivery isolation and properties of an extracellular nucleases of serratia marcescens guidance for industry on waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutics classification system issues in oral nanoparticle drug carrier uptake and targeting in vitro and in vivo models for the study of oral delivery of nanoparticles alginate/poly-l-lysine microparticles for the intestinal delivery of antisense oligonucleotides multi-compartmental oral delivery systems for nucleic acid therapy in the gastrointestinal tract extracellular ribonucleases from bacillus subtilis. i. crystallization and specificity intestinal delivery of non-viral gene therapeutics: physiological barriers and preclinical models first-pass elimination. basic concepts and clinical consequences key issues in non-viral gene delivery polymeric nanoparticle drug delivery technologies for oral delivery applications effect of sodium caprate on the intestinal absorption of two modified antisense oligonucleotides in pigs drug delivery of sirna therapeutics: potentials and limits of nanosystems oral delivery of antisense oligonucleotides in man the human microbiome project consortium. 2012 structure, function and diversity of the healthy human microbiome milk-derived exosomes for oral delivery of paclitaxel delivery of sirna to the mouse brain by systemic injection of targeted exosomes orally delivered sirna targeting macrophage map4k4 suppresses systemic inflammation oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models oral nucleic acid therapy using multicompartmental delivery systems oral delivery of rnase p ribozymes by salmonella inhibits viral infection in mice protection and systemic translocation of sirna following oral administration of chitosan/sirna nanoparticles interleukin 10 gene transfer prevents experimental colitis in rats inhibition of trkb limits development of the zebra finch song system commercial dairy cow milk micrornas resist digestion under simulated gastrointestinal tract conditions oral il-10 gene delivery in a microsphere-based formulation for local transfection and therapeutic efficacy in inflammatory bowel disease the role of nucleotides in human nutrition retroviral delivery of rna interference against marek's disease virus in vivo biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles systemic exosomal sirna delivery reduced alpha-synuclein aggregates in brains of transgenic mice pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver proteomics of extracellular vesicles: exosomes and ectosomes dietary yeast rna supplementation reduces mortality by aeromonas hydrophila in rohu (labeo rohita l.) juveniles effects of mannose density on in vitro and in vivo cellular uptake and rnai efficiency of polymeric nanoparticles peg-pla nanoparticles facilitate sirna knockdown in adult zebrafish heart exosome-mediated delivery of hydrophobically modified sirna for huntingtin mrna silencing oral delivery of small rna and dna colonic gene silencing using sirna-loaded calcium phosphate/plga nanoparticles ameliorates intestinal inflammation in vivo a randomized controlled trial of preoperative oral supplementation with a specialized diet in patients with gastrointestinal cancer a novel class of small rnas: trna-derived rna fragments (trfs) small non-coding rnas transfer through mammalian placenta and directly regulate fetal gene expression assessing the survival of exogenous plant microrna in mice effective detection and quantification of dietetically absorbed plant micrornas in human plasma human milk exosomes and their micrornas survive digestion in vitro and are taken up by human intestinal cells detection of plant mirnas abundance in human breast milk in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? detection of dietetically absorbed maizederived micrornas in pigs exosomal micrornas in giant panda (ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs unsuccessful detection of plant micrornas in beer, extra virgin olive oil and human plasma after an acute ingestion of extra virgin olive oil a novel chemopreventive strategy based on therapeutic micrornas produced in plants annotation of the goat genome using next generation sequencing of microrna expressed by the lactating mammary gland: comparison of three approaches expressional analysis of immune-related mirnas in breast milk identification of stable, high copy number, medium-sized rna degradation intermediates that accumulate in plants under non-stress conditions determination of the potential bioavailability of plant micrornas using a simulated human digestion process human breast milk mirna, maternal probiotic supplementation and atopic dermatitis in offspring the effect of dietary ginseng polysaccharide supplementation on the immune responses involved in porcine milk-derived esrnas immune modulatory function of abundant immunerelated micrornas in microvesicles from bovine colostrum uptake and function studies of maternal milk-derived micrornas mining of public sequencing databases supports a nondietary origin for putative foreign mirnas: underestimated effects of contamination in ngs literature review of information supporting food/feed risk assessment of rnai-based gm plants www.efsa.europa this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document alternative mirnas: human sequences misidentified as plant mirnas in plant studies and in human plasma transfer and functional consequences of dietary micrornas in vertebrates: concepts in search of corroboration: negative results challenge the hypothesis that dietary xenomirs cross the gut and regulate genes in ingesting vertebrates, but important questions persist real-time quantitative pcr and droplet digital pcr for plant mirnas in mammalian blood provide little evidence for general uptake of dietary mirnas: limited evidence for general uptake of dietary plant xenomirs the intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma caco-2 cells and rat small intestinal iec-6 cells the levels of human milk micrornas and their association with maternal weight characteristics detection of dietary plant-based small rnas in animals anomalous uptake and circulatory characteristics of the plant-based small rna mir2911 the atypical genesis and bioavailability of the plant-based small rna mir2911: bulking up while breaking down bioavailability of transgenic micrornas in genetically modified plants exogenous plant mir168a specifically targets mammalian ldlrap1: evidence of cross-kingdom regulation by microrna analysis of plant-derived mirnas in animal small rna datasets immune-related micrornas are abundant in breast milk exosomes honeysuckle-encoded atypical microrna2911 directly targets influenza a viruses one step forward, two steps back; xeno-micrornas reported in breast milk are artifacts small rnas from plants, bacteria and fungi within the order hypocreales are ubiquitous in human plasma dietary microrna database (dmd): an archive database and analytic tool for food-borne micrornas lack of detectable oral bioavailability of plant micrornas after feeding in mice survey of 800+ data sets from human tissue and body fluid reveals xenomirs are likely artifacts plant mirnas found in human circulating system provide evidences of cross kingdom rnai detection of plant mirnas abundance in human breast milk in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? optimization of enzymatic reaction conditions for generating representative pools of cdna from small rna computational characterization of exogenous micrornas that can be transferred into human circulation mining of public sequencing databases supports a nondietary origin for putative foreign mirnas: underestimated effects of contamination in ngs the complex exogenous rna spectra in human plasma: an interface with human gut biota? the spectrum of circulating rna: a window into systems toxicology detection of an abundant plant-based small rna in healthy consumers oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models exosomes as a nanodelivery system: a key to the future of neuromedicine? endogenous microrna can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen micrornas in serum of mice lack of detectable oral bioavailability of plant micrornas after feeding in mice high-throughput sequencing of rna silencingassociated small rnas in olive (olea europaea l.) plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic hjt-srna-m7 autosomal recessive hypercholesterolemia caused by mutations in a putative ldl receptor adaptor protein exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges extensive degradation and low bioavailability of orally consumed corn mirnas in mice an improved method to quantitate mature plant microrna in biological matrices using modified periodate treatment and inclusion of internal controls this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document cross talk between adipose tissue and placenta in obese and gestational diabetes mellitus pregnancies via exosomes grape exosome-like nanoparticles induce intestinal stem cells and protect mice from dss-induced colitis survey of 800+ data sets from human tissue and body fluid reveals xenomirs are likely artifacts cross-kingdom regulation of putative mirnas derived from happy tree in cancer pathway: a systems biology approach small non-coding rnas transfer through mammalian placenta and directly regulate fetal gene expression assessing the survival of exogenous plant microrna in mice reply to dr. witwer's letter to the editor effective detection and quantification of dietetically absorbed plant micrornas in human plasma plant mirnas found in human circulating system provide evidences of cross kingdom rnai detection of dietetically absorbed maizederived micrornas in pigs negligible uptake and transfer of diet-derived pollen micrornas in adult honey bees the transport mechanism of extracellular vesicles at the blood-brain barrier unsuccessful detection of plant micrornas in beer, extra virgin olive oil and human plasma after an acute ingestion of extra virgin olive oil a novel chemopreventive strategy based on therapeutic micrornas produced in plants interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles high-throughput assessment of microrna activity and function using microrna sensor and decoy libraries milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation corn rootwormactive rna dvsnf7: repeat dose oral toxicology assessment in support of human and mammalian safety a 28-day oral toxicity evaluation of small interfering rnas and a long double-stranded rna targeting vacuolar atpase in mice citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress cml xenograft growth by inducing trail-mediated cell death this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document ineffective delivery of diet-derived micrornas to recipient animal organisms adipose-derived circulating mirnas regulate gene expression in other tissues uptake and function studies of maternal milk-derived micrornas contamination or artifacts may explain reports of plant mirnas in humans alternative mirnas: human sequences misidentified as plant mirnas in plant studies and in human plasma real-time quantitative pcr and droplet digital pcr for plant mirnas in mammalian blood provide little evidence for general uptake of dietary mirnas: limited evidence for general uptake of dietary plant xenomirs detection of an abundant plant-based small rna in healthy consumers anomalous uptake and circulatory characteristics of the plant-based small rna mir2911 bioavailability of trasngenic micrornas in genetically modified plants exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in danio rerio molecular basis of mammalian transmissibility of avian h1n1 influenza viruses and their pandemic potential exogenous plant mir168a specifically targets mammalian ldlrap1: evidence of cross-kingdom regulation by microrna honeysuckle-encoded atypical microrna2911 directly targets influenza a viruses plant micrornas in larval food regulate honeybee caste development to evaluate oral toxicity of exogenous ncrnas in mice, petrick et al. administered for 28 days a repeated oral dose of sirnas and dsrna and evaluated several parameters including body weight, food consumption, clinical observations, clinical chemistry, haematology, gross pathology, or histopathology endpoints it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). signs (mortality, abnormalities, and sign of pain and distress), body weight and food consumption, haematology, organ weight, or pathology results. the minor differences observed in certain parameters were limited to single intervals, were not dose-related, and were attributed to interanimal variability torula yeast rna (rna negative control) at 100 mg/kg/day or a control vehicle were used. ten animals per sex and per group were used. no treatment-related effects were observed on clinical signs (mortality, or sign of pain and distress), body weight and food consumption, haematology, organ weight, or pathology results. minor changes in selected parameters in selected groups were observed, but these were attributed by the authors to normal variability and not treatment-related effects this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document ecological risk assessment for dvsnf7 rna: a plant-incorporated protectant with targeted activity against western corn rootworm literature review of baseline information on rnai to support the environmental risk assessment of rnai-based gm plants a comparative evaluation of the regulation of gm crops or products containing dsrna and suggested improvements to risk assessments endogenous small rnas in grain: semi-quantification and sequence homology to human and animal genes literature review of baseline information to support the risk assessment of rnai-based gm plants corn rootwormactive rna dvsnf7: repeat dose oral toxicology assessment in support of human and mammalian safety a 28-day oral toxicity evaluation of small interfering rnas and a long double-stranded rna targeting vacuolar atpase in mice rnai technologies in agricultural biotechnology: the toxicology forum 40th annual summer meeting no impact of dvsnf7 rna on honey bee (apis mellifera l.) adults and larvae in dietary feeding tests microstructure and ultrastructure of highamylose rice resistant starch granules modified by antisense rna inhibition of starch branching enzyme a 90-day toxicology study of high-amylose transgenic rice grain in sprague-dawley rats a three generation reproduction study with sprague-dawley rats consuming high-amylose transgenic rice high-amylose rice improves indices of animal health in normal and diabetic rats studies performed with human cells were mainly carried out ex vivo using lymphocytes and, to a significant extent, macrophages. from the 1730 studies retrieved during the screening phase on immune cells in relation to exogenous ncrna 7%) from rat and 348 (36.5%) from mouse. and within the 776 studies on immune cells other than lymphocytes (i.e. macrophages), the studies were allocated into 378 (48.7%) from human, 91 (11.7%) from rat, and 307 (39.6%) from mouse. all these data clearly show that studies retrieved in relation to ncrnas and www this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document immunological and metabolomic impacts of administration of cry1ab protein and mon 810 maize in mouse role of toll-like receptors in antisense and sirna recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 rna regulation of the immune system therapy of respiratory viral infections with intranasal sirnas cd14 is a coreceptor of toll-like receptors 7 and 9 a long noncoding rna mediates both activation and repression of immune response genes identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen micrornas in serum of mice identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen mirnas in serum of mice chemical modification patterns compatible with high potency dicer-substrate small interfering rnas is rna interference involved in intrinsic antiviral immunity in mammals? poly (i:c) induced immune response in lymphoid tissues involves three sequential waves of type i ifn expression structural basis for cytosolic double-stranded rna surveillance by human oligoadenylate synthetase 1 structural mechanism of sensing long dsrna via a noncatalytic domain in human oligoadenylate synthetase 3 rnas containing modified nucleotides fail to trigger rig-i conformational changes for innate immune signaling this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document identification of rna sequence motifs stimulating sequence-specific tlr8-dependent immune responses dsrna with 5′ overhangs contributes to endogenous and antiviral rna silencing pathways in plants a tim-3 oligonucleotide aptamer enhances t cell functions and potentiates tumor immunity in mice ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincrnas ribosome profiling provides evidence that large noncoding rnas do not encode proteins chromatin signature reveals over a thousand highly conserved large noncoding rnas in mammals lack of interferon response in animals to naked sirnas species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway sequencespecific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 lincrna-cox2 promotes late inflammatory gene transcription in macrophages through modulating swi/snf-mediated chromatin remodeling microrna 10a marks regulatory t cells the role of alternative polyadenylation in the antiviral innate immune response the 2'-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor 7 activation or inhibition design of noninflammatory synthetic sirna mediating potent gene silencing in vivo sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna immunostimulatory potential of silencing rnas can be mediated by a non-uridine-rich toll-like receptor 7 motif suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna sequence-and target-independent angiogenesis suppression by sirna via tlr3 microrna as a new immune-regulatory agent in breast milk this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document the effect of multigenerational diet containing genetically modified triticale on immune system in mice cross-kingdom regulation of putative mirnas derived from happy tree in cancer pathway: a systems biology approach double-stranded rna-mediated tlr3 activation is enhanced by cd14 molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor 7 the tlr3 signaling complex forms by cooperative receptor dimerization mir-181a is an intrinsic modulator of t cell sensitivity and selection assessing the survival of exogenous plant microrna in mice structural basis of toll-like receptor 3 signaling with double-stranded rna the host shapes the gut microbiota via fecal microrna in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? exosomal micrornas in giant panda (ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs microrna-30c promotes natural killer cell cytotoxicity via up-regulating the expression level of nkg2d neonatal immune activation during early and late postnatal brain development differently influences depressionrelated behaviors in adolescent and adult c57bl/6 mice small self-rna generated by rnase l amplifies antiviral innate immunity a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells cancer immunotherapy comes of age length of dsrna (poly i:c) drives distinct innate immune responses, depending on the cell type oligonucleotide-based pharmaceuticals: non-clinical and clinical safety signals and non-clinical testing strategies cytosolic viral sensor rig-i is a 5'-triphosphate-dependent translocase on double-stranded rna aptamers for cd antigens: from cell profiling to activity modulation cd28 aptamers as powerful immune response modulators influences of diet and the gut microbiome on epigenetic modulation in cancer and other diseases this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document aptamer-targeted attenuation of il-2 signaling in cd8+ t cells enhances antitumor immunity catalog of differentially expressed long non-coding rna following activation of human and mouse innate immune response. front immunol 8, 1038 long noncoding rna in hematopoiesis and immunity the effect of poly-l-lysine on the uptake of reovirus doublestranded rna in macrophages in vitro characterization of the mammalian rna exonuclease 5/nef-sp as a testis-specific nuclear 3' → 5' exoribonuclease microrna regulation of lymphocyte tolerance and autoimmunity advances in rna sensing by the immune system: separation of sirna unwanted effects from rna interference activation of the interferon system by short-interfering rnas type i interferons function as autocrine and paracrine factors to induce autotaxin in response to tlr activation antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals microrna expression in relation to different dietary habits: a comparison in stool and plasma samples interplay between dengue virus and toll-like receptors, rig-i/mda5 and micrornas: implications for pathogenesis exosome-mediated transfer of mrnas and micrornas is a novel mechanism of genetic exchange between cells dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects lps injection reprograms the expression and the 3' utr of a cap gene by alternative polyadenylation and the formation of a gait element in ciona intestinalis the complex exogenous rna spectra in human plasma: an interface with human gut biota? induced mir-99a expression represses mtor cooperatively with mir-150 to promote regulatory t-cell differentiation evolutionary dynamics and tissue specificity of human long noncoding rnas in six mammals toll-like receptor (tlr) 3 immune modulation by unformulated small interfering rna or dna and the role of cd14 (in tlrmediated effects) homology modeling of human tolllike receptors tlr7, 8, and 9 ligand-binding domains modulation of let-7 mirnas controls the differentiation of effector cd8 t cells this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document microrna regulation of ifn-beta protein expression: rapid and sensitive modulation of the innate immune response 2017. 5'-utr and 3'-utr regulation of micb expression in human cancer cells by novel micrornas microrna-889 is downregulated by histone deacetylase inhibitors and confers resistance to natural killer cytotoxicity in hepatocellular carcinoma cells ncrna-and pc2 methylation-dependent gene relocation between nuclear structures mediates gene activation programs plumbing the sources of endogenous mhc class i peptide ligands infalpha-2b inhibitory effects on cd4(+)cd25(+)foxp3(+) regulatory t cells in the tumor microenvironment of c57bl/6 j mice with melanoma xenografts stress-responsive regulation of long noncoding rnas' polyadenylation in oryza sativa exogenous plant mir168a specifically targets mammalian ldlrap1: evidence of cross-kingdom regulation by microrna antiviral activity of human oligoadenylate synthetases-like (oasl) is mediated by enhancing retinoic acid-inducible gene i (rig-i) signaling lipid-mediated delivery of rna is more efficient than delivery of dna in non-dividing cells ribose 2'-omethylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). (zhou et al., 2014) . twenty female and ten male rats per group in the first generation (f0) were fed the different diets. their pups, 20 weanling/female/group and 10 weanling/male/group were randomly selected as an f1 generation. the f2 generation was acquired by using the same procedure described above. at weaning, f3 generation rats were chosen, and 10 rats/sex/group provided the corresponding diets and observed for 13 weeks. no major differences in animal survival, health status, behaviour, body weight, food consumption, or reproductive capacity were observed with the different diets. some statistically significant differences were observed in animals given the transgenic rice diets compared to those receiving the standard diet for certain clinical chemistry parameters, in certain generations (alt, alkp, ldh, cholesterol, hdlc, ldlc). these changes were not considered adverse or biologically significant (zhou et al., 2014) . moreover, no evidence of altered incidence or altered severity of background changes was observed in any organ or tissues of the rats fed the three diets (zhou et al., 2014) . although minor changes in the mean relative weight of epididimides in male f3 adults was observed in rats fed the rice diets compared to the control group, no difference was observed between the transgenic rice fed rats and their near-isogenic line controls, suggesting overall that the transgenic line was unlikely to cause any risk to rat health in reproduction or development, even when consumed for up to three generations (zhou et al., 2014) . however, these studies did not evaluate any aspect related to rnai.heinemann et al. compared the history of risk assessment of gmos producing dsrnas in australia, new zealand and brazil, with a focus on regulatory context (heinemann et al., 2013) . the authors suggested some processes to properly assess the safety of dsrna-producing gm plants before their release or marketing. these include i) bioinformatics analysis to identify any likely, unintended targets of the dsrna in human and animals; ii) experimental procedures that would identify all new intended and unintended dsrna molecules in the gm product; iii) testing animal and human cells in tissue cultures for a response to intended and unintended effects of dsrnas from the product; iv) long-term testing on animals; and possibly v) clinical trials on human volunteers (heinemann et al., 2013) . in response to this article, the regulatory agency food standards australia new zealand (fsanz) published a document addressing regulation of gm crops and foods developed using gene silencing (http://www.foodstandards.gov.au/consumer/gmfood/pages/response-to-heinemann-et-al-on-theregulation-of-gm-crops-and-foods-developed-using-gene-silencing.aspx). this document criticised some of the comments of the above risk assessment review. some key points included the lack of weight of scientific evidence (published up to 2013) to support the view that small dsrnas in foods are likely to have adverse consequences in humans; the lack of a scientific basis for suggesting that small dsrnas present in gm foods have different properties than naturally-occurring ones; or the adequate acknowledge of many barriers (in the uptake, distribution and targeting) during oral development of small dsrna therapies, among others. the overall suggestion was that there was no need to consider additional studies as proposed by heinemann et al. (2013) .commentary documents reviewing scientific meetings in the context of rnai technologies in agricultural biotechnology discuss some of the aspects reviewed in this document .additional relevant documents of baseline information to support the risk assessment and environmental risk assessment of rnai-based gm plants can be obtained from efsa external scientific reports the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). key: cord-354592-vqws942c authors: cauvin, annick j.; peters, christopher; brennan, frank title: advantages and limitations of commonly used nonhuman primate species in research and development of biopharmaceuticals date: 2015-03-20 journal: the nonhuman primate in nonclinical drug development and safety assessment doi: 10.1016/b978-0-12-417144-2.00019-6 sha: doc_id: 354592 cord_uid: vqws942c nonhuman primates (nhps) have been used extensively during the past four decades for research and nonclinical development because they are close to humans in terms of genetics, anatomy, physiology, and immunology. they have been widely used in the development of infection models, leading to the generation of vaccines and drugs, as well as in the nonclinical pharmacologic and toxicologic assessment of biopharmaceuticals, especially in the fields of immunotherapy and oncology, despite the constant pressure to move to lower species. in many cases, nhps are the only species that allows a correct risk assessment for humans. nevertheless, limitations inherent to each species have to be considered before an investigation. this chapter shines some light on the respective interests and limitations of using cynomolgus monkeys, rhesus monkeys, and marmosets in medical research and nonclinical development, with a specific focus on reproduction and immunology. biopharmaceuticals, also known as a biologics or biologicals, differ from other pharmaceutical products, or small molecules, in that they are created using biological processes rather than being chemically synthesized. they include a wide variety of medicinal products such as vaccines, blood or blood components, somatic cells, gene therapy, tissue, recombinant introduction part 7 nhp-specific aspects of standard toxicology studies seasonal variations. some data can be generated from marmosets; however; their interpretation and their translatability to humans is questionable. one species may also be more predictive than another for a specific safety concern in humans. in addition, substrains of a given species from different geographical origins may also present genetic differences that may have a considerable impact on the outcome of biopharmaceutical evaluation. therefore selecting an nhp species for research or nonclinical development requires the careful evaluation of numerous ethical and scientific considerations. the possibility of using the clinical candidate is always an advantage. confidence in the similarity of target distribution, density, downstream signaling, and physiology are important. nevertheless, hazard identification also has to be taken into consideration, and in this respect, nhps are not always superior to other species, perhaps because of the limited number of individuals allocated to nonclinical studies. the purpose of this chapter is to help the reader understand how to best select the right nhp species or strain and to illustrate this with examples. several nhp species have been used for research and drug development in many research areas because of their close evolutionary relationship to humans. recent development of genomic resources also have boosted their value as model organisms. the old world primates rhesus macaque (macaca mulatta) and cynomolgus macaque (macaca fascicularis) are by far the most commonly used species. the new world primate marmoset (callithrix jacchus) is more evolutionary diverged from humans than old world monkeys but is sometimes used when the two other species are not pharmacologically relevant for the biopharmaceutical or when specific models exist only in the marmoset. each of these three species presents advantages and disadvantages for pharmacologic and toxicologic assessment of biopharmaceuticals, which are reviewed in this chapter. because of its relatively easy maintenance in captivity, wide availability, and anatomic and physiological proximity to humans, the rhesus macaque has been used extensively in medical and biological research on human and animal health-related topics and has given its name to the rhesus factor, one of the elements of a human blood group. the rhesus macaque is widely used as an nhp model to study infection and immunity because of the close genetic relationship with humans (93% average human-macaque sequence identity) and because of the homology between human and rhesus pathogen genomes. rhesus macaques have been used to study fundamental aspects of immunology, including the development and maintenance of t-cell memory, immune dominance, and the aging immune system. there also have been many studies of immune responses in rhesus macaque models of human infections such as human immunodeficiency virus (hiv), influenza virus, tuberculosis, epstein-barr virus (ebv), cytomegalovirus, smallpox, measles, and severe acute respiratory syndrome [7] . furthermore, rhesus macaques have been instrumental in designing and testing of vaccines against infections such as hiv and smallpox, in creating drugs to manage hiv/aids [7] , and in understanding the female reproductive cycle and development of the embryo and the propagation of embryonic stem cells [8] . the genome of the rhesus macaque was cloned in 2007 [9] . surprisingly, some normal gene sequences in healthy macaques cause profound disease in humans. for example, the normal sequence of phenylalanine hydroxylase in macaques is the mutated sequence responsible for phenylketonuria in humans. some gene families are conserved or under evolutionary pressure and expansion in humans, macaques, and great apes such as chimpanzees (e.g., cholesterol pathways), whereas some are uniquely under expansion in humans, chimpanzees, or macaques (e.g., major histocompatibility genes are more abundant in macaques) [9] . the rhesus macaque was instrumental in understanding key features of reproduction. however, males and females are only fertile 4-5 months per year; seasonality is influenced by the geographic origin and is still observed after several years of being housed in a research colony in western countries [10] . since the rhesus macaque is no longer available from india, it has started to be used less in research; there is often considerable lead time in sourcing these animals. in addition, it has been progressively replaced by the cynomolgus monkey in preclinical toxicology studies, in part because of its bigger size (requiring large amounts of a drug for dosing and making them more difficult to handle) and its seasonal reproduction, which makes its use in reproductive and developmental studies more difficult. this has resulted in less toxicology background data being available for interpretation of any findings. the cynomolgus monkey has become the most widely used primate species in preclinical toxicology studies. it is widely available, being purpose bred for laboratory use, is of a size that represents a good compromise with regard to the amount of drug required for dosing and the amount of blood/tissue that can be sampled since it is generally smaller than the rhesus macaque (table 19 .1). cynomolgus macaques are especially useful for reproductive and developmental toxicology when lower species are not pharmacology relevant or given their close resemblance to human. they are also good models for the development of locally administered drugs, such as those for use in specific ocular diseases or prostate disorders, and for inhalation. nevertheless, although specific information is easily obtained from numerous breeding colonies, the cynomolgus monkey has not been as extensively characterized as the rhesus macaque in terms of genomics; however, efforts are underway to comparatively screen rhesus, cynomolgus, and other macaque species genomes. both rhesus and cynomolgus macaque species exhibit substantial variations dependent on origin and genetic background. as an example, chinese-derived populations of rhesus macaques have been shown to be more resistant to simian immunodeficiency virus than indian-derived populations. hematology and clinical chemistry differences also have been demonstrated in these two populations. in rhesus macaques, such dependence on genetic background is understood and used to guide research. although cynomolgus monkeys are the most commonly used in the development of biopharmaceuticals such as mabs and recombinant proteins, there is relatively little information on their genome in public domain databases. nevertheless, cynomolgus monkeys originate from three different populations (e.g., indochina, insular asia and the philippines, and mauritius) with own genetic features. animals from mauritius and the philippines exhibit potentially lower genetic diversity, even for genes involved in immune response [11] . differences in the gene sequences of some target proteins have been encountered within cynomolgus monkeys sourced from different geographic origins (e.g., china, the philippines, mauritius), raising concerns that certain genetic variants might result in a loss or gain of pharmacologic responses in the cynomolgus monkey colony used for toxicology studies. phenotypic differences in cynomolgus monkeys of diverse geographic origin, in particular for immune system functions, are well established, as illustrated by the diverse major histocompatibility complex (mhc) genetics of cynomolgus monkey from different geographic origin [12, 13] . collectively, these observations suggest that a prospective assessment of target gene variation may be warranted for cynomolgus monkey part 7 nhp-specific aspects of standard toxicology studies (and other nhp species) pharmacology and toxicology studies. moreover, identifying a sequence variation within a target gene requires complementary target expression/distribution, epitope/binding, and/or functional readouts. c. jacchus (common marmoset) is one of the more primitive nhp species and is used widely in fundamental biology, pharmacology, and toxicology studies. utilizing this species may provide the optimal compromise between ethical considerations, disease understanding, and drug development. once the scientific rationale has been carefully considered and their use justified, there are several advantages to using the marmoset as a model in the nonclinical development of pharmaceutical products. there are a number of unique physiologic differences between old world primates such as rhesus and cynomolgus macaques and new world primates such as marmosets. for example, anatomic differences in placentation and frequent twinning lead to bone marrow chimerism of dizygotic twins. limited diversity at both mhc class i and ii loci has been identified in both tamarins and marmosets and may be responsible for differences in disease susceptibilities that are exploited in infectious disease studies. while the availability of research tools has been a limitation in the past, making marmosets less attractive than rhesus macaques as animal models, this is no longer the case in a number of key areas. experimental autoimmune encephalomyelitis (eae) is a well-established animal model of multiple sclerosis (ms). the disease induced in common marmosets more closely resembles ms than that in rodent or other primate models in terms of clinical signs, lesions, and evolution. the unique particularities of marmosets in terms of full bone marrow chimerism of littermates have been key in the understanding of disease pathogenesis. the eae model has been used in the preclinical evaluation of novel immunotherapies such as testing new anti-inflammatory agents, tolerance-based therapies, and costimulated target therapy [14] . marmosets also have been used widely in neuroscience research programs in models of cerebral vascular disease, tardive dyskinesia, and neurodegenerative diseases and in studies of normal neurophysiology [15] . common marmosets have been used extensively in infectious disease research in large part because of their unique sensitivity to a number of important human infectious agents, including viral, bacterial, and parasitic agents. in particular, marmosets are susceptible to a variety of herpes virus agents, including gammaherpes viruses, such as ebv and herpesvirus saimiri, herpes simplex virus, hepatitis a virus infection, junin virus, malaria, measles, and gb virus b, a surrogate model of human hepatitis c virus. the reason marmosets are highly susceptible to infection with many of these agents is not understood, but limited diversity at mhc class i and ii loci is hypothesized to play a role, as mentioned above. in particular, marmosets have been extensively used to investigate the molecular pathogenesis of viral-induced lymphoma and ebv and lymphocrypto virus (lcv) model of viral oncogenesis or viral persistence [15] . the marmoset also has been used as a nonrodent second species in drug safety assessment of new chemical entities (nces) and, more recently, of biopharmaceuticals based on side effects, findings of given drugs, and metabolizing enzymes or receptors found to be similar to humans [16] [17] [18] [19] ; because of the closer phylogenetic relationship to humans than other second species such as the dog, common marmosets may be more suitable for certain types of part 7 nhp-specific aspects of standard toxicology studies pharmacokinetic and toxicological screening. when the test item is a biopharmaceutical and the mechanism of action is dependent on specific receptors or epitopes, relevant species may be restricted to nhps. the small size of common marmosets may represent an additional advantage because it reduces the quantity of compound to be synthesized and may translate into shorter development times. however, their short stature, sensitivity, and activity level may also pose unique challenges [16, 17] . nevertheless, for some development programs, as for canakinumab, the marmoset may be the only suitable species [20] . in addition, marmosets are largely free of diseases harmful to humans. as a result, this species is being used in studies ranging from behavioral studies to reproductive and developmental protocols. however, this species poses unique challenges because their sensitivity, small size, housing conditions, and high activity level. many drugs in development are intended for chronic administration to infants, women of child-bearing potential (wocbp), fertile men, and infants, requiring the evaluation of hazard on fertility and/or early development of the offspring and juvenile toxicity. with biologics, often the only relevant species with which to assess these is the nhp. if for nces the reproductive strategies benefit from standard and well-validated study designs in rodents and in rabbits, for which extensive historical databases are available, some specific considerations for biopharmaceutical proteins have to be taken into account. they do not freely diffuse across membranes, including placenta, and are therefore not expected to affect early stages of development, especially embryogenesis, because the embryo/fetus is not directly exposed during early life [21] . although mabs and fc fusion proteins are also large proteins, they can be actively transported from mother to fetus in mammals via the neonatal fc receptor for immunoglobulin (ig) g (fcrn), with variable efficacy as a function of the igg subclass of antibodies from which they are derived, with igg 1 > igg 4 > igg 3 > igg 2 [21, 22] . the long half-life of many therapeutic iggs may result in prolonged pharmacological activity and effects on the developing fetus, including the immune system (developmental immunotoxicity). transfer of antibodies occurs mainly during the second and third trimester in humans, when fcrn starts being expressed on the placenta. all antibodies or recombinant proteins with an intact igg fc portion can be transported across placenta, whereas antibody fragments (fab, dab, scfv, f(ab)′ 2 ) without fc are not [23] . similar placental transfer of iggs has been demonstrated in humans and nhps [22, 24, 25] . because igg mabs are not transferred to the fetus until later in pregnancy when organ development has ended, the evaluation of the effects of therapeutic igg antibodies and fc fusion proteins on embryo-fetal development is not systematically required prior the enrollment of wocbp in phase ii clinical trials (as for nces), according to the revised ich m3 guideline [26] . this is because if a woman accidentally became pregnant while being administered an igg mab in a clinical trial, the risk of exposure to the developing offspring in the early weeks of pregnancy is very low. potential adverse effects to the fetus in these early stages are more likely to be due to secondary effects of maternal toxicity rather than direct effects. however, some cases of fetal abnormalities without maternal toxicity have been reported and considered secondary to altered placental function and/or reduced fetal growth, even if the direct inhibition of target signaling in the conceptus cannot be ruled out, as reported with anti-receptor for the insulin-like growth factor 1 (igfr) [27] . therefore the assessment should be designed based on the risks that may be related to target modulation in the mother or in the infant. a careful review of the distribution and the role of the target in different physiological conditions is important to appreciate risks and adapt the study design as a consequence. standard study designs for use with rodents and rabbits allow all steps of reproduction and development to be evaluated (as described in ichs5(r2)) and are generally predictive, provided the therapeutic mab does not modulate a pathway that is physiologically different between rodents/ rabbits and humans. conversely, study designs for use with nhps do not cover all aspects of reproduction (e.g., direct fertility assessment through mating or egg implantation-pregnancy cannot be confirmed until day 20 of gestation) and so may not allow a full evaluation of the specific risks in humans. however, the physiology of reproduction, pregnancy, and parturition, including hormonal control, are very close between human and nhps, allowing better identification of hazards, provided an appropriate study design is applied. unlike rodents, the relevance of the nhp model for developmental toxicity is well established [8] . the spontaneous incidence of congenital defects is low [28, 29] . clinical relevance and superiority over rodents has been demonstrated for thalidomide, vitamin a, and valproic acid [28] . choosing the nhp for assessing the effect of a therapeutic protein on reproductive and developmental functions may help identify hazards that would not be identified in other species and allows, in general, an evaluation of the clinical candidate. however, there are cases where no relevant nhp species is suitable for assessing reproductive toxicity of the clinical drug product. in those cases, mab surrogates are used for reproductive toxicity studies. well-known examples are infliximab (anti-tumor necrosis factor alpha (tnfα)), efalizumab (anti-cd11a), and eculizumab (anti-c5), respectively. among others, an important consideration is the choice of the nhp species or even substrain for reproductive and developmental testing (table 19 .1). the physiology of the menstrual cycle of old world monkeys closely resembles that of humans. for this reason, monkey species belonging to this group are preferred as experimental models in studies of human reproduction, pharmacology, and toxicology. especially for studies of sex hormones, synthetic steroidal compounds, or compounds that interfere with the hypothalamus-pituitary-ovarian (or testicular) axis, such monkey species are predictive [30] . because of its lack of reproductive seasonality, convenient body size, close resemblance to human physiology, and thanks to the availability of a large historical database, the cynomolgus monkey is the species of choice for reproductive toxicity studies. the average duration of the menstrual cycle in the female is 28 days, and the endocrinology of the ovarian cycle is similar to that in women, with one predominant follicle maturing to ovulation. markers of the menstrual cycle are also similar to those in women, and the cycle can be monitored by daily vaginal smears [31] . there are also strong similarities between the male cynomolgus monkeys and men in terms of endocrine control of testicular function (follicle stimulating hormone (fsh) is especially part 7 nhp-specific aspects of standard toxicology studies important for spermatogenesis and inhibin b is an important marker of testicular toxicity), prostate anatomy, and control of prostate function. fertility rates are typically around 40-70% and preimplantation loss is evaluated at 25% [10] . the most relevant period in which to evaluate adverse drug effects on organogenesis takes place mainly between gestation days 20 and 50, with palate fusion occurring around gestation days 45-50. pregnancy lasts around 160 days, with a range of 134-184 days. the maturation of different organs and systems evolves similarly to that of humans, with some noticeable differences, including earlier bone ossification, respiratory and urinary systems being mature at birth, and more rapid maturation of the digestive system [32] . twin pregnancies are extremely rare in macaques, with an overall twin live birth incidence around 0.1%. the low fertility rates and high abortion rates (10-30%) in this species limit the detection of infrequent risks [10] . nevertheless, its size, ease of housing, and extensive panel of reagents make it an attractive model. several marketed products have been tested in this species (basiliximab, adalimumab, rituximab, natalizumab, ustekinumab, alefacept, and omalizumab) for a partial or complete evaluation of embryo fetal and postnatal development. there is a much ethical pressure to limit the use of nhps, especially in reproductive toxicology. however, there are examples where rodents are not helpful in hazard identification, as illustrated in the two cases below. an igg4 directed against a soluble target for inflammatory indications was developed in the cynomolgus monkey and general toxicity studies were uneventful. the potential effects on the fetus and developing infant were addressed in a single enhanced pre and postnatal development study in the cynomolgus monkey. the decision was based on normal fertility, reproduction, and development in target knockout mice, or in mice treated with a therapeutic antibody blocking upstream signaling of the receptor, an based on an uneventful embryofetal development (efd) study in cynomolgus monkeys treated with a therapeutic antibody targeting the same signaling pathway from gestation days 20 to 50 and undergoing a cesarean delivery on gestation day 100. therefore, pregnant female macaques were administered the igg4 weekly from gestation day 20 to approximately gestation day 160 and were allowed to deliver their offspring naturally and raise their infants. maternal animals tolerated the antibody treatment as in general toxicity studies, without changes compared with control animals, and delivered healthy infants that developed normally with a functional immune system. however, an increased incidence of treatment-related perinatal mortalities was observed in infants. macroscopic examination of the decedents revealed skull bone luxation; subdural, cerebellar, or superficial brain hemorrhage, all suggesting difficult delivery; and an empty gastrointestinal tract, indicating the absence of suckling after birth. unexpectedly, treated maternal animals experienced a longer pregnancy than controls (up to a mean of 10 days) and a higher incidence of retained placenta, and prenatal maternal mortality associated in some cases with genital hemorrhages was observed. dystocia is one of the leading causes of late pregnancy loss in cynomolgus monkeys, rhesus monkeys, and baboons, often resulting in intracranial trauma/hemorrhages in the nonviable fetus/infant [33] [34] [35] . however, difficult delivery rarely results in maternal death and is not usually associated with important genital blood losses, even in the case of placental retention. a careful review of the literature in light of the findings of the study suggested that the soluble target may indeed play a role in parturition, as indicated by increased circulating concentrations of the target during the third trimester of pregnancy and increased messenger rna and protein concentrations in the cervix, myometrium, and choriodecidua during labor [36] . the target is thought to be involved in the local process of inflammation occurring at parturition, enabling recruitment of leukocytes in the cervix and uterine wall [37] to induce cervical ripening and dilation and probably resorption of the interface between the placenta and uterine wall [38, 39] . it also promotes the expression of the prostaglandin synthase type-2 in the cervix, myometrium, choriodecidua, and placenta [40] , enabling efficient contractions. although additional factors interfering with parturition cannot be ruled out, the inhibition of target signaling may explain the observations in maternal animals. maternal mortality was considered to be a direct consequence of the long and difficult labor associated in some instances with hemorrhage caused by incomplete resorption of the placenta/uterine interface and with genitourinary infections resulting from the prolonged labor. perinatal infant mortality was considered indirectly linked to the treatment, resulting from head injuries at parturition or lack of maternal care during the first critical hours after birth. such findings could not have been observed in mice, whose physiology of parturition is very different, or in monkey studies not specifically addressing parturition, but they highlight a risk for pregnant women still exposed to the mab by the end of the pregnancy based on the similar physiology of pregnancy and parturition in nhps and humans. this example illustrates the need to carefully evaluate target expression, the biology of the target, and its potential role in pregnancy, as well as the need to understand how close the test species and humans are for the pathway to be explored. another example in which the cynomolgus monkey was shown to be more sensitive than the rodent for detection of adverse effects on pregnancy is the case of the soluble receptor for interleukin-4 (il-4r) [41] . the treatment of pregnant cynomolgus monkeys with human soluble il-4r (0.2 and 2.0 mg/kg intravenous) during organogenesis (gestational days 20-51) resulted in increased incidence of spontaneous abortion, embryo/fetal death, and stillborn neonates. however, pregnant crl:cd1 mice treated with an intravenously soluble mouse (surrogate) il-4r during organogenesis presented no adverse effects up to the equivalent dose of 20 mg/kg. cd-1 mice may be th1-biased (like c57bl/6), a possible reason why no reproductive effects were seen in this mouse study-i.e., they already have more th1 bias, so tipping them further that way has little effect [42] . although the cynomolgus monkey is the established nhp model for reproductive and developmental toxicology, there are several advantages to conducting developmental toxicity studies using marmosets. considerations include specific cross-reactivity-particularly with biologics-and some specific teratogenic risks that are more difficult to demonstrate in species with only one offspring. from a reproductive point of view, marmosets are unique among nhps and differ from humans in some key respects. females have no menstrual bleeding, each cycle results in multiple ovulations, and postpartum conception is not uncommon. duration of the menstrual cycle varies from 24 to 30 days, with a long luteal phase, a mid-phase fsh peak, and simultaneous maturation of one to four eggs [43] . ovarian cyclicity can be controlled by monitoring progesterone after triggering luteolysis by administering prostaglandin-f2α. as a result, controlled efd studies are feasible. multiple, simultaneous ovulations result in litter part 7 nhp-specific aspects of standard toxicology studies sizes ranging from one to four births; twins (30-70%) and triplets (20-25%) are most frequent. interestingly, marmoset have a naturally high abortion rate to reduce the number of fetuses in utero, as they generally can nurse only two offspring. compared with old world monkeys, the father has to stay with the mother for the entire duration of the pregnancy to prevent abortion. gestation lasts between 140 and 150 days. the interbirth interval is approximately 6 months, but conception can occur 10-50 days postpartum. in contrast to humans, twins' placental disks are supplied by blood vessels of both fetuses. shared anastomoses in the chorion commonly occur, exhibiting a hematopoietic xx/xy chimerism in the case of twins of opposite sex. in addition, most marmosets are chimera from at least two fertilized eggs or early embryos fused together. the difference in phenotype may be subtle (hitchhiker's thumb and straight thumb, different hair growth on opposite sides of the body, etc.) or completely undetectable [43] . in marmosets, as in most new world monkey species, chorionic gonadotropin hormone plays the role of luteinizing hormone (lh). key differences are present in males too. the control of leydig cell function and testosterone production by lh is entirely different from that of humans. regulating genes on the y chromosome are different from those of male old world monkeys and humans, and some key fertility genes (e.g., daz) are absent. as a consequence, even if the organization of the testicular germinal epithelium is similar to that of man, the correspondence with the functional and hormonal spermatogenesis in human remains unknown. the reproductive cycle is considerably shorter than in macaques, and males attain fertility at 17-20 months of ages (for a review, see refs. 10, 16, 44) . this species is valid and relevant for efd studies (segment ii) in the event that cynomolgus monkeys cannot be used (e.g., a biopharmaceutical that is not pharmacologically active in cynomolgus or rhesus monkeys). effects of canakinumab on embryo-fetal development were assessed in marmosets using the drug product. the only finding associated with canakinumab treatment was a slight reduction in the number of fetuses at the high dose, which correlated with a slight trend toward decreased placental weight. there were no treatmentrelated effects on fetal development. despite the confirmation of placental transfer of igg at the time of cesarean delivery (gestational days 112-114), given that transfer of igg across the placenta starts late during gestation, it is unlikely that the fetus was exposed to canakinumab during organogenesis [22] . there are no other published examples of the use of marmosets in reproductive and developmental toxicology. the feasibility of the marmoset model for a full range of developmental studies, including segment iii pre/postnatal studies, must be more fully evaluated [10, 16, 44] . the high species specificity of the sequence/structure of most immune receptors targeted by mabs makes the selection of an appropriate animal model challenging. this is probably less true for small-molecule drugs. however, the nhp is the most commonly selected animal model for safety assessment of immunomodulators, given its genetic similarity to humans and the homology of its immune system to that of humans. for reasons of practicability/availability, the cynomolgus monkey is the preferred nhp species, although occasionally marmosets and rhesus macaques are used. nevertheless, there are safety-relevant functional differences between primate and human immune systems, despite a close evolutionary relationship. these include, for example, differences in mhc genes, distribution and function of fc receptors and toll-like receptors, and sensitivity to complement activation, which all need to be considered while assessing the safety of immunomodulatory compounds and, more specifically, agonists [45] . a range of potential liabilities associated with immune agonism may be explored in nhps [46] . the cynomolgus monkey has been used as a relevant toxicology species for immunostimulatory agents such as toll-like receptor agonists; however, while special recognition regarding the clinical risk of systemic cytokine release for certain targets is warranted, the nhp is not always predictive of this potential toxicity in humans. an important difference between humans and other nhps such as cynomolgus macaques is the stronger human t-cell proliferative responses upon activation via the t-cell receptor. this is the consequence of human-specific loss of t-cell siglec expression during the later stages of evolution. cd33-related siglecs, which are inhibitory signaling molecules expressed on most immune cells, downregulate cellular activation pathways via cytosolic immunoreceptor tyrosine-based inhibitory motifs. concordant with this species-related difference in siglec expression is the observation that several common human t-cell-mediated diseases, such as bronchial asthma, rheumatoid arthritis, and type 1 diabetes, have not been reported in chimpanzees or other great apes. the lack of siglec expression in humans may partially explain why the cytokine release syndrome (crs) in humans cannot be accurately predicted by nhps or rodents. the life-threatening cytokine storm effects induced by tgn1412 were a result of the unexpected tgn1412-mediated activation (proliferation and cytokine release) of cd28-expressing cd4 + cd45ro + effector memory t cells predominantly residing in tissue [47] . these effects were not observed in cynomolgus monkeys, which do not express cd28 on effector memory t cells, nor in mice, in which the rapid t regulatory cell expansion quenched the proinflammatory effects of the relatively small effector t-cell population in these animals compared with a mature human immune system [48, 49] . these human-specific differences in t-cell activation need to be carefully considered during safety assessment of immunostimulatory mabs against t-cell targets. immune genes are expanded in macaques. the macaque genome has 33 major histocompatibility genes, three times that of humans. this has clinical significance because the macaque is used as an experimental model of the human immune system. by contrast, marmosets exhibit limited diversity at mhc class i and ii loci [50, 51] . fc receptors are plasma membrane glycoproteins that bind to the fc region of antibodies. crosslinking of fc receptors by ab-opsonized antigen complexes initiate cellular immune responses, including phagocytosis, ab-dependent cell-mediated cytotoxicity, respiratory burst, release of cytokines and inflammatory mediators, and antigen presentation. binding human igg to cynomolgus monkey fcγrs is broadly similar to that observed with human fcγr [52] ; however, there are some important differences. in humans, fcγriiia (cd16a) is expressed on monocytes, macrophages, and nk cells, whereas the fcγriiib (cd16b) isoform is expressed on neutrophils, eosinophils, and other cells. in nhps there is only one cd16 gene, homologous to the human cd16a, which is restricted to nk cells and monocytes [53] . this difference necessitated the testing of an antihuman cd16 (fcγriiia and fcγriiib)-specific mab in human cd16 transgenic mice to assess the impact on neutrophils [54] . even greater differences in fc receptor expression, distribution, and internalization profiles between humans and mice also are observed [55] . anatomic differences in human and animal immune systems and their responses to immunomodulatory drugs have been reviewed [56] . the importance of ensuring that human-relevant fc binding and effector function activity of igg is occurring in the toxicology species is further illustrated by the case of hu5c8, an anti-cd40l igg1. development was stopped in clinical phase ii after it induced thromboembolism in patients [57] . no thromboembolisms were detected in preclinical studies conducted using cynomolgus monkeys. further investigations showed that treatment in rhesus monkeys, but not in cynomolgus monkeys, resulted in thrombi formation at various locations and vasculopathy characterized by intimal hyperplasia. the full-length igg1 induced platelet aggregation and serotonin release once bound to its target in humans and in rhesus monkeys. other constructs based on fab fragments or full-length antibody with aglycosyl fc did not cause platelet aggregation or activation in vitro, nor thromboembolisms or intimal vascular hyperplasia in vivo, while binding to the target with comparable affinity. the mechanism is not fully understood, but one hypothesis is that cd40 may play a role in hemostasis and platelet biology by functioning as a primary signaling receptor for cd40l and by serving as a docking molecule for cd40l immune complexes. cd40 may drive translocation of fcγriia (cd32a) to the platelet surface, enabling the fc part of antibodies bound to their target to be captured and inducing strong platelet activation [58] . based on these observations, the nonclinical development of a new anti-cd40l with a different format preventing fc interaction was therefore conducted in the rhesus monkey [59] . this case study also demonstrates that the rhesus monkey may for some toxicities be the most relevant nhp species. igg1 and igg3 antibodies are capable of mediating complement activation (binding to c1q). by contrast, igg2 shows little complement activation and igg4 shows none at all. therefore, for immunomodulatory mabs that bind to membrane-based targets, to avoid cell depletion, the igg2 or igg4 format or silent igg1 (with reduced fc effector function) is often used because of the absence of fc effector function. in addition to complement-dependent cytotoxicity, which is an intended pharmacological effect of certain mabs (e.g., direct killing of tumor cells), complement activation may lead to a number of safety-relevant consequences, including cytokine release, hematological effects, and/or potentially life-threatening hemodynamic disturbances. despites large concentrations of complement components in cynomolgus monkey serum compared with human serum, the hemolytic activity is comparable. however, mechanistic differences in terms of (cross-)species-specific igg/fc-mediated complement activation, particularly related to igg aggregation status, cannot be excluded and deserve further investigation [60] . immunophenotyping of peripheral blood cells in nhps is readily available for a variety of populations of lymphocytes and is usually included in the safety assessment of immunomodulators. a standard panel can include total t lymphocytes, helper t lymphocytes, cytotoxic t lymphocytes, b lymphocytes, and nk lymphocytes. populations that typically are not evaluated unless warranted to measure a specific pharmacodynamic response may include regulatory t helper lymphocytes, activated t helper lymphocytes, activated cytotoxic t lymphocytes, effector memory t helper lymphocytes, effector memory cytotoxic t lymphocytes, central memory t helper lymphocytes, central memory cytotoxic t lymphocytes, naïve t helper lymphocytes, and naïve cytotoxic t lymphocytes. the selection of such assessments should be driven by the biology of the drug target and should be considered as part of the safety assessment on a case-by-case basis [60] . some qualitative and quantitative differences exist with respect to surface markers on lymphocytes relevant for studies of immunotoxicity. for example, the marmoset lacks natural killer cells with typical markers found in humans (cd2 − cd56 + ); conversely, cytotoxic killer effector cd4 + cells (cd4 + cd56 + ) found in marmosets are not found in humans [17] . the t-cell-dependent antigen response (tdar) assay encompasses multiple aspects of the immune response (i.e., antigen capture and presentation, t-cell help, and antibody formation [including class switching]) or the ability to mount an immune response in one end point: antigen-specific antibody titers [61] . in toxicology regulatory guidance documents, the tdar is considered to be one of the functional assays of choice to evaluate potential immunotoxicity of investigational new drugs. however, the interanimal variability limits the sensitivity of the assay in studies powered for general toxicology evaluations, despite the similar behavior of sexes or animals from different countries of origin. inhibition of the tdar can be clearly evidenced with immunosuppressive agents in nhps, but demonstrating a lack of activity in the assay has been problematic and the definition of biologically irrelevant variations is not established. keyhole limpet hemocyanin, (klh), sheep red blood cells (srbc), and tetanus toxoid (tt) are the most commonly used antigens for challenge; klh and tt show similar interanimal variability and a trend to less variability than srbc. power analysis of numerous studies has demonstrated that animal group sizes of 8-12, combining males and females, could detect ≤3.1-fold differences in anti-klh igg responses. assay optimization and harmonization of the tdar in nhps remains a priority [60] . a functional ex vivo assay for nk cell-mediated lysis is available for nhps (using the k562 human myelogenous leukemia target cell line), but the development of a functional assay for ctl activity is needed and is the subject of significant effort. if there is generally a good concordance of pharmacodynamic effects in humans and nhps, there is often limited concordance for adverse effects when considering mabs or fusion proteins for cell surface targets or soluble targets [62, 63] . the rare but serious adverse events that are of greater concern for patients are usually not predicted in studies conducted in normal healthy animals. part of the reason why the animal studies fail to detect these effects is that nhp studies are not powered to detect rare events. also, serendipitous infectious challenge may not occur in a controlled laboratory environment, and animals are generally prescreened for certain infectious agents before inclusion in studies; only those animals that test negative are included in the study. laboratory animals do not receive comedications and are healthy when enrolled in toxicology studies. regarding cell surface targets, downstream signaling may also be different because the antibody or fusion protein tested does not bind to the same epitope. nhps are often the only relevant species for the development of biopharmaceuticals; however, a comprehensive assessment of immunotoxicity remains a challenge because of the interanimal variability associated with certain parameters. the stage of development of certain immune assays, the incomplete knowledge of target distribution or effector function compared with humans, and a greater understanding of the background incidence of certain pathogens in nhps are needed for proper interpretation of infectious agent-related findings that may be misinterpreted as treatment-related findings as a result of immunomodulation. several examples have demonstrated the need to use nhps, especially to identify hazards in the reproductive and developmental assessment of biopharmaceuticals. nevertheless, some important challenges that may impact safety and risk assessment for humans remain. these include limited numbers of animals and interanimal variability associated with some parameters, the stage of development of certain assays, the incomplete knowledge of target distribution or effector function compared with humans, and differences inherent to the species used or the provenance. preclinical safety evaluation of biotechnology-derived pharmaceuticals immunogenicity assessment in non-clinical studies immune function evaluation strategies in the nonhuman primate incidences and range of spontaneous findings in control cynomolgus monkeys (macaca fascicularis) used in toxicity studies spontaneous neoplasms observed in cynomolgus monkeys (macaca fascicularis) during a 15-year period background pathology of the common marmoset (callithrix jacchus) in toxicological studies extraction and characterization of the rhesus macaque t-cell receptor b-chain genes preclinical safety of biotechnology products intended for human use evolutionary and biomedical insights from the rhesus macaque genome reproductive/developmental toxicity assessment of biopharmaceuticals in nonhuman primates determining genetic background in captive stocks of cynomolgus macaques (macaca fascicularis) characterization of 47 mhc class i sequences in filipino cynomolgus macaques mhc class i a loci polymorphism and diversity in three southeast asian populations of cynomolgus macaque clinical, pathological, and immunologic aspects of the multiple sclerosis model in common marmosets (callithrix jacchus) white paper for complete sequencing of the common marmoset (callithrix jacchus) genome the common marmoset (callithrix jacchus) as a model in biotechnology overview of the marmoset as a model in nonclinical development of pharmaceutical products comparison of the formation kinetics of acetaminophen in chimpanzee, cynomolgus monkey, marmoset, rhesus monkey and human liver microsomes assessment of p450 induction in the marmoset monkey using targeted anti-peptide antibodies toxicology summary an interspecies comparison of placental antibody transfer : new insight into developmental toxicity testing of monoclonal antibodies fcrn: the neonatal fc receptor comes of age marginal transfer of reopro (abciximab) compared with immunoglobulin g (f105), inulin and water in the perfused human placenta in vitro the placental transfer of igg in the cynomolgus monkey placental transfer of fc-containing biopharmaceuticals across species, an industry survey analysis nonclinical safety studies for the conduct of human clinical trials with pharmaceuticals embryo-fetal developmental toxicity of figitumumab, an anti-insulin-like growth factor-1 receptor (igf-1r) monoclonal antibody, in cynomolgus monkeys frequency of spontaneous congenital defects in rhesus and cynomolgus macaques developmental and reproductive toxicology studies in nonhuman primates assessment of hormonally active agents in the reproductive tract of female nonhuman primates summary comparison of female reproductive system in human and the cynomolgus monkey (macaca fascicularis) developmental toxicity testing of biopharmaceuticals in nonhuman primates: previous experience and future directions frequency of prenatal loss in a macaque breeding colony stillbirths in macaca fascicularis the baboon model (papio hamadryas) of fetal loss: maternal weight, age, reproductive history and pregnancy outcome pro-labour myometrial gene expression: are preterm labour and term labour the same? leukocyte density and proinflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term the transcriptome of the uterine cervix before and after spontaneous term parturition role of cytokines and other inflammatory mediators myometrial prostaglandin e2 synthetic enzyme mrna expression: spatial and temporal variations with pregnancy and labor a comparison of effects on reproduction and neonatal development in cynomolgus monkeys given human soluble il-4r and mice given murine soluble il-4r th1/th2 cytokine balance as a determinant of acetaminophen-induced liver injury germ-line chimerism and paternal care in marmosets (callithrix kuhlii) feasibility of embryofetal development studies in the marmoset (callithrix jacchus) preclinical safety evaluation of biopharmaceuticals. a science based approach to facilitating clinical trials toxicity as a result of immunostimulation by biologics monoclonal antibody tgn1412 trial failure explained by species differences in cd28 expression on cd4 + effector memory t-cells rapid regulatory t-cell response prevents cytokine storm in cd28 superagonist treated mice predicting cytokine storms: it's about density loss of siglec expression on t lymphocytes during human evolution safety assessment and dose selection for first-in-human clinical trials with immunomodulatory monoclonal antibodies different adaptations of igg effector function in human and nonhuman primates and implications for therapeutic antibody treatment igg fc receptor iii homologues in nonhuman primate species: genetic characterization and ligand interactions nonclinical evaluation of gma161-an antihuman cd16 (fcγriii) monoclonal antibody for treatment of autoimmune disorders in cd16 transgenic mice fcgamma receptors as regulators of immune responses species differences in the structure and function of the immune system thromboembolic complications after treatment with monoclonal antibody against cd40 ligand the role of cd40 in cd40l-and antibody-mediated platelet activation an assessment of the thromboembolic potential of cdp7657, a monovalent fab' peg anti-cd40l antibody, in rhesus macaques regulatory forum opinion piece: immunotoxicology assessments in nonhuman primateschallenges and opportunities immunotoxicology strategies for pharmaceutical safety assessment concordance of preclinical and clinical pharmacology and toxicology of monoclonal antibodies and fusion proteins: soluble targets concordance of preclinical and clinical pharmacology and toxicology of monoclonal antibodies and fusion proteins: cell surface targets key: cord-016313-n4ewq0pt authors: baranyi, lajos; dropulic, boro title: advances in lentiviral vector-based cell therapy with mesenchymal stem cells date: 2012-09-27 journal: mesenchymal stem cell therapy doi: 10.1007/978-1-62703-200-1_14 sha: doc_id: 16313 cord_uid: n4ewq0pt the field of possible application of mesenchymal stem cells in medicine and research expanded tremendously with the advent of improved lentiviral-vectors capable of inserting stable copies of genes of interest and expressing proteins or biologically active rna species ad libitum, performing delicate gene editing or active gene silencing or serving as advanced drug delivery systems utilized in ex vivo cell therapy. the combination of these two fields has created a number of new areas of research in the landscape of modern medicine which are now extensively studied and discussed here. these areas include tissue engineering, tissue repair, wound healing and tissue implants, anticancer therapies, angiogenesis, myocardial infarction and repair as well as understanding and treating acute lung damage and injury. in addition, genetically modified, tagged mscs are being intensively deployed in research and therapeutic attempts of the various ailments of the central nervous system including parkinson’s disease, alzheimer’s disease, various phases of acute ischemia and trauma. the emergence of new and important data for type ii diabetes research is being followed with treatment suggestions and studies of senescence to find novel applications for genetically engineered mscs. we find in ­general that genetically modified mscs are at the cusp of breaking through from basic research into the next phase of clinical trials. stem cells, including the mesenchymal stem cells (mscs), are very close manifestations of plato's imagery of the shadows on the cave wall, since they are dif fi cult to study outside their intimate interactions with their microenvironment [ 288 ] . our observation methods change their responses and characteristics [ 9, 74, 82, 103, 145, 252 ] , as in quantum physics, when the observation changes the observed. with that caveat, we can admire the rapid development in stem cell research. alas, the dif fi culties in research are faithfully re fl ected in the confusion in the nomenclature used for describing and classifying stem cells, including the classes of stem cells of mesodermal origin. the recent de fi nition of mscs by dominici states that mscs are a stromal cell type, possessing the following characteristics and markers: plastic adherence in cell culture, speci fi c surface antigen expression of cd105(+), cd90(+), cd73(+), cd34(−), cd45(−), cd11b(−) or cd14(−), cd19(−) or cd79a(−), hla-dr1(−) and multi-lineage in vitro differentiation potential (osteogenic, chondrogenic, and adipogenic) [ 59 ] . however, this de fi nition would neatly exclude cd34+ hematopoietic stem cells (hscs ), while one also could argue that the hematopoietic stem cells are just a specialized subclass of the mesenchymal progenitors [ 180 ] . another subset of mscs that express hyaluronan (cd44), an adhesion molecule important for stem cell homing [ 14, 125, 281 ] , would also be excluded, but their perivascular equivalents could be considered to be true mpcs [ 180 ] . it becomes even more complicated if we include the results of (stem) cell reprogramming, when more or less differentiated cell types are regressed into less differentiated, pluripotent cell types [ 91, 185, 192, 239 ] , providing us with a never ending stream of novel biomarkers, more often represented by whole proteome analysis [ 1, 203 ] . time will tell, what are the biomarkers and criteria for properly characterizing the particular stem cell populations, but there is a functional de fi nition lingering around as a fi rm conceptual handle on the idea of cell plasticity of which stem cells are prominent representatives [ 21, 22 ] . the plasticity indicates the ability of matured or not fully differentiated cells to differentiate into novel cell types, or more accurately, it describes the existence of cells specialized into becoming progenitors of differentiated cells while sustaining their own type and maturation level. combining this with the embryology and origins of cell lineages from the three primordial "dermata" (ecto-, endo-, and mesoderm) provides us with a useful and generalized de fi nition of mscs being the pluripotent, self-renewing stromal cells of mesenchymal origin and allowing us to determine the speci fi c biomarkers later, at our convenience, as the state of affairs in mesenchymal stem cell biology progresses and solidi fi es. there is no doubt that we will fi nd the appropriate placement for the specialized subtypes as well as the proper and practical placement of some of the induced pluripotent cells (ipcs) in the realm of mscs. regardless of the exactitude of the classi fi cation, the (omni-) presence of the mesenchymal cell lineages in all of the organs and tissues [ 11, 102, 116, 132, 180 ] renders them good candidates not only for general stem cell therapy [ 22, 73, 102, 235 ] , but even more promising is the potential use of mscs for gene therapy [ 35, 45, 176, 196 ] , cell reprogramming [ 9, 36, 252 ] , delivery of bioactive molecules [ 163, 196 ] , and tissue engineering [ 4, 82, 153 ] . in addition, a number of new issues are arising from the results describing the importance of stem cells in inducing and sustaining the malignant phenotype and the potential therapeutic targeting of a wide range of the elusive cancer stem cell types [ 27, 183, 217, 244 ] . the genetic modi fi cation of mscs and associated cell types with lentiviral vectors opens their application beyond reliance upon the innate properties of the cells. expression of proteins that can modulate their biology or therapeutic properties enormously expands their utility for therapy. the lack of a crisp de fi nition of all the stem cell types affects targeting of lentiviral vectors to speci fi c subsets of stem cells. however, recent successful efforts in the pseudotyping of lentiviral vector is a step in the right direction. the use of the vsvg pseudotype expanded the tropism of the lentiviral vectors and, as a result, practically any cell type can be targeted and the narrowing of the tropisms by developing novel vector-pseudotypes will be addressed. the emergence of single chain antibodies as pseudotype indicates that we can expect a rapid expansion of this technology in the near future and will result in a precise tool for studying cell lineages. we focus and limit our review on the recent progress made in stem cell research using lentiviral vector-based gene delivery, a method that is emerging as the safest and most effective way to modify (stem) cells permanently or temporarily, if using non-integrating versions of the novel generations of lentiviral vectors, both of which have clear potential for a wide range of research application in preclinical studies as well as therapeutic applications. the most commonly used lentiviral vector framework is hiv-1 based although hiv2, siv (simian immunode fi ciency virus ), fiv (feline immunode fi ciency virus) have been successfully tested ; see review by dropulic [ 62 ] . the native hiv-1 is a human pathogen; but it had been modi fi ed to eliminate pathogenicity and increase safety before considering it as a broadly available tool for gene transfers. typically, lentiviral vector are generated by trans complementation , a process that separates the essential components of hiv (the genes encoding gag-pol , rev , and env ) into separate plasmids, which lack the packaging signal and , therefore, can never end up in a packaged vector unless they appear in a recombinant sequence [ 63 ] . the components (tat , vif, etc.) responsible for pathogenicity by upregulation of transcription [ 54 ] and export of genomic rna to the cytoplasm have been successively removed from the constructs. the potential for a recombination event is minimized, and for all practical purposes avoided, by carefully editing the genes and using codon degeneracy to reduce the chances of recombination with the wild-type virus. these separate plasmids are used to co-transfect a packaging cell, typically hek293, along with a payload plasmid that carries the packaging signal necessary for starting the envelope formation and encapsidation of the mrna that carries the payload gene (s) as well as the 5 ¢ and 3 ¢ long terminal repeats (ltr s) necessary for integration into the transcriptionally active regions of the host chromosomes. packaging is a delicate process, which ensures that with the rna, appropriate trnalys, protease, integrase, and reverse transcriptase enzymes are carried by the vector with the packaging elements necessary for successful cell entry, reverse transcription of vector rna to dna, transport of that dna into the nucleus, and the permanent integration of the dna into host chromosome. the env gene encodes the protein gp160 that is cleaved into trimer-forming gp120, which appears as spikes decorating the vector particle; and gp41, that carries a transmembrane region and a carboxy terminal sub-domain that interacts with the nucleocapsid within the envelope. the n-terminal domain has a fusogenic domain that facilitates cell entry by fusing the outer membrane of the vector with the cell membrane. a region further down to from the amino terminal region also binds to gp120 which in turn binds to primary hiv-1 receptors on the target cd4+ of t lymphocytes. this property if left unmodi fi ed would signi fi cantly limit the usability of the lentiviral vector, as very few cells types can be directly infected by hiv-1. pseudotyping overcomes this limitation and permits targeting to any mammalian cell. pseudotyping essentially replaces the original hiv env gene with a corresponding molecule from other viruses and carries over the cell-targeting speci fi city (i.e., the tropism of the virus) and obviates some of the safety concerns related to gp120 [ 258 ] . the list of successful pseudotypes and cell tropism is rather lengthy [ 18, 76-79, 105, 222 ] and growing. the most successful pseudotype so far uses the env from vesicular stomatitis virus (vsvg) that successfully broadens the tropism to cells in the brain, kidney, and liver amongst other. it extends to mesenchymal (stem) cells, even those in nondividing (resting) state [ 77, 136, 280 ] . filovirus env pseudotypes shift the tropism to a more limited set of cells, airway epithelial and endothelial cells [ 130, 148 ] . baculovirus gp64 and hepatitis c virus e1 and e2 pseudotypes redirect the vectors toward liver cells targeting their respective receptor, cd81 (tetraspanin) [ 19 ] . rabies virus env has been shown to ef fi ciently retarget the vectors to neuronal cells [ 162, 262 ] . rd114 env pseudotyped lentiviral vector show preference for hematopoietic cellular compartment [ 20, 56, 85, 115, 221, 268 ] . however, some applications require targeting a speci fi c cell type, which is not necessarily covered by the available pseudotypes listed above. in those cases, new targeting methods have been developed to further tighten the tropism of lentiviral vector by co-expressing cell-speci fi c coreceptors that recognize one of the cell-type speci fi c markers. the payload plasmid components providing the backbone for the transfer vector in the early hiv vectors were composed of a 5 ¢ ltr, followed by a major splice donor site, a packaging signal site encompassing the packaging signal components of the 5 ¢ region of gag (necessary for high ef fi ciency packaging and high vector titer) and a deletion of the rest of the gag gene. deletion of the u3 region from the 3 ¢ ltr promoters became also possible by relaying on constructing genes with their own promoter(s). the latest generation of lentiviral vectors carry an additional safety element, the self-inactivating ltr (sin lentiviral vector , [ 114 ] ) replacing the ltr with an hiv-independent promoter from cytomegalovirus (cmv). in these vectors the ltrs are modi fi ed in a way that upon integration they lose their intrinsic promoter ability reducing genotoxic potential. in addition the irreversible changes that occur during integration diminish the ability to mobilize after integration and to recombine with other elements to form a full-fl edged, replication capable virus [ 25, 279 ] . the formal proof of increased safety is still lacking, ironically, because of the inability to create and detect rcl capable viruses from lentiviral vector-treated cells [ 25 ] , indicating that this risk is mainly theoretical. the removal of rre and the associated splice donor and acceptor elements results in signi fi cant loss of transduction ef fi ciency of the vector [ 127 ] , while adding a 100 nucleotide central polypurine tract (central dna fl ap) restores the transduction ef fi ciency by improving the reverse transcription and nuclear transport ef fi ciency [ 47, 283 ] . the woodchuck hepatitis virus transcriptional regulatory element (wpre ) is another widely used regulatory element added to the lentiviral vector backbone to stabilize the transcription transgene mrna levels and improve transgene expression [ 292 ] . however, an open reading frame of the oncogenic whv-x element has been found within the native wpre sequence [ 128 ] , so the sequence has been modi fi ed to remove the translation start site [ 282 ] . further optimization continues to improve the safety of lentiviral vector, such as isolating the integrated vector dna to prevent translation beyond the vector boundaries by adding isolating elements. h owever, the insulators have themselves proven to be genotoxic in some instances, and no proof has emerged that such isolators are truly needed [ 100 ] . gene switches such as tet -on and -off have been added to subsequent generations of lentiviral vector and proven to be highly functional, operating with very low leakage [ 194, 260 ] . the cre-lox system has also been successfully implemented in the lentiviral context allowing high ef fi ciency engineering and sophisticated, site-speci fi c recombination techniques including the delivery and irreversible switching by small hairpin rna (shrna ) expression [ 135 ] , a tool extensively used in gene function analysis [ 37, 193 ] . a major concern regarding the safety of lentiviral vectors has been the potential genotoxicity resulting in oncogenesis, as observed previously during clinical trials for treating x-linked severe combined immunode fi ciency (x-scid) with transplanted hscs treated with murine oncoretroviral vectors carrying the gamma chain of il2r genes [96] [97] [98] [99] . the preferential insertion of oncoretroviral vectors in proximity of the lmo2 proto oncogene and the subsequent constitutive activation of the proto-oncogen driven by the enhancer element (ltr) in the vector resulted in uncontrolled cell proliferation. however, in a series of studies comparing oncoretroviruses and lentiviral vector, it has been shown that while the oncoretroviral vectors trigger a dose dependent acceleration of cancer onset in a mouse transplantation model sensitive to cancer-triggering genetic changes (cdkna2−/−), lentiviral vectors lacked such activity [ 172, 175 ] even though the vector integration rate was signi fi cantly higher. this important observation implying that a low level of insertional mutagenicity has been con fi rmed independently by several groups indicating a favorable safety pro fi le for lentiviral vectors while emphasizing the importance of vector design and avoidance of strong enhancers in the vectors [ 32, 169, 171 ] . recent clinical trials have supported the good safety pro fi le of lentiviral vectors . there have been no oncogenic effects reported in any of trials using lentiviral vectors to date [ 30, 83, 123, 143, 161, 195, 210, 276 ] . gene silencing by small interfering rna (rnai) is based on duplex formation between the mrna and a short complementary micro rna or small inhibitory rna, each having the ability to interfere with the protein synthesis and downregulate the expression levels of the targeted protein. a major problem with the inhibitory rna technologies is the short half-life and delivery of the rnai. this can be resolved using lentiviral vectors encoding arti fi cial genes with appropriate micro rna sequences that can be integrated into the host cell dna and ef fi ciently transcribed into primary micro rna that utilizes the natural intracellular processing by microprocessor complex formation with drosha to form small hairpin rna (shrna) in the nucleus. the exported mirna is subsequently cleaved by dicer and produces the complex forming inhibitory rnai. the process is rather complex, but ef fi cient to produce a signi fi cant blockade of protein expression that may be incomplete but readily achieves signi fi cant reduction, that is adequate for gaining insight into the function of the targeted protein and ef fi cient enough for phase i and ii clinical trials, though no therapeutic use has been approved by the fda. it is interesting to note that mscs are capable of secreting cholesterol-rich phospholipid microparticles encapsulating mirna, and therefore have the potential to facilitate intercellular communication and act as regulatory agents in their microenvironment [ 38 ] . an hematopoietic or general pluripotent stem cells are often selected targets for rna interference-based interventions and one of the promising efforts deal with creating arti fi cial virus resistance genes and virus resistant somatic cells. preventing hiv infection by reconstituting the immune system with such stem cell-derived virus-resistant progeny has been used as model system with signi fi cant clinical relevance [ 121 ] . the idea is that an ef fi cient hiv infection requires virus entry through the cd4 surface antigen and one or more virus co-receptors, among which ccr5 has been shown to play an essential role in the case of r5 tropic viral strains involved in primary hiv infection. clinical data indicate that ccr5 de fi ciency or certain mutations in this co-receptor protect the infected individuals from the onset of full blown aids, and the hope is that the arti fi cial knockdown of ccr5 using gene therapy and rnai will achieve similar protection [ 8, 57, 121, 146, 233 ] . the relative inef fi ciency of the ccr5 suppression remains a signi fi cant issue, but major improvement and complete knockdown of ccr5 have been achieved with somewhat longer (28 base instead of 23) shrna [ 7 ] . mesenchymal stem cell research is taking full advantage of the shrna techniques by characterizing the subtle, and not so subtle, changes induced by individual gene knockdowns. it is a long held view that mechanical stresses and mechanical characteristics of stem cells, as well as the microenvironment, can affect stem cell proliferation and differentiation. lentiviral vectors are excellent and ef fi cient targeting tools for these stem cells, even resting ones, and can deliver the shrna without causing major changes and stress that would otherwise change the stem cells on its own. chowdhury et al. studied the spreading response of mscs and showed that myosin ii , f-actin , src, or cdc42 were essential for cell spreading and changes in the mechanical characteristics ("softening") of the stem cells led directly to the downregulation of the oct3/4 gene. this indicates the possibility that small mechanical events may affect the embryo and developing tissues and even transplanted stem cells [ 41 ] . another area of ef fi cient use of lentiviral vectors and rnai technology in stem cell research is the production of transgenic embryos which carry knockdown genes. production of transgenic embryos is highly ef fi cient, and if the fertilized egg is transduced at a single cell stage, the entire germ line is affected, or partial chimerism can be achieved if multicellular embryos are treated with lentiviral vectors. an example of such a study is that by wang et al., in which they showed that the knockdown of runx1 in embryonal tissues and mscs by lentiviral vector-delivered interfering rna blocked chondrogenesis in limb buds [ 257 ] . the technique has been shown to be very ef fi cient for transgenesis, as high as 44% average rate of germ-line transmission can be achieved [ 227 ] , providing a new source of gene-modi fi ed mscs. a recent comprehensive review of the use of naturally occurring regulatory mirna technology in mesenchymal stem cell research has been written by guo et al. [ 93 ] , indicating that stem cells have discrete and distinct expression pro fi les that can account for intrinsic stem cell properties such as self-renewal and pluripotency, a property that cannot longer be overlooked by experts dealing with mscs. the accumulating data indicate that the progenitors and terminally differentiated mesenchymal cells can be tracked and de fi ned by function-related mirnas in addition to the already established sets of surface markers. the mirnas already identi fi ed affect osteogenic differentiation, chondric differentiation, adipogenic differentiation, myogenic differentiation, neuronal differentiation, wound healing, and replicative senescence. these advances open a wide array of possibilities to direct the differentiation patterns of the stem cell population temporarily by using non-integrating lentiviral vectors that are automatically lost from dividing cell populations and lead to the natural disappearance of control signal after a few cell division but potentially giving a push to the original stem cell population to develop in a preferred direction. extensive progress has been made in regards to the elucidation of the hedgehog signaling pathway in mscs using rna interference delivered with lentiviral vectors. the data suggest that at least some of the elements indeed act through the regulatory mirna network, by downregulating the cellular mirna levels. the data, however, also suggest signi fi cant off-target effects of the interfering rna molecules and indicate that we are a long way from the potential clinical use of the elucidated networks [ 124 ] . an ingenious method was devised by hu et al., to prepare the brain for traumatic interventions (surgery, extensive stem cell transplantations, etc.) by downregulating the cerebral matrix metalloproteinase 9 (mmp9) using lentiviral vector and mmp-9 shrna 2 weeks before the trauma. the knockdown of mmp-9 with the shrna proved to be an effective way to preserve the blood-brain barrier, and they achieved signi fi cant reduction of brain infarction volumes, reduction of brain water content and evans blue/igg extravasation (measure of edema formation) as well as a reduction in the neurobehavioral de fi cit in their rat brain trauma model [ 108 ] implying a potential for improved protocols for traumatic brain interventions needed for more extensive type of intracranial stem cell implantations. as mentioned earlier, lentiviral vectors provide a very ef fi cient method for generating transgenic embryos, signi fi cantly reducing the need for the generation of a high number of embryos to establish new sources of gene-modi fi ed stem cell lines, embryonic, or other [ 227 ] . the lentiviral technology is able to deliver a payload of 6-8 kb very ef fi ciently, but payloads of 10kb can be handled and delivery of 12-13 kb is possible, at a cost of lower ef fi ciency. this payload-carrying capacity allows the delivery of very large genes such as the gene encoding blood clothing factor viii, a 2,351 amino acid long protein together with its stabilizer, the von willebrand factor (2,813 amino acids in its native form) simultaneously or, one may need to use domain-engineered and shortened version of both; similarly it can be used to deliver all three chains of an igm molecule in a single, tri-cistronic complex. the implication is that the lentiviral vector system has suf fi cient payload capacity to deliver a number of relevant genes together with several supporting molecules envisioned for highly complex gene therapy scenarios currently outside the scope of monogenic gene therapy as practiced today. it may be used to target diseases with multi-gene disorders such as high blood pressure, arthritis, or diabetes in the future. zinc-fi nger nucleases (zfns) have the remarkable ability to (a) bind to a speci fi c location in the double-stranded dna; (b) break the double-stranded dna at that speci fi c location and, if an endogenous repair template is provided, (c) initiate homology-directed repair, restoring the integrity of the newly edited double-stranded dna. as their name implies, there is a speci fi c dna-binding part of this class of enzymes that consists of a tandem repeat of dna-binding zinc-fi nger motifs, hence the dna binding speci fi city and a catalytic domain, foki . for dna cleavage to occur, foki has to dimerize, one on the sense and the other on the antisense strand, while the zinc-fi nger domains attach to the right target half site and the left target half site. upon binding, a nick with a 5 ¢ overhang is initiated by foki between the target sites and the homology-directed dna repair mechanism is activated. what makes this con fi guration useful is that the spacer between the two target half sites can be several hundreds or even thousands of base pairs long and by providing a template for the activated repair mechanism, a novel dna sequence of equal length can be introduced into the dna; see a recent reviews by caroll [ 29 ] and others [ 50, 101, 117, 134, 246 ] . fundamentally, two factors determine the ef fi cacy of the dna editing or repair that the technology allows. the fi rst is the speci fi city of the zinc-fi nger binding, which also determines the length of the spacer and the proper speci fi city and uniqueness of the binding site and allows the minimization of the off-target effects that may be introduced by similar sites far away from the desired and targeted locus [ 101 ] . huge efforts are being made to tailor the zinc-fi nger nucleases for particular applications and improving the selectivity by successfully engineering the dna-binding speci fi city of the binding domain [ 3, 101, 158, 201, 220 ] . the second factor is the ef fi cient delivery of the zfns and the template dna by vectors. while the early attempts relied on retroviral vectors, adeno and adeno-associated vectors, and even baculovirus vectors, the recent advances in the fi eld clearly indicate that the lentiviral delivery system is considered to be a safer and more ef fi cacious route. as high as 50% conversion rate can be achieved with lentiviral delivery in a variety of cell lines and human embryonic stem cells [ 154 ] as compared with the earlier best rates of 18% with other methods in human and other species [ 3, 101, 197, 198, 201, 220, 245, 264 ] . one of the many holy grails of medicine, the ability to replace diseased tissue or even entire organs, seems to be hovering at the not too distant horizon. there is rapid progress in a wide range of areas, but at the center of the solution is almost always biocompatible scaffolding that is populated with a wide variety of cells. the strategically positioned cells fi nd their place within the 3d structure, propagate, differentiate, and fi ll the available space, while producing a structure that can replace or enhance the damaged tissue in the form of various implants or prosthetics. as for scaffolding, the options are quite numerous, including those obtained from cadavers or live organs (animal or human origin), by removing the cells while preserving the fi brous tissue that maintains the basic morphology of the organ. alternatively, a scaffold can be printed with various 3d printers [ 126, 159, 214-216, 229, 255, 261 ] . processed cartilage can also result in scaffold and it can be used to rebuild and regrow an implantable ear, nose, or cartilage for trachea reconstruction [ 174 ] . the culture, expansion, and differentiation of human mscs into arti fi cial tissues represent a very complex series of events and lentiviral vectors often serve as excellent research tools for marking, visualizing, and tracking the process [ 253 ] , or modifying the gene or protein expression patterns [ 68 ] . a number of tissue engineering attempts have reached the clinic and lentiviral vector have played various roles in the advancement of the technology. a very promising technology is the use of these scaffolded arti fi cial tissues employing mscs and lentiviral vectors for delivering biologics for prolonged times. van damme succinctly described the potential of these arti fi cal tissues built on scaffolds and providing arti fi cial implants for drug delivery. lentiviral vectors were used to transduce mesenchymal cells to express green fl uorescent protein (gfp) or fviii. expression was superior compared to oncoretroviral transduction, showing consistently higher transduction rates and expression remained high for several months post-transduction. the transduced cells retained their stem/progenitor cell properties, and they were still capable of differentiating along adipogenic and osteogenic lineages in vitro, while maintaining high gfp and fviii expression levels. implantation of lentiviral vector-transduced human bone marrow mesenchymal cells using collagen scaffolds into immunode fi cient mice resulted in ef fi cient engraftment of gene-engineered cells and provided sites for transgene-expression in vivo. in addition to the bone marrow-derived stem cells, adipose tissue-derived mesenchymal stem cells have been shown to be amenable to populate implantable scaffolds and retain the potential to differentiate into osteogenic cells. some of these scaffolds have been engineered for use in reconstructing craniofacial bone defects. lentiviral vector have been used to deliver fl uorescent proteins to track cells during manipulation such as osteogenic differentiation. the gfp-marked stem cells and their progeny remained fl uorescent over the 8 weeks of the study period. the gfp-marked stem cells were successfully induced into osteogenic cells both in monolayers and threedimensional scaffolds. quanti fi cation showed no decrease in staining of the osteoinduced stem cells indicating the ef fi ciency and durability of the labeling [ 256 ] . tissue engineered vascular grafts built on bilayered elastomeric poly (ester-urethane) urea scaffolds and seeded with pericytes have shown promise in the past. however, in vitro endothelialization is still an issue for the use of these types of grafts. doebis et al. reported in 2006 enhanced endothelialization using allogeneic endothelial cells or their precursors, expressing recombinant anti-alpha-mhc i single chain antibody to prevent rejection. the recombinant antibody was delivered ef fi ciently ex vivo using lentiviral vector, and has signi fi cantly reduced the mhc-1 expression levels as well as the killing of allogeneic cells by mhc-1 speci fi c cd* + t cells [ 58 ] . the results suggest that these allogeneic cells may provide a suitable alternative supply for the lining of vascular prostheses. endothelial cells and their precursors are attractive targets for gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. there have been a few reports which show lentiviral vector-mediated gene transfer ef fi ciency. sacoda and colleagues compared the effectiveness of lentiviral vector compared to adeno and oncoretroviral vectors. bovine aortic endothelial cells (baecs) were infected, in vitro, with these viral vectors. transduction ef fi ciency of beta-gal gene transfer in baecs by adenovirus, lentiviral vector, or retrovirus at a multiplicity of infection (moi) of 10 (determined on hela cells) was 69 ± 11, 33 ± 8, or 22 ± 6% respectively. at higher moi [ 50 ] both adenovirus and lentiviral vectors achieved an almost 100% transduction rate. however, retroviral vectors showed only 48 ± 6% at moi 50 and no increase at moi 100. the percentage of beta-gal positive cells decreased rapidly at longer passage of cells after being transduced by adenovirus. in contrast, lentiviral vector and retrovirus vectors mediated transductions showed sustained higher percentage of positive cells. furthermore, the transductions by lentiviral vectors had no signi fi cant effect on viability of baecs suggesting that for long-term cell therapy the lentiviral vectors have overall the best features [ 219 ] . expressing il10 in similar settings in the early, initiation phase, also inhibited and delayed the onset of the rejection process [ 287 ] . one of such cases in which the performance of the endothelial cells may need to be boosted is to increase the resistance to ischemia-reperfusion injury of the vascularized transplants and implants or normal tissues undergoing prolonged surgery. this is a condition which occurs too frequently and is responsible for devastating tissue injury caused by systemic activation of the complement system. lentiviral vectors can be used to force the over-expression of the anti-apoptotic gene, bcl-xl and indeed, it has shown signi fi cant protection from early apoptotic loss of vascular endothelial cells [ 286 ] . recently, tooth tissue engineering has attracted more and more attention. stem cellbased tissue engineering is thought to be a promising way to replace a missing tooth. the potential mscs for tooth regeneration mainly include stem cells from human exfoliated deciduous teeth (sheds), adult dental pulp stem cells (dpscs), stem cells from the apical part of the papilla (scaps), stem cells from the dental follicle (dfscs), periodontal ligament stem cells (pdlscs), and bone marrowderived mscs (bmscs). a recent review by peng et al. shows promising progress [ 190 ] . however, in practice, tissues other than bone marrow can serve as stem cell donors, including adipose tissue, periodontal ligament, and pulp for oral tissue regeneration [ 206 ] . the experimental data suggest that not only the stem cells ex vivo, but cells in the osteogenic tissue are amenable to direct transduction by lentiviral vector [ 259 ] . this opens up the periodontal reconstruction interventions to the bene fi cial effects of gene therapy enhancing the wound healing and improving engraftment by expressing growth promoters at low and slowly decreasing concentrations. estrela published an excellent review on the potential of mscs in regeneration of dental tissues [ 68 ] and rodrigues-loza reviewed the mesenchymal cell types recovered from dental tissues [ 208 ] . other data clearly show that the primary osteogenic cells are ef fi ciently transduced by lentiviral vector, and that their infusion into the mandible is a feasible method for locally delivering dna to primary osteogenic and bone cells in rat models [ 259 ] , indicating that future applications in vivo dental implant enhancement, using dental scaffolding, bone healing, and tooth regeneration may be feasible. recent efforts extend toward engineering dental repair by changing the expression of growth factors and bone morphogenic proteins leading to dentin formation, as discussed in a 2011 review by casagrande [ 31 ] and which seem to be amenable to cell therapy efforts with nonintegrating lentiviral vector. one of the tissues that is often injured but that presents dif fi culties when it comes to healing and repairs is the tendon. enhancing the healing process by in situ overexpression of helper factors such as il10 could reduce recuperation time and perhaps improve the quality of the repair. richetti et al. reported promising results in a murine model of patellar tendon injury after direct injection of an il10 transgene using lentiviral vector. although the tendons showed no obvious histological difference, the il-10-treated groups had superior mechanical characteristics by day 42 [ 205 ] . although the mechanism of wound healing in tendons is not yet understood, the involvement of mscs is suspected and delivery of additional factors that partake in healing process is discussed by meyerose and ashlan [ 13, 163 ] . recent fi ndings by shamis and colleagues [ 230 ] demonstrated that embryonic stem cells could be directed to speci fi ed and alternative mesenchymal cell fates whose function could be distinguished in engineered human skin equivalents. lentiviral shrna-mediated knockdown of hepatocyte growth factor (hgf) resulted in a dramatic decrease of hgf secretion from cell lines (edk cells) that led to a marked reduction in their ability to promote keratinocyte proliferation and reepithelialization of cutaneous wounds. in contrast, h9-mscs demonstrated features of mscs but not those of dermal fi broblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced signi fi cantly lower levels of hgf. characterization of these induced mesenchymal cells in 3d, engineered human skin equivalents demonstrated the utility of this tissue platform to predict the functional properties of stem cell-derived fi broblasts before their therapeutic use in reconstructive skin transplantation and wound healing. inhibition of hyper-keratinization by expressing a mutant form of tcgf beta3 that has lost its binding site for latency-associated peptide, reduced the re-epithelialization density and fi broblast/myo fi broblast trans-differentiation within the wound area [ 251 ] in a mouse skin wounding model. the expression of this mutated gene was achieved by injecting lentiviral vectors encoding the muttcgf beta3, into the regenerating tissue and the changes induced by this intervention predict a signi fi cant decrease in keloid formation and provide a potential model for preventing the painful dis fi gurement that follows the abnormally strong skin remodeling and scar tissue formation that oftentimes accompanies wound healing. the data indicate that future stem cell therapy with carefully designed interventions for patients prone to scar tissue formation could fi nd wide spread application. one of the causes of erectile dysfunction is the damaged penile cavernous smooth muscle cells (smcs) and sinus endothelial cells. song reports that it may be feasible to restore these cells by applying mscs to penile cavernous ecs or smcs. for this purpose immortalized (via lentiviral vector encoding v-myc) human bone marrow mesenchymal stem cell line b10 cells were transplanted into the cavernosum of sprague-dawley rats and harvested 2 weeks later. the expression of cd31, von willebrand factor (vwf), smooth muscle cell actin (sma), calponin, and desmin was determined immunohistochemically in rat penile cavernosum. multipotency of b10 to adipogenic, osteogenic, or chondrogenic differentiation was found. expression of endothelial cell-speci fi c markers (cd31 or vwf protein) and expression of smooth muscle cell-speci fi c markers (calponin, sma, or desmin protein) were demonstrated in grafted b10 cells indicating that human mscs may be a good candidates in the treatment of penile cavernosum injury [ 238 ] . angiogenesis requires the presence and active involvement of mscs and therefore mscs are ready to be recruited into the areas when there is a need for novel blood vessels: the in fl amed, hypoxic, tumor infested locations. gehmert et al. described an interesting model to study the migration of mscs. in their work, immunode fi cient mice were engrafted with human breast cancer cells (4t1) in the left mammary pad. a day later, the mice were injected ip with luciferase-labeled adipose tissue-derived mscs (using lentiviral vector technology). the mscs were found to rapidly migrate into the tumor, con fi rming the previous observations that mscs can be found within the tumor stroma and vasculature, even if the in fl ammation is not present, as the immunode fi cient mice lacked the in fl ammation signaling pathway. based on this result, it can be suggested that mscs can be attracted solely by the cytokines produced by the tumor. however, the power of in fl ammation has been clearly demonstrated in control animals, which received e. coli injections at contralateral locations and attracted all the mscs leaving the tumor implant msc-free [ 84 ] . elucidating the migratory mechanisms of the mscs seems to be an important step toward fi nding a delivery system to in fl ammatory sites and fi nding the conditions for clear migration into established tumors. even the simple marking of tumor tissue with fl uorescent proteins (such as gfp) holds important promise for surgeons, as delineating a breast cancer in situ during surgery would be possible by applying uv light and tracing the contours of the tumor. the technique already allows sophisticated molecular imaging combined with stem cell therapy [ 254 ] . wang and colleagues used the ability of mscs to differentiate into endothelial cells in vivo to establish whether the differentiated mscs persist in vivo and to determine if this potential persistence contributes to functional improvement after experimental myocardial infarction. they generated a lentiviral vector encoding two distinct reporter genes, one driven by a constitutive murine stem cell virus promoter and the other driven by an endothelial-speci fi c tie-2 promoter. the endothelial speci fi city of the lentiviral vector was validated by its expression in endothelial cells but not in undifferentiated stem cells. the lentivirus-transduced mscs were injected into peri-infarct are as of the hearts of severe combined immunede fi cient mice. persistence of injected cells was tracked by bioluminescence imaging (bli) and veri fi ed by immunohistochemical staining. the bli signal from the endothelial-speci fi c reporter revealed that the stem cells differentiated into endothelial cells 48 h after injection. however, both the constitutive and endothelial-speci fi c signals disappeared by day 50. nonetheless, the improvement in left ventricle ejection fraction with therapy persisted for up to 6 months. immunohistochemical staining showed that stem cell-derived endothelial cells integrated into endogenous cd31+ vessels. furthermore, stem cell-transplanted hearts had more cd31+ vessels and a lesser degree of cardiac fi brosis compared with the controls at 6 months. increased angiogenesis and decreased fi brosis were associated with cardiac functional improvement. similarly mscs double-marked with gfp-lentiviral vector and superparamagnetic iron oxide could be followed by mri for up to 8 months in a porcine model of infraction and revascularization [ 274 ] . endothelial cells respond to mild injurious stimuli by upregulating anti-apoptotic gene expression to maintain endothelial integrity. ec dysfunction and apoptosis resulting from ischemia/reperfusion injury may contribute to chronic allograft rejection. under optimized conditions for lentiviral vector transduction of rat aortic endothelial cells (raec) the delivery of the anti-apoptotic gene, bcl-xl, via lentiviral vector, protects raec from apoptotic death. the authors con fi rmed the damaging effect of the reperfusion phase. endogenous bax expression increased with i/r injury, whereas endogenous bcl-xl remained constant. raec transduced with lentiviral vector expressing bcl-xl were protected from early apoptosis caused by i/r injury, correlating with reduced cytochrome c release into the cytosol. this protective effect may be attributed to altering the balance of pro-and anti-apoptotic proteins, resulting in sequestration of the harmful bax protein, and may open up new strategies for controlling chronic allograft rejection [ 286 ] . inhibition of na+/h+ exchanger 1 (nhe1 ) reduces cardiac ischemia-reperfusion (i/r) injury as well as cardiac hypertrophy and cardiac failure. although the mechanisms underlying these nhe1-mediated effects suggest delay of mitochondrial permeability transition pore (mptp) opening, and reduction of mitochondrial-derived superoxide production, the possibility of nhe1 blockade targeting mitochondria has been incompletely explored. a short-hairpin rna sequence mediating speci fi c knock down of nhe1 expression was incorporated into a lentiviral vector (shrna-nhe1) and transduced into the rat myocardium. nhe1 expression of mitochondrial lysates revealed that shrna-nhe1 transductions reduced mitochondrial nhe1 (mnhe1) by approximately 60%, supporting the expression of nhe1 in mitochondria membranes. electron microscopy studies corroborate the presence of nhe1 in heart mitochondria. immunostaining of rat cardiomyocytes also suggests colocalization of nhe1 with the mitochondrial marker cytochrome c oxidase. to examine the functional role of mnhe1, mitochondrial suspensions were exposed to increasing concentrations of cacl 2 to induce mptp opening and consequently, rat heart mitochondrial swelling. shrna-nhe1 transduction reduced the cacl 2 -induced mitochondrial swelling by 64 ± 4%. whereas the nhe1 inhibitor hoe-642 (10 m m) decreased mitochondrial ca 2+ -induced swelling by only 37 ± 6. because mitochondria from rats injected with shrna-nhe1 present a high threshold for mptp formation, the bene fi cial effects of nhe1 inhibition in i/r resulting from mitochondrial targeting should be considered as a future target for cell therapy [ 250 ] oxidative stress is important in a number of pathologies, including cardiovascular diseases, such as atherosclerosis and cardiac ischemia-reperfusion injury. an important mechanism for adaptation to oxidative stress is the induction of genes through the antioxidant response element (are) which regulates the expression of antioxidant and cryoprotective genes via the transcription factor nrf2 (nuclear factor e2-related factor 2). as nrf2-regulated genes are induced during oxidant stress, occurring for example in reperfusion after ischemia, hurttila et al. took a novel approach to exploit are for the development of oxidative stress-inducible gene therapy vectors. to this end, one, two, or three are-containing regions from human nad(p)h: quinone oxidoreductase-1, glutamate-cysteine ligase modi fi er subunit and mouse heme oxygenase-1 were cloned into a vector expressing luciferase under a minimal sv40 promoter. the construct, which was the most responsive to areinducing agents, was chosen for further studies in which a lentiviral vector was produced for an ef fi cient transfer to endothelial cells. heme oxygenase-1 (ho-1), which has well-characterized anti-in fl ammatory properties, was used as the therapeutic transgene. in human endothelial cells, are-driven ho-1 overexpression inhibited nuclear factor-kappa b activation and subsequent vascular cell adhesion molecule-1 expression induced by tumor necrosis factor-alpha. they concluded that the are element is a promising alternative for the development of oxidative stressinducible gene therapy vectors [ 111 ] . progenitor cell therapy is a potential new treatment option for ischemic conditions in the myocardium and skeletal muscles. however, it remains unclear whether umbilical cord blood (ucb)-derived progenitor cells can be therapeutic in ischemic muscles and if yes, whether the ex vivo gene transfer can be used for improving the effect. the use of lentiviral vector led to ef fi cient transduction of both ucb-derived hscs and mscs resulting in long-term transgene expression. moreover, it did not alter the differentiation potential of either hscs or mscs. in addition, the therapeutic potential of cd133+ and msc progenitor cells transduced ex vivo with lentiviral vector encoding the mature form of vascular endothelial growth factor d (vegf-d ) or the enhanced green fl uorescent protein (egfp) marker gene achieved permanent gene expression. the transplantation of the progenitor cells into nude mice serving as mouse model of skeletal muscle ischemia enhanced the regeneration of ischemic muscles, but notably, without a detectable long-term engraftment of either cd133+ or msc progenitor cells. the results show that rather than directly participating in angiogenesis or skeletal myogenesis, the ucb-derived progenitor cells indirectly enhance the regenerative capacity of skeletal muscle after acute ischemic injury. however, rather counter-intuitively, the vegf-d gene transfer into the progenitor cells did not improve the therapeutic effect in ischemic muscles [ 131 ] . another cell type with improved adult stem cell functions has been discovered and cells have been isolated from the peripheral blood of young children. this clonally expandable, telomerase expressing progenitor cell type is distinct from hematopoietic or mesenchymal stromal cells and resembles that of embryonic multipotent mesoangioblasts. cell numbers and the proliferative capacity correlate with donor age, and express the pluripotency markers klf4 , c-myc , as well as low levels of oct3/4, but lack sox2 . overexpression of sox2 by lentiviral transduction of sox2 (sox-mabs) enhances pluripotency and facilitates differentiation to cardiovascular lineages. furthermore, the number of smooth muscle actin positive cells was higher in sox-mabs. in addition, pluripotency of sox-mabs was shown in a mouse model by demonstrating the generation of endodermal and ectodermal progenies and injection of sox-mabs into nude mice after acute myocardial infarction resulted in improved cardiac function compared to mice treated with control cells (cmabs). furthermore, cell therapy with sox-mabs resulted in an increased number of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells in vivo [ 133 ] . mesenchymal stem cell therapy emerges as a viable therapy in the context of acute lung injury /acute respiratory distress syndrome and chronic disorders, such as lung fi brosis and chronic obstructive pulmonary disease. there is evidence for bene fi cial effects of mscs on lung development, repair, and remodeling. the engraftment in the injured lung does not occur easily, but several studies report that paracrine factors can be effective in reducing in fl ammation and promoting tissue repair. mscs release several growth factors and anti-in fl ammatory cytokines that regulate endothelial and epithelial permeability and reduce the severity of in fl ammation, as reviewed by arboreau et al. [ 2 ] , suggesting that carefully controlled expression of these factors using transduced stem cells could enhance the bene fi cial effects of the mesenchymal stem cell therapy. this may be a risky proposal, however, since constitutive expression of tgf beta /tgf alpha in epithelial mscs generated breast cancer stem cells [ 12 ] . acute respiratory distress syndrome (ards) is a crippling disease with no effective therapy, and characterized by progressive lung damage followed by dyspnea. mscs have been proposed as a new therapeutic modality for ards because the stem cells can attenuate in fl ammation and repair the damaged tissue by differentiating into several cell types. the bene fi cial effect of the stem cells is still a minor mystery, as it is known that macrophages participate in the development of ards and that mscs can only weekly modulate macrophage function. the chemokine ccl2 is a potent inducer of macrophage recruitment and activation, and its expression is elevated in patients with ards. a set of mscs have been generated by transducing the cells with a lentiviral vector expressing 7nd, a dominant-negative inhibitor of ccl2, expecting enhanced therapeutic function of the mscs if the hypothesis is valid. the transduction was effective, and the stem cells produced a large amount of 7nd. after inducing lung injury by bleomycin treatment, the iv-injected mscs readily migrated into the site of injury as con fi rmed by immunostaining 24 h postinjection. this fi nding suggests that mscs could work as a drug delivery tool. mice treated with 7nd-expressing mscs showed signi fi cantly milder weight loss, suffered less severe lung injury, lower collagen content, lesser accumulation of in fl ammatory cells and in fl ammatory mediators, and ultimately showed signi fi cant gains in survival [ 218 ] . no evidence of 7nd-mesencymal stem cell-induced toxicity was observed during or after treatment. thus, inhibiting the effects of macrophages may greatly enhance the ability of mscs to affect lung repair in ards. direct transduction of lung tissues for gene therapy has always been an attractive proposal. the reoccurring problem, however, is that the airways are far less accessible to vector particles than hoped for and the depth of penetration of inhaled substrate ends in the branches which are larger than 100 m m in diameter [ 48, 263 ] . an attractive alternative delivery of gene therapy components could be the intrapleural injection of mscs. to enable tracking, the cells were labeled with green fl uorescent protein (gfp) using a lentiviral vector, and were found readily attached to the pleura of sprague-dawley rats. the isolated and recovered cells preserved the typical mesenchymal stem cell phenotype and could differentiate into adipocytes, osteoblasts, and chondroblasts in vitro. the highest number of the labeled cells was found to be adhered to the mediastinal pleura, but no labeled cells were detected in the lung parenchyma or other tissues/organs, such as the liver, kidney, spleen, and mesenterium, a remarkable compartmentalization of a stem cell transplant [ 200 ] . alzheimer's disease (ad) is one of the most devastating conditions and its prevalence is still rising paralleling the increase of average life expectancy. a hallmark of the disease is the accumulation of amyloid plaques and extensive neurodegeneration in the context of an intracerebral in fl ammation, leading to progressive dementia. over the years, a tripartite set of goals crystallized, when the potential treatments of ad were considered: (a) stop the progression of the disease by reducing/reversing the plaque formation; (b) stop the neurodegeneration that seems to be a consequence of both internal changes (neuro fi brillary tangle formation and related issues) and changes external to the cells, related to plaque formation and degeneration of the neuronal microenvironment; and (c) recover neurological function by replenishing the lost neuronal compartment [ 71, 81, 94, 122, 152, 188, 291 ] . interestingly, mscs and stem cell therapy are increasingly considered a potentially important part of the toolset to achieve these goals. the symptoms that are collectively categorized as ad often have different backgrounds, some of which seem to have roots implying genetic causes, such as improper processing of beta amyloid peptide. consequently, a disease-modifying therapeutic approach in alzheimer's disease aims to reduce the accumulation of neurotoxic beta amyloid aggregation peptides. habish et al. report new fi ndings for a potential autologous stem cell-based strategy for delivery of enzymatic activities against beta amyloid formation in the brain. f-spondin and neprilysin (cd10), genes expressed in adult mscs, are known to be involved in the formation and degradation of beta amyloid peptides, respectively. coincubation of the converted mscs with hek-293 cells stably expressing amyloid precursor protein (app) lead to a signi fi cant cell dose-dependent decrease of amyloid peptide release and deposition, indicating that mscs might be useful for delivering antiamyloid activity to treat ad [ 95 ] . this direction of research is gaining new momentum from the discovery of a new beta amyloid secretase and the tremendous progress gained in recent years in the fi eld of amyloid formation, its contribution to neurodegenerative diseases [ 122 ] and allowing new gene therapies to be conceived and tested. one effort has utilized human umbilical cord blood-derived mscs (hucb-mscs) which were transplanted into amyloid precursor protein and presenilin1 double-transgenic mice. this experiment resulted in signi fi cantly improved spatial learning and a decrease in memory decline. furthermore, beta amyloid peptide deposition, beta-secretase 1 (bace-1 ) levels, and the hyper-phosphorylation of the tau proteins were dramatically reduced in hucb-msc transplanted app/ps1 mice. interestingly, these effects were associated with reversal of disease-associated microglial neuroin fl ammation, as evidenced by decreased microglia-induced pro-in fl ammatory cytokines, reduction in the number of alternatively activated microglia , and decrease in anti-in fl ammatory cytokines. combining these fi ndings with the potential cell therapy targeting, these mscs are expected to produce a sustained neuroprotective effect by establishing a feed-forward loop engaging the alternative activation of microglia, thereby ameliorating disease pathophysiology and reversing the cognitive decline associated with amyloid deposition [ 139 ] . peng and colleagues report additional details on the use of lentivirus-expressed sirna as a method to ameliorate alzheimer disease neuropathology in app transgenic mice by reducing the levels of beta-site app cleaving enzyme 1, or bace1 [ 189 ] . a series of experiments demonstrated the potential of neural stem cells transduced by a multigenic lentiviral vector stably expressing recombinant human nerve growth factor in relevant amounts to exploit their ability for therapeutic applications. the multigenic lentiviral vector contained a tricistronic cassette to express simultaneously up to three independent genes: (1) rhngf (beta subunit); (2) egfp (enhanced green fl uorescent protein); and (3) neo (r) (neomycin antibiotic resistance gene). lentiviral vectors were released in culture media and subsequently used to transduce mouse stem cells. remarkably, the subsequent test revealed that engineered nscs were all positive for egfp and after 30 passages in vitro engineered cells maintained their multipotentiality to differentiate into neurons, astrocytes, and oligodendrocytes. furthermore, it was found that rhngf-stem cell-derived neurons expressed choline acetyltransferase and displayed an enhanced axonal growth. the stem cells showed an altered sphere forming frequency either in rhngf-nsc or in both groups of control nsc. lentivirus-mediated rhngf gene transfer into nsc was achieved without changes in the expression of neural differentiation markers, like microtubule-associated protein 2 (map2) (a/b), glial fi brillary acidic protein (gfap) and chondroitin sulfate proteoglycan [ 34 ] . secreted rhngf increased axonal sprouting by rhngf-nsc-derived neurons, which was associated with chat expression. rhngf-nscs may prospectively be a good candidate for the treatment of neurodegenerative diseases. a protein that has been shown to promote app accumulation is beta-secretase (beta-site app cleaving enzyme 1, or bace1). typically, a marked increase in the level of bace1 is found in the cerebrospinal fl uid of those affected with alzheimer's disease. through in vivo studies using app transgenic mice, it has been demonstrated that decreasing the expression of bace1 via lentiviral vector delivery of bace1 sirna has the potential for signi fi cantly reducing the cleavage of app, the accumulation of these products, and the consequent neurodegeneration. as such, lentiviral-expressed sirna against bace1 is a therapeutic possibility in the treatment of ad. neprilysin has recently been implicated as a major extracellular beta amyloid degrading enzyme in the brain. a unilateral intracerebral injection of a lentiviral vector expressing human neprilysin (lenti-nep) was tested in transgenic mouse models of amyloidosis reduced amyloid-beta deposits by half relative to untreated mice, indicating that neprilysin may have a role in alzheimer's disease treatment. that said, a more ef fi cient delivery system is likely required, a property that a neprilysin expressing stem cell could potentially provide [ 160 ] . gene transfer to the central nervous system provides a powerful methodology for the study of gene function and gene-environment interactions in vivo, in addition to a vehicle for the delivery of therapeutic transgenes for gene therapy. research has been signi fi cantly aided by successfully targeting speci fi c regions of brain, and for parkinson's disease, the substantia nigra. the key to success is the ease of pseudotyping lentiviral vectors, which makes it possible to change the patterns of tropism. cannon et al. used isogenic lentiviral vector particles encoding a gfp reporter and pseudotyped with envelope glycoproteins derived from vesicular stomatitis virus (vsv), mokola virus (mv), lymphocytic choriomeningitis virus (lcmv), or moloney murine leukemia virus (mulv). adult, male lewis rats were injected unilaterally with stereotactic infusions of vector into the substantia nigra. three weeks later, patterns of viral transduction were determined by immunohistological detection of gfp. different pseudotypes gave rise to different sites of transgene expression. vsv and mv pseudotypes transduced midbrain neurons, including a subset of nigral dopaminergic neurons. in contrast, lcmv-and mulv-pseudotyped lentiviral vector resulted in transgene expression exclusively in astrocytes. the restricted transduction of astroglial cells was not explained by the cellular distribution of receptors previously shown to mediate entry of lcmv or mulv. the availability of neuronal and astrocyte-targeting vectors will allow dissociation of cell autonomous and cell nonautonomous functions of key gene products in vivo. similar tissue and cell-speci fi c patterns can be achieved in stem cells using cell/tissue-speci fi c promoters and mirna [ 43, 79, 86, 177, 186, 199, 213, 232, 242, 266, 290 ] . multipotent mesenchymal stromal cells have raised great interest for brain cell therapy due to their ease of isolation from bone marrow, their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells, and for their ability to secrete a variety of growth factors and chemokines. a subpopulation of human mscs, the marrow-isolated adult multilineage inducible (miami) cells, when combined with pharmacologically active microcarriers (pams) have shown great promise in a rat model of parkinson's disease. pams are biodegradable and non-cytotoxic poly (lactic-co-glycolic acid) microspheres, coated by a biomimetic surface and releasing a therapeutic protein, which acts on the cells conveyed on their surface and on their microenvironment. in this study, pams were coated with laminin and designed to release neurotrophin 3, which stimulate the neuronal-like differentiation of miami cells and promotes neuronal survival. after adhesion of dopaminergic-induced (di)-miami cells to pams in vitro, the complexes were grafted in the partially dopaminergic-deafferented striatum of rats, which led to a strong reduction of the amphetamine-induced rotational behavior together with protection/repair of the nigrostriatal pathway. these effects were correlated with the increased survival of di-miami cells that secreted a wide range of growth factors and chemokines. moreover, the observed increased expression of tyrosine hydroxylase by cells transplanted with pams may contribute to this functional recovery [ 52 ] and provide an excellent new delivery system for genetically modi fi ed/enhanced cells into substantia nigra. lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by alpha-synuclein accumulation and includes gradually worsening dementia with lewy bodies (dlb) accumulating in neurons followed by advanced parkinson's disease (pd). recent evidence suggests that impairment of the lysosomal pathways (i.e., autophagy) involved in alpha-synuclein clearance might play an important role. for this reason, the expression levels of members of the autophagy pathway in brains of patients with dlb and alzheimer's disease and in alpha-synuclein transgenic mice were examined by immunoblot analysis. in dlb cases, the levels of mtor were elevated and atg7 were reduced compared to controls and ad. levels of other components of the autophagy pathway such as atg5, atg10, atg12, and beclin-1 were not different in dlb compared to controls. in dlb brains, mtor was more abundant in neurons displaying alpha-synuclein accumulation. these neurons also showed abnormal expression of lysosomal markers such as lc3, and ultrastructural analysis revealed the presence of malformed autophagosomes in abundance. similar alterations were observed in the brains of alpha-synuclein transgenic mice. intracerebral infusion of rapamycin, an inhibitor of mtor, or injection of a lentiviral vector expressing atg7 resulted in reduced accumulation of alphasynuclein in transgenic mice and amelioration of associated neurodegenerative alterations supporting the notion that defects in the autophagy pathway, and more speci fi cally in mtor and atg7, are associated with neurodegeneration. this supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects using genetically altered stem cells [ 44, 270 ] . although the advances in parkinson's disease research to date are signi fi cant, the lack of clinical use of genetically modi fi ed cells is a bit surprising and may indicate an oversight and underuse of the advanced tools provided by the combination of stem cells and lentiviral vectors. lasting cerebral ischemia is a frequent (~80%) consequence of stroke and, as a result, most of the stroke research is focusing on ameliorating the devastating consequences of ischemic events: endothelial damage, neurodegeneration, and breakdown of the blood-brain barrier (bbb) leading to dif fi cult-to-treat cerebral edema [ 108 ] . data indicate that transplantation of human umbilical cord stem cells helps to protect ischemic brain [ 149 ] , and the protection is partially attributed to cytokines and protective factors produced by these stem cells [ 10, 149 ] . another promising fi nding is that the mesenchymal and neuronal stem cells preserve their ability to differentiate into glial and neuronal cells [ 51, 119, 149, 231, 249, 275 ] . various studies on focal cerebral ischemic models have implicated the direct activation and expression of matrix metalloproteinases (mmps), especially mmp-9, as a key orchestrator of bbb disruption. moreover, studies have shown that mmp-9 sirna can protect the bbb from ischemia/reperfusion injury. one study investigated the neuroprotective role of a lentiviral vector-mediated mmp-9 shrna following focal cerebral ischemia [ 108 ] , indicating that it is possible to deliver mmp-9 inhibitors by genetically enhanced stem cells. this study also showed the ability to deliver the target deeper into the affected area normally not accessible by direct lentiviral vector infusion. the forerunner of such interventions is a study testing the hypothesis that transplantation of human neurotrophin-3 (hnt-3) over-expressing neural stem cells into rat striatum after a severe focal ischemia would promote functional recovery. the rat neural stem cells were transduced with a flag-tagged hnt-3 gene in a lentiviral vector. the stem cells were transplanted into the striatum ipsilateral to the injury of adult rats 7 days after 2 h occlusion of the middle cerebral artery from 3 days to 2 weeks after transplantation. the modi fi ed cells (nscs-hnt3, as de fi ned by flag immuno fl uorescence staining) that survived the transplantation procedures could secrete signi fi cantly higher levels of neurotrophin-3 protein in the graft sites than controls ( p < 0.001). furthermore, the rats that accepted nscs-hnt3 exhibited enhanced functional recovery on neurological and behavioral tests, compared with control animals transplanted with saline or untransduced stem cells, indicating that they might have value for enhancing functional recovery after stroke [ 285 ] . recovery from ischemic events is slow and rather unpredictable. however, there seem to be new therapeutical opportunities that could enhance the process such as using vegf-induction therapy [ 16 ] . there is accumulating evidence indicating that vegf has direct neuroprotective effects on various cultured neurons of the central nervous system. interestingly, in vivo vegf controls the correct migration of facial branchiomotor neurons in the developing hindbrain and stimulates the proliferation of neural stem cells in enriched environments and after cerebral ischemia. on the other hand, transgenic mice expressing reduced levels of vegf develop late-onset motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis (als). also, reduced levels of vegf have been implicated in a polyglutamine-induced model of motor neuron degeneration. intracerebroventricular delivery of recombinant vegf protein delays disease onset and prolongs survival of als rats, whereas intramuscular administration of a vegf-expressing lentiviral vector increases the life expectancy of als mice by as much as 30%. deciphering the precise role of vegf at the neurovascular interface promises to uncover new insights into the development and pathology of the nervous system and should be helpful to the design of novel strategies to treat (motor) neurodegenerative disorders [ 137 ] . vegf-expressing mscs have also been found bene fi cial in parkinson's disease [ 16, 271 ] . the development of lentiviral particles engineered for macrolide-responsive human vascular endothelial growth factor 121 (vegf121) expression will bring closer the in vivo use of inducible growth factor cell therapies, expressing the factors only in ischemic conditions using hypoxia-inducible erythropoietin promoter [ 6 ] . alternatively, the inducible vegf121 promoter system also compared favorably with isogenic streptogramin-and tetracycline-responsive con fi gurations and showed excellent growth-factor fi ne-tuning following transduction into a variety of mammalian cell lines and different human primary cells. chicken embryos transduced for macrolide-controlled vegf121 production can be fi ne-tuned to prime a dose-dependent neovascularization [ 168 ] . expression of survivin (svv) using an sin lentiviral vector carrying vascular endothelial growth factor further improved the expression of vegf and basic fi broblast growth factor in male sprague-dawley rats under hypoxic conditions. the in vivo experiment that produced this observation consisted of three groups of rats, one receiving intravenous injection of 500 m l of phosphate-buffered saline without cells (control group) and two groups administered the same volume solution with either three million gfp-mscs (group gfp) or svv/gfp-mscs (group svv). all animals were submitted to 2 h middle cerebral artery occlusion followed by reperfusion. modi fi cation with svv further increased secretion of both factors. the survival of the transplanted cells in the svv group was 1.3-fold higher at 4 days after transplantation and 3.4-fold higher at 14 days after transplantation, respectively, when compared with group gfp and reduced the cerebral infarct volume by 5.2% at 4 days after stroke and improved post-stroke neurological function at 14 days after transplantation. modi fi cation with svv could further enhance the therapeutic effects of mscs possibly through improving the mscs survival capacity and upregulating the expression of the protective cytokines in the ischemic tissue [ 151 ] . the identi fi cation of the genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. furthermore, the characterization of such pathways could facilitate the identi fi cation of novel targets for stroke therapy. one such novel approach was the ampli fi cation of the differential gene expression patterns in a primary neuronal model of stroke, by employing a lentiviral vector system to speci fi cally bias the transcriptional activation of hypoxically regulated genes. over-expression of the hypoxia-induced transcription factor subunits hif -1 alpha and hif-2 alpha elevated hypoxia-mediated transcription of many known hif-regulated genes well above control levels. furthermore, many potentially novel hif-regulated genes were discovered that were not previously identi fi ed as hypoxically regulated. most of the identi fi ed novel genes were activated by a combination of hif-2 alpha over-expression and hypoxic insult. these included several genes with particular importance in cell survival pathways and of potential therapeutic value. hypoxic induction of hif-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. further investigation of the genes identi fi ed in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia [ 202 ] and the genes need to be considered as useful targets in future mesenchymal stem cell therapies. however, the use of hypoxia-induced gene therapy has to be evaluated carefully in the light of recent provocative observations indicating that the hypoxic phenotype contributes to appearance of highly malignant cancer forms from the initial epithelial-mesenchymal transition to the ultimate organotropic colonization, and that can potentially be regulated by hypoxia, suggesting a master regulator role of hypoxia and hifs in metastasis [ 6, 155 ] . furthermore, modulation of cancer stem cell self-renewal by hifs may also contribute to the hypoxia-regulated metastasis program. the hypoxia-induced metastatic phenotype may be one of the reasons for the modest ef fi cacy of anti-angiogenic therapies and may well explain the provocative fi ndings that anti-angiogenic therapy increased metastasis in preclinical models [ 155 ] . the image of a wheelchair-bound superman exempli fi ed for all of us the tragedy that affects many of the victims of traumatic spinal cord injury and motivated research into protecting and restoring spinal-cord functionality beyond and above the usual efforts. the results are promising on many fronts [ 236 ] . on one hand, the intervertebral disk , cartilage, and bone injuries that threaten the integrity of the spinal cord can be almost completely healed and the healing can be facilitated and enhanced by stem cell therapies in most of the experimental models. the treatment often includes stem cells engineered with lentiviral gene transfer for enhancing and promoting wound healing and tissue restoration [ 13, 87, 89, 109, 247 ] . signi fi cant success has been achieved by expressing bone morphogenic proteins in the injured tissue [ 80 ] and observations that mechanical stimulation has a multiplying effect in bone regeneration will hopefully carry the research into clinical trials [ 140 ] sooner than later. probably, the fi rst trials will be done in well-designed spinal surgery, allowing even risky interventions, currently not practiced [ 13, 88, 163 ] . the progress is signi fi cantly slower when it comes to restoring the functionality of severed spinal cord, but successful demonstration that mscs migrate into the site of injury and differentiate into proper cell types needed for the healing [ 224 ] predicts potential breakthroughs. in this set of experiment, mesenchymal cells were labeled with green fl uorescent protein using lentiviral vector, were injected into the subarachnoid space, and their migration and differentiation was observed. cells were found on the surface of the injured spinal cord parenchyma, in deeper area of the perivascular spaces and some of them had been found deeply integrated into the parenchyma. immunostaining for nestin demonstrated that some gfp-positive cells differentiated into neural stem cells and mature neurons or glial cells in situ. lentiviral vectors pseudotyped with rabies env were successfully used to deliver genes into spinal cord and site of injury and showed successful retrograde transfer into deeper areas, indicating that gene therapy is possible and factors necessary for further differentiation of stem cells can be delivered [ 224, 241 ] . further advances in pseudotyping with rabies virus glycoprotein has a promise for more ef fi cient motor neuro-speci fi c delivery of transgenes and restoration of neuronal functions. as the examples indicate above, mscs have been recognized as promising delivery vehicles for gene therapy in the cns. a particularly unmet need is delivery of compounds that could help patients suffering from a particularly aggressive form of cancer, gliomas. a glimpse into a possible future can be gained from experiments in which stem cells were used to evaluate the antitumor effect of cytosine deaminase (cd) in a rat c6 glioma model . lentiviral vectors expressing cd and enhanced green fl uorescent protein (egfp) were constructed and transduced into rat mscs which were intracranially injected alone or in combination with c6 glioma cells supported by unlabeled parental mscs. the presence and effect of the engineered stem cells were then correlated with the possible effects on tumor growth, tumor cell apoptosis, tumor size, and rat survival in the presence of 5-fl uorocytosine (5-fc). fei et al. found that the cd/egfp cells were largely localized at the junction of the tumor with normal tissue. the mean survival time of rats co-injected with c6 glioma cells and mscs-cd/egfp cells was signi fi cantly extended to 45.9 days with tumor size reduction when compared with rats injected with c6 glioma cells alone surviving an average of 15.3 days, or those co-injected with c6 glioma cells and parental cells surviving only for 16.0 days. in addition, data suggest that msc-cd/egfpmediated gene therapy promoted tumor cell apoptosis in rat c6 gliomas [ 72 ] . without going into detail, hypoxia-induced genes seem to play an important role in the fate of mscs and require further studies, as modifying and preconditioning as well as changing their effects temporarily by gene therapy indicates a plethora of important insights into the potential use of this complicated class of stem cells in tumor therapy [ 142, 147, 150, 155, 267, 272 ] , and we expect rapid progress in this area in the near future. the rational is that tumor cells have signi fi cantly altered metabolism with a shift toward the anaerobic pathway and changes in the respective gene expression patterns providing novel targets and delivery methods for cancer therapy. transplantation of hscs to correct a series of lysosomal storage diseases and peroxisomal disorders has almost 25 years of history and involves over 20 diseases [ 23 ] . however, the success was limited to only a small subclass of diseases such as hurler syndrome, x-ald, and infantile krabbe disease. detailed studies are now available suggesting that hematopoietic stem cells are suitable only for a carefully selected cases, leaving open the fi eld for a more versatile mesenchymal stem cell therapy, especially those instances having neurological symptoms [ 69 ] . bone marrow-derived mscs are another promising platform for cell-and gene-based treatment of inherited and acquired disorders including a whole range of lysosomal storage diseases. several animal models exist to run preclinical studies [ 164 ] . human mscs distribute widely in a murine xenotransplantation model, and the human stem cells are amenable to lentiviral vector-mediated transduction to obtain expression of therapeutic levels of enzyme in xenotransplantation models of human disease (non-obese diabetic severe combined immunode fi cient mucopolysaccharidosis type vii [nod-scid mpsvii]) [ 164 ] . transduced mscs persisted in the animals that underwent transplantation and comparable numbers of donor mscs were detected at 2 and 4 months after transplantation. the level of circulating enzymes were suf fi cient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues providing additional evidence that transduced human mscs retain their normal traf fi cking ability in vivo and persist for at least 4 months, while able to deliver therapeutic levels of proteins in an authentic xenotransplantation model of human disease. similar results have been reported by muller and colleagues, who were able to restore aryl sulfatase and beta galactosidase levels in genetically de fi cient bone marrow mscs, and showed that untransduced cells from patients with metachromatic leukodystrophy , who are asa de fi cient, took up a substantial amount of asa that was released into the media from mscs [ 173 ] , an important milestone for future attempts to try stem cell therapy of metachromatic leukodystrophy. gm1 ganglyosiosys was successfully treated with mscs in a mouse beta-galactosidase knockout model indicating that autologous transplantation may be feasible using lentiviral-transduced mscs [ 228 ] . fabry disease affects an estimated 1 in 40,000-60,000 males, and far less frequently females. it is an inherited lysosomal disorder caused by a de fi ciency of alpha-galactosidase a (alpha-gal a). the systemic accumulation of globotriaosylceramide (gb3) results in gradual tissue deterioration leading to organ failure. there is a limited mouse model of the disease showing gb3 accumulation in an alpha-gal a-de fi cient mouse model. however, most of the important clinical manifestations are absent and the lack of relevant large animal model hinders the development of proper cell therapy. when compared to the human alpha-gal a, the porcine alphagal a showed a high level of homology in the coding regions. cell lysate and supernatants from fabry patient-derived fi broblasts transduced with a lentiviral vector carrying the porcine alpha-gal a cdna (lv/porcine alpha-gal a) showed high levels of alpha-gal a activity, and its enzymological stability was similar to that of human alpha-gal a. even more importantly, uptake of secreted porcine alpha-gal a by non-transduced cells was observed. furthermore, gb3 accumulation was reduced in fabry patient-derived fi broblasts transduced with the lv/porcine alpha-gal a. the fi nding that the porcine version of the gene is also x-linked (x22q) provides hope that a large animal (porcine) model of fabry disease can be constructed in the near future for use in testing a novel application of cell therapy using mscs [ 278 ] . the success of such model and eventually the feasibility of the treatment depends on the "bystander phenomenon ," i.e., the transduced mesenchymal cells intended for delivering the enzyme secrete the enzyme in abundance, but the defective cells in their microenvironment also must be able to take up the enzyme and utilize it. to facilitate the uptake, a fusion protein between gb3 and hiv tat protein has been made [ 104 ] . if successful, the range of enzyme replacement therapy approach could widen signi fi cantly. the data published by higuchi et al. indicate that indeed the tat's ability to penetrate the cell membrane was maintained in the recombinant fusion protein and it enhanced the enzyme uptake, as expected. since the different manifestations of the disease produce problems in different organs (brain, kidney, and heart), it seems to imply that mscs will be the best candidates for this enzyme replacement therapy as the earlier attempts to perform enzyme replacement therapy in mouse model showed insuf fi cient ef fi ciency [ 277 ] . the enormity of the problems posed by diabetes is re fl ected by the statistics published on the nih website ( http://diabetes.niddk.nih.gov/dm/pubs/statistics/#dd ). by the age of 65, almost one in four americans suffers from diabetes. the at-risk population of prediabetics is 37% of the population older than 20 years. the sheer number of patients indicates that restoring glucose metabolism by pancreas or pancreatic islet transplantation, even in the most severe cases, is just impractical if not impossible. the low engraftment rate makes the prospects of such treatment even worse, especially, as there never will be a suf fi cient number of donors. that leaves the stem cell technology as the major source of hope for solving the relevant issues in recovering regulated insulin production and glucose regulation functionality in diabetes. a large number of clinical trials using mscs are under way [ 75, 106, 120 ] , and a rather confusing sets of stem cell markers are listed in these studies indicating that there is a plurality of stem cells residing in different tissues, all of which have the potential to help pancreatic tissue regeneration. not surprisingly, the most obvious source of these stem cells could be the pancreas itself, from which the resting stem cells can be isolated, reactivated, and expanded by variety of stimulants. the data are still being evaluated, and need further con fi rmation, reproduction, and lineage tracing. the currently available datasets could not fi rmly substantiate the claims when using different markers ( carbonic anhydrase ii vs. hepatocyte nuclear factor 1 beta ) [ 61, 113, 237, 243 ] . since then, neurogenin 3 also was considered as a marker for endocrine type differentiation of proto beta cells [ 273 ] , leaving the subject as to whether well-de fi ned adult beta islet cell progenitors truly exist in signi fi cant numbers rather murky. the phenomenon of in vitro trans-differentiation of the acinar cells into beta cells upon exposure to egf, lif, notch1-inhibitors [ 15 ] looks promising, and recently zhou and colleagues added a more extensive study on in vivo reprogramming of adult pancreatic exocrine cells into beta cells [ 289 ] . however, the reported ef fi ciency was low and the progenitor cells remained elusive. this left the fi eld searching for other sources, including mscs from bone marrow, liver, intestine, and neural tissue (reviewed by efrat [64] [65] [66] and jones [ 118 ] ), that are capable of trans-differentiating into insulin-producing beta cells. with the available results, their ultimate hope was that these cells could be used to seed the pancreas with new sets of insulin producing islands. since lineage tracing was often omitted and the reproducibility of the results remained unsettled, the fi eld, despite its high importance, seems to be somewhat in shambles [ 106, 107 ] , ready for deployment of the novel, lentiviral vector supported techniques. szabat et al. report a signi fi cant set of results on beta-cell maturation using lentiviral vector-based lineage-study examining a novel pdx1/ins1 dual fl uorescent reporter vector. they con fi rmed that individual adult human and mouse beta-cells exist in at least two differentiation states, distinguishable by the activation of the ins1 promoter. they performed real-time imaging of the maturation of individual cultured beta-cells and followed the kinetics of the maturation process in primary human and mouse beta-cells and collected gene expression pro fi ling data as well. the gene expression pro fi ling of facs puri fi ed immature pdx1+ /ins1 (low) cells and mature pdx1 (high)/ins1 (high ) cells from cultures of human islets, mouse islets, and min6 cells revealed that pdx1+/ins1 (low) cells are enriched for expression of multiple genes associated with beta-cell development/progenitor cells, proliferation, apoptosis, as well as genes coding for other islet cell hormones such as glucagon [ 240 ] . it turns out that trans-differentiation can be successfully performed using mafa. mafa is a leucine zipper transcription factor from the maf family that can be activated by p38 map kinase. this protein is a known pancreatic transcriptional factor controlling the beta-cell-speci fi c transcription of the insulin gene [ 40 ] . expressing it using lentiviral vectors in placenta-derived multipotent stem cells (pdmscs) that constitutively expressed oct-4 and nanog resulted in signi fi cantly upregulated expression of a series of pancreatic development-related genes (sox17 , foxa2 , pdx1, and ngn3 ), similar to that of native pancreas and islet tissues. mafa increased the expression levels of the mrnas of nkx2.2 , glut2 , insulin , glucagons, and somatostatin , and further facilitated the differentiation of pdmscs into insulin + cells. importantly, the expression of mafa in pdmscs xenotransplanted into immunocompromised mice improved the restoration of blood insulin levels to control values and greatly prolonged the survival of graft cells in immunocompromised mice with stz-induced diabetes [ 40 ] . another successful lineage analysis and monitoring the induced trans-differentiation was reported by cheng et al., in which a relatively abundant epithelial cell source, fetal human pancreas, was used to assess the proliferation potential, changes in lineage markers during culture, and capacity for generating insulin-expressing beta cells from fetal epithelial cells. the fetal epithelial cells readily formed primary pancreatic progenitor cultures, although their replication capacity was rather limited. this was overcome by introduction and expression of htert (human telomerase reverse transcriptase) which greatly enhanced cellular replication in vitro. however, during culture the htert-modi fi ed pancreatic progenitor cells switched their phenotype gaining additional mesodermal properties. this phenotypic switching was inhibited when a pancreas-duodenal homeobox (pdx)-1 transgene was expressed with a lentiviral vector, along with inductive signaling through activin a and serum deprivation. this restored endocrine properties of htertmodi fi ed cells in vitro and were able to express insulin in vivo in immunode fi cient mouse model [ 39 ] . the complexities of these result indicate that a sophisticated multi-gene cell therapies may be needed to solve the issues of proper modulation of transdifferentiation pathways. other strategies using a lentiviral vector-based approach to achieve beta-cell proliferation through the beta-cell-speci fi c activation of the hepatocyte growth factor (hgf)/cmet signaling pathway are also being explored. one of these methodologies is based on the beta-cell-speci fi c expression of a ligand-inducible, chimeric receptor (f36vcmet ), under transcriptional control of the promoter from the human insulin gene, and its ability to induce hgf/cmet signaling in the presence of a synthetic ligand (ap20187) and result in speci fi c proliferation of human pancreatic beta-cells [ 182 ] . the selective, regulated beta-cell expansion may help to increase the availability of cells for transplantation in patients with advanced diabetes. these recent studies show that rapid progress may be achieved in this fi eld and lentiviral vectors may provide the necessary tools to analyze the issues. however, some of the notable efforts are made to avoid stem cell therapy altogether in certain types of diabetes. instead, choosing a more direct route, applying in vivo gene therapy for expressing insulin gene in cell types other than beta cells. ren et al. successfully restored near normal insulin levels for 500 days by expressing insulin in resting liver cells transduced with lentiviral vector using a rat diabetes model [ 204 ] . although monogenic disease appears to be the most obvious human diseases to treat with gene therapy, since they are caused by a single-gene defect, the progress in clinical studies has thus far been rather limited. explanations for the lack of success include inef fi ciency of transductions in vivo, dangers posed by vectors, the failure to permanently correct the gene defect in suf fi cient number of cells, or the rapid turnover of cells. alternative approaches therefore involve the search for and use of stem cell populations and depleting the active stem cell compartments ablation using cytostatic drugs to give chance if increased engraftment by transplanted stem cells. combining the versatility and availability of mscs, their ability to engraft, the use of autologous instead of allogeneic sources for safe transplantation, and the fact that the stem cell population can be expanded in vitro allows highly ef fi cient ex vivo gene therapy relying on latest generation of lentiviral vectors. cystic fi brosis (cf) is caused by a mutation in the gene for the cystic fi brosis transmembrane conductance regulator protein (cftr). the mutant form of the protein causes severe defect in mucus metabolism in the lungs and intestinal track that deteriorates into a life-long, deadly disease. cf is theoretically amenable to gene therapy. in spite of intensive research and a large number of clinical trials in the last 18 years, little practical success can be shown for treating cystic fi brosis [ 191 ] . the explanations include the fact that the deeper regions in the inner surface of the lung are not accessible to direct inhalation and direct treatment [ 48, 263 ] . in light of this fi nding, stem cells remain the most promising delivery vehicles. castellani et al. reviewed the recent attempts to identify lung-or bone marrow-derived populations of stem cells or progenitor cells and application of such cells, allogenic or gene-corrected autologous cells, to colonize the airways, while differentiating into functional respiratory columnar epithelial cells [ 33 ] . when the reporter gene expression was analyzed in trachea-lungs and bronchoalveolar lavage, 0.4-5.5% of stem cells survived in injured airways, but no stem cells survived in control, healthy airway, or in the epithelial lining fl uid [ 138 ] . the most successful approaches thus far appear to be obtained with bone marrow-derived mscs, although the trans-differentiation rate thus far has been limited to below 10-14% [ 26 ] . as an alternative, the proven multipotent nature of bronchoalveolar stem cells isolated from lung tissue may provide another promising approach for stem cell therapy. some additional improvement is expected from more ef fi cient targeting of lentiviral vectors. mitomo and colleagues built a sendai virus env-pseudotyped siv lentiviral vector that can be manufactured at high enough titer and is capable of transducing respiratory epithelium of the murine nose in vivo at levels that may be relevant for achieving clinical bene fi t to cystic fi brosis patients [ 167 ] . availability of novel cystic fi brosis gene-carrying stem cell lines derived from placental mesenchymal cells certainly will help to speed up the research [ 53 ] . however, much more needs to be known about the normal differentiation and functioning of the airway's basal cells and the differentiation and lineages of stem cells to have more ef fi cient treatment options both for gene therapy and for stem cell therapy [ 207 ] . we expect that the intensity of research and push for clinical trials will remain high as the outline of directions will become clearer. also the methods to derive respiratory cell types from stem cells will remain a critical piece [ 181 ] . this disease is an x-linked recessive disorder caused by a mutation in the dystrophin gene that destabilizes muscle cell membranes and causes muscle dystrophy in approximately 1 of 3,600 boys. the musculoskeletal abnormalities deteriorate to a fatal level and the average life expectancy is no more than 25 years, even with high quality care. the research is facilitated by the availability of dystrophin-de fi cient transgenic mice (mdx-mice ) and double knockout (utrophin/dystrophin-de fi cient mice) that can be used as experimental disease models [ 144, 269 ] . human immortalized pluripotent cell lines expressing the mutant dystrophin gene are also available [ 187 ] . the ability of mscs to differentiate into muscle cells places them on the top of the list of candidates that could be used to treat duchenne muscular dystrophy [ 157 ] . lentiviral vectors have been used in this fi eld for conditional immortalization of human cells for basic biologic studies. cudre-maroux et al. demonstrated that the lentiviral vector-mediated transduction of immortalizing genes into human primary cells is an ef fi cient method for obtaining such cell lines. for duchenne muscular dystrophy, the muscle satellite cell model was used to examine the impact of the transduced genes on the genotypic and phenotypic characteristics of the immortalized cells. the most commonly used immortalizing gene, the sv40 large t antigen (t-ag), was extremely ef fi cient at inducing the continuous growth of primary myoblasts, but the resulting cells rapidly accumulated major chromosomal aberrations and exhibited profound phenotypic changes. in contrast, the constitutive expression of telomerase and bmi -1 in satellite cells from a control individual and from a patient suffering from duchenne's muscular dystrophy yielded cell lines that remained diploid and conserved their growth factor dependence for proliferation. however, despite the absence of detectable cytogenetic abnormalities, clones derived from satellite cells of a control individual exhibited a differentiation block in vitro. in contrast, a duchenne-derived cell line exhibited all the phenotypic characteristics of its primary parent, including an ability to differentiate fully into myotubes when placed in proper culture conditions. this cell line should constitute a useful reagent for a wide range of studies aimed at this disease [ 46 ] . a realistic source of stem cells would be the adipose tissue-derived stem cells that can be enhanced for muscle repair. forced expression of myod using lentiviral vector in vitro strongly induced myogenic differentiation, while the adipogenic differentiation was inhibited. moreover, myod-expressing human multipotent adipose-derived stem cells had the capacity to fuse with dmd myoblasts and can restore dystrophin expression. importantly, transplantation of these modi fi ed human, multipotent, adipose-derived stem cells into injured muscles of immuno-depressed rag2(−/−)gam-mac(−/−) mice resulted in a substantial increase in the number of human multipotent adipose-derived stem cell-derived fi bers [ 92 ] . goncalves and colleagues went a step further and devised a technique to monitor the fusion events necessary for myoblast formation by using an elaborate bipartite genetic switch that relays on recombinaseinducible genetic switch that is activated after two cell types, one of which expresses cre and the other the rest of the elements with loxp1 sites, that switch on only upon fusion. this provides a sensitive tool to study the lineages and process of myocyte fusion in transgenic system [ 90 ] . ikemoto et al. used high transduction-ef fi ciency lentiviral vector-mediated gene transfer into freshly isolated autologous satellite cells. freshly isolated cells have better myogenic capability than satellite cell-derived myoblasts, and expansion of the satellite cells does not affect their regenerative potential. the transduced cells successfully regenerated the targeted muscle groups in mdx mice [ 112 ] . however, the vsvg pseudotyped lentiviral vector are inferior in transducing nondividing murine cells, and shunchang et al. demonstrated that by pseudotyping with feline immunode fi ciency virus env better transduction rates can be achieved [ 234 ] . we include this disease because of the challenges researchers faced in attempts to use cell-based therapies. granulomatous disease is a rather rare x-linked immunode fi ciency disorder caused by mutations in the cybb gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (nadph)-oxidase catalytic subunit gp91(phox). earlier attempts to restore the gene function with oncoretroviral vectors failed due to (a) gene silencing common for retroviral inserts, (b) high risk of genotoxicity that these oncoretroviral vectors pose [ 165 ] , and (c) low transduction ef fi ciency and inability to target appropriate cell lineage and practice differentiation-restricted gene expression [ 17 ] . the solution for the compound problem seems to lie in using a safer and more ef fi ciently targeting lentiviral vector system [ 17, 223 ] . it has been demonstrated and repeatedly con fi rmed that by using lentiviral vector it is possible to transduce hscs as well as differentiated neutrophiles from patients with x-linked chronic granulomatous disease (x-cgd) and correct the x-cgd-phenotype in the nod/scid model. the lentivector was a vsv-gpseudotyped, third-generation, self-inactivating (sin) lentivector encoding gp91 (phox). lentiviral vector ef fi ciently transduced cd34+ peripheral blood stem cells under ex vivo conditions nonpermissive for cell division and resulted in 54% of the cells expressing gp91 (phox). lentivector also achieved signi fi cant correction of differentiated human x-cgd neutrophils arising in vivo in nod/scid mice that underwent transplantation (20% and 2.4%, respectively). thus, third-generation sin lentivector-gp91 (phox) performs well as assessed in human x-cgd neutrophils differentiating in vivo, and the studies suggest that the nod/scid model is generally applicable for in vivo study of therapies evaluated in human blood cells expressing a speci fi c disease phenotype [ 165, 209, 226 ] . however, long-term solution can be expected from transducing hscs, and not the fully differentiated neutrophils that have only limited lifespan left and the lack of genotoxicity safety of the third-generation sin v lentiviral vector seem to address these requirements perfectly. wilson's disease is a genetic disease caused by a spectrum of mutations in the atp7b gene , whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. zhang and colleagues reported in 2011 an attempt to restore the normal phenotype by directed hepatocyte differentiation from human-induced pluripotent stem cells. the phenotype correction was achieved by chaperone drug curcumin that can reverse the functional defect in vitro in the case of the r778l chinese hotspot mutation in the atp7b gene. they propose this model system for correcting the gene using lentiviral technology [ 284 ] . atp7b gene seems to be relevant for the wider mesenchymal stem cell fi eld as over-expressing it protects mscs form copper toxicity . this in turn could be used as a selection advantage of transduced mscs over the non-transduced ones in copper-rich environment for enriching the transduced mesenchymal cell compartment before transplantation [ 225 ] . the image of the "fountain of youth" represents a mirage deeply engraved in the human psyche and expresses our fear and resentment of one of the inevitabilities of life: if we are lucky, we will get old, decrepit, suffer a lot from series of painful, chronic diseases, and fi nally succumb. the irony is that those who we consider unlucky, die young, but are saved from the long lasting predicaments of aging. certainly, the intricacies of the factors leading to longevity, or the lack thereof, keep generations of stem cell researchers awake and busy and for good reasons. a model for aging has been found in the condition known as progeria , or more precisely the hutchinson-gilford progeria syndrome , a rare disease affecting children of both sexes and which is caused by a mutant prelaminin a gene, encoding the lamin a-processing enzyme. prelaminin a that retains a farnesyl group, subsequently expressing its abnormal form, progerin. progerin in turn is anchored to the nuclear membrane and destabilizes the nucleus, limiting the ability of cells to divide and leading to premature cell death. unlike other accelerated aging diseases affecting dna repair (werner's and cockayne's syndrome), progerin may play role in normal aging process [ 211 ] and its production is slowly turned on in cells that have uncapped chromosomes, i.e., have truncated telomeres, resulting in premature depletion of stem cell compartments. see the popular ucsf website for details: http://www.ucsf.edu/news/2011/10/10766/aging-disease-children-sheds-lightnormal-aging . additional upregulation of multiple genes in major in fl ammatory pathways indicated an activated in fl ammatory response in progeria patients. this response has also been associated with normal aging, emphasizing the importance of studying progeria to increase the understanding of the normal aging process [ 178 ] . the progressing disease shows a pattern of tissue and organ degeneration that correlates with the depletion of a variety of stem cell compartments, a correlation fi rst pointed out by favreau [ 70 ] . the insight into the role of stem cell depletion in progeria accumulated rapidly in the last couple of years [ 170, 178, 179, 211 ] . this leads to the establishment of an animal model by creating zmpste24 knockout mice [ 67 ] in 2008, which showed premature senescence and progeroid symptoms. with the role of stem cells in aging and in progeria, the doors opened for studying stem cell renewal via dedifferentiation. autologous or heterologous transfer of native or lentivector-enhanced cells [ 129, 184, 212, 265 ] are being actively considered as a possible interventions to slow down progeria as well as natural aging [ 28 ] . however, in both cases, the changes are systemic and murine gene therapy data indicate that the therapy in lysosomal storage disease models, affecting large segments of the body, is more ef fi cient if done at an earliest possible age [ 28, 129 ] . this may have something to do with the limited availability of the microenvironment for the modi fi ed or transplanted stem cells. for this, the preexisting ones, even when "old" and malfunctioning, already occupy the microenvironment appropriate for stem cells. we already know that wound sites create new sites and attract mscs [ 5 ] . also, it is possible that cancer growth is able to generate and maintain an appropriate microenvironment for cancer stem cells [ 24 ] as well as mscs [ 42 ] (potentially for use as anticancer agents) but the normal tissue, even in aging, seems to be resilient in accepting externally provided stem cells. experiments are under way to create arti fi cial microenvironments using nanotechnology to deliver stem cells that produce therapeutical factors [ 52 ] , and 3d scaffolding mimicking bone marrow niches are being designed for similar purpose [ 55 ] and lentiviral vector are often used to deliver the genes of interest [ 60, 141, 166, 248 ] . virxsys pioneered the use of lentiviral vectors in phase i clinical trials to deliver antisense hiv genes as an antisense rna therapy for aids [ 110, 156 ] . this established an initial safety pro fi le for the ex vivo use of lentiviral vectors (see http:// clinicatrials.gov : identi fi er vrx496-usa-05-002). this phase i trial demonstrated the safety and tolerability of a single dose of approximately ten billion autologous hiv infected cd4+ t cells transduced with the lentivector vrx496 carrying a 937-base antisense targeting the hiv envelope. these encouraging results have led to design of a phase ii clinical trial to evaluate the safety, tolerability, and biological activity of four or eight repeated infusions of fi ve to ten billion autologous vrx496modi fi ed hiv+, cd4+ t cells. a major obstacle to completing this phase ii trial was manufacturing enough cells to administer multiple infusions in patients. for this study the safety issues were cleared successfully, opening the way for more extensive use of lentiviral vectors in clinical trials. currently, 16 lentiviral clinical trials are listed at the clinicaltrials.gov website. all of these trials are in early stage, phase i or ii. three of these trials have not yet started and 11 trials are still recruiting patients. most of the clinical trials are focused on hematopoietic stem cells, which are outside of the scope of this work. one trial targets netherton syndrome (clinicaltrials.gov: identi fi er: nct01545323) and attempts to restore lekti serine protease levels in an affected 5 cm 2 skin area, a proof of principle study that has the potential to utilize mesenchymal stem cells in the future. we witnessed a tremendous progress in characterizing and understanding stem cells, the factors needed for maintaining the stem cell phenotype as well as changing it in a predictable mode forcing the mesenchymal stem cells into various differentiation pathways. this progress provides a test bed for a higher level of bioengineering when the genetic buildup of the stem cells is changed to achieve well-de fi ned therapeutical goals. the overview of the recent literature presents a long list of "proof of concept experiments" in which tantalizing possibilities are validated as things that can be accomplished in a wide range of fi elds representing different pathologies: from the debilitating alzheimer's, parkinson's diseases; various traumatic neuronal injuries, diabetes, neuronal and cardiac ischemia; to agerelated tissue degeneration and tissue engineering or delivery of biologics for therapeutical purposes. in parallel, lentiviral vectors are becoming highly valued tools in this tedious work as they are highly ef fi cient vehicles for gene delivery to mark cells, express genes of interest, proteins, and various inhibitory rna species in stem cells. consequently these stem cells, especially the various forms of mscs, have been shown to be highly effective in delivering the targeted genes to dif fi cultto-reach tissues, including the cns. manipulating genes and gene expression, gene transfer has been made safe and ef fi cient by the recent progress in lentivector technology and merged successfully with the stem cell technology. this fi eld has reached an advanced stage, at which it has become feasible to use them safely in a clinical environment. more and more researchers as well as clinicians are becoming familiar with the power of these technologies both for ex vivo and in vivo cell therapy. it does not take a prophet to predict that advanced stem cell therapy has gaining a strong foothold, and even though a tremendous amount of work is needed to be done for it to become a everyday 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bm-failure-syndrome and in vivo in humanized mice advances in the fi eld of lentivector-based transduction of t and b lymphocytes for gene therapy measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both slam and cd46 entry receptors strategies for targeting lentiviral vectors enhancement of posterolateral lumbar spine fusion using low-dose rhbmp-2 and cultured marrow stromal cells peptides for therapy and diagnosis of alzheimer's disease mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration development of lentiviral gene therapy for wiskott aldrich syndrome limitation of in vivo models investigating angiogenesis in breast cancer rd114-pseudotyped retroviral vectors kill cancer cells by syncytium formation and enhance the cytotoxic effect of the tk/gcv gene therapy strategy tissue-speci fi c shrna delivery: a novel approach for 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ciency insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of scid-x1 a serious adverse event after successful gene therapy for x-linked severe combined immunode fi ciency optimized lentiviral vector design improves titer and transgene expression of vectors containing the chicken betaglobin locus hs4 insulator element zinc-fi nger nuclease based genome surgery: it's all about speci fi city role of mesenchymal stromal cells in solid organ transplantation immunomodulatory effect of mesenchymal stem cells alpha-galactosidase a-tat fusion enhances storage reduction in hearts and kidneys of fabry mice transgenic rabbit production with simian immunode fi ciency virus-derived lentiviral vector the quest for tissue stem cells in the pancreas and other organs, and their application in beta-cell replacement lineage tracing evidence for transdifferentiation of acinar to duct cells and plasticity of human pancreas lentivirus-mediated transfer of mmp-9 shrna provides 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progenitor cells enhance muscle regeneration in mouse hindlimb ischemia model resident vascular progenitor cells: an emerging role for nonterminally differentiated vessel-resident cells in vascular biology sox2 transduction enhances cardiovascular repair capacity of blood-derived mesoangioblasts methods in mammalian cell line engineering: from random mutagenesis to sequence-speci fi c approaches cre/loxp recombination system and gene targeting human mesenchymal stem cells (hmscs) expressing truncated soluble vascular endothelial growth factor receptor (tsflk-1) following lentiviral-mediated gene transfer inhibit growth of burkitt's lymphoma in a murine model vegf at the neurovascular interface: therapeutic implications for motor neuron disease developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury human umbilical cord blood-derived mesenchymal stem cells improve neuropathology and cognitive impairment in an alzheimer's disease mouse model through modulation of neuroin fl ammation cell therapy for bone regeneration-bench to bedside survival and neuronal differentiation of mesenchymal stem cells transplanted into the rodent brain are dependent upon microenvironment hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fi ber regeneration after ischemia through a wnt4-dependent pathway gene transfer in humans using a conditionally replicating lentiviral vector improved motor function in dko mice by intravenous transplantation of bone marrow-derived mesenchymal stromal cells hypoxia and hypoxia inducible factors in cancer stem cell maintenance inhibition of hiv-1 infection by a unique short hairpin rna to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction mesenchymal stromal cells expressing heme oxygenase-1 reverse pulmonary hypertension human fetal trachea-scid mouse xenografts: ef fi cacy of vesicular stomatitis virus-g pseudotyped lentiviral-mediated gene transfer human umbilical mesenchymal stem cells promote recovery after ischemic stroke hypoxia-inducible factor-1alpha is essential for hypoxia-induced mesenchymal stem cell mobilization into the peripheral blood effects of transplantation with bone marrow-derived mesenchymal stem cells modi fi ed by survivin on experimental stroke in rats neuro fi lamentopathy in neurodegenerative diseases arti fi cial cell microencapsulated stem cells in regenerative medicine, tissue engineering and cell therapy gene editing in human stem cells using zinc fi nger nucleases and integrase-defective lentiviral vector delivery hypoxia and hypoxia-inducible factors: master regulators of metastasis antisensemediated inhibition of human immunode fi ciency virus (hiv) replication by use of an hiv type 1-based vector results in severely attenuated mutants incapable of developing resistance sources of adult mesenchymal stem cells applicable for musculoskeletal applications -a systematic review of the literature design, engineering, and characterization of zinc fi nger nucleases biomimetic mineral-organic composite scaffolds with controlled internal architecture neprilysin gene transfer reduces human amyloid pathology in transgenic mice recent advances in lentiviral vector development and applications rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery mesenchymal stem cells for the sustained in vivo delivery of bioactive factors lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease site-speci fi c gene insertion mediated by a cre-loxpcarrying lentiviral vector targeting the tumor and its microenvironment by a dual-function decoy met receptor toward gene therapy for cystic fi brosis using a lentivirus pseudotyped 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system combining cell cycle-regulated and tissue-speci fi c transcriptional control dedifferentiation rescues senescence of progeria cells but only while pluripotent in vitro pathological modelling using patient-speci fi c induced pluripotent stem cells: the case of progeria origin of hematopoietic progenitors during embryogenesis deriving respiratory cell types from stem cells regulated expansion of human pancreatic beta-cells targeting the perpetrator: breast cancer stem cell therapeutics ef fi ciency of lentiviral transduction during development in normal and rd mice reprogramming to pluripotency: stepwise resetting of the epigenetic landscape targeting of therapeutic gene expression to the liver by using liver-type pyruvate kinase proximal promoter and the sv40 viral enhancer active in multiple cell types disease-speci fi c induced pluripotent stem cells glial cells in (patho)physiology lentivirus-expressed sirna vectors against alzheimer disease mesenchymal stem cells and tooth engineering stem cell therapy for cystic fi brosis: current status and future prospects progress in understanding reprogramming to the induced pluripotent state conditional mutagenesis in mice: the cre/loxp recombination system lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study lentiviral vectors approach the clinic but fall back: national institutes of health recombinant dna advisory committee review of a fi rst clinical protocol for use of a lentiviral vector mesenchymal stem cells as therapeutics and vehicles for gene and drug delivery plant biotechnology: zinc fi ngers on target chimeric nucleases stimulate gene targeting in human cells identi fi cation of the sensory neuron speci fi c regulatory region for the mouse gene encoding the voltage-gated sodium channel nav1.8 intrapleural delivery of mesenchymal stem cells: a novel potential treatment for pleural diseases zinc-fi nger nucleases for somatic gene therapy: the next frontier identi fi cation of potential stroke targets by lentiviral vector mediated overexpression of hif-1 alpha and hif-2 alpha in a primary neuronal model of hypoxia de fi ning pluripotent stem cells through quantitative proteomic analysis longterm correction of diabetes in rats after lentiviral hepatic insulin gene therapy effect of interleukin-10 overexpression on the properties of healing tendon in a murine patellar tendon model stem cell technologies for tissue regeneration in dentistry airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling mesenchymal stem cells derived from dental tissues third-generation, self-inactivating gp91(phox) lentivector corrects the oxidase defect in nod/scid mouse-repopulating peripheral blood-mobilized cd34+ cells from patients with x-linked chronic granulomatous disease current development of lentiviral-mediated gene transfer stem cell depletion in 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human skin equivalents mesenchymal stem cell transplantation modulates neuroin fl ammation in focal cerebral ischemia: contribution of fractalkine and il-5 superior tissue-speci fi c expression from tyrosinase and prostate-speci fi c antigen promoters/enhancers in helper-dependent compared with fi rstgeneration adenoviral vectors a highly ef fi cient short hairpin rna potently down-regulates ccr5 expression in systemic lymphoid organs in the hu-blt mouse model expression of truncated dystrophin cdnas mediated by a lentiviral vector mscs: biological characteristics, clinical applications and their outstanding concerns feasibility of a stem cell therapy for intervertebral disc degeneration pancreatic exocrine duct cells give rise to insulinproducing beta cells during embryogenesis but not after birth potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells induced pluripotent cancer cells: progress and application kinetics and genomic pro fi ling of adult human and mouse beta-cell maturation cervical spinal cord delivery of a rabies g protein pseudotyped lentiviral vector in the sod-1 transgenic mouse. invited submission from the joint section meeting on disorders of the spine and peripheral nerves micromanipulating viral-based therapeutics growth and regeneration of adult beta cells does not involve specialized progenitors tumor-initiating and -propagating cells: cells that we would like to identify and control highly ef fi cient endogenous human gene correction using designed zinc-fi nger nucleases genome editing with engineered zinc fi nger nucleases mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation development of an in vitro model for the simultaneous study of the ef fi cacy and hematotoxicity of antileukemic compounds mesenchymal stem cell transplantation changes the gene expression pro fi le of the neonatal 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proteins local gene transfer to calci fi ed tissue cells using prolonged infusion of a lentiviral vector harnessing hiv for therapy, basic research and biotechnology macroarchitectures in spinal cord scaffold implants in fl uence regeneration transduction patterns of pseudotyped lentiviral vectors in the nervous system establishment of an erythroid cell line from primary cd36+ erythroid progenitor cells targeted genome editing across species using zfns and talens in vivo gene transfer into adult stem cells in unconditioned mice by in situ delivery of a lentiviral vector combinatorial control of suicide gene expression by tissue-speci fi c promoter and microrna regulation for cancer therapy effect of hypoxia on the gene pro fi le of human bone marrow-derived mesenchymal stem cells the in fl uence of semen-derived enhancer of virus infection on the ef fi ciency of retroviral gene transfer microdystrophin delivery in dystrophin-de fi cient (mdx) mice by genetically-corrected syngeneic mscs transplantation long-term ef fi cacy and safety of human umbilical cord mesenchymal stromal cells in rotenone-induced hemiparkinsonian rats vegf-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for parkinson's disease mesenchymal stem cells promote cardiomyocyte hypertrophy in vitro through hypoxia-induced paracrine mechanisms beta cells can be generated from endogenous progenitors in injured adult mouse pancreas magnetic resonance evaluation of transplanted mesenchymal stem cells after myocardial infarction in swine transplantation of adipose tissue-derived stem cells for treatment of focal cerebral ischemia current advances in retroviral gene therapy correction of cardiac abnormalities in fabry mice by direct intraventricular injection of a recombinant lentiviral vector that engineers expression of alpha-galactosidase a sequencing and characterization of the porcine alpha-galactosidase a gene: towards the generation of a porcine model for fabry disease self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb glycosaminoglycans and pdgf signaling in mesenchymal cells validation of a mutated pre sequence allowing high and sustained transgene expression while abrogating whv-x protein synthesis: application to the gene therapy of was the hiv-1 dna fl ap stimulates hiv vector-mediated cell transduction in the brain rescue of atp7b function in hepatocyte-like cells from wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin transplantation of neural stem cells modi fi ed by human neurotrophin-3 promotes functional recovery after transient focal cerebral ischemia in rats lentiviral-mediated overexpression of bcl-xl protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis lentivirus-mediated gene transfer of viral interleukin-10 delays but does not prevent cardiac allograft rejection loss of proliferation and differentiation capacity of aged human periodontal ligament stem cells and rejuvenation by exposure to the young extrinsic environment in vivo reprogramming of adult pancreatic exocrine cells to beta-cells organ-speci fi c expression of the lacz gene controlled by the opsin promoter after intravenous gene administration in adult mice neurovascular pathways to neurodegeneration in alzheimer's disease and other disorders woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors key: cord-339386-sxyeuiw1 authors: mcintosh, kenneth; perlman, stanley title: 157 coronaviruses, including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) date: 2015-12-31 journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: 10.1016/b978-1-4557-4801-3.00157-0 sha: doc_id: 339386 cord_uid: sxyeuiw1 nan the family coronaviridae, within the order nidovirales, contains two subfamilies, the coronavirinae and the torovirinae. coronaviruses (covs) are a large group of viruses infecting mammals and birds and producing a wide variety of diseases. they have been divided into four genera, two of which contain viruses infecting humans (see later). all human coronaviruses (hcovs) are primarily respiratory pathogens. during the winter of 2002 to 2003, an alarming new disease appeared, severe acute respiratory syndrome (sars), which was quickly attributed to a new cov, the sars-cov. the outbreak originated in southern people's republic of china, with evidence that the virus was first derived from bats and was transmitted to man through an intermediate host, probably the palm civet (paguma larvata) or raccoon dog (nyctereutes procyonoides). [1] [2] [3] the sars epidemic was controlled through a massive effort at case identification and containment, and the last known case occurred in mid-2004. in retrospect, the emergence of sars is consistent with what is known about covs as a group: they are important pathogens in animals causing a wide variety of diseases through a wide variety of pathogenic mechanisms, and they have been noted to mutate frequently and infect new species. 4, 5 more recently, a related but different cov producing severe respiratory disease has emerged, the middle east respiratory syndrome coronavirus (mers-cov). mers-cov was grown in june 2012, from a sputum sample obtained from a man in saudi arabia who died of overwhelming pneumonia. the virus was quickly identified as a new cov most closely related to several bat covs. 6 this report was followed by a number of other reports identifying a total of 537 infected individuals, all of whom had acute respiratory symptoms, severe in most, and fatal in 145 (as of may 11, 2014) . 7 human-to-human transmission has been documented, especially in hospital settings, 7a but appears to be inefficient. in 1965, tyrrell and bynoe 8 cultured a virus obtained from the respiratory tract of a boy with a common cold by passage in human embryonic tracheal organ cultures. the media from these cultures consistently produced colds in volunteers. the agent was ether sensitive but not related to any known human virus. subsequently, electron microscopy of fluids from infected organ cultures revealed particles that resembled infectious bronchitis virus of chickens. 9 at about the same time, hamre and procknow 10 recovered a cytopathic agent in tissue culture from medical students with colds. the prototype virus was named 229e and was found on electron microscopy to have a similar or identical morphology ( fig. 157-1) . 9 using techniques similar to those used by tyrrell and bynoe, mcintosh and colleagues 11 reported the recovery of several infectious bronchitis-like agents from the human respiratory tract, the prototype of which was named oc43 (oc for organ culture). at much the same time, mouse hepatitis virus and transmissible gastroenteritis virus of swine were shown to have the same morphology on electron microscopy. 12, 13 shortly thereafter, the name coronavirus (the prefix corona denoting the crownlike appearance of the surface projections) was chosen to signify this new genus. 14 the number of animal covs quickly grew, including viruses causing diseases in rats, mice, chickens, turkeys, various other bird species, cattle, several wild ruminants, beluga whales, dogs, cats, rabbits, and pigs, with manifestations in the respiratory and gastrointestinal tracts, central nervous system (cns), liver, reproductive tract, and others. through sequencing and antigenicity studies, the animal and human covs (hcovs) initially were divided into three groups: group 1, which • covs are members of the nidovirales order, single-stranded, positive-sense rna viruses with a large genome. they mutate and also recombine frequently. • laboratory diagnosis is best accomplished by finding viral rna through polymerase chain reaction. • there are no accepted effective antiviral drugs for covs. • prevention is through epidemiologic methods. the sars epidemic was halted through careful case finding, quarantine, and use of barrier precautions. 15 a cov was independently and almost simultaneously isolated from sars patients by several laboratories and found by sequencing to be only distantly related to previously characterized covs. [16] [17] [18] [19] [20] the sars outbreak stimulated a rapid and intense public health response coordinated by the world health organization, and by july 2003, transmission had ceased throughout the world. despite this effort, however, 8096 probable cases had occurred in 29 countries, with 774 deaths. 15 with the identification of the sars-cov, the hcov field became much more active. sensitive molecular methods were developed to detect rna from viruses identical or closely related to hcov-229e and hcov-oc43 in the respiratory tract, and two new species were discovered: nl63, an alphacoronavirus, and hku1, a betacoronavirus. [21] [22] [23] hcov-nl63 was found independently by three groups, two in the netherlands and, somewhat later, the third in new haven, figure 157-2 phylogenetic relationships among members of the subfamily coronavirinae. a rooted neighbor-joining tree was generated from amino-acid sequence alignments of coronaviridae-wide conserved domains in replicase polyprotein 1 (adrp, nsp3; mpro, nsp5; rdrp, nsp12; hel, nsp13; exon, nsp14; nendou, nsp15; o-mt, nsp16) for 21 coronaviruses, each a representative of a currently recognized coronavirus species. five of the six known human coronaviruses (hcov-229e, hcov-nl63, hcov-hku1, sars-cov, and mers-cov) are indicated. hcov-oc43 is closely related to bovine coronavirus, which is shown in the figure. equine torovirus berne served as the outgroup. virus names are given with strain specifications; species and genus names are in italics as per convention. the tree shows the four main monophyletic clusters, corresponding to genera alpha-, beta-, gamma to be established numerous reports of cov-like particles (covlps) found by electron microscopy in human fecal matter, but these particles have been difficult to characterize further. more recent efforts to detect cov rna in feces using polymerase chain reaction (pcr) and primers for respiratory hcovs have had limited success and have failed to associate covs with gastrointestinal disease. 28, 29 toroviruses were, like covs, first described in animals. they were first detected in the feces of cattle (breda virus) and horses (berne virus). 30, 31 shortly thereafter, beards and colleagues 32 examined human fecal material and reported finding particles with a similar appearance that aggregated in the presence of antiserum to the bovine and equine viruses. the human toroviruses, like the bovine toroviruses, do not grow in tissue culture, and thus almost all existing information about them is based on electron micrographic data. unlike animal toroviruses, 33,34 pcr-amplified torovirus rna sequences have not been found in human stool samples. a report of genome sequences amplified from particles purified from human stool 35 was subsequently retracted and considered to reflect laboratory contamination from porcine strains. 36 at this time, there are no reports definitively showing the existence of human toroviruses. the cov nucleic acid is rna, approximately 30 kilobases in length, of positive sense, single stranded, polyadenylated, and infectious. the rna, the largest known viral rna ( fig. 157-3) , codes for (in order from the 5′ end) a large polyprotein that is cleaved by virus-encoded connecticut. 24 in all three cases, positive samples were from infants and children with respiratory disease. notably, hcov-nl63 and hcov-229e were estimated to originate from a common precursor and diverge approximately 1000 years ago. 25 hcov-hku1 was found in hong kong in an adult with respiratory disease. these two new hcov strains subsequently have been found worldwide and appear to have pathogenicity similar to that of hcov-229e and hcov-oc43, with the possible exception that nl63 is more frequently found in children with croup. 26, 27 the mers-cov was found when a man was admitted in june 2012 to a hospital in jeddah, saudi arabia with overwhelming acute pneumonia with renal failure, and a sample of sputum grew a cytopathic virus that, on sequencing, proved to be a cov, classified as a betacoronavirus, and most closely related to two bat covs, hku-4 and hku-5. 6 over the next 23 months 536 additional cases (145 fatal) were found, all but a few of them sporadic or hospital-based and in individuals living or traveling in the middle east. in the remainder of this chapter, the group of respiratory hcovs first discovered in the 1960s and containing hcovs 229e, oc43, nl63, and hku-1 are referred to as community-acquired hcovs to distinguish them from the sars-cov and the mers-cov. in view of the prominence of covs in animal enteric diseases, there have been extensive efforts to identify enteric hcovs. there are figure 157-3 genome organization of representative human coronaviruses. all coronavirus genomes have the same basic structure and mechanism of replication. 4 the 5′ end of each genome encodes a leader sequence, which is attached to each virus-specific messenger rna transcript by a novel mechanism of discontinuous replication. the first two thirds of each genome encode replicase-associated genes. gene 1 is translated as two large polyproteins, with the first expressed from orf1a and the second from orf1a/b following a −1 frameshift event. these polyproteins are then cleaved into individual proteins by two virus-encoded proteases. the major structural genes, the hemagglutinin-esterase (he), surface (s), envelope (e), transmembrane (m), and nucleocapsid (n) proteins, are indicated in green. the nonreplicase, accessory genes located at the 3′ end of the genome are indicated with open boxes. the functions of these proteins are largely not known, and there is no sequence homology between accessory proteins of different coronaviruses. some of these proteins are virion associated, but none are required for virus replication. the open reading frames (orfs) encoding these proteins are numbered in order of appearance from the 5′ end of the genome, with the exception that ns12.9 of hcov-oc43. i is an internal protein expressed from an alternative reading frame located within the n gene. it is equivalent to sars-cov-specific protein 9b and the mers-cov-specific protein 8b. (figure prepared by 4 (dpp-4), which, like ace2 and apn, is an ectopeptidase that is abundantly expressed in the respiratory tract. 46 all the community-acquired respiratory hcovs grow only with difficulty in tissue culture. despite this, both 229e and nl63 were discovered because they produced a detectable cytopathic effect, the first in human embryonic kidney 10 and the second in llc-mk2 cells. 21 both the sars-cov and the mers-cov were initially isolated and grew readily in vero cells. 6, 17 hcovs oc43 and hku-1 have been grown in tissue culture after laboratory adaptation or in primary human airway epithelial cells. [47] [48] [49] detection of all these viruses in clinical specimens is most conveniently and sensitively achieved using pcr. likewise, the enteric covs have been difficult to cultivate in vitro. all but a few strains have been detected only by electron microscopy of human fecal material. [50] [51] [52] 53, 54 some strains have been characterized by immune electron microscopy and found to be related to hcov-oc43. 55 two strains obtained from an outbreak of necrotizing enterocolitis in texas and passaged in intestinal organ cultures were reported to contain four or five proteins with apparent molecular weights similar to those of other covs but not related antigenically to known human strains or mouse hepatitis virus a59. 56 the evidence favors the view that these isolates, as well as particles antigenically related to hcov-oc43, are members of the family coronaviridae, although their association with human disease is not yet proven. surveys of children with diarrhea using pcr imply that diarrhea associated with the four well-described hcov strains is unusual. 28, 29 epidemiology evidence of community-acquired respiratory cov infections has been found wherever in the world it has been sought. in temperate climates, respiratory cov infections occur more often in the winter and spring than in the summer and fall. the contribution of cov infections to the total number of upper respiratory illnesses may be as high as 35% during times of peak viral activity. overall, the proportion of adult colds produced by covs may be reasonably estimated at 15%. early studies of hcov-oc43 and 229e in the united states demonstrated periodicity, with large epidemics occurring at 2-to 3-year intervals. 57 strain hcov-229e tended to be epidemic throughout the united states, whereas strain hcov-oc43 appeared in localized outbreaks. similar studies of nl63 and hku1 have not been done, but it seems from the available data that they also vary widely in incidence from year to year and place to place. reinfection is common and may be due to the rapid diminution of antibody levels after infection. 58 infection occurs at all ages but is most common in children. the ratio of symptomatic to total infections varies between 50% and 90%, depending on the age of the population studied, the method of virus detection, and the definition of "infection. " 59, 60 among adult volunteers 72% of those infected with hcov-229e developed colds. 61 the first report of a new cov causing severe pneumonia appeared in promed mail on september 20, 2012. a man from jeddah, saudi arabia had developed pneumonia in june and died of respiratory and renal failure, and a virus was grown from a sputum sample that was subsequently sequenced and found to be a betacoronavirus thought at the time to be most closely related to bat covs hku4 and hku5. 6 between then and may 2014, a total of 537 cases occurred, all infected by this virus, now termed the middle east respiratory syndrome coronavirus (mers-cov). one hundred forty-five of the cases were fatal. 7 more than 400 of these have been acquired and diagnosed in the kingdom of saudi arabia, with most of the remainder in the united arab emirates, qatar, jordan, and kuwait. cases originating in the arabian peninsula have also occurred in travelers to egypt, tunisia, germany, italy, great britain, malaysia, the philippines, and the united states, with a few secondary cases sometimes occurring in those locations through close family or hospital spread. in the united states,these include two unrelated mers cases, and a third case related to one of these. 61a while infections with severe respiratory involvement have proteases to form several nonstructural proteins, including an rnadependent rna polymerase, methyltransferases, and a helicase, followed by either four or five structural proteins intermingled with a variable number of nonstructural and minor structural proteins. 37 the first of the major structural proteins is a surface hemagglutinin-esterase protein, present on hcovs oc43 and hku1 and some animal betacoronaviruses, that may play some role in the attachment or release of the particle, or both, at the cell surface. this gene contains sequences similar to the hemagglutinin of influenza c virus, likely evidence of an interfamily recombinational event that occurred many years ago. the next gene encodes the surface glycoprotein that forms the petal-shaped surface projections and is responsible for attachment and the stimulation of neutralizing antibody. this is followed by a small envelope protein, a membrane glycoprotein, and a nucleocapsid protein that is complexed with the rna. there are several other open reading frames whose coding functions are not clear. the strategy of replication of covs is similar to that of other nidoviruses, in that all messenger rnas form a nested set with common polyadenylated 3′ ends, with only the unique portion of the 5′ end being translated. 4 as in other rna viruses, mutations are common in nature, although the mutation rate is much lower, approximately 2 × 10 −6 per site per replication cycle. 38 unlike other rna viruses, covs encode a 3′-5′ exonuclease that has proofreading activities, playing a critical role in maintaining replication fidelity in cell cultures and in animals. 39 covs are also capable of genetic recombination if two viruses infect the same cell at the same time. all covs develop exclusively in the cytoplasm of infected cells (fig. 157-4) . they bud into cytoplasmic vesicles from membranes of the pre-golgi endoplasmic reticulum. these virus-filled vesicles are then extruded by the exocytic secretory pathway. 40 the resultant virus particles have a diameter of 70 to 80 nm on thin-section electron microscopy and 60 to 220 nm on negative staining. they are pleomorphic, with widely spaced, petal-shaped projections 20 nm long (see fig. 157-1) . the cellular receptor for 229e and most other alphacoronaviruses is aminopeptidase n (apn). 41 interestingly, nl63, the other known human alphacoronavirus, uses as its cellular receptor angiotensinconverting enzyme ii (ace2), 42 the same receptor as is used by the sars-cov. 43 mouse hepatitis virus, a betacoronavirus related to strain oc43, uses as its receptor a member of the carcinoembryonic antigen family. 44 hcov-oc43 and bcov, which is closely related to hcov-oc43, bind to 9-o-acetylated neuraminic acid as part of the entry process. 45 the host cell receptor for mers-cov is dipeptidyl peptidase as 50%. there was no mortality in children or young adults younger than the age of 24 years. in response to the global spread and associated severe disease, the world health organization coordinated a rapid and effective control program that included isolation of cases, careful attention to contact, droplet and airborne infection control procedures, quarantine of exposed persons in some settings, and efforts to control spread between countries through travel advisories and travel alerts. presumably as a result of these efforts, global transmission ceased by july 2003. a few subsequent cases of sars were detected, but all were either a result of laboratory spread or individual cases related to presumed contact with civet cats or other intermediate hosts. the last known case occurred in mid-2004. spread of sars to humans is thought to have occurred primarily through droplet or contact transmission, with a possible role for fomites. in most instances, an individual case transmitted to very few others, although several well-documented instances of small-particle airborne transmission occurred, resulting in super-spreading events. 67 spread in hospital settings appeared to be surprisingly efficient, but it could be effectively suppressed with the enforcement of droplet and contact precautions. 68 containment measures were efficacious, in part, because patients were most contagious only after lower respiratory disease developed. 69 the chain of spread was finally broken in the people's republic of china, the last country to experience endemic spread, in june 2003. it now seems almost certain that the human epidemic began with the spread of a closely related bat virus first to palm civets or other animals sold in live wild game markets and then to humans in guangdong province in the people's republic of china, and that the virus adapted itself through mutation and possibly recombination, until it transmitted readily among humans. [70] [71] [72] the virus that spread worldwide came largely from a single infected individual who traveled from occurred at all ages, the elderly and those with underlying conditions (diabetes, renal disease, immunosuppression) have been most often severely or fatally affected. the who and the cdc have published a case definition, as well as surveillance instructions to aid in epidemiologic control of the mers-cov. 62, 63 the putative bat origin of this virus was strengthened by the finding that the virus grew readily in primary bat tissue culture. 64 nevertheless, and while bats sampled in the middle east, africa, and europe were found to carry viruses closely related to the mers-cov, 65 epidemiologic studies suggested there was likely to be at least one intermediate host. serologic and virologic studies indicated that camels in the middle east and africa were frequently infected by viruses very similar to some of those found in human mers cases. 65a acquisition of mers from camels appears likely, although the proportion of camel-acquired cases (versus those acquired from person-to-person contact or through another animal intermediate) is not clear. case clusters indicate that person-to-person hospital spread is more common than spread within families, and that casual-contact spread is unusual. 7a the sars epidemic began in guangdong province in the people's republic of china in mid-november 2002. 66 it came to worldwide attention in march 2003 when cases of severe, acute pneumonia were reported to the world health organization from hong kong, hanoi, and singapore. disease spread in hospitals to health care workers, visitors, and patients, among family members, and, on occasion, in hotels, apartment complexes, markets, and airplanes. worldwide spread was rapid but focal ( fig. 157-5) . the largest numbers of cases were reported from the people's republic of china, hong kong, taiwan, singapore, and toronto, canada. the overall case-fatality rates in these locations ranged from 7% to 17%, but persons with underlying medical conditions and those older than 65 years of age had mortality rates as high involvement in other organ systems, including diarrhea, leukopenia, thrombocytopenia, and, most notably, pan-lymphopenia. 83 virus has been detected in respiratory secretions, blood, stool, and urine specimens and tissue from the lung and kidney. on the basis of pcr testing, virus titer is highest during the second week of illness 84 and can often be detected into the third week of illness and sometimes for as long as several months. 18, 85 pulmonary symptoms may worsen late in the course of the illness, with the development of adult respiratory distress syndrome. 84 there may also be late evidence of liver and kidney involvement. the pulmonary pathology of infection by the sars-cov has been described extensively, 17,86,87 but little has been published about the pathology in other organ systems. [87] [88] [89] the extrapulmonary pathologic changes found most consistently at autopsy are extensive necrosis of the white pulp of the spleen and a generalized small vessel arteritis. 87, 88 in the lung, there is hyaline membrane formation, interstitial infiltration with lymphocytes and mononuclear cells, and desquamation of pneumocytes in the alveolar spaces. giant cells are a constant finding and usually have macrophage markers. in bronchoalveolar lavage, biopsy, and autopsy specimens viral particles have been noted in type i and ii pneumocytes. 90 at this point, little is known about the pathogenesis of mers-cov because the infection was only recently described and no pathological specimens are yet available. it is anticipated that the pathologic changes in the lungs of patients with severe disease will be similar to those observed in patients with sars or other patients with acute respiratory distress syndrome (ards). almost all the antigenically distinct respiratory cov strains that were isolated in the 1960s have been administered to volunteers, and all these produce illness with similar characteristics. 8,91 a summary of these characteristics is given in table 157 -1, in which a comparison is made with colds produced by rhinoviruses in similarly inoculated volunteers. the incubation period of cov colds was longer and their duration somewhat shorter, but the symptoms were similar. asymptomatic infection was sometimes seen and, indeed, has been a feature guangdong province to hong kong and infected a large number of individuals before himself succumbing to the disease. in contrast, the virus that was epidemic in the people's republic of china was more variable. although an etiologic role is not proven, enteric covs (or covlps) have been most frequently associated with gastrointestinal disease in neonates and infants younger than 12 months. particles have been found in the stools of adults with the acquired immunodeficiency syndrome. 73, 74 asymptomatic shedding is common, particularly in tropical climates 75 and in populations living in poor hygienic conditions. 76 the viruses can be detected for prolonged periods 51,53,77 and without any apparent seasonal pattern. 78 community-acquired respiratory covs (hcov-229e, oc43, nl63, hku-7) generally replicate in ciliated and nonciliated (hcov-229e) epithelial cells of the nasopharynx, 79 probably producing both direct cell degeneration 80 and an outpouring of chemokines and interleukins, with a resultant common-cold symptom complex similar to that produced by rhinovirus infection. 81 the incubation period is, on average, 2 days, and the peak of respiratory symptoms, as well as viral shedding, is reached at approximately 3 or 4 days after inoculation. 61 the pattern of virus replication of covs must be at least in part determined by virus-receptor interaction. the two best-defined receptors for the respiratory covs are aminopeptidase n for strain hcov-229e and angiotensin-converting enzyme ii for nl63. the pathogenicity of sars is more complex and involves systemic spread. the route of infection of the sars-cov is probably through the respiratory tract. after an incubation period that is usually 4 to 7 days, but can be as long as 10 to 14 days, the disease begins, starting usually with fever and other systemic (influenza-like) symptoms, with cough and dyspnea developing a few days to a week later. 82 although the lung is the focus of the disease process, there are often signs of levels decreased, suggesting that disease was partly immune mediated. 84 clinical improvement was associated with the onset of a virusspecific antibody response. 119 pediatric disease was, interestingly, significantly less severe than adult disease, although the features were similar. 121 disease during pregnancy was severe, with high mortality in both the mother and fetus. 122 congenital transmission was not described. the nature of the illness associated with enteric cov infection is much less clear. one study found a significant association of gastroenteritis in infants 2 to 12 months of age with the presence of covlps in the stool. 55 another study, confined to infants in a neonatal intensive care unit, found highly significant associations between the presence of covlps in the stool and the presence of water-loss stools, bloody stools, abdominal distention, and bilious gastric aspirates. 53 a further study of symptomatic infants shedding covlps pointed to possible differences between covlp-associated diarrhea and rotavirus diarrhea: although fever and vomiting were of similar incidence, stools were more often occult blood positive (18% in covlp-associated vs. 0% in rotavirus-associated disease), less often watery (66% vs. 92%), and more often mucoid (32% vs. 8%). 77 finally, covs have been associated with at least three outbreaks of necrotizing enterocolitis in newborns, 52, 53, 56 and the best characterized strains 56 were isolated in infants with this illness. surveys seeking hcov rna by pcr using primers that would detect the known community-acquired respiratory hcovs in stool have been quite disappointing. in one study of 878 fecal samples from children with gastrointestinal complaints tested over 2 years, all four hcov species were found, but in all but 4 of 22 hcov-positive cases, either rotavirus or norovirus was also present. 28 in addition, about half of the children in this survey had respiratory and gastrointestinal symptoms. in the same study, 112 asymptomatic children were sampled and 2 were positive. another study sampled 151 symptomatic children, and 2 were found to have hku1 rna. 29 molecular studies using primers that would broadly detect new covs should be considered to resolve questions about the role of covs in human gastrointestinal disease. 123 like many other viruses, covs have been sought as possible etiologic agents in multiple sclerosis. the search has been stimulated by the capacity of jhm, a well-studied strain of mouse hepatitis virus, to produce in mice and rats an immune-mediated chronic demyelinating encephalitis histologically similar to multiple sclerosis. 124 hcov-oc43 125, 126 and hcov-229e 127 have been detected in brain tissue from multiple sclerosis patients using virus isolation, 125 in situ hybridization, immunohistology, 126 and pcr. 127 moreover, t-cell lines established from patients with multiple sclerosis by stimulation with myelin basic protein or hcov-229e were found to be cross-reactive with the opposite antigen, suggesting that molecular mimicry might be a possible pathogenic mechanism for the disease association. 128 an adolescent boy with acute demyelinating encephalitis was found to have hcov-oc43 rna in both the respiratory tract and the cerebrospinal fluid. 129 despite these intriguing reports, compelling evidence is lacking to establish an etiologic or pathogenetic association of covs with cns disease in humans. although some human respiratory covs grow in tissue culture directly from clinical samples and although antigen detection systems have been developed for both hcov-oc43 and hcov-229e, 130, 131 laboratory diagnosis of cov respiratory infections is best accomplished by molecular methods. reverse-transcriptase pcr (rt-pcr) systems have been developed using many different primers and detectors. from a clinical point of view, a single generic test for respiratory covs would be desirable, and such tests have been developed. however, when tested side by side with specific systems, the generic systems have a somewhat lower sensitivity. 95 systems that combine primers and probes specific for several covs have also had considerable success. 132 of both serologic surveys and pcr-based studies of natural infection of infants, children, and adults. 92, 93 more serious respiratory tract illness is probably also caused by all four strains of community-acquired hcov. the evidence for this is not conclusive, but it seems likely that all strains can produce pneumonia and bronchiolitis in infants, 24, 27, 94, 95 otitis and exacerbations of asthma in children and young adults, [96] [97] [98] pneumonia in healthy adults, 99 exacerbations of asthma and chronic bronchitis in adults, 100, 101 both serious bronchitis and pneumonia in the elderly, 102, 103 and pneumonia in the immunocompromised host. 104, 105 hcovs are found in asymptomatic individuals of all ages, and, when accompanied by illness, are also sometimes accompanied by infections with other potential respiratory pathogens. these characteristics (infection without disease, coinfection during disease) are features of many respiratory pathogens, including particularly rhinoviruses, adenoviruses, human metapneumovirus, human bocavirus, and parainfluenza viruses, but also (although less frequently) respiratory syncytial virus and influenza virus. because infections with respiratory hcovs are so common, however, it is possible that they are responsible for a significant portion of these serious lower respiratory tract diseases, even though the basic pathogenicity of hcovs (judging from volunteer studies) is similar to that of rhinoviruses, and clearly less than that of respiratory syncytial virus, influenza viruses, and certain adenovirus types. there is some evidence that hcov-oc43 is more pathogenic in the elderly than hcov-229e 106 and also some evidence that infection with nl63 in children is different from the other respiratory hcovs in that several series have found an excess of children with croup. 26, 27 information about the clinical presentation of patients infected with the mers-cov is limited. it is clear that there is a spectrum of illness with some infections consisting of mild upper respiratory symptoms only, and others characterized by cough and fever with progression to respiratory failure over about a week. 6, 7, 62, 107 renal failure, as well as pericarditis and adult respiratory distress syndrome has been part of the reported clinical picture. the mers-cov host cell receptor, dpp-4, is expressed at high levels in the kidney, 108 raising the possibility that direct infection of this organ contributes to renal disease. a case definition that will lead to further epidemiologic studies has been published by the world health organization. 109 severe acute respiratory syndrome coronavirus the first symptom in most cases of sars was fever, usually accompanied by headache, malaise, or myalgia. this was followed, usually in a few days, but as long as a week later, by a nonproductive cough and, in more severe cases, dyspnea. approximately 25% of patients had diarrhea. interestingly, upper respiratory symptoms such as rhinorrhea and sore throat usually did not occur. 82, 84, [110] [111] [112] [113] the chest radiograph was frequently abnormal, showing scattered air-space opacification, usually in the periphery and lower zones of the lung. 114 spiral computed tomography demonstrated both ground-glass opacification and consolidation, often in a subpleural distribution. [115] [116] [117] lymphopenia was common, 84, 86, 110 with normal or somewhat depressed neutrophils. paradoxically, neutrophilia was associated with poor outcomes. 83 the decrease in lymphocytes in the blood was most marked for cd4 cells but was seen in all t-cell phenotypes, including cd3 and cd8, as well as natural killer cells. creatine kinase was often abnormal, as were lactic dehydrogenase and aspartate aminotransferase. levels of proinflammatory cytokines were elevated at early times during infection in patients with severe clinical disease 118 and decreased in those patients who resolved the infection. 119 approximately 25% of patients developed severe pulmonary disease that progressed to adult respiratory distress syndrome. adult respiratory distress syndrome with sars-cov infection was most likely to develop in patients older than 50 years or with underlying disease such as diabetes, cardiac disease, and chronic hepatitis. 84, 110, 111, 120 the overall mortality rate was between 9% and 12%, with the highest rates in the elderly and adults with underlying liver disease. in some patients, clinical deterioration occurred during the second week of illness, as virus ribavirin. 134 it is now known that ribavirin has little activity against sars-cov in vitro, and there is no evidence that either intervention improved outcomes. 135, 136 lopinavir/ritonavir and intravenous immune globulin were also used in some patients, again without conclusive evidence that they were helpful or harmful. there is anecdotal and at least partially controlled evidence of the benefit of either interferon-α or interferon-β treatment. further, treatment of sars-cov-infected cynomolgus monkeys with pegylated interferon-α resulted in improved outcomes, 137 lending credence to the use of this therapy if sars recurred. treatment of mers-cov infection depends entirely on supportive measures. no antiviral drugs are recommended, although several studies have indicated that mers-cov is more sensitive to interferonα or interferon-β than sars-cov. 138, 139 standard droplet precautions should be used, with aerosol precautions during certain high-risk procedures. 109 rigorous application of hospital infection control procedures, particularly those directed at contact and droplet spread, was shown to have a major beneficial effect on the spread of the sars-cov. 68 the containment of the global sars outbreak is a testament to the power of the cooperation and collaboration engendered by the world health organization to address a major public health threat. similar precautions are recommended for patients with suspected or confirmed mers-cov infections. 109 vaccines for animal covs have been developed and widely used with variable efficacy. in one instance, a vaccine for feline infectious peritonitis appeared to lead to enhanced disease with subsequent natural infection. if sars does return or mers reaches epidemic proportions, an effective vaccine would be extremely helpful in control efforts, and a variety of vaccination strategies, including inactivated, subunit, and live-attenuated vaccines, are being pursued. 135, 140 in addition, hospitals have been advised on improvement of infection control procedures so that in future epidemics of respiratory viruses, they will not be a major source of spread of infection, as occurred in the 2002 epidemic of sars. the mers-cov was originally isolated in vero and llc-mk2 cells, and there are several published methodologies for detection and identification by pcr. 133 current recommendations from who are that definition of a possible case should be immediately reported to national authorities, and clinical, epidemiologic, and microbiologic investigations should be carried out. 63 although sars-cov was grown from respiratory tract specimens in vero e6 and fetal rhesus monkey kidney cells, the more sensitive and rapid rt-pcr assays were most widely used to detect infection. virus was detected by rt-pcr in upper and lower respiratory tract, blood, stool, and urine specimens. early in the illness, specimens were found positive only in approximately one third of patients. 84 use of samples from multiple sources increased the yield. virus was detected most frequently during the second week of illness. 18, 84, 85 antibody tests have been developed using tissue culture-grown virus and indirect immunofluorescence or enzyme-linked immunosorbent assay. immunoglobulin m antibody can be detected in most patients for a limited period of time, and immunoglobulin g antibody appears first approximately 10 days after onset of fever in patients with good outcomes and becomes essentially universal after 4 weeks. 84 laboratory diagnosis of the gastrointestinal covs depends now entirely on electron microscopy of stool specimens and detection of characteristic particles in negatively stained specimens. such testing is best performed in laboratories with extensive previous experience. given the severity of sars, clinicians 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reversetranscription polymerase chain reaction development of a standard treatment protocol for severe acute respiratory syndrome treatment and vaccines for severe acute respiratory syndrome sars: systematic review of treatment effects pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus the spike protein of sars-cova target for vaccine and therapeutic development key: cord-341701-zropd3mo authors: adhikari, subash; nice, edouard c.; deutsch, eric w.; lane, lydie; omenn, gilbert s.; pennington, stephen r.; paik, young-ki; overall, christopher m.; corrales, fernando j.; cristea, ileana m.; van eyk, jennifer e.; uhlén, mathias; lindskog, cecilia; chan, daniel w.; bairoch, amos; waddington, james c.; justice, joshua l.; labaer, joshua; rodriguez, henry; he, fuchu; kostrzewa, markus; ping, peipei; gundry, rebekah l.; stewart, peter; srivastava, sanjeeva; srivastava, sudhir; nogueira, fabio c. s.; domont, gilberto b.; vandenbrouck, yves; lam, maggie p. y.; wennersten, sara; vizcaino, juan antonio; wilkins, marc; schwenk, jochen m.; lundberg, emma; bandeira, nuno; marko-varga, gyorgy; weintraub, susan t.; pineau, charles; kusebauch, ulrike; moritz, robert l.; ahn, seong beom; palmblad, magnus; snyder, michael p.; aebersold, ruedi; baker, mark s. title: a high-stringency blueprint of the human proteome date: 2020-10-16 journal: nat commun doi: 10.1038/s41467-020-19045-9 sha: doc_id: 341701 cord_uid: zropd3mo the human proteome organization (hupo) launched the human proteome project (hpp) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. during the subsequent decade, the hpp established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. on the occasion of the hpp’s tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. this knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases. a. establishing agreed, stringent, reliable standards, b. identifying >1 protein product from each proteincoding gene and c. detecting expression of the remaining missing proteins (see below). 2. to make proteomics an integrated component of multiomics studies to advance life sciences, biomedical sciences and precision medicine. comparisons with the hgp are numerous. both global projects are ambitious cooperative community efforts seeking to identify how genes (hgp) or proteins (hpp) help define the molecular mechanisms underlying health and disease. both groups have implemented exhaustive data sharing and stringent quality control efforts. however, we now know that sequencing human genomes is necessary but not sufficient to understand the complexity of human biology or pathology. knowledge of expressed proteins (including concentration, spatio-temporal localization, activities, protease-processed forms, transport, interactions, splice isoforms, ptms and the many proteoforms derived from the proteome) cannot be predicted by genome sequencing alone. hpp structure and achievements hupo launched the hpp in 2010 at the 9th hupo annual world congress in sydney, with gil omenn as the inaugural chair. the hpp started without long-term funding. financial support over the decade came through individual principal investigator projects, institutional infrastructure/core facilities and philanthropy, without large-scale, long-term, integrated multi-government strategic funding. the hpp grew from several archetypal hupo projects (plasma, liver, brain, cardiovascular, kidney/urine proteomes). at present, the hpp comprises two strategic initiatives-chromosome-centric (c-hpp; 25 teams) and biology/disease-centric (b/ d-hpp; 19 teams)-organized in a strategic matrix underpinned by four resource pillars: antibodies (ab), mass spectrometry (ms), kb and pathology (fig. 1a) . before explaining these elements in more detail, we will describe hpp's criteria underlying the establishment of the high-stringency human proteome. defining the human proteome at high-stringency. the hpp relies upon a coordinated system developed by nextprot and uniprotkb, which attributes five levels of supporting evidence for protein existence (pe) 6 . evidence at pe1 indicates clear experimental evidence for the existence of at least one proteoform, based on credible identification by ms, edman sequencing, xray, nuclear magnetic resonance (nmr) structure of purified natural protein, reliable protein-protein interaction and/or antibody data. pe2 indicates evidence limited to the corresponding transcript (cdna, reverse-transcriptase pcr, northern blotting). pe3 indicates the existence of orthologs in closely related species. pe4 refers to entries based on gene models without evidence at protein, transcript or homology levels. pe5 classifications indicate that coding evidence is doubtful and/or probably corresponds to an incorrect in silico translation of a non-coding element. as pe5 entries are largely non-coding, the hpp preferentially tracks only pe1,2,3,4 protein-coding entries. proteins classified as pe2,3,4 are colloquially referred to as missing proteins 7, 8 . since 2013, nextprot and the hpp (fig. 1b) have issued annual tallies of pe1,2,3,4,5 status (www.nextprot.org/about/protein-existence), which have been reported in annual collaborative metrics manuscripts (ref. 9 and references therein). the latest nextprot hpp reference release (https://www. nextprot.org/about/statistics, data release: 17-01-2020) designates credible pe1 evidence for 90.4% of the human proteome (17, 874 pe1s from the 19,773 pe1,2,3,4 protein entries excluding the dubious 577 pe5 entries). this leaves 1899 (9.6%) pe2,3,4 missing human proteome entries to be identified at highstringency. here, the term high-stringency refers to rigorous hpp standards for post-acquisition processing and any protein inference made from raw ms peptide spectral data 10 . this term avoids confusion with the pre-existing term 'high-accuracy' box 1 | hpp decadal achievements • generated a framework, plan and governance structure for community-based mapping of the human proteome. • confirmed nextprot as the hpp reference knowledgebase and supported creation and use of proteomxchange (px) to register and make proteomics raw mass spectrometry (ms) and metadata available and reusable under fair (findable, accessible, interoperable and reusable) principles. • engaged nextprot, peptideatlas, pride and massive as partners in the generation of annual nextprot hpp release and a high-stringency hpp knowledgebase (kb). • encouraged community support of high-stringency protein inference and proteomic data analysis. • built ms data interpretation guidelines that promoted the application of standardized analysis of community human ms and proteomic data to progressively complete the human proteome parts list. • aligned with the human protein atlasʼ (hpa) cell and tissue spatiotemporal maps in health/disease and supported community efforts to raise awareness of antibody specificity and quality assurance issues. • partnered with srmatlas to develop quantitative targeted proteomics assays for the analysis of key proteins and hallmark pathways/networks. proposed and built global collaborative initiatives to investigate the biology of human health and disease at a proteomic-wide scale. • raised the profile and visibility of proteomics, as an essential component of life sciences and biomedical research by promoting the development of instrumentation and methods for proteoform analysis, as well as activity/function that cannot be addressed by genomics. • initiated a programme to determine the biological function for uncharacterized pe1 proteins (see below) that currently lack functional annotation. • established a hupo early career researcher network to engage, mentor and highlight research from young scientists/clinicians, while actively promoting gender and regional balance. • establish a community initiative to systematically map all human proteoforms. • establish optimized workflows for human proteome detection, quantification and functional characterization, including low abundance and/or temporo-spatially restricted proteins. • continue to support and promote the provision of technical standards, metrics and stringent guidelines for confident protein identification and quantification. • create a comprehensive, accurate, publicly-accessible, reference human proteome knowledgebase, reusable under fair principles. • maintain education and training programmes in all aspects of proteomics including proteomic data analysis for early career researchers and clinical scientists. • be a focal point for life sciences researchers, pathologists, clinicians and industry communities seeking to translate and leverage proteomic and proteogenomic data to improve human health through: (i) greater understanding of the molecular mechanisms of common and rare diseases, (ii) identification of pathophysiological changes to generate disease and wellness diagnostic biomarkers, and (iii) development of new effective and safe personalized therapeutics. frequently used in ms parlance, which relates to the generation of high (mass)-accuracy spectra from modern instruments. the use of high-stringency influences the quality of all protein inferences derived from any raw ms data. the hpp routinely applies high-stringency protein inference analytics 10 , generated through the trans-proteomic pipeline 11 . claims for detection of new pe1s and/or detection of coding elements not previously in nextprot, should meet minimum evidence thresholds. the current hpp guidelines 10 require at least two uniquely mapping peptides (using nextprot's peptide uniqueness checker tool 12 ), which are at least nine amino acid residues long. peptides must be non-nested (i.e., one not fully contained within another) but may overlap partially so coverage exceeds >18 residues. the hpp also requires full declaration of false discovery rate (fdr) calculation procedures at peptide and protein levels, with a maximum allowable protein-level fdr of 1%. within hpp missing protein publications, further validation is required through synthetic peptide spectra matching 13 . going forward, the hpp also requests association of universal spectrum identifiers with every ms spectrum 10 . we emphasize that ms-based data from high-accuracy 14,15 ms instruments combined with subsequent high-stringency protein inference analysis provide definitive best-practice confirmation of protein identification and abundance. to ensure high-quality analyses, increasingly stringent criteria have been applied 10 spectral data findable, accessible, interoperable and reusable (fair) 17 . many previous studies use high (mass)-accuracy instruments with subsequent protein inference identifications undertaken at lower default settings, such as accepting single peptides or those only seven amino acids in length and/or not conforming to more rigid nextprot proteotypic analysis 18, 19 . these analyses can result in spurious identification of many more false positives 20 , with lower-quality single non-proteotypic spectra data colloquially referred to as one-hit wonders, better explained by sequence variation in other highly observed proteins 21 . chromosome-centric (c)-hpp. the c-hpp (https://www.hupo. org/c-hpp) aims to annotate all genome-encoded proteins 7, 8 in an unbiased and high-stringency manner. it explores proteins that have not previously been confidently observed by ms or other analytical methods 7, 8, 22 . international c-hpp teams are organized according to chromosomes (chrs), namely chrs 1-22, chr x, chr y and mitochondrial (mt) genome teams. from 2017, the c-hpp expanded its mission to include functional characterization of the 1899 pe2,3,4 proteins that have not been confidently observed and 1254 pe1s that have no nextprot curated function (uncharacterized pe1s or upe1s), cumulatively referred to as the dark proteome 23, 24 . biology/disease-centric (b/d)-hpp. the b/d-hpp (https:// www.hupo.org/b/d-hpp) measures and interprets human proteome data under a range of physiological and pathological conditions. it focuses on the following: (i) elucidating the hallmark protein drivers of biology/disease and (ii) promoting development of new proteomics analytical tools such as ab-based approaches and targeted selected/multiple/parallel reaction monitoring (srm/mrm/prm) assays. as an example, the initial hupo liver proteome project grew into a b/d-hpp team focussing on liver expression profiles, ptms, tissue expression, subcellular localization, interactions, physiology and pathologies 25 . the chinese cn-hpp have characterized four liver cell types, emphasizing benefits of acquiring cell-type specific maps to understand underlying biology/pathology 26 . in addition, they mapped landscapes of early hepatocellular and lung carcinoma, generating cancer subtype alterations where proteomic signatures identified poor prognosis patients and/or those benefiting from targeted therapy 27, 28 . in other studies, they analysed microdissected cell types with gross anatomical resolution using ms 26, 29 , revealing circadian cycles and spatio-temporal proteome expression in the liver, brain, heart and stomach 30 , providing resources to better understand organ biochemistry, physiology and pathology. significant discoveries continue to be made from all b/d-hpp teams across personalized cancer immunotherapy and therapeutic modalities (e.g., lymphoma 31 , ovarian 32 , liver 27 and lung 28 cancers), with ptms orchestrating many outcomes including response to therapy [33] [34] [35] . b/d-hpp resources include the human srmatlas 36 , a unique compendium of high-resolution spectra and multiplexed srm/ mrm/prm assays developed from 166,174 synthetic proteotypic peptides. this assay library enables targeted identification and quantification of a theoretical maximum of 99.7% of the human proteome 36 , provided proteins are expressed spatiotemporally at concentrations amenable to ms detection. for example, srmatlas supported the c-hpp chr x team's confident identification of missing proteins 37 . hpp resource pillars. the b/d-hpp and c-hpp are supported by four hpp resource pillars that ensure effective data generation, integration and implementation, including the establishment of metrics and guidelines, enhancement of technology platforms, reagent development and optimal use of existing and emerging data streams (fig. 1a) . the hpp ms resource pillar informs the community about ms technology/workflow advances, appropriate high-stringency standards and liaises with industry regarding instrument development, all leading to improved depth and accuracy of proteome identification, quantification and modification. these include methods like matrix-assisted laser desorption ionization time of flight (maldi-tof)-ms, electrospray-ms, bottom-up (shotgun) ms, data-dependent acquisition ms, data-independent acquisition (dia) ms, targeted srm/mrm/prm, top-down ms, crosslinking ms, ptm analysis, n-and c-termini measurement, ms data computational analysis and interactomics. the ms resource pillar previously undertook a swath/dia-ms reproducibility study 38 and are currently coordinating a phosphopeptide challenge involving >20 participating labs with partners synpeptide shanghai and resyn biosciences south africa, who have provided a human phosphopeptide standard set with unphosphorylated counterparts, as peptide mixtures spiked into a yeast tryptic digest background. this will result in a better understanding of phosphopeptide enrichment, ms data analytics and informatics tools. the hpp ab resource pillar, ostensibly led by the human protein atlas (hpa; www.proteinatlas.org), was initiated in 2003 and uses ab-based strategies to analyse spatio-temporal aspects of the proteome 39 . linking the identification of proteins with 'realtime' localization at tissue, cell and subcellular levels supports a more comprehensive understanding of biology, health and disease. this requires information at resolution not currently available by ms (see single-cell section below). approaches for spatio-temporal proteomics include single-cell in situ ms, fractionated cell lysates, proximity labelling or imaging-based proteomics 40, 41 . imaging-based proteomics has a clear advantage, namely analysing proteins in their native location at single-cell resolution. to this end, the hpa has developed industrial scale epitope-directed abs for community use. hpa also integrates multi-omics data. it contains extensive transcriptome data and nextprot pe assignments, and contributes to the open-access catalogue antibodypedia, containing >4 m abs (www.antibodypedia.org) 42 against >19,000 targets that assist the community to select application-appropriate abs. at hpp launch, the hpa had detected >50% of the proteincoding genome 43 . currently,~87% of the proteome is targeted by >1 hpa ab, detected though an encyclopaedia of >10 m annotated high-resolution digital images, partitioned into a number of sub-atlases that are interconnected [44] [45] [46] [47] [48] . these currently comprise the; tissue atlas (protein distribution across all major tissues), cell atlas (subcellular localization and heterogeneity in single cells), pathology atlas (correlations between gene expression and patient survival in major human cancer types), blood atlas (protein profiles across major immune cells and blood levels), brain atlas (protein distribution in the brain), and the metabolic atlas (various tissue metabolic enzymes localizations). over the decade, hpa's open-access database [44] [45] [46] [47] [48] has become one of the world's most visited biological resources (>3.6 m visits in toto annually). the hpa also plays an emerging role in establishing guidelines around the appropriate use of abs and ensuring immunoassay validation [49] [50] [51] . it recently spent considerable effort validating the selectivity of their abs, including championing efforts of the international working group for ab validation proposing many new approaches implemented across >10,000 abs 42 . in addition, hpa's massive collection of images has supported a multitude of publications and become a citizen science resource for developing ai classification learning models 52, 53 . the hpa is sustained perspective nature communications | https://doi.org/10.1038/s41467-020-19045-9 through community contribution to elixir 54 , that allows scientists from academia and industry to explore spatio-temporal aspects of the human proteome [55] [56] [57] . since 2018, the hpp pathology resource pillar has coordinated identification of areas of unmet clinical need, develops fit-forpurpose clinical assay guidelines/standards, promotes best-practice awareness, coordinates access to quality clinical samples/metadata and liaises with pathology organizations, diagnostic companies and regulatory agencies to promote professional application of proteomics in pathology. the hpp kb resource pillar captures, collects, collates, analyses and re-distributes all human proteome data. as cohesive knowledge transfer plays such a crucial role in big data science, including the hpp, the kb pillar's activities are addressed in the expanded section that follows. the human proteome in the nextprot hpp reference kb assembly and curation of nextprot. prior to the hpp, hupo established a protein standards initiative (psi; www.hupo.org/ proteomics-standards-initiative), emphasizing from the outset their priority for defining high-quality community standards, minimal requirements for experimental information 58 and highstringency data metrics. psi continues to work cooperatively with the hpp kb to inform hpp initiatives, pillars and teams. in 2013, nextprot 59,60 was officially designated as the hpp reference kb 61 . annually, a nextprot release is designated as the 'hpp release' and this serves as the basis for subsequent hpp high-stringency analyses, planning and reporting progress 10 . it receives and curates data from uniprotkb/swissprot 62 , adding ms evidence from peptideatlas 63 and since 2019 from mas-sive 64 . nextprot also curates ab-based, genome, transcriptome and other biological data to create an assembled snapshot of the human proteome 6, 65 . peptideatlas (http://www.peptideatlas.org) uses sequence search engines comet 66 , x!tandem 67 and spectrast 68 to reprocess publicly uploaded ms/ms data deposited through proteomexchange (px). data are aggregated using rigorous criteria including peptide spectral matching with fdr~0.0009% in the latest peptideatlas build to achieve ≤1% fdr at the protein level. massive searches public datasets using the ms-gf+ search engine 69 , also with strict criteria 70 to enforce global <1% fdr at the protein level for single-peptide identifications and stricter <0.01% fdr for proteins identified by >2 peptides. peptideatlas and massive peptide lists are integrated into current nextprot builds. nextprot then cross-references all peptides to protein entries and validates pe levels, requiring at least two msidentified uniquely mapping 9-mer non-nested peptides coming from either peptideatlas or massive. nextprot builds on uniprotkb/swissprot pe1 entries that include ms, partial or complete edman sequencing, x-ray or nmr structure, reliable protein-protein interaction data or ab detection by considering additional peptideatlas/massive data that meets minimum peptide uniqueness, number, length, nestedness and other requirements to upgrade entries to pe1. noticeably, nextprot is becoming more reliant on ms than non-ms data (e.g., 1860 non-ms data pe1s in 2016 down to 950 in 2020). to ensure only high-quality ab data are used, in 2018 nextprot/hpp ab pillar revised criteria to upgrade entries to pe1, including specificity and other rigorous criteria suggested by the international working group for ab validation 50 . as an example, nextprot recently analysed 41 ab-based publications and upgraded three pe2,3,4 entries to pe1. discussions regarding data provenance delivered by non-ms data for pe1 assignment actively continues in the hpp 71 . expansion of the high-stringency proteome. the baseline number of protein-coding genes in the reference proteome given by nextprot is managed by uniprotkb/swissprot. every proteincoding gene is assigned a protein entry (inclusive of all proteoforms), with chromosome location and other data organized under these entries. the number of protein-coding genes originally estimated by the hgp dropped from >100,000 72 to a relatively stable~19,700 at hgp draft release, a number that is close to the current nextprot's 2020 release of 19,773 proteincoding genes ( supplementary fig. 1) 60 . confident detection of the human proteome has consistently risen from 69.8% nextprot pe1 entries in 2011 to 90.4% in 2020 (fig. 2a) . however, progress has recently slowed (fig. 2a) , suggesting that it may be difficult to confidently identify all 1899 missing proteins. parenthetically, the hgp celebrated a provisional 90% completion ten years after its launch 5 and annotation of the human genome still remains incomplete or uncertain, especially in regions of repeat sequences and z-dna inserts. in each of the seven annual hpp metrics papers published to date ( 9 and refs therein), pe1,2,3,4,5 entries have been added, deleted, renamed, merged and/or de-merged, indicating ongoing fine-tuning of the hpp reference proteome. the sankey flow diagram 73 (fig. 2b) illustrates that significant fluidity has occurred across all pe classifications since 2011. the largest shifts occur with increases in pe1s (13,603 up to 17,874), followed by decreases in pe2s (5,587 down to 1,596), despite increasingly stringent hpp ms data guidelines adopted in 2015 (v2.1) and 2019 (v3.0). linear regression of pe1 increases against pe2 decreases results in a strong (r 2 = 97%) inverse correlation, suggesting new pe1s come from pe2s where mrna expression has been previously observed. extrapolation of this pe1 discovery curve suggests that 95% pe1s may be reached sometime between 2024 and 2027. minor conversions occurred between other pe categories, with downgrading of pe1 or pe2s and upgrading of pe4s, and even a few pe5s, to pe1s. although a plethora of studies show low linear correlations (40-60%) between mrna level and protein abundance 74,75 , our binary data (fig. 2 ) supports the contention that once a nextprot entry has mrna expression verified, those pe2s are amenable to upgrade to pe1, whereas pe3s, pe4s and particularly pe5s are more resistant to pe1 upgrade. missing protein analyses. nextprot protein descriptors and associated data can be used to analyse protein groups/families according to their chr location or pe classification. missing protein (pe2,3,4) analysis indicates that some groups/families have been upgraded to pe1 more successfully than others (fig. 3a) . for example, between 2011 and 2020, 372 zinc (zn) finger proteins, 171 transmembrane proteins, 93 carbohydrate metabolism proteins, 90 testes-, sperm-, prostate-associated proteins, 78 coiled-coil domain-containing proteins and 58 homeobox proteins have been upgraded from pe2,3,4 to pe1. these represent the six most prominent protein groups upgraded to pe1 over the decade (fig. 3a, green) . in contrast, two g protein-coupled receptor (gpcr) chemosensory families prove particularly resistant to pe1 upgrade: many olfactory receptors (ors) are still missing in 2020 76 (417 of the 2011 pe2,3,4 entries, including putative and uncharacterized ones) and 17 of 20 taste receptors remain pe2,3,4 s (fig. 3a, magenta) . in addition, some groups with a large number of pe1-upgraded protein entries still contain many pe2,3,4 entries (e.g., 85 non-gpcr transmembrane, 69 zn finger and 33 keratin-associated proteins remain pe2,3,4 s (fig. 3a) . when chr distribution of the most resistant (ors) or most discovered (zn-finger proteins) groups were plotted (fig. 3b) , difficult-to-find ors mapped mostly to chr 11 (~55%) and zn-finger proteins mapped mostly to chr 19 (~55%). our data demonstrate that 46% of all chr 19 proteins elevated to pe1 were zn-finger family members, illustrating this protein family has been highly amenable to confident ms detection over the decade. therefore, it was not surprising that most zn-finger protein members were coded on chr 19 (i.e., 255/698 total zn-finger genes), which has the highest chr pe1 reclassification rate over the decade (fig. 3b) . these data suggest that gene family duplication on particular chrs explains why some families are resistant or sensitive to pe1 reclassification. in agreement, pe1 discovery has occurred productively, but not uniformly, across all chromosomes (fig. 4) . missing protein decadal upgrade statistics range from 16% for chr y up to 29% for chr 1, with raw data representing 425 protein entries for chr 1 down to only 5 for chr y. a higher percentage of chr 19 proteins (29%) ascended to pe1 compared to those on chrs 11, 14, 21 or y (<17%). the hpa chromosome viewer (www.proteinatlas.org/humanproteome/proteinevidence) illustrates many recently-evolved protein family members are present on chr y, many ors on chr 11 and many keratinassociated proteins on chr 21. proteins on these three chrs have proven relatively resistant to pe1 reassignment. notably, there was just a single mt missing protein in 2011 and over the decade all mt protein-coding genes have now been identified. pe1 upgrade success by the 25 c-hpp teams could be predetermined by the presence of resistant or more easily-identified missing protein families on particular chrs. however, the current hpp kb processing pipeline utilizing peptideatlas, massive and nextprot (fig. 1b) makes it impossible to isolate decadal pe1 contributions from any particular c-hpp team as opposed to the overall community. although the hpp explores capturing full data provenance (i.e., from quantification/identification back to original data source) for fair data practices 17 , we can only historically estimate pe1s emanating from community deposition. in silico analysis reveals only 22 human proteins cannot produce the characteristic >2 high-stringency proteotypic peptides of the required length after tryptic digestion 36 . however, many missing proteins may be present at levels below detection limits or in under-studied cell types/tissues, expressed under particular conditions (e.g., stress/infection) or only found in developmental stages (e.g., embryo/fetus) 77 . equally, difficult-tosolubilize, hydrophobic multi-transmembrane domain membrane proteins may only generate short tryptic peptides that do not meet high-stringency guidelines or are indistinguishable from other family member sequences 76 . furthermore, transmembrane protein regions cut at single sites are unlikely to release embedded hydrophobic membrane-anchored protein strands 76 . we anticipate that finding the 9.6% remaining pe2,3,4 missing proteins will require exceptional future effort, including careful sampling of rare cells/tissues 78 combined with better sample fractionation and improved detection limits. low abundance proteins might be enriched using abs prior to ms. to this end, the hpa has developed abs against proteotypic sequences in many missing proteins 79 . several other labs are working on improved protocols for insoluble keratin-associated cross-linked missing proteins, non-tryptic or chemical digestion strategies to increase proteotypic peptide productivity 78,80 , higher efficiency search engines 81 , and compendia of missing protein biological evidence (e.g., missingproteinpedia) 71 . future hpp projects anticipate a shift to detection of biologically functional proteoforms 82 , noting their numbers are far larger and more difficult to measure 83 because of heterogeneous nuclear rna splicing, many ptms and detection of peptides with single amino acid variants (saavs). considerable ptm and splicing isoform data are already available through nextprot, including 190,938 ptm sites and 9,193,365 saavs 84 . community impact on the human proteome one barometer of community engagement in the hpp is the magnitude of investigator-submitted raw ms data that have been re-analysed. many journals require raw data submission and hpp action was a significant factor in journals adopting requirements aligned to hpp data deposition guidelines. raw data deposition occurs through px, which registers and standardizes capture/dissemination of public ms data from partner repositories, including founders pride 62 and peptideatlas and recent members mas-sive 64 , jpost 85 , iprox 86 and panorama public 87 . as of 2020, a total of 4634 human ms datasets have been received. each px dataset is branded with a unique pxd identifier with depositors, publications and voluntary metadata noted 88, 89 . illustrating the magnitude of this community data,~470 tbs of data have come from 5658 human datasets (~47% of 1 petabyte pride volume), with only 358 (6.3%) of these specifically tagged by depositors as from the hpp. the hpp encourages raw human ms data/metadata submission (including association to hpp) through px, and that journals request that pxd identifiers be published in accordance with fair principles 17 as discussed above. to provide an additional measure of global scientific impact, hupo commissioned the website construction of the human proteome reference library (hprl; https://hupo.org/hpp-hprl/), where all hpp-associated pubmed searches are hyperlinked and can be accessed and re-run routinely by the community. these hyperlinked searches automatically produce the latest pubmed outputs in a manner where all pubmed filtering, ranking and timeline tools can be applied subsequently by the hprl can also be used to measure impact (fig. 5) , where pubmed identifiers (pmids) from 2010 to 2019 references can be captured using web apis or software, e.g., ncbi e-utilities like https://www.ncbi.nlm.nih.gov/books/nbk25501 or the easy-pubmed r interface (www.rdrr.io/cran/easypubmed) that enable extraction and aggregation of pubmed bibliometric records. paralleling the low percentage of px datasets tagged as emanating from the hpp, hprl data show only~1000 pubmed title and abstract searches that specifically and cumulatively mention either the terms hpp, c-hpp and/or b/d-hpp (or their unabbreviated counterparts) out of a total of more than 50,000 'human and proteomics' title/abstract search outcomes over the decade. in contrast to this and more reassuringly, hprl searches of the community's biology/ or disease studies covered by nine of the b/d-hpp teams generated >5000 publications each since 2010 (fig. 5b) , with 'cancer proteomics' topping rankings at around 20,000 publications. although this may seem surprising, a pubmed search of 'human genome project' produced a similarly modest number of~1900 hits during the hgp's first decade. to visualize outputs from various hpp teams and the proteomics community in general, we have employed vosviewer 91 to construct and visualize bibliometric networks from collective pubmed searches (example shown in fig. 5c ). this particular analysis revealed a remarkable number of highly-interconnected relationships that have evolved between hpp teams, pillars and initiatives-providing evidence for the impressive level of established international collaboration developed over the decade. a key aspect of biomedical research lies in translating discovery into clinical use. protein assays remain a cornerstone of diagnostics. although individual proteins can be measured diagnostically with high precision (i.e., sensitivity and specificity), some assays suffer low specificity due to cross-reactivity with interfering substances including autoantibodies (e.g., thyroglobulin immunoassays). modern srm/mrm/prm assays allow multiple proteins to be measured simultaneously, accurately, sensitively and with high specificity. in addition, the use of liquid chromatography ms with immunocapture assays has been reported to eliminate interferences 92 . moreover, as most diseases are heterogeneous and multigenic, it is likely that multiplexed proteomic or multi-omics panels will achieve higher accuracy (e.g., optimized biomarkers for ovarian malignancy with adnexal masses 93 ). the hpp assists in the development of proteomic educational programmes with pathology societies to train the pathology community on the potential impact of proteomic technologies. below, in recognition of the impact human proteomics can and is having in precision medicine, we highlight examples demonstrating the role of the hpp and proteomics in tackling contemporary medical grand challenges. cancer precision medicine. as mentioned above, pubmed extracts~20,000 published human cancer proteome studies since 2011 (fig. 5b) . although genomics can routinely determine highrisk, predisposition and aspects related to tumour burden and recurrence, effective targeted cancer treatment is still not available for all cancers. for example, systematic genome-wide studies like the pan-cancer analysis integrated analyses of >2600 whole genomes from 38 tumour types with matching normal tissues, uncovered many cancer-associated genes 94 , chromosome rearrangements, some unknown drivers but few new therapeutic targets. this is mainly because mutations do not automatically cause predicted changes in the proteome, making it difficult to establish which changes are crucial biochemical drivers from those that are not. integrating genomic and proteomic data (i.e., proteogenomics) has the potential to provide insights into causes and mechanisms underlying diseases, including the hallmarks of cancer biology 95 . this can facilitate the implementation of effective therapeutic intervention. the value of a proteogenomic analysis of functional consequences of cancer somatic mutations has assisted in narrowing down candidate driver genes within large deletions and amplified regions 96 . reviewing the underlying causes of breast cancer also demonstrates that coupling genomic/transcriptomic data with proteomic/phosphoproteomic analysis was more insightful than any individual approach. of note, melanoma tumour genomic braf driver mutations match corresponding protein sequences 97 , illustrating that proteomic landscapes add value to genomic data, when considered with patient tumour histopathology and clinical metadata 98 . proteomics substantially benefits a comprehensive understanding of precision medicine. to illustrate this, nci's clinical proteomic tumor analysis consortium (cptac; proteomics. cancer.gov) in collaboration with the b/d-hpp cancer team, established guidelines, data sharing, and standards in analytical and computational workflows to ensure rigour in designing and performing research [99] [100] [101] [102] [103] . cptac applied these standards and workflows to tumours previously genomically characterized by the cancer genome atlas. in doing so, cptac pioneered the integration of proteomics with genomics (i.e., proteogenomics) to produce a more unified and comprehensive understanding of cancer biology and implemented its transition into cancer clinical research studies 96, 104, 105 . largely due to these efforts, nci hosts open-access repositories of unified proteogenomics datasets, assays and reagents, including the proteomic data commons (pdc.cancer.gov), a fit-for-purpose targeted assay site (assays. cancer.gov) 102, 106 and an ab portal (antibodies.cancer.gov) 107 . these activities empowered the recent creation of the international proteogenome consortium (icpc; icpc.cancer.gov) 108 . collectively, cptac and icpc collaborators have comprehensively characterized 13 cancer types at the proteogenomics level, with all datasets publicly accessible [109] [110] [111] [112] [113] [114] [115] [116] [117] . cardiovascular diseases. cardiovascular disease (cvd) research is challenged by daunting structural heterogeneity and molecular complexity. for example, cardiac circuitry function/dysfunction cannot be reduced to differentially expressed single genes. on the other hand, proteogenomics allows assessment of interactions, pathways and networks and informs diagnosis and therapy of multifactorial cvds. over the decade, cvd proteomics has broadened from identifying single canonical proteins to mapping proteoforms derived from combinations of alternative splicing, cleavage, and ptms 33, 83 . now, identification of proteoforms (e.g., genetic variations, alternatively spliced products, phosphorylation 118 , glycosylation 119 , oxidative 120 and other ptms 121 ) allow cvd sub-classification. in parallel, the heart is uniquely sensitive to alternative splicing (frequently altered in congenital heart diseases), explaining the b/d-hpp's interest in developing assays for splice isoform-specific changes in cardiomyocyte development and maturation 122, 123 . many technological developments have been prominent, including phospho-ptm analysis to identify pde5a targets in heart failure therapy 118 and proximity labelling to assess protein-protein interactions involved in β-adrenergic signalling of contractility in cardiac fight-or-flight responses 124 and evaluation of regenerative stem cell therapy efficacy in postinfarct hearts 125 . proteomic studies have also addressed the kilometres of vascular beds and extracellular matrix responsible for transporting blood, giving valuable insights into the molecular anatomy of aneurysms and atherosclerosis 126, 127 . targeted ms methods have been developed, again especially where interferences obfuscate immunoassays 128 or where additional biological context is required 129 . likewise, volumetric absorptive microsampling vams blood collection devices (e.g., mitra devices or dried blood spots) allow patients to mail samples from home to analytical labs to undertake srm/mrm/prm assays quantifying cvd risk-associated apolipoproteins 130 or other markers 131 . in summary, consumer-based cvd proteomics-based precision medicine testing services are now coming of age. microorganism detection. proteomics and the hpp have made fundamental contributions to understanding pathogenic infection, providing diagnostics and developing therapies 132 . the b/d-hpp infectious diseases team promotes international proteomics collaborations investigating viral, bacterial, fungal and parasitic diseases. maldi-time-of-flight (tof)-ms, once considered revolutionary, is now established as a routine tool in clinical microbiology 133 . classical phenotypic tests identify unknown and potentially pathogenic microorganisms, but may require incubation for several days, with misidentification resulting in adverse treatment consequences. maldi-tof-ms provides significantly shortened analyses (now minutes) with improved accuracy on single colony or bacterial pellets for difficult-todetect microorganisms, using automated spectra acquisition and extensive reference spectra databases 134 . minor spectral differences enable typing below species levels 135 , allowing subspecies identification through epidemiological analyses. bacteria and yeasts (most clinical identifications), mycobacteria 136 and moulds 137 can now be identified accurately and rapidly. further ms clinical diagnostic applications are being investigated (e.g., antibiotic resistance (art) and susceptibility testing (ast) based on hydrolytic β-lactamase activity 138 ), with kits under development commercially through star-carba, star-cepha and bruker daltonics. sars-cov-2 virology. the recent severe acute respiratory syndrome coronavirus 2 (sars-cov-2) outbreak that causes covid-19 disease represents a major threat to human health and our economies [139] [140] [141] . the pandemic underscores our need to understand virus pathobiology, identify host-pathogen interactions that support replication, find biomarkers correlative with clinical outcome and expand surveillance. many omics studies followed the 2003 sars-cov-1 and related mers and ibv coronavirus outbreaks [142] [143] [144] [145] [146] . the cell surface receptor for the cov-1 and cov-2 surface spike protein has been identified by affinity-ms to be angiotensin-converting enzyme 2 (ace2) 147 , which in a recent large-scale study based on antibodybased proteomics was shown to be mainly localized to the digestive system, kidney, heart, testis, placenta, eye and upper respiratory epithelia 148 . virus binding leads to proteolysis by the transmembrane serine protease tmprss2 expressed in airway epithelia 149 , thus a clinically approved tmprss2 inhibitor (camostat mesylate) is being investigated to block infections 150, 151 . furthermore, proteomics has characterized the infectious cov-1 viral particle 143 , temporal changes in host cells during infection 142 and virusinduced endoplasmic reticulum membrane remodelling into double-membrane vesicles 152,153 that house viral replication compartments 146 . proximity labelling revealed >500 host and 14 viral protein associations with the viral replicase nps2 146 , highlighting vesicular trafficking, autophagy and splicing proteins in coronavirus replication, which if shown to also be the case for cov-2, indicate potential drug targets. building on this knowledge of coronavirus infection, recent proteomics studies have focused on sars-cov-2, uncovering additional potential therapeutic targets 154, 155 . ms and array-based proteomics serology has screened for potential biomarkers and abs against infection 156, 157 . clinical isolate infection models have been developed using caco-2 cells 154 with temporal proteome changes identified during infection using multiplexed ms by combining metabolic labelling with tandem mass tagging methods. consistently, host vesicular trafficking, translation, rna splicing, nucleotide synthesis and glycolysis pathway proteins were upregulated following infection 143, 144 and targeting these processes with inhibitors revealed potential therapeutic targets 158, 159 . additionally, affinity-ms interactome studies examined 26 of 29 total sars-cov-2 proteins expressed within hek293t human cells 155 , suggesting 69 existing drugs merit further investigation. moreover, a recent phosphoproteome analysis pointed to the regulation of viral proteins through ptms 160 . the sars-cov-2 pandemic highlights the need for applying proteomic approaches to the development of serologic testing and preclinical and computational model systems to evaluate patient responses to infection 156 . serological biomarkers of asymptomatic/ symptomatic infection, disease severity, risk of re-infection and/or vaccine efficacy are being characterized 157, 161 . in addition to this accumulating omics knowledge, many aspects of sars-cov-2 pathobiology await further exploration including development of additional methods for clinical virus detection, identification of infection stage, and an in-depth understanding of functional spatiotemporal virus-host protein interactions and organelle remodelling [162] [163] [164] . for example, recent studies that utilize targeted ms for sars-cov-2 protein detection and proteomic characterization of serological immune responses 161,165-167 from patient samples may bolster pcr screening for the assessment of disease severity 168 . other proteomics approaches can also be deployed to further expand the understanding of sars-cov-2 biology. among these is the application of tails n-terminomics that promises to identify many sars-cov-2 protease substrates and those cellular pathways inactivated by viral proteolysis, as reported for other viruses 169 . in summary, proteomics plays increasingly important roles in understanding viral outbreak biology, accurate diagnosis and effective treatment and is positioned to continue to co-ordinate and drive international collaborations towards these goals. western and eastern cultures urge us to know thyself and thy enemy. these axioms resonate with precision medicine where future benefits arise from a detailed omics understanding of the hallmarks of health and disease. here, we reviewed the construction of a community-endorsed, high-stringency blueprint of the human proteome. the decadal nextprot hpp pe metrics shown here demonstrate the community's progressive success in pe1 identification from 13,588 in 2011 to 17,874 pe1s in 2020, marking the completion of >90% of the human proteome parts list (see strategic objective 1 above). we also present specific examples demonstrating proteomics will be an integrated component (with genomics and other omics) in future biomedical science discovery and precision medicine. hupo recommits to its original hpp strategic aims as well as the fair data principles 17 , while anticipating the following future priorities: 1. unearth credible proteomics data for the majority of current pe2 proteins: since most pe1 identifications come from former pe2s, our future strategy is to find credible data for 95-99% of current pe2s, allowing reclassification of these to pe1. pe3 also remain promising, as homologous proteins are detected in related species. 2. unravel currently unknown proteome functionalities: fill functional annotation gaps for all protein-coding elements, with a priority on credibly identified proteins 170 and develop, expand and apply function prediction tools 24, 171, 172 . 3. expand the hpp kb: maintain a sustainable knowledgetransfer hpp kb infrastructure with funding that captures/ displays high-stringency partner omics data streams and publication data (hprl) to researchers and the public in an accessible and compelling manner. 4. develop community-approved multi-omics technology guidelines: explore dia-ms, ab and aptamer-based multiplexed assays, top-down ms and other not yet invented multi-omics technologies. 5. champion collaborative multi-omics health/disease approaches: in addition to extensions in hpp kb partnerships, the hpp will collaborate with international and regional initiatives (including human variome, human cell atlas, hpop/ipop, motrpac, hubmap, cancer moonshot, htan, edrn, cptac, icpc and upcoming international initiatives) around multi-omics approaches to human disease processes, biomarker discovery and therapeutic development. 6. apply and improve single-cell proteomic technology: develop technologies that allow detection and quantification of proteomes in single cells to further understand cellular/tissue heterogeneity, differentiation, diseases and the intrinsic biological noise in health and disease 173, 174 . many single-cell proteomics advances will be explored to analyse cellular heterogeneity [175] [176] [177] [178] . sensitivity increases (analogous to pcr) and trade-offs maximizing coverage per cell with throughput/accuracy will be studied 179 . 7. champion dual ab-capture with ms identification: enhance the accuracy of ab-based epitope/antigen detection by confirmation using high-accuracy, high-stringency ms identifications across real-life spatio-temporal biological settings. collectively, there is recognition within the hpp that future stringent co-registration of ms with ab data are required to achieve the ultimate spatio-temporal human proteome expression atlas 180 . 8. exploit massively parallel ms 174 to increase throughput: for rapid, sensitive and higher-throughput ms analysis. 9. capitalize on human disease biobanks: the hpp will work with biobanking consortia to improve access for proteomics researchers to highly curated and accurately annotated clinical samples collected in a standardized manner. 10. encourage higher levels of community engagement: the hpp will continue to reach out to the community, supporting findable, accessible, interoperable and reusable (fair) data principles 17 , while encouraging and appropriately recognizing all contributions to the re-analysis of proteomics data. the post sars-cov-2 pandemic world will be different. it is likely that new paradigms to accelerate precision medicine will emerge. these will undoubtedly involve global collaboration (even between competing entities) using multidisciplinary approaches that enable the fast-tracking of novel diagnostic tests and precision therapeutics. almost certainly these outcomes will require knowledge involving the human proteome-celebrated here in the inaugural hpp high-stringency blueprint. received: 19 december 2019; accepted: 25 september 2020; a human proteome project with a beginning and an end building the 'practical' human proteome project-the next big thing in basic and clinical proteomics is a gene-centric human proteome project the best way for proteomics to serve biology first hpp publication after launch, outlining systematic global effort to map the human proteome (abundance, distribution, temporal-spatial and subcellular localisation, interactions and function) and decribing reource pillars (ms, ab and kb), with a biologically-driven and a chromosome-based mapping initiatives to deliver a protein parts list, reagents and tools human genome. finally, the book of life and instructions for navigating it metrics for the human proteome project 2013-2014 and 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performed on 103 lung adenocarcinomas, revealing many cancer-associated features (e.g., tumourassociated variants, patient clinical outcome correlation with egfr or tp53 mutational status, proteomic stratification of lung cancer subtypes and potential drug targets proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements a region-resolved mucosa proteome of the human stomach antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens the immunopeptidomic landscape of ovarian carcinomas precision profiling of the cardiovascular post-translationally modified proteome: where there is a will, there is a way in vivo phosphoproteome analysis reveals kinome reprogramming in hepatocellular carcinoma protein s-nitrosylation controls glycogen synthase kinase 3β function independent of its phosphorylation state generation and verification of a compendium of highly specific srm assays that enable quantification of >95% of all annotated human proteins. provides definitive ms data on 166,174 proteotypic peptides that facilitate the design of multiple, independent assays to quantify most human proteins, numerous splice-variants, non-synonymous mutations identification and validation of human missing proteins and peptides in public proteome databases: data mining strategy multi-laboratory assessment of reproducibility, qualitative and quantitative performance of swath-mass spectrometry a human protein atlas for normal and cancer tissues based on antibody proteomics spatial proteomics: a powerful discovery tool for cell biology getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles antibodypedia, a portal for sharing antibody and antigen validation data towards a knowledge-based human protein atlas a genome-wide transcriptomic analysis of protein-coding genes in human blood cells an atlas of the protein-coding genes in the human, pig, and mouse brain a subcellular map of the human proteome a pathology atlas of the human cancer transcriptome an atlas of human metabolism first of many encyclopedic hpa atlases that map the spatio-temporal expression human proteome based on integrated multi-omics data involving quantitative transcriptomics and ab microarraybased tissue immunohistochemistry and delivering spatial localisation of human proteins down to the single-cell level a proposal for validation of antibodies enhanced validation of antibodies for research applications deep learning is combined with massive-scale citizen science to improve large-scale image classification analysis of the human protein atlas image classification competition the elixir core data resources: fundamental infrastructure for the life sciences integration of transcriptomics and antibody-based proteomics for exploration of proteins expressed in specialized tissues cell type-specific expression of testis 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peptide identification from ms/ms ms-gf+ makes progress towards a universal database search tool for proteomics assembling the community-scale discoverable human proteome community encouragement to identify biological data that complement high-stringency ms strategies to accelerate discovery and understanding of human proteome pe2,3,4 missing proteins. database allows unpublished, preliminary or proprietary data (e.g., antibody, ms, cell biology and genetic studies chess: a new human gene catalog curated from thousands of large-scale rna sequencing experiments reveals extensive transcriptional noise dynamic visualization of multi-level molecular data: the director package in r can we predict protein from mrna levels? proteomic and interactomic insights into the molecular basis of cell functional diversity in silico peptide repertoire of human olfactory receptor proteomes on high-stringency mass spectrometry toward completion of the human proteome parts list: progress uncovering 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dissemination the proteomexchange consortium in 2020: enabling 'big data' approaches in proteomics structural genomics: keystone for a human proteome project software survey: vosviewer, a computer program for bibliometric mapping measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies the road from discovery to clinical diagnostics: lessons learned from the first fda-cleared in vitro diagnostic multivariate index assay of proteomic biomarkers pan-cancer analysis of whole genomes details the hallmarks of cancer acquired during multistep development of human tumors (i.e., sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion/ metastasis, genome instability, inflammation, reprogramming of energy metabolism and evading immune destruction). creates a roadmap for the understanding of 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rapidity at which modern proteomics can contribute to the understanding of a recent pandemic, revealing insights into viral transmission and therapeutic targets. indicates sars-cov-2 uses the receptor ace2 for human cell entry and serine protease tmprss2 for sars-cov-2 spike protein priming. a clinically approved tmprss2 inhibitor blocked virus entry and sera from convalescent sars-cov-2 patients crossneutralized viral entry making of viral replication organelles by remodeling interior membranes proteomic approaches to uncovering virus-host protein interactions during the progression of viral infection proteomics of sars-cov-2-infected host cells reveals therapy targets a sars-cov-2 protein interaction map reveals targets for drug repurposing sars-cov-2 proteome microarray for global profiling of covid-19 specific igg and igm responses proteomic and metabolomic characterization of covid-19 patient sera inhibitory effect of mizoribine and ribavirin on the replication of severe acute 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miniaturised microfluidic assay single-cell proteomics reveal that quantitative changes in coexpressed lineage-specific transcription factors determine cell fate transformative opportunities for single-cell proteomics investigating the applicability of antibodies generated within the human protein atlas as capture agents in immunoenrichment coupled to mass spectrometry was corresponding author, conceptualized hpp decadal blueprint, hupo proteomics timeline and hprl, and he designed/integrated all aspects of the manuscript made academic co-contributions to manuscript section drafting, editing, review and/or proof-reading health and human sciences australia. 3 institute for systems biology seoul 120-749, south korea. 8 faculty of dentistry usa. 12 science for life laboratory, school of engineering sciences in chemistry, biotechnology and health, kth royal institute of technology, 17121 solna, sweden. 13 rudbeck laboratory, department of immunology 23 cancer biomarkers research branch, national cancer institute, national institutes of health, 9609 medical center drive, suite 5e136 wellcome trust genome campus, hinxton, cambridge cb10 1sd, uk. 30 school of biotechnology and biomolecular sciences hupo acknowledges collaborators, proteomic scientists, independent partners, industry vendors and members of the scientific community who have contributed to the hpp. a full alphabetical listing of the human proteome project members appears in the supplementary information. in recognition of the many accomplishments, hupo has produced a publicly available hpp timeline available through https://hupo.org/ proteomics-timeline to be released with this hpp blueprint. parts of this work were supported by grants to s.r.p. is founder and chief scientific officer of atturos, a clinical diagnostics company. m.k. is an employee of bruker daltonics, a manufacturer of ms systems. all other authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-19045-9.correspondence and requests for materials should be addressed to m.s.b. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-338889-7hd3iibk authors: solbakk, jan helge; bentzen, heidi beate; holm, søren; heggestad, anne kari tolo; hofmann, bjørn; robertsen, annette; alnæs, anne hambro; cox, shereen; pedersen, reidar; bernabe, rose title: back to what? the role of research ethics in pandemic times date: 2020-11-03 journal: med health care philos doi: 10.1007/s11019-020-09984-x sha: doc_id: 338889 cord_uid: 7hd3iibk the covid-19 pandemic creates an unprecedented threatening situation worldwide with an urgent need for critical reflection and new knowledge production, but also a need for imminent action despite prevailing knowledge gaps and multilevel uncertainty. with regard to the role of research ethics in these pandemic times some argue in favor of exceptionalism, others, including the authors of this paper, emphasize the urgent need to remain committed to core ethical principles and fundamental human rights obligations all reflected in research regulations and guidelines carefully crafted over time. in this paper we disentangle some of the arguments put forward in the ongoing debate about covid-19 human challenge studies (chis) and the concomitant role of health-related research ethics in pandemic times. we suggest it might be helpful to think through a lens differentiating between risk, strict uncertainty and ignorance. we provide some examples of lessons learned by harm done in the name of research in the past and discuss the relevance of this legacy in the current situation. these are uncertain times-for all, wherever one lives, and whatever one aspires to know about the covid-19 pandemic. at present, there are so many things we do not (yet) know: how it will evolve and spread, when, if ever, it will end, how and why it started, whether infected persons develop permanent immunity, whether safe and effective cures and vaccines will be possible to develop, and last, but not least, what impact the pandemic will have on each and every one; be it individuals, families, societies, nations, regions, or globally. the aim of this paper is to address the ongoing debate about covid-19 chis and discuss the concomitant role of medical and health-related research ethics in the present situation the word pandemic comes from two ancient greek words (pan 'all' + dēmos 'people'), so literally speaking 'pandemic' means all the people, all the world. where there is an urgent need for developing effective methods of detection, treatment, and prevention to cope with the covid-19 pandemic and notably in ways that minimize harm and that benefit all human beings. although at present all attention and focus is on the covid-19 pandemic, it is key not to forget there have been pandemic surprises in the past and that future, unknown pathogens may lead to crises with unprecedented implications. the issues we discuss here are therefore relevant beyond the covid-19 pandemic; above all they concern how we should navigate (research ethics) in times of great threats to individual, public and population health and under conditions where different forms of uncertainty still prevail. 1 the british medical association's report on biomedical research and human rights states: «research is driven by a desire to understand the causes of disease or dysfunction and find effective methods of prevention and treatment." 2 however, the report continues, "even such humanitarian aims can be risky", in particular under circumstances perceived as extreme or exceptional. 3 in the report, nine risk-factors for abusive research are identified, of which three are of particular relevance in the present context: (1) the perception of an urgent and overriding scientific need; (2) the perception of a national necessity or government pressure to conduct research; and (3) the situation of contingent populations chosen as research subjects. 4 table 1 covid-19 and three forms of uncertainty (risk, strict uncertainty and ignorance) a goldstein and burstyn (2020) b sethuraman et al. (2020) c mutambudzi et al. (2020) d mcintosch et al. (2020) e hofmann (2020) f mcintosch et al. (2020) g yelin et al. (2020) h european group in ethics in science and technology (2020) i mcintosch et al. (2020) j hofmann (2020) k yelin et al. (2020) l kalil (2020) risk a,b,c,d (known outcomes and known probability distributions) test accuracy (sensitivity, specificity, predictive values) for the various tests in different contexts effects and side effects of new treatments prevalence of disease the risk of healthcare workers versus the risk of non-essential workers testing positive for covid-19 strict uncertainty e,f,g,h (known outcomes and unknown probability distributions) basic reproduction number (r) case fatality rate/infection fatality rate the precise interval during which an individual with sars-cov-2 infection can transmit infection the pathogenic effect of the sars-cov-2 in different age groups the extent to which transmission occurs from a-symptomatic or pre-symptomatic subjects and how much it contributes to the pandemic whether all infected patients mount a protective immune response and how long any protective effect will last reinfection how long sars-cov-2 can persist on surfaces whether pre-existing immune responses impact the risk or the severity of covid-19 and whether they will influence sars-cov-2 vaccine responses long-term sequelae and late-stage consequences of covid-19 ignorance i,j,k,l (unknown outcomes and unknown probability distributions) in a recent letter to the editor of the american journal of bioethics, stoeklé and hervé state that now is not the right time for ethics reflection, but rather for political action and for "indisputable confidence in medical care staff and scientists, not only in france, but everywhere around the world". 5 in a response to this view, and notably with the opposite title, the respondents state 6 : in times of crisis, like the current pandemic of covid-19, the perception that ethical standards can be relaxed due to the urgent need for solutions is growing, according to stoeklé and hervé. for them, ' ethics is only useful if you have the time, and right now, time is exactly what we do not have.' it is a misperception without any doubts. ethics has always preserved its identity as a rationalization of human action. therefore, ethical reflections to take decisions are useful all the time and must be reinforced in times of pandemic. another way of visualising this tension is by differentiating between the epistemological ethos of doing biomedical research, such as developing knowledge and skills for effective diagnostic, treatment and prevention, versus the ethical ethos of the same enterprise; the attempt to protect the interests and wellbeing of patients and healthy individuals involved in such research. ethos is here used in the meaning of 'accepted standards.' in research, two such normative standards or rules of play are used: epistemological rules of play (such as truth, probability, coherence, relevance, fruitfulness, interestingness, and utility), and ethical rules of play (such as autonomy, informed consent, justice, beneficence, non-maleficence, truthfulness, dignity, trust, vulnerability, and solidarity). the tension between these two normative standards is permanent, and one that probably never can be fully resolved. but in times of perceived urgency, the danger is increased tension, or worse, a disregard and violation of epistemological as well as ethical standards, and that shortsighted expediency with questionable results will ensue. in the pages to follow we argue in favour of an ethics of precaution, with particular emphasis on the role of research ethics in particularly challenging situations, such as pandemics. that is, we will analyse and critically assess the epistemological and ethical justification of several research initiatives that have been implemented or are at the planning stage. we claim that in the current situation of a palpable sense of medical and scientific urgency, and of national and global necessity, there should be no room for epistemological or ethical exceptions or shortcuts. on the contrary-and perhaps more than ever-there is a need to conduct biomedical and health-related research in compliance with existing rules of play and fundamental human rights commitments. faced with the "toxic brew of uncertainty" 7 the pandemic has caused, we suggest it would be helpful to differentiate between three different forms of uncertainty: risk, strict uncertainty, and ignorance or non-knowledge. risk represents a form of uncertainty with known potential outcomes, and, where the probability distribution is known. the plethora of uncertainties that the covid-19 pandemic are causing cannot, however, be addressed within this narrow framework of risk estimation; uncertainty considerations of the two additional kinds mentioned above should also be included. strict or fundamental uncertainty is a form of uncertainty where possible outcomes are known, but the probability distribution is unknown, while ignorance or nonknowledge represents forms of uncertainty where only some possible outcomes are known while the statistical likelihood of each of them is unknown. 8 the relevance of differentiating between these three forms of uncertainty in the present context is visualized in table 1 . in the aftermath of another global disaster, world war ii, several normative initiatives were taken to prevent a similar catastrophe to recur, such as the establishment of the united nations and the development of the universal declaration of human rights. in addition, more robust ethical standards for biomedical research were crafted in order to avoid inhuman research in the future-not the least in crisis-situations. the then first lady of the usa, eleanor roosevelt, served as the first chair of the un commission on human rights, which drafted the universal declaration of human rights (1948). the first paragraph in the declaration's preamble states that "the foundation of freedom, justice, and peace in the world" is the "recognition of the inherent dignity and of the equal and inalienable rights of all members of the human family". 9 of the 30 articles in the declaration article 1 and 5 are of particular relevance for research ethics; article 1 restates the freedom and equality of all human beings in terms of 5 stoeklé and hérve (2020) . 6 hellmann et al. (2020) . 7 outka (2020). 8 wynne (1992) , rørtveit and strand (2001) , nielsen and sørensen (2017) and hofmann (2020). 9 https ://www.ohchr .org/en/udhr/pages /udhri ndex.aspx. dignity and rights, and article 5 emphasizes the right to be protected from torture or cruel, inhuman or degrading treatment or punishment. the essence of these normative statements harkens back to the ethical code of medical research issued by the war crimes tribunal at nuremberg the previous year, in 1947. 10 of the 10 standards laid down in this code, and with which physician-researchers must comply when carrying out experiments on human subjects, standard 5, in particular, has become highly relevant these days due to pressure from influential medical stakeholders, agencies and bioethicists to permit the conduct of controlled human infection studies (chis), also labeled human challenge trials (hcts), or challenge studies (css) to possibly shorten the development time of vaccines to protect against covid-19 caused by the sars-cov-2 virus. 11 in the next paragraph of this paper we will examine and critique in detail four position statements advocating the epistemological and ethical justifiability of conducting covid-19 chi-studies: p. singer and r.y. chappell's, pandemic ethics: the case for experiments on human volunteers 12 ; the report, key criteria for the ethical acceptability of covid-19 human challenge studies, issued by a working group set up by who 13 ; the policy forum statement, ethics of controlled human infection to study by shah, miller, darton et al.; 14 and jamrozik and selgelid's statement, in addition, we will consider other recent papers advocating the use of covid-19 chis studies. in table 2 below we have made a summary of prevalent arguments for and against such studies. standard 5 of the nuremberg code reads: no experiment should be conducted where there is an a priori reason to believe that death or disabling injury will occur; except, perhaps, in those experiments where the experimental physicians also serve as subjects. 16 while repeating (in the first clause of article 7) that no one shall be subjected to torture or to cruel, inhumane or degrading treatment or punishment, the legally binding 1966 un international covenant on civil and political rights adds a second clause that "in particular, no one shall be subjected without his free consent to medical or scientific experimentation". 17 the prohibition is intended to prevent the recurrence of atrocities such as those that took place during wwii and the first decades after the war. even though this prohibition is implicit in the first clause, the matter was deemed so important as to require a specific and precise provision. 18 during the drafting of the covenant, there was a proposal that compulsory measures might be taken "in the interest of community health", but the proposal was rejected on the grounds that this might lead to abuse. 19 the consent requirement is thus formulated as absolute, without any exceptions. the human rights committee has reaffirmed what is already explicitly mentioned in article 4 (2), that even in situations of public emergency, no derogation from article 7 is allowed. 20 controlled human infection studies (chis) "are clinical studies that, as part of the protocol, deliberately expose trial participants to an infectious pathogen. these studies are often done in the context of vaccine development, with trial candidates exposed to a pathogen after being immunized with an experimental vaccine". 21 the main advantages of chis compared to large field trials are that they can generate data much faster, they are much less expensive and they do not require thousands of research participants, normally only between 10 and 50 participants. 22 the four position statements on chis referred to above all suggest that suitable candidates for such studies are "young people without underlying medical 13 who (2020). 14 shah et al. (2020) . 15 jamrozik and selgelid (2020a callaway (2020) , eyal (2020), eyal et al. (2020) , jamrozik and selgelid (2020a, b) , plotkin and caplan (2020) , schaefer et al. (2020) , shah et al. (2020) , , who (2020) and wolemonwu (2020) . 12 . the urgency of the current pandemic gives substantial weight to a challenge study j the endemic argument the probability of dying or developing disability if infected would be smaller in a chi trial k the risks in question do not entail a major net increase in risk (in light of background risks of infection) l participants face a background risk of infection in the community m only people with an especially high baseline risk of getting exposed during or soon after the trial period should be recruited (e.g., people residing in areas with high transmission rates) n the risk and safety argument participation in a covid 19-chi trial would be less risky than joining a standard efficacy trial for the same vaccine o covid-19 chis have a much higher risk than the minor risk threshold. p for a live sars-cov-2 challenge there are deadly risks q currently, we lack sufficient knowledge of sarscov-2 pathogenesis to inform inclusion and exclusion criteria for a sars-cov-2 chim r the medical benefit argument the probability of averting death in the event of infection would be substantially better inside a chi trial than outside s the altruistic argument it might seem that anybody volunteering to participate in such a study lacks capacity for rational decision-making. but humans do many important things out of altruism t volunteers who participate in the challenge trials should be motivated to advance human health and wellbeing rather than driven by their economic needs u the social value argument benefits to the subject + benefits to society > risks to the subject v,w given the risks to participants, sars-cov-2 challenge studies would need to demonstrate very substantial social value before proceeding. arguably, this bar might already be met given the high death toll and severe disruption caused by the pandemic x covid-19 chis could help prioritize among the almost 100 investigational vaccines and over 100 experimental treatments for covid-19 currently in development y young healthy adults may not generalize to older individuals and those with comorbidities who would most benefit from effective vaccines z conditions" 23 /"healthy young volunteers" 24 /"young healthy adults". 25 the last part of standard 5 of the nuremberg code deserves particular attention in this context because it rejects high risk experiments with human beings be they young or old, "except perhaps", in cases where physician researchers are willing to conduct such experiments on themselves. the proponents of starting such studies all admit that there remains "significant uncertainty" with regard to the pathogenic effects of sars-cov-2 in both elderly and young people, and that high or significant risk is involved in such studies with the potential of causing severe harm, or death. 26 even if the recommendations of the nuremberg code of letting researchers themselves serve as participants had been followed strong caution is still warranted for at least six reasons. first, as uncertainty still surrounds the pathogenic effect of the sars-cov-2 in different age groups, challenge-studies would not be justified because of the lack of robust knowledge and understanding of the short term consequences of the sars-cov-2, 27 and because of the almost complete absence of data on the long term effects of the virus. 28 for these reasons it would be very difficult to justify the inclusion even of the most knowledgeable individuals in such studies, i.e. covid-19 researchers, because even the most knowledgeable are currently deeply ignorant. 29 it is worth noting here that in a document on a eyal (2020) and callaway (2020) b the international alliance for biological standardization (2019), jamrozik and selgelid (2020a, b) , menikoff (2020) , eyal et al. (2020 eyal et al. ( , pp. 1753 eyal et al. ( -1754 who (2020) and jamrozik and selgelid (2020a) . 26 eyal (2020) , who (2020) . 27 in a recent paper reviewing and synthesizing available evidence on asymptomatic sars-cov-2 infection, subclinical lung abnormalities was found even among asymptomatic persons (for this see oran 2020) . 28 this additional form of uncertainty is admitted by jamrozik and selgelid (2020a, p. 4) . 29 jamrozik and selgelid (2020a, p. 4 ) briefly address the question whether "the permissibility of high-risk human challenge studies" would increase if researchers used themselves as research participants, but they warn against this, and notably for the reason that "clinical and research staff might feel pressure to participate". 23 shah et al. (2020) . 24 . challenge-studies for vaccine development adopted in 2016 by who's expert committee on biological standardization there is no talk about accepting such studies if the risk is of the size referred to in the who-working group's report of this year; i.e. "high" risk with the potential of causing "severe harm". 30 on the contrary, in the 2016 document emphasis is on "minimizing risks to subjects" and reference is made to "situations in which there may be greaterthan-minimal risk", or risks "…considerably greater than minimal", but still manageable as "…e.g. accepting that they [the trial candidates] will develop an acute, but manageable, disease that will resolve but in the meantime may cause considerable morbidity, such as severe diarrhea managed with fluid and electrolyte replacement". 31 however, risks "considerably greater than minimal" are still far from being equal to high risk with the potential of causing severe harm. further down the same paragraph of the 2016 who expert committee document the following is added: "however, accepting such risks requires absolutely that the elements of voluntary consent are based on truly being informed". 32 it is, however, difficult to see how it would be possible to comply with this condition when so much is still unknown with regard to both the short and long-term pathogenic effects of the sars-cov-2. a valid informed consent is a legally binding absolute human rights requirement. second, if something goes wrong after infecting healthy young volunteers with the virus, the treatment options at present are limited. accepting such studies in relation to the sars-cov-2 would therefore not be in compliance with two of the core conditions in current ethical frameworks for challenge studies: namely that the pathogen studied does not induce infections that may cause severe harm, or for which there exist no effective treatment. 33 despite this, challenge studies for covid-19 are proceeding and the recruitment of health volunteers has exceeded 30,000. 34 if something goes wrong, compensation to participants ought to be a major consideration for these trials. in a recent publication, carl elliot argues that although the focus of challenge studies is to recruit healthy volunteers, if the model is based on remunerating these volunteers, the studies may attract economically vulnerable volunteers who view participation as a means to an end. 35 and as stated by ruth macklin 36 : …given standard practice, it is virtually certain that monetary payment-which may be considerable-will serve as an inducement to enroll. the likely result is that a disproportionate number of volunteers would come from lower-income brackets, including many people who lost their jobs because of the pandemic. it is also likely that many volunteers would be members of racial and ethnic minorities, raising a serious question of social justice. economically vulnerable volunteers who become injured or "permanently disabled" may not receive compensation, especially in the us. 37 in contrast to other developed countries, research sponsors in the us are not under any legal obligation to pay for the medical care of research participants who become injured or ill, hence, few do. 38 furthermore, the us national vaccine injury compensation program excludes payment for experimental vaccines. 39 research participants in the us can therefore be left without compensation for injury caused by their participation in a challenge study. third, the technical label 'controlled human infection studies' is unfortunate, because it might give healthy young volunteers and other non-experts the impression of a kind of control that is misleading. fourth, at present one does not know whether controlled infections studies conducted with young and healthy adults (or covid19-researchers) will provide results that enhance survival and/or healing-rates of those most affected by sars-cov-2, i.e. old fragile persons with comorbidities. 40 hence, there is a significant problem of external validity. fifth, the argument that participants "might benefit from controlled infection and/or vaccination if they become immune to virus" 41 is also undermined by major uncertainty, as openly admitted by the proponents of such studies. 42 sixth, and, perhaps, most important, such studies violate the core ethical principle of human subjects research; i.e. the priority of the individual principle. this principle emerged as a normative response to the medical horrors that had been practiced during wwii in concentration camps in nazi germany and in japan. yet in spite of the universal declaration of human rights and the nuremberg 33 lynch (2020) . 34 cohen (2020)-for this, see also guarino and johnson (2020) . 35 elliott (2020) . 36 macklin (2020) . 37 elliott (2020) . 38 ibid. 39 ibid. 40 jamrozik and selgelid (2020a) . this concern is also raised by deming et al. (2020) , and by schaefer et al. (2020) . 41 shah et al. (2020) . 42 shah et al. (2020) , who (2020, note 27, p. 13 code, several ethically compromised studies involving vulnerable groups have been conducted after wwii. to underscore this point, and analyze their implications, allow us to describe two such examples. first, the hepatitis studies, conducted from 1953, at willowbrook state school, an institution for mentally disabled children on staten island, new york. more than 700 children, predominantly african-american and puerto-rican, were included in these studies, and a subgroup of almost 100 non-infected children were fed a suspension with the local strains of the hepatitis virus prepared from the stool of six children collected "during the first 8 days of recognized jaundice". 43 written consent from the parents of these children had been obtained by the chief investigator, dr. krugman. but later investigations indicated that the parents' consent might have been based on indirect coercion since volunteering their children to the infection study was allegedly put forward as a condition for admitting the children to care at willowbrook state school. krugman defended the contested infection and immunization studies his whole life with reference to their scientific merits; the "confirmation of two types of hepatitis, a and b, with different infection pathways (oral versus close contact), and the preparation of a "crude vaccine" containing the hepatitis b virus. 44 in a letter to the editor of the lancet in 1971 he justified the exposure of "a small number of newly admitted children to the willowbrook strains" of the hepatitis virus with reference to: (1) inevitability; the children were "bound to be exposed" to the same virus strains "under the natural conditions existing in the institution", (2) safety; they would be admitted to "a special, well-equipped and well-staffed unit", thus shielding them from exposure to other prevalent infectious diseases in the institution, (3) immunity; "they were likely to have a subclinical infection followed by immunity", and (4) informed consent; "only children with parents who gave their informed consent would be included". 45 the second study worth mentioning is the cancer injection study that took place in the early 1960s at the jewish chronic disease hospital in brooklyn, new york. the chief investigator, dr. chester m. southam, had since 1953 conducted research on the role of the immune system in protecting against cancer, using two different groups of research participants who were injected with a suspension of foreign cancer cells to study the difference in immunological rejection of the cells between the two groups. the first group was a cohort of patients at memorial hospital in ohio, in total 300, with different forms of widespread cancer, and the second group consisted of 300 healthy individuals from the ohio state penitentiary 46 ; i.e. a prison in downtown columbus, ohio. these studies documented that healthy individuals rejected the injected cancers cells faster than the cancer patients (4-6 weeks versus 6 weeks to 3 months). 47 in his presentation of southam's studies john d. arras labeled the second group of research subjects "healthy prison volunteers", and notably, without any reflection whatsoever either about the ethical justifiability of recruiting prisoners for a study that would not benefit them, or whether it is appropriate to consider prisoners to be free to volunteer. 48 on this point southam himself seems to have been, at least, partly aware of the dilemma of recruiting incarcerated individuals. in an interview in science he admitted that, although there was no theoretical likelihood that the injections would produce cancer, he had nonetheless been unwilling to inject himself, or his colleagues, when there was a group of normal volunteers at the ohio penitentiary fully informed about the experiment and its possible risks and nonetheless eager to take part in it: "i would not have hesitated", southam said, "if it had served a useful purpose. but to me it seemed like false heroism, like the old question whether the general should march behind or in front of his troops. i do not regard myself indispensable-if i were not doing this work someone else would be-and i did not regard the experiment as dangerous. but, let's face it, there are relatively few cancer researchers, and it seemed stupid to take even the little risk". 49 southam persuaded the then director of the jewish chronic disease hospital, emmanuel e. mandel, to permit, as a third part of his immune reaction studies, the injection of foreign cancer cells into 22 old patients with other debilitating chronic diseases than cancer. the scientific justification for this study was to get "direct evidence" that it was the cancer disease that caused the delay in rejection of the foreign cancer cells, and not the fact that most of the cancer patients he had studied were elderly, debilitated and with additional chronic diseases. 50 such evidence, he maintained, would be possible to establish by doing the same immune reaction study in a group of elderly patients with other chronic and debilitating diseases than cancer. in the letter southam wrote to mandel on july 5, 1964, he also discussed whether consent ("written permission") from the patients was warranted, something which he warned against, for two reasons; first, at memorial hospital they considered it a "routine study, much less hazardous than other routine procedures", and second, the only risk related to the use of cancer cells in these injections was the "phobia and ignorance surrounding the word "cancer". 51 in the same letter southam informed mandel that with regard to the 300 prisoners, "signed permits" had been obtained, but this, according to southam, was "because of the law oriented personalities of these men, rather than for any medical reason". 52 in 1966 the two doctors were found guilty of fraud, deceit and unprofessional conduct, and they were, in particular, criticized for assuming they were entitled to perform any kind of research without consent as long as the research in question was scientifically justified. two years later, however, southam, was elected president of the american association for cancer research for the period of 1968-1969. in 2001 miller and grady proposed a way of evaluating the ethical justifiability of planned challenge-studies by locating each candidate along a continuum from legitimate studies to clearly unacceptable ones. in the "border zone" between these two extremes they locate studies that are neither indisputably justifiable nor clearly unacceptable. among legitimate ones they count studies for common cold, cholera 53 and malaria, 54 while chi-models for lyme disease or helicobacter pylori are labeled "more controversial". 55 finally, among clearly unacceptable studies they mention two examples; hiv-chi-studies or chi-studies for hepatitis c virus. 56 their arguments for labeling these two chi-models unacceptable were twofold; "non-existent or ineffective" treatment, and "intolerable symptoms and/or the likelihood of serious morbidity or mortality". 57 two additional possible studies deemed unacceptable are referred to in cioms' commentary on guideline 4 of the international ethical guidelines for health-related research involving humans 58 : for example, a study that involves deliberately infecting healthy individuals with anthrax or ebola-both of which pose a very high mortality risk due to the absence of effective treatments-would not be acceptable even if it could result in developing an effective vaccine against these diseases. therefore, researchers, sponsors, and research ethics committees must ensure that the risks are reasonable in light of the social and scientific value of the research, and that the study does not exceed an upper limit of risks to study participants. an additional example-deemed unacceptable at the time of the evaluation-was a zika virus-chi-study. 59 the nih ethical review committee's reasons for deciding against the study were with reference to three kinds of uncertainty; uncertainty of the risk to research participants as well as to third parties (fetus and sexual contacts), uncertainty about the duration of protection needed, and, third, uncertainty of the study's societal value. 60 viewing the studies conducted by drs. krugman and southam in the light of miller and grady's differentiation between legitimate, more controversial and clearly unacceptable challenge-studies, and in view of current ethical standards pertaining to such studies, calls for reflection, not only for historical reasons; they may also be of help in investigating where the arguments in favor of chi-studies in the current context of the covid-19-pandemic differ from those of krugman and southam, and where these arguments seem to overlap. when it comes to locating the studies under discussion along the ethical line of decreasing permissibility proposed by miller and grady, southam's immune reaction studies on elderly, debilitated subjects arguably deserve the label ethically unacceptable, in spite of their scientific merits, for at least three reasons: (a) no consent was obtained, (b) 53 a chi-study in 197 healthy volunteers contributed to the development and licencing of the live oral cholera vaccine cvd 103-hgr study in 1993. for this, see: roestenberg et al. (2018, p. 4) . 54 in 2015, the first malaria vaccine allegedly achieved through the use of chi-studies gained ema approval. for this, see roestenberg et al. (2018, p. 4) . for this, see also: european medical agency: https ://www.ema.europ a.eu/en/docum ents/medic ine-outsi de-eu/mosqu irixsumma ry-publi c_en.pdf. 55 for such a study, see: graham et al. (2004) . 56 grady (2001, p. 1032) . 57 ibid., p. 1032. 58 cioms (2016, p. 10). 59 the international alliance for biological standardization (2019, p. 88). 60 ibid., p. 88. for a similar verdict concerning a zika chi-trial, see recommendation 2 in shah et al. (2017, p. 27) : "whether a zika virus human challenge trial has sufficient social value to proceed depends on the reasons for doing it and whether there are alternative ways to obtain the information. the most compelling rationale for conducting a zika virus human challenge trial, given the risks and uncertainty, would be if field trials were prohibitively difficult to conduct in light of a waning epidemic. this rationale is not currently met, but it could come to pass in the future. another valuable reason to conduct a challenge trial would be to accelerate the development of a vaccine that could prevent congenital zika infection. this rationale must be accompanied with strong evidence that results from a zika virus human challenge trial would be used by stakeholders (e.g., indication from regulatory agencies that finding a correlate would speed up the licensing of a vaccine). the committee did not hear sufficient evidence that this rationale is currently met. finally, using a challenge trial solely to learn about the pathogenesis and natural history of zika infection is unlikely to justify the risk involved given the alternative ways to obtain similar information". 51 southam in his letter to mandel. 52 ibid. the element of deceit involved and (c) the fact that participants did not stand to benefit in any ways from the studies. southam's reluctance against using himself and colleagues as study subjects was based on two considerations: first, exposure to possible risks caused by the injection of foreign cancer cells; second, since neither he nor his colleagues were physically debilitated they were unsuitable candidates for the study. in two of the position statements referred to above reservation is expressed against using researchers as studysubjects in covid-19 cih-studies, although for a different reason; "clinical and research staff might feel pressure to participate", 61 and "such individuals could feel pressured to participate (thereby undermining the voluntariness of informed consent". 62 krugman's challenge studies at willowbrook are more difficult to locate and label according to miller and grady's linear differentiation, not least because his defense of the studies has several striking similarities with arguments used in favor of conducting chi-studies to speed up the development of efficient vaccines against the sars-cov-2 virus. first the reference to the studies' scientific merit; the "confirmation of two types of hepatitis, a and b, with different infection pathways, and the preparation of a "crude vaccine" containing the hepatitis b virus. 63 a similar type of argument is used by and shah, miller, darton et al. 64 : for example, chis could clarify dynamics of infection, viral pathogenesis, and risk of vaccine pathogenesis or identify correlates of protection-all of which could inform the development and implementation of vaccines. second, their high social value. in his letter to the editor of the lancet in 1971 krugman emphasized the high social value beyond willowbrook of his studies: "it is unnecessary to point out the additional benefit to the world-wide population which have been plagued by an insoluble hepatitis problem for many generations". 65 in support of such arguments, shah, miller, darton et al. argue 66 : sars-cov-2 chis could have high social value in several ways. for example, they could help prioritize among the almost 100 investigational vaccines and over 100 experimental treatments for covid-19 cur-rently in development. chis could help identify the most promising agents, which would inform the design of larger trials, guide decisions to scale up manufacturing early, and thereby accelerate product development and implementation. in a similar vein, singer and chappel refer to the high social value, or in their wording-"the broader humanitarian benefits"-of such studies. 67 third, the exposure of a small number of individuals to risk for the sake of benefits to the rest. different versions of this argument are used in the four position statements, 68 and also by the international alliance for biological standardization. 69 fourth, the endemic argument-the children were "bound to be exposed" to the same virus strains "under the natural conditions existing in the institution". 70 two 2020-versions of the same argument read: "the risks in question do not entail a major net increase in risk (in light of background risks of infection", 71 and "participants face a background risk of infection in the community". 72 fifth, the safety-and better care argument. in krugman's wording the study subjects would be admitted to "a special, well-equipped and well-staffed unit", 73 to minimize risks to study personnel, participants should be in inpatient isolation, with contact reduced to the extent possible and robust personal protective equipment provided. and who's working group highlights the importance of "supportive care, including critical care" and "long-term follow-up" as two crucial risk-minimization strategies. 75 sixth, the immunity-argument; krugman argued that the children involved in his studies "were likely to have a subclinical infection followed by immunity" 76 , while who's working group refers to "[i]mmunity induced by experimental vaccines" as a potential benefit of study participants. 77 jamrozik and selgelid combine the safety-/better care argument with the immunity argument 78 : …potential direct benefits of being infected with sars-cov-2 in the course of human challenge studies would include participants being exposed to less infection-related risk than if they are infected in the community (e.g. because of early diagnosis and medical care) and gaining immunity to future infection in the context of a high background risk. seventh, and last but not least, krugman as well as the authors of the four position statements referred to above all emphasize the importance of informed consent. 79 this comparison between krugman's arguments and the arguments in the four position statements here subject of detailed analysis and in other recent papers advocating the use of covid-19 chis shows that their overall views are pretty much the same. in fact, the only difference in terms of substance is the use of vulnerable individuals or groups as study-participants which two of the four position statements warn against. 80 singer and chappel do not address this issue, while the authors behind the fourth position statement, jamrozik and selgelid, in a recent paper on cih studies in endemic settings, argue that sometimes it may be justifiable, in fact "ethically important" to include vulnerable populations: "…especially where the results of research in other populations are not likely to be generalisable to the vulnerable populations in question. this is one consideration that sometimes favours conducting (more) hcs in low-middle income countries (lmics)". 81 so where does all this lead us? should we accept the 'neo-krugman'ian' views advocated by proponents of covid-19 chis, or should we rely on the normative principles that were formulated as a reaction to the kind of studies southam and krugman had conducted? of these principles the priority of the individual principle is the most fundamental. the original formulation of this principle in biomedical research ethics occurred in the first, i.e the 1964-version of the declaration of helsinki as basic principle 5 and reads 82 : every biomedical research project involving human subjects should be preceded by careful assessment of predictable risks in comparison with foreseeable benefits to the subject or to others. concern for the interests of the subject must always prevail over the interests of science and society. this principle has been maintained in all versions of the declaration. in the latest version (wma 2013) it is included as general principle 8: while the primary purpose of medical research is to generate new knowledge, this goal can never take precedence over the rights and interests of individual research subjects. whereas the declaration of helsinki is a professional ethics norm, the principle was restated in article 3 of the une-sco universal declaration on bioethics and human rights, which was adopted by all member states of the united nations in october 2005, and notably with reference in the first section of the article to human dignity, human rights and fundamental freedoms: the interests and welfare of the individual should have priority over the sole interest of science or society. in their position statement singer and chappell claim that current research-ethical principles are based on "assumptions developed in calmer times when much less was at stake". 83 this claim is historically wrong, as this is not the first time the world has faced a public health crisis since the development of the core principles of research ethics. we have managed to live through those less calm times without 77 who (2020, p. 8). for the immunity argument, see also eyal (2020, p. 30) . 78 jamrozik and selgelid (2020a, p. 3) . a slightly different version of this argument reads thus: "in short, if researchers conducting challenge trials act as recommended, admittedly, the probability of getting infected would remain larger inside a challenge trial than either outside any trial or in a standard efficacy trial; but the probability of death or disability is likely to be much smaller inside a challenge trial than in these alternative scenarios. overall, a × b could be smaller for any individual inside the challenge trial than either outside any trial or in a standard efficacy trial. what the individual would lose in the probability of averting infection (with that probability rising) she could gain in better protection from death" (eyal 2020, p. 27) . 79 krugman (1971, p. 967) : informed consent by proxy; shah et al. (2020, p. 3): "robust consent"; jamrozik and selgelid (2020a, p. 4): "proper" or "adequate consent"; who, 220, p. 15: "sars-cov-2 challenge studies must involve rigorous informed consent". for this, see also bambery et al. (2020, pp. 97-98) , eyal (2020, p. 29 ), plotkin and caplan 2020, p. 3987), schaefer et al. (2020), and wolemonwu (2020, p. 3) . 80 shah et al. (2020, p. 3): «sites should be selected for sound scientific reasons while avoiding especially vulnerable populations"; who (2020, p. 13): "those whose background risk is high as a result of social injustice should be excluded from participation because their inclusion could be considered unethical exploitation (i.e., taking advantage of those who have already been wrongly disadvantaged. any prospective participants who could reasonably be perceived to be vulnerable in other ways that would undermine their consent or put them at greater risk (for example as a result of the mental health strain of inpatient isolation during the study) should also be excluded". 81 jamrozik and selgelid (2020b, p. 11 ). 82 https ://www.cirp.org/libra ry/ethic s/helsi nki/. 83 infringing those principles, and we have no particular reason to overstep them now. so to conclude our analysis and critique of controlled human infection studies (chis) with sars-cov-2: there is a consistent, historical line of research ethical principle from nuremberg, through the revisions of the helsinki declaration, and the successive un human rights declarations and other normative documents. this principle is that the interests of the individual research participant is paramount in research ethics, i.e. that if there is a conflict between the interests of society (e.g. in speeding up vaccine development) and the interests of the participants (e.g. in not dying or being permanently harmed) then the interests of society has to yield to the interests of the participant. there is no exception for times of crisis, or for instances where societal interests are large. one might argue against this principle, and some have done so, but well-founded and sustainable ethical principles can't be disregarded just because they seem inconvenient at a certain point in time. the concern here is not just an abstract philosophical principle, but a part of the normative core of all existing research ethics processes. if we take this principle seriously it prohibits the conduct of sars-cov-2 challenge studies at the present time where the challenge virus would be the native virus with full virulence and where there is no rescue treatment yet available. in a policy forum statement in science alex london and jonathan kimmelman, warn against "pandemic research exceptionalism" and against using crises as an excuse for lowering scientific standards. 84 they focus on five epistemological conditions of "informativeness and social value" that research should embody, even in times of emergency: first, importance 85 : trials should address key evidence gaps…as of this writing, more than 18 clinical trials enrolling more than 75,000 patients have been registered in north america for testing various hydroxychloroquine regimens for covid-19. this massive commitment concentrates resources on nearly identical clinical hypotheses, creates competition for recruitment, and neglects opportunities to test other clinical hypotheses. second, rigorous design. third, analytical integrity: "designs should be pre-specified in protocols, prospectively registered, and analyzed in accordance with pre-specification". fourth, complete reporting: "trials should be reported completely, promptly, and consistently with pre-specified analyses". fifth, and last, feasibility: "studies must have a credible prospect of reaching their recruitment target and being completed within a time frame where the evidence is still actionable". 86 similar warnings against research exceptionalism, and notably with reference to things that went wrong in the past when arguments in favor of this were used, are made by deborah doroshow, scott podolsky and justin barr 87 : the coronavirus disease 2019 (covid-19) pandemic has incited remarkable disruption in biomedical research. at academic institutions worldwide, laboratories have been forced to halt all but the most critical activities. clinical trials of novel agents for such diseases as cancer are temporarily suspended, limiting access to potentially life-prolonging medications […] although this boom has already begun to transform our response to the pandemic for the better, medical and scientific responses to past crises suggest that urgency may also result in compromised research quality and ethics, which may in turn jeopardize public faith in government and science, waste precious resources, and lead to the loss of human life. another problem with knowledge-production of the covid-19 pandemic has been the lowered standards of quality assurance of published research. it has been documented that the peer review process has been rushed ("express" or "opinion based peer review") 88 and so far (october 28, 2020) 37 research papers about covid-19 have been retracted. 89 a stunning example of this is an observational study based on the health records of almost 100,000 patients around the world published in the prestigious journal lancet in may 2020, which indicated that hydroxy-chloroquine had a sharply higher risk of death and heart problems compared to those who did not receive the drug, and that hydroxychloroquine did not provide any benefit. 90 on june 4, 2020 this study was retracted by the authors due to doubts about 84 london and kimmelman (2020, pp. 476-477) . 85 ibid., p. 476. 86 ibid, p. 477. 87 doroshow et al. (2020) . 88 ioannidis (2020) . 89 retraction watch. retracted coronavirus (covid-19) papers. https ://retra ction watch .com/retra cted-coron aviru s-covid -19-paper s/. 90 mehra et al. (2020a) . the veracity of the data used and the analyses conducted. 91 on june 16, 2020 preliminary clinical trial results from the recovery study of the university of oxford were broadcasted all over the world suggesting that a commonly used drugdexamethasone-reduced deaths among the sickest covid-19 patients by a third. 92 hopefully, the published study will prove this claim to be justified, but it is unfortunate when covid-19 researchers start "doing science by press release" 93 instead of following generally accepted publication procedures. as stated by dr. atul gawande at brigham and women's hospital in boston 94 : typically, researchers extensively detail their work in scientific journal articles. before publication, other scientists take an in-depth look at how the study was designed, who the patients were and whether any potential side effects were uncovered-a process called peer review. it takes time-weeks or months in some cases-for independent, unbiased experts to pore over the manuscripts, looking for any concerns. taking epistemic shortcuts inevitably produces poor evidence and often leads to bad decisions with potentially severe consequences for vulnerable persons. although tempting, we should avoid epistemic shortcuts, as high-quality evidence is needed in exceptional times, as otherwise. when taking chances, we must consider the risks of harm, not only the benefits. one such risk worth mentioning here is that persons selected for testing emergent vaccines become victims of enhanced disease, i.e. presenting worse symptoms from the effects of an unproven vaccine compared to persons catching e.g. the covid-19 flu through usual paths of contagion. potential covid-19 vaccine volunteers might e.g. end up with life-threatening complications (such as irreversible and untreatable clogged lungs) whereas, in the current situation most unvaccinated patients display only mild flu-indicators, if infected. as there as yet does not exist any known therapy, the perils these volunteers risk is ethically unacceptable. 95 two examples of enhanced disease precipitated by insufficiently proven vaccines occurred in connection with the inoculation of children against rsv (respiratory syncytial virus in the late 1960s. 96 similarly, from october 1976 to january 1977 more than 40 million adult citizens in the usa were vaccinated with a swine influenza virus vaccine. during the same period more than 500 vaccinated persons fell ill with a rare neurological illness (guillain-barré syndrome) and 25 of them died. 97 these unexpected events led to immediate cancellation of the vaccination program indicating that the effects of an insufficiently tested vaccine in some cases cause greater harm than benefit. hence, ethical reflection and compliance with epistemological rules of play are needed more than ever. in a paper reflecting on what might be learned from the 1976 swine flu vaccination program, sencer and millar warn against politicization of scientific information in a way that is well worth listening to also for today's public health leaders and their political peers 98 : while all decisions related to niip [the national influenza immunization program] had been reached in public sessions (publishing of the initial virus findings in cdc's weekly newsletter, the morbidity and mortality weekly report (mmwr); new york times reporter harold schmeck's coverage of the acip [the advisory committee on immunization practices of the united states public health service] sessions, the president's press conference, and 4 congressional hearings), effective communication from scientifically qualified persons was lacking, and the perception prevailed that the program was motivated by politics rather than science. in retrospect (and to some observers at the time), the president's highly visible convened meeting and subsequent press conference, which included pictures of him being immunized, were mistakes. these instances seemed to underline the suspicion that the program was politically motivated, rather than a public health response to a possible catastrophe. mehra et al. (2020b) . 92 university of oxford. dexamethasone reduces death in hospitalised patients with severe respiratory complications of covid-19. june 16, 2020. accessible at: https ://www.ox.ac.uk/news/2020-06-16dexam ethas one-reduc es-death -hospi talis ed-patie nts-sever e-respi rator y-compl icati ons. on october 16, 2020, who reported that "dexamethasone is the only effective drug for coronavirus". this information is accesible at: https ://www.aa.com.tr/en/lates t-on-coron aviru s-outbr eak/who-dexam ethas one-only-effec tive-drug-for-coron aviru s/20091 14. 93 the expression "doing science by press release" we have borrowed from an interview about the study with dr. george anesi, director of the medical critical care bioresponse team at the hospital of the university of pennsylvania. accessible at: https ://www.nbcne ws.com/healt h/healt h-news/scien ce-press -relea se-docto rs-view-covid -19-drug-resul ts-excit ement -n1231 183. 94 nbc news. doctors view dexamethasone results on covid-19 with excitement and skepticism. accessible at: https ://www.nbcne ws.com/healt h/healt h-news/scien ce-press -relea se-docto rs-view-covid -19-drug-resul ts-excit ement -n1231 183. 95 macklin (2020) . 96 acosta et al. (2015) . 97 langmuir (1979, p. 660) . 98 sencer and millar (2006) . rounding them should be communicated'. 99 this goal is much better accomplished if the explanations are communicated by those closest to the problem, who can give authoritative scientific information. scientific information coming from a nonscientific political figure is likely to encourage skepticism, not enthusiasm. another function of research ethics is to provide guidance on the "compassionate use" of drugs unapproved for covid-19. in the past months we have read about compassionate use access of covid-19 patients to hydroxychloroquine and remdesivir. 100 by compassionate use (otherwise known as expanded access), we refer to 101 : …a potential pathway for a patient with an immediately life-threatening condition or serious disease or condition to gain access to an investigational medical product (drug, biologic, or medical device) for treatment outside of clinical trials when no comparable or satisfactory alternative therapy options are available. in principle compassionate use must have regulatory oversight and, in some countries such as the us, spain, and italy, research ethics committee approval as well. 102 us regulations as well as those of individual eu member states stipulate the requirements before an investigational drug can be offered to either an individual, a limited group, or a wider population. 103 but what exactly is the role of research ethics? compassionate use is therapy in the sense that the purpose of it is to cure. however, compassionate use is not only therapy. 104 it is, after all, the provision of a drug with yet-to-be determined levels of efficacy and safety. considering the not very impressive success rate of an investigational drug of only 14% (i.e., from phase 1 to successfully being licensed for market distribution), 105 the risks of compassionate use, especially when doctors cannot be provided with definitive guidance on dosage or exclusion criteria, can actually be worse than the risks of a controlled clinical trial. this being the case, research ethics guidance is imperative for access to investigational drugs via compassionate use. also, faced with a surge of sick patients suffering from a new unknown disease, well-motivated clinicians and investigators all over the world, including drug companies and funding agencies, should adopt "a more integrated approach to learning while doing", and they should join forces, i.e. engage in collaborative efforts so as to increase the "exploitation/exploration trade-offs", and, hopefully, shorten "the period until effective treatments are discovered and implemented". 106 one of the tenets of research ethics is the provision of the benefits of research to the intended patient population. all major international ethics guidelines for research have provisions with different degrees of specifications. the declaration of helsinki article 34, for example, requires clinical trial "sponsors, researchers, and host country governments" to ensure post-trial provisions to participants who, at the end of the trial, might still need the trial intervention that has been "identified as beneficial in the trial". 107 unesco's universal declaration on bioethics and human rights provides more specificity and directive. article 15 says the following 108 : benefits resulting from any scientific research and its applications should be shared with society as a whole and within the international community, in particular with developing countries. in giving effect to this principle, benefits may take any of the following forms: (a) special and sustainable assistance to, and acknowledgement of, the persons and groups that have taken part in the research; (b) access to quality health care; (c) provision of new diagnostic and therapeutic modalities or products stemming from research; (d) support for health services; (e) access to scientific and technological knowledge; (f) capacity-building facilities for research purposes; (g) other forms of benefit consistent with the principles set out in this declaration. guideline 2 of cioms' international ethical guidelines for health-related research involving humans provides a similar specification in terms of provision of the fruits of research to the population or community where the research was carried out, most especially for research conducted in low-resource settings. 109 then, on guideline 20, it stipulates the applicability of guideline 2 for research during disaster and disease outbreaks. 110 during the covid-19 pandemic, we see these principles tested again and again. there were moments filled with high spirits and solidarity. the sequencing of the genome of sars-cov-2, as well as the identification of relevant proteins and enzymes, happened at lightning speed because of spontaneous collaboration between universities from different countries. 111 we also saw how public funds were earmarked covid-19 research and therapy development. 112,113,114 at the same time, we saw how public funds were used to secure advanced orders of potential covid-19 vaccines originally researched using public funds 115 and how pharmaceutical companies played hardball with the public for covid-19 testing kits. 116 there were real reasons to be worried, most especially if pharmaceutical companies continue to be granted free rein in pricing, which usually means paying "a small fortune" for new interventions. 117 to address potential access concerns brought about by patent market exclusivity, 118 costa rica spearheaded what is now the who solidarity call for a covid-19 technology access pool. 119 specifically, this is a call for "key stakeholders and the global community to voluntarily pool knowledge, intellectual property and data necessary for covid-19". 120 it was this same spirit of solidarity that spurred the sequencing of the genome of the sars-cov-2 and it is this same spirit of pooling, collaboration, and sharing of benefits that provides hope for timely and equitable access to much needed interventions. 121 research ethics has provided this foundation, as we saw above, and it remains research ethics' task to ensure that covid-19 drug discovery and development take this course. to date, 38 countries have signed the who solidarity call, which countries such as the us and the uk have yet to become signatories. the role of research ethics in research on and with those most vulnerated by the covid19-pandemic: the case of elderly people in nursing homes older people in nursing homes are among the most vulnerable in contemporary society. this has become very clear in several countries during the covid19-pandemic. in may 2020 a report from bergen (one of the largest cities in norway) revealed that 87% of the city's covid-19 deaths stemmed from patients living in nursing homes. 122 and in sweden nursing home residents account for nearly half of deaths linked to 123 while future estimates in england are that more than half of coronavirus-related deaths will affect people living in care homes. 124 research on how to prevent deaths in nursing homes during a pandemic, and ways of minimizing the risk of contagion represent issues of great ethical and health political urgency. there are, however, several research ethical challenges related to the inclusion of nursing home residents in research projects because the pandemic has rendered them victims of additional vulnerability. 125 the more vulnerable 109 'cioms' stands for council for international organizations of medical sciences. for this, see cioms (2016). 110 ibid. 111 editorial in nature. everyone wins when patents are pooled. nature, 2020; 581: 240. 112 aj impact/europen union (2020). 113 novavax (2020). 114 cnbc (2020a). 115 cnbc (2020b). 116 dutchnews.nl (2020). 117 lazarus (2020) . 118 hoen (2020) . 119 covid-19 technology access pool, 2020. https ://www.who.int/ emerg encie s/disea ses/novel -coron aviru s-2019/globa l-resea rch-onnovel -coron aviru s-2019-ncov/covid -19-techn ology -acces s-pool. 120 ibid. 121 emanuel et al. (2020) . 122 bergens tidende: accessible at: https ://www.bt.no/nyhet er/lokal t/i/70vxd 4/26-av-30-doede -var-sykeh jemsb eboer e. 123 bbc news. accessible at: https ://www.bbc.com/news/world -europ e-52704 836. 124 the guardian. accessible at: https ://www.thegu ardia n.com/socie ty/2020/jun/07/more-than-half-of-engla nds-coron aviru s-relat ed-death s-will-be-peopl e-from-care-homes . 125 the differentiation between vulnerability and being vulnerated is important to distinguish between on the one hand persistent and on the other variable forms of vulnerability (solbakk 2011, pp. 228-238) . the persistent form of vulnerability we all share, is part of the human condition, while the second form of vulnerability is contextdependent, in the sense that some people because of disease, poverty, lack of freedom etc. are vulnerated, i.e. harmed or wounded. this distinction points to the need for a differentiation between at least two distinct regimes of protection. firstly, a human rights-based regime aimed at protecting persistent or universal vulnerability. this regime requires negative action on the part of the state, in the sense that its responsibility is to guarantee basic liberties by securing a just social order that gives equal protection to the vulnerability of each citizen. these protective measures are, however, in need of being supplemented by additional measures of protection-of affirmative actionto cope with accidental states and situations when human vulnerabil-a research participant is, the greater the risk of causing harm. furthermore, the inherent asymmetric power relation between the researcher and his/her patients fuels powerimbalance, about which we as researchers should be ever more concerned, many older and vulnerable nursing home patients, many of whom either lack the competency to consent, or are verbally deficient. the principle of consent is one of the most basic research principles. the un human rights committee notes that special protection is warranted when including persons not capable of giving valid consent. 126 in some cases a next of kin may consent on behalf of the patient, but since we cannot be sure whether this consent reflects the wishes of the patient him/herself, this poses a problem. we should therefore be aware of both verbal and non-verbal signs and reactions from the participants' signs of discomfort and resistance throughout the research process. here we will argue that researchers need a moral sensitivity, not only to avoid physical harm or risks, but also to avoid psychological and social damage. 127 it is not sufficient to conform to "procedural ethics" at the onset of a researchproject or to gain/secure approval from a research ethical committee. moral sensitivity in research may be even more important during pandemics, when research protocols seem to be "rushed" and misgivings ignored. in addition to moral sensitivity as researchers, we may also argue for a kind of ethical calmness or what guillemin and gillam refer to as "ethical reflexivity" 128 throughout the research process. the arguments presented above suggest that the role of research ethics in pandemic times is exceptionally important, but not only in the sense of deviating from hard won core ethical and epistemological principles in the wake of wwii. on the contrary, perhaps more than ever, it is vital to restate the importance of human dignity, human rights and fundamental freedoms as the normative bedrock on which medical research involving human subjects should rely. there is no alternative pathway for research ethics that is viable; returning to the core values and principles enshrined in the universal declaration of human rights is urgently needed. we therefore share the views expressed by the european group on ethics in science and new technologies in a statement on the covid-19 pandemic 129 : it is natural in these circumstances of deep uncertainty to focus on immediate action and speed of measures. this must not, however, lead to a continuous suspension of rights and liberties. we therefore call for vigilance about the necessity, evidence, proportionality of any policy and technological intervention that, even temporarily, suspends fundamental rights. consideration needs to be given to the immediate and lasting impacts that such measures have on our societies. funding open access funding provided by university of oslo (incl oslo university hospital). open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. for the scientific merit argument, see also bambery for this argument, see also eyal jamrozik and selgelid performing chi is a way to learn and test, while minimizing the number of subjects for this argument, see also eyal also eyal (2020, p. 26) highlights the importance of covid-19-chi-trial participants having "access to standardof-care life-sustaining treatments accessible at: https ://www. pharm acyti mes.com/news/fda-annou nces-two-drugs -appro ved-forcompa ssion ate-use-in-treat ing-covid -19; and, fda is allowing two drugs to be used for here's what that means publi c-healt h-focus /expan ded-acces s. for this, see also reference is here made to: department of health and human services. annex 11: pandemic influenza response and preparedness plan brief history and characterization of enhanced respiratory syncytial virus disease aj impact/europen union. 2020. eu may deploy emergency funds in covid-19 vaccine race optimizing the trade-off between learning and doing in a pandemic the jewish chronic disease case article 7 (prohibition of torture, or other cruel, inhuman or degrading treatment or punishment) ethics, reflexivity, and "ethically important moments" in research. qualitative inquiry statement on european solidarity and the protection of fundamental rights in the covid-19 pandemic footnote 125 (continued) fallen vulnerability" is a metaphor that has been suggested to designate such situations ethical criteria for human challenge studies in infectious diseases ethics review in compassionate use the medical profession & human rights. handbook for a changing agenda should we infect healthy people with coronavirus? international ethical 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1976 swine flu vaccination program interpreting diagnostic tests for sars-cov-2 ethical considerations for zika virus human challenge trial ethics of controlled human infection to study covid-19 the principle of respect for human vulnerability and global bioethics pandemic ethics: the case for experiments on human volunteers covid-19: act first, think later the international alliance for biological standardization infectious hepatitis. studies of its natural history and prevention declaration of helsinki human challenge trials for vaccine development: regulatory considerations key criteria for the ethical acceptability of covid-19 human challenge studies human challenge trials for a covid-19 vaccine: should we bother about exploitation? uncertainty and environmental learning-reconceiving science in the preventive paradigm long-term consequences of covid-19: research need key: cord-350569-dtxtjtfo authors: kasoka, kasoka title: autonomy in hiv testing: a call for a rethink of personal autonomy in the hiv response in sub-saharan africa date: 2020-06-13 journal: med health care philos doi: 10.1007/s11019-020-09959-y sha: doc_id: 350569 cord_uid: dtxtjtfo the author reviews various conceptions of autonomy to show that humans are actually not autonomous, strictly speaking. he argues for a need to rethink the personal autonomy approaches to hiv testing in sub-saharan africa (ssa) countries. hiv/aids has remained a leading cause of disease burden in ssa. it is important to bring this disease burden under control, especially given the availability of current effective antiretroviral regimens in lowand middle-income countries. in most ssa countries the ethic or value of personal autonomy or self-determination is promoted as primary in hiv testing decision-making. ssa policymakers have an ontological and moral duty to adopt hiv testing policies that reflect human and medical realities, relationships, local contexts, and respect human rights for both individuals and others who are affected by hiv in society. without rethinking the value of autonomy in hiv testing decision-making, the article cautions that attainment of the sustainable development goal (sdg) 3 and the unaids fast-track strategy that explicitly call to end the epidemic by 2030 will not be feasible for ssa. the case of covid-19 pandemic has more than ever presented to the world that individual autonomy in society is at best conditional and relational. the coronavirus intrusive measures taken to contain the virus expose the fundamental problem in metaphysics-the issue of what makes a person the same over a period of time-and the moral dilemma posed in bioethics by the fact that an individual who lives in society with others is promoted as a sovereign of her own body, medical choices and life. this dilemma might otherwise be expressed: 'what makes a person the selfsame person today as who he or she was yesterday' (ndete 2016) , and what does autonomy mean to an individual whose exercise of self-governance has inescapable ramifications on the wellbeing and rights of others. thus, should there be a need to rethink the personal autonomy 1 premising of informed consent requirements in hiv testing in most sub-saharan african (ssa) countries, since informed consent requirements in hiv testing are mainly premised on personal autonomy? childress 1979/2000; fraser 2005; united nations 2009; naidoo and vernillo 2012) . or there isn't a need to rethink because humans are, as kantian and related notions of autonomy tell us, normatively and narratively sovereign selves? in this article i seek to show that personal autonomy, strictly speaking, is an illusion, and its primacy in healthcare ethics is morally problematic. in most of ssa countries the hiv prevalence has stabilised at high rates. the ssa region carries a disproportionate hiv burden, accounting for 70% of the worldwide burden of infection (bulstra et al. 2020) . hiv/aids is a leading cause of morbidity and mortality in the region (dwyer-lindgren, et al. 2019 ). 2 this is despite that major scientific breakthroughs (availability of effective hiv therapy) that have shifted the hiv paradigm that was once was considered a death sentence are now mostly available in the region. even with the availability of hiv medication in ssa countries like zambia, aids still remains the most common cause of death. 3 and yet, individual citizens are still celebrated and told through hiv laws or policies that they have a right to refuse hiv testing because they are sovereigns of their own health, life and destiny (castell v. de greef 1994; southern africa litigation centre 2012) . therefore, does this temptation and pursuit to view ourselves as autonomous hiv testing decision-makers blind us to an observable reality that shows that as humans we are interdependent and inextricably social and socialised beings? three decades into the hiv epidemic, hiv still poses a real health challenge from which no country in the world is immune, particularly ssa countries. according to unaids' factsheet, at the end of 2019 37.9 million people worldwide were living with hiv (unaids 2020a, b, c) . the same source indicates that 74.9 million people as of the end of 2018 became infected with hiv since the beginning of the epidemic. of the 74.9 million infected, 32 million have since died from aids-related illnesses. this makes hiv/ aids one of the major causes of morbidity and mortality in the world. hiv/aids is the second leading cause of death in ssa (business insider 2017). given the threat of the epidemic, it is not surprising that all the united nations member states agreed to reach certain targets in response to the disease (suthar et al. 2013 ). these targets were set to be achieved by 2015. some of the targets included the reduction of sexual and parental transmission of hiv by 50%, elimination of vertical hiv transmission, reduction of tb deaths among people living with hiv by 50%, and delivery of art to 15 million people (suthar et al. 2013; udjo and lalthapersad-pillay 2015) . it was noted that the achievement of such goals requires that people test for hiv since it is only through knowledge of one's serostatus that one can be linked to prevention, treatment and care services (suthar et al. 2013) . it is encouraging to note that such efforts have led to significant degrees of progress in response to hiv. overall, hiv infections are approximated to have been reduced by 40% between 2000 and 2013, with 13.6 million people globally reported to have been receiving art as of june 2014 (united nations 2015) . at present, new worldwide efforts are being made to end the epidemic by 2030. on 8 june 2016 united nations general assembly (unga) member states adopted, also in the light of the 2030 agenda for sustainable development and other, a new political declaration that seeks to end aids by 2030 (un general assembly 2016; un news centre 2016). the declaration includes a set of time-bound targets to fast-track response to reach three identified milestones by this year (2020). the 2020 milestones being: reduction of new hiv infections globally to less than 500,000; ensure that 90% of people infected with hiv know their hiv status, 90% of people who know their status are on put on art, and 90% of those on art have suppressed viral load (unaids 2015 (unaids , 2016 un news centre 2016) . 4 according to unaids, scale-up of art has put the reach of the global commitment to end hiv by 2030 on track (unaids 2016) . moreover, kharsany and karim state that the substantial declines in hiv infections are the result of hiv testing scale-up and widespread coverage of art (kharsany and karim 2016, p. 35) . thus, it is apparent that the above unga targets can only be achieved if there is an increased uptake of hiv testing and counselling (htc), and increased access to hiv prevention and care services (suthar et al. 2013, p. 23) . recognising the critical importance of hiv testing, indeed one of unaids' 90-90-90 2020 targets is that 90% of of all persons living hiv will know their hiv status (unaids 2020a, b, c) . as of 2018, 79% of all plhiv knew their hiv status (unaids 2020a, b, c) . the importance of hiv testing uptake cannot be overemphasised. its advantages include: being able to initiate early treatment for those who test positive at the time their immune system is still strong; early diagnosis improves long-term survival rates; knowledge of one's hiv status helps in prevention of hiv transmission to one's sexual partners, foetuses, babies, caregivers and other; knowledge of one's hiv status prevents re-infections, and; a positive test even for those who have been diagnosed later means one can still have access to life-saving treatment (st maarten aids foundation 2003) . put differently, 'an hiv test opens the door to accesing a range of hiv prevention options available depending on a person's hiv status to keep themselves and their loved ones hiv-negative' (unaids n.d.) . hiv testing is indeed critical as an entry point for hiv treatment and care. 5 unaids 4 one of the principles of the fast-track approach also calls for change; that is, among other things, stopping what does not work when it comes to the hiv response 'and scaling-up proven programmes' (unaids 2015, p. 6). 5 'hiv testing services (hts) are the entry point for diagnosis and access to life-saving antiretroviral therapy (art). early diagnosis and initiation of art have been shown to drastically decrease viral load, which reduces individual morbidity and mortality, and limits onward hiv transmission. hts can also offer a pathway for primary prevention interventions, including programs that deliver pre-exposure prophylaxis, voluntary medical male circumcision, and prevention of mother-to-child transmission ' (al., 2019) . and who have confirmed that adherence to an effective art regimen can result in reduction (by 96%) of the risk of transmitting the virus to an uninfected sexual partner, and can cause viral suppression that will lead to a plhiv living a normal lifestyle and longer life expectancy. without art, plhiv develop aids as a result of a compromised immune system, thereby exposing them to development of infections, certain cancers, and other severe clinical manifestations (unaids 2016; un-desa 2017) . in this vein, i am in favour of programmes such as the 'who test and treat' policy in ssa. those countries in ssa which have already adopted and implemented this approach are therefore to be commended. the introduction of 'test and treat' programmes have significantly contributed to an increase in the number of people accessing treatment (skovdal et al. 2016; avert, 2019a, b; unaids, 2020a, b, c) . ssa governments can alocate money, including requesting for hiv programmes funding from international donors, purchase mobile clinics and hire testing community workers to go door-to-door in communities and educate people about hiv prevention and the importance of hiv testing to an individual and common good, and then offer counselling and on-the-spot hiv testing and art initiation whenever individuals consent (egpaf 2017; sulat et al. 2018; silberner 2019) . this should be done in combination with routine hiv testing in healthcare facilities. without further delay, before the preparation of this article, i was aware that issues surrounding personal autonomy in hiv testing are multifaceted -therefore they cannot be all explored in this article. thus, the scope of this article is limited. firstly i have not explored various cultural conceptions of autonomy in this article (for such see the position of, mbiti 1970; gaylin and jennings 2003; woods 2002 woods -2004 traphagan 2013; song 2018) who have suggested that the primacy of the value of autonomy has its roots in western liberalism (particularly american). i have only mentioned in passing the ssa traditional ontological outlook and its potential impact on hiv healthcare ethics. secondly, the scope of the present review does not include a review of issues surrounding autonomy in clinical practice, including whether it is even possible to actualise informed consent requirements in medical practice (for this see, the position of manson and o'neill 2007) . thirdly, i have not explored in detail issues surrounding personal autonomy, hiv stigma and discrimination, and hence, some may argue, the importance of personal autonomy in healthcare practice. i am persuaded that the subject area of stigma versus personal autonomy deserves a critical exploratory article of its own. nonetheless, i have written this article having bourne in mind existing study findings and reports on stigma and discrimination (april 2009; stangl and grossman 2013) , 6 which, by implication, indicate that humans are social beings affected by the social environment. extensive studies that have found that fear for hiv stigma compromise hiv testing uptake and/or art adherence are also arguably an indictment on how respect to personal autonomy in hiv healthcare should be viewed. if indeed humans are self-rulers and rational players, why should social stigma prevent them from making self-rational and moral decisions in directing their own hiv medical therapy for their own good, and direct their lives as they deem fit? why should patients be declared and imputed with personal autonomy and yet at the same time blame the impact of social stigma on preventing potential hiv service-users from seeking medical attention? is this not conceptually and practically conflicting? and lastly, this paper does not explore whether the approach to hiv testing and treatment should be different from how we approach cancer, malaria, tuberculosis, covid-19, etc., even in light of hiv treatment universal health coverage. for my positions on this, i agree with hiv exceptionalism conclusions that criticise the subjectivism tendency to separate hiv response approaches from broader health systems (de cock and johnson 1998; april 2009; oppenheimer and bayer 2009; smith and whiteside 2010; benton 2015) . i agree that hiv exceptionalism was necessary before access to and effective art was made available in ssa. indeed, the hiv exceptionalism force is losing its original power as hiv has become less threatening (smith and whiteside 2010) due to accessible and effective hiv medication-treatment which has now been made available for the majority of affected populations in ssa. 'through the combined efforts of people living with hiv, national public health programmes, global donors and a broad community 6 basing autonomy on guarding against stigma can be problematic. among other things, april (2009) arguing for opt-out hiv testing versus the issue of stigma noted: 'under an opt-in programme, ….[a person] remains oblivious of her infection and avoids any immediate repercussions from her community. yet, this only delays the consequences -she will inevitably progress to aids. in societies with high hiv prevalence, as in much of sub-saharan africa, it is all but certain that her community will find out the cause of her suffering. it will be precisely at the time of her greatest physical ailing and need for emotional support that she will suffer the burden of hiv stigma and discrimination. in the case of an opt-out programme, although her decision to test may not have been borne of her own initiative, her decision not to decline testing will empower her to control the circumstances of her disclosure and formulate a plan for addressing her disease'. of stakeholders, the number of people on antiretroviral therapy (art) rose rapidly across ssa, going from about 100,000 in 2004 to 15.4 million by the end of 2017' (nash et al. 2018, p. 1). 7 hence, almost all countries in ssa have adopted hiv national policies to treat all persons, regardless of cd4 cell count (ssa has an hiv prevalence of 25.7 million people) (nash et al. 2018) . the massive expansion of art treatment in ssa has continued to save millions of lives, hence a need to even advance more appropriate hiv testing ethics. the scope of this article therefore concerns itself with a review whether a person who can test for hiv and have access to art is an autonomous patient who should be encouraged to choose her own medical therepy for her own good. the chief aim of this article is to show by reviewing procedural, substantive, ontological and socio-relational autonomy theories that capacity or achievement of autonomy in human society is an illusion, strictly speaking. thus, premising informed consent requirements of hiv testing testing on personal autonomy is problematic, philosophically and ethically. i have demonstrated that since humans don't exist in a vacuum, but are born into society and live in families and communities with fellow human beings, the promotion of primacy of personal autonomy over the common good 8 is inappropriate. the value of autonomy has been recognised as the core of medical ethics and has been validated by court judgments as a primary good in a free society (faden and beauchamp 1986; planned parenthood v. casey 1992; airedale nhs trust v. bland 1993; c v minister of correctional services 1996; lewanika v. frederick chiluba 1998; huri -laws v. nigeria 2000; diau v botswana building society 2003; southern africa litigation centre 2012) . for example, emphasising its primacy, the south african high court even held that '[i]t is, in principle, wholly irrelevant that [the patient's] attitude is, in the eyes of the entire medical profession, grossly unreasonable, because her rights of bodily integrity and autonomous moral agency entitle her to refuse medical treatment' (castell v. de greef 1994) . compounding this, the worldwide media through television dramas and documentaries foster this perception that individual choices or wishes are/ought to be sovereign in medical decision-making (english et al. 2006) . the individual or health service-user's medical judgment is celebrated as a right to be make one's own decisions for one's own health, body and life (southern africa litigation centre 2012). the individual is held to be a master of her own body and destiny, and is free to resist any violations to her autonomy (diau v. botswana building society 2003) against others of whom she lives with in society. behind the promoted freedom, autonomy, choice, and personal rights now stands a particular vision of what is entailed in being a human being; social relationships and arrangements are only recognised as far as they are able to nurture the atomistic self (gaylin 1996) . and behind this vocabulary of a sovereign human being is the belief that human behaviour is voluntarily chosen, and that other people's conduct can be modified through rational argument (gaylin 1996; gaylin and jennings 2003) . the case of the covid-19 crisis shows otherwise. 9 my article interrogates the personal autonomy arguments and reaches a conclusion that the philosophy surrounding the value is problematic, as well as, it is silent on the ethics of the actual implications of an autonomous decision in hiv testing (selemogo 2010) . this article is, therefore, composed of three themed sections. it begins by giving a brief overview of the origin of the word 'autonomy' and its evolution, and its relationship to informed consent in healthcare ethics. the second section analyses the various conceptions of autonomy. thirdly, an alternative conception of autonomy is reviewed and advanced (a review of whether autonomy is social as opposed to individual). the paper concludes by referencing the implication of a social view of autonomy on hiv testing in ssa where hiv is an epidemic. jones (2020) more 9 in discussing the impact of the coronavirus crisis, tobias jones observes: 'it has been fascinating to see the speed at which other attitudes have changed. the indignation expressed towards people not respecting social distancing (from those who would never normally describe themselves as moralists) has been understandably shrill: here too we've suddenly realised that the wellbeing of the group is endangered by indifferent individuals, and that community -for which we've yearned for so long -means originally simply a pooling of duties…. [t]he philosopher and activist simone weil wrote that "the notion of obligations comes before that of rights. it's a complicated, but convincing case, and it seems to me that in the last month there has been a radical shift in the balance between rights and responsibilities that has changed the timbre of our lives. i've never seen so many news items about applause, or so many social media posts accompanied by clappy emojis. before, "in the absence of adversity", the psychiatrist and philosopher iain mcgilchrist said this month: "we grew flabby, selfish. we manufactured grievances that now can be seen for what they were." now, when people meet their obligations to us we're obliged' (jones 2020) . 7 in east and southern africa, 67% of adults and 62% of children living with hiv are on art (avert 2019a, b) . 8 in this article, i have used the word 'common good' to mean 'the sum of those conditions of social life which allow social groups and their individual members relatively thorough and ready access to their own fulfillment' (velasquez et al. 2014 ). the ' "common good" refers to those facilities-whether material, cultural or institutional-that' individual 'members of a community provide to all members in order to fulfill a relational obligation they all have to care for certain interests that they have in common' (hussain 2018) . and as john finnis held, 'respect for human rights is a requirement of justice and that "the maintenance of human rights is a fundamental component of the common good"' (finnis 1980 , cited in hussain 2018 recently argued that what the coronavirus public health responses worldwide have shown us is that 'wellbeing isn't individual but social', and that humans 'are not actually independent at all'. the origin of autonomy, its relationship with informed consent, and hiv testing in ssa jackson (2013) explains that the word autonomy is from the greek words autos (self) and nomos (rule), which originally was used to refer to cities' independent self-rule. 10 feinberg (1986) , among others, also have noted that the word has a greek origin which comes from the greek for 'self' and 'law', meaning the making of one's own laws. in tracing the roots of personal autonomy, taylor (2005) suggests that individual self-authorship has its roots in the romantic liberalism of john stuart mill (2015) , in which free development of individuality was promoted. feinberg (1986) states that personal autonomy has interrelated meanings, from one's capacity to govern oneself, to the sovereign authority to govern oneself. o'neill (2003) provides that the original view of autonomy in antiquity-meaning self-legislation-never referred to persons, but to property. that is, ancient autonomous citystates made their own laws, colonies being given laws by the colonising parent cities (o'neill 2003, p. 3) . so, unlike its original cities' self-rule application, autonomy has now been extended to indicate the self-governance of rational independent individuals. this extension to an autonomous human has been adopted in medical ethics, and universalised, incuding in ssa. patient autonomy is promoted through informed consent requirements. informed consent has become one of the fundamental principles in medical ethics and law (dhai 2008) . individuals are held to have inviolable bodily and psychological integrity. this body-mind integrity right is violated when a patient is afforded medical intervention: without informing her in a language she understands the nature of the therapy; she has not been told about associated benefits and risks; available options in respect to an intervention have not been disclosed, and; she has not been informed that she has a right to refuse (mcquoid-mason 2008) . unlawful medical interventions that do not respect patient autonomy can constitute assault at law (dove et al. 2017) . in common law, free and informed consent means an inclusive recognition and respect of a: patient's capacity (and competence) to consent; disclosure of information to a patient related to the nature and extent of risks incident to a medical intervention; patient understands risks involved; patient is informed about available medical options, and; patient voluntariliy consents to (or to refuse) a medical intervention (beyleveld and brownsword 2007; mcquoid-mason 2008) . put differently, the legal and ethical elements of informed consent are: capacity; disclosure; understanding, and; voluntariness (dhai 2008) . a healthcare professional who by commission or omission fails to respect these requirements may be held responsible for any injury resulting therefrom. in this vein, '[t]he traditional hippocratic belief that one could do almost anything on a patient as long as the principles of beneficence (best interests) and nonmaleficence (no harm) were upheld has been considerably' stamped by rational and moral individuals whose autonomous actions or choices take precedence (dhai 2008, p. 27) . the principle of informed consent in clinical practice is primarily a negative right of non-interference (schermer 2002; clarke 2009) . '[i]t is closely connected to a particularly western, post-enlightenment idea that an adult person is a bounded individual who is able to live her life freely in accordance with her self-chosen plan, and ideally independently from controlling influences' (dove et al. 2017, p. 150) . its underpinnings can be traced to the influential kantian and millan conceptions of autonomy-or humans as rational self-legislators who are ends in themselves (selemogo 2010) . thus, 'in the domain of western biomedicine, the epitome of personal autonomy is a patient expressing a decision that she has come to autonomously and independently' (dove et al. 2017, p. 150) . to this effect, potential hiv service-users are told that they have the right to 'make choices that meet with their own interests according to their own will…[because humans have] capacity to understand substantial information, form a judgment according to their own values and communicate with the physician freely about their wishes' (song 2018, p. 296) . in this article, i have not questioned the importance of informed consent in healthcare conduct, and in hiv testing in particular. what i question is the premising of informed consent requirements on personal autonomy. i hold that grounding informed consent on autonomy distracts attention from observable human reality-which is that there are various important aspects of and factors in life that obstruct and pose challenges to realisation of personal autonomy in society. thus, instead of promoting personal autonomy, i advance a promotion of greater cooperation between patient, healthcare professional and one'a family and community. beauchamp and childress' definition of an autonomous patient who has freedom from controlling influences is mistaken. different schools of thought have advanced various conceptions of what autonomy means. o'neill (2003) has enumerated such definitions: dignity, integrity, individuality, independence, responsibility and self-knowledge; liberty; self-assertion; knowledge of one's interests; freedom from obligations; absence of external causation; choosing one's own moral position and accepting responsibility for one's own choices; self-mastery, voluntariness; privacy; and choosing freely. in the following review, much reference has therefore been made to killmister's (2013) thoughts on autonomy because the author identifies critical aspects of autonomy that have been overlooked in various theses by celebrated luminaries. killmister's structure and analysis of the subject matter is instructive for the present review. hence, the current analysis will be restricted to a critical review of feminist accounts; kantian accounts of autonomy which have been embraced by a number of feminist thinkers will therefore be reviewed. although feminists reject kantian and rawlsian notions of autonomy due to the former's argument that autonomy is relational, there are still different theses within the feminist school of thought which are arguably similar to kantian conceptions-the only major difference being that such feminist scholars advance a relational account of autonomy due to their rejection of self-sufficiency of human beings. besides marina oshana's socio-relational theory being arguably a more persuasive feminist account of autonomy, other accounts arguably embrace kantian and rawlsian rational choice theories. i commence by considering the reflective or historic endorsement accounts of frankfurt (1988) , dworkin (1988) and christman (2009) . the theses of these great thinkers indicate that an individual's capacity for autonomy lies in her psychological dispositions. nonetheless, christman's reflective endorsement theory has been credited as an account that is placed to address the problem of socialisation-it is argued that his theory overcomes the inadequacies identified in procedural models such as those of dworkin (1988) and frankfurt (killmister 2013) . in fact, christman has argued that gerald dworkin and harry frankfurt's first and second-order desire theories overlook the autonomycompromising nature of manipulation. being autonomous, according to dworkin and frankfurt, is synonymous with a capacity to critically evaluate firstorder values, desires and preferences by referencing them to one's second-order preferences, values, and desires. thus, one's capacity 'to accept or attempt to change these in light of higher-order preferences and values' makes one autonomous (dworkin 1988, p. 20) . 11 it is held that by exercising such a capacity under second-order desires, persons define their nature, give meaning and coherence to their lives, and take responsibility for the kind of person they are (dworkin 1988, p. 20) . under this theory, second-order values ought to take priority over first-order ones; second-order values are considered to be an individual's realm of autonomous decision-making. i wish to argue that dworkin and frankfurt's second-order accounts are philosophically and, consequently, morally problematic. philosophically, this rational thesis of autonomy fails to convincingly account for the problem of socialisation of values, desires and preferences, internalisations of which compromise second-order values. a person's socalled second-order desires can be a product of socialisation. effective manipulation can reach all the way to second-order desires. thus, indeed 'to judge as autonomous individuals who are hypnotized, brain-washed, or otherwise manipulated into developing second-order desires…is to misunderstand the very nature of autonomy' (killmister 2013, p. 98 ). holton has illustrated: 'to see this [philosophical problem of second-order thesis], suppose that i implanted a second order desire in you by hypnosis… then surely you wouldn't have free will if you got your desires to conform to that; but frankfurt's account seems to have the consequence that you would' (holton 2003, p. 2) . 11 'a first order desire is a desire for anything other than a desire; a second order desire is a desire for a desire. so, for instance, you might have a first order desire to smoke a cigarette; and a second order desire that you desire not to smoke a cigarette… thus [another example], i might wish that i wanted to give all money to charity, since i might think that having such a desire would show me to be an excellent person; but i might nonetheless not actually want that desire to be effective… but when a person does want the first order desire to be effective, when they want it to be their will, frankfurt calls this a second order volition' (holton 2003, p. 1) . it can be inferred from this thesis that a person can act autonomously through identification with second-order desires. that is, a person is seen to have autonomy in so far as she has second order volitions and can bring her first order desires into line with second order volitions or desires. thus, according to this account, dogs and children don't have autonomy because they lack second-order desires or volitions (holton 2003) . they don't have autonomous second-order desires which can be used to control first-order desires. first order values can, for example, be values which form the edifice of a given moral system. thus, common good morality can be situated in first-order values (ockham's beard 2010) . frankfurtian and dworkinian accounts possess a great potential to declare someone who is acting on internalised values to be autonomous. such accounts seem to neglect the fact that external conditions (in the form of, e.g., societal rewards and punishments, and limiting human biological factors) have a tendency to make humans conform to certain behaviours (gaylin and jennings 2003) . this can be seen in the present case of coronavirus where, due to fear of receiving fines or fear of being viewed as social deviant, among other reasons, people who would otherwise wish to open their entertainment venues, go to clubs, restaurants, bars and other social facilities, or go on holidays cannot now do so. internalised first-order desires have consequences on second-order desires. internalised ideas, fears, and values which we later mistakenly come to identify as our own have inescapable consequences on our daily choices or preferences (lanier and henry 2010) . 12 a good example of internalised preference can be inferred from the 18-year-old student theorised by benson (1991) . benson explores the case of an 18-year-old whom through internationalisation of the hollywood idea that appearance is a criterion of self-worth ends up regarding beauty and fashion as essential for her self-worth. first and second-order autonomy accounts fail to explain away this lived social reality. although it is possible to hold individuals to have acted autonomously by virtue of fulfilling frankfurtian or dworkinian first and second-order requirements, it can also be argued that morally this thesis of autonomy may be amoral because it has the potential to ignore that well-being can only be achieved when both individuals and others in society act responsibly towards each other. the common good values which sustain and advance the well-being of society can be denigrated through such outlooks, and the consequences of this would be the annihilation of millions of human beings and the end of human civilisation as we know it, as each individual acts according to their own second-order values. what about christman's theory of autonomy which killmister claims is more persuasive? does christman, who criticises frankfurtian theories of autonomy, provide a more realistic perspective? according to christman's view, for an individual to claim autonomy 'necessary endorsement must be directed at the process of desire-formation, rather than the desire itself' (christman 2009; killmister 2013, p. 98 ). christman's theory of autonomy to me is equally an internalist or rational theory of autonomy. stoljar explains: for christman, preferences or desires will be nonautonomous only if they fail either a competence condition or a hypothetical reflection condition. competency corresponds to the capacity of the agent to form effective intentions relative to a desire as well as to reflect critically about the desire. the hypothetical reflection condition employs the notion of non-alienation to characterize authenticity and hence autonomy. (stoljar 2014, p. 235 ). killmister has illustrated and differentiated christman's account from other procedural accounts of autonomy as follows: for instance, we can take an individual who desires to eat a slice of cheesecake. rather than asking, as dworkin and frankfurt would, whether the individual desires to desire to eat a slice of cheesecake, christman asks how she would feel about her desire, were she to be aware of how the desire were formed. if the desire were implanted by a hypnotist, and knowledge of this process caused the individual to feel alienated from the desire, then that desire [according to christman] would not be autonomous. (killmister 2013, p. 98 ). for christman, for an action to be autonomous the individual must personally identify with the desire through a feeling of non-alienation. like frankfurtian and dworkinian visions of autonomy, i wish to argue that christman's endorsement account is unpersuasive. endorsement accounts of autonomy fail to account for internalisation of norms, values which an individual may mistakenly endorse or identify to be her own. indeed, the problem with christman's theory is not the 'inclusion of a historical version of reflective endorse-ment…, but rather that this condition is insufficient to determine the autonomy of an individual' (killmister, 2013 p. 100 ). christman's hypothetical reflection account fails to appreciate that due to deeply ingrained traditional values, imparted through oppressive (and i add non-oppressive) ideological socialisation over many years of one's life, individuals can identify with deformed desires and not feel alienated (stoljar 2014) . effects of ideological socialisation and environmental changes can cause one to treat stereotypes and acquired feelings as natural to oneself and thus formulate one's desires and plans based on such ingrained nonautonomous desires (gaylin and jennings 2003) . recipients of socialised ideologies 'are unlikely to experience alienation from either the 12 'one of the more astonishing features of current political debates on autonomy and coercion is the naïve underlying assumption about rationality and human motivation. psychology -whether based on behaviourism or on its diametric opposite and antagonist, freudianism (or any of the splinter of these two dominant branches) -views few pieces of human conduct as rational choices selected at the moment. rather, modern motivational psychology tends to see most human behaviour as being "conditioned" or "unconsciously determined," a consequence or product of life experiences that tend to make responses to certain stimuli automatic and unchosen…' (gaylin and jennings 2003, pp. 16-17) . norms that they have internalized or the preferences formed on the basis of the norms' (stoljar 2014, p. 235 ). mele's (1995) external historical account is a hypothetical counter-example to procedural accounts (killmister 2013 ). mele dismisses psychological (internalistic) accounts of autonomy (christman 1999) as insufficient. he advances that, if all individual human beings were like athena or a god, procedural accounts of autonomy would indeed capture what amounts to autonomy, and therefore his (mele's) historical account of autonomy would be unnecessary. however, because humans are not athenas, he argues that his alternative account is therefore valid or convincing and necessary (mele 1995) . i actually find mele's account of autonomy more persuasive than frankfurt, dworkin and christman's accounts put together. for mele, for an agent to be autonomous her actions must not be compelled by another: it is a historical property of agents required for responsibility for the possession of a pro-attitude. a necessary condition of an agent s's authentically possessing a pro-attitude p (e.g., a value or preference) that he has over an interval t is that it be false that s's having p over that interval is, as i will say, compelled -where compulsion is not arranged by s. (mele 1995, p. 166 ). the implication of mele's theory is that pro-attitudes can violate an individual's autonomy. to this effect, mele's theory implies that second-order desires (explained above) must not be a product of pro-attitudes or compulsion. he predicates his theory on an understanding that humans are susceptible to acting on socialised values (compelled or instilled pro-attitudes 13 ). according to mele, compelled pro-attitudes, therefore, make individuals non-autonomous (mele 1995) . put differently, an agent's choices are nonautonomous, according to mele's exposition, when: an agent comes to have a pro-attitude because of an external force, rather than via exercise of her skills for critical reflection and evaluative judgment; (ii) the instilled pro-attitude is one she is unable (in the absence of radical counterfactuals) to eradicate or attenuate; (iii) she did not arrange the by-passing herself; and (iv) she does not, nor did she earlier, possess other pro-attitudes that would support her endorsing the instilled pro-attitude. (killmister 2013, p. 101) the implication of this understanding of autonomy is that human motivation is susceptible to socialised pro-attitudes that are difficult to efface. mele's exposition of autonomy makes more sense as it touches on external factors that internalist accounts of autonomy fail to account for. indeed, a fully competent individual who is able or capable of acting rationally upon desires (values which she has critically reflected upon) cannot necessarily by this very token be safely considered autonomous, as the pro-attitudes (which she considers to be her own) could have been socialised earlier in life. however, i do not completely agree with mele's thesis regarding what he then advances as constituting autonomy. this disagreement lies in mele's advancement of an alternative theory of self-control as being a mechanism through which one can achieve autonomy. this theory (capacity for autonomy by virtue of self-control) appears to water down the fact that both inculcated cognitive biases and environmental changes can compose the very mechanics of self-control. 14 mele's self-control theory does not satisfactorily explain how the problem of socialisation of pro-attitudes acquired since childhood can be qualified in the assessment of what amounts to one's autonomous self-control. studies have established that babies do not have competent mental capacities for reflection during early childhood-a period when they acquire pro-attitudes. 15 killmister has argued that there is a problem in mele's theory if it is to be used as a guide for identifying 'autonomy-inhibiting socialization' (killmister 2013, p. 101 ). mele's approach would rule out most early childhood education directed at developing the very necessary mental capacities. killmister postulates that 'if the relevant capacities are inoperative or not yet developed, as they will be in very young children, mele takes them to have been bypassed' (killmister 2013, p. 70) . 13 a pro-attitude is a person's mental attitude (a feeling or opinion about someone or something) directed toward an action under a certain description; pro-attitudes may include moral views, desires, urges, aesthetic principles and economic prejudices (bunnin and yu 2004) . 14 despite an individual 'being perfectly able to appraise critically and act upon her desires and values, the perfectly self-controlled person may nevertheless be acting on desires and values that have been implanted into her by artificial means, ones whose development has bypassed normal reflection and awareness' (christman 1999, p. 96) . 15 'human beings are born biologically premature, so that unlike most mammals, many of our neurological and physical functions and all of our behavioral capacities exist at birth in potential form only. to come to fruition, these potentials must be molded by a social environment' (gaylin and jennings 2003, p. 34) . the conditions which influence our everyday adult conduct are set by our parents and culture early in life through a conflation of coercion, parental encouragement, emotional intimidation, conditioning, and other mechanisms (gaylin and jennings 2003 ). mele has nonetheless acknowledged that bypassing is common in infants but argues that such bypassing is not sufficient for compulsion unless the pro-attitude is also practically non-sheddable. to this, killmister has responded that many people have pro-attitudes (traceable to early childhood socialisation) that they are practically unable to shed. she claims that even our attitudes towards evidence, reason, and reflection are both presumably inculcated and nearly unsheddable: the problem here is that one of the central roles of socialisation is to inculcate the very pro-attitudes that are required for the kind of self-control mele sees as necessary for autonomy. to be self-controlled is to believe and desire on the basis of an assessment of evidence. this will rely upon having the appropriate pro-attitudes towards evidence and reasons, which must at some point have been instilled in the child. (killmister 2013, p. 102) . in our early development each of us is subjected to physical and social forces of which we are largely ignorant, over which we have no control, yet from which we acquire values, beliefs, motivations, and capacities for rational evaluation that subsequently guide our choices and actions. these forces "destroyed" any capacity to become a different sort of person with selfcontrol regarding any unsheddable pro-attitude that we happen to have. consequently, every unsheddable proattitude is compelled, and anyone with firm unshakeable principles of action ends up being inauthentic and non-autonomous. (kapitan 2000, p. 89 ). stoljar rejects procedural approaches to autonomy by replacing them with a substantive account (stoljar 2000 (stoljar , 2014 . she argues that procedural conceptions fail to appreciate that people who have been subjected to false and oppressive internalised norms cannot be autonomous. according to her thesis, oppressive socialisation can lead an individual to internalise false beliefs which the victim may later come to endorse as her own. decisions made on the basis of internalised false beliefs are therefore not autonomous, she declares. stoljar's thesis indicates that mere possession of false/ oppressive beliefs makes an individual non-autonomous. i wish to agree with killmister's observation that stoljar's theory is initially promising as it acknowledges that 'the question for all theories is what kinds of socialisation are incompatible with autonomy' (killmister 2013, p. 104) . i, however, find stoljar's account unpersuasive. what is problematic about stoljar's theory is that it reduces autonomy to an individual who is free from false/ oppressive socialisation (killmister 2013) . such a perspective fails to consider that for an individual to be autonomous she does not only necessarily need to choose her own moral position but also enjoys the absence of any form of external influences on her self-determination capacity. that an autonomous individual is a self-ruler, or a law unto herself, no matter what form of socialisation she underwent, should not be ignored. an autonomous individual freely acts in accordance with a self-chosen plan by virtue of self-law and/ or morality (kant 1785; gaylin and jennings 2003) . what the preceding arguments entail is that even nonoppressive internalised norms can still be non-autonomous, provided they were instilled or regulated by another or something external to the individual claiming autonomy. if to be autonomous is to be self-governing or self-directing or, in other words, to act on motives, values, and reasons that are one's own, then how can we restrict non-autonomous action to only false/oppressive socialisation? moreover, the fact that the person who is supposed to be a law unto herself is at the mercy of false/oppressive socialisation shows a lack of sovereignty, like that of the christian god, hence the illusion of autonomy. the dialogical theses by westlund (2009) and benson (2011) view a person to be a social self. according to these accounts, a person's answerability to others becomes the key condition for autonomy. in other words, autonomy is synonymous with a person's ability to have normative power or authority over one's decisions (mackenzie 2008) . autonomy thus becomes dispositional. what is encouraging about dialogical approaches is that they indicate that human beings are interconnected-in other words, social selves. as will be shown below, however, the thesis of dispositional autonomy is unconvincing because this theory propounds that a capacity for autonomy is premised on an individual's mental capacity. westlund argues that autonomy should not be understood in terms of an individual's psychology or history; rather it is constituted by a particular kind of disposition. her theory suggests that an agent's answerability to others is a key condition for autonomy. she rejects psychological accounts which designate an autonomous agent as one who critically reflects in an appropriate way in evaluating one's preferences, desires and motives. in other words, westlund rejects rational choice accounts of autonomy because such theories merely argue that autonomy is achieved when an individual engages in critical reflection or if an individual acts or chooses to act in accordance with desires which she has self-reflectively endorsed (friedman 2003; christman 2009 ). according to westlund, one's autonomy is instead undermined if one is unable to give reasons in defence for one's actions or choices (westlund 2009; killmister 2013) . so, an individual who readily answers for herself constitutes a self-governor or autonomous person, she suggests. according to benson, an autonomous act is achieved when a selfregarding attitude is expressed in one's willingness to take responsibility for one's actions (benson 2011) . in this case, if an agent claims authority over her own actions, then such an agent can be considered autonomous. these accounts suggest that certain emotional states and attitudes towards oneself are necessary conditions for autonomy. such approaches appear to require that reliance on critical reflection and judgment for autonomy is only possible if a person can endure criticism of her own sense of basic competence and worth. i wish to state that dialogical accounts of autonomy are philosophically and morally problematic. they are philosophically unpersuasive because such approaches ground autonomy on a person's ability to offer reasons for her choices. and these accounts are morally problematic because they are individualistic in outlook-such outlooks appear to promote autonomy on the basis of meeting moral obligations to self. 16 although westlund criticises psychological accounts of autonomy, i wish to argue that dialogical accounts of autonomy are equally psychological or internalist in substance. thus, the criticisms of rational choice accounts of autonomy can to a larger degree equally be applied to dialogical ones; both approaches clearly ignore or brush aside the reality of socialisation and environmental conditioning. kantian psychological or rational conceptions of autonomy often erroneously portray agents as causally and psychologically independent of environmental conditioning. westlund regards someone as non-autonomous if they defer to something or someone rather than providing their own reasons in choice-making; however, she appears to ignore the fact that defending a choice with plausible reasons is not necessarily synonymous with being its author, or authentically identifying or individually valuing a given choice. an agent can defend, for example, oppressive values, not because that is what the agent desires, but because it is required. external circumstances can cause an agent to embrace oppressive values and yet still be in a position to stand behind a given decision as their own. thus, being answerable to one's choices or behaviour does not make one autonomous, but potentially makes one accountable to socialised values which one's external environment through interpersonal and environmental conditions have imposed upon or inculcated into the agent who is claiming autonomy. a person can give a convincing or persuasive reasoned answer to something not because she necessarily has chosen the value and identifies with it, but because of environmental conditioning and limitations. an agent is capable of defending inculcated or internalised oppressive values through adaptive preference formation (elster 2016) . 17 expressed differently, the problem with westlund and benson's conception of autonomy is that individuals can stand behind decisions or actions which are paradigmatically non-autonomous. this is especially true seeing that it is widely accepted that 'we humans are very good at inventing post-facto explanations of our actions, even to ourselves' (moll 2004 , cited in killmister 2013 . killmister (2013) concludes by suggesting that we do not have absolute autonomy, but 'degrees of autonomy'. i hesitantly agree! one agrees in a strict sense that 'degrees of autonomy' can be in the form of restricted autonomies in which individuals have been placed under laws where boundaries have been drawn on how far they can conduct themselves. one may as well call this 'permitted autonomy'. it is within the boundaries of laws (natural or positive), ethics, regulations and conventions where one can agree that 'degrees of autonomy' are apparently found. thus, yes, we humans have no absolute autonomy. for example, the current covid-19 lockdown and social distancing rules indicate that human autonomy is not absolute. being the law unto oneself is limited to what governments proclaim are boundaries within which individuals can be allowed to rule themselves. however, founding autonomy on limited or relative autonomy also crumbles when the whole picture of human motivations, in light of the ever-changing environment, is closely examined. thus, can killmister convincingly enumerate in what circumstances exactly an agent can enjoy this limited autonomy, circumstances which are excluded from the susceptibility to socialisation, internalisation, biological influences or environmental changes? gaylin (1996) has established that human motivation and behaviour is highly susceptible to environmental influences. therefore, he argues that protagonists of autonomy are mistaken in believing that human behaviour is voluntary. gaylin submits that present behaviour is moreover significantly determined by past treatment (gaylin 1996) . he suggests that human behaviour is less rational than we are willing to admit. he advances that our behaviours are influenced by our emotions, which are usually more effective than logical argument, and adds that shame, fear, guilt, greed and pride influence human behaviour. the implication of this analysis is that a combination of societal morality (morality which can be internalised through effective socialisation), the institution of law in society which defines the boundaries of human behaviour, and human natural susceptibility to emotions (as drivers of decisions) makes the argument for capacity for individual autonomy, even in limited circumstances, problematic. discussing the stifling nature of our biological make-up on human decision-making, morison (1984) has observed that besides historical influences human 'agency' is encumbered with genetic or environmental factors which subtly motivate our day-to-day choices. for morison, much of our human behaviour is significantly influenced by events over which we as individual have little or no control. he quotes melvin konner's the tangled wing: biological constraints on the human spirit (1982) 18 work as an extraordinary book which identifies, among others, the roles genes and the environment play towards behaviours and choice: it requires only a little reflection to realise… that our choice is strictly limited to the possibilities displayed before our conscious in real time including those we can call up from memory or create by synthesizing bits and pieces of previous experience… not nearly so clear is the degree to which the final choice is also so conditioned or "shaped" by genetic patterning or by previous experience of which we are no longer conscious. my hunch is that such influences are much more numerous than we imagine and that autonomy in any strict sense is an illusion. (morison 1984, p. 46 ). my review of autonomy so far agrees with feminist relational accounts to the extent that feminists generally highlight 'that agency cannot be separated from interdependence, since large swaths of our lives, especially during the formative years, are spent dependent on others, and those relationships function to inform our preferences, our values, and our understanding of our selves' (wenner 2020) . hence, i have contended above that autonomy cannot be conceptually explained without convincingly explaining away the impact of human social embeddedness and environmental change on human capacity for autonomy. i have thus shown that feminists' relational account analyses are only insufficient to the degree that they theorise that humans can be autonomous by virtue of a psychological state of mind. in the following discourse, i will show that autonomy is a social phenomenon. crittenden (1993) has observed that critics of liberalism, and even some liberal theorists, have criticised the emphasis of autonomy by liberalists. indeed, internalist conceptions of autonomy are not only conceptually unconvincing, but also, since they are associated with self-sufficiency, they exude 'the worst aspects of atomistic individualism, insulation, social isolation, and an indifference to society's values and interests' (crittenden 1993) . the fact that humans are born into society, are interdependent, co-exist, and therefore have to act in solidarity for the common good is almost ignored in rational theories. humans in psychological theories of autonomy become laws and morality unto themselves. crittenden, like gaylin, acknowledges that human beings by nature are interdependent and interconnected. hence, he argues that a conception of autonomy which is premised on self-sufficiency does not represent a fulfilment of autonomy but its abandonment. he submits that autonomy has a social nature and explains that autonomy develops through sociality, and it also requires sociality for its exercise. crittenden quotes anthony arblaster, who has noted that it is not normal or even desirable for a human to be self-sufficient (arblaster 1984) . he also invokes jennifer nedelsky, who submits that 'the perfectly autonomous man is thus the most perfectly isolated' (nedelsky 1989) . for crittenden, autonomy does not entail isolation from the presence of the influence of others or a necessary rejection of societal values, but the very concept of autonomy depends on these very factors. 'the social nature of autonomy appears to undergird a communitarian notion of the self as socially situated' (crittenden 1993, p. 37) . the author argues, among other reasons, that he believes autonomy is social because humans are not born with autonomy, but that autonomy requires psychosocial development. he also argues that 'autonomy is social because we can only be autonomous when we know we are acting autonomously; and we can only know that when we give an account to others of how we arrived at a decision or action' (crittenden 1993, p. 38) . according to crittenden, the only autonomous person is one who is able to remove herself from the social matrix. he argues that since no person is born autonomous; we may 18 konner has demonstrated that man's behaviour is influenced by nature and nurture (konner 1982) . only be autonomous through socially distancing ourselves from the social matrix. he suggests that self-autonomy, provided one was born and lives in society, is impossible: any kind of introspection or reflection must be done in and through language, and the language we use is itself a cultural or social inheritance. we do not create it, and in this sense also autonomy has a social side… to make our actions and judgments intelligible to ourselves, we not only translate them into language, but also form them through language. the only language we have available by and through which to think rationally and self-reflectively, is one based on cultural tradition… there is implicit in every language, as part of that cultural tradition, a system of norms and standards that determine proper use. (crittenden 1993, p. 44 ). i agree with crittenden's theory of social autonomy, even though there are issues which arise from a further reading of his account which i do not necessarily agree with. when crittenden suggests that an autonomous person is one who is able to distance oneself from the social matrix-a matrix which one earlier identified with-this perspective appears to suggest that an individual is capable of extricating herself from the social context, and from her genetic make-up. if crittenden means that an individual can extricate herself from the environment (a reality which has made that person who she is), i argue that doing so is not possible in reality due to biological constraints, interdependence, common human frailty, and socialisation. in other words, because all human beings have internalised pro-attitudes, it is impossible for them to disown or separate themselves from what they are personally disillusioned to believe are their own personal values and aspirations, if they later on choose to live by themselves. that is, even if a person later decides to live alone, like the asocial koala which only has social intercourse with other koalas during the breeding season, that person very likely already has embedded pro-attitudes which they will inadvertently carry with them, unless they were nursed by a koala as a baby and have since lived alone in the jungle. feminist theorist marina oshana grounds autonomy on a socio-relational premise. oshana submits that autonomy is equivalent to self-governance (she states that the synonyms for self-governing are: 'free-standing', to be independent (self-sufficient), to be separate, and to be self-ruling and sovereign) (oshana 1998) . she rejects psychological conceptions of autonomy and advances an externalist account. she invokes both the internalist (psychological) and externalist (socio-relational) perspectives in her conceptualisation of what autonomy is. oshana submits that individual self-governance is in reality social. for oshana, 'autonomy is a condition of persons constituted, in large part, by external, social relations people find themselves in (or in the absence of certain social relations)' (oshana 1998, p. 81) . in this sense oshana's conception is to some extent similar to crittenden's theory. however, unlike crittenden, oshana adds that manipulation, coercion, subjection to the dominant will of others, and also 'the internal phenomena native to the individual such as captivity to desires or physical impulses, psychological neuroses, or weakness of the will' compromise the capacity for autonomy (oshana 1998, p. 83) . oshana criticises dworkinian, frankfurtian and christmanian accounts of autonomy as inadequate because these accounts are internalist. indeed, as shown above, rational accounts of autonomy premise capacity for autonomy on the 'structural and/or historical character of a person's psychological states and dispositions, and on an agent's judgments about them' (oshana 1998, p. 83) . for oshana, an individual does not become non-autonomous merely by being born in an environment where one has no control, but one becomes non-autonomous by virtue of the effects of the conditions of a given environment. she maintains that choice does not guarantee autonomy. this is because a person who may be exercising what appears to be genuine choice may be compelled to do so by others, and endorsing such choices from within does not guarantee autonomous agency (oshana 1998) . in regards to the assumption that critical reflection in a procedurally independent fashion makes one's choice an autonomous one, oshana argues that 'being able to engage in critical reflection, to take stock of oneself and to shape oneself on the basis of this evaluation, does not guarantee that whatever state of affairs ensues from this activity will be one of autonomy' because autonomy does not exclusively depend on circumstances descriptive of an individual's psychology (oshana 1998, p. 89 ). oshana has therefore advanced four conditions necessary for autonomy: critical reflection, procedural independence, access to a range of relevant options, and the condition of social-relational properties. she argues that an individual cannot be autonomous if: (i) one cannot engage in critical reflection or objective appraisal of one's motives or actions, and the environment in which one's putative values develop; (ii) if she is, in fact, influenced or restricted by others in ways that constrain autonomy (such influences can be by way of manipulation or coercion); (iii) that autonomy becomes questionable when a person claims that their decision was autonomous in circumstances where they were lacking in access to an adequate range of options; and, (iv) an individual who is in society (having relations with others) does not limit herself to relations which enable her to pursue her goals in a context of social and psychological security (oshana 1998) . she submits that these four conditions for autonomy are not mutually exclusive; they must all be satisfied for an individual to claim autonomy. for oshana, her account of autonomy has three benefits: it recognises the human condition; it recognises the status of humans as moral agents; and, that a socio-relational account of autonomy 'can easily explain how persons might be selfgoverning even when manifesting external or communal virtues that might appear to reduce autonomy' (oshana 1998, p. 98) . the socio-relational conception of autonomy is more convincing, philosophically and morally. it not only appreciates that humans are unique, but also recognises that humans are only able to exercise their uniqueness or the 'self' 19 in a socialised world encumbered by fear, emotions, the need for social validation, a changing environment, and the need to act responsibly in order to protect and promote the common good for mutual well-being and survival. ravven (2013, pp. 336-411) has shown that mounting data from history of ethics and neuroscience demonstrate that a human being is in reality the 'i that is we'. she states that neurobiological and other evidence have shown that the body-mind boundaries ethics celebrated as inviolable by millan and kantian ontological theses are mistaken. ravven demonstrates that the dimension of the human self extend beyond the scope of the body-mind, and that human investment in others in the world is finally what ethics is all about, and should be acknowledged as such. we are made for togetherness. throughout, her analysis, she convincingly demonstrates how locating the self as distributed beyond our skins and brains can provide a more plausible and appropropriate ethical and moral approach to hiv testing decisionmaking ethics. for ravven, 'the scope of the self as a moral agent, of who is performing a given moral action, can be distributed beyond the individual to groups, and even extend at times to whole contexts'. this indicates that hiv testing decision-making is in reality complex. firstly, by citing the work of clark (2009), ravven has demonstrated that the self as we understand it is joined to society and is one with the world. this is in contrast to individualised informed consent requirements which advance a self self. according to clark (2009), reported by ravven, 'at least some aspects of human cognition… [are] realised by the ongoing work of the body and/or the extraorganismic environment,' so that the 'physical mechanisms of the mind… are not all in the head' or in other central nervous system. ' ravven explains that our attraction to selfhood fails to account for an observable reality that demonstrates that as humans we are not 'locked-in' agents-as beings whose minds and physical abilities are fixed quantities apt (at best) for mere support and scaffolding by…[our] best tools and technologies.' instead, 'our minds and bodies are essentially open to episodes of deep and transformative restructuring in which new equipment (both physical and 'mental') can become quite literally incorporated into the thinking and acting systems that we identify as our own minds and bodies.' she emphasises and advances that humans do not only discover the world within their selves, but also discover themselves as parts of it. this entails that humans ought not to conceive themselves as the whole story of the whole, but as part of the story that makes the whole story complete. this outlook is similar to ssa ontological perspective that situate a human as 'a part of the organic whole' (mbiti 1970; menkiti 1984; diop 1991; tutu 1999; nussbaum 2003; woods 2002 woods -2004 molefe 2019) . therefore, an hiv testing regime that locates hiv as a shared reality will also adopt appropriate ethics and human rights that intergrate this reality. current hiv testing ethics and human rights on decision-making in ssa have not reflected this human reality (eba 2015) . secondly, ravven shows how a person is an 'i that is we' by invoking co-consciousness empirical findings that have demonstrated that 'the self as a fully aware self-aware includes perspectives that were initially 'other': third person perspectives now taken in as one's own have relocated the self outside itself and in another'. she suggests that as humans we create a shared self-a self that is both mine and yours-when we unconsciously integrate our people's values and perspectives with our first person perspectives. she therefore concludes that humans are at best relational selves who discover the self through other's eyes with whom they have a shared reality, and a shared world. ravven indicates that humans share an environment of inescapable embeddedness. thus, ethicists, including hiv policymakers in ssa, should begin to acknowledge and accept that the self cannot exist in a social vacuum. the discovery that the self is not atomistic therefore 'heralds the end of the…cherished illusion… namely the illusory goal of independence, self-sufficiency, and free autonomy' promoted in hiv testing ethics which seek to disintegrate the self from her social embeddedness: at any given time the self is constructed by a relation to another person. 'people have as many selves selves as they have significant others.' these selves emerge from the early relationships with parents, siblings, extended family members, and others who have an impact on one's life, and they profoundly affect motivation and emotions. unconsciously perceived similarity triggers the relational patterns to take hold, without our conscious awareness or even the ability to control the process… the other and the relationships are necessary for the feeling of 'me' of this 'me'… heinz koht [a psychoanalyst]… held that it is through 'expanding its selfexperience to include the whole surround' that an infant comes to be able to (1) feel itself confirmed as a self, (2) pursue its ambitions, and (3) pursues its ideals. (ravven 2013, pp. 369-370) . thirdly, describing findings from neurobiological experiments, ravven reports that research by thomas metzinger and olaf blanke have also demonstrated that the boundaries of the self transcends the boundaries of our bodies and minds: we mistake the feeling of the self as our interior, a soul-thing, a solid bounded essence that is our true self that we alone know and disclose to the world. but that is a false picture. we are more like verbs than nouns; we make parts of the world feel like the self, and we fill our feeling with our engagemnets in the world. (ravven 2013, p. 372) . fourthly, ravven turns to neurochemistry discoveries, like the one by donald w pfaff, that have also illustrated that the self transcends body-mind. and suggests that ethics should be grounded on relational autonomy: pfaff shows that the feeling of the self and where we draw the line between the self and other, self and the world, depend upon particular neurochemicals. these chemicals that produce the self-other divide, he says, are sometimes turned off, and when they are we experience others as if they were ourselves. the neurochemistry that produces the feeling of self as extending into others, he argues, underlies ethics. he says the chemicals that turn off the feeling of self-other boundary operate in fear and in love, in empathy and in aggression, and in other situations. these chemicals create a sense of shared experience and even of merger. pfaff believes that this mechanism produces ethical action by creating empathetic responses to others. (ravven, 2013, p. 373 ). the dominant, individualistic understanding of autonomy that features in' bioethics and medical ethics underpin the idea that individuals are 'in their ideal form, independent, self-interested and rational gain-maximising decision-makers' (dove et al. 2017, p. 150) . my review of autonomy in this article demonstrates that such a view is erroneous as it does not convincingly account for both external and internal (influences of biology on decision-making) environmental factors. this individualistic conception of an autonomous patient is inconsistent with lived human life-it does not capture the breadth of lived human reality that shows the 'i that is we'. conceptions of autonomy, especially in clinical practice, 'should accommodate the fact that people are rarely, if ever, fully independent individuals' (dove et al. 2017, p. 151) , hence, as an example, the challenge posed by hiv stigma to hiv testing uptake. as humans, we are social, cultural and biological beings whose decisions, including in healthcare, are shaped by a conflation of powerful social, cultural and biological forces that often times are beyond us (konner 1982; gaylin and jennings 2003; ravven 2013; sapolsky 2017) . this is the more reason why we need each other as humans: as opposed to atomising ourselves. hiv testing ethics in ssa should be premised on the human reality of a self beyond itself, as this approach captures the physiological, biological, pyschological, sociological and cultural aspects of human condition; unlike the kantian and millan conceptions of personal autonomy which are largely abstract psychological ontologies. instead of advancing abstract ethics of a self within self and unto self, ssa hiv policymakers have also something to learn from ssa traditional and moral analysis that view a person as 'a person through other persons' (mbiti 1970; menkiti 1984 )-an outlook that was developed in the light of ssa's unique experiences and lived reality, besides ontological deliberations. this ssa outlook is consistent with the conclusion of my analysis in this article that suggests that humans are incapable of being autonomous-they are social creatures who constantly, also unconsciously, simulate the values of their socio-cultural environments as their own. drawing ethics that recognise and reflect environmental impacts, including of love, empathy, fear and hate, on individual decision-making, will therefore be a good starting point for reconfiguring individualised hiv testing informed consent requirements in ssa. hiv testing policymakers in africa should cease ignoring the impact of the illusion of personal autonomy on our shared humanity. rather, they should explore their own lived experiences and reality, review studies that indicate that as human we are part of the organic whole, explore potential ethics grounded on the recognition of social attachments as evidenced in sexual and parental love, and even in friendship, resist the illusion of autonomy, and advance realistic ethics where the individualised cold isolated person is restored to who she really is: a unique part of an organic whole. 'parent love and especially motherly love, 'take the blurring of identity between two living beings to new heights,'… [ -] the upshot of all this is that the various mechanisms that induce sexual and parental attachment can be harnessed and be recruited for all kinds of prosocial behaviours and attachments' (ravven 2013, p. 375) , as opposed to surrendering hiv testing ethics to the seduction of autonomy. to this end, i wish to invite ssa hiv policymakers to consider basing hiv testing on altruism, or what ravven (2013) terms, an ethic of: 'shared selves'-'a sefiness that loses the boundaries of the skin and is extended and distributed to others'. a self who is born into society, is interdependent, interrelated, and shares with others shared human frailty and vulnerability. recognising this kind self can lead to an appropriate reconfiguration of hiv testing ethics. hiv testing ethics, in particular informed consent requirements that are now premised on personal autonomy, should reflect a human being who is unique and yet a creature of the inescapable inculcating environment that makes her the 'i that is we'. according to ssa traditional and moral thought, 'i am because we are', and 'we are because i am'. ravven states: mounting data from neurosciences show that evil is rooted in the failure to see others as ourselves. in the case of genocides such as the holocaust, it is a collective failure of society-wide proportions. the self-other boundary beyond the skin is of central importance to rethinking of ethics, to a deeper understanding of both good and evil. (ravven 2013, p. 377) personal autonomy cannot provide a sufficient and persuasive starting point for bioethics (o'neill 2002 (o'neill , 2003 , in particular hiv testing decision-making. it is necessary for ssa countries to reconfigure their healthcare ethics and place them within the overall compass of human condition, and in the light of the setting of ssa's unique socio-economic and cultural background (wood 2002 (wood -2004 ravven 2013; joseph et al. 2018) . it is appropriate to get rid of abstract liberal and libertarian concepts of an autonomous patient and reconsider the observable and lived situation of the relationship between patients and their environments. the emphasis on primacy of patient autonomy distracts attention from important aspects of life and healthcare: 'wellbeing is social'. it is crucial to eradicate the man-made autonomy versus medical intervention crisis and move towards greater cooperation. hiv testing informed consent requirements should be premised on ethics of love, honesty, empathy, sympathy, friendship, togetherness and solidarity. personal autonomy is an illusion. a review of the universalised dominant western liberal view of personal autonomy demonstrates that the general agreement that informed consent in hiv testing is required to respect personal autonomy 'and that autonomy is a basic ethical value, is more apparent than real' (manson and o'neill 2007, p. 17) . personal autonomy, strictly speaking, is an illusion. human lived experience and reality show that our lives and decisions are encumbered by combined forces of socialisation, genetics and changing external environmental factors that make the idea of an ability or capacity for autonomy resemble a fantastic dream which, when we wake up, we realise was just a dream and not something achievable in a real, civilised and striving society. in this vein, universalised ethics which continue to premise informed consent requirements in hiv testing on personal autonomy are inappropriate. human beings are not asocial or atomistic that they can be a law unto themselves; they are individuals who are enmeshed in complex social realities of reciprocity, mutual obligations, and responsibilities for amicable co-existence, well-being and survival. moreover '[h]uman behaviour is less 'voluntary' than libertarians and theorists of autonomy would have" us believe (gaylin and jennings 2003, pp. 7-8) . as indicated in the analysis, "human behaviour is less rational than most of us would like to believe it is', in that 'fear, greed, shame, guilt, and pride fuel the machinery of' our decision-making and choices (gaylin and jennings 2003, pp. 7-8) . it is these aspects which should also be acknowledged and reflected in hiv testing informed consent requirements in ssa. like what has been shown in the covid-19 pandemic response, persons living in ssa countries should be made aware that as social beings and living in complex relations with others in society, they have a duty to test for hiv because hiv is an epidemic that affect the lives of both people living with hiv and others (especially, close family members). the hiv epidemic not only affects the person living with hiv, but causes suffering, and some instances even death, to family members and people in the community. hiv testing, like the involuntary covid-19 lockdowns and social distancing, is a common good. in other words, the effects of aids are often corporately felt within the arena of the 'i that is we'-especially in the ssa setting where relational autonomy is articulated (woods 2002 (woods -2004 . in conclusion, whenever we as humans are faced with the temptation to celebrate our 'individual autonomous right', we should pause, consider, and remember that we are because of the humanity of others: our birth came through others; our name was given to us by others; we have been educated by others; the respect we demand is given by others; the first bath we had was given to us by others when we were born; our last bath will be given by others; our funeral and potentially dignified burial will be organised and conducted by others; and everything we have owned will be inherited by others once we are dead. we are simply not autonomous as individuals; we are but interdependent and interconnected social creatures. therefore, we should act in solidarity towards each other by formulating appropriate hiv testing ethics and encouraging people to test for hiv so those who need it can access appropriate therapy. the covid-19 crisis has taught us that 'wellbeing isn't individual but social' (jones 2020) . this indicates that ssa countries may not be able to achieve the sustainable development goal (sdg) 3 (united nations 2019), together with the unaids' fast-track strategy that explicitly call to end the epidemic by 2030 (unaids 2014), if hiv testing policies in ssa continue to ignore that humans are social beings (gaylin and jennings 2003) and 'wellbeing isn't individual but social' 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without forgiveness assessing the achievements of the millenium development goals in southern africa resolution adopted by the general assembly on 8 un general assembly adopts popitical declaration to fast-track progess on ending aids fast-track: ending the aids epidemic by 2030 understanding fast-track: accelerating action to end the aids epidemic by 2030 unaids. 2020. 90-90-90: an ambitious treatment target to help end the aids epidemic unaids. 2020a. global hiv & aids statistics-2019 fact sheet test and treat showing results in uganda and zambia the benefits of knowing your hiv status report of the special rapporteur on the right of everyone to the enjoyment of the highest attainable standard of physical and mental health-a/64/272 united nations united nations division for social policy and development, indigenous peoples united nations department of economic and social affairs (un-desa) the common good non-domination and the limits of relational autonomy rights as slogans: a theory of human rights based on african humanism acknowledgements this paper was prepared thanks to study support from birkbeck, university of london, uk during the author's phd studies. the author is also grateful to prof. matthew weait, deputy vice-chancellor -university of hertfordshire for his supervisory support during research on the subject matter examined in this article. funding none.data availability not applicable.code availability none. conflict of interest the authors declared that they have no conflict of interest. key: cord-337659-x4oywbrj authors: wilson, brenda a. title: global biosecurity in a complex, dynamic world date: 2008-07-31 journal: complexity doi: 10.1002/cplx.20246 sha: doc_id: 337659 cord_uid: x4oywbrj biosecurity is emerging as a major global health priority for which innovative and unprecedented solutions are needed. biosecurity is a challenging biocomplexity problem involving multifaceted processes such as interactions between humans and nonhuman biota, anthropogenic environmental and ecological factors, and socioeconomic and political pressures. key to an effective biosecurity strategy will be fundamental understanding of evolutionary, anthropogenic and environmental driving forces at play in transmission and perpetuation of infectious diseases. biosecurity solutions will depend on increased support of basic biomedical research and public education, enhanced healthcare preparedness, alternative strategies for ensuringsafety, and improved interagency cooperation regarding global health policy. © 2008 wiley periodicals, inc. complexity, 2008. i t is widely accepted by historians that there are certain dates upon which history seems to pivot, turning points that forever change the course of future events. the momentous months of september and october 2001 mark such a critical period in our recent history. our world as we knew it shifted, not just for the united states, but all nations. as a consequence, defense against bioterror agents came to the forefront as a major health priority in the u.s. and elsewhere. indeed, we were still reeling from the impact when sars swept across the globe in 2002-2003, followed closely behind by the still ongoing worldwide spread of avian flu and the concomitant fear of it transforming into a human flu pandemic on the scale of that experienced in 1918. but, these events are only at the pinnacle of a mounting number of impinging natural and imposed biohazards (table 1) [1] [2] [3] [4] [5] . importantly, these manmade and natural events have revealed a number of glaring gaps in our knowledge about infectious diseases, their transmission and perpetuation, and how to effectively combat them. existing and looming biological threats have now made biosecurity, which includes biodefense, the most pressing global health priority. in a rapidly changing world, biosecurity is at the intersection of every sphere of medical, biological, ecological, socioeconomic, and political system. although one might argue that the principal difference in the infectious disease threat today versus say 10, 25, or 50 years ago is bioterrorism, the resources spend on preparing for a bioterror attack is viewed by most scientists as grossly exorbitant [6] , particularly considering the small numbers of individuals who have been or could be affected by this type of attack and considering the relatively low medical relevance or prevalence of the diseases caused by the limited number of highpriority bioterror bioagents, the socalled ''category a select agents.'' and, while admittedly the preparedness and surveillance measures put in place for one has certainly helped to protect against the other (the improved global response to and curtailment of sars coming after the anthrax bioterrorist attacks is a prime example of this), most scientists feel that the limited resources available from an already overburdened system should instead be used for studying and preparing against the looming and potentially more devastating infectious disease threats from natural or accidental exposure [7] , which could affect millions of people and animals and could have huge health and economic consequences. and thus, while the threat of bioterrorism must be considered, many scientists propound that the focus should be on the more urgent and dire problem of biosecurity, rather than just bioterrorism. it has been argued by many that there is no better creator of new highly potent biological threats than nature itself. however, there is also little doubt that anthropogenic environmental, socioeconomic, and ecological influences can have devastating impact on the extent and severity of the outcome of these natural biological hazards. risks to public health come from diverse scenarios ranging from epidemics to outbreaks during natural disasters to accidental exposures through poor food processing to deliberate releases or fear thereof ( table 2) . finding solutions to these challenging biocomplexity problems will require integrated, multilevel, flexible, and interdisciplinary approaches that stretch traditional concepts. key to an effective global biosecurity strategy will be improved detection, prevention, treatment, and management of infectious diseases, but also better understanding of the intrinsic and extrinsic factors that contribute to their virulence and influence their transmission, prevalence, and perpetuation. thus to achieve this, we first need a better understanding of the critical evolutionary, anthropogenic, and environmental driving forces that contribute to natural and man-made biological threats. to gain a sense of the potential impact of biological threats on biosecurity, it is best to begin by considering the source and nature of the biological agents that pose biosecurity risks (table 3) . manmade biological threats come in two flavors, deliberate and accidental. the concept of intentionally using biological agents as weapons is nothing new to warfare, and we are all aware of the american, russian, and other state-sponsored programs to develop biological agents as ''weapons of 22 cases/5 deaths total in 2001 from anthrax (perpetrator still at large) >2,000 hospitalized/18 deaths total in 1994-5 from sarin gas (aum shinrikyo) 751 cases/0 deaths total in 1984 from salmonella (rajneeshee) 1 death in 1978 from ricin (assassination of georgi markov) natural casualities: 300-500m cases/2 million deaths per year from malaria world-wide 76m cases/325,000 hospitalizations/5,000 deaths per year from foodborne illnesses in usa 8-10m cases/2 million deaths per year from tuberculosis world-wide >60m cases/>20m deaths total from hiv/aids world-wide, >0.5m deaths in usa 170m cases/10,000 deaths total from hepatitis c in usa mass destruction'' (wmd). these state-sponsored wmd programs have been largely dismantled [8] , and instead the concept of using biological agents as bioweapons has now been usurped by individuals or small groups acting as terrorists engaged in a different form of warfare, where these agents are perhaps more accurately described as ''weapons of mass disruption'' (still wmd). we experienced a vivid and horrible display of this new brand of wmd with the anthrax attacks of 2001, in which the u.s. postal system was utilized to dispense deadly disease, but even more notably, fear and turmoil. the cost of mounting a response to this new wmd has been enormous, not only in terms of billions of u.s. taxpayers' dollars, particularly directed toward biodefense (table 4 ), but also in countless man-hours expended in ramping up other areas of security and healthcare preparedness and in the astounding disruption of lifestyle (e.g. inconveniences caused by intensified travel-related security measures). a growing number of scientists feel that the bioterror threat is exaggerated [7] and that it is highly unlikely that any terrorist organization, foreign or domestic, could on their own develop from scratch a bioweapon capable of causing mass casualties. instead, it is more likely that the potential terrorists would steal or procure existing material and deploy it on a much smaller scale. in contrast, manmade threats resulting from inadvertent release, accidental contamination, or even from non-malicious intentional introduction of biological agents represent much more measurable concerns with known likelihood of risk. there have been a number of recent high-profile incidences that illustrate the havoc, alarm, and economic consequences that can result from widespread distribution of contaminated food because of accidental introduction of harmful microbes during food processing. what previously was seen only sporadically, such as at church socials, family gatherings or community picnics, moved abruptly into the public's eye with the largescale problem of undercooked fastfood hamburger meat contaminated with e. coli o157:h7, a toxin-producing bacterium that causes dysentery-like diarrhea and can cause kidney failure and death, especially in children and the elderly. this dangerous microbe has since been associated with over 400 multistate or multination outbreaks of contaminated food, including meat, radish sprouts, apple juice, let-tuce, and most recently spinach. late last summer, e. coli o157:h7 contamination of prepackaged fresh spinach led to 204 cases of illness across 26 states, with 104 hospitalizations, 31 kidney failures, and 3 deaths [9]. the outbreak, which was traced back to spinach obtained from a few fields in california [10] , shook consumer confidence and cost the industry an estimated $150m in economic losses [11] . there are many other examples of how the spread of natural threats can be greatly facilitated by our modern technologies, practices, and behaviors. viruses such as the marburg and ebola viruses, first discovered in the 1960s and 1970s, are examples of biological agents responsible for recently emerged diseases [12] . ebola and marburg viruses are considered to be zoonotic diseases that are transmissible by close contact with animal species, but their spread has been facilitated by conditions in the country of outbreak, including political upheavals, reuse of needles, and cultural burial practices. zoonotic diseases are often perceived as only a problem of developing countries, where there is much closer contact with animals, both domestic and wild. however, living with animals is not limited to the third world. consider how many americans alone live with pets, sleeping with them, and even kissing them. it is interesting to consider that measles virus is closely related to canine distemper virus [13, 14] , suggesting that at some point a dog-human transmission occurred or a common ancestral virus may have infected both. of particular and growing concern are high impact, foreign animal diseases, such as mad cow and foot-and-mouth diseases (table 5 ). bovine spongiform encephalopathy (bse), commonly known as mad cow disease, is a fatal, progressive neurodegenerative disease of cattle caused by an infectious form of misfolded protein called a prion [15] . transmission of bse occurs when healthy animals come in close contact, usually through ingestion, with prion-containing tissues from animals that have the disease. the first probable occurrence in cattle was in the early 1980s, possibly as a result of feeding cattle meat and bone meal that contained scrapie-infected sheep products. scrapie is a prion disease of sheep and goats. industrial cattle-farming practices in europe prior to 1987 used rendered meat and bone meal, instead of the more common soybean meal used elsewhere, as a protein supplement in cattle feed. in the early 1980s, a change in the rendering process in the u.k., in which a lower sterilization temperature was used for the steam boiling step in the process, is thought to be the major contributing factor to an increase in prions in the cattle feed that resulted in the bse epizoonotic outbreak. the uk epidemic peaked in 1993 with nearly 1,000 new cases per week, and by the end of 2005 there were over 184,000 cases of bse confirmed in the u.k. [16, 17] . bse attracted particular attention because it now appears that it can also be transmitted to humans that consume tainted meat. since the first reported case in 1996, at least 188 people, 160 in the u.k. and 28 elsewhere, have died of a disease with similar neurological symptoms to bse, called variant creutzfeldt-jakob disease (vcjd) [18] . for many of the vcjd cases, there is direct evidence that they had consumed tainted beef years before. the connection between bse and vcjd has a wider impact than just food safety--blood, tissues and organ donation programs are also affected and anyone having exposure to bse is a potential carrier [19] . because of the long incubation period for prion diseases (years to decades), the full extent of the human vcjd outbreak is still not fully known, although the number of new cases appears to be declining. the long incubation period also makes testing for the disease in animals difficult because most livestock are slaughtered long before noticeable symptoms occur or even before plaques in the brain can be readily detected during inspection by necropsy. although many foreign animal diseases are not of serious concern to human health (i.e. humans may be affected only very rarely through direct contact with infected animals), they do pose considerable threat to our agricultural and food industries and could cost millions or even billions of dollars in economic and trade losses (hence their status as ''high impact'' diseases). foot-and-mouth disease (fmd) is a highly contagious, sometimes fatal viral disease primarily of cattle and pigs, but it has a wide host range. fmd occurs worldwide, but a number of areas, including north america, australia, new zealand, japan, most of europe, and parts of south america have been fmd-free for some time, mainly due to eradication through rigorous vaccination and culling programs. however, in 2001, a major outbreak of fmd in the u.k. resulted in the slaughter of millions of animals, huge economic and trade losses estimated in the range of £20m, the temporary cancellation of sporting events and other outdoor events attended by farmers or those living in the country, and the implementation of strict policies on the sale and trade of livestock, as well as disinfection of all persons entering or leaving farming areas [20, 21] . countries are recognized to be in one of three fmd categories: fmd present with or without vaccination, fmd free with vaccination, and fmd free without vaccination [22, 23] . understandably, countries designated as fmd free without vaccination have the greatest export markets, so most developed countries have greatly enhanced their agricultural surveillance and trade policies to maintain their fmd-free status. innocuous introduction of foreign plants, animals, or insects can also have considerable ecological and economic impact on horticultural and agricultural industries. kudzu, a member of the pea family that is native to southeast asia, is an example of an invasive alien plant species that has caused considerable damage since its introduction to the southeastern regions of the u.s., where it is sometimes referred to as ''the plant that ate the south'' [24] . kudzu was first intro-duced from japan into the u.s. in 1876 at the philadelphia centennial exposition, after which it gained some popularity as an ornamental shade vine. but, from 1935 to 1953, the soil conservation service promoted its use as a means for controlling soil erosion. once established with a root system that can reach depths of up to 12 feet, kudzu vines grow as much as a foot per day during a season with lengths up to 100 feet. kudzu now covers over 1m hectares and poses a considerable threat to the otherwise high biodiversity of flora found in the south [25] . a tremendous amount of money and effort is spent each growing season to prevent the highly prolific kudzu from overtaking roads, bridges, powerlines, local vegetation, and even homes, barns and other buildings. it costs an estimated $500m annually in lost cropland and management resources [26] . for successful long-term control, the entire root system must be destroyed or the plant will grow back, and considerable effort has been made to find pesticides that can control this plant pest. key to an effective strategy to combat complex biological problems to ensure biosecurity will be a greater understanding of the driving forces that are important for transmission and perpetration of infectious disease and then the management and implementation of effective preventive or containment measures. the potential power of natural selection is obvious. time and again we have seen how rapidly microbes can evolve in response to selective pressure, such that certain behaviors or genetic traits tend to be eliminated from the gene pool, while others are maintained or changed. as a consequence, new biological threats are bound to emerge as we impose our influence on the environment. the goal of most pathogenesis research, of course, is to use our understanding of the ecology of host-microbe interactions and their role in pathogenesis to develop predictive models for disease progression and transmission. unfortunately, our current understanding is rudimentary at best, and we are just now beginning to tease out the intricacies of the co-evolution of pathogens with their hosts and environment and the role that these host-microbe interactions play in emergence of disease. human-induced evolution can be extraordinarily rapid and pervasive. the natural history of myxoma virus in american rabbits and its introduction into european rabbits as a means for controlling the rabbit populations in australia provides an interesting example of the co-evolution of a virus and its animal host [27] . it also provides a glimpse into understanding the emergence of infectious disease. shortly after european rabbits were first brought to the americas in 1895, they were found to succumb to a deadly and extremely infectious disease, which nearly half a century later was found to be caused by a vector-borne myxoma virus acquired through contact with the native, more resistant host, the common wild rabbit of south america. european rabbits were first introduced into australia in 1859 for hunting, but by the 1880s they had become a major pest. to help in controlling the rabbit population, rabbits infected with this deadly virus were introduced in 1950 and the favorable weather conditions for mosquitoes that year helped to rapidly spread the disease, killing millions of rabbits. but, evolution and the power of natural selection then came into play. the myxoma virus that was first introduced killed over 99.9% of infected rabbits, yet a few rabbits survived the exposure. within a year, new strains of the virus appeared that killed only 90% of infected rabbits, and in subsequent years even more attenuated viral strains appeared. under such strong selective pressure, the rabbits, too, evolved to gain increased resistance such that the original, highly lethal virus would no longer kill more than 30% of the rabbit offspring. clearly, genetic changes in both the viral and rabbit populations quickly altered the outcome of the disease in terms of severity and persistence. today, only 50% of infected rabbits succumb during a myxomatosis epidemic. one of the most devastating recently emerged diseases, whose initial and continuing spread can be attributed to human behavior, is that of hiv/aids. hiv is thought to have originated in nonhuman primates [28] , but has become established in humans and is now transmitted human-tohuman through unprotected sexual practices, reuse of needles, and at first (although no longer) through contaminated blood supplies [29, 30] . aids was first noticed in the early 1980s as unusual occurrences of a rare cancer, kaposi's sarcoma, in young homosexual men. the social stigma associated with the disease gradually shifted with the realization that other populations were also at risk, including heterosexual and bisexual women, drug addicts, hemophiliacs, blood transfusion recipients, and babies born to hiv-positive mothers. the annual death toll rose linearly from 1987 with over 13,000 deaths to a height of nearly 42,000 in 1995, before a noticeable decline was observed with the introduction in 1996 of a cocktail of three anti-hiv drugs. by 1997 the annual death toll was down to 15,500 and has since declined to around the 12,000 mark [31] . however, although the death rate has declined, the number of cases reported annually in the u.s. still hovers around 40,000 [4, 32] . host-microbe co-evolution over time appears to be in effect for hiv/ aids. there are now a considerable number of hiv-positive individuals who have survived for many years without acquiring aids. a large part of this is due to advances in anti-hiv medications and improved healthcare. however, even before the increased availability of hiv medications, there were a significant number of individuals engaging in high-risk behavior that appeared to be resistant to acquiring hiv. by examining these ''survivors,'' scientists found a genetic mutation (allele) in a surface receptor, called ccr-5, which prevents the hiv virus from entering host cells [33] . these individuals having the mutant receptor allele are mostly of european decent. in some parts of europe, up to 20% of the population carry at least one copy of the mutant receptor allele, while populations in the rest of the world do not carry the same allele [34] . it is believed that this allele might have arisen through selective pressure from previous exposure of the population to another plague (perhaps bubonic plague, although this is not certain). but, importantly, the strong selective pressure that the new antiviral medications have placed on hiv has led to an accelerated deadly arms race. current antivirals, at an annual cost of over $10,000 per person, are targeted mainly against the viral outer coat proteins gp120 and gp41, the processing protease, and the reverse transcriptase. although these viral protein targets are less variable than others in the viral genome, hiv has a high mutation rate and the strong selective pressure has caused the virus to rapidly evolve in response [29] . the ever-evolving hiv makes developing new drugs a constant, and costly, challenge [35] . other pathogens have been around for quite some time, but have recently acquired new properties making them increasingly more deadly. during the 1990s, staphylococcus aureus emerged as one of the most common causes of hospital-acquired infections in the us. drug-resistant infections increase the risk of death, as well as the cost and duration of hospital stays. over a relatively short period of time s. aureus acquired genes that increased the bacterium's resistance to antibiotics [36] . a timeline of the emergence of these new strains of s. aureus clearly demonstrates the evolution of a pathogen that is under strong selective pressure to survive (table 6 ). once acquired, antibiotic genes can be spread from one microorganism to another through a process known as horizontal gene transfer, which involves uptake or transfer of dna encoding those resistance genes within or between different bacterial species. importantly, the antimicrobial resistance is maintained even after the selective pressure is removed. this exchange of genetic information is believed to contribute to the alarming rise in multidrug resistant bacteria [37] . rapid development of antimicrobial resistance is forcing clinical and pharmaceutical researchers to devise alternative, innovative approaches to respond to this threat [38] . hospitals are thought to be a major source of multidrug resistant bacteria, but agricultural practices involving usage of antibiotics as prophylactics and growth promoters in feed and crops have also played a role in its emergence [39] . eye-opening evidence for just how prevalent genetic exchange occurs in natural environments is provided by the example of the substantial increase in antibiotic resistance among both community and clinical isolates of bacteria in the gut [40] . tuberculosis (tb) is the leading cause of death in the world, and after a century of decline in the u.s., tb is once again on the rise, and alarmingly multiple drug-resistant strains have emerged. this increase in cases worldwide is attributable to a number of complex factors, including changes in the social structure and socioeconomic upheaval, the hiv epidemic, and a failure in some countries to improve public treatment programs. multidrug resistance in tb is a growing international health concern, because it has dramatically increased the difficulty in controlling the spread of tb and because of the high mortality rate associated with co-infection with hiv [41] . co-infection of multidrug-resistant tb with hiv fuels the transmission of tb by accelerating the progression of latent tb into active disease because of the damage that hiv causes to the host immune system, which normally controls tb infection. individuals that test positive for both tb and hiv often die with 1-6 months. a major epidemic of tb-hiv infections has spread across the former soviet union due to socio-economic changes in the country that have led to overcrowded housing, and in particular overcrowded prisons [42] . unfortunately, the pharmaceutical industry has largely abandoned tb drug development due to perceived nonprofitable consumer market--at risk populations are also the poorest [43] . the spread of any disease that is transmitted from human to human is greatly facilitated under crowded conditions, which allow for efficient exposure to a higher initial inoculum of the infectious agent. sporting events, concerts, and other gatherings of large numbers of people in a confined area can promote human-to-human transmission, as well as exposure to new populations. legionnaire's disease, caused by the bacterium legionella pneumophila, was first recognized in 1976 when it struck a group of american legion conference attendees in philadelphia [44] . this bacterial pathogen normally lives in fresh water as a parasite of amoeba, but unfortunately for us it can also live and thrive inside one type of our immune cells called a macrophage. the disease is acquired through aerosol exposure from contaminated water in ventilation systems, such as the air conditioning units in a hotel, or through aspiration during nasogastric tube feedings diluted with contaminated potable water in hospital or nursing-home settings [45] . since the first widely publicized incident on the holland-america cruise line in 2002, there have been numerous reports of cruise ship passengers succumbing to acute gastroenteritis caused by the norwalk virus [46] . a cruise, where hundreds of passengers and crew mingle in close contact, can provide optimum conditions for a virus to spread through food, water, and direct contact. cruise ships are now required to report all gastrointestinal illnesses to the cdc before entering a u.s. port, especially if 2% or more of the passengers or crew are ill. what has been most economically troubling for the cruise line industry is the recalcitrant nature of the virus to decontamination efforts [47] . overcrowding often leads to poor sanitation, resulting in accumulation of refuse, sewage, and vermin that thrive under such unsanitary conditions. natural disasters, civil disturbances, and war have caused large population displacements, with millions of people (and their animals) worldwide today living in refugee camps, which are overcrowded with poor sanitation and often without adequate food or clean water. these crowded camps provide ideal conditions for brewing and transmitting new infectious agents in malnourished or immunocompromised populations of humans and animals. the norwalk virus reared its ugly head once again during the katrina crisis in 2005 [48] . of the estimated 24,000 evacuees sheltered temporarily at facilities in reliant park, a sports and convention complex in houston, texas, 1,169 persons reported symptoms of acute gastroenteritis during a 2-week period at the beginning of september. medical personnel, police, and volunteers having direct contact with patients also reported symptoms, suggesting secondary transmission. although the local public health officials and the cdc implemented extensive infection-control measures, including publicizing the need for enhanced hygiene techniques, the outbreak continued for an additional week before declining. habitat destruction (e.g. deforestation, slash-burn practices) and urban expansion can uncover natural reservoirs and expose humans and domestic animals to new disease-causing microbes. each year 100-200 zoonotic cases of the pneumonic disease tularemia, caused by the bacterium francisella tularenesis, are reported in the us, primarily in arkansas, missouri, and oklahoma [49] . transmission usually occurs through arthropod bites, especially ticks or deerflies, but it can also occur through inhalation of contaiminated aerosols. in the late 1930s, rabbits from arkansas and missouri were introduced to cape cod and martha's vineyard, and cases of tularemia in massachusetts were reported shortly thereafter. martha's vineyard experienced two larger outbreaks in 1978 and 2000, which were linked to outdoor activities of mowing lawns and cutting brush [50] . the humans were presumably infected by inhalation of microbe-contaminated animal remains mechanically aerosolized by the cutting action of the mowers or brush cutters. pollution and exposure to waste water or sewage can also lead to the emergence of new diseases. coral black-band disease is a globally distributed disease that has been causing the degradation of coral reef ecosystems. first reported in the 1970s, the disease is observed as a pathogenic microbial consortium (mat) that migrates from the top to bottom of healthy coral, leaving behind dead exposed skeleton that disrupts the ecological and geological structures of coral reefs (see figure 1 ) [51] . a factor that appears to be contributing to the development and spread of coral black band disease is the pollution of seawater from industrial, municipal, and other terrestrial waste sites near the coral reefs [52] . modern technologies have led to greater efficiency in production, marketing, and commerce of goods around the world. rapid transport of imported material and tourism related travel facilitate the spread of infectious diseases around the globe and are clearly contributing to the increased prevalence and severity of the diseases. exotic souvenirs, including wild animals and their associated microbes, have been imported illegally into the u.s. from various parts of the world. an outbreak of monkeypox in 2003 among residents of wisconsin, northern illinois, and northwestern indiana was the result of infection from prairie dogs bought at a pet shop in texas that became infected after contact with various exotic african rodents shipped from ghana and then distributed by other pet shop outlets in the midwest [53] [54] [55] [56] . rare zoonotic cases of monkeypox in humans had been reported previously only in remote villages of central and western africa near tropical rainforests where there is close contact with infected animals [57, 58] . recent studies suggest that exposure to monkeypox in these areas has increased due to encroachment of humans into animal habitats. the cdc and fda subsequently embargoed all african rodents into the u.s. and banned the distribution or sale of african rodents and prairie dogs in the u.s. [59] . trade routes and human practices have contributed to the spread of numerous diseases throughout history, but the speed with which they are spreading today have demanded the need for ever more rapid response and containment measures to be in place. an interesting example is that of cholera, caused by the cholera toxin-producing bacterium vibrio cholerae. in asia, cholera has been endemic for hundreds, maybe thousands of years, and cholera-like disease has been described in a number of ancient texts. the first well-documented epidemic in europe occurred in 1871. since 1871, seven major cholera pandemics have occurred [60] . the first six were caused by the classical o1 biotype, whereas the seventh, which began in 1961 and persists today, is caused by the el tor o1 biotype. in 1991, el tor reemerged in peru after a hiatus of over 100 years, and rapidly spread throughout central and south america over the following couple of years, with more than 1.5 million cases and over 10,000 deaths [61] . the spread of el tor in these countries could be traced along the major north-south coastal trucking route and is attributed to poor sanitation in these areas. the most recent cholera outbreaks have occurred in developing countries, such as angola, where civil strife has hindered water treatment and sanitation efforts [62, 63] . the extent of the global cholera burden has been grossly underreported [62] , in part due to limited resources, but also due to the detrimental effects such news can have on trade and travel to those regions. in some endemic areas, such as bangladesh, improved management strategies by the government and who, including aggressive rehydration therapy and antibiotics, have shortened the duration of illness and have reduced the fatality rates from natural cholera epidemics, which are largely seasonal in nature. in 1992, a new strain of vibrio cholerae, designated o139 or ''bengal,'' caused a massive cholera epidemic in south asia [64] . what was most disturbing about this new strain was its high prevalence in adults, suggesting that prior immunity gained during childhood through exposure to the classical or el tor o1 strains offered little or no protection against this new o139 strain. its subsequent spread to other asian countries lead some to worry that it may cause an eighth cholera pandemic, but luckily so far this has not materialized due to timely mobilization of effective control measures by researchers and healthcare officials. however, an emerging concern is the increased incidence of antibiotic resistant strains of vibrio cholerae in bangladesh. nearly all isolates are now resistant to the less expensive antibiotics, tetracycline, trimethoprinsulfamethoxazole, and erythromycin. although most are still sensitive to ciprofloxacin, the effective doses needed for treatment are increasing. seasonal changes in rainfall and sunlight can trigger periodic or transient emergence of some human pathogens such as cholera. an intriguing observation comes from the study of the annual epidemic profile of endemic cholera in the bengal region of bangladesh and india, where nearly all cases occur in a synchronized, explosive outbreak during major transitions of climate in the post-monsoon months of october and november [65] . as the rains decline and sunlight increases there is a burst of algal and zooplankton bloom. it has been proposed that the increased concentrations of these particles (surfaces to which the bacteria adhere) in drinking water sources consequently increase the rates of ingestion [66, 67] . during other times in the year, cholera cases occur only sporadically because the zooplankton sediment and there is less ingestion of bacteria-coated particles. recently, an additional factor has been credited toward the seasonal cholera epidemics, namely predation of the v. cholerae bacteria by bacteriophage (viruses that infect bacteria) due to amplification of the phage in the intestines of humans, followed by release into the environment [68] . support for this model comes from the inverse correlation of the phage count with the abundance of toxigenic v. cholerae in water samples and with the incidence rates of cholera [65, 69] . climate change can also dramatically alter the spread of arthropodborne diseases, which are most prevalent in a limited range of temperatures or environments preferred by these vectors. shifts in warming or cooling trends may extend or narrow the range of such vectors and the diseases they transmit. drought or flooding can also lead to spread of disease into new populations of animals or humans. the west nile virus is an example of a recently emerged vector-borne disease that has been introduced to a new geographic area. the virus was first isolated in uganda in 1937 and has since been known to cause disease in africa, west asia, europe, and the middle east [70] . until 1999 when it caused a deadly outbreak in the new york metropolitan area, it had never been observed in the u.s., but now it has spread to every state, except alaska and hawaii, as well as canada and mexico. as of march 2007, the cumulative number of human disease cases in the u.s. is 4,256 [71] . the west nile virus is usually transmitted between birds by mosquitoes, but can be transmitted to humans and other hosts, particularly during favorable seasonal conditions with a hot dry summer followed by a wet fall, as what occurred in the new york area in 1999. its introduction into the u.s. is thought to have occurred recently since the genetic profiles of the new york virus isolates suggest they came from a single source, which is related to a virus isolated in 1998 in israel [72] . although not known for certain, it is possible that an infected bird could have been imported or an infected mosquito or tick may have hitched a ride on an international flight or on a ship carrying old imported tires infested with mosquito larvae. large holding and storage facilities for meat, grains, dairy and produce provide new habitats and breeding grounds for insects and vermin such as mice and rats. humans can be infected with the deadly hantavirus through inhalation of aerosolized virus present in dried rodent urine in grains or feedstuffs. in 1993 the southwestern u.s. experienced a mysterious outbreak of a new deadly respiratory illness in healthy people, which within a couple of months was identified by the cdc as a previously unknown type of hantavirus [73, 74] . because the researchers knew that other hantaviruses were transmitted by rodents, they began trapping mice and rats in the area around the victims' homes and discovered that the deer mouse was the primary natural reservoir. further investigation revealed that there had been earlier unexplained deaths due to this hantavirus, but these cases were sporadic. the reason for the clustered outbreak in the 1993 could be connected to the unusually high numbers of mice in the area during that season [75, 76] . for several years, the region had experienced drought, but in early 1993, heavy snows melting and rainfall helped revive the flora and fauna in the region, such that the deer mice had plenty to eat. the mice increased dramatically in numbers, and consequently increased the likelihood of transmission to humans. intense cross and intraspecies interactions are conducive to transmission of a pathogen from one host to another. the rapidity with which microbes and viruses are able to evolve increases the likelihood of such close host-host contacts to cause the pathogen to ''jump'' across species barriers. like bse, chronic wasting disease (cwd) is a prion-mediated transmissible spongiform encephalopathy of cervids, such as mule deer, white-tailed deer, and rocky mountain elk [77] . the potential for cwd to similarly cross the species barrier from cervids to humans is considered unlikely. but, because bse has been transmitted from cattle to humans (as vcjd), it is feared by some that cwd might also ''jump'' the species barrier. although cwd can be transmitted to cattle, sheep, and goats by direct inoculation into the brain [78] , studies have not yet demonstrated that domestic livestock are susceptible via oral exposure, the presumed natural route of exposure to bse [79] . it is feared that homology within critical amino acid sequences of the human and cervid proteins might facilitate cross-species transmission of cwd to humans, as what appears to have occurred for bse. thus, understanding how prions overcome resistance to transmission between species is crucial if we are to prevent future epidemics. although surveillance efforts for cwd in captive and free-ranging cervids are continu-ing, eradication of cwd from wild populations of cervids is unlikely with currently available management techniques. the potential emergence of a new disease-causing zoonotic agent that is transmissible between humans is a major concern. constant exposure and certain behaviors increase the likelihood that a virus will ''jump'' species. we experienced a frightening example of this with the rapid worldwide spread of the sars virus. alarmingly, we are currently at the brink of experiencing another such emergence, which could have devastating consequences on the human population. already the current spread of avian influenza a virus has resulted in the death from disease or culling of over 300m domestic poultry in asia, with an estimated $10b in economic losses to the asian poultry sector (table 7 ) [5] . a question on many people's mind today is whether another pandemic flu like the one in 1918 is inevitable [80] . we already know that the circulating influenza h5n1 virus can ''jump'' from birds to humans [81] , but luckily we have not yet observed significant human-tohuman transmission other than a few cases through intimate unprotected contact with a critically ill index patient [82] . will this fine dividing line be crossed soon? or, will this threat diminish before it evolves into a more human-specific virus? the truth is that we know very little about the specific factors that trigger a ''jump'' between species or a transition into a rapidly transmissible virus [83] . we know even less about how to prevent these events from occurring or how to predict when they will occur. the current h5n1 strain, first limited to poultry, quickly spread to migrating birds, but has now emerged in mammals and humans mostly through zoonotic contact. previously, it was widely accepted that avian viral strains could only readily infect humans after first having undergone genetic shuffling within swine, but now it appears that direct transmission from bird to human can occur [84] . although its transmission from human-to-human is (luckily) still inefficient, the who, the cdc, and other organizations have already mobilized for just such an event [85] . distinguishing a deliberately introduced infectious disease from a naturally occurring or emerging infectious disease is inherently more difficult due to their ''dual-use'' nature. the good news, though, is that effective medical treatment and prevention strategies for combating a naturally occurring infectious disease will most likely work just as well for one that is deliberately introduced. the exponential advances that have been made in the life sciences, medicine, and biotechnology have not only dramatically enabled our ability to respond to biological threats, but, sadly they have also increased the potential risks of malevolent exploitation and inadvertent misuse. indeed, a report from the u.s. national research council and the institute of medicine concluded that the breadth of potential biological threats is far wider than is commonly appreciated and will continue to expand in the future [86] . the nih invests over $28b annually in medical research. since 2001, nih has directed over $10b toward countering bioterrorism alone and currently spends over $3b of its annual budget on infectious diseases with over $1.8b going toward emerging infectious diseases (table 4) [87] . considerable attention has been recently focused on developing better preparedness and surveillance (early warning) as strategies for more effective response to biological threats. this depends on having reliable, sensitive and rapid means for recognition of unusual events or unexpectedly high levels of common events. a number of animal and human health laboratory response networks have been established with the goal of maintaining an integrated national and international system for facilitating standardization and movement of information, for expansion of detection and diagnostics measures, for coordinating responses among federal, state, university and commercial clinical laboratories, and for identification of common source outbreaks. on the international front, the who and cdc have increased activities to build capacity for global disease detection and response, with immediate focus on and strengthening of influenza surveillance. the global alert and response network (goarn) was established in 2000 by who as a partnership of >140 institutions and networks to mobilize human and technical resources for the rapid identification and control of disease outbreaks that are of international importance [88] . the global livestock early warning system (glews) has been formed by the food and agricultural organization (fao) of the united nations and the world health organization for animal health (oie) to strengthen epidemiological analysis and prediction of major animal diseases and zoonoses and to improve reporting from a variety of data sources that might impact disease transmission from animals to humans [89] . the recent pandemics have also mobilized strengthening of the international health regulations, which were first established by who in 1969 to ensure maximum security against international spread of certain diseases (cholera, plague, yellow fever, and smallpox--although smallpox was removed from the list in 1981) with minimum interference of world commerce. in 2005 a revised set of regulations was adopted unanimously at the world health assembly to increase the roles and responsibilities of who and member states, including financing, developing, strengthening, maintaining and implementing core surveillance, and response capacities [90] . biosecurity requires multipronged, flexible, and interdisciplinary approaches to combat the perpetuation and spread of infectious diseases. in all, simultaneous use of multiple strategies will be necessary for effective infection control and disease management. one such strategy is to use evolutionary engineering (combining predictive evolution and genetic engineering) to design vaccines and drugs based on predictive targets. for example, an innovative approach for control of e. coli o157:h7 contamination of food and water was recently employed to eliminate the source for the organism. by vaccinating the animal reservoir--cattle--the researchers were able to prevent colonization of the cattle with the microbe, reducing the levels of bacteria shed in feces and thereby reducing the risk of human disease [91] . this strategy was shown to significantly decrease the prevalence of e. coli o157:h7 in a clinical trial conducted in a feedlot setting. improved sanitation and use of chlorinated drinking water has dramatically reduced the incidence of waterborne disease. evidence suggests that the cholera epidemic in south and central america was caused by a complex set of circumstances, including poor sanitation conditions, poor separation of drinking water and waste streams, and inadequate water treatment and distribution systems [61] . indeed, outside of peru's capital lima, chlorination of drinking water supplies at the time of the epidemic was limited at best. improved water quality and sanitation have since reduced the incidence of cholera in south american countries. another simple, yet surprisingly effective strategy recently implemented has been the use of filtering water through multilayered cloth filters to remove the plankton and other particles to which the vibrio bacteria adhere [92] . over the past 25 years an unprecedented mobilization of resources have been directed at stopping the hiv pandemic, ranging from preventive strategies for persons at high risk for contracting hiv, such as educational counseling, testing, and referral services, to treatment with multiple drug regimens, to management measures aimed at improving the healthcare of persons living with hiv and preventing further transmission [93] . although enormous success in prevention of hiv/aids in the u.s. has been achieved and we have learned a lot about how to approach rapidly evolving diseases from this experience, a number of prevention and treatment challenges remain. hiv prevalence remains high among homosexual men [94] and racial/ethnic disparities have increased, especially among african-american men and women [95] with prevalence among african-american men reported as high as 46% [96] . new programs are needed to more effectively reach these populations [93] . one approach toward improved treatment and reduction of drug resistance is the use of multidrug overkill (triple drug therapy). administration of just a single drug often leads to the development of resistance to that drug, but strong multidrug doses decrease the likelihood of multidrug resistance. for example, recent studies have shown that triple drug combination antiviral therapy in treating hivinfected persons offer superior viral suppression over other drug regimens [97] . when two or more drugs are used simultaneously, each helps prevent the emergence of resistance to the other drug. effective regimens for the treatment of tb must contain four different drugs to which the organisms are susceptible. to illustrate how this might be so effective, consider that mutation rates in the tb-causing bacterium lead to a frequency of resistance to isonazid of 1 in 10 8 , to streptomycin of 1 to 10 8 , to ethambutol of 1 to 10 7 , and to rifampicin of 1 to 10 9 . bacterial mutants resistant to any single drug are naturally present in any large bacterial population. an inactive tb granuloma contains 10 2 -10 4 bacteria, whereas an active tb lesion contains 10 7 -10 9 bacteria. this means that the chance of gaining resistance to any one of the drugs is relatively high in an active lesion, but the chance of gaining resistance to multiple drugs is considerably less [98] . the course of the four-drug treatment for tb usually lasts from 6 to 9 months. when adherence with the regimen is assured, the four-drug regimen is highly effective; however, a problem with tb treatment is that the drugs used are often counter-indicated, cause unpleasant side effects, and must be administered in series over a long period of time rather than simultaneously, which leads to problems with patient compliance [43] . nearly half of individuals with tb do not complete their treatments. reduction of noncompliance can be achieved by direct observation of the patient to ensure full dosage. in developed countries, such as the u.s., this is relatively easy to achieve with the use of healthcare workers and family members. however, in developing countries, there are many obstacles to adhering to treatment regimens, and alternative strategies to improve compliance are needed. in addition to direct observation of treatment, other tactics include sending reminder cards or phone calls, monetary incentives, health education and counseling, and making access to clinic facilities easier [99] . another strategy for reducing the prevalence of antimicrobial resistance is to remove the overall selection pressure by minimizing exposure to the drug, and especially withholding the most effective drugs, i.e. the ''drugs of last resort,'' until absolutely needed. this is becoming more and more difficult to accomplish with the accelerated rate of spread of antibiotic resistance through the overuse and over-prescription of antibiotics [37, 39, 100] . indeed, many researchers were dismayed at the large distribution of ciprofloxacin (cipro tm ) to treat over 60,000 people after the anthrax attack in 2001 and warned that this widespread use could lead to resistance in other bacteria [101] . the strain of bacillus anthracis strain used in the attack was equally sensitive to other less expensive and more commonly used drugs. ciprofloxacin is considered a ''drug of last resort'' because of its broad-spectrum and efficacy against many pathogenic bacteria, particularly those (such as staphylococcus) that are already resistant to other drugs. prescreening for sensitivity is another approach that allows for use of narrow-range rather than broad-spectrum antimicrobials, which further reduces the likelihood of resistance developing and spreading to other pathogens. management and implementation of effective preventive or containment measures in the event of natural or man-made biological threats will require increased infrastructure and diagnostic and surveillance capabilities. for example, the tremendous scale-up in food production, from the vast herds of cattle to the huge confined feedlots to the slaughterhouses to the many hundreds of distributors and supermarkets, has undoubtedly contributed to the emergence and prevalence of food-borne diseases such as that caused by e. coli o157:h7. the complexity of the modern food preparation and distribution process makes epidemiological tracking of the sources of contamination difficult, although there have been noticeable advances. in response to the need for improved agriculture and food biosecurity, congress passed the national agriculture and food defense act of 2007, which will require enormous effort and financial commitment on the part of the government and agricultural and food industries. the logistics involved are daunting, particularly in coordinating efforts among many different agencies. one example is the food-borne diseases active surveillance network (foodnet), which is a collaborative project of the cdc, the usda, and the fda to monitor and q 2008 wiley periodicals, inc. study the epidemiology of food-borne diseases [102] . in response to the growing need for better food surveillance, the fda implemented the hazard analysis and critical control point (haccp) program for prevention of food-borne diseases, which involves monitoring food distribution at critical control points where contamination is most likely to occur [103, 104] . in 2005 the food safety and inspection service (fsis) of the usda established the food emergency response network (fern), which will work with the fda and its haccp program to integrate a laboratory network across the u.s. that can quickly respond to food-related emergencies [105] . the fsis, as part of its task of protecting public health through food safety and defense, recently implemented a new assessment method, called carver1shock [106] , for identifying the most vulnerable target sites within a food processing system (table 8 ). in considering the gaps in the u.s. agriculture and food defense capabilities, the main question that must be addressed is: exactly what level of security do the people of the usa want the agricultural industry to achieve, and our government to enforce, in terms of food safety and biosecurity? it is clear that we (as a nation and international community) are not happy with the current security status of our food and agricultural supplies. the outbreaks and ensuing deaths, economic losses and drop in consumer confidence resulting from contaminated spinach by e. coli o157:h7 [11] , mentioned above, amply demonstrate this point. but, if we take this episode as an example, one must question exactly what could have been done to prevent or further mitigate this outbreak than was already done. by all accounts, the surveillance, detection, evaluation, containment, and recovery measures were remarkably fast, accurate, and as good as one could possibly hope, considering the circumstances-far better than in previous incidents of a similar nature, thanks to improved measures newly in place. this was considered an accidental outbreak, but what if it had been a deliberate attack? undoubtedly, the response would have been little different. yet, what happened is still deemed by most as unacceptable. so, how could this be improved? most current measures are aimed at further improvements to detect or con-tain a future incident by increasing surveillance at the front-end, increasing diagnostics or enhancing epidemiological monitoring capabilities once an incident has occurred. but, just how much of an improvement would that be? clearly, what the public wants is a near certainty that such an event as occurred with the spinach will not happen again. part of the difficulty in adequately addressing this need is that current efforts are focused on too broad and vast a target (there are just too many steps in the food processing, where things can go wrong and something could slip through the cracks). instead, the focus for ensuring near complete biosecurity should be at the very end of the food processing chain, namely at the packaging and delivery stage. while all the proposed surveillance, detection, evaluation, containment and recovery measure will significantly reduce the possibility of future contamination, and thus should be done to the extent possible, they will not ensure the desired near-complete protection from an incident (which is what the consumer is demanding). so, how can this be achieved? many scientists see food irradiation as one viable solution. food irradiation of already processed and packaged food through promising new technology can eliminate disease-causing microbes from foods. the effects of ionizing radiation on food and on animals and people eating the irradiated food have been extensively studied and deemed safe [107] [108] [109] , and indeed this technology has been implemented already for certain foodstuffs. yet, bringing this technology into use for most foods has been challenging, primarily due to misconceptions and fear about its safety on the part of the public and policymakers. closing this gap will require a ramping up of public engagement by the scientific community. somehow scientists must better communicate with the public and policymakers, convincing them of the benefit criticality-measure of public health or economic impacts of an attack to achieve terror goals accessibility-ability to physically access and egress from the target recuperability-ability of system to recover from an attack vulnerability-ease in accomplishing the attack effect-amount of actual direct economic loss from an attack as measured by loss in production recognizability-ease of identifying the target shock-the combined physical, health, economic and psychological effects of an attack the carver1shock method rates each of seven attributes on a scale of 1-10 for identification of vulnerable sites in food processing and distribution system that might be targets for attack or points of contamination. of this technology and at the same time alleviating their fears of its use. to achieve global health biosecurity it is critical that the communication and educational barriers between nonscientists and scientists are overcome and that efforts by public healthcare, scientific and security policy communities are better integrated. enhanced coordination between multiple agencies is essential for implementing and maintaining global disease surveillance systems, public healthcare, and diagnostic and basic research laboratories. thus far, many redundancies in effort remain and interagency cooperation is still fragmentary. international cooperation has improved with regard to regulatory and containment (import/ export) policies, particularly in the area of travel restrictions during pandemics, but cooperation is still a bit shaky in other areas where commerce and political issues are concerned. and, much work remains in the areas of regulation of agricultural, societal and medical practices, pollution control, and prevention of habitat destruction. strengthening national and international capacities to prevent and control disease epidemics will require continued promotion of international cooperation and technical partnerships with institutions and networks around the globe to mobilize resources for control of disease outbreaks. regulation and oversight of research and use of potentially dangerous bioagents (''select agents'' or highrisk or high-impact bioagents) is well underway, but there remain disparities between that conducted in the u.s. and that done elsewhere in the world. biosafety and biosecurity of pathogens in laboratories and healthcare settings will require enhanced education for better preparedness and increased confidence of the public and policy makers in science. many scientists feel that policymakers have not fully understood the difference between biosafety and biosecurity and have consequently imposed a number of mandates and regulations that equate enhanced need for security with enhanced danger, i.e. greater biosafety risk. balancing biomedical and biotechnological advancement with biosecurity will always be at odds due to the ambiguous definition of what constitutes a potential biosecurity threat, but improved education will help with making those decisions. although warning and prevention are preferable to coping with the consequences of an attack, an emphasis must also be placed on improving public healthcare and basic research and education. it is critical that we develop a homeland and global biosecurity strategy that is applicable to both intentional and unintentional disease outbreaks. the best defense against any microbial threat is a robust public healthcare system in regard to its science understanding, capacity, and practice. there has been considerable advancement in the area of prevention, with improved surveillance and detection, and with new drugs becoming available at a remarkably rapid pace, but we are still only at the tip of the iceberg in our understanding of pathogen evolution, post-exposure treatment and control, prediction of epidemic versus pandemic spread, rates of transmission, impact of climate change, and preparation for handling a biothreat agent of unknown origin, whether it be from natural, accidental, or deliberate exposure. h2n2) epidemic 1963 (h7n3) england, turkey 1902 (h3n2) 1966 (h5n9) canada, turkey 1918 (h1n1) ''spanish'' pandemic 1976 (h7n7) australia, chicken 1933 (h1n1) 1979 (h7n7) germany, chicken 1947 (h1n1) 1979 (h7n7) england, chicken 1957 (h2n2) ''asian'' 1983-1985 (h7n7) pennsylvania (17m killed) 1968 (h3n2) swine'' non-epidemic 2002 (h7n7) hong kong (>1m killed) 2003 (h7n7) netherlands (30m killed) 2004 (h7n3) canada (17m killed) 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in food safety irradiation of food-helping to ensure food safety food irradiation: a safe and useful technology key: cord-318407-uy0f7f2o authors: nara, peter l.; nara, deanna; chaudhuri, ray; lin, george; tobin, greg title: perspectives on advancing preventative medicine through vaccinology at the comparative veterinary, human and conservation medicine interface: not missing the opportunities date: 2008-11-18 journal: vaccine doi: 10.1016/j.vaccine.2008.07.094 sha: doc_id: 318407 cord_uid: uy0f7f2o abstract vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past 50 years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life—resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past 200 years, even in the past 10 years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train paraand professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it’s melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the 21st century. vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past 50 years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life-resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past 200 years, even in the past 10 years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it's melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the 21st century. published by elsevier ltd. the new millennium did not bring the anticipated global internet technology shutdown however, it has brought with and heralded a time of significant change, opportunity and challenges. i and my co-authors goal in this overview are to celebrate, provocate, instigate innovate and activate those in society who are in interested to contributing to the betterment of human and animal health through vaccination. for vaccination to have its greatest chance of working policy-relevant science for sustainable conservation in developing countries needs to address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the wellbeing of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the pre-ventative sciences and medicine lead to sustainable cultural public health and economic changes of the future is never more evident than today. if the article gets the attention of researchers, educators/teachers, funders, policy makers, economists and the general public in both developed and developing countries to become involved in finding collaborative solutions to the conservation crisis than we will consider it a success. vaccination has and remains one of the most cost-effective and safest forms of medicine toward improving health today. along with basic understanding of germ theory and sanitation, vaccination, over the past 50 years, has transformed lives in both rich and poor countries-resulting in the eradication of smallpox and huge reductions in the burden of previously common diseases such as polio, typhoid and measles. immunization is particularly well suited to all countries including those with weak health systems, because it requires relatively less training and equipment and does not depend on skilled diagnosis, long-term drug regimens or extensive medical care. immunization and sanitation remain as the most important public health modality responsible for improving the gnp of developing countries through additional gains in healthier children who are better educated and grow up to impact on their productivity. like schoolchildren, healthier workers have better attendance rates and are more energetic and mentally robust. workers in healthy communities, moreover, need to take less time off to care for sick relatives. body size, which is greatly influenced by one's health during childhood, has been found to have large impacts on long-term productivity. recent economists [1] have calculated that a 1-year increase in life expectancy improves labor productivity by 4%. despite the weakness of health systems in many developing countries, three-quarters of the world's children now receive a standard package of childhood vaccines through the who/unicef expanded program on immunization to protect them against diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis [2] . these vaccines currently save an estimated 3 million lives a year -almost 10,000 lives a day -and protect millions more from illness and permanent disability, thus providing as mentioned above a healthier cohort of people to contribute to the economic development of the nation. the full package of basic vaccines (diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis) costs less than $20 per year of life saved in poor countries. "life-years" and "year of life" consistently refer to disability-adjusted life-years (dalys). interventions are generally considered extremely costeffective if the cost per year of life is less than $100. by comparison, antiretroviral treatment for hiv/aids-an intervention that donors widely support in the developing world costs up to five times as much at $350 to $500 per life-year saved; by way of comparison, in the us and the uk medical interventions are considered cost effective at $50,000 to $100,000 per life-year saved [1, [3] [4] [5] . the week during the conference in amsterdam it was reported that "vaccine-preventable deaths reach new low in u.s." as reported in a federal report released tuesday, november 13, 2007 people readily associate the role of veterinarians with private veterinary practice focused on pets and farm animals, but the true dimensions and contributions of veterinary medicine are much broader and reflect expanding societal needs and contemporary challenges to animal and human health and to the environment [6] . veterinary medicine has responsibilities in biomedical research; ecosystem management; public health; food and agricultural systems; and care of companion animals, wildlife, exotic animals, and food animals. the expanding role of veterinarians at cdc reflects an appreciation for this variety of contributions. veterinarians' educational background in basic biomedical and clinical sciences compare with that of physicians. however, unlike their counterparts in human medicine, veterinarians must be familiar with multiple species, and their training emphasizes comparative medicine. veterinarians are competent in preventive medicine, population health, parsitology, zoonoses, and epidemiology, which serve them well for careers in public health. the history and tradition of the profession always have focused on protecting and improving both animal health and human health [7] . since 1892, a total of 14 diseases have been eliminated from equine, poultry, and livestock populations in the united states [8] . the elimination of these livestock diseases, along with outstanding research in animal health, is key to the remarkable gains in the efficiency of u.s. animal production [7] . partly as a consequence, u.s. residents spend only approximately 10% of their disposable income on food, whereas residents in other countries pay three or four times more [9] . although this achievement is recognized to have added billions of dollars to other parts of the u.s. economy, its success in allowing the u.s. public access to a nutritious, affordable, and sustainable food supply -also important for the public's health and well-being -is far less appreciated. the success of the national brucellosis and tuberculosis elimination campaigns has benefited not only the u.s. livestock industries but also human health by substantially reducing these zoonotic threats in animals. additional public health contributions can be attributed to the food safety and inspection service of the u.s. department of agriculture (usda), which has substantially reduced the burden of food-borne illnesses, improved food safety, and eliminated other zoonotic threats. over the years, cdc has worked closely with usda and the food and drug administration to improve the safety of u.s. foods and reduce antimicrobial resistance in pathogens that infect both humans and animals. veterinary scientist and veterinarians within the health and human services serve in many critical capacities. veterinary officers in the commissioned corps work throughout the u.s. department of health and human services and in other federal agencies. most veterinary officers are assigned to the cdc, nih, fda, usda, epa, ogha, ndms and state department. other veterinarians and veterinary scientists function as medical research scientists post-doctoral nih/nci fellows, principal investigators, some specializing in lab animal medicine and providing critical lab animal health infrastructure and support, design of animal models for human disease in most of the hhs institutes. some are part of the hhs national disaster medical services and were deployed as the veterinary medical assistant teams (v-mats) and supported the search and rescue in the world trade disaster. additionally, since 1986, the avma and the college of american pathologists have been working together to create a standard nomenclature that would allow veterinarians, physicians, and other medical professionals to create electronic medical records that use a common language. the systematized nomenclature of medicine, snomed, was initially created by the college for human medicine but has sincethrough the partnership with avma -expanded to include veterinary terms. on july 1, 2003, the department of health and human services announced it would make snomed available nationally at no charge, a step toward instituting a standardized electronic medical records system. the hhs signed a 5-year, $32.4 million contract with the college to license snomed and make it available nationally. the national library of medicine is administering the program. prior to the hhs's agreement with the avma, practitioners in areas of practice nationally on the front line of surveillance would have had to pay a $2000 to $3000 annual registration fee to access snomed. the cdc has expanded the role, scope, and influence of veterinarians and veterinary scientists and epidemiologists in public health since its inception in 1946 [10] . early in the history of cdc, veterinarians in the u.s. public health service and the cdc veterinary public health division helped reduce zoonotic diseases, especially rabies and food-borne illnesses [11] . today, 89 veterinarians serve throughout cdc in positions that address not only infectious diseases but also the entire spectrum of public health challenges: environmental health, chronic diseases, human immunodeficiency virus infection and acquired immunodeficiency syndrome, injuries, immunizations, laboratory animal medicine, global health, migration and quarantine, health education, and bioterrorism. veterinarians contribute as epidemiologists, laboratory scientists, policymakers, researchers, and surveillance experts and in environmental and disease prevention and control programs both domestically and globally. at cdc, 228 veterinarians have participated in the epidemic intelligence service since 1951 [12] . forty-one states now have state veterinary public health officials. in 2005, almost 300 students and faculty attended the first veterinary student day at cdc; in april 2007, cdc will co-host an inaugural conference with the association of schools of public health and association of american veterinary medical colleges. in addition, cdc has been recognized as a world association for animal health collaborating center for emerging and re-emerging zoonoses. the cdc publication, emerging infectious diseases, has highlighted zoonotic diseases in nearly every issue to zoonotic diseases and has devoted an annual issue in each of the previous 2 years. the cdc has provided an important scientific forum for zoonotic disease research and programs both domestically and globally and should serve as a template for the nih, as will be discussed later in this paper (section 5.1), for moving these highly trained and broad-based medical skill set professionals from a more decentralized setting into a central institute at the nih. benjamin franklin's famous quote, "an ounce of prevention is worth a pound of cure" was actually fire-fighting advice-he founded the first fire fighting organization in philadelphia, its obvious application to medicine, although obvious, has not been a mainstay of heavily invested research and development in human health practices. many of the leading causes of death and disability in the united states can be prevented [13] . primary prevention can prevent or arrest the disease process in its earliest stages by promoting healthier lifestyles or immunizing against infectious disease [72] . secondary prevention, by detecting and treating asymptomatic risk factors or early asymptomatic disease, can substantially reduce subsequent morbidity or mortality. the human and veterinary clinician plays a pivotal role in both primary and secondary prevention. health professionals deliver vaccinations, screen for modifiable risk factors such as high blood pressure and high cholesterol, counsel patients about smoking and other behavioral risk factors, provide screening tests for early detection of cancer and other chronic conditions, and advise patients about the benefits and risks of preventive therapies such as postmenopausal hormone replacement therapy. the preventative health care landscape has changed in some regards in the 17 years since the u.s. preventive services task force (uspstf/task force) was first established in 1984 to provide advice about prevention for health professionals. prevention became more of an integral component of primary health care [14] . delivery of clinical preventive services such as immunizations, mammograms, and cholesterol screening has risen steadily over the past two decades (national center for health statistics [15]). roughly 90% of employers now include well-child visits, childhood immunizations, screening tests, and adult physical examinations among covered health benefits, compared to less than half that did so in 1988 [16] . interest in prevention grew significantly among the public, clinicians, educators, employers, and policymakers [17] and health plans and individual clinicians were increasingly being held more accountable for the quality of the preventive care they provide to their patients [18] . at the close of the 20th century, health care costs in the united states continued to rise steadily, accounting for 13.5% of the gross domestic product in 1998 [19] , and debate on health care funding for the aging american population intensified. no doubt fueled by the incredibly imbalanced historic spending in preventative healthcare of 3 cents for every 97 cents spent for curative treatment [20] . numbers are harder to come by for recent years, but given the spiraling costs of treatment since 1988 it is likely that this ratio has gone down considerably since then-possibly grossly estimated to be closer to 1:99 today. in this environment, preventive services often compete with one another and with diagnostic-and treatment-oriented care for increasingly constrained resources [21] . while preventive services are often believed to save costs, delivery of most preventive services, with few exceptions (e.g. some immunizations), incurs net costs [22] . evidence that us society clearly favors the cure (or treat) approach to disease over prevention can be shown in the following ways. first, though there is a shortage of preventive medicine specialists (public health, general preventive medicine, occupational medicine, and aerospace medicine physicians), in the us the number of residents in training in 2004 was less than 0.4% of all residents, not sufficient for replacement or to fill the expanding demand for the specialty's skills and talents [23, 70] . second, preventive medicine residencies and subspecialties in human and veterinary medicine are generally found in only graduate medical education programs not financed by cms or mainstream academic training programs. third, we believe that our preventive acts are only statistical, whereas our curative acts are certain. this mistaken belief perhaps derives from our sense that we have more control over cure outcomes than prevention outcomes-we think that we do cure, whereas we only facilitate prevention. this notion of doing vs. facilitating is an important one, because if we believe that our curative actions are more effective than our preventive ones then we will more likely act toward the more effective. the editor of the british medical journal, fiona godlee, expressed this well when she states, "because it is acted on healthy people, preventive medicine needs even stronger supporting evidence on benefits and harms than therapeutic interventions" [24] . thus substantial gaps in the delivery of effective preventive care in the united states remained, however, because clinicians continued to face many of the same barriers that originally spurred the formation of the first uspstf. identifying effective interventions were and are difficult in prevention, where prospective controlled trials are often difficult to conduct. these studies come from the field of epidemiology which has changed remarkably during its growth in the past quarter century. one of those changes has been a mixed blessing of ever-increasing specialization among its practitioners at the cost of the generalist. this phenomenon has shaped the field and a partial explanation for this trend is found in the decline in the availability of training funds not focused on specific and general disease areas. without returning to the training of general conservation-medicine based epidemiologists, the needed trend associations and study designs that are needed to show the economic and public health returns related to preventative practices field will not be realized and in addition lose some of its ability to quickly respond to new and expected emerging public health challenges. conflicting recommendations from different organizations, further exacerbated by the advocacy positions of some groups, leave many clinicians uncertain about what to do. clinicians facing increasing time pressures in practice may question the value of some routine preventive interventions, as may employers and other payers struggling with accelerating health care costs. although more prevention information is reaching the public, the messages conveyed are often inconsistent and increasingly colored by commercial self-interest. clinicians may feel compelled to provide unproven or ineffective services because patients demand them or they fear being sued, but patients may find that insurance coverage for individual preventive services, especially new technologies, is inconsistent. the importance of clarifying what we know and do not know about the effectiveness of specific preventive services is as important in 2001 as it was in 1984. although the uspstf was disbanded in 1989 with the release of the guide, the need to keep pace with the rapid growth in scientific evidence led to convening a second panel in 1990. the second uspstf was smaller, with only 10 members, eight of whom were primary care physicians. it refined the previous group's methods for reviewing evidence and making recommendations, and expanded the scope of topics. it adopted policies for disclosure of conflicts arising from financial interests, funding sources, or other affiliations. the work of the second uspstf was marked by strengthened ties with both federal and nongovernmental partners, including primary care subspecialty societies. the work of the second uspstf culminated in the publication of the second edition of the guide in 1996, which covered over 200 interventions in 70 areas. by the time the second edition of the guide appeared, the environment for preventive medicine and evidence-based medicine had changed dramatically. managed care organizations, which had emerged as a dominant paradigm for delivering and paying for health care, included some preventive care among basic covered services more commonly than had traditional fee-for-service insurance. at the same time, the heightened competition spurred by managed care brought increased attention to costs and value of treatments with less attention given to prevention. the guide was frequently cited by health plans and systems of care in defending their health maintenance programs and benefits packages, and its recommendations informed many of the health plan employer data and information set (hedis) quality measures developed by the national committee on quality assurance for evaluating health plan performance but not integrated into cost saving practices by the insurance companies. the rapid progress towards universal vaccination coverage in the 1970s and 1980s has slowed in recent years. unicef funding for vaccination fell from $182 million to $51.4 million between 1990 and 1998 [25, 63] . global coverage of the diphtheria, tetanus, and pertussis (dtp3) vaccine has been at around 74% since 1990 [26] . fifty-seven developing countries have yet to eliminate neonatal tetanus, and 200,000 babies died of the disease in 2000. ten developing countries reported cases of polio in june 2005, despite the massive (and largely successful) global effort to eradicate the virus [27, 50, 51] . sixty-two percent of countries, meanwhile, had still not achieved full routine immunization coverage in 2003, with gavi estimating that at least 9.2 million additional infants need to be reached to achieve full coverage. there are several factors behind this loss of momentum. although dramatic progress has been made in increasing worldwide vaccination coverage from below 5% to above 70%, the task has inevitably become harder now that the easiest-to-reach populations have been vaccinated. because these communities are more elusive, the average cost per vaccination has increased, and it may be that other apparently cheaper health interventions have become more attractive. there are many practical problems impeding vaccine delivery. delivering vaccines to patients requires functioning freezers and reliable transport to move the vaccines from port to clinic; clinics refrigerators (which in turn require a constant supply of energy); good roads and with access to people who need to be immunized; parents who know the value of vaccination; trained medical staff to deliver the dose; and sterile syringes. only 16% of vaccineimporting countries could guarantee vaccine safety and quality [28] , while a further study of 19 developing countries found that at least half of injections were unsafe [64, [73] [74] [75] [76] . the third factor behind the lack of progress in recent years is political. political disruptions have affected coverage in some areas. in somalia and congo, for example, where vaccination rates have fallen rapidly in the past decade, war and social breakdown have impeded public health campaigns, despite "vaccination days" in congo that temporarily halted fighting. gauri et al. have found that the quality of institutions and governance are positively correlated with vaccination coverage [29, 62] . politics in the developed world have also played a part. according to a report by the us institute of medicine, in 1982 the us vaccine industry was forced to stop offering low-price vaccines to develop-ing countries following congressional hearings that "savaged" the industry for "allegedly subsidizing vaccines for the poor children of the world by charging high costs to us families and taxpayers". as the institute of medicine points out, this move was based on a flawed premise, as the us vaccines would have been developed anyway to protect american children and travelers. public perceptions of vaccination change-as coverage spreads through a community and it reaches a point at which those who are unvaccinated are highly unlikely to catch a disease because herd immunity has set in. at this juncture, it may be more rational for an individual to refuse vaccination in order to avoid any risk of side effects. with the oral polio vaccine, for example, there is a one in a million chance of paralysis, and in societies where mass vaccination has eliminated the disease, the risk of paralysis is greater than that of catching polio itself. what had once been a public and private good is now a public good but a private risk. as more and more people choose to avoid this risk, of course, overall coverage rates decline, and the community is once again exposed to the threat of the disease. public perceptions have been influenced by vaccine scares. controversy and the attendant bad publicity about the safety of vaccines have been abetted by incidents such as the withdrawal of half the us supply of flu vaccines in 2004 due to contamination at the manufacturer [60] . in addition, alarms over the safety of vaccines such as that for measles, mumps and rubella (mmr), which some believe to cause autism, have further fanned the anti-vaccine movement's flames [59] . in the us, disputes continue to rage about the scientific basis of such claims, but the preponderance of the evidence, according to the us centers for disease control (cdc), says that the mmr vaccine is safe [30] . in response to these types of controversies in the us, the institute of medicine has called for independent oversight of vaccine safety studies to ensure the fairness and openness of the vaccine safety datalink program, which is overseen by the cdc. as one can see there are many complex factors that have to be considered when bringing vaccination programs into existence. the impact of vaccination of animal diseases on agriculture is typically assessed in quantitative terms-lost revenues; costs of eradication, decontamination, and restocking; and the numbers of affected farms, animals and humans. this approach can be applied universally to all outbreaks in all countries because it normally reflects the hard data supplied by large commercial operations and the estimates by relevant governmental agencies of small farmer impact [31] . when used exclusively, however, it fails as a barometer, because it does not and cannot factor in the multi-dimensional character of major disease events-and the accompanying societal effects that often get lost when it comes to assessing the damage in developing countries. the quantitative approach must also be interpreted, and cannot be used "as is" for comparing impacts in developed and developing countries. further, while export trade losses in a developing country may be small in terms of the dollar amount, the impact upon its pre-epidemic market share is inevitably greater and more persistent. other impacts such as effects on human health and community stability tend to be more visible and last longer in developing countries, particularly at the village level where animal are husbanded primarily for the benefit of the immediate family, and often in impoverished circumstances [31] . the consequences of animal diseases in domesticated birds and livestock can be complex and generally go well beyond the immediate effects on affected producers. these diseases have numerous impacts, including: • productivity losses for the livestock sector (e.g. production losses, cost of treatment, market disturbances). • loss of income from activities using animal resources (in such sectors as agriculture; energy; transportation; tourism). • loss of well-being of human beings (morbidity and even mortality rates; food safety and quality). • prevention or control costs (production costs; public expenditure). • suboptimal use of production potential (animal species, genetics, livestock practices). the most direct economic impact of animal diseases is loss of production and/or productivity, and ensuing income losses for farmers [55] . however, if the economy depends on one or some of the vulnerable products, the impacts can be serious, and local food security can be threatened. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. if the farm economy is diversified or if there are other opportunities to generate income, the impacts can be mitigated. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. the loss of the farmer's "well-being" will generally be lower than the value of the lost product, except where the farmer has few alternatives or is wholly dependent on the affected product, which is quite often the case in developing countries. direct losses are the result of the disease itself (they may be very high when mortality rates are between 50 and 100%), or from animal health measures (stampingout policies) [31, 32] . in vietnam, one of the countries most seriously affected by the avian flu, almost 44 million birds -17% of the country's poultry population -had to be destroyed at an estimated cost of us $120 million (0.3% of gnp) [33] . the smaller scale producers lost the least in absolute terms, but the most in relative terms, as the outbreak resulted in losses equivalent to upwards of 50 times their daily income (from us $2 a day or less). in africa, abortions caused by the rift valley fever virus not only affect birth rates, but also push human consumption of milk downward in the year following an outbreak [34] . in the dairy farming sector in kenya, it is estimated that losses in milk production accounted for 30% of all losses caused by an outbreak of foot-and-mouth disease in the 1980s. direct costs are generally well below the indirect costs of animal diseases and are directly linked to the rapid containment of outbreaks: case studies have shown that early detection and the implementation of appropriate measures in the event of an outbreak are essential to help minimize direct losses as much as possible. conversely, inappropriate control and eradication measures are at the root of such endemic situations, which are much more difficult, and infinitely more costly, to keep under control or eradicate. the livestock sector plays a significant role in the economic development of many countries and vaccination can serve as one of the most important means of assuring its health. as such the cost of not developing new and important or properly applied vaccines can have tremendous economic consequences. the production of meat and other animal-based food items generates income, jobs, and foreign exchange for all stakeholders in the animal industries. consequently, an epizootic which could have been otherwise mitigated by vaccination can affect the industry's upstream (inputs, genetic resources) and downstream activities (slaughterhouses, butchering operations, processing, marketing) in terms of jobs, income for the stakeholders in the industry, or market access. a survey by the food and agriculture organization of the united nations (fa0) on avian flu revealed that in the most seriously affected regions of indonesia, 20% of permanent workers at industrial or commercial farms lost their jobs [33] . similarly, an outbreak of contagious bovine pleuropneumonia in botswana led to the destruction of more than 300,000 animals in the most seriously affected province, and the immediate closure of the export slaughterhouse, which employed 200 persons. owing to the catalyst role of livestock raising in the rural economy as a whole, the costs of the indirect effects of these measures were later estimated to be seven times higher than the costs caused by direct losses [32] . in vietnam, 60% of the poorest segment of the population, for which poultry farming accounts for 6-7% of household income, is particularly vulnerable to income losses caused by avian flu. the fao and world organization for animal health (oie) estimate that between one-third and one-half of the populations living in the most seriously affected areas of southeast asia depend on poultry farming for at least a part of their income [32, 35] . in france, the leading european poultry producer, it is estimated that farmers affected by the crisis lost 40% of their income in 3 months (between january and march 2006). the effects of the production losses are also linked to price variations, which are caused by supply and demand (im)balances. depending on the market, prices can rise sharply (consumer product on the domestic market) or plummet (product banned for export but cleared for consumption on the domestic market, product deemed too dangerous for human consumption or perceived as such). in brazil, where 30% of products are exported, the price of a day-old chick, an early indicator of a possible change in production, reportedly fell by 50%. and even in cases where the country is not infected, market uncertainties and the fall in prices prompted the largest producers to cut back production by 15% this year. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. in 1997/1998, the rift valley fever outbreaks in east africa seriously affected pastoral economies in somalia, with a decline of more than 75% in exports (which generate more than 90% of foreign exchange in "somali land"), following an embargo declared by saudi arabia on all animal products from the horn of africa [32, 36] . conversely, the world bank has reported that eradication of certain major diseases to facilitate access to "high value" export markets can provide considerable benefits. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. uruguay is a good example of a country that gained access to a lucrative market after eradicating foot-and-mouth disease. beef exports increased in volume by more than 100% and in value by 52% after the oie declared uruguay to be officially foot-and-mouth disease-free without vaccination in 1996. access to the u.s. market (where prices are double those of the domestic market) provides uruguay with additional revenue to the tune of us $20 million each year. a medium-term analysis showed that access to "pacific rim" markets would generate additional revenue of us $90 million each year, and yet, before the disease was eradicated, uruguay had been spending (only) us$8 million to us $9 million each year on vaccines to combat foot-and-mouth disease. in this case, control costs would account for less than 10% of the revenue generated by exports alone [32] . animal diseases that could be controlled by vaccination can have major effects on food availability and quality for poor communities. it is well known that agriculture plays an important role in the generation of income and jobs in other sectors but the closeness of this interdependence became particularly obvious during recent epizootics. for pastoral societies, animal husbandry contributes directly and indirectly to food security and to nutrition as a source of quality proteins, vitamins and trace elements, traction, and com-mercially tradable products [68, 69] . certain diseases could have significant repercussions on food supply and the nutrition of poor communities that do not have readily available substitute products, which could therefore lead to famine (rinderpest for example). poultry meat is the primary animal protein in africa (which has little to begin with) and the indispensable source of discretionary income for the survival of millions of small farmers. the high mortality rates as a result of avian flu, which is extremely pathogenic, and the sanitary slaughter of poultry would therefore have a negative impact on the food available to the entire population, as well as on rural revenue. furthermore, developing or transition countries which generally have poor public health systems are particularly at risk from zoonoses making vaccination against these diseases particularly important to target. in 1977/1978 a major rift valley fever epidemic in egypt resulted in 200,000 human cases and 600 fatalities [32, 36] . twenty years later, a new epidemic affected over 500,000 persons in east africa, and 500 persons succumbed to the hemorrhagic form of the disease. but zoonoses also affected industrialized countries with high health standards as was the case with the bovine spongiform encephalopathy crisis in europe [65] . food-borne diseases (over 200 have been classified) are a major source of acute gastroenteritis (which costs the netherlands us $27 million per year) and the cause of major morbidity with fatalities among children in the third world [32] . in the specific case of a pandemic, most of the economic loss is caused by the increase in morbidities and fatalities in the human population and its repercussions on the world economy. the most recent estimates suggest that the "spanish" influenza in 1918 caused the death of 50 million persons, that is, 2.5% of the population at the time. the most obvious economic losses were the reduction in quantity and productivity of the workforce, and according to the experts, in the case of a pandemic could represent 10 times more than all the other losses combined [33] . another category of economic impact is linked to individual strategies to avoid contamination-or to survive possible contamination. the example of the severe acute respiratory syndrome (sars) clearly shows the sharp drop in demand in the services sector (tourism, public transport, retail trade, hospitality and food services) resulting from the combined efforts of individuals to avoid any close contact [37] . based on the experience with severe acute respiratory syndrome in south-east asia, the world bank thinks that an avian flu pandemic could result in a 2% loss of the world's gross domestic product and cost the world economy us $800 billion in the space of 1 year. the losses are difficult to calculate and would undoubtedly be much more significant in light of the extremely high mortality rates in developing countries which do not have good health care systems. the impact of animal diseases on the tourism and leisure sectors could also be quite significant. the negative effect of foot-and-mouth disease in the united kingdom on these two sectors amounted to us $49 billion because of restrictions on access to rural areas and represented more than half of the total cost of the disease [32] . the federation of american scientists' animal health/emerging animal diseases (ahead) project proposed a major program in sub-saharan africa to detect and document the extent of infectious diseases shared by farm and wild animals, and to supply treatment, prevention and control services to remote communities that have previously been neglected by other programs, both national and international. this program, international lookout for infectious animal disease (iliad), was implemented in south africa [38] . at the core of iliad is the need for a permanent and sustainable regional program of in situ surveillance designed to detect, monitor, treat, prevent and control infectious diseases with the goals of increasing livestock production in remote farming communities, protecting the health of wild species, building indigenous physical and professional resources, and introducing communications and epidemiology information technologies. transmission of infectious diseases is rampant in remote communities in the sub-saharan region, just as they once were in the united states and as they always are wherever poverty and farming co-exist. diseases shared by wild, farmed and captive/bred animals, and by animals and humans, suppress food production, frustrate species preservation efforts and greatly affect public health. detection, prevention and control of these diseases are an essential element in expanding trade, improving nutrition, exploiting ecotourism and ensuring food security. iliad is structured in the investor mode-an international consortium of donor groups providing short-term developmental assistance with program direction and oversight provided by veterinary diagnostic, public policy and epidemiology experts representing the sub-saharan africa partnership members-the renowned onderstepoort veterinary and exotic disease institutes (ovi) and tuskegee university (tu), and fas-ahead. given positive assessments of the benefits of the program after 3 years, national or provincial institutions will integrate some or all of the activities into their official veterinary and agricultural activities. it is difficult to calculate the cost of the public's loss of confidence in animal industries in their countries, or of an importer country towards the veterinary services of the exporter country. animal diseases can have major effects on food availability and quality for poor communities. consumers' obsessive fear of bovine spongiform encephalopathy (mad cow disease), fed by the media and which a good communication strategy could have prevented, would have tremendous social repercussions on a europe still reeling from long term economic repercussions. in italy, the baseless perception of a food risk related to avian flu coupled with low confidence in public health services eventually resulted in a 70% reduction in the consumption of poultry and eggs. the loss of confidence by an importer country can trigger a lasting embargo and major economic and social repercussions (arabian peninsula embargo on the horn of africa, affected by the rift valley fever virus). loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. animal diseases might also have indirect long-term impacts, affecting deferred productivity. this is the case for example of the reduction in the fertility rate of long-cycle species, the effects of which span periods of 10-20 years [32] . in short, the long-term costs of a slow response are rarely taken into account. economic analyses focus primarily on the effects of the outbreaks and rarely take into account the long-term effects of an endemic situation (characterized by less virulent outbreaks which recur for several years). this is the case of classic swine fever in haiti where recurrent outbreaks reduced the usage rate by 10%, which for pig farmers meant a loss of revenue of us $2.7 million per year [31] [32] [33] . with major crisis, long-term impacts would make themselves felt, since the additional costs of financing prevention and control measures would lead to an equivalent reduction in savings and investments. for example, the analysis of the global impact of the avian flu crisis in europe was complicated by outbreaks of foot-and-mouth disease in brazil, the largest global exporter of beef and poultry. it is therefore easy to imagine what the combination of these two events would mean in terms of the upward push of prices of all meats, similar to what occurred in 2004 with north american beef and bovine spongiform encephalopathy. the european union, a net importer of beef, especially from brazil, would see an increase in the price of beef in its internal markets stemming from the embargo imposed on brazilian beef because of the foot-and-mouth disease. it must be pointed out that the crises could have a cumulative impact, particularly since they are amplified by the effects of globalization the following example therefore illustrates the ripple, spillover and remote effects: in the united states, where 62% of oleaginous and cereal production is geared towards animal production. an epizootic which reduces animal production by 10% would have the immediate consequence of the loss of 418,000 jobs, a surplus of 18.4 ton in cereals and oleaginous products, a 10% reduction in world trade and, crises in other producing countries. in 1900, nearly 800 americans out of every 100,000 died each year of infectious disease. laurie garrett, author of "the coming plague: newly emerging diseases in a world out of balance", writes that in the postwar environment, powerful medical weaponry (antibiotics, vaccines, water treatment, anti-malaria drugs) gave scientists confidence that they could significantly control and/or eradicate infectious disease from viral, bacterial or parasitical sources. in the late 1960s, the surgeon general of the usa, william h. stewart, said that ". . .it was time to close the book on infectious diseases and pay more attention to chronic ailments such as cancer and heart disease." a measure of that success came towards the end of the 1970s, when the world realized that smallpox had become the first disease to be eradicated from the human species. such halcyon days from the 1960s to the early 1980s are but a memory. by 1980, the numbers were down to 36 per 100,000. the "health for all" accord, signed in 1978, set a goal of the year 2000 for eliminating many international scourges. but amid all this optimism, the numbers started rising. in 1995, 63 people per 100,000 died and we know the rest of the story, . . . or do we? the grandiose optimism rested on two false assumptions; that microbes were biologically stationary targets, that for the most part human and other animal diseases were for the most part limited to those species and geographically sequestered. scientists have witnessed an alarming mechanism of microbial adaptation and change, anything but stationary, microbes and the insects, rodents and other animals that transmit them are in a constant state of biological, ecological flux and evolution [52] . according to the u.k. centre for tropical veterinary medicine, 60-70% of all the 1415 known species of infectious organisms that affect human health (causing a quarter of the world's deaths) can be transmitted by animals. approximately 175 of these infectious organisms are linked to diseases that have only recently emerged, or have increased in severity (and geographic distribution) in recent years. who averages 200 outbreak investigations every year, and around 50 will require an international response. more than 30 new and highly infectious diseases have been identified in the last 20 years. furthermore, 20 known strains of diseases such as tuberculosis, many species of gram positive and negative bacteria as well as many parasites, e.g. malaria, food animal coccidia have developed resistance to various classes of antibiotics, while old diseases have reappeared, such as cholera (in angola, with 1298 deaths), yellow fever (new cases recently reported in guinea, sudan, mali, and senegal), plague, dengue fever, meningitis, hemorrhagic fever, measles, mumps, rubella and diphtheria. there are 63 emerging diseases just among marine life, reports the book conservation medicine, and these include tuberculosis in fur seals and chlamydiosis in sea turtles. more staggering than these cited numbers in living animals including humans are those coming out of the microbial genetic sequencing and diversity studies involving the oceans. recent data in this field suggest that the oceans of the world contains approximately 10 (31) phage particles or virions (cf. 22 million metric tons), much of it turning over once per day and most likely be a regular source for current and future zoonotic and human infections. this vast mutation engine, even if one assumes a minimal mutation rate, generates the equivalent of hundreds of new complete human genomes per day. a 200-l sample of surface seawater was concentrated; ∼2 × 10 (12) viral particles, the dna once randomly sheared and cloned yielded 1,934 fragments for sequencing. data analysis showed that most of the sequences were from previously unknown viruses. approximately 3.5% of the total sequence samples overlapped, suggesting that the marine viral community was highly diverse. a unique mathematical analysis further suggested that approximately 10 (4) different new viral types may be present. it is obvious from this most recent description that complex and confounding zoonotic interactions can be expected to occur as ecosystems become concentrated and/or diluted during the upcoming environmental and ecological change. this will require a new breed of medical scientists that have foregone the days of super-and sub-specialization and are now grounded in both depth and broad general eco-and bio-medical systems training. few people recognize the broad range of clinical and basic veterinary research and its many important contributions to society in the realms of public health and food safety, vaccination, fertility, drug and vaccine development, surgical techniques and biodegradable materials, space medicine, animal health and welfare, and comparative medicine [66] . opportunities in veterinary research include comparative studies with animals that shed light on human health problems; the development of tools to better detect, prevent, and control zoonotic diseases (that spread from animals to humans); the establishment of scientifically based policies for the humane treatment of animals; and the development of measures to secure and protect the nation's food supply and farm-animal economy from a potential act of bioterrorism [67, 71] . the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-economic problems. the single profession that is most centered on the new paradigm is veterinary medicine. the three major disciplines within veterinary medicine and research -public health, comparative clinical and basic medicine, and animal health -are closely intertwined [49, 61] . for example, research in comparative medicine contributes to animal health through the development of preventive medicine and treatment. the study of wildlife diseases contributes not only to wildlife health and conservation, but also to public health because many animal diseases can spread to humans. therefore, collaborative and interdisciplinary research is crucial in translating scientific advances from one traditional discipline to another. however, interdisciplinary research is in many cases hampered by administrative, funding, and cultural barriers between institutions. furthermore, agencies that support veterinary research have specific missions. funding to support proposed interdisciplinary research can be difficult to obtain when it is partially related to the mission of several agencies but does not perfectly fit the mission of any one agency. the future requires the veterinary research community to encourage research funders to develop a long-term interagency strategy for veterinary research [53, 54, 57] . in 1858, rudolph virchow, the father of comparative medicine, stated, "between animal and human medicine there are no dividing lines-nor should there be. the object is different but the experience obtained constitutes the basis of all medicine" [39] . sir william osler (1849-1919), considered the best-known physician in the english-speaking world at the turn of the century, called the "most influential physician in history" is quoted as saying "veterinary medicine and human medicine complement each other and should be considered as one medicine." dr. calvin schwabe, a retired uc davis professor of veterinary medicine who pioneered the use of human disease tracking techniques in the study of animal illnesses, a global authority on animal diseases that are communicable to human beings and was an early visionary in a field that today is marked by the emergence of pathogens such as avian influenza, mad cow disease and sars. in 1966, dr. schwabe established the department of epidemiology and preventive medicine at the uc davis school of veterinary medicine-the first of its kind in the world at a vet school. the author of more than 200 publications, he promoted the concept of "one medicine," which attempts to bring the fields of human and animal health care together. to this goal in june of 2007 the american veterinary medical association (avma) announced that in partnership with the american medical association (ama) there was an adopted resolution calling for collaboration on a one health initiative. the two national, medical organizations will work collaboratively on areas of mutual medical interest, such as pandemic influenza, bioterrorism risks, biomedical research and will be charged with developing strategies to promote collaboration among the various health science associations, colleges, government agencies and industries. a quote from a ama board member, duane m. cady, md "new infections continue to emerge and with threats of cross-species disease transmission and pandemic in our global health environment, the time has come for the human and veterinary medical professions to work closer together for the greater protection of the public health in the 21st century," the avma policies supporting the concept of "one health" include: furthermore to strengthen this initiative and give it a renewable funding foundation, it would be recommended to move toward, the creation of: (1) a specific focus at the national institutes of health (nih) on integrated veterinary research via the roadmap initiativemore specifically to create or combine existing potions of institutes into an institute of comparative medicine (icm); (2) a joint interagency collaborative programs could also be established to enhance interdisciplinary collaborative research and have either re-routed an/or new congressionally supported intra-mural and extra-mural funding program; and (3) while working out and introducing the legislative changes for the icm the nih should create a veterinary liaison with all the current institutes like the veterinary-medicine and public-health liaison at the centers for disease control and prevention (cdc) in which a veterinarian, dr. lonnie j. king, was selected to head up the agency's national center for zoonotic, vector-borne, and enteric diseases-a relatively recent creation, the center is dedicated to understanding infectious disease ecology and will help to ensure integration of veterinary and human medical research. the american veterinary medical association, with information provided by many other organizations and institutions, conservatively estimates a current deficit of 1500 public health veterinarians (e.g. usda food safety and animal disease control, homeland security, research on domestic and foreign animal diseases, wildlife disease control, laboratory animal care and research) and is expected to increase possibly to 15,000 by 2025 as the human population increases without intervention. comparably the health resources and services administration (hrsa) in the u.s. department of health and human services (dhhs) released a report in 2006, projecting a shortfall of approximately 55,000 physicians in 2020 [56] . if current trends continue, the full time equivalent (fte) physician supply is projected to grow to 866,400 by 2020, while demand for physicians will increase to 921,500 due to the growth and aging of the u.s. population (physician workforce policy guidelines, 2005). the report projects shortages will be in greatest in non-primary care specialties. it has been over 30 years since the federal government has allocated funds to increase the number of veterinarians and physicians that graduate each year. to begin to alleviate this problem the veterinary workforce expansion act (s. 914/h.r. 2206), which is currently being considered by the united states house of representatives and senate would provide an average of $150 million per year for the next 10 years in the form of competitive grants to help increase the number of veterinarians entering public practice. if passed and funded, this bill would allow the nation's veterinary schools and other institutions training public health veterinarians to apply for competitive grants to increase capacity in the form of classrooms, teaching laboratories, research facilities, and administrative space. this bill would be vital to the nation's ability to protect human and animal health, as veterinarians are often the first line of defense for both. the text of the bill can be found at: http://thomas.loc.gov by searching by the bill numbers listed above. a list of co-sponsors to the bill can also be found via that site by selecting the link entitled "bill summary & status". over the past century, humanity has had a devastating impact on the earth's wildlife and ecosystems. we are in fact living through the largest mass extinction since the end of the dinosaurs 65 million years ago. unless effective solutions are found, this new century will see the demise of countless more species and pristine ecosystems, particularly in the tropics. the global society, and what surrounds and influences it, are in profound change. these changes will have very significant impacts on future veterinary medicine and veterinary medical education. there are major demographic, political, environmental, disease, technological, and economic influences, all forcing changes onto society. a few examples illustrate the point [40] . • with immigration into north america accelerating, combined with a declining birth rate, the ethnic diversity in society will continue to increase, with the associated impact on values. • in 2007, for the first time in history, urban people will outnumber rural people. • political destabilization, inflamed by bio-terrorism and religious fanaticism, is expected to increase. • changes in the atmosphere are causing powerful shifts in the environment (melting of the ice caps, rising sea levels) and in the climate (hurricanes, flooding). • global water shortages, especially in heavily populated areas, will soon approach critical levels. to information and service (http://www.jvmeonline.org/cgi/ content/full/34/1/1#b4#b4). • consumer spending power in emerging economies will go from $4 trillion to $9 trillion by 2015, but the gap between rich and poor is increasing. how will these changes alter the needs of society? how must academic veterinary medicine adapt to prepare veterinarians to respond to these new needs? clearly, humanity has yet to find a way to live on planet earth in a potentially sustaining manner where by stabilizing flora and fauna remain intact to promote healthy ecosystems diversity for all species including our very ownhumans. for example, it is estimated that the equivalent of six earths would be needed to sustain the current world's population if people everywhere consumed natural resources at the rate we do in the united states. it is interesting to think that vaccination directly or indirectly impacts six of the seven united nations millennium goals. understanding and coping effectively with an emerging crisis may sometimes require the birth of action-oriented "crisis disciplines." conservation medicine: ecological health in practice brings together an impressive group of experts from diverse specialties medicine, veterinary science, conservation biology, epidemiology, parasitology, public health, and others) to examine the links among human health, wildlife health, and ecosystem health and begin to address questions like: • how factors such as climate change, endocrine disruptors, and toxic microalgae affect wildlife and human health; • the importance of biodiversity for human health (as medical models, sources of medicines, factors in the ecology of infectious diseases, and indicators of environmental quality), with a review of 769 biodiversity-related biomedical research projects funded by the national institutes of health from 1995 to 1997; • how the health of rainforest-dwelling peoples depends on such diverse factors as forest integrity, floods, seasonality, community organization, education, gender dynamics, national budgets, and global markets; • how wildlife health relates to environmental security; • the health hazards of ecotourism; • the causes and impacts of emerging infectious diseases of humans and wildlife; • how the health of terrestrial and marine animals and ecosystems are monitored, and descriptions of innovations using stool dna and retrovirus evolution as markers of animal population dynamics, stool hormones to indicate species stress, and animal behaviors as proxies for the health of ecosystems; • how habitat fragmentation and reduced biodiversity can increase the risk of lyme disease infection; • how land use changes such as deforestation and water projects influence the ecology of malaria and other vector-borne infections; • how ecological health and wildlife disease are managed in national parks; • the role of zoos in the recovery and conservation of endangered species; • how reducing the burden of infectious disease among park workers in africa could prevent a devastating epidemic among the world's 650 remaining mountain gorillas; • how efforts to control livestock diseases are affecting wildlife health and ecosystems in botswana; • teaching ecosystem health in an undergraduate medical curriculum. more than one billion poor people in asia and africa are closely linked with animals for their livelihoods and they pay a really high tribute to different animal diseases-one can easily demonstrate that improving animal-health mechanisms at the national and local level, and decreasing this weight of animal diseases, will lead very quickly to the alleviation of poverty for this class of people. the world organization of animal health (oie) brings together chief veterinary officers from 172 countries in an effort to create global standards of animal health and animal welfare robust enough to withstand the daunting challenges of worldwide commerce. with an annual budget of around $20 million, it is a significantly smaller organization that its human-health counterpart, the world health organization, which has some $2 billion a year expenditure on its programs. last year, over 21 billion food animals were produced worldwide to help feed a population of 6 billion people, resulting in trillions of pounds of animal products distributed worldwide and projections for 2020 show that demand for animal products will increase by 50%, especially in developing countries. this poses unprecedented risks for human safety and will require a closer linking of the two agencies. along with those core concerns, oie also addresses farming practices, analyzes import risks, and mediates bilateral trade disputes among its member countries. among its most important achievements in recent years, has been the eradication of rhinderpest, a centuries-old intestinal disease also known as "cattle plague in some countires." in the early 1980s, rhinderpest wiped out upwards of $500 million worth of african livestock, contributing to widespread human famine. in general professional students of veterinary and human medicine are exposed more likely to a more scientifically exposed than a politically exposed culture. animal and human health policies must be science-based the rallying cry that is heard from animal and human health professionals around the world. the second verse of this mantra is often 'science, not politics', as if science is unquestionably 'good' and politics 'evil'. antipathy toward politics is worn as a badge of honor. the crowning moment frequently comes with the proclamation, 'i am a scientist, i want nothing to do with politics. ' the phrase 'science-based' infers that the underlying justification of animal/human health policy is derived from knowledge gathered through the systematic observation of, or experimentation with, phenomena. scientific knowledge implies the compilation and analysis of data by individuals with advanced education in specific disciplines. scientists seek facts, the fundamental truths which explain the world around us. at face value, the 'science, not politics' paradigm has a great deal of appeal. however, the very notion of fundamental truth is illusory, as scientific knowledge changes frequently with new observations and experiments. furthermore, conjecture and refutation characterize the scientific method, with disagreement and debate the recognized features of scholarly pursuit. scientists often reach conflicting interpretations of observational and experimental data. consequently, individual scientists may champion different, even diametrically opposed, sets of ideas and principles, so that any number of alternatives may be justified as 'science-based'. finally, animal health professionals typically consider only the biological and physical sciences as 'true sciences', dismissing the social sciences. politics reflect the human need for organization of authority, whether in public or private life. politics exist whenever two or more people come together. the terms 'office politics' and 'family politics' are recognized as clearly as the collective activities surrounding local or national governance. politics exist even in science, affecting scientific organizations, refereed publications and academe. indeed, politics are inescapable. all public courses of animal/human health action adopted by governments emerge from the interplay of science and politics. the policy-making process is governed by rules and regulations, affected by the organizational culture of the government agencies involved, and constrained by legal authorities, political correctness and resource availability. the animal/human health policy-making process involves consideration of current biological and physical scientific knowledge. policy decisions also consider social science factors including ideologies, economics and public opinion. hence the etiology of animal/human health policy is multifactorial. the current older traditional schools of veterinary and human medicine and future schools of "one medicine" will need to include leadership, economics and local, state, national and international governances courses and better training in mechanisms of public policies and rule making. epidemiologists accept the concept of multifactorial etiology as a basic tenet. we discuss disease in terms of agent, host and environment interactions. the practice of applied epidemiology demands a breadth of knowledge and the ability to work in interdisciplinary teams. the more complex the problem, the greater the demand for additional knowledge and insights. veterinary and human epidemiologists study risk factors for disease in animal/human populations and develop strategies for health promotion and disease control [58] . unfortunately these are done frequently in a vacuum when in fact the two are inter-related and contributing to the observed and at the time undiagnosed disease spectrum however, animal/human health problems cannot be resolved by consideration of biological and physical factors alone. the veterinary and human (someday to become one and the same) epidemiologist working with field problems soon recognizes the critical role played by people in animal/human health issues. applying epidemiological principles to animal/human disease prevention requires consideration of social issues such as attitudes toward animals, cultural and religious mores, and individuals' willingness and capability to implement the prevention strategies. implementing prevention on a national scale brings additional factors into the equation such as availability of resources, adequacy of veterinary services, and animal health infrastructure, among others. therefore, animal health policy development and imple-mentation require attention to macroepidemiology, the study of all of the economic, social and political inputs which affect the distribution and impact of animal or human disease at the national level [41, 77] . the low hanging fruit of yesterdays fields of microbiology, zoonotic and emerging infectious diseases, immunology, antibiotic sensitivity, vaccine development, oncology, anti-viral drugs, public health, ecology, environment, human and animal health, to name a few have been picked. today comparative and interdisciplinary research is critical to translating scientific advances from one discipline or species to another and providing new insights into human health problems. scientific fields such as laboratory animal medicine, pathology, immunology, biophysics, mathematics, bioinformatics, genetics, molecular biology and toxicology, when combined with veterinary medicine, have proven especially relevant to success in biomedical research. veterinarians also have contributed to public health through the care of companion animals. fifty-seven percent of all u.s. households own a dog, cat, or both. in addition, millions of exotic animals, birds, and reptiles are kept as pets [42] . although pets enrich the lives of humans, they also potentially can threaten public health. veterinarians help educate the public about prevention of zoonoses; vaccinate large numbers of pets for zoonotic diseases, such as rabies and leptospirosis; and reduce the level of ecoparasites that can transmit human diseases and intestinal worms, such as roundworms and hookworms, which can cause serious health problems in humans. the 60,000 private-practice veterinarians in the united states form a valuable front line for detecting adverse health events, reducing zoonotic diseases, and delivering public health education. because veterinarians work at the interface of human, animal, and environmental health, they are uniquely positioned to view this dynamic through the lens of public health impact. significant changes in land use, expansion of large and intensified animal-production units, and microbial and chemical pollution of land and water sources have created new threats to the health of both animals and humans [43] . because animals share human environment, food, and water, they are effective sentinels for environmental, human, and public health problems, including bioterrorism. concerns are increasing about antimicrobial resistance of pathogens, waste and nutrient management, and potential runoffs into streams, rivers, and oceans. food animal and wildlife populations are inextricably linked to some environmental problems. together these have led to creation of a new scientific discipline called conservation medicine and ecosystem health, and veterinarians are assuming a leadership role in the field [44] . several decades ago, special factors came together to create a new epidemiologic era characterized by increases in emerging and reemerging zoonoses [45] . humans, animals, and animal products now move rapidly around the world, and pathogens are adapting, finding new niches, and jumping across species into new hosts. in 2005, approximately 21 billion food animals were produced to help feed a world population of 6.5 billion persons; the united nations' food and agriculture organization estimates that demand for animal protein will increase by 50% by 2020, especially in developing countries [46] . the lessons learned from severe acute respiratory syndrome, west nile virus, monkeypox, and avian influenza are reminders of the need to view diseases globally; integrate animal and public health surveillance, epidemiology, and laboratory systems; and create new strategic partnerships among animal, human, and public health professions [47, 48] . veterinarians are essential to the detection and diagnosis of and response to these threats and are integral to first-line defense and surveillance for bioterrorism agents. the convergence of human and animal health drove creation of the newly proposed national center for zoonotic, vector-borne, and enteric diseases. plans are being completed to establish several multidisciplinary state-level zoonosis research and development centers. the veterinary profession has recently gained a important foothold on the health and human services government research institutes at the cdc and evolved in prominence as a member of the health professions and has established its importance and usefulness to human and public health. it is hoped and expected that with the developing visions and challenges outlined in this overview we will see the continued melding of veterinary and human medicine to create new educational programs and tools for developing future leaders for solving globally some of the most challenging public and animal health, ecosystem, and conservation problems of the 21st century. the value of vaccination world health organization. measles deaths drop dramatically as vaccine reaches world's poorest children the value of vaccination: a global perspective intervention cost-effectiveness: overview of main messages the costs and benefits of front-loading and predictability of immunization. cgd working paper 80 veterinarians in population health and public practice: meeting critical national needs cattle, priests and progress in medicine veterinary medicine: an illustrated history committee on assessing the nation's framework for addressing animal diseases. national research council. animal health at the crossroads: preventing, detecting, and diagnosing animal diseases veterinary medicine and public health at cdc the 50th anniversary of the veterinary medical corps officers of the u.s. public health service veterinarians and public health: the epidemic intelligence service of the centers for disease control and prevention 1951 actual causes of death in the united states primary care: balancing health needs, services, and technology mercer survey of employer-sponsored health plans recommendations to the congressional prevention coalition: nine high-impact actions congress can take to protect and promote the nation's health hedis 2000: the health plan employer data and information set and the health accounts team health spending in 1998: signals of change effectiveness in disease and injury prevention estimated national spending on prevention-united states disease prevention research at nih: an agenda for all. workshop j: prevention strategies, economic realities, and identification of prevention research needs dissecting cost-effectiveness analysis for preventive interventions: a guide for decision makers us graduate medical education editor's choice: preventive medicine makes us miserable immunization in developing countries: its political and organizational determinants progress towards immunization goals 2001: summary presentation of key indicators global market-global vaccines. immunization focus global market-global vaccines. immunization focus national academy of sciences. america's vital interest in global health vaccine safety: real and perceived issues the status and role of vaccines in the u.s. food animal industry: implications for biological terrorism economic and social consequences of animal diseases issue paper: global risks of infectious animal diseases tending animals in the global village: a guide to international veterinary medicine appendix a. global status of livestock and poultry species risk factors for human disease emergence summary of probable sars cases with onset of illness from lessening the impact of animal disease on developing country agriculture: a proposed program using developed country technologies sustainable agriculture solutions-action report veterinary medicine and human health envisioning the future of veterinary medical education: the association of american veterinary medical colleges foresight project, final report science, politics and animal health policy: epidemiology in action*1 the current and future market for veterinarians and veterinary medical services in the united states potential of cooperation between human and animal health to strengthen health systems emerging zoonoses and pathogens of public health concern microbial threats to health: emergence, detection, and response livestock to 2020: the next food revolution. food, agriculture, and the environment discussion paper 28 learning from sars: preparing for the next disease outbreak. workshop summary confronting zoonoses, linking human and veterinary medicine conservation medicine: ecological health in practice the burden of vaccine-preventable diseases making markets for vaccines: from ideas to action diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence committee on the national needs for research in veterinary science. national research council. critical needs for research in veterinary science committee on increasing veterinary involvement in biomedical research. national research council. national need and priorities for veterinarians in biomedical research animal agriculture and the global food supply. task force report 135. ames, iowa: council for agricultural science and technology council on graduate medical education. physician workforce policy guidelines for the u.s. for towards a better map: science, the public, and the media. economic and social research council the general epidemiologist: is there a place in today's epidemiology? misleading media reporting? the mmr story vaccine delays hit chiron's recovery. la times ten great veterinary public health/preventive medicine achievements in the united states advance market commitments: a policy to stimulate investment in vaccines for neglected diseases historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states priorities in developing countries surveillance and monitoring systems for animal health programs and disease surveys committee on assessing the nation's framework for addressing animal diseases, national research council. animal health at the crossroads: preventing, detecting, and diagnosing animal diseases. washington sustainable agriculture solutions (sas)-action report the annual report the annual report understanding and improving health and objectives for improving health. 2 vols an agenda for action: veterinary medicine's crucial role in public health and biodefense and the obligation of academic veterinary medicine to respond investing in health immunization at a glance immunization at a glance world development indicators polio eradication website, global case count measuring the national economic benefits of reducing livestock mortality the primary author wishes to acknowledge and dedicate this paper to the late dr.'s jonas salk and maurice hilleman whose lifetime experiences and contributions are unprecedented in medicine and vaccinology and whom personally encouraged and instilled in me during numerous public and private discussions and meetings the importance of working in and helping to advance the field of vaccinology for the betterment of global human and animal health. key: cord-005147-mvoq9vln authors: nan title: autorenregister date: 2017-02-23 journal: med genet doi: 10.1007/s11825-017-0126-6 sha: doc_id: 5147 cord_uid: mvoq9vln nan complex mechanisms of dosage compensation regulate the mammalian x chromosome due to the presence of one copy in males (xy) and two in females (xx). x inactivation silences one x chromosome in females in early development, leading to specific epigenetic and structural changes. the inactive x chromosome becomes condensed and forms a bipartite structure within the nucleus, as we have shown by chromatin conformation analyses. specific long non-coding rnas are implicated in the formation of this unique structure. the inactive x chromosome is preferentially located near the lamina or the nucleolus. genes that escape x inactivation tend to be located at the periphery of the condensed inactive x chromosome. such genes are more highly expressed in females, and thus associated with sex-specific differences manifested even in early development. we have found that significant sex bias in gene expression are associated with escape from x inactivation in human tissues from normal males and females, and in tissues from individuals with sex chromosome aneuploidy, including turner or klinefelter individuals. institute for genomic medicine, columbia university medical center, new york, usa a central challenge in human disease genetics is the identification of pathogenic mutations. one key approach to distinguishing benign and pathogenic mutations is to use population genetic data to identify regions of the human genome under purifying selection. here i describe how the residual variation intolerance scoring framework has been applied to identifying pathogenic mutations in and outside protein encoding regions of the genome. next i report how these are related approaches are being used to identify pathogenic mutations in large-scale scale studies in epilepsy and other neurodevelopmental diseases. finally, i discuss how the identification of genetic causes of disease can inform treatment choices. scents indicate things, make promises, attract attention and stimulate imagination, feed anxieties and hopes: they are the salt in the atmospheric soup. we regard seeing and hearing as more important sensory functions, because they contribute more to conscious, cognitive processes of perception -but at moments of the greatest enjoyment we close our eyes and taste the scent, smell the taste. before the spirit and beauty of a person can fascinate us, our nose must become infatuated. the olfactory system in the nose acts as a window, monitoring environmental chemical information and convert chemical stimuli in electrical nerve impulses which are conducted along the olfactory sensory neuron to their glomerular target in the brain. olfactory receptors (ors) activation shows the distinguished (camp-based) transduction pathway for odorant perception. in 1991 buck and axel discovered the olfactory gene family, the largest gene family in the human genome, and postulated an exclusive expression in the olfactory epithelium. however, recent whole genome sequencing data from our and other labs show that ors have been found in every tissue of human body which was analyzed by next generation sequencing. the importance of such ectopic expression of ors is raised since the physiological function of some of ors was characterized. when identifying additional expression profiles and functions of or in non-olfactory tissue, there are limitations posed by the deorphanization of ors concerning the activated ligands and by the small number of antibodies available. in contrast to the olfactory sensory neurons which are believed to express all 350 functional or genes (only one or type per cell), cells in non-olfactory tissues tend to express more than one individual or gene per cell. in addition, some of the signaling pathways in non-olfactory tissues seem to involve completely different components in comparison to the olfactory neurons. what is the functional role of these ectopically expressed olfactory receptors? evidences rapidly accumulate that ors participate in important cellular processes outside its primary sensorial organ where they function in odor detection and discrimination. in our lab the functional expression of the first was demonstrated in spermatozoa (2004) . in the meantime we could show the existence and function of ors in the cardiovascular system (heart, blood cells), the gastrointestinal system (small intestine, liver, pancreas), the genito-urinary system (kidney, testis, spermatozoa, prostate), the respiratory system (lung, smooth muscle cells), the skin (keratinocytes, melanocytes) and sensory organs (retina). interestingly we found a broad spectrum of important functions like cell-cell communication and recognition, tissue injury, repair and regeneration, cancer growth, progression and metastasis, nutrient sensing and muscle contraction. nevertheless the functional importance of ectopic ors is still not sufficiently understood. studies seeking to determine the function of ectopic ors are still in its infancy and require further intensive exploration. however, the potential of ors to serve a target for a wide range of clinical approaches is indeed given. this hold promises that the knowledge gained by future investigations would lead to deepen our understanding of or function in health and disease and may provide the basis for the development of applications in diagnosis and therapies in near future. enzyme replacement therapies have been developed over the last 25 years for several of the lysosomal storage disorders (lsd's). the success of enzyme replacement therapy for gaucher disease paved the way for the development of similar treatments for the mucopolysaccharidoses, fabry and pompe disease and lately also for neuronopathic lysosomal storage disorders by intrathecal or intracerebral injections. in addition, small molecule approaches have been developed including substrate reduction therapies and chaperones, which can be used orally. while in gaucher disease enzyme as well as substrate reduction therapy results in reversibility of disease manifestations, with decreases in hepatosplenomegaly, normalization of blood counts and prevention of skeletal disease, this is unfortunately not the case for all patients affected with other lysosomal storage disorders. an important concept is the "window of opportunity for treatment" which is different for these disorders. for example, in fabry disease, early fibrosis fects are well defined, neither the specific mechanisms underlying neurological abnormalities nor the role of decreased cholesterol versus sterol precursor accumulation in disease pathogenesis have been clearly delineated. to identify cellular phenotypes and causative signaling pathways, we derived induced pluripotent stem cells (ipscs) from slos and lath subjects to model these diseases in vitro. slos subjects were known carriers of the most common dhcr7 mutations, including the intronic splice acceptor mutation c.964-1g>c and the missense mutation p.t93m. while all ipscs demonstrated the expected biochemical defects due to dhcr7 or sc5d mutations, cellular assays uncovered a defect in neural stem cell maintenance resulting in accelerated neuronal formation in slos ipscs. further molecular and biochemical analyses demonstrated inhibition of cholesterol-wnt interactions and loss of wnt/β-catenin activity mediated cellular phenotypes. however, this cellular phenotype was exclusive to slos, as lath ipscs did not exhibit a neural progenitor defect or inhibition of wnt/β-catenin activity. while this work demonstrates the utility of ipscs for modeling rare diseases and identifies signaling deficits potentially underlying slos phenotypes, questions remain regarding cellular and functional consequences, the specificity of lipid-wnt interactions, and the role of other disrupted signaling pathways in mediating developmental and functional deficits in these diseases. unpublished work using a variety of approaches will be discussed comparing the specific effects of cholesterol synthesis mutations on cell fate, functional activity, and lipid modulated signaling pathways to more precisely define the consequences of cholesterol synthesis defects and identify potential targets for patient therapy. induced pluripotent stem cell (ipsc) technology has become one of the major approaches for disease modeling since its first report in 2006. the ability to reprogram cells from somatic into embryonic stem cell-like state and to differentiate them into desired cell types in the culture dish has allowed scientists to carry out the study of several diseases in cells such as neurons which, in the past, could not be isolated from living subjects. williams syndrome (ws), a genetic neurodevelopmental disorder where 25-28 genes are hemizygously deleted, is among those. despite cardiovascular abnormalities, its unique neurological phenotypes i. e. hypersociability is of our interest. for several decades, research on different neurological aspects of ws has been conducted in a variety of models such as patient-derived cell lines (lymphoblastoid cells and fibroblasts), post mortem tissue, and mouse models. however, the lack of physiologically relevant cell types such as neural progenitor cells (npcs) and neurons has left a critical gap in our knowledge the disease's cellular and molecular phenotypes. to fill this gap, we took the advantage of the reprogramming technology to capture the genomes of ws subjects in ipscs, which could be then differentiated into npcs and neurons, enabling evaluation of whether the captured genome with hemizygous deletion of those genes leads to relevant neuronal cellular phenotypes. dental pulp cells-derived ipscs of classical ws, rare ws and typical developing (td) subjects were neurally induced via dual-smad inhibition in order to generate npcs and neurons. we discovered that classical ws npcs exhibited increased apoptosis, and, therefore, doubling time, compared to td neurons. this could possibly contribute to the reduction in cortical surface area in classical ws individuals as assessed by magnetic resonance imaging. surprisingly, we found that rare ws npcs behaved similarly to td npcs rather than to classical ws npcs in terms of apoptosis. we confirmed that frizzled9, which is deleted in the classical ws but not in our rare ws genome, is responsible for such phenotype via gain-and loss-of-function assays. moreover, classical ws neurons in general showed increased frequency of activity-dependent calcium transient compared to td neurons. finally, classical ws neurons acid alpha-oxidation, and (4.) glyoxylate detoxification. with respect to peroxisomal fatty acid oxidation peroxisomes catalyze the chain-shortening of certain fatty acids including very-long-chain fatty acids, but requires the active help of mitochondria to catalyze the degradation of acetyl-coa and the reoxidation of nadh as produced in peroxisomes. furthermore, with respect to ether phospholipid biosynthesis peroxisomes heavily rely on the endoplasmic reticulum to complete formation of ether phospholipids whereas fatty acid alpha-oxidation also requires the functional interplay between peroxisomes and mitochondria and the same is true for glyoxylate detoxification. recent evidence holds that the interaction between peroxisomes and the different subcellular organelles, including mitochondria and endoplasmic reticulum, is mediated by specific tethering protein complexes which bring organelles physically together thereby allowing metabolism to proceed smoothly. the importance of peroxisomes in metabolism is stressed by the existence of a large group of single peroxisomal enzyme deficiencies of which x-linked adrenoleukodystrophy is best known. our current state of knowledge with respect to the role of peroxisomes in metabolism and the peroxisomal enzyme deficiencies will be presented at the meeting. huntington's disease: rna-sequencing, small rna-sequencing, chip-sequencing and gwas data. department of neurology, boston university school of medicine, boston, ma 02118, usa huntington's disease (hd) is a dominantly transmitted neurodegenerative disease of midlife onset. recently several different unbiased genome wide studies in hd have been performed. these analyses point to a variety of pathological pathways that are associated with important features of the disease, including age at onset, cag repeat size, and the extent of neuropathological involvement. genome wide association studies (gwas) have identified several regions of the genome that contain genes that are associated with the age at onset for hd. the strongest of these is located at 15q13.3 for rs146353869 for which a very rare allele (maf = 1.1%) is associated with an approximate 6-year younger age at onset for carriers of the minor allele. the same locus contains an independent effect for rs2140734 where a more common minor allele (maf = 30.2%) is associated with a 1. 4 year older age at onset for carriers of this allele. these single nucleotide polymorphisms are in the region of fan1, mtmr10 and several other genes; some of which are not expressed in brain and are not likely candidates for hd modification. eqtl analysis has not resolved which gene may be implicated. other gwas implicated loci include rs1037699 at 8q22.3 and rs144287831 at 3p22.2. we have sought to combine the information derived from multiple platforms to gain additional insight into the pathways that may be implicated in hd pathogenesis. in this strategy, we have performed mrna-sequencing, small rna-sequencing and chip-sequencing using the h3k4me3 mark for active transcription and the repressive mark h3k9me3 in human hd brain samples with gwas genotyping. while the striatum is most involved in hd, the extent of neurodegeneration in post-mortem tissue precludes meaningful comparison between disease and control samples, and consequently we studied prefrontal cortex (ba9). several common pathways were seen across these three platforms. mrna-sequencing and mirna-sequencing data identified altered transcriptional profiles implicating developmental pathways involving the hox genes and related homeo-box domain genes (e. g. pitx1, pou4f2, etc.) . notably, micrornas located in hox gene clusters were among those most increased and levels of these correlate with pathological involvement in the striatum. these genes, associated with early embryonic development, are commonly silent in normal adult brain, and were among the most differentially expressed genes in hd brain. these prominent statistical effects are driven by the near total absence of expression in normal brain. pathways implicated in mrna-seq and chip-seq studies, included immune function and regulation of gene expression. these associations were very strong, indicating a large immune reactive response in the hd in the heart is related to unresponsive disease and unfortunately fibrosis may occur already in an early stage, sometimes even without prior hypertrophy. whether earlier intervention will be beneficial is largely unknown. and then: what is early? many unresolved questions exist at this stage, including the following: -what is the natural history and "point of no return" for the different lsd's? -what is the natural history and "point of no return" for subgroups of patients within one lsd's? -what are the long term complications: treatments change the phenotype rather than cure the disease -what is the influence of antibody generation on clinical effectiveness? -how do we manage the extreme costs of these products, especially in light of the many unsolved issues with respect to effectiveness? surprisingly, so far healthcare professionals, governments and industry have failed to systematically address these issues, resulting in insufficient knowledge for potentially lifesaving treatments. early conditional access, followed by a strict, transparent, independent, collaborative evaluation in addition to fair pricing should be explored. after the recent explosion in sequencing throughput, variant interpretation has quickly become the bottleneck in our effort to usher in the era of genomic medicine. while homozygosity for apparently pathogenic variants in the context of disease states is a well-established phenomenon, homozygosity can uncover many medically relevant aspects of the human variome that are difficult to study otherwise. for example, seemingly benign variants may prove pathogenic in the homozygous state. this includes variants with benign prediction using in silico tools as well as variants in dominant genes with no phenotype in carriers because they represent bona fide recessive inheritance. variants that are associated with one phenotype in compound heterozygous states may express themselves quite differently phenotypically when homozygous. furthermore, previously reported pathogenic variants can be challenged when their presence in homozygosity is associated with no abnormal phenotype, thus improving the specificity of the annotation of the morbid genome. homozygosity for lof variants is a special scenario that allows us to study naturally occurring human "knockouts", a powerful tool to study the physiological context of genes in humans. finally, homozygosity in the context of autozygosity provides a robust mapping tool that can greatly aid in the identification of relevant variants, especially those that exert their pathogenic effect in ways that defy detection by our usual algorithms. by expanding the spectrum of phenotypes that are studied, one can unlock the full potential of homozygosity to understand the medical relevance of the human variome in it its full range from embryonic lethal to essentially benign. helmholtz zentrum münchen gmbh, institut für experimentelle genetik, ingolstädter landstr. 1, 85764 neuherberg, germany the inheritance of epigenetic information in mammals across generations has been controversial. some reports provided initial evidence that a paternal high fat diet may propagate obesity and glucose intolerance in offspring, but potential confounders such as molecular factors present in seminal fluid, paternal-induced alterations in maternal care or transmission of microbiomes were not ruled out in these studies. we have shown in mice that a parental high fat diet (hfd) renders offspring derived via in vitro fertilization (f1) more susceptible to develop excessive overweight and type 2 diabetes (t2d) in a gender and parent-of-origin specific mode. female, but not male, offspring from obese parents became significantly more obese during a hfd challenge than female offspring from lean parents. body weight trajectories and distribution patterns of individual body weights in female offspring from one obese and one lean parent demonstrate that paternal and maternal germline propagate obesity in a roughly equitable and additive fashion, but likely different mode of action. in contrast, a more deteriorated state of hfd-induced insulin resistance was observed in both f1 genders, albeit predominantly inherited via the maternal germline. towards the identification of epigenetic information in sperm and oocyte from hfd and low fat diet fed parents, we are currently analyzing their transcriptome and methylome signatures. the status of this analysis will be presented. we report for the first time epigenetic inheritance of an acquired metabolic disorder via mammalian oocytes and sperms excluding confounding factors. such an epigenetic mode of inheritance may contribute to the observed pandemic increase in obesity and t2d prevalence rates, especially in an environment where nutrition is abundant. brain which may be a major influence contributing to neurodegeneration. in many instances enrichment of h3k4me3 at transcription start sites was not accompanied by a corresponding increase in expression. the apparent inconsistency suggests that common regulatory mechanisms in the hd brain are disrupted and this may contribute to a complex interplay of factors contributing to the neurodegenerative process. often findings in human hd brain samples conflicted with those reported in hd transgenic mouse models, suggesting that one may wish to be cautious in interpreting the significance of either type of study in isolation. the causal pathogesis of huntington disease, new therapeutic approaches r. laufer senior vp discovery and product development global r&d, teva r&d product development mgmt. 12 hatrufa st, netanya, israel huntington disease (hd) is an autosomal dominant neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, cognitive decline and death 15-20 years after motor onset. hd is uniquely caused by a polyglutamine encoding cag expansion in the huntingtin gene (htt), which allows for identification of pre-manifest mutation carriers as much as decades before onset and should facilitate development of disease modifying therapies. yet over 20 years after identification of the hd mutation, available therapies offer only symptomatic relief and are fraught with side effects. development of safe small molecule therapies for hd has been hindered by difficulties identifying and validating tractable drug targets within the disorder's complex pathogenesis. teva pharmaceuticals is developing potential novel treatments based on a mechanistic understanding of disease pathways common to neurodegenerative diseases. the progress of these studies will be reviewed. rare complete gene knockouts in adult humans p. sulem statistics department, decode genetics, sturlugata 8, 105 reykjavik, iceland loss-of-function mutations cause many mendelian diseases. here have create a catalog of autosomal genes that are completely knocked out in humans by rare loss-of-function mutations. we sequenced the whole genomes of over 29,000 icelanders and imputed the sequence variants identified in this set into a total of 151 chip-genotyped and phased icelanders. of the genotyped icelanders, around 10% are homozygotes or compound heterozygotes for loss-of-function mutations with a minor allele frequency (maf) below 2% in close to 2000 genes (complete knockouts). genes that are highly expressed in the brain are less often completely knocked out than other genes. homozygous loss-of-function offspring of two heterozygous parents occurred less frequently than expected (deficit of 136 per 10,000 transmissions for variants with maf <2%, 95% confidence interval (ci) = 10-261). we are currently systematically phenotyping such human complete knock out. this phenotyping lasts 4 hours and attempts to cover most of the observable diversity in a non-invasive and cost efficient manner. i will demonstrate how using systematic phenotyping can advance the knowledge on individual gene knockout. we use results from in-house transcriptomics, existing animal models and complementary approaches to assess the observation in human. we will also discuss the scrutiny in other population in order to detect such complete knock-out. we will exemplify the impact of founder population and consanguinity in such an odissey. early onset and severe obesity can be inherited via loss of function mutations within the melanocortin pathway of hypothalamic body weight regulation. the most prominent player in this signalling pathway is the fat cell hormone leptin. leptin gene mutations were the first to be linked to monogenic early onset obesity. after binding of leptin to leptin receptors in the arcuate nucleus of the hypothalamus the neuropeptide msh is processed from the precursor pomc and acts as a ligand at the mc4 receptor. mutations in the leptin receptor gene, the pomc gene and the mc4 receptor gene were subsequently diagnosed in further patients with extreme early onset obesity. while leptin mutation patients can be treated with recombinant leptin -as shown already in the late 1990s -all other monogenic obesity forms are leptin resistants, and additional leptin failed to decrease body weight. only recently pomc gene deficient patients were successfully treated with the msh-analogue setmelanotide (kühnen et al. 2016) . common severe obesity is defined by the lack of disease causing monogenic defects. a plethora of gwas identified a large number of snps in common obesity associated with the individual bmi but only to a low amount of not more then 25%. however, almost all these common obese patients are characterized by high leptin levels suggesting sufficient generation of leptin in the increased fat tissue and a state of leptin resistance. several new data concerning the contribution of epigenetic and genetic variants in the pomc gene locus argue for a role of the melanocortin pathway also in common obesity and imply, therefore, a potentially new treatment option also in common obesity based on msh-analogues. genome-wide association studies have highlighted the role of genetic associations with susceptibility to common inflammatory diseases, highlighting potential new insights into disease pathogenesis and opportunities for therapy. however understanding the functional basis of these associations and delivering translational utility remains a significant challenge to the field. non-coding regulatory genetic variants are most commonly implicated in such studies. recent work highlights how such variants are also major drivers of diversity in the immune response transcriptome. this talk will discuss approaches we are taking to try and establish functional links between immune phenotype-associated regulatory genomic and epigenomic variation, and specific modulated genes and pathways. i will describe insights from the application of expression quantitative trait (eqtl) mapping to define genomic modulators of the global transcriptomic response in different primary immune cell populations and to specific innate immune stimuli in health and disease. this work highlights the extent of local and distant context-specific eqtl, enabling resolution of immunoregulatory variants and the identification of specific modulated genes involving disease associated loci. examples will be described showing how mapping trans-regulatory loci can be a powerful approach for discovery and dissection of gene networks informative for disease. i will also show how we have applied analysis of the genetics of gene expression in patients with sepsis admitted to intensive care, revealing new insights into disease pathogenesis. further progress in this area will require characterisation of associated variants in the context-specific disease relevant epigenomic landscape in which they may act, requiring careful consideration of relevant immune cell types and environmental modulators to study, to-abstracts aktuellen stellungnahme der deutschen forschungsgemeinschaft 1 sollen humane genomsequenzierungen die möglichkeit der rückmeldung von analyseergebnissen enthalten. als orientierung für einen verantwortungsvollen umgang mit dieser frage wird auf die projektgruppe eurat 2 verwiesen. dennoch bleibt das problem der einordnung mitteilungswürdiger ergebnisse aus forscher-und probandensicht und der bereitstellung der für aufklärungs-und rückmeldungsalgorithmen erforderlichen ressourcen. darüber hinaus ist bis heute nicht geklärt, welche kommunikativen prozesse eine ausreichende basis für ein informiertes einverständnis darstellen. diese und weitere fragen möchten wir mit frau prof. dr. med. dr. phil. eva winkler (nationales centrum für tumorerkrankungen, heidelberg), herrn dr. phil. martin langanke (theologische fakultät, universität greifswald) und herrn pd dr. phil. peter burgard (universitätskinderklinik heidelberg) kontrovers diskutieren. die organisatoren werden in die fragestellung einführen, anschließend sind impulsreferate (ca. 10 min) der referenten vorgesehen, bevor eine debatte mit dem publikum angeregt wird. fangerau, f. söhner (düsseldorf) in den 1960er jahren erlebte die humangenetik wie eine reihe anderer medizinischer disziplinen auch eine erhebliche institutionelle ausweitung. in den 1960er und 1970er jahren wurde beispielsweise an den universitäten der brd das gros der humangenetischen lehrstühle, in den ausgehenden 1970er und 1980er jahren auch in der ddr, eingerichtet. 1969 ging vom symposium "genetik und gesellschaft" im rahmen des marburger "forum philippinum" die initiative aus, in der ganzen bundesrepublik genetische beratungsstellen zu gründen und damit die genetische forschung (wieder) medizinisch nutzbar zu machen. in dieser phase der etablierung der humangenetik auf akademischer und praktischer ebene setzt das geplante zeitzeugenprojekt ein. es will die entwicklung der humangenetik in ihrem selbstverständnis als quer-und als längsschnittfach (pfadenhauer 2003:66) im deutschsprachigen raum ab den 1970er jahren mit hilfe von expertengesprächen dokumentieren und analysieren. im forschungsprojekt sollen zwei komplexe von fragestellungen bearbeitet werden: ein wissenschaftshistorischer, in dem die entwicklung und anwendung von diagnostischen und therapeutischen techniken im mittelpunkt steht, und ein sozialhistorischer, in dem es um die etablierung und den ausbau der institutionen der humangenetik sowie um den verlauf der das fach betreffenden gesellschaftlichen debatten geht. neben der gründung von instituten und der fachgesellschaft sowie der normierung der ausbildung für ärztliche und naturwissenschaftlich ausgebildete humangenetiker sollen die funktion der historischen reflexion und bearbeitung der facheigenen nationalsozialistischen vergangenheit in den 1980er jahren für die etablierung des fachs und der schwierige institutionelle trennungsprozess von der anthropologie mit ihren wirkungen auf das selbst-und fremdbild der humangenetik analysiert werden (weingart, kroll, bayertz 1992) . zusätzlich zur entwicklung in der brd sollen dabei auch die entwicklung des fachs in der ddr und mögliche deutsch-deutsche kooperationen zur sprache kommen (in jena befand sich z. b. in den 70er jahren das zentrale referenz-institut für genetische beratung in der ddr (vogel 1999:416) . das projekt kann sich auf zahlreiche arbeiten zur geschichte der deutschen humangenetik stützen (vgl. kröner 1997 , 1998 cottebrune 2006 cottebrune , 2008 weingart 1988; bennike 1992 dysmorphism carrying a pathogenic variant in the ebf3 gene detected by whole-exome sequencing. five missense, two nonsense, one 9-bp duplication, and one splice-site variant in ebf3 were found; the mutation occurred de novo in eight individuals, and the missense variant c. 625c>t [p.(arg209trp) ] was inherited by two affected siblings from their healthy mother who is a mosaic. ebf3 belongs to the early b-cell factor family (also known as olf, coe, or o/e) and encodes a transcription factor involved in neuronal differentiation and maturation. structural assessment predicts perturbing effects of the five amino acid substitutions on dna-binding of ebf3. transient expression of ebf3 mutant proteins in hek 293t cells revealed mislocalization of all but one mutant in the cytoplasm in addition to nuclear localization. by transactivation assays, all ebf3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene under control of the cdkn1a promotor that corresponds well with loose association of ebf3 mutants with chromatin as demonstrated by in situ subcellular fractionation experiments. finally, rna-seq and chip-seq experiments demonstrate that ebf3 acts as a transcriptional regulator at cis-regulatory sequences and ebf3 mutant had reduced function due to partial disruption of the dna-binding domain. these findings demonstrate that ebf3-mediated dysregulation of gene expression has profound effects on neuronal development in humans and add ebf3 to the growing list of genes in which mutations cause syndromic forms of intellectual disability. step, we performed wes in further unelucidated uhs cases and identified homozygous nonsense mutations in tgm3 (transglutaminase 3) and in tchh (trichohyalin), respectively. elucidation of the molecular outcomes of the disease causing mutations by cell culture experiments of padi3 and tgm3 and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild type proteins. by immunofluorescence analysis, we could demonstrate a diffuse homogenous cytoplasmic distribution of the wt padi3, whereas in the mutants the proteins were observed to form large aggregates throughout the cytoplasm. by use of human anti-citrullinated protein autoantibodies, we could show a strong labelling in the wt whereas the staining of the mutants was barely above background. in order to demonstrate the importance of padi3 in hair shaft formation, we generated padi3 knockout mice. electron microscopy observations revealed morphological alterations in hair coat of padi3 knockout mice. for tgm3, we performed a transglutaminase activity assay. the analysis results revealed that the wt had a significantly higher transglutaminase activity in comparison to the truncated protein. here, we report for the first time the identification of uhs causative mutations located in the three genes padi3, tgm3 and tchh. the two enzymes responsible for posttranslational protein modifications, and their target structural protein, are all involved in hair shaft formation through their sequential interactions. these findings provide valuable information regarding the pathophysiology of uhs and contribute to a better understanding of this protein interaction cascade. this could be of further value for cosmetics and pharmaceutics industries paving the way for development of novel products. deadenylases are best known for degrading the poly(a) tail during mrna decay. the deadenylase family has expanded throughout evolution and, in mammals, consists of 12 mg 2+ -dependent 3' end ribonucleases with mostly unknown substrate specificity. pontocerebellar hypoplasia type 7 (pch7) is a unique recessive syndrome characterized by neurodegeneration with ambiguous genitalia (mim%614969). we studied 12 human families with pch7, uncovering biallelic, loss of function mutations in toe1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed mid-and hind-brain degeneration, modeling pch-like structural defects in vivo. surprisingly, we found toe1 associated with incompletely processed small nuclear (sn)rnas of the spliceosome, which is responsible for pre-mrna splicing. these pre-snrnas contained 3' genome-encoded tails often followed by post-transcriptionally added adenosines. human cells with reduced levels of toe1 accumulated 3' end-extended pre-snr-nas, and immuno-isolated toe1 complex was sufficient for 3' end maturation of snrnas. our findings reveal the cause of a neurodegenerative syndrome linked to snrna maturation and uncover a key factor involved in processing of snrna 3' ends. the kidney maintains acid-base homeostasis and electrolyte balance through highly specialized cells. in the distal nephron acid secretion is mediated by type a intercalated cells (a-ics) , which contain v-type at-pase-rich vesicles that fuse with the apical plasma membrane on demand. intracellular bicarbonate generated by luminal h+ secretion is removed by the basolateral anion-exchanger ae1. dysfunction of type a intercalated cells results in distal renal tubular acidosis (drta) and human mutations in v-atpase subunits and ae1 are causative for this condition. for the ae1 r607h mutation a dominant-negative trafficking mechanism was proposed to explain ae1-associated dominant drta based on studies in mdck monolayers. to test this hypothesis in vivo and to test potential rescue strategies correcting this mistargeting defect, we have generated a r607h knock-in mouse strain, which corresponds to the most common dominant drta mutation in human ae1, r589h. heterozygous and homozygous r607h knock-in mice displayed incomplete drta characterized by compensatory upregulation of the na+/hco3-cotransporter, nbcn1. as expected for the r607h mutation, red blood cell ae1-mediated anion-exchange activity and surface polypeptide expression were unchanged. surprisingly, basolateral targeting of the mutant ae1 in a-ics was preserved in contrast to previous studies in mdck cells. instead, we found ae1 expression in a-ics strongly reduced in a r607h dosage-dependent manner. additional cell culture studies in two widely used immortalized renal cell lines verified that targeting and half-life time of mutant ae1 protein was indeed preserved. surprisingly, atpase expression was reduced and its plasma membrane targeting upon acid challenge compromised. ultrastructural analysis revealed a loss of apical vesicles in a-ics, while we observed lysosomal inclusions and multilamellar bodies. accumulation of p62-and ubiquitin-positive material in a-ics of knock-in mice suggest a defect in the degradative pathway, which may ultimately lead to loss of a-ics. highlighting the expression of ae1 specifically in a-ics, type b intercalated cells were unaffected. we propose that reduced basolateral anion-exchange activity in a-ics inhibits trafficking and regulation of v-type atpase, compromising luminal h+ secretion and possibly also lysosomal acidification. our findings illustrate the considerable, context-dependent complexity of ae1-related kidney disease. b. vona 1 , d. liedtke 1 , k. rak 2, 3 , r. katana 4 , l. jürgens 3 , pr. senthilan 5 , i. nanda 1 , c. neuner 1 , mah. hofrichter 1 , l. schnapp 1 , j. schröder 1 , u. zechner 6 , s. herms 7, 8, 9 , p. hoffmann 10 , t. müller 11 , m. dittrich 1, 11 , o. bartsch 6 , pm. krawitz 12 , e. klopocki 1 , w. shehata-dieler 2 , mc. göpfert 4 , t. haaf 1 although many genes have already been identified as causing non-syndromic hearing loss (nshl), diagnostic rates of approximately 50% among hearing impaired patients suggest that many more genes are remaining to be identified. nshl is the most common sensory deficit that has a prevalence between one and two per 1000 newborns. furthermore, it demonstrates classic genetic heterogeneity with as many as 1% of coding genes in the genome anticipated to be involved in non-syndromic forms of deafness. autosomal recessive (75-80%) and autosomal dominant (15-20%) forms dominate inheritance patterns of deafness; however, in a small fraction of cases, x-linked deafness (1-4%) can be observed. whole exome sequencing of a german family with diagnostically unresolved nshl revealed a novel missense variant predicted as pathogenic in the gene ferm and pdz domains containing protein 4 (frmpd4) on chromosome xp22.2. this gene, also known as preso1, was first described as a regulator of dendritic spine morphogenesis. previous screening of pathogenic cnvs in array based comparative genomic hybridization among families with heterogeneous x-linked intellectual disability (xlid) showed duplication of xp22.2 including part of frmpd4 which implicated the gene in xlid. interestingly, a segregating truncating and a de novo missense mutation in frmpd4 have associated this gene with xlid, a phenotype not observed in our family. mouse expression studies localize frmpd4 to spiral ganglion neuron peripheral dendrites of the developing cochlea. in addition, we analyzed frm-pd4 knockdown and loss-of-function zebrafish mutants for innervation and structural defects in the otic vesicle and lateral line neuromasts. posterior lateral line neuromasts are observed with reduced axonal outgrowth that is also likely reduced in the lateral line nerve. abnormal innervation is also apparent in the otic vesicle. fluorescent neuromast labeling marked a significant reduction of overall otic vesicle and lateral line neuromasts in mutants versus wild type zebrafish. scanning electron microscopy revealed a pronounced absence of kinocilia in posterior lateral line neuromasts of frmpd4 -/zebrafish. furthermore, adult frmpd4 mutants show significantly delayed acoustically evoked behavioural responses compared to wild type fish indicating hearing impairment. investigation of transgenic drosophila insertion mutants detected a mild auditory phenotype i. e. a reduction in mechanical amplification gain and associated reduction in antennal fluctuation power. our results associate frmpd4 with x-linked hearing loss and suggest mutations in this gene are correlated with pleiotropic effects abstracts has also been demonstrated to be an important pathobiochemical feature in rtt. to test whether common deficits in mtor signaling could be responsible for the molecular pathogenesis underlying both syndromes, we generated and studied a novel cdkl5 knockout (cdkl5 -/y) mouse model and performed in vitro experiments in human cells. in cdkl5 -/y knockout mice loss of cdkl5 is accompanied by reduced phosphorylation levels of critical components of the mtor signaling cascade. these findings point at a regulatory role of cdkl5/cdkl5 on mtor activity and function. to gain further insights into the possible mechanism through which cdkl5/pi3k interaction could regulate mtor signaling, we used hek-t cells as cellular model. following knock-down of cdkl5, the amount of pi3k protein was significantly reduced compared to controls. to evaluate the contribution of our findings to pathogenesis, we performed rescue experiments in cdkl5 knock-down hek-t cells using wild-type and patient-specific mutant cdkl5 constructs. further experiments are ongoing to clarify the molecular mechanism by which cdkl5 regulates pi3k protein level in the cells. inferring expressed genes by whole-genome sequencing of plasma dna medical university graz, graz, austria, 2 university of technology, graz, austria the analysis of cell-free dna (cfdna) in plasma represents a rapidly advancing field in medicine. cfdna consists predominantly of nucleosome-protected dna shed into the bloodstream by cells undergoing apoptosis. we performed whole-genome sequencing of plasma dna and identified two discrete regions at transcription start sites (tsss) where nucleosome occupancy results in different read depth coverage patterns for expressed and silent genes. by employing machine learning for gene classification, we found that the plasma dna read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. in patients with cancer having metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy. we were able to determine the expressed isoform of genes with several tsss, as confirmed by rna-seq analysis of the matching primary tumor. our analyses provide functional information about cells releasing their dna into the circulation. institute of human genetics, fau-erlangen-nürnberg, erlangen, germany, 2 institute of medical genetics, university of zurich, schlieren, switzerland rna-splicing is an important mechanism for eukaryotic gene expression and regulation. defective splicing significantly contributes to monogenic disease in humans. indeed, the mutational space for variants affecting splicing is larger than for coding variants. several computational methods have been developed to predict a variant's effect on splicing but lack predictive value outside the canonical splice sites and do not predict aberrant transcripts. thus the plethora of dna variants generated by recent advances in "next-generation" based sequencing (ngs) can be scored for a possible splicing effect, but a laborious wet-lab based confirmation and characterization is still required. rna-seq is widely used for quantification of gene expression and can be used to detect splicing events, but is limited for this use by the variable and often low read coverage of individual congenital anomalies of the kidneys and urinary tract (cakut) are the most common cause of chronic kidney disease in children. as cakut is a genetically heterogeneous disorder and most cases are genetically unexplained, we aimed to identify new cakut causing genes. using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (lifr) gene causing instability of the mrna in a patient presenting with bilateral cakut and requiring kidney transplantation at one year of age. lifr encodes a transmembrane receptor utilized by il-6 family cytokines, mainly by the leukemia inhibitory factor (lif). mutational analysis of 121 further patients with severe cakut yielded two rare heterozygous lifr missense variants predicted to be pathogenic in three unrelated patients. lifr mutants showed decreased half-life and cell membrane localization resulting in reduced lif-stimulated stat3 phosphorylation. lifr showed high expression in human fetal kidney and the human ureter, and was also expressed in the developing murine urogenital system. lifr knockout mice displayed urinary tract malformations including hydronephrosis, hydroureter, ureter ectopia, and, consistently, reduced ureteral lumen and muscular hypertrophy, similar to the phenotypes observed in patients carrying lifr variants. additionally, a form of cryptorchidism was detected in all lifr -/mice and the patient carrying the lifr frameshift mutation. altogether, we demonstrate heterozygous novel or rare lifr mutations in 3.3% of cakut patients, and provide evidence that lifr deficiency and deactivating lifr mutations cause highly similar anomalies of the urogenital tract in mice and humans. loss of cdkl5 associated with deficient mammalian target of rapamycin (mtor) signaling in mice and human cells we and other groups have shown that mutations in the x-linked cyclin-dependent kinase-like 5 (cdkl5) gene cause a severe neurodevelopmental disorder with clinical features including intellectual disability, early-onset intractable seizures and autism, that are closely related to those present in rett syndrome (rtt) patients. rtt is caused by mutations in the x-linked mecp2 gene. cdkl5 is a serine/threonine kinase and to date knowledge about its functional roles is scarce. we searched for cdkl5 interacting proteins by yeast-two hybrid screens. one of the candidates identified in these screens is a subunit of the phosphatidylinositol-4,5-biphosphate 3-kinase (pi3k). the results obtained in yeast could be confirmed in vitro in mammalian cells and in mouse brain by immunoprecipitation experiments and by co-localization studies. pi3k phosphorylates membrane lipids which act as docking sites to recruit targets upstream of mtor and thereby regulate among major cellular processes synaptic plasticity, which is the cellular basis for learning and memory. alteration of mtor signaling gene tubes for stabilizing rna immediately after drawing the samples. the subsequent rt-pcr analysis showed that 9 of the 11 variants located at potential splicing sites indeed affect splicing. thus 8 of these variants could be classified as deleterious (iarc class 5), while one chek2 variant could not be unequivocally classified as the rt-pcr analysis identified only 20% of the mutant transcript indicating continued usage of the constitutive splice acceptor site. this led to the classification as a probably hypomorphic allele. the variants in cdh1 and mlh1 did not affect splicing and were classified as benign (iarc class 1). none of the 5 rare synonymous and nonsynonymous exonic variants showed any effect on splicing. in conclusion, this analysis allowed the disambiguation of 10 out 11 vus at potential splice sites into a definite category (either iarc class 5 or 1). this work highlights the importance of computational splicing prediction and validation using rt-pcr of peripheral blood rna to assess the pathogenicity of vus. this in turn, allows more accurate genetic counseling and clinical management of affected families. gliomas present the major group of neoplasia in the central nervous system. they typically show invasive growth and high recurrence rate and are currently not curable. idh mutations are detected in nearly 70% of low grade gliomas and are considered to play a key role in low grade glioma development. while it is known that idh1/2 mutation leads to high-levels of 2-hydroxyglutarate (2-hg) that functions as an oncometabolite, little is known about the influence of idh1/2 mutations on energy metabolism and metabolic reprogramming in the tumor cells. since patient derived idh mutant cells do not grow in cell culture, previous studies from our group and others used transduced cell lines that overexpress idh1. in order to develop in vitro models with reduced side effects, we used crispr/ cas to introduce the idh1r132h mutation in a patient derived glioblastoma cell line. the edited cells expressed idh1r132h in western blot and expression levels of idh1 were comparable to the expression in wild type cells. the mutation was stable in long time culture experiments, without signs of senescence. moreover we found elevated 2-hg levels, proving that the idh1r132h neoenzymatic function is present in our cell lines. thus, we were able to edit and culture genomic idh1r132h mutated glioma cells for the functional analysis of the idh1r132h mutation for the first time without the effects of overexpression models. edited idh1r132h cell lines showed extended doubling times compared to wildtype cells. measurement of krebs cycle metabolites using mass spectrometry revealed elevated glutamate levels. we found enhanced atp-levels that could be a consequence of decreased atp consumption. additionally, the cells showed reduced viability compared to wildtype cells when cultivated in glycolysis inhibiting media, pointing out the enhanced dependency on glycolysis in idh1r132h cells. these results indicate changes in tumor cell metabolism and energy household induced by the idh1r132h mutation. since we and others could show that idh1r132h can alter nad+ and nadph levels, we tested if the idh1r132h mutated cells are more susceptible to selective inhibition of nad/p regenerating enzymes. esirna-silencing of nampt specifically decreased cell viability in idh1r132h but not wildtype cells with a concomitant increase of dead cells. in conclusion, we developed genes. thus, a standardized ngs based approach to characterize potential splice variants is lacking. hence we investigated the utility of hybridization based gene-panel enrichment and ngs of cdna. based on results of computational simulation we selected twenty rna-samples of patients with a known pathogenic splice-site variant in an inherited cancer predisposition gene. these variants were previously characterized by rt-pcr in our lab or in the literature. after rrna depletion and dna digestion we performed first and second strand cdna synthesis followed by "tagmentation"-based library preparation, targeted enrichment using the trusight cancer panel and sequencing on an illumina miseq platform. a computational pipeline was established to enable automated detection of aberrant splicing events by implementing different alignment and splice-junction detection algorithms together with filtering against control data sets. we also considered variant calling for the detection of allelic imbalance and gene-level expression analysis in this data. breast and ovarian cancer (bc/oc) predisposition has been associated with a number of high-and low-penetrance susceptibility genes. advances in sequencing technology has made multigene testing a practical option when searching for genetic variants associated with risk for bc/oc. variants of uncertain significance (vus), though, represent a major problem. we now studied 581 patients fulfilling criteria for brca1 and 2 testing using the next generation sequencing based trusight sequencing cancer panel on a miseq platform (illumina). data was analyzed after remapping with bwa to hg19 (grch37) using seqnext software (jsi) for variants in 14 known high and moderate penetrance susceptibility genes (brca1/2, atm, chek2, palb2, rad51c, rad51d, nbn, cdh1, tp53, mlh1, msh2, msh6, pms2) . besides 106 deleterious mutations we also identified 89 vus. of these, 11 variants (1 each in brca1, brca2, palb2, rad51c, rad51d, cdh1, and mlh1, and 2 in chek2 and mlh1, respectively) affect possible splicing sites. in addition, 5 synonymous and nonsynonymous variants outside the splicing sites (1 in brca1, brca2 and cdh1, respectively, 2 in rad51d) were not reported in exome variant server or exome aggregation consortium (exac) databases, so far. no families were available to study familial segregation. for all these variants a potential effect on splicing efficiency was predicted by three different computational algorithms (bdgp: splice site prediction by neural network, netgene2 server and the human splice finder (hsf 3.0) algorithm). we took advantage that these genes are ubiquitously expressed to investigate possible effects of these variants on mrna splicing using easily accessible peripheral blood. as mrna is notoriously unstable, we used pax-abstracts was evaluated for both the individual markers and their combinations derived from multiple algorithms. pronounced demethylation of all 3 markers was observed at baseline among cases compared to controls. risk of developing lc increased with decreasing dna methylation levels, with adjusted ors (95% ci) of 15.86 (4.18-60.17), 8.12 (2.69-4.48) , and 10.55 (3.44-32.31 ), respectively, for participants in the lowest quartile of ahrr, 6p21.33, and f2rl3 compared to participants in the highest 2 quartiles of each site among controls. the individual 3 markers exhibited similar accuracy in predicting lc incidence, with aucs ranging from 0.79 to 0.81. combination of the 3 markers did not improve the predictive performance (auc = 0.80). the individual markers or their combination outperformed self-reported smoking exposure particularly in light smokers. no variation in risk prediction was identified with respect to age, follow-up time, and histological subtypes. ahrr, 6p21.33, and f2rl3 methylation in blood dna are predictive of lc development, which might be useful for identification of risk groups for further specific lc screening, such as ct examination. over the past decades the search for disease causing variants has been focusing exclusively on the coding genome. this highly selective approach has been extremely successful however, recent data have revealed the importance of the non-coding genome in fundamental processes such as gene regulation, 3d chromatin folding, and pinpointed its role in disease. in this study, we systematically investigate the cis-regulatory landscape of pitx1, a homeodomain transcription factor that is exclusively expressed in the hindlimb. mutations and non-coding structural variations at the pitx1 locus have been shown to associate with a variety of congenital limb defects including club feet, polydactyly, and arm-to-leg transformation (liebenberg syndrome). we performed in vivo enhancer reporter essays in transgenic mice and identified several limb enhancer elements at the pitx1 locus; surprisingly they all showed both forelimb and hindlimb activity, although pitx1 is never expressed in the forelimb. capture hi-c experiments revealed a hindlimb-specific chromatin-organization at the pitx1 locus, which enables its promoter to contact several enhancers bearing a pan-limb activity only in the hindlimb. this tissue-specific chromatin folding plays a determinant role to refine the unspecific limb regulatory landscape toward a highly controlled and hindlimb delimited transcriptional output. to gain a better understanding of the pathology of pitx1 associated limb defects, we used crispr/cas9 to generate a set of deletions and inversions in the pitx1 cis-regulatory landscape in mice. genetic perturbations of the regulated 3d chromatin conformation lead to an ectopic forelimb expression of pitx1, resulting in an arm-to-leg transformation in mice and in human patients respectively. our data further highlight the role of non-coding mutations affecting chromatin folding in congenital disease and give new insights into the regulation of pitx1 during development and the pathomechanism of associated limb defects. hoxb13, a member of the embryonic homeobox transcription regulators, has been identified as the first susceptibility gene specific for prostate cancer (prca). the founder missense mutation g84e, which likely originated from finland, can be found in most populations of european ancestry. we determined the frequency of hoxb13 g84e for the german population, assessed in a cohort of 379 unrelated cases, each with positive family history of prca, 367 sporadic prca cases and in 1015 controls. additional 646 affected relatives from prca families were included to explore association with aggressive disease in subgroups with high gleason score (>7), advanced tumor stage, or psa at diagnosis >20 ng/ml. carriers of g84e were rare in controls (0.4%) and showed increased frequencies in both sporadic (1.6%) and familial prca cases (3.2%). estimated risks were or = 4.2 (p = 0.026) and or = 8.3 (p = 0.0003), respectively. the risk effect size increased with the number of affected individuals per pedigree: or = 12.6 (p < 0.0001) for 3 or more, and or = 14.4 (p < 0.0001) for 4 or more affected men. the strongest association with clinical features was observed between g84e and advanced tumor stage (or = 9.2; p < 0.0001). in conclusion, the observed frequency of hoxb13 g84e mutation carriers in our study cohort was intermediate as compared to the common prevalence in scandinavia and the rare occurrence in mixed european populations from the us. the risk estimates of hoxb13 g84e and the stronger effect sizes in families with increasing number of affected relatives were in line with a high penetrant germline predisposition. the association between g84e status and tumor stage may be of greater interest for clinical practice, but needs further validation. the absolute penetrance of the hoxb13 g84e mutation should be investigated in further studies in order to elucidate its suitability as a genetic predictor for prca. smoking-associated dna methylation markers predict lung cancer incidence homozygous smn1 loss causes spinal muscular atrophy (sma), the most common lethal genetic childhood motor neuron disease. smn1 encodes smn, a ubiquitously expressed housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. sma individuals harbor low smn expression from one to six smn2 copy genes, which is insufficient to functionally compensate for smn1 loss. however, rarely individuals with homozygous absence of smn1 and only three to four smn2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). previously, we identified plastin 3 (pls3) overexpression as an sma protective modifier in humans and showed that smn deficit impairs endocytosis, which is rescued by pls3 overexpression. here, we identify reduction of the neuronal calcium sensor neurocalcin delta (ncald) as a protective sma modifier in five asymptomatic smn1-deleted individuals carrying only four smn2 copies. we demonstrate that ncald is a ca 2+ -dependent negative regulator of endocytosis, as ncald knockdown improves endocytosis in sma models and ameliorates pharmacologically induced endocytosis defects in zebrafish. importantly, ncald knockdown effectively ameliorates sma-associated pathological defects across species, including worm, zebrafish and mouse. in conclusion, our study identifies a previously unknown protective sma modifier in humans, demonstrates modifier impact in three different sma animal models and suggests a potential combinatorial therapeutic strategy to efficiently treat sma. since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in sma and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies. mutations affecting coding or regulatory regions of smc2 cause dysregulation of condensins resulting in a phenotype reminiscent of cohesinopathies cornelia de lange syndrome (cdls) is a dominantly inherited malformation syndrome caused by mutations in genes encoding subunits (smc1a, smc3, rad21) or regulators (nipbl, hdac8) of the cohesin complex. this dna-bound complex regulates several chromatin-related processes such as chromosome segregation, dna-damage repair, transcription and chromatin structure. the project presented initially started with two children and their mother who showed clinical features reminiscent of cdls. while various sequencing approaches failed to identify the disease-causing mutation, a 60 kb spanning deletion co-segregating with the phenotype was identified by array-cgh. besides the last exons of cylc2, encoding a sperm head protein, no other genes were affected. subsequent in-silico analyses predicted the existence of a ~3 kb tissue-specific regulatory element within this region, located approximately 1 mb distant from the next protein-coding gene smc2, which encodes a subunit of the cohesin-related condensin complex. significant reduction of smc2 expression was verified in patient's fibroblasts by qpcr analysis. accordingly, a strong dysregulation of smc2 was observed in hek293 and sh-sy5y cells deficient for the putative 3 kb regulatory element, which was deleted by crispr/cas9 genome editing. reporter gene assays further highlighted the functional relevance of the identified regulatory element in regulating the smc2 gene promoter. interestingly, we could prove on protein as well as on mrna level that alterations in smc2 expression are correlated with the dysregulation of other condensin subunits such as smc4 in patient's samples as well as in cris-pr/cas9-generated cells. in a large exome sequencing project we have identified a smc2 frameshift mutation in an additional family with two patients who show clinical features overlapping with those seen in our initial family. quantitative pcr analyses in fibroblasts of both subjects also showed significant reduction of smc2 and smc4 expression, which is consistent with our findings in the first family. to further investigate whether alterations in condensin gene expression are specific for the dysregulation of smc2, we have decreased smc2 levels in different cell types by sirna. quantitative protein as well as mrna analyses revealed reduced smc4/smc4 expression. our data show for the first time the coordinated expression of different condensin subunits and its relevance for human disease. abstracts human pedigree. cardiac valves initially form through a process called endothelial-to-mesenchymal transition (emt) then subsequently elongate and mature during early juvenile life. expression analysis throughout embryonic and postnatal stages of adamts19-/-mice revealed an expression in all cardiac valves after valve formation. high resolution, digital echocardiography showed that mice without adamts19 expression develop dysfunctional aortic valves early in life, reminiscent of the human phenotype. notably, the expression of adamts19 in the valve was restricted to valvular interstitial cells and not observed in endothelial cells. functional analysis using proteomic approaches suggest that the presence of ad-amts19 is necessary to maintain extracellular matrix remodelling during valve development and its maturation. not only do the lof mice fully recapitulate the human phenotype, they also highlight adamts19 as a novel marker for valvular interstitial cells to specifically target initial post-emt processes as well as serve as an important model to understand an ageing valve phenotype in humans. exome sequencing of 55 bipolar disorder patients with rapid cycling implicates novel candidate genes in disease development bipolar disorder (bd) is a severe neuropsychiatric disorder characterized by recurrent episodes of mania and depression. bd has a lifetime prevalence of about 1% and a high heritability of about 70%. although recent genome-wide association studies identified the first susceptibility genes contributing to disease development, the cumulative impact of common alleles with small effect may only explain around 38% of the phenotypic variance (lee et al. 2011) . in consequence, rare variants of high penetrance have been suggested to additionally contribute to bd susceptibility. in the present study we focused on bd patients with rapid cycling (rc). rc is a course specifier of bd defined as having at least four recurrent episodes of acute illness within one year. since rc showed strong evidence for familiarity, we hypothesized that bd patients with rc might represent a more defined etiological subgroup and that rare variants of high penetrance might contribute to the development of rc in bd patients. we selected 55 unrelated bd patients with rc of german origin and performed exome sequencing using the illumina hiseq2500 platform. for data analysis, the varbank pipeline of the cologne center for genomics was used. we filtered for rare (minor allele frequency <0.1%), heterozygous and non-synonymous variants that were predicted to be possibly damaging or disease causing by at least 4 of 5 applied prediction tools. after these filtering steps, we identified a total of 110 different genes which harbored rare functional variants in at least three independent patients. gene set analysis for these genes using consensuspathdb revealed 165 decker 7 , g. nuernberg 8, 9 , d. hassel 2 , g. a. rappold 1, 10 mutations in the homeobox gene shox cause shox deficiency, the most frequent monogenic cause of short stature. the clinical severity of shox deficiency varies widely, ranging from short stature without dysmorphic signs to mesomelic skeletal dysplasia (léri-weill dyschondrosteosis, lwd). in rare cases, individuals with shox deficiency are asymptomatic. to elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with five affected individuals with lwd using whole genome linkage analysis and whole exome sequencing. the variant p.phe508cys of the retinoic acid catabolizing enzyme cyp26c1 co-segregated with the shox variant p.val161ala in the five affected individuals, while the shox mutant alone was present in three asymptomatic individuals. two further independent lwd cases with shox deficiency and damaging cyp26c1 variants were identified. the identified damaging variants in cyp26c1 affected its catabolic activity, leading to an increased level of retinoic acid. we also provide evidence that high levels of retinoic acid significantly decrease shox expression in human primary chondrocytes and zebrafish embryos. individual morpholino knock-down of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. together our findings demonstrate that shox and cyp26c1 act in a common molecular pathway controlling limb growth and describe cyp26c1 as the first genetic modifier for shox deficiency. heart valve dysfunction in men and mice is caused by loss of function mutations in adamts19, a novel marker for valvular interstitial cells on a global perspective defects of the cardiac valves are one of the most common heart abnormalities in humans, with a substantial number of them requiring surgical intervention at least once in their life. several mechanisms have been proposed ranging from acquired to developmental causes, but thus far the majority can not be explained on the molecular level. here we report on the identification of a unique human family affected by multiple dysfunctional cardiac valves early in life. genetic screening revealed a homozygous deletion of the first eight exons in ad-amts19, a novel candidate gene for valvular heart defects. to investigate its role in heart valve development, we designed a transgenic mouse model that reconstitutes the loss of function (lof) in adamts19 found in the statistically analyzing de novo mutations identified in >5,000 id patients highlighted ppm1d as a candidate id gene. ppm1d is a type 2c phosphatase that functions as a negative regulator of cell stress response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress induced apoptosis. we identified 14 patients with mild-moderate id and a de novo truncating ppm1d mutation. deep-phenotyping of the patients revealed in addition to id overlap for behavioural problems (adhd and anxiety disorder), hypotonia, broad based gait, facial dysmorphisms and periods of fever and vomiting. ppm1d is shown to be expressed during fetal (brain) development and in the adult brain. all mutations were located in the last, or penultimate exon, suggestive of escaping nonsense-mediated mrna decay. both ppm1d expression analysis and cdna sequencing in patient ebv-lcls support the presence of a stable, but truncated transcript, consistent with this hypothesis. exposure of patient's cells to ionizing radiation resulted in normal p53 activation suggesting that p53 signaling is not affected by the truncated protein. however, a cell growth disadvantage was observed. thus, we show that de novo truncating ppm1d mutations in the last and penultimate exon cause syndromic id which provides novel insights in the role of cell cycle checkpoint genes in neurodevelopmental disorders. de novo truncating variants in asxl2 are associated with a unique and recognizable clinical phenotype harvard stem cell institute, department of stem cell and regenerative enriched pathways (q < 0.05) including actin cytoskeleton and calcium ion binding. subsequently we applied the residual variation intolerance score (rvis) and identified 41 genes which were ranked among the 25% most intolerant genes in the genome. these genes included the previously reported genome-wide significant bd risk genes syne1 and mll2. in addition, we identified novel, promising candidate genes which have not previously been implicated in bd development such as ryanodine receptor 3 (ryr3, affected in six patients) and huntingtin (htt, 4 patients). both genes are ranked among the 0.5% most intolerant genes of the genome. ryr3 encodes a brain expressed intracellular cation channel that mediates the rapid release of ca2+ from the endoplasmic reticulum, thus making it a highly plausible candidate gene for contributing to rc. abnormal expansion of a trinucleotide repeat in the htt gene causes huntington disease which is a neurodegenerative disease characterized by motor, cognitive and psychiatric symptoms. the seven most promising genes are currently being followed up by resequencing in larger cohorts of 2500 independent bd cases (including 250 patients with rc) and 2500 controls of european ancestry using the single molecule molecular inversion probes (smmips) technology. de novo truncating mutations in the last and penultimate exon of ppm1d cause a novel intellectual disability syndrome abstracts with gα. signaling properties of g protein complexes carrying mutant gβ1 subunits were further analyzed by their ability to couple to dopamine d1r receptors by real-time bioluminescence resonance energy transfer (bret) assays. these studies revealed altered functionality of the missense mutations r52g, g64v, a92t, p94s, p96l, a106t, and d118g but not for l30f, h91r, and k337q. in conclusion, we demonstrate a pathogenic role of de novo and autosomal dominant mutations in gnb1 as a cause of gdd and provide functional evidence for a loss-of-function mechanism underlying the disease. comprehensive phenotyping and trio-exome analysis of 50 children with neurodevelopmental disease whole exome sequencing (wes) has been proven as a powerful analytical tool to dissect the genetic basis of human hereditary disorders. here, we report on a prospective deep phenotyping and trio-wes study of 50 children affected by previously undiagnosed and diverse complex neuropediatric disorders. all children underwent a standardised and comprehensive clinical work-up in a single centre that included detailed clinical evaluations by pediatricians and clinical geneticists, extensive laboratory and metabolic analyses, analyses of cerebrospinal fluid, mri of the brain and eeg, followed by trio-wes analysis. this systematic approach allowed to identify a pathogenic mutation in a known disease gene in altogether 21 children (42%) and discovered a convincing candidate disease gene in additional 22 children (44%). taken together, this translates into a successful genetic diagnosis of up to 86% in this cohort. in 3 children with mutations in a known disease gene (3/21 = 14.3%) the molecular diagnosis substantially influenced the clinical management and drug treatment. we further document an expansion of the phenotype in known disease entities in 4 individuals. the extraordinary high gene discovery rate in our cohort emphasizes the potential of trio-wes even in a clinically inhomogeneous group of individuals with likely genetic disease. however, this requires a multidisciplinary approach including deep and sometimes reverse phenotyping, research-based interpretation of trio-wes identified genetic alterations, extensive review of the literature, use of several mutation prediction and protein-modelling tools, as well as openness and exchange of data with national and international researchers and clinicians working on similar diseases. exome sequencing of pooled dna samples for large-scale screening in individuals with sporadic intellectual disability b. popp, a. ekici, s. uebe, c. thiel, j. hoyer, a. wiesener, a. reis, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germany high throughput sequencing has enabled identification of many novel disease genes and empowered diagnostic testing for heterogeneous disorders, especially for intellectual disability (id) where more than 1000 genes have been implicated. due to this extreme heterogeneity gene panels are ineffective, and expensive exome or genome sequencing is necessary. furthermore, many affected individuals have to be sequenced to confirm candidate genes and to refine the phenotypic spectrum. we now explored if pooling strategies could satisfy the need for a genome-wide, simple, cheap and fast screening technology. the asxl genes (asxl1, asxl2 and asxl3) participate in body patterning during embryogenesis and encode for proteins that are involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. germline de novo truncating variants in asxl1 and asxl3 have been respectively implicated in causing bohring-opitz and bainbridge-ropers syndromes, resulting in overlapping features of severe intellectual disability and dysmorphic features. to our knowledge, asxl2 has not yet been associated with a human mendelian disorder. in this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. all six had de novo truncating variants in asxl2. a careful review en abled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia and developmental disabilities. although overlapping features with bohring-opitz syndrome and bainbridge-ropers syndromes exist, features that distinguish the asxl2-associated condition from asxl1-and asxl3-related disorders are macrocephaly, absence of growth retardation and more variability in the degree of intellectual disabilities. we were also able to demonstrate with mrna studies that these variants are likely to exert a dominant negative effect, since both alleles are expressed in blood, with the mutated asxl2 transcripts escaping nonsense mediated decay. in conclusion, de novo truncating variants in asxl2 underlie a new neurodevelopmental syndrome, with a clinically recognizable phenotype. this work expands the germline disorders that are linked to the asxl genes. functional characterization of novel gnb1 mutations as a rare cause of global developmental delay over the past years, prioritization strategies that combined the molecular predictors of sequence variants from exomes and genomes of patients with rare mendelian disorders with computer-readable phenotype information became a highly effective method for detecting disease-causing mutations. the drawback of phenotype-based prioritization, however, is that they require a deep and comprehensive feature description to gain good performance. but in routine diagnostics, the naming of phenotypic features varies among clinicians, and sometimes a comprehensive phenotypic overview is not possible because of missing terminology. these gaps can be reduced by including a new layer of phenotypic information using facial recognition technology to detect dysmorphic features from two-dimensional photographs. automated image analysis is in principle able to identify any deviation from the norm and to quantify it objectively. we therefore developed an approach that combines facial dysmorphology novel analysis (fdna) technology with standard phenotypic and genomic features to identify pathogenic mutations in exome data. we have started collecting data from a diverse spectrum of patients with molecularly confirmed diagnoses in a multi-center study, and we present the current results. at the time of abstract submission more than 300 patients from over 10 contributing institutions were evaluated and used for simulation of a training set of exomes. automated facial recognition yields the correct diagnosis amongst the first ten suggested syndromes in more than two thirds of the cases and shows a high correlation with syndrome predictions that were based on expert annotated features. hereby, we could also confirm the diagnosis in cases with only subtle facial features. consequently, we used classical machine learning approaches to integrate scores based on the image analysis, phenotypic description and exome se-after initial evaluation of available computational methods by virtual pooling of exome data or simulated reads using different pooling fractions, we decided to exome sequence 96 individuals with sporadic id in 8 pools of 12 samples each. this was suggested to be the optimal combination with a 90% detection rate. dna was mixed in equimolar concentrations and submitted for exome sequencing. read data was aligned to the human reference, and variants were called using a ploidy of 24. resulting variant calls in 893 known id genes (sysid database) were then filtered for loss-of-function (lof) variants and for missense variants that were either previously reported as pathogenic or computationally predicted to be deleterious. furthermore, we screened 523 id candidate genes and 1694 haploinsufficiency intolerant genes for lof variants. subsequently, sanger sequencing was used to determine the individual carrying each variant in the respective pool and to test segregation in the parents. this approach resulted in the identification of 15 pathogenic variants (assumed or confirmed de novo) in known id genes (ahdc1, ankrd11, atp6v1b2, cask, chd8, kcnq2, kmt2a, kras, med13l, rit1, setd5, tcf4, wac, zbtb18), two pathogenic variants inherited from a symptomatic or healthy parent, respectively, (zmynd11, ifih1), and a homozygous variant in the recessive trappc11 gene. this included 13 loss-of-function and 5 missense variants. additionally, we identified 4 de novo variants in candidate genes. in our id cohort this resulted in a high mutation detection rate of 23%. thus, detection of rare variants from exome sequenced dna pools (pool-seq) is feasible and has a high detection rate similar other screening approaches. compared to affected-only exome sequencing this method can reduce costs by more than 90% with only marginal increase in sanger-sequencing costs and significantly speed up wet lab work with an acceptable increase in computational complexity. in contrast to targeted sequencing methods like molecular inversion probes or hybridization-based panels, our method has the advantage of allowing flexible re-analysis of the same data for new genes. in conclusion, we established exome pool-seq as a method for large-scale, cost-efficient and flexible sequencing in highly heterogeneous but well characterized disorders like id. three years of experience with targeted next-generation sequencing of developmental delay next-generation sequencing (ngs) has opened up new possibilities especially in the search for disease-causing mutations in disorders with common clinical features but a heterogeneous genetic background. the identification of the underlying genetic defect provides a clear diagnosis for patients more and more influencing their management and occasionally even their therapy, and it is the prerequisite for prenatal or preimplantation decisions in the affected family. ngs panels are used widely in clinical settings to identify genetic causes of various monogenic disease groups, such as intellectual disability (hu et al. 2015) , neurodevelopmental and neuromuscular disorders, among others. however, many new challenges have been introduced both at the technical level and at the bioinformatic level, with consequences including new requirements for interpretation of results, and for genetic counseling. we report on our experience with a targeted ngs panel comprising over 1200 brain related genes (mpimg-1-test) in the routine clinical diagnostics of patients with syndromic and non-sydromic forms of developmental delay as well as patients with neuromuscular disorders. 202 patients (age 1-71; mean 16) with syndromic (s) or non-syndromic (ns) developmental delay or with neuromuscular symptoms (nm), seen at the genetic counselling unit of our institute, were analyzed with targeted exon enrichment and ngs. chromosomal re-arrangements and copy number variations were excluded in all 202 patients previously by conven-it was recently shown that that clonal hematopoiesis can be driven by somatic point mutations. these acquired mutations occur with normal aging in up to 10% of older (>65y) individuals (1) (2) (3) and few reports in younger individuals. here we present a targeted re-sequencing assay that combines high throughput with ultra-high sensitivity based on single-molecule molecular inversion probes (smmip) (1) . we have now analyzed dna from 2000 healthy blood donors from 5 different age groups (20-29; 30-39; 40-49; 50-59 and 60-69y) , with no previous diagnosis of cancer, for somatic mutation in 100 loci. those loci included 50 known drivers of clonal hematopoiesis (2) (3) (4) and 50 novel or candidate loci. the improved assay allows low-frequency variant detection with variant levels down to <0.1%. this improved sensitivity allowed the identification of somatic mutations in a limited set of loci in >15% of old individuals, but also report those mutations in individuals of the youngest age group. most prevalent mutations include known hotspot mutations in dnmt3a and asxl1. here we show that somatic drivers of clonal hematopoiesis are more prevalent and occur in younger individuals than previously reported. these somatic events are age-related. however, the high prevalence and their occurrence in relatively young individuals implicates their origin as a common biological process involved in normal aging. a. m. nissen, j. graf, c. rapp, m. locher, a. laner, a. benet-pagès, e. holinski-feder medizinisch genetisches zentrum -mgz, munich, germany gene dosage abnormalities account for a significant proportion of pathogenic mutations in rare genetic disease related genes. in times of next generation sequencing (ngs), a single analysis approach to detect snvs and cnvs from the same data source would be of great benefit for routine diagnostics. however, cnv detection from exon-capture ngs data has no standard methods or quality measures so far. current bioinformatics tools depend solely on read depth which is systematically biased. we developed a novel approach based on: 1. utilization of five independent detection tools to increase sensitivity, 2. different reference sets for different kits and normalization against samples from the same sequencing run to improve robustness against workflow conditions, 3. definition of special quality thresholds for single exon events to minimize false negatives, 4. identification of reliable regions by assessment of capture efficiency using a reference set of cnv negative patients to minimize false positives. a cnv quence of the patients and could predict the pathogenic mutation among the top 10 positions in a prioritized exome in more than 90% of the monogenic cases in our cohort. hence, our results show that computer-assisted facial recognition is not only a promising technology that could be applied in the routine diagnostic workflow, but also a technology that allows diagnosis in cases with non-typical clinical presentation and boosts the diagnostic yield in exome studies. the added value of rapid exome sequencing in critical clinical situations for critical clinical situations, turnaround times (tats) of exome sequencing need to be fast in order to have an impact on clinical decision making. we therefore set out to develop a fast exome sequencing approach (max. 14 days). urgent exomes are preferably sequenced as trios to enable de novo analysis and assist data interpretation. dna library preparation is performed using the sureselect qxt protocol (v5, agilent), and sequencing is done on a nextseq500 (illumina) with a high coverage (200-300x). automated file handling allows rapid bwa mapping, gatk variant calling and annotation. a total of one-hundred samples have been sequenced until now using this rapid procedure: 28 trio's, 1x mother and child, 2x parents plus 2 children, and 6 single cases. six trios with known aberrations were used for experimental setup. of the remaining 31 families, in 14 families (possible) pathogenic snvs were identified, of which some still need further follow up, whereas 17 families remained negative after inspection of snvs and small indels. for cnv analysis, a trio based reference-free cnv approach is still under development. preliminary data show that all control cnvs (53kb-6mb) are detected correctly, and retrospective cnv analyses of the other samples identified three possibly de novo cnvs that need further follow up. shorter tats days were already beneficial for some patients, i. e. an adult male suffering from myelofibrosis and autoinflammatory symptoms. a sting-like phenotype (= stimulator of ifn genes) was suspected, with a possible involvement of the jak/stat pathway. urgent exome sequencing was performed and results were available within 9 days. interestingly, both a somatic variant in mpl (= trombopoetine receptor > myelofibrose) and a heterozygous variant in acp5 (trap, known immune dysregulation disorder) were identified, both fitting to the patients phenotype. based on these results the medication of the patient was changed, resulting in a substantial improvement of the patients constitution. in conclusion, we have implemented a rapid exome sequencing workflow for urgent cases. the rapid identification of pathogenic variants already had implications on patient treatment, underlying the added value of a fast genetic diagnosis. ement insertion, however, was spliced into the mrna of nabp1, leading to a frameshift mutation and a premature stop codon, potentially altering or abolishing gene function. in summary, we have shown that transposon insertions, both common variants as well as rare or de novo variants, can be detected in wes data. such insertions in coding or regulatory regions of disease-relevant genes might therefore explain some of the cases in which no pathogenic coding mutation can be identified by wes. the influence of human genetic variation on epstein-barr virus sequence diversity: a genome-to-genome approach c. hammer 1, 2 , a. loetscher 3, 4 , em. zdobnov 3, 4 genome-wide association studies (gwas) have identified common genetic polymorphisms that associate with clinical manifestation and immune response parameters of various infections. we here present an alternative approach, using variation in the virus sequence as phenotype, which is specific by nature and unique to genomic research in infectious diseases, for genome-to-genome (g2g) association studies. building on the unprecedented possibility to combine large-scale human and viral genomic data, we explored interactions between human genetic variation and viral sequence diversity in individuals infected with epstein-barr virus (ebv). the major goal is the identification of key genetic players in the evolutionary 'arms race' between pathogen and host. ebv is the pathogenic agent of infectious mononucleosis and is associated with a broad spectrum of lymphoid and epithelial malignancies, including lymphomas and nasopharyngeal carcinomas. there is also evidence for a role of ebv in the pathoetiology of multiple sclerosis. its genome is approximately 190 kbp long and encodes around 80 proteins, not all of which have been definitely identified or characterized. it is known that high loads of ebv are present in patients with advanced human immunodeficiency virus (hiv)-induced immunodeficiency. we therefore selected 780 immunosuppressed patients included in the swiss hiv cohort study (shcs) with low cd4+ t cell counts, and quantified ebv copy number in peripheral blood mononuclear cells (pbmcs). 290 cell samples contained more than 2,000 viral copies in total and were subjected to target isolation and subsequent enrichment using the sureselect method by agilent biotechnologies, followed by illumina whole-genome sequencing. after data processing and quality control, variable amino acids were called as binary variables, resulting in >200 variable positions per individual in average. the same patients also underwent genome-wide genotyping to obtain host genetic variation, followed by imputation based on the haplotype reference consortium reference panel. the association analyses are currently ongoing, and we will present the results at the conference. we use logistic regression to test for association between host single nucleotide polymorphisms (snps) and binary ebv amino acid variants. bonferroni correction is applied for multiple testing correction on the sides of both host and pathogen. stratification is taken into consideration by including principal components (pcs) for the host, and phylogenetic pcs for the virus. this project will offer a global description of the adaptive forces acting on ebv during natural infection. we have shown before for hiv that a virus genome associates much more strongly with human genetic variants than clinical endpoints. the analysis of all signals resulting from the interaction between human and viral genomes has the potential to identify novel host defense mechanisms, which could serve as future diagnostic and therapeutic targets. is called in a reliable region if at least two out of five tools are concordant for the respective cnv. the pipeline shows a sensitivity of 80% and a precision of 95%. within routine gene panel diagnostics we analyzed a total of 1088 patients indicated to have rare mendelian diseases for snv and cnvs. in 32 patients a cnv was detected in genes associated with the respective individual phenotype. interestingly, in several cases the cnv completed the patients report as it was detected in genes with a recessive mode of inheritance where previously only a heterozygous pathogenic snv was found. overall, with the additional analysis of cnvs we increased the diagnostic yield from 15% (class 4, 5 single nucleotide events) to 18%. however, there are still issues in the detection of cnvs from ngs data for routine diagnostics. cnv pipelines are very prone to errors caused by enrichment inconsistencies compared to snv detection tools. the assessment of sensitivity and specificity is difficult due to the lack of datasets to validate cnv detection pipelines. originally, the analysis of cnvs was performed mainly in patients with mental retardation disorders, resulting in a paucity of cnv data linked to other mendelian diseases. moreover, the identification of the actual size and thus the assessment of pathogenicity of a cnv is difficult, because targeted ngs gene panels do not cover all genes. in conclusion, ngs data is a suitable data source for the simultaneous detection of snvs and cnvs for clinical diagnosis; however, with the current tools it is only applicable in accurately validated regions. identification of transposon insertions in whole-exome sequencing data s. lukassen, n. übelmesser, ab. ekici, u. hüffmeier, ct. thiel, c. zweier, a. winterpacht humangenetisches institut, erlangen, germany 45% of the human genome consists of transposable element derived sequences, the most abundant of which are l1 and alu elements, followed by endogenous retroviruses. several hundred of these elements remain active, leading to insertion frequencies of up to one in 20 live births for alu elements and posing a threat to genome integrity. while most studies on transposons employ whole-genome sequencing (wgs) or target-enrichment based sequencing approaches, the most commonly used form of diagnostic high-throughput sequencing is currently whole-exome sequencing (wes). we were therefore interested in investigating transposon insertions in wes data as a possible source of disease causing mutations. we developed a software to call non-reference transposon insertions from single-end wes datasets by split-read mapping and analyzed 385 exomes this way. on average, 188 non-reference insertions were identified in each exome, with an average of 1.7 sites per patient identified in < = 0.5% of other patients. of these rare variants, 91% were deemed plausible by visual inspection. automated confidence calls of the software were concordant with visual inspection in 87% of cases. in 5% of cases a plausible insertion was awarded a lower score by the algorithm and in another 5% not called at all. in 1% of cases the automated call appeared to be falsely positive, in another 1% at the wrong position within the same 100bp window. laboratory validation of 11 convincing insertions revealed a 73% true positive rate, leading to an estimated specificity of 67%. when performing calls for reference l1 insertions on 10 exomes, 65% (49% -70%) of known elements whose flanking regions were covered by at least two reads were correctly identified, leading to a sensitivity of 65%. we thus estimate the average number of non-reference transposon insertions in our wes dataset to be 194 (153-235). 67% and 22.2% of sites identified were associated with alu and l1 elements, respectively, with the majority of calls stemming from evolutionary young transposons still assumed to be active. 10.3% of sites were located within the cds, 3.4% in the utrs of genes, 0.5% spanned an intron/ exon border, 48.5% were intronic and 37.3% of insertions were found in intergenic regions. we then chose 8 insertions within intronic (7) or utr (1) regions for further analysis. seven were not detected in the mrna. one intronic alu el-abstracts ws6-002 novel insights into male-pattern baldness pathobiology via integration of differential hair follicle mirna and mrna expression profiles with gwas data male pattern baldness (mbp) is a highly heritable condition and the most common form of hair loss in men. the phenotype is characterized by a distinct pattern of androgen-dependent progressive hair loss from the scalp that is restricted to hair follicles (hf) in the frontal and vertex scalp area. the molecular mechanisms that underlie this characteristic pattern and the differences in androgen-sensitivity between hf subpopulations in the frontal/vertex and the occipital scalp remain however elusive. to gain novel insights into the underlying biology and contributing genes and pathways, we systematically investigated for a differential expression (de) of mirna-and mrna-genes in hf samples from the frontal and occipital scalp area of 24 healthy male donors. array-based genome-wide mirna and mrna profiling revealed expression of 823 mirnas and 21,247 mrnas in human hf, of which 144 mirnas (17%) and 3,230 mr-nas (15%) showed a de between hf subpopulations. the strongest de mirnas included mir-4674, mir-6075 and mir-3185. among the strongest de mrnas were the wnt-signaling inhibitor dkk1, the protein kinase pak1 and the retinoid acid receptor rora. a subsequent pathway-based analysis in mirpathdb revealed that de mirnas targeted numerous interesting pathways. among them the wnt-and mtor signaling pathway which have been implicated in the control of hair follicle cycling, a mechanism that is disturbed in mpb affected hf and other plausible candidate pathways such as estrogen, thyroid hormone signaling or epidermal growth factor binding which have not yet been implicated mpb pathobiology. to yield further evidence for an involvement of de mirnas and mr-nas in the developement of mpb, we subsequently integrated our expression data with association data from a large gwas meta-analysis on mpb (n = 22,518). of the de mirnas and mrnas, only 1 mirna (mir-193b) and 49 mrnas were located within 1 mb of one of 63 genome-wide significant mpb risk loci. notably, the analysis revealed a co-localization of de mirna, de mrna, and nominally significant association signals (p < 10 -5 ) at 9 other genomic loci, pointing towards a role of these genomic regions in mpb pathogenesis. among them a locus on chromosome 3q22.2 that comprises the genes encoding the ephrin-type-b receptor 1 (ephb1) and the prostaglandin transporter slco2a1. interestingly, ephrins have been shown to be regulated by androgens and to play a role in hf formation, proliferation and hair cycling. and expression of prostaglandin d2, which is transported by slco2a1, has been found to be upregulated in balding scalp where it inhibits hair growth. in summary, our systematic analysis of differential mirna and mrna expression and the subsequent integration with genetic association data identified 9 novel potential risk loci for mpb and numerous candidate genes and pathways that are likely to play a role in mpb pathogenesis and emphasizes the importance of data integration of large-scale omic-analyses. palaeontological genomic analyses have shown that interbreeding between anatomically modern humans and neandertals occurred in europe and asia 40.000-50000 years ago. approximately 1.5-2% of the modern european and asian genome consists of introgressed dna from neandertals. some of these introgressed regions have been suggested to contribute to several traits and phenotypes including major depression and other mood disorders. in order to further assess the role of neandertal ancestry in cognition and the contribution of genetic risk for psychiatric disorders, we performed genome-wide analyses of neandertal alleles in publicly available psychiatric genomics consortium (pgc) gwas summary statistics with samples sizes ranging from about 6000 to 293723 individuals for the following phenotypes: educational attainment, attention deficit hyperactivity disorder (adhd), anorexia nervosa, anxiety disorders, autism spectrum disorder, bipolar disorder, major depressive disorder and schizophrenia. we estimated the proportion of heritability explained by snps in neandertal introgressed regions using stratified ld score regression (ldsc) and two sets of previously inferred neandertal introgressed regions. in a secondary analysis, we investigated whether specific functional annotations such as 3'utr, promoter regions or histone marks within neandertal regions were significantly associated with selected phenotypes. we identified a modest enrichment of heritability in neandertal introgressed regions in anorexia nervosa, autism spectrum disorder, bipolar disorder and major depressive disorder, although none of the results were statistically significant. several functional annotations, such as h3k4me1 histone marks within neandertal introgressed regions, appeared significantly enriched for snps contributing to the heritability of anorexia nervosa and autism spectrum disorder. in bipolar disorder, dnasei digital genomic footprinting regions, h3k9ac histone marks and super enhancer regions within neandertal regions appeared particularly enriched for heritability. on the other hand, both sets of neandertal regions were slightly depleted of snps contributing to the heritability of schizophrenia. for example, one set of neandertal regions that contained 33% of all analysed snps only contributed to 29% of the variance of risk (standard error: 0.04; p-value: 3.86 × 10 -3 ). in comparison to the rest of the genome, neandertal introgressed regions also contributed less to the heritability of educational attainment, adhd and anxiety disorders, although these findings were not statistically significant. to our knowledge this is the first study to systematically investigate the extent to which snps attributable to neandertal introgressed regions contribute to the heritability of several psychiatric/cognitive phenotypes. we are currently increasing our power to detect snp heritability in neandertal regions by applying the ldsc method to larger pgc datasets. harbor genes involved in the complement system, high density lipoprotein metabolism or extracellular matrix homeostasis. these pathways are known for their pleiotropic role in other conditions, such as cardiovascular disease, auto-immune diseases and cancer. here we aimed to investigate the extend of overlap between the genetic risk of various complex diseases and traits and the genetic risk for amd. methods: first, we catalogued 2,331 previously published, genome-wide significant variations associated with 82 complex diseases or traits. next, we computed a genetic score by calculating the (weighted) sum of risk increasing alleles for each disease or trait. consequently, a higher genetic score indicates that an individual has more risk/trait increasing alleles of a given disease or trait. for each score, we computed the association with late stage amd using a dataset provided by the international amd genomics consortium (iamdgc) including 16,144late stage amd cases and 17,832 controls. we also assessed the association of each variation individually with late stage amd risk in order to identify novel disease loci with strong evidence for pleiotropy. results: nineteen genetic scores of complex diseases and traits were significantly associated with amd risk (fdr < 0.01). most notably, all genetic scores related to autoimmunity were elevated in amd patients (p < 5.85×10-09 ), while scores related to cardiovascular disease were reduced in amd patients compared to controls (p < 3.10×10 -05 ). we also found that the genetic scores of melanoma and related malignancies were higher in amd patients (p < 8.43×10 -05 ). in addition, 32 out of 2,331 variants, which were used to compute the genetic scores, were significantly associated with amd (fdr < 0.01), implicating 25 novel, pleiotropic loci in amd risk. conclusion: our findings demonstrate a substantial overlap between the genetic risk of complex diseases/traits and the genetic risk for amd and provide evidence for 25 novel, pleiotropic loci associated with amd. while our findings highlight common disease pathways that may facilitate to develop multi-use drug targets, they also challenge the notion that gene/genome manipulation could be applied in general terms to eradicate risk for a defined complex disease. worldwide genetic association study of exfoliation syndrome and glaucoma identifies common genetic variants at five new susceptibility loci exfoliation syndrome (pex), a complex systemic disorder of the extracellular matrix, is the commonest cause of secondary glaucoma in aging population and thus a major cause of blindness globally, affecting 60-70 million subjects worldwide. inside a large, international collaboration project a genome-wide association study (gwas) was carried out on 9,035 pex cases and 17,008 controls, recruited from 24 countries across six inhabited continents, with replication in a further independent 4,585 cases and 92,829 controls from 15 countries. significant association was observed at seven loci, of which two confirmed the already known associated loci at the genetic markers mapping to loxl1-and cana1a-gene, five are new (p < 5×10 -8 ). the five new loci map to chromosomes 13q12 (rs7329408 near flt1-pomp-slc46a3, p = 9.41 × 10 -16 ), 11q23.3 (rs11827818 near tmem136-arhgef12, p = 1.21 × 10 -10 ), 6p21 (rs3130283 at agpat1, p = 2.12 × 10 -9 ), 3p24 (rs12490863 at rbms3, p = 3.9 × 10 -8 ) and 5q23 (rs10072088 near sema6a, p = 3.64 × 10 -8 ). to determine the pathophysiological role of the three most significantly associated loci (13q12, 11q23.3, 6p21), we investigated the expression and localization of the six related genes (flt1, pomp, slc46a3, tmem136, arhgef12 and agmale-pattern baldness (mpb) is characterized by a progressive hair loss from the frontal and vertex scalp that affects ~80% of men at the age of 80 years. epidemiological studies have shown positive associations between mpb and coronary heart disease (chd) and related phenotypes such as blood pressure (bp), diabetes (dm) or elevated blood lipid levels. the results however vary with regard to the associated pattern of hair loss (frontal or vertex) and the assessed endpoint measures for chd. and so far no study has investigated for a shared genetic determinant between the traits. using data from the heinz nixdorf recall study (n = 1,675 males) and a large meta-analysis on mpb (n = 22,518), we aimed at a systematic investigation of the association between mpb and chd on (i) an epidemiological and (ii) a genetic level. , men with vertex balding showed a higher bmi (β = 1.4 kg/m2), elevated fasting triglyceride (β = 8.0 mg/dl) and lower hdl-c levels (β = -2.7 mg/dl). to assess the genetic overlap between mpb and chd, we created a risk score (rs) from 63 mpb lead snps (p < 5×10 -8 ) and tested for association with chd and related traits phenotypes. no significant associations were observed. however, an age-stratified analysis revealed a 4% per allele risk increase for chd (hr = 1.04, 95%ci:0.97;1.17) and a decrease in fasting triglyceride levels (β = -0.5). we next used ld score regression analysis in to test for genome-wide genetic correlation between mpb and chd. the analysis revealed no significant correlations with cardiometabolic (n = 3), lipid (n = 4) or metabolic traits (n = 103). finally, to investigate for a genetic overlap at single loci, we compared the mpb risk loci with reported gwas signals for chd. the analysis identified seven overlapping associations between mpb and bp (n = 3); qt-interval length; atrial fibrillation; sudden cardiac arrest; and dm. for the majority of loci, the direction of effect differed between mpb and chd, opposing previous epidemiological findings. positive associations were identified between mpb and diastolic bp (fgf5, 4q21.21) and sudden cardiac arrest (atf1, 12q13.12). interestingly, fgf5 is known to stimulate cell growth and proliferation in multiple cell types, including cardiac myocytes and hair follicle (hf) cells, and atf1 is a hf expressed regulator of cell growth and differentiation that has been shown to prevent foam cell formation, which suggests that fgf5and atf1-signaling contribute to both traits. thus, our data support an association between mpb and chd related phenotypes and suggest that mpb deserves further evaluation as an additional risk factor for chd. pleiotropic effect of genetic variants associated with complex diseases and traits in age-related macular degeneration purpose: age-related macular degeneration (amd) is the leading cause of vision loss in western societies and is caused by both environmental and genetic risk factors. with regard to the latter, several associated risk loci abstracts otyping, results of which will be presented at the conference. of note, for nonsyndromic cleft lip with/without cleft palate (nscl/p), the most frequent form of orofacial clefting, 20 risk loci have been detected by gwas so far, with some of them reaching (nearly) genome-wide or significant p-values in samples much smaller than 550 cases. in the imputed nscpo dataset none of the presently known nscl/p risk loci showed a p-value < 10 -5 . our data so far confirm previous molecular and epidemiological findings, that nscpo is genetically distinct from nscl/ p. furthermore, the results indicate that common variants alone might not contribute to the same extent to nscpo as compared to nscl/ p. the correlation between defects at specific imprinted loci and distinct imprinting disorder (id) was accepted for a long time. however, it is now put into question because of a growing number of patients with multilocus imprinting disturbances (mlid), i. e. the aberrant methylation at more than one imprinted locus. in particular, mlid is present in individuals with silver-russell syndrome (srs) and beckwith-wiedemann syndrome (bws), and it has meanwhile turned out that patients with opposite phenotypes can share common epimutation patterns. on the other hand, mlid always occurs as mosaicism and varies in different tissues of the same individual. interestingly, the majority of mlid carriers show only one specific id phenotype, though loci of other ids are affected in addition to the one specific for the phenotype. we become aware of a growing number of patients with unexpected and even contradictory molecular findings in respect to the clinical diagnosis for referral. amongst others, we detected the srs specific icr1 hypomethylation in 11p15 in two of our patients referred as bws. in the first case, the icr1 hypomethylation was detected only in lymphocytes but was not present in buccal swab dna. the patient only had a slight asymmetry, but showed normal growth and did not exhibit any other feature compatible with bws, nor with srs. the reason for the lack of clinical features is unclear, but is comparable to the observation in monozygotic, but clinically discordant srs and bws twins. here the unaffected twin often carries the epimutation only in lymphocytes whereas the affected one shows the alteration in additional tissues. a reason might be sharing of hematopoietic stem. it can be postulated that the patient presented here is born after an (undetected) twin pregnancy with early loss of the affected twin. in the second case, the initial diagnosis of bws was made due to asymmetry, though overgrowth or other features were not present. further clinical ascertainment did not confirm this diagnosis, but growth of the patient was in the lower percentiles, in concordance with the icr1 hypomethylation. these cases as well as further cases in our cohort confirm that there is an urgent need to provide detailed clinical data upon requesting molecular diagnostics for imprinting disorders. in fact, the growing number of patients with unexpected results complicates the interpretation and illustrates the broad phenotypic range, but also provides further insights in the etiology of ids and setting of imprinting marks pat1) by qrt-pcr, immunohistochemical-and western-blot analysis in genotyped ocular tissues of pex and control patients. all six genes displayed moderate mrna expression in all ocular tissues analysed, with highest levels in iris, ciliary body, and retina. however, only pomp showed a trend towards reduced expression in the presence of the rs7329408 risk allele, in both pex and control patients. in general, both mrna and protein expression of pomp and tmem136 were significantly reduced up to 45% (p < 0.005) in anterior segment tissues in pex eyes compared to controls. no differences in mrna and protein expression were detected for the remaining genes analysed. immunofluorescence analysis showed that pomp, a proteasome maturation protein, is ubiquitously expressed in most ocular cell types and that tmem136, a transmembrane protein of unknown function, is primarily localized to endothelial cells of blood vessels and aqueous outflow structures. additionally, protein staining intensities for pomp and tmem136 were markedly reduced in anterior segment tissues of pex eyes compared to controls and co-localized to abnormal accumulation of pex material on ocular surfaces and in blood vessel walls. thus, at least two of the newly identified loci provide new biological insights into the pathology of pex syndrome/glaucoma and highlight a role for impaired proteasome function as well as vascular and trabecular endothelial dysfunction in the disease pathogenesis. nonsyndromic cleft palate only -evidence for a limited contribution of common variants in contrast to nonsyndromic cleft lip ± palate cleft palate only (cpo) is a common congenital malformation which might occur as part of a syndrome or in an isolated form, i. e., nonsyndromic cpo (nscpo). nscpo has a prevalence of 1:2500 and is considered multifactorial with genetic as well as environmental factors contributing to the disorder. in a recent study we identified the first genome-wide significant locus for nscpo which has been independently confirmed in another study. in order to discover more nscpo risk loci we performed a genome-wide imputation study with gwas data from 550 case-parent trios with european, asian and african ancestry which was retrieved from db-gap upon approved data access. notably, this gwas dataset had not yet been imputed, and we hypothesized that we can increase power to identify novel genetic associations by increasing the marker density and follow-up of suggestive findings by independent replication. genome-wide genotypes were imputed using impute2 based on 1000 genomes haplotypes, and snps were selected based on info-score > 0.4 and minor allele frequency > 1%. the imputation did not reveal any genome-wide significant snp, however, 83 snps at 26 loci showed p-values < 10 -5 . loci with more than two variants below this threshold (n = 19) were to be replicated using the massarray system (agena bioscience). three independent samples were used: two case/control replication cohorts from central europe (92 cases, 335 controls) and yemen (37 cases, 231 controls), and one european case-parent trio replication cohort (eurocran study; 223 trios). in a first round we genotyped 18 snps at eleven loci. one variant, rs6809420 at chr. 3q13, showed p < 0.1 in the replication cohort and after combining replication and gwas data, resulted in a decrease of p-value from 1.14×10 -03 to 3.22×10 -04 . this indicates that this locus, which includes candidate genes such as igs11, a known cell-adhesion molecule with yet unknown function in craniofacial development, might harbour a common risk variant with low effect size. we are currently performing a second round of gen-we report biallelic mutations in cad, encoding an enzyme of de novo pyrimidine biosynthesis, in four patients with developmental disability, epileptic encephalopathy, anaemia, and anisopoikilocytosis. two children died after a neurodegenerative disease course. treatment of two surviving children with oral uridine led to immediate cessation of seizures in both. a four-year-old girl, who was previously in minimal conscious state, started to communicate and walk with assistance after nine weeks of treatment. a three-year-old girl likewise showed developmental progress. blood smears normalised and anaemia resolved. our findings support the efficacy of uridine supplementation rendering cad deficiency a treatable neurometabolic disorder. delineation of the grin2a phenotypic spectrum alterations of the n-methyl-d-aspartate (nmda) receptor subunit glu-n2a, encoded by the gene grin2a, have been associated with a spectrum of neurodevelopmental, speech and epilepsy disorders. we identified 48 previously unreported patients with heterozygous pathogenic variants in grin2a, including 30 novel variants. after re-evaluation of all published grin2a cases, 104 previously reported patients met the acmg criteria for being pathogenic or likely pathogenic. thus, we are able to collectively review genotypes and phenotypes of 152 individuals with grin2a-related disorders. we show that the known phenotypic spectrum is expanded and ranges from near-normal development to severe and unspecific encephalopathy, comprising any disorder of speech development. furthermore, some patients do not display seizures. in contrast to previous reports, gri-n2a missense variants cluster within the functionally most relevant domains. we are the first to describe genotype-phenotype correlations in grin2a-related disorders, where carriers of pathogenic missense variants tend to have more severe neurodevelopmental phenotypes compared to carriers of truncating variants. the most severe end of the phenotypic spectrum was found to include novel features, such as infantile spasms and arthrogryposis and was associated with pathogenic variants in the pore-forming domain of grin2a. the eponymic name galloway-mowat syndrome (gamos; omim 251300) has been coined for the association of early-onset nephrotic glomerulopathy, microcephaly with variable brain anomalies, and facultative diaphragmatic hernia. it is supposed to be inherited as an autosomal recessive trait and clinical as well as genetic heterogeneity has been suggested. in 2014, wdr73 mutations were identified as a cause of gamos, but only a few cases have been reported to date. over the last 15 years, we have collected dna samples and clinical data from 56 unrelated families with one or more children affected by gamos or a gamos-like syndrome (glomerulopathy plus variable anomalies of brain morphology or function as inclusion criteria), including 15 consanguineous families. in this cohort, we performed whole exome sequencing followed by targeted analysis by sanger and ngs multigene panel resequencing. in a total of 25 families of this cohort (46%) the probable underlying genetic defect could be identified. in affected individuals from two consanguineous families, homozygous mutations of wdr73 could be found (vodopiutz et al., 2015) . thus, this gene accounted for only 4% of cases of our cohort. the affected child of another family had a novel homozygous mutation in arhgdia. this gene has previously been described in three families to cause early-onset steroid-resistant nephrotic syndrome (gupta et al., 2013; gee et al., 2013) , but there is some evidence that non-specific brain anomalies may also be part of the arhgdia-associated phenotype. fourteen and three index patients from unrelated families had mutations in one autosomal (osgep) and one x-linked gene (lage3), respectively, both encoding for components of the keops protein complex that has been implicated in transcription, telomere maintenance and chromosome segregation. no human phenotype has previously been assigned to mutations in this complex. notably, eight unrelated families with an identical mutation originated from the east asian population where the carrier frequency for this allele is 0.0008. in one consanguineous family with multiple affected children the disease segregated with a homozygous mutation in the sgpl1 gene encoding for sphingosine-1-phosphate lyase. in four families, the kidney phenotype could be attributed to mutations in genes for non-syndromic nephrosis (nphs1, plce1, one novel gene), while the brain phenotype was apparently independent. in conclusion, the molecular genetic findings in this cohort confirmed that gamos is exceedingly heterogeneous, and still in almost half of the patients with a gamos-like phenotype the genetic cause remained unclear. on the basis of our findings we are now able to define new biologic mechanisms that are critically involved in both, brain development and integrity of the glomerular filtration barrier. genotype phenotype correlations are emerging. finally, we demonstrate that gamos can also be inherited as an x-linked trait. abstracts taminase 1 gene (tgm1) is mutated in the majority of patients (around 30%), and its gene product, tgase1, is therefore primarily targeted in our approach for protein substitution. patients with arci have an impaired skin barrier function, most of them are born with a collodion membrane and suffer subsequently from varying degrees of hyperkeratosis, erythema, transepidermal water loss and infections. the disease can be life threatening neonatally but lacks a causative therapy and is still only treated symptomatically. therefore, our aim is to develop a personalised, causative therapy where the defective protein is substituted topically via a nanocarrier. therapeutic, human tgase1 was synthesized in hek 293 cells and assessed by western blot and flow cytometry analysis. enzyme activity was measured by in vitro assay. tgase1 was then coupled to a polyglycerol-based nanogel (dpg-ng) containing the thermoresponsive linker poly(n-isopropyl)acrylamide (pnipam), stabilising the enzyme as well as adding a thermal protein release trigger at 35°c, which is favorable for cutaneous applications. immunocytochemical stainings for tgase1 on monolayered basal keratinocytes that lack tgm1 expression confirmed the successful uptake of extrinsic tgase1 into the cells. further analysis over time showed that the enzyme was no longer detectable after 48 h and consequently led us to define a treatment schedule for the following experiments. 3d full thickness skin models were used as in vitro system to determine barrier function and enzyme activity after treatment with varying concentrations of the dpg-ng/tgase1 complex. three different sets of skin models were used for these experiments: normal models mimicking the healthy skin with an intact barrier function, models where tgm1 was knocked down, and models made with arci patient cells with tgm1 mutations. franz cell tests on treated skin models lacking intrinsic tgase1 confirmed the impaired barrier activity in disease models, demonstrated an improved barrier function after repeated treatments with dpg-ng/ tgase1 and showed restored tgase1 activity using an in situ assay. furthermore, first toxicity tests using mtt revealed high biocompatibility of dpg-ng/tgase1 after treatment of 2d and 3d cell cultures. these findings are successful steps for an advanced topical drug delivery system and are a promising approach for causative therapeutic intervention in arci. after further optimization concerning protein dosage and thorough toxicity tests, we will adapt this system also for the use with other proteins involved in arci. pigmentation disorders (pds) comprise a large group of rare and heterogeneous disorders that are mainly characterized by various coloration abnormalities affecting single parts of the body or the complete integument. the large group of pds includes the autosomal dominant inherited hyperpigmentation disorder dowling-degos disease (ddd). ddd is genetically heterogeneous, and to date causal mutations in three genes, namely krt5, pofut1 and poglut1 have been identified. after exclusion of mutations in these genes, we performed exome-and sanger-sequencing in six unrelated ddd-patients/families and identified six heterozygous truncating mutations in psenen encoding the presenilin enhancer protein 2. on closer examination of the histological sections, we came upon a novel feature that distinguished these individuals from previous ddd-cases by the presence of follicular hyperkeratosis. to assess the functional significance of psenen mutations in ddd pathogenesis, we performed mammalian cell culture based studies and knockdown experiments of psenen homolog psenen in zebrafish larvae (zfl). knockdown of psenen in zfl resulted in a phenotype with scattered pigmentation, which mimicked human ddd. in vivo-monitoring of pigment cells in the developing zfl suggested that disturbances in melanocyte migration and differentiation underlie ddd pathogenesis. interestingly, six of the psenen mutation carriers presented with co-morbid acne inversa (ai), an inflammatory hair follicle disorder. all individuals had a history of nicotine abuse and/or obesity, which are known trigger-factors for ai. although psenen mutations have been identified in a small subset (<1%) of familial ai previously and the co-manifestation of ddd and ai has been reported for decades, our study is the first to demonstrate experimentally that mutations in psenen indeed can cause co-manifestation of ddd and ai, most likely triggered by predisposing factors for ai. thus, the present report describes a clinically and histopathologically novel ddd subphenotype in psenen mutation carriers, which is associated with an increased susceptibility to ai. protein substitution therapy for autosomal recessive congenital ichthyosis (arci) overall burkitt lymphoma showed a low genomic complexity with a low number of snvs and svs. however, the integration of cnas, snvs and svs allowed us to identify recurrently affected genes, which are involved predominately in the pi(3) kinase pathway, tonic bcr signaling, and cell cycle regulation, chromatin composition and germinal center development. burkitt lymphoma (bl) is the most common mature aggressive b-cell lymphoma in childhood. the genetic hallmark of bl is a chromosomal translocation involving the myc oncogene and one of the immunoglobulin loci leading to myc deregulation. three epidemiologic variants of bl are differentiated: endemic (ebl), which occurs predominantly in equatorial africa and is associated with ebv-infection, sporadic (sbl), which occurs in westernized countries and immunodeficiency-associated. in addition, burkitt leukemia (b-al) is differentiated from bl in cases with more than 25% of the bone marrow cells being lymphoma cells. another rare bl-variant is myc-positive precursor b-cell acute lymphoblastic leukemia coexpressing tdt and myc (tdt+bl). finally, we recently described a myc-negative variant which shows a typical alteration on chromosome 11 (mnbll). the aim of the present study was to examine the epigenetic landscape of these bl variants. to this end, we analyzed the dna methylation of 116 bl (60 sbl, 29 ebl, 10 b-al, 15 mnbll, 2 tdt+bl) using the humanmethylation450 beadchip and contrasted the findings to 24 diffuse-large b-cell lymphoma (dlbcl) and 30 follicular lymphoma (fl). the majority of lymphoma were recruited in the framework of the icgc mmml-seq and mmml projects. the ebl were obtained from the nci ghana burkitt project. as controls, we used public available dna methylation data from 93 b-cell burkitt lymphoma (bl), including its leukemic variant burkitt leukemia (b-al), is the most common type of pediatric b-cell lymphoma accounting for 30-40% of new cases. its biological hallmark is the ig-myc translocation involving myc and mostly the immunoglobulin heavy (igh) locus or more rarely one of the immunoglobulin (ig) light chain loci. at the cytogenetic level the ig-myc translocation is the sole abnormality in around 40% of cases. overall, bl is characterized by a low genomic complexity. the aim of the present study was to analyze the genomic and transcriptomic landscape of pediatric/adolescent burkitt lymphoma by sequencing according to the guidelines of the international cancer genome consortium. a total of 39 samples of bl/ b-al from pediatric/adolescent patients entered this sequencing study. all patients were treated in population-based prospective clinical trials. inclusion criteria were besides availability of suitable materials, consent to participate in the study and appropriate diagnosis: age at diagnosis (≤ 19 years), the presence of ig-myc rearrangement detected by fish and/or whole genome sequence (wgs), absence of rearrangements of bcl2 or bcl6 genes. we performed wgs of tumor and matched control as well as transcriptome sequencing of the tumor cells according to the standards of the icgc (www.icgc.org). the pathognomonic ig-myc translocation was detected in 37of 39 of the cases using wgs, but was observed in all cases by fish. an igh-myc juxtaposition was detected in 34 patients and its variants igk-myc and igl-myc in 1 and 4 cases, respectively. we identified two different expression patterns of myc transcripts which were associated with the translocation breakpoint location. on the one hand the canonical myc transcript and on the other hand an alternative transcript with a transcription start site before the second exon. the latter produces an mrna which contains 486 nucleotides not included in the canonical transcript but nevertheless it encodes the identical protein. the integration of single nucleotide variants (snv) and copy number aberrations (cna) identified a total of 128 recurrently (≥2 samples) mutated genes. myc, id3, tp53, ccnd3, smarca4, arid1a, fbxo11, ddx3x were mutated in ≥20% of samples. in 33/39 (85%) cases, the id3/ abstracts ed. the annotation with the chromatin segmentation data of cd8 + t-cells from the blueprint project revealed enrichment of changes in methylation in distinct genomic regulatory elements in t-lgl. these differentially methylated functional regions were enriched for a set of transcription factor binding sites, known to be relevant in other lymphoid neoplasms. by bioinformatic analysis of methylation data and integration with gene expression data we identified hypermethylated and hypomethylated genes (e. g. bcl11b, themis, zeb2, hivep3) which point to candidate pathways potentially deregulated in the pathogenesis of t-lgl. conclusion: our study identified dna methylation changes in a set of candidate genes involved in various signaling pathways, which could potentially be used for diagnosis, prognosis and may become targets for novel treatment options. burkitt lymphoma (bl) is a mature aggressive b-cell lymphoma genetically characterized by a chromosomal translocation leading to ig-myc juxtaposition. treatment of bl is usually very successful particularly in children, with a cure rate of over 90% even among patients with advanced stage disease. however, the prognosis of the remaining patients experiencing disease progression and/or relapse is still very poor. bl has an overall low genomic complexity, thus secondary chromosomal changes in addition to the ig-myc translocation are rare. however, genomic complexity has been associated with aggressive disease and poor prognosis in various lymphomas including bl. because little is currently known about the underlying genetics of disease progression in bl we aimed at characterizing the molecular changes and characteristics that might lead to the relapse of bl. sequential tumor biopsies from initial diagnosis (id) and follow-up were available from a total of 8 patients (4-15 years at id), which were divided into two groups: five patients experienced a relapse from their initial bl diagnosed 58-210 days after id (group 1). in contrast, three patients developed twice a bl, i. e. presented with bl as secondary neoplasms diagnosed 3-5 years after id (group 2). dna extracted from archival formalin-fixed, paraffin-embedded (ffpe) tissue was used to analyze genome-wide copy number alterations (cna) using the oncoscan® platform (affymetrix) and mutational landscape by whole exome sequencing (wes). analysis of the cna in the 5 paired bl samples (group 1) revealed an increase in genomic complexity in 4/5 pairs as in id a mean of 8 cna was detected in contrast to 13.4 cna in relapse samples (p = 0.113). of note is that in all pairs, the relapse shared almost all cna which were present in id. wes analysis of group 1 showed similar results in all analyzed pairs. in total, 46.6% of mutations (median number of mutations = 106) were shared in id and relapse. nevertheless, a considerable amount of mutations were unique in id and relapse with a median of 18 (11.8%) and 51 (41.7%) mutations, respectively. on the other hand, mutations detected in samples populations of various differential stages. furthermore, we investigated whole-genome bisulfite sequencing (wgbs) data of 12 sbl and 6 b-al in comparison to 4 germinal center b-cell populations from healthy donors to decipher differentially methylated regions (dmr). these are defined as 10 or more cpgs differentially methylated between two groups. unsupervised dna methylation analysis of bl, fl and dlbcl revealed that all bl variants cluster apart from the non-bl cases. thus, supporting on epigenetic level that all analyzed bl samples are bl variants. multigroup comparison (σ/σ max = 0.4, q < 1e -13 ) separated the bl variants roughly in 3 groups: ebl, ebv-positive sbl and all other bl variants. furthermore, this analysis revealed ebl to harbor a massive hypermethylation in comparison to all other bl variants. comparison of the dna methylation using the humanmethylation450 beadchip data of sbl and b-al revealed 199 cpgs to be differentially methylated (σ/σ max = 0.4, q < 0.005). in contrast, using the wgbs data of the same samples a total of 4712 dmrs could be identified which were mostly located in enhancer and polycomb target regions. in conclusion, we show that all analyzed bl variants share a similar dna methylation profile. interestingly, dmrs between sbl and b-al were mainly located in enhancer and polycomb regions. in contrast, ebl showed a massive hypermethylation in comparison to the other bl variants. thus, the differences identified by dna methylation analysis can improve the understanding of the biological and clinical differences of the bl variants. dürig 1, 8 introduction: t-cell large granular lymphocytic leukemia (t-lgl) is a mature t-cell leukemia which often arises in the context of autoimmune disease. genetic changes like recurrent chromosomal aberrations are rare. recent studies identified somatic stat3 and tnfaip3 mutations in t-lgl cells. however, the molecular events driving leukemogenesis remain largely unknown. objectives: the goal of our study was to characterize the epigenetic basis of t-lgl to better understand leukemogenesis and potentially identify druggable pathways or diagnostic biomarkers for t-lgl. p. johansson 1,2 , l. klein-hitpass 2 , g. castellano 3 , k. kentouche 4 , f. nicolau 5 , i. oschlies 6 , e. carrillo-de santa pau 5 , m. przekopowitz 2 , a. queiros 3 , m. seifert 2 , a. valencia 5 , ij. martin-subero 3 , em. murga penas 7 , o. ammerpohl 7 , u. dührsen 1 , r. küppers 2 , j. we analyzed the dna methylome of facs sorted tumor cells of 11 t-lgl cases in comparison to benign αβ t-cell subsets. the infinium human methylation 450 bead chip was used for analysis. we annotated our data with the publicly available chromatin segmentation data of cd8 + t-cells from the ihec/blueprint project. the expression levels of selected genes were tested by reverse transcription real-time pcr. results supervised analysis of t-lgl compared to benign cd8 + memory cells resulted in 1,083 cpg loci significantly (q < 0.001) differentially methylatkrawitz 5, 6 , a. knaus 5, 6 , m. jäger 5, 6 , r. flöttmann 5 , t. eggermann 7 , b. hoechsmann 8 , h. schrezenmeier 8 paroxysmal nocturnal hemoglobinuria (pnh) is an acquired disorder of the blood-forming system. typically, affected hematopoietic stem cells (hscs) in pnh harbor a single somatic loss-of-function mutation in the x-linked piga gene. previously, a pnh patient with a different molecular etiology has been described and herein we report three more cases of this new subgroup: a predisposing germline mutation in pigt, which is an autosomal gene of the glycosylphosphatidylinositol (gpi)-anchor synthesis pathway, is followed by a second somatic hit. by means of deep sequencing and array-cgh, we observed acquired deletions of 8 mb to 18 mb on chromosome 20q in pnh cells that include pigt as well as a region that is commonly deleted in myeloproliferative neoplasms and myelodysplastic syndromes and that is known to be differentially methylated. this results in a complete loss of expression of certain genes at this locus which is also thought to contribute to the clonal expansion. the deficiency of gpi-anchored proteins on pnh cells results in a lack of the complement regulatory proteins cd59 and daf/cd55 on the cell surface and leaves them more vulnerable to the c5b-9 membrane attack complex. in contrast to classical pnh without any fully synthesized gpi-anchors, pigt mutations impair the transamidase that links the substrate to the anchor and thus result in an accumulation of unbound gpi molecules. this difference in the pathophysiology can also be visualized in flow cytometric analysis of peripheral blood: while cd55 and cd59 surface levels are reduced in all pnh cells, the atypical pnh cells due to a transamidase deficiency can be discriminated by a specific antibody, t5 mab, that binds free gpi anchors. besides the classical pnh symptoms of anemia, thrombosis, and hemolysis, patients with pigt mutations also manifest with additional autoinflammatory symptoms, such as urticaria, fever, arthralgia and meningitis, and it is hypothesized that the free gpi-anchor that accumulates in affected cells is causally related to autoinflammation. based on these findings, we propose the new entity of atypical pnh. background: the prevalence of metabolic disorders, in particular obesity has dramatically increased worldwide. genetic variants explain only a minor part of this obesity epidemics induced by physical inactivity and over nutrition. epidemiological studies in humans and animal models of di-from patients with secondary neoplasm (group 2) were mostly unique to id (= 109, 43.9%) whereas only 26.1% of all mutations were shared in id and secondary neoplasm samples (= 49). furthermore there were no shared cna in the corresponding samples identified by oncoscan® analysis. to sum up, the oncoscan® and wes analysis, of the paired bl group (1) provide strong evidence for a linear clonal evolution, meaning relapses may directly evolve from the previous lymphoma clone rather than a common precursor. in contrast, results obtained for patients with secondary neoplasm (group 2) showed no indication for linear but rather for divergent evolution. thus, analysis of recurrent mutations shared in id and second neoplasm samples can provide important information about disease progression and are therefore subject of ongoing analysis. y. murakami 1 , t. hirata 1 , s. murata 1 , t. kinoshita 1 , m. kawamoto 2 , s. murase 2 , h. yoshimura 2 , n. kohara 2 , n. inoue 3 , m. osato 4 , j. nishimura 4 , y. ueda 4 , y. kanakura 4 , p. m. in runx1 mutated aml the number of runx1 mutations, loss of the wild-type allele and the number and kind of additional mutations impact on prognosis a. stengel, w. kern, m. meggendorfer, k. perglerovà, t. haferlach, c. haferlach mll munich leukemia laboratory, munich, germany, 2 mll2, praha, czech republic aml with mutated runx1 show a distinct pattern of cytogenetic and molecular genetic abnormalities and an adverse prognosis. we analyzed the impact of multiple runx1 mutations and runx1 wild-type (wt) loss on associated genetic alterations and survival. for this, 467 aml cases with runx1 mutations (mut) were split in (1) runx1 wt loss (n = 53), (2) >1 runx1mut (n = 94) and 1 runx1mut (n = 323). 163 cases were selected for mutation analyses of 28 genes. in cases with 1 runx1mut, +8 was frequently found, whereas in wt loss +13 was the most abundant trisomy (+8: 66% in 1 runx1mut vs. 31% in wt loss, p = 0.022; +13: 15% vs. 62%, p < 0.001). cases with >1 runx1mut showed an intermediate distribution (+8: 44%, +13: 50%). missense mutations were the most abundant mutation type in wt loss cases (53% vs. 31%, p = 0.006), whereas in 1 runx1mut, frameshift mutations were found more frequently (45% vs. 28%, p = 0.016). in cases with >1 runx1mut, both were observed at similar frequencies (missense: 36%, frameshift: 38%). mutation analyses of 163 selected cases revealed 411 additional molecular mutations. 95% of cases showed at least one runx1-accompying mutation (range: 0-6). the median of accompanying mutations was n = 2 in the total cohort and in cases with 1 runx-1mut and >1 runx1mut, whereas it was n = 3 in runx1 wt loss. srsf2 (39%), asxl1 (36%), dnmt3a (19%), idh2 (17%), sf3b1 (17%), tet2 (17%) and bcor (16%) were revealed as most frequently mutated genes. cases with runx1 wt loss showed a higher frequency of asxl1mut compared to the other cases (50% vs. 29%, p = 0.009), while u2af1mut were absent from this group (0% vs. 10%, p = 0.019). median overall survival (os) in the total cohort was 14 months. wt loss (os: 5 months) and >1 runx1mut (14 months) showed an adverse impact on prognosis compared to 1 runx1mut (22 months; p = 0.002 and p = 0.048, respectively). mutations in asxl1 and kras and the presence of ≥2 additional mutations also negatively impacted os (10 vs. 18 months, p = 0.028; 1 vs. 15 months, p < 0.001; 12 vs. 20 months, p = 0.017). in univariate cox regression analysis runx1 wt loss (hr = 1.6; p = 0.024), ≥2 additional mutations (hr = 1.9; p = 0.019), asxl1mut (hr = 1.6; p = 0.030) and kr-asmut (hr = 4.4; p = 0.001) had an adverse impact on os. multivariate cox regression analysis revealed an independent adverse effect on os for runx1 wt loss (hr = 1.6; p = 0.039) and krasmut (hr = 4.2; p = 0.001). for 216/467 cases we received samples during course of the disease. in none of these cases, an evidence for a runx1 germline mutation was found by analyzing the mutation loads, thus all runx1 mutations are somatically acquired. taken together, we found strong differences between the subgroups in regard of cytogenetic and molecular genetic aberrations as well as regarding prognosis. thus, not only the presence and number of runx1 mutations but also the conservation of an intact runx1 allele as well as the number and kind of additional mutations is biologically and clinically relevant. abstracts different chromatin states, where methylation is inversely correlated with active histone marks. using the hardy-weinberg law, we estimate that there are 692 dmrs with a maf>0.05. we hypothesized that cis-acting dna polymorphisms could be responsible for the inter-individual variation of the dmrs methylation levels. we genotyped 2.5 million snps in the five donors and found that 82/157 (52%) dmrs have methylation levels highly correlated (>0.9) with the genotype of at least one nearby snp (± 6 kb window). this correlation was verified in 6/7 dmrs by targeted bisulfite sequencing in monocytes from 4 individuals used for wgbs and from 2 additional individuals. to validate our results in a larger population and possibly find correlating snps outside the ± 6 kb window for the remaining dmrs, we performed genome-wide association studies (gwas) using snp genotypes and illumina 450k cpg methylation data from blood samples of 1131 individuals from the heinz nixdorf recall study. these methylation arrays encompass only 51 cpgs contained in 30 of our dmrs, showing that they fail to identify a great number of potentially important regions. we certified that for these 51 cpgs, monocyte and whole blood dmrs methylation levels were correlated, and performed a gwas with ~500,000 snp for each of the 51 cpgs. for 48/51 cpgs, the correlation peak was near the cpg position. for each gwas, the snp with lowest p-value (in most cases p < 1e -200 ) was designated as lead-snp. snps in high linkage disequilibrium (r 2 > 0.8) to the lead-snps were located within the corresponding dmr or 16 bp to ~116 kb from it. many regions are bound by ctcf and other transcription factors. it is likely that snps affect the binding of these factors and thus the methylation state of the region. we conclude that these inter-individual differences in dna methylation are mainly driven by genetic factors. the dystonia 6 (dyt6) protein thap1 recruits the histone deacetylase hdac3 to mediate gene repression sektion für funktionelle genetik am institut für humangenetik, universität zu lübeck, lübeck, germany, 2 institut für neurogenetik, universität zu lübeck, lübeck, germany dystonia describes a heterogeneous group of neurological movement disorders characterized by contractions in various muscles resulting in abnormal postures, involuntary twisting and repetitive movements. dystonia 6 (dyt6), a primary torsions dystonia that first has an impact on cranio-cervical muscles causing problems with speaking and eating, is caused by mutations in the thap1 gene (thanatos-associated domain-containing apoptosis-associated protein 1). thap1 belongs to the family of thap proteins that are characterized by the presence of an evolutionarily conserved specific dna-binding thap zinc finger motif at their n-terminus. in humans 12 thap family members are known, designated thap0 to thap11. interestingly, most of the dyt6-causing mutations affect this thap domain. while we have previously described thap1-mediated repression of specific target genes, the molecular mechanisms how thap1 regulates promoter activity are rather unknown. it is known, that other members of the thap family such as thap7 and thap11 interact with the histone deacetylase hdac3 to mediate transcriptional repression. we have performed yeast-two-hybrid and gst pulldown assays to identify a specific interaction of thap1 with hdac3. by the use of truncated thap1 fragments we were able to narrow down hdac3 binding to the n-terminal thap-domain. for further functional characterization we have decreased hdac3 levels by sirna treatment or chemical inhibition and used taqman analyses to quantify the effect on thap1-target genes expression. thus, a significant increase of thap1-target genes expression was detected in those cells treated with hdac3 sirna. to further investigate whether the observed increase in gene expression is due to alterations of histone acetylation within the promoter regions we performed chromatin immunoprecipitation (chip) assays followed by qpcr using antibodies specific for different acetylated n-terminal residues of histone 3 as markers for transcriptional active promoters. by this, we detected an increased acetylation within the promoter regions of thap1 target genes that are dysregulated in cells treated with decreased hdac3 levels. et-induced obesity indicate that epigenetic changes associated with adverse parental and/or intrauterine factors may contribute to the missing heritability of metabolic disorders. possible adverse paternal effects are likely transmitted by the sperm to the next generation. to prove this hypothesis, we have systematically analyzed the effects of paternal obesity on the sperm epigenome and its implications for the next generation. results: to study the possible transmission of paternal bmi effects to the next generation, methylation levels of eight paternally expressed imprinted genes (peg1, peg3, peg4, peg5, peg9, peg10, nespas and igf2), two maternally expressed imprinted genes (meg3 and h19), and the obesity related gene hif3a were quantified by bisulphite pyrosequencing in sperm of 109 donors (undergoing ivf/icsi) and 121 fetal cord blood (fcb) of resulting offspring (conceived by ivf/icsi with the same sperm samples). hif3a showed a significant positive correlation between sperm methylation and paternal bmi. this effect on the sperm epigenome was replicated in an independent cohort of 188 sperm samples. for hif3a, paternal bmi also showed a significant positive correlation with fcb methylation. on the other hand, peg5/nnat exhibited a significant negative correlation between paternal bmi and fcb methylation. in contrast to pyrosequencing, deep bisulphite sequencing (dbs) allows one to study dna methylation at the single molecule level and enables us to distinguish between maternal and paternal alleles in fcb samples with an informative snp. epimutations which are defined as alleles showing >50% aberrantly (de)methylated cpg sites can also be identified with dbs. upon performing dbs on sperm samples, we observed a higher epimutation rate in the high bmi (28-40) group when compared to the low bmi (19-24) group across the four studied genes (peg1, hif3a, h19 and nespas). we are presently analyzing dbs data in selected cord blood samples with an informative snp to separately quantify methylation at the paternal and maternal alleles. it is important to decipher the methylation of the paternal allele when studying whether sperm methylation alterations are transmitted to the offspring. conclusions: our results suggest that male obesity is associated with modification of the sperm dna methylome, which may affect the epigenome (in fetal cord blood) of the next generation. allele-specific dna methylation occurs at functionally different regions: 1) at imprinting control elements, 2) on the silent x chromosome in females and 3) across the genome and probably dependent on the dna sequence in cis. the latter is termed haplotype-dependent allele-specific methylation and may contribute to inter-individual phenotypic variation. in a previous study on monocyte to macrophage differentiation, we showed that dna methylation differences between individuals were greater than between the two cell types. to study the genetic basis of these inter-individual differences in dna methylation, we analysed the methylome obtained by whole genome bisulfite sequencing (wgbs) of monocytes from five unrelated donors. for identifying differentially methylated regions (dmrs), we created two synthetic methylomes: one with the highest methylation values of each cpg in the five samples and one with the lowest methylation values. defining a dmr as a region of at least 4 cpgs with a methylation level difference of at least 0.8, we identified 157 dmrs, which cover 1165 cpgs and fall into cer, respectively. we show that ns-associated rit1 mutants intensified signal flux through the mek-erk pathway upon growth factor stimulation. by using heterologous expression systems, we identified the p21-activated kinase 1 (pak1) as novel effector of rit1. we found that rit1 interacts with the rho gtpases cdc42 and rac1, both of which are crucial upstream regulators of pak1. disease-causing rit1 mutations enhance protein-protein interactions and uncouple complex formation from growth factors. expression of both wild-type rit1 and its mutant forms resulted in dissolution of stress fibers and paxillin-containing focal adhesions from the cell center and increased cell movement. we conclude that rit1 is a potent regulator of actin dynamics, and dysregulated rac1/cdc42-pak1 signaling controlling cell adhesion and migration may be one aspect of the molecular basis of ns. medical systems biology, tu dresden, germany, 2 institute for clinical genetics, tu dresden, germany, 3 cancer science institute of singapore, national university of singapore, singapore, 4 institute of molecular biology, mainz, germany telomeres are short repetitive ttaggg sequences that cap the ends of chromosomes. these stretches of dna are covered by proteins and rnas which together protect the putative double strand break from dna repair mechanisms and facilitate replication. however, telomeres shorten with every cell division due to the end replication problem. the ribonucleoprotein telomerase counteracts this process by de novo elongation of telomeric repeats but its expression is mostly confined to the germ line and stem cells. even in the latter its activity is usually not sufficient to completely prevent telomere shortening. all cancer cells are also faced with this challenge and while the majority of cancer cells rely on telomerase, approximately 15% of cancers ensure sufficient telomere length via the recombination-based alternative lengthening of telomeres (alt) mechanism. to better understand telomere biology we aimed to identify novel telomeric factors by systematically screening for telomere-binding proteins in cell lines from 16 different vertebrates. here, we identified and characterized zbtb48, a zinc finger protein, as a novel direct telomere-binding protein across the vertebrate lineage. zbtb48 is directly binding to telomeric dna in vitro and it is localizing to telomeres in vivo via one specific zinc finger domain in both telomerase-and alt-positive cancer cells. interestingly, zbtb48 knock-out cells have longer telomeres, suggesting that zbtb48 limits telomere elongation. in addition, the combination of chipseq, rnaseq and proteome analysis revealed a transcription factor activity for a small, but specific set of target genes of zbtb48, linking its telomeric functions to mitochondrial metabolism. in conclusion, zbtb48 is a novel direct telomere binding protein with transcription factor activity that acts as negative regulator of telomere length. our data show for the first time a functional interaction of the 'dystonia 6 protein' thap1 with the histone deacetylase hdac3 and therefore give new insights into the molecular mechanisms of thap1-mediated gene repression. interestingly, previous functional studies as well as structure analyses revealed that only a subset of the dyt6-causing mutations affecting the n-terminal thap domain alter thap1-binding to dna. in ongoing studies we want to investigate the consequences of dyt6-causing mutations on thap1-hdac3 complex formation and its relevance in the molecular pathology of dystonia. reproductive homeobox (rhox) genes are clustered on the x chromosome and share a unique 60 amino acid helix-turn-helix dna binding homeodomain. they were identified in several species as having important roles in reproductive tissues, notably in the testis. the human rhox cluster is composed of three genes: rhoxf1 and two copies of rhoxf2 (rhoxf2a, rhoxf2b) which are referred to as rhoxf2/2b. rhox proteins are expressed exclusively by germ cells in human testis and aberrant rhox methylation is associated with several sperm parameters. because little is known about the molecular mechanism of rhox function in humans, the aim of the study was to identify target genes of human rhox proteins and to investigate the impact of rhox mutations on protein function. using gene expression profiling, we identified genes regulated by members of the human rhox gene cluster. some genes were uniquely regulated by rhoxf1 or rhoxf2/2b, while others were regulated by both of these transcription factors. several of these regulated genes encode proteins involved in processes relevant to spermatogenesis, e. g. stress protection and cell survival. one of the target genes of rhoxf2/2b is rhoxf1, suggesting cross-regulation to enhance transcriptional responses. the potential role of rhox in human infertility was addressed by sequencing rhox in a group of 250 patients with severe oligozoospermia. this revealed two mutations in rhoxf1 (c.515g>a and c.522c>t) and four in rhoxf2/2b (-73c>g, c.202g>a, c.411c>t and c.679g>a), of which only one (c.202g>a) was found in a control group of men with normal sperm concentration. functional analysis demonstrated that c.202g>a and c.679g>a significantly impaired the ability of rhoxf2/2b to regulate downstream genes. molecular modelling suggested that these mutations alter rhoxf2/f2b protein conformation. by combining clinical data with in vitro functional analysis, we demonstrate how the x-linked rhox gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility. colorectal cancer (crc). here, we proposed the possible molecular mechanisms responsible for crc initiation, progression and invasion using a network biology approach. materials and methods: in order to investigate the underlying crc pathogenesis, the dataset gse21510 consisting of normal tissues, stage i, stage ii, stage iii and stage iv of crc were obtained from gene expression omnibus (geo) and further examined. the differentially expressed genes (degs) were subjected to protein-protein interaction databases and a ppi network was constructed for each crc stage. topological analysis of resulted ppi networks revealed functional hub genes and involved in crc development. furthermore, the overlap genes between four studied crc stages were determined and deeply evaluated to identify deregulat ed biological networks during crc development. a standard real-time pcr was performed to validate the in silico findings utilizing sw620 and ncm460 cell lines. results: the most important hub genes (cdk1 for stage i, ubc for stage ii, esr1 for stage iii and atxn1 for stage iv) and sub-networks were identified in crc stages. moreover, several novel biomarkers were also introduced for each crc stage. gene ontology (go) and signaling pathway enrichment uncovered the important roles of wnt, mapk and jak-stat signaling pathways in regulation of crc pathogenesis. functional annotation of overlap genes revealed that cell cycle regulating genes are the most highly regulated genes during crc initiation, progression and invasion. in vitro analyses confirmed deregulation of atxn1 and cdk1, two hub genes of stage iv, in metastatic colon sw620 cells compared to normal colon ncm460 cell line. our study provides a new insight into the distinct molecular mechanisms underlying the pathogenesis of crc. the functional hub genes, sub-networks, prioritizes key pathways and novel crc biomarkers were also provided that can be useful in therapeutic programs. targeted next-generation sequencing approaches as well as next-generation whole exome sequencing are becoming more widespread in routine molecular diagnostics for patients with ataxia. however, since ngs at present is not suitable to detect (trinucleotide) repeat expansions, a pre-ngs testing for common polyglutamine expansion scas seems mandatory. but also sca subtypes caused by expansions in non-coding regions of genes like sca8, sca10, sca12, and sca36 as well as other ataxias known to be associated with repeat expansions like the fragile x-associated tremor ataxia syndrome (fxtas) should be taken into account before applying ngs-based diagnostics. in order to find an optimal diagnostic strategy in future more information about the frequency and phenotypic characteristics of rare repeat expansion disorders associated with ataxia would be helpful. we therefore analyzed a cohort of 441 patients with symptoms of cerebellar ataxia, dysarthria and other unspecific symptoms who were referred to our center for sca diagnostics and showed alleles in the normal range for the most common sca subtypes sca1-3, sca6, sca7, and sca17. these patients were screened for expansions in sca8, sca10, sca12, sca36 and fxtas as well as for the pathogenic hexanucleotide repeat in the c9orf72 gene. no expanded repeats for sca10, sca12 or sca36 were found in the analyzed patients. five patients with ataxia of unknown etiology showed sca8 cta/ctg combined alleles (83-129) that are discussed to be potentially pathogenic. one 51-year-old male patient with unclear dementia syndromes was diagnosed with a large ggggcc repeat expansion in c9orf72. and the analysis of the fmr1 gene identified one patient with a permutation (>50 cgg repeats) and seven patients poster *** = für den posterpreis nominiert preventive genetic counseling in neurogenetic disorders needs a better collaborative approach between genetic and neurology clinics -a report of four siblings with unverricht-lundborg disease: genetic counseling is the process of helping people to understand and adapt the medical, psychological and familial implications of genetic contributions to disease. for parents with a previous child or other family member with a known genetic syndrome expands options for preimplantation or prenatal diagnosis for the current or the future pregnancies. however, timely referral by health providers to genetic counselor and for discussing with couples regarding possible options is important. additionally, other factors such as personal decision making especially due to high price of some genetic services and uncertain results cause considerably delays to genetic testing. there are more than 200 various types of inherited neurological disorders in which alterations in genes lead to an inherited condition such as huntington disease, inherited forms of alzheimer disease, ataxia, muscular dystrophies and epilepsies. the knowledge of the causative gene mutations in the affected individual is critical in the possible prenatal diagnosis in other members of the pedigree. therefore a multidisciplinary care team, including neurologist and genetic counselor for the conditions diagnosed as inherited neurological disorders is critical in prenatal setting and consideration of an effective management. here, our report of four siblings affected by a rare form of inherited epilepsy (unverricht-lundborg disease) with an autosomal recessive pattern highlights the importance of the needs for a better collaborative approach in the neurogenetic setting. in fact, the birth of four successive siblings affected by similar neurogenetics disorders in a specific family is showing the need for more attention to this important issue, especially in terms of intersectoral collaboration. poorebrahim 6 hort: 6/10 differentially expressed transcription units). these differences in gene expression we detected did not correlate with dna methylation changes at the corresponding transcription regulatory sites. from our results we conclude that altered expression of imprinted genes indeed plays a role in tumorigenesis of germinal center derived b-cell lymphomas. however, the altered transcriptional regulation of these genes seems not to rely on the usual epigenetic mechanisms known from constitutional imprinting disorders. mf. abazari 1 , h. bokharaie 2 , m. asghari 3 , v. poortahmasebi 4 , h. askari 5 , m. investigating the expression of genes associated with autism spectrum disorders to identify sex related differences s. berkel, a. eltokhi, g. rappold institute of human genetics, heidelberg university hospital, heidelberg, germany neurodevelopmental disorders such as autism, attention deficit and hyperactivity syndrome as well as language problems and learning difficulties have a higher prevalence in male individuals compared to females. autism is characterized by impairments in social interaction, communication deficits and restricted and repetitive behaviors. boys are more frequently affected than girls; the ratio of affected boys compared to girls is 4:1 for autism and 11:1 for asperger syndrome. in this study we aim to elucidate the reason for this gender difference by following up two hypotheses: (1) risk genes for autism spectrum disorders (asd) might be expressed at different levels in males and females and (2) asd risk genes might interact with sexually dimorphic pathways. first, we investigated the expression of genes associated with autism spectrum disorders, including the shank gene family, in the brain of male and female mice to identify sex-dependent differences. the rna expression levels were analyzed in five different brain regions (cortex, hippocampus, striatum, cerebellum, thalamus) at different developmental stages (e15, e17, p1, p7, p12 and adult) in male and female mice. we identified a sex dimorphic expression of shank1 and shank3, but not of shank2. due to the fact that early brain development is strongly influenced by sex hormones (estrogen, testosterone), we further investigated the influence of these hormones on shank expression in human neuroblastoma cells (sh-sy5y) and primary mouse hippocampal neurons. a better understanding of the sex differences in the brain might help to explain the vulnerability for neuropsychiatric disorders like autism and paves the way to discover putative risk or protective factors for these disorders. imprinting defects in temple syndrome are caused by a failure in imprint establishment and/or maintenance j. beygo, c. mertel, g. gillessen-kaesbach, b. horsthemke, k. buiting institut für humangenetik, universitätsklinikum essen, universität duisburg-essen, essen, germany, 2 institut für humangenetik, universität zu lübeck, lübeck, germany temple syndrome (ts14) is a rare imprinting disorder characterised by low birth weight and height, muscular hypotonia and feeding difficulties in the infant period, early puberty and short stature with small hands and feet and often truncal obesity. in a subset of patients with ts14, the disease is caused by an imprinting defect (id) affecting the paternal allele of the imprinted region 14q32. the id results in aberrant methylation of the three known differentially methylated regions (dmrs), the germline-derived primary dlk1/meg3 intergenic (ig-)dmr (meg3/dlk1:ig-dmr), the postfertilization-derived, secondary dmr at the meg3 promoter (meg3:tss-dmr), and the postfertilization-derived, secondary intragenic meg8-dmr (meg8:int2-dmr). the meg3/dlk1:ig-dmr and the meg3:tss-dmr are methylated on the paternal chromosome and hypomethylated in patients with ts14 and an imprinting defect. the meg8:int2-dmr is unmethylated on the paternal chromosome and hypermethylated in these patients. both the meg3/dlk1:ig-dmr and the with alleles in the grey zone (41 to 54 cgg repeats), thus suggesting that individuals with fmr1 repeat expansions in the gray zone may also present with neurological signs. bernhart 3, 4, 5 , h. kretzmer 3, 4 , r. wagener 1 , c. mmml 6 some genes are subject to the mechanism of imprinting, i. e. their expression depends on parental origin. they primarily function in the control of proliferation, fetal development and cellular differentiation. constitutional imprinting disorders are in part also associated with an increased tumor risk. loss of imprinting has been also described as somatic event in tumorigenesis. while this phenomenon has been broadly analyzed in solid tumors, data on alterations of imprinting in lymphatic neoplasms are largely missing. we analyzed the rna expression of 321 transcription units/regions known or supposed to be subject to imprinting in two cohorts of normal b-cells and germinal center derived b-cell lymphomas. the first cohort (mmml) contains 686 samples: 56 burkitt lymphomas (bl), 600 non-burkitt lymphomas (non-bl, including various subtypes like follicular and diffuse large b-cell lymphoma) and 30 normal germinal center b-cell samples (gcbc, as controls). the second cohort (icgc mmml-seq) comprised 201 samples with 20 bl, 176 non-bl and 5 gcbc samples. gene expression was analyzed with affymetrix u133a genechips in the mmml cohort and by rna sequencing in the icgc mmml-seq cohort. results of the transcriptional analyses in the icgc mmml-seq cohort were compared to the dna methylation available from a subset of the analyzed samples (kretzmer et al., nat genet, 2015) . of the 321 transcription units 114 sites, corresponding to 64 transcription units, were present on the applied array used for the analysis of the mmml cohort. a two group comparison revealed 53 significantly differentially expressed sites corresponding to 31 transcription units between bl and non-bl including the plagl1 and peg10 genes. in total, 19 and 16 sites corresponding to 16 and 10 transcription units are differentially expressed between bl versus gcbc and non-bl versus gcbc, respectively. comparison of gene expression in the icgc cohort revealed 70 differentially expressed sites corresponding to 68 transcription units between bl and non-bl (overlap with mmml cohort: 24/31 differentially expressed transcription units), including again peg10 and plagl1, 37 differentially expressed sites corresponding to 37 transcription units between bl and gcbc (overlap with mmml cohort: 7/16 differentially expressed transcription units) and 44 differentially expressed sites corresponding to 39 transcription units between non-bl and gcbc (overlap with mmml co-abstracts maintenance dna methylation of l1 promoters, spoc1 could function in targeting g9a to l1 sequences. in conclusion our data implicate the epigenetic reader spoc1 in the suppression of line elements during germ cell development. s. bens 1 , j. kolarova 1 , m. kreuz 2 , sh. characterization of the expression of the imprinted kcnk9-gene in specific brain regions and the phenotypic analysis of kcnk9knockout mice a kcnk9/kcnk9 is a maternally expressed imprinted gene whose mutations are responsible for the maternally inherited birk-barel mental retardation dysmorphism syndrome. it encodes a member of the superfamily of k+channels with two pore-forming domains and is involved in the modulation of the resting membrane potential and excitability of neuronal cells. so far, only homozygous kcnk9 knockout mice with inactivation of both parental alleles were phenotypically characterized. these mice displayed cognitive deficits as well as a reduction of k+ leak current by 50%. in the light of maternal-specific imprinted expression of kcnk9/kcnk9 and the maternal inheritance of the birk-barel mental retardation dysmorphism, a thorough phenotypic analysis of heterozygous kcnk9 knockout mice with inactivation of only the maternally inherited allele is also warranted. as first aim of our study, we characterized the parental allele-specific expression of kcnk9 in various regions of the mouse brain. quantification of allele-specific expression by pyrosequencing (quasep) method was performed for different brain areas from several developmental stages of (c57bl/6xcast/ei) f1 hybrid mice. exclusive expression from the maternal kcnk9 allele was detected in the dentate gyrus, hippocampus, mesencephalon, medulla oblongata, thalamus and pons. biallelic expression with, however, a strong bias towards the maternal kcnk9 allele (94-99% of the transcripts) was observed in the olfactory bulbs, cortex, cerebellum, striatum and olfactory tubercles. as the second aim of our study, the phenotypes of wildtype, heterozygous kcnk9 knockout mice with maternal inherited knockout allele (kcnk9komat) and homozygous kcnk9 knockout mice (kcnk9kohom) were comparatively examined in a behavioral test battery. due to the already known cognitive defects of kcnk9kohom animals and especially the phenotype of the patients with birk-barel syndrome, it was assumed that kcnk9komat and kcnk9kohom animals show deficits in some of the tests. the spontaneous alternation in the y-maze test was significantly reduced by approximately 10-20% in kcnk9komat and kcnk9kohom mice compared to wildtype mice indicating a clearly impaired working memory. in addition, kcnk9komat and kcnk9kohom mice displayed a reduced prepulse inhibition of startle response compared to wildtype mice indicating an impairment of sensomotoric gating, a process to filter out irrelevant information. acoustic startle response as a measure of anxiety levels was also significantly decreased, but only in kcnk9kohom mice. our findings shall further elucidate the role of kcnk9/kcnk9 in brain physiology and pathophysiology and open new avenues for treatment of cognitive dysfunctions in birk-barel syndrome. meg3:tss-dmr act as imprinting control centres, although the meg3/ dlk1:ig-dmr functions as an upstream regulator of the meg3-dmr. so far, the function and regulation of the meg8-dmr is unknown. the hypomethylation of the paternal allele in ts14-id patients at the meg3/ dlk1:ig-dmr and the meg3:tss-dmr point to a failure in the establishment of the methylation imprint or to maintain the methylation imprint after fertilization. in this case, the incorrectly imprinted chromosome 14 would be inherited from either the paternal grandfather or grandmother. to prove this assumption we are investigating the grandparental origin of the affected chromosome 14 in our cohort of ten ts14-id families by studying the parent-of-origin specific methylation of the three dmrs in combination with informative single nucleotide variants (snps). at the moment we have identified three families informative for the meg3/ dlk1:ig-dmr, two families for the meg3:tss-dmr and two families for the meg8:int2-dmr. so far we have obtained results in two families for the meg3:tss-dmr. we found that in one case the allele harbouring the id was inherited from the paternal grandmother, but in the second case from the paternal grandfather, indicating that the id occurred after erasure of the parental methylation imprints. a complete lack of methylation observed in the majority of ts14-id patients is therefore likely due to a problem in establishing methylation on the paternal chromosome, whereas in rare cases with methylation mosaicism, the id is probably due to a problem to maintain the paternal imprint after fertilization. bosch, s. lukassen, j. kaindl, j. schwarz, c. nelkenbrecher, a. herrmann, a. reichel, a. ekici, t. gramberg, t. stamminger, a. winterpacht humangenetisches institut, erlangen, germany, 2 virologisches institut, erlangen, germany spoc1/phf13 is a gene located on human chromosome region 1p36.31 and mouse chromosome 4qe2. the protein was first described in patients with epithelial ovarian cancer, where its expression correlated with tumour progression and reduced survival time. spoc1 is a reader of the epigenetic mark h3k4me2/3, dynamically associates with chromatin during mitosis and plays a role in chromosome condensation. spoc1 deficient mice show a pronounced hypoplasia of the testis with a progressive loss of germ cells. although loss of spoc1 leads to a significantly reduced chromatin condensation of the sex chromosomes in meiosis, the protein is not expressed in spermatocytes but in the undifferentiated precursor cells, the spermatogonial stem cells (sscs). here, we present chip-seq data of mouse testis tissue demonstrating that spoc1 strongly binds to evolutionary young l1 elements in undifferentiated spermatogonia. we show that in hek cells overexpression of spoc1 leads to repression of transposition activity of line-elements strongly indicating a role of spoc1 in l1 element suppression. the cell has developed several lines of defence against retrotransposition to maintain genomic integrity, including dna methylation. these defence mechanisms are most elaborate in spermatogonial stem cells since transposition events in these cells would have a dramatic impact on the next generation. therefore, the repression of retrotransposition is of fundamental importance for germ cell development and ultimately the quality of the gametes. moreover, we present medip results showing that the methylation levels of l1 sequences decrease upon spoc1-knockout and demonstrate that the histone methyltransferase g9a is strongly upregulated in preleptotene meiocytes of spoc1 -/mice. g9a is expressed from spermatogonia until early meiosis where it regulates h3k9 di-methylation and has been shown to be involved in the repression of l1 element in mouse spermatogonia. we are able to demonstrate that h3k9me2 levels are unaltered in spoc1 -/mice, suggesting a potential functional link between g9a and spoc1 that does not affect the catalytic activity of g9a. since g9a can regulate de novo and medizinische genetik 1 · 2017 121 lated to investigate ciliogenesis. data resulting from rnaseq experiments are analyzed by established informatics tools (tophat, cufflinks and derivatives thereof). we will show results from our work in progress and we hope to convince people to intensify rna analyses even in routine labs to uncover hidden mechanism and/or mutations impacting mrna splicing and thereby causing human disease. telomeres are located at the ends of chromosomes and have an essential role in the maintenance of genome stability. after each cell division, a small part of this specialized sequence is lost. when telomeres reach a critically reduced length, the cell either dies through apoptosis or enters a state of permanent cell cycle arrest. it has been demonstrated that telomere biology is directly linked to basic biological phenomena such as aging, tumorigenesis and maintenance of dna integrity. it is known that oxidative stress accelerates telomere shortening in cells, resulting in premature cell senescence. shorter leukocyte telomeres have been observed in type ii diabetes or degenerative disease like dementia and alzheimer disease as well in chromosomal instability syndrome, such as fanconi anemia (fa) and nijmegen breakage syndrome (nbs). any link between telomere length and inflammation has not yet been extensively studied in autoimmune diseases. accelerated length shortening might be related to autoimmune disease predisposition. yet the reasons for this shortening are likely manifold, including the individual genetic background, oxidative stress and chronic inflammation. in order to shed light on these relationships, we investigate genomic dna extracted from blood of patients diagnosed with multiple sclerosis and from patients with huntington disease. the samples were divided into age groups. stepone q-pcr was applied to detect the relative telomere length as a function of age. initially identified differences in telomere lengths still have to be confirmed in larger cohorts. background: intrauterine exposure to gestational diabetes mellitus (gdm) confers a lifelong increased risk for metabolic and other complex disorders to the offspring. gdm-induced epigenetic modifications modulating gene regulation and persisting into later life are generally assumed to mediate these increased disease risks. to identify candidate genes for fetal programming, we compared genome-wide methylation patterns of fetal cord bloods (fcbs) from gdm and control pregnancies. methods and results: using illumina's 450k methylation arrays and following correction for multiple testing, 65 cpg sites (52 of which are associated with genes) displayed significant methylation differences between gdm and control samples. three of four candidate genes, atp5a1, prkch, and slc17a4, from our methylation screen and one, hif3a, from the literature were validated by bisulfite pyrosequencing. the gdm effect on fcb methylation was more pronounced in women with insulin-dependent gdm who had a more severe metabolic phenotype than women with dietetically treated gdm. however, the effect remained significant after adjustment for the maternal bmi and gestational week in a multivariate regression model. e. g. dekomien 1, 2 , b. bellenberg 2, 3 , n. trampe 2, 4 , r. schneider 2, 4 , c. prehn 2, 4 , c. krogias 2, 4 , r. kropatsch 1, 2 , m. regensburger 5, 6 , c. lukas 2, 3 , r. gold 2, 4 , 4 1 human genetics, bochum, germany, 2 ruhr-university bochum, germany, 3 radiology, st. josef-hospital, bochum, germany, 4 neurology, st. josef-hospital, bochum, germany, 5 molecular neurology, erlangen, germany, 6 university of erlangen, germany a pair of monozygotic 22-year-old twins suffering from hereditary spastic paraplegia 11 (spg11) is described. patients underwent thorough clinical examination and magnetic resonance imaging (mri) and mr-spectroscopy (mrs) at 3 tesla. genetic testing was performed by sanger sequencing and alternative splicing by rna analysis. clinically the patients presented a similar spectrum of symptoms with a higher level of disability in one of the patients. mri studies including morphometry and regional microstructural analysis by diffusion tensor imaging (dti) of the corpus callosum (cc) revealed marked thinning and corresponding increases of axial diffusivity (ad), radial diffusivity (rd) and apparent diffusion coefficient (adc) and reduction of the fractional anisotropy (fa) as compared to healthy controls in all cc sections, particularly in the anterior callosal body. there was marked supratentorial white matter reduction and to a lesser extent grey matter reduction in both patients. involvement of the cortico-spinal tracts was reflected by fa and rd alterations and cervical cord atrophy. the more strongly affected patient showed a higher degree of callosal microstructural damage and cervical cord atrophy. genetic testing of the spg11 gene revealed two mutations in compound heterozygous state, a known frameshift mutation as well as a novel synonymous exonic splice site mutation. this study shows similar but distinct clinical and imaging findings in monozygotic twins suffering from spg11, suggesting individual downstream genetic effects. targeted next generation sequencing techniques tremendously improved our ability to identify sequence variants. however fixing disease causing mutation still lack behind because of several reasons: inappropriate gene specific data bank, insufficient prediction tools, incomplete analysis and others. in addition identified sequence variants are a mixture of severe disease causing mutations and a myriad of variants of unknown pathogenicity. in addition an unknown number of silent mutations, neutral polymorphism and sequence variants deeply buried in introns might severely influence splicing of the premature rna molecule. by solely analysis of the dna sequence this impact onto the integrity of the mrna is completely ignored. in order to catalog the mrna isoforms derived from genes of our interest we started to set up rnaseq technologies in our routine lab. to reduce the amount of data, to improve the power of analyses and to identify rare isoforms of transcripts we use targeted rnaseq to characterize the mrna molecules originating from those genes we are interested in (e. g.: hereditary breast cancer core genes (10 genes), hereditary colon cancer (23 genes), primary ciliary dyskinesia (pcd)(40 genes). genes involved in pcd offer the invaluable advantage that the respiratory epithelium where these genes are normally expressed can be sampled from the inferior turbinate of the nose by brush biopsy either from healthy probands or from patients suffering from pcd. in addition to direct preparation of rna from these cilia, cilia carrying cells or tissue can be cultured and manipu-abstracts long-range pcr and direct sequence analysis. the comparative analysis of parental haplotypes with the sequences flanking the deletion breakpoints in the patients revealed the absence of any de novo mutations in breakpoint-flanking regions of 19 prs2-mediated and 6 prs1-mediated type-1 nf1 deletions. we conclude that although nahr is a mutational mechanism causing large nf1 deletions, there is no evidence for a local mutagenic effect of these recombination events. hence it is unlikely that nahr underlying type-1 nf1 deletions involves error-prone translesion polymerases that would increase the de novo mutation rate in breakpoint flanking regions. furthermore, the detailed haplotype analysis of prs2, a highly active nahr hotspot mediating the majority of large nf1 deletions, revealed that non-allelic homologous gene conversion (nahgc) between nf1-repa and nf1-repc, which results from non-crossover resolution of recombination intermediates, is the major driving force responsible for the haplotype diversity in this region. remarkably, the haplotype diversity patterns observed for nf1-repa and nf1-repc were markedly different indicating that during nahgc, nf1-repa is disporportionately more often the donor sequence used to repair mismachtes in heteroduplex regions than nf1-repc. we also noticed a correlation between haplotype diversity and the number of prdm9 a-allele binding sites suggesting that haplotype diversity and hence the nahgc rate within prs2 in nf-repa is regulated by prdm9. heidelberg center for personalized oncology dkfz-hipo dkfz, heidelberg, germany dna methylation aberrations at differentially methylated region of imprinted genes interfere with the naturally parental-specific mono-allelic expression. that leads to a bi-allelic or absent expression of the imprinted gene, a cause of imprinting disorders (ids). we aimed at analyzing the genome wide dna methylation pattern of two patients with ids, namely transient neonatal diabetes mellitus (tndm) and multi locus imprinting disturbance (mlid), and their respective parents. the dna methylation profiles of these individuals were obtained by whole genome bisulfite sequencing (wgbs) on b cells sorted by magnetic cell isolation. the sequencing libraries were prepared as described in kretzmer et al. [1] and sequenced on an illumina hiseq 2000 machine. wgbs data were processed with the methylctools toolkit. briefly, bisulphite-treated sequences were aligned with bwa-mem using a three-letter approach, and the methylation ratios were quantified for ~26.9 million out of 28.2 million cpg sites (coverage>5) genome-wide. quality control was performed to assess the quality of the dna methylation profiles and genetic fingerprinting was performed on the wgbs data confirming sample origin and family relationship. the wgbs data was further compared to already existing genome-wide human snp array 6.0 (snp array) and humanmethylation 450k bead array (450k) data, resulting in a good accordance with pearson's correlation coefficients >0.97. we detected an overall dna methylation level around 75% in all samples. already known dna methylation alterations, e. g. hypomethylation in plagl1 were validated by wgbs. by searching for differentially methylated regions (dmrs), defined as regions composed of at least five consecutive cpg loci showing a methylation difference between patient and corresponding parents above 30%, we identified 442 dmrs in the mlid our study supports an association between maternal gdm and the epigenetic status of the exposed offspring. consistent with a multifactorial disease model, the observed fcb methylation changes are of small effect size but affect multiple genes/loci. the identified genes are primary candidates for transmitting gdm effects to the next generation. they also may provide useful biomarkers for the diagnosis and prognosis of adverse prenatal exposures and assessing the success of interventions during pregnancy. the nuclease hsnm1b/apollo has a dual function in both dna-repair and maintenance of telomeres. as to the repair of dna interstrand crosslinks (icl), hsnm1b/apollo is linked to the fanconi anemia (fa) pathway and cells depleted for hsnm1b/apollo (sirna) resemble those from patients with fa. we have identified a single nucleotide polymorphism, rs6674384, which is associated with quantitative differences in hsnm1b/apollo expression (mrna). we analyze whether the differential expression relates to the degree of cellular sensitivity to the dna interstrand crosslinks inducing mutagen mitomycin c (mmc) and ionising radiation (ir), which induces, among other lesions, dna double strand breaks. all experiments are realized using lymphoblastoid cells derived from generally healthy donors. results of rt-pcr analysis of hsnm1b/apollo expression and of the cell viability assays will be presented and discussed in the context of the potential usefulness of considering rs6674384 in predicting individual sensitivity to mutagens relevant in anti-cancer treatment. p-basepi-014 nahr events causing type-1 nf1 microdeletions are not associated with an increased mutation rate in breakpoint-flanking regions m. hillmer 1,2 , a. summerer 1,2 , v. f. mautner 3, 4 , l. messiaen 5, 6 , h. 2 1 institute of human genetics, ulm, germany, 2 university of ulm, ulm, germany, 3 department of neurology, hamburg, germany, 4 university hospital hamburg eppendorf, hamburg, germany, 5 department of genetics, birmingham, usa, 6 university of alabama at birmingham, birmingham, usa large deletions of the nf1 gene and its flanking regions are the most frequent recurrent mutations in patients with neurofibromatosis type 1 (nf1). different types of large nf1 deletions have been identified which are distinguishable in terms of their size and breakpoint position. most frequent are type-1 nf1 deletions spanning 1.4-mb and characterized by breakpoints located within the low-copy-repeats nf1-repa and nf1-repc which exhibit 97.5% sequence homology within 51-kb. type-1 nf1 deletions are caused by non-allelic homologous recombination (nahr). two nahr hotspots have been identified termed prs1 and prs2 which encompass 5-kb and 4-kb, respectively. approximately 80% of all type-1 nf1 deletion breakpoints cluster within the prs1 and prs2 nahr hotspots. in this study, we analysed whether the nahr events causing type-1 nf1 deletions would be associated with an increased de novo mutation rate of sequences located in breakpoint-flanking regions. to do so, we sequenced the deletion breakpoint-flanking regions in the patients and compared these sequences with the homologous regions amplified from dna of the patients' parents who are not affected by nf1. however, in the germline of these parents, the deletions were mediated by nahr and then transmitted to their offspring. the parental haplotypes within the prs1 or prs2 regions of nf1-repa and nf1-repc were analysed by in the alpl gene and inherited as an autosomal dominant trait can cause milder forms. so far detailed knowledge of the milder forms is lacking. 39 patients with a mutation in the alpl gene were interviewed in a standardized questionaire concerning the different disease manifestations: teeth, bone fractures, pain of bones and muscles and quality of life. subgroups were formed with regard to the localization of the mutations in the three protein domains. patients with mutations clustering in the catalytic site of the molecule showed the most severe odontohypophosphatasia: one individual had premature primary tooth loss, 31% of patients showed adult tooth loss, 77% suffered from dental caries. the majority had the first manifestation before the age of 18. persons suffering from mutations in the two other domains reported a relatively high quality of live with low pain of muscles and bones. unexpectedly in all groups there was no significant difference in the portion of patients with bone fracture. conclusion: the clinical signs of dominant hpp are mostly unspecific. especially dental problems like severe adult teeth loss, an early manifestation of dental caries or enlarged pulp chambers can be a sign of odontohypophosphatasia and a dominant inherited mild hpp. mutations in the catalytic site of the alpl molecule are associated with a more severe odontohypophosphatasia. screening of non-neoplastic lymphatic tissues from children for the igh-myc fusion using a highly sensitive 4-color fish-assay burkitt lymphoma is a mature b-cell lymphoma which on the genetic level is characterized by the burkitt translocation t(8;14)(q24;q32) juxtaposing the igh locus in 14q32 next to the myc locus in 8q24. in a minor part of burkitt lymphomas, immunoglobulin light chain variants of the translocation result in overexpression of myc. despite being pathognomonic for burkitt lymphoma, the ig-myc juxtaposition alone is not sufficient on its own for a malignant transformation of the cell. other igh rearrangements like the igh-bcl2 fusion, typical for follicular lymphoma, were detected in a significant number of healthy individuals. for the igh-myc translocation, only scarce data in healthy individuals exist. this is most likely due to scattering of the breakpoints which are far more difficult to target by pcr than the igh-bcl2 translocation. therefore, we aimed at investigating if myc-translocation positive cells can also be detected in normal b-cell maturation. considering the epidemiology of burkitt lymphoma being the most common b-cell lymphoma in children, we focused on samples from young individuals. on the one hand, we analyzed non-neoplastic tissue specimen of bone marrow (n = 14) (age range 3-18, median age 8.5 years) and lymph nodes (n = 19)(age range 2-18 years, median age 12 years). on the other hand, considering the typical clinical presentation of burkitt lymphoma, we included non-neoplastic tissue specimen containing peyer patches (n = 25)(age range 48hours-20years, median age 17years). the specimen were analyzed using a four-color fluorescence in situ hybridisation (fish) assay with probes flanking the breakpoints on chromosomes 8 and 14. in this setting, a positive result comprised the break on both chromosomes (seen as signal split for each locus) and fusion of the involved genes (leading to two different fusion signals). the assay was first validated on controls of cells with a normal male karyotype from healthy individuals as well as on five burkitt cell lines and each five ffpe embedded t(8;14) negative and positive tissues as negative and positive controls. the assay was then applied for the screening of a igh-myc fusion in the above mentioned paraffin-embedded tissues. successful hybridizations of overall 9, 15 and 17 ffpe sections from bone marrow, lymph nodes and peyer patches respectively could be obtained. trio and 1776 dmrs in the tndm trio. in the mlid trio 238/442 dmrs showed increased and 204 dmrs decreased dna methylation in the patient's sample. of 442 dmrs, 325 are located in regions potentially associated with transcriptional regulation. further analysis revealed that 34/442 dmrs are associated with imprinted genes. in the tndm trio, we detected 1705/1776 dmrs to show hypermethylation in the patient compared to her parents and 71/1776 dmrs with lower dna methylation. in these trio 1166/1776 dmrs are associated with regions potentially correlated to transcriptional regulation and 13 dmrs with imprinted genes summarized, our results show that wgbs is a well suited and valid method for analyzing dna methylation. while the overall dna methylation levels does not differ between the analyzed patients and parents, a detailed analysis of smaller regions revealed the existence of 442 respectively 1776 differentially methylated regions between the analyzed mlid and tndm patient and their parents. supported by bmbf through fkz: 01gm0886 und 01gm1114 and 01gm1513 p-basepi-016 characteristic mutational profile in children of individuals exposed to ionizing radiation p. krawitz, h. manuel, a. knaus, g. hildebrandt, m. jäger, m. schubach, m. rodriguez de los santos, t. pantel, d. beule, s. mundlos, k. sperling institute for medical genetics and human genetics charite, berlin, germany the dna damaging effects of ionizing radiation are deliberately used in cancer therapy as well as feared in accidents related to nuclear technology. despite its influence on the exposed organism, irradiation was believed to have no major effect on succeeding generations, as irreparable dna damages were thought to result in cell death. recently, however, genome-wide mutation screenings in offsprings of male mice that were irradiated with high dosages showed an accumulation for certain de novo events. we therefore focused on these mutational classes in a small cohort of human individuals that were conceived while or after their fathers were exposed to high frequency radiation. in the whole genome sequences of 22 such offsprings we could confirm de novo rates for single nucleotide variants in the order of 10 -8 per base as previously reported. interestingly, however, we found de novo translocations of paternal origin as well as increased numbers of clustered de novo mutations that resemble the results from animal studies. this characteristic mutation profile might thus be used as an indicator of irradiation exposure in one of the individual's parents. from the upstream regular rb1 promoter. to test this hypothesis, we generated a genetic model carrying modifications in the rb1 promoter and in cpg85 using crispr/cas9 technology. data on the establishment of the model and first results will be presented. the gid/ctlh protein complex with its seven core protein members is conserved in all eukaryotic cells. in saccharomyces cerevisiae it functions as an ubiquitin ligase complex and regulates the metabolic switch from gluconeogenesis to glycolysis (1) . recently, we could show that the vertebrate gid/ctlh complex also functions as an ubiquitin ligase, however substrates and exact function remain unknown (2) . a growing number of components of the ubiquitin protein system (ups) are described to be regulators of ciliogenesis (3). defects in such genes are considered to cause ciliopathies, genetic disorders with typical phenotypic variations in patients and model organisms (4) . first data supports our hypothesis that the ctlh complex plays a major role in ciliogenesis, e. g. the ctlh subunit rmnd5a localizes to the basal body which is a modified centriole of primary cilia in nih-3t3 cells and rmnd5 knock down in xenopus laevis leads to defects in cilia formation of epidermal multiciliated cells. n. reich, m. sandbothe, r. buurman, b. schlegelberger, t. illig, b . skawran department of human genetics, hannover, germany background and aims: hepatocellular carcinoma (hcc) is characterized by genetic and epigenetic changes that lead to a deregulation of important tumor suppressors and oncogenes in a multistep process. one of these epigenetic changes is the elevated expression of histone-deacetylases (hdacs) which contribute to a transcriptional repression of certain genomic regions by remodeling the chromatin structure. thereby, not only the expression of tumor-relevant genes is affected, but also the expression of micrornas (mirnas). selected mirnas have been shown to play important roles in carcinogenesis. we aimed to identify mirnas deregulated by histone deacetylation and to understand their functional consequences in hcc tumorigenesis. methods: histone acetylation was induced by the global hdac inhibitor trichostatin a (tsa) in four hcc cell lines (hle, hlf, huh7, hepg2) and two immortalized liver cell lines (thle-2 and thle-3) in order to identify differentially expressed mirnas and messenger rnas (mrnas) by global expression profiling. findings were validated by transfection of microrna mimics and sirna-mediated knockdown in hcc cell lines, quantitative pcr, western blotting and luciferase reporter assays. results: after hdac-inhibition, hsa-mir-129-5p was significantly upregulated. the mir-129-5p holds tumor suppressive potential and its expression is reduced in different types of tumors. one predicted target gene of mir-129-5p is the hepatoma-derived growth factor (hdgf). this mitogenic growth factor is highly expressed in a variety of cancers, for example in hcc, and its expression correlates with a poor prognosis, irrespective of the tumor type. hdgf is a multifunctional protein that is involved in several signaling pathways, contributing to proliferation and metastasis of cancer cells, induction of angiogenesis and inhibition of apoptosis. incubation of hcc cells with tsa or transfection with mir-129-5p reduced expression of hdgf. luciferase assays indicate a direct regulation of hdgf by mir-129-5p. moreover, expression of the death receptor fas, which is a potential downstream target of hdgf, is also regulated by the mir-129-5p. a translocation t(8;14)(q24;q32) was not detectable in any of the investigated tissues. with the established assay we were able to provide a highly sensitive tool for the detection of the translocation t(8;14)(q24;q32). however, we did not detect normal b-cells carrying this translocation. this does not exclude that such cells exist. alternatively, the growth advantage conferred by myc may promote the acquisition of secondary genetic changes. this may result in a rapid tumorigenesis, that if occurring these cells only present as full blown burkitt lymphoma. myotonic dystrophy: links to the nuclear envelope p. meinke, s. hintze, s. limmer, b. schoser friedrich-baur-institute, munich, germany myotonic dystrophies (dm) are slowly progressing multisystemic diseases with a predominant muscular dystrophy -making dm the most frequent muscular dystrophy in adulthood. dm is caused by heterozygous dna-repeat expansions in the dmpk gene (dm1) or the cnbp gene (dm2). the repeat-containing rna accumulates in ribonuclear foci and splicing factors are sequestered to these foci, resulting in abnormal regulation of alternative splicing. dm patients show overlapping phenotype presentations with progeroid laminopathies, which are caused by mutations in nuclear envelope proteins. in search for molecular signatures of this overlap, we found an enrichment of nucleoplasmic reticuli in dm1 and dm2 patient myoblasts. additional, we found an alternative splicing of the lmna gene -both effects that are associated with progeroid laminopathies. this implies possible shared pathomechanism between dm and progeroid laminopathies. retinoblastoma is a tumor of the retina occurring in young children up to the age of five. it is caused by biallelic inactivation of the tumor suppressor gene rb1. we have shown that human rb1 is an imprinted gene and as such characterized by differential dna methylation of a cpg island (cpg85) in rb1 intron 2. cpg85 is not methylated on the paternal allele and acts as a promoter for the alternative rb1 transcript, rb1-e2b. it is argued that expression of rb1-e2b is causative of the observed skewing of regular rb1 expression in favor of the maternal allele. a true gametic differentially methylated region (gdmrs) is established in only one of the parental germ lines. it is supposed to be stable during early embryonic development and to be passed on to all daughter cells. we could show that cpg85 is free of methylation in human sperm. publicly available methylome data on oocytes revealed that cpg85 is fully methylated in human oocytes. these data are in agreement with cpg85 being a maternal methylated gdmr. we showed that the level of cpg85 methylation is 60 percent in blood, as expected. however, in eight tissues of three individuals we observed a gain of methylation at cpg85 ranging from 60 to 65 percent in liver and skin, and increasing to 70 to 85 percent in the other tissues (heart, kidney, muscle, brain, lung and spleen). interestingly, the degree of methylation was lower in fetal tissue than in adult tissue, as determined for brain and muscle. we also observed gain of methylation at cpg85 in two human embryonic stem cell lines and induced pluripotent stem cells. this is consistent with the finding of complete methylation at cpg85 in eight different retinoblastoma cell lines. we therefore conclude that cpg85 is an unstable dmr. in oocytes, dna methylation of gdmrs is established by transcriptional read-through from an upstream promoter. therefore, we hypothesize that gain of dna methylation at cpg85 is caused by run-through transcription erozygous state (nomenclature according to hgvs; reference sequence nm_000051.3). in silico-analysis by alamut (version 2.8.1) predict the loss of the donor splice site of intron 27 of the atm gene. cdna-analysis was performed and revealed the loss of exon 27 of the atm by a complex activation of two kryptic splice sites. a premature stop codon was generated giving rise to a truncated protein that leads to a pathogenic variant. the results of the genetic analysis are discussed in the context of the clinical findings. identification of the underlying genetic causes of gastric cancer will give a better view of the mechanisms that contribute to the pathophysiology of the disease. gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, 2 zentrum für diabetologie bergedorf, schwerpunktpraxis, hamburg, germany about 2-5% of all pregnant women develop gestational diabetes mellitus (gdm) during their pregnancies and diabetes complicating pregnancy is associated with adverse maternal and perinatal outcomes, notably, risk of fetal macrosomia and neonatal hypoglycemia and development of diabetes after pregnancy. gdm is considered to result from interaction between genetic and environmental risk factors. the case of a 35-year old female german patient with a novel mutation in the pax4 gene (rare mody gene type 9) as a cause of gestational diabetes mellitus is presented. we describe clinical, biochemical and genetic features of the patient, who developed gdm and gave birth to her child by cesarean section. mody genes type 1-11 were analyzed. sequencing the pax4 gene revealed a novel mutation in exon 8, pax4,c.778delc, p.(leu260cysfs*24); reference sequence nm_006193.2), a deletion of a cytosine leading to a truncated, non-functional protein. to date, no small deletion has been detected in the pax4 gene. identification of the underlying genetic causes of gdm will give a better view of the mechanisms that contribute to the pathophysiology of the disease. furthermore, early identification may improve options to prevent gdm and complications for the mother and her child. the results of the genetic analysis are discussed in the context of the clinical findings. the modulation of dna methylation is highly flexible and plays an important role during cell differentiation. furthermore, the dna methylome alters considerably during aging. age related changes in the dna methylation of regulatory genes are assumed to have a major impact on carcinogenesis (teschendorff, 2010) . moreover, it was demonstrated that the chronological age of a human donor can be predicted with high accuracy by analyzing the dna methylation of a specific minor set of cpg loci which are aberrantly methylated during aging (horvath, 2013) . hence, we intended to investigate the effect of epimutations identified in different lymphoma entities in comparison with the influence of epigenetic changes in sequential b cell differentiation stages on the epigenetic age. the altered expression of the tumor suppressor mir-129-5p due to chromatin remodeling may play a fundamental role in hepatocarcinogenesis. we expect that histone deacetylation and putative target genes of epigenetically deregulated mir-129-5p can be targeted by new therapeutic agents. the microrna-449 family inhibits tgf-β-mediated liver cancer cell migration by targeting sox4 introduction: modulation of microrna expression is considered for treatment of hepatocellular carcinoma (hcc). therefore, we characterized the epigenetically regulated microrna-449 family (mir-449a, mir-449b, mir-449c) with regards to its functional effects and target genes in hcc. methods: after transfection of mir-449a, mir-449b, and/or mir-449c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. binding specificities, target genes, and regulated pathways of each microrna were identified by microarray analyses. target genes were validated by luciferase reporter assays and expression analyses in vitro. furthermore, target gene expression was analyzed in 61 primary human hccs compared to normal liver tissue. results: tumor suppressive effects, binding specificities, target genes, and regulated pathways of mir-449a and mir-449b differed from those of mir-449c. transfection of mir-449a, mir-449b, and/or mir-449c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. importantly, mir-449a, mir-449b, and, to a lesser degree, mir-449c directly targeted sox4, which codes for a transcription factor involved in epithelial-mesenchymal transition and hcc metastasis, and thereby inhibited tgf-β-mediated cell migration. conclusions: this study provides detailed insights into the regulatory network of the epigenetically regulated microrna-449 family and, for the first time, describes distinct tumor suppressive effects and target specificities of mir-449a, mir-449b, and mir-449c. our results indicate that particularly mir-449a and mir-449b may be considered for mirna replacement therapy to prevent hcc progression and metastasis. novel mutation in the atm gene and activaton of two kryptic splice sites in an 52 year old female patient with gastric cancer gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, 2 schwerpunktpraxis, hämatologie, onkologie und palliativmedizin, hamburg, germany, 3 israelitisches krankenhaus, chirurgische klinik, hamburg, germany gastric cancer is a global public health concern, ranking as the third leading cause of cancer mortality. familial aggregation of gastric cancer is common in about 10% of the cases, and about half of these can be attributed to hereditary germline mutations. however, for most gastric cancer cases, whether genetic events contribute to cancer susceptibility remains unknown. here we present a case report of a patient with gastric cancer, a family history of breast cancer and a novel mutation leading to complex cryptic splicing in the atm gene. ngs panel sequencing and cnv/mlpa analysis of 18 genes associated with gastric and breast cancer were performed. sequencing revealed a novel mutation in intron 27 of the atm gene, atm,c.4109 + 1g>a in an het-abstracts tions remained unidentified since positive deletion-junction pcr products could not be amplified. to identify the breakpoints of the 15 deletions, we performed custom-designed microarray cgh analysis with a high resolution of probes located within and flanking the nahr hotspots prs1 and prs2. the array analysis suggested that 11 of the 15 deletions exhibit breakpoints within prs2, even although previously performed breakpoint-spanning pcrs with primers designed according to the reference sequence of the human genome have been negative in these 11 cases. since prs2 exhibits high sequence diversity resulting from frequent nonallelic homologous recombination events without crossover, we surmised that haplotype diversity is responsible for the failure of the breakpoint-spanning pcrs performed with primers designed according to the reference sequence. therefore we characterized the haplotype diversity of prs2 in 30 human individuals and designed deletion-junction pcr primers that facilitate the amplification of rare prs2 haplotypes. so far, we have identified the breakpoints of four of the 11 type-1 nf1 deletions predicted to have been mediated by prs2 according to the array results. we are confident to identify further breakpoints by extending these analyses using primers suitable to amplify rare prs2 haplotypes. our findings indicate that the characterisation of nahr hotspots in terms of haplotype diversity is a premise to identify the breakpoints of nahr-mediated microdeletions by means of deletion-junction pcrs. p-basepi-028 *** array-based dna methylome analyses of primary lymphomas of the central nervous system ulm university, ulm, germany, 2 university of cologne, cologne, germany, 3 christian-albrechts-university kiel, kiel, germany, 4 university hospital muenster, muenster, germany primary lymphomas of the central nervous system (pcnsl) are defined as diffuse large b-cell lymphomas (dlbcl) that are confined to the central nervous system (cns). although pcnsl cannot be distinguished from dlbcl by their morphology as well as their histology, they differ in prognostic outcome. the aim of the present study was to compare the epigenomic landscape of pcnsl and dlbcl. to this end, we analyzed the dna methylation of a total of 26 pcnsl (cryopreserved or formalin fixed and paraffin embedded (ffpe)) using the infinium humanmethylation450 beadchip array (illumina) and contrasted these findings to 79 dlbcl (kretzmer et al., 2015) . as controls, we used publicly available dna methylation data from a total of 50 normal brain samples derived from different regions of the cns (gilbert et al., 2105; jaffe et al., 2016; kurscheid et al., 2015; mur et al., 2013; wockner et al., 2014) . after normalization of the data we performed thorough filtering and removed the random snps, all loci located on gonosomes, as well as those loci with a detection p-value >0.01 in at least one of the samples, leading to 457,951 loci entering subsequent analyses. when comparing the dna methylation profiles of pcnsl versus dlbcl we identified 8279 differentially methylated loci (σ/σ max = 0.4; q < 1e-4). in order to remove those loci which represent a "brain signature", we compared dlbcl versus brain (σ/ σ max = 0.4; q < 0.05) based on the list of the previously identified 8279 loci. after removing this "brain signature", we ended up with 2231 loci that are differentially methylated between pcnsl and dlbcl. in a next step we wanted to make sure that the differences in methylation at these 2231 loci are not due to differences in starting material (cryopreserved versus ffpe) which is known to influence the outcome of the beadchip analysis. therefore, we compared the dna methylation profiles of five cryopreserved versus ffpe samples (derived from the same tissue samples) and identified 318 differentially methylated loci (σ/σ max = 0.4, q < 0.05). only five loci of both lists overlapped, which were subsequently removed from further analysis so that we ended up with a final list of 2226 loci which are differentially methylated between pcnsl and dlbcl. in order to analyze the biological implications of the differentially methylated loci we evaluated an enrichment of known functional groups (ku-additionally, our aim was to analyze whether entity-specific differences in the resetting of the epigenetic clock are generated during lymphomagenesis or derive from modified dna methylation in the germinal center b cells of origin. to address these issues, we performed dna methylation profiling (hum-anmethylation450 beadchip) of 72 burkitt lymphoma samples (age 2-76 yrs), 119 diffuse large b cell lymphoma samples (age 3-93 yrs) and 103 follicular lymphoma samples (age 22-80 yrs) from the icgc mmml-seq and mmml-consortium (kretzmer et al., 2015) and the hematopathology section kiel as well as 145 peripheral blood samples of healthy individuals (0-63 yrs) available from the same project and current publications of our group (kolarova et al., 2015; friemel et al., 2014) . in addition, we received 61 b cell subpopulation samples (0-66 yrs) covering different stages of b cell differentiation that were measured in the same way (kulis et al., 2015; lee et al., 2012) . the epigenetic age of the samples was predicted using the "online age calculator" accessible at https://dnamage.genetics.ucla.edu and compared with the corresponding chronological age of the donors. in fact, the epigenetic age of peripheral blood samples of healthy donors was in high accordance with their chronological age (pearson's r 0.968, p-value <0.001) while the correlation between epigenetic and chronological age of sequential b cell differentiation stages was slightly lower (pearson's r 0.893, p-value<0.001). in contrast, the predicted epigenetic age of the burkitt lymphoma samples was significantly higher than the corresponding chronological age. this deviation may be interpreted as "epigenetic pre-aging". nevertheless, the epigenetic age of diffuse large b cell lymphomas and follicular lymphomas tended to be less affected. in conclusion, we found significant epigenetic pre-aging in burkitt lymphoma samples that seems to be induced during lymphomagenesis and does not derive from altered dna methylation patterns in the germinal center b cells of origin. moreover, no significant shift of the epigenetic age was observed for the other lymphoma entities, healthy blood samples and b cells of sequential differentiation stages. identification of type-1 nf1 deletion breakpoints mediated by rare prs2 haplotypes a. summerer 1, 2 , m. hillmer 1,2 , v. f. mautner 3, 4 , l. messiaen 5, 6 , h. 2 1 institute of human genetics, ulm, germany, 2 university of ulm, ulm, germany, 3 department of neurology, hamburg, germany, 4 university hospital hamburg eppendorf, hamburg, germany, 5 department of genetics, birmingham, usa, 6 university of alabama at birmingham, birmingham, usa neurofibromatosis type 1 (nf1) is a hereditary cancer syndrome with an incidence of 1 in 3000. in 5% of all nf1 patients, large deletions encompassing the nf1 gene and its flanking regions are causing the disease. the majority of all large nf1 deletions are of type-1; they encompass 1.4-mb and are mediated by nonallelic homologous recombination (nahr) with crossover. the breakpoints of type-1 deletions are located within the lowcopy repeats nf1-repa and nf1-repc which exhibit high sequence homology to one another. previous studies suggested that type-1 deletion breakpoints cluster within the paralogous recombination sites prs1 and prs2 spanning 5-kb and 4-kb, respectively. in our present study, we investigated 218 patients with type-1 nf1 deletions using long-range pcrs to detect breakpoints located within prs1 or prs2. according to these analyses, 157 (72%) of the breakpoints are located within prs2 and 34 (15.6%) in prs1. however, 27 (12.4%) of the type-1 deletions were not positive for these deletion-junction pcrs. we surmised that some of these deletions may have breakpoints within the 14-kb region located between prs1 and prs2. this 14-kb region also exhibits high sequence homologoy between the nf1-reps which is a prerequisite for nahr. indeed, 12 of the 27 type-1 nf1 deletions exhibited breakpoints within this 14-kb region as determined by the analysis of seven overlapping deletion-junction pcrs. however, the breakpoints of 15 dele-esophageal adenocarcinoma (ea) represents one of the most rapidly increasing cancer types in high-income countries. barrett's esophagus (be) is a premalignant precursor of ea and has an estimated prevalence of 5-6% in the population. however, only 0.1 to 0.3% of be patients develop ea. within an international consortium, we carried out a gwas meta-analysis in 6167 be patients, 4112 ae patients and 17.159 controls (gharahkhani et al., lancet oncology, 2016) . in a comprised be/ae analysis, we identified 14 genome-wide significant risk loci, of which seven were previously unreported. the strongest associated new risk variant was identified for rs17451754 (p = 4.8×10 -10 ), which maps within intron 21 of the cftr gene. cftr encodes a protein that functions as a chloride channel and that is mutated in patients with cystic fibrosis (cf). mutations in cf lead to abnormal viscous secretions with altered chemical composition, resulting in dysfunction of the respiratory system and the gastrointestinal tract. the most common cf mutation is δf508, a deletion of three nucleotides in cftr that results in the loss of a single codon for phenylalanine on protein position 508. interestingly, cf patients show a highly increased incidence of gastroesophageal reflux, which represents the major risk factor for be and ae. in view of the phenotypic overlap for gastroesophageal reflux and cystic fibrosis, and for gastroesophageal reflux and both be and ae, combined with the association of cftr risk variants in patients with be and ae, it seems plausible that a common pathophysiological mechanism is triggered by cftr. in order to test this hypothesis, we analyzed the association of δf508 in a european case-control cohort with be and ae patients. for this, we performed a genotyping assay of δf508 in 1037 be patients, 1609 ae patients and 941 controls. we could not observe a significant association (p = 0.57). this might be (i) due to insufficient sample power or (ii) due to the fact, that not δf508 but other genetic variants at the locus might explain the underlying functional mechanism of the association. fine mapping of all genetic variation at the cftr locus and exten-lis et al., 2015). remarkably, cpg loci that are differentially methylated during normal b-cell maturation were significantly depleted. in turn we saw an enrichment of loci located in heterochromatin. in summary, we detected more than 2000 loci that are differentially methylated between pcnsl and dlbcl, which do not play a functional role in normal b-cell differentiation. replication study of gwas-identified genetic modifiers of age at huntington's disease onset although there is a strong correlation between cag repeat length and age at onset (ao) of motor symptoms, individual huntington disease (hd) patients may differ dramatically in onset age and disease manifestations despite similar cag repeat lengths. since the modifier variations described so far only account for a small fraction of the heritable contribution to the ao, the identification of loci and genes using genome-wide methods appears highly promising. against the background of incomplete understanding of the hd disease pathophysiology, the hypothesis-free approach of gwas offers an ideal starting point for the search of modifier genes. recently, a combined analysis of all gwa data to hd modifiers identified different loci with genome-wide significant signals for association to residual age at motor onset [gem-h. consortium]. interestingly, none of the most significant association signals and none of the trending snps in the european gwa analysis corresponded to any previously suggested candidate modifier genes. in order to be able to better assess these data, we tried to replicate the top ten associated gwas variants in a comprehensive cohort of 505 german hd patients. we only found modest association with one of the top ranked snps (rs72810940), all remaining variations showed no correlation with the ao. this inconsistency highlights once again the difficulties of modifier searching in hd or any other monogenic disorder, which faces the same challenges as the genetic characterization of complex disorders. with an incidence of 0.2-0.3/100.000 malignant tumors of the thymus are a rather rare type of cancer. here we report the case of a man of german descent, who presented with a thymoma at age 55. in the pathological report the thymus tumor was described as an extremely unusual thymoma with partial loss of keratin and massive proliferation of myoid cells. it was subsumed to a primary thymic, partially epithelial neoplasia, resembling an uncommon b2/b3-thymoma. after the patient's death his widow looked for genetic advice concerning the risk of disease for her children. detailed personal and familial history brought up surprising information: thymoma was one of four cancers in our patient. he developed adenocarcinoma of the colon at age 35, squamous cell cancer of the nose/upper lip at 54 years and in addition current cancer staging revealed a papillary renal cell carcinoma. according to family history his father and his uncle developed colon cancer with 58 and 66 years, the son of this uncle was diagnosed with colon cancer at age 47. this cousin of the propositus was referred to genetic counseling, because of msi-high-status and loss of mlh1 and psm2 in immunohistochemistry. he was found to have a deleterious mutation in exon 14 of mlh1gene (c.2644c>a, p.tyr548stop) resulting in a premature termination of mlh1-protein. our patient has never been tested for hnpcc. however a post mortem performed immunhistochemical examination of thymic cancer cells revealed an almost complete loss of mlh1 nuclear expression suggesting the presence of a mlh1 germline mutation and indicated hnpcc. considering the loss of mlh1 in tumor cells it is more than likely that the development of thymoma was the consequence of deficient dna mismatch-repair. there have been reports of rare tumors in hnpcc families in the last years (i. e. clear cell renal carcinoma and uterine sarcoma). pande et al. reported one case of thymoma in their registration of cancer occurrences in 368 mutations carries from 176 hnpcc families [1] . our case emphasizes the importance of detailed family history and contributes to the discussion of widening the inclusion criteria for genetic counseling and testing for hnpcc. to this day the revised criteria of bethesda are used to identify families at risk. we propose that the established criteria have to be revised and rare tumors should be included. unknown partner genes in leukemias with rare translocations can be identified using targeted rna sequencing c. haferlach, n. nadarajah, m. meggendorfer, n. dicht, a. stengel, w. kern, t. haferlach mll, munic, germany in hematological malignancies fusion genes play an important role and function as therapeutic targets, impressively shown for e. g. bcr-abl1 and etv6-pdgfrb. thus, the identification of fusion genes is the basis for precision medicine, selecting treatment based on genotype and providing markers for disease monitoring. the aim of this study was to test the value of targeted rna sequencing in a routine diagnostic work up. 51 cases were selected harboring rearrangements of kmt2a (n = 10), runx1 (n = 19), etv6 (n = 11), pdgfrb (n = 6), npm1 (n = 2), rara (n = 2) and jak2 (n = 1) identified by chromosome banding (cba) and fish analyses. in none of the cases the partner gene could be identified using standard methods. targeted rna sequencing was performed using the trusight rna fusion panel (illumina, san diego, ca) consisting of 7690 probes covering 507 genes known to be involved in gene fusions. this assay allows the capture of all targeted transcripts. sequencing was performed on nextseq (illumina, san diego, ca). analysis was performed with the rna-seq alignment app (basespace sequence hub) using star for alignment and manta for gene fusion calling with default parameters (illumina). sive functional analysis are needed to find the causal variant that explains how the cftr locus interferes with the pathomechanism of be and ae. a recent functional study indicated cftr as a tumor suppressor gene in murine and human intestinal cancer, providing further evidence for cftr as a true disease gene for be and ae. background: it has long been established that mutations in brca2 predispose for pancreatic adenocarcinoma with brca2 germline mutations identified in 6-10% of familial pancreatic cancer cases. consequently, screening for pancreatic cancer has been recommended for mutation carriers with an affected first-degree relative since early detection has been shown to significantly improve 5-year survival from 4-7% to 24%. for brca1 mutations, however, relevance in pancreatic tumorigenesis is still being discussed with several studies questioning an elevated risk of pancreatic cancer in families with brca1 mutations while others are suggesting that brca1 may also play an important role in predisposing to pancreatic cancer. clinical screening for pancreatic cancer commonly remains unavailable to brca1 mutation carriers and it has even been questioned whether brca1 should be analyzed in familial pancreatic cancer at all. clinical report: here we report on a 59 year old woman with metastatic pancreatic cancer whose sister had died of pancreatic cancer at 50 years of age. in this family we identified a pathogenic brca1-germline mutation (brca1: nm_007294.3:c.1292dupt,p.(leu431phefs*5)) by next-generation sequencing using a 94-gene panel. the index patients' tumor was available for genetic analysis and showed loss-of heterozygosity for brca1. this strongly suggests the brca1 mutation to be causative of the pancreatic cancer development in this patient. when the family was first introduced to genetic counselling there was no evidence of breast-or ovarian cancer in any relatives. only after identification of the mutation did the index person reach out to distant family members and it was thereby revealed that a distant branch of the family had independently been counselled for hereditary breast and ovarian cancer. in this part of the family, however, there had not been any cases of pancreatic cancer. subsequent predictive testing was offered to healthy family members and 3 further mutation carriers could be identified. two women were referred to breast cancer screening. additionally, the mutation was identified in a relative with recurrent metastatic breast cancer at the age of 38 years. for her and the index patient parp-inhibition therapy thus became a possible further treatment option. conclusions: in conclusion we propose next-generation sequencing approaches including the analysis of brca1 to be used in familial pancreatic cancer. we also argue that brca1 mutation carriers with pancreatic cancer cases in their family should be offered the same screening program as brca2 mutation carriers. within the framework of a study this could allow for more precise risk stratification in the future. contralateral dcis is unknown. only the male breast carcinoma was herceptin receptor positive, all other breast carcinomas of the chek2 mutation carriers were her2 negative. among our chek2 positive families we noticed the association with chek2 mutation and female breast cancer. we observed a contralateral breast cancer, male breast cancer and other tumors in our families as well. the majority of the observed breast cancers was estrogen and progesterone receptor positive and herceptin negative. while benign uterine smooth muscle tumors are among the most frequent human symptomatic tumors, their malignant or borderline lesions are only rare findings. both lesions can show somatic copy number alterations, but their patterns differ, thus constituting helpful diagnostic tools. aimed at an advanced classification of the lesions we have performed molecular inversion probe array analyses of these tumors. besides complex patterns of genomic alterations seen in nearly all cases, two of the lesions presented with copy number neutral uniparental disomies i. e. normal copy numbers with an apparent monoallelic origin. in one case, an upd of part of the long arm of chromosome 22 was detected in a uterine leiomyosarcoma. the tumor showed genetic heterogeneity with gains and losses. in addition, the 11.45 mb segment located at 22q12.1-q13.1 was clearly of monoallelic origin throughout all cells investigated. all other genetic alterations were restricted only to part of the cells of the sample thus reflecting the presence of tumor cells as well as normal bystander cells which in general characterizes mutations that had arisen during tumor development. in contrast, the upd that was detected in all examined cells clearly suggests its germline occurrence. the second tumor was a leiomyoma-variant of the type with bizarre nuclei. again, besides gains and losses an apparent germline upd was found that covered a 3.71 mb segment on chromosomal segment 8q11.21. upd for even the whole arm of chromosome 22 repeatedly has been reported not to coincide with phenotypic manifestations. nevertheless, the question arises whether or not the observed upds might be related to a familiar predisposition for uterine muscle tumors. of note, as a result of genome-wide association studies snps on 22q recurrently have been found to be significantly associated with fibroid development. triple negativity is an independent predictor of germline mutations in breast cancer predisposing genes breast cancer is the most common cancer in women. 12-15% of all tumors are triple-negative breast cancers (tnbc) lacking expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. so far, tnbc have been mainly associated with mutations in brca1, although recent studies also found mutations in other breast cancer susceptibility genes. a brca1/2-centered perspective thus may ignore the significance of other predisposing genes, whose relevance appears obvious as dna damage repair by homologous recombination is a complex process involving many proteins. in 32/51 cases with rearrangements involving kmt2a (n = 10), runx1 (n = 8), etv6 (n = 6), pdfgrb (n = 4), rara (n = 2), npm1 (n = 1) or jak2 (n = 1) the partner genes were identified. these were in kmt2a rearranged cases: mllt10 (n = 2), mllt1 (n = 2), itpr2, flnc, asxl2, dcp1b, maml1 and arhgef12. in runx1 translocated cases partner genes were plag1 (n = 2), prdm16, mecom, zfpm2, man1a2, n6amt2, and kiaa1549l. prdm1, mecom and zfpm2 have previously been described in the literature as runx1 partner genes but were not suspected in our cases as partner genes due to complex cytogenetic rearrangements. the other identified partner genes have not been described so far. interestingly, prdm1, mecom, zfpm2 and the newly identified plag1 are all members of the c2h2type zinc finger gene family. partner genes identified in etv6 rearranged cases were: abl1, ccdc126, clptm1l, erg, foxo1 and cflar-as1. wdr48, zbtb11, nfia and mprip were identified as partner genes of pdgfrb and rpp30 in an npm1-translocated aml. in an all patient a jak2-ppfibp1 fusion was identified leading to classification as a bcr-abl1-like all. in an apl patient showing an ins(17;11) (q12;q14q23) a zbtb16-rara fusion was identified and thus resistance to all-trans retinoic acid, arsenic trioxide, and anthracyclines can be predicted. further in a case with t(17;19)(q21;q13) an irf2bp1-rara fusion was detected. conclusions: targeted rna sequencing was able to characterize rare gene fusions and provided the basis for the design of rt-pcr based assays for monitoring mrd. targetable genetic aberrations were identified, which were not detected by cba enabling more individualized treatment. targeted rna sequencing may be a valuable tool in routine diagnostics for patients with rearrangements unresolved by standard techniques. female carriers of a pathogenic mutation in the chek2 gene are reported to have a life time risk of about 20-45% to develop breast cancer. there is evidence for increased risks for contralateral breast cancer, male breast carcinoma and other types of tumors. in addition to well-known mutation chek2:c.1100del, other pathologic mutations are being identified in the gene due to the inclusion of the gene in most breast cancer gene panels for dna testing. between 2012-2016 the center for hereditary breast and ovarian cancer regensburg cares for 10 families with pathogenic or probably pathogenic mutations in the chek2 gene affecting nine female patients and one male patient (6 × c.1100del, 1 × deletion of exon 10, and 3 × variants considered as likely pathogenic: c.1408g> c, c.1561c> t, c.1169a> c). the mean age of diagnosis of breast cancer (both sexes) was 45.1 years (range 25-61 years). the patient with the deletion of exon 10 was first diagnosed at 25 years of age and developed a contralateral breast carcinoma (dcis) at 35 years of age. the male patient was diagnosed with breast cancer at 58 years of age and at 59 years with a renal carcinoma. one patient was diagnosed with a papillary thyroid carcinoma at age 26 years and developed breast cancer with 35 years. in 8 out of the 10 families, breast carcinoma diagnosed with 51.3 years on average, was reported in the family history. in addition, there were additional malignancies such as prostatic carcinoma, thyroid carcinoma, colorectal cancer, gastric carcinoma, leukemia, cervical carcinoma and malignant melanoma. none of the affected family members was tested for the respective chek2 mutation. the tumors with an initial diagnosis at 25 years and 35 years were estrogen-receptor-negative and progesterone-receptor-negative. the other 8 of the 11 breast cancers were positive for the estrogen receptor, 7 of the 11 tumors were positive for the progesterone receptor. the receptor status of the abstracts it has been shown that in 3d culture hescs can be differentiated into neural retina containing organoids. we established this differentiation schedule and started comparative differentiation of wildtype h1 hescs and the rb1 null derivative (g4, rb1 mt/mt ) into neural retina. during the first weeks of differentiation into neural retina organoids generated from the rb1 mt/mt hescs have a smaller diameter and thinner retina layer compared to wildtype organoids. however, during the time-course the mutant organoids began to catch up. thus, at later stages no difference in size and thickness could be observed anymore. comparative immunostainings of cryosections at d19 show no difference in expression of the markers pax6 and sox2 between the wildtype and mutant hescs. further comparative immunostainings for markers specific for neural retina like e. g. rx and vsx2 at d19 and d33 are ongoing and will be presented. exome sequencing identified potential causative candidate genes for unexplained cowden syndrome purpose: cowden syndrome (cs) is a cancer predisposition syndrome characterized by the occurrence of breast cancer, epithelial thyroid cancer, endometrial carcinoma and various other findings such as mucocutaneous lesions and macrocephaly. cs belongs to the pten hamartoma tumor syndrome (phts) primarily associated with germline mutations in pten. in recent years, germline mutations in additional genes (sdhb, sdhc, sdhd, pik3ca, akt1, sec23b) have been described in few patients; however, to date, in 20-75% of patients meeting clinical criteria for cs the underlying cause remains unclear. methods: to uncover predisposing causative genes, the exomes of 11 clinically well characterized, mutation negative patients with suspected cs were sequenced (illumina hiseq) using leukocyte dna. assuming a monogenic disease model, the called variants were filtered for rare (minor allele frequency ≤1% for homozygous/compound heterozygous variants and ≤0.01% for heterozygous variants according to dbsnp, evs, and exac), truncating (nonsense, frameshift, highly conserved splice sites), and missense germline variants (predicted to be pathogenic by at least 2/3 in-silico tools). for data analysis and variant filtering the gatk software and the cartagenia bench lab ngs software were applied. all candidate genes were included in a pathway analysis (ingenuity). in a first preliminary analysis, we focused on known cancer genes and genes interacting with pten. results: after stringent filtering steps, comparison with large datasets from population-based controls, and detailed manual inspection to exclude artifacts, 75 genes were affected by presumed biallelic variants (16 homozygous and 59 putative compound-heterozygous), one of these is a known cancer gene (cbfa2t3); in 17 genes biallelic variants were found in 2-6 patients. heterozygous variants were found in 23 genes in 2-6 patients, but none of these are known cancer genes. in 132 genes, heterozygous truncating mutations occurred in only one patient, 4 of these are cancer genes (msh6, wrn, kdm5a, pml) . the phenotype of the patient with a msh6 frameshift deletion fulfilled key features of cs (early-onset metachronous papillary thyroid cancer, breast cancer, endometrial and colorectal cancer), however, the tumor spectrum is partly compatible with lynch syndrome/ hnpcc. examination of the colorectal cancer demonstrated microsatellite instability and a loss of msh6 protein expression. the pathway analysis of the remaining candidate genes identified several interacting partners of pten (grhl3, ehhadh, cstf3). conclusions: preliminary data indicate that exome sequencing might identify potentially relevant causative genes for cs, some of which are recurrently mutated. the present work-up consists of the inclusion of further non-cancer genes, validation of variants by sanger sequencing, testing of to determine the prevalence of mutations we performed panel-based germline mutation testing of 10 high and low-moderate penetrance breast cancer susceptibility genes (brca1, brca2, atm, cdh1, chek2, nbn, palb2, rad51c, rad51d and tp53) in 229 consecutive individuals affected with tnbc unselected for age at diagnosis or breast and ovarian cancer family history. age at diagnosis ranged from 23 to 80 years with an average of 50.2 and a median of 48 years. in 60 women (26.2%) we detected a pathogenic mutation, with a higher frequency (31.3%) in the group manifesting cancer before 60 years. deleterious brca1 mutations occurred in 14.8% of tnbc patients, predominantly frameshifting (24/34, 70.6%). the most frequent, both among brca1 mutations and in total, were the founder mutations c.5266dupc and c.2411_2412delag. deleterious brca2 mutations occurred in 5.7% of patients, all but one (c.1813du-pa) being unique. while no mutations were found in cdh1 and tp53, 15 mutations (25%) were detected in one of the six other predisposition genes (palb2, chek2, atm, nbn, rad51c, rad51d). no individual presented more than one mutation. almost half of all deleterious mutations (42.5%) were detected in very young women aged 35 years or less. the median age at diagnosis was significantly younger for brca1 (40 years) and brca2 (41.5 years) carriers compared to patients without a mutation (p = 2.746e-05; mann-whitney) or compared to non-brca1/2 mutation carriers (p = 0.022). in contrast, patients with non-brca1/2 mutations were not significantly younger than mutation negative women (p = 0.5288). interestingly, family history had an independent influence on age at diagnosis. taken as a whole, women with family history had a median age at diagnosis 6 years earlier than those without (p = 0.00057). this difference was lost in mutation carriers while it remained in cases without mutation. in summary, our data confirm and expand previous studies of a high frequency of germline mutations in genes associated with ineffective repair of dna damage by homologous recombination in women with tnbcs. many of these women would go untested with current restrictive criteria. in order that each patient receives therapies tailored to her genetic status, gene panel based mutation testing should be offered to all women diagnosed with tnbc, irrespective of age at diagnosis or family history. p-cancg-037 *** neural retina differentiation of hescs as an in vitro model for retinoblastoma d. kanber, m. hiber, d. lohmann, l. steenpass institute of human genetics, university hospital essen, university duisburg-essen, essen, germany retinoblastoma is the most common eye tumor of early childhood. inactivation of both alleles of the retinoblastoma gene (rb1) results in the development of retinoblastoma. our aim is to establish a human cell-based model for retinoblastoma. using the crispr/cas9 system we have generated human embryonic stem cells (hescs) carrying a mutation either on one or both rb1 alleles. all the detected mutations are located in exon 3 of the rb1 gene and close to the splice donor site of this exon. analyses on dna, rna and protein level were performed for three mutant and one double-mutant clone. the following genotypes were identified by deep sequencing (nm_000321.2(rb1_v001)): clone c2, c.364_380del, heterozygous; clones c7 and g3, c.372_378del, heterozygous; clone g4, c.[372_378del; c.367_368dup] (complex mutation on one allele), homozygous (loss of heterozygosity). the mutations of all four clones result in a premature stop codon in exon 4. on rna level we detected expression of mutant rb1 transcripts reflecting the genotype in all clones and an additional mutant rb1 transcript with skipping of exon 3 in three clones. as the heterozygous clones also showed expression of the wildtype rb1 transcript, rb1 protein (prb) could be detected for these clones (c2, c7, g3) by western blot analysis. however, the double-mutant clone g4 showed no expression of prb. so far, we have characterized 3 heterozygous and one homozygous clone. another three double-mutant clones are under investigation. the recurrent germline missense mutation g84e in the hoxb13 gene has been demonstrated to predispose to hereditary prostate cancer (prca), despite the underlying pathogenic mechanism is not yet understood. molecular examination of a first set of g84e positive tumors sought for somatic characteristics, and suggested that oncogenic ets gene fusions may appear at unusually low frequencies as compared to the general prevalence of ets fusions in prca (22% vs approx. 50%). hypothesizing that hoxb13 could predispose to ets fusion negative prca, we have analyzed 942 cases from three european ancestry populations (finland, germany and us) for the coincidence of hoxb13 g84e and the most common ets fusion, tmprss2:erg, in corresponding tumor samples. while the prevalence of tmprss2:erg fusions was similar among the three study groups (range: 56.5-60.7%), the frequency of g84e genotypes differed markedly between us (1.5%), german (3.6%) and finnish samples (8.3%). despite the expected frequency gradient among study populations, all subsamples showed a strong enrichment of g84e mutation carriers among tmprss2:erg fusion negative cases as compared to fusion positive cases (center adjusted or = 4.96; 95%ci = 2.30-11.9; p = 0.0001). consistent with the previous study, the crude frequency of the tmprss2:erg fusion in hoxb13 g84e carriers was 23.5% (range 16.7% -28.6%). examination of disease characteristics highlighted age at diagnosis to be associated with tmprss2:erg negative status (per year or = 1.04, p = 0,00007) and by trend, also with the presence of the g84e germline variant (per year or = 0.97, p = 0.14). within the subtype of tmprss2:erg fusion negative carcinoma carriers of g84e were diagnosed 3.5 years earlier as compared to non-carriers (61.6 ± 1.4 years versus 65.1 ± 0.4 years, p = 0.017). in conclusion, this study demonstrated a significant tumor subtype specific association for hoxb13 g84e mutation carriers having a higher frequency of tmprss2:erg fusion negative prca. meta-analyses from case control comparisons suggested that subtype specific risk of hoxb13 g84e for tmprss2:erg negative prca could be as high as or = 19.0, as compared to or = 9.9, when prca is regarded as one entity regardless of fusion status. finally, although tmprss2:erg negative prca is usually known to be associated with later ages of diagnoses, hoxb13 mutations may indicate a subgroup of earlier onset cases within the fusion negative entity. relatives to determine the phase of assumed biallelic variants and segregation with the phenotype where applicable. with 1 to 2 cases in 1 million inhabitants per year adrenocortical cancer (acc) is a rare disease. due to often late diagnosis and limited treatment options prognosis for patients are poor with a 5 year overall survival rate of 7 to 35%. though knowledge about molecular genetic events in acc increased over the last few years no reliable molecular prognostic factors, no effective targeted cancer therapy and no personalized treatment approach has emerged to date. that's why we intend to establish a reliable method to define a molecular signature of accs that could be used for a prognostic classification of adrenocortical cancers, for planning an individualized therapeutic approach and for the identification of known or potential targetable molecular events in the single patient. in a retrospective study dna from acc and matched blood samples is sequenced to detect somatic single nucleotide variants (snv), small insertions and deletions (indel) and copy number alterations (cnv). sequencing data are then compared to clinical data e. g. tumor stage, resection status, ki67-index and time of progression free and overall survival to define molecular prognostic factors. target enrichment of 160 genes that are known to be associated with different entities of cancer is performed with the human comprehensive cancer panel (qiagen) and sequenced on a nextseq500 (illumina). data are analysed with gensearchngs (phenosystems). znrf3, a gene that was also described to be involved in the development and the progression of acc a few years ago, is sequenced separately with sanger and analysed with gensearch (phenosystems). to date tumor samples and matched blood samples from 43 patients were analysed. one or more tumors comprise one or more snvs or small indels in 48 of 160 genes of the panel and in znrf3. snvs and small indels are most often found in tp53, ctnnb1 and znrf3 with frequencies of 28%, 26% and 19% respectively. in 37 of 160 genes cnvs -duplications and deletions -occur. cdk4 is duplicated in over 50% of the cohort. mdm2 gains are found in over 40%. one can also find three types of cn patterns: a quiet type with low number of copy alterations, a noisy one with high number of chromosomal breakages and a chromosomal one with high frequency of alterations of chromosomal arms. while no correlation between snvs and small indels and clinical outcome could be found so far, cn patterns of the accs seem to correlate with progression free survival and overall survival. patients with a noisy cn pattern have a shorter progression free and overall survival than patients with chromosomal and quiet type. though tendencies in the correlation of molecular markers and prognosis for patients suffering acc can be recognized, further samples need to be analysed to confirm the results. it is planned to sequence another 60 tumor samples and matched blood samples for this retrospective study and to validate the results in a prospective study with another 100 patients. expression of mir-371a-3p and mir-367-3p was analysed in serum samples by quantitative pcr. the cohort of 27 gcnis patients consisted of 11 patients with a solitary testicle, who had undergone orchiectomy for contralateral tgct, and 16 patients with two testicles, one of which with gcnis, but no concurrent tgct. twenty men with non-malignant testicular disease served as controls. additionally, in situ hybridisation (ish) with a probe against mir-371a-3p was performed on four testicular biopsy specimens known to harbour gcnis. sequential step sections of the corresponding tissue blocks were analysed immunohistochemically, using oct4 antibody to visualise gcnis. the median expression value of mir-371a-3p in gcnis-patients was 5.2 (interquartile range [iqr] = 35.8) which is significantly higher than the median expression of 0.0 (iqr = 0.0) in controls. both of the two gcnis subgroups had significantly higher mir-371a-3p levels than controls, with a median expression of 18.2 (iqr = 37.3) and 2.7 (iqr = 32.7), respectively. regarding mir-367-3p expression, there were no significant differences between gcnis and controls. using a relative quantity of 5 as a cut-off value, the mir-371a-3p was able to detect 51.2% (95% confidence interval [95% ci] = 31.9-71.3%) of gcnis, while only 5% (95% ci = 0.1-24.9%) of the controls were positive. in the subgroup with previous tgct 63.6% (95% ci = 30.8-89.1%) of gcnis could be detected and in the subgroup without previous tumour the rate was 43.8% (95% ci = 19.8-70.1%). the detection rates for all gcnis and for both subgroups were significantly higher than for the controls. ish staining demonstrated the expression of mir-371a-3p in gcnis cells in two of the four cases. in conclusion, this study indicates a new and minimal-invasive way of diagnosing gcnis by measuring serum levels of mir371a-3p. this approach is endorsed by the demonstration of mir371a-3p in gcnis cells by ish staining. however, the sensitivity is still low and thus, the method certainly needs refinement possibly by applying a panel of additional mi-crornas. nonetheless, measuring serum levels of mir371a-3p may constitute a valuable aid in clinical assessment of men afflicted with high-risk factors of tgct. p-cancg-043 *** the mir-371a-3p is a highly specific and sensitive serum-based marker for the diagnosis and follow-up of testicular germ cell tumours testicular germ cell tumours (tgct) are a paradigm of curable malignancies. clinical management largely relies on measuring the serum biomarkers. inopportunely, the markers beta-hcg, afp and ldh are only elevated in about 60% of patients. therefore, micrornas of the clusters mir-371-3 and mir-302/367 were proposed as novel serum-based markers. we evaluated four of the candidate mirnas (mir-371a-3p, mir-372-3p, mir-373-3p and mir367-3p) with regard to their usefulness as tgct markers. overall, serum samples from 166 tgct-patients and from 106 controls were analysed using quantitative pcr. the first 50 consecutive patients and 20 controls were analysed for all four mirnas. after roc-analysis only the marker with the greatest discriminative power was studied further. the decline of mirna expression after orchiectomy was quantified in 134 cases and in 27 metastasized cases the marker was analysed repeatedly during the course of chemotherapy. additionally 10 cases with relapsing disease were studied. the mir-371a-3p featured the highest discriminative power (area under the curve: 0.94; 95% confidence interval [95% ci]: 0.874-0.982). in the entire cohort, patients could be distinguished from controls with a sensitivity of 88.7% (95% ci: 82.5-93.3%) and a specificity of 93.4% (95% ci: neuroendocrine tumor of the adrenal gland: an unusual manifestation of tsc c. müller-hofstede, j. horvath, b. dworniczak, p. wieacker institute of human genetics, university of münster, germany we report on a young woman asking for the recurrence risk of the neuroendocrine tumor of her mother deceased at the age of 38. her mother clinically presented because of therapy-resistent hypertonia, dyspnoe, progressive edema in the legs and face and a caput medusae. mri scan revealed a tumor (11×12 cm) in the right adrenal gland with lymph node metastases compressing the v. cava inferior and synchronous metastases in lung and liver. laboratory examinations showed highly elevated levels of cortisol and adrenocorticotropin (acth). cerebral mri was normal suggesting an ectopic acth secretion by a non-pituitary tumor. histologically, an undifferentiated, largely necrotic tumor was described so that the neuroendocrine nature of the tumor could not be proven. she died within three weeks after diagnosis. on suspicion of multiple endocrine neoplasia type 1 we initially performed a sequence analysis of men1 on tumor dna by next generation sequencing without detection of a pathogenic mutation. thereupon the molecular genetic panel analysis (nf1, ret, sdhb-d,tmem127, tsc1, tsc2, vhl) uncovered the heterozygous mutation c.3379c>t (p.arg1127trp) in the gene tsc2. this mutation is already described as pathogenic (hu et al. 2014 ). in the tumor dna the allele frequency of the normal allele mounted up to 10%, whereas the allele frequency of the mutant allele came to 90% pointing to a loss of heterozygosity (loh). the mutation was confirmed by sanger sequencing. taken all together, we assumed, that the mutation in the gene tsc2 represents a germline mutation. mutations in the suppressor genes tsc1 and tsc2 cause tuberous sclerosis, an autosomal-dominant disorder, resulting in hamartomatous tumors in the heart, brain, kidneys, skin and other organs. once in a while it is discussed whether neuroendocrine tumors (nets) represent a characteristic of tsc. there are some case reports describing nets in the context of tsc, but mainly in connection with nets of the pancreas (e. g. insulinoma) or the pituitary. to the best of our knowledge there exists only one case report of a bronchial carcinoid as a result of a germline mutation in tsc1 (dworakowska et al. 2009 ) and no description of net of the adrenal gland due to a mutation in tsc1 or tsc2. ngs provides the opportunity of wide-spread testing, even post-mortem, in order to get clarification for the descendants. although in our case we could not distinguish if the mutation detected represents a germline mutation or a somatic mutation, we were able to offer a predictive testing to the daughter and other family members. we report on a rare case of net of the adrenal gland because of mutation in the gene tsc2. this case illustrates that in the differential diagnosis of nets, tsc genes should also be considered. germ cell neoplasia in situ (gcnis) is the precursor lesion of testicular germ cell tumours (tgct). if detected clinically, this lesion may herald a pending tgct. unfortunately, the only way of diagnosing gcnis is by testicular biopsy and subsequent immunohistochemical examination. therefore, non-invasive methods of diagnosis are required. mirnas of the mir-371-3 and mir-302/367 cluster had been suggested as serum biomarkers of full-blown tgcts. we aimed to explore the utility of these mirnas for the detection of the pre-invasive stage of tgcts and we looked to the expression of two mirnas in serum samples of 27 gcnis patients. psmc3ip located on chromosome 17q21 is a putative tumor suppressor gene that encodes for the nuclear psmc3 interacting protein. the protein functions as coactivator of steroid hormone mediated gene expression and is important for rad51 and dmc1-mediated homologous recombination during dna repair of double-strand breaks. recently germline variants in psmc3ip, also known as gt198, tbip, and hop2, have been identified with low frequency in early onset familial breast and ovarian cancer (hboc) patients and in a patient with apparently sporadic early onset breast cancer. somatic variants in psmc3ip are frequently observed in breast, ovarian, and fallopian tube cancers. in this study, we analyzed a cohort of 166 brca1/2 mutation-negative hboc (n = 158) or early onset sporadic breast cancer patients (n = 8) for variants in psmc3ip. we identified seven different heterozygous variants in 8 out of 166 index patients: c.-115g>a (rs191843707); c.-70t>a (rs752276800); c.-37a>t (rs199620968); c.-24c>g (rs200359709); c.519g>a p.(trp173*); c.537 + 51g>c (rs375509656); c.*24g>a. these variants were not listed or at very low frequency (<1%) in the exac database. carriers of psmc3ip germline variants were mostly (6/8) affected by early onset breast cancer (median age of onset 36 years). for three out of seven different variants (c.-115g>a, c.519g>a, and c*24a>g), a possible impact on psmc3ip expression or function was observed. the stop mutation c.519g>a p.(trp173*) was found in two sisters, which were both diagnosed with unilateral breast cancer at age 33. the premature stop codon is located within the dna-binding domain of psmc3ip and is predicted to induce nonsense-mediated mrna decay (nmd). remarkably, c.-115g>a was already described in familial breast and ovarian cancer, and was found once in this study in a female that developed unilateral breast cancer at the age of 33 years. the variant c.-115g>a (rs191843707) was shown to induce a slightly, albeit significant decrease of reporter gene expression. the c.24*g>a variant was identified in a woman diagnosed with unilateral breast cancer at the age of 36 years. luciferase reporter assays indicated an impaired effect of c.24*g>a on microrna binding. germline variants in psmc3ip are present in breast and ovarian cancer families. whether mutated psmc3ip is a new risk factor for early onset breast/ovarian cancer in families with hboc and/or apparently sporadic early onset breast cancer remains to be shown. 86.9-97.3%) with this marker. in patients without metastases the mir-371a-3p expression declined significantly after surgery. in metastasized cases the levels dropped sharply after chemotherapy. all of the 10 relapses had elevated mir-levels, and expression decreased upon chemotherapy. mir-371a-3p has significantly higher sensitivity than each one of the classical tgct markers and than a combined panel of beta-hcg, afp and ldh (87.8% vs 50.4%). in non-metastasised seminoma the mir-371a-3p expression depended significantly on tumour size. mir-371a-3p is highly sensitive and specific for tgct. it correlates with the stage of disease and with treatment effects and it therefore fulfils the prerequisites of a valuable serum-based biomarker. the significant association with tumour bulk in localised disease provides evidence for the tgct being the primary source of mir expression. the sensitivity of mir-371a-3p surpasses that of classical tgct markers by far, and thus it may become the new gold standard for serum diagnostics of tgct in the coming years. co-occurrence of radioulnar synostosis and amegakaryocytic thrombocytopenia (rusat) was initially described as an inherited thrombocytopenia syndrome that is caused by a mutation in hoxa11. in three simplex patients, de novo missense mutations in mecom were reported as an alternative origin of the disease (rusat2). mecom, identified as a common ecotropic viral integration site 1 (evi1) in murine myeloid leukemia, is known as a key transcriptional regulator in hematopoiesis and sporadic myeloid leukemia. we report here on a novel mecom mutation cys766gly (uniprot q03112-1) identified by whole exome sequencing in a family with rusat, hearing impairment, hand dysmorphisms, and patellar hypoplasia in four patients spanning three generations. notably, two of four affected individuals in our family developed a myeloid malignancy. the novel mecom missense mutation cys766gly affects a heavily conserved cysteine residue in c 2 h 2 -zinc finger motif 9 in the c-terminal zinc finger domain of mecom. this residue is crucial for the tetrahedral coordination of a zinc ion stabilizing the zinc finger conformation and thus, is essential for dna binding of the c-terminal zinc finger domain. our findings reconfirm the causality of mecom mutations and indicate that mecom mutations also need to be considered in familial rusat patients. in addition, we report for the first time that mecom germline mutations targeting the c-terminal zinc finger domain are associated with an increased risk for myeloid malignancies. this extends the rusat2-associated phenotype and proposes that mecom germline mutations can cause a genetic predisposition to myeloid malignancy. (z., b. and s., d. contributed equally to this work) many genes that harbor rare mutations which entail a medium or high risk for breast cancer (bc) belong to dna double strand repair and have been identified by linkage analysis or by sequencing of candidate genes in bc families. in addition, a considerable number of common but low risk germline variants have been found in genome wide association studies. however, these predisposing factors yet only explain a fraction of bc cases. with the intention to identify low frequency variants conferring an intermediate bc risk, we performed an association study using a candidate gene approach and testing dna repair capacity with the micronucleus test (mnt) as an additional second phenotype. rs3810813 in the slx4/fancp gene showed an association with bc that was pronounced in younger cases and was confirmed in a verification cohort (combined analysis of 1,448 cases, 2593 controls: or = 2.19 (1.45-3.30) , p = 0.00012 for cases ≤40 years). genotyping additional snps and imputation revealed a specific european haplotype of ca. 150kb length that spans slx4 and adjacent genes. it is tagged by 6 the observed mutual dependence of the two phenotypes allowed a considerably improved interpretation of the results: (i) the unknown causal variant on the haplotype can be assumed to be present mostly in cases, indicating a rare variant with a rather strong effect; (ii) using this information on the two phenotypes in the association between the mnt results and bc improved considerably the identification of the specific risk among cases (<60 years) who carried the haplotype. roc curves for bc depending on mnt results revealed that the stratification on carriers of the haplotype increased the auc from 0.65 (p = 0.0007) to 0.94 (p = 0.0001). both associations can be best explained by a risk variant carried by a fraction of the haplotypes that is enriched in early onset bc cases. slx4 is the only gene in the tagged region which can be functionally related to both associated phenotypes, while for the other genes no connection to bc or dna repair is reported. inherited dna repair mutations: are they modifiers of brca1 and brca2 penetrance and age at onset of hereditary breast and ovarian cancer? background: inherited mutations in brca1 and brca2 are the most common causes of hereditary breast and ovarian cancer (hboc). the risk of developing breast cancer by age 70 in women carrying a brca1 mutation is 57-65% and 45-55% in brca2 carriers. however, mutations in brca1 and brca2 only explain about 25% of all hboc cases. the lifetime risk varies between families and even within affected individuals of the same family. the cause of this variability is unknown but it is hypothesized that additional mutations or rare variants in genes that are possibly interacting with brca1/2 in different dna-repair pathways contribute to this phenomenon. methods: we obtained samples of 181 patients positive for brca1 or brca2 mutations and an age-of-onset (aoo) of breast cancer below 35 or above 60 years of age from the german consortium for hereditary breast and ovarian cancer. panel sequencing was done to screen germline dna for mutations in 311 genes involved in different dna-repair pathways. variants were classified into five classes according to a modified version of plon et al (2008) . only truncating mutations and known pathogenic missense mutations were considered pathogenic or likely pathogenic. results: the patient group with an early aoo (93 women) had developed breast cancer at a mean age of 26.4 years (± 2.06) and the control group (88 women) had developed breast cancer at a mean age of 69.5 years (± 7.05). a total of 4,293 variants were detected in all patients and 54 of these (1.3%; 95% ci, 0.96%-1.64%) were presumed to be deleterious. mutations were found in 45 genes other than brca1 and brca2. mutations were mainly found in single-strand break repair (ssbr 29%), double-strand break repair (dsbr 26%) and checkpoint factors (13%). the rest were found in genes with other functions such as brca1/2 interactors, centrosome formation, and signal transduction. the putative mutations were found in 26 women of the control group (29.5%; 95%ci, 20.3%-40.2%) compared to 36 women of the patient group (38.7%; 95% ci, 28.8%-49.4%). the incidence of germline mutations in dna-repair genes did not differ according to the age of onset (p = 0.2). prevalence of additional germline mutations in dsbr in patients (13%) was not significantly different from prevalence of dsbr mutations in controls 10.2% (p = 0.6) conclusions: the preliminary results failed to show a difference in mutation load between the two cohorts of brca1/2 carriers sorted by age of onset. larger studies are needed and may provide further insight into the role of mutation load in hboc age of onset of brca1/2 carriers. objective: glioblastoma stem-like cells (gscs) carry stem cell features and therefore seem to be responsible for tumor initiating, maintaining, recurrence and chemo-and/or radiotherapy resisting. the current knowledge on genetic and transcriptomic characteristics of these cells especially in comparison to glioblastoma tissue is still limited. the aim of this study is to compare the genetic and genomic profile of glioblastoma tissue and gscs. thereby, differences in involved genes and affected pathways on dna level as well as on gene expression level are identified. material and methods: peripheral blood and tumor tissue were obtained from patients with glioblastoma. tumor tissue derived explant cell culture and serum-free culture were established. based on multi-parameter magnetic-activated cell sorting (macs) technique, cd15 and cd133 labeled cell subpopulations of gscs could be isolated. the tumor tissue, serum-free culture, and the isolated cell subpopulations as well as blood were analyzed by snp array and gene expression (excluding blood) in a paired design. for preliminary characterization of gscs in the serum-free culture we confirmed the stem cell features of gscs by the expression of nestin, sox2, and cd133 (applying immunofluorescence staining). results: our results of snp array analyses showed genetic aberrations in all analyzed cellular entities (tumor tissue and cell subpopulations, e. g. gain of chromosome 7, loss of 10q23.31, loss of 10q11.1->q26.3, and complete loss of chromosome 10). furthermore, distinct genetic differences between the cell subpopulations and tumor tissue were observed (e. g. loss of chromosome 4 and segmental uniparental disomy of 9p24.3->p21.3, only in cancer stem-like cell subpopulations). in addition, we detected many possibly candidate cancer genes and pathways which may have an influence on tumorigenesis. gene expression analyses revealed strongest differences between fresh tumor tissue and serum-free culture based cells, where more than a third of investigated genes were affected. when contrasting fresh tumor tissue with stem cell marker positive serum-free cultured cells, 1,106 genes were upregulated in the stem cell marker positive cells, whereas 1,533 genes where upregulated in fresh tumor tissue. within these genes, strongest enriched pathways in stem cell marker positive cells included positive regulation of cell cycle and cancer-related pathways, whereas in fresh tumor tissue predominantly immune-related pathways were found, e. g. myeloid leukocyte activation, inflammatory response and phagocytosis. conclusion: differences between gscs and tumor tissue using snp array analyses and gene expression were detected. our results may help to get more information about the molecular pathomechanisms of glioblastoma. it still needs more investigations on the field of genetic and genomic analyses between gscs and glioblastoma tissue to identify novel potential targets for therapy development. background: fanconi anemia (fa) is a rare inherited chromosomal instability syndrome associated with bone marrow failure as well as myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). one-third of fa individuals exhibit bone marrow cytogenetic clones, notably gains of 1q and 3q and/or loss of 7/7q, and 15% to 30% of fa patients developed mds/aml. in recent years, the application of high throughput technologies has revealed recurrent somatic mutations in genes implicated in myeloid malignancies. as additional genetic maladies facilitating mds/aml development in fa is lacking, we aimed to elucidate whether these mutations would be present in fa patients with mds/aml. methods: using illumina trusighttm myeloid sequencing panel (san diego, ca), we performed next-generation sequencing (ngs) on dna extracted from bone marrow specimens from 17 fa mds/aml patients registered in the european working group of childhood mds. the sequencing panel targeted 54 genes frequently mutated in hematologic neoplasms. results:. ten of the 16 (62.5%) evaluable patients had 18 lesions (1 to 3 mutations per patient; 14 missense, 1 nonsense, 2 insertion and 1 duplication) in 13 genes. the presence of a somatic mutation did not appear to correlate with complex karyotype or −7/7q. all affected genes occurred in isolation with exception of runx1 and kras. while 13 of the mutations were pathogenic, 5 were variance of unknown significance. mutations in genes involved in epigenetics (dna methylation, chromatin maintenance and cohesin complex; idh2, tet2, dnmt3a, idh1, ezh2, rad21 and asxl1) and mutations in transcription factor genes (runx1, ikzf1 and etv6) represented the most frequently affected genes. this was followed by mutations of genes encoding signaling molecules including the ras pathway (kras and ptpn11). altered runx1 was the most common lesion and occurred in individuals with aml, raeb or raeb-t. one patient with refractory anemia with ring sideroblasts (rars) had a mutation in the spliceosomal gene, sf3b1. conclusions: while the most common mutations encountered in sporadic cases of mds were in genes involved in rna splicing and epigenetics, these two broad categories of genes appeared to have less influence in our fa patients. most mutations were nonrecurring suggesting that there is no specific mutation pattern of these genes in fa-related mds/aml. however, runx1 mutations and also mutations involved in genes of the ras pathway appear to play a pathogenic role in fa mds/aml development. taken together, the data suggests that mutations in genes that cause clonal hematopoiesis in the population at large do not contribute significantly to fa hematopoietic clonal disease; however, particularly acquisition of runx1 and ras pathway alterations promote malignant myeloid disease progression. more extensive studies analyzing more patients are necessary to further define the secondary hits leading to fa myeloid disease. chromatin remodeling is a complex process shaping the nucleosome landscape, thereby regulating the accessibility of transcription factors to regulatory regions of target genes and ultimately managing gene expression. the swi/snf (switch/sucrose nonfermentable) complex remodels the nucleosome landscape in an atp-dependent manner and is divided into the two major subclasses brahma-associated factor (baf) and polybromo brahma-associated factor (pbaf) complex. somatic mutations in subunits of the swi/snf complex have been associated with different cancers, while germline mutations have been associated with autism spectrum disorder and the neurodevelopmental disorders coffin-siris (css) and nicolaides-baraitser syndromes (ncbrs). css is characterized by intellectual disability (id), coarsening of the face and hypoplasia or absence of the fifth finger-and/or toenails. so far, variants in five of the swi/snf subunit-encoding genes arid1b, smarca4, smarcb1, arid1a and smarce1 as well as variants in the transcription factor-encoding gene sox11 have been identified in css-affected individuals. arid2 is a member of the pbaf subcomplex, which until recently had not been linked to any neurodevelopmental phenotypes. in 2015, mutations in the arid2 gene were associated with intellectual disability. in this study, we report on two individuals with private de novo arid2 frameshift mutations. both individuals present with css including id, coarsening of facial features, other recognizable facial dysmorphisms and hypoplasia of the fifth toenails. hence, this study identifies mutations in the arid2 gene as a novel and rare cause for css and enlarges the list of css-associated genes. the ubiquitin pathway is an enzymatic cascade including activating e1, conjugating e2, and ligating e3 enzymes, which governs protein degradation and sorting. it is crucial for many physiological processes. compromised function of members of the ubiquitin pathway leads to a wide range of human diseases, such as cancer, neurodegenerative diseases, and neurodevelopmental disorders. mutations in the thyroid hormone receptor interactor 12 (trip12) gene (omim 604506), which encodes an e3 ligase in the ubiquitin pathway, have been associated with autism spectrum disorder (asd). in addition to autistic features, trip12 mutation carriers showed intellectual disability (id). more recently, trip12 was postulated as a novel candidate gene for intellectual disability in a meta-analysis of published id cohorts. however, detailed clinical information characterizing the phenotype of these individuals was not provided. in this study, we present seven novel individuals with private trip12 mutations including two splice site mutations, one nonsense mutation, three missense mutations, and one translocation case with a breakpoint in intron 1 of the trip12 gene and clinically review four previously published cases. the trip12 mutation-positive individuals presented with mild to moderate id (10/11) or learning disability [intelligence quotient (iq) 76 in one individual], asd (8/11) and some of them with unspecific craniofacial dysmorphism and other anomalies. in this study, we provide detailed clinical information of eleven trip12 mutation-positive individuals and thereby due to its heterogeneous etiology, primordial growth retardation is often a challenge for geneticists and clinicians in respect of diagnosis, therapy and prognosis. thus, pinpointing its genetic origin is required for a personalized treatment and prognosis. one syndrome mainly characterized by intrauterine and postnatal growth is silver-russell syndrome (srs), a clinically and molecularly heterogeneous disorder with a considerable overlap with other syndromes. in only 60% of patients with the characteristic srs phenotype the diagnosis can be confirmed molecularly, but 40% of cases remain without molecular diagnosis. in fact, in clinically less well characterized patients referred for diagnostic testing, the detection rate is less than 20%. however, systematic investigations on the contribution of mutations in genes which may be considered in the differential diagnoses of srs are still missing. we examined 60 patients referred for molecular testing of srs but without molecular alterations associated with srs by ngs. a targeted ngs approach comprising 26 genes implicated in the differential diagnoses of srs or suggested as srs candidate genes was performed. in 5 patients fulfilling the criteria of srs accordingly to our recently developed clinical scoring system, disease-causing variants were found. these patients carried mutations in genes associated with bloom syndrome, mulibrey nanism, kbg syndrome, short syndromes or ig-f1r-associated short stature, respectively. indeed, some of the differential diagnoses detected in our cohort have a major impact on clinical management, including cancer screening because of a high risk for tumor development. furthermore, we did not identify any pathogenic mutation in one of proposed srs candidate genes (e. g. mest, grb10, copg2), thus raising the question whether these genes are indeed involved in the etiology of srs. we show that a (targeted) ngs approach is an important tool to identify the genetic cause in patients with unexplained growth retardation. furthermore, our data show (positive) clinical scoring in srs should not impede the consideration of differential diagnoses and other molecular causes. submicroscopic deletions of chromosome band 2p25.3 have been reported in more than 20 patients. common clinical features include intellectual disability/developmental delay, central obesity and behavioural difficulties. myt1l became the main candidate gene for id and obesity since it is deleted or disrupted in all published patients. however, reports of deletions affecting only this gene and even more so of deleterious myt1l sequence variants are very rare. to our knowledge, until now only two patients with de novo myt1l point mutations have been reported. in the present study, we analysed a cohort of individuals with intellectual disability of unknown aetiology and their unaffected parents by whole exome sequencing. we identified de novo myt1l sequence variants in two out of 311 patients. patient 1 carried a nonsense mutation (c.1531g>t, nm_015025.2; gly511*) whereas patient 2 carried a direct splice site mutation (c.2769-2a>g). according to prediction algorithms, both detected myt1l variants are deleterious (patient 1: sift score 0, cadd score 42; patient 2: cadd score 24.6). in addition, patient 2 carried a de novo splice site variant in setd1b. however, this variant is predicted to be benign (cadd score 2.5) as well as a known snv (rs749218728, maf 0.0000323). a comprehensive clinical characterisation of the two patients yielded only mild or moderate intellectual disability, behavioural problems and muscular hypotonia as common clinical signs. surprisingly, obesity was only present in patient 2. postnatal tall stature and transient microcephaly were present in one patient each. this clinical picture is compared to the published phenotypes of patients with myt1l point mutations, patients with microdeletions of only myt1l and patients with larger 2p25.3 deletions. with the reduced penetrance regarding obesity, the clinical picture of patients with myt1l mutations is becoming more and more unspecific. the retina and anterior neural fold homeobox gene (rax) controls the embryonic eye development and is involved in human autosomal-recessive microphthalmia. so far only a few compound heterozygous mutations in rax have been described in microphthalmia patients. we report a first case of microphthalmia caused by a novel homozygous mutation in rax. the 8-month-old patient was born to consanguineous parents and presented with extreme microphthalmia, panhypopituitarism and developmental delay. mri of the brain showed bilateral agenesis of the anterior visual pathway and tractus opticus. ocular ultrasound confirmed bilateral anophthalmia. additionally, dysgenesis of the corpus callosum and an abnormal pituitary gland have been detected. the first child of these parents, who died shortly after birth, had also been diagnosed with bilateral anophthalmia. using panel diagnosis of the disease associated genome, we identified the homozygous pathogenic variant c.112del, (p. 138fs) in rax. we performed a segregation analysis and confirmed that both parents are heterozygous for this variant. so far developmental delay and panhypo-expand the clinical spectrum of the trip12 gene in non-syndromic intellectual disability with or without asd. background: whole exome sequencing (wes) using next generation sequencing has proven to be a powerful tool in determining the underlying genetic cause of rare disorders. here, we show, that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering further disease-causing mutations. clinical report and genetic findings: we report follow-up data of a previously published consanguineous family with two children, a boy and a girl, suffering from severe encephalopathy, hypotonia, microcephaly and retinal dystrophy. wes had shown a homozygous intronic splice variant in pgap1 (c.1090-2a>g;p.?) causative for the symptoms. both parents were heterozygous carrier for the pgap1 variant (granzow, paramasivam et al, mol cell probes 2015) . in the next pregnancy, the unborn child presented hydrops fetalis, omphalocele, short tubular bones and cystic kidneys. chorionic villus sampling showed the fetus to be homozygous for the pgap1 variant. however, neither of these symptoms fit with a pgap1-associated disorder. additional wes of fetal dna and re-evaluation in the family showed a homozygous nonsense variant in ift140 (c.g3577t;p.e1193*) consistent with a diagnosis of mainzer-saldino syndrome (mss) which is characterised by the association of renal disease, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia, as a second diagnosis in the fetus. again, both parents were shown to be a heterozygous carrier for the ift140 variant. yet, as omphalocele was not accounted for by any of the identified conditions, a third genetic cause cannot entirely be excluded. alternatively, omphalocele may be a rare manifestation of mss, or be the result of a combination of both disorders. the couple opted for induced abortion. discussion: it is estimated that an individual carries multiple heterozygous variants for autosomal recessive disorders in his or her genome. especially in consanguineous families, this results in an elevated risk for children with more than one disorder. in recent publications of clinical exomes, double diagnoses have been reported in 0 to 12% of investigated subjects. thus, the possibility of more than one causative gene should be carefully explored when working with wes and re-evaluation in case of additional clinical symptoms within a family should be considered. also, follow-up of families with rare genetic disorders may lead the clinical geneticist beyond the assumed single cause to multiple single gene disorders in the same family. conclusion: using wes, we have identified two independent single gene disorders in a consanguineous family demonstrating that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering multiple disease-causing mutations in one family. we present a case study using molecular cytogenetic approaches on a 3 year old boy presenting with microcephaly-brachycephaly, macroglossia and absent speech development. the boy is the first child of healthy, consanguineous parents of pakistani origin. following an uncomplicated pregnancy, the hypotrophic newborn was delivered at 37 weeks weighing 2610 g. in the third month of life, the baby had viral meningitis. regular pediatric follow-up revealed psychomotor delay with hypotonia. creatine kinase, lactate and fibroblast growth factor 21 measured in serum were high. subsequent investigations at the age of 16 months included brain mri, electroencephalogram and muscle biopsy that gave hints of a mitochondriopathy or potential neuropathy of axonal type. due to the suspicion of a complex mitochondriopathy, whole exome sequencing was performed using a sureselect human all exon kit (agilent, 50mb v5) on a hiseq 2500 (illumina). the analysis revealed a heterozygous microdeletion of 121 kb on chromosome 9q34.3 which was classified as an unclear variant (uv), ehmt1-gen was not affected. re-examination of proband's dna using array cgh detected a larger 180 kb heterozygous deletion in the 9q34.3 region (arr[hg19] 9q34. 3(140,395,510-140,575,736) x1), encompassing exon 1 of ehmt1 (euchromatin histone methyltransferase 1; transcript nm_ 024757.4). haploinsufficiency of this gene results in kleefstra syndrome (omim 610253), a multisystem disorder due to either microdeletions in 9q34.3 encompassing ehmt1 or intragenic point mutations. mlpa analysis (ehmt1 mpla-kit p340) of parental dna further indicated a de novo origin of the deletion in our proband. a similar deletion has been described previously in a case presenting with clinical features of kleefstra syndrome [2] strengthening the importance to include the 5' part of ehmt1 in sequencing as well as cnv screening. in summary, our study clearly shows that array cgh is a valuable complementary approach to ngs especially for poorly covered regions in ngs i. e. exon 1 of many transcripts. we report on a three-generation family with variable manifestations including delayed and incomplete tooth eruption, early tooth loss due to short dental roots, acroosteolysis, osteoporosis, tendon ruptures, joint hypermobility, muscle weekness, glaucoma, neurological features, and psoriasis. after detection of elevated cd169/siglec1-expression on monocytes and an upregulation of interferon-stimulated gene transcripts, singleton-merten syndrome was diagnosed. the novel heterozygous mutation c.992c>g (p.thr331arg) in ifih1 was found in three affected family members. singleton-merten syndrome is a very rare autosomal dominant interferonopathy, so far described in not more than four families. until now, only two different gain-of-function mutations in ifih1 have been detected. mutations in ifih1 are also associated with aicardi-goutière syndrome and recently features of both conditions were found in the same family. our findings expand the mutational spectrum of singleton-merten syndrome and demonstrate the high intrafamiliar variability associated with mutations in ifih1. pituitarism have not been described in association with rax mutations. therefore we conducted array comparative genomic hybridization and karyotyping in the index patient. both tests gave normal results. prenatal diagnosis by chorionic villus sampling in the next pregnancy excluded a homozygous carrier status for this rax mutation. mutations in the phf6 gene are associated with borjeson-forssman-lehmann syndrome (bfls), an x-linked intellectual disability disorder affecting mainly males. female carriers usually show no or mild clinical signs. however, recent studies described females with de novo phf6 gene defects (mutations, deletions) and severe phenotypes resembling coffin-siris syndrome [1, 2] . here, we report on a girl with a maternally inherited phf6 mutation and a phenotype resembling those described previously in affected females. the mother had learning difficulties and mild dysmorphological features (hypertelorism, prominent forehead). when seen at age 12 months, the proposita showed muscular hypotonia, was unable to sit and had limited head control (developmental delay 6 months). dysmorphic features included scaphocephaly, hypertelorism, a small flat nose with anteverted nares, low set, prominent ears, a high, narrow palate, absent labia minora and linear skin pigmentation on the thighs. ophthalmologic investigation identified strabism convergens of the left eye, hyperopia and an excavated papilla with a pale optical nerve. ultrasound showed patent foramen ovale, tricuspid insufficiency and a unilateral incomplete duplication of the renal pelvis. karyotyping performed elsewhere was normal (46,xx). results of a genome-wide snp array analysis (affymetrix cytoscan hd) were also normal. using a targeted ngs approach for syndromic and non-syndromic developmental delay encompassing over 1200 brain related genes (mpimg-1-test), we identified a heterozygous lof-mutation c.88c>t (p.gln30*) in the phf6 gene (encoding phd finger protein 6). in addition, a human androgen receptor (humara) assay using blood dna showed a highly skewed x-inactivation (91:9). segregation analysis indicated a maternal origin of the variant. the mother also had skewed x-inactivation in blood. her husband and other daughter tested normal for the c.88c>t variant. here, we describe the first female patient with a maternally inherited phf6 mutation and a severe phenotype. the mild phenotype in the mother might be due to different patterns of x-inactivation or to (undetected) mosaicism. this report represents the 12th case of a severely affected female patient with a phf6 gene defect. findings support the assumption [1] that the female phenotypes of bfls might be more common than previously estimated. toms typical for ada2 deficiency such as fever, livedo racemosa, abdominal colics, arthralgias, and raynaud's phenomenon were observed months later. cecr1 sequencing (nm_001282225.1) revealed two previously described pathogenic missense mutations: c.140g>c, p.(gly47ala) and c.506g>a, p.(arg169gln). compound heterozygosity was confirmed by parental analysis. to the best of our knowledge, this combination of mutations has not been described until now. the p.(arg169gln) is considered as founder mutation in the dutch population, but first phenotype-genotype analyses did not allow further prediction of clinical outcomes. ada2 deficiency should be considered in patients with childhood stroke despite the absence of systemic inflammation and cerebral vasculitis. congenital hyperinsulinism (chi) has been described as heterogeneous entity caused by at least 9 different genes. in 1991, tein et al. first described a defective activity of l-3-hydroxyacyl-coa dehydrogenase in a 16-year old patient with hypoketotic hypoglycemic encephalopathy. biochemical markers of l-3-hydroxyacyl-coa dehydrogenase deficiency (schad) are high 3-oh-glutarate excretion in urine and c4-oh-carnitine in plasma. the clinical presentation is very heterogeneous with regard to age of onset, severity of symptoms as well as response to medical treatment and leucine-sensitivity. in some patients even a near total pancreatectomy was performed. schad dependent hyperinsulinism (hhf4) is a rare autosomal recessive disorder that is caused by mutations in the gene hadh. here we describe 4 patients from 3 unrelated families out of a cohort of 136 chi patients mainly from central europe. patients 1 and 2 are siblings from unrelated parents. the older brother manifested with hypoglycemic convulsions at the age of 5 weeks. a subtotal pancreatectomy was performed in an outside academic hospital. in further course he developed epilepsy and has been treated with diazoxide and anticonvulsants. the 2nd child was born with hypoketotic hypoglycemia and chi was diagnosed in first days of life. diazoxide treatment stabilized blood glucose and both children were referred to our pediatric endocrinology at the age of 8 years and 10 months, respectively. mutational analysis revealed the homozygous variant c.547-3c>g within the region of the splice acceptor site in intron 4 of the hadh gene in both affected children. this change is neither registered in exac nor described in the mutation databases hgmd or in the literature and was predicted to disrupt proper splicing. we then completed mutational analysis in unidentified patients of our chi cohort with diazoxide responsiveness and known or suspected consanguinity. patient 3 was born to consanguineous parents and chi manifested in the girl at neonatal age with hypoketotic hypoglycemia. she was successfully treated with diaxozide. later, she developed convulsions and statomotoric developmental delay. the homozygous splice mutation c.636 + 471g>t in intron 5 of hadh was detected in the child. the parents were identified as heterozygous carriers. in patient 4, a girl born to consanguineous turkish parents, chi manifested at the age of 6 months with hypoglycemic seizures. she responded well to diazoxide treatment. a homozygous missense mutation (c.406a>g; p.lys136glu) in exon 3 of hadh was detected in the patient and her parents were heterozygous carriers. hadh mutations in case 3 and 4 have been previously described in probands of turkish descent and appear to be founder mutations in the turkish population. in conclusion, we recommend hadh mutation analysis to be considered in chi children with unknown cause and known consanguineous pedigrees or originating from populations with higher prevalence of consanguinity. homozygous or compound heterozygous mutations in cecr1 (cat eye syndrome chromosome region, candidate 1) have recently been identified to causing deficiency of adenosine deaminase 2 (ada2; dada2) with childhood polyarteritis nodosa (pan) (omim # 615688). this inflammatory vasculitis affects the skin, and inner organs (predominantly kidneys and gastrointestinal tract) and also shows a high risk of ischemic stroke, brain hemorrhage as well as peripheral neuropathy. using whole-exome sequencing it was also found that the six adult patients (aged 20-48) described by sneddon in 1965 (sneddon syndrome, omim # 182410) likewise carried compound heterozygous cecr1 mutations. sneddon syndrome is characterized by a combination of dermatologic features (livedo racemosa) and ischemic brain infarctions. recently, clinical and genetic data of more than sixty ada2 patients have been reviewed and underlined the wide clinical variability in age at onset, clinical findings, outcome of neurological involvement, and additional hematological symptoms. typically, stroke has been reported to follow systemic inflammatory disease and predominantly affects posterior and central brain areas. here we describe one of the rare patients in whom acute mesencephalic stroke preceded systemic inflammation and presented as initial clinical symptom. symptriploidy is a recurrent finding in prenatal diagnostics. in a small number of individuals, correction of triploidy has been suggested based on the finding of (mosaic) genome-wide uniparental disomy (upd). we here investigated uncultured und cultured amniotic cells (ac) and placental tissue from a fetus, in which ultrasound examination in the 17 + 0th week of gestation revealed growth retardation, left diaphragmatic hernia with parts of stomach and bowel localized in the chest, dextrocardia, short nasal bone and single umbilical artery. these findings were confirmed at the 18 + 1th week when the pregnancy was terminated. the pregnancy was conceived spontaneously by a 28-year-old mother and a 31-year-old father, both healthy and with uneventful family history. the parents were non-consanguineous and carry a normal karyotype. microsatellite analyses of uncultured ac obtained at initial presentation showed for chromosomes 13, 18 and 21 a pattern suggesting triploidy with only biallelic presentation. while y-chromosomal sequences were lacking the x-chromosome showed, unexpectedly a rather disomic pattern. metaphase yield on cultured ac was low but showed a mosaic karyotype 48,xxx-,+10[20]/47,xxx[17], which was confirmed for several chromosomes by interphase fish. remarkably, a triploid clone was cytogenetically not detected. thus, we performed further analyses using microsatellite markers, oncoscan technology and fish. these studies unraveled in uncultured ac a pattern suggestive of triploidy with the supernumerary chromosomal complement derived from a maternal isodisomy with the notable exception of the x chromosome. in cultured ac and placental tissue for all chromosomes, except x and 10, a diploid pattern was observed with alleles from both parents identical to those in the uncultured ac. trisomy x was confirmed in both tissues with the supernumerary chromosome x being of paternal isodisomic origin. the trisomy 10 was seen only in cultured ac, and likely represents a pseudomosaic which nevertheless could not be proven due to insufficient yield of mitoses from the parallel cultures. finally, retrospective interphase fish on remnant uncultured ac showed two diploid clones, one disomic (approximately 40% of nuclei) and one trisomic (60%) for the x chromosome. the most likely explanation for the findings is a mosaicism for one diploid clone with genome-wide maternal isodisomy and a second diploid but bi-parental cell line with paternal trisomy x. given the identity of the (maternal) alleles in both clones our findings suggest that originally a triploid clone due to a maternal division error/inclusion of a polar body ii existed which underwent (erroneous) triploidy rescue resulting in one diploid biparental clone and one haploid clone of maternal origin that underwent haploid rescue resulting in genome-wide maternal isodisomy. the biparental clone with trisomy x either resulted from a sperm with two x-chromosomes or an erroneous x-duplication during trisomy rescue. since the introduction of non-invasive prenatal diagnosis (nipd, e. g. har-mony® test) in 2013, this test is frequently demanded and routinely applied in prenatal centers and medical practices. mostly, nipd is intended to detect autosomal trisomies (13, 18, 21), but also offers the possibility to analyze sex chromosomes. therefore, also sex chromosome aneuploidies (sca) (e. g. monosomy x (turner syndrome), triple x, xxy (klinefelter syndrome), xyy) are incidentally found. so far, in our prenatal center sca were detected in 7 pregnancies by har-mony® test, consisting of three pregnancies with monosomy x (turner syndrome) and two pregnancies with klinefelter syndrome (xxy). triple x and xyy were detected one time each. of the three cases with suspected monosomy x, the diagnosis of turner syndrome could only be confirmed in one case. this fetus also had a hydrops at week 10 + 0. for the other two fetuses, the chromosomal analysis of amniotic fluid revealed normal female karyotypes (46,xx). in both cases with suspected klinefelter syndrome, this diagnosis could be disproved by amniocentesis (karyotype 46,xy). in the pregnancies with assumed triple x and xyy, the true fetal karyotype was not further determined yet. from our experience, the rate of false positive results concerning the sex chromosome aneuploidies is noticeably higher than reported in two studies of nicolaides 2014 and hooks 2015. this has to be strongly considered in the counselling of patients who wish to know the fetal sex by nipd. congenital myopathies and congenital muscular dystrophies: will genetic testing replace muscle biopsy in the near future? congenital myopathies and muscular dystrophies are a group of inherited neuromuscular diseases with early onset and broad genetic and histopathological overlap. the diagnostic approach has considerably changed with next generation sequencing methods available. here, we describe the diagnostic value of genetic and histological methods in a cohort of 117 index patients and hence the efficacy of diagnostic procedures. 78 of 117 patients had a muscle biopsy as a first-tier approach. in 54 of 78 patients muscle biopsy was informative, leading to a classification in subgroups of cm or cmd. however, in only a few of these cases biopsy led to a specific diagnosis (e. g. merosin deficiency). in 55 of 78 patients genetic testing (candidate gene sequencing or ngs) was performed additionally to muscle biopsy as a second-tier diagnostic step, while 39 patients of the whole cohort received genetic testing only. in almost two-thirds of these 94 patients genetic testing identified known pathogenic or most likely pathogenic variations. these findings illustrate that genetic testing is superior to muscle biopsy in accurately diagnosing cm or cmd. in conclusion, we suggest that invasive muscle biopsy should be replaced by genetic testing as first-tier diagnostic procedure in patients with clinical signs of cm or cmd. nmd inhibition increases the amount of gaa-rna in patient's lymphocytes as well as in the cells of his parents. the residual function of the resulting protein has to be investigated. discussion and conclusion: rna analysis in lymphocytes with and without nmd inhibition is a simple method for analysing splice defects in all monogenic disorders with expression of the disease causing gene in lymphocytes. a further advantage for the patient is the use of blood cells instead of fibroblasts, because a skin biopsy can be avoided and analysis times are reduced. the exact characterization of pathogenic variants is an important aspect of diagnosis, prediction of disease severity and genetic counselling. in vitro nmd inhibition in lymphocytes of affected patients allows the characterization of splice defects. in the future successful inhibition of nmd in vitro might help to identify patients, who may profit from a therapeutic intervention with nmd inhibitors. even expression of a partial protein with low or no activity reduces the risk for the patient to develop antibodies hampering enzyme/protein replacement therapy. p-cling-067 12q14 microdeletion syndrome: a family with short stature and silver-russel (srs)-like phenotype. introduction: the silver-russel syndrome (mim 180860), first described independently by silver and russell in 1953, is a condition with intrauterine growth retardation, postnatal growth failure and other characteristic features, including relative macrocephaly (defined as a head circumference at birth ≥1.5 sd score (sds) above birth weight and/or length sds), prominent forehead, body asymmetry and feeding difficulties as recently defined in an international consensus statement. patients and methods: we report here on 3 first degree relatives with a silver-russel syndrome phenotype who presented with prenatal-and postnatal growth retardation, feeding difficulties, a prominent forehead and a failure to thrive. additional features such as dysmorphic facial features, periodically increased sweating, and scoliosis were present in one of the family members only, whereas learning problems and cardiac arrhythmia were present in one other. none of the patients had relative macrocephaly. high resolution array-cgh was performed to screen for cncs and mlpa to confirm the array-cgh result. results: no hypo-methylation of the imprinting center on 11p15 nor uniparental disomy of chromosome 7 and 14 were found in the index-patients. high-resolution array-cgh identified a 12q14.3 microdeletion of 1.67 mb (arr[grch37 12q14.3(65, 863, 528 ,640)×1). the heterozygous loss was confirmed by mlpa in the index patient and the other two affected family members (i. e. her brother and mother). the deletion includes the genes hmga2, llph, tmbim4, irak3, helb, grip1, and the pseudogene rpsap52. conclusion: to the best of our knowledge this is the first report on familial presentation of a silver-russel syndrome due to a microdeletion in 12q14.3. none of the patients had relative macrocephaly. supporting the hypothesis by takenouchi et al. that the causative gene for relative macrocephaly resides centromeric to hmga2, the region centromeric of hmga2 is not included in the deletion in our family. spastic ataxia of charlevoix-saguenay (sacs) is an autosomal recessive neurodegenerative disorder and is caused by homozygous or compound heterozygous mutations in the sacs gene. first symptoms of sacs are walking difficulties due to unsteady gait. further typical clinical features include spasticity, ataxia, pyramidal tract signs, nystagmus and dysarthria. here, we report on a 16-year-old female patient who initially presented with disturbances in motor abilities including frequent falls and high arched foot. cranial mrt was normal while nerve conduction velocity was significantly reduced. the patient's parents did not show any clinical features. since no pmp22 duplication was detected we performed a gene panel including 64 genes that are associated with hereditary motor and sensory neuropathies (hmsn) and related disorders by using targeted next generation sequencing. we identified the two heterozygous stop mutations c.9305t>a (p.leu3102ter) and c.9305dupt (p.leu3102phefs*8), located at the same position in the sacs gene. sanger sequencing did not enable us to properly display that there is a transversion and a duplication of the same nucleotide at two different alleles. this exemplifies that, in contrast to sanger sequencing, ngs can illustrate both alleles separately. to conclude, this case was only resolvable by ngs which makes this method appropriate for the detection of compound heterozygous mutations, especially in the rare event when two mutations occur at the same position. background: the precise identification and characterization of genetic variants in monogenic diseases has a wide influence on diagnosis and therapy. about 10% of pathogenic variants are splicing variants. due to the complex mechanism of splicing regulation it is difficult to predict the effects of variants on mrna splicing. possible consequences are exon skipping, intron retention, generation of novel splice sites or the utilization of a cryptic splice site. common consequences are a frame-shift and the generation of premature termination codon. this leads to rna degradation via the nonsense mediated decay (nmd) pathway. in a patient with the clinical symptoms of non-classical infantile pompe disease and a confirmed acid alpha-glucosidase (gaa) deficiency, we detected two novel, exonic variants in the gaa gene. both base pair exchanges suggested either an amino acid exchange or a splice defect as consequences. however, conventional investigation of the leucocyte mrna of the patient and his parents was inconclusive. degradation of the respective mutated rna by nmd was suspected. we developed an approach in order to characterize novel splicing mutations in a simple and non-invasive manner. material and method: isolated blood lymphocytes from patient and his parents were cultured in standard leucocyte medium supplemented with different concentrations of the nmd inhibitors ocadaic acid, anisomycin, and wortmannin for 24 h. cells were harvested and rna was isolated. the reverse transcribed cdna was amplified in allele specific pcrs and qpcr assays. results: compared to the non-stimulated lymphocyte controls nonsense mediated rna decay was inhibited by anisomycin. the consequences of aberrant rna splicing were detectable: the maternal mutation results in exon skipping, the paternal mutation in intron retention. furthermore vascular ehlers-danlos syndrome (type iv) is considered to be an autosomal dominant disorder caused by heterozygous mutations in col3a1, which are missense or splice site variants in about 95% of cases. we here report on a three-year-old female of non-consanguineous parents born with bilateral clubfoot as well as dysmorphic facial features, joint laxity, and mild contractures of finger joints. developmental delay became evident. after trauma at 2 years of age she developed brain haemorrhage. mri diagnosis at this age revealed an additional frontal aneurism as well as frontoparietal polymicrogyria. we identified novel compound heterozygous col3a1 mutations: the nonsense mutation c.1282c>t (p.arg428*) and the c.2057delc (p.pro686leufs*105) frameshift mutation leading to a premature stop codon. further studies showed that the mutations were inherited from each parent who had no features for ehlers-danlos syndrome type iv. only two other families have been reported so far with recessive mutations of this gene and a severe vascular phenotype and polymicrogyria. biallelic mutations of col3a1 seem to be accompanied with a significantly worse outcome compared with heterozygous mutations and polymicrogyria is an additional phenotypic feature. here we describe five patients with epidermolytic epidermal nevi in different degrees of severity with the mosaic mutation c.466c>t (p.arg156cys) in krt10 gene. the same mutation has previously been described in patients with ei (bygum et al. 2013) . we analyzed dna from peripheral blood and/or skin biopsies from affected and unaffected skin with next generation sequencing (ngs) and sanger sequencing methods. using ngs we found this mutation in blood in mosaic states ranging from 6% to 23%. the mosaic could only be confirmed by sanger sequencing in the patient with the highest mosaic frequency of 23%. in four of our patients we investigated skin biopsies from affected and unaffected skin. it is noteworthy, that only one of four patients showed the mutation in heterozygous state of 50% in the affected skin, whereas the other patients presented a mosaic state also in the affected skin. to exclude a recurrent sequencing artefact at this position, we examined 100 control patients for this mosaic mutation using ngs. in none of these patients we found the same dna change. patients with epidermolytic epidermal nevi have a higher risk to have children with a full-blown ei phenotype. our results show the importance of ngs as the method of choice to explore the molecular genetic basis of epidermolytic epidermal nevi. strikingly, all our patients carry the same mosaic mutation c.466c>t in krt10. we suggest that this position is a hotspot for postzygotic mutations in krt10. non-syndromic hearing loss (nshl), with presently around 100 associated genes, is one of the most genetically heterogeneous disorders constituting nearly 70% of genetic deafness with a predominantly recessive inheritance pattern. thirty percent of hearing loss (hl) can be connected as a part of over 600 distinct syndromes. next generation sequencing (ngs) technologies have revolutionized pathogenic variant identification. different strategies enhance pathogenic variant detection supporting detailed hl investigation to overcome the many ambiguities associated with clinical heterogeneity. detection of the disease causing variant in correlation with the phenotype can be challenging in small families, in situations with ambiguous clinical histories and allelic heterogeneity. using a clinical and whole exome sequencing approach, we tested over 130 probands as part of a multicentre iranian and german genetics of hl study that included 29 probands primarily with sporadic or dominant hl in a parent-child or parent-sibling trio context. the majority of these probands were pre-screened for defects in gjb2 and strc. libraries were prepared using trusight one and nextera rapid capture exome enrichment and sequenced using the miseq and nextseq 500 desktop sequencers (illumina). analysis was performed using gensearchngs and an inhouse exome analysis pipeline. around 40% of cases were resolved from phenotype matching and segregation analysis. interestingly, the fraction of resolved cases was much higher in our iranian cohort (>50%) compared to our german cohort (>30%) which may be attributed in part to increased consanguinity in the iranian families. we observed likely disease causing variants in syndrome-associated genes including eya1 causing branchio-oto-renal syndrome, a phenotype that was retrospectively confirmed by acquisition of additional clinical information. with few exceptions, we observed a diverse collection of affected genes in probands from our german collected cohort. contrastingly, the iranian cohort revealed frequent mutations in myo15a and otof. furthermore, co-segregation of variants in myo6 and tecta, with expected dominant hl phenotype, was a hindrance overcome by extensive segregation testing. familial locus heterogeneity was also observed by mutations in cib2 and slc26a4 segregating in different branches of the same extended pedigree. success in the identification of disease causing variants in known hl genes is contingent upon analysis strategy, clinical information and opportunity for segregation testing. the ability to retrospectively connect an already apparent syndromic phenotype to a syndrome-associated gene without prior knowledge is a powerful application of comprehensive analysis that is not restricted to nshl genes. this work provides an improved understanding of population-specific genetic epidemiology of hereditary hl and highlights the challenges in defining genetic causes in a highly heterogeneous disorder such as hl. vere disproportionate microcephaly (-15,6 sd), corneal clouding, myopia, teeth abnormalities and dysmorphism. panel diagnostic by next generation sequencing for primary microcephaly, including all known genes for seckel syndrome, was unremarkable. microarray analysis (affymetrix® cy-toscan hd) revealed a heterozygous 65 kb deletion, spanning the plk4 gene. this deletion was confirmed by mlpa (multiplex ligation-dependent probe amplification) analysis. subsequent sequence analysis of the plk4 gene showed a variant of unknown significance on the second allele. in silico analysis of this variant indicated a significant decrease of the relative splice efficiency at the splice donor site. rt-pcr analysis confirmed altered splicing, resulting in a predominant loss of exon 11 of the transcript and predicting truncation of the plk4 protein. interestingly, a residual wild-type transcript was also detectable in patient rna, implying that this variant effects splicing only partially. by analysis of the parents, the splice variant and the large deletion were proven to be compound heterozygous. discussion: up to now, only a few patients with plk4 mutations have been described in the literature. the phenotype comprises primary microcephaly, primordial dwarfism and chorioretinopathy (mccrp2). to our knowledge, we describe the first case of a plk4 heterozygous whole gene deletion and at least partial biallelic inactivation of the gene, therefore expanding the genetic background of this disorder. furthermore, we give a detailed phenotypic description of a further individual with plk4 alterations. the girl does not show retinopathy so far. while generalised retinopathy was discussed to be one of the most prominent distinctive features between mccrp2 and primary microcephaly/seckel syndrome, we consider plk4 rather to be a further candidate gene pointing towards seckel syndrome. additional investigations on centriole function in patient-derived cells are in progress. pathogenic variants of mitochondrial dna cause a wide range of severe congenital disorders with maternal inheritance and a high transmission risk for female carriers. we report on eight families with an index case presenting with the common pathogenic variant m.8993t>g (p.leu156arg) in the mt-atp6 gene in virtually homoplasmic form. in five families the mutation was detectable in peripheral blood from the mother in heteroplasmic form. in three families with a sporadic case of leigh syndrome the mutation was not detectable in peripheral blood (or urinary or buccal cells) from the mother, possibly indicating a de novo event. furthermore, one family presented with a de novo nonsense mutation in the gene mt-atp8, which was present in peripheral blood of the index case in about 70% and was not detectable in the mother or the unaffected sister. two female carriers with a heteroplasmy level of 50% asked for prenatal testing. both pregnancies showed an apparently homoplasmic load of the mutation. mutations in lztfl1 (bbs17) may be associated with a severe renal phenotype huntington's disease (hd) is a rare autosomal dominant neurodegenerative disorder caused by expanded cag repeats as diagnosed via direct dna analysis. for asymptomatic individuals, predictive testing (pt) can facilitate life planning and diminish uncertainty, but it is also associated with substantial social and psychological challenges. we present a prospective case series of counselees seeking predictive hd testing at the huntington centre north-rhine westphalia (bochum, germany) between 2010 and 2012. the international protocol including several pre-test sessions was followed throughout. the aim of this study was to prospectively follow the decision-making process of individuals at risk in our centre and explore their experiences following the decision as well as the impacts of mutation test results by means of standardized questionnaires and a semi-standardized telephone interview one year after the initial counselling session. 72 individuals participated in at least one of the three phases of the survey, including 31 individuals for the telephone interview. in our cohort, almost all interviewees reported a balanced emotional state one year after initial counselling, regardless of the decision for or against the test. the most important motivations for a decision in favor of pt were the ability to plan private life and to eliminate uncertainty. the most important motivations against pt were the fear of an increasing risk for others (e. g. offspring) and the fear to obtain an unfavorable htt mutation result, followed by the considered, willful decision for "wanting to not know". furthermore, we identified evidence for gender-specific aspects in decision-making in line with and expanding our previous observations. this study represents one of the few comprehensive prospective evaluations regarding decision-making and coping strategies related to predictive testing for huntington's disease. we submit that gender-related aspects should be heeded in genetic counselling during the predictive testing and counselling processes. our findings could serve as a basis for more extended prospective evaluations with higher numbers of participants and longer follow-up intervals. institute of human genetics, heidelberg, germany, 2 department of conservative dentistry, heidelberg, germany, 3 cegat gmbh, center for genomics and transcriptomics, tübingen, germany background: plk4 (polo-like kinase 4) has been designated as "master regulator" of centriole assembly. complete loss of plk4 is lethal in mice, whereas biallelic plk4 mutations with some retained function have been described in a few patients with microcephaly, growth failure and retinopathy (mccrp2, omim #616171). this is a heterogeneous entity overlapping with mcph (primary microcephaly) and seckel syndrome. during the last years several new genes have been discovered associated with this spectrum. clinical report and genetic findings: we report on a 4 year old female patient with intellectual disability, primordial dwarfism (-6,9 sd), most se-abstracts linked to rare autosomal recessive diseases with poor prognosis. we then compared couples and filtered for variants present in genes overlapping in both partners. putative pathogenic variants were tested for co-segregation in affected fetuses where material was available and in unaffected siblings. out of eleven couples of mediterranean and arabian ancestry (c:8, nc:3) and two non-consanguineous couples of european ancestry, we found five cases (5/13, 38%, c:4, nc:1) with both parents being heterozygous carriers of rare potentially deleterious variants in one or more overlapping genes. in four of these couples the underlying genetic cause for pre-or early postnatal child death could be established, in two of the families the diagnosis was confirmed by homozygous detection of the parental variant in the available dna of the affected child. in a consanguineous couple with pathogenic variants for a severe autosomal recessive disorder identified in both parents, the molecular diagnosis for their child that had died at 5 months of age could not be established. out of 9 couples in whom no causative diagnosis could be achieved 4 consented to undergo further wes analysis. identified variants are now used for preimplantation and prenatal diagnostics in all four families in which a causative diagnosis was established. our data show that ngs based gene panel sequencing of selected genes involved in lethal autosomal recessive disorders is an effective tool for carrier screening in parents and for the identification of recessive gene defects in families that have experienced early child death and/or multiple miscarriages. k. komlósi 1 , s. diederich 1 , d. l. fend-guella 2 , u. zechner 2 , s. schweiger 2 , o. bartsch 2 1 recently an x-linked syndrome with maternally inherited or de novo mutations in taf1 was described with global developmental delay, intellectual disability (id), delayed speech, characteristic facial dysmorphology, generalized hypotonia and variable neurologic features (mrxs33, mim: #300966, xlr). there have been only three publications of 14 unrelated families, 11 with single-nucleotide changes and 3 with gene duplications including taf1 (kaya et al., 2012; o'rawe et al., 2015; hu et al., 2016) . we identified a german family in which two brothers (21 and 5 years) showed severe intellectual disability, absent speech and understanding, and hypotonia but different neurologic and behavioral phenotypes. besides severe id the older brother also had postnatal short stature (-3 sd), a severe lennox-gastaut epilepsy and a neurodegenerative course. the younger brother showed autistic behavior and lost his very limited skills at age 3 to 3.5 years. both showed mild dysmorphic features (prominent supraorbital ridges, sagging cheeks, long philtrum, long face, thin upper lip, and high-arched palate), oropharyngeal dysphagia and generalized hypotonia. a gluteal crease with a sacral caudal remnant described as a characteristic feature was not seen in our case, and hearing impairment, microcephaly, dystonic movements or tremor were not observed either. the family history was highly suggestive of x-linked inheritance with an affected maternal uncle, a maternal aunt with multiple miscarriages, and two aunts with learning disability. since a previous analysis of 107 known x-linked mental retardation genes had not revealed the cause in the older brother, we used a targeted ngs approach (mpimg1-test: >1200 brain related genes) for the analysis of the younger brother. following enrichment a 300 bp paired end sequencing was carried out on an illumina miseq system with >95% of target covered >20-fold (hu et al, 2014) . only taf1 fitted the x-linked model and the phenotype. the unreported hemizygous sequence variant c.2833g>a (p.asp945asn) in exon 18 of taf1 was deemed pathogenic. it affected a highly conserved residue in the central "duf3591" domain, where 4/11 previously described mutations had clustered. segregation analysis confirmed hemizygosity in the older brother and heterozygosity in the mother with completely skewed x-inactivation (100:0). gonadism and learning disabilities. because of the delayed onset of symptoms, the diagnosis is often established during late childhood. however, in some cases renal morphological changes detected by ultrasound may resemble those usually seen in autosomal recessive polycystic kidney disease (arpkd). however, histologically bbs-kidneys differ distinctly from other polycystic disorders by cystic orientation, localisation, extension, structure and size. 21 bbs genes have been identified to date. mutations in bbs2, bbs4, bbs6 and bbs10 have been found to cause an antenatal presentation of bbs that may in some aspects mimic meckel gruber syndrome (mks). there is increasing evidence that bbs, at least in some families, shows an oligogenic mode of inheritance with three mutations at two bbs loci. yet, only three patients in two families with bbs caused by mutations in lztfl1 (bbs17) have been reported. their diagnosis was established in childhood and all patients had mesoaxial polydactyly as a distinct manifestation. in contrast to previous lztfl1 cases, in our family the diagnosis of arpkd was suspected sonographically at 27 weeks gestation (wg). pregnancy was terminated at 30 wg. autopsy revealed postaxial polydactyly of both hands, enlarged spongy kidneys, hemivertebra t6 and some features of potter's sequence. histological examination of the kidneys showed multiple, not radially oriented thin walled cysts, internally lined by thickened pas-positive basement membranes and microcystic dilatation of collecting ducts. cystic changes were accentuated in the renal medulla. corticomedullar differentiation was mainly preserved. the tentative diagnosis was bbs. fetal dna was investigated using a next generation sequencing panel which included 17 known bbs causing genes. hereby a heterozygous nonsense variant (np_065080.1: p.glu260*) inherited from the mother and a heterozygous missense variant (p.glu92lys) inherited from the father of the lztfl1 gene were identified. furthermore a maternally inherited heterozygous missense variant of unknown clinical significance in bbs4 was detected (np_149017: p.pro443ala). our case shows for the first time that mutations in lztfl1 can lead to a severe prenatal presentation of bbs due to profound renal manifestations with a kidney histology that is not considerably milder but distinct from that observed in mks. it is not clear to which extent the bbs4 variant may act as a disease modifier. this may challenge genetic counselling and prenatal diagnosis in a further pregnancy. furthermore our case shows that mesoaxial polydactyly is not always present in bbs patients with lztfl1 mutations and further studies are necessary to establish the frequency of mesoaxial polydactyly and other genotype phenotype correlations for bbs patients with lztfl1 mutations. p-cling-075 *** targeted next-generation sequencing analysis in couples at increased risk for autosomal recessive disorders k. komlósi, s. diederich, d. l. fend-guella, j. winter, o. bartsch, u. zechner, s. schweiger genetic childhood disorders leading to prenatal, neonatal or early childhood death are genetically heterogeneous. many follow autosomal recessive or x-linked modes of inheritance and bear specific challenges for genetic counselling and prenatal diagnostics. parents are carriers but unaffected and diseases are typically very rare but with recurrence risks of 25% in the same family. often, affected children (or fetuses) die before a genetic diagnosis can be established, post-mortem analysis and phenotype descriptions are insufficient and dna material of affected fetuses or children is not available for later analysis. a genetic diagnosis showing biallelic mutations or mutations on the x-chromosome in male fetuses or children is, however, the requirement for targeted carrier testing in parents, risk calculations, and prenatal and preimplantation diagnostics in further pregnancies. we employed targeted next-generation sequencing (ngs) for carrier screening of autosomal recessive lethal disorders in 8 consanguineous (c) and 5 non-consanguineous (nc) couples with one or more affected children. we searched for heterozygous variants (non-synonymous coding or splice variants as well as cnvs) in parents' dnas in a set of 430 genes r. kropatsch 1, 2 , j. preine 3, 4 , jt. epplen 1,2 1 department of human genetics, 2 ruhr university, bochum, germany, 3 charcot-marie-tooth disease (cmt), also commonly called as hereditary motor sensory neuropathy, is the most common monogenetic disease of the peripheral nervous system with significant clinical and genetic heterogeneity. the main clinical manifestations of cmt include progressive distal muscle weakness and atrophy, impaired distal sensation, depressed tendon reflexes and high-arched feet. based upon electrophysiological and histopathological features cmt can be divided into predominantly demyelinating or axonal forms. an intermediate form also exists characterized by evidence of both, demyelinating and axonal, impairments. genetically cmt can be caused by mutations in over 40 genes, including the dnm2 gene encoding dynamin 2 protein, a large gtpase primarily involved in receptor-mediated endocytosis and membrane trafficking. only a small number of mutations in dnm2 causing cmt have been described so far. we report the case of a 48-year-old man presenting with backache, ataxic gait and distal muscle weakness of the lower limb considered to be a consequence of the pre-diagnosed disc prolapse 3 years ago. over the past months bilateral progressive weakness of ankle dorsiflexion, foot drop and tingling paresthesia in stocking distribution have occurred. neurological examination disclosed depressed tendon reflexes of the upper and lower limbs. neurophysiologic investigations revealed an axonal sensible polyneuropathy with normal distal motor latencies and nerve conduction velocities. the sural nerve biopsy indicated single unmyelinated or thinly myelinated axons, loss of myelinated nerve fibers, numerous clusters of regenerating fibers without onion bulb formations suggesting an intermediate form of cmt. by using next generation sequencing (ngs) and a multi-gene panel, consisting of 751 inherited neurological disease-associated genes, we identified a heterozygous missense mutation c.439g>a, p.asp147asn (rs370086632) in the dnm2 gene. this particular mutation is located in a highly conserved nucleotide region encoding the catalytic n-terminal gtpase domain. this evidence suggests a pathogenic phenotype caused by the described mutation, which is being underlined by the following facts: public 1000 genome database covering harmless variants of the human genome does not report it. additionally, the exac browser with exome sequencing data of >60,000 unrelated individuals, lists the mutation and shows a low allele frequency of 0.000008243, corresponding to one known heterozygous mutation carrier. other online prediction tools like the mutation taster, polyphen, sift and provean categorize it as pathogenic. in conclusion, the novel dnm2 mutation is responsible with high probability for the late-onset form of intermediate cmt of the investigated patient. heterozygous mutations in pcdh19 cause an x-linked female-limited form of an early infantile epilepsy (juberg-hellman syndrome). the phenotype of this syndrome is variable, ranging from benign focal epilepsy to severe, serial seizures, repeating up to more than 10 times a day for several consecutive days. the intellectual outcome of affected patients ranges from normal to severe intellectual disability. psychiatric disturbances are frequent and manifest as autism, schizophrenia or aggressive behavior. neurological features such as ataxia may also be present. women with triple x syndrome usually show a normal physical development. cognitive deficits in vivo functional modeling of taf1 has already provided evidence for an effect on a neuronal phenotype. the phenotype in patients can be reminiscent of rett syndrome, but with milder regression and normal movements lacking a specific stereotypic pattern (no hand wringing). while severe neurodegeneration has been described in duplications, the present probands clearly showed developmental regression associated with a missense mutation. the analysis of further family members is pending. our case adds to the phenotypic spectrum of x-linked syndromic mental retardation type 33. targeted enrichment sequencing was successfully applied to identify greenberg dysplasia as cause of fatal anomalies in one fetus of a dizygotic twin pregnancy. p. m. kroisel 1, 2 , b. csapo 2, 3 , m. häusler 2, 3 , l. michelitsch 4, 5 , s. verheyen 1, 2 , p. klaritsch 2, 3 , k. wagner 1, 2 1 institute of human genetics, 2 medical university of graz, graz, austria, 3 department of obstetrics and gynecology, 4 gynecologist and obstetrician, 5 weiz, austria in the first pregnancy of a 29 year old kosovarian woman and her 36 year old husband during the 16th week of gestation one of her dizygotic twins showed a severe skeletal dysplasia with all long bones extremely shortened and partially bended. the thorax was short and narrow. in addition a ventriculomegaly of the brain and an increased nuchal translucency was noticed. a very bad prognosis was expected and an achondrogenesis was suspected clinically. the other twin appeared to be normal. an amniocentesis was performed to potentially identify the genetic basis of the disorder. qf-pcr to rule out common trisomy's and cytogenetics revealed normal results and following a normal agilent 60k array cgh analysis as a next diagnostic step next generation sequencing by using the trusight one gene panel focusing on three genes including slc26a2, trip11 and col2a1 was performed. since no pathogenic mutation was found by this approach, a more extended bioinformatics study was initiated. by filtering out common variants in the more than 4800 genes of the panel in our own database or in the exac-and 1000 genome databases our search was extended to genes with rare homozygous or compound heterozygous variants. by this strategy it was possible to reduce the potentially causative gene mutations dramatically and among those remaining genes for known very severe skeletal phenotypes just in the lbr gene the homozygous missense mutation c.1639a>g, p.asn547asp was identified in our fetus. since this particular mutation is already known to be pathogenic leading to the lethal greenberg dysplasia (clayton et al., nucleus. 2010 ) the diagnosis could be achieved in the affected fetus of the pregnancy of our patient still before completion of the 23rd week of gestation. both parents were found to be heterozygous for this mutation in the lbr gene. recently it was shown that different mutations of the very same gene can also lead to less severe forms of bone dysplasia. the couple was informed about our results and possible consequences were discussed and offered. the couple however came to the decision not to draw any consequences. both fetuses especially the affected one were well documented sonographically including in a series of 3d images. in the 27th gestational week during a sonographic investigation the affected fetus did not show cardiac function and an oligohydramnios was found. since development of the second non affected fetus was still within the normal range, we hope the now single pregnancy will carry on normal until birth. from our finding we would propose that our chosen strategy is straightforward and can be applied in a wide range of pregnancies to identify various up to severe and fatal single gene disorders associated with sonographic anomalies within a few weeks which should provide substantial benefits for these families. after the working diagnosis ps had been established, molecular analyses regarding the recurrent akt1 mutation (p.glu17lys) were performed by sanger sequencing in available affected tissue specimen of all three individuals. this revealed a high level of mosaic state for the akt1 mutation c.49g>a, (p.glu17lys) in affected tissues from bone and in meningiomas. re-evaluation of the ngs data from blood (individual i) confirmed the absence of that mutation in all reads, and no mutation was detected by sanger sequencing in dna from blood in individuals ii and iii. thus, a somatic mosaicism leading to a mild proteus phenotype could be confirmed as the underlying genetic cause in all three affected individuals. in conclusion, mild forms of proteus syndrome caused by the recurrent akt1 mutation in patients with limited regional involvement may be particularly difficult to diagnose and might be underdiagnosed. distal gne-myopathy: rare differential diagnosis of polyneuropathy here, we report a case of a 37-year-old patient with presumed polyneuropathy and elevated creatine kinase levels (400-800 u/l). clinical features included atrophic and bilateral paresis of lower legs of the frontal and rear compartment without high arched foot, while sensibility was not affected. additionally, a myopathic emg in m. tibialis anterior and a slight axonal damage in the motor neurography was detected. due to this overlapping neuromyological phenotype we performed a gene panel including 155 genes associated with neuromuscular diseases using targeted next generation sequencing. gene panel analysis revealed the homozygous mutation c.829c>t (p.arg277trp) in the gne gene. this mutation is described in the literature as cause of a distal gne-myopathy and was also detected in an affected brother (ck 1900 u/l), having consanguineous parents. the current case emphasizes that a large gene panel analysis is recommended in case of an overlapping neuropathological and myopathological phenotype. c. landgraf, g. schmidt, s. morlot, b. schlegelberger, b. auber institute of human genetics, hannover medical school, hanover, germany ehlers-danlos syndrome (eds) is a heterogeneous group of connective tissue disorders. according to the 1997 villefranche classification, eds comprises six major types as well as some rare specific entities. one of these has been referred to as "eds, musculocontractural type" (mc-eds), "adducted thumb-clubfoot syndrome" or "eds, kosho type". first described in 1959 as an eds vi subtype, it recently was identified to be caused by biallelic changes in either the chst14 or dse genes, resulting in a loss of dermatan sulfate (ds) biosynthesis. characteristic symptoms are multiple congenital malformations such as contractures (club feet, adducted thumbs), visceral and ocular anomalies, generalized joint laxity, scoliosis, muscular hypotonia, fragile, hyperextensible and bruisable skin, as well as a typical craniofacial appearance. distinctive features include hypertelorism, down-slanting palpebral fissures, bluish sclerae, micro-corneae, short nose with hypoplastic columella and long philtrum, thin upper lip vermillion, small mouth, retrognathia, low-set and posteriorly-rotated ears. the psychomotor development is delayed. 31 (chst14) and 3 (dse) patients have been reported as yet. the patient, a 30-year-old woman using a wheelchair, had club feet, surgically corrected asd ii, muscular hypotonia, the characteristic face and hyperextensible skin with atrophic scars; particularly visible were those and learning disabilities are more common than in the general population and compared to siblings. their motor skills are likely to be somewhat impaired and coordination problems are frequent. in some patients psychological problems were described. furthermore, eeg abnormalities are occasionally observed, with clinical seizures present in up to 15% of patients. here we report on a 15-year-old girl with a 47,xxx karyotype and early infantile, intractable epileptic seizures, beginning at the age of 9 months. about three years later, she developed severe, serial seizures often related to febrile infectious diseases. in adolescence, the epileptic symptoms became less intense. she additionally showed autistic features, mental deficiencies, hypermobility of the joints and ataxia. array-cgh, fragile x-analysis as well as sanger sequencing and mlpa of the scn1a and mecp2 genes revealed no additional abnormalities, besides the xxx karyotype. in the pcdh19 gene the heterozygous missense mutation c.695a>g (p.asn232ser) was identified, whereby the mutated allele seemed to appear in a 2:1 ratio compared to the wild type allele. this mutation has been previously described as disease causing. furthermore, three in silico prediction programs (sift, polyphen-2, mutationtaster) classified the mutation as pathogenic. the patient's asymptomatic mother had a normal 46,xx karyotype and was not a carrier of the pcdh19 mutation. pcdh19-related epilepsy exhibits an unusual mode of inheritance in which only heterozygous females are affected and hemizygous males are asymptomatic carriers. random x-inactivation in the brain of females with pcdh19 mutations causes a cellular mosaicism, which likely accounts for the pathogenesis by altering the cell-cell-interactions ("cellular interference"). however, the precise mechanism is still unknown. hypothetically, the wide range of phenotypic expressions may be explained by partially skewed x-inactivation and thereby limitation of the cellular interference. hence, an unequal ratio of mutated to wild type cells should give a milder phenotype compared to the fifty-fifty situation. in contrast to this hypothesis, the phenotype in our patient was rather severe. nevertheless, we cannot exclude that the triple x status contributes additionally to the observed phenotypic expression. proteus syndrome (ps, omim 176920) is a highly variable disorder with asymmetric and disproportionate overgrowth of the body, connective tissue nevi, epidermal nevi, dysregulated adipose tissue, and vascular malformations, caused by a somatic activating akt1 mutation. we report on three unrelated individuals (two adults and one 6 year old boy) who showed similar clinical findings that not fulfilled the rigorous clinical criteria for ps (biesecker, 1999) . beside an asymmetric hyperostosis of the skull or facial bones, all three had an ocular dermoid. two individuals developed alveolar hyperostoses and intracranial calcifying meningiomas. only one individual showed skin changes. all three had normal feet and no vascular lesions. molecular analyses in individual i performed in blood revealed normal results for array karyotyping and no relevant variant in whole exome sequencing (trio approach). introduction: incontinentia pigmenti (ip) is a rare x-linked male lethal genodermatosis that affects the neuroectodermal tissue and is always associated with a bullous rash of the skin along blashko lines in female neonates. it is caused by mutations in ikbkg which encodes the regulatory subunit of the ikb kinase complex required for nf-kb activation. ikbkg has a pseudogene (ikpkgp) with identical exons 4 to 10. the most frequent ip mutation is a recurrent exon 4_10 deletion due to non-allelic homologous recombination with the pseudogene. here, we report a novel deletion of exons 4 and 5 in the ikbkg transcript recognized by rna analysis. patient: we investigated a 9 yr old girl with typical erythematous rash after birth which resolved within 2-3 m. apart from a small hyperpigmented area around the right mammilla there were no skin alterations. she had few conically shaped teeth, normal nail and hair structure, no neurological manifestation and normal intelligence. only clinical sign were repeated vitreous hemorrhage of the left eye from age 5 m. family history was negative. methods: analyses on genomic dna extracted from blood included testing for the common ikbkg exon 4_10 deletion by long range pcr and mlpa (p073-a1, mrc holland), x inactivation analysis in the androgen receptor gene as described, and massively parallel sequencing (mpseq) of the ikbkg gene (trusightone, nextseq, illumina®; data analysis with nextgene/geneticist assistant [softgenetics®] and seqnext [jsi®]; reference sequence grch37, hg19). rna was extracted from cultured blood lymphocytes, and the entire ikbkg transcript (nm_003639.4, 10 exons) was sanger sequenced on cdna level (the a of the start codon is in exon 2); the results were analyzed with sequencepilot (jsi). result: genomic dna analyses including mlpa of ikbkg and ikbkgp specific probes in our patient did not reveal a putative mutation. there was a completely skewed x-inactivation pattern. cdna sequencing of ikbkg demonstrated skipping of exon 4 and 5 (r.400_671del) which is predicted to cause a frame shift starting from codon p.gln134, a premature stop codon 28 amino acids downstream (p.gln134glyfs*29) and complete loss of protein function. the loss of exons 4 and 5 is most likely due to an intronic splice variant in intron 6; investigations regarding the origin of this deletion are ongoing. conclusion: the presence of the high homologous pseudogene makes sequence analysis of ikbkg challenging. we report a deletion of exons 4 and 5 in the ikbkg transcript that required rna analysis for its identification. due to the skewed x-inactivation and typical clinical picture causality of the detected deletion is certain. the exact genomic cause of this alteration remains to be clarified. also in the era of mpseq, rna analysis may be necessary for detection of deep intronic mutations or the study of genes with homologous pseudogenes, as shown here in the case of ikbkg. primary congenital glaucoma (pcg) and early onset glaucomas are one of the major causes of of blindness in children and young adults' worldwide. both autosomal recessive and dominant inheritance have been described resulting from bowel surgeries due to colon perforation. her older sister who died aged 19 following an acute abdomen had club feet and the typical facial appearance, while three healthy sisters seem to be unaffected. her parents are first cousins of turkish origin and do not show eds symptoms either. the two affected sisters had been diagnosed with a syndromic disorder that could represent a rare form of eds. however, neither a confirmation of the suspected diagnosis nor a classification had yet been achieved. due to the distinctive symptom complex and the presumed autosomal recessive inheritance pattern, we strongly suspected this to be a case of mc-eds. sequencing of the chst14 gene (reference sequence: lrg_600) revealed a formerly undescribed homozygous variant (c.644c>t; p.pro-215leu). the variant changes the highly conserved pro215 residue which is located in the critical 3'-phospho-5'-adenylyl sulfate binding site and can be classified as likely pathogenic (acmg standards and guidelines, richards et al., genetics in medicine 2015) . the parents are heterozygous carriers of this variant, respectively. this case represents a unique entity within the umbrella term eds and illustrates the importance of clinical assessment leading to a diagnosis confirmed by genetic analysis. the underlying genetic defect in patients with mitochondrial peo is either a primary mutation of the mitochondrial genome (single, large-scale mtdna deletion or mtdna point mutation) or recessively and dominantly-inherited mutations in nuclear genes involved in mtdna maintenance leading to clonally-expanded multiple mtdna deletions in muscle. the nuclear disease genes are largely implicated in the replication and stability of mtdna, and as such a pathogenic mutation leads to secondary instability of the mitochondrial genome. causal mtdna deletions can be found in a heteroplasmic (mixture of mutated and wild type mtdna) state. however, each tissue/cell has its own biochemical threshold of mutant mtdna load which needs to be exceeded before focal respiratory chain deficiency becomes evident. to investigate this, muscle biopsies of 17 patients with genetically-and clinically-characterized mitochondrial disease of nuclear origin (9 polg, 5 twnk, 2 rrm2b and 1 slc25a4 (ant1)) and 4 healthy controls were analysed using quadruple oxphos immunohistochemistry, quantifying the biochemical phenotype in individual muscle fibres of patient muscle biopsies. this technique is based on quadruple immunofluorescence to detect structural components of complexes i (ndufb8) and iv (coxi), as well as porin (a marker of mitochondrial mass) and laminin (a cell membrane marker to define the boundaries of muscle fibres). further studies on 7/17 patients (3 polg, 2 rrm2b, 1 twnk, 1 slc25a4 (ant1)) included the correlation of the biochemical deficiency with the mtdna abnormality in individual cells, following laser microcapture and determination of the size and level of clonally-expanded mtdna deletion within fibres by real-time pcr. our preliminary data from quadruple immunocytochemical studies show that the muscle biochemical phenotype is different in patients with multiple mtdna deletions compared to other mtdna mutations; work is continuing to determine the exact size and level of clonally-expanded mtdna deletion in individual muscle fibres and correlate this with the observed biochemical defects and disease thresholds. abstracts these families, and functional studies, together with phenotype descriptions in the literature, are essential for pathogenic grading. however, several difficulties remain, such as the huge size of the ttn gene (>100kb) impeding functional studies, the wide spectrum of phenotypes and variants, the still small patient cohort, and often unspecific immunohistochemical abnormalities in muscle biopsies. the clinical evaluation of ttn variants thus presents a great challenge to the field of human genetics diagnostics. compound heterozygous variants in the qars gene (omim 603727) have been identified in only four patients with autosomal recessive progressive microcephaly with seizures and cerebral and cerebellar atrophy (mscca), to date. these patients showed severe developmental delay, progressive primary microcephaly, intractable seizures, hypomyelination or delayed myelination, thin corpus callosum, and small cerebellar vermis on brain imaging. here we report on two unrelated girls with progressive primary microcephaly, epilepsy and brain anomalies. trio exome analysis in each of the families revealed two different combinations of compound heterozygous variants in qars. all four variants are highly conserved throughout vertebrates, not reported in any database, yet, and in silico analysis predicted the variants as possibly damaging or deleterious. the first patient was born to non-consanguineous german parents. at birth, she was too short (−2.8 sd) and mildly microcephalic (−2.3 sd). she developed intractable seizures within the first hour of life. her growth continued to be mildly retarded (−2.8 sd at age 9 years) but microcephaly was progressive (−6.5 sd at age 9 years). she did not achieve any of the motor or cognitive developmental milestones, she did not have eye contact. the only interaction with her surrounding was a diffuse reaction to being touched. cranial mri showed no myelination of the supratentorial region, corpus callosum agenesis, simplified gyral pattern of frontal lobes, enlarged cerebral ventricles, and normal brain stem and cerebellum. trio-exome sequencing revealed the compound heterozygous qars variants c.1132c>t, p.(arg-378cys) and c.1567c>t, p.(arg523*). segregation analysis by sanger sequencing confirmed the heterozygous variants in the parents and two non affected sibling of the index patient. the second patient was initially evaluated at 11 days of age when she exhibited myoclonic seizures, intrauterine growth retardation, microcephaly, and elevated lactic acid. at birth, she was microcephalic (hc 29 cm) and microcephaly was progressive (−5.4 sd at age 19 months). cranial mri suggested undersulcation. she has required a gastrostomy feeding tube. trio-exome sequencing revealed the compound heterozygous qars variants c.40g>a, p.(gly14ser) and c.1573c>t, p.(arg525trp). segregation was confirmed by sanger sequencing analysis. together with the four previously described patients we conclude that compound heterozygous variants in qars are associated with a primary and progressive microcephaly, early onset of intractable seizures and severe developmental delay. brain imaging in the neonate can show simplified gyral pattern as an early characteristic feature. overlapping phenotypes are seen in patients with epileptic encephalopathy, lissencephaly and primary microcephaly. application of ngs panels or exome technology will allow for early diagnosis and further collection of patients for better delineation of the phenotype. with involvement of several genes including cyp1b1, foxc1, pitx2, myoc and pax6. however, mutations in these genes explain only a small fraction of cases suggesting the presence of further candidate genes. to elucidate further genetic causes of these conditions we performed whole exome sequencing in a patient with pcg and retinal detachment and identified compound heterozygous variants in col1a1 (p.met264leu; p.ala-1083thr). targeted col1a1 screening of 26 additional patients detected three further heterozygous variants (p.arg253*, p.gly767ser and p.gly-154val) in three distinct subjects: two of them were diagnosed with early onset glaucoma and mild form of osteogenesis imperfecta (oi), one patient had a diagnosis of pcg at age 4 years. all five variants affected evolutionary, highly conserved amino acids indicating important functional restrictions. molecular modeling predicted that the heterozygous variants are dominant in effect and affect protein stability and thus the amount of available protein, while the compound heterozygous variants act as recessive alleles and impair binding affinity to two main col1a1 binding proteins: hsp47 and fibronectin. dominantly inherited mutations in col1a1 are known causes of connective tissues disorders such as oi. these disorders are also associated with different ocular abnormalities, although the common pathology for both features is seldom recognized. our findings expand the role of col1a1 mutations in different forms of early-onset glaucoma with and without signs of oi. thus, we suggest including col1a1 mutation screening in the genetic work-up of glaucoma cases and detailed ophthalmic examinations with fundus analysis in patients with oi. the gene ttn encodes the largest known protein, titin, which plays a key role in structural, mechanical, developmental and regulatory functions of cardiac and skeletal muscles. accordingly, titinopathies are characterized by great clinical and genetic heterogeneity. the clinical spectrum ranges from severe phenotypes with cardiac involvement to pure myopathies at the milder end, including autosomal recessive and dominant inheritance patterns (chauveau et al. 2014, hum mutat; 35:1046) . next generation sequencing analysis identifies a large number of variants of unknown clinical significance; the potential clinical relevance of these variants cannot be assessed with certainty without further studies. three case reports highlight the difficulties in human genetics diagnostics concerning ttn. the first case is of a 46 year-old woman with proximal muscle weakness, slightly elevated ck, scoliosis, and no family history. a heterozygous known pathogenic variant was identified in mex1, associated with autosomal recessive congenital core myopathy combined with primary heart disease. additionally, an unknown variant was detected. both variants could be clinically relevant with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the second case is of a 7 month-old finnish girl who presented with severe muscle hypotonia at birth and mental alertness with normal brain mri and eeg. congenital fiber-type disproportion was suspected. a homozygous frame-shift mutation in mex1 was identified which to our knowledge has not yet been described in the literature. this variant is likely of clinical relevance with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the third case is that of a 34 year-old woman with suspected myofibrillar myopathy. a known pathogenic homozygous frame-shift mutation in mex6 was detected which is associated with autosomal recessive congenital myopathy with central nuclei. segregation analysis revealed that the healthy parents are heterozygous carriers of this variant. the clinical diagnosis of a ttn-associated disease could therefore be confirmed. ttn variants need to be assessed in combination with detailed clinical and muscle biopsy data. segregation analysis is necessary but not sufficient for the clinical grading of variants. identification of a variant in several independent families, segregation of the variant with disease phenotype in gous pathogenic smad4 variant c.719dupt;p.(ala241fs) (ncbi reference sequence nm_005359.5). gastric as well as colonic cancer and polyposis was present in the paternal family history. conclusions: ts14 and other imprinting disorders are likely underdiagnosed, as the main clinical features (e. g. growth retardation, hypotonia) are distinct but unspecific. as exome sequencing becomes a more frequent diagnostic procedure, imprinting disorders caused by mutations in imprinting centers will presumably be diagnosed more often. methylation defects, however, will remain underdiagnosed, without a specific clinical differential diagnosis, which would guide to appropriate analysis of the methylation status. a bowel invagination in early childhood due to a single polyp can be a symptom of jps, especially in the context of a paternal history of polyposis and intestinal cancer; thus, family history should be carefully obtained. in the outpatient clinic, child psychiatrists as well as neurologists thoroughly work up phelan-mcdermid patients according to a standardized protocol by taking medical history, performing physical examination, and, if needed, organizing further supplementary examinations. in addition, a genetic analysis and hair/tissue sampling is performed. since its foundation, a steadily increasing number of so far 70 patients from all over germany has been seen and treated. the outpatient clinic aims at facilitating and accelerating the diagnosis of phelan-mcdermid syndrome, improving medical support for affected patients of all ages, and, last but not least, fostering a better understanding of the causes and pathomechanisms leading to the symptoms of the disease. t. m. neuhann, l. neuhann, c. rapp, a. laner, a. benet-pages, e. holinski-feder medizinisch genetisches zentrum, münchen, germany congenital eye malformations, such as the microphthalmia-anophthalmia-coloboma (mac) spectrum, congenital cataracts, anterior segment dysgenesis (asd), and congenital glaucoma, affect more than 1:8.000 newborns. the phenotypic spectrum of the aforementioned entities is highly variable and partially overlapping. eye malfomations are very heterogeneous; to date causative mutations have been described in more than 100 next-generation-sequencing (ngs) technology has revolutionized genomic research and has transformed clinical diagnostics. ngs offers enormous potential for providing accurate diagnoses to individuals with previously unresolved syndromes. in the pediatric endocrine clinic, clinicians are often faced with the task of making a diagnosis in children with syndromic short stature. as there may be considerable clinical overlap between short stature syndromes, deriving a clinical diagnosis may prove challenging. furthermore, even if a clinical differential diagnosis is established, often several genes would need to be tested before a molecular diagnosis is made. as access to genetic testing is limited in algeria, we conducted a pilot study on 10 algerian patients with syndromic short stature using a combination of two different ngs modalities, namely whole-exome-sequencing (wes) and mendeliome sequencing (trusight one sequencing panel). a molecular diagnosis could be established in 9/10 patients, making the diagnostic rate in this initial cohort 90%. as 7 patients had novel mutations we could expand the mutational spectra of several genes, namely cul7, npr2, sos1, vps13b, and znf81. we could thus substantiate the clinical utility of wes and the mendeliome in patients with a diverse array of syndromic short stature syndromes. chromosome 14 harbours an imprinted locus at 14q32. maternal uniparental disomy of chromosome 14, paternal deletions and paternal loss of methylation at the intergenic differentially methylated region (ig-dmr) and the somatic dmr within meg3 are associated with temple syndrome (ts14, mim 616222). the phenotype of ts14 consists of pre-and postnatal growth retardation, early feeding problems and muscular hypotonia, joint laxity, motor developmental delay, premature puberty, and truncal obesity. juvenile polyposis syndrome (jps, mim 174900) is characterized by predisposition to hamartomatous polyps in the gastrointestinal (gi) tract, specifically in the stomach, small intestine, colon, and rectum, including the risk for gastrointestinal cancer. pathogenic variants in the bmpr1a and smad4 gene are identified in about 40-50% of affected families. we report on a family with two female children. the index patient, an 8-year-old girl, was diagnosed to have ts14 due to hypomethylation at the somatic dmr within meg3 with clinical features reminiscent of prader-willi syndrome in early childhood and milder clinical signs at further age (i. e. mild global development delay, muscular hypotonia, suspected central obesity, no prominent facial dysmorphisms). snp array-cgh analysis was unsuspicious and no deletion of the imprinting center was observed. thus, ts14 is caused by a sporadic imprinting defect in our patient. her 10-year-old sister was diagnosed with smad4-associated jps after an episode of intestinal invagination due to a polyp, histologically diagnosed as peutz-jeghers polyp, in early infancy. sequencing identified a heterozy-abstracts coding vmat2 have only very recently been described as causal for brain dopamine-serotonin vesicular transport disease in two families with multiple affected children (rilstone et al., n engl j med 368, 2013, 543-550; jacobsen et al., j inherit metab dis 39, 2016, 305-308) . the index case presented here is a 7-year-old girl with severe mental retardation and a dystonic movement disorder. she is the tenth child of a consanguineous arabic couple and was initially referred to neuropaediatric examination at the age of four months due to recurrent oculogyric crises and muscular hypotonia. blood metabolic testing and cerebrospinal fluid (csf) analyses were inconclusive. notably, biogenic amines were within their normal ranges and the differential diagnosis of aromatic l-amino acid decarboxylase (aadc) deficiency could not be confirmed. conventional cytogenetics, subtelomeric screening, array-cgh and different ngs panel analyses did not identify a causative mutation. both parents and all eight living siblings are obviously unaffected. a brother with a known hypotonic movement disorder died at the age of three years due to prolonged seizures with hyperthermia and cerebral edema. by utilizing whole-exome sequencing, we identified a homozygous substitution in the slc18a2 gene of the index case causing an amino acid change (c.710c>a; p.pro237his) in a conserved transmembrane domain of vesicular monoamine transporter 2 (vmat2). homozygosity for this missense change could also be verified in a dna sample of her deceased brother. an obvious reduction in frequency of oculogyric crises was observed in our index case under therapy with pramipexole already within 4 weeks after start of treatment. furthermore the patient shows less dystonic movements under therapy. the case presented here highlights the importance of considering brain dopamine-serotonin vesicular transport disease as differential diagnosis for early-onset extrapyramidal movement disorders combined with mental retardation even if neurotransmitters in csf are normal. for a large number of individuals with intellectual disability (id), the molecular basis of the disorder is still unknown. however, whole exome sequencing (wes) is providing more and more insights into the genetic landscape of id. in the present study, we performed trio-based wes in 311 patients with unsolved id and additional clinical features, and identified homozygous cplx1 mutations in three patients with id from two unrelated families. all displayed marked developmental delay and migrating myoclonic epilepsy, and one showed a cerebellar cleft in addition. the encoded protein, complexin 1, is crucially involved in neuronal synaptic regulation, and homozygous cplx1 knockout mice have the earliest known onset of ataxia seen in a mouse model. recently, a homozygous truncating mutation in cplx1 was suggested to be causative for migrating epilepsy and structural brain abnormalities. id was not reported. the currently limited knowledge on cplx1 suggests that complete loss of complexin 1 function may lead to a complex but variable clinical phenotype, and our findings encourage further investigations of cplx1 in patients with id, developmental delay and myoclonic epilepsy to unravel the phenotypic spectrum of carriers of biallelic cplx1 mutations. genes. due their heterogeneity, diagnostic testing for congenital eye malformations was limited in the pre-ngs era. we performed exome analysis in 30 patients with congenital eye malformation (mac spectrum, asd, congenital cataract, congenital glaucoma). primarily, a gene panel comprising 112 genes associated with eye malformations was evaluated. additionally the exome data was evaluated in selected patients as a second step. the panel analysis revealed pathogenic sequence variants in 10 patients and 8 genes (mab21l2, bcor, nhs, prss56, cyp1b1, foxc1, pitx2, gcnt2). putatively causative sequence variants were identified additional patients. the diagnostic yield of the panel was highest in patients with non-syndromic microphthalmia/coloboma and congenital cataracts, and lowest in patients with syndromic mac spectrum (i. e. additional systemic features/malformations). ngs based panel testing is a strong diagnostic tool to determine the underlying causes of non-syndromic congenital eye malformations. due to the partially overlapping phenotypes and high heterogeneity it is more sensible to perform large gene panel analysis, as opposed to smaller single phenotype based panels. superactivity of phosphoribosyl¬pyrophosphate synthetase i (prpps) is a rare inborn error of purine metabolism that is characterized by increased levels of uric acid in blood and urine (omim 300661). the disorder is caused by gain-of-function mutations in the x-chromosomal gene prps1. in male patients, disease manifestation is in early childhood. additional clinical characteristics include intellectual disability, hypotonia, ataxia and hearing loss. heterozygous female mutation carriers have a later age of onset and a less severe clinical course. only seven families with prps1 gainof-function mutations have been reported to date. we report on a 7-year-old boy with congenital hyperuricemia, urolithiasis, developmental delay, short stature, hypospadias and facial dysmorphisms. his mother also had hyperuricemia that was diagnosed at age 17 years but was otherwise healthy. a novel prps1 missense mutation (c.573g>c, p. leu191phe) was detected in the proband and his mother. enzyme activity analyses confirmed superactivity of prpp synthetase. the family reported here broadens the clinical spectrum of prpps superactivity and indicates that this rare metabolic disorder is associated with a recognizable facial gestalt. homozygous and compound heterozygous mutations of the rnu4at-ac gene are associated with mopd1 and roifman syndrome. mopd1 is characterized by severe microcephaly with brain malformations including abnormal gyral pattern, corpus callosum agenesis or hypoplasia, vermis hypoplasia and intracranial cysts, psychomotor retardation, short stature, skeletal dysplasia, dry skin, sparse hair, flexion contractures, round face with beaked nose and protruding eyes, and premature death with a majority of the patients who die before the age of 28 months for unknown reasons. roifman syndrome was first described as a novel association of antibody deficiency, spondyloepiphyseal chondro-osseus dysplasia, retinal dystrophy, poor pre-and postnatal growth, cognitive delay and facial dysmorphism including long eyelashes, downslanting palpebral fissures, a long philtrum and a thin upper lip. all patients with roifman syndrome reported so far lack brain malformations. the rnu4atac gene encodes a small nuclear rna (snrna), which is essential for minor intron splicing. homozygous (g.51g>a, g.46g>a) and compound heterozygous mutations (g.51g>a;g.55g>a, g.51g>a;g124g>a and g.40c>t;g.124g>a) have been described in mopd1. all mutations involve the 5' or 3' stem loop of the u4atac snrna. in contrast, all cases with roifman syndrome investigated so far showed compound heterozygous rnu4atac mutations with one allele harboring a mutation in the mopd1 associated 5' stem loop and the other allele showing a mutation in the stem ii site of the u4atac snrna, which has not been involved in mopd1, so far. thus, the different pattern of the mutations observed in mopd1 and roifman syndrome may contribute to the distinct features of both syndromes. however, our patient shows, that features of mopd1, i. e., brain malformations, may also be present in patients who show roifman syndrome associated rnu4atac mutations. this indicates that both syndromes may represent overlapping features of the clinical spectrum of rnu4atac mutations. h. roth, h. stöhr, b. h. f. weber institute of human genetics, university of regensburg, germany introduction: inherited retinal degenerations comprise a genetically heterogeneous group of eye diseases with overlapping clinical presentations. up to now, more than 200 genes have been associated with different forms of retinal dystrophies (rd) such as retinitis pigmentosa (rp) or cone-rod-dystrophies (crd) with mutations in 84 and 34 causative genes, respectively. here, we present the results from three patients with remarkable findings and discuss their implications for risk prediction and genetic counseling. methods: targeted next-generation sequencing (ngs) technology based on agilent custom designed gene panels (sureselect) has been established in our diagnostics department to identify causative mutations in a large patient cohort with approximately 200 rd patients. high-throughput sequencing data are routinely analyzed with the clc biomedical workbench. classification of variants was based on bioinformatic analyses using alamut visual software, mutationtaster, sift and polyphen-2 prediction programs, allele frequencies, amino acid conservation and literature. results. ngs analysis revealed two patients with rp and one patient with crd, each of whom carry putative causative mutations in several rd genes. first, a male patient with a family history of crd, is a carrier of a nonsense mutation p.(arg1144ter) in rims1 and two likely pathogenic missense mutations in aipl1 (p.(tyr134phe)) and guca1a (p.(pro-50leu)), each in a heterozygous situation. mutations in all three genes can cause adcrd. in addition, the patient carried a hemizygous nonsense mutation p.(glu1017*) in the x-chromosomal rpgr gene. secondly, a female patient with simplex rp was found to be homozygous for a frameshift-causing deletion p.(ser527leufs*28) in the impg2 gene causing arrp. she also carried three heterozygous, likely pathogenic missense mutations in crx (p.(tyr142cys)) causing adrp, in the x-chromosomal rpgr (p.(ala365val)) and in ush2a (p.(ile1621val)) associated m. s. reuter 1 , a. riess 2 , u. moog 3 , t. a. briggs 4, 5 , k. e. chandler 4 background: disruptions of the foxp2 gene, encoding a forkhead transcription factor, are the first known monogenic cause of a speech and language disorder. so far, mainly chromosomal rearrangements such as translocations or larger deletions affecting foxp2 have been reported. intragenic deletions or convincingly pathogenic point mutations in foxp2 have up to date only been reported in three families. we thus aimed at a further characterization of the mutational and clinical spectrum. methods: chromosomal microarray testing, trio exome sequencing, multi gene panel sequencing and targeted sequencing of foxp2 were performed in individuals with variable developmental disorders, and speech and language deficits. results: we identified four different truncating mutations, two novel missense mutations within the forkhead domain and an intragenic deletion in foxp2 in fourteen individuals from eight unrelated families. mutations occurred de novo in four families and were inherited from an affected parent in the other four. all index patients presented with various manifestations of language and speech impairment. apart from two individuals with normal onset of speech, age of first words was between 4 and 7 years. articulation difficulties such as slurred speech, dyspraxia, stuttering or poor pronunciation were frequently noted. motor development was normal or only mildly delayed. mild cognitive impairment was reported for most individuals. conclusion: by identifying intragenic deletions or mutations in fourteen individuals from eight unrelated families with variable developmental delay/cognitive impairment and speech and language deficits, we considerably broaden the mutational and clinical spectrum associated with aberrations in foxp2. h. rieder, f. beleggia, d. wieczorek we report on a 4-year-old boy with microcephaly, arachnoidal cysts, pachygyria, microgyria, and severe intellectual disability. he also had short stature including shortening and deformation of the femora, brachydactyly, and short ribs with costochondral dysplasia. he showed facial dysmorphism with narrow palpebral fissures, a short nose with a depressed nasal bridge, and a broad mouth with full lips. clinical laboratory investigations demonstrated persistently slightly elevated liver enzymes. exome sequencing revealed compound heterozygous mutations of the rnu4at-ac gene, g.51g>a;g.16g>a, which has been described in an individual with roifman syndrome. we report on a seven-year-old girl, first child of non-consanguineous italian parents, with developmental delay, muscular hypotonia and distinctive craniofacial features (epicanthus inversus, ptosis, broad nasal bridge, mild retrognathia, low-set posteriorly rotated ears and malpositioned teeth in the mandible). because of the tentative diagnosis of blepharophimosis-ptosis-epicanthus inversus syndrome (bpes), conventional cytogenetic analysis, sanger sequencing and mlpa (multiplex ligation-dependent amplification) of foxl2 were initiated and showed unremarkable results. microarray-cgh revealed a 414 kb microduplication of genetic material on 15q11.2: arr[hg19] 15q11.2(22765628_23179948)x3 encompassing the genes tubgcp5, cyfip1, nipa1 and nipa2 of maternal origin. patients with 15q11.2 microduplication have been described to be affected by developmental delay, motor and/or expressive language delay, epilepsy, learning disabilities and/or behavioral problems. however, genotype phenotype correlation is complicated by incomplete penetrance. healthy and mildly affected carriers are reported in the literature. we speculate that the microduplication might contribute but does not fully explain the phenotype of our patient, in particular concerning the craniofacial features. subsequent trio whole-exome sequencing identified a de novo heterozygous mutation in setbp1 (c.3909t>a/ p.tyr1303*) leading to a premature stop codon and most probably resulting in a truncated and functionally impaired protein. mutations in the set binding protein 1 gene (setbp1) on 18q12.3 have been identified to cause schinzel-giedion syndrome (sgs, omim 269150), a rare autosomal dominant disorder characterized by postnatal growth failure, severe developmental delay, seizures, facial dysmorphism, genitourinary, skeletal, neurological, and cardiac defects. chromosomal deletions in 18q including setbp1 have been reported to cause a milder phenotype known as "autosomal dominant mental retardation-29" (mrd29, omim 616078). these observations suggest that the severe sgs phenotype might be the consequence of a gain-of-function or dominant-negative effect of the mutations and that setbp1 haploinsufficiency results in a different, milder phenotype. so far, the function of the set-bp1 protein is unknown. the presented case adds up to the yet small number of reported cases of mrd29 and thereby contributes to the clinical spectrum of setbp1 haploinsufficiency. this work was supported by "förderstiftung des uksh" (project number: 006_2016). the demand for genetic counseling had been constant, in germany, over many years. from 1996 to 2004 around 47.000 cases per year on the average and with minor fluctuations were reimbursed by the german sickness funds (public health insurance system; pabst and schmidtke, gendiagnostik in deutschland, bbaw, p. 195-203, 2007) . in connection with the "genbin2"-project, a new nationwide survey was initiated regarding with arrp. finally, in another female rp patient with no family history of rd, we detected a nonsense mutation p.(trp558ter) and a likely pathogenic splice site change (c.6078 + 3a>g) in the arrp gene eys, assuming compound heterozygosity. in this patient we also identified two heterozygous, likely pathogenic missense mutations in hmcn1 (p.(pro2226thr)) and cep290 (p.(arg2210cys)) underlying dominant and recessive forms of rd. in all three cases, specific mutation(s) could not be uniquely identified as causative. conclusion. results in the three rd cases emphasize that ngs can generate unexpected results that are difficult to interpret, particularly in the absence of segregation analysis and functional data on pathogenicity. the implications for genetic counselling and predictive testing will be discussed. smart qnipt study -detection of fetal trisomy 21 based on methylation-specific quantitative real-time pcr m. sachse, s. werler, j. bonnet, u. neder, h. sperling, s. busche, s. grömminger, w. hofmann lifecodexx ag, konstanz, germany objectives: current non-invasive prenatal testing (nipt) methods for the detection of fetal trisomy 21 (t21) are primarily based on next generation sequencing (ngs) strategies which are quite costly in clinical application and hence are limited to patients who can afford the testing. here, we describe the results of a blinded study with respect to the test accuracy of a newly developed nipt assay based on quantitative real-time pcr (qpcr) for prenatal testing of fetal trisomy 21 (qnipt). methods: in the study maternal plasma samples were collected from 1,044 pregnant women and blinded by an independent contract research organization. after extraction of cell-free dna using qiasymphony instrument and methylation-specific digestion of dna samples a multiplex qpcr was performed. the primary qpcr data were finally evaluated with our ce marked data analysis software. results from this analysis and from confirmatory ngs testing were compared with nipt results using ngs. the study results of successfully analysed maternal plasma samples (n = 966) demonstrated a positive percentage agreement (ppa; equates to sensitivity) of 100% (lower 1-sided 95% confidence interval of 91.8%; n = 35/35) and a negative percentage agreement (npa; equates to specificity; n = 931/931) of 100% compared to ngs-based results. the negative predictive value (npv) for the novel qnipt and confirmatory ngs testing was 100% (lower 1-sided 95% confidence interval of 99.68%). the average fetal fraction of the 966 examined blood samples was 8.1%. the qnipt assay provided reliable test results in 54 blood samples with a fetal fraction below 4% and as low as 2.4%. conclusion: our results suggest that the proprietary qnipt assay is a very reliable and robust method suitable for clinical routine in accordance with international medical associations. the assay represents a more cost-efficient solution over ngs testing and will also be able to provide results in the shortest possible time. while current nipt methods require a minimum fetal fraction of 4% in blood samples from singleton pregnancies, we could demonstrate in the study that our smart qnipt assay can be employed on blood samples with a fetal fraction of as low as 2.4%. in summary, the application of smart qnipt could have the potential to become a nipt solution on a global scale for pregnant women of all ages and risk groups. further studies which aim to include the determination of trisomy 13 and trisomy 18 are currently underway. with respect to the developmental delay of our index patient, chromosome analysis and array-cgh were performed. a microduplication in 3p26.2 (app. 50kb) of unknown significance and a microduplication in xq27.3 (app. 550kb), which comprises the fmr1-gene, were identified and shown to be of maternal origin (arr[hg19] 3p26.2 (2, 811, 861, 170)x3, xq27.3(146, 663, 212 ,089)x3). fmr1 is associated with fragile x syndrome, which is one of the most common causes for x-linked mental retardation. cgg-trinucleotide repeat expansions in the 5' untranslated region (>200 repeats) lead to aberrant hypermethylation of the fmr1-promotor and silencing of fmr1 expression. in contrast, premutations (55-200 repeats) lead to a higher expression of fmr1 and cause a clinical syndrome that is characterised by late progressive cerebellar ataxia (fxtas). in line with this gain-of-function mechanism, we hypothesize that the xq27.3 duplication, which could lead to an increased gene dosage of fmr1, causes a fra(x)-/fxtas-like syndrome and explain the clinical findings in our family. vengoechea et al. described a patient with a similar duplication, who was affected by developmental retardation, epilepsy and hyperactivity. they discussed the microduplication, which arose de novo in their patient, as the cause for the boy's symptoms (vengoechea j. et al., eur j hum genet., 2012 nov; 20(11):1197-200) . in conclusion, we assume a fmr1-duplication syndrome in our family with variable expressivity and a different impact on male and female patients. to further prove this hypothesis, we are planning to perform a segregation-analysis within the whole family. background: congenital myasthenic syndromes (cms) are a genetically heterogenous group of disorders leading to weakness of skeletal muscles -especially ocular, bulbar and limb muscles -with onset mostly at birth or in early childhood. the severity of cms can vary significantly ranging from death in early childhood due to respiratory insufficiency to only mild muscle weakness in adulthood. more than 25 genes that are highly expressed in the neuromuscular junctions are associated with cms. mutations in the chrne gene on chromosome 17p13.2 are responsible for about one half of genetically solved cms cases. they can cause different subtypes of cms with either autosomal dominant or autosomal recessive inheritance. results: here, we report a 3-year-old boy who was born with bilateral eyelid ptosis and congenital vertical talus of the right foot that needed surgical correction. the boy displayed muscular hypotonia with a myopathic facial expression and delayed motor development. ophthalmologic examination revealed external ophthalmoplegia. a next generation sequencing based gene panel for congenital myopathies detected the homozygous frameshift mutation c.750_769dup (p.leu257profs*50) in the chrne gene in the boy. gene dosage analysis did not show an exonic deletion in the chrne gene. sanger sequencing confirmed the mutation in a heterozygous state in the boy's father. however, his mother did not carry the mutation in the chrne gene. conclusions: these results suggest the rare event of a (partial) paternal uniparental isodisomy of chromosome 17 as cause of the homozygous c.750_769dup (p.leu257profs*50) in the chrne gene in the boy. further experiments are currently undertaken to confirm this hypothesis. the annual reimbursement frequencies of the relevant entries in the ebm fee schedule, 08572, 01792, 01837 and 11232, for which only specialists in human genetics and subspecialists in medical genetics can account, from 2009 until 2014. contrary to the findings in the earlier period the demand for genetic counseling has risen sharply: 41,243 (of a total of 54,360) cases in 2009; 45,525 (58,341) in 2010; 46,691 (59,724) in 2011; 51,316 (63,242) in 2012; 54,739 (69,732) in 2013; and 61,308 (74,780) in 2014. we speculate that the temporal correlation of the rise of genetic counseling demand with the enactment of the german act on testing (february 1, 2010) is not coincidental. further factors that might contribute to the increase in demand are the ensuing guidelines of the german commission on genetic testing and cme activities related to attaining a qualification for genetic counseling for specialties other than human genetics. in the course of these activities the awareness for the importance of genetic counseling delivered by specialists in human genetics and subspecialists in medical genetics may have risen. acknowledgements: the "genbin2"-project supported by the robert koch-institute through funding from the german federal ministry of health. we gratefully acknowledge the collaboration with dr. michael erhart, zentralinstitut der kassenärztlichen bundesvereinigung (zi-kbv), berlin, germany it is well known that duplications of down syndrome critical region (dscr) on chromosome 21q22 can cause down syndrome whereby the distinct phenotype is associated with the involved genes and the size of duplication. however, in literature are hardly any cases with mosaic duplications of dscr described. here we report on a 6 year old boy with some clinical features of down syndrome including distinctive craniofacial dysmorphism, simian crease and sandal gap as well as delayed motor and speech development. no other organ abnormalities are known. conventional chromosome analysis showed no numerical or structural aberration whereas interphase fish analysis revealed three signals for dscr in approx. 40% of lymphocytes and in approx. 80% of buccal mucosa cells. array-cgh analysis on dna from peripheral blood confirmed a 2,56 mb duplication of chromosome 21q22.13q22.2. the duplication involves among others the gene dyrk1a which is reported as a candidate gene for down syndrome. this case presents one of the smallest known duplications within dscr which causes even in a mosaic state a mild phenotype of down syndrome. 4-year-old boy was referred to our outpatient clinic due to global developmental delay mainly affecting his speech and his fine motor development. in addition, muscular hypotonia and an abnormal gait were reported by the referring paediatrician. his mother, his maternal grandmother, and numerous relatives are affected by gait ataxia. no causative mutation was detected in the maternal grandmother by means of a multi-gene panel for spinocerebellar ataxia encompassing 118 genes. genetic testing for friedreich ataxia was also without pathological findings. cer, but the patient mother's grandfather had a cancer of unknown origin and died at the age of 50 years. because of the suspicion of having a lynch syndrome an immunohistochemistry and microsatellite analysis have been performed on the tissue of the colorectal cancer and the hepatic metastasis. all four mmr proteins were properly expressed in immunohistochemistry in colorectal cancer. just the expression of mlh1 protein in the hepatic metastasis was focally weakened and inhomogeneous. the microsatellite markers bat25, bat26, d5s346 (apc), d2s123 and d17s250 (mfd) were all stable, a a146t-kras mutation was found. after performing a multi-gene panel (ngs, next-generation sequencing), a gross heterozygous deletion of exon 1 in msh6 gene has been found in the cnv analysis of the ngs data. this mutation was confirmed with a mlpa and quantitative real time pcr analyses. furthermore, rna expression of msh6 was reduced to 50% in blood lymphocytes in comparison to control samples pointing to a potential role of msh6 loss in the patient's tumor development. we are observing more and more patients with probably pathogenic and pathogenic mutations in one of the mmr genes with normal immunohistochemistry and microsatellite analysis. therefore, we propose that the criteria for performing a molecular genetic analysis of hnpcc/lynch syndrome should be revised. exome sequencing reveals gata1 mutation in a patient with partial delta-storage pool deficiency and mild thrombocytopenia objectives: we report about a 35-year-old male patient of russian background with severe and frequent epistaxis and hematoma since infancy. he presented with mild thrombocytopenia and increased mean platelet volume. von willebrand's disease and subhemophilia had been excluded. previously, he was diagnosed with immune thrombocytopenic purpura. he never underwent elective surgery. his parents were asymptomatic. however, his 4-year-old daughter also suffers from severe bleeding symptoms (multiple, light red hematoma in consequence of minimal trauma). methods: whole exome sequencing (wes) was carried out for the patient, his asymptomatic wife, his symptomatic daughter and her asymptomatic 8-year-old brother. platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. lysates of gel-filtered platelets were analyzed for total granule p-selectin, cd63 and von willebrand factor (vwf) content by western blotting and for serotonin levels by elisa, respectively. results. platelet function and characterization of the patients granula suggested a delta-storage pool disease (spd). in most cases delta-spd occurs as part of a syndrome, e. g. combined with albinism, immunodeficiency or a thalassemic-like blood disorder. as the patient and his daughter did not show any conclusive phenotype, their dna was subjected to wes. exome sequencing revealed a not yet described gata1-mutation close to two zinc finger domains (znf1 and znf2) in a highly conserved region of the gata1 gene in the 4-year old daughter (c.886a>c, p.t296p, heterozygous) and her father (c.886a>c, p.t296p, hemizygous). this mutation was absent in 150 wildtype-controls but could also be demonstrated in the indexpatients' asymptomatic mother. only a few mutations are known to be located in this c-terminal region to date. mutations in gata1 may lead to different clinical presentations, depending on their location within gata1 (e. g. diamond-blakfan anemia (exon2), x-linked thrombocytopenia (znf1), transient myeloproliferative disorder (intron 1, exon 2, exon 3) and acute megakaryoblastic leukemia (intron 1, exon 2, exon 3) in case of down-syndrome). significantly increased hbf-levels (reference level: ≤0,8%) in the affected family members of 13,5% (4-year-old daughour proposita is a 42 years old woman, who was transferred to our genetic counselling department for the suspicion of m. osler (hereditary hemorrhagic teleangiectasia, hht). during routine check up an anemia was diagnosed. a tumor search was initiated and unexpectedly the ct of the abdomen showed a suspect coin lesion of about 2.5 cm in diameter localized in the basal part of the right lung. further investigations revealed a pulmonary arterio-venous malformation which was hemodynamically relevant and already led to chronic right heart overload. a coil embolization was performed. retrospectively, medical history of the patient included episodes of severe epistaxis in childhood and a neurosurgical intervention for intracerebral bleeding at the age of 16 years without permanent neurological deficits. during genetic counselling our proposita mentioned that her 8 years old daughter also suffered from anemia due to multiple polyps of the colon. after polypectomy her hemoglobin values normalized. although histologically the polyps appeared as juvenile ones a mutation search in the apc-gene was initiated by the gastroenterologists without identifying a pathogenic mutation. combining the two pieces of information, we offered a mutation search in the smad4 gene and a pathogenic mutation c.1081c>t (p.arg361cys) was found in both patients in heterozygosity. colonoscopy in the mother did not show juvenile polyps or gastrointestinal vascular malformations. vice versa, no cerebral or pulmonary arteriovenous malformations could be detected in the daughter. our family illustrates, that the same mutation within a family may phenotypically appear as different diseases. a careful taking of medical history and the knowledge of all relevant diagnostic findings (in this case e. g. the histology of the polyps) can enable the geneticist to offer a precise differential diagnosis leading to a well-directed molecular testing. to our opinion this is still relevant even in the era of ngs-based panels because the more precise the clinical diagnosis and the choice of the genes to analyse the less problems with unclassified variants will arise. the hereditary non-polyposis colon cancer (hnpcc, lynch syndrome) is caused by pathogenic germline mutations in mismatch repair genes (mlh1, msh2, msh6 and pms2) causing microsatellite instability (msi) and decreased or lost expression of the appropriate mismatch repair protein (mmr) in the immunohistochemistry (ihc) on tumor material. thus, ihc and msi testing help to identify the mmr gene, which most likely harbors a germline pathogenic variant. msi and ihc testing prior to germline analysis are specified in the s3 guidelines of hnpcc and if negative normally preclude further genetic analyses. here, we present a 51 year old patient with a synchronous colonic (ceacum) and renal cancer at the age of 50 years. at the time of the diagnosis hepatic metastases of the caecal adenocarcinoma have already been present. histologically, the colonic tumor was a poorly differentiated adenocarcinoma (pt3pn) with lymphangiosis and haemangiosis carcinomatosa. the renal cancer showed histology of a moderately differentiated clear cell renal carcinoma. in the family history the 50 year old sister and the parents are healthy. the twin sister of the patient's mother had a collateral breast cancer at the age of 46 years and died two years later. the mother's grandparents had no canmedizinische genetik 1 · 2017 155 p-cling-109 pitfalls in molecular genetic diagnostics a. tibelius, e. fey, k. hinderhofer institute of human genetics, heidelberg, germany probably every clinical laboratory geneticist may look back on at least one case in his career which has caused him quite a headache. this means those cases with completely unexpected and at a first glance implausible results which could be interpreted correctly only after intensive enquiry and additional testing. here, we report on three of such pitfall cases from our routine diagnostics. case 1: a 30-year-old woman, pregnant with monozygotic twins, was referred to prenatal cystic fibrosis diagnosis. she and her partner were carriers of mutations in the cftr gene (621 + 1g>t and g542x, respectively). prenatal molecular diagnosis demonstrated that both fetus had inherited only the paternal mutation. routinely, we performed maternal cell contamination analysis by comparing polymorphic microsatellite loci between the maternal and fetal dna. surprisingly, 12 of 16 tested microsatellite loci revealed a discrepancy between the maternal and fetal genotypes, meaning neither of both maternal alleles was present in fetal dnas. a potential confusion of samples was excluded. moreover, the presence of paternal mutation in fetal dnas indicated a correct genetic relationship between awaited children and the partner of pregnant woman. the only one plausible interpretation of the obtained result was a pregnancy by egg donation. afterwards, this suspicion was confirmed by the couple. case 2: we present a 38-year-old man with infertility resulting from azoospermia. conventional chromosomal analysis and an additional fish analysis using y probes indicated a 46,xx karyotype with no detectable sry. in parallel, a molecular azf (azoospermia factors) diagnostic was performed by a standard multiplex pcr. by this method the absence of sry region and a deletion of regions azfb and azfc, was identified, explaining the observed azoospermia. interestingly, the pcr showed that the azfa region was still present in patient chromosomes, contradicting cytogenetic and fish results. thus, a complementary fish analysis was performed in order to reveal a low-grade y-mosaicism and sry material was detected in 3% of the cells (a result under the threshold level). based on this observation, pcr conditions for the azf diagnostic were modified and a very weak sry-specific pcr product detected. case 3: a molecular diagnostics for frax (fragile x syndrome) was requested for a 4-year-old boy with a slight delay in speech development. his brother was already molecular-genetically diagnosed as having frax. the analysis by southern blot hybridisation in the patient revealed a smear of methylated fragments characteristic for an expanded allele in the full mutation range. surprisingly, two fragments of normal length, methylated and non-methylated, could also be detected in patient's dna. a subsequent aneuploidy mlpa confirmed a supernumerary x-chromosome in the patient consistent with a klinefelter syndrome. these results were verified by an independent cytogenetic analysis. the syndrome of congenital symmetric circumferential skin creases (cscsc1 and cscsc2) replaces the old term michelin-tire-baby syndrome (mim 156610) and is characterized by congenital circumferential skin folds, primarily of the limbs, facial dysmorphism, cleft palate and intellectual disability. mutations in the β-tubulin encoding gene tubb or in the microtubule end binding family member mapre2 are the underlying genetic cause. ter) and 2,8% (35-year-old indexpatient) suggested dyserythropoiesis, although thalassemic features of the blood count were lacking. conclusion: we describe a gata1 mutation as the cause of a delta-storage pool disorder. imbalanced x-chromosome inactivation might explain the different phenotypes of the gata1 mutation carriers and will be investigated through allele-quantification based on rna isolated from whole blood and from platelet rich plasma in case of the index patient and different family members. we saw three siblings (aged 62, 61, 54; two female, one male) with variable signs of retinal disease. all three developed night blindness till the 3rd decade, furthermore near-sightedness and deterioration of the visual acuity to a various degree at age 35-45. the clinical diagnosis was given as stargardt's disease, fundus flavimaculatus or unspecified retinal degeneration respectively. two more siblings (aged 59 and 57) and the mother (died age 62) were clinically unaffected. the father (died age 69) was reported to have had bad eyesight beginning in the 5th decade, his father similarly (no further information available). the three affected siblings have a total of five children (age 25-39), none of them showing clear signs of retinal disease up to now. considering the clinical diagnosis stargardt's disease we analysed the genes abca4, elovl4, prom1 and cngb3 by sanger sequencing. no pathogenic mutation could be detected. afterwards we performed next generation sequencing of several genes associated with retinal dystrophies and found a novel splice site mutation in the prph2 gene (mim #179605). the mutation was confirmed in all three affected siblings by sanger sequencing. considering that mutation pathogenic we could re-diagnose our patients with patterned macular dystrophy type 1 (mim #169150). that disease is inherited in an autosomal dominant fashion, corresponding to the pattern of inheritance evident in our family. the etiology of epileptic encephalopathies, characterized by severe, early-onset seizures accompanied by developmental delay or regression, is highly heterogeneous. in recent years, next-generation sequencing approaches have led to the discovery of numerous causative genes; however the spectrum of associated phenotypes still needs to be further explored for many of these genes. we performed multi-gene panel analysis in a little boy of german non-consanguineous parents who showed severe early-onset infantile epileptic encephalopathy and almost absent neurological development. in this patient we identified a novel heterozygous missense mutation in the gabrg2 gene which was absent in the parents. in silico analyses strongly suggested a pathogenic relevance of this sequence variation which resides within a highly conserved region. so far, gabrg2 mutations have mainly been associated with milder types of epilepsy such as febrile seizures and childhood absence epilepsy. therefore, our findings extend the phenotypic spectrum associated with mutations in this gene at the severe end. referring on a case report by al marzouqi et al. (2014) , who reported on a girl with bilateral amastia in the context of skewed x-inactivation, we want to underline the importance of mlpa analyses in the case of negative sequencing of the eda gene, as whole exon duplication can be the cause of hypohidrotic ectodermal dysplasia. to our knowledge, the report of al marzouqi et al. has been the only case about this genetic alteration in the literature so far. novel pogz mutation in a patient with intellectual disability, microcephaly, strabismus and sensorineural hearing loss we report on a 3-year-old male patient with severe intellectual disability, microcephaly, sensorineural hearing loss, ocular abnormalities (strabismus and hyperopia), congenital heart defect (atrial septum defect and pulmonary stenosis) and minor facial abnormalities (thin upper lip, frontal upsweep). next-generation sequencing analysis revealed a novel heterozygous de novo mutation in pogz: c.2703_2710del, p.(thr902serfs*39) . the clinical problems of this patient are in accordance with the findings in the previously reported pogz mutation carriers. reports of additional patients with pogz mutations will be needed to establish detailed phenotype-genotype correlations of this novel and probably underdiagnosed syndrome. novel clinical and molecular aspects in two patients with kleefstra syndrome d. wand, b. seebauer, k. heinimann, i. filges medical genetics, university hospital, basel, schweiz the phenotype of kleefstra syndrome is clinically variable but characterized by facial dysmorphism, intellectual disability, childhood hypotonia and variably associated other malformations. haploinsufficiency of ehmt1, caused by either heterozygous microdeletions in 9q34.3 or sequence mutations in ehmt1, has been identified to be the underlying causal mechanism. here we present two girls from two unrelated families with clinical signs of kleefstra syndrome. besides the main features such as facial dysmorphism, intellectual disability/developmental delay and childhood hypotonia, the 17 years old girl presented with additional accelerated growth whereas the other girl, 2 years old, showed failure to thrive. both children have no heart defect, renal or urogenital anomalies or severe respiratory infections. we identified two rare variants likely to be causal: a novel heterozygous splice site ehmt1 variant and a heterozygous microdeletion in chromosome 9q34.3, including exons 26 an 27 of the ehmt1-gene . these patients broaden the spectrum of kleefstra -associated ehtm1 causes, contribute to novel aspects of genotype-phenotype correlations and a better understanding of the clinical variability of the disorder. here, we present two girls from two non-consanguines families showing clinical aspects of the syndrome of congenital symmetric circumferential skin creases. additional features are present in both girls. patient 1 is the first child of healthy parents. she was born at 39 + 6 weeks of gestation with a weight of 2660 g, a length of 49 cm and a head circumferences of 34 cm. respiratory distress, a cleft palate, a heart defect (atrial septum defect), an anogenital malformation and facial dysmorphism were diagnosed after delivery, additionally to the skin creases phenotype. conventional karyotyping performed on blood lymphocyte cultures and cgh-array analysis showing normal results. patient 2 is the second child of unrelated healthy parents. her older sister is healthy as well. because of an intrauterine growth retardation an amniocentesis with chromosomal analysis was performed, showing a normal karyotype of 46,xx. patient 2 was born at 38 + 3 weeks of gestation by caesarean section. the birth weight was 2450 g, the birth length was 47 cm and the head circumferences was 34 cm and the apgar score was 1/3/5. due to respiratory distress and hypoxia a tracheotomy was initiated. she also presented with cleft palate, feeding difficulties, a heart defect (atrial and ventricular septum defect), asplenia and facial dysmorphism. the skin phenotype was remarkably similar to that of patient 1 with a prominent neck fold and skin creases mainly on the back part, but also at the limbs. in both children the skin folds gradually diminish over the time without any intervention like it was described for michelin-tire-baby syndrome/ cscsc1 and cscsc2 patients. for these reason, a disease-causing mutation in the genes mapre2 and tubb were excluded in both children. to identify a genetic cause, we performed a trio whole-exom sequencing, including the healthy parents and the affected child, in both families. a de novo stop mutation was detected in patient 1, while no promising results could be detected in patient 2. further studies, especially functional in vivo studies and analysis of further patients with a similar clinical presentation will answer the question if the above described phenotype is an expanded variant of the congenital symmetric circumferential skin creases or a unique new syndrome. clinical findings in a family with x-linked hypohidrotic ectodermal dysplasia due to a duplication of exon 2 in the eda gene -a case report we want to summarize the phenotypic spectrum in one affected three generation family with x-linked hypohidrotic ectodermal dysplasia. we report on one strongly affected male and 2 slightly affected female carriers, carrying a duplication of exon 2 in the eda gene. reason for genetic assessment was the request of a female for evaluation of a possible risk for occurrence of the familial disorder in a further planned pregnancy. the woman reported an inability to breastfeed and she and her daughter showed conical teeth, a dry skin and sparse hair. the affected male showed absent deciduous teeth, hypodontia of permanent teeth, missing regulation of the temperature due to a lack of sweat glands, bilateral nipple hypoplasia, a dry and wrinkled skin, missing eye lashes, eyebrows and scalp hair and sparse body hair. the fingernails were inconspicuous, whereas the nails of the toes, particularly the nails of the hallux were yellowish and thickened. the affected male had an operation due to a gallstone and cysts were found in one kidney. there was no increased predisposition to infections. the suspected diagnosis of x-linked hypohidrotic ectodermal dysplasia was confirmed by genetic analysis of the eda gene (sequencing and mlpa analysis). a duplication of exon 2 was detected in the affected male patient and was confirmed in the two mildly affected female relatives by realtime-pcr. terminator sequencing mix v1.1 on an abi3130xl genetic analyzer (applied biosystems) and detected an insertion of 77 bp between the exons 7 and 8. we located the insertion's sequence in intron 7 of the dmd gene and sequenced flanking sequences of gdna to find the underlying mutation causing the insertion. two hemizygous single nucleotide variants (snvs) surrounding the inserted fragment could be identified. the first variant (c.650-39575 a>c) is a common polymorphism (maf according to 1000 genomes project: 14.97%) at the position of an existing acceptor splice site. the second variant (c.650-39498a>g) is novel and creates a new cryptic donor splice site with high probability. these two cryptic splice sites create the formation of a 77 bp pseudoexon which produces a frameshift and a premature stop codon (p.asp217alafs*) in the dmd gene. in summary, we could genetically confirm the clinical diagnosis of a dystrophinopathy by two divs in dmd. although the insertion of the pseudoexon creates a premature stop codon the patient's clinical phenotype indicates a milder type of becker muscular dystrophy. this contradiction could be explained by the remaining existence of dmd wild type mrna most likely due to a not constantly active cryptic splice site. most interesting about the present case is the fact that a common snv facilitates the creation of a pseudoexon. this makes the region a potential hotspot for divs in the dmd gene which would be worthwhile further investigations. with more than 90% of all humans preferring to use their right hand, handedness is the most noticeable functional expression of cerebral lateralization in humans. however, the precise molecular mechanisms that regulate handedness and other related forms of cerebral lateralization remain largely elusive. therefore, the question which genetic, epigenetic and environmental factors contribute to human handedness is one of the central questions in research on lateral asymmetries. handedness is a complex, heritable trait, for which polygenic inheritance is assumed, meaning that a large number of genetic factors with a small additive effect contribute to the observed variance in hand preference. to date, genetic association studies have implicated only a few specific genes influencing handedness. particularly interesting is the association between the human androgen receptor (ar) gene and different aspects of handedness, since the interrelationships constitute a conceptual bridge between the theories that invoke testosterone as a factor in the development of cerebral asymmetries with theories proposing that the x chromosome contains a locus that influences the direction of hand preference. in an initial large association study in 1057 samples of healthy adults we already demonstrated that handedness in both sexes is associated with the ar cag-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. in addition, we have performed a second association study in an independently collected healthy cohort of more than 1000 test persons with comprehensive data on the handedness phenotype. we were able to replicate the association with longer cag repeats being related to a higher incidence of non-right-handedness, especially in females. since longer cag-repeat blocks have been linked to less efficient ar function, these results implicate that differences in ar signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization. dej. waschk, ac. tewes, p. wieacker, s. ledig institute of human genetics, münster, germany the mammalian female and male reproductive tracts develop from the paramesonephric ducts (müllerian ducts, md) and mesonephric ducts (wolffian ducts, wd), respectively. in the absence of testicular differentiation and anti-müllerian hormone, the wd regress and the md give rise to the fallopian tubes, uterus, cervix and the upper part of the vagina. disorders of normal md development in females can manifest as fusion anomalies of the uterus such as septate uterus, bicornuate uterus, unicornuate uterus and uterus didelphys, or more complex malformation patterns like mayer-rokitansky-küster-hauser syndrome (mrkh) or herlyn-werner-wunderlich syndrome. mrkh is characterized by congenital absence of the uterus and the upper two-thirds of the vagina in individuals with a normal female karyotype, most of whom show normal ovarian function. mrkh can further be associated with additional malformations e. g. of the kidneys and the skeletal system. herlyn-werner-wunderlich syndrome is characterized by uterus didelphys, obstructed hemivagina and ipsilateral renal agenesis. despite anomalies of the md occur quite frequently, the etiology of most cases with these disorders remains unknown. the homeodomain transcription factor emx2 (empty spiracles homeobox 2) was found to be critical for urogenital and central nervous system development. previous studies showed that in emx2 mutant mice, the kidneys, ureters, gonads and genital tracts were completely missing. in order to elucidate whether mutations in emx2 are causative for md anomalies in humans, we performed sequence analyses of emx2 (genbank nm_004098.3) in our study group of 142 female patients with clinically characterized disorders of the md including 62 patients with mrkh and 7 cases with herlyn-werner-wunderlich syndrome. we found the heterozygous intronic mutation c.592-17c>a twice in this cohort. this variant has been described earlier (rs41308651) and is listed in the exome aggregation consortium exac variant with a minor allele frequency of 0.23%. in silico analysis revealed no significant changes for the correct splicing of emx2. we therefore consider this variant to be a benign polymorphism. we conclude that mutations in emx2 are not causative for disorders of the md in our cohort. a. zaum, w. kreß, a. gehrig, s. rost institute of human genetics, würzburg, germany dystrophinopathies are x-linked muscle diseases caused by mutations in the dmd gene (omim: 300377). due to the huge size of this gene, the detection of mutations is sometimes challenging. despite multiplex ligation-dependent probe amplification (mlpa) and sequencing of all 79 exons, about 7% of patients do not show any mutations in coding regions and therefore remain without molecular diagnosis. we assume that the majority of these patients have deep intronic variations (div) which are not detectable by standard diagnostic techniques. the index patient analysed is a twelve-year-old boy who was by chance diagnosed with elevated ck levels (up to 15,000 iu/i) at eight weeks of age. today he is still able to walk without walking aids but needs assistance when climbing stairs. in 2008, a muscle biopsy revealed complete absence of dystrophin which established the diagnosis of dmd. for the molecular diagnosis, standard diagnostics ascertained no causative mutation. therefore we decided to search for deep intronic mutations. we isolated mrna from muscle tissue of the patient and amplified overlapping cdna fragments using rt-pcr. the fragments were analysed by gel electrophoresis for size differences compared to an unaffected control. the cdna product comprising exons 6-12 revealed an augmented fragment size compared to the control and the expected product size (about 1100 bp instead of the expected 1007 bp). we sequenced the altered cdna product using bigdye recent research in psoriasis has identified pustular psoriatic manifestations as either mendelian traits or as major genetic risk factors in contrast to the numerous associated snps in classical plaque psoriasis vulgaris. autosomal recessive mutations in il36rn have been identified in ~25-40% of patients with generalized pustular psoriasis (gpp), a rare, severe pustular variant of psoriasis. in addition, heterozygous missense variants in card14 and ap1s3 have been associated to pustular psoriasis as well and shown to be functionally relevant. in order discover how relevant those genes were in our large gpp cohort of 61 patients recruited all over germany, we screened them for coding variants in il36rn, card14 and ap1s3 by sanger sequencing and for quantitative aberrations by mlpa. we identified homozygous or compound heterozygous il36rn mutations in 15 of 61 gpp patients (25%) and single heterozygous mutations in 5 patients (8%). the most common mutations were c.338>c>t/ p.ser113leu and c.227c>t/ p.pro76leu, present on 49%/20% of mutated alleles, respectively. we also identified three so far unreported mutations: c.338c>a/ p.ser113x, c.295-300delacct-tc/ p.thr99_phe100del and c.130g>a/ p.val44met. according to molecular modeling, c.338c>a/ p.ser113x resulted in a shortened, de-stabilized protein analogous to c.280g>t/ p.glu94x. the other two mutations were also predicted to result in destabilized, likely disease-relevant, loss-of-function proteins. heterozygous ap1s3 mutations were detected in two gpp patients, both of whom carried additional homozygous or compound-heterozygous il-36rn mutations. 4 gpp patients were heterozygous carriers of rare missense variants in card14 (7%); of note, two of these patients carried additional mutations in il36rn. our genotype-phenotype correlation revealed a similar gender distribution in carriers of il36rn mutations and wildtype carriers, but a strong association between bi-allelic mutations in il36rn and early age of manifestation (p = 7.4x*10-04). as in other autosomal recessively inherited mutations, the frequency of parental consanguinity was significantly higher in patients with two il36rn mutations compared to non-carriers. overall, our genetic studies suggest a lower impact of variants in ap1s3 and card14 in pustular psoriasis than of those in il36rn. interestingly, the combination of il36rn mutations with either ap1s3 or card14 congenital prosopagnosia (cp), also known as developmental prosopagnosia or face blindness, describes the inability to recognize faces. cognitive functions such as intelligence as well as the sensory visual capabilities are usually not impaired but people with prosopagnosia are negatively affected in their social life because individuals with the disorder have difficulty in recognizing family members, close friends or colleagues. although the prevalence of cp is estimated at 2.5% and it appears to run in families, the contexts, which genetic, epigenetic and environmental factors contribute to this trait, are largely unknown. therefore we started to establish a large, well-characterized cp cohort for genetic studies. we hypothesize that rare highly penetrant non-synonymous genetic variants could explain some cases of cp. as part of a larger genetic study of patients with cp, we performed family based whole-exome sequencing and targeted re-analysing on four individuals from two families with multiple affected members. by obtaining samples from affected and unaffected members of the same family, we hope to effectively identify de novo and inherited variations. variations are considered on the basis of allele frequency, mutation type, literature and mutation prediction tools, thus generating a list of candidate variations/genes for each patient that is amenable to interpretation and further analyses in the extended cohort. through this approach, we hope to identify causal variations/genes in families and isolated patients with cp. erythrokeratodermia variabilis (ekv) is a rare, autosomal dominant genetic skin disorder with a highly heterogeneous phenotype. to date, three causative genes (gjb3, gjb4 and gja1) are described but further genetic heterogeneity is expected. card14 mutations are only described for psoriasis vulgaris, generalized pustular psoriasis and pityriasis rubra pilaris. for the first time, we present disease causing card14 mutations in patients with an "ekv-like" phenotype. it refers to one familial case with two affected individuals, with an autosomal dominant transmission from the mother to the daughter and one independent sporadic case. all patients present typical ekv symptoms. a rash of well-demarcated erythematous and scaly plaques interspersed with distinct islands of uninvolved skin or small reddish papules coalescing into large reticulated scaly plaques and palmoplantar keratoderma. to confirm the suspected diagnosis of ekv, we analyzed a custom designed multi-gene panel by next generation sequencing with 74 genes associated to hereditary skin diseases (agilent haloplex technology). the sequencing results did not reveal any mutation in the genes gjb3, gjb4 and gja1, but we found two pathogenic mutations in card14. in the familial case c.467t>c p.leu156pro (rs387907240, enst00000570421.5) was detected, while the sporadic case carries c.371t>c p.leu124pro. we hypothesize that different genetic and environmental factors are involved in the evolution of the phenotype in patients with card14 mutations. our cases show that classification of unusual skin phenotypes can be challenging without genetic testing. therefore, gene panel sequencing is a cost-efficient and time-saving solution for solving difficult cases with sometimes unexpected genetic background. bipolar disorder (bd) is a complex psychiatric disorder affecting more than 1% of the world's population. the highly heritable disease is characterized by recurrent episodes of manic and depressive symptoms. as the cumulative impact of common alleles with small effect may only explain around 38% of the phenotypic variance for bd, rare variants of high penetrance have been suggested to contribute to bd susceptibility. in the present study we investigated 226 individuals of 68 large multiply affected families of european origin using whole exome sequencing (wes). in each family, two to five affected individuals with bd or recurrent major depression were selected for sequencing. wes was performed on the illumina hiseq2500 platform and the varbank pipeline of the cologne center for genomics was used for data analysis. all identified variants shared within each family were filtered for a minor allele frequency <0.1% and potentially damaging effects predicted by at least four of five different bioinformatics tools. we identified a total of 1214 rare, segregating and functional variants implicating 1122 different genes, of which 903 were brain expressed. subsequently, we applied the residual variation intolerance scores (rvis, petrovski et al., 2013) and identified 294 genes that were ranked among the 20% most "intolerant" genes in the genome. gene enrichment analysis of these genes showed a significant enrichment for a total of 18 pathways (p < 0.001) including neuron projection, axon development and cell adhesion. for follow up analyses, we prioritized genes that were either found in at least two unrelated families in the present study or that were previously reported in next generation sequencing or gwas studies of bd. in addition, we enclosed the genes that were predominantly driving the significant pathways in the above mentioned gene enrichment analysis. the different approaches of prioritization yielded 42 candidate genes that are currently being followed up by resequencing in cohorts of about 2500 independent bd cases and 2500 controls of european ancestry. the candidate genes include cdh22 that encodes a calcium-dependent cell adhesion protein that may play an important role in the morphogenesis of neural cells during the development and maintenance of the brain. for resequencing we use the single molecule molecular inversion probes (smmips) technology that enables multiplex targeted resequencing in large cohorts. the smmips sequences were designed with an empirically variants in several patients indicated an oligogenic inheritance rather than a purely monogenic one. moreover, the oligogenic basis of this group of inflammatory diseases might currently be underestimated, as our study suggests that genetic risk factors other than il36rn mutations remain to be identified in the majority of gpp patients. exome sequencing of 46 multiply affected schizophrenia families provides new insights into the pathogenesis of the disorder schizophrenia (scz) is a multifactorial psychiatric disorder with a lifetime risk of ~ 1% and a heritability of about 60-80%. analysing multiply affected families using whole exome sequencing (wes) is a very promising approach to identify new scz risk factors. in these families, individuals are affected with scz over several generations. it is likely, that in multiply affected families genetic variations with particularly strong effect co-segregate with the disorder and contribute to the development of the psychiatric symptoms. to our knowledge, the present study is the largest study analysing multiply affected scz families using wes worldwide so far. we included 46 families with at least 3 affected members each. from each family, 3-5 individuals were exome sequenced on an illumina hiseq 2500 and analysed using the varbank pipeline of the cologne center for genomics (http://varbank.ccg.uni-koeln.de) and the clc bio biomedical genomics workbench. we included rare (allele frequency ≤ 0.1% in the exome aggregation consortium dataset) variants that were predicted to be pathogenic (combined annotation dependent depletion score ≥ 15; cadd.gs.washington.edu), confirmed by sanger sequencing and co-segregating with the disorder. in total we identified potentially pathogenic mutations in ~ 880 genes. a substantial proportion of these will not contribute to the pathogenesis of scz. in order to further tease out the most promising candidate genes we applied multiple strategies: (i) screening our mutations in independent patient and control cohorts through international cooperations (access to more than 3,000 scz patients), (ii) gene-based tests, (iii) pathway-and network-analyses, (iv) gene expression analyses and (v) sequencing of the candidate genes in 2,500 scz patients and 2,500 controls. analyses are ongoing and will be presented at the upcoming conference. abstracts and our evolution, we know very little about the different mutagenic processes in our germline. of particular interest are a handful of highly recurrent dnm associated with congenital disorders and/or rasopathies, that have been described as driver mutations expanding in the male germline. the mutation itself causes a change in the tyrosine kinase receptor/ras/ mapk pathway, which in turn confers the spermatogonial stem cell a proliferative advantage. selfish or driver mutations are quite common in cancer, but we still know very little about the selfish expansion in the male germline. the reason might be that mutations in the human germline are very rare, and it is rather difficulty to directly measure such rare events. most of our knowledge on germline mutagenesis comes from indirect sequence comparisons or whole genome sequencing of pedigree families, but it renders little information about individual mutagenic events. for this reason, we have adapted an ultrasensitive, next generation sequencing (uss) technology for the measurement of rare mutations to study the expansion of selfish genes in the male germline. as a proof-of-principle, we have sequenced at an extremely high coverage exon 10 and 15 of the fgfr3 gene in young and old sperm donors. we found an increased mutation frequency for the loci associated with achondroplasia and thanatophoric dysplasia ii in sperm of older donors. our results also show that we can distinguish ultra-rare mutations occurring at a frequency of one in hundred thousand wild type; thus, making this method ideal to discover potential driver dnm that might be expanding with paternal age. s. sivalingam 1, 2 , a. j. forstner 1, 2, 3 , s. herms 1, 2, 4 , a. maaser 1, 2 , c. s. reinbold 4, 5 , t. andlauer 6 , j. frank 7 , h. dukal 7 , d. schendel 7 , 2 , p. hoffmann 1, 2, 4 , t. kircher 8 , u. dannlowski 8, 9 , a. krug 8 , a. cichon 1, 2, 4 , s. witt 7 , m. rietschel 7 , m. m. nöthen 1, 2 introduction: affective disorders such as major depressive disorder (mdd) and bipolar disorder (bd) are genetically complex and heterogeneous disorders. both genetic and environmental risk factors contribute to the etiology of the diseases. however, the neurobiological correlates by which these risk factors influence the disease development are hardly understood. increasing evidence suggests that epigenetic modifications such as dna methylation have important implications on the development of psychiatric disorders including mdd and bd. several studies revealed that alterations in the dna methylation can modulate gene expression in response to the environment. to investigate this, genome-wide dna methylation analysis of 66 female individuals from three extreme groups (genetic-, environmental risk and healthy controls) was performed. methods: for the genome-wide dna methylation analysis we selected: (i) 22 individuals with genetic risk (at least one 1st degree relative with a life-time diagnosis of mdd or bd), (ii) 22 individuals with environmental risk (maltreatment in the childhood trauma questionaire) and (iii) 22 matched healthy controls. all individuals were of european origin. processing was done according to the manufacturer's protocol using the infinium methylationepic beadchip (illumina, san diego, ca, usa) covering more than 850.00 methylation sites at the life & brain center (bonn, germany). state-of-the-art data processing protocols, including correction for blood cell type heterogeneity, color correction, eliminating probes containing snps and cross reactive probes were used. after quality control trained design algorithm mipgen (boyle et al., 2014) and sequencing is currently performed on the illumina hiseq2500 platform. our preliminary results strongly suggest that rare and highly-penetrant variants in neuronal and cell adhesion genes contribute to bd etiology. the results of resequencing of a large case/control sample will provide further evidence for an involvement of particular pathways. the use of zebrafish as model system to quantitatively assess the impact of risk variants in non-coding regions in vivo s. l. mehrem 1, 2 , b. nagarajan 3 , n. ishorst 1, 2 , a. c. böhmer 1, 2 , e. mangold 2 , b. odermatt 3 , k. u. ludwig 1, 2 1 department of genomics, life & brain center, university of bonn, bonn, germany, 2 institute of human genetics, university of bonn, bonn, germany, 3 institute of anatomy, university of bonn, bonn, germany most human malformations occur early in embryonic development and are present immediately after birth. one common human birth defect is nonsyndromic cleft lip with or without cleft palate (nscl/p), affecting about 1 in 1,000 newborns. nscl/p has a multifactorial background with a strong genetic component. recent genome-wide association studies identified several loci as risk factors for nscl/ p. notably, most of them map to non-coding regions and are expected to have a functional impact through regulatory mechanisms. given the early developmental time point of facial development and the resulting lack of accessible human tissue, follow-up analyses of risk variants are challenging. we are hypothesizing here that we might be able to quantitatively detect differences in regulatory activity between wildtype and risk variants located in predicted enhancers by using the zebrafish as model organism. the advantages of using the zebrafish are (1) ex utero development, (2) transparency of the fish, (3) easy manipulation and (4) relatively short generation times. we applied a dual-luciferase assay plasmid system which is based on a sequential measurement of two luciferases (firefly and sea pansy luciferase) in fish lysates upon injection of a single plasmid. this plasmid, which contains a minimal promoter (minp) and the enhancer region of interest, is microinjected into zebrafish eggs of one-cell stage. after three days, fish are collected, lysed and luciferase activity is measured using a luminometer. for our proof-of-principle analysis we analyzed an nscl/p risk locus on chromosome 13q31 (ludwig et al. 2012) . through database research one enhancer was predicted that contained two strongly associated risk variants. in vivo fluorescence analysis using egfp in zebrafish embryos revealed this enhancer to be active in craniofacial development, but qualitative differences in activity were not observed by eye. upon cloning of the enhancer in the dual-luciferase system, our injection results in vivo indicate that the system is working in principle. however, a high standard deviation between single replicate measurements was observed, probably due to variability in transfection efficiency. we therefore are planning to adapt the protocol in order to screen for positively injected fish embryos. we are currently investigating the functionality of these screening constructs in zebrafish embryos. results will be presented at the meeting. once successful, our approach represents a practical method to quantify the activity of regulatory elements in real time in vivo. this will be of particular importance in the functional follow-up of genetic findings in non-coding regions for the majority of birth defects. previously genome-wide association methods in patients with classic bladder exstrophy (cbe) found association with isl1, a master control gene expressed in pericloacal mesenchyme. this study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. genotypes of 12 markers obtained from 268 cbe patients of australian, british, german ital-and normalization 780,467 cpg-sites were tested for genome-wide dna methylation by a linear regression model while accounting for biological as well technical covariates. results: the genome-wide dna methylation analysis of the three extreme groups revealed 39 cpg sites (p < 1×10 -04 ) in the subgroup analysis "environmental risk vs. controls" and 35 cpg sites (p < 1×10 -04 ) in the analysis "genetic risk vs. controls". in addition, we identified 48 cpg sites (p < 1×10 -04 ) in the comparative analysis of "genetic risk vs. environmental risk". none of these cpgs showed significant differential dna methylation after correction for multiple testing. however the hierarchical clustering of the differentially methylated sites provided some evidence for differentially methylated patterns between the subgroups. discussion: our genome-wide dna methylation analysis of the extreme groups provided some evidence for differentially methylated cpg sites which unfortunately did not withstand correction for multiple testing. this may reflect at least in part that the sample size of the present study was too small to detect differentially methylated cpgs at the genome-wide level. a. tafazzoli 1, 2 , t. vaitsiakhovich 3 , l. pethukova 4, 5 , 2 , s. redler 1, 6 , r. kruse 7 , b. blaumeiser 8 , m. böhm 9 , g. lutz 10 , h. wolff 11 , , am. christiano 5 , p. kokordelis 1 , mm. nöthen 1, 2 , rc. betz 1, 2 1 alopecia areata (aa) is a common hair loss disorder that occurs in both sexes and all age groups. aa is thought to be a tissue-specific autoimmune disease directed against the hair follicle. both, gene-based and genome-wide association studies have identified more than 10 susceptibility loci for aa; however, a large percentage of the overall heritable risk still awaits identification. to provide further insight into the immune related nature of aa, we and our us collaborators had each performed an immunochip-based analysis. we recently performed a meta-analysis, combining the data from both studies, and are now aiming to follow-up the best results in an additional german cohort by use of a sequenom assay to identify novel susceptibility loci. we conducted the meta-analysis by using data from the above mentioned two studies on illumina beadchip arrays including a total of 1,096 cases and 3,176 controls of central european origin. method of synthesis of regression slopes (msrs) was used for the analyses which are implemented in metainer software package. for follow-up step, we chose the most promising candidate snps. these will be examined with the sequenom massarray iplex platform in an independent aa sample comprising 1,459 cases and 970 controls. by use of the meta-analysis combining data from the us and our sample, we identified 49 novel loci with a suggestive p-value of pbecker-wu ≤ 10 -3 (phet ≥ 0.01). among them, nfkb is the most significant locus (pbecker-wu = 1.5 × 10 -07 ). in order to achieve genome-wide significance, we plan to follow-up the most promising snps in an independent german sample. we considered the 19 most significant loci (lower p-becke-wu value) for the replication step. the experiments are ongoing and results will be presented at the meeting. despite the recent identification of susceptibility loci for aa, our understanding of the genetics of aa is incomplete. identification of new loci may provide novel insights into biological pathways and a better elucidation of disease pathophysiology. 22q13.1 encompassing only one gene: kcnj4. the duplication segregates with the phenotype in the family. kcnj4 belongs to the same subfamily of potassium channels as the known disease gene for cooks syndrome kcnj2 and both share several biological functions. recent data show that gain of function mutations in another potassium channel kcnh1 cause zimmermann-laband syndrome, a congenital malformation syndrome also associated with hypoplasia or aplasia of nails and terminal phalanges. therefore we propose that duplications of kcnj4 may also cause tissue specific misregulation resulting in digit and nail defects. taken together we show in a three generation pedigree that cooks syndrome is associated with a duplication of kcnj4. our data further highlight the emerging role of potassium channels in congenital digit and limb anomalies. case report: deletion of the terminal short arm of chromosome 5 (chromosome 5p deletion syndrome) without 5q-duplication with a familial history of a large pericentric inversion of chromosome 5 b. gläser, n. hirt, e. botzenhart, j. fischer, m. leipoldt institut für humangenetik, universitätsklinikum freiburg, freiburg, germany we report on a male patient referred as a newborn with typical clinical features of chromosome 5p deletion syndrome. conventional karyotyping of lymphocyte cultures confirmed a deletion of the terminal short arm of chromosome 5 with breakpoint in 5p14. the size of the deletion (22mb) could be refined by microarray analysis and assigned to pos 5:16497-22278242. parental cytogenetic investigations showed a normal karyotype in the mother whereas the father was revealed to be carrier of a large pericentric inversion of one chromosome 5. odd crossing-over in the inverted segment of a pericentric inversion in a parent can lead to unbalanced offspring caused by gametes with a terminal deletion of the p-arm together with a duplication of the q-arm or gametes with a duplication of the p-arm together with a deletion of the q-arm. in order to find out, whether the 5p-deletion in the child is the result of an independent event or if it is related to the structural chromosomal aberration of the father a microsatellite analysis is going to be performed p-cytog-130 a small supernumerary marker chromosome of the pericentric region of chromosome 8 in a child with intellectual disability: case report and literature review b. hoffmann, g. gillessen-kaesbach, i. hüning institut für humangenetik, universität zu lübeck, lübeck, germany small supernumerary marker chromosomes (ssmc) are reported in 0.043% of newborn infants. we report on a girl, which was born preterm at 28 weeks of gestation via cesarean section due to pathological ctg. the pregnancy was complicated by gestational diabetes. she presented with muscular hypotonia, multiple hemangiomas, dysmorphic features and feeding problems. the body measurements were in normal range. the feeding problems made a tube feeding necessary until the age of four months. facial features consisted in epicanthus, high palate, broad nasal tip and broad nasal root. a brain mri showed periventricular leukomalacia and hypoplasia of corpus callosum. drug-resistant seizures with hypsarrhythmia started at the age of ten months. the affected girl was the only child of healthy non-consanguineous parents. the father also presented with a few small hemangiomas in the face. there was no history of intellectual diasability in the extended family. karyotyping showed ssmc in mosaicism. molecular characterization by array-cgh showed that the ssmc consists of pericentric chromosomal material derived from chromosome 8 (arr [ h g 1 9 ] 8 p 1 1 . 1 p 2 1 . 3 ( 2 2 , 8 1 6 , 5 2 7 -4 3 , 3 9 6 , 7 7 6 ) x 2~3 , 8 q 1 1 . 1q11.21(47,673,716-52,164,874) x2~3 dn (grch37/hg19). ian, spanish and swedish origin and 1,354 ethnically matched controls and from 92 cbe case-parent trios from north america were analysed. only marker rs6874700 at the isl1 locus showed association (p = 2.22 × 10 -08 ). a meta-analysis of rs6874700 of our previous and present study showed a p value of 9.2 × 10 -19 . developmental biology models were used to clarify the location of isl1 activity in the forming urinary tract. genetic lineage analysis of isl1-expressing cells by the lineage tracer mouse model showed isl1-expressing cells in the urinary tract of mouse embryos at e10.5 and distributed in the bladder at e15.5. expression of isl1 in zebrafish larvae staged 14 hpf to 24 hpf was detected in the developing pronephros region. our study supports isl1 as a major susceptibility gene for cbe and as a regulator of urinary tract development. to date, seven patients with interstitial deletions at chromosome 8q22.2-q22.3 have been described in the literature. all patients reported had moderate to severe intellectual disability and a characteristic facial phenotype including blepharophimosis, telecanthus, epicanthus, flat malar region, and a thin upper lip vermillion. six of the seven patients had epileptic seizures. by analyzing the deletion's overlaps, two distinct critical regions have been suggested for the facial phenotype as well as for intellectual disability and seizures. here we present another patient with a de novo 3.6 mb deletion in 8q22. 2-q22.3 . the patient shows moderate mental retardation. he has an abnormal eeg, however, only one episode of clinical seizures has been observed so far. the facial gestalt resembles the typical dysmorphic features of the patients with 8q22.2-q22.3 deletions reported previously. minor anomalies were short fingers and toes, and a single palmar crease. our report supports the assumption that deletions in 8q22.2-q22.3 cause a distinctive and clinical recognizable microdeletion syndrome with characteristic facial features and intellectual disability. since the patient's deletion overlaps with most of the critical region for the dysmorphic phenotype but only with parts of the intellectual disability critical region, the molecular data presented here further narrow down the critical region for the intellectual disability seen in patients with 8q22.2-q22.3 microdeletions. p-cytog-128 *** cooks syndrome is associated with a duplication of the potassium channel kcnj4 mundlos 1, 2, 4 , d. horn 1 , m. spielmann 1, 2, 4 1 institute for medical genetics and human genetics, charité universitätsmedizin berlin, berlin, germany, 2 max planck institute for molecular genetics, berlin, germany, 3 northern ireland regional genetics service, belfast city hospital, belfast, ireland, 4 berlin-brandenburg school for regenerative therapies -bsrt, berlin, germany cooks syndrome (mim106995) is a rare autosomal dominant disorder classically characterized by onychodystrophy, and anonychia, with absence or hypoplasia of the distal phalanges of the hands and feet. large duplications including kcnj2 were shown to be causative for cooks syndrome. recently mouse studies revealed that tissue specific misregulation of kcnj2, a potassium channel of the subfamily j, cause hypoplasia of nail beds and abnormal distal phalanges, thus resembling the cooks phenotype. here we report on a three generation pedigree with typical features of cooks syndrome that was negative for kcnj2 testing. we performed high resolution array-cgh and identified 80 kb duplication on chromosome p-cytog-132 chromosome 17q23.1-q23.2 deletion syndrome -an additional case with sensorineural hearing loss. s. leubner, c. hennig, a. junge mitteldeutscher praxisverbund humangenetik, dresden, germany the chromosome 17q23.1-q23.2 deletion syndrome (mim #613355) is a contiguous gene syndrome caused by a deletion encompassing the chromosome region 17q23.1-q23.2. initially, it has been described by ballif et al. (2010) . up to now, only a few cases with a microdeletion 17q23.1 q23.2 have been reported. most of them carry a microdeletion with recurrent breakpoints and similar size of about 2.2 mb. the common clinical features of cases with the chromosome 17q23.1-q23.2 deletion syndrome comprise mild-to-moderate developmental delay, microcephaly, postnatal growth retardation, heart defects, limb anomalies, and hearing loss. we present an additional male patient with a 2.2 mb deletion of chromosome 17q23.1-q23.2, detected by array cgh (cytochip isca 4×180k v1.0, illumina). our index patient shows typical symptoms of the chromosome 17q23.1-q23.2 deletion syndrome like developmental retardation (in particular speech delay), a head circumference at the 3rd centile, postnatal growth retardation with a body height at the 4th centile, heart anomalies (right-sided aortic arch, patent ductus arteriosus), and sensorineural hearing loss on both sides. our data improve the characterization of the typical phenotype caused by a chromosome 17q23.1-q23.2 deletion and reinforce the suspicion that this region might be associated with sensorineural hearing loss. partial, homozygous deletions of ahi1 gene causes joubert syndrome type 3 d. meier, a. behnecke, jwg. janssen, u. moog, k. hinderhofer institute of human genetics, university hospital heidelberg, heidelberg, germany we describe a patient who is second child of consanguineous healthy parents from turkey. he was born after 37 weeks of gestation with normal birth parameters (2850 g, 51 cm, ofc 32 cm). at the age of three months atypical eye movements became apparent. psychomotor development was delayed from the beginning, he presented with hypotonia and later developed ataxia. an abnormal breathing pattern was not noticed. an abnormal mri with hypoplasia of the cerebella vermis at the age of 21 months and the clinical signs described above led to a clinical diagnosis of joubert syndrome. at that time no diagnostic testing was available. he returned to the outpatient clinic of the institute of human genetics at the age of 22 years having developed retinopathy in the meantime. his height was below average (~3 cm, <p3) but still within his family's range. aside from the known irregular eye movements he showed nail hypoplasia of the thumbs, and small hands and feet. he was working at a sheltered workshop. the year before, the pediatric department of the university hospital heidelberg had initiated testing of 7 of the by then known genes causative for joubert with no mutation being found (nphp1, tmem216, cc2d2a, mks3, rp-grip1l, ofd-1 and mks4). the gene associated with joubert syndrome type 3 (omim #608629) was not included though. with the external mri not being available at that time we decided to first perform chromosome and array analyseis. the conventional chromosome analysis was without pathological findings. subsequent array analysis by affymetrix® cytoscan hd oligo/snp showed an 11 kb homozygous deletion in 6q23.3 within the ahi1 gene, the gene for joubert syndrome type 3. the deletion encompasses exon 10 to 13 which includes parts of the wd-repeats (tryptophan-aspartic acid (w-d) dipeptide) on the protein level. the homozygous deletion was confirmed by mlpa in the patient and both parents identified as heterozygous carriers. so far, alterations in ahi1 leading to joubert syndrome comprise homozygous point mutations or small heterozygous deletion/duplications in combination with a second pathogenic mutation. so we here present the first case of a large homozygous deletion in the ahi1 gene, being causative for joubert syndrome type 3. we conclude that in un-a review of the literature showed that the few patients with a ssmc of the pericentric regions of chromosome 8 described in literature show early feeding problems, developmental delay, delay in acquired language, difficulties in social skills, difficulties in attention and activity levels or autistic-like behavior. the facial dysmorphic features were not specific. multiple hemangiomas have been reported in some cases. west syndrome or seizures have not been reported so far. the phenotype of our patient is consistent with the literature data, however there are no patients with a ssmc showing the same breakpoints reported so far. especially due to the mosaicism and the rare number of patients described in the literature, no precise clinical prognosis for this patient was possible. the generation of patient-specific induced pluripotent stem cells (ipscs) by reprogramming of adult somatic cells (e. g. skin fibroblasts) represents a novel technology for studying disease mechanisms. for generation of ipscs several reprogramming methods, including the use of integrating vectors and non-integrating vectors, have been developed. since genetic abnormalities can arise during the reprogramming process, genetic quality assurance is indispensable. the aim of this analysis was to compare the genetic stability of ipscs generated using either a dna-integrating or a dna-non-integrating reprogramming technique in order to reveal possible method-specific differences. we studied ipscs derived from patients with parkinson's disease (pd), the second most common neurodegenerative disease worldwide. to monitor the genetic stability, we investigated copy number variants (cnvs) in addition to conventional chromosome analyses in ipscs of ten pd patients and six healthy control individuals. using high-resolution chromosomal microarray analysis (cma), we monitored the formation of relevant cnvs during reprogramming to pluripotency by comparing fibroblasts and ip-scs of the respective individual. to date, fibroblast cultures of all 16 selected probands as well as 47 retroviral reprogrammed ipsc clones and eight sendai-viral reprogrammed ipsc clones were analyzed. aneuploidies were detected in three fibroblast cultures (19%), in five retroviral ipsc clones (11%), and in none of the sendai ipsc clones. de novo cnvs were identified in 33 retroviral ipscs (70%) -19 clones showed one (40%), nine clones showed two (19%), four clones showed three (9%), and one clone showed four newly arisen cnvs (2%). in seven of eight sendai clones, de novo cnvs were detected (88%). two de novo cnvs were identified in three clones (38%), three cnvs in two clones (25%), four in one clone (13%), and even six de novo cnvs were detected in one clone (13%). additionally, a mosaic gain of the whole long arm of one chromosome 9 was identified in one sendai clone. only 14 of the retroviral reprogrammed clones (30%) as well as one sendai reprogrammed clone (13%) showed no newly arisen cnvs. the cnvs were between 106 kb and 6.4 mb in length in retroviral clones and between 106 kb and 926 kb in sendai clones. because almost all cnvs contained genes, including genes involved in neurodevelopment and transcriptional regulation, a biological relevance seems highly probable and changes in cell phenotype cannot be excluded. since ipscs and cells derived thereof -independent of reprogramming methods -often contain aberrations not detected by standard chromosomal analysis, our findings highlight the importance of high resolution cma procedures. in spite of differences in the analyzed number of clones, our results indicate more and larger cnvs in sendai clones than in retroviral reprogrammed clones. supported by forips, bavarian research network induced pluripotent stem cells. abstracts mother's lymphocytes showed a balanced form of the aberrant karyotype found in her son. the healthy mother possesses the insertion ins(12;4) (q24.33;q24q28), the simultaneous resection of the 12q2?3-12q24.3 material and generation of the additional ring chromosome with this material. during meiosis a normal chromosome 4, the derivative chromosome 12 and the ring chromosome were passed on to the son leading to the unbalanced karyotype as described above. further analysis with pan-centromeric probes and immunohistochemistry with an antibody against cenp-a might confirm the existence of a neocentromere on the ring chromosome which was meiotically transmitted from the mother to the son. the mtor (mechanistic target of rapamycin) kinase is the most important regulator of local dendritic and presynaptic protein translation in the brain. it has been shown to fundamentally influence ampa receptor activity by shifting glua1 to glua2 balance and to control the synthesis of several proteins involved in synaptic function and plasticity. both, loss and gain of mtor activity lead to significant disturbances of brain homeostasis and neuronal function resulting in intellectual disability (id), epilepsy, autism and behavioural alterations. in order to analyse mechanisms of homeostatic regulation of mtor-dependent synaptic function we are using a heterozygous tsc2 knockout (ko) mouse model for tuberous sclerosis (ts). tsc2 (together with tsc1) is part of a complex that inhibits the mtor kinase. mutations in either of the two genes result in increased mtor activity and upregulated downstream signalling and are causative for autosomal dominant ts. it had been shown previously that mtor hyperactivity leads to a set point shift and significantly influences neuronal homeostasis by altering cell excitability and e/i balance in heterozygous tsc2ko animals. according to these studies we hypothesize that this shift significantly leads to synaptic connectivity and plasticity as well as network alternations that result in progressive development of cognitive dysfunction, epilepsy, autism and behavioural alterations over time in ts. we have established a behaviour battery, which consists of a novel object recognition test (nort), a morris water maze test (mwm) and an evaluation of nest building and social interaction to study social behaviour and cognitive performance in heterozygous tsc2ko mice in different age groups. interestingly we found that, while two months old animals do not show any alterations, three to four months old animals develop significant disturbances in social behaviour in the social interaction test and in nest building. furthermore, we could show significant changes in cognitive performance of seven to eight months old heterozygous tsc2ko mice in the nort compared to three to four months old heterozygous tsc2ko animals. these data show that the ts phenotype builds up on shifts of the e/i balance that develop at a very early stage. pharmacological intervention with e/i balance is a promising therapeutic strategy to prevent brain damage caused by congenital hyperexcitability and homeostatic dysfunction. clear cases of a joubert phenotype an array-cgh is recommended besides sequencing to clarify the molecular basis of the phenotype. recently, a new syndrome of intellectual disability (id) with dysmorphisms due to deletions within the chromosomal band 3q26.32 harboring the tbl1xr1 gene was described. additionally, a specific point mutation in tbl1xr1 causes pierpont syndrome, a distinct mental retardation syndrome. we report four patients in which array-cgh analysis and real-time quantitative pcr of genomic dna revealed tbl1xr1 microduplication. adjacent genes were not affected. the microduplication was verified as de novo by real-time pcr of parental dna in one patient. the other three cases occurred in two generations of a second, unrelated family. we compare the remarkably similar clinical findings in tbl1xr1 microdeletion, point mutation and microduplication cases and expand the tbl1xr1-associated phenotypic spectrum. among others, intellectual disability, hearing loss and autism spectrum disorders are common features of tbl1xr1-associated diseases. our clinical observations add to the increasing evidence of tbl1xr1's role in physiological brain development and simultaneously demonstrate that opposing genetic disease mechanisms affecting tbl1xr1 can lead to similar id phenotypes. in conclusion, the observed phenotypic overlap indicates that this novel microduplication is a genomic sister-disorder to the 3q26.32 microdeletion syndrome. neocentromere formation has been reported in more than 100 small supernumerary marker chromosomes (ssmc). here we report the formation of a putative neocentromere within a supernumerary ring chromosome derived from chromosome 12 in a five year old patient with generalized developmental delay, microcephaly and hexadactyly. chromosomal analysis (gtg-banding, resolution 550 bands) of the boy showed an aberrant karyotype with a derivative chromosome 12 demonstrating additional chromosome 4 specific material inserted into the distal part of the long arm (ins(12;4)(q24.33;q24q28)), a simultaneous deletion of the chromosomal region 12q2?3-q24.33 on the same homolog and an additional supernumerary small marker chromosome, presumably a ring chromosome in all metaphases tested. in parallel, array-cgh was performed and revealed a gain of 22.6 mb in chromosomal region 4q24-q28.1 without a detectable imbalance of chromosome 12 material within the limits of the method (400k array). fish analysis with wcp probes and telomeric probes for chromosomes 4 and 12 confirmed the interstitial insertion of chromosome 4 material into the long arm of the derivative chromosome 12. the marker chromosome stained completely with a wcp12 probe but centromere probes for chromosomes 4 and 12 were both negative suggesting the formation of a neocentromere within region 12q2?3q24.33. in total, the complex rearrangement resulted in a partial trisomy 4q in the boy as a presumable explanation for the phenotype. the mother has experienced one abortion and the parents have two more children who show a normal development so far. both parents were karyotyped. the analysis of the father was normal (46,xy). konventional chromosomal analysis of the in flvcr1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to hsan. using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the flvcr1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans. autosomal-dominant familial hypercholesterolemia (fh, omim 143890) is one of the most common genetic diseases with an estimated prevalence of 1: 200 to 1: 500. three major genes have been associated with the disease: low density lipoprotein receptor (ldlr), apolipoprotein b (apob) and proprotein convertase subtilisin/kexin 9 (pcsk9). we have recently published data on the mutation spectrum in these three genes in about 200 fh patients from germany, however, this screen for mutations was negative in about 40% of the cases. stap1 (signal transducing adaptor family member 1) was recently suggested to be the fourth fh gene. we therefore screened the stap1 gene for disease causing variants in a group of 70 fh patients negative for mutations in the three established fh genes by direct dna sequencing (sanger). the newly identified variants will be presented including a phenotype-genotype correlation. phenotype: we present a 13-year-old girl with developmental delay, craniofacial anomalies, congenital eye anomalies (4.5 dpt), short stature and behavioral disorder with auto-aggressions. preliminary examinations (array-cgh analysis, karyotyping, diagnostic of metabolism, eeg, cmrt, sequencing of srcap-gene) revealed normal results. methods: in course of the midas-project, we sequenced the index patient and her healthy, non-consanguineous parents on a nextseq500 platform (illumina, san diego, ca, usa) to perform a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed with a sureselect human all exon kit v6 (agilent, santa clara, ca, usa), targeting coding exons and conserved splicing sites of over 20.000 genes. the library of the index patient was sequenced with a 285-fold mean coverage as 151-bp paired-end reads. 94% of the target region were covered 20-fold or higher. data analysis and variant evaluation was done using the clc genomic workbench 9.0 (qiagen, hilden, germany) and annotations from commercial as well as public databases. results: the trio analysis of whole-exome sequencing data from the index patient and her parents revealed 58 variants. we identified the disease-causing de novo adnp mutation c.2188c>t (p.arg730*, adnp), resulting in the introduction of a stop codon in exon 5/5 and truncation of the corresponding protein. both of the parents did not carry this mutation. adnp is part of the atp-dependent baf chromatin-remodeling p-monog-137 the challenge to insert costello syndrome causing hras mutations into human keratinocytes using the crispr/cas9 editing technology. l. brandenstein, k. kutsche, g. rosenberger university medical center hamburg-eppendorf, hamburg, germany germline missense mutations in the hras gene cause costello syndrome, a rare developmental disorder characterized by a typical facial gestalt, postnatal growth deficiency, intellectual disability, and predisposition to malignancies as well as skeletal, cardiac and dermatological abnormalities. the molecular pathophysiology caused by heterozygous hras gain of function mutations has been analysed in various tissues and cell types, however, up to date the molecular basis for cutaneous manifestations in costello syndrome is largely unknown. to address this question in an appropriate model system, permanent human keratinocyte (hacat) cells carrying costello syndrome-associated mutations in the endogenous hras gene should be generated by using the crispr/cas9 technology. double strand breaks induced by cas9 can be repaired in two ways: the error-prone non-homologous end-joining pathway for the generation of knockout models or the homology directed repair pathway, which allows precise editing. the latter enables the introduction of specific point mutations into a cell line by using a single stranded dna (ssdna) as repair template. however, we found that this is a very rare event and its efficiency depends on various factors including used cell line, selected guide rna, length and amount of ssdna and also cas9 variant. nonetheless, by using cas9 wildtype, we could insert the disease-associated c.35g>t (p.g12v) mutation into genomic hras both in hacat cells (8 positive clones out of 800) and hek cells (3 positive clones out of 15). in contrast, using the cas9 nickase protein variant that prevents off target effects, did not result in positive clones. taken together, in 10 months of working with crispr/ cas9 we gradually gained experience with many problems and pitfalls of this technology and, finally, now we are able to introduce point mutations in cell lines. next, we will use the mutant hacat cell line to gain deeper insight into the function of hras for epidermal homeostasis and its deregulation in costello syndrome. ccdc66 could also play a role in prenatal development of the mouse retina and brain. embryonic stages of interest comprise <e10 at the initial development of the eye, e13 after formation of nasal pits and prominent brain growth, when brain vesicles are initially visible and e18 where the eye and nasal brain develops. this study aims at characterizing ccdc66 rna and ccdc66 protein expression during key steps of prenatal development in the wt mouse and respective ccdc66 reporter gene expression in the ccdc66 -/model. methods: ccdc66 rna expression was analyzed by quantitative ccdc66 qrt-pcr and ccdc66 protein expression was determined by sds page and western blot in eye and whole brain homogenates of the mouse at selected embryonic stages (<e10, e13 and e18). in addition, 15 µm sagittal cryosections of mouse embryos were hematoxylin and eosin stained and analyzed for ccdc66 reporter gene expression by x-gal staining or antibody staining followed by light or fluorescence microscopy. results: ccdc66 rna and ccdc66 protein is detected in the wt mouse at different developmental time points in the eye and brain tissue. accordingly, ccdc66 -/reporter gene expression is evident at prenatal developmental stages in cryosections of whole embryos. conclusion: expression studies of ccdc66 gene and products in the eye and brain in wt and ccdc66 -/mice in different developmental embryonic stages are of crucial interest, and the role of ccdc66 gene and products in prenatal development has to be evaluated in detail in further studies. nn. hauer, b. popp, e. schoeller, ab. ekici, a. reis, ct. thiel institute of human genetics friedrich-alexander-universität erlangen-nürnberg, erlangen, germany the application of next generation sequencing technologies (ngs) has been extremely successful in the identification of the underlying causes of genetic disorders. whereas, most variants are located in coding regions, variants within splice-sites that might lead to alternative splicing affecting protein function or causing mrna decay are less well characterized. even for canonical splice-sites, prediction of the splicing effects for an identified variant is cumbersome. this becomes even more apparent as ngs based exome and genome sequencing tremendously increase the number of variants potentially affecting splice-sites. in our study to identify the underlying causes of idiopathic short stature by whole exome sequencing in more than 200 patients, we observed 44 variants in consensus splice-sites defined as up to 12 bases from the 5' and 5 bases from the 3' end of an exon. we used several computational methods (splice site finder, maxentscan, nnsplice) to calculate their potential effect on splicing. these results were often inconclusive and did not predict the definite effect on transcripts. therefore we performed rtpcr amplification of the respective regions, electrophoretic evaluation of fragments and sanger sequencing. in case of a suspected alternative splicing the evaluation of the distinct resulting isoform, though, was often hampered by the overlap of sequences. this was especially severe when multiple naturally occurring alternative transcripts were present. to overcome these issues, we established a deep sequencing approach (targeted rtpcr-seq) to identify all present isoforms in the affected patients compared to controls. for this method the 44 rtpcr fragments were normalized to generate an equimolar mixture. libraries for patients and controls, respectively, were generated by ligation of distinct index adaptors for both amplicon pools and subsequently sequenced on an illumina miseq platform. the resulting reads were aligned and visualized as sashimi plots. observed splice-junctions in patients were compared to the controls and public databases. we found concordant results between standard rtpcr evaluation and our rtpcr-seq approach for 40 variants (alternative splicing for 8 variants). for these confirmed variants the specific effect was more obviously ascertained by the ngs based approach. for 1 variant only rtpcr-seq was able to separate multiple co-occurring distinct isoforms, both in controls complex, which is involved in the regulation of gene expression. an influence on neuronal cell differentiation is postulated. mutations in the adnp gene were previously reported in 10 patients with autism and mild to severe intellectual disability. all reported adnp mutations are characterized by de novo mutations that are clustering in exon 5 and severely disrupting the protein-coding sequence, either by the introduction of a stop codon or a frameshift induction. by additionally considering the clinical symptoms, we were able to diagnose the helsmoortel-van der aa-syndrome (hvdas; omim id: 615873). discussion: the challenge of next-generation sequencing (ngs) approaches in diagnostics is no longer the technical feasibility or costs but the medical interpretation of the large sequencing data sets. besides a robust genetic analysis with focus on disease-associated genes, family anamnesis and detailed information of clinical symptoms in a standardized scheme are important for a reliable diagnosis. here, we present one of over 50 cases that will help to establish the multiple integration of data annotation software (midas), which will enable phenotype-genotype-correlations to enhance the validity of results of genetic analysis, minimize the risk of misinterpretation and enable faster diagnosis in routine patient care. peripheral neuropathies are a group of clinically and genetically heterogeneous disorders, mutations in a large number of genes have been described so far. among patients with charcot marie tooth disease (cmt)/hereditary motor and sensory neuropathies (hmsn), a common duplication of the pmp22 gene on chromosome 17p12 is by far the most frequent mutation; sequence variants in the genes gjb1, mpz and mfn2 account for a considerable proportion of patients but all other associated genetic variants are extremely rare. deletions of the pmp22 gene lead to hereditary neuropathy with liability to pressure palsies (hnpp) but transition between the different phenotypes may be rather fluid in some instances. next generation sequencing (ngs) has revolutionized cmt diagnostics as a large number of genes can be investigated at a time but it also leads to challenging questions. use of ngs has certainly improved the diagnostic yield in cmt diagnostics and it has repeatedly resulted in unexpected findings. broad molecular testing has already disclosed overlaps between clinical conditions and more surprising results are yet to come. here we report our recent experiences with the diagnostics of neuropathies. our results, different testing strategies, implications for clinical management and genetic counselling will be discussed and possible further steps suggested. (gerding et al., hum mol genet., 2011) . retinal ccdc66 protein expression in the wildtype (wt) mouse demonstrates highest levels shortly after birth. moreover, ccdc66 protein can also be detected early postnatally in mouse brain tissue. therefore, there is strong evidence that p-monog-145 *** individuals with hereditary trichilemmal cysts present a combination of an inherited high-risk plcd1 allele (including two sequence variants) and additionally acquired somatic plcd1 variants in the cysts. trichilemmal cysts are benign tumors derived from the outer root sheath of the hair follicle. they occur most often solitary or multiple on the scalp but can also be found on other body parts. the cysts can develop sporadically or in an autosomal dominant mode of inheritance with incomplete penetrance. in 2005, the hereditary cyst form was mapped to the tricy1 locus on chromosome 3 p24-p21.2 in a familial genome-wide linkage study. we analyzed 5 affected individuals from 4 different tunisian families by whole-exome sequencing. the bioinformatic evaluation revealed no rare, disease causing mutations but the sequence variant c.1379c>t, p.s460l (maf = 0.025, rs75495843, enst00000334661.4) in the phospholipase c delta 1 (plcd1) gene within the tricy1 locus. furthermore, all five individuals present a second variant c.903a>g, p.p301p (maf = 0.27, rs9857730) in the same gene. plcd1 is a member of the phospholipase c family. the enzyme is involved in calcium-dependent intracellular signal transduction and catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate into the second messenger diacylglycerol and inositol triphosphate. homozygous knockout-mice have hair defects and show aberrant skin development with increased progression of skin tumors and intradermal hair-follicle derived cysts. in humans, plcd1 is highly expressed in the hair follicle but so far only nonsense mutations have been described causing hereditary leukonychia totalis without any skin or hair abnormalities. a segregation analysis of plcd1 in a tunisian family cohort and one german family (13 families, 64 individuals, 38 affected) showed that all affected individuals contain the same two sequence variants. based on these results, we propose that plcd1 is responsible for the cyst phenotype. cdna sequencing from three different cysts revealed additional acquired somatic sequence variants in plcd1. we found c.2234c>t p.s745l in two cysts and the two variants c.2129c>t, p.s710f and c.2132c>t p.s711f in the third one. all three somatic variants are not described in the exac database and the 1000 genomes project. allele-specific rt-pcrs were performed with cyst cdna and we could show that the somatic variants are on the same allele as the inherited variants. the acquired somatic cyst sequence variants lie within or respectively near the c2 domain of the plcd1 protein. the c2 domain is involved in the calcium-dependent binding to membrane-integrated phospholipids. depletion of the domain leads to decreased membrane association and protein activity. we assume that the allele with the variants c.1379c>t and c.903a>g is a risk factor for hereditary trichilemmal cysts and that additional acquired rare sequence variants are the genetic trigger for the development of the cysts. and patients. discordant results for 4 variants proposed a higher specificity of the rtpcr-seq. thus, we established a simple amplicon based deep sequencing approach for standard rtpcr fragments to ascertain the effects of specific splice site variants. this technique has proven to be highly scalable, fast and efficient to analyze splice-site variants. p-monog-144 eif2s3 mutations associated with severe x -linked intellectual disability syndrome mehmo mehmo (mim %300148), is a rare x-linked syndrome characterized by profound intellectual disability, epileptic seizures, hypogonadism, hypogenitalism, microcephaly, and obesity. in 1998 steinmüller and colleagues described a large family with mehmo syndrome with five affected males in two generations and assigned the disease locus to the short arm of chromosome x (xp11. 3-22.13 ). we took advantage of massively parallel sequencing in four families with mehmo syndrome, including the family reported by steinmüller et al. to identify the underlying genetic cause if this severe disorder. we here show mehmo syndrome is associated with mutations in the x chromosome gene eif2s3. in three families we identified a c-terminal frameshift mutation (p.ile465serfs) and in an unrelated boy who is less severely affected, we identified a novel maternally inherited missense mutation (p.ser108arg) in eif2s3. eif2s3 encodes the gamma subunit of the eukaryotic translation initiation factor 2 (eif2). eif2 is essential for eukaryotic translation initiation and regulation of the integrated stress response (isr). subsequent studies in patient fibroblasts (p.ile465serfs) showed increased isr activation due to the mutation and functional assays in yeast demonstrated that the p.ile465serfs mutation impairs eif2 gamma function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the affected males with ile465serfs mutation. our results suggest that more severe mutations in eif2s3 cause the full mehmo syndrome, while less deleterious mutations are associated with a milder form of the syndrome with only a subset of the symptoms. introduction: larger structural genomic duplications or deletions (copy number variations = cnvs) are routinely detected by array comparative genomic hybridization (acgh). while acgh has been established as a robust and effective approach for cnv screening, it remains expensive and is limited by resolution. in addition, commercial mlpa testing today allows the identification of exon deletions or duplications for a limited number of core genes. more recently, massive parallel sequencing of multi gene panels (mgps) has been introduced as a fast and cost-effective tool in routine genetic diagnostic testing to identify causal intragenic sequence alterations not only in core genes but also in those with small contribution to the respective phenotypes. the obtained mgps data may also be bioinformatically assessed to detect exon deletions or duplications within the analyzed genes panels and thus can be expected to further improve the diagnostic yield. methods: more than 100 patients were sequenced with phenotype specific gene panels on a miseq platform (illumina) and analyzed with our bioinformatic diagnostic workflow including a quantitative data assessment using our in house java based bioinformatic script to search for gene or exon deletions. detected deletions were confirmed by an independent method (e. g. mlpa, pcr amplification of the junction fragment or linkage analysis), if available. results: we here report details for six suspected deletions in seven patients detected by our in house bioinformatic workflow: heterozygous gene deletions of pafah1b1, spastin or arfgef2, respectively; two heterozygous cftr deletions of exon 2 and 3, one homozygous partial sftpb deletion and a homozygous ispd exon deletion. the complete gene deletions of pa-fah1b1 and spastin as well as the cftr deletion of exon 2 and 3 could be confirmed by mlpa. for the partial sftpb deletion both breakpoints could be precisely located within the readout, allowing determining correct deletion size and design of primers to amplify the junction fragment. the homozygous deletion of exon 6 in the ispd gene could be confirmed by pcr and linkage analysis. discussion: diagnostic multi gene panel sequencing after nextera enrichment allows sufficient homogeneity of the obtained patient and control data per target to quantitatively search for constitutional deletions covering two or more adjacent targets. combined data assessment considering the individual clinical data will not only further increase the diagnostic yield but can also be expected to further delineate the mutational spectrum for specific phenotypes by the simultaneous detection of clinically relevant sequence variants as well as cnvs. mgps sequence assessment may also allow to gain new insights into the genomic architecture and origin of target regions and haplotypes, involved in common structural variations. c. niemietz, v. sauer, l. fleischhauer, s. guttmann, s. reinartz groba, a. zibert, h. schmidt universitätsklinikum münster, klinik für transplantationsmedizin, münster, germany various types of somatic cells have been reprogrammed to induced pluripotent stem cells (ipsc) followed by differentiation into hepatocyte-like cells (hlc). recently, cells that shed from the renal epithelial system were shown to be a suitable and convenient source for ipsc generation. in the current study, urine-derived cells (ucs) were isolated from urine donations of patients having familial amyloid polyneuropathy (fap), a neurodegenerative disease caused by mutation of the transthyretin (ttr) gene and wilson disease (wd), a genetic disorder of atp7b causing copper accumulation, predominantly in liver and brain. patient-specific hlcs were differentiated in order to study disease-specific mechanisms and to investigate the efficacy of novel compounds. for isolation of renal epithelial cells, urine of fap and wd patients was processed. ucs were reprogramed into ipscs using plasmids resulting in transient expression of factors sox2, oct3/4, klf4, and c-myc. after characterization of ipsc that expressed high levels of pluripotency markers, like oct4 and nanog, a 3-step hepatocyte differentiation protocol was performed. ipscs were subjected to a treatment with growth factors (activin a, wnt3a, fgf2, hgf) for 14 days. the hepatic, patient-specific character of differentiated hlcs was assessed by functional analysis, gene expression profiling, genotype analysis, and immunostainings. therapeutic oligonucleotide efficacy targeting ttr was determined by immunocytochemistry, qrt-pcr and western blot analysis. ttr-stabilizing activity of tafamidis was investigated by means of thermal shift assay and western blot analysis. copper chelation by methanobactin was determined by atomic absorption spectroscopy. reprogramming of ucs resulted in stable ipsc lines with characteristic pluripotent marker expression. differentiated hlcs showed high similarity to human hepatocytes in terms of genetic profile and functional activity. small-interfering rnas (sirnas), antisense oligonucleotides (aso) (niemietz et al. 2016, plosone 11(9) :e0161455), and the ttr stabilizing compound tafamidis that are currently assessed in clinical studies were studied in hlcs derived from fap patients. a novel chelator was used to determine intracellular copper accumulation in hlcs derived from wd patients (lichtmannegger et al. 2016, jci 126 (7):2721-35). fap-specific hlcs revealed differently expression of key regulators of the protein quality control (pqc) system. our results demonstrate that ipsc derived from urine are excellently suited to study hereditary liver diseases. hlcs could be investigated in the patient-specific genetic background. the efficacy of novel compounds was assessed and individual responses were monitored. involved in gene regulation. by parent-patient trio whole-exome sequencing, we were able to characterize the unterlying genetic cause in the patient, who has undergone multiple diangostic test before without receiving a diagnosis. discussion: although for many congenital syndromic diseases the disease-associated genes are known it remains difficult for physicians to interpret the highly variable phenotypes as well as their variable nomenclature in order to request the appropriate molecular analysis. here we present one of more than 50 cases that will help to establish a database connecting specific phenotypes, using the human phenotype ontology terms, with the each corresponding disease-causing genes (midas). novel compound heterozygous nalcn variants in two brothers with muscular hypotonia and global development delay l. segebrecht 1 , c. weiß 2 , m. jäger 1,3 , t. zemojtel 1, 4, 5 , d. horn 1 , n. ehmke 1, 6 1 institute of medical and human genetics, charité -universitätsmedizin berlin, berlin, germany, 2 spz dpt. pediatric neurology, charité -universitätsmedizin berlin, berlin, germany, 3 berlin-brandenburg center for regenerative therapies -bcrt, charité -universitätsmedizin berlin, berlin, germany, 4 institute of bioorganic chemistry, polish academy of sciences, poznań, poland, 5 labor berlin -charité vivantes gmbh, berlin, germany, 6 berlin institute of health, berlin, germany we report on two brothers aged two and three years, with muscular hypotonia, global development delay, abnormal respiratory rhythm, mild facial dysmorphism, recurrent respiratory infections, and failure to thrive. sequencing of 3089 disease related genes identified compound heterozygosity for two novel mutations in nalcn: c.4281 c>a (p.phe1427leu) and c.4103 + 2 t>c. nalcn encodes a voltage-independent, non-selective cation channel, which is involved in regulation of neuronal excitability. the missense variant c.4281 c>a affects the highly conserved amino acid position phe1427 which is located in segment s6 of domain iv in the pore-forming unit of nalcn. the variant c.4103 + 2 t>c alters the donor splice site in intron 36 and is predicted to cause skipping of exon 36, resulting in loss of function of nalcn. biallelic mutations of nalcn are associated with infantile hypotonia, psychomotor retardation and characteristic facies (ihprf1), whereas heterozygous de novo mutations cause congenital contractures of the limbs and face, muscular hypotonia, and global developmental delay. the clinical features of our patients resemble mild ihprf1, caused by a biallelic missense mutation in segment s3 of domain iv. it has been suggested that variants in or close to s5 and s6 of the pore-forming domains lead to the above mentioned autosomal dominant condition whereas variants in other regions or loss of function mutations result in autosomal recessive inheritance. this is the first report of a mutation in a s6 segment, inherited in autosomal recessive manner. our findings indicate that phenotype-genotype correlations in nalcn are more complex than suggested so far. phenotype: in a young undiagnosed patient with developmental delay, intellectual disability and craniofacial dysmorphic anomalies, whole-exome sequencing (wes) identified a de novo insertion in the gene arid1b, known to cause the rare congenital coffin-siris syndrome and nicolaides-baraitser syndrome, respectively. methods: as part of the midas genotype-phenotype-correlation project, we sequenced the index patient and his healthy parents on a next-seq500 platform (illumina, san diego, ca, usa) and performed a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed using the sureselect human all exon kit v6 (agilent, santa clara, ca, usa) to target most of the over 20.000 genes. the libraries were sequenced to approximately 190-fold mean coverage as 151bp paired end reads. 92% of the target region was covered 20-fold or higher. data analysis and variant evaluation was performed using the clc genomic workbench 9.0 (qiagen, hilden, germany) and annotations from commercial as well as public databases (dbsnp, hgmd, clinvar, exac). results: we identified a de novo 1bp insertion in the arid1b gene, causing a frameshift mutation that leads to the truncated protein. arid1b is part of the atp-dependent chromatin remodeling baf-complex, which is abstracts tient affected by psoriasis carried the surrogate marker snp rs4406237-a for the psors1 risk variant hla-cw 0602 haplotype homozygously; and the same snp was found heterozygous in his prp-affected father. neither the pathogenic variant in card14, nor the risk variants for psoriasis described above were found in the healthy mother. whole exome sequencing revealed genetic variants, predicted to have serious consequences in further genes involved in the nf-κb as well as the notch pathway. these variants either segregate with prp or are present in the psoriasis affected individual only. the presence of an individual carrying the same card14 mutation as his prp-affected relatives but suffering from psoriasis instead strengthens the relation between prp and psoriasis, which has been repeatedly suggested in literature. we propose a balance between familial prp and psoriasis in the family investigated in this study and present genetic variants, which might influence this balance in addition to variants in card14. wilson's disease (wd) is an autosomal recessive disease resulting from copper (cu) excess due to mutations in the atp7b gene coding for a cu-transporting atpase. wd pathogenesis, however, can not only be explained by gene coding mutations since phenotypes exhibit strong variations despite the same exonic dna makeup in the gene. also in several patients with clinical wd symptoms no gene coding variants are detectable. our former studies revealed decreased liver atp7b mrna expression in some wd patients. this decrease was not only observed in patients with nonsense atp7b mutations leading to rapid mrna decay, but also in patients with missense mutations and also in some patients with suspected wd without atp7b mutations. patients with low atp7b expression presented with a more fulminant disease progression. however, we could not detect mutations in the atp7b promoter region (c.-900 to atg) in those patients. there are possibly other deregulating mechanisms responsible for decreased atp7b mrna expression. up to now, atp7b transcriptional regulation is only poorly characterized. it is known, that four metal responsive elements (mre a, c, d and e) are located within the atp7b promoter. gene regulation through mres is often metal-dependent. liver atp7b mrna expression revealed also to increase under cu addition in several species by an unknown mechanism. up to now, only one atp7b transcription factor (tf), the mrea binding ku protein, is known. the aim of our work was to further analyze the regulation of the atp7b gene, especially through mres. to screen for tf-mre interactions and to narrow down the binding site of tf, we performed electrophoretic mobility shift assays (emsa) by incubating nuclear extracts of the liver cell line hle with probes corresponding to atp7b mrec, d and e. to identify mre-binding tf matinspector analysis was performed. identified candidate tf were coexpressed with atp7b promoter-driven reporter gene to evaluate their impact on reporter gene expression. one in the reporter assay positively tested tf was validated by different emsa experiments. further it was overexpressed with and without addition of metal ions in hle to investigate the impact on endogenous atp7b expression. we showed that tf mtf1 is able to bind to mree within the atp7b promoter and significantly increases atp7b promoter driven reporter gene expression. mtf1 binding was primarily mediated by the first three bases of the mre consensus sequence. also for mrec and mred specific protein interaction could be shown and the protein binding site was narrowed down by emsa with protein identification still pending. fur-polyglutamine diseases is the formation of the so called neuronal intranuclear inclusion bodies (nii). as ataxin-3 is predominantly located in the cytoplasm, the formation of protein aggregates in the nucleus require a nucleocytoplasmic shuttling of ataxin-3. we already demonstrated in vivo using transgenic mouse models that the toxicity of expanded ataxin-3 depends on its intracellular localization: while nuclear ataxin-3 gave rise to a strong phenotype with a high number of protein aggregates, purely cytoplasmic ataxin 3, however, even with a highly expanded polyglutamine repeat (148 glutamines), was not able to induce a phenotype and even did not aggregate. we further identified and characterized intracellular transport signals (two nuclear export signals, nes, and one nuclear localization signal, nls) within the coding sequence of ataxin-3. therefore, it is evident that proteins involved in the nucleocytoplasmic transport machinery recognize these localization signals, control the intracellular localization of ataxin-3, thereby influence the toxicity and aggregation of ataxin-3 and, thus, the pathogenesis of sca3. we now screened a library of transport proteins in order to identify the transport protein which is critically involved in the nucleocytoplasmic shuttling of ataxin-3. we indeed identified a transport protein which modifies both the formation of aggregates and the intracellular localization of ataxin 3. while the overexpression of this protein moved ataxin-3 into the nucleus, its downregulation kept it out of the nucleus. we replicated this correlation in vivo in drosophila and observed in addition to this again a clear link between the intracellular localization of ataxin-3 and its toxicity i. e. its ability of induce neurodegeneration and a behavioral phenotype. likewise we even confirmed in a mouse model of sca3 the importance of the identified transport protein as its knockout largely prevented ataxin-3 from aggregating and alleviated behavioral and movement deficits. understanding the mechanisms behind the intracellular transport of ataxin-3 could give us clues into the pathogenic functions of expanded ataxin-3 and ways to mediate the progression of neuronal degeneration in sca3. evidence for genetic factors outside card14 influencing the phenotype of a family with familial pityriasis rubra pilaris and psoriasis familial pityriasis rubra pilaris (prp) is an erythematous inflammatory skin disease caused by heterozygous activating mutations in card14, a known activator of the nf-κb pathway. different genetic variants within card14 have been associated with psoriasis. the purpose of our study was to clinically and genetically investigate affected as well as unaffected members of a family with prp in order to determine the mutation responsible for this severe skin disease in the three affected family members. a father, three of his adult children as well as the mother of one child affected by prp were investigated clinically. in addition we extracted genomic dna from the blood of each individual and performed whole exome sequencing as well as direct sequencing of single genes. clinical investigation confirmed that the father and two of his children were affected by familial prp, with the skin showing the characteristic pattern of prp, early onset and chronic course. a third child was unaffected by prp, suffered however from psoriasis. the mother of one child affected by prp showed no sign of skin disease. genetic investigation revealed a heterozygous missense mutation in exon 4 of card14, c.[371c>t], p. present in all investigated individuals with prp or psoriasis. the same mutation has been described before as being pathogenic in a different family with prp. regarding genetic variants associated with psoriasis, we found the risk alleles of three coding variants in card14: rs2066964, c. short stature is a common condition of great concern to patients and their families. in most cases it is genetic in origin but the underlying cause often remains elusive due to clinical and genetic heterogeneity. in an unbiased approach we carefully phenotyped 565 patients and randomly selected 200 for whole exome sequencing. sequence variants were analyzed for pathogenicity and the affected genes characterized regarding their functional relevance for growth. all patients received extensive clinical and endocrinological examinations, careful clinical genetic phenotypic evaluation followed by targeted diagnostic assessment for suspected diagnoses. we identified a known disease-cause in only 14% of patients, the most common causes being cnvs found in 7%, followed by syndromic monogenic causes in 5% and turner syndrome in 2%. whole exome sequencing identified additional mutations in known short stature associated genes (27) in 17% of patients who manifested only part of the symptomatology precluding an early clinical diagnosis. here, heterozygous carriers of recessive skeletal dysplasia alleles (acan, npr2) were a surprisingly frequent cause of idiopathic short stature found in 3.5% of cases. we next selected known short stature genes with mutations for pathway analyses of the affected proteins and found that 54% are involved in the main functional categories cartilage formation, chromatin modification and ras-mapk signaling. in addition we identified 37 further strong candidate genes, of which seven had deleterious mutations in at least two families. interestingly, 48% of these candidate genes are involved in the 10 main functional categories already identified for the known short stature associated genes further supporting their pathogenicity. finally, in 16% of the 200 sequenced individuals our findings were of significant clinical relevance regarding preventive measures, symptomatic or even targeted treatment. besides evaluation for orthopedic or developmental issues especially screening for neoplasias (trim37, ptpn11, nf1), symptomatic treatment for chronic kidney disease (clcn5) and targeted treatment for severe hypertension (pde3a) were of clinical relevance for the affected individuals. these results demonstrated that deep phenotyping combined with targeted genetic testing and whole exome sequencing is able to increase the diagnostic yield in short stature up to 31% with concomitant improvement in treatment and prevention. rigorous variant analysis considering phenotypic data further led us to the identification of further 37 probable novel candidate genes. thermore, we found the endogenous atp7b mrna expression to be significantly increased in hle after cu treatment or cu treatment and concurrent mtf1 overexpression. sole mtf1 overexpression did not alter atp7b expression. we newly identified mtf1 to bind mree within the atp7b promoter. its in vivo role in the pathogenesis of wd needs to be further elucidated. modifying genes have been identified for lung function in cystic fibrosis [1] , disease severity [2] and several comorbidities [3] . within the european cf twin and sibling study, we focus on genes that modify the basic defect, assessed as defective chloride conductance in cftr-expressing epithelia, which we could describe by an association study on patient cohorts selected for informative endophenotypes among f508del-cftr homozygous patients [2, 4, 5] . as a first example, rs7901656 in fas modifies fas gene expression (p = 0.0009, data from 16 intestinal biopsies [6] ), cf disease phenotype (praw = 0.0039, comparing 13 concordant mildly affected f508del homozygous sib pairs and 12 concordant severely affected sib pairs; praw = 0.0047, comparing 20 unrelated f508del homozygous index cases without residual chloride secretion by nasal potential difference measurement (npd) to 13 patients with cftr-mediated chloride secretion; [2] ) and alters binding affinity for the transcription factors nf-kbp50, nf-kbp65 and hif1a [7] which govern the cellular response to infection and hypoxia. as a second example, the transcription factor ehf, derived as a positional candidate from a north american genome wide scan [1] , is associated with 2 npd-defined phenotypes (praw = 0.0082, comparing 17 index cases with high response to amiloride in npd to 13 index cases with low response to amiloride in npd; praw = 0.0268, comparing 14 index cases without chloride secretion in intestinal current measurement (icm) to 9 index cases with cftr-mediated residual chloride secretion in icm [5] ) and affects the transcriptome of cf patients' intestinal biopsies in favor of a better processing of f508del-cftr [5] which we could confirm in epithelial cell lines as sirna provided against ehf results in a downregulation of mgat2 and mgat4, both of which are key enzymes for the complex glycosylation of proteins such as cftr. these examples indicate that small, albeit carefully selected subpopulations facilitate the identification of genetic variants by an association study that can be validated in functional assays. furthermore, we suggest that while the selection of subsamples within a population with a rare disease such as cystic fibrosis results in a loss of power, findings obtained for more than one endophenotype are indicative for a true-positive finding of a modifying gene. finally, transcriptional regulation influences the cf basic defect. interference with these pathways may results in better f508del-cftr maturation, leading to better cftr function in patient's tissue and thereby promoting health in cystic fibrosis. funding by: deutsches zentrum für lungenforschung dzl; mukoviszidose institut ggmbh abstracts at presentation at age 21 years her head circumference was on the 75th centile, her height below the 3rd centile, and her weight on the 3rd centile. she showed a disproportional short stature and mild skeletal signs like clinodactyly of the 5ths fingers. minor facial dysmorphisms included an oval face, epicanthic folds, upslanting palpebral fissures and a bulbous nose. karyotype was normal and copy number variants were excluded by high-resolution cma. as no aetiological diagnosis could be made clinically we performed whole exome sequencing (wes) and detected a homozygous splice site mutation (c.1640 + 1 g>t) in pik3c2a (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha) (nm_002645). sequencing of rt-pcr products from cdna of patient's fibroblasts showed in-frame skipping of exon 5 and 6, equally affecting all known isoforms. phosphatidylinositol 3-kinases (pi3ks) are lipid kinases involved in a large set of biological processes, including membrane receptor signaling, cytoskeletal organization, and endocytic trafficking. pi3kc2a is ubiquitously expressed and has been proposed to play an important role in clathrin-mediated endocytosis and regulation of phosphatidylinositol 3-phosphate (ptdins3p) levels. furthermore pik3c2a has been implicated in the biology of the primary cilium. the patient's distinct phenotype resembles the previously described phenotype in pik3c2a hypomorphic mice with pre-and postnatal growth retardation and a broad spectrum of renal abnormalities. the complete knockout of mouse pik3c2a showed embryonic lethality. ongoing studies on the exact consequences of the splicing defect will determine if and how much residual wild-type transcript is retained and if this is a hypomorphic variant. this case is to our knowledge the first description of a pik3c2a human phenotype. adaption of the crispr/cas genome editing system as a platform for the mutation of nipal4 as a representative of arci-associated genes in hela cells n. ballin, m-a. rauschendorf, j. fischer institute of human genetics, freiburg, germany autosomal recessive congenital ichthyosis (arci) is a rare genetic disorder with known disease-causing mutations in 9 genes. functional implementations of identified mutations are in most cases still unknown, which is amongst others due to the limited amount of skin biopsies of arci-patients. thus, suitable cell culture models for the investigation of keratinocyte differentiation are highly needed. during the last years the prokaryotic clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated (cas) system has been turned into a potent tool in the field of genome engineering. the cas9 endonuclease is directed by a short guide rna (grna) to its target sequence, where it generates a dna double strand break (dsb). as cellular repair mechanisms often fail in reconstituting the original sequence, insertion/ deletion (indel) mutations occur potentially leading to a complete gene knock-out (ko). consequently, the crispr/cas system offers a simple rna-programmable tool for in vitro mutation of arci-associated genes. hence, this system was applied in hela cells to target nipa like domain containing 4 (nipal4), the second most frequently mutated gene in arci patients. in this context, functional studies were used to validate nipal4 as suitable target in hela cells. successful application of the crispr/cas system lead to the generation of a new clonal hela cell line carrying a one basepair insertion in nipal4 exon 1 (c.106insg (ccds 47328.1) ). this mutation was further characterized and potentially results in a complete ko of nipal4. this system can now be used as a basis for targeting further arci-associated genes and for transferring the system into keratinocytes, which are the amyotrophic lateral sclerosis (als) is a late-onset progressive, neurodegenerative syndrome. most als cases are sporadic (≈90%). in familial forms, mutations in several different genes have been identified with a repeat expansion in c9orf72 and mutations in sod1 being the most prevalent. no non-genetic cause of als has been identified. since there are no overt clinical or pathoanatomical differences between sporadic or familial cases, de novo mutations have been suggests in disease pathogenesis and two previous studies provided some evidence for this hypothesis. we present data of 82 patient-parents trios from an international collaborative study. by whole exome sequencing we identified 69 non-synonymous de novo mutations in the patients, however all of them occurred in different genes. there was no concordance between the mutated genes found in our trio set and the two earlier smaller trio studies. in silico analyses suggest that none of the here identified mutations are part of any of the previously postulated molecular pathways. also, gene-gene-interaction analyses failed to find an enrichment of interacting genes. lastly, we demonstrate that the de novo mutations in als patients in this and the two earlier studies are located in genes prone for de novo mutations in general. our results thus indicate that, in contrast to previous reports, de novo mutations do not seem to be a major contributor for als. disproportioned short stature and multiple anomalies in a patient with a homozygous pik3c2a mutation -a new ciliopathy? we present a 21 year old female with disproportional growth retardation, eye and renal anomalies. she was born small for gestational age as the first child of healthy, consanguineous tunisian parents at 37 weeks after an uneventful pregnancy (2470 g, 47 cm). during her first year of life, there were severe feeding problems with regurgitation and she developed postnatal growth retardation. developmental milestones were normal. during the second year the girl developed strabismus in both eyes. later on, a cataracta polaris anterior, anomalies of the cornea, hyperopia and progressive retinal degeneration led to profound visual impairment. furthermore, a right kidney agenesis and a complex tubulopathy, as well as increased echogenicity of the single left kidney were diagnosed. r. brumm, m. schmuck, y. dinçer, s. schulz, s. wilson, i. rost, hg. klein, sh. eck center for human genetics and laboratory diagnostics dr. klein, dr. rost and colleagues, martinsried, germany next-generation sequencing may lead to a significant improvement in diagnostic yield for rare, heterogeneous disorders through the ability to simultaneously sequence all genes contributing to a certain indication at a cost and speed that is superior to traditional sequencing approaches. on the other hand, the practical implementation of ngs in a clinical diagnostic setting involves a variety of new challenges which need to be overcome. among these are the generation, analysis and storage of unprecedented amounts of data, strict control of sequencing performance, validation of results, interpretation of detected variants and reporting. especially the variant interpretation emerges as current bottleneck of the diagnostic workflow. here we present midas (multiple integration of data annotation software), a central software system for data integration in a diagnostic laboratory. the goal of midas is to construct a modular software system to integrate data from laboratory information management system (lims), data from the routine sanger sequencing workflow as well as ngs sequencing results and correlate the identified variants with the patients' phenotypical features to aid in variant interpretation and accelerate reporting. the phenotype is systematically recorded using the human phenotype ontology standard nomenclature. in particular, genotype-phenotype correlations identified in one patient are made available for all other cases, to aid the interpretation and build a comprehensive knowledge base. the midas software may thus serve as central information system all diagnostic patient data. midas is implemented in java using direct database access via jdbc, and javafx as graphical user interface. its architecture is designed modular including a dynamic module loader, a user management with ldab connection and basic search functionality. according to available modules the user management and search form are adjusted; granting access for module specific views. as an advantage of this architecture, other molecular diagnostic data, such as arraycgh or mlpa results can easily be integrated by implementing new modules. midas aims to aid molecular diagnostics by simplifying and accelerating data analysis and interpretation, improving patient care. midas is being developed as part of a prospective multicentric study including clinical, diagnostic and software development partners. a grant by the bavarian ministry of economy, media, energy and technology is used to fund this effort. institute of human genetics -medical faculty -rwth aachen university, aachen, germany, 2 izkf aachen -rwth aachen university, aachen, germany with the implementation of next generation sequencing (ngs) based assays as key tool in dna-sequencing, conventional sanger-sequencing has become a standard method to verify single nucleotide polymorphisms (snps) of interest. however, designing specific pcr-and sanger-sequencing primers has always been a rather time consuming task in terms of searching the right genomic sequence, considering known snps that could result in allele-specific amplification, and converting selected primer-sequences to upload them to web-based services. to circumvent this, we developed optimus primer, a python script, that automatically designs respective primers. the script uses the database of the ucsc genome browser to download positional information of genomic sequence (refseq gene: hg38) and common snps (snp147common: hg38) in the region of interest. the genomic sequence is downloaded via the ucsc primarily affected cell type in the skin of arci patients. furthermore, it is only a small step to expand the system from generating gene ko to gene editing allowing the introduction of patient-specific mutations in non-patient derived keratinocyte cells. hence, setting-up the crispr/cas system to target an arci-associated gene is an important starting point for future studies to investigate pathogenic effects of arci-causing mutations and to understand arci pathogenesis on a molecular level. in this context, the transfer of the established protocol into keratinocytes offers the possibility to generate 3d cell culture models of mutations in arci-associated genes and allows in vitro investigation of their implications on the differentiation process. this system would further represent an organotypic model of arci-disease with potential application in screening and identification of chemical compounds for the complementation of existing clinical therapies. background: telomeres cap and protect chromosome ends from degradation and fusion, and are therefore essential for maintaining chromosome stability and genomic integrity. they have a length of 5 to 15 kb (depending on age, sex and cell populations). due to the end-replication problem there is a continuous telomere loss of 50 to 150 bp/cell cycle and thus throughout lifetime. in laboratory practice different methods of telomere length measurement are used to identify patients with bone marrow failure syndromes (e. g. dyskeratosis congenita, aplastic anemia), hematological diseases, or other telomeropathies. each telomere length measurement method has its advantages and disadvantages regarding material required the complexity and feasibility of the method and other parameters. methods: in this study we compared and validated four different methods for telomere length measurement, i. e. southern blot analysis, quantitative pcr (qpcr), quantitative fluorescence in situ hybridization (t/ c-fish) and flow cytometry-fish (flowfish). whenever possible, edta and/or heparin blood samples were collected from a population of 154 healthy individuals of different age groups (newborn -81 years). depending on the method dna (southern blot and qpcr), metaphases (t/ c-fish) or rather vital cells (flowfish) were analyzed. results: comparison and validation of the telomere length measurement methods allowed us to calculate percentiles for all age groups. percentile curves could be used in diagnostic to identify patients with short telomeres. all methods showed acceptable accuracy, but equally imply the necessity of validation and appropriate controls in each experiment. here, flowfish was the most precise, accurate and reproducible method compared to the other methods. discussion: our study emphasizes the influence of expertise and experience that is required in order to produce robust and reliable telomere length analyses. here, we provide advice on how to choose the appropriate method in general and for individual cases to safely discriminate between natural variability and pathological telomere shortening in individual cases. in the next generation sequencing age, the strongest challenges have shifted from genotyping to handling the myriad of variants detected. whole exome sequencing yields tens of thousands of coding and non-coding variants, a number increased to millions if the whole genome is sequenced. to master this mountain of data, computer-based identification of potential disease mutations is absolutely indispensable. however, current computational strategies are usually generated by computer scientists without integrating human geneticists into software development. this often leads to tools which fulfil their main purpose but are not ideally suited to the needs of human geneticists. to assess the relevance of a suggested disease mutation, geneticists need detailed information about the effect on the protein and about the gene or protein itself. to close this gap, we have developed mutationdistiller, a web-based tool for user-driven variant prioritisation based on biological disease properties. its analysis includes the potential role of a mutated gene in pathogenesis as well as the estimated effect of a variant on gene/protein function. thus, mu-tationdistiller allows human geneticists to use every piece of information they consider relevant. unlike similar tools, its input goes beyond the human phenotype ontology and can include complete diagnoses, biological pathways, gene expression, and gene function. potentially harmful variants are identified by mutationtaster, which provides a deleteriousness score together with data on the actual effect of the variants. mutationdistiller is not restricted to non-synonymous variants but can also handle intragenic non-coding or synonymus variants. moreover, the program incorporates the known modes of inheritance of disease genes and the genotype of the queried variants (including compound heterozygosity). the output page provides all information on one site: the core component is a concise overview table of the most likely disease genes and variants. here, the most crucial information such as the variants' effect on the protein, frequencies in polymorphism databases and known diseases caused by mutations within this gene and their mode of inheritance are listed. more detailed gene and disease information and hyperlinks to external data are provided below, hence offering a comprehensive overview of the available knowledge. this allows geneticists to draw their own conclusions without any tedious collection of relevant information from the internet. a beta-version of the program is freely available at http://www.mutationtaster.org. m. jäger 1,2 , m. schubach 1 , t. zemojtel 1 , k. reinert 3 , d. m. church 4 , p. n. robinson 1, 5, 6 1 institute for medical and human genetics, charité, berlin, germany, 2 bcrt, berlin, germany, 3 institute for bioinformatics, department of mathematics and computer science, freie universität, berlin, germany, 4 10x genomics, pleasanton ca, united states, 5 department of mathematics and computer science, freie universität, berlin, germany, 6 the jackson laboratory, farmington ct, united states with the grch37 human genome release in 2009 the genome resource consortium extended the previous linear, "golden-path" paradigm of the human genome and introduced a more graph-like model in the sense of regions with alternate loci, representing common alterations in sequence and structure. in whole-genome sequencing (wgs) these stretches of sequence are largely but not entirely identical between the primary assembly and an its corresponding alternate locus can result in multiple variant calls against regions of the primary assembly. this results in characteristic and recognizable patterns of variant calls at positions that we term alignable scaffold-discrepant positions (asdps). we developed an algorithm (asdpex) that analyzes these patterns in the 178 structurally variable regions of the current grch38 genome assembly. a heuristical approach then infers whether the pattern of variant calls of a sample contains sequences from the primary assembly, an alternative locus, or their heterozygous combination at each of these 178 regions. api and common snps are annotated. in the following step primers are picked using primer3 (whitehead-institute). the target sequence is either the snp-containing exon in case of exons smaller than 300 bp, or, the genomic region flanking the snp of interest. the positional information of the snp is gathered using the mutalyzer api (lumc). the last two bases of each primer are checked for all snps in the 3' end (snp147: hg38). the script will be integrated into a website for free easy access and usability (www.optimus-primer.com). the only information needed is the refseq id, the exon, and the cdna position (eg: nm_000.249.3:c.464t>g). the script optimus primer will provide the user with primers in the widely used primer3 format. the users are also able to download the source from our website, and to run it on a local unix system as a command line tool. mutationtaster3: moving towards a comprehensive evaluation of disease causing mutations o. ebner, jm. schwarz, d. hombach, m. schuelke, d. seelow charitè universitätsmedizin, berlin, germany mutationtaster is a free and user-oriented application for comprehensible evaluation of non-synonymous and synonymous as well as non-coding dna sequence variants. as 1500+ citations show, the software has been strongly embraced by the clinical and research community. however, its capacities for assessing the functional consequences of non-coding variants are still limited. while mutationtaster is able to predict the variant's effect on major intragenic regulatory features such as splice sites or polyadenylation signals, many other potential effects of intronic or utr variants are not covered by the software yet. variants in the non-coding region of the genome are frequent and thought to cause a substantial part of yet unsolved mendelian diseases. these variants can occur either in extragenic regulatory regions (see abstract on regulationspotter) or in the untranslated regions, including introns, of a gene. unfortunately, their effects are much harder to predict than those of non-synonymous variants. therefore, only few disease mutations outside the coding sequence have hitherto been found and experimentally confirmed. to close this gap, we are advancing the current version of mutationtaster to improve the analysis of intragenic non-coding variants. we do so by implementing additional tests for variants in the 5' and 3'utr to determine their effect on au-rich elements, microrna binding sites and the influence of secondary structure on gene expression. moreover, we plan to shift from the analysis of exclusively monogenic to complex diseases with cancer being the first disease model to be integrated into mutationtaster3. already implemented enhancements of mutationtaster3 comprise the improved splice site analysis using maxentscan and the integration of mitochondrial polymorphisms from the human mitochondrial genome database. the addition of links to the integrative genomics viewer and the lof-metrics of the exac browser (exome aggregation consortium) streamline the usage of the mutationtaster3 results and enable researchers to get a detailed view of the relevant variants and associated genomic sequences. taken together, mutationtaster3 will offer much better capabilities to predict the disease-causing potential of intragenic variants than its predecessor. the software is freely available at http://www.mutationtaster.org neurocure exzellenzcluster, berlin, germany, 2 neuropädiatrie charité-universitätsmedizin, berlin, germany, 3 medizinische genetik charité-universitätsklinikum, berlin, germany, 4 berliner institut für gesundheitsforschung bih, berlin, germany ifications, or genome-wide interactions. all variants which have the potential to influence one or several candidate genes are presented in a graphical interface and ranked according to their predicted effect on the target gene(s). instead of giving scores, we present the potentially affected regulatory features in an intuitive graphical matrix. the software can freely be used at http://www.mutationtaster.org p-techno-179 genecascade2017 -a one-stop shop for finding disease mutations d. seelow 1, 2 in the last years, our genecascade software suite for the elucidation of rare diseases has seen some major extensions, mainly for the discovery of non-coding mutations. the website contains a number of tools focusing on the different steps in studying genetic disorders. all applications are web-based, have easy-to-use interfaces and are aimed directly at human geneticists, without any need to install software, use the command line or to try to explain clinical features to it specialists. we think that software should adapt to the user -not the other way around. and, unlike other web shops, its use is completely free. homozygositymapper finds disease-linked regions in consanguineous families. users can upload genotypes from snp chips as well as wes and even wgs data. we display likely disease loci in intuitive graphical interfaces. the results can also be used in our 'downstream' applications such as genedistiller, mutationtaster, or mutationdistiller to identify the actual disease mutation. genedistiller provides a user-driven way to find the best candidate gene for a genetic disorder. users can specify various aspects of the patients' phenotypes and the results are presented in a comprehensive way without the need to manually query other internet resources to collect further information relevant to asses the disease potential. mutationtaster evaluates the disease potential of non-synonymous as well as of non-coding and synonymous dna variants within genes. it does not only display a prediction but also detailed information on the variants' likely effect on mrna and protein. it can either analyse single variants as found by sanger sequencing or complete vcf files from wes or wgs projects. regulationspotter lists information about potentially regulatory dna variants outside of genes. variants are ranked according to their predicted effect on gene regulation. in addition to a score, we provide a user-friendly summary of the functional effects a variant may have. mutationdistiller combines genedistiller and mutationtaster in a single application for convenient variant and gene prioritisation. it offers human geneticists much more freedom to enter the phenotype than similar applications and provides deeper information about the variant and the gene. cnvinspector is aimed at the study of copy number variants. users can upload their patients' (or cohorts') cnvs and compare them with their own healthy controls or public data stored in our database. we include deci-pher to indicate known diseases caused by cnvs at the same position. epossum examines the effect of dna variants on transcription factor binding. as tf binding site prediction is notoriously unreliable, we also give an indication of the statistical relevance of a result. the genecascade website can be accessed at http://www.mutationtaster.org. we investigate 121 in-house wgs datasets and found that on average 51.8 ± 3.8 of the 178 regions correspond to an alternate locus rather than the primary assembly sequence. filtering these genomes with our algorithm identified around 7900 variant calls per genome that colocalized with asdps. our findings suggest the potential of fully incorporating the resources of graph-like genome assemblies into variant calling. our algorithm already uses the information contained in the 178 structurally variable regions of the grch38 genome assembly to avoid spurious variant calls in cases where samples contain an alternate locus rather than the corresponding segment of the primary assembly. the human phenotype ontology project provides three resources, the ontology of clinical features, disease-phenotype associations and algorithms that enable analysis of data that is described using hpo. this resource is being used for computational deep phenotyping and precision medicine. it enables clinical data integration for translational research. the hpo is being increasingly adopted in software, research projects and companies world-wide. we will discuss the progress and recent developments that the hpo project has made since 2008. this will include the expansion of hpo for common (complex) diseases, novel algorithms for phenotype-driven analysis of genomic variation, cross-species mapping of phenotype data, translation of hpo into several languages and also the addition of a more patient-friendly terminology for hpo terms. millions of patients worldwide suffer from a rare genetic disease. to date, the omim database lists more than 8000 diseases with proven or suspected mendelian basis, but the molecular cause is known for less than 60%. although next generation sequencing has drastically facilitated the discovery of disease mutations, a substantial fraction of whole exome sequencing (wes) projects fail to identify the causal variant. this may be due to the fact that disease-causing mutations are not always located within the coding sequence. whole genome sequencing (wgs) could solve this problem, but we are not yet readily prepared to handle the vast amount of variants which are generated by this technique, as intuitive and reliable software solutions for variant evaluation are missing. the currently existing approaches are not well-suited for a diagnostic setting, because they output a battery of different scores whose interpretation is left to the users. however, human geneticists are specialists for the assessment of symptoms of genetic diseases -not for the analysis of numerical scores indicating different likelihoods of the occurrence of regulatory elements. this is especially true when it comes to the hundreds of thousands of extragenic variants found by wgs, for which effect predictions always face a high level of uncertainty. we consider it crucial to provide geneticists with the information they need to determine the significance of a variant, not to flood them with scores whose meanings are difficult to grasp. to facilitate the interpretation of extragenic variants, we are developing regulationspotter. in contrast to other approaches such as genomiser or cadd, we integrate as much knowledge as possible in a user-friendly fashion to pinpoint the variants which are most likely to disturb the expression of candidate genes. regulationspotter includes different data on regulatory dna elements such as dna methylation, transcription factor binding sites, histone mod-the human exon 1 in the 5' end of the murine huntingtin gene. by using a novel object recognition test with a 24 h interval between sample and test phase we have found a profound deficiency of hippocampus dependent long-term memory in heterozygous transgenics. this phenotype was detected as early as 12 weeks of age and is complementary to deficits that we have identified in the hdhcag111 mouse model previously. motor deficits as well as intranuclear aggregates are described at much later stages in both of these models. we have shown previously that in hd patients, mediated through mtor signaling, translation of mrna carrying expanded cag repeats is elevated (krauss et al., 2013) . we have also seen that the biguanid metformin antagonizes mtor signaling in neurons in-vitro and in-vivo (kickstein et al., 2010) . we show here that metformin, by interfering with the mtor kinase and its opposing phosphatase, pp2a, regulates local protein synthesis in the brain and is able to suppress the production of disease making protein in early hd. furthermore metformin leads to a significant improvement of movement abilities in a c-elegans model for hd and to a rescue of early cognitive symptoms in the hdhcag150 animal model. these data suggest that metformin is a very promising candidate for early phase treatment of hd patients. allele-specific suppression of dominant-negative bestrophin 1 mutations a. milenkovic, lm. braun, f. grassmann, b. h. f. weber institute of human genetics, regensburg, germany purpose: retinal pigment epithelium (rpe) differentiated from human induced pluripotent stem cells (hipsc) demonstrated degradation and mislocalization of mutant bestrophin 1 (best1) protein in autosomal dominant best disease (bd). importantly, mutated alleles revealed a dominant-negative effect leading to an impairment of volume-regulated chloride transport, the basic function of the homo-pentameric best1 channel. here, our study aimed for a proof-of-concept to treat bd by selectively eliminating best1 mutant transcripts in patient-derived hipscs prior to rpe differentiation via the crispr/cas9 genome editing technology. methods: adult human dermal fibroblast were obtained from skin biopsies of bd patients and reprogrammed into hipscs. single guide rna sequences (sgrna) targeting 6 best1 mutations were selected by the "optimised crispr design tool" (zhang lab, mit 2015) . editing efficiency and specificity of designed sgrnas were tested in hek293 cells using an established fluorescence-based assay. after transfection of hipscs the percentage of indel formation of on-and off-targets was determined by a pcr-based crispr/cas cleavage detection kit. cas9-treated stem cell populations were analyzed for pluripotency and selected for full genome sequencing before differentiation to rpe cells. results: computational design of 6 disease-causing best1 variants (n11k, v86m, s108r, q238r, a243v, and i295del) offered at least one sgrna with predicted high quality per mutant allele. the guide sequences were cloned into the cas9-expressing plasmid px459 and co-transfected with pcag-egxxfp plasmids containing genomic fragments of ~500 bp of either the mutated or wildtype sequence to the corresponding sgrna. as targeted cas9 cleavage results in reconstitution of the egfp expression cassette by homology dependent repair, the efficiency and specificity of cas9 cleavage was evaluated on a plate reader by quantifying egfp fluorescence after 48 h of transfection. as a result, 5 out of the 6 sgrnas tested showed high allele-specificity and are now used for targeted genome editing in hipscs. conclusion: so far, there is no treatment for bd although the molecular pathology of best1 has recently been established. our proof-of-concept study aims to determine whether haploinsufficiency of normal best1 protein is sufficient to fully or partly restore cellular function in cells of primary bd pathology, namely the rpe. to this end, we will determine the degree of rescue (i. e. reconstitution of volume-regulated chloride conductance) by whole-cell patch-clamp analysis. if successful, our crispr/ cas9-driven approach will be useful to treat other diseases with dominant negative effects of the mutated allele. p-therap-184 *** metformin rescues early cognitive symptoms in the hdhcag150 mouse model and is therefore a promising candidate for treatment of hd patients. huntington's disease (hd) is an autosomal dominant neurodegenerative disorder that is caused by an unstable glutamine (cag) trinucleotide repeat expansion within exon 1 of the huntingtin gene and leads to cognitive decline and affects motor abilities. in the prodromal phase of the disease patients develop mood swings, personality changes and subtle cognitive impairment. close understanding of clinical signs and molecular mechanisms behind this early stage of hd is an important step for the development of a causal therapy. we have analysed a knock-in mouse model that carries 150 cag repeats and single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation age-related clonal hematopoiesis associated with adverse outcomes clonal hematopoiesis and bloodcancer risk inferred from blood dna sequence age-related mutations associated with clonal hematopoietic expansion and malignancies cancer spectrum in dna mismatch repair gene mutation carriers: results from a hospital based lynch syndrome registry update on kleefstra syndrome characterization of a novel transcript of the ehmt1 gene reveals important diagnostic implications for kleefstra syndrome national institute of child health and human development abstracts p-monog-147 novel mutation in hoxc13 expand the mutation spectrum of pure hair and nail ectodermal dysplasia abstracts in conclusion, targeted sequencing represents an effective, fast, cost-efficient and flexible method, since new candidate genes can be added easily to the panel, for the sequential analysis in chh patients. furthermore, panel sequencing alleviates the uncovering of oligogenic inheritance in genetic traits like chh abstracts p-monog-159 neurodegeneration in the olfactory bulb and olfactory impairment in the ccdc66 -/-mouse model for retinal degeneration s. schreiber 1 , e. petrasch-parwez one hallmark of sca3 and other mechanisms that govern ccdc66-deficient degeneration, a detailed evaluation is performed in order to reveal processes that contribute to retinal degeneration during early postnatal development and adulthood. methods: the functional role of ccdc66 deficiency is investigated by two independent approaches: 1) gene expression profiles at p10 and p28 are analyzed in retinal tissue of cccdc66-/-and wildtype mice (genechip mouse gene 2.0 st array, affymetrix) followed by quantitative real-time pcr (qrt-pcr) and 2) potential retinal interaction partners of ccdc66 protein are identified by yeast-two hybrid screening in the wildtype mouse and further analyzed by immunohistochemistry. results: using two screening methods (rna-expression profiles and protein interaction partners), our results indicate that 1) the ccdc66 deficient mouse model reveals early changes in retinal rna gene expression already at p10 and the highest number of genes differential expressed at p28. most expression differences were related to genes associated with the extracellular matrix. further genes are involved in retinal degeneration, angiogenesis, transcription factors and proteolysis. 2) the ccdc66 protein interacts with the proteins eps8 (epidermal growth factor receptor kinase substrate 8) and mpdz (multiple pdz-domain protein). both proteins are expressed in several retinal layers of the retina, confirmed by immunohistochemistry. moreover, mutations in the mpdz gene were already identified in patients with retinitis pigmentosa/leber congenital amaurosis. conclusion: expression profiles reveal expression changes at an early time point of retinal degeneration in the ccdc66-/-mouse model enabling further studies on the role of genes and processes involved in early retinal degeneration. in addition, the interaction partners of ccdc66, eps8 and mpdz, are the basis for further studies examining the pathways of retinal degeneration in the mammalian retina including man -and possibly contribute to future studies in man and human disease. ccdc66 null mutation causes retinal degeneration and dysfunction of medical and molecular genetics and skeletal dysplasia multidisciplinary unit hospital unit of genetics of neurodegenerative and metabolic diseases sel-002, ws7-005, p-compl-125 ws3-004 p-cling-051, p-cling-052 ws1-003, ws4-006, p-cling-056, p-cling-068, p-cling-075 ws7-001, p-cling-050, p-cling-074, p-monog-141 p-cancg-042 p-cling-060, p-cling-073, p-cling-086 p-cling-063, p-cling-065 ws7-005, ws8-004, p-cling-088 ws7-001, p-cling-110 p-cancg-044, p-cancg-046, p-cling-082 ws2-001 medizinische genetik 1 · ws3-002, p-cling-052 ws5-001, p-cling-055 ddd study 0. p-cling-095 ws3-006, ws6-001, p-cling-054, p-compl-120 ws4-006, p-cling-056, p-cling-075 distelmaier f. ws7-003 ws9-004, p-basepi-010, p-cancg-041 ws7-001, ws8-006, p-cling-050, p-cling-067, p-cling-074 ws5-001, p-cling-058, p-monog-162 ws2-002 ekici ab. ws5-005, p-monog-143 sel-001 p-cancg-048, p-techno-171 plen 2, ws4-002 golla a dfg-workshop göpfert mc sel-004, sel-004 haferlach c. ws8-005 haferlach t. ws8-005 ws3-006, ws6-001, p-compl-120, p-compl-121 p-cancg-032, p-cancg-039, p-cancg-046 gilissen c. ws3-003 ws4-003, p-basepi-006, p-cling-089, p-cling-099 p-cling-063, p-cling-065, p-cling-081, p-cling-095 ws7-001, p-cling-050 p-cling-051, p-cling-052, p-cling-054 ws3-006, p-compl-121 p-monog-155 medizinische genetik 1 · ws2-002 hüffmeier u. ws5-005 ws4-003, p-cling-099 seq consortium ws8-002 ws1-003, ws3-006, p-compl-121 p-cling-053, p-cling-059, p-cling-072, p-cling-109 ws2-005, p-cancg-040 ws1-003, ws3-006, ws9-002, p-compl-121 p-cancg-044, p-cancg-045 ws3-003 ws5-001, p-cling-055, p-cling-058, p-cling-069, p-cytog-128 ws6-003, ws6-006, p-compl-120, p-compl-124, p-compl-125 ws2-003, p-cling-095 p-cling-052, p-cling-080 p-cling-063, p-cling-065 p-cling-050, p-cling-060, p-cling-074 ws9-004, p-cancg-042 ws4-006, p-cling-075 ws2-002, ws2-003, p-cancg-036 khor cc. ws6-005 sel-001, ws8-001 ws4-003 medizinische genetik 1 · p-cling-053, p-cling-072, p-cling-095 ws3-006, p-compl-120, p-compl-121 ws2-005, p-cancg-040 ws6-006, p-cling-054 meggendorfer m. ws8-005 ws7-004, p-cancg-046 p-cling-051, p-cling-052, p-cling-080 p-cling-081, p-cling-090 ws2-005, p-cancg-040 ws2-002, ws4-005, p-monog-143 ws8-001 ws3-004 ws2-002, p-cling-095 pasutto f. ws6-005 nanda i. ws1-003 nöthen mm. ws3-006, ws6-001, ws6-002, ws6-003, ws6-006 ws3-003 ws3-003, ws3-006, ws7-005, ws8-004, p-cling-088, p-compl-120, p-compl-121 sel-004 medizinische genetik 1 · p-cancg-044 schnapp l. ws1-003 ws2-004, p-cancg-031, p-cancg-046, p-cling-092 ws4-006, p-basepi-008, p-cling-056 p-cling-066, p-cling-106 p-cancg-031, p-cling-092 salaverria i. ws8-002 ws3-006, p-compl-121 ws2-003, ws4-005, ws6-005, p-cancg-036, p-cling-085, p-cling-095, p-cytog-131, p-monog-143, p-monog-167 p-cancg-044, p-cancg-045, p-cling-067, p-cling-089 ws9-005 sel-002, ws7-005, p-compl-125 the international gamos consortium e ws2-002, ws4-005, ws5-005, p-cancg-036, p-monog-143, p-monog-167 sel-002, ws3-006 ws9-004, p-cytog-127 p-cancg-031, p-cling-092, p-cling-112 p-cancg-044, p-cancg-045, p-cling-067 stengel a. ws8-005 p-cling-085, p-cling-094 ws3-002, ws4-004, ws7-003, p-cling-051, p-cling-052, p-cling-054, p-cling-080, p-cling-093 p-cancg-047, p-cling-051 ws8-003 ws2-001, p-cling-111 ws7-005 witsch-baumgartner m. p-cling-084 p-cancg-039 p-cancg-044 wunderle m. ws2-003 zechner u. ws1-003, ws4-006, p-basepi-008, p-cling-056, p-cling-068, p-cling-075 ws6-004, p-cancg-046, p-cling-097 edu 2, p-cling-051, p-cling-052, p-cling-054, p-cling-080, p-cling-094, p-cling-096 p-cling-051, p-cling-052, p-cling-054 wiesener a. ws4-005, p-monog-167 ws4-005, ws6-005, p-cancg-036, p-cling-085, p-compl-119, p-monog-167 ws2-002 ws2-005, p-cancg-040 autorenverzeichnis zenker m. ws7-002, ws9-005, p-cling-061, p-cling-080 dialog mit shg, p-cling-074 ws4-005, ws5-005, p-cling-095, p-monog-151 ac. koller 1,2 , j. strohmaier 3 , ku. ludwig 1,2 , fc. degenhardt 4 , m. wulff 1,2 , d. breuer 1,2 , l. winkler 1,2 , f. neukirch 1,2 , a. maaser 1,2 , a. forstner 1,2 , s. sivalingam 1,2 , a. reif 5 , a. ramirez 6 , w. maier 6 , d. rujescu 7 , i. giegling 7 , h. thiele 8 , p. nürnberg 8 ,2 , a. fischer 9 congenital hypogonadotropic hypogonadism (chh) is a rare and clinically and genetically heterogeneous disorder. chh is characterized by incomplete or absent puberty caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (gnrh) neurons, disturbed secretion or action of gnrh, or both. chh is often associated with anosmia and is then termed kallmann syndrome (ks), as well as with other phenotypes like unilateral kidney agenesis, skeletal abnormalities, midline malformations, and hearing loss. x-linked, autosomal-dominant, and autosomal-recessive, as well as di-and oligogenic inheritance has been described for chh. in the meantime a multitude of genes has been reported to be associated with chh. actually, in fewer than 40% of the chh cases the underlying genetic cause can be identified. we analyzed a total of 19 patients with chh (4 female/15 male) by using targeted sequencing of 16 chh-associated genes kal1, chd7, fgf8, fgfr1, fshb, gnrh1, gnrhr, hs6st1, kiss1, kiss1r, nsmf, prok2, prokr2, spry4, tac3 and tacr3 and identified pathogenic mutations in 8 chh patients of our cohort. mutations were detected in kal1 (3 patients), tacr3 (2 patients), prokr2 (1 patient), hs6st1 (1 patient) and gnrhr (1 patient). furthermore, we found in two patients with described pathogenic mutations (one patient with prokr2 mutation and the other with kal1 mutation, respectively) additional mutations in nsmf gene and tacr3 gene, respectively, suggesting digenic inheritance in these cases. pain is necessary to alert us to actual or potential tissue damage. specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (hsan). although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the flvcr1 (feline leukemia virus subgroup c receptor 1) gene, which encodes a broadly expressed heme exporter. different flvcr1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. mutations institute of human genetics, bonn, germany, 2 the rild institute, wonford, exeter, uk introduction: ectodermal dysplasias (eds) are a large group of heterogeneous genetic disorders characterized by abnormal development in ectoderm-derived tissues and organs including skin, hair, and nails. among the eds, pure hair and nail ectodermal dysplasia (phned) is a rare genodermatosis characterized by nail dystrophy and sparse or absent hair on the scalp. materials and methods: a family of iranian origin was enrolled in this study. two children from a consanguineous marriage are affected from phned. in addition, the father has alopecia areata (aa) but does not show any nail dysplasia. the mother is unaffected. the paternal and maternal grandfathers had nail dysplasia almost similar to the siblings but did not manifest any hair loss or aa. homozygosity mapping and genedistiller analysis were performed to identify candidate genes. sanger sequencing was used to localize the mutations. results: in total, we identified 10 homozygous regions with almost 700 candidate genes. among these genes were also krt85 and hoxc13, already known to be related to the phenotype of our patients. therefore, we focused our additional analyses on krt85 and hoxc13. sanger sequencing showed a so far unknown homozygous insertion of 28 bp in exon 2 of hoxc13. conclusion: we identified an unknown mutation for phned which expands the spectrum of mutations for phned. the hair loss of the father seems rather be due to a distinct type of hair loss, namely aa, which is quite common in the general population. however, the nail dysplasia from both grandfathers is unclear and cannot be examined anymore. it still remains unclear if the nail dysplasia in the grandfathers was due to the same mutation in hoxc13 or is based on a different mutation. generation of a cell culture model for epidermodysplasia verruciformis by knock-out of ev3, a novel gene involved in this genodermatosis e. imahorn 1 , m. aushev 2 , sj. de jong 3 , e. jouanguy 4, 5, 6 , jl. casanova 4, 5, 6 , ph. itin 1, 7 , j. reichelt 2, 8 epidermodysplasia verruciformis (ev) is a rare hereditary skin disease leading susceptibility to certain types of cutaneous human papilloma viruses, mainly β-hpv, and a high risk for development of cutaneous squamous cell carcinoma. homozygous or compound heterozygous loss of function mutations either in tmc6 or in tmc8 have been described in several ev patients, but more than 1/3 of affected families do not have mutations in one of them. a third gene (ev3) has recently been identified to be mutated in ev patients without tmc6 or tmc8 mutation. investigations on the function of this gene may elucidate pathomechanisms of ev. we aim to generate an ev3 deficient model cell line to study the effects of ev3 despite the scarcity of patient material. detection of deletions during diagnostic massive parallel ngs gene panel sequencing neuronal differentiation at a later time point. a novel mutation c.4a>g, pser2gly in des gene in a family with catecholamine polymorphic ventricular tachycardia s. komatsuzaki, p. villavicencio lorini, k. hoffmann institute for human genetics, university hospital halle, germany background: myofibrillar myopathy (omim 601419) is a heterogeneous neuromuscular disease characterized by progressive muscle weakness, partially with cardiomyopathy and/or arrhythmia. pathohistological examinations show desmin-positive protein aggregations. the mutations in des, cryab, bag3, myot, ldb3, flnc, fhl1, and dnajb6 have been identified in patients with myofibrillar myopathy. desmin is a intermediate filaments and composed of non-helical n-and terminal head domain and a central a-helical rod domain. till now more than 67 mutations myofibrillar myopathy were identified in patients with and a genotype-phenotype was reported: mutations in rod domain tend to cause neuromuscular symptoms, and mutations in head domain is associated with cardiac symptoms. the head-domain is a serine rich domain and phosphorylated by protein kinase c. almost all reported mutations in head-domain in des are at serine residues (e. g. s2i, s7f, s13f, s46f, s26y, s26t). these findings suggested that the serine residues in head-domain could play an important role in biological function of desmin. here we report a novel mutation c.4a>g, ps2g in des in a family with catecholamine polymorphic ventricular tachycardia. case: a 58 years old patient developed a syncope in a cold winter. the cardiological investigations revealed a diagnosis of catecholamine polymorphic ventricular tachycardia and the implantation of permanent pacemaker was indicated. his older brother, father and paternal uncle suffered from arrhythmia and all of them received a permanent pacemaker implantation at around 60 years of age. his cousin was died at age of 24 years due to sudden cardiac arrest. we performed a genetic analysis for ryanodin-rezeptor 2-gen and lamin a gene and no mutation in these gene was identified. next, we performed an exome sequencing. here a novel heterozygous mutation c.4a>g, ps2g in des was identified. this mutation was also identified in uncle of index patient. the c.4a>g, ps2g in des was not reported neither in 1000 human genome project, nor in exome aggregation consortium. a mutation at serine 2 (p.s2i) was previously reported in patients with myofibrillar myopathy with cardiac symptoms. based on these findings, it was strongly supposed that the p.s2g in des gene is a causative mutation for myofibrillar myopathy with arrhythmia in our patients. discussion: recent studies suggest that the mutations in des influence not only on muscle stability and myocardial force generation, but also impaired ubiquitin proteasome system, which could be caused by aggregated desmine. generally, phosphorylation of serin and threonine in head-domain of desmin is related to disassembly of filaments. therefore the p.ser-2gly mutation in des could cause altered phosphorylation of desmin. the functional abnormalities caused by pser2gly in des gene need to be studied. gene dosage manipulation of the chromatin organizer ctcf in the nervous system of drosophila melanogaster results in neurological and morphological phenotypes e. konrad, a. gregor, m. brech, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germanythree-dimensional organization of eukaryotic genomes is crucial for temporal and spatial regulation of gene expression. architectural proteins, like the ccctc-binding factor ctcf are responsible for establishing and maintaining this organization. ctcf is involved in virtually all chromatin regulating processes including enhancer function, insulation, alterna-for this purpose we used the crispr/cas9 system to delete the ev3 coding sequence in an immortalized keratinocyte line. after isolation by facs, single cell clones have been expanded. we screened 41 clones for deletion of the whole gene as well as for the expression of ev3. three clones showed no detectable gdna sequence or expression of the ev3 gene. we found only one wildtype clone without deletion of ev3 or alterations near the cas9 cut sites. all clones have been characterized by a snp-array as well as sequencing of the knockout site and the most probable offsite targets. these ev3 deficient keratinocyte lines are the first cell culture model for ev. it will be a valuable tool to identify cellular pathomechanisms of the disease and allow insight into the control of β-hpv in the general population. hemizygous loss of function of single x-chromosomal genes is the most frequent cause for genetic intellectual disability (id). while a long list of gene mutations have so far been described to be responsible for the disease phenotype, little is known about the underlying neuronal mechanisms. reprogramming of somatic cells into stem cells (ipscs) followed by differentiation into neuronal precursors (npcs) is an important tool for translating research allowing an understanding of network dysfunction in id patients. opitz bbb/g syndrome (os) is characterized by a number of ventral midline defects combined with learning disability, developmental delay and intellectual disability. it is caused by mutations in the x-linked mid1 gene, which, as we have shown previously, regulates mtor dependent local protein synthesis. in a mouse model, loss of mid1 function leads to significant disturbance of axonal outgrowth. we have generated ips cells from several patients with os and from one mother that carries a loss of function mutation in the mid1 gene. by sorting cell clones after reprogramming we have established ips cell clones from this female carrier of a 4bp deletion in the mid1 gene (c.1801_1804delctcc) either expressing from the mutated x-chromosome or the non-mutated x-chromosome and have shown that indeed the generated ipsc-clones express either 0% of the mutated or 0% of the wildtype mid1. we determined this directly using an allele-specific mid1-rt-pcr and indirectly by comparing the methylation of humara-alleles. comparison of mutation expressing ipsc-clones with non-mutation expressing ipsc-clones showed that the mid1 mutation results in significantly smaller cells with reduced s6 phosphorylation supporting aberrations in the mtor/pp2a signaling cascade. when differentiating ipscs into neuronal precursor cells, significantly bigger embryoid bodies (ebs) can be detected in the mutation expressing clones, while the total eb number is higher for the non-mutated mid1 expressing clones. when further differentiated, ebs from all ipsc-clones form neuronal rosette structures expressing beta-iii-tubulin, with a lower rosette structure count in the mutated mid1 expressing cells. these data clearly hint a defect in neurogenesis in cells with hemizygous mid1 mutations. interestingly, while ipscs stably kept the x-incativation pattern of the original fibroblast, during differentiation x-inactivation was lost leading to biallelic expression from both x-chromosomes in the npcs pointing towards an as yet unknown reactivation mechanism of the inactive x-chromosome. we are currently analyzing x-inactivation throughout the differmutations in the aminoacyl-trna-synthetase genes sars and wars2 are associated with autosomal recessive intellectual disability l intellectual disability (id) is the common feature of a very heterogeneous group of disorders, which comprises a broad variety of syndromic and non-syndromic phenotypes. here we present mutations in two aminoacyl-trna synthetases that are associated with id in two independent iranian families. in the first family, we found a missense mutation (c.514g>a, p.d172n) in the cytoplasmic seryl-trna synthetase (sars) gene that affects the enzymatic core domain of the protein and impairs its enzymatic activity. this probably leads to reduced trnaser concentrations in the cytoplasm. in silico analyses predicted the mutant protein to be unstable. this prediction could be experimentally substantiated by results obtained through studies with ectopic mutant sars in transfected hek293t cells. in the second family, we identified a compound heterozygous genotype of the mitochondrial tryptophanyl-trna synthetase (wars2) gene, consisting of a nonsense mutation (c.325dela, p.ser109alafs*15), which very likely leads to nonsense-mediated mrna decay, in combination with a missense mutation (c.37t>g, p.w13g). the p.w13g mutation affects the mitochondrial localization signal of wars2, leading to mislocalization of the mutant protein. thus, when taking aimp1 into account, which we have recently implicated in the aetiology of id as well, there are now three genes with a role in trna-aminoacylation that are associated with this condition. hence we propose that the functional integrity of t-rnas in general is an important constituent in the development and maintenance of human cognitive functions. dravet syndrome is a rare autosomal dominant genetic disorder with early-onset epileptic encephalopathy is mainly caused by different de novo mutations of the scn1a gene encoding the type 1 subunit of voltage-gated sodium channel. whole exome sequencing(wes) enables scanning a large number of genes which not only can confirm the diagnosis but also helpful in understading for possible relationship between clinical manifestations and mutaion. the authors investigate wes in a 18 months term boy with hypotonia and convulsion of normal and relative parent. she had uncontrolled sizure without fever, and developmental delay from 6 months.in treatment protocol anti covulsants changes to valorate, clonazepam and stiripentol as well. a deleterious novel heterozygous splice site mutation in scn1a tive splicing, imprinting, v(d)j recombination, chromatin loop formation and defining topologically associated domains (tads). recently, we identified de novo mutations in ctcf in patients with a surprisingly mild phenotype of variable developmental delay or intellectual disability, mild short stature and microcephaly, and behavioural anomalies. apart from observing brain malformations and early lethality or learning deficits in two conditional knockout mouse models, little is known about the role of ctcf in neuronal development and preservation so far. therefore, we utilized the model organism drosophila melanogaster to further explore the role of ctcf in cns development and function. similar to observations in knockout mice, complete knockout or ubiquitous knockdown of ctcf is embryonic lethal in drosophila. we therefore utilized the uas/gal4 system to induce tissue specific knockdown or overexpression of ctcf in the fly nervous system. we first investigated development and morphology of the larval neuromuscular junctions (nmjs), an established model for synaptic development. while pan-neuronal overexpression of ctcf showed no morphological nmj alterations, pan-neuronal knockdown resulted in fewer nmj branches than in a specific control. additionally, we observed a reduced number of active zones in a hypomorphic mutant line compared to a wildtype control. using the negative gravitaxis assay to examine gross neurological function, we found a highly significant impairment of geotaxis behavior in flies with ctcf knockdown in neurons, motoneurons and muscle and in flies with overexpression of ctcf in glia cells, muscle and motoneurons. currently we are testing learning and memory behavior with the courtship conditioning paradigm. our findings of various neurological and morphological anomalies upon manipulation of ctcf dosage in the fly nervous system emphasize the role of ctcf in nervous system development and function and provide a basis to further study the molecular mechanisms underlying cognitive dysfunction caused by ctcf-deficiency. congenital or early-onset nystagmus (cn) is characterized by involuntary eye movements and shows enormous clinical and genetic heterogeneity. cn may be an ambiguous sign of many different diseases, including retinal dysfunction/degeneration, ocular/oculocutaneous albinism, and severe central nervous system disorders, such as pelizaeus-merzbacher or pelizaeus-merzbacher-like diseases (pmld). due to enormous heterogeneity found among the diseases leading to cn, whole exome sequencing (wes) and panel-based bioinformatics was considered as an approach to rapidly identify disease-associated genetic sequence variants. we have analyzed 9 families with 16 cn-affected patients. herein, we present three clinically different patients who were initially affected with cn, but developed further clinical symptoms of various severities. one of these patients showed features of retinal dysfunction, including night blindness and myopia, while two other patients developed severe phenotypes including mental retardation or pmld. wes identified four genetic variants in genes associated with cn. the first patient showed a hemizygous splice-donor variant (c.2576 + 1g>a) in the calcium channel voltage-dependent alpha-1f subunit (cacna1f) gene, the second patient carried a hemizygous variant (c.1403g>a, p.r468h) in the ferm domain-containing protein 7 (frmd7) gene, and the third patient showed two heterozygous variants (c. 291c>g, p.y97* and c.716t>c, p.v293a) in the gap junction protein gamma-2 (gjc2) gene. sanger sequencing confirmed the identified variants in the index patients and verified co-segregation in several family members. our results suggest a beneficial role of wes to identify the molecular causes of cn and to rapidly confirm an initially unclear clinical diagnosis. especially, patients with rare and severe disorders (e. g. pmld) will benefit from a wes analysis performed in the early stage of the disease. the ccdc66-deficient (ccdc66-/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al., hum mol genet., 2011) . in order to determine whether ccdc66 gene expression might also play a role outside the retina, this study aimed at characterizing ccdc66 protein expression during early postnatal development of the mouse brain. furthermore, morphological and behavioral impact of ccdc66 deficiency in the mouse brain was analyzed. methods: ccdc66 protein expression was determined by sds page and western blot in whole brain homogenates and in selected brain regions of interest (olfactory bulb, hippocampus, cortex, striatum, cerebellum, brain stem) during early postnatal development and in adult wildtype (wt) mice. in addition, cryosections of the ccdc66-/-olfactory epithelium and bulb (during postnatal development) and the rostral migratory stream (in adult) were analyzed for ccdc66 reporter gene expression by x-gal staining. selected brain regions were additionally analyzed by electron microscopy. in order to correlate anatomical with behavioral data, olfactory performance was studied in aged ccdc66-/-mice compared to ccdc66+/+ controls by an olfactory habituation/dishabituation test (yang and crawley, curr protoc neurosci., 2009) , where olfactory exploration-time during the presentation of neutral and social odors is examined. results: ccdc66 protein was detected throughout the early postnatal development of the wt mouse brain, decreasing after birth. amongst analyzed brain regions, highest expression of ccdc66 protein was detected in the olfactory bulb exhibiting similar ccdc66 levels to retinal expression. accordingly, ccdc66 reporter gene expression was demonstrated in the mature olfactory bulb glomeruli, the adjacent olfactory epithelium and along the rostral migratory stream in the ccdc66-/-mouse brain. interestingly, strong ccdc66 reporter gene expression in glomeruli of the ccdc66-/-olfactory bulb was correlated with signs of degeneration in the ccdc66-/-mouse, but not in controls. the degeneration was also reflected by olfactory impairment in ccdc66-/-mice, which spent significantly less time for sniffing at initial presentation of unknown, neutral odors and barely responded to social odors. conclusion: besides the retina, ccdc66 protein plays a crucial role in the olfactory system as shown by its expression there as well as by ccdc66 deficiency resulting in neurodegeneration and alteration of olfaction-related behavior in the ccdc66-/-mice. as impairment of the olfactory sense in multiple neurodegenerative disorders is a common finding, the ccdc66-/mouse model is not only restricted to study retinal degeneration but possibly also degeneration of the central nervous system. background: the ccdc66-deficient (ccdc66-/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al. 2011) . in order to gain insights into the molecular disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. here we report a novel homozygous missense variant (c.223 g>a; p.g75r) in the arl13b gene, which we identified by whole exome sequencing of a trio from a consanguineous family with multiple affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. the same variant was also identified in a second family. we saw a striking difference in the severity of ataxia between affected male and female individuals in both families. functional analysis demonstrated that dihydrotestosterone treatment of sh-sy5y cells induced a down regulation of arl13b expression. both arl13b and arl13b-p.g75r expression rescued the cilia length and shh defects displayed by arl13bhennin (null) cells, indicating that the mutation did not disrupt either arl13b function. in contrast, arl13b-p.g75r displayed a marked loss of arl3 guanine nucleotide-exchange factor activity, despite retention of its gtpase activities, highlighting the correlation between its loss of function as an arl3 guanine nucleotide-exchange factor and joubert syndrome. s. renner, a. busch, t. bierhals, j. butter, v. kolbe, g. rosenberger institute of human genetics, university medical center hamburg-eppendorf, hamburg, germany taad (thoracic aortic aneurysm and dissection) is a heterogeneous disease that often remains silent until a life-threatening complication occurs. it belongs to the connective tissue disorders and causes 1% of death in industrial countries. several disease genes have been already identified; however, about 50% of patients with taad-associated syndromes do not show a mutation in these genes. thus, further heterogeneity is obvious. since individual risk stratification and therapeutic options highly depend on the individually mutated gene, it is very important to identify more disease genes which are aimed to be found by whole exome sequencing (wes). many of the known disease genes encode for proteins that are important for the structure and stabilization of the extracellular matrix as well as for the contraction of vascular smooth muscle cells. one central pathway is the tgf-beta signaling which functions among other proteins via the tgf-beta receptor, its ligand and its downstream target smad2/3. our project plan includes (i) exome sequencing both in affected individuals within families as well as in sporadic patients, (ii) filtering of raw data and prioritization of sequence variants by using a bioinformatic in house pipeline, (iii) verification of novel putative disease genes in a cohort of 200 mutation-negative patients with taad spectrum disease and (iv) functional analyses to gain deeper insight into the pathobiology of taad. in a first round of wes analysis and variant prioritization, we identified a highly conspicuous sequence variant in three family members with taad. screening of the respective gene in a large cohort of mutation negative patients revealed another variant in two siblings with taad. structural and functional considerations strongly support deleterious effects for both identified putative pathogenic missense variants that affect a novel cell cycle-and/or apoptosis regulating protein. functional analyses did not show an involvement of this protein in tgf-beta signaling. in ongoing experiments, we focus on mutation-induced consequences on cell proliferation, cell cycle progression and apoptosis. indeed, we found inhibitory effects of the missense variants on proliferation by affecting the cell cycle key protein cdkn1a. we hypothesize that dysregulation of proliferation and/ or apoptosis of specific cells, e. g. smooth muscle cells, underlies taad.abstracts for screening. positively tested compounds were reanalyzed by whole-cell patch clamp recordings and cell volume measurements. results: the halide assay revealed reproducible halide permeability across wells and, as a control, reliably detected mdckii cells overexpressing wild type best1 by a decrease of yfp fluorescence to 70% following 60 seconds iodide stimulation. cells harboring mutant best1 showed 85% of default yfp fluorescence after the same time interval. conclusion: the current study established an assay appropriate for high and small-scale compound screening targeting best1 localization and function. this assay will be used to screen for compounds in mutant cells lines best1-t6p and best1-y227n for their ability to improve trafficking to the pm or correcting protein folding to enhance ion permeability. background: neuropeptide y-y2 receptor (y2 receptor), an auto-receptor of neuropeptide y (npy) and attractive guanine nucleotide (g) protein-coupled receptor target, has been implicated as a potential therapeutic target for many clinical conditions, including epileptic seizure, depression, pain, and alcoholism. in huntington's disease (hd) patients and animal models of hd, npy-expressing striatal interneurons are selective preserved and increased with advancing disease. however, the potential role of y2 receptor in hd pathology remains under-explored. aims: to investigate whether activation of y2 receptor using npy and selective y2r ligands could ameliorate behavioral deficits and neuropathology in r6/2 mouse model of hd. methods/techniques: npy and selective y2 receptor agonist npy13-36 were intranasally administered to r6/2 mice, five days in a week, beginning from 4 weeks of age until 12 weeks of age. in the second study, r6/2 mice received daily intraperitoneal administration of selective non-peptide y2 receptor antagonist (sf-31) to selectively block y2 receptor. results/outcome: intranasal application of npy showed significant increase in rotarod performance compared to the saline and sf-31 treated r6/2 mice (*p < 0.05 and **p < 0.01 at 8 and 12 weeks of age respectively, n = 12). however, treatment with npy13-36 showed a clear trend towards increased rotarod performance at 12 weeks of age compared to the saline and sf-31 treated r6/2 mice but the difference did not reach significance. also, treatment with npy and npy13-36 showed no significant effect on body weight loss in r6/2 mice, contrasting with previous data obtained with single intracerebroventricular (icv) injection of npy in r6/2 mice. furthermore, intranasal application npy or npy13-36 led to decrease in mutant huntingtin (htt) aggregation and mediated increase in dopamine-and camp regulated phosphoprotein (darpp-32) and brain derived neurotrophic factor (bdnf) levels. additionally, we found that npy and npy13-36 attenuate microglial activation, inducible nitric oxide synthase (inos) expression, and proinflammatory cytokines production in r6/2 mice compared to the saline and sf-31 treated r6/2 mice. conclusion: taken together, our findings suggest that targeting npy-y2 receptor might be a potential neuroprotective therapy for hd and other neurodegenerative diseases. best of both world's: a novel, rapid capture protocol that overcomes drawbacks associated with dna fragmentation in established methods j. seggewiß, c. ruckert, p. wieacker institute of human genetics, westfaelian wilhelms-university of muenster, muenster, germany rapid capture protocols are an attractive proposition for clinical sequencing labs, as they enable quicker sample-to-sequencing turnaround times. the fragmentation of input dna for the construction of pre-capture libraries is a bottleneck in established protocols. mechanical shearing is the gold standard, but is laborious using single-tube covaris instruments; and higher-throughput instrumentation is cost-prohibitive to many smaller labs. "tagmentation"-based methods (e. g. the nextera rapid capture system from lllumina, or agilent's sureselect qxt system) employ transposases for fast ond simple library construction. however, these protocols are associated with significant sequence bias, especially with low-quality ffpe samples and are extremely sensitive to dna input -thus requiring meticulous quantification of viscous, high-molecular weight dna. here we describe a newly-developed rapid capture protocol that combines the kapa hyperplus kit (kapa biosystems) with integrated, low-bios enzymatic fragmentation, and agilent's proven sureselect xt target enrichment technology. the streamlined method follows for the preparation of high-quality, sequencing-ready libraries in one working day. the novel enzymatic fragmentation reagent does not require careful quantification of input dna, yielding reproducible fragmentation profiles optimal for capture over the 5fold range-tested (50-250 ng) the single-tube kapa hyperplus protocol results in very efficient conversion of input dna to precapture library thereby decreasing duplication rates and increasing the complexity of the library going into the modified, 90min sureselect fast xt hybridization protocol. our protocol represents a significant improvement for fast routine diagnostics, where robust and reproducible pipelines ore needed to support timely treatment decisions. lmj. braun, a. milenkovic, bhf. weber institute of human genetics, regensburg, germany purpose: human bestrophin-1 (best1) is a chloride channel controlled by ca 2+ and cell volume and is localized at the basolateral membrane of the retinal pigment epithelium (rpe). so far, there is no therapy for the best1-associated diseases, of which the most common is best vitelliforme macular dystrophies (bvmd). in this study, we developed an assay applicable for high and small-scale compound screening targeting best1 localization and function. methods: to assess best1 channel function we developed a halide assay. briefly, mdckii cell lines were established, stably expressing wildtype best1 or bvmd-associated best1 mutants together with a yellow fluorescent protein (yfp)-based halide sensor. in polarized mdckii cells, wildtype best1 and best1-r218c localize regularly at the basolateral plasma membrane (pm) while best1-l224m and best1-y227n appear significantly reduced in quantity and grossly mislocalized to cytosolic compartments. cells were stimulated with extracellular addition of iodide known to pass the pm through anion cannels and, as a consequence, intracellularly quench yfp fluorescence. variations in yfp fluorescence levels as a marker for best1 function were recorded in 96 well plates by a plate reader setup. a small-scale 2,560 compound library, commercially available as spectrum collection (microsource discovery systems, gaylordsville, usa) was used key: cord-352798-rb2ggonx authors: chaber, anne-lise title: the era of human-induced diseases date: 2017-11-21 journal: ecohealth doi: 10.1007/s10393-017-1299-9 sha: doc_id: 352798 cord_uid: rb2ggonx nan a global political and research infrastructure has grown around the fight against ''zoonoses,'' or diseases transmitted between vertebrates and humans. the one health or ecohealth approach and its integration of human, animal, and environmental health and the concomitant critique of the epistemological fragmentation of the western scientific tradition can be applauded, but scientific and political focus needs to shift from zoonotic disease to human-induced disease. human-induced disease includes both infectious-i.e., zoonoses and disease from animal origin-and non-infectious anthropogenically driven diseases. human-induced disease as the label for diseases-both infectious and non-infectious-caused by human activities and their environmental impact emphasizes the role of the human in disease transmission and could serve reshaping our approach to disease management and prevention. zoonotic diseases have been at the cornerstone of the one health/ecohealth approach. zoonoses incurred an estimated $20 billion in direct costs and over $200 in indirect losses in the last 10 years (world bank 2010). these events generate moral panic, legitimate new spheres of legal activity, and spur new arguments for the funding of research. the arsenal of disease-combatting weapons grows constantly, evidenced by mass immunization or eradication of animal reservoirs. this dynamic derives from an anthropocentric perception and the psychologically useful imagination of a species barrier. most viruses and bacteria are not pathogenic but symbiotic, with humans hosting some 100,000 billion bacteria compared to their own 10 billion cells. bacteria have a vital role in human bodily function and are integral elements of human cells (mitochondrion), and viruses constitute at least 10% of human dna. humans share most of the viruses, bacteria, and fungus with the rest of the animal kingdom, and thus it should come as no surprise that zoonotic pathogens were the cause of more than 65% of emergent infectious disease events in the last 60 years, with 75% of these originating in wild fauna (keusch et al. 2009 ). the biologically relevant question is what allows or prevents infection by microorganisms, whether or not the infected species is human. the direct link between zoonosis and human activities and demographic growth is established. land use modification for urbanization, food production, and agricultural change accounts for around 50% of all zoonotic emerging infectious diseases (eids) (keesing et al. 2010 ). demographic, societal, and behavioral change gave rise to human immunodeficiency virus (hiv/aids) (de sousa et al. 2010 ) and outbreaks of syphilis (d' angelo-scott et al. 2015) . anthropogenic environmental change leads to the emergence of infectious diseases in wildlife (daszak et al. 2001) the advent of mass travel exacerbates the historically established dissemination of infectious disease along pathways of migration. examples are bubonic plague (yersinia pestis), cholera (vibrio cholerae), seasonal influenza, severe acute respiratory syndrome (sars), and malaria (plas-modium falciparum) (tatem et al. 2006) . the trade in goods and animals is directly linked to outbreaks such as human monkeypox virus in north america (karesh et al. 2005) or h5n1 in the united arab emirates (naguib et al. 2015) . it is nevertheless common that cause is attributed to the animal and not to human behavior as evidenced, for example, by the mass culling of badgers and the ibex to control tuberculosis or prevent brucellosis in europe. this also evidences the diminished value to humans of wild animals and comes despite the universal declaration of animal rights (unesco 1978 and revised, 1989) which states that ''action endangering the survival of a wild species, and every decision leading to such act form a genocide, i.e. a crime against the species'' (article 8). when human health is concerned, even hypothetically, such principles are quickly abandoned in the urge to displace consequence as cause. is reducing the risk of transmission by tackling human activities amplifying disease transmission really out of our hands? human-induced diseases do not only encompass infectious disease such as zoonoses whose spread is enhanced by human activities, but it also includes chronic disease linked to environmental perturbation caused by human activities. environmental degradation is a sensitive topic since environmental health is a concept that is still lacking definition, consensus and true global liability and accountability. the world health organization (who) estimates that ambient air pollution caused 3.7 million deaths throughout the world in 2012 (who and others 2014). the organization for economic co-operation and development (oecd) claims this death rate increased by 5% in china, 12% in india (oecd 2014), and 4% worldwide between 2005 and 2010. it estimated that the annual economic cost of illness and premature mortality linked to air pollution is $3600 billion (oecd 2014)-a figure that is 85% of the world's annual public budget for human health. the international monetary fund reported the use of fossil energies costs $4900 billion a year (parry et al. 2014) in disease, premature death, and environmental damage. this figure is 1.2 times more than the combined public health budgets of 193 countries and yet industries that exploit fossil energies are subsidized directly and indirectly by more than $500 billion annually (imfdirect-the imf blog 2016). environmental degradation is estimated to be the cause of 40% of world mortality (pimentel et al. 2007 ). human activity gives rise to multiple forms of toxic pollution affecting both our health and the environment. for example, production of garments for international markets takes place mainly in developing countries and involves unregulated tannery operations that generate chromium pollutants known to have carcinogenic effects on both animals and humans. in 2013 industrial plants with poor waste treatment and disposal infrastructure were responsible for the lead pollution linked directly to the death from chronic illness of 853,000 people living mainly in low and middle income countries (harris and mccartor 2011) . the institute for health metrics and evaluation also estimated that lead exposure accounted for 9.3% of the global burden of idiopathic intellectual disability, 4% of the global burden of ischemic heart disease, and 6.6% of the global burden of stroke. unesco's world water assessment programme estimates that industry is responsible for the annual accumulation of 300-500 million tons of sludge, heavy metals, and other toxic wastes, and that 70% of untreated industrial waste in developing countries is dumped directly into water systems (united nations educational, scientific and cultural organization 2016). [for people of] poor countries, resource degradation is the most tragic, forced as they are to overuse natural resources from which depends their survival. they are driven to sacrifice their future to ensure precarious everyday life. that is why in many countries, acting against poverty includes protecting the environment.-un secretary general, 4th june 1992, rio da janeiro. a plethora of examples is already linking human diseases-both infectious and non-infectious-to environment perturbation in developed and developing countries. concerns are also raised regarding the impact of climate change on human disease burden. climate change is shifting ecological dynamics leading to potential increase in vector-borne disease such as lyme disease (brownstein et al. 2005) or malaria (martens et al. 1995) , zoonotic diseases like hantavirus infection (tian et al. 2017) , and non-infectious disease such as ciguatera fish poisoning (gingold et al. 2014) . even though environmental health and human health are deeply connected, environmental health is difficult to protect without consensus on definitions, common objectives between beneficiaries, and long-term planning. con-ventions of international environmental law introduced the concept of legal protection for species, sites, habitats and ecosystems. three concepts of nature coexist: it is a law project (unesco 2016) in which the common heritage dimension is underlined; it is a legal subject (united nations 2016); or it is a law purpose (unep 2016)? the international criminal court announced in september 2016 that it will now prioritize crimes that result in the ''destruction of the environment'', ''exploitation of natural resources'', and the ''illegal dispossession'' of land (international criminal court 2016) . yet legality and illegality are often controversial. policies intending the liberalization of international trade do not take into account resource consumption, pollution, or negative societal impact in the producing and exporting countries (pearson 2000) . traded goods are not priced to incorporate such costs, and as such benefits of trade are distorted. a multitude of supranational environmental agencies, commissions, programs, and secretariats exist, but there is no global authority able to levy an appropriate pollution tax or fines on national governments or economic agents. it is becoming increasingly apparent to many that solutions and actions cannot be left to government or supranational government agencies. consumers are boycotting products with negative social, environmental, and health impacts such as slavery in clothing factories, harvesting of exotic timber, or methylmercury contamination in seafood. environmental citizenship is a recent and theoretically complex concept (bell 2005) . it is defined by the united nations environmental programme as an ''attempt to make environmental conservation and sustainability an important duty of citizenship that citizens all over the world should be aware of'' (unep). many multinational enterprises are now engaged in corporate citizenship programs to promote sustainable development, including the simultaneous consideration of economic growth, environmental protection, and social equity in business planning and decision-making. yet the willingness and ability of governments to reflect environmental values of their citizens vary greatly among countries (pearson 2000) . the individual remains the fundamental element of society. how powerful can one individual be? the internet and its powerful networking effect is beyond control of established institutions. it creates opportunities for horizontal communication, develops new forms of democracy and social participation, and could bring people closer to having impact on issues of common concern such as health, social justice, and the environment (barcena 1997) . could billions of individuals, scattered through the world but connected via the internet, become the strongest and most active pillar of environmental protection and thus enhance health and improve social justice? the term human-induced diseases might have the potential to put the human back into perspective and unify concerns and efforts. human-induced disease as the label for diseases (infectious and non-infectious) caused by human activities emphasizes the role of human and could serve bringing together scientists, politicians, industrials and laymen in common pursuit. human-induced diseases need to be named in order to be collectively claimed. effect of climate change on lyme disease risk in north america anthropogenic environmental change and the emergence of infectious diseases in wildlife high gud incidence in the early 20th century created a particularly permissive time window for the origin and initial spread of epidemic hiv strains ciguatera fish poisoning and climate change: analysis of national poison center data in the united states global energy subsidies are big-about us$5 trillion big, imfdirect-the imf blog the world's worst toxic pollution problems report international criminal court, the office of the prosecutor (2016) policy paper on case selection and prioritisation wildlife trade and global disease emergence impacts of biodiversity on the emergence and transmission of infectious diseases sustaining global surveillance and response to emerging zoonotic diseases potential impact of global climate change on malaria risk outbreaks of highly pathogenic avian influenza h5n1 clade 2.3. 2.1 c in hunting falcons and kept wild birds in dubai implicate intercontinental virus spread the cost of air pollution: health impacts of road transport ecology of increasing diseases: population growth and environmental degradation global transport networks and infectious disease spread anthropogenically driven environmental changes shift the ecological dynamics of hemorrhagic fever with renal syndrome who, others (who) (2014) burden of disease from ambient air pollution for towards a one health approach for controlling zoonotic diseases unesco world heritage centre-convention concerning the protection of the world cultural and natural heritage i would like to acknowledge dr. françois moutou and prof. claude saegerman for their advices and inspiring comments. my sincere thanks also go to fleur toulat, anya stafford, and rachel johnson for editing and polishing the english language. key: cord-354651-bxm9yxjm authors: zeng, yawen; pu, xiaoying; du, juan; yang, xiaomeng; li, xia; mandal, md. siddikun nabi; yang, tao; yang, jiazhen title: molecular mechanism of functional ingredients in barley to combat human chronic diseases date: 2020-03-30 journal: oxid med cell longev doi: 10.1155/2020/3836172 sha: doc_id: 354651 cord_uid: bxm9yxjm barley plays an important role in health and civilization of human migration from africa to asia, later to eurasia. we demonstrated the systematic mechanism of functional ingredients in barley to combat chronic diseases, based on pubmed, cnki, and isi web of science databases from 2004 to 2020. barley and its extracts are rich in 30 ingredients to combat more than 20 chronic diseases, which include the 14 similar and 9 different chronic diseases between grains and grass, due to the major molecular mechanism of six functional ingredients of barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch). the antioxidant activity of barley grass and grain has the same and different functional components. these results support findings that barley grain and its grass are the best functional food, promoting ancient babylonian and egyptian civilizations, and further show the depending functional ingredients for diet from pliocene hominids in africa and neanderthals in europe to modern humans in the world. this review paper not only reveals the formation and action mechanism of barley diet overcoming human chronic diseases, but also provides scientific basis for the development of health products and drugs for the prevention and treatment of human chronic diseases. global cost of five chronic diseases (diabetes, cardiovascular disease, mental illness, chronic respiratory disease, and cancer) treatment to reach $47 trillion from 2011 to 2030 [1] . the intake of high sodium with low whole grains and fruits was the top most dietary risk factors for deaths and disability-adjusted life years globally and in many countries [2] . diabetes in 11 production regions of polished rice with high glycemic index (gi ≥ 70) caused the biggest reduction in health-adjusted life expectancy at birth in 21 regions in 187 countries from 1990 to 2013 [3] . the micronutrients deficiencies at the highest risk are fe, zn, and vitamins (v b1 , v b2 , v b12 , and v c ) [4] . the outbreak of human chronic disease is due to taste pursuit that changes a healthy diet, i.e., the ancients switched from brown rice (gi ≤ 55, high k and high micronutrients) and barley (gi ≤ 25) or its grass flour (k/na ≥ 10) as staple foods to modern polished rice (gi ≥ 87) and wheat white flour (gi ≥ 86) with low and low micronutrients as staple foods [3, 5] . barley grass is not only the best functional food for cell nutrition and detoxification in human body but also the most abundant bioactive ingredients for lots of health-promoting effects [6, 7] . it can combat more than 20 chronic diseases due to gaba, flavonoids, sod, k-ca, vitamins, and trypto-phan mechanism in barley grass ( figure 1 ) [6] . the sustaining major foods+barley grass powder can achieve the who's intake target of low sodium (<2 g) with high potassium (>3.5 g) every day [8] . more than 30 functional ingredients in barley grass can combat over 20 chronic diseases, and 15 functional ingredients in barley grains may prevent 11 chronic diseases [9] . barley enhanced the sterols accumulation through ltp2 gene action that take part in the abiotic stress reaction of mediating intracellular lipid transport [10] . barley straw (2.0~8.0 g/l) for phenolic acid in degradation inhibited the alga (m. aeruginosa) blooms of aquatic eutrophication by cell shrinkage of metabolic activity and chlorophyll a fluorescence decay [11, 12] . therefore, barley grass powder plays an important role for solving human chronic diseases. barley grains have the highest functional value (low gi with high β-glucans and resistant starch) and antioxidant properties among cereal crops. the soluble fiber β-glucans is a group of polysaccharides found in barley, oats, mushrooms, yeasts, and seaweed [13] . hulless barley variety zangqing 320 has a 4.84-gb sequence with 46,787 genes in seven chromosomes [14] ; three hvcslf genes take part in (1,3; 1,4)-β-glucan synthesis [15] . qingke (hulless barley) is a major food for tibetan people and an important livestock feed in the qinghai-tibetan plateau, which has lots of gene family related to stress reactions [16] , especially different antioxidant capacities due to some polysaccharide and phytochemical compositions [17] . regular daily consumption of whole barley flour can prevent chronic diseases, especially diabetes, colonic cancer, hyperlipidemia, high blood pressure, and gallstones [18] . although barley grains have played an important role in health effects of human being, health contribution and different major mechanisms from barley grass for preventive human chronic diseases and functional ingredients in barley grains are unclear. [21, 29, 39] resistant starch (%) whole grains 3:63 ± 2:32 0.2~24.0 [73] [74] [75] arabinoxylan (%) endosperms 0:67 ± 0:06 0.53~0.90 [55] barley bran 4:66 ± 3:35 1.97~8.42 [29, 30] grains flour 1:31 ± 0:73 0.70~2.13 [29, 30] polyphenols (mg/100 g) whole grains 231:61 ± 34:26 150.0~300.0 [37, 118] barley bran 421:84 ± 24:46 376.1~443.5 [30] grains flour 140:41 ± 10:21 129 .9~160.7 [30] phenolic acids (mg/100 g) whole grains 414:70 ± 32:86 336.29~453.94 [39] total flavones (mg/100 g) whole grains 80:64 ± 17:15 37.93~236.91 [37, 39, 75] flavonoids (mg/100 g) whole grains 12:51 ± 10:14 6.20~30.08 [18] catechin (mg/100 g) whole grains 2:25 ± 0:94 0.90~4.27 [18, 227] quercetin (mg/100 g) purple grains 3:51 ± 2:24 2.00~6.08 [18, 227] kaempferol (mg/100 g) whole grains 3:66 ± 14:87 1.27~6.31 [18, 227] myricetin (mg/100 g) whole grains 11:07 ± 22:25 0~73.30 [227] total alkaloid (mg/100 g) whole grains 25:96 ± 1:41 6.36~44.63 [228] total anthocyanin (mg/100 g) whole grains 35:50 ± 23:82 4.9~103.7 [229] barley bran 256:05 ± 137:67 158~353.4 [54] refined flours 39:15 ± 25:67 21.0~57.3 [54] proanthocyanidin (mg/100 g) whole grains 6:97 ± 3:84 1.58~13.18 [18] total tocols (mg/100 g) whole grains 5:85 ± 3:51 0.85~12.49 [21, 66, 70, 71] antioxidant activity (%) whole grains 41:55 ± 7:82 24.10~82.00 [37] gaba (mg/100 g) whole grains 8:00 ± 3:92 0.10~30.67 [75] protein % whole grains 14:92 ± 0:13 9.51~20.46 [135] folates (mg/100 g) whole grains 71:24 ± 16:62 51.8~103.3 [18, 23] phytosterols (mg/100 g) whole grains 91:13 ± 21:14 76.1~115.3 [18] p (mg/kg) whole grains 2,592:9 ± 1,045:5 936~6538 [24] k (mg/kg) whole grains 4,801:7 ± 1,839:2 207~9162 [24] ca (mg/kg) whole grains 568:3 ± 235:1 68.4~1150.0 [24] mg (mg/kg) whole grains 1,249:8 ± 392:7 308.4~2164.0 [24] fe (mg/kg) whole grains 52:7 ± 31:3 8.8~156.1 [24] zn (mg/kg) whole grains 39:5 ± 15:5 9.4~76.2 [24] cu (mg/kg) whole grains 14:1 ± 10:3 0.6~68.0 [24] mn (mg/kg) whole grains 29:3 ± 24:8 5.8~120.0 [24] na (mg/kg) whole grains 190:5 ± 104:7 6.7~611.5 [24] s (mg/kg) whole grains 1,505:2 ± 262:8 686.0~2363.5 [24] 3 oxidative medicine and cellular longevity and metal chelating activity 16.6-43.2%) than that of plains (97~126 m altitude), but the soluble β-glucan and arabinoxylan content ranged from 2.0% to 2.8% and 0.08% to 0.19%, respectively [25] . 2.1. β-glucan. β-glucan in barley is the most abundant group of polysaccharides in cell wall. the molecular weight of β-d-glucan in hulless barley grains is 571.4 kda, which composes of (1 → 4)-and (1 → 3)-glucopyranosyl residues, especially its trisaccharide and tetrasaccharide accounted for 66.6% of total cellulosyl units [26] . β-glucan content (%) in naked, malt, black, waxy-naked, and blue barley is 3.44, 3.46, 6.08, 6.75, and 5.91, respectively, especially waxy-naked barley flour has the highest extraction rate (95.49%); however, gi in vitro starch digestibility was lowered by adding β-glucan [27] . the fatty acid derived flavouring substance (dodecanoic acid, octyl butanoate, ethyl decanoate, and decyl acetate) in beer has important role in the aggregation behavior of barley β-glucan [28] . the β-glucan concentrations in the six hulless barley grains varied from 4.96% to 7.62%, among shorts (8.12~13.01%)>bran (6.15~7.58%)>flour (2.48~2.95%) [29] . the total β-glucan contents in the nine hulless barley are 4.7~6.3% in bran and >3.4~4.4% in refined flour [30] . barley. phenolic compounds have the antioxidant, anti-inflammatory, and antitumor potentials [31] . there are the most abundant polyphenols in barley grains, especially p-hydroxybenzoic (17.6%), pcoumaric (15.2%), and ferulic acids (54.4%) [32] . the most abundant polyphenol in barley extract include free polyphenols (98:0 ± 10:0 mg/100 g) and bound polyphenols (51:0 ± 2:0 mg/100 g), especially ferulic acid (27.77 mg/100 g) and >procyanidin b (7.37 mg/100 g) [33] . phenolic acids such as ferulic acid and p-coumaric acid were 0.215 mg/100 g and 0.110 mg/100 g in barley grains and 0.407 mg/100 g and 0.144 mg/100 g in malt, respectively [34] . the extraction polyphenols yield of barley lactobacillus fermented solution of 60% ethanol concentration was 1.809%, main components (mg/100 g) including rutin (3.508), vanillic acid (2.128), ferulic acid (1.938), coumaric acid (1.136), gallic acid (0.680), protocatechuic acid (0.299), and p-coumaric acid (0.083) [35] . compared to the raw barley extract, the protein, total phenols, and βglucan of fermented barley extract with lactobacillus plantarum dy-1 can significantly increase to 34.94%, 13.61 mg/g, and 13.44%, respectively [36] . there were larger genetic variations in the contents of total polyphenol (203:314 ± 34:256 mg/100 g), total flavonoid content (88:042 ± 14:343 mg/100 g), and antioxidant activity (41:55 ± 7:82%) among the 223 barley genotypes; however, major qtls between bpb-0572 and bpb-4531 control phenolic compounds in tibetan wild barley, especially the udp-glycosyltransferase gene with biosynthesis of flavonoid glycosides was colocated with bpb-4531 [37] . barley. nacl stress increased the phenolic compounds (vanillic acid, p-coumaric acid, ferulic acid, and sinapic acid) accumulation and synthesis by upregulating the gene expression of phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme a ligase, p-coumaric acid 3-hdroxylase, and caffeic acid/5-hydroxyferulic acid o-methyltransferase of germinated hulless barley [38] . the blue hulless barley grains have larger variation on phenolic compounds and antioxidant activity, such as the free, bound, and total phenolic acids varied between 166.2~237.6, 170.1~240.8, and 336.3~453.9 mg/100 g, respectively, where the major phenolic compounds include quercetin, rutin, naringenin, hesperidin, (+)-catechin, gallic acid, benzoic acid, syringic acid, and 4-coumaric acid [39] . the anthocyanin and total phenolic contents in hulless barley grains are higher for high altitude, and the contents in its refined flours were 0.39~0.76 mg/100 g and 129.9~160.7 mg fae/100 g and in its bran were 1.85~2.65 mg/100 g and 367.1~443.5 mg fae/100 g, respectively [30] . whole grain hulless barley had high contents of total phenolic (259.90 mg/100 g), total pentosan (10.74 g/100 g), and orac values (41:805 ± 0:565 mol/100 g) [40] . the bran extract of hulless barley rich in phenolic acids on the nε-carboxymethyllysine formation during processing biscuits, which can reduce glycation and benefiting health [41] . total polyphenols (291.7 mg/100 g) and proanthocyanidins (163.0 anthocyanin for human health belongs to flavonoids, which is a secondary metabolite that plants adapt to harsh environments. ant2 gene (2hl) with a bhlh domain control purple grain and ant1 gene control red leaf sheath and pericarp in barley, r2r3-myb (ant1)+bhlh (ant2) complex promotes the synthesis by affecting expression of the anthocyanin biosynthesis structural genes (f3 ′ h) and ans genes [49] . the 2hl alleles from barley purple pericarp synthesis the peonidin-3glucoside [50] . the anthocyanin synthesis hvmyc2 gene is the major variant factor for blue aleurone of barley [51] . barley anthocyanins take part in the amino acid biosynthesis, carbon metabolism, phenylpropanoid biosynthesis, and metabolic pathways [52] . flavonoid 3 ′ -hydroxylase (f3 ′ h) and flavonoid 3 ′ , 5 ′ -hydroxylase (f3 ′ 5 ′ h)-coding genes take part in anthocyanin synthesis in barley [53] . the anthocyanin bran-rich fractions of yellow (158.7 mg/100 g, 9 anthocyanins) and purple barley (353.4 mg/100 g, 15 anthocyanins) are 6 times higher than that of the whole grain flours (21.0 and 57.3 mg/100 g), especially cyanidin 3-glucoside, delphinidin 3-glucoside, petunidin 3-glucoside, delphinidin 3-rutinoside, and cyanidin chloride [54] . arabinoxylan in barley is the second highest cell wall polysaccharide [55] . arabinoxylan in barley plays an important role in quality traits of malt and beer product [56] ; however, arabinoxylan arabinofuranohydrolase i can be used as novel enzyme products in the beer industry [57] . the starch degradation for seedling relies on cell wall degradation, where the iminosugar 1,4dideoxy-1,4-imino-l-arabinitol inhibits dextrinase and arabinoxylan arabinofuranohydrolase but permits rapid diffusion of αand β-amylase [58] . arabinoxylan contents in barley grains range from 4.2% to 5.4% [59] where it ranged from 0.53 mg/100 g to 0.90 mg/100 g at an average value of 0.67 mg/100 g in barley endosperm in 128 spring 2-row barley; its two qtls include glycosyltransferases and glycoside hydrolases [55] . arabinoxylan accounts for 45% of total polysaccharide [60] and 50~83% of total monosaccharide in barley husk [61] . arabinoxylan contents in hulless barley are in bran 8.42%>in shorts 4.08%>in flour 2.13% [29] which differ from the report of moza et al. [30] (in bran 1.97~3.6%>in flour 0.7~1.1%). 2.4. phytosterols. phytosterols in plant membrane are similar in structure to cholesterol [62] . higher phytosterols are found in the outer layers of barley grains and ranged between 82.0 mg/100 g and 115.3 mg/100 g, among which β-sitosterol is 47:6 ± 0:1 mg/100 g and campesterol is 18:1 ± 0:2 mg/100 g. the other phytosterols include stigmasterol (3.9 mg/100 g), brassicasterol, δ5-avenasterol, stigmastanol, stigmastadienol, and other minor sterols (δ5-and δ7-avenasterols, δ7-stigmastenol, and stigmastadienol: 8:6 ± 0:1 mg/100 g) [18] . . vitamin e is the major lipid-soluble antioxidant for human health, which has eight different stereoisomers [63, 64] by three chiral centers in tocopherols from barley. spring barley has higher α-tocotrienol content in four tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, and α-tocopherol) [65] . [67] . a tocochromanol in barley grains ranged from 162.0 to 185.2 mg/100 g, but which is much higher than in oat (4.5 mg/100 g) and triticum (107.0 mg/100 g) [68] . tocochromanols content in barley is 50% in pericarp, >37% in endosperm, and >13% in germ; about 85% of the tocochromanols were tocotrienols, and tocopherols in germ (80%) was higher than that in pericarp (20%) [69] . the hulless barley especially with waxy, double waxy and tercel cultivars have the highest tocols content. tocol in whole grain was 5.38 mg/100 g to 12.49 mg/100 g, and in pearling flour was 19.5 mg/100 g to 36.3 mg/100 g; however, the ratios of total tocotrienols to total tocopherols ranged from 1.6 to 3.9 [70] . the highest content of tocols (6.03~6.76 mg/100 g) and vitamin e concentrations (1.80~2.01 mg/100 g) was found in the waxy barley, especially in the hulless waxy washonubet (tocols 6.76 mg/100 g and α-tocotrienols isomer 4.21 mg/100 g) [71] . 2.6. resistant starch. resistant starch (rs) can prevent dietrelated chronic diseases such as diabetes and colon cancer. rs in hulless barley grains is related with b-type granules and the amylopectin f-iii fraction; however, sequential rate of enzymatic hydrolysis in diets is waxy>normal>high amylose barley [72] . rs of 209 spring barley cultivars approved and popularized during the past 100 years in 5 oxidative medicine and cellular longevity europe, in which rs content ranged from <1% to >15% [73] . rs content of unprocessed grains of high-amylose, normal, and waxy barley is 24:0 ± 0:8%, 17:0 ± 0:0%, and 9:2 ± 0:7%, respectively, but slowly digestible starch in unprocessed grains of normal (41:6 ± 0:1%)>high-amylose (23:5 ± 0:5%)>waxy barley (20:8 ± 0:2%) and rapidly digestible starch in normal (6:6 ± 0:1%)<high-amylose (10:7 ± 0:4%)<waxy barley (16:3 ± 0:5%) [74] . the rs content (mg/100 g) of 629 accessions barley grains was 1:56 ± 1:22%, with a highest content up to 9.0% [75] . snps (i.e., 10th exon g(3935)-to-t and fifth exon c(2453)-to-t) in three exons play different roles on the expression of the waxy transcript, granule-bound starch synthase i (gbss i), protein, amylose, and starch properties of hulless barley [76] . the chronology of rs contents in different barley diets is ground pearled barley (9.4%)>pearled barley flakes (8.1%)>whole pearled barley (7.4%)>cut barley 2.7. gaba and linoleic acid. gaba increased α-amylase gene expression by treating barley aleurone with exogenous gaba, especially α-amylase activity began to rise after about 24 h and reached a peak at 48 h [78] . the gaba content (mg/100 g) in 629 accessions of barley grains is 8:00 ± 3:92 mg/100 g, the highest up to 30:67 8:00 ± 3:92 mg/100 g [75] . the gaba has a very important role in mediating nacl stress phenolic compounds accumulation in germinated hulless barley [38] . linoleic acid content increased from 51.74% to 56.56% and oil from 1.73 to 2.13%, while oleic content decreased from 19.94% to 15.62% and palmitic acid from 18.53% to 17.33% during barley malting process [23] . 2.8. phytases. hydrolyze phytate in barley associated the bioavailable nutrient elements (p, fe, and zn), which exists as a single gene (paphy_a) in barley, but as two or three homeologous copies in wheat [79] . the improvement of hvpaphy_a transformed barley showed phytase activity increases up to 110-fold in green leaves, 19-fold in grains, and 57-fold in dry straw [80] . barley grains for preventive chronic diseases 3.1.1. antidiabetic properties. diabetes is a chronic metabolic disease with high mortality rates; therefore, search for novel natural inhibitors has gained much attention [81] . major antidiabetic elements in barley are β-glucan, phenolic compounds (phenolic acids and flavonoids), phytosterols, tocols, arabinoxylan, and resistant starch ( table 2) . oxidative stress not only leads to insulin resistance, impaired glucose tolerance, b-cell dysfunction, ultimately diabetes but also can treat diabetes and obesity by phytochemicals (phenolic acids, flavonoids, phytosterols, and tocols) in barley [18] . chronic consumption of foods with high β-glucans in barley can improve insulin resistance and lower the postprandial glucose response and increase satiety [82] . the β-glucan in hulless barley reduced the insulin resistance, arterial sclerosis, serum glucose, and serum lipid in high-fat mouse [83] . high phenolic content (168.7 mg/100 g) and low rapidly digested starch (38.7%) make barley muffin to modulate glycemic response [84] . the hypoglycemic effect of ethanol extract polysaccharide from barley malt is better for decreased fasting plasma glucose of the diabetes mice than that of water extract [85] . the boiled barley kernels evening meal can facilitate glucose regulation, increase the release of glucagon-like peptide-1, and reduce energy intake and fasting serum free fatty acids, mediated through gut microbial fermentation of the indigestible carbohydrates [86] . the glycemic index (gi = 82:8) of all-wheat bread is higher than that (gi = 57:2) of 60% wheat+40% barley flour (6.0% β-glucan) [87] . gi for barley with 4.6% β-glucan and oat tempe are 30 and 63, respectively [88] . the hulless barley can reduce postprandial glucose and improved insulin sensitivity by amino acid and biogenic amine profiles [89] . 3.1.2. antiobesity. major antiobesity components in barley are β-glucan, resistant starch, polyphenols, dietary fiber, arabinoxylan, tocols, and phytosterols ( table 2) . β-glucan in barley significantly treats obesity that reduced low-density lipoprotein, total cholesterol, and serum p-cresyl sulfate levels and increased flow-mediated dilation [90, 91] . barley β-glucan can prevent visceral fat (≥100 cm 2 ) obesity and increase faecal scores, but decreased nutrient digestibility and antiobesity [92, 93] . rs and β-glucans as well as soluble arabinoxylan were utilized mainly in the caecum, especially rs shifted the utilization of other polysaccharides to more distal parts of the colon of pigs [94] . obesity and insulin resistance associated with bile acid changes and lower dietary fiber (β-glucan) in barley diet [95] . the aqueous extract of fermented barley has antiobesity effects due to β-glucan and phenolic acids (vanillic acid and ferulic acid) [36] . β-glucans in black and blue hulless barley for preventive obesity were very higher than that of white one, based on its molecular weights, particle sizes, viscosities, binding capacities (fat, cholesterol, and bile acid), and inhibiting activities on pancreatic lipase [96] . the polyphenols extracted in black hulless barley show notable decreases in total cholesterol, low-density lipoprotein cholesterol, and atherosclerosis, but significant increase in high-density lipoprotein cholesterol levels [97] . barley β-glucan can reduce low-density lipoprotein cholesterol and non-highdensity lipoprotein cholesterol as well as alters the gut microbiota for preventing cardiovascular disease (see table 2 ) [98] . oxidative stress and inflammation are two important factors of atherosclerosis, and polysaccharide extracts with antioxidation and anti-inflammation of hulless barley prevent cardiovascular diseases [17] . some other functional components of barley have been associated with 6 oxidative medicine and cellular longevity cardiovascular health, such as polyphenols, phytosterols, lignans, tocols, and folate [18] . major anticancer elements in barley are β-glucan, phenolics, arabinoxylan, phytosterols, lignan, and resistant starch ( table 2 ). functional ingredients of barley with antioxidative and immunomodulatory activities are associated with anticancer effects [18] . barley with high dietary fiber (β-glucan) has an important role for the prevention of colon cancer and cardiovascular diseases [99] ; low molecular weight β-d-glucan can enhance antioxidant and antiproliferative activities [100] . aqueous extract of fermented barley can induce subcutaneous transplantation tumor apoptosis that can be used for a nutrient supplement in the treatment of human colon cancer [101] . β-glucans in hulless barley has anticancer activities in vitro, but its anti-inflammatory activities increased as their molecular weights decreased [42] . the bound phenolics in dehulled hulless barley have excellent antioxidant and antiproliferative effects to human liver cancer cells [102] . a water soluble polysaccharide (glucose : xylose : arabinose : rhamnose = 8.82 : 1.92 : 1.50 : 1.00) from hulless barley can inhibit colon cancer, which induce ht-29 apoptosis through ros-jnk and nf-κb-regulated caspase pathways [103] . 3.1.5. antioxidation. antioxidants are compounds that remove reactive oxygen species from cells, which play a dual role in aggravating and preventing diseases [104] . major antioxidants in barley are phenolic compounds (phenolic acids, flavonoids, and anthocyanin), tocols (vitamin e), polysaccharide (arabinoxylan), dietary fiber, and phytic acid ( table 2) . antioxidant effects of polyphenols in barley are flavanols>flavonols (quercetin)>hydroxycinnamic acids (ferulic, caffeic, and coumaric acids) [33] . malt has higher phenolic content (sinapinic acid and epicatechin) than its barley grains, which plays a key role on antioxidant stability of beer [105] . the antioxidant activity for anthocyanin in barley bran was markedly higher than that of whole grains flour [54] . five of the seven associations in barley were with markers near genes associated with the tocochromanol (vitamin e with the most powerful antioxidants) pathway [106] . overexpression of homogentisate geranylgeranyl transferase for barley enhanced the tocotrienol levels (δ-, β-, and γ-tocotrienol 10-15%) and antioxidant capacity (radical scavenging activity 17-18%) in barley seeds [107] . the antioxidant effects of dietary fiber in hulless barley bran were associated with total phenolic concentration, which had the dpph (1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)-hydrazyl) radicalscavenging activity and ferric-reducing antioxidant power 7 oxidative medicine and cellular longevity [108] . gaba induces the accumulation of proline and total phenolics and enhances the antioxidant system in germinated hulless barley under nacl stress [38] . the chapatti quality score reduced by 15% and its phenolic concentration increased from 23.7 to 28.7 mg/100 g, while biscuit spread factor reduced by 33% and its β-glucan concentration increased from 0.60 to 2.4% as well as phenolic content increased from 6.3 to 13.5 mg/100 g after blending of 30% hulless barley flour, especially markedly increased antioxidant activity [109] . 3.1.6. anti-inflammation. major anti-inflammatory ingredients in barley are β-glucans, lignans, vanillic acid, arabinoxylan, and so on ( table 2) . endothelial cell adhesion molecules were identified as an early step in inflammation and atherogenesis; barley β-glucans not only have a maximum anti-inflammatory activity at mw~1:40 × 10 5 , especially the inhibition of tnf-α-induced expression of vascular cell adhesion molecule was stronger than that of oat β-glucans, but also have a higher ratio of 3-o-β-cellobiosyl-d-glucose to 3-o-β-cellotriosyl-d-glucose oligomers in the polymeric chains [110] . the molecular weights of β-glucans in hulless barley increased that add inhibitory abilities on α-amylase and pancreatic lipase, but the antiinflammatory abilities decreased, especially the low intrinsic viscosity and high solubility of β-glucans acid hydrolysis for 20 min might contribute to its higher anti-inflammatory activity, which significantly affected their bioactivities (e.g., anticancer), which was beneficial for a better understanding of their structure-function relationships [111] . the fermented barley extracts (vanillic acid) downregulate glucose consumption and reducing proinflammatory cytokine secretion [112] . the anti-inflammatory property of malt and wholegrain barley is due to the formation of short chain fatty acid (scfa) and changes in microbiota composition [113] . 3.1.7. immunomodulation. major immunomodulatory substances in barley are β-glucans, arabinoxylan, and so on ( table 2 ). the immunomodulatory activity of barley β-glucans insolubility associated with its particle size, granule conformation, and particulate homogeneity. all β-glucan fractions can induce more cytokines in bone marrowderived dendritic cells than their oat equivalents; however, the insolubility of β-glucan affects its immunomodulatory activity, which is related to its particle size, particle configuration, and particle uniformity [114] . a water-soluble polysaccharide (bp-1, molecular weight 6:7 × 10 4 da) from hulless barley can improve the immune ability of immunosuppressive mice through increasing the serum levels of il-2, tnfα, and ifn-γ, such as bp-1 (80 mg/kg and 160 mg/kg) can not only significantly increase the number of bone marrow cells and peripheral blood white blood cells, as well as enhance the production of il-2, tnf-α, ifn-γ, igg, and igm in the spleen and serum levels for improving the immune function, but also promote the proliferation and phagocytosis activity of macrophages as well as repair the damage induced by ctx in the spleen cells of immunosuppressive mice [115] . table 2 ), which shows a 109% survival rate after cardiac ischemia (30 min)/reperfusion (60 min) injury, reduces left ventricular anion superoxide production (62%) and infarct size (35%), and increases the capillary (12%) and arteriolar density (18%) and vegf expression (87.7%) of hearts in mice [116] . the products of barley grains can reduce the cardiometabolic risk and regulate the blood glucose and appetite hormones in 11-16 h after intake; however, its mechanisms are gut fermentation of indigestible carbohydrates [117] . 3.1.9. hypocholesterolaemic effects. cholesterol is a synthesis lipid in the body [118] . dietary β-glucan of hulless barley reduces the plasma ldl cholesterol content (see table 3 ) by promoting the excretion of faecal lipids and regulating the activities of 3-hydroxy-3-methyl glutaryl-coenzyme a 8 oxidative medicine and cellular longevity reductase and cholesterol 7-αhydroxylase in hypercholesterolaemic rats [119] . barley bran 5% and 10% in diet to the hypercholesterolaemic rats improved the level of lipids, lactate dehydrogenase, liver enzymes, and creatine kinase-mb [120] . whole grain hulless barley has hypocholesterolaemic effects by promoting bile acid synthesis and reabsorption, controlling cholesterol synthesis and accumulation in peripheral tissue, decreasing the expression of 3-hydroxy-3methylglutaryl coenzyme a reductase, while increasing the hepatic expressions of amp-activated protein kinase α, cholesterol 7α-hydroxylase, ldl receptor, liver x receptor, and pparα [118] . 3.1.10. blood pressure regulation. higher consumption of barley β-glucan is associated with lower systolic and diastolic blood pressure (see table 2 ), i.e., diets rich in β-glucans reduce systolic blood pressure by 2.9 mmhg (95% ci 0.9 to 4.9 mmhg) and diastolic blood pressure by 1.5 mmhg (95% ci 0.2 to 2.7 mmhg) for a median difference in β-glucans of 4 g [121] . the consumption of high molecular weight barley β-glucan can reduce blood pressure [98] . 3.1.11. bowel health improvement. gastrointestinal tract disease is a major global health problem. β-glucan in hulless barley has protective effects to the gastrointestinal tract (see table 2 ) [122] . the dietary fiber in hulless barley improved indices of bowel health compared with refined cereal foods, especially himalaya 292 possesses high amylose and resistant starch due to lacking activity of a key enzyme responsible for starch synthesis; however, consumption of himalaya 292 foods resulted in 33% higher faecal weight, a lowering of faecal ph from 7.24 to 6.98, a 42% higher faecal concentration, a 91% higher excretion of butyrate, a 57% higher faecal total scfa excretion, and a 33% lower faecal p-cresol concentration [123] . fermented barley extract (10 mg/100 g) can act as a promising laxative agent to cure spastic constipation [124] . butyric acid for improving the colonic health is produced by degradation of barley dietary fiber by microbiota [125] . 3.1.12. gastroprotective effects. β-glucan from hulless barley can mitigate the gastric lesions and gastric mucosal damage as well as gastric oxidative stress injury through decreasing the level of malondialdehyde [122] . the oral administration of fermented barley extract had strong gastroprotective effects through strengthening antioxidant defense system and anti-inflammatory effects, as well as decreasing lipid peroxidation and cat activity by increasing the gsh levels and sod activity in the body, and the 200 mg/kg dose of fermented barley extract was similar gastroprotective as the 10 mg/kg dose of omeprazole, which indicates that this dosage can be used for patients suffering from different levels of gastric damages [124] . 3.1.13. reduce chronic kidney disease. barley β-glucans is associated with a saccharolytic shift in the gut microbiota metabolism by a reduction of pcs toxin blood levels and an increase of scfa production at colonic site, which can reduce the microbial-derived uremic toxin and cardiovascular complications in end-stage renal disease (see table 3 ), especially chronic kidney disease [90] . the total cholesterol and triglycerides were reduced, and hdl cholesterol increased in 10% and 20% barley intervention in breakfast diet; however, barley in the diet of stage 3 chronic kidney disease patients has significantly improved the nutritional status and renal functions [126] . 3.1.14. improve metabolic syndrome. barley β-glucan can improve postprandial glucose response and cholesterol levels as well as the metabolic syndrome based on individual gut microbiota composition (see table 3 ) [127] . tibetan hulless barley can reduce insulin resistance, dyslipidemia, and body weight gain, which can diminish the prevalence of metabolic syndrome induced by high-fat-sucrose diets, i.e., rats fed with tibetan hulless barley can increase the assessment of insulin resistance scores (body weight, abdominal fat deposition, liver weight, liver fat deposition, triglyceride, fasting blood glucose, and serum fasting insulin) and decrease lowdensity lipoprotein cholesterol levels compared to rats fed with a basal diet [128] . 3.1.15. hepatoprotective effect. β-glucans in barley can decrease fatty liver in diabetes with obesity (see table 2 ) [129] . the free phenolic extract in barley added the hepatic levels of antioxidant enzymes [130] . whole grain hulless barley had significantly lower liver lipid levels (total phenolic and pentosan) [118] . barley sprout extract protects liver cells under oxidative stress by activating nrf2 and adding glutathione synthesis, especially against alcohol-induced liver injury, as it inhibits glutathione depletion and hepatic lipid accumulation, reduces serum biochemical markers of liver injury, and inhibits inflammatory responses [131] . 3.1.16. wound healing acceleration. nowadays, β-glucans represent effective topical agents for the treatment of chronic wounds and burns due to the activation of the immune and cutaneous cells (see table 3 ), which increase wound repair by enhancing the infiltration of macrophages and promote tissue granulation, collagen deposition, and reepithelialization based on inducing the proliferation and migration of keratinocytes and fibroblasts through specific receptors such as dectin-1, cr3, or tlrs [132] . barley β-glucan in vivo promotes the wound closure in mouse skin by promoting the migration and proliferation of human dermis fibroblasts [133] . 3.1.17. heart failure prevention. barley β-d-glucan is a natural activator of mnsod expression, which can prevent heart failure (see table 3 ) [134] . the food and drug administration has made a health claim between β-glucan and reduced risk of coronary heart disease, diabetes, and heart-related problems [135] . barley products can prevent and reduce the risk of coronary heart disease, which is associated with the constituents like β-glucan, phenolics, tocols, linoleic acid, and folate [18, 136] . 3.1.18. atopic dermatitis alleviation. the allergen produced by barley and the protein expressed in insect cells induce the same amount of ifn-γ and il-4 in pbmc from vaccinated horses (see table 2 ) [137] . fermented barley extract 9 oxidative medicine and cellular longevity p reduced skin lesions by inhibiting inflammatory cytokines [138] , but gaba alleviated atopic dermatitis by suppressing serum immunoglobulin e and splenocyte interleukin production [139] . barley intake reduces the risk of total stroke by significantly increasing the medium and small particle sizes of high-density lipoprotein cholesterol (see table 3 ) [140] . increasing the expression of antioxidant genes in the liver and the regulation of nrf2 played a role in the regulation of metabolic diseases in stroke-prone spontaneously hypertensive rats consuming a fermented barley extract p diet [141] . 3.1.20. allergic rhinitis alleviation. ma-al-shaeer formulation based on barley can treat for allergic rhinitis, especially reduced the nasal congestion, post nasal drip, and headache (see table 3 ) [142] . fermented barley extract alleviated allergic rhinitis in ova-sensitized mice by regulating cytokines (ifn-γ or il-17) related to chronic inflammation [138] . in addition, the common edible whole-barley flour can reduce the risk of hyperlipidemia and cholelithiasis [18] , antiaging [140] as well as increase body strength and increase the content of linoleic acid and linolenic acid in pigs, cattle, sheep, and geese. in a word, there are more than 20 kinds health effects for barley grain preventive chronic diseases according to the summary of current retrieval literature. the descriptions of some literatures for barley preventive chronic diseases (antidiabetes, antiobesity, anticancer, antioxidation, anti-inflammation, hypocholesterolaemic effects, blood pressure regulation, cardioprotection, immunomodulation, improve gastrointestinal, hepatoprotection, bowel health, cardiovascular disease prevention, atopic dermatitis alleviation, wound healing acceleration, heart failure prevention, and so on) are relatively sufficient, but some literatures for barley preventive chronic diseases (antiaging, reduce cholelithiasis, and increase body strength) are relatively less. these results fully demonstrate the health contribution relationship between barley functional food and human chronic disease prevention. the efficacy of preventing chronic diseases is related to barley genotype and its location, composition, extraction, and compatibility, and with the in-depth study of barley grain in prevention and treatment of human chronic diseases, the novel mechanisms for the prevention of chronic diseases may be ascertained as well as its putative role, either at major or minor level, would be further validated. grains for preventive chronic disease 3.2.1. β-glucans mechanism. β-glucans can be used as candidates for the medication in the treatment of human chronic diseases [133] . β-glucans have many bioactivities including antidiabetes; anticancer; antiobesity; anti-inflammation; immunomodulation; cardioprotection; lower cholesterol and lower blood pressure; improve bowel health, gastroprotection, and hepatoprotection; reduce chronic kidney disease and metabolic syndrome; prevent the risk of heart and cardiovascular diseases; and accelerate wound healing activities (figure 1 , tables 2 and 3 ) [13, 18, 98, 111, 116, 122, 123, 127, 133, [143] [144] [145] [146] [147] [148] [149] . the major mechanisms of β-glucans of barley involve in the prevention of chronic diseases are as follows: β-glucans can interact with intestinal lipids and bile salt to reduce cholesterol levels and subsequently prevent diabetes, hypertension, cardiovascular disease, and metabolic syndrome [143] . barley β-glucans not only control appetite and improve insulin sensitivity by gut hormone secretion via microbiota produced scfa [144] due to its high molecular weight and high viscosity but also increase their antigen ability and enhances the proinflammatory cytokines, which can be degraded by macrophages and natural killer cells mediating its cellular cytotoxicity opsonized tumor cells [145] . β-glucans of barley are the major regulators of adipogenesis, especially markedly downregulated the target genes in the adipose tissue including adipocyte fatty acid-binding protein, lipoprotein lipase, uncoupling protein-2, and glucose transporter 4 in 3t3-l1 cells [146] and also have the effect of inhibiting the α-amylase and pancreatic lipase [111] . barley β-glucan can reduce blood pressure and cardiovascular diseases that alters the composition of gut microbiota, decrease body mass index, waist circumference, and triglyceride levels [98] and also reduce the systemic inflammatory profile, prevent alveolar bone loss, and improve βcell function in diabetic animals [147] . barley β-glucans can not only regulate immune responses and connect innate and adaptive immunity [13] but also have cardioprotective mechanism of promoted angiogenesis through endothelial upregulation of the vascular growth factor [116] . the hypocholesterolaemic effect of β-glucan in barley is due to the increased bile acid synthesis [148] and improved bowel health by inhibited feed intake and increased cecal fermentation [149] . barley β-glucan not only has the gastroprotective effects by increasing the sod and cat activity, decreasing the gastric ulcer index, and increasing prostaglandin e2 and nitric oxide in laboratory rodents [122] but also affects lipid metabolism and scfa production, lowering microbes in patients with metabolic syndrome [127] and improving chronic kidney disease by reducing the microbial-derived uremic toxin. chronic consumption of barley β-glucans can decrease fatty liver by increasing small intestinal contents viscosity and improving glucose, lower glycated hemoglobin and relative kidney weights [129] , strengthen the angiogenic ability of ros-exposed endothelial cells for preventive heart disease [123] , and accelerate the wound closure by promoting the migration and proliferation of human dermal fibroblasts [133] . barley polyphenols have lots of bioactivities including antidiabetes, antiobesity, anticancer, antioxidant, anti-inflammation, hepatoprotection, and prevention of cardiovascular and heart diseases ( figure 1 , tables 2 and 3 ). barley lignan as natural polyphenols has anticancer, antioxidant, anti-inflammation properties, and preventive role for cardiovascular diseases. anthocyanin belongs to flavonoids, and flavonoids belongs to polyphenols. black, purple, and blue barley grains have gained much attention recently because their anthocyanins have anticancer, glycemic and body weight regulation, antioxidation, anti-inflammation, neuroprotection, hypolipidemia, retinal protection, hepatoprotection, and antiaging effects [150, 151] . several mechanisms of barley polyphenols for preventing chronic diseases have been documented so far. zhang et al. [36] has verified the molecular mechanism of insulin resistance by fermented barley extract vanillic acid through regulating mir-212 expression. the polyphenols with antiobesity from black hulless barley has strong superoxide radical, hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl radicalscavenging activity, ferric reducing antioxidant power, and moderate metal ion-chelating activity [97] . an efficacious antiproliferation capacity in caco-2 cells of black barley malt free extract was predicted due to its phenolic constituents which have cellular antioxidant and oxygen radical absorbance as well as peroxyl radical scavenging activities, dpph, and abts radical scavenging assays [152] . black barley sprouting stimulates the phenolic biosynthesis by upregulating proline-associated pentose phosphate pathway to support structure of sprouts with antioxidant capacity [153] . total phenolic and pentosan in hulless barley grains have antioxidant activity by downregulated expression of heat shock protein 60 and phosphatidylethanolamine binding protein 1 but upregulated expression of enoyl-coenzyme a hydratase and peroxiredoxin 6 [118] . the total polyphenol (flavonoid) content and the 2,2-diphenyl-1-picrylhydrazyl and abts radical scavenging abilities increased as the barley added to the food mixture [154] . lignan (-)-7(s)-hydroxymatairesinol inhibited tumor necrosis factor-α stimulated endothelial inflammation by inhibiting nf-κb activation and upregulating nrf2 antioxidant element signaling pathway [155] . lignans in barley have high anti-inflammatory abilities in endothelial cells by reducing nuclear factor-κb and extracellular signal as well as regulating kinase phosphorylation [156] . the polyphenols such as (+)-catechin, protocatechuate, and quercetin in barley not only have hepatoprotective effect [130] but also prevent coronary heart disease by reducing oxidativeinduced tissue damage through modulating intracellular signaling pathways [157] . polyphenols play an important role in alleviating cardiovascular diseases due to their antiradical scavenging abilities [18] . barley arabinoxylan has a lot of health benefits, which include antidiabetes, antiobesity, anticancer, lowering cholesterol, immunomodulation, antioxidant, cardiovascular diseases prevention, and so on (figure 1, tables 2 and 3 ). arabinoxylan in barley is the most abundant polysaccharide that has the capacity of lowering cholesterol and glucose as well as antioxidant activities [55] . the major mechanisms of barley arabinoxylan for preventing chronic diseases are as follows: arabinoxylan can improve 21 urinary metabolites associated with diabetes by improvement of carbohydrate and lipid as well as amino acid metabolism [158] . the antioxidant and antiobesity as well as immunomodulation of arabinoxylans associated with prebiotic effects and short-chain fatty acids production by interaction of gut microbiota and arabinoxylans [122] . arabinoxylan rice bran can increase anticancer effects in the older population by increased nk activity [159] . the arabinoxylan in barley with the immunomodulatory activity consisted of a xylan backbone with acetate, arabinose, galactose, glucuronic acid, and 4-o-methylglucuronic acid [160] . the arabinoxylan diet led to a lower postprandial blood for glucose-dependent insulinotropic polypeptide response, especially fat oxidation has an important role in the antiobesity and in the prevention of cardiovascular diseases [161] . barley polysaccharide prevent cardiovascular diseases by the vasodilatory effect of controlling angiotensin-converting enzyme production [17] . tables 2 and 3 ). barley grains contain phytosterols that can esterify to fatty acids, phenolic acids, steryl glucosides, or acylated steryl glycosides [18] . phytosterols significantly inhibited the ability of oxysterols to activate the liver x receptors transcription in modulating cancer cell behavior [62] , which are thought to influence multiple processes related to cancer, such as carcinogen production, cancercell growth, angiogenesis, invasion, metasis, and cancer-cell apoptosis [162] . for the prevention of cardiovascular diseases, barley phytosterols can compete with cholesterol for micelle formation, inhibiting cholesterol absorption in intestine and lowering cholesterol in central nervous system of the brain [18, 162] . the effect of plant sterols on neurodegenerative diseases is due to its passage through the blood-brain barrier, modulating cholesterol metabolism and inflammation in the central nervous system process in the brain, which involve low-density-lipoprotein, apolipoprotein e, and scavenger receptor class b type 1 [162] . the lowering of cholesterol and prevention of cardiovascular disease are due to the fact that the phytosterol structure in the barley membrane is similar to the configuration of different cholesterol [18] , but functionally similar to precursors in phytohormone synthesis, but lowering the blood concentration of cholesterol is beneficial to reduce the risk of cardiovascular disease. extremely rich in content in barley embryos. barley tocols are the best in cereals due to a high concentration and favorable distribution of eight active vitamers [18] . barley tocols have anticancer, antiobesity, and antioxidant effects and can lower cholesterol level, reduce the risk of stroke, and prevent cardiovascular and heart diseases ( figure 1 , tables 2 and 3 ) [18, 136] . tocotrienols can suppress various cancers (breast, lung, ovary, prostate, liver, brain, colon, myeloma, and pancreas) by its molecular mechanisms of cellular proliferation, apoptosis, angiogenesis, metastasis, and inflammation [63] , which are associated with human intake of whole grains, especially barley and rye. tocotrienol is a functional food of obesity and diabetes by regulating adipogenesis and increase apoptosis of adipocytes and improve glucose homeostasis through 11 oxidative medicine and cellular longevity suppression of inflammation and oxidative stress [64] . tocol is one of the most powerful antioxidants that has the ability to interact with polyunsaturated acyl groups and scavenge lipid peroxyl radicals and quench reactive oxygen species, thus protecting fatty acids from lipid peroxidation [163] . all barley pitas had the greatest antioxidant and vitamin e levels from barley malt flour [66] . the antioxidant properties of barley tocols due to its ability to inhibit lipid peroxidation in biological membranes induce the immune system, promote apoptosis induction, and reduce the risk factors of cardiovascular diseases and stroke by atherosclerotic blockages in the carotid artery [18] . barley rs has many immune properties like antidiabetes, antiobesity, anticancer, and so on ( figure 1,table 2 ). ceramide can promote lipid storage, impaired glucose utilization, and inhibited enzyme dihydroceramide desaturase 1, which can treat hepatic steatosis and metabolic disorders [164] . antidiabetes of rs can be increased by suppressing amylopectin synthesis through silencing of starch branching enzymes in barley [165] . t cell development and gut iga production suppress host lipid absorption by modulating cd36 expression [166] to achieve the effect of antiobesity of barley rs. gbss i is mainly responsible for amylose synthesis whereas sss i and sbe ii for amylopectin synthesis in amyloplasts [167] . barley with high β-glucan and moderate rs may benefit hyperglycemia-impaired lipid metabolism [168] . the blending of barley starch citrate with resistant starch iv up to 20% can produce noodles of acceptable quality and numerous health benefits [169] . early hominids used fruits/vegetables and leaves rich in polyphenols and k-ca as well as vitamins as staple foods to increase the dependence of the human body on these functional ingredients (see figure 2 ). diet played an important role in early hominids evolution [170] , but no reports have been delivered to date for diet in coevolution of human chronic diseases. miocene (23.0~5.3 ma) apes had a variety of foods that included folivory, soft-fruit eating, and hard-object feeding [170] . the diet of pliocene (5.3~2. 5 ma) and early pleistocene (2.5~1. 4 ma) hominids in africa was mainly fruits and leaves of c3 plants (trees, bushes, shrubs; 5.3~4.1 ma) which was gradually transformed into grass (c4 plant/tropical grasses and sedges) and hard-object (seeds and nuts) (4.0~1.4 ma) [170] [171] [172] , which was due to low availability of fruits in dry and active glacier (1.81~1. 55 ma) as well as migration to warm grasslands. ethiopia's pliocene lucy is one of the oldest and most complete fossils in hominid bones, her death due to fall out vertically and live on tall tree [173] . early hominids and australopithecines inhabited forests and savannas for collinearity oxidative medicine and cellular longevity found between tasty fruits (fructose/sucrose, quinine, and tannins) and primate sensory perception, which offered evidence of the two-direction evolutionary trend determining taste sensitivity [174] . our ape ancestors possessed a digestive dehydrogenase enzyme capable of metabolizing ethanol about 10 ma that they began using fruits fermentation from the forest floor [175] . there were differences in the proportion of meat and vegetables between the early hominids australopithecus and paranthropus; paranthropus ate more hard food than australopithecus [176] . early hominid australopithecus africanus, like chimpanzees, are dominated by fruit, leaves, and carbon-13-enriched foods about 3 ma [177] . the worldwide daily consumption of fruits and vegetables as well as tea has become the main tool for prevention of cardiovascular disease, stroke, cancer and diabetes due to their polyphenols modulate tau hyperphosphorylation and beta amyloid aggregation [178] . the anthocyanins and polyphenols for major functional ingredients in blueberry played a key role in preventing 15 chronic diseases [151] . the organopolysulfides and quercetin for major functional ingredients in allium genus played a key role in preventing 10 chronic diseases [179] . baobab was cultivated from seeds from 11 countries in east and west africa, its leaves had the highest vitamin b2 content (1:04 ± 0:05 mg/100 g) from senegal, adult leaves provided the highest ca content (3.373%) and young leaves with the highest ca and k content of nankoun in burkina faso [180] . the diets of early hominids related with five center of crop origin (mediterranean, middle east, central asia, indo-burma, and china-korea); however, the rich food structure maintained the survival and development of early hominids who lacked survival competition and migration could not improve their intelligence. therefore, polyphenols and k-ca as well as vitamins ingredients in fruits/vegetables and leaves as well as grass/seeds (such as gramineae and its ancestor species of barley) for preventive chronic disease are the results of long-term dependence for diet from pliocene hominids in africa to modern human beings ( figure 2 ). ingredients for neanderthals. neanderthals used mushrooms and nuts rich in polysaccharide and phytosterols as well as linoleic acid as staple foods to increase the dependence of the human body on these functional ingredients (see figure 2 ). neanderthals as well as early homo sapiens show high dietary variability in mediterranean evergreen habitats, but less diet in high latitude steppe or coniferous forests [181] . the steppe-like neanderthals of belgium feed on the meat of the woolly rhinoceros and wild sheep, while the neanderthals of the spanish forest feature root feed on mushrooms and pine nuts [182] ; neanderthals in northern spain roasted vegetables and used medicinal plants about 2.5~5.0 ma [183] . neanderthals ate meat (high chloroprostol) and plants (5β-sitosterol) as staple foods [184] . plant foods of neanderthals from iraq and belgium had the typical modern human diets, which include palms, date, legumes, and grass seeds [185] . 130 medicinal functions (major polysaccharide) in medicinal mushrooms and fungi can prevent and treat more than 10 chronic diseases [186] ; however, β-glucans in barley preventive 16 chronic diseases. the total polysaccharide content in morchella sp. reached up to 18.4% of dried biomass in a mixture of 1 : 1 of wheat grains and potato peels [187] . β-glucans are group of polysaccharides found in mushrooms, yeasts, seaweed, barley, and oats [13] . macrofungal β-glucans are major β-1,3and β-1,6-glycosidic bonds, which have immunomodulatory, anticancer, and antioxidant properties; total β-glucan content varied from 13.5% in a. bisporus to 40.9% in t. rutilans [188] . phytosterols are diet ingredients found in an array of nuts, seeds, and vegetables which have anticancer activities via their interactions with the plasma cell membrane [189] . oil palm (elaeis guineensis jacq.) is one of the highest oil-yield crops in the world; however, palm oil is the largest variety of plant oil produced, consumed (30%), and internationally traded in the world, rich in linoleic acid (10%) that is associated with egfad12 gene [190] . 50~55% carbohydrate diets (especially whole-grain breads, vegetables, and nuts) had minimal risk of mortality [191] . fruits, vegetables, and whole grains were indispensable among four top diets (mediterranean diet with five best ranked first, dash diet with lower blood pressure, flexitarian diet with lose weight and mind diet with brain health). the diets of neanderthals was not correlated with the complete center of crop origin, poor diet (meats, mushrooms, and pine nuts) and lack of migration lead to extinction ( figure 2 ). these results reveal the importance of whole grain and vegetables or fruits in human health; however, lack of three categories of food suggesting the cause of the neanderthal destruction. therefore, polysaccharide (βglucan) and phytosterols as well as linoleic acid in mushrooms and nuts as well as palm oil for preventive chronic disease are the results of long-term dependence for diet from neanderthals in europe to modern human beings ( figure 2 ). ingredients for homo sapiens. homo sapiens not only used grass and seeds rich in gaba and enzymes as well as resistant starch as staple foods to increase the unique dependence of the human body on these functional ingredients (see figure 2 ) but also inherited the staple foods of early hominids and neanderthals. feeding and diet played key roles in human evolution, especially homo sapiens have a relative masticatory structure similar to that of other primates [192] . homo sapiens moved from africa into the middle east about 120 ka, according to fossils at skhul and qafzeh caves in israel [193] . the aba-mirna-9497 in belladonna with highly homologous to homo sapiens mirna 378 can target and downregulate human brain-enriched transcription factor (znf-691) and gene expression in the human central nervous system [194] . for homo sapiens, foragers have greater complexity than farmers or pastoralists; meanwhile, the old world foragers had significantly higher anisotropy values than new world foragers, but similarity between hard food foragers and hard food farmers [195] . the meat diet abuse by a herbivorous homo sapiens can lead to atherosclerosis [196] . the diets of african homo sapiens associated with center of crop origin in ethiopia, their migration along eight center of crop origin changed the fate of mankind [9] . variation in cranial robusticity from 14 geographical homo sapiens associated with cranial shape, size, climate, and neutral genetic distances; however, cranial robusticity may be an adaptation to cold and harsh environments as well as masticatory differences in diet [197] , especially higher gaba (such as barley grass 271.3 mg/100 g) grass diets in crop suiting environmental extremes improved intelligence [9] . gaba contents in picked tea leaves under anoxic treatments at 4 h and 6 h are 16:12 ± 1:05 and 17:13 ± 0:80 mg/kg (fresh weight) [198] . gaba a receptors modulate vigilance, emotions, cognition, and muscle tension, and they are the targets of anxiety-reducing and sedative-hypnotic benzodiazepines and some general anesthetics [199] . shisa7 regulates gaba ar trafficking, function, and pharmacology, especially modulates benzodiazepine action in the brain [200] . barley can be grown in four seasons (spring, summer, autumn, and winter) at 1,900-2,300 m in yunnan province of china which may be associated with harboring 300 enzymes in barley grass [6] . the diet high in sodium and low four diets (whole grains, fruits, vegetables, nuts, and seeds) were major dietary risk factors for deaths and disability-adjusted life-years globally and in many countries [2] ; however, the whole grain is the manifestation of resistant starch type i surrounded by protein matrix and bran layer for making the starch unavailable for enzymes. the least chronic disease of ancient humans due to replacing the salt with seasoning crops, such as onions, ginger, garlic, coriander, pepper, chili, and so on [201] . the coevolution of the preventive human chronic diseases are related to major diets of vavilov's eight crop origin centers (ethiopia, mediterranean, middle east, central asia, indo-burma, china-korea, mexico-guatemala, and peru-ecuador-bolivia) [8] . human chronic diseases are related with six dietary structures (fruits/vegetables, young grass/barley grass, carnivorous, cereals crop, polished rice/wheat flour, and polished rice/-wheat+grass powder), but polished rice/wheat+barley grass powder is the most major healthy dietary guidelines for modern humans; therefore, it is necessary to unravel coevolutionary mechanism between preventive chronic diseases and human diet for functional foods [202] . these results support homo sapiens used grass/seeds (rich in gaba and enzymes as well as resistant starch), fruits/vegetables and leaves (rich in polyphenols and k-ca as well as vitamins), mushrooms, and nuts (rich in polysaccharide and phytosterols as well as linoleic acid) as staple foods to increase the dependence of the human body on these functional ingredients. barley is the oldest and more important cereal crop with the utmost dietary fiber in the world; its malt, as a functional food, is not only the largest beer raw material in the world but also one of the 300 most commonly used chinese herbal medicines [6] . our review point out that barley grass has antidiabetic, anticancer, antidepressant, antioxidant, fatigue, anti-inflammatory, hypolipidemic, antigout, calcium supplementary, and antiacne/detoxifying effects; promotes sleep; regulates blood pressure; enhances immunity; protects liver; reduces hyperuricemia; alleviates atopic dermatitis; improves cognition, constipation, gastrointestinal function; and prevents hypoxia, cardiovascular diseases, and so on [6] . barley grass powder is known to play a pivotal role in prevention of 20 chronic diseases that involves six molecular mechanism of gaba, flavonoids, sod, k-ca, vitamins, and tryptophan [202] ; however, barley grains play key roles in prevention of 20 chronic diseases that involves six molecular mechanism of β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch. modern humans had originated in the progeniture of african homo sapiens with cognitive hominin [203] . the staple foods of modern human are the synthesis of homo sapiens that inherited early hominids and neanderthals, which carry the neanderthal dna due to interbreeding between homo sapiens and neanderthal took place in the middle east. human flt3 ligand isolated from transgenic barley seeds is a glycoprotein including α(1,3)-fucose and α(1,2)xylose, which showed expression of human growth factor in barley grains with active protein [204] . the peptide ll-37 is a component of the human innate immune system, it accumulated 0.55 mg/kg in the barley grains [205] . human fgf-1 gene was fused with barley α-amylase signal peptide dna sequence and expressed in transgenic salvia miltiorrhiza plants driven by 35s promoter; however, recombinant fgf-1 in leaves was 272 ng/ fresh weight [206] . therefore, functional ingredients in barley grass and grains are essential for the health contribution of modern human (homo sapiens), neanderthals, and early hominids staple food to prevent and treat human chronic diseases. barley has health beneficial properties and was part of the modern hominid diet; thus, it has evolved in its functional ingredients and contributed to a reduced risk of diseases. it is amazing that barley grains and malt as well as grass powder can prevent or treat more than 20 human chronic diseases. we think the major scientific basis for that is as follows: first, barley prevent over 20 humans chronic diseases which associated with the similar origin and evolution center of barley and human beings: ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley ( figure 2) [8, 170, 207] . ethiopia, morocco, fertile crescent, and tibet of china have been proposed as centers of barley origin and the primary habitat of wild barley (figure 2 ) [207] . wild barley is a selfing annual grass of predominantly morocco and irano-turanian and israel-jordan in arid desert or salt environments, the cold region in tibet of china and ethiopia, which has accumulated abundant functional ingredients for drought, salt, and cold resistances. the earliest modern human originate from ethiopia and morocco are dated to~190 ka and~315 ka (figure 2) , respectively [208] . the earliest human occupied high-altitude habitats in the andes and the tibetan plateau, especially late pleistocene humans adapted to the severe environments of these glaciated above 4,000-meter elevation 14 oxidative medicine and cellular longevity in the bale mountains of ethiopia ( figure 2 ) [209] . neanderthals in modern-day iraq and belgium ate grasses, cooked barley grains, and others. second, barley grass powder plays a key role in the promotion of human intelligence in the early stage: the incremental evolution of globular braincase associated with diets of brain health from ethiopia and middle east as well as from mediterranean center of crop origin, especially the highest gaba in crop diets suiting environmental extremes improved intelligence. brain development is a self-reinforcing process in which brain cells proliferate, differentiate, migrate, and connect functional neural circuit, especially primate-specific features of gabaergic interneuron development [210] on the basis of gaba content in diet. survival depends on the selection of behaviors adaptive for environment; however, stimulation of dorsal raphe gaba neurons promoted movement in negative but not positive environments to promote environment-specific adaptive behaviors of serotonin [211] . barley is a major crop in many developed countries [9] ; gaba in barley grass suiting environmental extremes (cold, arid, and salt) can significantly increase, which can improve cognition and prevent 12 chronic diseases [6] . the average content of gaba in 31 cultivars that we bred is 271.3 mg/100 g which is 1.8 fold higher than that of other barley crops around the world [9] . third, healthy effects of functional ingredients of barley grass and grains are the sum of staple foods for early hominids and neanderthals as well as homo sapiens: early hominids used fruits/vegetables and leaves rich in polyphenols (flavonoids) and k-ca as well as vitamins (tocols); neanderthals used mushrooms and nuts rich in polysaccharide (β-glucans and arabinoxylan) and phytosterols as well as linoleic acid; homo sapiens used grass and seeds rich in gaba and enzymes (sod) as well as resistant starch; modern human used barley grass rich in gaba, flavonoids, sod, k-ca, vitamins and tryptophan; however, barley grains rich in β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, resistant starch, and so on. therefore, barley played an important role in solving the problem of depending functional ingredients of homo sapiens and hominids as well as neanderthals, especially food safety in the process of migration and evolution from ancient humans to modern people. migration. food shortages and survival struggles caused by climate change were the causes of early human evolution of suiting environmental extremes from africa to asia and later to eurasia (figure 2 ) [8, 212] . barley is not only the most widely used cereal crop with comprehensive utilization of forage, materials for intoxicating liquor, functional food, stable food, ornamental weaving, and chinese medicines but also is the crop with the strongest resistance to stress (drought, cold, and salt) for the highest content of functional components, especially the growth period of barley varies from 70 to 200 days, which can be grown in four seasons in the world or at 1,900~2,300 m in yunnan province of china. these excellent characteristics become the best food for human migration. interestingly, there is a striking similarity between the human migration route and barley translocation/evolution route (see figure 2 ). in [215] . the spread of farming peoples of eurasia from the near east (8.0 ka), with movements both westward and eastward, especially ancestor of modern south asians is a mixture between early holocene populations of iran and south asia; however, yamnaya in the bronze age of europe moved both westward and eastward from north of the black sea [216] . discovery of different crushing apparatuses in mountains of iran revealed that people were grinding wheat and barley about 11,000 years ago [217] . these results support the healthy food contribution between human migration and barley translocation/evolution, especially climate change increased functional ingredients in barley for preventive chronic diseases. civilization. this point of view reveals the evolution of human skull morphology on the basis of the hybridization between h. sapiens and other hominin species in morocco at 300,000 years ago, all of which are descendants of the african homo sapiens population [203] . all living people in europe and asia carry the same amount of neanderthal dna due to the interbreeding between homo sapiens and neanderthal that took place in the middle east [193] . fertile crescent is the concentrated area of wild barley [214] and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam ( figure 2 ). barley is one of the oldest crops used by ancient farmers, its cultivation has been optimized by modern humans in the ancient era for less shattering, higher yield and better grains; however, the sharp awn of barley has become an important guarantee for the most resistant birds trouble and largescale farming civilization in cereal crops. climate change stimulated agricultural innovation and exchange across asia between 5,000 and 1,500 years ago; sorghum and millet made their way from china to central asia; wheat and barley moved from central asia to the far east and became a staple food in the north of china at 1.8 ka, which exchanges across central and high-altitude asia coalesced to form the silk road (2.114 ka~1.873 ka) and grand canal for traffic great artery of north-south in ancient china (2.486 ka) [215] . tibetan barley (qingke) is derived from eastern domesticated barley, north pakistan, india, and nepal between 4,500 and 3,500 years ago, which supports a feral or hybridization origin for tibetan weedy barley [218] . the rise of barley against stress (drought, cold, salt, and bird) to staple food has increased functional ingredients (especially gaba) in diet to prevent chronic diseases and promoted human civilization. gaba-mediated inhibitory interneurons control memory-encoding ca1 neurons; nucleus incertus (ni) establishes gabaergic inhibitory synapses on interneurons; ni gabaergic cells can regulate hippocampusdependent episodic memory formation bidirectionally, and its dysfunction may contribute to anxiety-like syndromes [219] . cities and words and metallurgy as well as complex ceremonial buildings are the four standards of world civilization; fertile crescent for one of centers of barley origin is closely related to ancient babylonian and ancient egyptian civilizations, especially ancient babylonian civilizations have the earliest human civilization (agriculture 11 ka, cities 8~10 ka, metallurgy 6~7 ka, words 5.2 ka, calendar 5 ka, and systematic religion 4 ka) in the world; however, ancient egyptian civilizations has the earliest empire (mathematics 5.2 ka, geometry 5 ka, and writing tool 5 ka). the ancestral blocks of the domesticated barley genomes were descended from all over the fertile crescent, especially levantine (western) and zagros (eastern) clusters of the origin of agriculture for nine wild barley populations, i.e., carmel and galilee, golan heights, hula valley and galilee, judean desert and jordan valley, lower mesopotamia, negev mountains, north levant, sharon, coastal plain and judean lowlands, and upper mesopotamia [220] . the neolithic crops facilitated the early agricultural establishment; the barley evolution followed the agricultural development in the near east [213] . for humans from hunter gathering to agriculture about 12 ka of the levant in near east, barley was a founder crop for converting the brittle floral axis of the wild-type into a tough and nonbrittle spike, which made a major contribution to the emergence of early agrarian societies [221] . for ancient indian civilization, the earliest known farming cultures in south asia emerged in the hills of balochistan in pakistan about 9.2 ka; however, seminomadic peoples domesticated wheat, barley, sheep, goat, and cattle [217] . for ancient chinese civilization, the earliest beer recipe in china included broomcorn, millet, barley, job's tears, and tubers around 5,000 y ago, which may have motivated the initial translocation of barley from the western eurasia into the central plain of china [222] . diseases. western medicine solves the issue of human organs, and traditional chinese medicine solves the issue of human body system; however, functional food is to address the problem of human cells. barley grains and its grass not only are the best functional food that provides nutrition and eliminates toxins from cells in human beings, which are rich in 30 ingredients to combat more than 20 chronic diseases but also have all the nutrients needed for cell nutrition and detoxification, which is the result of the long-term coevolution of the dietary structure of ancient apes for plants and early homo sapiens with the staple food of barley. interestingly, the types of prevention and treatment of human chronic diseases by key functional components in barley grain were in order: β-glucans (16)>polyphenols (13)>arabinoxylan (7) = tocols (7)>phytosterols (5)>resistant starch (4), but gaba (13)>flavonoids (11)>sod (8)>k-ca (7) = vitamins (7)>tryptophan (3) in barley grains. the 12 key functional components of barley grains and its grass not only play an important role in the prevention and treatment of more than 20 chronic human diseases but also interact with other 30 nutritional functional components to provide the possibility to solve hundreds of human diseases caused by cell undernutrition and their detoxification disorders. the unique theory and practice system of the whole regulation improve the immunity of chinese medicine to prevent and treat human diseases. with advances in science and technology, barley will also find many behavioral mechanisms that combat human diseases, such as barley malt has been used in many prescriptions for the prevention and treatment of covid-19 in lots of provinces in china, which includes jiangxi, guangdong, gansu, guizhou, and jiangsu province in china [223] . dietary restriction is an activator of prolongevity molecular pathways based on an escape from costs incurred under nutrient-rich conditions [224] . chronic disease deaths due to heredity, environment, and lifestyle are as high as 41 million, accounting for more than 70% of all deaths worldwide [225] . these results support that human has only new cell disease theory, which are made up of sixty million cells, more than 1,000 diseases are due to cell nutritional deficiencies and detoxification disorder caused by the disease [226] . ingredients of barley were preserved in the chinese crop genebank. the detection of functional ingredients in grains and grass powder of more than 10,000 accessions barley gremplasm especially combinated with high-density snp markers can not only reveal the molecular mechanism of functional ingredients and their molecular breeding and gene editing techniques but also make great contributions for improving human health by increasing functional ingredients in barley grains and grass powder. ingredients in barley. some excellent barley varieties have very high functional ingredients both in grains and grass powder, which are the result of adaptation to high and low temperature, drought and waterlogging, long and short sunshine, and day and night changes caused by altitude, latitude, and seasonal differences. first of all is the breeding of high functional ingredients in barley grains. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (β-glucan >8.5%, resistant starch >10%, arabinoxylan >2%, or polyphenols >0.2%) and cultivated barley at low temperature and drought as well as high altitude, such as yunke 1 and zangqing 25 with high β-glucan, yungongmai 1, and yungongmai 2 with high vitamin c. secondly is the breeding of high functional ingredients in barley grass. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (gaba >0.2%, k >3.0%) and cultivated barley at low temperature and drought as well as high altitude. the average content of gaba in 31 cultivars that we bred is 271.3 mg/100 g which is 1.8 fold higher than that of other barley crops around the world, especially barley grass powder of yungong brand contains 62 times gaba (327.5 mg/100 g) and 99 times ca as well as 31 times k than those of polished rice [9] . in addition, the functional ingredients of barley grain and grass powder can also be greatly improved by gene editing technology, which needs to be further studied and enriched in the future. functional ingredients of barley. functional ingredients of barley grain and its grass powder have larger variation due to latitude, altitude, season, light, temperature, water, day, and night. all the high-altitude (1,200~3,500 m) hulless barley can increase higher functional ingredient content than that of plains (97~126 m altitude) [25] . therefore, it is necessary to promote the functional components of the barley grain and its grass powder under the best ecological conditions, and we also make a useful exploration of the functional components at different altitudes and in different seasons. in addition, the functional ingredients of different parts of barley grain (see table 1 ) and the functional ingredients of different grass cutting stages were different. barley is the oldest and the richest functional food among global cereals. its grains are rich in β-glucan; polyphenols (phenolic acids, flavonoids, and anthocyanins), polysaccharide (arabinoxylan), phytosterols (β-sitosterol, campesterol), tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, α-tocopherol), resistant starch, alkaloid, gaba, folates, linoleic acid, phytate, and so on. this review paper summarizes the obvious efficacy of barley grains that includes antidiabetes, antiobesity, anticancer, antioxidants, anti-inflammation, immunomodulation, cardioprotection, gastroprotection, and hepatoprotection properties, and also, barley grains can lower blood pressure; prevent cardiovascular diseases; optimize cholesterol; improve bowel health and metabolic syndrome; prevent heart disease; reduce chronic kidney disease; decrease stroke; alleviate allergic rhinitis and atopic dermatitis; and accelerate wound healing activities. barley grains, grass, straw, husk, bran, and fine powder are rich in 30 ingredients and food structure to defeat chronic diseases during human migration, especially molecular mechanisms of six functional ingredients barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch) involve to combat more than 20 chronic diseases. these results suggest that barley plays an important role in a healthy diet and in the promotion of early human intelligence. in particular, the healthy effects of functional components of barley grains and grass are the result of longterm continuous evolution of early hominids (fruits/vegetables and leaves rich in polyphenols, k-ca, and vitamins), neanderthals (mushrooms and nuts rich in polysaccharides, phytosterols, and linoleic acids), and homo sapiens (grasses and seeds rich in gaba, enzymes, and resistant starch), which associate with modern humans originating in the progenitor of african homo sapiens with cognitive hominin, especially after interbreeding between homo sapiens and neanderthals that took place in the middle east. the migration route from africa to asia and then to eurasia is basically consistent with the origin and spread of barley and its domestication path, which indirectly supports that barley against stress (drought, cold, and salt) enriched with functional ingredients prevented chronic disease from ancient humans to modern people. fertile crescent is the concentrated area of wild barley and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam. these results indirectly support this great contribution of barley for promoting world civilization. the polyphenols in fruits/leaves and polysaccharide in mushrooms/nuts as well as gaba in grass/seeds for prevention of chronic disease are associated with depending functional ingredients for diet from pliocene hominids in africa to modern humans. ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley. food shortages and survival struggles caused by climate change were the causes of early human evolution associated with gaba in the barley grass increased 17 oxidative medicine and cellular longevity sharply under environmental extremes from africa to asia and later to eurasia, especially gaba in crop diets suiting environmental extremes improved intelligence. these results support findings that barley and its grass may be the best functional food crop, especially barley prevents over 20 human chronic diseases based on six functional ingredients of barley grass and grains due to three aspects of the scientific basis, i.e., the similar origin and evolution center of barley and human, the promotion of human intelligence in the early stage, and the sum of staple foods for early hominids and neanderthals as well as homo sapiens. we put forward the strategy of increasing the functional ingredients of barley grain and its grass powder that is as follows: (1) exploration of excellent germplasm with high functional ingredients in barley; (2) breeding excellent cultivars with high functional ingredients in barley; (3) optimization of ecological conditions for high functional ingredients of barley. in addition, the functional ingredients of different parts of barley grain and the functional ingredients of different grass cutting stages are different. although therapeutic mechanisms of functional ingredients in barley grains and grass powder for prevention of human chronic diseases seem a very complicated task, and functional food for therapeutic interventions opens up new ways, it is necessary to find further scientific evidence that demonstrates the health effects of functional ingredients of barley and their extracts from barley on the treatment of chronic diseases. barley is one of the most exciting potential natural sources for the development of functional foods and new drugs with improved efficiency and safety. although we have found some relationship of origin and migration between human and barley, especially preventive role of barley for chronic diseases of human beings, it is necessary to conduct more systemic studies to unravel coevolutionary interconnection mechanism between chronic diseases prevention and human diet for barley functional foods. unfortunately, so far there, 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7(s)-hydroxymatairesinol protects against tumor necrosis factor-α-mediated inflammation response in endothelial cells by blocking the mapk/nf-κb and activating nrf2/ho-1 lignans 7-hydroxymatairesinol and 7-hydroxymatairesinol 2 exhibit anti-inflammatory activity in human aortic endothelial cells hyperglycemia-induced oxidative stress and heart disease-cardioprotective effects of rooibos flavonoids and phenylpyruvic acid-2-o-β-d-glucoside arabinoxylan attenuates type 2 diabetes by improvement of carbohydrate, lipid, and amino acid metabolism evidence-based review of biobran /mgn-3 arabinoxylan compound as a complementary therapy for conventional cancer treatment structure elucidation of an immunostimulatory arabinoxylan-type polysaccharide prepared from young barley leaves (hordeum vulgare l.) dietary steamed wheat bran increases postprandial fat oxidation in association with a reduced blood glucose-dependent insulinotropic polypeptide response in mice the impact of phytosterols on the 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for human health human diet and health how did homo sapiens evolve? barley as a green factory for the production of functional flt3 ligand molecular farming in barley: development of a novel production platform to produce human antimicrobial peptide ll-37 ectopic expression of human acidic fibroblast growth factor 1 in the medicinal plant, salvia miltiorrhiza, accelerates the healing of burn wounds genebank genomics highlights the diversity of a global barley collection new fossils from jebel irhoud, morocco and the pan-african origin of homo sapiens middle stone age foragers resided in high elevations of the glaciated bale mountains, ethiopia assembling human brain organoids intense threat switches dorsal raphe serotonin neurons to a paradoxical operational mode die ersten menschen co-evolution of methods and thoughts in cereal domestication studies: a tale of barley (hordeum vulgare) genomic analysis of 6,000-year-old cultivated grain illuminates the domestication history of barley climate change stimulated agricultural innovation and exchange across asia the formation of human populations in south and central asia world history / ancient civilizationsseptember 2019 origin and evolution of qingke barley in tibet conducting memory formation targeted resequencing reveals genomic signatures of barley domestication evolution of the grain dispersal system in barley revealing a 5,000-y-old beer recipe in china remarkable efficacy of covid-19 treatment by traditional chinese medicine the hidden costs of dietary restriction: implications for its evolutionary and mechanistic origins are noncommunicable diseases communicable? never be sick again: health is a choice, learn how to choose it genetic analysis of four main flavonoids in barley grain analysis of functional ingredients in barley grains from different regions between southwest china and icarda nutrition analysis and food process of barley the research and publication of this article was funded by china agriculture research system (cars-05-01a, cars-05-02b) and yunnan provincial expert grass-roots scientific research workstation. key: cord-252810-rko3e5va authors: basil, maria c.; katzen, jeremy; engler, anna e.; guo, minzhe; herriges, michael j.; kathiriya, jaymin j.; windmueller, rebecca; ysasi, alexandra b.; zacharias, william j.; chapman, hal a.; kotton, darrell n.; rock, jason r.; snoeck, hans-willem; vunjak-novakovic, gordana; whitsett, jeffrey a.; morrisey, edward e. title: the cellular and physiological basis for lung repair and regeneration: past, present, and future date: 2020-04-02 journal: cell stem cell doi: 10.1016/j.stem.2020.03.009 sha: doc_id: 252810 cord_uid: rko3e5va the respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. in this review and as members of the nih/nhlbi-supported progenitor cell translational consortium, we discuss key aspects of lung repair and regeneration. we focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. we also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues. the respiratory system is organized into multiple integrated compartments comprising multiple tissues that perform gas exchange between the blood and the external environment. the various anatomical regions of the respiratory tract are populated by numerous types of unique epithelial, vascular, mesenchymal, and immune cells critical for the functioning of each particular compartment. historically, the development of the respiratory system has been thought to involve several discrete morphogenetic steps including lineage specification, branching morphogenesis, sacculation, and alveologenesis (morrisey and hogan, 2010) . while these steps were previously conceived of in terms of distinct temporal stages of development, more recent evidence has suggested that there is overlap between these stages and particular events such as cell specification and commitment, which are now thought to occur very early and coincident with the basic patterning of the respiratory airway tree (frank et al., 2019) . the branched network of airways and gas exchange surfaces co-develops with the cardiovascular system to bring both organ systems into intimate proximity for full functionality. more details on these important developmental events can be found in several recent reviews (herriges and morrisey, 2014; hines and sun, 2014; morrisey and hogan, 2010; nikoli c et al., 2018; whitsett et al., 2019; zepp and morrisey, 2019) . the culmination of these events is the generation of an extensive surface area for efficient gas exchange that in the human lung comprises approximately 70 m 2 . this review will focus on how the mature respiratory system maintains its normal homeostatic structure and function and how it responds to injury and regenerates itself. we will explore the cellular constituents of the two major compartments in the lungs-the gas exchange alveoli and the conducting airways including the trachea-and describe established and emerging techniques to explore human lung regeneration. the lung alveolus is composed of multiple epithelial, endothelial, and mesenchymal cell types (figure 1 ). in addition to these resident cell types, the alveolus also is inhabited by several immune cell lineages, including alveolar macrophages, interstitial macrophages, and dendritic cells and several recent datasets have shown this diversity of cells at single-cell resolution in both animals and humans (guo et al., 2019; travaglini et al., 2019; vieira braga et al., 2019) . emerging data suggest there is some degree of inter-cellular communication between the lineages in this niche, but our understanding of the crosstalk among alveolar cell lineages during homeostasis or regeneration remains poor. the alveolar compartment remains largely quiescent in the uninjured lung, and most cells within this niche exhibit a relatively slow turnover. after lung injury, multiple alveolar cell types are able to proliferate, and when repair is effective both alveolar structure and function are restored. this ability to react to injury involves both activation of self-renewal as well as differentiation into more mature cell lineages. the self-renewal and differentiation of various lung epithelial cells are modulated by a growing list of cell types that includes neighboring epithelial cells, mesenchymal cells, airway smooth muscle, neurons and neuroendocrine cells, endothelium, and various leukocyte populations (barkauskas et al., 2013; cao et al., 2017; lechner et al., 2017; lee et al., 2017; rafii et al., 2015; zepp et al., 2017) . these studies have highlighted recurrent themes regarding the signals that can drive alveolar epithelial regeneration, including wnt signaling. alveolar epithelial response to injury. there are two primary lineages in the alveolar epithelium: the alveolar epithelial type 1 (at1) and type 2 (at2) cells (figure 1 ). at1 cells cover 95% of the alveolar surface area and are closely juxtaposed with the capillary plexus. at2s are responsible for generating pulmonary surfactant, which is essential for reducing the surface tension of the alveolar surface area to prevent the lungs from collapsing upon every breath. at1 and at2 cells are specified very early in lung development and at2 cells do not appreciably generate figure 1 . alveolar cell lineages involved in lung repair and regeneration (a) the human distal airways connect with the alveolar niche through a transitional respiratory airway (also called the respiratory bronchiole or rb) region. the rb is lined with a simple but poorly characterized cuboidal epithelium while the more intermediate airways exhibit a pseudostratified epithelium containing secretory, goblet, and ciliated cells that may exhibit as yet distinct heterogeneity. of note, basal cells are found in human intermediate and respiratory airways. (b) mice do not have respiratory bronchioles and transition from the intermediate airways, which exhibit a pseudostratified nature but lack basal cells, into the alveolar region. the distal badj region in the mouse lung, which is not found in the human lung, contains the basc population. the architecture and cell lineages found in both the mouse and human lungs are very similar and contain both at1 and at2 epithelial lineages as well as various mesenchymal lineages and vascular endothelial cells. (c) the various cell types found in the distal airways and alveolus of the human and mouse lung. at1 cells during early postnatal lung growth (frank et al., 2016 (frank et al., , 2019 . however, in adult mice, at2 cells can act as both a selfrenewing stem cell like population and regenerating at1 cells after injury (barkauskas et al., 2013) . a sublineage within the at2 cell population that expresses the transcriptional target of wnt signaling, axin2, has been shown to play a dominant role in repairing the lung alveolus after acute injury (nabhan et al., 2018; zacharias et al., 2018) . these cells, which have been called alveolar epithelial progenitors or aeps, preferentially re-enter the cell cycle after injury, self-renew, and regenerate mature at1 and at2 cells. aeps appear primed to enter the cell cycle and they respond robustly to wnt and fgf7 signaling (zacharias et al., 2018) . in alveolar development, wnt-responsive at2 cells selfrenew in the presence of wnt signaling and differentiate in its absence (frank et al., 2016) , but it is not yet known whether these same signals drive renewal versus differentiation during regeneration. a recent report also shows that aeps promote metastasis in models of lung cancer (laughney et al., 2020) . aeps have been identified in the human lung and are responsible for generating the majority of at2 cell growth in human alveolar organoids (zacharias et al., 2018) . in contrast to at2s and aeps, the at1 cell contribution to alveolar epithelial regeneration is thought to be very limited (jain et al., 2015) . following the surgical removal of lung tissue in adult mice and other model organisms, new alveoli are formed to compensate for lost alveolar surface area (buhain and brody, 1973) . this compensatory lung growth after partial pneumonectomy is commonly used as a ''sterile'' model of lung regeneration. not surprisingly, this regenerative response involves the coordinated actions of nearly every cell type in the lung including epithelial cells, endothelial cells, mesenchymal cells, and leukocytes (chen et al., 2012; jain et al., 2015; lechner et al., 2017; rafii et al., 2015) . in this model, a small number of cells expressing hopx + , a marker for the at1 lineage, were found to both proliferate and, in rare instances, give rise to sftpc + at2 cells (jain et al., 2015) . a recent study revealed heterogeneity within the at1 cell population where expression of igfbp2 marked the most mature at1 cell subtype that lacks differentiation capacity following pneumonectomy . although cells expressing at1 markers proliferate during compensatory lung growth after partial pneumonectomy, whether bona fide at1 cells are able to contribute to repair after acute lung injury is largely unknown. alveolar endothelial response to injury. effective gas exchange is dependent upon at1 cell and pulmonary capillary endothelial cell (pcec) proximity, and successful alveolar regeneration requires re-establishment of this spatial relationship. following lung injury in rodents, there is rapid proliferation in the microvasculature, with expansion of resident microvascular endothelial progenitor cells . these cells are marked by cd34 and cd309, and in organoid culture demonstrate significant vasculogenic capacity. while both the mechanisms of pcec regeneration and cellular identities within this compartment are incompletely understood, endothelial cells expressing sox17 have recently been shown to play a role in endothelial regeneration after endotoxic-induced vascular injury (liu et al., 2019a) . additionally, pcecs enhance alveologenesis following injury, with pcec-derived vegfr2 and fgfr1 mediating epithelial proliferation (ding et al., 2011) . this signaling is thought to be through mmp14, and possibly co-mediated through platelet activation of endothelial sdf-1 receptors (rafii et al., 2015) . significant additional heterogeneity is thought to exist within the distal lung endothelium, with differential vasculogenic capacities and crosstalk with the epithelium , and this has recently been revealed in single-cell transcriptomic studies of the homeostatic lung and its response to acute injury (niethamer et al., 2020) . this study demonstrated the presence of multiple microvascular endothelial subsets including one which expresses high levels of car4 and cd34. this study also showed that a population of proliferating endothelial cells emerges soon after influenza injury and single-cell informatic trajectory analysis suggests that these cells arise from multiple endothelial subsets. future work is needed to define the functional role for these endothelial subsets in both normal alveolar homeostasis and the response to injury. alveolar mesenchymal response to injury. the lung alveolus is also home to a complex mixture of mesenchymal cell types, many of which are in close physical association with both alveolar epithelial and endothelial cells and play an active role in alveolar epithelial regeneration. pioneering electron microscopy studies demonstrated direct and extensive contacts between lung fibroblasts and at2 cells (sirianni et al., 2003; walker et al., 1995) . mesenchymal cells expressing the platelet-derived growth factor alpha (pdgfra) are often found in close association with at2 cells (barkauskas et al., 2013; green et al., 2016; zepp et al., 2017) . the first functional evidence of trophic interactions between these populations was the observation that pdgfra + fibroblasts support the growth and differentiation of at2s in an alveolar organoid co-culture assay (barkauskas et al., 2013) . alveolar organoids have been recently used to provide functional evidence that multiple signaling pathways originate in pdgfra + cells to influence at2 cell self-renewal and differentiation into at1 cells including fgf7, bmp, and il6 (chung et al., 2018; green et al., 2016; zepp et al., 2017) . as molecular techniques have evolved, an emerging literature has revealed molecular and functional heterogeneity of lung alveolar mesenchymal cells. in particular, accumulating data demonstrate the previously underappreciated heterogeneity of cells within the pdgfra + population of fibroblasts in the adult (green et al., 2016; zepp et al., 2017) . single-cell transcriptome analysis combined with spatial distance mapping recently demonstrated that one subpopulation of fibroblasts, defined by the co-expression of axin2 and pdgfra, is localized particularly close to at2 cells and provides signals including il6, fgfs, and bmp antagonists that promote the selfrenewal and differentiation of at2s (zepp et al., 2017) . this fibroblast population has been named the mesenchymal alveolar niche cell or manc and these cells are thought to have a critical role in homeostatic alveolar regeneration following injury (zepp et al., 2017) . immune response to alveolar injury. resident and circulating leukocytes are thought to also play a critical role in alveolar repair and regeneration, with elegant in vivo and in vitro studies demonstrating that ''inflammatory'' cytokines have direct effects on the proliferation and differentiation of both airway and alveolar epithelial cells (danahay et al., 2015; katsura et al., 2019; kuperman et al., 2002; tadokoro et al., 2014; xie et al., 2018) . however, our understanding of the interactions between alveolar epithelial cells and resident or circulating leukocytes is in its infancy. macrophages, the primary resident immune cell of the alveolus, begin to populate the lung during embryonic lung development (tan and krasnow, 2016) . lps stimulation of these early resident macrophages leads to impaired branching morphogenesis, attributed to changes in integrin, bmp, and wnt signaling (blackwell et al., 2011) . whether unstimulated resident macrophages have a normal role in branching morphogenesis or the differentiation of alveolar epithelial cells remains to be elucidated. however, there are multiple lines of evidence indicating that leukocytes play important roles in adult alveolar regeneration, mediated at least in part through bidirectional intercellular communication with alveolar epithelial cells. for example, following either chemical or infectious lung injury, resident alveolar macrophages can stimulate epithelial proliferation through the production of wnt ligands (hung et al., 2019) . a subset of alveolar macrophages can also act as memory macrophages and help guide the rapid activation of multiple chemokines including those that stimulate neutrophils . compensatory lung growth after partial pneumonectomy provides a relatively simple model to study pro-regenerative epithelial-immune interactions without the confounding effects of infection and inflammation. this model was used to demonstrate that platelets can initiate a regenerative cascade by secreting sdf1 after pneumonectomy. this stimulates capillary endothelial cells to express mmp14, releasing egf ligands from the extracellular matrix and subsequently promoting at2 cell proliferation and differentiation (rafii et al., 2015) . following partial pneumonectomy, local production of the chemokine ccl2 leads to the recruitment of ccr2 + monocytes to the lung (lechner et al., 2017) . these monocytes and resident macrophages can be polarized by il13 that is secreted by type 2 innate lymphoid cells, and loss of this axis impairs optimal compensatory lung growth after pneumonectomy. data from an in vitro co-culture assay suggest that macrophages can directly modulate at2 cell survival and self-renewal (lechner et al., 2017) . recruited ccr2 + monocytes have also been implicated in dysplastic alveolar repair following both bleomycininduced lung injury and other models of lung fibrosis (misharin et al., 2017; venosa et al., 2019) . these studies highlight the context-dependent roles of ccr2 + monocytes in both normal and abnormal lung regeneration. in infectious and more destructive lung injury models, it can be difficult to discern the inflammatory roles of leukocytes from regenerative roles, if such a distinction exists. nevertheless, data from a growing number of contexts have shown that resident and recruited immune cells are essential for resolution of lung injury. for example, the depletion of macrophages during the resolution phase of bleomycin-induced lung injury prolonged the fibrotic response and impaired resolution (gibbons et al., 2011) . this effect was attributed to a decrease in the clearance of accumulated extracellular matrix but could also involve disrupted communication with epithelial progenitor cells or other populations. other immune populations are activated or recruited to the alveolar niche following lung injury. we have nascent understandings of the inflammatory cellular diversity and intercellular communications that determine normal versus abnormal lung regeneration in response to lung injury. understanding this diversity and improving model systems, including more precise animal models and organoid and lung-on-a-chip models that incorporate immune cells, will allow us to study the contributions of immune cell communications that drive lung repair (gkatzis et al., 2018) . maintenance and regeneration of the proximal airway epithelium. the proximal airways in mice and humans are exposed to frequent insults from the environment and serve as the first line of immune and toxin defense in the lung, warming and filtering the air as it passes to more distal regions. much of what is understood about airway regeneration comes from studies of the mouse trachea, which most closely resembles the structure of human proximal airways. there are many parallels between the murine trachea and the human intrapulmonary airways, with the most relevant for this discussion being the presence of basal cells. in mice, basal cells reside in the trachea and proximal main stem bronchi; however, in humans this population extends for several airway generations (figures 1 and 2) . murine intrapulmonary airways are not pseudostratified and do not contain basal cells which are the primary stem cell population in human airways. thus, intrapulmonary mouse airways should not be used as a model system for the study of human airways. the pseudostratified upper airway and tracheal epithelium exhibits very slow turnover during health but several of the mature lineages are capable of re-entering the cell cycle to replenish loss of neighboring cells and maintain an epithelial barrier. basal cells in the proximal airways are the major stem cell population that selfrenew and when necessary give rise to multiple cell types such as secretory, goblet, and multi-ciliated cells (figure 2 ; hegab et al., 2012; hong et al., 2004; rock et al., 2009 rock et al., , 2011 . this process is critical for both maintenance and cellular regeneration after significant injury and is controlled by notch signaling (mori et al., 2015; rock et al., 2011; ruiz garcã­a et al., 2019; stupnikov et al., 2019) . although it was once thought that basal cells were a rather homogeneous population, recent findings reveal more complexity and have demonstrated their early origins in development . careful lineage tracing of cytokeratin 5 (krt5) basal cells over time indicated that at least two populations of basal cells exist in the upper airway, with one acting more as a self-renewing stem cell, and the other committed to luminal differentiation (watson et al., 2015) . consistent with this paradigm, depending on enhanced notch2 signaling or c-myb expression, basal stem cells directly give rise to secretory cells or multi-ciliated cells, respectively (pardo-saganta et al., 2015) . recent studies have uncovered additional complexity within the pseudostratified airway epithelium of mouse trachea and human large airway, with the description of new, rare cell types including cftr-rich ionocytes plasschaert et al., 2018) . multiple studies using lineage tracing analysis combined with single-cell transcriptomic work confirmed that the krt5 basal cell population was capable of giving rise to all the observed cell types within the airway, including the newly identified ionocyte, and other rare epithelial cell subsets such as the tuft cell, which is not normally present in uninjured mouse airways mori et al., 2015; rane et al., 2019; rock et al., 2011; ruiz garcã­a et al., 2019) . moreover, tuft cells, also known as solitary chemosensory cells, ectopically emerge after influenza injury in the mouse and may play a role in post-injury dysplastic remodeling of the lung (rane et al., 2019) . some of these lineage relationships in mice can be modeled in vitro with primary human cells in culture, highlighting the shared regenerative potential across species (rock et al., 2009) . while basal cells are a main driver of regeneration after airway injury, other cell types have been shown to contribute as facultative progenitors. lineage tracing analysis of airway scgb1a1 + cells revealed that secretory cells proliferate to help maintain the club cell population ( figure 2 ; rawlins et al., 2009; van keymeulen and blanpain, 2012) and are a major source of multi-ciliated cells in normal airways, particularly in the more distal murine airways where basal cells are not typically found. in addition, subsets of secretory cells expressing upk3a in mice, so-called variant-club cells, have been shown to be localized near neuroendocrine bodies, suggesting a possible niche, and are capable of giving rise to both secretory and ciliated cells in development; however, a role in the response to injury is not yet clear (guha et al., 2012 (guha et al., , 2017 . additionally, neuroendocrine (ne) cells also can function as facultative progenitors after airway injury, interact with immune lineages during expansion, and may harbor sublineages with enhanced progenitor capacity in ne bodies (branchfield et al., 2016; garg et al., 2019; ouadah et al., 2019) . in extreme situations, mature secretory/club cells (defined by scgb1a1 expression) have been reported to dedifferentiate in the setting of marked basal cell loss, and contribute to the basal stem cell pool, although the homeostatic or physiologic role of this in injury is not clear (tata et al., 2013) . in addition, a novel pathway of basal cell repletion from the submucosal gland was also reported by two different groups using lineage tracing and multiple airway injury models (lynch et al., 2018; tata et al., 2018) . these investigators found migration of glandular myoepithelial cells into the airways with subsequent differentiation to mature airway lineages including basal cells. a number of distinct, small populations of progenitor cell types have been reported to contribute to regeneration of distal airway epithelia after injury in mice, which lacks basal cells (barkauskas et al., 2013; bertoncello and mcqualter, 2010; chen, 2017; perl et al., 2005) . one of the first was a variant club/ secretory cell (v-club cells) defined by its location near neuroendocrine bodies and low cytochrome cyp2f2 expression, making these cells resistant to naphthalene injury and thus a source of regenerative cells after this injury (giangreco et al., 2009; hong et al., 2001) . more recently upk3a was identified as a unique marker for v-club cells. a upk3a creer lineage trace (guha et al., 2017) demonstrated that these cells give rise to club cells and ciliated cells during homeostasis and after naphthalene airway injury, as predicted from prior studies (giangreco et al., 2009; hong et al., 2001; volckaert et al., 2011) . although seemingly limited, in these studies some vclub/secretory cells were also noted to differentiate into at2 cells during bleomycin-induced alveolar injury, implying a capacity for mobilization and distal migration. a second small population of stem/progenitor cells was identified at the branch point between distal murine airways and alveolus and was termed bronchoalveolar stem cells (bascs). bascs were initially characterized by dual expression of the secretory cell marker scgb1a1 and the at2 marker sftpc by immunostaining and their localization at the bronchioalveolar duct junction (badj) (kim et al., 2005) . stem cell antigen-1 (sca1) was also proposed as a marker for these cells and has been used in flow cytometry to purify these cells for in vitro culture studies (raiser and kim, 2009 structure of the pseudostratified large airways and trachea of the mouse lung showing submucosal glands which are lined with both myoepithelial cells and other luminal secretory lineages. the smaller airways of the mouse lung contain various luminal secretory and ciliated epithelial lineages as shown. some of these including the bascs and the h2-k1 high secretory cell subtypes have been proposed to generate alveolar epithelium after severe injury. after naphthalene injury and peri-junctional at2 alveolar cells after bleomycin injury or influenza injury (liu et al., 2019b; salwig et al., 2019) . one of the limitations of these studies is the use of scgb1a1 expression to mark bascs, as this gene and protein are known to be expressed in a subset of at2 cells, which could confound such lineage tracing . while the genetic depletion of the basc population resulted in a delay of the murine regenerative response to injury, full regeneration was eventually observed, suggesting that bascs are not absolutely required for lung regeneration. an additional distal airway stem/progenitor cell population with regenerative potential was identified descriptively as lineage-negative epithelial progenitors (lneps), also referred to as distal airway stem cells (dascs). these cells are distinct from v-club/secretory cells and bascs. lneps were originally defined as distal airway integrin b4 + /cd200 + cells without discernible mature lineage markers by protein immunostaining (vaughan et al., 2015) . these cells are also sox2 + , as expected for an airway epithelial cell. subsequently it became apparent that lneps/dascs are composed of both trp63 + cells and trp63 ã� cells. the trp63 + cells appear to be holdovers from embryonic trp63 + basal cells and some 20% of these cells could be traced with the scgb1a1 creer mouse line . although true basal cells are not normally found in the distal mouse airway, the rare krt5 + /trp63 + lneps/dascs expand and mobilize after influenza injury to generate collections (or pods) of krt5 + basal-like cells throughout the heavily damaged areas of influenza-injured mice (vaughan et al., 2015; xi et al., 2017; zuo et al., 2015) . this mobilization is initially protective to the mouse but ultimately is a dysplastic response as krt5 + basal cells have limited potential to differentiate into at2 cells and the mouse becomes permanently burdened with airway-like cystic structures throughout the alveolar compartment. appearance of these permanent cystic structures was linked with activated notch signaling, and blockade of hypoxemia response via deletion of hypoxia inducible factor 1 (hif1a) in airway cells promotes the contribution of airway cells to regeneration of at2s and subsequent improvement in oxygen saturation (xi et al., 2017) . contribution of distal airway progenitors to alveolar repair. although endogenous at2s represent the primary regenerative responders to various alveolar insults and regenerate the overwhelming number of new at2 and at1 cells after limited alveolar damage, several lines of evidence implicate activation of distal airway epithelial cells as an alternative source of alveolar epithelial cells following certain types of severe lung injury (barkauskas et al., 2013; chapman et al., 2011; xi et al., 2017) . recently, rare mhc high (h2-k1 high ) club cell-like progenitors have been described within the larger scgb1a1 lineage-traced population of all lung cells. these cells have proliferative capacity, appear to give rise to at2 and at1 cells following bleomycin-induced lung injury, and can be purified by flow cytometry using anti-h2-k1 antibodies (kathiriya et al., 2020) . h2-k1 high cells are a subpopulation of b4/cd200 cells, express low or no mature lung epithelial lineage markers (e.g., scgb1a1 or sftpc) at the protein level, and represent $5% of all scgb1a1 creer -labeled lung cells, but exhibit clonogenic potential to generate these broader populations in cell culture outgrowth assays (kathiriya et al., 2020) . h2-k1 high cells were found to survive and differentiate into at2 and at1 cells in vivo after intra-airway transplanta-tion into bleomycin-injured mice. these cells were also found to be specifically targeted by h1n1 pr8 influenza virus, consistent with a prior report showing b4 + /cd200 + cells as a primary target of the viral injury (quantius et al., 2016) . the alveolar epithelial differentiation capacity of h2-k1 high progenitors after bleomycin injury contrasts sharply with the differentiation repertoire of the rare trp63 + cells which predominantly gives rise to dysplastic krt5 + pods, but virtually no alveolar epithelial cells (ray et al., 2016; vaughan et al., 2015; xi et al., 2017) . additional studies combining injury models with modern cell lineage tracking techniques and single-cell analysis are needed to clarify the injuryspecific activation of airway cells and their relative contributions toward alveolar regeneration. importantly, whether bascs, lneps, and h2-k1 high progenitors represent similar or overlapping populations of cells remains unclear. future studies will need to directly compare these distal airway cells to the resident at2 and aep cell populations, to more fully assess their ability to repair and regenerate the alveolar niche after acute injury and in chronic diseases. contribution of the mesenchyme to airway regeneration. a series of complex and interconnected interactions are employed to maintain distal airway epithelium during quiescence and after injury within the local niche. the local niche is comprised of the extracellular matrix (ecm) and several mesenchymal cell types. specifically, the mesenchyme plays a critical role in function of distal airway epithelium by providing a number of signaling cues that ultimately determines the stem cell response during injury. much of what is known about the interplay in adults between the mesenchyme and the epithelium has been derived from studying lung development. these developmental signaling pathways include wnt/b-catenin, fibroblast growth factor 10 and its receptor fgfr2 (fgf10/fgfr), retinoic acid, and sonic hedgehog (shh) members of transforming growth factor b (tgfb) superfamily including bone morphogenetic proteins (bmp), hippo/yes-associated protein (yap), and notch signaling. for example, airway smooth muscle cells promote epithelial repair through the production of fgf10 volckaert et al., 2011 ) and a population of pdgfra + fibroblasts promote the differentiation of multi-ciliated cells through the production of il6 (tadokoro et al., 2014) . in addition to signaling to the airway epithelium during regeneration, the airway mesenchyme can undergo a phenotypic change following injury that drives abnormal regeneration. a recently identified mesenchymal subpopulation identified by axin2 expression but lacking pdgfra expression, the axin2 + myogenic progenitor or amp, becomes activated after naphthalene airway injury, begins to express acta2, and contributes the airway fibrosis in the naphthalene model (zepp et al., 2017) . a more thorough review on these pathways and their cell-specific functions can be found elsewhere (kotton and morrisey, 2014; leach and morrisey, 2018; lee and rawlins, 2018; zepp and morrisey, 2019) . the human lung contains many structural and anatomic differences which make it unique from its murine counterpart. the murine lung has a 6,000-fold smaller tidal volume, an 8,000 times smaller surface area, and roughly half the generations of airways compared to its human counterpart (irvin and bates, 2003; knust et al., 2009; thurlbeck, 1967 ). this significant difference in organ size poses distinct challenges for gas distribution, immune function, and gas exchange. these differences highlight the critical variances that need to be accounted for as the field moves from bench to bedside. the alveolus is one of the most architecturally conserved regions between the murine and human lung (figure 1 ). conservation of the cellular populations between mouse and human alveoli have been demonstrated through multiple techniques and include the above-described at1 and at2 epithelial lineages and the aep sublineage. furthermore, the regenerative capacity of the alveolus appears to be retained across species, as discussed above. the cellular anatomy of trachea and the most proximal airways in mice and the large airways in humans is very similar. the cells of the murine trachea and their progenitor and regeneration capacity appear to be closely aligned with in vitro models of human proximal airway regeneration. however, the distal conducting airway anatomy of the mouse is quite different from humans. in mice, the intrapulmonary airways are almost completely devoid of cartilaginous rings, bronchial blood support, submucosal glands, and pseudostratified epithelium, all of which is in direct contrast to their human counterparts. furthermore, murine conducting airways terminate directly into alveolar sacs at the site of the badj. humans do not have a badj, and instead have a distinct distal airway compartment which includes the respiratory bronchioles. these distal respiratory bronchioles are interdigitated with alveolarlike structures that leads into the larger alveolar compartment. there is no analogous counterpart to the human respiratory bronchioles in mice. terminal and respiratory bronchioles in humans contain krt5 + basal cells that are generally absent in the distal airways of mice. to date, a basc-like cell population has not been found in humans, perhaps due to the lack of the corresponding badj anatomical niche. thus, the cellular origins of distal airway repair and the origin of airway-based stem/progenitors mobilized during alveolar injury in humans are likely different from that of mice. given the significant differences between the human and mouse lung, especially in the distal airways, greater focus is needed to study the human lung, both through descriptive assessment using new techniques including single-cell analysis as well as more sophisticated assays including organoids, ex vivo lung explants, and pluripotent stem cell-derived human lung lineages. idiopathic pulmonary fibrosis (ipf) is the most common adult interstitial lung disease (ild), a class of pulmonary diseases pathologically defined by interstitial fibrosis, inflammation, or the combination of fibrosis and inflammation (lederer and martinez, 2018) . a recognized theory of ipf pathogenesis places the initial site of ''micro-injury'' in the alveolar space with the at2 cell holding an important position in disease development. recent transcriptional interrogation of the distal epithelium in ipf identified activation of cell stress and senescence pathways, and murine modeling of at2 cell dysfunction from expression of either mutant sftpc, loss of telomere function, and increased mechanical tension have provided in vivo proof of concept that disruption of at2 cell homeostasis is a driver of lung fibrosis (katzen et an emerging hypothesis of ipf pathogenesis is that the dysfunctional at2 cell loses its facultative progenitor capacity creating a regenerative void for lung repair. in support of this hypothesis, a cardinal feature of the pathobiology of ipf is bronchiolization, a term coined decades ago to codify the observation by chest pathologists that epithelial cells with airway markers accumulate in the fibrotic regions of human lungs, appearing to extend the junctional region between distal airways and remaining alveoli (chilosi et al., 2002) . micro-honeycomb cysts, another cardinal feature of fibrotic remodeling in humans, are mainly lined by epithelial cells with airway markers, including basal, goblet, and ciliated markers (seibold et al., 2013) . the realization that distal airway stem/progenitors robustly mobilize in mice to occupy alveolar surfaces suggests a parallel process in humans. while potentially protective against loss of tissue integrity, the progenitors arising from distal airways are also subject to signals that direct their differentiation to airway rather than alveolar phenotypes. these include hypoxia and notch signaling (chen, 2017) . once differentiated to airways cells, differentiation to normal at2 or at1 cells may be very difficult and inefficient, potentially accounting for the dysplastic structures that dominate the pathobiology of lung fibrosis (kumar et al., 2011; vaughan et al., 2015) . in this paradigm, the dysplastic epithelial cysts accumulating progressively in fibrotic human lungs represent remnants of failed repair, as discussed above. future efforts to both better understand the development of at2 cell progenitor dysfunction and to minimize the pathway of airway differentiation of activated progenitors within alveoli of humans could be therapeutic. chronic obstructive pulmonary disease chronic obstructive pulmonary disease (copd) is a leading cause of morbidity and mortality worldwide, and prevalence continues to increase across the globe with the continued rise in cigarette use and toxic biomasses (burney et al., 2015) . in contrast to ipf, which is characterized by robust cellular production at the site of injury resulting in a dysplastic, fibrotic pulmonary parenchyma, the cardinal feature of copd in the alveolar space is cellular loss and alveolar simplification, known as emphysema. mechanistic studies in copd, however, have failed to generate novel therapeutics aimed at actual lung regeneration. while much of the work on the deleterious effects of toxin exposures on airway cells has been done in bronchial or upper airway cells, careful anatomic studies have suggested that the site of obstruction is localized to the distal airways and in particular the respiratory airways (mcdonough et al., 2011; koo et al., 2018) . the cellular structure and composition of the distal airway region of the human respiratory system, in particular the respiratory bronchioles, is poorly understood. the final endpoint of emphysema, however, is marked by loss of the alveolar epithelium, which can be studied in murine models. studies have pointed to an increase in senescence in at2 cells and the associated endothelium as a mechanism for the development of copd in certain patient populations and the increased prevalence of the disease with aging (gao et al., 2017) . together with the presence of increased senescence in lung fibroblasts from patients with copd, these studies may suggest an exhausted phenotype in a final common pathway downstream of repeated alveolar repair in the setting of recurring toxic exposure (mâ�¬ uller et al., 2006) . furthermore, defective alveolar epithelial repair has been associated with reduced wnt signaling in the copd microenvironment, and this defective response has been suggested to be downstream of a shift in canonical to noncanonical wnt signaling in the presence of toxic stimuli such as cigarette smoke (baarsma et al., 2017) . whether this change in wnt signaling is associated with an aberrant or absent response by the wnt-responsive aep sublineage in the at2 cell population, remains unclear. more work is needed to understand how toxic injuries incite distal epithelial cell responses in copd, in order to be able to develop therapies aimed at bona fide repair of the alveolus, and therefore clinical improvement. deriving lung epithelium de novo via directed differentiation of escs/ipscs the fact that most types of primary lung epithelial cells have been difficult to propagate as stable phenotypes in cell culture has raised significant hurdles for performing basic mechanistic studies in vitro for human lung lineages. a potential solution to these challenges has emerged with the discovery of techniques to differentiate pluripotent stem cells (pscs) in vitro into a diversity of lung lineages. embryonic stem cells (escs) and their engineered equivalents-induced pluripotent stem cells (ipscs)represent the gold standards of pscs, and a rapidly emerging literature has gradually led to successful methods for controlling their differentiation in cell culture. directed differentiation of pluripotent stem cells. the in vitro differentiation of either escs or ipscs into specific tissue lineages can be guided by adding combinations of growth factors or small molecules to media at specific times during culture (figure 3) , in order to recapitulate the signaling pathways that regulate in vivo organ development (murry and keller, 2008) . the process of recapitulating development in vitro to sequentially pattern pluripotent stem cells toward desired fates is termed ''directed differentiation'' and has been successfully applied for deriving multiple cell types from escs/ipscs, including lung epithelia . to date most of the cell types produced from esc/ipsc have an immature phenotype and are not yet ready for clinical applications. initial attempts at deriving lung epithelium from pscs were inefficient, stochastic, used incompletely defined media, or relied on the presence of drug-resistance genes (coraux et al., 2005; van haute et al., 2009; wang et al., 2007) . the difficulty in part was due to a lack of information regarding normal lung development in vivo. as basic mechanisms were discovered that regulate the formation of definitive endoderm, an ability to derive lung epithelia from pscs in vitro via sequential differentiation into definitive endoderm and subsequent patterning into foregut endoderm followed (green et al., 2011; kubo et al., 2004) . the most successful of these strategies to date has involved the exogenous addition of growth factors and inhibitors at specific times and concentrations to mimic the progressive cell signaling between the endoderm and mesoderm that specify definitive endoderm, pattern the endoderm anteriorly and then ventrally, ultimately inducing nkx2-1 + respiratory progenitors that differentiate into lung epithelium (green et al., 2011; longmire et al., 2012; mou et al., 2012; rankin et al., 2016) . a variety of studies to date indicate that the key step of initial specification of the lung epithelial lineage from foregut endodermal precursors generated from pscs in vitro can be monitored by assessing the kinetics and efficiency of expression of nkx2-1 or reporters for this locus (gotoh et al., 2014; green et al., 2011; longmire et al., 2012; mou et al., 2012; rankin et al., 2016) . these studies indicate that lineage specification requires the precise temporal activation of wnt, bmp, and ra signaling (huang et al., 2014; rankin et al., 2016; serra et al., 2017) . although a high degree of agreement exists on strategies to generate lung-specified anterior foregut endoderm (afe), the generation of mature lineages is less straightforward. similar to other organs and tissues, the generation of fully mature cells remains challenging and is a major limitation to the application of human psc-derived cells for disease modeling and regenerative medicine (lancaster and knoblich, 2014) . nevertheless, substantial progress has been made in recent years and some guiding principles have emerged. in initial studies, differentiation of psc-derived lung progenitors was performed in adherent 2d the key signaling factors and the main stages of the in vitro derivation of lung epithelial cells are provided. following lung specification, late withdrawal of chir gives rise to mature at2 cells (dark red). early withdrawal of chir gives rise to airway progenitors (light blue) or a mixture of mature airway cells (dark blue) and at2 cells, depending on the protocol used. cultures, both in mouse (longmire et al., 2012; mou et al., 2012) and human (firth et al., 2014; huang et al., 2014 huang et al., , 2015 mou et al., 2012) . this led to cultures containing a mixture of cells of undetermined maturity, but including cells expressing markers of at2 cells. however, expression of sftpc, one of the most specific markers of at2 cells, was sparse. culture of the same cells on thin slices of human decellularized lung matrix induced strong expression of sftpc, suggesting that sfptc expression and hence emergence of at2 cells was facilitated by a 3d environment (huang et al., 2014) . furthermore, cultures in 3d conditions, typically matrigel, induced sftpc expression accompanied by emergence of a mature at2 program that includes production of surfactant proteins and phospholipid (gotoh et al., 2014; jacob et al., 2017; yamamoto et al., 2017) . culture systems have now been developed where at2 cells can be propagated as spheroids in 3d matrigel cultures in presence and absence of feeders consisting of pulmonary fibroblasts yamamoto et al., 2017) . similarly, airway progenitors generated from developmental lung progenitors specified to an anterior fate can be grown as epithelial spheres containing ciliated and secretory cells in 3d cultures (konishi et al., 2016; mccauley et al., 2017) . single-cell rna sequencing profiles of these spheres (mccauley et al., 2018) has confirmed the derivation of airway lineages, but has also raised a cautionary note as non-lung endodermal lineages (e.g., hepatic or gut epithelia) also appear within these spheres over extensive culture periods, representing an ongoing challenge to the field. further efforts have resulted in the generation of more complex lung organoids from pscs. lung organoids grown in matrigel droplets can generate airway structures after xenografting into mice, while the bud tip organoids could only generate small pockets of ciliated cells after transplantation into naphthaleneinjured lungs of immunodeficient mice (dye et al., 2015 (dye et al., , 2016 miller et al., 2018 miller et al., , 2019 . in a second model, psc-derived anterior foregut endoderm cells were grown in suspension culture and as spheres containing respiratory endoderm, and the endodermal cells expressed sonic hedgehog (shh), while the mesenchymal component expressed shh targets. these organoids were termed lung bud organoids . most of the cell types in these psc lung organoid assays have been shown to consist of fetal stage cells, again illustrating the challenges associated with achieving full maturation dye et al., 2015 dye et al., , 2016 miller et al., 2018 miller et al., , 2019 . the origin of the mesenchymal cells that appear to co-develop along with endoderm in these psc lung organoid cultures to date remains unclear. the second emerging principle arising from experience with ipsc models is a need to better understand the temporal developmental signaling pathways required for lung lineage specification. for example, bmp inhibition is required for afe specification (green et al., 2011) . however, consistent with mouse genetic models, subsequent bmp agonism promotes lung fate from afe (huang et al., 2014; longmire et al., 2012 ). an important component for the differentiation of at2 cells is dexamethasone, 8-bromo-camp, and isobutylmethylxanthine (dci), fgf7 or 10, and gsk3b agonism using the small molecule chir9902 (huang et al., 2014 longmire et al., 2012; serra et al., 2017; yamamoto et al., 2017; miller et al., 2018) . withdrawal of gsk3b inhibition, which results in reduced wnt signaling, appears to induce a proximal fate in lung progenitors . conversely, maintenance of chir9902 in 2d cultures and subsequent plating in 3d cultures in the presence of chir9902 yielded at2 spheroids that could be replated and expanded for many passages . interestingly, full morphological maturation accompanied by reduced proliferation of at2 cells could be accomplished by withdrawal of gsk3b inhibition in the 3d cultures . in some 3d models, withdrawal of chir9902 led to both proximal and distal maturation, with the generation of ngfr + postnatal basal cells, at2 cells, and cells expressing markers of at1 cells (de carvalho et al., 2019) . gsk3b inhibition was also required to generate renewing distal tip progenitors (miller et al., 2018) . together, these studies indicate that further analysis of the temporal activation and repression of wnt signaling in driving lung endoderm fate is needed. importantly, gsk3b integrates multiple inputs and affects a wide variety of signaling pathways and cellular process, and some of its effects may be independent of wnt signaling (patel and woodgett, 2017) . additional signaling pathways including notch firth et al., 2014; mccauley et al., 2017; konishi et al., 2016) and retinoic acid are important for promoting lung endoderm cell fate (miller et al., 2018) . a third principle guiding effective directed differentiation of pscs into lung epithelium is the proper initial establishment of lung progenitor fate. the purity of the psc-derived lung progenitors, which can vary from line to line, remains a problem . one solution is the use of reporters, such as fluorochrome constructs targeted to nkx2.1, sftpc, or scgb3a2 loci, which allow flow cytometric purification of desired cell types (gotoh et al., 2014; hawkins et al., 2017; mccauley et al., 2017 mccauley et al., , 2018 serra et al., 2017) . these cell lineage-specific reporters have been useful in defining the differentiation strategies of various cell types in the lung. however, a drawback of reporter lines is that this strategy is unlikely to be approved for the purification of human cells for clinical therapies. a second solution is the use of surface markers to enrich for lung endoderm progenitors such as carboxypeptidase m or cd47 hi cd26 lo cells (gotoh et al., 2014; hawkins et al., 2017; korogi et al., 2019) . lung disease modeling via pluripotent stem cells. one of the major applications for ipsc-derived lung epithelial lineages is providing an in vitro platform for studying human pulmonary diseases. early applications of ipscs attempted to model cystic fibrosis (cf), a disease caused by mutations in the anion channel protein, cftr (cystic fibrosis transmembrane conductance regulator), which leads to progressive lung damage due to impaired mucociliary clearance, inflammation, and recurrent respiratory infections. wong et al. (2012) developed the first ipscderived model of cf using a 2d system in which cf-ipscderived epithelial cells exhibited lower cftr expression and reduced anion transport in response to forskolin, a known cftr activator. a subsequent 2d airway model was published by firth et al. (2014) , in which the authors demonstrated the presence of functional cftr channels, with anion currents that could be modulated by both forskolin and a cftr inhibitor. crispr correction of a cftr mutation in ipscs by two separate groups then demonstrated an in vitro rescue of channel function (crane et al., 2015; firth et al., 2015) . finally, recent work from mccauley et al. (2017) used ipsc-derived airway epithelial spheres to demonstrate that crispr correction of cftr mutants can rescue defects in forskolin-induced organoid swelling, providing an easily visualized read-out for future high throughput screens of airway cftr function. more recent applications of ipsc-derived lung models have focused on a broad diversity of genetic lung diseases, lung cancer, or platforms for the study of viral respiratory infections. for example, 2d differentiation of hpsc-derived lung progenitors allowed modeling influenza susceptibility in ipscs from a patient with a genetic deficiency in irf7 (ciancanelli et al., 2015) and from a patient with tlr3 mutation (lim et al., 2019) . the importance of these findings lies in the fact that they showed that not only interferon production by immune cells was relevant to the increased susceptibility of these patients to lethal influenza infection, but that interferon production within distal lung epithelial cells is likely involved as well. in branching 3d organoids, the pathological changes associated with respiratory-syncytial virus-associated bronchiolitis could be reproduced and infection with clinical isolates of parainfluenza virus could be modeled (porotto et al., 2019) . in 2d cultures, through genetic manipulation of rb and p53 expression in notch-inhibited epithelium enriched in neuroendocrine cells, a human in vitro model for small cell lung cancer was developed . for modeling genetic diseases affecting the distal lung, distal specification and generation of expandable at2 cells in 3d allowed modeling of sftpb deficiency, which was corrected by crispr/cas9-mediated gene editing . recessive mutations in some genes implicated in hermansky-pudlak syndrome (hps) cause hps-associated interstitial pneumonia (hpsip), a clinical entity similar to idiopathic pulmonary fibrosis (lederer and martinez, 2018) . introduction of hps mutations associated with hpsip in escs promoted fibrotic changes in branching lung organoids , while deletion of hps8, which is not associated with hpsip, did not . further studies revealed an essential role for il11 in the fibrotic process, suggesting that il11 is a potential therapeutic target in this intractable disease . additional work using at2 cells generated from patient-specific ipscs profiled the at2 cell dysfunction that results from hps1 mutations (korogi et al., 2019) . many false starts and claims of engraftment of exogenous cells into injured murine lung tissue have confused the field for the past 20 years, with an extensive literature emerging during the controversial claims of bone marrow plasticity during the 1990s (reviewed in kotton, 2012) . more recently a few groups have begun to publish more convincing evidence in support of the capacity of epithelial stem/progenitor cells, of both mouse and human origin, to survive in injured mouse lungs after intratracheal or intravenous delivery (farrow et al., 2018; miller et al., 2018; nichane et al., 2017; rosen et al., 2015; vaughan et al., 2015) . there is also recent evidence of more mature at2 cells of both mouse and human origin surviving transplantation into injured mouse lungs (hillel-karniel et al., 2020; weiner et al., 2019) . whether these cells are functionally integrated into host lung tissue, are long-lived, and retain expression of a complete lung transcriptomic program are still open questions that will need to be addressed before these cells can be referred to as ''engrafted.'' for example, only one study to date has attempted to profile the transcriptomes of grafted cells at single-cell resolu-tion (nichane et al., 2017) . the use of lineage tracing techniques and clonality studies are notably absent from most prior reports, raising uncertainty about the cellular origins of the source cells and preventing assessment of the stem cell properties of transplanted cells. importantly, physiologic improvement of injured mouse lungs after cell transplantation as described in some of the above reports might be explained by paracrine effects or transient secondary reparative effects on recipient lung tissue rather than by direct, durable replacement of the epithelium with functional cells. in addition to alveolar cell therapy, a recent study demonstrated that it is possible to correct cf mutations using primary airway basal cells, which could be used for engraftment into patients (vaidyanathan et al., 2020) . many additional challenges remain in the translation of experimental cell therapy in mice to humans with major lung injury, including the issue of allogeneic rejection, possibly circumvented by the development of autologous, syngeneic ipscderived stem/progenitors if these were to have engraftable potential mccauley et al., 2017; miller et al., 2018) . the limitations listed above have also resulted in a dearth of information regarding the mechanisms or microenvironmental niche interactions that might regulate homing, survival, proliferation, or functional activation of potentially engrafted cells. more sophisticated methods are needed to quantify the contributions of exogenously delivered cells, relative to either endogenous cells or competing alternative source cells. additional studies are needed to resolve the ongoing debate over which of many candidate lung cell populations, from resident progenitors or ipsc derivatives to common mature airway or alveolar cells, might have the best relative potential to reconstitute an injured epithelium after transplantation. the extensive cellular heterogeneity within the respiratory system and the lack of insight into the phenotype differences in these purported cell lineages make single-cell genomics a very useful tool in the study of lung repair and regeneration. this spatial diversity in architecture and cellular heterogeneity reinforces the importance of sampling at multiple regions along the respiratory airway axis to obtain a true cell atlas. advances in scrna-seq techniques allow for prediction of cell state, origins, and trajectories without requiring permanent genetic labeling of cells. new tools enabling data analysis, visualization, and interpretation are being developed at a rapid pace and are readily available as ''open source'' tools. likewise, data web portals provide the research community with open access to rich diversity of rna, protein, and lipidomic and imaging data related to the lung. examples of this include the lung gene expression atlas and breath databases (https://research.cchmc.org/ pbge/lunggens/mainportal.html and https://www.lungmap.net) (du et al., 2017) . while time series of single-cell transcriptomic data are most useful in identifying progenitor cells and their trajectories, these processes can be identified in pseudotime using data derived from a single time point when large numbers of cells are in transition; for example, during organ formation, tissue repair, or during active disease. likewise, single-cell transcriptomic data generated with embryonic stem cells or induced pluripotent stem cells as they differentiate into distinct lung cell types have provided new insights into the identity of lung cell progenitors and the regulatory networks controlling cell fate decisions hawkins et al., 2017; mccauley et al., 2018; nikoli c et al., 2018) . trajectory inference algorithms, or pseudotime analysis, reconstruct continuous cell state transitions from ''snapshots'' of single-cell transcriptomic data, providing insights into gene expression kinetics and regulatory dynamics of biological processes (gerber et al., 2018; guo et al., 2017; hawkins et al., 2017) . wanderlust (bendall et al., 2014) , monocle 1 (trapnell et al., 2014) , waterfall (shin et al., 2015) , and tscan (ji and ji, 2016) are among the early cell trajectory inference algorithms using scrna-seq data. the general analytical workflow of a pseudotime analysis algorithms includes (1) modeling individual cells as data points in high-dimensional gene expression space, where each dimension is defined by the expression of a gene; (2) projecting cells onto a lower-dimensional space using a dimension reduction method, e.g., principal component analysis, which finds a smaller set of new dimensions, each being a combination of original input genes, to represent major variances in the original gene expression space, reduce noise, and enable data visualization; (3) determining cell trajectories in the reduced dimensional space; (4) ordering single cells by progress along the cell trajectories; and (5) identifying gene expression patterns along cell orderings. with the evolution of single-cell technologies and exponential growth in numbers of cells collected from single-cell experiments (svensson et al., 2018) , scrna-seq datasets have captured complex cell trajectories that often contain multiple cell type decision branches. monocle 2 (qiu et al., 2017) , wishbone (setty et al., 2016) , dpt (haghverdi et al., 2016) , slice (guo et al., 2017) , slingshot (street et al., 2018) , and urd (farrell et al., 2018) can be used to infer tree-structured cell trajectories from scrna-seq data, reconstructing branched cell transitional paths from a progenitor toward multiple cell fates. to infer a robust tree-structured cell lineage model, slingshot (street et al., 2018) combines the use of multiple techniques recently developed for processing highly noisy scrna-seq data and allows users to specify initial and terminal cells to supervise parts of the tree construction, which might lead to more accurate dataspecific lineage tree inference. monocle 2 (qiu et al., 2017 ) is a comprehensive computational pipeline for scrna-seq analysis, including gene expression modeling, preprocessing, cell clustering, differentiation expression analysis, and cell trajectory inference analysis. recently developed methods, including paga (wolf et al., 2019) and monocle 3 (cao et al., 2019) , can be used to infer more complex, non-tree-based cell trajectories. together with a number of implementation improvements in computational efficiency and memory usage, paga and monocle 3 can be applied to infer complex lineage relations from large scrna-seq datasets with more than 1 million cells, for example inferring the lineage relations of cells from a whole adult animal (plass et al., 2018) or the reconstruction of 10 disjointed major cell trajectories from $1.5 million mouse embryonic cells during organogenesis (cao et al., 2019) . most of the trajectory inference methods pseudo-temporally order cells purely based on transcriptomic similarity without estimating cell states (e.g., differentiation states). they require the use of external knowledge, such as time information, cell identity, marker gene expression, or user inputs, to determine the start and end points and the directions of inferred cell trajectories. stemid (grâ�¬ un et al., 2016) , slice (guo et al., 2017) , and rna velocity (la manno et al., 2018) provide the capability to estimate cell differentiation states, predicting progenitor cells and directions of cell differentiation transitions. stemid (grâ�¬ un et al., 2016) and slice (guo et al., 2017) both exploit the concept of entropy for predicting cell differentiation states from single-cell transcriptomic profiles. rna velocity showed that scrna-seq data can provide not only static information on mrna abundance of each gene in a single cell but also dynamic information on how the expression of each gene in a single cell is changing through annotating the ratio of unspliced and spliced transcripts of each gene in each cell, predicting the future state of each cell (la manno et al., 2018) . single-cell experiments can be performed at multiple time points relevant to biological processes, such as lung development, regeneration, or disease progression, generating singlecell time-course data. trajectory inference methods, including stitch (wagner et al., 2018) and waddington-ot (schiebinger et al., 2019) , utilize the time of collection to supervise the analysis and improve the accuracy of pseudotime analysis using timecourse scrna-seq data (wagner et al., 2018) . since data collected at multiple time points can be influenced by technical variations in cell isolation, library generation, and sequencing, stitch (wagner et al., 2018) analyzed time-course scrnaseq data by first constructing a single-cell graph from each time point and then joining pairs of graphs from adjacent time points by connecting similar cells (wagner et al., 2018) . waddington-ot, on the other hand, directly estimates a transitional probability between any two cells from consecutive time points in a time-course scrna-seq data without explicitly inferring a lineage topology (schiebinger et al., 2019) . given a set of cells at a time point, waddington-ot predicts the most likely ancestor cells in earlier time points and descent cells in later time points based on the derived cell-cell transition probabilities, reconstructing a cell transitional path across time points. importantly, these cellular trajectory models require experimental validation either in the form of cell-type-specific genetic lineage tracing in mice or the use of cellular barcoding strategies in non-murine systems such as was performed in a recent study to predict the differentiation of lung epithelial progenitors from pluripotent stem cells (hurley et al., 2020) . single-cell analysis has been used extensively to assess both mouse and human lung development and disease. improved scrna-seq methods carefully coupled with cell-type-specific lineage tracing has uncovered new developmental and regenerative origins for multiple lung cell lineages. the early specification of at1 and at2 cells, coincident with such early and basic tissue patterning processes such as branching morphogenesis, and the heterogeneity of embryonic alveolar epithelial progenitors, was recently uncovered using scrna-seq methods (frank et al., 2019; guo et al., 2019) . the heterogeneity within the adult lung mesenchyme was recently described and revealed the importance of the mesenchymal niche in alveolar homeostasis and regeneration (guo et al., 2019; lee et al., 2017; zepp et al., 2017) . human lung scrna-seq has been reported in both ''normal'' lungs and in diseased lungs such as idiopathic pulmonary fibrosis (reyfman et al., 2019; xu et al., 2016) . scrna-seq was also used to identify the ionocyte, which is thought to play a key role in cystic fibrosis plasschaert et al., 2018) . there are multiple consortia that are working to establish a human lung cell atlas including the nih-supported lungmap and the chan-zuckerberg-supported human cell atlas. one of the major hurdles in using single-cell techniques to map the human lung is the high level of cell heterogeneity that exists in the organ, which means that isolation procedures including both upstream and downstream processing are going to be highly variable depending on the site and research group involved. these issues are revealed in many experiments that lack significant representation of fragile cells such as at1 cells and over-representation of immune cells. in the absence of procedures and protocols that generate highly reproducible isolation of all known cell types, both qualitatively and quantitatively, across most if not all lung samples, the resulting datasets are unlikely to provide an accurate cell atlas. moreover, the respiratory system is highly complex and contains many spatially restricted compartments and niches which need to be uniquely sampled, possibly using different techniques for each region. thus, much work remains to be done to ensure that such single-cell mapping efforts can reproducibly isolate and characterize all resident and immune cells within the human respiratory system. bioengineering of the lung has made remarkable progress in the recent years, motivated by strong clinical needs and enabled by advances in tissue engineering and stem cell biology. as many as 25 million people suffer from end-stage lung disease in the united states alone, with $400,000 patients dying each year, a third of these from nonmalignant diseases (optn, 2012; morrisey and hogan, 2010; petersen et al., 2010) . worldwide, lung disease remains the third leading cause of death (murphy et al., 2018; rabe et al., 2007) . notably, most lung diseases affect epithelium, including the acute respiratory distress syndrome (ards), chronic obstructive pulmonary disease (copd), emphysema, cystic fibrosis (cf), and pulmonary fibrosis. lung transplantation, the only definitive treatment option for end-stage lung disease, remains hampered by a severe shortage of donor organs. in addition, a majority of donor lungs are deemed unacceptable for transplantation at the time of receipt (ware et al., 2002) , making lung the least utilized solid organ and necessitating the use of extracorporeal membrane oxygenation (ecmo) as a bridge to transplant for critically ill patients (fuehner et al., 2012; javidfar and bacchetta, 2012; klein et al., 2010; pomfret et al., 2008; ware et al., 2002) . to overcome this crisis, there are major efforts to increase the number of transplantable lungs, including (1) criteria expansion, i.e., accepting older donors, lungs donated following cardiac death (bittle et al., 2013; elgharably et al., 2015; van raemdonck et al., 2009 ), (2) ex vivo lung perfusion (evlp) to recover marginally unacceptable donor lungs (cypel and keshavjee, 2015) , (3) bioengineering of functional lungs by populating decellularized lungs or scaffolds with epithelial and vascular cells (ott et al., 2010; petersen et al., 2010; rosen et al., 2015; wagner et al., 2013; wobma and vunjak-novakovic, 2016) , and (4) utilization of xenogeneic lungs from swine or non-human primates (cooper et al., 2012; laird et al., 2016) . thus far, the number of lung transplantations remains steady, resulting in the increasing waitlist mortality (valapour et al., 2015) . new strategies are being developed to increase the numbers of lungs for transplantation by recovering marginal quality donor lungs and developing physio-logical ex vivo platforms for modeling lung disease. the enormous complexity of the lung, with its hierarchical architecture, more than 40 cell types, and a very large area ($70 m 2 ) for gas exchange, presents major challenges to lung recovery (crapo et al., 1982; massaro and massaro, 1996; weibel, 1973) . finally, generation of humanized whole lungs through stem cell complementation in other species could lead to a new source of transplantable lungs . ex vivo lung perfusion (evlp) and cross-circulation with a living host. among current approaches, evlp holds the strongest potential for immediate clinical impact by recovering marginally unacceptable donor lungs, which has been already implemented and is being further evaluated in clinical trials (popov et al., 2015) . notably, many of the conditions that render donor lungs unacceptable for transplantation (e.g., aspiration, infection, pulmonary contusions) could be reversible. however, conventional methods of donor lung preservation that involve cold static ischemia preclude endogenous repair and recovery (guibert et al., 2011; pinezich and vunjak-novakovic, 2019) . the field of ex vivo lung perfusion (evlp) is now addressing this limitation by providing initially unacceptable donor lungs with physiologic conditions of normothermia, perfusion, and ventilation, to recover function outside the body to a level acceptable for transplantation (makdisi et al., 2017; tane et al., 2017) . since the introduction of evlp by steen and colleagues in 2001 (steen et al., 2001) , evlp platforms have successfully demonstrated short-term support and recovery of marginal quality donor lungs in pre-clinical and clinical settings (cypel et al., 2011; warnecke et al., 2018) . a major limitation of evlp is the lack of whole-body homeostasis that requires renal, hepatic, pancreatic, and neurohormonal functions. the current durations of clinical usage of evlp systems are too short for advanced therapeutic interventions (e.g., immunomodulation, cell therapy) (warnecke et al., 2012) . moreover, current evlp systems cannot recover the majority of unusable donor lungs, due to the lack of appropriate physiologic milieu for endogenous repair. using a clinically relevant swine model, a cross-circulation platform has been established with the recipient support enabling 36 h of normothermic perfusion for the maintenance and recovery of injured lungs . in recent studies, the duration of lung support on cross-circulation has been extended to 4 days (hozain et al., 2019) . cross-circulation is actually an old technique that has been used in the past to support patients suffering from a critical but potentially reversible illness given sufficient time for recovery (e.g., hepatic insufficiency, uremia, eclampsia) (burnell et al., 1965 (burnell et al., , 1967 eschbach et al., 1964 ) by a healthy individual. for extracorporeal lungs, cross-circulation was used to extend the duration of lung maintenance ex vivo from hours to days, by providing metabolic clearance and systemic factors to the perfused and ventilated lung (figure 4 ; o'neill et al., 2017) . this method allowed time for multiscale therapeutic interventions, with the aid of real-time theranostic (therapeutic + diagnostic) imaging (kim et al., 2015b . by the end of cross-circulation support, the lungs that were severely damaged by ischemia or gastric aspiration exceeded transplantation criteria, and the recipients tolerated the procedure without significant changes in physiologic parameters. these findings suggest that cross-circulation could enable extended support necessary for gene and cell therapies of the extracorporeal lungs. lung bioengineering by complete decellularization and recellularization of the lung parenchyma and vasculature. the niklason and ott laboratories have pioneered a methodology that involves removal of all cells from the lung and subsequent infusion of epithelial cells into parenchymal region and endothelial cells into the vascular region of the lung (ott et al., 2010; petersen et al., 2010) . however, these bioengineered lungs fail shortly upon transplant, due to incomplete regeneration and leaky vasculature causing alveolar edema and thrombosis (ott et al., 2010; petersen et al., 2010 petersen et al., , 2011 petersen et al., , 2012 . while this highly innovative approach has advanced through many meritorious studies, three limitations remain: (1) the inability to restore gas exchange, (2) the inability to maintain vascular function upon transplantation, and (3) the need for very high cell numbers (billions) to repopulate the lung (petersen et al., 2010; song et al., 2011) . targeted treatment of lung epithelium with the preservation of lung vasculature. in principle, the limitations of full decellularization/recellularization of the lung could be overcome if acutely injured donor lungs are treated to replace only the epithelium in the most injured regions of the lung, while preserving the integrity of the lung vasculature. because acute injury tends to affect some regions of the lung and not the entire lung (raghavendran et al., 2011) , a targeted treatment could help preserve much of the existing lung function and facilitate regeneration of the injured regions. an alternate approach to lung bioengineering has been developed that is based on removing damaged epithelial cells from distal lungs while maintaining the integrity of the basement membrane, surrounding lung cells and matrix, and the functionality of lung vasculature. denuded regions in the lung airway are repopulated with epithelial progenitors. the maintenance of an intact vascular network was considered critical for maintaining the blood-gas barrier as well as for supporting survival of newly delivered cells. this airway-specific approach was first demonstrated in the rat model, resulting in vascularized lung grafts that supported the attachment and growth of human adult pulmonary cells and stem-cell-derived alveolar progenitor cells . targeted decellularization/recellularization of the lung epithelium was achieved by introducing soluble reagents (such as the solutions for cell removal, suspensions of therapeutic cells) in micro-volume liquid plugs to the targeted branches of the pulmonary airway: upper airways, small airways (bronchioles), or the most distal lung (alveoli). liquid plugs (only <1 ml in volume) were instilled into the upper airway and pushed into a specific more distal airway to form liquid film covering lung epithelium, using programmed ventilation of the lung in conjunction with radiation-free transpleural imaging (kim et al., 2015a) . combination of long-term lung support ex vivo with the targeted treatment of lung epithelium. cell replacement therapy offers compelling prospects for the treatment of injured lungs, if the extracellular matrix (including the basement membranes) could be preserved. a new methodology for lung regeneration is now under development through an integrated use of three components: (1) extended duration of lung support ex vivo (several days) by cross-circulation with a living host, (2) targeted treatment of the injured regions of the lung (from conducting airways to distal regions), with the preservation of the surrounding lung parenchyma, and (3) selective removal and replacement of lung epithelium with the preservation of lung vasculature (for immediate blood supply for lung survival and function) (figure 4) . the same issues regarding whether newly delivered or replaced cells merit use of the controversial term, ''engraftment,'' (indicating true, durable integration of functional cells) apply to this novel approach as discussed above (see cell-based therapy for the respiratory system) and have not yet been addressed. the utility of this approach was demonstrated in clinical-scale swine models for recovering lungs unsuitable for transplant due to acute injury by ischemia and gastric aspiration (guenthart et al., 2019) ( figure 5 ). in both cases, the lung epithelium was removed from injured regions while preserving the surrounding cells, matrix, and intact lung vasculature , and the denuded epithelial regions were repopulated with pulmonary progenitor cells. in one study, the injured lungs were maintained on cross-circulation support without decline in lung function and were subjected to therapeutic interventions that enabled cellular regeneration and improved function over the course of 36 h (guenthart et al., 2019) . regeneration of severely damaged lungs to the levels necessary for meeting the transplantation criteria would help significantly increase the pool of donor lungs available for transplant, for example by recovering lungs rejected because of gastric aspiration damage. on cross-circulation, the injured lungs recovered their mechanical compliance to 68% of that of uninjured control lungs, corresponding to a 6-fold improvement relatively to the levels measured post-injury. a lung injury scoring rubric that has been developed in these studies (guenthart et al., 2019; o'neill et al., 2017) was also used to assess the extent of lung injury over 4 days of lung maintenance and recovery (hozain et al., 2019) . the lung injury scores decreased across all categories, with the greatest improvements observed in alveolar cells functionality, lung edema, and cell apoptosis. the recovered lungs displayed regeneration of cell surface markers in the pulmonary epithelium, endothelium, tight and gap junctions, recovery of subcellular structures (e.g., intracellular vesicles), and cellular function and integrity (guenthart et al., 2019) . throughout the duration of extracorporeal support, the recipient tolerated cross-circulation with no significant changes in physiologic parameters. after 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abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional cftr protein progressive pulmonary fibrosis is caused by elevated mechanical tension on alveolar stem cells local lung hypoxia determines epithelial fate decisions during alveolar regeneration single-cell deconvolution of fibroblast heterogeneity in mouse pulmonary fibrosis single-cell rna sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis long-term expansion of alveolar stem cells derived from human ips cells in organoids spatial-temporal lineage restrictions of embryonic p63 + progenitors establish distinct stem cell pools in adult airways induction of autonomous memory alveolar macrophages requires t cell help and is critical to trained immunity regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor cellular crosstalk in the development and regeneration of the respiratory system distinct mesenchymal lineages and niches promote epithelial self-renewal and myofibrogenesis in the lung p63(+)krt5(+) distal airway stem cells are essential for lung regeneration the following authors were supported by funds from the national institutes of key: cord-017686-127xfkse authors: bindenagel šehović, annamarie title: human rights and state responsibilities date: 2018-01-14 journal: reimagining state and human security beyond borders doi: 10.1007/978-3-319-72068-5_2 sha: doc_id: 17686 cord_uid: 127xfkse this chapter lays out an argument that citizens’ human rights are the responsibility of the corresponding state, meaning that citizens of a territorial state claim particular rights that state is obliged to deliver. in return, in an aspect which is often neglected in analyses of human security, citizens also owe allegiance to the state. citizens’ rights have been expanded to encompass not only physical protection within a territory but also a host of economic and welfare provisions. despite the increasingly international discourse on human security rights, their legal home remains with the national state vis-à-vis its citizens. the chapter argues that the rules of the state-based order are shifting, with no clear loci of responsibility and accountability for human security. the introduction sketched the origins and elements of human security. this chapter lays out one argument to make the case that citizens' human rights are the responsibility of the corresponding state, meaning that citizens of a territorial state claim particular rights that state is obliged to deliver. as outlined in the introduction, these rights have been expanded to encompass not only physical protection within a territory but also a host of economic and welfare provisions (hösle 2003; slaughter 2004; cescr 1966; icpcr 1966) . however, especially with regard to the latter, not all of these rights are equally or legally encoded into national law. thus despite the increasingly international discourse on human security rights, their legal home remains with the national state vis-à-vis its citizens. in return, in an aspect which is often neglected in analyses of human security, citizens also owe allegiance to the state. 1 this includes submitting to civic codes such as police ordinances and taxation, as well as to the military draft when instituted 2 : without such a reciprocal relationship between states and citizens, it might not be possible to guarantee territorial or other human security protections. of course, the necessary existence of such a relationship does not preclude its potential for abuse by either party (see also howell 2014) . this reciprocal relationship is based on a state-citizenship centric order, and that not only at the national level, but also internationally. in other words, citizenship here is dependent upon its conferral by a territorial state, which derives its contours from its citizenry. this chapter thus assumes that the current national/international governmentality order continues to be based upon this state-citizens relationship, with a twist. that is, while the national/international legal order rests upon the pillars of state-citizen reciprocity with regard to rights and obligations, this exchange does not reflect the more complicated reality. that is, the rules of the state-based order are shifting, with no clear loci of responsibility and accountability for human security. the hypothesis presented here argues that a bifurcated evolution wherein rights have ascended up the international agenda but not necessarily at the national level, and state or sovereign obligation has been diffused between state and nsas without clarifying where the locus of the final guarantee of protection lies, describes the current status. this has led to a diffusion of the guarantor status of the national state, with elements of power in governance-agency, scope, mechanisms, and normative context-diverging. this leads to two questions: first, if state a acts as a guarantor to the human security of citizens of a, the same holds for state b and citizens of b; but what happens to citizens of state a residing in state b, or vice versa? second, what are the consequences of human security provision to citizens of a or b by nsas, notably when nsas go bankrupt or depart? both of these questions point again to the need to clarify the relationship between citizens and states in order to conceive of suitable answers. as outlined in the introduction, the idea of human security as the remit of the state is inextricable from the notion of state sovereignty. sovereignty as a concept has been debated since its inception, and each idea of it has made various assumptions as to what it entails and what it excludes. the majority of scholars (krasner 1999) of westphalia-influenced definitions of sovereignty include requisites such as the state enjoying a monopoly of power capable of defending its territorial borders against external aggression; even these have rarely been absolute in practice (krasner 1999, 8-9, 42) . in describing this 'compound' myth, anne-marie slaughter defines westphalian sovereignty as "the right to be left alone, to exclude, to be free from any external meddling or interference" (slaughter 2004, 284 ). yet the same sovereignty that offers the option to opt out is also the ticket to inclusion in the inter-national community of (equal) states. the westphalian definition also invokes "the right to be recognized as an autonomous agent in the international system, capable of interaction with other states and entering into international agreements" (slaughter 2004, 284) , the responsibility for whose implementation resides squarely with those signatory states. this reveals the schism between what robert keohane (1995) called formal and 'operational' sovereignty, and what i referred to as the divergent 'final guarantee' and 'functional' sovereignty with regard to the governance accountability problem (gap) (šehović 2014). it acknowledges that westphalian sovereignty is not absolute, and rather that "it is now a platitude that the ability of governments to attain their objectives through individual action has been undermined by international political and economic interdependence" (keohane, quoted in slaughter 2004, 283) . the eu exemplifies political and economic interdependence, a model partially replicated to differing degrees by the african union (au), asean (association of southeast asian nations), and mercosur (the common market of select south american states) in a quest to confront threats and maximize opportunities. both in theory and in practice then, this means that, on the one hand, states increasingly cannot-and often do not want to-fully guard against external interference. on the other hand, states (should also) acknowledge that the sources of such interference include not only other states but also activities of nsas, from crime syndicates and cyber surveillance and mercenaries to human rights' campaigners, as well as cross-border challenges such as (infectious) disease spread and migration. these interdependencies and their potential both for cooperation and for conflict directly influence a state's ability not only to control its own territory (see krasner, interdependence sovereignty, pages 12-14) but also to the "security, economic stability and a measure of prosperity, clean air and water, and even minimum health standards" (slaughter 2004, 283) that are the hallmarks of hösle's expanded definition of sovereignty (hösle 2003) and the integral components of human security. human security's main argument places the emphasis of security on the human as opposed to the state. the central assumption underscoring human security is that "when a human faces a threat, so does international security" (burgess and gräns 2012, 101; kerr 2007, 92; undp 1994 ). yet the two are necessarily in dialogue with each other: first of all, states in the inter-national remain the arbiters of human security (hösle 2003; un declaration 1948; undp 1994) , regardless of whether the point of departure is human-or state-centric; and second, as members of the international community of (equal) states, these are themselves increasingly subjected to trial by their peers. "states can no longer assume that if they refrain from interfering in the affairs of other states they will remain free from interference themselves" (slaughter 2004, 284) . furthermore, governments increasingly understand that they often cannot afford to look the other way; that fundamental threats to their own security, whether from refugees, terrorists, the potential destabilization of an entire region, or a miasma of disease and crime, may well have their origins in conditions once thought to be within a state's exclusive domestic jurisdiction. (slaughter 2004, 284) as the post-cold war era has shown, both intra-and inter-state conflict have coincided with the spread of disease. this has been evident in the former yugoslavia, in rwanda and somalia, in iraq and syria (intrastate conflict by the numbers 2013; human security centre). these conflicts have seen the increase in cross-border spread of disease such as evd, h5n1, hiv, measles (notably in continental europe, and the us), mers-cov, and sars, to name a few examples. this incidence salience of the insight that: "states can only govern effectively by actively cooperating with other states and by collectively reserving the power to intervene in other states' affairs" (slaughter 2004, 285) . it has been backed up by the normative evolutions first from rights to responsibility, to the r2p, to, arguably at this moment, the responsibility to respond. this captures the essence of a continual conversation between human security and sovereignty. therein, "internally, a government has a responsibility to respect the dignity and basic rights of its citizens," and "externally, it has a responsibility to respect the sovereignty of other states" (slaughter 2004, 287) , except when a state heeds the (r)evolution rewriting sovereignty as control to sovereignty as responsibility. daniel philpott describes this shift as part of an ongoing process. he attributes this revolution in sovereignty to "prior revolutions in ideas about justice and political authority" (philpott 2001, 4) . the post-cold war reordering of the world proffers a multitude of examples of this progress: from emergent multipolarity (flockhart 2016) to the rise of nongovernmental organizations (ngos) and nsas, from the human rights debates to gain access to hiv treatment to those to usher in the r2p (iciss 2001), reconceptualizations of internal and external state responsibility have been pitted against each other. though the state remains legally dominant, theoretical and philosophical evidence underscored by empirics points to two key unresolved tensions: the locus of the responsibility for human security and the scope of human security, particularly in reaching non-citizens. on the theoretical side, foucault presciently identified emergent 'governmentality' (faubion and rabinow 1994), anticipating the collaborative governance that would emerge as states and nsas sparred and cooperated in response to ever more global challenges to human security. the 1990s, amid the (western) euphoria of the 'end of history' (fukuyama 1989) , witnessed an initial acknowledgment that states alone could not meet the rising number of international and increasingly global challenges-from the multiplication of intra-state conflict and the proliferation of weapons to water management. rosenau introduced the idea of 'governance without government' (rosenau and czempiel 1992), maintaining that governance 'regimes' composed both of states and nsas would form to tackle specific issues in the international realm. nsas have long been engaged in shoring up or tearing down state sovereignty, with (hösle 2003) or without the consent of the state. while on the one hand a tension exists between theory and practice of state sovereign obligation with regard to human security, it also means that though threats to human security abound on the part of both state and nsas, precedents likewise exist for mitigating these to the benefit of human security. to a large extent, rosenau has been proven correct: if nsas are included, then a plethora of organizations exist dedicated to treating hiv/aids, providing water and sanitation, and even administering public transportation in municipalities around the world. however, these are not regimes in the sense that they have a central organizational structure, that their interventions are legally binding, or that any mechanisms are in place to ensure the continuation of their work if and when they opt out. this is not a central theme of risse's work, which focuses on 'areas of limited statehood' (risse 2007) . here nsas might perform functions theoretically if not in practice associated with state responsibility for human security. yet they are not bound to such actions, for instance, of service delivery and health care. critically, instead of shoring up states' lack of capacity, nsas have contributed to the fragmentation of their power-including their ability to guarantee traditional and human security: ngos' [nongovernmental organizations'] role and influence have exploded in the last half-decade. their financial resources and-often more importanttheir expertise, approximate and sometimes exceed those of smaller governments and of international organizations. "we have less money and fewer resources than amnesty international, and we are the arm of the u.n. for human rights," noted ibrahima fall, head of the u.n. centre for human rights, in 1993 . "this is clearly ridiculous." today ngos deliver more official development assistance than the entire u.n. system (excluding the world bank and the international monetary fund). in many countries they are delivering the services-in urban and rural community development, education, and health care-that faltering governments can no longer manage. (matthews 1997) nonetheless, risse assumes that nsas will continue their activities. that these nsas might be accountable not to the human beings they serve, but otherwise, or that they might be dependent upon funding sources whose priorities are prone to shift, remains under-analyzed. it leaves unanswered the questions of what happens to the state-citizen relationship when they do not. krasner attempts to corral some of these disparate responses to the sovereign redrafting by delineating four elements of sovereignty: westphalian, juridical, domestic, and interdependence (krasner 1999; czempiel and rosenau 1992) . none directly deal with the engagement between sovereignty and human security explicitly, yet they are critical in highlighting their exchange. whether the four 'sovereignties' can be meaningfully divorced from one another and applied in an empirical sense to state or human security remains unproven: while theory must conform to practice, so, too, must practice inform theory (see box 2.1). while keohane's divide between formal and functional sovereignty alludes to some of the problems with distilling sovereignties listed above, they are not thereby resolved (keohane 1995) . similarly, slaughter's network theory, taking nsas into account, revives some of the same solutions put forward by foucault and rosenau. likewise my 2014 gap thesis, while identifying the lack of accountability between state and nsas with regard to the guarantee of human security to citizens, it did not deal with the same responsibility to non-citizens. this points to a new stage in philpott's (r)evolutions in ideas: while each of the conversations between sovereignty and human security introduced above acknowledges the limits of westphalian absolutism, each fails to account for their (re)imagining beyond borders. on the philosophical side, scholars have wrangled with this conceptually in various terms. the human rights agenda, which both precedes and parallels that of human security, is itself an outgrowth of a historical trajectory of political theology. referring to "to the connections between religion (in the broadest sense, including philosophy as well) and legally structured power," political theology is of "special importance in the western world and influenced the development of juristic concepts, especially those concerned with public law" (hösle 2003, 467; schmitt 2007) . public law, inextricable from the relationship between states and subjects, then states and citizens, is vested with antecedents of values-with morals and their changing interactions with politics (hösle 2003, 21; carlson and owens 2003) . • what is the value of westphalian sovereignty where a state cannot control its territory? • what role does juridical sovereignty play when a state is only partially recognized by its peers? (see kosovo) • what does domestic sovereignty mean if (a) a portion of the citizenry is excluded from, for instance, health care? (b) non-citizens have no recourse to rights (to education, health, justice)? • what is interdependence sovereignty if borders are porous or surveillance systems are technologically or politically incompatible? christianity in the west, particularly after the treaties of westphalia largely ended internecine wars on the european continent, contributed immensely to the conversation and construction of sovereignty, as related to human rights and human security. hösle argues that christianity estranged citizens from their state and universalized their rights' claims. through the idea of all human beings as god's children, a broader as well as existentially deeper diffusion of the universalistic and individualistic ideas of hellenism-and thereby eliminated a possible identification with any state that does not include all human beings and is not constituted in accord with the principles of christianity. (hösle 2003, 22) christianity can arguably be made responsible for two things (hösle 2003, 24) : first, a politics free of religious and ritual considerations, taken further through the enlightenment; and second, an intensive moralization of the religious, demanding "an influence on politics that went far beyond what was conceivable for ancients" (hösle 2003, 24) . the latter finds its echo in the articulation and demand for individual human rights delivered by the state. thus although the notion of a christian theocracy likewise receded with the secularization of westphalia, the ideas of universal human rights and of a universal claim to human security have wound their way through various (r)evolutions in sovereignty right up to this present reimagining. returning to the core of the conversation between sovereignty and human security, theory and philosophy back up the urgent need to practically respond to the three main tenets of state and human security: (1) ensuring the territorial and physical security of citizens; (2) protecting lives and livelihoods through basic economic stability, health, and welfare; and (3) bearing accountability internally and to the international community (hösle 2003; risse 2007) . assuming that states remain the final arbiter of such securities, articulating, delegating, and assuming respective state and human rights and responsibilities are key to reimagining and implementing human security beyond borders. states, sovereignty, human security-all are predicated upon a relationship of rights and responsibilities between citizens and states. the tension in this reciprocal relationship is not new. it can be divided into three broad shifts dating from westphalia through to the last major global reordering in the 1990s, which ended the second wave of democratization (strand et al. 2012 ) and inaugurated the third wave of liberal, democratic capitalism based on state sovereignty. the first shift, demarcated but by no means consolidated with the treaties of westphalia in 1648, ordered responsibility, for territorial and physical protection in the name of state sovereignty, at the level of the state. the second shift, from circa the 1960s, occurred at the height of the second wave of democratization, and in the name of 'self-determination' (unpo 2006) . this meant on the one hand that especially newly minted states could cling in particular to the westphalian notions of 'nonintervention,' a stance reemphasized by both blocs at the height of the cold war. on the other hand, however, the existence of the post-world war ii un and its emerging norms and values spread the notion that state responsibility includes human rights discourse if not its translation into practice. this pre-exposure arguably paved the way for the 1990s shift, which, in the words of the independent commission on human security, refers to the 'vital freedom,' explicitly tied state responsibility to "protecting people from severe and pervasive threats, both national and societal, and empowering individuals and community to develop the capabilities for making informed choices and acting on their own behalf" (ogata and cels 2003, 274) . what remained formally the same throughout these transitions, and became all the more pronounced as state subjects/constituents became citizens, is the onus placed on the state to assume responsibility for the security of those citizens. while a constituent refers to a voter within a particular area, a citizen is a (political) member of a state. this has two implications: first, a constituent must not be a citizen. indeed, a constituent might receive physical security within a territory in return for heeding the obligation to serve that same territorial state's security in the event of war. this leads to the second point: a citizen might have more privileges, such as the right to vote. yet the obligation to serve the state-by taxation and/or by (required) military service-remains. so, too, does the threat of the revocation of citizenship if an individual serves in the armed forces or swears allegiance to another state. as is quoted on the inside of every us passport: 13. loss of u.s. citizenship: under certain circumstances, you may lose your u.s. citizenship by performing, voluntarily and with the intention to relinquish u.s. citizenship, any of the following acts: (1) being naturalized in a foreign state; (2) taking an oath or making a declaration to a foreign state; (3) serving in the armed forces of a foreign state; (4) accepting employment with a foreign government; or (5) formally renouncing u.s. citizenship before a u.s. consular officer overseas. (authors' passport) (generous) provisions do exist that allow dual citizenship. some states, the us among them, allow citizens to renounce their citizenship. others, such as iran, do not. while citizenship obligation has long been linked to a measure of state responsibility for protection, such as consular services overseas, it has not been synonymous with citizenship rights. by its very exclusivity, citizenship does not and cannot confer universal, inalienable rights. the concept and enactment of human security attempt to rebalance those obligations into an equation wherein state sovereign responsibilities meet individual human rights (bergman 2010; kerr 2007; nef 1999; undp 1994) . the revolution of human security and rights-based development lies in their universalism. states become the bastions not only of ultimate responsibility for the extent of the provision of rights for what is possible within their capacities but also, arguably, for the highest standard internationally. president franklin d. roosevelt's now-famous "four freedoms speech" of 1941 preceded the call for human security in the 1994 undp and again in the 2003 publication of the report "human security now" by the commission on human security (roosevelt 1945; ogata and cels 2003) . from the very beginning of the post-world war ii period, article 1 of the un charter and article 25 of the un universal declaration of human rights (udhr) encoded the principles of human security, including an emphasis on the right to health, which is central to the case studies presented in chaps. 5 and 6: everyone has the right to a standard of living adequate for the health and well-being of himself and of his family, including food, clothing, housing and medical care … and the right to security in the event of … sickness, [and] disability … motherhood and childhood are entitled to special care and assistance. (udhr 1948) the centrality of health among global policy priorities is reiterated in the constitution of the world health organization (who) in 1948; the international covenant on economic, social and cultural rights (icescr); the 1994 undp; and the adoption of the ihr in 1969 and most recently updated in 2005. the icescr-as well as the convention on the elimination of all forms of discrimination against women (cedaw), the convention on the rights of the child, and the world trade organization's doha declaration on "trade-related aspects of intellectual property rights," which allows for the production of generic versions of essential medicines under certain conditions before patent protection runs outappears to provide an implicit obligation on the part of states to improve health and to establish and secure health as a human (security) right. however-and crucially-none of them prescribes an explicit obligation. similarly, the ihr emphasize the universal and expanding right of each individual citizen (of the world) to the highest standard of health. in fact, the ihr, having gone into effect in 2007, require their 196 signatory state parties to "develop public health capacities to detect and respond to public health emergencies of international concern (pheic), with states required to cooperate in building these capacities" (who 2008) . "however, the regulations do not provide incentives, sanction states for failing to cooperate, or allocate responsibility" (gostin and friedman 2014, 1323) . no specific or enforceable obligation to ensure that individuals attain physical and mental health and no guidelines for how the state's obligations are to be discharged exist (davies 2010) . this situation obviously creates problems for the implementation of the right to health within the remit of a state's responsibility to provide (human) security. nonetheless, these agreements have transformed normative ideas into principles of action (icescr 1966, article 12 ). yet real implementation lags, lost in the opaque realm between theoretical and practical responsibility. the consequences are particularly obvious with regard to states' responses to threats to human security of, but not only of, health. in the narrow sense, human security is limited to physical protection and the creation of conditions conducive to human welfare, but stops short of full protection and provision. whether or not defending those values parallels interests that reach to the hindu kush (löfflmann and vaughan-williams 2017; maull 2006) , the assertion of which resulted in the then german defense minister struck (2002 struck ( -2005 tendering his resignation, remains a point of contentious debate, not just in germany. kaldor et al. (2007) attempt to work this into an especially value-based foreign policy strategy for the eu that nonetheless takes state interests into account. this morphs into the broader conceptualization of human security, wherein an equal level of priority is given to any type of threat (thakur 2004, 37) . critics argue that such prioritization of all is equal to prioritization of none. liotta and owens present one attempt to differentiate between threats, risks, and vulnerabilities as part of this debate (liotta and owens 2006) . they arguably all converge and infringe upon human security which demands a response. a gap emerges between theory of protecting human security and its practice. it begs the questions: for whom? how far? by whom? the (inter)national system based on sovereign states continues to operate under the assumption that "governments have a responsibility for the health of their peoples which can be fulfilled only by the provision of adequate health and social measures" (who 1948) . critically, "while only states are parties to the covenant, and thus ultimately accountable for compliance with it, all members of society-individuals, including health professionals, families, local communities, intergovernmental and non-governmental organisations as well as the private business sectorhave responsibilities regarding the realisation of the right to health" (who, ihr 2005) . as lake notes with regard to judicial processes in the congo, "the de facto assumption of power by these diverse sets of actors has created opportunities through which non-state actors can enter and influence juridical processes by engaging in tasks normally reserved for representatives of the sovereign government. these activities would not be possible in contexts where the state had greater reach" (lake 2014, 519) . this exacerbates the problem of responsibility because merely counting the number of convictions of a prioritized crime or the number of people inquiring about health treatments and antiretroviral medications for hiv, for example, "tells us little about the dynamics of power" that determine the necessary response to the problem (including the problem definition) at hand (lake 2014, 523) . lake notes that "on a broader scale, it could also be argued that the involvement of international actors in micro-level governance activities in dr [democratic republic of] congo has served not to build capacity but in fact to further relieve the congolese state of its responsibilities to provide basic goods and services to its citizens." indeed, because a litany of "international and domestic organizations ready to engage in this work, there may be little incentive for the central government to re-invest its own time and resources into developing a functional state apparatus" (see also keohane 1995; lake 2014, 524) . such developments actively undermine state's sovereignty and capacity to exercise responsibility, leading to absurdities such as indonesia's claim to 'viral sovereignty'-the idea that viruses belong to the state in which they originate. it was invoked to prevent and delay sharing data and sam-ples of h1n1 influenza also due to the anticipated costs of being branded a state of contagion amid exclusion from research and treatment benefits. indonesia's was an ill-fated attempt by the state to seize control over information pertaining to the outbreak, its domestic response, and its interdependence sovereignty-notably its ability to regulate any potential medical interventions and possible patents created externally and sold (back) to indonesia. these examples all iterate the theory and practical reality in the still state-centric international system that there are roles that only the state-at least among today's polities-can perform. states are the only nonvoluntary political unit, the one that can impose order and is invested with the power to tax…. moreover, it may be that only the nation-state can meet crucial social needs that markets do not value. providing a modicum of job security, avoiding higher unemployment, preserving a livable environment and a stable climate, and protecting consumer health and safety are but a few of the tasks that could be left dangling in a world of expanding markets and retreating states. (matthews 1997) assuming then the necessary vitality of a responsible sovereign state to the guarantee of access to rights, any reworking of state and human security must take states into account even while rising to the challenge of responding to and guaranteeing human security beyond states. placing the responsibility for human security beyond states requires flexible relocation of that responsibility itself. although state sovereignty continues to be the building block of local, national, and international relations and global governance, its real power to enact responsibilities and assume accountability for the provision of the rights of its citizens has arguably waned-not uniformly but almost regardless of whether the state in question is considered consolidated, fragile, or failing/failed. consequently, the ostensibly sovereign state is ultimately responsible for the traditional, territorial security and physical security of the populace within its borders. in addition, it is accountable for both of these securitizations both internally and externally (i.e., within the international community of states). however, the same state is increasingly confronted with nsas that both demand its action and assume some of its functional responsibility-but not state(-citizen) accountability. as such, the statecentric international governance system faces the challenge of responding to both internal and external rights' demands and responsibility duties. the next chapter will further explore these conceptual challenges and analyze possible levels of such a reordering of human security responsibilities beyond borders. see also the cambridge dictionary entry for "citizenship: the status, rights and duties of a citizen, especially of a particular country on concepts and paradigms in mixed methods research human security the sacred and the sovereign global politics of health the coming multi-order world the end of history and the last man. the national interest (spring) morals and politics international commission on intervention and state sovereignty (iciss). 2001. the responsibility to protect the international relations and security network intrastate conflict by the numbers human security: a new strategic narrative for europe hobbes's dilemma and institutional change in world politics: sovereignty in international society human security sovereignty: organized hypocrisy organizing hypocrisy: providing legal accountability for human rights violations in areas of limited statehood ebola: a crisis in global health leadership why human security? the whitehead journal of diplomacy and international relations winter/spring narrating identity, border security, and migration: critical focus groups and the everyday as problematic power shift germany's uncertain power: foreign policy of the berlin republic human security and mutual vulnerability: the global political economic of development and underdevelopment revolutions in sovereignty: how ideas shaped modern international relations paradoxien der souveränität: die konstituive norm, auf der die heutige staatenwelt gründet-dass nämlich staaten souverän sind-gilt uneingeschränkt nicht mehr. was heisst das? internationale politik state of the union address (the four freedoms) governance without government: order and change in world politics human security-protecting and empowering the people the concept of the political hiv/aids and the south african state: the responsibility to respond sovereignty and power in a networked world order democratic waves? global patterns of democratization international covenant on civil and political rights unrepresented nations and peoples organization (unpo) constitution of the world health organization world health organization key: cord-257597-jy4a8al8 authors: von essen, erica; lindsjö, johan; berg, charlotte title: instagranimal: animal welfare and animal ethics challenges of animal-based tourism date: 2020-10-08 journal: animals (basel) doi: 10.3390/ani10101830 sha: doc_id: 257597 cord_uid: jy4a8al8 simple summary: animals of countless species, wild as well as tame, can now entertain tourists on their holidays. the popularity, however, of animal-based tourism comes with significant risks for the welfare of these animals. many animals are kept in small confinements, are broken down to interact obediently with tourists, or are made to perform, entertain, transport or even give their lives for human leisure. in this paper, the challenges of animal-based tourism are presented from the perspectives of interdisciplinary researchers. the challenges are discussed based on a two-day symposium with workshop sessions. we bring attention to the problem of cultural relativism and the difficulty of imposing universal standards of animal welfare. we conclude that reforms and individual travel decisions as a result of biosecurity concerns will impact animal welfare. in addition to this, we observe that technology has a dual role to play in enhancing edutainment but also potentially inviting new challenges. in the end, we declare some possibilities for compassionate animal based tourism. abstract: by animal-based tourism, a host of activities offering passive viewing or active interaction with wild, semi-wild or captive animals is included. the multibillion dollar industry is on the rise globally today, offering modes of engagement with animals that trade on increasingly embodied close encounters with non-human animals. as new modes of animal-based tourism proliferate, such as sloth selfies, visiting cat cafes, swimming with sharks and agri-tourism petting zoos, animal welfare standards risk deteriorating. in the following paper, we collate concerns over animal welfare into a discussion on the challenges facing animal-based tourism. our synthesis is the first to consider the full spectrum of such animal-based tourism: across agri-, hunting, zoo and safari tourism, to name a few, and crossing consumptive and non-consumptive boundaries. a literature review is first provided. findings are then presented thematically following workshops at an international interdisciplinary symposium of leading tourism, animal welfare, ethics and leisure sciences scholars together with practitioners of the industry. it discusses macrolevel drivers to animal-based tourism as an industry, the problem of cultural relativism and the role of technology in enhancing or promoting the experience. we indicate ways forward toward implementing a compassionate animal-based tourism. taking selfies with animals and uploading them to instagram may be considered tacky to some but is part of a growing suite of activities in late modernity that sell embodied encounters with animals [1, 2] . and indulgence which should not be judged by the ethical criteria deployed in daily life" [33] . nevertheless, it also appears that some tourists are increasingly discerning consumers, whose choices and preferences on holiday signal identity. this means that changes in visitor tastes may be used to improve animal welfare standards in the future [34] . certainly, there is appeal for many western tourists of attaching oneself to practices with epithets such as "sustainable", "eco" and "ethical" tourism [35] . nevertheless, there is cause also to be wary of eco-labeling and green-washing in the industry, and even words like animal "sanctuaries" [31] as these, too, function as neutralising cognitive dissonance. in light of such profound challenges to animal lives and welfare, what is the future of animal-based tourism? can we expect to see a diversification in the sorts of animals commoditised, the destinations offered and array of interactions with animals available? parallel to this and as a result of a few notable scandals (such as cecil the lion, blackfish, and exposures of phajaan, breaking of the will of elephants to get them docile in thailand), can we also expect increased scrutiny of the industry in terms of its treatment of animal workers? a doctrine of cultural relativism prevents legislation from managing all animal-based recreational practices too uniformly, but consumers themselves may, as indicated, become more selective across practices that receive negative reviews or public naming and shaming. the landscape for animal-based tourism is shifting unpredictably, however, in light of covid-19 of 2020, which saw a simultaneous drop in tolerance for practices involving interactions with wild animals and an increase in media stories on how animals in captivity were coping without visitors everyday-sometimes poorly, prompting concerns they were lonely and liked people to visit them. the lack of tourists due to the covid-19 pandemic has had two other contradictory impacts; on the one hand, animals seem to benefit from abandoned natural areas, e.g., national parks and beaches, where they increase their presence, prompting the atlantic to call the coronavirus "the biggest conservation action" of this time [36] . on the other hand, fewer tourists mean fewer resources and less incentive to protect wild animals and biological diversity, which may come with its own risks. in namibia, the world travel and tourism council estimates a loss of us $3.2 million in annual tourism revenue following covid-19, an additional us $3.5 million loss of staff salaries, and increased poaching by locals who have lost their livelihoods in tourism businesses [37] . setting out to answer some of the most burning questions on the future of animal-based tourism, we arranged a two-day interdisciplinary symposium at the swedish university of agricultural sciences in august 2019, titled: instragranimal: animal welfare and ethical challenges of animal-based tourism. the following report is a synthesis of its discussions and next steps in research, policy and practice. the symposium featured some fifty participants across disciplines and sectors, involving veterinarians, ethologists, ecologists, animal and environmental ethicists, philosophers, sociologists and tourism scholars on the one hand, and on the other hand practitioners from animal-tourism ventures and animal welfare and animal rights non-governmental organisations (ngos). the spread of invited presenters was global, calling for researchers at the forefront of tourism studies in leading hubs in australia and new zealand, as well as ethologists based at the swedish university of agricultural sciences. the symposium marked the gathering of these people for the first time, and also the first time that animal-based tourism was approached holistically and not divided across, e.g., prior consumptive/non-consumptive, captive/wild axes, geographical regions or segregated across industries such as hunting, agri-or ecotourism. the rationale for this was to identify common challenges across these dimensions. in what follows, we synthesise the discussions that were held during this symposium into four themes. the discussion was open to the public on the first day. on day two, it was organised in closed workshops for registered participants. these sessions sought to collate the topics and reflections of the two days into action points or themes for future research. the themes were chosen together with participants in plenary. they build on the ideas presented in the above introduction, discussing new arenas for animal tourism, societal drivers behind the phenomenon and compassionate animal tourism. at the end of each of these four themes, we present a short section containing next steps on three levels: directives to legislation and policy, guidelines to tourists and calls for further research. the themes reflect the main aims of the symposium, which were to look toward the future as to: 1. identify challenges in animal welfare and animal ethics in tourism; 2. identify new animal tourism developments conceptually or empirically; 3. explore and suggest needed regulative responses on the part of governments, international bodies or pressure from animal protection and rights ngos and consumers of tourism, to secure the development or enforcement of welfare standards; 4. to develop calls for future research on animal-based tourism, both intradisciplinary and across disciplines. the selection of invited presenters at the symposium started with erica von essen and johan lindsjö conducting a literature review and survey of tourism research centers globally that showcased prominent researchers writing about animal-based tourism, animal welfare and ethics. a list of fifteen researchers was generated at first stage and presented as part of the application for the grant supporting the symposium. these researchers were then reached out to and personally invited by email. a desire was to span the three contexts of animal tourism: ecotourism, hunting tourism and agritourism. media were also present during the two days and followed up on the findings afterwards, in radio, tv and a hunting journal. in the following section, we present four themes of animal-based tourism that merit more discussion, research and/or legislation. one working group at the symposium outlined the societal drivers and global processes that promote animal-based tourism on the one hand, and on the other hand the types of drivers and processes that may mediate people's tolerance to animal suffering in these settings. here, world events, paradigms and value shifts were discussed as to their impact on the industry going forward. a key premise in this theme may be said to be the alleviation of responsibility from the individual tourist or tourism operator, to consider macrolevel drivers responsible for the industry's appearance today. what will be the long-term impact of the so-called flight shame movement on animal-based tourism abroad? it has been speculated that a decline in long-distance travel, exacerbated also by covid-19 in 2020, may give rise to new local forms of tourism with animals. on this argument, the proximate and the everyday in the animal context may be exoticised and commoditised in, for example, staycations and day trips. this may partly account for the popularity of local agri-tourism, where nearby farms are visited [38] . these sites offer reconnecting also with local economies and pastoral culture-something that no doubt would have seemed absurd only a few decades ago. in sweden, "cow releases" are the new agri-tourism happenings that bring families and urban residents to see local farms put their cows out to pasture for the summer grazing period, freeing them from the confinement of the winter barn the pastiche of rural life is at once two forms of "liberation": a physical one for the cows to be enjoyed as spectacle, in terms of relieving the cows from indoor confinement and giving them the opportunity to realise themselves as bovines in the grassy field; and a spiritual one for urban visitors, who may experience the temporary alleviation of alienation from the modes of production of their dairy that they consume daily. however, the turn to local tourism need not necessarily be in the service of a rural renaissance. some suggest that given the urban is becoming a significant haven for wildlife, intuitively following greater urbanisation [39] and its concentration of human-animal interaction [40] . today, several animal tourism activities take place within the city: cat cafes and zoos, tours of the city, animal walks, but also less organised experiences such as spotting urban wildlife: pigeons in venice, rhesus monkeys in indian cities and wild boars in berlin. while there has traditionally been a strong focus on experiencing pristine nature, in particular in so-called marketed wilderness tourism and last-chance tourism, which includes endangered species, the future may have to adapt to a "messier" nature that is characterised by multispecies interfaces, including a strong human presence [41] . insofar as the city may be a location for such tourism, of course, there are biosecurity concerns regarding the conditioning of wildlife to feeding, trespassing human-inhabited areas, affecting sanitation and increasing zoonotic disease risk [42] . a future-oriented research agenda on animal-based tourism needs to consider the flows of people, the push and pull factors present in various parts of the world at any given time and may even need to attend to the long-term changes in tourism circulation, such as climate change opening up routes in the arctic following the melting of ice caps. the increase of invasive species, posing a threat to pristine tourism based on native flagship species, may need to be problematised and reconsidered as a potential tourism revenue. we recognize that tourist routes may be shifted with climate change and geopolitical processes, but tourist preferences may shift independently of the physical, in longer term value shifts. as we enter a post-industrial society, there may be less emphasis on the accumulation of wealth (as has characterised agricultural societies) and more on experiences and how they contribute to a person's identity. given this, one might ask whether travel may increase, but travel-associated accumulation (such as physical souvenirs) may decrease. as tourism becomes a ritual context for showing identity-based goods, moreover, animal tourism experiences may be valuable to establishing a person's status [43, 44] . within this, last-chance tourism, danger tourism and slum tourism in relation to interacting with animals may feasibly be on the rise among some tourists [3, 45, 46] . although our symposium took place before the outbreak of covid-19, this is a world event which will likely have profound impacts on human mobility more broadly, and where and how tourists choose to engage with animals on holiday. biosecuritisation of borders will mean a changed travel landscape. the pandemic helped to shed a light on animal malpractices in parts of the world and generally opened the public's eyes about the risks of human-wildlife interfaces and the dangers of keeping several animal species in confined or shared spaces for human consumption or leisure. on a fundamental level, the spatial concentration of animals into confined areas for tourism consumption involves heightened biosecurity risks [47] . since many enterprises in animal tourism, including zoos, keep animals in these ways, change may be afoot. this may extend also to the practices of tourists, who may be compelled to adopt more clinical, hygienic ways of interacting with animals and animal-derived products, involving the washing of hands, wearing of gloves or masks, quarantining in time and space following certain encounters and handling animals or products of animals with greater care or sanitation (such as the meat from hunting trips). research should begin to apprehend how the idea of increased biosecurity risk is impacting animal tourism, not merely from a legislative point of view, but how such risks add to, detract from or otherwise impart changes in clients' experience of the tourism activities. recognising that part of the drivers for animal-based tourism is in self-fulfillment and escape of "inauthentic" lives [48] , a discussion also needs to be held on the various extreme forms that help to realise such self-fulfillment. within this, gender and masculinities play a role in enacting animal tourisms. indeed, animal tourism may be an arena in which ideas of gender can be played out and negotiated, given that touristic settings are a liminal space partly freed from everyday constraints [49] . moreover, that animal interactions can inform one's gender identity seems apparent with an entertainment industry that readily commodifies macho, primeval and atavistic encounters with wild animals, where nature is marketed as a kind of antidote to the feminising influence of modern city life [50] . the popularity of survival shows featuring wilderness rangers, bear grylls and survival and self-sufficiency guides appears to testify to a masculine domain of taming the wild. hunting packages such as the manly-titled scottish, and alaska: rampage, invite a particular clientele (see, for example, the macho "shoot-outs". often, the animals involved in these trips are dangerous-predators or game that fight back [51] . oppositely, contexts involving care and nursing relations with animals on holiday, including bottle-feeding baby animals or volunteering at shelters, may be both a female domain and a context in which alternative notions of masculinity can be played out. bertella [43] suggests that direct experience of wild animals may contribute to emotional and cognitive capacities that support caring attitudes, but there is a possibility these engagements may also be pastiches-exaggerated or contrived. below we present a simplified table 1 that addresses next steps for this theme. table 1 . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on the impact of broader societal structures on animal-based tourism. develop, review and ensure implementation of animal welfare legislation and "best practice"-guidelines in animal-based tourism, nationally and internationally. go local and explore animal-friendly and ethically justifiable animal tourism venues at home before flying across the world. be a responsible tourist-inform yourself, contact travel retailers and tour operators, demand animal-friendly and ethically justifiable approaches to animals in tourism. society s view of animals' roles in animal-based tourism-how do the perceptions, values and attitudes of tourists correspond to those of tourism operators and animal welfare organizations? possibilities to stimulate local, animal-welfare-friendly and ethically justifiable animal-based tourism. how gender is performed, contested and negotiated in animal encounters in animal-based tourism. local customs and universal animal welfare standards sometimes clash. in many cases, the animal tourism industry is a significant source of income livelihood for local communities. this means that when external pressures are put on destination communities to restrict or prohibit traditional animal uses, we are presented with ethical dilemmas between human and cultural sustainability and animal welfare. oh and jackson [52] write that these instances present a dilemma between two both dominant cultural scripts in late modernity: on the one hand, one pertaining to multiculturalism, individual choice and cultural rights, and on the other hand, one pertaining to animal rights. both, in effect, carry weight today and represent directions for policy and legislation. the latter script pertaining to animal welfare is frequently accused of cultural imperialism or ethnocentrism [53] , as cultural rights and freedom of choice to animal leisure and cuisines may be a point of pride for many nationals. one can also see the dilemma as between two other cultural scripts: one of cultural globalisation and commercial homogenisation [52] and one of cultural protectionism, respectively. in a world where we try to simultaneously adhere to these opposing scripts, increased market and legislative pressure is placed upon parts of the world such as china where animal tourism practices are generally more permissive [54] . however, accommodating changes toward greater welfare is sometimes seen by critics as caving to international pressures [52, 55] . oppositely, scholars observe how the presumption "in favour of right to culture" [53] that endorses the multiculturalism script now presents " . . . a real danger of exoticising and marginalising immigrant minorities, placing them outside of the circle of moral dialogue, criticism and community" (p. 244). a related human rights concern that may challenge animal welfare is the increased catering to persons with disabilities. for the most part, this poses no additional problems for animals, but when animal treks are made to transport obese persons across long distances, animal welfare problems may arise. such abuse has been documented in the case of donkey rides in seaside tourism in several parts of europe, notably greece and portugal [56] . the dilemma raises questions about the expediency of hard versus soft regulatory mechanisms to implement animal welfare regulations globally, relating to both governmental legislation and voluntary standards [57] . a neoliberalisation of government policy may exacerbate the establishment of regulation. in these cases, should we trust to market and, e.g., corporate social responsibility to push through improved standards of animal welfare in tourism? where market demand is too slow in bringing about change, additional questions are raised as to who exactly will regulate, who will inspect and whom this will affect (travelers, countries, operators, etc.). destination marketing organisations (dmos) and ngos may be able to operationalise locally universal codes of conduct. this "aspirational" universal code must be somewhat flexible to allow for local context. lovelock and lovelock [32] write that this is in line with the multifunctionality of codes of conduct, which are to educate, aspire and regulate. moreover, this aspirational code needs to be dynamic also across time, allowing for constant evolution as conditions and priorities change. hultsman [58] points to a needed separation between a paradigmatic (aspirational) code of ethics and an operational code of ethics. further, without either of these codes worked out in connection with local communities with, e.g., ngos and dmos, such regulation is likely to do more harm than good and be seen as imperialist. the above presuppose a dilemma between animal welfare and human sustainability, but ways forward may include a stronger emphasis on the interrelation of human and animal welfare, as in the "one health", "one welfare" concept [59] . otherwise stated, we need to appreciate that animal welfare and human rights are not diametrically opposed. addressing animal welfare within the sustainable development goals [60] specifically may be a potential way forward in acknowledging the intersectionality of human and non-human oppressive conditions in tourism. indeed, pitting these against one another is likely to undermine not only the welfare of both, but human-animal relations, as protected animals often become the subject of resentment among locals. following the killing of cecil the lion and the uproar of the western world, some zimbabweans expressed concern that people in the west cared more about lions than the predicament of poor zimbabweans. it is also important to note that western culture does not have a monopoly on animal welfare and rights, or compassionate animal practices. hence, attempts to implement a typically western model for other countries may be counterproductive insofar as cultures may come with their own existing and historically grounded repertoires of animal ethics, notions of stewardship and benevolence [32] . following this, codes of conduct pertaining to animals would do well not only to list prohibitions, but to emphasise cultural virtues in dealing with animals. it may then be more effective to build on these than to implement top-down directives. it must also be recognised that there are substantial differences in moral values and animal practices also within societies [32] . as covid-19 intimated, many chinese may already be opposed to wildlife markets [61] . below in table 2 , we present an overview of next steps for research and practice in regard to the cultural relativism challenge meeting animal-based tourism. table 2 . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on cultural relativism. develop, review and ensure implementation of animal welfare legislation and "best practice" guidelines in animal-based tourism among travel retailers, tour operators and animal users, emphasising the benefits from a sustainability and human perspective as well. develop, review and implement legislation and guidelines about information, certification and labeling, implementation of codes of conduct, also including benefits from a sustainability and human standpoint. be a responsible tourist-inform yourself, contact travel retailers and tour operators, demand animal-friendly and ethically justifiable approaches (compassion-do no harm) to animals, humans and the environment in tourism (one welfare). push for certification, labeling and information before and during traveling, a code of conduct, based on one welfare. attitudes and compliance of certification, labeling and information about animal-based tourism-potential cultural differences. the roles and responsibilities of humans in animal-based tourism. impact on un sustainability goals from animal-based tourism-and its interconnection. animal-based tourism from a one welfare perspective. technology can powerfully mediate distance and interactions with animals, changing the way we see and think about animals [62] . it can bring us into heretofore unmatched proximity; new techniques such as drone-based thermal images and camera trapping invite us squarely into the everyday lives of wild animals in dens and burrows [63] . no doubt, the success of bbc's planet earth series owes much to the advances in technology that enable us to learn more and get closer and into areas or animal behavior previously hidden from view. for this reason, it should be asked to what extent a fully or semi-virtual animal-based tourism may be on the rise today. we may also ask the extent to which this may replace or complement "real" encounters, thus taking some pressure and stress off the animals in their habitats, enclosed or wild. in times where crowding and overtourism is a real concern [64], not just for the "victims" of tourism (animals, locals, cultural heritage and property) but also as something that denigrates the tourism experience also for the clients, more remote viewing appears promising. indeed, the use of binoculars to improve views, trail and surveillance cameras, sometimes even mounted in the nests and dens of animals, allow intimacy without getting civilians physically close to animals. the use of drones capturing footage, which can now come extremely close to many wild animals without or at least cause less disruption to their behavior, may hence allow for remote viewing close-ups. to this end, this provides only visual satisfaction for tourists, and the thrust of the embodied turn in animal-based tourism is that of advocating for a multisensory engagement that transcends mere passive viewing, involving tactile and auditory senses [6, 65, 66] . one striking development that has effected human-wildlife relations is the democratisation of the access and scope of technology used to document animal encounters. no longer the purview of professional photographers, amateurs can capture footage of wild animals with their smartphones, camera traps and take part in apps that tell them where animals are. such technology has formed the basis for several citizen science monitoring programs [67] . mobile phone technology can thus bring power down to the individual level, permitting decidedly personal encounters memorialised in custom photos. taken to its extreme, the technology could also be brought down to the animal level where animal-mounted go-pro cameras show animal points of view and agency. the ability of ordinary people to autonomously zoom in on cameras affords a potentially personalised, intimate view of animal lives that is not captured in edited documentaries that often focus on grand spectacle. moreover, future research needs to consider the use of technology from the animal side, in terms of using apps and programs to communicate their needs to us, or games on, e.g., ipads for stimulation in enclosures. recently, for example, virtual reality goggles fitted for cows were devised to stimulate green pastures for the cows when in reality, their habitats were confined indoors. could this technology be used to contribute to animal welfare goals in animal-based tourism or would it perhaps make it worse? technology also provides a divisive subject in mediating the human-animal experience in animal-based tourism. discussing "cheater technology" [68] , critics have argued that too sophisticated equipment detracts from the animal encounter. hence, an app that lures birds to a site may be frowned upon by dedicated birders; heli-hunting is seen as "not real hunting" by hunting tourists, and the addition of technological "comforts" in nature-based trips, such as ipads and microwave ovens, may be criticised by tourists who value authenticity. use of and acceptance of technology will vary across animal-based activities, across demographics and between different types of technology. as contended, technology used in the service of promoting the virtues and authenticity of the animal experience may be permitted, while ones that do away with these virtues may be criticised and prohibited or phased out. this coheres with scholars' assertion that technology is neither good nor bad, instead showing an ambivalent face, being "empowering and hindering at the same time" [69] . just as technology may alter or distort a tourism experience from some ideal type, technology may also serve to reproduce certain representations of animals as caricatures and facilitate the continued consumption of these animals in particular ways. burt [70] writes that technology and visuals do not merely reflect but constitute animal ethics. the way we film particular species, and what we leave out for viewers, serve to shape our notions of animals. hansen et al. [71] suggest that the public's vocabulary for communicating about the environment is predominantly visual. technology has thus meant greater scope for manipulating animals into televisual commodities " . . . packaged for the purposes of eliciting donations, membership monies, and repeat visits" which is reflected in our treatment and legislation of them. social media are an intuitive context and platform for both tourism advertising and generating expectations on animal encounters and for clients potentially disseminating critical reviews and exercising moral reflection [49] . in a time of perpetual documentation of our experiences on holiday and our life achievements generally [48] , animal tourism experiences are lived out again on social media and reviewed on travel and booking platforms-making animal tourism also a digitally reproduced endeavor. here, the influence of "intermediaries" between tourists and the industries, including expedia and tripadvisor, play a critical role. influencers on instagram showing close contrived encounters with wild animals, including #slothselfies and cuddling with tiger cubs at thai "sanctuaries", are now recognised as an increasingly harmful driver to animal tourism, insofar as it mischaracterises animal interactions. concepts such as "social envy", stemming from viewing others' holiday experiences online, and e-lineation, describing a myopic or outright harmful representation of the real thing, are now explored in relation to animal tourism [48] . instagram has now taken action to delete images associated with certain hashtags associated with poor animal welfare, or advancing alerts for them [72] . lastly, technology provides opportunities for edutainment in animal tourism: clients learning about the animals, their biology and ecology, not only through footage captured by sophisticated camera technologies, but interactively on digital platforms. they can also feature a kind of learning with animals, as through the anthropomorphising of particular animal into pedagogical figures for edutainment. many zoos today feature interactive quizzes and ways to educate and entertain visitors. indeed, verma, van der wal and fischer [67] discuss whether technology may be part of an important bridging communication tool between policy-makers, the media, conservation practitioners and the general public. cloke and perkins [73] suggest cetacean encounters in dolphin tourism, both to entertain and to educate, heavily rely on an assemblage of technology: sonar, telecommunications, radar, spotter planes and more, which may be obtrusive to the dolphins. here, a recurring challenge may be achieving a balance between presenting animal lives through "simulated spectacle" and the "objectivity of science" [74] , balancing the public's investment from emotion and cognition, respectively. digital learning is not necessarily without its risks. within the context of many action-based tourism activities such as hunting and fishing, one now no longer learns skills from family mentors to the same extent as in the past, relying instead increasingly or at least in large part on influencers and guides on social media platforms such as youtube, for good and bad. as contended earlier, moreover, technology also allows for enhanced manipulation through editing and effects. grazian [75] notes that animal tourism educators still have significant power in selecting the parts to be displayed and often mute key aspects of animal lives to suit the particular audience, something which they can easily do with technology. insofar as the representation of animals and the gaze with which they are represented can powerfully constitute ethics and human-animal relationships, care needs to be taken to not misrepresent reality. below in table 3 , an overview is provided in a table format on suggested next steps and topics for a research agenda on the role of technology in animal-based tourism. we also outline ways forward for policy and practice. table 3 . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on the role of digital technology in animal-based tourism. outreach and education about animal welfare and ethical challenges, resulting in guidelines for web-based platforms and influencers. certification and labeling on internet-based platforms (websites, social media) informing about and promoting animal-based tourism activities. implement codes of conduct. promote development and implementation of virtual animal-based tourism (see 3rs in section 1.4.4). develop and implement legislation/guidelines about using animals first when technology cannot replace use of real animals. require that web-based platforms and influencers consider the animals' situation and ethics surrounding animal use, demand that they take a standpoint (a condition for your attention, you following them, etc.). require tour operators to consider and implement technical development replacing, reducing and refining animal use. the impact of web-based platforms, including influencers, on animal-based tourism and how they can promote animal-friendly and ethically justifiable tourism. a moral dilemma that meets many forms of animal-based tourism is that the lives of individual animals are essentially "sacrificed" to benefit the species. this recurs across several tourism sectors: in hunting tourism perhaps most palpably, trophy hunting has been promoted on the basis of securing biodiversity conservation [76] . it is now a prevalent slogan across hunting tourism campaigns to "be the savior" of a species by killing its individual specimens: the "kill it to save it" narrative [5] , in which the revenues from hunting tourism are said to ensure the survival of endangered species. according to many hunters, therefore, the benefits outweigh the potential harms in canned hunting [77] . although here animals individually pay the ultimate price for the survival of their wild cousins, other forms of animal tourism showcase a similar logic in which the welfare of the individual animal may suffer so that this particular individual can serve as a flagship animal for its species [78] . thus, zoo tourism is now promoted as a necessary evil in which the lives of charismatic megafauna in particular are devoted to ambassadorships for the greater good of their species [79] . the thrust of this dilemma is a trade-off between individual welfare of sentient beings and species level welfare, often conceptualised as a conflict between the sentientistic and ecocentric. the difference in moral patients across these two points of departure underpins a range of conservation conflicts today. animal welfarists accuse ecological managers of allowing the sacrifice or suffering of individual animals to save the whole. this is especially so when there is epistemic uncertainty about the consequences of our interventions on the ecosystem. managers, on the other hand, express frustration with what they see as the short-sightedness of the welfare perspective [80] . the ecocentric, holistic perspective runs into objections on the difficulty of speaking on behalf of the good of a whole species or ecosystem, in which seemingly grandiose claims can be made that sanction the killing of individual members [81] . nonetheless, the sentiensistic perspective comes with obvious limitations in prescribing no particular moral obligation to endangered species or the preservation of wild habitats for the sake of species conservation alone (only insofar as its members are happy): a cow has the same right to life, and living a good life, as a white rhino. moreover, if one white rhino were to be used for spreading knowledge and awareness about the plight of his species, by being placed in a life of captivity, this cannot be condoned on the sentientistic deontological rationale. given the ostensible incompatibility of these two points of departure, which one ought we to prioritise when there is a conflict? in animal-based tourism, which level of being should be ascribed moral priority? frustrated with the trade-off, a growing body of scholars have embraced so-called compassionate conservation, which promotes the consideration of animal welfare in conservation, benefitting both individuals, species and conservation outcomes compassionate conservation [82] is debated, mainly because of the inherent conflict between the cost for (welfare of) the individual animal and the greater good for a population or species (i.e., is it possible or not to apply compassion in successful conservation activities). burns [83] extracted valuable lessons from an approach that embodies compassionate conservation in wildlife tourism. we further extend the concept of compassionate conservation to compassionate animal-based tourism, i.e., the welfare of the individual animal within all kinds of tourism; eco-, hunting and agritourism. hence, we argue that individual animal welfare is compatible with responsible tourism. to this end, two approaches may be used to practice compassionate animal-based tourism: first, the 3rs (replacement of animals with alternative methods, reduction of the number of used animals and refinement of the methods, including housing and care, to mitigate suffering and promote animal welfare), initially developed to improve animal welfare for animals used in research [84] , can be applied in other areas as well, including animal-based tourism. the initial question is: is there a need to use animals at all? is the interest for the tourism (and thus society) bigger than the cost of the individual animal? from a compassionate perspective, are there any activities where the use of animals can never be accepted? animals can be replaced with virtual reality or completely replaced by tourist activities without any animal theme. if animals are involved, how many animals need to be involved in a given activity? in line with reduction, zoos, amusement parks, elephant and horse riding camps and farms may exhibit fewer species or fewer individual animals within a species, fewer species or individuals have to be affected by safari or trophy hunting activities, catch and release fishing, etc. refining the treatment of animals used in tourism will ensure a good welfare and compassion for these animals. housing (captive animals), exposure, care and handling that enable natural behavior, health and positive feelings will not only benefit the individual animal, but also groups, populations and species, especially if these are small or otherwise vulnerable. compassionate animal-based tourism relies on animal protection, i.e., what we do, or ought to do, to provide a good animal welfare through legislation, but also education, policy making and, importantly, information that reaches out to tourists, industry and decision makers. the second approach emphasises the need of information to the tourists. certification and labeling of products and services, including the introduction of codes of practice, are well-known strategies to inform consumers. we believe that this is one strategy to help tourists to make animal-welfare-friendly and compassionate choices when traveling. to achieve credibility in society, this needs to be based on scientific knowledge and collaboration between ngos, academia, industry and local people involved in, or otherwise affected by, tourism. another way to reach out to tourists is to provide information about animal-based tourism at travel hubs, i.e., airports, ferry terminals, train and bus stations, car rentals, etc. to ensure effective communication, pr strategists and experts in advertising should be involved. the benefits for animals, humans and the environment from a responsible tourism need to be communicated. the final table 4 summarises next steps for a compassionate-based animal tourism. table 4 . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on compassionate animal-based tourism. policy: develop, review and ensure implementation of animal welfare legislation and "best practice" guidelines (based on research on animal health, physiology, behavior, emotions and natural living) among travel retailers, tour operators and animal users. ban non-acceptable animal activities in tourism. develop, review and implement legislation and guidelines about information, certification, labeling and introduction of codes of conduct. be a responsible tourist-inform yourself, contact travel retailers and tour operators, demand animal-friendly and ethically justifiable approaches (compassion-do no harm) to animals in tourism. push for certification, labeling and information before and during traveling. require tour operators to include a 3r approach, replacing, reducing and refining animal use. attitudes and compliance of certification, labeling of and information about animal-based tourism-potential differences between activities, species and demography. health, physiology, behavior, emotions and natural living with regard to different species and if and how they are suitable in animal-based tourism. knowledge, attitudes, identification and implementation of compassion and the 3rs in animal-based industry. we began by highlighting current debates in the field of animal-based tourism as this industry is gaining in popularity and scope. animal welfare challenges were identified and traced to cognitive dissonance among tourists and to structural barriers of the industry. we noted that animals become laborers in a global capitalist economy when they are conscripted into the service of the tourism industry. the form of labor that they provide varies. in the above sections, animals featured in various roles, including: • a kind of spiritual commodity [73] , including serving as totem animals; • a mode of transportation, such as donkey treks or dog-sledding in the arctic; • a laborer in the background to a more center-stage tourism activity [32] , such as how research has found that having wolves in an area where hunting tourists hunt herbivores adds to the overall experience [45] , or goats in the background to a farmers' market; • a culinary delight [30] , such as alligator meat in louisiana [5] ; • a front-stage performer, such as animals doing tricks in circuses [27] ; • a marker of place, such as the kangaroo or koala for australia [11] ; • a "facilitator" of leisure [13] , such as an animal trained to serve drinks; • the face of a souvenir or toy [5, 43] ; • the ultimate sacrifice as a game animal to be hunted as part of the big five. our symposium, devoted to identifying the main challenges for this industry from an ethical and welfare perspective, generated four overlapping themes worthy of further exploration: macro processes as drivers to animal-based tourism, cultural relativism as a potential challenge to implementing universal animal welfare standards, the role of technology in enhancing, promoting or even replacing animal-based tourism, and the potential for a compassionate animal-based tourism that could reconcile animals' well-being with tourists' interest. these are themes that point, above all, to a complex and changing landscape for animal-based leisure, with an uncertain future. a shift in public perception toward either greater reverence for animal welfare and condemnation of unethical practices, or increased concern toward host destinations and locals needing to make a living in an uncertain future, will make or break some animal-based tourisms. likewise, based on present trends and values, we suggested that global travel patterns and pandemics will significantly impact the future of certain tourism activities. in addition to the above cross-cutting themes, we noted also tension between needing to balance the educational and entertainment functions of animal-based tourism, respectively. "edutainment" seeks to provide both, but there are inherent challenges to finding an ethical, workable and profitable balance. in this regard, technology may aid in bringing us closer into animal lives and facts without becoming unduly obtrusive. this is in line with the 3rs (replacement, reduction and refinement). however, we also noted some risks related to technology with regard to tourists' experience and insights. for example, online reproduction of animal-based tourism and its tendency to convey partly selective or false impressions may hence build expectations and contribute to a culture of commodification. furthermore, in the suggested next steps for each of these themes or challenges for animal-based tourism, there are two levels to contend with: the structural level and that of individual clients. as evidenced, both literature and policy struggle to determine which of these levels is the most expedient to try to impart changes on. a third and final tension identified by the scholars of our symposium was that of reconciling sentientistic welfare perspectives-valuing the well-being of the individual animal-and ecocentric perspectives. indeed, this cut across multiple tourism sectors. there is as of yet no consensus on whether it is morally right to "sacrifice" animal lives to benefit their wild species kin. ultimately, this raises a larger discussion on the value of the wild, the loss of value of ex situ confined animals in zoos or parks. in the end, the future of animal-based tourism may be somewhat uncertain. one might anticipate that its growing popularity may paradoxically undermine its success, as it is often predicated on selling the rare and exotic. hence, if these products become too commonplace, animal engagement may lose part of its appeal in the future. nevertheless, as citizens, we have a moral obligation to travel with responsibility and use compassion for animals involved in tourism. facing the wild: ecotourism, conservation, and animal encounters staging tourism: bodies on display from waikiki to sea world tiger tourism: from shooting to petting an analysis of zoo visitors' favourite and least favourite animals consuming the king of the swamp: materiality and morality in south louisiana alligator tourism neo-darwinian leisures, the body and nature: hunting and angling in modernity the cow goes moo: farm animal and tourist interactions on long island's north fork nature-based tourism animals & modern cultures-a sociology of human-animal relations in modernity volatile ecologies: towards a material politics of human-animal relations multispecies leisure: human-animal interactions in leisure landscapes animals in the tourism and leisure experience exploring the boundaries of a new moral order for tourism's global code of ethics: an opinion piece on the position of animals in the tourism industry fear of the animal planet: the hidden history of animal resistance animal resistors: on the right of resistance and human duties of non-return and abolition tourism and animal welfare towards an ethical framework for animal-based attractions captive wildlife at a crossroads-sanctuaries, accreditation, and humane-washing asian elephant (elephas maximus), pig-tailed macaque (macaca nemestrina) and tiger (panthera tigris) populations at tourism venues in thailand and aspects of their welfare the customer isn't always right-conservation and animal welfare implications of the increasing demand for wildlife tourism animal welfare in tourism services: examples and practical tips for the well-being of animals used for tourism in lapland tourists' attitudes toward the use of animals in tourist attractions: an empirical investigation circus and zoo animal welfare in sweden: an epidemiological analysis of data from regulatory inspections by the official competent authorities farmed animal sanctuaries: the heart of the movement? politics anim biological impacts of ecotourism-tourists and nesting turtles in behavioural effects of tourism on oceanic common dolphins, delphinus sp., in new zealand: the effects of markov analysis variations and current tour operator compliance with regulations beasts and tourists: human-animal relationships in tourism ethical fading: the role of self-deception in unethical behavior the attitude-behavior gap in sustainable tourism abstracting animals through tourism unethical use of wildlife in tourism: what's the problem, who is responsible, and what can be done? animal ethics, dietary regimes and the consumption of animals in tourism wildlife tourism the importance of the aesthetic lockdowns could be the 'biggest conservation action' in a century the coronavirus threat to wildlife tourism and conservation agri-tourism: in between rural change, tourism restructuring and environmental imperatives a new urban dispositif? governing life in an age of climate change public perceptions and implications for species management. hum. dimensions wildl animal geographies i: hearing the cry and extending beyond biosecurity and the topologies of infected life: from borderlines to borderlands an eco-feminist perspective on the co-existence of different views of seals in leisure activities leisure, labor, and the complexity of culture: an anthropological perspective the competitive consumption and fetishism of wildlife trophies the reflexive tourist distribution and interspecies contact of feral swine and cattle on rangeland in south texas: implications for disease transmission not such smart tourism? the concept of e-lienation neo-colonialism and greed: africans' views on trophy hunting in social media tourism: an introduction a room of (his) own: italian and italian-american male-bonding spaces and homosociality animal rights vs. cultural rights: exploring the dog meat debate in south korea from a world polity perspective multiculturalism goes imperial: immigrants, animals, and the suppression of moral dialogue cultural variation, animal welfare and telos current status of animal welfare and animal rights in china the oppression of donkeys in seaside tourism just tourism: an ethical framework one welfare-a platform for improving human and animal welfare what we've got wrong about china's 'wet markets' and their link to covid-19 digital technology and the conservation of nature what do we know about wild boar in iberia? are tourists interesting? ways of knowing for 'response-ability' in more-than-human encounters: the role of anticipatory knowledges in outdoor access with dogs becoming tourist: renegotiating the visual in the tourist experience microscope and spectacle: on the complexities of using new visual technologies to communicate about wildlife conservation troubled shooting-the ethics of helicopter-assisted guided trophy hunting by tourists for tahr animals in film researching visual environmental communication exclusive: instagram fights animal abuse with new alert system cetacean performance and tourism in kaikoura seeing green: knowing and saving the environment on film where the wild things aren't selling the serengeti: the cultural politics of safari tourism the seven sins of hunting tourism killing with kindness: when hunters want to let you know they care problematic animals in the zoo: the issue of charismatic megafauna alternative facts and alternative views: scientists, managers, and animal rights activists animal liberationism, ecocentrism, and the morality of sport hunting promoting predators and compassionate conservation ethics and responsibility in wildlife tourism: lessons from compassionate conservation in the anthropocene the principles of humane experimental technique; methuen: london, uk, 1959. © 2020 by the authors we want to sincerely thank all the participants of our symposium for contributing their insights and experiences on animal-based tourism during the two-day event. we also wish to thank some key helpers without whom the event could scarcely have proceeded: jennie persson assisted during the planning and execution of the event. adélaïde fouache kindly took notes for us during the proceedings, providing the basis for this report. lara tickle supplied the catchy title back in june. finally, anna martin headed up the graphic design for the symposium poster, which we retained as our aesthetic template. there is no conflict of interest to report. key: cord-346245-o9hvuwvq authors: harvey, david j. title: analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: an update for 2009–2010 date: 2014-05-26 journal: mass spectrom rev doi: 10.1002/mas.21411 sha: doc_id: 346245 cord_uid: o9hvuwvq this review is the sixth update of the original article published in 1999 on the application of maldi mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2010. general aspects such as theory of the maldi process, matrices, derivatization, maldi imaging, arrays and fragmentation are covered in the first part of the review and applications to various structural typed constitutes the remainder. the main groups of compound that are discussed in this section are oligo and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. many of these applications are presented in tabular form. also discussed are medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. © 2014 wiley periodicals, inc. mass spec rev 34: 268–422, 2015. this review is a continuation of the six earlier ones in this series (harvey, 1999 (harvey, , 2006 (harvey, , 2008 (harvey, , 2009 (harvey, , 2011 (harvey, , 2012 on the application of matrix-assisted laser desorption/ionization mass spectrometry (maldi) mass spectrometry (ms) to the analysis of carbohydrates and glycoconjugates. it is intended to bring the coverage of the literature to the end of 2010. although the intention is to be a comprehensive as possible, there is an increasing tendency to publish maldi data in supplementary information. because of the ever-increasing number of papers and journals, it has not been possible to check all supplementary information and, consequently, papers that do not refer to maldi in the main text may well have been omitted. also omitted are papers that simply report the mass of glycoproteins and those concerned with nucleotides and nucleosides: the latter compounds, although containing carbohydrates are considered to be different types of compound. maldi continues to be a major technique for the analysis of carbohydrates; figure 1 shows the year-by-year increase in papers reporting use of the technique for the period 1991-2010. because the review is designed to complement the earlier work, structural formulae, etc. that were presented earlier are not repeated. however, a citation to the structure in the earlier works is indicated by its number with a prefix designating the review containing the structure (i.e., 1/x refers to structure x in the first review and 2/x to a structure in the second). a large number of books and review articles directly concerned with, or including maldi analysis of carbohydrates and glycoconjugates, have been published during the review period. those of a general nature are listed in table 1 ; those concerned with specific carbohydrate types are listed in the appropriate sections. details of the maldi process are still not fully understood and several investigators have attempted to obtain greater understanding. although not all of these studied have involved carbohydrates, they are included here because maldi processes for different compounds are likely to be similar. one of the commonly accepted models for the formation of analyte ions in maldi-ms assumes a primary ionization of the matrix, for example, by photoionization leading, among other things, to stable protonated and deprotonated matrix ions. peptide and protein ions are then envisaged as being formed by secondary proton transfer reactions in the expanding matrix plume. this model has been checked experimentally by hillenkamp et al. (2009) by comparing the yield of positive to negative ions of three peptides (bradykinin, angiotensin i and fibrinopeptide a) and six matrices a-cyano-4-hydroxycinnamic acid (chca, 1/23), 2,5dihydroxybenzoic acid (dhb, 1/26), 6-azo-2-thiothymine (att, 1/45), 4-nitroaniline (4-na, 3/3), 2-amino-5-nitro-4-picoline (anp, 1) and 5-aminoquinoline (5-aq, 2), differing in gas-phase basicity by about 100 kj/mol. it was shown that the observed ion yields cannot be explained by any single and consistent set of parameters such as gas-phase basicity or acidity of the analyte and matrix. it was concluded that the existing simple model needs be modified to fully explain the experimental findings. liu et al. (2009a) have used synchronized dual-polarity maldi ms to demonstrate incoherent production of positive and negative matrix ions. in both positive and negative ion modes, matrix ions were found to appear from thin, homoge-neous dhb matrix films at different threshold laser fluences. the presence of singly charged molecular matrix ions suggests that the existence of dhb ion-pairs may not be a prerequisite in the maldi process. photoelectrons induced by the laser excitation may assist the production of negative dhb ions, as shown in experiments conducted with stainless steel and glass substrates. at high laser fluences, the relative yield of positive and negative matrix ions remained constant when homogeneous matrix films were used, but the yield fluctuated significantly with inhomogeneous crystal morphology. this result was also inconsistent with the hypothesis that matrix ion-pairs are essential primary ions. thus, results from both low and high laser fluences suggest that the production of positive and negative matrix ions in maldi may occur via independent pathways. the same authors have examined the initial ionization reaction in maldi based on the appearance of photoelectrons. the threshold laser fluence for the ejection of photoelectrons from dhb, sinapinic acid (1/48) and 2,4,6trihydroxyacetophenone (thap, 1/44) on stainless steel targets was found to be 0.05, 0.41, and 8.39 mj/cm 2 , respectively. these values were considerably lower than those for maldi ions, indicating that the electron detachment probably precedes other ionization reactions. the stainless steel target was thought to general review with a section on glycolipids glycan analysis by mass spectrometry short review, maldi and esi, applications to n-linked glycans 20 (sekiya & iida, 2008) deciphering carbohydrate structures by ion mobility ms short general review of glycomics and ion mobility. applications to flavonoids, gags and glycoproteins 96 modern maldi-tof mass spectrometry development of tof mass spectrometers since the introduction of maldi 26 (vestal, 2009) maldi mass spectrometry of carbohydrates short general review (in chinese) play an insignificant role in the production of photoelectrons because suspended dhb produced a photoelectron signal similar to dhb on the surface. in addition, decreasing the dhb thickness on the target reduced the photoelectron intensity. for crystalline dhb and sinapinic acid, the photoelectron intensity was found to increase with the laser fluence (nitrogen laser at 337 nm) in less than a second order relationship, suggesting considerable reductions of ionization potentials in comparison with free molecules. according to ab initio calculations, the ionization potential of dhb clusters was found to reduce as the cluster size increased from monomer to octamer. the paper discusses the impact of these abundant electrons on ion production in maldi. the earlier rate equation model for maldi ion formation and reaction (knochenmuss, 2002 (knochenmuss, , 2003 , has been extended to include positive and negative ions of both matrix and analyte (knochenmuss, 2009) . the resulting positive/negative ratios of secondary analyte ions show that a recent static equilibrium approach is not adequate for quantitative analysis of maldi experiments. although the ion ratios remain close to unity whenever the reaction free energies are at least moderately favorable, deviations from this condition result in unequal ratios of oppositely charged ions and show once again that the dynamic aspects of maldi cannot be neglected. molecular dynamics simulations of maldi have been performed to investigate laser pulse width and fluence effects on primary and secondary ionization process. at the same fluence, short (35 or 350 psec) pulses were found to give much higher initial pressures and ion concentrations than longer ones (3 ns). these differences were found not to persist because the system relaxes towards local thermal equilibrium on a nanosecond timescale. higher fluences were found to accentuate the initial disparities. axial velocities of ions and neutrals were found to span a wide range and to be fluence-dependent. the total ion yield was found to be only weakly dependent on the pulse width and to be consistent with experimental estimates. secondary reactions of matrix cations with analyte neutrals were efficient even though analyte ions were ablated in clusters of matrix (knochenmuss & zhigilei, 2010) . lai et al. (2010) have employed transition state theory for modeling the desorption of surface ions, assuming chemical and thermal equilibrium in the solid state prior to desorption. the method was different from the use of conventional models that assume chemical equilibrium in the gas phase. this solid-state thermodynamic interpretation was used to examine the desorption of thap and of an angiotensin i/thap mixture. it successfully described the changes in ion yield with the effective temperature under various laser fluence and initial temperature conditions. the analysis also revealed the key role played by ion concentration in the modeling used to provide the best fit of the model to observations. divergence of the ion beam with laser fluence was also examined using an imaging detection method and the signal saturation normally seen at high fluence was appropriately reduced by ion focusing. simplified but deceptive theoretical interpretations were obtained when the analysis was conducted without adequate calibration of the instrument bias. the laser plume produced by several ionic liquid matrices has been studied by a post-ionization approach in which the neutrals in the ablation plume were ionized with a second laser pulse. it was found that after the initial event that produced the ions, a second, time-delayed, ablation event occurred in which the plume contained only neutral molecules. the presence of these neutral molecules was explained by a reflected-shockwave model in which the shockwave emerging from the laser ablation is reflected from the sample plate behind the sample. it was assumed that when the shockwave arrived at the sample surface it caused a second ablation of the neutral molecules (hellwig et al., 2009) . the 355 nm multiphoton dissociation and ionization of 2,5-dihydroxyacetophenone (dhap, 1/43) has been studied (dyakov et al., 2009) . the experimental results indicate that photoionization that occurs in the gas phase after dhap vaporizes from the solid phase may not play an important role in the maldi process. a combined ir-maldi ion source with an electrospray ionization (esi) emitter for post desorption ionization has been described (sampson, murray, & muddiman, 2009) . the source produced multiply charged ions from proteins but singly charged ions from carbohydrates (o-glycans cleaved from mucin (muc) were tested) and avoided the fragmentation produced by some other techniques. ir-maldi ms with a laser emission in the 6 mm wavelength range, which utilized energy absorption at the co double-bond stretch region, has been investigated for analysis of several types of biomolecule. the softness of ir-maldi ms was evident in the negative ion mode where abundant [màh] à ions were obtained for acidic biomolecules with sulfate, phosphate, or carboxylate groups. better sensitivity was obtained than with ultraviolet (uv) maldi ms. furthermore, there was no substantial loss of sialic acid due to the prompt fragmentation from sialylated glycoconjugates such as gangliosides. such loss is a common problem with maldi analysis of sialylated carbohydrates. the technique was used in conjunction with a potent new matrix, oxamide (3), resulting in the use of low laser fluence, and removing one of the limitations of maldi ms for biomolecular analysis of uv-maldisensitive molecules (tajiri, takeuchi, & wada, 2009a) . tu and gross (2009) have reviewed methods for miniaturizing sample spots for maldi analysis. topics include minimizing sample dispersion by target modification, the use of hydrophobic materials as maldi-plate surfaces, the use of microdispensing devices such as piezoelectric dispensers and the use of droplet charging by induction or polarization. a simple device for maldi sample preparation, based on the spraying of matrix/sample solution through a stainless steel sieve, has been used for the preparation of maldi samples of peptides, polysaccharides (pss) and high molecular weight (mw) proteins (cristoni et al., 2009) . the spectra obtained by laser irradiation of the resulting microspots exhibit resolution and sensitivity higher than those achieved by the commonly employed dried droplets method. furthermore, the target surface was more homogeneous than those obtained by the dried droplet method. chca and super-dhb (dhb þ 2-oh,5-ome-benzoic acid) (s-dhb) matrices were used and applications were to insulin and dextran oligomers. ultrasound produced by a simple piezoelectric device has been used as an alternative method for soft ionization of biomolecules. cavitation was proposed as the major mechanism producing the ions and the technique was applied to carbohydrates, proteins and fatty acids. however, although an abundant ion, said to be the [mþh] þ from the high-mannose glycan man 8 glcnac 2 (5/20), was obtained in the presence of dhb, thap, or sinapinic acid, neither the mass nor the stated elemental composition were consistent with this structure . the recent availability of commercial ion mobility instruments offers another dimension to carbohydrate analysis by providing the ability to separate by molecular shape and offering the possibility of rapid isomer differentiation. however, resolutions on current instruments are comparatively poor and do not match those that can be obtained with high performance liquid chromatography (hplc). a comparison of three types of ion mobility ms (field asymmetric waveform ion mobility (faims), drift tube and traveling wave ion mobility spectrometry (twims)) for separation of chiral molecules with applications to monosaccharides has been reported (enders & mclean, 2009) . data are best reported as rotationally averaged collisional cross-sections. these can be obtained directly with drift tubes and indirectly with the twims instruments. obtaining such measurements with faims instruments, however, is more challenging. collisional-cross sections have been measured for a large number of biologically relevant molecules including oligonucleotides (n ¼ 96), carbohydrates (n ¼ 192), lipids (n ¼ 53), and peptides (n ¼ 610). collisional cross sections increased with mass but were found to be different for each molecular type in the order oligonucleotides < carbohydrates < peptides < lipids. the specific correlations were best described by logarithmic regressions. thus, the technique was able to separate compounds of different structures but with the same or similar mws. in addition, some separation of compounds with the same mass within a particular class was possible. the latter point was demonstrated by separations of isobaric oligonucleotides, which were interpreted by molecular dynamics simulations mclean, 2009) . have used ion mobility to extract carbohydrate ions from incubation mixtures obtained from protein-n-glycosidase (pngase) f release of n-glycans. glycoproteins such as ribonuclease b (rnaseb) were first digested with trypsin followed by pngase f and then analyzed directly by ion mobility ms with a waters synapt instrument. the carbohydrate ions showed different mobilities from peptide and other contaminating ions allowing them to be extracted directly from the crude mixtures. both esi and maldi ion sources were used; the maldi ion source was better at ionizing the larger carbohydrates and did not suffer from the problem of producing both [mþh] þ and [mþna] þ ions that were seen in the esi source. the technique would appear to have great potential for rapid glycan analysis, particularly as the synapt instrument also offers the ability to fragment the isolated ions. hossain and limbach (2010) have reviewed matrices used for maldi analysis of several compound classed including carbohydrates. the review covers common matrices such as dhb and chca and less common systems such as liquid matrices and carbon nanotubes (138 references). the use of ionic liquid matrices has also been included in a review by liu et al. (2009e) although the bulk of this review discusses the use of ionic liquids for sample preparation. the thermal stability of several commonly used crystalline matrices, 2,5-dhb, thap, chca, sinapinic acid, nor-harmane (1/35) and harmane (1/34) has been studied by heating them at their melting point and characterizing the remaining material by a variety of spectroscopic and chromatographic techniques. in general, all compounds, except for chca and sinapinic acid, remained unchanged after fusion. chca showed loss of co 2 , yielding a trans-/cis-4-hydroxyphenylacrylonitrile (4) mixture. sinapinic acid showed mainly cis-to-trans thermal isomerization and, with very poor yield, loss of co 2 (tarzi, nonami, & erra-balsells, 2009) . 1h-pteridine-2,4-dione (lumazine, 5) has been described as a good matrix for phospholipids where the presence of cationcontaining compounds suppresses signals from neutral compounds. phosphatidyinositol was reported to give a signal that was an order of magnitude higher than that obtained from dhb (calvano, carulli, & palmisano, 2010) . a number of papers have reported the use of various nanoparticles as matrices. unlike traditional chemical matrices, these materials generally produced little or no signal in the low mass region, thus making them ideal for analysis of small carbohydrates. titanium dioxide micro-and nano-particles, prepared by hydrolysis of ti butoxide and maintained in aqueous solution, have been evaluated as matrices for the detection of several small molecules. most of the reported applications were to lipids but the nanoparticles were also applied to flavonols/anthocyanins and their glycosides in rose petals. the spectra showed ions from many more small molecules than did spectra recorded from dhb, particularly in the region of the dhb matrix ions. one advantage of the nanoparticles is that their small size (average of about 200 nm) facilitates their penetration into tissue for in situ imaging (lorkiewicz & yappert, 2009) . titanium dioxide (tio 2 ) has also be used by kawasaki, okamura, and arakawa (2010) for ionization of several types of compound, including carbohydrates, with a similar absence of low mass matrix ions. in a study of the most suitable crystalline form, the anatase-type tio 2 , was shown to be the best. nanoparticles of diamond, tio 2 , titanium silicon oxide, barium strontium titanium oxide, and silver have been examined for their potential as maldi matrices for direct laser desorption/ionization of carbohydrates, especially fructans, from plant tissue. two sample preparation methods including solventassisted and solvent-free (dry) deposition were compared. all examined nanoparticles except ag were found to desorb/ionize standard sucrose (6) and fructans in both positive and in negative ion mode. in positive ion mode, sugars gave [mþna] þ and/or [mþk] þ ions depending on the ionic composition of the sample spot, and [màh] à ions in negative mode. ag nanoparticles yielded good signals only for non-salt doped samples that were measured in the negative ion mode. when used to study fructans in plant tissues all the nanoparticles except ag could desorb/ionize carbohydrates in both ion modes. nanoparticles gave similar limit of detection (lod) for standard fructan triose (1-kestose, 7) in the positive ion mode and better lods in the negative ion mode than those given by the common crystalline organic maldi matrices such as dhb, nor-harmane or carbon nanotubes. although lower signal-to-noise ratio (s/n) signals were obtained from the tissues with solvent-free matrix deposition than when solvent was used, the reproducibility averaged over all sample was more uniform (gholipour et al., 2010) . maldi detection at the level of several hundred zmoles has been achieved by the addition of gold nanoparticles (aunps) with a diameter of several tens of nanometers into a sample solution (shibamoto et al., 2009) . n-acetyl-tetraose 1 fmol/ml gave a strong signal in the presence of 50 nm aunp (4.5 â 10 7 particles). the mechanism appears to be related to surface plasmon (sp) excitation of the aunps. citrate-capped aunps have been shown to act as matrices for the determination of several types of biomolecule, including carbohydrates such as starch, dextrins, and glycosphingolipids (gsls) in the presence of high concentrations of salt (wuhrer, koeleman, & deelder, 2009b) . with the gsls, however, some loss of sialic acid (1/11) was found. a combination of immobilized silica and dhb on iron oxide magnetic nanoparticles has been shown to give a clean background and to be appropriate for the analysis of a range of small molecules, including glycolipids . the ratio between sio 2 and dhb was found to affect the surface immobilization of dhb on the nanoparticle, critically controlling the ionization efficiency and background. enhancements of molecular ion signal over those produced from dhb alone were noted and high quality product-ion spectra were obtained. fullerene (8)-derivatized silica (pore size 30 nm) also appears to be good for small molecules including monosaccharides (szabo et al., 2010b) . multifunctional zro 2 nanoparticles and zro 2 -sio 2 nanorods have been successfully used as matrices for cyclodextrins in atmospheric pressure and vacuum maldi. the lod for cyclodextrins was found to be 7.5-20 fmol and the matrices were used to analyze cyclodextrins in urine samples (kailasa & wu, 2010) . manganese dioxide nanoparticles have been used to ionize ginsenosides with the advantage that the spectra lacked matrix ions in the low mass region allowing low-mass postsource decay (psd) ions to be clearly visible. the technique was named nanoparticle-assisted laser desorption/ionization (nano-paldi) (sahashi, osaka, & taira, 2010) . single-crystalline euf 3 hexagonal microdisks with hollow interiors have been prepared as background free matrices. the long-lived excited state of europium ions is capable of transferring energy to the molecules allowing the microdisks to act as matrices. they were successfully used for analysis of small peptides, amino acids and hydroxypropyl b-cyclodextrin (9) (chen et al., 2009d) . another new matrix with low background is mesoporous silica, sba-15, functionalized with quinoline . the material was obtained by using calcined . compared with dhb and sba-15 itself, the modified material demonstrated several advantages in the analysis of small molecules such as less background interference ions, high homogeneity, and better reproducibility. detection limits in the region of 8 pmol were reported. in a more traditional approach for reducing low-mass matrix ions, fujita et al. (2010) have used b-cyclodextrin (4/6) mixed with thap or 2,4-dhap. the latter compounds, in particular, formed inclusion complexes with the cyclodextrin as demonstrated by the lack of a similar effect when the corresponding linear carbohydrate, maltoheptaose (11) was used as a co-matrix. a method for recording negative ion spectra that is suitable for small molecules in that it produces no matrix-related ions in the low mass region uses the proton sponge, 1,8-bis(dimethylamino)naphthalene (dman, 12) as a solution in ethanol as the matrix. this compound has very high proton affinity and can take up protons from even weak acids to form deprotonated anions. moreover, dman in solution has a strong uv absorption band in the region 330-350 nm, fully compliant with frequencies of nitrogen and neodymium-doped yttrium aluminium garnet (laser) (nd:yag) uv lasers. the matrix was found to be suitable for a range of small molecules including fatty acids, carbohydrates and prostaglandins (shroff & svato, 2009; shroff & svatos, 2009; . the authors proposed to name the technique as matrix-assisted ionization/laser desorption, abbreviated as maild, ms . n(ch 3 ) 2 (ch 3 ) 2 n 1,8-bis(dimethylamino)naphthalene, 12 zhang et al. (2010i) have reported a new method for the analysis of small molecules by using matrices such as metalphthalocyanines (mpc, 13) which form matrix-analyte adducts and shift the molecular ions into a high and interference-free mass range. the mass of the target analyte was calculated by subtracting the mass of mpc from the mass of the mpc-analyte adduct. the mpcs themselves were also detectable and could serve as internal standards. various mpcs with aromatic or aliphatic groups and different metal centers were explored. aluminum-phthalocyanines (alpcs), gallium-phthalocyanines (gapcs), and indiumphthalocyanines (inpcs) were found to be efficient matrices for a large number of compound types and formed mpc-analyte adducts in either the positive or negative ion mode. the detection limits varied from 17 to 75 fmol, depending on analyte. mass spectrometry reviews doi 10.1002/mas graphene has also been reported to be a good matrix for low mw compounds with essentially no ions from the matrix (dong et al., 2010) . colloidal graphite has also been used for atmospheric pressure maldi and has been shown capable of producing both [mþh] þ and [màh] à from many types of small molecules, including glycosylated flavonoids (perdian, schieffer, & houk, 2010) . two reviews on ionic liquids, including their use as maldi matrices have been published (soukup-hein, warnke, & armstrong, 2009; sun & armstrong, 2010) in an extensive study to find new liquid matrices 114 matrices were tested and 105 new ionic liquids were prepared (crank & armstrong, 2009) . both the anionic and cationic moieties were varied systematically to find a matrix with the best physical properties, analyte signal intensity and widest mass detection range. the developed matrices showed a wide mass detection range (<1,000 da to >270,000 da) for proteins and peptides with greater s/n ratios than solid matrices and could effectively ionize proteins of large mass without disrupting noncovalent interactions between monomers. it was found that both the proton affinity and pk a of the cation have a large effect on the ability of the matrices ionize the analyte. the matrices could be used to detect carbohydrates with fewer degradation products than solid matrices. n,n-diisopropylethylammonium a-cyano-4-hydroxycinnamate and n-isopropyl-nmethyl-t-butylammonium a-cyano-4-hydroxycinnamate were the best matrices for proteins and peptides, while n,n-diisopropylethylammonium a-cyano-4-hydroxycinnamate and n,ndiisopropylethylammonium ferulate were found to be the best matrices for carbohydrates. another novel ionic liquid matrix has been made from the 1,1,3,3-tetramethylguanidinium (14) salt of thap and found to be well suited for the maldi analysis of glycopeptides and glycans, particularly as it appeared to overcome the well-known ionization suppression of carbohydrates in the presence of peptides. for example, even glycopeptides containing short peptide backbones and large carbohydrate moieties gave high signal intensities when using this matrix even though they did not produce spectra directly from thap. in a second experiment, glycans were released with pngase f from total tryptic digests derived from glycoproteins and analyzed by maldiquadrupole ion trap (qit)-time-of-flight (tof). when using the liquid matrix, it was possible to detect the glycans with high intensities in the presence of the tryptic peptides, whereas, once again, glycan ionization was completely suppressed when measured with thap alone. the extent of metastable decay of glycopeptides was also found to be reduced when using the liquid matrix (ullmer & rizzi, 2009) . ionic liquid matrices have shown considerable advantages over conventional matrices for maldi-ms analysis of polysulfated carbohydrates such as heparin and heparin sulfate. these compounds are not easily analyzed by uv-maldi ms analysis because of the thermal lability of their o-and n-so 3 moieties. two new ionic liquid matrices based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (haba, 1/32) with 1,1,3,3-tetramethylguanidine or spermine (1/30) have been successfully applied to the analysis of these compounds (przybylski et al., 2010b) . these matrices gave improved signalto-noise ratios as well as a decrease of fragmentation and desulfation. sulfated oligosaccharides were detected with higher sensitivity than with the usual crystalline matrices, and their intact sulfated ions [mna] à were easily observed. maldi-ms characterization of challenging analytes such as heparin octasaccharide carrying eight o and four n-sulfo groups, and heparin octadecasulfated dodecasaccharide was also successfully analyzed. in a paper published in 2007, gomenez et al. (2007) reported that dhb, with vacuum drying to improve target spot homogeneity, was a better matrix than sinapinic acid for obtaining reliable molecular mass values of intact glycoproteins because it prevented sugar fragmentation. in a follow-up to this work, the group have investigated the use of liquid matrices prepared from dhb and sinapinic acid with the aim of avoiding the vacuum drying step (giménez et al., 2010) . the best results were obtained with a variety of glycoproteins such as bovine a1acid glycoprotein, bovine fetuin and human transferrin (tf) from matrices prepared by adding an equimolar amount of butylamine (64.1 ml) to 3,242 ml of a 200 mm solution of sinapinic acid or dhb in meoh. the mixture was vortexed and sonicated for 1 min, evaporated to approximately 100 ml with air, and finally reconstituted with 100 ml of etoh or mecn, for dhb and sinapinic acid mixtures, respectively. furthermore, it was noted that both matrices gave the same masses for the tested glycoproteins that agreed with literature values, suggesting that no fragmentation of the carbohydrate moieties had occurred. a matrix consisting of chca and aniline (15) has also proved to be successful for a number of different compounds, including raffinose (16) (calvano, carulli, & palmisano, 2009) . although it was noted that this matrix gave a stronger signal than chca alone, the latter matrix is not very efficient in ionizing carbohydrates. a comparative investigation of several matrices for analysis of the small carbohydrates, glucose (glc, 1/4) and sucrose (6) has been performed by yang et al. (2010c) . of the matrices studied, sodiated dhb, carbon nanotubes, an ionic liquid matrix of dhb-pyridine, a binary matrix of dhb-aminopyrazine (6/7), zinc oxide nanoparticles and aunps, the best sensitivity was provided by the carbon nanotubes. the detection limit was 3 pmol. both carbon nanotubes and the ionic liquid matrix exhibited the highest reproducibility. tzeng, zhu, and chang (2009) have investigated doping various maldi matrices with alkali metal hydroxides. it was found that for neutral underivatized oligosaccharides, the addition of 2% naoh to dhb caused partial alkaline degradation by glycosidic cleavages upon laser desorption. the effect intensified when nonacidic compounds such as thap or 5-amino-2-mercapto-1,3,4-thiadiazole (amt, 4/7) were used as the matrix. the degradation allowed identification of the reducing end residue of the analyte and facilitated its structural characterization by psd tof-ms. use of matrices consisting of lioh and thap or amt led to the production of singly as well as multiple lithiated ions. multiple lithiation appeared to occur with carbohydrates containing free 3-oh groups. up to 6 li atoms were found to be incorporated for maltoheptaose, b-cyclodextrin, and dextran 1500. such a "lithium tagging" technique makes it possible to differentiate positional isomers of milk-neutral oligosaccharides, lacto-n-difucohexaose i and ii (lndfh-i (17) and lndfh-ii, (18)), without the need of chemical derivatization or tandem ms analysis. lndfh-ii, 18 = glcnac, = galactose, = fucose, = glucose (for details, see (harvey, et al., 2009c) choi and lee (2009) have studied the ionization efficiencies of maltooligosaccharides (degree of polymerization, dp 1-7) with the cations na þ , li þ , and k þ in salts containing tfa à , cl à , and oh à with dhb as the matrix. the signal strength rose with the number of glucose units with sodium consistently giving the most intense signals. the nature of the anion also affected the ionization with the tfa à salts generally being the most effective. a sample preparation method developed by nishikaze and amano (2009) has been compared with the conventional drieddroplet or ethanol (etoh) recrystallization methods and reported to give superior results in terms of ion yield and signalto-noise ratio. the method, named the "reverse thin layer method" consists of first, complete drying of the oligosaccharide solution on the maldi target plate and then deposition of the matrix dissolved in a small amount of meoh. using this method, a relatively homogeneous matrix crystal was generated and higher yields of both positive and negative ions were obtained. the authors comment that the method could be applied to various other matrices including both solid and liquid matrices. a method for direct archaebacterial glycolipid and lipid analysis by maldi ms in intact membranes, without prior extraction/separation steps, has been developed by angelini et al. (2010) . the purple membrane isolated from the extremely halophilic archaeon halobacterium salinarum was used as a model, lyophilized and ground with 9-aminoacridine (9-aa, 6/ 18). the mixture was crushed in a mechanical die press to form a thin pellet, small pieces of which were attached directly to the maldi target. in parallel, solution spectra of individual phospholipids and glycolipids, were analyzed by maldi-tof/ ms using 9-aa as the matrix. results show that 9-aa is a suitable matrix for the conventional maldi-tof/ms analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized membranes. a new technique termed matrix-free material-enhanced laser desorption/ionization mass spectrometry (mf-meldi-ms) has been described which employs a single compound prepared by immobilizing bradykinin on silica gel coupled to 4-(3triethoxysilylpropylureido)azobenzene (19) for both the maldi matrix and a stationary phase for thin-layer chromatography (tlc). the technique was applied to the analysis of carbohydrate reference standards such as glucose, sucrose, raffinose and plant extracts from quercus robur (oak). carbohydrates formed [mþna] þ and abundant [mþk] þ ions. the meldi material adsorbs the laser energy sufficiently for desorption and ionization and delivered excellent results in respect to signal-to-noise (s/n) ratio (s/n ratio: >9/1) and sensitivity with a lod in the low ng/ml range. for preparation of the tlc plates, the modified silica gel was suspended in methanol, acetone, acetonitrile or acetonitrile/water (1:1) for 3 min. about 15-20 ml of the suspension was applied in narrow channels cut into a stainless steel target in the form of a thin layer and dried at room temperature. the sample was placed on this layer for subsequent tlc in n-buoh/acetone/acetic acid/ h 2 o (35:35:10:20) as mobile phase (qureshi et al., 2010) maldi imaging has seen many developments during the review period and several reviews and related articles have been published; details are in table 2 . a mass spectrometer with ion mobility separation capability (waters synapt) has been used by snel and fuller (2010) to produce high spatial resolution images of glucosylceramide (20) in the spleens of mouse models of gaucher disease. the matrix (chca) was applied to the tissue sections with an airbrush. for data acquisition, the laser was continually fired at one position until no more ions were observed and then the sample was moved by 15 mm (laser diameter about 150 mm). ions were generated from only the un-irradiated surface at each of these positions achieving an effective spacing of 15 mm. at this spacing, it was possible to visualize macrophages enriched in glucosylceramide which could be distinguished from other cell types in the spleen. current maldi mass spectrometric imaging (msi) spatial resolution is typically limited by the diameter of the laser spot-size, which is usually between 50 and 100 mm, covering an area equivalent to tens of mammalian cells. perdian and have addressed the problem of acquiring high resolution imaging with high resolution ms on an orbitrap mass spectrometer. at a spatial resolution of 100 mm, the orbitrap mass analyzer is able to image a 2,000 â 2,000 mm 2 sample area in 7-14 min with one scan per step, depending on the resolution. if the spatial resolution is increased to 5 mm, the same size sample area will take more than 44-88 hr to complete the experiment, a time that is not practical in the normal laboratory. however, because the orbitrap also has a linear ion trap (it) analyzer, this was utilized, together with a two-motion plate movement to reduce the time while maintaining the resolution. thus, for every pixel position on the target, the laser spot was moved in a spiral fashion such that both orbitrap and ms/ms data were acquired. with a fast nd:yag laser the data acquisition time was decreased by 43-49% compared to that from orbitrap-only scans; however, 75% or more time could be saved for higher mass resolution and with a higher repetition rate laser. using this approach, a high spatial resolution of 10 mm was maintained at it imaging, while orbitrap spectra were acquired at a lower spatial resolution, 20-40 mm, all with far less data acquisition time. furthermore, various ms imaging methods were developed by interspersing ms/ms and ms fragmentation n times (ms n ) it scans during orbitrap scans to provide more analytical information. the method was applied to several flavonol glycosides from an arabidopsis flower petal in which ms/ms, ms n , it, and orbitrap images were all acquired in a single data acquisition. spectra were acquired in negative mode and no matrix was required. for uv-absorbing compounds such as flavonoids and their glycosides, a matrix is not necessary for imaging as demonstrated by imaging at the single cell level of secondary metabolites in arabidopsis thaliana and hypericum species (hölscher et al., 2009) . the highest spatial resolution achieved, 10 mm, was much higher than that achieved by commonly used maldi) imaging protocols. a new matrix-free technique called nanostructure-initiator mass spectrometry (nims) has been developed for the analysis e c n e r e f e r s n o i t a t i c s t n e t n o c t c e j b u s general review short article, mainly applications 50 (murayama et al., 2009) general review methods and applications to peptides and lipids (including glycolipids) 96 general review short general review (award lecture) 47 general review from origins to state of the art 41 (francese et al., 2009a) concise review of mass spectrometry imaging (sugiura et al., 2010b) and tissue imaging of carbohydrates and steroids (patti et al., 2010) . analysis was accomplished by spray depositing nacl or agno 3 with a fused-silica picotip emitter onto a porous silicon surface to provide a uniform layer rich with cationization agents prior to desorption of the fluorinated polymer initiator. upon laser irradiation, carbohydrates produced [mþna] þ ions whereas steroids formed [mþag] þ . for glucose, a plot of concentration against the 12 c/ 13 c 6 ratio was linear with a correlation coefficient r 2 of 0.9975 over the range 1-200 mm. carbohydrates and steroids could be detected down to the 800-amol and 100-fmol levels, respectively. the ability of the method to perform tissue imaging was demonstrated by imaging the distribution of sucrose in a gerbera jamesonii flower stem and the distribution of cholesterol (21) in a mouse brain. the flower stem and brain sections were placed directly on the ion-coated nims surface without further preparation and analyzed directly. no deposition of a matrix onto the sample surface was needed. a similar matrix-free method, termed nano-assisted laser desorption-ionization (naldi) ms for tissue imaging has been demonstrated by vidová et al. (2010) . commercially available nano-structured surfaces were used as substrates for imprinting tissue sections. the lithographic transfers were washed and the lipids were imaged by laser desorption mass spectrometry (ldms). the naldi imaging of lipid transfers was compared with standard maldi imaging of matrix-coated (chca) tissue sections and the resulting images were found to be of the same quality with no spatial information being lost due to the imprinting process. naldi imaging was reported to be faster due to the absence of the time-consuming matrix deposition step, and the naldi mass spectra were less complex and easier to interpret than maldi spectra. the method was applied to the analysis of phospholipids, gsls and glycerophospholipids in mouse kidney slices. naldi ms was able to identify the same lipid species as maldi and was reported to provide better distinction between kidney and adrenal gland tissues based on the lipid analysis. miura et al. (2010a) have developed a maldi imaging system that is claimed to be able to image from single cells in thin tissue sections. an indium tin oxide-coated glass slide marked with a 50 mm-wide mesh work to enabled matching of optical and ms images was used for mounting tissue sections. a suspension of hela cells in culture medium was mounted onto the slide and incubated for 6 hr at 37˚c. cells were then washed with phosphate-buffered saline (pbs) and the appearance of single cells adhering to the glass was observed by optical imaging. the single cell-adhered glass slide was then mounted onto maldi sample plate with a parallel experiment involving brain tissue slices and the matrix, 9-aa, was applied with an airbrush. several metabolite peaks were detected at the position of the single cell. adenosine triphosphate (atp, 22; m/z 505.99, identified by comparison with a standard sample) was identified with a good signal-to-noise ratio. peaks were also obtained from other metabolites such as fructose-1,6-bisphosphate (23) a major hurdle for imaging gangliosides in tissue using ms is that sialic acid residues can be dissociated in the ionization process. chan et al. (2009a) have investigated the ionic liquid matrix chca/1-methylimidazol (6/62) (1:1 w/w) and have found that it produces excellent sensitivity for ganglioside detection without significant loss of sialic acid residues. the matrix was applied to the tissue samples with an airbrush; the method best adapted to handling a mixed matrix whose components have different volatilities. image analyses of mouse brain tissue sections demonstrated that the n-fatty acyl chains of gangliosides were differentially distributed in mouse hippocampal regions. gangliosides with n-c18 acyl chains were enriched in the ca1 region, while gangliosides with n-c20 acyl chain were enriched in dentate gyrus. colsch and woods (2010) have developed a method for imaging sialylated gsls in mouse brain. the total glycolipid profile was obtained by maldi-tof following solvent extraction and then individual species were mapped from frozen brain slices by maldi using a linear tof/tof system in negative ion mode. the matrix, which consisted of saturated 2,6dihydroxyacetophenone (dhap, 1/49) dissolved in 50% ethanol with the addition of ammonium sulfate (125 mm) and 0.05% of heptafluorobutyric acid (hfba) was applied with a chip-1000 chemical inkjet printer with a piezoelectric head. twenty-eight nanoliters of matrix were deposited per spot with the distance between two spots of 240 mm. the ammonium sulfate in the matrix mixture limited the formation of salt adducts, while the addition of hfba increased the stability of dhap in the vacuum and reduced its rapid sublimation. some sialic acid loss was noted, particularly with gd1 and gt1, which were detected as the ganglioside ( the results showed differences in gsl localization in several brain regions depending on the sialic acids and the ceramide (cer). imaging of lipids, including gsls, has been reported by landgraf et al. (2009) using a hybrid linear it/orbitrap mass spectrometer that allowed the acquisition of ms/ms spectra. a dramatic improvement in mass spectral resolution and a decrease in background were observed from lipids distributed within nerve tissue when compared with results obtained from fragmentation within the linear it. the dhb matrix was applied to the dried tissue samples with an epson inkjet printer and the maldi ion source was operated at a pressure of about 75 mtorr. the technique was used to image lipids from rat spinal cord sections. goto-inoue et al. (2009) have imaged the glycolipid, seminolipid (24) in mouse testis. this sulfated glycolipid comprises more than 90% of the glycolipid in mammalian testis and disruption of the gene catalyzing transfer of galactose (gal) results in male infertility due to the arrest of spermatogenesis. different molecular species are defined by fatty acid composition. tissue imaging was performed from thaw-mounted tissue slices that had been sprayed with dhb with an airbrush. it was found that the major molecule (c16:0-alkyl-c16:0-acyl) was expressed throughout the tubules: some (c16:0-alkyl-c14:0acyl and c14:0-alkyl-c16:0-acyl) were predominantly expressed in spermatocytes and the other (c17:0-alkyl-c16:0acyl) was specifically expressed in spermatids and spermatozoa. this is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation. experimental details for performing imaging of glycolipids using the airbrush application method for applying dhb, as used by this group, has been published separately (goto-inoue, hayasaka, & setou, 2010a) . the distribution and localization of gsls present in mouse brain sections have also been investigated using nanoparticleassisted laser desorption/ionization imaging ms. aunps modified with alkylamine were used as a new matrix to maximize the detection of gsls. the mouse brain was frozen in liquid nitrogen, and serial sections were thaw-mounted onto indiumtin oxide (ito)-coated glass slides. a thin layer of aunps or dhb matrix was applied to the surface with an airbrush. ims analyses were performed by raster-scanning in the x-axis with a scan pitch of 200 mm in the y-axis. sulfatides and gangliosides were detected in mouse brain sections with the new matrix whereas it was difficult to detect them using dhb (goto-inoue et al., 2010c ). an oscillating capillary nebulizer (ocn) was also used by chen et al. (2010g) for analysis of sphingolipids in tissue slices in tay-sachs/sandhoff disease. in addition to the above-mentioned matrix-free methods, jung et al. (2010) have reported imaging of cellulose in poplar (populus deltoids) stem using more traditional techniques. analysis of microcrystalline cellulose was performed first to determine the best matrix. dhb gave much stronger signals than matrices such as chca or sinapinic acid and was applied to poplar cellulose with an ocn. ions at m/z 1, 500, 2, 472, and 3, 120 (dp 9, 15, and 19) were monitored with a voyager de str instrument and produced images that closely resembled the optical image. taira et al. (2010a) , on the other hand used an airbrush with chca to image ginsenosides in cross-sections of panax ginseng root and used ms/ms to obtain detailed structural information. ginsenosides were found to be located more in the cortex and periderm than in the medulla and that they were at greater concentration in the root tip than in the center of the root. several carbohydrates including hexoses (hex) and d-fructose 6-phosphate have been imaged in eggplant (solanum melongena, also known as aubergine) fruits from dhb although the paper was mainly concerned with imaging of g-aminobutyric acid (gaba, 25) (goto-inoue, setou, & zaima, 2010b) . li, bohn, and sweedler (2010h) have compared two ms imaging methods, maldi and sims, for glycan detection in the stems of the perennial grass miscanthus â giganteus. several methods of sample preparation were investigated for maldi. a thin (2 nm) gold coating provided high quality signals of oligosaccharide-related ions and dhb also showed high efficiency for ion production. on the other hand, chca produced only very weak spectra, consistent with its use as a stand-alone matrix for carbohydrates. direct laser ablation of untreated sections gave high resolution images, although coating the sections with a nanometer thick layer of gold greatly enhanced the quality of the sims images. two reviews describing derivatization reactions have been published (busch, 2010; ruhaak et al., 2010b) , the second (207 references) is more comprehensive and deals specifically with n-linked carbohydrates. these derivatives are most often used for introducing fluorescent tags for chromatographic detection but also find use in ms. 2-aminobenzamide (2-ab, 1/56) and 2-aminopyridine (2-ap, 1/52), favored by japanese investigators, are the derivatives most frequently encountered; use of the latter derivatives has been reviewed by hase (2010) . several new derivatives (or labels) have been reported. thus, 5-amino-2-naphthalenesulfonic acid (ansa, 26) has been used to derivatize n-glycans by reductive amination for capillary electrophoresis (ce), hplc, and maldi-tof analysis (briggs et al., 2009) . the derivative was reported to give superior resolution in both ce and hplc analysis to the wellused 8-aminopyrene-1,3,6-trisulphonic acid (apts) derivatives and in maldi-tof analysis, the negative charge enabled both neutral and acidic glycans to be examined simultaneously. 3-amino-9-ethylcarbazole (6/19) (mou et al., 2009) , another new derivatizing agent, has been reported to increase sensitivity of ms detection. 2-picoline-borane (27) has been proposed as an alternative to toxic sodium cyanoborohydride for the reductive amination reaction (ruhaak et al., 2010a) . pabst et al. (2009) have compared the preparation and performance of 15 different labels for n-glycan analysis. several cleanup procedures were developed for cleaning these derivatives before analysis, of which hydrophilic interaction solidphase extraction (spe) appeared to be the most widely used. however, the cleanup was laborious and a better method was sought. simple addition of acetone resulted in the formation of a precipitate, which turned out to be the labeled oligosaccharide. a single addition and removal of acetone reduced the amount of reagent to approximately 0.2% (measured with 2-ab). two further extractions of the pellet with acetone reduced the excess of amine reagent by at least as much as clean-up with a cyano-spe cartridge. in addition, reduction of the schiff base of 2-aplabeled glycans proceeded faster and/or required less reagent when the samples were pre-purified before the addition of reducing agent. acetone extraction was successfully applied to many other labels such as 2-ab and 3-aminobenzoic acid (2-aa) (1/57). with respect to the performance of the individual labels, procainamide (28) emerged as more sensitive than 2-aa for normal-phase hplc, but its chromatographic performance was not convincing. 2-ap gave the lowest retention on reversedphase and graphitic carbon columns and, thus, appeared to be most suitable for glycan fractionation by multidimensional hplc. most glycan derivatives performed better than native carbohydrates in maldi and esi ms, but the sensitivity gain was small and hardly sufficient to compensate for sample loss during preparation. amano et al. (2009a) have labeled oligosaccharides with a pyrene derivative in order to acquire negative ion maldi-qit-tofms n spectra. the oligosaccharides were reacted with pyrene butanoic acid hydrazine (6/21) and then reduced with nabh 4 and cleaned with a small c 18 column. the derivatives gave intense and stable molecular ions in both positive and negative ([màh] à ions) modes and as little as 10 fmol of pyrene-labeled oligosaccharides, such as monofucosyllacto-nhexaose (29) gave sufficient signals for analysis. although some fucose loss by in-and post-source occurred in positive ion mode, this loss was significantly less in negative mode. a method, termed glycan reductive isotope labeling (gril) has been introduced for glycan quantitation. free glycans or those released from glycoproteins, were derivatized by reductive amination with either [ 12 c 6 ]aniline or [ 13 c 6 ] aniline. these dual-labeled aniline-tagged glycans could be recovered by reversed-phase chromatography and could be quantified by uv absorbance or ms. one labeled variety was used as the reference standard against which the test glycan, labeled with the other isotope was measured. unlike previously reported isotopically coded reagents for glycans, the derivatives did not contain deuterium, which can sometimes be chromatographically resolved. the technique allowed linear relative quantitation of glycans over a 10-fold concentration range and could accurately quantify sub-picomole levels of released glycans (xia et al., 2009) . on-target derivatization with the matrix 3-aminoquinoline (1/24) has yielded schiff bases which could be measured directly in positive and negative ion mode from one single spot. the optimal conditions were 20 mg/ml of 3-aq in a mecnwater mixture (1:2 v/v) with 0.07% of an inorganic acid to give a ph of 5. in negative ion mode, spectra from chloride adducts of the derivatives were acquired from 1 fmol of oligosaccharide. furthermore, psd fragmentation in positive and negative ion mode was enhanced, providing information on oligosaccharide sequence, linkage, and branching. the method was applied to trifucosyllacto-n-hexaose (30) and trifucosyl-para-lacto-n-hexaose (31), two isomers occurring in human breast milk samples, which were easily identified and distinguished (rohmer et al., 2010) . tfplnh, 31 (symbols as defined for structures 17 and 18. anomericity not specified) reductive amination derivatization has also been exploited in other areas, combined with their use in ms. binding of sugar chains to proteins, viruses and cells is conveniently monitored by the surface plasmon resonance (spr). key to this method is the use of linker compounds for immobilization of the sugar chains to the gold-coated spr chip. sato et al. (2009a) have developed a novel linker molecule, named "f-mono," which combines a linker with a fluorescent moiety to allow high sensitivity monitoring of the glycans. since the molecule (32) contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved by the reductive amination reaction. the mass spectra of thee compounds contained a peak 2 da higher than the molecular ion due to the reduction of the thioctic acid moiety providing a convenient method for identifying the glycans even using unfractionated crude samples. immobilization of the derivatives onto gold-coated chips, and their subsequent spr analyses were successively accomplished. use of hydrazone formation removes the need for a reductive step to stabilize the derivative as required by schiff base formation from primary amines. experimental details for synthesis of the phenylhydrazone derivatives discussed in earlier reviews (lattova & perreault, 2003a,b; lattova, perreault, & krokhin, 2004) have recently been reported in an edition of methods in molecular biology devoted to glycomics . small oligosaccharides and n-glycans from chicken ovalbumin have been converted into their biotin derivatives by incubating them with biotinamidocaproyl hydrazide (bach, 33) (kapková, 2009) . the derivatives imparted improved mass spectral sensitivity over that of the free glycans and, because the labeling reagent contained a biotin handle, it allowed the interaction between lectins and biotin-derivatized oligosaccharides to be investigated. fragmentation of the n-linked glycans was dominated by y and b/y-type glycosidic ions. han et al. (2010a) have used a new substituted hydrazine, 1-(4-cyanophenyl)-4-piperidinecarbohydrazide (34) and produced a derivative that increased detection sensitivity by about 10-fold over that from the underivatized glycan. the observation of [mþna] þ ions rather than the expected [mþh] þ species suggested that the sensitivity increase was the result of increased hydrophobicity. maldi analysis employed dhb and super-dhb; recrystallization of the super-dhb sample with acetonitrile substantially improved the signal-to-noise ratio and reproducibility. girard's t reagent (1/55), with its constitutive cationic charge, has been used in quantitative measurements, as described below and for measuring a-galactose-containing n-glycans in porcine pig corneal endothelial cells and keratocytes (kim et al., 2009c) . because of the in-built charge, signal strengths from glycans of different structures were thought to be equal. this is not necessarily the case for formation of [mþna] þ ions. rather than reacting the reducing-terminal aldehydes of carbohydrates with amines or hydrazines, the reverse reaction can be used if the glycan is first converted into its glycosylamine (35). in fact, this type of reaction can be used directly on pngase f-released n-glycans because these are released in this form. chemical formation of glycosylamines generally utilizes the kochetkov reaction in which the glycan is treated with an excess of ammonium carbonate. unfortunately, this reaction is slow and can take up to five days for completion. to overcome this problem, liu, salas-solano, and gennaro (2009h) have used microwave assistance to speed the reaction up to as little as 90 min. following amidation the glycans were reacted with tris-(2,4,6-trimethoxyphenyl)phosphonium acetic acid n-hydroxysuccinimide ester (36) to introduce a permanent charge on the glycan and the investigators were able to detect derivatized maltoheptaose at 2 fmol/ml by maldi-tof ms using dhb and chca matrices. glycans from rnaseb, chicken ovalbumin and asialofetuin were also detected at high sensitivity. a potential problem arises from possible cleavage of the reducingterminal n-acetylglucosamine (glcnac) residue as such a reaction has recently been reported as a by-product of the kochetkov reaction when, for example, ammonium bicarbonate at 37˚c was used (murase & kajihara, 2010 several carbohydrates, including maltoheptose, blood type b antigen, pullulan and the glucan of ganoderma lucidum have been converted into their naphthimidazole (naim) derivatives (37) in high yields by the iodine-promoted oxidative condensation (scheme 1; lin et al., 2010b) . the reaction took about 6 hr to go to completion giving derivatized carbohydrates that gave enhanced maldi signals ([mþh] þ ions) compared with those from the free carbohydrates or their 2-ab derivatives with dhb or thap as matrices. less than 1 pmol of carbohydrate could be detected. furthermore, the derivatives could easily be hydrolyzed to the parent glycans. a further series of such derivatives has been synthesized by condensation with diamines such as substituted benzene-1,2-diamine (38) in order to increase hydrophobicity and detection sensitivity (lin et al., 2010c) . using maltotriose (11, n ¼ 1), derivatives with pyrimidine-4,5diamine (39), pyridine-3,4-diamine (40) and 1,2,5-oxadiazole-3,4-diamine (41) b. derivatives of other sites permethylation has long been used in carbohydrate analysis, most frequently for linkage determination by gas chromatography/mass spectrometry (gc/ms) following hydrolysis and acetylation (permethylated alditols acetates). however, there appears to be an increasing trend to employ the reaction for maldi analysis. the advantage of permethylation is that it increases sensitivity and several investigators employing its use appear to detect larger glycans, particularly n-glycans, than by the use of underivatized glycans. however, sample clean-up of the highly basic reaction mixtures can be a problem with, in some cases, losses offsetting any gain in sensitivity. introduction of solid-phase permethylation has improved the situation. experimental details of the solid phase permethylation method (methyl iodide on sodium hydroxide beads) (kang, mechref, & novotny, 2008; kang et al., 2005) that is capable of permethylating very small amounts of carbohydrate have been published in methods in molecular biology (mechref, kang, & novotny, 2009a) . on the negative side, it has been reported that permethylation can lead to loss of o-linked acetyl groups from certain sialic acids (liu & afonso, 2010 ). an extension of the solid-phase method to allow sulfated glycans to be analyzed by maldi-tof ms has been developed (lei, mechref, & novotny, 2009) . sulfated glycan samples were permethylated followed by methanolytic cleavage of the sulfate groups. the desulfated, permethylated glycans were then subjected to another permethylation step using deuteromethyl iodide to label the hydroxyl groups that were exposed by removal of the sulfates. the number of attached sulfate groups could be calculated from the mass-shift caused by the presence of the deuterium label and the position of the sulfate substitution could be determined by collision-induced dissociation (decomposition) (cid). the method was validated with linear standard glycans and used to identify sulfated n-glycans released from bovine thyroid-stimulating hormone (btsh). yu et al. (2009c) have shown that it is possible to permethylate sulfated glycans with methyl iodide and sodium hydroxide (ciucanu and kerek method) (ciucanu & kerek, 1984) , without loss of the sulfated glycans. the trick is to avoid the normal chloroform/water partition stage, which results in much of the sulfated material (unmethylated) partitioning into the aqueous phase. instead, clean-up employed c18 reversedphase spe cartridges and microtips self-packed with nh 2 -beads. the methylation reaction was capable of methylating phosphates but not sulfates, allowing them to be readily identified. formation of methyl esters by reaction of the sodium salt with methyl iodide has frequently been used to stabilize sialic acids to maldi analysis by eliminating the labile proton on the acid group. an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the maldi spectrum has been published by wheeler, domann, and harvey (2009) (fig. 2) . the sugars were desalted, dried, dissolved in methanol and treated with 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-ethylmorpholinium chloride (dmt-mm, 5/12). after removal of the solvent, the products were transferred directly to the maldi target and examined from dhb. however, for the analysis of small amounts of n-glycans derived from biological sources, the method benefited from an additional clean-up stage involving the use of a nafion 117 membrane. unlike the reaction with methyl iodide with the sodium salt that produced a single peak from each sialylated glycan, irrespective of the linkage, the reaction with dmt-mm produced different derivatives from a2 ! 3-and a2 ! 6-linked sialic acids. the a2 ! 6-linked sialic acids produced only methyl esters whereas a2 ! 3-linked sialic acids were converted into their lactones providing a 32 da difference in mass, thus enabling the linkage to be determined directly from the maldi-tof spectrum (fig. 2) . negative ion cid mass spectra of these neutralized glycans provided information, in many cases, on the antenna of n-linked glycans to which the variously linked sialic acids were attached. in an application of the method to the glycoprotein carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1), it was shown that both a-2 ! 3-and a-2 ! 6linked sialic acids were present (harvey, baruah, & scanlan, 2009a) . 4-(4,6-dimethoxy-1,2,3-triazil-2-yl)-4-methylmorpholinium chloride in the presence of ammonium chloride, converts sialylated glycans into amides with the same linkage-specific reactivity difference. thus, the a2 ! 3-linked sialylated glycans yield lactones, as above, whereas the a2 ! 6-linked compounds form amides. have used this reaction to examine blood serum glycoproteins but their technique differed from the above methyl ester formation in that the glycans were permethylated after reaction with dmt-mm. this reaction formed the methyl ester from the a2 ! 3-linked compounds as the result of the opening of the lactone ring, whereas the amide that was derived from the a2 ! 6-linked compounds was converted into the corresponding dimethylamide with a 13 da increase in mass over that of the methyl ester. 4-(4,6-dimethoxy-1,2,3-triazil-2-yl)-4-methylmorpholinium chloride can also be used to form substituted amides directly and has been used by endo et al. (2009) to link the fluorescent derivative 2-(2-pyridylamino)ethylamine (paea, 42) to the carboxy group of sialic acids in sialo-oligosaccharides and gangliosides. the derivative gave good hplc and tlc sensitivity and possessed the following advantages for maldi analysis: suppression of preferential cleavage of neu5ac; enhancement of molecular-related ion intensities; simplification of ms spectra; and finally, since paea-amidation did not cleave the linkage between sugar and aglycon, allowed maldi-tof-ms and ms/ ms analyses to reveal the complete structure of the molecule. 4-(4,6-dimethoxy-1,2,3-triazil-2-yl)-4-methylmorpholinium chloride has also been used to form amides from hexuronic acids (hexas) in an investigation of the n-linked glycosylation of structural subunit rvh2 of rapana venosa hemocyanin . as well as containing the rather unsusal (for n-glycans) hexuronic acid, the glycans also contained naturally methylated hexoses and internal fucose residues. gil et al. (2010) have stabilized sialic acids by formation of amides with acetohydrazide under mild acidic conditions in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (edc, 43) . glycoproteins were first reduced and alkylated and then the sialic acids were amidated. glycans were released with pngase f and a permanent charge was attached to the reducing terminus by further reaction with girard's t reagent. alternatively, derivatization with 2-aa was used and the products were examined both by hplc and maldi-tof ms. the amidation reaction was performed on the glycoprotein because acetohydrazide would also have reacted with the aldehyde function of the released glycan, precluding derivatization with an amine more suited to the detection method used for monitoring the glycans. the method was applied to the analysis of n-glycans from transgenic pig-derived human factor ix. another method for synthesis of methylamides has been reported by liu et al. (2010i) . sialylated glycans were reacted with methylamine in the presence of (7-azabenzotriazol-1yloxy)trispyrrolidinophosphonium hexafluorophosphate (pyaop, 44) and n-methylmorpholine (45) for 30 min at room temperature. after methylamidation, sialylated glycans were analyzed by maldi-tof and esi ms without loss of the sialic acid moiety. both a2 ! 3-and a2 ! 6-linked sialic acids were quantitatively converted to their methylamides. this method was validated with n-glycans released from the well-characterized glycoproteins, fetuin, human a1-acid glycoprotein, and bovine a1-acid glycoprotein and was used to study n-glycans from serum glycoproteins from human, mouse, and rat. both neu5ac and oacetylated analogues were stable under maldi conditions. glycopeptides tend to produce weaker signals than nonglycosylated peptides and it is frequently difficult to observe their molecular ions in samples rich in unglycosylated peptides. have developed a highly sensitive on-plate pyrene derivatization method using 1-pyrenyldiazomethane (46) for acquisition of maldi ms n spectra of glycopeptides in amounts of less than 100 fmol. unusually, the pyrene groups were easily released from glycopeptides during ionization when dhb was used as a matrix. thus, most ions were observed in their underivatized form. at the same time, pyrene derivatization was found to reduce the ionization of peptides and to produce signals from the glycopeptides that were strong enough for acquisition of ms n spectra that contained ions from both glycan and peptide. when the liquid matrix, 3aq-chca, was used, the sialic acid linkages of the pyrene sialylated glycopeptides were found to be stable because of inefficient release of the pyrene group allowing ms n spectra of the intact glycans to be obtained. the method was used to examine glycopeptides from 1 ng of prostate specific antigen. ohara et al. (2009) have developed a method for analysis of sulfated carbohydrates by forming complexes with 1-(pyren-1ylmethyl)guanidine (pmg, 47) from a matrix consisting of this compound and p-nitroaniline (3/3). two types of sulfated carbohydrate were examined, chondroitin sulfate (48) and carrageenan (49). the pmg complexed with the sulfate group and was eliminated under maldi conditions such that the molecules produced a ladder of peaks separated by masses corresponding to losses of each complexed sulfate (mass difference 353 u). in positive ion mode, the molecular ions from the chondroitin sulfates contained one more pmg molecule than the number of sulfate groups. one pmg group was presumed to bind to a carboxylate group. in negative mode, one less pmg molecule was bound. carrageenans showed a slightly different pattern in that the number of pmg molecules found in the positive ion spectra equaled the number of sulfates. sialic acids are classically analyzed by hplc after formation of fluorescent 1,2-diamino-4,5-methylenedioxybenzene (dmb, 50) derivatives. these derivatives require an a-keto acid group in the sialic acid. galuska et al. (2010b) have developed a method for specifically measuring nucleotide-activated sialic acids in the presence of unactivated acids by first reducing the keto group that is present only in the non-activated acids. under the conditions of the derivatization reaction, the nucleotide group was removed leaving, in the case of the activated acids, only, an intact a-keto acid group that reacted with the dmb reagent. subsequent analysis was by hplc and maldi-tof. maldi ms is used extensively in analyses with glycan arrays as summarized in recent reviews (see table 3 ). table 4 lists glycans that have been used in array construction. most of the above work has been with carbohydrate preparations prior to array construction. however, maldi ms has also been used to interrogate arrays directly. as an example, a new type of glycan array covalently or non-covalently attached to aluminium oxide-coated glass slides has been developed for studies of enzymatic reactions and protein binding . the array was prepared by tagging glycans with a polyfluorinated hydrocarbon (51) tail and spotting them robotically onto the aluminium oxide-coated slide surface which contained a layer of polyfluorinated hydrocarbon terminated with phosphonate. after incubation and washing, the noncovalent array was characterized by maldi-tof at a low laser energy without addition of matrix. a cellotetraose (d-glc-(b-(1 ! 4)-d-glc) 2 -b-(1 ! 4)-d-glc) array was developed to study the activity and specificity of different cellulases and to differentiate the exo-and endoglucanase activities. compared to the preparation of glycan arrays on glass slides and other surfaces, this method using phosphonic acid reacting with aluminium oxide-coated was said to be more direct, convenient and effective and represented a new platform for the highthroughput analysis of protein-glycan interactions. polyfluorinated hydrocarbon tag, 51 in another application, a ligand affinity capture (lac) method for detection of biotinylated biomolecules has been developed by jørgensen, juul-madsen, and stagsted (2009) . metal-coated glass slides were treated with amino-silane and derivatized with biotin followed by avidin. biotinylated biomolecules could then be captured and detected in the low femtomole to low picomole range by maldi-tof ms using chca in a dried droplet method. the technique was used to detect biotinylated lipopolysaccharide (lps) and its binding to the antagonist polymyxin b. the a-mannose-specific lectin concanavalin a (con a) has been bound to polydopamine-modified gold, indium, and iridium surfaces and its activity was demonstrated with rnase b using spr spectroscopy. surface-maldi-tof ms experiments revealed that the affinity of the immobilized con a depended on the oligosaccharide structure and composition. thus con a was found to bind certain man 7 -(4/20), man 8 -(5/20), and man 9 -glcnac 2 rnase b glycoforms more strongly than man 5 -and man 6 -glcnac 2 glycoforms (morris, peterson, & tarlov, 2009) . concern has frequently been expressed about the ability of maldi-tof ms to provide quantitative information. fortunately, this concern appears to be unjustified as two recent studies have shown. thus, a systematic study of widely different glycopeptides was performed by thaysen-andersen, mysling, and højrup (2009) to determine the relationship between the relative abundances of the individual glycoforms and the maldi-tof ms signal strength. glycopeptides derived from glycoproteins containing neutral glycans (rnaseb, igg, and ovalbumin) were profiled and yielded excellent and reproducible quantitation (correlation coefficient r ¼ 0.9958, n ¼ 5) when evaluated against a normal-phase hplc 2-ab glycan profile. similarly, precise quantitation was observed for various forms of n-glycans (free, permethylated, and fluorescence-labeled) using ms. in addition, three different sialo-glycopeptides from fetuin were site-specifically profiled, and good correlation between peak intensities and relative abundances was found with only a minor loss of sialic acids (r ¼ 0.9664, n ¼ 5). for glycopeptide purification, a range of hydrophilic and graphite materials packed in microcolumn format was evaluated and proved capable of desalting without loss of quantitative information. thus, maldi-tof ms signal strength of glycopeptides has been found to accurately reflect the relative quantities of glycoforms, enabling rapid and sensitive site-specific glycoprofiling of n-glycan populations. the second study relates to the concern that has often been expressed over possible losses of fucose residues when glycans are ionized by maldi. tajiri, kadoya, and wada (2009b) have recently assessed possible fucose loss and found it to be insignificant. fucose (fuc) is known, however, to migrate within [mþh] þ ions particularly when these are derivatized by reductive amination. experiments on fucose loss were conducted with fucosylated glycopeptides from tf and haptoglobin. studies with increasing collision energy on the [mþh] þ ions showed that the major fragmentation was cleavage at glcnac residues. biantennary glycans containing a1,3/4-antenna fucose or a1,6-core fucose showed different fragmentation behaviors in experiments. stability was dependent on peptide backbone sequences. cleavage of the glcnacb1 ! 2man linkage occurred at a slightly lower activation energy than for the core fucosylated (cf) species, while the linkage of a1,6core fucose was more stable than that of antenna a1,3/4 fucose. however, these fragmentations only occurred at relatively high collision energy. consequently, the authors concluded that quantitation of fucosylated glycans by ms of glycopeptides, without collisional activation, was justified. the fucosylation levels calculated from the signal intensities in nanospray ionization and uv maldi mass spectra were essentially the same. the mass spectrometric profiling of glycopeptides from tf from patients with congenital disorders of glycosylation (cdg-ia and cdg-iic) demonstrated that the elevation or reduction of fucosylation in pathological conditions can be reliably determined by ms of glycopeptides. in spite of these reassurancies, it is possible that for mixtures of compounds, complex suppression effects may degrade quantitative results. consequently some investigators prefer lc/ms methods because the lc step removes, or minimizes, the effect that other compounds in a mixture have on the ionization of the target compounds. instability of sialylated glycans under maldi conditions complicates quantification and errors can possibly also be introduced by unequal ionization of glycans in mixtures. to overcome these problems with n-glycans from a therapeutic glycoprotein from a chinese hamster ovary (cho) cell line, jang et al. (2009a) first formed methyl esters of the sialic acids to stabilize them and then converted the glycans to their girard's t derivatives. these derivatives have a constitutive positive charge, thus overcoming the problem of unequal ionization. percentages of sialylated glycans were measured at 22.5 and 5.2% in two cell lines. the results were comparable to those obtained by np-hplc combined with fluorescence detection using 2-ab or 2-ap derivatization. girard's t derivatives have also been used by kim et al. (2009a) to quantify glycans released from gsls originating from miniature pig endothelia and islet cells. a method using a deoxyribonucleic acid (dna) sequencer has been described for the quantitative analysis of plant nglycans released with pngase a or f and derivatized with apts (1/59). maldi-tof analysis was used to confirm structures with the aid of exoglycosidase digestions (lee et al., 2009i) . a method for the quantification of fructo-oligosaccharides using maldi tof ms with dhb as the matrix, has been developed with the fructan, raftilose, a partially hydrolyzed inulin with a degree of polymeration 2-7 as the test compound (onofrejová, farková, & preisler, 2009) . nystose (2/11), which is chemically identical to the raftilose tetramer, was used as the internal standard. two mathematical approaches, conventional calculations and artificial neural networks (ann), were compared for data processing. the conventional method relied on the assumption that a constant oligomer dispersion profile will change after the addition of the internal standard. on the other hand, ann was found to compensate for a non-linear maldi response and variations in the oligomer dispersion profile with raftilose concentration. as a result, the application of ann led to lower quantification errors and excellent day-to-day repeatability compared to the conventional data analysis. this reproducibility was satisfactory for ms quantification of raftilose in the range of 10-750 pg with errors below 7%. the method was applied to measurements of the content of raftilose in dietary cream and it was stressed that no special optimization of the maldi process was carried out. maldi-tof ms with dhb, thap or p-nitroaniline (3/3) has been used to determine the concentrations of the unsaturated disaccharide from chondroitin sulfate (48) obtained by enzymatic digestion of native chondroitin sulfate with chondroitin abc lyase. the signal-to-noise (s/n) ratio in the spectrum was used as a quantitative measure: amounts of chondroitin sulfate (measured as the disaccharide) down to at least 500 fmol could be detected and there was a direct correlation between the s/n ratio and the amount of chondroitin sulfate between about 2 and 200 pmol although the curve was sigmoidal. the influence of different parameters such as the matrix, the applied laser intensity and different methods of data analysis were also tested. advantages and drawbacks of this approach are critically (song et al., 2009d) bacillus anthracis tetrasaccharide with thiol linker maldi for attachment to a maleimide functionalized microarray to study of carbohydrate-antibody interactions (oberli et al., 2010) glycodendrimers with n 3 group terminating in α-man, β-glcnac or β-gal tof immobilized on an acetylenyl-terminated gold substrate via click chemistry high-mannose glycans -oxime linked tof used to probe binding to malectin muc1 glycopeptides tof synthesis on an amine-reactive hydrogelcoated microarray glass surface. to detect autoantibodies in breast cancer correction: (blixt et al., 2011) n-glycan-asn-fmoc conjugates from chicken ovalbumin, bovine fetuin, and horseradish peroxidase (hrp) tof/tof printed onto commercially available nhydroxysuccinimide (nhs) -activated glass slides after deprotection. glycans interrogated using plant lectins and antibodies in sera from mice infected with schistosoma mansoni (song et al., 2009e) n-glycan clusters tof (dhb) biantennary and high-mannose n-glycans linked to non-reducing terminus of man 3 glcnac 2 core, plus biotin (song et al., 2009f) thiol-terminated nonamannoside tof coupling of a thiol-terminated mannoside to maleimide-functionalized glass surfaces derived from γ -aminopropyl silane slides (dietrich et al., 2010) various oligosaccharides derivatized with 4-(2aminoethyl)aniline by reductive amination reagent has amine groups at both ends allowing the modified carbohydrates to be covalently attached to an amino-reactive nhs-activated glass surface by formation of stable amide bonds (seo et al., 2010) discussed in the paper. finally, the method was validated by the determination of the chondroitin sulfate content in samples of known concentration as well as in enzymatically digested bovine nasal cartilage and compared with two further established methods of chondroitin sulfate determination (the carbazole and alcian blue methods) . positive ion fragmentation of carbohydrates is fairly well understood with two types of cleavage, glycosidic cleavage that occurs between the sugar rings and involve hydrogen migration and cross-ring cleavages that involve the rupture of two of the bonds forming the rings. glycosidic cleavages revealing sequence information predominate in positive ion spectra whereas negative ion spectra frequently contain very abundant cross-ring product ions that provide information on linkage. the nomenclature introduced by domon and costello (1988) is universally used to describe the ions. the stabilities of glycosyl bond linkages in various carbohydrates have been investigated by computational calculations to find general rules of fragmentation of sodiated oligosaccharides . the calculations revealed that a-glucose, a-galactose, b-mannose, a-fucose, b-glcnac and b-galnac linkages were cleaved more easily than b-glucose, b-galactose, and a-mannose linkages because the transition states of the former were stabilized by the anomeric effect. the 1-6 linkage was more stable than the others whereas the sialyl bond was the most labile of all the linkages investigated. comparison of activation energies and binding affinities to the sodium cation revealed an increase in activation energy in proportion to the increment in binding affinity. the calculated stabilities of glycosyl bonds were: a-man (mana1 ! 3, 4 or 6man ) > a-neunac (neunaca2 ! 3 or 6); this result was close to the experimentally deduced trend. in-source decay frequently accompanies ionization of permethylated glycans. although the presence of fragments can lead to complex spectra, they can also be used to obtain pseudo-ms 3 spectra. smargiasso and de pauw (2010) have investigated matrices and conditions for optimal production of such ions and have concluded that dhb was the most versatile matrix; the presence or absence of isd fragments could be controlled, depending on the location of the laser shots. spectra obtained from the center of dhb targets produced mainly [mþna] þ ions that did not yield isd fragments, whereas spectra from the crystals surrounding the target yielded mainly [mþh] þ ions that fragmented readily. 9-aa (6/18), on the other hand, formed homogeneous matrix spots and did not induce isd. soltwisch and dreisewerd (2010) have noted that collisional cooling in an orthogonal tof mass spectrometer stabilizes fragment ions that are generated in-source and that by varying the buffer gas pressure, production of isd and post-source (psd)-type ions could be varied. under high-pressure conditions, isd-type fragments of o-linked glycosylated peptides were generated that retain the glycan. psd fragments, on the other hand, showed partial loss of the glycan from y-type peptide fragments. the detailed positive ion psd and isd fragmentation of deprotonated d-ribose (1/1) and d-fructose (1/16) has been studied with the aid of the isotopically labeled analogues, [1-13 c]-d-ribose, [5-13 c]-d-ribose and [c-1-2 h]-ribose (bald et al., 2009) . the fragmentation was compared with fragmentation through dissociative electron attachment (dea). fragmentations of deprotonated monosaccharides formed in the maldi process, as well as their transient molecular anions formed upon electron attachment, were characterized by loss of different numbers of h 2 o and ch 2 o units. two different fragmentation pathways leading to cross-ring cleavage were identified. metastable decay of deprotonated d-ribose proceeded either via an x-type cleavage yielding fragment anions at m/z ¼ 119, 100, and 89 (dominant ion, c 3 h 5 o 3 ), or via an a-type cleavage resulting in m/z ¼ 89, 77 and 71. this result is in contrast to previous cid studies where only a-type cross-ring cleavage was proposed. it was found that the heavier fragment anions at m/z ¼ 119 and m/z ¼ 100 generated via metastable decay exclusively arise from an x-type cleavage whereas the smaller fragment anions at m/z ¼ 89 and 71 arise predominantly from an a-type cleavage. a fast and early metastable cross-ring cleavage of deprotonated d-ribose observed in isd was dominated by x-type cleavage leading mainly to m/z ¼ 100 and 71; the latter ion is not the same as that found in psd. for dea of d-ribose, a sequential dissociation was identified that included metastable decay of the dehydrogenated molecular anion leading to m/z ¼ 89. the most dominant fragment ions in dea were due to faster decompositions occurring within several hundred nanoseconds (as in isd) and, thus, sequential reactions including an initial dehydrogenation could be excluded. several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kand i-carrageenans obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides, have been used as model compounds to study prompt (isd) and psd fragmentation. sulfated alditols gave [màh] à ions in negative-ion mode together with prompt fragment ions produced mainly by desulfation. sulfated aldoses gave mainly prompt fragmentation ions (c-cleavages and desulfation). non-sulfated aldoses and alditols only gave ions ([mþna] þ ) in positive ion mode with no prompt fragmentation. the aldoses yielded cross-ring fragmentation in the psd mode. several different matrices (dhb, norharmane (1/35), ferulic acid (1/41) and the ionic liquid matrices dhb/acid-n-butylamine and ferulic acid/n-butylamine) were investigated; the best results were obtained with dhb and nor-harmane (fatema et al., 2010) . cid on tof/tof and magnetic sector instruments have been compared with several types of biomolecule . the sector instruments produce high-energy collisions (8-20 kev) yielding more structural information from many compounds than instruments producing only low energy (1 kev) collisions. the case with different tof/tof instruments is less clear-cut because the collision energy is spread over a wide range. fewer differences were seen with carbohydrates than with some other compound types because most fragmentations (except formation of x-type cross-ring cleavages) occur at low energies. high-energy fragmentation in positive ion mode generally enhanced the relative abundance of cross-ring cleavage fragments, particularly x-type ions and has been used with hplc (offline) to provide a powerful method for glycoprotein analysis . full experimental details are given in the paper. experimental details for obtaining infrared multiphoton dissociation (irmpd) spectra from carbohydrates have been described . the technique offers the advantage that, because both parent and product ions can absorb ir photons, the spectra can be richer in information than those obtained by cid. in the same publication, the authors discuss sustained off-resonance irradiation-collision-induced dissociation (sori-cid) implemented with a maldi-ft-icr mass spectrometer which produced similar spectra to irmpd. experimental details are described. sori-cid with maldi-ft-icr has also been applied to muc-type o-glycans . the 157 nm photodissociation of n-linked glycopeptides has been investigated in a modified maldi tof/fof instrument by irradiating the ions within the collision cell. singly charged glycopeptide ions from horseradish peroxidase (hrp) yielded abundant peptide and glycan fragments. the peptide fragments included a series of x-, y-, v-, and w-ions with the glycan remaining intact. these ions provide information about the peptide sequence and the glycosylation site. the glycan fragmented to give both glycosidic fragments and abundant cross-ring fragments that were not observed in low-energy cid spectra. doubly charged glycopeptides generated by nanospray in a linear it mass spectrometer also yielded peptide and glycan fragments. however, the former were dominated by low-energy fragments such as b-and y-type ions while the glycan was primarily cleaved at glycosidic bonds (zhang & reilly, 2009 ). maldi-lift-tof-ms/ms and esi tandem mass spectrometry (esi-it-ms/ms n ) have been used for the characterization of free oligosialic acids and those derivatized with dmb, as well as partially o-acetylated derivatives. electrospray required the acids to be lactonized but the larger lactones could only be detected by maldi-tof. the fragmentation spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. however, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-and longchained species. furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues (galuska et al., 2010a) . the effect of the pressure of cooling gas in the ion source of an orthogonal-tof ms has a strong influence on the extent of analyte ion fragmentation. soltwisch et al. (2009) have investi-gated the effect of this parameter on peptide and oligosaccharide fragmentation using substance p and the milk sugar, lnfp-ii (52), respectively in both uv-and/or ir-maldi. a range of pressures, from 0.05 to 1.8 mbar was used in combination with seven different buffer gases (he, ne, ar, n 2 , co 2 , ch 4 , and isobutane). the influence of the ion extraction voltage on the analyte fragmentation was also investigated for a selected set of gas parameters. fragment ions exhibited characteristic fragment yield-pressure dependencies that were classified into three groups. for lnfp-ii, the yield of molecular ions rose with pressure until at the higher pressures, it was similar to that from substance p. the authors speculated that the consistently lower ion yields reported from oligosaccharides could be attributed to the generally low pressures used when recording their spectra. with respect to fragmentation, glycosidic fragment ions (termed type i) ions dominated the spectra at low pressure but their relative abundance fell dramatically as the pressure rose. the ions resembled species that are also found in maldi-psd ms. the abundance of type ii ions, which resembled typical isd fragments, and consisted mainly of cross-ring products, rose with pressure, probably because of a reduction in the secondary fragmentation process that resulted in loss of fucose. a few other ions, termed type iii ions did not show such dramatic changes with pressure. comparing the yields of fragmentation for the different buffer gases revealed a correlation between their internal degrees of freedom and their collisional cooling efficiency. changing the buffer gas pressure and/or extraction field provided an easy means to influence analyte ion fragmentation and to switch from the primary production of one type of fragment species to another. hexose rearrangements of protonated molecules of n-glycopeptides and reductively aminated n-glycans have been observed by maldi-tof/tof-ms/ms and esi-it-ms/ms (wuhrer, koeleman, & deelder, 2009b) . fragmentation of proton adducts of 2-ab and 2-aa-labeled high-mannose n-glycans from rnaseb resulted in transfer of one to five hex residues to the fluorescently labeled innermost glcnac residue. glycopeptides from various biological sources containing high-mannose glycans were likewise shown to undergo hex rearrangement reactions, resulting in migration of one or two hex moieties to the chitobiose core. tryptic fc-glycopeptides from igg peptides containing biantennary n-glycans were also shown to undergo hex rearrangements. such rearrangement products can cause major problems with the interpretation of unknown glycans but, fortunately, do not appear to occur from [mþna] þ ions, the major ions in maldi spectra of most neutral carbohydrates. the use of computer software for analyzing carbohydrate spectra is not as advanced as that for proteins; nevertheless, many investigators have developed software for spectral interpretation, composition calculations, and have constructed databases, usually for specific glycan types. much of this software is applicable to spectra generated from the majority of common ion sources. a good source of information is the book bioinformatics for glycobiology and glycomics: an introduction by the late claus-wilhelm von der lieth, luetteke, and frank (2009) . in addition, various tools for glycomics and available databases are covered in a comprehensive review by frank and schloissnig (2010) . the simplest of these algorithms calculates compositions from m/z values. although such software is extremely valuable, a composition is not a structure and such programs should not be used as the basis of labeling spectra unless the proposed structures are confirmed by suitable techniques. one such tool reported in the review period is called lipid and calculates molecular compositions for glycerophospholipids, gsls, fatty acids and small oligosaccharides from m/z values entered as single values or as mass lists. the user-extendable software is a microsoft excel add-in developed using visual basic for applications and is compatible with all versions of ms excel since ms excel 97 (hübner, crone, & lindner, 2009) . kronewitter et al. (2009) have constructed a library of possible n-glycan masses by successive dismantling of tetraantennary hybrid and high-mannose glycans. these calculations gave the possible masses that would be expected in a glycan mixture. three hundred thirty one distinct neutral compositions were obtained but many of these will represent several isomeric glycans. the smallest mass difference that was observed was 0.37 da. however, many of the masses coincided with isotope peaks from ions of different compositions meaning that, without deisotoping, a resolution of at least 12,500 would be needed to resolve all peaks. the theoretical masses were matched against measured masses from n-glycans released from human serum glycoproteins and 78 discrete compositions were detected. in a similar way, an in silico glycan database of possible nglycan compositions has been constructed by addition of known monosaccharide residues, such as those in a neu5ac-gal-glcnac antenna, to the common trimannosyl chitobiose core. the derived masses were then matched to the experimental mass and the software, named glyquest, predicted compositions and possible structures. next, it calculated possible glycosidic fragments from the proposed structures, matched these to the experimental mass spectrum and constructed a spectrum labeled with the proposed structures (gao, 2009) . the software could also be applied to glycans containing fluorescent labels such as 2-ab but was not applicable to glycopeptides with unknown modifications. however, as with much of the software developed for this work, the detailed structural details such as the linkage of each monosaccharide are not available and the software must be regarded as a guide to the total structure. a similar "branchand-bound" algorithm developed by peltoniemi, joenväärä, and renkonen (2009) starts with the trimannosyl-chitobiose core and then constructs n-type glycans in an iterative process until the target carbohydrate composition is reached. the algorithm identified several glycans from tf and human serum samples. glycospectrumscan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. two experimental data sets are required as inputs: (1) oligosaccharide compositions of the n-and/or o-linked glycans present in the sample and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of nand/or o-glycosylation sites. glycospectrumscan uses ms rather than ms/ms data, to identify glycopeptides and determine the relative distribution of n-and o-glycoforms at each site. it can be used to assign monosaccharide compositions on glycopeptides with either single or multiple glycosylation sites. the algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both esi and maldi data from all mass spectrometer platforms. low resolution data from, for example, its are heavily smoothed to yield the average mass whereas data from high resolution instruments receive a milder smooth and deisotoping to give the monoisotopic mass. the software was used to characterize the n-and o-linked glycopeptides from human secretory iga (siga), consisting of secretory component (7 n-linked sites), iga1 (2 n-linked, 5 o-linked sites), iga2 (4 n-linked sites) and the j-chain (1 n-linked site). glycospectrumscan is freely available at www.glycospectrumscan.org (deshpande et al., 2010) . prediction of glycosylation sites is another area of software development. a program that predicts n-and o-glycosylation sites based on local information, general protein information, sub-cellular localization and binding specificity of glycosyltransferases has been developed and was claimed to be about 90% accurate (sasaki, nagamine, & sakakibara, 2009a) . however, as with all predictive programs that are not 100% accurate, results should only be taken as a guide for designing appropriate location experiments. software that attempts to predict structures from spectra is possibly the most active area in computer applications. simglycan 1 is one such tool (apte & meitei, 2010) . the software accepts raw or standard experimental ms data files, matches them with its own database of theoretical fragments and generates a list of probable candidate structures. each structure is scored to reflect how closely it matches the experimental data. the software also predicts novel glycan structures by drawing a glycan and mapping it onto the experimental spectrum. other biological information is also available for easy reference. the program can be downloaded from http://www.premierbiosoft. com/glycan/index.html. another software platform for carbohydrate assignment is sysbioware, developed by vakhrushev, dadimov, and peter-katalinić (2009) and designed to work directly from raw ms data. the data are first imported into the spectrum browser, baseline corrected and denoized. peak detection is based on shape matching and the software detects monoisotopic m/z values and charge states. a biological filter is used during compositional analysis of the monoisotopic ions. the software was successfully tested with human urine. sysbioware, a software platform developed for ms data evaluation in glycomics, has been applied to the interpretation of spectra from human serum gsls. the masses of predicted ions arising from cleavages in the glycan and the ceramide moieties were calculated, thus enabling structural characterization of both entities. the calculated masses were then used to match with those in the spectra for structural identification . böcker, kehr, and rasche böcker (2009) have presented an algorithm for calculating glycan structures from tandem mass spectra. twenty-four spectra (of [mþh] þ ions) of 2-ap-labeled n-glycans obtained from batroxobin (from bothrops moojeni venom) were used as test compounds, the spectra were measured with a tof/tof instrument with a maldi ion source and the algorithm rapidly predicted possible topologies. biological restraints needed to be used to limit the predictions to reasonable structures. goldberg et al. (2009) have compared three algorithms, "max subgraph," "parsimony," and "randomwalk" that make inferences about glycan synthesis from biological knowledge for their ability to assign structures from 71 single-ms spectra from a variety of tissues and organisms, containing more than 2,800 manually annotated peaks. max subgraph behaved better than the other two but only uniquely assigned the correct structure to about half of the peaks in 41 out of the 71 spectra. a computer model that predicts n-linked glycan profiles based on cellular enzyme activities has been developed (krambeck et al., 2009) . the paper describes the expansion of a previously developed detailed model for n-linked glycosylation (krambeck & betenbaugh, 2005) with the further application to analyze maldi-tof mass spectra of human n-glycans. the glycosylation reaction network is automatically generated by the model, based on the reaction specificities of the glycosylation enzymes and allows prediction of the complete glycan profile and its abundances for any set of assumed enzyme concentrations and reaction rate parameters. a predicted mass spectrum of model-calculated glycan profiles is obtained and enzyme concentrations are adjusted to bring the theoretically calculated mass spectrum into agreement with that obtained experimentally. the result is a complete characterization of a measured glycan mass spectrum containing hundreds of masses in terms of the activities of 19 enzymes. in addition, a complete annotation of the mass spectrum in terms of glycan structure is produced, including the proportions of isomers within each peak. the method was applied to mass spectrometric data obtained from normal human monocytes and monocytic leukemia (thp1) cells. a kinetic model originally developed for the prediction of peptide cid spectra has been extended to predict the cid spectra of n-glycopeptides. the model was trained with 1831 cid spectra obtained with an ion trap and was able to predict cid spectra with excellent accuracy in ion intensities for n-glycopeptides up to 8,000 da in mass. the program is said to be capable of predicting up to 524 common glycoforms including high-mannose, hybrid and complex n-glycans with two to four antennae (zhang & shah, 2010) . spencer et al. (2010) have devised a computational approach to predict the fine structure and patterns of domain organization of heparan sulfate (hs). analysis uses chemical composition data obtained after complete and partial enzymatic digestion of mixtures of hs chains and produces populations of theoretical hs chains with structures that meet both biosynthesis and enzyme degradation rules. the program was used to analyze hs from various cell types and good agreement was found between experimental data and computer predictions. glycoviewer (http://www.systemsbiology.org.au/glycoviewer) is a web-based tool that can visualize, summarize, and compare sets of glycan structures. it takes as its input a list of glycan structures in international union of pure and applied chemistry (iupac) format or glycan structures constructed with a sugar structure builder. the output is a graphic, which summarizes all salient features of the glycans according to features such as the nature and length of any chains and the types of terminal epitopes. the tool can summarize several hundred glycan structures in a single figure. the report contains an example of use of the tool for analysis of normal and disease associated glycans from the human glycoproteome (joshi et al., 2010) . several glycan databases are available. the kyoto encyclopedia of genes and genomes (kegg) glycan databases contain useful information on glycan structures and metabolic pathways (hashimoto & kanehisa, 2009) and data mining the protein data bank for glycol-related data using the glycosciences. de internet portal has been discussed in methods in molecular biology (lütteke & von der lieth, 2009) . ito et al. (2010a) have synthesized n-and o-linked glycan libraries (named glycan mass spectral database, gmdb) and constructed a library of their positive ion ms 2 , ms 3 and ms 4 fragmentation spectra. n-glycans were in the form of their 2-ap derivatives whereas oglycans that were released by b-elimination were not. the library was said to be accessible on-line at http://riodbdev.ibase. aist.go.jp/rcmg/glycodb/ms (however, attempts by the reviewer to connect to the site have failed.) it can be searched either by mw of glycan composition in terms of isobaric monosaccharides and instructions on how to use the software are given in the paper. although such databases and tools for glycomics are readily available on the web, these have, until now, been isolated. this unfavorable situation has been discussed (von der lieth, 2007) and has been largely overcome by ranzinger et al. (2009) who have developed glycomedb, a meta-database for public carbohydrate sequences. at the time of publication (2009) it contained 35,056 unique structures in glycoct (www. glycome-db.org) encoding, referencing more than 100,000 external records from 1,845 different taxonomic sources. a user-friendly, web-based graphical interface has been developed which allows taxonomic and structural data to be entered and searched. the structural search possibilities include substructure search, similarity search, and maximum common substructure. a novel search refinement mechanism allows the assembly of complex queries. with glycomedb, it is now possible to use a single portal to access all digitally encoded, public structural data in glycomics and to perform complex queries with the help of a web-based user interface. a list of databases is given in aoki-kinoshita (2010). n-and o-glycan structures are usually depicted with small cartoons in which each constituent monosaccharide is shown by a symbol. unfortunately, there is no consensus on which symbol to use for any particular monosaccharide with most investigators preferring to develop their own system. several years ago, the consortium for functional glycomics (cfg) attempted to redress the problem with the introduction of a system that has since been adopted by several laboratories. unfortunately, this system has several major drawbacks: (a) it does not diagrammatically show linkage or anomericity and (b) it uses color to differentiate hexoses, thus causing problems when structures are printed in black and white and making the system difficult to use with pen or pencil on paper. a new system that overcomes these problems has recently been introduced (harvey et al., 2009c) . monosaccharides are shown as shapes with various additions to indicate functional groups (e.g. an inclusive dot to indicate a deoxy-sugar and a filled shape to code for an n-acetyl sugar). linkage is shown by the angle of the lines linking the sugar symbols and anomericity is shown by the type of line (full for a b-bond and broken for an a-link). examples can be seen below. although color is not used to define monosaccharides, the cfg colors can be used with the oxford symbols. unfortunately, color was omitted from the table of symbols in the original article but was published later as an erratum (harvey et al., 2009b) . the article received comments from the authors of the cfg system (varki et al., 2009) and discussion is continuing. this scheme and others are compared in a review by frank and schloissnig (2010) . two tools for displaying n-glycans found in the mammalian cho cell line have been developed (mcdonald et al., 2010) . both take as input the 9-digit identifier devised by krambeck and betenbaugh (2005) that uniquely defines each structure assuming the existence of the trimannosyl-chitobiose core. the first of these tools, glycoform, is designed to display a single structure from an identifier entered by the user. the display is updated in real time, using symbols for the sugar residues, or in text-only form. the two symbol sets discussed above are used, the symbols and layout devised by the cfg http://www. functionalglycomics.org/static/consortium/nomenclature.shtml or the alternative "oxford" system used by glycobase, a relation database of 2-ab labeled n-glycans (campbell et al., 2008) . however, although glycoform can display structures using the oxford system, unfortunately, it does not display the correct linkage information that is inherent to the full oxford system. in addition, glycoform can display the name of the glycan as used by glycobase. glycobase formalism does not yet handle nacetyllactosamine (naclac, 53) repeating units and is, therefore, currently limited to structures with one gal residue per branch. structures can be added to a library, which is recorded in a preference file and loaded automatically. individual structures can be saved as image files either portable network graphics (png), jpeg or windows bitmap (bmp) formats. the second program, glycologue, reads a file containing columnar data of nine-digit codes, which can be displayed on-screen and printed at high resolution. both programs, for windows, mac os x and linux x86 gtk can be downloaded from http://www.boxer.tcd. ie/gf. a major problem with the analysis of monosaccharides or small oligosaccharides by maldi is the presence of very abundant ions from matrices such as dhb in the low mass region of the spectra. various methods for overcoming this disadvantage are discussed above. nevertheless, maldi with conventional matrices has produced results and the technique works well for the larger oligosaccharides. thus, oligosaccharides from dextran, alginate, hyaluronan and chondroitin sulfate have been characterized by maldi-tof ms directly from a tlc plate after soaking it in the dhb matrix. the plate had a metal backing to ensure electrical contact. the tlc solvent system was n-butanol/formic acid/water (3:4:1, v/v/v). it was found that the high content of formic acid caused few problems but was responsible for partial formylation of glycosaminoglycans (gags) and minor n-acetyl loss from hyaluronan and chondroitin sulfate . a comparative study of maldi and a new technique, electrospray droplet impact secondary ion mass spectrometry (edi/sims) has been applied directly to fruits such as bananas, apples, grapes and strawberries. the major constituents, fructose (1/16), glucose (1/4), sucrose (6) and organic acids gave abundant [m þ k] þ ions positive mode and cf 3 coo à adducts in negative mode (the cf 3 coo à ions came from cf 3 cooh in the esi spray. these negative ion spectra were almost free of background ions. maldi from dhb, on the other hand, although producing positive ions gave virtually no ionization in negative ion mode (asakawa and hiraoka, 2010) . reviews of the analysis of polysaccharides are listed in table 5 . large polysaccharides need to be hydrolyzed, often enzymatically but sometimes chemically, to smaller fragments before they are amenable to maldi analysis. one chemical method involves the selective cleavage of the rhap-(1 ! 4)-a-galap linkage in rhamnogalacturonans. enzymic cleavage of this linkage is often ineffective, especially in highly branched rhamnogalacturonans but deng et al. (2009) have developed an improved chemical fragmentation method based on belimination of esterified 4-linked galacturonic acid (galpa, 54) residues that overcomes the problem. at least 85% of the carboxyl groups of the galpa residues in a. thaliana seed mucilage were converted to methyl or hydroxypropyl esters and b-elimination was found to be more extensive with hydroxypropyl-esterified than with methyl-esterified rhamnogalacturonans. the non-reducing 4-deoxy-b-l-threo-hex-4-enepyranosyluronic acid (55) residue formed by the b-elimination reaction was removed by treatment with aqueous n-bromosuccinimide. this method was used to fragment the branched rhamnogalacturonan from peppergrass seed mucilage with product characterization by maldi-tof ms, glycosyl residue composition analysis, and 1 and 2d nmr spectroscopy. the results showed that the most abundant low-mw fragments contained a backbone rhamnose (1/10) residue substituted at o-4 with a single side-chain, and suggest that peppergrass seed mucilage rhamnogalacturonans is composed mainly of the another chemical method for analysis of rhamnogalacturonan ii makes use of mild acid hydrolysis to hydrolyze the acidlabile monosaccharides 3-deoxy-d-manno-oct-2-ulosonic acid (kdo, 1/13), 3-deoxy-d-lyxo-2-heptulosonic acid (dha) and apiose (api, 56) at the branchpoint between the sidechains and the oligogalacturonide backbone to release short polysaccharide chains that were analyzed by esi and maldi-tof ms (séveno et al., 2009) . the method was optimized using citrus pectin and then applied to other plant species. experimental details for examination of extracellular polysaccharides (epss) from plants following digestion with a variety of endoglycosiodases and maldi-tof analysis from dhb has been reported by günl, gille, and pauly (2010) . other examples are listed in tables 6 (plants) and 7 (bacteria). ionization efficiencies of cyclodextrins and corresponding linear compounds (maltohexaose and maltoheptaose) have been compared together with differences in the ionization efficiencies of aand b-cyclodextrins (4/6) . alkali metal salts of li þ , na þ , k þ , and cs þ were used as the cationizing agents to enhance the ionization efficiency. relative ion intensities of the cyclodextrins were much larger than those of the linear carbohydrates and the difference showed an increasing trend with the size of the alkali metal cation. b-cyclodextrin had higher ionization efficiency than a-cyclodextrin (4/24) and the difference increased with increasing size of the alkali metal cation. the ionization efficiency was also found to be affected by the counter anions. the higher ionization efficiencies of cyclodextrins were explained with the number of coordination sites and the binding energies. analysis of milk oligosaccharides appears to be receiving increasing attention. reviews of mass spectrometric methods for their analysis have been published by niñonuevo and lebrilla (2009) , kolarich and packer (2010) and urashima et al. (2009) . wu et al. (2010b) have developed an annotated library of neutral human milk oligosaccharides with characterization by hplc, maldi-ft-icr ms and exoglycosidase digestion. pyrene labeling (amano et al., 2009a) has been used by amano et al. (2009b) to enable neutral carbohydrates from human milk to be observed by negative ion maldi-tof ms n . the neutral oligosaccharides from the milk of a woman (blood type a, le bþ ) were obtained by anion-exchange column chromatography after the removal of lipids and proteins. further fractionation was performed by means of aleuria aurantia lectin-sepharose column chromatography and reversed-phase hplc after labeling. twenty-two oligosaccharides with decaose cores were identified and, of these 21 had novel structures. locascio et al. (2009) have published a method for measuring the consumption of human milk oligosaccharides by 12 strains of bifidobacteria. oligosaccharides were quantified with deuterated and reduced oligosaccharide standards that were added after bacterial growth and results were processed with inhouse software called glycolyzer after removal of contributions from 13 c isotopes. high growth was found for bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains showed low or only moderate growth ability. other examples of the use of maldi analysis of milk glycans are listed in table 8 . glycoproteins and their attached n-and o-glycans is possibly the largest group of compounds that have been analyzed by maldi, catalyzed largely by developments in the biotechnology industry. analysis of these compounds has been reviewed by many authors (table 9) . n-glycans are normally attached to an asparagine residue in a asn-(xxx)ser-(thr) consensus sequence where xxx is any amino acid (xxx) except proline however, asn-linked n-glycans have recently been found at the 0.5-2.0% level on a non-consensus amino acid sequence (tvswn 162 sgal) in the c h 1 domain of human antibodies and on igg1 (valliere-douglass et al., 2009b). many investigators have published methods for glycoprotein enrichment. solid-phase glycan/glycoprotein capturing methods have become popular in recent years and some of these have been highlighted in an article by . (kooy et al., 2009) upis ceramboides (alaskan beetle) xylomannan first identification of a nonprotein anti-freeze compound (walters et al., 2010) commercial chitosan oligosaccharides analysis of commercial samples and preparation of sample with glcnac 5-12 not stated but has been found in cryptococcus neoformans human macrophage activation shown to be triggered by chitotriosidase-mediated chitin and chitosan degradation (gorzelanny et al., 2010) 1 format (not all items present): maldi method (matrix), compounds run (derivative), other methods. enrichment strategies for glycopeptides based on lectinaffinity chromatography and polysaccharide hydrophilic affinity physicochemical chromatography have been discussed by ito, hayama, and hirabayashi (2009b) . the combined use of these techniques effectively removes non-glycosylated peptides. the ability of boronic acids to form cyclic derivatives with the cis-dihydroxy groups present in most glycans has been extensively used. thus, 3-aminophenylboronic (apb) acid (5/42)functionalized beads, mesoporous silica, and nanodiamonds have been developed to enrich glycosylated peptides and proteins but the direct immobilization of the apb group was found to be insufficient to suppress nonspecific adsorption/ adhesion. consequently jang et al. (2009b) have designed a selfassembled monolayer (sam)-based plate, which contained a spacer group such as oligo(ethylene glycol) to reduce the nonspecific adsorption/adhesion, for direct detection of glycoproteins after affinity-capture (or enrichment) on the plate. the utility of the plate was demonstrated with model glycoproteins such as ribonuclease g and tf. a two-stage glycopeptide enrichment technique using boronate-functionalized beads has been developed by chen et al. (2010f) . samples were incubated with the functionalized magnetic beads in slightly alkaline conditions at room temperature for about 1 hr with gentle shaking. the beads were washed and the enriched glycoproteins/peptides were eluted under acid conditions and dried in a speed-vac evaporator. the glycoproteins were then either dissolved in 50 mm ammonium bicarbonate and digested by lys-c overnight or separated by sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis (page) and the resulting gel-bands were digested in-gel by lys-c overnight. the compounds were then ready for a second enrichment. alternatively, digestions could be carried out with trypsin. analysis was by maldi-tof. boronic acid functionalized nanoparticles have also been used to concentrate antibodies by capturing the carbohydrates attached to the fc region of igg . chalagalla and sun (2010) have prepared a boronic acid-containing polymer capped with biotin (57) for linkage to a magnetic bead and used the product for glycan capture and lin et al. (2010f) have constructed magnetic nanoparticles with immobilized apb acid for glycoprotein capture. "sno 2 @poly(hema-co-st-co-vpba)" core-shell nanoparticles containing boronic acid groups have been prepared by of copolymerization between 2hydroxyethyl methacrylate grafted (58) on sno 2 nanoparticles, styrene, and 4-vinylphenylboronic acid (vpba, 59). they have been used to extract tryptic peptides from hrp, bovine asilotf and human serum glycoproteins. analysis was by maldi-ms/ ms using an axima qit instrument (sheng, xia, & yan, 2010 survey epo and of several analytical methods including maldi, chromatography 175 (reichel & gmeiner, 2010) application of proteomics in biomarker discovery mainly proteins and glycoproteins. a novel boronic acid functionalized mesoporous silica, which holds the attractive features of high surface area and large porosity has also been used to concentrate glycopeptides. in comparison to direct (traditional) analysis, this method was stated to enabled two orders of magnitude improvement in the detection limit of glycopeptides irrespective of the nature of the attached glycans . the same group has also synthesized boronic acid functionalized core-satellite composite nanoparticles that possess both the superparamagnetic properties of magnetic nanoparticles and the surface chemistry of aunps. glycoproteins or glycopeptides could be obtained in high yield by use of a magnet. the composite nanoparticles were used to enrich glycosylated proteins from human colorectal cancer tissues for identification of n-glycosylation sites. in all, 194 unique glycosylation sites mapped to 155 different glycoproteins were identified, of which 165 sites (85.1%) were new. boronic acid functionalized gold-coated si wafers have been used as maldi plates to isolate and enrich glycopeptides . this method was claimed to be beneficial for several reasons. thus, solution transfer and eluting steps required in conventional enrichment strategies were not needed, thereby reducing sample loss. secondly, the lower limits of detection of glycopeptides were said to have been increased by two orders of magnitude. thirdly, non-specific bindings were not detected even when non-glycopeptides were 100 times more concentrated than glycopeptides. furthermore, glycopeptides could be detected in the presence of 200 mm ammonium bicarbonate or the physiological buffer, pbs. in a related method (tang et al., 2009a; yao et al., 2009) , aunps were first spotted and sintered onto a stainless steel plate, then modified with 4-mercaptophenylboronic acid (60) to provide a porous substrate with a large surface for capturing glycopeptides from peptide mixtures. the captured peptides were then analyzed by maldi-tof ms simply by deposition of a dhb matrix. the technique enabled sample enrichment, washing and detection steps to be fulfilled on a single maldi target plate. well-characterized glycoproteins, such as hrp and asialofetuin, were employed as standards to investigate the enrichment efficiency. fe 3 o 4 @c@au magnetic microspheres functionalized with 4-mercaptophenylboronic acid have been synthesized by the same group (qi et al., 2010) and successfully used for enrichment of glycoproteins and glycopeptides. a polyfunctional device has been constructed from macroporous silica foam (mosf) containing boronic acid (bmosf) or amino groups (nh 2 -mosf) and used to immobilize enzymes such as trypsin and selectively enrich glycopeptides. use of the device considerably speeded up hydrolysis times as demonstrated with glycopeptides from hrp (qian et al., 2010) . tang et al. (2010a) have immobilized the lectin con a on apb acid-functionalized magnetic nanoparticles using methyl a-d-mannopyranoside as a linker. the selective capturing ability of the con a-modified nanoparticles was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7,703. regeneration of the protein-immobilized nanoparticles could easily be performed by utilizing the reversible binding between the boronic acid and the sugar. cona has also been used in conjunction with hollow fiber flow field-flow fractionation (hf5) to preconcentrate high mannose type nlinked glycoproteins from bacterial lysates as exemplified by glycoproteins from streptococcus pyogenes . the specificity of datura stramonium agglutinin (dsa) for triand tetra-antennary glycans has been utilized to enrich human liver glycoproteins containing these larger glycans which were then separated and identified by sds-page followed by maldi-tof analysis (sun et al., 2009b) . the performance of chromatographic columns consisting of agarose-bead-bound 3-aminophenyl boronic acid, agarosebound wheat-germ agglutinin (wga) or a mixture of both compounds (boronic acid lectin affinity chromatography, blac) has been evaluated for glycoprotein enrichment using the model proteins of rnaseb and trypsin inhibitor in the presence of the non-glycosylated proteins, myoglobin (neutral) and lysozyme (basic) over a wide temperature range (5-65˚c). the results showed that glycoaffinity micropartitioning at 25˚c provided the highest recovery rate for glycoprotein enrichment. a large amount of lysozyme was present in the elution fractions of the 3-aminophenyl boronic acid-containing micropartitioning columns due to an ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase. at 65˚c, nonspecific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate (olajos et al., 2010) . a novel boronate affinity monolith, poly-(3-acrylamidophenylboronic acid-co-ethylene dimethacrylate) (61) has been prepared in 530 mm capillaries by a one-step in situ polymerization procedure . the monolith was used to separate glycopeptides from peptides produced from hrp and to separate this glycosylated protein from non-glycosylated bovine serum albumin (bsa). the maldi-tof spectrum of the hrp peptides showed little evidence of the presence of glycopeptides before passage through the capillary but revealed abundant glycopeptide ions after treatment. mass spectrometry reviews doi 10.1002/mas b. other solid-phase methods titanium dioxide (tio 2 ) microspheres, synthesized by a sol-gel method, have a high affinity for the acid groups of sialic acids and peptides. they have successfully been used for simultaneous enrichment of glycopeptides and phosphopeptides from, for example bovine rnaseb and human igg . detection was by esi but the method would be equally applicable to maldi-tof analysis. mysling et al. (2010) have used zic-hilic in a microcolumn format for spe and glycoprotein enrichment involving trifluoroacetic acid (tfa) ion pairing to increase the hydrophilicity difference between glycopeptides and non-glycosylated peptides. three mobile phases were investigated: 2% formic acid, 0.1% tfa and 1% tfa all containing 80% acetonitrile and experiments were conducted on single glycoproteins, a five-glycoprotein mixture and depleted plasma. the presence of tfa, particularly at the 1% level, in the mobile phase significantly improved the glycopeptide enrichment (3.7-fold) as evaluated by maldi-tof ms and rp-lc-esi-ms/ms. four types of hydrazine functionalized carboxyl and epoxysilanized magnetic particles (hfmp) have been developed by sun et al. (2010a) for isolation of glycopeptides. particles prepared by adipic dihydrazide functionalization from carboxylsilanized magnetic particles yielded the maximum capture capacity. the method was verified by successful isolation of all formerly glycosylated peptides from standard glycoproteins (fetuin, rnaseb, and human serum albumin (hsa)) and by identification of their glycosylation sites. c. other techniques maldi-tof ms has been used by carvalho et al. (2009) to characterize the aand b-subunit of recombinant and pituitary glycoprotein hormones that have been separated by a new method of incubating the glycoproteins overnight with acetic acid (0.5-3.0 m) at 37˚c. a. use of mass spectrometry to detect glycosylation of proteins a simple method to detect glycosylation is to measure the mass of a glycoprotein and then to repeat the measurement after incubation with pngase f to remove the n-glycans. the method has been used to detect the presence of n-glycans in recombinant bovine cd38 expressed in pichia pastoris (muller-steffner et al., 2010) . the molar masses of non-glycosylated (29,343 da) and penta-glycosylated thermomyces lanuginosus lipase (40,906) as measured by maldi-tof ms have confirmed glycosylation and shown that each glycan-moiety adds approximately 2,000 da to the molar masses (pinholt et al., 2010) . in another example, cu/zn superoxide dismutase monomer was determined to have a mass of 17,097 da before deglycosylation and 15,871 da afterwards giving a mass for the glycan of about 1,200 (nedeva et al., 2009) . the difference between the sequence mass (33,768 da) and measured mass of about 44,604 da of the peroxidase from royal palm tree (roystonea regia) together with the fact that the amino-acid sequence includes 12 possible n-glycosylation sites, suggests heavy glycosylation. glycosylation sites were identified, in this case, by n-terminal sequencing and maldi-tof-ms analysis of tryptic peptides . b. mass spectrometric detection of glycoforms of intact glycoproteins although resolution of glycoforms by maldi-tof ms is generally inferior to that obtained by esi, glycoproteins with masses in the region of 60 kda, containing only a limited number of glycoforms have been resolved successfully as illustrated by the resolution of four glycoforms of antithrombin in a study of altered glycosylation causing antithrombin deficiency . glycans appeared to be sialylated biantennary (residue mass 2,204 da). ogawa et al. (2009) have observed resolution of glycoforms of stereum purpureum endopolygalacturonase i produced in p. pastoris (36.5 kda). three main ion peaks corresponding to the protein with the high-mannose glycans man 8-10 glcnac 2 together with some minor unresolved ions were observed. c. detection of glycosylation sites and site occupancy the most common method for detecting site occupancy is to utilize the conversion of the asn to which the n-glycans are attached to aspartic acid (asp) when the glycans are released with pngase f. the increase by one mass unit is readily detected by ms. the method has been used, for example to confirm occupancy of six of the seven potential n-linked glycosylation sites in the envelope glycoprotein gp116 and three of the four potential sites in the gp64 protein of the yellow head virus from the penaeus monodon shrimp (soowannayan et al., 2010) . periodate oxidation and hydrazide capture on a solid support have been used by lewandrowski and sickmann (2009) to study glycosylation sites in human platelet proteins. the bound glycoproteins were sufficiently stable to allow washing, following which the proteins were hydrolyzed. glycopeptides remained bound to the solid support through the glycan moiety from where they were released with pngase f and the glycosylation site was identified by means of the asn to asp conversion. a new method using tandem 18 o stable isotope labeling (tosil) to quantify the n-glycosylation site occupancy has been reported (liu et al., 2010k) . glycoproteins were digested with trypsin and pngase f in the presence of either h 2 18 o or h 2 16 o. three 18 o atoms were introduced into n-glycosylated peptides, two at the carboxyl terminus of all peptides and the third at the n-glycosylation site. the samples were mixed to give pairs of ions in the resulting maldi or esi spectra. a unique mass shift of 6 da was produced by n-glycosylated peptide with a single glycosylation site, whereas non-glycosylated peptides produced an ion pair spaced by only 4 da. intensity ratios could be used to monitor site occupancy in various physiological and disease conditions. the method yielded good linearity within a 10-fold dynamic range with the correlation coefficient r 2 > 0.99. the standard deviation (sd) ranged from 0.06 to 0.21, for four glycopeptides from two model glycoproteins. the method was used to monitor glycoproteins in the sera from a patient with ovarian cancer and healthy individuals. eighty-six n-glycosylation sites were quantified and n-glycosylation levels of 56 glycopeptides showed significant changes. a similar 18 o-labeling technique was used by alvarez-manilla (2010b) to identify n-glycosylation sites in con-a-extracted glycopeptides from pluripotent murine embryonic stem cells. glycopeptides rather than glycoproteins were extracted from tryptic digests to avoid false positives produced from non-glycosylated proteins that were bound to extracted glycoproteins if no digestion had been performed. segu et al. (2010b) have published a method for detecting sites occupied by glycans carrying a fucose residue attached to the core. the method made use of the endoglycosidase m which, like pngase f has a broad spectrum of activity with the exceptions that (a) it is not active on core-fucosylated glycans and (b) it shows reduced activity with larger glycans. the second exception was overcome by conducting incubations in the presence of sialidase, b-galactosidase and b-n-acetylhexosaminidase, which reduced the size of the glycans. then, the results were compared with the products of digestions performed with pngase f allowing the core-fucosylated sites to be determined. analyses were by lc/ms but the technique would be equally applicable to maldi-tof analysis. d. analysis of glycopeptides because many glycoproteins are too large and heavily glycosylated for direct analysis by ms, much work is performed on derived glycopeptides, most commonly tryptic glycopeptides. tryptic cleavage of glycoproteins is frequently hindered by steric hindrance imposed by the glycans but improvements can be made by the use of heat to increase the rate of proteolysis. segu, hammad, and mechref (2010a) have used microwaveassisted enzymatic digestion to achieve higher sequence coverage of several model glycoproteins such as fetuin, tf, and fibrinogen. efficient digestion was achieved in 15 min at an optimum temperature of 45˚c; there was no apparent loss or partial cleavage of the glycans. signals from glycopeptides are often weak or absent from the spectra of mixed peptides and glycopeptides, a situation that can be improved by fractionation of the two compound classes. wohlgemuth et al. (2009) have investigated several techniques and have shown that hydrophilic interaction chromatography (hilic) chromatography with zic-hilic and tskgel amide-80 are very specific at capturing glycopeptides from mixtures. sialylated glycopeptides could also be enriched with tio 2 . capture using a hydrazide column resulted in lower recovery and involved a more complex enrichment scheme. a new material for glycopeptide concentration, termed "click maltose," has been synthesized by linking the alkynyl-derivatized maltose chain to the azide derivatized silica gel through click chemistry. unlike the rigid structure of sepharose, the saccharide chain of click maltose exhibits a certain amount of flexibility, which provides a sufficient number of hydroxyl groups for the effective formation of hydrogen bonds with the glycans attached to glycopeptides. the material was used to isolate glycopeptides from igg, rnaseb, and agp . cellulose columns have also been used for concentration of glycopeptides (snovida et al., 2010) . a method for detecting core-fucosylated (cf) glycoproteins for screening purposes has been reported by jia et al. (2009) . after igg depletion, fucosylated plasma proteins were enriched by use of lens culinaris lectin and the bound glycoproteins were digested by trypsin. these compounds were enriched by use of a 3,000 da cut-off filter, a procedure that also combines de-salting and concentration. the recovered glycopeptides were then treated with endoglycosidase f3, which specifically cleaves the glycosidic bond between the two proximal (core) glcnac residues and leaves the fucosyl-glcnac residues attached to the peptides. four standard glycoproteins, apo-tf, fetuin, rhepo, and rnaseb, was used to illustrate the method. in addition, a part of the untreated tryptic peptides was treated with pngase f in order to locate the glycosylation site by the asn to asp conversion. products were detected by maldi-tof. in a related method using an ion trap, a neutral loss scan for fucose (146 da) was also used to detect fucosylation. the methods were applied to the detection of fucosylated glycoproteins in the plasma of healthy subjects and subjects with hepatocellular carcinoma. over 100 fucosylated glycoproteins and attachment sites were identified, and over 10,000 mass spectra of cf glycopeptide were analyzed. a method termed the sulfate emerging method has been described for specifically extracting sulfated glycopeptides (toyoda, narimatsu, & kameyama, 2009 ) from mixtures. the method overcomes the often negative contribution from other charged groups in the molecules. to accentuate the negative charge on the sulfate group, basic amino acids were eliminated and carboxylic acids were neutralized as follows: the protein was first hydrolyzed with trypsin and then the positively charged c-terminal lysines (lyss) and arginines (args) were eliminated by incubation with carboxypeptidase b. the negative charges of the carboxylic acid groups on the peptides were then neutralized by chemical modification with acetohydrazide using the recently reported quantitative modification of carboxyl groups in sialic acid using this reagent and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (43). the sulfated glycopeptides in the mixture were then captured by anion exchange resin with a basic buffer (ph 8.6) in which protonation of histidine residues was suppressed. finally, the sulfated glycopeptides were eluted from the anion exchange resin by increasing the ionic strength of the elution buffer for analysis by ms. rather than trypsin, pronase has been used as a nonselective enzyme to reduce the protein to a single amino acid (asn) or short peptide attached to n-glycans. these compounds are generally smaller than those obtained from o-linked glycopeptides, probably because o-glycans lie closer to the peptide backbone than n-glycans and protect the polypeptide from enzymatic digestion. n-glycans usually rise above the peptide backbone exposing the polypeptide to enzymatic digestion. dodds et al. (2009) have described immobilized pronase which retains its activity after repeated use for at least 6 weeks. use of negative ion detection has been reported by nwosu et al. (2010) as providing distinct advantages over detection in positive ion mode for the detection of glycopeptides produced by pronase digestion. analysis in positive ion mode, although most commonly used for glycopeptide characterization, is hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides carrying sialic acids. furthermore, cid spectra of glycopeptides in the positive ion mode predominantly yields glycan fragments with minimal information on the peptide moiety. in the study by nwosu et al., which employed bovine lactoferrin for detection of n-glycosylation and k-casein for o-glycosylation, 44 potential n-linked glycopeptides were detected in the positive ion mode whereas 61 potential n-linked glycopeptides were detected in negative ion mode. analysis of k-casein, which contained mainly sialylated glycans, yielded improved results in negative mode. experimental details for peptide mass fingerprinting and identification of glycosylation sites have been published by wilson, simpson, and cooper-liddell (2009) . unlike the case with released glycans, where a relatively low mass accuracy measurement is usually sufficient to determine the composition in terms of the constituent monosaccharides, the situation is very different for measurements of glycopeptides. desaire and hua (2009) have examined the accuracy required and have concluded that in only a few cases can the mass accuracy provided by most commercial instruments be sufficient to unambiguously assign compositions to all glycopeptides in a mixture. e. n-glycan release once glycosylation sites have been determined, detailed structural analysis of the attached glycans is more conveniently carried out after releasing the glycans from the protein or peptide. both chemical and enzymatic methods are available but although, in the past, chemical release with hydrazine was popular, most investigators now prefer enzymatic methods. i. enzymatic release pngase f. peptide n-glycosidase f (pngase f), an amidase, is the most popular enzyme for cleaving n-glycans from their asn linkage site. it shows a broad range of substrate specificity with the exception that it does not release glycans bearing a fucose residue attached to position 3 of the reducingterminal glcnac; in these cases, pngase a is appropriate. pngase f cleaves the entire glycans, which are released as the corresponding glycosylamines. these compounds rapidly hydrolyze to the glycan with retention of the reducing terminus. this method is, thus, distinctly advantageous to techniques such as b-elimination, popular with o-glycans (see below) because this site can conveniently be used to attached tags for fluorescence or other detection methods. wang et al. (2009i) have recently found that the activity of the enzyme towards denatured glycoproteins can be enhanced by removal of the nterminal h1 helix from the enzyme. bereman et al. (2009b) have studied methods for optimizing the release of glycans with this enzyme. dialysis of plasma prior to incubation was found to have little or no effect. however, microwave-assisted glycan release was found to be beneficial; 20 min at 20˚c with approximately 250 w was found to give optimal results. surprisingly, no protease digestion was required as needed with standard incubation methods, and it was found that an 18-hr incubation with no detergent (np40) led to the greatest ion abundance of glycans from plasma glycoproteins. data could be obtained in less than 1 day from raw plasma samples utilizing microwave irradiation. pngase f-glycan release from human serum glycoproteins has been achieved in 10 min by using a constant microwave power of 20 w, giving a temperature of 44˚c (kronewitter et al., 2010) . in this study, the glycans were recovered by spe using a robotic liquid handler and examined by maldi with an ft-icr instrument from dhb. replicate analysis gave coefficients of variation of less than 0.2. the standard protocol for n-glycan release from glycoproteins requires relatively long deglycosylation times (from several hours to, usually, overnight) and relatively high enzyme concentration (from 1:250 to 1:500 enzyme/substrate ratio). szabo, guttman, and karger (2010a) have used a high-pressure method, both to reduce the reaction time and the amount of enzyme required. thus, a pressure-cycling device was use to cycle the pressure from atmospheric to as high as 30 kpsi. greater than 95% release of the asn-linked glycans from bovine rnaseb, human tf, and polyclonal human immunoglobulin was achieved in only a few minutes using as low as 1:2,500 enzyme: substrate molar ratio. a reactor with immobilized pngase f on a monolithic polymer support in a capillary has been developed that allows fast and efficient release of n-linked glycans. performance was determined with rnaseb, chicken ovalbumin, and human igg with detection by maldi-tof ms. the optimized reactor was integrated into a multidimensional system comprising on-line glycan release and hydrophilic interaction liquid chromatography (lc) followed by esi-tof ms detection. using this system, human igg was deglycosylated at room temperature in 5.5 min to an extent similar to that achieved with the soluble enzyme after 24 hr at 37˚c (krenkova, lacher, & svec, 2009) . immobilization of pngase f on detonation nanodiamonds has resulted in glycan release from glycoproteins in less than 10 min . the method, using trypsin immobilization, also gave good results and proved to be better for proteolysis than the use of commercial immobilized trypsin beads. artefacts associated with pngase release of n-glycans. for solution release of glycans, the glycoprotein is usually denatured by reduction and alkylation or by use of detergents or other compounds such as urea. in general, low concentrations of urea (<3 m) do not usually cause irreversible protein denaturation. indeed, pngasef itself is stable in 2.5 m urea at 37˚c for 24 hr and still possesses about 40% activity in 5 m urea. however, other glycoproteins appear more susceptible. for example, analysis by sds-page and maldi-tof ms have revealed that additional 2.5 kda of glycans can be released by pngase f if the deglycosylation is conducted in 2 m urea suggesting that urea treatment exposes a glycosylation site that was previously inaccessible to pngasef . use of urea, however, can cause problems with the released glycans because it has been reported to compete with water for hydrolysis of the initially formed glycosylamines with the formation of a urea complex (omtvedt et al., 2004) . another artefact that has been found in glycans released with pngase f involves the reaction of the glycosylamines with h 2 s to form the glycan-sh analogue. the h 2 s arises from dithiothreitol (dtt, 6/44), a reagent used for protein alkylation and present in some commercial preparations of the reagent. the consequence of this reaction is an increase in 16 da giving the impression, from a simple mass measurement, that the glycan has an additional oxygen atom. addition of 16 da can also be observed in maldi spectra as a consequence of the formation of [mþk] þ rather than [mþna] þ ions, but this possibility can be excluded by formation of [mþcs] þ ions whereupon the 16 da mass increase will still be present. the reaction with h 2 s was first noted with glycans from human igg and negative ion ms/ms of the artefactual products located the 16 da to the reducing-terminal glcnac residue. the negative ion fragmentation pattern was the same as that expected for a glycan with a hexose attached to the 6-position of the residue rather than fucose (deoxy-hex) that was actually the case. this result emphasizes how careful one must be, not only in deducing compositions from glycan masses but also in interpreting their ms/ms spectra . another artefact of the pngase f release step, detectable by ce but not by ms has been identified as the product of epimerization of the terminal glcnac residue to n-acetylmannosamine (mannac, 62) under the slightly basic conditions usually employed in the release reaction. reducing the ph to 5.5 effectively removed the by-product (liu, salas-solano, & gennaro, 2009h) . other endoglycosidases. another popular enzyme for n-glycan release is endo h which cleaves the chitobiose core of high-mannose and hybrid glycans but not complex ones, leaving a glcnac residue, with any linked fucose, attached to the protein. pace et al. (2009) have made use of this property to release and identify minor glycans in igg without interference from the more abundant complex glycans. endo f1 has a similar specificity and has been used by, for example, voutilainen et al. (2010) to detect glycosylation in talaromyces emersonii cellobiohydrolase cel7a produced in the yeast saccharomyces cerevisiae. ammonium hydroxide/carbonate-based chemical deglycosylation and pngase a enzymatic release have been compared for glycan release from a plantibody produced in tobacco plants (triguero et al., 2010) . although both methods gave similar profiles as evaluated by hplc of 2-ab derivatives, the main drawback of the chemical release method was that it induced degradation of a1,3-fucosylated n-glycans. ii. extraction and purification of released glycans. cleanup of samples prior to ms is crucial to obtaining good spectra. many methods are in use; porous graphatized carbon (buser et al., 2010) is popular and we have found nafion membranes (börnsen, mohr, & widmer, 1995) to be convenient at removing both salts and some hydrophobic compounds. avoiding the introduction of contaminants is also important. disposable plasticware such as plastic test tubes that are normally used to process samples have been shown to be a major source of contamination. the contaminants, which produce ions, mainly prompt fragments across the entire mass range to about m/z 3,000, originate from polymers that are used to protect the plastic against oxygen or uv light degradation. such compounds are hindered amine light stabilizers (halss) used in modern polyolefin (polypropylene, polyethylene) stabilization. the polymeric agent: poly-(n-b-hydroxyethyl-2,2,6,6-tetramethyl-4-hydroxy-piperidinyl succinate, 63), known as tinuvin-622 or lowilite 62, has been found to leach from laboratory polypropylene or polyethylene plastic test tubes into solvents used for sample preparation. 1.5 ml plastic tubes were found to be the major source of the contamination but the authors of the paper found that this could be minimized by using large solvent volumes of, for example, matrix solution (sachon et al., 2010) . amano and nishimura (2010) have used two kinds of hydrazide-functionalized glycobeads, termed glycoblot h and glycoblot abc of which the latter carries an additional fluorescent probe, to extract pngase f-released n-glycans from solution. sialic acids were then converted into methyl esters using the 3-methyl-1-p-tolyltriazene (mtt, 6/23) reagent de-scribed by miura et al. (2007) and the products were examined by maldi-tof ms after release of the glycans from the beads by mild acid hydrolysis. the method was used to examine human serum glycoproteins for cancer biomarkers. full experimental details of the extraction and derivatization procedure are given in the paper. enrichment of serum and cellular glycoproteins with glycoblot h beads has also been used for o-glycan analysis . glycans were released from human milk osteopontin and urinary muc1 glycoproteins with ammonium carbamate and the method was proposed as ideal for identification of biomarkers. a method for sequentially enriching sulfated glycans by strong anion-exchange chromatography according to their degree of sulfation has been described by lei, novotny, and mechref (2010) . the method is based on modifying the binding ability of strong anion-exchange material with different sodium acetate concentrations, thus enabling selective binding and a subsequent elution of different glycans according to their degree of sulfation. before this enrichment, the negative charge on any sialic acid was eliminated by permethylation. the method was initially optimized using sulfated oligosaccharide standards and then used to examine the sulfated n-glycans from btsh, a glycoprotein possessing mono-and disulfated n-glycans. f. analysis of released glycans ahn et al. (2010) have obtained good resolution of n-glycans as their 2-ab derivatives using hilic columns packed with 1.7 mm sorbent. glycans were released from rnase b with pngase f and extracted using a microelution hilic spe 96well plate. the labeled glycans were also extracted from the preparative reagents using the same plate and their integrity was checked by maldi-tof ms. in another technique, guillard et al. (2009) have experimented with optimizing a linear ion trap instrument for automated measurement of permethylated n-glycans in serum. glycans were released with pngase f, cleaned with graphitized carbon and permethylated with the sodium hydroxide method. dhb, although the favored matrix for carbohydrates, failed to give the necessary reproducibility because of the large crystal size. chca, on the other hand, proved to be satisfactory. full experimental details for analysis of o-and n-linked glycans have been published by several authors (azadi & heiss, 2009; morelle et al., 2009a; north et al., 2010b) . g. total methods for glycoprotein structure there have been many reports of methods for total glycoprotein analysis of which the following are representative. a small-scale method for n-glycan release and analysis from plants used to produce recombinant glycoproteins has been described . concentration, protease digestion and deglycosylation are carried out in a single concentrator unit mass spectrometry reviews doi 10.1002/mas without the need for intermittent purification. this approach minimized adsorptive losses and facilitated handling. the plant protein was concentrated in a unit with a 5 kda cutoff and after buffer exchange, pepsin digestion was carried out in the concentrator overnight. deglycosylation was carried out with pngase a for 24 hr. released n-glycans were purified using reversed-phase and cation exchange chromatography in micro-columns and analyzed by maldi-tof ms without derivatization. a chip-based reversed-phase lc/ms method for n-glycan analysis suitable for biomarker discovery has been developed by . n-glycans were released from bovine fetuin as a model glycoprotein and human serum glycoproteins with pngase f and reduced to alditols with an ammonia-borane complex. the glycans were then permethylated in dimethylformamide to avoid artefacts in ms measurements and their structures were checked by maldi-tof measurements. reversed-phase microfluidic lc of the permethylated n-linked oligosaccharide alditols was then performed and was shown to resolve some closely related structures. optimized lc gradients, together with nanospray ms were then used with human serum samples to distinguish breast cancer patients from control individuals. a previously established two-dimensional hplc technique has been adapted as a hplc-maldi ms method for n-glycan analysis by gillmeister et al. (2009) . glycans were released from glycoproteins with pngase f purified with graphitized carbon and fluorescently labeled with 2-ap. the labeled glycans were analyzed on a 2-mm reversed phase (rp) hplc column and spotted onto a maldi-tof ms plate together with the dhb matrix using an automated plate spotter. the method gave a 100-fold reduction in the required amounts of starting protein compared with the earlier procedure. the entire process could be carried out in 2-3 days for a large number of samples as compared to 1-2 weeks per sample for previous two-dimensional hplc methods. the modified method was verified by identifying n-glycans from an igg antibody from human sera samples and applied to analysis of tissue plasminogen activator (tpa) from cho cell cultures under varying culture conditions. kim et al. (2009b) have released glycans on a polyvinylidine difluoride (pvdf) membrane with pngase f and cleaned them with graphitized carbon contained in a 96-well plate before converting them into girard's t derivatives to introduce a constitutive cationic charge for quantification. analysis was by maldi-tof ms and the robust method was used to profile n-glycans from ovarian cancer patients using as little as 5 ml of serum. a high-throughput method for the analysis of human plasma glycomes using a 48-channel multiplexed capillary gel electrophoresis (cge) dna sequencer with laser-induced fluorescence detection (cge-lif) system has been described with maldi-tof ms used to provide structural information (ruhaak et al., 2010c) . glycans were released from plasma glycoproteins in a 96-well plate using pngase f and converted into apts derivatives with the help of 2-picoline borane (27) as the reducing agent. analysis by cge-lif using the dna sequencer allowed 96 samples to be analyzed in only 2.5 hr (the experimental time was longer because of two overnight incubations). the method was applied to a study of glycosylation patterns during first, second, and third trimesters of pregnancy, as well as 6 weeks, 3 months, and 6 months postpartum. although analysis of glycoproteins carrying neutral glycans is now routine, analysis of glycoproteins with sialylated glycans is more difficult. hao, ren, and xie (2010) have approached the problem by first performing a tryptic digestion to give peptides and glycopeptides. peptide mass fingerprinting was performed on the peptides in order to identify the protein. the glycopeptides, separated by hilic chromatography, were examined by maldi-tof ms and ms/ms and treated with pngase f to release the glycans, which, together with the resulting peptides were again examined by ms. the asn to asp conversion in the peptide fraction enabled the glycosylation site to be identified. finally, the glycans were desialylated with dilute hcl and again analyzed by ms. the technique was applied to glycoproteins from human serum separated by 2-d electrophoresis and the differences in n-glycosylation were successfully determined for a1-antitrypsin between different gel spots. in another method, sialylated glycoproteins have been selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of the sialic acid glycosidic bonds. mass spectrometric fragment analysis allowed identification of glycan structures and additional fragmentation of deglycosylated ions yielded peptide sequence information which allowed glycan attachment sits to be identified together with identification of the protein. using this method, the investigators identified 36 n-linked and 44 o-linked glycosylation sites on glycoproteins from human cerebrospinal fluid . related to this method is one developed by klement et al. (2010) for enrichment of o-glcnac-modified proteins. glycoproteins were again oxidized with periodate and captured by hydrazide resin capture. rather than release of the peptide enzymatically, the glycopeptide was released by hydroxylamine treatment which also converted the aldehyde groups of the oxidized glycan to oximes. the open nature of the carbohydrate ring, following oxidation, lead to the production of characteristic fragment ions facilitating both glycopeptide identification and site attachment. the method was applied to analysis of a-crystallin a and the drosophila proteaosome. h. comparisons of methods for n-glycan analysis a comparative study of three techniques, maldi-tof, sds-page and cge-on-a-chip, for measuring the mws of large glycoproteins has been reported by müller et al. (2010b) . it was found that all three techniques were capable of determining the mw of all high mw (glyco)proteins tested. the non-commercial cge-on-a-chip assay allowed electrophoretic separation of proteins in the mw range from 14 kda to 1 mda. mw assignment was limited to 500 kda in the case of sds-page but with the proper matrix (thap for most glycoproteins, sinapinic acid for a2-macroglobulin) and sample preparation, analysis with a standard maldi-tof-ms provided accurate mws for all high mw proteins up to 1 mda. three methods for n-glycan characterization, namely maldi-ms of glycopeptides from tryptic digestion, negativeion esi-ms/ms of released n-glycans, and normal-phase hplc of fluorescently labeled glycans, in combination with exoglycosidase sequencing, have been evaluated for glycan identification using monoclonal antibodies expressed in tobacco plants as model compounds (triguero et al., 2010) . the ms methods identified the major glycans, but the hplc method was found to be the best for identification and relative quantitation. negative-mode esi-ms/ms easily provided direct identification of features such as the linkage position of the fucose residue linked to the inner core glcnac residue. grey et al. (2009) have developed a high-performance ion exchange chromatographic method for n-glycan analysis and have shown that it gives very similar results to analysis by maldi-tof. a series of standard glycans was examined and the method was extended to the analysis of n-glycans released from igg1. an inter-laboratory study involving eleven uk laboratories using their routine glycan analysis procedures looked at reproducibility on glycan profiling from n-glycans released before the study from four glycoproteins, human and bovine agp, bovine pancreatic rnaseb and human serum immunoglobulin g (higg). data interpretation focused on the relative amounts of different glycan structures present, the degree of sialylation, galactosylation profiles, fucosylation, and bisecting glcnac content and the number of glycan components identified. all laboratories found high levels of sialylation for human and bovine agp, but varying amounts of di-, tri-, and tetra-antennary glycans. values obtained from mass spectrometric and chromatographic methods clustered separately. the proportion of the major man 5 glcnac 2 from rnaseb was between 29% and 62%. proportions of fucosylated and bisected glcnac glycans from higg were between 58% and 96% and 9% and 23%, respectively. mass spectrometric approaches consistently identified more glycan species, especially when both n-glycoylneuraminic acid (neu5gc) and neu5ac were present (thobhani et al., 2009) . a recent test of the ability of several laboratories to identify n-glycans released with pngase f from a mixture of four glycoproteins, asialo-fetuin, chicken ovalbumin and both human and bovine agp has also yielded some alarming results (orlando, leymarie, & keck, 2010) . although 18 of the 19 laboratories detected the presence of fucosylated complex nglycans, 14 of them incorrectly located the fucose to the core glcnac of human agp rather than to an antenna. all nine of the labs using ms (not ms 2 ) misidentified the site and all five of laboratories relying on software to identify the site also reported it incorrectly. although the ionic charge was not specified, it is assumed to be positive because it would be impossible to make this mistake with negative ion fragmentation. features such as the position of fucose residues produce diagnostic cross-ring fragments whose mass depends on the location of the fucose residue . such mis-identifications are particularly worrying because serum agp is elevated in inflammatory disease and is of potential use as a biomarker. many laboratories using this potential biomarker in serum also report the structure incorrectly. another problem in the survey arose with n-glycoylneuraminic acid (5/38), present in the bovine version of a1glycoprotein. this carbohydrate is antigenic and is of concern to pharmaceutical companies producing pharmaceuticals in organisms that utilize this sialic acid. in the survey, eight of the laboratories, including seven of the eight participating industrial laboratories failed to detect its presence. all of the four laboratories that used a fluorescence tag, failed to detect this sialic acid. slightly better results were obtained by laboratories using maldi; of ten labs that used this technique, only two failed to detect this sialic acid. most laboratories that successfully detected this carbohydrate, permethylated their samples, which, of course, would stabilize it to maldi conditions. with mixtures containing different quantities of glycans, no lab correctly detected either three or four of the changes, one lab identified two of the four changes, seven labs identified one change but the 11 other labs failed to identify any changes correctly. clearly, current analytical methods leave much to be desired. i. applications of maldi to the detailed structural determination of n-linked glycans a large number of reports have appeared on the applications of the above techniques to analysis of n-glycans from specific glycoproteins. these are summarized in tables 10 and 11 . other examples can be found in the tables on medical applications of maldi ms (table 23 ) and biopharmaceuticals (table 24) . some of the more unusual structures to be discovered are tetraantennary glycans with poly-n-lactosamine extensions with up to nine fucose residues in human seminal plasma , a man 5 glcnac 2 glycan with a bisecting glcnac residue (64) (buser et al., 2010) ; a man 8 glcnac 2 -type glycan with two bisecting glcnacs (65) proposed from dictyostelium discoideum feasley et al., 2010) and an unusual glycan with internal fucose and glucuronic acid (glca) from rapana venosa hemocyanin (velkova et al., 2009) . however, in the latter case, the structure was based on evidence from only one positive ion ms/ms spectrum and is open to alternative interpretations. symbols as defined for structures 17 and 18 plus = mannose j. miscellaneous studies among other studies, maldi-tof ms has been used to confirm the glycan compositions of several well-known glycoproteins in a study showing that glycosylation protects proteins against free radicals generated from toxic xenobiotics (martínek et al., 2010) the rice-derived recombinant human transferrin (rhtf) has been shown to be non-n-glycosylated by maldi and pngase f enzyme digestion . structural determination (highmannose, with bisect on branching mannose and 6antenna) (continued) (continued) sodium hydroxide followed by reduction to prevent a peeling reaction is the most common method but has the disadvantage of eliminating the reducing terminus, thus preventing tagging with fluorescent or other tags. some investigators have, thus, investigated the use of milder bases with the aim of avoiding the reductive stage and retaining the reducing terminus. zheng, guo, and cai (2009) have compared ammonia, methylamine and dimethylamine at 55˚c for 6 hr for release of n-acetylgalactosamine (galnac) from a small glycopeptide. b-elimination with dimethylamine and methylamine resulted in the conversion of the glycopeptide to 69.2% of the dimethylamine derivative at m/z 550.32 and 61.5% of the methylamine derivative at m/z 543.33, respectively. however, the incubation of the glycopeptide with ammonia only resulted in 8% production of the product. the authors concluded that elimination with dimethylamine was the most efficient for release the o-linked glycans. release with methylamine was used by sun et al. (2010b) to determine the glycosylation sites in human protein c inhibitor by the 13.03 mass increment introduced by the reaction. release with ammonia has been investigated in detail by yu et al. (2010a) for o-glycan chains with b1,3-linked cores. in contrast to b1,4-linkages of the n-glycan-type, which were shown to be stable under the ammonium-based alkaline conditions, the b1,3-linkage was found to be labile and to give considerable peeling . the results indicated that complete prevention of peeling under nonreducing alkalicatalyzed hydrolysis conditions remains difficult. the yields of o-and n-glycans from bovine fetuin released by conventional means (pngase f and reductive b-elimination with naoh) were found to be greater. it was concluded that great care should be taken when employing such non-reducing alkaline conditions in glycomic analysis and in obtaining some o-glycans for functional studies. because the hydroxide ion appears to cause the unfavorable peeling reactions, miura et al. (2010b) have investigated the use of the ammonium salt, ammonium carbamate for glycan release. the efficiency of release with ammonium carbamate was compared with a common conventional procedure, namely saturated ammonium carbonate/aqueous ammonia with bovine submaxillary mucin (bsm) as the test compound. release with ammonium carbamate did not exhibit significant loss of glcnac-b1,3 (neu5aca2,6)galnac or glcnacb1,3(neu5gca2,6)galnac structural determination (highmannose, bisected high-mannose glycans). development associated with alteration of fucosylated glycans structural determination (highmannose, hybrid, complex glycans). poly-fucosylation human (ht-29 5m12 colon cancer cells) pngase f, tof, glycans (per-me) pattern of n-glycosylation serves as a recognition signal for galectin-4. high-man, hybrid, bi-, tri-tetra-antennary. (stechly et al., 2009) human (cytolytic t lymphocytes) pngase f, tof/tof (dhb), glycans (per-me) loss of effector function shown to be accompanied by major changes in nand o-glycosylation (antonopoulos et al., 2012) marmota monax (woodchuck with liver cancer), gal-α-(1→3)-gal in mouse epidermis but galnac-β-(1→4)-glcnac in human. high-mannose, hybrid complex (uematsu et al., 2009) pngase a pngase f, tof/tof (dhb/3% plants engineered to produce lewis x epitopes. (paucimannosidic glycans) mass spectrometry reviews doi 10.1002/mas and the profile of the major o-glycans was similar to that obtained following conventional reductive amination with naoh/nabh 4 . on the other hand, glycans obtained by treating bsm with ammonium carbonate/28% aqueous ammonia and analyzed by maldi-tof ms showed a significant increase of the disaccharide components, neu5aca2,6galnac and neu5-gca2,6galnac, suggesting the presence of a peeling reaction. use of ammonium carbamate, thus, appears to produce efficient release without concomitant peeling. the release was performed by addition of powdered ammonium carbamate and incubation for 20 hr at 60˚c. yamada et al. (2009) have used a recently developed automated release apparatus using lithium hydroxide to obtain o-glycans from leukemia and epithelial cancer cells. because these cells usually contain free glycans, the investigators first reduced these with sodium borohydride and then labeled the released glycans with 2-aa in order to avoid interference. a new release method reported by goetz, novotny, and mechref (2009a) combined enzymatic and chemical techniques and used b-elimination to cleave glycans from serine (ser) and threonine (thr) but not asn. the method involved first a nonspecific proteolysis with pronase, followed by solid-phase hydrazine, tof (dhb), glycans (2-ap) structural determination. high-man, hybrid, bi-, tri-, tetra-antennary complex (sumiyoshi et al., 2010) rabbit and chicken (erythrocytes) tof, glycans (2-ap) to study alterations in receptor-binding properties of h1-type swine influenza viruses in embryonated chicken eggs. high-man, hybrid, bi-, tri-antennary (takemae et al., 2010) rat ( structural determination (highmannose, hybrid, complex glycans) girard's t for quantitation (kim, et al., 2009c) sus domestica (respiratory epithelial cells) pngase f, tof, tof/tof (dhb), glycans (per-me) influenza virus shown to utilise α2→6linked neu5ac to infect cells permethylation with sodium hydroxide. the basic sodium hydroxide caused the glycans to be released by b-elimination and these were immediately permethylated. this combination of the enzymatic and chemical procedures was reported to give a substantial improvement in sensitivity and analytical reproducibility over existing methods by minimizing sample losses. moreover, the approach was reported to extend the cleavage protocols to large glycoproteins where small oligosaccharides were not accessible to conventional chemical treatment. the method was developed with fetuin and used to identify new o-glycans from bile salt-stimulated lipase (bssl). maniatis, zhou, and reinhold (2010) have released o-glycans with aqueous dimethylamine in the presence of sodium borohydride by use of a microwave oven. the release was performed at 70˚c and, for a heptapeptide carrying a glcnac group attached to thr, was complete in 70 min. the reaction also labeled the site of detachment with a dimethylamino group. use of a 1:1 mixture of dimethylamine and [ 2 h 3 ] 2 nh produced doublets in the peptide mass spectrum separated by six units, allowing the glycosylation sites to be readily identified. n-glycans are frequently removed from glycoproteins before o-glycan removal by b-elimination. however stone et al. (2009) have reported improved recovery of o-glycans from murine tissues without prior n-glycan removal. kbh 4 and koh were used to remove the o-glycans and possible low levels of concomitantly released n-glycans were tolerated. ii. use of hydrazine. hydrazine has also been used to release these glycans with a new gas-phase method using anhydrous hydrazine being evaluated by goso, tsubokawa, and ishihara (2009) with muc-type oligosaccharides from porcine gastric mucin (pgm) and bovine fetuin. released glycans were examined by hplc and maldi-tof as 2-aa derivatives. glycans obtained by the treatment with hydrazine at 65˚c for 6 hr resembled those obtained by b-elimination, except for the additional disaccharide fractions derived from the core 1 side of the oligosaccharides by further degradation. the other degraded products derived from the core 2 side could not be derivatized by 2-aa, therefore, were not visible by fluorescence detection. release of the glycans was incomplete after 6 hr but almost complete liberation was achieved by extending the treatment to 18 hr. however, degradation increased. in this case, the addition of barium oxide to the reaction vessel decreased the degree of further degradation. results similar to pgm were obtained from bovine fetuin, but with less degradation. application of this method to the analysis of rat gastric mucin (rgm) showed that rgm has a large oligosaccharide portion on the core 1 side. iii. other methods. a new method for o-glycan removal for study of the residual deglycosylated protein has been reported . desialylated glycoproteins whose sugar chains consisted of gal-galnac, were immobilized on alkali-stable, reversed-phase poros 20 beads and treated with periodate to oxidize the cis-glycol groups in the gal residue. the resulting aldehydes were then susceptible to b-elimination under mild (nh 3 ) basic conditions. the remaining galnac residue, which now contained a cis-glycol group, was oxidized with further treatment with periodate and removed with base. although the number of cycles required depended on the number of gal-galnac repeats, large core 2-type glycans that usually only have gal attached to the 3-position of the core galnac, could be deglycosylated in only two steps. o-linked glycosylation often occurs in muc-type domains that are heavily and heterogeneously glycosylated. several strategies to determine the heterogeneity of these domains have recently been investigated with four glucanases secreted in large quantities from trichoderma reesei, all of which contained heavily o-glycosylated muc-like linker regions, being used as models. the strategies involved monosaccharide compositional analysis and identification of the released glycans by hpaecpulsed amperometric detection (pad) and carbon-lc esi-ms/ ms. glycosylated peptides were generated by different protease digestions (trypsin, papain, asp-n, pretaq) and enriched by hilic microcolumns. the complex o-glycan heterogeneity was determined by maldi-ms and esi-ms, but the dense o-glycosylation in the muc-type domains conferred high resistance to protease cleavage. etd-ms/ms of the glycopeptide-enriched protease digests was unsuccessful for the assignment of o-glycosylation at individual sites within the muctype domains but allowed several previously unknown o-linked sites outside the defined linker region to be found on two of the four glucanases (christiansen et al., 2010) . iv. comparison of methods. three samples of iga1 isolated from the serum of patients with multiple myeloma have been distributed on behalf of the human proteome organization human disease glycomics/proteome initiative to 15 laboratories for comparative analysis of their o-glycans. a range of techniques was used; the two strategies that yielded the best data were direct positive ion ms analysis of permethylated glycans and lc-ms analysis of native reduced glycans in negative ion mode. the studies reinforced the pre-eminent performance of ms techniques for o-glycan profiling (wada et al., 2010b) . several reviews and general analytical methods have been reported; these are listed in table 14 . loss of sulfate is a major problem in the maldi analysis of these compounds but this can be avoided by use of desorption esi (przybylski et al., 2010a) . little use appears to have been made of the peptide binding method for stabilization of sulfate groups in these compounds as reported by juhasz and biemann (1994) . one report concerns the identification of a pentasulfated hexasaccharide responsible for binding to the growth factor pleiotrophin . the compound was complexed with (arg-gly) 15 and identified by maldi-tof from dhb. in another study , a pentasulfated hexasaccharide with a novel structure (d 4,5 hexaa1-3galnac(4s)b1-4idoa(2-s)a1-3galnac(4s)b1-4idoa(2s)a1-3galnac(4s)) has been isolated from the chondroitinase ac-i digest of shark skin. again, (arg-gly) 15 was used as the complexing agent. bultel et al. (2010) have developed a method for analysis of heparin oligosaccharides by using controlled nitrous acid degradation followed by high-performance anion exchange chromatography (hpaec) separation and uv-maldi-tof analysis. the use of three different matrices, dhb, chca, and nor-harmane were investigated but only dhb and nor-harmane were needed to assign the position of sulfate groups. dhb allowed the molecular ion to be detected in nearly all cases and gave fragments arising from the loss of sulfate groups. nor-harmane, in contrast, produced mainly fragments. in all cases, ions retaining the sulfate groups were observed making these fragments essential for assigning the sulfated positions of each residue. while nor-harmane was not able to produce enough analyte desorption/ionization, fragments useful for structural assignment were produced by the addition of butylammonium formate to the dhb matrix. a method for analysis of gpi anchors on the "proteomic" scale has been described . partially purified proteins were separated by sds-page and then blotted onto a pvdf membrane. following identification of the protein, the gpi anchor was analyzed by three methods. first, the compound was hydrolyzed with hcl in the presence of [1,2,3,4,5,6-2 h 6 ]myo-inositol and the hydrolysate was analyzed as trimethylsilyl (tms) derivatives by gc/ms. next, the phosphate bonds were cleaved and the carbohydrate structure was elucidated by electrospray or maldi-tof ms. finally, the diacylglycerol-attached myo-inositol moiety was detached by nitrous acid deamination and analyzed by negative ion electrospray. bütikofer et al. (2010) have studied lipid remodeling of gpi glycoconjugates in procyclic-form trypanosomes and shown that, in trypanosoma congolense, the steady-state lipids consist of lyso-(acyl)phosphatidylinositol (pi, 66), deacyl-pi and deacyl-(acyl)pi species, where (acyl) indicates an acyl group attached to the inositol moiety. maldi-qit-tof in negative ion mode from thap was used to analyze the pi species after deamination with nitrous acid. structural determination (core 1, core 2). samples from geographically remote labs. similar glycans (babu, et al., 2009) human, (enterocytelike ht-29 cells) β-elimination, tof, glycans (per-me), gc/ms structural determination (core 1) (morelle, et al., 2009b) human ( absence of fucose on core 3 glycans impairs baba-mediated adhesion to gastric mucosa (magalhães et al., 2009) (continued) although many bacteria produce s-layer proteins with glycosylation, qazi et al. (2009) have used maldi-tof ms to show that those from clostridium difficile are not glycosylated. this topic has been reviewed by zhang et al. (2009f) and capote and sanchez (2009) . maldi is used mainly to determine the extent of glycation of intact proteins or to determine the sites of attachment of the covalent glycans. a. specific methods for glycated peptides amadori peptides have been enriched with boronate affinity tips for measurement by maldi-tof/ms. the tips showed the highest binding efficiency for glucose at ph 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mm. the bound constituents were released by sorbitol (1/ 42) or formic acid. using sorbitol for elution required desalting prior to analysis. of three different sorbents tested: fullerenederivatized silica, ziptips (c18), and c18 silica, fullerenederivatized silica and ziptips showed the same performance with respect to the numbers of glycated peptides and gave better performance than c18 silica. fewer glycated peptides were detected by lc-ms/ms than by maldi . a novel fullerene(c60)-derivatized silica material has been compared with octadecyl(c18) and triaconthyl(c30)-silicas for their ability to recover peptides from digests of hsa and fibrinogen. c30-and particularly the c60(30 nm)-spe materials were found to be the two most effective. after glycation the digests of fibrinogen and hsa were also separated. this new method made the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. for hsa, 10 new sites of glycation at lys and arg residues were found . a mass spectrometric method for screening large tandem mass spectrometric (ms/ms) datasets for protein glycation with glucose (1/4), lactose (67) and maltose (68) has been developed (montgomery, tanaka, & belgacem, 2010) . control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in ms/ms analysis for glucose, lactose and maltose condensed with peptide. for glucose glycation, neutral losses of 36, 120, and 162 da were observed in accordance with previously published reports. the neutral loss patterns for lactose and maltose were found to be identical, with characteristic losses of 162, 198, 282, and 324 da. these signature losses were observed irrespective of the maldi mass spectrometer used and were valid in both tof-tof and qit-tof instruments. these neutral loss signatures were then applied to elucidation of modified peptides from a complex hsa digest glycated with each of the proposed sugars. screening of these large datasets o-labeled digests indicated that lyss 525 and 439 also had significant degrees of modification. the modifications that occurred at these sites were variations of fructosyl-lys and advanced glycation end products (ages) which included 1-alkyl-2formyl-3,4-glycoyl-pyrole (4/45) and pyrraline (4/44). the fragmentation behavior of the peptide ac-paapaa-papaektpv-oh (human histone h1.4, positions 6-20) glycated via its lys residues to adp-ribose has been studied by fedorova, frolov, and hoffmann (2010a) . under maldi conditions, the adp-ribosyl group was cleaved, almost completely at the pyrophosphate bond by isd and psd. however, this cleavage was very weak in esi-ms. the remaining phospho-ribosyl group was stable, providing a direct and reliable identification of the glycation site via the b-and yion series. as well as being associated with health problems, in for example, diabetes, protein glycation is important in the food industry in, for example, the browning of food during cooking. the reaction is also being used to attach carbohydrates to proteins to improve technological and biological functionalities. in relation to this latter use, corzo-martnez et al. (2010) have used maldi-tof ms to study the reaction between b-lactoglobulin and the sugars galactose and tagatose (69) and found that the reaction can be competitively moderated with pyridoxamine (70). other reports of the use of maldi to study glycation of specific proteins are summarized in table 15 . typical structures consist of glcnac-murnac (71) disaccharides cross-linked by short peptides. they are usually analyzed as muropeptides following enzymatic digestion. reports of the use of maldi to study peptidoglycans and muropeptides are summarized in table 16 . this is a very large group of compounds but most work involving maldi has been concentrated on the lps from bacteria and the gsls. work, mainly involving structural determination, on the many other types of glycolipids found in bacteria and similar organisms, is summarized in table 17 . a general review on analysis of microbial glycopolymers has been published by , and fuchs and schiller (2009) phenol/chloroform/petroleum ether and the molecules are frequently split into their component parts by mild acid hydrolysis for further structural analysis. dephosphorylation and deacylation are also common. reviews on the structural investigation of bacterial lps by ms and ms/ms have been published by banoub et al. (2010) and by grice and wilson (2009) . lipid a from coxiella burnetii, the causative agent of q fever has been discussed by toman, skultety, and ihnatko (2009) and a more general review of the core region and lipid a of lps has been published by holst and molinaro (2010) . the first structures of lps to be elucidated from cyanobacteria has been reported by snyder et al. (2009) . two strains of marine water-soluble proteins glucose l-tof (2,6-dhap) use of maldi-tof to study glycation during malting synechococcus, wh8102 and cc9311, were used and were shown to have very simple structures without the complex o-chain found in most proteobacteria. the lps (72) of these cyanobacteria did not contain phosphate, heptose (hep) or kdo (1/13) but instead possessed 4-linked glucose as their main saccharide component, with low levels of glcn and galacturonic acid (gala). maldi-tof ms of the intact minimal core lps revealed triacylated and tetraacylated structures with a heterogeneous mixture of both hydroxylated and nonhydroxylated fatty acids connected to the di-glcn backbone. in contrast to enteric lipid a, which can be liberated from lps by mild acid hydrolysis, lipid a from these organisms could be produced only by two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. unique caryophyllose (a-3,6-dideoxy-4-c-(d-altro-1,3,4,5tetrahydroxyhexyl)-d-xylo-hexopyranose, 73)-containing cell wall glycolipids have been identified in lps from mycobacterium marinum (rombouts et al., 2009) . maldi-tof spectra were obtained from dhb and esi-ms/ms was used to elucidate the glycan sequence. b. lipid a sample preparation has been shown to be critical for assessing the true composition of these compounds. in a comparative study, kawasaki (2009) have shown that maldi-tof ms analysis of lipid a prepared using a commercial "tri-reagent"based procedure with a 5-chloro-2-mercaptobenzothiazole (cmbt) (1/33) matrix gave the best results for compounds with a phosphoethanolamine (petn) modification. in contrast, the analysis of lipid a prepared using an lps extraction kit-based procedure with dhb was preferable for the detection of an aminoarabinose modification. for isolation of lipid a from yersinia enterocolitica, pérez-gutiérrez et al. (2010) used an ammonium hydroxide-isobutyric acid method. lyophilized crude cells were incubated with isobutyric acid and ammonium hydroxide (5:3, v/v) at 100˚c for 2 hr, washed twice with methanol and the insoluble lipid a was solubilized in chloroform-methanol-water (3:1.5:0.25, v/v/v). analysis was by maldi-tof ms from dhb in negative ion mode because of the presence of two phosphate groups. the lipid as a contained both four or six fatty acyl chains whose (boniface et al., 2009) ratio changed with growth temperature. a similar method has been used by march et al. (2010) to study lipid a from acinetobacter baumannii. the molecules were found to have from four to seven acyl groups. a microwave-assisted method for obtaining lipid a from helicobacter pylori has been developed by zhou et al. (2009a) . lyophilized cells were suspended in sodium acetate buffer (ph 4.5) containing proteinase k and subjected to microwave irritation at 50 w for 5 min at 58˚c and then kept for 1 hr at 100c . after centrifugation and washing, the dried supernatant was examined by maldi-tof/tof from cmbt. the reliability of the technique was demonstrated by analysis of the lipid a from bacterial cells of different h. pylori strains. the phosphorylation and acylation patterns could be elucidated using material from a single colony. furthermore, the investigators found unusual heptaacyl lipid a species present in low abundance in h. pylori mutant that have not been previously reported. the study was claimed to provide the first characterization by ms of the lipid a component from a single bacterial colony. the mass spectrometric behavior of lipid a is highly dependent on both the matrix and phosphorylation patterns. zhou et al. (2010a) have investigated the effects of different matrices and co-matrices using lipid a from escherichia coli o116 as a model system. good results were obtained with cmbt with added edta (5/43) ammonium salt as the matrix. this matrix system was found to enhance the sensitivity of the detection of diphosphorylated lipid a by more than 100-fold and, in addition, provided tolerance to high concentrations of sds and to both sodium and calcium chlorides at mm concentrations. the method was evaluated for analysis of lipid a with different phosphorylation patterns and from different bacteria, including h. pylori, salmonella enterica serovar riogrande, and francisella novicida. an lc/ms-based assay has been developed for the quantitation of aminosugars, including glcn (74), galactosamine (galn, 75) and aminoarabinose (aran, 76) together with ethanolamine (etn), present in lipid a and has been applied to the analysis of lipid a isolated from several biosynthetic and regulatory mutants of s. enterica serovar typhimurium and francisella tularensis subspecies novicida characterized by maldi-tof. lipid a was treated with tfa to liberate and deacetylate individual aminosugars and mass tagged with 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate (77), which reacts with primary and secondary amines. the derivatives were separated using rp-chromatography and analyzed with a quadrupole ms to detect quantities as small as 20 fmol. galn was detected only in francisella and aran only in salmonella, while glcn was detected in lipid a samples from both species (kalhorn et al., 2009) use of grazing-incidence x-ray scattering and monte carlo simulation to investigate physics of bacterial survival against cationic antimicrobial peptides . (continued) c. core oligosaccharide during an analysis of the permethylated derivative of the core oligosaccharide from aeromonas hydrophila, prepared by the hakamori method, ions appearing at 78 mass units higher than the [mþh] þ ions were observed. these ions were determined to be dimethylsulfoxide (dmso) covalent addition products resulting from the michael addition of the dimsyl anion on the c-2-c-3 double bond of a 4,8-anhydro kdo (1/13) residue followed by an addition of a proton on the double bond. corresponding ions were also seen in methylations performed using the naoh technique and represent the first characterization of these addition products (sioud et al., 2010) . typical pre-maldi techniques for the analysis of these compounds include separation by tlc and cleavage of the cer portion so that the glycan can be analyzed without the heterogeniety produced by the lipid. reviews on the analysis of gsls are summarised in table 18 . a. analysis of intact compounds stübiger et al. (2009) have separated lipids, including gsls by high-performance tlc, stained them with coomassie blue and analyzed them either directly from the tlc plates or, after their removal, with an appropriate solvent. thap (1/44) was used as the matrix with acetone as the solvent for the on-plate analysis because its high volatility minimized sample spreading. a method for structural profiling of individual gsls in a single thin-layer chromatogram by multiple sequential immunodetection has been developed by souady et al. (2009) . structures of the antibody-detected gsls were determined by direct coupling of tlc with ir-maldi after treatment of the tlc plate with glycerol. this combined technique was used to demonstrate structural gsl profiling of crude lipid extracts from human hepatocellular cancer. a new method for analysis of gsls involves selective ozonolysis of the c-c double bond in the ceramide moiety of biological samples and subsequent enrichment of the generated gsl aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticles (aognp). the gsl-bound nanoparticles were removed by ultrafiltration and the gsls were analyzed by maldi-tof and -tof/tof ms from dhb. the method was used for structural profiling of mouse brain gangliosides such as gm1, gd1a/gd1b, and gt1b for adult or gd3 in the case for the embryonic mouse. because the saturated acyl groups remained intact, the spectra provided information on both the carbohydrate and fatty acyl moieties (nagahori, abe, & nishimura, 2009) . the direct structural characterization of microbial gsl receptors by use of the tlc overlay assay combined with ir-maldi-o-tof-ms has been described (müsken et al., 2010) . glycan mixtures were separated by tlc in three parallel lanes. one lane was stained with orcinol and a second was overlayed with gsl-specific bacteria. the bound microbes were detected with primary antibodies against bacterial surface proteins and the relevant gsls were detected in the third lane by ir-maldi-tof. the combined method worked on the microgram scale of gsl mixtures and was successfully applied to the compositional analysis of globo-series neutral gsls recognized by p-fimbriated e. coli bacteria, used as model microorganisms for infection of the human urinary tract. (niedziela et al., 2010b) incubation of botulinum neurotoxin serotype d with the gsl, gt1b has produced a complex (mw 51,921) that was detected intact by maldi-tof ms from sinapinic acid and provided evidence that the toxin attacks neurons in a ganglioside-dependent manner (strotmeier et al., 2010) . a method for generation of novel fluorocarbon derivatives from gsls has been described by li et al. (2010f) . the derivatives had high affinity for fluorocarbon phases allowing them to be recovered from biological matrices by fluorous solid phase extraction (f-spe). sphingolipid ceramide n-deacylase was used to remove the fatty acid from the ceramide moiety, after which the fluorocarbon-rich substituent (f-tag, 78) was linked to the free amine. finally, the molecules were permethylated for ms analysis and the method was used to examine a crude ganglioside mixture extracted from bovine brain. in addition, the flourous tag was used in a microarray format to fix f-tagged gm1 ganglioside to a fluorous glass surface, with the glycan intact and available for interaction with a fluorescent derivative of cholera toxin b chain. cheng et al. (2010a) have used 9-aa (6/18) as a matrix for quantitative analysis of sulfatides in biological samples. the matrix was said to promote selective ionization of sulfatides in negative ion mode with a detection limit in the high attomole range. experimental details have been published for highperformance tlc separation of glycolipids followed by blotting to a pvdf membrane in a technique termed far-eastern blot with analysis by maldi-tof ms (taki et al., 2009) . b. studies on the glycan moiety following removal of the ceramide for studies of the carbohydrate portion of these molecules, lipid heterogeniety is frequently reduced by removing the ceramide with enzymes such as rhodococcal endoglycoceramidase or leech ceramide glycanase. li et al. (2009e) have described the preparation of the intact oligosaccharides from gm1 (neuacggose 4 cer) and gbose 4 cer as examples to show the use of ceramide glycanase and have optimized the specificity and detergent requirements of rhodococcal endoglycoceramidase for the release of glycan chains from various gsls. a novel method of detecting 6-gala series gsls (those possessing an r-galb1-6galb1-1-cer, group) has been reported (ishibashi et al., 2009) . the method used the specificity of endogalactosylceramidase, an enzyme that is capable of hydrolyzing 6-gala series gsls to produce intact oligosaccharides and ceramides but which also catalyzes transglycosylation reactions. in the latter reaction, the enzyme transferred oligosaccharides from the gsls to acceptors such as fluorescent 1-alkanols. in this application, 7-nitro-2,1,3-benzoxadiazole pentanol (nbd-pentanol, 79) was used as an acceptor. the fluorescent products, nbd-pentanol-conjugated-6-gala oligosaccharides, were separated and detected by tlc or hplc with a fluorescent detector and characterized by maldi-tof ms. the method could also be applied to glycoglycerolipids and digalactosyldiacylglycerol and was successfully applied to detect 6-gala series gsls in the fungus, rhizopus oryzae and the parasite, taenia crassiceps. other applications of maldi to the analysis of these compounds is summarised in table 19 . rhamnolipids are glycolipids of the type 80 produced by pseudomonas spp. maldi-tof ms approaches have been developed for high-throughput screening of naturally occurring (fungus), mycelia tof (chca), psd ident. of novel neogala-series glycosphingolipids with terminal man and glc (tani et al., 2010) human (mesenchymal stem cells from bone marrow) macrobdella decora endoglyco-ceramidase, r-tof/tof, ft-icr (dhb), glycans (per-me) structural determination (heiskanen, et al., 2009) human (colonic adenocarcinoma cell lines) ceramide glycanase, tof/tof, q-tof (dhb), glycans (per-me) enhanced expression of β3-gal-t 5 activity induces in vivo synthesis of extended type 1 chains on lactosyl-cer human (umbilical vein endothelial cells) tof (chca) acyl mainly 24:0. may act as biomarker for inflammation, gsl = gb4 (okuda et al., 2010) human (umbilical vein endothelial cells) tof (chca) structural determination and dynamics of globotetraosylceramide under tnf-α stimulation (okuda, et al., 2010) human (kidney and colon) tof identification of gsls that bind shiga toxin from e. coli human (embryonic stem cells) switching of the core structures of gsls from globo-and lacto-to ganglio-series on cell differentiation miniature pig (endothelial and islet cells) ceramide glycanase, r-tof (dhb), esi (-ve), glycans (per-me and girard's t) structural determination. identification of neu5gc epitopes ) (krambeck, et al., 2009) mouse (thymus) tof/tof (dhb), esi structural determination and natural killer t cell development (li et al., 2009f) mouse (liver and serum) tof (-ve) acute kidney injury down-regulates gene expression of hepatic cerebroside sulfotransferase. quant. by maldi-tof. (zhang et al., 2009h) mouse ( use of 9-aminoacridine (9-aa) as matrix, structural determination -phospholipids, gal-cer-sulfate, gb5 trichoderma viride (fungus) l-tof (7-nh 2 -2-mecoumarin) ident. of phosphocholine-containing glycosyl inositol phosphoceramides respectively. the method was validated by compositional analysis using gc/ms, fractionation by rp-hplc and analysis by 1 and 2d nmr applications of maldi to the analysis of other glycolipids are listed in table 20 . although much work has been published on glycosides during the review period, maldi appears to occupy a relatively minor position with fast atom bombardment (fab) and, particularly esi being the preferred techniques. most work has been on the identification of glycosides from various plant sources using a variety of techniques such as nuclear magnetic resonance (nmr), uv and ir spectrometry. applications of maldi to the analysis of glycosides and other natural products are summarised in table 21 . several investigators have reported that the main fragmentation pathways of flavonoids are apparently independent of the ionization mode (esi, atmospheric pressure chemical ionization (apci), or maldi) or the types of analyzers used to acquire the spectra (triq, it, or qtof) as reported in a review of the structural characterization of flavonoid glycosides by multistage ms (vukics and guttman, 2010) . maldi-tof (dhb) was used by bankefors, nord, and kenne (2010) to examine saponins from quillaja saponaria bark extracts in connection with the development of a multidimensional method using hplc and esi-it ms n for profiling complex mixtures of natural products. increasing use of maldi has been made in the characterization and detection of disease and in the identification of biomarkers. some of the glycan biomarkers reported for, for example, cancer, however, appear to be more associated with glycopro-teins involved in inflammation and are, thus, secondary to this disease. blomme et al. (2009) have noted that "although individual liver diseases have their own specific markers, the same modifications, hyperfucosylation, increased branching and a bisecting glcnac, seem to continuously reappear in all liver diseases." increases in fucosylated triantennary glycans from agp is a case in point. several reviews have appeared and are summarised in table 22 . practical details for the characterization by maldi-tof and esi-ms of n-linked glycosylation on recombinant glycoproteins produced in p. pastoris and for detecting potential cancer biomarkers in various cell lines and sera from patients have been published. bereman, williams, and muddiman (2009a) have developed a nano-lc linear trap quadrupole (ltq) orbitrap method for analysis of released n-glycans and compared the spectra with those obtained by maldi-ft-icr. whereas the maldi spectra showed much loss of sialic acids from the sialylated glycans, the orbitrap spectra showed no decomposition. the method was applied to glycans released from plasma glycoproteins in benign gynecologic tumors and from epithelial ovarian cancer patients. one of the biantennary glycans found to be down-regulated in the cancer patients was a fucosylated biantennary glycan in which the fucose was unusually shown attached to a gal residue rather than to the core as determined by ms/ms. the compounds were ionized as [mþh] þ species suggesting that this might be an erroneous structure and the result of an internal rearrangement that is known to occur under these conditions. a detailed statistical analysis has been performed on eight data sets of n-glycans released from serum glycoproteins from prostate, breast and ovarian cancer patients (barkauskas et al., 2009) and measured by maldi-ft-icr ms. significant differences between control and cancer groups were found in all eight datasets. two structurally related compounds were found to be significantly different between control and cancer groups in all three types of cancer. these compounds had compositions of hex 3 -hexnac 4 -fuc 1 and hex 5 -hexnac 4 -fuc 1 and were probably from igg rather than being produced by the cancer cells. narimatsu et al. (2010a) have developed a high-throughput method for detecting cancer biomarkers in early stages of the disease. briefly, the method consisted of the extraction of tissue mrnas and measurement of the expression by quantitative realtime polymerase chain reaction (pcr). the results suggested that different glycan structures were synthesized in different cell lines. secreted proteins from the same cancer cells were collected from serum-free culture and then applied to a lectin microarray to select lectin(s) that showed differential binding to glycoproteins secreted from each cancer cell line. after selection of a specific lectin, isotope-coded glycosylation sitespecific tagging (igot) was used to identify core proteins that carry an epitope bound by a specific lectin. each candidate biomarker was immunoprecipitated from serum using commercially available antibodies and their glycan structures were profiled by lectin microarray, and finally determined by ms n technology with measurements by maldi-qit-tof ms. other applications are listed in table 23 . α-glc(1-7)-α-hep-(1-5)-α-kdo4p-(2-6)-β-glcn4p-(1-6)-α-glcn1p arthrobacter globiformis and a. scleromae diglycosyl glycerol ft-icr (dhb), nmr structural determination. mannose and galactose (paściak et al., 2010b) aspergillus fumigatus glycoinositolphospho-ceramides tof the mita gene shown to be required for mannosylation (kotz et al., 2010) hymenobacter sp. carotenoids maldi identification of 2'-methyl and 1'-xylosyl derivatives of 2'hydroxyflexixanthin (klassen et al., 2009) leishmania major glycoinositolphospholipids tof (chca), esi-ms n demonstration of a udpglucose independent udpgalactose salvage pathway (lamerz et al., 2010) lipoteichoic acid tof (dhb) identification of two enzyme systems involved in biosynthesis phosphatidyl-myoinositol mannosides (pims) glycopeptidolipids shown to mask pims in cell wall (rhoades et al., 2009) there is currently great interest in the production of therapeutic antibodies and maldi-tof ms is frequently used in the analysis of their attached glycans. reviews have been published on methods for the production and ms analysis of igg (huhn et al., 2009) therapeutic antibodies (beck et al., 2008; del val, kontoravdi, & nagy, 2010; higgins, 2010; zhang, pan, & chen, 2009i ) and on the humanization of recombinant glycoproteins expressed in insect cells (tomiya, 2009) . practical details for characterization of antibody glycans have been published by several investigators janin-bussat et al., 2010a ,bjanin-bussat et al., 2010a . a discussion, with experimental details, of methods based on blot detection with glycan-specific probes, ms of released glycans and lc/ms detection of glycopeptides with the aim of determining whether, how and where plant-derived biopharmaceuticals are glycosylated has also been published . several new methods have been reported. thus: a highthroughput method for monitoring igg glycosylation using a 96well plate format with iggs purified from 2 ml of human plasma has been developed by selman et al. (2010) . iggs were extracted using immobilized protein a, cleaved with trypsin and the resulting glycopeptides were purified by reversed-phase or hydrophilic interaction spe. glycopeptides were analyzed by intermediate pressure maldi-fticr-ms using both dhb and chca, both of which produced signals from sialylated as well as nonsialylated glycopeptides. the method showed an interday variation of below 10% for the six major glycoforms of both igg1 and igg2 and was found to be suitable for isotype-specific high throughput igg glycosylation profiling from human plasma. the method was applied to the igg glycosylation of 62 human samples. two lectin-affinity chromatography techniques, con a and lens culinaris agglutinin, have been used to enrich, by removal of high-mannose glycans, the nonfucosylated n-glycans from igg with product detection by maldi-tof following pngase f digestion (tojo et al., 2009) . prien et al. (2010) have used a stable isotopically labeled derivative for rapid glycan screening of biotherapeutics. glycans were labeled with either [ 12 c 6 ]-or 13 c 6 ]-2-aa for both maldi-tof or lc-ms analysis. the 2-aa label provided high sensitivity detection in negative ion mode and the mass separation of six units between the isotopically labeled variants eliminated problems arising from isotopic overlap. pegylation of proteins is frequently used to prolong the serum half-life time of recombinant proteins but their very high mws put many of them outside the mass range of commercial maldi-tof systems using conventional secondary electron multiplier (sem) detectors. seyfried et al. (2010) have investigated the use of a high mass (hm) detector combined with a maldi linear tof ms system for the detection of pegylated (glyco)proteins in the range of 60-600 kda. the system consisted of a shimadzu axima cfrþ instrument equipped with both a conventional detector and additionally, with an inline moveable hm ion conversion detector (icd hm1, from covalx). spectra were run from sinapinic acid in the linear positive ion mode and were obtained from small (interferon a2a), middle (hsa) and high (coagulation factor viii and von willebrand factor (vwf), both heavily glycosylated proteins) molecular mass proteins. the particular challenge was the heterogeneity of the (glyco)proteins in the high mw range in combination with heterogeneity added by the pegylation, nevertheless, the performance of maldi linear tof ms was found to be superior to that of other methods. although the sem was able to obtain information about protein pegylation in the mass range up to 100 kda (e.g., pegylated hsa), the hm system was crucial for detection of ions from the larger compounds, the masses of which sometimes exceeded 0.5 mda. detection of these compounds was impossible with the standard sem. the particular challenge for the analysis was the heterogeneity of the (glyco)proteins in the high mw range in combination with additional pegylation, which even introduced more heterogeneity and was more challenging for interpretation. nevertheless, the performance of maldi linear tof ms with both detector systems in terms mw and heterogeneity determination depending on the m/z range was superior to the other methods. isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility (rooney et al., 2009) proposed as a member of a new genus and species (golyshina et al., 2009) aplysia kurodai (sea hare) glycosaminoglycans tof, ms/ms structural determination (yoon et al., 2010a) arthrobacter crystallopoietes n-08 α1,α1-trehalose tof, esi, nmr first report that trehalose can be produced from maltose in this bacterium beverage from fermented plant (gülcemal et al., 2010) radix puerariae (kudzu) tof identification from cultivated kudzu root (nguyen et al., 2009b) red wine procyanidins and anthocyanins tof (indole-3-acrylic acid) phenolic extracts shown to induce nitric oxide (no)mediated vasoprotectivity (auger et al., 2010) salmo salar ( a novel method for the determination of aminoglycoside antibiotics used surface-assisted laser desorption/ionization mass spectrometry (saldi ms) with the aid of silver-coated gold nanoparticles (au@agnps) capped by anionic citrate. these nanoparticles were used both as concentrating agents and as matrices in saldi ms. the lods at signal-to-noise ratio of 3 were 3, 25, 15, 30, and 38 nm for paromomycin, kanamycin a, neomycin, gentamicin and apramycin respectively. the lods of the first four of these antibiotics in plasma samples were 9, 130, 81, and 180 nm respectively. recoveries of the antibiotics from plasma were about 80% (wang et al., 2009h) . further examples of the use of maldi ms in the analysis of therapeutics are given in table 24 . applications in this section mainly involve the use of maldi to investigate products of newly isolated enzymes. these are summarised in table 25 . other studies are aimed at elucidating enzyme activity as illustrated by the development of a method to determine the cleavage site in small oligomannoses that has been developed by hekmat et al. (2010) . enzymatic cleavages were performed in 18 o-labeled water, conditions that introduced 18 o into the anomeric position of the cleaved glycans. thus, measurements by maldi-tof could determine if a product arose from the non-reducing end of the original oligomannose by its incorporation of the label. the addition of a small amount of various ionic liquids has been found to modify the activity and regioselectivity of different immobilized preparations of rhizomucor miehei lipase that catalyzes the hydrolysis of hexa-o-acetyl lactal in aqueous media (filice, guisan, & palomo, 2010) . the activity of the enzyme glcnac-transferase vb, which transfers glcnac to the 6-position of the 6-antenna in n-glycans has been compared with that of gnt-v. one unusual product found after 8 hr was the addition of glcnac to the 6-position of both antennae (alvarez-manilla et al., 2010a). relevant reviews on carbohydrate synthesis are summarised in table 26 principal component analysis, shows glycobiological differences between normal and cancer. hybrid, complex glycans (goetz et al., 2009b) breast cancer cd98hc clycoprotein pngase f, tof/tof (dhb), complex glycans (per-me) identification of the glycoprotein that binds to galmbp (fragment of mannose-binding protein) (powlesland et al., 2009) breast cancer serum trypsin, tof/tof (dhb), glycopeptides expression of helix pomatia lectin binding glycoproteins in women with breast cancer in relationship to their blood group (welinder et al., 2009) breast cancer cells β-elimination (nh 4 carbamate), r-tof/tof (dhb), glycans, ms/ms use of new release method using glycoblotting and ammonium carbamate for β-elimination, core 2 o-glycans breast cancer serum set of glycans identified that can be used as biomarkers (hammoud et al., 2010) (continued) changes in 57 glycans . three n-glycans sufficient for detection with 90% accuracy hepatocellular carcinoma serum glycoproteins tof/tof (dhb), glycans (per-me) analysis using hierarchical clustering analysis. 7 potential glycan markers identified. man 5 glcnac 2 , bi-, tri-antennary (tang et al., 2010c) hepatocellular carcinoma serum and liver glycoproteins of rats pngase f, r-tof (dhb), glycans (per-me) increase in core-α-1,6fucosylated glycoproteins. possible biomarker hepatocellular carcinoma suggests shiga toxin as potential therapeutic agent (distler et al., 2009) enhanced expression of monosialylated triantennary glycans in cancer (yoon et al., 2010b) stomach cancer mkn45 cells, serum automated β-elim. (matsuno et al., 2007) , tof (dhb), glycans (phenylhydrazones) mkn45 cells found to express characteristic trisialopolylactosamine-type glycans. reduced triantennary complex and increased biantennary glycans at asn-143 in patients table 29 and general reactions in table 30 . methods to change the glycosylation of a glycoprotein are common for recombinant antibiotic production as outlined above. a general method for producing homogeneous glycoproteins with eukaryotic n-glycosylation has been reported and involves the transfer of the campylobacter jejuni glycosylation machinery to e. coli and production of glycosylated proteins with the key glcnac-asn linkage. the bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to give a eukaryotic-type n-glycosylation (schwarz et al., 2010) . a method for immobilization of unstable membrane-bound enzymes to a commercially available sepharose support for glycan synthesis has been published ito et al. (2010c) . it involves modification of the protein c-terminus and a transpeptidase reaction by staphylococcus aureus sortase a (srta) has been developed. recombinant human b1,4-galactosyltranseferase or recombinant h. pylori a1,3-fucosyltransferases were bound with simple aliphatic amino groups displayed on the surface of the solid support and were shown to have the required glycosyltransferase activity. as with previous reviews in this series, two types of compound appear to be particularly suited to maldi-tof analysis; namely glycodendrimers and carbohydrate/protein complexes. lysosomal proteins in mouse model in contrast to previous assumptions, cho cells shown to be capable of adding antigenic α-gal residues to n-glycans (bosques et al., 2010) α1-antitrypsin (in human age1hn cells) tof to characterize novel human cell line (northoff et al., 2010) α1-proteinase inhibitor (in aspergillus niger) trypsin, pngase f, βelimination, tof/tof (dhb), glycans (per-me), gc/ms production, purification, and characterization, glycans = high-mannose (large antennae) (chill et al., 2009) anti-cd20 igg1s, anti-cd20 igg1 rituximab mutant (s239d/s298a i332e), (human) removal of fucosylated antibody ingredients from therapeutics shown to elicit high antibody-dependent cellular cytotoxicity in blood by two mechanisms anti-egfrxanti-cd3 bispecific igg ( model for comparison of release and glycan analysis methods (triguero, et al., 2010) centellosides (in centella asiatica plant cell cultures) tof to obtain a more efficient production system. α-amyrin, converted into centellosides by c. asiatica cells introduction of bisecting glcnac suppresses 1→3-fucosylation and xylose attachment to form paucimannosidic glycans (karg, et al., 2010) α-l-iduronidase (human in seeds of brassica napus and nicotiana tabacum) tof (sinapinic), glycoprotein. gc/ms of monosaccharides attached carboxy-terminal er-retention motif, sekdel, reduces xyl and fuc in n-glycans but has little effect on enzyme activity (galpin et al., 2010) iga1 and iga2 (in murine melanoma cells) pngase f, l-, r-tof (dhb, thap), glycans structural determination. hybrid, bi-, tri-, tetra-antennary complex. igg ( use of glycosidase inhibitor to produce igg with man 9 glcnac 2 . effect on xtal structure and effector functions (crispin et al., 2009a) igg (mouse in tobacco by2 cells) hydrazine, tof (dhb), glycans (2-ap) use of an er retention signal (kdel) increased high-mannose glycans but did not eliminate paucimannosidic glycans igg (human 29ij6 in silkworm larva hemolymph) pngase f, tof, glycans (2-ap) produced 8 mg per kg of larvae with recovery of 83.1%. paucimannosidic glycans igg (in cho cells) pngase f, tof, glycans production of non-fucosylated glycans by mutation (glycans -high-man, biantennary complex) (von horsten et al., 2010) igg (in cho cells) pngase f, tof chromatographic method to enrich nonfucosylated glycans (tojo, et al., 2009) igg ( effect of culture medium components on product yield. glycans: g1f, g2f (paul et al., 2009b) a. synthesis of multivalent carbohydrates, dendrimers, and glycoclusters an extensive review of dendrimers (astruc, 2010 #7260) , with a section on glycodendrimers, illustrates the breadth of this subject. masses of the larger compounds are frequently in the range 20-50 kda. the largest compound, based on a polyglycerol scaffold contained an estimated 222 mannose residues but the authors had difficulty obtaining a maldi spectrum because of the high mw. syntheses frequently involve huisgen-type click chemistry because high-yield reactions are essential when so many carbohydrate molecules have to be attached. table 31 lists papers reporting syntheses of glycodendrimers and similar compounds. among the largest of these compounds to have been reported during the review period has involved conjugation of lpsderived oligosaccharides to diphtheria toxin crm 197 protein in an attempt to develop a vaccine against invasive meningococcal disease. conjugates with a mass of 102 kda were analyzed by maldi-tof from sinapinic acid, the most widely used matrix for compounds of this type. conjugates can be characterized by maldi-tof ms but for large molecules the resolving power of most instruments is insufficient to distinguish each product and only a broad peak is observed. whereas the center of the peak represents the mean copy number of ligands per protein, information on the dispersity of the sample is usually neglected. patel et al. (2010) have produced a mathematical approach for calculating dispersity. by simply measuring the width at half maximum of the broad peaks that usually arise from carbohydrate-protein complexes and from the unmodified proteins, they were able to calculate the product distribution variance. furthermore, since the area between ae2s equates to 95% of the total, m ae 2s represents the range within which 95% of adducts exist, this gives a direct measure of dispersity. as one example of the type of work involved with this type of compound, the glycosylation sites of o-specific polysaccharide of vibrio cholerae o1, serotype ogawa linked to bsa via squaric acid chemistry have been determined by maldi-tof/ tof ms (from chca). the spectra showed the presence of hapten-bsa neoglycoconjugates with different hapten-bsa ratios (4.3, 6.6, and 13.2). sites of glycation were determined by comparison of the masses of the peptides resulting from the digestion of the bsa glycoconjugates and bsa itself using tandem ms/ms with a high-collision energy cell. the spectra improved secretion of molecular chaperone-assisted igg. no alterations in n-glycans (dojima, et al., 2010) interferon-γ, (human in cho cells) tof, glycans (per-me) study of intracellular glycosylation activities. effects of nucleotide sugar precursor feeding (wong et al., 2010b) interferon ( enzyme, which usually fucosylates core of n-glycans, shown to fucosylated chitooligosaccharides (ihara et al., 2010a) galactofuranosyltransferase glft2 (recombinant in m. tuberculosis h37rv) l-, r-tof (chca) polymer length shown to be controlled by a template-independent polymerase (may et al., 2009) β-d-galactoside-α1,4-gal-transferase and β-d-galactoside-β1,4-galtransferase from pigeon two novel enzymes catalyzing the formation of galα1→4galβ1→-galβ1-4glcnac galnac transferase 2 (human) tof/tof (dhb) adds galnac to several unnatural peptides (yoshimura et al., 2010) galnac transferase 10 (human) tof (dhb) enzyme found to have a unique galnac-o-ser/thr-binding site (perrine et al., 2009) galnac transferase 20 (human) tof found in testis and brain (peng et identification of gal-transferase adding gal to core fuc. (titz et al., 2009) gnt-i fuzed to maltose binding protein from nicotiana tabacum tof/tof recombinant expression and characterization (dohi et al., 2010) gnt-v and gnt-vb (recombinant human. incubations in 293t cells) maldi enzymes transfer glcnac to 6position of 6-antennae in n-glycans. comparisons of enzymes (alvarez-manilla, et al., 2010a) gpi-modifying β1-3-glcnac transferase from trypanosoma brucei enzyme characterization (izquierdo et al., 2009) glycosidases penicillium canescens tof (dhb) isolation and biochemical characterization, (burtseva et al., 2010) cellulase cel45a from trichoderma reesei tof/tof (dhb, thap), esi-ms/ms enzyme shown to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (enebro et al., 2009b) chitinase-a from cycas revolute (cycad) tof (dhb) biochemical characterization, cdna isolation, and posttranslational modification (taira et al., 2009b) chitinase-a and b from serratia marcescens strain bjl200 expressed in e. coli top10 effects on inhibition efficacy of allosamidin, a general competitive inhibitor of family 18 chitinases. (zakariassen et al., 2010) chitinase and n-acetylhexosaminidase from verticillium fungicola tof (dhb) effect of ph on the production of enzymes in submerged cultures cellulomonas fimi tof (dhb) use of mutations to affect mannose binding (hekmat, et al., 2010 ) exocellulase cel6b from thermobifida fusca tof proposal of a novel hydrolysis mechanism involving proton-transfer er glucosidase ii from rat liver tof found to be a broad specificity hexosidase (miyagawa et al., 2010) family gh38 α-mannosidase from streptococcus pyogenes tof, high-man glycans (per-me) characterization. α1→3mannosidase (suits et al., 2010) β-galactosidases from arabidopsis subfamily a1 tof comparative characterization (gantulga et al., 2009) (continued) β-galactosidases 4 and 5 from tomato tof enzymatic activity and substrate specifcity (ishimaru et al., 2009) β-galactosidases (bgac protein) from streptococcus pneumoniae specific for galβ1-3glcnac moiety (jeong et al., 2009) α-glucosidase from aspergillus niger r-tof (dhb) monitoring hydrolysis and transglycosylation activity (shimba et al., 2009) α-glucosidase from aspergillus niger tof (thap) activity towards dextrin and starch (ota et al., 2009) β-glucuronidase from aspergillus niger tof isolation and substrate specificity for transglycosylation (kiryu et al., 2009) shown to be involved in the maintenance of chronic infections (domenech et al., 2009) cholinephospho-transferase from mycoplasma fermentans tof/tof (dhb) molecular cloning and expression of enzyme involved in glycol-glycerophospholipid biosynthesis hemicellulase from chrysosporium lucknowense c1 tof investigation of the fungus for hemicellulase production (hinz et al., 2009) showed the presence of three conjugation sites on lys residues 235, 437, and 455, assumed to be the most accessible. the identification of y-series product ions was found to be useful for sequencing of various peptides and the a-and b-product ions confirmed the sequence of the conjugated peptides . other examples are listed in table 32 . maldi-tof analysis, combined with ir spectroscopy has demonstrated covalent modifications of chitin with silk-like proteins in the formation of shells in the mollusc mytilus galloprovincialis (weiss et al., 2009) . maldi-tof ms has also been used as a standard against which to evaluate and optimize a fluorophore-assisted carbohydrate electrophoresis (face) method for analysis of pectic oligosaccharides (sun et al., 2009a) (article in chinese). koroleva et al. (2010) have used maldi-tof ms to identify all the major glucosinolates that accumulate in s-cells of arabidopsis leaves and flower stalks. cell sap was diluted in 10 ml methanol and mixed 1:1 with chca before being spotted onto the maldi target plate. the analysis was performed in negative-ion mode using an ultraflex tof/tof instrument. finally, mun, rho, and kim (2009) have used maldi to confirm the molecular sizes of commercial cycloamyloses and patsos et al. (2009) have used it to detect aryl glycans in cells treated with inhibitors of o-glycan processing. maldi continues to be a major technique for carbohydrate analysis although electrospray is more widely used. a major advantage of maldi is that is gives a cleaner profile of glycan mixtures because of the absence of multiple charging. new techniques, such as ion mobility have emerged to complement both maldi and electrospray but there is still much scope for improvement in carbohydrate analysis. surveys during the review period have highlighted the fact that many laboratories still make mistakes when assigning structures. much of this can be attributed to assumptions made between a simple mass (urch et al., 2009) s-adenosyl-l-methionine dependent methyltransferase from haloferax volcanii tof (chca) enzyme shown to methylate n-linked pentasaccharide tlmk from strepto-alloteichus hindustanus ft-icr functional characterization in tallysomycin biosynthetic pathway (wang et al., 2009g) trehalose synthase from thermotoga maritima dsm3109 tof molecular cloning and characterization (ryu et al., 2010) udp-arabinopyranose mutase from rice tof (sinapinic), glycoprotein arg residue shown to be reversibly glycosylated with single glycosyl residue; residue needed for activity (konishi et al., 2010b) (gao et al., 2009b) aminated xyloglucan (from tamarind) tof (dhb) physico-chemical properties of aminated xyloglucan extracted from tamarind seed (simi & abraham, 2010c) bridged c-furanosides tof intramolecular nucleophilic attack of bzo group in a triflated cyclooctenol (jürs & thiem, 2009 ) 1-deoxynojirimycins with dansyl capped n-substituents tof as probes for morbus gaucher affected cell lines (fröhlich et al., 2010) 2,3-dibromo-3-methyl-1phenylphospholane 1-oxide tof as novel anticancer agent (yamada et al., 2010b) synthesis of polysaccharide intracellular adhesins using an acid reversion reaction of n-acetylglucosamine in hf-pyridine (leung et al., 2009) amino-bridged oligosaccharide mimetics r-tof (dhb), fab synthesis using reductive amination. glycomimetic target structures as potential ligands for the receptor protein nkr p1 of natural killer cells (neumann & thiem, 2010) amylose chains tof from starch by action of phosphorylase. preparation of a-type crystals (montesanti et al., 2010) bi-fluorescently-labeled maltooligosaccharides tof for amylase assays (oka et al., 2010a) bis-hydrazides tof for conjugation with proteins etc. (adak et al., 2010) blood group tetrasaccharides b (types 1, 3 and 4) tof 3-aminopropyl glycosides of tetrasaccharides synthesised using acetylated galα(1→3)-(fucα(1→2))gal trichloroacetimidate as a glycosyl donor (korchagina et al., 2009) 3,6-branched arabinogalactan-type tetraand hexa-saccharides tof for investigation of monoclonal antibodies raised against arabinogalactan proteins from pressed juice of echinacea purpurea. carboxymethylated cyclosophoraose tof as a novel chiral additive for the stereoisomeric separation of flavonoids by ce (jeon et al., 2010) chitooligosaccharides tof by the enzymatic hydrolysis of chitosan ) chitooligosaccharides tof by the enzymatic hydrolysis of chitosan (xu et al., 2010a) chitooligosaccharides tof/tof (dhb) glycans (amac) characterization of family 46 chitosanase from streptomyces coelicolor a3(2) and use for degradation of chitosans chitooligosaccharides tof to study the antibacterial activity against bifidobacteria (šimůnek et al., 2010) chitosan and chitooligosaccharides adsorption properties for uranium (paper in chinese) (jiang et al., 2010b) chitosans tof synthesis from lobster chitin by naoh deacetylation and enzymatic hydrolysis. to protect crops from main pathogens (falcón et al., 2010) chito-tetrasaccharide β-1,4-glcnac-β-1,4-glcn repeat tof (dhb) by condensation of two disaccharides deacetylated chitohexaose tof hydrolysis of chitosan. could limit cell proliferation of breast cancer cells (xiong et al., 2009 ) 2-deoxy cyclic and linear oligosaccharides tof (dhb) synthesis by oligomerization of monomers (paul et al., 2009a) β-d-fructopyranosyl-(2→6)-d-glucopyranose tof synthesis from d-glucose and d-fructose by thermal treatment (yamamori et al., 2010) galactofuranose oligomers tof/tof (chca) to probe mechanism by which polymer length is controlled in mycobacteria (splain & kiessling, 2010) galactomanno oligosaccharides tof from hydrolysis of guar gum by βmannosidase (paper in chinese) (zhao, et al., 2009b) galactooligosaccharides ft-ms, gc/ms synthesis by acid hydrolysis of the polysaccharides from nerium indicum mill α-glucan pentasaccharide from aconitum carmichaeli use of chiral-auxiliary-mediated 1,2-cisglycosylations for the solid-supported synthesis (boltje et al., 2010) glucan with α -(1→6) linkages and α -(1→3) and α -(1→4) branch points produced from glucan of leuconostoc mesenteroides nrrl b-742 by microwave assisted hydrolysis (majumder et al., 2009) glucosylated raffinose tof (dhb) use of glucansucrase (alternansucrase) for synthesis glc-glc, gal-gal, gal-glc, gal-gal disaccharides tof (dhb) 16-member library containing all regioisomers. solid-phase synthesis (ágoston et al., 2009b) linear isomaltooligosaccharides (dp2-10) tof one-step synthesis using synthetic fusion enzyme of dextransucrase and dextranase (kim et al., 2009e) macrocyclic oligosaccharides tof copper(i)-catalyzed huisgen's 1,3-dipolar cycloaddition of alkyne and azide provides size-defined macrocyclic carbohydrates (muthana et al., 2009) mannose-capped disaccharide with a thiol terminus to provide a tethered sugar for attaching to gold nanoparticles to mimic carbohydrateinvolved cell-surface interactions (continued) chemical stereochemically controlled synthesis (pastore et al., 2010a) nigerose-containing oligosaccharide tof/tof (chca) escherichia coli non-glycosidically linked pseudodisaccharides tof (thap) thioethers, sulfoxides, sulfones, ethers, selenoethers, and their binding to lectins oligosaccharides from red seaweed polysaccharides tof (chca), esi efficient conversion of galactans into cglycosyl aldehydes (ducatti et al., 2009) oligosaccharide mimics of sialyl lewis a trisaccharide. neu5ac and fuc replaced with hso 3 and d-ara respectivly (jakab et al., 2010) pentasaccharide anticoagulant (idraparinux) maldi compound is fully o-sulfated, o-me mimic of antithrombin iii binding domain of heparin ) n-quaternary chitosan derivatives tof synthesis, characterization and antibacterial activity (rúnarsson et al., 2010) sialylated lactosides tof (dhb), per-me for coupling to bsa by huisgen reaction. as glycocongugate antigen (mosley et al., 2010) sialyllacto-n-tetraose and sialyllacto-n-neotetraose tof use of α2-3and α2-6-sialylated lactosaminide precursors obtained by enzymatic procedures with glycosylations employing triflic acid/n-iodosuccinimide (schmidt & thiem, 2010) sucrose-based guanidinecontaining g7 molecular transporters tof show different patterns of intracellular localization depending on the nature of the linker chains as well as the fluorescent dyes (lee et al., 2009l) sulfated oligofucosides maldi synthesis of sulfated octyl tetra-to octaoligofucosides with different sulfation patterns (zong et al., 2010a) α to achieve steric instead of electrostatic stabilization. two-step synthesis (kloser & gray, 2010) cyclic β-glucan r-tof/tof (chca) synthesis using laminarinase 16a glycosynthase mutant from the basidiomycete phanerochaete chrysosporium (vasur et al., 2010) dextrin-hydroxyethylmethacrylate and dextrinvinyl acrylate hydrogels tof/tof (dhb) for the determination of biocompatibility and biodegradability in mice (moreira et al., 2010) epoxy-starch derivatives tof (dhb) synthesis by epoxidation of allylated starch (huijbrechts et al., 2010) poly-n-acetyllactosamine tof (dhb, chca) chemo-enzymatic synthesis. characterization for cgl2-galectin-mediated binding of ecm glycoproteins to biomaterial surfaces (sauerzapfe et al., 2009) triblock co-oligomers of tri-o-methylated and unmodified cello-oligosaccharides: tof (dhb) synthesis and structure-solubility relationships (kamitakahara & nakatsubo, 2010) xylan-based polysaccharides tof (dhb) amino-modified low mw xylan reacted with unmodified xylan and cellodextrins ) glycosaminoglycans and related compounds n-acetyl-heparosan oligosaccharides tof digestion of n-acetyl-heparosan with heparitinase. for study of enzymology (sugiura et al., 2010a) 2,3-desulfated heparin maldi for control of inflammation by inhibition of e-selectin (lakshmi et al., 2010) heparan sulfate oligosaccharides tof/tof (dhb), esi use of modular building blocks for preparation of a library of 12 oligosaccharides used to probe the structural features of hs for inhibiting the protease, bace-1 (arungundram et al., 2009) heparan sulfate oligosaccharides tof synthesis and inhibition of endothelial cell functions essential for angiogenesis (cole et al., 2010) hyaluronan tof for nmr study of chemical proton exchange over the β(1→3) glycosidic linkage (nestor et al., 2010) hyaluronic acid decasaccharide maldi chemical synthesis using preactivation-based chemoselective glycosylation strategy hyaluronic acid subunit and fully protected oligomers maldi tetra-, hexa-and octa-saccharides. multigram synthesis (virlouvet et al., 2010) isosteric sulfonate analogues of at-iii binding domain of heparin tof (thap) d-glca-and l-idoa-containing disaccharide. related to antithrombin-binding pentasaccharide of heparin. one sulfate ester replaced by na sulfonato-me moiety bisected octasaccharide maldi chemical synthesis (wang et al., 2009e ) galβ(1→3)[neuacα(2→6)] glcnacβ(1→2)man motif tof chemical synthesis as molecular probe (bao et al., 2010) glucuronyl oligosaccharides tof, glycans (2-ap) synthesis of glucuronyl and sulfoglucuronyl oligosaccharides from hnk-1 glycoprotein (yagi et al., 2008) high-mannose glycans tof (dhb) related to hiv gp120. one-pot catalytic glycosidation/fmoc removal (pastore et al., 2010b) high-mannose glycans with glc 3 units tof (dhb) study of the conformational properties of the glc 3 man unit (mackeen et al., 2009) high-mannose glycansmethotrexate derivatives tof identification of the recognition motif of the glycoprotein-folding sensor enzyme, udp-glc: glycoprotein glucosyltransferase biantennary and high-mannose n-glycans linked to non-reducing terminus of man 3 glcnac 2 core, plus biotin for arrays n-glycan library tof (dhb), glycans (2-ap) enzymatic construction of library for building ms database phosphorylated highmannose glycans tof high-mannose glycans from ribonuclease. incubation with recombinant glcnac phosphotransferase. (bohnsack et al., 2009) (continued) (song, et al., 2009f) sialic acid containing complex-type n-glycan tof new solid-phase synthesis. stereoselective βmannosylation, microfluidic α-sialylation and glycosylation of n-phf 3 acetimidate on jandajel resin thiol-terminated nonamannoside tof for synthesis of microarrays (dietrich, et al., 2010) various complex glycans tof development of hek393t expression system for human glycosyltransferases enzymatic construction of library for building ms database glycosylated amino acid derived from psgl-1 tof use of trichoroacetimidate donors and thioglycosyl acceptors (vohra et al., 2009) kl-6 antigen tof/tof (dhb) library of glycans to determine binding to anti-muc1 antibody aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. synthesis and anti-influenza activity androgenic gland hormone of the woodlouse, (armadillidium vulgare) tof with various glycans. showed that thermodynamically most stable form not most active: result of disulfide pairing (katayama et al., 2010b) antifreeze glycopeptide analogues tof (dhb, chca) microwave-enhanced synthesis and functional studies (heggemann et al., 2010) β-hfsh with chitobiose units at the natural linkage sites tof use of the sinaÿ radical glycosidation for simultaneous installation of biantennary chains and glycal chemistry to construct the tetrasaccharide core (nagorny et al., 2009) β-sheet-rich protein plus glcnac at various positions tof effect of glcnac position on protein folding kinetics and thermodynamics (price et al., 2010a) biantennary n-glycans plus peptide tof/tof (dhb) solid-phase peptide synthesis. glycans linked -nh-co-ch 2 -s-peptide (murase et al., 2009) bivalent glycopeptide tof mannosides linked with squaric acid (lindhorst et al., 2010a) cd52 glycopeptide antigens tof (dhb, chca) chemo-enzymatic synthesis of glycopeptide with n-and o-linked glycans (huang et al., 2010c ) c-glycosyl β 2 -and β/β 2peptides maldi solution-phase synthesis. 3-8 amino acids (inaba et al., 2009) c-linked antifreeze glycoprotein analogues tof (dhb) effect of the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone influences ice recrystallization inhibition (tam et al., 2009) c-mannosylated peptides tof (dhb, chca) peptides shown to interact with hsc70 to modulate its signaling in raw264.7 cells ) cyclic antifreeze glycopeptides tof exhibited antifreeze activity by forming hexagonal-bipyramidal ice crystals (hachisu et al., 2009) cyclic neoglycopeptides maldi (chca) for binding to adjacent protein binding sites in wheat germ agglutinin (schwefel et al., 2010) dihydrofolate reductase tof glycans = glcnac, lactose, maltotriose analysis as tryptic peptides. for study of effects of glycosylation on stability (tey et al., 2010) mass spectrometry reviews doi 10.1002/mas microwave-assisted synthesis. 5(6)-carboxyfluorescein shown to be stable fmoc-threonine bearing a core-2 glycan tof as building block for psgl-1 via fmocassisted solid-phase peptide synthesis (krishnamurthy et al., 2010 ) o-fucosylated epidermal growth factor-like repeat 12 of mouse notch-1 receptor chemical synthesis and studies on folding (hiruma-shimizu et al., 2010) galactosylated naringinase tof (sinapinic) modification of glycosylation to effect deglycosylation of rhamnosylated drugs (garnier et al., 2010) gal-β-3galnac-α-lys 5 tof immunogen design for tumor t antigen immuno-targeting (sendra et al., 2009) glycopolypeptide-based cholera toxin inhibitors maldi effect of peptide charge and glycan linker length on activity (maheshwari et al., 2010 ) glycopeptide carrying tetra-n-ac-lactosamine containing core 2 decasaccharide tof (dhb) solid-phase synthesis (ueki et al., 2010) glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan tof to investigate structures that best serve as a hexosamine acceptor for enzymatic glycosyl transfer (tamura et al., 2010) glycosulfopeptides from nterminus of human endoglycan tof/tof (thap) containing tyrosine sulfate residues and sialyl lewis x in core 2 o-glycans and bind to human l-selectin (leppänen et al., 2010) glycosylated peptoids tof by on-resin click (huisgen reaction) glycoconjugation (norgren et al., 2009 ) glycosylated cell-penetrating peptide (r6/w3): ac-rrwwrrwrr-nh 2 tof one, two, or three galactose(s), with or without a spacer introduced via a triazole link (dutot et al., 2010) grgdy grafted to chitosan tof (chca) linked with sulfosuccinimidyl-6-[4'-azido-2'nitropheny-lamino]hexanoate as drug carrier n-glycosylated insulin l-tof (dhb) addition of three glcnac residues at nh 2 groups on peptide. use of endo m to transfer glycan to one of them. (tomabechi et al., 2010a) n-glycoproteins carrying intact natural n-glycans tof/tof (dhb) enzymatic synthesis of biantennary glycans (huang et al., 2009b) glycosylated analogues of glucagon-like peptide 1 tof, lc-ms to improve proteolytic stability and blood glucose-lowering activity. sugars = glcnac, lacnac, sialyl lacnac synth. by chemoenzymatic approaches (ueda et al., 2009b) glycosylated neurotensin analogues tof containing o-linked β-melibiose and α-tf antigen or β-lactose units linked by a peg3 spacer. exhibit subpicomolar anticonvulsant potency in model of epilepsy (lee et al., 2009f) glycosylated tetracontapeptide with acidlabile sialyl-t n antigens maldi (dhb) 20-residue glycopeptide-α-thioester and 20residue glycopeptide with cys at n-terminal. solid phase synthesis; coupled by cys ncl ser . o-glycopeptide mimetics tof (dhb, chca) aziridine ring opening as regio-and stereoselective access to o-glycosyl amino acids and their transformation into oglycopeptide mimetics (schäfer et al., 2009) heptasaccharide from campylobacter jejuni plus acra61-210 tof/tof (chca) development of nmr method for 3d structural determination of glycoproteins using enzymatically synthesised glycoprotein (slynko et al., 2009) (continued) 4-oh-proline oligomers q-tof (dhb) compounds form very stable polyproline ii helices (owens et al., 2010) iga-hinge peptide tof to study effect of glycosylation on cis/trans isomerization of prolines (by nmr) man 5-9 glcnac 2 -asn-n-14 c r-tof (dhb) for micro-method for determining precise oligosaccharidic specificity of mannosebinding lectins (debray et al., 2009) mannosylated lysine derivative tof bivalent carbohydrate branching unit. suitable for solid-phase peptide synthesis chemical synthesis, for enzymatic study in dictyostelium (see table 12 ,o-glycans) (wang, et al., 2009k) s-linked glycopeptides maldi thioglycosylated building blocks prepared from per-ac sugars via glycosyl iodides in one-pot fashion and used in sub-monomer solid phase strategy (comegna & de riccardis, 2009) synthesis using using the newly developed n-troc-protected gm3 and galn intermediates (komori et al., 2009) anchor with n-terminal cys maldi general method for producing proteins containing a natural gpi anchor using expressed protein ligation (schumacher et al., 2010) anchor with unsaturated lipid chains tof to investigate mechanism of gpi anchoring (swarts & guo, 2010 bearing pyrazolines, isoxazolines, and dihydropyrimidine-2(1h)-thiones. as antibiotics (el-sayed et al., 2009) hydroquinone fructoside l-tof synthesis using leuconostoc mesenteroides levansucrase hydroquinone glucoside l-tof (dhb) synthesis using leuconostoc mesenteroides levansucrase lobatoside e-related triterpene glycoside maldi gold(i)-catalyzed glycosylation with glycosyl ortho-alkynylbenzoates as donors man-α-(1→6)-man-o-octyl analogues maldi synthesis and evaluation as potential substrates and inhibitors of a ppm-dependent α-(1→6)-mannosyltransferase involved in lam/lm biosynthesis (tam & lowary, 2010) mannosylated pyreneperfluoroalkyl lipid maldi to study multivalent binding on the lateral phase separation of adhesive lipids (liem et al., 2010) mannosyl glycolipids with perfluoroalkyl membrane anchors maldi to assess the cluster glycoside effect during the binding of concanavalin a to mannosylated artificial lipid rafts (noble et al., 2009) mesogenic azobenzenes tethered to sugar alcohols (gretskaya & mikhalyov, 2007) cholesterol plus linear glucose tof (dhb) synthesis and gelling properties 1,2-dipalmitoyl-3-(npalmitoyl-6'-amino-6'-deoxyα-d-glucosyl)-sn-glycerol tof glycoglycerolipid of a marine alga with a high inhibitor activity against human myt1kinase. synthesis starting from α-me-glcp enzymatic synthesis from 1,2dipalmitoylglycerol in one pot reaction hydroquinone galactosides l-tof (dhb) a potential skin whitening agent. synthesis by reaction of lactase with lactose as donor. fluorescently labelled synthetic ionophore synthesises in 12 steps with 26% yield (coppola et al., 2010) phosphatidyl-myo-inositol mannosides tof to study mannosyltransferase corynebacterium glutamicum pimb' (batt et al., 2010) puerarin-cycloamylose inclusion complex l-tof (dhb) enzymatic synthesis using 4-αglucanotransferase and maltogenic amylase (choi et al., 2010) sialic cinnamoyl-α-cyclodextrin tof initiates polymerization of δ-valerolactone (osaki et al., 2009) cinnamoyl α-cyclodextrin tof cds self-organize to give different supramolecular complexes in aqueous solutions (tomimasu et al., 2009) (continued) synthesized via hydrosilylation reaction under thermal or uv-activated polymerization (tian et al., 2009) cd-based telluronic acid plus mn(iii)meso-tetra[1-(1adamantyl methyl ketone)-4pyridyl] porphyrin maldi as artificial enzyme with superoxide dismutase and glutathione peroxidase activities (yu et al., 2010d) cyclodextrin dimers tof one homo-dimer of β-cd and two heterodimers of α-cd and β-cd as hydrolases (ikeda et al., 2010) cyclodextrin dimers and trimers tof/tof, esi bridged cds with links at different positions formed supramolecular adducts with shapespecific ligands (aime et al., 2009) β-cyclodextrin cyclic-nitrone conjugate maldi with superoxide radical anion and dodecyl chain for membrane insertion α-, βand γ-cyclodextrinesters (acrylate, pent-4-enoate and undec-10-enoate) synthesis with nitrophenol esters (nielsen et al., 2010a) β-cd plus hydroquinone-αglycoside tof synthesis and doxorubicin-inclusion abilities (oda et al., 2009) β-cd monoalkyn tof (dhb) for conjugation to long-chain thiols for selfassembled monolayer prep. (dubacheva et al., 2010) cyclodextrin-polyester polymers tof ring-opening polymerization -heating cyclic esters and cds (harada, 2009) γ-cds possessing an azido group and a triisopropylbenzenesulfonyl group as useful synthetic and authentic intermediates for unsymmetrically functionalized derivatives (himeno et al., 2009) β-cd-ended linear poly(nisopropylacrylamide) (β-cd-pnipam) tof (thap, dctb) self-assembly of pnipam-based amphiphiles formed by inclusion complexation (zou et al., 2009) β-cd with octadecyl-linked perylene bisimide maldi self-assembled amphiphilic perylene-cd conjugate for vapor sensing of organic amines (jiang et al., 2010a) [6-deoxy-6-(1-h-1,2,3triazol-4-yl)(me)6-(4-omebi-ph-4'-yloxy) hexanoyl]-βcyclodextrin tof (dhb) synthesis, liquid-crystalline properties, and supramolecular organization for controlled release application. (adrian et al., 2009) inclusion complexes of β-cd with bipyridine guests tof with 4,4'-vinyl-enedipyridine, 2,2'vinylenedipyridine, 1-(2-pyridyl)-2-(4pyridyl)ethylene, 4,4'-ethylene-dipyridine, 4,4'-dithiodipyridine, and 2,2'dithiodipyridine insulated molecular wire with highly conductive πconjugated polymer core tof rod-like -ph-c=c-ph-core coated with α-cds (terao et al., 2009b) ionic-liquid-functionalized βcyclodextrin tof as bonded chiral stationary phases for hplc (zhou et al., 2010b ) linear α-cyclodextrin oligomers tof controlled synthesis using copper-catalyzed huisgen 1,3-dipolar cycloaddition (rawal et al., 2010) oligothiophene derivatives bearing β-cyclodextrin tof (dhb) 2t-β-cd 2 and 3t-β-cd 2 , with bithiophene and terthiophene with β-cd at each end form supramolecular assemblies in aqueous solns. (sakamoto et al., 2009b) perylene bisimide-bridged bis-(permethyl-β-cds) ft-icr as solid-state fluorescence sensor for vapor detection perylene-bridged bis(βcyclodextrin) ft-icr (kitano et al., 2009) glycodynamers maldi dynamic polymers bearing oligosaccharide residues such as glc 6 obtained from α-cd (ruff et al., 2010) membranes (cross-linked) tof (dhb) based on acrylated cyclodextrins and polyethylene glycol dimethacrylates (rölling et al., 2010) multivalent galactotrehaloses tof α,β-gt isomers converted into vinyl monomers then radical copoly-merization with 4-acrylamidophemyl-β-glc or β-glcnac) with acrylamide (miyachi et al., 2009b) pentafluorostyrene copolymers with glucose tof synthesis by nitroxide-mediated polymerization and "click" chemistry (becer et al., 2009) polydioxanone with a protected monosaccharide end-group l-tof (dithranol) ring opening polymerization of p-dioxanone using a protected monosaccharide (1,2;3,4-di-o-isopropylidene-α-d-galactopyranose) /al(oipr) 3 initiator system (sugih et al., 2009) polyfluorene derivative with 20 mol% 2,1,3benzothiadiazole plus α-man on an amine-reactive hydrogel-coated microarray glass surface. to detect autoantibodies in breast cancer correction: (blixt, et al., 2011) nanofibres tof nanofibers formed through π· · ·π stacking of the complexes of glucosyl-c2-salicyl-imine and phenylalanine (continued) for study of carbohydrate-protein interaction oligosaccharides from plants tof (dhb) for construction of microarray to screen for plant transglycosidases activity (kosík, et al., 2010 ) 6-sulfo-n-acetyl-dglucosamine containing, on gold tof to study mechanism of amyloidosis of amyloid β peptides (fukuda et al., 2010b) miscellaneous β-d-allopyranoside-grafted ru(ii) complex tof synthesis and both acid-base and dnabinding properties azo-sugar nucleotides plus many alkynes tof (dhb) click chemistry (huisgen reaction) to produce inhibitors of glycosyltransferases 1-deoxy-1-nitropiperidinoses maldi synthesis from a protected galactooctopyranose (collin & vasella, 2010 ) 4,4-di-f-5,7-di-me-4-bora-3a,4a-diaza-sindacene-3propionic acid derivative of man 5 glcnac 2 . (bodipy) for development of fluorescence assay (haga et al., 2009) carbohydrate-functionalized salphen-metal complexes tof complexes with peripheral glucose and galactose substituents. self-assembled supramolecular structures were produced (hui et al., 2009) dioxolane-type (9'anthracenyl)methylene acetals tof synthesis, regioselective hydrogenolysis, partial hydrogenation, conformational study (jakab et al., 2009) (kim et al., 2009f) amino-sugars tof (chca), esi deprotection method for the 2,2,2trichloroethoxycarbonyl (troc) group using tetrabutylammonium fluoride 1,6-anhydrosaccharides tof use of 7 mol % of aubr3 to catalyse glycosylations of 6-oh-propargyl/me mono-, diand tri-saccharides (thadke & hotha, 2010) colored carbohydrates tof addition of a fmoc analogue protecting group based on guaiazulene (aumüller & lindhorst, 2009) (bindschädler et al., 2009) glycoamino acid building blocks tof use of staudinger ligation glycopeptides tof use of pyruvoyl as a novel protecting group for solid-phase synthesis (katayam et al., 2009 ) gpi-anchored proteins tof sortase a-catalyzed transpeptidation of gpi derivatives for chemoenzymatic synthesis (wu et al., 2010f) heptasaccharide asparagine building block tof (dhb) use of one-pot catalytic glycosidation/fmoc removal (mezzato & unverzagt, 2010) homolinear α(1→6)-linked octamannosyl thioglycosides maldi imidazolium cation-tagged mannosyl fluoride and thiomannoside using block couplings. efficient alternate approach for oligosaccharide synthesis (yerneni et al., 2009) homooligosaccharides tof (dhb) (per-ac, per-me) base-promoted glycosylation of unprotected glycosyl fluorides (steinmann et al., 2010) lipophilic thioglycosides tof use of heavy lipophilic tag to assist biphasic liquid-liquid separation (encinas & chiara, 2009) macrocyclic neoglycoconjugates tof (chca) macrocyclization of linear d-galacto-2heptulopyranose-containing oligoketosides by intramolecular glycosidation and ring-closing metathesis mucin-type oglycopeptides and oligosaccharides tof (dhb, thap, chca) using transglycosylation and reverse-hydrolysis activities of bifidobacterium endo-α-nacetylgalactosaminidase neu5gc-containing glycans tof sialylation with n-glycolylneuraminyl phosphite (hanashima et al., 2009 ) n-linked glycopeptides tof condensation of glycosylamines with asn. demonstrated with man 8 glcnac 2 . (chen & tolbert, 2010) measurement and structure, particularly when structures are selected directly from databases. at present, no one mass spectrometric technique can identify all structural features of carbohydrates. sialylated and sulfated glycans still remain a problem although sialylated glycans can be handled after suitable derivatization, particularly by permethylation or simply by methyl ester formation. permethylation appears to be becoming more popular, particularly for quantification and maldi. the next few years are expected to bring many developments, particularly in instrumentation and ionization techniques, that will possibly address some of the problems with carbohydrate analysis highlighted above. 2-ab 2-aminobenzamide 2-aa 3-aminobenzoic acid 9-aa 9-aminoacridine aboe aminobenzoic acid octyl ester aeab 2-amino-n-(2-aminoethyl)-benzamide age advanced glycation end products agp a1-acid glycoprotein alpcs aluminum-phthalocyanines a-tf a-thomsen-freidenreich antigen all allose amac aminoacridone amt 5-amino-2-mercapto-1,3,4-thiadiazole ann artificial neural networks anp 2-amino-5-nitro-4-picoline ansa 5-amino-2-naphthalenesulfonic acid 2-ap 2-aminopyridine apb aminophenylboronic apci atmospheric pressure chemical ionization api apiose apts 8-aminopyrene-1,3,6-trisulphonic acid aq aminoquinoline (3-or 5-) use of genetic engineering to produce glycoproteins in e. coli which are then enzymatically remodelled (see text) (schwarz, et al., 2010) o-sulfated trisaccharyl glycopeptide tof (dhb) solid-phase synthesis. use of new benzyl protection method. (kawahira et al., 2009) oligosaccharides tof (dhb) rate acceleration on stereoselectivity and velocity of o-glycosylation reactions (ishiwata et al., 2010) pseudooligosaccharides maldi use of cross-metathesis reaction between sugarolefins, followed by intramolecular cyclization (ronchi et al., 2009 ) s-linked glycoconjugates tof, esi by-thiyl glycosylation of olefinic proteins (floyd et al., 2009) sialylated glycans tof use of koenigs-knorr reaction (with ag 2 co 3 ) (pazynina et al., 2010) thioglycosides r-tof/tof solvent-free synthesis by use of ball-milling (patil & kartha, 2009 ) trisaccharide libraries tof use of linker-tagged building blocks imobilized on a soluble polymeric support (elsayed, 2009) acceptor specificity in transglycosylation reactions using endo-m l-tof (dhb), esi (tomabechi et al., 2010b ) acetolysis of 6-deoxyhexosemethyl glycosides. role of sugar configuration tof (dhb) (cirillo et al., 2009 ) acid-catalyzed hydrolysis of β-1,4-glucan, including cellobiose and crystalline cellulose with so 3 h-bearing amorphous carbon tof (suganuma et al., 2010) allyloxycarbonyl group removal provides a practical orthogonal protective strategy for carbohydrates tof (zong et al., 2009 ) amadori ketoses synthesis in microwave field via mo vi -catalyzed stereospecific isomerization of 2-c-branched sugars bearing azido function tof (hricovíniová, 2010 (camponovo et al., 2009) benzhydrylamine-lysine capped with 2 to 64 mono-, di-and tri-α-d-manp residues tof reactive n-oh-succinimide esters to ensure complete reaction of dendrimer amines (g3 mass = 13,841 da) (greatrex et al., 2009) 4,7-bis(9,9-bis(2-(2-(2azidoethoxy)-ethoxy)ethyl)fluorenyl)benzo-thiadiazole with 4 mannose residues tof (dhb) as an intelligent energy transfer pair for label-free visual detection of concanavalin a boltorn h30 (commercial polymer) with 27 gal-cer groups tof binds hiv-1 gp120 optically pure fullerodendron formed by diastereoselective diels-alder reaction calixarene-based glycocluster oligonucleotide with 4 galactose residues tof click chemistry. triazole-tethered glycoclusters with 3 arrangements. affinities towards pa-il and rca 120 with dna-based glycoarray. (moni et al., 2009) carbosilane with 3, 4 or 6 sialyl-α-(2→3)-lactose residues tof for anti-influenza properties carbosilane with 3, 4, 6 or 12 neu5ac residues tof, fab, esi influenza neuraminidase inhibitors. dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties resistant to neuraminidases (sakamoto et al., 2009a) carbosilane with 8 or 16 mannose residues tof (dithranol) hydrosilylation of allyl tetra-ac-man with carbosilane dendrimers containing monohydrosilane end groups and the subsequent deacetylation (ortega et al., 2010) carbosilane with 4 sialyl-nacetyllactosamine groups tof combined chemical and enzymatic synthesis (matsuoka et al., 2010) cyclic α-(1→6)-octaglucoside with from 2-7 mannose residues tof oxidation of vic-diols, reductive amination with 2-aminoethylmannoside β-cyclodextrin with 27 mannose or glucose residues tof (dhb) heteroglycoclusters. 7 antennae each with two mannose and one glucose. for lectin binding (gómez-garcía et al., 2010) β-cyclodextrin with dextran tof (chca) synthesis by click chemistry using huisgen reaction (nielsen et al., 2010b) cyclopeptide with 4 mannose residues tof synthesis by click chemistry and molecular recognition study by surface plasmon resonance (chen et al., 2009c) dihydroxy-benzamide based with 2-8 mannose, galactose, lactose, glucose or glcnac groups tof (chca) octa-dendrimers. for screening of lectins for multivalency effects. click chemistry (pera et al., 2010b) diphenyldisulfide with 4 glucose residues and others without sugars tof, gc/ms use as catalyst to convert allyl alcohols into carbonyl compounds (tsuboi et al., 2009) cysteine with 3 or 4 (from dicysteine) mannose residues tof as inhibitors of type 1 fimbriae mediated bacterial adhesion (schierholt et al., 2010) ethylene glycols with 2, 3 or 4 galactose,lactose, maltose or lacnac groups ferrocene with one or two glucose, mannose or lactose residues tof/tof as electrochemical probes for molecular recognition studies (casas-solvas et al., 2009b) polyglycerol substituted phenylboronic acid tof dendrimer formed adducts with, fru, d-(+)-gal, d-(+)-glc, d-(+)-man, and me-α-d-man, by removal of four h 2 o (hashidzume & zimmerman, 2009) fullerene. sugar balls with 12 iminoglucose residues tof synthesis by click chemistry. glucosidase inhibition shown with resulting iminosugar balls (compain et al., 2010) fullerene. sugar balls with 12 glucose or galactose residues tof, esi, fab 12 residues on 6 arms, synthesis by click chemistry gd-diethylenetri-aminepentaacetic acid with 2 glucose residues tof synthesis, in vitro and in vivo studies of gd-dtpa-xda-d1-glc(oh) complex as a new potential mri contrast agent (ozaki et al., 2010a) hydroxy benzenes and naphthalenes with 2, 4 or 8 mannose residues tof synthesis by click chemistry (rajakumar et al., 2009 ) l-lysine plus gd chelates and 4 galactose residues tof/tof as liver imaging probes (luo et al., 2009) tetramer of glycodecapeptide from muc1 with sialyl t n antigen as vaccine candidate (keil et al., 2009) peptide/tris-oh-methylmethylamine. 9 galactose or mannose residues attached to β-cd containing doxorubicin tof for uptake studies in the human hepatocellular carcinoma cell line hepg2 (bernardes et al., 2010) peptide/tris-oh-me-methylamine/ [ru(bipy) 3 ] 2+ with 18 galactose or mannose residues maldi to study lectin interactions by monitoring change in fluorescence quantum yield of ru(ii). (kikkeri et al., 2010a) phloroglucinol, triazyne, tetrachlorosilane, pentaerythritol, myo-inositol with 2, 3, 4 or 6 clinked sialic acids tof synthesis by huisgen cycloaddition of azide and alkyne (click chemistry). to explore sialic acid binding to cell surfaces (papin et al., 2009) phthalocyanine with 4 α-galp residues tof potential application as photosensitizers in photodynamic therapy (soares et al., 2009) pentaerythritol, methyl α-dglucopyranoside, d-glucose, and dmannitol with 4, 5 or 6 mannose tof (dhb) synthesis by click chemistry, measurement of binding affinities (sattin et al., 2010) poly-glycerol with 8 mannose residues tof maldi poor because of high mw (up to 493 kda) (kizhakkedathu et al., 2010) poly-lysine with 16 biantennary nglycans tof synthesis by click chemistry. effect of sialic acid linkage on in vivo dynamics. mass around 40 kda (tanaka et al., 2010b) poly-lysine on tris-(2-ethylamino)amine with 3 aulfated cellobiose groups tof (sdhb) synthesized by sulfation of polylysinedendritic cellobiose (prepared from cellobiose and polylysine dendrimer generation 3) (han et al., 2010b) porphyrin with 8 lactose glycans tof synthesis by huisgen click cycloaddition of azide and alkyne (okada et al., 2009b) porphyrin with galactose or lactose maldi synthesis by click chemistry. activity on carcinogenic hep2 cells activators of natural killer lymphocytes (renaudet et al., 2010) ruthenium(ii) bipyridine with 6 or 18 mannose or galactose residues tof as probes to study lectin-carbohydrate interactions and to measure mono and oligo-saccharide concentrations electrochemically (kikkeri et al., 2010b) ruthenium porphyrins with 4 glucose residues tof water-soluble catalyst for carbenoid transfer reactions tetrabenzo-porphyrins with 4 glucose residues tof to improve targeting of cancer cells (ménard et al., 2009) tetraphenyl-ethylene with 4 lactose or sialyl-lactose residues tof/tof synthesis by click chemistry. as fluorescent probes for detection of influenza virus tetraphenyl-ethylene with 4 or 8 mannose residues tof (dhb) for fluorescence turn-on sensing of lectins based on aggregation-induced emission (sanji et al., 2010) dihydroxybenzamide base with 4 galabiose residues tof (chca) for isolation of pathogenic streptococcus suis bacteria (pera et al., 2010a) 1,3,5-tris-(2-propynyloxy)-benzene with 3 β-cyclodextrin rings tof (dhb) synthesis by microwave-assisted click chemistry as fluorescent tripod detection system for pesticides (mallard-favier et al., 2009) trimesic acid and others with 2, 3 or 4 phosphocholine residue related to glycol-sphingolipid from earthworm pheretima hilgendorfi tof for enhanced immune responses when compared to their monovalent counterparts various amino-alkyl with 2, 3 or 4 lactose residues tof (chca) synthesis from carbamate-linked lactose ) zinc(ii) phthalocyanines with 8 glc, gal or cellobiose residues tof (dhb) chemical synthesis as photosensitizer in photodynamic therapy (iqbal et al., 2009a ) zinc(ii) phthalocyanines with 8 glc, gal, cellobiose or maltose residues tof (dhb, chca) eight residues. for photodynamic therapy (iqbal et al., 2010 ) zinc(ii) naphthalocyanines with 4 glucose residues tof as photosensitizer in photodynamic therapy (iqbal et al., 2009b ) zinc(ii) naphthalocyanine with 8 glucose or galactose residues tof, esi ex post glycoconjugation (berthold et al., 2010) free amino group on lps used for site-specific conjugation. towards bivalent immunogens (grandjean et al., 2009) 4-amino-4-deoxy-larabinose (ara4n) maleimideactivated bsa potent immunogen (müller et al., 2010a) n-acyl-modified sialylated glycans hsa tof squaric acid chemistry. as inhibitors of adenoviruses causing epidemic keratoconjunctivitis biantennary gal, 2-keto-gal + fluorescent probe single-chain antibody tof method for drug targeting using antibodies galactose bsa tof new photoinduced thiol-ene coupling galactose hsa tof two step synthesis of gal 28 as optical imaging agent for peritoneal carcinomatosis (regino et al., 2010) ginsenoside rg3 bsa tof (sinapinic) generation and characterization of monoclonal antibody to ginsenoside rg3 (joo et al., 2009) α-glc, α-man, β-gal, αto study role on antigen in immune system interaction (tefsen et al., 2009) heparin tetra-saccharide complement factor h tof (sinapinic, chca) study of interaction between protein and tetrasaccharide by cross-linking (blaum et al., 2010) (hirata et al., 2010) α-mannosides (bi-and penta-) bsa tof immunogenicity and induction of candidacidal activity methyl glyoxal (advanced glycation end product, polysialic acid insulin maldi shown to increase insulin lifetime in vivo (bezuglov et al., 2009) serogroup 6 pneumococcal oligosaccharides bsa seldi-tof, fab synthetic carbohydrate conjugates express epitopes found in native capsular polysaccharides (parameswar et al., 2009) tn antigen hsa maldi to study effects of hapten density on induced antibody repertoire tumor-associated muc1 glycopeptides to study multivalent binding to human α-defensin (hd5) (lehrer et al., 2009) faims field asymmetric waveform ion mobility spectrometry fc fragment (crystallisable) region of igg fmoc 9-fluorenylmethoxycarbonyl fru fructose f-spe fluorous solid phase extraction ft fourier transform fuc fucose g0 (g1, g2) biantennary glycans with 0, (1 or 2) galactose residues g3ca coumaric 1,1,3,3,-tetra-methylguanidine gaba gamma-aminobutyric acid gags glycosaminoglycans gal galactose gala galacturonic acid galn galactosamine galnac n-acetylgalactosamine g3ca coumaric 1,1,3,3,-tetra-methylguanidine (liquid matrix) gapcs gallium-phthalocyanines gc/ms gas chromatography/mass spectrometry production of chitooligosaccharides and their potential applications in medicine synthesis of biotinylated sialoside to probe cd22-ligand interactions nanofibers formed through p…p stacking of the complexes of glucosyl-c2-salicyl-imine and phenylalanine: characterization by microscopy, modeling by molecular mechanics, and interaction by a-helical and b-sheet proteins bishydrazide glycoconjugates for lectin recognition and capture of bacterial pathogens the applicability of enzymes in cellulose ether analysis electron impact ion fragmentation pathways of peracetylated c-glycoside ketones derived from cyclic 1,3-diketones inclusion complexes of gcyclodextrin and carboxyl-modified g-cyclodextrin with c60: synthesis, characterization and controlled release application via microgels biochemical characterization of two xylanases from yeast pseudozyma hubeiensis producing only xylooligosaccharides synthesis of 4-o-glycosylated 1,5-anhydro-d-fructose and of 1,5-anhydro-d-tagatose from a common intermediate 2,3-o-isopropylidene-d-fructose solid-phase random glycosylation comparative solution and solid-phase glycosylations toward a disaccharide library mutational and functional analysis of large in a novel cho glycosylation mutant separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 mm sorbent depolymerization of sodium alginate under hydrothermal conditions cell wall b-(1,6)-glucan of saccharomyces cerevisiae. structural characterization and in situ synthesis new cyclodextrin dimers and trimers capable of forming supramolecular adducts with shape-specific ligands development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage reflection colour changes in cholesteric liquid crystals after the addition and photochemical isomerization of mesogenic azobenzenes tethered to sugar alcohols preparative enzymatic synthesis of polyprenyl-pyrophosphoryl-n-acetylglucosamine, an essential lipid intermediate for the biosynthesis of various bacterial cell envelope polymers plant cell wall proteomics: mass spectrometry data, a trove for research on protein structure/function relationships introducing capillary electrophoresis with laser-induced fluorescence detection (ce-lif) for the characterization of konjac glucomannan oligosaccharides and their in vitro fermentation behavior introducing capillary electrophoresis with laser-induced fluorescence (ce-lif) as a potential analysis and quantification tool for galactooligosaccharides extracted from complex food matrices nmr spectral mapping of lipid a molecular patterns affected by interaction with the innate immune receptor cd14 bioconjugation of d-glucuronic acid sodium salt to well-defined primary amine-containing homopolymers and block copolymers glycomic analysis of sialic acid linkages in glycans derived from blood serum glycoproteins chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated n-linked glycans: a potential methodology for cancerbiomarker discovery plasticity of xyloglucan composition in bean (phaseolus vulgaris)-cultured cells during habituation and dehabituation to lethal concentrations of dichlobenil comparison of the substrate specificities and catalytic properties of the sister n-acetylglucosaminyltransferases, gnt-vand gnt-vb (ix) glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides negative-ion maldi-qit-tofms n for structural determination of fucosylated and sialylated oligosaccharides labeled with a pyrene derivative structural determination by negative-ion maldi-qit-tofms n after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk derivatization with 1-pyrenyldiazomethane enhances ionization of glycopeptides but not peptides in matrix-assisted laser desorption/ionization mass spectrometry large-scale glycomics for discovering cancer-associated n-glycans by integrating glycoblotting and mass spectrometry a concise review of mass spectrometry imaging a glycomics approach to the discovery of potential cancer biomarkers determination of glycosylation sites and site-specific heterogeneity in glycoproteins glycomics and disease markers structural analysis of a fucoidan from the brown alga fucus evanescens by maldi-tof and tandem esi mass spectrometry structural analysis of a highly sulfated fucan from the brown alga laminaria cichorioides by tandem maldi and esi mass spectrometry n-glycan targeted gene delivery to the dendritic cell sign receptor oxazole-modified glycopeptides that target arthritis-associated class ii mhc aq and dr4 proteins glycocluster design for improved avidity and selectivity in blocking human lectin/plant toxin binding to glycoproteins and cells carbamate-linked lactose: design of clusters and evidence for selectivity to block binding of human lectins to (neo)glycoproteins with increasing degree of branching and to tumor cells maldi-tof/ms analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine structural characterization of cell wall polysaccharides from two plant species endemic to central africa, fleurya aestuans and phragmenthera capitata disease-associated glycosylated molecular variants of human c-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and indian visceral leishmaniasis the n domain of human angiotensin-i-converting enzyme: the role of n-glycosylation and the crystal structure in complex with an n domain-specific phosphinic inhibitor, rxp407 loss of effector function of human cytolytic t lymphocytes is accompanied by major alterations in n-and oglycosylation glycome informatics: methods and applications 5a-carba-glycopyranoside primers: potential building blocks for biocombinatorial synthesis of glycosphingolipid analogues identification, characterization and immunogenicity of an o-antigen capsular polysaccharide of francisella tularensis characterization of oligomeric xylan structures from corn fiber resistant to pretreatment and simultaneous saccharification and fermentation dense-shell glycodendrimers: uv/vis and electron, paramagnetic resonance study of metal ion complexation bioinformatics in glycomics: glycan characterization with mass spectrometric data using simglycan tm functional identification of the proteus mirabilis core lipopolysaccharide biosynthesis genes three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures lactosomes: structural and compositional classification of unique nanometer-sized protein lipid particles of human milk the pleurotus ostreatus hydrophobin vmh2 and its interaction with glucans comparison of the water-soluble carbohydrate composition and fructan structures of agave tequilana plants of different ages synthesis, conformation, and biological characterization of a sugar derivative of morphine that is a potent, long-lasting, and nontolerant antinociceptive modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies direct profiling of saccharides, organic acids and anthocyanins in fruits using electrospray droplet impact/secondary ion mass spectrometry syntheses of mucin-type o-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of bifidobacterium endo-a-nacetylgalactosaminidase recovery of water-soluble compounds from ganoderma lucidum by hydrothermal treatment binding and cellular activation studies reveal that toll-like receptor 2 can differentially recognize peptidoglycan from gram-positive and gram-negative bacteria grafting of aminated oligogalacturonans onto douglas fir barks. a new analysis of carbohydrates and glycoconjugates & route for the enhancement of their lead (ii) binding capacities defining criteria for oligomannose immunogens for hiv using icosahedral virus capsid scaffolds dendrimers designed for functions: from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine the red wine extract-induced activation of endothelial nitric oxide synthase is mediated by a great variety of polyphenolic compounds coloring carbohydrates: investigation of azulene derivatives as blue protecting groups differences in the sialylation patterns of membrane stress proteins in chemical carcinogen-induced tumors developed in balb/c and il-1a deficient mice mass spectrometry of n-linked glycans structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology structural characterization of non-reducing oligosaccharide produced by arthrobacter crystallopoietes n-08 fragmentation of deprotonated d-ribose and d-fructose in maldi-comparison with dissociative electron attachment multidimensional profiling of components in complex mixtures of natural products for metabolic analysis, proof of concept: application to quillaja saponins structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry probe design and synthesis of galb(1 ! 3)[neuaca(2 ! 6)]glcnacb(1 ! 2)man motif of n-glycan glycoprofiling bifidobacterial consumption of galacto-oligosaccharides by mass spectrometry reveals strain-specific, preferential consumption of glycans n-glycosylation of plant recombinant pharmaceuticals synthesis of thiourea-tethered cglycosyl amino acids via isothiocyanate-amine coupling permeate from cheese whey ultrafiltration is a source of milk oligosaccharides detecting glycan cancer biomarkers in serum samples using maldi ft-icr mass spectrometry data quantitative analysis of glycation sites on human serum albumin using 16 o/ 18 olabeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry characterization of wrss2 and wrss3, new second-generation virg(icsa)-based shigella sonnei vaccine candidates with the potential for reduced reactogenicity mass spectrometry in the characterization of human genetic n-glycosylation defects combined treatment of human mcf-7 breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays ex vivo assays of cem cells cultured and treated in the three dimensional cultures glycan analysis and influenza a virus infection of primary swine respiratory epithelial cells: the importance of neuaca2-6 glycans acceptor substrate discrimination in phosphatidyl-myo-inositol mannoside synthesis. structural and mutational analysis of mannosyl transferase corynebacterium glutamicum pimb synthesis and antimicrobial evaluation of amphiphilic neamine derivatives clicking pentafluorostyrene copolymers: synthesis, nanoprecipitation, and glycosylation trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and fc-fusion proteins chemical structure of bacteriovorax stolpii lipid a core chirality based tailoring of the liquid crystalline properties of supermolecular tetrapedes development of a nanolc ltq orbitrap mass spectrometric method for profiling glycans derived from plasma from healthy, benign tumor control, and epithelial ovarian cancer patients development of a robust and high throughput method for profiling n-linked glycans derived from plasma glycoproteins by nanolc-fticr mass spectrometry genomic and biochemical analysis of n-glycosylation in the mushroom-forming basidiomycete schizophyllum commune the structurally similar, penta-acylated lipopolysaccharides of porphyromonas gingivalis and bacteroides elicit strikingly different innate immune responses design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting ex post glycoconjugation of phthalocyanines modification of recombinant proteins by covalent polysialation illustrated with the example of human insulin influence of protein molecular mass on the glycation historical overview of glycoanalysis synthesis of differentially protected glucosamine building blocks and their evaluation as glycosylating agents de novo synthesis of differentially protected l-iduronic acid glycosylating agents chemical analysis of flavonoid constituents of the seagrass halophila stipulacea: first finding of malonylated derivatives in marine phanerogams substantial spatial flexibility and hydrogen bonding within the catalysis exerted by cyclodextrin artificial glycosidases lysine and arginine side chains in glycosaminoglycanprotein complexes investigated by nmr, cross-linking, and mass spectrometry: a case study of the factor h-heparin interaction glycomic analysis of n-linked carbohydrate epitopes from cd24 of mouse brain o-glycosylation pattern of cd24 from mouse brain a high-throughput oglycopeptide discovery platform for seromic profiling a high-throughput oglycopeptide discovery platform for seromic profiling-correction alteration of protein glycosylation in liver diseases investigating the molecular basis for the virulence of escherichia coli k5 by nuclear magnetic resonance analysis of the capsule polysaccharide monitoring of malting process by characterization of glycation of barley protein z determination of glycan structure from tandem mass spectra use of fullerene-, octadecyl-, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen site specific conjugation of fluoroprobes to the remodeled fc n-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection innovative approach for producing injectable, biodegradable materials using chitooligosaccharides and green chemistry reciprocal principle of molecular recognition in supramolecular chromatography-highly selective analytical separation of cyclodextrin congeners on a silica-bonded [60]fullerene stationary phase cation-independent mannose 6-phosphate receptor. a composite of distinct phosphomannosyl binding sites synthesis of lacdinac-terminated glycoconjugates by mutant galactosyltransferase-a way to new glycodrugs and materials new 4,4-difluoro-3a,4a-diaza-sindacene (bodipy)-labeled sphingolipids for membrane studies solid-phase synthesis of a pentavalent galnac-containing glycopeptide (tn antigen) representing the nephropathy-associated iga hinge region chiral-auxiliary-mediated 1,2-cis-glycosylations for the solid-supported synthesis of a biologically important branched a-glucan multimeric bivalent immunogens from recombinant tetanus toxin hc fragment, synthetic hexasaccharides, and a glycopeptide adjuvant the elucidation of the structure of thermotoga maritima peptidoglycan reveals two novel types of cross-link a m23b family metallopeptidase of helicobacter pylori required for cell shape, pole formation and virulence lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of hiv-1 ion exchange and purification of carbohydrates on a nafion(r) membrane as a new sample pretreatment for matrix-assisted laser desorption-ionization mass spectrometry chinese hamster ovary cells can produce galactose-a-1,3-galactose antigens on proteins characterization of atll, a bifunctional autolysin of staphylococcus lugdunensis with nacetylglucosaminidase and n-acetylmuramoyl-l-alanine amidase activities dimeric architecture of the hendra virus attachment glycoprotein: evidence for a conserved mode of assembly unusual molecular architecture of the machupo virus attachment glycoprotein synthesis of polymerizable cyclodextrin derivatives for use in adhesion-promoting monomer formulations physicochemical properties of microbial glycopolymers an analytical system for the characterization of highly heterogeneous mixtures of n-linked oligosaccharides site directed processing: role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type o-glycan core 2 high content phenotypic cell-based visual screen identifies mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling strategies for analysis of the glycosylation of proteins: current status and future perspectives characterization of irx10 and irx10-like reveals an essential role in glucuronoxylan biosynthesis in arabidopsis uv-maldi-tof mass spectrometry analysis of heparin oligosaccharides obtained by nitrous acid controlled degradation and high performance anion exchange chromatography distribution of o-glycosylhydrolases in marine fungi of the sea of japan and the sea of okhotsk: characterization of exocellular n-acetyl-b-d-glucosaminidase of the marine fungus penicillium canescens derivatization in mass spectrometry identification, characterization, and biosynthesis of a novel n-glycan modification in the fruiting body of the basidiomycete coprinopsis cinerea lipid remodelling of glycosylphosphatidylinositol (gpi) glycoconjugates in procyclic form trypanosomes: biosynthesis and processing of gpis revisited caenorhabditis elegans n-glycan core b-galactoside confers sensitivity towards nematotoxic fungal galectin cgl2 novel mannosidase inhibitors probe glycoprotein degradation pathways in cells chitosan oligosaccharides modulate the supramolecular conformation and the biological activity of oligogalacturonides in arabidopsis efficient synthesis of a 6-deoxytalose tetrasaccharide related to the antigenic o-polysaccharide produced by aggregatibacter actinomycetemcomitans serotype c aniline/a-cyano-4-hydroxycinnamic acid is a highly versatile ionic liquid for matrix-assisted laser desorption/ionization mass spectrometry 1h-pteridine-2,4-dione (lumazine): a new maldi matrix for complex (phospho)lipid mixtures analysis characterization of acp, a peptidoglycan hydrolase of clostridium perfringens with nacetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis glycobase and autogu: tools for hplc-based glycan analysis carrageenans: biological properties, chemical modifications and structural analysis-a review click" glycodendrimers containing 27, 81 and 243 modified xylopyranoside termini the plasma von willebrand factor o-glycome comprises a surprising variety of structures including abh antigens and disialosyl motifs analytical progress for protein glycosylation in china strategies for proteomic analysis of nonenzymatically glycated proteins preparation of saturated and unsaturated fatty acid hydrazides and long chain c-glycoside ketohydrazones glycoproteome study in myocardial lesions serum by integrated mass spectrometry approach: preliminary insights changes of serum-associated fucosylated glycoproteins and changes in glycosylation of iga in human cirrhosis efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones ferrocene-cyclodextrin conjugates: synthesis, supramolecular behavior, and use as electrochemical sensors ferrocene-carbohydrate conjugates as electrochemical probes for molecular recognition studies characterization of main anthocyanins extracted from pericarp blue corn by maldi-tof ms highly glycosylated human alpha interferon: an insight into a new therapeutic candidate the protease-associated domain and c-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (slsbt3) design and creativity in synthesis of multivalent neoglycoconjugates 3-deoxy-dmanno-octulosonic acid (kdo) hydrolase identified in francisella tularensis, helicobacter pylori, and legionella pneumophila synthesis and characterization of biotin chainend functionalized boronic acid-containing polymer (boropolymer) as functional glyco-affinity macroligand maldi mass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix das181 inhibits h5n1 influenza virus infection of human lung tissues synthesis, characterization, and self-assembled nanofibers of carbohydrate-functionalized mono-and di(2,2 0 :6 0 ,2 00 -terpyridinyl)arenes developmental regulation of oligosialylation in zebrafish glycan array on aluminum oxide-coated glass slides through phosphonate chemistry mannose receptor interacts with fc receptors and is critical for the development of crescentic glomerulonephritis in mice efficient synthesis of idraparinux, the anticoagulant pentasaccharide the efficient total synthesis of bisglycosyl apigenin from naringenin: a greener way a mixed cyclodextrinbiphenyl thermotropic liquid crystal: synthesis, liquid-crystalline properties, and supramolecular organization boronate affinity monolith for highly selective enrichment of glycopeptides and glycoproteins application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms) in preparation of chitosan oligosaccharides (cos) with degree of polymerization (dp) 5-12 containing well-distributed acetyl groups study of on-resin convergent synthesis of n-linked glycopeptides containing a large high mannose n-linked oligosaccharide facile synthesis of cyclopeptide-centered multivalent glycoclusters with 'click chemistry' and molecular recognition study by surface plasmon resonance glycosite analysis in glycoproteomics by mass spectrometry combination of matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry for the analysis of intact glycopeptides from horseradish peroxidase an introduction to sphingolipid metabolism and analysis by new technologies one-pipeline approach achieving glycoprotein identification and obtaining intact glycopeptide information by tandem mass spectrometry imaging maldi mass spectrometry of sphingolipids using an oscillating capillary nebulizer matrix application system single-crystalline euf3 hollow hexagonal microdisks: synthesis and application as a background-free matrix for maldi-tof-ms analysis of small molecules and polyethylene glycols selective desorption/ ionization of sulfatides by maldi-ms facilitated using 9-aminoacridine as matrix tethered derivatives of d-glucose and pentacyclic triterpenes for homo/heterobivalent inhibition of glycogen phosphorylase total synthesis of a furostan saponin, timosaponin bii subcompartment localization of the side chain xyloglucan-synthesizing enzymes within golgi stacks of tobacco suspension-cultured cells carbohydrates as recognition receptors in biosensing applications expression system for human glycosyltransferases and its application production, purification, and characterization of human a1 proteinase inhibitor from aspergillus niger direct amidation of aldoses and decarboxylative amidation of a-keto acids: an efficient conjugation method for unprotected carbohydrate molecules novel succinylated and large-sized osmoregulated periplasmic glucans of pseudomonas syringae pv. syringae chiral separation of hesperetin and hesperetin-o-glycoside in capillary electrophoresis using microbial b-1,2-glucans structural modification and characterization of rice starch treated by thermus aquaticus 4-a-glucanotransferase enzymatic biosynthesis of a puerarincycloamylose inclusion complex by 4-aglucanotransferase and maltogenic amylase analysis of mixture of maltooligoses using maldi-tofms: influence of cationizing agent types comparison of ionization behaviors of ring and linear carbohydrates in maldi-tofms challenges of determining o-glycopeptide heterogeneity: a fungal glucanase model system mutational analysis of bacillus megaterium qm b1551 cortex-lytic enzymes mass spectrometric imaging for biomedical tissue analysis synthesis of muc1 peptide and glycopeptide dendrimers synthesis and biological evaluation of multivalent carbohydrate ligands obtained by click assembly of pseudo-rotaxanes pseudomonas aeruginosa exploits lipid a and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection straightforward synthesis of novel akt inhibitors based on a glucose scaffold the role of sugar configuration in the acetolysis of 6-deoxyhexose methyl glycosides synthesis of a b-glcn-(1 ! 4)-murnac building block en route to n-deacetylated peptidoglycan fragments a urea-linked glucosamine dimer as a building block for the synthesis of linear and cyclic neosaccharides a simple and rapid method for the permethylation of carbohydrates trypanosoma brucei amp-activated kinase subunit homologs influence surface molecule expression porphyromonas gingivalis resistance to polymyxin b is determined by the lipid a 4 0 -phosphatase, pgn_0524 a new archaeal b-glycosidase from sulfolobus solfataricus. seeding a novel retaining b-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase cba2 b-glycosyl azides as substrates for a-glycosynthases: preparation of efficient a-l-fucosynthases synthetic heparan sulfate oligosaccharides inhibit endothelial cell functions essential for angiogenesis towards the synthesis of 1-deoxy-1-nitropiperidinoses synthesis and evaluation of s-and c(1)-substituted analogues of lincomycin localization and imaging of sialylated glycosphingolipids in brain tissue sections by maldi mass spectrometry an efficient modular approach for the assembly of s-linked glycopeptoids glycosidase inhibition with fullerene iminosugar balls: a dramatic multivalent effect modification of gastric mucin oligosaccharide expression in rhesus macaques after infection with helicobacter pylori identification of novel contributions to high-affinity glycoprotein-receptor interactions using engineered ligands synthesis and conformational analysis of a novel carbohydrate-fused bis-crown ether: crown-cyplos design, synthesis and characterisation of a fluorescently labelled cyplos ionophore application of liquid chromatography-tandem mass spectrometry for the characterization of galactosylated and tagatosylated b-lactoglobulin peptides derived from in vitro gastrointestinal digestion effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine b-lactoglobulin role of pyridoxamine in the formation of the amadori/heyns compounds and aggregates during the glycation of b-lactoglobulin with galactose and tagatose acceptor products of alternansucrase with gentiobiose. production of novel oligosaccharides for food and feed and elimination of bitterness glucosylation of raffinose via alternansucrase acceptor reactions investigating the candidacy of lps-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading investigating the candidacy of lps-based glycoconjugates to prevent invasive meningococcal disease: immunology of glycoconjugates with high carbohydrate loading towards a second generation of ionic liquid matrices (ilms) for maldi-ms of peptides, proteins, and carbohydrates sinorhizobium fredii hh103 cgs mutants are unable to nodulate determinate-and indeterminate nodule-forming legumes and overproduce an altered eps carbohydrate and domain architecture of an immature antibody glycoform exhibiting enhanced effector functions a human embryonic kidney 293t cell line mutated at the golgi a-mannosidase ii locus sieve-based device for maldi sample preparation. ii. instrumental parameterization a link between the assembly of flagella and lipooligosaccharide of the gram-negative bacterium campylobacter jejuni nonglycosidically linked pseudodisaccharides: thioethers, sulfoxides, sulfones, ethers, selenoethers, and their binding to lectins stable expression of a human-like sialylated recombinant thyrotropin in a chinese hamster ovary cell line expressing a2,6-sialyltransferase impaired lysosomal trimming of n-linked oligosaccharides leads to hyperglycosylation of native lysosomal proteins in mice with a-mannosidosis biochemical and immunocytological characterizations of arabidopsis thaliana pollen tube cell wall engineering of n. benthamiana l. plants for production of n-acetylgalactosamine-glycosylated proteins-towards development of a plant-based platform for production of protein therapeutics with mucin type o-glycosylation xylan-based nanoparticles: prodrugs for ibuprofen release towards unnatural xylan based polysaccharides: reductive amination as a tool to access highly engineered carbohydrates a simple micromethod for determining precise oligosaccharidic specificity of mannose-binding lectins functional and morphological adaptation to peptidoglycan precursor alteration in lactococcus lactis towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns molecular characterization of a putative sucrose: fructan 6-fructosyltransferase (6-sft) of the cold-resistant patagonian grass bromus pictus associated with fructan accumulation under low temperatures biomarkers and diagnosis of congenital disorders of glycosylation improved procedures for the selective chemical fragmentation of rhamnogalacturonans when can glycopeptides be assigned based solely on high-resolution mass spectrometry data? mmps4 promotes glycopeptidolipids biosynthesis and export in mycobacterium smegmatis glyco-spectrum-scan: fishing glycopeptides from ms spectra of protease digests of human colostrum siga common sialylated glycan in actinobacillus suis the major surface carbohydrates of the echinococcus granulosus cyst: mucin-type o-glycans decorated by novel galactosebased structures surface analytical characterization of carbohydrate microarrays shiga toxin receptor gb3cer/ cd77: tumor-association and promising therapeutic target in pancreas and colon cancer synthesis of some quaternary n-(1,4-anhydro-5-deoxy-d,l-ribitol-5-yl)ammonium salts a barley cellulose synthase-like cslh gene mediates (1,3;1,4)-b-d-glucan synthesis in transgenic arabidopsis analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics recombinant expression and characterization of n-acetylglucosaminyltransferase i derived from nicotiana tabacum comparison of the n-linked glycosylation of human b1,3-n-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae improved secretion of molecular chaperone-assisted human igg in silkworm, and no alterations in their n-linked glycan structures structure of glycosylated cu/znsuperoxide dismutase from kluyveromyces yeast nbimcc 1984 glycan structures and antiviral effect of the structural subunit rvh2 of rapana hemocyanin baca, an abc transporter involved in maintenance of chronic murine infections with mycobacterium tuberculosis a systematic nomenclature for carbohydrate fragmentations in fab-ms/ms spectra of glycoconjugates transformation of linear oligoketosides into macrocyclic neoglycoconjugates a new ligation strategy for peptide and protein glycosylation: photoinduced thiol-ene coupling graphene as a novel matrix for the analysis of small molecules by maldi-tof ms envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens clinical collection and protein properties of expressed prostatic secretions as a source for biomarkers of prostatic disease mycobacterium marinum mmar-2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan electrochemically controlled adsorption of fc-functionalized polymers on b-cd-modified selfassembled monolayers production of carbohydrate building blocks from red seaweed polysaccharides. efficient conversion of galactans into c-glycosyl aldehydes polysaccharide mimicry of the epitope of the broadly neutralizing anti-hiv antibody, 2g12, induces enhanced antibody responses to self oligomannose glycans glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies 355 nm multiphoton dissociation and ionization of 2,5-dihydroxyacetophenone lack of complex n-glycans on hiv-1 envelope glycoproteins preserves protein conformation and entry function prebiotic oligosaccharides: in vitro evidence for gastrointestinal epithelial transfer and immunomodulatory properties c-furyl glycosides, ii: synthesis and antimicrobial evaluation of c-furyl glycosides bearing pyrazolines, isoxazolines, and 5,6-dihydropyrimidine-2(1h)-thiones structural characterization of bordetella parapertussis lipid a polysaccharide pharmacokinetics: amphotericin b arabinogalactan conjugate -a drug delivery system or a new pharmaceutical entity hexosamine template. a platform for modulating gene expression and for sugar-based drug discovery combinatorial syntheses of trisaccharide libraries on a soluble polymeric support lipophilic thioglycosides for the solution-phase synthesis of oligosaccharides using biphasic liquid-liquid separation chiral and structural analysis of biomolecules using mass spectrometry and ion mobility-mass spectrometry fluorescent labeling of a carboxyl group of sialic acid for maldi-ms analysis of sialyloligosaccharides and ganglioside liquid chromatography combined with mass spectrometry for the investigation of endoglucanase selectivity on carboxymethyl cellulose investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques cellulosic graft copolymer: poly(methyl methacrylate) with cellulose side chains radially oriented cellulose triacetate chains on gold nanoparticles amphiphilic carbohydrate-phthalocyanine conjugates obtained by glycosylation or by azide-alkyne click reaction different and new nod factors produced by rhizobium tropici ciat899 following na þ stress identification of the polyketide synthase involved in the biosynthesis of the surface-exposed lipooligosaccharides in mycobacteria bioanalysis of recombinant proteins and antibodies by mass spectrometry lectin and carbohydrate microarrays: new high-throughput methods for glycoprotein, carbohydrate-binding protein and carbohydrate-active enzyme analysis chitosans as bioactive macromolecules to protect economically relevant crops from their main pathogens serum n-glycome biomarker for monitoring development of dena-induced hepatocellular carcinoma in rat secondary cell wall formation in cryptococcus neoformans as a rescue mechanism against acid-induced autolysis identification and quantification of protein posttranslational modifications matrixassisted laser desorption/ionization time-of-flight (maldi-tof) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides glycopeptidome of a heavily n-glycosylated cell surface glycoprotein of dictyostelium implicated in cell adhesion fragmentation behavior of amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with adp-ribose in tandem mass spectrometry effect of 1,3;1,6-b-d-glucan and the products of its enzymatic transformation on the formation of germs of buckwheat fagopyrum esculentum mönch synthesis of 3,6-branched arabinogalactan-type tetra-and hexasaccharides for characterization of monoclonal antibodies phosphoglucomutase of yersinia pestis is required for autoaggregation and polymyxin b resistance mass spectrometric investigation of molecular variability of grass pollen group 1 allergens cyclodextrin aldehydes are oxidase mimics simultaneous glycoproteomics on the basis of structure using ion mobility-mass spectrometry characterizing ion mobility-mass spectrometry conformation space for the analysis of complex biological samples effect of ionic liquids as additives in the catalytic properties of different immobilized preparations of rhizomucor miehei lipase in the hydrolysis of peracetylated lactal synthetic pseudopterosin analogues: a novel class of antiinflammatory drug candidates purification, characterization and in vivo studies of salmon heparin thieme chemistry journal awardees-where are they now? synthesis of the marine glycolipid dioctadecanoyl discoside thiyl glycosylation of olefinic proteins: s-linked glycoconjugate synthesis synthesis of the lewis b pentasaccharide and a hsa-conjugate thereof maldi mass spectrometry imaging, from its origins up to today: the state of the art detection of honeybee venom in envenomed tissues by direct maldi msi bioinformatics and molecular modeling in glycobiology expression of rat b(1,4)-n-acetylglucosaminyltransferase iii in nicotiana tabacum remodels the plantspecific n-glycosylation glycoprotein expression in human milk during lactation 1-deoxynojirimycins with dansyl capped n-substituents as probes for morbus gaucher affected cell lines new secoiridoid glucosides from ligustrum lucidum induce erk and creb phosphorylation in cultured cortical neurons application of maldi-tof mass spectrometry in lipidomics an update of maldi-tof mass spectrometry in lipid research maldi-tof-ms directly combined with tlc: a review of the current state isolation and structural analysis in vivo of newly synthesized fructooligosaccharides in onion bulbs tissues (allium cepa l.) during storage maldi mass spectrometry using 2,4,6-trihydroxyacetophenone and 2,4-dihydroxyacetophenone with cyclodextrins: suppression of matrix-related ions in low-molecular-weight region change in glycosylation pattern with extension of endoplasmic reticulum retention signal sequence of mouse antibody produced by suspensioncultured tobacco by2 cells chemical characterization of the oligosaccharides in bactrian camel (camelus bactrianus) milk and colostrum dendritic sugar-microarrays by click chemistry aggregation of alzheimer amyloid b peptide (1-42) on the multivalent sulfonated sugar interface scope and limitations of imidazolium-based ionic liquids as room temperature glycosylation promoters the carboxy-terminal erretention motif, sekdel, influences the n-linked glycosylation of recombinant human a-l-iduronidase but has little effect on enzyme activity in seeds of brassica napus and nicotiana tabacum mass spectrometric fragmentation analysis of oligosialic and polysialic acids quantification of nucleotide-activated sialic acids by a combination of reduction and fluorescent labeling synaptic cell adhesion molecule syncam 1 is a target for polysialylation in postnatal mouse brain comparative characterization of the arabidopsis subfamily a1 b-galactosidases preparation and gelling properties of sugar-contained low-molecular-mass gelators: combination of cholesterol and linear glucose generation of asparagine-linked glycan structure databases and their use the first total synthesis of 7-o-b-dglucopyranosyl-4 0 -o-a-l-rhamnopyranosyl apigenin via a hexanoyl ester-based protection strategy efficient synthesis of 4-amido-n 5 -acetyl-4-deoxyneuraminic acid and its application to the c-4 modification of sialic acids endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation lectin-directed enzyme activated prodrug therapy (leapt): synthesis and evaluation of rhamnosecapped prodrugs characterization of a new b(1-3)-glucan branching activity of aspergillus fumigatus enhanced production and partial characterization of an extracellular polysaccharide from newly isolated azotobacter sp. ssb81 phase diagrams of monoacylated amide-linked disaccharide glycolipids diamond, titanium dioxide, titanium silicon oxide, and barium strontium titanium oxide nanoparticles as matrixes for direct matrix-assisted laser desorption/ionization mass spectrometry analysis of carbohydrates in plant tissues synthesis of a tetrasaccharide corresponding to the teichoic acid from the cell wall of streptomyces sp. vkm ac-2275 synthesis of the hexasaccharide repeating unit corresponding to the cell wall lipopolysaccharide of azospirillum irakense kbc1 sialic acids in different leishmania sp., its correlation with nitric oxide resistance and host responses 9-o-acetylated sialic acids enhance entry of virulent leishmania donovani promastigotes into macrophages high throughput quantification of n-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-offlight mass spectrometry gdp-d-mannose 3,5-epimerase (gme) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynthesis in tomato an hplc-maldi ms method for n-glycan analyses using smaller size samples: application to monitor glycan modulation by medium conditions towards a reliable molecular mass determination of intact glycoproteins by matrixassisted laser desorption/ionization time-of-flight mass spectrometry ionic liquid matrices for maldi-tof-ms analysis of intact glycoproteins synthesis of pegylated lactose analogs for inhibition studies on t. cruzi trans-sialidase glycosylation site-specific analysis of clade c hiv-1 envelope proteins enzymatic/chemical release of o-glycans allowing ms analysis at high sensitivity glycomic profiling of invasive and non-invasive breast cancer cells assessment of chemoselective neoglycosylation methods using chlorambucil as a model glycan family analysis for deducing n-glycan topology from single ms detection of hepatocellular carcinoma using glycomic analysis discovery of rifampicin as a new anti-glycating compound by matrix-assisted laser desorption/ionization mass spectrometry-based insulin glycation assay first total synthesis of 1,2-dipalmitoyl-3-(n-palmitoyl-6 0 -amino-6 0 -deoxy-a-dglucosyl)-sn-glycerol-a glycoglycerolipid of a marine alga with a high inhibitor activity against human myt1-kinase acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family ferroplasmaceae isolated from a hydrothermal pool, and transfer of ferroplasma cupricumulans to acidiplasma cupricumulans comb. nov synthesis of polyhydroxy [n]-polyurethanes derived from a carbohydrate precursor characterization of n-linked glycosylation on recombinant glycoproteins produced in pichia pastoris using esi-ms and maldi-tof dna-templated homo-and heterodimerization of peptide nucleic acid encoded oligosaccharides that mimick the carbohydrate epitope of hiv human macrophage activation triggered by chitotriosidase-mediated chitin and chitosan degradation evaluation of conditions for release of mucin-type oligosaccharides from glycoproteins by hydrazine gas treatment imaging mass spectrometry of glycolipids visualization of spatial distribution of g-aminobutyric acid in eggplant (solanum melongena) by matrix-assisted laser desorption/ionization imaging mass spectrometry the specific localization of seminolipid molecular species on mouse testis during testicular maturation revealed by imaging mass spectrometry the detection of glycosphingolipids in brain tissue sections by imaging mass spectrometry using gold nanoparticles practical heavy fluorous tag for carbohydrate synthesis with minimal chromatographic purification production and nglycosylation of recombinant human cell adhesion molecule l1 from insect cells using the stable expression system. effect of dimethyl sulfoxide glyco-sams by 'dual click': thiourea-bridged glyco-oeg azides for cycloaddition on surfaces mass spectrometry in the elucidation of the glycoproteome of bacterial pathogens investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of vibrio cholerae o1, serotype inaba the synthesis and immune stimulating action of mannose-capped lysine-based dendrimers some patterns in dimer ii formation in bodipy-fl-labeled lipids bodipy-labeled ganglioside probes for membrane and biological studies development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides analytical approaches towards the structural characterization of microbial wall glycopolymers differential effect of plasma or erythrocyte age-ligands of rage on expression of transcripts for receptor isoforms biosynthesis of a new udpsugar, udp-2-acetamido-2-deoxyxylose, in the human pathogen bacillus cereus subspecies cytotoxis nvh 391-98 effective enlargement of fluorescence resonance energy transfer of poly-porphyrin mediated by b-cyclodextrin dimers total synthesis of the heptasaccharide repeating unit of the iron-binding exopolysaccharide secreted by klebsiella oxytoca bas-10 mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry oxidative modifications in glycated insulin new insights into the early steps of phosphatidylinositol mannoside biosynthesis in mycobacteria. pimb' is an essential enzyme of mycobacterium smegmatis fatty acyl structures of mycobacterium tuberculosis sulfoglycolipid govern t cell response automated measurement of permethylated serum n-glycans by maldi-linear ion trap mass spectrometry monoterpenoid glucoindole alkaloids and iridoids from pterocephalus pinardii production, refining, structural characterization and fermentability of rice husk xylooligosaccharides oligo mass profiling (olimp) of extracellular polysaccharides facile synthesis of three bidesmosidic oleanolic acid saponins with strong inhibitory activity on pancreatic lipase sortase-catalyzed peptide-glycosylphosphatidylinositol analogue ligation one-pot synthesis of cyclic antifreeze glycopeptides syntheses of glycoclusters containing a phosphocholine residue related to a glycosphingolipid from the earthworm pheretima hilgendorfi establishment of a real-time analytical method for free oligosaccharide transport from the er to the cytosol purification, characterization and molecular cloning of a novel endo-b-n-acetylglucosaminidase from the basidiomycete, flammulina velutipes comparative glycomic profiling in esophageal adenocarcinoma derivatization and analysis of oligosaccharides by matrix-assisted laser desorption/ionization time of-flight mass spectrometry sulfated oligosaccharide cluster with polylysine core scaffold as a new anti-hiv dendrimer lipophilic b-cyclodextrin cyclic-nitrone conjugate: synthesis and spin trapping studies sialylation using n-glycolylneuraminyl phosphite donors to synthesize neu5gc-containing glycans chemical de-oglycosylation of glycoproteins for application in lc-based proteomics secondary acylation of vibrio cholerae lipopolysaccharide requires phosphorylation of kdo rapid characterization of n-linked glycans from secreted and gel-purified monoclonal antibodies using maldi-tof mass spectrometry synthesis of porphyrin-carbohydrate conjugates using "click" chemistry and their preliminary evaluation in human hep2 cells an improved protocol for n-glycosylation analysis of gel-separated sialylated glycoproteins by maldi-tof/ tof artificial polymerases and molecular chaperones interaction of nectarin 4 with a fungal protein triggers a microbial surveillance and defense mechanism in nectar n-glycans on the link domain of human hare/stabilin-2 are needed for hyaluronan binding to purified ecto-domain, but not for cellular endocytosis of hyaluronan analytical characterization of monoclonal antibodies: linking structure to function identification of rhodococcus equi lipids recognized by host cytotoxic t lymphocytes matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period 1999-2000 analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities, and biological applications analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for the period analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for identification of by-products formed during the release of n-glycans with protein n-glycosidase f in the presence of dithiothreitol application of negative ion ms/ ms to the identification of n-glycans released from carcinoembryonic antigen cell adhesion molecule 1 (ceacam1) structural and quantitative analysis of n-linked glycans by maldi and negative ion nanospray mass spectrometry proposal for a standard system for drawing structural diagrams of n-and o-linked carbohydrates and related compounds fragmentation of negative ions from n-linked carbohydrates, part 4. fragmentation of complex glycans lacking substitution on the 6-antenna identification of high-mannose and multiantennary complextype n-linked glycans containing a-galactose epitopes from nurse shark igm heavy chain o-glycoside biomarker of apolipoprotein c3: responsiveness to obesity, bariatric surgery, and therapy with metformin, to chronic or severe liver disease and to mortality in severe sepsis and graft vs host disease pyridylamination as a means of analyzing complex sugar chains switching the selectivity of a polyglycerol dendrimer monomolecularly imprinted with d-(à)-fructose kegg glycan for integrated analysis of pathways, genes and glycan structures sphingolipidomics: methods for the comprehensive analysis of sphingolipids antifreeze glycopeptide analogues: microwaveenhanced synthesis and functional studies degradation of chitosans with a family 46 chitosanase from streptomyces coelicolor a3(2) glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage rational engineering of mannosyl binding in the distal glycone subsites of cellulomonas fimi endo-b-1,4-mannanase: mannosyl binding promoted at subsite -2 and demoted at subsite -3 uv-laser ablation of ionic liquid matrices structure analysis of n-glycoproteins toward synthesis of the isosteric sulfonate analogues of the at-iii binding domain of heparin plant immunity induced by oligogalacturonides alters root growth in a process involving flavonoid accumulation in arabidopsis thaliana conversion of a-amyrin into centellosides by plant cell cultures of centella asiatica carbohydrate analysis throughout the development of a protein therapeutic positive and negative analyte ion yield in matrix-assisted laser desorption/ionization revisited g-cyclodextrins possessing an azido group and a triisopropylbenzenesulfonyl group as useful synthetic and authentic intermediates for unsymmetrically functionalized derivatives hemicellulase production in chrysosporium lucknowense c1 genetically engineered mannosylated-human serum albumin as a versatile carrier for liverselective therapeutics free oligosaccharides to monitor glycoprotein endoplasmic reticulumassociated degradation in saccharomyces cerevisiae chemical synthesis, folding, and structural insights into o-fucosylated epidermal growth factor-like repeat 12 of mouse notch-1 receptor a water-soluble ruthenium glycosylated porphyrin catalyst for carbenoid transfer reactions in aqueous media with applications in bioconjugation reactions synthesis and self-assembled nanostructures of novel chiral amphiphilic liquid crystals containing b-d-galactopyranoside end-groups synthetic antitumor vaccines from tetanus toxoid conjugates of muc1 glycopeptides with the thomsen-friedenreich antigen and a fluorine-substituted analogue matrix-free uv-laser desorption/ionization (ldi) mass spectrometric imaging at the singlecell level: distribution of secondary metabolites of arabidopsis thaliana and hypericum species core region and lipid a components of lipopolysaccharides matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms) application in carbohydrate analysis glycoproteomic analysis of wgabound glycoprotein biomarkers in sera from patients with lung adenocarcinoma synthesis, characterization and in vitro pharmacology of novel pregabalin derivatives an efficient approach to the discovery of potent inhibitors against glycosyltransferases electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities, and biological applications design, synthesis, and immunochemical characterization of a chimeric glycopeptide corresponding to the shigella flexneri y opolysaccharide and its peptide mimic mdwnmhaa antiproliferative cardenolide glycosides of elaeodendron alluaudianum from the madagascar rainforest the influence of microwave irradiation on stereospecific mo(vi)-catalyzed transformation of deoxysugars a new approach to amadori ketoses via mo vicatalyzed stereospecific isomerization of 2-c-branched sugars bearing azido function in a microwave field substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase hvxet6 from barley (hordeum vulgare l.) determination of n-glycosylation site and glycan structures of pectin methylesterase in jelly fig (ficus awkeotsang) achenes the composition of polysaccharides in primary walls of litchi chinensis sonn xyloglucans of monocotyledons have diverse structures the effect of acid dextrinisation on enzyme-resistant starch content in extruded maize starch new oligosaccharides prepared by acid hydrolysis of the polysaccharides from nerium indicum mill and their anti-angiogenesis activities lectin microarray widely applicable deprotection method of 2,2,2-trichloroethoxycarbonyl (troc) group using tetrabutylammonium fluoride hydrolytically stable bioactive synthetic glycopeptide homo-and copolymers by combination of nca polymerization and click reaction chemoenzymatic synthesis and lectin array characterization of a class of n-glycan clusters expeditious chemoenzymatic synthesis of cd52 glycopeptide antigens arthrobacter endo-b-n-acetylglucosaminidase shows transglycosylation activity on complex-type n-glycan oxazolines: one-pot conversion of ribonuclease b to sialylated ribonuclease c glycosynthases enable a highly efficient chemoenzymatic synthesis of n-glycoproteins carrying intact natural n-glycans synthesis of oleanolic acid saponins mimicking components of chinese folk medicine di wu lipid-a software tool for automated assignment of lipids in mass spectra a lipid profile typifies the beijing strains of mycobacterium tuberculosis. identification of a mutation responsible for a modification of the structures of phthiocerol dimycocerosates and phenolic glycolipids igg glycosylation analysis supramolecular assembly of carbohydrate-functionalized salphen-metal complexes synthesis and application of epoxy starch derivatives an echinococcus multilocularis coproantigen is a surface glycoprotein with unique o-gycosylation glycoproteomics in neurodegenerative diseases structural elucidation of a novel b. cenocepacia et-12 lipooligosaccharide isolated from a cystic fibrosis patient after lung transplantation against the rules: a marine bacterium, loktanella rosea, possesses a unique lipopolysaccharide first structural characterization of burkholderia vietnamiensis lipooligosaccharide from cystic fibrosis-associated lung transplantation strains fucosylation of chitooligosaccharides by human a1,6-fucosyltransferase requires a nonreducing terminal chitotriose unit as a minimal structure c-mannosylated peptides derived from the thrombospondin type 1 repeat interact with hsc70 to modulate its signaling in raw264.7 cells two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (adcc) efficacy of non-fucosylated therapeutic antibodies in human blood production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons homotropic cooperativity of cyclodextrin dimer as an artificial hydrolase glycomimicry: display of the gm3 sugar epitope on escherichia coli and salmonella enterica sv typhimurium enzymatic conversion of diacetylated analysis of carbohydrates and glycoconjugates & sophoroselipid into acetylated glucoselipid: surface-active properties of novel bolaform biosurfactants preparation and conformational analysis of c-glycosyl b 2 -and b/b 2 -peptides the multiplicity of n-glycan structures of novine milk 18 kda lactophorin (milk glycam-1) synthesis of an octasubstituted galactose zinc(ii) phthalocyanine anomerically glycosylated zinc(ii) naphthalocyanines synthesis of octaglycosylated zinc(ii) phthalocyanines. synthesis sesquiterpene coumarins from ferula gumosa transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of mycoplasma fermentans enzymatic synthesis of mycoplasma fermentans specific glycoglycerophospholipid from 1,2-dipalmitoylglycerol isolation and structural determination of reducing fructooligosaccharides newly produced in stored edible burdock antiallergic potential of oligomannose-coated liposome-entrapped cry j 1 as immunotherapy for japanese cedar pollinosis in mice enzymatic activity and substrate specifcity of recombinant tomato b-galactosidases 4 and 5 development of highly efficient and stereocontrolled o-glycosylation methodologies and its application to the construction of bacterial glycans effects of frozen conditions on stereoselectivity and velocity of o-glycosylation reactions specificity analysis of lectins and antibodies using remodeled glycoproteins production, purification and structural characterization of an exopolysaccharide produced by a probiotic lactobacillus plantarum mtcc 9510 in vitro and in vivo enzymatic syntheses and mass spectrometric database for nglycans and o-glycans strategy for glycoproteomics: identification of glyco-alteration using multiple glycan profiling tools enrichment strategies for glycopeptides photooxidative mineralization of microorganisms-produced glycolipid biosurfactants by a titania-mediated advanced oxidation process highly oriented recombinant glycosyltransferases: sitespecific immobilization of unstable membrane proteins by using staphylococcus aureus sortase a a first total synthesis of ganglioside hlg-2 identification of a glycosylphosphatidylinositol anchormodifying b1-3 n-acetylglucosaminyl transferase in trypanosoma brucei direct profiling of tissue lipids by maldi-tofms glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the o-ps of vibrio cholerae o1, serotype ogawa, and bsa revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry contribution of porphyromonas gingivalis lipopolysaccharide to periodontitis synthesis of new sulfonic acid-containing oligosaccharide mimetics of sialyl lewis a synthesis, regioselective hydrogenolysis, partial hydrogenation, and conformational study of dioxane and dioxolanetype (9 0 -anthracenyl)methylene acetals of sugars mass spectrometric quantification of neutral and sialylated n-glycans from a recombinant therapeutic glycoprotein produced in the two chinese hamster ovary cell lines method development for direct detection of glycoproteins on aminophenylboronic acid functionalized self-assembled monolayers by matrix-assisted laser desorption/ionization mass spectrometry antibody glycans characterization structural characterization of antibodies by mass spectrometry mucin-lectin interactions assessed by flow cytometry mucin-type o-glycosylation-putting the pieces together carboxymethylated cyclosophoraose as a novel chiral additive for the stereoisomeric separation of some flavonoids by capillary electrophoresis characterization of the streptococcus pneumoniae bgac protein as a novel surface b-galactosidase with specific hydrolysis activity for the galb1-3glcnac moiety of oligosaccharides characterization of n-linked protein glycosylation in helicobacter pullorum a strategy for precise and large scale identification of core fucosylated glycoproteins self-assembly of amphiphilic perylenecyclodextrin conjugate and vapor sensing for organic amines structures of chitosan and chitooligosaccharides and their adsorption toward radionuclide uranium a high-throughput purification of monoclonal antibodies from glycoengineered pichia pastoris a bifunctional enzyme in a single gene catalyzes the incorporation of glcn into the aeromonas core lipopolysaccharide genetics and proteomics of aeromonas salmonicida lipopolysaccharide core biosynthesis selective monitoring of rutin and quercetin based on a novel multi-wall carbon nanotube-coated glassy carbon electrode modified with microbial carbohydrates a-cyclosophorohexadecaose and succinoglycan monomer m3 utilizing the o-antigen lipopolysaccharide biosynthesis pathway in escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach design, synthesis, and evaluation of n-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis natural phosphoryl and acyl variants of lipid a from neisseria meningitidis strain 89i differentially induce tumor necrosis factor-a in human monocytes profiles of structural heterogeneity in native lipooligosaccharides of neisseria and cytokine induction golgi targeting of drosophila melanogaster b4galnactb requires a dhhc protein family-related protein as a pilot strong igg antibody responses to borrelia burgdorferi glycolipids in patients with lyme arthritis, a late manifestation of the infection generation and characterization of monoclonal antibody to ginsenoside rg3 a novel, simple and sensitive ligand affinity capture method for detecting molecular interactions by maldi mass spectrometry glycoviewer: a tool for visual summary and comparative analysis of the glycome mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from deinococcus geothermalis and neisseria polysaccharea direct analysis of cellulose in poplar stem by matrix-assisted laser desorption/ionization imaging mass spectrometry superior in vivo efficacy of afucosylated trastuzumab in the treatment of her2-amplified breast cancer benzyl ethers as nucleophiles: from hydroxy cyclooctanes toward bridged c-furanosides multifunctional zro 2 nanoparticles and zro 2 -sio 2 nanorods for improved maldi-ms analysis of cyclodextrins, peptides, and phosphoproteins a synthetic vaccine consisting of a tumor-associated sialyl-tn-muc1 tandem-repeat glycopeptide and tetanus toxoid: induction of a strong and highly selective immune response fully synthetic vaccines consisting of tumor-associated muc1 glycopeptides and a lipopeptide ligand of the toll-like receptor 2 ghrelin-like peptide with fatty acid modification and o-glycosylation in the red stingray, dasyatis akajei synthesis of glycopeptides reactivity of rare sugar d-allose during glycation of human serum albumin arabidopsis thaliana alg3 mutant synthesizes immature oligosaccharides in the er and accumulates unique n-glycans two arabidopsis thaliana golgi a-mannosidase i enzymes are responsible for plant n-glycan maturation a sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid a selfpoisoning of mycobacterium tuberculosis by targeting glge in an aglucan pathway definitive evidence that a single n-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding aglj adds the first sugar of the nlinked pentasaccharide decorating the haloferax volcanii s-layer glycoprotein aba-and bab-triblock cooligomers of tri-o-methylated and unmodified cello-oligosaccharides: syntheses and structure-solubility relationship lectin-based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation: application to streptococcus pyogenes synthesis and characterization of hydroquinone fructoside using leuconostoc mesenteroides levansucrase high-throughput solid-phase permethylation of glycans prior to mass spectrometry solid-phase permethylation of glycans for mass spectrometric analysis antibacterial activity of a disaccharide isolated from streptomyces sp. strain jj45 against xanthomonas sp insight into the regulation of glycan synthesis in drosophila chaoptin based on mass spectrometry a tandem mass spectrometric approach to the identification of o-glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in pichia pastoris mass spectrometric analysis of carbohydrates labeled with a biotinylated tag localization of the attachment site of oligoglucans to mesorhizobium loti hambi 1148 murein synthesis of a heptasaccharide fragment of the mannan from candida guilliermondii cell wall and its conjugate with bsa synthesis of 3,6-branched oligomannoside fragments of the mannan from candida albicans cell wall corresponding to the antigenic factor 4 reduction of n-linked xylose and fucose by expression of rat b1,4-n-acetylglucosaminyltransferase iii in tobacco by-2 cells depends on golgi enzyme localization domain and genetic elements used for expression a small-scale method for the preparation of plant n-linked glycans from soluble proteins for analysis by maldi-tof mass spectrometry analyses of biologically active steroids: antitumor active osw-1 and cardiotonic marinobufotoxin, by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry structural analogues of diosgenyl saponins: synthesis and anticancer activity pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction chemical synthesis of mouse pro-opiomelanocortin(1-74) by azido-protected glycopeptide ligation via the thioester method correct disulfide pairing is required for the biological activity of crustacean androgenic gland hormone (agh): synthetic studies of agh loss of udp-galnac: polypeptide n-acetylgalactosaminyltransferase 3 and reduced oglycosylation in colon carcinoma cells selected for hepatic metastasis development of tetraphenylethylene-based fluorescent oligosaccharide probes for detection of influenza virus further structural study of the xyloglucanase-derived eggplant xyloglucan oligo-saccharides initiation of methylglucose lipopolysaccharide biosynthesis in mycobacteria synthesis of a chito-tetrasaccharide b-1,4-glcnac-b-1,4-glcn repeating unit a novel polycondensation method for the synthesis of a two-faced b-1,4-glucan solid-phase synthesis of o-sulfated glycopeptide by the benzylprotected glycan strategy salmonella enterica serovar typhimurium lipopolysaccharide deacylation enhances its intracellular growth within macrophages influence of crystalline forms of titania on desorption/ionization efficiency in titania-based surfaceassisted laser desorption/ionization mass spectrometry alternative procedures for analysis of lipid a modification with phosphoethanolamine or aminoarabinose recognition of endogenous ligands by ctype lectins: interaction of serum mannan-binding protein with tumorassociated oligosaccharide epitopes highly fucosylated n-glycan ligands for mannan-binding protein expressed specifically on cd26 (dppvi) isolated from a human colorectal carcinoma cell line structural analysis of two trisaccharides isolated from fermented beverage of plant extract phosphorylase-catalyzed n-formyl-a-glucosaminylation of maltooligosaccharides gold-catalyzed glycosidations: unusual cleavage of the interglycosidic bond while studying the armed/ disarmed effect of propargyl glycosides dendrimers of vaccines consisting of tumor-associated glycopeptide antigens and t cell epitope peptides bacillus anthracis surfacelayer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csab and tago sialic acids acquired by pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated hostcell recognition mass spectrometric analysis of sulfated n-and oglycans steroidal monoglycosides from the far eastern starfish hippasteria kurilensis and hypothetic pathways of polyhydroxysteroid biosynthesis in starfish two new steroid glycosides from the far east starfish hippasteria kurilensis lectin biosensing using digital analysis of ru(ii)-glycodendrimers ru(ii) glycodendrimers as probes to study lectin-carbohydrate interactions and electrochemically measure monosaccharide and oligosaccharide concentrations enzymatic synthesis and characterization of hydroquinone galactoside using kluyveromyces lactis lactase click synthesis of estradiol-cyclodextrin conjugates as cell compartment selective estrogens grandidentatin isomer from bark of suwon poplar (populus alba l. â populus glandulosa uyeki) biotransformation of mulberroside a from morus alba results in enhancement of tyrosinase inhibition glycosylation pattern of humanized igg-like bispecific antibody produced by recombinant cho cells mass spectrometric analysis of the glycosphingolipid-derived glycans from miniature pig endothelial cells and islets: identification of neugc epitope in pig islets rapid and high-throughput analysis of n-glycans from ovarian cancer serum using a 96-well plate platform identification of a-gal and non-gal epitopes in pig corneal endothelial cells and keratocytes by using mass spectrometry qualitative and quantitative comparison of n-glycans between pig endothelial and islet cells by highperformance liquid chromatography and mass spectrometry-based strategy construction of a fusion enzyme of dextransucrase and dextranase: application for onestep synthesis of isomalto-oligosaccharides enzymatic synthesis of alkyl glucosides using leuconostoc mesenteroides dextransucrase alteration in the glycan pattern of pilin in a nonmotile mutant of synechocystis sp. pcc 6803 suppression of b-n-acetylglucosaminidase in the n-glycosylation pathway for complex glycoprotein formation in drosophila s2 cells carbohydrate moieties contribute significantly to the physicochemical properties of french bean 7s globulin phaseolin structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatographymatrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion phenylethyl glycosides from digitalis lanata acceptor and substrate specificity of b-glucuronidase with transglycosylation activity from aspergillus niger kinetic study on the binding of lectin to mannose residues in a polymer brush high molecular weight polyglycerol-based multivalent mannose conjugates 0 -methyl and 1 0 -xylosyl derivatives of 2 0 -hydroxyflexixanthin are major carotenoids of hymenobacter species modifications of human total serum n-glycome during liver fibrosis-cirrhosis, is it all about immunoglobulins? immunoglobulins are the major glycoproteins involved in the modifications of total serum n-glycome in cirrhotic patients enrichment of o-glcnac modified proteins by the periodate oxidation-hydrazide resin capture approach surface grafting of cellulose nanocrystals with poly(ethylene oxide) in aqueous media a quantitative model of ultraviolet matrix-assisted laser desorption/ionization a quantitative model of ultraviolet matrix-assisted laser desorption/ionization including analyte ion generation a bipolar rate equation model of maldi primary and secondary ionization processes, with application to positive/ negative analyte ion ratios and suppression effects electrospray and maldi mass spectrometry: fundamentals, instrumemtation, practialities and biological applications molecular dynamics simulations of maldi: laser fluence and pulse width dependence of plume characteristics and consequences for matrix and analyte ionization enzymatic polymer synthesis: an opportunity for green polymer chemistry synthetic studies on the carbohydrate moiety of the antigen from the parasite echinococcus multilocularis mass spectrometry for the analysis of milk oligosaccharides amphiphilic block copolymers based on cyclodextrin host-guest complexes via raftpolymerization in aqueous solution synthesis and conformational analysis of (a-d-galactosyl)phenylmethane and a-,b-difluoromethane analogues: interactions with the plant lectin viscumin structures of aldouronic acids liberated from kenaf xylan by endoxylanases from streptomyces sp study on systematizing the synthesis of the a-series ganglioside glycans gt1a, gd1a, and gm1 using the newly developed n-troc-protected gm3 and galn intermediates characterization of sialylated and fucosylated glycopeptides of b2-glycoprotein i by a combination of hilic lc and maldi ms/ms glycopeptide profiling of beta-2-glycoprotein i by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome application of deep sea yeast-production of biosurfactants biosurfactant-producing yeast isolated from calyptogena soyoae (deep-sea cold-seep clam) in the deep sea an arginyl residue in rice udp-arabinopyranose mutase is required for catalytic activity and autoglycosylation structural identification of the main ellagitannins of a boysenberry (rubus loganbaccus â baileyanus britt.) extract by lc-esi-ms/ms, maldi-tof-ms and nmr spectroscopy quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis block synthesis of blood group tetrasaccharides b (types 1, 3 and 4) advances in the separation, sensitive detection, and characterization of heparin and heparan sulfate glucosinolateaccumulating s-cells in arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts synthesis of a neoglycoconjugate containing a chlamydophila psittaci-specific branched kdo trisaccharide epitope the mita gene of aspergillus fumigatus is required for mannosylation of inositol-phosphorylceramide, but is dispensable for pathogenicity sulfated oligosaccharides: new targets for drug development stability of 5 (6)-carboxyfluorescein in microwave-assisted synthesis of fluoresceinlabelled o-dimannosylated peptides triterpene glycosides from agrostemma gracilis porphyrin-cyclodextrin conjugates as a nanosystem for versatile drug delivery and multimodal cancer therapy a mathematical model of n-linked glycosylation a mathematical model to derive n-glycan structures and cellular enzyme activities from mass spectrometric data glycosyl azides: novel substrates for enzymatic transglycosylations multidimensional system enabling deglycosylation of proteins using a capillary reactor with peptide-nglycosidase f immobilized on a porous polymer monolith and hydrophilic interaction liquid chromatography-mass spectrometry of glycans glycomic analysis: an array of technologies synthesis of an fmoc-threonine bearing core-2 glycan: a building block for psgl-1 via fmoc-assisted solid-phase peptide synthesis the development of retrosynthetic glycan libraries to profile and classify the human serum n-linked glycome human serum processing and analysis methods for rapid and reproducible n-glycan mass profiling shigella sonnei oligosaccharide-protein conjugates immunochemical studies of shigella flexneri 2a and 6, and shigella dysenteriae type 1 o-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates a,a-trehalose-based polyacetals and macrocyclic acetals application of spectroscopic methods for structural analysis of chitin and chitosan the molecular basis of inhibition of golgi a-mannosidase ii by mannostatin a glycomics and proteomics analyses of mouse uterine luminal fluid revealed a predominance of lewis y and x epitopes on specific protein carriers productionof sophorolipid biosurfactants by multiple species of the starmerella (candida) bombicola yeast clade four new triterpenes from anchusa azurea var. azurea sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates dethreading of deoxyribonucleotides through a-cyclodextrin efficient guest inclusion by b-cyclodextrin attached to the ends of dna oligomers upon hybridization to various dna conjugates determination of sialic acid and gangliosides in biological samples and dairy products: a review solid-phase thermodynamic interpretation of ion desorption in matrix-assisted laser desorption/ionization controlled release of 2,3-desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting e-selectin deletion of udp-glucose pyrophosphorylase reveals a udp-glucose independent udp-galactose salvage pathway in leishmania major imaging of lipids in spinal cord using intermediate pressure matrix-assisted laser desorption-linear ion trap/orbitrap ms utilization of the linear mode of maldi-tof mass spectrometry in the study of glycation during the malting process profiling of n-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry matrix-assisted laser desorption/ ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of n-linked oligosaccharides from ovalbumin method for investigation of oligosaccharides using phenylhydrazine derivatization n-glycomic changes in human breast carcinoma mcf-7 and t-lymphoblastoid cells after treatment with herceptin and herceptin/lipoplex mass spectrometric study of n-glycans from serum of woodchucks with liver cancer chemical characterization, antiproliferative and antiadhesive properties of polysaccharides extracted from pleurotus pulmonarius mycelium and fruiting bodies polymerizable vancomycin derivatives for bactericidal biomaterial surface modification: structure-function evaluation the chap domain of cse functions as an endopeptidase that acts at mature septa to promote streptococcus thermophilus cell separation biorecognition of chemically modified bovine serum albumin with lactose prepared under different conditions conjugates of bovine serum albumin with chitin oligosaccharides prepared through the maillard reaction characterization of 4-a-glucanotransferase from synechocystis sp. pcc 6803 and its application to various corn starches effects of differential glycosylation of glycodelins on lymphocyte survival targeted molecular imaging of vegf receptors overexpressed in ischemic microvasculature using chitosan-dc101 conjugates the poplar gt8e and gt8f glycosyltransferases are functional orthologs of arabidopsis parvus involved in glucuronoxylan biosynthesis the f8h glycosyltransferase is a functional paralog of fra8 involved in glucuronoxylan biosynthesis in arabidopsis down-regulation of pogt47c expression in poplar results in a reduced glucuronoxylan content and an increased wood digestibility by cellulase the arabidopsis family gt43 glycosyltransferases form two functionally nonredundant groups essential for the elongation of glucuronoxylan backbone glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein isolation and tandem mass fragmentations of an anti-inflammatory compound from aralia elata high-throughput quantitative analysis of plant nglycan using a dna sequencer synthesis of selenium nanowires morphologically directed by shinorhizobial oligosaccharides core 3 o-glycan synthase suppresses tumor formation and metastasis of prostate carcinoma pc3 and lncap cells through down-regulation of a2b1 integrin complex core2 o-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-iv during intestinal cell differentiation synthesis and cellular uptake properties of guanidine-containing molecular transporters built on the sucrose scaffold tracing the development of structural elucidation of nglycans synthesis of undecaprenyl pyrophosphate-linked glycans as donor substrates for bacterial protein nglycosylation deficiency of dol-p-man synthase subunit dpm3 bridges the congenital disorders of glycosylation with the dystroglycanopathies n-glycosylation patterns of hemolymph glycoproteins from biomphalaria glabrata strains expressing different susceptibility to schistosoma mansoni infection multivalent binding of carbohydrates by the human a-defensin, hd51 structural analysis of sulfated glycans by sequential double-permethylation using methyl iodide and deuteromethyl iodide sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements a new allergen from ragweed (ambrosia artemisiifolia) with homology to art v 1 from mugwort an unusual galactofuranose lipopolysaccharide that ensures the intracellular survival of toxin-producing bacteria in their fungal host human l-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl lewis x in core 2 o-glycans efficient synthesis and protein conjugation of b-(1-6)-d-n-acetylglucosamine oligosaccharides from the polysaccharide intercellular adhesin steroid compounds from two pacific starfish of the genus evasterias n-glycosylation site analysis of human platelet proteins by hydrazide affinity capturing and lc-ms/ ms phosphoethanolamine substitution of lipid a and resistance of neisseria gonorrhoeae to cationic antimicrobial peptides and complement-mediated killing by normal human serum infrared multiphoton dissociation mass spectrometry for structural elucidation of oligosaccharides collision-induced dissociation tandem mass spectrometry for structural elucidation of glycans pancreatic cancer serum detection using a lectin/ glyco-antibody array method structure of pleiotrophin-and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches overexpression and topology of bacterial oligosaccharyltransferase pglb effects of hapten density on the induced antibody repertoire advances in the glycosylation of milk protein light-switchable janus [2] rotaxanes based on a-cyclodextrin derivatives bearing two recognition sites linked with oligo(ethylene glycol) maldi-tof-ms analysis of small molecules using modified mesoporous material sba-15 as assisted matrix preparation of homogenous oligosaccharide chains from glycosphingolipids mass spectrometry of fluorocarbon-labeled glycosphingolipids gold(i)-catalyzed glycosylation with glycosyl ortho-alkynylbenzoates as donors: general scope and application in the synthesis of a cyclic triterpene saponin immunologic glycosphingolipidomics and nkt cell development in mouse thymus comparison of sample pre-treatments for laser desorption ionization and secondary ion mass spectrometry imaging of miscanthus â giganteus glycan array: a powerful tool for glycomics studies switching of the core structures of glycosphingolipids from globo-and lacto-to ganglio-series upon human embryonic stem cell differentiation the effect of multivalent binding on the lateral phase separation of adhesive lipids 5-n,4-o-carbonyl-7,8,9-tri-o-chloroacetyl-protected sialyl donor for the stereoselective synthesis of a-(2 ! 9)-tetrasialic acid enhanced expression of b3-galactosyltransferase 5 activity is sufficient to induce in vivo synthesis of extended type 1 chains on lactosylceramides of selected human colonic carcinoma cell lines a new naphthimidazole derivative for saccharide labeling with enhanced sensitivity in mass spectrometry detection i 2 -catalyzed oxidative condensation of aldoses with diamines: synthesis of aldo-naphthimidazoles for carbohydrate analysis in vivo protection provided by a synthetic new alpha-galactosyl ceramide analog against bacterial and viral infections in murine models fabrication of oriented antibody-conjugated magnetic nanoprobes and their immunoaffinity application characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot-plant synthesis of magnetic nanoparticles with immobilized aminophenylboronic acid for selective capture of glycoproteins the grignard reaction of cyclodextrin-6-aldehydes revisited: a study of the stereoselectivity upon addition of organometallic reagents to aldehydes and ketones a bivalent glycopeptide to target two putative carbohydrate binding sites on fimh en route to photoaffinity labeling of the bacterial lectin fimh muropeptide rescue in bacillus subtilis involves sequential hydrolysis by b-n-acetylglucosaminidase and n-acetylmuramyl-lalanine amidase incoherent production reactions of positive and negative ions in matrix-assisted laser desorption/ionization initial ionization reaction in matrix-assisted laser desorption/ionization isolation and identification of two novel sds-resistant secreted chitinases from aeromonas schubertii norclerodane diterpenoids from rhizomes of dioscorea bulbifera an apple oligogalactan prevents against inflammation and carcinogenesis by targeting lps/tlr4/nf-kb pathway in a mouse model of colitis-associated colon cancer phosphoryl moieties of lipid a from neisseria meningitidis and n. gonorrhoeae lipooligosaccharides play an important role in activation of both myd88-and trif-dependent tlr4-md-2 signaling pathways efficient synthesis of flaccidoside ii, a bioactive component of chinese folk medicine di wu concise synthesis of two natural triterpenoid saponins, oleanolic acid derivatives isolated from the roots of pulsatilla chinensis ionic liquids in sample preparation albizosides d and e, two new cytotoxic triterpene saponins from albizia chinensis cytotoxic oleanane triterpene saponins from albizia chinensis electrospray ionization mass spectrometry as a critical tool for revealing new properties of snake venom phospholipase a2 18 f-labeled galacto and pegylated rgd dimers for pet imaging of a v b 3 integrin expression alteration of n-glycome in diethylnitrosamine-induced hepatocellular carcinoma mice: a noninvasive monitoring tool for liver cancer is permethylation strategy always applicable to protein n-glycosylation study? a case study on the o-acetylation of sialic acid in fish serum glycans microwave-assisted kochetkov amination followed by permanent charge derivatization: a facile strategy for glycomics methylamidation for sialoglycomics by maldi-ms: a facile derivatization strategy for both a2,3-and a2,6-linked sialic acids investigation of sample preparation artifacts formed during the enzymatic release of n-linked glycans prior to analysis by capillary electrophoresis supramolecular assembly of perylene bisimide with b-cyclodextrin grafts as a solid-state fluorescence sensor for vapor detection elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry tandem 18 o stable isotope labeling for quantification of n-glycoproteome plant-expressed recombinant mountain cedar allergen jun a 1 is allergenic and has limited pectate lyase activity modular approach to triazole-linked 1,6-a-d-oligomannosides to the discovery of inhibitors of mycobacterium tuberculosis cell wall synthetase glycosylation of b2 subunits regulates gaba a receptor biogenesis and channel gating lipidomic analysis of porcine olfactory epithelial membranes and cilia a versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides the egmur3 xyloglucan galactosyltransferase from eucalyptus grandis complements the mur3 cell wall phenotype in arabidopsis thaliana morphological, toxicological and molecular characterization of a benthic nodularia isolated from atlantic estuarine environments enthalpic studies of xyloglucan-cellulose interactions titania microparticles and nanoparticles as matrixes for in vitro and in situ analysis of small molecules by maldi-ms reactivity-based onepot synthesis of immunosuppressive glycolipids from the caribbean sponge plakortis simplex chemical synthesis of a hyaluronic acid decasaccharide a yeast glycoprotein shows highaffinity binding to the broadly neutralizing human immunodeficiency virus antibody 2g12 and inhibits gp120 interactions with 2g12 and dc-sign fatal outcome due to deficiency of subunit 6 of the conserved oligomeric golgi complex leading to a new type of congenital disorders of glycosylation two kdo-heptose regions identified in hafnia alvei 32 lipopolysaccharide: the complete core structure and serological screening of different hafnia o serotypes structural analysis of the lipid a isolated from hafnia alvei 32 and pcm 1192 lipopolysaccharides functional l-lysine dendritic macromolecules as liver-imaging probes structural characterization of n-linked oligosaccharides of defibrase from agikistrodon acutus by sequential exoglycosidase digestion and maldi-tof mass spectrometry studying a cell division amidase using defined peptidoglycan substrates data mining the pdb for glyco-related data facile preparation of fluorescent neoglycoproteins using p-nitrophenyl anthranilate as a heterobifunctional linker synthesis of the pentasaccharide repeating unit of escherichia coli o128 antigen a b-d-allopyranoside-grafted ru(ii) complex: synthesis and acid-base and dna-binding properties new phosphane based on a b-cyclodextrin, exhibiting a solventtunable conformation, and its catalytic properties structural analysis of the o-specific polysaccharide isolated from plesiomonas shigelloides o51 lipopolysaccharide the conformational properties of the glc 3 man unit suggest conformational biasing within the chaperone-assisted glycoprotein folding pathway euscaphinin, a new ellagitannin dimer from euscaphis japonica (thunb.) kanitz study of lysozyme glycation reaction by mass spectrometry and nmr spectroscopy fut2-null mice display an altered glycosylation profile and impaired babamediated helicobacter pylori adhesion to gastric mucosa aglp is a s-adenosyl-l-methionine-dependent methyltransferase that participates in the n-glycosylation pathway of haloferax volcanii manipulation of electrostatic and saccharide linker interactions in the design of efficient glycopolypeptide-based cholera toxin inhibitors chemical synthesis and proinflammatory responses of monophosphoryl lipid a adjuvant candidates glucooligosaccharides production from glucan of leuconostoc mesenteroides nrrl b-742 by microwave assisted hydrolysis isorhizochalin: a minor unprecedented bipolar sphingolipid of stereodivergent biogenesis from the rhizochalina incrustata development of continuous flow type hydrothermal reactor for hemicellulose fraction recovery from corncob phenolic compounds from bursera simaruba sarg. bark: phytochemical investigation and quantitative analysis by tandem mass spectrometry iodine-hexamethyldisilane (hmds)-mediated anomerization of peracetylated 1,2-trans-linked alkyl and aryl glycosides a simple, mild, and regioselective method for the benzylation of carbohydrate derivatives promoted by silver carbonate efficient synthesis of a fluorescent tripod detection system for pesticides by microwaveassisted click chemistry highly efficient deletion of fut8 in cho cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies rapid de-o-glycosylation concomitant with peptide labeling using microwave radiation and an alkyl amine base dissection of host cell signal transduction during acinetobacter baumannii-triggered inflammatory response consumption of human milk oligosaccharides by gutrelated microbes analysis of protein posttranslational modifications using mass spectrometry interactions of lipopolysaccharide and polymyxin studied by nmr spectroscopy amino-acetone-bridged cyclodextrins-artificial alcohol oxidases a systematic approach to protein glycosylation analysis: a path through the maze flavonoids with prolyl oligopeptidase inhibitory activity isolated from scutellaria racemosa pers glyconanoparticles: polyvalent tools to study carbohydrate-based interactions glycosylation protects proteins against free radicals generated from toxic xenobiotics gold manno-glyconanoparticles: multivalent systems to block hiv-1 gp120 binding to the lectin dc-sign antithrombin murcia (k241e) causing antithrombin deficiency: a possible role for altered glycosylation a series of 2-o-trifluoromethylsulfonyl-dmannopyranosides as precursors for concomitant 18 f-labeling and glycosylation by click chemistry synthesis and delivery activity of new cationic cholesteryl glucosides mutational deglycosylation of the fc portion of immunoglobulin g causes osulfation of tyrosine adjacently preceding the originally glycosylated site biochemical and immunological characterization of the plantderived candidate human immunodeficiency virus type 1 mucosal vaccine ctb-mpr 649-684 supported molecular matrix electrophoresis: a new tool for characterization of glycoproteins development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans synthesis of sialyllactosamine clusters using carbosilane as core scaffolds by means of chemical and enzymatic approaches novel synthesis of functional mucin glycopeptides containing both n-and o-glycans functional neoglycopeptides: synthesis and characterization of a new class of muc1 glycoprotein models having core 2-based o-glycan and complex-type n-glycan chains a tethering mechanism for length control in a processive carbohydrate polymerization structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass extracellular monoenzyme deglycosylation system of 7-o-linked flavonoid brutinosides and its disaccharide transglycosylation activity from stilbella fimetaria biodistribution and excretion of monosaccharidealbumin conjugates measured with in vivo near-infrared fluorescence imaging glycoform and glycologue: two software applications for the rapid construction and display of n-glycans from mammalian sources site-directed mutagenesis to probe catalysis by a thermobifida fusca b-1,3-glucanase (lam81a) the mass-mobility correlation redux: the conformational landscape of anhydrous biomolecules glycomic analysis by capillary electrophoresis-mass spectrometry solid-phase permethylation for glycomic analysis high-sensitivity analytical approaches to the analysis of n-glycans quantitative serum glycomics of esophageal adenocarcinoma and other esophageal disease onsets the effect of glycation on foam and structural properties of b-lactoglobulin structural characterization of glycans on omega-1, a major schistosoma mansoni egg glycoprotein that drives th2 responses proteomic scale high-sensitivity analyses of gpi membrane anchors glycotyping of trypanosoma brucei variant surface glycoprotein mitat1 studies on the boronation of methyl-b-d-cellobioside-a cellulose model assessment of heat treatment of dairy products by synthesis of tetraglucosyl-and tetrapolyamine-tetrabenzoporphyrin conjugates for an application in pdt synthesis of an fmoc-asn-heptasaccharide building block and its application to chemoenzymatic glycopeptide synthesis fluorescent bodipylabelled gm1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions helicobacter pylori binding to new glycans based on n-acetyllactosamine transcriptomic analysis of arabidopsis developing stems: a close-up on cell wall genes glycoforms of human endothelial cd34 that bind l-selectin carry sulfated sialyl lewis x capped o-and n-glycans chemical structure analysis of starch and cellulose derivatives genetic and structural characterization of l11 lipooligosaccharide from neisseria meningitidis serogroup a strains the n-glycolyl form of mouse sialyl lewis x is recognized by selectins but not by heca-452 and fh6 antibodies that were raised against human cells 1-(d-glucopyranosyl-2 0 -deoxy-2 0 -iminomethyl)-2-hydroxybenzene as chemosensor for aromatic amino acids by switch-on fluorescence ultrahighly sensitive in situ metabolomic imaging for visualizing spatiotemporal metabolic behaviors rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry glycoblotting-assisted o-glycomics: ammonium carbamate allows for highly efficient o-glycan release from glycoproteins synthesis and absolute structures of mycoplasma pneumoniae bglyceroglycolipid antigens multivalent galacto-trehaloses: design, synthesis, and biological evaluation under the concept of carbohydrate modules promiscuous activity of er glucosidase ii discovered through donor specificity analysis of uggt novel rhamnosyltransferase involved in biosynthesis of serovar 4-specific glycopeptidolipid from mycobacterium avium complex systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide: n-glycanase structural and functional characterization of recombinant human serum transferrin secreted from pichia pastoris the cytotoxic activity of linum grandiflorum leaves new acylated flavone and cyanogenic glycosides from linum grandiflorum synthesis of novel gluco-and galacto-functionalized platinum complexes design of triazoletethered glycoclusters exhibiting three different spatial arrangements and comparative study of their affinities towards pa-il and rca 120 by using a dna-based glycoarray capsule polysaccharide conjugate vaccine against diarrheal disease caused by campylobacter jejuni a-type crystals from dilute solutions of short amylose chains the n-glycosylation of classical swine fever virus e2 glycoprotein extracellular domain expressed in the milk of goat n-glycosylation pattern of e2 glycoprotein from classical swine fever virus glycation pattern of peptides condensed with maltose, lactose and glucose determined by ultraviolet matrix-assisted laser desorption/ionization tandem mass spectrometry regulated and aberrant glycosylation modulate cardiac electrical signaling synthesis of hyperbranched b-galceramide-containing dendritic polymers that bind hiv-1 rgp120 in vivo biocompatibility and biodegradability of dextrin-based hydrogels analysis of glycosylation and other post-translational modifications by mass spectrometry analysis of n-and olinked glycans from glycoproteins using maldi-tof mass spectrometry glycosylation pattern of brush borderassociated glycoproteins in enterocyte-like cells: involvement of complex-type n-glycans in apical trafficking anthocyanin components and mechanism for color development in blue veronica flowers biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain isolation of basidiomycetous yeast pseudozyma tsukubaensis and production of glycolipid biosurfactant, a diastereomer type of mannosylerythritol lipid-b production of glycolipid biosurfactants, mannosylerythritol lipids, by a smut fungus, 7 nbrc 32730 production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by pseudozyma parantarctica and its interfacial properties coupling of planar chromatography to mass spectrometry selective binding of rnase b glycoforms by polydopamine-immobilized concanavalin a glycosylated cu/zn-superoxide dismutase from kluyveromyces yeast, determined by mass spectrometry chemoenzymatic synthesis of conjugatable oligosialic acids analysis of recombinant cd24 glycans by maldi-tof-ms reveals prevalence of sialyl-t antigen separation and identification of oligosaccharides labeled with 3-amino-9-ethylcarbazole using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry o-acetylation of peptidoglycan in gramnegative bacteria. identification and characterization of peptidoglycan o-acetyltransferase in neisseria gonorrhoeae identification of the n-glycosylation sites on recombinant bovine cd38 expressed in pichia pastoris: their impact on enzyme stability and catalytic activity efficient synthesis of 4-amino-4-deoxy-l-arabinose and spacerequipped 4-amino-4-deoxy-l-arabinopyranosides by transglycosylation reactions molecular weight determination of high molecular mass (glyco)proteins using cge-on-a-chip study of inclusion complexes of cycloamylose with surfactants by isothermal titration calorimetry analysis of n-and o-linked protein glycosylation in children with prader-willi syndrome supramolecular nanocycles comprising b-cyclodextrin-click-ferrocene units: rings of rings of rings unique cleavage of 2-acetamido-2-deoxy-dglucose from the reducing end of biantennary complex type oligosaccharides efficient and systematic synthesis of a small glycoconjugate library having human complex type oligosaccharides imaging mass spectrometry: principle and application application of thin-layer chromatography/infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry to structural analysis of bacteria-binding glycosphingolipids selected by affinity detection chemoenzymatic synthesis of a new class of macrocyclic oligosaccharides advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry shiga toxins, glycosphingolipid diversity, and endothelial cell injury heparin/heparan sulfate 6-o-sulfatase from flavobacterium heparinum. integrated structural and biochemical investigation of enzyme active site and substrate specificity heparin/heparan sulfate n-sulfamidase from flavobacterium heparinum. structural and biochemical investigation of catalytic nitrogen-sulfur bond cleavage utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle synthesis of urea tethered glycosylated amino acids and glycopeptides mediated by dppa employing na-fmoc-asp/glu-5-oxazolidinones toward fully synthetic homogeneous b-human follicle-stimulating hormone (b-hfsh) with a biantennary n-linked dodecasaccharide. synthesis of b-hfsh with chitobiose units at the natural linkage sites experiments on the synthesis of carotenoid glycosides glycomic analyses of glycoproteins in bile and serum during rat hepatocarcinogenesis transferrin receptordependent cytotoxicity of artemisinin-transferrin conjugates on prostate cancer cells and induction of apoptosis synthesis, structural analysis and application of novel acarbose fructoside using levansucrase extraction and characterization of native heteroxylans from delignified corn stover and aspen a strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics effect of glycosylation on cis/trans isomerization of prolines in iga1-hinge peptide proteomic approaches to study structure, functions and toxicity of legume seeds lectins. perspectives for the assessment of food quality and safety engineering host cell lines to reduce terminal sialylation of secreted antibodies purification and partial characterization of cu/ zn superoxide dismutase from kluyveromyces marxianus yeast experimental evidence of chemical exchange over the b(1 ! 3) glycosidic linkage and hydrogen bonding involving hydroxy protons in hyaluronan oligosaccharides by nmr spectroscopy synthesis of amino-bridged oligosaccharide mimetics production of chitin oligosaccharides with different molecular weights and their antioxidant effect in raw 264.7 cells genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of pseudomonas syringae pv. tabaci identification of a naturally-occurring 8-[a-d-glucopyranosyl-(1-6)-b-d-glucopyranosyl] daidzein from cultivated kudzu root novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium pseudomonas aeruginosa strain ny3 novel o-antigen of hafnia alvei pcm 1195 lipopolysaccharide with a teichoic acid-like structure structures of two novel, serologically nonrelated core oligosaccharides of yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution syntheses and characterisation of novel cyclodextrin vinyl derivatives from cyclodextrin-nitrophenol-derivatives use of b-cyclodextrins to control the structure of water-soluble copolymers with hydrophobic parts facile synthesis of b-cyclodextrin-dextran polymers by "click" chemistry fullerene sugar balls gold nanoparticle arrangement on viral particles through carbohydrate recognition: a non-cross-linking approach to optical virus detection enrichment of glycopeptides for glycan structure and attachment site identification characterization of the quantitative relationship between signalto-noise (s/n) ratio and sample amount on-target by maldi-tof ms: determination of chondroitin sulfate subsequent to enzymatic digestion differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate identification of o-glycosylated decapeptides within the muc1 repeat domain as potential mhc class i (a2) binding epitopes mass spectrometric methods for analysis of oligosaccharides in human milk preparation and characterization of branched bcyclodextrins having a-l-fucopyranose and a study of their functions reverse thin layer method for enhanced ion yield of oligosaccharides in matrix-assisted laser desorption/ionization assessing the cluster glycoside effect during the binding of concanavalin a to mannosylated artificial lipid rafts systematic screens of a candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity onresin click-glycoconjugation of peptoids mass spectrometry in the analysis of n-linked and o-linked glycans glycomics profiling of chinese hamster ovary cell glycosylation mutants reveals n-glycans of a novel size and complexity mass spectrometric analysis of mutant mice characterization of the novel human age1hn cell line for production of recombinant proteins production and characterization of monoclonal antibodies specific to lactotriaosylceramide repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (g418) with enhanced suppression of diseases-causing nonsense mutations development of novel aminoglycoside (nb54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry structural characterization of an oligosaccharide made by neisseria sicca electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities and biological applications microanalysis of plant cell wall polysaccharides molecular analysis of carbohydrate-antibody interactions: case study using a bacillus anthracis tetrasaccharide endo-b-n-acetylglucosaminidasecatalyzed polymerization of b-glcp-(1-4)-glcpnac oxazoline: a revisit to enzymatic transglycosylation syntheses and doxorubicininclusion abilities of b-cyclodextrin derivatives with a hydroquinone a-glycoside residue attached at the primary side synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat a2,6-sialyltransferase expressed in bmnpv bacmid-injected silkworm larvae expression, purification, and analyses of glycosylation and disulfide bonds of stereum purpureum endopolygalacturonase i in pichia pastoris chemically responsive supramolecular assemblies of pyrene-b-cyclodextrin dimer matrix-assisted laser desorption/ionization mass spectrometric analysis of polysulfated-derived oligosaccharides using pyrenemethylguanidine structual analysis of neutral and acidic xylooligosaccharides from hardwood kraft pulp, and their utilization by intestinal bacteria in vitro structural study of the n-glycans of intercellular adhesion molecule-5 (telencephalin) an essential epitope of anti-muc1 monoclonal antibody kl-6 revealed by focused glycopeptide library simple and conveniently accessible bi-fluorescence-labeled substrates for amylases syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl a (2 ! 3) lactose moieties as inhibitors for human influenza viruses characterization of endoplasmic reticulum-localized udp-dgalactose: hydroxyproline o-galactosyltransferase using synthetic peptide substrates in arabidopsis structural analysis of three novel trisaccharides isolated from the fermented beverage of plant extracts novel fructopyranose oligosaccharides isolated from fermented beverage of plant extract convenient approach to access octa-glycosylated porphyrins via "click chemistry n-glycosylation engineering of lepidopteran insect cells by the introduction of the b1,4-n-acetylglucosaminyltransferase iii gene efficient substitution reaction from cysteine to the serine residue of glycosylated polypeptide: repetitive peptide segment ligation strategy and the synthesis of glycosylated tetracontapeptide having acid labile sialyl-tn antigens structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under tnf-a stimulation boronic acid lectin affinity chromatography (blac). 3. temperature dependence of glycoprotein isolation and enrichment physical mechanisms of bacterial survival revealed by combined grazing-incidence x-ray scattering and monte carlo simulation use of ftir and mass spectrometry for characterization of glycated caseins artefacts formed by addition of urea to n-linked glycans released with peptide-n-glycosidase f for analysis by mass spectrometry okicamelliaside, an extraordinarily potent antidegranulation glucoside isolated from leaves of camellia japonica quantification of fructooligosaccharides based on the evaluation of oligomer ratios using an artificial neural network quantitative glycomics results of the 2010 glycoprotein research group's (gprg) study on quantitative glycoprotein analysis click multivalent heterogeneous neoglycoconjugates-modular synthesis and evaluation of their binding affinities globular carbosilane dendrimers with mannose groups at the periphery: synthesis, characterization and toxicity in dendritic cells biosynthesis and structure of the burkholderia cenocepacia k56-2 lipopolysaccharide core oligosaccharide. truncation of the core oligosaccharide leads to increased binding and sensitivity to polymyxin b switching of polymerization activity of cinnamoyl-a-cyclodextrin action of a-dglucosidase from aspergillus niger towards dextrin and starch contiguous ogalactosylation of 4(r)-hydroxy-l-proline residues forms very stable polyproline ii helices microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies synthesis, in vitro and in vivo studies of gd-dtpa-xda-d1-glc(oh) complex as a new potential mri contrast agent structural characterization and hypoglycemic effects of arabinogalactan-protein from the tuberous cortex of the white-skinned sweet potato (ipomoea batatas l.) comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method characterization of minor n-linked glycans on antibodies using endo h release and maldi-mass spectrometry methods in molecular biology, glycomics: methods and protocols the caenorhabditis elegans bus-2 mutant reveals a new class of o-glycans affecting bacterial resistance surfactant-assisted lipopolysaccharide conjugation employing a cyanopyridinium agent and its application to a competitive assay multifaceted approaches including neoglycolipid oligosaccharide microarrays to ligand discovery for malectin glycoproteomic profile in wine: a 'sweet' molecular renaissance mass spectrometry based targeted protein quantification: methods and applications convergent synthesis of a common pentasaccharide-repeating unit corresponding to the o-specific polysaccharide of escherichia coli o4: k3, o4: k6, and o4: k12 synthesis of penta-and hexasaccharide fragments corresponding to the o-antigen of escherichia coli o150 analysis of the human seminal plasma glycome reveals the presence of immunomodulatory carbohydrate functional groups fast access to robust c-sialoside multimers synthesis, conjugation, and immunological evaluation of the analysis of carbohydrates and glycoconjugates & serogroup 6 pneumococcal oligosaccharides human igg1 expression in silkworm larval hemolymph using bmnpv bacmids and its n-linked glycan structure the synthesis and spectral properties of an encapsulated aminoazobenzene dye o-glcnacylation disrupts glyceraldehyde-3-phosphate dehydrogenase homo-tetramer formation and mediates its nuclear translocation immunochemical studies of trehalose-containing major glycolipid from tsukamurella pulmonis structural characterization of the major glycolipids from arthrobacter globiformis and arthrobacter scleromae bioconjugation and detection of lactosamine moiety using a1,3-galactosyltransferase mutants that transfer c2-modified galactose with a chemical handle muropeptides trigger distinct activation profiles in macrophages and dendritic cells rapid assembly of gp120 oligosaccharide moieties via one-pot glycosidation-deprotection sequences one-pot catalytic glycosidation/fmoc removal-an iterable sequence for straightforward assembly of oligosaccharides related to hiv gp120 analysis of the dispersity in carbohydrate loading of synthetic glycoproteins using maldi-tof mass spectrometry solvent-free synthesis of thioglycosides by ball milling synthesis of mycobacterial triacylated phosphatidylinositol dimannoside containing an acyl lipid chain at 3-o of inositol o-glycan inhibitors generate arylglycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines detection of carbohydrates and steroids by cation-enhanced nanostructure-initiator mass spectrometry (nims) for biofluid analysis and tissue imaging protein glycosylation analysis with capillary-based electromigrative separation techniques glycoprotein analysis using protein microarrays and mass spectrometry synthesis of 2-deoxy cyclic and linear oligosaccharides by oligomerization of monomers maintaining product titer while replacing undefined components in a cho culture system model a-mannoside conjugates: immunogenicity and induction of candidacidal activity hydrodynamic properties of cyclodextrin molecules in dilute solutions koenigs-knorr glycosylation with neuraminic acid derivatives synthesis of the core structure of the lipoteichoic acid of streptococcus pneumoniae de novo glycan structure search with the cid ms/ms spectra of native n-glycopeptides identification of a novel human udp-galnac transferase with unique catalytic activity and expression profile molecular engineering of exocytic vesicle traffic enhances the productivity of chinese hamster ovary cells the vesicletrafficking protein munc18b increases the secretory capacity of mammalian cells detection of pathogenic streptococcus suis bacteria using magnetic glycoparticles rapid screening of lectins for multivalency effects with a glycodendrimer microarray imaging ms methodology for more chemical information in less data acquisition time utilizing a hybrid linear ion trap-orbitrap mass spectrometer atmospheric pressure laser desorption/ionization of plant metabolites and plant tissue using colloidal graphite click multivalent homogeneous neoglycoconjugates-synthesis and evaluation of their binding affinities role of lipid a acylation in yersinia enterocolitica virulence glycopeptide-preferring polypeptide galnac transferase 10 (ppgalnac t10), involved in mucin-type o-glycosylation, has a unique galnac-o-ser/thr-binding site in its catalytic domain not found in ppgalnac t1 or t2 steroidal glycosides from the leaves of ruscus colchicus: isolation and structural elucidation based on a preliminary liquid chromatography-electrospray ionization tandem mass spectrometry profiling off-line coupling of microcolumn separations to desorption mass spectrometry selective discrimination of cyclodextrin diols using cyclic sulfates 2d-hplc and maldi-tof/tof analysis of barley proteins glycated during brewing synthesis of cationic derivatives of quil a and the preparation of cationic immunestimulating complexes (iscoms) cardenolides from pergularia tomentosa display cytotoxic activity resulting from their potent inhibition of na þ /k þ -atpase structural determination of the o-chain polysaccharide from the haloalkaliphilic halomonas alkaliantarctica bacterium strain crss structure of the core region from the lipopolysaccharide of plesiomonas shigelloides strain 302-73 (serotype o1) structural characterization of the core region from the lipopolysaccharide of the haloalkaliphilic bacterium halomonas alkaliantarctica strain crss the complete structure of the core of the lps from plesiomonas shigelloides 302-73 and the identification of its o-antigen biological repeating unit enhancement of repair of radiation induced dna strand breaks in human cells by ganoderma mushroom polysaccharides structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by raoultella terrigena ez-555-6 isolated in the chernobyl exclusion zone a protein important for antimicrobial peptide resistance, ydei/omda, is in the periplasm and interacts with ompd/nmpc role of e-cadherin n-glycosylation profile in a mammary tumor model influence of glycosylation on the adsorption of thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces diazo transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives with high antibacterial and anti-influenza virus activity: an aggregation and receptor binding study high-energy collision induced dissociation of biomolecules: maldi-tof/rtof mass spectrometry in comparison to tandem sector mass spectrometry polymer structure of commercial hydrolyzable tannins by matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry design, synthesis, and evaluation of novel fluoroquinolone-aminoglycoside hybrid antibiotics cycloartane-type glycosides from astragalus amblolepis triterpenoid saponins from astragalus wiedemannianus fischer mechanisms involved during the ultrasonically induced depolymerization of chitosan: characterization and control oligonucleotide carbohydrate-centered galactosyl cluster conjugates synthesized by click and phosphoramidite chemistries substrate recognition and hydrolysis by a fungal xyloglucan-specific family 12 hydrolase the structural elucidation of glycosaminoglycans chemical modification and biological evaluation of new semisynthetic derivatives of 28,29-didehydronystatin a1 (s44hp), a genetically engineered antifungal polyene macrolide context-dependent effects of asparagine glycosylation on pin ww folding kinetics and thermodynamics maldi-tof mass spectrometry of naturally occurring mixtures of monorhamnolipids and dirhamnolipids functionalized c-glycoside ketohydrazones: carbohydrate derivatives that retain the ring integrity of the terminal reducing sugar mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics desorption electrospray ionization mass spectrometry of glycosaminoglycans and their protein noncovalent complex haba-based ionic liquid matrices for uv-maldi-ms analysis of heparin and heparan sulfate oligosaccharides mannose-substituted conjugated polyelectrolyte and oligomer as an intelligent energy transfer pair for label-free visual detection of concanavalin a development of a ce-ms method to analyze components of the potential biomarker vascular endothelial growth factor 165 lipopeptides produced by a soil bacillus megaterium strain mass spectrometric analysis of the s-layer proteins from clostridium difficile demonstrates the absence of glycosylation facile synthesis of mercaptophenylboronic acid-functionalized core-shell structure fe 3 o 4 @c@au magnetic microspheres for selective enrichment of glycopeptides and glycoproteins a smart glycol-directed nanodevice from rationally designed macroporous materials secondary disorders of glycosylation in inborn errors of fructose metabolism online coupling of thin layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: synthesis and application of a new material for the identification of carbohydrates by thin layer chromatography/matrix free material enhanced laser desorption/ionization mass spectrometry acylated triterpenoidal saponins and cytokinins from gleditsia aquatica click chemistry approach for the synthesis of water-soluble glycodendrimer with triazole as building unit synthesis and evaluation of n-acetyl-2-amino-2-deoxy-a-d-galactosyl-1-thio-7-oxaceramide, a new analogue of a-d-galactosyl ceramide mass spectrometry for pectin structure analysis multiple site-specific in vitro labeling of single-chain antibody the effect of ph on the production of chitinolytic enzymes of verticillium fungicola in submerged cultures glycome-db. org: a portal for querying across the digital world of carbohydrate sequences iodine-sodium cyanoborohydride-mediated reductive ring opening of 4,6-o-benzylidene acetals of hexopyranosides glycosylation patterns of hiv-1 gp120 depend on the type of expressing cells and affect antibody recognition the glycosylation of myeloperoxidase the glycosylation and characterization of the candidate gc macrophage activating factor controlled synthesis of linear acyclodextrin oligomers using copper-catalyzed huisgen 1,3-dipolar cycloaddition two-step synthesis of galactosylated human serum albumin as a targeted optical imaging agent for peritoneal carcinomatosis handbook of experimental pharmacology 195, doping in sports synthesis of multivalent glycoconjugates containing the immunoactive lelte peptide: effect of glycosylation on cellular activation and natural killing by human peripheral blood mononuclear cells golgi function and dysfunction in the first cog4-deficient cdg type ii patient mycobacterium abscessus glycopeptidolipids mask underlying cell wall phosphatidyl-myo-inositol mannosides blocking induction of human macrophage tnf-a by preventing interaction with tlr2 capsule anchoring in bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of o-antigen repeating units identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein identification of n-glycans from ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry synthesis, characterization, and immunogenicity in mice of shigella sonnei o-specific oligosaccharide-core-protein conjugates 3-aminoquinoline acting as matrix and derivatizing agent for maldi ms analysis of oligosaccharides cross-linked membranes based on acrylated cyclodextrins and polyethylene glycol dimethacrylates for aromatic/aliphatic separation mycobacterium marinum lipooligosaccharides are unique caryophyllose-containing cell wall glycolipids that inhibit tumor necrosis factor-a secretion in macrophages structural analysis of an unusual bioactive n-acylated lipo-oligosaccharide los-iv in mycobacterium marinum exploiting the cross-metathesis reaction in the synthesis of pseudo-oligosaccharides isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility characterisation of haptoglobin in the blood plasma of harbour seals (phoca vitulina) n-glycan moieties of the crustacean egg yolk protein and their glycosylation sites synthesis of lewis x epitopes on plant n-glycans glycodynamers: dynamic polymers bearing oligosaccharides residues -generation, structure, physicochemical, component exchange, and lectin binding properties oligosaccharide analysis by graphitized carbon liquid chromatography-mass spectrometry 2-picoline-borane: a non-toxic reducing agent for oligosaccharide labeling by reductive amination glycan labeling strategies and their use in identification and quantification optimized workflow for preparation of apts-labeled n-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed cge-lif decreased levels of bisecting glcnac glycoforms of igg are associated with human longevity antibacterial activity of n-quaternary chitosan derivatives: synthesis, characterization and structure activity relationship (sar) investigations the presence of monoglucosylated n196-glycan is important for the structural stability of storage protein, arylphorin molecular cloning and characterization of trehalose synthase from thermotoga maritima dsm3109: syntheses of trehalose disaccharide analogues and ndp-glucoses maldi tof-tof characterization of a light stabilizer polymer contaminant from polypropylene or polyethylene plastic test tubes quantification of soyasaponins i and bg in italian lentil seeds by solid-phase extraction (spe) and high-performance liquid chromatography-mass spectrometry (hplc-ms) nutrition analysis by nanoparticle-assisted laser desorption/ionisation mass spectrometry isolation and characterization of antibodies against three consecutive tn-antigen clusters from a phage library displaying human single-chain variable fragments o-glcnac modification of the extracellular domain of notch receptors systematic syntheses of influenza neuraminidase inhibitors: a series of carbosilane dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties supramolecular assemblies of oligothiophene derivatives bearing b-cyclodextrin 1h-1,2,3-triazol-1-yl thiodigalactoside derivatives as high affinity galectin-3 inhibitors synthesis and structural characterization of sialic acid-glutamic acid hybrid foldamers as conformational surrogates of a-2,8-linked polysialic acid the remarkable stability of chimeric, sialic acid derived a/d-peptides in human blood plasma lack of a-xylosidase activity in arabidopsis alters xyloglucan composition and results in growth defects intact and top-down characterization of biomolecules and direct analysis using infrared matrix-assisted laser desorption electrospray ionization coupled to ft-icr mass spectrometry triumfettosterol id and triumfettosaponin, a new (fatty acyl)-substituted steroid and a triterpenoid 'dimer' bis(b-d-glucopyranosyl) ester from the leaves of wild triumfetta cordifolia a. rich. (tiliaceae) fluorescence turn-on sensing of lectins with mannose-substituted tetraphenylethenes based on aggregation-induced emission synthesis of tri-and pentasaccharide fragments corresponding to the o-antigen of shigella boydii type 6 biorecognition of escherichia coli k88 adhesin for glycated porcine albumin synthesis of a single-molecule l-rhamnose-containing three-component vaccine and evaluation of antigenicity in the presence of anti-lrhamnose antibodies glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis support vector machine prediction of n-and o-glycosylation sites using whole sequence information and subcellular localization silkworm expression and sugar profiling of human immune cell surface receptor, kir2dl1 high levels of e4-pha-reactive oligosaccharides: potential as marker for cells with characteristics of hepatic progenitor cells highsensitivity analysis of naturally occurring sugar chains, using a novel fluorescent linker molecule di-tert-butylsilylenedirected a-selective synthesis of p-nitrophenyl t-antigen analogues analysis of the human cancer glycome identifies a novel group of tumor-associated n-acetylglucosamine glycan antigens the n-glycome of human embryonic stem cells inhibition of dc-signmediated hiv infection by a linear trimannoside mimic in a tetravalent presentation chemo-enzymatic synthesis of poly-n-acetyllactosamine (poly-lacnac) structures and their characterization for cgl2-galectin-mediated binding of ecm glycoproteins to biomaterial surfaces aziridine ring opening as regio-and stereoselective access to o-glycosyl amino acids and their transformation into o-glycopeptide mimetics identification of a polyprenylphosphomannosyl synthase involved in the synthesis of mycobacterial mannosides reductive amination of the lysine n eamino group leads to a bivalent glyco-amino acid building block suited for spps utilizing staudinger ligation for the synthesis of glycoamino acid building blocks and other glycomimetics cysteinebased mannoside glycoclusters: synthetic routes and antiadhesive properties development of dictyostelium discoideum is associated with alteration of fucosylated n-glycan structures tlc/ hptlc with direct mass spectrometric detection: a review of the progress achieved in the last 5 years o-glycosylation modulates proprotein convertase activation of angiopoietin-like protein 3. possible role of polypeptide galnac-transferase-2 in regulation of concentrations of plasma lipids inhibition binding studies of glycodendrimer/lectin interactions using surface plasmon resonance self-assembling carbohydrate-functionalized oligothiophenes chemical synthesis using enzymatically generated building units for construction of the human milk pentasaccharides sialyllacto-n-tetraose and sialyllacto-n-neotetraose epimer hydroxypropyl-substituted b-cyclodextrins: influence of degree of substitution on the thermodynamics of complexation with tauroconjugated and glycoconjugated bile salts site-specific analysis of nlinked oligosaccharides of recombinant lysosomal arylsulfatase a produced in different cell lines synthesis of a gpi anchor module suitable for protein post-translational modification a combined method for producing homogeneous glycoproteins with eukaryotic n-glycosylation structural basis of multivalent binding to wheat germ agglutinin mass spectrometric characterization of the surface-associated 42 kda lipoprotein jlpa as a glycosylated antigen in strains of campylobacter jejuni probing the lactose gm3 carbohydrate-carbohydrate interaction with glycodendrimers identification of n-glycosylation in prolyl endoprotease from aspergillus niger and evaluation of the enzyme for its possible application in proteomics rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion assigning nglycosylation sites of glycoproteins using lc/msms in conjunction with endo-m/exoglycosidase mixture glycan analysis by mass spectrometry immunoglobulin g glycopeptide profiling by matrixassisted laser desorption ionization fourier transform ion cyclotron resonance mass spectrometry glycan bioengineering in immunogen design for tumor t antigen immunotargeting characterisation of cell wall polysaccharides from okra (abelmoschus esculentus (l.) moench) okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups synthesis and characterization of hydroquinone glucoside using leuconostoc mesenteroides dextransucrase lipoprotein lipase and hydrofluoric acid deactivate both bacterial lipoproteins and lipoteichoic acids, but platelet-activating factor-acetylhydrolase degrades only lipoteichoic acids a functional carbohydrate chip platform for analysis of carbohydrate-protein interaction structural and immunological characterization of the n-glycans from the major yellow jacket allergen ves v 2: the n-glycan structures are needed for the human antibody recognition identification of glycosyltransferase 8 family members as xylosyltransferases acting on o-glucosylated notch epidermal growth factor repeats developments and applications of mass microscopy characterization of a putative 3-deoxy-d-manno-2-octulosonic acid (kdo) transferase gene from arabidopsis thaliana structural characterisation of the pectic polysaccharide rhamnogalacturonan ii using an acidic fingerprinting methodology maldi linear tof mass spectrometry of pegylated (glyco) proteins synthetic glycosides of ent-caurene series containing substituents with benzyl, phenoxyl, and uracyl fragments sno 2 @poly(hema-co-st-co-vpba) core-shell nanoparticles designed for selectively enriching glycopeptides followed by maldi-ms analysis mass spectrometry based analysis of protein o-glycosylation synthesis and characterization of watersoluble conjugated glycopolymer for fluorescent sensing of concanavalin a laser desorption ionization mass spectrometry by using surface plasmon excitation on gold nanoparticle the n-linked oligosaccharide at fcgriiia asn-45: an inhibitory element for high fcgriiia binding affinity to igg glycoforms lacking core fucosylation monitoring the hydrolysis and transglycosylation activity of aglucosidase from aspergillus niger by nuclear magnetic resonance spectroscopy and mass spectrometry n-linked glycan analysis of glycoproteins secreted from rice cell suspension cultures under sugar starvation characterization of receptor proteins using affinity cross-linking with biotinylated ligands proton sponge: a novel and versatile maldi matrix for the analysis of metabolites using mass spectrometry 1,8-bis(dimethylamino)naphthalene: a novel superbasic matrix for matrix-assisted laser desorption/ionization timeof-flight mass spectrometric analysis of fatty acids acid-base-driven matrixassisted mass spectrometry for targeted metabolomics phenolic compounds in the leaves of populus ussuriensis and their antioxidant activities phenolic compounds from populus davidiana wood the structure of the carbohydrate backbone of the lipooligosaccharide from the halophilic bacterium arcobacter halophilus the structure of the carbohydrate backbone of the lipooligosaccharide from an alkaliphilic halomonas sp delineation of the roles of fadd22, fadd26 and fadd29 in the biosynthesis of phthiocerol dimycocerosates and related compounds in mycobacterium tuberculosis biodegradable biocompatible xyloglucan films for various applications transparent xyloglucan-chitosan complex hydrogels for different applications physico chemical properties of aminated tamarind xyloglucan fractionation of sugar beet pulp by introducing ion-exchange groups the antimicrobial action of low-molar-mass chitosan, chitosan derivatives and chitooligosaccharides on bifidobacteria structural details and composition of trichomonas vaginalis lipophosphoglycan in relevance to the epithelial immune function determination of distinctive carbohydrate signatures obtained from the aeromonas hydrophila (chemotype ii) core oligosaccharide pinpointing the presence of the 4-o-phosphorylated 5-o-linked kdo reducing end group using electrospray ionization quadrupole orthogonal time-offlight mass spectrometry and tandem mass spectrometry biosynthetic origin of the galactosamine substituent of arabinogalactan in mycobacterium tuberculosis aftd, a novel essential arabinofuranosyltransferase from mycobacteria nmr structure determination of a segmentally labeled glycoprotein using in vitro glycosylation the role of peptides and proteins in melanoidin formation optimization of matrix conditions for the control of maldi in-source decay of permethylated glycans high-spatial resolution matrix-assisted laser desorption ionization imaging analysis of glucosylceramide in spleen sections from a mouse model of gaucher disease a simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano lc coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry structure of compositionally simple lipopolysaccharide from marine synechococcus synthesis of water-soluble phthalocyanines bearing four or eight d-galactose units discrimination of isobaric leucine and isoleucine residues and analysis of post-translational modifications in peptides by maldi in-source decay mass spectrometry combined with collisional cooling effect of gas pressure and gas type on the fragmentation of peptide and oligosaccharide ions generated in an elevated pressure uv/ir-maldi ion source coupled to an orthogonal time-of-flight mass spectrometer formation and structure elucidation of n-(2,3,4-tri-o-acetyl-b-d-glucopyranosyl)-n 0 -acetylthiourea disaccharide-modified liposomes and their in vitro intracellular uptake carbohydrate arrays: recent developments in fabrication and detection methods with applications discovery of the first series of small molecule h5n1 entry inhibitors transglycosylation properties of maltodextrin glucosidase (malz) from escherichia coli and its application for synthesis of a nigerose-containing oligosaccharide fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays novel fluorescent glycan microarray strategy reveals ligands for galectins generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (fmoccl) as a cleavable fluorescent tag glycan microarray analysis of ptype lectins reveals distinct phosphomannose glycan recognition development and application of mass spectrometric methods for the analysis of progranulin n-glycosylation francisella tularensis blue-gray phase variation involves structural modifications of lipopolysaccharide oantigen, core and lipid a and affects intramacrophage survival and vaccine efficacy glycosylation of gp116 and gp64 envelope proteins of yellow head virus of penaeus monodon shrimp structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with direct ir-maldi-o-tof mass spectrometry software utilities for the interpretation of mass spectrometric data of glycoconjugates: application to glycosphingolipids of human serum ionic liquids in analytical chemistry isolation of b-mannanase from cocos nucifera linn haustorium and its application in the depolymerization of b-(1,4)-linked d-mannans n-glycan trimming by glucosidase ii is essential for arabidopsis development immuno-maldi-tof ms: new perspectives for clinical applications of mass spectrometry a computational approach for deciphering the organization of glycosaminoglycans synthesis of galactofuranose-based acceptor substrates for the study of the carbohydrate polymerase glft2 analysis of n-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses the mechanism of boron mobility in wheat and canola phloem removal of the outer kdo from helicobacter pylori lipopolysaccharide and its impact on the bacterial surface galectin-4-regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells characterization of an immunodominant cancer-specific o-glycopeptide epitope in murine podoplanin (ots8) formation of homooligosaccharides using base-promoted glycosylation of unprotected glycosyl fluorides glycosyltransferase function in core 2-type protein o-glycosylation xyloglucan endotransglycosylases (xets) from germinating nasturtium (tropaeolum majus) seeds: isolation and characterization of the major form botulinum neurotoxin serotype d attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner the chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides analysis of human plasma lipids and soybean lecithin by means of high-performance thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry chemoenzymatic synthesis of a glycolipid library and elucidation of the antigenic epitope for construction of a vaccine against lyme disease acylated cholesteryl galactosides are specific antigens of borrelia causing lyme disease and frequently induce antibodies in late stages of disease detailed n-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation synthesis and acid catalysis of cellulose-derived carbon-based solid acid experimental studies on the ring opening polymerization of p-dioxanone using an al(o i pr) 3 -monosaccharide initiator system glucuronyltransferase activity of kfic from escherichia coli strain k5 requires association of kfia. kfic and kfia are essential enzymes for production of k5 polysaccharide, n-acetylheparosan current imaging mass spectrometry for metabolite molecules imaging mass spectrometry for visualization of drug and endogenous metabolite distribution: toward in situ pharmacometabolomes imaging mass spectrometry protocols for mass microscopy structure and kinetic investigation of streptococcus pyogenes family gh38 a-mannosidase strategic glycan elution map for the production of human-type n-linked oligosaccharides: the case of hen egg yolk and white comprehensive analysis of n-linked oligosaccharides from eggs of the family phasianidae pectic oligosaccharide analysis by fluorophore-assisted carbohydrate electrophoresis ionic liquids in analytical chemistry dsa affinity glycoproteome of human liver tissue isolation of n-linked glycopeptides by hydrazine-functionalized magnetic particles further insight into the roles of the glycans attached to human blood protein c inhibitor synthesis of 6-petn-a-d-galpnac-(1 ! 6)-b-d-galp-(1 ! 4)-b-d-glcpnac-(1 ! 3)-b-d-galp-(1 ! 4)-b-d-glcp, a haemophilus influenzae lipopolysacharide structure, and biotin and protein conjugates thereof computationally and experimentally derived general rules for fragmentation of various glycosyl bonds in sodium adduct oligosaccharides molecular cloning of pigeon udp-galactose: b-d-galactoside a1,4-galactosyltransferase and udp-galactose: b-dgalactoside b1,4-galactosyltransferase, two novel enzymes catalyzing the formation of gal-a1-4gal-b1-4gal-b1-4glcnac sequence structural analysis of n-glycans from gull egg white glycoproteins and egg yolk igg synthesis of a glycosylphosphatidylinositol anchor bearing unsaturated lipid chains rapid release of n-linked glycans from glycoproteins by pressure-cycling technology laser desorption/ionization mass spectrometric analysis of small molecules using fullerene-derivatized silica as energy-absorbing material synthesis of three regioisomers of the pentasaccharide part of the skp1 glycoprotein of dictyostelium discoideum b-glycosidation of sterically hindered alcohols an aeromonas caviae genomic island is required for both oantigen lipopolysaccharide biosynthesis and flagellin glycosylation new triterpene glycoside from cyclamen adzharicum tubers mycobacterium leprae phenolglycolipid-1 expressed by engineered m. bovis bcg modulates early interaction with human phagocytes defects in flagellin glycosylation affect the virulence of pseudomonas syringae pv. tabaci 6605 mass spectrometric imaging of ginsenosides localization in panax ginseng root thermosensitive hydrogels composed of cyclodextrin pseudorotaxanes. role of [3]pseudorotaxane in the gel formation hydrogels composed of organic amphiphiles and a-cyclodextrin: supramolecular networks of their pseudorotaxanes in aqueous media a plant class v chitinase from a cycad (cycas revoluta): biochemical characterization, cdna isolation, and posttranslational modification distinct features of matrix-assisted 6 mm infrared laser desorption/ionization mass spectrometry in biomolecular analysis dissociation profile of protonated fucosyl glycopeptides and quantitation of fucosylation levels of glycoproteins by mass spectrometry loose-fit polyrotaxane composed of g-cyclodextrin and single poly(ethyelene glycol) chain: making room in g-cd cavity for additional inclusion complexation optically pure fullerodendron formed by diastereoselective dielse-alder reaction physiological and glycomic characterization of n-acetylglucosaminyltransferase-iva and -ivb double deficient mice synthesis and ring-opening polymerizations of novel s-glycooxazolines enrichment of amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography alterations in receptor-binding properties of swine influenza viruses of the h1 subtype after isolation in embryonated chicken eggs tlc blot (far-eastern blot) and its applications epimeric and amino disaccharide analogs as probes of an a-(1-6)-mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesis solution conformation of c-linked antifreeze glycoprotein analogues and modulation of ice recrystallization application of proteomics in biomarker discovery: a primer for the clinician synthesis of the glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan, which serve as a hexosamine acceptor for enzymatic glycosyl transfer baca is indispensable for successful mesorhizobium-astragalus symbiosis synthesis of a sialic acid containing complex-type n-glycan on a solid support a combined 6p-azaelectrocyclization/ staudinger approach to protein and cell engineering: noninvasive tumor targeting by n-glycan-engineered lymphocytes noninvasive imaging of dendrimer-type n-glycan clusters: in vivo dynamics dependence on oligosaccharide structure synthesis of non-natural xyloglucans by polycondensation of 4,6-dimethoxy-1,3,5-triazin-2-yl oligosaccharide monomers catalyzed by endo-b-1,4-glucanase novel dialkoxytriazine-type glycosyl donors for cellulase-catalysed lactosylation on-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct maldi-qit-tof ms analysis concanavalin a-immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line biomaterials from sugars: ring-opening polymerization of a carbohydrate lactone synthesis of a monophosphoryl derivative of escherichia coli lipid a and its efficient coupling to a tumor-associated carbohydrate antigen identification of n-glycan serum markers associated with hepatocellular carcinoma from mass spectrometry data novel neogala-series glycosphingolipids with terminal mannose and glucose residues from hirsutella rhossiliensis, an aureobasidin aresistant ascomycete fungus functional characterization of tlmh in streptoalloteichus hindustanus e465-94 atcc 31158 unveiling new insight into tallysomycin biosynthesis and affording a novel bleomycin analog the effect of temperature on the stability of compounds used as uv-maldi-ms matrix: 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, a-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, nor-harmane and harmane towards an integrated proteomic and glycomic approach to finding cancer biomarkers chemoenzymatic synthesis of multivalent neoglycoconjugates carrying the helminth glycan antigen ldnf fragment-based development of triazole-substituted o-galactosyl aldoximes with fragmentinduced affinity and selectivity for galectin-3 tempo oxidation of gelatinized potato starch results in acid resistant blocks of glucuronic acid moieties molecular sieves provoke multiple substitutions in the enzymatic synthesis of fructose oligosaccharide-lauryl esters synthesis of organic-soluble conjugated polyrotaxanes by polymerization of linked rotaxanes insulated molecular wire with highly conductive p-conjugated polymer core reaction of the antitumor antibiotic olivomycin i with aryl diazonium salts. synthesis, cytotoxic and antiretroviral potency of 5-aryldiazenyl-6-odeglycosyl derivatives of olivomycin i highly site-selective stability increases by glycosylation of dihydrofolate reductase gold-catalyzed glycosidations: synthesis of 1,6-anhydro saccharides site-specific glycoprofiling of n-linked glycopeptides using maldi-tof ms: strong correlation between signal strength and glycoform quantities purification and characterization of a new recombinant factor viii (n8) identification and quantification of n-linked oligosaccharides released from glycoproteins: an inter-laboratory study determination of nlinked sialyl-sugar chains in the lungs of domestic cats and dogs in thailand susceptible to the highly pathogenic avian influenza virus (h5n1) cyclodextrin-based hyperbranched polymers: molecule design, synthesis and characterization glycoproteomics: past, present and future molecular basis for galactosylation of core fucose residues in invertebrates. identification of caenorhabditis elegans n-glycan core a1,6-fucoside b1,4-galactosyltransferase galt-1 as a member of a novel glycosyltransferase family glycoproteomic analysis of abnormal n-glycosylation on the kappa chain of cryocrystalglobulin in a patient of multiple myeloma solvolytic depolymerization of chondroitin and dermatan sulfates a chromatographic approach for elevating the antibody-dependent cellular cytotoxicity of antibody composites structural characterization and surface-active properties of a succinoyl trehalose lipid produced by rhodococcus sp. sd-74 chemo-enzymatic synthesis of glycosylated insulin using a glcnac tag acceptor specificity in the transglycosylation reaction using endo-m coxiella burnetii glycomics and proteomics-tools for linking structure to function social self-sorting: alternating supramolecular oligomer consisting of isomers humanization of recombinant glycoproteins expressed in insect cells inactivation of mycobacterium tuberculosis mannosyltransferase pimb reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death the recognition motif of the glycoprotein-folding sensor enzyme udp-glc: glycoprotein glucosyltransferase enrichment method of sulfated glycopeptides by a sulfate emerging and ion exchange chromatography enzymatic solubilization of brewers' spent grain by combined action of carbohydrases and peptidases poly(n-vinylpyrrolidone) bearing covalently attached cyclodextrin via click-chemistry: synthesis, characterization, and complexation behavior with phenolphthalein chemical and enzymatic n-glycan release comparison for n-glycan profiling of monoclonal antibodies expressed in plants analysis of carbohydrates on proteins by offline normal-phase liquid chromatography maldi-tof/tof-ms/ ms carbohydrate structural analysis of wheat flour arabinogalactan protein the fine structure of neisseria meningitidis lipooligosaccharide from the m986 strain and three of its variants dihydrobenzoic acid modified nanoparticle as a maldi-tof ms matrix for soft ionization and structure determination of small molecules with diverse structures photoinduced isomerization of allyl alcohols to carbonyl compounds using dendrimer disulfide as catalyst miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry glycomics profiling of heparan sulfate structure and activity alkali-hydroxide-doped matrices for structural characterization of neutral underivatized oligosaccharides by maldi time-of-flight mass spectrometry phosphocholinecontaining glycosyl inositol-phosphoceramides from trichoderma viride induce defense responses in cultured rice cells targeted serum glycoproteomics for the discovery of lung cancer-associated glycosylation disorders using lectin-coupled pro-teinchip arrays chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucoselowering activity daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis solid-phase synthesis of glycopeptide carrying a tetra-n-acetyllactosamine-containing core 2 decasaccharide glycosylation specific for adhesion molecules in epidermis and its receptor revealed by glycoform-focused reverse genomics isolation and identification of arabinose mycolates of cell wall skeleton (cws) derived from mycobacterium bovis bcg tokyo 172 (smp-105) synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-dna conjugates with different carbohydrate presentation use of a novel ionic liquid matrix for maldi-ms analysis of glycopeptides and glycans out of total tryptic digests efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline milk oligosaccharides structural and functional characterization of a putative polysaccharide deacetylase of the human parasite encephalitozoon cuniculi glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction an oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides deciphering carbohydrate structures by ims-ms. applications to biological features related to carbohydrate chemistry and biology software platform for high-throughput glycomics preparation and properties of polyurethanes based on castor oil chemically modified with yucca starch glycoside synthesis and characterization of neurostatin-related compounds with high inhibitory activity of glioma growth o-fucosylation of an antibody light chain: characterization of a modification occurring on an igg1 molecule asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the c h 1 domain of human antibodies biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (tafi) glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase. glycosylation is required for optimal enzymatic activity n-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human igg immunoglobulin g galactosylation and sialylation are associated with pregnancyinduced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study structural and functional analysis of glycosphingolipids of schistosoma mansoni elucidation of molecular diversity and body distribution of saponins in the sea cucumber holothuria forskali (echinodermata) by mass spectrometry qualitative and quantitative saponin contents in five sea cucumbers from the indian ocean localization of secondary metabolites in marine invertebrates: contribution of maldi msi for the study of saponins in cuvierian tubules of h. forskali elevated sulfatide levels in neurons cause lethal audiogenic seizures in mice rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility glycome profiling using modern glycomics technology: technical aspects and applications symbol nomenclature for glycan representation synthesis of cyclic b-glucan using laminarinase 16a glycosynthase mutant from the basidiomycete phanerochaete chrysosporium purification and nglycosylation analysis of melanoma antigen dopachrome tautomerase rapana venosa hemocyanin with antiviral activity stable isotope-enhanced two-and threedimensional diffusion ordered 13 c nmr spectroscopy (sie-dosy 13 c nmr) modern maldi time-of-flight mass spectrometry orthogonal activation of propargyl and n-pentenyl glycosides and 1,2-orthoesters laser desorption-ionization of lipid transfers: tissue mass spectrometry imaging without maldi matrix characterization of branched polysaccharides using multiple-detection size separation techniques multigram synthesis of a hyaluronic acid subunit and synthesis of fully protected oligomers finding new posttranslational modifications in salivary proline-rich proteins enzymatic glycosylations on arrays rapid assembly of oligosaccharides: a highly convergent strategy for the assembly of a glycosylated amino acid derived from psgl-1 databases and informatics for glycobiology and glycomics production of nonfucosylated antibodies by co-expression of heterologous gdp-6-deoxy-d-lyxo-4-hexulose reductase characterization of the acidic n-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach pectin, a versatile polysaccharide present in plant cell walls expression of talaromyces emersonii cellobiohydrolase cel7a in saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity structural characterization of flavonoid glycosides by multi-stage mass spectrometry the absence of an identifiable single catalytic base residue in thermobifida fusca exocellulase cel6b quantitation of saccharide compositions of o-glycans by mass spectrometry of glycopeptides and its application to rheumatoid arthritis comparison of methods for profiling o-glycosylation. human proteome organisation human disease glycomics/proteome initiative multi-institutional study of iga1 fluorinated glycosyl amino acids for mucin-like glycopeptide antigen analogues a nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant alaskan beetle upis ceramboides glycans on influenza hemagglutinin affect receptor binding and immune response synthesis of a mannose-capped disaccharide with a thiol terminus steroidal saponins from the rhizomes of polygonatum odoratum oxidative stress-induced peptidoglycan deacetylase in helicobacter pylori convenient synthesis of an n-glycan octasaccharide of the bisecting type matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms). application in carbohydrate analysis molecular aggregation behavior of perylene-bridged bis(b-cyclodextrin) and its electronic interactions upon selective binding with aromatic guests functional characterization of tlmk unveiling unstable carbinolamide intermediates in the tallysomycin biosynthetic pathway silver-coated gold nanoparticles as concentrating probes and matrices for surface-assisted laser desorption/ionization mass spectrometric analysis of aminoglycosides conversion of squid pen by pseudomonas aeruginosa k187 fermentation for the production of nacetyl chitooligosaccharides and biofertilizers n-terminal deletion of peptide: n-glycanase results in enhanced deglycosylation activity a new chalcone glycoside, a new tetrahydrofuranoid lignan, and antioxidative constituents from the stems and leaves of viburnum propinquum cosmc is an essential chaperone for correct protein o-glycosylation role of a cytoplasmic dual-function glycosyltransferase in o 2 regulation of development in dictyostelium crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (roystonea regia) two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in listeria monocytogenes n-glycan analysis of recombinant l-selectin reveals sulfated galnac and galnac-galnac motifs glycosylation of the phosphate binding protein, psts, in streptomyces coelicolor by a pathway that resembles protein o-mannosylation in eukaryotes immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis comparative glycoproteomics: approaches and applications covalent modification of chitin with silk-derivatives acts as an amphiphilic self-organizing template in nacre biomineralisation expression of helix pomatia lectin binding glycoproteins in women with breast cancer in relationship to their blood group phenotypes analysis of site-specific glycosylation of renal and hepatic g-glutamyl transpeptidase from normal human tissue release and characterization of single side chains of white cabbage pectin and their complement-fixing activity characterization of wbpb, wbpe, and wbpd and reconstitution of a pathway for the biosynthesis of udp-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid in pseudomonas aeruginosa maldi-tof ms and ce-lif fingerprinting of plant cell wall polysaccharide digests as a screening tool for arabidopsis cell wall mutants branched arabino-oligosaccharides isolated from sugar beet arabinan derivatization of sialic acids for stabilization in matrix-assisted laser desorption/ionization mass spectrometry and concomitant differentiation of a(2-3) and a(2-6) isomers glycomic characterization of prostate-specific antigen and prostatic acid phosphatase in prostate cancer and benign disease seminal plasma fluids synthesis of bifunctional peptide derivatives based on a b-cyclodextrin core with drug delivery potential glycoprotein production for structure analysis with stable, glycosylation mutant cho cell lines established by fluorescence-activated cell sorting triple recognition of b-dna by a neomycin-hoechst 33258-pyrene conjugate introductory glycosylation analysis using sds-page and peptide mass fingerprinting synthesis and cytotoxic activity of g3pamam-nh 2 dendrimer-modified digoxin and proscillaridin a conjugates in breast cancer cells quantitative site-specific analysis of protein glycosylation by lc-ms using different glycopeptide-enrichment strategies profiling of n-glycosylation gene expression in cho cell fed-batch cultures an investigation of intracellular glycosylation activities in cho cells: effects of nucleotide sugar precursor feeding vitro bacterial polysaccharide biosynthesis: defining the functions of wzy and wzz ultrasound ionization of biomolecules new development of glycan arrays gold nanoparticles as assisted matrices for the detection of biomolecules in a high-salt solution through laser desorption/ionization mass spectrometry development of an annotated library of neutral human milk oligosaccharides synthesis of monomeric and dimeric repeating units of the zwitterionic type 1 capsular polysaccharide from streptococcus pneumoniae chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored glycopeptides synthesis of mangiferin, isomangiferin, and homomangiferin sortase a-catalyzed transpeptidation of glycosylphosphatidylinositol derivatives for chemoenzymatic synthesis of gpi-anchored proteins synthesis of talosin a and b, two bioactive isoflavonoid glycosides direct labelling of peptides with 2-[ 18 f]fluoro-2-deoxy-d-glucose ([ 18 f]fdg) structural glycomics using hydrophilic interaction chromatography (hilic) with mass spectrometry hexose rearrangements upon fragmentation of n-glycopeptides and reductively aminated nglycans ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry glycan reductive isotope labeling for quantitative glycomics core 3-derived o-glycans are essential for intestinal mucus barrier function rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies preparation of chitooligosaccharides by the enzymatic hydrolysis of chitosan potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro synthesis of well-defined 7-arm and 21-arm poly(nisopropylacrylamide) star polymers with b-cyclodextrin cores via click chemistry and their thermal phase transition behavior in aqueous solution chitooligosaccharides protect human embryonic hepatocytes against oxidative stress nduced by hydrogen peroxide on-plate enrichment of glycopeptides by using boronic acid functionalized gold-coated si wafer highly specific enrichment of glycopeptides using boronic acid-functionalized mesoporous silica n-glycosylation profiling of turtle egg yolk: expression of galabiose structure development and application of high performance liquid chromatography map of glucuronyl n-glycans comparative studies on the structural features of o-glycans between leukemia and epithelial cell lines hyphenated technique for releasing and maldi ms analysis of oglycans in mucin-type glycoprotein samples preparation and characterization of phospha sugar analogues, 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide derivatives, as novel anticancer agents synthesis of b-d-fructopyranosyl-(2 ! 6)-d-glucopyranose from d-glucose and d-fructose by a thermal treatment switching from altro-a-cyclodextrin dimer to pseudo[1]rotaxane dimer through tumbling a molecular reel: shuttling of a rotor by tumbling of a macrocycle selective enrichment of glycopeptides/phosphopeptides using porous titania microspheres synthesis of di-and tetrasaccharide containing 6-deoxytalose from the o-antigenic polysaccharide of b. pseudomallei strain 304b mechanism of mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide repeats leading to a complete series of exclusively oddnumbered oligosaccharides heterogeneous components of chitosans glucopyranoside-incorporated n-heterocyclic carbene complexes of silver(i) and palladium(ii): efficient water-soluble suzuki-miyaura coupling palladium(ii) catalysts detection of small neutral carbohydrates using various supporting materials in laser desorption/onization mass spectrometry synthesis of a novel class of glycocluster with a cyclic a-(1 ! 6)-octaglucoside as a scaffold and their binding abilities to concanavalin a isolation and structural characterization of a polysaccharide fcap1 from the fruit of cornus officinalis expression, glycoform characterization, and antibody-binding of hiv-1 v3 glycopeptide domain fused with human igg1-fc synthesis of kaempferol 3-o-(3 00 ,6 00 -di-o-e-p-coumaroyl)-b-d-glucopyranoside, efficient glycosylation of flavonol 3-oh with glycosyl odlkynylbenzoates as donors total synthesis and structural revision of tmgchitotriomycin, a specific inhibitor of insect and fungal b-n-acetylglucosaminidases chemoselective glycosylation of carboxylic acid with glycosyl ortho-hexynylbenzoates as donors homogeneous synthesis of grgdy grafted chitosan on hydroxyl groups by photochemical reaction for improved cell adhesion facile synthesis of 4-mercaptophenylboronic acid functionalized gold nanoparticles for selective enrichment of glycopeptides new triterpenoid saponins from the roots of gypsophila paniculata l the crystal structure of a xyloglucan-specific endo-b-1,4-glucanase from geotrichum sp. m128 xyloglucanase reveals a key amino acid residue for substrate specificity imidazolium cation supported solution-phase assembly of homolinear a(1 ! 6)-linked octamannoside: an efficient alternate approach for oligosaccharide synthesis growth phasedependent expression of proteins with decreased plant-specific nglycans and immunogenicity in tobacco by2 cells oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method-precursors for the synthesis of labeled conjugates developmental changes in glycolipids and synchronized expression of nutrient transporters in the mouse small intestine differences in n-glycan structures found on recombinant iga1 and iga2 produced in murine myeloma and cho cell lines extraction of glycosaminoglycan from sea hare, aplysia kurodai, and its functional properties 2. structural properties of purified glycosaminoglycan synthesis of dopamine and l-dopaa-glycosides by reaction with cyclomaltohexaose catalyzed by cyclomaltodextrin glucanyltransferase n-glycosylation status of b-haptoglobin in sera of patients with prostate cancer vs. benign prostate diseases unexpected tolerance of glycosylation by udp-galnac:polypeptide a-n-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups modification-specific proteomics in plant biology effect and limitation of excess ammonium on the release of o-glycans in reducing forms from glycoproteins under mild alkaline conditions for glycomic and functional analysis hydrophilic interaction chromatography based enrichment of glycopeptides by using click maltose: a matrix with high selectivity and glycosylation heterogeneity coverage construction and transmembrane dissociation behavior of supramolecular assembly of quinolinocyclodextrin with porphyrin metabolic labeling of glycoconjugates with photocrosslinking sugars identification of blood group a/ a-leb/y and b/b-leb/y active glycotopes co-expressed on the oglycans isolated from two distinct human ovarian cyst fluids a supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans synthesis and 13 c nmr spectroscopy of model compounds for the microstructure analysis of poly(vinyl glycoside)s production of anti-carbohydrate antibodies by phage display technologies. potential impairment of cell growth as a result of endogenous expression precise structure of acidic polysaccharide present in salvia hydrogels n-glycosylation in archaea: on the coordinated actions of haloferax volcanii aglf and aglm on-line separations combined with ms for analysis of glycosaminoglycans mass spectrometry and glycomics matrix-assisted laser desorption/ionization imaging mass spectrometry effect of enzyme processivity on the efficacy of a competitive chitinase inhibitor catalytic properties of endo-1,3-b-d-glucanase from the vietnamese edible mussel perna viridis cerebrocostomandibular-like syndrome and a mutation in the conserved oligomeric golgi complex, subunit 1 a new mutation in cog7 extends the spectrum of cog subunit deficiencies clickable lipids: azido and alkynyl fatty acids and triacylglycerols expression, purification, and characterization of recombinant human transferrin from rice structural characterization and anti-fatigue activity of polysaccharides from the roots of morinda officinalis a modified synthesis and serological evaluation of neoglycoproteins containing the natural disaccharide of pgl-i from mycobacterium leprae synthesis of novel aminoglycosides via allylic azide rearrangement for investigating the significance of 2 0 -amino group development of a plate-based scintillation proximity assay for the mycobacterial aftb enzyme involved in cell wall arabinan biosynthesis transient expression and purification of chimeric heavy chain antibodies extracting both peptide sequence and glycan structural information by 157 nm photodissociation of n-linked glycopeptides synthesis of neu5ac-gal-functionalized gold glyconanoparticles total synthesis of apigenin-4 0 -yl-2-o-(pcoumaroyl)-b-d-glucopyranoside recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics specific enrichment methods for glycoproteome research boronic acid functionalized core-satellite composite nanoparticles for advanced enrichment of glycopeptides and glycoproteins condensed tannins from mangrove species kandelia candel and rhizophora mangle and their antioxidant activity lew3, encoding a putative a-1,2-mannosyltransferase (alg11) in n-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in arabidopsis thaliana a perspective on the maillard reaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease a novel strategy for maldi-tof ms analysis of small molecules steroidal saponins from the rhizomes of paris delavayi acute kidney injury induced by proteinoverload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides prediction of collision-induced dissociation spectra of common n-glycopeptides for glycoform identification mass spectrometry for structural characterization of therapeutic antibodies analysis of protein glycosylation and phosphorylation using liquid phase separation, protein microarray technology, and mass spectrometry preparation and characterization of galacto-mannan-oligosaccharides hydrolyzed from guar gum by bmannanase inclusion behavior of bcyclodextrin with bipyridine molecules: factors governing host-guest inclusion geometries combination of b-elimination and liquid chromatography/quadrupole time-of-flight mass spectrometry for the determination of o-glycosylation sites study of matrix additives for sensitive analysis of lipid a by matrix-assisted laser desorption ionization mass spectrometry microwaveassisted sample preparation for rapid and sensitive analysis of h. pylori lipid a applicable to a single colony bc10, a duf266-containing and golgi-located type ii membrane protein, is required for cell-wall biosynthesis in rice synthesis of ionic liquids functionalized b-cyclodextrin-bonded chiral stationary phases and their applications in high-performance liquid chromatography rapid identification of gallotannins from chinese galls by matrix-assisted laser desorption/ ionization time-of-flight quadrupole ion trap mass spectrometry engineered nanoparticle surfaces for improved mass spectrometric analysis convenient synthesis of sulfated oligofucosides synthesis of a mannose hexasaccharide related to the cell wall mannan of candida dubliniensis and trychophyton mentagrophytes synthesis of the 6-deoxytalose-containing tri-and hexasaccharides of the o-antigen polysaccharide from mesorhizobium huakuii ifo15243t highly efficient removal of allyloxycarbonyl (alloc) function provides a practical orthogonal protective strategy for carbohydrates dual reversible self-assembly of pnipam-based amphiphiles formed by inclusion complexation cytotoxic triterpenoid saponins acetylated with monoterpenoid acid from albizia julibrissin key: cord-006230-xta38e7j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: 2012-02-22 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-012-0736-0 sha: doc_id: 6230 cord_uid: xta38e7j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, 2.4 mg/kg/d 4 days) via minipumps. chronic iso increased mrna levels of ndpk c by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within 3 hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a 1.5-fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~50%. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. 1, 2 , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line hct116 and the human hepatoma cell line hepg2 as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h 2dcf-da assay in hct116 cells) decreased with increasing methyl groups in the b-ring. myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct116 cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly389arg variant of the β1-adrenergic receptor (β1ar) on receptor conformation we used β1ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β1ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg389-β1ar did not show altered activation kinetics after prestimulation, the gly389-β1ar became slower compared to the initial stimulation suggesting that β1ars possess a memory of previous activation. we then permeabilized β1ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β1ar variants did not display receptor memory, suggesting that the β1ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna-145 or mirna-33a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. 1 micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir-145, which shows decreased levels in colon carcinoma. in contrast, while mir-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir-33a and identify the protooncogenic kinase pim-1 as a target of mir-33a. pim-1 harbours a highly conserved mir-33a binding site within its 3'-utr, and seed mutagenesis of this target sequence abolishes the mir-33a-mediated downregulation of pim-1. the knockdown of pim-1 by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir-33a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna-145 through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk5. likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir-33a. this is due to the mir-33a-mediated downregulation of pim-1 expression and resembles the pim-1 knockdown through rnai / pim-1 sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir-145 and mir-33a may be promising mirnas in colon carcinoma therapy. md 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md 288 to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md 288 was also investigated. the potential of md 288 to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v79 lung fibroblasts (v79 cells). v79 cells were treated with 0.4 µm, 0.8 µm and 1.5 µm md 288 or the positive control, the direct mutagen 4-nitroquinoline-n-oxide (nqo, 1 µm) for 24 h. on day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . whereas at 1 µm chloroquine, a 38% decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, 10 µm md 288 were needed to observe a similar decrease (28%) in fluorescence intensity of eb. therefore, md 288 is 10fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells was 9 ± 2. as expected, 1 µm nqo caused a significant increase in the mutant frequency (mf, 168 ± 9) . in contrast, mf was not significantly affected by treatment with md 288 at both noncytotoxic (0.4 µm: 5 ± 4) and cytotoxic (0.8 µm: 9 ± 7) concentrations. in conclusion, md 288 is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md 288 does not cause gene mutations in the hprt test. since current studies show that various metabolites of md 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca 2+ entry or depolarisation. recently, we showed that trpm4 acts as a ca 2+ -activated non-selective cation channel and critically determines the driving force for ca 2+ influx in mast cells following fcεri-stimulation (1) . in addition to trpm4 we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc2, trpc3, trpc5, trpc6 and trpm7. in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc1, trpc4, trpc5, trpc6, trpm2, trpm4 and trpm5. to identify the functional role of those trp channel proteins for mast cell activation we analysed ca 2+ signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc6 channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp-9 and flufenamic acid (ffa), but could not evoke a rise in the [ca 2+ ]i in both pmc and bmmc. sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate trpc5 channels, induced only minor rise in [ca 2+ ]i in bmmcs, respectively. here, we will present our analysis of ca 2+ signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance p and compound 48/80 in bmmcs and pmcs derived from mouse lines with inactivation of trpc1, trpc3, trpc4, trpc5 or trpc6 since specific antagonists are still lacking for these trp channels. the α2a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α2a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α2a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α2a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α2a-ar activation with very high temporal resolution, we took advantage of the previously described α2a-ar flash/cfp sensor [1] . the results indicate that in our system the efficacy of ne in eliciting α2a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. the ec50 values decrease in an exponential manner and arrive at ~ 2 µm with a half-life of ~ 330 ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α2a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [2] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine-1-phosphate (s1p) is an immune modulator produced by sphingosine kinase 1 (sphk1) and sphingosine kinase 2 (sphk2) and de-phosphorylated or degraded irreversibly by s1p phosphatases and a lyase, respectively. we recently showed that tlr4-induced il-12p70 is selectively counter regulated by sphk1, s1pr1 and its extracellular ligand s1p. on the other hand, spiegel et al. have demonstrated that specific, sphk1-dependent, binding of s1p to traf2 enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s1p. in a first approach we focused our investigations on sphk1 effects on both traf2 and traf6 stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr4 or cpg-tlr9 signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il-12p70 secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf2 activated via tnf-alpha interacted with the traf6 pathway to reduce il-12p70. further series with dcs derived from sphk1-deficient mice confirmed our former results that il-12p70 in contrast to other cytokines is specifically sensitive to sphk1-s1p feedback, but did not change the effects of tnf-alpha on wt dcs il-12p70 release. in comparison, cpg-tlr9-induced il-12p70 release reached only 20% of lps-induced il-12p70 levels and was less sensitive to tnf-alpha costimulation. however, sphk1-deficiency strongly augmented cpg-dependent il-12p70 production. in ongoing experiments we started to analyze the details of traf2/rip1 and traf6/tak1 activation by ubiquitination blots in wt, sphk1-and s1plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s1p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. 1 , besche v. 2 , bros m. 2 , li h. 1 , handler n. 3 , bauer f. 3 , erker t. 3 , behnke f. 4 , mönch b. 5 , förstermann u. 1 , dirsch v. m. 6 , werz o. 5 , kleinert h. 1 , pautz a. university of vienna department of pharmacognosy, althanstr. 14, 1090 wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin 1 (sirt1) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld-1 cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna-155 function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p38 mapk activation or activity. in addition we have evidence that sirt1 is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. 1 the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all 16 nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude 61, 66421 homburg, germany tmem2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek293 cells. tmem2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura-2 revealed that the elevation of the cytosolic ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem2-deficient acinar cells. tmem2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem2 fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek293 cells the tmem2-eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem2-eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem2-eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) 0,79 ± 0,03, n= 4 using lysotracker ® dye). analysis of subcellular localization with independent tmem2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem2 and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem2 -/mice. the small molecule bcl-2/mcl-1 inhibitor tw-37 shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. 1 , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl-1 or bcl-2, with high expression correlating to high risk phenotype. co-expression can be detected in approximately 10% and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl-2 and other proteins, but not or to a lesser extend mcl-1, elucidated the importance of mcl-1 inhibition for cytotoxicity in nb. tw-37 is a small molecule inhibitor that showed almost equal affinity to bcl-2 and mcl-1. to explore the effect of combined bcl-2/mcl-1 inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr-5, sy5y and kelly) were treated with tw-37 and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw-37. using sirna, we investigated the functional relevance of mcl-1 and bcl-2 in kelly cells. for in vitro cell viability we observed ic50 values of 0.59 ± 0.39 µmol/l. on treatment with 1 µmol/l dose of tw-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl-2 as well as mcl-1 knockdown induced apoptosis in kelly cells. interestingly, tw-37 was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl-2 and mcl-1 using tw-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl-2/mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. 1 , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e1s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein 1 (oscp1) and the organic anion transporting polypeptides oatp6a1 and oatp1c1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek293 cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek293 cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ 3 the hepato-intestinal induction of the detoxifying enzymes cyp3a4 and cyp3a5 by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp3a4, cyp3a5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp3a5 promoter we demonstrate that the cyp3a5 expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang 1 (yy1)-binding site from the cyp3a5 promoter which occurred in haplorrhine primates. this yy1 site is conserved in cyp3a4, but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy1 binding site from promoters of the cyp3a5 gene lineage during primate evolution may have enabled the utilization of cyp3a5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp3a5 and cyp3a4. serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. 1 , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap-1) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant 0315370d) . barlow s. harrington house, 8 harrington road, brighton, bn1 6re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. in 1995, from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in 2004, kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova (1 mg/ml) was dissolved in sodium borate buffer (0.1m, ph 9.4) . for chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (dnfb; sensitizer) or 2,4-dichloro-1-nitrobenzene(dcnb; non-sensitizer) was added and stirred for 2 hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with 100 µg/ml ova and blocked with 10% fcs in pbs. ova samples (native or conjugated; 0.6 -10 µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for 30min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace 50% of ab to plate-bound ova (ic50) was calculated (minimum of n=3 independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic50 value, whereas mock treatment resulted in comparable ic50 values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic50 value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. 2´-deoxy-camp in human cell lines: another second messenger? beckert c. 1 , hinz c. we have shown that 2´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~10 was observed. as a remarkable exception, in hl-60 promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than 3-fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek293 cells after 24 hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek293 cells ac assays (mn 2+ /forskolin-or mn 2+ /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde3 and pde4 hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek293 cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h1r -h4r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h1r and h4r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h1r and h4r. in order to analyze these signaling pathways, we established a cellular model using transfected hek 293 cells which stably express recombinant mh1r or mh4r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca 2+ concentration ([ca 2+ ]i) in cells expressing either of both, mh1r (pec50 = 8.2) or mh4r (pec50 = 6.9). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh1r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh4r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h1r-expressing cells were blocked by the h1r antagonist mepyramine ([ca 2+ ]i: pkb = 8.6) and those in the h4r-expressing cells by the h4r antagonist jnj7777120 ([ca 2+ ]i: pkb = 8.8) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj7777120, which behaves as a partial mh4r agonist in the steady-state gtpase assay using membranes of infected sf9 cells, was without effect on [ca 2+ ]i and forskolin-induced camp-accumulation in the mh4r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h1r and the h4r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk1/2, p38, jnk, creb, pkb (akt), and mkk 3/6 were detected in cells expressing either of both, mh1r and mh4r. in summary, the hek 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels belkacemi a. 1 cavβ subunits of voltage-activated ca 2+ channels are required for trafficking the poreforming cavα1 subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih 3t3 fibroblasts do express cavβ2 and cavβ3 subunits, but we could not detect any voltage-activated ca 2+ influx. whereas in mouse cardiomyocytes or hek 293 cells coexpressing cavβ3 and cavα1 subunits a dihydropyridin-sensitive voltage-activated ca 2+ influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca 2+ influx. among the proteins potentially interacting with cavβ3 are the inositol 1,4,5-trisphosphate receptors (ip3rs) [1, 2] . we therefore coexpressed mouse cavβ3 and mouse ip3r type 1, 2 or 3 in cos-7 cells and found coimmunoprecipitation of ip3rs using an antibody for cavβ3 and vice versa. to study the release of ca 2+ from ip3-sensitive stores we performed fura-2 measurements on fibroblasts isolated from wild type and cavβ3-deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par-1, lpa or bradykinin. receptor-activated ca 2+ release was more pronounced in β3-deficient mefs and cfs, whereas thapsigargin-induced ca 2+ release was the same in cells from both genotypes. in addition, ip3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of ip3-releasable ca 2+ . [1] berggren, yang, murakami, et al., removal of ca channel β3 subunit enhances caoscillation frequency and insulin exocytosis. cell 119, (2004) 273-284. [2] müller, haupt, bildl, et al., quantitative proteomics of the cav2 channel nanoenvironments in the mammalian brain. pnas 107, (2010) 14950-14957. bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc42 by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat-1-mediated arginine transport by pma (vina-vilaseca et al, j biol chem 2011 286:8697) . this mechanism requires nedd4 e3 ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u373 glioblastoma cells overexpressing hcat-2a-egfp. in addition, sirna-mediated knock down of cdc42 prevented the decrease of hcat-2amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc42 is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. 1 parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase 1. by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp1-dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap1. whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap1 is a specific target of the kinase pink1 (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase 1. we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table 1) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek293 cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac1, mac3, mac9, soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca 2+ homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca 2+ -channels and the expression of ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (ddd mode) for one year. 7 minipigs were paced from the rvfw and 7 minipigs from the lva, respectively. 7 minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca 2+ currents. western-blots were carried out to investigate the expression of the ca 2+handling proteins l-type ca 2+ -channel, serca2 and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca 2+ -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca2-expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca 2+ -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. 1 the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like 3d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd 431) and irritation (oecd 439) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp 1a, 2b and 3a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo 1/3 activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [1] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [2] or propanal (aldh) oxidation [3] . nat1 activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project (0315226d). signalling pathway indicates that in b104 cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to 58% of the tolerable weekly intake (twi) of 2.5 µg/kg bw for cd defined by the european food safety authority (efsa) in 2009. beverages and vegetables are the food groups most relevant for exposure to pb. about 3.7 µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with 94%. for pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα12/13 and gαq via specific rhogefs. the p63rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p63rhogef and characterize the dynamics of p63rhogef-gαq-interaction in single living cells. the fusion of p63rhogef with venus resulted in a functional gαq-regulated p63rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h1and cholinergic m3-recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p63rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p63rhogef was verified by introducing point mutations rendering p63rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p63rhogef and gαq upon cotransfection of rgs2, suggesting a very short lifetime of the p63rhogef-gαq-complex or the ability of rgs2 to bind to p63rhogef-associated gαq. taken together, fret-based imaging of the interactions between p63rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs2. toxicity of silver nanoparticles in intestinal cells boehmert l. 1 the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco-2 is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco-2 cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a4f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco-2 cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. 1 , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp2a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06112 halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2a (pp2a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n=3) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein 25 (hsp25) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp25 protein was increased (p<0.05, n=7-8) but not camkii (p>0.05). protein phosphatase type 5 (pp5) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p<0.05, . in contrast, pp5 protein was upregulated (p<0.05, in tg compared to wt. for comparison the regulatory a-subunit of pp2a and hsp90 were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp2a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h2s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. 1 , hassan m. there is increasing evidence that hydrogen sulfide (h2s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e2-related factor 2 (nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf2 -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h2s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf2 protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf2 may constitute a protective mechanism during glomerular inflammatory disease. rac1 knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. 1, 40225 düsseldorf, germany rac1 belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac1 is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap1. anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac1 in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac1 may be involved in the ddr. to study the in vivo function of rac1 we used an inducible cre-based knockout mouse model (rac1 flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh2ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone 3 as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac1 the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp1 mrna expression was reduced in the liver of rac -/animals as compared with rac1 proficient animals. in addition we found more apoptotic cells in rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gh2ax foci in rac1 -/animals was reduced in comparison to rac1 +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac1 -/mice. none of these protective effects resulting from rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. there were no significant differences in the number of gh2ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac1 -/animals. furthermore the number of mitotic events was almost two times higher in the rac1 -/mice compared to the rac1 +/+ mice. summarizing, our findings show that impaired hepatic expression of rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse 56, 72074 tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine 3t3 cells with tbhq in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf2 pathway. s14 056 angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type 1 receptor brand s. 1 , amann k. 2 , schupp n. 1 1 universität würzburg institut für pharmakologie und toxikologie, versbacherstr. 9, 97078 würzburg, germany 2 universität erlangen-nürnberg institut für pathologie, krankenhausstraße 8, 91054 erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c57bl/6 mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c57bl/6 mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c-3t3 cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm 15, 30173 hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c-3t3 cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c-3t3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c-3t3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c-3t3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s9. in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b 1 and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c-3t3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac5 and ac6 being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac5 and ac6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac5, as well as overexpression of ac6 both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac5 ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from 16-20 week old homozygote ac5 knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine 5'-triphosphate (gtp) and 5'-o-(3thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac1-9, gs-, gi-a and β1-, β2-ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac5 knockout, mrna expressions analysis of ac1-9, gs-, gi-a and β1-, β2-ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac5. whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac5 and ac6, or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk-1/2 and lkb-1 in hypothalamic gt1-7 cells breit a., ellen d., gudermann t. goethestrasse 33, 80336 münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin-4 receptor (mc4r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt1-7 cells) and monitored ampk phosphorylation at thr 172 which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr 172 and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser 79 . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases-1/2 (erk-1/2), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk-1/2 was blunted by pka inhibitors, we propose that erk-1/2 serves as a link between pka and ampk in gt1-7 cells. furthermore, down-regulation of liver kinase b-1 (lkb-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk-1/2 and lkb-1 in gt1-7 cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than 100 candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav1.2 gene (cacna1c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell 2004; 119:19-31) . functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (herzig et al., faseb j. 2007; 21:1527-38; jangsangthong et al., pflugers arch. 2010; 459:399-411) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (cacnb2) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb2 in 155 patients with asd. we found three rare missense mutations in asd patients, but not in 259 unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav1.2 subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β2 subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav1.2. β2 subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. (2010) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta (1) subunits. pflugers arch 459, 399-411. herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. (2007) . mechanism of ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. faseb j 21, 1527-1538. splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. (2004) . ca(v)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell 119, 19-31. trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. (2006) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] background: brain serotonin (5-ht) has been implicated in the regulation of food-intake. the ingestive effects of 5-ht are mediated by a number of different receptor subtypes under which the 5-ht1a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by 5-ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei or by postsynaptic 5-ht1a heteroreceptors in serotonergic terminal structures. methods: the effect of the 5-ht1a receptor agonist 8-oh-dpat (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l35 mice. l35 mice are characterized by an overexpression of postsynaptic 5-ht1a receptors. results: the administration of the 5-ht1a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the 5-ht1a receptor in food intake. further, we make the assumption that the hyperphagic effect of 8-oh-dpat is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei which decreases 5-ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-ht1a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc2 (mrp2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) 1β on abcc2 mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc2 expression while inflammation. three different cell lines a) hepg2 cells (liver tissue) and b) caco2 (colon tissue) both without naïve il1β expression and c) skhep1 cells representing physiological liver tissue with naive il1β expression, were stimulated with different concentrations of il1β (range 10 pg/ml to 10 ng/ml). over a period of 48h samples were taken at defined time points. abcc2 mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p38 mapk, akt, erk1 and jnk) we analysed the signal transduction pathways associated with il1β-mediated transcriptional abcc2 regulation. on abcc2 mrna level an up-regulation in caco2 cells (1, 35 fold) and hepg2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml il1β was shown. in contrast skhep1 cells demonstrated a decreased abcc2 mrna expression (0,59-0,62 fold) in comparison to unstimulated controls. the abcc2 protein expression exhibited a time and il1β dependent regulation as well. the analysis for the signal transduction showed for p38mapk a moderat time dependet down regulated phosphorylation (15%) in hepg2 cells whereas it showed no effect in caco2 cells. concluding, the expression of abcc2 is regulated moderately by il1b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc2 expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from 21 cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of 667 micrornas. statistical analysis using the 2 -∆ct method was performed. micrornas showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan 1 , mirdb 2 , pictar 3 , microcosm 4 , diana microt 5 ), selected putative target genes were further functionally investigated by the david bioinformatics database 6 . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad4, nras, rb1, raf1). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and 12 micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta2-adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. 1 , buschauer a. activation of the β2-adrenergic receptor (β2ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β2ar ligands on cyclic adenosine 3',5'-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o2 •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o2 •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o2 •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β2ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β2ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β2ar antagonists in the camp assay and the o2 •assay are different. such differences were previously reported for β2ar antagonists in other test systems [4] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o2 •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human 5-ht2a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße 44, 39120 magdeburg, germany the serotonin 2a (5-ht2a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of 5-ht2a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of 5-ht2a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged 5-ht2a receptor by internalization. using immunocytochemical techniques in stably transfected hek293 cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the 5-ht2a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized 5-ht2a receptors colocalize with a488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-ht2a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces 5-ht2a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-ht2a internalization to occur. overall, we conclude that the internalization of the human 5-ht2a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of 5-ht2(a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-ht2a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung 4, kurt-georg-kiesinger-allee 3, 53175 bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma 2007). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma 2010) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/100). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method 4.1.1) and for atropine (ph. eur. method 3.1.1 or 4.1.1) based on lhrd leads in each case to a suggested fsd of d7 related to 10 g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed fsd of d6 related to 10 g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in c. elegans büchter c. 1 , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin (100 µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h 2dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives (100 µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf-16, the nad + -dependent protein deacetylase sir-2.1 and the heat-shock transcription factor hsf-1, respectively. lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf-1 loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1. in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma 2 bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany activation of phospholipase c-γ2 (plcγ2) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ2 is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology 2 (sh2) domains and one src homology 3 (sh3) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ 2 is also regulated by autoinhibitory elements within its specific array (walliser et al., 2008; everett et al., 2011) . our recent data demonstrated that plcγ2 is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh2 (sh2c) and the sh3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ2. plcγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon bcr stimulation (kim et al., 2004) . both tyrosines are located in the linker between the sh2c and the sh3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ2. interestingly, a novel phosphorylation site in plcγ2 was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue 733 (rikova et al., 2007) . in this work, we demonstrate, for the first time, the activation of plcγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr733 is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ2 at tyr733. molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. 8-10, 10589 berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg2 was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between 5 µm and 25 µm. at concentrations higher than 25 µm the substance was cytotoxic to the cells (ic 50 47µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (hnf4α) by incubating the cells already with moderate concentrations of pfoa at a level of about 1 µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s17 070 human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. 1 , bumke scheer m. (kao et al., 1995) . two daughter cell lines were developed from m13sv1 after x-ray irradiation (m13sv1 r2) and an additional transfection with a mutated erbb2 oncogene (m13sv1 r2-n1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., 2010) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using 2d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. (1995) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h1-receptor (h1r) shows no ameliorating effects on asthmatic symptoms, h4r antagonists may be new drugs for asthma therapy. in addition to the h3r-and h4rselective antagonist thioperamide, the selective h4r antagonist 1-[(5-chloro-1h-indol-2yl)carbonyl]-4-methylperazine (jnj7777120) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj7777120 as well as the selective h3r antagonist 1-({4[3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (jnj5207852) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 m znso4 using 30 µl of plasma. analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp 80a mercury column (10 x 2mm; 4µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph 5) and methanol at a flow rate of 0.4 ml/min. the analytical run-time was 5 minutes. for plasma samples the assay was linear over a concentration range of 0.078 -40 µg/ml for jnj7777120 and jnj5207852, and 0.313 -40 µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, jnj7777120 and jnj5207852 in a range of 0.004 -2 µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk5 plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk5, one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk5 is already expressed during embryogenesis and we can detect it in the developing heart. however, grk5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk5 on cardiac signalling and development. tools for grk5 specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk5 in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk5 may potentially serve as a candidate gene for congenital heart disease. identification of a hcn3 interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn1, hcn2 and hcn4 channels have been studied in quite some detail there is only little information on the particular role of the hcn3 channel. as an important step towards achieving a better understanding of hcn3 function we set out in this study to identify proteins that are assembled with hcn3 in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn3 c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn3 using heterologous coexpression in hek293 cells. here, we provide an in-depth analysis of the functional interaction between hcn3 and one of the identified interacting proteins (hip3.1). we show that hip3.1 physically binds to hcn3 channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc1 and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn1 and generated a series of tpc1 antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc1 knock-out model by our self-generated tpc1 antibodies. tpc1 knockout mice are viable and do not show any obvious deficits. to isolate tpc1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc1 by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc1. foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf-7 breast cancer cells and mcf-7 cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo4 overexpressing cells was a20 a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a20 is a direct target of foxo4, a luciferase reporter containing a 1.2 kb of the a20 promoter was co-transfected with different amounts of foxo4 wild-type expression construct. foxo4 induced a dosedependent increase in a20 promoter activity, supporting the assumption that a20 is a direct transcriptional target of foxo4. overexpression of foxo4 led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf-7 cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a20, an ubiquitin modifying enzyme, as a novel foxo4 target gene. our data implicate that sustained foxo4 expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm7 is essential for motility, proliferation and cell growth. up-regulation of trpm7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm7 channel is inhibited by the known modulators of sk1-3 channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns8593, ska31, ucl 1684. the most potent of these compounds, ns8593 (ic50 1.6 µm), interferes with the regulation of trpm7 by cytosolic mg 2+ . here we show that ns8593 (10 µm) fully and reversibly inhibits native trpm7-like currents in hek 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm7 currents by ns8593 would impact cellular processes known to be affected by a genetic inactivation of trpm7. we found that ns8593 (10-30 µm) suppressed motility of hek 293 cells without a detectable effect on cell viability. taken together, our findings indicate that ns8593 is a potent and reversible inhibitor of endogenous trpm7 currents and may be a good candidate drug for pharmacological targeting of trpm7. sulfur mustard (2,2'-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance 2-chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed 10 minutes after treatment with 3 mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp-1 becomes activated upon treatment with mustards. parp-1 is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp-1 is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape1 and ercc1, i.e. endonucleases involved in ber and ner, respectively. the reduction of ape1 expression had no effect on parp activity. surprisingly, the knockdown of ercc1 almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc1-parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt-888. expression and activity of g protein coupled receptor kinase 2 (grk2) are elevated in several conditions of compromised heart function. although grk2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk2inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf1 but it also acts as a physiological inhibitor of grk2 upon phosphorylation by pkc at serine153. a detailed understanding of the rkip/grk2 interaction may help to identify inhibitory compounds for grk2. since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf1-into a grk2-inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk2, we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk2 association. this implicates, that dimerization of rkip is essential to bind grk2. to determine whether rkip dimers consequently inhibit grk2 activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk2 inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk2 binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk2 interaction and will help to develop specific grk2 inhibitors. expression and function of trpm3 ion channels in epithelial mdck2 cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. 1, 66421 homburg, germany trpm3 proteins build ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . trpm3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . however, northern blots and in situ hybridization experiments revealed that trpm3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm3 is also expressed in madin-darby canine kidney2 (mdck) cells. quantitative analysis of trpm3 expression by qrt-pcr revealed a ~ 5 fold upregulation in mdck2 cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm7, a related ion channel described as regulator of proliferation in other cell types, remained constant. hek293 cells overexpressing trpm3 channels did not proliferate in the presence of the trpm3 agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck2 cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca 2+ imaging experiments using fura2 revealed that pregnenolone sulphate induces ca 2+ entry in well separated mdck2 cells but not in cells growing in confluency. we hypothesize that trpm3 might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck2 cells. inhibition of grk2 by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase 2 (grk2). grk2 initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk2 are upregulated in human heart failure, it has been proposed that grk2 inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk2 inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r9c ). pln r9c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r9c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r9c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r9c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi2 and gαi3 with gαi2 as the predominant isoform. gene-targeted mice lacking either gαi2 or gαi3 were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi2-deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi2-deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi2deficiency. to assess the pathophysiological consequences of platelet gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi3deficient mice were strongly protected from injury. thus we suggest that gαi2 and gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b16 melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog 8-br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b16 melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk1/2 and p38 phosphorylation, growth and migration of b16 melanoma cells. similar results were obtained with wm1205 human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav2.3 (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) 1 , which is associated with absence epilepsy. cav3.2 is one of the two t-type calcium channels known to be expressed in the rt 2 . it has been demonstrated that ablation of either cav2.3 or cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav2.3(-|-), cav3.2(-|-) and cav3.2(-|-)xcav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav2.3 and cav3.2 calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip1/rip3-dependent necroptosis in neuronal cells diemert s. 1 , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase 1 dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring 15, bldg. n, 54286 trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat1 enzyme activities after incubation of thp-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat1 protein inhibition was observed for 100 and 1000 µm cyanamide using over-expressed human recombinant nat1 (insect cell cytosol containing recombinant human nat1*4). however, no inhibition was found in the presence of recombinant nat2*4. as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat1 or nat2 cytosol, by thp-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat1. further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat1, leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat1, which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat1 dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc6 a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca 2+ influx and the subsequent contraction of pasmc [2] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc6 channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . trpc-expression in freshly isolated and passaged pasmc cultured in low (5%) and high fetal bovine serum (25%) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc1 and trpc6 the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek293 cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc6 and inducing acute hpv in pasmc will be presented. references [1] staub, n. c. (1985) . site of hypoxic pulmonary vasoconstriction, chest 88, 240s-245s. [2] weissmann, n. et al. (2006) . classical transient receptor potential channel 6 (trpc6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. 103, 19093-19098 trpv5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv5 is assumed to contribute to the maternal-fetal calcium transport. most probably trpv5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv5 protein in the placenta that might contribute to the regulation of the trpv5 protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv5 (aa 1-330 and 571-723) as trpv5-gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv5 interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv5 protein is calpain 6. in contrast to the classical calpains (calcium activated cystein proteases), calpain 6 is unique in that it lacks the cysteine residue in the active site. calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv5 and calpain 6 was confirmed in reciprocal pulldown experiments and the trpv5 binding region for calpain 6 was narrowed down by using overlapping n-terminal trpv5-gst fusion proteins. after injection of trpv5 crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain 6 crna. in further experiments we want to study potential regulatory effects of the trpv5 protein on the calpain 6 function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. 45, 48149 münster, germany it has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. 2011 , j toxicol. 2011 we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g2/m-and s-phase arrest as well as subg1 peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg1, caspase 3 activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h2o2 induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h2o2 induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse 27, 4012 basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr5/app/ps2) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. at 12 months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c57/bj6-thy1-appsl (ad-mice) mice that were fed with 600 mg olesoxime/kg feed for 3 months. drug plasma levels reached approx. 600 ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca 2+ -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca 2+ -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca 2+ -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the 7th framework program for rtd -project mitotarget -grant agreement health-f2-2008-223388. several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µm) for 10 min. thereafter, the cell lysates were subjected to diagonal 2d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine 157 of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf2-keap1-antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf2 pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq1. the lusens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. 1 ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il-5 and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße 1, 30625 hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd16antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. we stimulated eosinophils with varying concentrations of il-5, eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of 2-4 pm/1.5x10 6 eosinophils. neither il5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation. our results suggest that chemotactic molecules like eotaxin and il-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse 38, 67056 ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg 427 or 428. however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. after 6h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a549 lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p53 into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a549 and saos osteosarcoma cells. the internalized biotin-p53 also partially co-localized with early endosomes. importantly, the delivery of biotin-p53 into p53-deficient saos cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. 1 , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb126. activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb126. thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb126 leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a (1, 2) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb126. already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the 69 genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch-223191 and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq1as a novel ahr target protein in rat liver oval cells. although the function of foxq1 is poorly understood, it has been shown very recently that foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h1 and h3 receptors in the brain can be easily deduced since h1 receptor antagonists of the first generation have a sedative effect and an inverse h3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h2 and h4 receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked 3 h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h2 agonist impromidine 10 µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pec50 was 6.9 and the pa2 of the h2 antagonist ranitidine against impromidine was 7.2. with respect to h4 receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h4 receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h4 agonist 4-methylhistamine 10 -30 µm but inhibited by histamine 10 µm via h3 receptors by 28 and 55 %, respectively. in mouse cortex membranes, 4-methylhistamine 30 µm also failed to affect 35 s-gtpγs binding although r-α-methylhistamine 3 µm, acting via h3 receptors, increased it by 20 %. in conclusion, h2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h4 mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 s-gtpγs binding (mice), could not be shown. murine cx40 promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany connexin 40 (cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx40 gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx40 down-regulation, suggesting that creb related transcription factors are involved in cx40 gene regulation. in order to study the functional role of creb in the regulation of the cx40 promoter we have generated a luciferase based murine cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx40 promoter activity (cacreb 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dncreb 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18). the activity of the murine connexin 40 promoter is modulated by creb. both cacreb and dncreb led to cx40 down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf1 transcription factor family, which in turn could suppress cx40 promoter activity. (supported by the dfg) results: fxa increased par-2 mrna, protein and cell-surface expression and augmented par-2-mediated mitogenesis. par-1 expression was not influenced. the regulatory action of fxa on par-2 was concentration-dependent and mimicked by a par-2 selective activating peptide. the thrombin inhibitor argatroban or par-1 gene silencing did not influence fxa-stimulated par-2 expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox-1 in smc. nox-1 gene silencing prevented fxa-stimulated par-2 regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par-2 expression and mitogenic activity. fxa induced nuclear translocation and par-2 dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par-2 expression. in separate studies, fxa promoted par-2 mrna stabilisation through increased human antigen r (hur)/par-2 mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par-2 expression. conclusion: expression and mitogenic activity of vascular par-2, but not par-1, is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox-1-containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor 4 aim: we recently reported that functional expression of par-4 thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par-4. methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par-4 mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose (25 mm vs 5.5 mm) slowed par-4 mrna degradation in the presence of actinomycin d. par-1 mrna decay was not affected. hur binding to par-4 mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par-4 mrna. hydrogen peroxide (h 2o2) also induced cytosolic hur shuttling and increased par-4 mrna and total protein expression. the role of endogenously generated h2o2 in the regulatory effect of high glucose on par-4 expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h2o2 generation) and cell-permeant catalase (to degrade cellular h2o2). both approaches prevented the stimulatory effect of high glucose on par-4 expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par-4 transcript stability. cicaprost also attenuated high glucose-induced hur binding to par-4 mrna and as a consequence normalised par-4 expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par-4 thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h2o2, increase par-4 expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. 72h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . a significant reduction of recovery was observed in the ndpk b depleted mice at days 3 and 7. thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than 50% in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for 4 hours to constant atmospheric et concentrations of 5, 20, or 50 ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of 2 between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw11 on human dendritic cells fink c. 1 , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw11 on the expression of maturation related genes (cd1a, cd83), cytokines (tnfα, il-4, il-12), chemokines (ccr7), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr2, tlr4). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in 6 well plates. non-adherent cells were eliminated. to induce idc development, 50 ng/ml il-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day 6, maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of 1µg/ml and cultured for additional 3 days. after incubation with different concentrations (0. in idc, compared to control, we found a significantly increased expression of cd83 (2.1-2.6 fold) and tnfα (2.6-3.3-fold) genes after treatment with 0.1-5% stw11, respectively, but no effect was found on the expression of cd1a, il-4, il12, adam19, cd11c, cd40,tlr2, tlr4, mhc-ii and ccr7. in summary, these data demonstrate a stimulatory effect of stw 11 in idc and especially in mdc, concerning an increase of various genes related to maturation (cd83), immunomodulation (tnfα, cd40), adhesion (ccr7) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring 20a, 76131 karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of 17-beta-estradiol (e2) and estrone (e1). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p450-catalyzed metabolic activation to catechols (wang et al., chem res toxicol 23, 1365 . like e2 and e1, zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res 53, 1123 . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of 8-oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp1a2, and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of 8-oxo-dg could be demonstrated with each extract. the levels of 8-oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-hydroxylated zen/zel, which is the major catechol, was more reactive than 13-hydroxylated zen/zel to form 8-oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e2 and e1. the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me 574/32-1) and "food and health" of kit. thrombin regulates expression of sphingosine kinase-1 (sphk-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk-1 expression and atherosclerotic burden in vivo flößer a. 1 results: thrombin induced a time-and concentration-dependent (1-100 nmol/l) increase in sphk-1 mrna and protein expression in human saphenous vein smc, n=6-7. this was mimicked by a synthetic par-1 ligand. inhibition of sphk-1 attenuated thrombin-induced smc proliferation but not smc migration (n=5). the regulatory action of thrombin on sphk-1 expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk-1 mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk-1 mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). conclusions: thrombin induces sphk-1 expression and s1p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk-1 expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to 0.4 µg/g don and 0.5 µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t-2 toxin, ht-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to 6 µg/g don, 7 µg/g ennb, 16 µg/g ergotamine, 50 ng/g ht-2 toxin) exert pronounced cytotoxicity in v79 cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp1a1. recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp1a1 and proinflammatory cox-2, respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc 2544) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 3'methoxy-4'-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase 1 (chk1), an important cell cycle regulator that arrests the cell in g2/m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk1 in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk1 client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: 50.0 ± 5.8 min. vs. mpa: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase 9 (mmp-9) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg 18, 42096 wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a 96-well plate format using glutamate/malate (20 mm/0.5 mm) and succinate (25 mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. (2008) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., 2007) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), 1-octyn-3-ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd86 expressed on dcrc (ec50 = half maximal effective concentration). a pronounced induction of cd86 at low concentrations was seen with la, lna and oa (ec50: 3, 5 and 5 µmol/l, respectively). ua exhibited an intermediate response (ec50: 307 µmol/l). with oc and ma, we observed effects at higher concentrations only (ec50: 1340 and 2293 µmol/l). no significant increase of cd86 was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd86 was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd86 at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd86 (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd86 was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (oct1) and oct2, respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph 7.4 and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least 4 million ddd each) for their inhibitory interaction with oct1 and oct2. human embryonic kidney (hek) cells stably overexpressing oct1 or oct2 and the prototypical oct substrate 1-methyl-4phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at 20 µm, 59% and 51%, respectively, of the tested compounds significantly decreased oct1-and oct2-mediated uptake of mpp+. the most potent inhibitors (inhibition >75%) of oct1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct2. in contrast, neither at 20 µm nor at 200 µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct1 or hek-oct2 cells. there was a significant correlation between the degree of oct1 and oct2 inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µm in humans, inhibitory interactions of these compounds with hepatic oct1 have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: 58±3 %, n=3) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: 63±7 %, n=3) and failing patients (ef: 28±1 %, n=3). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation450 beadchip (illumina). this microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified 1280 cpg sites in hypertrophic samples and 1365 cpg sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, 496 sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`utr regions. specifically 9 probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase 1 (srpk3) showed diminished cpg-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh6 or myh7. these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only 15 months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln18, u87-mg and gl261. the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic50 values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. 1 brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat 1 . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora1, a2a, a2b and a3. they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora1 or a2a, respectively 2 . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. 3 abundance of adora1, a2a, a2b and a3 mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora2b being the most abundant. adora1, adora2a and adora3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). adora2a is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). adora3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). in contrast to this, ador2b was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora2a activation by cgs21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. moreover, adora1 antagonist psb36 increased lipolysis by 35% (+/-0.05%) (n=4). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora2a agonist or adora1 antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio 2 or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified 39 peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin-2 (myoz2) and g protein signaling modulator 1 (gpsm1, also termed ags3) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa (4-methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: 10 cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below 10 µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged maa elimination half-life of up to 12 hours. in one case with manifest hepatic insufficiency an maa concentration of more than 200 µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than 10 µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b 0+ amino acid transporters recognize fet as substrate (lat1 and 2, y + lat2, and b 0+ at, respectively). in contrast, y + lat1 and atb 0,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat1 is the dominant amino acid transporter in all glioblastoma cells investigated (ln229/u373/u87mg/u251/a172/t98g). a strong lat1 expression was also shown on the protein level. to find out whether lat1 is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln229 glioblastoma cells. [ 3 h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat1 in ln229 cells led to a concomitant decrease of lat1 mrna and tyr transport (down to 3% and 20%, respectively). these results indicate that tyr is exclusively transported by lat1 in ln229 cells. we are currently performing transport studies using [ 18 f]fet to investigate whether fet transport is also exclusively mediated by lat1 in glioblastoma cells. a further question is if lat1, a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat1 substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at1 receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht-29 cells, and their doxorubicin ic50 was four times higher. comparative protein expression levels showed that the primary cells had less rad 51 and 52 as well as less topoiiα, while ku70 and 80 levels were similar. another very interesting protein is the mrn (mre11-rad50-nbs1) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre11. the toxicity of mirin in ht-29 cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of 100 µm and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [1] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [2] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e1 and hdac 1 expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e1 protein level in the human colon carcinoma cell line ht29 after 24h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ 50 µm. hdac 1 expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac-1 and sumo e1. these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. 1, 35034 marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia2 rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into 17-oh-pregnenolone, the placenta tissue highly depends on the supply of c-19 steroids for their conversion into c-18 estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e 1) and estradiol (e2), while 16αoh-dheas supplied by the fetus contributes to over 90% of placental estriol (e3) synthesis. soat, a member of the slc10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e1s), and pregnenolone sulfate (pregs) [1] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg-3 as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg-3 cell line that transformed dhea into e2 and 16αoh-dhea into e3. by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg-3 cells. currently we investigate the transformation of dheas of these soat-jeg-3 cells by lc-ms-ms. we could demonstrate transport of 16αoh-dheas for stably transfected soat-hek293 cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [1, 2] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [3] for polymer toxicity testing. fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (fig.1 ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative (0.9 % nacl) and positive controls (1 % sodium dodecyl sulphate (sds) and 0.1 n naoh) were investigated. additionally ld 50 values for different cationic polymers have been determined. within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally 613, based on toxicological testing in animals (munroe et al., 1996) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., 1978) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn 2008) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of kibra into the pgl3-vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy5y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 5'cdna ends (5'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p1 was separated by ~1060 bp into two distinct regions, promoter p1a and p1b. deletion constructs harbouring promoter p1b were transcriptionally active only in ihke cells. 5'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_015238). exclusively in ihke cells, two additional tss were detected in intron 1, resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p2 and p3) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf7l2 (transcription factor 7-like 2 [t-cell specific, hmgbox]) resulted in a ~3-fold increase of promoter p1a and intron promoter p2 ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p1 and three alternative promoters p1b, p2 and p3. the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon 3 could result in truncated kibra protein isoforms. tcf7l2 is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg 428 and a corresponding technical guidance document (gd 28) give technical guidance how to perform valid experiments 1, 2 . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: -0.07 -6.2). finite dose experiments using rat and human skin were performed with 14 c-labeled testosterone, caffeine, mcpa (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester mcpa-2ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h2o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the 14 c-penetrants. teer, tewl and ³h2o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h2o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. 1 background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status 5'-upstream of dna regions encoding for selected candidate mirnas. results: out of 754 mirnas, 150 were detected in both tissue types. the expression of one mirna was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox11, mecp2, bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm61390. recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il-21-and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c57bl/6 b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/2, cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt1 mice hamann m. 1 early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt1 gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. (2005, j. neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (dyt1 mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in dyt1 mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in dyt1 mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt1 and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt1 mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt1 mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd332991 which inhibits cdk4 and cdk6 is currently tested in patients with solid tumors such as glioma. we found that pd332991 suppressed il-1-induced expression of il-8 suggesting that cdk4 or cdk6 may have unknown anti-inflammatory properties. to study the effects of cdk6 on the il-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk6, cdk6 s178p, in asynchronized hela cells. cdk6-expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk6 s178p enhanced il-1-induced il-8 and il-6 mrna expression. moreover, shrna-mediated suppression of endogenous cdk6 confirmed a role of this kinase in regulation of maximal il-1-induced gene expression of il-8. these data also revealed that the contribution of cdk6 to inflammatory gene expression is highest in g1, when activity of endogenous cdk6 is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g1, g1/s, g2 or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk6 in hela fucci or inhibition by pd332991 suppressed inducible il-8 expression. microarray experiments identified many additional genes that required active cdk6 for maximal il-1-or tnf-inducible gene expression. we also found that cdk6 co-immunoprecipitated with p65 nf-κb, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal il-8 promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc6 channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc6 mutations. since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc6 mutations, we have analysed all mutated trpc6 channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring 20a, 76131 karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h4r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. 1, 30625 hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h4-receptor (h4r) is involved in acute inflammation and th2 cytokine production. consequently, we intended to analyze the role of h4r in a murine th2 lymphocyte transfer-based model of asthma. specifically the ability of h4r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd4 + t cells were polarized in vitro under th2-favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h4r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il-4 production in t cells polarized in the presence of h4r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h4r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th2 phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h4r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about 40% eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h4r -/-dcs yielded only about 10-20% eosinophils in bal fluids. in summery, the h4r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h4r on dcs reduced their ability to stimulate proper th2 polarization of cd4 + t cells, we conclude that ha via the h4r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf2 (skn-1) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box 101007, 40225 düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf2 signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn-1 is the nrf2 homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf2::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf2 in cells, it has no effect on skn-1 localisation. further the effect on the nrf2 protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf2 signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are 36 and 26 µg/(kg d), resp. for children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). the adult exposure is almost entirely (82%) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed 0,9 µg/(kg d). high exposures were estimated (95th percentile) up to 10,8 µg/(kg d). on average people in germany are exposed to dehp below the current tdi of 50 µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) 3707 61 201). on the basis of the available measurements of dehp, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±sem; creb-ko 4871±258 vs. wt 3396±171; n=68/69 cells, n=4 mice, p<0.01 vs. ctr.). the nuclear factor of activated t-cells c3, nfatc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc3 signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc3 on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an 8.0±1.5 fold increase of the nfat model promoter activity (n=36; n=6 transfections), but had no impact on kv4.2 promoter activity which is known to be regulated by nfatc3 (1.2±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). nfatc3 overexpression led to a 4.5±0.7 fold increase of the nfat-dependent model promoter activity (n=12; n=2) and to an inhibition of kv4.2 promoter activity (0.8±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc3 might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p2x7-rezeptors -perazine-type neuroleptic drugs are potent modulators of p2x7 receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany p2x7 receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p2x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p2x7 is a promising target for pharmacological intervention. accordingly, p2x7 blockers are currently tested in phase ii clinical trials. in an attempt to identify p2x7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek293hp2x7 cell line. with ic50 values of 1-2 µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp (1 mm)-triggered increases in the intracellular ca 2+ concentration ([ca 2+ ]i) that was mediated by human p2x7 (hp2x7). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp2x7mediated ca 2+ influx. whole-cell inward currents, measured at -60 mv, were blocked by more than 60% by 3-10 µm prochlorperazine. the inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the p2x7 protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro-1 uptake when preincubated before p2x7 stimulation with 1 mm atp or when applied subsequent to the agonist. interestingly, when added to a hek293 cell line expressing the rat p2x7, perazines potentiated the atp-induced increase in [ca 2+ ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca 2+ ]i, changes in yo-pro-1 permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p2x7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p2x7. similarly, pre-treatment with lov also lowered dox-induced stabilisation of p53 and phosphorylation of chek1 and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h9c2), it decreased dox-and eto-induced phosphorylation of histone h2ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il6, ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p21, wee1, cjun/fos and hmox-1 following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb1 and abcg8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). moreover, cyp7a1 mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc2a9, abcg2, npt1, and urat1 as predicitive for the serum levels of urate 1 . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc2a9 have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc2a9. therefore, the aim of our study was to investigate which sequences in the slc2a9 gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc2a9 gene. dna from human kidney samples was then genotyped for rs6449237 being part of this region. next total slc2a9 mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs6449237 showed lower slc2a9 mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc2a9, urat1, npt1, and oat10, respectively was observed. conclusion. our data suggest that the slc2a9 snps rs6449237 and rs74794351 might influence slc2a9 mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc2a9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. 1 methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p450 enzymes yielding 1´-hydroxymethyleugenol (1´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)-1´-ohme (separated into its enantiomers) served as test compound. we could show that hsult1a1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult1a1-proficient and sult-deficient bacteria was examined after incubation with 7 µm of (+)-or (-)-1´-ohme. for selective detection and quantification of me-derived 2´-deoxyadenosine (da) and 2´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult1a1. the concentration dependence of adduct formation in hsult1a1-proficient bacteria was examined for (+)-1´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-ohme (i.p.) to mice carrying the hsult1a1/1a2 gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy926) and in human kidney cell lines (hek293t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy926 cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha1 and beta1 and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx1) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse 17, 91054 erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx1) to pacemaking. ncx1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx1 in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx1 knockout (cpncx1ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx1ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx1 results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx1ko animals as compared to controls. in conclusion, these initial results establish ncx1 as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg2). the ultimate carcinogenic phase i bp metabolite anti-bp-7,8dihydrodiol-9,10-epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco-2 cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco-2 monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc1, abcc2 and abcc3 knockdown cell lines were generated allowing to demonstrate that abcc1 mediates the basolateral, abcc2 the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco-2 cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. 31, 93053 regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura 2-am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of 8-br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without 8-br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. 1 tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (fsk) in immortalized rat vsmcs (13.7 ± 0.93; n=12 from 3 isolations). in a7r5 rat smooth muscle cells the icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of fsk stimulation (n=20 from 4 isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of 8 members (tmc1-tmc8) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc1 cause deafness in human and mice whereas tmc6 and tmc8 (also referred to as ever 1 and 2) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc6 and tmc8 in hek293 cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc6 or tmc8, stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc6-or tmc8-expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d1er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc6 or tmc8. recently, it has been reported that tmc6 and tmc8 might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc6 and tmc8 mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc8, hacat cells revealed the same phenotype as described above for the hek293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between 0.1 thz and 10 thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around 100 ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 thz, 0.380 thz and 2.520 thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at 0.106 thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t-84 by resazurin fluorescence and compared it with the placebo supplement. additionally, the t-84 cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il-1-beta. after the cytokine stimulation the mrna expression of ip-10, il-8 and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t-84 cells increased the expression of ip-10 (gamma-interferon inducible protein 10), tnf-alpha and il-8. by preincubation with fn at 10 µm the mrna expression of ip-10 was strongly reduced (log2-ratio -14). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [1] h. hoensch, b. groh, l. edler, w. kirch (2008) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, 14, 2187-2193. the cxcr7 c-terminal domain mediates efficient cxcl12 uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. 1, 07747 jena, germany cxcl12-signaling mediated by the g protein-coupled cxcr4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl12-receptor cxcr7 modulates cxcl12/cxcr4-signaling by acting as a cxcl12-scavenger. given the distinct functions of cxcr4 and cxcr7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek293) with respect to trafficking, stability, 125 i-cxcl12 radioligand degradation, and g protein-coupling. we found that the 24 c-terminal residues of cxcr7 were sufficient for cxcr7 to undergo rapid spontaneous internalization. replacement of the cxcr7 c-terminal domain with that of cxcr4 (cxcr7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr4 c-terminal domain by that of cxcr7 caused ligand-independent internalization of cxcr4. receptor-mediated 125 i-cxcl12-uptake, release of 125 i-cxcl12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr7 c-terminus. while the cxcr7 c-terminus was sufficient to abolish g protein coupling in the cxcr4-7tail mutant, replacement of the cxcr7 c-terminus, cxcr7 second intracellular loop or both domains with the corresponding cxcr4 domain did not generate a g protein-coupled cxcr7 chimera. taken together, we provide evidence that the cxcr7 c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr7-c-terminal domain may effectively control cxcr7 functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. 1 brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, 2009 ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap2 and bat marker genes ucp1, pgc1α, cidea. the α2 and β1 isoforms of sgc were highly expressed in bat whereas α1 sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pcpt-cgmp. in contrast, staining was lower in sgcβ1 -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in 56-40% decrease in c/ebpα, pparγ and ap2 expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ1 -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately 80% in sgcβ1 -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. 1 carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. 2004 , muller et al. 2005 , ma-hock 2009 , pauluhn 2010 . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. 165 synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße 26, 80336 münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1h-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( 1 h, 13 c, cosy, hmqc, hmbc). for synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty 81/1-1). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon1, pon2 and pon3. while pon1 is found predominantly in the circulation, pon2 and pon3 are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon2 and pon3 are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon2 and pon3 were shown to interact with coenzyme q10 which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon2 and pon3 reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon2 caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon2 and pon3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. 5, 64295 darmstadt, germany stw 5 (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw 5 on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco-2 cells after stimulation with lps (10 ng/ml) for 2 hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps (100ng/ml) stimulation of differentiated thp-1 cells was measured using a commercially available elisa. stw 5 (62.6 -500.5 µg/ml) reduced lps (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw 5 to different extents. the maximum inhibition differed in a wide range between the components. stw 5 (500.5 µg/ml) reduced significantly the release of tnf-α by 87 % in lps (100 ng/ml)-stimulated differentiated thp-1 cells while having no effect in untreated cells. in concentrations equivalent to stw 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw 5, indicating a possible synergistic effect. our results indicate a multi-target effect of stw 5. the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw 5. immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. 1 , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp11a1, encoding p450ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 hduring the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of 365 mirnas in placentas of different gestation times revealed a significant expression of 277 mirnas by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). when comparing the analyzed groups, 106 of these mirnas expose a continuous up-(including mir-20a, mir-379) or downregulation (including mir-9, mir-200a). while comparing placentas of early gestation to term placentas, 30% (e.g. mir-9, mir-429) were up-and 70% (e.g. mir-126, mir-373) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, 60% of mirnas showed higher (e.g. mir-107) and 40% lower expression (e.g. mir-627, mir-191). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir-9 and mir-429. specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir-9 (93%) and mir-429 (94%) in term placentas, while crh is upregulated 114fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the 3´-utr sequence complementary to crh was exhibit for the predicted mir-9 binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted mir-429 binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a1) and experimental (a2,a3 ) of rats weighing 350-450g local application of dichloromethane extract of s.i in vehicle olive oil preparations of 50 and 100mg were used. animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. group a3 showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a2. the macroscopic ulcus dimensions were better in group a3 while the microscopic view were similar in a2 and a3 and better in comparison to control a1. a tendency to trauma remodelling was observed, without statistical significance (t =0.3) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the 5xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female 5xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the 5xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf (9772-365), the dfg graduate school grk1033 and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi3k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi3k/akt, as activation of pi3k/akt using a pi3k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase-3 activation in tcsl-treated fibroblasts. conclusions: 1. rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. 2. the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi3k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a4 (anxa4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca2+ binding anxa4 undergoes conformational changes, which lead to oligomerization of anxa4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa4 on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa4, there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic50 value of 6.787 µm [5.745 to 8 .017] which is much higher than ic50 values of sirolimus described in literature [0.1nm to 1nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf9 and ifitm1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße 1, 07747 jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (s363) threonine 370 (t370) and serine 375 (s375) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s363a/t370a/s375a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s363a/t370a/s375a/t376a/t379a/t383a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t376 and t379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s375 was the primary phosphorylation site, whereas t370, t376 and t379 were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s375 but not of t370, t376 of t379. our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. 1 methylene dianiline (mda; cas no. 101-77-9) is on the usepa list 2 for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of 0.01 mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at 0.2 mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: 7.5 mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: 9 mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp1 controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. 1 background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein 1 (arfrp1) is a member of the arf-family and controls the arf-like 1 (arl1)-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp1 in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp1 flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp1 expression was suppressed by sirna in caco2-cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco2-cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp1. in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp1 vil-/mice. arfrp1 vil-/enterocytes as well as arfrp1 mrna depleted caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp1 vil-/epithelium and was predominantly colocalized with rab2. our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp1 is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. 1 the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the 2d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the rhoa knockdown cells reattach, but only 5% of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by 33%. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung 1, albertstraße 25, 79104 freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the 146-kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα13 and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. 2007; 3: 707) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor 16 (pi16). here we report the generation of a mouse line where pi16 can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi16 allele. global pi16 deficiency (pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi16 +/+ = 5.4 ± 0.2 (n = 6), pi16 lox/lox = 5.9 ± 0.7 (n = 4), p > 0.05; fibrosis (%): pi16 +/+ = 3.3 ± 0.2 (n = 7), pi16 lox/lox = 3.9 ± 0.8 (n = 5), p > 0.05; fractional shortening (%): pi16 +/+ = 29.8 ± 0.7 (n = 7), pi16 lox/lox = 26.4 ± 2.5 (n=5), p > 0.05). in addition we carried out an immunohistochemical analysis of pi16 expression using pi16-deficient mice as negative controls. pi16 localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi16 preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi16 and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [1, 2, 3] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec 3.1.1.10) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [3] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [3] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [3] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [5] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β1-adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase 2 (grk2) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk2 and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on 7-week-old c57/bl6 mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice 3 weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after 3 weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c18 eps 2, c18 sh 2, phenyl 2, cn 2, and c30 were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a1, an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp90 as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [1] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp90 and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp90 and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp90/cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk506-binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [1] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. (2011) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h441) on top and microvascular endothelial cells (iso-has-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, 30 nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin-1 and -2 labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il-8 increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp-1 (prestimulated with 8 nm pma for 4d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h441/iso-has-1 with thp-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il-8 release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. in these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 mio daily doses have been sold, corresponding to more than 33 mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). from the packages sold in germany in the years between 2005 and 2009, 48.1 mio were attributable to self-medication, 10.8 mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on 9 may 2010. she has initiated this work. prucalopride was introduced as a new selective 5-ht4 receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that 5-ht4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other 5-ht4 receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human 5-ht4a receptor. isolated electrically driven (1 hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven (1 hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr113808, a 5-ht4 receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec50 of -6.8 and -7.1, respectively (p<0.05 vs. wt, n=3). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by 10 µm gr113808. we could demonstrate that prucalopride acts as an agonist at the 5-ht4 receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw 5 (a 9 component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome (3, 4) ). in one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) (6, 7) . adult animals were restrained for 90 min/ day for 1 week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw 5 in restoring the deranged parameters. a group of animals received stw 5 orally once daily starting treatment 1 week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw 5 in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine dna glycosylase (ogg1), which generates harmful repair intermediates [1, 2] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodg) varies between the opposing dna strands of the gene [3] . we now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single 8-oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single 8-oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for 8-oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of 8-oxodg present in the 5′-versus 3′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of 8-oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg1 protein. the results thus support the initiatory role of ogg1 in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg1 suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg 5, 55128 mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . an efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg1 [2] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing 8-oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a 5′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites (5′-gcaatgnn). adapters containing the restriction sites for directional cloning into the 5′-or the 3′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single 8-oxog in the same sequence context into opposing dna strands in the 5'utr and in the 3'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°c. less is known about the influence of cholesterol on trpv3 signalling. we modified the cholesterol content of hek293 cells stably transfected with trpv3 and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv3 by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv2, another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv3 -/mice, our results imply that a cholesterol-regulated trpv3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution (100 ml min -1 ). after equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for 4 h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po2 was adjusted to 120 mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po2 of 50 mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc6 blocker larixol acetate (5 µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. this weight gain was inhibited by supplementation of the trpc6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the trpc6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). we conclude that a trpc6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. 67, 55101 mainz, germany the transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human dna codes for much more (450.000 ?) non-coding rnas 1 . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il-1β-induced inos expression by an antisense rna (as-3-utr-rna) in rat hepatocytes 2 . a promoter located on the antisense strand 3' to the last exon (exon 27) of the rat inos gene drives the expression of an as-rna complementary to the 3'-utr of the rat inos mrna. using different sense primers with homology to the 3'-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as-3-utr-rna in human cells. in addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld-1 cells revealed no promoter activity of these sequences. korneev et al. described the expression of a 2 kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells 3 . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld-1 cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld-1 cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna 14, 2030 rna 14, -2037 rna 14, (2008 . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse 190, 8057 zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s341, s343, t353, t354, t356 and t359, which in turn leads to a robust endocytosis of the sst2 receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s341 and s343. somatoprim and ke108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. this approach allows detection of receptor activation by phospho-specific sst2 antibodies. compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. ke108 and pasireotide are partial agonists at the sst2 receptor. pasireotide, ke108 and somatoprim show comparable effects on sst5 receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. 1, 2 , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where 17beta-estradiol (e2) and cdcl2 modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi 10 .1007/s00204-011-0787-x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium (2mg/kg b.wt cdcl2 (cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (ee2)) on ahrassociated gene expression (i.e., ahr, cyp1a1, gsta2, nqo1) in the small intestine of rats after oral exposure (3 days gavage). the animals were also co-treated with cd2 and pure anti-estrogen (2.5 mg/kg b.wt zk191703 (zk)) or ee2, to asseess whether ermediated processes are involved. we also measured cyp1a1 mrna expression in two estrogen receptor negative colon cancer cell lines (ht-29 and caco2) treated for 5 days with cdcl2 (1µm) and e2 (0.01µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt cdcl2 equivalent to 5, 50, and 150 ppm) and ee2 (0.08 mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o 6 -methylguanine-dna methyltransferase (mgmt), o 6 meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o 6 meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u-87 mg and ln-229 glioma cell lines. the maximum amount of autophagy was observed several days (96 h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o 6 meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o 6 meg, are repaired preferably by homologous recombination (hr) ln-229 cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o 6 meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf (02nuk016). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb1a) have been found in patients suffering from rp. we used cngb1-deficient (cngb1 -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb1a cdna (approx. 4 kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype 8) into the subretinal space of 2-week-old cngb1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb1 -/mice expressed full-length cngb1a and cnga1, which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb1 -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h295r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on 14 reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category 1) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg 405) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . for many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [4] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco-2 cells by activated pmn and this effect increased with increasing pmn to caco-2 cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco-2 cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h 2o2 was increased in buthionine sulphoximine (bso) pre-treated caco-2 cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco-2 cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p450 monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg2 cells incubated with oa in the absence and presence of s9 mix. pure oa, as well as oa pre-activated with liver homogenisates (s9 mix) were used to treat human hepg2 cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s9 fraction plus cofactors of phase i enzymes. the cell viability was measured after 4 h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s9 mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s9 mix was toxic at 75 nm oa. strong effects could be observed when oa was pre-activated with human s9-mix at a concentration of 50 nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg2 cells treated with oa in the presence of all investigated s9-mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg2 cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse 10, 10589 berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in 2004 the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october 2011. here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. 44, 39120 magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p38 mapk and phospholipase d2. in line with this, we observed marked phosphorylation of p38 mapk and activation of phospholipase d2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to 100 fold induction of il-4, which was dependent on p38. in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il-4. by inducing il-4 opioids significantly modulate the t helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, t helper cell type 1-mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse 150, 44780 bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde5, the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde1, the ca2+/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca2+ concentrations. and lastly pde3, the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde3. cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has1-3). the has3 gene is alternatively spliced. has3 transcript variant 2 (has3v2) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has3 transcript variant 1. the aim of the present study was to investigate whether has3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has3v2 mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has3v2 fusion protein and a ddk-tagged has3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has3v2 mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has3v2 protein expression in vsmc and platelets. in vsmc has3v2 mrna expression was strongly up-regulated in response to interleukin-1β (il-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-bb (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. transfection of hek cells with eyfp-has3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has3v2 proteins were precipitated together suggesting formation of multimeric has3v2 complexes. transfection of has3v2 did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has3v2 is present in vascular cells and responds to inflammatory cytokines such as il-1ß. because of the intracellular localization and the lack of ha secretion in has3v2 transfected cells, has3v2 may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th2 cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of 1 mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p15, in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig)2-vpgkg-(vpgig)2, were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p15 peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde2a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). notably, pde2a protein abundance is 4-fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). to this end we tested whether pde2a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde2a overexpression induced α-sma expression (3.2-fold p<0.05, n≥8) and to a lower extent ctgf synthesis (1.5-fold, p<0.05, n≥5). mechanistically, pde2a showed preferential subsarcolemmal localisation with diminished total cgmp levels (-56%, p<0.05, n≥5). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde2a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde2a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). these data implicate pde2a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde2a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. 2005) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the np (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. the organic pigment was a poorly soluble pyrrol with an intense orange color. the np (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. they were tested at 1, 3, 10 and 30 mg/m3 for the np and 3, 10 and 30 mg/m3 for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso4, zno, ceo2, al-doped ceo2, zro2, amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio2 and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso4 as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. 1 , wächter t. the eu reach regulation no 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go3r, accessible free of charge at www.go3r.org, has been created to allow quickly finding relevant hazard information and data. go3r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go3r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid 5 chapters. hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go3r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h2-receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. 1, 30625 hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of aml (1) . it is assumed that histamine exerts its effects by activating the histamine h2-receptor (h2r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation (2) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml (3), we initiated a study to explore the possibility that h2r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl-60 cells. in hl-60 cells, histamine and various h2-receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h2r conformations. h2r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h2r agonists showed no signs of cytotoxicity. intriguingly, following h2r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) 4, indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h2r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb1 receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca 2+ release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca 2+ imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/11 proteincoupled metabotropic glutamate receptor 1 (mglur1) was activated by superfusion of (rs)-3,5-dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca 2+ concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur1 antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb1 receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca 2+ stores by thapsigargin, cyclopiazonic acid and ip3. the results indicate that after activation of the gαq/11 protein-coupled metabotropic glutamate receptor 1 (mglur1) of the postsynaptic neuron ca 2+ is released from the endoplasmic reticulum. this ca 2+ release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb1 receptors. the guanine nucleotide exchange factor dock9 controls reelin dependent cdc42effects on radial migration pichler m. 1 the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi2-deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi2-deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi2deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low (3 mm) and high (16 mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi2 proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi2-deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi2-deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi2-targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r7260 (molecular probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). the affinity of compounds in radioligand binding was slightly higher in sur2b than in sur2a-type channels, but the enantiomeric ratio in sur2a channels matched that one determined for the sur2b-type indicating some conformity of the binding pockets of sur2a and sur2b-proteins. surprisingly, however, the membrane potential tests revealed that the (3r,4s)-enantiomer acted as agonist (a) whereas the (3s,4r)-enantiomer acted as antagonist (b): (3r,4s)-bms-191095 induced membrane hyperpolarisation whereas (3s,4r)-bms-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms-191095 is not selective for sur2a as compared to sur2b-type k atp channels. its enantiomers activate and block sur2-type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur2b-type katp channels act as partial agonists at cardiac sur2a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. 1, 40225 düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur1-type katp channels (nielsen et al., j med chem 45: 4171, 2002) were characterized in sur2b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal 375, r45, 2007) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek 293(kir6.2/sur2a)-cells and compared to hek 293(kir6.1/sur2b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye r7260 (molecular probes). standard-kco induced hyperpolarisation with ~10-fold smaller potency (pec50) in sur2a. ttd-compounds with ch3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur2a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur2a and sur2b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca2 channels in nonneuronal cells. the aim of this study was to investigate the expression of kca2 channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca2 channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca2.2 and kca2.3 channel activator cyppa (25 µm) and specific inhibitory peptides (50 µm) were applied to distinguish effects mediated by the kca2 channel subtypes. all kca2 channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps (200 ng/ml) induced microglial proliferation. the kca2.2/kca2.3 channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca2.3 channels, but not of kca2.2 channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il-6, but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il-6 production while tnf-a production was not affected. moreover, using inhibitory sk3/kca2.3 channel peptides, we demonstrated that sk3/kca2.3 channels modulate lps-induced cytokine il-6 production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca2.3 channel stimulation reversed microglial activation. thus, kca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. (3r,4s)-(3s,4r)intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. 1, 30625 hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r.8 to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is 10 in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase 5 (cdk5) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk5 in cancer. cdk5 was shown to regulate tumor growth, and our group discovered that cdk5 regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk5 is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk5 in hcc. histology of tissue micro arrays indicates an increased expression of cdk5 in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk5 in hcc, we analyzed the impact of both pharmacological inhibition of cdk5 and specific downregulation of cdk5 with rna interference on hcc cells. pharmacological inhibition of cdk5 with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g2/m cell cycle arrest and cell death, and reduced motility of huh7 and hepg2 cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk5 also reduced proliferation, clonogenic survival, migration and invasion of huh7 cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat3 at ser727 that is directly phosphorylated by cdk5, and tyr705. in line with this, the expression of the antiapoptotic protein mcl-1 is reduced by inhibition of cdk5. our results point to an important function of cdk5 in hcc and suggest cdk5 as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c3 toxin lillich m. 1 , chen x. clostridium botulinum produces the adp-ribosyltransferase c3, which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . thus, c3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c3bot mutant (c3mut) and streptavidin. the c3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c3mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j774a.1 macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c3mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c3mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j774a.1 macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c3mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j774a.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c3mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h4iie rat hepatoma cells. in this context cw60 (a diphosphane ligand with azoyl substituents r2p(ch2)2pr2, r= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an ic50 value of 6,5mm (24h incubation). further investigations revealed that cw60 induced an apoptotic cell death in h4iie demonstrated by the activation of caspase 3/7 (48h incubation with 10mm cw60). in addition the induction of apoptosis was confirmed by the dna ladder formation (24h incubation with 5mm cw60). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi3k/akt but after 24h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw60 shows strong toxic effects in h4iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for 923, biedersteiner str 27, 80802 münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc6-, and trpc3-, and trpc3/c6-double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc6-, and trpc3/c6-double knock-out mice as compared to aorta from ctr and trpc3-knock-out mice indicating a functional link between cgki and trpc6 channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by 8-br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc6-, trpc3-, and trpc3/c6-double knock-out mice. no effect of 8-br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc3-, and trpc6-knockout mice. trpc3 could be detected in both smooth muscle and endothelial cells whereas trpc6 was only detected in endothelial cells. the results suggest that absence of cgki or trpc6 impairs the vasodilator tone induced by endothelial no production but that cgki and trpc6 channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc6 -/-, and trpc3 -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb 153 in human hepatic cell models lohr c. 1 , neser s. after the treatment with tcdd, however, a total of 281 genes were more than 2-fold up regulated in hepg2 e.g. cytochrome p450 1a1 (cyp1a1) (32-fold) a sensitive marker for ahr activation. additional up regulated genes in hepg2 were; arylhydrocarbon receptor repressor (ahrr) 15-fold, aldehyde dehydrogenase 3 a1 (aldh3a1) 11-fold, and cytochrome p450 1b1 (cyp1b1) 10-fold. 44 genes were more than 2-fold down regulated in hepg2 cells e.g. -proprotein convertase subtilisin/kexin type 9. markedly different findings were obtained in hheps, i.e., 117 genes were up regulated, the highest up regulated gene was cyp1b1 with a 95-fold increase in gene expression, followed by cyp1a1 (41-fold) and aldh3a1 (21-fold). only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only 30 in common genes were up regulated, including cyp1a1, cyp1a2, cyp1b1 and aldh1a3. only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg2 and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg2 cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. 1 , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb 815/a14). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac 1, expressed in sf9 insect cell membranes, with cam, cam-206, -213, -214 and -215 by measuring the catalytic activity of ac 1. cam-mutants show a 3-4-fold lower potency than cam, but they are more efficacious than cam. most prominently, cam-215 was 133 % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam-215, it is more hydrophobic than cam and this leads to a better ppi with ac 1. in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac 1 interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac 1. because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac 1 interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee 114-116, 14558 potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about 20-30% non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p450 2e1 and 4a1 isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep3 receptors for prostaglandin e2 convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was (1) the pharmacological characterization of ep3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l-826266 served as the ep3 antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep1/ep3 agonist sulprostone (1 nm -100 mm) concentrationdependently contracted human pulmonary artery rings (pec50 and emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by kcl 60 mm). the concentrationresponse curve of sulprostone was not affected by the ep1 antagonist sc 19920 (10 µm) but shifted to the right by l-826266 (10 µm) (apparent pa2 6.22). extending the exposure time to l-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (schild plot-based pa2; concentrations 0.1, 1 and 10 µm). in pithed rats electrical stimulation (0.66 hz, 1 ms, 50 v for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (hr) by 55 beats/min. sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. l-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%. in conclusion, ep3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. -5-bromo-n[3-(5voltage-gated ca 2+ channels of the central nervous system control a multitude of ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav2.1 ca 2+ channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca 2+ -channels. ca 2+ influx via cav2.1 ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav2.1 ca 2+ channels play a crucial role in synaptic transmission. the global cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav2.1 ca 2+ channels for learning and memory. therefore, we crossed a floxed cav2.1 mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav2.1 for learning and memory. helicobacter hepaticus-infected rag2 -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., 2009 , pnas 106: 1027 -1032 . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag2 -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by 20 weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. 1 recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [1] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the drosophila protein. it covers 171 amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c90rf7 and the ca v2.2 channel in xenopus oocytes reduced the amount of the α1b and cavβ3 subunits of the ca 2+ channel in the plasma membrane but did not affect the gating properties of the cav2.2 channel. expression of c90rf7 alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c9orf7-fusion in e.coli (yield 2mg at 1mg/ml). we are currently preparing the c9orf7 part of the his-sumo-c9orf7fusion protein by ulp1-protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. we could not identify any homologues of c9orf7 in the mouse genome and to analyze its function we are currently generating c9orf7 deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c9orf7 by expression of the bgalactosidase gene under the control of the endogenous c9orf7 promoter. by southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of 17β-estradiol in cultured v79 cells expressing human cytochrome p450 1a1 martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany oxidative metabolism of the female sex hormone 17β-estradiol (e2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e2 undergoes metabolic activation by cytochrome p450-dependent monooxygenase (cyp) isozyme 1a1 to 2-hydroxy-(2-ho) and to a lesser extent to 4-ho-e2. if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e2 was determined in male chinese hamster lung fibroblasts (v79 cells) expressing human (h) cyp1a1 and (ii) the metabolite profile of e2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp1a1 and dimethylsulfoxide as solvent control. v79 hcyp1a1 were treated with 100 nm e2 for 3 weeks and the resulting 6-thioguanine (6-tg) resistant mutants selected at weeks (w) 2 and 3. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). as expected, 0.25 µm bap induced a significant increase in mutant frequency (mf, 266 ± 13, w2 and 132 ± 46, w3) . treatment with 100 nm e2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in mf, suggesting slight mutagenic activity. in culture medium of v79 hcyp1a1 treated with 100 nm e2, 2-ho-e2, 2-methoxy-(meo)-e2, 3-o-methyl-2ho-e2 and 4-meo-e2 (suggesting intracellular formation of 4-ho-e2) were detected. while 4-meo-e2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-ho-e2, correlating with the maximum increase in mf, observed after 2 weeks as well. in conclusion, e2 possessed a slight mutagenic potential after hcyp1a1-mediated activation to 2-, 4-ho-e2 and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., 10589 berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg-3 and transactivation via estrogen receptors α and β in stably-transfected hek 293 cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of jeg-3 cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. 1 trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm4 and trpm5 have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm4 regulates catecholamine release. besides trpm4 we recently identified transcripts encoding additional trp channels including trpc5 and trpc6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc1/c5/c6 triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp4 (ec 50 0.32 vs. 2.8 µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t0070907 but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at1a and at1b receptors as mechanosensors in myogenic vasoconstriction blodow s. 1 , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/11-proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/11-coupled receptors such as angiotensin ii at1b, vasopressin v1a, endothelin eta and etb and α1a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v1a receptor and α2a and α2ab adrenoceptors showed no differences of myogenic tone, blocking of at1a and at1b receptors by losartan and candesartan, eta receptor by bq123 and α1a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at1a -/mice with and without additional blocking of at1b receptors by candesartan suggested that especially at1b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at1b -/mice. these findings suggest that mechanosensitive gq/11-protein coupled receptors, especially at1b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . they encode a substantial variety of isoformes and so far we have identified more than 20 distinct trpm3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . these variants differ enormously in their biophysical properties. for example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of trpm3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant trpm3 proteins. however, the frequency of these different isoformes in trpm3 expressing tissues is completely unknown. to get insight into the significance of the different trpm3 isoformes we investigated the abundance of alternative trpm3 transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. furthermore we analyzed the trpm3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm3 transcripts are most abundant. for that purpose we sequenced more than 120 clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon 13. in cells of the choroid plexus nearly all (124 /126 clones) carried the short ca 2+ permeable pore. furthermore, we identified seven variants spliced in exon 20 encoding truncated trpm3 proteins. however, the composition of the trpm3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm3 function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of 0.15 µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has1, -2, -3), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at 14 weeks coincided with ha deposition and induction of has3 in aortic root plaques. in human symptomatic carotid artery plaques has3 was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has3 was specifically induced via activation of nfkb by interleukin-1β (il-1β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay 11-7082. has3 was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il-1β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f4/80 positive macrophages as shown by immunohistochemistry. to study the effects of has3 mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has3 were employed. overexpression of has3 resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has3 were mediated by both pi3k signaling and mapk signaling via hyaluronan receptors cd44 and rhamm. conclusion: the present results suggest that has3-dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has3-mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn3-deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. 5-13, 81377 münchen, germany hcn3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn3 -/and hcn3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn3 in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn3 knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn3 -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. 1 , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, 2008) silva et al.,1993) . we simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 l of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation "1 to 2 glasses of wine on occasion" (agence nationale d'accréditation et d'évaluation en santé, assante 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. (1993) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type 1 (gαi1) and adenylyl cyclase type v (ac5). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a2a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai1-cfp and yfp-ac5. after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. 2003) . to further analyze the properties of the dissociation between gai1 and ac5 we measured in parallel the offset kinetics of the interaction between gai1-yfp and gβg-cfp (gi1-fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs4 accelerated the deactivation of gi proteins and the dissociation of gai1-cfp from yfp-ac5. these experiments revealed that in the absence of rgs4 the dissociation of gai1 from ac5 after agonist withdrawal takes about 3 times longer than the deactivation of gi proteins. in the presence of rgs4 this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai1 from ac5 was only marginally accelerated by rgs4. these observations lead us to hypothesize, that ac5 might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai1-ac5 interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi0/0) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by 65% ± 1% in pkgi0/0 as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha (19% ± 4.8%), ppargamma (66% ± 2.9%) and ap2 (37% ± 10.6%) expression in pkgi0/0 cells as compared to pkgifl/fl. treatment of 3t3-l1 cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in 3t3-l1 cells. concomitant activation of pkgi in 3t3-l1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (ucp-1) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser-188 in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp-1 promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex 1 and 2, mtorc1 and 2. under amino-acidrich conditions, activated mtorc1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc1 function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc1 function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc1 translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl 2 in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc1.2 bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the 1,4-bz flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas 4-cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. the difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the 1,4-bz could be mediated by the recognition sites targeted by the 2,3-bz, i.e. the gria2-encoded ionotropic glutamate receptor ampa2, we investigated by quantitative pcr whether hmc1.2 cells express gria2, tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra3, gabrb3, gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra2, gabra4, gabrb2, gabrg3 and gabrr2. expression of gria2 was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee 114-116, 14558 nuthetal, germany 5-hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v79 cells genetically engineered for expression of human sult1a1 suggesting that hmf is converted into the reactive 5sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (n 6 -ffmda) and n 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (n 2 -ffmdg). these adducts were also detected in dna from v79-sult1a1 cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta100 engineered for expression of human sult1a1. the putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta100-sult1a1 and in dna of liver, lung and kidney of fvb/n mice that had received about 390 mg ffa/kg body weight per day via the drinking water for 28 days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp2 (methyl cpg binding protein 2) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp2 is known to be a target gene for several micrornas including the cluster mir-132/212 in the brain. recently, our group could show that mecp2 expression is downregulated in human heart failure suggesting that mecp2 might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp2 by the cluster mir-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir-132/212 expression, we treated cultured nrcms with 40 µm norepinephrine for 72 hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of mir-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated mecp2 mrna and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). to check whether mecp2 downregulation also occurs by direct mir-132/212 activation we increased levels of mir-132 and mir-212 in cardiac myocytes by transfecting precursor mir-132 and mir-212-3p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp2 mrna and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after mir-132 overexpression. similar results were obtained by overexpression of mir-212-3p. to test the effects of adrenoceptor activation on the mir-132/212-mecp2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). after 7 days, cardiac ventricles were analyzed. nppa gene expression (2.81 ± 0.32 -fold of control animals), mir-132 and mir-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while mecp2 protein levels decreased to 77% (p<0.05) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir-132/212 expression and to downregulation of mecp2 in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at-1 receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a 30% increase of control (pec30%) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec30%=8.6±0.2 (mean±sem, n=18); maximum increase=110±8%]. neither rosiglitazone nor pioglitazone (0.3-3 µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µm of rosiglitazone, respectively, n=4-6, each). in contrast, pioglitazone in concentrations up to 3 µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer 1 (dlc1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc1 loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia 1 and 2 (mkl1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc1. moreover, dlc1 loss and mkl1 nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl1 in dlc1-deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl1 phosphorylation. dlc1 loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr61, myl9 and myh9. furthermore, we identified a novel target gene, integrin a5, with a key role in cell migration and metastasis, that exhibited a dlc1-and mkldependent regulation. depletion of mkl1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc1 loss. our data provide insight into the mechanism by which dlc1 loss initiates tumorigenesis. as mkl1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, 53121 bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p2y receptors (p2y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or p2x receptors (subunits: p2x1-7). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p2y and p2x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p2y and p2x receptors [1] [2] [3] [4] [5] [6] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm2.5 has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. for the pm2.5 concentration the following values were calculated: cumulative pm2.5 concentration as auc-pm2.5 (µg/m3/sec), peak pm2.5 concentration as cmax-pm2.5 (µg/m3) and average pm2.5 concentration cmean-pm (µg/m3). in closed door condition both cigarettes produced particulate auc-pm2.5 values of 59 000 ± 15 000 µg/m3/sec (rc) to 85 000 ± 31 000 µg/m3/sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin-6 (il-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has 1-3) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il-6 participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il-6. therefore, the aim of the present study was to elucidate whether il-6 regulates the expression and function of ha-matrix in cfs. cells were isolated from c57bl/6j mice and used during passage 2-3 for experiments. cfs were stimulated with il-6 or hyper-il-6 which is a fusion protein of il-6 and soluble il-6 receptor (sil-6r). after 10 and 30 min signal transducer and activator of transcription 3 (stat3) was phosphorylated in response to hyper-il-6 but not in response to il-6. rt-pcr revealed rapid upregulation of has 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to hyper-il-6. has2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas has3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). in contrast, il-6 had no effect on transcript levels of has isoenzymes. in turn, expression of has1 and has2 in response to hyper-il-6 was inhibited by ag490, which indicates the involvement of stat signaling. interestingly, despite induction of has1 and has2 the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il-6. this may indicate that il-6 regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il-6 trans-signaling (hyper-il-6) via the soluble il-6r and subsequent stat3 signaling with increased ha-synthesis. the fact that il-6 had no significant effect suggests that the expression of the non-signaling membrane-bound il-6 α-receptor (il-6r) in cultured murine cardiac fibroblasts is not sufficient to induce has 1 and -2 gene expression. therefore, il-6 trans-signaling mediated by il-6 and the circulating sil-6r might be necessary to mediate the il-6-induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße 25, 53127 bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura-2 fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ 3 h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified 3-dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec50 165 µm). fura-2 fluorimetry showed an increase in intracellular ca 2+ levels by 3-dl-aminoisobutyric acid (300 µm). moreover, 3-dlaminoisobutyric acid reduced the forskolin-induced camp production by up to 65 % (ec50 195 µm). in addition to 3-dl-aminoisobutyric acid, we identified 3-dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of 4.9 (nfat assay), thioridazine with an apparent pkb of 5.4 (nfat assay) and 5.3 (camp assay) and rimcazole with an apparent pkb of 5.2 (nfat assay) and 5.2 (camp assay). in conclusion we show for the first time that 3-dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf-1-chemokine receptor cxcr4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr4 is involved in disease states including inflammation and cancer. it is well established that sdf-1-stimulated cxcr4 receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr4 c-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr4. here, we analyzed the phosphorylation pattern of cxcr4 at 3 c-terminal sites after stimulation of the receptor with sdf-1 and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s324/325, s338/339 and s346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with sdf-1 or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf-1-induced phosphorylation at s324/325, s338/339 and s346/347 was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s346/347 and then at s324/325 and s338/339. taken together, these results indicate that the c-terminus of cxcr4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: published data [2] indicate that levels of ota in milk are roughly one tenth (0.1) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [1] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was 235 ± 129 ng/l, and no major variations were observed over time (p = 0.19). on the other hand, ota levels found in colostrum (85 ± 60 ng/l) were higher than in mature milk (p < 0.05). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p 0.43 ± 0.26) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [3] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [2] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= 28) and turkey (n=10, only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [3] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [4] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of 5 ng/kg-bw/day set for adults [1] . in both cohorts, most of the urine samples tested positive for ota (chile 72%, turkey 80%). ota levels observed in urine samples from the turkish infants (range: 116 -1,361 ng/l) were 10 fold higher than levels found in chilean samples (range positive samples: 17 -320 ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to 30% of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p450 system. using the estrogen receptor response assay (e)-4-hydroxyclomiphene and (e)-4-hydroxy-n-desethylclomiphene (ec50: 2.5 and 1.4 nm, respectively) turned out to be 100 times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp2d6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. n-deethylation was catalysed by cyps 3a4/5, 2d6, 2c19 and 2c8. rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional cyp2d6 genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp2d6 genes (poor metabolizers) cmax of (e)-4-hydroxyclomiphene and 4-hydroxy-ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional cyp2d6 allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µm. compounds 2 and 3 showed significant toxic effects at a concentration of 5 µm. the compound 4 showed no toxicity up to a concentration of 100 µm. all derivatives 1, 2 and 3 have a ec50 between 10 and 15 µm. we further proved the induction of apoptosis by apo-one assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb 153 using whole genome microarray analysis neser s. 1 , lohr c. 1 , van ede k. i. 2 , andresen k. 3 interaction with the aryl hydrocarbon receptor (ahr), with 2,3,7,8-tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p450 isoenzymes (cyps), i.e., cyp1a1, 1a2, and 1b1. one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c57bl/6 mice were treated with single doses of six dl-congeners (tcdd, pcb 118, pcb 126, and pcb 156) , and the 'non-dioxin-like' (ndl) pcb 153. quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array (4x44k) system. the quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated 89 genes, and down-regulated 66, 4-pncdf-treatment had impact on 3208 (up), and 2396 (down). treatment with pcb 118 led to marginal numbers of 16 up and 6 down-regulated genes. with 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between tcdd-and 1-pncdd-treatment. no overlap was found due to treatments with the ndl pcb 153 (21 up, 1 down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. while pcb 118 regulated only genes involved in drug metabolism, omission of pcb 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb 153 was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, 4454 enquiries were registered in total. in 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). the majority of the requesting patients (61.5%) was born before 1970. a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and 14 c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over 120 min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter 2, oat2 (slc22a7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat2 localization in human liver. because interindividual variability of oat2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat2 expression. methods: an expression profile of oat2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat2 mrna expression was analyzed in well-characterized human liver samples from 150 caucasians that were accompanied by detailed demographic and clinical data. oat2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat2 protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc22a7 gene region. results: oat2 mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat2 was identified in human liver. hepatic expression of full-length oat2 mrna and oat2 protein varied 37-fold and 41fold, respectively. oat2 mrna and protein levels did not correlate with each other. oat2 was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the slc22a7 gene were identified. univariate analysis showed that oat2 mrna is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat2 expression. this indicates consequences for hepatic drug elimination of and response to oat2 drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [1] . we investigated the interaction of mb327 and several structure analogous at the orthosteric binding site of human α7 nachr (hα7 nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα7 nachrs were investigated with radioligand binding experiments performed as high-throughput method [2] . membrane preparations of gh 4c1 cells stably expressed hα7 nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb442, mb456 and mb770 exhibited ki values at micromolar concentrations while three other compounds, mb327, mb583 and the pharmacological inactive mb424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb442 which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku80 mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh2ax and 53bp1 foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku80 formed at early times after acnu treatment less γh2ax and 53bp1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku80 mutant cells show foci levels similar to the wt indicating restitution of h2ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh2ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh2ax foci increased significantly in the s-phase and remained at a high level during g2 where a fraction of cells remained arrested. rad51, atm, mdc-1 and rpa-2 foci were also formed and shown to co-localize with γh2ax. these foci were ameliorated significantly in s-and g2-phase, which was similar to the time course of γh2ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk2 and less phosphorylation of chk1 in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb1 globally knock-out animal as background, we induce the expression of cb1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb1 analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb1 receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c5a, fmlp, and leukotriene b4 are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi2 and gαi3 isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi2 was upregulated in gαi3-deficient macrophages. vice versa gαi3 was upregulated in gαi2-deficient macrophages. concerning gβ isoforms, both gβ1 and gβ2 were strongly reduced in the gαi2-deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi3-deficient macrophages showed reduced gβ1 protein levels only which caused a change in the gβ2/ gβ1 quotient in favour of gβ2. we are currently establishing an in vivo infection model with l. monocytogenes in gαi2and gαi3-deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr.9, 97078 würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin2, in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin2. upon β2-adrenergic receptor (β2ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin2flash2), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin2 relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin2/β2ar interaction on a timescale of seconds. a β2ar mutant that was previously shown not to interact with β-arrestin2 was utilized as control and did not induce a conformational change in the β-arrestin2 molecule. our data provide evidence that β-arrestin2 changes it`s conformation upon binding to the activated β2ar in living cells. the β-arrestin2flash2 sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a4 in the heart nunes f. 1 the calcium binding protein annexin a4 has been examined in the context of heart failure in the past. annexin a4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res 2000). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a4 gene trap model (gt) in which the annexin a4 protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 hz, 1hz and 2 hz as well as an increased sarcomere shortening at 1 hz and 2 hz in anxa4 gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. wt; n=14-18/5). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca 2+ -atpase (serca2a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n=7). however, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with hax1, which acts as a repressor of serca2a (n=4). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 m-10 -5 m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at 10 -4 m iso [%]: wt: 433±76; gt: 699±63 *= p<0.05 vs wt; n=8-10). in conclusion, annexin a4 contributes to the regulation of cardiomyocyte contractility. the anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. 2000 mar;45 (4) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr1i subfamily of nuclear receptors. based on a 3-dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr1i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of 250 µl serum with oasis hlb cartridges allowed a reliable quantification down to 5 ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp18 column (55 mm x 2 mm; 4 µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai 7, 60590 frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl11(4-79) has antagonistic function for cxcr3, cxcl12(p2g2) is able to inhibit cxcr4 and the herpesvirus 8 encoded protein vmip-ii interferes with ccr1, -2 and -5 as well as with cxcr3, -4 and cx3cr1. their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl11(4-79), cxcl12(p2g2) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th-1 cell clone if12 with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl11(4-79), cxcl12(p2g2) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. small heterodimer partner 1 (shp-1) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp-1 is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp-1 expression, suggesting that shp-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp-1, including hnf4α, lrh1, lxr, fxr, srebp1c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp-1 and to determine their impact on the transactivation of shp-1. 120 dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely -594t>c (rs71636795), -413g>c, -423 c>t (rs78182695) and del-195ctga (rs145613139). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh1, lxr, fxr, srebp1c and pparγ) of shp-1 expression. only the transactivation by hnf4α was decreased in the presence of the -423 c>t polymorphism to 69% and the -413g>c polymorphism to 75%. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about 1 in 20.000 individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [1] . mutations in the pkhd1 gene cause arpkd. more than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [1] [2] [3] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [2, 3] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [4] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda 1-1874 ) and tcdb (tcdb 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda 1-1874 and tcdb 1-1852 induced time and concentration dependent cell rounding and rac1-glucosylation. however, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh3-only protein bid, a member of the bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi6c9 in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi-6c9 were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht-22 cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in ht-22 cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. 1 , selinski s. in the 1990s, an uncommonly high percentage of glutathione s-transferase m1 (gstm1) negative bladder cancer cases (70 %) was reported in the greater dortmund area (golka et al., 1997) . the question arose whether this uncommonly high percentage of gstm1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. in total 196 bladder cancer patients from the st.-josefs-hospital dortmund-hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm1, gstt1 and the n-acetyltransferase 2 (nat2) tag snp rs1495741. the frequency of the gstm1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). nat2 genotypes were distributed equally among cases and controls (63% slow acetylators). less gstt1 negative genotypes were present in cases (17%; controls 20%). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht-22 neurons. silencing of aif expression by sirna (20nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone (20nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht-22 cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca 2+ influx through cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2a (csnb2), aland island eye disease (aied) and cone-rod dystrophy type 3 (cordx3). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav1.4 function we analyzed the visual phenotype of cav1.4-deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav1.4-deficient mice. heterozygous cav1.4 mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav1.4 channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. 25, 79104 freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase 2b15 enzymes (ugt2b15) revealed that ugt2b15.2 and ugt2b15.5 had markedly lower intrinsic clearance as compared to ugt2b15.1 (hanioka et al., 2011) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, 2009, mielke et al., 2011) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt2b15.2 variant (v2) was 5 fold and those of the ugt2b15.5 variant (v5) was 7 fold higher than that of the ugt15.1 variant. with dermal exposure at a relevant exposure level, the cmax values were 1.4 (v2) and 1.6 fold (v5) of ugt15.1 variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (v2) and 3.2 fold (v5) higher cmax values as compared to ugt15.1 variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose 5.7 fold (v2) and 8.6 fold (v5), for relevant dermal exposure 1.4 fold (v2) and 1.6 fold (v5), and for combined exposure 1.9 fold (v2) and 2.5 fold (v5) of ugt15.1 variant. from the results we conclude: (1) polymorphism of udpglucuronyltransferase (2b15.2 and 2b15.5) has an impact on the blood concentrations which, however, is less than 10 fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. (2) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase 2b15 polymorphism on bisphenol a glucuronidation. arch toxicol. 2011, 85(11) :1373-81. mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. 2009 8; 190(1) :32-40. mielke h, partosch f, gundertthe heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway (1) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde5 specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation (1) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. 2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion (2) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde5) is expressed in cm (2). sil may act on other pdes, such as pde1c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase (7days of angii infusion at 2mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (t333) and 347 (t347), which enabled us to selectively detect either the t333-or the t347-phosphorylated form of sst5. we show that agonist-mediated phosphorylation occurs at t333, whereas t347 is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand l-817,818 but not octreotide or ke108 were able to promote a clearly detectable t333 phosphorylation. however, none of these compounds was able to stimulate t333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. agonist-induced t333 phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase 2 (grk2). like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. however, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. we also identify protein phosphatase 1g (pp1g) as sst5 receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t333. in addition, we identify grk2-mediated phosphorylation and pp1g-mediated dephosphorylation of t333 as key regulators of rapid internalization and recycling of the human sst5 receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß2 adrenergic receptor, d1 dopamine receptor, parathyroid hormone receptor 1, thromboxane a receptor and the vasopressin receptor 1. however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase 1beta (pp1ß) as the phosphatase for the cluster of phosphorylated threonines (353ttetqrt359) within the sst2a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase 1gamma (pp1γ) as mor phosphatase that catalyzed t370 and s375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst2a chimera with the rsst2a c-terminal tail and a rsst2a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c7-carbonyl function of pat. the exocyclic amino group of the dna base is linked to c1 of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of 10 top prescribed psychiatric drugs. for 7 drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in 40 spcs 2220 individual data points (55.5±17.4 per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. the respective spcs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the 28 spcs of the 7 drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. protein kinase ck2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . elevated ck2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . ck2 is a heterotetramer consisting of two catalytic subunits (ck2α) attached to a dimer of regulatory subunits (ck2β) [4] . ck2β stabilizes ck2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . due to the relatively small and hydrophobic ck2α-ck2β interface (832 å²) [4] , low molecular weight inhibitors are able to interfere with the ck2 subunit interaction and thus affect the kinase activity [6] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [3] . we have developed an elisa-based ck2α-ck2β binding assay using recombinant human ck2 subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck2 subunit interaction. primary hits were further characterized by determination of the parameters ic50 and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy926 were exposed for 14 days to 100 ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of matrigel-coated inserts. differential expressions of 20 phosphoproteins were found in cb-treated cells while an altered expression of 33 phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [1] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße 8-10, 10589 berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in 1996. since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in 2007 by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: 1. applicability domain. the chemical space of the ttc dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz 1, 44789 bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-bpde)-dna adducts as well as cytochrome p450 (cyp) 1a1/1b1 enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a549 lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after 24 h and 48 h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp1a1/1b1 activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-bpde/10 8 nucleotides after incubation with 1 µm (24 h) and 3 µm b[a]p (48 h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after 24 h than after 48 h. increased cyp1a1/1b1 activities were observed at > 0.1 -1 µm (24 h) and > 0.1 -3 µm (48 h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µm b[a]p and after 48 h with ≥ 1 µm b[a]p. overall, incubation of a549 cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a549 cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav1.2 channel complex has been shown to play a key role in cav1.2 channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb2 gene coding for cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase a or ca 2+ /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav1.2. potential phosphorylation sites are ser478 and ser479 (in the β2 subunit in rat, perez-reyes et al. 1992 a ) and ser1928 in the c-terminus of the poreforming α1c subunit (lemke et al. 2008 b ) . in cardiomyocytes camkii regulates ca 2+ release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav1.2 channel complex and camkii are thr498 in the β2 subunit (in rat, grueter et al. 2006 c ) and ser1512 and ser1570 in the cterminus of the α1c subunit (blaich et al. 2010 d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon 14 after aa pro501. this mutation prevented translation of the cavβ2 c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ2stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ2stop mice with s1928a mice (lemke et al. 2008 b ) lacking the pka phosphorylation site s1928 in the α1c subunit (s1928aβ2stop mouse) or with sf mice (blaich et al. 2010 d ) lacking the camkii phosphorylation sites s1512 and s1570 in the α1c c-terminus (sfβ2stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s1928aβ2stop mice. electrophysiological investigations on cardiomyocytes of s1928aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav1.2 channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: 43.6 ± 4.6 pf, n=33; caf: 54.7 ± 5.1 pf, n = 17) and membrane potential (sr: -21.0 ± 4.3 mv, n = 14; caf: -27. 4 ± 4.8 mv, n = 16) . in both groups, we observed fast activating outward currents with a mean threshold at -20 mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: 23.8 ± 4.2 pa/pf, n = 15; caf: 6.1 ± 1.0, n = 6; p < 0.05). when maintained in culture for 3-5 weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: 38%; sr: 15%). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: 6.1 ± 2.0 pa/pf, n = 5; caf: 17.4 ± 4.4 pa/pf, n = 6; p < 0.05). na + currents were not altered by 100 nm tetrodotoxin (ttx), but 10 µm ttx reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav1.5. conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha2a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., 35043 marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha2a-adrenergic receptor. hek293t cells were transfected with yfp-tagged alpha2a-adrenergic receptor and the three subunits of gi1. the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha2aadrenergic receptor recruits gi with a half-life of around 150 milliseconds. when arrestin3 and grk2 were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around 2 seconds. there seemed to be no effect of grk2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk2 alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha2a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin3-cfp to yfp-tagged alpha2a-adrenergic receptors in the presence of grk2. upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin3, confirming previous observations that the alpha2a-adrenergic receptor recruits arrestin3. co-transfection of the three subunits of gi1 had no effect on the kinetics of arrestin3 binding to the alpha2a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha2a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b1, b2, b3) in detrusor and urothelium. we have shown previously that only b3-ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with 1 µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment 10 µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b3-ar in the absence of urothelium. but in intact detrusor strips b2-ars seem to have an additional inhibitory effect. affinity of the selective b3-ar antagonist l748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator 1 (crtc1) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc1 is involved in this process, we investigated the expression of crtc1 in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc1 protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc3 mutant mice the expression of crtc1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). in both forms of human hypertrophy, we found a significantly 5-fold upregulation of crtc1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). conclusions: crtc1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse 190, 8057 zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at1-receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for 3 months with telmisartan (tel 8mg/kg/d) or amlodipine (aml, 12 mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after 6 months by more than 60 g. tel normalized sbp whereas it remained >200 mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β2-adrenoceptor -camp mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., 53105 bonn, germany based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. mrc-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mrna determination by qpcr. β2-adrenoceptor mrna decreased with a half-life of 25 min after inhibition of mrna synthesis with actinomycin d (30 µm), but increased by 333±85%, 502±52% and 640±165% (means±sem) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µm). the β2-adrenoceptor agonists formoterol and olodaterol (1-100 nm) induced a rapid increase in β2-adrenoceptor mrna (maximally within 1 h by 100±19% and 110±19% at 10 nm, resp.). however, after 4 h exposure to 10 nm formoterol or olodaterol a reduction in β2-adrenoceptor mrna by 59±8% and 58±6%, resp., was observed. both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µm, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). the formoterol-induced up-regulation of β2-adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. et-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et-1 expression in hlf was explored. mrc-5 hlf were cultured for up to 24 h in absence or presence of test substances, followed by prepro-et-1 (ppet-1) mrna determination by qpcr. the muscarinic agonist oxotremorine (10 µm) induced an increase in ppet-1 mrna by 180%, an effect prevented by 10 nm tiotropium. the β2-adrenoceptor agonist olodaterol (up to 100 nm) caused a reduction of ppet-1 mrna expression by 45%. the effect of 10 nm olodaterol was prevented by ici 118,551 (1 µm), but not affect by cgp 20712 (3 µm) . the pka agonist 6-bnz-camp (500 µm) caused a reduction in ppet-1 mrna expression by 65%, whereas the epac agonist 8-cpt-2'-o-me-camp (100 µm) caused only a marginal inhibition by 22%. olodaterol (10 nm) strongly opposed the stimulatory effect of 10 µm oxotremorine. an increase in ppet-1 mrna expression by 185% caused by 0.3 ng/ml tgf-β was effectively opposed by 10 and 100 nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet-1 mrna caused by a maximally effective concentration of tgf-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nm olodaterol. likewise, the pkaagonist 6-bnz-camp (500 µm) opposed the increase in ppet-1 mrna expression caused by 0.3 ng/ml tgf-β, but not that caused by 1 ng/ml tgf-β. tgf-β caused, with an ic 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mrna expression, maximally by 90% within 6 h. et-1 expression in hlf is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. the effect of β2-adrenoceptors may be mediated via pka. et-1 expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β2-adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β2-adrenoceptors. since et-1 can promote pro-fibrotic features in hlf, inhibition of et-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h9c2 embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv4.3 and kv4.2, the cytosolic β-subunit kchip2 and the transmembrane subunits dpp6 and dpp10 using rt-pcr. treatment with doxorubicin (2 µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h9c2 cardiac myoblasts. while kv4.3 was detected in h9c2 cells and hearts from human and rat, kv4.2 mrna was only expressed in adult rats. during hypertrophy kv4.3 was downregulated in human tissue as well as in doxorubicin-treated h9c2 cells compared to the controls. in rat hearts kv4.2 expression was increased after doxorubicin treatment. interestingly, kv4.2 was also found to be up-regulated in rat heart tissues and h9c2 cells after treatment with doxorubicin. as previously shown, kchip2 mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip2 mrna was upregulated in hypertrophic rat hearts and h9c2 cells. the expression of dpp6 and dpp10 was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp6 and dpp10 were not expressed in the adult rat heart or h9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h9c2 cells, the mrna of dpp6 and dpp10 was up-regulated. h9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h9c2 cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp6 and dpp10 in doxorubicin-induced hypertrophic h9c2 cells. in preliminary experiments specific short-hairpin rna, targeting dpp6 and dpp10, has been designed and tested in heterologous expression systems. the nonsynonymous c.521t>c germline genetic variation in the liver-specific organic anion transporter slco1b1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. 1 background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc2, slco19a1, and slco1b1, have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc2, slco19a1, and slco1b1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c24h, mtx auc24-48h, and mtx clearance cl) in an unselected population of 419 children with all from the all-bfm 2000 trial (clinicaltrials.gov: nct00430118) who received 1676 courses of mtx at 5 g/m 2 as 24 h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. (1998) . abcc2 c.-24c>t (rs717620), slco19a1 c.80g>a (p.his27arg, rs1051266), slco1b1 c.521t>c (p.val174ala, rs4149056) and slco1b1 388a>g (p.asn130asp, rs2306283) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c24h (50.95 ± 24.15 µmol/l), auc24-48h (57.44 ± 37.52 h*mg/l), and cl (390.72 ± 223.95 ml/min/m²). the rgc values of c24h, auc24-48h, and cl ranged from 0.61-0.71 suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco1b1 c.521t>c snp was significantly associated with c24h (p<0.001), auc24-48h (p<0.001), and cl (p=0.011) of mtx. compared with the wildtype genotype, in patients with the tc genotype c24h and auc24-48h increased by 18% (p=0.009) and 28% (p=0.003), respectively, whereas cl significantly decreased by 15% (p=0.012). pharmacokinetic variables significantly changed with increasing number of variant c.521t>c alleles (p<0.02, jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc2 c.-24c>t, slco19a1 c.80g>a, and slco1b1 388a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c.521t>c polymorphism in slco1b1 contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase-2 (cox-2) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a549, h358, h460), the present study investigates the contribution of cox-2 and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µm), whereas structurally-related cox-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox-2 inhibitor ns-398, the pparγ antagonist gw-9662 and by sirna targeting cox-2 or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox-2 and pparγ at both mrna and protein level. using an established cox-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns-398 was shown to suppress celecoxib-induced cox-2 activity when added prior to arachidonic acid. among the cox-2-dependent pgs analyzed, pgd 2 and its dehydration product 15-deoxy-∆ 12,14 -pgj2 were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns-398 which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox-2-and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox-2 and pparγ and a subsequent nuclear translocation of pparγ by cox-2-dependent pgs. uncoagulated ppp contained 40-60 nm thrombin throughout. fxa was initially measured in clots at 10 nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for 24h only marginally influenced smc apoptosis but increased mitogenesis over 15-fold. this was reduced by all 4 inhibitors. clot-stimulated induction of tnfα and interleukin-6 mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx9065a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef17 mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. 1 , stephan-schnatz k. the guanine nucleotide exchange factor rhogef17 is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef17 by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef17 influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef17 depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the rhogef17 knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef17 was paralleled by a loss in rhoa and cadherine expression. as rhogef17 mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon (8-pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first 12 hours. in contrast, the adhesion of rasmc after rhogef17 knockdown was no longer stimulated by cgmp. as these data indicate that rhogef17 mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef17 depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef17, we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef17 knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h2 receptors (h2r). activation of fpr leads to a release of reactive oxygen species (ros). h2r activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h2r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites (100 mg) were incubated in gc vials with 1 ml dest. water or 1 ml methanol, each at 37 °c for 72 hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites 18 different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram (5 ± 2 µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr4 is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr4 is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr4 in tumor metastasis is discussed. subsequently, detection of cxcr4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr4 antibody umb-2. methods: in comparison to negative and positive control samples (cxcr4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr4 was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr4 positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb-2 may prove to be of great value in the assessment of cxcr4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced o2 β2-adrenoceptor (adrb2)-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb2 gene contains a total of 49 polymorphisms. this variability could cause part of the ~70 % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb2agonists for each individual. (ortega et al. 2007; chung et al. 2011) our present study connects sequence data with pharmacological data of prototypical adrb2-ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. characteristic pharmacological values (pic50, emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb2-sequence variant is determined by sanger-sequencing. complete determination of a 1,490 bp sequence, including the entire adrb2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (camp) and cyclic guanosine 3',5'-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cump). 2 these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using 2'-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde3a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine 3',5'-monophosphate (cimp). 2 using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde3a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde3a. this finding is consistent with data published by hardman and sutherland 3 who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde3a. as cump has furthermore been proven to be present in mammalian cells 4 , to differentially activate camp-and cgmp-dependent protein kinases 5 and to be synthesized from utp by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde3a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat3 and identify signal proteins involved in this pathway. rhoa-dependent stat3 stimulation requires rock and junkinase activation as well as ap1-induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl1/i-309 as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat3-dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. 1 christian-albrechts universität institut für immunologie, michealisstrasse 5, 24105 kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about 50%. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt=8ml/kg and f=180min -1 with fio2=0.3 and peep=2cmh2o while lung mechanics were followed. half of the mice received 50µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. 1 cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht22 neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht-22 cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high 4525 µmol; medium 2219 µmol; low 1053 µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium) and 14.6 ± 6.8% (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nm*h -1 (high); 299.3 ± 79.6 nm*h -1 (medium) and 222.8 ± 91.3 nm*h -1 (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p2x3 ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany the homomeric p2x3 receptor (p2x3r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p2x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p2x3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p2x3 receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p2x3 wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek 293 cells, heterologously expressing the human p2x3 receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p2x4r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p2x3 agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany purinergic p2x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p2x receptor family, the p2x3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p2x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p2x3 receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp2x3 model that we developed from the known zebra fish p2x4 crystal structure in the closed state. voltage-dependent modulation of alpha2a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. 1, 35034 marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α2a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek 293 cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations (500 nm: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µm: 9 % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine (1 µm, vm=-90 mv) resulted in a fret response that was inhibited by 40 % at +60 mv. in contrast to ne, strong receptor inhibition at +60 mv was present even at super-saturating concentrations of clonidine (100 µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a2a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = +60 mv. therefore we conclude that negative membrane potentials promote active conformations of the a2a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has3, in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. 1 , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e2) mediated effects on ha-rich ecm during actinic aging. 10 weeks of uvb irradiation (3 x 1 med (80 mj/cm 2 ), weekly) of hairless skh-1 mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e2) by means of controlled release pellets abolished these effects confirming the stimulatory role of e2. the increase of dermal ha correlated with induction of ha synthase has3 by e2. in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e2. however in cultured skin fibroblasts e2 reduced the expression of versican and had no effect on has3. therefore, direct upregulation of has3 and versican in fiborblasts by e2 was excluded. however, e2 increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has3 and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e2 treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e2 during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. 1 , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno3 solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio2 samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. 2, 10115 berlin, germany c3 exoenzyme (c3bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c3bot to the hippocampus-derived ht22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c3bot. the binding assays established that c3bot bound in a concentration-dependent manner to ht22 cells. in the overlay assay we detected one clear band of 55 kda. to elucidate whether glycosylation is important for the c3bot-protein interaction, ht22 cells were incubated with glycosidase f resulting in a decreased binding of c3 to the 55 kda band. to explore the involvement of phosphorylation in the binding of c3 to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c3bot. pretreatment greatly reduced the c3bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c3botprotein interaction in the following overlay. thus, interactions between c3bot and ht22 cell proteins may require phosphorylation. to further characterize the 55 kda band as binding target of c3bot, the 55 kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this 55 kda single gel band 141 proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c3bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c3bot-protein interaction. s76 335 solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. 24, 50931 köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, vmax and clint with regard to the 1-hydroxylation of midazolam via cyp3a4 and the cyp1a2 catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er81 rommel c. 1 , rösner s. introduction: the transcription factor er81 (ets related 81) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er81 is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er81 mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er81 expression. thus, the aim of the present study was to investigate the cardiac function of er81 in genetically modified mouse models. we previously generated transgenic mice overexpressing er81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day 5 after birth (p5) and in adult mice (3 months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day 5 after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er81 αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er81 αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine 16 of phospholamban (pln) was reduced in er81 αmhc mice. in addition, protein phosphatase 1 (pp1) expression was significantly increased in er81 αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca2a protein in er81 αmhc atria. electron microscopy revealed the significant structural remodeling of er81 αmhc atria at 3 months of age. conclusion: increased cardiac expression of the ets-transcription factor er81 leads to structural and electrical remodeling of the atria. thus, er81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06097 halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p2-purinoceptors which are further divided in p2x1-7 and p2y1-14-receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µm atp as well as utp transiently increased phosphorylation of erk 1/2 and p38 mapk with a maximum effect at 5 to 10 minutes after application of atp and utp in neonatal cardiac myocytes (n=3 preparations each). the maximum phosphorylation of p38 increased with atp to 284% ± 68% (p<0.05) at 10 minutes and with utp up to 204% ± 21% (p<0.05) at 5 minutes. the phosphorylation with erk 1/2 mapk increased with atp to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with utp at 10 minutes of basal values, respectively. after 20 minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk 1/2 and p38 mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. 1 , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students (5 th or 10 th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of 60 points could be obtained as a sum of both tests. results: in the means 40% of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk1/2 ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany background and aims: the extracellular regulated kinases 1 and 2 (erk1/2) play an important role in cardiac hypertrophy and cell survival. erk1/2 are phosphorylated at the so-called tey motif, which in turn activates erk1/2. hypertrophic stimuli lead to an additional autophosphorylation threonine188 (thr188). this autophosphorylation of erk1/2 stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr188 phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk1/2 activity and prevention of erk thr188 phosphorylation, we either inhibited erk activity with pd98059 or overexpressed a mutant of erk2, which cannot be phosphorylated at thr188 phosphorylation. while inhibition of overall erk activity with pd98059 attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (erk2 t188a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk2 t188a significantly reduced tac-induced hypertrophy compared to wild-type erk2 overexpressing mice. in line with the in vitro experiments, erk thr188 inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk1/2. therefore, specific targeting of erk thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde10, a pde in principle capable of hydrolysing camp as well as cgmp. pde10 contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde10 was mostly based on the analysis of mrna distribution, we generated antisera against pde10 to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde10 displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde10 inhibitor papaverine as well as specific pde10 inhibitors suggest pde10 as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde10 to camp degradation in striatum, to identify the physiological pathways pde10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse 31, 93053 regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp 3r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp3r-ii and the insp3r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the 52 amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca 2+ -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp3r interactions. the insp3r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp3r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp3r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp3r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp3r-i and insp3r-iii but not with insp3r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type 1 enhancer binding protein 1 (hivep1) is regulated by proinflammatory stimuli and statins salomon a. 1, 2 , schmitz b. objective: hivep1 binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the hivep1 gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, 2010; plos one, 2011) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy926) with proinflammatory cytokines or statins (24h). serial hivep1 promoter deletion constructs were cloned into the pgl3-basic vector, a potential enhancer fragment, harbouring rs169713c/t, into the pgl3-promoter vector. in ea.hy926 cells and thp1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy926 cells, endogenous hivep1 expression was increased by proinflammatory cytokines tnfα and il-1β. simvastatin (1.2 and 2.4 µm) and atorvastatin (9 µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep1 expression. the construct harbouring rs169713t exerted significantly higher transcriptional activity (ta) compared to rs169713c (p<0.001). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep1 expression in ea.hy926 and thp1 cells. cotransfection of sp1 and egr1 led to an increase in ta, while wt1 exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp1 to the 5'-flanking region and the intronic modulator of hivep1. conclusion: increased hivep1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep1 expression is regulated by sp1 combined in a transcription factor module with egr1 and wt1 under basal and/or inflammatory conditions. the rs169713 site harbours potential activational capacity for hivep1 gene transcription and may communicate with the sp1/egr1/wt1 module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv7.2/3 channels, we examined the acute effects of the preferred kv7.2/3 channel opener ica 27243 on lid in this animal model. four weeks post 6-ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg l-dopa and 15 mg/kg benserazide for 20 days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. for drug testing, ica 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ica 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ica 27243. a higher dose of 15 mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v7 channel opener retigabine was based on its action on striatal kv7.2/3 channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n=4-5). myotoxic cerivastatin (0.01, 0.1, 1 µm for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in inos transcript (mean±sem: 0.01 µm 4±0.9-fold, n=3 p<0.05; 0.1 µm 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µm 5.1±2-fold, n=4 p<0.05; 0.1 µm 7.8±0.8-fold, n=3 p<0.05). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with 1400w, a specific inos inhibitor. we applied 5 µm of 1400w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n=9-15). also, l-name (10 mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp (10 µm) caused a reduction of contractile force. combined treatment with cerivastatin and 0.1 µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with 3m syndrome and mutations in the cul7 gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany 3m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul7 (76%) or obsl1 (16%) gene, respectively. cul7 constitutes an e3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (igf-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (irs-1). to investigate the role of cul7 mediated irs-1 degradation in the pathogenesis of 3m syndrome. primary skin fibroblasts of seven 3m syndrome patients (six with cul7 mutations, one with a obsl1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs-1 protein concentrations and activation of the igf-1 signaling pathway (western blot). the proliferation rate of 3m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs-1 protein levels and activation of the pi3k/akt and erk mapk pathway were only increased in a subset of 3m cells that carried cul7 mutations, but not in cells from a patient with the obsl1 mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs-1 levels to the observed phenotype, human imr90 fibroblasts were stably transfected with retroviral vectors encoding irs-1. despite 20-fold overexpression of irs-1 compared to empty vector controls, no significant effect of igf-1 stimulation on proliferation rate or pi3k/akt and erk mapk signaling was observed. skin fibroblasts of 3m patients with cul7 mutations displayed an increased proliferation rate and enhanced activation of the igf-1 signaling pathways. despite accumulation of irs-1 in fibroblasts from a subset of 3m patients with cul7 mutations, no pathomechanistic role for irs-1 could be demonstrated. collectively, our data indicate that a dysregulated igf-1 signaling may contribute to the pathogenesis of 3m syndrome, yet in an irs-1 independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was 10 days. to date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak1 sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. 43, 61231 bad nauheim, germany tgf-β activated kinase 1 (tak1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin-1 or tnfa to ikk, p38 and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t2 with animals carrying floxed alleles of the tak1 gene. adipoqcreer ; tak1 fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak1 knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin-6. stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak1 deficient adipocytes show reduced activation of jnk and p38 which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/11 and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/11-regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk2 and subsequently the erk1/2 cascade. the role of pyk2 for the growth of sclc cells was further supported by the fact that inhibition of pyk2 using a sirna approach or a novel specific inhibitor, pf431396, exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g 12/13 signaling by sirna-mediated g(alpha)12 or g(alpha)13 knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk2 versus the role of calcium-independent signaling via g12/13, we tested the effect of pyk2 inhibition in cells with impaired g12/13 signaling. notably, pyk2 and g12/13 double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk2 activation or g12/13-dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp-1 were used for 7 days and stimulated with various cytokines (il-4, gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd11c, cd209, cd83) and by immunfluorescence compared to monocytes. the bronchial tract contains up to 800 dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 hz and the transepithelial resistance values were stable between 600 to 800 ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git1 and cavb3 schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, 66421 homburg, germany high-voltage activated ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, cavβ1-β4 and, maybe, α2δ1-δ4. by a cavβ3-specific antibody based affinity chromatography the cavβ3 protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ3 were identified by mass spectrometry (lc-esi-ms/ms) and include α1-subunits, α2δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ3 protein, including the g protein-coupled receptor kinase-interactor 1 (git1). the 770aa git1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git1 and cavβ3 proteins were co-immunoprecipitated by the antibodies for cavβ3 and git, respectively. we cloned the git1 cdna from mouse brain and co-expressed it with the cavβ3 subunit in hek cells. like in brain lysates the git protein was retained by cavβ3 precipitated by the antibody for cavβ3 and cavβ3 was retained by the git1protein precipitated by the antibody for git1. both proteins, cavβ3 and git1 are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ3-git1 interaction in the absence of functional cav channels. in addition, using mefs from cavβ3-deficient mice enables us to control the impact of cavβ3 on git1 function. vice versa down-regulation of git1 by specific sirnas might allow to control the impact of git1 on cavβ3 function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine-1-phosphate and fty720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine 1-phosphate (s1p) is a mediator that modulates various physiological functions of skin cells. s1p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s1p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s1p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s1p, but not fty720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s1p via the s1p2 receptor, which is not activated by fty720. there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s1p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s1p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s1p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s1p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il-6). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp-1) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd206) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo-1) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above 50 µm are accompanied with inhibition of nat1 activity in human keratinocytes [1] . in the following we investigated the impact of ppd on nat1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp-1 cells. measured nat1 activity of thp-1 was comparable to those found in primary keratinocytes. a 24h treatment of thp-1 cells with physiologically relevant concentrations of ppd (10-200 µm) led to a 47% reduction of nat1 activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already 8h after application of ppd or mappd while nat1 mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat1 activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat1 protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat1 protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat1 protein after 2h. the greatest inhibition was found for oxidised ppd (up to 50%). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat1 enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat1 in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec 3.2.1.22). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened 4011 bp of the 5'-flanking region of gla in 60 patients with fabry's disease and 60 controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy926) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 5'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). we identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to 5.3-fold (p<0.001). in ihke cells, insertion of the minor t allele (rs2071225) significantly enhanced basal ta of the proximal promoter region (p=0.0006), while insertion decreased basal ta (p<0.0001) of the distal promoter portion. the combined insertion of the minor c alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p=0.0037). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c16:0-cer levels were 1.9 fold increased in ms patients. this translates into the finding that c16:0-cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c16:0-cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) 6 in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers6 and its product c16:0-cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c16:0-cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers6 plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers6 and therefore to limit the inflammatory effects of c16:0-cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. 1 cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc1 or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm22 promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm22 promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a549 lung epithelial cells, hepg2 liver epithelial cells and hk-2 proximal tubule epithelial cells. the used nanomaterials incorporated five tio 2 samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst-1 assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp7 project enpra (fp7-nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v79 cells expressing human cytochrome p450 1b1 schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v79 lung fibroblasts (v79 cells) represents a widely-used mammalian test system to detect gene mutations. since v79 cells do not express any cytochrome-p450dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v79 cells expressing human (h) cyp isozymes have been commercialized. to test these v79 cells for their use in the hprt test, v79 h1a1 and h1b1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (smf), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v79 cells (w2:13±4; w4:2±1) and v79 h1a1 (w2:17±4; w4:3±0) only varied within the range of historical controls, smf of v79 h1b1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) although the smf of v79 and v79 h1a1 were similar, the mutational spectrum of v79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of v79 h1a1 was characterized exclusively by transversions and deletion of exon 7+8. surprisingly, with 57 out of 59 cdnas derived from v79 h1b1 mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v79 h1b1, cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between 0.7±0.6 and 1.2±0.0. yet its smf was unstable reaching up to 104±6. in conclusion, the mutational spectrum differed between the v79 cell lines. furthermore, h1b1 expression seemed to enhance smf in v79 cells. even though a temporary reduction of the smf by cloning was possible, smf of v79 h1b1 cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with 20µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open cc on silica gel. the most active fractions 11-17 were separated again yielding 10 subfractions. this afforded 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol-3-o-β-dglucopyranoside, the structures of which were determined by esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats (10-12 weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca 2+ selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai1 and trpc3 in plasma membrane microdomains of rbl-2h3 mast cells. orai1-mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc3. this negative impact of trpc3 on icrac was independent of channel function as the trpc3 pore dead mutant (e630k) inhibited icrac to a similar extent as wild type trpc3. importantly, despite a reduction in icrac, nfat translocation in trpc3 overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc3e630k but substantially reduced by trpc3 mutants with either i) eliminated fkbp12/calcineurin binding (p704q) or ii) deficiency in pkc phosphorylation (s712a). moreover, inhibition of pkc phosphorylation by (gfx109203x; 3 µm) strongly suppressed nfat signaling. we suggest trpc3 as a scaffold that links orai-mediated ca 2+ -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. 1 , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf96365 were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to 39±26% of the initial airway area (%-iaa) and 63±21%-iaa, respectively. in frequency response curves half maximal response was found at 7.0±1.2 hz for guinea pig pcls and 8.1±0.8 hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to 18±15%-iaa. 60% of human pcls were responsive to capsaicin and airways contracted to 72±10%-iaa, respectively. in gp ruthenium red and skf96365 blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk1/2-pathway is involved in pkc-induced nox4 up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55131 mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox4 is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy 926 endothelial cells with phorbol 12myristate 13-acetate (pma) for 48 h leads to an up-regulation of nox4 expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö 6983 and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox4 up-regulation can be attenuated by pd 98,059 (an erk1/2 inhibitor), but not by sp 600125 (a jnk inhibitor), indicating in the involvement of erk1/2. consistently, pma treatment leads to a sustained activation of erk1/2, and sirnamediated knockdown of erk1/2 markedly reduces the pma-induced nox4 up-regulation. h89, an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox4 expression, indicating that msks are not the target molecules of erk1/2 in this scenario. on the contrary, knockdown of the transcription factor elk-1 by sirna significantly reduces the pma-induced nox4 up-regulation. in conclusion, erk1/2 and elk-1 are involved in the pkcα-induced nox4 up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, 97074 wuerzburg, germany annually, over 57,000 women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p53 gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with 2 g of normal mammary gland tissue a smf of 2.2±1.4x10 -7 per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (il-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il-8 mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p65 nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac1 expression, but did not affect epac2 and pka expression. importantly, epac1 expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il-8 release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac1. polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt4) exposed to 0.5 µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt4) were exposed to 0.5 µm bap for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. 1) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mrna (20 %) and protein metabolism (25 %). this pattern changed after 8 wk of treatment. then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins 1 and 2, are currently being validate by pcr. fused master gel : representative 2-de gel of rt4 cells exposed to 0.5 µm bap for 8 wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. 1 , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd (0.01 -3 µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am-630 (cb2 receptor antagonist) and by o-1602 (g protein-coupled receptor [gpr] 55 agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) 1/2. blockade of erk activation by pd98059 prevented cbd-stimulated msc migration, whereas inhibition of p38 mapk by sb203580 was inactive in this respect. furthermore, am-630 and o-1602 were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c57bl/6 wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c57bl/6 wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. 1 hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße 1, 91054 erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr1i2) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco1a2 and slco1b3 and a significantly decreased expression of transporters such as slco2b1, slco2a1 and abcc3 in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco2b1 mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ 2 =6.59; p=0.010; n=34). bisulfite sequencing revealed that promoter cpg islands of abcc3 and slco2a1 were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc-1 and scc-15 cell lines, transcript expression of transporters (e.g., abcc3, slco2a1; p<0.001) and pxr (nr1i2; p<0.001) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc3 and slco2a1 expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco2b1 may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc-1 and scc-15 cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn2 in neuropathic and inflammatory pain schnorr s. 1 , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn1-4) and contributes to cellular excitability in the heart and the nervous system. hcn1 and hcn2 are the two most abundant hcn subunits in peripheral sensory neurons with hcn2 being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn2 by using a nociceptor specific cre-transgene driven by the nav1.8 promoter. the nociceptor-specific knockout of hcn2 in drg neurons (nosphcn2ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn2 was mainly restricted to small sized drg neurons. the conditional loss of hcn2 resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn2ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn2 protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn2 might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy5y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa1 channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv1, trpv2, trpv3, and trpv4 and the cold receptor trpm8 were insensitive towards apomorphine treatment, trpa1 could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa1 channels in a stably transfected cell line (hek293-trpa1), as well as natively expressed trpa1 in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa1 activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa1 expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [1] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [2] , agonists of the muscarinic m2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m2 receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m2 receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m2 receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day 15 the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp1a1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p450 (cyp) 1a1 and 1a2. the most prominent regulator of cyp1a1 is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp1a1 induction in murine hepatoma cells (hepa-1c1c7). indeed, aoh and ame enhanced the levels of cyp1a1 in hepa-1c1c7 cells but not in cells with inactivated ahr (hepa-1c1c12) or arnt (hepa-1c1c4). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp1a1 expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. the protein sequence database uniprotkb stores over 10,500,000 sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go3r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go3r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β1-adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca 2+ -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. 150, 44781 bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca 2+ concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca 2+ concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc1, with angiotensinii (1.44 mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc1 ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. 43, 61231 bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc1), b16f10 melanoma cells or human neuroblastoma cells (sh-sy5y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc13-4 deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc1 and b16 cells was indistinguishable between wild-type mice and animals lacking munc13-4, the number of metastases in the lung was strongly reduced in munc13-4-deficient animals. the strong decrease in formation of metastases in munc13-4 deficient mice was also observed after i.v. injection of llc1 and b16f10 cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol schumacher f. 1 , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that 1-methoxy-3-indolylmethyl (1-mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [1] . we have identified and synthesized the major dna adducts n 2 -(1-mim)-dg and n 6 -(1-mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-mim glucosinolate. the peak levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite 1-mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered 1-mim alcohol generated significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [1] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., 192 (2011) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. 1, 2 , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb1) and multidrug resistance protein 2 (abcc2), and the nuclear pregnane x receptor (pxr/nr1i2), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n=178) dgf occurred in 27.5 %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs2276707 8055tt genotype was significantly associated with dgf (recessive model: or, 9.0; 95%ci, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 holm-adjusted). when we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs2276707 tt genotype of the donor (recessive model: or, 16.08; 95% ci, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). for abcc2 variants only the donor rs17222723 3563t>a genotype correlated with dgf by univariate (or, 4.65; 95%ci, p=0.005 unadjusted) as well as by multivariate analysis (or, 5.5; 95%ci, p=0 .01; table 4 ) but not after correction for multiple testing (p=0.12). for variants in the abcb1 gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant 03is2061c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs1/fe-10). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. 25, 79104 freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas 2011) . in the cytosol, cdt adp-ribosylates actin at arg 177, thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog 2009 ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp2 and acf7. besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim1 that usually functions as a calcium sensor in the er and activates the store operated orai1 calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after 60 min. when bilobalide (10 µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. in the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of 16% of baseline. direct addition of bilobalide (10 µm) to post-ischemic mitochondria caused a 2-fold increase of complex i activity in vitro. pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before mcao caused a significant, 3-fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as 2-hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod-1), catalase (cat) or glutathione peroxidase (gpx1/2). raw246.7 mouse macrophages were exposed to hema (0-8mm) for 24h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine (50µm bso), or enhanced by 2-oxothiazolidine-4-carboxylate (5mm otc) and n-acetyl cysteine (10mm nac). expression of sod-1 found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod-1 expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx1/2 was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h2o2 decomposition. the inhibitory effect of hema on gpx1/2 expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h2o2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h2o2 production leads to the activation of cat expression and a feed-back inhibition of sod-1 expression. the hema-induced reduction of gpx1/2 expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx (0.75 µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime (10 µm) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 hz to 1.67 ± 0.24 hz (control 1.79 + 0.22 hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. muscle force production after indirect stimulation was stable for 6 hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a 1.3 fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n=10 from 6 isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling 5 (rgs5) and peptidylprolyl isomerase a (ppia) were 1.5-2.5 fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n=6). transcripts of rgs5 (2.6 fold, ) and ppia (1.8 fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n=10-12). we conclude that crem deficiency is associated with transcriptional changes of rgs5, pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by gβγ and h-ras shymanets a. 1 phosphoinositide 3-kinase γ (pi3kγ) controls a plethora of cellular responses. pi3kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by gβγ and ras. earlier we speculated that p101 binds to gβγ to translocate cytosolic pi3kγ to the plasma membrane, enabling direct activation of p110γ (brock et al., j. cell biol. 2003) . however, the p87 subunit does not function as gβγ adapter (kurig et al., pnas 2009) . since the impact of each non-catalytic subunit in regulating p110γ by gβγ and ras still remains elusive, we studied their role in detail. gβ1γ2 variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf9 cells and tested for their ability to activate pi3kγ. we observed that p101, but not p87, was able to rescue the stimulatory activity of gβ1γ2 mutants incapable to activate p110γ (shymanets et al., biochem. j. doi:10 .1042/bj20111664). to further study the functional impact of the non-catalytic subunits on pi3kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi3kγ variants. although p87/p110γ exhibited stronger sensitivity to gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of gβ1γ2. to study an effect of ras proteins on pi3kγ variants, recombinantly expressed h-ras was purified from sf9 cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of pi3kγ variants in different signalling pathways. taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of pi3kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive 1999/45/ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, 2008) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive 1999/45/ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, 2010) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. 50 % of all saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (february to may) and a 25 to 40 % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. 25, 79104 freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα12/13 and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα12/13 family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st-2 cells and in primary osteoblasts from rat calvaria. st-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st-2 cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c57bl/6 and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c57bl/6 or balb/c were subjected to high (25cmh 2o) or low pressure (8cmh2o) ventilation for 240 minutes. after 180 minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another 60 minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c57bl/6 mice showed higher tnf, il-1β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl-1, cxcl-2) and il-6. kinase activities (jnk, akt, erk1/2, p38 map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by 50% and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c57bl/6 and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m2 macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh2 nanoparticles for up to 6 days. analysis of cell viability revealed that ps-nh2 but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh2 trigger apoptosis in macrophages. we further polarized macrophages to either m1 or m2 using ifn-γ and lps or il-4, respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m1 and m2 macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp1313. loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. 20, 89081 ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, thp-1 monocytic leukemia cells, and by pmadifferentiated thp-1 cells via different mechanisms. in buffer, macrophages and thp-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp-1 cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp-1 cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh 2 were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp1313. specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh2) of ~100 nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp3 inflammasome activation and the subsequent release of il-1β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp3 protein resulting in a conformational change of the pyrin domain of the nlrp3 protein as predicted by molecular modeling. txnip interaction with nlrp3 led to assembly of the nlrp3 inflammasome complex, to caspase-1 activation, and release of il-1β. using an in vitro knockdown approach, we showed that ps-nh 2 induced activation exclusively of nlrp3, whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of il-1β caused by ps-nh2 nanoparticles. these data reveal a novel mechanism of the nlrp3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp1313. the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a2 and s100a10. addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h 2dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p65/rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph 7.4, helenalin interacts with rela (k d = 4.8 µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic50 of 5.0 µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine 38 by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg35, lys37, gly44 and ile118 of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of 24 µm, though no significant interaction was observed at ph 7.4. thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp1313. prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v79. induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a2 (pga2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the 15dpgj2 and pga2 -induced significant decrease in the tgsh level in v79 may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd86. determination of the concentration required to cause a half-maximal increase in cd86-expression (ec 50) allowed a quantitative evaluation. the irritative potential of the substances was assessed by 7-aad (7-amino-actinomycin d)-staining. the concentration required to devitalize 50 % of the examined cells compared to a zero control was termed ec50%. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). due to a pronounced cytotoxicity (ec50% = 70.94 µmol/l), we could not estimate an ec50-value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin 4 by protein kinase dyrk1a at serine 107 affects protein stability soppa u. 1 septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin 4 (sept4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept4 by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase 1a (dyrk1a). dyrk1a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept4 and overexpression in hela cells we identified serine 107 as the major phosphorylation site of dyrk1a and generated a phosphospecific antibody. transient coexpression of sept4 and dyrk1a in hela cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk1a mutants k188r and d287n did not increase serine 107 phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk1a inhibitor harmine reduced phosphorylation of exogenous sept4 at serine 107 about 25% in hela cells. furthermore, down regulation of dyrk1a by rna interference lead to decreased phosphorylated serine 107. these results indicate that endogenous dyrk1a contributes to sept4 phosphorylation in hela cells. finally we analyzed protein stability of wild type sept4 compared to the phosphorylation resistant s107a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept4 s107a mutant is more stable than wild type sept4. in summary, our results suggest phosphorylation at serine 107 by dyrk1a as a novel mechanism to regulate sept4 stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, 89069 ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee 3, 53175 bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. 2003) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca 2+ influx. in the present study we analyzed whether kca channels of sk4 and bk type play a role in sustaining ca 2+ oscillations at g1-to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk4 modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca 2+ ]i oscillations and peak amplitude were determined using ca 2+ indicator fura-2am. results: sk4, but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk4 inhibition by tram-34 dose-dependently (0,1 to 10 µm) inhibits the growth of primary mammary tumor cells probably by a g1 cell cycle arrest. ca 2+ oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk4 channels. -dependent cell cycle progression is dependent on sk4 activity. blocking sk4 disrupts a feed-forward loop that coordinates ca 2+ influx via trp or crac channels in tumor cells. the consequences of sk4 inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline n-oxide stolze k. 1 esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap 5,5-dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. 9, 97078 würzburg, germany type 2 diabetes mellitus (dm2) is a growing health problem affecting more than 150 million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o 2-) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm2 complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc1 is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn 2+ quenching and qpcr analysis were applied. furthermore, the effect of trpc1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc1 was not able to function as a homomeric channel. instead, trpc1 subunits formed functional receptor-operated heteromeric channel complexes with trpc3, 4, 5, 6, and 7. heteromers containing trpc1 subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc1 further reduced calcium permeability. in gnrh neurons endogenously expressing trpc1, 2, 5, and 6, downregulation of trpc1 by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc1 was not involved in store-operated cation influx in gnrh neurons. moreover, trpc1 suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc1 and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin-3 (trpm3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm3 blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic50 values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm3 blockers. furthermore, we could show that these blockers are effective on endogenous trpm3 in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm3 activity in vivo. in sensory neurons, trpm3 may exert similar functions as trpv1. thus, trpm3 blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec50) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): 1 /25× ec50, 1 /10× ec50 , 1 /3× ec50, and 1× ec50. each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative γh2ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec50 values of substances were found (mmol/l;mean +/-sem): tmp(eo)9ta a, b 0.087 ± 0.011; 1,6-hddma b, c 4.500 ± 0.700; etma a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-hddma, b significantly (p < 0.05) differently to etma, c significantly (p < 0.05) differently to tmp(eo)9ta. after six hours of exposure with tmp(eo)9ta at 0.00348 mm there were induced 0.55 γ-h2ax foci-formations in hgfs, at 0.00870 mm 0.67 foci, at 0.02900 mm 0.86 foci and at 0.08700 mm 0.97 foci. after exposure with 1,6-hddma at 0.180 mm there were induced 0.45 γ-h2ax foci, at 0.450 mm 0.64 foci, at 1.500 mm 0.94 foci and at 4.500 mm 1.32 foci. after exposure with etma at 0.48 mm there were induced 0.43 γ-h2ax foci, at 1.2 mm 0.50 foci, at 4.0 mm 0.61 foci and at 12 mm 0.71 foci. the negative controls dmso and medium cultures displayed 0.31 -0.34 γ-h2ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-hddma > tmp(eo)9ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. 1 , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative analysis of the γ-h2ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec 50 (mmol/l) of triethylenglykol dimethacrylat (tegdma) and 2-hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < 0.05) higher dsbs compared to hema (1.91 ± 0.04 vs 1.66 ± 0.02). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed (2.02 ± 0.06), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed (1.89 ± 0.07), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed (1.64 ± 0.04), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed (0.76 ± 0.02), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys 101 was replaced by ala (c101a) resulting in destabilization of enos has been generated (enos-c101a). transgenic mice carrying c101a were generated on a c57bl/6 background using the endotheliumspecific tie-2 promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c101a (enos-ko/enos-c101a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c57bl/6, enos-ko, and enos-ko/ enos-c101a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c101a-tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of enos on ser1176/79 (470±47%) as compared to c57bl/6 (p<0.05, n=8). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c101a-tg (p<0.05, n=5-8), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c101a as a source of elevated radical generation. endothelium-specific introduction of enos-c101a at ~ 35% of c57bl/6 level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc)2 indicated that endothelium-dependent relaxation in enos-ko/enos-c101a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p<0.05, n=4-7). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n=4-7, p<0.05). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c101a-tg was substantially lower then in enos-ko and tended to be similar to c57bl/6. conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. 1 , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle-6tn and 16hbe14o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk1/2 were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn3o4 or mno2 with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. 1 numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g12/13, or gq/11 g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga11 selectively in nociceptors to investigate the contribution of the entire gq/11signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/11 deficient mice, suggesting a novel function for gq/11 in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/11, whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/11 branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany ap duration and ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for 1 second (5hz; 10hz; 5hz + s2-stimulus after 50-80ms) followed by a 4s rest period. the resting-membrane-potential (rmp) was observed over 30 cycles. we observed rmp-fluctuations of different length (amplitude <5 mv; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mv; 92%, 5.1) and spontaneous aps (62%, 1.9). intracellular ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/am. after preconditioning (10 min/1 hz) cells were measured under basal and continuous isoprenaline (10 -6 m) stimulation (iso). a 4 min pacing period was followed by a 1 min interval of no pacing. pacing frequency was reduced after each cycle (1 hz, 0.5 hz, 0.25 arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca 2+ overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord1, bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi500 and cgi6000 with apparent cgmp affinities of 500 nm and 6000 nm, respectively. one mouse line expresses cgi6000 driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm22alpha promoter directs cgi500 expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and aif). exposure of hepatocytes to α-ama at concentrations of 0,2 µm, 0,5 µm and 1 µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µm and 1mm, silibinin at 50 µm and 100 µm and a combination of both (30 µm benzylpenicillin and 50 µm silibinin, 1mm benzylpenicillin and 100 µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept 50 µm silibinin at 0,2 µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations (0,5 µm and 1 µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy 926) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh 4), we analyzed the amount of bh4 in ea.hy 926 by hplc. a concentration-dependent reduction of bh4 and also bh2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh4 can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh4 involving dihydrofolate reductase, dhfr). treatment of ea.hy 926 cells with dex decreased mrna and protein expression of both gch1 and dhfr. because bh4 is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh4 which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh10tert foreskin fibroblasts (behaving like primary cells) and sv40-immortalized gm637 fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap-1 binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after uvc exposure) resulted in c-fos protein expression, 18-48h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p38k and erks) were immediately induced upon uvc exposure and stayed active for at least 24h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed 24-40h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch 665/2-1). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p450 (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp2c19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg qd), albeit carriage of cyp2c19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp2c19 genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type 2 (octn2/slc22a5) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc22a5 gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn2 expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn2 expression in monocytes and thp-1 cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn2 expression was characterized on monocytes and thp-1 cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn2 specific signals as well as a localization in the plasma membrane. following thp-1 cells were activated using lps (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased tnfα (24fold induction) and il-1β (37fold induction) mrna levels. in addition, octn2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. in parallel activated thp-1 cells were coincubated with increasing concentrations of the octn2 substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by 64% in the presence of 50mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp-1 cells represent a useful model to study octn2 expression and function. in addition, we demonstrate immunosuppressive effects of octn2 substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg-01 cells to ricinoleic acid caused an increase in [ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca 2+ ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e2 receptors ep3 and ep4 as mediators of ricinoleic acid-induced effects. to test if ep3 and ep4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep3-deficient (ep3 -/-) or ep4-deficient mice (ep4 -/-). while ep4 -/mice responded similarly to the wt mice, ep3 -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep3 receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep3, consistent with a preferential expression of the ep3 receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep3 receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep3 receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected 5 drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an 8-times (cipralex ® : 5 → 40) and 5-times (onbrez ® : 2 → 10), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : 34 of 40; onbrez ® : 10 of 10). an additional amount of 22 conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc1). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in 24h as measured by an ha-binding protein linked assay (starved, 0g/l glucose: 100±4.7%; 1g/l: 185.7±44.8%; 4g/l: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). surprisingly, total ha concentrations were about 2.2-2.8 fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/l insulin for 24h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, 1g/l glucose: 100±6.5% vs 67.83±4.4%; 4g/l: 100±2.2% vs 71.8±6.6%). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc1 cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut1, glut4) were analyzed by qrt-pcr. the relative abundance was 24.32±3.49 in favor of glut1 indicating the presence of insulin-independent glucose transport. conclusion: in osc1 the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße 56, 72074 tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb1-mutated tumours. larch-derived diterpenes are potent and selective trpc6 blockers urban n. 1 , kübler w. the transient receptor potential channel trpc6 is a poorly ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc6 activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc6 and trpc3. indeed, turpentines and resins, but not coniphere oils blocked trpc6 and trpc3 in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc6-prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc6-selective block. larixol acetates displayed an ic50 towards the dag-or receptor-stimulated trpc6 activity of about 0.3-1 µm, but did not strongly inhibit a number of other trp channels, including trpv1, trpm2, trpm3, trpm8, or trpa1. selectivity for trpc6 compared to its closest relative, trpc3, was about 30-fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc6. electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc6 -/mice. we conclude that trpc6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt4 cells were cultured in mccoy's 5a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) 50 mg of protein sample was mixed with 5% of coomassie blue g-250 (cbb g-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a 12% sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina3n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. 1, 2 , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina3n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina3n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany introduction. activated β1-adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk1/2 at threonine 188 (erk thr188phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ 3 h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk2 showed a significant increase in [ 3 h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr188 -phosphorylation deficient mutants (erk2 t188a and erk2 t188s ) or pretreatment with the erk inhibitor, pd98059, significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type erk2 t188t or erk2 t188s . echocardiography and histological analyses revealed that erk t188s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk1) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk2 t188a or erk2 t188s are retained in the cytosol and prevented elk1-phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr188 -phosphorylation. conclusion. taken together, gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing erk thr188 -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. 1 , burke m. introduction: alpha-toxin, a 34 kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets (2) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp1, pp2 or su6654 (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr-416-phospho-src from calbiochem and cell signaling, respectively (3). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). pre-incubation with 3 src inhibitors (pp1, pp2 or su6654) reduced α-toxin-induced platelet aggregation by about 50%. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr-416 followed by a fast and complete dephosphorylation within 10 min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr-416 phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin (1µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr-416 typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m2 and m4 prefer coupling to gi proteins, whereas the odd-numbered receptors m1, m3 and m5 prefer coupling to gq proteins. with respect to ligand binding and m2 receptor activation, the conserved epitope trp 7.35 at the beginning of tm7 displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m2 receptor [1] . in the inactive m2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . in the active receptor, m2 trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] . to study the role of trp 7.35 for m3 receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm3 wild-type-cells and hm3 trp 7.35→ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m3 trp 7.35→ala relative to m3 wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m2 and the corresponding mutant. in the case of pilocarpine, replacement of trp 7.35 by alanine in m3 did not reduce intrinsic efficacy. this finding is in contrast to m2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m3 trp 7.35→ala mutant relative to m3 wild-type. this finding is also in contrast to m2, at which pilocarpine's potency was not sensitive to the trp 7.35→ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp 7.35→ala mutation between the m3 and the m2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human 3dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these 3d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such 3dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of 20 substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in 3t3 and a549 cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the 3d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca 2+ handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. 1 background: in atrial myocytes ca 2+ entry through l-type ca 2+ channels (ica,l) triggers a larger ca 2+ release (ca 2+ transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, 1µm) and the nonselective phosphodiasterase (pde) inhibitor 3-isobutyl-1-methylxanthine (ibmx, 10µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l (3.3±0.3pa/pf, n=12/6 [myocytes/patients] vs 6.2±0.8 pa/pf, n=15/10, p<0.01) and cat (182.2±26.8nm vs 307.5±30.9nm, p<0.01) were lower than in ctl myocytes, whereas diastolic [ca 2+ ]i levels were unchanged (caf, 312.3±56.7nm; ctl, 305.3±43.6nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to 10.4±2.0pa/pf (n=7/4) in caf and to 14.3±1.7pa/pf (n=9/7) in ctl. the corresponding cats increased to 493.0±132.3nm in caf and to 545.0±79.0nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca 2+ ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, +202.3±47.3% vs ctl, +98.4±16.1%, p<0.05) and cat (caf, +215.4±57.8% vs ctl, +101.9±20.3%, p=0.06) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, 9.9±1.5pmol/mg, n=6 vs ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. 1 g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o 6 -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o 2 -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, g-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g2. molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac2 and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s13 and n62) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz3/bcl6 or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac2-mediated phospholipase c-β 2 (plcβ2) and plcγ2 activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ2, plcβ2, and plcδ1, but that it had little or no effect on the activity of plcγ1 and plcε. the amino acid residues s13 and n62, likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac2 caused inhibitory effects on rac2-mediated plcβ2 and plcγ2 stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ2 distinct from those which are necessary for rac2 interaction, as the split pleckstrin homology domain of plcγ2, which is essential for its interaction with activated rac2, is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ2 and plcγ2, both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ2 activation. effect of rac1 inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse 1, 40225 düsseldorf, germany background: the small gtpase rac1 is a well characterized member of the rashomologous (rho) family. rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap1). furthermore, rac1 can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac1 is still unclear. here, we address the question how rac1 influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac1, human hepatoma cells were pretreated with the rac1-inhibitor eht 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac1 inhibitor showed a higher viability, less phosphorylation of h2ax (s139) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac1 resulted in a reduced phosphorylation of topo iiα (s1106) and an increased interaction of topo iiα with hsp90 in doxorubicin treated cells. the data indicate that inhibition of rac1 protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg2 cdna-clone sequences were submitted to the ncbi genebank (eu570105, gq141082 and gq241418). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst 33342 was measured and the selectivity was determined by the bcrp inhibitor ko143. inhibition studies using hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. 52, 67663 kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . as an α,b-unsaturated aldehyde, ac forms 1,4-michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-(3-hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-(2-carbamoylethyl)-cysteine (aama), and (n-acetyl-s-(2-hydroxy-2-carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . nevertheless, in a pilot study in humans urinary 3-hpma excretion of 14 non-smokers was reported to be about three fold higher, as compared to aama [3] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg aa (absolute amount), together with an as yet unknown amount of acrolein [4] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to 24 h after test meal uptake. the results demonstrated kinetics of 3-hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of 3-hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [1] stevens, j.f. and maier, c.s. (2008) molecular nutrition & food research 52; 7 [2] osorio, v. m. and de lourdes cardeal, z., (2011) journal of chromatography a 1218; 3332 [3] schettgen, t., musiol, a., and kraus, t., (2008) rapid communications in mass spectrometry 22; 2629 [4] ewert, a., granvogl, m., and schieberle, p., (2011) lebensmittelchemikertag 2011 450 protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str.31, 78457 konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase 1 (parp-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine 3':5' monophosphate (camp) and guanosine 3':5' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [2] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr1. we chose this gene because it is regulated by numerous stimuli including camp [3] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr1 gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr1. esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am (3, 10, 33, 100 µm) over 15 to 240 min 24 h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr1 expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb 153, sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr1 expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr1 expression in hela cells. methyl-cpg-binding protein 2 (mecp2) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp2 is diminished in murine and human heart failure. prevention of mecp2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp2 gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp2. in order to investigate the cardiac function of mecp2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp2 in cardiac myocytes under control of the tetracycline-system (mecp2-tg) and mice with targeted ablation of mecp2 in myocytes (mecp2 mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp2 did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp2 expression could be completely prevented by the mecp2 transgene. cardiac contractility and relaxation were significantly decreased in mecp2-tg animals. upon electron microscopical investigation, transgenic mecp2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp2 caused a rightward shift in the size distribution of mitochondria as compared with mecp2-tg hearts. epigenetic processes, including the recognition of dna methylation by mecp2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren2 rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass 9, 97078 würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren2 rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of 13 and 32 weeks. untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h2ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren2 rats compared to control rats. as another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d541a pore mutation leads to complete inactivation of trpv6 channels in epididymis weißgerber p. 1 , kriebs u. replacement of aspartate residue 541 by alanine (d541a) in the pore of trpv6 channels in mice disrupts ca 2+ absorption by the epididymal epithelium resulting in abnormally high ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv6 d541a proteins (sci signal 4, ra27, 2011) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv6 d541a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv6 -/and trpv6 d541a/d541a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv6 -/and trpv6 d541a/d541a males. the profound reduction in ca 2+ uptake by the epididymal epithelium was identical in trpv6 -/and trpv6 d541a/d541a males. this direct comparison of these parameters indicate that deleting trpv6 does not further aggravate the phenotype observed in trpv6 d541a/d541a mice, and -in our opinion -allows the conclusion that the d541a pore mutant of the trpv6 protein leads to complete inactivation of the trpv6 channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n996i) on herg channel function in hek293 cells sellmaier v. 1 , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome 2 (lqt2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh2 or kv11.1). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing 2 of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg1 gene (n996i) in a patient with lqt. both herg1a and herg1b subunits were cloned from a human heart cdna library and the specific n996i mutation introduced by site directed mutagenesis. hek 293 cells were transiently transfected with equal amounts of mutated herg1a and wild type (wt) herg1b cdnas and the resulting potassium current compared to herg1a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mv for wt and -29 mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in wt channels, determined from the tail current at -40 mv after a 5-second pulse to + 50 mv. the half-maximal steady-state inactivation of the tail current was shifted by 20 mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc2 is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc2 polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc2 haplotypes by the influence of micrornas. methods: abcc2 cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 3'utr sequence of abcc2 whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc2 were identified form an mirna array after rifampicin stimulation of hepg2 cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'utr vs. mod. 3'utr) concerning the -24c/1249g/3972c (cgc) abcc2 wt in hepg2 cells. using the fl vector construct, the expression level of cac haplotype protein was increased (136,15% ± 20%). transfection assays with the mir-397, which was identified as a candidate mirna targeting abcc2 mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir-379 was able to down regulate the abcc2 protein expression. there was no significance in downregulation for one abcc2 haplotype, respectively (modified 3'utr: cgc 22-34%; cac 20-40%, fl 3'utr: cgc 20-25%; cac 17-44% down regulation compared to mir-negative control). discussion: differences of abcc2 protein expression level are due to the epigenetic regulation of abcc2 haplotypes. to further characterize the effect of mir-379 on tcg, tgt and cgt abcc2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg 14, 07743 jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk886 effectively reduced ltb 4 pleural levels in female but not in male rats treated with carrageenan, and mk886 increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk886. in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, 21:102, 2009 ): male wistar rats were exposed to the test items for 6 h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of 60-120 nm. in the last 2 years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd 427) with 65 zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v79 cells, both showing negative results whereas the mouse lymphoma mutation test / l5178y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of 1.5 mg/m3 in the 90-day study. in a prenatal developmental toxicity study according to oecd tg 414, with repeated inhalation exposure to female wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. (1996) . the initial report used a database of 613 organic substances compiled from publicly available sources. in total, 2941 noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per 3 november 2011, containing data for more than 3900 registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to 6 were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the 5 th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than 1000 substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. 1 , schumann b. b-and l-moc can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h322 to tpm (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. in a549 viability was 67% after 72h with tpm (30 mg/l). the same dose of tpm lead to a decrease in viability to 82% (24h) or 25% (72h) in nhbec and to 74% (24h) or 28% (72h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm (5 mg/l). in h322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. while in a549 it was 1.3 (24h) and 1.4 fold (72h). in nhec and plc tpm (5 mg/l) did not have a significant effect on gsh-levels after 72h. in n-moc tpm (5 mg/l) also did not modulate gsh after 24h, but it diminished gsh-level by 0.5 fold upon prolonged contact (72h). in b-moc gsh concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. 1 , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by 20 % (wt 0,25 ± 0,01 mg/g vs. tg 0,30 ± 0,02 mg/g, p<0.05, n=12). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, 100 µl 1 mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. after 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and qtc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in tg (n=7) compared to wt (n=5) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße 9, 97078 würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca 2+ -atpase serca2a, thereby decelerating cytosolic ca 2+ clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca 2+ transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l37a, i40a and c41f) or favor pentamer formation (i45a, v49a). transfected hek293 cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with 0.25µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with 2.5µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek293 cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. 1, 2 , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a 5-fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. 1 camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r2) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s49 wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s49 kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n 6 -mb-camp and into 2'-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) 1 . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s49 wt und s49 kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s49 wt nor in s49 kincells. interestingly, we observed apoptosis in s49 wt and s49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. (1) wolter s, golombek m, seifert r (2011) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s49 cells : induction of apoptosis in s49 wt and in s49 cells lacking the catalytic subunit of pka (s49 kin-) with a) db-cnmps and b) cnmp-ams for 72h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. 67, 55131 mainz, germany nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. 48 spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam-1, vcam-1) and pro-inflammatory cytokines (e.g. il-6). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde4 inhibitors using two novel pde4 reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg 18a, 42096 wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde4 reporter cell lines. plasmid constructs encoding human pde4b1 or pde4d3 were stably co-transfected with the beta1-adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde4 and non-pde4 inhibitors, we could show that our novel pde4b1 and pde4d3 reporter cell lines specifically monitor pde4 inhibition with high sensitivity. pde4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant pde4 expression, pde4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde4d3 from cell lysates. two different groups of pde4 inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d159153, showed high cellular activity and much better inhibition of full-length versus truncated pde4d3. the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde4d3. the results imply that these novel pde4 reporter cell lines are well-suited for the characterization of the cellular activity of pde4 inhibitors and may also support a better understanding of the complex pde4 pharmacology. plexin-b2 is required for kidney regeneration after acute renal failure xia j. 1 , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b2 during kidney regeneration we generated mice lacking plexin-b2 specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b2 conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within 3 weeks after injury, plexin-b2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b2 might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small gtpase rac1. we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox2 and nox4 in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac1 membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (sirt1). we then wanted to find out whether the effect of resveratrol on rac1 was mediated by sirt1. sirt1 is a histone/protein deacetylase. in vitro incubation of rac1 with sirt1 led to a reduction of lysine acetylation. deacetylation of rac1 on lysine 166 could be identified by mass spectrometry analyses. the lysine 166 lies within the p67phox-binding region of rac1. consistently, in vitro incubation of rac1 with sirt1 markedly reduced its binding activity to p67phox. in conclusion, we provide evidence that rac1 is a direct target molecule of sirt1. sirt1 deacetylates rac1 on lysine 166 and thereby inhibits its interaction with p67phox. this is a novel mechanism of nadph oxidase inhibition by sirt1/resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany dexrazoxane (drz, icrf-187) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top2). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top2b, the dominant cardiac top2 isoform. this was investigated using top2b mutants resistant to drz, which were expressed in cells depleted of wild-type top2 isoforms. top2b-mediated double-strand dna breaks were assessed as γ-h2ax. the levesl of dsb generated by the top2b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top2b. preincubation with drz depleted wild-type top2b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top2b depletion nor the reduction of dsb by drz was seen in drz-resistant top2b mutants. furthermore, the cardially ineffective drz analog icrf-161, capable of iron chelation but not of top2 binding, affected neither the stability of top2b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top2b rather than by iron chelation. they also suggest a cardioprotective function of top2b, which is currently under investigation using cardiomyocyte-specific top2b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw3 were cultivated for 3, 6, and 9 days in dmem supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). we determined the expression of two junctional proteins (connexin 43, ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested (30 mg/l) connexin 43 and n-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). no change was recognized for vimentin (102±16%). effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; n-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; n-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). after incubation for 6 and 9 days the effects occurred in the same range. the substances showed no influence on the viability of serw3 sertoli-cells up to 300 mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin 43 and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ 50% inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s700e) in bk-strex, resulted in a ~50% reduction of basal channel activity, whereas the s700a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c12:13a) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-bp). both, mutation and pretreatment with 2-bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ 50% inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s700a channel mutant pretreated with 2-bp. in a clonal rat somatomammotroph pituitary cell line (gh3b6), in which pcr products without (zero) and with the 174 bp strex exon could be identified, the pkc activator pma blocked channel activity by ~25 %. this inhibition was increased to over 50% when gh3b6 cells were pretreated with 2-bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser700, probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met-5a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. 1 , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. 03x0109a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv40transformed, non-malignant pleural mesothelial cell line met-5a was chosen as the main in vitro model in this project. in the present study part, met-5a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in met-5a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg1modified comet assay. thus, met-5a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met-5a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met-5a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm7 channels zierler s. 1 transient receptor potential melastatin 7 (trpm7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm7. waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg 2+ ) concentration. mutating a mg 2+ -sensitive site on the trpm7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg 2+ . waixenicin a failed to inhibit the closely homologous trpm6 channel and did not significantly affect trpm2, trpm4, and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm7 ion channels. consistent with trpm7 inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg 2+ -dependent block of trpm7, waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg 2+ . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße 4-6, 30449 hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total 16 water pipe strands of 11 domestic installation systems were examined. they comprised 379 single water samples and 5306 single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc4 protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. 1, 66421 homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical 4 (mtrpc4) were identified. the trpc4 protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc4 gene exist, trpc4a (974 aa) and trpc4b (890 aa), trpc4b lacks aa 781 to 864 of the trpc4a variant. both trpc4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc4-interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc4a (aa 1 to 324; aa 622 to 974). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc4 in hek 293 cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc4. in addition, changes of cytosolic calcium were monitored and trpc4 currents were recorded in hek 293 cells expressing the candidate cdnas and stably expressing the trpc4a or trpc4b and the muscarinic receptor type 2 cdnas. the tarbp2 protein, one of the candidates shown to interact with trpc4, changed calcium influx when coexpressed with trpc4. in order to identify the domains of trpc4 responsible for its interaction with the tarbp2 protein, six trpc4-gst-fusion proteins covering the carboxyl terminal 353 aa of trpc4a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc4 could be identified to interact with tarbp2. one of these domains is well conserved within the trpc5 protein, corresponding to the result, that trpc5 and tarbp2 effectively co-immunoprecipitate, too. the tarbp2 protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer1, eif2c2/ago2 and tarbp2 (chendrimada et al. 2005) . by its interaction it may link trpc4 to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c57bl/6-mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600ng/kg · min during 28 days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type 1 (at1) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at1 receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at1 receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at1 receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine 3´, 5´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek 293 cells that stably express hcn1, hcn2, hcn3 or hcn4, respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn2 and hcn4 channels, ccmp shifted the membrane potential for half maximal activation (v0.5) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec50 for ccmp was 30 µm which is about 5 times higher than the ec50 of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only 65 % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of 1 mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v0.5 of ih by about +5 mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about 20 % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a 3d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. 1 the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [1] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line scc25, in normal human keratinocytes and fibroblasts [4] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the 3d construct of scc developed by höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc12 seeding to grow an aklike construct with scc12 cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki-67, marker for scc: cytokeratin-10, axl, marker for invasion: mmp2, marker for apoptosis: caspase-7, nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. efficacy of a 0.05% oxbu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [4] . -therefore, these 3d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3d models of skin diseases currently gaining increased interest as test platforms. ima910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd8+ and cd4+ t-cell responses associated with improved survival walter s. 1 , kuttruff s. ima910 is a novel peptide-based vaccine consisting of 10 hla-a*02 and 3 hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima910 was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima910 vaccinations in combination with low-dose gm-csf (cohort 1; n=66) or ima910 / gm-csf plus topically applied imiquimod (cohort 2; n=26). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd8 + t-cell responses and by ics assay for cd4 + tcell responses. as immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (mdsc1-6) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple cd8 + and cd4 + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd8 + cell responses as detected by ics (p=0.016). multiple cd8 + and multiple cd4 + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd8 + and multiple cd4 + responses. these patients had significantly higher dcr at 6 months (p=0.002), improved ttp (p=0.006) and improved pfs (p=0.009) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p=0.088, hazard ratio 0.53). in the study population, levels of 5 different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc4 and mdsc5 high frequencies were associated with shorter os (p=0.007 and p=0.019, respectively). to summarize, both hla-a*02 and hla-dr restricted peptides in ima910 were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima910. acrylamide (aa), a genotoxic carcinogen (iarc class 2a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp450 2e1 into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n7 of guanine (n7-ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-(2-carbamoylethyl)-cysteine (aama), and n-acetyl-s-(2-hydroxy-2carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n7-ga-gua dna adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of n7-ga-gua was reached. the untreated control group was found to excrete about 0.8 nmol (aama plus gama) in the urine (16 h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of 0.1 µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n7-ga-gua adduct levels in any organ tested (limit of detection, lod, 0.2 adducts/10 8 nucleotides). at the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. at 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg aa/kg bw (about 1-2 adducts/10 8 nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to n7-ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. 27, 01307 dresden health insurance costs in germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn eur in 2009. while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. the data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. 1, 2 , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than 60 million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations. 3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav 2.3 e/r-type voltage-gated calcium channel transcriptional upregulation of cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav3.2 t-type calcium channels histidine residues in the is3-is4 loop are critical for nickel-sensitive inhibition of the cav2.3 calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct2 (slc22a2) functional characterization of the human organic cation transporter 2 variant p.270ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp-2 tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs5 domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc-631570 (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago2 for microrna processing and gene silencing index a 009, 019, 035 011, 014 003, 044 objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr3c2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels >140 mmol/l were defined as 'high'. after application of 5 µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). both nr3c2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. the rs2070951 c allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 a allele only showed a trend towards stronger amiloride effects (p=0.06). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr3c2 variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. 55, 45122 essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb1 (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p450 (cyp) enzymes, including cyp2e1. cytotoxicity of acetaminophen (aap) is based on its cyp2e1-catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp2e1-overexpressing ctnnb1mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb1-mutated tumors. administration of a single dose of aap (300 mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb1-mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp2e1-overexpressing hepatoma. release of 5,6-epoxyeicosatrienoic acid (5,6-eet) upon neuronal activity induces trpa1-dependent mechanical pain hypersensitivity sisignano m. 1 , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp450) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of 5,6-eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception 5,6-eet-levels increased in the drg and it is released from activated sensory neurons in vitro. 5,6-eet potently induced a calcium flux [10 nm] in cultured drg-neurons which was completely abolished when trpa1 was deleted or inhibited. in spinal cord slices 5,6-eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, 5,6-eet-induced enhancement of sepsc frequency was abolished in trpa1 null mice, suggesting that 5,6-eet pre-synaptically facilitates spinal cord synaptic transmission via trpa1. finally, intrathecal injection of 5,6-eet caused mechanical hyperagesia in wild type but not trpa1 null mice. we conclude that 5,6-eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa1 at central afferent terminals in the spinal cord. sisnaiske j. 1 , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep2010/002811). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy5y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca2+, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox4 by histone deacetylases siuda d. 1, 2 , zechner u. 3 , prawitt d. 4 nox4 is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox4-mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox4 transcription by histone deacetylase (hdac). in cultured human ea.hy 926 endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox4 mrna expression. a similar down-regulation of nox4 mrna expression can be achieved with sirna-mediated knockdown of hdac3. hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox4 promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox4 promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox4 transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox4 mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox4 promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox4 promoter is significantly inhibited as well, which may explain the reduction in nox4 transcription. in conclusion, hdac inhibition decreases nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox4 promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v79 cells solecki g. m. 1 key: cord-006229-7yoilsho authors: nan title: abstracts of the 82(nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the 18(th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: 2016-02-06 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-016-1213-y sha: doc_id: 6229 cord_uid: 7yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise 80% of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only 6.5% of the volume, but 40% of the total cell number (kmiec 2001) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. 2010) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr3 breast cancer cells were exposed to the anti-cancer drug herceptin (20μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm-13 cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around 35% of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr3 cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm-13 cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu 7th health program grant agreement no 279288. deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases 1 and 2 (erk1/2) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk1/2 activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine 188 as a prerequisite for nuclear erk1/2 signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk1/2 signaling: since activation of erk1/2 triggers erk dimerization, a prerequisite for erk t188autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t188 -phosphorylation. first, we investigated the impact of monomeric erk2 on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk2 ∆174-177 and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk2 ∆174-177 overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆174-177 in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk2 and prevents its dimerization. moreover, the peptide effectively inhibited erk t188 -phosphorylation and nuclear translocation of yfp-tagged wild-type erk2 after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype 9 (aav9)-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i2knockout on cardiac function and survival in a murine heart-failure model of cardiac β 1adrenoceptor overexpression. methods and results: β 1 -adrenoceptor overexpressing mice lacking gα i2 (β 1 -tg/gα i2 -/-) were compared to wild-type (c57bl/6) mice and littermates either overexpressing cardiac β 1 -adrenoceptors (β 1 -tg) or lacking gα i2 (gα i2 -/-). at 300 days of age mortality of mice only lacking gα i2 was higher compared to wild-type or β 1 -tg, but similar to β 1tg/gα i2 -/mice. beyond 300 days mortality of β 1 -tg/gα i2 -/mice was enhanced compared to all other genotypes (mean survival time: 363±21 days). echocardiography revealed similar cardiac function of wild-type, β 1 -tg and gα i2 -/mice, but significant impairment for β 1 -tg/gα i2 -/mice (e.g. ejection fraction 14±2% versus 40±4% in wild-type mice). significantly increased ventricle-to body-weight ratio (0.71±0.06% versus 0.48±0.02% in wild types), left-ventricular size (length 0.82±0.04 cm versus 0.66±0.03 cm in wild types) and anp and bnp expression (mrna: 2819% and 495% of wild type, respectively) clearly indicated hypertrophy. gα i3 was significantly upregulated in gα i2 -knockouts (protein compared to wild-type mice: 340±90% in gα i2 -/and 394±80% in β 1 -tg/gα i2 -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β 1adrenoceptors in β 1 -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than 2-fold higher (b max : 1425 ± 68 fmol/mg) than on the otherwise used c57bl/6 background. accordingly, fvb/n-based β 1 -tg mice showed a significantly impaired cardiac function at an age of 300d while c57bl/6-based β 1 -tg mice did not. conclusions: gα i2 -deficiency combined with cardiac β 1 -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c57bl/6 background β 1adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day 300 while there was overt cardiomyopathy in mice additionally lacking gα i2 . we propose an enhanced effect of increased β 1 -adrenergic drive by lack of protection via gα i2 . the observed gα i3 upregulation was not sufficient to compensate for gα i2 deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i3 is currently addressed in a subsequent study using β 1 -tg and gα i3deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; 10 mg/kg per d) for 6 and 24 hours and 7 days. overall 7 days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; 7d iso 156±1) vs. wt cardiomyocytes (7d iso 143±2). at this time point a 29 % decrease of cardiac output and a 16 % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after 6 h (75-fold), and declined after 24 h (29-fold) to 2.5 fold increase after 7 days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: 55 regulated genes (up-regulated: 45 %) in untreated; 103 altered genes (up 35 %) after 6h; 1437 changed genes (up 97%) after 24 h and 131 altered genes (up 19%) after 7 days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after 7 days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at 24 h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m3 receptors, while m2 receptors appear to exert a heart-specific function only at later stages. continuous m3 blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m3 inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m3 receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m3 receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m3 inhibition. taken together, we postulate that muscarinic m3 receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m3 receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir-21 has proved effective against fibrosis of the myocardium and in other organs. mir-21 has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir-21 in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir-21 fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir-21 in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav9-icre vector allowed for specific and almost complete deletion of mir-21 in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir-21 in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir-21 gene in mice. aav9-mediated deletion of mir-21, albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir-21 demonstrated that mir-21 exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir-21 therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c81 acts as a multikinase inhibitor. results of a thermal shift assay revealed that c81 shows by far the highest binding affinity to the bmp-2-inducible kinase (bmp2k/bike). bmp2k represents an as yet largely uncharacterized protein, which is not regulated by bmp-2 in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c81 and (ii) the role of bmp2k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c81 affected the viability of human umbilical vein endothelial cells (huvecs). both c81 and the knock-down of bm2k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec-1). also the proliferation of hmec-1 was reduced by c81 treatment (ic 50 : 7 µm). a tube formation assay on matrigel demonstrated that c81 significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c81 treatment nor by bmp2k gene silencing. in regard to inflammatory processes, c81 treatment or bmp2k silencing of huvecs decreased the adhesion of thp-1 cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c81 or bmp2k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c81 treatment, the knock-down of bmp2k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p65 translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c81 in vitro. the precise role of bmp2k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp2k silencing and c81 treatment will be further elucidated. moreover, since the inhibition of bmp2k seems not to be responsible for all actions of c81, we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). cxcr4 seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr4 agonists or antagonists could result in reduced tlr4 signaling. however, the role of cxcr4 and the influence of different cxcr4 ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr4 activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose (5 mg/body weight) in mice. the plasma stable cxcl12 analog ctce-0214d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. 24 hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr4, cxcl12 and tlr4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce-0214d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately 35%, 50% or 70%, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce-0214d diminished the lps induced expression of cxcr4, cxcl12, tlr4, nf-κb, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce-0214d in all organs. the cxcr4 is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce-0214d attenuated tlr4 signaling, induced heme oxygenase 1 activity and mitigated apoptosis. thus, the administration of cxcl12 analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c57bl/6j mouse strains with a loxp flanked exon 4 of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t2 ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef 2-1 (fxn -/-) and fx-mef 2-8 (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over 12 days, with medium and substance changes at day 4 and 8, which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day 12 of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day 3, 5, 7 and 10 and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into 96well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor 2 which leads to disruption of protein synthesis and finally causes cell death [1] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c2 toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [2, 3] . in detail, we confirmed the involvement of the host cell chaperone hsp90 and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [4, 5, 6] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp40 and fk506-binding protein (fkbp)51 as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp90, cypa, cyp40, fkbp51 and fkbp52. the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [1] murphy (2011) toxins 3, 294-308. [2] barth (2011) naunyn-schmied arch pharmacol 383, 237-245. [3] kaiser et al. (2012) cell. microbiol. 14, 1193-1205. [4] dmochewitz et al. (2011) cell. microbiol. 13 , 359-373. [5] ratts et al. (2003) j. cell biol. 160, 1139-1150. [6] schnell et al. (2015) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins (1). prompted by these result, we found that only human α-defensin-1 (hnp-1) but not human β-defensin-1 (hbd-1), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin-1 prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp-1 causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy1-asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a 42.6% knockdown (p<0.05 student's t-test, n=6) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n=9). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p450 (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s9 mix), as was the synthesized ba-epoxide (-s9 mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type 2 diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o 6 -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr5-positive colon stem cells, which coincided with atr-chk1-p53 signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h2ax, suggesting the usefulness of γ-h2ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [2] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type 2 diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines (1). in particular, the chemokines monocyte chemoattractant protein (mcp-1, ccl2), interleukin-8 (il-8/cxcl8) and stromal cell-derived factor 1 (sdf-1a/cxcl12) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl14 was shown to attenuate obesity in mice (2) . the cc chemokine ccl2 is known to stimulate ccr2 receptors, and the cxc chemokines cxcl8 and cxcl12 to activate cxcr1 and cxcr2 or cxcr4 and cxcr7, respectively. the receptor(s) activated by cxcl14 is currently unknown. a role of cxcl14 in modulating cxcr4 signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr1 and the related receptor fprl1, and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr2 receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr2 receptors were absent (3) . yet, the analysis revealed appreciable levels of mrna encoding ccl2, cxcl12, and cxcl14, and ccr1, cxcr2, cxcr7, fpr1, and fprl1, and cxcr4. of interest, cxcr4-and cxcr7-mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl2, cxcl12, cxcl14. while sgbspreadipocytes and adipocytes did not accumulate ccl2-mcherry upon stimulation, they showed a small accumulation of cxcl12-mcherry, and a strong accumulation of cxcl14-mcherry in the endosomal compartment. similar results were obtained in murine 3t3l-1 preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl14-binding putative receptor protein(s) in murine 3t3l-1 preadipocytes. (1) makki, k. et al., (2013) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic 3', and to some extent of the corresponding cytidylyl analogue ccmp 1,2 . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown 3 . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp 1 . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity 2 . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac1-camps 3 in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. 1 kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell 157, 163-186 (2014) . 2 koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. 14 (2): p. 190-9 (2010) . 3 nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem.279, 37215-37218 (2004) cardiovascular pharmacology 039 hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel (70%:30%:0.4 mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology (1% strain, 1 hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen 0.03; collagen + cms 0.03; collagen + cms + cfbs 0.09, collagen + cms + ffbs 0.2. we next generated ehm with cms and ffbs or cfbs. after 4 weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p<0.01) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm 2 : 1.8±0.1, n=29 vs. 0.3±0.1, n=20). cross sectional area (csa) of tissues was greatly increased (p<0.01) in cfb-ehms (csa in mm 2 : 1.6±0.1, n=20 vs. 0.6±0.03, n=30) and nonmyocyte content was higher in cfb-ehms (5.6±0.7, n=9 vs. 3.1±0.4, n=15; x10 5 cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase 2 (has2) enzyme was significantly (p<0.01) upregulated. based on these findings, we pharmacologically intervened with has2 mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all 4 weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p<0.01) reduced csa (csa in mm 2 : control 1.8±0.4, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.9±0.2, n=4) with a concurrent, statistically significant (p<0.01), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm 2 : control 0.2±0.05, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.4±0.08, n=4). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type 2 receptor (vegfr2), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac1 and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with 5 µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with 30 µm of the camp analog 8-pcpt-2'-o-camp (007) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac1 in huvec decreased the basal and vegf-induced sprouting. surprisingly, 10 µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than 007, indicating an additional role of pka. in accordance, 1 µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, 50 ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac1 and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh5cre ) were created using the cre/loxp system. mr cdh5cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received 1 % nacl with drinking water for 6 weeks. the mineralocorticoid deoxycorticosterone acetate (doca, 2.5 mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype (141.7 ± 8.0 vs. ctrl 97.8 ± 3.2 mmhg, p<0.001) and mr cdh5cre (150.0 ± 3.0 vs. ctrl 104.1 ± 4.7 mmhg, p<0.001) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh5cre mice (ventricle weight 162.4 ± 4.2 mg vs. mr wildtype 189.0 ± 10.9 mg, p<0.05). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh5cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam1) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: 2.4 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) and mr cdh5cre (2.3 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) mice. mrna expression of the fibrosis marker gene col3a1 (mr wildtype 4.3 ± 0.9-fold; mr cdh5cre 3.9 ± 1.1-fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype 20.4 ± 5.7fold; mr cdh5cre 34.3 ± 12.5-fold vs. ctrl). in the kidney vcam1 mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca 2+ concentration due to ca 2+ entry from the extracellular space is critical for platelet activation and aggregation. ca 2+ entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a 2 (txa 2 ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca 2+ entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca 2+ entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc3 and trpc6 in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca 2+ signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc3/c6 proteins for procoagulant activity. in addition, we detected the expression of s12 trpc1 transcripts in mouse platelets. therefore, we tested trpc1/c3/c6-deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc1/c3/c6 inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc1/c3/c6-deficient mice and we observed that the aggregation was reduced after adp (3µm) or txa 2 -analogue (1µm) stimulation, but not after collagen stimulation (10µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca 2+ response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/0 mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/0 mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/0 mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/0 mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/0 mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp2b10 (1200-fold in females, 700-fold in males) in 6-day old offspring of adult female mice exposed one week prior mating to tcpobop (3 mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop (3 mg/kg) prior to mating. the analysis of hepatic cyp2b10 expression in 6-day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a 700-fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e18.5 followed by crossfostering proved transmission to be mediated predominantly via lactation (f1 hepatic cyp2b10 induction 3000-fold) and only to a minor part via intra-uterine exposure (300fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f0 and f1 generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp2b10 catalytic activity. in both tcpobop-pretreated f0 and in their f1 descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by >50%. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by 45% in 12-weeks-old offspring. in summary, the car-mediated cyp2b10 induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb 761 ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with 96 alzheimer´s disease patients indicated that the combined treatment with donepezil and egb 761 ® had less side effects than donepezil alone (yancheva et al., 2009) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb 761 ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment (15 days of pretreatment) with donepezil (1,5 mg/kg p. o.), egb 761 ® (100 mg/kg p. o.), or the combination of the two drugs, or vehicle in 18 -20 month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally 30 minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb 761 ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb 761 ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. (2009) . ginkgo biloba extract egb 761(r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, 13 (2) , [183] [184] [185] [186] [187] [188] [189] [190] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations (3-28 µm; just et al. expert opin emerging drugs 20: 161-164, 2015) observed in patients treated with dantrolene. although not yet proven directly, oat3 may be involved in renal secretion of dantrolene and 5-oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp4. since oat3 was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. 483: 133-138, 2004) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr1, by influx through oat3, where it inhibits calcium efflux by ryr1 thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat3. in addition, we identified, besides dantrolene and 5-oh dantrolene, several further fdaapproved drugs such as tyrphostin ag 1478, ceefourin 1, glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat3. controls 1 with other defects and controls 2 with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥50mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with 95% confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls 1 and additionally adjusted for maternal age in controls 2. results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were 65/464 (14.0%) for cases, 512/6033 (8.5%) for controls 1 and 130/1564 (8.3%) for controls 2 and adjusted ors (95%cis) were 1.71 (1.29-2.26) and 1.77 (1.27-2.46), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-(2.41 vs. 1.61 for controls 1, and 2.55 vs. 1.67 for controls 2). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being 2 and 1.6 times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac1 protects non-transformed cells from topoisomerase ii-(top2)-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac1-mediated regulation of topoisomerase ii activity. however, the link between rac1 and top2-poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top2-poison-induced cytotoxicity. here, we investigated the relevance of rac1-regulated actin cytoskeleton integrity as well as mitochondrial integrity in top2-poison-induced dna damage responses as well as cytotoxicity under situation of rac1 inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h5v) as well as cardiomyocytes (h9c2) were chosen as in vitro model systems for top2-poisoning. the cells were pre-treated with rac1 inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh2ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top2-poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc1-assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac1 inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h2ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac1-inhibitor. top2-poison-induced raise in mitochondrial mass was slightly enhanced by the rac1-inhibitor and statins. interestingly, inhibition of rac1 counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top2-poisoninduced cytotoxicity in h9c2 and h5v cells. the data point to a role of rac1-regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top2-poisons. poly(adp-ribose) polymerase 1 (parp1) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [1] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp1 as well as by non-covalent poly(adp-ribose)-wrn interaction [2] [3] [4] . furthermore, pharmacological parp inhibition as well as a genetic parp1 ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [5] . here we report a novel role for parp1 and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h 2 o 2 -induced oxidative stress, parp1 is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp1 activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp1 interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp1 and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp1-dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp1 in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [1, 2] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [3] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [4] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [5 -7] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases 5methylcytosine and 5-hydroxymethylcytosine as well as their derivatives (5formylcytosine and 5-carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene 9, , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb2, xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the 17 members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp1 is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle 2011, mangerich and bürkle 2015) . here we generated a genetic knock out of parp1 in one of the most widely used human cell systems, i.e. hela parp1 ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp1 ko cells with a series of artificial and natural parp1 variants, we analyzed structure-function relationships of parp1 in a cellular environment without interfering with endogenously expressed wt-parp1. we confirmed that the parp1e988k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp1l713f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp1-par interaction via at least one specific par binding motif located in zinc finger 2 of parp1. expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp1l713f itself triggered apoptosis, parp1e988k expression led to a strong g2-arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt888 mitigated effects of the e988k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp1 ko cells with a natural cancer-associated parp1 snp variant (v762a), as well as a newly identified parp1 mutant present in a patient of pediatric colorectal carcinoma (f304l-v762a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp1-dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp1 variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle (2011) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer128 (2): 251-265. mangerich, a. and a. bürkle (2015) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: 125-179. leukemic cells frequently overexpress the transcription factor wilms tumor 1 (wt1) and the persistence of wt1 expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt1 determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt1 and cell death. in addition, we show that an rna interference-mediated elimination of wt1 sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt1 suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt1 also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt1 is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt1 is a novel biological target of hydroxyurea and they suggest that wt1 has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß 1 ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res 97:472, 2013) and stably transfected to hek 293 cells (hekß 1 -fret). to monitor the camp response the epac1-based fret sensor of camp, was stably transfected alone (hekwt-e1) and together with a moderate level of native ß 1 ar to hek293 cells (hekß 1 -e1; nikolaev et al. jacc 50: 423, 2007) . fret-activity was measured with recently developed fluorescence detectors (12 channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using 438 nm for excitation and 483/540 nm for the emmission ratio. cells were cultivated in 96-format 12-well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß 1 -fret cells exhibited ec 50 -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: 6.9±0.1, adrenaline 5.7±0.1, noradrenaline 6.2±0.1). ßar-expression levels were controlled by radioligand binding with [ 3 h]-(-)-cgp12,177 resulting in different densities of ~4x10 6 and ~1.4x10 4 receptors/cell in hekß 1 -fret and hekß 1 -e1, respectively, whereas in hekwt-e1 cells only ~1000 ß 2 ar were found. surprisingly, the low level of ßar in hekwt-e1 cells allowed the measurement of the action of ß 2 -sympathomimetics (ß 2 sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~6 .7) to an effective regulation of fret activity in the presence of 0.06 mm ibmx (pec 50~8 .3±0.1), nearly matching the ~100-fold amplification of iso (pk d~6 .9; pec 50~8 .9). in order to determine ß 1 ar-mediated side effects of ß 2 sym, hekß 1 -e1 cells characterized by a 14-fold higher level of ß 1 ar over ß 2 ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec 50~8 .4 whereas the high affinity ß 2 sym salmeterol acted a partial agonist (~75% of iso maximum, pec 50~8 .6), both compounds being rather insensitive against the highly effective ß 1 ar-blockade with 1 µm cgp 20,712 a. for that reason hekß 1 -fret cells were used characterizing fenoterol as partial agonist (~25%) whereas salmeterol activated less than 10% of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß 2 ar present in hekß 1 -e1 cells precludes the exact determination of ß 1 ar-mediated side effects of ß 2 sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β 1 -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β 1 -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek293 cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek293 cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β 1 -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β 2 -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β 2 -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β 2 ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. the affinity of arrestin-3 to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin-3 and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin-3 dissociation from the β 2 ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin-3 into the bleached region was prolonged between two-and fourfold for the β 2 ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β 2 ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek293t cells transfected with the appropriate cdnas. here we focused on muscarinic m1-, m2-, and m3-receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/11-proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m3-and m1-achrs was found to be 10-fold faster in comparison to gq. similarly, we observed a 15-fold right shift of the concentration-response curves of go proteins binding to m3-achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m3-achr by using a fret-based assay. our results showed that gq activation required 10-fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m2-achr for gi family members by comparison of dissociation kinetics of gi1-, gi2, gi3-, and go-proteins from m2-achr under nucleotide depleted conditions. k off of gi1 and gi3 were found to be two-fold higher in comparison to gi2 and go proteins, indicating the higher affinity of the latter ones to m2-achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising 15 receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone 1 receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone 1 receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine 375 that is predominantly catalyzed by g protein-coupled receptor kinase 5. as a consequence, the selective morphine-induced s375 phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s375 but also drive higher order phosphorylation on the flanking residues threonine 370, threonine 376, and threonine 379 in a hierarchical phosphorylation cascade that specifically requires grk2 and grk3 isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels 061 hcn4 in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn1 and hcn2 isoforms for brain function and epilepsy has been demonstrated, however the role of hcn4, the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn4 in controlling oscillations in the thalamocortical network. hcn4 is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn4-deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn4 null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn4 is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc4 and 5 channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc3/6/7 channels are directly activated by diacylglycerol (dag), trpc4 and 5 channels are commonly regarded as daginsensitive. in contrast to trpc3/6/7 channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) 1 and 2. interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc5 confers dag-sensitivity on trpc5 channels. trpc5 channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip 2 depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf1 and 2 proteins and overexpression of a nherf1 mutant incapable of trpc5 binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip 2 depletion itself caused slight trpc5 current increases while during pip 2 depletion, the membrane permeable dag analogue oag evoked even higher trpc5 currents suggesting that pip 2 depletion induces an active and dag-sensitive channel conformation. receptor mediated pip 2 depletion also resulted in dissociation of nherf1 and 2 from the c-terminus of trpc5 thereby eliciting a dag-sensitive trpc5 channel conformation. thus, our findings suggest that dag-sensitivity of trpc5 is the result of an activation cascade starting with pip 2 depletion and subsequent dynamic dissociation of nherf1 and 2 from the c-terminus of trpc5. altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm3 is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm3-deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm3 may exert antinociceptive properties. fluorometric ca 2+ assays and a compound library containing approved drugs were used to identify trpm3 inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura-2-loaded single cells was applied to monitor [ca 2+ ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm3 cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm3 inhibitors. the compounds exhibited half-maximal inhibitory concentrations of 0.6-6 µm. the selectivity profiles of maprotiline and primidone for trpm3 were promising with no inhibitory effects on trpm2, trpm8, trpa1, trpv1, trpc5, trpc6 and p2x7 receptor channels. primidone inhibited pregs-induced [ca 2+ ] i signals in rat drg neurones, indicating a block of native trpm3 channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm3 at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm3-related effects by acutely challenging the channel´s function. pharmacological interference with trpm3 applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm3 in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il-6) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e103/k308 can be crosslinked efficiently in both states, the closed-and open state of the p2x2r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e84/r290, e103/k308, e167/r290 and e167/k308 and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p2x2r. the comparison of the closed-state and open-state model of the rat p2x2r showed that atp promotes a marked rearrangement of the side chains of the residues r290 and k308 to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r290 and k308 with atp competitively releases e84, e103 and e167 from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. 1 in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem1, tmem2 and tmem4. expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem2 is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem2 proteins exhibit 6 to 10 plasma-membrane spanning domains, but fluorescently labeled tmem2 fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem2-eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem2 -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem2-deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura-2 or indo-1 indicate higher resting ca ++ concentrations in tmem2-/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem2-yfp-knock-add-on mice we identified tmem2 in organelles of the apical acinar cell pole and a partial colocalisation with lamp2 proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh 4 cl application. the role of tmem2 for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin 2pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck-8 stimulation indicating that tmem2 inactivation leads to an enhancement of the globalization of cck-8 evoked ca2+ release from intracellular organelles. taken together, our study identifies tmem2 as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [1] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the 3rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [1] gundert-remy, u. et al. (2015) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol 89: 1881-93. during the past decades, considerable progress has been made in implementing 3r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster 3r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. 3r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes (4 x 4 gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed 24 h after the last irradiation by γh2ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht1864, a small molecule inhibitor of the small rho-gtpase rac1, was applied in vivo, pointing to an involvement of rac1 in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas9 is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas9 allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh3-interacting domain death agonist) knockout cell line in neuronal ht-22 cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas9-knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht-22 cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin-1 and the bid inhibitor bi-6c9 on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas9-bid-knockout cells. bi-6c9 inhibited glutamate-induced morphological changes of ht-22 cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin-1 in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi-6c9 abolishes lipid peroxide formation in the erastin model and ferrostatin-1 in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi-6c9 and ferrostatin-1 in the respective model systems. investigating the crispr/cas9-bid-knockout ht-22 cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type 2 (aav2) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange 13 and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of 1 mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange 13 at the azo-and other weak bonds in our experimental set-up (fig.1a) . amongst other substances, the carcinogens aniline (max. conc. 1.01 ± 0.12 µg/ml) and 3,3-dichlorobenzidine (max. conc. 0.88 ± 0.18 µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. 1b) . here, 1,2-benzenedicarbonitrile (max. conc. 1.11 ± 0.12 µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. 35.8 ± 4.32 µg/ml) and the human carcinogen benzene (max. conc. 0.19 ± 0.06 µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥50 µg/ml evolving upon ruby laser irradiation of >1.5 mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference 1 schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep 5, 12915 (2015) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween 20 and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco-2 model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). 2015; 10 (21) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [1, 2] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > 120 °c [3] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [4] . we here present results of a nine-day human intervention study with 14 healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [5] . the study design included washout periods with an aa-minimized diet (21 -41 ng /kg bw), a low aa intake day (0.6 -0.8 µg /kg bw) as well as a high aa intake day (1.3 -1.8 µg /kg bw). after a three-day washout period an aama baseline level of 93 ± 31 nmol/d was determined. low aa intake led to an aama excretion within 24 h of 225 ± 37 nmol/d, high intake to 404 ± 78 nmol/d corresponding to an aama excretion rate of about 30% of the ingested aa dose within 24 h, whereas aama output within 72 h corresponded to 58% of the respective aa intake, the aama baseline after 3 days washout corresponds to a net exposure level of 0.2 -0.3 μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [1] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste 2013 , 2013 doi: 10.1002 iarc, iarc monographs on the evaluations of carcinogenic risks to humans 1994, 60. [3] tareke et al., j. agric. food chem. 2002, 50, 4998-5006. [4] efsa panel on contaminants in the food chain (contam), efsa journal 2015; 13(6):4104 [321 pp.] . [5] ruenz et al., arch. toxicol. 2015 doi: 10.1007 /s00204-015-1494 heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct116 colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf2/skn-1, daf16), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + 57%). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn-1 (homolog to mammalian nrf2), but not daf-16 (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct116 cells, baicalein activates nrf2; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf2. the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf-16 transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn-1 but not daf-16. experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf2/skn-1 activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [1] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in 12 healthy postmenopausal women (50-70 yrs).in a randomized doubleblind protocol each subject received four treatments, 2 doses of intranasal progesterone (4.5mg mpp22; 9.0 mg mpp22), 10mg of zolpidem and placebo. the 4 conditions consisted of 2 experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from 23:00 to 07:00. simultaneously blood was collected every 20 min between 22:00 and 07:00 by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after 4.5 and 9.0mg mpp22. univariate f-tests in the manovas pointed to the following results (significant p-values at α=0.05). sei was higher after zolpidem than after the other three treatments. after 9.0mg mpp22 sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after 9.0mg mpp22 and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after 9.0mg mpp22 and after zolpidem. after all active treatments with mpp22 and zolpidem the time spent in sleep stage 2 was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp22 resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after 9.0 mg mpp22. no other changes of hormone secretion were found. our study show sleep promoting effects of 9.0 mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after 9.0 mg mpp22. no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january 2013 and june 2015 from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of 2 or 3 and possible anticholinergics with a score of 1). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of 3. a patient's individual score of 3 or higher is considered to be clinically relevant. in total, 130,186 geriatric patients (median age 82 years, 66.3% female, median no. of drugs 9) were evaluated. of these, 41,456 (31.8%) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in 10,941 (26.4% of the patients taking anticholinergic drugs) patients. 11,241 (27.1% of the patients taking anticholinergic drugs) patients had a score of 3 or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine (58), amitriptyline/quetiapine (41) and amitriptyline/carbamazepine (35). 2,885 (7.0%) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct2 and mate1 expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. 1,2 although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to 3 the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. 4 however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α 2 -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct1 (encoded by slc22a1), oct2 (slc22a2), oct3 (slc22a3) and mate1 (slc47a1). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek293 stably expressing the organic cation transporters oct1, oct2, oct3 or mate1. 5 intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct2 and mate1 in a time-and concentration-dependent manner, but not by oct1 or oct3. timolol is only transported by mate1, but not by the octs. as shown by immunolocalization studies, the oct2 and mate1 transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct2 and mate1 may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: 1. zhang et al., nat rev drug discov. 2012 jun 15; 11(7) :541-59. 2. lavik et al., eye (lond). 2011 may; 25(5):578-86. 3. gaudana et al., pharm res. 2009 may; 26(5):1197 -216. 4. kraft et al., invest ophthalmol vis sci. 2010 51(5):2504 -11. 5. nies et al., plos one. 2011 6(7) :e22163. supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz 1 , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel 600mg, prasugrel 30mg or prasugrel 60mg (n=300). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day 1 after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s =0.18; p=0.002), hipf (r s =0.18; p=0.002), ipc r s =0.26; p<0.001). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p<0.001). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p450 2c19 polymorphisms (*2 and *17), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s21 was the strongest predictor of on-treatment platelet reactivity (partial η 2 =0.045; p<0.001) followed by intrinsic platelet reactivity (partial η 2 =0.034; p < 0.002). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp 2c19*2 polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [1] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [2] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [3] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki-67 index 23-fold in the cscc model (p£0.001). while the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [4] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for 2.6-fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [2] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb3r with integrated graduate education was launched in 2014. the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the 3rs and to find alternatives to animal experimentation within the next years. a panel of 3r experts has been set up to provide advice and assistance and to raise awareness in society for 3r-related issues. research in bb3r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb3r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on 3r including modules on ethics and legislation. currently, 14 phd students are qualified for management positions in professional areas related to the 3rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb3r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd 442c and 442d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz-3-derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after 14 days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer 2,4-dinitrochlorobenzene (dncb) induced il-6 and il-8 secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd83 and pd-l1 in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine (3rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the 21st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than 10,000 compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing 647 compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. 20 %) fail in the late drug development due to toxicity and adverse effects [1] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about 51,000 compounds and 115 cancer cell lines from the national cancer institute's (nci) nci-60 growth inhibition data [2] with focus on five immune cell lines (rpmi-8226, ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [1] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [2] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [3] , and encourage the reduction of in vivo testing according to the principles of the 3rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype 1e1 activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure 1 ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult1e1 ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for 3d pharmacophore development. eight specific 3d pharmacophores were created that allow identification of sult1e1 substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over 6,500 experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult1e1 inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult1e1 for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. (2011) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. (2011) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p=0.013). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p=0.010). 50% of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in 2013. by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like 3d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the 2-organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd86 and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex 3d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the 2-organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused 2-organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd86-values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the 2-organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past 10 years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [1, 2] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) 14 clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck14, which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il-1 alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [1] vávrová, k., henkes, d., strüver, k., sochorová, m., školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3d skin construct. the journal of investigative dermatology. 134, 746-753 (2014 bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive 2010/63 has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the 3-rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the 3-r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c57/bl6 j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for 45 minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored 30 and 150 minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for 24 hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the 82 nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb3r is funded by bmbf. in 1959 the 3r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the 3r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the 3r-concept is the overall goal of scientific animal welfare. article 4 of the 'directive 2010/63/eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [1] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately 70 % [2] [3] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [4] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from 0.05 to 2.5 mg/kg per bodyweight [5] [6] [7] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [8] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [1] rl 2010/63/eu. [2] bmel (2014) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [3] eu commission (2013) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [4] carbone l (2011) pain in laboratory animals: the ethical and regulatory imperatives. plos one 6, e21578. [5] gv-solas, a.f. a.d. (2013) . empfehlung zur schmerztherapie bei versuchstieren. [6] carpenter, j.w., t.y. mashima, and d.j. rupiper (2001) . exotic animal formulary, 2nd edition, w.b. saunders co., phila. [7] flecknell, p. (1996) . laboratory animal anaesthesia, 2nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a 64-slice scanner were collected from 18 female minipigs, consisting of 6 animals aged 12 months (group 1, n=6) and 12 animals (group 2; n=12) examined at the age of 17 and 21 months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than 50 parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to 65% where the largest volume was 13,4 ml and the lowest 4,7 ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between 12 and 17 months of age. comparing 17 and 21 months old minipigs, no significant difference in distance could be observed. from the age of 17 months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between 12 and 17 months of age indicates ongoing anatomical changes of this parameter until the age of 17 months. therefore animals should be older than 17 months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [1]. worldwide it is estimated that over 85 million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [2] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [3] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals (3r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin (5-ht) system, in particular the 5-ht 1a receptor, playing a major role. activating 5-ht 1a receptors by the 5-ht 1a receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic 5-ht 1a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the 5-ht 1a receptor in serotonergic projection areas, bert et al. (2008, pmid: 18396339) revealed exaggerated 8-oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic 5-ht 1a receptors to thermoregulation, more precisely to the hypothermic effect of 8-oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of 8-oh-dpat (0.1 mg/kg -4 mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the 5-ht synthesis inhibitor parachlorophenylalanine (pcpa; 100 mg/kg, i. p. on four consecutive days) would alter the effects of 8-oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the 5-ht 1a receptor. results: 5-ht 1a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: 36.0 °c; nmri wild-type mice: 37.4 °c). in both genotypes, systemic administration of 8-oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (-2.8 °c compared to -1.5 °c in nmri wild types). dose response curves of 8-oh-dpat revealed an ed 50 = 0.4 mg/kg in transgenic and an ed 50 = 0.57 mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to 8-oh-dpat in mice. the dose-response curves indicate a higher potency of 8-oh-dpat in transgenic mice. the exaggerated hypothermic response to 8-oh-dpat in mutant mice implies that postsynaptic 5-ht 1a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that 8-oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing 5-ht 1a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg389gly in the human beta1-adrenoceptor (adrb1). the adrb1 carrying arginine at position 389 in helix 8 in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta1-adrenoceptor variants arg389-adrb1 and gly389-adrb1 is poorly understood. despite its hyperfunctionality, we found the arg389-variant of the adrb1 to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly389-variant. using adrb1 sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg389-adrb1 exerted faster activation speed and arrestin recruitment than the gly389-variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb1 mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb1 finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb1 variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved 10-residue sequence, 370 trehpstant 379 , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine 375 (s375) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase 5 (grk5). by contrast, high-efficacy opioids not only induce phosphorylation of s375 but also drive higher-order phosphorylation on the flanking residues threonine 370 (t370), threonine 376 (t376), and threonine 379 (t379) in a hierarchical phosphorylation cascade that specifically requires grk2/3 isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk2 and grk3 to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk2/3. in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk2/3 isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk2 or grk3. conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk2 or grk3. mutation of s375 to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all 11 carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/11, and g12/13, which interact with distinct cellular effectors to shape the final cellular response 1 . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs 2 . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation 3 . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/11/14 inhibitor fr900359 4 on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m1 acetylcholine receptor (cho-m1). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor 1,5 . we show that fr900359 can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m1 membranes. in contrast to this, bim-46187, a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation 3 . interestingly, inhibition of high affinity agonist binding by fr900359 was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr900359, combined uncoupling of both gi and gs proteins from m1 was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m1 receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [1] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [2] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m1 machr which preferentially activates g proteins of the g q/11 type but can also promiscuously stimulate g s proteins. g q/11 -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m1 machr in ip1 and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper-6-naph completely lost intrinsic activity for both g q/11 and g s activation at the m1 machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m1 machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/11 -mediated signaling, activation of the g s protein in m1 machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m1 machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades 3-(1h-imidazol-4-yl)propylguanidine (sk&f-91,486 (1) [1] ) is known as the prototypic pharmacophore of highly potent histamine h 2 -receptor (h 2 r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [2] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f-91,486, we characterized 78 newly synthesized derivatives including several bivalent compounds (e.g., 2) by using different pharmacological in-vitro methods. [3] the potential h 2 r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf9 cells [4] (hh 1,2,3,4 r). compounds were also functionally characterized in the [ 35 s]gtpgs assay (hh 2 r, sf9 cell membranes). [5] selectivity vs. hh 1,3,4 r was determined for selected derivatives also using this technique. organ bath studies (gph 1 r (ileum), gph 2 r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f-91,486 analogues displayed partial or full agonism via hh 2 r and gph 2 r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine 2, was a partial agonist (e max = 88%) and 250-times as potent as histamine vis-à-vis the gph 2 r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa 2 values for the antagonist (5.87 -6.38) which are in accordance with literature data. however, in the functional in-vitro assay on gph 2 r, the positive chronotropic response evoked by sk&f-91,486 was surprisingly resistant to antagonism by cimetidine and other typical h 2 r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h 2 r agonist. this behaviour is unique within the large series of sk&f-91,486 analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [1] parsons, m.e. et al.; agents actions 1975, 5, 464. [2] buschauer, a.; j. med. chem. 1989 , 32, 1963 -1970 [3] pockes, s.; dissertation, univ. regensburg 2015. [4] seifert, r. ; j. pharmacol. exp. ther. 2003, 305 (3) , 1104-1115. the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine 351 (s351) and threonine 362 /serine 363 (t362/s363), which enabled us to selectively detect either the s351-or the t362/s363-phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch221510 and ro64-6198 induce a stably phosphorylation at s351 and t362/s363 followed by a profound internalization of the receptor. the nociceptin-induced s351 and t362/s363 phosphorylation can be blocked by selective nop receptor antagonists such as j113397 or sb612,111. nnc63-0532, buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s351 phosphorylation occurred at a faster rate than phosphorylation at t362/s363 indicating that s351 is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol 12-myristate 13-acetate facilitated receptor phosphorylation only at s351 but not at t362/s363, indicating that s351 can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t362/s363 in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch221510 failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling (1). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation (2) . in man, two cc chemokine receptors ccr2a and ccr2b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin (2) . interested in differential signaling of ccr2a and ccr2b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk1/2) in the presence of ccl2, ccl8, ccl7, and ccl13. all these chemokines have been shown to interact with human ccr2 receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr2 receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr2b receptors by ccl2 induced both rho-dependent srf activation and receptor internalization, while ccl8-stimulation resulted in srf but little if any receptor internalization. in contrast, ccr2a-expressing cells showed ccl2dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr2 receptors for coupling to g proteins revealed that arginine 313 within the putative 'eighth helix' of the carboxyl-terminal portions of ccr2a and ccr2b is not involved in ga i -mediated induction of erk1/2 and plays a minor role in ccr2b receptor internalization, but is specifically required for the ccr2a/ and ccr2b/ga q -mediated activation of srf. serotonin 5-ht 2c receptors (5-ht 2c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). 5-ht 2c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express 5-ht 2c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa2-10 cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where 5-ht 2c r in vivo expression has been detected. thus, we tested mhypoa2-10 cells for putative 5-ht 2c r expression by performing single cell calcium imaging. we observed that serotonin and the specific 5-ht 2c r agonist, way161,503 induced robust calcium transients, which were strongly inhibited by two unrelated 5-ht 2c r-selective antagonist (sb206,553, rs102,221). serotonin and way161,503 also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases-1/2 in a sb206,553 and rs102,221 sensitive manner, providing further evidence for functional 5-ht 2c r expression in mhypoa-2/10 cells. intrinsic activity of way161,503 ranged between 0.3 and 0.5 compared to serotonin in all assays, defining way161,503 as a partial 5-ht 2c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa-2/10 cells endogenously express 5-ht 2c r and thus represent the first cell line to analyse 5-ht 2c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub 1 1 walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with 3 h-adenine and calculation of the conversion rate to 3 h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α 2a or µ 1 opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek293 cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc5 or heteromeric kir3.1/kir3.2 or kir3.1/kir3.4 channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp2a mice leading to double transgenic mice (h 2 xpp2a = dt). pp2a mice (j biol chem 2004; 279:40827) overexpress the catalytic subunit of protein phosphatase 2a (pp2a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about 240 days we noted reduced ef in pp2a (33.1 ± 3.5 %, n=16) compared to wt (54.4 ± 4.5 %, n=10) and tg (59.8 ± 2.9 %, n=10). interestingly, in dt the ef (43.9 ± 4.9 %, n=11) was higher than in pp2a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp2a is involved in h 2 -receptor signaling and we tentatively conclude that the h 2 -receptor is able to ameliorate systolic but not diastolic cardiac function of pp2a mice. serotonin (5-ht) can exert positive inotropic and chronotropic effects in humans via 5-ht 4 -receptors. we have generated transgenic mice (tg) which overexpress the human 5-ht 4 -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with 30 µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and 3, 7 and 24 hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to 58.8 ± 20.7% seven hours after lps in wt and to 61. [4] [5] [6] [7] [8] [9] [10] [11] [12] p<0 .05 vs pre-lps value). however, 24 hours after lps, diastolic function, measured as e/a, amounted to 1.86 ± 0.43 in p<0 .05 tg vs. wt). moreover, after 24 hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after 3 and 7 hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from 1158.3 ± 212.2 to 782.5 ± 154.3 in wt and from 1208.4 ± 235.1 to 798.8 ± 170.1 in tg (tg vs. wt, p>0.05, n=10-12; after 7 hours). we tentatively conclude: 5-ht 4 -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec 50 = 34 nm) or amthamine (ec 50 = 10 nm), a more selective and potent h 2 -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to 44 mm potassium chloride. thereafter, histamine (100 µm) induced a pie (31.1 ± 10.4 % of control, n=7) in tg but not in wt preparations whereas isoprenaline (10 µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound 48/80, a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine (10 µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h 2 -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk2, a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek293t cells transfected with yfp-tagged µor and mturquoise-tagged grk2 as well as non-fluorescent gα i1 , gβ and gγ subunits showed a robust increase in fret upon superfusion with 10 µm [d-ala 2 , n-mephe 4 , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine (30 µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately 15-20% of that of the corresponding damgo signal. grk2 binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk2 affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i1 and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i1 and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk2 on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk2 interaction with that of the µor-gβγ interaction in the presence of grk2 revealed similar time constants both for the on-and off-kinetics (grk2: k on 0.16 s ). this suggests that, in the absence of grk2, the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk2, however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk2-gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor in the absence and presence of grk2 and compared the kinetics with the kinetics of grk2 binding and unbinding to these receptors. in both cases, we found that grk2 and gβγ in the presence of grk2 associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c57bl/6 mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin-1 (et-1) for 90 sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in 3d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et-1 treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et-1 had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin-3 was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn1 and ccn2 to be moderately up-regulated after 24h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et-1 treatment. finally, we could show that the perinuclear localization of cav3 and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production (1). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs (2) (3) (4) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac1-camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine 1-phosphate (s1p) is generated by sphingosine kinase (sk)-1 and -2 and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s1p 1-5 . after activation, s1p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s1p 1 mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru-486. in addition, in vivo studies showed that dexamethasone downregulated s1p 1 expression in glomeruli isolated from c57bl/6 mice treated with dexamethasone (10 mg/kg body weight). functionally, we identified s1p 1 as a key player mediating s1p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s1p-induced migration of mesangial cells. this effect was again reversed in the presence of ru-486. conclusion: we suggest that dexamethasone inhibits s1p-induced mesangial cell migration via downregulation of s1p 1 . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. 2015; 396: 803-12) . the g protein-coupled receptor mrgd is a receptor for angiotensin-(1-7) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-(1-7) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-(1-7). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek293 cells, we provided final pharmacological proof for mas to be a receptor for ang-(1-7). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-(1-7). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type 2 receptor at2 as a receptor for the heptapeptide, but discovered that the at2 blocker pd123119 can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-(1-7) but excluding the at2 receptor, and by enforcing the revisit of such publications which concluded at2 function by using pd123119 as a specific at2 blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within 1 ms 1 . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells 2 . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between 30-80 ms 3 . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor 1 (mglur1) sensors by fret after uncaging of an inert glutamate derivative. 4-methoxy-7-nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur1s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur1 protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur1 activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α 2aadrenergic receptor, among others receptors, has been extensively studied in single cell assays (1) (2) . the activation kinetics of the labeled α 2a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α 2a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology (3) . additionally, we used halotag in combination with the nanoluc (4) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α 2a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec50 value in accordance with the previously published data. this suggests the functionality of the fret-based α 2a -halo/cfp sensor. similar results were obtained with the α 2a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α 2aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin2 biosensors. upon stimulation, β2-adrenergic receptors bound β-arrestin2 with a time constant τ = 1.3 ± 0.17 s, indicating that β-arrestin2 binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin2 with a τ = 2.2 ± 0.22 s. stimulation of β2-adrenergic vs. m2 muscarinic or ffa4 receptors resulted in different patterns of conformational changes in the various β-arrestin2 sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin2 activation. upon agonist removal, first the interaction (delay = 1.9 ± 0.51 s) and only then the active state of β-arrestin2 (delay = 4.2 ± 0.85 s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β2-adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about 1,000 fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi10463 (historical strain), strain 1479 (serotype f, variant tcdbf), strain r20291 (hypervirulent strain, ribotype o27), and strain r9385 (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain 1470) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi10463. in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p63rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in 2d and 3d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p63rhogef-knockout mice were adenovirally transduced for 48 to 72 h with recombinant adenoviruses or directly used. for 2d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p63rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p63rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p63rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p63rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p63rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p63rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p63rhogef implies that p63rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p63rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p63rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il-6 and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/11 , g 12/13 and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr1 (r206h), a bmp (bone morphogenic protein) type 1 receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c2c12 cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp4-induced osteoblast differentiation in c2c12 cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr1 r206h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p63rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p63rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p63rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for 48 h in culture. for activation of the g q/11 -signaling the cells were treated with endothelin-1 (et-1), angiotensin ii (ang ii) or phenylephrine (pe) for 90 s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p63rhogef is involved in the regulation of the et-1-induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p63rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor 3 (kdelr3) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p63rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin-3 the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p63rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p63rhogef in the transport of membrane proteins, we overexpressed p63rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr3 and its co-localization with p63rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr3 was found in control-transduced cells. further, p63rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p63dn and detected similar changes with respect to kdelr3 localization and golgi structure suggesting that this regulatory function of p63rhogef is not dependent on its activity. conclusion: p63rhogef mediates the activation of rhoa from gpcrs coupled to g q/11 proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/11, g12/13) exist in mammals and these must suffice to convey signals from about 800 g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. 1 understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr900359, a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/11/14 over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek293 cells that lack functional gq/11 alleles by crispr-cas9 genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/11 using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") 2 . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. 2009 ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept2, 6, and 7 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc42 and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb1 thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc42, borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [1] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [1] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [2] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at 24 hours post fertilization, larvae at 5 days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [3] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac10). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [4] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [1] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences 40, 8-15 (2015) . ccmp, 3',5'-cump and 3',5'-cimp were ineffective. to further characterize the action of 3',5'-cgmp on hut-78 cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). 3',5'-cgmp significantly increased apoptosis (ec 50 = 75 µm) and inhibited proliferation (ec 50 = 63 µm) of okt3-activated hut-78 cells. interestingly, also 2',3'-cgmp exhibited comparable effects on apoptosis and proliferation with ec 50 values of 92 µm and 75 µm, respectively, while 2',3'-camp, 2',3'-ccmp and 2',3'-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested 3',5'-cgmp degradation products under the same experimental conditions and found that both 5'-gmp and guanosine increased apoptosis and inhibited proliferation with ec 50 -values between 50 and 100 µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of 3',5'-cgmp, 2',3'-cgmp, 5'-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [1]. 3',5'-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut-78 cells (e.g. enpp1), yielding 5'-gmp, which could be further degraded to guanosine by the 5'-ekto-nukleotidase cd73. a similar pathway may lead from 2',3'-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms 3a and 3b as well as the cgmp-selective pde9a also degrade the emerging second messenger cump [1, 2] . we analyzed the enzyme kinetics of pde3b-mediated cump-hydrolysis using recombinant gst-tagged pde3b and a highly sensitive and specific hplc-ms/ms method. our data show that pde3b is a low-affinity enzyme for cump with a cump k m -value of >100 µm. the pde3-selective competitive inhibitor milrinone inhibited pde3b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde3b is highly expressed in adipose tissue [3, 4] . thus, we differentiated murine 3t3-l1-mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde3b expression and basal cnmp-concentrations. in both differentiated and undifferentiated 3t3-l1 cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by 66 % and 60 %, respectively, while ccmp was reduced by 78 % and cump even by 85 %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde3b was upregulated ~1000-fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde9a-mediated cump hydrolysis. pde9a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi500. they comprise a "green" membrane-targeted version (mcgi500) and a "red" variant (red cgi500) that contains the fluorophores tsapphire and dimer2. methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi500 sensor showed a good signal-to-noise ratio, an ec 50 value of ≈1.1 µm for cgmp, and a high selectivity for cgmp over camp (>100-fold). flincg3, a non-fret-based cgmp sensor, showed similar properties as cgi500. the new membrane-targeted mcgi500 and the new red cgi500 displayed ec 50 values of ≈0.4 µm cgmp and a high selectivity for cgmp over camp (>300-fold). in vsmcs, the red cgi500 showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de5. the "green" fret-based camp sensor, epac1-camps, showed a signal-to-noise ratio comparable to that of cgi500, an ec 50 value of ≈3 µm for camp and a selectivity for camp over cgmp of ≈6-fold. finally, imaging of cells expressing both the epac1-camps and the red cgi500 demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi500 would be useful to investigate membrane-associated cgmp compartments and red cgi500 to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at1-receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [1] . whether this causes an activation of cgks is not known. these enzymes exist in 3 different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [2] , [3] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet (0.02% na + ) and enalapril (10mg/kg/d) for 3 weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay41-8543, a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw1/lrmp (lymphoid-restricted membrane protein) is a type 2 membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [1] , [2] . jaw1 and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw1 and irag share the highest homology [3] , [4] . the coiled coil domains of irag and jaw1 are important for the interaction with ip 3 rs. as already known, irag forms a trimeric complex with cgkiβ and ip 3 r1 and gets phosphorylated by cgkiβ [5] . hence we examined if jaw1 is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw1 possible phosphorylation sites can be found. our in vitro studies with jaw1 and cgki showed that jaw1 gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw1 was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw1 and cgkiβ was detected. to examine the importance of jaw1 in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw1. mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw1 was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw1 influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw1. hence, the function of jaw1 in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (camp) and guanosine 3',5' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp 1 and 2, hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine 3',5'-cyclic monophosphate (ccmp) and uridine 3',5'-cyclic monophosphate (cump) also fulfill the criteria of second messengers [1, 2] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [3] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap9 (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [4] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap9) were examined as potential ccmp and cump target proteins. hek293 cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor 2 (npr2, also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase 2a (pde2a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde2a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor 3 (npr3) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e12.5 embryos. because npr3 binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr3 could lower the activity of the cnp-npr2-cgmp signaling cascade in drg neurons. in the absence of npr3, the majority of sensory axons showed normal bifurcation, but »13 % of the axons turned only in rostral or caudal direction. this study shows (1) that pde2a is important for the degradation of cgmp in embryonic drg neurons, (2) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde2a, and (3) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi500). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d-2-amino-5-phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac1-camps fused to a phosphodiesterase (pde4a1). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than 100 different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families 3, 4 and 5) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde4. however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde2a3 (v max = 120 µmol/min/mg) to a camp fret sensor (epac1-camps) we monitor camp concentrations in direct vicinity of pde2a3. in comparison to pde4, we show that pde2a3, albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j774 macrophages, arglabin strongly induced accumulation of lc3 type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc3-ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp3 and pro-interleukin (il)-1β, we further analyzed whether arglabin inhibits the nlrp3 inflammasome. arglabin reduced expression of nlrp3 and pro-il-1β, inhibited activation of caspase-1, and release of mature il-1β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp3 inflammasome. intraperitoneal injection of arglabin into female apoe2.ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il-1β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) 2 stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par2 activating peptide sligrl (100µg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), losartan (3 mg/kg) and furosemide (40 mg/kg) in conscious wild-type (wt) and par2-deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet (0.6% nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par2-deficient mice than in wild-type mice (190 ± 35 versus 380 ± 91). renin mrna expression was 50 ± 9% of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par2-deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par2 (-/-) and wt mice. administration of sligrl increased renin secretion about 6-fold (p<0.01). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par2 (-/-) and wt mice. the absolute changes (delta prc) were similar (3780 ± 770, 2230 ± 400, 2780 ± 70, 1390 ± 460 in wt, and 2320 ± 270, 2530 ± 250, 3010 ± 210, 1230 ± 470 in par2 (-/-)). in conclusion, chronic absence of par2 reduces basal renin expression and renin release. however, par2-deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par2 does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca 2+ sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin-3-gallate (egcg), which has been shown to decrease myofilament ca 2+ sensitivity, on cardiac myocyte contractility and force-ca 2+ relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc3-targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca 2+ transients at 37 °c under 1 hz pacing using the ionoptix system in the absence or presence of egcg (1.8 µm). furthermore, force-ca 2+ relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg (30 µm). results: in baseline settings and absence of fura-2, ki cardiomyocytes displayed higher sarcomere shortening ( type-1 serine/threonine protein phosphatase (pp1) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca 2+ handling and contractility. all pp1 heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor 2 (i-2). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i-2 resulted in an improved basal ca 2+ handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i-2, regulated by gsk3-dependent phosphorylation at thr 72 , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, 30 mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with 0.9% nacl (n=5). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca 2+ transients were measured in isolated, indo-1-loaded myocytes. the peak amplitude of [ca] i was reduced by 39% in tg nacl compared to wt nacl (p<0.05), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by 25% in tg iso compared to wt iso (p<0.05). consistently, sr ca 2+ load was diminished by 28% in tg nacl compared to wt nacl (p<0.05). chronic iso stimulation led to an unchanged sr ca 2+ content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than 4-fold higher phospholamban phosphorylation at ser 16 in tg (p<0.05). thus, these findings suggest that overexpression of i-2 is able to reduce the progression of heart failure by an improvement of myocyte ca 2+ handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., 168 derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of 56 virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp-1 macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a1 (abca1) expression in human thp-1 macrophages by this leoligin derivative. the transporters abcg1 and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock1 and rock2 are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock1 and rock2 are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock1 and rock2 by 75% was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with 10 μm fasudil or 3 µm h1152p for general rock inhibition and 3 µm slx-2119 for inhibition of rock2. protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock1 and rock2 influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock1 and rock2 in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock2, but not of rock1 led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock2 in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h1152p and qpcr analysis of cf with a downregulation of rock1 and rock2 showed that both rocks are involved in the regulation of ecm proteins, such as collagens 4a2, 6a, and 8a1, biglycan, decorin, elastin and its respective degrading enzyme mmp12. conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, 2011] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, 2014] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p1 mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm-1 as a nucleus-specific marker was used to enrich cm nuclei to >95%. proteins were extracted from nuclear lysate with 2% sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from 3 murine cell lines served as silac (lys8/arg10)-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified 1041 proteins, 688 of which are annotated to the nucleus. 21%/23% (adult / p1) of nuclear proteins are annotated as dna-binding. 1.5%/1.4% belong to transcription factor complexes; 7.3%/7.5% are able to bind transcription factors. 7.9%/8.3% have chromatin modification functions; 4.3%/4.4% modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. 71% of proteins are shared between p1 and adult nuclei. 142 proteins are exclusive to p1, 34 are only found in adult. 93 proteins are enriched (1.3-fold increase in abundance) in p1 hearts, 89 proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p21 cip1/waf1 , senescence-associated ß-galactosidase (sa-ß-gal) and p16 ink4a were increased 2-, 8-and 20-fold (n=5-7; p < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β1-adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin (92 ± 0.9%), platelet derived growth factor receptor α (94 ± 0.9%) and α-smooth muscle actin (71 ± 2.3%), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor 1 (cb1) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb1 receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, 1,5 mg/kg/day) with osmotic minipumps for 14 days. treatment with cb1 antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb1 antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb1 antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb1 ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna-181a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb1 antagonist treatment and foxb1 (a direct target of mir-181a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb1 receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb1 antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir-181a/b family seems to be sensitive to cb1 antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb1 inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f2α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type 2 diabetes mellitus (t2dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t2dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: 10-week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or respective placebo for 20 weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in 3t3-l1 cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd11c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il-6 from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using 3t3 l1 cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca 2+ -atpase (serca2a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca2a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. 1 using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca 2+ cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca 2+ kinetics were measured in isolated, fura2-loaded and electrically paced (0.5hz) cardiomyocytes as the time to 50% decay of the ca 2+ signal (t 50% ). the time to 50% baseline of sarcomere length (t 50% baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed 3.3-fold stronger phosphorylation signals if pentamers were absent (p<0.0001). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t 50% [ms]: 92±8 and 122±8, respectively, p<0.05). notably, t 50% of pln-ko myocytes was 79±5ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca 2+ clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t 50% baseline [ms]: 32±8 in tgpln versus 7±4 in tgafa-pln and 5±7 in pln-ko, p<0.05). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca 2+ kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the 3' end of mrnas (3' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous 3' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih-3t3 fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor-2 (par2) regulates inflammatory responses including monocyte migration and cytokine release. par2 is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine-1-phosphate (s1p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s1p on the expression of par2, tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp1 and u937 cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par2, tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with 50 ng/ml pma (phorbol 12-myristate 13-acetate) over 72 h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp1 and u937 cells s1p induced a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of par2 mrna and total protein expression. par2 total protein was upregulated maximally (about 1.5-fold, n=7) with 1 µm s1p after 16 h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp1 monocytes and tm was not regulated by s1p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s1p resulted in a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of tf expression within 3 to 6 h of incubation. conversely, par2 total protein expression was reduced by about 50% after 24 h s1p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp1 cells as tf source. timedependent incubation with s1p (1 µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from 8.0±1.6 to 6.5±1.4 min and elevated total the thrombin generation capacity from 814±130 to 1286±129 nm. peak thrombin formation was elevated from 66±31 to 130±30 nm/min (control versus s1p for 6h, mean±sd, n=5, respectively). these data suggest that s1p induces an enhanced expression of par2 in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s1p does upregulate tf expression but attenuates par2 levels. since par2 is involved in regulation cell migration, s1p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin-6 (il-6) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il-6 expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il-6 mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp-1 cells was determined by western blot. immunostainings for il-6 and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa (30 nm) induced a time-dependent (3 -16 h) increase in il-6 mrna expression. maximum expression was observed within 6 h to a 7.2±3.3 fold increase (mean±sd, n=5, p<0.05). incubation with an inhibitor of p38 map kinase (sb203580, 10 µm) or pi3k (ly294002, 10 µm) significantly attenuated fxa-induced il-6 mrna expression (n=5). inhibition of p42/44 mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for 24h resulted in a markedly increased il-6 secretion into the smc culture media from 0.6±0.2 to 1.1±0.3 ng/ml (p<0.5, n=4). stimulation of thp-1 cells with il-6 (1 ng/ml) induced a time dependent (6 -24 h) increase of up to 2.1±0.6 fold (p<0.05, n=5) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il-6 expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il-6 regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il-6 expression and secretion in venous smc which may be regulated via p38 and pi3k signaling. il-6 enhanced tf expression in thp-1 monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il-6 release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p300). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week 5-16 with vpa (0.71% in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week 7 and 16). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week 7: tg veh : 33±2%, wt veh : 47±1%; week 16: tg veh : 19±2%, wt veh : 44±1%, p<0.05). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week 7: 40±2%, tg vpa at week 16: 34±1%, p<0.05 vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p<0.05) 998 proteins were significantly changed while 854 proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p<0.05). 109 proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p<0.05), whereas vpa affected 525 proteins in tg atria (tg vpa vs. tg veh , p<0.05), out of which 43 proteins were common. 295 prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h 2 -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine (1 mm), shifted the concentration response curves for the pie of histamine from ec 50 = 110 nm to 37 nm (p<0.05). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine (10 µm), shifted the pie of histamine from ec 50 = 70 nm to 38 nm and increased the efficacy of histamine for the pie (p<0.05). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp1, pp2a, pp2b (calcineurin) and pp5 in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase 2cβ (pp2cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp2cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased (4.24 ± 0.14 mg/g) compared to wild type (wt) littermates (3.78 ± 0.20 mg/g; p<0.05) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg (4.78 ± 0.17 mg/g) vs. wt (4.13 ± 0.22 mg/g; n=8-12; 10-11 months of age; p<0.05). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: 58.33 ± 3.16 % (tg) versus 76.94 ± 0.92 % (wt); n=12-16; 8-11 months; p<0.05 and fractional shortening: 30.84 ± 2.02 % (tg) versus 44.94 ± 0.87 % (wt); n=12-16; 8-11 months; p<0.05). the left ventricle was dilated (systolic diameter: 2.78 ± 0.21 mm (tg) versus 1.84 ± 0.06 mm (wt); n=12-16; 8-11 months; p<0.05; diastolic diameter: 3.97± 0.19 mm (tg) versus 3.33 ± 0.07 mm (wt); n=12-16; 8-11 months; p<0.05). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp2cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein 5 (igfbp5) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein 5 (igfbp5) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp5 functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp5 in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp5 in murine cardiac tissue at different developmental stages by qpcr normalized to tpt1 (tumor protein, translationally-controlled 1). this analysis showed temporal changes of cardiac igfbp5 expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp5 expression in different heart cells showed a very low igfbp5 in adult cardiomyocytes in contrast to a high expression in undifferentiated sca-1 positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp5 was found up-regulated (p<0.05). further we found an increased igfbp5 expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p<0.01). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp5 upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p<0.05). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp5 expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp5 in angiotensin 2 or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin 2, showed the opposite results, namely a reduction in its expression (p<0.01; p<0.05, respectively). summary and conclusion: our results show active igfbp5 transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp5 in a conserved manner. we hypothesize that igfbp5 may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp5 is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c57bl/6j mice fed a high-fat diet (hfd) for 22 weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last 4 weeks with crataegus extract ws® 1442 (150 mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® 1442, neither in pvat nor in aorta. phosphorylation at serine 1177 is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine 1177 in pvat, but not in aorta. ws® 1442 treatment significantly improved enos serine 1177 phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine 1177 phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® 1442 treatment. in addition, ws® 1442 treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin-1 (sirt1), known also as a longevity gene. the activity of sirt1 depends, among others, on the intracellular content of its cofactor nad. ws® 1442 treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt1 is enos. deacetylation of enos at lysine residues 497 and 507 by sirt1 enhances the activity of the enos enzyme. currently, we are studying the effect of ws® 1442 on nad synthesis, sirt1 activity, and enos (de)acetylation. in conclusion, crataegus extract ws® 1442 reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine 1177 and upregulation of sirt1. the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk2) and the raf-erk1/2 pathway. these two functions of rkip could counteract each other. while grk2 inhibition is cardio-protective, inhibition of the pro-survival erk1/2 axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. 1). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp1) under control of the alpha-mhc promoter. two different rkip-transgenic lines with 2.7-fold and 3.4-fold increased cardiac rkip protein level were generated (ref. 2, and jax id number 911819). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as 10 weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of 32.9±2.3 % (n=6; ±s.d.) were documented at an age of 8 months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk2 and raf-erk1/2 axis by the human rkip gene (pebp1) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp1), which does not seem to inhibit the raf-erk1/2 axis in vivo but instead confers grk2 inhibitionheterodimerization between the at1 receptor (at1r) for the vasopressor peptide, angiotensin ii, and the b2 receptor (b2r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at1r--b2r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at1r--b2r heterodimer in vivo, we generated a transgenic model of at1r--b2r heterodimerization (tg-b2r+) by transgenic expression of the b2r gene (bdkrb2) in the b2r-deficient tg-b2r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at1r and b2r at a distance of less than 9 nm in tg-b2r+ mice whereas the at1r--b2r heterodimer was absent in tg-b2r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b2r in the aorta of tg-b2r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b2r. the efficient at1r--b2r heterodimerization in tg-b2r+ mice was accompanied by an enhanced angiotensin ii at1r-stimulated vasopressor response relative to that of tg-b2-/-mice, which lack the at1r--b2r heterodimer. as a control, the endothelin-1-stimulated vasopressor response mediated by the etar, which did not dimerize with b2r, was not significantly different between tg-b2r+ and tg-b2r-/-mice. together these findings provide strong evidence that at1r--b2r heterodimerization occurs in vivo and enhances the angiotensin ii at1r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase 2 (grk2) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf24 extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk2, grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk1/2 axis and involved the erk1/2-dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine-273. as a consequence of erk-dependent phosphorylation of pparg on serine-273, the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine-273 phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine-273a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk2 inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as 5 weeks of age in homozygous mice and 10-12 weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac2 inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from 12 weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for 7 weeks with vpa (0.4 mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by 7 mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of 90.2±8 v/s (tg: n=33 cells) vs. 109.8±5.1 v/s (wt: n=30, p=0.05). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at 50% (tg: 11.6±1.4 ms, n=35 vs. wt: 6.9±0.5 ms, n=30, p<0.01), at 70% (in ms: 21.2±2.5 vs. 13.6±1, p<0.05) and at 90% (in ms: 46.8±4.5 vs. 34±2.5, p<0.05) repolarization. vpa treatment reduced ap amplitude in wt mice by 6 mv (n=22, p<0.05) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced (50%: 7.3±0.8 ms, 70%: 13.8±1.5 ms, 90%: 30.9±3.2 ms, n=21, p<0.05 vs. untreated tg), the amplitude was increased by 5 mv (n.s.) and the slope of depolarization was increased by 21% (p=0.16, n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: 104±7.2 pf, n=27 vs. wt: 55±5.4 pf, n=30, p<0.001), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: 64.8±7 pf, n=17 n.s. vs. wt; tg-vpa: 88±7 pf, n=14, p<0.05 vs. wt-vpa; p<0.01 vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac2 inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca 2+ exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca 2+ imaging revealed that the transduction rate of spontaneous sub-threshold ca 2+ -waves into supra-threshold transient-like ca 2+ -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca 2+ transport rate (r serca ) was enhanced in tg vcms potentially limiting ca 2+ extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during 10 -6 m isoproterenol challenge, tg: 3.65*, ctl: 0.4; n=20/condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx1, serca2a and decrease of kchip2 protein levels. however, the respective mrna levels (slc8a1, atp2a2 and kcnip2) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir-369 (fold change in tg: 0.04*) and mir-1 (fold change in tg: 0.13*) (n=10/condition). atp2a2 is a predicted target of mir-369 and mir-1 has recently been shown to regulate ncx1 and i to related potassium channel subunits. (*p<0.05 vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. 8 days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from 6.2(mg/g) in untreated control animals to 10.9(mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of 7.7 (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr (8.4 mg/g) compared to saline treated animals (6.8mg/g) and pka mutants (7.1 mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: 6.5 vs 7.9 mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh6/7 ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged (6.9 mg/g) or a strongly elevated hbr (13.5 mg/g). however, additional hypertrophic factors including anp, myh6/7 ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of 100 µm after 24 and 48 h (ctb assay) nor increase in the apoptosis rate after 24, 48, or 72 h up to 100 µm. measuring adenosine triphosphate (atp) levels revealed that 30 µm soraphen a does not alter the atp levels in huvecs after 24 h treatment. in contrast, a 48 h treatment significantly lowered the atp levels by 12 %. also gene silencing of acc1 in huvecs attenuated the atp levels by 11 %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels (10 %) in soraphen a-treated cells after 24 h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic 50 value of 34 µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g 0 /g 1 phase by 26 % and increases the number of cells in the g 2 /m phase by 50 %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, 30 µm soraphen a lowered the migration of endothelial cells by 65 %. gene silencing of acc1 in huvecs strongly decreased endothelial migration, whereas a knockdown of acc2 had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by 34 %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by 60 % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type 9 (pcsk9) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october 2015. we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor 15 (klf15) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf15 and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w2 domain containing protein 2 (bzw2), a phylogenetically conserved protein, as a β-catenin and klf15 interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw2 mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw2 is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p<0.05, cm n=4, cfb n=4, cpc n=2), and skeletal muscle. bzw2 was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw2, containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw2 protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf15 knockout (ko) mice **p<0.01 n=3, and in a cardiac specific β-catenin stabilized mouse model, **p<0.01 n=6). a mouse model with constitutively bzw2 loss of function (bzw2 ko) showed cardiac specific upregulation of β-catenin on rna level (***p<0.001, p<0.05, ctrl n=4, bzw2 ko n=5) and on protein level (**p<0.05, ctrl n=7, bzw2 ko n=9). echocardiography analysis in eight-weeks-old bzw2 ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw2 expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p<0.05 ctrl n=4, angii n=3) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p<0.05 ctrl n=3, dcm n=11, icm n=10). conclusion: these data demonstrated that bzw2 is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine-1-phosphate (s1p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s1p lyase [1] . mice that lack s1p lyase are characterized by the accumulation of s1p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [2] [3] [4] . on the other hand, embryonic fibroblasts from s1p lyase-deficient mice (sgpl1 -/--mefs) are resistant to chemotherapy-induced apoptosis [5] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [6] . interestingly, s1p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [7] . the aim of the present study was to analyze the link between s1p lyase deficiency and altered cholesterol homeostasis using sgpl1 background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine-2 (p-ser2) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser2-pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after 4 weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm1 antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> 0.062 fpkm, gene expression rank < 12,653) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser2-pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type 1 (tnni1) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni1 gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin 1 (acta1) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta1 after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase 1 is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt1 in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt1 gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt1 fl/fl mice with heterozygous dnmt1 fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl7-cre) resulted in embryonic deletion of dnmt1 (ko). embryos without myl7-cre served as controls (ctl). embryos were dissected and genotyped at e11.5, e12.5, e13.5 and e14.5. rna-seq and pyrosequencing of genomic dna was performed on e11.5 hearts, histology on e12.5 hearts and electron microscopic imaging on e13.5 hearts. for deletion of dnmt1 in adult mice, homozygous dnmt1 fl/fl mice expressing an inducible cre recombinase (myh6-mcm) were given tamoxifen i.p. over 4 days. homozygous dnmt1 fl/fl mice not carrying myh6-mcm as well as myh6-mcm carrying mice without dnmt1 fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl7-cre mediated loss of dnmt1 resulted in progressive embryonic lethality with absence of living ko embryos after e14.5. ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt1 in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt1 in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt1 is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet3 involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet3. methods: female mice homozygous for tet3 flox and male mice heterozygous for tet3 flox and heterozygous for myl7-cre were mated and offspring were genotyped after weaning. mice homozygous for tet3 flox and heterozygous for myl7-cre (ko) or homozygous for tet3 flox without myl7-cre (controls) were sacrificed at 12 weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly 50 %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet1 and tet2. cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp2a2 gene and at the binding site of the transcription factor gata4 but not near the promotor and the binding site of the transcription factor tbx5. higher methylation levels were not accompanied by changes in atp2a2 expression. however, both myh7 and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet3 is involved in dna demethylation in cardiomyocytes. loss of tet3 expression resulted in embryonic lethality. compensatory upregulation of tet1 and tet2 isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet3 in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor 16 (pi16). a recent gwas analysis showed an association of a genetic variant in the pi16 genomic locus (rs1405069) with chemerin plasma levels. here we tested the hypothesis that pi16 determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi16, which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi16-deficient animals compared to littermate controls. to investigate whether pi16 and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi16 to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi16 would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi16. when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi16 to potently inhibit cathepsin k activity. taken together, we propose pi16 to act as a regulator of chemerin processing. the transient receptor potential canonical 6 (trpc6) is a second messenger-gated cation channel, which mediates depolarization and ca 2+ entry. it is known to be activated by diacylglycerol derivatives (dag, 1-oleoyl-1-acetyl-sn-glycerol oag) [1] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc6 gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [2] thus, the discovery of potent inhibitors of trpc6 may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc6. [3] larixyl acetate, another component of the resin showed an even higher potency in trpc6 inhibition (ic 50 = 0.26 µm) and a 12-fold selectivity compared to trpc3. these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc6. hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c1 position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc6 with similarly low ic 50 values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c1 position, however, seems to play no important role for the inhibition of trpc6 currents. larixyl methylcarbamate lead to trpc6 inhibition with an ic 50 value of 0.15 ± 0.06 µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc6, this compound bears the benefit of high subtype selectivity towards trpc3. even with concentrations up to 50 µm of larixyl methylcarbamate, no complete inhibition of the ca 2+ influx via trpc3 channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc6 in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α7 nachr using whole-cell patch clamping under voltage-clamping conditions (-70 mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα7 nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α7 nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, 4-tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes (1). among the 28 mammalian trp channels, the canonical channel 5 (trpc5) is a ca 2+ -permeable ion channel, which is predominantly expressed in the brain (2) . many aspects of trpc5 function are still elusive although behavioral experiments with trpc5-knock-out mice suggest a role in innate fear-response (3) and some studies indicate a trpc5-mediated down regulation of neurite outgrowth in nerve cells (4, 5) . to elucidate trpc5 function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc5 modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo (6, 7) . thus, there is still a need for identifying novel and efficient trpc5 modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc5 activator. hek293 cells heterologously expressing trpc5 upon tetracycline induction (hek trpc5 ) show a btd-induced concentration-dependent activation in both ca 2+ assays (ec 50 = 0.71 µm) and in electrophysiological whole cell patch clamp recordings (ec 50 = 1.1 µm). btd elicits currents with an n-shaped i/v curve, typical for trpc5. the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc5 inhibitor (8) . mtt assays revealed that incubating hek trpc5 cells for 24h with btd concentrations above 1 µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca 2+ -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to 10 µm. ca 2+ assays showed no influence of btd on closely related trpc4 channels, as well as trpc3/6/7 at concentrations up to 10 µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc5 subunits can also assemble to heteromeric channel complexes with their closest relatives trpc1 and trpc4 (9) . trpc1/5 and trpc4/5 heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc5 subunit. transient receptor potential canonical channels 3/6 and 7 are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc3, based on the recently available structural information on trpv1, we performed structure-guided mutagenesis and identified a single residue in transmembrane domain 6 (g652), which is conserved within the canonical family of trp channels. single point mutations at position 652 in trpc3 largely eliminated lipid sensitivity. trpc3 g652a expressed in hek293 cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc3/6/7 channels (gsk1702934a) activated wild-type trpc3 and trpc6 channels as well as the respective lipid insensitive mutants (trpc3 g652a, trpc6 g709a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g652 to gsk recognition by trpc3. our results demonstrate the existence of two different mechanisms of trpc3/6 activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc3/6 involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s6 provided by g652. lipids and synthetic activators of trpc3/6 may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb327 (1,1'-(propane-1,3-diyl) bis (4-tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c 1 -c 10 ) of mb327 were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by 100 µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c 7 ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of 100 µm carbamoylcholine in the functional assay. mb327 and several bispyridinium structure analogues (mainly c 2 -c 4 linker) exhibited no regular displacement curves at [ 3 h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the 4-tert-butyl-substituted bispyridinium compound mb327 showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb327 or mb420, could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations (1-300 µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field (20, 50, 100 hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb327 (1,1′-(propan-1,3-diyl) bis (4-tertbutylpyridinium) di(iodide)) and mb420 (1,1′-(propan-1,3-diyl) bis (2-ethylpyridinium) di(iodide)) in the tested concentration range (1-300 µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low (20 hz) and high (50 and 100 hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p2x3 receptor-induced current by the opening of asic3 channels g. stephan 1 , p. illes 1 1 universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p2x3 receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel 3 (asic3) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p2x3 and asic3 subunits has to be taken into consideration. we transfected rat (r)p2x3r and rasic3 constructs individually or together in a ratio of 1:1 into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p2x3r-and asic3-constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from 7.4 to 6.7 caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to 6.5. the opening of asic3 channels failed to facilitate the p2x3r current when 2-guanidine-4-methylquinazoline was used to stimulate a non-proton ligand-sensor of asic3. then, we substituted ca 2+ in the extracellular medium by ba 2+ or decreased the intrapipette concentration of egta, to modify the free intracellular ca 2+ concentration. in cells individually transfected with the receptor-channels, external ba 2+ increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p2x3r-and asic3-specific currents in a similar manner as external ba 2+ . in cells co-transfected with p2x3r/asic3, the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic3 activation. taken together, our results suggest that p2x3r and asic3 interact with each other, since the activation of asic3 had a marked impact on the p2x3r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca 2+ and the proton sensor of asic3 appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned-19. in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc1 specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc1 null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned-19 support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv4 channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv4 has been identified in endothelial cells (1), epithelial cells (2) and arterial smooth muscle cells (3) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in 4). it is a matter of debate however if trpv4 activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc6 (5) which is also expressed in both cell types. to analyze the potential role of trpv4 on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after 90 minutes of ischemia in trpv4-deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv4/trpc6 double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc6 deficiency protected lungs from iri-induced edema (5) . moreover, we identified reduced expression levels of aquaporin 5 in trpv4-deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv4 might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily 3 (trpm3) has been shown to be expressed in human kidney 1, 2 . using newly developed anti-trpm3 antibodies, we are able to visualize trpm3 protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm3 (trpm3 the atp-gated p2x7 receptor (p2x7r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p2x7 -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p2x7r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p2x7r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p2x7r are overexpressed under the control of the endogenous p2x7 promoter. the egfp-tagged p2x7rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p2x7-egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map2-and neun-positive cells indicating that under non-pathological conditions, the p2x7 receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd68 protein was observed in the p2x7r-overexpressing mice. these results suggests that overexpression of p2x7rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd68 primarily localizes to lysosomes and endosomes, this further supports a role of the p2x7r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc1 and tpc2 -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca 2+ channels or such as trp channels. due to their biophysical properties, tpc1 and tpc2 are assumed to be involved in the efflux of ca 2+ from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc1 for vesicle fusion & fission processes we investigated a potential impact of tpc1 on the uptake of bacterial toxins. first we determined the precise localization of tpc1 in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc1 deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc1-deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm3 ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland 1,2 . the trpm3 gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists 3, 4 . variations include the presence or absence of five stretches of 10 to 25 amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini 5 . furthermore, three different aminotermini of human and mouse proteins have been described 5 , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified 12 variants that differ in exons 8, 13, 15, 17 and 20 . however, only variants bearing the short, ca 2+ permeable pore loop were detected. 3´ rapid amplification of cdna ends (3´race) revealed that trpm3 proteins of the pituitary gland carry truncated c-termini exclusively. 5´race identified five independent regions of transcription initiation within the trpm3 gene implying the presence of five independent promotors. the different transcripts encode four trpm3 amino termini α, β, γ and δ of 1-155 amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~76 %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to 16 different bilayers at a time. here we utilize the "orbit" multichannel system with a meca16 chip by ionera to measure tpc1 channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav1) and potassium channels (like kv1, kv4). the voltage-gated na + channel nav1.5 initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv4.3 is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav1.5 and kv4.3 have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) 10 is known to interact with the kv4.3 channel complex, modulating kinetics and voltage dependence. the overexpression of dpp10 in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv4 channels. to study the potential influence of dpp10 on nav1.5 channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k1 cells, expressing scn5a alone or with dpp10. surprisingly, we observed significant effects of dpp10 on nav1.5 channel voltage dependence and kinetics. thus, the co-expression of dpp10 significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav1.5 channels alone. in addition we analysed the effects of dpp10 on the kinetics nav1.5 currents. while time to current peak was not affected in cells co-expressing nav1.5 and dpp10 compared to nav1.5 alone, dpp10 slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav1.5 and dpp10 compared to nav1.5 alone. in summary, we provide first evidence that dpp10 not only interacts with kv4 channels, but also influences nav1.5 channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv4.3 and nav1.5 behave very differently depending on the expression of its associated proteins like dpp10. cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, 2009 ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip 3 ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc1/c4-deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca 2+ -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc1 and trpc4 (camacho londoño et al., 2015) . nevertheless, the role of trpc1/c4 for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal 6 days. our results show that there is no difference in 100 nm at-ii induced ca 2+ -release or ca 2+ -entry in trpc1/c4-deficient cf compared to wt. to evaluate whether the lack of trpc1/c4 can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca 2+ -release and ca 2+ -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca 2+ ] i like isoproterenol, 5-ht, thrombin and endothelin-1. cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv7.1 channels, which are known to be expressed in vascular smooth muscle cells. k v 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l3 (k v 7.1 opener), hmr1556, chromanol-293b (k v 7.1 inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq1-/-mice to determine whether k v 7.1 plays a role in the regulation of arterial tone. we found that r-l3 produces similar concentration-dependent relaxations (ec 50 ~1,4 µm) of wild-type (kcnq1+/+) and kcnq1-/-arteries pre-contracted with either phenylephrine or 60 mm kcl. this relaxation was not affected by 10 µm chromanol-293b, 10 µm hmr1556 or 30 µm xe991 (pan-k v 7 blocker). the anti-contractile effects of pvat were normal in kcnq1-/-arteries. chromanol-293b and hmr1556 did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq1-/-mice exhibited normal peak k v currents. the k v 7.2-5 opener retigabine caused similar relaxations in kcnq1-/-and wild-type vessels. we conclude that k v 7.1 channels are apparently not involved in the control of arterial tone by alpha 1 adrenergic vasoconstrictors and pvat. in addition, r-l3 is an inappropriate pharmacological tool for studying the function of native vascular k v 7.1 channels in mice. introduction: according to international guidelines [1] [2] [3] [4] [5] [6] [7] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [8] [9] . material and methods: tissue the skin samples are descended from female pigs of german landrace (50 kg weight, approx. 4 month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from 10 different skin area (fig. 1) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of 1.000 µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of 30 mm were punched out from the frozen skin stripes and stored at -20°c. hplc and skin absorption waters corporation hplc containing 2767 sample manager, 2545 binary gradient pump, 2998 pda detector (optional: 3100 electron spray mass spectrometer); column: nucleodur® 100-5 c18 ec 50mm x 4.0 mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, 6-cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of 48 h at 32°c. the dosage compartments of each cell were filled with approximately 300 µl of the caffeine solution (10 mg/ml). samples of 1 ml are taken after 0, 12, 24, 36 and 48 hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. 2) . thickness was in the order of magnitude between 897 to 1.230 µm and intra variations were less than 10 %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [10], demonstrating intact barrier properties of the membranes after 3 month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps1000 provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in 2012, the mak commission derived a new threshold value of 0.3 mg/m 3 for gbs with a density of 1, recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio 2 dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day 3, 28 and 90) and the lung toxicity after intratracheal instillation in rats (day 3 and 28): tio 2 (rutile, micro), tio 2 (anatase, nano), eu 2 o 3 (micro-nano mixed), baso 4 (micro), zro 2 (micro) and amorphous sio 2 (nano). two doses of 0.5 and 1.5 µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio 2 (rutile) and baso 4 (pmn < 5% after 3 days in the low dose group; < 15% in the high dose group; full recovery after 28 days). in contrast, the tio 2 (anatase) showed a stronger response (pmn > 30% after 3 and 28 days). the rare earth eu 2 o 3 (micro-nano) dust showed the strongest effect (approx. 40% pmn after 3 and 28 days) including a red-coloured lung lavage fluid. µ-zro 2 and amorphous sio 2 showed a strong acute response after 3 days, however, mostly complete recovery after 28 days. the low solubility criterion was met by the following dusts: tio 2 (both) and zro 2 . -similar volumetric lung burdens were deposited in a parallel validation experiment (14-day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group 1 carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, 25 µm of pyr and 20 µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp1a1 gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp2b6 in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p450 enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p450 enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use 8 compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the 3 test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from 1) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and 2) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of 10 µm (bpa), 3 µm (fen) and 0.01 µm (keto), for concentrations in target organs resulted in estimated oral loels of 16, 4 and 0.04 mg/kg. when calculations were made for plasma levels oral loels were estimated to be 608, 77 and 16 mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of 375 mg/kg bw day for bpa (1), 50 mg/kg day for fen (2) and 6 mg/kg day for keto (3) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal4/uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp3a4 promotor activity was investigated using a pxr-dependent cyp3a4 reporter gene assay. cyp3a4 induction was analyzed at the mrna and protein levels in hepg2 cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp3a4 promotor activity could only be detected for echimidine. cyp3a4 induction by echimidine was verified at the mrna and protein level in hepg2 wildtype and hepg2 pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases (7 isoforms are known, nox 1-5, duox 1 & 2) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox 1-, nox 2-and nox 4-deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600 ng/kg during 28 days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox 2 and nox 4 could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox 1-knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox 1-knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox 4-knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox4. conclusion: separate eliminations of 3 nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last 50 years about 60 drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across 5 species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin 2, podocin, synaptopodin, retinol-binding protein 4, clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of 1,2-intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c3 exoenyzme (c3) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c3 promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c3 lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c3. knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c3. moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c3. proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c3-induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c3 compared to wild type cells. as vimentin knock out did not completely inhibit c3 uptake into cells, an additional uptake mechanism or additional receptor for c3 is likely. nevertheless, our data reveals that c3 employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl 2 on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl 2 was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl 2 . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an 80 compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for 24 h before toxicants were added to the cells. migration and viability of the cells were then measured after another 24 h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, 26 compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed 23 of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein 1 (rip1) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) 1 , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht-22 cells erastin promotes activation of rip1 and subsequent rip1-rip3 necrosome formation which has been investigated as a hallmark of regulated necrosis 2 . in fact, silencing of rip1 by sirna or by the rip1 inhibitor necrostatin-1 prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin-1 fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein-1 (rip1) and rip3 kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) 2, 3 . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip1/rip3-dependent necroptosis 4 and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip1-rip3 complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns5a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns5a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns5a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi-6202, was from medchem express. murine embryonic stem cells (es-d3) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of 0.1% in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after 10 days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between 0.1 and 30 mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of 10 mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in 23, 22 and 2 wells out of 24 wells in total at concentrations of 1, 3 and 10 mg/l. at 30 mg/l no differentiation was observed. effects on cell viability were observed at 30 mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of 1 mg/l this nsa5-inibitor showed a clear impact on differentiation with 6 out of 24 wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi-6206 at concentrations up to 30 mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg 416) and developmental toxicity studies (oecd tg 414) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the 2-generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some 21-days old pups. for all effects clear noaels were determined. in the 2-generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies (1-generation toxicity study and a 3-week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in 2006, dimoxystrobin was classified with r63 (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r63 (which has been translated into the ghs classification repr. 2, h361d) was based on offspring body weight and heart effects seen in the 2-generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in 2009 and a restriction program for pfoa was initiated in 2013. therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek-293t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist 17β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of 17β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by 17β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t47d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a 6-fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a 6-fold repeated estrogen response element (ere). both constructs were stably transfected into t47d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e2 and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone-2 and 4-mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of 17betaestradiol (e2) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) 81. furthermore, rats fed idd until pnd74 were fed ird for 7 days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e2 and estrone (e1) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e2 and diet (idd vs ird and idd vs idd+ird). e2 levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e1 and ratios e2/e1 in mg and ratio of e2 levels in pl/mg were not affected by diet. in contrast, diet tended to affect e2 concentrations in pl (p=0.1211) due to an increase in the ird group (p=0.0056) whereas e2 levels in the idd+ird group only tended to be elevated (p=0.0788). in mg, ird and idd+ird increased e2 levels only weakly (p=0.0788 each). likewise, besides significant changes in transcript levels of cyp1a1 and 1a2, putatively decreasing oxidation of e2 to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e2 levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp53, mt1a) was observed in the idd+ird group. e2 mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e2 levels but not diet were significantly associated with gata3 transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt4) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e2 (increase in gdf15, hgf, igf1r, wnt5a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e2 and even decreased not significantly by idd+ird. taken together, despite an increase in e2 levels in pl, less er activation was observed after dietary exposure to if. whereas e2 and transcript levels of enzymes involved in e2 (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le 1329/10-1. august copenhagen irish (aci) rats with 17β-estradiol (e2)-releasing implants are an accepted model to study the etiology of breast cancer, but neither e2 (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) 75 and 175, placebo (-e2) or silastic implants containing 4 mg e2 were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd 285. palpable mg tumors (pt) and 1-2 mg per animal without pt were characterized histologically and categorized into normal (-e2 group, n=12), hyperplasia and non-pt and pt with and without solid tumors (+e2 group, n=32). e2, estrone (e1), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e1 and e2 in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of 49 transcripts involved in (biotrans)formation of e2 and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e2, plasma e2 as well as e1 and (borderline) e2 levels in mg were higher in ird. plasma e2 as well as e1 and e2 levels in mg were lower in the -e2 group than that in the +e2 group. e2 levels as well as e2/e1 and e2 mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki67). ird increased e2/e1 ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata3) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd1) tissues. levels of e1 and 2-meo-e1 were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd17b2 (conversion e2 to e1) and cyp1a1. transcript levels of gstm1 and gstm2 were decreased in the whole +e2 group and of gstt1 and gstt3 in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp53 and mt1a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of 2-meo-e1 nor cellular stress (gclc, mt1a, tp53). of note, neither 4-meo-e1, nor e1 catechols, nor e2 catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e1 or e2 were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e1 or e2. taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e1 and cellular (oxidative) stress, pt exhibited highest e2 levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e2/e1 ratio in pt. supported by dfg le-1329/10-1. level of 17beta-estradiol (e2) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from 37 mammoplasty specimen obtained from women (age 18-66 years old) not taking estrogen active drugs. 14 of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for 7 days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n=12) and bacterial metabolites (n=7) as well as the estrogens estrone (e1)-sulfate, e1, e2 and 2-methoxy-e1 were determined by uhplcand gc-ms/ms, respectively and transcript levels of 19 enzymes involved in e2 (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e2/e1 ratio with agl(+con) was observed in glandular tissues (r=0.49, p=0.002), accompanied by a significant negative correlation of e1 levels with agl (r=-0.35/p=0.032), possibly due to reduction of 17beta-hydroxysteroid dehydrogenase 2 (conversion of e2 to e1) expression as indicated by a weak negative correlation of transcript levels of 17beta-hydroxysteroid dehydrogenase 2 with agl+con (r=-0.25, p=0.080). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e1/e2 ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le-1329/10-1. allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp-1 cells were used as a model system and treated with the sensitizer 2,4dinitrochlorobenzene (dncb; 5, 10 and 20 µm) and the irritant sodium dodecyl sulfate (sds; 100 µm). samples were taken after 4, 8 and 24 hours. thp-1 activation was analyzed by measuring the established activation markers cd86 and cd54 after 24 hours. a targeted lc-ms/ms approach was used to analyze 188 metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., 2015) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp-1 were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp-1-macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp-1 cells. evaluation of hepatic cell stress showed the strongest effect on thp-1-macrophages when phh were incubated with apap or dic for 4 h.treatment of kc and thp-1-macrophages with supernatants of phh challenged for 4 h with hepatotoxic compounds indicates that thp-1-derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp-1 derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of 4 h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a 90day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to 0, 0.1, 0.3, 1 and 3 mg/cm³ ceo 2 as well as 50 mg/m³ baso 4 nanoparticles (6 h/day, 5 days/week, 13 weeks). animal dissections were conducted at five time points (exposure day 1 and 28; recovery day 1, 28 and 90) aiming for endpoints mandatory according to oecd guideline 413. additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo 2 and baso 4 retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to 90 days was detected by balf and histopathology examination. based on our current results effects of ceo 2 nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt-90 project is funded by the german federal ministry of education and research (bmbf) -03x0149a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco-2 model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco-2 cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana 2.0 expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the 25 market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana 2.0 project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana2.0 provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana 2.0 is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around 13% of total pm10 emissions are emitted by wood burners (uba 2006) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a549) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted 1:40 and directly delivered to the exposure chamber. after 4h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp9 cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. 2014 part fibre toxicol 11:59), or amosite asbestos at 3 µg/cm2 for 24 h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed 3788 significantly differentially regulated genes for mwcnt a, 1680 for mwcnt d, 145 for amosite, and 4 for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht29). for this purpose ht29 cells were exposed for 24 hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of 2-10 µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of 1.39 pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to 40 % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about 50 % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about 12%; control about 6%), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about 20%; control about 6%) for concentrations above 8 µg/ml. induction of apoptosis was determined for silver particles (up to 24% early apoptotic and 20% late apoptotic cells) as well as for titanium dioxide and zinc oxide (10% each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to 90%). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to 100nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [1] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of 45nm and 16µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in 48-well plates and exposed to doses of 1, 5, 10 and 25µg/cm² growth area of the respective cnt type for 24h. the wst-8 assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h 2 so 4 /hno 3 mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [2] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph 2 the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to 48h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union 7 th framework program (grant agreement no. 310187), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of 50µg/cm 2 (24h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il-8 liberation (elisa) as primary endpoints, and quartz dq12 and al 2 o 3 as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) 2 nanoplatelets (mg1; mean lateral size: 1,5-2 µm; mean thickness: 15-20 nm) represented the least, and pristine few layers graphene nanoplatelets (gr1; mean lateral size: 2 µm; mean thickness: 3 nm; graphene layers: 8 ± 0.5) the most biologically active nm. clear concentration dependencies were detected for gr1 in follow-up experiments. mg1 and gr1 were further tested in other lung-relevant cell types, i.e. mrc-5 primary human lung fibroblasts and a549 lung adenocarcinoma epithelial cells. interestingly, mrc-5 cells were less sensitive towards biological effects of gr1, compared to am, whereas a549 cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for 24 h to 12.5 or 25 µg/cm 2 of gr1 on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr1, i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc-5 cells and limited uptake in a549 cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc-5 cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) for two years; a control group was exposed to clean air. after one year exposure, 42 µg/lung was found in animals exposed to 0.1 mg/m³ and 2.6 mg/lung in animals exposed to 3 mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of 0.1 and 0.3 mg/m³ ceo 2 . neither pre-neoplastic nor neoplastic changes were observed after 12-months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) and barium sulfate (nm-220; 50 mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and 50 animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally 6 additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo 2 lung burdens were maximally 3.5 mg/g lung tissue at the highest exposure concentration of 3 mg/m³. in comparison, highest ceo 2 burdens in organs remote to exposure were liver and spleen with maximally roughly 1 x 10 -3 g/g tissue. in brain, maximum ceo 2 levels were 7x10 -6 mg/g lung tissue. baso 4 lung burdens were comparatively low (1 mg/g) within the first 13 weeks of exposure and steeply increased to 6 mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df4nano) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df4nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df4nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. the df4nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df4nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df4nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df4nano for 24 nanomaterials were validated against grouping by results of existing in vivo data and demonstrated 23 concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a549 cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo 2 ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo 2 nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo 2 nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo 2 np with an average diameter of 15-30nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations (0.01, 0.1, 1 and 10%) and were used to expose human lung adenocarcinoma cells (a549) for up to 24 hours. at 100µg/ml ceo 2 np showed little to no toxic effecton growth arrested a549 cells at fcs concentrations of 1% or below, but cell viability was decreased to about 80% in proliferating cells in cultures with 10% fcs. the proinflammatory effect of ceo 2 np was investigated through measurement of il-8 mrna expression after 3 hours and cellular il-8 secretion after 24 hours. the qrt-pcr showed that the expression of il-8 mrna in cells treated with 100 µg/ml ceo 2 np in 10% fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il-8 secretion, which showed a stronger increase by cells treated at 10% fcs. differences in cellular uptake of ceo 2 np was determined by fluorescence-activated cell sorting (facs) after 2 and 24 hours of exposition. after 2 hours, the cells treated with ceo 2 np in 10% fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after 24 hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo 2 np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about 1030nm (10% fcs) to 4090nm (0.01% fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr8383 rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio 2 np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr8383 rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase-1 (ho-1) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)-1β, il-6, il-18 and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il-1β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio 2 np by wst-1 assay overall revealed only minimal toxicity in comparison to sio 2 np which were used as reference material. ho-1 and g-gcs mrna analyses indicated that specific tio 2 np triggered a moderate induction of oxidative stress. il-6 was only induced after sio 2 treatment, whereas il-18 was not affected by any of the tested np. in contrast, various tio 2 np caused a significant induction of il-1β mrna expression. however, no significant induction of il-1β and tnf-α protein secretion was observed for any of the tio 2 np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio 2 np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp7 programme. specific types of tio 2 particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase 1 (nat1) activity j. lichter 1 , b. blömeke 1 1 trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase 1 (nat1), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat1 enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat1 has not been analyzed yet. to address whether human nat1 is a target of silver nanoparticles and released irons on protein, purified nat1 was exposed to silver ions (agno 3 ) and silver nanoparticles (ag10-cooh, average size 10 nm, carboxyl functionalized), and nat1 enzyme activity was analyzing the n-acetylation of the nat1 substrate para-aminobenzoic acid (paba). therefore, purified nat1 (1 ng/µl) was co-exposed for 20 min to paba (1 mm) and agno 3 or ag10-cooh (0.01, 0.1, 1, 10 and 100 µg/ml each), resulting in a nat1:silver relation of 1:0.01-100 (w/w). both, agno 3 and ag10-cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat1 (w/w, 1 µg/ml) enzyme activity was reduced about 98±0.2% (agno 3 ) and 82±0.6% (ag10-cooh). the lowest concentration analyzed (0.01 µg/ml) reduced nat1 activity about 24±5% (agno 3 ) and 17±5% (ag10-cooh). fifty percent activity reduction was caused by 0.11 ± 0.01 µg/ml of agno 3 and 0.21 ± 0.04 µg/ml of ag10-cooh, which is 10-fold lower compared to the published ic50 values for other metal oxide nanoparticles (3-15 µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat 28-days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of 60 and 6 mg/kg body weight for nanosilver and corresponding amounts of silver acetate (9.3 and 0.93 mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by 2-d gel electrophoresis and differentially expressed spots were identified by maldi-ms. 374 unique proteins, showing a log 2 ratio of ≤ -0.3 for downregulation and ≥ 0.3 for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf2-mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated 14-3-3 protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso 4 -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso 4 -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below 40 % for 1 mg/ml after 72 h). in general the effects of both nps were almost similar. additionally the effects of the baso 4-nps were compared more in detail. uptake of the baso 4 was quantified by icp-ms after 24 h and 72 h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso 4 of plc and nhbec to 0.1 mg/ml over 24 h and 72 h leads to ~200 µg baso 4 /1 mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il-8 and il-33 could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after 72 h. summing up baso 4 -nps showed toxic effects in primary human lung cell cultures after 72 h for concentrations under 1 mg/ml. c3 exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht22 revealed a c3-mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., 2012) . former results of studies on map kinase signalling indicated c3-induced modulations of downstream signalling modules. therefore, ht22 cells treated with 500 nm c3 for 48 h were applied for screening of the activity of 48 various transcription factors followed by luciferase reporter assays. five transcription factors namely sp1, atf2, e2f-1, cbf, stat6 were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp1 target genes p21 and cox-2 as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd153, a target gene of atf2, was decreased. our results suggest that c3 is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of 1.077g/cm 3 , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure 1 ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from 400 to 500 xg resulted in higher yields. in general, large variations in yield (46.8% ± 17.1%) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl-2-naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or 2-tert-butyl-4-methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule 1 chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine 34 -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system (4000 qtrap) and a hybrid quadrupole time-of-flight instrument (tt5600 + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the 20 th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group 2a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. (2011 ) chem res toxicol 24, 1957 resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d 7 -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of 5 fmol per injection (5 pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and 3-monochloropropane-1,2-diol (3-mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and 3-mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and 3-mcpd. the metabolite 2,3-dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is 3-mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary 3-mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound 13 c 2 -dhpma. the limit of quantification is 10 µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = 45) of one male volunteer collected over ten days was 154 ± 21 µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing 1.1 mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c 3 -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b6c3f1 mice and nasal neoplasms in male f344/n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic 2-sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n 2 -[(furan-2-yl)methyl]-2′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase 2 (nat2) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat2 genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in 772 bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n=207), dortmund (n=167) and neuss (n=398). nat2 was genotyped using seven characteristic polymorphisms (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931). haplotypes were reconstructed using phase v.2.1.1. we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat2*5b/*5b and *5b/other slow haplotypes as well as between the ultra-slow *6a/*6a and *6a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to 5 yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of 371 (49%) patients showed a relapse within the first 5 yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators (51% vs. 46%, p=0.154). this frequency is even higher in ultra-slow acetylators (61%, or=1.81, p=0.019) but not in slow *5b/*5b genotypes (49%, p=0.609). ultra-slow acetylators had a significantly shorter relapse free time within 5 yr after bc diagnosis than rapid acetylators (median 0.66 vs. 0.94 yr, hr=1.57, p=0.009). this trend was not that pronounced in all slow acetylators combined (0.78 yr, hr=1.19, p=0.127) nor in the subgroup of nat2*5b/*5b genotypes (0.79 yr, hr=1.20, p=0.255). the effect of ultraslow nat2 is even more pronounced in smokers (hr=1.78, p=0.003) but absent nonsmokers (hr=0.89, p=0.781). ultra-slow nat2 seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat2 in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand 17β-estradiol (e2). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf-7 cells treated with e2 or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e2 and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [1]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; 2011/12). urine specimens from children aged from 20 to 80 months from 23 different day-care centers were analyzed. in total, 253 preschool children were recruited with mean age of 54 months. our study results show that nearly all children (>95 %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, 5oh-mehp, 5oxo-mehp, 7oxo-minp), for bisphenol a and methylparaben. triclosan was detected in 16 % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about 5-50 µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about 1000 µg/l. median urinary concentration for methylparaben and bisphenol a were about 48 µg/l and 2 µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were 1770 µg/l, 72.4 µg/l and 55,6 µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study (2014/2015) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of 2 µg/kg benzo[a]pyrene and 12 µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [1]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl 3 ). first, the time-dependent internalization of hemin and fecl 3 into hct116 cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after 1 h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to 24 h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco-2 and hct116-cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion 8-oxoguanine (8-oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue 1 (mth1), which protects the nucleotide pool by hydrolysis of 8-oxogtp, 8-oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct116 and caco-2 cells using western blot analysis, which was followed by a reduction in cell viability at high doses after 72 h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o 6 -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o 6 -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between 0.3% and 95%. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [1, 2] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [3] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta 100 for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for 24 h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [4] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [1] göggmann, schimmer (1983) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. 121, [191] [192] [193] [194] wiseman et al. fatty acid esters of 3-chloro-1, and of 2-chloro-1, are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free 3-mcpd and 2-mcpd. 3-mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class 2b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which 10 mg/kg b.w./day of either 3-mcpd or 2-mcpd were orally administered to rats over a period of 28 days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis (2de). among others, the redox sensor protein dj-1 was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with 3-mcpd or 2-mcpd. up to six different isoforms of dj-1 were identified by 2de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either 3-mcpd or 2-mcpd predominantly led to a shift of the abundance between two dj-1 isoforms in the rat tissues. this effect was more pronounced with 3-mcpd compared to 2-mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys106) of dj-1 to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either 3-mcpd or 2-mcpd. dj-1 is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys106 was shown to be crucial for the activity of dj-1, and the irreversible oxidation of cys106 to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of 3-mcpd and 2-mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. 1,2-unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure (24h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure (14d) comparatively to short-term exposure (24h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after 14d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp7a1 (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in 1998 in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab 5 protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp78, which results in cell stress and caspase 3/7dependent cell death. recently, we discovered that higher concentrations of suba (10 mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [1] . in contrast to hela and caco-2 cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of 3-mcpd and its esters. high amounts of 3-mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related 2-mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of 3-mcpd, pointing to kidney and testes as main target organs. 3-mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for 2-mcpd, showing similar organ toxicity compared to 3-mcpd and identifying heart as additional target organ. no such data exist for 2-mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for 3-mcpd, 2-mcpd and their esters. hence, an oral 28-days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by 3-mcpd and 2-mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of 3-mcpd + 2-mcpd (10 mg/kg body weight), and 2-mcpd-dipalmitate (53 + 13.3 mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by 2-d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of 270 unique proteins deregulated at a log 2 ratio of ≤ -0.5 for downregulation and ≥ 0.5 for upregulation at p ≤ 0.05 were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by 2-mcpd versus 3-mcpd. dipalmitate treatment even caused more deregulation than 2-mcpd. upregulated cytochrome b-c1 complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after 2-mcpd and 2-mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after 3-mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after 3-mcpd treatment. protein dj-1 (park7), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with 3-mcpd and the higher dose of the 2-mcpd diester. tropomyosin beta chain (tpm2) was commonly upregulated after 3-mcpd and 2-mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of 2-mcpd-and 2-mcpd dipalmitate-induced proteomic changes exist compared to 3-mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as 7α-hydroxy-chol (7α-ho-chol), 7β-hydroxy-chol (7β-ho-chol), 7-keto-chol (7-o-chol), 5,6α-epoxy-chol (α-epoxy-chol) and 5,6β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where 7β-ho-chol (milk) and 7-o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: 5α-cholestan-3β-ol), respectively in breast adipose tissues of 48 healthy women undergoing mammoplasty. furthermore, tissue levels of 25 fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: 0.36 nmol/g tissue, range: 0.06-1.55 nmol/g), followed by α-epoxy-chol > 7-o-chol > 7α-ho-chol> 7β-ho-chol (0.009 nmol/g, range: 0.002-0.11 nmol/g). tissue levels of chol (2.8 micro mol/g, range: 1.88-3.87 micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of 7α-ho-, 7β-ho-, and 7-o-chol (r = -0.29, p=0.024-0.044) median oxychol/chol ratios ranged from 0.0119 (5, to 0.0003 (7β-ho-chol). 7-o-chol and 7-ho-chols correlated strongly with each other (r=0.81-0.91, p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, 7β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r=0.30, 0.30, and 0.31, respectively, p=0.037, 0.034, 0.028, respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products (1) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin (2) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them (3) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation (4) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument (3). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently (1,2). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points (3). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas 134-62-3), icaridine (cas 119515-38-7) and ebaap (cas 52304-36-6) (4). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than 10% as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than 1,000 international publications have been transferred to a database, with more than 120,000 entries. according to the database, pharmaceuticals have been found in the environment of 71 countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than 600 active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in 50 countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than 33000 discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, 3585 chemicals (ca. 11%) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a 4 and 32 days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet (1 -100 mg/l) and els study (0.0003 -0.03 mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat-1 (slc7a1) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat-1 for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat-1 expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat-1 in human ea.hy926 endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat-1 mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb (5,6-dichloro-1-β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat-1 gene responsible for this regulation. to our surprise and in contrast to data published by others 1 the chromosomal region up to 5 kb upstream of the first hcat-1 exon exhibited no promoter activity in either endothelial or dld-1 colon carcinoma cells that exhibit a very strong endogenous hcat-1 expression. we could however identify a promoter element within the first intron of the hcat-1 gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat-1 expression. our results indicate starvation-induced transcriptional regulation of hcat-1 through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc47-family consisting of the two multidrug and toxin extrusion transporters mate-1 (slc47a1) and mate2-k (slc47a2). while mate-1 is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate2-k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with 26 different anti-neoplastic agents comparative transport experiments with the model substrate 1-methyl-4-phenylpyridinium (mpp) have been carried out. therefore stably transfected hek293-cells expressing mate-1 or mate2-k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek293-cells were used. the interaction analyses were carried out by determining the uptake of [ 14 c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) 2b1 that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp2b1 substrate estrone 3sulfate (e 1 s), and observed a significantly lower k m for mdck-hoatp2b1 compared to .00 ± 10.23 µm, *p<0.05 student´s t-test) whereas there was no difference in v max (mdck-hoatp2b1 119.4 ± 11.3 fmol/min/µg protein; mdck-roatp2b1 102.3 ± 12.8 fmol/min/µg protein). the human oatp2b1 exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [1] . to identify whether rat oatp2b1 owns the same characteristics, the cellular accumulation of 50 µm e 1 s (low affinity site) or 0.005 µm e 1 s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp2b1. as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e 1 s in mdck-hoatp2b1 cells (110.0 ± 14.6 fmol/min/µg protein vs. 60.7 ± 7.5 fmol/min/µg protein, *p<0.05 student´s t-test), there was no inhibition of e 1 s accumulation in mdck-roatp2b1 cells (53.2 ± 13.2 fmol/min/µg protein vs. 49.0 ± 17.4 fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp2b1 while human oatp2b1 is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per 1000 ng of total rna) in different tissues of rat revealed a high expression of oatp2b1 in liver (159.6 ± 45.5), a moderate expression in small intestine (14.9 ± 4.0) and colon (57.0 ± 12.3), and a low expression level in kidney (2.3 ± 0.8) . the latter is in accordance with previous findings showing that oatp2b1 is abundant in rat kidney as quantified by absolute proteomics technics [2] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of 10 drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of 754 mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v.5, targetscan7.0) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of 754 mirnas, 248 were detected in all tissue types. out of ten transporters five showed significant inverse correlations with 10 putatively targeting mirnas (e.g. pept1 and hsa-mir-27a, r= -0.498, p=0.002). reporter gene assays indicated interactions of mir-193a/b with pept1 3'-utr (p = 0.0025 and p = 0.0012) as well as of mir-27a with abcb1 3'-utr (p = 0.0006). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept1 and abcb1. background: the human uptake transporter nact [gene symbol slc13a5; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc13a5 gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc13a5 gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g219r, -p.t227m and -p.l488p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of 24 tested) inhibited nact-mediated citrate transport at the tested drug concentration of 100 µm. from these, benzbromarone shows the strongest inhibition with an ic 50 value of 83.2 µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g219r, -p.g219e,p.t227m, -p.l420p and -p.l488p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc13a5 gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member 1 (abcb1) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb1 in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb1 deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol-4,5-bisphosphate (pip 2 ) is a known regulator of abcb1. the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip 2 . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb1 activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht-29 served as a model. we employed various in-vitro methods for the measurement of abcb1 activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb1 activity and re-sensitize our cell model against chemotherapeutic agents like 5-fluorouracil which are known to be substrates of abcb1. taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb1 might be a new approach to target resistant tumors without interfering with the natural function of abcb1 in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ 3 h]-labeled homoarginine as substrate and human embryonic kidney (hek293) cells stably overexpressing human cat1 [gene: slc7a1 (solute carrier family 7)], cat2a (slc7a2a) or cat2b (slc7a2b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat1 (7-fold), cat2a (1.6-fold) or cat2b (2.2-fold) after 2.5 min using 100 µm substrate (each p<0.001). apparent k m values for cellular net uptake of homoarginine were 174.6 µm for cat1, 3640 µm for cat2a and 522.8 µm for cat2b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat1, cat2a and cat2b. compared to other cat substrates, the cat1-and cat2b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter 1 (oct1, alternative name slcc22a1) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b1), known oct1 substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct1 has been shown to have strong inter-species differences. in rodents oct1 is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct1 is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct1 orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct1 orthologs. methods: overexpression of oct1 orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ 293. the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct1 orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct1. human oct1 showed a 5fold higher v max for the uptake of asp + and 2-fold increase of v max for sumatriptan in comparison to the rodent oct1 orthologs. conversely, human oct1 showed a 4-fold lower v max for the uptake of fenoterol compared to rodent oct1s. there was no difference between rodent and human oct1 in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct1 constructs and by comparing the effects of key point mutations in mouse and human oct1 orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct1 orthologs. therefore oct1 pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct1. ranitidine is a histamine h 2 -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately 30% of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct1 [1]. oct1 is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct1 alleles are known [2] . thereof 12 alleles confer partial or complete loss of oct1 activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct1 on the uptake of ranitidine. we used hek293 cells stably transfected to overexpress wild type or variant oct1 isoforms. the variant oct1 alleles oct1*1a (met408val), oct1*1c (phe160leu), oct1*1d (pro341leu/met408val), oct1*2 (met420del), oct1*3 (arg61cys), oct1*4 (gly401ser), oct1*5 (gly465arg/met420del), oct1*6 (cys88arg/met420del), oct1*7 (ser14phe), oct1*8 (arg488met), oct1*9 (pro117leu), oct1*10 (ser189leu), oct1*11 (ile449thr), oct1*12 (ser29leu) and oct1*13 (thr245met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct1 isoforms. wild type oct1 showed a time and concentration dependent uptake of ranitidine with a k m of 62.91 ± 4.32 µm and a v max of 1125.41 ± 86.12 pmol/min/mg protein. variants oct1*5, oct1*6, oct1*12 and oct1*13 completely lacked ranitidine transport activity. variants oct1*2, oct1*3, oct1*4 and oct1*10 showed v max values decreased by 64, 77, 91 and 50 %, respectively. oct1*8 variant showed an increase of v max by 25 %. there was no significant changes in ranitidine uptake by variants oct1*1a, oct1*1c, oct1*1d, oct1*7, oct1*9 and oct1*11 compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct1 substrate and demonstrated that genetic polymorphisms in oct1 can strongly affect ranitidine uptake. the effects of oct1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of 5-ht 2a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate 5-ht 2a receptors, we here compare lsd and dmt with regard to processes of 5-ht 2a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the 5-ht 2 selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective 5-ht 2a antagonists ketanserin and mdl100907. after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ 35 s]gtpуs binding) is paralleled by a desensitisation of frontocortical 5-ht 2 receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha-5-ht 2a transfected) hek293 and (endogenously 5-ht 2a expressing) c6 glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise 5-ht 2a receptors, fails to activate the phospholipase d (which is needed for 5-ht 2a internalisation), and fails to inhibit the synthesis of 5-ht 2a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of 5-ht 2a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of 5-ht 2a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., 2010) and sunset (schmidt et al., 2009) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div 60. in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor-2 kinase inhibitor a 484954 and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a 484954 and spermidine had no effect on de novo protein synthesis in juvenile neurons (div 20) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div60). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin 1a receptors (5-ht 1a r) apparently play a pivotal role in the development of depression. the involvement of an altered, 5-ht 1a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located 5-ht 1a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic 5-ht 1a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic 5-ht 1a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment (15 days) with the selective 5-ht 1a r agonist 8-oh-dpat (0,5 mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; 50 mg/kg/day). mice are sacrificed either one day (proliferation) or 21 days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic 5-ht 1a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia (33 days), but not after spontaneous remission (age 90 days). as indicated by unaltered homeoboxprotein nkx 2.1 (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc2 and cavii mrna, used as markers for the gaba-switch, were unchanged in 18 and 33 day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha1 subunit). by stereological determination, we found a 46% decrease in alpha1 subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in (32%) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha1 gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem (2.0 and 10.0 mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam (0.5 and 1.0 mg/kg i.p.) in the dt sz hamster. examinations of αlpha2 gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha1 gaba a r subunits. changes in αlpha1 gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. 2005) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: 250 µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh-018 (5 x 10 -6 m) and jwh-210 (5 x 10 -6 m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh-018 was not affected by pregnenolone (10 -7 m), the inhibition by jwh-210 was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone (10 -7 m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ 9 -tetrahydrocannabinol (2 x 10 -5 m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ 9tetrahydrocannabinol were not changed by pregnenolone (10 -7 m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, 10 -6 m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol 521: 437-449, 1999) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ 9 -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β3-antagonist sr59230a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor 1 (trh-r1) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp1 mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr59230a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr59230a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par-1, par-3 and par-4. in human adipose tissue, par-1 is found in vascular stromal cells and par-4 in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in 3t3l1 cells. results: differentiation of 3t3l1 preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par-4 is time-dependently upregulated in maturing cells while par-1 expression is detectable but not altered. in preadipocytes, thrombin (3 u/ml) activates the mitogenic kinase erk1/2, promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki67 and the inflammatory cytokine interleukin (il)-6 are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par4-activating peptide (gypgkf, 100 µm) and cathepsin g, a proteolytic par-4 activator released from neutrophils and mast cells. repeated exposure of maturing 3t3l1 cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il-6 expression. pretreatment of mccm with a par-4 inhibitor further drives lipid accumulation, the induction of il-6 by contrast is suppressed. conclusion: par-4 activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par-4 expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr4 antagonist amd3100 exerts deleterious effects in endotoxemia in vivo s. seemann 1 , a. lupp the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). although a blockade of the cxcr4/cxcl12 axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr4 seems to be part of the lipopolysaccharide sensing complex, cxcr4 agonists or antagonists may have a positive impact on tlr4 signaling. additionally, cxcr4 is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr4 blockade on endotoxemia by applying a sublethal lps dose (5 mg/body weight) in mice. the selective cxcr4 inhibitor amd3100 was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. 24 hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp91 phox as well as of heme oxygenase 1 in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd3100 plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than 200% or 35% when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about 11% in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd3100 and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp91 phox to occur above average, whereas heme oxygenase 1 expression was distinctly decreased. our results indicate that a blockade of the cxcr4 in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd3100 and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr4 antagonist cannot be recommended, hence indicating the activation of cxcr4 to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr2 by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr2 expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr2, tlr1 and tlr6 mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr2 expression in a time-dependent and concentration-dependent manner and reduced tlr1/6 expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il-1β further increased tlr2 mrna levels. gene expression of mapk phosphatase-1 (mkp-1) was also upregulated by dexamethasone. glucocorticoid-induced tlr2 expression was negatively regulated by p38 mapk signalling under inflammatory conditions through mkp-1 induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr2 signalling as demonstrated by reduced il-8, il-1β, mmp-1 and mcp-1 levels. however, the expression of traf6, a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr2 agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il-1 cytokines and t helper 17 (th17) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th17 activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il-1β, propranolol induced the th17 priming cytokines il-23 and il-6 in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il-36γ and lps failed to increase il-23 release in molc and modc in the presence of propranolol but further induced secretion of il-1β. autophagy has been linked with the secretion of il-1 family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p62 and lc3-i to lc3-ii conversion, induced accumulation of lc3 positive vesicles, as well as expression of il-1 signalling downstream adapter molecule traf6, indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz-3 (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr2 expression and responded robustly to tlr2 engagement, confirmed by increased cd83 and cd86 expression, upregulated il-6, il-12p40 and il-23p19 mrna levels and il-8 release. tlr2 activation reduced ccr6 and elevated ccr7 mrna expression and induced migration of mutz-lcs towards ccl21. similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il 1β whereas ligands of tlr3 and tlr4 failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr2-activated mutz-lcs, co culture with naive cd4+ t cells led to significantly increased ifn-γ and il-22 levels indicating th1 differentiation independent of il-12. tlr2-mediated effects were blocked by the putative tlr2/1 antagonist cu-cpt22, however, no selectivity for either tlr2/1 or tlr2/6 was observed. computer-aided docking studies confirmed non-selective binding of the tlr2 antagonist. taken together, our results indicate a critical role for tlr2 signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep19-2.5, a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep19-2.5 and the structurally related compound pep19-4lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr2 (fsl-1) but not tlr4 (lps) activation by increased il-8 production, as determined by elisa. western blot analysis showed that both salps inhibited fsl-1-induced phosphorylation of nf-κb p65 and p38 mapk. furthermore, the peptides significantly reduced il-8 release and gene expression of il-1β, ccl2 (mcp-1) and hbd-2 as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below 10 µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il-6 secretion, downregulated expression of the maturation markers cd83 and cd86, as analysed by flow cytometry, and inhibited ccr7-dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il-6 levels and cd83/cd86 expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep 19-2.5 affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as 1 ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf2 exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf2 in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf2-deficient (nrf2 -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for 30min (v t =16ml/kg, f=90min -1 , peep=2cmh 2 o, fio 2 =0.3), before 50 µl hydrochloric acid (hcl) with ph=1.5 or ph=1.3 were instilled intratracheally, controls received nacl. mice were then ventilated for further 6h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph=1.5 induced mild lung injury, indicated by hypoxemia (po 2 /fio 2 ~300mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf2 -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf2 -/mice. in contrast, hcl ph=1.3 caused more severe lung injury (po 2 /fio 2 ~200mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf2 -/mice. conclusion: we conclude that the presence of nrf2 augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany 2 fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [1] [2] [3] [4] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [5] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [6] . adma is degraded by dimethylarginine dimethylaminohydrolase 1 and 2 (ddah) enzymes [7] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide-3 kinase (pi3k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh1] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah1 and ddah2 enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi3k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah 1 and 2 enzymes. the pi3k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-(1-7) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-(1-7) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-(1-7) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-(1-7) infusion provided best protection from experimental ali when administered by continuous infusion starting 30min after oa infusion till the end of the experiment (30-240min). both pretreatment (-60-0min before oa) and short-term therapy (30-90 min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-(1-7) 90min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-(1-7) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y-27632 and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of 10 µm y-27632 under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens-1 (zo-1). under conventional culture, y-27632 induced higher growth rate of primary ebec and increased the passage number up to 5 passages with retained epithelial cell behavior. in the presence of y-27632, ebecs under ali showed higher teer values. expression of zo-1 correlated with the increase in teer, but in y-27632-treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y-27632 facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic 50 values for the viability in the low micromolar range [1] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe 2+ . the radicals were trapped by 2-methyl-2-nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = 16.8 g, and a h = 1.8 g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic 5,5-dimethyl-1pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe 2+ a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = 16.1 and a h = 24.6 g. according to previously published data [2] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [1] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine 21:1048-1052, 2014. nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca 2+ -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the 5' untranslated region (5'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld1. using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 5'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld1 cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c101a-enos). normal enos (enos-tg) or a novel dimer-destabilized c101a-enos described previously (antioxid redox signal. 2015 sep 20; 23(9) :711-23) were introduced in c57bl/6 in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for 4 weeks or treatment with sod mimetic tempol. results: c101a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos 1176/79 phosphorylation and amp kinase (ampkα) phosphorylation at thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c101a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd31. exercise training increased phosphorylation of enos at ser 1176/79 and of ampkα at thr 172 in wt but not in c101a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c101a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c101a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp-1 cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka (0.1 mg/ml) and mya (1 mg/ml) treatment down to 24% and 16% resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka (3 mg/ml) treatment by 26% and 25% resp.; after mya (0.1-1mg/ml) treatment by 17% and 44% resp. lps-induced tnfα release from thp-1 cells was inhibited concentrationdependently by my (ic50 = 60.65 μg/ml; 97% inhibition), ka (ic50 = 439 μg/ml; 71% inhibition) and cc (ic50 = 1886 μg/ml; 44% inhibition). furthermore, ka (200 µg/ml) and cc (500 µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to 100-fold (for cxcl13). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a2a receptors (a2ar) and adenosine a2b receptors (a2br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a2ar agonist (cgs 21680) and an a2br antagonist (psb-1115) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw 5 with a2ar as a possible mechanism of the protective effect of stw 5 in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a1, a2b, and a3 receptor with specific agonists reduced the acetylcholine (ach, 10µm)-induced contractions, while activation of a2br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs 21680 (10µm) or with psb 1115 (100µm). the same effects with smaller intensity were found for stw 5 (512 µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw 5 interact with the a2ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw 5 are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw 5 as a therapeutic approach in ibs. (allescher 2006) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw 5(ottillinger et al. 2013) . stw 5 (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw 6) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw 5 and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than 150 pharmacological tests, is needed. all data from studies including stw 5 alone, or stw 5 and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of 2d histograms (lorkowski et al. 2015) . results: more than 150 pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw 5 contribute differently to the overall effect of stw 5. altogether, the evaluation of the data shows that stw 5 is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw 5 manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw 5 ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw 5 (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw 5 consists of a mixture of 9 standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw 5 ii lacks the last 3 components. stw 5 was shown to be effective clinically to treat functional dyspepsia (1) and irritable bowel syndrome (2) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis (3) and in the management of ulcerative colitis (4) . the present study was initiated to show whether stw 5 ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with 5% dextran sodium sulfate in drinking water for 1 week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw 5 in doses of 2 and 5 ml/kg or stw 5 ii in a dose of 2 ml/kg for 1 week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of 300 mg/kg. conclusions: the modified preparation, stw 5 ii thus proved to be as effective as stw 5, thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. (1) schmulson mj (2008) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor 1 (maropitant) and serotonin receptor 3 (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk1) and channel opening (activation of 5ht3) on the intracellular calcium homeostasis (using fluorometric ca 2+ analysis) and cell proliferation rates in endogenously nk1 and 5ht3 receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk1 or 5ht3 cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk1 receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk1 and 5ht3 receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every 4-5 days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b2 and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b2 in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b2 signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal1 controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal1 in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal1 gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal1 in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh1 promoter, further corroborated that rasal1 expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal1 as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa-5ht], urb597) on a549 lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, 2-ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (timp-1). using sirna approaches, a causal link between the timp-1-upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp-1 expression. inhibitor experiments point toward a decisive role of cb 2 and transient receptor potential vanilloid 1 in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa-5ht and urb597. as underlying mechanism of its anti-invasive properties an upregulation of timp-1 was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb597 and arachidonoyl serotonin (aa-5ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p42/44 mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p42/44 mapk activation, a blockade of pparα left p42/44 mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p42/44 mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes 9 and 22 leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k562. an inverse correlation of mir-212 expression and protein levels of the efflux transporter atp-binding cassette transporter g2 (abcg2) was observed in cells resistant to different im-concentrations, pointing to a relation of mir-212 to im-resistance. hence, we investigate in current studies, how the influence of mir-212 on im-sensitivity could be explained. methods: we transfected k562 cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir-212 or inhibitory anti-mir-212, challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst-1-, caspase glo 9-assay and cell counting. in addition, we analyzed changes in abcg2 expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k562 showed an effect of mir-212-inhibition using anti-mir-212. this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p<0.01) and cell membrane integrity and reduced capase-9 activity (p<0.05). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg2 expression, we found that 2 µm im-resistant k562 do not express higher amounts of abcg2, but showed higher transport rates of im or the abcg2-substrate hoechst 33342. conclusions: overall, these experiments indicate that mir-212 does not only affect abcg2-expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg2. in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than 50 % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe(1%)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of 1-3 gy using a 120 kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around 1 nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [1] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [2] 1 to 5 % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to 30% of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h 2 s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h 2 s production in the nociceptive system are only poorly understood. here we investigated the expression of the h 2 s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h 2 s. this work was supported by the deutsche forschungsgemeinschaft (sfb815-a14) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany 2 heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt-112) and mesenchymal-like (j-82) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt-112 r , j-82 r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh2ax and 53bp1 foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j-82 and j-82 r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt-112 cells showed an about 2-3-fold higher resistance to cispt than j-82 cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt-112 r and j-82 r revealed a 2-3-fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt-112 r cells disclosed a reduced apoptosis and enhanced g2/m arrest following cispt exposure as compared to rt-112 wild-type cells. by contrast, induction of cell death was similar in j-82 and j-82 r cells. notably, j-82 r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j-82 r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt-112 r and rt-112 cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j-82 r and rt-112 r . unexpectedly, j-82 r and rt-112 r shared the upregulation of xaf-1. treatment of j-82 r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk-1 and, moreover, re-sensitization to cispt by chk-1 inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt-112 cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j-82 cells. furthermore, our findings indicate that chk-1 might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her3 expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her3 impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein 1 (satb1) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb1 in the upregulation of her3 after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb1 expression in gastric cancer cells, and the downregulation of satb1 by sirnas completely prevented the induction of her3 upon c-met inhibition. in contrast, her1 or her2 expression levels were not affected by satb1-specific sirnas. the function of satb1 as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her3 expression after c-met inhibition. interestingly, the upregulation of her3 in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb1 and pkc are critically involved in the regulation of her3 expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her3 plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb1 may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb-231 breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki-67 proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb-231 breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to 47 h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for 1406, pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb-231) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam-1, vcam-1, e-selectin, n-cadherin, and galectin-3. moreover, the chemokine system cxcl12/cxcr4 was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl12 in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl12 could be excluded, since blocking the cxcl12 receptor cxcr4 on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β1-integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β1-integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse (1). based on an evolutionary cancer model (2) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc9 and hcc827 cell lines; both erlotinib sensitive) (3). plasma concentrations from two single-dose groups, 30mg/kg and 200mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens (15mg/kg daily, 30mg/kg daily, 200mg/kg every 2 days, 200mg/kg every 4 days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem 7.3. results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the 200mg/kg dose. a 1-compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: 13 and 52 days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec 50 =0.53μm for both cell lines), and on the partially sensitive transit phase (ec 50 =1.24μm and 3.00μm, for hcc827 and pc9 cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and 5-lipoxygenase (5-lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of 30 min whereas the increase in vocalisation threshold persisted over a period of 90 min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox-2 and 5-lo but not of cox-1. we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox-2 and 5-lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. 2,5 million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., 2001 and buchner et al., 1998 documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > 0,6. in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over 7000 purified compounds and over 500 extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of 0,05 %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d 2 receptor partial agonist 2bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than 21 million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d 2 receptor partial agonist 2-bromoterguride (2-bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [1]. to determine its atypical character in vivo, the ability of 2-bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of 2-bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: 10 week old male sprague-dawley rats were injected with 2-bt (0.1 or 0.3mg/kg; i.p.) followed by pcp (1.5mg/kg; s.c.) or apomorphine (0.5mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of 2-bt (0.1 or 0.3mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp (5.0mg/kg; i.p. twice daily for 7 days) was assessed in the nor task consisting of a 3min acquisition trial and a 3min retention trial separated by a 1h inter-trial interval. clozapine (5.0mg/kg; i.p) or haloperidol (0.1mg/kg; i.p) were used as positive controls. results: the dopamine d 2 receptor partial agonist 2-bt (0.3mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly 2-bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that 2-btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that 2-bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of 2-bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of 2-bromoterguride [1], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of 2-bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb2 possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb2 for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in 1. 2-1.8 year old cb2 -/and control mice, fed with a standard diet and assessed effects of the cb2 agonist, hu308 during high fat diet in 12-16 week old mice. results: the cb2 -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb2 agonist hu308 prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m1-like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb2 agonists may fortify cb2-mediated anti-obesity signaling without the risk of anti-cb1 mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic 50 : xan 23 µm, res 161 µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic 50 : xan 7 µm, res 14 µm) while had less influence on the viability of pmm (ic 50 : xan 68 µm, res > 438 µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin (5-ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph1), or the raphe nuclei of the brainstem (tph2) and the respective 5-ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral 5-ht is mainly produced by tph1-expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, 5-ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about 37000 small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic 50 in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral 5-ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco2 permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg2 as a hcc tumor cell line and the fibroblast cell line fi301 as stroma simulation. additionally caco2 cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of 0.01 -100 µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: 0.5 µg/ml -cr: 1 µg/ml -es: 5 µg/ml. performance of the caco2-transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with 5-fluorouracil (5fu), which is used as a chemotherapeutic agent, caused a reduction of its ic50 values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of 5fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin-1, -3, -5, -12. claudin-5 is known to be the key tj protein tightening the bbb. therefore, claudin-5 has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin-5 and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin-5 containing barriers. the claudin-5 targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin-5 and immortalized brain endothelial cells (bend.3). the peptides decreased the amount of claudin-5 and zo-1 at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein (376 da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, 547 da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin-5 (cldn5) which tightens the bbb for molecules <800 da. consequently, cldn5 is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn5 assay, the barrier opener 1 (bo1) was identified as a cldn5 modulator. initially, a significant removal of cldn5 from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw 521 da) demonstrated the effect of bo1. concentration dependent treatment (50-150 µm) of cell monolayers with bo1 reduced tightness of the tjs between some hours and 24 h. applying 2-hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo1 became detectable in mice. due to short stability (< 2 h) of bo1 in the bloodplasma repeated administration (1.5 mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw 376 da). the small molecule bo1 is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo1 is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about 16 h of incubation with concentrations varying from 100 to 150 µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow (457 da) and fitc-dextran (10 kda); the localization of tric elongated from ttjs towards btjs and cldn1 was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c57bl/6 mice. c57bl/6 mice without transplantation were used as normal controls. donor c57bl/6 mice to recipient c57bl/6 mice were served as the isograft group. 42 days after transplantation, mice were scanned using an in vivo small animal µct (skyscan 1176). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf-1 pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf-2 (igf1r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf-1 pathway is the transcription factor daf-16 (hfoxo3a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf-1 pathway. recently, a cohort study showed that a decreased mortality rate in humans between age 78 -90 correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac1 or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about 20%) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf-16 overactivity. consequently we found an increased nuclear localization of daf-16 in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf-16-ko mutant background. rt-qpcr brought jnk-1, a known activator of daf-16, into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf-16 dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf-1 pathway and particularly the transcription factor daf-16. the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. (2005) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance (1). in hfd/stz model, the rats are fed with high-fat diet for 2-4 weeks or for a relatively long time (≥ 3 months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz (30-35 mg/kg), some of the diabetic rats receive treatment (2) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for 4 weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats (234.40 ± 42.71 mg/dl vs. 464.00 ± 23.88 mg/dl, p < 0.05). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats (249 ±6.00 gvs. 288.00 ±4.41 g, p < 0.05). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for 4 weeks. further details can be found in table 1 . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency (3) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type 2 diabetes (4). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type 2 diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: 1. srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. (2005) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. pharmacol res 52 (4): 313-320. 2. oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. (2015) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem 90 (7): 506-515. 3. franz, m. j. (2007) . the dilemma of weight loss in diabetes. diabetes spectr 20 (3) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt1 dystonia, which is caused by a gag deletion in tor1a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d2 receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d2 receptor mediated acetylcholine release has not been examined yet. the aim of present study was to (1) generate more powerful tests to detect behavioural alterations in the dyt1 knock-in mouse and to (2) examine the behavioral effects of the d2 receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt1 knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt1 knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole (0.25 -1 mg/kg i.p.) were tested in dyt1 knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt1 knock-in mice showed increased thigmotaxis at a dose of 0.5 mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt1 mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt1 model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt1 knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt1 dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d2 dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay 41-8543 after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay 41-8543 was (4mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay 41-8543 administration influenced the activity of the ecm degrading matrix metalloproteases (mmp2 and mmp9) and their inhibitor timp-1, the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor-1 (pai-1)) and the secretion of cytokines, e.g. il-6. thereby, bay 41-8543 increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay 41-8543 might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly1 (sh3-domain protein expressed inlymphocytes 1) is essential for the generation of a fully functional t-cell response. the lack of sly1 leads to reduced survival rates of infected mice. the increased susceptibility of sly1 ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly1 ko t cells displayed a dysregulation of forkhead box protein o1 (foxo1) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. interestingly, we observed a similar regulation for the adapter protein sly1, where tcr stimulation results in sly1 phosphorylation and sly1 export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly1 to 14-3-3 proteins. altogether, this study describes sly1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly1 regulates t-cell proliferation (schäll et al., eur j immunol. 2015) . the catalytical isoforms p110γ and p110δ of phosphatidylinositol-4,5-bisphosphate 3kinase γ (pi3kγ) and pi3kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p110γ and p110δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi3kγ and pi3kδ in p110γ ko δ d910a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. here we analysed p110γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il-5 and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p110γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p110γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p110γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p110γ and p110δ causes eosinophilia and ige hyperproduction, p110γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type 2 receptors (b2) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med 2015:372; 418-425) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually 20-30 min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a 200,000 kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately 200 µm below the surface. after stabilization of the green fluorescent signal, a 10,000 kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b2 changes physiologic endothelial permeability. this newly developed transgenic mouse line (b2 tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment. we observed that b2tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the 10,000 kda dextran at each time point (fig. 1) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b2 increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type 2 (b2). using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b2 was generated and backcrossed to c57bl/6 for more than 10 times (b2 tg ). lung mrna using primers specific for the human or the mouse b2 cdna revealed a 12.5-fold stronger expression of human b2 in b2 tg (n=6), while the expression of murine b2 mrna was unchanged and similar to transgene negative littermates (b2 n ). we have evaluated the specificity of several antibodies directed against b2 and found that a rabbit monoclonal anti b2 antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b2 -/mice. however, this antibody primarily stains rodent b2 and has only little cross-reactivity to human b2. hence, we were not able to detect a significant increase of b2 protein in tissues of b2 tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at 1 µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b2 activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b2 n , we found a significant icatibant sensitive aortic dilation in b2 tg following preincubation with diclofenac which indicates functional overexpression of b2 in conductance vessels of b2 tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of 30 µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching 4.41±0.11 fold at 18.9 nmol bradykinin in c57bl/6 (n=6 each, p<0.0001 vs. vehicle). a similar increase was found in b2 n (4.45±0.25 fold, n=7, p<0.0001 vs. vehicle), while there was a stronger response in b2 tg (5.50±0.16 fold, n=7, p<0.0001 vs. vehicle) which was significantly different to b2 n (p<0.01) and c57bl/6 (p<0.01). in another set of experiments the specific and ace-resistant b2 agonist labradimil (1.89 nmol) was used instead of bradykinin. labradimil increased extravasation by 3.736±0.121 fold in c57bl/6 (n=6 each, p<0.0001 vs. vehicle), by 4.51±0.11 fold in br2 n (n=7 each, p<0.0001 vs. vehicle) and 4.88±0,21 fold in b2 tg (n=6, p<0.0001 vs. vehicle) which was significantly different to c57bl/6 (p<0.01) but not to b2 n (p>0.05). the effects of bradykinin and labradimil were largely blocked by 10 nmol icatibant (i.v.) in c57bl/6, b2 n and b2 tg mice (p<0.0001) and hence mediated by activation of b2. these data suggest that overexpression of b2 in b2 tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b2 tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller 1 1 universitätsklinikum, innere 1, nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = 0 a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = 0.5772 …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd19+ and cd20+ b-cells completely persists for 6 month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = 100 mcg/ml and the infusion time t = 2 hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t1/2 = 2 hours (not shorter than the time t of infusion). with the terminal half-life of 460 hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t1/2 < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: 1) are the observed metabolites ruminant specific and formed directly in the rumen? 2) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro >7 days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph 6.6) and redox potential (~-300 mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh3 and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least 7 days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating 14c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within 72 h to 1.2.4.-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides (12c-polydatin, octyl-14c-β-d-glucopyranosid) were cleaved within 1 hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd503 and contribute significantly to animal welfare (3r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. (1982) and including an empiric correction factor, mrd for the training set was 2.49 whereas bias was 2.80 µmol/l. by applying the developed pbpk model to the test set the respective values were mrd=3.92 and bias=1.43 µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco-2 cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco-2 cell system under different experimental conditions. the permeation characteristics of hypericin (5 µm) in absence or presence of hypericum extract 145, 62.5 µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from 0 to 7.5 %). the majority of hypericin was found after cell extraction (44% in absence and 76% in presence of the extract). the recovery was in the range of 90 %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in 10 healthy foals (5 ♂ and 5 ♀, age: 42-63 days, body weight: 100-177 kg) which were treated once with rif alone (10 mg/kg s.i.d., p.o., a) followed by the administration of gam (6 mg/kg once weekly, i.v., b) for 3 weeks. study period 3 ("rif-gam acute", c) includes the administration of gam and rif for 7 days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for 2 weeks. all periods were completed with blood sampling for pharmacokinetic analysis for 48 ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c 24 h : 1.2 ± 0.5; balc/c 24 h : 2.01 ± 1.24). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c 24 h -ratio was unaffected (peters et al. 2011) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c 24 h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. 1 1 institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp2d6. direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp2d6. however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct1 and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about 9% of caucasians are compound heterozygous carriers of loss-of-function oct1 polymorphisms. therefore, oct1 polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct1 function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct1 alleles. methods: 50 mg desvenlafaxine (pristiq®) was orally administered to 41 healthy subjects preselected according to their oct1 genotypes. oct1*1 allele was regarded full active, oct1*2 to *6 alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to 60 hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the 41 subjects 14 carried two active, 13 one active, and 14 zero active oct1 alleles. age, height and weight were 26.9 ± 6.4 years (mean ± standard deviation), 1.75 ± 0.11 m and 70.9 ± 12.1 kg with no significant differences among the oct1 genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc 0-infinity of desvenlafaxine varied between 52.8 and 282.2 min*mg/l and auc 0-infinity of didesmethylvenlafaxine between 3.5 and 30.7 mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct1 genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average 14% greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct1 genotype groups. conclusions: our results suggest that oct1 genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct1 genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly (1). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression (2) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september 2013 and december 2013 using the medical records of elderly patients (≥65 years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss 16.0 statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of 541 elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n=135). median age was 72 (min:65, max:98), 84 patients were female (62.2%). according to medical records of 135 patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug (69.6% vs. %30.4) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics (65.4% vs. 33.3%) and angiotensin receptor blockers (36.9% vs. 23.5%) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women (58.8% vs. 38%, 68.6% vs. 20.2%, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over 20% of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e14 guideline [1], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [2] the ability to detect qtc-prolongations of about 10 ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [3, 4] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like-1 (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin-12, -13 and -17. apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in 23 patients with nstemi, 4 stemi patients and 14 controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd62 and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap1) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for 30 sec to 5 min reduced platelet aggregation in lta studies by up to 20 % for apelin-17. this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name (300 µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group (56,84% ± 9,28 % in mi versus 100 % ± 19,35 %in controls; p = 0.029). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin-17 in patients with mi (14.95 ± 0.6 pg/ml versus 16.98 ± 0.6 pg/ml; p = 0.035). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = -0.46; p = 0.03). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin-17. upon platelet activation, apj is internalized and surface expression is reduced by about 50 %. in mi patients, plasma levels of apelin-17 and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin-17 levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m 1 receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m 1 muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m 1 receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from 2004 to 2011. inclusion criteria comprised the initial absence of dementia and age of 75 years or older in 2004. anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m 1 receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn 1,2 , s. schmiedl 3, 4 , k. janhsen 3, 5 , p. thuermann 3, 4 , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of (1) drugs with a known association to a certain adverse event (=positive controls) and (2) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for 100% of the positive control drugs, and for none of the negative controls. ryan et al. 2013 developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. 2013 ;36 suppl 1:s33-47) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in 2010 to 2013 were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of 50% (relative risk 1.5) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with 80% statistical power. results: from a total of 182 index drugs in the reference sets of ryan et al., 142 (78.0 %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of 5,485,722 index drug initiators were included in the analysis. for a total of 16 index drugs, a relative risk of 1.5 could be detected with 80% power. the numbers of index drugs for each of the outcomes of interest were: ami (3 positive controls; 6 negative controls); aki (3 positive controls; 1 negative control); ugib (2 positive controls; 1 negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with 80% power to detect a relative risk of 1.5 was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of 11-nor-9-carboxyadministration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ 9tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study 11-nor-9-carboxy-∆ 9 -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of 16 patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > 0.05). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of 440 lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term 2014/15) at technische universität münchen (tum). the course consisted of a 28-day teaching period, followed by a 12-day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of 224 out of 393 (57%) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. 1). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n=194 3 rd -year medical students, n=213 4 th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of 60 items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the 60 items were presented as statements for restudy, 20 items were tested using single-choice questions, and 20 items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of 30 single-choice questions, 15 questions of which had not been used before (as a standard of comparison). the remaining 15 questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of 5 of these reworded 15 questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [1] . in order to address the persisting prescribing deficiencies [2] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [3] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of 67 indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least 0.3% of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [4] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising 8 indications, 28 drugs representing 25 mechanisms, and up to 120 adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july 2014 by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november 2015 and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are 15 chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in 2014 1 it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. 1907/2006 chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above 1000 tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, 1932 dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, 43% to 82 % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., 2015) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category 4. this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of 10000-100 000 t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to 800 ml/m³ and in mice in response to 200 ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category 4 with a mak-value of 5 ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl-60 cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm (900 mhz) and umts (1950 mhz). additionally, lte (2.535 mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from 0 to 4 w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in 2014 who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ 10% (m) in 5% (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in (1) a simple non-probabilistic way, (2) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or (3) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector (2) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between 0.1 and 3.0 µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox21, which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s81 assays in addition to the h295r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some 60 substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than 750 different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized 4 week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than 80 control groups with each 10 males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., 2012) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately 1000 to 3000 mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca 2+ -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of 389 and 778 mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca 2+/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: 610,8 mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to 3000 mg per day over the period of up to 12 weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with 24 male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of 3000 mg of hca for 12 weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer 1 1 universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include 0 and 1, respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to 100 µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only 10 µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg 431 and 439 especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt4 to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt4 cells were either continuously incubated for 72 h (approximately 1.5 cell cycles duration) with methyl methanesulfonate (mms; 50-200 µm), benzo[a]pyrene (b[a]p; 0.1-1 µm), vincristine (3-20 nm) , and colcemid (10-20 nm) or cells were irradiated with xrays (0.5-2 sv) and then cultured for 72 h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation (75 w, 20 min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for 3 out of the 5 treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤2-fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt4 cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt4 cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk-52e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s139 phosphorylated h2ax (γh2ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk-52e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p53, p-chk1, p-chk2 and p-kap1. furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk-52e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann 1 , g. fritz 1 1 institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a 3´-5´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic 10 ) and moderate toxic (ic 40 ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy926 cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic 10 and ic 40 were determined after 24h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy926 cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, 24h after permanent tbhq treatment. pictures were taken at the time points 0h, 4h and 8h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht-29 cell line on the ea.hy926 monolayer was investigated. wrn-deficient or non-deficient cells were treated with 100 µm and 160 µm tbhq or with tnfα. results: for ea.hy926 cells, 100 µm and 160 µm tbhq were determined as ic 10 and ic 40 , respectively. performing the migration assay, ea.hy926 cells showed 75% gap closure, whereas wrn-deficient cells showed a closure of only 35% after 8h. the gap was closed of 65% and 55% after 100 µm and 160 µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after 100 µm tbhq treatment, whereas the treatment with 160 µm tbhq showed a slight decrease in migration of about 10% compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after 160 µm tbhq treatment a slight increase of adhesion was detected in ea.hy926 cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy926 cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht-29 adhesion to ea.hy926, which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl60, jurkat and tk6) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk6 cells showed similar increasing trends, whereas hl60 cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration (100 nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl60 cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk6 cells. the results for the highest investigated concentrations (31.6 nm and 100 nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk6 cells. in contrast the hl60 cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p53 status of hl60 (null), jurkat (multiple mutations), tk6 (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc-1 were applied. accumulation of p53 and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p53 and pakt analysis, and quantification of the oxidized dna base 8-oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than 1000 bacterial species and population densities of up to 10 7 cfu per cm 2 . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., 2014) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of 20-30 hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and 6-4 photoproducts (6-4pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [1] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc3b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct116. similar results were obtained in sw48 and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p62 as observed by flow cytometry and western blot analysis in hct116, sw48 and hcec. cdt-and irinduced autophagy rates in hct116 increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein 1 (lamp-1) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser473. pharmacological inhibition of akt in hct116 cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc3b and lamp1 after 48 h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after 72 h and significantly lower apoptosis/necrosis rates after 48 h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n7-guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative 2chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n7-ethylthioethyl-2´desoxyguanosine (n7-ete-dg) and n3-ethylthioethyl-2´desoxyadenosine (n3-ete-da) and their thermal hydrolysis products n7ethylthioethyl-guanine (n7-ete-gua) and n3-ethylthioethyl-adenine (n3-ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh2ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone 2ax (gh2ax), which can be visualised as gh2ax foci by immunocytochemistry. in the present study, we tried to assess if gh2ax is a reliable biomarker for detecting the cellular response to dna damage. we selected 14 well-characterised genotoxic compounds and compared them with 10 non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh2ax by manual and automatic scoring of γh2ax foci, and by flow cytometry counting of γh2ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh2ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh2ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh2ax positive cells showed a significant correlation. further we compared dna damage induced by 4 selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh2ax assay detects a specific response to dna damage which can persist longer. the γh2ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of 3 stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh2ax), nuclear division (phospho-h3, dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk6 cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for 4 and 24 hrs in triplicates on a 96 microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide 3-chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c6 (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h3 events at 4 and 24 hrs and polyploidy at 24 hrs time point. eto induced a clear increase of yh2ax with a simultaneous reduction of phospho-h3 at 4 and 24 hrs. finally, cccp caused a reduction of phospho-h3 events, increased cleaved parp events and did not influence γh2ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh2ax and phsopho-h3 pilot study results as well as cleaved parp with caspase 3/7. in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage 2, additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih3t3, mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g0/g1 = ~70 %) and semi-confluent cultures, similarly arrested in the g0/g1 phase by serum-starvation or the mek inhibitor u0126, ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc6 staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk2. however, inhibition of atm, the kinase responsible for chk2 activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip1, since the chemical rip1 kinase inhibitor necrostatin-1 (nec-1) blocked cell death up to averagely 50 %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp-1 and p53 are probably not involved. deeper analyses could give evidence that nec-1 did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec-1, possibly rip1, acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca 2+ signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc1 and mtorc2) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c 3t3 cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for 3 days with the tumour initiator mca (3-methylcholanthrene) followed by 13 days with the promotor tpa (12-o-tetradecanoylphorbol-13-acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with 10 nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi-027. surprisingly, an incubation with 3 µm osi-027 led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s6. our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi-027, which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi-027 by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past 50 years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type 2 diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c 3t3 cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator 3-methylcholanthrene (mca) and the tumour promotor 12-otetradecanoylphorbol-13-acetate (tpa). in the first experiment several metformin concentrations (0.1-1 mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using 1 mm metformin. this effect was not caused by growth inhibition of metformin itself since 1 mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the 2 phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using 1 mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, 55131 mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to 5, 10, and 20 ppm for 2 years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [1]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: 0.5 ppm) after whole body exposure to vapor [2] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of 0, 5, 10, 20 ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate 200 mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp4a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, 2-pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [3] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day 0 (e0) at 37.5°c and 82% humidity. on e3, the eggs were cracked open and transferred into petri dishes. on e10, cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to 48 hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with 3-methylcholanthrene as a tumour initiator and 12-o-tetradecanoylphorbol-13-acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in 96-well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march 2013). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between 5-10%. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's (3r), described by russel and burch in 1959. in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches 1 , resulting in a refinement of in vitro testing. in this study, human tk6 cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of 19.3 (± 1.7) h was observed while fbs supplemented cells showed a doubling time of 13.5 (± 0.8) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg 442c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by 1) determining reactivity class cut offs based on published data on 199 substances for the dpra performed according to oecd 442c to predict un ghs sensitizer classes, 2) a variant of the dpra assessing reaction kinetics (time and concentration) for 12 substances or 3) an extended protocol testing several test substance concentrations for 50 reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of 6.38% (ec6.38%). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes 1a and 1b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between 5 min and 24 hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the 3 rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. 1, 10, and 100 mm) indicated that potency classes could be assigned using the ec6.38% value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec6.38% value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase 1 (nat1) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u937 and thp-1) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p450-dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of 27 putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). 18 of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or 5-amino-2-methylphenol, respectively. in contrast, covalent peptide adducts were identified for 12 putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a '2 out of 3' prediction model provided a high sensitivity of 81% for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the '2 out of 3' prediction model in a set of 95 direct haptens was comparable (sensitivity = 87% when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than 1 tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf2 luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and 86 two-test and 360 three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. 1) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [1] [2] [3] [4] . in recent supplement, 2-methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [5, 6] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [7] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of 1 mg/ml (1%), finite dosing and 30 min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [8] and electrostatic potentials using gamess [9] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. 1). toluene-2,5-diamine shows a higher accumulated mass due to increased lipophilicity (fig. 1) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [1] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [1] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in 2004 the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba1] contains data for about 600 substances on dermal absorption which has been found through the echemportal [3] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. 165 chemicals have been investigated in a comparable test system; from these 79 were shown to have a low dermal absorption of less than 1% and 51 compounds showed a high absorption rate of more than 50%. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for 15 substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than 1% to more than 50%. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in 2015 [5] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [1]who (2006) ehc 235 dermal absorption [2] scholz et al (2014) naunyn-schmiedeberg´s arch pharmacol 3 (suppl 1):s86 [3] www.echemportal.org [4] http://edetox.ncl.ac.uk [5] jung et al (2015) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p450 variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb3r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett 233:172-86, 2015) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology 79: 63-73, 2004) . we modelled the concentration time profile exploring the dose which would lead to a concentration at 1 hour and at 24 hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between 3 and 15 µm 3 /sec (truisi et al., 2015) and an absorption of 100% and an absorption rate of 1/h (cristofoletti and dressman, j pharm sci 103: 3263-75, 2014). results: the data of the in vitro study with 100 µm ibuprofen could well be modelled. when assuming a clearance of 15 µm 3 /sec the dose of 1480 mg resulted in an 1 hour concentration of 66.5 µm in the hepatic vein of pbhkm equal to 133.0 nmol/well (volume of the well = 2 ml) in the in vitro study in which the measured concentration was 138.75 nmol/well. the concentration at 24 hours of 8.7 µm (equal to 17.4 nmol/well) corresponded with the in vitro concentration (16.5 nmol/well). the modelling approach was less successful with in vitro dosing of 1000 µm. the 10 fold higher dose of 14800 mg lead to nearly double the concentration at 1 hour than measured in vitro. with a dose of 8500 mg/kg an approximation was feasible resulting in 396.7 µm in the hepatic vein at 1 hour which is equal to 793.4 nmol/well whereas the measured concentration in vitro was 782.85 nmol/well. even with a clearance value as low as the 2.5 percentile (3 µm 3 /sec), the concentration at 24 hours was modelled to be lower than the in vitro measured value (in vivo model: 98.7 µm which corresponds to 197.4 nmol/well; measured in vitro concentration: 979.2 nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro (100 µm) corresponds to a dose of 1480 mg (note: the highest approved daily dose is 2,400 mg). for the high in vitro dose modelling was successful only for the concentration 1 hour after dosing and a dose of 8,500 mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and 3.5 fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up 12 acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score (4 positive out of 8 possible genotoxicity categories) was assigned to 9 of the 12 tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [1], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of 12 acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even 200 years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin 1 (trpa1) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk1/2, are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa1 activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk1/2 and increased protein biosynthesis of sre-regulated genes in hek293 cells overexpressing htrpa1. cees increased perk1/2 levels already after 2.5 min which could be prevented by the trpa1 blocker ap18. activation of target genes through perk1/2 signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa1 channel activation. thus, trpa1 might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst-1) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort ("3rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a549), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a549 cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a549 cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg2 is often used as a model for liver toxicity studies. however, under two-dimensional (2d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for 21 days in a three-dimensional (3d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg2 cells. in our present study we extended previous studies and compared hepg2 cell cultivation in three different 3d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p450 monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. our results show that the previously reported increase of metabolic competency of hepg2 cells is not primarily the result of 3d culture but a consequence of the duration of cultivation. hepg2 cells grown for 21 days in 2d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all 3d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg2 is not due to 3d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about 55 different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [1]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [2] , whereas asfas were less toxic [3] . a substantial transfer across an intestinal barrier model (caco-2) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [4] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [5] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after 48 h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic 50 ~ 7-12.5 µm) in a concentration range comparable to that of arsenite (ic 50 ~ 7.5 µm), whereas asfas were less cytotoxic (ic 50 > 100 µm). after incubation with ashcs the cellular arsenic concentrations increased 10-20fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [1, 2] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic 50 values of 0.67 ± 0.02 µm for hache and 81.28 ± 0.04 µm for hbche. ic 50 values with chloroquine were 28.37 ± 0.02 µm for hache and 55.62 ± 0.02 µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after 60 minutes, only paraoxon-inhibited hache (50%) and cyclosarin-inhibited hbche (10%) were reactivated by 500 µm chloroquine. on the contrary, 10 µm amodiaquine reactivated all tested ops after 60 minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with 100 µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [1] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., 2012, 49, 229-238. [2] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., 2015 , 16, 2205 -2215 bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional (3d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a 3d model was used consisting of the permanent breast epithelial cell lines mcf10a (er -, estrogen receptor negative) and mcf12a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the 3d culture of mcf10a and mcf12a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf12a) and er-independent (mcf10a and mcf12a) effects. the 3d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg2) cells and (b) co-culture systems of human monocytic or macrophage-like (thp-1) and hepg2 cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg2 and thp-1 monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about 20% cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg2. we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator (3-methylcholanthrene) and promotor (12-o-tetradecanoylphorbol-13-acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day 42, morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that (1) several substances have a different impact on the transformation process, (2) it is possible to identify for each substance the phase with the greatest effectiveness and (3) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c 3t3 assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et50 protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg 405) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg 437) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg 438) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et50 (exposure time reducing viability of treated tissue to 50%) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg 438, one of the five severe ocular irritant formulations (un ghs cat 1) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat 1 formulations was just above the un ghs cat 1 classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et50 predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et50 protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins-1e) and exocrine (ar42j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, 2014 . 9(12): p. 2809 . briefly, pancreas was infused and embedded with agarose. tissue sections of app. 200 µm were prepared using a vibratome and maintained in cell culture medium for up to 6 days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in 10 % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins-1e cells) or amylase/lipase (ar42j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins-1e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar42j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of 6 days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg 1 , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd86 assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers (2,4-dinitrochlorobenzene, 2-mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd86 on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd86 after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology 2008; 249:146-1529) . dendritic cells generated in fcs supplemented medium were cd1a+/cd1c+, whereas dendritic cells from serum free culture conditions were cd1a−/cd1c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd1a+/cd1c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp-1 cells, as model for dc, enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p450 (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp-1 cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp-1 cells by microarray technology. we identified 6 differentially expressed mirnas in cocultured thp-1 cells compared to monocultured thp-1 cells irrespective of the treatment (medium, 0.2% dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after 48h) 8 additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp-1 cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp-1 cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp-1 and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa1 expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm5 and 6. trpa1 has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in 1). although recently two reports identified trpa1 in pulmonary epithelial and endothelial cells (2, 3) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa1 protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa1-deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa1 on a cellular level we cultured a hek293 cell line stably expressing trpa1 (4) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca 2+ -influx in this cell line, which was blocked by the specific inhibitor a96079. in the future, we will utilize the isolated perfused lung model (5) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa1-deficient mice after exposure to potential toxic inhalation hazards (tih see 6) to challenge the hypothesis of trpa1 as an important toxin sensor in the lung. by this strategy we hope to understand trpa1 function in lung cells and to evaluate trpa1 proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole-3-acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole-3-propionic acid for aminoglycosides, taurine for fluoroquinolones, 3indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., 2011; luo, schomaker, houle, aubrecht, & colangelo, 2014; yamazaki et al., 2013) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low (30 mg/kg) and a high (80 mg/kg) dose. rats were sacrificed following 3, 7, or 14 consecutive daily doses, or after recovery 10 days following 14 consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) 4 and 15 days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide 1 (oatp1) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/11 proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c58 protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann 1 1 background: in a 28-day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp2r1, gc, cyp3a, ugt1a) in liver was also affected, while initial indications for modulation of renal cyp24a1 and renal and hepatic cyp27b1 could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n=5 for single substances, n=10 for combinations) were treated for 28 days at dose levels tested based on noaels from 90-day subchronic feeding studies and ranged from noael/100 to noaelx10. quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total (25-oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n=30) were 64.7±8.3 ng/ml (min/max: 48/78 ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c (1000 ppm in food, n=5), there was a statistically nonsignificant reduction of vitd levels to 71.3±17.6% of the concurrent control (n=5). however, for 4 of 5 animals of this group, measured vitd level were below the range observed in pooled controls (n=30). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p21 for c, e and pz; cdk1 and gadd45a for e; cdkn1c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp3a1/2 and ugt1a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with 1000ppm cyproconazole, there were indications for a moderate reduction of 25-oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a 26-year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the 9th week, and she has been taking a multidrug therapy (dapsone 100 mg/day, rifampicin 600 mg/month, clofazimine 50 mg /day and clofazimine 300 mg/month) for the past 8 months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about 20 weeks. in the 8th, 22nd, 28th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at 28 weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the 39th week of gestation without perinatal complications. the baby was also healthy (apgar 8-9,3300 g,51 cm), and its growth and development were normal during a 6-month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was 15 months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after 3 months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in 2009 in berlin, germany, to evaluate kidney function in people ≥ 70 years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure 1 illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table 1 shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january 2014 until april 2015). the probability of dabigatran use rose with increasing age (+12%), and the probability of phenprocoumon use rose in case of egfr < 60 ml/min/1.73m 2 (+54%) or male sex (+82%). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ 65 years in 12 nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > 20 mmhg and/or dbp decrease of > 10 mmhg within 3 min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > 139 mmhg and/or mean dbp >89 mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with 96 nhr (mean age = 84.5 ± 7.3 years). in total, 15 subjects (15.6%) had oh. the mean change in sbp from sitting to standing was 19.2 ± 15 mmhg (range +8.5 to -52.5 mmhg) in patients with oh and 1.5 ±10.9 mmhg (range +44.5 to -17 mmhg) in patients without oh. mean sbp was significantly higher (143.6 ± 17.1 mmhg) in people with oh than in those without (131.5 ± 20.1mmhg). all of the nhr with oh were hypertensive compared to 89% of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p>0.05). medication data was available for 89 patients. all individuals with oh and 60 nhr without oh (80%) had antihypertensive medication. more than 2 different antihypertensive drugs were present in 11 patients with oh (78.5%) and in 43 patients without oh (57.3%). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than 75% of patients in both groups reached 80 points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around 50% of patients with oh as well as patients without oh needed more than 19 sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz 1 , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than 1000 metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ 30 %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer (60:40) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from 50 mg/d clom citrate to up to 150 mg/d in case of non-ovulation. however, therapy outcome is variable and approximately 10 -30% of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via 4-hydroxylation of trans-clom by the highly polymorphic cytochrome p450 (cyp) 2d6 leading to the major active metabolite trans-4hydroxyclomiphene (trans-4-oh-clom) [1] . recently, we identified a less active trans-3-oh-clom which is also formed by cyp2d6. besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of 100 mg clom citrate. results: incubations with human liver microsomes revealed an almost 60-fold higher glucuronidation rate for trans-3-oh-clom, which is exclusively catalyzed by ugt2b7, compared to the more potent trans-4-oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans-4-oh-clom to its sulfate is 16-fold higher compared to 3-oh-clom. for both metabolites a participation of sult1a1 and sult1e1 was identified. these results were in line with previous studies, which identified the same sults [2] and ugts [3] responsible for the conjugation of the structurally related trans-4-hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans-3-oh-clom and trans-4-oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom-3-o-glucuronide. conclusion: our results suggest a new metabolic route via trans-3-oh-clom which appears to be a potential inactivation pathway of clom. 1 1 institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of 0.07 µm sm (ic 10 ). today the cells are able to tolerate a concentration of 7.2 µm sm (ic 90 ), which reflects to a concentration of which 90% of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h 2 o 2 , glutathione, ca 2+ -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening 49 significant up-regulated and 20 significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of 11 different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than 35 weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg2 cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp1a1 expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm1-mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg2 cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka 1 1 institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp7b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp7b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in 2006 as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in 2008. the program was accredited in 2011 by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates 30 places per year to an average of 60 applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about 75% of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler 1 , m. hermanns-clausen 1 1 uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from 2010 to 2015 in total 85 cases of human exposures to chlorine developed from mixtures of hypochlorite and acids (0.8 of 1000 cases) were registered. in 55 cases the exposure was due to mixtures of products intended for domestic use. 94 % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough (45 %), dyspnea ( 33 %), irritated upper airway (26 %), abdominal discomfort (pain, nausea, vomiting) (21 %), thoracic pain (20 %), irritated eyes (11 %), dizziness (8%), and bronchospasm (6 %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in 12 patients (14 %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( 83% ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were 42 patients in the same period exposed to hypochlorite evaporation alone. 36 of them (86 %) had symptoms of which in 30 cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway (33 %), nausea or vomiting (30 %), cough (23 %), irritated eyes (20 %). dyspneoa was less fequent than in the mixture group (10 %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh2ax assay for genotoxic and nongenotoxic agents: comparison of h2ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant 1322-530 is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [1] mürdter t, et al. hum mol genet, 2011, 21:1145-54 [2] nishiyama t. et al., biochemical pharmacology, 2002, 63:1817 -1830 [3] sun d. et al., drug metabolism and disposition, 2007, 35:2006 background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: 10 bd patients (5/5 m/f, 43.4±5.3y, tbsa 40 .9±5.9%) and 5 patients (74.4±3.9y) from the icu were included in this observational study. blood samples were taken within the 3 rd interval of the 8h-lasting dosing period of piperacillin/tazobactam (4/0.5g within 0.5h) at 1, 4 and 7.5h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least 1xmic but preferably 4xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic 16 mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at 1, 4, and 7.5h, were as follows: c max 69.6±7.9 vs.116.3±10.4 mg/l, p<0.05, half-life 1.8±0.3 vs. 2.3±0.3h, p>0.05, clearance 12.6±1.9 vs. 6.5±0.4 l/h, p<0.05, volume of distribution 27.8±2.0 vs. 20.9±1.3l, p<0.05. free concentrations (which were included in tdmx calculations) were 87±2 vs. 81±2% (p<0.05) of total concentrations. duration per day while concentrations exceeded 1xmic (15.6±1.9 vs. 22.0±1.1h, p<0.05) or 4xmic (5.4±1.3 vs. 9 .4±0.7h, p<0.05) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for 4xmic could be enhanced to 16.2±1.5h if the piperacillin amount will be increased to 4x8 g/d and the infusion duration to 3h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as >1000mg mtx/m 2 bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the 1950s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years 2004-2009 were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. 29 patients aged 2-18 years (male = 19, female = 10) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, 103 mtx treatment cycles corresponding to 300 mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph <6.5, trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [1] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [2] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [3] , we speculated that repression of gene expression as the predominant transcriptional response to ota [4, 5] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using 13 c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [6] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk-2) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h2a, h2b, h3 and h4. we further selected acetylation at histone h3 lysine 9 (h3k9), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [7] and which was reduced from ~ 3% in controls to < 0.1% in response to ota, to establish a link between loss of h3k9 acetylation and expression of genes consistently shown to be down-regulated in response to ota [4, 5] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h3k9 acetylation at promoter regions of the selected genes (% of controls: amigo2: 45%, clasp2: 60%, ctnnd1: 54%). overall, these data provide first evidence for a mechanistic link between h3k9 hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays (7 most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of 3 ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced 3d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from 500 in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to 80-90% (average prediction accuracy was 70%). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> 30 µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [1] [2] started in 2010 and is a public-private partnership project within the european innovative medicines initiative (imi) [3] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to 4 week) repeated dose studies, mostly in rodent. overall more than 7000 study reports for approximately 1400 investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [4] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than 13 million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately 150 prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [5] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than 400 active substances approved for being used in pesticides and over 100000 chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg2, heparg, rat: h4iie) and adrenal gland (human: h295r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a 28-day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg2, heparg, hek293). cytoxicity has been analysed by wst-1 and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p450 (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp-1 as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens (hennen et al., 2011) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp-1 with b[a]p and eugenol in coculture with hacat cells augmented cyp1a1 and/or cyp1b1 mrna levels, while this was not found for thp-1 monoculture. augmentation of cyp1a1 mrna needed continuous presence of hacat cells. in coculture, levels of 3-oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase 1 enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of 3-oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp-1 cells by 2,4-dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp-1 cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci 2011 123:501-510.toxicology -toxic pathway analysis/aop 395 background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel 600mg or prasugrel 60mg (n=200). adp-induced platelet reactivity was assessed by impedance aggregometry 30 to 120 minutes and day 1after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at 120min (r=0.21; p<0.001). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations 120min/day 1: p=0.57 for clopidogrel, p=0.76 for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so1861) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc-3 and miapaca-2 cells in comparison to the non-target cell line nih3t3 with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc-3 cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so1861 by subcutaneous injection at the neck. after the tumor reached a diameter of 2 to 3 mm in size 6 treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so1861 in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k1492). complete blood count analysis was done by labor 28 gmbh, berlin. results: the adenine release mediated by dianthin-egf was 47.8 pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic50 value of approximately 1 nm for egf receptor expressing miapaca-2 and bxpc-3 cells as compared to 100 nm for non-target nih3t3 cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from 10 pm to 1 µm. toxicity studies in balb/c mice revealed 0.4 µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas 40 µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of 28 days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be 80% less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet 50% less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc-3 cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so1861 proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp3a4 and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp3a4 at different concentrations with and without nadph to determine the ic 50 shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr1 -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version 2014.11 and admetsar. results: fractions (f1-f3) indicated ic 50 shift reduction ≥5 post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = 2.46) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic 50 = 35.45 µg/ml) across mdr1 -mdckii cells. niaziminin indicated 85.57% bioavailablity via the human intestinal membrane with 61% chance of inhibiting cyp3a4. βsitostenone showed strong p-gp inhibition (83.27%) with 100% absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp3a4 or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in 2000 to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than 180 departments of all 51 berlin hospitals from october 2002 until december 2011. through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than 2 mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all 198 cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases (5.1%). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table 1 . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient 1, pelargonium sidoides in patient 6), and eight times as possible (valeriana in patients 3, 4, 8, 9, 10 , mentha piperita in patient 5, hypericum perforatum in patient 2, eucalyptus globulus in patient 7). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient 6). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil 2 as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. 1). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. 1, fig. 1 ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil 2 for one of our patients ( fig. 2 & 3 ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for 61 drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for 20% of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, 3rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, 2015) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg 428 under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from 4 different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n=4 per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at 32°c throughout the experiment and electrical resistance barrier integrity testing was performed at the start (0 h) and end of the experiment (24 h). four test preparations at final bpa concentrations of 2.4, 12, 60, and 300mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ 14 c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca 1%, v/v), uridine 5'-diphosphoglucuronic acid (udpga, 2 mm) and 3'-phosphoadenosine-5'-phosphosulfate (paps, 40 µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with 20 successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration (300 mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between 1.7 and 3.6% of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between 16 and 20% of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z 225 and 237 instead of the molecule ions (m/z 294 and 306) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were 1.4 ng/ml and 0.5 ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over 24 hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter 4 (mct4), encoded by the slc16a3 gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct4 and its epigenetic regulation by slc16a3 promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct4/slc16a3 might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct4 protein expression was analysed in 130 paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v.3.6 (definiens ag). dna methylation in the slc16a3 promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in 82 paraffin embedded metastasis samples by maldi tof-ms. mct4 protein expression data and dna methylation at the specific cpg site in the slc16a3 promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct4 protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately 28% and 14% in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc16a3 promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the 5'-regulatory region of mct4 may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) 4-hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in 390 postmenopausal breast cancer patients of european origin who were treated with 20 mg/day of tamoxifen for at least 6 months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method 1 . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of 313 premenopausal breast cancer patients mainly of european descent 2, 3 . twelve snps close to or above genome-wide significance (p <5e-08) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes 1, 2 and 16 for (e) metabolite e and to chromosomes 15 and 18 for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < 5% were confirmed as putative candidates: rs662106 was associated with a 21-39% variant allele-dependent increase of (e) and (z) metabolite e isomers (p< 0.05), and rs3731872, mapping to a gene encoding zinc finger protein znf124, was associated with increased risk of reccurrence or death (hr carriers 2.6, 95% ci: 1.3 -3.4; p < 0.005). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of 15 known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water (1:1) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from 7.8 nm to 10000 nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the 15% tolerance range for all analytes in all matrices. matrix effects were between 39-104% (plasma) and 31-95% (urine), apparent recoveries in plasma were above 97%. basal plasma ba level (mean ± sd) in fasting healthy subjects were 667 ± 574 nm (unconjugated bas), 935 ± 629 nm (glycine-conjugated bas) and 104 ± 62 nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were 193 ± 225 nm (unconjugated bas), 89 ± 29 nm (glycine-conjugated bas) and 6 ± 2 nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord-282925-efkb8hc7 authors: braidotti, r. title: “we” are in this together, but we are not one and the same date: 2020-08-25 journal: j bioeth inq doi: 10.1007/s11673-020-10017-8 sha: doc_id: 282925 cord_uid: efkb8hc7 the covid-19 pandemic is a man-made disaster, caused by undue interference in the ecological balance and the lives of multiple species. paradoxically, the contagion has resulted in increased use of technology and digital mediation, as well as enhanced hopes for vaccines and biomedical solutions. it has thereby intensified humans’ reliance on the very high-tech economy of cognitive capitalism that caused the problems in the first place. this combination of ambivalent elements in relation to the fourth industrial revolution and the sixth extinction is the trademark of the posthuman condition. this essay explores this condition further, offering both critical and affirmative propositions for moving forward. the covid-19 pandemic is a man-made disaster, caused by undue interference in the ecological balance and the lives of multiple species. paradoxically, the contagion has resulted in increased use of technology and digital mediation, as well as enhanced hopes for vaccines and biomedical solutions. it has thereby intensified humans' reliance on the very high-tech economy of cognitive capitalism that caused the problems in the first place. this combination of ambivalent elements in relation to the fourth industrial revolution and the sixth extinction is the trademark of the posthuman condition. the underlying mood during this pandemic is affective. it involves complex and internally contradictory alternation of emotions-that mark what i have called the posthuman convergence (braidotti 2013 (braidotti , 2019 ). an intense sense of suffering alternating with hope, fear unfolding alongside resilience, boredom merging into vulnerability. excitement and exhilaration in view of the advanced technologies that drive the fourth industrial age, flip into anxiety and fear at the thought of the huge costs and damages inflicted by the sixth great extinction, on both the human and non-human inhabitants of this planet. although climate change has come to represent this danger in an almost emblematic manner, and the nuclear threat is far from abated, in the current state of emergency, the centre of all concerns is the covid-19 pandemic. even before the rigours of the lockdown, the closing of the borders and the rising death toll, the intensity and spread of that negative affective economy was palpable. exhaustion and fatigue-a recurrent sense of hopelessness or impossibility-have become prominent features of the contemporary psychic landscapes, across the urbanized over-developed world. they are witnesses to the daily and nightly struggles to come to terms with what our world has become and the complexities of our historical context. it is not easy to address these issues. words, in so many ways, falter and fail. one can only talk about fear, sorrow, and exhaustion in a language reduced to its ossified minimal components, a language that has reached the edge of what it can express, approximating silence but not falling into it just yet. spiritual fatigue almost longs for a neutralized style that has perfected ways of de-linking from the grand statements of high theory. it would be obscene and unethical to theorize about the epidemiological catastrophe that is unfolding under our very eyes. this is not a time for grandiose theorizing but for collective mourning, affective resistance, and regeneration. we need to mourn the dead, humans and non-humans and not build theories on their dead bodies-that would be a shameless abuse of intellectual power. but over and above all else, we also need to develop different ways of caring, a more transversal, relational ethics that encompasses the non-humans. there is so much that we need to both embrace and resist: the wave of collective and personal despair at the loss of lives, the hardship of the socio-economic consequences of this man-made disaster, the awareness of all that was wrong with the old world and which has now become manifest. so the first challenge is to find the appropriate language for such an endeavour, a language that remains critical but in a modest capacity. we need to understand and account for the pain of the world, for what this damaged planet is going through, but such knowledge production has to remain de-linked from the pretentions of some sovereign idea of the thinker, the scholar, and the writer as the master signifier. the affective and social climate we are in calls for humility, cooperation, and is antithetical to syntheses and to authoritarian anthropocentric injunctions. the thoughts that come to mind in attempting to account for our predicament have almost the form of fragments of meditation upon the sorrowful present. to begin with: viruses born of human interference with animals and environmental sources, such as covid-19, are anthropogenic and hence discriminate just as much as humans do. they act as indicators of massive social inequalities, which dominant neo-liberal political classes were intent on denying. now the horrid truth about the consequences of their austerity policies is hitting them in the face: public health is an intensely political issue. if it is undeniable that the "capitalocene"-the greed of consumers' society-is responsible for the abuse of animal life that produced the infections of the bats and generated covid-19, it is equally true that neoliberal governance has laid the foundations for the spread of the contagion by exacerbating socio-economic power differences. secondly, not all humans are equal and the human is not at all a neutral category. it is rather a normative category that indexes access to privileges and entitlements. appeals to the "human" are always discriminatory: they create structural distinctions and inequalities among different categories of humans. humanity is a quality that is distributed according to a hierarchical scale centred on a humanistic idea of man as the measure of all things. this dominant idea is based on a simple assumption of superiority by a subject that is: masculine, white, eurocentric, practicing compulsory heterosexuality and reproduction, able-bodied, urbanized, speaking a standard language. this subject is the man of reason that feminists, anti-racists, black, indigenous postcolonial and ecological activists have been criticizing for decades. exposing the power-ridden assumptions of the dominant category of the human also results in relocating the subjects who have come to represent the dialectical opposites and opponents of this dominant and normative vision of the human. these are the less-than-human others, dehumanized or excluded from full humanitythese qualitatively minoritarian subjects actually very often are quantitively the majority. historically, they have been the sexualized others (women, lbgtq+); the racialized others (non-europeans, indigenous); and the naturalized others (animals, plants, the earth). in a concomitance of events that marks the extraordinary period we are going through, the voices, experiences, and perspectives of these multiple others are exploding all around us. the power of viral formations has become manifest in the pandemic, stressing the agency of non-human forces and the overall importance of gaia as a living, symbiotic planet. but at the same time a global revolt again endemic-and indeed viralracism has also exploded in this fateful june 2020, led by the "black lives matter" movement. as these multiple crises unfold, the politics of the sexualized, racialized, naturalized others are moving centre stage, pushing old anthtropos off-centre. thirdly, it is important to keep in mind that the binary distinctions between nature and culture, humans and non-human have been foundational for european thought since the enlightenment and that many cultures on earth do not adopt such a partition (gibson, rose, and fincher 2015) . this is the strength of the insights and understandings that can be learned from indigenous epistemologies and cosmologies, postcolonial and decolonial thought and african philosophy. many of them pose a "multinatural" continuum across all species, all of which partake of a distributed idea of humanity. this means that all entities are considered as being endowed with a soul, which assumes a commonly shared human nature that includes the nonhumans. to call this approach "animism," as colonial powers did, is to miss the point, adding to this ignorance an uncommon dose of epistemic violence. when it comes to human/ non-human relations, it is time to start learning from the south. fourthly, some critical self-restraint may be needed. a pandemic on the scale of covid-19, brings home to the western world an ancient truth: that "we" are all in this planetary condition together, whether we are humans or others. but it is also high time for this heterogeneous and collective "we" to move beyond the eurocentric humanistic representational habits that have formatted it. dislodging also the philosophical anthropocentrism they entail and enforce. this shift of perspective inaugurates critical posthuman thought. nowadays we can no longer start uncritically from the centrality of the human-as man and as anthropos-to uphold the old dualities. this acknowledgment, however, does not necessarily throw us into the chaos of nondifferentiation, nor does it awaken the spectre of extinction. it rather points in a different direction, towards some other middle-ground, another milieu, which expressed the awareness that "we"-all living entities-share the same planetary home. yes, we are connected, that is to say ecologically interlinked through the multiple interconnections we share within the nature-culture continuum of our terrestrial milieu. but we differ tremendously in terms of our respective locations and access to social and legal entitlements, technologies, safety, prosperity, and good health services. the posthuman subjects of today's world may be internally fractured, but they are also technologically mediated and globally interlinked. it is important to stress the materially embedded differences in location that separate us but also to stress the shared intimacy with the world that creates a sense of belonging together, within webs of ever-shifting relations. fifth insight: feminist theory is of great assistance to think equality with difference, multiple belongings and power rifts, because it stresses the embodied, embedded, and sexed roots of all material entities, humans included, and their unexplored resources. the relevance of feminist thought in times of crisis is to emphasize the multiple perspectives that emerge from attention to embodiment and lived experience. but is also adds an intersectional approach that stresses the inclusion of axes of analysis such as race, age, class, and able-bodiedness. stressing corporeality, embodiment, and interconnection is one of the strengths of feminist philosophies, which have replaced discriminatory unitary categories, based on eurocentric, masculinist, anthropocentric, and heteronormative assumptions, with robust alternatives. the embedded and embodied empiricism at work in feminist theory acts as the source of counter knowledges, methods, and values. they are needed more than ever today. sixth insight: post/de-colonial and indigenous theories have a great deal to teach us. not only do they stress that for most people on earth, the nature-culture distinction does not hold but also that the fear of death and extinction is an integral part of colonized cultures. for many indigenous people on earth, epidemics, dispossession, and environmental devastations were the mark of the colonial conquests and of the europeans' appropriation and destruction of first nations cultures. catastrophes on this scale are for many people on earth an everyday reality-whether we think of climate change, inter-generational transmission, or public health issues. europeans have a lot to answer for. in light of these insights, i would reach a preliminary conclusion that we need to be lucid about the affects involved in our current predicament and relativize them a bit as well. we need to resist with equal lucidity the pull of apocalyptic thinking as well as the abyss of selfpity: this is a time to organize, not to agonize. the current crisis can make us more intelligent about what we are ceasing to be and who we are capable of becoming. it enables subtler and more complex cartographies of powers and discourses at work in our societies, that is to say a more adequate rendition of where we are at (braidotti and hlavajova 2018) . these accounts have to start by questioning who "we" might be to begin with and whose anxiety is taking centre-stage in public debates about the crisis. accepting our shared exposure to environmental and public health man-made risks is the starting point for a process of assessing these risks and dealing with them collectively and socially. this approach expresses a sort of epistemological humility that reiterates the neverending nature of the processes of becoming-humans. an adequate response to a crisis on the scale of covid-19 calls for community-based, experiments to see how and how fast we can transform the way we live. that means facing up to the negative conditions, the social and environmental inequalities and the collective responsibility towards exposed or vulnerable populations. this praxis of forging communal solutions through the confrontation of uncomfortable truths is central to the ethics of affirmative ethics. it is a praxis that promotes action and critical self-knowledge, by working through negativity and pain. this pro-active activism manifests the living beings' shared ability to actualize and potentiate different possibilities. this transformative energy is the core of affirmative ethics, which stresses the inexhaustible potential of all living organisms-humans and non-humans-to generate multiple and yet unexplored interconnections. this is the immanence of a life that can only be coconstructed and jointly articulated in a common world. what is inexhaustible is not some transcendental and abstract notion of life with capital letters but rather the more patient task of socially co-constructing one's life, alongside so many others. just one life, following the formula of the ancient stoics, can only be predicated in a constant, friendly companionship with pain and suffering. this in turn means that ethics is the practice of extracting knowledge and wisdom from the reworking of pain. pushed to the extreme, it brings us face to face with mortality, the extreme manifestation of vulnerability. death is the painful event par excellence, but it is also the event that marks our inscription into the time of our life. we need to make friends with death. at the level of awareness, it is the event that has always already happened, because to be born means to become mortal. as such, it is a strangely impersonal event. death marks the outer boundary of the limited time we have at our disposal. being aware of this limit can be an energizing thought, not a catastrophe. affirmative ethics encourages us to train for making the most of one's powers and capabilities, so as to become the most affirmative possible version of what one could be, through the pain and the acknowledgment of mortality. posthuman resistance must mobilize for the compositions and collective construction of affirmative forms of action and solidarity and can activate their own generative force. many lives today are the object of biopower's thanato-politics, doomed to ethnic cleansing or slaughter, to being killed without their killer being held accountable. think of the refugees dying on the edges of fortress europe or on manus island. we are vulnerable to viruses and other illnesses, to the effects of climate change and other devastations-many of these exposed lives are not human. but our shared life as an inhuman, non-anthropocentric force (which i call zoe)-exceeds these negative conditions, because zoe exists independently of humans. many of us are struck about how, in the middle of this pandemic, spring is advancing, flowers are blooming, the earth keeps on growing-regardless. humans are not the centre of creation. this is the greatness of affirmative thought as a secular, materialist philosophy of becoming. it is an inexhaustible generative force that potentially can transmute lives into sites of resistance-all lives, also the non-human. life is a generative force beneath, below, and beyond what we humans have made of it. zoe/geo/techno-perspectives at the core of this heterogeneous definition of life are sites of resistance. they provide multiple alternatives to the devastations of necropolitics and the entrapment of biopolitical management of life as capital. but what a huge task that is! fatigue, fear, and despair overlap and accumulate to produce a feeling of utter impotence. this closing down of the horizon of possible actions is the symptom of the negativity of our times. negativity expresses itself in a social and psychological dimming of a sense of possibility, which triggers a systemic fragmentation and a shattering of our relational capacity. this weakening of the desire to act often feeds an appeal to external powers to take over the task of organizing how to live our lives. this negativity ultimately brings about a shrinking of our ability to take in and on the world that we are in, simply because it hurts too much to take it in and on. we have to dose how much of it we can take, till it gets too much. too-much-ness is one of the sources of exhaustion, which marks so much of or current predicament. what is inexhaustible, however, is our desire to persevere in living, against all odds. this is the innermost essence or potentia of all living entities: the life in me that does not answer to my name. this vital sense of life is not to be taken for granted or to be sacralized in religious terms. it remains materialist and secular. "just a life" expresses a deep sense of belonging to a common world, the one world we have in common. the desire to get on with it, is the fragile yet irrepressible bond that interconnects all living entities. this produces a roar of energy that is mostly unperceived and imperceptible, yet indispensable. what is inexhaustible is our capacity, our power even, to differ within ourselves, as well as between us. we can extract ourselves from this sad state of affairs, work through the multiple layers of our exhaustion, and co-construct different platforms of becoming. this transformative praxis can only be enacted collectively, together, as transversal subjects of posthuman times. shared exhaustion actually unfolds upon a deeper wisdom about what it is exactly that one knows, when one is facing momentous changes in unfamiliar territories. one knows that life lives on regardless of human pretensions and expectations. "we" can only intervene in this as transversal ensembles, acting collectively: "we"-who-are-not-one-and-the-same-but-are-in-thisconvergence-together. melbourne artist patricia piccinini (2020) made this point clearly with her new public art campaign about the bat-boy are we capable of becoming this kind of posthuman caring hearts? are we prepared to steer this path together? to become a "we"-the missing people? open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. the posthuman posthuman knowledge posthuman glossary manifesto for living in the anthropocene embracing the future. arken useum of modern art and institut fur kulturaustausch publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-289626-8oldaa8i authors: murray, kris a.; olivero, jesús; roche, benjamin; tiedt, sonia; guégan, jean‐francois title: pathogeography: leveraging the biogeography of human infectious diseases for global health management date: 2018-04-19 journal: ecography (cop.) doi: 10.1111/ecog.03625 sha: doc_id: 289626 cord_uid: 8oldaa8i biogeography is an implicit and fundamental component of almost every dimension of modern biology, from natural selection and speciation to invasive species and biodiversity management. however, biogeography has rarely been integrated into human or veterinary medicine nor routinely leveraged for global health management. here we review the theory and application of biogeography to the research and management of human infectious diseases, an integration we refer to as ‘pathogeography’. pathogeography represents a promising framework for understanding and decomposing the spatial distributions, diversity patterns and emergence risks of human infectious diseases into interpretable components of dynamic socio‐ecological systems. analytical tools from biogeography are already helping to improve our understanding of individual infectious disease distributions and the processes that shape them in space and time. at higher levels of organization, biogeographical studies of diseases are rarer but increasing, improving our ability to describe and explain patterns that emerge at the level of disease communities (e.g. co‐occurrence, diversity patterns, biogeographic regionalisation). even in a highly globalized world most human infectious diseases remain constrained in their geographic distributions by ecological barriers to the dispersal or establishment of their causal pathogens, reservoir hosts and/or vectors. these same processes underpin the spatial arrangement of other taxa, such as mammalian biodiversity, providing a strong empirical ‘prior’ with which to assess the potential distributions of infectious diseases when data on their occurrence is unavailable or limited. in the absence of quality data, generalized biogeographic patterns could provide the earliest (and in some cases the only) insights into the potential distributions of many poorly known or emerging, or as‐yet‐unknown, infectious disease risks. encouraging more community ecologists and biogeographers to collaborate with health professionals (and vice versa) has the potential to improve our understanding of infectious disease systems and identify novel management strategies to improve local, global and planetary health. in a globalized world where the spread of infectious diseases appears to ignore all boundaries and the risk of emerging pathogens is on the rise (jones et al. 2008 , fisher et al. 2012 , there has been a resurgence in interest by academics, the general public and both national and international government authorities in the geography of human infectious diseases at all spatial scales. since most endemic and emerging human pathogens utilise non-human animal species at some stage in their transmission cycles (e.g. reservoir and intermediate hosts and vector species) (taylor et al. 2001, woolhouse and gowtage-sequeria 2005) , biogeographers and community ecologists are increasingly involved in this pursuit. their contributions have yielded a range of novel and complementary insights on the spatial and temporal patterns of infectious disease occurrence, emergence and burden, their underlying ecological processes, and their surveillance and management (guernier et al. 2004 , smith et al. 2007 , peterson 2008 , reperant 2010 , johnson et al. 2015 , murray et al. 2015 , stephens et al. 2016b . medical geography has a long and rich history (barrett 2000 , cliff et al. 2000 , rogers et al. 2002 , cliff and haggett 2004 and its methods and objectives have numerous parallels to those of modern biogeography, with its broad aim of determining how multiple processes (e.g. speciation, adaptation, extinction, ecology, geology, climate) interact with one another to produce distributional patterns in the world's biota (myers and giller 1988) . for infectious diseases, this history stretches as far back as to the time of the debate between contagionists and anticontagionists, when experts disagreed about the very existence of infectious disease-causing agents; for example, several 'spot maps' in the context of yellow fever in the us were developed in the late 18th and early 19th centuries to identify patterns and attempt to infer environmental causes for the disease, well before pasteur and koch's eventual and definitive development of the 'germ theory of disease' in the late 1800s (howe 1989 , jones 1990 , lederberg 2000 . however, the largely correlative methods and findings of medical geography seemed to lose ground as modern medicine developed in favour of a relatively narrow focus on molecules, individuals, individual diseases or sub-components thereof, and small and homogenous populations and areas (e.g. cohort studies, randomized-and case-control trials, small area health statistics), in which causality is presumed easier to stalk (schwartz 1994, mclaren and hawe 2005) . as a consequence, and despite the availability of theory and methods in other disciplines to overcome, negate or manage key issues related to correlation and scale complexity (chesson 2012), modern epidemiology has been arguably caught off-guard in a rapidly changing world. so called 'prisoners of the proximate', in reference to a limiting preoccupation with direct individual-level disease risk factors, mcmichael (1999) suggested that modern epidemiologists and public health managers have been sluggish and ill-equipped to recognise, prepare for and proactively respond to some of the most pressing and emerging health challenges of the 21st century, such as climate change, habitat alteration and degradation, biodiversity loss, invasive species including vectors, demographic shifts, migration and epidemiological transitions. although many global health metrics, such as life expectancy and childhood mortality, have nevertheless continued to improve, researchers from a range of disciplines are increasingly forecasting a collision between ongoing improvements in human health and a range of large-scale and accelerating global change processes, particularly those relating to environmental factors and declining environmental quality (foley et al. 2005 , mea 2005 , raudsepp-hearne et al. 2010 , suk and semenza 2011 , costanza et al. 2014 , watts et al. 2015 , whitmee et al. 2015 . health funders are also beginning to recognise these complex risks to human health (e.g. wellcome trust  https:// wellcome.ac.uk/what-we-do/our-work/our-planet-ourhealth , rockefeller foundation  www.rockefellerfoundation.org/our-work/initiatives/planetary-health/ ). given that this type of multi-scale, multi-disciplinary complexity is commonplace in biogeography, and much precedence exists from the study of parasitism, plant and animal diseases and global change ecology, there has never been a better time for biogeographers and ecologists to contribute their knowledge, theory and methods to public and global health. critically, where strong links between human health and the environment are identified and quantified, such collaborations could stimulate novel streams of funding and yield cost-effective co-benefits for health and the environment (myers et al. 2013 , waldron et al. 2017 . this is particularly salient for research on human infectious diseases, most of which involve animal hosts and vectors in shaping their distributions and are therefore influenced by many of the same ecological processes that govern biodiversity patterns more generally (guernier et al. 2004 , murray et al. 2015 . analogous to its utility for understanding wildlife distributions, biodiversity patterns and improving conservation management, biogeography has the potential to improve our understanding of infectious disease distributions, describe and explain patterns and processes underlying pathogen diversity ('pathodiversity') and contribute to infectious disease forecasting, risk management and threat abatement. through the analysis of historical disease, host and/or vector occurrence and co-occurrence patterns, biogeographic approaches could even yield some of the earliest, and in some cases the only, insights into poorly known, burgeoning and future infectious disease risks (murray et al. 2015) . here, we review the building blocks of biogeography and illustrate how it has and could continue to provide novel insights for the study and management of human infectious diseases, an integration we refer to as 'pathogeography' (resurrecting a term coined by plant pathologist israel reichert; reichert and palti 1967) . better understanding of pathogeography among ecologists and improved biogeographic knowledge among veterinary and medical scientists and public health managers should help improve disease surveillance, combat the global burden of human infectious diseases, and improve environmental management. it may even help rebridge a divide that has opened between the medical and ecological sciences, two powerful, explanatory and potentially predictive disciplines that ultimately share common roots in basic scientific inquiry. the overarching objectives of pathogeography, following biogeography (myers and giller 1988) , are to determine how the interactions among varying biotic (e.g. speciation, adaptation, extinction) and abiotic (e.g. topography, geology, climate) factors and processes combine to produce distributional patterns in infectious diseases through time. johnson et al. (2015) and stephens et al. (2016b) recently reviewed the community ecology and macroecology of infectious diseases, respectively, and a natural intersection between these disciplines and the biogeography of infectious diseases occurs where these fields become spatially explicit. macroecology deals with ecological questions that demand large-scale analysis (brown 1995 , brown and lomolino 1998 , cox et al. 2016 . the limits between biogeography and macroecology are fuzzy. they converge when biogeography is focused on the study of how population-or community-level parameters vary along geographic dimensions (lomolino et al. 2006) , or when macroecology deals with spatial patterns (e.g. the geographic distribution of a certain pathogen species or diversity patterns). the divergence occurs when spatially explicit components are not crucial to answering questions being addressed by macroecology (blackburn and gaston 2006) , and when biogeography does not invoke structural and functional patterns of ecological systems (e.g. areography and some evolutionary biogeography approaches) (morrone 2009 ). by contrast, community ecology offers further insights on the mechanisms and processes that bridge fine scale processes of individuals and populations with the ecology and evolutionary processes of species and disease distributions at larger spatial scales (johnson et al. 2015) . whereas biogeography is concerned with the analysis of spatial patterns of biological diversity, pathogeography (i.e. the biogeography of pathogens) is concerned with the analysis of spatial patterns of pathogen (or disease) diversity ('pathodiversity' being the obvious analogue, but also referred to as the 'pathogen pool', 'pathogen community', 'pathosphere' or 'pathobiome'). taylor et al. (2001) constructed the first list of distinct species at a global scale known to be infectious to and capable of causing disease in humans under natural conditions, tallying 1415 species (217 viruses and prions, 538 bacteria and rickettsia, 307 fungi, 66 protozoa and 287 helminths). discovery and recognition of new human pathogens occurs regularly (woolhouse et al. 2008) , with the most recent comprehensive inventory that we are aware of listing 2107 pathogens in humans (274 viruses, 1003 bacteria, 447 fungi, 82 protozoa and 301 helminths) (wardeh et al. 2015) . a comprehensive global database of clinically relevant human infectious diseases, gideon ( www.gideononline. com/ ), tracks more than 350 of these pathogens (berger 2005, victor and edberg 2005) . surprisingly, the distributions of most of these are very poorly known. as recently as 2013, only 7 infectious diseases were considered 'comprehensively mapped' (including old world coltiviruses, plasmodium falciparum and p. vivax, monkey pox, dengue, lassa fever and mayaro) (hay et al. 2013) , fundamentally limiting the ability of public and global health resources to be systematically and efficiently directed towards precise geographic areas and populations at highest risk from other impactful diseases. indeed, distributional patterns of human infectious diseases are generally far more poorly compiled and characterized (e.g. often at only country or regional level and as coarse presence vs absence data) than many plant and animal species, for which numerous global stock takes, status assessments, occurrence databases and detailed distribution maps exist following a long tradition of biogeographic study (wallace 1876 , murray et al. 2015 ) (see also supplementary material appendix 1 table a1 ). however, with the development of big data approaches and curated databases, resources are slowly improving for human infectious diseases (wardeh et al. 2015 , stensgaard et al. 2017 . data may even on occasion be far richer or more geographically precise than for many plant and animal species (especially invasive species) owing, for example, to notifiable disease reporting requirements, such as those in place in eu member states for reporting human cases of certain diseases to ecdc and zoonotic and food-borne diseases to other eu registries (e.g. haemorrhagic cases of dengue and rift valley fever, crimean congo haemorrhagic fever, west nile fever, cholera, campylobacteriosis) (lindgren et al. 2012 ). in addition, databases for specific groups of diseases (e.g. helminths, neglected tropical diseases) and host-pathogen (including human) associations are increasing (e.g.  www.thiswormyworld.org/ ). in global studies that have classified pathogens according to their epidemiological traits, the majority of known human pathogens (58-61%) are classed as zoonotic, defined as diseases and/or pathogens that are naturally transmissible from vertebrate animals to humans, and 14% are arthropod vector-borne (who 1959 , palmer et al. 1998 , taylor et al. 2001 , woolhouse and gowtage-sequeria 2005 . the close association between animals and human pathogens means that the diversity of potentially pathogenic microorganisms that occur in animals including wildlife is also of major interest for human health (morse 1995, murray and daszak 2013) . however, at present, the full dimensions of this broader 'pathogen pool' are almost entirely unknown. for example, estimates of the total number of viruses within just nine target viral families from the first intensively sampled wildlife species (the natural host of nipah virus, fruit bat pteropus giganteus) suggest that the number of known human pathogens is just a tiny fraction of the total viral diversity that occurs in wildlife (anthony et al. 2013 ). the 'operational taxonomic unit' (otu) of pathogeography may differ somewhat from conventional otus in biogeography (e.g. genes, species). while infectious diseases are all caused by specific pathogenic microbial species (which could, or perhaps should, themselves be the relevant otu), it is their infection/presence in human and in some cases livestock or wild animal hosts or vectors that is of primary interest to health stakeholders and the usual target of surveillance programs. 'occurrence' is the presence of a disease or its causative agent in a particular place at a particular time. this can in turn be represented as a presence (i.e. in a human host or a specific geographic unit) or some measure of relative abundance either within single hosts (e.g. the infection load, particularly for macroparasites, such as helminths), within a defined geographic area (e.g. density), time period (incidence) or within the host population (e.g. prevalence). it might also be represented to reflect the process of transmission itself (e.g. 'force of infection'). with some information on the average impact of infections in humans, public health practitioners often also describe spatial and temporal patterns of disease in terms of 'burden', with various available measures that broadly seek to capture the loss of healthy life (e.g. death and disability) attributable to the presence of certain diseases within a population (e.g. disability adjusted life years, dalys) . conversely, the absence of disease is of equal interest for pattern and process analysis, but harder to obtain given the sampling difficulties of asserting freedom from disease (cameron 2012 ). furthermore, occurrence records (and its derivatives) will generally be a subset of actual occurrences, because in most cases they will be heavily influenced by observation effort. given the diversity of health studies, all of these epidemiological metrics could be potentially relevant for pathogeographic analysis. the same units of measure (occurrence, abundance, density) for known or potential hosts and vector species are also relevant and will already be familiar to ecologists. some estimates of disease 'risk' (or, perhaps more accurately, 'hazards') are based on these (e.g. tick nymphal abundance as a measure of lyme disease risk) and their ecologies may contribute directly or indirectly to disease patterns (civitello et al. 2015) , such that data on their potential occurrence can improve biogeographically-informed risk mapping of disease outcomes in humans (messina et al. 2015 , pigott et al. 2016 , olivero et al. 2017a . one major challenge for biogeographic analyses of human infectious diseases, however, is the availability and utility of appropriate data, which we discuss further in box 1. supplementary material appendix 1 table a1 provides some example data types, and some useful databases and sources relevant to biogeographic analyses of human infectious diseases. the study of infectious disease spatial distributions is not new (barrett 2000) , nor are integrated approaches to studying infectious disease distributions, whether they are labeled biogeographic (peterson 2008) or not (lambin et al. 2010) . indeed, the emergence of informatics, geographic information systems (gis) and satellite technology has increased the availability of tools and high quality spatial datasets relevant to both ecologists and medical geographers, driving a recent convergence in the data and in some cases the methods used to examine the distributions of wild species and infectious diseases alike and for developing or evaluating causal hypotheses underpinning them (hay et al. 2006 , kraemer et al. 2016 . such developments have facilitated the study of the spatial structure of some infectious diseases in unprecedented detail ( fig. 1) . in some cases, there has been innovation in the integration of these approaches with mechanistic models traditionally used to explore the population dynamics of infectious diseases (redding et al. 2016) , and some analyses have been developed and updated in close to real time (e.g. during the west african ebola outbreak) (pigott et al. 2014 (pigott et al. , 2016 . these advances complement an already strong suite of tools already used in epidemiological studies, which might equally flow the other way into the toolboxes of ecologists (magalhães et al. 2011 , caprarelli and fletcher 2014 , bhatt et al. 2017 . peterson (2008) defined the first explicit 'biogeographic framework' for human infectious diseases. drawing on the work of soberón (2007) on the grinnellian niche and geographic distributions of species, the framework is characterized by considerations of a pathogen's dispersal ability combined with the abiotic and biotic factors that interact to determine whether a disease is able to fulfill its full geographic potential. although not explicitly demonstrated, peterson (2008) emphasizes that a key difference between pathogens and diseases in comparison to free ranging wild species (with the exception of invasive species) is the relative unpredictability of dispersal events (e.g. rapid, long distance introductions), and the relatively lower importance of abiotic and relatively higher importance of biotic factors. this applies particularly to the high degree of inter-specific interactions, from the infection of a pathogen in a host, to the numerous other species that may be involved in pathogen transmission cycles. the distribution of an infectious disease is thus defined by the joint distributions of all species involved in its transmission cycle as dictated by the suitable ecological conditions and dispersal limitations for each. in contrast, lambin et al. (2010) present an alternative framework for understanding generalized disease risks across landscapes, drawing on principals from 'spatial epidemiology' (ostfeld et al. 2005) and 'landscape epidemiology' (pavlovsky 1966) . pavlovsky (1966) proposed that infectious disease occurrence is determined by 'a continuous interaction' of five landscape factors: 1) animal 'donors' (e.g. reservoir hosts), 2) vectors, 3) animal 'recipients' (including humans), 4) an infective pathogen, and 5) environmental factors that are conducive to transmission. lambin et al. (2010) emphasise the use of biogeographical methods for examining the distributions of human infectious diseases will be dependent on the degree of existing knowledge (e.g. about the epidemiology of a particular pathogen or pathogen assemblage in a particular geographic context), data availability (e.g. from publicly accessible databases, surveillance data) and the potential for new data collection (e.g. targeted field collections). these elements could range from no knowledge, no data and large barriers to the collection of new data (e.g. for many emerging infections in developing country contexts, such as at the beginning of the 2014 ebola outbreak in west africa), to high degree of knowledge from existing scientific studies, well developed and accessible databases on relevant geographic/environmental, reservoir host, vector, pathogen, human and disease data, and existing structures to streamline the collection of new data (e.g. high impact diseases in developed country contexts, such as malaria in the eu). data on human infectious diseases are normally collected for the needs of a particular discipline or research focus, which may typically limit the extent to which it can be used laterally or opportunistically for answering non-target questions. this may be particularly the case for macroecological and biogeographical studies, which are often data intensive and conducted at scales that may be difficult or impractical to undertake new data collection. there is thus a clear need to expand the scope of research programmes on infectious diseases to encompass the geographic, biological and temporal scales relevant to biogeographical analysis. this involves informing monitoring and surveillance activities on what types of data would be most useful and urgent. improving data capture and quality with standardized sampling methodologies could help lead to analyses and discoveries that transcend the specific epidemiological details of a single site, geographical context or disease. to this end, the following information would be helpful to allow coherent data collection and analysis of infectious disease occurrence and transmission in space and time: • operational taxonomic units: potential complications for biogeographic pattern and process analysis may arise when the 'presence' of a specific disease type is in fact confounded. this could occur, for example, if multiple causal agents result in disease complexes (e.g. leishmaniasis) or if the causal agent is unknown and diseases are instead classified by their symptoms (e.g. syndromes). distributional data and databases on infectious diseases should thus strive for the highest 'taxonomic resolution' possible (murray et al. 2015 , stensgaard et al. 2017 ). • observation effort: a persistent issue in robustly quantifying occurrence, in any form, is its relationship to observation effort (allen et al. 2017) . observation effort could vary spatially, temporally or taxonomically. presence, prevalence, burden and diversity patterns of infectious diseases, hosts and vectors may all increase proportionally to observation effort, and confidence surrounding reported absences also increases with observation effort. failing to account for this has the potential to introduce biases in biogeographic studies. numerous studies have taken steps to incorporate measures of observation effort to limit the potential effects of observation bias in biogeographic studies of infectious diseases, typically by including factors hypothesised to be related to observation effort, such as sampling intensity, gdp, health expenditure or publication output, in statistical models (jones et al. 2008 , hopkins and nunn 2010 , yang et al. 2012 , murray et al. 2015 ) (see also o in box 2). • sampling protocols: a lack of communication between biostatisticians and field workers in both ecological and health fields before collecting samples can lead to a breakdown in robustness of subsequent analyses. for example, sampling too many host individuals of one species could be as problematic as not sampling enough from a target host (e.g. humans), particularly when research resources are constrained. best practices involving probability sampling should be pursued where possible (nusser et al. 2008 ). • geographic coverage and resolution: sampling should cover a sufficiently large area(s) to reproduce in space what really exists in the field; for example, metapopulation or metacommunity geographic distributions with sources and sinks of pathogen transmission. the effects of uneven sampling across space and one-shot sampling should be avoided (peterson 2014) . at the other extreme, improving precision on available data points is a high priority for developing robust geo-referenced databases of disease (or pathogen, host, vector) occurrence. all data should be collected and stored for subsequent access at the highest spatial resolution possible (i.e. gps coordinate locations). • temporal coverage: infected hosts including humans may travel long distances during disease incubation periods, introducing uncertainty in the attribution of the geographic location of infection (peterson 2014) . surveillance and sampling strategies should thus allow the capture of appropriate temporal scales (i.e. matching scale of disease-relevant processes) so that data can be better aligned with covariate information such as environmental variables and human activities. • phenology: animal and plant phenology should be monitored; the behavior and biology of most species, including humans, are influenced by often relatively predictable annual changes in climate that determine when they start or finish natural events, such as flowering and fruiting, breeding and mass gatherings. many of these activities and departures from norms due to, for example, unusual weather events have implications for disease risks and spatio-temporal distributions, and could have large implications when considering the influences of large scale change (e.g. climate change). • humans as hosts and dispersers: humans will often serve not only as hosts but also as effective dispersal mechanisms for infectious agents. although this can lead to unpredictability and extreme long distance invasion events (peterson 2008) , data on human populations and their movement are being increasingly well resolved at both fine and coarse spatial scales and this is proving invaluable in decomposing biogeographical components of many infectious disease systems including emergence risks (e.g. use of flight or road traffic data, mobile phone use data, social media) (colizza et al. 2007 , balcan et al. 2009 , wesolowski et al. 2012 , jurdak et al. 2015 ). • concurrent covariate sampling and time-lagging: ideally, assessment of environmental and social variables should be conducted at the same time as human or wildlife disease sampling (if it is not available at the appropriate scale retrospectively i.e. from remote sensing data). peterson (2014) discusses a range of issues relevant to modeling the distribution of infectious diseases, including quality control of input occurrence data, sampling design with the reduction of oversampling in certain areas, and design of analysis (see also hosseini et al. 2017 ). the dynamic nature of the spatial and temporal interactions between these 'prerequisites' at multiple scales when assessing the impact of landscape changes on vector-borne and zoonotic diseases. although they did not identify their study explicitly as 'biogeographic', there are clear parallels with peterson's (2008) framework (as well as many others, such as plowright et al.'s (2017) recent treatment of spillover ecology). we develop these ideas further in box 2, focussing on the interactions between a number of inter-dependent elements, including: physical geography (g), environment (e), reservoir host(s) (r), vector(s) (v), pathogen(s) (p), human factors (h), and the management (m) and observation (o) landscapes, the latter serving as the lens through which all other elements and disease distributions (d) are ultimately observed. from this framework, we can envisage how these elements may act and interact to influence both the real and observed distributions of specific diseases in space and through time, as well as multidisease patterns that may emerge at higher levels of organization (see 'emergent patterns and multiple diseases' below). depending on epidemiological characteristics, not all of the elements illustrated in box 2 will be relevant for all human infectious diseases, resulting in a range of 'biogeographic complexity' among human infectious diseases. whereas g and e will almost always have some kind of modifying influences, single-element transmission source systems (i.e. diseases only involving human-human, environmental, single reservoir species or single vector species transmission) represent the least biogeographically complex examples in this framework. examples include tetanus (e), measles (h), and lassa fever (r). the more biogeographically complex diseases involve multiple elements; for example, multiple reservoir host or vector species (r n and v n , respectively) in addition to human-human or environmental transmission (e.g. dengue (hv n r n ); infection with mycobacterium ulcerans (ev n r n )). we describe the potential utility and demonstrate an application of this general framework further in box 3, by undertaking a 'disease trait profiling' exercise whereby we classify a large number of clinically relevant human infectious diseases according to their transmission sources (i.e. the e, h, v and r elements described in box 2). the trends that emerge illustrate how certain disease characteristics are far more common than others (e.g. transmission from animal reservoirs vs arthropod vectors) and the extent to which mapping efforts are already underway. however, the analysis also highlights some important gaps. for instance, 31.9% of diseases with a strong rationale for mapping (as rated by hay et al. 2013) have not been mapped in any study (box 3, fig. panel f) , and declining overall quality of mapping efforts correlates with increasing biogeographic complexity (box 3, fig. panel g) , highlighting a need to expand the breadth of ecological interactions considered within disease systems to improve mapping quality in future studies. an additional consideration not explicitly included in box 2 is that changes in interactions through time can influence the occurrence, transmission and emergence of many diseases. for example, short or longer term changes in land-use and climate (both components of e) can influence d directly or through their influences on v, r and/or h (epstein 2001 , patz et al. 2004 , nakazawa et al. 2007 , jones et al. 2013 , hoberg and brooks 2015 , mackenstedt et al. 2015 . ebola virus disease (evd) outbreaks, for example, have been closely associated with inter-annual anomalies in meteorological seasonality, whereby sharply drier conditions at the end of the rainy season seem to favour the occurrence of outbreaks (pinzon et al. 2004 ). more recently, ebola outbreaks have been linked to fragmentation (rulli et al. 2017 ) and recent ( 2 yr) deforestation (olivero et al. 2017b ). a promising approach emerging from ecology warranting additional attention here is the increasingly widespread use of species distribution modelling (sdm) (also called ecological niche modelling (enm)). when applied to pathogens, hosts and/or reservoirs and vectors, sdm is useful for understanding risk factors conditioning the distributions, emergence or the accumulation of new outbreaks of disease. in sdms, the available information on the presence or the incidence of disease is linked to a diverse set of environmental variables and allows the evaluation of the degree to which certain environments are favourable for the occurrence of disease, even in areas where it has not been detected before. in the absence of, or in combination with, local-scale data suitable for (pigott et al. 2016) . illustrating the detail of modern cartographic projections of disease risk based on models of environmental suitability for the zoonotic transmission of ebola virus (shaded colours) and the spatial variation in disease risk that would be masked from country-level chloropleths (thick black lines). dotted lines indicate regions that have reported no index cases to date but are predicted to be partially at risk based on environmental suitability models that utilize a thresholding approach on the model output that captures 95% of the occurrence records used for model fitting (see also fig. 6 ). boosted regression trees were used to develop the model based on georeferenced records of ebola virus disease outbreaks in humans and infection records in bat reservoirs and a range of environmental covariates. the scale reflects the relative probability that zoonotic transmission of ebola virus could occur at these locations; areas closer to 1 (dark red) are more environmentally similar to locations reporting ebola virus occurrences; areas closer to 0 (light yellow) are least similar. mechanistic models, sdms can take advantage of the large geographic scale to explore macroecological processes that are able to explain and predict the occurrence of disease. this can reveal emergent patterns and processes not perceptible at the local scale (brown 1995) , and can help lay a foundation for hypothesis testing or provide a geographic context for further studies on the ground (allen et al. 2017) . the outputs provided by sdms can be summarized in three main categories: probability (e.g. logistic regression, generalized additive models, random forests, boosted regression trees), suitability (e.g. maxent, garp) and favourability (e.g. favourability function). suitability is an idiosyncratic way of ranking local sites according to their capacity to hold the species or pathogen that is not directly related to the probability of occurrence (guisan and zimmermann 2000, royle et al. 2012) . in contrast, favourability values can be obtained from probability and prevalence (here defined as the proportion of presences in the set of observations) (real et al. 2006 ). probability and suitability are biased in their outputs when working with samples differing in prevalence, which is not the case with favourability (acevedo and real 2012 ). this quality of favourability enables direct comparison and combination we may represent the challenge of simultaneously understanding patterns and processes of infectious disease systems with respect to a series of interacting elements; including g, the physical geography context (e.g. topography) and e, the abiotic (e.g. climate) and biotic (e.g. habitat) environment; r n and v n , the single or multiple (denoted by superscript n) species of reservoir hosts or vectors; p, the pathogen being transmitted; h, the human population itself; o, the observation effort that may apply to each of the other elements (e.g. surveillance and data collation from existing sources); and m, the management landscape (e.g. interventions). the combinations of these elements ultimately give rise to d, the observed disease distributions. where these elements have consistent effects across multiple diseases, 'higher order' biogeographic patterns emerge; for example, we can observe biogeographic regionalization as a consequence of the more pervasive effects of components of g (e.g. ocean or mountain barriers to dispersal) or e (e.g. unsuitable climates), while the effects of components of h (e.g. population growth, global movement, socio-economic status, immunity heterogeneity) and m (e.g. health infrastructure, vaccination) have fundamentally reshaped the global diversity and burden patterns of infectious diseases. in addition, each layer potentially has additional modifying elements that could further shape disease distributions and diversity patterns, such as temporal fluxes (see main text), or lifehistory and epidemiological traits of hosts, vectors and pathogens (smith et al. 2007 , woolhouse et al. 2016 . for pathogeographic analyses and as a starting point for risk assessments, a series of 'profiling' steps could help integrate existing information at a scale relevant to a particular research question. this could include geographic and/or environmental profiling (e.g. detailed assessments of g and e for diseases/hosts/vectors in regions of interest relevant to single diseases or disease assemblages) (eisenberg et al. 2007 ), disease trait profiling (classifying epidemiological features of diseases, such as the presence or absence of the e, v, r and h elements as sources of pathogen transmission (see box 3), and the species known to be implicated in each), and human profiling (assessments of the human population distribution, density and movement patterns and the existing management and observation landscapes). intersection of these geographic, disease trait or human profiles will ultimately yield yet deeper understanding or management relevant insights (semenza et al. 2016). when several species are involved in the analytical design; for example, when using models for deriving indices based on multiple species (estrada et al. 2008) , and for the study of biogeographical relationships between species (real et al. 2009 , acevedo et al. 2010 , including relationships between pathogen and host complexes (olivero et al. 2017a) . sdm approaches represent one of the major recent advances in infectious disease cartography (hay et al. 2013 , peterson 2014 , kraemer et al. 2016 , producing a diverse range of predictions on the presence or risk of human infectious diseases or their causative organisms (peterson et al. 2004 , peterson 2006 , neerinckx et al. 2008 , reed et al. 2008 , samy et al. 2014 , zhu and peterson 2014 , and their animal hosts and vectors (sweeney et al. 2006 , de oliveira et al. 2013 , giles et al. 2014 . modelling the distribution of biotic interactions is a relatively recent advance in macroecology and biogeography (kissling et al. 2012 , wisz et al. 2013 , ovaskainen et al. 2016 ) and could similarly provide a further way forward for the analysis of pathogens with zoonotic cycles based on joint distributions and multispecies assemblages. other methods of representing potential interactions at a community level (e.g. network modeling) or inferring potential hosts/vectors and host/ vector ranges from geographic co-occurrence are similarly being developed alongside (or in some cases integrated with) niche modeling approaches to yield novel insights disease trait profiling could help synthesize and summarize the range of potential ecological interactions of diseases and highlight important priorities or gaps for biogeographic analyses. to illustrate, we classified all clinically relevant diseases within the gideon database with single causative pathogens (n  186 diseases) into each of the combinations possible considering whether disease transmission sources included the e, h, v and r elements outlined in box 2 (note, although h is by definition always involved for human infectious diseases, here it is treated more specifically as a transmission source i.e. human-human transmission). we then examined how variations in the complexity of diseases, as indicated by the number of elements involved in their transmission (complexity score, cs), was related to geographic range size (as broadly indicated by the number of countries in which the disease is present), the rationale for mapping, whether mapping efforts had already taken place, and the quality of existing mapping as rated by hay et al. (2013) . consistent with other studies, 71.0% of diseases in our dataset involved (but do not necessarily require) transmission to humans from animal reservoirs (zoonotic) (fig. a) . human-human transmission was also common (40.3%), while diseases including potential transmission from vectors (31.7%) or environmental sources (21.5%) were less common. for diseases involving vectors or reservoirs, the great majority involved multiple vector (95%) or reservoir (85%) species rather than single species (fig. a) . diseases ranged considerably in their degree of 'biogeographic complexity', but only 15 of 35 possible combinations were represented in our dataset (fig. b) . the least complex examples involved transmission from the environment (e) only, a single reservoir (r) only, or humanhuman (h) only (cs  1), while the most biogeographically complex diseases involved multiple vectors, reservoirs and could include either environmental (evnrn) or human-human (hvnrn) transmission as well (cs  8). on average, diseases including humanhuman transmission were the least biogeographically complex ('h' mean cs  3.0), followed by diseases including transmission from reservoirs ('r' cs  4.0), while diseases involving transmission from the environment ('e' mean cs  4.3) and vectors ('v' mean cs  5.2) were more complex. on average, complexity was not obviously related to commonness (fig. c) , but more complex diseases tended to be more geographically restricted (fig. d) , and had both a stronger rationale for mapping (fig. e) as well as a higher proportion of diseases that had already been mapped (fig. f) , particularly when human-human or environmental transmission were never involved (traits that can radically increase their distributions to the point of making them essentially ubiquitous). however, the quality ratings of existing mapping efforts for more complex diseases were considerably lower on average than for simpler diseases (fig. g) . these trends illustrate the extent to which mapping efforts are already underway for clinically relevant infectious diseases but also highlight some important gaps. for instance, as of hay et al.'s (2013) study, 31.9% of diseases with a strong rationale for mapping had not been mapped in any study (fig. f) , and declining overall quality of mapping efforts for more restricted and biogeographically complex diseases highlights a clear need to address the breadth of ecological interactions within disease systems in future studies. on the spatial distribution of some infectious diseases (stephens et al. 2009 (stephens et al. , 2016a . returning to the framework in box 2, g, e together with time (t) could each have more pervasive effects through their simultaneous influences on the other elements, giving rise to higher-order biogeographic patterns that are defined by multiple diseases, such as co-occurrences (including co-infection patterns), chorotypes, diversity gradients, or biogeographic regionalisation. while biogeography has already made significant contributions to providing a generalized framework for disease mapping (peterson 2014, escobar and craft 2016) , we emphasise that it is the comparative power of biogeography that could help take pathogeography a step further, diverging more radically from medical geography, to address the factors that govern the structure, assembly, dynamics and spatial patterns of multiple or entire assemblages of human infectious diseases over a more diverse range of spatial and temporal scales. below we provide some illustrative examples relevant to what has or potentially could be applied to human infectious diseases. a chorotype is a type of distribution pattern that is followed by one or several species (baroni urbani et al. 1978 , real et al. 2008 , passalacqua 2015 . as chorotypes represent the shared geographical, ecological and evolutionary context of several species (real et al. 2008 , fattorini 2015 , these patterns could be useful for generating hypotheses about the causes and origins of host, reservoir and pathogen distributions. although not yet widely explored for infectious diseases, chorotype analysis could significantly contribute to the study of the complex interactions characteristic of human infectious disease systems (peterson 2008 , roche et al. 2013 . in the analysis of disease distributions, the relative importance of these interactions, compared to the relevance of other environmental factors, is variable. some studies have raised this issue through the comparative analysis of pathogen models and host models based on their respective responses to environmental conditions (maher et al. 2010, costa and peterson 2012) . however, with the exception of using host distributions as a simple proxy for the potential distributions of pathogens (daszak et al. 2012) , the distribution of reservoir species has only recently been used as an explanatory variable, together with other environmental descriptors, to define a pathogen distribution model (e.g. compare peterson et al. walsh and haseeb, and pigott et al.' s models for ebola virus) (peterson et al. 2004 , walsh and haseeb 2015 , pigott et al. 2016 . although we are aware of no studies examining chorotypes of human infectious diseases, mammalian chorotypes have been recently incorporated into an infectious disease distribution model (olivero et al. 2017a) . when the ecology of a pathogen is complex and unresolved (e.g. ebola virus, leroy et al. 2004 , groseth et al. 2007 , olival and hayman 2014 , imposing restrictions to the selection of host or vector species considered in a model might under-represent the zoological substrate conditioning a pathogen's transmission and distribution (roche et al. 2012) . olivero et al. (2017a) thus addressed the mapping of favourable areas for the ebola virus in the wild by combining two biogeographical approaches: sdm and chorotype analysis. mammalian chorotypes in africa were employed as surrogates of the types of distributions shown by reservoirs and any wildlife species implicated in the virus spillover cycle. olivero et al. (2017a) found that a model based on a number of diversity patterns, each one associated with a different mammalian chorotype, defined favourable areas for the presence of ebola virus with higher accuracy than did a model based on environmental variables alone (i.e. climate, forest type), concluding that mammalian biogeography contributes significantly to explaining the distribution of ebola virus in africa. in addition, vegetation was identified as a factor placing clear limits to the presence of the virus. favourable areas for ebola virus were thus determined from information provided by both models (fig. 2) . it is now widely recognized that multiple pathogens may act independently or interact through a variety of different mechanisms to influence disease outcomes in human populations (pederson and fenton 2006 , jolles et al. 2008 , scholthof 2011 , and yet studies of human infectious disease, host and vector diversity or community assembly patterns are rare in comparison to distributional studies of single infectious diseases (see 'single diseases' above). characterizing diversity patterns can thus provide a range of insights on the distributions and processes underlying multiple species of pathogens or diseases. based on components first proposed by whittaker (1960) , inventory diversity quantifies diversity within an environment, where alpha (α) diversity is used to refer to diversity at the local scale (i.e. smallest scale being measured). α-diversity of human infectious diseases has been analysed in a number of studies, typically by comparing the number of diseases occurring in different countries at a global or continental scale due to limited comparative data availability at higher spatial resolutions. although strongly heterogeneous (fig. 3 ) (see also stensgaard et al. 2017) , some striking patterns suggest that human infectious disease communities are shaped by the same ecological processes that shape the diversity of life more generally (guernier et al. 2004) . this is evident, for example, from observations of a clear latitudinal gradient in disease diversity and disease range sizes, whereby disease richness decreases and range size increases towards the poles (fig. 4) (guernier et al. 2004, guernier and guégan 2009) . other patterns are consistent with island biogeography theory, such as a positive relationship between land surface area and disease richness (smith and guégan 2010) , and reduced richness on smaller islands and with distance to the nearest mainland (jean et al. 2016 ). together, these findings likely explain why the strongest predictor of human infectious disease richness known to date is wildlife richness (dunn et al. 2010) , while some vector groups show similar patterns (foley et al. 2007 ). these patterns also illustrate that a correlation between human disease diversity and wildlife/ vector diversity may not necessarily imply direct causation. nevertheless, other evidence points to the importance of animals, particularly mammalian and bird wildlife, as a key source of endemic and emerging human pathogens (taylor et al. 2001 , woolhouse and gowtage-sequeria 2005 , jones et al. 2008 , allen et al. 2017 . in addition to the causal links, such parallels in patterns of human disease and other taxa reinforce the importance and potential utility of considering wildlife and vector biogeography alongside or as a central component of studies of infectious diseases, including distributional and diversity studies of known or potential zoonotic hosts and vectors (foley et al. 2007 , cooper and nunn 2013 , han et al. 2016 , olivero et al. 2017a . in contrast to inventory diversity, proportional diversity measures the difference in diversity between environments or across gradients of habitats, commonly expressed as beta (β) diversity (whittaker 1960 , jost 2007 . β-diversity patterns can be expressed in a number of ways, such as biogeographic regionalisations, which define biotic boundaries according to between-area gaps in species composition, and biotic regions based on biotic similarities (olivero et al. 2013) . such approaches, however, are yet to be widely applied to human infectious diseases. in one study, β-diversity patterns of human infectious diseases appear to parallel patterns in other taxa, consistent with patterns identified to date for α-diversity (richness); murray et al. (2015) show that human infectious disease assemblages exhibit biogeographic regionalisation reminiscent of zoogeographic patterns, particularly for zoonotic (fig. 5a, b) , vector-borne and parasitic diseases, and that mammalian assemblage similarity is consistently among the strongest predictors of human infectious disease assemblage similarity among countries (fig. 5c ). such an effect is very likely predictive of as-yet undescribed patterns of microbial diversity, such as the geographic structure recently demonstrated among novel coronaviruses detected in wild bat hosts (anthony et al. 2017) . in addition to this dominant explanatory effect of biodiversity, other factors, including environmental (climate, land area, population size), social (human connectivity, health expenditure, observation effort) and epidemiological characteristics (e.g. pathogen type, transmission mode) also affect infectious disease αand β-diversity patterns (fig. 5c ). the strongest β-diversity patterns, for example, can be observed in zoonotic, vector-borne and parasitic infectious diseases, likely due to a more dominant role of environmental factors and persistence of historical dispersal barriers limiting their geographic distributions, while patterns of humanspecific diseases are far more homogenous at the global scale (smith et al. 2007 , dunn et al. 2010 , just et al. 2014 , murray et al. 2015 , jean et al. 2016 ) (see also box 3 fig. panel d) . guernier et al. (2004) ); (c) nestedness: a hierarchical pattern of human pathogen composition with the pathogen species found at higher latitudes (darker bars) constituting nested subsets of those in progressively richer communities at lower latitudes (lighter bars) (adapted from guernier et al. (2004) ); (d) disease range size: narrower distributional ranges occur in the tropics (darker circles) for human pathogens compared to higher latitudes (lighter bars) (adapted from guernier and guégan (2009) ). β-diversity can be further decomposed into two separable components, nestedness and turnover, which may further help characterize the processes driving differences in the composition of assemblages between sites (harrison et al. 1992 , baselga 2010 . nestedness occurs where species assemblages are subsets of the biotas at more diverse sites (wright and reeves 1992, ulrich and gotelli 2007) , and indicates a non-random process arising from any factor that promotes the orderly disaggregation of assemblages (gaston et al. 2000) . the latitudinal gradient of human infectious diseases exhibits such a pattern, with disease assemblages occurring at higher latitudes being subsets of those occurring closer to the equator (fig. 4c ) (guernier et al. 2004 ). in contrast, turnover indicates the replacement of some species by others as a consequence of environmental sorting or spatial and historical constraints (qian et al. 2005 , baselga 2010 ). in the only assessment of nestedness vs turnover undertaken so far for human infectious diseases that we are aware of, both nestedness and turnover appear to contribute to overall differences in infectious disease assemblages among countries at a global scale, with the relative contribution varying between major epidemiological classes (murray et al. 2015) . for example, nestedness dominates differences in human-specific diseases, whereas turnover dominates differences in zoonotic and vector-borne diseases. biogeographic methods and outputs have already contributed and continue to show great promise for a number of health management or research applications on infectious diseases, which could help direct the allocation of scarce public and global health resources more efficiently and effectively. as our abilities to assemble ecological datasets and conduct infectious disease surveillance and analyses are steadily improving, multidimensional ecological data can be mapped and relationships can be identified as data accumulate in close to real-time, providing decision-relevant information for health managers and researchers to respond to. this has already lead to rapid advances in improving disease mapping for single infectious diseases and a closing of the gap between the data types and methods used to characterise disease and species distributions by medical geographers and ecologists, respectively. many other applications are conceivable albeit so far poorly explored. for example, taking inspiration from conservation and ecological applications, diversity analyses and biogeographic regionalisation could be used to test and propose hypotheses about ecological factors and historical events that could underlie the current organization of disease assemblages or . colours represent statistically supported groups (n  11 groups) of countries that share similar diseases, as derived by evaluating results from the silhouette, elbow, ch index and gap statistic tests; (b) global pathogeographic realms for zoonotic diseases derived from (a) (colours for mapping match country clusters identified in (a)). legend labels indicate the statistically supported regions (first column) and how these align with 'classic' zoogeographic realms (second column) (note although the realm label for nearctic includes greenland for illustration purposes, the 'islands' group (dark blue) actually includes a large number of small islands plus a few other countries scattered globally (a) that may cluster on the basis of being a depauperate or data deficient group); (c) the relative explanatory value of a range of social and environmental covariates for explaining these global patterns in disease beta diversity, illustrating that mammalian biodiversity is the best predictor of zoonotic disease diversity at a global scale (as derived from a relative importance analysis following multiple regression on distance matrices controlling for the effects of spatial autocorrelation (following murray et al. (2015) ). combined disease risks (e.g. identifying processes of disease dispersal, establishment, and extinction and the 'upstream' risk factors or drivers of novel health threats) (following carmona et al. 2000 , báez et al. 2005 ; to define contexts for representativeness (e.g. improved disease surveillance design) (following austin and margules 1986 , carey et al. 1995 , mackey 2008 ; to provide consistent units for environmental management and for sampling stratification (e.g. for the optimal discovery of novel pathogens) (following bunce et al. 1996 , wright et al. 1998 ; and as geographic contexts for imputation/extrapolation or forecasting when data from a unit within a region is missing or unavailable (e.g. where disease surveillance coverage is low or patchy) (cooper and nunn 2013) . biogeographic approaches may also be useful for examining the risks associated with emerging infectious diseases (eids), since data are often extremely limited on eids and yet the priorities for management revolve around anticipating (through forecasting) when, where and why emergence of pathogens in human populations occurs (peterson 2008 , morse et al. 2012 . large-scale demographic and environmental factors and changes in these factors are increasingly being recognized as key drivers underlying disease emergence, with shifts in the distributions of disease hosts and vectors being central to this process (jones et al. 2008 , semenza et al. 2016 . given that most pathogen distributions are very poorly characterized or completely unknown (hay et al. 2013) , and that most of the microbial diversity from which novel and potentially pathogenic agents could originate are as yet undiscovered (anthony et al. 2013) , biogeographic pattern definition and process identification based on historical patterns of disease occurrence, recent emergence events, or proxy taxa (e.g. mammalian or arthropod vector biodiversity) may provide some of the earliest and in some cases the only insights into such burgeoning or future disease risks (fig. 6a , b) (murray et al. 2015) , which may give way to more refined models as data quality or availability increases (fig. 6c, fig. 1 ). increasing pathogeographic awareness, participation and collaboration among ecologists, biogeographers and veterinary and medical practitioners could thus contribute to closing the gap between environmental and health management, increased inter-disciplinary research and management efficiency, and reductions in the global burden of disease. data available from the dryad digital repository:  http:// dx.doi.org/10.5061/dryad.p1n10dv  (murray et al. 2018) . fig. 1 for raw model output and description). yellow indicates countries that contain some environmentally suitable areas for ebola. darker colour indicates countries that do not contain areas predicted to be suitable for ebola. the overlap in top 22 priority countries between (b) and (c) is ~70%. the approach taken in (a/b) requires no specific 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discovery of human viruses assessing the epidemic potential of rna and dna viruses. -emerg host range and emerging and reemerging pathogens. -emerg on the meaning and measurement of nestedness of species assemblages ecoregions as a level of ecological analysis global distribution of outbreaks of water-associated infectious diseases potential geographic distribution of the novel avian-origin influenza a (h7n9) virus supplementary material (appendix ecog-03625 at  www. ecography.org/appendix/ecog-03625 ) key: cord-274756-nnm1n09a authors: varadé, jezabel; magadán, susana; gonzález-fernández, áfrica title: human immunology and immunotherapy: main achievements and challenges date: 2020-09-02 journal: cell mol immunol doi: 10.1038/s41423-020-00530-6 sha: doc_id: 274756 cord_uid: nnm1n09a the immune system is a fascinating world of cells, soluble factors, interacting cells, and tissues, all of which are interconnected. the highly complex nature of the immune system makes it difficult to view it as a whole, but researchers are now trying to put all the pieces of the puzzle together to obtain a more complete picture. the development of new specialized equipment and immunological techniques, genetic approaches, animal models, and a long list of monoclonal antibodies, among many other factors, are improving our knowledge of this sophisticated system. the different types of cell subsets, soluble factors, membrane molecules, and cell functionalities are some aspects that we are starting to understand, together with their roles in health, aging, and illness. this knowledge is filling many of the gaps, and in some cases, it has led to changes in our previous assumptions; e.g., adaptive immune cells were previously thought to be unique memory cells until trained innate immunity was observed, and several innate immune cells with features similar to those of cytokine-secreting t cells have been discovered. moreover, we have improved our knowledge not only regarding immune-mediated illnesses and how the immune system works and interacts with other systems and components (such as the microbiome) but also in terms of ways to manipulate this system through immunotherapy. the development of different types of immunotherapies, including vaccines (prophylactic and therapeutic), and the use of pathogens, monoclonal antibodies, recombinant proteins, cytokines, and cellular immunotherapies, are changing the way in which we approach many diseases, especially cancer. the knowledge of human immunology has improved exponentially in recent years, and more advances in the near future are certainly imminent. the immune system is extremely complex, but we are now developing new tools and skills to study it. several factors have been involved in these advancements, and the most important ones include the development of thousands of different monoclonal antibodies that allow the identification of a large variety of cell subpopulations and the functional analysis of immune cells. these tools, together with new and sophisticated technologies, such as single-cell analysis, imaging techniques, omics (including massive dna-rna sequencing, proteomics, and metabolomics data and new tools for processing these data, such as artificial intelligence and machine learning approaches, mathematical modeling, etc.), newly designed animal models (using conventional transgenic/knockout/knock-in mice or new technologies such as crispr-cas9 (clustered regularly interspaced short palindromic repeats-crispr-associated protein 9), are increasing our knowledge about how our immune system functions. the study of the interaction between the immune system and other systems, such as the nervous and endocrine systems or the microbiome, in several illnesses has produced interesting results with important clinical applications. all of these advances can be applied to several immunemediated pathologies, but overall, the success achieved with some types of immunotherapies in recent years is revealing new ways to explore and manipulate the immune system for our benefit. writing a review about human immunology is a significant challenge, but we have attempted to bring together recent knowledge about the immune system, immune-mediated illnesses and types of immunotherapies. the last two decades have witnessed a major revolution in the field of immunology. the traditional classification of the immune system into two different arms, namely, innate and adaptive components that collaborate to respond to foreign antigens or to perform self-/nonself-discrimination, has become much more complex. the development and application of new technologies have provided new findings and created a new landscape in which the immune system establishes cross talk, not only between immune components but also with commensal microorganisms 1, 2 and other important systems, such as the endocrine and nervous systems [3] [4] [5] . these developments have forced immunologists to reformulate the immunological architecture that confers protection, which has made the study of the immune system especially attractive. moreover, these advances have led to an increased interest in better understanding, managing, and manipulating the immune response in both health and disease. the characterization of new immune cell subsets has been a constant feature in the immunology field. this evolution is clearly reflected in the discovery of an innate counterpart of t lymphocytes, collectively named innate lymphoid cells (ilcs) 6 , and in the identification of different types of effector cd4 and regulatory t cells 7 . innate lymphoid cells (ilcs). ilcs are lymphocytes, but in contrast to adaptive immune cells, they can colonize lymphoid and barrier tissue sites during fetal development, do not undergo somatic recombination and do not express antigen-specific receptors 8, 9 . in addition to lymphoid organs, ilcs are enriched in barrier tissues, such as the gastrointestinal tract, airways, and skin 10, 11 . these innate cells have been considered to be tissue-resident cells, but recent studies suggest that ilcs can migrate through the lymphatic system during homeostasis or enter into the circulation upon infection and inflammation 6, 12 . currently, five different ilcs are defined on the basis of their transcription factor expression, different cytokine production and/or developmental patterns 6 : natural killer (nk) cells (discussed below), lymphoid tissue inducer cells (ltis) and three subsets of helper-like ilcs (ilc1s, ilc2s, and ilc3s), which are considered to be the innate counterparts of t helper (th) 1, th2, and th17 cells, respectively. the main focus of this review is ilcs. ilc1s are dependent on the t-box transcription factor t-bet and produce interferon gamma (inf-γ), but they differ in the expression of eomesodermin transcription factor 13 . ilc1s express cd127 in humans and cd200r in mice, but the natural cytotoxicity receptor nkp46 (also known as ncr1) is expressed in both species 14, 15 . ilc2s constitute the most homogeneous class of ilcs; they are dependent on gata3 and rorα, and they produce type 2 cytokines, mainly interleukin 5 (il-5) and il-13. ilc2s are involved in immune responses to parasite infection, and in humans, they express chemoattractant receptor-homologous molecule expressed in t h 2 cells (crth2) and high levels of cd161, whereas most mouse ilc2s express st2 (a member of the il-1 receptor family) 14, 15 . the development and function of ilc3s depend on the transcription factor rorγt. both human and mouse ilc3s can produce granulocyte macrophage colony-stimulating factor (gm-csf), il-17, and/or il-22 16, 17 . in humans, two major ilc3 subsets can be distinguished on the basis of the expression of the natural cytotoxicity receptor nkp44 (also known as ncr2) 14, 15 . both types can produce il-17, but the production of il-22 is mainly confined to nkp44 + ilc3s. extensive research has focused on deciphering the role of ilcs to ensure the maintenance of tissue homeostasis and immune protection 11, 18 . ilcs express particular sets of receptors in a tissuespecific manner, and these allow the detection of host-derived signals (including those from alarmins, neuronal mediators, microbia, and the diet) 19 . the integration of these endogenous signals is essential for the maintenance of tissue homeostasis, but dysregulation of ilc responses leads to inflammation and disorder 12, 20 . ilc are mainly involved in early protection against viruses and bacteria 13, 21 , but their response to dysregulated local proinflammatory cytokine production in adipose tissues leads to the development of metabolic disorders and obesity 20 . il-5 and il-13 produced by ilc2s induce goblet cell differentiation and the recruitment of eosinophils, basophils, and mast cells 22 , which are involved in protection against infection by helminths and viruses, but when uncontrolled, these cells drive allergic responses and metabolic disorders. moreover, the depletion of ilc2s in animal models suggests a role for these cells in atopic dermatitis and asthma 23 . ilc3s are abundant in mucosal tissues, and ncr2 + ilc3s have been proven to be essential for regulating the balance between commensal and pathogenic bacteria through the production of il-22 24 . in contrast, ncr2 − ilc3s can promote colitis in a model of inflammatory bowel disease 25 . the lack of immunodeficiency in ilc-deficient patients led to the proposal that ilcs are dispensable in the presence of functional t cells and b cells 26 . however, recent studies support the idea that ilcs cannot be considered to have functions that only duplicate those of the adaptive immune system. in addition to those showing the essential role of lti cells in the formation of secondary lymphoid organs during embryogenesis and the postnatal development of intestinal lymphoid clusters, recent studies also provide evidence that subsets of ilcs express multiple factors that modulate the adaptive immune response in health and disease 27, 28 . in particular, ilc2s and ilc3s modulate the t-cell response. studies in mice suggest that in healthy intestine, ilc3s express major histocompatibility complex (mhc) class ii molecules but lack the expression of costimulatory molecules; therefore, they inhibit microbiota-specific t-cell responses, thus preventing intestinal inflammation 29 . it seems that the interaction between ilc3s and tfh cells limits il-4 secretion and the production of iga by mucosal b cells 30 . studies with murine models have significantly contributed to the classification and understanding of the role of ilcs in the immune system, especially since similarities have been observed between ilcs identified in mice and humans 15 . however, the differences between these two species present real challenges 15, 31 because human ilcs have unique attributes that are only now being elucidated, with further work required in this exciting field. the roles of ilcs in immunity and their cross talk with other components of the immune response await further analysis. detailed coverage of this topic is beyond the scope of this review, and we refer the reader to recent reviews that provide more information on the biology of human 32 and mouse 33, 34 ilcs. t cells and plasticity. t cells are categorized as tα/β and tγ/δ cells, depending on the type of t-cell receptor (tcr) that they express 35 . human tγ/δ cells, similar to their murine counterparts, are a minor population (1-10% of nucleated cells) in peripheral blood, but are especially abundant in barrier tissues such as the epidermis [35] [36] [37] . the three main subsets of t cells carrying α/β receptor are the cd4+t helper cells and cd8+cytotoxic and cd4+ cd25+ regulatory t cells 38 . new effector cd4+ helper t-cell subsets (initially classified as th1 and th2) 39, 40 have been recently described, and at least six human th cell subsets have been identified to date: th1, th2, th17, tfh, th9, and th22 cells 38, 41 . all of these cells recognize foreign peptides presented by class ii mhc molecules on antigenpresenting cells (dendritic cells, macrophages, and b lymphocytes). th1 cells are required to activate macrophages and cellmediated immunity to kill intracellular pathogens 42 , whereas th2 cells are important in facilitating eosinophils to fight against parasitic helminths and b cells for antibody production and antibody class-switching to generate iga or ige 43 . th17 cells are required to mobilize neutrophils for the clearance of fungi and extracellular bacteria, and they are also involved in mucosal protection 44 . th9 and th22 cells are also involved in mucosal immunity; th9 cells protect against parasites 45, 46 , and th22 cells prevent microbial translocation across epithelial surfaces and promote wound healing 47, 48 . as mentioned in the introduction to ilcs, studies on human th cells isolated from lymphoid organs and blood samples, along with recent observations on the developmental mechanism of distinct th cell subsets, have revealed both similarities and differences of human and mouse th cells 41, 49, 50 . tfh cells are very important for germinal center reactions, antibody class switching, affinity maturation, and the development of high affinity antibodies and memory b cells 51, 52 . at the surface marker level, tfh cells are generally characterized by the expression of cxcr5, the chemokine receptor for cxcl13, which is highly expressed on b-cell follicles for expressing inducible t-cell costimulator (icos) and programmed death protein 1 (pd-1) 53,54 , which enable their involvement in the interaction of tfh cells and b cells 55 . the definition of a given t cell lineage is based on its ability to sense different inductive cytokines, to produce particular cytokines or to express a lineage-specifying transcription factor. th1 cells produce ifn-γ and express t-bet 56 ; th2 cells are characterized by il-4, il-5, and il-13 production and gata-3 expression 57,58 ; ptregs, which are induced in the periphery from naïve precursors, produce tgf-β and express foxp3 (tr1 cells are il-10-secreting tregs that do not express foxp3) 59 . th17 cells produce il-17a, il-17f, and il-22 and express rorγt 60,61 , and tfh cells produce il-4 and il-21 and express the bcl6 transcription factor. in addition, th22 cells, which produce il-22 and express the aryl hydrocarbon receptor (ahr) 47, 62 , and th9 cells, are characterized by the expression of il-9 and the transcription factor pu.1 63 . additional levels of regulation, such as the differential expression of micrornas, long noncoding rnas (lncrnas), and protein stability and function, have been found to control various aspects of th cell differentiation and effector function 64, 65 . cd8+ cytotoxic t cells express the dimeric cd8 marker and have specific lytic capacity to target cells through several mechanisms, including the release of cytotoxic granules, secretion of cytokine tumor necrosis factor alpha (tnfa) and interferon gamma, and the induction of cell death through the interactions of fas and the fas ligand 38, 66 . their tcrs are restricted to interactions with peptides presented by class i mhcs. regulatory t cells (tregs) include thymically derived and peripherally induced regulatory t cells (ttregs and ptregs, respectively), and they produce either il10, tgf-beta, il-35 or combinations of these proteins 67 . ttregs express the transcription factor foxp3 and secrete il10 and tgf-β; ptregs, which are induced in the periphery from naïve precursors, can also be subdivided into il-10-induced tregs [tr1 cells] (which secrete large amounts of il-10 and moderate levels of tgfβ), th3 cells (which produce il-10 and tgf-β), and tgfβ-induced tregs [itregs], which may or may not express foxp3. moreover, new subsets of regulatory t cells have been described. they include follicular regulatory t cells (which express foxp3 and bcl-6 and cxcr5), which modulate the function of tfh cells and fine-tune the germinal center response [68] [69] [70] , and a il-35dependent regulatory population of cells (referred to as itr35 cells), which show potent suppressive potential in several mouse disease models 71 . other regulatory populations have also been described, including bregs and cd8+ tregs, which are the analogous counterparts of tregs [72] [73] [74] . recent studies have revealed the capacity of differentiated t cells, particularly th17 cell and ptreg subsets, to change their phenotype in response to changing contexts [75] [76] [77] [78] [79] . becattini et al. 78 found that human memory cd4 t cells primed in vivo by pathogens (e.g., candida albicans and mycobacterium tuberculosis) or vaccines (tetanus toxoid) are highly heterogeneous, both at the population and clonal levels. with respect to studies on human arthritis, nistala et al. 79 proposed that th17 cells are recruited to the joint and converted to th17/1 or th1 cells in response to local il-12 levels. this plasticity has also been observed with in vitro assays under conditions that mimic a disease site, namely, low tgf-β and high il-12 levels 79 . these results are inconsistent with the original idea of th lineage stability and provide new possibilities for disease treatment aimed at inducing particular th subsets to modulate the immune response against pathogens or to control detrimental immunity 76, 77, 80 . trained and adaptive immune memory other classical concepts in fundamental immunology, such as immune memory, are also changing. the specificity and the capacity to generate long-lived memory cells are two properties that have been classically used to distinguish innate immunity from adaptive immunity. adaptive immunity is clearly based on the specific recognition of antigenic determinants by somatically diversified receptors (b cell and t cell receptors (bcr and tcrs, respectively)) and on its capacity to respond more effectively to restimulation with the same antigen. in contrast, innate immune responses have traditionally been considered nonspecific and without the capacity to adapt 81 . however, the discovery of germline-encoded pattern recognition receptors (prrs) and the "trained innate" immunity (or innate immune memory) have provoked a shift in our understanding of the immune response. in 1997, medzhitov et al. demonstrated that pattern recognition receptors (prrs) expressed on innate cells recognize invariant molecular structures expressed by invading pathogens 82 . after the interaction, prrs trigger the expression of costimulatory molecules and activate important signaling pathways to induce the activation of innate and adaptive immune cells. prrs mainly belong to four families: toll-like receptors (tlrs), nod-like receptors (nlrs), c-type lectin receptors (clrs), and peptidoglycan recognition proteins (pgrps) 83, 84 . the profiles of prrs expressed by innate cells can lead to partially specific recognition of a type of microorganism; e.g., innate cells can distinguish between gramnegative and gram-positive bacteria and modulate the immune response based on this recognition, although they cannot differentiate between bacterial species 85 . the idea that only jawed vertebrates developed immunological memory has also been challenged by the observation of resistance to reinfection in organisms that lack an adaptive immune response, such as plants 86 and invertebrates 87, 88 . recent studies have shown that monocytes and macrophages exposed to candida albicans or β-glucans exhibited an enhanced secondary response 89 . in addition, immunization of mice with bacillus calmette-guérin (bcg, the tuberculosis vaccine) induces t cellindependent protection against secondary infections by candida albicans, schistosoma mansoni or influenza virus [90] [91] [92] [93] . thus, organisms are protected not only against the original microorganism but also to unrelated pathogens. the mechanisms underlying the establishment of this innate immune memory differ from those involved in adaptive immune memory 81 . after infection or vaccination, innate immune cells (such as monocytes and macrophages) display long-term functional changes through epigenetic and metabolic reprogramming, including histone acetylation, methylation and modulation of noncoding rnas [94] [95] [96] . in turn, the faster and more pronounced reactivity of adaptive immune cells (t and b lymphocytes) upon reinfection is characterized by permanent changes in the genome of cells, such as mutations, gene rearrangement, clonal expansions, as well as epigenetic modifications, all of which ensure a more persistent effect than is endowed by trained immunity 81, 94, 95 . other cells for which immunological memory has been described include tγ/δ cells 97 and innate lymphoid cells 98 . recently, some authors have proposed that nk cells are also capable of immunological memory [99] [100] [101] [102] . antigen-specific recall responses by human nk cells were observed by nikzad et al. 103 in humanized mice and in varicella zoster virus (vzv)-exposed adult human volunteers, in which cytotoxic nk cells were recruited to sites of an vzv test antigen challenge on the skin. sensitization with haptens using mice lacking t cells and b cells led to the generation of hapten-specific memory nk cells 99 . the recall response persisted for more than four months after priming, and was adoptively transferred to naïve mice 100 . interestingly, nk cells exhibit memory that is not only specific to a given virus, such as cytomegalovirus 101, 102 , but that is also induced in the absence of a defined antigen 104, 105 . furthermore, new studies suggest that trained immunity is not a phenomenon that is restricted to immune cells, because epithelial stem cells also retain memory of previous inflammatory challenges by displaying an enhanced wound-healing capacity upon skin damage 106 . given the data outlined above, immunological memory is now recognized to be highly diverse and not restricted to b cell-or t cell-mediated adaptive immunity. much remains to be learned in this field, but the different manifestations of immunological memory described above offer an important basis for clinical applications, such as the development of novel vaccination strategies 107 or new therapies for pathological situations in which immunological memory can be detrimental, such as allergies or autoimmune diseases 94, 108, 109 . interaction of the immune system and the microbiome the immune system has evolved in the presence of commensal microorganisms that colonize barrier surfaces of vertebrates and invertebrates 1, 110 . the cross talk between the natural host microbiome and immune system is particularly interesting in the gastrointestinal tract, where the density and diversity of indigenous bacteria, viruses and fungi are greatest compared to those of other anatomical sites 111 . in the literature, reports of observed changes in microbial community composition during diseases are diverse and include those in inflammatory bowel disease (ibd), obesity, metabolic syndrome, and multiple sclerosis [112] [113] [114] [115] [116] . however, the microbiome can be influenced by different factors, such as the specific niche that it occupies, diet, stress, environmental factors, and host genetics, and a specific correlation does not necessarily infer causation. the presence of these commensals in mucosal tissues has been known since before metchnikoff, but the current knowledge on the role of the microbiome in shaping the immune system throughout life came mostly from the development of next-generation sequencing (in particular, the reduction in the cost of 16s ribosomal rna gene sequencing) and the use of germ-free animal models, which can be colonized even with human microbiota 117 . germ-free mice are characterized by atrophy of peyer's patches with few germinal centers and isolated lymphoid follicles, a lower number of b, t, and dendritic cells and a decreased level of immunoglobulins, particularly iga and igg 118 . these effects are observed at the mucosal and systemic levels, and they can be reversed within weeks after the colonization of germ-free mice with commensal bacteria 119 . moreover, colonization with commensal bacteroides fragilis revealed the immunomodulatory effect of bacterial polysaccharides in restoring systemic cells and the differentiation of cd4+ t cells into regulatory t cells (foxp3+ tregs), which in turn favor mucosal immunomodulation 120 . the induction of th17 cell maturation by segmented filamentous bacteria has also been reported 121 . these important examples emphasize the major roles of the commensal microbiome in the maturation of mucus-associated lymphoid tissue and the systemic immune system. the development of new technologies to better track the locations and activities of distinct microbial populations is essential to elucidate host-microbe interactions, through which other systems, such as the nervous system, seem to play important roles 2,122-125 . the better characterization of some immune cell subsets, trained immunity, and host-microbiome interactions provides a few very good examples that prove the maturation of immunology in the last few decades. in this sense, studies with mouse models have significantly contributed to the increase in our fundamental knowledge; however, the differences between murine and human immunology are notable, and conclusions drawn from mouse studies are sometimes not fully translated to humans 31 . if we want to fully exploit the power of the immune system for human health, greater effort is required for understanding human immunology. immunologists, in cooperation with experts from other fields, have developed a variety of protocols and tools to achieve greater selectivity in the identification and analysis of human cell subsets, types of cytokines and receptors, chemokines, etc. these tools range from biological approaches that rely on next-generation sequencing, mass spectrometry, and bioinformatics to immune monitoring technologies based on multiparameter flow cytometry and single-cell gene expression analysis. although not without limitations, these techniques provide a much better picture of the whole immune system than individual and independent approaches. immune-mediated illnesses comprise a wide variety of diseases characterized by the dysregulation of a normal immune response. most of these illnesses are complex disorders believed to arise from a combination of genetic and environmental factors 126 . infectious diseases infectious diseases are caused by pathogens (viruses, bacteria, fungi or parasites that infect the host body), and they remain a leading cause of mortality worldwide. prominent examples include illnesses produced by mycobacterium tuberculosis, human immunodeficiency virus (hiv), plasmodium falciparum or the current coronavirus disease 2019 (covid-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which has already infected millions of people and produced thousands of deaths in many countries. for a number of years, many people believed koch's postulates, which implied that virulence traits reside solely in the pathogen. however, recent advances in molecular biology have shown that host genes play major roles in infection, together with a wide range of environmental variables 127 . to date, six gene products endowing infectious disease susceptibility have been validated in the literature: (1) hemoglobin subunit beta; (2) band 3-anion transport protein; (3) duffy antigen/ receptor, which is associated with plasmodium spp. infections; (4) the prion protein associated with creutzfeldt-jakob disease; (5) fucosyltransferase 2 and 3, which is associated with norwalk virus infections; and (6) c-c motif chemokine receptor 5 (ccr5) coreceptor, encoded by an immune-related gene and leads to the impairment of the entry of the human immunodeficiency virus (hiv) into helper t cells, thus avoiding/decreasing the progression to acquired immunodeficiency syndrome 128 . another gene associated with infectious disease and the immune system is the natural-resistance-associated macrophage protein (nramp1), which encodes an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. it is a susceptibility locus for increased ratios of infection with leishmania spp. parasites and certain strains of salmonella spp., mycobacterium bovis and mycobacterium tuberculosis 129, 130 . in addition, it has been suggested that functional variants of immunoglobulin fc gamma riia (cd32) are related to the development of invasive encapsulated bacterial infections 131 . moreover, because of recently acquired genomic data, new human polymorphisms have been discovered, some of which play roles in changing immunoglobulin levels, seroconversion rates or the intensity of antigen-specific immune responses. in addition, they also contribute to human susceptibility to infection by viruses such as influenza, rhinovirus and respiratory syncytial virus 132 . these polymorphisms are mapped within the mhc (hla-dqb1*03, hla-drβ1, or hla-dpβ1), natural killer cell immunoglobulin-like receptors 1 and 4 (kir3dl1 and kir2ds4) and natural killer lectinlike receptor d1 (kldr-1) 133 . several recent studies available as preprints have analyzed certain genes that may explain the differences in the variable expression of and susceptibility to covid-19 by patients, either by affecting the host receptor for the virus (angiotensin i converting enzyme 2 (ace-2)) 134 , immune genes (tlr7 and others) or blood groups (group o seems to be the most protective) 135 , and more extensive omics studies are now underway with larger numbers of patients. in 1901, the physician paul ehrlich first used the term "horror autotoxicus" to describe the way autoimmunity contradicts the natural aversion to self-injury ("living with the enemy", reviewed in 136 ). currently, according to the american autoimmune related disorders association, more than 100 autoimmune diseases have been identified. historically, these diseases were considered to be rare, but current epidemiological data have shown that they affect approximately 3-5% of the population worldwide. some of the most common autoimmune diseases include type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease (https://www.aarda.org/diseaselist/). although significant progress has been made in understanding the mechanisms of autoimmune diseases and the nature of selftolerance, these disease remain major burdens on health systems around the world. autoimmune diseases arise when the immune system attacks normal components of the body 137 . the concept of immune tolerance is defined as the ability of the immune system to prevent the targeting of self-molecules, self-cells or self-tissues. on the other hand, the failure to distinguish self from nonself is often termed a break of tolerance, and it is the basis for an autoimmune disease 138 . what are the mechanisms that lead to a break in tolerance? autoimmune diseases are complex disorders that are believed to arise from a combination of genetic (mutations and higher inheritance frequency of some types of major histocompatibility complex alleles), epidemiological (age and sex) and environmental (infections, microbiota, tobacco, chemicals and pharmaceutical drugs). factors these factors trigger a break in self-tolerance with the activation of self-reactive lymphocytes through several mechanisms, such as molecular mimicry, the overexpression and abnormal expression of mhc class ii molecules in peripheral tissues, thymic aging, and immunodeficiencies (discussed below) and many others. some lymphocytes escape control due to polymorphisms in several genes that affect the routes of lymphocyte activation. other causes may include defective antigen presentation by some mhc variants with specific polymorphisms. therefore, the self-reactive lymphocytes that have escaped control and react against self-constituents initiate the autoimmune process 139 . although a large number of genome-wide association studies (gwas) have led to the identification of hundreds of polymorphisms associated with the development of different autoimmune diseases, it has proven difficult to define the role of most of these polymorphisms in the breakdown of tolerance to a selfantigen [139] [140] [141] [142] [143] [144] [145] . it is worth highlighting, however, that the mhc remains the main genetic factor associated with human autoimmunity 138, 139 . other gene variants identified are common to many autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type i diabetes, ulcerative colitis, autoimmune hepatitis and numerous other autoimmune diseases. for example, the protein tyrosine phosphatase nonreceptor type 22 (ptpn22) gene encodes a protein that inhibits t-cell activation in the adaptive immune system, whereas it promotes myeloid cell activation; interferon regulatory factor 5-transportin 3 (irf5-tnpo3) is involved in the accumulation of lymphocytes within lymphoid organs and failed elimination of autoreactive naïve t cells; btb domain and cnc homolog 2 (bach2) has a critical role in immunoglobulin class-switching recombination, somatic hypermutation of immunoglobulin encoding genes and the activation of tissue macrophages. a more complete list of genes associated with autoimmunity can be found in the review by wang et al. 138 researchers are currently looking for the missing heritability in autoimmune diseases by focusing on the study of methylome profiles, genetic cargos in extracellular vesicles, genetic alterations, and ways in which the microbiome may affect these diseases. immune-mediated rejection of tissue allografts was first described in 1945 by the british immunologist peter medawar 146, 147 . only three years later, george snell described the mhc, which carries the histocompatibility genes, and one decade later, jean dausset described the human leukocyte antigen (hla); each of these scientists was recognized with the nobel prize in physiology and medicine 148 . since its discovery, mhc has emerged as the most polymorphic gene locus in eukaryotes with 24093 hla and related alleles, more than 362709 nucleotide variants reported in the individual-participant data-international immunogenetics/ human leukocyte antigen (ipd-imgt/hla) work group database (https://www.ebi.ac.uk/ipd/imgt/hla/), release 3.39.0, 2020/01/20 149 . although the main barrier for long-term organ and tissue grafting is driven by hla incompatibilities, other important players play roles in transplant rejection. in particular, minor histocompatibility antigens, which are peptides derived from allelic variants of normal cellular proteins, presented by class i or ii mhc antigens induce cellular immune responses in hla-matched individuals who lack the same allelic variant 150 . natural killer (nk) cells also play important roles in transplantation through their killer cell immunoglobulin-like receptors (kirs), which are receptors for hla class i molecules. nk cells expressing an inhibitory kir-binding self-hla can be activated when exposed to allografts that lack a ligand for the inhibitory receptor 151 . the locus that codifies these receptors displays a considerable degree of polymorphism, with 1110 alleles reported in the individual-participant data-international/killer cell immunoglobulin-like receptors (ipd/kir) work group database, release 2.9.0, 2019/12/11 149 . more recently, we have begun to appreciate the importance of non-hla genetic factors in the development of transplant rejection; examples include polymorphisms in the genes encoding cytokines, such as tumor necrosis factors (tnf), interleukins (il-1, il-6 and il-10), interferon gamma (ifn-γ), and transforming growth factor-β3 (tgf-β3). other genes encode pathogen recognition receptors, with nucleotide-binding oligomerization domaincontaining 2 (nod2 (card15)) being the most widely studied, although conclusive data have not been obtained to date 148 . primary immunodeficiencies (pids) comprise a heterogeneous group of more than 400 genetic disorders that result in defects in the immune response 152 . pids are considered mendelian disorders because they are mainly autosomal recessive disorders that often display incomplete penetrance, which affects the severity and onset of the disease. with the exception of immunoglobulin a (iga) deficiency, pids are considered to be rare disorders, as their prevalence worldwide ranges from 1 to 9 among 100,000 people 153 . unsurprisingly, these types of diseases are not uncommon in highly consanguineous populations such as those in the middle east/northern africa (mena) region. the incidence of consanguinity marriage in these areas ranges between 20 and 56%, which leads to a unique population in which autosomal recessive diseases arise, with the prevalence of pid in these countries as high as 30 in 100,000 people 154 . although more than 400 genes have been described for pids, approximately 60% of the causal genes remain unknown, and next-generation sequencing studies performed in mena populations are contributing to the search for currently unknown genes that cause pids 155 . a complete and updated list of pid-causing genes and diseases can be found at the european society for immunodeficiencies (esid) webpage (https://esid.org) 156 . clinical manifestations of pids are highly variable; many disorders involve an increased susceptibility to several types of infections, but some patients develop autoimmune diseases. patients usually present recurrent sinus or ear infections or pneumonia within a one-year period; other indicators are failure to thrive, poor response to prolonged use of antibiotics, and persistent thrush or skin abscesses 153 . depending on the affected pathway, pids are associated with varying levels of severity, times of onset, and risks of infection by certain groups of microorganisms. according to the international union of immunological societies (iuis) (https://iuis.org/ committees/iei/), 430 inborn errors of immunity can be classified as follows: (a) immunodeficiencies that affect cellular and humoral immunity; (b) combined immunodeficiency (cid) with associated or syndromic features; (c) predominant antibody deficiencies; (d) diseases of immune dysregulation; (e) congenital defects of phagocyte number, function, or both; (f) defects in intrinsic and innate immunity; (g) autoinflammatory disorders; (h) complement deficiencies; and (i) phenocopies of a pid 156, 157 . however, pids are broadly classified as follows according to the component of the immune system affected: • t-cell immunodeficiency, e.g., defects in the ifn-γ/il-12 pathway and mutations in the autoimmune regulator (aire) gene. • b-cell (antibody-mediated) immunodeficiency: gamma-globulinemia, x-linked common variable immunodeficiency (cvid), selective iga deficiency, specific antibody deficiency, and igg subclass deficiency. • combined immunodeficiency: wiskott-aldrich syndrome, ataxia telangiectasia, digeorge syndrome and severe combined immunodeficiency (scid). • phagocyte defects: chronic granulomatous disease, hyperimmunoglobulin e (ige) syndrome and leukocyte adhesion deficiency. • complement defects (deficiency in early, late or regulatory complement components) 158 . autoinflammatory diseases systemic autoinflammatory diseases (aids) are characterized by recurrent acute inflammatory episodes secondary to a dysregulated inflammatory process that typically develops during childhood, with recurrent episodes of fever, rashes, and disease-specific patterns of organ inflammation. genetically speaking, these are hereditary disorders, andto date, more than 40 genes (table 1) have been identified as causes of aids, which can be grouped according to the pathway that is altered 159 . (1) inflammasome. the inflammasome is a multiprotein intracellular complex that detects pathogenic microorganisms and stressors and activates the highly proinflammatory cytokines il-1β and il-18. genes affected in this group are mefv (mediterranean fever pyrin innate immunity regulator), which is related to familial mediterranean fever (fmf); nlrc4 (nlr family card domaincontaining 4); nlrp1 (nlr family pyrin domain-containing 1) and wdr1 (wd repeat domain 1) 159 . (2) type-i interferon (ifn)-mediated disorders. these disorders are characterized by the upregulated expression of genes induced by ifn. the gain of function by variants of tmem173 (transmembrane protein 173) is the core manifestation of this disorder group, but other genes have been identified, including ddx58 (dexd/h-box helicase 58), dnase2 (lysosomal deoxyribonuclease 2), pola1 (dna polymerase alpha 1 subunit) and usp18 (ubiquitin-specific peptidase 18) 159,160 . (3) ubiquitination disorders. ubiquitination is a process that marks proteins for degradation via the proteasome, which is required for the processing of intracellular antigens (such as virus proteins or mutated tumor proteins) and their presentation by class i hla molecules. ubiquitination involves three main steps: activation, conjugation and ligation, which are performed by ubiquitin-activating enzymes (e1s), ubiquitin-conjugating enzymes (e2s), and ubiquitin ligases (e3s). ubiquitination disorders are caused by variants of the psmb8, psmb9, psma3 and psm4 genes (proteasome 20s subunit beta 8, subunit beta 9, subunit alpha 3 and subunit alpha 4, respectively), affecting the proteasome subunits, proteasome maturation protein gene (pomp) and/or proteasome assembly chaperone 2 (psmg2), by encoding proteasome assembly molecules 161 . in addition, other genes in this group, such as otulin (otu deubiquitinase with linear linkage specificity), encode ubiquitin peptidases, i.e., proteins involved in ubiquitination assembly complexes, such as hoil-1 (hemeoxidized irp2 ubiquitin ligase 1) and hoip (nhp2-like protein 1 homolog). finally, the loss of function due to variants of the tnfaip3 (tnf-alpha-induced protein 3, also known as a20) gene, which encodes a protein with ubiquitin ligase and ubiquitinase activity, has also been described 159 . (4) inflammatory or innate immune regulators. a large number of genes have been found to affect the pathways/ mechanisms involved in macrophage and b-cell differentiation and lymph node development, among many functions. genes in this group include ada2 (adenosine deaminase 2), tnfrsf11a (tnf receptor superfamily member 11a), adgre2 (adhesion g protein-coupled receptor e2), trnt1 (trna nucleotidyltransferase 1), lacc1 (laccase domain-containing 1) and ap1s3 (adaptor related protein complex 1 subunit sigma 3) 159 . allergy allergic diseases can be termed complex diseases that involve both genetic and environmental factors, and they influence not only the development of ige-mediated sensitivity in the case of hypersensitivity type i allergies but also the subsequent development of clinical symptoms in a range of tissues, including skin, nose, and lung tissue 162 . since the first report of a link between chromosome 11q12 and atopy in 1989 163, knowledge about the common risk variants for allergic diseases has increased exponentially, mainly because of gwas. most allergic diseases have allergy-related traits such as asthma, with the strongest association mapped to chromosome 17q21. however, the disease-associated gene at this locus remains unclear; one of the candidate genes is ormdl3 (sphingolipid biosynthesis regulator 3) due to its role in sphingolipid synthesis and the regulation of eosinophils. other genes associated with asthma are interleukin 33 (il33) and its receptor, il1rl1 (interleukin 1 receptor-like 1), hla region, smad3 (sma-and mad-related protein 3) and il2rb (interleukin 2 receptor subunit beta) 164 . as asthma and other allergic-associated traits could be present in patients without allergies, some researchers performed gwas analysis on cohorts of patients who had high levels of allergenspecific immunoglobulin e (ige) or a positive skin prick test. as a were identified, and the strongest association was on chromosome 11q13. this locus has been associated with two genes: c11orf30 (emsy transcriptional repressor, brca2 interacting), a potential regulator of interferon-stimulated gene, and lrrc32 (leucine rich repeat-containing 32), which is involved in transforming growth factor beta (tgfβ)-signaling in t regulatory cells. the rest of the associated loci involved in the pathogenesis of allergy highlight the importance of the th2 responses (stat6 (signal transducer and activator of transcription 6), tslp (thymic stromal lymphopoietin), bcl6 (b-cell lymphoma 6 protein), il1rl1 (interleukin 1 receptor-like 1), il33 (interleukin 33), gata3 (transacting t-cell-specific transcription factor binding protein 3)); innate immunity (tlr1/6/10 (toll-like receptor 1/6/10)); tgfβ-signaling (lrrc32 (leucine rich repeat-containing 32), smad3 (mothers against decapentaplegic homolog 3)); t-cell (il2 (interleukin 2), ptger4 (prostaglandin e receptor 4)) and t regulatory box (lrrc32 (leucine rich repeat-containing 32), il-2, nfatc2 (nuclear factor of activated t cells 2), foxa1 (forkhead box a1)) 164 . in the last two years, researchers have focused on epigenomewide association study (ewas) of allergy processes. the epigenetic landscape is specific for a given cell; thus, ewas requires careful selection of the relevant cell type for a given biomedical condition. for allergies, ewas has mainly been performed on nasal mucosal cells and whole blood (although the result was later normalized by the number of circulating eosinophils). nasal mucosal cells comprise cd8 + t cells, cd4 + t cells, myeloid cells, innate lymphoid cells, b cells, double-negative t cells, granulocytes, cd117 + cells, and plasma cell populations 165 . in all of these studies, 36 cpg-associated regions were identified, from which the smad3 gene, coding for an important regulator of t-cell differentiation, was replicated in three independent cohorts 166 . of all of the genes in whole blood identified using ewas, only the acot7 (acyl-coa thioesterase 7), epx (eosinophil peroxidase), gja4 (gap junction protein alpha 4) and mettl1 (methyltransferase-like 1) genes were confirmed in the nasal cell populations 167 . in 1909, ehrlich proposed the idea that mutant cells arise continuously and that the immune system scans for and eradicates these mutant cells before they manifest clinically 168 . however, immune surveillance remained a controversial topic until its acceptance in the 1990s 169 . immune surveillance is the recognition and elimination of cancerous cells by lymphocytes, which act as sentinels that recognize transformed cells. ultimately, during tumor progression, cancer cells show low immunogenicity and resistance to immune effector cells, thus expanding and escaping immune control. the way in which cancer cells modify the immune system has been called immune editing 169 . the key of immunosurveillance is cancerous cell expression of tumor antigens that can activate various immune cell phenotypes; for simplicity, any overexpressed, mutated, dysregulated, or rearranged gene product expressed by a cancerous cell may be considered a tumor antigen. it is critical to consider that most of these proteins, except those derived from virus-infected cancer cells, are primarily self-proteins, but they are expressed with mutation(s) or minor changes in their antigenic structure 170 . one mechanism by which cancer cells escape from immune recognition is antigenic modulation. for example, the loss of mhc class i molecule expression leads to aberrant antigen masking, which is one of the mechanisms described for tumor cells that escape specific antitumor t-cell immune responses 171 . in addition, the mhc-peptide-t cell receptor complex elicited by a tumor antigen shows weak stability, since high-affinity t-cells tend to be rendered tolerant to these antigens 172 . another mechanism is the direct inhibition induced by cancer cells due to their interaction with surface regulatory molecules, table 1 . also called checkpoint molecules. these molecules include programmed cell death-1 (pd1) and cytotoxic t-lymphocyteassociated protein 4 (ctla-4), which induce the inhibition of host t cells. although these checkpoints usually help conventional immune responses control immune activation, they can also be used by tumor cells to inhibit antitumoral t-cell responses 173 . pd1) is a transmembrane protein expressed on t, b, and nk cells, and it binds to pd1 ligands (pd-l1 and pd-l2) on target cells. when it binds to its ligand on tumor cells, pd1 inhibits tumor cell apoptosis, causes peripheral effector t-cell exhaustion, and promotes the conversion of effector t cells into regulatory t cells 172, 174 . ctla4 is also a physiological negative regulator of t-cell activation. the interaction with cd80/cd86 in the tumor leads to the inhibition of t-cell function and suppressed effector activity 175 . knowledge of these two checkpoint inhibitors has opened the door to new antitumoral therapeutic approaches, such as the use of monoclonal antibodies that block the aforementioned interactions (anti-pd1, anti-pd-l1, or anti-ctla-4), which are called checkpoint inhibitors 176 . in addition, tumor cells create an inhibitory microenvironment around them. malignant cells can recruit other cells, such as immune cells and fibroblasts, which can be corrupted by tumor cells. the interaction between tumor and nontumor cells creates the tumor microenvironment, which is mostly driven by the dynamics of the tumor promoting the proliferation/expansion of cancer cells. for example, tumor and stromal cells release multiple factors, such as the chemokine ccl28 (c-c motif chemokine ligand 28), which inhibits effector t-cell functions and attracts tregs to the microenvironment 172 . tumor cells use different mechanisms to promote cancer progression and further metastasis. the complete immunological eradication of cancer is the goal of antitumoral immunotherapy and is discussed later in this review. immunosenescence and inflammaging aging is accompanied by the decline and dysregulation of immune efficacy, which results in an increased vulnerability to infectious diseases, diminished responses to vaccination, and reduced tumor clearance. immune alterations mainly manifest as a reduction in the number of naïve peripheral blood cells and a relative increase in some types of memory cells 177 . natural aging causes progressive atrophy of the thymus, which is called thymic involution. the endpoint is a significant decrease in naïve t cells, which reduces the diversity of the t-cell antigen receptor (tcr) repertoire and culminates in disrupted t-cell homeostasis 178 . the cellular and molecular hallmarks of aging have been described as genomic instability, telomere attrition, epigenetic alterations, sarcopenia, changes in intracellular communications, cellular senescence, immunosenescence and mitochondrial dysfunction 179 . the process of aging alters the innate and adaptive immune systems. in terms of innate immunity, aging results in a decreased number of circulating monocytes and dendritic cells, reduced phagocytic properties of macrophages and neutrophils, and impaired antigen presentation by dendritic cells 179 . as mentioned above, aging also generates a reduction in the t-cell and b-cell receptor repertoire due to the accumulation of senescent or exhausted lymphocytes, together with a decrease in the number of circulating naïve t and b cells 178, 179 . on the other hand, nk cell cytotoxicity is maintained in centenarians, and an increase in the number of these cells is observed in healthy aging people 177 . moreover, cd4+ t cells exhibit cytotoxic features in centenarians; this is an acquired characteristic for cd4+ t cells that usually have helper, but not cytotoxic functions under physiological conditions 180 . in addition to these features, chronic inflammation is considered the key that underlies the phenomenon called 'inflammaging', which is related to elevated self-reactivity and results in the typical chronic low-grade, systemic inflammatory phenotype observed in the elderly in the absence of acute infection. currently, it is believed that self-reactive t cells are the main contributors to this process. it has been proposed that this basal inflammatory state contributes to the development of some diseases, such as type ii diabetes, alzheimer's disease and atherosclerosis 178 . understanding the mechanisms of age-related disorders in immune regulation is important for identifying more efficient strategies of immune rejuvenation and for the effective induction of vaccination-mediated immunity in older individuals 177 . immunotherapy includes the use of certain components of the immune system (antibodies, cells, cytokines, etc.) for the treatment of various cancers and autoimmune diseases and the manipulation of the immune system through vaccines for the prevention and treatment of infectious and allergic diseases (fig. 1) . immunotherapy using microorganisms or their components in vaccines was first practiced centuries ago; soluble substances such as poly-and monoclonal antibodies, as well as cytokines, have been used for many years, but recently, cellular immunotherapy has emerged in clinical practice. although immunotherapy can be used for many diseases (infections, autoimmune diseases, macular degeneration, allergic diseases, etc.), it is being used most expansively in the cancer field. the main goal is to destroy the tumor, either directly or indirectly (by enhancing the patient's immune system), while offering greater specificity and fewer side effects than conferred by conventional therapies. pathogens and vaccines for infectious diseases immunotherapy associated with pathogens was first linked to the prevention of infectious diseases, starting from variolization (in the x century), followed by edward jenner's vaccination against smallpox (in the xviii century) and subsequently many other preventive vaccines for infectious diseases. the great advances in the knowledge about infectious diseases took place in the nineteenth century, but the xx and xxi centuries are clearly the vaccination centuries, as many new successful vaccines (with attenuated or dead pathogens, subunits, recombinant proteins, carbohydrates or dna) introduced against a variety of pathogens. more recently, and with the increased knowledge of the human microbiome, the use of microorganisms in therapy has seen a resurgence. some intestinal infections, such as those produced by clostridium difficile, can be cured with the transfer of intestinal bacteria from healthy people (feces transplantation) 187 . numerous other attempts to use microorganisms to cure inflammatory illnesses (crohn's disease, ulcerative colitis, etc.) have met with limited success 188 , which indicates that this type of therapy is much more complex than initially anticipated. as a consequence, many more studies are required to ensure that this approach can be used for curative immunotherapy. researchers are also working on genetically modified or artificial bacteria (e.g., based on salmonella enterica, listeria monocytogenes or lactobacillus lactis), but only limited effects have been observed to date 189 . oncolytic viruses (ovs). although the use of bacteria in antitumoral therapy has been largely restricted, the use of therapeutic viruses is increasing. virus-based therapy was introduced in the 1990s with the use of adenovirus, but only in recent years has it been used in practice in the clinic. oncologic viruses 190 have the capacity to attack tumor cells in a preferential manner and induce immunogenic cell death (icd) and host antitumor immunity (fig. 2) . the first virus approved for use in therapy was a recombinant oncolytic adenovirus named h101, which was licensed in 2005 by the china food and drug administration (cfda) for treating head and neck carcinoma in combination with chemotherapy 191 . ten years later, the oncolytic attenuated-modified virus herpes simplex i-talimogene laherparepvec (t-vec, imlygic®) was approved by both european (emea) and american (fda) agencies for the treatment of melanoma 192 . the virus is modified by the insertion of human gm-csf and deletion of the icp47 gene. since the approval of t-vec, a new era has dawned on the use of ovs in cancer therapy 193, 194 . currently, oncolytic viruses from the adenoviridae, herpesviridae, picornaviridae, reoviridae and poxviridae families are in different phases of clinical studies for several types of tumors 194, 195 . for example, reovirus against brain tumors (alone or combined with other therapies) 196 or maraba virus against triple-negative breast tumors 197, 198 offer some hope to patients with these types of cancer. viral sequences can be modified by genetic engineering techniques, thus making the virus more prone to infect some cells and enhancing viral infiltration and tumor tropism. combinations with other components (immunomodulators, drugs, and cytokines) are also being explored to suppress antiviral immunity and enhance antitumoral cytotoxicity 199 . vaccines for cancer prevention. it is clear that certain viruses and bacteria play roles in cancer development. viruses such as genital herpes, hepatitis b, epstein barr or human papilloma and bacteria such as helicobacter pylori have been associated with cancers of the uterus and liver, in burkitt's lymphoma, and oral/genital and stomach cancers, respectively 200 . therefore, immunization against these pathogens offer protection not only from infection but also from cancer. therapeutic vaccines. once an illness has developed, the intention of a therapeutic vaccine is to eliminate or decrease its pathology. thus, vaccines are used for cases of allergies, cancers and autoimmune diseases. allergy (type 1): allergen-specific immunotherapy (ait) aims to modulate the immune system against an allergen, thus modifying the natural course of the allergic disease and conferring longlasting benefits 201 . the basic ait involves the introduction of repeated doses of allergen (either injectable or sublingual allergen extract tablets) and often in escalating doses in a controlled manner, followed by a maintenance phase. in cases for which long-lasting tolerance is acquired, therapy may be discontinued. allergen extracts can be obtained from different sources, such as cat hair and pelt, mites, different types of pollen, venom protein, foods, etc. allergy vaccines are currently the only effective therapy that can stop the progression of the illness because treatment with anti-inflammatory drugs, such as anti-histaminic drugs or corticoids, mitigates the symptoms of the allergic processes but does not modify the natural course of the disease 202, 203 . ait has been shown to induce the activation of antigen-specific tregs and il-10-producing bregs (br1) subtype cells, which is combined with anergy caused by th2 cells 201 and the production of allergen-specific igg antibodies that can compete with ige for binding to allergens 204 . in the past, most vaccines were developed using natural allergen extracts. however, significant progress has been made in recent years to correctly characterize the allergen at the molecular level, and some of these allergens are now being produced by recombinant technologies, nucleic acid-based strategies, or synthetic peptide chemistry 205 . cancer: another therapeutic approach for vaccines is in the field of cancer. therapeutic cancer vaccines that contain self-or nonself-patient tumor lysates, viral vectors, mutated tumor proteins or peptides, among other types 206 administered in the presence of adjuvants can activate the immune system to induce antitumoral responses 207 . the goal is to activate the th and tc cell compartments to expand specific cytotoxic t and nk cells directed against tumor cells. some vaccines are more immunogenic than others, and this effect can be related to several factors, such as the types/numbers of genetic mutations in the tumor, expression of neoantigens, production of viral proteins, an immunosuppressive environment, lack of expression of histocompatibility complex molecules, etc., which together may explain the large variability in tumor elimination 208 . therapeutic cancer vaccines are generally very safe, and major secondary effects have not been observed, although large differences in patient responses are detected. moreover, this strategy may be used in conjunction with other complementary therapies 209 , such as monoclonal antibodies, chemotherapy or cellular therapy 209, 210 . several patients are currently taking part in clinical trials and are receiving therapeutic cancer vaccines against different types of tumors, such as lung (clinicaltrials.gov identifier: nct04397926), prostate (clinicaltrials. gov identifier: nct03525652) or pancreas (clinicaltrials.gov identifier: nct04161755), using individual or combined therapies. autoimmunity: in the case of therapeutic vaccines for autoimmune diseases, such as multiple sclerosis, diabetes, myasthenia gravis or guillain barré syndrome, the intention is to induce tolerance to self-antigens through the activation of regulatory cells (tregs and bregs) and tolerogenic dendritic cells, thus avoiding the immune response to self-components 211 . due to the large variety of autoimmune diseases, the different etiologies and extensive variability, even in the same type of disease, designing a vaccine that can be useful for a wide range of patients is very difficult. however, several researchers are obtaining good results in animal models with nanostructures and peptides that induce specific tolerance, and it is predicted that, in the near future, these types of therapies will be applied to patients suffering from autoimmune diseases (reviewed by serra and santamaria 212 ). polyclonal antibodies (pabs)-serotherapy the discovery of antibodies by dr. e. von behring and kitasato 213 at the end of the xix century highlighted the potential of antibodies to neutralize tetanus and diphtheria toxins. this discovery opened the way to exploring the potential clinical applications of conventional antiserum-containing polyclonal antibodies from immunized animals/humans 214 . this "serotherapy" was initiated by dr. roux and dr. yersin, who used antidiphtheria serum to treat several children 215 . after this initial success, the use of serotherapy was increased for use against diphtheria and other diseases but also led to the identification of problems, such as immunogenicity with the formation of immune complexes (arthus reaction), the variability and limitation of the antibody batches, the content of a mixture of classes and subclasses of antibodies with different biological activities, and their temporal effects. for all of these reasons, therapy with polyclonal antibodies was very much restricted to special cases, such as the use of gamma-globulins for the prevention of rhesus (rh) maternal-fetal incompatibility and tetanus or snake venom toxicity 216 . with the identification of gamma-globulin-deficient patients by dr. bruton in 1952 217 , the use of immunoglobulins as therapeutic molecules for the treatment of humoral immunodeficiencies was initiated. however, some problems were encountered in the initial phases, mostly related to the serum preparation and aggregation/ fragmentation of antibodies. since their initial use, several efforts have been made to avoid impurities and to improve the purification process, and several commercial products are now available (as intravenous or subcutaneous preparations). currently, many patients with humoral immunodeficiencies are successfully being treated to prevent them from catching infectious diseases. more recently, the therapeutic applications of immunoglobulins have expanded to other diseases, such as against covid-19 caused by sars-cov-2 infection (see below), autoimmune disorders and kawasaki syndrome in children 218 . the beneficial effects seem to be mediated by several immunological mechanisms, including viral neutralization, inhibition of inflammatory cells and activation of immune regulators 214 . monoclonal antibodies (mabs) the development of monoclonal antibodies (mabs) by c. milstein and g. köhler in 1975 219 (nobel prize winners for physiology/ medicine in 1984) changed medicine and immunology completely, along with many other disciplines. monoclonal antibodies are produced from the fusion of two cells to generate a hybrid cell or hybridoma with two characteristics, i.e., the production of one specific antibody and immortality. dr. milstein is considered to be the father of modern immunology for his crucial contribution 220 . the development of many different mabs has enabled the identification of new molecules and the development of more accurate diagnostic approaches; specific, fast and inexpensive technologies; processes for the purification/concentration of compounds; and better and more specific therapy. mabs can now be used against specific targets according to the concept of the "magic bullet", a term coined by dr. paul ehrlich at the beginning of the xx century (reviewed in ref. 221 ). numerous different mouse and rat mabs were produced against several molecules, but due to their murine origin, patients treated with these mabs suffered from hypersensitivity and immune responses 222, 223 . thus, most mabs currently used in clinical applications are linked to radioactive elements and used for diagnostic purposes (table 2) . in an effort to avoid immunogenicity, mabs were subsequently modified by genetic engineering approaches to carry mostly sequences of human origin. several research groups and companies developed chimeric and humanized mabs ( table 2) , and these mabs included additional modifications, such as changes in the carbohydrates (glycosylation) and/or antibody regions, with the aim of improving their therapeutic action [224] [225] [226] [227] [228] . moreover, fragments of recombinant antibodies (fabs, single-chain fvs, different v regions, fusion proteins, smaller antibodies, etc.) increased the variability of these potential therapeutic agents. the generation of fully human mabs took more time due to technical difficulties and ethical issues; therefore, researchers sought alternative methods to conventional approaches, such as the development of transgenic animals carrying human immunoglobulin genes using minilocus vectors, artificial yeast/human chromosomes or p1 vectors. the generation of knockout mice (in which mice lack ig genes) and further crosses with transgenic mice carrying human antibody sequences led to the generation of mouse strains that were able to produce fully human mabs 229, 230 . other initiatives, such as the generation of immunodeficient mice in which human bone marrow or libraries of recombinant phages carrying human variable genes were reconstituted, allowed the development of more fully human antibodies ( table 2) . sir greg winter, nobel prize winner in chemistry in 2018 231, 232 , became the pioneer of mab humanization through the genetic engineering of an antibody (campath 1), later developing a fully human antibody (antitumor necrosis factor alfa, tnf-a) using recombinant phage technology 225, 233, 234 . several companies are currently developing human antibodies using these and new technologies (reviewed in 225, 227, 233, 234 ) . since 1975, the list of approved mabs for human therapy has continued to increase (table 2) , and many more mabs are in clinical trials [235] [236] [237] . the versatility of mabs is based on a different mechanism of action 238 : human immunology and immunotherapy: main achievements and challenges j varadé et al. -neutralization/blocking of soluble elements. for example, the neutralization of cytokines (tnf-α) and growth factors (vascular endothelium growth factor) prevents the exhibition of their effects, i.e., inflammatory and angiogenic effects, respectively 239, 240 . -complement activation. igg/igm antibodies activate the complement cascade by the classical route, which leads to the death of the target cell 241, 242 . -cytotoxicity mediated by nk cells. nk cells can facilitate mab killing of target cells. the mab, after binding to a target cell, can attach to fc receptors on the surface of nk cells to trigger the release of granzymes and perforin, thus inducing cell target death 243, 244 . -induction of cell death by apoptosis. certain mabs directed against some membrane molecules can directly activate apoptosis 243 . -blocking activation signals. antibodies can block some membrane receptors and avoid cell activation/proliferation activation/proliferation 243, 245 . -carriers of toxins, pro-drugs, enzymes, and radioactive elements. mabs are able to concentrate select compounds around target cells, providing a much more selective therapy than conventional chemo-or radiotherapy 244 . -check point inhibitors. leading to a recent revolution in cancer therapy, the identification of several inhibitory molecules can be blocked by mabs, thus leading to the activation and proliferation of antitumoral t cells. molecules such as ctla-4 and pd1 and its ligand pdl-1, maintain immune cells under controlled conditions. however, it is possible to reactivate the antitumoral immune responses by blocking some of these molecules with mabs, either directed to only one of them or by using various antibodies in combination (for example, against ctla-4 and pd1) 246 . the results obtained with these therapeutic mabs against checkpoint inhibitors in some types of cancer have been amazing. for their contribution to the understanding of the roles of ctla-4 247 and pd-1 248 , the swedish academy gave the nobel prize in 2018 to dr. j.p. allison and dr. t. honjo, respectively 249 . however, this therapy is not efficacious in all types of cancers for several reasons, such as the expression of these and other checkpoint inhibitors in immune cells, the number of antitumoral cells in each patient, an immunosuppressant microenvironment, the rate of cancer mutations, and the expression of histocompatibility molecules. there is a large list of recombinant proteins that are currently being used for human therapy, including interleukin 2 (il-2), interferons (ifns) and gm-csf. il-2 was identified in 1976 as a growth factor for t lymphocytes, and soon after dr. rosenberg started to use it in antitumoral therapy 250, 251 . years later, in 1991, il-2 was approved by the fda for patients with metastatic renal cancer and in 1998 for the treatment of metastatic melanoma 251 . interferon (ifn) was described in 1957 by isaacs and lindenmann 252 . the interferon family is the largest family of cytokines and is classified into three different types (i, ii, and iii). type i ifns (including ifn-α and ifn-β) exhibit several molecular actions that may be very relevant for use in therapy for a range of pathologies (such as autoimmune diseases and cancers) 253 . in 1986, the fda approved human ifn-α2a and ifn-α2b for patients with hairy cell leukemia and later on for patients with multiple sclerosis. since their initial use, these interferon species have been approved for many other diseases, including chronic hepatitis b and c, lymphoma, advanced melanoma, and as adjuvants together with other therapies for several types of cancers 254, 255 . another cytokine is gm-csf, which activates the production of granulocytes and monocytes from bone marrow myeloid progenitors and has shown adjuvant antitumoral effects 256, 257 . other cytokines, such as il-5, il-7, il-12, il-15, il-18, and il-21 258, 259 , are being tested in several clinical trials, and it is expected that some of them, either alone or in combination, can be used in future antitumoral therapy. other recombinant proteins are already on the market, some of which are derived from antibodies, with some advantages such as small size, low immunogenicity and general ease of production. examples are etanercept and abatacept (ctla-4 ig), which were approved by the european medicines agency in 2000 and 2007, respectively. the former is a chimeric protein that carries the external portion of the tumor necrosis factor (tnf) receptor linked to the igg fc region, which captures soluble tnf to block its inflammatory effects 260 . the latter example is a fusion protein that combines the extracellular portion of human ctla-4 and igg1 fc. abatacept is a competitive inhibitor that blocks t-cell activation and can be used in the treatment of inflammatory illnesses such as rheumatoid arthritis 261 . natural killer (nk) and lymphokine-activated killer (lak) cells. natural killer (nk) cells were described in the 1970s based on their capacity to eliminate tumor cells without prior sensitization, with differences observed compared with specific cytotoxic t cells (which are activated based on the recognition of the target cells) 262, 263 . in 1985, piontek et al. reported that nk cells have the ability to preferentially kill cells that had lost the expression of the major histocompatibility complex class i molecules 264, 265 . lymphokine-activated killer (lak) cells are a heterogeneous population that includes not only nk but also nkt and t cells, which can be generated in an in vitro culture of peripheral blood mononuclear cells (pbmcs) in the presence of il-2 266 . dr. rosenberg and collaborators carried out studies using these cells in the presence of il-2 (reviewed by rosemberg 251 ). these lak cells showed good antitumoral responses in 22% of the melanoma patients who received them as therapy 250 . however, secondary effects such as liver toxicity and the expansion of the treg population limited their therapeutic effect. researchers started to design new recombinant il-2 with some mutations to avoid the activation of tregs 267 , with linking it to polyethylene glycol (peg) to increase its half-life and efficacy 268 . another cytokine described later, il-15, showed similarities to il-2 in many respects 269 , and it had some unique advantages, such as the capacity to activate nk and cytotoxic t cells (tc) but not tregs. il-15 is being used in different versions (alone, as a heterodimer with receptor il-15/il15ra or il15rα igfc, or in an agonist complex with alt-803) 269 and in combination with other therapies in several clinical trials (examples: nct01021059, nct03905135, and nct03759184). more recently, researchers have focused their attention on other cytokines and combinations (such as il-15, il-12, and il-18) 270 , which are able to activate nk cells in vitro and induce a good responses in animal models. in some human clinical trials, remission has been observed for patients with acute myeloid leukemia 271, 272 , which broadens the options for the use of nk cells in the treatment of this pathology. the properties of nk cells reveal their versatility as treatments against tumors. nk cells are able to kill tumors through several mechanisms, including receptor-mediated cytotoxicity, antibodydependent cell-mediated cytotoxicity (adcc) and death receptormediated apoptosis, but they also secrete cytokines such as interferon gamma that enhance the antitumoral adaptive immune response. nk cell adoptive transfer (either autologous or allogenic nks) is currently being tested in clinical trials for hematological diseases and solid tumors, and numerous research groups have recognized their potential in other situations, such as transplant rejection and pregnancy. nk cell lines, memory-like nk cells and stem cell-derived nk cells are additional types of cells that can be used for tumor immunotherapy 273 . regarding other cellular therapies, nk cells as substitutes for t cells for use upon transformation with an chimeric antibody receptor (car) are being explored (see below). dendritic cells. paul langerhans identified dendritic cells in human skin in 1868 274 , but these cells were not named until 1973 by dr. ralph m. steinman (nobel prize in 2011) and dr. zanvil a. cohn, who chose the term because the cell morphology, with long extensions, resembles that of neuronal dendrites 275 . in humans, dendritic cells are obtained from different sources that vary in origin, maturation state and tissue distribution (skin, lymphoid tissue, circulating cells). among the main types of dendritic cells, plasmocytoids are conventional myeloid dc1 and dc2, pre-dc and monocyte-derived dendritic cells. in the epidermis, there are three types: langerhans cells (lc), monocyte-derived lc-like cells and inflammatory dendritic epidermal cells (idecs) 276 . as indicated above, dcs are antigenpresenting cells and are the only cells that are able to activate naïve t lymphocytes. a subpopulation of dcs also carries out a process known as cross-presentation. in this way, they facilitate the activation of both helper and cytotoxic t lymphocytes 277 . in addition to their participation in the immune response, they can be used in antitumoral therapeutic vaccines 277, 278 . it is possible to generate a type of blood monocyte-derived dendritic cell in the presence of a mixture of cytokines in culture 279 -a process that induces their subsequent maturation and activation in the presence of tumor antigens (cell lysates, recombinant or purified antigens, peptides, rna, dna, and viral vectors 280 ). these cells can also be obtained from bone marrow hematopoietic cd34 + progenitor cells 281 . other sources, such as circulating or skin dendritic cells, are relatively scarce and are therefore not usually used. after their differentiation and activation in vitro 278, 282 , dcs are exposed to tumor antigens and infused back into the patient (either by blood infusion or injected into areas near the lymph nodes or even directly into them) to reach the secondary lymphoid organs as soon as possible, at which point they can present antigens to the t cells. this approach is a type of individualized therapy and is therefore expensive. the first approved vaccine in which autologous dendritic cells were used was sipuleucel-t (provenge) 283 , which was a treatment for prostate cancer refractory to hormonal treatment. immunotherapy with dendritic cells is currently being tested in more than 200 clinical trials for various tumors: brain, pancreas, mesothelioma, melanoma and many others (clinicaltrials.gov identifiers: nct01204684, nct02548169, nct02649829, and nct03300843, respectively). the data indicate that the therapy is well tolerated and has led to increased patient survival in some trials. furthermore, complete cure and partial remission outcomes have also been observed. the lack of efficacy on other tests was probably due to the presence of immunosuppressive factors in the tumor environment. another therapeutic use of dendritic cells involves their induction of immunosuppression both in transplants and in autoimmune diseases 284 . in an autoimmune pathology such as multiple sclerosis, the intention is to achieve stable tolerogenic dendritic cells that can act against some autoantigens (such as myelin peptides) in the presence of vitamin d3, dexamethasone, or other agents 285 . phase i clinical trials have generally shown good tolerance to this therapy without serious adverse effects 286 . however, greater control of this treatment is necessary in several respects to obtain the best therapeutic results 284 ; e.g., the human immunology and immunotherapy: main achievements and challenges j varadé et al. type of dendritic cells and ex vivo differentiation, the antigens used, and the injection route are important considerations. gamma/delta t cells (tγ/δ). human t cells expressing γ/δ tcr cells have interesting properties, including the capacity to kill a broad range of tumor cells. the advantages of these cells in cancer therapy are based on their independence from mhc expression on tumor cells and that their relative insensitivity to some inhibitor molecules (such as pd-1). the initial clinical application, with the adoptive transfer of autologous vδ2+ cells after ex vivo expansion, showed only sporadic responses 287 , and different exploratory studies are currently being carried out to increase their clinical therapeutic use. allogeneic vδ2+ cells are also being explored in cancer therapy; e.g., they are being used against refractory hematological malignancies 288 and advanced cholangiocarcinoma 289 . although the basis of immune regulation was suggested centuries ago, regulatory t cells were described by sakaguchi et al. as cd4+ cd25+ natural regulatory t cells 290 that expressed the forkhead box p3 transcription factor (foxp3) 291 . later, induced or adaptive regulatory t cells were also identified, including different subsets that carry several phenotypic markers and express various cytokine secretion profiles 292 . all of these factors play crucial roles in the maintenance of immunological self-tolerance by suppressing autoreactive t cells. the manipulation of tregs to achieve therapeutic outcomes is a field of great interest, because of both their expansion and activation in diseases, such as allergic and autoimmune diseases, and as a potential targets for cancer immunotherapy 293 . tumor-infiltrating lymphocytes (tils). lymphocytes that infiltrate solid tumors are called tumor-infiltrating lymphocytes (tils). in 1957, thomas and burnet proposed that the immune system performs tumor immune vigilance, with lymphocytes as sentinel cells leading to the elimination of somatic cells transformed by spontaneous mutations 294, 295 . since the end of the 1980s, dr. rosenberg has been trying to prove and improve the effective use of tils. the process starts with surgery and the isolation of tils from a tumor, followed by til activation in culture in the presence of cytokines, cellular expansion and, finally reinfusion into the patient. since its inception, this therapy has been improved markedly, with an increase in optimal responses from less than 30% to the current 50-75%, in some cases. these higher success rates are due, in particular, to the prior preparation of the patient, including the depletion of lymphoid tissues, to avoid an expansion of regulatory cells 296 , myeloid suppressor cells and other cells that can compete with the transferred tils. currently, there are more than 200 trials in which tils are being used alone or in combination with other immunotherapies on several tumors, such as melanoma, metastatic colorectal cancer, glioblastoma, pancreatic cancer, hepatobiliary cancer, ovarian cancer and breast cancer. this individualized therapy has limitations; it can only be used on solid tumors, and the number and specificity of the tils and the type of tumor and microenvironment make standardizing this therapy difficult. chimeric antigen receptor (car). since tils include a variety of t lymphocytes with different specificities, the next step was to obtain t cells of a single type (monoclonal cells) carrying a clonal receptor capable of recognizing tumor antigens. this effort was carried out for the first time in mice and subsequently, in 2006, in humans with a transgenic tcr against the mart-1 melanoma antigen 297, 298 . these types of receptors are known as ttcrs, but their ability to recognize antigens is restricted since they can only identify the peptides presented by antigen-presenting cells on self-histocompatibility molecules. this situation changed because of one of the latest revolutions in antitumor therapy, the development of t lymphocytes that carry a chimeric antigen receptor (car) based on a specific antibody directed to a target surface molecule 299, 300 . these modified t cells can directly recognize tumor cells without required antigen processing or presentation by professional antigen-presenting cells. moreover, the car includes all of the necessary elements for intracellular signaling and activation of helper and cytotoxic t lymphocytes. car therapy was developed by one of its pioneers, dr. carl june at the university of pennsylvania in the united states 300 , who used modified t lymphocytes that carried a chimeric antigen receptor to target cd19+ leukemic b cells. after interacting with cd19+ cells, these modified car t cells were activated and able to proliferate and exert cytotoxic functions against target cells. in this case, both tumor and healthy b cells were affected. although bone marrow continues to produce b lymphocytes, in cases of severe b lymphopenia, it is possible to provide exogenous immunoglobulins periodically. the whole process of the current car t-cell therapy begins with blood donation, from which lymphocytes are purified and genetically modified in vitro by a viral vector, which carries the genes coding for the chimeric antigen receptor. the cells are expanded in the presence of cytokines in culture and are subsequently reinfused into the patient. this type of cellular immunotherapy is individualized for each patient, with his/her car t cells ultimately destroying the tumor. since the first generation of cars appeared, namely, a chimeric receptor composed of an anti-cd19-specific single-chain variable fragment linked to a transmembrane domain and intracellular signaling domain of the t cell receptor (cd3 ζ chain), researchers started to modify the original design. new generations of cars, including the cd3 ζ subunit together with other signaling domains, such as cd28, cd134, cd137 (4-1bb), cd27, and icos, or combinations (cd3 ζ, cd28, and cd134) 301 , have been developed in the second and third generations of cars to improve several aspects, such as the activation, proliferation and survival of car t cells. the fourth generation of cars show improved the antitumoral effects by carrying additional molecules (such as cytokines or drugs), improvements to the safety of car tcell therapy through the use of suicide genes 301 and many new designs, such as dual cars or the so-called split universal and programmable (supra) car system 302 . in addition to t cells, other types of cells, such as nk cells, are now being explored for use in antitumoral responses 303 . in an effort to avoid using personalized treatment, researchers are now working on universal cars that may be used on many different patients without inducing the problem of rejection [304] [305] [306] [307] . the encouraging results obtained with this therapy have led to interest from companies, and some commercialized examples are available, although many more "in-house" or academia-produced cars are in clinical trials. car t-cell therapy was initially designed for use against hematological cancers (leukemia and lymphomas), but many new opportunities have been opened for its use against solid tumors 308 , infectious diseases (such as hiv) 309 , allotransplantation, autoimmune diseases 310 and severe allergies 311 . china and the usa are the leading countries in producing car t-cell therapy, and numerous clinical trials are underway. immunotherapy for covid-19 patients coronavirus disease 2019 (covid-19), which is produced by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), affects millions of people in many countries. most of the infected patients (80-85%) are asymptomatic or have mild symptoms, but the disease in some patients progresses to a moderate or severe illness that requires hospitalization in intensive care units because of respiratory distress, multiorgan failure, and/or other pathologies, and more than one-half million fatal cases have been human immunology and immunotherapy: main achievements and challenges j varadé et al. reported worldwide. the most vulnerable population includes aging patients and those with comorbidities such as hypertension, diabetes and cardiovascular diseases. there are several aspects of the covid-19 pathogeny that suggest an overreaction of the immune system in severely ill patients, with increased levels of inflammatory cytokines such as il-6, il-1 and others (creating the so-called "cytokine storm"), together with blood lymphopenia and cd8 t cell and nk cell exhaustion. special therapies have not yet been identified to cure these patients, and preventive vaccines are not yet available, but some immunotherapies have been proposed as adjunct therapies, and some of these are currently in different phases of clinical trials 312 . the immunotherapeutic strategies include the following: future challenges in immunotherapy immunotherapy has been used for centuries, but only in recent years has this area expanded rapidly in several respects, mostly by the use of soluble elements (monoclonal antibodies and cytokines) and, more recently, with immune cells (cellular immunotherapy). there are many fields in which immunotherapy faces a range of challenges: vaccines. 1. researchers are working on reducing the number of injections by employing a combination of vaccines. several current vaccines contain components from 3-6 pathogens in a single injection, and these are able to provide adequate protection against all of these pathogens 315 . 2. researchers are developing more stable and durable vaccines. improvements in the half-lives of vaccines, for example, by lyophilization, while maintaining immunogenicity is expected to reduce current problems, especially those involved in the transportation of vaccines to remote areas 316 . in this respect, nanotechnology can help in the design of more stable vaccines that lead to slow antigen release and improved immunogenicity 317 . 3. researchers are working on vaccines that confer protection against all serotypes of a specific pathogen (universal). this outcome is especially important for pathogens with high variability (such as the influenza virus). researchers are designing vaccines that can protect against several variants by using common regions that can induce protective immune responses to all or most of the variants 318 . 4. researchers are developing alternative routes of administration (e.g., oral, inhaled, intranasal, skin, rectum, vagina) as substitutes for intramuscular or subcutaneous injections. one of the problems to be solved is the immune tolerance developed to elements delivered by the oral route, but some vaccines are already effectively administered by this route (such as the oral polio, cholera, typhoid fever and rotavirus vaccines). the intranasal route has also proven effective for some vaccines (nasal influenza vaccine), and vaccines administered through other routes are under investigation. 5. researchers are seeking the early protection of newborns 319 . newborns are very susceptible to infections due to their immature immune system 320 . moreover, the protection exerted by maternal antibodies transferred through the placenta during pregnancy against some pathogens interferes with the development of the newborn's own immune response. greater knowledge on ways to activate the immature immune system early will enable the development of vaccines for newborns. moreover, immunization of pregnant women may help to enhance neonatal protection against several pathogens 321 . 6. researchers are developing new and more effective vaccines. this effort is crucial for very prevalent pathogens such as mycobacteria tuberculosis, hiv virus or plasmodium falciparum. although there are treatments against these pathogens, most are not curative-as in the case of hiv; prevention is the best way to stop their spread. 7. researchers are working to address emerging pandemics. in the case of new pathogens, such as sars-cov-2, which has produced a recent global outbreak, effective vaccines are urgently required 322 . new technologies for vaccine formulations and routes of administration, the identification of immune-related factors of protection and modifications to the governmental regulatory and approval process for vaccines for emerging pathogens are challenges that must be faced to achieve a rapid vaccination procedure for outbreaks. hundreds of vaccines against sars-cov-2 (using different strategies such as live attenuated or inactivated pathogens, viral vector-based, viral rna, dna, recombinant proteins, peptides, etc.) 323 are now under development, and some are in clinical trials. however, the need to develop a new vaccine in a short period of time should not negate the main principles of vaccination use: safety and immune protection. 8. researchers are working on genetic (rna, dna) vaccines because they have great advantages, including no requirement for growing a pathogen. genetic vaccines can be obtained in a much shorter time, with much faster and safer production processes, and can be transported much more easily. however, the immunogenicity of these vaccines must be improved, and other problems need be avoided, such as the potential deleterious effects of integrating vaccine sequences into cells 324 . 9 . researchers are developing safer and more powerful adjuvants. many years ago, the only adjuvant authorized for vaccines was aluminum hydroxide (alum), but currently, several adjuvants are on the market 325 . the use of ligands that activate the innate immune response, such as those linked to tlrs or nanostructures with adjuvant effects, is currently under study. 10. researchers are boosting trained immunity, a new concept related to the innate immune memory-like described for nk cells (expansion) and macrophages (epigenetic modifications). knowledge of how to handle trained immunity will enable better vaccine design and more effective secondary responses 326 . 11. researchers are seeking to eradicate diseases from the earth. the greatest challenge, eradicating disease is possible in the short term for some pathogens, such as poliovirus. very few cases of polio have been recently reported, and these reports came from only three countries; therefore, it is feasible that this disease can be eradicated in the near future. human immunology and immunotherapy: main achievements and challenges j varadé et al. 12 . advances are challenged by the anti-vaccine movement. paradoxically, there are people who doubt the beneficial effects of vaccines, and they are putting the health of their own children and society in general at risk 327 . the effectiveness of community protection conferred through vaccinated people is disrupted by decreased numbers of immunized persons. this lesser coverage enables pathogens to infect the most susceptible people, such as small children, elderly patients and those who cannot be vaccinated due to certain pathologies or because they are undergoing immunosuppression treatment. thus, news about the return of illnesses that were nearly forgotten, such as tetanus in italy (the first case in 30 years), the death of a child in catalonia from diphtheria, or the exponential increase of measles cases (already counted in the thousands) worldwide 328 , should make parents think carefully about the risks of not protecting children by vaccines. the world health organization (https://www.who.int/topics/vaccines/en/) argues that anti-vaccine movements can roll back all the achievements thus far in this field and have cited this issue as one of the main challenges to be resolved. addressing the antivaccine movement requires a coordinated effort of professionals to inform parents adequately and perhaps other types of coercive measures that some countries are already applying (financial fines, denial of access to public assistance in childcare units, removal of authorization to travel/live in some countries, new laws, and so on). antibodies and cytokines. immunotherapy with monoclonal antibodies has been a true revolution for many pathologies, as has the use of certain cytokines and recombinant fusion proteins. it is therefore predicted that these approaches may have a bright future, and regulatory agencies are expected to authorize many more mab-based therapies in the coming years, especially given the good results obtained in clinical trials. complete antibodies or those modified to increase their functionality or decrease their immunogenicity, combinations of antibodies and cytokines, antibody fragments, etc., are only some of the many possibilities for this type of product, which will expand the range of therapeutic options. one of the main problems regarding the use of antibodies in therapy, especially in cancer, is based on their often unpredictable efficacy. large variability in terms of remission and durable clinical benefits between patients is observed (for example, in the antitumoral responses by antibodies directed to the checkpoint inhibitors). thus, the main challenge is to understand the situations in which an antibody will have the desired effect. it is crucial to find validated biomarkers (with predictive and/or prognostic value) that can help to stratify or select patients for the best immunotherapy. a better understanding is also required for tumor heterogeneity, resistance to some drugs and immunosuppressive microenvironments 329 . an in-depth immunological study, together with a personalized approach, is certainly the way to improve the success of these types of therapies. in combination with conventional therapies (radiotherapy, chemotherapy, and surgery), other immunotherapeutic drugs or cellular immunotherapies can also help to maximize the efficacy of this immunotherapy, but increases in toxicity will be another challenge to face. pathogens. the use of oncolytic viruses (ovs), bacteriophages that selectively infect bacteria, modified pathogens for vaccines or for antitumor immuno-activation, and the manipulation/ modification of the microbiota are some of the therapies that are being considered. ovs are designed to kill tumor cells and to activate the immune system against those cells. however, many of ovs have shown limited therapeutic effects when applied in monotherapy; therefore, much more work is required to improve their systemic antitumor effects and avoid the attenuation of the virus, which limits the viral replication. several obstacles, such as low viral delivery and spread, resistance to therapy and antiviral immunity, have been observed 330 . thus, the main challenges with oncolytic viruses are addressed by improving their antitumoral efficacy, including the optimization of viral delivery, the development of ovs engineered to activate the immune system (e.g., by releasing cytokines), and their use as adjuvant therapies or in combination with other immunotherapeutic agents, such as immunomodulators 331 . regarding gut microbiota manipulation as a therapeutic approach, fecal microbiota transplantation is an effective therapy for recurrent clostridium difficile infection 332 and is now being investigated for other indications, such as inflammatory bowel disease and cancer. some of the challenges facing microbiome transplantation are the lack of precise knowledge about the complete microbiome and the mechanisms of action involved in its therapeutic capacity, the large variability of its effectiveness and the external factors that affect it. more studies are centered on understanding how to manipulate bacterial colonies, the discovery of therapeutic molecules, nutrient competitions, etc., that are required for successful application. the best type of therapy (either individual or the combination of bacteria) is also under debate, along with how to reach the market by translating this individualized therapy into commercial scale products. the safety and potential adverse long-term effects are also being assessed. other components (nanomaterials and small molecules). nanomaterials. to obtain approval for the use of other elements from incipient fields, such as the use of different types of nanostructures, either alone or in combination with other immunotherapies, it is important to resolve certain issues. in the case of nanoparticle use, a better understanding of the interaction between nanomaterials and biological media; nanoparticle biodistribution, metabolism and biocompatibility; and the reproducibility of the synthesis and scaled up production of nanomaterials are among the issues to address. small molecules. a greater knowledge of several molecules involved in the immune system has led to the development of new therapeutic agents, which have been synthesized by traditional chemistry and block or activate intracellular signaling. the low cost of production of these molecules, along with the scaling and reproducibility of small-molecule batches, has attracted the attention of pharmaceutical companies interested in a whole set of new immunomodulatory drugs. a better understanding of the mechanism of action of small-moleculebased drugs and proof that they offer higher efficacy than existing therapies, either in monotherapy or in combination therapy, are challenges that face those seeking to engineer new types of targeting molecules. cellular immunotherapy. to date, cellular immunotherapy has been an individualized therapy with high production costs, and it requires the involvement of multidisciplinary groups in hospitals. a real challenge in the field of cellular immunotherapy is the acquisition of universal off-the-shelf cell therapies to replace those currently made to order in a very personalized manner. the development of universal cells, for example, in the case of car tcell therapy, would increase the number of patients who could benefit from this treatment at thus reduce the costs. other challenging aspects of cellular immunotherapy are the life-threatening toxicity of induced and their lack of effect on solid tumors, which is mostly due to the immunosuppressive tumor microenvironment. this approach requires new strategies to overcome these difficulties. in addition to cancer, cellular immunotherapy has a long history of use against autoimmunity, infectious diseases, allergies and transplantation rejection. it is also important to find biomarkers for prognosis/prediction that can help to optimize this method. other therapies that involve the use of activated nk cells, tumor-infiltrating lymphocytes, vaccination with dendritic cells, etc., are having partial clinical success. similar to other treatments, these approaches require further study, but it is feasible that they may become reality in the near future. greater knowledge of the immune system, especially concerning the variety of cellular and humoral components and the close regulation among them, the interaction with other systems or with elements such as the microbiota, will allow the development of new types of therapies that may be safer, more effective and specific but with much lower toxicity than found in current therapies. this long journey has been possible due to the efforts of numerous researchers (throughout the centuries), who have contributed with their work, creativity, successes and failures 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partially vaccinated children and adults in the united states and canada cancer immunotherapy, part 3: challenge and future trends oncolytic viruses: overcoming translational challenges oncolytic virus immunotherapy: future prospects for oncology the role of gut microbiota in clostridium difficile infection a.g-f conceptualized the study and conceived the project, and all the authors participated in writing the paper. competing interests: a.g-f is a copromoter of the spin-off nanoimmunotech. there are no competing financial interests in relation to the work described. key: cord-031907-ilhr3iu5 authors: nan title: isev2020 abstract book date: 2020-07-15 journal: nan doi: 10.1080/20013078.2020.1784511 sha: doc_id: 31907 cord_uid: ilhr3iu5 nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb 2015) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used 4t1 mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld1. as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan8 introduction: tetraspanin tspan8 is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan8 in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan8 are yet not fully understood. methods: to study the function of tspan8, we have established a fibrosarcoma model consisting of the parental cell line (ht1080) and its derivatives expressing tspan8 (ht1080-tspan8) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan8. co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan8. small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan8 expression. remarkably, among 20 of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht1080-tspan8 cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan8 is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan8 is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan8, indicating their interaction. summary/conclusion: our data show that the expression of tspan8 in the tumour cells greatly impacts ev cargo. moreover, localization of tspan8 on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan8. introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd63 and cd63-rfp expressing hek293 t cells by ultracentrifugation. gfp-cd63 and cd63-rfp evs were added to hek293 t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin-3, respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a1 and u18666a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin-3 which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin-3 recruitment to endosomes in presence of cd63-rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd63 ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, (1) a significant number of healthy donors should be included, (2) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, (3) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: (1) to establish a clinical reference, we collected blood from 224 healthy volunteers and prepared platelet-free plasma. (2) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. (3) we measured all samples by flow cytometry (apogee a60-micro) and used custom software (matlab r2018b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm2) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from 10^5 to 10^6 per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first 21 donors analysed, show that within the ev size range of 162-1,000 nm, the median concentration of cd61+ evs is 3.9•10^8 per ml (apc>150 mesf), cd62p+ evs is 1.1•10^7 per ml (pe>83 mesf), cd235a+ evs is 5.2•10^7 per ml (pe>123 mesf), and cd45+ evs is 1.8•10^7 per ml (apc>91 mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to 224 samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~8 μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd63 positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip 1660177) to j.l.m and t.r.g., department of defence (ca170373) to j. l.m., and the national institutes of health (r35gm119623) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using 50 ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was 35-78%, however particle: protein ratio (indicating ev purity) was approximately 10x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at 20 nucleotides (nt) and 60 nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~60 nt, whereas the protein factions contained small rna of 20 nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the 20 nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~60 nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af4 introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, 2017) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm 2019) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af4). methods: an af4 long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of 350 µm and a regenerated cellulose membrane of 10 kda were used. pbs supplemented with 0.02% nan3 was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af4 technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step 4*10^10 rev isolated from conditioned medium by a velocity gradient were injected in the af4 system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with 1*10^11 rev and injected in the af4 column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af4 correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af4 protocol for characterization of ev by af4 as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af4 has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev2018 guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about 5 times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were 6-10 times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading 9ug of total protein for western blot, cd9, cd81, cd63 and flot1 could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about 4 times higher than those of the rest two methods. the total protein amount of precipitation was 3 times higher than exodisc and sec, 10 times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were 100 times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was 8.6%, 31%, 50.1% and 42%, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov3 cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than 5 mins and 10 μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a 1 cm pet c-cp fibre tip was found to be~7e11 particles (50%). the release of evs was confirmed using dot blot analysis for cd9, cd81, and cd63 tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her2, cd24, egfr, epcam, ca125). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek293 s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd9, cd63, and cd81 and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with 20 tb positive and 20 tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag85. using a cut-off above the negatives, the auc of roc curves were 0.9975 and 1.0, respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l1 or glypican-1 on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t181 (p-tau t181) in human donor plasma were detected. in 8 ad and 5 healthy donor samples, p-tau t181 signal increased 134% in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the 3d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas9. results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn 118018, by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since 90% of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ6 integrin, a surface receptor for fibronectin and vitronectin. the αvβ6 integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ6 in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ6-negative endothelial cells (hmec1) were incubated with αvβ6-positive sevs from prca cells to evaluate the transfer of αvβ6 by immunoblotting (ib) and facs. the effect of αvβ6-positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec1. results: we demonstrate for the first time that the αvβ6 is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ6-positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec1. mechanistically, we demonstrate that hmec1 treatment with sevs from pc3 cells that endogenously express αvβ6, decreases pstat1(y701), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec1 treatment with sevs isolated from pc3 cells harbouring crispr/cas9-mediated downregulation of β6, or shrna-mediated downregulation of β6, results in increased levels of pstat1(y701). this sev treatment also results in a decrease of survivin in sevs and hmec1. summary/conclusion: overall, our findings show that αvβ6 in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r01-224769 (lrl); p01-140043 (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc1 (inflammationinducing rarely metastasizing murine hnscc line) and moc2 (highly metastasizing murine hnscc line) were used for this study. moc1 and moc2 cells were transplanted into mice tongues orthotopically, and moc1/moc2 derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc1 into moc2 tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc2 into moc1 tumours didn't promote metastasis. cd4 positive t-cell distribution in moc2 tumour was significantly changed by moc1 sev injection. t-cell deprivation treatment using anti-cd3 antibody increased the frequency of metastasis in moc1-sev treated moc2 tumours. from the result of proteomics analysis on moc1 and moc2 sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc1 sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r01ca163592 and r01ca206458 to aw. desmoglein 2 enhances squamous cell carcinoma tumour development through extracellular vesicles in an il-8/mir-146a-dependent mechanism introduction: the cadherin dsg2 is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg2 activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg2 enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg2 or a palmitoylation deficient mutant, dsg2cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of 106 cells in scid mice and monitored up to 4 weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg2 enhanced ev release by 40% and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single 20 µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg2, reduced ev release (~55%) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg2-evs were enriched with pro-inflammatory cytokines including il-8, a potent chemotactic and angiogenic factor. most importantly, il-8 was surface-bound on evs. furthermore, rnaseq revealed mir-146a, a negative regulator of il-8, to be significantly downregulated in response to dsg2. treatment with mir-146a mimic or mir-146a inhibitor decreased or increased, il-8 expression in scc cells, respectively. summary/conclusion: in summary, dsg2 plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir-146a, which in turn upregulates il-8 synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r01 introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for 24 hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas9 technology. results: we found enhanced phosphorylation of erk1/2 and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of 1411 ev-associated proteins, with 79 proteins uniquely expressed in control-evs. another 68 proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc-2016-cog-725229 evicare grant. hypoxia enhances the therapeutic potential of human cd34+ stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd34+ cell-derived exosomes (cd34exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd34exo. methods: cd34exo were isolated from primary human cd34+ stem cells cultured under hypoxia (1.5% o2, or normoxia (20% o2, n-cd34exo) using density gradient ultracentrifugation. cd34exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd34exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd34exo was evaluated via lc-ms/ms and 2-d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd34 cells. both h-and n-cd34exo expressed cd9, cd81 and cd63 surface markers. interestingly, h-cd34exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd34exo. in the same line, h-cd34exo also significantly improved perfusion (ratio: 0.93 ± 0.05 v 0.77 ± 0.02) and prevented ischaemic limb amputation (0% v 37.5%) as compared to n-exo (p < 0.05; n = 7-8) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir-210) that could be responsible for the proangiogenic and beneficial function of h-cd34exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd34exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd34exo, and thus, may represent a more efficient clinical strategy for cd34exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir-192 and −432 are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. 2ʹ-o-methylated rna duplexes inhibited mir-192 and −432 expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp1 cells with/without tnfα exposure were treated with exosomes and the expression of il-6, −8, and −10 was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir-192 and −432 expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp1 exposed to inhexos for 4 h. however, tube formation capacity is slightly decreased (~20%) by inhexo compared to norexo. summary/conclusion: exosomes from mir-192 and −432-depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd63. uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe3 and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd9 antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab7+ late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd9 (referred hereafter as cd9 fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd9-gfp fusion protein from femx-i melanoma, sw480 colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from 72 h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd9 fab was prepared from 5h9 hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd9 both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd9 fab significantly reduced the cellular uptake of cd9-gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd9 antibody stimulated both events. summary/conclusion: the effect of cd9 fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd9 both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd9 fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd63 allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m153 r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m153 r-cd63. cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m153 r-cd63 were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m153 r-cd63 and phluo_m153 r-cd63-mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m153 r tag is located on the surface of small evs. live cell imaging reveals phluo_m153 r-cd63-positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd63, alix, and tsg101 but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m153 r-cd63 construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r01gm117916, r01ca206458, 3u19ca1795 14-05s1, r01ca216248. uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas9-based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas9-based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas9-based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas9-based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited (1% spontaneous rate at 1h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr18-ce15-0008-01 and arc pja 20171206453 and pga1 rf20180206962. introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a 3-channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed >95% encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < 30 for 300 evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~150 bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po1 ca069246, ro1 ca204019, r21 ca236561 quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h9 (high cd81, low cd63) and promonocytic u937 (low cd81, high cd63) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev2018 guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. nta detected a population of particles with a mode diameter above 100 nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh118164 and ug3ca241694. importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r100 (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov-3 cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd9, anti-cd63, or anti-cd81. chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r100. following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~50 nm) distinguished 10x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced 10x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd9 ab significantly decreased detection of cd81 with less impact on cd63. this discrepancy indicated possible spatial patterning of tetraspanins with cd9 and cd81 closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase 3 knockout (casp3-/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type 2 diabetes (t2d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp3-/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp3-/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp3-/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t2d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on 17 rabbits. immediately after surgery and at days 7 and 14 post-surgery, 9 rabbits received 1ml injections of 200 µg msc-sevs and ha in both knees, and 8 rabbits received 1-ml injections of ha in both knees. at 6 and 12 weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at 6 weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from 6 to 12 weeks. by 12 weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score (10.33 vs 7.5; p < 0.001) and histological score (21.87 vs 11.11; p < 0.001). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre (13.41 vs 3.95mpa; p = 0.001) and overall periphery (12.22 vs 3.37mpa; p = 0.012], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, 75% o2) at birth and returned to room air at postnatal day (pn) 14. mice received a bolus mex dose at pn4. adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn4, 7, 14, or 28 to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd45+ immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr2-monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r01hl146128 (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered 0.5 h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day 3. haemodynamic changes and right ventricular hypertrophy were evaluated at 3 weeks. results: msc-sevs were 72 nm in size with cd63/81 expression. pkh26-labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il1b in balf peaked at 24h post-lps, whereas il6 peaked at 10h. histology showed significant intra-alveolar cell infiltrate at 72h. msc conditioned media attenuated il1b in balf, whereas a trend towards reductions in il1b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered 1 h post-lps as compared to 5 h, 10 h or 24h. for pah rats, sevs reduced right ventricular systolic pressure (51.4 ± 8.3 mmhg) as compared to control (68.1 ± 0.4 mmhg; p = 0.012), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at 21%o2, 5%o2 and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify 92 inflammatory proteins in the evs. results: there was no difference in evs cultured at 21%o2, 5%o2 or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd9/63/81) and internal proteins (alix, hsp70). evs from primed cells showed a > twofold increase of 5 cc chemokines and a > sixfold increase in cxcl5 and csf-1. protein cargo did not differ in evs from 21%o2 and 5%o2. summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of 4 2xtg-ad, 15 3xtg-ad, and 9 wild type (total of 28 mice; 14 female, 14 male; age: mean = 8.17, sd = 1.61, 5-11 months) . plasma samples were collected retro-orbitally for 5 weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde1) and the single endpoint (nde2) were immunocaptured by targeting the neuronal marker l1cam. we measured human total tau and pthr181-tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = 0.6885 and 0.7026, p < 0.0001, cortex vs nde1 and nde2; r = 0.4958, p = 0.0073, hippocampus vs nde1; r = 0.6058, p = 0.0006, hippocampus vs nde2). levels of p-tau in nde1 showed positive correlations with levels in the cortex (r = 0.4640, p = 0.0155) and hippocampus (r = 0.6086, p = 0.0008); however correlations were not observed for nde2 (r = 0.1152, p = 0.6273 vs cortex; r = 0.0531, p = 0.7926 vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. (5xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model (5xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than 2 million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in 48 patients with ms and 20 healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd14 + (monocyte), cd61+ (platelet), cd66+ (neutrophil), cd25+ (t-reg), and cd105+ (endothelium). pm10 and pm2.5 concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd14+ (p < 0.05), cd61+ (p < 0.003), cd66+ (p < 0.009), cd25+ (p < 0.003), and cd105+ (p < 0.003) in patients compared with controls. in cases, pm was inversely associated with cd14+ evs (pm2.5, β = −0.03; p < 0.05), cd66+ evs (pm2.5 β = −0.03; p < 0.03), and cd25+ evs (pm10 β = −0.02; p < 0.04; pm2.5, β = −0.03; p < 0.02). on the contrary, in controls pm was positively associated with cd14+ evs (pm10 β = 0.02; p < 0.03; pm2.5, β = 0.03; p < 0.02). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for 3 years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l1cam (l1 cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd9) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l1cam and glast) and immunogold labelling (cd9, cd63 and l1cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h9c2 cardiac myoblasts and raw264.7 monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da049267, da017763. vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top 10 causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale (30-200 nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot08.3 introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u937 monocytes (exu) and infected u937 (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd63, tsg101, . immune responses of naïve u937 cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of 173 host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il-6, il-8 and il-10 cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il-6-dependent manner. specific host signalling molecules are strongly modulated by the exi, including p38, jak2 and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p38 to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il-6-dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas9 based genome editing was performed to knockout cd11b in human monocytic thp-1 cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ-1. release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr3 receptor on monocytes as demonstrated by crispr-cas9 based cr3 genome editing in thp-1 cells, and by using cr3 knock out mice. isolated tgf-β1-transporting vesicles reduced the inflammatory response in human m1 macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β1transporting vesicles is investigated in detail and results in inhibition of il-1β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β1-transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr3-dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet 124 projects c4, c5, c6 and z2. introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, 7sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd47 is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin-1 and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd47 is associated with a poor prognosis for some cancers. conversely, cd47 blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd47 as a diagnostic and prognostic biomarker and as a therapeutic target. cd47 is also expressed on extracellular vesicles (evs), and we reported that cd47 expression identifies a distinct population of evs from those that express the traditional ev markers cd63 or mhc1. cd47-, cd63-and mhc1-enriched vesicles contain distinct small rna populations (pmid: 29416092), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd47 directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd47 regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd47-deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd47 and exportin-1/ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd47-deficient human t cells and in cd47-/-mouse plasma were enriched in 5ʹ-7-methylguanosine-capped micrornas and mrnas that depend on the exportin-1/rangtp pathway. knockdown of cd47 in wild type cells or thrombospondin-1 treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd47 in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd47 with components of the exportin-1/ ran nuclear export complex and its known cargos and between the cd47 cytoplasmic adapter ubiquilin-1 and the exportin-1/ran complex. interaction of cd47 with exportin-1 was inhibited by leptomycin b, which inactivates exportin-1 and increased levels of cap-dependent rnas in ev released from wild type but not cd47-deficient cells. summary/conclusion: these findings indicate that cd47-dependent thrombospondin-1 signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin-1-and gtpdependent physical interactions with the exportin-1/ ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc 009172). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein 2 (dsg2), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg2 promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg2 promotes ev release, but the mechanism by which dsg2 enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors 2-bromopalmitate, gw4869, and bafilomycin a1 were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg2/gfp, or palmitoylation deficient dsg2cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg2 on ev release was reduced by the palmitoylation inhibitor 2-bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg2cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg2 to the lysosomes for degradation. dsg2 increased expression and subcellular localization of flot1, a membrane lipid raft protein critical for membrane invagination. dsg2 also altered membrane localization of several early (eps15 and eea1), but not late (rab7, rab11, and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg2cacs mutants abrogated these effects. finally, dsg2-induced ev release was abrogated by the sphingomyelinase inhibitor gw4869 or augmented by the v-atpase inhibitor bafilomycin a1. summary/conclusion: the combined results of the drug treatments and functional mutations of dsg2 suggest that dsg2 plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif4e (4e) is an oncogenic protein that is upregulated in 30% of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif4e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d1, and mcl1. furthermore, our lab pioneered studies demonstrating that a subset of 4ehigh aml patients is clinically responsive to treatment with a 4e inhibitor (ribavirin) indicating the importance of 4e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of 4e-whether the oncogenic role of 4e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify 4ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether 4e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif4e upregulates cellular protein levels of the vesiculation marker cd63 by increasing its nuclear export. in addition to increased cellular expression, cd63, cd81, cd9, and flotillin-1 proteins are elevated in evs released from 4e-high cells. this is also associated with an increased release of vesicles that are 50-200 nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from 4e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that 4e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the 5xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to 3-months old 5xfad mice (~75 billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker 5ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin-1 beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) ot10.2 the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips 2.0 sendai reprogramming kit -protocol: clementino fraga filho uh 38583914.7.0000.5257/933.018). the ev were isolated from ipsc supernatants (cultured 24 h in mtesr-1 medium) by ultracentrifugation (100,000 g for 2 h at 4°c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia (1% o2). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping (45 min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro -043/19). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed-1). modulation of rna levels was assessed by rt2 profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk1, sod1, sod3, txn1 and txnrd2. characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin-1. summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to 1) evaluate which rna species were responsible for ph rescue, and 2) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). to identify the mediators of afsc-evs, we used deseq (fdr<0.01) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir17~92 and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated 35 genes, which formed 415 mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir17~92 cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße 29, 69120 heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße 29, 69120 heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a549 with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp46. results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of 69 lncrnas were significantly changed within sevs isolated from a549 cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha6-1, acted as a competing endogenous rna (cerna) for hsa-mir-4485 which subsequently up-regulate the natural cytotoxicity receptor (nkp46) expression. furthermore, we verified over-expression of linc-epha6-1 significantly enhance the cytotoxicity of nk cells against zika virus-infected a549 cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha6-1 wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc3 in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc3 during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a549 with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha 1b (defa1b) expression was significantly increased within evs isolated from zikv infected a549 cells. additionally, we found that the extracellular defa1b but not the intracellular defa1b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa1b can retard cell cycles of host cell. we verified defa1b could bind with the origin recognition complex 1 (orc1) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa1b decreased the orc1 level in nuclear. interestingly, evs with defa1b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa1b wrapped in evs, and defa1b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa1b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after 4 hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and 40 were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box 1 (hmgb1), neurofilament (nf-l) and phosphorylated tau-181 (p-tau) proteins. results: using 3 different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of 0.82 ± 0.16. using 4 different combinations of clinical data and the 3 nev protein targets, selected clinical data and hmgb1 best predicted cognitive impairment (auc = 0.82). the most important features included cd4 count, hmgb1, nf-l and education. summary/conclusion: specific clinical features plus nev hmgb1, an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau-181 elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv-1, htlv-1, rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected (100%) cells. these infected cells were initially put in g0 quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release (20% fbs + pma/pha) at 0, 3, 6, 12, and 24 hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd63, cd81, and cd9) at 6 hours and a gradual decrease of all ev associated proteins by 24 hours. however, the ev from hiv-1 infected cells showed all three tetraspanins present at 3 hours and expression gradually increased up to 24 hours. when compared to htlv-1 infected cells, the three tetraspanin proteins peaked at 6 hours and expression continued to decrease up to 24 hours. htlv-1 infected cells also showed a unique pattern of cd81 expression. autophagy associated proteins (lc3a, lc3b and p62) from uninfected cells and htlv-1 infected cells plateaued at 6 hours, whereas in hiv-1 infected cells their expression continued to increase and peaked at 24 hours. hiv-1 viral proteins (p24, gp120, nef) expression was present at 6 hours and continued to increase and peaked at 24 hours. htlv-1 proteins (p19 and gp46/61) peaked at 6 hours and gradually decreased overtime. hiv-1 and htlv-1 rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from 6-and 24hour supernatants were performed on three naive cells. hiv-1 supernatant 6-hour sample was found not to be infectious. however, hiv-1 was successfully rescued from 24-hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3-colour panel, which included annexin v (for the majority of circulating evs), cd41 (for platelet-derived evs) and cd105 (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273. prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p) , were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of12 symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at 10 min and 3 hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [18 f]fluorodeoxyglucose (18 f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked 18 f-fdg in vivo by pet-scan, within seconds of ev, rbc or free 18 f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free 18 f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" 18 f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the 3ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the 3ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p22. cryo-tem was used to determine the morphologies of mvs and phage p22. the purity of mv isolates was validated by measuring the particle concentration before and after triton x-100 treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p22 was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto 82. results: by incubating mvs and af532-labelled p22, the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p22 attachment. the binding ability of p22 to mvs is greater than that of bacteria. we confirmed that p22 can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto 82, we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants 21934004, 21627811 and 21475112). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi293 f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi293 f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium111(in111)-dtpa or xenolight dir post-ev isolation. ct26 tumour-bearing balb/c mice were intravenously dosed with 1 × 1011 evs followed by imaging at 1 h, 4h and 24h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to 24h post-injection. results: xenolight dir and (in111)-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in111-dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at 24h post-dose, with less than 5% ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in111-dtpa and nluc evs showed rapid clearance of vesicles from circulation, with 15% of injected dose detected in blood after 30 min and less than 5% after 16 h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs1 instrument (spectradyne llc, usa). pe-cd235a (anti-glycophorin a) and alexa647-wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn 5/200 glass column filled with sepharose cl-2b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of 163.5 ± 0.7 nm and standard deviation of 30.6 ± 0.6 nm. the concentration of revs measured by mrps was 3.14*10e11 particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd235a and with alexa647-wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from 10e9 particles/ml to 10e11 particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was 10e9 particles/ml for alexa647-wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. 9, 19868, 2019) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd 121326 and nvkp_16-1-2016-0007. zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which 1-2 μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < 50 ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below 20%. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes (1-2 μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon 2020 -future and emerging technologies (h2020-fetopen), id: 801367. marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd9/anti-cd63/anti-cd81antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. 2018-1720) and index (european union's horizon 2020 research and innovation programme under grant agreement n°766466) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size (30-2000 nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than 70 nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs >80 nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h9c2 cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg101, syntenin1, cd63 positivity while grp94 was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd63, cd9 and cd81. cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h9c2 treated with evs showed activation of pfak after 10ʹ of treatment, this induction was 1.5 times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y1, y3, y4) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density 1.11-1.18 g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir-122), in both ev and lpp (e.g. mir-16 and mir-21), or in ev (e.g. mir-150). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y4/y3 ratios and pbmcspecific y3/y1 ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type 1 diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included 75 type 1 diabetic individuals (41 normoalbuminuric, 15 microalbuminuric and 19 macroalbuminuric). urine samples were collected either overnight (n = 38) or during 24-hours (n = 37) and uevs were isolated from 20-30 ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq 2000 (illumina) pair-end protocol. the association between kidney specific gene expression levels (>4 fold higher compared to other tissues, n = 53) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with >5 million pair end reads. we detected on average expression of 12,642 genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and 24-hour urine collections. comprehensive look-up of 53 kidney-enriched genes revealed expression of >73% (total 39) of these genes in urine evs with high expression of five kidney-specific genes (slc12a3, slc12a1, nphs2, aqp2 and slc22a12). pc analysis combining the impact of 39 kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc1 axis, and the axis also correlated with the albumin-to-creatinine ratio (p = 0.0004) explaining 11% of the variance (p = 0.005) in the whole data set. the pc1 axis also showed correlation with hba1c (p = 0.003), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek-293 t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav9 or eaav6 and standard aav9 or aav6 encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = 3) after glenn procedure and swine (n = 3) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded 6 and 12-months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed (98%, fold-change<2). plsr reduced the dimensionality of the swine mirna dataset to 50 mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > 0.9). summary/conclusion: these findings support the use of: (1) swine pab model for rv failure in hlhs, (2) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and (3) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi29 prna three-way junction (3wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail 3wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc5 sirnas loaded evs (fig.1 ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: 1) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. 2) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. 3) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. 2018 , 13:82. li z et.al. sci rep. 2018 introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with 3 differently coloured monoclonal antibodies per well in a 96 well plate format (n = 242 separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = 36) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed 10 cases of cancer and 26 screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < 0.05, with aucs >0.80), including epcam+/cd63+ events likely from cancer cells and cd41+/cd62p+/cd9+ microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best 4 markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best 4 surface markers to distinguish sclc subtypes were ceacam5, fam174a, lrfn1, epha2. immunoblot analysis validated ceacam5 and epha2 but not fam174a or lrfn1. we also found that ncam1, a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd24 is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam5 and epha2 corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd24 expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u01 ca224276 and nih u54 ca217450. leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood1. while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes)2 is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna)3,4, a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her2, egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl60) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs (100,000g ultracentrifugation) and soluble plasma (sup)5. results: hras-mediated cellular transformation (in ras-3 cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly6g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from 60 participants, including 31 early-stage crc patients, 19 adenoma patients, and 10 normal controls (nc), and characterized according to misv2018 guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of 120 additional participants. results: a total of 1615 rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, 888 rna species were differentially expressed between plasma sevs in adenoma and nc participants. 1160 rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into 6 modules, which exhibited different expression trends during the carcinogenesis of crc. a 60-gene combined tsne model consists of the top 10 genes in each module could perfectly classify crc, adenoma, and nc samples. a 6-gene combined tsne model consists of the top 1 gene in each module could roughly distinguish crc and adenoma from nc, with only 1 sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of 120 participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago2 protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq2500. our nanofluidic pillar array is manufactured in an sio2 mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately 1-3% of prostate tissue-specific rna were discovered in evs; iii) over 77% (17 of 22 rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over 50-70% of noncoding rna (40-60% was mirna), while tissue predominantly yielded rrna (>60%); v) finally, gene set analyses generated that over 90% of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of 68 prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, 80% of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to 1. diagnose pdac patients from healthy and disease controls and 2. distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = 29). subsequently, we trained our panel of biomarkers using a training set of n = 47. finally, we validated the performance of our platform using an independent blinded test set of n = 145. results: the results of a blinded test set achieved an accuracy = 92% and an auc = 0.95 on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = 0.85 and accuracy = 89% with sensitivity of 90% and specificity of 88% on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca19-9 for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m0) from distant metastasis patients(m1) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove26 type 1 diabetic mice, and their wild-type (wt) littermates. at 20 weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove26 mice or their wt littermates. (144.2 ± 7.6 vs 123.5 ± 4.9 [ove26] vs. 114.6 ± 5.7 mmhg [wt], p < 0.05). ms identified 297 independent proteins with at least 2 peptides per protein. of these, 163 proteins were found in all groups studied, 48 were exclusive to wt mice, 23 were exclusive to ove26 mice and 4 were exclusive to ttrhren mice. in addition, 22 proteins were observed with >1.5 fold change (fc) compared to wild-type mice, and 68 proteins were reduced by >50%. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove26 and ttrhren mice compared with wild-type controls. similarly trem-like transcript 1, sarcoplasmic/endoplasmic reticulum calcium atpase 3 and junction plakoglobin were all downregulated in both ove26 mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~1-5um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase 1 (rock1), a process that can be pharmacologically inhibited using a rock-1 inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp-1 and il-8, suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt1141732, gnt1140187) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for 8-weeks (038/15; a02/16-61-15). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd68+ cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp-1 and pai1 and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col1a1, col4a1) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the 8-weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling 3 (socs3) protein. uptake of socs3-containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription 3 (jak-stat3) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs3 is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of 2-deoxy-d-glucose (2-dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without 2-dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (>100-kda) socs3 secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg12d mice bearing lung tumours 16 weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of 2-dg. vesicular (>100-kda) socs3 secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas 2-dg enhanced, socs3 and ev release by mh-s cells. treatment of rat ams with cse (1%) attenuated secretion of socs3, an effect that coincided with increases in glycolytic activity, and co-treatment of ams with 2-dg abrogated the inhibitory effect of cse on socs3 release. finally, ams collected from lsl-krasg12d mice exhibited a deficiency in socs3 secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of 2-dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs3 is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (δtolc) in the model cyanobacterium synechocystis sp pcc6803 (s6803), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation (4400 g, 10 min) and filtration (0.2 µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of 100 kda), and later ultracentrifuged for 3 h at 100000 g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s6803 δtolc. both wild-type (wt) and δtolc cells were then challenged with different metals. compared to the wt, δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s6803. in addition, cu could also be detected in isolated δtolc-ev. in addition, more ev were detected when s6803 wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/130478/2017; feder-compete2020-poci-fct project: poci-01-0145-feder-029540. juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects 1/800 and is caused by mutations in pkd1 and pkd2, which encode polycystin-1 and −2. in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin-2 motors and kinesin-3 klp-6/kif28 transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab11a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab11-exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct116 colorectal and hela cervical cancer cell lines to identify rab11-exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab11-exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp5, chmp1 and ist1, are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab11-exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt-0 components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab11-exosomes. in flies the escrt-0, hrs, localises to the limiting membrane of rab11-endosomes. comparative proteomics reveals other proteins enriched in rab11-exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab11-exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional (3d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in 2d display decreased exosome release and increased uptake compared to 3d cultured cells. exosome release in 3d culture was inhibited with the exosome release inhibitors brefeldin a and gw4869, but was not significantly altered by knockout of rab27b. in addition, disruption of polarity signals provided by 3d culture did not impact exosome release or uptake in 3d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi3 k signalling. summary/conclusion: release and uptake of exosomes is altered in 3d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r345 w-fibulin-3 induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin-3 versus rpe cells expressing r345 w-fibulin-3 and investigate the effects of these evs on rpe cell differentiation. methods: arpe-19 cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin-3 or luciferase-tagged r345 w-fibulin-3. evs were isolated from the media of arpe-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe-19 cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around 30 nm and a large group with diameters around 100 nm. moreover, tem and cryo-em showed an increased amount of small evs (~30 nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r345 w-fibulin-3 causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe-19 cells overexpressing wt-fibulin-3 contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r345 w-fibulin-3 contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad4, leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63+ evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op2.06 = pf17.06 heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of evintroduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir-29a-3p (mir-29a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir-29a-3p (mir-29a). in situ hybridization of the frontal brain area revealed that mir-29a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir-29a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir-29a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir-29a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir-29a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r01da042379 blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)-1β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, 0.1-1 µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (<100 nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il-1β content of mps increased 68-fold. administration of an il-1β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor-2 to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il-1β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n00014-16-1-2868 from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c9orf72 als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod1, fus and tdp43 are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g4c2 aberrant nucleotide repeat expansion in c9orf72 gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at 21,000xg and the smaller evs at 100,000xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc34 were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc34 transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc34 were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc34 with gw4869. to understand the downstream effect of polygr+ evs in recipient cells we studied tdp43 mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting 2.3 million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human 16-17-week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c57/b6 female mice with mog35-55 peptide. results: we found that hasc-evs expressed high levels of galectin-1 and promoted a significant increase of the immunoregulatory enzyme indoleamine 2,3dioxygenase-1 enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper 17 (th17) cells, and increased percentage of regulatory t cells (treg-foxp3+) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l1 cell adhesion molecule (l1cam) from serum samples of 146 wrap participants who were cognitively unimpaired at baseline (mean age 62.4 ± 6.3 years old; 71.2% females; 42.5% apoe4 carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p181 and p231) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p181-(p = 0.07) and p231-tau (p = 0.05) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau231 (p < 0.01), total tau (p < 0.001) and ptau181 (p < 0.05) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap-2 mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap-2 transgene was inserted at the mouse h11 locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a 3ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap-2 mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that 93% of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim1215 crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus 16 (hpv16) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in 15 (88%) saliva samples collected from hpv16-driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv-16 dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv16-driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd9, cd63 and cd81) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv-16 dna. the proteomic signature of salivary exosome was significantly (p < 0.01) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase 1 (pgk1), glyceraldehye-3-phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev2018 guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ3 integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c4-2b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du145 cells treated with sevs expressing or lacking the αvβ3 integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c4-2b cells that express αvβ3, but not from the control cells that lack αvβ3, induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ3 integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ3 integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p01-140043, r01-224769 to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b16f1 melanoma cells and ht1080 fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab27a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv-1 tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir-7 was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv-1 protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin 5, resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir-7 was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir-7 was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir-7 oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir-7 with downregulation of claudin-5 was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv-1 tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir-7 as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely (1) mechanical extrusion, (2) freeze-thawing, or (3) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying (1) extent of fusion between evs and synthetic calceinloaded liposomes, (2) efficiency of calcein loading, and (3) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg-19-116-01-cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm3 inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein 3 (ifitm3), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm3-containing evs (ifitm3-exos) were overall safe to the animals and suppressed zikv viraemia by 2 log10 s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm3 protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm3 was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm3, an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec9a scfabs to target conventional type 1 dendritic cells (cdc1s), anti-cd3 scfabs to target t cells, and cd40 ligand to target b cells. in mice, systemic administration of anti-clec9a evs resulted in a 75% increase in the percentage of cdc1 cells that take up evs over controls. anti-cd3 evs resulted in both an increase in the percentage of ev positive t cells (3.75 and 3-fold for cd4+ and cd8+) and the number of evs per cell (15 and 7-fold for cd4+ and cd8+) in the blood. furthermore, in primary mouse dendritic cells, anti-clec9a evs loaded with sting agonist achieved a 15fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec9a evs reduce the required sting agonist dose 10-fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to 1) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; 2) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd63, hsp70, flo-1, and tsg101 (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e20.5. ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after 24h, dams and pups were sacrificed. 3d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = 27) and nitrofen-exposed (n = 45) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os22.5 introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves4us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves4us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles 2018 (misev-2018 guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened 20 strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev-2018. our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves4us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon 2020 research and innovation programme under grant agreement no 801338. sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with 1000-10,000 higher levels than in parental cells. contrarily, cell-tropic mirnas were only 10-100 times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in 0.001 mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min6 β-cell line treated with diabetogenic cytokines (tnfα, il-1β, ifnγ, 48h). evs were also isolated from human type 2 diabetic (t2dm) and lean non-diabetics (lnd) plasma. gw4869 (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min6 β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t2dm plasma revealed a~twofold increase (p < .05, vs. control (ctl) and lnd ev). immunofluorescence staining of cd9 and cd63 expression was significantly elevated in human t2dm pancreas (p < .05, vs. lnd). while acute inhibition of ev formation with gw4869 (5 µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~7 and 2 fold vs. cytokines alone, p < .05). moreover, functional assessment of mouse islets exposed to cytoev (48h) resulted in suppression of gsis (~55%, vs. untreated, p < .05). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl10 (~40 fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl10-cxcr3 signalling (p < .001) and downstream pathways (e.g. nfκb; p = .016 and jak/stat; p = .021). furthermore, inhibition of cytoev (gw4869) with cytokines markedly decreased cxcl10 (~30%) and cxcr3 receptor (~65%) expression in min6 β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl10 expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl10 consequently activating cxcr3 signalling and downstream pathways to impair β-cell function in diabetes. synergy between 15-lipoxygenase and secreted pla2 promotes inflammation by formation of tlr4 agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor 4 (tlr4), but the activating ligands were not fully determined. additionally, several enzymes, among them 15-lipoxygenase (15-lo) and secreted phospholipase a2 (spla2) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek293 cells exposed to 10um a23187 and isolated with ultracentrifugation. 20:4 lysopi was oxidized for 10 min with 15-lo. additionally, synevs were prepared from phospholipids (pls), oxidized with 15-lo and hydrolysed with spla2. activity was measured by qpcr and elisa on wt and tlr4-ko macrophages. 15-lo oxidized 20:4 lysopi was analysed by mass spectrometry. spla2 activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr4 ko mice (c57bl/6 mice) with spla2-iia injection was used (approval no. u34401-14/2019/8 by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr4 with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, 15-lo oxidized synevs, but only 15-lo oxidized lysopls activated cytokine expression through tlr4/md-2. hydroxy, hydroperoxy and keto products of 20:4 lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla2 activity, which we detected in the synovial fluid from patients, promoted formation of tlr4 agonists after 15-lo oxidation. injection of spla2-iia into mice promoted k/bxn serum induced arthritis in tlr4-dependent manner. summary/conclusion: both 15-lo and spla2 are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr4 signalling. the formation of tlr4 agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h2020-msca-itn project tollerant (grant no. 642157), slovenian research agency (project no. j3-9257 to mmk, research core no. p4-0176 to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl2. injured and uninjured muscles were dissected 24h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m1, m2, and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m1, m2, and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m1 and m2. indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv2 cells. we showed that m-evs are internalized by both mouse bv2 and human c20 microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc3b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c20 microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, 510515, p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = 48): half dying within 2 years (short-lived group) and half surviving ≥10 years (long-lived group) after the blood draw; all matched for age (median age 77.3 ± 1.7 years, range 72-80), gender (50% female), and race (50% white/50% black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd81, cd9, cd29, cd63, cd8, cd4, cd68, cd14, cd56, cd15, cd19, cd235a, cd41a, cd34, cd31, hla-abc, hla-g, hla-drdpdq, cd90, cd73, cd105, m cadherin, ryr1, ryr2, fabp4, dlk1). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x-20 flow cytometer as we recently reported. graphpad prism 8.0 software was used for statistical analysis. results: we found significantly increased percentages of cd9+, hla-abc+, cd31+ and cd41a+ large evs (1000-6000 nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium (100-1000 nm) or small (<100 nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd9 is expressed by hscs and immune cells. cd9 regulates homing of human cord blood cd34+ hscs, and delivers a potent cd28independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd31 is expressed by hscs, immune cells and epithelial cells, and cd31 + plasma evs declined with age in healthy people. cd41a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd9+, hla-abc+, cd31+, cd41a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after 10 days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after 10 days of msc expansion under dynamic conditions, we collected 1.3 l of conditioned medium with approximately 0.5 million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd9, cd63, and cd81. simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than 100 times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht29 and sw403. methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of 38 conditions. a431 evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp90 and membrane marker egfr, both with high expression in a431 evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht29 and sw403 evs through probing of trios of surface (2) and internal (1) protein targets. results: the selected tevam protocol successfully maximized hsp90 signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than 450 combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht29 and sw403 evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc2.4 mouse dendritic cells were cultured in flasks containing a total of 1.2 l of ev-depleted media (10% fbs, centrifuged 18 hr. x 100,000 g.) when cells reached 80% confluency, conditioned media was collected, depleted of debris with two 10 min. x 2,500 g spins, and concentrated down to~5 ml using a pall jumbosep 100 kda mwco filter. the ev concentrate was purified from protein using an izon qev-10 column, with 5 ml fractions collected. the protein content of the ev-containing fractions was analysed by a280, pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc2.4 preparation, one highly abundant population with a power-law distribution, whose peak diameter is below 60 nm, and a second, less abundant population with a peak diameter at approximately 140 nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, 40 μl of~108 particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of 8 clinical samples (7 ovca and 1 non-malignant control) were tested in combination with ovca skov-3 cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov-3 cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov-3 evs from both ldl and vldl. pca also differentiated skov-3 evs incubated with ldl from skov-3 evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein 3 (vamp3, cellubrevin) on evs. conversely, knocking-down vamp3 in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level 4 (low, no matches in databases) to level 2 (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from 10 ma-act, and 10 controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd9, cd63, and cd81), platelet evs (cd41), pro-coagulant evs (annv), and red blood cell evs (cd235). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v3.0. fold change was calculated by δδcq between ma-act and ctl for mirna expressed in ≥ 60% of samples in at least 1 group. we identified the top 20% of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) 1.2 fold change in expression, (ii) area under the receiver operating characteristic curve of 0.75, and (iii) glass's δ of 1. for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~110 nm) and concentration (~7.5 x 1010 particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd41+ evs, and to a much lesser extent cd235+ evs. of the 207 mirna expressed in ma-act and/or ctl plasma evs, there were 110 mirna that have at least a 1.2 fold increase or decrease in ma-act. 10 mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, 5 of the 10 mirna correlate with a ma associated variable, including frequency of use and age of first use. together the 10 mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the 10 mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old (24-26month old) and young (6-8-week old) c57/bl6 mice and collected bm-evs by differential centrifugation (2000 × g for 30 min, supernatant collected and centrifuged 100,000 × g for 1 hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with 2 x 10^9 young bm-evs via tail vein, daily x 3 days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca-1+/cd150+; lsk-slam) and tested stem cell function in competitive bone marrow transplants (4-5 recipients/group). results: at 6 months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: 15% ± 5% (young evs) vs. 61% ± 14% (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: 7% ± 4% (young evs), 27% ± 10% (old evs), 15% ± 2% (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately 7-fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants 5p20gm119943, dk112808-01a1 and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op3.04 = ps15.04 combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op3.08 = ps15.08 introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by 5-aminolevulinic acid (5-ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd63+/cd81+ evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using 5-ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between 100-300 nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of 658 proteins were detected in fgr evs and, according to prediction software effectorp, 12.5% of these were potential effectors. similarly, 70% of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a 39% overlap in proteins identified in the 465 cd and the 658 sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with 15.4% and 12.9% of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op3.11 = ps15.11 commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately 60 usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just 4.8 million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in 2017 with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since 2017 the hevi has organized 2 research symposia, 2 hands-on training days, hosted 2 international students as well as providing one-on-one training for 41 individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported 38 people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with 10 grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of 4 researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on 25 october 2019, we organized the 1st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg1, introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg2 was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg2 shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h1975 as well as the ovarian cancer cell line skov3 were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb2/her2 and insulin-like growth factor 1 receptor (igf1r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants (293 t, a431, u87, thp-1, sh-sy5y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v6 (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd9, cd63, cd81, cd82, cd151, cd53, cd231) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd9, cd63 or cd81) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd9 on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than 50% of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from 27 donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a 15-fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd63 immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased 5.1x (p < 0.01; 15/30 vesicle-related top go terms) in proteins common to intact arteries and valves (n = 1,411). proteomics found 24 ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified 1,104 proteins and 123 mir cargoes linked to 5,182 target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. 179 proteins and 5 mirs were significantly altered between artery and valve evs (q < 0.05); multi-omics integration found that evs differentially modulated cellular contraction and p53mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r01 hl136431, r01 hl141917 and r01 hl147095 (ea). mir-20a regulates exogenous cd82 expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir-20a regulating the expression of exosome cd82 on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd82 in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc-803 was used as the research object. the exosomes were extracted from the culture supernatant of mgc-803 by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd82 in exosomes was detected by elisa. the expression of cd82 in exosomes and cd82 in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir-20a lentivirus experimental group (mir-20a group). the lentivirus control group (mir-20a/con) was transfected into cells. qrt-pcr was used to verify the status of mir-20a after transfection; western-blot was used to detect the expression of cd82 and downstream erk1/2, akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd82 in exosomes was detected by elisa and western blot. the expressions of cd82, akt, erk1/2 and m tor in mir-20a group were significantly lower than those in mir-20a/con and mock groups. cd82 protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir-20a group were significantly lower than those of mir-20a/con group and mock group. the weight of the nude mice in the mock group and the mir-20a/con group decreased, while the weight loss in the mir-20a group was not significant. the tumours in the mir-20a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir-20a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir-20a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd82. streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time (1, 2, 3, 5, 9, 24, 48 or 120hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of 1:10 and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x-100 labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and >5-hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to 24 hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core 700/superose 6 resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to 24 hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd 1 and 2 in tasmanian devils. methods: exosomes were isolated from three different dftd-1, dftd-2 and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd-2 abundantly express the inhibitory checkpoint molecule cd200 relative to exosomes from dftd-1 cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [1] , artificial coiled-coil peptides with receptor targeting [2] , and cell-penetrating sc18 peptides [3] derived from cationic antimicrobial protein, cap18 for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [4] . methods: [methods] cd63 (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [5] . we developed the methods for modification of arginine-rich cpps or sc18 peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan-4) and macropinocytosis signal transduction [1] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [2] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [4] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [1] sci. rep. 6, 34937 (2016), [2] chem. commun. 53, 317 (2017), [3] chrmmedchem. 12, 42 (2017) [4] anticancer res. 39, 6701 (2019), [5] sci. rep. 5, 10300 (2015) os28.3 multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of 300-1200 nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, 1) mcmv bud from localized sites on the endothelial pm, 2) mcmv contain mvb, and 3) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated >200 distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained >200 distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans (70-100pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [1] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [2] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science 2018 361 (6409)). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. 2019 87 (2); infect immun. 2016 84 (2861)). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression >200-fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd4 + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd4 + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd4 + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir-142-3p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir-142-3p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # 2016/r/10 and # 2018/r/4 revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = 10) and control (n = 5) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., 2017) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate 3000 rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of 16 statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx30, stau1 and vcam1, respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct01860794). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir-451a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir-451a had anti-inflammatory effect on cells of msa-p condition such that the level of cx3cl1 expression and its receptor, cx3cr1 were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir-451a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., 2019) . the aim of this study was (1) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and (2) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase-1 and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase-1 were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of 60 ms patients (45 with relapsing remitting, 15 with progressive ms) and 31 healthy controls, targeting the neuronal-specific marker l1cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c1q, c3, c3b/ic3b, c4, c5, c5a, c9, factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced (2.5-fold; p < 0.0001 for both), whereas multiple complement components in ms aevs were markedly increased (c1q: 2.5-fold change; c3: 1.25-fold change; c3b/ic3b: twofold change; c5: 1.4-fold change; c5a: 1.4-fold change; factor: 1.5-fold change; p < 0.0001); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c1q, r = −0.7 and p < 0.0001; c5, r = −0.6 and p < 0.0001; factor h, r = −0.46 and p < 0.0002/synaptophysin vs: c1q, r = −0.75 and p < 0.0001; c5, r = −0.65 and p < 0.0001; factor h, r = −0.52 and p < 0.0002), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-(1-carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go (0.5 mm) for 24 hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. 2019 ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp9, angptl-4, lcn2, ptx2, s100b, rage, dj-1 and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ1-40, aβ1-42, ptau t181 and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx-2, lcn-2, dj-1, on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl-4 while the expressions of mmp-9 and angpl-4 were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn-2 by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp9, agntl-4, ptx-2, dj-1, aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u87 mg), microglia (sv40), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either 0% or 50% cse for 24 hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions 1-6), ev (7-10), and protein (11-14) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd63, cd81, and tsg101) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da040385, da047807, and ai144997. a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles 30-150 nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from 8 patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of 30 samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of 14.83 ng (range 1.72-137.17 ng) and no samples fell below the threshold required for library preparation and sequencing (10 pg) determined by inhouse optimization on the smart-seq v4 ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia 30 years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from 19 participants with ad and 14 non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r2 = 0.98). in serum, 189 micrornas were differentially expressed by a fold change ≥|1.1| in the ad and control groups (p ≤ 0.1) and 251 micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to 145 diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the 189 differentially expressed micrornas in serum, 6 were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = |0.488-0.609|). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p50ag005136) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd144, cd105), platelets (cd31, cd62p, cd41a, cd42b), and erythrocytes (cd235) as well as brain biomarkers (s100b, uchl-1, gfap, tau and nse) and t-lymphocytes (cd3, cd4, cd8, cd31). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: 13/17 study patients and 16/20 controls were male. 76% of study patients presented with a glasgow coma scale of 15. in the hypercoagulability panel, of the 10 subsets with statistically significant differential expression, 4 involved s100b+ and were elevated in patients. platelet-derived cd41a evs and uch-l1 evs were significantly elevated in controls in 7 ev subsets identified in the brain-specific panel. finally, cd3+/31+ evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s100bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir-451a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [13 c]-sucrose was increased in the forebrain by 14fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir-451a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir-451a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by 15-, 1.6-and twofold respectively. qpcr analysis of mrna from reported mir-451a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir-451a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for 5 days (subcutaneously; 10 mg/kg, day 1, 20 mg/kg days 2-3, and 40 mg/kg days [4] [5] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le7106, harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from 10 healthy subjects (hc) and 10 ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m1 (gm1), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente 2017-2018 to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of 18 healthy subjects (hc) and 22 pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev2018 guidelines, raman analysis was performed. the raman microspectroscope was used with a 532 nm laser in the spectral ranges 600-1800 cm-1 and 2600-3200 cm-1. data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of 71%. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente 2017 to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag1 or rag2 gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de ética no uso de animais -ufrgs; n 29818) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats (3 and 21 months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed 48 differentially expressed micrornas (p < 0.05; fold change of ≥ |1.1|); 18 mirnas were up-regulated and 30 were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < 0.0001; z-score: 3.413). the differentially expressed mirnas impacted 32 molecules in the neuroinflammation pathway. interestingly, the ion channel grin2b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin2b was previously related to neurodegenerative diseases. moreover, let-7a-5p is predicted to be downregulated and target all the 32 molecules of the neuroinflammation signalling pathway. previous studies have correlated let-7a-5p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = 6). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of 94.3%. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by 80% in epidermis compared to control (tlnκ/sicontrol)(n = 10). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day 10, suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il-6, cox-2, inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir-30b in mi-exo and vegf mrna expression confirmed. results: mi-exo using 1% acetic acid extraction showed the highest yield. the average size of the exosomes is approximately 110 nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of 10^10 particles showed about 65% antioxidant activity. when 10^10 particles were treated, rna expression of cytokines showed about 2 times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of 10^10 particle = 4.7%). and mir-30b in milk exosomes was detected at ct value = 22. summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when 10^10 particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions 8 and 9. the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd9, cd63 and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine (6-4) prymidinone photoproducts (6-4pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm10, malvern). nhdfs were exposed to uvb (1 mj/cm2) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration (200-400 nm). when nhdfs were added with adens and incubated at 37°c for 48 h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and 6-4pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir-29 c and mir-107, while increasing vegf and hif-1a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < 0.01). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a 900-fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < 0.01) than compared to control msc evs (p < 0.05). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small (30-150 nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u87 cells in culture and incubated for 3 h. they were then fixed by 4% paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u87 cells took up exosomes from other u87 cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u87 exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award 1457888. loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir39 or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at 4oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir39 specific qpcr. curcumin was measured using a spectrophotometer at 420 nm. results: evs isolated from whey had an average size of 120 nm, were positive for hsp70, cd9 and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir39 in relation to control (loaded with shame) and the negative control (p < 0.05). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd81. the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd81-snorkel-tag under p16ink4a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd81-snorkel-tag under p16ink4a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w1224). engineering exosomes with gata-4 jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles (30-150 nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata-4, a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata-4 protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata-4 by infusing gata-4 with exosome targeting peptide. methods: the open reading frame of mouse gata-4 cdna was ligated to xpack lentivirus vector (xpack-gata-4) and plvx-ef1-ires-pouro lentivirus vector (plvx-gata-4), respectively. hek 293 cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek293 cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata-4 was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend3 cells with exosomes pre-labelled with pkh26. introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd81 was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd81-gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than 7 million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point (25-50 µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd1 mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized 4-and 24-hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces 24-hours post-treatment compared to 4-hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd68 + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types 24-hours post-treatment compared to 4-hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution 24-hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd44v6 and tspan8. results: the majority (95%) of patients with paca and patients with nonpa-malignancies reacted with anti-cd44v6 and anti-tspan8. serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = 4, 14) and controls (n = 5, 12) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the 95 proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha-3(vi) chain (col6a3), −1(vi) chain (col6a1) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these 3 proteins in plasma ev can distinguish former nfl players from controls with 85% accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col6a3, reln and col6a1 in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = 4), and urine from men with pca prior to (n = 12) and after local treatment (n = 10), benign prostatic hyperplasia (n = 12) and other urological cancers (n = 11). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of 6 healthy controls (hc), 6 patients with active crohn disease (cd) and 6 cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate 1*109-5*109 particles/ml from 500 mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: 60% of the total population of mvs derived from hc were coated, 20% from remissive cd patients, and <5% of active cd patients; and for selected g+ ve derived mvs: 55% of the total population of mvs derived from hc were coated, 34% from remissive cd patients, and 25% of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c4-2, lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi-001) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c4-2 prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: 1) monitoring response to therapy and, 2) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately 30 million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified 153 proteins from neuronderived ev (nde), 215 proteins from microgliaderived ev (mde) and 380 proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, 15, 48 and 251 proteins are present individually in ndes, mdes and ades. among them, high levels of atp1a3 and syt1 in ndes, itgam and cd300a in mdes, and eaat1 and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are 1 trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p53, total p53, cleaved caspase 3, erk1/2 and phospho-akt in whites compared to aas. higher ev levels of phospho-igf-1r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = 3) was collected into k3-edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for 3 days. microev were collected from platelet poor plasma by 10,000 g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd61) and red blood cells (cd235a). the increase in associated protein was seen more in the k3-edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa1 and hla-dqb1, to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = 30) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd63 and cd9), and transmission electron microscopy. exosomes were also studied for their interleukins (il-6 and il-1ra) content. results: among the studied population, 86% present at least one of the alleles leading to cd development and 60% carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il-1ra content (108.8 ± 15.91 to 148.8 ± 12.37), as the persons were not following any treatment. however, values of il-1ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il-1ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al 2018) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol 1) or a combination of density cushion and density gradient (protocol 2) or immune-capturing (anti-cd63, anti-cd9 and anti-cd81 beads) (protocol 3). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol 1 and the majority of the vesicles were cd9/ cd41a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd9/cd41a positive evs where also identified with protocol 3. furthermore, western blot showed increased amount of cd41a in plasma samples in protocol 2. when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci-60 panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α4, αl, and β2 are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p450 and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p450 2a6 (cyp2a6), antioxidant enzymes (aoes), sod1 and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv-1 pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp2a6, sod1, and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd9 and cd63. the results showed menthol and av together increased the levels of cd9 (p ≤ 0.05), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod1 (p ≤ 0.05), which showed a pattern of increased levels of catalase and cypa6, though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp2a6 (p ≤ 0.01), sod1 (p ≤ 0.05), and catalase (p ≤ 0.05) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp2a6 (p ≤ 0.01), and catalase (p ≤ 0.05). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp2a6 in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da047178, da-047638) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: 1) protein association network analysis interaction (string) and 2) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of 343 proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l1-cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < 0.01) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l1cam expression was found to be significantly elevated in smokers (p < 0.05). both gfap and l1cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l1cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da047178) for supporting our work. . electrochemical detection of mirna-21-5p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna-21-5p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna-21-5p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna21-5p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna21-5p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn)6]4-/[fe(cn)6]3-. results: the results indicated that the electrodeposition of gold was more effective for −1.5 v for 600 s and could lead to better signals upon anti-mirna-21-5p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna-21-5p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range (50 nm -400 nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about 150 nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as 150 nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev2018 guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is 50 nm to 500 nm, 10e7 to 10e10 1/ml, and 1.35-1.45, respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect 50 nm particles with ri = 1.35 with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from 10 molecules of equivalent soluble fluorochromes (mesf) to 10 000 mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with 50 nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below 100 copies per ev with 50 nm diameter, i.e. mesf values above 100 are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under 18hlt01 metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon 2020 research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from 3d hollow fibre bioreactor and 2d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish (2d) versus hfbr (3d). methods: human clonal afsc were derived from patient-consented amniotic fluids. 1x10e6 hafsc were seeded in 2d (145cm2), and 1.6x10e7 hafsc on a small 20kd mwco hfbr (fibercell-c2025d, 450cm2) with fibronectin coating; both cultured in chang medium with 20% of es-fbs, starved for 24 hr and then evs collected. the effect of harvest frequency was tested (8hrs, 24 hr, 72hrs,1 wk). 2d-evs and 3d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: 2d production was~5.5x10e9 ev/ml/24 hrs while 3d was~2.8x10e10 ev/ml (first four 24 hrs) and 4.4x10e10 ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~130 nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. 3d-evs trapped vegf (an in vitro established potency assay) as efficiently as 2d-evs, and expressed cd9, cd81, cd63, cd80, cd86 and vegfr1 as 2d-evs. summary/conclusion: 3d-evs had comparable properties and bio-activity to 2d-evs, but the hfbr produced 10x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available 1.2 m2 cartridge provides a 50x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional 2d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from 30 to 100 nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub-100 nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at −80°c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a60-micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased 27% in plasma (p = 0.04; mean of the 3 cryopreservation agents) and 35% in urine (p = 0.05) after one day of storage. after 5 months of cryopreservation, the concentration of plasma evs decreased 2% (dmso and exocap) and 8.5% (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased 6% (exocap) and 18% (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to 5 months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in 2021. funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon 2020 research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h2b-rfp-nanoluc(h2b). results: hek293 t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells 96 hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h2b for 72 hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h2b which was blocked by cyclohexamide, confirming translation of the mrna (2.2kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for 72 hrs. all extracellular evs were removed and cells were trypsinized (0.25% for 30 min) to remove any non-internalized cox protein. 48 hrs later, evs (cd63+ and cd9+) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise2 (n65) or mscarlet-i(66 c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek293, u87, hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at 4°c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht29 cell culture supernatant by differential centrifugation, aliquoted and kept at −80 c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at +4 c. the kinetics of vesicles loss was measured by anti cd9 immunomagnetic capturing followed by cd81, epcam and cd166 staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to 2x10^5 for cd81+, 5x10^5 for epcam+ and 2x10^8 for cd166+ vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for 3 days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd63, cd81, and cd9 confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from 80-150 nm in size and were positive for cd63, cd9, and cd81 proteins. these sevs could be stored at −80°c for >4 months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let7a, mir21, and mir26a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as 6 h after seeding, which were not observed in control huvec cultures until 15 h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = 5) supplemented with msc-sevs versus control, with the former lasting >60 h and the latter lasting <50 h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = 3) were able to expand robustly with a doubling time of 1.1, 2.1 and 8.9 days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a50 and a60 micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed3/303570/7/ ncbr/2017) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles 30-150 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af4), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > 1 × 1010 exosomes/ml and average protein equivalent of more than 1 mg/ml. more than 90% of the exosomes population had an average size distribution of 50-200 nm and median size of 110 nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd63, cd81, cd9) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla6, a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg101, and cd63. surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla6 was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (>150 nm) and exosomes (<150 nm) by size distribution and the concentration of evs (1x108 particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg101, and cd63 was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd326) and stem cell markers (cd133/1) that depict donor cell origin and functional proteins including integrin-binding (cd29) and tgf-beta receptors (cd105). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla6 expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in 96-well plate in with concentration of 5,000 cell/well. cells were incubated for 24 hours and fasted for another 16 hours using only dmem. cells were treated with complete medium consist of dmem and 10% fbs. culture medium were collected after 5, 12, and 24 hours after treatment. cell were culture in 37ºc dan 5% co2. vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture 5, 12, and 24 hours were 74.022 pg/ml, 76.67 pg/ml, and 93.58 pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a 100 kda cut-off. proteomic analysis by tmt mass spectrometry identified 1262 unique proteins. 90% of the exocarta top 100 were identified from this list. additionally, we identified the mirna cargo within exosomes and found 27 highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = 3.1; p < 0.0001) and transforming growth factor-beta (tgfb) signalling (fold enrichment = 4.1; p < 0.0001). exosome size was quantified to be 119.7 ± 3.4 nm with a modal average of 98 nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited 32.2% less collagen than vehicle controls (p = 0.036). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb-231 and hs578t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using 2d cultures. moreover, we also used 3d culture to assess the exosomes uptake and to observe their capability of internalization into a 3d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines (2d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using 3d culture we provided a comprehensive understanding regarding exosomes internalization in 3d structures, which closely mimics in vivo conditions, compared to 2d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk3.3, derived from normal human lymphocytes. nk3.3 evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k562 leukaemia cells were treated with nk3.3 evs in vitro and analysed for proliferation and markers of cell death. results: nk3.3 evs contain ev-associated proteins alix, cd63, hsp70, and tsg101, nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf19b, an e3 ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir-186. nk3.3 ev treatment of k562 significantly decreased its expression of proliferation markers cd71 and ki67, and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases −3 and −7. non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk3.3 ev treatment significantly reduced the frequency of k562 cells highly expressing aldh, a csc marker. summary/conclusion: nk3.3-derived evs have a robust anti-tumour effect on k562 myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk3.3 evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. >150x expansion within 10 days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both 2d flask and 3d bioreactor culture and generated over 2,000 particles per cell in 2d and over 4,000 particles per cell in 3d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased >2x after 24 hrs. alternatively, ev productivity was also increased >2x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt0ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such 2d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that 3d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the 3 to 15 l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in 2d and then seeded on three different microcarriers and cultured in a bioreactor for 5 days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for 24 hours, then in ev collection media for 3 additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts 24 hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following 3-day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells (2e9 particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a 3d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd105, cd 90, and cd73, but they were negative in expression of cd45 and cd34. the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b16f10 cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein1(tyrp-1), and tyrp-2. in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government (2019m3a9h1030682). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells1-3. therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair4. lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these 3d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai078859, ai074410, ai127351-01, ai043894, and ns099029 to fk and r33 ca206937 and r01ar068436 to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f12:neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day 46 and 60), in dmem/f12:neurobasal or in opti-mem, with and without 3-hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions 7-10, which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions 13-17 in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: 8.30e+08 particles/ ml, opti-mem: 9.61e+08 particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, 3-hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: 5.28e+10 particles/ml, non-stimulated: 1.43e+11 particles/ml, p < 0.02). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a6) could have a role in immune cell regulation. specifically, we have investigated the possible a6 ev effect on murine macrophages (raw264.7) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a6 evs were collected from conditioned milieu by ultracentrifugation. raw 264.7 cell proliferation with or without a6 evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw264.7 cell phagocytosis they were treated with 2 μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a6 evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw264.7 migration ability. furthermore, a6 evs are able to increase macrophage phagocytic activity. as it is known that hsp70 is involved in for macrophagic activity increase and a6 evs express hsp70 on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr2, tlr4 and cd91. our data demonstrated that a6 evs increase phagocytosis through hsp70 and its receptors. we have also proved that a6 evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw264.7 cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m2 phenotype. summary/conclusion: in conclusions, our data show that a6 evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. 1 and 2) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd2 (yet preserve its channel function) also cause cysts. besides cilia, pkd2 is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd2 trafficking and localization leads to the kidney pathology remains unknown. pkd2 is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd-2 is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd-2-containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin-2 cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd-2 plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd-2-associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd-2 carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd-2-carrying evs that differ in their densities: 1.11-1.12 versus 1.14 g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd2 plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd-2 ev signalling will inform on the interactome of human pkd2 and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm-1 localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm-1 in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm-1 and gfp-tagged pkd-2 (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm-1::tdtomato and pkd-2::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm-1 subpopulation is formed, we are investigating candidate genes. anoh-1, a homolog of the ca2+ scramblase tmem16f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh-1 mutants, the number of clhm-1::gfp evs released is significantly decreased but the number of pkd-2::gfp evs does not significantly change. in addition, i am using facs to isolate clhm-1 and pkd-2 containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm4b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane-4b (laptm4b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm4b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy5y cells were treated with cer (porcine brain-derived cer or synthetic d18:1/c2:0~c24:0 cer) for 24 h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd63 and rab11 immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c16 and c18 fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm4b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd63-positive mvb with rab11-positive recycling endosomes, further demonstrated that laptm4b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm4b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg8 proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg8 s and their adaptor proteins in sev. cq-induced enrichment of atg8 s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute 2 (ago2) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago2 in evs and it is postulated that ago2 is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago2 that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago2 in sevs. methods: methods: wildtype kras colorectal cancer cells, dks8, were conditioned with 3 different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks8 total cell lysates, large evs and density gradient fractions was performed, probing for ago2 and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions 6 and 7, as assessed by western blot analysis. ago2 was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago2 was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago2 was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago2 in evs has been controversial. multiple factors may affect the ability to detect vesicular ago2, including serum and growth factors in the conditioned media that may provide sources of extravesicular ago2 and also regulate the trafficking of ago2 into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim4-beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with 1 mm methyl β-cyclodextrin. results: using 4 melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between 100 and 200 nm. protein amounts in ecvs were measured, showing heterogeneous levels at 10~80 μg/100 ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim4beads and flow cytometry. gd3 and gd2 were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd3, showing increased levels of integrins in ecvs from gd3+ cells compared with those from gd3-cell lines. integrin levels in cell lysates from these cells (gd3+ and gd3cells) were almost equivalent. treatment of a gd3-expressing melanoma cell line by 1 mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg101 in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd3 + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from 4t1 breast cancer cells cultured in normoxia (21% o2) and hypoxia (5% o2) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd63 and mcherry-cd63, separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs (10k fraction) and small evs (100k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd9 and cd63 (yoshioka et al., nat commun., 2015) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort1 and lamp2 genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort1 or lamp2 could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with 10% fbs and 1× antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy5.5 for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea1, anti-rab7 and anti-gm130. in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system 7900. lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language 3.3.3 results: we determined that rab7, located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to 3 orders of magnitude (the order of the size of giant phospholipid vesicles is between 1 and 100 micro metres, in erythrocytes it is about 5 micro metres and in evs it is about 100 nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc4 (arrestin domain containing protein 4) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd4 family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc4 in the trafficking of the divalent metal ion transporter dmt1 to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc4. we then generated arrdc4 wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified 5 potential ubiquitinated lysine residues. out of these, lysine 270 appeared to be the most important for arrdc4 function. arrdc4k270 r mutation caused a decrease in the number of ev released by the cell compared to arrdc4 wt, and a reduction in trafficking of dmt1 to evs. furthermore, we also observed a decrease in dmt1 activity and an increase in its intracellular degradation in the presence of arrdc4k270 r. k270 also appeared to be ubiquitinated with k29 polyubiquitin chains by the ubiquitin ligase smurf1. summary/conclusion: our data suggests that k29 polyubiquitin chains are the signal for arrdc4mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a 3d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available 3dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, 2019, then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing:3986565. instructions for 3d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel 3d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this 3d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d492 and d492her2+ breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d492 and d492her2+, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d492 and d492her2+ was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep 16/60 sephacryl s-400 column run with äkta start (280 nm), 240 min runs. each fraction (4-5 ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns300, camera gain 10, static mode, capture time 90 sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with 2% formaldehyse and stained with 2% uranyl acetate, run at 80kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f1-f3, 40-130 min) were collected from d492her2+ cell culture medium. interestingly, both nta and tem suggest that f2 and f3 contained evs as the isolated particles measured 65 and 58 nm, respectively and tem revealed spherical particles 20-50 nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram (180 min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in 2016, is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, 2019) and evs in immunology (buenos aires, 2020) introduction: since its founding in 2012, isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, 2012. in december, 2019, the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, 2020, the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the 2019 and 2020 isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, 2019 workshop) and evs in immunology (buenos aires, argentina, 2020 workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the 2019 and 2020 workshops assembled a total of more than 100 individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, 2019) and evs in immunology (buenos aires, 2020) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid-19 kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in 2016, featuring lunch and presentations by academic and industry participants. when covid-19 prevented in-person meetings beginning in march, 2020, the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid-19 shutdown in the host country (us). an audience of between 80 and 150 individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm10 and pm2,5, chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev9 signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v3-v4 16 s rrna gene regions amplified from upper-airway tracts of 25 ar cases and 25 healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm10 and pm2,5 concentrations at the municipality of residence in the 7 days preceding nasal sampling (i.e. day −1 to day −7) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria (50.8%) was the most represented, followed by firmicutes (34.7%) and proteobacteria (12.8%) while in cases proteobacteria were 38.8%, actinobacteria were 37.1% and firmicutes were 23.4%. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd61 +), activated endothelium (cd62e+), monocytes (cd14+), eosinophils (cd294+), neutrophils (cd177+), mastocytes (cd203 c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased 2.5fold in cases versus controls (p < 0.001). evs were modified by pm exposure at several time lags. in particular, a negative association between pm10 and eosinophil evs was observed (beta = −0,016; pvalue = 0,017). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th17 cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th17 cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th17 cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th17 cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c57/bl6 and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved 048/2012). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th17 cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th17 cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th17-derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th17 cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at 4°c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan3) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph 5.5; in this condition nano2 releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with 100 nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration (100 µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg 1, il-12 and il-23 genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x 00102/6. immuno-toxicological evaluation of human mesenchymal stem cellintroduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; 0.4 mg/kg), or low-dose (1x10e8 paticles/head) and high-dose (1x10e9 paticles/ head) of hevs every other day for 10 days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for 3 days with low conc. (1x10e8 particles/ml), high conc. (1x10e9 particles/ml), pma/ionomycin as a cell activator or cpt (10 μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about 2~7% in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant (18172mfds173) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). 1 x 10(8) human or murine msc-evs were administered intravenously into 4t1 breast tumour bearing balb/c mice (n = 6) and healthy controls (n = 6). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: 30-150 nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd63, cd81, cd82; cytosolic protein tsg101 and were negative for calnexin. ev concentrations ranged from 4.08 x 10(9) -6.6 x 10(9)/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd4+, cd8+, cd25+), neutrophils (gr-1+, ly-6 c+), dendritic cells (cd11 c+), macrophages (cd11b+, mhci+, mhcii+), nk cells (cd27+) and b cells (cd19+) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook 7h9 medium (m7h9) supplemented with 10% (v/v) oadc enrichment and 0.5% (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin-1β (il-1β), terleukin-6 (il-6) and interleukin-12 (il-12). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd42b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna 4.0 array. results: microrna from 9 sepsis patients (median age 7.0 years; iqr:1.2-13 and 77% female) with a median psofa score of 6 (iqr:2.5-10) and from 5 healthy controls (median age 9 years; iqr:6.5-13.5 and 20% female) was isolated and compared. in septic vs. healthy patients 30 mirnas were differentially expressed (false discovery rate (fdr)<0.05; fold change ≥|1.5|) affecting 921 mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥2. summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir-142-3p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir-142-3p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir-142-3p involved in salivary gland (sg) physiology was performed with mirdip 4. serca2b, ryr2 and ac9 were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca2b, ryr2 and ac9 were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir-142-3p in relation to the expression levels of serca2b, ryr2, and ac9, a double fluorescent in situ hybridization was performed. ca2+ signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir-142-3p is over-expression in the sgs of ss-patients. next, we demonstrated that mir-142-3p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir-142-3p which transfer into glandular cells and affecting intracellular ca2+ signalling, camp production and protein production by mir-142-3p targets (serca2b, ryr2 and ac9). summary/conclusion: this study provides evidence for a functional role of the mir-142-3p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il35-coated extracellular vesicles promote infectious tolerance (p35) subunits, yet the forms that il35 assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il35-producing t regulatory (treg) cells in tregebi3 wt c57bl/6 reporter mice, and identify il35 producers by expression of ebi3tdtom gene reporter, plus ebi3 and p35 proteins. results: curiously, both subunits of il35 were displayed on the surface of tolerogen-specific foxp3 + and foxp3neg (itr35) t cells. furthermore, il35 producers, although rare, secrete ebi3 and p35 on extracellular vesicles (ev) targeting a 25-to 100-fold higher number of t and b lymphocytes, causing them to acquire surface il35. this surface il35 is absent when ev/exosome production was inhibited, or if ebi3 is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il35, promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r01-ai119140-03 (to w.j.b.), r01 ca203689 and p30 ca047904 (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p30 ca014520. unique formulated dual targeting antigen specific and delivered mirna-150 gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd8 + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna-150 to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna-150-pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac-1 integrin (cr3 receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac-1 activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c3bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c3bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c3bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac-1 activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw4869 and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw4869 exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for 8 or 48hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at 48 hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il-6 and il-8, with il-8 release reaching statistical significance at 0.1%, 0.5%, and 1% de concentrations (p = 0.0367, <0.0001, and <0.0001 respectively) and il-6 trending a similar dose response but only statistically significant at 1% de (p = <0.0001). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the 5 and 10% iodixanol fractions (p = <0.0001 and 0.0219, respectively) at 8 hr following de treatment. alternatively, there were significantly less ev in the 20 and 40% density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs >100-fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to (1) develop a faster method to avoid swarm detection, and (2) increase the number of detected evs per second. methods: we measured serial dilutions of cd61stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a60-micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of 4,100-4,200 events/s. the cd61+ evs concentration scaled linearly over 1.5 orders of magnitude of the dilution and most donors required >1000-fold dilution to avoid swarm detection, thereby reducing cd61+ ev counts. for fluorescence triggering, the cd61+ evs concentration scaled linearly over >3 orders of magnitude of the dilution. for all donors, swarm detection was absent after 32-fold dilution (relative to pure plasma). the count rates of cd61+ evs were 30-100-fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥32-fold and compared to scatter triggering, count rates of cd61 + evs were 30-100 fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n30 nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd9, cd81, and cd63. however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd171, snap25, α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek293 t cells through transient transfection of plasmids (origene). media was conditioned for 24-48 hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r100 system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of 3 labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > 50 nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects (40-70y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd41 for platelet-derived evs and cd105 for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap-6) and u46619. the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of 71 nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = 0.568; p = 0.01) and clot size at 30 min (r = 0.480; p = 0.002). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = −0.410; p = 0.009), whereas they were found positively correlated with lysis progression (r = 0.417; p = 0.007). both mean and mode size of cevs, detected by nta, were significantly correlated with u46619-induced platelet aggregation (r = −0.338; p = 0.033, r = −0.382; p = 0.015, respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a 3d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd63 and cd81. ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh26-labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a1 is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a1 is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa1-sirna were enumerated and phenotyped using the exoview r100 platform. evs with surface expression of cd9, cd81, cd63, and annexin a1 were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a1 allowed us to assess the specificity of the selected annexin a1 antibody. results: the ev fraction was positive for cd9, and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a1 both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a1+ evs were captured on the annexin a1 spots from the control cm, and there was a marked decrease in capture and labelling from anxa1-sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd9, then cd63 and cd81. there was a significant population of annexin a1+ evs that was negative for tetraspanins. summary/conclusion: annexin a1 is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a1+ evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl136432 and hl147095. possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (<40 mmhg) and angiography. ten preoperative clti patients and 10 control patients without pad were analysed (all patients with diabetes and 50% of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into 20 micrornas for esrd patients and 23 micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir-21 and mir-133a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t0) and six months after surgery (t1). isolated pexos were quantified by nanoparticle tracking analysis and mir-1, mir-21, mir-133a, and mir-208a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = 8). local ethical committee approved the study (emigrate study, approval n°1529) and informed consent was obtained from all patients. results: pexos levels at t0 were lower (−32%, p = 0.02) in patients with worst postoperative lv function, while they were higher at t1 (+31%, p = 0.03) in patients with reversed lv remodelling after surgery. at t1, the increase in pexos levels was associated to decreased heart mass index (−13%, p = 0.02) and higher levels of exosomal mir-21 (+78%, p = 0.02) and mir-133a (+69%, p = 0.05) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir-21 and 133a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin-2 (lcn2) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc19606) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between 100-250 nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness (0.5-20 kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of 8 surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin-5 and the tetraspanin cd63. young melanocytes produced more annexin-5 positive evs and evs larger than 500 nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb-231 breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over 600 proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over 400 ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd11b+ and f4/80 + cells, but not b220+ cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as 1 hour after msc-ev injection and was not significantly different between 2 and 24 hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants 5uh2tr000880, 3uh3tr000880-03 s1, 5p20gm11 9943, and 5t32hl116249. linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw480 and ht29 human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to 80% reduction in lipids observed at the 50 µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il-6 and il-8 by over 3 fold and 10 fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) (30-120 nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg101 and cd63), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein 3 (ifitm3), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm3 contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence1. bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh7 cells were cultured on acrylamide gels with the stiffness was modulated to 500 pa (soft) or 10 k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin8 phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: (1) stiff matrix promoted exosomes secretion. (2) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin8-rab8 pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick 3d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used 3dimensional (3d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional (2d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a 3d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of 3d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< 50 nm) to small (< 200 nm) and medium and large (> 200 nm) evs and larger evs (> 50 nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco2a1 and clic1 expression on non-tropic evs conferred lung-tropism, while cd13 gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev-293 t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles (30-120mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb-231 luciferase (luc) expressing cells or ht-29-luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of 1x10^7 msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. 8x10^7 evs were incubated with the radionuclide, technetium-99m tagged duramycin (99 mtc-dur) for 30 minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the 99 mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: 18 hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected 10 minutes and 4 hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia 1 ; aejez sayeed 2 ; rachel derita 1 ; shiv ram krishn 3 ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using 10 gy, for 5 consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from 50-100nms to 70-250nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = 20) and control (n = 18) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps396 tau and aβ1-42 were significantly increased in ad evs. annexin a5 (anxa5), neurosecretory protein vgf, neuronal membrane glycoprotein m6-a (gpm6a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these 4 proteins were confirmed to predict ad with the 88% accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa5, vgf, gpm6a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide 6 (trap; n = 7), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = 3), and (iii) stored at room temperature or at 4°c for 6 hours (n = 3). the concentrations of evs from platelets (cd61+), activated platelets (p-selectin+), erythrocytes (cd235a+), and leukocytes (cd45+) were determined by flow cytometry (apogee a60-micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased 5.8-fold and 10.4-fold in citrate-anticoagulated blood, compared to 1.4-fold and 2.0-fold in edta-anticoagulated blood (edta vs citrate: p = 0.018 and p = 0.043, respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. 5 μl of exosome samples from cancer cells (a549 and h3255) or normal cells (mrc5) were injected into the pdms microfluidic device and incubated for 30 minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed 26.6 folds absolute cd peak value change and 3.41 folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than 10 mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using 7 blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [1] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [2] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over 90% of separation efficiency at 225 µl/min flow rate and exosomes were collected continuously with high recovery (~70%). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was 1x10^6 exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , 1800528, 2019) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd63. we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to 2 µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h2020-eu.1.2.1-fet open programme via the grant agreement 801338. immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with 600 nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (>25x) and three-dimensional pore density (>1000x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with >100x enrichment from input and 49x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard 150 mm petri dishes were patterned with 2 µm tall polystyrene microtracks spaced by 5, 10 and 20 µm across a 100 mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb231 cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final 48 hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the 5 µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < .05). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir-21 excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir-21 as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir-21 recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h2020, through mindgap/fet-open/ga829040 project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = 31). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and 10 ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine 37 distinct uev surface markers of cd9+/cd63+/cd81+ vesicles in a multiplexed bead-based manner including respective controls. the accuri c6 (bd) was utilized for data acquisition. for further misev2018-compliant characterization, cd9+/cd63+/cd81+ uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd209, all other 36 surface markers could be identified. the most abundant markers were cd9 and cd63, which were detected in 93% of samples, followed by cd133/1 (84%), cd326 (81%), cd81 and cd24 (77%, each). cd3 (42%), cd9, cd45 (39%), cd49e (32%) and cd81 showed similar relative median fluorescent intensities (rmfi), while cd63 yielded significantly higher (p = 0.009) and all other markers significantly lower rmfi (p < 0.011). tem and f-nta revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 e + 10 particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg101, cd9, cd63 and cd81 without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = 40) aged 40-70 yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a 3colour panel, which included annexin v (for the majority of circulating evs), cd41 (for plateletderived evs) and cd105 (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and 10-yr cvd risk detected by qrisk2. results: ev numbers, as determined by nta, were strongly associated with bmi (r = 0.602, p < 0.001), blood pressure (systolic bp: r = 0.359, p = 0.023; diastolic bp: r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = 0.330, p = 0.038). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining 49.4% of the variance for total ev numbers. an additional 9.3% of the variance in total ev numbers was predicted by diastolic bp. ancova of the 10-yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with 10-yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd4 + t cells were isolated from secondary lymphoid organs of foxp3-rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th1, th2, and th17), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, 15-day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for 5 days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th1, th2, th17) showed either no change in proliferation (th1) or decrease in proliferation (th2, th17), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < 0.0001). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th1), il4 and il13 (th2), or il17a and il17 f (th17). in contrast, exposure of tregs to cdc-evs resulted in~1000-fold increase in expression of il10, a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il-10 in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il-10. this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t32hl116273 prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from 42 patients treated with prostanoids (study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with prostanoids (control group; n = 38, 55 ± 19 years, 65% female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; 0.5 mm), adenosine diphosphate (adp; 6.5 µm) and thrombin receptor-activating peptide (trap; 32 µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd61+ and cd62p+; pevs), leukocytes (cd45+, levs) and endothelial cells (cd146+, eevs) were measured in plateletdepleted plasma by flow cytometry (a60-micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = 0.01) and lower concentrations of pevs and levs (p ≤ 0.05). furthermore, thrombus formation was delayed (p ≤ 0.003) and thrombus size was decreased (p = 0.008) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd61+ pevs (p = 0.04). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ 0.05) and the concentrations of pevs (p ≤ 0.08). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ 0.04). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium 2018/31/n/ nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from 17 cf children (<6 years of age). for nanoflow cytometry, evs were stained with di-8-anepps, and with epcam, cd66b and cd115 (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = 0.003, spearman's rho = 0.689). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = 0.001, rho = 0.857). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv15a0), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: 142 individuals were enrolled into our study, subdivided into a training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer n = 20, pneumonia n = 18, sepsis n = 37). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq2) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed 29 significantly regulated mirnas in pneumonia compared to volunteers, and 25 mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by 12 mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992). mir-193a-5p (log2fc = 1.86, padj = 1.49e-6) and mir-542-3p (log2fc = 1.67, padj = 3.29e-5) differentiated between pneumonia and volunteers and mir-1246 (log2fc = −2.41, padj = 1.78e-04) between pneumonia and sepsis. expression levels of mir-193a-5p and mir-1246 were related to disease severity. mir-542-3p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was 73.33%. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study,3healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd63 and cd81 were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and 9 mirnas (mir-103a-p, mir-191-5p, mir-320a, mir-200b-3p, mir-185-5p, mir-223-3p, mir-21-5p, mir-155-5p, let-7 g-5p), were evaluated by rt-qpcr. after the optimization of the methodology, 10 healthy adults subjects and 10 patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd63 and cd81. however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna-320a showed a significant increased (p = 0.02) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir-320a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf2-active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid 2-related factor 2 (nrf2) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf2-active exosomes, mscs were incubated with potent nrf2 activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive msc markers (cd44, cd73, cd90, and cd105) and <5% express negative markers (cd45, cd31, cd14, cd19, or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd81, cd9 and tsg101. nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of 168.0 ± 6.5 nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf2-active human mscs increase exosome secretion by 54%, compared to nrf2-baseline mscs (p < 0.05). both nrf2-baseline and nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). this reduction eliminated the delay in closure time compared to wounds of c57/b6 mice. nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated c57/b6 wounds. at day 10, exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd31+ vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf2 function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist1 is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist1. methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc3-ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc3-ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc3-ml lines stably overexpressing or deficient in twist1. results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc3-ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc3-ml overexpressing twist1 showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist1 expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following 6-week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial #02204670). eleven sedentary obese youth (15.7 ± 0.5 years, bmi ≥ 95th percentile underwent an acute bout of ae (60% heart rate reserve, 45 min). blood was collected before [time (at) −15, 0 min], during [at15, 30, 45 min], and after [15 min (at60), 75 min (at120)] exercise. afterwards, youth participated in 6-week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below 50 percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at0), immediately after ae (at45) and after recovery (at120). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~141.1 nm) as opposed to nrs (~97.3 nm) at at0 (p < 0.05) and this pattern was maintained at at45 and at120, though not significant (p = 0.1). nrs displayed differential ev size distribution (peaks at 100 nm or 300 nm), while ev distribution was highest at 250 nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~35% of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~5mm chunks, placed in serum-free dmem and incubated for 24 hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases >100 times more evs than adipose tissue per unit of mass (p < 0.01 using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (<0.1% of events). evs from mg/mt mouse skm overwhelmingly expressed mg (>95% of events) with negligible (< 1%) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~3%) or mt (~4%). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: 1) modification of evs post-secretion, 2) clearance of fluorescent evs by the liver or 3) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association (18ipa34110052) to dsl. endothelial cd36 delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd36 facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd36 controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd36-facilitated fa uptake is unknown. here we examined the role of ec cd36 in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute (4 hr) cold exposure (4°c) with core temperature monitored every 30 minutes. after 4 hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd31 magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa 568. for radioactive studies, primary lung ec were serum starved for 5 hrs and incubated overnight with 3h-oleic acid bound to fa-free bsa (2:1 ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd36 (eccd36-/-) compared to controls (cd36fl/fl). however, during cold exposure at 4°c , eccd36-/-are unable to maintain body temperature (p < 0.001). plasma free fa are higher in cold exposed eccd36-/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd36-/-and cd36fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav-1 phosphorylation. the internalized cav-1 and cd36 positive vesicles containing fas are released as sevs. to determine the dependence of cd36 on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd36-/-cells contain less labelled-fa (p = 0.05). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk111175 and dk056341. introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco-2 cells+ht-29 cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for 12 weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with 150 micrograms/day for 4 weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat2, angptl4 and dgat1, but a decreased expression of fabp2, fatp4, mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl4 mrnas, 2 therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn1) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn1 are present in vertebrates, of which the soluble plasma fn1 is derived principally from hepatocytes, while cell-associated fn1 is produced by numerous cell types. here we describe a novel function of plasma fn1 in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml12 hepatocytes, fn1 ko aml12 cells in which fn1 was ablated using crispr-cas9, primary human or mouse hepatocytes, or human hepg2 cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh26-labelled evs from parental (ev-hep) or fn1 ko (ev-hepfn1 ko) aml12 cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl4 for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn1 ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn1 ko were 50-200 nm in diameter and positive for common ev markers (cd63, cd9, flotillin-1). mass spectrometry showed that fn1 was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn1 was verified by western blot and co-immunoprecipitation with anti-cd9 or antiflotillin-1. western blot showed that fn1 was also abundant in evs from primary human or mouse hepatocytes, hepg2 cells, and human or mouse serum. fn1 and ev-hep co-sedimented at a density of~1.15 g/ml. ev-hepfn1 ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn1-independent. as compared to ev-hep, the binding of ev-hepfn1 ko to target cells was highly reduced whereas ev binding was independent of fn1 expression by the target cells themselves. both ev-hepfn1 ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen 1⍺1 expression in mouse hscs in vitro. summary/conclusion: fn1 is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn1 in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type 2 diabetes and islet transplantation introduction: type 2 diabetes (t2d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t2d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type 1 diabetes (t1d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = 4 donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = 4/ group) were cultured in normal (5.5 mm) or elevated (11.1 mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t2d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~35% of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c57bl/6 j mice (n = 6) or female mt/mg mice (n = 6) was cut into~5 mg pieces and added to 12 well plates (~50 mg/well) filled with 1 ml of serum-free dmem and placed in a cell culture incubator at 37 c for 24 hours. skm from male mice was treated with 2-deoxyglucose (2-dg) (0.1 nm -100 mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with 10um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged (3,000 x g for 15 min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer 3000. ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for 2-dg experiments were made using a repeated measures 1-way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = 0.07) as a function of 2-dg treatment, but no effect on ev diameter (p = 0.37). bleb treatment did not alter ev abundance (p = 0.69), but significantly reduced ev mean diameter (p = 0.007; 11% decrease; dmso: 114.9 ± 5.1 vs. bleb: 103.9 ± 4.2). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with 2-dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa34110052). introduction: extracellular vesicles (evs, exosomes) are nanovesicles (30-200 nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [1] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [2, 3] . in this study, we newly developed evs decorated with cellpenetrating sc18 or (sc18)2 peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap18, because the peptides can be efficiently internalized by breast cancer cells. [4, 5] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd63-gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc18 peptide is known as one of cell-penetrating peptides, and branched structure of sc18 peptides, (sc18)2, further enhances the cellular uptake [5] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc18 or (sc18)2 peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc18)2 peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc18 peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc18)2 peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc18)2 peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas9 based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc3-dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb-231 cells expressing either a targeting or non-targeting control sgrna and applied to hek293 t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek293 t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were 140 ± 7 nm in diameter as measured by dls and possessed a negative surface charge of −25.3 ± 3.3 mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only 1 in 3.8e05 ± 3.5e05 evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were 51 ± 0.6 nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between 4 to 54 sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately 9e03 copies per cell. summary/conclusion: mda-mb-231 evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp-1 cells. after 48 h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il-17 in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas9 protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. 2012) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas9 protein conjugated to the highly positively charged protein. post-uptake of these cas9-loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of 65 ± 15 nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s2r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp53 are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p53-mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p53 proteins with novel activities termed gain-of-function (gof). even though mutations in tp53 are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p53. methods: p results: in the colon, tumour cells harbouring mutp53 were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp53, monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p53 affected tam were characterized as tnf-αlow/il-10 high, over expressing cd-206 and cd163, with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp53 tumour cells to macrophages, revealed a mutp53-specific mirs signature led by mir-1246 promoting the tam phenotype and creating an invasive front together with tumour cells. mir-1246 was also found to be the top mutp53-associated mir in a cohort of 57 human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p53 mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p53. we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p53. omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd47 expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p53 mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p53 facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order 3d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). 38 sera from infected and 32 from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh16.5 tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of 239 proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg101, alix, cd63). two parasite proteins, enolase and the fh16.5 tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh16.5 protein generated a highly significant difference between the two groups (p value = 0.003614); as did enolase (p value was 0.02294). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il-10 and il-12 release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ1-4glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ1-4(fucα1-3) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from 5-week-old melon plants, either uninfested or infested with adults of a. gossypii (n = 15, 4 replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of 381 different proteins were identified in phloem sap evs, including 152 present in exosome databases (exocarta). on top of that, 44 differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < 0.05). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and 24% of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s5™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir-21-5p, mir-23a-3p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) 0.92. to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir-21-5p and mir-23a-3p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir-30a-5p and two isomirs (mir-574-3p and mir-205-5p) . the auc of the panel index was 0.95. we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes (40-120 nm), microvesicles (50-1,000 nm) and apoptotic bodies (500-2,000 nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than 90%. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c57bl/6 j male mice (12 weeks) were orally dosed with ccl4 (vehicle, 7, 70 mg/kg). serum were separated from blood after 2, 4, 8 and 24 hours after ccl4 administration. the serum was centrifuged at 10,000 x g to remove cellular debris and subsequently ultracentrifuged 100,000 x g. the pellet is resuspended in pbs and ultracentrifuged 100,000 x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than 10 novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl4: 7 mg/kg & 70 mg/ kg). well known hepatotoxicity biomarkers, mirna-122 and mirna-192 were upregulated more than 1000-fold in the administration of 70 mg/kg ccl4, but not responded in the administration of 7 mg/kg ccl4. summary/conclusion: these results suggest that mir-122 and mir-192 are mainly released from liver to blood directly only in the administration of 70 mg/kg ccl4, while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both 7 mg/kg and 70 mg/kg ccl4 should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing (1500-2000x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of >1 micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (>10,000 bp versus~170 bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l858 r or the resistance mutation t790 m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled 10 patients with metastatic lung ac: 8 with egfr mutations and 2 without (control). from the patients with egfr mutant lung ac, we processed 25 blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon 19 deletion or l858 r mutations. results: from 6 patients with egfr exon 19 deletion mutations, we detected identical mutations in exorna from 15/19 samples. exorna based mutational load increased and mirrored clinical progression in 2 patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at 2 points and was therefore not plotted. exorna from 2 patients with l858 r and t790 m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from 3 patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in 2/4 patients. summary/conclusion: in summary, we detected egfr mutations in 19/23 exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of 3.2 per 100,000 people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from 8 patients at the time of initial diagnosis, and 8 matched controls by sex and age. ev-associated rna was isolated from 1-2 ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed 95 mrnas as differentially abundant between gbm and control samples, with 82 mrnas enriched in gbm samples and 13 mrnas enriched in control samples (p < 0.05). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. 4 million americans are estimated to be suffering from the disease, 90% of which are women with an average age of 40. overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of 3 years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from 7 ss patients and 7 healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over 80% of reads mapping to the transcriptome. further rna biotype analysis showed over 75% of transcriptome reads mapped to protein coding genes and lncrnas. we detected over 10,000 mrnas and approx.1000 lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified 455 upregulated genes, including 415 mrnas and 37 lncrnas (p < 0.05). 120 genes were found to be downregulated in ss, including 114 mrnas and 6 lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = 15) and unsuccessful (n = 15) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± 10 nm dihydrotestosterone (dht) ± 10 µm enzalutamide (enz) for 48 h. evs were isolated using sequential ultracentrifugation (2000 g for 20 min, 10,000 g for 30 min, 100,000 g for 2 h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of 50 nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie2. snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg38 using bowtie2. results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p53 mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l1 blockade, is a method to eliminate cancer cells. ectopic expression of pd-l1, on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l1 is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l1, and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l1 expressing cancer cell lines were used to generate evs. pd-l1-myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l1 clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl-1 or ev associated markers. exoview r100 reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl-1 on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l1-myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l1 positive evs from different cancer cell lines, only 28.8 and 73 10 clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l1, however, only clone 28.8 was able to immobilize these evs. the results suggested that clone 28.8 could be a potential pd-l1 antibody to detect pd-l1 positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l1 antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in 2017, there were over 49,000 new cases and 9,700 deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the 5-year overall survival rate for hnscc is only 40-50%. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used 8 representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag-10plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd9 and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat3, hla-a, tenascin, e-cadherin, β catenin, cytokeratin 19, epha2, and cd59, and hpv-related markers p16, p53, rb, cyclin d1, and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat3, hla-a, and tenascin. only kert19 demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p53, rb, cyclin d1 and p16, only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha2 and cd59 showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be 1) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or 2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf7 cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc-3 cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd63, cd81, cd9, flotillin1 (flot1), alix and tumour susceptibility gene 101 (tsg101), negative marker calnexin (canx), and contaminant markers apo-a1 for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot1 is membrane protein that forms microdomains in the plasma. alix and tsg101, an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a1 (apo-a1) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd63, cd9, cd81 and flot1 were positive and canx and apo-a1 were negative. in urine evs, cd9, cd63, flot-1, alix and tsg101 were positive and canx and thp were negative. in ccm evs, cd9, cd63, flot1, alix and tsg101 were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from 3 sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir-21-5p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir21-/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt (10-plex) label of the total mir21-/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer 2.2 (thermo) was used for protein processing and quantification. results: a total of 1.861 proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes. we found out several protein modulated by mir21 and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir21-/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the 6th most common cancer worldwide and the 3rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from 20 healthy subjects, 19 subjects with cirrhosis, and 19 subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, 39 samples were eligible for analysis after low expression filtering. we used pair-wise ratios of 34 cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of 0.93 and 0.92 respectively using 6 mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified 10 protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek293 cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic 96 well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd9, cd63, cd81, adrβ2, hsp70, epcam and flotilin-1) followed by binding of cy5-labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta-2 receptor (adrβ2-receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and 24 h collections.the aim of this study is to compare uevs excreted in all single voids during a 24 hour collection period and compare it with 24 hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the 24 hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg101, alix and cd9). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel-39 mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin-2 and uevs pan tetraspanins (cd9 + dc63 + cd81) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the 24 hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a 24 hour urine collection reflected the average results of all single voids over a 24 hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus 24 hour uev analysis. we concluded that the 24 hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, 36 out of 79 detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d16. identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of 20 genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger4) and up-regulated (e.g., lifr) genes were found (p < 0.05); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc4-/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc4-/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc4-/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc4-/-epididymal epithelial cells, and addition of wild type evs to arrdc4-/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to 25% of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at 24-32 weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = 7) compared to gdm-aga (n = 13; p > 0.05). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = 7/group; >twofoldchange; p < 0.05), including four skeletal muscle-specific "myomirs": mir-1-3p, mir-133a-3p, mir-133b, and mir-499a-3p (all increased). all four myomirs were present in placenta but only mir-1-3p was significantly altered in gdm-lga compared to gdm-aga (n = 12-14/group; p < 0.05). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir-1-3p regulates vascular development in other systems, so we propose that mir-1-3p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm4 sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm4 sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter 200 nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc1 or tsc2 genes. together, the tsc proteins regulate mtorc1 activity. all tsc patient post-mortem samples exhibit renal disease and 40% of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas9 to disrupt the tsc2 gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc2 gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately 90% of the evs were in the 100-250 nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg101, the transmembrane proteins cd63, cd81 and cd9, and the primary cilia-related hedgehog signalling-related proteins arl13b. proteomic analysis of evs identified a significant difference between the tsc2-intact and tsc2-deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified 675 intact proteins and rna-seq data identified 2,535 mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the 100 most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic 3d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro 3d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro 3d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is 8th most common cancer in men and 10th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns300 and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing 90% of all bladder tumours. at diagnosis 75% of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly 35-80% of these patients will experience disease relapse and 10-20% will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from 9 uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from 50nm to 400 nm with characteristic ev morphology, express ev-associated markers as cd63 and hsp70 and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t98 & u138) gbm cells were "primed" using evs derived from mesenchymallike (u87 & ln18) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd44 was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y27632 (to block those budding at cell membrane) and gw4869 and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs578ts(i)8, using a discontinuous optiprep density gradient, after the cells were treatment for 48 hrs with the compounds listed above. evs (pooled fractions 3-9 with a density range of 1.03-1.16 g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs578t and mda-mb-468 cells using the ev to cell ratios of 1 × 105 evs/3x103 cells, 1 × 106 evs/3x103 cells, 1 × 107 evs/3x103 cells to evaluate doseresponse. ev-track id ev190109 (score of 88%). results: gw4869 significantly (p = 0.035) decreased ev release from hs578 ts(i)8 cells. manumycin a and a combination of calpeptin and y27632 (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y27632 actually increased ev release; but not significantly. of the reduced numbers of evs released following gw4869 treatment, hla-dr+ evs were significantly (p = 0.032) enriched. none of the evs analysed significantly changed hs578 t or mda-mb-468 growth rates. however, evs from cells treated with calpeptin (p = 0.007), gw4869 (p = 0.025), manumycin a (p = 0.01) and combo (p = 0.001) caused significant reduction in mda-mb-468 migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw4869 (p = 0.011), and combo (p = 0.018) caused significant reduction in hs578 t migration. summary/conclusion: while gw4869 was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw4869 or calpeptin and y27632 significantly reduced migration of both recipient cell lines. funding: phd funding: 1252 tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a 16-day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as 90% of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu6) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas-2b), but are also capable of disrupting a 16-day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas-2b and calu6 evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas-2b cells were cultured in transwell for 16 days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas-2b monolayers were treated with calu6 evs at 1, 10 and 20 μg/ml for 24 hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas-2b cells treated with calu6 evs at 1 and 10 μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after 48 hrs. results: beas-2b and calu6 evs were enriched in 50-200 nm size range, and cd9 and cd81 were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp96. calu6 evs significantly impaired 16day mature beas-2b monolayer's barrier properties, which at the highest dose caused 33% reduction in teer from 27.0 ± 2.5 to 18.2 ± 3.2 ω.cm2 (n = 4). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in 30 min (14.8 ± 7 ng/ml in control vs 87.3 ± 51 ng/ml in treated) (n = 3) and complete loss of e-cadherin expression at cell-cell tight junctions (n = 3). at the transcript level, calu6 evs induced significant downregulation of e-cadherin by 36% and upregulation of vimentin (mesenchymal marker) twofold (n = 3) in beas-2b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp78 plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein 78 (grp78), a chaperone protein of the hsp70 family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration (0.22 µm) and differential ultracentrifugation. grp78 surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp78 was identified on the surface of evs from irradiated cells. the increase in ev grp78 correlated with increased grp78 expression at the donor cell surface. the grp78 content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp78 in radiation resistance we overexpressed grp78. here the modest (3x) overexpression of grp78 was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp78-dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp78 in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp78 levels, and subsequent ev-mediated transfer leads to enhanced grp78 levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. 3d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from 2d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from 3d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma 22-rv1 line were cultured in 2d and 3d conditions, using 3d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the 40 fractions of 4 ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd63 and cd9 were used as ev markers, apob and apoa1 for lipoprotein contaminants control, and cd3, cd41 and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in 3d conditions, the 22-rv1 cells produce up to 1000-times higher ev number than in 2d. size and density distribution of evs derived from 3d cultures but not of 2d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under 3d conditions. the profiles of ev subtypes produced by cancer cells growing in a 3d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over 80,000 cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok111, vok130, and vok151 were cultured in flasks containing 500 ml of ev-depleted media (10% fbs, centrifuged 18 hr x 100,000 g). when cells reached~75% confluency, the conditioned media was collected and spun at 2,500 g for 10 mins two times to deplete any remaining debris, leaving~450 ml of media. this media was concentrated to a final volume of~7 ml using a pall jumbosep 100 kda mwco filter. this concentrate was purified from protein by using an izon qev-10 column, collecting 5 ml fractions. protein content of each fraction was analysed using a280 absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~190 µl using the pall microsep 100 kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the 11 detection antibodies used across 39 capture beads, cd276, cd26, cd82, beta-2 microglobulin, and cd151 were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd63, cd81, and cd29. summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct116 and ht29, were cultured in serum-free medium for 24 hours. conditioned medium was concentrated by ultrafiltration (mwco 10 kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff-1 fibroblasts were used as recipient cells. the uptake of evs (pkh26 fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff-1. results: nta showed a higher concentration of evs released by ht29. hct116 and ht29 evs displayed a mean diameter around 140 nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd81 and tsg101 and negative for gm130 a non-evs marker. ht29 lineage as well as derived-evs are lmwptp-enriched in comparison to hct116 cells and evs. upon incubation, fluorescently hct116 and ht29 derived evs were internalized into hff-1 cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff-1 cells. intriguingly, evs derived from ht29 promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff-1 fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir-181a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir-181a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir-181a expression and silencing of exosomal mir-181a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r181a expression in all. methods: jm1, sup-b15, and nalm-6 leukaemic cell lines were treated in vitro with vincristine (0.1 to 4.0 µm) and prednisone (0.1 to 12.0 µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir-181a analysis. expression of mir-181a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm1 and sup-b15) in a dosedependent manner. both cellular and exosomal mir-181a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm1, sup-b15, and nalm-6). these observations demonstrate that cellular mir-181a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir-181a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for 24 h either under normoxia or hypoxia (1% oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq550 (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin-1 function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., 2017) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her2 mirna and engineered to adhere to her2 on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging 100-150 nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her2 (human epidermal growth factor receptor 2) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her2 both in cells constitutively expressing her2 and in cells transfected with a plasmid encoding her2. secondly, we report that the mirna targeting the synthesis of her2 reduced the viability of her2 positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her2 on the surface of tumour cells. the 293-mir-her2 exosomes package with mirna designed to block her2 synthesis and deliver to cells. these exosomes kill cancer cells dependent on her2 for survival but have no effect on cells lacking her2 or which were engineered to have her2 but do not depend on it for survival. the 293-mir-xs-her2 exosomes carry in addition a peptide which enables the exosome to adhere her2 on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her2 on their surface. the exosomes with 293-mir-xs-her2 are significantly more effective in shrinking the size of her2-positive tumours implanted in mice than the 293-mir-her2 exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her2 have no effect on her2 negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her2 mirnas by her2-positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her2 on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her2 by her2 positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj20170411 094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd8 + t cell activation introduction: cd8+ tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd8+ til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd8+ til such that specific mirnas contained within modulate physiological processes that inhibit cd8 + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd8 + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after 3 days of co-incubation of tumour derived exosomes, cd8 + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day 0 and day 3. rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd8 + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd8 + t cell activation owing to different donors. exosomes derived from peo.1 and ovca432 cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo.1 remained a low ifn-gamma expression and from ovca432 stayed at relatively high level. small rnas profiling analysis between the two cell lines identified 56 mirnas (p < 0.05), and 13 mirnas have been reported with validated targeting information, and 10 out of 13 have targets involved in immune signalling. 210 mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top 20 enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd8 + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose (8gyx3) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [1] [2] [3] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated (8gyx3) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [4] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [4] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by 5-methyl cytosine dna elisa kit. results: dna fragments with size between 60 and 250 bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than 99% of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of 60-250 bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [5] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire1) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire1 inhibitor mkc8866 and cells were analysed for immune modulatory surface markers, like hla-i, b7-h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd54) are downmodulated by ire1 inhibition across different cell lines. cell surface expressed cd63 and b7-h3 show cell line specific downmodulation profiles upon ire1 inhibitor treatment. other immunomodulatory marker such as b7-h1 and b7-h4, integrin cd29, cell adhesion-promoting cd146 and stemness/metastasis marker (cd44 and ssea) are unaltered on ire1 treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd9, cd63, cd81), similar in number and showed differential expression of immune markers upon ire1 treatment. mitochondrial and lysosomal activities were unaltered under ire1 inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cúram research centre, 13/rc/2073), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = 3) following wisgrill et al., (2016) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, 31x106, 8,5x106 and 9,7x106 events/ml mean, eevs was 6,4x106, 1 × 106 and 4,3x106 events/ml mean, levs was 2x106, 6 × 105 and 1,3x106events/ml mean and mevs 5,7x105, 6,5x105 and 3,7x105 events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp 2014/00984-3. gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens 1, zo-1) and gap junctions (con-nexin43, cx43) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for 48 h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo-1 at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx43, evs isolated during an episode of acs caused loss of cx43 from the plasma membrane. the integrated intensity of cx43 membrane staining was decreased bỹ 20% following treatment with acs evs. cx43 protein decreased on average by 32%, cx43 mrna levels by 21% and neurobiotin transfer by 67-94% in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx43 are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx43-mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul1 tr000430, comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited 7 day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to 10 days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation (100,000xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and 50% lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd41 as a marker of platelet origin and cd9 as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < 0.001) when compared to the control plasma. pevs also had a significant effect (p < 0.01) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) 30-100 nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor 5-year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from 12 paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the 12 paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from 30 nm to 300 nm with an intact morphology, expressing ev-associated markers such as hsp70, cd63, cd81 and cd9. size-resolved evs also had a differential expression of mitofilin, actinin-4, syntenin-1 and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc3-epi and pc3-emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc3 cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at 1000 × g for 20 min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at 10,000 × g for 20 min at 4°c. the remaining supernatant was then centrifuged to isolate the evs at 100,000 × g for 120 min at 4°c. the evs pellet was further washed in 1× pbs followed by a second centrifugation at 100,000 × g for 120 min at 4°c. the final evs pellet was resuspended in 1× pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of 770 selected mrnas. the nanostring ncounter low rna input kit with the multiplex 770gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all 12 samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc3-epi and pc3-emt cells were significantly different. clec2b, kdr, crip2, il13ra2, cc2d1b were significantly upregulated in pc3-emt cells, while cxcl8, epcam, esrp1, tgfb2, cdh1, s100a14, ovol2 were significantly downregulated in pc3-emt cells. the expression patterns between pc3-epi and pc3-emt evs were also significantly different. tbx1, cav1, col4a1, slc35a3, myc, itgb2, timp4, camk2b, ptgds, p3h2, itgb6, vim, stat3 were all significantly downregulated in pc3-emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta6 integrin regulate cd163, il-6 and il-10 levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta6 integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc3 prostate cancer cells. we have also shown in the past that alphav beta6-positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m2 markers, such as cd163 and cd204. finally, we have demonstrated that down-regulation of alphav beta6 integrin up-regulates the stat1-interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat1 has a causal effect in pbmc polarization, we down-regulated alphav beta6 and stat1 in prostate cancer cells derived sevs using sirna as well as crispr-cas9 strategies. the sevs isolated from these cells were used to analyse m2 polarization by measuring the levels of cd163 in pbmc. the results show that sevs lacking alphav beta6 inhibit cd163 levels in pbmc in a stat1-independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta6, show that pbmc selectively up-regulate the levels of il-10 and il-6, which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta6 do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta6 integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd163, il-6 and il-10 levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, 2016) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b6 splenocytes in vitro. the transfer of donor mhc class i and ii on b6 cells was analysed by imaging flow cytometry. next, t cells from b6 mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b6 spleen cells crossdressed with allogeneic balb/c mhc. alternatively, 2 × 109 balb/c or b6 bm derived evs or 20 × 106 balb/c bm cells were injected iv to b6 mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r01dk115618. antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c3 against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c3 produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. 2018) . we show here that the more biologically relevant surface-associated c3 exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c3 as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c3 culture on agar by similar methods to meers et al 2018. omv were stained in some cases with fluorogenic syto 9 dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c3 was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa-555 and washed 4x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto 9-stained vesicles from surface-adherent c3 were similar to previously observed~130 nm vesicles (meers et al., 2018) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c3 cultures grown on filters with 400 nm pore size but not 30 nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c3 on filters with a 200 nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c3 omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c3 specifically bind and inhibit the growth of fungal hyphae of various species without direct c3 cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt549, and human and rodent endothelial cells, with a varying degree of icam-1 expression, were used to determine the effect of lfa-1 expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam-1 and lfa-1. in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf-1 syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of 45 cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for 5 days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf-1 was the cytokine detected in mvs samples in large amount (0.88 ± 0.63 pg/µg proteins in mvs). fibroblasts treated with mvs or plgf-1 significantly stimulated pro-collagen i level production with a fold change of 1.80 ± 0.18 and 2.07 ± 0.18. moreover, the neutralization of plgf-1 present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf-1 signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin2014-04404); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size (30 to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation (2000 g for 20 minuntes followed by 13,000 g for 2 minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd41, anti-cd63, anti-cd81 and anti-cd9 were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd41/cd9 population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd81 positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank3 animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank3 mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank3 homozygotes mice model of autism. cultures were treated with msc-exo (10^7 particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of 372 mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around 90% of detected mirnas were found in both blood products, whereas only 30% of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir-101 were consistently depleted in evs compared to the corresponding blood product, others like mir-27a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing 3 (nlrp3) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp3 inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp3 inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp3 inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp3 inflammasome because s-elns had no impact on activation of the absent in melanoma 2 (aim2) inflammasome. s-elns also inhibited the secretion of interleukin (il)-6 and both protein and mrna levels of the il1b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp3 inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated 3 different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of 30 different proteins found in bovine serum were quantified. results: six of 30 proteins were not detected in any sample. 8 of 24 array antibodies were found to cross react with human antigens. of the 16 bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained 15 of 16 proteins,13 of 16 proteins, and 0 of 16 proteins, respectively. concentrations of individual bovine proteins ranged from 0.2 to 1,220.1 ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: 1) methodology, 2) study design, 3) intervention details and 4) efficacy/ adverse events. results: after screening 754 articles, 206 studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal (16%), neurological (12%) and cardiac (10%) conditions. benefits were described in 95% of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~30-150 nm, microvesicles~150-1000 nm) only 60% of studies used appropriate nomenclature. ultracentrifugation (70%) and isolation kits (23%) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein (68%), particle number (16%) or cell count (4%), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only 12 and 4 studies met the characterization standards recommended in the misev 2014 and 2018 guidelines, respectively. finally, 50% of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately 150-200 nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta2, fn, tnc, snai2, col1a1 and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad2/3 pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata12: umo-2016/23/d/nz3/01310 from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all 50-200 nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd63, tsg101 and hsp70. in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir17-92a cluster and mir302-367 cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after 48 h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after 4 weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox-2, pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at 3 consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls-1-1-051 g. introduction: malnutrition impacts approximately 50 million children worldwide and is linked to 45% of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; 30-150 nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control (18%) or a low protein (1%) diet for 14 days to induce malnutrition. from day 10 to 14, they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day 15, 4 hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of 3d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how 2d and 3d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for 2d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for 3d culture, mscs were aggregated into spheroids using non-adherent 96-well plates and cultured with addition of 0.25% methylcellulose. to collect conditioned media, both 2d and 3d mscs were cultured using serum free medium for 4 days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd81/cd63/cd9. cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β1 to induce fibroblast activation, subsequently exposed to 2d and 3d evs, and collagen production was measured. further, 2d and 3d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: 2d and 3d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd81/cd63/cd9 and average particle diameters of <100 nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in 2d and 3d mscs. 2d and 3d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both 2d and 3d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in 3d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for 3d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > 20 fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around 30-50 nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip56 that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a 64kda omp, predicted to be involved in iron acquisition, and 5 other omps (18-34kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to 30 ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to 0 gy or 13 gy x-rays to the whole body except the hind leg. starting with either 500 µl or 1000 µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a 3rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as 500 µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from 20 ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from 20 µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from 20 ml culture medium or 20 µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks (2,427 m/z, 3,596 m/z, 4,458 m/z, 4 ,537 m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks (2,268 m/z, 5,713 m/z, 6,255 m/z, 6,337 m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd63, cd81, cd9 protein markers using western blot analysis. fraction no.8-10 of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in 2-d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using 2-d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly 200 protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for 2-d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: 1) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and 2) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir-93, lncrna al137186.2 and ac108050.1 were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of 3 ibd patients and 1 healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd3-cd28 beads for 6 days in serum-free media. size and concentration were analysed using a nano sight 300 instrument, while the presence of known evs markers (cd63, cd81, hsp70) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of 1299 proteins in the four groups. of those, 673 (51.8%) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd9, tubulin, and serpins among others. patients' evs shared 46 proteins in common such as proteasome subunit beta type-10, t cell receptor beta, and the amine oxidase containing copper 3. interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type-3, and signalling lymphocytic activation molecule (slamf1). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about 75% of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top 3 go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top 3 biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = 5 subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from 0.5, 0.5 and 2.7 ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l1cam antibody. in total and l1cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd81, cd63 and cd9 on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < 0.0001) and protein concentrations (p < 0.001). l1cam evs immunocaptured after exq showed 16-fold higher particle (p < 0.001) and fivefold higher protein (p < 0.01) concentrations compared to l1cam evs after exu, and 41-fold higher particle (p < 0.001) and 11-fold higher protein (p < 0.001) concentration compared to l1cam evs after qev. l1cam evs after ev precipitation (exq) showed 48, 35 and 30-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after exu, and 59, 43 and 36-fold higher cd81, cd63, and cd9 concentrations (p < 0.001) compared to l1cam evs after qev. l1cam evs following 3 different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l1cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l1cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a549 and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd63 and cd81. protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range (45-100 nm), protein content (20-40 ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = 20) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd62p using flow cytometry. the resulting platelet releasate (500 ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for 16 hours before treatment with pevs (15 µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be 100-300 nm in size. the final product was positive for platelet markers (cd41, cd62p) and key ev markers (tsg101, alix, cd63). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over 48 hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev 2018 guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex1) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually 50-150 nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af (1.23 ± 0.03) is significantly lower than that in mj (1.27 ± 0.01), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs (1.23 ± 0.03 and 1.29 ± 0.02, respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj (173.0 ± 21.3 nm), af (154 ± 11.0 nm), af-derived evs collected at 40,000 × g and 10,000 × g (108.5 ± 2.4 nm and 95.7 ± 3.1 nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only 500 µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture 6 classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed 1.02 × 1010 particles per ml from a 1 ml fplc fraction of evs measured from 25,000 events by mrps, confirming that evs are being captured by this method. there were also 1.26 × 1010 particles/ml and 2.70 × 1010 particles/ml in the two subsequent 1 ml fractions that are known to contain lipoprotein particles, though these were measured from 1,500 events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single 500ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r01ag062240; nih ug3 ca241694-01 optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a 3d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd9, cd63, and cd81, calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd9, cd63, and cd81 and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after 10 weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses (1,000 to 750,000 da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek293 cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > 10,000 da and linear pei with mw > 25,000 da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir-142-3p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: 2-3 ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into 3 batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of 6 adherent cell lines were established in celline ad 1000 bioreactors and propagated for up to 19 weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation (2000 g -10,000 g -100,000 g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt20 and bt474 consistently shed mainly dead cells (60-100%), as opposed to mcf7 and mda-mb-231 which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each 3-4 day culture averaged 2-7 × 1010 particles, representative of yields obtained from cells grown in 8 to 28 conventional t175 tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to 19 weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the 5ʹ-end and cholesterol at the 3ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio-66-nh2 and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with 78% isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in 40 minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin 12 (il12) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs1 locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at 0, 1, and 2 months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il12 and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il12 and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp2, a highly pathogenic strain, and 33384, a natural commensal strain. af488-labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto-1. results: using flow cytometry, we observed jp2 omvs were delivered to 33,384 cells, and at a lesser amount to jp2 cells. alternatively, 33,384 omvs associated readily with jp2 cells, more than to 33,384 cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r03de025275 & r21de027769. utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap1) modified via crispr-cas9 to model gaucher's disease in vitro. these cells contain a 479bp insertion in the 6th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc9) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were 130 nm in size with a typical lipid bilayer structure, and were positive for cd63, cd81, flotillin and negative for annexin a1 confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph 5.5) compared to drug release at ph 7.2. cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a549 lung cancer cells compared to mrc-9 cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a549 tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il4, are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv2) was engineered to stably overproduce endogenous il4. this cell line was cultured in exosome-depleted rpmi and stimulated with pma(20 mg/ml) for 30 min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of 300 g,10 min, rt and 2000 g,20 min, 4°c. the resulting supernatant was filtered (5 µm) and ultracentrifuged at 100,000 g for 2h at 4°c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of 60-80 nm and a broad zeta potential ranging from −10 mv to −60 mv, while the second population has a mean diameter of 120-140 nm and a zeta potential of −10/-20 mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il4 inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il4 internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il-3rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin-3 acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il-3rα blockade on tec changes tec-ev (anti-il-3r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il-3r blockade on tec impacts tumour progression via their unique ev cargo. methods: 27 human tnbc samples, mda-mb-231, mda-mb-453 and mcf10 cell lines were evaluated for the expression of il-3rα. nevs and anti-il-3r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from 55.5% of the human tnbc samples expressed il-3rα. mda-mb-231 and mda-mb-453, but not mda10 cells, expressed il-3rα. in vitro, nevs provide survival and migratory signals, while anti-il-3 r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il-3 r-ev treatment induced vessel regression in established tumours formed of mda-mb-231 cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist1 were found in tumours from animals treated with anti-il-3 r-evs. in addition, anti-il-3 r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb-231 cells. nevs that were depleted of mir-24-3p (antago-mir-24-3p-evs) were effective as anti-il-3 r-evs in down-regulating twist1 and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il-3 rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il-3 rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya8 ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya8 cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either 3d migration or boyden chamber invasion assays. previous to exosome isolation, heya8 cells were treated with simvastatin (5um, 24 h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg101), its trafficking (rab7a, rab27a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya8 cells or hgsocs enhance the metastatic potential of heya8established spheroids in 3d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya8-derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg101 and an increase in the inactive forms of rab7a and rab27a in heya8 cells. we also observed a decrease in emmprin levels in heya8-derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw620 colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw480 carcinoma cells expressing er-gfp and rab7-rfp were exposed to evs from sw620 cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp3 and rab7 orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp3 or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta1 are co-immunoisolated with orp3, vap-a and rab7 suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p21 and p27. bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g1 checkpoint proteins, p21 and p27. we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk1/2. because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r00 ca178177 and commonwealth of pennsylvania -department of health sap 4100072566 for kmb. phosphorylation of tyrosine 23 in annexin a2 is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for 15%-20% of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a2 (anxa2), a ca+2-dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa2 is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) 23 in anxa2 has been implicated in regulating several anxa2 activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa2 at tyr23 is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa2 as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn1-egfp plasmids expressing the constitutive phosphomimetic (anxa2-y23e) and non-phosphomimetic mutant (anxa2-y23 f) gene expressing mutation at tyr23 site were overexpressed in mda-mb-231 tnbc cells. mutant cells were experimentally validated for anxa2 specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa2-y23e-gfp) and non-phosphomimetic (exo-anxa2-y23 f-gfp) cells and were analysed for exosomal surface expression of anxa2 by immunoprecipitation and flowcytometry. cal-148 breast adenocarcinoma epithelial cells were treated with exo-anxa2-y23e-gfp and exo-anxa2-y23 f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa2 in cal-148 was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb-231 phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa2-y23e-gfp had elevated surface anxa2 expression compared to exo-anxa2-y23 f-gfp. cal-148 cells treated with exo-anxa2-y23e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa2(tyr23), p-src(tyr416) and p-paxillin(tyr31) compared to exo-anxa2-y23 f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr23 in anxa2 in mda-mb-231 tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro1ca220273 and nimhd's u54md006882 to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from 15 patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from 500 µl of plasma using a commercially available qev 35 size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd61 magnetic beads were used to fractionate the evs into cd61+ (platelet derived) and cd61-(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd9 and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd61+ and cd61fractions in all patient samples. the average ± standard deviation of total evs obtained from the 15 patients was 6.39x10^11 ± 4.60x10^11 evs/ml of plasma (coefficient of variation [cv] : 72%) while the average and standard deviation of cd61-evs was 1.24x10^10 ± 3.37x10^10 (cv: 271%). the cd61-ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from 0.0001% to 32.21%. multiparameter flow cytometry showed that over 80% of total evs were cd9+ and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd61-fraction (5% -30%) as compared to the cd61+ fraction (0.04% -1%). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd61-ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp2 and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than 10% 5-year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp2 and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp2 functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp2 and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < 0.01). notably, functional exosomal mmp2 and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ 3.8-fold (p < 0.01). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp2 and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp2 and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm23-h1, product of first identified metastasis suppressor gene nme1, is significantly downregulated under metastatic conditions. nm23-h1 serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab5, rab7 and rab27) in the biogenesis of exosomes. in addition, bacterial homolog of nm23 has been shown to interact with rab5 and rab7. however, experimental evidence supporting a relationship between exosomes and nm23-h1 is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb-231 cells) on lower/non metastatic cells (mcf-7 cells). nme1 was overexpressed in mda-mb-231 cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb-231 cells and mda-mb-231/nme1 cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm23-h1 in the exosomes isolated from mda-mb-231 cells upon overexpression of nme1. proteinase k protection assay confirmed the packaging of nm23-h1 inside the exosomes isolated from mda-mb-231/nme1 cells and excluded the possibility of membrane association of nm23-h1. additionally, overexpression of nm23-h1 led to a significant reduction in the ability of mda-mb-231 exosomes to stimulate movement of mcf-7 cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd63, cd9 and alix in the exosome fraction isolated from mda-mb-231/nme1 cells as compared to mda-mb-231 cells. interestingly, rab7a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm23-h1 overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm23-h1 via exosomes. these findings support a regulatory role of nm23-h1 in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm23-h1 which affects the biogenesis of exosomes as well as to assess the effect of nm23-h1 overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c57bl/6 mice performed moderate treadmill exercise (10 m/min, 4-wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day 2 by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day 28. the correlations of cepc-exs and their carried mir-126 with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir-126 level, apoptosis, axon growth ability and gene expressions (cas-3, bdnf and akt) were measured. results: 1) exercised mice had a smaller infarct volume on day 2, which was associated with decreased cell apoptosis and cleaved cas-3 level, and a higher microvessel density than those in control; 2) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day 2. the upregulated mir-126 level positively correlated with the numbers of tepc-exs in ischaemic brain; 3) the numbers of cepc-exs and their carried mir-126 level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day 2; 4) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day 28, accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; 5) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir-126 and pi3 k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir-126. funding: this work was supported by american heart association (18post33990433) and nih (1r01ns 102720). syndecan-1 regulates alveolar type 2 epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion1 zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type 2 epithelial cell (at2) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan-1, a heparan sulphate proteoglycan, is overexpressed by at2 cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan-1 is profibrotic. moreover, syndecan-1 deficient (sdc1-/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc1-/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir-34b-5p, −142-3p, −144-3p, and −503-5p) compared to the sdc1-/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan-1 altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan-1 regulates at2 senescence through evs. methods: bleomycin was intratracheally given into wt and sdc1-/-mice. at day 21, lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a549) and mouse (mle-15) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at2 cells of wt mice, these genes were significantly downregulated on sdc1-/-at2 cells. senescence proteins, p16 and p21, were also less expressed in the lungs of sdc1-/-than of the wt mice by wb. to determine the functional effects of evs in bal, a549 cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p21, by wb on mle15 cells whereas sdc1-/--bal-evs inhibited p21 expression. summary/conclusion: our data indicate that syndecan-1 regulates lung fibrosis through the senescence signalling pathway on at2 cells. furthermore, syndecan-1 controls at2 senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for 24 hrs in serum-free basal media, in hypoxic culture. after 24 hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, 8-10 week-old wistar-kyoto female rats received 10^10 imcdc-ev, imev-pi, scramble or vehicle intracoronary 20 minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at 24 and 48 hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were 35x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at 48 hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at 24 hrs and an increase at 48 hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles (30-200 nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from 100 to 500 nm, with the majority of them measuring 100-200 nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms (2d static tissue culture flask vs 3d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in 2d t-flasks and 3d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both 2d and 3d culture systems. msc-ev were characterized according to misev 2018 guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the 3d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = 6 in each group) were used to isolate sevs with 3 different isolation methods including commercial exoquick ultra kit, qev 35 and qev 70 size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd9 and cd81 commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev35 demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev70. furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev 70 method demonstrated better performance in isolating relatively pure sevs from human plasma; qev35 has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev35 but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to 5 channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of 10%. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd9 and cd63. the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd9, cd63 and cd81, it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: 1) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and 2) the assessment of subpopulation target enrichment relative to the population average by leveraging tim4 as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr3 (her2+), t47d and mcf-7 (er+pr+), bt549 and mda-mb-231 (triple negative) breast cancer cell lines, as well as five mda-mb-231-derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her2 was broadly detected in her2+ skbr3 evs. interestingly, her2-t47d and mcf-7 evs also expressed her2 where it was highly enriched in its epcam+ subpopulations. itg α6, β3 and β4 were only found in triple negative and organotropic evs with itg β3 and β4 differentially enriched based on the organotropism. the population average of mda-mb-231 and lung-tropic evs had high expression of itg β4, where subpopulations of cd44+ evs showed positive enrichment while cd9+ and cd63 + evs showed negative enrichment. itg α5, β3 and β4 were absent in the bone-tropic cd81+ ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb-231 evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du145 prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified 49 targets that bind to hs-gag chains, and also 108 different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of 48 patients (15 highgrade serous, 24 low-grade, 7 benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = 100) was used for validation. results: an oc biomarker panel of 10 micrornas (voom: 96.55% accuracy; random forest: 88% accuracy) was generated. the panel consists of members from the mir-200 family and the mir-16 family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh7.5cyp2e1 [hepatoma cells stably transfected with cyp2e1 designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from 4 groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs 2018 (misev2018). the human hepatic stellate lx-2 cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il-1β, il-6, tnfα, and fibrotic markers acta2, and timp1 using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror 2.0 and david bioinformatics resources 6.8. results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately 85-90% of particles distributed in the range of 50 to 200 nm, with a peak at~90 nm. enriched expression of hiv protein p24 was observed in fractions f9-f12. western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg 101 and cd9 specifically in f2-f8 fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il-1β, il-6, tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih-1 r01 aa027189-01a1. antimicrobial peptide ll-37 induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll-37, a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll-37 and functions of ll-37-induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll-37-stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll-37 augmented the level of evs. evs from ll-37-injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll-37 stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll-37-induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll-37 induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december 2019, and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top 10, which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir-140-3p, and hsa-mir-423-3p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with 5 × 105 or 107 evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in 3 doses during 45 days (15 days interval). after immunization, mice were infected intra-peritoneally with 500 trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f4/80+ cd11b+ and cd80/cd86 expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab5075. seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a549. results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a549 cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict 2017-3536) medicine, nanfang hospital, southern medical university, guangzhou, 510515, china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw 264.7 murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of 30 to 200 nm. t. marneffei evs were internalized by raw 264.7 murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m1-polarization marker cd86 and diminish that of the m2 markers cd206. incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin-1β(il-1β), interleukin-6 (il-6) and interleukin-12 (il-12). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m1 polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about 8 million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation (2,000 × g, 10,000 × g) and concentration (100 kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h2 s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy4137 treated (gyy-evs) a549 cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a549 cells were cultured for 24 hours in the presence or absence of control-or gyy-evs, then infected with rsv for 24 hours. viral titres by plaque assay were measured in recipient infected a549 cells. results: we confirmed the presence and purity of our evs. we found that gyy4137 reduced the particles number of evs, but did not change ev size. a549 cells treated with gyy4137 showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd63 expression, a mvbs marker, compared to untreated cells. recipient a549 cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h2 s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd45+ cells included b (30%), cd4 + t (16%), cd8 + t (10%), nk (6%) and nkt (2%) lymphocytes. myeloid cells comprised neutrophils (16%), monocytes (2%) and dcs (0.3%). erythrocytes represented 70% whereas, unexpectedly, reticulocytes were 0.93% of total cells. in addition, we also detected hscs, which accounted for 0.02%. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd63, cd5 l and cd71 markers. interaction studies showed an increased proportion of t cells (cd4 + 3-fold and cd8 + 4-fold), monocytes (1.5-fold) , b cells (2.3-fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir-375 promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below 200 nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf7 breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb-231 triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf7 cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb-231 significantly reduces the young's modulus values of mcf7 cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf7 cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf7 cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over 50% of the crc patients develop liver metastasis (lm) and 90% will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only 20% of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only 10-15% of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms 2 distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double 5-year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: 1. text mining of published ev mirna from crclm patients; and 2. differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing 50-60 patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach 2, where we have classified the lesions in the patients. preliminary data on 10 patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein 1 (cdcp1), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp1 was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp1 exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp1 in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr4 in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr4, known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr4/cxcl12 signalling. overexpression of cxcr4 was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr4 has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr4 in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht29 and sw620, which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh67 fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr4 in ht29 and sw620 by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr4 in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht29 and sw620 cell lines. the expression of cxcr4 in ht29 was increased after the uptake of p-evs. additionally we observed that migration rate of ht29 cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr4 receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht29 cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht29 clones stably overexpressing snail and from control ht29-pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after 24 h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ 50 reads, two fold up or down regulation between 2.5 − 7.5 and adjusted pvalue ≤ 0.05. the uptake of pca-sevs was performed. three candidate mirnas (has-mir-200 c-3p; has-mir-1275; has-mir-383-5p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < 0.001), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw4869. the uptake of pca-evs by mc3t3-e1 was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx2, osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china (81872347); xijing hospital science and technology foundation project (xjzt19ptk14). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from 4 rbt (hsjd-rbt1, hsjd-rbt2, hsjd-rbt5, hsjd-rbt14) and 3 rbvs (hsjd-rbvs1, hsjd-rbvs3, hsjd-rbvs10) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of 5666 proteins were identified. among these, 5223 and 3637 were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin-5, tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over 300 cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from 3 independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc 1000 and q exactive instruments. tune (version 2.9) and xcalibur (version 4.1) was used to collect data while proteome discoverer (version 2.2) was used to analyse data. protein expression lists were created by merging the 3 sample replicates together and commonly expressed proteins were determined using vinny 2.0 vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of 5.24x10^11 particles/ml (n = 12) with a mean mode size of 125.2nm (n = 12). surface marker analysis confirms the presence of exosome associated proteins cd63, cd81, and cd9 in addition to a high expression of cd133 (n = 3). the completed analysis revealed 1225 commonly detected proteins across 3 products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p53 pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca1. summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd19-targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a 3-day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm-6 (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at 1000 particles/target cell for 24-hours. total viable cell counts were assessed via imaging-based cytometry (nc-3000) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm-6 as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd19 car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm-6 cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks (50 billion per administration) starting from 18.5 months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin-1 beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin-10 and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last 2 decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c2c12 myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after 5 days incubation. treatment with bmp-4 was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for 3 days. qpcr analysis of mrna expression of inflammatory cytokines (il-6, il1-beta, il-8) metalloproteinases (mmp3 and adamts4), and apoptotic genes (bax, bcl2) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd81, cd63 and cd9). the mean number of exosomes per microgram of protein was 3.31 ± 2.33 x 108 indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c2c12 cells with bmp-4, 2 x 108, 1 x 109, 2 x 109, 5 × 109 or 1 × 1010 exosomes alone was 28. 5, 1.0, 3.7, 7.4, 11.8 and 27.6 respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba-1 and cd68), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank3 gene. shank3 is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank3 mutated mice. the first is a complete deletion of exon 22 (22q13.3) and the second is a specific insertion mutation of guanine to 3680 position in the gene (insg3680) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the 2018misev guideline recommendations. each animal received an intranasal administration of 20ul containing 107 exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in 100µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within 72 hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses (6, 19 and 23 years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd9, cd63) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < 0.0071). the gap closure achieved with msc-evs was slower than with msc-cm (p < 0.7985) but faster than with cm of tenocytes (p < 0.8289). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd-63 (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c57bl/6 mice in chf received echoguided myocardial injection of pbs vehicle control (60ul, n = 11), cpctx-ev (60ul, n = 11), or cpcres-ev (45ul, n = 11). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~72 nm) at a concentration of~1.6e11 particles/ml (~2,600 particles/cell) and~0.975mu cd63/ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by 4.75x, 1.4x, and 2x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased >14%) than pbs control mice (pbs vs cpctx-ev, p < 0.05; pbs vs cpcres-ev, p < 0.056), and the average ejection fraction of the pbs group deteriorated 2.5x more than the cpctx-ev group (−4% vs −1.6%, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds (3-mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for 4 and 8 weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp7 (osterix) was evaluated at 4 and 8 weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the 50ng bmp-4 control. dose escalation from 1.5, 2, and 4e9 resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at 4 or at 8 weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp 1, 2, 3, 4 and 6, tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il-6 r, il-16, were also detected. expression of pro-inflammatory cytokines mcsfr, mip-1a; anti-inflammatory cytokines tnf-ri, tnf-rii, il-1 ra; and homoeostatic cytokines timp-1 and timp-2 were observed. cytokines associated with wound-healing, icam-1, g-csf, gdf-15, and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range (30-150 nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly 50-200 nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at −20 c. calvarial defects of 8-mm diameter were surgically created on thirty-two 10-week-old male sprague-dawley rats. these rats were then randomly assigned to 2 groups (n = 16 rats/group): defects treated with collagen sponge containing 100 μg of sevs in 100 μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at 1 and 8-week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at 1-week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of 5.7 ± 2.4% than that of cs/control group (2.1 ± 1.3%; p = 0.003). cd31+ microvessels at sizes ≤50 µm and >50 µm in cs/sevs group (34.4 ± 2.9 and 8.6 ± 2.1 microvessels/hpf) were also significantly higher than that of cs/control (18.8 ± 1.1 and 5.2 ± 1.4 microvessels/hpf; p = 0.002 and p = 0.038 respectively). by 8 weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of 3.9 ± 0.2 which was significantly better than that of cs/control group (2.5 ± 0.8; p = 0.001). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < 0.01), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of 5.4 ± 1.0 than that of cs/control (2.6 ± 1.8; p = 0.001). moreover, cs/sevs group showed significantly higher ocn+ area of 48.9 ± 7.9% than that of cs/control group (20.4 ± 4.4%; p = 0.004). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r221000114114, national medical research council singapore, r221000123213. immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to 12 different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for 5 days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd14+ (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd4 and cd8 t cells expressing activation markers like cd54 and cd25. summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd4 cell types, i.e. th1, th2, th17 and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct04281901). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from 29 blood samples of volunteers with no record of disease. methods: plasma obtained from 20 ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = 0,70, p < 0,001) and to the concentration of evs (r = 0,82; p < 0,0001). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = 0,75, p < 0,001). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon 2020 research and innovation program under grant agreement no. 801338 (ves4us project) and slovenian research agency (arrs, grants p2-0232, p3-0388, j1-9162). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c2c12 and primary dmd myotubes. methods: scs from c57 mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c2c12 myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c2c12 myotubes were treated with 50 µm h2o2 for 24 h followed by treatment with 3.12x108 sc-evs for 24 h. separately, cultured dystrophic myotubes were treated with 3.12 × 108 evs every 24 h for 72 h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < 0.05). results: based on estimated cell number and ev quantification, each sc released approximately 2.35 × 105 ± 3.10x104 evs/day. evs delivered protein cargo into myotubes within 2 h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h2o2 resulted in a 42% decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a 76% increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a 78% increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of 180 nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of 80-8,000 ng/ml and 4-800 µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r2 = 0.92). importantly, cholesterol quantification on purified ev samples, ranging from 1e11 to 4e13 particles/ml, correlated well with nta measurements (r2 = 0.96). both 45 µm filtration or an additional 16,000 rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx220 (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd9, cd81, cd63). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly 100% of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around 40-50% of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than 50 nm cannot be detected, while fluorescently stained ev smaller than 50 nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small (30-150 nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di-8-anepps (di-8) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di-8). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo 357 pancreas carcinoma cells based on size and mass. after 10,000x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from 80 to 40 nm in size. interestingly, tetraspanin markers cd9 and cd81 were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant 180802), leo pharma germany (grant 180208). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd9, cd63, cd83) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat 120 min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs (8.3 particles/μl) method was estimated 3000 times superior to the lod of conventional elisa (26,000 particles/μl). the spfs calibration curve showed a wide dynamic range at least over 5 logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about 10 times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim4. by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about 1,600 compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd63, cd9 and flotillin-1 were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas 2016) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb-231-luc-d3h1, -d3 h2ln and -bmd2a were cultured in ev-depleted media. microvesicles (16,500 x g, 20 min) and exosomes (118,000 x g 2.5 h) were isolated using a combination of differential ultracentrifugation and a density cushion (~1.1 g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = 3 x 3 cell lines). results: in total 6493 proteins were quantified, with 4851 being quantified in all samples. in total 818 and 1567 proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin-1 was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam10 and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to 10 minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo (2018/7633081117). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered 2,5% glutaraldehyde/2%formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the 3d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd171 has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at -80 degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with 405 instead of 488 nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r100 platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd9, cd63, cd81) and the neural cell adhesion molecule l1 (cd171). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd9, cd63, cd81). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd171 binding. interestingly, the cd171 positive evs were not positive for any of the classic exosomal markers (cd9, cd63, and cd81). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd171 could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε2/ε3 (n = 5), ε3/ε3 (5), ε3/ε4 (6), ε4/ε4 (6)] and non-ad controls (n = 7) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev2018. proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s5™ using ion™ 540 chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among 28 proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin-6. regarding apoe genotypes, 16 proteins were differentially expressed between ε4 carriers (ε3/ε4 and ε4/ε4) with non ε4 carriers (ε2/ε3 and ε3/ε3). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε2/ε3 and ε4/ε4 groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh118164. efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing 300 pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of 18 months-old c57bl/6 female mice. 4.5 months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca1 pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at8. evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca3 and ca1 subregions. at8 was localized primarily in gad67 + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca1 pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf1ag054199, nih r01ag054672, nih r56ag057469, cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd63 (cd63 + ev), cd81 (cd81+ ev), and neural cell adhesion molecule (l1cam+ev) were analysed in young, male athletes with or without mtbi (18-29 yo, n = 6 per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within 48-hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at −80°c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size (179-304 nm) cd63 + ev, cd81+ ev and l1cam+evs were significantly higher in mtbi athletes (mean rank: 8.0, 9.5, 9. 3) than controls (mean rank: 3.6, 3.5, 3.7) (u = 3.0, p = 0.03; u = 0.0, p > 0.01; u = 1.0, p > 0.01, respectively). population percentages of large size (500-800 nm) cd63+ ev, cd81+ ev and l1cam+evs were also significantly higher in mtbi athletes (mean rank: 8. 2, 9.2, 9.5) than controls (mean rank: 3.4, 3.8, 3.5) (u = 2.0, p = 0.02; u = 2.0, p > 0.01; u = 0.0, p > 0.01, respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd144+ ev) or platelets (cd42a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from 27 pd, 19 matched healthy controls (hc), 9 ap with multiple system atrophy (msa) and 9 ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of 37 ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort (10 pd, 20 hc, 5 msa, 5 ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc 0.908; sensitivity 96.3%, specificity 78.9%) and msa (auc 0.974; sensi-tivity100%,specificity94.7%)andgoodaccuracyforap-tau (auc 0.718; sensitivity 77.8%, specificity 89.5%). a diagnostic model based on ev markers expression, cor-rectlyclassified88.9%ofpatientswithreliablediagnostic performance after validation in an external cohort (77% of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off 3,000), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions (3 + 4) after the sec void volume as evaluated by detection of cd63 and cd9 markers (immunoblotting) and annexin a2 (peptide mapping by nanolc-ms/ms). there, nanoparticles around 150 nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between 55 and 165 nm, with average diameter 94 ± 31 nm. cd63 was visualized by immunocytochemistry at the surface of ev around 80 nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin-3 binding protein (lgals3bp or 90 k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those nanoparticles had ring-like appearance. occasionally 90 k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of 90 k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from 90 k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted 2, 4, 8, 16 and 32-fold and activated by 30 μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and 10-fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd61 + and cd61+, cd62p + >1000 nm) and activated platelets (cd61+, cd62p+ >1000 nm) were measured by flow cytometry (apogee a60-micro). platelet aggregation was assessed using impedance aggregometry. results: a 10-fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd61 + 2.2-fold and cd61+-cd62p + pevs 7.2-fold. the concentration of cd61+ (r2 = 0.71) and cd61+-cd62p+ (r2 = 0.78) pevs as well as platelet aggregation (r2 = 0.8) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd61+ (r2 = 0.80) and cd61 +, cd62p+ (r2 = 0.64) pevs in undiluted wb, which was absent in 10-fold diluted blood (r2 < 0.05). summary/conclusion: the absence of aggregation and pev release upon platelet activation in 10-fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium 2018/31/ n/nz7/02260. evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni 15924. age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at 5~7-(young) and 15-(aged) week-old. large evs were isolated from the plasma by centrifugation (10000 x g). sevs were isolated by ultracentrifugation (164,071 x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than 150 nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de ética no uso de animais -ufrgs; nr. 29,818) approved all animal procedures and experimental conditions. male wistar rats of 3-and 21-month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna 4.0 arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed 728 micrornas. of these micrornas, 48 were differentially expressed between aged and young-adult animals, 18 micrornas were significantly upregulated and 30 were downregulated in aged animals compared to young adult (p < 0.05; fold change of |1.1|). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < 0.0001). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin-1 signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule-1 (vcam-1) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) 1 and hmgb2. pharmacological blockade of hmgb1 and hmgb2 and sirna depletion of hmgb1 in smooth muscle cells attenuated nfkb (p65) phosphorylation and nuclear translocation, vcam-1 expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled 330 patients undergoing elective cabg. of these, 179 underwent coronary computed tomography angiography 18 months post-surgery showing 24% graft occlusion. flow cytometry mv analysis was performed in 60 patients (30/group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = 0.020) and four-fold (p = 0.042) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < 0.05). this mv signature predicted graft occlusion (auc of 0.897 [95%ci: 0,77-0,96], p = 0.02). by using a mv-score (0-6), the or for re-occlusion for a score above 3 was 16.3 (95% ci 4.1-65.3, p < 0.001). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme 2019). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg2 knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for 4 weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at 4 weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction 73 ± 2 vs 59 ± 5% in vehicle mice, p = 0.03), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration (4.0 ± 2.0 ms imexo vs 6.5 ± 2.9 ms vehicle, p = 0.02) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction (10.0 ± 32% vs 55.0 ± 52% in vehicle, p = 0.031). in vitro study showed nrvm exposed to imexos for 2 days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by 10-minute illumination with 254 nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r01 hl124074 (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf2/ are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf2/antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf2/are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf2 protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf2-targeting mirnas. nrf2-targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf2-targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf2-targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf2/ are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf2/are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx43, the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin43 (cx43) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx43. results: release of cx43 was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx43 in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p62 partially restored the secretion of cx43, suggesting an interplay between ischaemiainduced cx43 degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx43 into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor 4 (tlr4) and its downstream regulators (tnfα and il-6) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek293 cells was induced by calcium-ionophore a23187. evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h9c2 and ac16 cell lines. results: a23187 induction of hek293 cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to 16 h ischaemia followed by 2 h reperfusion in h9c2 and ac16 cells in a dose-dependent manner. evs activated tlr4 and its downstream signalling pathway in h9c2 and ac16 cells as well as the expression of cytoprotective haem oxigenase 1 (ho-1) in h9c2 cells. summary/conclusion: a23187-induced evs exert cytoprotection in h9c2 and ac16 cells by inducing tlr4 signalling and ho1 expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will (1) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and (2) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = 126) was calculated for a study power of 90% and α error of 5% for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = 63) and malignant (n = 63) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd63, cd81 and cd9. ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (>200nt) and short rnas were isolated from ev enriched urine of pregnant (n = 5) and non-pregnant (n = 5) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v4 ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v2 -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v.2.7.3.a. results: overall, rna isolation using mircury followed by the smart-seq v4 library preparation kit yielded the highest % of mapped reads: 42% in pooled non-pregnant, 27% in individual non-pregnant, and 31% in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v4 libraries had higher % of mapped mrna reads compared to pico libraries (p < 0.05, t-test). in contrast for mirneasy advanced it was reversed (38% vs 21%). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v4 kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih 7 k99 hd096125-02, nih u01 hl126494, and a ucsd igm-illumina mini-grant. il-2 mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin-2 (il-2) is essential for pan t cell proliferation and performance, low dose il-2 treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt101 is a novel il-2 mutein fc fusion molecule (il-2 m) designed to selectively engage with tregs. using a humanized nod-scid il2rn-null (nsg) mouse model we have shown that pt101 expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt101-dosed humanized mice exhibit increased expression of foxp3 and cd25, and demethylation of foxp3 and ctla-4 genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt101-or vehicle-treated mice in order to identify treg specific mirnas from the il-2 m treated animals. methods: cd34+ haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt101 or vehicle. plasma samples from 8 mice were collected at day 7 and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with 75bp single end reads at an approximate depth of 10-15 million reads per sample. raw sequences were mapped to human genome grch37 and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il-2 m-treated groups was mostly comprised of mirna and trna. plasma was pooled from 8 animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt101 treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt101treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv2 cells). a cd63 antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f4/80) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem119 and iba1) were identified in exosomes isolated from bv2 cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type 1 diabetic patients (t1d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from 10-50 ml of 24 h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v4 ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq 2000 (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq 4000 platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > 5 m) were closely similar. instead, rna reads were lower than 2 m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns300. the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over 90% of dna in plasma and urine exist as a free circulating pool, while in saliva up to 30% is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are 30-150 nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn96-isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn96 is a 27 amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn96 peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within 30 minutes. total nucleic acid (tna) was extracted from either vn96-isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn96 isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including 100% specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn96 peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn96-based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h660 and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: 10 plasma samples of hepatocellular carcinoma (hcc) patients, 10 plasma samples of patients with liver cirrhosis 3-4 on the hepatitis c virus (hcv) background, 5 healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd63, cd9) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b2 m, actb, tuba1a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for 50 genes in hcc and liver cirrhosis groups (increasing up to x200 compared to control samples and decreasing up to no detected expression). in 6 out of 10 patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in 8 out of 10 patients with hcc increased expression for mrna line-1 was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds "5-100" circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into 4 groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma (38 patients in total) and rna from plasma in a separate group (24 patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir-1285-5p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir-375 in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir-375) and ev-based (mir-1285-5p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after 2x3,000 g centrifugation, followed by differential centrifugation at 5,000 g and 10,000 g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd3 and anti-cd28 antibodies. after 24 h t cell activation was measured by il-2 cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il-2 production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il-2 production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon 2020 framework programme under the grant fetopen-801367 evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev2018. methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, 38 ml samples were centrifuged at 12 k g, 35 k g, 75 k g, 100 k g and 200k g sequentially for 75 min each and pellets re-suspended in 1 ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. 100 μl of the sample was loaded and the pbs buffer was used as eluent. 24 factions were collected and analysed by cd9/cd9 sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than 99.8% of proteins were removed while the recovery of extracellular vesicles was close to 70%, which is much higher than 32% of the commercial product. the total separation time was less than 15 min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~100 µl or > 100 ml) while concomitantly removing 99.9 % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: 50 ml of cell culture media (ccm), 0.5 ml serum, and 100 ml urine samples were processed with the isuf platform and recovered in 100 µl. for all cases, evs were enriched with recovery efficiency greater than 95%. the processing time for a 100 ml sample was 120 min with over 99% of purity. we compared ev concentration and purity isolate from 0.5 ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = 10), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from 10 metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her2, cd24 and mir21 biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug3tr002884 (e.r.); r01hl 126945, r01hl138116, and r01eb021926 (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated 3 commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs5 treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between 3 to 5 hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin-1 and cd81 by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of 1.07-1.11 g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd81 immunocapture. the resulting sev proteome was enriched 1.5-to 4-fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare 3 evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of 600 and 20nm. methods: a431 cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of 1 h10 at 110,000 g at 4 degrees celsius (rotor type 70ti, beckman floor ultracentrifuge optima l90 k), qev size exclusion chromatography columns from izon (qevoriginal/70 nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns500), trps (izon), nanodrop (spectrometernd1000), tem (fei tecnai 12 120 kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of 600 nm and 20 nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to 10 times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized 3 purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of 10, a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about 100-500 nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [1] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase 3 and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir-455-3p and hsa-mir-92a-3p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib49 cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir-146a and mir-155, play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg2, a stem cell marker, modulates ev release. dsg2 is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg2 were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch37 alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed 87 cellular and 15 ev mirnas that were differentially expressed in response to dsg2 with 7 overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p53 signalling pathways. interestingly, mir-155 was upregulated while mir-146a was dramatically downregulated in evs. targets of mir-146a, icam-1, il-6, and il-8, cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg2 contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r01 cancer cells grown in 3d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional (3d) cancer cell cultures. methods: human prostate cancer pc3 cells were grown in 3d cultures using ecm-based hydrogel, in standard 2d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that 3d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive 3d cultivated pc3 organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a 3d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of 3d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y2763 (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw4869 (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc3 (pc3 rd) cells after 48 h incubation in dfbs-medium with or without the 4 compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. 1.03-1.16 g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at 1x105evs/3x103cells, 1x107evs/3x103cells), were evaluated on recipient du145 and 22rv1 cells. evtrack id ev190113, score 88% results: calpeptin and y27632, alone and in combination, did not significantly affect quantities of evs released. however, gw4869 significantly (p < 0.004) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp94 compared to control evs (p < 0.006). this effect was reverted when gw4869 was combined with manumycin a (p < 0.004). following all compounds treatments, 1x105evs/ 3x103cells inhibited 22 rv1 proliferation (p < 0.0014), while at 1x107evs/3x103cells only evs from manumycin a (p < 0.05) and y27632 (p < 0.00036) treated cells reduce 22 rv1 proliferation. evs following gw4869 treatment significantly (p < 0.001) inhibited du145 migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < 0.001). summary/conclusion: while none of the 4 proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low 5-year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the 3d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp53 mutation contained not only ki67+ proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki67+ proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_16-0007 (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated 3 hours after r and incubated with 3t3 l cells for 24 hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of 17 genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified 11 mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir-23a/27a/24-2 family members are the most markedly increased mi-rnas in mi/r exo. incubation of 3t3 l cells with mi-r27a mimic significantly downregulated edem3, dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r27a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem3 and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r27a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of 3t3 l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir27a mimic. finally, in vivo administration of gw4869 (exo biogenesis inhibitor) or mir27a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir-23a/27a/ 24-2. funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the 3rd leading cause of cancer-related deaths by 2030. patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a 5-year survival ratẽ 9%. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are 90% non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc-1, sw-1990, capan-1 and miapaca-2) and patientderived xenograft (ppcl-46 and ppcl-68) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h6c7) were co-cultured with cancer cell evs for 24-48 hours. metabolic activity was measured using a mito stress test on a seahorse xfe96 extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on 24 plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev-24 mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma (80% of correlation with msc classifier). mscpos-evs stimulated 2d and 3d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a549 lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir-126 modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m2 polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir-320 is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir-126-evs from endothelial cells and mir-320-evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd63 tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd44, cd47, met and nrp2 which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b16f10 lineage. methods: b16f10 cells were cultured in dmem supplemented with 10% fbs and antibiotics. the cells supernatant were harvested each 24 hours, filtered through 0.22 µm and ultracentrifuged at 110000 x g for 90 min at 4ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd63, cd9 and cd81 on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd63 molecule in comparison to cd9 and cd81. we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd44, cd47, met and nrp2. summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b16f10 lineages harbouring shrna vectors, in order to knockdown gene expression of cd63, cd44, cd47, met and nrp2 to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b16 f10-derived cells. introduction: overexpression of her2 occurs in2 0% of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her2, potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to 70% of patients with her2-overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her2 status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her2-positive cell lines (hcc1954 and skbr3) and their neratinib-resistant counterparts (hcc1954-nr and skbr3-nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs578 ts(i)8, were evaluated as negative control for her2. in brief, cm was centrifuged at 300 g, for 10 min x3 to get rid of any cells. the supernatant was then centrifuged at 200,000 g for 6 h at 4ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in 100 μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev190112). results: neratinib-resistant cell line variants were found to have reduced her2 protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her2, while those from drug-resistant cells lacked her2 (similar to the evs from the tnbc cells); reflecting the her2 status of their cells of origin. summary/conclusion: this study indicates that a reduction in her2 protein expression is a mechanism by which cancer cells manifest resistance to her2-targeted drugs (i.e. by making fewer her2 receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her2 seems to reflect the her2 status of their cells of origin. this suggest that analysis of her2 on evs in the peripheral circulation may help predict response to her2-targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab27a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than 50% of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab27a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc38 tumour cell line was established by repeated injections with ohsv in mc38 tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. results: 1) the lower expression level of rab27a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. 2) furthermore, we established an ohsv resistant mc38 tumour cell line by repeated injections with ohsv in mc38 tumour-bear mouse model. similarly, in ohsv resistant mc38 cell line, rab27a expression was lower than parental mc38 cells. and the release of exosomes and virus was decreased in ohsv resistant mc38 cell line. these results were confirmed by rab27a sirna knockdown. 3) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab27a and overexpression of rab27a can rescue ohsv replication. summary/conclusion: downregulation of rab27a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab27a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd2015071414385495, shenzhen science and innovation commission project grants jcyj201 70411094933148, jcyj20180306173333907 to shenzhen international institute for biomedical research. inactivation of emilin-1 by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b16-f1), high metastatic potential (b16-f10) and lymph node metastasis (b16-f1 r2). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin-1 secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw4869. cell viability and cell cycle assays with an overexpression model (b16-f1-emilin-1) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin-1 expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b16 mouse melanoma model. among them, we found that emilin-1, a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin-1 is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin-1 has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin-1 overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin-1 are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin-1 by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf2014 54541 r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb2 gene and the consequent overexpression of the encoded protein her2 have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her2 monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb2 gene amplification and her2 protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb2 gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol1 that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb2 amplification and consequent her2 overexpression on a limited volume of plasma (1.5 ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: 1) we detected erbb2 amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb2 positive patients; 2) we confirmed her2 overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her2 protein on the surface of the isolated evs; 3) we succeeded in the quantification of her2 transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = 20) and on a cohort of her2 positive (n = 20) or her2 negative breast cancer patients (tnbc; n = 20). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb2 detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse 2014-2020) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in 4% paraformaldehyde or 2% glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l1cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a 100x oil immersion objective. sevs were imaged with a 3d-sim acquisition protocol. tem was performed on 300 mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd63. samples were counterstained with uranyl acetate and observed under a jeol 1010 ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from 180 to 190 nm were detected, as the final resolution achieved was 160 nm. instead, for tem, sevs under 100 nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd63 or l1cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from 30 to 100 nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from 30-1000 nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd63, cd81, cd9), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by 9 healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, 2 and 24 hr post-exercise. perceived muscle pain (pmp) was assessed at 2 and 24 hr post-exercise. large evs were isolated using a 10 000 g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions (7-10) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = 1.17 x 1010 particles/ml plasma). mean ev diameters were 194 ± 22 nm across all timepoints. no change in ev size nor number was seen over time, however, mir-31 decreased at 24 hr when compared to 2 hr in the small ev isolate only. plasma displayed an immediate increase in myomirs-1 and −206 at 2 hr, which returned to baseline at 24 hr. in contrast, myomirs-208b and 486 remained elevated over the 24 hr period. myomir-133b and 486, as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir-31 in small evs at 24 hr is consistent with previous data. no decrease in mir-31 in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national 11 university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either 3d or 2d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either 2d or 3d platforms displayed distinct magnetic sensitivities, whereby mscs grown in 2d or 3d platforms responded most favourably to pemf exposure at 2 mt and 3 mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses 1 in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd14 positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m0 macrophages and activated into m1 macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il1β and tnfα levels increased. as control, thp1 monocytes were used. patient-derived oa chondrocytes were grown in 6 well plates and co-cultivated with activated m1 macrophages which were seeded into thincerts and added to the 6 wells representing the inflammation model. furthermore, cells were treated for 48 hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp1 and primary monocytes) into m1 macrophages was demonstrated by elevated levels of the inflammatory cytokines il1β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m1 macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il1β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst3-f-5030664/003-2017. 1α,25(oh)2d3 regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small (50-150 nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. 1α,25 (oh)2 vitamin d3 (1α,25) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir-22, mir-122 and mir-451. the aim of this study was to determine the regulatory role of 1α,25 in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from 5 wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with 10-8 m 1α,25 or vehicle for 24 h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = 3 per variable. significant differences (p < 0.05) were determined using r v 3.6.2. results: 1α,25 treatment altered expression of 30 mv mirs compared to control mvs, whereas 5 cell mirnas were differentially expressed. 62.1% of significantly up or down regulated mir found in mvs overlapped between 1α,25 and vehicle groups with the remaining being uniquely differentially expressed. 1α,25 increased mv mir-150 and decreased mir-384-3p two mirs known to regulate osteoblast proliferation (150 increases, 384 decreases). summary/conclusion: 1α,25 regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that 1α,25 regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of 5%. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/31651/2017, uidb/04565/2020). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a60 platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a 96 well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods (5-30 minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was 2-8% c.v. detected bead concentrations were linear with flow rates between 0.75 and 10.5 ul/min. double well aspirations provided similar data with aspirations between 60-80ul. valid data was achieved for a low abundant target (~200-400 events/ul) after only 30 s, <10%c.v. detection sensitivity was determined to be~1/400,000. carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par20-053, is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par16-267/ 277, successfully funded 7 r01 and 4 r21 grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, 2018); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, 2019). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, 2019). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on 5 january 2020. this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of 7 protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores (600 nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the 7 protein biomarkers (tau, uchl-1, nfl, gfap, il6, il10, and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl1 were elevated in mtbi patients compared to controls (p < 0.05), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il6 and tnf-with tau from glur2+ evs showed 88% accuracy with 80% sensitivity and 100% specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps15.05 = op3.05 l1cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l1cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l1cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l1cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l1cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd9, cd63 and cd81) as well as l1cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l1cam appears. we also immunocapture l1cam from csf and plasma and perform western blots for the internal and external domains of l1cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l1cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l1cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l1cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l1cam in plasma is likely not coming from the brain. this data call into question the utility of l1cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant 3d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed 3d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in 6-month-old wt mice, (n = 6/groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for 15% of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy5y cells with n-myc amplified sk-n-be2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps15.08 = op3.08 results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: 0.15 m-0.3 m nacl) and high salt (h-s: 0.3 m-0.5 m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. 9: 435, 2018) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup5: kitani et al., results immunol. 4: 68-74. 2014 ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: 2019 #04) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a64)) and the japan science and technology agency (crest [jpmjcr17h2]). clic4 is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic4, a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic4 expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic4 in the host or tumour cells to develop metastases. results: experimentally, the essential clic4 host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic4 was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic4 in comparison to those from mice bearing tumours that lack clic4. tumour cells released evs that induced myofibroblast conversion of wildtype but not clic4 ablated lung fibroblasts. summary/conclusion: these results illuminate clic4 expression as a prognostic marker for breast cancer patients, and experimentally, clic4 is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc 005445. the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via 3d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage 3 via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg101, alix, cd63) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp2016-2), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps08: separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps10: diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of 400-1800 cm-1. we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of 204.7 ± 96.5 nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb2 detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean 142 nm vs 75 nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean 125.7 nm vs 122 nm nta can only detected vesicles above 70 nm and exoview™ only measures vesicles between 50-200 nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc9gr) and mrc5 (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with 1 μm osimertinib or pbs for 3 days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc9gr during 6 days. we will investigate how mpa-exsomes activate erk signalling pathway in pc9gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc9gr cells by more than 50% compared to control pbs. pc9gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc9gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t790 m mutation.these data show that "mrc5-pc9gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of 1um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc5 lung fibroblast increased cell viability and exosomal mir-21 through the tgf-ß pathway in treatment osimertinib. exosomal mir-21 up-regulation in cocultured nsclc with mrc-5 induced drug resistance to drug-induced apoptosis. thus, exosomal mir-21 expression in co-cultured nsclc with mrc5 may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd9, cd63, cd81, and alix with a mean size of 145 nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified >1,000 proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke (1r01ns106907-01 to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within 6 hours (kodali et al., int j mol sci, 2019) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately 10 billion evs were dispensed bilaterally into both nostrils of young f344 rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba-1 (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba-1 and cd68 dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca1 subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are 30-150 nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over 21 days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected 274 gene products. of these, 24 were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component 5a (c5a) in sevs from sni model. intercellular adhesion molecule 1 (icam-1), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of 40 als patients and 30 healthy volunteers by differential centrifugation and characterized by nanosight ns300. cd45, cd31, cd61, cd235a and annexin v were used for flow cytometry. sod1, tdp43, fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf2 slide and raman spectra were acquired using a 633 nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod-1, tdp-43 and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps05.07 introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab27b or with shrnas against the exosome secretion and biogenesis regulators rab27b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) 6 or spines at div12. for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div9 cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab27b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab27b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from 0.5 ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > 5 ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with 2.3 ml of samples were centrifuged at 186 k g for 18 h. fractions were then pooled based on densities (1.1-1.2 g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev190096). results: immunoblotting showed absence of actinin4 from all samples, while cd63 and tsg101 were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~10^14/ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < 0.05) more cd81-positive particles/ml of milk vs all other guc and 200 kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < 0.01) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb-231 and skov-3 cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd9, cd63, cd81 or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh67 and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh67 and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh67 ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb-231derived evs isolated based on cd9 and cd81 expression were taken up more efficiently by hmec-1 and mda-mb-231 cells than evs isolated based on presence of cd63 or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. 17296) of the dutch research council (nwo). aml were harvested from tib49 cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca1+ ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor 1 (ifnar1) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h2ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib49 ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg15, il-6, ifnα, ch25 h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar1-null hspcs as well as the lack of evs-induced stat1 phosphorylation or ifnar1 downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor 2 (batf2) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf2 has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf2 inhibitory effects of mdscs recruiting. we used transwell assay to determine batf2-positive evs (evs-batf2) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf2 model to demonstrate evs-batf2 crosstalk with distant tissues. amd3100 blocking in tumour model confirms that evs-batf2 dominated by the sdf-1a/cxcr4 signalling pathway. in addition, exo-counter detection of evs in 32 pairs of gliomas in different stages proposes plasma-evsbatf2 (plevs-batf2) as a prognostic marker. results: we found that tumour-derived evs-batf2 regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf2 can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf2 injection significantly reduced sdf-1α expression in the tumour tissues. after blocking sdf-1α signalling by amd3100, the inhibitory effects of batf2 overexpression on mdscs recruitment were rescued. evs-batf2 inhibit mdscs recruiting and secreting mmp2, mmp9, and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in 32 pairs of gliomas in different stages reveals that the number of plevs-batf2 can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf2 may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf2 in regulating tumour microenvironment and propose plevs-batf2 as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited 100 nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled 50~100 nm pores in the si3n4 membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd63-phluorin) and extrinsic (lipophilic dye, di-8-anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of 20-150 features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from 50 to 100 nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = 3) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in 10 × 10 s acquisitions. two microfluidic cartridges were used to study a dynamic range of 50-450 nm. samples were diluted into phosphate buffered saline with different concentrations of tween 20 or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of 50 or 65 nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within 99% confidence. summary/conclusion: we recommend to use 0.1% w/ v bsa in dpbs as sample diluent, because tween 20 affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to 12 h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp-2), which provides an active surface for probe immobilization. then, antibodies against cd9, cd81, cd63 markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd9, cd81, and cd63 markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than 500 billion of people around the world within upcoming 20 years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about 10% of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from 400 cm-1 to 4000 cm-1 and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in 100 mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~2 to~90 mda and z~50 to~1300 e and are highly dependent upon the conditions used to extract and isolate the evs. in total, 57,350 particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s1, s2, and s3 families that are centred at~3.5,~5.9, and~8.3 mda, respectively, appear too small to be ascribed to exosomes. the remaining 45,229 (79%) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r01 gm131100-01). bab is supported by indiana university quantitative chemical biology fellowship (t32gm131994). in situ detection of exosomal microrna-10b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna-10b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb-231, mirna-10b-encapsulated anionic liposomes, and mirna-10b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna-10b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna-10b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna-10b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna-10b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma (12,000xg, 34 min/0.22 ) m filtration/110,000xg, 5 h)(n = 7) and lymphoma conditioned medium(300xg, 10 min/200xg, 20 min/1000xg, 30 min/120,000xg 1.5 h/120,000xg, 1 h) (n = 3). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd63/cd9/flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k (1 mg/ml/56°c/10 min) and rnase a (2 mg/ ml/37°c/20 min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = 0.005, n = 4) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a 13-fold and 200fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of 66 ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for 66 putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd9, cd63 and cd81) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above 0.9, indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered 0.5-4 ml plasma or 2-20 ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction (4 μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam10 proteolytic activity might be more informative than the level of adam10 protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of 70 different al-pha bead biosensors were designed. a control, tev-specific biosensor detected 0. introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom20. mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the 374 isev2020 abstract book biogenesis of evs is neutral sphingomyelinase 2 (nsmase2), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase2 is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase2 inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo [1,2-b] pyridazin-8-yl) pyrrolidin-3-yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated 5xfad mice with 10 mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, 5xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting 20% of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of 20 mdd subjects and 20 hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd9. tem images showed the typical cup-shaped morphology with sizes mostly between 30 and 200 nm. preliminary sequencing results revealed that mir-33a-5p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir-33a-5p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using 5-ala, fluorescently labelled ev (cfda-se, cd81) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd81) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that 58% are tenascin c positive, 2.7% are egfrviii positive and 1.6% are 5-ala positive. there was only a minor overlap (<16%) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs (2.5-fold), tumour specific evs (2.8-fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll 2 receptor (tlr2) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il-12 and il-6, which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af4) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp-1) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk-2 epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with 50 to 50 nm (ev1) and another with 100 to 120 nm (ev2). ev1 induced more tnf-alpha, il-6, ip-10 and ccl20 than ev2. it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and 0.2um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid50 assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps15.12 = op3.12 kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of 11.5 µg/ml of plasma, as geometric means ≥11.5 µg/ml were nearly equal. hevs were detected at 48 µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f200x electron microscope and measured between 40-90 nm, and hevs were between 60-160 nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl20 chemokine, thus contributing to npc immune escape (mrizak et al., jnci, 2015) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress 2017). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl20 in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd3 t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl20 (ccr6).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl20dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than 20 times at over 97% purity without leaking by 750 kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, 180-1300 nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation (18,900 g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤15%) and precision (cv ≤5%) across both platforms used. we defined two ev size gates: a "latex" gate (300 to 1100 nm polystyrene latex beads), and a "silica" gate (180 to 1300 nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv <20%) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from 10 hnc patients were taken before (a) and after (b) rt. 10 healthy volunteers were used as a control group (c). ev were isolated from 1 ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h2o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/493/4/06/1/2016/g). results: an untargeted gc-ms-based approach allowed the detection of 189 metabolites in serum samples and 50 exosomal small molecules, of which 32 joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were 49 metabolites which levels discriminated compared groups (a,b,c) of serum samples and 12 compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from 3 human pancreatic cancer cell lines (capan-2, mia paca-2, and panc-1) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of 4,907 unique proteins were identified confidently across the combined samples. the proteins present in all four sev types (1,135 proteins) consist of general housekeeping proteins. 348 proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/3 t3 cells. conversely, there were 313 proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd9 show an increased signal for the luminal protein tsg101. ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg101 in a lower extent indicating a subpopulation of evs. the microarray uses less than 100 µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within 3 h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for 6 different surface marker from 16 samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of 120 ml were collected from 5 patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and 100 ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus-70, 10 k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of 50-200 nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles 385 comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida 2019. the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the 20-120 nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad2 accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc97 h cells and incubated with low metastatic hcc97 l cells. in other experiments, cpe-shna loaded exosomes from hek293cells were incubated with hcc97 h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc97 h cells revealed cpe-wt mrna and protein. exosomes released from hcc97 h cells were able to enhance proliferation and invasion of hcc97 l cells. when cpe expression was suppressed in the hcc97 h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek293 (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified 386 isev2020 abstract book exosomes were used to transfer cpe-shrna to hcc97 h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d1 and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. 20892. stress hormones promote prostate cancer aggressiveness through modulation of mir-628-5p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir-628-5p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe-1, and pca cells pc3, lncap and mda-pca-2b. proliferation of cells treated with leptin in the presence or absence of mir-628-5p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir-628-5p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir-628-5p in rwpe-1 cells.co-treatment with mir-628-5p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir-628-5p target jag1 and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc3 cell exosomes with a 2.6-fold increase at 5 nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at 5 nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir-628-5p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek293 t cells and mir-146a-5p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r01 and t32 (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed 200 nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd63, a protein marker of ev. we modified 5ʹ-terminal and 3ʹ-terminal of the cd63 aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd63 aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd63 aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd63 aptamer for specifically binding with cd63 in evs. in addition, we designed cd63 aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample (70 µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex1). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx0e03). methods: t-flask expansion of ctx0e03 cells was performed to yield batches of 5-15 l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of 0.22 µm dead-end filtration prior to concentration in a 300kda hollow-fibre tff system. tff retentate was loaded onto 1 ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd 81 and cd 63. exosomes recovered in this manner could achieve full wound closure in vitro over 72 hours, when dosed at 20 µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of 1e10-5e10 particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir-126 were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate-1 (irs-1). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from 0.5 ml of plasma from 104 pd-n, 83 pd-mci, and 39 pd-d patients, we immunocaptured nevs using anti-l1cam antibody. then, irs-1pser312 and irs-1ptyr20 and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < 0.005) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = 0.005). irs-1pser312 was lower in pd-d than in pd-n. irs-1ptyr20 significantly decreased with increasing mds-updrs iii score (p < 0.005). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs-1ptyr20 were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample (25 ml) was collected from 7 patients and processed within 4 hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation (2.000 x g, rt) for cell removal and storage at −80°c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through 0.22 µm pores was used for large particle removal, while centrifugal 100 kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a280) were pooled and concentrated to a volume of 70 µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was 129,9 nm with concentration range of 1 × 109 particles/ml of starting urine. uevs were positive for the tested marker proteins hsc70, flotillin, tubulin, gadph and cd63. qpcr verified mirna presence in uevs, with ct for mir let-7i at 20. summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin-4 1 and epha2 that were enriched in adev-atp. integrin-4 1 facilitated binding of adevs to the neuronal surface, and epha2-receptor signalled through ephrin to the tyrosine kinase erbb2/ 4 that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf3. this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc1, arc, and cplx3 to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and 3d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a 3d-printed scaffold-perfusion bioreactor using a flow rate of 5 ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il-6, quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with 100 µm ethanol for 24 hours prior to ev collection results in a strong inhibition of il-6 production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor (1.40e12 ± 7.92e10) when compared with flasks (2.28e10 ± 2.81e9). protein measurements also indicated that ev production in the bioreactor (~7600 µg) was much greater compared with production in the flasks (~2400 µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr1 and hsp90) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp90 was detected in subpopulations 3 and 4 of both pf-evs and ui-evs, whereas sr1 only in subpopulation 3. isev2020 abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam 550 fib-sem gemini ii (zeiss, germany) and jsm-6500 f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at 22°c and 37°c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x-100 and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord-015394-uj7fe5y6 authors: nan title: scientific abstracts date: 2008-12-23 journal: reprod sci doi: 10.1177/19337191080150020102 sha: doc_id: 15394 cord_uid: uj7fe5y6 nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day 6 , wistar rats were randomised into: normoxia (21% o 2 7 litters), hypoxia (14% o 2 8 litters) and hypoxia + vit c (14% o 2 + 500mg.100 ml -1 vit c in water, 6 litters). on day 20, dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein 70 (hsp70; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by 70 % of baseline; a similar increment to human trials (poston et al. 2006) . hypoxia elevated placental hsp 70 and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. 21 normal (n) and 21 gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n=3274±62; gdm=3381±111 g) while placental weights were significantly different (n=474±16; gdm=571±35 g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: 245.3±19.0 in n and 242.2±18.5 ml/min in gdm; qumb/kg: 74.6±5.0 in n and 71.4±4.6 ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o 2 sat, o 2 cont and po 2 and a significant increased lactate conc was found in both uv and ua compared to n (table 1) . o 2 umb uptake (n=0.78±0.08; gdm=0.56±0.08 mmo/l/min) and o 2 umb uptake/kg (n=0.24±0.02; gdm=0.16±0.02 mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f0) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f2 offspring with no manipulation of the f1 pregnancy. methods: pregnant guinea pigs (f0) were subcutaneously injected with betamethasone (beta; 1mg/kg) or vehicle (veh) on gestational days 40/41, 50/51 60/61. adult f1 female offspring from each group were mated with control males. hpa function was assessed in adult f2 offspring by non-invasive measurement of salivary cortisol concentrations: 1) under basal conditions, 2) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, 3) following dexamethasone suppression. results: there was no effect of beta (f0) on bodyweight from birth to adulthood in f2 offspring. basal salivary cortisol in the betaf2 females was lower than in the vehf2 group in the morning but not the afternoon; there were no differences in male f2 offspring. in contrast, both male and female betaf2 failed to mount adrenocortical responses to psychological stress compared to vehf2 offspring that mounted robust responses (p<0.02). swim stress induced robust adrenocortical responses in all groups (p<0.0001), however, the response was consistently lower in betaf2 offspring. beta exposure also led to a significant difference (p<0.003) in the cortisol response to dexamethasone suppression in female (f2) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f0) causes transgenerational programming of adrenocortical function in adult f2 offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf2 female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using 40 mhz ultrasound biomicroscopy in control (c57bl/6j) and enos -/-mice at 17.5 days of pregnancy (n=6 mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe-1 and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was 55% lower (p<0.05) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (-33%, p<0.001) and mean velocity (-17%, p<0.05). uterine arterial resistance index was 52% higher (p<0.001), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated (4%, p<0.05) and capillary lobule length was reduced by (-28%, p<0.05). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin 5 methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium 2 with 2% of fcs and 50 ng/ml vegf for 14 to 21 days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin 4 , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t2dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age 31 years) with regular menses (27-29 days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days 1-3, 6-9, 13-15, 20-22) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd133, kdr and cd34) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was >30 nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being 3-fold higher in the follicular compared to periovulatory phase (p<0.05). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg1 fc (fvii/igg1 fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of 1.0 mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein (10 ug) was delivered i.p. once a week for 4 weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with 10ug of icon abolished all lesions in 7 of 10 mice and reduced both size (2.33 to 1.4mm) and number of lesions (1.9 to 1 per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of 87% +/-26% (p=0.002, mean +/-sem, n=3) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp3 demonstrated increased secretion of crisp3 in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp3, is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa 10 regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa10, treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment (31%, p=0.049) or hoxa10 transfection (11%, p=0.027). coadministration of progesterone along with increased hoxa10 transfection had no additive effect on the expression of gaba-a pi receptor mrna (p=0.98). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa10 or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa 10 each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa 10 expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated 40,000 women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase 2 trial, gog 229e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr2). ic 50 experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic 50 of 1.15 m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by 50% using concentrations up to 2.5 m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of 0.2 mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna21, which was significantly down-regulated. microrna21 is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, 840 data points for each gene from 420 arrays have been collected from 42 patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove 13 arrays that deviated from twice the inter-quartile range calculated from the 420 array signals. results: the custom microarray profiling identified 38 genes that were differentially expressed between normal and fibroid tissues (p <0.01; fdr<0.13). among them, 12 genes encode receptor tks, 15 genes encode tk ligands, and 11 genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log2 ratios of these 38 genes has led to the division of the 42 patients into two major groups. thirty four (81%) belong to a group characterized by the significant downregulation of the cyr61 (ccn1) gene in fibroid tissues. the cyr61-down group can be further divided into two sub-groups based on the expression of another tk ligand, efn4a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr61 and efn4a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, 1 arthur c fleischer, 2 brian day, 1 stephanie v blank, 1 bhavana pothuri, 1 robert wallach, 1 john p curtin, 1 david a fishman. 1 1 gynecologic oncology, new york university school of medicine, new york, ny, usa; 2 radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the 4th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu22 scanner after intravenous injection of 1 g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of 93 ovaries have been analyzed, 78 benign and 15 malignant. of the malignancies (3 fallopian tube, 12 ovarian: 3 stage i, 12 stage iii), 12/15 women were correctly identified using conventional tv imaging and 3 others were identified using mvi 15/15. all benign lesions were correctly identified by mvi 78/78 while tv detected 81 normals (3 false negatives). the lesions detected as malignant by mvi were 2-stage i fallopian tube and 1-stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in (26.1 ± 6.3 vs 24.9 ± 7.6, p = 0.7), greater peak enhancement (23.3 ± 2.8 vs 12.3 ± 3.9, p < 0.01 ), longer wash-out (139.9 ± 43.6 vs 46.3 ± 19.7 (p < 0.01), and greater perfusion (2012.9 ± 532.9 vs 523.8 ± 318, p < 0.01) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir-199a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir-199a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: 11 human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex 200 system. ikk transfection was done with roche fugene6 transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir-199a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk 3'-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified 18 mirnas differentially expressed in type i versus type ii cells, one of which, mir-199a, had 3 putative binding sites in the 3'-utr of ikk mrna. mir-199a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's 3'-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir-199a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd55 in the rat uterus by nitric oxide and the involvement of p13 kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd55 is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd55 levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd55 in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats (200g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at 10 or 50/mg/rat/day in saline or saline alone. uteri were obtained from these rats at 48 or 72 hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name (3mm), diethylenetriamine-no 1mm) or worthmanin (0.1 m) in mem without phenol red for 24 hours. uterine tissues were homogenized in trizol and mrna levels for cd55 were measured using rt-pcr and expressed as a ratio to 18s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd55 mrna levels in a time and dose dependent manner with maximal responses seen with 50mg l-name and at 72 hours. in vitro studies show that deta-no suppressed cd55 mrna levels in the rat uterus. both l-name and pi3 kinase inhibitor, worthmanin caused increases in cd55 levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd55 levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi3 kinase appears to be involved in cd55 regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, 1 daniel b hardy, 2 carole r mendelson. 2 1 dept of pediatrics; 2 depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during 95% of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p 4 ). we previously observed that p 4 acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase-2 (cox-2) expression (hardy et al., 2006) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., 2004) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p 4 also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p 4 (1 mg/ml) from 15 to 19 days post-coitum (dpc). as expected, treatment with p 4 delayed parturition by 24-48 h. this also was associated with a decrease in uterine cox-2 mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p 4 treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at 19 dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p 4 -injected mothers, as compared to controls. this was associated with an inhibitory effect of p 4 treatment on cox-2 protein and mrna levels in the fetal lungs. these findings were of interest, since cox-2 expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., 2005) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., 1990) . collectively, these findings suggest that maternal p 4 treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p 4 inhibits an important fetal signal leading to labor. supported by nih p01 hd011149; nih r37 hl050022. recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl-2 is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl-2 gene expression in myometrium during term and ru486-induced preterm labor. also ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl-2 as a mediator of this stretch response. we also studied the effect of progesterone (p4) on the myometrial secretion of ccl-2. methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl-2 gene and protein induction by static mechanical stretch. ccl-2 gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl-2 production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for 2-24 hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch (25% elongation for 24 hours) caused a significant accumulation in ir ccl-2 which was repressed by pretreatment with p4 (1 um). the rise in ccl-2 protein levels was preceded by a transient increase on ccl-2 mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl-2 significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p4 offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, 1,2 guadalupe estrada-gutierrez, 1 lourdes vadillo-perez, 1 felipe vadillo-ortega. 1 1 direction of research, instituto nacional de perinatologia, mexico city, df, mexico; 2 escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: 1) term without labor (n=4) and 2) term with spontaneous labor (n=4). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd45, cd56, cd14, cd19 and cd3 subpopulations of leukocytes were identified. intracellular mmp-9 was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd3+, cd+14 and cd56+ cells, both in the chd and inside the fm. mmp-9 was associated to cd 56+ cells. cd3+ exhibited a more widespread localization in the fm and cd56+ cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp-9, which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n=45), iugr (n=53) and uncomplicated pregnancies (n=106) were recruited at a mean follow up time of 10.8 years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p <0.05) as well as higher mean indices of body mass, fat mass and lean mass (p< 0.05). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p<0.001) as well as excess of android fat mass and increased android-to-fat ratios (p<0.05). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p <0.001). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p <0.001) and waist-to-hip ratios (p <0.001) as well as smaller hip circumferences (p < 0.001). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, 1 timo h ekhart, 2 robert aardenburg, 2 louis lh peeters. 2 1 obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; 2 obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least 20 years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in 20 formerly preeclamptic women (pe) and 24 healthy parous controls (control) who were normotensive at 6 weeks post-partum follow up, we assessed at least 20 years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/1.73m 2 ) after which we calculated total peripheral vascular resistance (.100 dyne.s/cm 5 ) and renal vascular resistance (.100 dyne.s/cm 5 ). data were analyzed parametrically (p<0.05). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, 40% of formerly pe women and 13% of control were hypertensive (p<0.05). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about 20% higher and erpf 15% lower in formerly pe women as compared to control. conclusion: at least 20 years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe 5.0±0.6 397±61* 18±2* 120±29* control 4.7±0.8 469±80 15±2 97±25 * = p<0.05 101 pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, 1,2 marilyn j cipolla. 1 1 neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; 2 ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from 4 groups of sprague dawley rats: np (n=7), lp (d20; n=7), ovariectomized and implanted with 17 -estradiol (0.5mg, 21-day release) and estriol (5.0mg, 21-day release) pellets for 14 days (ovx+e; n=6), and ovariectomized and implanted with placebo pellets for 30 days (ovx; n=6). pcas were isolated, pressurized in an arteriograph, and perfused with 0.5mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from 60-200mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p<0.05; figure 1 ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p 150% in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was 256% greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, 1 marc ea spaanderman, 1 dorette a courtar, 2 robert aardenburg, 2 ben j janssen, 3 fred k lotgering, 1 louis lh peeters. 2 1 obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; 2 obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; 3 pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a 3 times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in 24 non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i 125 indicator dilution method) at least 6 months post partum. we intravenously infused 500 ml of iso-oncotic fluid over 30 minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg 2 , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within 1 year and were evaluated at 12 weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p<0.05). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv (17/24) and npv (7/24). in npv, lfsys decreased 28%, but only 7% in lpv (p<0.05). anp remained unaltered in npv, but increased in lpv. in the pregnant group, 4 women had lpv and 4 had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt-1 was increased, and plgf reduced at all gestations (p<0.0001). seng levels were also increased in pe. after 28 weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt-1 and seng, in pe only. the concentrations of both sflt-1 and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p=0.0002). only sflt-1 was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after 28 weeks' gestation is associated with a significant fall in serum sflt-1 and seng, and in placental sflt-1. these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor 1 pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, 1 debbie a sanders, 2 olivera spasic-boskovic, 1 graham j burton, 1 d stephen charnock-jones. 2 1 dept of pdn, university of cambridge, united kingdom; 2 dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase-1 (sflt-1, svegf-r1), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt-1 and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt (50-250 ng/ml) in a basic 2% fbs-dmem medium for 24 hr. on day 3, low doses of tnf-(0.5 ng/ml) were applied to pre-treated cells for 6 hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody (5-10 g/ml), anti-kdr antibody (1 g/ml), anti-vegf antibody (5 g/ml), or vegf receptor inhibitor su5614 (5 m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in 2% pfa for immunofluorescence or incubated with labelled hl60 leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl60 leukocytes and increased icam-1, vcam-1, endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su5614, anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su5614 or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term (37-41 wks) from lean kg/m 2 ), overweight (25-29.9 kg/m 2 ) and obese (30-40 kg/m 2 ) patients (n=5 or 6/group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using 2,4-dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab (1:150) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p<0.003, anova) with expression in tissue from obese women being significantly increased compared to lean (p<0.005) and overweight (p<0.05, tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p<0.02, mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and 19.2% coverage as 3-beta hydroxysteroid dehydrogenase (3bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. 3bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor 1 . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer 2 . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from 29 teenagers (15-18 years) and 21 adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of 14 c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (>2mm increase in kneeheight per 90 days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p<0.05). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p<0.01). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight 3550g and 3290g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p<0.01), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate (30s and 20 min respectively) as na + -dependent 14 cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles (0.165mm) or fragments (0.0085mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: 20mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p<0.05) inhibited na + -dependent 14 c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n=9, kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib (0.05-20mm) caused a dose-dependent inhibition of na + -dependent 14 c-meaib uptake into mvm with ec50 values (in mm; mean ± se) of 0.53 ± 0.04, 0.27 ± 0.02, and 1.13 ± 0.12 respectively, n=6). na + -dependent 14 c-meaib uptake into fragments was reduced substantially in the presence of 10mm l-hcy or dl-hcy causing a 85 ± 13 and 88 ± 12% reduction (mean ± sd, n=2-3) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. 110 glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, 1 theresa l powell, 2 thomas jansson. 2 1 department of physiology, institute of neuroscience and physiology, gothenburg, sweden; 2 department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at 66 hours. cells were cultured for an additional 24 hours in culture media containing 0.5 mm, 4.5 mm, or 16 mm glucose (control), which corresponds to standard culture media. at 90 hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s6k1 phosphorylated at thr-389, the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of 3h-leucine. results: as compared to control, phospho-thr-389-s6k1 expression was reduced by 24% in cells incubated in 0.5 mm results: fgr pregnancies were delivered earlier (39±0.2 v 32±0.6w; p<0.01), weighed less (3.5±0.2 v 1.2±0.1kg; p<0.01) and had higher s/d ratios (2.2±0.1 v 5.3±1.1; p<0.01). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p<0.05) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all 9 eaa for both groups. amino acids fell into 3 groups for fgr pregnancies: 1) his (ratio 1.38), 2) leu, phe, met (ratios 1), and 3) val, thr, ile, trp, lys with intermediate ratios ( 0.6 ns conclusion: this is the 1 st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a 43-year old female were used. cells were treated with estradiol [e2], equilin [eq] (0.1 nm) or genistein [gen] (5 micromolar) for 48 hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three 2.63, 2.06 and 16.74 for e2, eq and gen respectively. similarly nfkb1 was also reduced 2.62, 2.07 and 6.33 fold compared to controls. significant reduction of ccl2 was demonstrated with eq (3.11 fold) and gen (5.49 fold). tnfreceptor1a and 1b were only significantly decreased by gen. vcam-1 which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced (25.95-fold) with genistein, and a slight reduction was seen with e2 and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e2 has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin-1 (et-1). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et-1 pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged (12 month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , 17 -estradiol [ovx+e 2 ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm6001, 10μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et-1 (ec 50 =0.07±0.01μm in ovx versus 0.15±0.04μm in ovx+e 2 or 0.16±0.03μm in ovx+etan groups; p<0.02). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p<0.05) role of mmps in big et-1-induced vasoconstriction in the ovx+e 2 group (reduction in max constriction=70.3±11.8%) compared to ovx (40.7±9.1%) and ovx+etan groups (35.4±3.9%). conclusions: both estrogen and tnf inhibition reduced big et-1 vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et-1 vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et-1 vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and 8 women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of -246 to +74 was amplified by pcr and then cloned into pcr2.1-topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh5a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of 66 methylated cpg sites were found in the patient group with pelvic organ prolapse while only 1 methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of 57,585 parous women participating in the nurses' health study ii from 1997 to 2005. our primary outcome was incident premenopausal breast cancer. results: during the study period, 621 cases of premenopausal breast cancer were diagnosed during 355,871 person-years of follow-up. women who had ever breastfed had a 24% lower incidence of premenopausal breast cancer (95% confidence interval [ci] 5-40%) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age 18, family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p=0.52). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p=0.05); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a 46% (95%ci 23-63%) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p=0.03). among women who had never used suppressive medication and reported a family history, those who had breastfed had a 67% lower covariate-adjusted risk of premenopausal breast cancer (95% ci 38-82%) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio 0.68, 95% ci 0.42-1.09). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, 1 rachael a clark. 2 1 brigham women's hospital, boston, ma, usa; 2 harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd4 + cd25 + t cells has been documented but it is unclear if these represent foxp3 + t cells (tregs) . the possibility also exists that other cd3 + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd4 + cd25 + cells are true foxp3 + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median 33%) of cd25 + t cells were true foxp3 + regulatory t cells. from 65 donors, foxp3 + tregs accounted for a median of 7.1% of all cd4 + cells. these cells were memory cd45ro + t cells lacking ccr7, and l-selectin but expressing cd25, ctla-4, gitr, and hla-dr. additionally we found that a median 21% of cd3 + t cells expressed the nkt marker cd161. these cells were a mixture of cd4 and cd4 -cd8 -t cells, with variable numbers of cd8 t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd1d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire (0.4% iv24jq) and also expressed cd69, hla class ii, cd45ro but were ccr7 and l-selectin low. comments: we find that only a minority of cd25-expressing t cell in the decidua are true foxp3 + tregs. because much of the work on treg in pregnancy has used cd25 as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, 1 kathleen a connell, 1 richard bercik, 1 lloyd g cantley. 2 1 obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy-1 is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy-1 expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy-1 expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy-1 as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy-1, asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy-1, asma and desmin in vaginal tissue from women with pop and controls. further, thy-1 mrna expression was downregulated 57% in women with pop compared to controls (p = 0.004). a parallel decrease in thy-1 protein was seen in women with pop compared to controls (p=0.02). although a 44% and a 37% decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy-1 expression in vaginal tissue from women with pop compared to controls. differential expression of thy-1 in prolapsed vaginal tissues suggests that thy-1 may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the 3' untranslated region (3'-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n=14) and ectopic (n=7) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e2; 10 -8 m), or progesterone (p, 10 -8 m) for 24 and 48h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases (157.5 ±11.08 and 190.0 ±15.2, respectively), compared to the mid-late proliferative (270.0±8.0) and early-mid secretory phases (256.6±20.2 )(p<0.01). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p<0.01). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both 24 and 48 h compared to control (p<0.05) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp19a1 gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp19a1 gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c57bl6 mouse tissues. 5'-rapid amplification of cdna ends (5'-race) was used to determine the transcriptional start sites of cyp19a1 transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp19a1 transcript was isolated using 5'-race and this novel gonadal fat-specific exon i of cyp19a1 mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to 75 kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine 3t3-l1 cells with these plasmids showed that promoter activity was conferred by the sequence located at -1 to -343 bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp19a1 transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp2, bmp4, bmp7, gdf5, gdf8 (myostatin), gdf11 and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m108a and sb431542) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m108a (25nm) and sb431542 (10 m) significantly reduced decidualization (60% and 77% respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp2, gdf5 and tgf 1 protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp2 and tgf 1 secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp2 and tgf 1 in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with 4% pfa, permeabilized with 0.5% triton x-100 for 15 min, and immunostained against a-tubulin and the nucleus for 1 hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h3k9 modifications were measured in nondiabetic and diabetic denuded murine oocytes at 44 hours post pmsg (0 hour) and at six hours post hcg (6 hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in 4%pfa and permeabilized with 0.5% triton x-100. they were stained via immunohistochemistry against histone protein h3 at lysine 9 (h3k9me2) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately 10/50 (20%) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about 1/70 normal oocytes (1.4%). western blot demonstrated 3 times higher expression of dimethylated chromatin in diabetic oocytes at time 0 compared with nondiabetic oocytes. at time 6 hours, diabetic oocytes had significantly fewer h3k9 modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h3k9me2 by immunohistochemistry, we demonstrated h3k9me2 expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc1 mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc1 expression in human prophase i (pi) and metaphase ii (mii) oocytes, 8-cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a 625 aa protein with 77% identity and 84% similarity to mouse epab and contains 4 rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in 8-cell embryos or blastocysts. pabpc1 mrna is ubiquitously present in all tissues as well as 8-cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc1 following zga, which occurs at 4-to 8-cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh2-terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh (100miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with 100miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp600125. results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig 1a) . likewise by if, phospho-jnk expression was significantly increased in sigc 1 hour post fsh exposure (fig-1a) . similarly on wb, phospho-cjun expression increased as early as 30 min after fsh exposure and peaked at 4 hrs. cjun phosphorylation was abolished 1 hr after treatment with sp600125 (50mm) (fig 1b) . facs analysis showed that the inhibition of jnk by sp600125 resulted in cell cycle arrest at g2/m transition in a dose dependent fashion (fig 1c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd 043339-01). the induction of 17{alpha}-hydroxylase (cyp17) expression in granulosa cells. satin s patel, 1 victor e beshay, 1 william e rainey, 2 bruce r carr. 1 1 reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; 2 physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein-1 (ap-1) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp17 in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp17 expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp17 expression in granulosa cells. transformed human luteinized granulosa (hgl5) cells were utilized for all experiments. hgl5 cells were cultured in monolayer for 72 h. cells were treated for 48 h with and without pd98059 (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp17. cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for 48 h. rna was isolated and rt-pcr was also performed for cyp17. immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp17. treatment of hgl5 cells with the mapkk inhibitor pd for 48 h, resulted in a 10-fold increase in cyp17 mrna expression compared to basal conditions. in cfos gene silenced cells, cyp17 mrna expression also increased by 10-fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp17 showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp17. conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp17. these results suggest that the ap-1 transcription factor, cfos, may play a role in the inhibition of cyp17 expression in granulosa cells. this may provide an explanation for the lack of cyp17 expression in granulosa cells. the g2/m stages of the granulosa cell cycle. as clip-170 has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g2 and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip-170, leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip-170 and mtor were evaluated. computational analysis of potential clip-170 phosphorylation sites and comparison with residues on known mtor targets were performed. clip-170 threonine 182 and serine 186 were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip-170 function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip-170 phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip-170 and a peptide substrate. results: clip-170 was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip-170, mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip-170 phosphorylation supported thr 182 as a bona fide mtor target. conclusions: clip-170 was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip-170 and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, 1 thomas m goodwin, 2 gorm greisen, 3 morten hedegaard, 4 joan-carles arce. 5 1 warwick medical school, university of warwick, coventry, united kingdom; 2 maternal-fetal medicine, university of southern california, los angeles, usa; 3 dept of neonatology, rigshospitalet, copenhagen, denmark; 4 dept of obstetrics, rigshospitalet, copenhagen, denmark; 5 clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v 1a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in 6 countries. a total of 163 women between 34+0 and 35+6 weeks gestation, and with 6 uterine contractions of 30sec duration during 30min, cervical length 15mm, and cervical dilatation >1 and <4cm were randomised to receive a single intravenous bolus dose of either barusiban 0.3mg, 1mg, 3mg, 10mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within 48h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within 48h (72% in the placebo group and 65% to 88% in the barusiban groups). there was no dose-effect relationship nor an effect at 12 or 24h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to 48h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range 0.3-10mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v 1a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients (45.1%) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage (20.3 +/-3.4 weeks vs. 23.5 +/-3.5 weeks, p=0.0019). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation (59.4 +/-41.3 days vs. 25.9 +/-30.1 days, p=0.0016). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at 24 hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied 52 singleton pregnancies with pprom at <25 weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: 11 patients showed an amniotic fluid pocket (afp) persistently 2 cm and did not undergo amnioinfusion (group b) whereas 41 had a maximum afp < 2 cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in 17 patients of group a amnioinfusion was successful (afp 2 cm for 48 hours following the procedure: group a1) whilst in 24 it was unsuccessful (afp<2 for 48 hours: group a2) and repeated. results were analized with the student t test for unpaired samples and with the 2 when appropriate. p values <0.05 were considered significant. results the group where amnioinfusion was not successful (group a2) showed the worst outcome (see table) . there were 6 intrauterine deaths, all in this group. pulmonary hypoplasia was present in 11/52 (22.5%) newborns (both survived and deceased) newborns, 10/11 in group a2. no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a1 (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi 2004; 11:478-82) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a 0.6g weight with those connected to a 2.4g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, 100nm or 1000nm). following the 3 day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under 2g tension and responses obtained to 50mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium (0.6g weight = 24.6 [5.3]; 2.4g weight = 43.1 [6.5], n=6, p=0.01) and oxytocin (0.6g weight = 30.4 [8.0]; 2.4g weight = 50.3 [7.6], n=6, p=0.01). in strips with a 0.6g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = 30.1 [5.5]; mpa = 23.2 [5.2], n=5, p=0.02) and there was a trend towards a reduced maximum response to oxytocin (vehicle = 37.6 [8.2]; mpa = 31.5 [7.2], n=5, p=0.1). in strips with a 2.4g weight, incubation in mpa for three days had no effect on either the maximum response to potassium 1. prolonged stretch increases human myometrial contractility in vitro. 2. prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of 17oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: 17 pregnant preterm labor women were included. uterine electromyography (emg) was measured for 30 minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g1: tocolysis, n= 4; g2: no tocolysis, n=13). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p<0.05 significant. results: there was a significantly higher uterine emg activity (pdspf: 0.453 ± 0.051 vs. 0.379 ± 0.016), but no difference in cd (5.125 ± 1.727 vs. 3.714 ± 1.976), for patients delivering within 6 days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table 1) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within 6 days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r01-hd037480. objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n=20). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide (100mmol) followed by cumulative additions of levosimendan in the concentration range of 1 nmol/l to 100 mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n=6, p<0.05). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: 46 term patients were included in the analysis: 27 were in labor (group 1), while19 were non-labor (group 2). 30 minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of +1 means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < 0.05 considered significant). results: the mean and sd of the lyapunov exponent for all the patients was 0.0955 ± 0.0701. moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r01-hd037480. comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: 20 pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at 100hz and band-pass filtered in the 0.34 to 1.00 hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of "1." in-between contraction periods were assigned a "0." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. 1) . corrected opa, ppa, and npa were high at 90.68%, 84.52%, and 95.77%, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. 2) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r01-hd037480. introduction -this is a secondary analysis of women participating in a 14 center randomized placebo controlled trial (rct) evaluating the impact of 17-ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma 17-ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of 17-ohpc. methods -women with twins were randomized between 16-21 weeks to receive weekly im injections of either 17-ohpc (250 mg) or placebo until 35 weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between 24-29 weeks. the sample drawn just prior to the next scheduled injection was analyzed for 17-ohpc by hplc-ms in a blinded manner. the lower limit of quantification of 17-ohpc was 1 ng/ml. we conducted univariate analyses to assess the association of 17-ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb<35 weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results -96 women assigned to 17-ohpc were included; all received all of their scheduled injections. the concentration of 17-ohpc was significantly higher in women delivering < 35 weeks compared with those women delivering >35 weeks (p=0.002, table) . concentration of 17-ohpc was significantly correlated with ga at delivery (r = -0.38, p=0.0001). each unit increase of 17-ohpc was associated with a 30% increased odds of delivering < 35 weeks (odds ratio 1.3, 95% ci,1.14-1.50, p=0.001) and a 12% increase in hazard of spontaneous delivery (hazard ratio 1.12, 95% ci, 1.05-1.19, p=0.0004) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < 35 weeks (n=34) 13.9 (5.9) >35 weeks (n=62) 9.4 (3.5) conclusion plasma 17-ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since 17-ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between 23.7 and 34.7 weeks' gestation (cases) or term controls (37-41 weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within 60 minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: 20 cases and 20 controls were studied (see table for comparisons). controls p value ga at enrollment, weeks 29.4 ± 3.2 39.3 ± 1.1 <0.0001 interval to delivery, days (median, range) 2 (0 -25) 0 (0 -1.4) <0.01 maternal progesterone at enrollment, ng/ml 125 ± 87 185 ± 64 <0.02 maternal progesterone after delivery, ng/ml 55 ± 57 47 ± 27 0.6 cord progesterone, ng/ml 878 ± 602 762 ± 457 0.5 among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes (475 ± 372 ng/ml, n = 8), as compared to prom (952 ± 417, n = 12), p<0.02. this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches (1990 ( -april 2007 and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever >3 replication data sets were available results: a total of 5422 abstracts were reviewed and 88 were selected for full text review. data were extracted from 50 articles. over 100 associations were reported between snps on various candidate genes and ptb; however only 32 had replication dataset. meta-analysis documented significant association between pon2a148g (odds ratio [or]=1.64 (95%ci 1.18-2.26), pon1(rs#662)(or=1.14; ci-1.007-1.34), tnfrsf6-670a/g (fas) (or=1.52; ci-1.11-2.08) and ptb. two snps pon2s311c (or=0.56; ci-0.36-0.85) and ifn gamma (rs2430561; or-0.62; ci-0.43-0.91) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, 1 mounira habli, 2 jeff livingstone, 2 william polzin, 3 foong lim, 1 timothy crombleholme. 1 1 pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; 2 obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; 3 obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from 2004-2007 was performed. results among 254 cases of ttts 11/254(4.3%) patients with high order gestations were identified (n=11) with a mean ga at diagnosis of 20.3±0.8 weeks. 9 pregnancies (81.8%) were dichorionic triamniotic and 2(18.1%) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n=3), moderate (stage iiib, n=1) and severe (stage iiic or iv, n=3) categories. 4/11 (36.3%) were treated with amnioreduction alone (ar), 2/11(18.1%) with selective fetoscopic laser photocoagulation (sflp) alone, 2/11(18.1%) with ar followed by sflp and 1/11(9.09%) with ar followed by intrafetal radio frequency ablation (rfa). 1/11(9.09%) patient had a cervical cerclage. 2/11(18.1%) patients were treated but remain undelivered. mean ga at delivery was 29.5±1.04 weeks. overall survival was 21/27(77.7%) with bystander survival was 9/9(100%), donor survival 6/9(66.6%), recipient survival was 6/9(66.6%). conclusion triplet pregnancies treated for ttts have 100% survival rate for bystander fetuses and have 66.6% survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis 20.3±0.8weeks cincinnati modification of quinterro 1(i), 1(ii), 3(iii), 3(iiia), 1(iiib), 2(iv) ga at delivery 29.5±1.04 weeks live birth -donor 6/9(66.6%)* # -recipient 6/9(66.6%)*# -bystander 9/9(100%) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of 170 methadone dependent mother and infant pairs. patients were categorized into 3 groups based on maternal dose at time of delivery: group 1: dose 50 mg, group 2: dose 51-100mg, group 3: dose >100 mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were 2 scores of 8. neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the 3 groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between 26-29 weeks gestational age (ga) and fetoscopic balloon removal around 34 weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in 23 consecutive pregnancies but not in the first 28 pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [28.2 (1.8) vs. 27.5 (1.5) weeks; p= ns] and balloon removal [33.9 (0.7) vs. 33.7 (1.1) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was 2/23 in the study group compared to 9/28 in the control group (p<0.05). mean (sd) ga at delivery was 37.0 (1.8) weeks in the study group, compared to 36.4 (2.0) in the control group (p=0.28). bleeding from the trocar insertion site occurred in 4 cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in 4 patient in the treatment group compared to 6 in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between 1995 and 2005 was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period 1995 to 2005 there was a total of 60 pregnancies affected by hydrops (incidence 0.2 per 1000 births). the average maternal age was 29 years. in 5 cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was 18 weeks (range 11-36 weeks). in just over half (53%) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but 2 of the babies not born alive. edema was present in at least 3 cavities in over half of cases (n=36). chromosomal anomalies included trisomy 21, trisomy 18 and turners syndrome. in all cases of infection, parvovirus b19 was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in 33 cases an interruption of pregnancy was performed at a mean gestational age of 19 weeks. of those who did not elect to terminate the pregnancy, there were 15 fetal deaths in utero, 11 live borns with 1 neonatal death. for the live borns, the median gestational at delivery was 36.3 weeks (range 33 to 40.7 weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n=3), infection (n=2), chromosomal abnormality (n=1), unknown (n=4) and other (n=1 (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed 900 fetal brain exam performed between 2001 and 2007, seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at 1.5 tesla, using surface abdominal coils and single-shot fast spin-echo t2-weighted 3-4 mm thick sections, with 1.1-1.25 mm in planar spatial resolution. we found 6 cases with dmv anomalies (tab.1). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that 1 in 220 after 1 st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than 35 attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september 1, 2003 to july 1, 2007 was retrieved from our database. information concerning women younger than 35 years of age at the estimated date of delivery was extracted and tabulated. results: during the study period 591 women had genetic counseling for positive ft. thirteen patients were excluded from further analysis (8 had incomplete clinical documentation and 5 had spontaneous miscarriages prior to 16 weeks gestation). four hundred and twenty-five patients were older than 35 and 153 were younger than 35 at the estimated date of delivery. table 1 depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately 25% of this younger group of women opted for chorionic villous sampling (cvs), 40% for amniocentesis and more than 34% declined prenatal genetic testing. moreover, this data also suggests that: 1) these women opted for cvs when the ft risk (mean = 1 in 58) almost doubled the cvs procedure related risk quoted at 1% and, 2) when the ft risk is between 1 in 100 and 1 in 220 almost half (53 out of 115) declined not only the 1st trimester cvs but also the 2nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than 35 to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline (1966 medline ( -2007 , embase (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) and the cochrane library (2004 issue 2). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of 8581 articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all 12 examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of 0.6, 1.0, 1.4, 1.6. in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr >1.0 compared to <1.0 provided the strongest association with perinatal survival (peto or 5.73, 95% ci 3.38-9.71). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or 0.25, 95% ci 0.16-0.39). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > 1.0 compared to <1.0 was or 7.65, 95% ci 3.42-17.10). data was also available for liver up and lhr > 1.4 which had a peto or of 2.2 (95% ci 0. 83-5.82 ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from 2000 to 2007 was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as 2x2 tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and 15 could be pooled in the meta-analysis giving a sensitivity of 0.75 (0.63-0.86) and a specificity of 0.89 (0.84-0.95) for 1078 cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of 0.20 (0.10-0.35) and a specificity of 0.28 (0.18-0.41) for 112 cases. three studies had data for liver length measurements and two were pooled. the sensitivity was 0.16 (0.05-0.36) and the specificity was 0.77 (0.46-0.95) but only 38 cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of 0.73 (0.61-0.83) and a specificity of 0.54 (0.33-0.73) with 97 cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving 52 cases. the sensitivity was 0.95 (0.83-0.99) and specificity was 0.85 (0.55-0.98). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr 0.71; 95% ci 0.57, 0.89) and neonatal deaths (rr 0.30; 95% ci 0.17, 0.56) than amnioreduction ( figure 1 ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at <2 years rr 0.70, 95% ci 0.57, 0.86)( figure 2 ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage 1 and 2) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol.2007; 110 (3):687-94). the west midlands is a large region within the uk containing approximately 12% of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were 371 first trimester cvs performed, by ten operators, in the west midlands (uk) in 2005. this equates to 5.4 procedures per 1000 births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was 35 (range 9-81). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan (24.25%) ii) abnormal (>1 in 300) risk on combined first trimester screening (22.4%) iii) molecular genetic diagnosis (18.1%) and iv) maternal request (35.25%). using a combination of first trimester scanning and cvs, 33% had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was 3.6% (95thci 1.1-6.0%) up to 7 days postnatal (perinatal loss) and 2.7% (95thci 0.60-4.8%) with 28 days of procedure. the proportion of cvs in which an adequate sample was not obtained was 1.1% (95th ci 0.0-2.1%). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a 27 yo who underwent a duodenal switch procedure in 2005. she was discovered to be pregnant during evaluation for persistent malnutrition in 2006. multiple prenatal ultrasounds were performed; the first at 15 weeks gestation was unremarkable. the 19 week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. 3d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs 6 weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the 33 wk sonogram. delivery by cesarean section was at 33 5/7 weeks secondary to nonreasuring fetal status. the birthweight was 1468gm; apgars were 1, 4, and 6. postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until 12 weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <.03 ng/dl at time of delivery(normal > 0.10). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n=228) and term controls (n=563) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median =2; mean =3.6) versus those born to women with uncomplicated term deliveries (median =2; mean =2.8, p<0.001). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p<0.001). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor=2. 16 [1.36-3.43 ], p=0.001) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor=8 [1. 43-44.6 ], p=0.018) and cpap use (aor=2.6 [1. 23-5.32 ], p=0.012) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, 1 gh schuring-blom, 2 kd lichtenbelt, 2 jse laven, 3 d van opstal, 4 mjc eijkemans, 1 nick s macklon. 1 1 reproductive medicine gynaecology, university medical centre, utrecht, netherlands; 2 clinical genetics, university medical centre, utrecht, netherlands; 3 obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; 4 clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( 36 years at 18 weeks of gestation) in the netherlands between 1995 and 2005. results: from a total of 322246 pregnancies, 20885 cvs results were analysed: 235 in the ivf/icsi group and 20650 in the control group. the mean age of women in both groups was 38.4 years (mean difference -0.08, 95% ci -0.35 -0.18). foetal karyotype was missing in 152 cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, 1.3% vs. 2.2% (odds ratio 0.58, 95% ci 0.18 -1.81) whereas the incidence of foetal chromosomal anomalies was increased 4.3% vs. 2.4% (odds ratio 1.82, 95% ci 0.96 -3.46) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, 1 stacey l jeronis, 2 john p gaughan, 3 enrique hernandez, 2 ossie geifman-holtzman. 2 1 obstetrics and gynecology, duke university, durham, nc, usa; 2 obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; 3 biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol 2006; 195:1163-73) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, 95% confidence intervals for a proportion were calculated and compared between groups. we found 37 english-written publications reporting non-invasive rhd genotyping and 4 reporting non-invasive rh ce genotyping from maternal blood. fourteen (37.8%) of the rh d articles and three (75%) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: 67 .7 % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in 11/11 (100%, 95% ci 70.0, 100), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy 18 and trisomy 21. we present data showing that aneuploidy can be detected even when 90% of the starting dna sample was derived from a euploid source and only 10% was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp65 and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a 20bp region of the promoter between -712 to -692 from tss. we now report that this sequence binds both nf-bp65 and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il-1 in vivo. materials and methods: emsa studies were performed using a 33 bp oligonocleotide sequence (-712 to -672) , containing the 20 bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il-1 1ng/ml for 6 hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p65, c/ebp and h4 (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region -744 to -593, which includes the 20 bp response sequence. to further confirm the interaction between nf-bp65 with c/ebp a 6xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp65 or c/ebp . results: specific nf-bp65 and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp65 and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p65 and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p65 and c/ebp to this region of the otr promoter following stimulation with il-1 . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the 20 bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the 20bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il-1 leads to binding of nf-b and c/ebp to the 20bp region. regulation of pro-labour genes by c/ebp, nf-b and ap-1 in human uterine myocytes. suren r sooranna, 1 shirin khanjani, 2 yun s lee, 2 phillip r bennett, 2 mark r johnson. 1 1 imperial college parturition research group, imperial college, london, united kingdom; 2 irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p65) and ap-1(c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il-8, pghs-2 and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il-8, pghs-2, otr, connexin-43 and fp. methods: primary cultures of human uterine myocytes (n=6) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in 24 well plates to 80% confluence at which point expression constructs for c/ ebp (lap), nf-b(p65), ap-1 (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg5 was used as a filler construct. cells were cultured for 24 and 72h after which culture medium was collected for elisa and cells frozen at -80°c prior to rna extraction. copy numbers of il-8, pghs-2, otr, fp, connexin-43 and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: 24h post transfection with c/ebp (lap), nf-b (p65), c-fos and c-jun alone or in combination showed no significant changes in pghs -2, otr and connexin-43 expression. over expression of p65 alone or together with either c-fos or c-jun increased fp expression by 56, 76 and 73% respectively (p=0.028). nf-bp65 consistently increased il-8 expression either alone (by 1108%; p=0.018) or in combination with lap, c-fos or c-jun (by 1110, 1385 and 527% respectively; n=6; p=0,028). rel a, lap, c-fos and c-jun together also increased il-8 expression (by 502%; p=0.018) and a small but significant increase was seen with a combination c-fos and c-jun (by 137%; p=0.018). the changes observed in il-8 expression were reflected in the medium il-8 concentration at 72h post transfection. in the presence of rela and c-fos il-8 increased from 3.7 ± 0.2 to 71.0 ± 16.7pg/ml of culture medium (mean ± sem; n=4). conclusions: nf-b (p65) consistently increased fp and il-8 expression in human myometrium. the data suggest that pghs-2 activation has greater dependence upon other transcription factor(s) in addition to p65. true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine 165). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t47d contains a 60 kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met595) which serves as a translation initiation site for the generation of a pr isoform of 60 kda. based on such findings condon et al (mol endo 2006) have focused on the possible role of 60kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc-538 santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg5 expression vector. in vivo translation produced a protein of only 39kda. furthermore overexpression of pr-c595 did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a 60 kda protein. we constructed potential pr isoforms in psg5 vector with initiation sites at met 289 and 301. in vivo translation produced proteins of approximately 70 and 62 kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c289, pr-c301 and pr-c595 with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c595 both pr-c289 and pr-c301 significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr-312 (nc) and the sc-539 (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc-538 antibody detected only pr-a and pr-b very poorly. our data suggests that the sc-538 antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the 60kda but not the 39kda isoform might inhibit pr-b function. tnf receptor 1 antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u0126) (p<0.01), but not by tnf receptor 2 antibody (tnf rii ab), p38 mapk inhibitor (sb203580) and jnk inhibitor (sp600125). by western blot analysis, we found that the protein level of tnf receptor associate factor 2 (traf2) was higher than that of tnf receptor associate factor 1 (traf1) (traf2>traf1) in untreated ct cells. however, after tnf treatment for 4h to 24h, traf1 protein level was increased, but traf2 protein level was reduced (traf1>traf2). the increase of traf 1 and decrease of traf2 were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within 5-30 min) and significantly increased phosphorylation of ikk , erk1/2 and jnk1/2/3 and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf1 and decreased traf2 in ct cells (traf1>traf2) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf2>traf1). nuclear localization of nf-b p65 in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp-9 production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p38-ap-1 pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp-9 expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected <21 weeks gestation (mean 10.2 weeks, sd 3.9) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) <34 weeks (n=32) and 34-<37 weeks (n=72) were compared to women with term births >=37 weeks (n=215). high tat was defined as >5.2 ng/ml (highest quartile among women with term births) and high crp was defined as >=8 ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (<34 weeks, 53.1%; 34-<37 weeks, 30.7%; >=37 weeks 24.11%, p<0.01). women with high tat concentrations had a 3.3-fold (95% ci: 1.5-7.3) increased risk for sptb <34 weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb 34-<37 weeks (or 1.2, 95% ci 0.7-2.3). additional adjustment for elevated crp (>=8ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (<34 weeks, or 2.9 [1.1-8.0]; 34-<37 weeks, or 2.7 [1.3-5.6]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb <34 weeks (or 8.3, 95% ci 1.8-38.1). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb <34 weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of 3,073 participants, plasma crh and cortisol concentrations at 24 and 28 weeks gestation were available in 178 controls who delivered after 37 weeks and 159 cases who delivered before 37 weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at 24 or 28 weeks among women above or below the lowest quartile on the asaps (controls:p=0.17-0.66 cases:p=0.47-0.97). greater psychological stress was not associated with higher concentrations of crh or cortisol at 24 or 28 weeks(controls:p=0.33-0.63 cases:p=0.17-0.78). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at 24 and 28 weeks were lower in black women than non-black women (controls:p<0.01 cases:p< 0.01). the median cortisol concentration among control black women was 11.0 g/ml at 24 weeks compared to 14.4 g/ml among non-black women and 14.4 g/ml compared to 18.6 g/ml at 28 weeks. black women reported less psychological stress than non-black women (p= 0.001) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp-9 in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp-9.several evidences suggest that mmp-9 is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp-9 within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp-9 in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for 96 h. relative expression of mmp-9 by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp-9 through the fm, fluorescein-conjugated prommp-9 was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp-9 was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp-9 was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp-9 in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since 24 h (p<0.05). experiments using labeled mmp-9, repeatedly showed that after 12 h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp-3 significantly decreased the activation of pro-mmp-9. conclusions. our results demonstrate that the increased secretion of mmp-9 by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp-9 can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp-9 depends mainly on mmp-3, which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n=18). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of 1 nmol/l to 100 mmol/l and vpa (100nmol/l-1mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were 65.35% for spontaneous contractions (n=6; p< 0.05), and 53.53% for oxytocin-induced contractions (n=6; p<0.05). the mmi values for vpa were 36.57% for spontaneous contractions (n=6; p< 0.05), and 35.20% for oxytocin-induced contractions (n=6; p<0.05). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin-6 expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin-6 (il-6) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il-6 expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in 10 -8 m estradiol (e2) of e2 +10 -7 m mpa for 7 days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after 24 hours, il-6 levels in conditioned dm were measured by elisa and western blotting. in parallel 6-hour incubations, il-6 mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il-6 levels were similar in cultures maintained in e2 alone (0.43 ± 0.14) and e2 + mpa (0.33 ± 0.06 pg/ml/ug protein; mean ± sem; n = 9). the addition of thrombin (2.5 u/ml) enhanced secreted il-6 levels by 14.3 ± 2.4 fold (p<0.05) in incubations with e2 and by 8.2 ± 1.4-fold (p<0.05) in incubations with e2 + mpa. the inhibitory effect of mpa was statistically significant (p<0.5). in confluent dcs incubated with e2 + mpa, exogenous thrombin (0.05-2.5 u/ml) elicited a concentration-dependent increase in secreted il-6 levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il-6 secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il-6 m-rna levels corresponded to protein changes. conclusion: thrombin enhances il-6 mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il-6 may contribute to the protection against ptd, and may explain the reported protective effects of administration of 17 -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, 2-hydroxyestrogen (2ohe2), to 2-ethoxyestrogen (2meohe2). the comt substrate, 2-ohe2, can exhibit an anti-estrogenic effect in multiple biologic assays while the 2methoxyestrogen (2-meoe2) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites (2ohe 2meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited 3 folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi 2007) . objective: to investigate the impact of comt inhibition on prostaglandin e2 (pge2) production by human amniotic membrane explants. study design: explants consisting of 2-cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at 37ºc. after a 4-hour incubation, explants were treated with the selective comt inhibitor ro 41-0960, at 10 and 50 m concentrations. the incubation media was harvested after 6 and 24-hour intervals. the levels of prostaglandin e2 (pge2) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro 41-0960 comt inhibitor induced major reductions in pge2 production in media collected from amnion explants of human fetal membranes. in the group treated with 10 m of ro 41-9640 for 24 hours there was 72%±8% reduction of pge2 after 24 hours compared to untreated control (p<0.001). in the amnion explants treated with 50 m of ro41-9640, there was 74%±9% after 6 hours and 84%±11% after 24 hours of treatment compared to untreated control (p<0.001). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge 2 production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge 2 . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from 5 non-pregnant women, 5 women during elective cesarean section, 6 woman after spontaneous parturition and 4 after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s 35 during 24 hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately 50% in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around 40% and perlecan are significantly increased by around 60% compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge 2 production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox-2). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge 2 release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for 6 hrs (ot: 10-7 m; n=3; triplicate samples; 40 fold increase p<0.05). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n=3; duplicate samples; p<0.001). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il-1b resulted in a significant upregulation of otr which peaked with a 17 fold induction after 6 hours (p<0.001). il-1 caused a 16 fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il-1 to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o 2 -), hydrogen peroxide (h 2 o 2 ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod-2 (3.5 fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n=7; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod-2, serum/glucocorticoidinduced protein kinase-1 (sgk-1) and the dna repair enzyme gadd45a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women 1 whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway 2 . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in 20 women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix 3cm dilated; contractions every 3-4 minutes), a second sample was collected. results: seventeen (85%) of the subjects were multigravida. the inductions were for postdates(n=7); decreased fetal movements(n=3), fetal growth restriction(n=2), symphysis pubis dysfunction(n=2), diabetes mellitus(n=1), pre-eclampsia(n=1), fetal cardiac complication(n=1), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n=1). plasma aea levels increased significantly once labour was established (figure) and demonstrated in 19 (95%) of the 20 cases. the median (interquartile range) plasma aea level increased from 1.35nm (0.99-1.61) at induction to 2.24nm (1.49-2.82); *p=0.01; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r 2 =0.41, p=0.06 pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p65.the amount of dna was constant and psg5 was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg5. affymetrix genechip u133 microarray was performed (n=3, each group). we found that 33 genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and 5 were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin 8 demonstrated the highest, 18 fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip3), chemokine ligand 2 (ccl2), chemokine ligand 5 (ccl5), pentaxin related gene (ptx3), interleukin 6 (il6) and superoxide dismutase (sod-2). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand 20 (ccl20), chemokine ligand 11 (ccl11), chemokine ligand 2 (cxcl2) and il1 . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p65 subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p65 nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd68 positive cells. treatment with il-1 or tnf-induced nuclear translocation of p65 in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, 1 niccolo bosso, 2 marianna andreani, 1 agnese pizzardi, 1 fulvio magni, 2 anna locatelli. 1 1 obstetrics and gynaecology, university of milano-bicocca, monza, italy; 2 experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang 2005) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group 1, n=10) and term prom (group 2, n=10). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of 17 -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from 52 women who delivered preterm (median[range], ga: 27 [18-33] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n=17) within one week prior to amniocentesis were matched to 35 controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of 3 or 4 confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin-6 (il-6) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr 3 or 4 (n=18) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il-6 levels compared to unexposed women (progestins (n=6): 77 vs. crl (n=12): 12 [0.3-94] ng/ml, p=0.027). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il-6. conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r5020 on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n=6/group) were treated with zk (3 mg/rat, s.c.) alone or vehicle (controls) on day 17 of gestation. other groups of rats were treated with zk in combination with various doses of r5020 (0.3, 1 or 2 mg/rat s.c) with and without ng (2 mg s.c. pellet) from days 18 to 19 of gestation. all rats were sacrificed on day 20 of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p<0.05 was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p<0.05) treated with vehicle only (ca. 3% fetuses delivered). ng treatment alone did not affect the delivery rate (p>0.05) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p>0.05). however r5020 dose dependently reduced (p<0.05 at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r5020 plus ng (p<0.05). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r5020, a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r5020 and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, 1 jp rasanen, 1,2 dw sadowsky, 1 m bertolino, 3 m carbonatto, 3 e gillio tos, 3 s canali, 3 j lacy, 4 a chollet, 4 mj novy. 1 1 reprod sci, oregon primate res ctr, beaverton, or; 2 ob/gyn, oregon health sci univ, or, usa; 3 rbm, merck serono, italy; 4 merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as606521, a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n=3) and pregnant (120-150 dga, term 165d; n=5) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as606521, and ot rescue therapy. ot infusions (2-64 mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as606521 was administered p.o. at 5, 10, 20mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p2x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days 15, 18, 20 and 22. the expression of each subtype of p2x and trpc channels was measured by real time rt-pcr (abi7700) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; 0.2 mg/kg) was injected into intraperitoneal cavity at day 18 and the tissue was sampled after six hours. results; p2x4 and p2x7 were determined to be dominant subtypes of p2x channel. the expression of p2x4 was increased by 70% at day 22, compared with day 15 and that of p2x7 was enhanced by 90%. on the other hands, trpc3 and trpc4 were detected dominantly. the expression of trpc3 was increased three times in the late stages of gestation. however, trpc4 was suppressed by 57%. in the lps treated rat myometrium cox-2 mrna expression was measured to be 52 fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p2x4, p2x7 and trpc3 were enhanced by 7.4, 18.6 and 24.7 times, but trpc4 was not changed. the mrna expression of p2x4, p2x7 and trpc3 channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin (10 nm) and carbachol (100 μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p<0.05). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp1-4 consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp1, 3 and 4 as well as hdac1 and 2 form complexes with biotin-labelled fragments relating to sp1-4 elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor 5-azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp1-4 elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as 6h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component -1 (pgrmc-1). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n=4), preterm and not in labor (n=7) and term and in labor (n=5). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc-1 and gapdh. abundance of pgrmc-1 protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc-1 in the human pregnancy myometrium. results: pgrmc-1 protein was identified in each biopsy specimen. there was a 2-fold increase in pgrmc-1 protein in term compared with preterm biopsies (relative to gapdh p<0.04). the relative level of pgrmc-1 protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p=0.8). pgrmc-1 immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc-1 protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc-1 is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, 2008) . the aim was to examine the mechanism of p4 inhibition. methods: uterine tissues from women (n=55) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p4 alone (10 -10 to 10 -3 m) or p4 bound to bovine serum albumin (bsa/p4, 10 -6 to 10 -3 m p4), a progestin with low affinity to mpr (r5020, 10 -7 -10 -3 m), or a non-sex steroid (cholesterol, 10 -5 to 10 -2 m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq 22536, 10 -5 m), guanylate cylase (odq, 10 -5 m), phosphodiesterase (rolipram, 10 -5 m), nitric oxide (no) synthases (l-name, 10 -4 m) or a nuclear p4 receptor antagonist (mifepristone, mif, 10 -5 m), followed by p4. data were analyzed by anova for statistical differences (p<0.05). results: p4 rapidly (<1 hour), effectively (100%) and dose-dependently inhibits spontaneous and kclinduced contractility (ed 50 of <10 -5 m incubation of strips with zd-6416, at concentrations up to 1 m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l-798106 with a concentration of 100nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge 2 (10 -10 to 10 -6 ) were added to l-798106 (100nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd-6416 (100nm) pre-treated strips. since the reduction in contractility caused by l-798106 was greater than that caused by zd-6416, it is likely that the stimulating effect of pge 2 acts predominantly via ep3 receptors. taken together with our previous data showing an increase in ep3 at labour, this data shows that targetting an ep3 receptor may be a useful strategy in managaing pre-term labour. recently a truncated 46kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this 46kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n=4) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/10% fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with 0.05% typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f-10 domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated (46kda) and the full length (66kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the 46kda erisoform, yet the expression of the 66kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a 46kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f 2 (pgf 2 ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf 2 downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf 2 treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/-10 -7 m or 10 -6 m pgf 2 for 1, 3, 6 or 24 hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/-10 -6 m pgf 2 and +/-10 m myristoylated pkc inhibitor (20-28) and examined for ptgfr cellular localization as described above. results: pgf 2 treatment resulted in a dose dependent decrease in ptgfr membrane signal at 1, 3, 6 and 24 h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor (20-28) prevented the pgf 2 induced decrease in membrane ptgfr at 1 h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf 2 decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf 2 induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi-17, promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia (0.5% o2) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of 10 nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca 2+ ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca 2+ have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for 40-60 minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine (1um) rapidly abolishes spontaneous contractions, subsequent addition of 100nm oxytocin produced an initial, transient rise in force, approxiamately 20% compared to oxytocin alone, followed by high frequency oscillations in >50% of strips. calcium-free solutions were used to confirm that oscillations were due to ca 2+ entry. disabling the sr store using thapsigargin (1um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil (1um ) showed no inhibition of oscillations. an ip 3 receptor and store-operated calcium channel inhibitor, 2-aminoethyldiphenylborate (2-apb) 50um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf-96365 (10um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca 2+ entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, 1 mark r johnson, 2 rachel m tribe. 1 1 division of reproduction and endocrinology, king's college london, london, united kingdom; 2 department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il-1beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim1, stim2, orai1 and orai2 in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at -80 o c or used for cell culture. rna was extracted from whole tissue (n=5), primary cultured myometrial cells (n=4) and passaged (p2) myometrial cells (n=5) and stim1, stim2, orai1 and orai2 mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim1 and stim2 mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p<0.05). however, there was no significant difference in either orai1 or orai2 expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim1/2 and orai1/2 mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg (2007) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin-1 (dallot et al., 2003) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock-1 inhibitor y-27632, oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells (150,000 per well) passage 3-8, were embedded in 1.5 mg/ml collagen, in 0.5 ml aliquots, in dmem-f12 media on 24 well plates (invitrogen) (dallot et al., 2003) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek293 cells, did not. kcl (20nm) stimulated an 11% increase in contractility (n=3, p=0.0262) while 10 nm tnf resulted in an 8.4% increase (n=3, p=0.0205), in comparison to unstimulated cells embedded in gel. the rock 1 inhibitor y-27632 (10 m) inhibited contractility, with a 17.5% decrease in collagen gel contractility (n=3, p=0.011). a 13% increase (n=4, p= 0.0146) in utsmc embedded collagen contractility was observed with 10 nm oxytocin, which was antagonized by atosiban (1 m) (n=4). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using 3d power doppler angiography (pda). nw jones, 1,2 nj raine-fenning, 2 h mousa, 1 mj taggart, 3 k jayaprakasan, 2 gj bugg. 1 1 nottingham university hospitals nhs trust, united kingdom; 2 nottingham university, united kingdom; 3 university of newcastle upon tyne, united kingdom. methods 3d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) 1 in a volume of myometrium at the uterine fundus of 20 nulliparous women at term, in the first stage of uncomplicated spontaneous labour. 3d data sets were obtained during a single cycle of uterine relaxation (r1), contraction and subsequent relaxation (r2). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r1. data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm 3 ) of interest for r1 was 17.9cm 3 (12.6-24.4cm 3 ), for the contraction was 17. (fig.1) . the % change for all three indices between r2 and r1 was not significantly different. however, there was a significant difference between the contraction and r2 (p< 0.01). the mean intra-class correlation coefficient and 95% confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were 0.99 (0.98-0.99), 0.99 (0.98-0.99) and 0.98 (0.96-0.98), indicative of good inter-observer reliability. conclusion 3d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. 1. raine-fenning nj, et al. the inter-observer reliability of 3d pda acquisition within the female pelvis. ultrasound obstet gynecol. 2004; 23:501-8. 196 the role of pgf2 on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, 1 andre chollet, 2 phillip r bennet. 1 1 imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; 2 merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using 5x2mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph 800ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of 5-6g. a novel antagonist to pgf2 (fpa) was studied in this model. the fpa has a ki of 6nm for fp and is 10-100 fold selective for fp compared with other prostanoid receptors. results: addition of pge 2 and pgf2 after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge 2 was being greater than that of pgf2 . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to 1 m (10 -6 ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa (10 -8 to 10 -5 ) decreased the total work done by 5-fold on strips treated with pgf2 beforehand in comparison the pgf2 -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf2 induced but not spontaneous contractions, it is likely that pgf2 does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from 4 groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women 30-34 weeks' gestation, and term between 38 and 41 weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by 18s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all 4 groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt 1020675). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n=6) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence (10d), the cells were incubated (6h) with 100nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< 0.05) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. 2004; 23:501-8. conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage 28) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after 7 days. single cell clones of stably transfected cells were further isolated by plating in 96 well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage 37, ultr-ht cells grew 3.1 fold (p<0.0001) and 26.9 fold (p<0.0001) faster than ultr cells at passages 28 and 34, respectively. currently 8 single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm22, and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at1/2), nox family (nox1, 4, 5 and duox1), nox-associated genes (p22phox, p47phox, p67phox, and rac1/2), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca 2+ ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca 2+ ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the 18 days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca 2+ ] i were measured simultaneously in fura-2 loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol 12,13-dibutyrate (pdbu) applied at 10 -7 m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca 2+ ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca 2+ ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca 2+ ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca 2+ ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca 2+ ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs-2 and il-8. on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr-397, creating a src binding site. this promotes further fak phosphorylation at tyr-576, 577 and 925, enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek 1/2 in order to test the affect of fak kinase activity on expression on pghs-2 mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto 6-well flexible bottom plates coated in type i collagen. cells were subjected to 11% static stretch for up to 60 minutes. cells were also incubated with either the rho kinase inhibitor y27632 or the mek 1/2 inhibitor u0126 prior to being stretched. western blots were performed using antibodies to fak phospho-397, fak phospho-925 and erk 1/2 phospho-202/204, -actin was used as a loading control. rna was also extracted and levels of pghs-2 measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr-397 and tyr-925 with the greatest increases occurring at 30 and 60 minutes. however the increase at tyr-397 appeared to be greater than at tyr-925. incubation with the rho kinase inhibitor reduced phosphorylation of fak-397, however this did not affect phosphorylation of erk 1/2 or stretch induced up regulation of pghs-2 mrna. in contrast incubation with the mek 1/2 inhibitor reduced erk 1/2 phosphorylation and expression of pghs-2 mrna, whilst also reducing fak phosphorylation (n=4; p=0.05). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs-2 expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs-2 expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and 17 hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section 37 weeks gestation. strips of myometrium 15 mm long ,2 mm wide were cut and suspended under a resting tension of 20 mn in organ baths of krebs gassed with 95% o 2 / 5% co 2 within 12 hours of collection. progesterone or 17 hp were added in a cumulative manner at 20-minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for 2 hours, myometrial strips contracted in a rhythmic manner (amplitude 91.6 ±16.1 mn, n=8 pairs). progesterone (10nm-30 m) produced a concentration dependent inhibitory effect on myometrial contractions (fig1), which was greater than that of vehicle (p<0.05). maximum inhibition measured 93.3 ± 2.0% and 67.2 ±14.2% for progesterone and vehicle, respectively. 17 hp exerted an inhibitory effect, this was not significantly different from the vehicle (p>0.05). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[3]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of 17 hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [4] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than 17 hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; 2 pathology, ramathibodi hospital, mahidol university, bangkok, thailand; 3 obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) 1 level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp 1 in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = 5 each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp 1. reverse transcriptionpolymerase chain reaction (n = 10 each) using gene specific primer against mcp 1 and mcp 1 receptor was performed to identify mcp 1 messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp 1 in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp 1 was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp 1 and mcp 1 receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of 4075 fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within 6 days from delivery. we considered fetal macrosomia a birthweight 4000g and big babies a birthweight 4500g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was 38 +3 weeks (3 +3 sd). 1015 neonates weighted less than 2500 g, 2818 had a weight range between 2500 and 3999 g and 242 weighted more than 3999 g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > 350 mm which allowed to select 867 fetuses. analysing this population we found 3183 true negative cases, 25 false negatives, 650 false positives and 217 true positives, with a sensitivity of 89.6% and a specificity of 83%. to detect big babies the roc curve analysis identified a cut off of ac > 361 mm (n.452 fetuses), reaching a sensitivity of 100% and a specificity of 90%, without false negative cases and with 413 false positives (39 true positives, 3623 true negatives). the 141 cases that weighted between 4000 and 4499 g, were included in the 413 cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score 2.0) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed 320 genes up-and 354 down-regulated (n=3,3; p<0.05). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed 525 genes up-and 869 down-regulated (n=3,3; p<0.05). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at 0.9g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at 0.5g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =-7(30.4) <(u.)) 14(48.1) )(6.7)"" 1'0~.1) !(11.7)' d.'cbom< 7(104) 2f(81 ))""" s(17l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors 1,2 . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother 3 . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from 1 st to 3 rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n=369, 45% non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)-1, igf binding protein(bp)-1 and leptin, were measured in maternal plasma (28 weeks gestation). results maternal growth (defined as increase in knee-height >2mm/90days) was detected in 36% of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p<0.05). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p<0.01). these parameters correlated positively with maternal igf-1 and leptin but negatively with the igf inhibitor, igfbp-1 (p<0.001 for all). subjects delivering sga infants gained significantly less weight (p<0.01) and had lower igf-1 levels (p<0.01) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf-1 and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf-1 and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o 2 umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c=38.9±0.2 and iugr=32.0±0.5 wks). an ultrasound examination was performed within 4 hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * 0.5. blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o 2 umb uptake = qumb*(uv-ua) o 2 content. results. as expected, average fetal and placental weights were significantly different in the studied groups (1287±83 and 254±32 g in iugr vs 3274±62 and 474±16 g in n) . iugr pregnancies showed a significant reduction in qumb (98.5±6.6 vs 234.8±12.1 ml/min; p<0.01) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o 2 sat, o 2 cont and po 2 in both uv and ua compared to n. (uv-ua)o 2 content (1.79±0.72 vs 3.3±0.24 mmol/l; p<0.001) and o 2 umb uptake/kg (0.12±0.01 vs 0.24±0.01 mmol/min/kg; p<0.01) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that 50% on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il-6 in pregnancies with iugr. calvin j hobel, 2 chander p arora, 1 adegoke adeniji, 1 priya arora, 1 susan e jackman, 1 olga miadel, 1 baldjyan lilit. 1 1 ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; 2 university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency (37.5-80 nmol/l) or even deficiency of vitamin d (<37.5 nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin -6 (il-6) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il-6 and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for 25(oh)d using elisa. the samples were also analyzed for il-6 at three stages of pregnancy: t1 (18-20 weeks), t2 (28-30 weeks) and t3 (34-36 weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in 18 of 528 women with available samples from a behavior in pregnancy study (bips) . out of these subjects, 22 were selected as matched case controls. circulating levels of vitamin d (25(oh)d) were significantly lower in iugr cases at each visit (p<.001). the levels indicated deficient (29.4±4.6nmol/l) vitamin d in iugr group at t1 but sufficient vitamin d levels (83.8±5.8 nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t2: 19± 3.1 nmol/l vs 45±3.6nmol /l; t3: 22 ± 4.1nmol/l vs 50± 2.9nmol /l). at all three time intervals, significantly (p<.001) higher levels of il-6 were associated with the iugr cases (415pg/ml, 1329pg/ml and 2526 pg/ml respectively) as compared to the controls (60pg/ml, 330pg/ml and 1240pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il-6 expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global 30% maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology 165:2). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology 140:10). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n=12) or 70% of wt adjusted ctr diet (mnr, n=11) from 0.16 gestation (g) and fetuses were recovered at c-section under general anesthesia at 0.5 (n=9; 5 ctr and 4 mnr) and 0.9g (n=14; 7 ctr and 7 mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x 100%) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at 0.5g there was no difference in igf-ii or insulin fraction or density between groups. at 0.9g, igf-ii fraction (2.57 ± 0.42 vs 1.44±0.27,p<0.05) and density (1.02x10 7 ±1.90 x10 6 vs 5.19x10 6 ±9.50x10 5 , p<0.05) and insulin fraction (2.51 ± 0.25 vs 1.58 ± 0.05, p=0.05) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at 0.9g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut1? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut1, 11 hsd1, the gluconeogenic and glycolytic enzymes pepck and g3pdh and the transcription factors pgc1 and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in 19 non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in 19 pr and 9 control (c) fetuses at 103-117d and arterial blood samples were collected for blood gas analysis. mean gestation po 2 <17mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at 140-145d. hepatic mrna expression of glut1, 11 hsd1, pepck, g3pdh, pgc1 and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po 2 and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po 2 (r 2 =0.67, y=0.21x+0.5, p<0.01). liver weight was significantly lower in a cohort of pr-h fetuses (c, 24.1±1.6; pr-n 22.9±0.9; pr-h 22.0±1.6; pr-h-rlg 15.8±0.3g:kg; p<0.05). glut1 expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of 11 hsd1, pgc1, ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p<0.05). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in 11 hsd1 expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc1 and ppar . interestingly the compensatory increase in hepatic glut1 expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, 1 jeffrey gornbein, 2 calvin j hobel. 3 1 obstetrics gynecology, los angeles, ca, usa; 2 biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; 3 obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at 1 st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and 4 sonographic fetal biometric parameters at 18-20 weeks, 28-30 weeks, and 34-36 weeks. the rate of change in fetal weight was defined as 60 x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were 454 multi-ethnic women included in the study, after 112 were excluded. they underwent a total of 1,277 exams. fetal gender (p=0.0048), maternal weight at 1 st exam (p=0.0055), and cumulative maternal weight gain at exam 3 (p < 0.001) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of 46.9 grams per 60 days higher than females. the rate of fetal weight change increased by an average of 8.8 grams per 60 days for each 10 lb increase in maternal weight at the 1 st exam. the fetal growth rate increased 2.2 grams per 60 days for each 1 lb of cumulative weight gain at the 3 rd exam. conclusions: in this study, maternal weight at the 1 st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. 216 high risk patients. other predictors include the known risk factors of age <15 and >40, single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes,2005) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b1-ar. methods: baboons were fed as ad lib controls (ctr) or 70% of wt adjusted ctr diet (mnr) from 0.16 gestation(g) with fetuses retrieved at c-section under general anesthesia at 0.5 or 0.9g. protein expression determined by immunohistochemistry for b1-ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x 100%. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at 0.05. results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at 0.5g = 197.5 ± 1.7 vs. 162.8 ± 11.7%, p< 0.05 . b1-ar fraction was lower following mnr at 0.5g and at 0.9g (p< 0.05; fig 1) conclusions: mnr decreased b1-ar over 50% at 0.5g and 20% at 0.9g. decreased fetal liver b1-ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (<150d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (>150d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) 1, blood cell mrna was collected from pi steers (n=2; confirmed by virus isolation), or uninfected control steers (n=3) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp2, bvdv naïve pregnant heifers (n=6 per group) were not infected (control) or infected with ncpbvdv on d.75 or d.175 of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d.190; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed 294 genes that were differentially regulated in pi vs. controls (p<0.05, >1.5 fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg 2006-03907 from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients >14 weeks of gestation were prospectively enrolled into an observational study (08/2002 to 06/2007). transvaginal sonographic examinations were performed every 2 weeks until delivery. the shortest cervical length between 14-32 weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: 303 asymptomatic patients were studied. 19.8% of patients delivered an sga neonate (60/303). the median cervical length was 21 mm (0 to 55mm); 184 patients had a cervical length <25mm. of these, 17% (32/184) delivered an sga neonate. similarly, 17% (17/98) patients with a cervical length <15mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < 25mm and <15 mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [17% (32/184) vs. 24% (28/119); p=0.34]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: 1) preconception start of folic acid use; 2) start of folic acid use in first ten weeks of gestation; 3) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age 18-25 and >25 weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from 6,719 pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of 6 grams (95%ci 2.92-9.01, p<0.001) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of 5 grams (95%ci 2.19-7.88, p=0.001) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of 69 grams (95%ci 39-99) and 51 grams (95%ci 23-80), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than 2500 grams when folic acid was not started preconceptionally (or 1.5, 95%ci 0.9-2.4). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in 17 severely premature iugr fetuses (estimated fetal weight < 10 th percentile and umbilical artery pulsatility index > 95 th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < .05 was considered statistically significant. results: all fetuses were delivered at < 32 weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than -1.8 resulted in iufd, whereas a score greater than -1.8 resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than 28 weeks resulted in nd and those fetuses at or greater than 28 weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a 50% food restricted diet (mfr; n=5) or were fed ad libidum (control; n=5) from d10 until d21 of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from 3 randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n=5) p-value litter size (median, range) 11 (8,15) 12 (9,15) 0.59 fetal weight (mean ± sd) g 3.29 ± 0.12 4.0 ± 0.1 <0.001 placental weight (mean ± sd) g 0.73 ± 0.05 0.90 ± 0.03 0.007 collagen i (mean ± sem) ct 17.9 ± 0.13 18. we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in 6 non-pregnant ewes to induce pr. vascular catheters were inserted in 6 pr and 4 control (c) fetuses at 103-117d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt-1 levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt-1, in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method 42 maternal peripheral venous samples were collected antenatally from two groups of pregnant women between 25-40 weeks of gestation. group a: uncomplicated normal pregnancies (n = 20) and group b: pregnancies complicated by isolated placental vascular disease( n = 22) as defined by birth weight less than 10 th centile for gestation and umbilical artery doppler s:d ratio above the 95 th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt-1 levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt-1 levels (figure1) in group a: normal pregnancies increased with gestation (p < 0.001). the sflt-1 levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = 0.002) and did not show a significant variation with gestation ( p = 0.47). the plasma vegf levels were below the detectable levels of the assay in all samples except 3 normal pregnancies. the significantly increased maternal plasma sflt-1 levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of 5 -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of 5 -reductases (5 r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al 2007 neurosci 146:1726 . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al 2007 repro sci 14 #381). the aim of the present study was to determine the effects of suppression of 5 -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term 68d). sham operated or gr dams received finasteride (a 5 r inhibitor; 25mg/kg/day) during late gestation (55d until term). activated caspase-3, a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, 5 r1 and cytochrome p450 side chain cleavage (p450scc) were measured by real time pcr and western blotting in fetuses (65d) and neonates 24h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by 30% whilst sparing brain weight. the number of activated caspase-3 positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase-3 activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p450scc mrna and 5 r1 protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from 21 fetuses ranging from 27 to 39 weeks gestational age for a total of 69 recordings. 29 measurements were taken from 10 mothers with a history of smoking (sm) and 40 from 11 mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of 20 recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in 35 out of 40 fetal recordings in the ns group and 23 of 29 in the sm group. the neonatal response rate was 90% for each group. the latencies were divided into three components: c1 (100-300 ms), c2 (301-600 ms) and c3 (601-900 ms). in both fetuses and neonates as well, there was a statistically significant difference (p<0.05) between the two groups in the c2-component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a1 and a2a receptors. objective: this study was designed to test the hypothesis that ado a1 and a2a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (>0.8 term). intravascular infusion for 1 h of dpcpx (a1 receptor antagonist) or zm241385 (zm, a2a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for 1 h by having the ewe breathe a hypoxic gas mixture (fio2 = 0.10). data were analyzed by two-way repeated measures of anova. results: blockade of ado a1 receptors (n=8) increased significantly (p < 0.05) fetal concentrations of glucose [control (c): 15.6 ± 1.0 (se)]; experiment (e): 17.38 ± 1.1 mg/dl] and lactate (c: 16.5 ± 2.4; e: 22.5 ± 4 mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a2a receptors (n=6) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n=11), which did not alter pao2 or paco2, increased concentrations of glucose (c: 18.0 ± 0.8; e: 22.6± 1.3 mg/dl) and lactate (c: 12.2 ± 1.0; e: 20.4 ± 1.6). zm (n= 7), but not dpcpx (n=9), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao2 13 torr), which increases (2-3 fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: 7.331 ± 0.016; e: 7.140 ± 0.032), and increased fetal levels of glucose (c: 16.2 ± 1.3; e: 33.7 ± 7.8 mg/dl) and lactate (c: 13.3 ± 1.4; e: 97.3 ± 7.7 mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: 1) a1 receptors modulate plasma levels of glucose and lactate in normoxic fetuses; 2) a2a receptors mediate the adoinduced rise in plasma glucose and lactate; and 3) a1 and a2a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd-18478. objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at 72 hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at 72h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at 72h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within 3 hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and 7-day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr139317 (12 mg/kg/day; et a antagonist) or 4.2% nahco 3 vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day 14 (term=22 days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day 7. oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days 1 and 7. results are presented as means ± se. newborn 7-day neonate vehicle 92.6 ± 0.9 94.4 ± 0.8 eta antagonist 94.7 ± 0.8 91.6 ± 1.1 there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day 14 through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, 1 jan b derks, 1 martijn a oudijk, 1 avnesh s thakor, 2 tereza cindrova-davies, 2 frank van bel, 1 gerard ha visser, 1 graham j burton, 2 dino a giussani. 2 1 perinatal center, university medical center utrecht, netherlands; 2 department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child 91:163, 2006) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at 0.8 of gestation,10 surgically instrumented sheep fetuses were submitted to i/r (5 x 10 min repeated ucc) under maternal allopurinol (n=5) or saline vehicle (n=5) infusion. fetal hearts were collected 48h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from 5 uninstrumented fetal sheep at 0.8 gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox-2, enos, hsp90 and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. 1 objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day 50 for 14d, breathing either room air (nmx), or 10.5% or 12% o 2 (12% o 2 hpx or 10.5% o 2 hpx). on day 65 (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by 2d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs800 densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the 4700 proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a 2x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein 70 (hsp70), actin (actg1), interleukin-1 (il-1), and glutamine synthetase (gs) were each up regulated, while cofilin-1, brain-type creatine kinase (bb-ck), and -1 gtp binding protein were each down regulated. all protein changes were proportional to the hpx (12% o 2 hpx vs nmx, 12% o 2 hpx vs 10.5% o 2 hpx, p<0.05). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il-1, and gs have each been previously investigated, the potential roles of actg1, bb-ck, beta-1 gtp binding protein, and cofilin-1 in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r01 hl049041-12, cpw) and cdc grant (u7dp00187-03, cpw). intermittent umbilical cord occlusion occurs in 5% of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a 4 day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting 1 min/ hour (mild group; n=5), 2 min/hour (moderate group; n=4), 3 min/hour (severe group; n = 6) or no occlusion (control group; n=7). fetal arterial blood samples were drawn 5 min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < .01). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < .01).elastin content of the superior mesenteric artery did not differ between the 3 experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control 5.7 ± 0.4 4.0 ± 1.7 -mild 7.0 ± 0.7 21.06 ± 2.5 ** 10.39 ± 0.6 moderate 7.4 ± 0.7 32.4 ± 1.6 ** † 14.3 ± 0.5 † severe 9.5 ± 1.0 * 34.5 ± 0.6 ** † † 17.4 ± 1.2 † † * p < .05; ** p < .01: experimental groups compared to control † p < .05; † † p < .01: compared to mild group the max in map was positively correlated with elastin content (r 2 = .56, p < .05). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, 1 roy mansano, 2 michael g ross, 2 robert gagnon, 1 bryan s richardson. 1 1 obgyn, chri, univ of western ontario, london, canada; 2 obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n=10) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild (1min every 5min), moderate (1min every 3min) and severe (1min every 2min) ucos with each series lasting 1h or until fetal arterial ph decreased to <7.0. fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph 7.36±0.03 to 6.91±0.12, p<0.001). 52±13 min prior to fetal ph drop <7.0, the sef of ecog began to increase abruptly during each fhr deceleration from 3±1 hz up to 23±2 hz (p<0.001) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p<0.001). conclusion: our findings suggest that in the animal model studied (1) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; (2) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph <7.0. these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e2) and progesterone (p4). however, the roles of e2 and p4 in da regulation have not been studied. objective: we hypothesized that: e2 and p4 have opposite effects in the da; that rising e2 and falling p4 levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e2 and p4. methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc-1, -2, and serbp-1 was examined by rt-pcr and qpcr on days 15, 17, 19 (term), and p1. the effects of e2 and p4 (10 -9 -10 -4 m) on the d19 fetal da were examined in a cannulated microvessel myography system. e2 and p4 effects were also studied in the presence of receptor antagonists (ici 182780, ru486) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc-2 expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o2 conditions, e2 induced rapid, concentrationdependent dilation at 10 -6 -10 -4 m (n=11); p4 induced progressive vasodilation at all doses (n=10). e2 and p4-induced dilation occurred within 60-180 seconds of exposure. while e2-dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e2 (n=10) suggesting antagonism of e2 effects. the vasodilatory effects of e2 were partially inhibited by pretreatment with l-name. p4-dilated das did not respond to ru486, whereas ru486pretreated das showed a small, significant response to p4 (n=10), suggesting partial antagonism or signaling via alternative, ru486-independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p4. conclusions: contrary to our expectation, both e2 and p4 have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po 2 (tpo 2 ), and electrocorticographic (ecog) activity to arterial o 2 tension (pao 2 ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o 2 probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = 8) we measured laser doppler cbf (ld-cbf), tpo 2 , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency-90% (sef 90 ) in response to 40 min moderate isocapnic hypoxia. results. ld-cbf, cerebral o 2 delivery, tpo 2 , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo 2 decreased briefly to 7±1 from a control value of 10±1 torr; however, as ld-cbf increased 18±5%, and sef 90 increased to14±2 from 7±1%, tpo 2 returned to near normal value. with acute hypoxia (pao 2 = 12±1 torr) when in the lvhf state ld-cbf increased only 23±14%, as opposed to a 52±10% increase when in hvlf ecog state. with this degree of hypoxia, tpo 2 decreased to 3±1 torr, sef 90 remained at 8±2%, and cerebral metabolic rate for o 2 (cmro 2 ) decreased 32±5% (p<0.05). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo 2 , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo 2 , cmro 2 , and ecog state, with increased ld-cbf failing to restore cortical tpo 2 . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd-03807). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, 1 kanchan m kaushal, 1 malgorzata mlynarczyk, 1 kimberly hyatt, 2 dean a myers. 2 1 ctr. for perinatal biol., loma linda univ., loma linda, ca; 2 ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude (3,820 m) from day 40 to 130-131 of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po 2 was maintained at a level comparable to that observed at altitude ( 60 mmhg) by nitrogen infusion. on 132 dg, lth (n = 4) and age-matched, normoxic control (n = 4) fetuses were implantated with vascular catheters. each fetus received a 15 min infusion of either saline vehicle, 100 ng/kg of ovine crh or 20 ng/kg of avp (estimated body weight/min) in a randomized order over 3 consecutive days (137-139dg). blood samples were collected at 0 min (baseline prior to infusion), 15, 30, 60 and 90 min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate 22 kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at 90 min) were significantly higher in the lth group compared to control (240±17 vs. 110±14, respectively; p<0.01). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p<0.01). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control (209±51 vs. 67±20 respectively; p<0.01), however peak levels were reached at between 15 and 30 min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels (102.1±27.9 vs. 66.1±10.2, p<0.05, lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd33147 and hd31226). martin g frasch, 1 roy mansano, 2 michael g ross, 2 robert gagnon, 1 bryan s richardson. 1 1 ob/gyn, chri, university of western ontario, london, canada; 2 ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep (125 ± 2dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild (1 min every 5 min), moderate (1 min every 3 min) and severe (1 min every 2 min) uco each lasting 1 h or until ph decreased to < 7.0. fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph (7.36 ± 0.03), fhr (163 ± 5 bpm) and bp (45 ± 4 mmhg) were within the physiological range. average depth of fhr dec for all uco was 93 ± 11 bpm and increased with higher lactate concentrations (r = 0.40, p < 0.05) and lower ph (r = 0.34, p = 0.08). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = 0.49, p < 0.05). bp increased 11 ± 2 mmhg (p < 0.05) during mild uco (ph to 7.32 ± 0.03), 4 ± 3 mmhg during moderate uco (ph to 7.19 ± 0.04, p < 0.05) and 2 ± 4 mmhg during severe uco (ph to 6.91 ± 0.12, p < 0.05). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde5 in placental dysfunction following fetal bypass. mitali basu, 1 r scott baker, 1 christopher t lam, 1 kenneth e clark, 2 pirooz eghtesady. 1 1 cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; 2 ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at 100-110 days gestation were placed on bypass for 30 minutes and followed post-bypass for 2 hours. placental samples were collected immediately prior to bypass and at 30 and 120 min post-bypass (n=7) and compared to a group of similarly instrumented controls, (n=6). placental enos, inos and pde5 protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p=0.05). pre-bypass protein expression did not differ between groups. pde5 protein levels and phosphorylated pde-5 expression were both elevated 30 min post bypass, and reduced 120 min post bypass compared to sham, (p=0.01). enos levels in the bypass group increased linearly from pre-bypass to 120 min postbypass (p<0.01), and were also elevated compared with shams (p<0.01), while shams had declined significantly by 120 min post bypass, (p<0.01). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to 120 min post-bypass, (p=0.058), while shams trended towards decline by 120 min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at 30 and 120 min post-bypass, (p<0.04). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde5, phosphorylated pde-5, enos and ostensibly phosphorylated enos. increased pde5 expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde5 and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that 5 days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at 125 days of gestational age, pregnant sheep were submitted to 5 days of hypoxemia (75% of fetal arterial po 2 ; at day 5 fetus 14±1 vs 20±0.5; mother 67±5 vs 116±3 mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n=10) and hypoxemic (n=10) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using 125 i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of 125 i-sarthran in nts and dmnx resulting from an increased expression of at1 receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; 8.5±0.6 vs. 11.5±1.2, p<0.05). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: 0.04-0.15 hz) and high frequencies (hf: 0.15-0.4 hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf 3.3±0.4 vs 2±0.5, p<0.05; dbp lf/hf 2.7±0.3 vs 1.8±0.2, p<0.05). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf 9.2±1 vs 15.3±2.6, p<0.05; alpha hf 8.5±1.4 vs 19.8±5.3, p<0.05). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat1, may have potential long-term consequences for the development of hypertension. hd37885;hd047584;hl51952. objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from 256 range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses (2.5±1.6 , rs=0.51 p<0.01) with linear regression with standardized coefficient of 0.4 (p<0.01). in contrast, wsr (421±97) is unchanged during the first , second and third trimesters (p=0.8).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin-1 (et-1)-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing 10% serum collected from ovine fetuses (n=3) that underwent 30 min of cpb, then were maintained for 120 min. serum was collected before cpb, from pump prime before initiation of cpb, 30 min on cpb, or 30 and 120 min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested 24 and 48 hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et-1 was measured in the culture media by elisa. results: no production by huvec after 24 and 48 hr was stimulated above control levels by fetal serum collected during and up to 120 min after fetal cpb (p< .01). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<.01). stimulation of et-1 after 24 and 48 hr of huvec culture peaked with serum collected at 30 min after fetal cpb (p<.01 compared with control), but was elevated above control levels at each collection time point (p<.05, table 1 ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et-1 production during and for at least 120 min after cpb. although the specific regulatory proteins remain to be identified, the no and et-1 pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n6-(1-iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; 21%o 2 ) or 10.5%o 2 (hypoxia) in a hypoxic chamber for 14 days prior to term (term=65d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of 1-2mg/kg/d for 10 days. at 60d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n 2 and stored at -80 o c until ready for analysis. the effect of total no product (no 2 and no 3 -, nox) of left ventricles of fetuses exposed to normoxia (n=4), hypoxia (n=5) and hypoxia plus l-nil (n=4) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by 18% and increased placenta/fetal body wt by 33% as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by 4%. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia (68.4+11.2) by 1.8 fold compared to normoxic controls (37.3+12.1). l-nil significantly decreased (p<0.05) nox levels in hypoxic hearts by 58% (68.4+11.2 vs 29.0+9.3; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep (125±2dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild (1min every 5min), moderate (1min every 3min) and severe (1min every 2min) uco with each series lasting 1h or until fetal arterial ph decreased to <7.0. fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at 20min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r=0.92, p<0.001). bd ecf correlated strongly with bd blood (r=1, p<0.001). bd ecf increased by 0.55±0.03 mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. 1) . lactate, bd blood , bd ecf and bd gap correlated to ph (r=0.86, r=0.95, r=0.95 and r=1, respectively, all p<0.001) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor-1 (sdf-1), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf-1 in brain hemispheres with or without hypoxic-ischemic lesion. sdf-1 expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf-1 expressing brain regions and we were able to show that these cells express the sdf-1 receptor cxcr4 on their surface. the functional implication of sdf-1 in directing hucb cell migration was determined by application of neutralizing sdf-1 antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf-1 in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd-1 mice on e15 receive intrauterine injection of lipopolysaccharide (lps) or saline. 6 hours after injection, fetal brains from the left upper horn were harvested. 2 fetal brains were removed from each dam with 6 dams per treatment group. 12 separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe 430av2 chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n=10-14 per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. 542 genes were significantly differentially expressed between lps and saline brains (p<0.01). with a p value of <0.0001, 40 genes were differentially expressed. pathway analysis revealed significant involvement of 1) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score 4.03) and 2) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score 2.2). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in 6/10 genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx 32 weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice 0-28 days after birth (p0-p28) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b7.2, colocalised with standard microglial marker iba1. results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha-5, alpha-6, alpha-m, alpha-x, beta-2 and b7.2. the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p0, decreased linearly over p3 and p7 and disappeared at p10. this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p0 cortical microglia and formation of stubby processes in p3 fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p0-p7 swm, roughly equivalent to early 3 rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. 1997, pp. 13-25) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia 2005) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol 1967) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci 14.1, 2007, 248a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in 47 healthy fetuses at 0.27 (n=6), 0.40 (n=5), 0.53 (n=5), 0.63 (n=6), 0.75 (n=12), 0.87 (n=9) and 0.93 (n=4) of gestation (term 150 days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image 6.21, nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at 0.53 of gestation followed by the cortical white matter structures at 0.63 of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, 1969) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a 55-mhz transducer at hh stages 18, 21 and 23 which are comparable to humans at 5 to 8 weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure 1a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure 1b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro 2 ), cortical tissue po 2 (tpo 2 ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin-1, which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin-1 modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from 50d to 65d (term) with either room air (nmx) or 10.5% or 12% o 2 . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin-1. total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin-1, bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using 18s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek1, the upstream activator of erk1/2, which was under the control of the pan-neuronal talpha1 alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p7 c576bl/6 mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with 8% oxygen for 60 mins at 36°c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek1 dn was associated with a reduction in erk1/2 activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek1dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, 60 min hi insult (*p<0.05, **p<0.01, anova). similar protective effects of mek1dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek1 and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, 2004) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day 15, wistar rats were divided: control (21% o 2 ) + melatonin (5 g.ml -1 drinking water), hypoxia (10% o 2 ) + melatonin, and undernutrition (40% reduction in food intake) + melatonin (n=7 per group). on day 20, dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: 3.73±0.05g; brain/body weight ratio: 46.2±1.2mg/g; n=76), both hypoxic (2.94±0.03g; 58.4±1.1mg/g, n=80) and undernourished pregnancy (3.05±0.04g; 53.0±1.1mg/g, n=80) promoted asymmetric iugr (p<0.05), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic (0.43+0.01, n=95), hypoxic (0.44±0.01, n=98) and undernourished (0.41±0.01, n=81) pregnancies relative to untreated controls (0.48±0.01, n=76; p<0.05). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig.1 objective: midkine (mk) is a 13-kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: 1) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term (3-fold: 16wks vs. 37wks). 2) tissue transglutaminase known to polymerize mk was abundant in af. 3) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). 4) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan-4 were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan-3 were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav8 hfix vector (1 -9 x 10 12 p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = 3) or late (n = 4) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected 18 days after late ip injection (44% and 28% normal human levels). early gestation ip injection gave 8.7% and 1.8% at 3 and 21 days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels (0.7%) 1 year after birth in early and 4 months after birth in late gestation injected lambs. up to 1 year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to 1 year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor 1 has a positive effect on estrogen production throughout pregnancy. urocortin 2 (ucn 2), a novel peptide of the crh family binding exclusively crh receptor 2, is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn 2 on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn2 in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p450arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn2, in presence of dheas significantly increased e2 levels at 4, 8 and 12 hours treatment, while in presence of androstenedione and testosterone an increase in e2 secretion was detected only at 12, 24 and 36 hours (at different ucn2 doses). both p450arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn2 caused a synergistic increase. anti-sauvagine 30 (a crh type 2 receptor antagonist) resulted in a significantly attenuated ucn2 effects on e2 secretion and on p450arom mrna and protein expression. conclusions: the present study supports a possible role of ucn2 on placental e2 biosynthesis: e2 secretion and p450arom transcript and protein expression were significantly increased after ucn2 treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn2. a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss-1 gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss-1, a 54-amino-acid peptide encoded by a putative metastasis suppressor gene. kiss-1 acts on its placental g protein-coupled receptors (kiss-1r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss-1 in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss-1 at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss-1 protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss-1 mrna expression was significantly higher (p<0.0001) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss-1 levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss-1 mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss-1 on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( 2-8 % o 2 ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk1/2, which stabilizes hypoxia-inducible transcription factor-1 (hif-1 ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf2-and vegf-stimulated cell proliferation partly via activating erk1/2 and stabilizing hif-1 protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( 20% o 2 ) or hypoxia (3% o 2 ) for 2 days were treated with fgf2 and vegf. after 3 days, the number of cells was determined. activation of erk1/2 and levels of hif-1 protein were determined by western analysis. results: under normoxia, fgf2 and vegf stimulated (p < 0.05) cell proliferation and induced ( 5 min, p < 0.05) erk1/2 phosphorylation in hpae and hpme cells. hypoxia promoted (p < 0.05) fgf2-and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < 0.05) such actions in hpme cells. hypoxia enhanced fgf2-, but not vegf-induced erk1/2 phosphorylation in hpae cells. in contrast, hypoxia promoted (p < 0.05) vegf-, but not fgf2-induced erk1/2 phosphorylation in hpme cells. hypoxia increased (p < 0.05) hif-1 levels in the hpae cells: first detected at 0.5 hr and maintained up to 24 hr. hpme cells had high basal levels of hif-1 (3 folds; p < 0.05) compared with hpae cells. hypoxia did not alter hif-1 levels up to 4 hr and decreased (p < 0.05) hif-1 levels at 24 hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf2-and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif-1 protein levels. however, inhibition by hypoxia on fgf2-and vegf-stimulated hpme cell proliferation is associated with relatively high hif-1 levels in the first 4 hr and decreased hif-1 levels at 24 hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif-1 levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day 12 of gestation (term=22 days), housed in under 12h-12h light-dark conditions (lights on 0600). lab chow and water were continuously available. on day 18, pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at 0545-0600hr (zeitgeiber time, zt0) or 1245-0100hr (zt6). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of 339 bp size was consistently identifiable in placenta harvested at zt6 but not at zt0. nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt6 in giant trophoblast (gt) cells (gt-crf: zt0= 0.176 ± 0.122 au, zt6= 0.272 ± 0.014 au, p=0.007) but not in spongiotrophoblast cells (stc) (stc-crf: zt0 = 0.218 ± 0.025 au, zt6= 0.162 ± 0.011au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt0= 0.148 ± 0.003, zt6 = 0.149 ± 0.015 au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt6. these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp1, expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp1, and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr-8/svneo cells and term placenta-like trophoblast carcinoma cell line jeg-3. cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of 0.5, 5 and 50 m for 2 hrs (aqp1 mrna expression) and 20 hrs (aqp1 protein expression). for time course experiments, cells were incubated with 5 μm of either sp-camp or forskolin for 2, 10 and 20 hrs at 37°c, 5% co 2 . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp1 mrna expression in both cell lines after 2 hrs (p<0.05) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. 5 m of sp-camp and forskolin stimulated aqp1 mrna expression after 2 hrs in htr-8/svneo cells and after 10 hrs in jeg-3 cells (p<0.05). 5 m of either sp-camp or forskolin stimulated aqp1 protein expression in both cell lines after 10 hrs (p<0.05) and the expression remained high at 20 hrs. conclusion: aqp1 gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a 37 kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin-1 in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n=20) collected from sprague-dawley pregnant rats on day 21 of gestation (term=22) were either fixed in 4% paraformaldehyde solution (ph 7.4) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc-1824 santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro 4.01 software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: 0.191±0.010, giant trophoblast cells: 0.139±0.029, spongiotrophoblasts: 0.120±0.003, fetal membranes: 0.128±0.009. pcr analyses identified a single 434 bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per2 clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene 2 (per 2), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day 13 of gestation and housed in a controlled environment (0600-1800h lights on) with free assess to food and water. placentas collected on days 20 and 22 of gestation (term=22days) between 0930 and 11.30 am were fixed in 4% paraformaldehyde and paraffin embedded. for each gestational ages a total of 12 placentas from 6 different pregnant rats were used. paraffin sections (3 sections per placenta) were subjected to immunohistochemical analysis with antibody to per2 (1:200, santa cruz biotechnology, ca) by abc technique and 3, 3'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro 4.01 software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < 0.05 as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per2 clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day 22 of gestation, we conclude that fetalmaternal interactions in per2 regulation disappear at term (or at the time of birth.). our findings imply that per2 may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n=6) on day 21 of gestation were given a single subcutaneous injection (at 5:45 am) of betamethasone (350μg/kg body weight) while control pregnant rats (n=6) received saline. dams were exposed to isoflurane anesthesia at 10:45 am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc-8992, santa cruz biotechnology), with immunoreactive material identified by abc technique using 3, 3' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro 4.01 plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p<0.05 considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (<1%) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p<0.05). among the cells, gt (69±10%) demonstrated a greater percentage of cells expressing gc-nr than did st (34±5%) or (lb=38±5%) (p<0.05), though there was similar immunoreactive intensities (gt: 0.216±0.017, st: 0.238±0.031, lb: 1802±0.022 au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on 118±2 days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf(1:300-1:500, phoenix pharmaceuticals, ca,) crf binding protein (1:100-1:200, sc 20630) and gr (1:100-1:200, sc: 8992, santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro 4.01 plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf (0.363±0.033 au), crf-bp (0.505±0.003 au) and nuclear-gr (0.383±0.015au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at118 days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn-1), a 40 amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn-1 protein and ucn-1 mrna. a number of published studies indicate that ucn-1 is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn-1 protein and ucn-1 mrna. methods: placenta (n=18) collected from pregnant rats at 21 day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn-1 (1:500 to 1:750, sigma) by standard abc technique. control sections were incubated with omission of ucn-1 antibody. immunoreactive materials on the sections were identified using 3, 3' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn-1 polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: 0.334±0.319 au, spongiotrophoblast cells: 0.318±0.013 au and labyrinth cells: 0.233±0.139 au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< 0.01). pcr analyses revealed the presence of a single band of 379 bp, consistent with ucn-1 mrna. conclusion: similar to human placenta, rat placenta expresses ucn-1 protein and ucn-1 mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from 12 weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from 6 oc and 7 normal pregnancies (np), and 3 human livers were collected and preserved in rnalater. explant cultures were prepared from a further 4 np placentas and cultured for 6 days at 8% oxygen. on day 5 they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for 24 hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l19. results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr3 and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, 1 jenny chambers, 1 jo wyatt-ashmead, 2 kishore bhakoo, 1 catherine williamson. 1 1 imperial college, london, united kingdom; 2 hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects 0.7% of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from 15 oc and 5 normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further 4 np placentas, and cultured for 6 days at 8% oxygen. on day 5 a subset were treated with udca overnight, and on day 6 the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for 6 hours prior to fixing. 5 m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p=0.024). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge 730) placental volume measurement from art pregnancies (n=26; 10-13 wks) were compared with data from normal controls (n=26) matched for gestational age. data were analysed with software stata 9. results. mean placental volume was 46.2 ml (sd±23.9) in art pregnancies and 58.4 ml (sd±22.9) in normal controls. mean difference resulted in 12.12192 ml (-1-25.19835) and was statistically different (p=0.047). multiple linear regression analysis showed a statistically significant interaction (p=0.04) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets (39.1 weeks ± 1.6) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: 2945 g (sd±537) vs 3226.(sd±294.4) (p=0.044). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by 3d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor-1(ad-igf-1) in trophoblast cells. mounira habli, 1 datis alae, 2 foong yen lim, 2 jignesh parvadia, 2 timothy crombleholme. 2 1 obstetrics and gynecology; 2 pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf-1 corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf-1;we examined the effect of ad-igf-1 on trophoblast proliferation, differentiation and invasion. methods: rcho-1 trophoblast cell line derived from rat choricarcinoma was used. ad-igf-1 and galactosidase transgene (ad-lacz) were given at moi of 10:1 and 100:1 in all the experiments.transduction efficiency was assessed 24 hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho-1 was measured by using bdmatrigel invasion chamber kit. rcho-1 proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho-1 differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen-1 antibodies(specific hormone produced by giant cells). results: 100% efficiency of gene transfer of ad-lacz to rcho-1 cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at 12 and 24 hrs. ad-igf-1 induced morphologic differentiation of trophoblast cells compared to control (fig1) at 24 hrs.ad-igf-1 induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group(16% at 100moi vs 0.8% in control p<0.008)(fig2). conclusion: ad-igf-1 gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin-1 is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin-1 action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach3p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons 2-5. the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional 3 4-deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a 3-deletion variant and a novel splice variant, which was identified as partial 2 -partial 3 deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv40-transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin-1 to induce different effects on the two major cell types of the placenta. (supported by grant 11258, jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, 1 katia pacioni, 1 emanuela marinoni, 1 giovanna corona, 1 massimo moscarini, 1 alfredo patella, 2 romolo di iorio. 1 1 department of gynecology, perinatology and child health, university "la sapienza", rome, italy; 2 department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n=7) were cultured in presence or absence of hypoxia (3% o 2 ) and treated with or without betamethasone at the dose of 10 -5 , 10 -6 , 10 -7 m. media and cells were collected at 48h and at 2, 8 and 24h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates 5-8% of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with 95% o 2 /5% co 2 at 37°c. viability was demonstrated by contraction to 110mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf2 , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf2 at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf2 and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in 1,000 pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within 15 minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of 1000 ng/ml and 5000 ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every 10 minutes for total 60 minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p53, bax, bcl-2, aif, and pro-caspase-3) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was 0.67 +/-0.01 ml/min and 1.04 +/-0.15 ml/min at the low and high carboplatin concentrations, respectively. an estimated 50% increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from 60 to 300 ng/ml. the placental perfusion experiments conducted at carboplatin 1000 and 5000 ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy 60 women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= 0.0025) in women delivering preterm (n=20; ucn= 1.23 ± 0.07 pg/ml) (m ± se) than in those delivering at term (n= 40; ucn= 0.77 ± 0.11pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, 1 elisabetta federico, 1 francesca caprio, 1 maria teresa schettino, 1 gabriele coppola, 2 antonio de luca, 2 luigi cobellis. 1 1 department of obstetrics and gynecology, second university of naples, naples, italy; 2 department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n=15), first trimester abortion (miscarriage) (n=15), delivery section (n=15), spontaneous birth (n=15), preeclampsia (n=15) and diabetes (n=15). the gestation period ranged from 5 to 40 weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations (6% 1%) to mimic physiological and non-physiological intervillous o 2 tensions. the d4 media was concentrated, immunodepleted to remove fibrinogen (using beckman igy-12 columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a 4800 maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot 2.0 (applied biosystems). results: when matched pooled (n=6) media samples were subjected to our proteomics workflow over 590 proteins were identified with a calculated false positive identification rate of <1%. of these proteins a total of 29 display a statistically significant difference in protein levels between the 1% 6% o 2 samples (p=<0.001). from a list of 40 proteins of interest we selected 7 proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin 8 is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il-8 (pg/ml) in 6% o 2 : 43.7, range 30.8 to 47.3; 1% o 2 : 103.0, range 57.6 to 141.0; values are median with interquartile range. *** p<0.0001, mann-whitney test (n=40) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., 2007) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c2c12 myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n=8) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg-3) and other cell lines (mrc-5, hl-60, huvec) were cultured in the absence or presence of 20 μm forskolin or solvent control for 0-3 days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg-3), cultured endothelium (huvec), and a fibroblast cell line (mrc-5), but was not detected in a leukemic cell line (hl-60). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin-1 levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of 3 days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, 1 violetta kolesnikova, 1 carri tillotson, 2 jean-marie guise, 3 terry morgan. 1 1 pathology; 2 center for biostatistics; 3 obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta (2001), 22:399-404) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of 242 consecutive singleton placentas submitted to ohsu pathology (2005-06), excluding elective terminations and spontaneous abortions before 22 weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: 1) neonatal sex; 2) weight (iugr calculated by routine methods), 3) trimmed weight of the placenta (sga calculated by routine methods), 4) gross placental infarctions, 5) gestational age at delivery, and 6) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by 2 analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( 2 30.5; p <0.0001). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n=5), f (n=3) and m blood (n=3) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg-6)beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip 6´2 protocol. samples were scanned on the illumina beadarrayer 500gx reader using illumina beadscan image data acquisition software (ver. 2.3.0.13). differential gene expression data analysis was performed using illumina beadstudio software (ver. 1.5.0.34). results. the detection level of gene transcripts using illumina methodology with control human rna was 6000-7000 at p<0.0001. 4329 gene transcripts were detected in f blood and 2957 in maternal blood. 1452 transcripts were uniquely expressed in fetal blood. 130 transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was 4825 and of these 2953 genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of 353/7069 (5%) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, 193(55%) had placental histologic examination performed, and in 50/193 the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score 0 to 3. results: mean interval between meconium appearance and delivery was 136.7± 126.7 min (range 10-510). after exclusion of 6 cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in 40/44 cases at the level of amniochorionic membranes, in 34/44 cases at the placenta, and in 18/44 cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p=0.53), in the placenta (p=0.78), in the cord (p=0.71), or score of severity of meconium uptake (p=0.76). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to 8.5 hours. transfer of bisphenol a across the human placenta. biju balakrishnan, 1,2 kimiora henare, 1,2 eric thorstensen, 1,2 murray d mitchell. 1,2 1 the liggins institute, university of auckland; 2 national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa (10ng/ml), antipyrine (ap) (40μg/ml), and fitc dextran (fitc-dx) (12.5μg/ml) were added to the maternal perfusate, and perfusion was continued for 4 hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the 4-hour perfusion period. the physical parameters observed (fetal pressure <50 mmhg, ph range 7.2-7.6) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of <5ml/hr, and by <2% materno-fetal transfer of fitc-dx. an observed ap transfer of 20-30% further validated our model. bpa first appeared in the fetal compartment within 30 minutes of perfusion and reached a peak of about 25-30% of maternal concentration within 3 hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, 1 katsu naruse, 1,2 barbara a innes, 1 stephen c robson, 1 judith n bulmer. 1 1 institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; 2 obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent (8-10 and 12-14 weeks gestation, n=12 each group). 0.2×10 6 cd56 + unk cells were positively selected from decidua and co-cultured with evt (0.2×10 6) or cytotrophoblast (ctb; 0.2×10 6 ) purified from the same placenta for 24h in direct or indirect contact (n=6 each group). angiogenin, ang2, pdgf-bb, fgf-basic, timp1, icam1 and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang1 and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at 8-10 weeks gestation unk secretion of ang2 (p=0.03), icam1 (p=0.05) and plgf (p=0.03) was increased in the presence of evt compared with ctb. no differences were observed at 12-14 weeks gestation. in addition, at 8-10 weeks gestation unk secretion of ang2 (p=0.004), vegf-c (p=0.007) and ang1 (p=0.004) was increased in direct co-culture with evt compared with indirect co-culture. at 12-14 weeks gestation unk secretion of timp-1 (p=0.03) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. 3-d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a 5% haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between 1 and 10 ml/min, whilst maternal-side flow was held at 14 ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = 0.6hz, wall motion filter = low 1, and gain = 0.0). three 3-d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure 1 ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, 1 brian a kilburn, 1 anelia petkova, 1 samuel s edwin, 2 roberto romero, 2 d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n=6). chorionic villous explants were prepared and cultured at either 20% or 2% o 2 and treated with the hbegf antagonist crm197 or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki67 and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm197 (10 mg/ml) increased trophoblast cell death after culturing villous explants 8 h at 20% o 2 (p<0.05), but only slightly affected proliferative capacity. culture at 2% o 2 increased trophoblast cell death 100% above explants incubated at 20% o 2 (p<0.05). trophoblast cell proliferation decreased after 24 h in explants cultured at either 20% or 2% o 2 (p<0.05). exogenous hbegf (1 nm) prevented the elevation of cell death during hypoxia (p<0.05) and maintained nuclear ki67 expression at 20%, but not 2%, o 2 . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o 2 compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, 1 emily king, 1 carrie tillotson, 2 jean-marie guise, 3 terry morgan. 1 1 pathology; 2 center for biostatistics; 3 obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately 30% of placentas routinely submitted to pathologists (beebe et al. obstet gynecol 1996; 87:771-8) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least 3 hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of 250 consecutive singleton placentas submitted to ohsu pathology (2005-06) was performed, excluding abortions before 22 weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: 1) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), 2) gestational age, 3) maternal diagnosis, 4) apgar scores, and 5) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: 1) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least 1/ hpf), 2) chorioamnionitis, and 3) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x 2 analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common (86/250, 34%) and was significantly more frequent in c-sections performed for fetal distress (19/35 cases, 54%) (x 2 7.1; p-value <0.01). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams 50% food-restricted from day 10 of gestation. placentas were harvested at embryonic days 16 and 20 (n=3). placentas were fixed in 4% pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at 1:100 dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated 9.29% relative apoptotic index when compared to 2.95% in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of 0.112 + 0.025 in food restricted placentas as compared to 0.049 + 0.003 per μm2 surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e16 than at e20, with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, 1 avish arora, 1 lilit baldjyan, 2 sharon k sugano, 1 olga miadel, 2 adegoke adeniji, 2 michael g ross, 1 calvin j hobel. 2 1 ob-gyn, harbor-ucla medical center, torrance, ca, usa; 2 ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a 37kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the 37kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm (28 to <37 weeks, n=6) umbilical cord (6 pieces of 3mm thickness taken at 1-2 cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at -80c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong 50kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of 37, 25, 20kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the 50kda major band is either glycosylated crf-bp precursor or glycosylated 37kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature 37da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a1, a2a, a2b, and a3. recently our study has shown that increased a3 receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a3 receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a3 receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n=10) and preeclamptic (n=10) pregnancies were cultured at high (20%) and low (3%) oxygen levels for 5 days. explants were analyzed for mmp-2/-9 and timp-1/-2 by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a3 receptor agonist, cl-ib-meca and a3 receptor antagonist, mre. mmp-2/ -9 expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp-2/-9 activity was evaluated by zymogram assay. results: there were significantly increased a3 receptor intensity and reduced mmp-2/-9 and timp-1/-2 expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp-2/-9 and timp-1/-2 expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp-2/-9. this cl-ib-meca-induced expression of mmp-2/-9 was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a3 receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a3 receptors in the trophoblast and suggest that adenosine a3 receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, 1 timothy a sato, 1 anderson wang, 1 jeffrey a keelan, 1 anna p ponnampalam, 1 michelle glass. 2 1 liggins institute; 2 department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids 2-arachidonoyl glycerol (2ag) and anandamide (aea) and with the synthetic cannabinoid cp55,940, to determine the ability of these substances to modulate prostaglandin e 2 (pge 2 ) production. pge 2 was measured by radioimmunoassay. the explants were also treated with cp55,940 in the presence of either sr141716a (a selective antagonist of the cannabinoid receptor cb1) or ns398 (a cox-2 inhibitor), to determine whether any observed stimulation of pge 2 production was mediated through cox-2 activity and/or the cb1 receptor. cox-1, cox-2, cpla2 and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge 2 production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge 2 production caused by cp55,940 was abrogated by co-treatment with either sr141716a or ns398, illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb1 agonism and cox-2. preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox-2 expression. however, there was no change in cox-1 expression and no evidence either for up-regulation of cpla2 or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor-2, but not vegfr-3 or vegfr-1, during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, 1 hongyan tang, 1 raul gomez, 1 bronislaw pytowski, 2 daniel j hicklin, 2 jan kitajewski, 1 mark v sauer, 1 ralf c zimmermann. 1 1 division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; 2 imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr-1, r-2, and r-3 are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr-1, r-2, and r-3 function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed 3.75, prior to embryonic expression of these receptors. embryonic development was evaluated on ed 10.5, blood vessel density and apoptosis on ed 7.5, and cellular proliferation on ed 5.5 (n=5 per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr-2 and vegfr-3, but not vegfr-1 resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr-3 mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr-2 pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed 10.5 control anti r-1 anti r-2 anti r-3 no. of implantation sites 10.7 +/-1.2 11 +/-0.9 11.0 +/-1.1 14.0 +/-0. hoxa10 encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa10 in multiple cellular model-systems. using microarray technologies, we identified putative hoxa10 target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa10 was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified 82 statistically-significant genes regulated by hoxa10 overexpression of which 57 genes were down-regulated greater than 2-fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the 3-phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa10 during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a 2fold expression decrease when hoxa10 is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to 40% when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa10 is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa10 is also expressed in leukocytes. here we hypothesized that hoxa10 would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa10 in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa10 mrna in u937 cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa10 is expressed in leukocytes in vivo, peripheral leukocyte hoxa10 mrna expression was measured over sequential estrus cycles in mature cd1 nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa10 mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa10 mrna expression levels in u937 cells. in a murine model, we demonstrated that hoxa10 mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa10 expression is prior to proestrus and increased up to 2-fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa10 mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa10 expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa10 transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa10 expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa10 mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods 210 women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. 16 known soluble mediators of implantation were measured using a multiplex immunoassay, namely il1ß, il5, il6, il10, il12, il15, il17, il18, tnf , vegf, ifn , eotaxin, mcp-1, ip-10, dkk-1 and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p<0.1) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the 17 analysed were detectable in 90-100% of the samples. il15 was detectable in 76% of samples, dkk-1 68%, il-10 56%, il-17 54%, il-5 35% and hbegf was detectable in 23% of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p =0.036). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n=12) were treated with daily leuprolide (1.0 mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, 0.2 mg/d) for 20 days. after day 10 of e, subjects also received daily i.m. injections of p, randomized to 10 mg/d (sub-physiological) or 40 mg/d (physiological). endometrial biopsy was performed after 10 days of combined e and p treatment. additional untreated women had biopsies performed 10 days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the 40 mg/d p group were 18.2(5.1) and 9.4(4.8) ng/dl, respectively, while those in the 10 mg/d group were 7.0(3.0) and 3.4(1.0) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was 25.7(0.8), 24.4(0.9), and 24.8(1.3) for the 10 mg, 40 mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, 3 integrin subunit, osteopontin, cyr61, egr-1, fkb52, c-fos, and cd55), although variability of gene expression was greater in those who received 10 mg/d p than in those who received 40 mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u54 hd-35041). endometrial ip10 attracts trophectoderm through cxcr3 interaction. simcha yagel, 1 caryn greenfield, 1 hen sela, 1 jacob hanna, 2 irit manaster, 2 orna singer, 3 ronit haimov-kochman, 1 shira natanson-yaron, 1 diana prus, 1 benjamin reubinoff, 2 debra s goldman-wohl, 1 ofer mandelboim. 2 1 obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; 2 lautenberg center for immunology, jerusalem, israel; 3 human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and 1 st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec-1 cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr1 and cxcr3. immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr3 and cxcr1 as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp-4 and jeg cells, and blastocyst trophectoderm expressed principally cxcr3. elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip10 and il8. migration assays demonstrated that ip10 significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr1 and cxcr3, and il8 and ip10. these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip10-cxcr3 is the most relevant to early implantation as only cxcr3 is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. 2d page analysis with dige minimal dye labelling was conducted across the pi range 4-7 on endometrial tissue from either the mid-proliferative or midsecretory phases (n=4/group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of 1010 spots were detected: 196 were differentially expressed (p < 0.05) with 17 spots having an overall false discovery rate <5% (q <0.05). hierarchical clustering analysis revealed that these 17 proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural (7), transport (4), regulatory (2), membrane (2), enzyme (2), motor (1) and others (2). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, 1 hn jabbour, 2 p lourenco, 1 s wright, 2 s battersby, 2 arw williams, 1 hod critchley. 1 1 reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; 2 mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n=18) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin 1 (pk1) and prokineticin 1 receptor (pkr1) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk1 and pkr1 protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk1 and pkr1. conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e2) inhibits glycogen synthase kinase 3 beta (gsk-3 ), resulting in nuclear accumulation of cyclin d1 and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk-3 inhibitor, licl, results in epithelial cell proliferation in the absence of e2 (zhu, pollard. pnas. 2007; 104:15847) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged 18-45. immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after 6 weeks of out-growth, mice were ovariectomized, and replaced with e2 or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: 40-50% of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst 33258 staining confirmed that xenografts were made up of human cells ©. e2 (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki67, with li of 25.9±1.3 and 8.8±3.3, respectively, p =0.02). minichromosome maintenance-2, a protein involved in dna replication licensing, was more sensitive for e2-treated cells synthesizing dna than was ki67 staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e2. licl (f) induced proliferation similar to e2 and greater than control (li =18.4±6.4, p= 0.2 for e2 vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e2 induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for 24h. the cells were treated with estradiol (e 2 , 10 nm, 24h), progesterone (p4, 10 nm, 24h), gnrha (la 1 μm, 24 h), e 2 (12h) followed by p4 (last 12h) and gnrha plus e 2 plus p4 where, gnrha added either first, second or last in order at 8h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il-1 , il-1 , il-2, il-4, il-5, il-8, il-10, il-12p35, il-12p40, il-15, ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il-2, il-4, il-5 and ifn-. treatment with e 2 and p4, either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e 2 and p4 significantly diminished e 2 , p4 or e 2 plus p4 induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd37432. introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a 230 kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor-2 function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, 1,2 cobi heijnen, 1 esther baart, 1 karima amarouchi, 1 carolien boomsma, 1 janet carver, 2 helen mardon, 2 annemieke kavelaars, 1 nick macklon. 1 1 reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; 2 nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: 97 human embryos, cryopreserved at day 4 after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from 18 donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day 8, the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il-1 , il-5, il-6, il-10, il-12, il-15, il-17, il-18, tnf-, mcp-1, ip-10, eotaxin and hb-egf using a multiplex immunoassay. day 8 supernatants from 29 culture systems in which no embryo had been placed were also analysed as controls. results: 11 out of 42 (26%) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day 8 and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod 2007), we pre-treated human ishikawa cells with or without ovarian steroid hormones (17 -estradiol + progesterone; ep), fa-5 (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa-5 resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and 2-arachidonoyl glycerol (2-ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and 2ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n=43). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn-1-diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for 2ag; and monoacylglycerol lipase (magl) and cyclooxygenase-2 (cox2), the degradative enzymes for 2ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, #10005430). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and 2ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p=0.004) was seen. magl gene expression was higher in the secretory phase than the pro phase (p=0.004). cox2 gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p<0.05 for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, 2ag's degradative enzymes increase in the secretory phase, suggesting that lower 2ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin-1 beta (il-1 ) regulates il-6 signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, 1 cf yen, 2 cp chen, 3 f schatz, 1 cj lockwood. 1 1 obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; 2 ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; 3 ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il-6 levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il-6 contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il-6 promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il-6 may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: 1) localized il-6 mrna levels in preeclamptic versus normal decidual sections; 2) evaluated mechanisms regulating il-6 synthesis by targeting intracellular signaling pathways with specific inhibitors; 3) identified potential il-6 targets by immunolocalizing the il-6 receptor (il-6r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il-6 mrna in normal versus preeclamptic decidua. il-6r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e2 and mpa ± il-1 (1 ng/ml) ± an inhibitor of p38 mapk (sb203580) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for 24 hrs. an elisa measured secreted il-6 levels. results: il-6 mrna was present primarily in decidual cells with increased il-6 mrna levels observed in pe. preferential expression of the il-6r was observed on decidual cells in placental bed biopsies. compared with basal il-6 levels (0.22 ± 0.14 pg/ml/ g cell protein) by decidual cells, il-1 enhanced il-6 output by decidual cells (364.55 ± 160.29 pg/ml/ g cell protein). only the p38 mapk inhibitor significantly reduced this output to 39.50 ± 8.09 pg/ml/ g cell protein (n=4, p<0.05). our results indicate that inflammatory cytokine enhances il-6 synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p38 mapk. such il-6 is likely to act as an autocrine/paracrine effector via decidual cell-expressed il-6r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, 1 shira natanson-yaron, 1 caryn greenfield, 1 achinoam lev-sagie, 1 lichtenstein michal, 2 haya lorberboum-galsky, 2 israel vlodavsky, 3 simcha yagel, 1 arye hurwitz. 1 1 ob/ gyn, jerusalem, israel; 2 cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; 3 cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec1a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased 100fold in secretory phase (d21) compared to proliferative phase (d10) endometrium. heparanase transcript levels increased 14fold during 8 hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec1a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin-18 (il-18) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, 1,2 tien-hung huang, 2 chin-jung li, 1 chyi-long lee, 1,2 hsin-shih wang, 1,2 yung-kuei soong. 1,2 1 obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; 2 obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il-18 system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il-18 system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n=7) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n=4) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh (94 bp), il-18 (144 bp), il-18bp (188 bp) and il-18r (307 bp). quantitative il-18 and il-18bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il-18 system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il-18 and il-18bp as well as il-18r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and 2 -ct method, a significantly higher il-18 expression in tubal ectopic implantation and lower ratio of il-18 antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p<0.05). immunoreactive il-18 system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il-18 system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like 3 (fstl3). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl3 in ovariectomized rats. adult female wistar rats (n=21) were ovariectomized and received one week later a single dose of estradiol benzoate (1.5 mg/kg body weight, i.m. injection), either alone (n=7) or associated with depot medroxyprogesterone acetate (2.4 mg/kg body weight, i.m. injection, n=7), or oil vehicle (control group, n=7). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p0 (rpp0) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone (7.4 fold increase over controls, p<0.05) and to the same extent in rats receiving estradiol plus medroxyprogesterone (6.1 fold increase over controls, p<0.05). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone (0.5 fold compared to controls, p<0.05) and estrogen plus progestin (0.4 fold compared to controls, p<0.05), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl3 expression was similar in the 3 groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, 1 irit manaster, 2 jacob hanna, 2 ronit haimov-kochman, 1 miri godin, 2 yuval bdolach, 1 caryn greenfield, 1 shira natanson-yaron, 1 arye hurwitz, 1 debra s goldman-wohl, 1 ofer mandelboim. 2 1 obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; 2 lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp44, nkp30 and nkp46, collectively known as natural cytotoxicity receptors (ncrs), and nkg2d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days 10 and 21. a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd56, cd16, nkp44, nkp30, nkp46 and nkg2d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd2-ig and nkg2d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il-8 with and without il-15 were performed. results: endometrial lymphocytes of day 10 and 21 in these women are mostly cd56 bright cd16-nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp30, nkp44. nkp46 and nkg2d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp30, nkp44 and nkg2d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, 1 amy o robb, 1 imogen b smith, 1 nicholas l mills, 2 hilary od critchley, 1 david e newby. 2 1 centre for reproductive biology; 2 centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: 10 healthy, nulliparous, pre-menopausal, non-smoking women (mean age 31 years), with regular menses (27-29 days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days 1-3, 6-9, 13-15 and 20-22). cpmas (monocytes positive for the platelet marker cd42a) were measured by flow-cytometry. psp-selectin expression was calculated on cd42a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was >30 nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle (25.59±2.15 vs. 16.65±2.88%, p=0.01 and 3.95±0.77 vs 2.10±0.37%, p<0.05, respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p=0.8). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during 1 st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd34-immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt-1, kdr, angiopoietin-1, angiopoietin-2, tie-2 and the membrane-type matrix metalloproteinases mt1-, mt2-, mt3-and mt5-mmp were determined at mrna and antigen level and cd56-positive unk cells, cd68-positive macrophages, proliferation (ki67) and apoptosis (activated caspase-3) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt1, kdr, mt2-and mt5-mmp was increased at the implantation site of cases. ki67 and active caspase-3 showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, 1 hong y wen, janice endsley, 2 alison hogg, 2 hui-qun wang, 3 manubai nagamani, 1 chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd56 decidual nk cells were purified by magnetic bead isolation. results: 1) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; 2) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd56 staining, express adm receptor components crlr/ramp 2 /ramp 3 and their mrna expressions were futher confirmed by rt-pcr; 3) k562 target cell killing assay indicates that adm inhibits cytokine il-12/il-15-induced decidual nk cell cytotoxicity; and 4) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at 12-14 weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at 12-14 weeks gestation (not at 8-10 weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd56 + unk cells were isolated from early pregnancy decidua (8-10 and 12-14 weeks gestation, n=10 each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid 721.221 transfected with either hla-g1 (221g) or a mock cdna (221cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n=5 each group and each gestational age) for 24 hours. cell supernatants were analysed for cytokines using a fastquant® th1/th2 multiplex protein assay (il-6, il-1 , il-10, il-2, il-5, tnf-, il-13, il-4, ifn-) or by standard elisa (tgf-1). the effect of direct co-culture of unk cells with 221g compared with co-culture with 221cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with 221g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the 221g or 221cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either 221g or 221cdna cells at either gestational age. in addition, direct or indirect co-culture of 221g or 221cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either 8-10 or 12-14 weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg2d receptor pathway plays a fundamental role in target elimination through binding nkg2d ligands on the cell surface. reduction in the nkg2d ligand, ulbp2, expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg2d pathway in this process. material and methods: expression of nkg2d ligands; ulbp2, ulbp3, mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd56+cd3-) from three healthy volunteers were isolated using macs system with purity greater than 90% and kept in interleukin 15 overnight. fibroblast elimination with and without ulbp2 blocking was investigated following 3-hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg2d ligands (ulbp2, ulbp3, mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp2 expression (p = 0.039). western blot analysis also revealed a lower ulbp2 protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was 20% lower in at in comparison with np fibroblasts. blocking ulbp2 expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by 27%, supporting the role of nkg2d receptor pathway in the process. conclusions: our results demonstrate that nkg2d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin-1 is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin-1 is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals1 gene in primates. methods: seven primate nucleotide sequences were generated, aligned to 27 vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin-1 expression was studied by immunohistochemistry and western blot. results: 1) the lgals1 gene had high sequence identity among all investigated species. 2) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals1 gene in placental mammals (dn/ds=0.11); 3) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. 4) immunostaining showed a uniformly abundant and ubiquitous galectin-1 expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals1 gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin-23 in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, 1,2 william murk, 1 umit a kayisli, 1 aydin arici. 1 1 department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin-23 (il-23) is a recently discovered heterodimeric cytokine, comprised by a novel p19 subunit and a p40 subunit shared by il-12. it has biological activities that are similar to but distinct from il-12, and is known to be involved in th1/th2 cell class switching and the regulation of cytokines such as ifn-gamma, il-12, tnf-alpha, and il-17. early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il-23 in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n=44) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n=8) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il-23p19 antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p<0.05 considered significant. results: il-23 immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il-23 immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il-23 staining compared to escs from non-pregnant endometrium (p<0.001). il-23 immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p<0.001). moreover, egcs from the early secretory phase (p<0.05) and first trimester tissues (p<0.01) showed higher il-23 immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il-23 expression in the human endometrium and decidua. these results suggest that il-23 has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il-2 production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat 6.1 t cells, initially grown in estrogen free media, were incubated in 4pm (representing postmenopausal levels) or 40pm (premenopausal levels) of estradiol (e 2 ) for 48 hours. cells were either resting or activated with a phorbol ester, 4 -phorbol 12 -myristate 13 -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il-2. expression of signaling protein components, cd3 and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd3 , jak2, and jak3 gene expression. a p value of <0.05 was considered significant. results: jurkat cells exposed to 4pm e 2 and activated exhibited significantly diminished numbers of il-2 producing colonies compared to t cells exposed to 40pm (55.7±1.2 vs. 75.3±0.7 colonies, p<0.0001). analysis of cellular cd3 and jak2 protein demonstrated that jurkat cells incubated in 4pm e 2 expressed a 1.48-fold decrease in cd3 and 1.64-fold decrease in jak compared with cells incubated in 40pm e 2 (p<0.001). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd3 , jak2 and jak3 were significantly decreased in jurkat cells incubated in low levels of estrogen (11.3, 213.3, and 13.3 fold, respectively, compared to 40pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il-2 following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd3 , jak2 and jak3 gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of 2 novel cytokines (il-24 and 29) in human fetal membranes. judith eckardt, 1,2 stephen j fortunato, 1 holger maul, 2 ramkumar menon. 1 1 the perinatal research center, nashville, tn, usa; 2 womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) 24 and 29 are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il-24 and il-29 in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n=12) from cesareans at term (normal, not in labor) were placed in an organ explant system for 48 hours and were stimulated with lps for an additional 24 hrs. fetal membranes were also collected (n=10) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n=200) were collected to document the role of il-24 and il-29 in ptb. tissue expressions of il-24 and il-29 were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il-24 and il -29 expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il-24 after lps stimulation (lps-65.5 vs. 7.7 pg/ml; p=0.006) whereas no difference was noticed in il-29 concentration (lps-22.8 control-19.5 pg/ml; p=0.5) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit 7.65 pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il-24 production was stimulated by lps whereas il-29 was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il-24 concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd4 + cd25 bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, 1,2 dave l roelen, 2 barbara j van der mast, 1 godelieve m de groot, 1 carin kleijburg, 1 sicco a scherjon, 1 frans hj claas. 2 1 department of obstetrics, leiden university medical centre, leiden, netherlands; 2 department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd4 + cd25 + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd4 + cd25 bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy (1) . in this study we examined the phenotypic and functional properties of cd4 + cd25 bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd4 + cd25 bright t cells from maternal peripheral blood demonstrates regulation to a 3rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd4 + cd25 bright foxp3 + and cd4 + cd25 bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd4 + cd25 bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. (1) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd4 (+) cd25 (bright) and cd8 (+) cd28 (-) t-cells in decidua and maternal blood during human pregnancy. placenta. 2006 apr;27 suppl a:s47-53. 3 alicia del toro-arreola, 2 lourdes nunez-atahualpa, 4 juan velazquez-rodriguez, 5 laura gonzalez-lopez, 6 jorge i gamez-nava, 3 adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to 10 women not using hormonal contraceptives in the day 5th of the follicular phase and 21st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= 0.802, p<0.05 and rho= 0.757, p<0.05, respectively) with cd94/nkg2 in t cells and negative correlated (rho= -1.00, p<0.01 and rho= -0.857, p<0.05, respectively) with nk cells. meanwhile cortisol was positive correlated (rho= 0.857, p<0.05) with the receptor nkg2d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd94/nkg2 inhibitor receptor and nkg2d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: 1) term not in labor (tnl; n=20); 2) term in labor (til; n=31); 3) preterm labor without histologic chorioamnionitis (ptl; n=30) and 4) preterm labor with histologic chorioamnionitis (ptl-hc; n=27). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: 1) the af sp-d concentration increased as a function of gestational age (mean, til: 52,055.7 ng/ml vs. ptl: 31,371.0 ng/ml; p<0.05); 2) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: 375.8 ng/ml vs. ptl: 672.9 ng/ml; p<0.05, and til: 366.9 ng/ml vs. ptl: 1606.8 ng/ml; p<0.001, respectively); 3) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: 506.1 ng/ml vs. 1054.4 ng/ml; p<0.001); 4) however, sp-d mrna expression in maternal leukocytes was 4.8 fold higher than that of fetal leukocytes (p<0.001); 5) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: 1) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; 2) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; 3) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from 2004 to 2006 675 patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by 65.8% of the couples. the global pregnancy rate was 15.4%. the pregnancy rate for the couples who had intercourse was 13.3% and 19.5% for those who did not have intercourse (p<0.05). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p<0.01). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n=463) was similar (16.1% vs 19.6% for positive and negative intercourse, respectively). the average number of ovulatory follicles was 2.12 + 1.1 for the group that had intercourse and 2.17 + 1.3 for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost 66% of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were 35 subjects with 73 observations. for the fmc analysis, there were 68 subjects with 145 observations. table 1 provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida 1 gravida 2 or more adjusted* or (95%ci) mmc 7/14 (50%) 5/48 (10%) 0.19 (0.04-0.79) fmc 13/30 (43%) 36/115 (31%) 0.49 (0.21-1.17) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th1/th17 gene expression. tommie g nguyen, 1 eileen d gallery, 1,2 jonathan m morris. elevated t-helper cell type-1 (th1) and type-17 (th17) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th1/th17 responses toward a th2 response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th1/th17 gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for 5 hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j1 mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given 3 daily doses of 10 mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = 10 per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th1 immunity), tnf-(a classical pro-inflammatory th1 cytokine), and il-17 (a critical proinflammatory th17 cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il-2 (two classical th1 cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group (7.8 ± 1.1, mean ± s.e.m), preventive group (5.4 ± 1.2) and therapeutic group (3.5 ± 1.3) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il-17 in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg1 anti-human beta 2 -glycoprotein i monoclonal antibodies (mabs), designated id2 and iic5, were used in these studies. the first trimester trophoblast cell line, htr8, was incubated with either medium, a mouse igg1 control, id2 or iic5 (5-40 g/ml), in the presence or absence of unfractionated heparin (100ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a 48 hour incubation, significant trophoblast cell death was induced by iic5 (34.5 ± 2.8%) and id2 (46.4 ± 3.2%) at the high dose of 40 g/ml, when compared to the medium and mouse igg controls (p<0.001). hoechst staining showed that id2-and iic5-induced trophoblast cell death was a result of apoptosis. moreover, id2 and iic5 induced a significant increase in caspase-3, -8 and -9 activity (p<0.001). treatment of trophoblasts with heparin significantly inhibited the effects of iic5 and id2 on cell death by 46.5 ± 12.8% and 52.6 ± 8.6% %, respectively (p<0.005). following a 72 hour incubation at lower concentrations (20 g/ml), treatment of trophoblast cells with id2 or iic5 resulted in a significant upregulation of il-8, mcp-1, gro production (p<0.05), and a significant reduction in il-6 secretion (p<0.05). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of 40, affecting approximately 1% of women <40 years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n=6), pcos (n=4) and pof (n=5). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common 6 antigenic proteins (row 2, antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of 25% annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd3-and jak3 were tested. these markers were chosen since they are central to t cell signaling. cd3-is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak3 upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat 6.1 (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for 72 hours. these cells were then exposed for 48 hours to 0 pm, 4 pm (postmenopausal), or 40 pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein (0, 0.4, 1.5, 6.25, 25, and 100 m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd3-and jak3 expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd3-reaching a maximum of 20 fold. although cultivation of t cells in 40 pm of estradiol significantly increased the levels of cd3-and jak3 relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il-6 as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, 1 juan deleon-luis, 2 isabel galan. 2 1 obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; 2 amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at 36-44 days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and 24 hours after celocentesis for quantification of hormones and il-6 using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p<0.05). results: all 9 animals survived their pregnancies. seven animals receiving <5% concentration of nk delivered at term ( 180 days gestation) while 2 animal receiving more than 7% concentration of nk had dead fetuses on ultrasound evaluation 24 hours after celocentesis. table 1 depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure 1 shows mean il-6 changes from baseline in continuing (n.s.) and rejected pregnancies (p<0.05). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il-6 might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis (9) the activity of cytotoxic cd8+ t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd8+ t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd8+t cells. methods: female c57bl/6 mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd8 and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd8+tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day 5 of pregnancy (n=5), the number of brdu positive cd8+ t cells was two fold higher than that found in um (n=12, p<0.01). the number of proliferating cd8+ t cells continued to be non-significantly elevated at day 8 (1.6x, n=5), day 10 (1.4x, n=7), and day 12 of pregnancy (1.5x, n=6) compared to um. by day 15 of pregnancy (n=5) the number of proliferating cd8+ t cells returned to the um level, however by this time the total number of splenic cd8+ t cells was 1.5 fold higher than um (n=12 p<0.001). on gestational day 18, the number of proliferating cd8+ t cells declined further (0.3x, n=4), and the number of splenic cd8+t cells returned to the um level (n=12, p>0.05). compared to um mice, there was no significant difference in the number of cd8+ t cells undergoing apoptosis at any gestational day examined (0.7-0.9x, p>0.05). in normal murine pregnancy, the number of cd8+ t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd8+ t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro1hd043-185 niht32ai055402. arturo cerbulo-vazquez, 1 cun li, 1,2 gene hubbard, 2 natalia e schlabritz-loutsevitch, 1,2 peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf-1 (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at 0.6, 0.65, and 0.7 gestation (g) alters fetal lymphocyte populations at 0.95 (g) (j repro immunol, 2006, 69:149) . we determined if fetal gc exposure alters thymic 1) structure; 2) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n=6) or betamethasone i.m. (175 μg/kg daily for two days at 0.6, 0.65 and 0.7g; gc group; n=6), c-sectioned at at 0.9g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf-1, b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c57bl/6 (b6) or recombinase deficient c57bl/6 mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at 2 and 6 hours after lps injection in pregnant b6 and rag-ko mice. methods: b6 and rag-ko mice were mated and on gestation day 15, females were injected intraperitoneally with 3μg lps in 200 l saline (pbs) or 200 l pbs alone. euthanasia and uterine harvest occurred 2 or 6 hours after injection. frozen uterine sections were stained with the macrophage marker f4/80 or an isotype-matched control followed by an alexafluor 546-conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f4/80+ dapi+ and total dapi + cells were counted in 3 areas of 1 representative section and the percentage of f4/80+ dapi+ cells was calculated. the mean percentage for at least 6 representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b6 and rag-ko mice injected with pbs (b6, n=3, 2.9±1.6; rag-ko, n=3, 2.7 ± 1.2 % + per area). lps injection increased macrophages (p<0.05) in both strains (b6, n=3, 4.4±2.9; rag-ko, n=3, 5.5 ± 1.8,); no difference was evident between strains. the percentage of f4/80+ cells was similar 6 hours post-injection (b6, n=3, 3.4± 1.9 v. rag-ko, n=3, 3.6± 2.8), and not elevated relative to the 2 hour time point. objective. decay-accelerating factor (cd55), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide 3-kinases (pi3ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins (3,4,5) p3. akt, a serine-threonine kinase acts downstream of pi3k and regulates cell survival, growth and proliferation. the pi3k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi3k-akt activity affects the expression of cd55 in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi3k activity were used in this study. ishikawa and rl95-2 endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec-1-a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd55 was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi3k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi3k-akt pathway on cd55 expression was evaluated in cells treated with wortmannin (400 nm), ly294002 (25 mm), or with akt inhibitor sh5 (5 mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd55 expression correlates with the status of pi3k-akt pathway. the cd55 expression was lowest in hec-1-a, ishikawa and rl95-2 cells which constitutively express p-akt. higher cd55 expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were 3.4-fold higher for kle cells and 12-fold higher for hela cells compared to hec-1-a cells. treatment of cells with akt inhibitor led to 1.1-1.4-fold increase in cd55 expression. the 1.1-5.7-fold increase following treatment with pi3k inhibitor wortmannin was found in ishikawa cells, rl95-2 and kle cells. conclusions. human endometrial cell lines with elevated pi3k-akt activity express lower level of cd55 compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi3k-akt pathway are involved in the downregulation of cd55. endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase-9. while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl95-2, hec-1-a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, (1ug/ml) or nocodazole (1mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin 18 mouse antibody followed by anti-mouse alexa 488 conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa 549 conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa 488 and alexa 459 were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec-1-a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin-18 and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl95-2 cells (0.87) followed by ishikawa (0.79) and hela cells (0.72). lowest correlation was found for kle (0.65) and hec-1-a cells (0.59) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase-9 and caspase-3 in the early apoptotic stages. background: cd4+ cd25+ t regulatory cells (t-reg), express foxp3,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd4+ cd25highfoxp3+) was assessed in the peripheral venous blood of 25 non-pregnant, 63 pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n=26), second (n=16) and third (n=5) trimester of human pregnancy. paraffin sections were immunostained for foxp3 and cd25. foxp3 +cells were quantified in 5 x400 fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd4+ cd25highfoxp3 t-cells in pregnant (3.06% [range 1.21-4.8%]) vs. non-pregnant controls (2.58% [range 1.1 -5.03%]; p<0.05). a progression from 1st, 2nd and 3rd trimesters indicated the % of cd4+ cd25highfoxp3 t-cells was 3.01%, 2.88% and 3.14%, respectively. low numbers of foxp3+ cells were detected in all decidua samples and their distribution mirrored that of cd25+ cells. in 1st trimester samples, foxp3+ cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp3+ cells were detected in 1st (21.9±4.3) compared with 2nd trimester decidua (9.9±3.2; p<0.05) but there was no difference between 1st and 3rd trimester (12.0±3.1), nor between 2nd and 3 rd trimester decidua. in 1 st trimester decidua, numbers of foxp3+ cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd4+ cd25highfoxp3 t-cells. the presence of foxp3+ cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr2 and tlr4, whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin 2 release by keratinocytes, via tlr2 and tlr4. methods: the induction of -defensin 2 production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr2 and tlr4 in -defensin 2 production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin 2 production following hyaluronic acid treatment in human keratinocytes. the -defensin 2 production induced by hyaluronan was abolished following block of tlr2 and tlr4 by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il-8, tnf-, il-1 and il-6. conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin 2 production in keratinocytes, via tlr2 and tlr4. this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, 1 serena wu, 1 mary d stephenson. 1,2 1 obstetrics and gynecology, university of chicago, chicago, il, usa; 2 obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between 1992 -2004 (stephenson, 1996 . couples who met aps criteria (wilson et al, 1999) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from 1999-2004. secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies 24 weeks, regardless if they ended in success or demise. pearsons 2 test with yates continuity correction was applied. results: 388 subjects were identified. 226 subjects had 319 prior live births of known gender (174 male/145 female), giving a ssr of 1.20. there were also 25 prior fetal demises 24 wks of known gender (13/12) giving a sex ratio for all prior pregnancies at 24 wks of 1.19. 252 subjects delivered 301 subsequent live births of known gender (158/143), giving a ssr of 1.08. there were also 6 subsequent fetal demises (3/3) giving a sex ratio for all subsequent pregnancies of 1.10. 112 subjects delivered both prior and subsequent live births. the ssr was 1.36 (83/61) among their prior and 1.24 (68/55) among their subsequent live births. including fetal demises, 119 subjects had ongoing prior and subsequent pregnancies. the sex ratio was 1.32 among their prior pregnancies and 1.10 among their subsequent. as the control, a ssr of 1.06 (124,982/118,093) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than 30 passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p26-34) of ultr and ultr-ht (p31-38) cells were grown on either plastic or collagen iv (cn4)-coated surfaces. ultr-ht cells were further stimulated with angii (0.1 um) for 24 hrs. expression of nadph oxidase core (nox1-5 and duox1/2) and associated subunits (p22phox, p47phox, noxo1, p67phox, noxa1, p40phox, and rac1/2), and angii receptors at1/2 was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox1, p22phox, and noxa1 genes, and an up-regulation of at1/2. overexpression of htert did not reverse nox1, p22phox and noxa1 expression while cell division rate was increased. however, there was a down-regulation of nox4, at1/2 and rac2. plating ultr-ht cells on cn4 induced nox4/5 down-regulation and up-regulation of duox1/2, with no apparent change of at1/2. however, exposure to cn4 re-directed cellular response to angii such that only nox5 was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox1, 4, 5, and duox1/2 are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox5 mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, 1 masahiro nakayama. 2 1 women's health, national center for child health and development, tokyo, japan; 2 aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under 34 years old, and the old group over 35 y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p<0.05. [results] 1) srage in plasma, 1517±642 pg/ml (mean±sd), n=40 in the young group was significantly higher than 1268±530 pg/ml, n=51 in the old group. there was no significant difference in plasma vegf. 2) vegf in follicular fluid was 45±22 pg /mg protein, n=37 in the young, and 56±27 pg /mg protein, n=63 in the old was increased significantly. 3) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir-21 which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir-21 in lsmc, msmc, t-lsmc and sklm. we found that mir-21 is expressed and regulated by 17b-estridiol and medroxyprogesterone acetate (10 -8 m) in these cells (p<0.05). we further assessed the regulatory function of gain of and loss of function of mir-21 on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir-21 oligonueclotides resulted in a significant increase and/or inhibition of mir-21 expression in these cells, respectively as determined by realtime pcr (p<0.05). over-expression of mir-21 resulted in a significant reduction, while transfection with antimir-21 increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p<0.05). we concluded that mir-21 is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd37432). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin 1 (ucn1), ucn2, ucn3 and crf -r1 and crf-r2 receptor subtypes together with crfbinding proteins locally. ucn2 and ucn3 are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro 4.01 in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens (3-4mm thickness, 6 pieces per umbilical cord) collected at preterm and term (n=6 each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf (1:300), ucn1 (1:250), ucn2 (1:250) and ucn3 (1:250) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using 3, 3'-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro 4.01 software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= 0.0451±0.0043 vs crf-t= 0.1027± 0.0242 (p<0.5); ucn1-pt=0.1736 ± 0.0108 vs ucn1-t= 0.0959± 0.0116 (p<0.01); ucn2-pt= 0.0964± 0.0063 vs ucn2-t= 0.0724 ± 0.1632 (p=ns); ucn3-pt= 0.1214 ± 0.0034 vs 0.0732± 0.0050 (p<0.05). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn1 and ucn3 contents significantly decreased and no significant change occurred in ucn2 content. based on the opposing changes in crf vs ucn1 and ucn3 immunostaining, we speculate that crf, but not ucn1 or ucn3, is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn1 and ucn3 may perform cytoprotective functions at preterm. physiol. 1947; 150: 511-519, am j physiol. 1952; 170:72-76) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr (40%, nia protocol as described in j geront. 1999 54a:b492-b501) was initiated in c57bl/6 female mice at 4 months and continued until 15.5 months of age, at which time al feeding was resumed. matings were initiated at 10 months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day 21) were recorded. results: fertility was lost in 3 of 11 al-fed females by 10 months of age and continued to decline through 15.5 months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of 9 pregnancies achieved out of 12 females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at 15.5 months of age. while only 1 out of 8, al-fed females achieved a pregnancy between 15.5-18.5 months of age, 6 out of 10 age-matched cr-then-al-fed females achieved a total of 10 pregnancies in this 3-month time. notably, 79% of the pups born to cr-then-al-fed females between 15.5-18.5 months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r37-ag012279). bile acids in human ovarian follicular fluid. laura p smith, 1,2 kaila deiorio-haggar, 1 jason reindollar, 3 alan s penzias, 1,2 anny usheva-simidjiyska. 3 1 ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; 2 boston ivf, boston, ma, usa; 3 endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and 36 hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from 0 to 15 micromoles/l. bile acids are present in follicular fluid with a mean concentration of 15.28 micromoles/l in large follicles and 14.22 micromoles/l in small follicles (p=0.108). pre and post h-cg serum bile acid concentrations differed significantly (13.88 micromoles/l vs. 7.85 micromoles/l, p=0.004). there was a trend toward higher bile acid concentration in large follicles of young patients < 35 years old compared to older patients 40 years old (16.22 ± 5.55 vs. 13.56 ± 2.26, p=0.051). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients (4.47 ± 1.92 vs. 2.90 ± 0.72, p=0.079). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient 0.678, p=0.005). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in 201 women undergoing ivf/icsi treatment blood samples were collected on cycle day 2 and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal'5'phosphate (plp) concentrations were determined. women with a serum folate 22.5 nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p 0.001). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p 0.001). in the total group, a two-fold increase of thcy in follicular fluid resulted in a 0.06 mm decrease of the follicular diameter (p 0.05). in non-supplemented women this decrease was 1.64 mm (p 0.01). in supplemented women a twofold increase of follicular fluid folate resulted in a 0.74 mm decrease of the follicular diameter (p 0.05). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. 2), post-fixed for 40 rain in 2% w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, 0.23 m/min); a soluble guanylyl cyclase inhibitor, 1h-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (odq, 100 m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, 1 mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p<0.05). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p<0.05) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from 201 women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range 1-5; with 1 being best quality). secondary endpoint was the occurrence of pregnancy. results 67% of the included women used a folic acid supplement (serum folate 22.5 nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate -0.87; p 0.05) and higher thcy levels (median 7.1 mol/l, range 4.0-47.0) correlated with a worse embryo quality (estimate 1.01; p 0.05). in supplemented women higher follicular fluid thcy (median 6.4 mol/l, range 3.5-73.6) correlated with better embryo quality (estimate -0.58; p 0.05). the follicular fluid folate level of oocytes that did not fertilize was 1.1-fold higher than in the fluids of a fertilized oocyte (95% ci 1.00 -1.18; p 0.05). a two-fold increase of follicular folate corresponded with a 3.3 higher chance to achieve pregnancy (95% ci 1.09-9.71). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, 2007) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in 2002. we defined a cycle with hloi as having >50% immature oocytes (>2 sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= 35% (1sd above median) immature oocytes on all cycles. results: from 450 subjects, we identified 8 cases of recurrent hloi (comprising 19 icsi cycles) and 240 control subjects. at baseline, cases had more oocytes retrieved per cycle than controls (22.5 vs. 16.3, p<.0001). all other baseline characteristics were similar. all case subjects were younger than 37. outcomes are shown in table 1 . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during 19 cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, 23 were performed for an initial diagnosis of premature ovarian aging (poa), 6 for the diagnosis of premature ovarian failure, and 11 for repeated pregnancy loss. in women under age 42, b-fsh levels over 12 miu/ml and up to < 50 miu/ml denoted a diagnosis of poa, while levels of 50miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day 3 fsh levels ranged from 5.6 to 30.0 miu/ml (mean 13.6 ± 7.2 miu/ml). amh levels ranged from < 0.1 to 2.7 ng/ml (mean 0.8 ± 0.8) and allele repeats ranged from 17 to 49, (mean for allele-1, 27.2 ± 4.3; allele-2, 32.0 ± 6.4). mixed model analysis of variance for all patients, adjusted for age, race, and allele-1 revealed a statistically significant correlation between b-fsh levels and number of repeats in allele-2 ( figure 1 ; p < 0.01). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above 32 were significantly associated with serum amh levels less than 1 ng/ml (chi square = 0.04). conclusions: triple repeats above 30, a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm1 gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, 4.5 mg/kg cisplatin or 6.0 mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized 54 hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p<0.001). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p=0.001 and p<0.001 respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p=0.001 for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas 23a, 23b, 17-5pp, 211 and 542-3p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir-23a, mir-23b, mir-542-3p, mir-211 and mir-17-5p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)-2, il-1 , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from 26 women (age range 23-42; mean age 34.8 ± 0.5) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir-23a, mir-542-3p, mir-211 and mir-17-5p displayed a significant and positive correlation with the expression of cox-2. moreover, mir-23b significantly correlated with star mrna expression, but not with other genes tested (p<0.05). with advancing age, the expression of mir-17-5p and cox-2 was significantly decreased, with cox-2 expression displaying a positive correlation with aromatase, star and er mrna expressions (p<0.05). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of 31.5 y best predicted whether mir-17-5p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir-23b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir-17-5p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant 37432. introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e2 levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: 8 infertile women (<38 years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh<10 dor, fsh >10miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u133 plus 2.0 genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > 2 fold was considered biologically relevant. results: of the 8,846 genes identified to be differentially expressed in young women with dor compared to normal reserve, 215 genes demonstrated consistency of expression across five different normalization schema; 94 were down regulated and 121 up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated 3-fold over two probe sets (-3.08) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (-5.33) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf-9 which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k24cd41978. nih 5k12rr17672. ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp19) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl5). the granulosa cells were cultured +/-dibutyryl camp (1 mm) and incubated +/-the gnrh antagonist, ca, for 24-48 h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp19 expression. materials and methods: kgn cells were cultured in f-12dmem and were starved for 48 h before experiments. subsequently they were treated for 48 h with vehicle (control), fsh (100ng/ml), and/or bpa (20, 40, 60, 80, 100um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a 10-fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp19 production, with the greatest effect at 100um (p<0.001), resulting in 91+/-3 % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at 100um (p<0.01) resulting in 90+/-5% decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor-1 and gata-4, mimic the pattern of cyp19 expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing 822 cylindrical round bottom recesses 200 m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of 800,000 cells per gel were incubated for up to 10 days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least 10 days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for 24h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa1 and saa2 mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa1 and saa2 in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase 2 (sod2), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin 4.5 mg/kg or cisplatin 6.0 mg/kg as 2 weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod2. the anti-sod2 antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod2 following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod2 in antral follicles and in corpora lutea (p<0.01 for both). no change was found in the h score of sod2 in other ovarian structures. western blot analysis of sod2 in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod2 in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod2 in antral follicles and in copora lutea. cisplatin decreases the amount of sod2 available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e 2 ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged 53 ± 2.7 yr (range 48-56) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than 40 u/l and/or amenorrhea greater than 12 months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of 0.05 ng/ml and 7 pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: 1) six patients had no detectable follicular activity by amh and inhibin b levels. 2) one patient demonstrated detectable inhibin b levels with an 7-fold gradient between ovarian venous effluent (226 pg/ml) and peripheral blood (31 pg/ml), however no amh was detected. 3) in one patient, aged 52 and 120 months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at 912 pg/ml. amh was detectable at levels of 0.95 ng/ml. conclusions: 1) in the majority of patients, continued e 2 and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. 2) some patients have evidence of follicular function up to ten years after menopause. 3) since e 2 is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f2 (pgf2 ) mediates luteal regression, this study examined the role that the transcription factor yin yang 1 (yy1) and histone deacetylase 1 (hdac1) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p450 side chain cleavage enzyme (p450scc) expression. rat luteal cells (10 days post-ovulation) were treated with pgf2 (24 hr) and trichostatin a (tsa; 24 hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek 293t cells treated with yy1, myy1, a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf2 caused a significant 2-fold decline in star (p<0.005), and smaller declines in sr-bi and p450scc which occurred concomitantly with an increase in yy1 (4-fold, p<0.001) and intracellular lipid staining (20% increase). in contrast, 100 nm tsa treatment caused a dose dependent increase in sr-bi, star, and p450scc protein levels, 3.5-fold (p<0.005), 1.4-fold (p<0.05), 4.1-fold (p<0.005), respectively, and a 2.5-fold decline (p<0.05) in intracellular lipid levels. tsa prevented the pgf2 -induced decline in sr-bi, star, and p450scc expression. promoter analysis demonstrated that wildtype yy1, but not myy1, repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy1 plays in pgf2 induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy1-mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd35163 and nih hl78817. regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: 1) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp1 and ramp3 mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; 2) to demonstrate immunohistolocalization of imd, crlr, ramp1, ramp2 and ramp3 in rat ovary. methods: groups of 4 sprague-dawley non-pregnant and pregnant rats on day 18 of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase1 before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp1, ramp2 and ramp3 proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day 18. results: 1) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; 2)expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; 3) expression ramp1 mrna is higher in proestrus compared to diestrus and estrus (p<0.05) but there is no significant change in the ramp 3 expression during the estrus cycle and ; 4) imd, ramp1, ramp2 and ramp3 are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, 1 ap goud, 1 mp diamond, 1 b gonik, 2 hm abu-soud. 1 1 div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; 2 div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set 1, oocytes were retrieved from the following superovulated b6d2f1 mice: (a) young breeders (yb, n=109); (b) retired breeders (rb, n=40), and © old animals (oa, n=16), aged 6-12, 45-52, and 70-75 weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [1], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set 2 (n=56), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m-16, 0.23 m no/min , n=23, 3 h, 37°c, 5% co 2 ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [1]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set 1, a significantly fewer oocytes were retrieved per animal (mean) in rb (10) and oa (4) compared to yb (27.3, p<0.05). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p<0.01). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p<0.05). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p<0.01). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set 2, exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca2+ in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from 16 patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all 18 gaba-a receptor subunits, gad65 and gad67 plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca2+ study, gc were cultured on coverslips. gc were loaded with fura-2-am and changes in ca2+ concentration of gc were studied using a dynamic digital ca2+ imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, 10 m bicuculline, were perfused 1 min. prior to and during application of muscimol. the responses were represented as changes in the 340/380 nm fluorescence ratio over time. 50 m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the 5 expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were 3, 3 and 2. gad67 isoenzyme was more abundantly expressed in cumulus and mural gc than gad65. ca2+ imaging showed that muscimol, had the ability to increase ca2++ in gc, about 19% gc (n=6) cells responded to muscimol. muscimol increased intracellular ca2+ in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from 19% to 1 % (n=6, p< 0.05). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad67 expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca2+ response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p38 mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis 34 guinea pig fetuses at 35 and 60 days gestation were assigned to normoxic (nx, n=6), hypoxic (hx, n=6) and hypoxic pretreated with nos inhibitor (hx+l-name, 30mg/kg i.p., n=5) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio 2 of 0.07 for 60 min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p38 antibodies. in the 35days gestation group: expression of p-p38 was 39.5±3.4 (nx), 79.1±2.8 (hx) 47.1±4.1 (hx+l-name). p-erk expression was 59.7±3.2 (nx), 109.6±6.3 (hx), 72.4±2.7 (hx+l-name). p-jnk expression was 68.3±7.2 (nx), 94.6±3.8 (hx), 55.8±9.0 (hx+l-name). in the 60 days gestation group: expression of p-p38 was 108.7±3.6 (nx), 231.1±8.3 (hx), 116.5±9.8 (hx+l-name). p-erk expression was 94.1±12.9 (nx), 226.5±9.1 (hx), 139.1±12.6 (hx+l-name). p-jnk expression was 83.2±9.5(nx), 218.2±6.9 (hx) 105.3±10.8 (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p38, erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p38, erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p38, erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p38, erk and jnk in the guinea pig fetus. (nih-hd 20337, nih-hd 38079 and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint 1 . high levels (28nm) in culture are associated with embryo death 2 while plasma levels (>4nm) at 6 weeks in women undergoing ivf-et are associated with failed pregnancy 1 . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method 3 at 6-12 weeks gestation in 45 women (nonsmokers, bmi <30kg/m 2 ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the 45 women subsequently had a miscarriage. the plasma aea levels in those women who had live births was 2.85-fold lower than that in those who subsequently miscarried (1.21 ± 0.12nm versus 3.45 ± 0.31nm, mean ± sem; respectively; p<0.0001 unpaired student's t-test). the roc analysis revealed an area under the curve of 0.972 ± 0.025 with a sensitivity of 100% (95%ci of 66.3% to 100.0%) and a specificity of 94.4% (95% ci of 81.34% to 99.3%). using an aea level of 2.0nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of 100% and a specificity of 94% with a negative predictive value of 100% and a positive predictive value of 78% for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products (3 exon deletions and 1 intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon 2 and deletion of exon 6, showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek293 cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon 2, found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected 2 and 7 days after urinary lh surge in the same menstrual cycle from three fertile women (n=3). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using 2-d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, 24 cm ipg-strips in the nonlinear range of ph 3-11 were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v6.5 and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table 1 displays the differentially protein abundance between days lh+2 and lh+7 (> 2-fold change). of these proteins, 10 were increased at lh+7 in comparison with lh+2, whereas only 2 proteins were decreased. stathmin was found more than 2-fold decrease in lh+7 compared to lh+2 in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases 1 and 2 (erk1/2) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk1/2 is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk1/2 activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n=20) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen (10-8m), and progesterone (10-8m) for 5 and 15 minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p<0.05). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within 5 and 15 minutes (p<0.05) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin-1 (et-1), soluble intercellular adhesion molecule-1 (sicam-1), soluble vascular cell adhesion molecule-1 (svcam-1) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et-1 (p < 0.05), sicamp-1 (p < 0.05), svcam-1 (p < 0.001) and hscrp (p < 0.001) as compared with age-matched controls, respectively. positive correlations were evident between et-1 and fai (r = 0.41; p < 0.01) but et-1 negatively correlated with shbg (r = -0.36; p < 0.05). svcam-1 positively correlated with total t (r = 0.62; 0.001), hscrp correlated with: bmi (r = 0.73; p < 0.001), and homa (r = -0.39; p < 0.05), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. (1), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation (2), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from 47 women, median age 34yrs (range 20-45) and bmi 22kg/m 2 (range 19-27), with regular menstrual cycles, with no medical problem and not on any medication for the preceding 6 months. the menstrual cycle was divided to early follicular d2-7(n=11), late follicular d8-11(n=7), ovulatory d12-15(n=10), early luteal d16-22(n-10) and late luteal phases d23-28(n=9). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. (1 , 5, 10, 15, 20, 25, 50 and 120, as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp1,2,3,4,5,6) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp2 elevate in fetal and newborn rat and graduately decline to a lower level at day 15 and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp2 was not detected by wlb assay. the igfbp-2 cleaved products were observed after rat day age day 15, which associated with a decrease in full length of igfbp-2, suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp-3, with heterogeneous glycosylation, were appeared after age day 25 and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp3 has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp5 were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp2 suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp2 in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp-2 at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged 18-44years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and 17 steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api 5000). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e2), estrone (e1), progesterone (p), 17-hydroxyprogesterone (17ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, 11-deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d3 were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: 1.32, 0.86, and 1.02miu/ml respectively. similarly, mean t4 9.8, 12.23 (23% increase) (p=0.05), 9.41μg/dl (-7%) in active smokers; (active vs. exposed p=0.03). shs increased dhea levels (33% higher, p = 0.02), dheas (23% higher, p = 0.07), cortisol (21% lower, p = 0.01), aldosterone (63% lower, p = 0.02) and androstenedione (20% higher, p = 0.01). these data suggest that active smoking and shs can have a profound effect on serum t4, adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: 300 with pcos and 300 age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between 8:00-11:00, after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft 4 , 17-ohp, 4-a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos (53.5%, 95% ci: 37.6-61.2%) as compared with women without pcos (14.7%, 95% ci: 10.2-18.6%) (p<0.000). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p<0.001). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p<0.001). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c (83.1 ± 10.5%); increased triglycerides (53.4 ± 7.7%); and increased bmi (38.2 ± 4.6%), respectively. the prevalence of mbs from lowest to highest tertile of free t level was 20.1, 33.7 and 54.2%, respectively; in women with both pcos and mbs. in women with pcos, 9% exhibited all 5 components of mbs; 14.1% had 4 components, and 40.5% had 3 components. conclusions: women with pcos exhibited significantly higher prevalence of mbs (3.6-fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is 1 in 20,000 live male births. we present a case of complete androgen insensitivity in 2 members of the same family with differing residual mullerian tissue. sister a presented at age 17 for evaluation of primary amenorrhea. a karyotype revealed 46,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at 2.91 ng/ml. other laboratory evaluation showed fsh 4.5 miu/ml, lh 7.04 miu/ml, free testosterone 3.9 pg/ml, and estradiol 24.6 pg/ml. approximately two years later sister b presented at age 17 for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed 46,xy. laboratory evaluation demonstrated fsh 1.0 miu/ml, lh 13.28 miu/ml, estradiol 37.3 pg/ml, total testosterone 9.25 ng/ml, and free testosterone 11.1 pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb1 and cb2 and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea 1 are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at -20°c prior to extraction. an internal standard of 2.5pmol of deuterated aea (aea-d 8 ) was added to each plasma sample and aea extracted from 5 aliquots on each occasion over three days using the lpe 1 and spe methods. spe was performed with waters oasis hlb 1cc/30mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with 40% methanol at 1ml/min. aea was eluted with 1ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in 80 ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was 3-fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method 1,2 . methods: authentic non-labelled and deuterium-labelled aea (aea-d8) diluted in acetonitrile were maintained at 4°c before analysis and separation by uplc on a c 18 (2.1 x 50mm) column maintained at 40°c using a gradient of 80% a, 0.5min: 80% a, 1.5min: 0% a, 2.5min: 80% a, 3.5min: 80% a with a flow rate of 0.7ml/min. the mobile phases were a (2mm ammonium acetate, 0.1% formic acid) and b (acetonitrile, 0.1% formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were -2, 17 and -17ev, respectively. calibration curves were performed in triplicate with 2.5pmol aea-d8 as the internal standard. transitions employed were 348.3>62.3 and 356.3>63.3 for aea and aea-d8, respectively. results: calibration curves (1.66 to 133fmol aea on column; n=15) were linear, producing a mean (±sd) gradient of y = 2.48±0.14x, crossing the y-axis very close to the origin (0.004±0.04 units). variability was limited, with an r 2 =0.999. measurements were precise, 133fmol aea produced a cv of only 3.7%, and the retention time was consistent at 1.67±0.0009min (n=20). the limit of quantification (signal/noise>10) was 0.22fmol on column and the limit of detection (lod) was 0.055fmol on column (signal/noise=3). accuracy for 3.33fmol, 6.65fmol and 133fmol aea was 97.5±9.5%, 98.5±6.1% and 104.5±3.2%, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods 1,2 with a lower lod [0.055fmol v. 25fmol 1 or 43fmol 2 ] and shorter run time [4min v. 15min 1 or 5.4min 2 ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor-1 (sf-1), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: 1) total rna was extracted from the gonadotrope cell line, l t2, and analyzed by standard rt-pcr analysis. 2) the cv-1 fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat -207/+5 lh promoterreporter vector as well as cmv-driven expression vectors for gata-2, gata-3, dngata-3, fog-2 and/or sf-1. results: gonadotrope l t2 cells were found to express transcripts encoding gata-2, gata-3, and gata-4 as well as fog-1 and fog-2. gata-2 and gata-3 stimulated lh gene promoter activity by approximately 5-fold (p<0.05) and synergistically increased the sf-1 response (40-fold versus 10-fold for sf-1 alone; p<0.05). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog-2. similarly, co-transfection with a gata-3 dominant negative construct blunted wild-type gata-3 effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n=119) were randomized to control (2ml saline/ewe) or dex treatment (0.14mg/kg ewe weight) consisting of four intramuscular injections at 12-hourly intervals over 48 hours on 40-41 days of gestation (dg). at 50, 100, 125, and 140dg fetal weights were recorded. i 125 -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at 100dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc-1 mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at 50dg*, was similar at 100 and 125dg, then elevated at 140dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at 140dg* were associated with increased fetal adrenal expression of p450c17 and 3bhsd mrna in females, reduced expression of mc2r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p<0.05. synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, 1 beom su kim, 1 amanda biehl, 1 blenna cl bett, 1,2 john yeh. 1 1 gynecology-obstetrics, university of buffalo, buffalo, ny, usa; 2 physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh (1:200-chemicon international) and with a rabbit polyclonal antibody against synaptophysin (1:100-santa cruz biotechnology) for 16 h. after incubation the sections were washed and incubated with a mixture of alexa 488 conjugated goat antimouse and alexa 594 conjugated goat antirabbit (1:1000; molecular probes) for 2 h. slices were visualized with confocal microscopy (zeiss lsm-510). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control (2 ml saline/ewe) or dex groups (0.14 mg/kg), consisting of four at 12 h intervals on days 40-41 of pregnancy. at 7 months postnatal age, catheters were implanted; a bolus injection of crh (0.5 mg/body weight) and avp (0.1 mg/body weight) were administered and arterial blood samples were taken at -30, -15, 0, 5, 10, 20, 30 60, 90, 120 and 180 min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to 18s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n=5) were lower than in dex males (n=7) from 30 to 90 min (p 0.05) and lower than in control females (n=6) at 30 minutes (p 0.04), also in the dex-m group were higher than in control males (n=5) at 60 min (p 0.04). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at 7 months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control (2 ml saline/ewe) or dexamethasone treatment (0.14 mg/kg) consisting of four injections at 12 h intervals on days 40-41. hippocampal was collected from the offspring at 7 months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to 18s rrna. results: dexamethasone-treated male animals (n=7) had significantly higher levels of mr gene expression than both control males (n=5; p=0.028) and females (n=6; p=0.037). gr gene expression levels were higher in treated vs. control males (p=0.043), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women <35 with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from 1/2005-6/2007 with follicle stimulating hormone (fsh) levels 10 based on age <35 or 35. results: 49 so cycles were performed in 18 women. no differences were noted in clinical pregnancy. women <35 were more likely to have inseminates with a lower mean tms/ins, median tms/ins was 71.0 among pregnancy cycles compared to 28 for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women <35, mean number of follicles 15 mm peak e2 were significantly higher in women <35. so ivf measures are in tables 1 2, respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women <35 required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women <35 with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about 50 l containing 3x10 9 pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e2 and p4 measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p<0.02). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average 18 follicles/ovary were observed in ad-hfshr-treated group of which 4 follicles were at the antral stage while only 2 follicles observed in ad-lacz control group, with zero follicles at antral stage. a 2.5 to 3 folds increase in e2 and about 50% decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and 16 samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day 3, and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential (0.66±0.13) compared to those that failed to implant (0.33±0.16) (p<0.001). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of 96% and a specificity of 77.8%. quantification by integration showed significantly decreased glutamate levels (p=0.002) and a trend toward an increase in pyruvate levels (p=0.1) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single 18-20 mm follicle was obtained from 24 women (age 29-43) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p<0.001 for all analytes) and the levels were strongly correlated (r2>0.45, p<0.003 for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte (0.08 +/-0.04 g/ml vs. 0.01 +/-0.01 g/ml, p=0.015). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of 23°c. thermistors, pinhead sized sensors with an accuracy of 1%, were used to monitor temperature differences between control fluid (cf) (sterile water 37ºc) and aspirated fluid. data was recorded using an 8-channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a 33cm, 17 gauge, single lumen needle connected to 70 cm of tubing was used to aspirate into the empty collection tube. temperature was recorded 2x/second, each experiment was repeated 3 times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group (32.99±1.39°c vs 36.61±.16°c, p=.016). two seconds after aspiration, the lowest aspirate temperature was observed, (hh 30.34±.14°c, hb 30.48±.23°c, p>.05) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh 34.57±.38°c, hb 34.86±.33°c, p>.05). the hb group took an average of 5.28 min to return to baseline (37°c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of 6.59±.19°c corresponding to 30.41°c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, 1 luis roca, 1 niki b istwan, 2 debbie j rhea, 2 gary j stanziano, 2 victor hugo gonzalez-quintero. 1 1 obstetrics and gynecology, university of miami medical center, miami, fl, usa; 2 clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n=1421). eligible for inclusion were patients later hospitalized with acute rptlsx (n=862). included were those <35 weeks' gestational age (ga), with intact membranes, remaining undelivered for >48 hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n=418) following hospitalization were compared to those having an alteration in treatment (alttx group, n=238) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at <0.05. results overall, 862 (60.7%) of twin pregnancies prescribed nt experienced rptlsx; 206 (23.9%) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during 2005. stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of 666 elective cesarean deliveries (cd), 348 underwent routine cervical dilatation. the overall rate of febrile morbidity was 4.2%. no significant differences in postpartum febrile morbidity were noted between the groups (5.1 and 3.1%; p=0.071). in addition, hospitalization duration did not differ between the groups (4.1±1.4 and 4.1±2.0 days p=0.95 ). about 31 % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin 9.50 ±0.73 mg and 9.54±0.65mg p= 0.91 ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or=2.2; 95% ci 0.8-6.4; p=0.161). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or=5. the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between 25 and 32 weeks of gestation were chosen for this study. eighteen patients (group 1) were treated with amoxicillin and erythromycin for 7 day period while the other 18 patients (group 2) were treated with 3 rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day 3, and day 7 after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the 2,4-dinitrophenylhydrazine method. results: 1. the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy (4.77±0.36 vs 7.11±0.41 nmol/mg protein, p<0.01), (3.55±0.22 vs 5.69±0.30 nmol/mg protein, p<0.01). 2. there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). 3. there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group 1 among before the antibiotics administration, day 3, and day 7 after the antibiotics administration. 4. the protein carbonyls formation in the venous plasma of group 2 was significantly decreased at day 3 and day 7 after the antibiotics administration than that of before the antibiotics administration (6.04 ±0.44, 5.53±0.37, 7.04±0.51 nmol/mg protein, p<0.01). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of 3 rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between 24 and 41 weeks gestation in 664 consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< 0.74 or >/= 0.74 mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than 0.74 mom (5.5 days, interquartile range 2-10) in comparison with the women with mca pi higher or equal than 0.74 mom (22.5 days, interquartile 5-37.5 days (p<0.001, mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< 0.001, exp(b) 2.77, ci 95% 1.95-3.90). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n=16). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of 1 nmol/l to 100 mmol/l. two sets of control experiments were performed simultaneously as follows: 1. strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or 0.5 nmol/l oxytocin; 2. strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: 45.34 ± 5.92% for spontaneous contractions (n=6; p< 0.05) and 41.88 ± 5.40% for oxytocin-induced contractions (n=6; p<0.05). no significant difference was found between control 1 and control 2 for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: 11 pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all 11 patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < 0.05 considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure 1) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table 1) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r01-hd037480. results: 68% of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p<0.04); after a failed instrumental delivery, 74% obstetricians would recommend labour but only 44% would choose that option for themselves (p<0.01). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently (66% and 57% respectively) than chosen for personal care (57% and 52%). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at 39 weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of 2.06/100,000, 5.8/100,000 and 18.2/100,000 for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. 2,500,000/annually in the u.s.) would be delivered at 39 weeks by either an elective c/s or an induction of labor. we further assumed that 50% of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at 39 weeks would be 145 and 253 for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds 77%, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at 39 weeks is relatively low (approximately 12.5/100,000 deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below 77%, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: 29 providers (21 residents, 5 midwives, 3 nurse practitioners) and 51 patients participated. providers asked about smoking, alcohol and drug use in all 51 (100%) visits. 25 patients reported being smokers, 4 reported alcohol use, and 11 reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women (20 with actual psychiatric disorder, 20 with stressful life events, and 40 healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at 20 (+1), 28 (+1) and 34 (+1) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at 18-20 weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, 1998) ; moreover, the 17-item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight (3084 + 414 g) and higher percentage of birth weight below the 10 th centile for gestational age (30%) than infants of healthy mothers (3408 + 453 g and 5%, respectively). there was also a trend towards lower mean birth weight (3242 + 400 g) and higher incidence of birth weight below the 10 th centile (10%) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from 26 weeks-delivery are generally recommended for patients with hcg levels > 2.0 mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > 2.0 mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) <10%ile for the estimated gestational age (ega). sga was defined as an actual birthweight <10%ile for the ega at the time of delivery. results: from 1999-2007, there were 659 subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of 1708 ultrasound examinations were performed. the median number of examinations per subject was 3 with a range from 1-5 examinations per subject. the incidence of iugr and of sga were 5.0% (n=33/659) and 7.3% (n=48/659), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified 31.3% (n=15/48) of sga infants. however, the sensitivity for the detection of sga was 100% when an efw cut-off of 75% was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > 75%ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, 1 christiaan v hulzebos, 2 gerda g zeeman. 1 1 obstetrics and gynecology, university medical center groningen, groningen, netherlands; 2 pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first 24-48 h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach 32 wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > 32 wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > 32 wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between 24-32 wks, who deliver > 32 wks (latency > 24 h) , during the 4-yr period of 2002-2006 at a single academic center. data were extracted from medical records and electronic department databases. results:108 pprom pregnancies were identified. all but 2 received at least 1 full course of steroids. 27 (25 %) delivered > 32 wks. 3 newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > 32 wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n =10). tertiary care center (n = 8) to determine if an association exists between macrosomia (birthweight > 4000g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of 36,241 singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p<0.05 to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than 4000 grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was 10%. methods: we prospectively analyzed the outcomes of 410 pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi <30 kg/m 2, absence of major risk factors for diabetes and of pregestational diabetes. 50 g 1-hour oral gct was performed at 24 -28 weeks of gestation. women were subdivided into 3 groups according to the result of the gct: group 1= negative (load glucose >10% and <140 mg/dl) 327 women (79.8%); group 2=flat (load glucose 10% than basal and <140 mg/dl) 51 women (12.4%); group 3=positive gct/negative ogtt, 32 women (7.8%). data are mean ± sd. differences were calculated with the student t test for unpaired samples and 2 test. regression analysis were performed by the least squared method. p-values were considered significant at p<0.05. results: the characteristics and obstetric outcomes in the three groups are presented in the table 1 . in all patients there was a significant linear relationship between the load and basal glucose values (load value=66.8+0.6 basal value; r=0.2; p<0.001) and between birthweight and load values (birthweight=2978.3+2.8 load value; r=0.15; p<0.02). the relationships were significant also in group 1 and 2 separately but not in group 3. in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table 1. the probability to be primiparous and to deliver babies <10° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies >90° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p<0.001) and to start pregnancy without any medical and obstetrical risk but obesity (85.6% vs 90.5 and 90.2%; p<0.01). african women exhibited the highest bmi and the highest rate of obesity (table 2 ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than 50% have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c57bl/6j female mice were fed control (c, 11% of energy from fat) or isocaloric high fat (hf, 32% of energy from fat) diets ad libitum for 8 weeks prior to mating and during gestation. at gestational day 18.5 maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using 3 h-meaib, and 14 c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e18.5 or litter size between treatment groups. fetal weight was increased in the hf group by 45% (p<0.05). maternal adiponectin levels were significantly (p<0.01, n=18) decreased (hf 45 ± 8, c 69±15 g/ml) and leptin levels increased by 60% in animals fed a high fat diet, but this difference did not reach statistical significance (n=6). adiposity (fat pad weight) was increased by 7% (p<0.05, n=10 in the dams fed high fat diet, however no difference was observed in maternal il-6 levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least 50% in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased 11 hsd1 expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of 11 hsd1 in the lamb before and after birth. methods: ewes were provided with either 100% (control,c) or 160% (well fed, wf) of maintenance energy requirements from 115d gestation until delivery. post-mortem was performed on either 139±141d gestation (c=6,wf=8) or and postnatal day 30 (c=12,wf=9). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of 11 hsd1 and reference gene rpp0 were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at 30d (p<0.05). the expression of 11 hsd1 mrna was significantly higher in the postnatal compared to the fetal lamb (p<0.001) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of 11 hsd1 mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of 11 hsd1 in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first 24hrs after birth (r 2 =-0.78, p=0.002). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between 11 hsd1 mrna expression in the liver at 30d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic 11 hsd1. we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven (38%) of mothers were classified as obese (bmi>30) based on their first pregnancy weight. glycemic control at 36 weeks was superior in the non-obese group (table 1) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table 1) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table 1) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight (3675g+593 vs. 3434g+520, p=.005) and higher rate of macrosomia (11 vs. 9%, p=0.04). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr-3 by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr8, was treated with polyi:c. c57b/6 wild type and tlr-3 knock out mice were injected intraperitoneally with polyi:c at 16.5 gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex100 is. results: polyi:c induced cytokine (il6, il1 and il1 ) and chemokine (il8, rantes, gro , mcp1 and mip1 ) secretion and production by human cultured trophoblast in a time (24-48 h) and a dose (2-25 g/ml) dependent manner. injection of polyi:c to c57b/6 wild type mice induced preterm delivery within 24 h at a dose of 9 mg/kg body weight. no effect was observed in tlr-3 knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed 2 and 4 h following polyi:c treatment. trophoblast cell cultures from tlr-3 ko mice confirmed that the response to polyi:c is tlr-3 dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr-3. our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr-3 mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: 2 mouse models were utilized: (1) a model of intrauterine inflammation (lps into uterine horn) (n=9); controls received intrauterine saline (n=9) and (2) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) 110 will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of 6 lps infected animals and 3 controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+20.4 %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by (1) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or (2) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr-1 supernatant is able to antagonize the actions of lps on cytokines and ptgs2 in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs2 expression levels were measured by western blot analysis and tnf-, and il-10 concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-(30.9 pg/ml to 1175.3 pg/ml, n=11, p<0.05), an effect that was greater in placentae of the male fetuses (1516.6 pg/ml, n=6) compared to female fetuses (562.5 pg/ml, n=5). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il-10 production (n=5, p<0.05) and ptgs2 expression (n=4, p<0.05). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n=8) by 63%, and this effect was greater in placentae of the female fetuses (n=4). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs2. conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , 2007) . this study investigates whether iai is associated also with altered expression of neuropilin-1. methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n=3 for each). neuropilin-1 expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n=6), purified, and depleted of leukocytes. after treatment with 10 -8 m estradiol (e2), 10 -7 m medroxyprogesterone acetate (mpa), both, or vehicle for 7 days, dscs were stimulated with il-1b (1-10 ng/ml), tnfa (1 ng/ ml), or thrombin (2.5 iu/ml) for 24h. since no elisa exists for neuropilin-1, protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il-1b on neuropilin-1 mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin-1 expression in term decidua was increased in tissues with iai vs controls (p<0.05), and localized primarily to dscs. using icc, an increase in neuropilin-1 was noted after stimulation with il-1b and tnfa, but not thrombin. il-1b increased neuropilin-1 mrna expression in dscs by 6.5±1.6-fold (from 28.0±17.7 to 240.9±189.4 neuropilin-1 mrna/b-actin mrna; p=0.031). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin-1, in term decidual tissues. il-1b and tnfa (but not thrombin) stimulated neuropilin-1 expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr1 and cxcr2 ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr1 and cxcr2. methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n=8; cervix n=9) and in labour (il) (myometrium n=8; cervix n=9). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in 10 randomly selected high-powered fields (400x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr1 and cxcr2. results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median 11.9, iqr 6.7 -19.8; il median 26.9, iqr 16.1 -40.2, p=0.025). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median 19.0, iqr 4.6 -35.5; il median 7.9 iqr 3.5 -10.4, p=0.17). analysis of chemokine receptor expression demonstrated co-localisation of cxcr1 to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr1, the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage 1-2) and wish cells were serum starved for 24hrs and treated with 17-hydroxyprogesterone (17-hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru486 (a pr/gr antagonist) or org 31710 (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with 17-hp and p (1 and 10 m) and dex (10nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p (100nm to 10 m), and decreased sequentially within cytoplasmic fractions. in the presence of org31710 gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p>0.05) within both cells types in response to p. conclusion: in this study we show that gr is activated by 17-hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and 17-hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: 6 hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd-1 mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n=6 per group) each with 2-4 gs with15 fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata-1 (erythroid precursors), cd3, and bm8 (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h2ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p=0.02). in lps-exposed, fetal mp were increased in the placenta (p=0.007), fetal extremity mesenchyme (p=0.018), fetal liver (p=0.005) and leptomeninges (p=0.013) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. 69% of lps-exposed fetuses and 0% of saline-exposed had liver hemorrhages (p< 0.001). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p=0.004). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem-1 (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem-1 in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology 2006). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem-1 expression in the uterus, cervix, and placenta across gestation and to determine if trem-1 levels are altered by intrauterine inflammation. methods: in cd-1 mice, trem-1 was investigated in non-pregnant (np) and throughout gestation e 15, e 18 (n=3 -6 per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected 6 hours after intrauterine infusion of saline (n=3) or lipopolysaccharide (lps) (n=3). for a non-pregnant model, using cd-1 mice, lps (n=3) or saline (n=3) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested 6 hrs later. quantikine ® mouse trem-1 immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p<0.05) results: trem levels are significantly different between np and pregnant uterine tissues (p=0.002). e15 and e18 trem expression is significantly increased 2.3 and 2.5-fold compared to np (p=0.004 and 0.001 respectively). trem-1 levels in the placenta and cervix were not significantly different between e15 and e18. trem levels increased about 4-fold in the uterus after intrauterine infusion of saline or lps compared to e15 controls. in nonpregnant, trem levels were significantly different (p=0.05) with a 17-fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem-1 in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, 1 adegoke adeniji, 1 susan e jackman, 1 babak forooghi, 1 isaac mostadim, 1 phillip yadegari, 1 calvin j hobel. 2 1 og-gyn, cedars-siani medical center, los angeles, ca, usa; 2 university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (<37.5nmol/l), insufficiency (37.5 to 80 nmol/l) and sufficiency (>80nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of 25(oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, 626 ethnically diverse women were evaluated at 18-20 weeks (t1), 28-30 weeks (t2) and 34-36weeks (t3). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups (58 with no infection during pregnancy, 101 with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for 25(oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of 25(oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<.001). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy (46.2 nmol/l ± 6.8 at t1, 28.4 nmol/l ± 4.1 at t2 and 30.2 nmol/l ± 3.6 at t3; all p<.001) but not subjects with sufficient vitamin d at t1(87.3 nmol/l ± 5.4 at t1, 27.9 nmol/l ± 3.8 at t2 and 32.8 nmol/l ± 4.6 at t3; all p<.001). conclusion the results reveal a positive association between 25(oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected (7.5x10 8 ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in 14.5 day pregnant c57b/l6 mice. the mice were euthanized at 5 hr (n=4) and 14 hr (n=6), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase 3 activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n=3), dmso (n=4) or pancaspase inhibitor z-vad-fmk (n=6) prior to hk-gbs and observing the animal for delivery for 48 hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase 3 activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase 3 and caspase independent m-calpain in the placenta. z-vad-fmk (10 mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and 25 (3.2%) had a late sptb. the mean + sd gestational age at blood draw was 12 + 2 weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= 0.047 for trend). women with bb in the top quartile were 4.7 times more likely to have an early sptb as compared with women who had lower levels of bb (95% ci 1.5 to 14, p = 0.003). there was no association between bb and late sptb (rr= 0.8, 95% ci = 0.3 to 2). the adjusted or of an elevated bb for early sptb was 4.3 (95% ci = 1.3 to 14, p= 0.02). when the analysis was restricted to the 38 women with sptb the rr of an elevated bb for early sptb was 3.1 (95% ci 1.3 to 7.5, p = 0.03). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding 30 weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day-15 pregnant cd-1 mice underwent intrauterine injection of 250 g lps. subsequently, the mice were euthanized at 0, 2, 6, 12, 18 and 24 hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism 7000 multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a 2% agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all 12 of the reported mouse tlr isoforms. these tlr amplicons range in size from 83 to 200 bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at 6 hours after lps injection, tlr3 increased 3-fold and tlr6 increased 2-fold (both p<0.05). in contrast, the expression of tlr4 (the ligand for lps) remained stable during the 24 hours after lps; the expression of tlr1, 5, 7, 8, 9, and 13 also remained stable. tlr2 expression trended upward and tlr11 trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all 12 tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd044747). udp-glucose and its receptor p2y14 as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p2y14, a g protein-coupled p2y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p2y14 have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p2y14 in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il-8 mrna expression. we also measured the il-8 secretion using elisa. small interfering rna was used to knock down p2y14 expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip-2, the il-8 homologues in mice. results: p2y14 was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il-8 in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p2y14 knockdown abrogated udp-g-induced il-8 production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il-8 neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip-2 mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il-8 production via p2y14 in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p2y14 may be involved in the trans-species mucosal immune system in frt. (jsgi 2007; 14, 2(suppl) , abst #31) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar 1; 7-14 x 10 7 cfu) at 130 ± 1.2 days gest. age (dga, mean ± sem, term=167 d). six of these animals received maternal i.v. azi (12.5mg/kg q12h or q6h for 10 d) either alone (n=3) or in combination (n=3) with dexamethasone (dex; 4mg/kg/d i.v. for 4d) and indomethacin (indo; 100mg/d p.o for 5d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent (0), minimal (1), moderate (2) and severe (3). results. inoculation-to-delivery interval was 18-25d for combined treatment groups and was similar to long duration infection without treatment (15-21d). treatment effects were tabulated as mean scores and compared as follows: control (n=3), score 1; short duration infection (3-8d; n=4), score 8; long duration infection (15-21d; n=3), score 12; short duration infection + treatment (n=5), score 4; long duration infection + treatment (n=1), score 5. conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd06159, rr00163. brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor-4 (tlr4). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr4 signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr-4 specific lps to cd1 pregnant mice at 16 day of pregnancy caused preterm delivery after 18 to 24 h with 70% pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk3 was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p<0.007) lps-induced preterm delivery and reduced pup mortality up to 15%. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox-2, cxcl1 (il-8 equivalent) and ccl2 in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin 15-deoxy-12,14 -prostaglandin j 2 delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor-4 (tlr4) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin 15-deoxy-12,14 -prostaglandin j 2 (15d-pgj 2 ) inhibits il-1b-induced nf-kb activity and cox-2 expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of 15d-pgj 2 can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr4 specific lps to cd1 pregnant mice at 16 day of pregnancy caused preterm delivery after 18 to 24 h with 70% pup mortality. co-administration of 15d-pgj 2 with lps to pregnant mice delayed significantly (p<0.008) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to 5%. we have looked at the expression profile of several labor associated genes in myometrium 6 hours after lps administration. (otr, connexin 23 and 46, cox-1, cox-2, cxcl1 (il-8 equivalent) and ccl2). we have found that treatment with lps significantly up-regulated cox-2, cxcl1 and ccl2 and this is significantly suppressed after with co-administration of 15d-pgj 2. western analysis for ser536-phosphorylated p65 and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both 1 h and 6 h. co-administration of 15d-pgj 2 was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion 15d-pgj 2 may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr-2 and tlr-3: mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, 1 emmet hirsch. 1,2 1 department of ob/gyn, evanston northwestern healthcare, evanston, il; 2 department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr-2 responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr-3 is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr-3 ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr-2 and tlr-3. here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw 264.7) were treated in tissue culture with either pgn (1 g/ml), poly(i: c) (10 g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for 5-10 hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin 1 (il-1 ), tumor necrosis factor (tnf), the chemokine rantes and tlr-2, normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw 264.7 cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il-1 , tnf and rantes (p<0.05 for all) at 5 and 10 hours . sequential stimulation with either pgn or poly(i:c) for 5h followed by incubation for an additional 5h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for 5h followed by medium for 5h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr-2 mrna after 5h but not 10h. tlr-3 mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr-2 by either tlr-2 or tlr-3 activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il-6 by elisa. paired maternal serum il-6 levels from 34 subjects were divided into 2 groups: il-6 levels obtained 12-120 hours prior to completion of antibiotics and those obtained 12-120 hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p<0.05. of the 34 pprom subjects, the maternal age was 26.8 yrs; gestational age at admission was 26.7 weeks; latency was 13.1 days; gestational age at delivery was 30 weeks; and infant birth weight was 1409 grams. median maternal serum il-6 levels obtained off antibiotics were significantly higher when compared to those on antibiotics (4.9 vs. 2.7 pg/ml, p<0.02). the results of this investigation suggest that maternal serum il-6 levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in 2005. placentas with histologic chorioamnionitis were divided into two cohorts: group 1-maternal inflammatory responses only, and group 2-maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of 351 consecutively submitted placentas, 210 had histologic chorioamnionitis: 155 with maternal inflammatory responses only (group 1) and 55 with both maternal and fetal inflammatory responses (group 2). fetal inflammatory responses observed in group 2 were associated with higher stages of maternal inflammatory responses (p<.001). 80% of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group 2 patients were more likely to have had amnioinfusion (29% v 13%, p=.006) and less likely induction of labor (22% v 39%, p=.02). group 2 was more likely to have had intrapartum fever (56% v 40%, p=.04) and maternal tachycardia (58% v 39%, p=.02). newborns from group 2 were more likely to have been observed for sepsis (69% v 37%, p <.001) and have an apgar score 6 at 5 minutes (7% v 1%, p=.02). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or 6.3) and amnioinfusion (or 2.9) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin-8 (il-8) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il-8 production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il-8 production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il-8 production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with 0-1.6 mm sasp overnight and then stimulated with 9 ccu/ml ureaplasma parvum, 10 8 cfu/ml escherichia coli, or 2 x 10 6 cfu/ml gardnerella vaginalis for another overnight incubation in 96-well cultures. conditioned medium was then harvested and production of il-8 was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated 3 times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il-8 production by endocervical cells not treated with bacteria. at the highest concentration tested (1.6 mm), sasp significantly inhibited il-8 production by cultures stimulated with e. coli (p<0.001), u. parvum (p<0.001), and g. vaginalis (p<0.001). viability of the cells, however, was significantly reduced by sasp at 0.8 and 1.6 mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il-8 production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of 17 -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: 17 -hydroxyprogesterone caproate (17-ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that 17-ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, 17-ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, 17-ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if 17-ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of 10 pregnant women between 16 and 24 weeks. pregnant women had no prior history of preterm birth. cells were treated with 17-ohpc (1 m) and escherichia coli lipopolysaccharide (lps, 1 g/ml) alone or in combination. after 6 and 24 hours of culture, supernatants were collected and tested for tnf-and il-6 levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < 0.05 was considered significant. results: pbmc exposed to lps significantly increased tnf-and il-6 secretion compared to untreated controls (p=0.008 and p=0.008). tnf-concentrations were not significantly different between lps and lps+17-ohpc treated cells at both 6 and 24 hours (p=0.86 and p=0.17). il-6 production was significantly increased after 6-hour treatment with lps+17-ohpc compared to lps alone (2,730 [2,150-5,090] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps (1 g/ml)+/progestin (50nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to 17-hydroxyprogesterone (17-hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and 17-hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase 2 [cox-2], toll-like receptor 4 [tlr-4]) was evaluated. cox-2 and tlr-4 were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox-2 and tlr-4 proteins were significantly inhibited by both p (p<0.01 and p<0.05, respectively) and 17-hp (p<0.01 and p<0.05, respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr-2, tlr-4 and tlr-9 at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr-2, tlr-4 and tlr-9 within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps (1mg/ml) or lta (10 mg/ml) for 0, 2, 4, 6, 8 and 24 hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr-2, tlr-4, tlr-9 and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr-2 with lta was observed in choriocarcinoma cells (6-fold, 2h), and endometrial mesoderm cells (19-fold, 4h). tlr-4 induction with lps was observed in ectocervical cells (4-fold, 4h), choriocarcinoma cells (4-fold, 4h) and endometrial mesoderm cells (7-fold, 24h). tlr-9 induction with lps was observed in choriocarcinoma cells (32fold, 4h) and neonatal epithelial cells (11-fold, 4h). all cell lines showed at least constitutive levels of transcription for tlr-2, tlr-4 and tlr-9. these data demonstrate that tlr-2, tlr-4 and tlr-9 are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at 50 days of gestation (d, term=150 d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) 2x10 7 colony forming units (cfu) of u parvum (serovar 3, n=7; serovar 6, n=8), (b) 2x10 4 cfu of u parvum (serovar 3, n=6; serovar 6, n=8) or (c) media control (n=6). at 125 d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to 40 cmh 2 o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with 4% paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar 3 or 6 (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p<0.05). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p<0.05). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n=3) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than 90% making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p65, the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in 4 cohorts: preterm no labor (pnl, n=20), preterm labor (ptl, n=20), spontaneous term labor (stl, n=19), and term no labor (tnl, n=22). subjects were delivered between gestational ages 26-36 weeks (preterm) and 37-44 weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p65 and microscopically examined. myometrial samples were categorized in a blinded fashion to 3 groups of staining: no nuclear p65 (-), rare nuclear p65 (+), and >50% nuclear p65 (++). a p65 nuclear labeling index (nli; % nuclear p65/total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of 5 non-overlapping low-magnification fields. results: nuclear p65 labeling was rare in amnion and inconsistently present in chorion. in decidua, p65 nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl-40% (24-55%); ptl-45% (39-57%); tnl-37% (25-43%); stl-41% (30-59%); all values are median (iqr)]. in myometrium, ++ p65 nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl-26%; stl-50%). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p65 labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of 40% (32-55%) [median (iqr)], 37% (28-47%), and 56% (40-58%), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p65 immunolabeling was observed in myometrial cells following labor. nuclear p65 labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin-1 and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il-1 levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with 10 -8 m estradiol (e2) + 10 -7 m medroxyprogesterone acetate (mpa) for 7 days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il-1 . after 24h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from 6h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were 0.13± 0.06 pg/ml/ug cell protein [mean ± sem, n=8] and did not differ from e2+mpa basal levels. the addition of 1ng/ml of tnf-or il-1 significantly elevated g-csf output to 2.22 ± 0.97 and 454 ± 122 respectively (p<0.05), representing more than a 10-fold and 4,000-fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il-1 from 0.01 to 10.0 ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il-1 may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr2 and tlr4 in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. 3. to compare tlr2 and tlr4 protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n=18) and pregnant (n=11) human cervical cells were obtained by smear and tlr2 and tlr4 expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr2 and tlr4 protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr2 expression in late pregnant compared with non-pregnant cervical epithelium (p=0.0001), whilst tlr4 did not appear to differ. there was an apparent up-regulation of tlr2 protein expression in response to 17beta-oestradiol (n=5) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: 4-hour urine samples from 28 pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n=10), b: cranberry in the am, then placebo at dinner (c, p; n=10), c: placebo two times daily with meals (p, p; n=8). we identified 15 non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected 4-hour urine specimens from them. the ph of all urine specimens was adjusted to ph=7 and filtered. aliquots of each were independently inoculated with overnight culture of 10 2-3 cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after 24 hours of incubation for e. coli and k. pneumoniae, and 48 hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r21dk65827-01; clinical trials registration nct00506025. ...,.. e. coli 1.2x 10 8 .:!: 8.5x 10 8 1.2 x 10 8 .:!: 3.0 x 10 8 group a (c, c) group b ( c, p) 2.3x 10 8 + 2.7x 10 8 9.4 x 1 0 7 + 4.8 x 10 8 .33 .81 .83 group c (p, p) 1.1 x 1o"::!>s x 1o" 1.1 x 10 8~ 1.ox:1o• k. ~neumoniae group a ( c, c) 5.3 x 10 7 + 3.4 x 10 7 9.2 x 10 7 + 6.7 x 10 7 .88 .87 .91 group b (c, p) 6.0 x 10 7 + 4.8 x 10 7 6.3 x 10 7 + 3.0 x 10 7 group c (p, pl 4.7 x 10 7~2 .3x 10 7 6.9x 10 7~4 .6x 10 7 c. al bicans group a (c, c) 1.1 x 10 '+1.8x 10 7 1.9 x 10 8 + 1.0 x 10 8 group b (c, p) 1.5x 1o•+ 1.0x 10 7 2.4 x 10 8 + 1.1 x 10 6 .75 . 87 .60 group c (p, p) 4.7x 10 7~2 .3x 10 7 2.1 x 1 0 7~5 .9 x 10• objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected 4-hour urine specimens from 34 pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n=11), b: cranberry in the am, then placebo at dinner (n=12), c. placebo two times daily with meals (n=11). we identified 14 non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n=6, group b: n=3, group c: n=5), and collected 4-hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as 0, 50, and 100%. results: when combining all subjects and dosing regimens, the sensitivity of the assay was 25%, range 17% in the pregnant group assigned single daily dose of cranberry to 100% in the non-pregnant group assigned to multiple daily doses. the specificity ranged from 63% to 100%. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r21dk65827-01. clinical trials registration nct00093938. objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor 2 and 4 (tlr2 and 4) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from 37 pregnant women: 28-36 weeks gestation (w) preterm not in labour (ptnl) n=10; 28-36 w preterm in labour (ptl), n=7; 37-42 w term not in labour (tnl) n=10; and 37-42 w term in labour (tl) n=10. leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il-8, icam-1, mcp-1, tlr2 and tlr4 was significantly increased in tl compared to gestation matched tnl. the expression levels of il-1b, il-8 and tlr-2 were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr2 gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr-2 agonists: macrophage-activating lipopeptide (malp-2), pam 3 csk 4 , and peptidoglycan. methods. cecs were isolated by smears and compared between 7 pregnant (3 rd trimester) and 7 nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr2 primary antibody and its igg 1, isotype control, to determine tlr-2 protein expression. a further 7 nonpregnant smear samples were each prepared, and seeded at a concentration of 100,000 cervical epithelial cells/ml into 24-well cell plates. cells were incubated at 37°c in 5% co 2 overnight with the tlr2 agonists malp-2 and pam 3 csk 4 (at concentrations of 10 and 1000ng/ml), peptidoglycan(50ng/ml), il-1 (10ng/ml, positive control) and rpmi 1640 + 1-glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at -80°c until the concentration of il-8 was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr2 (specific mean fluorescence 4.7 vs 3.3 respectively) but did not differ (p = 0.2). unstimulated cells incubated with buffer alone demonstrated high il-8 levels (6602 pg/ml), which did not differ following stimulation with malp-2 (6217 pg/ml), pam 3 csk 4 (6449 pg/ml) or peptidoglycan (4867 pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr2 agonists are pending. conclusion. high basal il-8 levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr-2 agonists did not lead to further release of il-8 may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega-3 fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega-3 fatty acids on producing pge2 and il-6, in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega-3 fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for 24 hours in 37°c, co 2 incubator. after that, 10 g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for 8 hours. the cell media were collected, and pge 2 and il-6 concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)-2 were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge2 (p<0.05) and il-6(p<0.01) significantly in elisa. cox-2 expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration (50, 100 m) in the western blotting (p<0.01). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p<0.01). conclusion; this study offers some scientific mechanisms, by which omega-3 fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox-2, nf b and pparare related. (2mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: 2-aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il-10 production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il-1b and il-6. we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d89-90 of gestation. fetuses received repeated iv injections of lps (100ng/kg estimated fetal weight/day) between d95 and d99. plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d94 and d104. at d105 ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days 97 and 103 (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n-3 and n-6 polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n-3 and n-6 pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid (18:1n-9), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid (18:1n-9), a trans-monosaturated fa, induced tnf production by 300-fold compared to vehicle (fig. a) . likewise, palmitic acid (18:0), a saturated fa, induced placenta tnf production by 800-fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid (22:6n-3, dha) reduced placenta tnf production by up to 90% following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid (18:2n-6, la) or arachidonic acid (n20:4n-6, aa) suppressed tnf production by up to 87% and 98%, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n-6 fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n-3 fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk1 mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk1 have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day 10 to term gestation, pregnant rats received either ad libitum food and water (control, n=7), or wr to produce an increment of 6 meq/l in plasma sodium (n=7). following delivery, all dams received ad libitum food and water. at d1 after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk1, mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to 18s). at d21 after birth, offspring glomerular number were determined in formalin fixed 5 m sections using histomorphometric analysis. values are means±se. results: wr offspring (day 21) had higher glomerular number than control (wr 25±1, control 22±1 per mm2, p<0.01). flk1 mrna expression was increased in wr offspring kidneys (wr 6.4±2.3, control 1.0±0.3, p<0.05) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr 0.9±0.1, control 1.0±0.2, p=0.8). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk1, increased receptors (flk1) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk1 expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day 10 to birth) reduces rat kidney glomerular number by 19% at 3 weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e16-e20, suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e16 fetal kidney explants from dams with mfr. methods: e16 fetal kidneys were collected from 50% mfr pregnant rats and ad libitum control rats (n=3 per group and time point), incubated in dmem/f12k medium for 0, 1, 2, 3, and 4 days, and fixed with 4% paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p<0.05. results: mfr ex-vivo kidneys at day 0 demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: 78±9 vs c: 86±3 terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p<0.05) increases in branching in explant culture to maximum values at 4 days (mfr: 147±16 vs c: 217±16). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of 9% at day 0, 14% at day 1, 24% at day 2, 20% at day 3, and 32% at day 4 (p<0.05, mfr vs c at day 4). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e16) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr91 and gcr99) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr91 knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr91 and gcr99 are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, 20% casein) or low protein (lp, 8% casein) diet throughout gestation. kidneys were collected on embryonic day 19 (e19), post-natal day 30 (d30) or 130 (d130), n 3. real-time pcr was used to compare gcr91, gcr99 and renin expression. data were standardized to a housekeeping gene (s15) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p<0.05). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr91 and gcr99 were significantly elevated compared with controls (3.6 fold and 4.7 fold respectively; p=0.02), whereas renin was 0.78 fold lower. on post-natal d30, there was a trend towards increased expression of both gcr91 (1.7 fold; p=0.07) and renin (1.6 fold; p=0.09) in offspring from lp dams. at d130, there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr91/ 99 pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 to produce iugr newborns. all pups were nursed by control dams and weaned at 3 weeks to ad libitum feed. at 1 day and 9 months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at 9 months, hepatic triglyceride content was determined enzymatically. results: at 1d of age, iugr pups showed significant downregulation of ppar (0.2-fold) and ppar (0.4-fold) expression, though crp expression was significantly upregulated (4-fold). findings persisted at 9 months of age, with continued downregulation of ppar and ppar (0.5-fold) and upregulation of crp expression (5-fold). furthermore, iugr adults had significantly increased hepatic triglyceride content (600±60 vs. 373±28 mg/g liver, p<0.05). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, 1 ederlen casillas, 1 guang han, 1 darran n tosh, 1,2 michael g ross. 1 1 dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat3 and pi3k pathways, whereas insulin activates pi3k pathway by binding to its receptor (ir) and substrate (irs2). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat3 and decreased expression of ir and irs2, suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21. 1 day old male offspring were given saline or leptin (1 g/g, i.p). hypothalamus was dissected at 15, 30 and 45 minutes and protein expression of total stat3, phosphorylated stat3 (p-stat3), inhibitor of leptin signal (socs3), ir, irs2, total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. (1) jak-stat3: leptin-treated controls show progressively increased pstat3 (4-fold) with initial suppression of socs3 (0.5-fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat3 expression (0.3-fold) with failure to downregulate socs3. (2) pi3k pathway: leptin-treated controls showed a significant reduction in irs2 (0.3-fold) and pakt (0.5-fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs2 (3-fold) and pakt (2-fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat3 (p-stat3) response in conjunction with an enhanced socs-3 response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, 2007) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from 3 week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from 3 week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus (106-109 viral infective particles) or a -galactosidase-adenovirus as a control. after 24 hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with 107 viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use 3 types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with 30% of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at 12, 24 and 60 weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day 28 after delivery (p<0.01). at 12 weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all 12, 24 and 60 weeks in offspring of b > c >a. (p<0.01, vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n=91). these women were divided into controls, ri < 0.78 (n=74) and cases, ri 0.78 (n=17), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching (48% vs, 55% p<0.001 and 22% vs. 68%, p<0.001, respectively). in contrast to that observed in patients of heterogenous parity previously, ri 0.78 was not associated with adverse pregnancy outcomes despite an average ri of 0.86, significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt-1 over-expression during pregnancy determines higher reactivity in the offspring airways at 6 months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt-1 induced preeclampsia at 1 year of age and compare responses between the two age groups. methods: cd-1 mice at day 8 of gestation were injected via the tail vein with adenovirus carrying sflt1 (adsflt1, 10 9 pfu/100 l) or mfc (admfc, 10 9 pfu/100 l). mice were allowed to deliver. tracheas were isolated from female and male offspring at 6 months and 1 year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, 60 mm), the mast cell degranulating agent compound 48/80 (48/80, 100 g/ml), and concentration-responses curve to acetylcholine (10 -9 -10 -5 m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound 48/80 between 1 year old offspring born to the sflt1 and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt1-treated group were significantly higher in 1 year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, 1 year old male offspring had higher responses to compound 48/80 and acetylcholine than 6 months old males. responses to kcl were significantly higher in 6 months old males than 1 year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where 6 months old offspring demonstrated significantly higher responses to acetylcholine compared to 1 year old offspring. conclusions: our findings did not show that airways of 1 year old offspring born to mice with a preeclampsia-like syndrome induced by sflt-1 over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf-1, which modulates growth and is secreted by the liver, may contribute to these morbidities. igf-1 is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf-1 mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day 10 to 21 and lactation, whereas study dams were 50% fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at 1 day and 9 months, male livers were analyzed for igf-1 mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h3k4 trimethyl, and associated levels of each igf-1 species were measured by pcr. results: at 9 months, obese fr/adlib males showed increased mrna levels of igf-1a, igf-1b, igf-1 exon 1, and igf-1 exon 2 as compared to controls (134±5, 165±6, 149±6, and 146±7 %). comparing fr/adlib 9 month to newborn offspring, h3/k4 was increased at igf1-promoter 1, promoter 2, exon 5, utr#3 and utr#4 (610±157, 731±167, 345±66, 1597±412, and 794±208 %), though there was no differences between control 9 month and newborns. in contrast, 9 month fr/fr males had comparable mrna levels to the controls except for igf-1b (% of control: 147±19). further, fr/fr 9 month h3/k4 was only different from newborns at utr#4 (% of newborn: 289±69). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf-1 mrna levels, likely a result of igf-1 histone and chromatin structure modifications to h3k4 trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf-1 epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; 156-amino acid peptide) which is then processed into its active forms: "big" igf-ii (1-87) and mature igf-ii (1-67). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats (200-250g) were given nicotine (1mg/kg/day) or saline for 14 days prior to mating, during pregnancy, and throughout lactation. at gestational day 15, 18 and 21, dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd1) and weaning (pnd21). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day 21. however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day 21. nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day 21. in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd21). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar 2), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp1c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to term. adipocytes from 1 day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following 100% confluence, iugr and control adipocytes were treated to two doses (1 and 10 m) of either rosiglitazone or badge for 24h. mrna and protein was extracted for expression of ppar , c/ebp , srebp1c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar (3.5-fold) and c/ebp (2-fold) as compared to control adipocytes, though srebp1c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation (125% of control, p<0.05) and showed greater response to rosiglitazone (2.5-fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf-1 is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf-1 mrna levels, likely a result of igf-1 histone and chromatin structure modifications to h3k4 trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf-1 changes which are distinct from diet-induced obesity. we determined igf-1 hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were 50% maternal food restricted from day 10 to 21. all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, 16%) diet whereas iugr were weaned to normal ad libitum diet (fat, 9%) to produce diet-induced (dio) and programmed obese groups, respectively. at 9 months, male hepatic igf-1 were analyzed for igf-1 mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h3k4 dimethyl and h3k4 trimethyl, and associated levels of each igf-1 species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf-1a, exon 1 and exon 2 (336±4, 267±4, 143±5%). chip with h3k4 dimethyl showed increased igf-1 exon 6 (215±20%) and with h3k4 trimethyl, increased igf-1 promoter 1 and promoter 2 (282±26, 1183±23%) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf-1a mrna and h3k4 dimethylation and trimethylation of igf-1 than dio controls. changes in igf-1 in adulthood from a prenatal insult thus suggest that igf-1 is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, 1 pandu gangula, 2 chandra yallampalli. 1 1 obstetrics gynecology, university of texas medical branch, galveston, tx, usa; 2 internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed 6% protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: 1) changes in estrogen receptor (er) angiotensin ii subtype receptor 1 (at 1 -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either 20% protein (ctrl) or 6% protein (lpd) from day 1 of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at 1 -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or 18s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at 1 -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at 1 -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at 1 -r or enos. no changes in at 1 -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at1-r or enos in females, and the decrease in enos but not at 1 -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with 30% of the normal gestation-matched dietary intake from day 17 of gestation to delivery (group b) and 30% restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at 12 and 24 weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at 8 weeks, were equilibrated at 2 g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with 5%co2 in air (37°, ph 7.4) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type-1 receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to 10-5m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride (60 mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day 1 (p<0.01). at 12 weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both 12 and 24 weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n=6). maximal contractions to ne were significantly stimulated by l-name in a (p<0.05), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p<0.05), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use 5 types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with 30% of the normal gestation-matched dietary intake from day 17 of gestation to delivery (group b), 30% restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally 30% restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at 12, 24 and 60 weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day 1 (p<0.01). at day 28 after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p<0.01). at 12 weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at 12 weeks in offspring of d> b > e > c >a. (p<0.01, vs. a). at 24 weeks, hypertensive offspring as b>d>e>c>a (p<0.01, vs. a). at 60 weeks, d> e > b > c >a (p<0.01, vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f1 blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day 30 in female offspring of rats exposed to hypoxia (14,000 ft; 11.6% po 2 ) from days 12 thru 15 of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n=15) were hypoxic from 12-15 dga with additional control dams either fed ad libitum (al, n=10), pair-fed with the hx dams throughout gestation (pg, n=12), or only pair-fed during the window of hypoxia (ph, n=12). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n=8/litter) by 48 h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p<0.05) than that of al pups, and hx pups were lighter (p<0.05) than ph pups. weight of hx offspring remained lower (p<0.05) than al pups until the termination of the study at pn120, while the pg and ph pups reached weights comparable to the al offspring by pn90. relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p<0.05) in the hx offspring at pn120 as was rv/lvs (p<0.05). cardiac function was evaluated by echocardiography at pn174. rv wall thickness was 47% greater (p<0.05) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were 36%, 14%, and 31% higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p<0.05) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p<0.05) in the hx pups. conclusion: prenatal hypoxia from 12-15 dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd48902. reproductive sciences; 2 pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp2 in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx (10.5%o2 in chamber) for 14d prior to term (65d). in two separate groups, nic was also added to the drinking water ( 18 mg/kg/d) for 10d at a dose that generates fetal nic levels (88ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( 63d) were excised and weighed. left ventricles of hearts were obtained and frozen at -80c for storage. mmp2 protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p<0.05) fetal body wt by 12%, increased (p<0.05) the relative fetal brain wt (brain wt/fetal body wt ratio) by 14.2% and had no effect on relative placental or fetal heart wts. hpx alone decreased (p<0.05) fetal body wt by 20.3%, increased the relative fetal brain wt by 12% but, in contrast to nic alone, increased relative placental wt by 33.8%. both mmp2 protein levels (mmp2/a-actin density values) and activity (clear band density) were increased (p<0.05) by nic alone (by 1.6 and 1.8 fold, respectively) and hpx alone (by 1.5 and 1.3 fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by 1.6 and 1.3 fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp2 expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide-3 kinase (pi3k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi3k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate 2 (irs-2), and phosphorylating pi3k. activated pi3k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk1/erk2) signaling pathway likely involves irs-1. further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi3k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n=5) food, whereas study dams were 50% mfr (n=5) from pregnancy day 10 to 21 to produce iugr newborns. at day 1, hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi3k (ir, irs2, pi3k and akt) and mapk (irs1, erk1, erk2) pathways, and pten. data is presented as fold difference normalized to beta-2-microglobulin. results: at 1d of age, iugr pups exhibited downregulation of the entire pi3k pathway with significantly decreased (p<0.05) mrna levels of ir (0.6-fold), irs-2 (0.5-fold), pi3k (0.6-fold) and akt (0.6-fold). further, iugr pups showed similar decreased mrna expression of erk1 (0.6-fold) and erk2 (0.5-fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi3k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day 18 of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day 1 (pn1). post mortem was carried out on pn1 (c and r) and pn7 (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn1 and pn7 (p<0.05). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn7. male, but not female, relative bmp4 mrna expression on pn1 was higher in r than c (p<0.05) while gdnf, tgf 1 and at1 receptor were not different. on pn7, wnt4 (but not at1r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p<0.05) when compared to c-on-c (p<0.05). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p<0.05) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn7. higher bmp4 mrna expression indicates impaired branching morphogenesis in pn1 r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn7 there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn7. amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa10 is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f2 hoxa10 gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b6d2f1 (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa10 expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n=5, 4.7±3.3 pups; n= 4, 8.7±2.3 pups, respectively; p<0.01). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa10 mrna and protein expression were decreased in the uterus of the f2 females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with 1nm to 100nm ghrelin resulted in a 30 to 80% increase (p<0.01) in hoxa10 expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa10 expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos3) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos3. in this study we aimed to determine hepatic and renal histopathologic damage in nos3 deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos3 +/+wt and nos3 -/-ko mice during their first (p0) or second (p1) pregnancy were sacrificed at day 18 of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos3 +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos3 -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was 44% and 49%, respectively. in nos3 -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were 6% and 19%, respectively (p<0.05). this correlation persisted when we analyzed the relative severity of hepatic lesions between p0 and p1 animals. although a similar trend was observed, the difference between p0 and p1 animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p1 versus p0 nos3 -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, 1,2 r de jonge, 3 wa helbing, 2 bd van zelst, 3 j lindemans, 3 eap steegers, 1 rpm steegers-theunissen. 1,2,4,5 1 obstetrics gynecology; 2 pediatrics; 3 clinical chemistry; 4 epidemiolog y biostatistics; 5 clinical genetics; 6 erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at 16 months after the index-pregnancy. case-children (n=24) were included if they had ds and chd. children (n=251) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or(95%ci) of the biomarkers are shown in table 1. an increase of the sam/sah ratio with 1 unit decreases the risk of a child with ds and chd with 30 percent. moreover, every increase of 1 mol/l of homocysteine 1.1 fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon 5 of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c57bl/6 female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting 18% protein or 9% protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at -80c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct-4 and sphk-1 gene upstream regions in the mouse liver was analyzed. hepatic oct-4 or sphk-1 promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct-4 and sphk-1 gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both 24 and 48 hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp-3) were downregulated at both 24 and 48 hours after infection. sdf-1, which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam-1, contrasted by decreased amounts of vcam-1, indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as 24 hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n=6 in each group) with twin fetuses at 118-120 days (very preterm) and 140-142 days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at -80c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b90: 7.2 ± 1.5 pg/ml) and cortisol (diagnostic laboratory, b90: 1.2 ± 0.4 ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable (33±4 ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: 86±6 pg/ml; cortisol: 55±1 ng/m) and fetal plasma (crf: 730±68 pg/ml; cortisol: 5±2 ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p <0.001). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin objectives: interleukin-6 (il-6) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il-6 may be a marker of maternal obesity and in utero fetal programming. our hypotheses were 1) il-6 correlates with maternal obesity and 2) il-6 mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il-6 levels from 173 non-diabetic women who underwent a live birth. the independent variable was il-6 (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< 25 kg/m 2 ); overweight (25-30 kg/m 2 ); and obese (> 30 kg/m 2 ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il-6 levels were compared across bmi categories using anova. the association of il-6 levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il-6 levels were significantly higher in obese women (2.6± 1.6) compared to overweight (1.2± 0.4) and normal/underweight women (1.1± 0.7) [p< 0.001]. after adjustment, il-6 levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc)0.12; 95% ci: (0.05, 0.2)]. as shown in the table below, elevated levels of il-6 were statistically significantly associated with a 0.5 gm higher infant birth weight after full adjustment. each 1 unit increase in pre-pregnancy bmi was associated with a 8 gm higher infant birth weight. table 1 . association of il-6 with infant birth weight characteristic regression coefficient 95% confidence interval interleukin-6 0.5 0.02, 0.7 pre-pregnancy bmi 8 0.7, 38 gestational weight gain -3 -15, 10 bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il-6 and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il-6 or an alternative independent pathway. we have demonstrated that 9 -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line 1 . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase 3 (hdac3). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at 1 or 4 x 10 5 cells /well to 96-well and 6-well plates for growth and rna experiments, respectively. after growing to 70-80% confluence, cultures were treated with varying concentrations of aea up to a maximum of 30 m for 48hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac3 and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of 3 m where confluency was significantly reduced from 75% at 3 m to 50-60% at 15 m and 30 m (*p<0.05; one-way anova with tukey's hsd test; n= 4). cultures treated with 15 m and 30 m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac3 mrna expression with a significant effect at 0.3 m (*p<0.05; oneway anova with tukey's hsd test; n=9). at this dose, the effect of aea had reached an effective maximum decrease in hdac3 mrna levels (48%) because hdac3 mrna levels were not decreased further by either 3 m aea (45%) or 30 m aea (49%). the alteration of hdac3 gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the 1 st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as 1 st trimester miscarriages. (1) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb1 and cb2 with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb1, cb2 and faah constitute the endocannabinoid system and previous studies have shown that faah and cb1 are expressed in term human placenta(1) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb1 and cb2 expression in the placenta during the first trimester of pregnancy. methods: first trimester samples (7 to 12 weeks gestation) were fixed in 10% neutral-buffered formalin for 4 days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of 1:2000 in pbs, cb1 at 1:4000 and cb2 at 1:500. transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb1 and cb2 were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the 7th and 10th gestational week and by week 11 faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb1 and cb2 localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb1 immunoreactivity showing diminished intensity after week 10, although this did not reach significance at the transcript level. cb1 immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb1 and cb2 immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb2 immunoreactivity in the syncytiotrophoblast differed from that of cb1 and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb2 levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb1 expression from the syncytiotrophoblast after the 10th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., (2003) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb-131 complete solution containing cgrp (10 -8 m), cgrp plus its antagonist cgrp 8-37 (10 -7 m), in 6 well plates with 2x10 5 cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein 1 (ramp1) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: 1) immunofluorescent staining showed that cgrp receptor components crlr and ramp1 are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; 2) cgrp dose-dependently (10 -10 to 10 -7 m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp 8-37; 3) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and 4) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp (10 -8 m) was significantly increased over that of the untreated controls. this increase was observed at 12 hours of treatment and further increase was noted at 24 hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs-3 gene promotes il-10 production by jeg-3 trophoblast cells. qin dong, 1,2 ruping fan, 2 yang gu, 2 david f lewis, 2 yuping wang. 2 1 biochemistry, harbin medical university, harbin, heilongjiang, china; 2 obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling-3 (socs-3) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il-10 expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs-3 in placental trophoblasts could promote il-10 production. methods: full-length socs-3 open reading frame (socs-3 cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen1-n1 vector, which encodes a green fluorescent protein zsgreen1. successful socs-3 cloning was confirmed by sequencing. for transfection, jeg-3 cells were placed into 6 well/cluster plates at a concentration of 1.2 x105/well. the tranfection was carried out with 1.0 g of socs-3/zsgreen1 plasmid (psocs-3/zsgreen1) for 6 hours when cell reached 45-50% confluence. siport lipid were used. jeg-3 cells transfected with zsgreen1 plasmid (pzsgreen1) only was used as control. after approximately 60 hours of transfection, cells were treated with il-6 at 1 and 10ng/ml. medium was then collected and measured for il-10 by elisa. il-10 production was calculated as the percentage of increase by psocs-3/zsgreen1 transfected cells compared to the cells transfected with pzsgreen1 only. result: il-10 production was increased by psocs-3/zsgreen1 transfected jeg-3 cells compared to the cells transfected with pzsgreen1 only when stimulated by il-6, control: 9.9% increase; 1ng/ml il-6: 28.5% increase and 10ng/ml il-6: 140% increase, p< 0.05, respectively. data are means from three independent experiments. conclusion: over-expression of socs-3 gene could promote il-10 production by placental trophoblast cells. reduced sil-6r release and increased ratio of sgp130/sil-6r production by placental tissues from women with preeclampsia. shuang zhao, 1 ruping fan, 1 jingxia sun, 2 yang gu, 1 david f lewis, 1 yuping wang. 1 1 obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; 2 obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il-6, il-8 and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il-6 receptor (sil-6r) and membrane receptor il-6/gp130 complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp130 (sgp130) is an antagonist for il-6/il-6r trans-signaling. this study was undertaken to determine sil-6r and sgp130 production by villous tissues from normal and pe placentas. methods: placentas delivered by 8 normal and 9 pe pregnant women were used in this study. placental explants were incubated with dmem for 48h. the culture medium was collected. placental villous tissue productions of sil-6r and sgp130 were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level <0.05 was considered statistically different. results: placental tissues from pe produced significantly less sil-6r than tissues from normal pregnancies, 3.56±0.61 vs. 5.84±0.34 pg/mg of wet tissue, p<0.01. soluble gp130 production was relatively compatible between pe and normal placental tissues: 162.29±21.71 vs. 162.83± 5.56 pg/mg of wet tissue. the ratio of sgp130/sil-6r release was significantly higher in pe than in normal placentas, 49.84±6.93 vs. 28.62±0.02, p<0.01. conclusion: reduced sil-6r production and/or increased ratio of sgp130/sil-6r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, 1 jing dai, 2 frank z stanczyk, 2 daniel r mishell, jr. 2 1 obstetrics and gynecology, university of louisville, louisville, ky, usa; 2 obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from 137 healthy pregnant women and 170 diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs-180 and immulite systems. data was grouped in 4-week intervals until 16 weeks of gestation and 8-week intervals thereafter, with 21 to 27 samples in each interval for healthy women and 6 to 56 samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs-180 system, mean hcg levels (miu/ml) for healthy pregnant women were 64,489 at 5-8 weeks, 100,421 at 9-12 weeks, 52,724 at 13-16 weeks, 16,741 at 17-24 weeks, 16,580 at 25-32 weeks, and 22,535 at 33-41 weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were 45,165, 59,899, 45,448, 19,920, 20,372 and 19,525 , respectively. between 9-12 weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p=0.02) compared to healthy controls. 3) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/4) and ace knockout (4/4) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type 1 receptor (at1r), ang type 2 receptor (at2r), ace, and ace-2 in the three genotypes (n=3/group). placental sections were blinded to genotype; a score range of 0-4 was used. the 2 test was used (spss version 15.0) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of 4/4 mice compared to wt/wt and wt/4 mice (at1r p<0.05; at2r p<<0.001; ace p<<0.001; ace-2 p<<0.001). at2r and ace-2 displayed increased staining in the fetal vascular endothelium of 4/4 mice (at2r p<<0.001; ace-2 p=0.05), and in the cells lining the maternal central artery (at2r p<<0.001; ace-2 p<<0.001). ace-2 expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in 4/4 placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at2rs is vasodilatory and ace-2 catalyses production of the vasodilatory ang (1-7); placental blood flow was presumably well-maintained and pregnancy outcome is normal in 4/4 mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of 17 alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect 17 alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with 17 alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of 17 alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase 1 (cox-1), cyclooxygenase 2 (cox-2), and 15-hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with 17 alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox-1 was relatively unchanged and pgdh was only slightly increased, but cox-2 was noticeably decreased in the treated slides. these results were time dependent. this data suggests that 17 alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase 2 enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf1r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp-2 in this pathway. methods: amaxa nucleofector technology was used to deliver shp-2 or scrambled sirna (100nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for 72 hours, then treated with igf-i or igf-ii (10nm) for a further 24 hours before immunohistochemical (ihc) analysis for cell proliferation (ki67, brdu) or apoptosis (m30). results: sirna-mediated knockdown of shp-2 in bewo cells (95% reduction on western blot) demonstrated that igf-induced proliferation was reduced from 67.3±3.5% to 41.9±2.4% (p<0.001, n=5). ihc analysis of tissue demonstrated that shp-2 is localised to ct. following knockdown (93% decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by 60.02±7.4% and 55.9±9.6 % respectively (p<0.001, n=4). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m30 staining) was reduced by 48.3±10.1% and 39.1±7.9% respectively (p<0.05, n=4) after shp-2 knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp-2. exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp-2-dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr1) and estrogen receptor-beta (esr2). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr2 has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr2 knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol (10 -9 m to 10 -6 m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr2 is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase-2 (cox-2) mrna levels 3-to 6-fold, as quantified by real-time pcr (p<0.001). conversely, there was no effect of estradiol on cyclooxgenase-1 (cox-1). conclusion: these results suggest that estradiol and esr2 are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u54-hd40093. previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx40, her-1 and alpha5 integrin protein expression) to an invasive phenotype (characterized by her-2 and alpha1 protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her-1 receptor antagonist, ag1478. furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag1478. thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her-1 ligand, resulted in the downregulation of her-1 and an upregulation of her-2 expression, as well as an induction of alpha1 integrin expression. pre-treatment of placental villous explants with ag1478 blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her-1 and upregulating her-2 expression, effects that were both blocked when jar cells were pre-incubated with ag1478. in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her-1/her-4 heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her-4 in evt outgrowths and in jar cells; her-4 expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her-4 signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr-8/svneo cells). methods: htr-8/svneo cells cultured at 37º c and 5%co2 in rpmi-1640 medium supplemented with 5% fbs were transiently transfected with 100nm of three different sirna sequences of pthrp, si1, auaccuaacucaggaaacuuu; s i 2 , g a g c u g u g u c u g a a c a u c a u u ; a n d s i 4 , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at 90% confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent 24 h after transfection, and protein was extracted using lysis buffer 72 h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either 18s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si4 sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c57bl/65 mice were fed a hypercaloric diet (20% fat, 38% sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day 18 gestation) the dams (4 controls, 4 overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse 430a_2.0 array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. 41 probe sets, corresponding to 37 genes showed differential expression (p < 0.01); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd-03807, earnest eu framework 6, tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing 50% less protein than normal chow (10% vs. 20% protein content) from embryonic day 10.5 (e10.5) to e17.5. following placental rna extraction, we used the affymetrix mouse 430a_2.0 array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e17.5 pups were decreased 15% (p<0.05). 244 probe sets, corresponding to 235 genes, were regulated by protein restriction (p<0.001); ninety-one being up-regulated and 153 down-regulated. of particular note, several genes related to the p53 pathway were up-regulated. along with p53 itself, positive regulators of p53 (zmiz1, jmy, hipk2) and genes activated by p53 (inpp5d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p53 pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p53 pathway. (supported by usphs hd-03807 and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of 1-chloro-2,4-dinitrobenzene (cdnb) conjugate 2,4-dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within 30 minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m199 medium to 48hr. at 2, 4, 6, 10, 24, and 48hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to 100 μm cdnb, rinsed and incubated in buffer at 37°c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp1-1, abc transporter isoforms b1, c2 and g2 (abcb1, abcc2, and abcg2, resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to 48hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p1-1 (gstp1-1) and abcg2 remained unchanged over 48hr in culture. expression of abcb1 and abcc2, and total tissue glutathione decreased with culture time. ldh release was unchanged up to 24hr and increased at 48hr, while mtt incorporation remained constant to 10hr and decreased at 24 and 48hr suggesting a decline in tissue integrity and viability at 48hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through 48hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by 70.5 ±6.90% (p<0.05), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp1-1 and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor 2 (eif2 ) is a subunit of eif2 which is required for initiation of translation however when phosphorylated, eif2 is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif2 . we hypothesized that hypoxia would increase the levels of phosphorylated eif2 in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for 18 hr in atmospheres of 1, 3, 5 or 10% o 2 or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, 0.02-2.0 mm) in 20% o 2 . cell extracts were analyzed by western blotting to determine the degree of eif2 phosphorylation. results: incubation in 1, 3, or 5% o 2 did not increase eif2 phosphorylation relative to the 10% o 2 control (n=4, separate placental preparations). incubation in dmog concentrations up to 0.5 mm did not affect eif2 phosphorylation however incubation in 2.0 mm dmog increased eif2 phosphorylation by 58 ± 15% (p < 0.05, n=3). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif2 regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif2 phosphorylation does occur, we did not observe changes at dissolved oxygen levels of 1%. these data suggest that a reduction in syncytial protein synthesis via the eif2 pathway takes place only under severe hypoxic stress. (supported by nih hd46982). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat1, 2 and 4 which mediate neutral aa transport. snat2 is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat2. methods: nonpregnant ewes were randomly assigned to a control (c, 100% of nrc recommendations) or obesogenic (ob, 150% of nrc) diet from -60 to 75 days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at -80 0 c for western blot analysis. results: fetuses from ob ewes were 26% heavier (p<0.05) than those from c ewes at 75 dg (234± 7 vs. 186±7g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p<0.05), or tended to be higher (met and lys, p<0.10) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p<0.05) in ob compared to c ewes. snat2 content in cot tissue was reduced in ob when compared to c ewes (0.5±0.1 vs. 1.0±0.2 arbitrary units; p<0.05). conclusions: maternal obesity in pregnancy reduced expression of placental snat2 protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre 1p20rr16474. early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o 2 and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o 2 uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n=9) or normothermic (con; n=6) environment at 40 days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until 55 dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate (153.3±5.4 vs 91.6±9.2 breaths/min) and arterial po 2 (91.0±1.2 vs 85.8±1.0 mmhg) were increased (p 0.01), whereas maternal heart rate (74.3±1.83 vs 88.8±0.03 beats/min), blood pressure (83.7±1.3 vs 94.0±3.2 mmhg) and arterial pco 2 (30.2±1.1 vs 35.6±0.9 mmhg) were reduced (p 0.01) in fgr pregnancies. at 55 dga, fetal weight was not different (p 0.10), but placental (total placentome) weight (85.5±15.1 vs 146.7±27.0 g) was reduced (p 0.05) in fgr pregnancies. while uterine artery (pregnant horn) flow (115.5±13.4 vs 178.4±54.3 ml/min) tended (p=0.064) to be reduced in fgr pregnancies, relative uterine artery flow (4.6±0.5 vs 5.8±0.5 ml/min/g fetus; 157.6±25.8 vs 128.1±17.3 ml/min/100g placenta) was not different (p 0.10). uterine o 2 uptake (mmol/min), relative uterine o 2 uptake (ml/min/g fetus or ml/min/100g placenta) and uterine o 2 extraction (%) were not different (p 0.10) between fgr and con pregnancies. at 55 dga, umbilical vein po 2 (mmhg), o 2 content (mm) and o 2 capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o 2 uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o 2 to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd43089. barton c staat, 1 anna maria marconi, 2 cinzia paolini, 2 alex cheung, 1 henry l galan, 1 frederick c battaglia. 1 1 obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; 2 dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with 13 c labeled isotopes of glucose, myo-inositol and mannose over 2 hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at 1 hour (h), 1.5h and 2h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of 10 polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose (0.985 ± 0.05, p=0.03) and glucose (0.917± 0.06, p=0.002) were not significantly different from 1.0, consistent with transplacental supply. the feto-maternal ratio for myo-inositol (0.1 ± 0.05, p=0.02) indicates little transplacental flux (10% of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, 1 manuela augsten, 1 christian guelly, 2 ursula hiden, 1 ingrid lang, 3 manfred moertl, 1 uwe lang, 1 gernot desoye. 1 1 clinic ob/gyn; 2 center of med res; 3 inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser1177-phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with 125 i-labelled hdl. hdl from human donors was used to stimulate ser1177 enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for 4 cpg-rich promoter regions of sr-bi. pdzk1 a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after 7 passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was 2-fold higher in eca than in ecv (89 ± 0.4 vs 49 ± 4.1 ng hdl/mg prot). hdl did not induce ser1177 enos phosporylation in eca or ecv, which was stimulated by ionomycin about 3-fold in both cell types. pdzk1 was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk1 may account for the failure of hdl to activate enos. (grants 10053, 10896, 11165 oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p70 and the 4ebp1proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p70 and 4ebp1 in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia (2% o 2 ) or normoxia (21% o 2 ) for 24 and 48 hours. western blot for p-mtor, mtor, p-p70, p70, p-4ebp1, and 4 ebp1 were done for each time studied. results: trophoblast cells demonstrated: 1) positive staining for cytokeratin, 2) non significant differences for mtor at either 24 (1.5-fold; p= 0.128971) or 48 hours (1.4-fold, p= 0.13181), 3) no differences in p70 protein at 24 (1.1fold; p= 0.128971) or 48 hours (1.3-fold, p= 0. 0.153458), 4) no differences for 4ebp1 at either 24 or 48 hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r01 hl071990-01a1). increased expression of phospho-mtor, phospho-p70, phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p70 are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p70, 4ebp1, erk and akt will be phosphorylated (activated) in the placentae of 130 age (dga) animals. study design: 4 ewes were exposed to ht conditions for 80 days to induce iugr and 4 were placed in ambient conditions. at necropsy (130 dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p70, p70, (p) 4ebp1, 4ebp1, (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses (2914±201g v. 1718±433g; p=0.03) and smaller placentae (349±21g v. 169±22g; p=0.03) at 130 dga. fgr cotyledon showed an increase in p-mtor (1.8-fold; p=0.01), p-p70 (1.8-fold; p<0.008), p-erk (1.4-fold; p<0.008) and p-akt (2.6-fold; p<0.02). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p70 but not 4ebp1 while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation (95 dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r01 hl071990-01a1). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for 2 hours with uric acid (8.3 mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid (10 m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [14c] methyl-amino-isobutyric acid, was measured over 20 minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by 33% (±0.06%, p<0.05). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as 10 weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid (4.5 mg/dl, 6.4 mg/dl and 8.3 mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr8-svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within 48 hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p<0.05), with decreases of 29% (± 5.6%), 58% (± 3.6%) and 71% (± 2.2%) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor 2 (smurf2), a hect type e3 ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf2 has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf2 has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf2 in human placental villi during pregnancy. we have also investigated the possible function of smurf2 in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr8/svneo. our results showed that expression of smurf2 in placental villi was the highest during the first trimester and the expression decreased in the 2nd trimester. expression of smurf2 was lowest in placental villi at parturition. overexpression of smurf2 in htr8/svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf2 resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad2, another potential target of smurf2, was unchanged. in conclusion, the present study suggests that smurf2 participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp-26 in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp-26 expression were examined in an immortalized human cytotrophoblast cell line, b6tert-1 that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp-26 mrna and protein levels in b6tert-1 cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp-26 expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp600125, an inhibitor of a jnk kinase, reversed the up-regulation of mmp-26 induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk1/2 pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp-26. during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than 5%. however, the majority of studies on primary trophoblast cell development have been performed at 20% oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd-9 negative selection and cultured at 20%, 5% or 2.5% oxygen tension for up to 5 days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at 20% oxygen 60% of the cells remain in culture; at 2.5% oxygen tension 80% of the cells remained. the cells at 2.5% oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing 822 cylindrical recesses 200 m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels (800,000 cells per) for up to 10 days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days 3, 7 and 10 revealed central vacuolization forming a trophoblast vesicle with an outer rim 12.3 m (+/-1 m) thick. this rim size remained constant for at least 20 days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase-3 staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within 7 days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing-1 (gcm1) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm1 mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase-4 (timp-4) in the highest (4-fold) upregulated group of genes. confirmatory rtpcr demonstrated a 7-fold mrna induction. in placental villi, gcm1 acts as a transcription factor promoting expression of the fusogenic protein syncytin1 that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp4 and gcm1 in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in 8% oxygen with prior exposure to either gcm1 sirna or anti-sense oligo-nucleotides to gcm1. gcm1 inhibition in the bewo system was associated with a 50-70% increase in timp-4 protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in 3% oxygen) to test the hypothesis that gcm1 mediates metallo-proteinase expression and evt invasion via timp-4. presently we conclude that gcm1-mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp-4. funding: cihr. scott h purcell, 1 jeremy d cantlon, 1 virginia d winn, 2 russell v anthony. 1,2 1 colorado state university, fort collins, co; 2 university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr15. objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin-7. cytotrophoblasts from first trimester pregnancies (n=5) were subjected to an in vitro invasion assay and rna was harvested following 0, 3, and 12 h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs15 mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for 72 h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from 0, 3, to 12 h in all samples; while hypoxia decreased expression at 18 h of invasion compared to 18 h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at 2, 18, 83 and 97% of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than 98% mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd 17-47 ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr-8/svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek1/2/ erk1/2 phosphorylation. objectives: 1) to asses the effect of imd on migrating capacity of htr-8sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u0126 and manumycin a, respectively ; 2) to assess the effect of imd peptide on phosphorylation of mek1/2 and erk1/2 in first trimester htr cells 3) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr-8sv/neo cells were used to assess the effect of imd (10 -8 m) on the expression of hla-g mrna and phosphorylation of erk 1/2and mek1/2 protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to 18s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, 1) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u0126 and manumycin a, respectively; 2) imd (10-8 m ) stimulates phosphorylation of erk1/2 and mek1/2 proteins in htr cells, 3) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt-1. yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor-1 (sflt-1) in the maternal circulation during pregnancy. increased placental release of sflt-1 is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt-1 release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt-1 release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = 5) were used. we tested if chymotrypsin could promote sflt-1 release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at 1.0, 2.5, and 5.0 g/ml for 6 hours. the culture medium was then collected for measuring sflt-1. we then determined the specificity of chymotrypsin induced sflt-1 release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt-1. soluble flt-1 was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < 0.5 is considered as statistically different. results: 1) sflt-1 concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt-1 release induced by chymotrypsin was in a concentration-dependent manner: control: 8.25±1.39; 1.0 g/ml: 10.51±1.89; 2.5 g/ml: 13.03±2.06; and 5.0 g/ml: 16.12±2.23 (p<0.01) pg/mg tissue/hour. 2) ci could attenuate sflt-1 release. this inhibitory effect was also revealed in a concentration-dependent manner: control: 5.67±0.92; ci 0.5 g/ml: 4.96±1.30; and ci 5.0 g/ml: 3.08±0.47 (p<0.05) pg/mg tissue/hour. conclusions: increased placental sflt-1 release stimulated by chymotrypsin and decreased placental sflt-1 release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt-1 generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt-1 production in pe. (supported nih grants hl65997 and hd36822). the change of autophagy-related proteins, lc3 and beclin-1, by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc3, but not beclin-1, was increased in placentas from pregnancies complicated by severe preeclampsia (sgi 2007abstract #209) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl 2 for a given time and the changes of beclin-1 and lc3 were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc3-ii in cultured primary trophoblasts while decreasing the expression of beclin-1 (p<0.05 for each). however, cocl 2 stimulation did not induce a significant change of both lc3-ii and beclin-1. conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct8, recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t3. we previously reported that mct8 expression in n-tera-2 (nt2) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct8-null jeg-3 choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s448x mct8 mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l471p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct8 via sirna and evaluate the effects of cell proliferation (mtt and [ 3 h]-thymidine assays) and tri-iodothyronine (t 3 ) uptake. results: repression of endogenous mct8 expression in nt2 cells by 90 % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t 3 concentration (8.6 %, 7.2 % and 8.3 % induction at 0, 10 and 50 nm t3, n=3, p<0.02). we also sought to examine the role of mct8 in t3 uptake. in jeg-3 cells, wild type mct8 induced a 1.7-fold increase in the uptake of 125 i-labelled t 3 . by contrast, mutants s448x and l471p failed to significantly augment t 3 uptake, though r271h caused a mild but significant 1.17 fold induction in uptake, hence retaining approximately 25% of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t 3 binding protein, resulted in a similar increase in t3 uptake compared with control (1.9-fold; n=6; p<0.001), implying that mct8 plays only a minor role in thyroid hormone efflux in jeg-3 cells. mutants s448x, l471p and r271h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct8 in the modulation of cell proliferation, independent of t 3 transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between 1987-2001 with a singleton pregnancy >=24 weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included 1,123 preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as 10.5% (p=0.01) among multiparous women 24-28 weeks ga to as high as 70.8% (p<0.01) among nulliparous women with diabetes. a total of 1,019 women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt1, endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in 30 women with uncomplicated normotensive pregnancies compared to 20 women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average 10.6±6.2 months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p<0.05. results: the mean concentration of sflt1, endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih-5mo1-rr00056 and nih-2po1-hd30367. (ajp, 2007) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had 50% mfr from day 10 of gestation until delivery. mfr and control offspring were sacrificed on day 1 of life (p1). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into 1-2 mm explants and incubated with leptin (25-100ng/ml) in dmem (10% fbs). after 24 hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr1 and r2, and ob-ra, stat3 and socs3 (18s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p1 mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr1 and vegfr2 mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat3 and socs3 mrna in the control tissues. in contrast, in mfr explants only the 50ng/ml concentration of leptin induced an increase in stat3 mrna, and the magnitude of socs3 mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a 10-year interval, 243 primiparous women with a history of early-onset preeclampsia (delivery <34 weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of 374 healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was 30.5 years for cases compared to 28.3 years for controls (p<.001). after adjustment for age, we observed significantly increased mean values for weight (p=.002), body-mass index (p<.001), systolic blood pressure (p<.001), diastolic blood pressure (p<.001), total cholesterol (p=.006), ldl cholesterol (p<.001), triglycerides (p=.027), fasting blood glucose (p<.001), and lower hdl cholesterol (p<.001) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated 10-year risk of first cvd events by framingham risk scores remained <10% for all women (low-risk). nonetheless, at mean (sd) 0.7 (1.0) years after early-onset preeclampsia, 15% of women met the criteria for metabolic syndrome, 89% of women exhibited >=1, 51% of women >=2 and 19% of women >=3 major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited 129 women with hypertensive disorders in pregnancy (63 pe and 66 non-proteinuric hypertension [ht]) and 129 matched normotensive controls. inhibin a and activin a levels, before and 24-48 hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in 84 placentas delivered at cesarean section at similar gestational age (29 pe, 24 ht and 31 controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p<0.0001). activin a (but not inhibin a) level was also increased at all gestations in ht (p<0.0001). after 28 weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p<0.0001). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after 28 weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase 1 (sflt-1) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = 29), ht (n = 24) and normotensive pregnancies of similar gestational age (n = 32). these samples were stored at -80ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt-1 and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p=0.0002). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p<0.0001); there was no significant difference between the levels in pe and ht (p=0.3). the fact that placental concentrations of sflt-1 and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt-1 was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt-1 in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved 2831 asymptomatic pregnancies scanned at 11 +0 -13 +6 week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n=43) and normotensive controls (n=129) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at -84 o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p<0.05 was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was 13% and 22% significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt1 (1762.1 ± 397.5 vs. 1169.4 ± 71.1 pg/ml, p<0.05) and placenta growth factor (22.0 ± 3.5 vs. 49.8 ± 5.4 pg/ml, p<0.016) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate 56% with 10% false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt 1050482. currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin-6 (il-6) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il-6 levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery <34 weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il-6 at baseline, 1.3 days and 3.3 days after vaccination. results: median baseline crp and il-6 levels of women with a history of early-onset preeclampsia were comparable to controls (1.6 versus 0.8 mg/l; p=0.44 and 5.0 versus 3.4 pg/l; p=0.34, respectively). however, high crp and il-6 responses to vaccination were more common in cases (ors for response >75th, >80th, >85th, >90th and >95th percentile based on the distribution of control values of 2.3, 2.7, 3.1, 4.3 and for crp [p for trend 0.11] and of 0.9, 1.4, 1.9, 2.6 and 4.5 for il-6 [p for trend 0.043], respectively). the relationship between high il-6 responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend 0.048). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor 4 (tlr4) and nucleotide-binding oligomerization domain 2 (nod2), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr4 (d299g and t399i) and nod2 (r702w, g908r and l1007fs) in 340 primiparous women with a history of early-onset preeclampsia, of whom 177 women developed hellp syndrome and in 113 women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il-6, sicam-1, fibrinogen and von willebrand factor in a subset of 214 women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr4 were more common in women with a history of early-onset preeclampsia than in controls (or 2.9 [95% ci 1.2-6.7]). highest frequencies for tlr4 variants were observed in women who developed hellp syndrome (adjusted or 4.1 [95% ci 1.7-9.8]). in addition, high levels of il-6 and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr4 and nod2 allelic variants and high levels of il-6 was 6.9-fold more common in women with a history of early-onset preeclampsia (95% ci 2.1-23.2) compared to controls. we observed an association of common tlr4 and nod2 gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples (68) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n=45), mild pe (n=8 bp; 140/90-160/110, proteinuria; trace to 1+ or 5-300mg/24hr urine) and severe pe (n=15 bp; >160/110, proteinuria; >2+ or >5g/24hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n=4) and with pe (n=5) were fixed and embedded in paraffin. sections (7um) were immunostained with pbef antibody 1/250 (pheonix) and treated with abc reagent (vector labs) followed by dab (0.5% mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between 5-150 ng/ml and were significantly higher in those patients with mild pe (p=0.027) and further significantly elevated in those with severe pe (p=0.003) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u54rr14607-08 to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: 25 healthy, non-smoking primigravida with an uncomplicated pregnancy and 16 primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at 16, 24, 32,37 wks in controls, at time of diagnosis in pe cases and 6 wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd40 expression (mcd40) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd40 expression and pp-sel increased with gestation and decreased post-natally (table 1) . for pe analysis, data was divided into pre-term (sampled at mean 30 wks), and term (mean 38 wks). pp-sel was lower in pre-term pe than control (normal pregnancy 32 wks; p=0.03) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase 1 (sflt1), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt1 as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between 11+2 and 13+6 weeks of gestation from 46 women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt1 were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, 5.57 ± 1.18 ng/ml versus 5.02 ± 1.01 ng/ml, p = 0.009, unpaired mann-whitney test). similarly, the serum levels of sflt1 were higher in women later developing late-onset pre-eclampsia (1764 ± 757 ng/ml) when compared to controls (1537 ± 812 ng/ml, p = 0.036). sensitivities and specificities for predicting pre-eclampsia were 63% and 57% for seng and 64% and 56% for sflt-1, respectively. the combination of the two markers by multiplication yielded a sensitivity of 64% and a specificity of 55%. conclusion: seng and sflt1, showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, 1 adrienne schonberg, 2 gary j badger, 3 ira m bernstein. 2 1 biochemistry; 2 obstetrics and gynecology; 3 medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd63 expression (%cd63) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in 16 healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day 7.5+0.9). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, 11-16 wks) and late pregnancy (lp, 31-34 wks). five of these women were diagnosed with hypertensive complications (4 hypertension, 1 preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p<0.05 was accepted for significance. results subjects were 29.1+0.9 years old with a bmi of 23.5+0.9 kg/m 2 at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p=0.015). there was little change in the %mp formed between pp and ep (pp, 6.0±3.8 % and ep, 10.4±3.8 %, p=0.384). however, the %mp increased significantly in lp (20.9±3.8 %) as compared to pp (p=0.005) and ep (p=0.041). this increase occurred independent of the development of hypertensive complications (p=0.657) and independent of pp platelet activation status. although statistically significant increases in cd63 expression were not observed, the change in cd63 expression over pregnancy correlated with the change in %mp over pregnancy (r=0.56, p=0.024) and cd63 expression correlated with %mp in lp (r=0.57, p=0.020). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd63 expression, during pregnancy. supported by nih hl 71944. backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day 22 + 5) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of 25 or 100 mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at 1 and 3 hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n=5) versus sc (n=6). p<0.05 was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc (68% increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young (29.1 + 2.9 years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day 7.5 + 3.6) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian (15/16) with a mean prepregnancy bmi of 23.5 + 3.5 kg/m 2 . plasma volume was re-estimated at 32-34 weeks gestation. all patients were placed on sodium and total calorie balanced diets for 3 days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was 2,868 + 446 ml or 123 + 18 ml/unit bmi. third trimester plasma volume was 4,306 + 663 ml representing a 51 % increase (p<0.001). the range of plasma volume expansion was 20-84% dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= 0.65 (p=0.006). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately 42% of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl 71944. background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt-1 (soluble vegfr-1) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt-1 in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n=2). the first set delivered at 23 weeks due to excessive bleeding. the second set delivered at 26 weeks due to severe iugr and elevated blood pressure. mrna level of sflt-1 was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt-1 in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt-1. immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl-1. results: mrna level of sflt-1 was increased by 2.3 fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited 2.7 fold increase compared to controls. sflt-1 protein expression in the molar placentae was increased by 7.7 fold compared to controls, while the co-twin placentae exhibited a 1.4 fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt-1 in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt-1 expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt-1 in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n=145); 2) neonates of patients with pe (n=104); and 3) sga neonates (n=48). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration <5 th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: 1) cord plasma pz concentration differed significantly among the 3 study groups (kruskal wallis, p<0.001); 2) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median 0.45 g/ml, range 0.05-1.68, p<0.001; sga: median 0.46 g/ml, range 0.06-3.56, p=0.011; normal pregnancy: median 0.6 g/ml, range 0.04-1.17); 3) there were no differences in the rate of pz deficiency among the groups; and 4) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: 1) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; 2) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n=26,133) reported upon enrollment at 10.8 weeks (sd 3.6) the number of weeks of regular multivitamin use during a 12 week periconceptional period (lmp-4 to lmp+8). preeclampsia cases were identified using icd-10 codes (n=605, 2.3%). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+2] and post-conception [lmp+3 to lmp+8]). results were stratified a priori by overweight status. results: overall, 18,551 women (71%) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (<4 weeks of use) had no relation to risk (or 1.15; 95% ci 0.92,1.44) but regular use (>=8 weeks) was associated with modestly reduced risk (0.84 (0.67,1.06). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or 0.83, 95% ci 0.67-1.02). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi <25 kg/m², or 0.74; 95% ci 0.55-1.01), with no apparent effect among overweight women (bmi 25 kg/m², or 0.93; 95% ci 0.68-1.26). a total of 7,332 women reported regular mv use in both the preconception and post-conception periods, and 5,630 women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or 1.29, 95% ci 0.89-1.85). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or 0.56, 95% ci 0.39-0.82). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the 9,160 women who gave birth at yale-new haven hospital (ynhh) during 2004 and 2005, and a 90 person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for 30 years and is internationally known. results: during 2004 during -2005 , the pe rate for the general population at ynhh was 7.8%. for the pa group, the rate was 2.2%. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, 1 esther streefland, 2 pieter koolwijk, 3 frans m helmerhorst, 1 jan jaap hm erwich. 2 1 department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; 2 department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; 3 department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt-1, kdr, angiopoietin-1 (ang-1), angiopoietin-2 (ang-2) and tie-2 mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang-1, ang-2 and tie-2 appeared increased in fgr cases compared to matched controls. in addition, plgf, ang-1 and tie-2 mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in 1 st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early 1 st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, 1 adrienne schonberg, 2 gary j badger, 3 ira m bernstein. 2 1 biochemistry; 2 obstetrics and gynecology; 3 medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam-1 and svcam-1, beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam-1 and svcam-1 were measured in 16 healthy, nonsmoking women (cycle day 7.5 + 0.9, prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, 11-16 weeks) and late pregnancy (lp, 31-34 weeks). five of these women developed hypertensive complications (4 gestational hypertension, 1 pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p<0.05 was accepted as significant. results subjects were 29.1+0.9 years old with a mean bmi of 23.5+0.9 kg/m 2 at the time of prepregnancy studies. significant differences in sicam-1 levels as a function of pregnancy were observed (p=0.046) and are outlined in table 1 p=0.662 differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam-1 levels in ep (p=0.024) followed by an increase in sicam-1 levels in lp (p=0.044). in women with hypertension in pregnancy, these differences in sicam-1 levels were not evident (p=0.66). there were no differences in sicam-1 levels comparing women with or women without hypertensive complications prior to pregnancy (p=0.971). in contrast to sicam-1, we observed no significant differences in svcam-1 levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam-1 change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam-1. supported by nih hl 71944. yuval bdolah, 1 uriel elchalal, 1 shira natanson-yaron, 1 hadas caspi, 1 tali bdolah-abram, 1 angelika bord, 1 caryn greenfield, 1 debra goldman-wohl, 1 ariel milwidsky, 1 franklin h epstein, 2 s ananth karumanchi, 2 simcha yagel, 1 drorith hochner-celnikier. 1 1 ob/ gyn, jerusalem, israel; 2 ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to 4-5 times higher than multiparous pregnancies. soluble fms-like tyrosine kinase-1 (sflt1), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt1 and sflt1/plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt1 (or sflt1/plgf). methods: maternal serum samples from nulliparous (n=68) and multiparous (n=159) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt1 and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt1 levels were 13,385 ± 960 and 10,584 ± 722, (p=0.021), the mean serum plgf levels were 215 ± 15 and 249 ± 14 (p=0.117), and the mean ratios of sflt-1/plgf were 98 ± 13 and 65 ± 6 (p=0.025), accordingly. in a subgroup of 10 multigravidous nulliparous pregnancies, sflt1 levels were 14,063 ± 2295. correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a 5-10 years interpregnancy interval compared with a 1-5 years interval. conclusions: in nulliparous pregnancies, circulating sflt1 levels and sflt1/ plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even 5-10 years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt-1), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from 34 placental abruption cases and 250 normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: 7.95 ± 0.48 ng/ml vs. 6.48± 0.15 ng/ml, p <0.05. there were no significant difference in serum levels of plgf, 40.31± 6.22 ng/ml versus 40.04± 1.79 ng/ml, p=ns, although sflt1 levels were lower in cases: 1.86 ± 1.7 ng/ml vs. 2.7±0.98 ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio 1.3, 95% ci 1.1-1.6) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or 7.4, 95% ci 1.6-33.5) and third (or 8.8, 95% ci 1.9-39.1) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or 13.1, 95% ci 1.3-129.09) and third (or 13.3, 95% ci 1.9 95.3) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. 4 ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at 12 and 32 weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at 12 and 32-34 weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of 60/sec. results: compared to the proliferative phase viscosity was decreased at 12 weeks and more so at 32 weeks gestation (p<0.05). 0.096 ± 0.002 0.096 ± 0.002 nsd 0.13 ± 0.004*** 0.17 ± 0.008*** ua velocity (cm/sec) 9.3 ± 0.57 15.3 ± 1.0*** 31.4 ± 3.0*** 55.7 ± 7.7*** ubf (ml/min) 15.0 ± 0.9 27 ± 2*** 101.8± 12.9*** 294.2 ± 40.3*** shear stress (dynes/cm 2 ) 20.7 ± 1.5 37.8 ± 2.62*** 37.1 ± 3.8*** 47.9 ± 6.8*** internal ua radius was not altered by the menstrual cycle, and was greater at 12 weeks (+35%) and 32 weeks (+77%). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory (80%) and did not rise further in early pregnancy but by 32 weeks shear stresses was further elevated 131%. conclusions: equivalent rises in shear stress during the secretory phase and 12 weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl49210, hd38843, hl63101 background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day 20) rats were used: control (n=5), hypertensive (0.5g/l l-name in the drinking water, n=9), and treated with l-name and hydralazine (also in the drinking water, 0.272 g/l, n=5) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (<150 m) were mounted in a pressure arteriograph and equilibrated at 60 mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from 20 to 200 mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with 0.1mm papaverine and 10 m diltiazem). results: myogenic tone developed in controls (46±5% maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone (33±4% maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group (401 vs. 425mg, p<0.05) was restored by hydralazine (433mg, p<0.05 vs. l-name). average fetal weights were also reduced in the l-name group (1.96 vs. 2.22g, p<0.001), but only partially restored by hydralazine co-treatment (2.11g, p<0.05 vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises 30-fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi 2005) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp 1992) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only 25% (jci 1996) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: 11 pregnant ewes were studied at 100-148d gestation age (ga). 4 had doseresponse studies with uterine artery l-name infusions to achieve 0.025-1.0 mg/ml over 30min. 7 had 72h arterial l-name infusions to achieve and maintain levels of 0.01 mg/ml at 107 (n=6), 122 (n=7) and 135d (n=7) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during (2,4,6,24,30,48,54 and 72h) and after (72, 96h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b 0-20%, but was not doserelated. 72h arterial l-name infusions decreased upbf b by 2-6h at each ga (p 0.025, anova) and values returned to baseline by 96h postinfusion. sensitivity did not differ between ga (p=0.1, anova), upbf b falling 20-30% at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at 107 and 122d ga, p 0.03; but were unaffected at 135d ga. venous-arterial cgmp concentration differences were seen at 107d and absent at 72h of l-name infusion, p=0.04. conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b 30%, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose 5-10%, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises 40-fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi 2007) and inhibition with tetraethylammonium ( 0.3 mm) dose-dependently decreases basal upbf 80% in the last third of pregnancy (jsgi 2005) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory 1-subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of 2 nd generation uterine arteries obtained from nonpregnant (n=3), pregnant (n=14, 56-145d gestation; term 150d) and postpartum (n=8, 2-56d) sheep were used to measure expression of bk ca -and regulatory 1-subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the 100 kda -subunit, rose 70% with placentation (p<0.05) and was unchanged thereafter. expression of the 39 kda regulatory 1-subunit paralleled the rise in bk ca density during placentation, increasing 50% (p<0.001), but increased another 2-fold and exponentially in the last third of pregnancy (p<0.001, r 2 =0.99, n=13). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p=0.8, r=0.08, n=14), pkg expression rose 2-fold (p=0.002, r=0.86, n=11) and gradually returned to nonpregnant levels by 30d postpartum (p=0.008, r=0.83, n=9). uvsm cgmp is being measured. conclusions: these are the 1 st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory 1-subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for 5 days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio (0.13±0.01 vs 0.19±0.01 μg collagen/μg protein; mean±sem; p<0.02) as well as a significant increase in smc density (6.1±0.1 vs 5.0±0.1 cells/1000 μm 2 ; p<0.001) compared to control mice with no significant change in elastin content (104±4 vs 97±14 μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio (0.25±0.02 vs 0.28±0.04 μg collagen/μg protein), smc density (3.9±0.2 vs 4.0±0.2 cells/1000 μm 2 ) or elastin content (89±12 vs 83±5 μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr7, and not the lgr8 receptor. both lgr7 and lgr8 receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for 5 days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a 20 mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr7 wild-type mice showed inhibited myogenic reactivity with a 6.1 ± 0.5% increase in diameter whereas the arteries from rhrlx-infused lgr7 knock-out mice exhibited robust myogenic reactivity with only a 1.2 ± 0.4% change in diameter (p=0.001). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr8 knock-out and wild-type mice. the veh-treated lgr7 and lgr8 mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr7wild-type, but not from lgr7 knock-out mice (p=0.01). in contrast, the arteries from rhrlx-infused lgr8 wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr7, and not the lgr8 receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c57bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for 4 hours/day. smoke exposure was increased from day 4 of gestation until late pregnancy (day 16-19) with mean total suspended particle levels of 63 mg/m3, representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n=3-6 in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses (0.88 ± 0.1g vs 1.0 ± 0.08g, p=0.02), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice (51.9 ± 9.2 vs 4.2 ± 0.8ng/ml for virgin mice and 33.2 ± 21 vs 2.8 ± 0.4ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented 10 rats for measurement of renal function. five were ovariectomized, and 5 sham-ovariectomized. after 7 days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg (25 iu/min) was implanted s.c. and renal function was again assessed 2 and 5 days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p<0.001 vs. baseline), but not ovariectomized (p=ns) rats on both days 2 and 5 of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p<0.001 vs baseline). plasma hcg concentrations were 2,500 miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, 1-100 ng/ml), but not hcg (2,500-200,000 miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, 1,2 michele brochu, 2 jean st-louis. 1,2 1 research centre, chu ste-justine, montréal, qc, canada; 2 pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol (17 -e2) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of 17 -e2, we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. 17 -e2 and 17 -e2 were equipotent in relaxing phe (1 μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of 17 -e2, tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici 182,780 (estrogen receptor modulator), and kt5720 (pka inhibitor) did not significantly influenced the response to 17 -e2. rp-8-pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to 17 -e2 to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for 17 -e2 concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of 17 -e2 in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered (20 mg/kg/day) to pregnant rats from the day 15 to 22 of gestation. rats were sacrificed at day 17, 19 and 22 of gestation together with untreated and day 14 pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring (2-3mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day 17 (2 days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day 17 of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day 14 of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c57bl6/j mice (streptozotocin; 200 mg/kg) prior to mating. mice were culled at day 19 of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to 55mmhg and equilibrated (37°c; 5%co 2 /air). arteries were constricted with phenylephrine (10 m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; 0.1nm-1 m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; 10 m), a cyclooxygenase inhibitor (indomethacin; 10 m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec 50 31.4 ± 8.0nm vs 41.9 ± 15.9nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at 1 m ach, edhf-mediated relaxation was significantly reduced (p=0.03, one-way anova) compared with controls (13.17 ± 4.87 vs 67.23 ± 13). in comparison, no-mediated relaxation was significantly increased (p=0.04, one-way anova) compared with controls (64.88 ± 10.78 vs 17.54 ± 5.77%). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf-1 serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine (1) if raf kinase contributes to phenylephrine (pe)induced contractile response, (2) the role of raf kinase inhibitor (gw5074) in regulating vascular tone during pregnancy, and (3) mechanism by which gw5074 produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw5074. pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw5074 shifted the pe-induced dose response to the right in both np and p rats. gw5074 itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw5074 did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw5074 produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw5074 were greater as the pregnancy progressed from day 18 to day 20. raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek1/2 in a time-and dose-dependent manner. measurement of [ca 2+ ] i with fura-2 showed that gw5074-mediated inhibition of pe-induced contraction was not associated with decrease in [ca 2+ ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt1 and p-mlc20 which was inhibited by gw5074. conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine 1) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, 2) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and 3) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: 1) mean arterial pressure (map) was measured in conscious np and p rats. 2) isometric tension was measured in mesenteric arteries of np and p rats. 3) expression of contractile proteins, p-mlc20 and p-mypt1 were studied in cultured vascular smooth muscle cells (vsmcs). 4) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: (1) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. (2) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( 10 μm) compared to p (1 -3 μm) rats. (3) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc20 and mypt1 in mvsmcs. (4) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, 1 arianne van koppen, 2 marc ea spaanderman, 1 paul abm smits, 2 frederik k lotgering. 1 1 obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; 2 pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e 50 (flow inducing 50% dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to 40% precontraction with u46619. results: in nw rats rlx decreased e 50 and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e 50 and increased e max , and lower myogenic reactivity). in ow rats rlx increased e 50 to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p<0.05 between nw/ow, # p<0.05 within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e 2 ). methods: uaecs were cultured until 80% confluence, and then subjected to 0 (static control), 3 or 15 dynes/cm 2 for 48hrs in the absence or presence of e 2 (10nm). western analysis was used to compare optical densities of ser635-penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by 3 and 15 dynes/cm 2 (from 1.26 + 0.5 to 2.4 + 0.008 and 2.56 + 0.06, respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels (2.2 + 1.5) and this was further increased 1.5-1.8 fold with 3 dynes/cm 2 (3.2 + 0.8) and 15 dynes/cm 2 (3.9 + 0.38). as seen with fol uaecs, preg uaec static penos (1.8 + 0.4) appeared higher than lut uaec, but unexpectedly, neither 3 nor 15 dynes/cm 2 significantly raised these levels of penos (1.76 + 0.05 and 1.6 + 0.04). regardless of shear stress level, e 2 replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine635 in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine635 phosphorylation of enos at either 3 or 15 dynes/cm 2 . in contrast to our hypothesis, chronic treatment with e 2 for 48hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl49210, hd38843, hl87144. cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with 1 mm of no donor gsno or dea nonoate, estradiol-17 (10 nm) in the presence of l-name (1 mm) for 30 min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( 3x10 6 /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol-17 also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro1 hl70562 and 74947). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for 2 3 days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for 24 hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of 400 μm on a spectrophotometer. results: a dominant band of renin at approximately 47 kd was detected in all samples. when compared with the control (i.e. 0%), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, 1 karoline pickel, 1 daniela ulrich, 1 philipp klaritsch, 1 isa alkan, 2 uwe lang, 1 manfred g moertl. 1 1 obstetrics and gynecology, medical university of graz, graz, styria, austria; 2 cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: 20 patients with uncomplicated pregnancy were measured with the taskforce® monitor 2040i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and 16.15±5.54 [>30 wks]) (figure 1). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure 2). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, 1 manfred g moertl, 1 karoline pickel, 1 philipp klaritsch, 1 isa alkan, 2 uwe lang, 1 dietmar schlembach. 1 1 obstetrics and gynecology, medical university of graz, graz, styria, austria; 2 cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: 20 healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor 2040i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at 8, 10, 12, 14, 16, 28, 36 weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n=8). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels (65+/-2 pg/ ml, mean+/-sem) to 83+/-2 pg/ml by 10-week lmp. gnrh ii concentrations continued to increase through 16 weeks gestation over the 10-week levels, although a significant increase was only attained by 28 weeks. gnrh ii continued to increase in late term, attaining levels of 99+/-1 pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to 106+/-2 pg/ml, i.e., 1.5 times that of 8-week gestation (73+/-4 pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at 8-weeks lmp (85+/-3 pg/ml) as compared to normal pregnancies (73+/-4 pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. 1 high plasma aea levels at 6 weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy 2 . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from 45 women in early pregnancy (5-12 weeks) of whom 25 had a viable pregnancy and 20 had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar (29 ± 4.9 versus 28 ± 4.7 years; mean ± sd; p=0.45; student's unpaired t-test and 24 ± 2.7 versus 25 ± 2.9 kg/m 2 ; p=0.91) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies (1.68 ± 0.77 versus 1.15 ± 0.30nm; p=0.007). there was no correlation found between aea levels and hcg or progesterone levels p=0.272 and r=0.274; p=0.075, respectively) , despite progesterone being significantly lower in the non-viable group (40.0 ± 35.1 versus 61.0 ± 26.3ng/ml; p=0.027). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. 6n-3) and arachidonic (aa, 20:4n-6) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of 5d, 6d and elongases 2 and 5 (elov 2 and 5) in the maternal liver rat. methods. pregnant rats were fed control (20 % casein; c) or restricted (10 % casein; r) isocaloric diets. at day 19 gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver 5d, 6d, elov 2 and 5 mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat (760 ± 39 vs 543 ± 22 mg) serum leptin (5 ± 0.1 vs 7 ± 0.7ng/ml) and insulin (0.2 ± 0.04 vs 0.9 ± 0.09 ng/ml) differed in c and r respectively (p<0.03). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig 1) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from 1993 to 2002 were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* 89% of patients received blood products and 40% received clotting agents. the average blood loss was 2650 cc. there were no maternal deaths and 4 injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while 90% of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc7) determined blood pressure of 120-139/80-89 in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of 40 patients who were diagnosed in the first and early second trimester (<15 weeks) with blood pressure of 120-130/80, and a control group consisting of 87 patients with blood pressure < 120/80 at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: 10% versus 2% of the study and control group pregnancies, respectively, experienced preeclampsia (p=0.07); and 14% versus 0% of the study and control group, respectively, were associated with gestational hypertension (p=0.0005). 10% of the control group deliveries were associated with admission to the nicu, compared to only 3% in the control group (p=0.16); 17% versus 10% of the study and control group deliveries, respectively, were preterm (p=0.36); twenty-nine percent of the study group were cesarean deliveries, compared to 38% in the control group (p=0.05). on admission mean sbp and dbp was 132 and 76 mmhg, respectively, in the study group, compared to 122 and 70 mmhg in the control group (p=0.0003 for sbp and p=0.03 for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at 24-32 weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of 1-10, is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from 2002-2007. pain at various dilatations (3-10cm) of women who were laboring and then delivered at 24-32 weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results 99 laboring patients between 24-32 weeks gestation and 409 at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at 3-4 cm dilatation (4.8 vs. 6.3‚ p=0.01) and significantly greater at 9-10 cm (4.2 vs. 2.3‚ p=0.03). pain reported at 5-6 cm and 7-8 cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between 24-32 weeks gestation may perceive less pain at 3-4 cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately 30 minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of 0.34 to 1.00 hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see 1) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; 2) the results suggest that uterine electrical activity is altered in women affected by asthma; 3) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from 9 women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n=7) and also from a woman with no sons and a previous miscarriage of undetermined gender (n=1). no male cells were observed in the control (n=1), a woman with 3 daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd3+ and cd19+ lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i 2 and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the 24 integrin (i) heterodimers, 1 1, 2 1, 10 1 and 11 1 are primary receptors for collagen. the predominant cervical collagen is collagen type 1. the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day 12, 16, 18, 20, 21 and 22 animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i 2 mrna expression increased through mid-gestation but decreased to np levels on day 22. a similar pattern was seen with the integrin beta 1 subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha 1, 10, 11 and integrin beta 1 were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the 1 subunit paralleled col 1 2 expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p20 rr018728 p20 rr017695. background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in 11 pregnant baboons at a mean gestational age of 134 (+/-10) days. detailed ultrasound examination (biometry, doppler, screening) was performed 3 days before, at and 1 day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood (2ml) and amniotic fluid (4ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized 24h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all 11 mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but 3/11 (27%) fetuses died within 24h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of 50 ultrasound examinations were performed in 11 fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was 16.8kg (range 14.0-20.2) and the estimated fetal weight was 405gr (range 317-484). sixteen out of 18 cordocenteses (89%) and all 18 amniocenteses (100%) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, 17-hydroxyprogesterone (17ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after 8 weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to 17ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, 17ohp, cortisol ©, estradiol (e2) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or 17ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: 43 hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these 5 hormones are depicted in figure 1 (maternal serum, from conception to term 180 days) and figure 2 (newborn serum) conclusion: this study provides reference values for 5 hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and 17ohp administration during pregnancy. (1)non-pregnant women eligible for ivf treatment and (2)pregnant women receiving prenatal care. the pre-9/11 samples were drawn in the year prior to 9/11/01; the post-9/11 samples were drawn within 6 months after. the pre-9/11 and post-9/11 normal pregnant samples were drawn at 20+2 weeks' gestation. the non-pregnant samples were from ivf candidates with serum e2 levels<75 newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels<12.5 miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp70 and hsp90 were measured by commercially available elisa. these results were compared in the patient populations before and after 9/11. t-tests were used for analysis. statistical significance was set at p<0.05. results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp70 pre-and post-9/11 (table 1) . only serum hsp90 levels were significantly increased in the pregnant women post-9/11.* mean serum acth, cortisol, hsp70, and hsp90 levels were all significantly different in non-pregnant subjects pre-and post-9/11. conclusions: except for hsp90, serum markers for stress were unchanged in pregnant subjects after the stress of 9/11/01 in comparison to non-pregnant women whose serum hsp90, hsp70, cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that 11ß hsd-2 activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on 1301 women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after 37 weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the 1301 women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r=0.12, p=0.00001; r=0.10, p=0.0002; and r=0.09, p=0.0009, respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r=0.08, p=0.006; r=0.07, p=0.009 and r=0.06, p=0.03, respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower 11ß hsd-2 activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting 2-10% of the us. our prior studies found that 29% of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a 2-year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every 6 months for 2 years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results 2907 women with fi were surveyed and of those, 1247 (43%) returned at least 1 survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, 51% felt frustrated due to fi, 26% reported fi impacted their emotional health, 18.5% reported fi impacted child-caring abilities, and 16.7% reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were 4-7 times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. 33.9% felt their stool was stored elsewhere before bowel movements, 13.9% reported using digital defecation and more than half (52%) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only 10% of women at 6 months, 13.5% at 1 year, and 16.7% at 2 years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n=26,781) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or 0.53, 95% ci 0.47-0.61) and blacks (0.32, 95% ci 0.27-0.37) had statistically significant lower rates of breastfeeding, while latinas (or 1.18, 95% ci 0.99-1.41) showed a trend towards higher rates of breastfeeding. in latinas, we found that 3 rd and 4 th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table 1) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; 4 pediatrics, erasmus medical centre, rotterdam, netherlands; 5 public health, erasmus medical centre, rotterdam, netherlands; 6 clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april 2002 and january 2006 information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from 6,940 pregnant women were available. of all women 37% adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or 9.5, p<0 .001), nonwestern ethnicity (or 3.5, ci 2.9-4.3, p <0.001) and a low educational level (or 2.5, ci 1.8-3.6, p<0.001). other risk factors were single marital status, smoking, multiparity (all p<0.001) and alcohol use (p<0.05). prior spontaneous abortion was associated with increased adequate folic acid use (p<0.001). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since 2005, little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least 70% of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the 219,335 pregnant admissions captured between 1994 and 2005. logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of 0.1 considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from 8.1% in 1994 to 24.5% in 2005. although white women had 3 times the odds of using ma in 1994 (adjusted or (95%ci) 3.2 [2.8, 3.8]), this had dropped to 1.9 [1.7, 2.1] by 2005, mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled 1,006 women who delivered a live born singleton infant between 23 and 27 completed weeks gestation at 14 tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor (40), pprom (23), preeclampsia (18), placental abruption (11), cervical insufficiency (5), and fetal indication/iugr (3). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, 1 sarah wagner, 1 sean keeler, 1 may tam, 1 julian mierlak, 1,3 aaron caughey. 2 1 obstetrics and gynecology, nyu school of medicine, new york, ny; 2 obstetrics and gynecology, ucsf, san francisco, ca; 3 obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november 1, 2001 through june 30, 2006. data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the 4,049 pregnancies, asian and hispanic race/ethnicities accounted for 50.4% and 44.0% of the population, respectively, with 11.2% being us-born. the mean age was 27.02 years and the overall ppd+ rate was 50.3%. there was 5% non-compliance with ppd testing, and 5% of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women >40 years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and 95%ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted 45 minutes, and each focus group lasted 60-90 minutes. we recruited 20 patients (10 hispanic, 10 aa) and 20 providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd050121-02; r21 ca126450 (spring). population. kristine beckers, 1 isabelle guelinckx, 2 greet vansant, 2 roland devlieger. 1 1 obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; 2 clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in 605 belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was 14.8 ± 4.7 kg (32.63 ± 10.36 lbs). mean weight gain was 15.4 ± 4.1 kg (33.95 ± 9.04 lbs) in the underweight population, 15.1 ± 4.5 kg (33.29 ± 9.92 lbs) in the population with normal weight, 13.7 ± 5.3 kg (30.20 ± 11.68 lbs) in the overweight population and 12.0 ± 5.9 kg (26.46 ± 13.01 lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e2 and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c566t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at 6-10 weeks of age were randomized into treated versus control groups.bm from 1 syngenic female donor was injected into the tails of 2 recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at 0, 1, 2, 3, and 4 weeks post bmt. all organs were weighted and examined by h e. fsh, e2, and p4 were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in 1% agarose gel. results: total body weight significantly increased in treated animals(p< 0.02). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p<0.03 and p< 0.002). six out of 8 treated animals showed estrogenic changes in daily vaginal smear. e2 level increased 2.5-7.5 times and fsh level dropped to 50% in treated animals. normal (donor allele) fshr gene was amplifiable in 6 out of 8 recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of 3 cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional (2d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional (3d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc12 cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the 3d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that 3d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc12 cells. conclusions: improvement of the 3d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal 3d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c57bl/6j female mice and in vitro fertilized with sperm from male c57bl/6j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice 4 days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf4 and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf4 and heparin, fixed on day 8, stained with -tubulin and zo-1 antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, 1 cisca wijmenga, 2 ruben van 't slot, 2 lude franke, 2 bart cjm fauser. 1 1 reproduction gynecology, university medical centre, utrecht, netherlands; 2 complex genetics group, university medical centre, utrecht, netherlands. introduction: around 1% of women have a post-menopausal hormonal profile before age 40, referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of 10mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to 50 kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included 108 caucasian, 46,xx patients with spontaneous secondary amenorrhea before age 40, fsh > 40 iu/l and absence of (low) 45x/46 xx mosaicism and fmr1 premutations. dna analysis was performed using illumina 370k cnv arrays containing 370,404 probes. 12,556 probes were located on the x chromosome ( 1 probe for every 12 kb), of which 1,348 probes were specifically designed to detect known cnvs. ten samples with call rates <99% were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( 60 kb) using an algorithm detecting at least five consecutive probes with intensities (logr > -0.20) below the mean probe intensity. we identified a total of 183 x chromosomal microdeletions, divided in 84 loci and varying between 60-200 kb in size. 46 of the 97 samples showed at least one deletion on the x chromosome. 27% of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found 26 coding regions containing 38 genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, 1 patrizio antonazzo, 1 sabrina cozzolino, 1 stefania calabrese, 1 lia crotti, 2 francesca ferrari, 3 roberto insolia, 2 fabio facchinetti, 3 peter j schwartz. 2 1 dept. of obst/gynecol, univ. of milano, milano, italy; 2 molec cardiol lab, policlinico san matteo, pavia, italy; 3 mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly 1 in every 200 pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, 25-50% of cases remain unexplained. we recently demonstrated that 10% of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: 60 patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at 22 weeks or more of gestation according to the definition of late fetal death of the who. 32 out of 60 cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq1, kcnh2, scn5a, kcne1 and kcne2 were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of 122 caucasian women with uneventful pregnancies. preliminary data on the first 17 cases (gestational age of death: 23-38 weeks) are reported. results: a total of 3 missense mutations were identified in 3 of 17 stillbirths (18%), two on scn5a and one on kcnh2. the two mutations on scn5a (v1951l; p2006a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh2 is a novel genetic variant absent in 244 reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at 33 weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon 10 (cex10del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon 9 (c1290a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon 9 followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon 9 snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon 9 snp, cex10del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon 9 snp, the cex10del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon 10 (c1383g/c) that co-segregated with the exon 9 snp. presence of c1383g led to the formation of an ese that binds splicing regulatory protein srp40, leading to efficient exclusion of exon 10. real time pcr revealed a five-fold increase in the expression of the cex10del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon 10 snp compared to those that did not (homozygous c/c) at the same locus (p < 0.001). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, 1,2 joop se laven, 1 robert jan galjaard, 2 j hikke van doorninck. 1,2 1 obstetrics gynaecology; 2 clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., 2006) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone 4, lysine 12 acetylation (h4k12) at the germinal vesicle stage and complete deacetylation at the mii stage in 45% of the oocytes, while 55% keep high levels of h4k12 acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, 80% of oocytes with naturally remaining h4k12 acetylation showed abnormal spindle figures. in contrast 80% of the normal de-acetylated oocytes showed normal spindles (p=0.002). this suggests that defective de-acetylation of h4k12 in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth <36 weeks) control study (term birth > 37 weeks) maternal and fetal dna from 370 (172 cases and 198 controls) c and 279 (82 ptb and 197 term) aa were collected. a high-throughput candidate gene association study was performed examining 1442 snps in 130 genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in 23 genes associated with ptb (p<0.01) were common between races with both maternal and fetal dna analyses. however, snps in 24 genes in c and 32 in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c-4443t; rs879293) at both allelic (p = 2.00x10 -3 ) and genotypic (p=2.0x10 -6 ) level with an odds ratio (or) of 2.80 ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin-10 receptor antagonist gene (a18792g; rs17121510) for both allele (p=0.01) and genotype (p=3.34x10 -4 ), or 1.92 ). in aa, the strongest associations were in interluekin-15 (c13929t-rs10833, allele p=2.91x10 -4 , genotype p=2.0x10 -3 ) in maternal dna with an or=0.54 [ci 0.37-0.78] and in fetal dna interleukin-2 receptor b (a14481g; rs84460, allele p=1.4x10 -4 , genotype p=6.3x10 -4 ) with an or 2.36 ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t2dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type 2 diabetes. as risk factors are similar between t2dm and gestational diabetes (gdm), we investigated a possible association of those 18 snps with gdm. study design: blood was collected from caucasian (100 cases, 364 controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi 2 contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type 2 diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t2dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox-2 in human myometrial and amnion-derived cells [reproductive sciences, vol 14, no 1 (supplement, #9)]. we have also shown that expression profiles for hdacs (1-6, 8) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac 1, 2 and 8 during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac1, 2 and 8. the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac 1 and 2 appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac8 appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that 1) myometrial hdac1 appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; 2) hdac2 appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; 3) no changes in phosphorylation levels were observed for hdac8 using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b1-adrenergic receptor (adrb1) located on chromosome 10 have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= 1315) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs1801253 single nucleotide polymorphisms (snps). genotype frequencies were also determined in 100% complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs1801253 snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the 100% pacific islander group was significantly different from the caucasians (p=0.04) and asians (p=0.008) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, 1 p antonazzo, 1 s tabano, 2 f colleoni, 1 a martinelli, 1 s calabrese, 1 c benedetto, 3 l marozio, 3 f facchinetti, 4 m miozzo, 2 i cetin. 1 1 inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; 2 dept. biology and genetics, univ. milan, italy; 3 dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add1) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add1 g460w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add1 g460w polymorphisms yet remains to be investigated. population association study we genotyped 2 polymorphisms (ace i/d and add1 g460w) in 497 women: 197 with pe (119/197 with severe pe) and 300 controls. moreover, we investigated a subset of their fetuses: 24 from severe pe, 17 from mild pe and 300 controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add1 genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add1 snp. in mother-fetus genotype couples, neither ace nor add1 polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent (8% vs 13%; p<0.05) and ace i-d is significantly more frequent (59% vs 42%; p<0.02) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls (37% vs 38%). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb-1 in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, 1-cell, 2-cell, 4-cell, 8-cell embryos and blastocysts. in human, pi and mii oocytes, 8-cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and 1-cell and 2-cell embryos, and became undetectable in 4-cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in 8-cell embryos or blastocysts. zga occurs at late 2-cell stage and 4-to-8-cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou5f1 (oct3/4) and caudal-homeobox domain 2 (cdx2) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct3/4 in mice prevents icm development, while cdx2 knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct3/4, nanog and cdx2 was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected 144 hours post insemination, then fixed prior to incubation with primary antibodies directed against oct3/4, nanog and cdx2. following incubation with cy3 conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx41 fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct3/4 protein was detected in both the icm and te, based on two different antibodies. the expression of cdx2 was localized specifically to the te. conclusions the ubiquitous pattern of oct3/4 expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct3/4 protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants 1r01hd045966 1r21rr021881, and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated 3 experimental groups of b6 mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd1 foster mothers and one cohort of animals fertilized in vivo and transferred to cd1 foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b6 foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to 18 weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was 6.3 (in vivo), 3.7 (fb) and 3.5 (ivf). birth weight of male mice both ivf (1.57 mg) and fb (1.73 mg) was larger than male in vivo mice (1.30 mg) (p<0.05). ivf mice tended to be smaller than fb mice in both sexes (p= 0.0533). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p<0.0001) and more importantly from ivf growth curves (p=0.0516) (fig. 1) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, 1 luke carrey, 1 nancy valego, 1 philip deibel, 1 james perrott, 1 jorge figueroa, 1 mark chappell, 2 james c rose. 1 1 obestetrics and gynecology, wake forest university, medical school, nc, usa; 2 hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at 1.5 years of age which were exposed to either vehicle or betamethasone at 80-81 days gestation. we implanted vascular and bladder catheters and then allowed the animals a 5-7 days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl (0.0275 meq/kg/min at 0.55 ml/min) for 60 minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults (2.267 ± 0.10 ml/min/kg) compared with the vehicle animals (1.935 ± 0.08 ml/min/kg) (p=0.028). rpf was similar in the vehicle and steroid exposed group (15.483 ± 0.40 ml/min/kg vs 13.561 ± 0.80 ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group (16.60 ± 5.37 μmol/h vs 14.37 ± 2.46 μmol/h). this similarity was also present after normalization by body weight (0.278 ± 0.09 μmol/h/kg vs 0.234 ± 0.04 μmol/h/kg). the vehicle group excreted 53.65 ± 9.71% of the dose of na during the experiment while the betamethasone exposed group excreted only 37.07 ± 4.42% (p<0.05). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd 47584, hl 68728 and hd17644. characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst 33342 dye efflux. this ability to efflux hoechst dye is due to expression of abcg2 (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: 15 human fetal hearts were collected aged between 6-16 wk. we used rt-pcr, using primers for several stem cell (abcg2, cd34, cd45) and cardiac specific (mlc-2v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg2, cd34, cd45, islet1). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg2 mrna was highly expressed in all regions of the fetal heart between 7-10 wk. expression was down regulated at 11-15 wk especially in the la and lv. a similar pattern of expression was observed for cd34. cd45 showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at 13-15 wk. mrna and protein analysis showed similar results. whilst protein expression of abcg2 was robust at 7 wk (approximately 3-6%), it declined with increasing gestational age. cd34 was also strongly expressed at 7 weeks of age. there was no co-localisation between abcg2 and islet1. conclusion: abcg2 and cd34 are both expressed between 7-10 wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg2 and cd34 expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog 120:817,1974) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev 6:17, 1994) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi 2006) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, 8 sheep at 0.8 gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to 2 h normoxia, 0.5 h hypoxia (mat fio 2 to reduce fetal pao 2 to ca. 10 mmhg) and 1 h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low (30 mg.kg -1 over 20 min) or high (150 mg.kg -1 over 30 min ) doses. treatments finished 100 min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child 91:163, 2006) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps (50 microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following 28 days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa10, proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa10 and pcna compared to control (p<0.01). lower, but increased, levels of hoxa10 and pcna gene expression were seen in the specimens exposed solely to estradiol (p<0.05). those specimens exposed to bpa alone demonstrated a small induction in hoxa10 expression (p<0.05) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa10 in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, 1 john d richard, 1 guo l liu, 1 sharon k sugano, 1 ahmet karadag, 2 michael g ross. 1 1 dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: 118-120 d gestation), preterm (pt: 130-132 d), near term (nt: 140-142 d) and term (t: 146-147 d) (n=6 fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody (1:200, sc-8992, santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by 3,3'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at 10 different fields at 400x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r1, increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r1. method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: 118-120 days), preterm (pt: 130-132 days), near term (nt: 140-142 days) and term (146-147 days) ovine fetuses (n=6 at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies (1:800 to 1:1000) with abc reagents and examined at 400x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r1 receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: 55±3, pt: 53±3, nt: 54±3, and t: 57±3 %) than myenteric neurons (vpt: 35±3, pt: 42±3, nt: 38±4, t: 31±2 %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r1 receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r1 mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, 1 guo l liu, 1 john d richard, 1 sharon k sugano, 1 raina khan, 1 octavio balbuena, 1 kimberly chap, 1 virender rehan, 2 michael g ross. 1 1 dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n=6 for each gestational ages) at very preterm (vpt: 118-120 days gestation), preterm (pt: 130-132 days), near term (nt: 140-142 days) and term (t: 146-147days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos (10 field per colonic ring, 6 rings each gestational age) taken at 400x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt=0.130 ±0.009, pt=0.154±0.002, nt=0.151±0.005, t=0.169±0.016; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = 49±7, pt = 27±5, nt = 19±3, t = 22±1%; p<0.05), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n=4) and second (n=5) trimesters, paired upper and lower segment myometrium from preterm (22-28 wks, n = 4), term not in labour (n = 10), term spontaneous labour (n = 8) and non pregnant (n = 10). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins (90-120kda/60-70kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < 0.001) and in labour (p < 0.001) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods 86 women undergoing ivf were classified as: 1. "no ovarian factor", (tubal or male factor and egg donors, nof;n= 37); 2. endometriosis (endo; n=17); 3. poor responders (pr; n=19); 4. polycystic ovary syndrome (pcos; n=13). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day 3 fsh and e2 and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd45 immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof (137±33.6) was statistically significantly higher than in pr (57±15.2) and lower than in pcos (290±77.2). both endo (85±30.2) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r=0,26; p< 0,05) and mii (r=0,35; p< 0,01) oocytes and negatively with the units of fsh (r=-0,31; p< 0,01) and lh (r=-0,24; p< 0,05) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day 3 fsh levels in all patients except in pcos (r=-0,25; p<0,05). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, 1 zhi-bin tong, 1 eric widra, 2,3 alan h decherney. 1 1 reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; 2 department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; 3 shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with 2 or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into 2 groups. in group 1, the oocytes were placed in 50 l of ultrapure water and kept at -80 o c for further atp analysis. in group 2, the oocytes were vitrified using 15% ethylene glycol, 15% dimethyl sulphoxide and 0.5 m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for 5-7 days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after 90 minutes of incubation then oocytes were placed in 50 l of ultrapure water and kept at -80 o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from 23 women. 47 oocytes were vitrified/thawed before measuring for atp content. the other 44 oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is 72.34% (34/47). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table 1). coefficient of variation of luciferin-luciferase bioluminescent assay is less than 5%. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl1, kl2, fshr, papp, p450) and its relationship with ivf outcome. materials and methods 89 patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day 3 fsh and e2 and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed 36 hours after administration of 10.000 iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd45 immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl2 expression correlates positively with kl1 levels (r=0,86; p< 0,01) and with genes implicated in granulosa cell function such as fshr (r=0,36; p< 0,01), papp (r=0,54; p< 0,01) and p450 (r=0,39; p< 0,01). the number of mii oocytes obtained is positively correlated with both fshr (r=0,35; p< 0,01) and papp (r=0,28; p< 0,05) expression, but not with the other genes. kl2 expression in women who became pregnant during the ivf cycle studied was higher (25,8± 10) than in non pregnant women (16,6 ± 3,1).(n=80, mann-whitney u p<0,05). conclusions patients who become pregnant have increased expression of kl2, which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: 150 subjects undergoing first cycle of ivf had 3d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. 50 of them developed ovarian hyper-response, defined as retrieval of 15 oocytes and ohss. 100 subjects had normal ovarian response with retrieval of 4 to 15 oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi (9.64 ±7.94 vs. 8.64 ± 7.31), fi (37.95 ± 5.5 vs. 37.95 ± 5.5) and vfi (3.77 ± 2.68 vs. 3.51 ± 3.1) were similar in both the groups. afc and ov were significantly higher (p<0.01) in the ohss group (28.90 ± 14.55 and 10.40 ± 3.77 cm 3 respectively) than in the control group (19.23 ± 9.91 and 8.94 ± 4.7 cm 3 respectively). afc was the only significant (p=0.001) predictor of ohss on multiple regression analysis (table 1) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by 3d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at 48h, media was collected and progesterone (p4) and estradiol (e2) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp19) and cholesterol side-chain cleavage (cyp11a1) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp19 and cyp11a1 promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p4 and e2 were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp19 and cyp11a1 mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp19 and cyp11a1 gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp19 and cyp11a1 gene promoters. our studies demonstrate that human cumulus cells are sites of significant p4 and e2 biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf-1mice and fertilized in vitro with cauda epididymal sperm from b6d2f1/j mice. fertilized eggs were cultured in whitten medium under 5% co2 in humidified air at 37 °c for 96 h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within 24 hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of <0.05 was considered significant. results: there were total of 12 newborn animals: 5 females (2 ivf, 3 controls) and 7 males (3 ivf, 4 controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf (0.145 mm 2 , 0.69%) animals compared to female controls (0.079 mm 2 , 0.97%) and higher in male ivf (0.019 mm 2 , 0.40%) animals compared to controls (0.138 mm 2 , 0.25%). the average of males and females was slightly lower for ivf (0.082 mm 2 , 0.54%) than controls (0.109 mm 2 , 0.61%). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a 29 year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a 39 year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was 9 and 6, maturation rate after 24 hours was 33% (2/6) and 44% (4/9), fertilization rate after 24 hours was 100% (2/2) and 25% (1/4), maturation rate after 48 hours was 50% (2/4) and 40% (2/5) and fertilization rates after 48 hours 50% (1/2) and 50% (1/2), for case 1 and case 2 respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from 2002 to 2007 were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at 6-7 weeks gestation) in women who received intravaginal micronized progesterone (200 mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of 182 cycles were evaluated in 72 women treated with clomiphene citrate. clinical pregnancies were documented in 19.5% (18/93) of cycles in the progesterone group compared to 9.9% (9/91) of the non-progesterone group (p < 0.08). forty-three cycles were evaluated in 24 patients treated with letrozole. clinical pregnancies were documented in 17.2% (5/29) of cycles in the progesterone group compared to none (0/14) in the non-progesterone group (p < 0.02). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi (7.5 ± 5.3 vs. 8.6 ± 7.9), fi (38.9 ± 6.9 vs. 37.9 ± 6.5) and vfi (3.2 ± 2.6 vs. 3.5 ± 3.4) were similar in both poor and normal responders. afc and ov were significantly lower (p<0.01) in poor responders (9 ± 3.3 and 6.3 ± 3.5 cm 3 respectively) than in normal responders (19.2 ± 9.9 and 8.9 ± 4.7 cm 3 respectively). afc was the best (p<0.001) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was 40 years. all eight couples were in a committed relationship and had been together for an average of 7.3 years. five of the couples (69%) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples (75%) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, 1 az steiner, 2 rh fogle, 1 mj kalan, 1 rj paulson. 1 1 ob/gyn, usc keck school of medicine, la, ca, usa; 2 ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day #3 a baseline ultrasound was performed and serum fsh and e2 levels were measured. subjects were given 100mg of clomiphene daily from days 3-7 and then returned for serial ultrasounds on day #8. when at least 2 follicles 12mm were present, 200 iu of hcg im daily was initiated. cycles were monitored by ultrasound every 2 days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: 5 subjects, aged 33.2±2.5 yrs (mean±sem) and bmi 25.8±1.6 kg/m 2 with 7.6±3.2 yrs of infertility were enrolled. the mean fsh and e2 levels were 8.9±1.4 miu/ml and 37.4±5.9 pg/ml, respectively. there was an average of 6.2±0.2 days from starting clomiphene to the attainment of 2 follicles 12mm. 4 out of the 5 subjects had 2 follicles 12mm by day #8. a mean of 10.4±0.7 days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance (2.8±0.4 follicles, measuring 20.6±0.9 mm in the micro-dose hcg cycles, compared to 1.4±0.3 (p=0.05), measuring 18.6±2.4 mm (p=0.47) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: 8.4±2.0 mm vs. 10.0±2.3 mm (p=0.30), respectively. all subjects had at least 2 ovulatory sized follicles in the study cycles. 40% of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: 1) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates 2) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their 1st or 2nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than 40 years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table 1) . maternal antenatal and intrapartum complications were similar in both groups (23.4% vs 39.4%, p=0.23). there were more singletons in the study group (84.9% vs 63.8%, p<0.05). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table 2) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table 1 lupron (n=33) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in 1-5% of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at 5% (icu admission) and 10% for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $8,399 to a high of $14,615. the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $940 to a high of $2,060. this resulted in an estimated cost burden of $7,459 to $12,555 for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed 321 ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were 149 gt, 145 cc and 27 ai cycles. age, gravida, day 3 (d3et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d3et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < 0.001). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = 0.311; p < 0.001) rather than with mcet (r = 0.168; p = 0.002). there was a reverse relationship between the age of the patients and ep (r = -0.215; p < 0.001). a negative correlation was also found between the gravida and ep (r = -0.28; p < 0.001). pregnancy rate was significantly correlated with ep (r = 0.193; p < 0.001). pregnancy rate was 2% in type a ep, 5.2% in b, 16% in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in 52% type c pattern in contrast to 33% with cc (p = 0.014 for ai vs. cc; p = 0.05 for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < 0.001). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, 1,2 desiree mccarthy-keith, 1,2 barbara stegmann, 1,2 alicia armstrong. 1,2 1 reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; 2 reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january 2000 to 2007, followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day 3 and 10 fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was 35 and 36 pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) 35 +3.6 36 +3.6 day 3 fsh (u/l) 6.9 + 1.7 6.1 + 0.96 day 10 fsh (u/l) 6.1 +1.9 6.2+ 2. objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in 30 cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and 450 miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert4d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl-10-14400 active mis/amh elisa. statistical analysis was performed using spss version 15.0. continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = 0.036). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of 1377 ivf cycles performed from january 1997 to july 2007 resulting in 665 pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day 14 (post-oocyte aspiration) hcg level, and day 16 hcg levels were significant in predicting pregnancy outcome. day 14 and day 16 hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day 14 hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day 14 hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. 99% of ongoing pregnancies had hcg level > 23 miu/ml. conclusions: a single day 14 post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u0126 (10 m)) reduced fsh stimulated cyclin d2 mrna expression by 50%. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with 10nm rapamycin for 15 min significantly reduced fsh-mediated cyclin d2 mrna expression. dht exposure for 24 hours also inhibited fsh stimulated mtor signaling, as shown by a 40% reduction in the phosphorylation of its down stream target p70s6kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc-2 phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc-2 phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated 5 reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd-38424). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, 1 annemarie g mulders, 1 aida bertoli-avella, 2 ben a oostra, 2 joop se laven. 1 department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; 2 department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: 72 pcos patients (2003 rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± 20.000 inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus 141 unaffected first-degree family members were genotyped using 366 polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md-10 (average spacing 10 cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in 11 clusters of 2 or more closely related patients (simwalk2). results: there was an average number of 10.1 alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome 6 (d6s262 and d6s308) that showed significant association with pcos (p 0,01). other markers that showed significant association were positioned on chromosomes 1 (d1s450), 3 (d3s1565), 4 (d4s406) , 7 (d7s530), 8 (d8s264) and seventeen (d17s784) (all p values 0.01). linkage analysis in 11 sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome 19 (d19s884) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of 10 cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal 2d and 3d ultrasound was performed in 36 women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table 1 ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with 3d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, 1 jill blaine, 1 anuja dokras. 2 1 obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; 2 obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n=130) and controls (tubal infertility, n=130) after in vitro fertilization at university of iowa from 1994-2006. the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was 18% in women with pcos and 15% in controls. after logistic regression analysis pcos was not associated with miscarriage (p=0.495). the prevalence of gestational dm (gdm) was similar in both groups 12% pcos vs 11% controls. pcos was not associated with gdm after adjusting for age and bmi (p=0.678). however, bmi was significantly associated with gdm after adjusting for age and pcos (p=0.012). risk of both pre-eclampsia and pih was 10% in pcos and 5% in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p<0.01). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the 2 groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of 21-33%, with 7-24% among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their 20s found no increase in bmi or fasting insulin with pcao but regular menses. participants: 222 women aged 25-45 with regular cycles (every 25-35 days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of 12 on one ovary or ovarian volume > 10cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table 1) . of the 69 women with pcao, 62% met the afc criterion only, while 19% met the volume criterion only, and 19% met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in 1986, have led to an increased prevalence of pcao among women with regular menses, over 1/2 of women in their 20s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl-2 fraction. this is an atherogenic profile; in fact, studies suggest low hdl-2 may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl-2 levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl-2 and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of 42 obese women newly diagnosed with pcos was determined. they were then placed on a high protein (80-100 g/day), low carbohydrate (30-50 g/day) diet with or without metformin (76 and 24%, respectively) and followed monthly for an average of 13 months (range 5-31). results: at diagnosis, 66% had low hdl and 89% had low hdl-2; only 29% had elevated tgs. on the diet, the patients demonstrated an average weight loss of 24.7 lbs (212.01 to 187.4, p<0.001) and decreased bmi of 4.2 kg/m2 (34.2 to 30.0, p<0.001). hdl levels increased significantly (18% increase from 46.6 to 55.0, p<0.001), especially the hdl-2 fraction (34% increase from 10.9 to 14.6, p<0.001). triglycerides decreased as well (135.0 to 107.2, p<0.027). ldl decreased but did not reach statistical significance. 29 resumed menstrual cycles, 11 were started on oral contraceptives, and 1 had a hysterectomy. 2 pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl-2 at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl-2 levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for 3 days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and 1, 2 and 3 hrs after 75g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels (24.9 + 2.2 μu [±se] /ml) and insulin response to oral glucose (auc) (527.9 + 52.2μu/ml) were higher (p < 0.001) in women with pcos compared to controls. all were insulin resistant with homa-ir value > 3.9 mol x μu / l 2 . resistin levels in women with pcos (15.6 ±1.3 ng/ml) was significantly (p < 0.02) higher compared to control women (10.7 ± 1.4 ng/ml). crp levels in women with pcos (9954.2 ± 918 ng/ml) was also significantly (p < 0.001) higher than the controls (2396.2 ± 579 ng/ml). there was a significant positive correlation between resistin and crp levels (r = 0.362, p < 0.04). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that (1)women with pcos and insulin resistance have increased resistin levels, (2) there is a strong association between resistin and crp (3) elevated resistin and crp levels may predict women who are at increased risk for chd (3) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, 1 enrico carmina, 2 prati vardhana, 1 michel ferin, 1 rogerio a lobo. 1 1 department of obstetrics and gynecology, columbia university, new york, ny, usa; 2 department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these 2 phenotypes, we studied 48 hyperandrogenic women with pcos (age 27.3±6.4, bmi 27.1±5.6) and 20 age matched controls (age 27.7±4.5, bmi 26.8±3.9). women with pcos were divided into weight matched (ov) n=14 and (c) n=34 groups. fat and weight distribution were assessed by dexa (total fat, r1 fat, trunk fat) as well as fasting levels of lh, e2, mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein-4 rbp4) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference (93.2 14.9 vs. 86 85 cm, p<0.05) trunk fat, r1 fat and %trunk and %r1 fat compared to ov-pcos (p<0.05). leptin, rbp4 and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p<0.05). quicki was significantly lower in c-pcos (0.33±0.02 vs. 0.35±0.02, p<0.05) and insulin was higher (14.9±5.76 vs. 9.7±3.5, p<0.01). serum lh was also higher (11.3±7.8 vs. 6.5±1.7, p<0.05) but kisspeptin, testosterone and estradiol were similar. mis/amh (8.3±5.2 vs. 6.8±4.9 ng/ml, not significant) and crp (2.78±1.5 vs. 1.3±0.5, p<0.05) were higher in c-pcos. significant correlations (p<0.05) were noted among kisspeptin/rbp4 (r 0.426), mis/testosterone (r 0.451), insulin/ %trunk fat (r 0.616, p<0,01), total fat/leptin (r 0.772, p<0.01), %trunk fat/adiponectin (r -0.522). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida 4 gene (zp4) is recently identified gene, which belongs to zona pellucida genes such as zp1, zp2 and zp3. the role of zp4, contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp4 coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from 29 patients (mean age 24,2 +/-3,23 years; mean bmi 31,4 +/-4,54 kg/m 2 ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from 2003. dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp4 gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of 29 patient samples with pcos, we identified 5 nucleotide changes in the zp4 coding seguence : 4 silent nucleotide changes in exons 1,2,5 ,7, and 1 nucleotide change in the exon 5 ( position 114, t>g). the mutation in exon 5 ( t>g ) results in substitution of cystein for glycin of amino acid in position 223 of zp4 protein. in summary, our data demonstrate that zp4 nucleotide changes account for 15% of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and 1, 2 and 3 hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels (16.4 + 2.4μu/ml) vs 9.9 + 1μ u/ml [+ se]) (p < 0.03) and area under the curve (auc) of insulin (267.9 + 33.0 vs 134.0 + 12.2μu/ml) (p <0.001) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < 0.001) increased in women with pcos (6.5 + 0.9 ng/ml) compared to controls (1.9 + 0.2 ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = -0.822, p< 0.0001). there was significant negative correlation between mis and fasting insulin levels (r = -0.462, p < 0.03) and insulin auc during the ogtt (r = -0.485, p <0.02). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, (1) there is an increase in secretion of mis, (2) higher insulin levels are associated with lower mis levels, (3) acute increase in insulin levels has no effect on mis levels, (4) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: 40 infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent 75g oral glucose tolerance test (75ggtt, sampling at 0, 60, 120 minutes), and 2-step elisa for amh (sensitivity 0.7 pmol/l, inter intra-assay c.v. :12.3%, 14.2%, respectively). p<0.05 was considered as statistical significance. results: average amh level of the subjects (age:31.9+3.9 years, bmi: 21.1+3.0 kg/m 2 , mean+s.d.) was 50.4+31.1pmol/l, which was higher than normo-ovulatory women (16.7+10.5 pmol/l, n=16). amh levels had significant positive correlation with total ovarian volume by ultrasound (18.2+11.4 cc), but not with bmi, waist-hip ratio (0.85+0.06), and levels of serum lh (5.5+3.6 iu/l), total testosterone (67.3+32.8 ng/dl). although fasting glucose levels (83.1+4.9 mg/dl) were positively correlated with total ovarian volume (r=0.41), amh levels had no significant correlation with fasting insulin (4.3+2.3 iu/l), fasting glucose/insulin ration (22.8+8.4), homa-ir (0.90+0.51), and the sum of insulin levels (98.6+42.1 iu/l)during 75ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with 2 different fragments of the mouse gnrh gene promoter (-3446/+28 and -249/+28 bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, 5 male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the -3446luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the -249luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from -3446luc mice confirmed gnrh promoter activity in the prostate (3876 ± 624 rlu) and penis (576 ± 151 rlu) and absence in the testes (39 ± 11 rlu). in the -249luc mice, however, luciferase activity was detected only in the testes (1283 ± 97 rlu) and not in the prostate (65 ± 25 rlu) or penis (48 ± 10 rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal -249 bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while -3446 bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g2/m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio 2. upon meiotic reactivation, pumilio 2 loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, 1-cell, 2-cell, 4-cell, 8-cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a1 and a2) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a (1 and 2) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g2/m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type 2 diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of 146 women diagnosed with type ii or gdm initially treated with glyburide at a single institution from 2001-2004. maternal characteristics, adequate glycemic control defined as more than 66% of fasting glucose <95 mg/dl and one-hour post-prandials <135 mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the 146 women initially treated with glyburide, 25 (17%) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose (20 mg/d) reached (56%), maternal hypoglycemia (28%) and other side effects (16%). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes (75% vs. 60%, p=0.25), prior history of gdm (24% vs. 32%, p=0.63) and macrosomia (>4000 g) (28% vs. 24%, p=0.80) or 1 hour glucose challenge test (204+51 vs. 180+36mg/dl, p=0.07). non-responders had a higher rate of obesity (bmi>30) (71% vs. 44%, p=0.02) and earlier gestational age at initiation of therapy (24+7.7 vs. 29+7.7 wks, p=0.01). there were no differences between the groups in the mean 36-week fasting (96+17 vs. 90+14 mg/dl, p=0.15) and post-prandial glucose values (133+26 vs. 127+19 mg/dl, p=0.87). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the 100gram, 3-hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the 100gm, 3-hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the 50-gm, 1-hour glucose challenging test (gct) for initial screening and the 100gm, 3-hour gtt as confirmatory tests for the diagnosis of gdm between 1988 and 2001. various combinations of the 100gm, 3-hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: 2,535 women had 100gm, 3-hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure 1) . summation of the 1-hour and 2-hour gtt results with a diagnostic threshold of 334mg/dl yielded the most optimal balance between sensitivity (82.1%) and specificity (93.2%). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of 1-hour and 2-hour gtts had higher odds of operative vaginal delivery (aor=1.81, 95% confidence interval [ci] 1.03-3.27) and neonatal birthweight >4000gm (aor=1.91, 95% ci 1.03-3.57). conclusion: using only the summation of only the 1-hour and 2-hour gtt results of the 100gm 3-hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight >4000gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of 973 women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low 5' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period 973 women delivered in our institution following prom and induction of labor , 49 of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group (28% vs 17%, p<0.05). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for 43% and 27% (p<0.05) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low 5' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco 2 levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january 2003 and march 2006. computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 1'and 5', umbilical cord venous and arterial ph and base excess. results: during the period between january 2003 and march 2006 there were 1411 singleton elective cesarean sections, 404 of them were breech and 1007 vertex. the mean age, gravida and parity were significantly different between groups (33.5 vs.31.8, 2.95 vs. 2.19. and 1.26 vs.0.64 respectively, p<0.001). the birth weight was significantly different -3414.8 gram for the vertex and 3189.9 gram in the breech deliveries (p<0.001) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term (38-40+6w). venous and arterial po 2 and pco 2 levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, 1 amalia levi, 2 yaron zaulan, 1 avi harlev, 1 eyal sheiner. 1 1 obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; 2 epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years 1988-2005 were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before 22 weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, 175,093 singleton deliveries occurred in our institute. of these, 2010 (1.1%) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption (63.5%; n=1276) and placenta previa (36.5%; n=734). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= 1.6; 95% confidence interval [ci] 1.2-2.0; p=001 and 1.5;1.2-1.8;p<0.01 respectively ), suspected intra uterine growth restriction (iugr, 3.2; 2.6-4.0; p<.001), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, 72.9% vs. 12.1%, or=19.5; 95%ci 17.6-19.5; p<0.001). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<.001). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or 95% ci p crude or for perinatal mortality 7.9 6.6-9.4 <0.001 or adjusted for: <0.001 iugr 7.5 6.3-9.0 <0.001 oligohydramnios 7.6 6.3-9.1 <0.001 premature rupture of membranes 7.9 6.6-9.7 <0.001 the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than 0.5 indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p<0.001). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and 95% confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below 2.5 cm (before 25 weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, 104 pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, 52 of whom had delivered before 32 weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and 18 women underwent a vaginal cerclage. eighty-four women (80.8%) delivered at 37 weeks or beyond. one-hundred and three women (99.0%) delivered no earlier than 30 weeks, and only one woman delivered at 23 weeks. two women gave birth to a twin (at 30 and 35 weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from 2 tertiary healthcare systems from 2002-2007 experiencing pprom 24.0 weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within 12 hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of 92 women carrying 117 fetuses (73 singleton, 15 twin, 2 triplet, 2 quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of 20.7 (+/-2.6, range 13.6-24) weeks. the average latency period was 31.8 (+/-29.4, range 0.5-105) days, with a mean delivery gestational age of 25.2 (+/-4.3, range 15.4-34) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure 2 : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of 25% in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was 39.9 years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, 1 jane khoury, 2 padmini c sekar, 3 ralph c buncher. 3 1 obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; 2 center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; 3 environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per 1990, who estimates 1% of maternal mortality occurs in developed countries 1 . this rate has established without much decline in recent years. the mmr for ohio for 1987 to 1996 was reported by cdc to be 6.3 per 100,000 live births with 95% confidence interval 5.1 to 7.6. objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last 20 years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd9 codes 630 to 677 and icd10 codes o0 to o9. five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of 20 in 1981 to a low of 4 in 1996 and 1997. in general the five-year mortality rate was decreasing over time until the last four years 2000 to 2003, when we may be seeing an upward trend compared to 1995 to 1999 (p=0.07). the rates and associated 95% confidence intervals (ci) are shown in the table below. years mmr 95% ci 1980 ci -1984 ci 7.9 4.6-11.1 1985 ci -1989 6.0 3. 2-8.8 1990-1994 5.8 3.0-8.6 1995-1999 4.5 1.6-7.4 2000-2003 8.4 3.3-13.5 the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period 2002-2007 we studied 750 singleton deaths > 20 weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g20210a were tested at induction of labour. panel classification of cause according to the tulip classification 1 was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the 689 women tested for factor v leiden, 46 (6.7%) were carriers. of the 261 deaths caused by "maternal and fetal placental circulation disorders" 18 (6.9%) mothers were carriers of factor v leiden. of the 428 deaths with another cause of death 28 (6.5%) mothers were carriers of factor v leiden (p=0.88). of the 691 women tested for prothrombin g20210a, 21 (3.0%) were carriers. of the 264 deaths caused by "maternal and fetal placental circulation disorders" 9 (3.5%) mothers were carriers of prothrombin g20210a mutation. of the 427 deaths with another cause of death 12 (2.8%) mothers were carrier of prothrombin g20210a mutation (p=0.65). in women with iufd and factor v leiden or prothrombin g20210a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; 2 pathology, university medical center groningen, groningen, netherlands; 3 trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; 4 hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from 2002-2007 we studied 750 singleton deaths > 20 weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g20210a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the 642 men tested for factor v leiden, 26 (4.0%) were carrier versus 618 (96.0%) non-carriers. prevalence of factor v leiden in the normal population is 5% (p=0.27). 642 men were tested for prothrombin g20210a, 6 (0.9%) were carriers and 636 (99.1%) non-carriers. all were heterozygous. prevalence of prothrombin g20210a mutation in the normal population is 3% (p=0.002). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g20210a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied 898 consecutive gdm women with singleton childbirth and 2 or 3 abnormal values in 2-h ogtt with 75 g of glucose. abnormal plasma glucose values of the ogtt were: fasting 5.1, 1-h 10.0 and 2-h 8.7 mmol/l. insulin treatment was started when 2 values were 5.5 preprandially or 7.8 mmol/l postprandially in the 24-h glucose profile done within 7 days of diagnosis. if the same woman had more than 1 childbirth during the study period, only the last pregnancy was included. the control group consisted of 798 women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) >2.0 sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw >4000g than controls. insulin was started in 367 gdm women (41.9%). c/s rate was 18.7% in controls, 27.0% in diettreated and 42.0% in insulin-treated gdm women. macrosomia rate was 2.3% in controls, 4.6% in diet-treated and 18.4% in insulin-treated gdm women (p<0.001 compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in 0.3% of controls, in 1.7% of diet-treated and in 1.6% of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p=0.013). by regression analysis, previous child with bw >4000g (p<0.0001), previous c/s (p=0.0006), mother's age (p=0.012), bmi (p=0.015), insulin treatment (p=0.02) and fasting plasma glucose of the 24-h profile (p=0.027) were significant independent predictors of fetal macrosomia. conclusions the 24-h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. 0.264 1'-:·:tpartq,-, 1'-:·:tpartq,-, 1'-:·:tpartq,-, 25% p=0.001). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri 0.63 vs. 0.67, p=0.39; pi 1.29 vs. 1.44, p=0.42) . however, bilateral notching was much more common in the cohort of prior adverse outcomes (22% vs. 45% p=0.05) as well as in patients destined to have a subsequent poor outcome (p=0.001). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from 1996 through 2007. these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, 500 ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples (28-32 weeks' gestation; n=305 subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was 17.8 years (iqr:17.1-18.4). outcome data was available for 478 subjects. 214 (44.8%) had uncomplicated pregnancies. median birthweight was 3,200g (iqr: 2,831-3,530g) and median birthweight centile was 37.8% (iqr: 14.3-64.2%). 84 (17.6%) infants were born sga and 43 (9.0%) were preterm. spontaneous vaginal deliveries occurred in 71.0% of cases. there were 14 (2.9%) cases of pre-eclampsia and 34 (7.1%) admissions to neonatal care. 31.9% of subjects reported smoking at booking. iron deficiency anaemia was prevalent in 49.4% of subjects by 28-32 weeks and was strongly associated with higher infant birthweight centile (p<0.0001). 48.4% had serum 25-hydroxy vitamin d concentrations <15ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p=0.001), low serum folate (p=0.032) and high serum homocysteine (p=0.027) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight <2500gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight <2500gm, and apgar score <7 at 5 minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to 150-200ml with incremental increases of volume by 50 ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash 3000). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p1: the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p2 (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p3). we calculated the correlation coefficient between p3 and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; 3 women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last 5 years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the 19,426 births (2003) (2004) (2005) (2006) , accurate paired results were available on 64% of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p<0.05). the median (2.5 th , 97.5 th centile) values for cord arterial blood gases were: ph 7.27 (7.08,7.37); po2 16.3mmhg (4.6,31.0); pco2 55.1mmhg (39.0,79.0); base excess -3.0 (-10.8, 2.7); and lactate 3.7mmol/l (1.8,7.8). there was a progressive improvement in all blood gas measures over the 4 years of this study (all p<0.05). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < 7.0 ph < 5th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to <4h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (<4h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered 5 million units (mu) of intravenous pcn to be followed by 2.5 mu every 4 hours until delivery (cdc 2002) . subjects with renal disease, multiple gestation, and preterm delivery (<37wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were <3%. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: <1 h, 11.6±4.5 g/ml (n=10); 1-2h, 9.7±3.4 g/ml (n=15); 2-3h, 6.6±3.8 g/ ml (n=15);3-4h, 3.6±1.8 g/ml (n=17);4-8h, 6.9±3.7 g/ml (n=11);>8h 4.1±2.6 g/ml (n=24); and for those without a second dose after 4h, 2.3±0.9 g/ml (n=6). fetuses exposed to short duration (<4h) had higher levels of pcn than those exposed to >4 h (p=0.003). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until 1 hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs (0.1 g/ml)(p<0.002). furthermore, every sample individually remained 10-179 fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to <4h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: 3-oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of 3-oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n=18, and term elective cesarean section n=8) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and 3-oh aa (isocratic hplc using fluorometric detection with assay sensitivity at 1 pmol). results: 3-oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table 1) . for labouring group patients, the 3-oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of 3-oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of 3-oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; 2 department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all 3 conditions). results: among term births with ipd, 22.6% presented as maternal disease only, 69.5% as fetal disease, and the remainder (7.4%) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; 2 dept ob/gyn, wayne state univ, detroit, mi, usa; 3 dept pathology, wayne state univ, detroit, mi, usa; 4 dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: 1) mid-trimester (n=60); 2) term not in labor (tnl) (n=21); 3) term in labor (tin) (n=47); 4) preterm labor (ptl) who delivered at term (n=89); 5) ptl without iai (n=74); 6) ptl with iai (n=78); 7) preterm prelabor rupture of membranes (pprom) with (n=48) and without iai (n=48). af fetal hg concentrations were determined by elisa. results: 1) fetal hg was detected in 80.4% of all af and 31.7% of mid-trimester samples; 2) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester (19.5 ng/ml, iqr 0-49 vs 0.0 ng/ml, iqr 0.0-19.9, p=0.008); 3) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: 19.1 ng/ml, iqr 0-36.2; p=0.4); 4) median af fetal hg concentration was not significantly different among the 3 ptl subgroups [ptl with iai: 114.6 ng/ml, ptl who delivered preterm: 109.6 ng/ml, ptl without iai who delivered at term: 134.21 ng/ml, p=0.71(kruskal wallis) ]; 5) in pprom, there were no differences among patients with and without iai (148.4 ng/ml, respectively; p=0.074) ; 6) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p<0.001 for all comparisons). conclusions: 1) immunoreactive af fetal hg increases with gestational age; 2) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; 3) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, 1 deborah penava, 1 barbra de vrijer, 1 orlando da silva, 2 bryan s richardson. 1 1 obstetrics and gynaecology; 2 pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom >25 and <37 weeks gestation, singleton and no major anomalies, delivering between january 1, 1996 and december 31, 2005. patients were grouped according to ga at pprom stratified for latency time <72 hrs vs >72 hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were 1535 patients who met the inclusion criteria of whom 10%, 19%, and 71% had pprom at 25-28 wks , at 29-32 wks, or at 33-36 wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery >72 hrs, at 67%, 42%, and 10%, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table 1 . conclusion: despite a 2 to 3 fold increase in the incidence of chorioamnionitis with latency to delivery >72 hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until 32 weeks, and moderate infant morbidity until 36 weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso 4 ), nifedipine, terbutaline and 2 tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and 2 . comparisons were expressed as odds ratios (95% ci). results: in a 9-year period, 183 twin gestations were admitted in ptl and administered 1 tocolytic agent(s) with mean gestational age of 28.8 weeks. when 2 tocolytic agents were used, maternal complications were: 7 (9.5%) with intact membranes and 1 (2.8%) with prom had pulmonary edema (or=2.67, ci 0.58-12.29); 11 (14.1%) with intact membranes and 2 (5.6%) with prom had chorioamnionitis (or=1.3, ci 0.41-4.15); 2 (2.6%) with intact membranes and 2 (5.6%) with prom had postpartum hemorrhage (or=0.43, ci 0.05-3.62). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using 2 tocolytic agents. similarly, there is no difference between the 2 groups with latency period 48 hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of 2 tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥48 hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: 344 women were included (non-labor group (n=146), labor group (n=120), non-labor pre (n=78)). the median and mean hscrp levels were 6 and 9 and 15 and 21 in the non-labor and labor groups respectively (p=0.005). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p=0.3), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p=0.67), mode of delivery (p=0.3), or admission to the nicu (0.96). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean 34.5 vs.20.1, median 27 vs.6, p<0.001). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with 95% cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of 351 submitted placentas, 210 had histologic chorioamnionitis (cases) and 141 did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p .01). the cases were more likely to have had epidural anesthesia (or 2.9), internal monitoring (or 2.2), fever (or 4.8), maternal tachycardia (or 1.9) and fetal tachycardia (or 3.8) and less likely to have had induction of labor (or 0.49). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or 3.8 severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with 2 or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age=23.6± 7.6 years) with a mean gestational age at delivery 29.5±8.9 weeks. etiologies were pyelonephritis(n=8), septic abortion(n=4), endomyometritis (n=3), chorioamnionitis (n=3), ruptured appendix(n=3), pneumonia( n=1) and one unknown. eighteen (78%) were diagnosed during antepartum and 5 (22%) postpartum period.there were 2(9%) maternal deaths and high rate of major morbidities (table) . among the 18 antepartum patients,there were,4 abortions,4 iufd,2 neonatal death for a perinatal survival rate of only 39%. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru486 inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru486 (1.5 um= dose used in patients, 0.5 um, 0.05um) at different time points during infection (before, at the time of or after nf-kb activation). ru486 degradation kinetics was also included. the effect of ru486 on nf-kb induced il-6 and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il-6 and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il-6 and tnfa levels. in the presence of ru486, il-6 and tnfa levels continued to rise. the effect of ru486 on nf-kb induced il-6 and tnfa was dose dependent and was more prominent in slow metabolizers who had ru486 in the system for a longer time. addition of ru486 after the onset of nf-kb activation led to increased il-6 and tnfa levels above those observed without ru486. the addition of misoprostol (prostaglandin e1 analogue), at the concentrations used together with ru486 for medical abortion, did not add to the effect of ru486 on nf-kb induced il-6 or tnf. conclusions: ru486 has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld 50 that is 5 orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp-1 and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c 4-6 week old adult mice and 3-5 day old neonatal mice. liver and spleen was collected at 0, 24, 48 and 72 hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f4/80 rat antimouse igg, a marker for macrophages) and secondary antibodies (cy-3 goat anti-rabbit igg; af488 goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp-1 and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at 24 hours post-infection with listeria, adult liver and spleen showed increased staining for f4/80 and class ii, which increased further and became confluent surrounding microabscesses at 48 and 72 hours. in contrast, the listeria infected neonatal mouse showed some increase in f4/80 around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp-1 and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of 2 macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from 1992 to 2006. prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over 25%. results: a total of 22.4% (10,966) of 48,913 deliveries from 1992 to 2006 were by cesarean. annual cesarean rates increased by an absolute rate of over 10%; while, post-cesarean endometritis rates decreased from 23% to 2%. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure1) or nadirs. the month of july accounted for only 4 out of a total of 40 peaks for all years. the adjacent months of june and august accounted for only 3 each. the month with the highest number of peaks was april with only 7. these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results 1,030 patients were evaluated (439 cases and 591 controls). 85% and 15% of cases and 74% and 26% of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was 6.15% and 2.65% (p=0.09). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery <34 wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v.11.5. results: the immediate anatomic cod could be determined in 33.5% of all infants examined. in over 50% of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women (40 and older at their due date) who delivered at or after 32 weeks. non-exposed women delivered from july 2003 to december 2004 (when at for ama was not routinely recommended); exposed women delivered from july 2005 to december 2006 (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of 20%, we had 80% power to detect an increase to 35% after the introduction of at. results: 276 women met the inclusion criteria: 147 delivered before the introduction of at (non-exposed=before at) and 129 delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group (92% vs 27%; p<0.0001). 86 women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table 1) . the corrected iufd rates were similar in both groups (8/1000 vs 7/1000). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix <4 cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr 1.7, . physicians in solo practice had higher rates of elective inductions (p<0.0001), but there was no association between cesarean and practice type. conclusions: inductions accounted for 13.1% of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to 41 weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd40l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: 1) women with normal pregnancy (n=71) and 2) patients with fetal demise without disseminated intravascular coagulation (n=50). plasma concentrations of scd40l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: 1) patients with fetal demise had a higher median maternal plasma scd40l concentration than women with normal pregnancy (median 1213.3 pg/ml, range 118-3818 vs. median 369.5 pg/ml, range 63.5-1848.7, p<0.001); 2) there was no significant difference between the groups in the median maternal plasma tf concentration and 3) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median 45.6 ng/ml, range 13.6-115.2 vs. median 66.7 ng/ml, range 37.4-86.5, p<0.001). conclusions: 1) a change in the plasma concentration of tf was not demonstrated; 2) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; 3) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and 4) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase 9 production, activity and prostaglandin e 2 secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) 9 activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp9 activity/protein and pge 2 secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid (0-1mm/6h), then with increasing doses of tnf (0-50ng/ml/24h). medium and cells were analyzed by gelatin zymography/western blotting for mmp9/mmp2 activities/protein. pge 2 output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp9 production, secretion and activity in amnion epithelial cells. tnf (50ng/ml) induced an 18 fold increase in cellular active mmp9 production and 3 fold increase in secreted mmp9 enzyme activity by amnion epithelial cells. these increases were reduced 62-100% following 6h pre-treatment with 0.5-1.0mm -lipoic acid. mmp2 protein/activity and pge 2 secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp9 protein/activity. mmp2 protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge 2 production (13 fold increase/50ng/ml tnf/24h) that was inhibited by 70%-100% following 0. objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il-8 gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the 2 kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl3 luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek293 cells. at 48 h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to 11 eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes 3 -983 to -977 and 9 -329 to -324 ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity 4-to 5-fold. whereas mutations in ebox 3 or 9 resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il-8 gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd44 in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd44, a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd44 expression by immune cells is important in extravasation of leukocytes into tissue. cd44 may also be required for ha catabolism through the action of hyaluronidases 1 and 2. in the cervix, cd44 is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd44 in cervical remodeling, mice with a null mutation for cd44 (cd44 -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd44 -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd44 plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd44 -/mice. this suggests that postpartum breakdown of ha in cd44 -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase 2 (hyal-2) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd44 -/cervix was similar to wt. conclusion: these studies provide evidence that cd44 may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal2 may compensate to allow ha removal. furthermore, the activity of hyal-2 may be dependent on cd44. little difference in the recruitment of immune cells between cd44 and wt animals suggest that cd44 expression is not required for this process. these experiments provide a greater understanding for the role of cd44 and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: 221 term fm had the following characteristics: maternal age 26±6.4 yr, gravida 3.1±2.2, gestation 39±1.1 wks, elec. cs 10.0%, duration of rom 403±417 min, duration of contractions 642±440 min, african american 45%. 40% of the fm had <10 % separation; 11% had more than 95 % separation. srom fm with >10% separation (vs. <10%) had significantly shorter duration of rom (p=0.03) and admission to birth (p=0.05) times. srom fm with >95% separation (vs. <10%) had even shorter rom (p=0.006), duration of contractions (p=0.002) and admission to birth (p=0.0003). the >95% group (vs. <10%) was further along, gestationally (p=0.05). srom fm (vs. arom) had shorter admission to birth (p=0.008), but longer rom to birth (p<0.0001) times. absence of epidural (p=0.001), srom mode of rupture (p=0.04), svd (vs. elec. c/s) (p=0.06), and the presence of meconium (p=0.002), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, 1 shirin khanjani, 1 yun s lee, 1 tg teoh, 1,2 philip r bennett. 1 1 institute of reproductive and developmental biology, imperial college, london, united kingdom; 2 maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase-2 (cox-2), which is essential to the labour process. cox-2 mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox-2 expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from 20 women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p65 was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p65 protein concentrations were used for whole genome analysis using affymetrix u133 arrays. results and conclusions: we identified 919 significantly regulated genes. the gene with the highest fold change was cox-2 (x44.4) followed by oxytocin receptor (x24.1), ch10orf (x19.6), integrina2 (x17.7), and connective tissue growth factor (x14.9). other significant genes included interleukin-8 (il-8) (x6.3). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox-2 and il-8 expression. to prove that cox-2 is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p65 sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p65 associated with inhibition of cox-2 demonstrating that nfkappab is essential for cox-2 expression. objective: premature birth is a major public problem accounting for over 13,000 deaths and 30,000 surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r 2 = 0.99) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih 48476. the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase 2 (has2) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has2 are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon 1 (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has2 during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has2 gene. the regulation of has2 was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has2. regulation of has2 expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has2 mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon 1. two transcripts of approximately 4.1kb and 2.8kb were detected that arise from use of 2 polyadenylation sequences. as compared to np, the expression of has2 is increased 4, 23, and 9 fold on gestation days 15, 18 and shortly postpartum respectively. has2 mrna expression is increased upon treatment of caski cells with epidermal growth factor (30ng/ml) and is suppressed in cells treated with ag1478 (20 m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at 4 and 8 hours of treatment. conclusion: has2 is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has2 gene. in vitro studies using caski cells suggest has2 is regulated in part by the egf signaling pathway resulting in a several fold increase in has2 expression. these results provide an understanding of has2 gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately 30% of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from 1% to 100% in 96-well plates. cell viability was determined at 24, 48 and 72 hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at 48 hours in both cell types (figure 1 ). at 48 hours, chorion exhibited greater percent cell death compared to decidua at concentrations of 20, 40 and 60% cse (p=0.003, 0.019, and 0.069, respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure (36, 48 and 72 hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from 20-60% compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, 1 tae-bok song, 1 cheol hong kim, 1 jong woon kim, 1 moon kyoung cho, 1 sung yeul yang, 2 bong whan ahn. 2 1 obstetrics gynecology, chonnam national university medical school, gwangju, korea; 2 biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n=20) and pregnant women with pprom (n=20) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n=20) and pregnant women with pprom (n=20) were determined by the 2,4-dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to 5 hours with 0.2ml of 1mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: 1. the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy (9.74±0.48 vs. 7.20±0.38 nmol/mg protein, p<0.01). 2. the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy (13.0±0.33 vs. 11.27±0.17 nmol/mg protein p<0.01). 3. the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level (12.08 ± 0.81 vs. 9.74±0.48 nmol/mg protein, 20.08 ± 0.66 vs. 13.0±0.33 nmol/mg protein, p<0.01). 4. the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level (5.04 ± 0.33 vs. 9.74±0.48 nmol/mg protein, 9.76 ± 0.35 vs. 13.0±0.33 nmol/mg protein, p<0.01). 5. there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il1 . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) 2 and 9, which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp-2, mmp-9, and vimentin (a dc marker) was performed on cam-complicated (n=5) and gestational age-matched control decidua (n=5), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with 10-8 m estradiol (e2) or e2 + 10-7 m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e2 +/-mpa with or without 1 ng/ml of il1 or tnf . secreted mmp-2 and mmp-9 levels were measured by elisa (n=8), and quantitative rt-pcr assessed mmp-2 and mmp-9 mrna levels (n=4). results: tissue staining revealed that mmp-2 and mmp-9 levels in cam-complicated decidua (hscore mean±sem: 203±14 and 198±12, respectively) were significantly higher than in control decidua (136±6, and 114±6 respectively; p<0.05). in cultured term dcs incubated with e2, tnf and il1 significantly increased secreted levels of mmp-9 compared to e2 alone (pg/ml/ g protein: 162.3±110.1 and 20.4±6.5, respectively, vs. 0.5±0.2; p<0.05). in parallel incubations with e2+mpa, basal mmp-9 output was lowered by 50%, and tnf -and il1 -elicited mmp9 levels were blunted by 70% and 40%, respectively. rt-pcr confirmed that tnf and il1 increased mmp9 mrna levels (p<0.05), although mrna levels in e2+mpa incubations were not different from those of e2 alone. mmp2 levels in all treatments were similar. conclusions: mmp-2 and mmp-9 are elevated in cam decidua compared to controls. our in vitro results suggest that mmp-9 expression is enhanced by the high levels of il1 and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp-1 and mmp-3 and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, 1 patama promsonthi, 1 wasun chantratita, 2 mana rochanawutanon, 2 patcharee karnsombut, 2 chutatip srichunrusami, 2 stephen j lye, 3 boonsri chanrachakul. 1 1 obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; 2 pathology, ramathibodi hospital, mahidol university, bangkok, thailand; 3 obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) 1, a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp 1 in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from 4 groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in 10% formal saline for paraffin section. immunohistochemistry (n = 5 each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp 1. the mcp 1 messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp 1 and mcp 1 receptor (n = 10 each). results: immunohistochemistry demonstrated mcp 1 in cervical tissues from all four groups of women. mcp 1 was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp 1 and mcp 1 receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp 1 and mcp 1 receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from 3 groups of cd-1 mice. group 1: mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group 2: e15 dams representing preterm controls. group 3: e18.5-19 dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n=6 dams/treatment group. 18 separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of 64-fold change with p value of <0.0001, 368 genes in the cervix were differently regulated between the 3 groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: 1) in preterm cervical ripening (e15 lps vs e15): immune and inflammation response, defense response 2) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, 1 valentina ciani, 2 gunvor e ekman. 1 1 obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; 2 obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase-3 and the inflammatory cell marker cd 45 was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase-3 staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p=0.002) with unripe cervix and from non-pregnant patients (p=0.00067). the inflammatory cells staining for cd 45 increased in post partal and term pregnant sections compared to non-pregnant (p=0.016). there was a higher frequency of caspase-3 positive cells in the post partal tissue than of cd45 positive cells (p=0.00011). the localization of cd-45 positive staining was highest in the epithelia and basal lamina while caspase-3 staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days 12, 16, 20, 21, and 22) sprague-dawley rats underwent tensile testing using an instron 5800 material testing system. the testing regimen consisted of tissue extension to near maximal strain over 60 seconds followed by a period of constant strain for 60 minutes, then return to rest over 60 seconds. this cycle was repeated 2 additional times with a 60 minute rest period between cycles. strain and force measurements were recorded at 1 second intervals. 4-6 animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli 1.2x 10 8 .:!: 8.5x 10 8 1.2 x 10 8 .:!: 3.0 x 10 8 group a (c, c) group b ( c, p) 2.3x 10 8 + 2.7x 10 8 9.4 x 1 0 7 + 4.8 x 10 8 .33 .81 .83 group c (p, p) 1.1 x 1o"::!>s x 1o" 1.1 x 10 8~ 1.ox:1o• k. ~neumoniae group a ( c, c) 5.3 x 10 7 + 3.4 x 10 7 9.2 x 10 7 + 6.7 x 10 7 .88 .87 .91 group b (c, p) 6.0 x 10 7 + 4.8 x 10 7 6.3 x 10 7 + 3.0 x 10 7 group c (p, pl 4.7 x 10 7~2 .3x 10 7 6.9x 10 7~4 .6x 10 7 c. al bicans group a (c, c) 1.1 x 10 '+1.8x 10 7 1.9 x 10 8 + 1.0 x 10 8 group b (c, p) 1.5x 1o•+ 1.0x 10 7 2.4 x 10 8 + 1.1 x 10 6 .75 . 87 .60 group c (p, p) 4.7x 10 7~2 .3x 10 7 2.1 x 1 0 7~5 .9 x 10• introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd-1 mice were used in these two models of ptb: 1) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n=6); controls for this model received intrauterine saline (n=6) and 2) a non-infectious model of ptb using ru486 sq (150ugrams/dam) (n=5); controls for this model received no intervention (n=6) were used for these studies. for both models, 6 hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il-6 and tnf-alpha in the uterine tissue. mrna expression of ifn , il-6, il-1 , il-10, tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il-6 and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p<0.001) (figure 1). in cervical tissue, an increase in il-6, il-1beta and tnf mrnawas observed in both models, while ifn-gamma and il-10 were only increased in the lps model. (figure 2 ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f4/80. the density of adherent f4/80 + cells greatly increased in equivalent volumes of af between 15.5 and 18.5 days postcoitum (dpc) (19.5 dpc = term). interestingly, the f4/80 + cells at 18.5 dpc were highly similar in morphology to those present in 18.5 dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f4/80 and for cd45, a pan-leukocyte marker. we observed that af from 15.5 and 18.5 dpc mice contained two sub-populations of cd45 + f4/80 + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox-2, and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from 9 nonpregnant (proliferative, n = 4; progestin treatment, n = 5) and 35 pregnant women undergoing hysterectomy (eg, n = 7; term before ripening, n = 10; after ripening, n = 7; in labor, n = 11). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase (47.6 ± 8.7 pg/ug cdna) and decreased 80% by progestins (8.6 ± 2.7 pg/ug cdna). this level was maintained in stroma from pregnant women before labor (6.6 ± 1.3, eg.; 9.9 ± 1.8 before ripening; 12.5 ± 2.9 after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix (2.4 ± 0.6 pg/ug, p < 0.05). interestingly, whereas pr-b was 30 ± 2% that of total pr in the nonpregnant cervix, pr-b mrna levels were 52 ± 4% to 70 ± 6% in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr1294), pr-b immunoreactivity was 50% that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from 44 ± 9 to 19 ± 8 units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il-8, mcp-1, tlr-2, cd62l, cox-2, and s100a9 mrna (all p < 0.05, anova) but not cd11b or tlr-4. pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox-2, expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox-2 but not il-8, mcp-1, or toll-like receptors. ifn-y il-6 il-1,6 il-10 tnf-01 "p value <0.05 lps/saline ru486/controls 10.3"" 0.63 68" 6.6" 15.7* 24" 12" 5.9* 1.8 3.1" 722 association between interleukin-6 (il-6) and ilto examine association of amniotic fluid interleukin 6 (il-6) concentration with il-6 and its receptor il6-r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [<36 weeks]) and control (term [>37weeks]) amniotic fluid (af) il-6 concentrations were analyzed for association with haplotypes of the il-6 and il6r genes in aa and c separately. in il-6, eight, and in il-6r, 22 single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa:35 maternal:cases-57 controls-34 fetal: cases-54 fetal controls-; c: maternal cases-88, controls-40, fetal:cases-86; controls=36). haplotype associations were performed by using a sliding window with outcome il-6 concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il-6r rather than in il-6. in c fetal dna il-6r haplotypes defined by markers 16311-21909 bp from the transcription start site associated most strongly with af il-6 concentrations (global p=1.6x10 -3 ) and in aa maternal il-6r haplotype markers at 16311-21909-22214-26274 (global p=2.30x10 -3 associated with il-6 concentrations. in the c fetal cases the 16311-21909 haplotype with the highest concentration was a-g (log(cytokine) = 3.67 pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at 16311-21909-22214-26274.this was seen in both cases and controls at (case log (cytokine) = 3.84; control log(cytokine) = 3.49 pg/ml). significant associations from haplotype analyses converged on three regions of the il-6r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il-6 that associated in cases with miac but not in cases without miac (-661 and -636; -636 and -237)(both with p < 1x10 -3 ) conclusion: differences in the af il-6 concentration in ptb do not result from single snp effect on il-6 but are a result of complex relationships between il-6 and il-6r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, 1 amanda tattersall, 1 mark tattersall, 1 suren sooranna, 1 peta grigsby, 2 leslie myatt, 2 mark johnson. 1 1 obstetrics gynaecology, imperial college, london, united kingdom; 2 obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il-1 or il-8 on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further 6 samples were obtained before the onset of labour (n=6) from which myocytes were isolated and cultured in 6-well plates. when cells were 85-95% confluent either tnf-at a concentration of 1ng/ml was added to the cells for 7, 15, 30, 60 and 120min and the cells analysed by western blotting or tnf-at concentrations of 0, 0.1 and 1ng/ml was added either alone or in combination with similar concentrations of il-1 or il-8 to cells for 6 hours and mrna was extracted and converted to cdna to determine il-8 and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr1 and tnfr2 mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-(1ng/ml) activated all three mapk substypes: erk, jnk, and p38, activation peaked between 7-15 minutes. preliminary data suggest that tnf-induces il-8 mrna expression but exposure to il-1 or il-8 itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il-8 mrna expression. this enhanced mapk-dependent il-8 expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, 1 anna-marie fairhurst, 2 mala s mahendroo. 1 1 obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; 2 immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil 7/4). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with 2-4h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d15 mice were treated for 13h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day 18, d18.75) and remain high through postpartum (pp). macrophages (mø) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d15 resulted in a premature increase in mo but not neutrophils or mø. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d18.75 and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mø numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until 90-95% confluent and stimulated with 1ng/ml il-1 prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp65, which pulled out nf-bp65 and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table 1 shows the different tfs interacting with nf-bp65 based on the degree of binding stimulated by il-1 compared to no-il-1 control. conclusion: these data show that il-1 stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap-1, which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il-1 stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table 1 high ap-1, c/ebp, cbf, creb , c-myb, e2f-1, ets, ets-1/pea3,fast-1, gas/isre, hse, mef-1, mef-2, myc-max, nf-1, nfatc, nf-e1, nf-e2, pax-5, objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi 10:323,2003; reprod biol endo 3:39,2005) . although parturition is associated with systemic or local progesterone withdrawal (ajog 196:289,2007) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c57bl6 mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod 12:602,1997) . after 15 days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day 17 (e-p) and groups killed on days 18 and 19. cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n=6/group/day; 3 sections/ cervix; biol reprod 61: 879,1999; jsgi 12:578,2005) . stained macrophages and neutrophils were counted (image pro-plus 6, media cybernetics). results: e+p treatment for 15 days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for 15, 18, or 19 days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days 18 and 19 (p<0.05, e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at 24 and 48 h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t47d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf-10a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf-10a breast epithelial cells and a673 rhabdomyosarcoma cells by the change in fluorescent emission of jc-1. total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with 30-60 min treatment with progesterone and r5020 which were inhibited by a specific pr antagonist, rti-6413-049b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r5020. progesterone/progestin treatment for 120 min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge 2 is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge 2 receptors, ep-1 and ep-3 mediate contractions whilst ep-2 and ep-4 mediate quiescence. ep-4 contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep-4 expression. myocytes plated in 6 well plates were treated with il-1 (1ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p65 sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p65. rna was extracted for amplifying ep-4 using quantitative rt-pcr with amplification of l19 as a control to normalise data. il-1 caused an increase in expression of ep-4. knock down of nfkappab using si-rna resulted in a further increase in ep-4. this data suggests that although il-1 stimulates expression of ep-4 it does so through an nfkappab independent mechanism. the upregulation of ep-4 with sirna knock down of p65 suggests that activation of nfkappab would inhibit ep-4 expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il-6 is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il-6 gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f 2 receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)-2, oxytocin receptor (otr) and connexin-43 (cx-43), suggesting multiple physiological effects for il-6 in term delivery. objective: to examine the effect of il-6 deficiency on the peri-partal uterine mrna expression of the pge 2 receptors (ep) 2, 3 and 4; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p 4 ) concentrations. methods: gestational length was observed in pregnant c57bl/6 wt (n=61) and ko (n=39) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d15 through delivery and up to 24h postdelivery. serum p 4 was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p<0.05. results: birth was delayed in the il-6 ko mice (20.7±0.2d vs. 19.7±0.1d), and this affected the timing of peri-partal changes for all uaps similarly. both ep2 and ep4 (relaxatory receptors) were elevated at d18 in ko dams, but returned to low levels before delivery and were elevated at delivery (ep2) or afterwards (ep4). ep3 levels did not change. otr increased several hours before delivery in all dams. cx-43 increased at delivery, then fell, while pghs-2 increased at delivery and remained elevated afterwards. ptgfr mrna increased 3-4-fold at delivery and a further 2-3-fold after delivery, suggesting loss of pgf 2 permitted enhanced ptgfr expression. p 4 serum concentrations fell pre-partum in both groups. conclusions: il-6 ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p 4 fall, suggesting a potential ovarian effect. a uterine role for il-6 in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, 1,25-dihydroxyvitamin d 3 (1,25(oh) 2 d 3 ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme 1 -hydroxylase (cyp27b1) which catalyzes synthesis of 1,25(oh) 2 d 3 from the inactive pro-hormone 25-hydroxyvitamin d 3 (25ohd 3 ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp27b1, 25ohd 3 and 1,25(oh) 2 d 3 on innate immune responses in trophoblastic cells. methods: 3a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for 24 hrs with various concentrations of 1,25(oh) 2 d 3 (1-100 nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp27b1 was affected by pathogenic stimuli, 3a cells were treated with ligands for toll-like receptor (tlr) 1-9, cyp27b1 expression (real time pcr) and 25ohd 3 utilization were assessed. results: 3a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp27b1 with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for 24 hrs with 1,25(oh) 2 d 3 showed dose-dependent induction of the antimicrobial defensin cathelicidin expression (3.5-17 fold induction), whilst cells treated pro-hormone 25ohd 3 (100 nm) showed 2.5-fold induction of cathelicidin expression. activation of tlr3 (poly i:c) and tlr4 (lipopolysaccharide) enhanced the expression of cyp27b1 (2-and 2.5-fold) and increased the sensitivity to 25ohd 3 as a consequence. conclusion: these data show that autocrine synthesis of 1,25(oh) 2 d 3 from 25ohd 3 can stimulate trophoblast immune responses in a similar fashion to macrophages. as 25ohd 3 is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent 12-kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il-1 on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in 6 well plates and when cells were 70-80% confluent they were serum starved overnight and incubated with 1 m 6methyl-17 -hydroxy-progesterone acetate for 48h and with or without 1ng/ml il-1 for a further 6 hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n=6 for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: 6 h incubation of uterine myocytes with 1ng/ml il-1 caused a marked increase in slpi by 201% (n=12; p<0.015). incubation of uterine myocytes with 1 m progesterone for 48h also increased slpi by 139% (n=12; p<0.05). incubation with il-1 for 6h in the presence of 48h with progesterone increased slpi: gapdh mrna ratio from 10.50 ± 3.37 to 33.33 ± 10.72 (mean ± sem; p<0.004). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by 50-and 200-fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p<0.05). these data show slpi is present in human myometrium and that it is increased by il-1 . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap-1 and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science 2005: 307; 426-430) . its levels are elevated in obese patients, those with type 2 diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef (100ng/ml) for 1h, lysed and the resultant proteins labeled with cy3 or cy5 (amersham). there were used on a protein microarray containing 64 constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap-1, cre, hse and gre response elements using the exgen500 reagent. the cells were treated with rhpbef (0.1, 1.0, 10, 100 ng/ml) 4 hrs, lysed and 50ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv40 luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated 20 mapk signaling components including 9 functioning as part of the erk pathways and 5 belonging to p38 signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated 3 signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of 4 functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il-8, pghs-2, otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in 6 well plates until 70-80% confluent. cells were serum starved overnight and incubated with 1μm methyl progesterone, 0.2mm sodium 8-bromo-camp, 0.1mm forskolin, 1μm kt5720, 75ng/ml brefeldin a and 0.1mm 8-pmeopt-2'o-me-camp either alone or in combination for 48h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n=6 for ptnl, ptl, tnl and l). copy numbers of epac1, epac2, il-8, pghs-2, otr, fp, gapdh and -actin were measured by qpcr. results: epac1 and epac2 were present in the upper and lower segment of myometrium with epac1 levels being some 10-fold higher than those of epac2. there was no change with the onset of labour at or before term. treatment with il-1 for 6h significantly increased epac2 (n=12; p<0.012) but had no effect on epac1. when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs-2 and fp mrna expression (n=6; p<0.05), but had no effect on il-8 and tended to reduce otr. the il-1 -induced increase in pghs-2 and il-8, but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs-2 and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n=4) and second trimester myometrium (n=5), and in paired upper and lower segment pregnant (non-labouring, p, n=8) and labouring (l, n=8) myometrium; as compared to non-pregnant (np, n=8) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at 90-95 kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p<0.001); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p<0.05). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p<0.05). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: 1) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u937 cells at epithelial/macrophage cell ratio of 5:1 was employed. 2) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of 1, 10, 100 and 1000 nm for 2 hrs followed by 2 day stimulation of 10 g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il-1 , il-6 and il-8, il-10 and tnf were determined using elisas. 3) the co-culture was pre-treated with 100nm of each pas for 2 hours prior to lps stimulation at 10 g/ml for 0.5, 1.5, 6, 12, 24 and 48 hours. the phosphorylation of p38 mapk and gr and the expression of mapk phosphatase 1 (mkp1) were determined by western-blotting. results: il-1 , il-6 and il-8, il-10 and tnf were elevated by 3.8 fold, 1.7 fold, 1 fold, 4.9 fold and 4.6 fold in the co-culture model in response to lps(p<0.001 for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il-6, but not on il-1 , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp1. p38 mapk was activated by lps for up to 24 hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p38 mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p4) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p4 effect and optimize concentrations, cells were pretreated with p4 (10 -4 m-10 -6 m), dexamethasone (3um) or vehicle for 1 hour prior to overnight lps (10ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p4 incubation and lps stimulation to determine whether the p4 effect was reversed by a camp antagonist. results: p4 significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at 10 -4 m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig 1) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig 2) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p4. objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the 4th day in culture, nonadherent cells were removed. on the 7th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp1 water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp1 gene expression using the first trimester-derived extravillous htr-8/svneo cells and the term placenta-like trophoblast carcinoma cells jeg-3. methods: cultures of both cell lines were treated with a physiological concentration of avp (0.1 nm) to determine aqp1 mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with 1% fbs without avp. to determine whether avp regulation of aqp1 occurs by a camp signaling pathway, jeg-3 cells were preincubated with 100 μm 9-(tetrahydro-2'-furyl) adenine (sq22536), a cell-permeable camp inhibitor, before being treated with avp (0.1 nm). cells were incubated for 10 hrs for aqp1 mrna expression and for 20 hrs for protein extraction at 37°c. after harvest, real time pcr and western blotting analysis were used to detect the aqp1 mrna and protein expression levels, respectively. results: avp increased aqp1 mrna expression in both cell lines by 1.8 and 2.8 fold after 10 hrs. aqp1 protein expression paralleled the increase seen in the mrna (p<0.05). pretreatment of jeg-3 cells with sq22536 inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp1 gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp1 gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp1 gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium (1,2). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone (0, 100nm, 1mm, 10mm, 100mm) for 2, 6 and 24hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a 60kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru486 and organon 31710 in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. 1. taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at 0.75 g, the equivalent of 30 weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at 104 days g (dg). experiments started at 110 dg in 8 dex (2 mg) and 8 saline treated controls (ctr), each receiving 4 injections 12 h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres (1.86x10 6 total; sterispheres, biopal) were injected at 11:00am into fetal jugular and femoral veins before maternal i.m. injection (t0) and 2 h after the 3rd injection (t3) in both groups. tissues were collected 12 hours after the 4th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex (6.8+1.5 mmhg; p=0.007) with no change in ctr (-0.1+0.4 mmhg). regional bf distribution is presented as a percent of the total counts per 100 g tissue (table 1) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table 2 . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r2 is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r1 and crf-r2 receptors in mouse fetuses. methods: time-dated cd-1 pregnant mouse were anaesthetized and fetuses (n=10) were collected at e17 (term = e19). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r1 and crf-r2 by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using 3',3 diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro 4.01 plus software. results: immunostaining for both crf-r1 and crf-r2 was seen in the smooth muscle layers of all lumens examined (7 lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r1 (maximal iod: 0.369±0.024 au and minimal iod: 0.177±0.13au) and for crf-r2 (maximal iod: 0.318±0.015 au and minimal iod: 0.240±0.020 au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r1 and crf-r2 receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r1 or r2 receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a 20-30% reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (ctr) 24-hs apart at 80 days gestational age and allowed to deliver at term. at 1.5 yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a 48-hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl 68728; hd p01 hd04784. clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin 1-7 in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n=4) or vehicle (n=3) at 80-81 days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after 5 days recovery the sheep received an infusion of ang 1-7(1ng/kg/min) with or without its antagonist, [d-ala 7 ]-ang-(1-7)(d-ala, 10ng/kg/min), into the renal artery for 24 hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang 1-7 infusion. however, the infusion of ang 1-7 resulted in a decrease (change from baseline) (p=0.029) in na + excretion of 0.41±0.15 meq/kg/24h in the vehicle animals and 0.12±0.05 meq/kg/24h in the b animals. there was no significant change in gfr during ang 1-7 infusion. gfr was lower in the b group during the combined ang 1-7 and d-ala infusion (91.5±3.5ml/min vs 117.0±15 in vehicle group, p=0.03). intrarenal infusion of ang 1-7 at 1ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang 1-7 may be mediated by a receptor other than the mas receptor for ang 1-7 and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine (0.1-0.6 g/kg/min) and to an l-name (2 mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max 142±4 vs 101±5 % of baseline, p<0.05, figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure 1 right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl 68728. women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone (0.17 mg/kg) or vehicle on days 80 and 81 of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between 6 and 9 months and cells were dispersed and plated at a density 2×10 5 cells per well in 48 well plates. after 48h, cells were stimulated with normal medium, 10nm arginine vasopressin (avp), 100nm avp, 1nm corticotropin releasing factor (crf) or 10nm crf for 2h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following 10nm avp (21.9 ± 4.3 vs. 20.4 ± 6.6) and 1nm crf (10.0 ± 2.8 vs. 10.7 ± 5.0). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells (24.1 ± 4.9 and 11.8 ± 5.9 respectively), but was significantly decreased in betamethasone cells (9.9 ± 7.5 and 2.4 ± 4.3 respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd47584 and hd11210. lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first 24-48 h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n=6) were exposed to a paradigm of graded, stepwise hypoxia on day 22 (term=22) (pediatr. res. 61: 176-179, 2007) . control pregnant rats were exposed to 21% oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in 4% paraformaldehyde and paraffin embedded. sections (n=6 per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro 4.01 software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua (0.206±0.015 au) and in all three major placental cells types (giant trophoblast cells: 0.143±0.019 au, spongiotrophoblast cells: 0.133±0.003 au and labyrinth cells: 0.083±0.008 au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua (0.197±0.006 au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et-1 and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (v) 24 hours apart at 80 days of gestational age and allowed to deliver at term. at 1.5 yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f=5.166, p=0.030) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f=7.05; p=0.01). conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects. the activation of etb receptor by et-1 inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl 68728; hd p01 hd04784. in the placenta, 11 -hydroxysteroid dehydrogenase type 2 (11 hsd2) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate 11 hsd2 expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect 11 hsd2 expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received 4 intramuscular injections consisting of either saline (2ml saline/ewe) or dex (0.14 mg/kg) given 12 hours apart starting on 40 days of gestation (dg). the animals were sacrificed at 50, 100, 125, and 140dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure 11 hsd2 placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental 11 hsd2 mrna expression across gestation. however at 50dg, there was a significant increase in 11 hsd2 expression after dex (p=0.026). there was an increase in placental 11 hsd2 mrna progressively from 50dg (mean = 0.45) to 140dg (mean =1.48), independent of treatment. overall, a positive correlation between 11 hsd2 and fetal weight (r 2 =0.43) was apparent at 50dg and at 140dg (p=0.065) in both control (p=0.01) and dex (p=0.13). significant positive correlations between placental 11 hsd2 and fetal weight (r 2 =0.45) were found in females (p=0.01) and a similar trend was found in male fetuses (p=0.07). we conclude that in the sheep placenta, 11 hsd2 expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between 11 hsd2 and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental 11 hsd2 is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; 1996; 64:412) and increases femoral vascular resistance in the lamb (jphysiol; 2003; 547:61) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between 3mo and 2,5y of age (p<0.01). vascular relaxation to acetylcholine (ach) but not to pge2 decreased during the same time (p<0.01). vascular contractility of the renal artery (ra) increased in response to k + (p<0.05). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol;2002;283:r441) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem;1997;82:234) increase stroke mortality for unknown reasons (amjpsychiatry; 2003; 160:1823) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n=6) or 170μg/kg dexamethasone (n=6) i.p. at day e19/20 equivalent to 2x12mg dexamethasone administered to a 70kg pregnant woman. at 2.5 years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p<0.05). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p<0.05). relaxation to ach and pge2 was similarly diminished in both vessels after precontraction with na (p<0.05). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n=7) or with two doses of bmz (n=6), 0.17mg/kg, 24 hours apart at 80 days of gestation. renal cortex from the male offspring was harvested at 19-24 months of age. cortical membranes were then solubilized and incubated at 37°c with either 125 i-ang i or 125 i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln4760 to inhibit ace2 activity, or sch39370 to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p<0.05 was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p=0.0073). ace2 and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace2 activity, bmz treated animals exhibited a 2.5 fold greater proportion of ace activity versus control animals (p=0.04). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd47584, hd17644. we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n=6) were dissected from term ovine fetuses (146-147 d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n=3) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n=3) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r2 receptors. both ucn-1 and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r2 receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn-1 functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: 118-120 days gestation), preterm (pt: 130-132 days), near term (nt: 140-142) and term (t: 146-147days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn-1 (1:500 sigma) by abc system. intensity of ucn-1 immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p<0.05). conclusion: ucn-1 immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn-1 inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n=3) or vehicle (n=3) at 80-81 days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after 5 days of recovery, the sheep were infused with ang ii (1 ng/kg/min) with or without candesartan (10 ng/kg/day) or pd123319 (pd 500 g loading dose and 10 ng/kg/min infusion) into the renal artery over 24 hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p=0.03). ang ii decreased the nae in both groups(p=0.0007). candesartan increased nae among controls(controls 0.16 and b 0.56 meq/kg/h, p=0.048). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p=0.0172). during ang ii infusion there was a decrease in gfr among control compared to b sheep (-71.17 vs. -18.46 ml/min, p=0.0081). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at2 receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type 1 receptor (crhr-1) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, 24 hour incubation with the specific crh-r1 antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r1, a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by 24 hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short (2.5 hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ 200000 cells/well. after 48 hours in culture, wells were rinsed 2x and refilled with serum-free dmem/ f12 containing vehicle (dmso) with or without ant. after 2.5 hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem,264:21474,1989) completed. triplicate vehicle or ant wells were treated with i 125 -tyrosine 23 human acth (1-39) with or without 10 -6 m acth (for non-specific binding). after 1 hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk (10 -6 m) was added to the wells. after 2.5 hours, medium was removed and stored @ -80ºc for camp eia and cortisol ria. results (mean±se) of 2.5 hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n=4 3024±415 2966±463 cortisol (ng/ml/2.5hr) n=7 144±20 126±19* (p=.012) camp (pmol/ml/2.5hr) n=12 9.1±2.0 4.8±1.1* (p=.003) * indicates significant difference from vehicle alone. conclusion: the crh-r1 antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive 2 doses of 0.17 mg/kg of or vehicle, at 80 and 81 days of gestation; the offspring were studied at 6 and 18 months of age. in 17 female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term (142-148 days of gestation) were brought from the farm at 12-32 months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl (0.0275 meq/kg/min at 0.55 ml/min) was given for 60 min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males (17.74± 1.15 vs 8.90± 1.46 p<0.0001) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males (33.9± 12.7 vs 13.88± 3.06 p<0.0001) during the na infusion experiment, the exposed females had lower arc than did the control females (0.40± 0.08 vs 1.28± 0.20 p<0.05). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd 47584 and hd17644. background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin-2 receptor; mc2r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth 1-39 yet decreased expression of key steroidogenic enzymes and the mc2r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with >90% homology to the human mrap n-terminal 68 residues, with partial homology to the beta isoform in the carboxyl region ( 65%). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from 105-120 (n=5) and near term (142-145; n=5) days of gestation (dg) as well as from lth (n=6) fetuses (exposed to high altitude hypoxia from 40 to 139-141 dg) and age-matched normoxic controls (n=6). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/50 ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between 105-120 dg (11.72 ± 4.2) and increased (p<0.05) approx. 5-fold near term (140-145 dg; 50.6 ± 7.3). levels of mrna for mrap were highly correlated to cyp17 mrna in individual samples. mrap expression in control (25.1 ± 2.5) and lth (26.5 ± 4.2) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace2 and neprilysin, ang (1-7) and ang (1-4), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n=5) and female (n=8) sheep were harvested at 15-31 months of age. the tissue membranes were solubilized and incubated at 37°c with either 125 i-ang i or 125 i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln4760 to inhibit ace2 activity, or sch39370 to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p<0.05 was considered significant. results ace activity was over 4 times greater (1282 ± 103.5 vs. 303.3 ± 118.4 fmol/mg/min, p<0.0001), ace2 activity was 2.5 times greater (1826 ± 312.5 vs. 721.2 ± 86.4 fmol/mg/min, p=0.02) and nep activity was nearly 2 times greater (761.3 ± 73.09 vs. 401.5 ± 39.81 fmol/mg/min, p=0.0081) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd47587, hd17644. in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine 1) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and 2) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (ctr) 24-hours apart at 80 days gestational age and allowed to deliver at term. at 9 mo of age, female sheep were randomly allocated to be fed at either 100% of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a 0.25 g/kg glucose bolus was used and for the hec 4mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > 50% of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f=18.982;p<0.001). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl 68728. syngergistic induction of 11 -hydroxysteroid deyhdrogenase type 1 expression by cortisol and interleukin-1 in human fetal lung fibroblasts. z yang, 1 p zhu, 1 cm guo, 1 l myatt, 2 k sun. 2 1 school of life sciences, fudan university, shanghai, china; 2 obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce 11b-hydroxysteroid dehydrogenase type 1 (11b-hsd1) which converts inactive cortisone to active cortisol, but their effect on 11b-hsd1 expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin-1b (il-1b) effect on 11b-hsd1 in human fetal lung fibroblasts (hfl-1 cells). methods: the expression of 11b-hsd1 in hfl-1 was examined with immunocytochemistry and pcr. 11b-hsd1, prostaglandin h synthase-2 (pghs-2) and cytosolic phospholipase a2a (cpla2) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il-1b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il-1b were studied using a gr antagonist (ru486) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv500-a/cebp). results: hfl-1 cells expressed a high level of 11b-hsd1. both cortisol (10 -8 -10 -6 m) and il-1b (0.1-10 ng/ml) induced 11b-hsd1 mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor 5,6-dichlorobenzimidazole riboside (75 μm). ru486 (10 -6 m) blocked the induction of 11b-hsd1 by cortisol. induction of 11b-hsd1 mrna expression by cortisol (10 -6 m) was synergistically increased by co-treatment with il-1b (0.1-10 ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla2 and pghs-2 expression by il-1b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of 11b-hsd1 mrna expression by either cortisol or il-1b. these data suggest induction of 11b-hsd1 expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of 11b-hsd1 expression by il-1b. conclusions: cortisol and il-1b synergistically induce 11b-hsd1 expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental 11 -hydroxysteroid dehydrogenase (11 hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, 1 pl van zijl, 2 cw rumball, 2,3 al jaquiery, 2,3 je harding, 2,3 mh oliver, 2,3 fh bloomfield, 2,3 jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of 11 hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of 11 hsd-1 and -2 activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from 60 days before until 30 days after mating to reduce maternal body weight by 15%, with ad libitum feeding thereafter. placentomes were collected on days 50, 85, 120, and 131 of gestation (term, 147d) and 11 hsd-1 and -2 activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower 11 hsd-2 activity at d50 (p=0.1), a significant reduction at d85 (p<0.05), but no difference at d120 or d131. 11 hsd-1 activity was not different between n and un animals at any time. there was no effect of twinning on 11 hsd-1 or -2 at d50. however, with twins both 11 hsd-1 (p=0.01) and -2 (p<0.05) activities increased at d85. 11 hsd-1 activity was reduced in twins (p=0.06) at d120 but was higher than any singleton pregnancies at d131 (p=0.06). 11 hsd-2 was not different between singletons and twins at either d120 or d131. overall, 11 hsd-2 was lower in placentae of male compared to female fetuses in late gestation; 11 hsd-1 was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental 11 hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had 50% mfr from day 10 of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day 1 of life (p1) and at 6 months (n= 6-8 per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels (18s as control). we evaluated the expression of 11-beta hydroxylase (cyp11b1), aldosterone synthase (cyp11b2), acth receptor (mcr2), p450 side chain cleavage enzyme (cyp11a1), star protein, 11 -hydroxysteroid dehydrogenase type 1 (hsd1) and type 2 (hsd2), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr3c2). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp11b1 (p=.01), cyp11b2 (p=.04), hsd2 (p=.03), p450 (p=.05), acth receptor (p=.05), star (p=.01) and nr3c2 (p=.02) mrna in p1 mfr offspring, with no changes in hsd1 and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd1 and gr were significantly upregulated with a trend towards an increase in acth receptor (p=.07) whereas in the female mfr the expression of acth receptor (p=.02) was increased and nr3c2 (p=.03) and cyp11b2 were decreased (p=.04). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=.02) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd1 indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth (40-90d), quiescence (100-120d) followed by reactivation (>135d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf2 mrna expression between male and female fetuses. methods: a total of 37 twin fetuses were used in this study. post mortem was performed at either 110-112d, 125-134d or 137-145d gestation. adrenal mrna expression of igf1, igf2, igf1r, igf2r and cyp17 was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p<0.0001) with increasing gestational age. the relative adrenal weight was lower however after 125d when compared to the earlier gestation age group (p<0.0001). adrenal igf1 mrna expression was lower (p<0.0001) at 137-145d when compared to the earlier gestation age groups. interestingly, igf1r expression was highest at 125-134d (p<0.0001). there was an interaction between the effects of age and gender on adrenal igf2 and igf2r mrna expression such that the expression was higher in males compared to females at 125-134d (p<0.01), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp17 mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf2. in this study we have demonstrated there is an increase in both igf2 and igf2r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf2 expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude (3,820 m) from day 30 to near term. on days 138-141 (term = 146 days), fetal adrenal glands were collected from lth (n=6) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase1/2 (erk1/2) and p38 map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk1/2 and p38 at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n=24) and with preeclampsia (n=8) between 31 and 40 weeks of gestation. we evaluated the expressions and phosphorylations of erk1/2 and p38 map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p38 and phospho-p38 map kinase were not detected in invasive trophoblasts in cases or controls. erk1/2 and phospho-erk1/2 were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk1/2 was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk1/2 in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g1 phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n=60) and from severe early onset preeclamptic (n=20), and age-matched control placentae (n=15). protein expression of cyclin e1, cyclin d1, cyclin d3 and cell cycle inhibitors, p15, p16, p21, and p27 was assessed by western blot analysis. spatial and temporal localization of cyclins e1, d1, d3, p21 and p27 was determined by fluorescence immunohistochemistry. expression of cyclin e1 and cyclin d1 mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e1 and cyclin d3 to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e1 was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d1 expression was predominantly restricted to the villous stroma. cell cycle inhibitor p27 was expressed in both ct and syncytiotrophoblast (st) cells whereas p21 was restricted to the st. during normal placentation, levels of both cyclin e1 and cyclin d3 were high in the first trimester and decreased with advancing gestation. the expression of cyclin d1, p27 and p16 showed an inverse correlation to cyclins e1 and d3 whereby their expression increased towards term. levels of p21 and p15 remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e1 and p27 and a decrease cyclin d1 and p15 expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e1 positive progenitor ct cells in the floating villi and an increased expression of p27 in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g1 phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif-1-mediated placental and circulating sflt-1 at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt-1 with maternal and fetal oxygen tensions. we hypothesized that circulating sflt-1 and free vegf would be increased at 3600 m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from 77 and 65 healthy mother-infant pairs living at 400 m and 3600 m respectively. plgf, free vegf and sflt-1 were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf (7±2 pg/ml vs. 12±2 pg/ml, p<.05) and sflt-1 (25.7±2.4 vs. 31.4 ±2.2 ng/ml) were increased at 3600 m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (-35±3% plgf; -85±5% free vegf; -24±2% sflt-1, p<.001). cord blood free vegf was 100-fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in 95% of the babies. similar results were obtained for sflt-1. thus, while free and total maternal circulating vegf and sflt-1 are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n=6) and hs (n=2) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g2505b scanner. images were analyzed using genepix 5.0 (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were 310 differentially expressed genes between spe and hs (p<.05). among these candidate genes, 233 were up-regulated and 77 were downregulated. the most up-regulated genes are 5(3)-deoxyribonucleotidase (dnt-2), superoxide dismutase 3, hydroxy-delta-5-steroid dehydrogenase, caspase 9, and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. (14), severe preeclampsia (10), hellp syndrome (4) and eclampsia (4) and control group (29 patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to 2 (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf-3 is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk1-7) have been identified. our aim was to investigate the expression pattern of alk1 and alk5 receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk1 and alk5 mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk1 and alk5 antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk1 expression increased with advancing gestation. in preeclamptic placentae alk5 expression was significantly increased compared to preterm and term controls, whereas alk1 expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad1, a tgf signaling molecule, which is activated by alk1 and increased phosphorylation of smad2, which is triggered by alk5. the expression of alk1 and alk5 in iugr placentae differed from that of preeclampsia as both alk1 and alk5 mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk5 expression was significantly increased in iugr placentae at the protein level, while no differences in alk1 protein levels were noted between iugr and controls. conclusions imbalance between alk1 and alk5 signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk1 or alk5 has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). (14), severe preeclampsia (10), hellp syndrome (4) and eclampsia (4) and 29 patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd45a and its downstream signal proteins--phospo-p38, phospho-mkk3/mkk6 were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd45a and sflt-1 proteins in placenta tissue sections using abc method. huvec cells were cultured to 80-90% confluence and were divided into 3 groups: control, stress induction (sorbitol, 0.3m, 4h), p38 inhibition (sb-203580, 1ug/ml, 5ug/ml and 10ug/ml, 1 hour) + sorbitol (0.3m, 4h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd45a and phospho-p38. supernatant sflt-1 was measured with an eia kit and the results were read at 450nm wavelength. results: gadd45a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk3 and p38) activation compared with control. overexpression of gadd45a and sflt-1 in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd45a was induced by sorbitol, triggering the activation of the downstream p-38 pathway and the accumulation of sflt-1 in the supernatant. the up-regulation of sflt-1 secretion by inducing gadd45a was depleted when treated with p-38 inhibitor. conclusions: our study reveals that gadd45a and its down stream p-38 pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt-1, which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt-1 secretion via the gadd4. trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, 1 jennifer a doll, 2 emily j su, 1 serdar e bulun, 1 mona cornwell, 2 susan e crawford. 2 1 obstetrics and gynecology, northwestern university, chicago, il, usa; 2 pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n=5) and those complicated by severe preeclampsia (n=5) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored (1=none, 2=spotty, light, 3=consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (>2/3 positivity for factor 8 antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas (3.0 + 0 standard deviation versus 2.3 + .39, p = 0.018). endothelial expression was not significantly different in preeclamptic (2.6 + .41) versus normal placentas (2.3 + .39, p>0.05). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od 450nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < 0.05 was set for statistically different. results: 1) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p<0.01; 2) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; 3) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p<0.05. conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd36822 and hl65997). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from 4 groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n=3, ptspe, 28.9 weeks); (ii) preterm labor (n=3, ptl, 30.9 weeks); (iv) term no labor (n=3, tnl, 39.6 weeks); (v) term labor (n=3, tl, 39.7 weeks). microarray gene profiling was performed using affymetrix human genome u133 plus 2.0 arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly 2-fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r01 hl049041-12, cpw) and cdc grant (u7dp00187-03, cpw). the expression of gp130 at implantation site: implication for the pathogenesis of preeclampsia. objective: gp130 is a common shard signal transducing subunit of the il-6 cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp130 at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = 9, respectively) were immunostained for gp130. the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp130was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value 180 [142.5-185.0] and 120 , respectively (p<0.05). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast (40 [30-50]; p<0.05). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp130 expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for 2 3 days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia (10% or 3% oxygen) in a serum-free medium for 24 hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately 47 kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. 10% and 3% oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd71 antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for 24 hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for 7 days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor-2 (hif-2 ) and the receptor svegf-r1/sflt-1: implication for phatophysiology of preeclampsia. julio e valdivia-silva, 1,3 juan c gonzalez-altamirano, 2 keisy lopezthe trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr-1/sflt-1, hif-1 and-2 . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r1 selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r1) of the cts was diminished. on the other hand, the expression of the arnm of hif-1 or -2 in cells was comparable in all the types of placentas, nevertheless, the protein hif-2 was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif-2 and the increase of the receptor svegfr-1, we used sirna-hif2 . in response to the inhibition, the expression of the receptor svegf-r1 diminished dramatically. the blockade of hif-2 did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif-2 is one of the molecules involved in the selective expression of the receptor svegf-r1 in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt-1/svegfr-1 after transfection with sirna-hif2 in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting 30% of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e 2 ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e 2 increases synthesis of vwf, we treated heecs with e 2 at 0.001μm, 0.01μm and 0.1μm for 24 hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e 2 can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) activity results in increased amounts of active vwf. rrelease of activated adamts13 will be determined by fret. to ascertain if e2 regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici 182,780. elisa and fret will assess the release of active vwf and adamts13, respectively. results: western blotting demonstrated e 2 increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e 2 acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e 2 increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e 2 increases synthesis of vwf in heecs in vitro. if e 2 increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e 2 to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, 1 jacqui f donoghue, 1 marc g achen, 2 steven a stacker, 2 jane e girling. 1 1 obstetrics gynaecology, monash university, melbourne, victoria, australia; 2 ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium 1 . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c57bl/ 6jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human 293ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after 4 weeks. serial sections were immunostained with lyve-1 and/or vegfr3 (lymphatic endothelial cell markers), cd31 (blood endothelial cell marker), mab286 (human vegf-d), mab1278 (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in 24% of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. 293ebna tumours formed inside and outside the uterine horn of both the control (n=4 of 6) and vegf-d group (n=3 of 6). localization of 293ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected 293ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d 293ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n=5) and without (n=5) abruption. purified term dscs were passaged until >99% free of cd45+ cells by facs. confluent dscs were primed with 10 -8 m estradiol (e2), 10 -7 m medroxyprogesterone acetate (mpa), both, or vehicle for 7 days. after 24h incubation in defined medium with corresponding steroids ± thrombin (0.5-2.5 iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, 2.17-fold stimulation by 2.5 iu/ml thrombin from 5.05±1.23 to 12.62±3.01 pg/ml per mcg protein for e2+mpa; p=0.034). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, 2.9-fold for e2+mpa; p<0.05). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert (6 well/plate, 8 micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of 0.25, 0.5, 1.0, 2.5, and 5.0 g/ml were added to the upper chamber of the cell insert. after 24 hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < 0.05 was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: 1.016 ± 0.065 g/ml; chy 0.25: 1.284 ± 0.281 g/ml; chy 0.5: 1.204 ± 2.06 g/ml; chy 1.0: 2.726 ± 0.829 g/ml; chy 2.5: 4.499 ± 1.467 g/ml (p<0.01); chy 5.0: 5.159 ± 1.087 g/ml (p<0.01), respectively. data are means from 6 independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd36822 and hl65997). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk2/1) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv40-of to test whether: 1) vegf-activated erk2/1 signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk2/1 activation and; 2) caveolin-1, the structure protein of caveolae, regulates vegf-stimulated erk2/1 phosphorylation. methods: ofpaec or sv40-of cells were cultured in mcdb-131/10% fbs/antibiotics. serum-starved subconfluent ( 80%) cells were treated with rhvegf (0 to 100 ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, 10 mm, 60 min) or caveolin-1 scaffolding domain (cav-sd, 5 m, 2 hr). sv40-of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient (45%/35%/5%) ultracentrifugation. activation of erk2/1 signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk2/1 phosphorylation in a time-and dose-dependent manner. erk2/1 phosphorylation maximized by vegf (10 ng/ml) at 5-10 min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk2/1 signaling module, plc 1, pkc , src, ras, raf-1, mek2/1 and erk2/1, were detectable in purified caveolae membranes positive for various markers including caveolin-1, enos, flotillin-1, and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk2/1 without altering total erk2/1 in a time-dependent manner similar to that in total cell extracts, which also maximized at 5-10 min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk2/1 phosphorylation in caveolae. conclusion: vegf activates the erk2/1 signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk2/1 signaling pathway. we conclude that caveolae/caveolin-1 serves as a platform for compartmentalizing the vegf-induced erk2/1 signaling pathway in placental ec (hl74947 and hl70562). hypoxia upregulates gcm1 in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm1. gcm1 is also expressed in the human placenta. placental gcm-1 protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm1 we hypothesised that gcm1 expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm1 expression in the human placenta. two model systems were used; (1) free floating villous explants and (2) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm1 protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n=6 experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm1 protein was detectable at a low level in villous explants maintained for 7h in 20% o 2 . a striking increase in gcm1 protein was observed when villous explants were incubated for 1h in 0% o 2 (p<0.002). incubation of villous explants for 1h in 0% o 2 followed by re-oxygenation for 6h in 20% o 2 resulted in a marked decline in gcm1 protein (p<0.002). expression of gcm1 was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in 18% o 2 or reduced oxygen (2% o 2 ) conditions. gcm1 protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm-1 protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm-1 in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm1 warrant further investigation. the effect of acute alcohol exposure on histone3-lys9 modification in the mid-gestation embryonic lung. xiangyuan wang, 1 debra wolgemuth, 1,2,3 laxmi baxi. 1 1 ob/gyn; 2 genetics development; 3 human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within 12 hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone3 at lysine9 (ach3lys9) in the mid-gestation embryonic lung. pregnant c57bl/6j mice at day 13.5 of gestation (e13.5) were injected intraperitoneally with 2 doses of 25% ethanol (3.75g/kg ), 4 hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved 12 and 24 hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach3ly9 antibody. the entire lung tissue field was evaluated for the levels of ach3lys9 staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e13.5, the morphology of ae embryonic lungs was normal. however, high levels of ach3lys9 were detected in 50% of the bronchial epithelial cells and 50% of the mesenchymal cells. the expression level in both lineages decreased 24 hr after alcohol exposure. in the controls, the expression of ach3lys9 was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e13.5 lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme 16 hours after alcohol treatment. we now observe that elevated expression of ach3lys9 in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h3 could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at 15 to 20 weeksgestation and randomly assigned to receive either 1000 mg of vitamin c and 400 iu of vitamin e (study group, n=20) or placebo (control group, n=20) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: 1. the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group (2.22±0.06 vs. 2.56±0.12 nmol/mg protein, p<0.05). 2. the orac values in the umbilical venous plasma of study group were significantly higher than that of control group (15010.1±649.4 vs. 11804.6±463.7 u/ml, p<0.05). 3. the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group (130.2±10.5 vs. 78.7±4.4 nmol/ml, p<0.01). 4. there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr5, is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n=16) and with pre-eclampsia (n=16) at 35-40 weeks gestation. protein expression levels and tissue localisation of trail and dr5 were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr5 protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr5 within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr5 was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [391.6 (312.4-646.8) , median (interquartile range), pg/ml, n=6]. however, there was no significant increase either in pregnancy [372.3 (319.6-442. 3) pg/ml, n=12] or pre-eclampsia [301.8 (209.0-435.5 ) pg/ ml, n=13] (kruskal-wallis test, p>0.05). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr5 in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr5 in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n=3). following staining with mayer's hematoxylin (n=3), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from 10-15 villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately 37 kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately 5000 (lane 1), 2000 (lane 2), 1000 (lane 3), and 500 (lane 4) nuclei revealed that fasl could be reliably detected in specimens containing as few as 1000 nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd33909. the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin-1) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n=3), mild (n=2) and severe (n=3) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, 1 tamao yamamoto, 1 yoshimasa watanabe, 2 takeo itoh, 2 hidetaka izumi. 3 1 obstetrics and gynecology, nagoya city university, nagoya, japan; 2 pharmacology, nagoya city university, nagoya, japan; 3 obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o 2 -) and nitric oxide (no), is strong oxidant as well as o 2 -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women (7/10), although it was not seen from normotensive pregnant women (0/8). the localization was also seen in placentas from 5 of 7 severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar (0.788±0.161 for sever preeclampsia and 0.984±0.209 for normotensive pregnant women). from these results, it was suggested that an increase in o 2 production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il1 and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) 1 and 3 in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp-1, mmp-3, and vimentin (a dc marker) was performed on decidua from normal (n=5) and preeclamptic (n=4) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with 10-8 m estradiol (e2) or e2 + 10-7 m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e2 +/-mpa with or without 1 ng/ml of il1 or tnf . secreted mmp-1 and mmp-3 levels were measured by elisa (n=8) and confirmed by western blotting. quantitative rt-pcr assessed mmp-1 and mmp-3 mrna levels (n=3). results: tissue staining revealed that mmp-1 and mmp-3 levels in preeclamptic decidua (hscore mean±sem: 217±41 and 205±11, respectively) were significantly higher than in normal decidua (140±22 and 148±25, respectively; p<0.05). in cultured first trimester dcs incubated with e2, tnf increased secreted mmp-1 and mmp-3 levels by 13±2 and 8±2-fold, respectively, while il1 increased them by 17±3 and 28±13 fold, respectively (p<0.05). in parallel incubations with e2+mpa, basal mmp-1 and mmp-3 output were lowered by approximately 70% and 60%, respectively, while tnf -and il1elicited mmp-1 and mmp-3 levels were blunted by 40-60%. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp-1 and mmp-3 secreted protein levels. conclusions: over-expression of mmp-1 and mmp-3 in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il1 and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il1 -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce 2-methoxyestradiol (2-me), a compound with diverse biological activities including inhibition of hif-1 , a transcription factor that mediates cellular response to hypoxia. circulating levels of 2-me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of 2-me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon 4 of the comt gene (rs4818), which does not change amino acid sequence (136leu136), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (>98%) african-american. the frequency of the minor rs4818 "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: 19%; controls: 23%, p=0.5), but was significantly greater in fetal dna from 35 pregnancies complicated by preeclampsia compared to 55 control pregnancies (g allele frequency cases: 34%; control: 16%; p<0.001). likewise, fetal carriage of the rs4818 "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: 1.74; 95% c.i.: 1.22, 2.48, p<0.0045). there was also a significant discordance between paired maternal and fetal rs4818 genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: 3.17; 95% c.i.: 1.19, 8.49). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of 2-me. supported by nih p60md002256. smoking is associated with elevated adma in preeclampsia. michael p frank, 1 robert w powers. 1,2 1 magee-womens research institute, pittsburgh, pa, usa; 2 obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of 120 women with uncomplicated pregnancy (controls), and 57 women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p<0.05 results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p<0.001). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p<0.05). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n=16) and lp (d19-20; n=16) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of 70kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of 15 m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po 2 >100mmhg, pco 2 =35-45mmhg). results: although the pressure change was similar between np and lp ( 83 and 85 mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from 86 ± 10 to 242 ± 22 ml/100g/min in np (181%) and 94±9 to 318±31ml/100g/min in lp (238%; p<0.05 vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np (0.7±0.07 vs. 0.5±0.05 mm hg/(ml/100g/min); p<0.05). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: 113±50 vs. 362±87 for anterior (p<0.05) and 167±58 vs. 545±140 for posterior (p<0.05). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf-165 are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = 6 each): delivering maternal side inflow perfusate at oxygen concentrations of 15.6 % and 8 %, respectively, distributed via 22 cannulae; and fetal side inflow perfusate oxygen concentration of 2-3 % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein-1 alpha (mip-1 alpha) (reference oxygen sensitive hormones), free vegf, svegfr-1 and plgf. in separate perfusions, fetoplacental vasodilation in response to 550 pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u46619, (n = 6 each). results: maternal-side mip-1 alpha release was higher in the 'hypoxic' than the 'normoxic' system (680 ± 119 and 531 ± 93 pg/ml, respectively, at 6 hours, mean ± se; 2-way anova: p<0.01). maternal-side epo and fetal-side soluble free vegf and svegfr-1 release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group (0.13 ± 0.10 and 0.03 ± 0.01 pm, respectively, at 6 hours, mean ± se; 2-way anova: p < 0.05). there was no difference in the vasodilatory response to vegf-165 in the fetoplacental vasculature between the groups (64.8 ± 5.2 and 63.5 ± 6.0 % change in fihp, mean ± se). discussion: differences in mip-1 alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr-1 release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at4r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at4r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at4rs; it also stimulates nf-kappa beta and modulates glut-4. to date at4r expression has not been investigated in the placenta. we propose that at4r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at4r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at4r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from 4 np and 4 pe at delivery (gestational ages: 39 and 38 weeks respectively). samples were taken from 3 areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade 0 = no positive labelling, 1 = 1 -25%, 2 = 26 -49%, 3 = 50 -74% and 4 = 75 -100% of cells positively labelled. median (max, min) are shown. results: 1) at4r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. 2) at4r positivity was reduced in near cord pe samples (2.2 (2.4,1.2)) compared to np (3.3 (4.0,2.5) p=0.029). conclusion: we have shown for the first time, dense at4rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at4r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, 1 hm boelens, 1 jj hm erwich, 1 j heimweg, 2 s van der heide, 2 ajm van oosterhout, 2 aej dubois, 3 jg aarnoudse. 1 1 obstetrics, university medical center groningen, groningen, netherlands; 2 laboratory of allergology and pulmonary disease, university medical center groningen; 3 pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th2 immune profile. treg cells are a subpopulation of cd4+ lymphocytes and are specifically characterized by the lineage specific transcription factor foxp3. treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, 18 women with pregnancies complicated by pre-eclampsia and 26 healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since 13 women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd4+ foxp3+ treg cells (4.5 vs 8.8%; p<0.05). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd4 + foxp3 + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd4 + foxp3 + treg cells. no difference was found in umbilical cord blood (7.2 vs 7.9%). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh4), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh4 and sepiapterin (sep, a precursor for bh4 biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups (4-6) of shr pregnant rats were either treated with bh4, sep (20 mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day 12 of pregnancy until day 20 of gestation. animals were sacrificed on day 20 of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p<0.05) elevations in blood pressure (bp, mmhg) were observed in shr (shr, 141±1.42 vs. wky, 106 ± 3.04 mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh4 or sep (128±2.19), significantly (p<0.05) reduced elevated bp beginning from day 14 of pregnancy. fetal but not placental weights were significantly (p<0.05) reduced in shr (2.39±0.03 grams) compared to wky (3.10±0.04) rats. bh4 (2.51±0.04) treatment partially (p<0.05) increased fetal weights compared to the shr group. vascular enos expression is significantly (p<0.05) elevated in shr (1.0±0.07) compared to wky rats (0.56±0.15). further, treatment with bh4 (0.58±0.1) but not sep (0.92±0.1) significantly (p<0.05) reduced elevated enos protein expression in shr rats. conclusions: bh4 may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t-786->c and missense glu298asp variants. toshihiro yoshimura, 1 michihiro yoshimura, 2 masafumi nakayama. 3 1 obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; 2 cardiovascular medicine, jikei university school of medicine, tokyo, japan; 3 cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu298asp missense variant in exon 7 and a t-786-->c variant in the 5'-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu298asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t-786-->c and severe preeclampsia. materials and methods: the study participants included 62 patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the 345 general normal population. results: the analyses revealed that the frequency of the missense glu298asp variant (n=18/62, 29%) was significantly higher than the general population (n=44/345, 13 %), as we previously published. however, the frequency of the t-786-->c variant (n=9/62, 14%) was not different from the general population (n=61/345, 18 %). interestingly, only one patient had both t-786-->c and missense glu298asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t-786-->c variant is associated with severe preeclampsia. the t-786-->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t-786-->c may have some reproductive significance in combination with missense glu298asp variant, however huge number of patients would be needed to analyze such rare (2.5%) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, 1 jong kwan jun, 2 chan-wook park, 2 joong shin park, 2 bo hyun yoon, 2 hee chul syn. 2 1 obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; 2 obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and 199 normotensive pregnant women were included in this study between jan 1998 and jul 2004. diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early (30 weeks or before) and late-onset (31 weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c677t, angiotensinogen (agt) m235t, vascular endothelial growth factor (vegf) c936t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of <0.05 was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: 1.82, p<0.05) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only 20 out of possible 27 genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels (10-200 μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining (64.6 ± 4.2 vs. 40.4 ± 3.9 vs. 21.8 ± 4.7, p<0.001); % vsm staining (30.2 ± 6.1 vs. 20.0 ± 5.3 vs. 4.0 ± 2.6, p<0.01). for mmp8, obese and overweight patients had a greater (p<0.001) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining (63.0 ± 5.5 vs. 55.2 ± 4.3 vs. 26.0 ± 3.0); % vsm staining (36.8 ± 2.9 vs. 27.4 ± 3.1 vs. 2.8 ± 1.2). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp8. speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp8. hl069851. collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t-25 flasks (40,000 cells/flask) and grown for 3 days to 70% confluence. the cells were treated for 24 hours with medium control, ros (hx, 0.05 mm + xo 0.003 u/ml), tnf-(1 ng/ml); and neutrophils (60,000) activated with arachidonic acid, 50 μm, (1:16 ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt2 profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group 1 was the control and group 2 was the experimental treatment. results: table 1 . conclusions: neutrophils, ros and tnf increased mmp1 expression. interestingly, genes involved in collagen synthesis (col1a1) or inhibition of mmp-1 activity (timp1) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl069851, fogarty 5d43tw007692, p60md002256. objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio 2 . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n=4) were cultured 24 h in 1% and 20% oxygen in dmem with 10% human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed 6-12 h to confluent, phorbol myristate acetate differentiated u937 macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin 7 filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with 1% oxygen was 56.2%, not different from the 52.6% from cultures exposed a 1% fio 2 and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd29190 and hd045675. is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, 1 laura j minchell, 1 jenny e myers, 1 philip n baker, 1 carl a hubel, 2 ian p crocker. 1 1 maternal and fetal health research group, the university of manchester; 2 obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for 24h with pooled plasma (2%) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal (100, 113, 37 m, ratio 0.9) and pe pregnancies (140, 172, 53 m, ratio 1.6) 1,2 . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc-1 assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p<0.01, wilcoxon signed ranks, n=9). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p<0.05, n=7). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p<0.01, n=8) and a reduction in jc-1 fluorescence (p<0.05, n=7), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p<0.05, n=9; jc-1 assay: p<0.05, n=10). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. 1 lorentzen (1995) bjog; 2 endresen (1992) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time 3. in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time 3 compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: 207/276 (75%) of patients has 'none-mild' pain, and 69 (25%) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' 30.5+7.7 kg/m2; 'moderate-severe' 30.6+7.6 kg/m2, p=0.95), nor was pain severity (p=0.58). pain prevalence was significantly associated with ac (mean: 'none-mild' 37.1+7.7 inches; 'moderate-severe' 40.2+7.0, p=0.004). a borderline positive association exists between ac and pain severity (p=0.077). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin 2 release in keratinocytes has been recently shown. methods: we report preliminary data regarding 20 women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg800 (10 patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle (10 patients). patients were asked to apply the cream to the vulva twice-daily for 7 days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg800 report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg800 also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg800 seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin 2 production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg800 might be linked to a reduced infective charge, attributable to the activity of -defensin 2. background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in 60 to 70% from matched unrelated donors. genital cgvhd complicates about 25% of hsct and may uncommonly result in labial fusion. case: 22 year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at 13 years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a 1 cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after 2 weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at 3 months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was 4 cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: 413 women with a diagnosis of an ectopic pregnancy were studied, with 90 surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p<0.05. results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or 0.98, p=0.96) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or=6.8, p<0.001), a large volume of cul-de-sac fluid (or=4.2, p<0.001), and increasing pain (or=3.5, p=0.004) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods]197 female patients (average age: 44.6 years old) who consulted a physician and agreed to answer about the questionnaires during september, 2006 -june, 2007 . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in 2005. and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] 52 in 197 patients had migraine (26.4% average age: 40.6 years old). prevalence were 33.3% in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome 67%, dysmenorrhea 52.6%), sterility (42.4%), and severe menopausal disorder (80%). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of 3 doses of estradiol (e 2 ) gel 0.1% (divigel ® ), a novel formulation of e 2 consisting of 1 mg e 2 per 1 g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: 488 postmenopausal women were evaluated in a 12-week study comparing placebo to e 2 gel 0.1% at doses of 1.0 g/day, 0.5 g/day, and 0.25 g/day with estimated nominal daily deliveries of 0.027 mg, 0.009 mg, and 0.003 mg of e 2 respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e 2 gel 0.1% showed statistically significant improvements from placebo gel as early as week 2 (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all 3 doses of e 2 gel 0.1% compared to placebo. conclusion: e 2 gel 0.1% significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e 2 gel 0.1% offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied (0.25 mg e 2 , delivering 0.003 mg e 2 /day), to treat vasomotor symptoms associated with menopause. estradiol (e 2 ) gel 0.1% (divigel ® ) is a novel formulation of e 2 consisting of 1 mg e 2 per 1 g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e 2 gel 0.1% were evaluated in a large placebo-controlled trial. design: 488 postmenopausal women participated in a 12-week study comparing placebo to 0.25 g/day, 0.5 g/day, and 1.0 g/day of e 2 gel 0.1%. circulating e 2 and estrone (e 1 ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e 2 gel 0.1% produced physiologic e 2 :e 1 ratios similar to those seen in premenopausal women. e 2 :e 1 increased from a baseline mean of 0.17 to 0.49, 0.69, and 0.95 with, respectively, the 0.25, 0.5, and 1.0 g/day doses of e 2 gel 0.1%. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e 2 gel 0.1%. shbg levels remained unchanged after 12 weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after 4, 8, and 12 weeks of treatment were 0.0 for all treatment groups except for a mean value of 0.1 ± 0.34 for the 0.5 g dose group after 4 weeks of treatment. conclusion: e 2 gel 0.1% is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e 2 (0.25 mg to 4.6 mg), with the lowest systemic daily delivery of e 2 attained by the divigel ® 0.25 g dose. following 12 weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from -6.0 (divigel ® 0.25 g) to -11.1 (estrasorb ® ); change in severity scores for msvms ranged from -0.3 (divigel ® 0.25 g) to -1.7 (divigel ® 1.0 g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in 5% of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e 2 non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb-2914 (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short (1.0-1.5 cm) lengths of silastic tubing (od 2.4 mm), either empty (n=3),or filled with silicone rubber matrix containing 50% of micronized cdb (n=5), were inserted into the uterine lumens of 8 ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, 2002) . after 3.5 cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole 3.5 cycles, animals bearing a blank ius bled for an average of 11.66 ± 0.88 days while cdb treated animals bled for an average of only 1 ± 0.45 days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of 409 ± 112 mg while the cdb-treated endometria weighed only 188 ± 30 mg. the proliferation markers ki-67 and phospho-h3 were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr00163, hd 43209 and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were 1) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and 2) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n=6); estradiol (e2,n=3); medroxyprogesterone acetate (mpa,n=6) or e2+mpa (n=3). after 21 days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by 8isoprostane and 8-oh-deoxyguanosine (8oxog). apoptosis was investigated by the tunel method. statistical analysis was by 1-way and 2-way anova. results: gp uteri were enlarged by both e2 (p<0.001) and mpa (p=0.025). effects of mpa on uterine weight differed significantly depending on e2 levels (p<0.001), where mpa opposed the e2 effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p=0.036) and bv average area (p=0.002). the presence of e2 significantly decreased these parameters (bv density mean± sem: crl: 9.4±1.0%, e2: 10.3±1.6%, mpa: 13.6±1.1%, e2+mpa: 6.0±0.7%, p=0.002). these changes were associated with highly elevated 8-isoprostane content in e2+mpa-treated uteri compared to all other groups (p<0.001). abnormalities in the e2+mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear 8oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over 24 mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with 20 micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of 604 women reported the presence of 16 symptoms prior to initiating contraception (217 dmpa, 216 oc, 171 nh) and every 6 mo thereafter for 24 mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in 6 mo increments for 24 mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= 0.7), cramping (or=0.5), hair loss (or=0.6), acne (or=0.4), nervousness (or=0.5) and mood swings (or=0.7). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within 6 mo in nearly 50% of those with it at baseline, but no significant resolution after 6 mo. also mastalgia (29% at baseline) was less likely to persist at 18 and 24 mo. bleeding between menses was reported for the first time at 18 mo by 11% of oc users. dmpa users of all races had an increased risk of missed periods (or=95.1), bleeding between menses (or=3.6), bleeding >20 d (or=13.6) and loss of libido (or=2.2) relative to nh users. it reduced cramping (after 12 mo) and bloating (all 24 mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after 18 mo while resolution often occurs within 6 mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing 2 months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers 35 micrograms of ee; the weekly tc contains .75 milligrams of ee. each treatment was followed by 2 months of washout and 2 months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < .05. results: after two months of treatment the mean (+/-sem) ee levels for tc = 81.9 pg/ml (+/-12.0) and oc = 94.1 pg/ml (+/-9.8). the ee level is not significant different for the two medication (p = 0.42). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined 35 patients per arm were needed to demonstrate a significant difference of 2 in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under 12 weeks and general anesthesia for over 12 weeks. thirty-nine patients were randomized to receive a paracervical block with 0.5% bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, 30 and 60 minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, 1 mika gissler, 2 satu suhonen. 1 1 ob gyn, university of helsinki, helsinki, finland; 2 national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from 30 to 38% in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of 1269 women undergoing medical abortion between august 2000 and december 2002. the subjects were followed by means of finnish registry on induced abortions until december 2005; the follow-up time (mean ± sd) was 49.2 ± 8.0 months. results altogether 179 (14.1%) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january 2003 through june 2007. patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were 316 essure procedures attempted at the dmc from january 2003 through june 2007. of the 316 attempted procedures, there were 25 failures (7.9%). 13 of the 25 failures were attributed to difficulty visualizing the ostia (52%). other causes of failure included expulsion of the device (2), tubal spasm (1), uterine perforation (1), and tubal ostia too large for the device (1). there were 3 cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and 3 post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure (40.6 vs 26.6 min, p = 0.004), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia (33.3% vs 4.7%, p = 0.003). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is 7.9% with a pregnancy rate of 0.95%. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january 2003 through june 2007. follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in 83 uhc patients of which 79 were successfully completed. of the 79 patients, ten underwent a post-essure hsg (12.7%). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only 12.7%. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus 1 thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, 1 dong zhang, 3 salama salama, 1 cheryl walker, 2 hala el-mazar, 1 ayman al-hendy. 3 1 ob/gyn, utmb; 2 md anderson; 3 ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex-1-thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; 10, 20 and 30 days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by 75% ±16, 59% ±6 and 68%±28 of pretreatment volume at days 10, 20 and 30 respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by +26%±10, +66%±23, +102% ±38 while receiving media continue to grow by + 20% ± 6 , +70% ± 8 and +110% ± 15 at same time points. ad-tk/gcv induced significant increase in caspase 3 activity, bax expression, decrease in bcl2 and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d1and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd4 and cd8 expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv1tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, 1 dong zhang, 3 hala el-mazar, 1 cheryl walker, 2 ayman al-hendy. 3 1 ob/gyn, utmb; 2 md anderson; 3 ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro 41-0960) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro 41-0960 150 mg /kg ,twice/ day for 28 days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at 2 and 4 weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. 24 hours urine samples were collected for evaluation of estrogen (e2) metabolites and bone resorption marker. results:animals treated with ro 41-0960 exhibited significantly lower uterine fibroid tumor burden (86% and 105%) of pretreatment volume at 2 and 4 weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached 300 %, and 300 % of pretreatment size at the same time points. ro 41-0960 treatment resulted in an increase in urinary 2/16 hydroxy e2 metabolite ratio. in addition ro 41-0960 increased bax expression and decreased parp , pcna and cyclind1 experssions . all ro 41-0960 treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro 41-0960, a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d1). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite 2 hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf-3, focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf-3, or anti-tgf-3 antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf-3 (2.96±0.71 fold), retinoic acid metabolizing protein (cyp26a1; 16.81±4.50), and versican variant v0 (7.75±2.2 fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v0 decreased to levels found in myometrial cells (0.96±0.04 fold). conversely, when treated with tgf-3, expression of versican variant v0 increased 2.98±0.22 fold. to confirm that tgf-3 was central to the overexpression of v0, we treated leiomyoma cells with anti-tgf-3 antibody, and found that baseline over-expression of v0 template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf-3 expression, demonstrating that tgf-3 template decreased to levels comparable to myometrial cell expression (0.84±0.12 fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf-3, providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of 10% serum until 70% confluent. for the next 72 h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at 0, 10, 20, 30, 60 m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma 20 m after serum replacement. pr-a and pr-b were detectable at 10, 20 and 30 m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: 10 microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u133a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the 3 specimens per subject and 5 subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base 2. confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. 45 genes were differentially expressed when we used a 0.1 q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp19 was detected and we report for the first time that scc-112 (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin-1 (tsp-1) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp-1 plays an important role in angiogenesis and is an activator of tgf -3. in a previous study, we found that differential expression of tsp-1 in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp-1 by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to 70% confluence. then smc were serum deprived for 72 h and treated back with 10% serum for 10, 20, 30, and 60 m. cells were collected for rna and protein, and tsp-1 expression was evaluated. in addition, cells were stained using the combination of anti-cd45 and anti-smooth muscle -actin or combination of anti-cd42d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp-1 mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for 60 m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd45 and platelet marker cd42d suggesting that the myofibroblasts cells themselves were the source of the tsp-1. conclusions: unlike skin wounds, where tsp-1 is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp-1. elucidating the roles of tsp-1 in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp-1 in the activation of tgf -3 are indicated. phospholipid s1p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine-1-phosphate (s1p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s1p enhances while high dose s1p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s1p affects invasion, using specific inhibitors. methods: dov13 eoc cells were pretreated for 4-hrs with vehicle, 0.5 m or 20 m s1p and with inhibitors for gi, p38-mapk, rac and rock, thereafter cells were detached and tested for invasion towards 40 m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p=0.019) and 20 m s1p treated cells ( p=0.0002). inhibition of both p38-mapk and rac did not affect 20 m s1p treated cells, in contrast invasion of control cells was mildly increased (p=0.025, 0.034). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and 20 m s1p treated cells (8 fold, p=0.0035, 23 fold, p=0.00037). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s1p (p=0.002, 0.001). ptx fully inhibited gelatinase activity of control and 0.5 m s1p treated cells, and partially of the 20 m s1p treated cells (p<0.00007). 0.5 m s1p significantly increased upa activity of attached (p=0.011) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. 20 m s1p inhibited upa in both attached and invading cells (p=0.0004), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and 20 m s1p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by 0.5 m s1p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine-1-phosphate (s1p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s1p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov13 eoc cells were pretreated for 4-hrs with vehicle, 0.5 m or 20 m s1p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc23766, (rac-i)), and cells were detached and assayed for invasion towards 40 m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p=0.004) increase of invasion by 0.5 m s1p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p=0.0024-0.015). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with 20 m s1p. control cells invasion was increased (2.3 fold, p=0.0019) by continuous rac-i treatment. 0.5 m s1p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated 3 and 2 fold gelatinase activity of control and 20 m s1p treated cells. in contrast upa activity was inhibited by both 0.5 m and 20 m s1p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with 0.5 m s1p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with 20 m s1p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s1p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp-2, and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp-2 activation. lpa-dependent pro-mmp-2 activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp-2 activation. in the current study, inhibition of rho-kinase/rock with y-27632 reversed the repression of lpa-stimulated pro-mmp-2 processing observed with treatment of the egfr kinase inhibitor, ag1478. correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp-2 processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov13), breast cancer (mda mb 231) and mesothelioma (hp-1) cells were grown in media supplemented with 10% fbs. after serum deprivation, 10% vesicle-free fbs was added for 4 hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with 1 and 10 g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp-2 and 7. fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at 1 g/ml they induced significantly high levels of invasion in all cell types tested. at 10 g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr-2 as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr1, vegfr2, co-receptor nrp1 and nrp2) in established eoc cells lines (dov13, r182, ovca429, skov3) and an immortalized normal ovarian epithelium (iose-29). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr-2 silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr-4-myd88-nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il-6, il-8, mcp-1, and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for 48h. migration assay was done using an in vitro cell culture insert with 8 m-size pore and pkh26 red fluorescentlabeled thp-1. cytokine profile of thp-1 cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp-1 cells with eoc cell cm for 24 h prior to exposure to apoptotic bodies (1:1 ratio with thp-1 cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il-6, il-8, mcp-1, and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il-6, il-8, mip-1 , mip-1 , and gro (18-fold, 2.5-fold, 2-fold, 3-fold, and 11-fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above 80%, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al 2007 clinical cancer research) and evaluated the ability of the test to monitor ct response methods: 55 patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at 1) baseline (mean 35 days after surgery), prior to the first cycle of ct, and 2) after 3 cycles of ct. 20 μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in 96 % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, 15/20 patients (specificity 75 %) had a negative test after 3 cycles. from the patients with short ttp, 13/14 had a positive test (sensitivity 93 %) p=0.000096 ( ²). the risk of experiencing a ttp shorter than 6 months when having a positive test after 3 cycles of ct was 72 %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry-520) as an alternative for paclitaxel in myd88-positive epithelial ovarian cancer cells. ki h kim, 1 ayesha b alvero, 1 yanhua xie, 1 david trollinger, 2 gil mor. 1 1 obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr-4, but not the adaptor protein myd88. when treated with paclitaxel, a known tlr-4 ligand, myd88-positive eoc cells exhibited nf-b activation, increased secretion of il-6, il-8, mcp-1, and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry-520, can be an alternative for paclitaxel in myd88-positive ovarian cancer patients. methods: myd88-positive and myd88-negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry-520 (3 to 3000 nm) or paclitaxel (0.2 to 20 m) for 24, 48, and 72 hours and cell viability was determined using the celltiter 96 aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry-520 and paclitaxel exhibited the same cytotoxic effect on myd88-negative and positive eoc cells. the ic 50 at 48h for myd88-negative eoc cells was 0.003 m and 0.2 um for arry-520 and paclitaxel, respectively. for myd88-positive eoc cells, the ic 50 at 48h was 3 m and 20 m for arry-520 and paclitaxel, respectively. however, unlike paclitaxel, arry-520 did not induce nf-b activation or enhance the secretion of il-6, il-8, mcp-1, and gro , and did not induce erk phosphorylation on myd88 positive cells. conclusions: administration of paclitaxel to patients with a myd88-positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry-520 has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd88positive eoc cells, and therefore, the ksp inhibitor arry-520 may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter 96 cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic50 of 0.5-1μm in 48 hours and was associated to increasing levels of caspases 8,9 and 3 activity. intracellular changes induced by erib included: 1) inhibition of nf-b activity; 2) decrease in cytokine production; 3) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and 4) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc4 was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc4 and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh2 is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah 2774 and skov-3) were treated with serial dilutions of ritonavir (1-10 mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit-8, annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with 0.5 mm etoposide treatment for 16 hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad51 foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh2 and lower levels of rad51 and xrcc2 as compared to controls and in turn, had lower prevalence of rad51 repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh2 and an increase in expression of rad51 and xrcc2 when compared to untreated eoc cells. after induction of dsb, the rad51 repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g1 cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase 4 and 6. ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, 1 tamer seckin, 1 noa feldman, 1 susan jormark, 2 michael divon. 1 1 department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; 2 department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n=18), lmp-mp (n=9) and lgsc (n=11). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u133 plus 2 arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from 1/1/05 -4/30/06. pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: 850 patients were included. adenomyosis was confirmed by pathology in 375 patients, an incidence of 44.1%. incidence was not significantly different among races after controlling for parity. 46.3% in african americans, 35.3% in caucasians, and 40.9% in others. incidence was not statistically different between uninsured(28.0%), privately insured (47.7%), and medicaid (41.9%) patients. p=.065. incidence of adenomyosis was not different between smokers (38.9%) and nonsmokers (44.6%). p= .386. table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in 44.1% of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of 848 women age 42-52 was studied. each woman collected daily first void urine samples for one complete menstrual cycle or 50 days (whichever came first) once a year for 3 years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of 47 and with 73% in the early perimenopause at the start of the study. 21% of all cycles were anovulatory. short cycle intervals (< 21 days) were associated with the early perimenopause (or 5.2, ci 2.1, 12.9) and with anovulation (or 20.3, ci 7.2, 56.8). long cycle intervals (36+ days) were associated with late perimenopause (or 8.7, ci 1.0, 79.1)and with anovulatory cycles (or 2.6, ci 1.9, 3.5). both short (1-3 days) and long (8+ days) duration of menstrual bleeding were significantly associated with anovulation (or 2.4 and 2.6, respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or 4.7, ci 2.6, 8.5) and with the self-reported presence of fibroids (or 4.1, ci 1.8, 9.5). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: 10 postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score 0-3; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after 1 month of daily treatment with 300 mcg of tt. data was assessed by t test and fischer's exact test; significant p < .05. results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline (16 +/-0.8) to treatment ( 38 +/-10.9); the difference was not significant; p = .8, although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness (2.2 vs. 0.75; p = .03) and dyspareunia (2.3 vs. 1; p = .02 saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e2) levels. however, particularly in postmenopausal women, the low salivary e2 levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e2 assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n=4,651) and postmenopausal women (n=1,770) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n=119; premarin, n=439), oral micronized e2 (estrace, n=145; compounded e2, n=1618), transdermal e2 patches (climara, n=623; vivelle, n=1619); or topical e2 cream (compounded e2, n=107). e2 levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be 0.8 pg/ml, compared with >2 pg/ml without extraction. 3.9 0.5 5.7 1.0 9.6 1 oral conjugated equine estrogens; 2 oral e2; 3 e2 patch; 4 topical cream salivary e2 levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e2 levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin-3 in pelvic organ prolapse. david d rahn, 1 jesus f acevedo, 1 patrick w keller, 1 lihua marmorstein, 2 r ann word. 1 1 ob-gyn, ut southwestern, dallas, tx, usa; 2 visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin-5 (fib-5) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= 0.01). lox, loxl1, 3 and 4 proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: (1) stratified squamous epithelium; (2) the lamina propria and (3) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl1. patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa11: molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa11 is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp2. we have also shown that hoxa11 is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa11 regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa11 expression on fibroblast proliferation, and its potential role in pop. methods: nih 3t3 cells, a murine fibroblast cell line, were seeded onto a six well plate (1x 10 5 cells/well) and transfected with either a vector carrying a hoxa11 cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b1 and phospho-histone 3. results: constitutive expression of hoxa11 in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa11 had a mean brdu incorporation of 40.8+ 8.8 cells/ 100 cells compared with 32.2 + 7.5 cells/ 100 cells in controls (p=0.03). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b1 and phospho-histone3 expression was not significantly different. cyclin b1 and phosphor-histone 3 were expressed in comparable numbers of cells in both groups. conclusion: hoxa11 is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa11 may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa11 mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: 5k12hd047018-03. connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, 1 gennaro acone, 1 sara d'avino, 1 marco torella, 1 gennaro trezza, 2 annamaria marenna, 1 nicola colacurci, 1 luigi cobellis. 1 1 department of obstetrics and gynecology, second university of naples, naples, italy; 2 loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of 25 premenopausal women with uterine prolapse and 16 controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of 8 virgin, 8 late pregnant , 8 immediate and 8 late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of 33.1 ± 6 mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ (124mm), virgin vagina generated the greatest contractile force (43.92 ± 3 mn/g) vs. late pregnant vagina (22.6 ± 2.4 mn/g, p<0.001) or immediate post partum vagina (26.81 ± 4 mn/g, p=0.033). four week postpartum k+ induced forces were similar to virgin levels (43.89 ± 3 mn/g). sm force generation (difference in active vs. passive length tension curves at 3mm of displacement) was decreased in late pregnancy (15.3 ± 4 mn/g) compared with virgin (112 ± 29 mn/g, p < 0.023) and 4 week postpartum vagina (172 ± 34 mn/g, p < 0.004). the expression of sm -actin was lowest in late pregnancy (15 ± 1.5, p < 0.001) and immediate postpartum vagina (23 ± 1.5, p < 0.001) relative to virgin (33 ± 3) with a return to virgin levels 4 week postpartum (56 ± 2). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by 4 weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, 2006) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp-3 expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u54hd052668. k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, 1,2 di licence, 1,2 cristin g print, 1,3 d stephen charnock-jones. 1,2 1 pathology, university of cambridge, cambridge, united kingdom; 2 obstetric gynaecology, university of cambridge, cambridge; 3 department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v12+/-/cre +/+ /rosa26r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, 16 mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice 60 hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c57bl/6 recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n=6 in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible 28 days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that 220 transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra6 is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra6, a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra6. here we investigate the differential expression of stra6 in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra6 expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with 10 -7 m estradiol (e 2 ), 10 -7 m r5020, a progesterone agonist, or vehicle for 24 hours. real-time pcr was employed to quantify stra6 mrna levels. we treated cells for 48 hours and quantified protein levels by immunoblotting. results basal mrna of stra6 levels in endometrial cells (n=8), were >1,000 fold higher than those in vehicle-treated endometriotic cells (n=8, p<.001). in endometrial stromal cells (n=8), r5020 stimulated stra6 mrna levels by 1.7-3 fold. r5020 induced stra6 protein levels in endometrial stromal cells (n=3). conclusions stra6 is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra6 may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf-1 has been suggested to play an important role. given that the aromatase substrate androstenedione (a4) is the predominant steroid in peritoneal fluid, we aimed to determine whether a4 has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a4 (5-10 nm) for 24 h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their 5'-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a4 (5-100 nm) revealed that 10 nm a4 maximally upregulated expression of both aromatase and sf-1. when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf-1. treatment of hes cells (24 h) with tritiated a4 demonstrated its metabolism to estradiol (e 2 ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a4, t and e 2 upregulated aromatase and sf-1 mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici 182,780, markedly diminished aromatase and sf-1 mrna expression induced by a4. finally, chromatin immunoprecipitation revealed that a4 significantly enhanced recruitment of sf-1 to its response element (-136 bp) upstream of cyp19 exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c 19 -steroids may cause an acute upregulation of cyp19 gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r01-dk031206. the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, 1 b frank, 2 j gemmill, 1 g chrousos, 1 m ballweg, 3 p stratton. 1 1 rbmb, nichd/nih, bethesda, md; 2 som, wake forest university, winston-salem, nc; 3 endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged 18-50, with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection (1 mcg/kg), serial blood samples (-5, 0, +15, +30, +45 minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > 55 pg/ml without a rise in cortisol levels. 4 subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: 31 women (21 cpp, 10 controls) had a mean age of 33.5 ± 9.1 years (range: 21-47). 18 women responded normally (10 pnr, 8 npnr) and 13 women responded abnormally (11 par, 2 npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: 85.2 a vs 33.5 pg/ml n, p<0.001; absolute: 51.2 a vs 27.5 pg/ml n, p=0.024). there was a significant difference within groups: peak acth: 88.5 par vs 37.1 pg/ml pnr, p=0.001; 67.5 npar vs 20.3 pg/ml npnr, p<0.001; absolute acth: 72.2 par vs 28.2 pg/ml pnr, p=0.001, 56.1 npar vs 20.4 pg/ml npnr, p<0.002). however, total cortisol level was not different between or within the 2 groups (23.2 a vs 20.0 mcg/dl n, p=0.08). mean depression score did not differ among cpp patients (34.7 par vs 35.2 pnr, p=0.95), but differed among controls (25 npar vs 4.3 npnr, p=0.01). anxiety scores did not differ between or within groups (29.5 npar vs 8.4 npnr p = 0.12; 40 par vs 39.7 pnr, p=0.66). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of 8-week-old cd1 female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd1 mice. seven controls received sham surgery only. after 14 weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa10, hoxa11, bteb1, emx2, igfbp1, integrin -3, total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa10, hoxa11, bteb1, emx2, and igfbp1 mrna expression showed 0.57-fold (p=0.046), 0.25-fold (p<0.001), 0.50-fold (p=0.043), 0.23fold (p=0.038), and 0.09-fold (p<0.001) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed 2.7-fold (p=0.016) and 8.39-fold (p=0.006) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups (20.98 vs. 7.53 , endometriosis vs. control, p>0.05). there was no significant change in integrin -3 mrna expression (1.09-fold, p>0.05). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, 1 felice petraglia, 2 gerson weiss, 1 stefano luisi, 2 pasquale florio, 2 jeff gardner, 1 andrea wojtczuk, 1 laura t goldsmith. 1 1 obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; 2 pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr7 receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n=8) and eutopic (n=11) endometrium of patients with endometriosis and from endometrium of normal controls (n=12), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr7 relaxin receptor mrna, h2 relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method (2 -ct ) determined relative lgr7 and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from 9 of 12 (75%) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [9 of 19 (47.4%)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [2 of 8 (25%)] than in eutopic samples [7 of 11 (63.6%)]. lgr7 relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr7 mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls 5.9-fold higher levels in the sp than pp; and in endometriosis patients 3.6-fold higher levels in the sp than pp. in both phases, lgr7 mrna levels were lower in ectopic samples than in either eutopic samples (6.2-fold lower in pp and 19.0-fold lower in sp) or endometrium from normal controls (6.5-fold lower in pp and 15.6-fold lower in sp). eutopic endometrium had similar lgr7 mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january 2003 and september 2007 was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > 15mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was 4.1±2.4 in the control ovary and 1.1±1.4 in the previously operated ovary(p=0.031). the mean(±sd)number of follicles with a mean diameter > 15mm was 5.6±2.5 in the control ovary and 1.8±1.1 in the previously operated ovary(p=0.0004). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o 2 ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in 1% o 2 . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o 2 tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o 2 tensions. methods placental villous explants were cultured with either 10% serum (n=10) or in serum-free conditions (n=6) for 96h in 20%, 6% and 1% o 2 . after 96h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in 50% ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at 20%, 6% and 1% o 2 were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of 0.001 was considered significant. results the mean intra-assay variability was 9.1%. there were no differences in the metabolic profile of conditioned culture medium from 6% and 20% o 2 . several metabolites differed in culture medium from 1% compared to 6% and 20% o 2 including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in 1% o 2 could be completely discriminated from 6% and 20% o 2 . deoxyribose was also elevated in tissue lysate from 1% o 2 compared to 6% o 2 . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in 1% o 2 . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n=5) were cultured for 96h in 1%, 6% and 20% oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at 1%, 6% and 20% were compared to identify differences in the metabolic profiles. this procedure was repeated for 6 different explants and the results compared across the 2 studies in order to validate initial findings. approximately 350 unique peaks were detected in both sample sets in negative ion mode and 300 peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval<0.01) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others 2-deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o 2 1% and o 2 6%. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) 14 to 18. the number of brdu positive cells was also compared on e14 and e16. brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat3 phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat3 pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e16 and e18, and in the cortical plate on e18. the number of brdu-positive cells was reduced in vz of e14 and e16 lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by 2 days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes1 mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat3 nor that of stat3 was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes1 expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat3 pathway does not mediate the leptin signal in undifferentiated neural lineage cells. 311a vaginal wall, and >90% of fib-5 knockout (ko) mice develop pelvic organ prolapse by 20 weeks of age. in contrast, fib-1 and -2 deficiencies do not result in prolapse, and fib-4 kos die shortly after birth. herein, we evaluated the role of fib-3 in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in 227 fib-3 ko (fib3 -/-) and in fib3 -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was 37 wks. fib3 -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp2 (2-fold, p=0.03), active mmp2 (2-fold, p=0.06), and prommp9 (3-fold, p=0.05) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib-5 and tropoelastin (1.7-fold, p<0.001). regardless of prolapse maternal morbidtties tf23 ards-n(%) 12 (52) acute ren~m f11lure-n (%) 10(43) car~tovascular ~ysfunrtion-n ("/o) 15 ( 65) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) 6 (26) durallon of !cu suy (days. mean+sd) 9 21:t 12 .8 hospital my (days.. me411+sd) 2. condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. (2006) mol endocrinol 20(4), 764-775. objective: innervation of the uterine cervix is important for the process of parturition (physiol beh 63:929,1998; j histochem cytochem 52:1249 ,2004 jsgi 12:578,2005) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day 15 post-breeding. live pups were born spontaneously by all rats at term on days 22-23 post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, 1,2 della m yates, 2 carolyn m mitchell, 2 jonathan j hirst, 1,2 tamas zakar. 2,3 1 school of health sciences, university of newcastle, newcastle, nsw, australia; 2 mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; 3 obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. 109:1417) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta 17:299) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h2 synthase-2 (pghs-2) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs-2 mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were 13.8 times higher in decidua and 4.0 times higher in chorion and placenta than in amnion (p<0.001). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were 50% of those in amnion (p=0.02). this low level of prorenin receptor mrna in decidua persisted after labor (p<0.001). pghs-2 mrna expression was highest in amnion; in decidua and placenta it was only 6 and 4% of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs-2 expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, 1 xiangying xue, 2 christine n metz. 2 1 obstetrics and gynecology, nyu school of medicine, new york, ny, usa; 2 feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p4) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p4 action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p4 (10 -4 m) 1 hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+10% fbs, b)'yellow' media: phenol red-free rpmi+10% fbs, and c)'clear' media: phenol-free rpmi+10% dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p4 suppressed tnf production in all medias. tnf levels were suppressed to 39.4% (p<0.06) of controls using 'red' media and to 42.0% (p<0.03) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were 64.5% (p<0.02) of control. (fig 1) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig 2) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p4 effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p4 to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. 743 ovine fetal regional blood flow following prenatal dexamethasone (dex) at 0.75 gestation (g). antonine d van heesewijk, 1 jan g nijhuis, 1 susan l jenkins, 2 peter w nathanielsz, 2 mark j nijland. 2 1 ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; 2 ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n=6). dispersed adrenal cortical cells (2.5 x10 5 cells/tube; in duplicate) were challenged with 10 -8 m acth. samples were collected at time 0 (baseline), 5, 15, 30, and 60 min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/2.5x10 5 cells) was higher in the lth group compared to the control, (p< 0.01) at 15min (3.3 ± 0.5 vs. 1.0 ± 0.1), 30min (7.7 ± 0.6 vs. 1.9 ± 0.1), and 60min (10.9 ± 0.7 vs. 2.5 ± 0.1). peak camp levels (fmol/2.5x10 5 cells) were observed at 30 min but did not differ between control (81.8 ± 10.4) and lth (77.9 ± 17.8) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p<0.05) at 0 min (1.02 ± 0.07 vs. 0.59 ± 0.05), and at 60 min (1.26 ± 0.04 vs. 0.65 ± 0.05), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd31226, hd33147 and llu-nih imsd 2r 25gm060501-05). expression. hiroaki itoh, 1 makoto kawamura, 2 shigeo yura, 2 haruta mogami, 2 tsuyoshhi fujii, 2 norimasa sagawa. 3 1 obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; 2 gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; 3 obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-hsd2) expression. methods: the 30 % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; 20% casein protein; spd-un) or high protein diet (hpd;40% casein protein; spd-un). in some groups, these pregnant mice were sacrificed at 18.5 d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at 16 wks. fetal and maternal corticosterone levels were measured by elisa. the placental 11beta-hsd2 gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at 16 weeks (106 mmhg) was higher than that in spd-nn offspring (99.3 mmhg). by contrast, systolic blood pressure in the hpd-un offspring (97.5 mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring (151 ng/ml) was lower than those in spd-un offspring (208 ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental 11beta-hsd2 gene expression in the hpd-un was 72% higher than that in spd-un, suggesting that maternal hpd augmented placental 11beta-hsd2 gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental 11beta-hsd2 gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, 11 hsd2, including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru26752 (mra, 1.68 mg/h over 12h) into 120-130 day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at 10-12h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru26752, the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses (34.7±1.6 vs 30.2±1.7). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at 12h (657±262 vs 171±20 pg/ml); in the mra-treated fetuses, acth concentrations at 12h were greater than in the same fetuses at 0h (124±16 pg/ml), wheras there was no increase in plasma acth in the control fetuses (0h: 100±19 pg/ml). similarly, plasma cortisol concentrations at 11 and 12h were greater in the mra infused fetuses than in control fetuses (12h: 18.2±5.5 vs 7.0±1.4 ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses (0h, mra: 4.7±1.5, control: 2.9±0.5 ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at 9-12h of infusion of mra (12h: 37±1 vs 32±1 %) and fetal pco2 was significantly greater at 12h of infusion of mra as compared to control fetuses (61.0±2.0 vs 53.5±0.8). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase-2 (cox-2) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox-2 in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox-2 inhibitor, was infused (1 mg/day, n=9), and vehicle (50% dimethylsulfoxide, n=6) was infused. arterial blood samples were drawn from fetuses at 48 hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p<0.001 by anova) before spontaneous parturition at 146±0.3 days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p<0.001 by anova) and the fetuses were born spontaneously on 148±0.3 days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p<0.01 by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e2, demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox-2 dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase1/2 and p38 mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin-1 (1mm) on inos protein expression (30 min, 4 and 8 h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p<0.05) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by 35% in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin-1 treatment decreased inos protein abundance at 4 and 8 h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin-1 treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: 8-isoprostane (8-iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by 8-iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled <26 weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for 8-iso by ultra-sensitive elisa. elevated 8-iso was defined by a value 75 th %tile. maternal factors associated with elevated 8-iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios (95 th %ci) for elevated 8-iso. results: 791 women had complete data for analysis. the median 8-iso level (iqr) was 1,806 (16 -81,870 pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated 8-iso. the logistic regression model for elevated 8-iso is shown below. adjusted or (95 th ci)* mild periodontal disease 1. maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a 23-year-old primiparous woman was referred to our hospital for prenatal care after 12 weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be 5,500/ l; hemoglobin, 8.0 gm/dl; and platelet count, 38,000/ l. her bleeding time was normal (3 min). until the 34th week, the patient's pregnancy was uneventful. during the 35th week, however, the patient's platelet count declined to 16,000/ l, when proteinuria became evident. her bleeding time was prolonged to 13 min. the coagulation system was normal. by the 37th week, the patient's blood pressure was elevated to 150/100 mmhg, and her urinary protein excretion exceeded 2 g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after 38 weeks. at the same time, the patient was transfused with 10 units of platelets, but her platelet count remained unchanged (20,000/ l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses (400 mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets (15 units) were again transfused, this time raising the platelet count to 110,000/ l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within 2 days after delivery, but gradually increased to the prepregnancy level by 2 weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, 1 juan c gonzalez-altamirano, 2 julio e valdivia-silva. 1,3 1 oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; 2 cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; 3 inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day 1 postpartum. 96 live singleton infants were born after preeclamptic pregnancies and compared those with other 50 control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a 8% (95% confidence interval [ci] 6%, 9%) reduction in birth weight. 4 women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = 3.4; 95% ci 2.8, 7.0) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = 1.8; 95% ci 0.7, 2.5). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il-1 and il-1 expression in 3 preterm placentas with severe pe+iugr, and 3 idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n=5) were treated with il-1 and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups (28.3±2.2 vs 27.6±2.8 wks). ihc of pe+iugr samples revealed intense villus core staining of il-1 adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il-1 staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il-1 action, primary cultures of fibs and scts were incubated for 48 h in serum-free medium ± 1 ng/ml il-1 . by elisa we observed that il-1 treatment of fibs increased il-8 levels from 4±1 to 2161±346 pg/ g protein (p<0.001). similarly, multiplex array revealed that levels of il-4, il-6, and il-11 were induced 24-, 269-, and 12-fold, respectively in fibs. the presence of 200 ng/ml il-1 receptor antagonist reduced the il-1 -mediated increase in il-8 levels 95±3% (p<0.001), indicating that this response was mediated through il-1 receptor. in marked contrast, il-1 treatment did not significantly affect levels of il-4, il-6, il-8 or il-11 in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il-1 in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week 10, promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases 1 . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin 1 . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp (750 g/kg bw; n=9) or saline (n=5) on day 14 of pregnancy (permanent jugular vein cannula/1 hr infusion). non-pregnant rats (n=8) were infused with atp identically. one day before and 1, 3, 6 days after infusion, 24 hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig 1) . wbc were also increased only in pregnant rats infused with atp (days 15 and 17 vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r 2 = 0.66). staining of glomeruli for the angii receptor (at-1r) showed decreased at-1r expression in control pregnant rats as compared to non-pregnant rats, while at-1r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days 17 and 21 in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor-1 (tgf-1), that is known to promote progressive fibrosis in human tissues. moreover tgf-1 plays a major role in trophoblast growth and differentiation. tgf-1 inhibits hgf expression as well as hgf inhibits tgf-1 expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf-1 are differently expressed in tissues of normal and pe placentas. methods: we studied 13 placentas from pregnancies complicated by pe (7 with normal fetal growth and 6 with fgr) and 5 placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf-1 in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf-1 and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf-1, that promote fibrosis. placental expression of hgf, c-met and tgf-1 is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase 8 (mmp8), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp8, this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp8 as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from 5 normal weight, 5 overweight and 5 obese women. formalin fixed, paraffin embedded 8 μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp8. data were evaluated for intensity of vessel staining by visual score (0-4), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n=9) or pe (n=10) pregnancies were explanted for 4 days at 6% oxygen. conditioned 24h medium (day 3-4) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on 80% matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin-1, prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of 15 chemokines/cytokines. results: in the angiogenesis assay, tubule length (p<0.05, mann-whitney u test) and number (p<0.05) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p<0.05). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)-6, il-8, gro-, monocyte chemotactic protein-1 and macrophage inflammatory protein-1 (mip-1 ). higher levels of both il-8 and gro-were present in the normal medium compared to pe (both p<0.0001). mip-1 was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p120) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: 1) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p120 was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p-120. 2) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: 1) ve-cadherin and p120 are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p120 is lost in ecs exposed to pe cm. 2) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl65997 and hd36822). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately 7% neurons were colon-specific and 11% were uterus-specific. among these uterus-or colon-specific neurons, up to 5% of labeled drg neurons in the l1-s3 levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of 548 hysterectomies performed for benign indications from june 2001 to june 2006. institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at 4 time points: time 0 (immediately postoperatively), time 1 (2 weeks to 5 months postoperatively), time 2 (5 months to 1 year postoperatively) and time 3 (1 to 2.5 years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p<0.05) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time 3 compared to baseline in all women (31.9 to 33.6). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr-2 were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr-1, vegfr-2, nrp-1 and nrp-2), vegfr-2 was the predominant receptor that expressed in the more invasive cell lines (dov13, skov3 and r182). lpa, at 10-20 m, significantly induced vegfr-2 expression in dov13 and skov3 cells (p<0.05), without significantly affecting vegfr-1 expression. lpa (1-20 m) also significantly induced the expression of vegf 121 and vegf 165 (p<0.05) in dov13 and skov3 cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr-2 expression could significantly decrease dov13 cells' invasiveness (p<0.001) and moderately decrease skov3 cells' invasiveness. moreover, silencing of vegfr-2 by sirna significantly suppressed lpa-induced dov13 and skov3 invasion. conclusion: these results suggest that knocking down vegfr-2 expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp-1 and mmp-7, has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp-1, mmp-7 and osteopontin expression with tumor stage in 41 ovarian tumor tissues and 7 normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = 7) and ovarian tumors (n = 41) of different stages (stage i, n = 16; stage ii, n = 3; stage iii, n = 19; stage iv, n = 3) was amplified by real time pcr using gene specific primer pairs for mmp-1, mmp-7, and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp-1, mmp-7 and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as 1. the average expression of mmp-7 in ovarian tumor tissues (14630 ± 30606) is 49 fold of that in normal ovaries (301 ± 641, p = 0.0047). using an arbitrarily set standard, 1 of 7 normal ovaries (14.3%) has elevated mmp-7 expression; in ovarian tumor tissues, the percentage is 82.9% (34 of 41), significantly higher than in normal tissues (p = 0.0008). in early stage tumors (stage i/ii, n = 19), 17 of them have elevated mmp-7 expression (89.4%, p = 0.0008 vs. normal ovaries), whereh the average expression of mmp-7 is 63 fold of that in normal ovaries (p = 0.069) and 1.8 fold of that in late stage tumors (stage iii/iv, n = 22). the mmp-1 expression in ovarian tumors (n = 41, 154 ± 632) is 28 fold of that in normal ovaries (n = 7), however, the difference is not significant (p > 0.05) due to the extremely high expression of mmp-1 in two tumor tissues. osteopontin expression in ovarian tumor tissues is 11.2 fold of that in normal ovarian tissues (11 ± 12.51, n=7 vs. 123.6 ± 160.0, n = 41, p<0.0001). in early stage (i/ii) ovarian tumors, osteopontin expression is 8.1 fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is 13.9 fold of that in normal ovaries (p<0.05), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp-1, mmp-7 and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac6 inhibitor trichostatin a (tsa), anti-metabolite fluorouracil (5fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic 50 or 10-fold ic 50 of each agent, and incubated at 37°c for 1 h, 24 h and 48 h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation (2.5-fold) was observed with vinblastine at 10-fold ic 50 after 48 h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression (8.5 fold) was observed with 10-fold ic 50 of vinblastine at 48 h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after 1 h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p53 plays an important role in the induction of apoptosis by a variety of anticancer drugs. p38 mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p38 and p53 activation induces apoptosis, we hoped to evaluate the relationship between p38, p-p53 and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob (5 to 200nm) for 16h. parp cleavage product p85 as well as p-p38, p53, phospho-p53 (ser-15), p21 and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without 5 m sb203580, a specific inhibitor of p-p38, followed by treatment with epob (20 or 50 nm) for 16h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p38 and anti-phospho-p53 antibodies. results: epob induces p53 activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p53 precedes phosphorylation of p53 in hey cells. the expression of p53 targeted gene, p21 (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p38 markedly inhibited the p53 phosphorylation at serine 15. conclusions: 50 nm epob (a concentration that triggered mitotic arrest) causes a decrease in p21 expression and an increase in survivin expression. epob induces parp cleavage. p38 inhibitor sb203580 inhibits epo-b -induced p53 phosphorylation. these results suggest that phosphorylation of p38 may lead to p53 activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin2 is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: 1) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; 2) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and 3) to analyze the effect of imd on the expression of tnf-alpha and il-1beta in human epithelial cervical carcinoma cells (hela). methods: groups of 4 sprague-dawley, non-pregnant and pregnant rats on day 18 of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to 80% confluency in rpmi medium supplemented with 10% fbs. the cells were starved in 1%fbs for 4 hrs followed by treatment with imd (10 -8 m) in presence or absence of imd antagonist, imda (10 -5 m). cells were further incubated for 24hrs and total rna was extracted using trizol reagent. rna was treated with dnase1 before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of 18s. results: 1) imd mrna is expressed in rat and human cervix and in hela cells. 2) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. 3) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il-1beta mrna and, 4) expression of imd mrna is significantly elevated (p< 0.05) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya-8 and skov3-ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel (20 mg/kg) and carboplatin (50 mg/kg) delivered on timeevery 21 daysachieved better tumor response in both aggressive (heya-8) and metastatic (skov3-ip1) human ovarian tumors compared to the non-treatment and vehicle groups. in heya-8 mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim (1000 μg/kg) and darbepoetin (10μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya-8 model. for skov3-ip1 mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< 0.01). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< 0.05). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. -415) . median age at recurrence was 73 (33-104), and median interval to recurrence 13.5 months (1-415). 81 (56%) patients died of recurrent disease and 10 (7%) of other causes. site of recurrence and was local in 92, groin in 30 and distant in 22. 58 of the local recurrences were managed with wide local excision (wle) 12 radical surgery (6 radical excisions with skin flaps, 4 posterior exenterations, 1 anterior exenteration, 1 total exenteration), 10 wle and radiotherapy (rt)/chemort, 6 chemo/chemort/rt, and 6 palliation. 12 groin recurrences were managed surgically (5 adjuvant rt), 3 rt alone, 1 chemotherapy alone, and 1 palliation. 2 patients with distant metastases were managed surgically, 10 with rt/chemo/chemort, and 9 palliation. overall median survival after recurrence was 17 months. median survival (months) by site of recurrence was: local 56, groin 12, distant 4, groin or distant 6. there was a significant difference in survival in local vs groin node recurrence (p<0.0001), local vs groin and distant (p<0.0001), and groin node vs distant (p 0.0075). 3 and 5 years after recurrence survival rates were: local 56% and 46%, groin 13% and 13% distant 0,0. conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that 2 edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf7 (breast) cells were cultured in steroid-free, phenol-free media with bpa (25 m), des (5 x 10-8 m) or vehicle for 48 hours. pregnant cd-1 mice were treated with intraperitoneal injections of des (10 mg/kg) or vehicle on days 9-16 of gestation. 2 weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: 3 dnmts (dnmt1, 3a, and 3b) and 2 hmts (mll and ezh2) were screened after in vitro exposure to edcs (table 1) . ezh2 mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh2 expression was seen in mcf7 cells treated with des or bpa. due to the consistent induction of ezh2 in all cells with either treatment, we examined the effect of des exposure on ezh2 expression in vivo. in adult mice exposed to des in utero, ezh2 expression was persistently increased (3.0±0.66 fold, p<0.05). conclusions: breast and uterine cell lines show increased expression of ezh2 in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh2 has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh2 expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., 2007) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov1 cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov1 cancer cells (ic 50 =10um). treated cells showed a 2-3 fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, 1 faheem m shaikh, 2 john a lucas iii, 1 susan l bellis. 2 1 obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; 2 physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st6gal i, which is responsible for the 2,6 sialylation of 1 integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st6gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because 1 integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st6gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st6gal i into the ov4 ovarian tumor cell line, which we found to be lacking the st6gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st6gal i expressing (st6) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov4-st6gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < 0.05), increased haptotactic collagen i cell migration (p < 0.01), and increased invasiveness (p < 0.05) in the st6 cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st6gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted 47 probe sets as differentially expressed between lmp and lmp-mp, 134 probes between lmp and lgsc, and 180 probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk1/3 pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated 2 members of the mapk1/3 pathway-poly adp ribose polymerase1 (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the 3 tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk1/3 and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately 50% of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) (95%), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone (50%). recent studies have shown that immunostaining for p16 ink4a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p16 or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente (2005-07). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n=90) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic 0.85). the correlation between p16 and proexc scores was moderate (kappa 0.40). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p<0.001 jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e 2 ) and progesterone (p 4 ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and 21-day e 2 and p 4 treated 30-day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for 24 hrs either with no hormone or with a single or a combination of 100 pg/ml of e 2 or p 4 , 100 ng/ml of hcg. then mmp-2, -7 and -10 mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp-2 mrna levels were unaffected, mmp-7 mrna levels significantly decreased and mmp-10 mrna levels significantly increased in lhrko animals as compared with wt siblings. e 2 and p 4 treatment reversed mmp-7 and mmp-10 changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp-10 mrna levels, but it did increase mmp-7 mrna levels. this increase was synergistic with both e 2 or p 4 . conclusion: while lh/hcg do not regulate uterine mmp-2 and mmp-10, they seem to co-regulate mmp-7 with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from 8 pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity (1-4) and percentage of positive cells (1-4) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; 4 with emca and 4 with aeh. three pts had no response, 1, a partial response and 4, complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean 2.8 vs. 8; p<0.01) and the stroma (mean 2.3 vs. 5; p<0.05). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean 4.6 vs. 2.7; p=0.14), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean 7.4 vs. 3; p<0.05). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean 7.4 vs. 4.6; p=0.07). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o 6 -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included 77 endometrioid endometrial cancers, 43 endometrial tumors of adverse histologic type, and 6 endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the 120 endometrial cancers evaluated or the 6 endometrial cancer cell lines. one of the 21 ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas (1% of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor 5-years overall survival (< 10-30%). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages (1 to 13) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin-1 and -actin. this cell line expresses low er and pr levels. trail receptors (r1 and r2) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin (5um for 24 h) produced low cell cytotoxicity (< 20%). trail (200 ng/ml for 18 h) induced about 30% cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to 80%), this was demonstrated by a significant increase in the sub g0/g1 region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt 1050744). defining the tumor initiating capacity of cd133 + human endometrial cancer cells. anne m friel, 1 petra a sergent, 1 christine l cummings, 2 rosemary foster, 3 current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd133, a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd133-expressing enca cells. to evaluate the tumorigenic potential of cd133 + cells, the tumor initiation capacity of xenograft derived cd133 + and cd133cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd133 + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb2, whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n=60; mean age 50± 2.7) with endometrial hyperplasia (n=20), polyps(n=20), and cancer(n=20), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n=20) were obtained from menopausal women (n=20; mean age 51± 2.5), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm1) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad-2 and reduction of proliferation of phm1 cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her2neu over-expression and pi3kinase/akt pathway activation in paget's disease of the vulva. amy stenson, 1 bita behjatnia, 1 jaime shamonki, 1 jianyu rao, 1 amer karam, 1 jonathan berek, 2 oliver dorigo. 1 1 ob/gyn and pathology, ucla, los angeles, ca, usa; 2 ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her2neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her2neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her2neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of 0 (negative) to 3 (strongly positive). clinical data was retrieved via chart review. results: between 1995 and 2007, 25 patients with vulvar paget's were identified. median age was 65 yrs (36-83 yrs). a family history of cancer was found in 14/25 (54%), 7/25 (28%) were smokers and 13/18 (72%) had a history of hormone use. intraepithelial lesions accounted for the majority (15/25, 60%), while 6/25 (24%) demonstrated invasion and 4/25 (16%) were associated with underlying gi malignancy. 16/25 (64%) had at least one recurrence, with median time to recurrence 24 months. so far, 13 specimens were stained for her2neu and pakt. overexpression of her2neu was found in 10/13 (77%.) positive staining for pakt was evident in 10/13 (77%.) statistical analysis suggested a correlation between her2neu and pakt expression. conclusions: our study demonstrates that overexpression of her2neu and activation of the pi3kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her2neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her2neu, or a pi3kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to 70% of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at 37ºc in a humidified atmosphere of 5% co 2 -95% air in smbm medium supplied with 5%fbs, 0.1% insulin, 0.2% hfgf-b, 0.1% ga-1000 and 0.1% hegf(lonza). the cells were plated at density of 2×10 5 cells/well in 6-well plates and grown under the same conditions above. the monolayer cultures at approximately 70% confluence were treated with various concentrations (0, 0.1, 1.0, 10, 50, 100 and 200μm) of egcg (sigma) for 7 days. a fluorometric assay using hoechst 33258 dye (sigma) for dna quantitation was conducted at the following designed time points, day 1, 3, 5, and 7 post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution (1μg/ml). fluorescence was measured after excitation at 355nm and emission at 458nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at 10 μm and peaked at 100 μm concentration. the difference was statistically significant (p = 0.002). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp-1 and mmp-7), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg-3. design: in vitro experiments using the jeg-3 cell line. materials and methods: after plating jeg-3 cells in a 24 well tray overnight in rpmi media containing 10% fbs, cells were washed twice with pbs and then cultured in 500 ul of serum-free rpmi media containing one of four treatments: 1) saline; 2) hcg (50 iu); 3) hcg (50 iu) plus hcg-ant (100 iu); or 4) hcg-ant (100 iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp-1, mmp-7, and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg-3 cells with hcg-ant vs. hcg alone reduced expression of mmp-1 (51.95 ±15.51 vs. 85.99 ±10.56) and . hcg-ant reduced mmp-1 and mmp-7 expression by 40% and 43%, respectively (p<0.05). treatment with hcg-ant vs. hcg increased metastin expression (5.44 ±2.29 vs. 4.63 ±1.32). metastin expression was increased by 17% following hcg-ant treatment. conclusion: treatment of the jeg-3 choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp-1 and mmp-7 following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids (9-cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over 60% using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g1/g0 cell population by 20% and decreased s phase cells by 20%, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately 4 to 6 ng/ml/10 5 cells (n=5). although there was less than 0.25 ng/ml/10 5 cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, 1 frank z stanczyk, 2 stan patel, 2 alison edelman. 3 1 ob/gyn, university of north carolina, chapel hill, nc, usa; 2 ob/gyn, usc keck school of medicine, los angeles, ca, usa; 3 ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages 18-35 years, of normal (< 25 kg/m 2 ; n = 10) and obese (> 30 kg/m 2 ; n = 9) body mass index (bmi) received a low dose oc (20 mcg ethinyl estradiol/100 mcg levonorgestrel) for two cycles. serum was obtained at three time points: after 21 days of active pills (cycle 1, day 21), at the end of the 7-day hormone-free interval (cycle 1, day 28), and during the first week of active pills in cycle 2 (cycle 2, day 5,6, or 7). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = 0.90) or by active versus placebo pill use (2.79 ng/ml ± 1.3 vs. 2.85 ng/ml ± 1.4, p=0.93) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = 0.04), but not by use of active or placebo pill (1.63 ng/ml ± 1.2 vs.1.71 ng/ml ± 1.2, p = 0.88). across the cycle, cv(standard deviation/mean) averaged 20.2%± 3.1 in the obese and 8.8%±1.3 in the normal bmi women ( p=0.003). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = 0.02) and lower amh levels in the obese group (p<0.001). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after 7 days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within 3 years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a 10-item questionnaire assessed the participant's level of comprehension. a score of 80% was adequate to complete the informed consent process. those scoring <80 % were re-counseled and retested. demographic variables determining the likelihood of an initial score >80% were evaluated by univariate and multivariable analyses. results: 32% (24/76) scored >80% on initial testing. all women scored >80% after re-counseling and retesting. likelihood of an initial response >80% correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of 8 women whose furthest educational level was high school scored >80% on initial testing, significantly less than those with a college education (p=0.04). a higher proportion of college graduates (13/32) scored >80% compared to those attaining further education (11/35) (p=0.43). comprehension was greatest for study purpose and duration (64/76 and 70/76 correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd (41%) or breast cancer (50%) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k24-hd41978 to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, 1 alex j polotsky, 1 aileen p mcginn, 1 carol a derby, 1 rachel p wildman, 1 lhasa ray, 1 kavitha t ram, 1 gerson weiss, 2 nanette f santoro. 1 1 albert einstein coll of med, bronx, ny; 2 univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over 75% of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited 277 women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from 1 (not at all) to 5 (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation (4-item scale: marin, hisp j behav sci 2:183, 1987) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or=2.2, 95%ci: 1.1-4.3) and headaches (or=2.7, 95%ci:1.4-5.3), dominican women were most likely to report vaginal dryness (or=2.8, 95%ci: 1.1-7.1) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr004061; ag012505, ag012535, ag012531, ag012539, ag012546, ag012553, ag012554, ag012495 and cd41978). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged 65 years or older. participants and methods: annual modified mini mental state (3ms) examinations were used to assess global cognitive function among the 7,212 women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and 300 women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline 3ms testing. associations between baseline cognitive risk factors common to both trials and baseline 3ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial 3ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in 3ms scores of 0.5 units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p=0.003) but were also evident for raloxifene (p=0.06) and cee (p=0.02). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied 25 postmenopausal women (pm) within 4 years of menopause (age 58.4±1 yrs) of normal weight (bmi 26.3±1) and compared them to 20 weight matched (bmi 27.4±1) premenopausal (pre) controls (age 26.4±1yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein 4 (rbp4.) while total fat was similar in the 2 groups, pm had more trunk and abdominal fat (10516±800; 818±67g) compared to pre (8626±493g p<0.05; 577±33g p<0.01) pm also had greater %trunk fat and %central abdominal fat compared to pre, p<0.01.serum leptin (31.8±0.5 vs 28±5 pg/ml) and visfatin ( 10.4±1 vs. 9.4±0.5 ng/ml) were similar but adiponectin (17±1 vs. 13.3±1 g/ml) and rbp4 (28.7±2 vs. 22 .3±1 ng/ml) were higher, p<0.05 in pm. while in pre, abdominal fat correlated negatively with adiponectin (p<0.01) in pm only leptin correlated with various parameters of fat mass, p<0.02, and adiponectin did not correlate but correlated positively with age (r 0.47, p<0.05.) as expected, pm had reduced bmd at the lumbar spine and hip (0.84±0.025 vs. 0.99±0.025 g/cm2; 0.72±0.025 vs. 0.87±0.025 g/cm2, p<0.01) but there was a correlation between total and trunk fat in pm and lumbar bmd (r 0.69, 0.66, p<0.01) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r 0.47, p<0.02) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp4 are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant (18) and 20 pregnant (d14) wt mice underwent either vaginal distention with a 750μl balloon x 40 min (to simulate parturition) or sham procedure. tissues were obtained at 24 and 48 h for immunoblot analysis, zymography, and histology. distention was also performed in 30 young fbln5 -/-, fbln5 +/-, and wts, and prolapse was quantified for 8 weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp9, 5.6-fold; active mmp9, 3.5-fold; prommp2, 2.4-fold; active mmp2, 3.7-fold; all p < 0.05 compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin-5 in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin-5 and tropoelastin), however, distention resulted in 3-fold upregulation of both proteins (p<0.05). distention also induced increased mmps in pregnant animals (mmp-9, 1.7-fold; prommp-2, 1.7-fold; active mmp-2, 2.3-fold; all p < 0.05). thirteen young fbln5 -/-(4-6 wks prior to age of prolapse development), 11 het siblings, and 6 age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within 3 d) in fbln5 -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln5 -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade 3 by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n=7, controls: n=6) to study the protein expression of mmp-1, -12, timp-1, -2, -4. gelatin zymography was used to quantify the activity of mmp-2 and -9. immunohistochemical analysis for timp-2 and -4 was performed on pfa-fixed vaginal biopsy tissue (n=3) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp-12, and mmp-1. the protein expression of the 45 kda active form of mmp-12 was significantly increased in patients with pop compared to controls (p=0.03). zymography detected the enzymatic activity of the proform and active form of both mmp-2 and mmp-9. we found a significant increase in gelatinolytic activity of both 68 kda pro-form and 62 kda active forms of mmp-2 (p=0.01 and p<0.001 respectively) as well as 87 kda active form of mmp-9 (p<0.001) in patients with pop compared to controls. timp-1 protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p=0.002). timp-2 and -4 immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl1, loxl3 and loxl4 proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade 3 by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n=7, controls: n=6). pfa-fixed vaginal biopsy tissues (n=3 for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox (47 kda pro-form and 35 kda active form), loxl1 (47 kda pro-form and 35 kda active form), loxl3 (83 kda) and loxl4 (84 kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in 38 women (aged 34.4 years; range 22-45 years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp9.5, using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all 23 of the women in whom endometriosis was surgically diagnosed and in none of the 15 women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of 100%. the density of nerve fibers in the endometriosis cases was 3.4 per mm²± 4.3 (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk1/2 are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e2), vegf, and cytokines such as il-8 and mcp-1, stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n=24) and ectopic (n=25) endometrium were immunostained for total-(t-) and phospho-(p-) erk1/2 and cd34 (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e2 (10-8m), il-8, mcp-1, and vegf (all 2 ng/ml) for 5 15 min, and western blotted for p-/t-erk (n=3). heec were treated with peritoneal fluid (pf; diluted 1:3 in basal media) from normal (n=4) and endometriotic (n=4) women, with or without pd98059 erk1/2 inhibitor (20 m) for 48 h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd34+ endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p<0.05). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after 48 h compared to normal pf (p<0.05). this effect was abrogated by erk1/2 inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in 5 and 15 min (p<0.05), while e2, il-8, and mcp-1 had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk1/2 is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk1/2 inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal (20% o 2 ) and hypoxia (2% o 2 ) conditions for 24 and 48 hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod (49.7 pg/ grna, p<0.05) as compared to adhesion fibroblasts (38.1 pg/ grna, p<0.05). short exposure to hypoxia resulted in a significant increase in sod mrna levels to 63.3 and 52 pg/ grna in normal and adhesion fibroblasts respectively, p<0.05. in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to 41.5 and 25.7 pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p<0.05. conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp-3. recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp-3. methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in 1 nm estradiol (e) for 24 hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e (8 μg, silastic capsule implants) and simvastatin (5-25 mg/kg/day) by gavage for 10 days beginning 1 day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e (1 nm) or e+medroxyprogesterone acetate (mpa; 50 pm) with and without simvastatin (1-10 μm). some cultures additionally received interleukin-1 (il-1 , 200 ng/ml). conditioned media was subjected to western analysis for expression of mmp-3. results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin (25 mg/kg/day), the number of endometrial implants was decreased by 83% and the volume by 98% in comparison to the control group. isolated stromal cells expressed abundant levels of mmp-3 following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il-1 induced a dramatic increase in mmp-3 secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp-3 induction by il-1 . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a 26 year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around 2 weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a 3 cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of 9 biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p38, mek, pi3k and ikk. after a period of 24 or 48 hours, concentrations of il-6, mcp-1, and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp-1 and gm-csf were 2-3 times higher, while il-6 was 10 times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il-6 levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il-6 and mcp-1 following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig-6, a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig-6 knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig-6 expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n=20) women and pcos (n=15) women. regulation of mig-6 was studied in ishikawa and ecc-1 endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag2-(c) immunocompromised mice, treated with e 2 or e 2 + p pellets to mimic a natural cycle. results: mig-6 protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig-6 protein and mrna than normal endometrium (p < 0.01). xenografts using pcos samples had paradoxically elevated expression of mig-6 compared to normal, and appeared unresponsive to steroid treatments. mig-6 expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e 2 or e 2 + p. conclusions: mig-6 expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig-6. reduced expression mig-6 provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u54-hd35041(sly), u54hd055764 (lcg) and u54hd77495 (fjd). could androgens influence human luteal cells function? anna tropea, 1 mariateresa orlando, 1 maria francesca gangale, 1 federica romani, 1 isamaria loiudice, 1 federica tiberi, 2 stefania catino, 3 antonio lanzone, 3 rosanna apa. 1 1 cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; 2 istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; 3 istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge2 and pgf2a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and 17--estradiol on p, vegf, pge 2 and pgf 2 secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for 24 h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from 10 -10 to 10 -6 m) or in presence of increasing concentrations of estriol, estrone and 17--estradiol (from 0.1 to 10 ng/ml). moreover, testosterone and androstenedione 10 -7 m were tested in presence of exemestane (from 4 to 400 nm). in the culture medium vegf, p, pge 2 and pgf 2 secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf 2 secretion, while an increase was observed on pge 2 production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge 2 production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf 2  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf 2 release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, 1 mariateresa orlando, 1 maria francesca gangale, 1 federica romani, 1 isamaria loiudice, 1 federica tiberi, 2 stefania catino, 3 antonio lanzone, 3 rosanna apa. 1 1 cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; 2 istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; 3 istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e 2 and increase pgf 2 luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf 2 and pge 2 production was also analysed. highly purified human luteal cells were incubated for 24h with medium alone (control) or with hcg (100 ng/ml) or with cocl 2 (10 μm) or in presence of increasing concentrations of unacylated ghrelin (1-100 nm) and obestatin (1-500 nm). in the culture medium vegf, pgf 2 , pge 2 and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge 2and to reduce pgf 2 release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform 3 (sk3) in transgenic mice compromised parturition, suggesting a role for sk3 channels in this process. based on these findings, we hypothesized that sk3 channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk3 channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk3 (sk3 t/t ) mice to wild-type (wt). in wt mice, sk3 transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk3 t/t mice on the day of delivery was significantly less than wt or sk3 knockout control (sk3 dox ), and the contractile force reached wt levels by application of the sk3 channel inhibitor, apamin. moreover, lipopolysaccharide and ru486, which induce pre-term labor in wt mice, failed to result in completion of delivery in sk3 t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk3 channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk3 gene promoter region contains two specificity protein (sp) binding sites; 1) sp1, is a transcription factor known to enhance the transcription of genes in response to estrogen, and 2) sp3, which competes for the same binding motif as sp1, reduces gene expression. sp1 protein expression in mice uteri dramatically decreases in late pregnancy, while sp3 protein level remains consistent. our data indicate that sk3 channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk3 channel expression may be transcriptionally regulated by sp1 and sp3.